Indian Journal of Research in Pharmacy and Biotechnology

Krupa Rani Gorthi and Aravind G
ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology
QUANTITATIVE ANALYSIS OF ACETAMINOPHEN IN NUROMOL TABLETS BY HIGH PRESSURE LIQUID CHROMATOGRAPHY

Krupa Rani Gorthi1* Aravind G2 1. Sheffield Hallam University, Sheffield, United Kingdom 2. Nimra College of Pharmacy, Ibrahimpatnam, Vijayawada *Corresponding author: Email:krurani@gmail.com ABSTRACT In this study quantitative analysis of Acetaminophen in Nuromol tablets by High pressure liquid chromatography were reported and discussed. The mobile phase as a mixture of the acetonitrile and water 25:75 v/v, the pH was adjusted to 2.5 with the help of phosphoric acid and the flow rate was set to 1.4ml/min; the UV detection was set to 207nm. The column used for the separation of the compounds was the C18 column. The results showed that the calibration curve was linear when plotted and was tested by running the standards from 1 to 10 micrograms/ml and best fit line was obtained. Accuracy achieved is 86.21% and different parameters for validation of a method like precision, limit of detection, limit of quantification, linearity, robustness, repeatability and reproducibility were calculated and the results were within the limits. It is therefore reasonable to conclude that the method can be used for quantification of the acetaminophen in Nuromol tablets by high pressure liquid chromatography. KEYWORDS: Acetaminophen, quantitative analysis, high pressure liquid chromatography 1. INTRODUCTION Nuromol is a painkiller contains combinations of 200mg of ibuprofen and 500mg of acetaminophen. It is being used for the temporary relief of backache, migraine, headache rheumatic, dental and non dangerous arthritis, cold and fever. Acetaminophen is one among the most used non-narcotic analgesic - antipyretic agents. Ibuprofen is also called as an anti inflammatory and non steroidal drug. Pharmacologically it inhibits action of cyclo oxygenase which produces prostaglandins. Prostaglandins are produced in body when an injury occurs and cause inflammation, pain and swelling, therefore the ibuprofen reduces the pain and inflammation. By minimising the prostaglandins ibuprofen reduces the fever which raises the temperature in the body (Rang and Dales, 2007). The objective of the present scientific method is to develop and validate a simple, reliable, and accurate HPLC method for acetaminophen and ibuprofen. 2. MATERIALS AND METHODS Chemicals that were used for the experiment were Triethylamine, which is ion pair reagent, purity of 99.6% (from VMR international ltd), Acetonitrile, is HPLC grade which is stored at <30 degree centigrade (from Fisher scientific UK ltd), Methanol, which is HPLC grade (code: M/4056/17), orthophosphoric acid is from sigma Aldrich. The HPLC used was Perkin Elmer and series 200 pump and series is 200 UV/visible detector. The column used was C18 and the drugs that were used in the experiment were acetaminophen and ibuprofen (Nuromol) tablets. Chromatographic conditions that were used for the analysis is the mobile phase as a mixture of the acetonitrile and water 25:75 v/v, the pH was adjusted to 2.5 with the help of phosphoric acid and the flow rate was set to 1.4ml/min; the UV detection was set to 207nm. 2.1. Preparation of the standard solutions: Different standards ranging from 1, 2, 3, 4 and 5 microgram/ml from a stock solution of 100 microgram/ml concentration. All the standards were run for several times the mean of the peak area was calculated. The calibration curve was plotted and it was a good best fit line. Again the standards were prepared ranging from the 1 to 10 microgram/ml to see the linearity, the standards were run through the HPLC in triplicate and the mean of the peak area was calculated and plotted the calibration curve, by taking the peak area on Y-axis and the concentration on X-axis it was a good linear curve.
Acetaminophen
600000
y = 110288x R2 = 0.9987
500000 400000
Peak area
Series1 300000 Series2 Linear (Series1) 200000 100000
0 0 1 2 3 Concentration 4 5 6
Figure: 1 Chromatogram of Acetaminophen Volume 1 Issue1
Figure: 2 Calibration curve for Acetaminophen Page 1
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Table: 1 Calibration values for Acetaminophen: Experimental data showing the mean of the peak area of Acetaminophen of standards Concentration Peak area of Concentration Peak area of (microgram/millilitre) acetaminophen (microgram/millilitre) acetaminophen 1 117933.98 4 438583.07 118541.26 448428.59 117043.20 428049.60 118232.15 431510.70 117937.64 x 436642.99 231130.10 5 550008.12 233739.96 549109.25 234718.30 548903.80 232514.80 539045.50 x 233025.79 x 546766.66 3 329190.94 339694.16 328068.20 338290.80 x 333811.02 2.2. Preparation of sample solutions: The sample was weighed about 0.1248grams and was taken in a conical flask added 50ml of methanol and was kept in the ultrasonic bath for 15 min to dissolve completely. The solution was filtered into a 100ml volumetric flask and was made up to the mark with the mobile phase. From this solution again 1ml was pipetted out in to a 100ml volumetric flask and was made up to the mark with the mobile phase. The sample was injected in to the HPLC with the help of a micro syringe and chromatograms were recorded for four times, the mean of the peak area was calculated and plotted against the absorbance for finding out the concentration. The concentration of the acetaminophen was 4.88 microgram/ml. Table: 2 Acetaminophen: Experimental data showing the mean of Acetaminophen, of the Sample and their recovery from 0.1248 grams of the sample Sample Powder Peak area of Recovery weight(0.1248grams) Acetaminophen 546059.52 520399.38 537560.90 549073.80 x 538273.4 86.21% 3. RESULTS AND DISCUSSION Method validation is carried out to assert the analytical procedure applied for a test is suitable. The results obtained from the validation method are being used to justify the reliability, consistency and quality of the analytical results. The parameters for the method validation are accuracy, precision, repeatability, reproducibility, linearity, and limit of detection, limit of quantification, robustness and specificity. The method had been validated by calculating the above parameters Table: 3 Validation parameters of acetaminophen R.S.D L.O.D L.O.Q Linearity x 3xS.D/slope 10xS.D/slope 117937.64 0.17 0.01 0.05 0.9987 233025.79 0.66 0.04 0.14 333811.02 1.54 0.13 0.46 436642.99 1.43 0.17 0.56 546766.66 0.94 0.14 0.46 Accuracy: The above results show that the accuracy obtained is 86.21%. Precision: precision was calculated by finding the standard deviation was obtained using regression values and it was found to be less than 5%. The values were close to each other. Reproducibility: The method was repeated several times and on repeating the recovery obtained was the same. Volume 1 Issue1 January February 2013 Page 2 x 2
Linearity: The linearity obtained was 0.9987. Limit of detection: The limit of detection was found to be 0.01microgram/ml. Limit of Quantification was at 0.05microgram/ml. Robustness: The different operational parameters such as pH, column temperature, Wavelength, flow rate, mobile phase composition and the volume injected had a tolerance that is specified. 4. CONCLUSION All statistical values Limit of detection (L.O.D), Limit of quantification (L.O.Q) and Relative standard deviation (R.S.D) were within the limits. This method could be more suitable for routine analysis acetaminophen in Nuromol Tablets. Further research can be performed by developing a method for combination of acetaminophen and ibuprofen with other drugs. Raman micro spectroscopy can be used for the evaluation of acetaminophen and ibuprofen and its combinations. By using mass spectroscopy molecular weight of acetaminophen and ibuprofen and its combinations can be identified. Pre-treatment of the samples can be done to minimise the errors, and to achieve the complete extraction different methods of extraction steps can be used. REFERENCES Brent D Karcher, A 21st Century HPLC Workflow for Process R&D, Journal of the Association for laboratory automation, 10(6), Dec 2005, 381-393. http://www.drugbank.ca/drugs/DB00341 Ghada M. Hadad, Development and validation of a stability indicating RP-HPLC method for the determination of Paracetamol with Citirizine and pseudoephedrine in two pharmaceutical dosage forms, 79(5), 2009, 1360-1367. Franeta J T, HPLC assay of Acetylsalicylic acid, Paracetamol, Caffeine and Phenobarbital in tablets, 57(9), 2002, 709-703. Linker R, Soil identification and chemometrics for direct determination of nitrate in soils using FTIR-ATR midinfrared spectroscopy, Chemosphere, 61 (5), 2005, 652-658. Marika Kamberi, A validated, sensitive HPLC method for the determination of trace impurities in acetaminophen drug substance, 34(1), 2004, 123-128. Marin E, Validation of a HPLC quantification of Acetaminophen, Phenylephrine and Chlorpheniramine in pharmaceutical formulatins: Capsules and sachets, 29(4), 2002, 701-714.
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Harish Gopinath et. al
FORMULATION AND EVALUATION OF CAPTOPRIL MICROENCAPSULES: A SUSTAINED RELEASE APPROACH Harish Gopinath1*, Koteswararao Pasupuleti1, Debjit Bhowmik, Duraivel S1 Department of Pharmaceutics, Nimra College of pharmacy, Vijayawada *Corresponding author: E.Mail:harishgopinath4u@gmail.com ABSTRACT The aim of the present study is to formulate and evaluate Captopril micro-encapsules. Microspheres and micropellets of captopril prepared with different polymers through the techniques of microencapsulation were found to have a good spherical shape, and were non-aggregated exhibiting good flow properties. In addition, all the formulations prepared, showed good drug incorporation efficiency and an extended release of the drug, thereby enhancing the duration of action. The different techniques used in the preparation such as, emulsion-phase separation, solvent-evaporation and ionotropic-gelation techniques were found to be simple and reproducible. All the polymers used viz., chitosan, ethyl cellulose, sodium alginate and Hydroxy propyl methyl cellulose (HPMC) were economic, easily available and biocompatible. The industrial applicability of the methods would be simple and rapid, provided the work being extended to in-vivo studies. It is obvious from the above work that, the study has engineered a drug delivery profile in which the drug release is controlled to a great extend and that the formulations in therapy can minimize untoward side effects, thus improving patient compliance. Keywords: Captopril, Microencapsulation, Sustained release 1. INTRODUCTION The advance drug delivery techniques aim to improve overall drug performance and efficacy. There is now a growing realization that innovative delivery of drugs would not only increase safety and efficacy levels, but also increase the overall performance of the drug. The advances in drug delivery systems are also expected to offer a host of additional advantages such as ease of administration, increased patient compliance, decreased side effects and cost reduction. Moreover, novel drug delivery techniques are value added features for which companies can charge a premium due to the increased convenience they provide to patients. In the study, the method adopted for sustaining the drug release was microencapsulation. The different techniques of Microencapsulation and the polymers used in the study were Emulsification-Phase separation using chitosan, Solvent evaporation using ethyl cellulose, ionotropic gelation using sodium alginate and combination of sodium alginate with HPMC. The process of microencapsulation enables us to achieve the taste-masking, selective sorption and sustained release, reduced gastric irritation and conversion of liquid to solid form for stabilization, reduction of volatility, stabilization to oxidation. The drug used in the study was Captopril, a widely used antihypertensive drug, having a shorter half life of 2 hrs and with a bioavailability of 75% and the daily dose being 75 to 150 mg in divided doses, the drug satisfies all the criteria needed for a drug to be formulated into a sustained release system. 2. MATERIALS AND METHODS 2.1 Material: Captopril obtained as a gift sample from Wockhardt Laboratories Ltd. Chitosan from Cochin Fisheries, Ethyl cellulose (EC) and Sodium alginate (SA) from Loba Chemie and HPMC from Kemphasol Acetone, chloroform, Petroleum ether and Whatmann filter paper was obtained as gift sample from Qualigens, Calcium chloride from Ranbaxy, Glacial acetic acid from S.D Fine Chem. Ltd., Liquid paraffin (light) from Fisher Ltd., Glutaraldehyde from Kemphasol Ltd., Hydrochloric acid from Chemspure Inorganics & Aromatics. 2.2 Pre Formulation Studies 2.2.1 Drug- excipients compatibility studies: Before formulation of drug substances into a dosage form, it is essential that it should be chemically and physically characterized. Pre-formulation studies give the information needed to define the nature of the drug substance and provide a framework for the drug combination with pharmaceutical excipients in the fabrication of a dosage form. In this Compatibility studies one of the requirements for the selection of suitable polymers or carriers for pharmaceutical formulation is its compatibility. Therefore in the present work, a compatibility study was carried out by using an infrared spectrophotometer to find out if there is any possible chemical interaction between Captopril and the polymers (Chitosan, Ethyl Cellulose, Sodium Alginate and HPMC). Weighed amount of the drug (3mg) was mixed with 100mg of potassium bromide (dried at 40-50C), which was then compressed under 10-tonn pressure in a hydraulic press to form a transparent pellet. Similarly, was prepared the pellets of individual polymers and that in combination with the drugs which was then scanned from 4000-400cm-1 in IR spectrophotometer. 2.2.2 IR Spectral Analysis: Using FTIR 410 PC spectrometer carried out the compatibility studies between the drugs and the polymers. There was no appearance or disappearance of any characteristic peaks, which confirmed the absence of any chemical interactions between the drug and polymer. The results are showed in figures 2(a) to 2(j). 2.2.3 Captopril pure drug analysis: The absorbance of the prepared solutions was checked using a UV spectrophotometer at 226.6nm. 0.1N acetic acid was used as the blank. The procedure was repeated with pH 1.2 buffer. The results were tabulated table number 2. Volume 1 Issue1
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2.3 Method of Preparation 2.3.1 Chitosan microspheres: Chitosan microspheres containing Captopril was prepared by phase separation emulsion technique. Light liquid paraffin and petroleum ether was used as the external phase and a solution of chitosan in acetic acid as the dispersed phase. In this Procedure a weighed quantity of chitosan was added to 6% acetic acid and the resulting solution was stirred for one hour to form a gel. Captopril was added and stirred for fifteen minutes to get a gelled mixture. The gelled chitosan mixture was added dropwise into a dispersion medium containing liquid paraffin and petroleum ether. The resulting dispersion was stirred using a stainless steel remistirrer at 1000rpm for 10minutes.1ml of glutaraldehyde saturated with toluene was then added and stirring was continued for 1hr.The microspheres formed were filtered and washed several times with petroleum ether and dried overnight. 2.3.2 Ethyl cellulose microspheres: Ethyl cellulose microspheres were prepared by emulsification solvent evaporation method. In this Procedure a Weighed amounts of the drug and the polymer was dissolved in chloroform and added drop wise to 200ml liquid paraffin containing 2% w/v of span 80 and stirred at 1000 rpm for 3 hrs. The formed microspheres were filtered by vacuum filtration and washed several times with petroleum ether, and water and then air dried. 2.3.3 Sodium alginate microcapsules: Sodium alginate microcapsules were prepared by Ionotropic gelation technique. In this Procedure a Weighed quantity of the drug was added to sodium alginate solution and mixed at 500rpm. Resultant solution was extruded drop wise with the help of a syringe and needle into 2%calcium chloride solution and stirred at 100rpm. Stir for 10 minutes; the formed microcapsules were separated, washed with water and dried at 700C for 6hours in an oven. Another set of microspheres was prepared with a combination of sodium alginate and HPMC. 2.4Evaluation Studies of Formulated Microspheres and Microcapsules 2.4.1 Drug content analysis: UV spectrophotometeric method was used to analyze the drug content in the prepared microspheres and micropellets of varying drug-polymer ratios. Drug was extracted from the microspheres and micropellets with phosphate buffer pH 7.2 and absorbance was measured using UV spectrophotometer at 226.6nm. The amount of drug in the prepared microspheres and microcapsules was estimated using the standard graph. 2.4.2 Entrapment efficiency: The entrapment efficiency of Captopril in the prepared microspheres and micropellets was determined from the ratio of weight of Captopril incorporated to the weight of Captopril initially taken. % =
2.4.3 Micromeritic studies: In this size, shape, entrapment efficiency, bulk density, true density and porosity (), angle of repose and Hausners ratio were studied. The results were tabulated table number 2. 3. RESULTS AND DISCUSSION The microspheres and micropellets were evaluated for drug content, and incorporation efficiency, Scanning Electron Microscopy, and particle size analysis, Micromeritic properties and in-vitro dissolution profiles. Chitosan microspheres had a good drug incorporation efficiency that ranged from 60.0% to 66.0% while that of ethyl cellulose microspheres was found to be between 68.0 to 72.0%. The entrapment efficiency of sodium alginate micropellets was between 69.2% to 78.7% where as that prepared with a combination of sodium alginate and HPMC showed an efficiency ranging between 72.0% to 79.4 SS%. The result was tabulated table number 2. 3.1 Scanning electron microscopy (SEM): The surface morphology of the prepared microspheres and microcapsules was shown to be spherical by the SEM photographs. The SEM image was shown in Figure 1. 3.2 Particle size analysis: The particle size analysis was carried out using an optical microscope. The arithmetic mean particle size of various microspheres and micropellets prepared are shown in tables 5 to 20. The particle size distribution of chitosan microspheres ranged between 223.4 m to 286.2 m, whereas that of ethyl cellulose microspheres was between 226.8 m to 289.5 m. The sodium alginate microcapsules showed a particle size distribution between 281.4 m to 369.9 m whereas that of microcapsules prepared with a combination of sodium alginate and HPMC showed a particle size distribution that ranged between 280.1 m to 362.4 m. 3.3 Micromeritic properties: The various Micromeritic properties of the prepared microspheres and microcapsules were studied. Acceptable range of angle of repose is between 20 o-40o and all the formulations showed an angle of repose within the range. Formulations C1, C2, E1, E2, S3 and SH1 showed an angle of repose less than 25o and hence exhibit excellent flow properties. Acceptable range of Haus ners ratio is upto 1.25 and all the formulations had a value less than 1.25, thereby exhibiting good flow properties. The results were shown in Table 2. 3.4 In-vitro release studies: Captopril release from the formulations was studied in acid buffer (pH 1.2) for 2 hours and in phosphate buffer (pH 7.4) for the next 6 hours. The release pattern was slow and spread over Volume 1 Issue1
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extended periods of time. In the case of chitosan microspheres, C3 had the highest drug incorporation (68%) and showed a drug release of 11.70% in acid buffer and 54.36% in phosphate buffer after 8 hours. Among the ethyl cellulose microspheres prepared, E3 showed the highest drug incorporation (77.6%) and exhibited a drug release of 7.92% in acid buffer and 52.02% in phosphate buffer. Formulation S4 of the sodium alginate microcapsules showed the highest drug content (78.7%) which exhibited a drug release of 7.22% in acid buffer and 57.41% in phosphate buffer. For the micropellets prepared with a combination of sodium alginate and HPMC, formulation SH4 showed maximum drug content (79.4%) with a drug release of 4.81% in acid buffer and 46.98% in phosphate buffer. The results were shown in Table 3. 4. CONCLUSION The microspheres and micropellets of captopril prepared with different polymers through the techniques of microencapsulation were found to have a good spherical shape, and were non-aggregated exhibiting good flow properties. In addition, all the formulations prepared, showed good drug incorporation efficiency and an extended release of the drug, thereby enhancing the duration of action. The different techniques used in the preparation such as, emulsion-phase separation, solvent-evaporation and ionotropic- gelation techniques were found to be simple and reproducible. All the polymers used viz., chitosan, ethyl cellulose, sodium alginate and HPMC were economic, easily available and biocompatible. And hence, the industrial applicability of the methods would be simple and rapid, provided the work being extended to in-vivo studies. It is obvious from the above work that, the study has engineered a drug delivery profile in which the drug release is controlled to a great extend and that the formulations in therapy can minimize untoward side effects, thus improving patient compliance.
Table.1.Standard graph of captopril at 226.0nm

Concentration (g/ml) 5 10 15 20 25 Absorbance Std Graph at pH 1.2 Std Graph at pH 7.4 0.1720 0.1910 0.3641 0.3954 0.5601 0.5764 0.7421 0.7752 0.9310 0.9612
Table.2.Evaluation of formulation and micromeritic properties

S. No Formulatio n code Formulation characters Entrapment Arithmetic mean efficiency (%) particle size Bulk density (g/ml) Micromeritic Properties Angle of True density Porosity (%) (g/ml) repose () Hausners ratio
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
C1 C2 C3 C4 E1 E2 E3 E4 S1 S2 S3 S4 SH1 SH2 SH3 SH4
60 64 68 66 68 72.8 77.6 72 69.2 73.6 77.6 78.7 72 77.6 77.9 79.4
223.425 m 261.99 m 270.12 m 286.2 m 226.8 m 250.425 m 262.275 m 289.575 m 281.475 m 294.49 m 332.5 m 369.9 m 280.15 m 300.375 m 326.17 m 362.473 m
2.176 1.947 1.714 1.626 1.19 1.123 0.909 1.9433 0.9433 0.8928 0.8982 0.8620 1.2081 1.2341 1.18 1.06
2.968 2.4 2.007 1.828 1.31 1.25 1.162 1.063 1.010 0.9615 0.9433 0.8928 1.4310 1.3891 1.21 1.18
27 20.8 22.7 11.11 15.38 12 18 11.3 6 7.29 5.3 5.6 14.2 7.3 8.3 10
23.97 21.96 29.28 30.96 22.9 23.4 29.7 28.9 28.2 26.2 24.2 30.2 24.8 27.9 26.5 29.9
1.3 1.26 1.176 1.125 1.18 1.93 1.22 1.12 1.06 1.07 1.05 1.23 1.16 1.08 1.09 1.11
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Figure 1. SEM of the Captopril microcapsules
Table.3. In-vitro Dissolution Studies

S. No Formulation code C1 C2 C3 C4 E1 E2 E3 E4 S1 S2 S3 S4 SH1 SH2 SH3 SH4 Percentage cumulative amount of drug release pH 1.2 pH7.4 1hr 2hr 3hr 4hr 5hr 6hr 7hr 10 15.3 23.04 35.32 42.6 48.2 55.64 7.74 12.24 18.18 23.74 37.58 45.6 53.64 4.15 11.7 20.88 27.36 35.22 43.01 49.8 1.34 5.5 10.26 18.18 24.84 33.82 42.66 7.56 14.42 24.62 31.86 42.03 50.9 59.74 6.36 12.24 18.84 26.82 37.36 48.96 55.26 1.53 7.92 12.96 18.21 27.9 36.36 46.44 0.80 4.32 9.12 16.56 24.48 32.04 41.76 8.46 14.95 24.3 32.01 46.40 57.96 61.42 7.02 11.7 18.9 25.02 38.88 48.96 55.6 4.68 10.8 18.4 30.24 37.08 46.98 53.04 1.2 7.22 14.22 21.24 32.54 44.46 50.01 10.8 15.48 24.3 30.42 42.52 49.5 57.96 8.82 12.96 15.3 26.28 37.26 46.16 56.16 3.78 10.98 14.76 20.88 39.1 41.94 36.62 1.08 4.81 12.24 18.9 28.16 33.12 42.66 8hr 59.04 57.60 54.36 51.84 62.82 60.8 52.02 47.7 66.24 64.08 62.52 57.41 62.64 60.48 54.36 46.98
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Figure 2(a) FTIR studies of pure drug Captopril
Figure 2(b) FTIR studies of Chitosan
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Figure 2(c) FTIR studies of Ethyl cellulose
Figure 2(d) FTIR studies of Sodium alginate
Figure 2(e) FTIR studies of HPMC
Figure 2(f) FTIR studies of HPMC+ Sod.Alginate
Figure 2(g) FTIR studies of Captopril + chitosan
Figure 2(h) FTIR studies of Captopril + Ethyl cellulose
Figure 2(i) FTIR studies of Captopril + Sod.alginate
Figure 2(j) FTIR studies of Captopril + Sod.alginate+ HPMC
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REFERENCES Barry Bleidt and Michael Montagne, Clinical research in pharmaceutical development, Marcel Dekker Inc, New York, 75, (2000), 23-25 Bhagwatwar H P, Biodegradable microparticles of peptide drugs using poly lactide polymers in advances in controlled and novel drug delivery, N K Jain, 1st Edn, CBS Publishers and Distributors, Delhi (2001), 12-14 Garima Chawla, Piyush Gupta and Aravind K Bansal., Gastro retentive drug delivery systems: progress in controlled and novel drug delivery systems, N K Jain, CBS Publishers and Distributors, Delhi, 1st Edn, 2004,76-98 Gwen M Jantsen and Joseph R Robinson, Sustained and controlled drug delivery systems in modern pharmaceutics, Gilbert S. Banker and Christopher T Rhodes, Marcel Dekker, Inc., New York, 14th Edn, 1996, 501-529. Jane E Fraser and Gordon F Bickerstaff, Entrapment in calcium alginate, Methods in BiotechnologyImmobilization of Enzymes and Cells, G F Bickerstaff (ed.),Human pres Inc., Totowa, New Jersey (1998),61-63. Jayakrishnan A and Latha M S, Biodegradable polymeric microspheres as carriers in controlled and novel drug delivery, N.K.Jain, CBS Publishers and Distributors, Delhi, 1st Edn, 1997, 327-357. Praveen R. Chaturvedi, Pharmacokinetics of microparticulate systems for the delivery of proteins and vaccines in microparticulate systems for the delivery of proteins and vaccines, Smadar Cohen and Howard Bernstein(eds.), ,Marcel Dekker, Inc., New York, 77, 1996, 321-349. Robert Langer and Joseph Kost, Real time response polymeric drug delivery systems in temporal control of drug delivery, William I.M.Hrushesby, Robert Langer and Felix Theuwes, New York Academy of Sciences, 68, 1991, 330-335 Shein Chung Chow and Jen Peiliu, Statistical design and analysis in pharmaceutical science-validation, process controls and stability, Marcel Dekker, Inc, New York, 143, 2001, 271-292. Shrikanth V Dighe and Wallace P Adams, Bioavailability and bioequivalence of oral controlled release products in pharmacokinetics, regulatory, industrial, academic perspectives, G Welling and Francis L S, Marcel Dekker, Inc.,New York, 33, 1988, 307-337 Stephen D Bruck, Controlled drug delivery-clinical applications, Vol., CRC press, Inc., Florida (2 000), 1-15 Sucker H., Pharmacokinetics and pharmaceutical technology in pharmacokinetics-A modern review, Leslie Z. Benet, Gerhard Lery and Bobbea L.Ferrailolo (eds.), Plenum press, New York (1984), 97-105 Victor A. Crainich., Microencapsulation: scale -up considerations and production technology in specialized drug delivery systems-manufacturing and production technology, Praveen Tyle, Marcel Dekker, Inc, New York, 41, 1990, 221-257 Vyaas S P and Khur R K, Targeted and controlled drug delivery-novel carrier systems,1st Edn, CBS Publishers and Distributors, Delhi, 2002, 417-458 Wilfred J Westlake, Bioavailability and bioequivalence of pharmaceutical formulations in biopharmaceuical statistics for drug development, Karl E Peace, Marcel Dekker Inc, New York, 86, 2000, 329-353
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Harish Gopinath et.al.,
FORMULATION, CHARACTERIZATION AND IN-VITRO DISSOLUTION STUDIES OF DICLOFENAC SODIUM TABLETS THROUGH SOLID DISPERSION USING POLY ETHYLENE GLYCOL
*Harish Gopinath1, Haritha M1, Duraivel S1, Debjit Bhowmik1, Sangeetha S2 Department of Pharmaceutics, Nimra College of Pharmacy, Vijayawada, India 2 Departments of Pharmaceutics, SRM College of Pharmacy, SRM University, Chennai, India *Corresponding Author: E.Mail: harishgopinth4u@gmail.com
1
ABSTRACT
In the present study the formulation of Diclofenac sodium tablet from poly ethylene glycol solid dispersion has been carried to enhance the bioavailability and solubility of the drug, Diclofenac. The post compression studies were carried out for the prepared solid dispersion and the compressed tablets and, it was found that all the parameters were in the limits. All the formulations showed rapid release when compared to Diclofenac sodium immediate release marketed tablets. The drug release after 90 minutes of dissolution was 28.35% for marketed tablet and for tablets formulated with solid dispersion was 95.62% with Diclofenac sodium: PEG-6000 combination. Keywords: Diclofenac sodium (DS.), Solid dispersion (SD), Poly ethylene glycol (PEG), 1. INTRODUCTION The oral absorption and bioavailability of a drug can be increased by Salt formation, Solublisation, particle size reduction, complexation, solid solution and solvate formation. However there are practical limitations to these techniques. These limitations can be overcome by formulating a drug as a Solid dispersion. Solid dispersion refers to the dispersion of one or more active ingredients in an inert carrier on matrix at solid state prepared by solvent method. Different types of Solid dispersions are simple eutectic mixtures and solid solutions (Christopher B, 2000). These solid solutions are further classified based on the way in which the solute molecules are distributed in the solvent as substitutional crystalline solid solutions, interstitial crystalline solid solutions and amorphous solid solutions. The various carriers used for solid dispersions are poly ethylene glycols, polyvinyl pyrrolidine, cellulose derivatives, polyacrylates and polymethacrylates, urea, sugar polyols and their polymers and organic acids and their derivatives. Solid dispersions are prepared by techniques like Fusion process, Solvent evaporation method and Melting solvent method (Faiez Zannad, 2000) (Fang Gao, 2010). Different methods that have been used to characterize solid dispersion are thermo analytical methods, Differential thermo analysis, Hot stage microscopy, Spectroscopic methods, Microscopic methods, colourimetric analysis of the solution, Dissolution testing. In this topic a solid dispersion of a non steroidal anti inflammatory drug (NSAIDS) Diclofenac Sodium was prepared and characterized. NSAIDS are preferred because they relieve pain without interacting with opioid receptors and they possess anti-inflammatory activity and uricosuric property. They also reduce the elevated body temperature. (Giuseppe Mancia, 1999) (Graham A, 2000) 2. MATERIALS AND METHODS Diclofenac Sodium was received as a gift sample from Ind-Swift and poly ethylene glycol-6000 was obtained from Loba Chemicals, Mumbai, Polyethylene glycol 4000 was received from Sisco Research Laboratories Pvt. Ltd., Mumbai, Methanol was received from Fischer In-organics, Chennai. 2.1 Preparation of Solid Dispersion of Diclofenac Sodium: Solid dispersion can be prepared by prepared by kneading method and Solvent evaporation method. The various carriers used are PEG 4000, PEG 6000 alone and PEG 4000 and PEG 6000 in combination at equal ratio in each formulation. (Herbert A, 2000) (Hideki Igata, 1998) (H L Sharma, 2007).
Table 1: Drug carrier ratios in respective amount taken for solid dispersion Drug : Carrier ratio Drug (mg) Carrier (mg) 1000 1000 1:1 1000 2000 1:2 1000 3000 1:3 1000 4000 1:4 1000 5000 1:5
2.2 Evaluation of Diclofenac Sodium Solid Dispersion 2.2.1 Drug Content Uniformity: Accurately weighed amount of solid dispersion (equivalent to 100 mg of Diclofenac Sodium) was dissolved in minimum quantity of methanol and made up with 0.1 M Ph 7.4 phosphate buffer solution and distilled water is added to make up to 100 ml in a standard flask. The stock solution after suitable dilution was measured for absorbance at 276 nm using JASCO-V-530 UV Spectrophotometer and noted. 2.2.2 In-vitro Dissolution Studies of Different Solid Dispersions of Diclofenac Sodium: Dissolution rate studies of various solid dispersions were carried out in 0.1M.PH 7.4 Phosphate buffer using XXII Dissolution rate apparatus (Electro Lab). Paddle type stirrer was used which is adjusted to 100 rpm. Temperature was maintained at 37.4 0C and absorbance was measured at 276 nm. Volume 1 Issue 1 January February 2013 Page 10
3. RESULT AND DISCUSSION 3.1. Evaluation of Tablets: The formulated Diclofenac sodium tablets were subjected for the following IPQA tests such as hardness, friability and weight variation and the results were within the results. 3.2. Dissolution rate studies: Dissolution of each formulated Diclofenac tablet was studied in USP paddle type dissolution rate apparatus. 500ml of 0.01M pH 7.4 buffer solution containing each of the formulated Diclofenac sodium tablets was placed in the dissolution medium. The paddle type of stirrer was adjusted to 100rpm the temperature was maintained at 37.40C, 5 ml aliquot of dissolution media was withdrawn and replaced with fresh quantity of dissolution media. The samples were analyzed for Diclofenac sodium by measuring absorbance at 276 nm using JASCO 530 UV-Visible spectrometer. pH 7.4 is used as the blank. The percentage of Diclofenac sodium dissolved at various intervals was calculated and plotted against time. The results are shown in the Table 3.
Solid dispersion Table 2. Drug content uniformity Drug: carrier Amount of SD Expected taken amount of DS in SD(mg) 1:1 100 50 1:2 150 50 1:3 200 50 1:4 250 50 1:5 300 50 1:1 100 50 1:2 150 50 1:3 200 50 1:4 250 50 1:5 300 50 1:1 100 50 1:2 150 50 1:3 200 50 1:4 250 50 1:5 300 50 % of DS estimated by UV spectrophotometer 98.50 99.00 98.25 99.87 99.17 99.13 98.75 99.35 98.83 99.93 99.20 98.94 98.76 99.50 99.27
Diclofenac sodium PEG-4000
Diclofenac sodium PEG-6000
Diclofenac sodium (PEG-4000 & PEG6000)
Table 3. Dissolution of Diclofenac sodium from SD with PEG-6000 in different Drug:Carrier ratios Time in min Percentage of Diclofenac sodium dissolved from 1:1 1:2 1:3 1:4 1:5 Marketed formulation (MD) 10 1.35 1.80 10.17 3.33 6.75 2.70 15 4.50 7.20 27.00 12.33 20.25 10.35 30 10.35 15.75 56.25 29.25 38.25 18.00 45 14.85 27.90 65.25 42.75 54.00 34.65 60 25.65 36.00 72.00 54.90 65.25 42.75 90 28.35 43.65 87.75 67.50 76.50 55.35
4. FORMULATION OF DICLOFENAC SOLID DISPERSION TABLETS Diclofenac Sodium Solid dispersions at a drug carrier ratio of 1:2 was formulated into tablet and evaluated. 4.1 Direct Compression Method: In this method powdered material was directly compressed into a tablet modifying the physical nature of material itself thus hydrolysis of water sensitive drugs can be avoided. In this method a direct compression vehicle (DCV) having good flow and compressibility is used. 4.2 Preparation of Diclofenac solid dispersion Tablet: Tablet containing 50 mg was prepared using Solid dispersion and additives along with ascorbic acid as antioxidant. The total weight of tablet is 600 mg. the following materials such as diclofenac Sodium Solid dispersion, Microcrystalline cellulose, Magnesium Stearate, Lactose were used for the preparation of the tablet (Ketelhut, 2010), (Karin Skov, 1998) (Jennifer Wang., 2010). 5. Evaluation of Tablets The formulated Diclofenac sodium tablets were subjected for the following quality control tests .Hardness, Friability, weight variation, Drug content uniformity and Dissolution rate studies USP Dissolution rate apparatus (Electro lab) is used. Dissolution medium used is 500 ml. of 0.01M, P H 7.4 buffer solution. Paddle type of stirrer was adjusted to 100 RPM; Temperature was maintained at 37.4 0 C. The absorbance of samples was measured at 276 nm using JASCO 530 UV-Visible Spectrometer. The solid dispersion prepared was studied for the drug release characteristics and based on the drug release 1:5 (Drug: Carrier) ratio of DS: PEG-6000 was selected for tablet formulation. The dissolution of Diclofenac sodium from tablet formulation based on solid dispersion was found to be fast and rapid when compared with marketed formulation. The additives added have not hindered the Volume 1 Issue 1 January February 2013 Page 11
dissolution of Diclofenac from the solid dispersion. All the formulations showed rapid release when compared to Diclofenac Sodium marketed tablet.
100 80 60 40 20 0 0 20 40 60 80 100 Time (Minutes) MD 1:1 1:2 1:3 1:4 1:5
Percentage drug release
Figure 1: Dissolution of Diclofenac sodium from solid dispersion of Diclofenac sodium by PEG-6000 in different drug: carrier ratios Table.4. Ingredients for tablets of Diclofenac sodium: Polyethylene glycol solid dispersion S.No Ingredient Quantity in mg/ tablet
1 2 3 4 5 6 Amount of SD taken Microcrystalline cellulose Lactose anhydrous Magnesium state Crosscormellose sodium Colloidal silica Total weight of the tablet 300 50 18 8 20 4 400
Table.5. Percentage of Diclofenac Sodium Dissolved From Tablets

Time(minutes) 10 15 30 45 60 90 Percentage of Diclofenac sodium from
Marketed product Tablet of Diclofenac sodium:PEG6000 SD
1.35 4.50 10.35 14.85 25.65 28.35
5.025 22.50 45.00 73.50 84.37 95.62
6. CONCLUSION Hence the solid dispersion can be mixed with various other additives required in the tablet formulation without losing their rapid dissolution properties. All the formulations showed rapid release when compared to Diclofenac sodium marketed tablet. Hence solid dispersion technique can be employed to enhance dissolution of Diclofenac sodium. REFERENCES Christopher B Granger, Georg Ertl, Jerzy Kuch, Aldo P Maggioni, Randomized trial of Candesartan cilexetil in the treatment of patients with congestive heart failure and a history of intolerance to angiotensin- converting enzyme inhibitors. American Heart Journal, 139, 2000, 609-617. Faiez Zannad, Preserving Target-Organ Function with Candesartan Cilexetil in patients with Hypertension, Infroma health care, Blood pressure, 9, 2000, 36-39. Fang Gao, Zhiwen Zhang, Huihui Ba, Yan Huang, Zhiwei Gao, Jianan Shen, Chunjie Zhao, Yaping Li, Nanosuspension improves the oral absorption of candesartan cilexetil in rats : Performance and mechanism, Journal of controlled release, 1, 2010, 1-7 Giuseppe Mancia, Raffaella Delioro, Carljurri, Guido Grassi, Comparison of angiotensin II Receptor blockers: impact of missed doses of Candesartan cilexetil and lorsartan in systemic hypertension. The American journal of cardiology, 84, 1999, 28-34 Graham A Macgregor, J Reuven Viskoper, Tarek F T Antonios, Feng J He, Efficacy of Candesartan cilexetil alone or in combination with Amlodipine and Hydrochlorothiazide in Moderate to severe Hypertension, Journal of Hypertension., 36, 2000, 454-460
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ISSN: 2320 3471(Online) Harish Gopinath et.al., Indian Journal of Research in Pharmacy and Biotechnology Herbert A Libermann, Leon Lachmann, Joseph B Schwartz, Pharmaceutical dosage forms, Tablet vol I, second ed. revised and expanded, 2000, 106-160 Hideki Igata, Takahito Kitayoshi, Yoshiyuki Inada, Effect of Candesartan cilexetil an Experimental congestive heart failure in Rats and Dogs, Journal of cardiac failure, 4, 1998, 66-69 H L Sharma, K K Sharma, Principles of Pharmacology, Paras Medical Publisher, Hyderabad, First ed, 2007, 250-260. Ichiro Naeshiro, Keijchiro Sato, Fumio Chatani, Shuzo Sato, Possible mechanism for the anemia induced by Candesartan cilexetil (TCV-116), an angiotensin II receptor antagonist, in rats, Eur. J. Pharmcol, 354, 1998, 179-187. Jennifer Wang, Hong Wen, Divyakant Desai, Lubrication in tablet formulation, Eur.J.Pharmcol, 75, 2010, 1-15. Karin Skov, Susie Mogenses, Michael J Mulvany, Persistent effect of treatment with Candesartan cilexetil on blood pressure in spontaneously hypertensive rats, European journal of pharmacology, 315: 1998, 156-157. Ketelhut, Reinhard, Bramlage, Peter, Candesartan cilexetil/ Hydrochlorothiazide Treatment in high- Risk patients with Type 2 Diabetes Mellitus and Microalbuminuria: The CHILI T2D Study, Clinical Drug investigation,5,2010, 301-311.
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Dayananda Bhowmik et.al.,
STUDY OF THE ANALGESIC ACTIVITY OF METHANOLIC EXTRACT OF JASMINE ROOT (Jasminum sambac)
Dayananda Bhowmik1*, DP Chatterjee1, Arunabha Mallik2, Amit Roy3 1. Oriental College of Pharmacy, Raisen Road, Bhopal (M.P.), India 2. Bansal College of Pharmacy, Kokta, Bhopal (M.P.), India 3. Columbia Institute of Pharmacy, Tekari, Raipur (C.G.), India *Corresponding Author: E-mail: dbhowmik2005@yahoo.com; Phone: 9893571085
ABSTRACT The present study was done to evaluate analgesic activity of methanolic extract of root of Jasmine (Jasminum sambac Linn Aiton). Methanolic extract of root of Jasmine was given in the dose of 200 and 400 mg/kg in Wister albino rats and mice of either sex in tail flick and acetic acid induced writhing method respectively. The extract was compared with standard drugs like Buprenorphine (0.05 mg/kg sub cutaneously) and Aspirin (100 mg/kg intra peritonially).The preliminary studies have shown that the methanolic extract of Jasmine root has significant analgesic activity and needs further investigations. Keywords: Jasmine, Jasminum sambac, root, analgesic, writhing, tail flick. INTRODUCTION Jasminum sambac (Linn) Aiton, Family: Oleaceae; commonly known as Jasmine, is an evergreen, somewhat climbing shrub. Leaves are opposite, 3-10 cm long, variable in shape, usually ovate, acute or acuminate or obtuse, entire, glabrous. Flowers are white, very fragrant, solitary or usually in 3-flowered terminal cymes. Jasmine is a vine or shrub reaching up to 1-3 m tall, widely distributed and cultivated throughout India, Myanmar and Bangladesh. It is also distributed and cultivated more or less throughout Srilanka, Pakistan, Nepal, Malaysia, China, Indonesia, France, Spain, Hawaii and tropical Australia (Kirtikar K R and Basu B D, 1987). The traditional use of this plant suggests analgesic, antidepressant, anti-inflammatory, antiseptic, aphrodisiac, sedative, expectorant, anti-spasmodic effects. Essential oil of Jasmine is used as fragrance for skin care products. Leaves and flowers are used as antipyretic and decongestant; roots as analgesic, flowers as lactifuge, flower extract as deodorant. In China, the root is used to treat headaches, insomnia, and pain due to dislocated joints and broken bones; it is reported to have anesthetic properties also (Kirtikar K R and Basu B D, 1996) ( Khare C P, 1998). MATERIAL AND METHODS Plant material: Roots of Jasmine were collected from Medicinal garden of Oriental College of Pharmacy, Bhopal, India in the month of January. The identification and authentication was carried out at Department of Pharmacognosy, Oriental College of Pharmacy, Bhopal by Dr. DP Chatterjee (Taxonomist and Pharmacognosist). A voucher herbarium specimen (OCP/2012/04) of the crude drug material was deposited at the herbarium and museum of the institute. Preparation of extract: Fresh roots of Jasmine were washed and shade dried in department of pharmacology. Shade dried roots were powdered using mixer and grinder, then passed through a #60 mesh sieve and were extracted with methanol in a Soxhlet apparatus by continuous heat extraction. The extract was concentrated in a rotary flash evaporator at a temperature not exceeding 50C. The methanol extract was prepared in distilled water containing 2% v/v Tween 80(as a suspending agent) for experimental purpose. Phytochemical analysis: Phytochemical analysis of different extracts was carried out by successive solvent extraction. Weighed quantity of air dried powdered roots was extracted in soxhlet apparatus successively with solvents started with petroleum ether (60-80C) followed by benzene, chloroform and methanol. After extracting with each solvent, the marc was dried in hot air oven below 50C; finally the marc was macerated with chloroform water for 24 hour. Each extract was concentrated by distilling off the solvent and evaporating to dryness. The dry extracts were subjected to preliminary phytochemical screening for detection of various phytoconstituents. (Harborne J B, 1998). Experimental animals: Albino rats of either sex (200-250 grams) and Albino mice of either sex (30-50 grams) were used in this study. The animals were procured from Jawaharlal Nehru Cancer Hospital and Research Centre, Bhopal and housed in the animal house maintained under standard hygienic conditions, at 20 20 C, humidity (60 10%) with 12 hour day and night cycle, with food and water ad libitum. The study was carried out as per CPCSEA (Committee for the purpose of Control and Supervision of Experiments on Animals) norms after obtaining approval from the Institutional Animal Ethical Committee (Approval No. 1349/ac/2012/02). Acute toxicity studies: The acute oral toxicity studies were performed to study the acute toxic effects and to determine minimum lethal dose of the drug extracts. Swiss albino mice of either sex weighing 18-25 g were used for the study. The methanolic extract was administered orally to different groups of overnight fasted mice at the doses of 30, 100, 300, 1000 and 3000 mg/kg body weight. After administration of the extracts, animals were observed continuously for the first three hours for any toxic manifestation. Thereafter, observations were made at regular intervals for 24 hrs. Further the animals were under investigation up to a period of one week (Ghosh M N, 1984).
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Chemicals, drugs and Instrument: Buprenorphine (Buprinor 2 ml injection, Astra Zeneca) and Tablet Aspirin 75 mg in each tablet (Ecosprin-USV) were purchased from Medical Store. Acetic acid was purchased from S.D. FineChemical Ltd. For tail flick method we used Analgesiometer from Inco (Ambala). Analgesic activity: Analgesic activity of methanolic extract of Jasmine was evaluated by using tail flick and acetic acid writhing method. Tail flick method: Albino rats of either sex were fasted for 12 hours. Basal reaction time to radiant heat was taken by placing tip (last 1-2 centimeters) of the tail on nicrome wire of analgesiometer. Tail withdrawal from the heat is taken as end point. Normal reaction time was 3-4 seconds. Animals failing to withdraw their tail within 3-4 seconds were discarded. Cut off time was taken as 10 seconds to avoid excessive damage to the tail. Animals were divided into 4 groups (each containing 6 rats). Group I (Control) received 2 ml of distilled water containing 2%v/vTween 80 orally. Group II (Standard) received Buprenorphine (0.05 mg/kg sub cutaneously) (Flecknell P A, 1996) 30 minutes before exposure to radiant heat. Group III and IV received methanolic extract of root of Jasminum sambac (Linn) Aiton in the dose of 200 and 400 mg/kg orally respectively 60 minutes before exposure to radian heat. Reaction time was taken at 15, 30, 60 and 90 minutes (Davies O L, 1946). Acetic acid writhing method: Mice of either sex were fasted for 12 hours. Acetic acid (0.6%) 0.1 ml was injected intraperitoneally in all mice. Response was observed as abdominal contraction and relaxation with hind limb extension for 20 minutes. Mice failed to produce writhing were discarded. Then animals were divided into 4 groups similar to tail flick method. Group I (Control) received 2 ml of distilled water containing 2% v/v Tween 80 orally. Group II (Standard) received Aspirin (100 mg/kg orally) (Kulkarni S K, 1999) 60 minutes before acetic acid injection. Group III and IV received methanolic extract of root of Jasminum sambac (Linn) Aiton in the dose of 200 and 400 mg/kg orally respectively 60 minutes before acetic acid injection. Mice were observed for 20 minutes to count number of writhings in each group (Gerhard Vogel, 2002). Statistical Analysis: All the values ware statistically analyzed by one-way analysis of variance (ANOVA) followed by Tukey- Kramer multiple comparison test. Comparison between control and drug treated groups were considered to be significant. All values are expressed as mean SEM. Results In tail flick method, Buprenorphine showed increase in reaction time at 15 minutes and maximum reaction time at 90 minutes. With Jasminum sambac (L.) Ait. root extract 200 mg/kg there was no significant analgesic effect. But with dose 400 mg/kg showed maximum increase in reaction time at 60 minutes which was statistically significant (Probability < 0.05). Table 1: Analgesic effect of methanolic extract of root of Jasminum sambac (Linn)Aiton in albino rats (Tail flick method)
Groups(Drug) (n=6) I (Control) II (Buprenorphine) III (Methanolic extract) IV(Methanolic Extract) Dose Before Drug Administration No. of tail flicking after Drug Administration 15 min 30 min 60 min 90 min
2 ml 3.350.13 3.130.17 3.230.17 3.310.25 3.280.27 0.05 mg/kg 3.310.38 5.800.22** 7.050.75** 8.050.5** 8.660.3** 200 mg/kg 3.460.16 4.300.40 5.080.51 6.280.21 6.700.2 400 mg/kg 3.180.23 4.610.26* 5.810.54* 6.760.5* 7.410.3* * P < 0.05, ** P < 0.01, *** P < 0.001 MeanSEM (P for Probability) In acetic acid writhing method, Aspirin (100 mg/kg) showed significant activity (p < 0.01) in comparison to control. Jasminum sambac (Linn) Aiton root extract in the dose of 200 mg/kg and 400 mg/kg showed significant (p < 0.01) analgesic activity in comparison to control.
Table 2: Analgesic effect of methanolic extract of root of Jasminum sambac (Linn) Aiton in albino mice (Acetic acid writhing)
Groups (Drug) (n=6) Dose Writhing response Number of writhings % of inhibition
I(Control) 2ml 43 0.71 -II (Aspirin) 100 mg /kg 16 0.65 62.79** III (Methanolic Extract) 200 mg / kg 35 0.71 18.60** IV(Methanolic Extract) 400 mg / kg 310.33 27.90** * P < 0.05, ** P < 0.01, *** P < 0.001 MeanSEM (P for Probability) DISCUSSION Analgesic activity of the methanolic extract of roots of Jasminum sambac (Linn) Aiton was tested by tail flick method in rats and acetic acid induced writhing model in mice, analgesic effect of Jasmine root was seen in both tail flick method and acetic acid writhing suggesting that it has central as well as peripheral mechanism of action (Khanna N, 1995). In our study we got analgesic activity of Jasmine root extract in both peripheral and central Volume 1 Issue 1 January February 2013 Page 15
mechanism animal models. In comparison to standard drugs it was showing significant analgesic action. Acetic acid, which is used to induce writhing, causes analgesia by liberation of endogenous substances, which then excite the pain nerve endings (Taesotikul T, 2003). The extract produced significant writhing inhibition comparable to standard drug aspirin. Based on this, it could be concluded that it might possess analgesic activity. CONCLUSION Jasmine root extract has analgesic activity with central and peripheral mechanism. Further studies are required to find out active constituents responsible for analgesic action. REFERENCES Davies O L, Raventos J, Walpole L, A method for evaluation of analgesic activity using rats. British Journal of Pharmacology, 1, 1946, 255-264. Flecknell P A, Laboratory Anaesthesia, An introduction for research workers and technicians, Academic Press 2nd edition New York 1996. Gerhard Vogel, Wolfgang H Vogel, Bernwad A, Scholkens Sandow Drug discovery and evaluation Pharmacological Assay, 2nd Edition, Springer-Verlag Berlin Heidelberg, 2002, 716 Ghosh M N, Fundamentals of Experimental Pharmacology, 2nd rev. ed. Calcutta, Scientific Book Agency, 1984, 144-158. Harborne J B, Phytochemical methods. 3rd ed. London, Chapman and Hall, 1998, 160-178. Khanna N, Goswami M, Sen P, Antinociceptive action of A. Indica in mice, A possible mechanism involved., Indian Journal of Experimental Biology 33, 1995, 848-850. Khare C P, Indian Herbal Remedies, 1st edition, Germany, Springer-Verlag Berlin Heidelberg, 2004, 269-271. Kirtikar K R and Basu B D, In: Indian medicinal plants, 2nd edition, Dehradun, India, International Book Distributors and Book sellers, 1987, 37275. Kulkarni S K, Handbook of Experimental Pharmacology, Vallabh Prakashan, 3 rd Edition, 1999, 190. Nadkarni K M and Basu B D, Jasminum sambac, In: Indian Materia Medica, 1st edition, Mumbai, India, Popular prakashan Ltd, 1996, 120-123. Taesotikul T, Panthong A, Kanjanapothi D, Verpoorte R, Scheffer JJC, Antiinflammatory, antipyretic and antinociceptive activities of Tabernaemontana pandacaqui, Poir. J. Ethnopharmacol, 84, 2003, 31-33.
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Adesh A Bawane et.al.,
PHARMACOGNOSTICAL AND PHYTOCHEMICAL STUDIES ON LEAVES OF STEVIA REBAUDIANA LINN

*Adesh A Bawane1, B. Gopalakrishna2, Kusum S. Akki3, Supriya Das1, Manish K Gupta1 1. Institute of Pharmacy, H C P G College, Azamgarh Road, Varanasi. 2. RR College of Pharmacy, Bangalore. 3. KLEs College of Pharmacy, Vidyanagar, Hubli * Corresponding author: adeshab@rediffmail.com, supriohere@gmail.com ABSTRACT Stevia rebaudiana (Bert) is a plant of compositae family and native to Paraguay, its sweetness and calorie free property increased its demand tremendously. It not only imparts the sweetness but also maintain the normal blood sugar level thats why leaves are being used in homemade recepies and also in allied industries for diabetics. The leaf of Stevia contain more sweetness and having many other uses due to this importance leaf was analyzed for preliminary phytochemical studies including physical constants such as moisture content, total ash, acid insoluble ash, water soluble ash, sulphated ash, extractive values in different solvent and phytochemical test. Key words: Stevia rebaudiana, Phytochemical studies, Pharmacognostical studies INTRODUCTION Stevia rebaudiana (Bert) is a plant of compositae family native to Paraguay, whose leaves have been used for centuries as a sweetener (Viana A M and Metivier J, 1980). Stevia is a natural herb, low calorie sweetener. It not only imparts sweet taste but also maintain the normal sugar level. Although there are more than 180 species of stevia plant only stevia rebaudiana gives sweetest essence due to the fact that these leaves accumulate eight sweet diterpene glycosides (Soejarto D D, 1983). Stevioside is one of the principle diterpene glycosides having a sweetness of 30 - 320 times than sucrose. The crude stevia leaves and herbal green powder is 10-15 times sweeter than sucrose (Crammer and Ikan R, 1986) it contains several diterpene glycosides, which are non glycemic, yet ranges in sweetness from 30 320 times than that of sucrose. Four major diterpene glycosides are reported in the literature: stevioside, rebaudioside A, C and dulcoside. Besides these it also contains sterols, triterpenoids, flavonoids, caumarins (Samulsson and Gunnar, 1992) (Kinghorn AD and Soejarto DD, 1985). It has a wide range of applications in pharmaceutical and allied industries and also has been found to be calorie free and non toxic. Various diseased conditions are associated with free radical oxidative stress (Devasgayam TPA, 1996). Stevia leaf containing free radical scavengers and are well known for their therapeutic activity. Hence the present study was aimed to study pharmacognostical and phytochemical studies on stevia leaf. MATERIAL AND METHODS The leaves of Stevia rebaudiana (Bert) were collected in well cultivated land from Belwatgi farm in Dharwad district and were authenticated by Dr. B D Huddar, professor and Head, Department of Botany, Shri Kadsiddheshwar Arts College and H S Kotambari Science Institute, Vidyanagar, Hubli, Karnataka. It was dried under shade, pulvarised and passed through sieve no. 40 and stored in a closed vessel. Organoleptic Evaluation: Morphology is the study of the form of an object whilst morphography is the description of that form. The application of morphology in the drug analysis lies in the field of crude drugs, where the material is known to occur in original form. The majority of the natural products used as drugs are derived from the plants or the parts of plants (Brandle JE, 1998). The macroscopical characters such as color of untreated leaves were examined under a diffuse day light and artificial light source with wavelength similar to those of day light may be used. Odour was examined by taking a small piece of leaf in the palm of hand and also pieces of it slowly and repeatedly inhaled the air over the leaf also crushed the leaf between the thumb and index figure. Taste is extremely sweet. Size and shape was examined by placing the leaf of various size and shape on graph paper and trace the outline of leaf. Leaf constant evaluation: Leaf constant such as stomatal no, stomatal index, vein islet no, vein termination no and palisade ratio were studied (Brandle JE, 1998) (Kokate CK, 2006) (Mukharjee Pulok K, 2008). Proximate value determination of stevia leaf: In proximate values extractive, moisture and total ash, acid insoluble ash, water soluble ash and sulphated ash were studied (Evans WC and Trease, 1983) (Indian Pharmacopoeia, 1996). Fluorescence analysis: Fluorescence characters of stevia leaf material with different chemical reagents were determined under ordinary and ultraviolet light (Chase CR and Pratt RJ, 1949). 1 mg of stevia leaf sample was taken in a glass slide and treated with various reagents for the presence of their fluorescence characters under ultraviolet lamp. Preliminary phytochemical analysis: Preliminary qualitative analysis of all extracts was carried out by employing standard conventional protocols (Sazada S, 2009).
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ISSN: 2320 3471(Online) Adesh A Bawane et.al., Indian Journal of Research in Pharmacy and Biotechnology RESULT AND DISCUSSION Leaf colours of both surfaces are found changed. Macro- Morphological studies of leaf was studied for various parameters. Colour change found in dry leaf and the sweetness was more than the fresh leaf; results were shown in table no 1, 2 and 3. Anomocytic stomata were found on both the surface and were found more on the upper surface as compared to lower surface. Results were on table no 4. The Alcohol soluble exhaustive extractive value, Water soluble extractive values were 31.0196 and 40.210 (%w/w) respectively. Moisture content was 6.9397 - 7.2964(%w/w). Acid insoluble ash value was lesser than water soluble ash value. The values are on table no 5. The result of fluorescent studies of the powdered plant material using different chemical reagents were studied and given in table no. 6. Fluorescence is an important phenomenon exhibited by various chemical constituents present in the plant material (Kamil MS and Paramjyothi B S, 2010). If the substance themselves are not fluorescent, they may often be converted into fluorescent derivatives by reagents hence some crude drugs are often assessed qualitative in this way and it is an important parameter of pharmacognostic evaluation. Water, ethanol and ethyl acetate extracts were found sticky and butanol and petroleum ether were found waxy. Extract colour is different and their % dry weight was shown on Table 7. Stevia doesnt contain alkaloids it mainly contain glycosides, flavonoids, sterol, triterpenoids and others are given on Table 8. Table1.Organoleptic evaluation of fresh leaf Parameters Organoleptic Parameters Colour: Upper surface Green Lower surface Light green Odour Characteristic Taste Extremely sweet taste Texture Rough Table2. Macro-morphological evaluation of fresh leaf Morphology Parameters Morphology 3.8- 7.3 cm Leaf base Acute (Young leaf) & attenuate (Mature leaf) 1.2- 2.6 cm Surface Pubescent on both surface Simple leaf. Leaf shape Oblanceolate Pinnatifid Petiole Absent Reticulate Stipulate Exstipulate Serrate with ciliate, Teeth Sheath Absent 5-11 (left) and 5-10 (Right) Acute Insertion Connate Phylotaxis Opposite
Parameters Length Breadth Composition Incision Venation Margin
Apex
Table no. 3. Organoleptic features of dried leaf Parameters Organoleptic characters Colour Upper surface Light green Lower surface Whitish green Texture Rough on both surface Odour Characteristic Taste Extremely sweet taste Sweeter than fresh leaf Shape Oblanceolate # Maximum leaves were curved at its margin part on dorsa side. # Some leaves were slightly curved at its lower end. # 3 Nerves were found prominently on lower and upper surface of leaves.
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Adesh A Bawane et.al.,
ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology Table no. 4. Leaf constant values of the Stevia leaf Palisade ratio Stomatal index Stomatal no. 2.5 3.75 5.0 2.5 3.0 3.5 21.05 25.00 29.41 10.00 11.42 13.33 100-125 25-50
Sr. no. 1 2
Surface view Upper surface Lower surface
VIN 225-300
VTN 200-275
Sr.No 1 2 3 4 5 6 7
Table no. 5. Proximate values of Stevia leaf Parameter Value determined in (%w/w) Alcohol soluble extractive value 31.0196 Water soluble extractive value 40.210 Total ash 8.2904-10.2030 Acid insoluble ash 1.2716-2.647 Water soluble ash 4.2944-6.0444 Sulphated ash 12.04 13.34 Moisture content 6.9397 - 7.2964 Table no. 6. Fluorescence study of dry stevia leaf Powder drug Visible/ day light Ultra violet light Powder Greenish brown Greenish brown Powder + FeCl3 Dark grey Dark grey Powder + conc HCl Orange yellow Grayish yellow Powder + 10% HNO3 Orange Greenish yellow Powder + 10 % K2Cr2O7 Yellow Green Powder + 1M NaOH Brownish yellow Green Powder + AgNO3 Greenish brown Light brown Powder + conc HNO3 Orange Fluorescent yellow Powder + conc H2SO4 Orange Fluorescent green Powder + Br2 water Brown Light brown Powder + 5 % H2O2 Brown Greyish green Powder + CCl4 Green Greenish brown Powder + Methanol Dark Brown Brown Powder + CH3COOH Orange Brown Dark green Powder +Xylene Grey Orange green Powder + NH3 Green Fluorescent green Powder + I2 Dark green Grey
Table no. 7. Phytochemical investigation Extract Color Odor Alcoholic 47.328 Blackish green Characteristic Aqueous 41.766 Yellowish brown Characteristic Successive Extraction Pet. ether (40-600C) 4.067 Yellowish green Characteristic
% dry weight
Consistency Sticky Sticky Waxy Waxy Sticky Sticky Sticky
Butanol Ethyl acetate Ethanol Water
40.291 0.755 11.63 30.321
Blackish green Greenish brown Yellowish brown Blackish green
Characteristic Characteristic Characteristic Characteristic
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ISSN: 2320 3471(Online) Adesh A Bawane et.al., Indian Journal of Research in Pharmacy and Biotechnology Table no 8. Preliminary phytochemical screening of various extracts of stevia leaf Phyto -Constituents Alcoholic Aqueous Successive Extractions PE B.Nol EA Ethanol Water Extract Extract Carbohydrates + + + + Flavonoids + + + Alkaloids Steroids + + + Triterpenoid + + Tannins & Phenolic gr. Proteins & amino acid Glycosides + + + + + + + + + -
Keywords: PE = Petroleum Ether Extract (40-600C) + = Present. B.Nol = Butanol Extract - = Absent EA = Ethyl Acetate Extract CONCLUSION An attempt was made to standerdise stevia leaf by using various parameters Viz. Organoleptic evaluation, leaf constant, proximate values determination, fluorescence study and identification of various phytoconstituents present in the leaves. Further studies are needed to qualify the chemical constituents present in the leaves to use in pharmaceuticals. REFERENCES Brandle JE, Starratt AN, Gijzen M, Stevia rebaudiana: its agricultural, biological and chemical properties, Can J Plant Sci, 78 (4), 1998, 527-536. Chase CR and Pratt RJ, Fluorescence of powdered vegetable drugs with particular reference to development of a system of identification, J Amr Pharm Assoc, 38, 1949, 324- 331. Crammer and Ikan R, Sweet glycosides from the stevia plantChem Brit, 22, 1986, 915-917. Devasgayam TPA, Kamat JP, Mohan H, Kesvan PC, Biochem Biophys Acta, 1282, 1996, 63-70. Evans WC and Trease, Pharmacognosy, Elsevier publication, 19 th Edn, 1983, 96-98. Fujita H and Edahiro T, Safety and Utilisation of Stevia Sweetener. The food Industry, 22(22), 1979, 1-8. Indian Pharmacopoeia, Govt of India Publication. India. 1996, 2 nd Edn, A- 89. Kamil MS and Paramjyothi B S, Preliminary pharmacognostical and phytochemical evaluation of Portulaca quadrifida Linn, Int J PharmTech Res, 2(3), 2010, 1699- 1702. Kinghorn AD and Soejarto DD, Economic and Medicinal plant Research, Academic Pres, New York, USA, 1985, 1-51. Kokate CK, Purohit AP and Gokhale SB., Pharmacognosy. Nirali prakashan. Aug 2006, 35 th Edn: 99100. Mukharjee Pulok K. Quality control of Herbal drugs. Business horizons pharmaceutical publishers. 2008; 3rd reprient, 176- 178. Quality control methods for medicinal plant materials. World Health Organisation, Geneva, 1998, 11- 21. Samulsson and Gunnar, Drugs of National Origin, Swedish Pharmaceutical Press, Stockholm, Swedam, 1992. Sazada S, Arti V, Ayaz A, Faraha Z and Maheshwari MK, Preliminary phytochemical analysis for some medicinal and aromatic plants, Adv, In Biological Res, 3 (5), 2009, 188-195. Soejarto D D, Compardre C M, Medon PJ, Kamath SK and Kinghorn AD, Potential sweetening agents of plants origin II field search for sweet tasting stevia species, Econ Bot, 37(1), 1983, 71-75. Viana A M and Metivier J, Changes in the level of total soluble proteins and the sugar during leaf ontogeny in Stevia rebaudiana Bert. Annals Botany, 45, 1980, 469-474.
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PRELIMINARY PHYTOCHEMICAL SCREENING AND STANDARDISATION OF LEAVES OF CATHARANTHUS ROSEUS (L.) G. DON
Shambaditya Goswami Department of Pharmacy, ITM, Gorakhpur, UP, India Email:shamba_mpharm@rediffmail.com ABSTRACT Catharanthus roseus (L.) G. Don (Periwinkle) belongs to the family Apocyanaceae, is a bushy perennial herb and evergreen shrub. More than 100 alkaloids and related compounds have so far been isolated and characterized from the plant. Main alkaloids are vincristine and vinblastine which are responsible for anticancerous activity. The present study deals with phytochemical screening, standardization and transverse section of the plant leaf. The dried leaves of Periwinkle were subjected for different standardization parameters like Total ash (0.4%), Acid insoluble ash (0.68%), Water soluble ash (1.68%), Sulphated ash (4.12%), Water soluble extractive (6.34%), Alcohol soluble extractive (4.8%), Moisture content (10.09%), Loss on drying (5.01%), Foaming index (0.9cm height) and Swelling index (0.8g) for their purity and strength as per WHO guideline. The Preliminary phytochemical screening report reveals the presence of Alkaloids, Glycosides, Terpenoids, Flavonoids, Phenols, Tannins, Carbohydrates, Saponins, Phytosterols, Protein and amino acids in ethanolic extract of the leaf of Catharanthus roseus (L.) G. Don . The transverse section of the plant exhibits small layer of rectangular cells covered with thick cuticle, uni-cellular covering trichome and vascular bundle present in the middle of midrib region and cruciferous stomata. This phytochemical standardization study will improvise the further research on this plant. Key Words: Catharanthus roseus (L.) G. Don, Apocyanaceae, Standardization, WHO guideline, Phytochemical Screening, Transverse section INTRODUCTION Periwinkle or Catharanthus roseus (L.) G. Don (Family Apocyanaceae), commonly known as Nayantara or Sadabahar, is an erect bushy perennial herb and evergreen shrub. The species was formerly known as Vinca rosea. The native of Periwinkle is mainly Madagaskar. This plant is grown commercially for its medicinal uses in Australia, Africa, India and Southern Europe. Except the highly alkaline or water logged soil, Periwinkle does not require any special conditions of soil. It favourably grows in light sandy soil, rich in humus. The rainfall of about 100 cm is most suitable for it. The leaf is simple, opposite, exstipulate, petiolate (Kokate C K, 2009). More than 100 alkaloids and related compounds have so far been isolated and characterized from the plant. The alkaloid contents in different parts show large variations as roots 0.14-1.34%, stem 0.074-0.48%, leaves 0.321.16%, flowers 0.005-0.84%, fruits 0.40%, seeds 0.18% and pericarp 1.14%. Dry leaves contain vinblastine (vincaleucoblastine or VLB) 0.00013-0.00063%, and vincristine (leurocristine or LC) 0.0000003-0.0000153% which have anticancerous activity (PP Joy, 1998). Vinblastine and vincristine (the antileukemic agents) were isolated, in a pure form, from Catharanthus roseus by several chromatographic techniques. All the isolated mixtures were evaluated by HPLC and HPTLC analyses (Khaled A Shams, 2009). Phytochemical studies have attracted the attention of plant scientists due to the development of new and sophisticated techniques. These techniques played a significant role in the search for additional resources of raw material for pharmaceutical industry (phytochemicals) (Arvind J Mungole, 2008). The present study deals with the macroscopy, microscopy, standardization for purity and strength as per WHO and preliminary phytochemical screening of the leaf of Catharanthus roseus (L.) G. Don. MATERIALS AND METHODS Collection of plants:The plant leaves of Catharanthus roseus (L.) G. Don were collected from the area of ITM, campus (Gorakhpur, Uttar Pradesh). The plant was authenticated and deposited in Department of Pharmacy, ITM, GIDA. Macro morphology of leaf: The parameters studied were form, shape and surface characters of drug (Pulok K. Mukherjee, 2008). Fluorescence analysis: Many herbal drugs when exposed to illumination emit light of different color. The fluorescence analysis helps to identify the drug with specific fluorescence. It also helps to detect fluorescent impurities. This method can be used as a diagnostic tool for testing adulteration (Madhavan V, 2009) This method has been done by treating the leaf powder along with 1N HCl, 1 N NaOH, 50% HCl, 50% H2SO4, 50% HNO3 and Methanol was observed under the short UV light (254nm) and long UV light(365nm). Transverse section of root: The leaves were taken cleaned and fixed in formalin, acetic acid and ethanol. After 24 hours of fixing the specimens were dehydrated with graded series of tertiary butyl alcohol. The sections were stained with toluidine blue, safranin, fast green and iodine. Temporary preparations of the slide were made by mounting on glycerin.
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Powder microscopy: Shade dried leaf of the plant was powdered with the help of a electric grinder till a fine powder was obtained .This fine powder was subjected to powder microscopy using different staining reagents as mentioned above. Determination of physico chemical parameter: The dried leaves of Catharanthus roseus were subjected for different standardization parameters for their purity and strength. Different parameters like Ash Value, Moisture content Loss on Drying has been performed according to WHO guideline, 1998. Extraction: The leaf powder (250g) was packed in a soxhlet apparatus and was subjected to successive extraction using increasing order of polarity solvents like petroleum ether, acetone, chloroform, ethanol and water. The temperature was maintained 20-250c and 24 hrs to 72 hrs time has been consumed to complete each extraction under ideal condition. After completion of extraction, the solvent was removed by distillation. All the extracts were then concentrated except petroleum ether. The residues were then stored in dessicator. Preliminary phytochemical Screening: All the extracts of Catharanthus roseus (L.) G. Don were subjected to qualitative tests for the identification of various active constituents by different chemical tests. The tests for carbohydrates, glycosides, fixed oil and fat, proteins & amino acids, saponins, tannins, alkaloid, phenolic compounds and flavonoids have been performed. RESULT AND DISCUSSION Macro morphology of leaf: Morphology of leaf implies that the leaves are simple, petiolate, ovate and oblong. The colour, odour, shape, margin and appearance is shown in Table 1. Fluorescence analysis: The fluorescence analysis of the leaf powder of Catharanthus roseus (L.) G. Don is given in Table 2. Transverse section of leaf: TS of the fresh leaf of Catharanthus roseus (L.) G. Don shows that the presence of Epidermis which is single layered and covered with thick cuticle. The trichomes are unicellular and covered. Parenchyma layer which consists of Palisade and Spongy Parenchyma, is present just below upper epidermis and it is single layered, elongated and closely packed. Xylem and Phloem are present in the middle of midrib region. The diagram by observing TS has been drawn and photomicrographs of TS are shown in Figure 1 and 2 respectively. Powder microscopy: Powder microscopy reveals that there is presence of cruciferous stomata and unicellular covering trichome. Calcium oxalates are absent. (Figure 3 and 4) Physicochemical parameter: All the physicochemical parameters like ash value, water and alcohol soluble extractive, moisture content, loss on drying, foreign matter, foaming index, swelling index are within the limit prescribed by WHO and has been tabulated. (Table 3) Extractive Value: Successive extractive values were reported in Table 4, which implied that ethanolic extract has greatest values than aqueous one. Preliminary Phytochemical Screening: All the extracts were subjected for different chemical tests and ethanolic extracts showed the presence of most chemical constituents. Alkaloids, Glycosides,Terpenoids, Flavonoids, Phenols, Tannins, Carbohydrates, Saponins, Phytosterols, protein and amino acids were showed their presence in ethanolic extract. Also it has been reported that in winter season total phenolic and flavonoid showed more content in the plant and that total flavonoids and total phenolics in this plants increased by increasing pollution loads across the sites (Qayoom A, 2009). Table 1: Macromorphology of the leaf of Catharanthus roseus (L.) G. Don Properties Inference Colour Green Odour Characteristic Taste Bitter Shape Petiolate, Ovate or Oblong Margin Centric Appearance Glossy Apex Acute Table No.2: The fluorescence analysis of the leaf powder of Catharanthus roseus (L.) G. Don Reagents Used Day light LowerUV (320-400nm) Short UV(280-320nm)
Powder Drug Powder+1N HCl Powder+1 N NaOH Powder+ 50% HCl Powder+ 50% H2SO4 Powder+50% HNO3 Powder+ Methanol Light Green Pale Yellow Red Pale Yellow Brownish Black Reddish Orange Yellowish Orange Dark Green Pale Green Greenish Yellow Pale Green Brown Dark Green Yellowish Green Black Dark Green Pale Green Greenish Blue Dark Brown Pale Green Pale Green
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Table 3: Physico-chemical constants Parameters Value obtained Total Ash 0.4% w/w Acid insoluble ash 0.68% w/w Water soluble ash 1.68% w/w Sulphated ash 4.12% w/w Solubility Water soluble extractive 6.34% w/w Alcohol soluble 4.8% w/w extractive Moisture content 10.09% w/w Loss on drying 5.01% w/w Foaming index 0.9cm height Swelling index 0.8 g
Table No 4: Extractive Values Extraction Method Values w/w Petroleum Hot 0.3% ether Percolation Acetone Hot 1.2% Percolation Chloroform Hot 1.05% Percolation Ethanol Hot 3.45% Percolation Aqueous Cold 2.1% Maceration
Test Alkaloid Glycoside Terpenoids, Flavonoids Phenols Tannins Carbohydrates Saponins Phytosterols protein and amino acids Fixed oil and fats Resin
Table 5: Phytochemical screening Petroleum ether Acetone Chloroform + + + + + -
Ethanol + + + + + + + + + + -
Aqueous + + + + + + + -
Figure1: Diagram of TS of Catharanthus roseus (L.) G. Don
Figure 2: Photomicrograph of TS of Catharanthus roseus (L.) G. Don
Figure 3: Trichome CONCLUSION Volume 1 Issue 1
Figure 4: Cruciferous Stomata
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The phytochemical screening and standardistion study is not only an important tool for identification of plant part but also it informs quality control and product formulation development. As Catharanthus roseus (L.) G. Don is an medicinally important plant which possesses anticancerous (El-Sayed A and Cordell GA, 1981) (ElSayed A, 1983) antidiabetic (both ethanolic and aqueous) (Singh SN, 2001), anti-inflammatory (Chattopadhyay RR, 1992), antioxidant (Zheng W and Wang SY, 2001) activity, this study will improve the identification criteria and research validation scheme which finally will be useful in alternative medicine. REFERENCE Arvind J. Mungole, Ravi Awat Alka Chaturvedi and Prakash Zanwar, Preliminary Phytochemical screening of Ipomoea obscura (L): A hepatoprotective medicinal plant, International Journal of PharmTech Research, 2(4), 2008, 2307-2312. Chattopadhyay RR, Sarkar SK, Ganguli S, et al. Antiinflammatory and acute toxicity studies with leaves of Vinca rosea Linn in experimental animals, Indian J Physiol Pharmacol, 36, 1992, 291292. El-Sayed A, Cordell GA, Catharanthus alkaloids, Catharanthamine, a new antitumor bisindole alkaloid from Catharanthus roseus J Nat Prod, 44(3), 1981, 289-293. El-Sayed A, Handy GA, Cordell GA, Catharanthus alkaloids, Confirming structural evidence and antineoplastic activity of the bisindole alkaloids leurosine-N'b-oxide (pleurosine), roseadine and vindolicine from Catharanthus roseus, J Nat Prod, 46(4) (1983), 517-27 Khaled A Shams, Naglaa M Nazif, Nahla S Abdel Azim, Khaled A Abdel Shafeek, Mostafa M El-Missiry, Shams I Ismail, and Medhat M Seif El Nasr,Isolation and Characterization of Antineoplastic Alkaloids from Catharanthus Roseus L. Don. Cultivated in Egypt, Afr J Tradit Complement Altern Med, 6(2), 2009, 118122. Kokate C K, purohit A P, Gokhale S B, A Textbook of Pharmacognosy, Nirali Prakashan, 2009, 27- 28. Madhavan V, Hema Basnett, Gurudeva MR, Yogonarasimhan SN, Pharmacognostical evaluation of Drosera BurmanniiVahl(Droseraceae). Indian journal of traditional knowledge, 8 (3), 2009, 326-333. P. P. Joy, J. Thomas, Samuel Mathew , Baby P. Skaria ,Medicinal Plants, Kerala Agricultural University, Aromatic and Medicinal Plants Research Station Odakkali, Kerala, India , 1998 Pulok K. Mukherjee, Quality control of Herbal Drugs- An approach to evaluation of Botanicals, New Delhi: Business Horizons, 2008. Qayoom A. Mir, T. Yazdani, S. Ahmad and M. Yunus, Total flavonoids and phenolics in Catharanthus roseus L. and Ocimum sanctum L. as Biomarkers of Urban Auto Pollution, Caspian, J. Env. Sci, 7, 2009, 9-16. Quality control methods for medicine plant materials, World Health Organisation, Geneva, 1998. Singh SN, Vats P, Suri S, Effect of an antidiabetic extract of Catharanthus roseus on enzymic activities in streptozotocin induced diabetic rats, J Ethno pharmacol, 76, 2001, 269-277. Zheng W, Wang SY, Antioxidant Activity and Phenolic Compounds in Selected Herbs, J Agric Food Chem, 49, 2001, 5165-5170.
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Haritha et.al.
A BRIEF INTRODUCTION TO METHODS OF PREPARATION, APPLICATIONS AND CHARACTERIZATION OF NANOEMULSION DRUG DELIVERY SYSTEMS
Haritha*, Syed Peer Basha, Koteswara Rao P, Chakravarthi Vedantham Nimra College of Pharmacy, Vijayawada, India *Corresponding author: Email:haritham29@yahoo.com ABSTRACT Nanoemulsions have attracted great attention in delivery of therapeutically active agents since approximately 40% of new chemical entities are hydrophobic in nature and the delivery of these poorly water soluble drugs is a challenge for delivery of drugs. The emulsions and Nanoemulsions differ mainly in the size and shape of the particles dispersed in the continuous phase. The particle size in nanoemulsions is (10-200nm) and those of conventional emulsions are (1-20m). Nanoemulsions are prepared by high energy emulsification methods like micro fluidic and ultrasonic methods, these methods rupture large micro droplets into nano scale droplets. Oil in water (O/W) and water in oil (W/O) nanoemulsions were prepared by aqueous phase titration method. Nano emulsions can be evaluated for their morphology, droplet size, viscosity, PH, optical clarity, zeta potential, conductivity, transmission electron microscopy and polydispersity. Nanoemulsions find application in controlled drug delivery, targeted drug delivery, nutraceuticals, food products, transdermal and colloidal drug delivery. This article includes preparation, characterization, evaluation and application of nanoemulsions. Key words: Nanoemulsion, co surfactant, phase inversion, microfluidization 1. INTRODUCTION The term Nanoemulsion refers to a thermodynamically stable isotropically clear dispersion of two immiscible liquids, such as oil and water stabilized by an interfacial film of surfactant molecules. Nanoemulsion is considered to be a thermodynamically or kinetically stable liquid dispersion of an oil phase and water phase in combination with a surfactant. The dispersed phase droplet size is about 5 nm-200 nm and should have very low oil/water interfacial tension. Cosurfactant or cosolvent is used in many cases in addition to the surfactant, the oil phase and the water phase. 1.2. TYPES OF NANOEMULSION Depending on the composition there are three types of nanoemulsions: 1. Oil in water nanoemulsions wherein oil droplets are dispersed in the continuous aqueous phase. 2. Water in oil nanoemulsions wherein water droplets are dispersed in the continuous oil phase. 3. Bi-continuous nanoemulsions wherein micro domains of oil and water are interdispersed within the system. The main difference between emulsions and nanoemulsion are that even though emulsion is having kinetic stability they are thermodynamically unstable. Emulsions are cloudy but nanoemulsions are clear and translucent. They also differ in their method of preparation. 1.3. ADVANTAGES OF NANOEMULSION 1. Increase the rate of absorption. 2. Eliminates variability in absorption 3. Helps solubilize lipophilic drug 4. Increases bioavailability 5. Various routes like topical, oral and intravenous can be used to deliver the product. 6. Helpful in taste masking. 7. Rapid and efficient penetration of the drug moiety. 8. Liquid dosage form increases patient compliance. 9. Nanoemulsions are thermodynamically stable system and the stability allows self emulsification of the system. 1.4. DISADVANTAGES OF NANOEMULSION 1. Use of a large concentration of surfactant and cosurfactant necessary for stabilizing the nano droplets. 2. Limited solubility capacity for high melting substances. 3. The surfactant must be non toxic for using pharmaceutical applications. 4. Nanoemulsion stability is influenced by environmental parameters such as temperature and PH. 1.5. COMPONENTS OF NANOEMULSION Nanoemulsions contain three main components. 1. Oil 2. Surfactant/cosurfactant 3. Aqueous Phase
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1.6. SURFACTANTS USED IN NANOEMULSION Surfactants used for stabilizing the systems may be 1. Non ionic 2. Zwitterionic 3. Cationic 4. Anionic 1.7. CLASSIFICATION OF SURFACTANTS 1. Nonionic - Fatty alcohols, Glycerol esters, Fatty acid esters. 2. Anionic contain - Carboxylate groups, Soaps, Sulfonates, Divalent ions 3. Cationic Amines and quaternary ammonium compounds. Surfactants used in nanoemulsions S.No Surfactants Capryol 90 1 Gelucire 44/14, 50/13 2 Cremophor RH 40 3 Imwitor 191, 308 (1) 380, 742, 780 4 k, 928, 988 Labrafil M in 1944 CS, M, 2125 CS 5 Lauroglycol 90 6 PEG MW > 4000 7 Plurol Oleique CC 497 8 Poloxamer 124 and 188 9 Softigen 701, 767 10 Tween 80 11 Oils used in nanoemulsions S.No Oils Captex 355 1 Captex 200 2 Captex 8000 3 Witepsol 4 Myritol 318 5 Isopropyl Myristate 6 Cosurfactants used in nanoemulsions S.No Cosurfactants Transcutol p 1 Glycerin, Ethyleneglycol 2 Propylene glycol 3 Ethanol 4 Propanol 5
2. PREPARATION OF NANO EMULSION The drug is dissolved in the lipophilic part (oil) of the nanoemulsion. The aqueous phase is combined with surfactant and a cosurfactant. The aqueous phase is added at a slow rate with gradual stirring until the system is transparent. The amount of surfactant and cosurfactant that can be incorporated shall be determined with the help of pseudo ternary phase diagram. Finally ultra sonicator can be used to achieve the desired range of dispersed globules. Then it is allowed to equilibrate. Gel may be prepared by adding a gelling agent. Most widely used gelling agent is carbomer. Factors to be considered during preparation of nanoemulsion: The prime requirement in nanoemulsion production is an ultra low interfacial tension should be attained at the oil water interface, so surfactants must be carefully chosen. Concentration of surfactant must be high enough to provide the number of surfactant molecules needed to stabilize the nano droplets. The interface must be flexible to promote the formation of nanoemulsion. 2.1. METHODS OF PREPARATION OF NANOEMULSION Several methods have been suggested to prepare nanoemulsion. Formation of nanoemulsion system required a high amount of energy. This energy can be provided either by mechanical equipment or the chemical potential inherent within the component. Some methods used for the preparation of nanoemulsion are 2.2. Phase Inversion Method: Fine dispersion is obtained by chemical energy resulting of phase transitions occur through emulsification method. The adequate phase transitions are produced by changing the composition at constant temperature or by changing the temperature at constant composition. The phase inversion temperature (PIT) method was introduced based on the principle of changes of solubility of polyoxyethylene type surfactant with temperature. This surfactant becomes lipophilic with increase in temperature because of dehydration of polymer chain. At low temperature the surfactant monolayer has a great positive spontaneous curvature forming oil swollen micellar solution phase. 2.3. Sonication Method: In this method the droplet size of conventional emulsion are reduced with the help of sonication mechanism. Only small batches of nanoemulsion can be prepared by this method. 2.4. High Pressure Homogenizer: This method is performed by applying a high pressure over the system having oil phase, aqueous phase and surfactant or co-surfactant. The pressure is applied with the help of homogenizer. Some problems associated with homogenizer are poor productivity, component deterioration due to generation of much heat. With this method only Oil in water (O/W) liquid nanoemulsion of less than 20% oil phase can be prepared and cream nanoemulsion of high viscosity or hardness with a mean droplet diameter lower than 200 nm cannot be prepared. Volume 1 Issue 1 January February 2013 Page 26
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2.5. Microfluidizatiion: Microfluidization technology makes use of a device called MICRO FLUIDIZER. This device uses a high pressure positive displacement pump (500-200 PSI) which forces the product through the interaction chamber, consisting of small channels called micro channels. The product flows through the micro channels on to an impingement area resulting in very fine particles of submicron range. The two solutions (aqueous phase and oily phase) are combined together and processed in an inline homogenizer to yield a course emulsion. The course emulsion is into a micro fluidizer where it is further processed to obtain a stable nano emulsion. 2.6. Production with high amplitude ultrasound: This method is a viable alternative to high pressure homogenization. Intense shear forces necessary for the nanoemulsification are generated by ultrasonic cavitation which produces violently and asymmetrically imploding vacuum bubbles and break up particles down to the nanometer scale. This method is successfully used in small scale production of nanoemulsions. 3. CHARACTERIZATION OF NANOEMULSIONS 3.1. Phase behavior study: This study is a characterization and optimization of ingredients (surfactant, oil phase and aqueous phase). Generally the study is necessary in case of nanoemulsion formulation prepared by phase inversion temperature method and self emulsification method in order to determine the phase of nanoemulsion and dispersibility. Study is done by placing the different ingredients of nanoemulsion by varying the concentration in glass ampoules and thoroughly homogenized at a certain temperature for a time until equilibrium anisotropic phase can be identified by polarized light. 3.2. Particle size analysis: Generally in case of nanoemulsion dynamic light scattering (DLS) method is used for the measurement of particle size and their distribution. 3.3. Surface Charge Measurement: The surface zeta potential of nanoemulsion is predicted with the help of mini electrode. 3.4. Transmission Electron Microscopy: This method is used to observe the morphology of nanoemulsion. 3.5. Viscosity: Viscosity will be measured to ensure the better delivery of the formulation. 3.6. Stability of Nanoemulsions: Stability studies are performed on nanoemulsions by storing them at refrigerator and room temperatures over a number of months. The viscosity, refractive index and droplet size are determined during this period of storage. Insignificant changes in these parameters indicate formulation stability. Accelerated stability studies can also be performed. In this case, nanoemulsion formulation is kept at accelerated temperatures and samples are withdrawn at regular intervals and analyzed for drug content by HPLC. The amount of drug degraded and remaining in nanoemulsion formulation is determined at each time interval. 4. APPLICATIONS OF NANOEMULSION Nanoemulsions containing pharmaceutically active agents can be utilized for the production of pharmaceutical preparations. If desired a special galenic form can be imparted to the mixture. Ampoules, especially sterile injection and infusion solutions; solutions, especially oral liquids, eye drops and nose drops which can contain various auxiliary substances can be formulated in the form of nanoemulsion; aerosols without metering feature and dosing aerosois, which can contain propellant gas and stabilizers besides the nanoemulsion; hydrophilic and hydrophobic gels and ointments containing the nanoemulsion; o/w or w/o creams containing the nanoemulsion; lotions and pastes containing the nanoemulsion are available in the market. 4.1. Ocular delivery: Oil in water emulsions are being explored for improved topical lipophilic drug delivery to the eye. Examples: Piroxicam, Pilocarpine, Indomethacin, cyclosporine A 4.2. Percutaneous route: Many drugs exhibit low skin permeation, which results in poor efficacy. Common chemical skin penetration enhancers, organic solvents are generally associated to some degree with skin irritation, toxicity and sensitization. A solvent free topical vehicle based on drug entrapment in the o/w emulsion droplets of submicron size is more efficacious in terms of percutaneous absorption with possibly devoid of adverse effects. Examples:NSAIDs, Diazepam, -tocopherol antifungal drugs (Econazole or Miconazole nitrate) EMLA (Eutectic Mixturees of local anaesthetic) have proven to be useful medication by this route. 4.3. Nasal route: The nasal route has received great attention due to number of advantages over parenteral and oral administration especially by bypassing the liver. Nanoemulsions increase absorption by solubilizing the drug in the inner phase of an emulsion and prolonging contact time between emulsion droplets and nasal mucosa. Examples: Lipid soluble renin inhibitor was incorporated into an O/W emulsion, insulin and testosterone can also be delivered by this route. 4.4. Use of nanoemulsion in cosmetics : Nanoemulsions are recently becoming increasingly important as potential vehicles for the controlled delivery of cosmetics and for optimized dispersion of active ingredients into skin. Nanoemulsion gain increasing interest due to their own bioactive effects. Nanoemulsions are acceptable in cosmetics because there is no inherent creaming, sedimentation, flocculation or coalescence observed with macro emulsions. 4.5. Antimicrobial Nanoemulsions: Antimicrobial nanoemulsions are oil in water droplets with size range from 200-600n.m. The nanoemulsion particles are thermodynamically driven to fuse with lipid containing organisms. When enough nano particles fuse with the pathogens, they release part of the energy trapped with in the emulsion. Volume 1 Issue 1 January February 2013 Page 27
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Both the active ingredient and the energy released destabilize the pathogen lipid membrane, resulting in cell lysis and death. Nanoemulsion has broad spectrum activity against bacteria (e.g. E. Coli, Salmonella, S. aureus) enveloped viruses (e.g. HIV, Herpes Simplex), Fungi (e.g. Candida, Dermatophytes) and spores (e.g. anthrax). 5. CONCLUSION Although high energy emulsification method is traditionally used for the preparation of nanoemulsion formulation but low emulsion emulsification method now create an attraction due to their wide application and advantages as a formulation and stability aspects. The applications of nanoemulsion are limited by the instability. Stability of formulation may be enhanced by controlling factors such as type and concentration of surfactant and cosurfactant, type of oil phse, methods used, process variables and addition of additives. Overall nanoemulsion formulation may be considered as effective, safe and patient compliance formulation for the delivery of pharmaceuticals. 6. REFERENCES Gasco M R, Gallarate M, Pattarino F, In-vitro permeation of azelaic acid from viscosized microemulsions, Int J Pharm, 69, 1991, 193196. Kreilgaard M, Pedersen E J, Jaroszewski J W, NMR characterization and transdermal drug delivery potential of microemulsion systems, J Control Release, 69, 2000, 421-433. Kriwet K, Mller-Goymann C C, Diclofenac release from phospholipid drug systems and permeation through excised human stratum corneum, Int J Pharm, 125, 1995, 231242. Ktistis G, Niopas I, A study on the in-vitro percutaneous absorption of propranolol from disperse systems, J Pharm Pharmacol, 1998, 50, 413418. Shinoda K, lindman B, organized surfactant systems micro emulsion Langmuir, 3, 1987, 135-149. Shinoda K, Lindman B, Organised surfactant systems: microemulsions, Langmuir, 3, 1987, 135149. Tenjarla SN; Microemulsions, An overview and pharmaceutical Therapeutic Drug Carrier Systems, 16, 1999, 461521. applications, Critical Reviews in
Tenjarla SN, Microemulsions: An overview and pharmaceutical applications, Critical Review s in Therapeutic Drug carrier, systems 16, 1999, 461-521. Trotta M, Influence of phase transformation on indomethacin release from microemulsions, J Control Release 60, 1999, 399405.
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FORMULATION AND EVALUATION OF CHEWABLE TABLETS OF MONTELUKAST

Sree Giri Prasad B *, Pradip Das, Raghu Kiran CVS,Vasia Teegala Krishna Reddy college of Pharmacy, Meerpet, Hyderabad-500097, AP, India * Corresponding author: Email:pradipceutics@gmail.com ABSTRACT The objective of the present study is to develop chewable tablets containing different pharmaceutical compositions with simple manufacturing procedures using different excipients. Mannitols, L-HPC 11, Aspartame, Crospovidone, Crospovidone, Aerosil, and Magnesium Stearate are used as excipients for effective formulation of anti-asthmatic drug Montelukast. Montelukast is a selective, orally acting leukotriene receptor antagonist that is used for the treatment of asthma and seasonal allergic rhinitis. Montelukast chewable tablets were prepared by both wet granulation and Direct Compression methods using suitable excipients. The chewable tablets were better presented using artificial sweetener Aspartame as flavouring agent. A total of eight formulations were prepared and the granules were evaluated for pre-compression parameters. The formulated tablets were evaluated for post-compression parameters. The results showed that all the physical parameters were within the acceptable limits. I.R spectral studies revealed that there was no interaction between the drug and excipients. The in vitro release study of formulation F7 showed 98.85%drug release at the end of 30 min. The stability studies for the formulation F7 showed no significant changes and the study concludes that formulation F7 showed better characteristics of chewable tablet. Key Words: Chewable Tablets, Mannitol, Crospovidone, Aerosil, Magnesium Stearate. 1. INTRODUCTION Today in the world of pharmacy around 90% of the tablets manufactured are ingested orally (Deepak Parasar, 2012).Administration of drugs through oral route is the most common and the easiest way to administer a drug3. However, pediatric, geriatric and bedridden patient shows inconvenience swallowing conventional tablets or capsules due to difficulties in swallowing with lesser amounts of water with the medication, unable to tolerate the taste of many drugs when formulated as liquid dosage forms, resulting in poor patient compliance. The rationalized approach in case of medication leads to the development of chewable tablets. These are formulated and manufactured so that they may be chewed in the mouth producing a pleasant tasting residue in the oral cavity that is easily swallowed and does not leave a bitter a unpleasant taste. Chewable tablets are the tablets which are required to be broken and chewed in between the teeth before ingestion. These tablets are given to the children who have difficulty in swallowing and to the adults who dislike swallowing. Successfully tablet formulation development involves the careful selection of ingredients in order to manufacture a robust solid dosage form. Choosing the appropriate excipients to perform a specific function in a tablet formulation, such as disintegration or lubrication can be critical to achieving acceptable manufacturing performance. Sweeteners, both naturally occurring and synthetic, are one type of functional excipients commonly used in chewable tablet formulations to mask unpleasant tastes and facilitate pediatric dosing (Mullarney, 2003). Ideally chewable formulations should have smooth texture upon disintegration, pleasant taste and no bitter or unpleasant after taste. Upon chewing, they are broken down in the mouth and release their ingredients in the process and therefore, do not have much lag time as required for the disintegration of tablets before absorption from stomach (Biradar, 2006). Montelukast is an oral leukotriene receptor antagonist that is used for the treatment of asthma and seasonal allergic rhinitis. Leukotrienes are a group of naturally occurring chemicals in the body that promote inflammation in asthma and seasonal allergic rhinitis. It is used for the treatment of asthma, seasonal allergic rhinitis, and prevention of exercise induced bronchospasm and begins working after 3 to 14 days of therapy. It should not be used for the treatment of an acute asthmatic attack. The recommended dose of montelukast in adults is 10 mg daily for treating asthma and allergic rhinitis and 10 mg two hours before exercising for prevention of exercise induced bronchospasm. Montelukast should be taken in the evening with or without food when used for asthma or allergic rhinitis. The 4 and 5 mg tablets are used in children. Children find it difficult to swallow the normal tablets of Montelukast. So in order to avoid this problem, chewable tablets are most preferable. Hence it was decided to formulate montelukast chewable tablet to improve the compliance in children. Additionally, chewable tablets facilitate more rapid release and hence more rapid absorption of active ingredients and provide quick onset of action (Jagdale 2010). 2. MATERIALS AND METHODS Montelukast was obtained from Aurobindo Pharma. Ltd. Hyderabad, L-HPC 11, Aspartame and Crospovidone were procured from Hetero Drugs, Mannitol, Aerosil, Magnesium Stearate were of Analytical Grade. 2.1. Identification and flow property of montelukast: Montelukast was identified by organoleptic evaluation, U.V Spectroscopy, FTIR Spectroscopy and Differential Scanning Calorimetry. Flow properties of Montelukast were determined by Carrs Index, Hausners Ratio and Angle of Repose.
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2.2. Drug excipient compatibility study: The drug and excipient are checked for their compatibility by Fourier Transform Infrared Spectroscopy (FTIR). Infrared spectrum peaks of pure drug and drug excipient are evaluated by the use of FTIR Spectrophotmeter (Bruker series) by potassium bromide (KBR) pellet method. Depending on the favourable compatibility test the formulation is designed and the formulation aspects are initiated (Kathiresan, 2010). 2.3. Preparation of montelukast chewable tablets: Chewable tablets containing 10mg of Montelukast were prepared with a total tablet weight of 150 mg by Wet Granulation Method. Exactly Weighed quantities of Mannitol (intragranular), Montelukast, L-HPC 11 and aspartame were weighed and were passed through #30 mesh (Table 1). Add little water to the dry mix and knead to form granules. Pass the wet mass through #30 mesh and dry the granules in hot air oven. Diluents were weighed according to ratio and sifted through #30mesh and added to the above and mixed for 5 m. Sift the dried granules through #30 mesh along with the remaining quantity of Mannitol (extra granular) Superdisintegrant crospovidone weighed was also added. Sweetener and flavor were weighed and passed through #30 mesh and added to the above mixture. The lubricant Magnesium separate was weighed and sifted through #30 and added to the above mixture and blended with the mixture for 1 minute. The final blend was mixed thoroughly for 2-3 minutes in polybag; tablets were compressed in 7 mm round flat punches. Table 1: Composition of Various Formulations of Montelukast Chewable Tablets S. No INGREDIENTS F1 F2 F3 F4 F5 F6 F7 F8 Intragranular 1 Montelukast 10 10 10 10 10 10 10 10 2 Mannitol 75 100 100 100 75 75 75 75 3 L-HPC 11 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 4 Aspartame 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 Crospovidone/ 5 5/0 5/1 Aerosil Extragranular 6 Mannitol 50 25 50 25 25 50 50 7 Aspartame 2.5 2.5 2.5 5 2.5 2.5 2.5 2.5 8. Crospovidone 5 5 7.5 5 7.5 5 5 5 Magnesium 9. 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 stearate Total weight (mg) 150 148 175 125.5 125.5 123 153 153 2.4. Physical Characteristics of Montelukast Chewable Tablets: 2.4.1. General Appearance, Diameter and Thickness (Aulton ME, 1998): The general appearance of all tablets, its visual identity and overall elegance is essential for consumer acceptance. The formulated chewable tablets were evaluated for size, shape, organoleptic characters such as, colour, odor and taste. The diameter and thickness of the tablets were measured by using Vernier caliper. 2.4.2. Hardness (Rudnice, 1990): Hardness is a force required to break a tablet across the diameter. The hardness of a tablet is an indication of its strength. The hardness was measured using Monsanto Hardness tester. The values were expressed in Kg/cm2. 2.4.3. Weight variation (Rudnice, 1990): Twenty tablets of each formulation were selected at random and weighed individually. The weight of individual tablets was noted. Average weight was calculated and the individual weights were compared with the average weight. The weight of not more than two tablets must not deviate from the average weight by more than 5%. 2.4.4. Wetting Time (Rudnice, 1990): A glass Petridish was partially filled with water and tablet was placed on the surface of a band of filter paper supported on a glass slide. The uptake of water occurred from lower surface of the tablet. Time required for water to reach the center of upper surface of tablet was noted as wetting time. 2.4.5. Friability (Indian Pharmacopoeia, 2007): The friability of tablets was determined by using Roche Friabilator. Ten tablets were weighed and placed in friabilator and rotated at 25 rpm for 4 minutes. Then the tablets were taken out, dusted and reweighed. The percentage friability of the tablets was calculated by the formula: Percentage Friability = [(Initial Weight Final Weight)/ Initial Weight] 100 2.4.6. Disintegration Time (Indian Pharmacopoeia, 2007): Disintegration test was carried out by using Disintegration test apparatus. One tablet is placed in each tube, and the basket rack was positioned in a 1-litre beaker of water, at 37C 2C. A standard motor-driven device is used to move the basket assembly containing the tablets up and down through a distance of 5 to 6cm at a frequency of 28 to 32 cycles per minutes. The time taken for the tablet to disintegrate completely was noted. 2.4.7. Drug Content Estimation (USP, 2007): Drug content in all formulations was estimated by U.V Spectrophotometric method. Two tablets from each batch were taken into 0.5% SLS containing in 100ml Volumetric Flask. Finally the volume was made upto 100ml with 0.5% SLS. It was filtered through whatman filter Volume 1 Issue 1
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paper no: 41. First10ml was discarded. The clear filtrate was collected and diluted suitably with 0.5% SLS and measured at 350nm. 2.4.8. In Vitro Dissolution Studies (Indian Pharmacopoeia, 2007): Dissolution test has been performed by using dissolution apparatus USP Type II with a paddle. The dissolution fluid was 900ml of distilled water with 0.5% SLS and a speed of 50 rpm and a temperature of 370.5C. The samples of dissolution medium (5ml) were withdrawn through a filter of 0.45m at different time intervals, suitably diluted and assayed for Montelukast by measuring absorbents at 350nm. Samples withdrawn were analyzed for the percentage of drug released. 2.4.9. Stability Analysis (Suryakanth B, 2011): The formulation F8 was subjected to stability studies, by storing at 40o2oC/755%RH for a period of 30 days. At the optimized period, samples were analyzed for drug content, disintegration time and in vitro dissolution studies. 3. RESULTS AND DISCUSSION Montelukast found to be white to off-white crystalline powder, tasteless, odorless, in organoleptic evaluation. U.V Spectroscopy study showed that the maximum absorbance at 350nm, after a baseline correction in the U.V Spectrophotometer, when a 6mcg/ml solution of Montelukast in 0.5% SLS was scanned between 200 to 400nm. IR Spectrum of Pure drug montelukast, Montelukast with L-HPC11 and Montelukast with crospovidone has exhibited IR spectra indicating presence of hydroxylic of carboxylic acid and hydroxyl of tertiary carbon. Table 2: Post - compression parameter of montelukast Parameters Thickness (mm) Hardness (kg/cm2) Friability (%) (%) Drug content Disintegration time (sec) Wetting time (sec) F1 0.71 3.5 0.23 98.02 22 25 F2 0.71 3.4 0.15 97.23 18 22 F3 1 3.5 0.16 99.21 15 19 F4 0.64 3.6 0.28 99.01 29 32 F5 0.64 3.5 0.24 99.89 12 15 F6 0.63 3.6 0.11 100.2 26 28 F7 0.72 3.5 0.62 100.03 10 13 F8 0.72 3.5 0.44 99.24 13 15
Hence it exhibited broad band around 3451 cm- indicating overlapping of these peaks. Peak due to C-H peaks has appeared as shoulder between 2900 cm-and 3100 cm-.The C=O peak has appeared at 1636 cm- along with a merged peak 1636.66 cm-.This is due to the complex structure of drug molecule. These are the characteristic absorption peak of montelukast which shows compatible with excipients. Depending on the above favorable compatibility test the formulation is designed and the formulation aspects are initiated. The various Post Compression parameters that lie within the limits as per individual evaluation parameter. The flow properties of the granules are essential as per the handling and filling is concerned. The formula is optimized based on over all evaluation. The composition of chewable tablets is presented in Table1. Different concentrations of superdisintegrants were investigated. The final blend of drug and excipient of all the formulations were evaluated for flow properties and was found to be good. Micrometric properties of all formulations showed an excellent angle of repose i.e. <25 except the formulation F1. Percentage Compressibility was found to be less than 21, ensuring good flow properties and results are presented in Table 2. Hausners ratio values were well below 1.15. All the formulations were subjected to physical-chemical evaluations like weight variation, thickness, hardness, friability, dr u g cont ent, disintegration test, modified disintegration test, and wetting time were carried out in order to assess the suitability of the formulations with respect to the dosage form and intended therapeutic purpose. All the tablet formulations passed all tests. The Disintegration time evaluation parameter as it forms the bases for developing orally disintegrating tablets. It shows the wetting time of most of the formulation and lied within the range i.e 13-32 seconds. The lowest (13 seconds) was obtained with formulation F7.The comparison of all the wetting time of formulation is given above. The disintegration time for each tablet was found to be less than a minute and the results ae shown in Table 2. The tablet containing crospovidone 10mg showed lowest disintegrant time of 10seconds when compared to other formulations. Montelukast chewable tablets from all formulations and marketed sample were subjected to in-vitro release studies. Figures 6 & 7 shows release studies of F1, F2, F3, F4, F5 & F6 formulations, prepared by wet granulation technique and crospovidone was not added intra-granularly, hence % drug release is slow. In F2 and F3 more amount of mannitol was added in intra-granular, thus showing slow dissolution. In F3 more amount of crospovidone is added in extra-granular thus initially showed fast drug release but later slow drug release due to more amount of mannitol. Mannitol is a slow dissolving diluent. In the formulation F4 mannitol was not added in extra-granular so that it is gets dissolved faster but the drug release was slow as even the amount of crospovidone in extra-granular was reduced. In F5 amount of mannitol was reduced and the amount of crospovidone was increased thus showing a good drug release. In F6 formulation there was no significant Volume 1 Issue 1
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release.Both these formulations (Fig - 8 & 9) were prepared by the addition of crospovidone intra-granularly and Aerosil was added in F8 formulation. F7 formulation showed excellent %drug release compared to all other formulation. Both the formulations were prepared by wet granulation containing 10 mg of pure drug in the formulation.The optimised formulation F7 containing the drug, mannitol, LHPC11, cross-povidone, aspartame, magnesiumstearate showed bio-equivalent %drug release with the marketed product which was formulated by the addition of mannitol, MCC, HPC, Red Ferric oxide, Crosscaramellose Sodium, Cherry Favour, Aspartame and Magnesium Stearate.
Figure 1: FTIR Spectra of pure drug Montelukast.
Figure 2: FTIR Spectra of Montelukast and L-HPC 11
Figure 3 : FTIR Spectra of Montelukast and Crospovidone

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Wetting time (seconds)
30 25 20 15 10 5 0 f1 f2 f3 f4 f5 f6 f7 f8 Formulations
Figure 4 : Disintegration time of formulation F1 to F8
Figure 5 : Wetting time of formulation F1 to F8
Figure 6 & 7: Dissolution Profile of Batches F 1, F2, F 3, F4, F5 & F6 Volume 1 Issue 1
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120 100 80 60 40 20 0 0 10 20 TIME(min) 30 40 F7 MARKETED
Figure 8: Dissolution Profile - Batch F7 and F8.
Figure 9: Dissolution Profile - Batch F 7 anMarketed product.
Figure 10: Dissolution Profile - Batch F7 and F8. 3.1. Stability studies results: The optimized formulation F7 was subjected to stability studies. The tablets were stored at 400 C / 75% RH and room temperature in a closed high density polyethylene bottles for three months. The bottles were properly labeled and the kept for three months under these conditions and evaluated for appearance, disintegration time, % drug content and % drug release. The stabilized tablets were checked for their appearance and color and recorded. The optimized tablets were subjected to disintegration time test to check for any changes in their disintegration times. Percentage drug content at different conditions and dissolution profile were conducted and presented in the Table 3 and Table 4 respectively. 4. SUMMARY AND CONCLUSION In the present study Montelukast chewable tablets were by using to different concentrations of crospovidone. In the present research work all tablets met the compedial limit in terms of physicochemical parameter, disintegration and dissolution. From the study, the optimized chewable tablets of Montelukast to be a promising alternative for the effective management of acute Asthma. Crospovidone is the only superdisintegrant employed. Optimized batch F7 shows better in-vitro drug release in comparison to the other formulation batches prepared with the batches prepared with the second polymers. It was concluded that the rate of dissolution was found to be increased by increasing the concentration of crospovidone. The Chewable tablets of F7 batch containing 10mg of Montelukast, 125mg of mannitol,10mg of crospovidone,2.5mg of L-Hpc11, 5mg of aspartame,0.5mg of magnesium stearate considered to be the best among all other eight batches of tablets
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Time in Days 0 30
Table 3: General appearance and drug content after stability studies Appearance 400 C / 75 % RH Room Temp. Temperatures - % Drug Content Room temperature 400 C / 75 % RH Batch F7 Batch F7 Batch F7 Batch F7 Batch F7 white 10(sec) 10(sec) 100.03 100.03 white 11(sec) 10(sec) 100.01 100.0 Table 4: In - vitro dissolution profile for batch F7. % Drug release Time in minutes Initial 30 days 5 75.84 75.21 10 85.43 84.87 15 90.21 89.91 20 96.40 96.01 30 98.85 97.63
5. REFERENCES
Agarwal SP, Ragesh Khanna, Physical Pharmacy, New Delhi, CBS Publishers and distributors, 2nd edn, 2000, 247. Aulton ME, Wells TI, Pharmaceutics, The Science of Dosage Form Design, Churchill Livingstone, Vingstone, London, 1988, 168. Biradar, Formulation and Evaluation of chewable tablets, Int J Pharmacy and Pharm Sci, 2, 2006, 461 464. Deepak Prashar, Sanjay Saklani. Formulation and Eavaluation of Anthelmintix Chewable Tablets, Internationale Pharmaceutica Sciencia, 2(1), 2012, 13-16. http://www.medicinenet.com/montelukast/article.html Jagdale, Formulation and Evaluation of chewable tablets of Levamisole, Int J Res Pharm Sci, 1, 2010, 282-289. Kathiresan, Formulation and Evaluation of Loratadine chewable tablets, Res J Pharm Biological Chem Sci, 1(4), 2010, 763- 774. Lachman L, Lieberman HA, Kanig JL, The Theory and Practice of Industrial Pharmacy, 3rd Edition, 1991; 293. Mullarney , The powder flow and compact mechanical properties of sucrose and three high intensity sweeteners used in chewable tablets, Int J Pharmaceutics, 257, 2003, 227-236. Nayankumar C, Ratnakar , Bhupendra G,Prajapati, Der Pharmacia Sinica, 2 , 2011, 333-340. Rippe E, Swarbrick J, Encyclopedia of Pharamaceutical Technology, Marcel Dekker Inc, Newyork, 3, 199, 149-166. Rudnic E, Schwartz JB, Oral Solid Dosage forms In: Remingtons Pharmaceutical Sciences, 18th edition, Mack publishing Company. Easton, Pennsylvania, USA; 1990, 1633-1665. Sukhbir, Formulation Development and Evaluation of Chewable Tablet of Albendazole by Different Techniques, Int J Pharmacy and Pharm Sci, 4(1), 2012, 461 464. Suryakant B, Jadhav , Dinesh M, Sakarkar, Datta R, Kaudewar, Der Pharmacia Sinica, 2, 2011, 623-631. The Indian Pharmacopoeia, Ministry of Health and Family welfare, Govt of India, Controller of Publications, New Delhi, 2007, 2, 973, 1377,144,736. United States pharmacopoeia (USP 29-NF 24), The official compendia of standards twin brook parkway, rockvillle. Asian Edition, 3, 27, 60-62, 2007, 2675, 2505.
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Sampath Kumar KP et.al.
TRANSDERMAL IONTOPHORESIS TECHNIQUE-A POTENTIAL EMERGING DRUG DELIVERY SYSTEM

Sampath Kumar KP1, Debjit Bhowmik2*, Jyoti Jaiswal3 1. Department of Pharmaceutical Sciences, Coimbatore medical College, Coimbatore 2. Karpagam University, Coimbatore 3. Institute of clinical research of India, New Delhi * Corresponding author: Email: debjit_cr@yahoo.com ABSTRACT The objective of delivery system is to achieve optimum therapeutic management. But, it still remains a challenge in the field of pharmaceuticals for delivery of ionic species such as proteins and peptides. Development of iontophoretic system is a breakthrough in this field designed to improve the delivery rate of ionic compounds. This technique generates an electrical potential gradient that facilitates the movement of solute ions across the membrane. Moreover with the advent of more sophisticated techniques available for the production of recombinant proteins and peptides, there is an ever-increasing demand of novel delivery systems that could effectively deliver these ionic species at the specific site. Iontophoresis seems to be an ideal candidate to sort out the limitations associated with the delivery of ionic drugs. This delivery system utilizes electric current as a driving force for permeation of ionic and non-ionic medications. The rationale behind using this technique is to reversibly alter the barrier properties of skin, which could possibly improve the penetration of drugs such as proteins, peptides and other macromolecules to increase the systemic delivery of high molecular weight compounds with controlled input kinetics and minimum inter-subject variability. The controlled or feed back iontophoretic drug delivery may include the use of polymeric system responsive to an oscillation magnetic field, temperature sensitive polymers, polymers responsive to externally applied ultrasound and chemically sensitive polymers. The iontophoretic delivery of macromolecules will open the doors to non-invasive transdermal delivery of peptide-based pharmaceuticals, following the advances in recombinant DNA technology, which are the wonder drugs of tomorrow. Key words: Iontophoresis, Transdermal therapeutic system, Drug delivery, clinical application. 1. INTRODUCTION The first transdermal drug delivery system was introduced in the United States over 20 years ago. The technology generated tremendous excitement and interest amongst the major pharmaceutical companies in the 1980s and 90s. Excitement dwindled to disappointment, when the limitations of the existing transdermal technology became evident and the numbers of drug candidates were limited to very few molecules. Factors limiting the success of transdermal technology included such things as local skin irritation associated with certain drugs and formulations, limitation on the dose of drug that could be delivered transdermally, a lag time associated with the delivery of the drug across the skin, variation of absorption rate based on site of application, skin type and patient age, and variation in adhesive effectiveness across skin types. These limitations, in addition to the rise in other non-oral drug delivery systems, such as pulmonary delivery systems, caused interest in transdermal technology to decline. Without the interest of big pharma and the funding partnerships that it provided, few transdermal drug delivery companies could sustain themselves without a large pipeline that led products to the marketplace. By the mid to late 1990s, the trend of TDS companies merging into larger organizations (i.e., J&J acquiring ALZA and a part of Cygnus, Watson acquiring Theratech, and Elan acquiring Sano) combined with the increasing number of mega-pharmaceutical mergers, left fewer companies that wanted to develop transdermal products. Acceptance of transdermal technology by larger pharmaceutical companies became more conservative, and development efforts remained focused on oral drug delivery (Chein YW, 1992). Interest in transdermals has increased on several fronts over the past several years. Technology companies have generated additional clinical data demonstrating the potential of advanced transdermal technology. More than 20 transdermal patches are available, with 13 drug molecules are already available in the market.(Pellet M, 2003) However the small group of marketed products has represented drugs of many important classes; antianginal, (nitroglycerin, isosorbide dinitrate), antihypertensive (clonidine), antiemetics (scopolamine), hormones (estrodiol, testosterone), urinary antispasmodic (oxybuyrin), local anesthetic (lidocaine) and CNS drugs (fentanil, nicotine). Although there are only a limited number of marketed transdermal patches available, many others are in development or awaiting FDA approval. Eg.: Noven Pharmaceuticals has recently submitted a new drug application for a methylphenidate transdermal patch. There is also a great deal of investigation into an insulin transdermal delivery system. Some other drugs that are being investigated for transdermal use include albuterol, enalapril, dronabinol, ketorolac, alprazolam, cytarabine, atenolol, buprenorphine, selegiline, isosorbide dinitrate, and prazosin. Improvement in physical and chemical permeation enhancement technologies also led to renewed interest in transdermal drug delivery. Products have already reached the US market using iontophoresis (eg. lidocaine in the Iomeds Iontocaine and epinephrine in the Phoresor iontophoresis system), which is marketed for local dermal Volume 1 Issue 1 January February 2013 Page 35
analgesia. Similarly, Vyteris is awaiting approval for its iontophoretic system, which also delivers lidocaine for dermal anesthesia in children. Several other companies have completed various stages of clinical iontophoresis studies, most notable among them being ALZA with its E-TRANS system using fentanil for the management of postoperative pain.Companies, such as Altea Therapeutics, Transpharma, and ALZA are using various microporation technologies to transdermally deliver peptides and proteins, vaccines, and various pain medications. Initial clinical results are encouraging and have helped bring greater attention to the potential of active transdermal technology. Clinical development continues in systems utilizing electroporation, sonophoresis, and electronic component integrated technologies (Chein YW, 1992). Transdermal Technology ensures as much as 95% of a supplement reaches the cells where it is needed. Doctors around the world are calling Transdermal delivery "the delivery system of the future" and found fantastic alternative to pills and tablets. The iontophoretic delivery of macro-molecules allows the strategy for non-invasive transdermal delivery of peptide-based pharmaceuticals, and contributes to further future advancement toward recombinant DNA technology. Although iontophoresis provides many benefits and seems to be more effective than other techniques, there is a need for further research and judicious use of technology with microelectronics devices and to make it available for commercial application. Thus, iontophoresis may prove to be a potential emergence to transdermal drug delivery. 1.1. Transdermal drug delivery system: The novel drug delivery systems (NDDS) are being investigated so as to alter the body distribution of drug(s) with a view to reduce the toxicity of drug and /or deliver them more efficiently to their site of action or to improve therapeutic index. Research soon followed on ways the drug levels could be modulated and extended to derive more benefits and lower the toxicological risks. Transdermal drug delivery systems are ideally suitable for drugs that need to be administered for diseases those are chronic in nature. Hence cardiovascular agents of both therapeutic and prophylactic importance have been investigated for its dermal penetration extensively. Although a large number of bioactive agents have been studied but only few drugs could meet the required permeability target for clinical benefit. Then, commercially successful transdermal delivery systems of the worldwide transdermal patch market approach 2 billion and are growing rapidly (Williams AC, 2004). Iontophoresis is the technique which facilitates the movement of ions across a membrane under the influence of an externally applied electrical potential difference. Iontophoresis dramatically enhances both the rate of release and the extent of penetration of the salt form of the drugs. Without iontophoresis, such charged species are largely incapable of transdermal penetration due to the skin's lipophilic nature. Iontophoresis is gaining wide popularity as it provides a non invasive and convenient means of systemic administration of drugs with poor bioavailability profile, short half life and with multiple dosing schedules. Iontophoresis, in comparison to oral route, definitely provides benefits of improved efficacy and/or reduces adverse effects. Skin is one of the most extensive and readily accessible organs of the human body. In modern-day pharmaceutical practice, therapeutic compounds are applied to the skin for dermatological (within the skin), local (regional) and transdermal (systemic) delivery. For transdermal delivery of drugs, stratum corneum is the main barrier layer for permeation of drug. So, to circumvent the stratum corneum and to increase the flux through skin membrane, different approaches for enhancement of penetration are used. Among other things, the skin serves as a port of entry into the body for drug administration to providing continuous transdermal infusion into the systemic circulation. As previously mentioned, it is one of the most extensive and readily accessible organs of the human body. As both the largest and most visible organ of the body, the skin is of unequalled importance in portraying an individuals state of being. The delivery of therapeutic agents to systemic circulation via skin requires a delivery mechanism, and many drug delivery mechanisms utilize alternative forms of energy to facilitate permeation of drugs through the skin. Thus, to provide continuous drug infusion through intact skin, transdermal drug delivery system (TDDS) has been developed for topical application onto the intact skin surface, which can deliver medicines via skin portal to systemic circulation. Delivery via the transdermal route is an interesting option in this respect since it is convenient and safe (Chong S, 1989). 1.2. Advantages of TDDS (Chong S, 1989): Transdermal drug delivery offers several important advantages over more traditional dosage forms. These includes The steady permeation of drug across the skin allows for more consistent serum drug levels, often a goal of therapy. Intravenous infusion also achieves consistent plasma levels, but it is more invasive than transdermal drug delivery. The lack of peaks in plasma concentration can reduce the risk of side effects. Thus, drugs that require relatively consistent plasma levels are very good candidates for transdermal drug delivery. In addition, if toxicity were to develop from a drug administered transdermally, the effects could be limited by removing the patch. Volume 1 Issue 1 January February 2013 Page 36
Another advantage is convenience, especially notable in patches that require only once weekly application. Such a simple dosing regimen can aid in patient adherence to drug therapy. Transdermal drug delivery can be used as an alternative route of administration to accommodate patients who cannot tolerate oral dosage forms. It is of great advantage in patients who are nauseated or unconscious. Drugs that cause gastrointestinal upset can be good candidates for transdermal Drugs that are degraded by the enzymes and acids in the gastrointestinal system may also be good targets. First pass metabolism, an additional limitation to oral dosage drug delivery, can be avoided with transdermal administration. Continuity of drug administration permitting the use of a drug with short biological half-life 1.3. Disadvantages of TDDS (Chong S, 1989): One of the greatest disadvantages to transdermal drug delivery is the possibility that a local irritation will develop at the site of application. The drug, the adhesive, or other excipients in the patch formulation can cause erythema, itching and local edema. For most patients, site rotation can minimize irritation. The skins low permeability limits the number of drugs that can be delivered in this manner. Many drugs with a hydrophilic structure permeate the skin too slowly to be of therapeutic benefit. Table No 1. Currently Available In Market Product Name Active Ingredient Category E Trans Fentanyl Analgesia Catapres TTS Clonidine Antihypertensive Lidoderm Lidocaine Post-herpaticneuralgia Nitro-Dur Transderm-Nitro Nitroglycerine Angina Transderm Scop Scopolamine Motion sickness 1.4. Selection criteria for drug candidate: Transdermal route of drug administration has certain inherent difficulties that make it unsuitable for a large number of drugs. The selection of suitable candidates is an important step for success of transdermal research. Ideal characteristic drug should possess for the successful delivery through this approach includes A TDDS should not cover an area more than 50 cm2 and the daily dose is of order of a few mg. The effective concentration of the drug should be low, presumably in the ng/ml. The half life (t1/2) of the drug should be short. The active ingredients should not cause any skin toxicity or irritation. As the diffusion of drug through polymer as well as skin is dependent on molecular size, the drug of low molecular size is preferred. The drug should have a low melting point so that it acts on normal body temperature. Drugs, which degrade in the GI tract are inactivated by hepatic first-pass effect, are suitable candidates for transdermal delivery. Tolerance to the drug must not develop under the near zero-order release profile of transdermal delivery. The candidate drug should have adequate hydrophilic and lipophilic balance to negotiate the lipid barrier of stratum corneum before being partitioned into the aqueous viable tissue. 1. 5. Approaches to enhance drug delivery through skin: The intrinsic skin permeability of most drugs is inadequate to meet the therapeutic demand. Since novel technologies have been investigated to enhance the permeation rate, discussed below are the major. 1.5.1. Prodrugs for dermal delivery: It involves the chemical modification of a known pharmacologically active compound into a bioreversible form, with the aim of changing its pharmaceutical and / or pharmacokinetic character and thereby enhancing its delivery, efficacy and therapeutic value. The particular combination of functional groups introduced into the parent drug to give the prodrug composes the promoiety. The important parameters, which influence the activity of prodrugs, are physicochemical properties, biopharmaceutics and pharmacokinetics properties, as well as the toxicity and bioactivity. The skin is a highly active metabolic organ. It contains a multitude of different enzymes that may metabolize a wide range of synthetic and naturally occurring xenobiotics. This capacity of skin is utilized by the prodrugs to revert back to the active parent drug. 1.5.2. Chemical potential adjustment: The vehicle plays an important role in permeation and achieving a thermodynamic activity that favors partitioning of the drug to the skin is one of the approaches used. Artificially designed supersaturated solutionare used to enhance the permeation rate (Pellet M, 2003). 1.5.3. Ion pairs: Charged molecules do not readily penetrate stratum corneum. One technique forms a lipophilic ion pair, by adding an oppositely charged species. The complex partitions into the SC lipids, as charges temporarily neutralize. The ion pair diffuses to the aqueous viable epidermis, there it dissociates into its charged species, which partition into the epidermis and diffuse onward (Brown M V, 1992). 1.5.4. Eutectic systems: The melting point of a drug influences solubility and hence skin penetration. According Volume 1 Issue 1 January February 2013 Page 37
to regular solution theory, the lower the melting point, the greater is the solubility of a material in a given solvent, including skin lipids. The melting point of a drug delivery system can be lowered by formation of a eutectic mixture: a mixture of two components which, at a certain ratio, inhibit the crystalline process of each other, such that the melting point of the two components in the mixture is less than that of each component alone. EMLA cream, a formulation consisting of a eutectic mixture of lignocaine and prilocaine applied under an occlusive film, provides effective local anaesthesia for pain-free venepuncture and other procedures. The 1:1 eutectic mixture (M.P. 18oC) is oil, which is formulated as an oil-in-water emulsion thereby maximizing the thermodynamic activity of the local anaesthetics. A number of eutectic systems containing a penetration enhancer as the second component have been reported, for example: ibuprofen with terpenes, menthol and methyl nicotinate, propranolol with fatty acids; and lignocaine with menthol. In all cases, the melting point of the drug was depressed to around or below skin temperature thereby enhancing drug solubility. However, it is also likely that the interaction of the penetration enhancer with SC lipids also contributed to the increased drug flux (Tsai J C, 1998). 1.5.5. Liposomes and other vesicles: Liposomes are colloidal particles, typically consisting of phospholipids and cholesterol, with other possible ingredients. These lipid molecules form concentric bimolecular layers that may entrap and deliver drugs to the skin. It is a localizing effect whereby vesicles accumulate drugs in SC or other upper skin layers. Generally, liposomes are not expected to penetrate into viable skin, although occasional transport processes were reported (Tsai S C, 1998). 1.5.6. High velocity particles: The solid particles can be directly injected to the skin using the supersonic shock wave of helium gas (e.g. Powder Jet). The advantages of the system include pain-free delivery, improved efficacy and bioavailabilty, targeting to a specific tissue, sustained release, or fast release, accurate dosing, overcomes needle phobia and safety. However, there have been problems with bruising and particles bouncing off skin surfaces. Regulatory authorities will need convincing that high velocity particles smashing through the SC really do no damage to this elegant structure, which is not readily repaired, nor do they carry surface contaminants such as bacteria into viable skin layers (Williams A C, 2004). 1.5.7. Chemical penetration enhancers: Substances temporarily diminishing the barrier of the skin, known also as accelerants or sorption promoters. They can enhance drug flux. Many enhancers, such as Azone, DMSO, alcohols, fattyacids and terpenes, have been shown to increase permeability by disordering or fluidising the lipid structure of the stratum corneum. In some cases the enhancers penetrate into and mix homogeneously with the lipids. However, others such as oleic acid and terpenes, particularly at high concentration, pool within the lipid domains to create permeable pores that provide less resistance for polar molecules (Williams A C, 2004). 1.5.8. Microneedle array: SC can be bypassed by injection and one development of this approach is a device of 400 microneedles, which insert drug just below the barrier (Elias P M, 1983). The solid silicon needles (coated with drug) or hollow metal needles (filled with drug solution) penetrate the horny layer without breaking it or stimulating nerves in deeper tissues. Flux increases up to 100000-folds are claimed. The technique may also be combined with iontophoresis. 1.5.9. Skin abrasion: The abrasion technique involves the direct removal or disruption of the upper layers of the skin to facilitate the permeation of topically applied medicaments. Some of these devices are based on techniques employed by dermatologists for superficial skin resurfacing, e.g. microdermabrasion. Microdermabrasion uses a stream of aluminium oxide crystals. Adhesive tape, which removes SC prior to drug application, and a microinfusor device, has also been proposed to deliver drugs through transdermal route. On the other hand a blister by suction, an epidermatome removes the raised tissue, after which a solution delivered directly to the exposed dermis (Williams A.C. 2004). 1.5.10. Medicated tattoos: A related means of delivering compounds transdermally has been developed by Lipper-Man Ltd. There is no predetermined duration of therapy for Med-Tats. Instead, it provides a color chart that can be compared to the color of the patient's tattoo to determine when the tattoo should be removed. This visual comparison, which relies on dyes incorporated into the patch, introduces a significant amount of inter patient variability (Audit M.C. 2001). 1.5.11. Ultrasound (phonophoresis, sonophoresis): This technique, used originally in physiotherapy and sports medicine, applies a preparation topically and massages the site with an ultrasound source. The procedure was extended to transdermal drug delivery studies. The ultrasonic energy (at low frequency) disturbs the lipid packing in SC by cavitation. Shock waves of collapsing vaccum cavities increase free volume space in bimolecular leaflets and thus enhance drug penetration into the tissue (Elias P M, 1983). 1.5.12. Electroporation: Skin electroporation (electropermeabilization) (Prausnitz, 1993) creates transient aqueous pores in the lipid bilayers by application of short (micro- to millisecond) electrical pulses of approximately 100 1000 V/cm. These pores provide pathways for drug penetration that travel straight through the horny layer. Significant movement can also occur between pulses by simple diffusion due to relatively persistent changes in the SC lowering its resistance. The process may also transport into the integument, vaccines, liposomes, as well as nanoparticles and microspheres. Electroporation may combine with iontophoresis to enhance the penetration of peptides such as vasopressin, neurotensin, calcitonin and LHRH (Schreier H, 1994). Electroporation has also been Volume 1 Issue 1 January February 2013 Page 38
combined with ultrasound (Schreier H 1996). 1.5.13. Magnetophoresis: Limited work probed the ability of magnetic fields to move diamagnetic materials through skin. Langer discussed the interesting idea of employing intelligent systems based on magnetism or microchip technology to deliver drugs in controlled, pulsatile mode (Cere G, 2003). 1. 5. 14. Laser radiation: This method involves direct and controlled exposure of a laser to the skin, which results in the ablation of the SC without significantly damaging the underlying epidermis. Removal of the SC using this method has been shown to enhance the delivery of lipophilic and hydrophilic drugs (Tsai J C, 1998). 1.5.15. Iontophoresis: The idea of transmitting therapeutic agents by means of electrical current has a history of more than 250 years. The first suggestions for the use of electricity for drug transport to be reported in the literature date from the mid-18th century and not all of them seem to be reliable. In a publication dated 1747 (7), the Italian librarian Giovanni Francesco Pivati (1689-1764) that the smell of Peruvian balsam hermetically sealed in a glass cylinder became apparent in the room after applying electrical current and could even be transmitted to another room by wire. The Turin anatomist Giovanni Battista Bianchi (1681 1761) observed that purgatives held in the hand of a person during electrification had the same laxative effect as when administered orally. However, the data observations were of theoretical interest. The use of electricity in the transdermal delivery was revived some twenty years ago when it was thought that the technique can be useful for the systemic delivery of macromolecules. Iontophoresis involves application of low intensity electric current (usually 0.5 mA/cm2). The electrode was placed in a drug reservoir to the surface of the skin. A repulsion effect was developed between the electrode and the charged drug moiety, which drives the skin. Positive charge drug is delivered from anode while the negative charged drugs from the cathode. The technique offers number of advantages like, the benefits of bypassing hepatic first pass effect, higher patient compliance, delivery of ionized and unionized drugs, enabling continuous or pulsatile delivery of drug, permitting easier termination of drug delivery, offering better control over the amount of drug delivered, restoration of the skin barrier function without producing severe skin irritation, improving the delivery of polar molecules as well as high molecular weight compounds, ability to be used for systemic delivery or local (topical) delivery of drugs and reducing considerably the inter and intra-individual variability (Ceve G, 2003). a) Iontophoretic system: An iontophoretic drug-delivery system has three basic components: the source of electric current, which usually consists of a battery and control electronics; an active reservoir system, which contains the ionic therapeutic agent; and an indifferent or return reservoir system, which contains an electrolyte and serves to complete the electric circuit. When the active and indifferent reservoir systems are placed on the skin, the current source causes electronic current to flow to the active reservoir where the electronic current is transformed to ionic current. The ionic current flows through the active reservoir, through the skin, beneath the skin towards the indifferent reservoir, and back through the skin into the indifferent reservoir. At the indifferent reservoir, it is transformed back into electronic current completing the circuit at the opposite pole of the current source (Godin.B 2003). Fig: 1 Depicts iontophoresis using an Ag/AgCl electrode system b) Current induced drug transport: The increased flux during iontophoresis would include, flux due to the electrochemical potential gradient across the skin, change in the skin permeability due to the electric field applied, and electro-osmotic water flow and the resultant solvent drag (Ceve.G 2003). Jionto = Jelectric + Jpassive + Jconvective
Where Jelectric is the flux due to electric current application Jpassive is the flux due to passive delivery through the skin convective J is the flux due to convective transport due to electro osmosis c) Pathways of iontophoresis: Skin appendages, which include sweat glands and hair follicles, are postulated to be involved in the major pathways of drug transport during iontophoresis. Other pathways, which have been shown to be involved in iontophoretic drug delivery, include paracellular transport especially for water and uncharged Volume 1 Issue 1 January February 2013 Page 39
polar solutes, artificial shunts due to temporary disruption of the organized structure of the SC and potentialdependent pore formation (Ceve G, 2003). d) Factors affecting iontophoresis: The technique of iontophoresis depends upon several physicochemical variables apart from the factors, which affect the skin uptake of drugs during passive diffusion. Current intensity, vehicle pH, type of electrode, co-ions, concentration, conductivity of drugs and skin impendence affect the transport of drugs by inotophoresis (Barry .B.2002). i. Current intensity: The electromigratory contribution to iontophoretic flux is directly proportional to the applied current provided when the respective ion concentrations are kept constant. In general, this holds true both for small molecules and peptides. Although increasing the current produces an increase in iontophoretic transport, at higher current levels a saturation level is obtained. After a limiting transport number is achieved, further increase in current has no effect (FDA 2002). ii. pH: This affects iontophoresis in two ways. The pH of the donor solution influences the pH of the skin and thus makes the skin a permselective membrane especially if the pH of the skin raises above 4.This causes the carboxylic acid moieties in the skin to become ionized and the ca ionization of the drug itself. Thus a weakly basic drug will be ionized to a lower extent at pH higher than its pKa and will not permeate by electromigration in presence of tionic drugs are promoted. The pH of the donor solution also affects the iontophoresis. The drug will be more dependent on electro-osmosis to travel across the skin (Ceve G, 2003). iii. Type of electrode: Type of electrodes used also affect the iontophoretic delivery. Electrodes Ag/AgCl are the most preferred as they resist the changes in pH which are generally seen during the use of platinum or zinc/zinc chloride electrodes. The following reactions typically occur at the anode. Ag + ClAgCl + eThe electron is released to the circuit and insoluble AgCl precipitates at the anode surface. In the case of other metals like platinum, the chloride ion at the anode will be converted to Cl2, which will in turn react with water to generate hydronium ions. These then migrate to the donor solution and compete with similar-charged drug ions and being highly mobile enters the skin thus reducing drug transport and simultaneously causing skin irritation. iv. Co ions: The presence of a co-ion (ion with the similar charge as the drug) results in competition between the drug and the co-ion. It reduces the fraction of the current available to the drug and thus causes reduction in drug flux. A most common source of co-ions is the buffer added to control the pH of the donor medium. v. Drug concentration: Drug concentration and its impact on iontophoretic flux is one of the most commonly studied experimental parameters. It suggests the existence of a fairly straightforward correlation between the amount of drug in the formulation and the observed flux. However, the experimental results differ from the theory. Increasing the amount of drug in the formulation may not increase the number of molecules in the membrane; second, the concentrations and mobilities of competing ions play an important role; and third, structure and physicochemical properties of specific drugs may influence its ability to permeate through iontophoretic transdermal pathways (Barry B, 2002). vi. Resistance of skin: Under electricity, the dermal resistance de creases. However, it is desirable that once the current is removed, the skin resistivity is restored. vii. Drug salt form: It has been reported that different salt forms have different specific conductivities and that conductivity experiments in vitro will provide information concerning the general suitability of a drug for iontophoresis. The salt form of the drug must be considered along with the pH of the solution for determining the amount of drug in the ionized state (Finin CB, 2003). viii. Stability of the drug during the iontophoresis process: The drug undergoing iontophoresis must be stable in the solution environment up to the time of iontophoresis and also during the iontophoresis process. Drugs which are easily oxidized or reduced must be appropriately formulated. This is important because oxidation or reduction of a drug not only decreases the total drug available but the degradation compounds, if they possess the same charge as the drug ion, will compete with the drug ion and reduce the overall transmembrane rate of the drug (Kumar P, 2004). ix. Molecular size: It has been shown that the permeability coefficients in positively charged, negatively charged and uncharged solutes across excised human skin are a function of molecular size. When the molecular size increases, the permeability coefficient decreases. However, there are certain solutes with a relatively high molecular size (e.g. insulin, vasopressin and several growth hormones), which have also been shown to penetrate the skin barrier into the systemic circulation (Finin C B, 2003). 2. CLINICAL APPLICATION 2.1. Dentistry: Dentistry, probably to an even greater extent than physical therapy, has used iontophoresis with patients prior to oral surgical procedures. Treatment of hypersensitive dentin (e.g. in teeth sensitive to air and cold liquids) using negatively charged fluoride ions;Treatment of oral ulcers ("canker sores) and herpes orolabialis lesions ("fever blisters") using negatively charged corticosteroids and antiviral drugs, respectively; andThe application of local anaesthetics to produce profound topical anaesthesia, as is done in some physical therapy applications. Volume 1 Issue 1 January February 2013 Page 40
2.2. Dermatology: Iontophoresis has many uses in the field of dermatology. Except for the use of lidocaine for anaesthesia and the treatment of patients with hyperhidrosis, most uses of iontophoresis in dermatology have largely been abandoned. Iontophoresis with tap water or anticholinergic compounds has been used for the treatment of patients with hyperhidrosis of the palms, feet, and axillae. 2.3. Otorhinolaryngology: Iontophoresis is a preferred method for obtaining anaesthesia of the tympanic membrane prior to simple surgical procedures involving that structure. Iontophoresis of zinc has also been used for the treatment of patients with allergic rhinitis. 2.4. Ophthalmology: Iontophoresis has been used experimentally to deliver antibiotics into the eye. The principal disadvantage of this technique is the time required for direct contact of the electrode with the eye. 2.5. Diagnostic Applications: Iontophoretic application of the drug pilocarpine produces intense sweating, allowing sufficient amounts of sweat to be collected and analyzed. This is now accepted as the primary test in the diagnosis of cystic fibrosis. 3. CONCLUSION Iontophoresis is a technique used to enhance the transdermal delivery of compounds through the skin via the application of a small electric current. By the process of electromigration and electro-osmosis, iontophoresis increases the permeation of charged and neutral compounds, and offers the option for programmed drug delivery. Interest in this field of research has led to the successful delivery of both low (lidocaine) and high molecular drugs, such as peptides (e.g., luteinising hormone releasing hormone, nafarelin and insulin). Combinations of iontophoresis with chemical enhancers, electroporation and sonophoresis have been tested in order to further increase transdermal drug permeation and decrease possible side effects. In addition, rapid progress in the fields of microelectronics, nanotechnology and miniaturisation of devices is leading the way to more sophisticated iontophoretic devices, allowing improved designs with better control of drug delivery. REFERENCES Ansel H C, Loyd A V, Popovich N G, Pharmaceutical dosage system, ed 7, Lippincott Williams and Willkins publication. Audet M C, Evaluation of Contraceptive Efficacy and Cycle Control of a Transdermal Contraceptive Patch vs. an Oral Contraceptive: A Randomized Controlled Trial, JAMA, 285(18), 2001, 2347-2354 Barry B. Transdermal drug delivery, In:Aulton, E M.(ed).Pharmaceutics, The science of dosage forms design, ed 2, Churchill Livingstone, Newyork,Harcourt publishers, 2002, 499-33. Brown MB, Jones SA, Hyaluronic acid: a unique topical vehicle for localized delivery of drugs to the skin, JEDV, 19, 308-318. Cevc G, Transferosomes, liposomes and other lipid suspensions on the skin: permeation enhancement, vesicle penetration and transdermal drug delivery, Crit Rev Ther Drug Carrier Syst, 13, 1996, 257-388. Cevc G, Transferosomes: Innovative Transdermal Drug Carriers, In: Rathbone MJ, Hadgraft J and Roberts M S, eds, Modified Release Drug Delivery Technology, Marcel Dekker, 2003, 533-560. Chien YW, Novel drug delivery systems, Drugs and the Pharmaceutical Sciences, Marcel Dekker, New York, NY, 50, 1992, 797. Chong S, Fung HL, Transdermal Drug Delivery Systems: Pharmacokinetics, Clinical Efficacy, and Tolerance Development, In: Hadgraft J, Guy, RH, Eds, Transdermal Drug Delivery: Developmental Issues and Research Initiatives, Marcel Dekker, New York, 1989,135. Electronic Orange Book, Food and Drug Administration, www.fda.gov/cder/ob, IMS, 2002 Elias PM, Epidermal Lipids, Barrier Function and Desquamation, J Invest Dermatol, 1983, 80, 44-49. Finnin C B, Transdermal drug delivery-What to expect in the near future. Business briefing: Pharma tech.2003, 192-93. Godin B and Touitou E, Ethosomes: New prospects in transdermal delivery, Crit. Rev. Ther. Drug. Carrier, 20, 2003, 63-102. Kumar P, Sankar C, and Mishra B, Delivery of macromolecules through skin. The Indian Pharmacist 2004, 7-17. Liu JC, Sun Y, Siddique O, Chien YW, Shi Wm Li, J Int J Pharm, 44, 1988, 197. Masada T, Higuchi W I, Srinivasan V, Rohr U, Fox J, Behl C, Pons SS, Int J Pharm, 49, 1989, 57. Pellet M, Raghavan, SL, Hadgraft J and Davis AF, The application of supersaturated systems to percutaneous drug delivery, In: Guy RH and Hadgraft J, Eds, Transdermal drug delivery, Marcel Dekker, 2003, 305-326. Volume 1 Issue 1 January February 2013 Page 41
Roberts MS, Targeted drug delivery to the skin and deeper tissues: role of physiology, solute structure and disease, Clin Exp Pharmacol Physiol, 1997 Nov, 24(11), 874-879. Schreier H and Bouwstra J, Liposomes and Niosomes as Topical Drug Carriers: Dermal and Transdermal Drug Delivery, J Control Rel, 1994, 30, 1-15. Siddiqui O, Sun Y, Liu JC, Chien YW, J Pharm Sci, 76, 1987, 341. Singh J, Maibach HI, Dermatology, 187(4), 1993, 235-238. Singh P, Maibach HI, Crit Rev Ther Drug Carrier Syst, 11(2-3), 1994, 161-213. Srinivasan, V, Higuchu WI, Su M H, J Controlled Release, 10, 1989, 157. Tsai JC, Guy RH, Thornfeldt CR, Gao WN, Feingold KR and Elias PM, Metabolic Approaches to Enhance Transdermal Drug Delivery, Effect of lipid synthesis inhibitors, J Pharm Sci, 85, 1998, 643-648. Turnell W J, Proc Royl Soc Med, 14, 1921, 41-52. Williams AC and Barry BW, Penetration Enhancers, Adv Drug Del Rev, 56, 2004, 603-618.
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Debjit Bhowmik et.al.
MICROEMULSIFYING DRUG DELIVERY SYSTEM-NEW ERA OF DRUG DELIVERY SYSTEM Debjit Bhowmik1*,Harish G1, S.Duraivel1, Aravind.G1, KP Sampath Kumar2 1. Nimra College of Pharmacy, Vijayawada, India 2. Department of pharmaceutical sciences, Coimbatore medical college, Coimbatore, India *Corresponding author: Email: debjit_cr@yahoo.com ABSTRACT Micro emulsions are isotropic, thermodynamically stable transparent system of oil, water and surfactant, frequently in combination with a co-surfactant. These versatile systems are currently of great technological and scientific interest to the researchers because of their potential to incorporate a wide range of drug molecules (hydrophilic and hydrophobic) due to the presence of both lipophilic and hydrophilic domains. They have been used to improve the oral bioavailability of various poorly soluble drugs.The discoveries of micro emulsions have attained increasing significance both in basic research and in industry. Due to their unique properties namely ultralow interfacial tension, large interfacial area, thermodynamic stability and the ability to solubilise other immiscible liquids, uses and applications of micro emulsions have been numerous. In topical formulations they have been proved to increase the cutaneous absorption of both lipophilic and hydrophilic APIs. In this type of applications the micro emulsions attribute to the performance to generally a higher solubility of the APIS of micro emulsions, generating increased concentration gradient towards skin. Key words-Microemulsion,Oral administration, Bioavailability, solubility 1. INTRODUCTION Oral administration study of microemulsion formulations has lot of importance, because of their capacity to improve the bioavailability of poorly soluble drugs. Microemulsion is considered to be a thermodynamically or kinetically stable liquid dispersion of an oil phase and a water phase, in combination with a surfactant. The dispersed phase typically comprises small particles or droplets, with a size range of 5 nm-200 nm, and has very low oil/water interfacial tension. The term "microemulsion" refers to a thermodynamically stable isotropically clear dispersion of two immiscible liquids, such as oil and water, stabilized by an interfacial film of surfactant molecules. A microemulsion is considered to be a thermodynamically or kinetically stable liquid dispersion of an oil phase and a water phase, in combination with a surfactant. The dispersed phase typically comprises small particles or droplets, with a size range of 5 nm-200 nm, and has very low oil/water interfacial tension. Because the droplet size is less than 25% of the wavelength of visible light, microemulsions are transparent. The microemulsion is formed readily and sometimes spontaneously, generally without high-energy input. In many cases a cosurfactant or cosolvent is used in addition to the surfactant. Three types of microemulsions are most likely to be formed depending on the composition:Oil in water microemulsions wherein oil droplets are dispersed in the continuos aqueous phase Water in oil microemulsions wherein water droplets are dispersed in the continuous oil phase; bi-continuous microemulsions wherein micro domains of oil and water are inter dispersed within the system. In all three types of microemulsions, the interface is stabilized by an appropriate combination of surfactants and/or co-surfactants. The key difference between emulsions and microemulsions are that the former, whilst they may exhibit excellent kinetic stability, are fundamentally thermodynamically unstable and will eventually phase separate. Another important difference concerns their appearance; emulsions are cloudy while microemulsions are clear or translucent. In addition, there are distinct differences in their method of preparation, since emulsions require a large input of energy while microemulsions do not. The latter point has obvious implications when considering the relative cost of commercial production of the two types of system. By many estimates, up to 40 percent of new chemical entities discovered by the pharmaceutical industry are poorly soluble or lipophilic compounds. Solubilization of hydrophobic drugs with low aqueous solubility has been a major area of interest in recent years. Various solubilization techniques involve the use of co-solvents and surfactants along with pH adjustments. Applications of microemulsions have also drawn attention in the field of solubilization techniques. Microemulsions are optically isotropic and thermodynamically stable systems consisting of water, oil, a surfactant, and a co-surfactant and are known to enhance the bioavailability of drugs via topical and systemic routes. Parenteral delivery of the hydrophobic drugs is a very challenging task. The conventional approaches such as use of co-solvents, oily vehicles and modern approaches such as mixed micelles, liposomes, complexation with cyclodextrins and emulsions have several limitations. Microemulsions have evolved as a novel vehicle for parenteral delivery of the hydrophobic drugs. Their interesting features such as spontaneity of formation, ease of manufacture, high solubilization capacity and self-preserving property make them the vehicle of choice. The use of microemulsions as drug delivery vehicle has been an exciting and attractive area of research because of its many potential and extraordinary benefits. Microemulsions offer an interesting and potentially quite powerful alternative carrier system for drug delivery because of their high solubilization capacity, transparency, thermodynamic stability, ease of preparation, and high diffusion and absorption rates when compared to solvent without the surfactant system. The oral efficacy of microemulsion has already been proved by cyclosporine formulation
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(Neoral), but apart from oral route, microemulsions for other routes like dermal, transdermal, ocular, vaginal, rectal, buccal, periodontal, parenteral, and nasal delivery routes have also been developed. 2. ADVANTAGES OF MICROEMULSION BASED SYSTEMS Microemulsions exhibits several advantages as a drug delivery systems: 2.1. Microemulsions are thermodynamically stable systems and the stability allows self-emulsification of the system whose properties are not dependent on the process followed. 2.2. Microemulsions act as supersolvents of drug. They can solubilize hydrophilic and lipophilic drugs including drugs that are relatively insoluble in both aqueous and hydrophobic solvents. This is due to existence of microdomains of different polarity within the same single-phase solution. 2.3. The dispersed phase, lipophilic or hydrophilic (oil-in-water, O/W, or water-in-oil, W/O microemulsions) can behave as a potential reservoir of lipophilic or hydrophilic drugs, respectively. The drug partitions between dispersed and continuous phase, and when the system comes into contact with a semi-permeable membrane, the drug can be transported through the barrier. Drug release with pseudo-zero-order kinetics can be obtained, depending on the volume of the dispersed phase, the partition of the drug and the transport rate of the drug. 2.4. The mean diameter of droplets in microemulsions is below 0.22 mm; they can be sterilized by filtration. The small size of droplet in microemulsions e.g. below 100 nm, yields very large interfacial area, from which the drug can quickly be released into external phase when absorption (in vitro or in vivo) takes place, maintaining the concentration in the external phase close to initial levels. 2.5. Same microemulsions can carry both lipophilic and hydrophilic drugs. 2.6. Because of thermodynamic stability, microemulsions are easy to prepare and require no significant energy contribution during preparation. Microemulsions have low viscosity compared to other emulsions. 2.7. The use of microemulsion as delivery systems can improve the efficacy of a drug, allowing the total dose to be reduced and thus minimizing side effects. 2.8. The formation of microemulsion is reversible. They may become unstable at low or high temperature but when the temperature returns to the stability range, the microemulsion reforms. 3. DISADVANTAGES OF MICROEMULSION BASED SYSTEMS 3.1. Use of a large concentration of surfactant and co-surfactant necessary for stabilizing the nanodroplets. 3.2. Limited solubilizing capacity for high-melting substances 3.3. The surfactant must be nontoxic for using pharmaceutical applications 3.4. Microemulsion stability is influenced by environmental parameters such as temperature and pH. These parameters change upon microemulsion delivery to patients. 4. THEORY OF MICROEMULSION Microemulsions are clear, transparent, thermodynamically stable dispersions of oil and water, stabilized by an interfacial film of surfactant frequently in combination with a co-surfactant. Recently, there has been a considerable interest for the microemulsion formulation, for the delivery of hydrophilic as well as lipophilic drug as drug carriers because of its improved drug solubilisation capacity, long shelf life, ease of preparation and improvement of bioavailability. In this present review, we discuss about the various advantages of microemulsion in pharmaceuticals, along with its composition variables, physicochemical characterisation etc.Various theories concerning microemulsion formation, stability and phase behavior have been proposed over the years. For example, one explanation for their thermodynamic stability is that the oil/water dispersion is stabilized by the surfactant present and their formation involves the elastic properties of the surfactant film at the oil/water interface, which involves as parameters, the curvature and the rigidity of the film. These parameters may have an assumed or measured pressure and/or temperature dependence (and/or the salinity of the aqueous phase), which may be used to infer the region of stability of the microemulsion, or to delineate the region where three coexisting phases occur, for example. Calculations of the interfacial tension of the microemulsion with a coexisting oil or aqueous phase are also often of special focus and may sometimes be used to guide their formulation. 5. SIGNIFICANCE IN MICROEMULSION OVER OTHER DOSAGE FORMS Increase the rate of absorption Eliminates variability in absorption Helps solubilize lipophilic drug Provides a aqueous dosage form for water insoluble drugs Increases bioavailability Various routes like tropical, oral and intravenous can be used to deliver the product Rapid and efficient penetration of the drug moiety Helpful in taste masking Provides protection from hydrolysis and oxidation as drug in oil phase in O/W microemulsion is not exposed to attack by water and air. Liquid dosage form increases patient compliance. Less amount of energy requirement. Volume 1 Issue 1 January February 2013 Page 44
6. PREPARATION OF MICROEMULSION The drug is be dissolved in the lipophilic part of the microemulsion i.e. Oil and the water phases can be combined with surfactant and a cosurfactant is then added at slow rate with gradual stirring until the system is transparent. The amount of surfactant and cosurfactant to be added and the percent of oil phase that can be incorporated shall be determined with the help of pseudo-ternary phase diagram. Ultrasonicator can finally be used so to achieve the desired size range for dispersed globules. It is then be allowed to equilibrate. Gel may be prepared by adding a gelling agent to the above microemulsion. Carbomers (crosslinked polyacrylic acid polymers) are the most widely used gelling agent. 6.1. Construction of phase diagram: Pseudo-ternary phase diagrams of oil, water, and co-surfactant/surfactants mixtures are constructed at fixed cosurfactant/surfactant weight ratios. Phase diagrams are obtained by mixing of the ingredients, which shall be pre-weighed into glass vials and titrated with water and stirred well at room temperature. Formation of monophasic/ biphasic system is confirmed by visual inspection. In case turbidity appears followed by a phase separation, the samples shall be considered as biphasic. In case monophasic, clear and transparent mixtures are visualized after stirring; the samples shall be marked as points in the phase diagram. The area covered by these points is considered as the microemulsion region of existence. 7. CHARACTERIZATION OF MICROEMULSIONS Microemulsions have been characterized using a wide variety of techniques. The characterization of microemulsions is a difficult task due to their complexity, variety of structures and components involved in these systems, as well as the limitations associated with each technique but such knowledge is essential for their successful commercial exploitation. Therefore, complementary studies using a combination of techniques are usually required to obtain a comprehensive view of the physicochemical properties and structure of microemulsions. At the macroscopic level viscosity, conductivity and dielectric methods provide useful information. 7.1. Phase Behavior Studies: Phase behavior studies are essential for the study of surfactant system determined by using phase diagram that provide information on the boundaries of the different phases as a function of composition variables and temperatures, and, more important, structural organization can be also inferred. Phase behaviour studies also allow comparison of the efficiency of different surfactants for a given application. In the phase behaviour studies, simple measurement and equipments are required. The boundaries of one-phase region can be assessed easily by visual observation of samples of known composition. The main drawback is long equilibrium time required for multiphase region, especially if liquid crystalline phase is involved. Other useful means and ways of representing the phase behaviour are to keep the concentration of one component or the ratio of two components constant. As the number of components increases, the number of experiments needed to define the complete phase behaviour becomes extraordinary large and the representation of phase behaviour becomes extremely complex. 7.2. Scattering Techniques for Microemulsions Characterization: Small-angle X-ray scattering (SAXS), smallangle neutron scattering (SANS), and static as well as dynamic light scattering are widely applied techniques in the study of microemulsions. These methods are very valuable for obtaining quantitative information on the size, shape and dynamics of the components. The major drawback of this technique is the dilution of the sample required for the reduction of interparticular interaction. This dilution can modify the structure and the composition of the pseudophases. Nevertheless, successful determinations have been carried out using a dilution technique that maintains the identity of droplets. Small-angle X-ray scattering techniques have been used to obtain information on droplet size and shape. Static light scattering techniques have also been widely used to determine microemulsion droplet size and shape. In these experiments the intensity of scattered light is generally measured at various angles and for different concentrations of microemulsion droplets. Dynamic light scattering, which is also referred as photon correlation spectroscopy (PCS), is used to analyse the fluctuations in the intensity of scattering by the droplets due to Brownian motion. The self-correlation is measured that gives information on dynamics of the system. 7.3. Nuclear Magnetic Resonance Studies: The structure and dynamics of microemulsions can be studied by using nuclear magnetic resonance techniques. Self-diffusion measurements using different tracer techniques, generally radio labeling, supply information on the mobility of the components. The Fourier transform pulsedgradient spin-echo (FT-PGSE) technique uses the magnetic gradient on the samples and it allows simultaneous and rapid determination of the self-diffusion coefficients (in the range of 10-9 to 10-12 m2s-1), of many components. 7.4. Interfacial Tension: The formation and the properties of microemulsion can be studied by measuring the interfacial tension. Ultra low values of interfacial tension are correlated with phase behaviour, particularly the existence of surfactant phase or middle-phase microemulsions in equilibrium with aqueous and oil phases. Spinning-drop apparatus can be used to measure the ultra low interfacial tension. Interfacial tensions are derived form the measurement of the shape of a drop of the low-density phase, rotating it in cylindrical capillary filled with high-density phase.
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7.5. Viscosity Measurements: Viscosity measurements can indicate the presence of rod-like or worm-like reverse micelle. Viscosity measurements as a function of volume fraction have been used to determine the hydrodynamic radius of droplets, as well as interaction between droplets and deviations from spherical shape by fitting the results to appropriate models (e.g. for microemulsions showing Newtonian behaviour, Einsteins equation for the relative viscosity can be used to calculate the hydrodynamic volume of the particles). 7.6. Predicting Microemulsion Type: A well-known classification of microemulsions is that of Winsor who identified four general types of phase equilibria Type I The surfactant is preferentially soluble in water and oil-in-water (O/W) microemulsions form (Winsor I). The surfactant-rich water phase coexists with the oil phase where surfactant is only present as monomers at small concentration. Type II The surfactant is mainly in the oil phase and water-in-oil (W/O) microemulsions form. The surfactantrich oil phase coexists with the surfactant-poor aqueous phase (Winsor II) Type III A three-phase system where a surfactant-rich middle-phase coexists with both excess water and oil surfactant-poor phases (Winsor III or middle-phase microemulsion). Type IV A single-phase (isotropic) micellar solution, that forms upon addition of a sufficient quantity of amphiphile (surfactant plus alcohol). 7.7. Simple tests used are 7.7.1. Dye Solubilization: A water soluble dye is solubilized within the aqueous phase of the W/O globule but is dispersible in the O/W globule. A oil soluble dye is solubilized within the oil phase of the O/W globule but is dispersible in the W/O globule. 7.7.2. Dilutability Test: O/W microemulsions are dilutable with water whereas W/O are not and undergo phase inversion into O/W microemulsion. 7.7.3. Conductance Measurement: O/W microemulsion where the external phase is water are highly conducting whereas W/O are not, since water is the internal or dispersal phase. To determine the nature of the continuous phase and to detect phase inversion phenomena, the electrical conductivity measurements are highly useful. A sharp increase in conductivity in certain W/O microemulsion systems was observed at low volume fractions and such behaviour was interpreted as an indication of a percolative behaviour or exchange of ions between droplets before the formation of bicontinuous structures. Dielectric measurements are a powerful means of probing both structural and dynamic features of microemulsion systems. 7.8. Electron Microscope Characterization: Transmission Electron Microscopy (TEM) is the most important technique for the study of microstructures of microemulsions because it directly produces images at high resolution and it can capture any co-existent structure and micro-structural transitions. There are two variations of the TEM technique for fluid samples, the Cryo-TEM analysis and the Freeze Fracture Tem technique. 8. IMPORTANT CHARACTERISTICS OF MICROEMULSIONS Particle size < 200 nm Thermodynamically stable Optically clear Surface area increased High solubilizing capabilities 9. APPLICATIONS OF MICROEMULSIONS Pharmaceutical Applications Parenteral delivery Oral drug delivery Topical drug delivery Ocular and pulmonary delivery Microemulsions in biotechnology 9.1. Parenteral Delivery: Parenteral administration (especially via the intravenous route) of drugs with limited solubility is a major problem in industry because of the extremely low amount of drug actually delivered to a targeted site. Microemulsion formulations have distinct advantages over macroemulsion systems when delivered parenterally because of the fine particle microemulsion is cleared more slowly than the coarse particle emulsion and, therefore, have a longer residence time in the body. Both O/W and W/O microemulsion can be used for parenteral delivery. The literature contains the details of the many microemulsion systems, few of these can be used for the parenteral delivery because the toxicity of the surfactant and parenteral use. An alternative approach was taken by Von Corsewant and Thoren in which C3-C4 alcohols were replaced with parenterally acceptable cosurfactants, polyethylene glycol (400), polyethylene glycol (660) and 12-hydroxystearate, while maintaining a flexible surfactant film and spontaneous curvature near zero to obtain and almost balanced middle phase microemulsion. The middle phase structure was preferred in this application, because it has been able to incorporate large volumes of oil and water with a minimal concentration of surfactant.
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9.2. Oral delivery: Microemulsion formulations offer the several benefits over conventional oral formulation for oral administration including increased absorption, improved clinical potency,and decreased drug toxicity. Therefore, microemulsions have been reported to be ideal delivery of drugs such as steroids, hormones, diuretic and antibiotics. Pharmaceutical drugs of peptides and proteins are highly potent and specific in their physiological functions. However, most are difficult to administer orally. With on oral bioavailability in conventional (i.e. nonmicroemulsion based) formulation of less than 10%, they are usually not therapeutically active by oral administration. Because of their low oral bioavailability, most protein drugs are only available as parenteral formulations. However, peptide drugs have an extremely short biological half life when administered parenterally, so require multiple dosing. A microemulsion formulation of cyclosporine; named Neoral has been introduced to replace Sandimmune, a crude oil-in-water emulsion of cyclosporine formulation. Neoral is formulated with a finer dispersion, giving it a more rapid and predictable absorption and less inter and intra patient variability. 9.3. Topical delivery: Topical administration of drugs can have advantages over other methods for several reasons, one of which is the avoidance of hepatic first pass metabolism of the drug and related toxicity effects. Another is the direct delivery and targetability of the drug to affected area of the skin or eyes. Both O/W and W/O microemulsions have been evaluated in a hairless mouse model for the delivery of prostaglandin E1. The microemulsions were based on oleic acid or Gelucire 44/14 as the oil phase and were stabilized by a mixture of Labrasol (C8 and C10 polyglycolysed glycerides) and Plurol Oleique CC 497 as surfactant. Although enhanced delivery rates were observed in the case of the o/w microemulsion, the authors concluded that the penetration rates were inadequate for practical use from either system. 9.4. Ocular and pulmonary delivery: For the treatment of eye diseases, drugs are essentially delivered topically. O/W microemulsions have been investigated for ocular administration, to dissolve poorly soluble drugs, to increase absorption and to attain prolong release profile. The microemulsions containing pilocarpine were formulated using lecithin, propylene glycol and PEG 200 as co-surfactant and IPM as the oil phase. The formulations were of low viscosity with a refractive index lending to ophthalmologic applications. The formation of a water-in-HFA propellent microemulsion stabilized by fluorocarbon non-ionic surfactant and intended for pulmonary delivery has been described. 9.5. Microemulsions in biotechnology: Many enzymatic and biocatalytic reactions are conducted in pure organic or aqua-organic media. Biphasic media are also used for these types of reactions. The use of pure apolar media causes the denaturation of biocatalysts. The use of water-proof media is relatively advantageous. Enzymes in low water content display and have Increased solubility in non-polar reactants Possibility of shifting thermodynamic equilibria in favour of condensations Improvement of thermal stability of the enzymes, enabling reactions to be carried out at higher temperatures. Many enzymes, including lipases, esterases, dehydrogenases and oxidases often function in the cells in microenvironments that are hydrophobic in nature. In biological systems many enzymes operate at the interface between hydrophobic and hydrophilic domains and these usually interfaces are stabilized by polar lipids and other natural amphiphiles. Enzymatic catalysis in microemulsions has been used for a variety of reactions, such as synthesis of esters, peptides and sugar acetals transesterification; various hydrolysis reactions and steroid transformation. The most widely used class of enzymes in microemulsion-based reactions is of lipases. 10. IN VITRO DRUG PERMEATION STUDIES 10.1. Determination of permeability coefficient and flux: Excised human cadaver skin from the abdomen can be obtained from dead who have undergone postmortem not more than 5 days ago in the hospital. The skin is stored at 4o C and the epidermis was separated. The skin is first immersed in purified water at 60C for 2 min and the epidermis then peeled off. Dried skin samples can be kept at 20o C for later use. Alternatively the full thickness dorsal skin of male hairless mice may be used. The skin shall be excised, washed with normal saline and used. The passive permeability of lipophilic drug through the skin is investigated using Franz diffusion cells with known effective diffusional area. The hydrated skin samples are used. The receiver compartment may contain a complexing agent like cyclodextrin in the receiver phase, which shall increase the solubility and allows the maintenance of sink conditions in the experiments. Samples are withdrawn at regular interval and analyzed for amount of drug released. 10.2. Determination of residual drug remaining in the skin on tropical administration: The skin in the above permeation studies can be used to determine the amount of drug in the skin. The skin cleaned with gauze soaked in 0.05% solution of sodium lauryl sulfate and shall bathed with distilled water. The permeation area shall be cut and weighed and drug content can be determined in the clear solution obtained after extracting with a suitable solvent and centrifuging.
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10.3. Pharmacological studies: Therapeutic effectiveness can be evaluated for the specific pharmacological action that the drug purports to show as per stated guidelines. 10.4. Estimation of skin irritancy: As the formulation is intended for dermal application skin irritancy should be tested. The dorsal area of the trunk is shaved with clippers 24 hours before the experiment. The skin shall be scarred with a lancet. 0.5 ml of product is applied and then covered with gauze and a polyethylene film and fixed with hypoallergenic adhesive bandage. The test be removed after 24 hours and the exposed skin is graded for formation of edema and erythema. Scoring is repeated 72 hours later. Based on the scoring the formulation shall be graded as non-irritant, irritant and highly irritant. 10.5. Stability studies: The physical stability of the microemulsion must be determined under different storage conditions (4, 25 and 40 C) during 12 months. 11. APPLICATION OF MICROEMULSIONS IN DERMAL AND TRANSDERMAL DRUG DELIVERY Microemulsions are thermodynamically stable colloidal dispersions of water and oil stabilized by a surfactant and, in many cases, also a cosurfactant. In the pharmaceutical field, microemulsions have been used as drug carriers for percutaneous, ocular, oral and parenteral administration. This review discusses some of the applications of microemulsions specifically for topical and transdermal applications. Microemulsion nomenclature and composition, with particular emphasis on choice of surfactant and cosurfactant, is discussed. Methods used to characterize microemulsions are reviewed. Microemulsion formulations for dermal and transdermal delivery of pharmaceutical agents with particular emphasis on anti-inflammatory and anaesthetic agents are critically evaluated. Microemulsions are potential drug carrier systems for oral, topical, and parenteral administration. They offer the advantage of spontaneous formation, ease of manufacturing and scale-up, thermodynamic stability, and improved drug solubilization and bioavailability. Preparing a pharmaceutically acceptable dosage form demands a clear understanding of the microemulsion structure, phase behavior, factors leading to its thermodynamic stability, factors influencing drug release from the formulation, requirements of ideal microemulsion excipients, and the potential uses and limitations of the microemulsion system. Knowledge of the various methods available to thoroughly characterize a microemulsion system is essential. An overview of these factors is presented in this review. While microemulsion is used in several fields, in this review the pharmaceutical applications are emphasized. Several references are cited, but the list is by no means exhaustive. The review is written so that a newcomer to the field can easily grasp the important facts pertaining to this novel delivery system. 12. CONCLUSION The thrust for finding newer drug delivery systems for exiting therapeutic molecules has opened a wide window for colloidal systems. Due to the presence of different domains of variable polarity in the microemulsion systems, they show a huge potential to be used as drug delivery vehicles for a variety of drugs. The use of microemulsion as drug delivery vehicles through a number of routes has engaged a large number of research groups in this area. Microemulsion media finds several applications ranging from drug delivery to drug nanoparticle templating due to its ability to enhance solubility, stability and bioavailability. Microemulsions (socalled due to their small particle size; 5-100 nm) have found application in a wide variety of systems, such as pharmaceutical and oil recovery, but their application in food systems has been hindered by the types of surfactant permissible for use in food. The objective of this review is to provide an overview of the structures and phase behavior of microemulsions. REFERENCES Bianca BM, Arturo LQ, Begona M, Delgado C, Anna MF, Jose BM, Micro emulsions for topical delivery of 8methoxsalen, J Contr Release, 69, 2000, 209218. Brisaert M, Plaizier JA, Vercammen, Investigation on the photo stability of tretinoin in creams, Int J Pharm, 334, 2007, 56-61. Chong KK, Zhong GG, HanGC, Hee Jong S, Kyung MP, Soo Jeong, Physicochemical characterization and evaluation of a micro emulsion system for oral delivery of cyclosporine A, Int J Pharm, 161, 1998, 7586. Derle DV, Sagar BSH, Pimpale S, Micro emulsion as a vehicle for transdermal permeation of nimesulide, Ind J Pharm Sci, 68(05), 2006, 622-625. Donentella P, Cinzia AV, Steven N, Giovanni P, Massimo F, Lecithin micro emulsions for the topical administration of ketoprofen; percutaneous adsorption through human skin and in vivo human skin tolerability, Int J Pharm 44, 2002, 2131. Jitendra B, Raj K.K, Arvind Y, Methoxsalen loaded chitosan coated micro emulsion for effective treatment of psoriasis, Int J Drug Del, 2, 2010, 159-167.
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Kashyap N, Manikandan R, Sebastian B, Naruka PS, Design and development of micro emulsion drug delivery system of isotretionin for improvement of bioavailability, Int J Pharm, 2(08), 2010, 13. Lawrence MJ, Gareth D, Micro emulsion-based media as novel drug delivery systems, Adv Drug Delivery Rev 45, 2000, 89121. Sedigeh D, Behzad S, Makhmal Z, Rahim F. Preparation and evaluation of the self emulsifying drug delivery system containing Loratadine, Int J Adv Pharm Sci 2010; 239-248. Sevgi A, Gulten K, Ozguney I, Yesim K, Comparison of different Water/Oil micro emulsions containing diclofenac sodium preparation, characterization, release rate, and skin irritation studies, AAPS Pharm SciTech 8 (4), 2007, 91. Suman R, Manish K, Vikas C, Vandana G. Formulation development and characterization of micro emulsion for topical delivery of glipizide, Der Pharmacia Lettre, 2(3), 2010, 33-42. Surjyanarayan M, Snigdha DM, Naazneen S, Vandana P, Development of Micro emulsion formulation for the Solubility enhancement of flunarizine, Der Pharmacia letter, 2(3), 2010, 227-36. Sushama T, Adnan A, Farhan JA, Roop KK, Shadab AP, Zeenat IK, Micro emulsions: A novel approach to enhanced drug delivery, Bentham Science Publishers Ltd, 2, 2008, 238-257.
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Shameer H et.al.
SYNTHESIS, CHARACTERIZATION AND ANTI-INFLAMMATORY ACTIVITY OF NOVEL N-SUBSTITUTED-5-OXA-IMIDAZOLE

Shameer H1, Nageswara Rao R1, Vijay Kumar MMJ2, Jayachandran E3*, Sreenivasa GM3 1. Jaya prakash naryana educational socities, Mahabubnagar, India 2. S.J.M. College of Pharmacy, Chitradurga, India 3. S.C.S. College of Pharmacy, Harapanahalli, India * Corresponding author: E Mail: sameer2349@gmail.com, Mobile: +919886061429 ABSTRACT Various substituted 2-(2'-phenyl-4'-p-dimethylaminobenzylidenyl imidazolin-5'-one)-6-fluoro-7substituted (1,3) benzothiazoles and 2-(2'-phenyl-4'-o-hydroxy benzylidenyl-5'oxo imidazolin-1-yl amino) 6-fluoro-7-substituted (1,3) benzothiazoles containing different functional groups have been synthesized by treating substituted 2-aminobenzothiazoles with oxozolone in presence of dry pyrirdine. The identity of compounds were confirmed on the basis of their spectral (UV-Visible, IR, 1H NMR and Mass) data. Further, they have been screened for their antimicrobial, antiinflammatory, anticonvulsant and anthelmintic activities. KEY WORDS; Fluorine, Benzothiazole, Imidazole, anti-inflammatory activity. 1. INTRODUCTION We report here in the new and unreported yet the synthesis of fluoro benzothiazoles comprising imidazole derivatives of pharmacological activity of clinical importance in the areas of antibacterial, antifungal, antituburcular, MAO inhibitors, anthelmintic, anticonvulsant, anti-cancer, analgesic and anti-viral. The chemistry and pharmacology of imidazole have been of great interest because, of its various biological activities, so that the biological and pharmacological activity of imidazole with fluoro benzothiazoles may be taken into account for synergism. It is well known that the introduction of fluorine atom into an organic molecule causes dramatic changes in its biological profile, mainly due to high electro negativity of fluorine, the strong carbon-fluorine bond and increased solubility in lipids. Therefore it was thought worthwhile to synthesize better kinds of drugs by incorporating imidazole in benzothiazole moiety. In search for new biodynamic potent molecule, it was thought worthwhile to incorporate some additional heterocyclic moieties in the imidazole nucleus and study their biological and pharmacological activity. The review of literature prompted us to synthesize substituted Fluorobenzothiazole, imidazole compounds and those will be screened for anti-inflammatory activity. 2. MATERIALS AND METHODS 2.1. Chemicals and Reagents: 4-fluoro-3-chloro aniline, Potassium thiocyanate , Glacial acetic acid , Bromine, Hippuric acid, Pyridine, N N amino Benzaldehyde , Ethanol, Ammonia , Benzene, Conc.Hydrochloric acid, Sodium acetate, N,Ndimethyl formamide (DMF), Primary and secondary aromatic amines. 2.2. Experimental Section: Step I: 4-fluoro-3-chloro aniline was treated with potassium thiocyanate (KSCN) in presence of glacial acetic acid and bromine to get 2-amino-6-fluoro-7-chloro-benzothiazole. Step II: N, N'- Dimethyl amino benzaldehyde treated with benzoylglycine (Hippuric acid) in presence of dry acetic acid and anhydrous sodium acetate to get 2-phenyl-4-p-dimethylaminobenzylidenyl-5-oxazolone. StepIII: 2-amino-6-fluoro-7-chloro-benzothiazole treated with 2-phenyl-4-p-dimethylaminobenzylidenyl-5oxazolone, in presence of Pyridine to get 2-(2'-phenyl-4'-p-dimethylaminobenzylidenyl imidazolin-5'-one) 6fluoro- 7-chloro, (1, 3) benzothiazole. Step IV: 2-(2'-phenyl-4'-p-dimethylaminobenzylidenyl imidazolin-5'-one)-6-fluoro-7-chloro, (1,3) benzothiazole was treated with various primary and secondary aromatic amines in presence of N N' dimethyl formamide (DMF) yields various 2-(2'-phenyl-4'-p-dimethylaminobenzylidenylimidazolin-5'-one)-6-fluoro-7-substituted (1,3) benzothiazoles. 2.3. General Procedures: Melting points were determined in open capillaries and are uncorrected. IR spectra (KBr pellet technique) were recorded using a Perkin-Elmer 237 spectrophotometer. 1H NMR spectra were recorded on Bruker AM 400 instrument (at 400 MHz) using tetramethylsilane (TMS) as an internal standard and DMSO-d6 as a solvent. Chemical shifts are given in parts per million (ppm). Splitting patterns are designated as follows: ssinglet, d- doublet, t- triplet, q- quartet and m-multiplet. Mass spectra (MS) were recorded on Schimadzu GC-MS operating at 70eV. All the synthesized compounds were purified by recrystallization The reactions were followed up and the purity of compounds was monitored on pre-coated TLC plates and visualizing the spots in ultraviolet light. 2.4. Study of anti-inflammatory activity (In-Vitro models): The synthesized compounds are screened for antiinflammatory activity by using inhibition of albumin denaturation technique which was studied according to Muzushima and Kabayashi with slight modification. The standard drug and test compounds were dissolved in minimum amount of dimethyl formamide (DMF) and diluted with phosphate buffer (0.2 M, pH 7.4). Final concentration of DMF in all solutions was less than 2.0%. Test solution (1 ml) containing different concentrations of drug was mixed with 1 ml of 1% mM albumin solution in phosphate buffer and incubated at 27 010C in BOD
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Shameer H et.al.
incubator for 15 min. Denaturation was induced by keeping the reaction mixture at 60010C in water bath for 10 min. After cooling the turbidity was measured at 660 nm (UV-Visible Spectrophotometer SL-159, Elico India Ltd.). Percentage of inhibition of denaturation was calculated from control where no drug was added. Each experiment was done in triplicate and average was taken. The Diclofenac sodium was used as standard drug. Results are tabulated. Vt % of inhibition = 100 1 Vc Scheme
C6H5 NH2 F Cl KSCN :Br2/AcoH NH 3 F Cl S N NH2
+
HC
Oxazolone
CHO HN
C 6H 5
C6 H5
+
N H3C CH3
O OH O
(CH 3 CO) 2 O CH 3 COONa
N HC
N H3C CH3
Hippuric acid
N, N'- dim ethy l am ino benzaldehy de
H3C N CH3
6Hrs reflux in dry pyridi ne
Ox azolone
N F Cl O N S
C6H5 N
CH
NH2
R Reflux for 2hrs in DMF R

1
N H3C CH3
N F NH N S O R
C6H5 N F R1 CH S
N N
C6H5 N
CH
R= o,m,p, ni troanil ine o,m,p, chl oroani li ne anil ine o,m,p, ani sidi ne o,p, tolui dine 2 ethyl ani li ne PABA
N H3C CH3
R =morpholi ne piperzine methyl piperzine dimethyl ami ne diethylamine
N H3C CH3
Table No. 1 Anti-inflammatory activity (In-Vitro model)

Name Compounds Absorbance value (Mean SE) Inhibition of denaturation(%) Name Compounds Absorbance value (Mean SE) Inhibition of denaturation(%)
Control D1 D2 D3 D4 D5 D6 D7 D8 D9 D10
0.087 0.001 0.120 0.003 0.122 0.004 0.125 0.003 0.130 0.003 0.138 0.008 0.122 0.008 0.127 0.004 0.138 0.003 0.128 0.006 0.125 0.003
37.93% 40.22% 43.36% 49.42% 58.62% 40.22% 45.97% 58.62% 47.12% 43.67%
D11 D12 D13 D14 D15 D16 D17 D18 D19 Diclofenac sodium
0.124 0.006 0.122 0.003 0.120 0.008 0.120 0.008 0.129 0.005 0.128 0.006 0.135 0.004 0.128 0.002 0.127 0.006 0.1630.003
42.52% 40.22% 37.93% 37.93% 48.27% 47.12% 55.17% 47.12% 49.97% 87.35%
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Shameer H et.al. Sl. No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Compound Code D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 D14 D15 D16 D17 D18 D19 M.P/ B.PC 176 186 162 182 178 146 172 175 120 135 130 138 150 168 134 164 158 166 180 % Yield 78% 82% 75% 72% 74% 73% 76% 65% 69% 83% 77% 85% 86% 78% 80% 78% 76% 72% 82%
Table No. 2 Analytical data

MOL. FORM C31H23O3SN6F C31H23O3SN6F C31H23O3SN6F C31H23OSN5FCl C31H23OSN5FCl C31H23OSN5FCl C31H24OSN5F C29H26O2SN5F C29H27OSN6F C30H29OSN6F C37H28OSN5F C27H24OSN5F C32H26O2SN5F C32H26O2SN5F C32H26O2SN5F C32H24O3SN5F C32H26O2SN5F C27H24OSN5F C32H26O2SN5F M.Wt. 578 578 578 568 568 568 533 527 526 540 609 485 563 563 563 577 563 485 563 Calculated % C 64.35 64.35 64.35 65.54 65.54 65.54 69.77 66.02 66.14 66.65 72.89 66.78 68.19 68.19 68.19 66.54 68.19 66.78 68.19 H 4.01 4.01 4.01 4.08 4.08 4.08 4.53 4.97 5.17 5.14 4.63 4.98 4.65 4.65 4.65 4.19 4.65 4.98 4.65 N 14.52 14.52 14.52 12.33 12.33 12.33 13.12 13.27 15.96 15.54 11.49 14.42 12.53 12.53 12.53 12.12 12.43 14.42 12.43
Table No. 3 Characteristics IR absorption bands

Comp ound C-S (in cm-1) 715 720 715 710 720 715 730 720 725 720 730 725 730 725 725 720 725 730 Imidazoli ne ring corbonyl (in cm-1) 1640 1650 1655 1650 1645 1645 1640 1620 1685 1650 1645 1640 1655 1650 1640 1640 1645 1640 C=N Stretchin g (in cm-1) 1510 1515 1510 1515 1525 1535 1520 1520 1610 1590 1610 1525 1525 1530 1525 1535 1535 1520 C=C Stretching (in cm-1) 1750 1765 1765 1760 1770 1755 1755 1700 1690 1765 1760 1765 1760 1765 1760 1760 1755 1745 NO2 (in cm-1) C-F (in cm-1) C-Cl (in cm-1) Sec.Ar. Amine (in cm-1) 1310 1310 1305 1300 1310 1305 1320 Ter.Ar.A mine (in cm-1) 1370 1380 1375 1370 1380 1365 1370 1370 1365 1380 1380 1390 1380 1390 C-O-C
D1 D2 D3 D4 D5 D6 D7 D8 D9 D11 D12 D13 D14 D15 D16 D17 D18 D19
1375 1370 1370 -
1160 1160 1165 1160 1165 1165 1160 1120 1265 1160 1165 1165 1170 1260 1160 1270 1185 1185
820 820 815 -
1190 1200 1250 -
Compound Code D9
D16
Table:4 NMR Spectral Data Hydrogen Multiplity (ppm) -Ar-H7.1-7.5 Multiplet -saturated H 2.7-4.0 Multiplet -NH- proton 3.8 Doublet -Ar-H7.0-7.8 Multiplet -NH- proton 8.1 Doublet -CH33.0 Singlet Vinylic Proton 6.2 Multiplet
Solvent CDCl3
CDCl3
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Shameer H et.al.
Table: 5. Mass spectra Compound Mol. formula Mol. weight M/Z M+1 M+2 M+3 Code D9 C29H27OSN6F 526.62 526.62 379.1 293.1 165.7 D16 C32H24O3SN5F 577.62 577.62 478.62 292 237.8 3. RESULTS AND DISCUSSION Sythesised compounds of 2-(2'-phenyl-4'-p-dimethylaminobenzylidenyl imidazolin-5'-one)-6-fluoro-7substituted (1, 3) benzothiazoles and 2-(2'-phenyl-4'-o-hydroxy benzylidenyl-5'oxo imidazolin-1-yl amino) -6-fluoro-7substituted (1, 3) benzothiazoles were tested for anti-inflammatory activity by invitro method compared to standard Diclofenac showed significant anti-inflammatory activity. All the compounds tested are showed better antiinflammatory activity compare to standard Diclofenac sodium. 4. CONCLUSION Result of present study demonstrate that, a new class of different aromatic aniline, anisidine, PABA, piperzine, encompassing imidazole derivatives were synthesized and evaluated as anti-inflammatory activity. The newly synthesized heterocyclics exhibited better anti-inflammatory activity by using inhibition of albumin denaturation technique which was studied according to Muzushima and Kabayashi with slight modification. It can be concluded that this class of compounds certainly holds better activity towards good active leads in medicinal chemistry. A further study to acquire more information concerning pharmacological activity is in progress. 5. ACKNOWLEDGEMENT The authors are thankful to Shri. Sha. Bra. Chandramouleshwara Shivacharya swamiji, President, Sri. T. M. Chandrashekhariah, Secretary, T.M.A.E. Society, Harapanahalli, for providing necessary facilities through the Principal, S.C.S. College of Pharmacy, Harapanahalli to carry out this work. 6. REFERENCES Anjani Solankee, Kishor Kapadia, Jayesh Patel, Smurti Lad, Indrajit Thakar. Synthesis and antimicrobial activity of 1-(4-N-acetyllamino) phenyl-2-phenyl-4-benzylidine/substituted benzylidine-imidazoline-5-ones; Oriental Journal of chemistry 2003; 19(2): 477-80 Biological assays, Indian Pharmacopoeia Published by Govt. of India, 2, 1996, 88 Biplab de and Ramasarma have reported Determination of Pka and correlation with analgesic activity of 5oxoimidazolylaminopyrazole-4-carboxaldehyde and their schiffs base, Indian drugs, 36(2), 1999, 583 Conte L, et al. Liquid-phase Fluorination of aromatic compounds by elemental fluorine; J Fluorine Chem, 70, 1995, 175-179 Desbois Michel, Eur Pat, APP.l EP. 248746 (IPC Co7Co85/00), Synthesis of 4-fluoro-3-chloroaniline from 4-halo nitrobenzene, Chem Abstr, 108, 1987, 159896 Farzin Hadizadeh and Razieh Ghodsi, Synthesis of novel N-substituted imidazolecarboxylic acid hydrazides as MAO inhibitors IL Farmaco, 2005(60), 237-240 FIiller R, Biologically-Active fluorochemicals, J Fluorine Chem, 33, 1986, 361-375 Hirpura S B, Parikh K A, Merja B C, Parekh H H, Synthesis of thiazoles and imidazolinone possessing multiple biological activities, Indian Journal of Chemistry 42, 2003,1172-1175 Purohit D M, Shah V H, Synthesis of arylidene methyl dichloro nitro phenyl imidazolinones as possible antimicrobial agents; Indian Journal of Heterocyclic Chemisty 8, 1999, 213-216 Shah M D, Desai N C, Keshav K Awasthi, Anil K Saxena, Synthesis and biological activities(Anticancer and antitubercular activity) of thiadiazoles and imidazolinones, Indian Journal of Chemistry, 42, 2003, 1172-1175 Solankei, Kapadia K, Upadhyay K Patel, Synthesis and anticancer activity of pyrazoline substituted benzylidene imidazolinones, Oriental Journal of chemistry, 2001, 17(2), 315-316 Sreenivasa Rao D, Jayachandran E, Sreenivasa G M and Shivakumar B, Inhibition of albumin denaturation and anti-inflammatory activity of 2-[N-p-Tolyl sulphon hydrazino]-6-fluoro-7-substituted (1,3) benzothiazoles Oriental Journal of Chemistry, 21(1), 2005, 113-116 Srinivasa GM, Jayachandra E, Shivakumar B, Sreenivasa Rao D, Synthesis and pharmacological screening of 2-[3amino,5-s-met carboxamido pyrazol-1-yl]6-fluoro,7-substituted (1,3)benzothiazole, Oriental Journal of Chemistry, 20(1), 2004, 103-110 Turner RA, Anti-epileptic drugs, in screening methods in pharmacology, XV edn.(Ed. R.A. Turner), Academic Press, New York 1965; 165-172.
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Peer Basha Syed et.al.
SCREENING OF BIOACTIVE ACTINOMYCETES FROM SUGARCANE SOILS Peer Basha Syed, Chakravarthi V,Venkateswarlu B,Koteswararao P, Farooq Ahmed Department of Pharmaceutics, Nimra college of pharmacy, Vijayawada, India Corresponding author: Email:peerbasha2005@gmail.com ABSTRACT Actinomycetes and Streptomyces are once considered to be microbiological curiosities of no great economic importance, but have become the subjects of intensive searches for source of new biologically active compounds. The demonstrated ability of these microorganisms to produce useful antibiotics and to carry out other transformations of commercial interest as focused attention of factors bearing on their isolation from their natural habitat. It would be desirable, therefore to be able to isolate aerobic soil inhabiting actinomycetes with a minimum of interference from associated bacteria and higher fungi. There have been a number of reports in the literature dealing with selective isolation of the higher fungi and the expense of bacteria or the expense of moulds. Several investigators used antibiotics as the selective agents. In view of the above systematic screening was undertaken to isolate bioactive actinomycetes from natural sources. Key words: Actinomycetes, Streptomyces, Anti-bacterial activity, Anti-fungal activity, B.subtilis, E.coli. 1. INTRODUCTION Actinomycetes are best known for their ability to produce antibiotics and are gram positive bacteria which comprise a group of branching unicellular microorganisms. They produce group of branching mycelium which may be of two kinds, substrate mycelium and aerial mycelium. Among Actinomycetes, the Streptomycetes are the dominant organisms. In addition inhalation of some thermophillic actinomycetes such as Micropolysporafaeni and Thermo actinomycetes species may cause allergic alveolitis. Most of actinomycetes are soil saprophytes and others are commensals of mouth of both man and animals. The actinomycetes causes a disease known as actinomycosis in humans it is usually caused by A.israelii. Actinomycetes are gram positive, filamentous bacteria intermediate in properties between true bacteria and fungi. Like bacteria, they posses cell wall containing muramic acid, prokaryotic nuclei and are susceptible to anti bacterial antibiotics, whereas like fungi they form a mycelial network of branching filamentous. Hence, the actinomycetes are true bacteria with a superficial resemblance to fungi. They are related to Corynobacterium and Mycobacterium. They are non motile, nonsporing, non capsulated filamentous that break up into small bacterial fragments and like freely in nature, particularly in the soil. Actinomycetes are unicellular organisms that mass together to form filaments called hyphae. Colonies of Actinomycetes can then forms a mass of inter wined hyphae called a mycelium. These filaments are long and it may fragment into much smaller units and less broad than that of the fungal mycelium usually 0.5-1.0 m in diameter but sometimes reaches to 2 m in few cases. A chain of sexual spores called conidia are produced on their hyphae, and few of the Actinomycetes (genera) found in soil bear the sporangium containing spores. The colonies resemble powdery mass over the surface of culture media. The zone of inhibition may be small or the Actinomycetes colony may be completely surrounded by an area free of growth by other organisms. 2. METHODOLOGY 2.1. Collection of soil samples: Two different soil samples were collected and kept in sterile containers. 2.2 Determination of pH of Soil Samples: The pH of the soil sample was determined by taking 1 gm of soil in 250 ml Erlen meyer flask contained 50ml of sterile water then the flask was kept on a rotary shaker for half an hour. Then the pH of the supernant liquid was determined by using pH paper. 2.3. Isolation of Actinomycetes by using selective method: Starch casein agar was prepared and adjusted the pH to 7.0 -7.2. Then the medium was sterilized by autoclave at 121C with 15 lb/in2 pressure for 20 min.1gm of each soil sample was weighed separately and transferred into 9ml of sterile water and serial dilutions were performed (10-1 to 10-10).1ml of soil suspension was withdrawn from each selected dilutions (10 -5 to 10-7) and they were added to above three media at 45Cusing 1mlstirle pipette under aseptic conditions. Then the media was mixed thoroughly and poured into sterile Petri plates and they were allowed to solidify. Then the plates were incubated at 28C for 7 days. The Petri plates were examined carefully by holding them close to the light, individual actinomycetes colonies were observed and they were sub cultured onto the same medium and the inoculated slants were incubated at 28C for 7 days. A total of 11 isolates were obtained from the above three soil samples. In the next step all the11 isolates were tested for their enzymatic and anti bacterial activities 2.4. Determination of Enzymatic activity 2.4.1. Determination of Amylase activity: Starch is an insoluble polymer of glucose which has a source of carbon for microorganisms which have an ability to degrade them. Starch degrading micro organisms transport the degraded form across the cytoplasmic membrane of the cell and some bacteria possess the ability to produce amylase that breaks starch into maltose. The amylase is an extra cellular enzyme which is released from the cell wall of micro organisms. Starch agar medium was prepared and sterilized. The sterile molten medium was poured into
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sterile petri plates and allowed to solidify. Then all the 11 isolates were streaked on solidified medium separately under aseptic condition. Then the inoculated plates were incubated at 28C for 7 days. The amylase activity was performed by the addition of 3-4 drops of grams iodine solution. If blue-black color appears it indicates the presence of starch-iodine complex if the area around the isolate remains clear it indicates the degradation of starch has occurred due to production of amylase by the isolates. The hydrolyzing zone (HZ) and growth zone (GZ) were measured. The ratio of HZ /GZ was calculated as given in table 1. Figure1. Soil Actinomycetes grown on Figure 2. Screening of Actinomycetes Starch casein agar medium from soil samples
2.4.2. Determination of Caseinase activity: The milk casein agar medium was prepared and sterilized by auto clave at 121C with 15 lb/in2 for 20 min. Then the molten medium was mixed with skimmed milk and poured into sterile petri plates. Then the medium was allowed to solidify. Then each isolate was streaked on solidified medium separately under aseptic condition. The inoculated plates were incubated at 28 C for 7 days. The results are given in table 2. In subsequent studies we have determined the anti bacterial activity and biochemical test such as nitrate reduction test. Table 1. Amylase activity test Isolate Hydrolysing zone width (mm) 36 19 22 36 15 17 16 14 17 11 9 Growth zone Width(mm) 15 12 17 9 13 11 4 11 8 8 5 Ratio Isolate Table 2. Caseinase activity test Hydrolysing zone width (mm) 17 16 15 18 14 20 22 15 14 25 16 Growth zone Width(mm) 13 8 10 7 8 11 8 9 8 19 10 Ratio
SSS1 SSS2 SSS3 SSS4 SSS5 SSS6 SSS7 SSS8 SSS9 SSS10 SSS11
3.2 1.5 1.2 4 1.1 1.5 4 1.2 2.1 1.3 1.8
SSS1 SSS2 SSS3 SSS4 SSS5 SSS6 SSS7 SSS8 SSS9 SSS10 SSS11
1.3 2.0 1.5 2.5 1.75 1.81 2.75 1.66 1.75 1.31 1.6
2.5. Determination of anti bacterial activity 2.5.1 Anti-bacterial activity by cross streak method: The antibacterial activity studies were performed on nutrient agar medium. The nutrient agar medium was prepared and autoclaved at 121 0C with 15 lb/in2 for 20 min then the medium was transferred into sterile petri plates separately, and allowed the medium to solidify. After setting the medium each isolate was picked up by using the inoculating needle from the slants and it was streaked on solidified agar medium this procedure was repeated for all isolates then they were incubated at 28 0C for 7 days. B.subtilis, E.coli were used as test organisms. Then they were incubated at 370C for 24 hours. The length of inhibition was measured as shown in table 3. 2.6. Determination of anti fungal activity 2.6.1 Anti-fungal activity by cross streak method: The antifungal activity studies were performed on potato dextrose agar medium. The potato dextrose agar medium was prepared and autoclaved at 121 0C for 20 min then the medium was transferred into sterile petri plates separately, and allowed the medium to solidify. After setting the medium each isolate was picked up by using the inoculating needle from the slants and it was streaked on solidified agar medium. Then they were incubated at 280 C for 7 days. This procedure was repeated for all isolates.
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Aspergillus niger was used as test organism. The inoculated plates were incubated at 280C for 4 days. The length of inhibition was measured as shown in Table.4. Table 3. Anti-Bacterial Activity test Table 4. Anti Fungal Activity Isolate No Length of inhibition(mm) Isolate No Length of inhibition(mm) E.coli* B.Subtilis* A.niger* A.niger* (7 days (4 days 8 5 SSS1 incubation) incubation) SSS2 2 SSS1 17 15 SSS3 4 SSS 11 13 2 SSS4 12 4 SSS3 19 20 SSS5 3 SSS 16 14 4 SSS6 2 SSS 9 10 5 SSS7 10 8 SSS6 15 17 SSS8 4 3 SSS 18 17 7 SSS9 2 SSS 10 12 8 SSS10 6 5 SSS 17 15 9 SSS11 5 3 SSS10 13 12 * Test organism in both the tables 3 and 4 15 17 SSS11 2.7 Nitrate reduction test: Certain micr oorganisms use nitrate (NO 2 ) in place of oxygen as an external terminal electron acceptor. In the beginning, nitrate can easily be reduced to nitrite. In case of aerobic micro organisms, oxygen is first used to prevent nitrate reduction and then utilize nitrate .The nitrite may further give rise to nitrogen, ammonia, nitrogen oxide (N 2 O). The enzyme reaction is catalyzed by nitrate reductase as given below + NO3 + 2H +2e NO2 +H2O Sterile nitrate broth was prepared and dispersed 5ml quantities into each test tube. Then all the test tubes were plugged tightly with cotton and they were sterilized by autoclave. The sterile nitrate broth tubes were inoculated with all eleven teen isolates and incubated at 28C for 7 days. Clear broth was separated and transferred into porcelain dish and it was tested by the addition of three drops of nitrate test reagent along with one drop of H 2SO4. If blue color appears it indicates that nitrate is produced if no blue color develops either due to no occurrence of nitrate to nitrite reduction or all the nitrite so formed must have been further converted to other products Nutrient agar medium Caseinase activity of SSS7 Beef extract Peptone Agar-Agar NaCl Distilled water PH 10gm 20gm 20gm 5gm 1.0L 7.0
3. CONCLUSION A total of 11 isolates were screened. All the 11 isolates exhibited amylase and caseinase activities. Isolate SSS4 and SSS7 has shown good amylase activity Isolate SSS7 and SSS4 has shown excellent caseinase activity where as isolate SSS2 and SSS6 has shown good activity. All the 11 isolates have shown poor anti bacterial activity by cross streak method. All the 11 isolates has shown good anti fungal activity among them SSS 7, SSS3, SSS1 shown good results as compared with others All the 11 isolates have shown positive results in case of nitrate reduction test. Isolate SSS 7has shown good enzymatic activities particularly the caseinase activity. REFERENCES Balagurunathan, Isolation of thermotolerant actinomycetes strains from the himalayan mountain, 2003, IJB volume 4, april 2005, pg 271-276 Benedict RG, Pridham TG, Lindenfelser LA, Hall HH, Method for the preferential isolation of Actinomycetes from soils, 34(4), 1946, 375-384 Brown JA, Enhanced enzyme production by the cellulo lytic fungus, 9, 1987, 176-178
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K Kathiresan, Fungisidal activity of marine actinomycetes against phytopathogenic fungi, 4, 2004, 276-281 Karadi R V, Jaiprakash B, Protease from an actinomycetes species: production, optimization an d characterizationIndian drugs 46(10) oct 2009, 33-39 Kunio ohnishi, Lipase production of Aspergillus oryza, 1994, 77 no 5 490-495 Kalakoutskje L V, Prokofyeva-Belgovskaya; the structure and development of actinomycetes, 221, 1963, 44 Mustranta Annikka, Applications of biotechnology, 38, 1992, 61-62 Emerson RL, Almaj Whiffen, Nester Bohonos, Studies on the production of antibiotics by actinomycetes and Molds, 52(3), 1946, 357-366 Yamanda K, Studies on production of lipase by micro organisms on medium composition of candida cylindraceae, 37, 1963, 645-648
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Suruchi Singh et.al.
PLANTS USED IN HEPATOPROTECTIVE REMEDIES IN TRADITIONAL INDIAN MEDICINE

Suruchi Singh1*, Maryam Bincy Thomas, Sharada Pal Singh, D.Bhowmik 2 1. Sunderdeep Pharmacy College, Ghaziabad, Uttar Pradesh 2. Nimra Pharmacy College, Vijayawada, Andhra Pradesh * Corresponding author: Email-suruchibpharm89@gmail.com ABSTRACT There is no plant in this Universe which is non-medicinal and which cannot be made of use for many purposes and by many modes. This definition rightly suggests that in principle all plants have a potential medicinal value. Medicinal plants have been considered as important therapeutic aid for alleviating ailment of humankind. Search for eternal health and longevity and to seek remedy to relieve pain and discomfort prompted the early man to explore his immediate natural surroundings to develop a variety of therapeutic agents using natural resources. Herbal plants or botanical medicines have been used traditionally by herbalist worldwide for the prevention and treatment of liver disease. Medicinal plants play a key role in human health care. About 80% of the world population relies on the use of traditional medicine, which is predominantly based on plant material. The present review discusses different types of medicinal plants containing hepatoprotective activity. Keywords: Herbal drugs, therapeutic agents, hepatoprotective, medicinal plants, traditional medicine, 1. INTRODUCTION It is estimated that about 7,500 plants are used in local health traditions in, mostly, rural and tribal villages of India. Out of these, the real medicinal value of over 4,000 plants is either little known or hitherto unknown to the mainstream population. The classical systems of medicine such as Ayurveda, Siddha, Amchi, Unani and Tibetan use about 1,200 plants. A detailed investigation and documentation of plants used in local health traditions and pharmacological evaluation of these plants and their taxonomical relatives can lead to the development of invaluable plant drugs for many dreaded diseases. Random screening of plants has not proved economically effective. Liver is a vital organ play a major role in metabolism and excretion of xenobiotics from the body. Liver injury or liver dysfunction is a major health problem that challenges not only healthcare professionals but also the pharmaceutical industry and drug regulatory agencies. Liver cell injury caused by various toxic chemicals like certainanti-biotic, chemotherapeutic agents, carbon tetrachloride (CCl4), thioacetamide (TAA) etc, excessive alcohol consumption and microbes is well studied. The available synthetic drugs to treat liver disorders in this condition also cause further damage to the liver. Hence, Herbal drugs have become increasingly popular and their use is widespread. Herbal medicines have been used in the treatment of liver diseases for a long time. A number of herbal preparations are available in the market. The present review is aimed at compiling data on promising phytochemicals from medicinal plants that have been tested in hepato toxicity models using modern scientific system. Medicinal plants play a key role in the human health care. About 80% of the world population relies on the use of traditional medicine which is predominantly based on plant materials. The traditional medicine refers to a broad range of ancient natural health care practices including folk/tribal practices as well as Ayurveda, Siddha and Unani. These medical practices originated from time immemorial and developed gradually, to a large extent, by relying or based on practical experiences without significant references to modern scientific principles (Bagepalli Srinivas and Ashok Kumar, 2011). 2. HEPATOPROTECTIVE HERBS Herbal-based therapeutics for liver disorders has been in use in India for a long time and has been popularized world over by leading pharmaceuticals. Despite the significant popularity of several herbal medicines in general, and for liver diseases in particular, they are still unacceptable treatment modalities for liver diseases. The limiting factors that contribute to this eventuality are a) Lack of standardization of the herbal drugs b) Lack of identification of active ingredient(s)/principles(s) c) Lack of randomized controlled clinical trials (RCTs) d) Lack of toxicological evaluation The use of natural remedies for the treatment of liver diseases has long history, starting with the Ayurvedic treatment, and extending to the Chinese, European and other systems of traditional medicines. The 21st century has seen a paradigm shift towards therapeutic evaluation of herbal products in liver disease models by carefully synergizing the strengths of the traditional systems of medicine with that of the modern concept of evidence based medicinal evaluation, standardization and randomized placebo controlled clinical trials to support clinical efficacy. A large number of plants and formulations have been claimed to have hepatoprotective activity. Nearly 160 phytoconstituents from 101 plants have been claimed to possess liver protecting activity. In India, more than87 plants are used in 33 patented and proprietary multi ingredient plant formulations. In spite of the tremendous advances made, no significant and safe hepatoprotective agents are available in modern therapeutics. Therefore, due importance has been given globally to develop plant-based hepatoprotective drugs effective against a variety of Volume 1 Issue 1 January February 2013 Page 58
liver disorders. The present review is aimed at compiling data based on reported works on promising phytochemicals from medicinal plants that have been tested in hepatotoxicity models. The hepatoprotective activity is probably due to the presence of flavonoids in all few herbal plants. The results of this study indicate that extracts of leaves and plants extracts of some medicinal plant have good potentials for use in hepatic disease. The present review study give evidential explore mechanism of action of medicinal plants against experimentally induced hepatotoxicity. Hence the review study is concluded that the herbal drug possesses hepatoprotective activity and it has been proved by different animal models give many links to develop the future trials (Venkatesh P, 2011). 2.1. Grape Seeds: Hepatoprotective effect of Grape Seed extract (GSE) on hypercholesterolemia, where, Wistar rats fed a cholesterol rich diet (hypercholesterolemic group-HCD) and to see the effect of GSE, another group fed on cholesterol-rich diet enriched with 0.3% GSEW/W-PG for 8 weeks. Serum lipid levels, serum antioxidant status, liver and kidney function were analysed in addition to histopathological examination of the liver. Furthermore, the liver function expressed as glutamic pyruvate transaminase (GPT) and albumin serum levels, decreased significantly and reached to normal level in case of oral administration of GSE. Histological examination of liver sections confirmed the serum analysis where GSE had a protective effect on animals fed on HCD, the liver of these animals showed mild affection in the form of microvesicular vacuolation of hepatocytes in the peripheral zone of the hepatic lobule (<50%) in comparison to the fatty change observed as microvesicular and macrovesicular vacuolation in >50% and <70% of the liver sections in HCD group. 2.2. Annona squamosa: The hepatoprotective effect of alcoholic and water extract of Annona squamosa (custard apple) hepatotoxic animals with a view to explore its use for the treatment of hepatotoxicity in human. These extracts were used to study the Hepatoprotective effect in isoniazid + rifampicin induced hepatotoxic model. There was a significant decrease in total bilirubin accompanied by significant increase in the level of total protein and also significant decrease in ALP, AST, ALT and -GT in treatment group as compared to the hepatotoxic group. In the histopathological study the hepatotoxic group showed hepatocytic necrosis and inflammation in the centrilobular region with portal triaditis. The treatment group showed minimal inflammation with moderate portal triaditis and their lobular architecture was normal. It should be concluded that the extracts of Annona squamosa were not able to revert completely hepatic injury induced by isoniazid + rifampicin, but it could limit the effect of these drugs in liver. The effect of extracts compared with standard drug silymarin (Fasalu Rahiman and Rupesh Kumar M, 2011). 2.3. Apium graeolens Linn: The hepatoprotective activity of the Apium graeolens Linn (Apiaceae) against CCl4 induced hepatotoxicity in albino rats. The degree of protection was measured by using biochemical parameters like serum transaminases (SGOT and SGPT), alkaline phosphatase, total protein and albumin. The methanolic extracts showed the most significant hepatoprotective activity comparable with standard drug silymarin. Other extracts namely petroleum ether and acetone also exhibited a potent activity (Patil Prakash, 2011). 2.4. Fumaria indica: Fumaria indica (Fumariceae) were studied for their hepatoprotective activity against carbon tetrachloride, paracetamol and rifampicin-induced heptatotoxicites in albino rats. The petroleum ether extract against carbonetrachloride, total aqueous extract against paracetamol and methanolic extract against rifampicin-induced hepatotoxicities showed similar reductions in the elevated levels of some of the serum biochemical parameters in a manner similar that of silymarin indicating its potential as a hepatoprotective agent (Patil Prakash, 2011) 2.5. Wedelia calendulacea (Bhanra): Hepatoprotective activity of the ethanol leaf extract of W.calendulacea (Asteraceae) (EEWC) was studied by estimating serum enzyme activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), protein and bilirubin. The treatment with EEWC showed a dose-dependent reduction of CCl induced elevated serum levels of enzyme activities with parallel increase in total protein and bilirubin, indicating the extract could preserve the normal functional status of the liver (Oluseyi Adeboye Akinloye and Moshood, 2011). 2.6. Spermacoce hispida: Ethanolic extract of the Spermacoce hispida. Linn (SHE) was used against carbon tetra chloride (CCl4) inducd hepatotoxicity in rats. Liver functions were assessed by the determination of SGOT, SGPT, ALP and bilirubin. Histopathological studies were carried out. The serum biochemical analysis results suggest that the use of Ethanolic extract of Spermacoce hispida. Linn exhibited significant protective effect from hepatic damage in CCl4 induced hepatotoxicity model. Histopathological studies revealed that concurrent administration of the extract with CCl4 exhibited protective effect on the liver, which further evidenced its hepatoprotective activity (Suman Pattanayak and Siva Sankar Nayak, 2011). 2.7. Juncus subulatus: The volatile oil, ethyl acetate, n-butanol and total alcoholic extracts of J. subulatus were evaluated for their hepatoprotective and antioxidant activity in female rats against ethanol induced hepatic injury. Serum Liver enzymes (AST, ALT and ALP), total protein, albumin, cholesterol, triglycerides, nitric oxide (NO), malondialdhyde (MDA) and total antioxidant capacity (TAC) were measured colorimetrically. The results showed that all extracts of Juncus subulatus exhibited hepatoprotective activity in the Volume 1 Issue 1 January February 2013 Page 59
following order: volatile oil extract > ethyl acetate extract > n-butanol extract > total alcoholic extract (Balakrishanan N and Balasubaramaniam A, 2011). 2.8. Mamordica subangulata and Naragamia alata: The hepatoprotective activity of Mamordica subangulata (leaf) and Naragamia alata (whole plant) suspension was studied using paracetamol overdose induced liver damage in rats. The effect of the plant suspensions on bile flow was studied in anaesthetised normal rats by surgical cannulation of bile duct with polyethylene tubing. The drug was given intraduodenally after 1 hour bile collection. Mamordica subangulata leaf suspension (500mg/kg,fresh weight;50 mg/kg, dry weight) protected rats from paracetamol induced liver damage as judged from serum marker enzyme activities. It also stimulated bile flow in normal rats. Naragamia alata was inactive in protecting rats from paracetamol induced hepatotoxicity. A suspension of Mamordica subangulata leaf (dry or fresh) can protect rats from paracetamol induced hepatotoxicity (Kuppan Nithianantham and Murugesan Shyamala, 2011). 2.9. Leucas Aspera: The effect of Leucas aspera leaves fresh juice against carbon tetrachloride (CCl4) induced liver damage. The evaluation markers used were GOT, GPT, Alkaline phosphate, glucose, bilirubin, cholesterol and total protein. These biochemical parameters were significantly changed due to single dose of CCl4, but the treatment of Leucas aspera leaves fresh juice significantly recovers all markers to normal levels. In this study silymarin was used as a standard for comparison. The observation of markers as well as Light and electron microscope photographs supports the regeneration of liver parenchyma. This proves overall promising effect against liver disorders (Kalpana Patila and Shaikh Mohammed Imtiaza, 2011). 2.10. Plumbago zeylanica: Petroleum ether extract of root of Plumbago zeylanica was investigated for hepatoprotective activity against paracetamol induced liver damage to evaluate the hepatoprotective activity of ethanolic extract. In serum total bilirubin, total protein, aspartate transaminase, alanine transaminase, alkaline phosphatase, lactate dehydrogenase, -Glutamyl transferase, Total Cholesterol and serum triglycerides were determined to assess the effect of the extract on the paracetamol induced hepatic damage. The study was also supported by histopathology of liver sections. Results of this study revealed that the markers in the animals treated with paracetamol recorded elevated concentration indicating severe hepatic damage by paracetamol, whereas the blood samples from the animals treated with petroleum ether extract of roots showed significant reduction in the serum markers indicating the effect of the plant extract in restoring the normal functional ability of the hepatocytes. The dosage of extract of plant roots used was 300 mg/kg bodyweight of rat. The present study reveals that the petroleum ether root extract of Plumbago zeylanica could afford a significant protection against paracetamol-induced hepatocellular injury (Kavita Suryawanshi and Sanjay Khakre,2011). 2.11. Leucas ciliata leaves: Hepatoprotective activity of the ethanolic extract of Leucas ciliata leaves extract was evaluated by carbon tetrachloride (CCl4) induced liver damage model in rats. The extract demonstrated a significant dose dependent antioxidant activity comparable with ascorbic acid. In hepatoprotective activity study, CCl4 significantly increased the levels of serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP) and total bilirubin. Pretreatment of the rats with ethanolic extract of L.ciliata (100, 200 and 400mg/kg po) inhibited the increase in serum levels of SGPT, SGOT, ALP and total bilirubin and the inhibition was comparable with silymarin (100mg/kg po). The present study revealed that L. ciliata leaves have significant hepatoprotective activity (Amol Bhalchandra Deore, Vinayak D, 2011). 2.12. Coptidis Rhizoma (Huanglian): Berberine is an active compound in Coptidis Rhizoma (Huanglian) with multiple pharmacological activities including antimicrobial, antiviral, anti- inflammatory, cholesterol-lowering and anticancer effects. The hepatoprotective effects of berberine on serum and tissue superoxide dismutase (SOD) levels, the histology in tetrachloride (CCl4)-induced liver injury. Sprague-Dawley rats aged seven weeks were injected intraperitoneally with 50% CCl4 in olive oil. Berberine was orally administered before or after CCl4 treatment in various groups. Twenty-four hours after CCl4 injection, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, serum and liver superoxide dismutase (SOD) activities were measured. Histological changes of liver were examined with microscopy. The present study demonstrates that berberine possesses hepatoprotective effects (Merlin NJ and Parthasarathy V, 2011). 2.13. Careya arborea: The methanol extract of Careya arborea bark, (myrtaceae) was tested for antioxidant and hepatoprotective activity in Ehrlich ascites carcinoma (EAC) tumor-bearing mice. Tumor control animals inoculated with EAC showed a significant alteration in the levels of antioxidant and hepatoprotectiven parameters8. The extract treatment at 50, 100 and 200 mg/kg body weight doses given orally caused a significant reversal of these biochemical changes towards the normal in serum. Liver and kidney when compared to tumor control animals indicating the potent antioxidant and hepatoprotective nature of the standardized extract (Babalola O, 2011). 2.14. Cassia fistula (Amaltas): Hepatoprotective activity of the n-heptane extract of Cassia fistula (Fabaceae) leaves was investigated by inducing hepatotoxicity with paracetamol in rats. The extract at a dose of 400 mg/kg body wt. exhibited orally, significant protective effect by lowering the serum levels of Volume 1 Issue 1 January February 2013 Page 60
transaminases (SGOT and SGPT), bilirubin and alkaline phosphatase (ALP). The effects produced were comparable to that of a standard hepatoprotective agent (Mohammad N, 2010). 2.15. Cleome viscose Linn (Tickweed): The hepatoprotective activity of the Cleome viscosa Linn (Capparidaceae) extract was assessed in CCI4 induced hepatotoxic rats. The test material was found effective as hepatoprotective, through in vivo and histopathological studies. The extract was found to be effective in shortening the thiopental induced sleep in mice poisoned with CCl4. The hepatoprotective effect of ethanolic extract was comparable to that of silymarin, a standard hepatoprotective agent (Rajeswari H and Vasuki R, 2011). 2.16. Morinda citrifolia (noni): The hepatoprotective effects of Noni juice (NJ) (Rubiaceae) against CCl(4)induced chronic liver damage in female Sprague Dawley (SD) rats. Histopathological examination revealed that liver sections from the NJ + CCl(4) appeared similar to controls, whereas typical hepatic steatosis was observedmin the placebo + CCl(4) group. Serum alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine transaminase (ALT), total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) levels were increased in the placebo group compared with the NJ group. In contrast, high-density lipoprotein (HDL) was increased in the NJ group and decreased in the placebo group. Thus, NJ juice appears to protect the liver from chronic exogenous CCl(4) exposures (Shivananda Nayak B and Julien R.Marshall, 2011). 2.17. Phyllanthus amarus (Bhuiamala): Ethanolic extract of Phyllanthus amarus (euphorbiaceae), at (0.3g kg (-1) BW 0.2 ml (-1) day (-1) was given to all groups except control groups (gp. I and gp. V), after 30min of aflatoxin administration. The entire study was carried out for 3 months and animals were sacrificed after an interval of 30 days till the completion of study. Phyllanthus amarus extract was found to show hepatoprotective effect by lowering down the content of thiobarbituric acid reactive substances (TBARS) and enhancing the reduced glutathione level and the activities of antioxidant enzymes, glutathione peroxidase (GPx), glutathione- transferase (GST), superoxide dismutase (SOD) and catalase (CAT). (Samy M. Mohamed and Emad M. Hassan, 2011) 2.18. Sargassum polycystum: The protective effect of ethanol extract of Sargassum polycystum was evaluated in D-galactosamine-induced hepatitis in rats. Prior oral administration of S.polycystum extract [125mg/kg bodyweight/day for 15 days] significantly attenuated (P<0.05) the D-galactosamine-induced increases in the levels of diagnostic marker enzymes (AST, ALT and ALP) in plasma of rats. It has also demonstrated antioxidant activity against D-galactosamine-induced hepatitis by inhibiting the activation of lipid peroxidation and by preserving the hepatic enzymatic and non-enzymatic antioxidant defense system at near normal. The antihepatotoxic potential of S. polycystum might possibly due to its antioxidant property and membrane stabilizing action (Rupesh Kumar M and Pasumarthi Phaneendra, 2011). 2.19. Prostechea michuacana: Methanol, hexane and chloroform extracts of Prostechea michuacana (PM) were studied against CCl4-induced hepatic injury in albino rats. Pre-treatment with methanolic extract reduced biochemical markers of hepatic injury levels demonstrated dose-dependent reduction in the in vivo peroxidation induced by CCl4. Likewise, pretreatment with extracts of PM on paracetamol-induced hepatotoxicity and the possible mechanism involved in this protection were also investigated in rats after administering the extracts of PM at 200, 400 and 600mg/kg. The degree of protection was measured by monitoring the blood biochemical profiles. The methanolic extract of orchid produced significant hepatoprotective effect as reflected by reduction in the increased activity of serum enzymes, and bilirubin. These results suggested that methanolic extract of PM could protect paracetamol-induced lipid peroxidation thereby eliminating the deleterious effects of toxic metabolites of paracetamol. This hepato-protective activity was comparable with sylmarin. Hexane and chloroform extracts did not show any apparent effect. The findings indicated that the methanolic extract of PM can be a potential source of natural hepatoprotective agent (Subash KR and Ramesh KS, 2011). 3. CONCLUSION A phytotherapeutic approach to modern drug development can provide many invaluable drugs from traditional medicinal plants. Search for pure phytochemicals as drugs is time consuming and expensive. Numerous plants and polyherbal formulations are used for the treatment of liver diseases. However, in most of the severe cases, the treatments are not satisfactory. Although experimental evaluations were carried out on a good number of these plants and formulations, the studies were mostly incomplete and insufficient. The therapeutic values were tested against a few chemicals-induced subclinical levels of liver damages in rodents. Development of such medicines with standards of safety and efficacy can revitalise treatment of liver disorders and hepatoprotective activity.
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Botanical name Amaranthus caudatus Linn Anisochilus carnosus Linn Asparagus racemosus Linn Azima tetracantha Calotropis procera R.Br Cajanus cajan Linn Cajanus scarabaeoides Linn Carissa carindas Linn Clitoria ternatea Linn Cucumis trigonus Roxb Family Amaranthaceae Lamiaceae Asparagaceae Salvadoracaeae Asclepediaceae Leguminosae Fabaeceae Apocyanaceae Fabaceae Cucurbitaceae Parts used Whole plant Stems Roots Leaves Root bark Pigeon pea leaf Whole plant Root Leaves Fruit Solvent used Methanol Ethanol Ethanol Ethanol Methanol ethanol n-butanol, ethanol Ethanol Methanol Pet ether, chloroform, alcohol, aqueous Methanol Ethanol Ethanol, chloroform Aqueous Ethanol
Table 1: List of Hepatoprotective plants

Chemical constituents Flavonoids, saponins, glycosides Alkaloids,flavonoids, glycosides Phenols, coumarins Flavonoids, triterpenoids Terpinoidsglycosides, flavonoids Flavonoids, stibenes Flavonoids Alkaloids, tannins, steroids Phenolic flavonoids Flavonoids Screening method Carbon tetra chloride induced Carbon tetra chloride induced Paracetamol induced Paracetamol induced Carbon tetra chloride induced D-galactosamine Paracetamol induced Carbon tetra chloride induced Paracetamol induced Carbon tetra chloride induced Reference Venkatesh P, 2011 Venkatesh P, 2011 Fasalu Rahimom, 2011 Arthika, 2011 Patiprakash, 2011 Oluseye Ade Boye Akinloye, 2011 Suman Pattanayak, 2011 Balkrishnan, 2011 Yengchen, 2011 Mohammad Imtiaz, 2010
Ficus religiosa Linn Garcinia indica Linn Gmelina asiatica Linn Hyptis suaveolens linn Leucas cilita Linn
Moraceae Clusiaceae Verbenaceae Lamiaceae Lamiaceae
Stem bark Fruit rind Aerial parts leaves Whole plant leaves Fruit Leaves Leaves
Glycosides, steroids, tannins Benzophenones, garcinol Flavonoids Flavonoids Flavonoids
Melia azhadirecta Linn Morinda citrifolia Linn Myoporum lactum Linn Myrtus communis Linn Solanum nigram Linn
Piperaceae Rubiaceae Myoporaceae Myrtaceae
ethanol Aqueous Methanol, nbutanol Silymarin
Spectro photo metric method Saponins, triterpins, steroids Flavonoids Flavonoids, terpenoids, steroids Flavonoids, terpenoids
Paracetamol induced Carbon tetra chloride induced Carbon tetra chloride Acetaminophen induced Carbon tetrachloride induced Carbon tetra chloride, silymarin Streptozotocin induced Profenofos induced Paracetamol induced Carbon tetra chloride
Kavitha Suryawanshi, 2011 Amol Bhalchandra, 2011 Parthasarathy, 2011 Babalola, 2011 Qureshi, 2010
H Rajeswary, 2011 Shivananda Nayak, 2011 Mohammad, 2011 Pasumarthi Phaneendra, 2011 Subash, 2011.
Solanaceae
Fruits
Ethanol
REFERENCES Amol Bhalchandra Deore, Vinayak D Sapakal, Nilofer S Naikwade, Antioxidant and Hepatoprotective activity of Garcinia indica Linn fruit rind, Pharmacie Globale International Journal of Comprehensive Pharmacy, 2(6), 2011, 1-5. Arthika S, Shanthammal Y, Sheryl Igal N, Elankini P, Pramod Reddy G, Gaidhani SN, Ganesan R, Hepatoprotective activity of the ethanolic extract of Azima tetracantha against paracetamol-induced hepatotoxicity in wistar albino rats, Journal of Advances in Pharmacy and Healthcare Research, 1(2), 2011, 1420. Babalola O, Ojo E, Oloyede F, Hepatoprotective activity of aqueous extract of the leaves of Hyptis suaveolens (L.) on acetaminophen induced hepatotoxicity in rabbits, Research Journal of Chemical Sciences, 1(7), 2011, 85-88. Bagepalli Srinivas, Ashok Kumar, Kuruba Lakshman, Paresandra Avalakondarayppa Arun Kumar, Gollapalle Lakshminarayana Shastry Viswanpha, Veeresh, Prabhakar Veerapur, Boreddy, Shivanadpea, Thippeswamy, Bachappaa,Manoj, Hepatoprotective activity of methanol extracts of Amaranthus caudatus Linn. against paracetamol-induced hepatic injury in rats, Journal of Chinese Integrative Medicine, 9(2), 2011, 194-200.
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Balakrishanan N, Balasubaramaniam A, Sangameswaran B, Bhaskar VH, Hepatoprotective activity of two Indian medicinal plants from Western Ghats-Tamil nadu, Journal Of Natural Pharmaceuticals 2011, 2( 2), 9298. Fasalu Rahiman, Rupesh Kumar M, Tamizh Mani T, Mohamed Niyas K, Satya Kumar B, Phaneendra P, Surendra B, Hepatoprotective activity of Asparagus racemosus root on liver damage caused by paracetamol in rats, Indian Journal of Novel Drug Delivery, 3(2), 2011, 112-117. Gupta Amartya K, Ganguly Partha, Majumder Upal K, Ghosal Shibnath, Hepatoprotective & antioxidant effect & stereoidal saponins of solanum of Solanum xanthocarpum & Solanum nigrum in Paracetomol induced hepatotoxicity in rats, Pharmacology online, 1, 2009, 757-768. Kalpana Patila, Shaikh Mohammed Imtiaza, Anoop Singha, Varsha Bagewadia, Shaikh Gazib, Hepatoprotective activity of Cucumis trigonus Roxb. fruit against CCl4 induced hepatic damage in rats, Iranian Journal of Pharmaceutical Research 2011,10(2), 295-299. Kavita Suryawanshi, Sanjay Khakre, Ashish Chourasia, Chaurasiya PK, Pawar RS, Deenanath Jhade, Hepatoprotective activity of stem bark extracts of Ficus religiosa Linn in rats, International Journal of Biomedical Research, 2(8), 2011, 466-475. Kuppan Nithianantham, Murugesan Shyamala, Yeng Chen, Lachimanan Yoga Latha, Subramanion L. Jothy, Sreenivasan Sasidharan, Hepatoprotective potential of Clitoria ternatea leaf extract against paracetamol induced damage in mice, Molecules 2011, 16, 10134-10145. Merlin NJ and Parthasarathy V, Antioxidant and Hepatoprotective activity of chloroform and ethanol extracts of Gmelina asiatica aerial parts, Journal of Medicinal Plants Research, 5(4), 2011, 533-538. Mohammad N Qureshi, Bhanudansh S Kuchekar, Nadeem A Logade, Majid A Haleem, In-Vitro antioxidant and in-vivo hepatoprotective activity of Leucas ciliata leaves, Academy of Chemistry of Globe Publications, 4(2), 2010, 124-130. Oluseyi Adeboye Akinloye, Moshood Olajire Olaniyi, Hepatoprotective effect of Cajanus cajan on tissue defense system in D-galactosamine-induced hepatitis in rats, Turk J Biochem 2011, 36(3), 237241. Patil Prakash, Prasad K, Nitin M, Vinay Kumar M, Sreenivasa Rao K, Evaluation of hepatoprotective effect of Calotropis procera R.Br root extract against CCL4 induced hepato-oxidative stress in albino rats, International Journal in Ayurveda & Pharmacy 2011, 2(1), 319-324. Rajeswari H, Vasuki R, Samudram P, Geetha A, Hepatoprotective action of ethanolic extracts of Melia azedarach Linn and Piper longum Linn and their combination on CCl4 induced hepatotoxicity in rats, Indian Journal of Experimental Biology, 49, 2011, 276-281. Rupesh Kumar M, Pasumarthi Phaneendra, Surendra Bodhanapu, Fasalu Rahiman OM, Mohamed oiyas K, Tamizmani T, Antioxidant and Hepatoprotective activity of the aqueous extract of Myrtus communis (Myrtle) Linn leaves, Pharmacologyonline, 1, 2011, 1083-1090. Samy M Mohamed, Emad M Hassan, Khaled A Abd Elshafeek, Azza M Mohamed, Investigation of flavonoidal constituents and hepatoprotective activity of Myoporum lactum, International Journal of Academic Research, 3(3), 2011, 528-533. Shivananda Nayak B, Julien R.Marshall, Godwin Isitor, Andrew Adogwa, Hypoglycemic and hepatoprotective activity of fermented fruit juice of Morinda citrifolia (Noni) in diabetic rats, Evidence-Based Complementary and Alternative Medicine 2011, 1-5. Subash KR, Ramesh KS, Binoy Vargheese Charian Francis Britto, Jagan Rao N, Vijayakumar, Study of Hepatoprotective activity of Solanum nigrum and Cichorium intybus, International Journal of Pharmacology, 7(4), 2011, 504-509. Suman Pattanayak, Siva Sankar Nayak, Durga Prasad Panda, Subas Chandra Dinda, Vikas Shende, Amol Jadav, Hepatoprotective activity of crude flavonoids extract of Cajanus scarabaeoides (L) in paracetamol intoxicated albino rats, Asian J Pharm Biol Res 2011, 1(1), 22-27. Venkatesh P, Dinakar A, Senthilkumar N, Hepatoprotective activity of an ethanolic extract of stems of Anisochilus carnosus against carbon tetrachloride induced hepatotoxicity in rats, International Journal of Pharmacy and Pharmaceutical Sciences, 3(1), 2011, 243-245.
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A Ravi Kumar et.al.
PHARMACOGNOSTICAL STUDIES ON ARTEMISIA ANNUA

A Ravi Kumar1, K M Subbu Rathinam2 1. Department of Pharmacognosy, Bapatla College of Pharmacy BAPATLA 522 101 (A.P.) INDIA. 2. Department of Zoology, Serfoji Govt. College (Autonomous) Thanjavur -613005 (T.N.) INDIA. * Corresponding Author: aravikumar@india.com ABSTRACT With increasing demand in the field of herbal medicines and cosmetics, it has become necessary and pertinent to probe into the area of systematic knowledge about herbal drugs. There is a need for the application of this knowledge in authentification, detailed study and practical utilization of crude drugs. The present paper deals with the taxonomy, anatomy, powder study pertaining to organoleptic, microscopic, fluorescence and physical constant evaluations of Artemisia annua Keywords: Artemisia annua, pharmacognostical, studies 1. INTRODUCTION Pharmacognosy is the study of the structural, physical, chemical and sensory characters of crude drugs of animals, plants and mineral origin. The search for biologically active compounds from natural source has always been of great interest to researchers looking for new source of drugs useful in infectious diseases. Higher plants have played a vital role as the source of important therapeutic agents. Only a small percentage of higher plant species have so far been exploited and much remains to be done. A. annua belongs to the family Asteraceae. Literature survey of this plant indicates its high medicinal value. Artemisia annua L. (annual wormwood, sweet wormwood, sweet annie), a highly aromatic annual herb of Asiatic and eastern European origin, is widely dispersed throughout the temperate region. The species has naturalized in the United States and is sold on a limited scale as a dried herb for the floral and craft trade where it is used as an aromatic wreath. The plant has traditionally been grown in India China as a medicinal and, more recently in Europe for its aromatic leaves which are used in flavoring beverages. Recent research in the Peoples Republic of China with traditional herbal medicine has brought attention to A. annua, the source of Qinghaosu (artemisinin), a compound that shows promise as an anti-malarial agent Artemisinin has also been reported to be a potent plant inhibitor with potential as a natural herbicide Artemisinin and its derivatives, artemether and artesunate, have been studied for their efficacy as antimalarial agents. In in vitro trials conducted in China, all three compounds have been effective against the erythrocytic stages of two chloroquine-resistant Hainan strains of Plasmodium falciparum, the malarial parasite, at lower minimum effective concentrations than chloroquine, the most commonly used drug. Artemisinin and its derivatives have effectively treated malaria and cerebral malaria in human subjects with no apparent adverse reactions nor side effects With P. falciparum developing resistance to chloroquine and pyrimethamine/sulfonamide alternative treatments based on new compounds such as artemisinin and its derivatives are actively being sought. While artemisinin and its derivatives may be synthesized the synthetic compounds are unlikely to be economically competitive with the naturally derived plant products The relatively low content of artemisinin in cultivated European and New World types of A. annua has been a limiting factor for the isolation and evaluation of artemisinin on a technical scale. Artemisinin yields of 0.06% have been extracted from samples of A. annua collected in the United States which are low for commercial exploitation. Yields of extracted artemisinin from the above-ground portions of the plant have ranged from 0.01% to 0.5% (w/w) in the People's Republic of China, although artemisinin yield varies with environmental and management conditions, specific effects are unknown. The extent of genetic variation on artemisinin content was also poorly understood. Essential (volatile) oil composition was characterized in order to evaluate A. annua as a source of aroma chemicals for the fragrance industry. 2. MATERIALS A. annua is a biennial herb. Disease free plants were collected from Vegetative parts of the plant was identified and authentified and preserved in crude drug museum, Department of Pharmacognosy, Bapatla College of Pharmacy, Bapatla. 3. PREPARATION Removed adhering dust and then dried under shade. It was finally powdered with the help of pulvarizer. This powder was used for further studies. Morphological characters of plants like color, surface texture, taste and odor were examined. Free hand sections were taken, cleared with chloral hydrate and treated with phloroglucinal and mounted in glycerin. Organoleptic evaluation, histochemical color reactions, fluorescence evaluations, behavior of the powder with different chemical reagents, Ash values, and preliminary phytochemical analysis were determined. 3. RESULTS AND DISCUSSION 3.1. Macroscopic characters: Herbs of strong- scented leaves of alternate arrangement, entire and incised. Petiolated leaves and obscurely lobed hoary on both surface. Broadly hemispheric pedicellate second nodding distant in lax long racemes terminating the branches, outer involucres bracts green hoary, inner broadly scarious, receptacular hairs straight, outer flowers female, seriate, fertile, inner flowers bisexual fertile or sterile, disk-flowers Volume 1 Issue 1
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fertile, bracts glabrous. Anther bases obtuse, yellow tubular small flowers. Fruit are very small achenes. The taxonomic features collected from the species have been confirmed with the flora of presidency of Andhra Pradesh and Authentified. 3.2. Microscopic characters 3.2.1.T.S.of leaf: Epidermis of leaf is single layered, cuticularised and glandular hairs present in upper and lower side of epidermis. Mesophyll consists of palisade and spongy parenchyma. Palisade cells are single layered and closely arranged with chlorophyll. These are elongated columnar cells. Spongy tissues are seen scattered and loosely arranged in the mesophyll region. Vascular bundle consists of well developed midrib portion. Collenchyma cells can be seen in the upper and lower epidermal regions. Thin parenchymatous ground tissues are seen. Vascular bundles are conjoint, collateral and one in number. Bundle sheath is also present. 3.2.2. T.S of stem: The A. annua stem shows circular shape with numerous epidermal hairs. Epidermis is single layered, with upper most cuticles and multicellular glandular epidermal hairs. Cortex consists of chlorenchymatous and parenchymatous cells. Endodermis is s ingle layer ed a nd parenchymatous with characteristic casparian thickening. Pericycle consists of sclerenchymatous and parenchymatous cells.Vascular bundle is separated by wide medulary rays. There are distinct cambial strips in between xylem and phloem. 3.3. Physico chemical evaluation tests: Color, odour, taste, texture and special features are recorded in Table 1. Phytochemical screening was conducted and presented in Table 4. Behavior of the powder with different chemical reagents is presented in Table 3. Total ash values, NaOH insoluble ash, ethanol insoluble ash, acid insoluble ash (HCl), sulphated ash are presented in Table 2. A. annua plant powder and the extracts of the powder on various solvents were examined under ordinary light and ultraviolet light (365 nm). Table.1. Organoleptic evaluation of A. annua Particulars Observations Colour of Powder Pale green Odour Aromatic and pleasant Taste Bitter and astringent Texture Smooth Special features Snake shaped leaves which are pinnately compound 3.4. Phytochemical screening: The phytochemical test performed and results obtained are presented in Table 5. The macroscopic and microscopic characters, fluorescence analysis, phytochemical characters can be used as a diagnostic tool in the correct identification of plants. The adulterants if any in the plant material can also easily identified by these studies. Table.2. Ash values of Artemisia annua Parameters Ash values* Total ash value 11.59 Sodium hydroxide insoluble PPin% 1.72 Ethanol (insoluble ash) 2.4 ash (NaOH) Acid insoluble ash (HC1) 3.45 Sulphated ash (H2SO4) 6.47 * The values are average of three replicates. Values are expressed in percentage on dry weight basis. Table.3. Fluorescence behavior of the powder of Artemisia annua with different chemical reagents Test observations Inference Powder + picric acid Yellow color Presence of alkaloids Powder + Cone. Sulphuric acid Reddish brown color Presence of steroids Powder + aqueous ferric chloride Green fluorescence Presence of flavonoids Powder + iodine solution Blue color Presence of starch Powder + ammonia solution Pink color Presence of anthraquinone Powder + aqueous silver nitrate White precipitate Presence of protein Powder + aqueous potassium hydroxide Yellow color Presence of flavonoids 4. CONCLUSION Morphological characters of Artemesia annua like color, surface texture, taste and odor were examined. Organoleptic evaluation, phytochemical color reactions, fluorescence evaluations, behavior of the powder with different chemical reagents, ash values, and preliminary phytochemical analysis were determined. Other Pharmacognostic data of Artemesia annua were examined. There is a need of important medicinal plants study in examination in investigation of new species for further studying the plant biological activities.
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Table.4. Comparison of light discharge behavior of the powder of Artemisia annua with different chemical reagents under visible and UV lights Treatment visible UV (365nm) Treatment visible UV (365nm) Benzene Pale green Light pink Methanol Olive green Pinkish Petroleum Ether Pale green Reddish Hydrochloric acid (HC1) Pale green Pinkish Ethanol Olive green Colorless Sulphuric acid Light green Pink (H S0 ) Solvent Ether Pale green Light pink Sodium 2 4 hydroxide(NaOH) Olive green Colorless Chloroform Olive green Rose N- Propanol Olive green Reddish Acetone Pale green Reddish Powder as such Pale green Whitish green Water Dull green Colorless Table.5. Phytochemical screening of A. annua Types of compounds Methanolic extract Types of compounds Methanolic extract Alkaloids +++ Tannins and phenolic compounds +++ Saponins +++ Steroids and sterols +++ Carbohydrates +++ Fixed oils and fats +++ Glycosides +++ Triterpenoids +++ Flavonoids +++ Resins +++ Gums and Mucilage ++ Santonica/Artemisinin +++ Proteins and Amino acids +++ +++ High presence of the particular chemical; ++ moderate presence the particular chemical REFERENCES Acton N and DL Klayman, Artemisinin, a new sesquiterpene lactone endoperoxide from Artemisia annua. Planta Medica, 47, 1985, 442-445. Acton N, DL Klayman and IJ Rollman, Reductive electrochemical HPLC assay for artemisinin (Qinghaosu), Planta Medica 47, 1985, 445-446. Brinda P, Sasikala B and K K Purusothaman, Pharmacognostic studies on Merugan kizhangu Bull, Medico Ethno Botanical Res, 3(1), 1981, 884-896. Carles DJ, Simon JE, Wood KV and Heinstein P, Germplasm variation in artemisinin content of Artemisia annuaL. using an alternative method of artemisinin analysis from crude plant extracts, J Nat Prod, 53, 1990, 157160. Chase CR and Pratt, Fluoresence of powder vegetable drugs with particular reference to development system of identification, Mourn Am Assoc, Sci Ed, 38, 1949, 324-331. Croteau R, Biochemistry of monoterpenes and sesquiterpenes of essential oils. In: Craker, L.E. and J.E. Simon (eds.). Herbs, spices, and medicinal plants: Recent advances in botany horticulture and pharmacology. Oryx Press, Phoenix, AZ, 1, 1986, 81-133. Duke SO, KC Vaughn, EM Croom Jr. and Elsohly HN, Artemisinin, a constituent of annual wormwood (Artemisia annua),is a selective phytotoxin, Weed Sci, 35, 1987,499-505. Henderson W, JW Hart, P How and J Judge, Chemical and morphological studies on sites of sesquiterpene accumulation in Pogostemon cablin (Patchouli), Phytochemistry, 9, 1970, 1219-1228. Kelsey RG and F Shafizadeh, Glandular trichomes and sesquiterpene lactones of Artemisia nova (Asteraceae), Biochem Syst Ecol, 8, 1980, 371-377. Klayman DL, AJ Lin N, Acton JP, Scovill JM, Hock WK, Milhous and AD Theoharides, Isolation of artemisinin (qinghaosu) fromArtemisia annuagrowing in the United States, J Nat Prod, 47, 1984,715-717. Klayman DL, Qinghaosu (Artemisinin): an antimalarial drug from China, Science, 228, 1985, 1049-1055. Schmid G and W Hofheinz, Total synthesis of qinghaosu, J Amer Chem Soc, 105, 1983, 624-625. Simon JE and J Quinn, Characterization of essential oil of parsley, J Agric Food Chem, 36, 1988, 467-472. Xu, Xing-Xiang, J Zhu, Da-Zhong Huang and Wei-Shan Zhou, Total synthesis of arteannuin and deoxyarteannuin, Tetrahedron 42, 1986, 819-828. Zhou, Wei-Shan, Total synthesis of arteannuin (quinghaosu) and related compounds. Pure Appl. Chem. 58, Volume 1 Issue 1
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MECHANISM OF DRUG LOADING, EVALUATION AND APPLICATIONS OF ERYTHROCYTES AS CARRIERS FOR DRUG TARGETTING
Sandeep Kumar Singh*, Shailesh Kumar Yadav, Ajay Kumar, Amit sankar Dutta R.K.Pharmacy College, Sathion, Azamgarh, Uttar Pradesh * Corresponding author:Email:Sandeepcyra07@gmail.com ABSTRACT Resealed Erythrocytes are of appropriate size and shape to carry drugs. They are biocompatible and they have minimum toxic side effects. Minimum leakage can be observed before they reach the target site and they are able to carry broad spectrum of drugs. Carrier erythrocytes and resealed erythrocytes are being used for continuous implementation of safe and effective delivery of various drugs for passive and active targeting. Further optimization is required to become a routine drug delivery system which has been extended to the delivery of biopharmaceuticals and to be explored continuously regarding the potential of encapsulated erythrocytes. Resealed erythrocytes and loaded erythrocytes allow controlled drug release and increased specificity of delivery to the targeted organ. This review focus on various methods of encapsulated erythrocytes preparation, techniques of drug loading such as electroencapsulation, chemical perturbation, entrapment by endocytosis etc, mechanism of resealed erythrocyte release and evaluation resealed erythrocytes. Keywords: Resealed erythrocytes, controlled drug release, electro-encapsulation, chemical perturbation. 1. INTRODUCTION Drug delivery is now entering quite an exciting and challenging era. Significant high costs involved in the development of new drug molecule has compelled scientists all over the world to search for alternative ways of administering the existing drug molecules with enhanced effectiveness. Improper drug administration inside the biological system not only causes distress to other body tissues but also demands more therapeutic molecules to elicit the appropriate response. Among the various carriers used for targeting drugs to various body tissues, the cellular carriers meet several criteria desirable in clinical applications, among the most important being biocompatibility of carrier and its degradation products. Leucocytes, platelets, erythrocytes, nanoerythrocytes, hepatocytes, and fibroblasts etc. have been proposed as cellular carrier systems (Gothoskar AV, 2004) (Rossi L, 1996). Among these, the erythrocytes have been the most investigated and have found to possess greater potential in drug delivery. Therapeutic uses of a variety of drug carrier systems have significant impact on the treatment and potential cure of many chronic diseases, including cancer, diabetes mellitus, rheumatoid arthritis, HIV infection, and drug addiction. Erythrocytes are natural products of the body, biodegradable in nature, isolation of these is easy and large amount of drug can be loaded in small volume of cells, non immunogenic in action and can be targeted to disease tissue or organ, prolong the systemic activity of the drug while residing for a longer time in the body (Lejeune A, 1997), protest the premature degradation, inactivation and excretion of proteins and enzymes, act as a carrier for number of drugs, target the drugs within the reticuloendothelial system (RES) as well non RES organs/sites. Moreover, the possibility of targeting carrier erythrocytes to non-RES organs has been exploited in recent years, e.g., using homing devices such as IgG or IgM. Also these cells are non-immunogenic and biodegradable; they freely circulate throughout the body and offer ease of preparation; they have the capacity to carry large amounts of drug; and can behave as a slow-release long-acting system (Al-Achi A, 1990). Also, lungs to tissues and the CO2 produced in tissues back to lungs. Thus, erythrocytes are a highly specialized O 2 carrier system in the body. Because as aging erythrocytes are normally phagocytized by cells of the reticuloendothelial system, thus, these cells could serve as a natural target for delivery of their pay load to these organs. Potential clinical indications for RES targeting include iron over-storage diseases, parasitic diseases, hepatic tumors (Jain S, 1997) (Jain S, 1995) (Guyton CA, 1996) and lysosomal storage diseases. Erythrocytes, also known as red blood cells, have been extensively studied for their potential carrier capabilities for the delivery of drugs and drug-loaded microspheres. Such drug-loaded carrier erythrocytes are prepared simply by collecting blood samples from the organism of interest, separating erythrocytes from plasma, entrapping drug in the erythrocytes, and resealing the resultant cellular carriers. Hence, these carriers are called resealed erythrocytes. Upon re injection, the drug-loaded erythrocytes serve as slow circulating depots and target the drugs to a reticuloendothelial system (RES). Disadvantages include the possibility of leakage of drug from the cells & dose dumping. 2. SOURCE AND ISOLATION OF ERYTHROCYTES Various types of mammalian erythrocytes have been used for drug delivery, including erythrocytes of mice, cattle, pigs, dogs, sheep, goats, monkeys, chicken, rats, and rabbits. To isolate erythrocytes, blood is collected in heparinized tubes by venipuncture (Gaudreault B, 1989). Fresh blood is typically used for loading purposes because the encapsulation efficiency of the erythrocytes isolated from fresh blood is higher than that of the aged blood. Freshly collected blood is immediately freezed to 4 0C and stored. It can be used for a period of two days. The erythrocytes are then harvested and washed by centrifugation. The washed cells are suspended in buffer
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solutions at various hematocrit values as desired and are often stored in acidcitratedextrose buffer at 40C for a period of 48 hr before use (Banker GS, Rhodes, 2002). 3. ERYTHROCYTES CAN BE USED AS CARRIERS IN TWO WAYS 3.1. Targeting particular tissue/organ: For targeting, only the erythrocyte membrane is used. This is obtained by splitting the cell in hypotonic solution and after introducing the drug into the cells, allowing them to reseal intospheres. Such erythrocytes are called Red cell ghosts. Ghosts do not remain in the circulation for a long time as they are quickly sequestered and rapidly phagocytosed by the reticuloendothelial cells in the liver and spleen. The disadvantage of using ghosts is that they can be targeted only to those tissues which contain phagocytic cells (liver and spleen) and not to all other tissues in the body. 3.2. Continuous or prolonged release of drugs: Alternatively, erythrocytes can be used as a continuous or prolonged release system, which provides prolonged drug action. There are different methods for encapsulation of drugs within erythrocytes. They remain in the circulation for prolonged periods of time (up to 120 days) and release the entrapped drug at a slow and steady rate (Mitchell DH, 1990). 4. VARIOUS METHODS OF PREPARATION OF RESEALED ERYTHROCYTES Methods of Drug loading in Resealed Erythrocytes
Membrane Perturbation
Electroencapsulation
Hypo osmotic Lysis
Lipid fusion endocytosis
Dilution Method
Dialysis method
Preswell method
Osmotic lysis method
When erythrocytes are osmotically lysed and then resealed, there is an exchange of intracellular and extracellular solutes. Therefore, a drug added during the lysis procedures will be encapsulated within the membrane envelope of erythrocytes. 4.1. Methods of drug loading: Several methods can be used to load drugs or other bioactive compounds in erythrocytes, including physical (electrical pulse method) osmosis-based systems, and chemical methods (chemical perturbation of the erythrocytes membrane). Irrespective of the method used, the optimal characteristics for the successful entrapment of the compound requires the drug to have a considerable degree of water solubility, resistance against degradation within erythrocytes, lack of physical or chemical interaction with erythrocyte membrane, and well-defined pharmacokinetic and pharmacodynamic properties (Pitt E, 1983). Table 1: Comparison of various hypo-osmotic lysis methods Method Percent Advantages Disadvantages loading Dilution 30-40% Fastest and simplest especially for low Entrapment efficiency is less method molecular weight drug Dialysis 30-45% Better in vivo survival of erythrocytes Time consuming hetero- genous size better structural integrity of membrane distribution of resealed erythrocytes Preswell dilution Isotonic Osmotic lysis 30-90% Good retention of cytoplasm constituents and good survival in vivo Better in vivo surveillance Impemeable only to large molecules, process is time consuming
4.2. Hypotonic hemolysis: This method is based on the ability of erythrocytes to undergo reversible swelling in a hypotonic solution. Erythrocytes have an exceptional capability for reversible shape changes with or without accompanying volume change and for reversible deformation under stress. An increase in volume leads to an initial change in the shape from biconcave to spherical. This change is attributable to the absence of superfluous membrane; hence the surface area of the cell is fixed. The cells assume a spherical shape to accommodate additional volume while keeping the surface area constant. The volume gain is ~25 50%. The cells can maintain their integrity up to a tonicity of ~150 milli osmoles /kg, above which the membrane ruptures, releasing the cellular contents. At this point (just before cell lysis), some transient pores of 200 500 are generated on the membrane.
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After cell lysis, cellular contents are depleted. The remnant is called an erythrocyte ghost. The principle of using these ruptured erythrocytes as drug carriers is based on the fact that the ruptured membranes can be resealed by restoring isotonic conditions. Upon incubation, the cells resume their original biconcave shape and recover original impermeability (Alvarez, F.J., 1998). 4.3. Use of red cell loader: Novel methods of entrapment of non-diffusible drugs are used to load in to erythrocytes. Piece of equipment called a red cell loader. With as little as 50 mL of a blood sample, different biologically active compounds were entrapped into erythrocytes within a period of 2h at room temperature. The process is based on two sequential hypotonic dilutions of washed erythrocytes followed by concentration with a hemofilter and an isotonic resealing of the cells. There was ~30% drug loading with 3550% cell recovery. The processed erythrocytes had normal survival in vivo. The same cells could be used for targeting by improving their recognition by tissue macrophages (Gutierrez Millan, C, 2004). 4.4. Hypotonic dilution: Hypotonic dilution was the first method investigated for the encapsulation of chemicals into erythrocytes and is the simplest and fastest. In this method, a volume of packed erythrocytes is diluted with 2 20 volumes of aqueous solution of a drug. The solution tonicity is then restored by adding a hypertonic buffer. The resultant mixture is then centrifuged, the supernatant is discarded, and the pellet is washed with isotonic buffer solution. The major drawbacks of this method include low entrapment efficiency and a considerable loss of hemoglobin and other cell components. This reduces the circulation half life of the loaded cells. These cells are readily phagocytosed by RES macrophages and hence can be used for targeting RES organs. Hypotonic dilution is used for loading enzymes such as - galactosidase and - glycosidase, asparginase,and arginase, as well as bronchodilators such as salbutamol (Tajerzadeh, H., 2000). 4.4. Hypotonic dialysis: Several methods are based on the principle that semipermeable dialysis membrane maximizes the intracellular: extracellular volume ratio for macromolecules during lysis and resealing. In the process, an isotonic, buffered suspension of erythrocytes with a hematocrit value of 70 80 is prepared and placed in a conventional dialysis tube immersed in 1020 volumes of a hypotonic buffer. The medium is agitated slowly for 2 h. The tonicity of the dialysis tube is restored by directly adding a calculated amount of a hypertonic buffer to the surrounding medium or by replacing the surrounding medium by isotonic buffer. The drug to be loaded can be added by either dissolving the drug in isotonic cell suspending buffer inside a dialysis bag at the beginning of the experiment or by adding the drug to a dialysis bag after the stirring is complete. In this method, the erythrocyte suspension and the drug to be loaded were placed in the blood compartment and the hypotonic buffer was placed in a receptor compartment. This led to the concept of continuous flow dialysis, which has been used by several other researchers. This method has been used for loading enzymes such as -galactosidase, glucoserebrosidase,asparginase, inositol hexaphosphatase, as well as drugs such as Gentamicin, Adriamycin, Pentamidine and Furamycin, Interlukin-2, Desferroxamine, and human recombinant erythropoietin (Hamidi M, 2003). 4.5. Hypotonic preswelling: This method is simpler and faster than other methods causing minimum damage to cells, drug in capsulated in erythrocytes. This method was developed by Rechsteiner in1975 and was modified by Jenner et al. for drug loading. The technique is based upon initial controlled swelling in a hypotonic buffered solution. This mixture is centrifuged at low g values. The supernatant is discarded and the cell fraction is brought to the lysis point by adding 100120L portions of an aqueous solution of the drug to be encapsulated. The mixture is centrifuged between the drug-addition steps. The lysis point is detected by the disappearance of a distinct boundary between the cell fraction and the supernatant upon centrifugation. The tonicity of a cell mixture is restored at the lysis point by adding a calculated amount of hypertonic buffer. Then, the cell suspension is incubated at 37 C to re anneal the resealed erythrocytes. Such cells have a circulation half life comparable to that of normal cells. Drugs encapsulated in erythrocytes using this method include Propranolol, Methotrexate, Insulin, Metronidazole, Levothyroxine, Elaprnailat, and Isoniazid (Jaitely V, 1996). 4.6. Isotonic osmotic lysis: This method, also known as the osmotic pulse method, involves isotonic hemolysis that is achieved by physical or chemical means. The isotonic solutions may or may not be isoionic. If erythrocytes are incubated in solutions of a substance with high membrane permeability, the solute will diffuse into the cells because of the concentration gradient. This process is followed by an influx of water to maintain osmotic equilibrium. Chemicals such as urea solution, polyethylene glycol, and ammonium chloride have been used for isotonic hemolysis. However, this method also is not immune to changes in membrane structure composition. In 1987, Franco et al. developed a method that involved suspending erythrocytes in an isotonic solution of dimethyl sulfoxide (DMSO). The suspension was diluted with an isotonic-buffered drug solution. After the cells were separated, they were resealed at 37oC (Pei L, 1994). 4.7. Chemical perturbation of the membrane: This method is based on the increase in membrane permeability of erythrocytes when the cells are exposed to certain chemicals. In 1973, Deuticke et al showed that the permeability of erythrocytic membrane increases upon exposure to polyene antibiotic such as amphotericin B. In 1980, this method was used successfully by Kitao and Hattori to entrap the antineoplastic drug Daunomycin in human and
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mouse erythrocytes. Lin et al used halothane for the same purpose. However, these methods induce irreversible destructive changes in the cell membrane and hence are not very popular (Li, L.H., 1996). 4.8. Electro-insertion or electro encapsulation: In 1973, Zimmermann tried an electrical pulse method to encapsulate bioactive molecules. Also known as electroporation, the method is based on the observation that electrical shock brings about irreversible changes in an erythrocyte membrane. In 1977, Tsong and Kinosita suggested the use of transient electrolysis to generate desirable membrane permeability for drug loading. The erythrocyte membrane is opened by a dielectric breakdown. Subsequently, the pores can be resealed by incubation at 37 oC in an isotonic medium. The procedure involves suspending erythrocytes in an isotonic buffer in an electrical discharge chamber. A capacitor in an external circuit is charged to a definite voltage and then discharged within a definite time interval through cell suspension to produce a square-wave potential. The optimum intensity of an electric field is between 110 kW/cm and optimal discharge time is between 20-160s.An inverse relationship exists between the electric-field intensity and the discharge time. The compound to be entrapped is added to the medium in which the cells are suspended from the commencement of the experiment. The characteristic pore diameter created in the membrane depends upon the intensity of electric field, the discharge time, and the ionic strength of suspending medium. The colloidal macromolecules contents of the cell may lead to cell lysis because of the increase in osmotic pressure. This process can be prevented by adding large molecules (e.g., tetrasaccharide stachyose and bovine serum albumin) and ribonucleose.One advantage of this method is a more uniform distribution of loaded cells in comparison with osmotic methods. The main drawbacks are the need for special instrumentation and the sophistication of the process. Entrapment efficiency of this method is 35%, and the life span of the resealed cells in circulation is comparable with that of normal cells. Various compounds such as sucrose, urease, Methotrexate, Isoniazid, DNA fragments, and latex particles of diameter 0.2m can be entrapped within erythrocytes by this method. Mangal and Kaur achieved sustained release of a drug entrapped in erythrocytes with the use of electroporation. 4.9. Entrapment by endocytosis: This method was reported by Schrier et al. in1975. Endocytosis involves the addition of one volume of washed erythrocytes to nine volumes of buffer containing 2.5 mM ATP, 2.5 mM MgCl2, and 1mM CaCl2, followed by incubation for 2 min at room temperature. The pores created by this method are resealed by using 154 mM of NaCl and incubation at 37oC for 2 min. The entrapment of material occurs by endocytosis. The vesicle membrane separates endocytosed material from cytoplasm thus protecting it from the erythrocytes and vice-versa. The various candidates entrapped by this method include primaquine and related 8 aminoquinolines, Vinblastine, Chlorpromazine and related Phenothiazines, Hydrocortisone, Propranolol, Tetracaine, and Vitamin A (Alvarez-Guerra M, 1998). 4.10. Loading by electric cell fusion: This method involves the initial loading of drug molecules into erythrocyte ghosts followed by adhesion of these cells to target cells. The fusion is accentuated by the application of an electric pulse, which causes the release of an entrapped molecule. An example of this method is loading a cell-specific monoclonal antibody into an erythrocyte ghost. An antibody against a specific surface protein of target cells can be chemically cross-linked to drug-loaded cells that would direct these cells to desired cells (Hamidi M, 2003). 4.11. Loading by lipid fusion: Lipid vesicles containing a drug can be directly fused to human erythrocytes, which lead to an exchange with a lipid-entrapped drug. This technique was used for entrapping Inositol monophosphate to improve the oxygen carrying capacity of cells. However, the entrapment efficiency of this method is very low (~1%). 5. EVALUATION 5.1. In-vitro properties of loaded erythrocytes 5.1.1. Cell counting and cell recovery: This involves counting the number of red blood cells per unit volume of whole blood, usually by automated counting. Red cell recovery may be calculated on the basis of the differences in the hematocrit and the volume of the suspension of erythrocytes before and after loading. The goal is to minimize the loss during the encapsulation procedure to maximize cell recovery (Hamidi M, 2001). 5.1.2. Morphological aspect: The morphological examination of these ghost erythrocytes is undertaken by comparison with untreated erythrocytes using either transmission (TEM) or scanning (SEM) electron microscopy. By means of electron microscopy observation may be made of the morphological changes in the erythrocytes induced by osmosis-based encapsulation methods, when they are subjected to solutions of different osmolality. Thus, when rat erythrocytes are subjected to isotonic solutions (300 milli osmoles / kg) they reveal the typical morphology of discocyte (biconcave). This evolves to a morphology of stomatocyte (uniconcave) when they are subjected to solutions of 200 milli osmoles/kg, attaining the spherocytic shape (the most fragile of the three) when the solution is of 150 mosM/kg (Gupta A, 2010). 5.1.3. Osmotic fragility: Osmotic fragility is a test to detect abnormal fragility of red blood cells. Untreated or loaded erythrocytes are tested by exposure to hypotonic solutions, making them swell, in order to determine the relative fragility of the red cells (Gupta A, 2010).
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5.1.4. Turbulence shock: Turbulence shock enables an evaluation to be made of the stability of the loaded erythrocytes against the turbulence stress exerted by the cells against in-vivo circulation turbulence (De Loach R, 1983) (Gupta A, 2010). Where by the suspension of cells are passed several times through a 22-gauge needle. 5.1.5. In-vitro drug or peptide release: The encapsulation of many drugs in erythrocytes can give rise to a sustained release of the drug that influences the pharmacokinetic behaviour in vivo of the loaded erythrocytes. Invitro leakage of the drug from loaded erythrocytes is tested using autologous plasma or an iso-osmotic buffer at 37C with a hematocrit adjusted between 0.5 % and 50 %. The supernatant is removed at the time intervals previously programmed and replaced by an equal volume of autologous plasma or buffer. Certain authors recommend performing in vitro the release studies from loaded erythrocytes using a dialysis bag. The molecular weight and liposolubility of the substance constitute two factors that have a decisive bearing on the release profile of the active principle from the loaded erythrocytes. Liposoluble drugs may be released from the red cells by a mechanism of passive diffusion. Other drugs may become attached to cell structures and are not released by the diffusion mechanism, requiring the lysis of the cell. Band 3 and glycophorin A are proteins present in high density on the extra-cellular surface of erythrocytes and which may act as potential targets for anchoring via covalent bond formation with different substances. Band 3 plays an important role as a carrier protein for anions (Jaitely V, 1996). 5.1.6. Haemoglobin release: The content of hemoglobin of the erythrocytes may be diminished by the alterations in the permeability of the membrane of the red cells during the encapsulation procedure. Furthermore, the relationship between the rate of hemoglobin and the rate of drug release contributes to interpreting the mechanisms involved in the release of the substance encapsulated from the erythrocytes. The hemoglobin leakage is tested using a red cell suspension by recording the absorbance of supernatant at 540 nm on a spectrophotometer (Vyas SP, 1999). 5.1.7. In vitro stability: The stability of the loaded erythrocytes is assessed by means of the incubation of the cells in autologous plasma or in an iso-osmotic buffer, setting the hematocrit between 0.5% and 5% at temperatures of 40 and 370C. The mechanism of resealed erythrocytes shows potential for a safer and sure delivery of various drugs for active and passive targeting. The authors have reviewed the explanations of the different method of drug loading and their characterization parameters for resealed erythrocytes. However more research and inputs are required for the cell based drug delivery systems to open a new perspective to the possibility of using cells for therapeutic purposes. The authors are confident that erythrocytes have great potentialities in the field of drug delivery, since the key to success of much therapeutics greatly depends on the development of novel technologies to improve and control the delivery of drugs (Kravtzoff R, 1990). 5.1.8. In vitro characterization: The in vivo performance of resealed erythrocytes is affected to a great extent by their biological properties. Hence, in vitro characterization forms an important part of studies involving such cellular carriers. 5.1.8.1. Physical characterization: Shape, surface morphology, vesicle size and size distribution of drug loaded erythrocytes can be studied by using transmission electron microscopy, scanning electron microscopy, phase contrast microscopy, optical microscopy (DeLoach, 1983). Drug release from the erythrocytes can be carried using diffusion cell method. Surface charge of the erythrocytes can be measured by zeta potential measurement (Vyas SP and Khar RK, 2002). 5.1.8.2. Cellular characterization: Percentage Hb content is determined by deproteinization of cell membrane followed by hemoglobin assay. Percentage cell recovery is carried out by Neubaurs chamber and hematological analyzer. Osmotic fragility test involves stepwise incubation with isotonic to hypotonic saline solutions and determination of drug and hemoglobin assay. Osmotic shock is determined by dilution with distilled water and estimation of drug and hemoglobin. Turbulent shock is determined by Passage of cell suspension through 30gauge hypodermic needle at 10 mL/min flow rate and estimation of residual drug and hemoglobin, vigorous shaking followed by hemoglobin estimation Erythrocyte sedimentation rate by ESR methods (Schrier SL, 1975). 5.2. Biological characterization: Biological characterization of the developed erythrocytes includes sterility test, pyrogenicity test, LAL test and toxicity tests. The morphology of erythrocytes decides their life span after administration. Light microscopy reveals no observable change in resealed cells but in few cases spherical erythrocytes (spherocytes) are detected. Scanning electron microscopic studies will show that a majority of the cells maintain their biconcave discoid shapes after the loading procedure, and few stomatocytes, a form of spherocytes. In some cases, cells of smaller size (microcyte) are also observed. Shape change (deformability) is another factor that affects the life span of the cells. This parameter evaluates the ease of passage of erythrocytes through narrow capillaries and the RES. It determines the rheological behavior of the cells and depends on the viscoelasticity of the cell membrane, viscosity of the cell contents, and the cellular surface-to-volume ratio. The deformability is measured by passage time of definite volume of cells through capillary of 4 m diameter or polycarbonate filter with average pore size of 45 m. Another indirect approach is to evaluate chlorpromazine induced shape changes turbidimetrically. The osmotic fragility of resealed erythrocytes is an indicator of the possible changes in cell membrane integrity and the
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resistance of these cells to osmotic pressure of the suspension medium. The test is carried out by suspending cells in media of varying sodium chloride concentration and determining the hemoglobin released. In most cases, osmotic fragility of resealed cells is higher than that of the normal cells because of increased intracellular osmotic pressure. The turbulence fragility is yet another characteristic that depends upon changes in the integrity of cellular membrane and reflects resistance of loaded cells against hemolysis resulting from turbulent flow within circulation. It is determined by the passage of cell suspension through needles with smaller internal diameter(e.g., 30 gauges) or vigorously shaking the cell suspension. In both cases, hemoglobin and drug released after the procedure are determined. The turbulent fragility of resealed cells is found to be higher. Routine clinical hematological tests also can be carried out for drug-loaded cells, including mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin content. Studies have shown that the average size and hemoglobin content of resealed cells is lower than that of normal cells (Bhaskaran, S., 1995). 5.3. Drug release kinetics: The most important parameters for evaluation of resealed erythrocytes are the drug release pattern. Hemoglobin is also invariably released because drug release involves the loss of cell membrane integrity indicating hemolysis. On the basis of the various in vitro release experiments carried out on these cells, three general drug release patterns are observed: The rate of drug release is considerably higher than that of hemoglobin. In other words, drug diffuses readily. Such a pattern is shown by lipophilic drugs, including Methotrexate, Phenytoin, Dexamethasone, Primpquin, and Vitamin B12. Cell lysis is not essential for the release of such drugs. The rate of drug release is comparable to that of hemoglobin. This indicates that cell lysis is essential for drug release and drug cannot be released by mere diffusion. Polar drugs such as Gentamicin, Heparin, and Enalaprilat, and enzymes such as Asparginase, Peptides, including Urogasterone and l-lysine-lphenylalanine follow such pattern. The rate of drug release lies between the above mentioned two extremes; for example, propranolol, isoniazid, metronidazole, and recombinant human erythropoietin. The two factors that determine the drug release pattern are size and polarity of the drug molecule. The release rate can be modified by cross-linking cell membrane with gluteraldehyde, which results in a slower drug release. This can also be achieved by entrapping biodegradable prodrug such as o-acetyl propranolol, o-pivaloyl propranolol, cortisol-21- phosphate, prednisolone-21-sodium succinate, and cytosine arabinoside monophosphate. The complexation of a drug with macromolecules such as dextran and albumin also retard the release rate. 5.3.1. Mechanism of release of resealed erythrocytes: The rate of diffusion depends upon the rate at which a particular molecule penetrates through a lipid bilayer. Many substances enter cells by as pecific membrane protein system because the carriers are proteins with many properties analogous to that of enzymes, including specificity e.g. nucleotides and nucleosides. Release of drugs from erythrocytes is rapid followed by sustained release profile and rate of exit is proportional to the instantaneous intra cellular drug concentration (first order kinetics). By incorporating polymer into erythrocytes, the release pattern may be modified. The drug, however, could be resealed from macrophages after phagocytosis if the linkage is susceptible to lysosomal enzymes (Rossi, L., 2005). Phagocytosis Diffusion through the membrane of cell By using a specific transport system. 6. IN VITRO STORAGE The success of resealed erythrocytes as a drug delivery system depends to a greater extent on their in vitro storage. Preparing drug-loaded erythrocytes on a large scale and maintaining their survival and drug content can be achieved by using suitable storage methods. However, the lack of reliable and practical storage methods has been a limiting factor for the wide-spread clinical use of the carrier erythrocytes. The most common storage media include Hanks balanced salt solution and acidcitratedextrose at 4 oC. Cells remain viable in terms of their physiologic and carrier characteristics for at least 2 weeks at this temperature. The addition of calcium-chelating agents or the purine nucleosides improve circulation survival time of cells upon reinjection. Exposure of resealed erythrocytes to membrane stabilizing agents such as dimethyl sulfoxide, dimethyl, 3,3-di-thio-bispropionamide, gluteraldehyde, toluene-2-4-diisocyanate followed by lyophilization or sintered glass filtration has been reported to enhance their stability upon storage. The resultant powder was stable for at least one month without any detectable changes. But the major disadvantage of this method is the presence of appreciable amount of membrane stabilizers in bound form that remarkably reduces circulation survival time. Other reported methods for improving storage stability include encapsulation of a prodrug that undergoes conversion to the parent drug only at body temperature, high glycerol freezing technique, and reversible immobilization in alginate or gelatin gels (Price RJ, 1998). 7. CROSSLINKING, STABILITY AND IN-VIVO SURVIVAL OF RESEALED ERYTHROCYTES The cells treated with dimethyl sulphoxide (DMSO), toluene 2, 4-di-isocyanate (TD1) and gluteraldehyde are even resistant to sonication, freezing and thawing. Chemically cross linking of erythrocytes renders a yield of 55-97% of non-lysed cells. An attempt was made to get drug loaded cells in lyophilized form. The dried powder was filled in amber color glass vials and stored at 4C for one month. Improvement in shelf -life of the carrier
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erythrocytes was achieved by storing the cells in powder form ready for reconstitution at 4C. This is important in the large scale manufacturing of drug loaded erythrocytes (Mangal PC, 1991). 8. IN VIVO LIFE SPAN The efficacy of resealed erythrocytes is determined mainly by their survival time in circulation upon reinjection. For the purpose of sustained action, a longer life span is required, although for delivery to targetspecific RES organs, rapid phagocytosis and hence a shorter life span is desirable. The life span of resealed erythrocytes depends upon its size, shape, and surface electrical charge as well as the extent of hemoglobin and other cell constituents lost during the loading process. The various methods used to determine in vivo survival time include labeling of cells by 51Cr or fluorescent markers such as fluorescin isothiocyanate or entrapment of 14C sucrose or gentamicin. The circulation survival kinetics of resealed erythrocytes show typical bimodal behavior with a rapid loss of cells during the first 24 h after injection, followed by a slow decline phase with a half life on the order of days or weeks. The early loss accounts for ~15 65% loss of total injected cells. The erythrocytic carriers constructed of red blood cells of mice, cattle, pigs, dogs, sheep, goats, and monkeys exhibit a comparable circulation profile with that of normal unloaded erythrocytes. On the other hand, resealed erythrocytes prepared from red blood cells of rabbits, chickens, and rats exhibit relatively poor circulation profile (Talwar N, 1992). 9. APPLICATIONS OF RESEALED ERYTHROCYTES Resealed erythrocytes have several possible applications in various fields of human and veterinary medicine. Such cells could be used as circulating carriers to disseminate a drug within a prolonged period of time in circulation or in target-specific organs, including the liver, spleen, and lymph nodes. A majority of the drug delivery studies using drug-loaded erythrocytes are in the preclinical phase. In a few clinical studies, successful results were obtained (Moorjani M, 1996). 9.1. Slow drug release: Erythrocytes have been used as circulating depots for the sustained delivery of antineoplastics, antiparasitics, veterinary antiamoebics, vitamins, steroids, antibiotics, and cardiovascular drugs (Magnani M, 1998). 9.2. Drug targeting: Ideally, drug delivery should be site-specific and target-oriented to exhibit maximal therapeutic index with minimum adverse effects. Resealed erythrocytes can act as drug carriers and targeting tools as well. Surface-modified erythrocytes are used to target organs of mononuclear phagocytic system/ reticuloendothelial system because the changes in the membrane are recognized by macrophages. However, resealed erythrocytes also can be used to target organs other than those of RES (Hamidi M, 2007). 9.3. Targeting RES organs: Damaged erythrocytes are rapidly cleared from circulation by phagocytic Kupffer cells in liver and spleen. Resealed erythrocytes, by modifying their membranes, can therefore be used to target the liver and spleen. The various approaches to modify the surface characteristics of erythrocytes include Surface modification with antibodies, Gluteraldehyde, Sialic acid, Sulphydryl and Surface chemical cross-linking e.g. delivery of 125 I-labeled carbonic -anhydrase loaded in erythrocytes cross-linked with sulfosuccinmidyl propionate (Hamidi M, 2001). 9.4. Targeting the liver- enzyme deficiency/replacement therapy: Many metabolic disorders related to deficient or missing enzymes can be treated by injecting these enzymes. However, the problems of exogenous enzyme therapy include a shorter circulation half life of enzymes, allergic reactions, and toxic manifestations. These problems can be successfully overcome by administering the enzymes as resealed erythrocytes. The enzymes used include -glucosidase, -glucoronidase, -galactosidase. The disease caused by an accumulation of glucocerebrosides in the liver and spleen can be treated by glucocerebrosidase- loaded erythrocytes (Hamidi M, 2001). 9.5. Treatment of hepatic tumors: Hepatic tumors are one of the most prevalent types of cancer. Antineoplastic drugs such as methotrexate, bleomycin, asparginase and adriamycin have been successfully delivered by erythrocytes. Agents such as daunorubicin diffuse rapidly from the cells upon loading and hence pose a problem. This problem can be overcome by covalently linking daunorubicin to the erythrocytic membrane using gluteraldehyde or cis-aconitic acid as a spacer. The resealed erythrocytes loaded with carboplatin show localization in liver (Hamidi M, 2001). 9.6. Treatment of parasitic diseases: The ability of resealed erythrocytes to selectively accumulate within RES organs make them useful tool during the delivery of antiparasitic agents. Parasitic diseases that involve harboring parasites in the RES organs can be successfully controlled by this method. Results were favorable in studies involving animal models for erythrocytes loaded with antimalarial, antileishmanial and antiamoebic drugs. 9.7. Removal of res iron overload: Desferrioxamine-loaded erythrocytes have been used to treat excess iron accumulated because of multiple transfusions to thalassemic patients. Targeting this drug to the RES is very beneficial because the aged erythrocytes are destroyed in RES organs, which results in an accumulation of iron in these organs.
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9.8. Removal of toxic agents: Cannon et al. reported inhibition of cyanide intoxication with murine carrier erythrocytes containing bovine rhodanase and sodium thiosulfate. Antagonization of organophosphorus intoxication by resealed erythrocytes containing a recombinant phosphodiestrase also has been reported. 9.9. Targeting organs other than those of RES: Recently, resealed erythrocytes have been used to target organs outside the RES. The various approaches include Entrapment of paramagnetic particles, photosensitive material along with the drug and Antibody attachment to erythrocyte membrane to get specificity of action. Zimmermann proposed that the entrapment of small paramagnetic particles into erythrocytes might allow their localization to a particular location under the influence of an external magnetic field. Jain and Vyas reported entrapment of the antiinflammatory drugs Diclofenac sodium and Ibuprofen in magnetoresponsive erythrocytes. Photosensitized erythrocytes have been studied as a phototriggered carrier and delivery system for Methotrexate in cancer treatment. Rossi L et al have reported in-vitro targeting of erythrocytes to cytotoxic T-cells by coupling of Thy-1.2 monoclonal antibody. Price et al. reported delivery of colloidal particles and erythrocytes to tissue through microvessel ruptures created by targeted microbubble destruction with ultrasound. IV fluorescent erythrocytes were delivered to the interstitium of rat skeletal muscle through microvessel ruptures by insonifying microbubbles in vivo. This technique provides a noninvasive means for delivering resealed erythrocytes across the endothelial carrier to the target tissue. Other approaches for targeting organs outside the RES include the preparation of carrier erythrocytes fused to thermoresponsive liposomes and their localization using an external thermal source, intraperitoneal injection of resealed erythrocytes for drug targeting to peritoneal macrophages, and lectin pretreatment of resealed cells loaded with antineoplastic drugs to improve targeting tumor cells. 9.10. Delivery of antiviral agents: Several reports have been cited in the literature about antiviral agents entrapped in resealed erythrocytes for effective delivery and targeting. Because most antiviral drugs are nucleotides or nucleoside analogs, their entrapment and exit through the membrane needs careful consideration. Nucleosides are rapidly transported across the membrane where as nucleotides are not, and thus exhibiting prolonged release profiles. The release of nucleotides requires conversion of these moieties to purine or pyrimidine bases. Resealed erythrocytes have been used to deliver Deoxycytidine derivatives, recombinant herpes simplex virus type 1 (HSV1) glycoprotein B, Azidothymidine derivatives, Azathioprene, Acyclovir, and Fludarabine phosphate. 9.11. Enzyme therapy: Enzymes are widely used in clinical practice as replacement therapies to treat diseases associated with their deficiency (e.g., Gauchers disease,galactosuria), degradation of toxic co mpounds secondary to some kind of poisoning (cyanide, organophosphorus), and as drugs. The problems involved in the direct injection of enzymes into the body have been cited. One method to overcome these problems is the use of enzymeloaded erythrocytes. T hese cells then release enzymes into circulation upon hemolysis act as a circulating bioreactors in which substrates enter into the cell, interact with enzymes, and generate products or accumulate enzymes in RES upon hemolysis for future catalysis. The first report of successful clinical trials of the resealed erythrocytes loaded with enzymes for replacement therapy is that of -glucoserebrosidase for the treatment of Gauchers disease. The disease is characterized by inborn deficiency of lysosomal -glucoserebrosidase in cells of RES there by leading to accumulation of -glucoserebrosides in macrophages of the RES. The most important application of resealed erythrocytes in enzyme therapy is that of asparginase loading for the treatment of pediatric neoplasms. This enzyme degrades aspargine, an amino acid vital for cells. This treatment prevents remission of pediatric acute lymphocytic leukemia. There are reports of improved intensity and duration of action in animal models as well as humans. To treat lead poisoning, the concentration of -aminolevulinate dehydrogenase (ALAD) in erythrocytes decreases. This leads to an accumulation of -aminolevulinic acid in tissues, blood, and urine. This state leads to acute porphyria and CNS related problems. An injection of resealed erythrocytes loaded with ALAD to lead intoxicated animal significantly reduces toxic manifestations. Other enzymes used for loading resealed erythrocytes include urease, galactose-1-phosphate uridyl transferase, uricase and acetaldehyde dehydrogenase. 9.12. Improvement in oxygen delivery to tissues: Hemoglobin is the protein responsible for the oxygen-carrying capacity of erythrocytes. Under normal conditions, 95% of hemoglobin is saturated with oxygen in the lungs, whereas under physiologic conditions in peripheral blood stream only ~25% of oxygenated hemoglobin becomes deoxygenated. Thus, the major fraction of oxygen bound to hemoglobin is recirculated with venous blood to the lungs. The use of this bound fraction has been suggested for the treatment of oxygen deficiency. 2, 3Diphosphoglycerate (2, 3-DPG) is a natural effector of hemoglobin. The binding affinity of hemoglobin for oxygen changes reversibly with changes in intracellular concentration of 2, 3-DPG. This compensates for changes in the oxygen pressure outside of the body, as the affinity of 2, 3-DPG to oxygen is much higher than that of hemoglobin. Other organic polyphosphates can serve as allosteric effectors of hemoglobin with binding affinities higher than those of 2, 3-DPG and can compete with 2, 3-DPG for binding to hemoglobin. Inositol hexophosphate (IHP) is one of the strongest effectors of this type. However, because of its ionization at physiologic pH, it cannot enter erythrocytes. Hence, it is entrapped by the electroporation process. Upon encapsulation, IHP irreversibly binds to hemoglobin, thereby decreasing the oxygen affinity to hemoglobin and subsequent shift of oxygen binding isotherm to the right. As a result, the oxygen pressure corresponding to 50% of the total binding capacity of
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hemoglobin to oxygen (P50 value) increases from 2627 mm Hg to 50mm Hg. In the presence of IHP encapsulated in erythrocytes, the difference between the oxygen bound fraction of hemoglobin in lungs and tissues increases, thereby increasing the oxygen concentration in tissues. Also, the extent of carbamate formed in the N-terminal amine group of -chain of hemoglobin decreases, which is compensated by an uptake of H + and CO2 that leads to increased formation of bicarbonate ion. Intravenous (IV) injection of IHP-loaded erythrocytes to piglets led to a decrease in cardiac output with a constant oxygen consumption by animals. This indicates that because of an increased extraction ratio of oxygen by tissues, a given amount of oxygen can be delivered in lower blood flow. In addition, these erythrocytes reduce ejection fraction, left ventricular diastolic volume, and heart rate. An isolated perfused-heart model showed reduction in coronary blood flow with increased oxygen consumption by myocardium upon administration of IHP-loaded erythrocytes. The same results are reported when intact animal models were used. An application of IHP-loaded erythrocytes for improved oxygen supply is beneficial under the following conditions, such as high altitude conditions where the partial pressure of oxygen is low, reduction in the number of alveoli, where exchange surface of the lungs is decreased, increased resistance to oxygen diffusion in the lungs, reduction in oxygen transport capacity, mutation or chemical modification, which involves a decrease in oxygen affinity for hemoglobin, increased radiosensitivity of radiation-sensitive tumors, restoration of oxygen-delivery capacity of stored blood and ischemia of myocardium, brain, or other tissues. 9.13. Microinjection of macromolecules: Biological functions of macromolecules such as DNA, RNA, and proteins are exploited for various cell biological applications. Hence, various methods are used to entrap these macromolecules into cultured cells (e.g., microinjection). A relatively simple structure and a lack of complex cellular components (e.g., nucleus) in erythrocytes make them good candidates for the entrapment of macromolecules.In microinjection, erythrocytes are used as microsyringes for injection to the host cells. The microinjection process involves culturing host eukaryotic cells in vitro. The cells are coated with fusogenic agent and then suspended with erythrocytes loaded with the compound of interest in an isotonic medium. Sendai virus (hemagglutinating virus of Japan, HVJ) or its glycoproteins or polyethylene glycol have been used as fusogenic agents. The fusogen causes fusion of cosuspended erythrocytes and eukaryotic cells. Thus, the contents of resealed erythrocytes and the compound of interest are transferred to host cell. This procedure has been used to microinject DNA fragments, arginase,proteins, nucleic acids, ferritin, latex particles,bovine and human serum albumin, and enzyme thymidine kinase to various eukaryotic cells. Advantages of this method include quantitative injection of materials into cells, simultaneous introduction of several materials into a large number of cells, minimal damage to the cell, avoidance of degradation effects of lysosomal enzymes and simplicity of the technique. Disadvantages include a need for a larger size of fused cells, thus making them amenable to RES clearance, adverse effects of fusogens and unpredictable effects on cell resulting from the co-introduction of various components. Hence, this method is limited to mainly cell biological applications rather than drug delivery (Kravtzoff R, 1990). 10. NOVEL APPROACHES 10.1. Erythrosomes: These are specially engineered vesicular systems that are chemically cross-linked to human erythrocytes support upon which a lipid bilayer is coated (Vyas SP and Dixit VK, 1999). This process is achieved by modifying a reverse-phase evaporation technique. These vesicles have been proposed as useful encapsulation systems for macromolecular drugs. 10.2. Nanoerythrosomes: These are prepared by extrusion of erythrocyte ghosts to produce small vesicles with an average diameter of 100 nm (Jaitely V, 1996). Daunorubicin was covalently conjugated to nanoerythrosomes using gluteraldehyde spacer. This complex was more active than free Daunorubicin alone, both in vitro and in vivo. 11. CONCLUSION The use of resealed erythrocytes looks promising for a safe and effective delivery of various drugs for passive and active targeting. However, the concept needs further optimization to become routine drug delivery system. The same concept also can be extended to the delivery of biopharmaceuticals and much remains to be explored regarding the potential of resealed erythrocytes.
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REFERENCES Al-Achi A and Boroujerdi M, Pharmacokinetics and tissue uptake ofdoxorubicin associated with erythrocytemembrane: erythrocytes-ghosts vs erythrocytes-vesicles, Dev Ind Pharm, 16, 1990, 21992219. Alpar HO and Irwin WJ, Some Unique Applications of Erythrocytes as Carrier Systems, Adv Biosci, 67, 1987, 19. Alvarez FJ, Jordn JA, Calleja P, Lotero LA, Olmos G, Dez JC, Tejedor MC, Cross-Linking Treatment of Loaded Erythrocytes Increases delivery of Encapsulated Substance to Macrophages, Biotechnol Appl Biochem, 27, 1998, 139143. Alvarez Guerra M, Nazaret C and Garay RP, The Erythrocyte Na, K, Cl CoTransporter and Its Circulating Inhibitor in Dahl Salt- Sensitive rats, J Hypertens, 10, 1998, 14991504. Bhaskaran S and Dhir SS, Resealed Erythrocytes as Carriers of Salbutamol Sulphate, Indian J Pharm Sci, 57, 1995, 240242. De Loach R, Encapsulation of Exogenous Agents in Erythrocytes and the Circulating Survival of Carrier Erythrocytes, J Appl Biochem, 5 (3), 1983, 149157. Fraternale A, Rossi L and Magnani M, Encapsulation, Metabolism, and Release of 2-Fluoro-Ara-AMP from Human Erythrocytes, Biochim Biophys Acta, 1291(2), 1996, 149154. Gaudreault B, Bellemare, Lacroix J, Erythrocyte Membrane Bound Daunorubicin as a Delivery System in Anticancer Treatment, Anticancer Res, 9 (4), 1989, 1201-1205. Gothoskar AV, Resealed erythrocytes: A Review, Pharm Tech, March 2004, 140-158. Gupta A, Cell Based Drug Delivery system Through Resealed Erythrocytes: A Review, Int J Parma Sci and Drug Research, 2(1), 2010, 23-30. Gutierrez Millan C, Zarzuelo Castaneda A, Sayalero Marinero ML, Lanao JM, Factors associated with the performance of carrier erythrocytesobtainedby hypotonic dialysis, Blood Cells Mol Dis, 33, 2004, 132140. Guyton CA and Hall JE, Red Blood Cells, Anemia and Polycytemia, Textbook of Medical Physiology (W.B. Saunders, Philadelphia, PA, 1996, 425433. Hamidi M and Tajerzadeh H, Carrier Erythrocytes: An Overview, Drug Delivery, 10, 2003, 920. Hamidi M, Tajerzadeh H, Carrier erythrocytes: an overview, Drug Deliv, 10, 2003, 920. Hamidi M, Tajerzadeh H, Dehpour AR, Ejtemaee Mehr S, ACE Inhibitionin Rabbits Upon Administration of Enalaprilat Loaded Intact Erythrocytes, J Pharm Pharmacol, 53, 2001, 12811289. Hamidi M, Tajerzadeh H, Dehpour AR, Rouini MR, Ejtemaee Mehr S, In vitro Characterization of Human Intact Erythrocytes Loaded by Enalaprilat, DrugDelivery 8, 2001, 231237. Hamidi M, Zarei N, Zarrin AH, Mohammadi Samani S, Preparationand in vitro characterization of carriererythrocytes for vaccine delivery, Int. J.Pharm, 338, 2007, 7078. Jain S and Jain N K, Engineered Erythrocytes as a Drug Delivery System, Indian J Pharm Sci, 59, 275281. Jain S, Jain SK, Dixit VK, Erythrocytes Based Delivery of Isoniazid: Preparation and In Vitro Characterization, Indian Drugs 32, 1997, 471476. Jain S, Jain SK, Dixit VK, Magnetically Guided Rat Erythrocytes Bearing Isoniazid: Preparation, Characterization, and Evaluation, Drug Dev Ind Pharm, 23, 1997, 999-1006. Jain SK and Vyas SP, Magnetically Responsive Diclofenac Sodium- Loaded Erythrocytes: Preparation and InVitro Characterization, J Microencapsul, 11 (2): 1994, 141151. Jaitely V, Kanaujia P, Venkatesan N, Jain S, Vyas SP, Resealed erythrocytes:carrier potentials and biomedical application.Indian Drugs, 33, 1996, 549589. Jaitely V, Kanaujia P, Vyas SP, Resealed Erythrocytes: Drug Carrier Potentials and Biomedical Applications, Indian Drugs, 33, 1996, 589594. Kravtzoff R, Ropars C, Laguerre M, Muh JP, Chassaigne M, Erythrocytes as Carriers for L-Asparaginase: Methodological and Mouse In-Vivo Studies, J Pharm Pharmacol, 42, 1990, 473476.
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Lejeune A, Poyet P, Gaudreault RC, Gicquaud C, Nanoerythrosomes, A New Derivative of Erythrocyte Ghost: III. Is Phagocytosis Involved in the Mechanism of Action? Anticancer Res, 17, 1997, 5A. Lewis DA, Red Blood Cells for Drug Delivery, Pharm J, 233, 1984, 384385. Li LH, Hensen ML, Zhao YL, Hui SW, Electrofusion between Heterogeneous Sized Mammalian Cells in a Pellet: Potential Applications in Drug Delivery and Hybridoma Formation, Biophys J, 71 (1), 1996, 479-486. Magnani M and Rossi L, Erythrocyte Engineering for Drug Deliveryand Targeting, Biochem, 28, 1998, 113. Biotechnol, Appl
Magnani M, Bianchi M, Rossi L, Stocchi V, Acetaldehyde Dehydrogenase Loaded Erythrocytes as Bioreactors forRemoval of Blood Acetaldehyde, Alcoholism, Clin Exp Res, 13, 1989, 849859. Mangal PC, and Kaur A, Electroporation of Red Blood Cell Membrane and its Use as a Drug Carrier System, Ind J Biochem, Biophys, 28 (3), 1991, 219-221. Mitchell DH, James GT, and Kruse CA, Bioactivity of Electric Field-Pulsed Human Recombinant Interleukin2 and Its Encapsulation into Erythrocyte Carriers, Biotechnol Appl Biochem, 12(3), 1990, 264275. Moorjani M, Lejeune A, Gicquaud C, Lacroix J, Poyet P, Gaudreault RC, Nanoerythrosomes, A NewDerivative of Erythrocyte Ghost II: Identification of the Mechanism of Action, Anticancer Res, 16 (5), 1996, 28312836. Pei L, Omburo G, Mc Guinn WD, Petrikovics I, Dave K, Raushel FM, Wild JR, De Loach JR, Way JL. Encapsulation of Phosphotriesterase within Murine Erythrocytes, Toxicol Appl Pharmacol, 124 (2), 1994, 296301. Pitt E, Johnson CM, Lewis DA, Jenner DA, Offord RE, Encapsulation of Drugs in Intact Erythrocytes: An Intravenous Delivery System, Biochem Pharmacol, 22, 1983, 33593368. Price, R. J., Skyba, D.M., Kaul, S., Skalak,T.C. Delivery of Colloidal Particlesand Red Blood Cells to Tissue throughMicrovessel Ruptures Created by TargetedMicrobubble Destruction with Ultrasound,Circulation 98(13), 1998, 12641267. Rossi L, Serafini S, Pierige F, Antonelli A, Cerasi A, Fraternale A, Chiarantini L and Magnani M, Erythrocytebaseddelivery, Exp Opin Deliv, 2, 2005, 311322. Schrier SL, Junga I, Johnson M, Energized Endocytosis in HumanErythrocyte Ghosts, J Clin Invest, 56 (1), 1975, 822. Tajerzadeh H and Hamidi M, Evaluation of the Hypotonic PreswellingMethod for Encapsulation of Enalaprilat inHuman Intact Erythrocytes, Drug Dev Ind Pharm, 26, 2000, 12471257. Talwar N and Jain NK, Erythrocytes as Carriers of Metronidazole: In-Vitro Characterization, Drug Dev Ind, Pharm, 18, 1992, 17991812. Talwar N and Jain NK, Erythrocytes as Carriers of Primaquin Preparation: Characterization and Evaluation, J Controlled Release, 20, 1992, 133142. Torotra GJ and Grabowski SR, The Cardiovascular System: The Blood, Principles of Anatomy and Physiology Harper Collins College Publishers, New York , NY , 7th ed, 1993, 566590 Vyas SP and Dixit VK, Pharmaceutical Biotechnology, CBSPublishers & Distributors, New Delhi, 1999, 655. Vyas SP and Khar RK, Resealed Erythrocytes in Targeted and Controlled Drug Delivery: Novel Carrier Systems, CBS Publishers and Distributors, India, 2002, 87416.
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EVALUATION OF ANTI-DIARRHOEAL ACTIVITY OF ETHANOLIC LEAF EXTRACT OF SCOPARIA DULCIS LINN ON WISTER ALBINO RATS
Amitabha Dey1,2, Ghanshyam Panigrahi1, Lokesh Deb2,*, Kh. Nongalleima 2, Pratap Patra3 1. Department of pharmacology, Royal College of Pharmacy and Health Sciences, Berhampur, Odisha, India. 2. Pharmacology Laboratory, Medicinal Plants & Horticultural Resources Division, Institute of Bioresources & Sustainable Development, Department of Biotechnology, Government of India, Takyelpat, Imphal, Manipur, India. 3. College of Pharmaceutical Sciences, Mohuda, Berhampur (Ganjam), Odisha -760001, India. *Corresponding address: Email: lokeshdeb.ibsd@nic.in ABSTRACT The objective of the study was to find out the ethanolic extract of the leaves of Scoparia dulcis Linn for antidiarrhoeal effects on Wister albino rats. The extract was evaluated for castor oil- induced diarrhoea and intestinal transit in rats by charcoal meal. S. dulcis Linn. Significantly (p<0.05) and dosedependently reduced frequency of stooling in castor oil-induced diarrhoea and intestinal motility in rats. The oral LD50 values obtained were greater than 2000mg/ kg in Wister albino mice. These findings suggest that the ethanolic extract of the leaves of S. dulcis Linn may contain some biologically active ingredients that are useful for the treatment of diarrhoea in Indian herbal traditional medicine. Keywords: Scoparia dulcis, Antidiarrhoeal activity, Castor oil, Charcoal meal 1. INTRODUCTION Diarrhoeal disease is an important cause of morbidity worldwide and it represents a leading cause of childhood death in the developing world (Jebunnessa, 2009). Diarrhoea is characterized by increased frequency of bowel movement, wet stool and abdominal pain. Many public and private research institutions trying to control this disease, but the rate of diarrhoeal death incidence is still high in developing countries (Mukherjee J, 1995) (Synder JD and Merson MH, 1982). Despite massive technological advancement in modern medicine, many people in the developing countries still depend on the healing practices of use of medicinal plants for their daily health care needs (Jebunnessa, 2009). To fight this problem, the world health organization (WHO) has started a diarrhoea disease control program to study traditional medicine practices and other related aspects, together with the evaluation of health education and prevention approaches (World Health Organization, 2006) (Akuodor GC, 2011). Plants have been a valuable source of natural products for maintaining human health for many years. More recently, there has been a greater search for natural therapies. The WHO suggested that medicinal plants would be the best source from which a variety of medications can be developed. About 80% individuals from developed countries receive traditional medicines including formulations derived from medicinal plants. Such medicinal plants can be demoralized since it has been shown that they are important sources of new chemical substances with potential therapeutic effects(Farnsworth NR, 1989) (Eisner T, 1990). Scoparia dulcis Linn commonly known as Mithi patti (Hindi), is distributed in the tropical region of India. It grows as a wetland herb. The traditional healers have developed its many promising traditional uses. The leaves of this plant used traditionally for abortion, menstrual irregularities and used as female contraceptive. It also used against stomach aches, injuries, wounds, bronchitis, coughs, diarrhoea, eye infection, fever, and kidney failure and liver diseases. This has been used in case of infections such as gonorrhoea, skin infections and warts (Atta AH and Mouneir SM, 2004) (Mishra MR, 2011) (Murti K, 2012) (Orhue NE J, 2006). The purpose of the present study was to find out the scientific basis for the efficacy of antidiarrhoeal property of ethanolic leaf extract of S. dulcis Linn. 2. MATERIALS AND METHODS 2.1. Plant collection and preparation of extract: The plant S. dulcis Linn. was collected from Jajpur District, Odisha, and was authenticated by Prof (Dr.) S. K. Dash, Head of the Department, P.G. Bioscience, College of Pharmaceutical sciences, Mohuda, Berhampur (Ganjam), Odisha. Approximately 500g of fresh leaves of S. dulcis Linn. was cleaned; shade dried and reduces into coarse powder in an electrical blender. The powdered material was then subjected to soxhlet extraction with petroleum ether as solvent in 1:4 (w/v) ratios to remove wax and extracted three times with ethanol (99.5%) v/v at room temperature in a cycle of 48h. The ethanol extracts were concentrated in Rotavapour (Buchi Rotavapor R-210) at reduced pressure and temperature below 40C and dried in vacuum desiccators. After drying the product was stored in refrigerator (8 2C) and same was used for in-vivo studies. 2.2. Preliminary Phytochemical Screening: The crude extract of S. dulcis Linn. was subjected to qualitative phytochemical screening according to standard methods. The presence of phytochemicals such as carbohydrates (Fehlings test, Benedicts test), alkaloids (Mayers test, Draggendorffs test), flavonoids (Shinodas reaction), saponins (Frothing test), tannins (5% w/v alcoholic ferric chloride), terpenoids (2, 4-Dinitro phenyl hydrazine), glycosides (Fehlings test), steroids (Libermanns Burchard test) and anthraquinone (Borntragers test) (Trease GE, and Evans MC,1996) (Kokate CK, 1994) were screened. 2.3. Animals: Albino male rats (Wistar) weighing 150-200g and Wistar albino male mice weighing 20-25g were used in this study. They were brought from animal house of Royal College of Pharmacy and health Sciences Volume 1 Issue 1 January February 2013 Page 78
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(RCPHS), Berhampur (Ganjam), Odisha. The animals were acclimatized for one week under laboratory conditions. They were housed in polypropylene cages and maintained at 27C 2C, under 12 hr dark / light cycle. They were fed with standard pellet diet and water ad libitum. The litter in the cages was renewed daily to ensure hygienic condition and maximum comfort for animals. Ethical clearance for handling the animals was obtained from the Institutional Animals Ethical Committee (IAEC), RCPHS, Berhampur, Odisha (approval No.-RCPHS/03/2008) prior to the beginning of the experimental works. 2.4. Determination of acute toxicity: The acute toxicity for ethanolic extract of S. dulcis Linn. was determined in Wister albino mice, maintained under standard conditions. The animals (n=3) in each group was fasted overnight prior to the experiment. Fixed dose (OCED Guideline no. 420) method of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) was adopted for toxicity studies. The extract and fractions in suspension was administrated orally. The mortality and abnormality was observed after administering samples at a dose of 2000mg/kg in all animals (Veerarghavan P, 2001) (Silva MGB, 2011). 2.5. Castor oil-induced diarrhea: Overnight fasted Albino rats of either sex (200-250g) were divided into four groups of six animals each. They were allowed free access to water. Animals of group 1 were treated with 0.5ml of normal saline, which served as control; Group 2 animals received standard drug (Loperamide 3mg/kg B. W.) and served as standard group. Test groups 3 and 4 animals received different doses of the extract (200 and 400mg/kg B.W.) respectively. All doses were administered orally. The animals were then housed singly in cages lined with transparent paper. Thirty minutes after pre-treatment with the extract, the animals were challenged with 1 ml of castor oil orally. The numbers of both wet and dry diarrhoeal droppings were counted every hour for a period of 4 hours. At the end of 4th hour, the cumulative wet and dry weight of stool mass was weighed. Percent reduction in stool mass was calculated (Jebunnessa, 2009) (Akuodor GC, 2011). 2.6. Study of small intestinal transit: This was done according to the method previously described in Akuodor et al. 2011 (Akuodor GC, 2011), using charcoal meal as a diet marker. Albino rats of either sex (200-250g) were randomly divided into four groups of six rats each. They were fasted for 24 hours prior to the test, but were allowed free access to water. The first group was orally administered with 0.2ml normal saline. The second group orally received atropine sulphate (3mg/kg BW). The third and fourth groups orally received different doses of the extract (200 and 400mg/kg BW). Thirty minutes after drug administration, 2ml of charcoal meal (5% w/v activated charcoal in 10% w/v aqueous tragacanth) was administered to all the animals in the study and thirty minutes later, all the rats were sacrificed and the abdomen opened. The small intestine was dissected out and the distance covered by the charcoal meal in the small intestine from the pylorus to the caecum was measured and expressed as a percentage of the distance traveled. 2.7. Statistical Analysis: Data was expressed as mean Standard Error Mean (SEM). Differences was considered significant at ***P<0.001, or **P < 0.01 or * P<0.05 when compared test groups v/s control group. For numerical results, one-way analysis of variance (ANOVA) with Dunnett test (compare rest vs. control) was performed using GraphPad InStat Version 3 (GraphPad Software). 3. RESULTS Phytochemical analysis of the crude extract gave positive reaction for each of the following secondary metabolites: Tannins, Saponins, Terpens, Steroids, Alkaloids, Flavonoids and Carbohydrates. There was no mortality observed in the mice upon oral administration, even at doses as high as 2000mg/kg, signifying that the LD50 was greater than 2000mg/kg. Apart from sedation and weakness, the ethanolic extract of S. dulcis Linn. did not produce any major clinical signs of toxicity in mice during 72hr observation period. Both doses of ethanolic extract significantly decreased (p < 0.05) the total number of wet faeces produced by administration of castor oil. The effect of Ethanolic extract of S. dulcis Linn. was similar to that of the standard drug, Loperamide (3 mg/kg) which produced an inhibition of 70.38 %. The average weight of faeces in the control group was 7.38 gm. Treatment with both doses of Ethanolic extract of S. dulcis Linn. significantly reduced (p < 0.05) the weight of faeces to 5.04 gm (Table 1). The ethanolic extract of S. dulcis Linn. also slowed down the forward motion of charcoal meal through the gastrointestinal tract when compare to the control group. The percentage of intestinal length travelled by charcoal meal in ethanolic extract of S. dulcis Linn. pre-treated (200 and 400mg/kg B.W.) groups and control group rats was 66.93%, 51.64% and 74.54% respectively. Atropine pretreated group produced a marked decrease in the propulsive movement and the intestinal length travelled by charcoal meal was 40.32 % (Table 2). 4. DISCUSSION Diarrhoea is usually considered a result of altered motility and fluid accumulation within the intestinal tract. Many anti-diarrhoeal agents act by reducing the gastrointestinal motility and / or the secretions. Castor oil causes diarrhoea due to its active metabolite ricinoleic acid, which stimulates peristaltic activity in the small intestine, leading to changes in the electrolyte permeability of the intestinal mucosa. Its action also stimulates the release of endogenous prostaglandin. Prostaglandin contributes to the pathophysiological functions in gastrointestinal tract (Jebunnessa, 2009) (Akuodor GC, 2011). In this study, the ethanolic extract of S. dulcis Linn. exhibited a Volume 1 Issue 1 January February 2013 Page 79
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significant anti-diarrhoeal activity. The results were similar to that of the standard drug loperamide (3 mg/kg) with regard to the severity of diarrhoea ethanolic extract of S. dulcis Linn significantly reduced intestinal transit as observed by the decrease in intestinal motility of charcoal meal. Phytochemical screening revealed the presence of tannins, sterol and /or triterpenes and reducing sugars. Earlier studies showed that anti-dysenteric and antidiarrhoeal properties of medicinal plants were due to tannins, alkaloids, saponins, flavonoids, sterol and/or triterpenes and reducing sugars (Atta AH and Mouneir SM, 2004). Hence, tannins, reducing sugars, sterol and/or triterpenes may be responsible for mechanism of action of ethanolic extract of S. dulcis Linn anti-diarrhoeal activity. This can be due to the fact that the extract increased the reabsorption of water by decreasing intestinal motility as observed in the decrease intestinal transit by charcoal meal. Loperamide, apart from regulating the gastrointestinal tract, is also reported to slow down transit in the small intestine, reduce colon flow rate, and consequently any effect on colonic motility (Atta AH and Mouneir SM, 2004). Atropine significantly reduced intestinal transit time. This is possible due to its anticholinergic effect. Furthermore, a decrease in intestinal transit time with atropine could also be due to reduction in gastric emptying. 5. CONCLUSION In conclusion, the present study revealed that ethanolic extract of S. dulcis Linn. contains pharmacologically active substances effective for management of diarrhoea. This study also scientifically justifies the traditional claim of usefulness of this plant against diarrhoea. Further investigation is necessary for isolation, identification and characterization of different active compounds from the extract and for elucidating their mode of action, responsible for these properties on different biological systems. 6. ACKNOWLEDGEMENTS Authors thank Dr. N. C. Talukdar, Director and ScientistF, Institute of Bioresources and Sustainable Development (IBSD), Dept. of Biotechnology, Govt. of India, Imphal, Manipur and the technical staffs of Pharmacology Laboratory, IBSD, Imphal, Manipur and Prof (Dr.) P. N. Murthy, Director and Principal, Royal College of Pharmacy and health Sciences (RCPHS), Berhampur (Ganjam), Odisha, for providing all possible facilities to successfully complete this research work. Table 1: Effect of ethanolic extract of S. dulcis Linn on Castor oil induced diarrhoea in rats Treatment Dose Total number Total number % inhibition Total weight % inhibition of faeces of Diarrhoeal of Diarrhoeal of faeces (gm) total weight faeces faeces of faeces Control 0.5 ml 24.834.53 20.835.115 0 7.382.30 0 (Normal Saline) Standard 3.0 mg/ml 9.830.93* 6.171.60* 70.38 1.760.83* 76.83 (Loperamide) Test Group 1 200 mg/ml 17.502.43* 4.332.65* 79.22 5.041.47* 31.69 (EtOH Ext) Test Group 2 400 mg/ml 15.804.37* 7.171.32* 65.58 5.040.58* 31.71 (EtOH Ext) All data were expressed as mean Standard Error Mean (SEM). Differences were considered significant at * P<0.05 when compared test groups Vs Control group (n = 6). Table 2: Effect of ethanolic extract of S. dulcis Linn on intestinal motility of rats by charcoal meal Treatment Dose (mg/ml) Distance travelled by charcoal meal % Inhibition Control (normal saline) 0.2 74.54 9.90 0 Standard (Atropine) 3.0 40.32 4.25* 45.93 Test Group 1(EtOH Ext) 200 66.93 7.78* 10.26 Test Group 2(EtOH Ext) 400 51.64 4.25* 30.75 All data were expressed as mean Standard Error Mean (SEM). Differences were considered significant at * P<0.05 when compared test groups Vs Control group (n = 6).
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REFERENCES Akuodor GC, Muazzam I, Usman-Idris M, Megwas UA, Akpan JL, Chilaka KC, Okoroafor DO, Osunkwo UA. Evaluation of the Antidiarrhoeal Activity of Methanol Leaf Extract of Bombax Buonopozense in Rats. Ibnosina J Med BS 2011, 3(1), 15-20. Atta AH, Mouneir SM. Antidiarrhoeal activity of some Egyption medicinal plant extracts. J Ethnopharmacol, 2004, 92, 303-309. Eisner T, Chemical prospecting, A call for action, In: Borman FH, Kelleet SR, editors. Ecology, Economic and Ethics: The Broken Circle. New York: Yale University press, 1990. Farnsworth NR, Screening plants for new medicines. In: Wilson EO, editor. Biodiversity. Washington: National Academy press, 1989, 83-97. Jebunnessa, Uddin SB, Mahabub-Uz-Zaman M, Akter R, Ahmed NU. Antidiarrhoeal activity of ethanolic bark extract of Mitragyna diversifolia, Bangladesh J Pharmacol, 2009, 4, 144-146. Kokate CK, Practical Pharmacognosy, 4th ed. New Delhi, Vallabh Prakashan, 1994, 107-111. Kumar JJ, Joytsna T, Kumar PV, Elayaraja A, Rahman SA, Pharmacognostical study on hole plant of Scoparia dulcis L. J Pharm Res, 2012, 5(2), 1221-1223. Mishra MR, Behera RK, Jha S, Panda AK, Mishra A, Pradhan DK, Choudary PR. A Brief Review on Phytoconstituents and Ethnopharmacology of Scoparia Dulcis Linn, Int J Phytomedicine, 2011, 3, 422-438. Mukherjee J, Das R, Balasubramania K, Saha M, Pal BP. Anti-diarrhoeal evaluation of Nelumbo nucifera rhizome extract. Indian J pharmacol 1995, 22, 262-264. Murti K, Panchal M, Taya P, Singh R. Pharmacological properties of Scoparia Dulcis: Review. Pharmacologia 2012; 3(8): 344-347. Orhue NE J, Nwanze EAC. Scoparia dulcis reduces the severity of Trypanosoma bruceiinduced hyperlipidaemia in the rabbit, Afr J Biotechnol, 2006, 5 (10), 883-887. Silva MGB, Aragao TP, Vasconcelos CFB, Ferreira PA, Andrade BA, Costa IMA, Costa-Silva JH, Wanderley AG, Lafayette SSL. Acute and subacute toxicity of Cassia occidentalis L. stem and leaf in Wistar rats. J Ethnopharmacol 2011, 136, 341-346. Synder JD, Merson MH. The magnitude of the global problem of acute diarrhoea disease. A review of active surveillance data. Bull WHO 1982, 60, 605-13. Trease GE, Evans MC, editors. Textbook of Pharmacognosy 14th ed. London: Bailliere Tindal, 1996, 565-566. Veerarghavan P. Expert consultant: Committee for the Purpose of control and supervision of Experiments on Animals (CPCSEA). Animal Welfare Division, Government of India. 2001, (Guideline No. 423, Annexure-2d of OECD). World Health Organization, Country Health System Fact Sheet, 2006.
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B S Venkateswarlu et.al.
DESIGN AND IN VITRO EVALUTION OF SUSTAINED RELEASE FILM COATED TABLETS OF VERAPAMIL HYDROCHLORIDE
BS Venkateswarlu*, B Jaykar, Pasupathi A, R Margret Chandira, Palanisamy P Vinayaka missions college of pharmacy, Vinayaka missions university, Salem, Tamil Nadu
* Corresponding author: Email: palanisamy2907@gmail.com
ABSTRACT An attempt was made to formulate the sustained release tablet of verapamil hydrochloride by using the wet granulation method for the treatment of hypertension. In the present formulation the tablets releases the loading dose by immediate drug release and maintenance dose up to 15 hours by extended release. The drug excipient compatability study was carried out with HPLC method and there was no interaction found. Immediate release fraction was formulated by using croscarmellose sodium as a disintegrating agent and extended release fraction was formulated by using Hypromellose E4 as a rate controlling polymer. The granules were evaluated for pre and post compressional character which showed satisfactory results. In vitro dissolution study was carried out for 15 hrs using USP dissolution apparatus type II with 0.1 N HCl and 7.4 pH phosphate buffer as dissolution medium. From the dissolution profile, F2 & F3 values were calculated which were within the specification. Stability study was carried out for the optimized formulation at 40C/75% RH for 1 month, the result showed that there was no significant change in physical and chemical parameter of the tablet. Key words: Verapamil Hydrochloride, Sustained release tablet, Hypromellose E4. 1. INTRDUCTION Verapamil Hydrochloride is a calcium channel blocker and class IV antiarrhythmic agent used in the supraventricular arrhythmias, and in the management of angina pectoris, hypertension and myocardial infarction. Hypertension, commonly referred to as high blood pressure, is a medical condition where the pressure is chronically elevated is one of the commonly found diseases, affecting most of the populations in the world. So, for treating hypertension effectively is main criterion of study. For treating hypertension, commonly used drugs include ACE inhibitors, alpha blockers, beta blockers, calcium channel blockers, diuretics and combination of any of these categories in immediate action required. The objective of this study is to develop immediate and sustained release formulation of Verapamil hydrochloride coated Tablets. 2. MATERIALS AND METHODS 2.1. Materials: Verapamil HCL was procured by Cadila pharmaceutical Limited, Ahmedabad, Lactose monohydrate, Microcrystalline cellulose 102, Hypromellose (Methocel E4 Premium), Hypromellose (Methocel E5LV Premium) and PVPK-30 was gifted by Feicheng Rutai Fine Chemicals Co Ltd, Mannitol SD-102 was gifted by Arihant trading Co. Ltd, Sodium alginate, Magnesium Stearate, Crosscarmellose sodium (ac-di-sol), Opadry Blue OY-30944 and Talc was gifted by Relience cellulose Pvt Ltd, Purified Water was gifted by Loba Chemie Pvt Ltd. 2.2. Formulation of Verapamil SR Tablet: a) Co mixed ingredients verapamil hyochloride and mannitol SD 200 geometrically and sifted through 40 mesh b) Co mixed other granulation ingredients in a polybag c) Mixed steps a and b in RMG (Rapid Mixer and Granulator) for dry mixing for 10 minutes. d) Binder was dissolved in water to get clear binder solution. e) Binder solution was added to the dry mixed ingredients in RMG with both mixing and chopper blades on initially at high speeds and finally at low speed for 5 minutes. f) Dried the granules in FBD (fluidized bed drier) at 60oc for 30 min and then milled through 2.0 mm screen in multimill and passed oversized through sieve 20 mesh g) Dried the bulk of step (f), for another 15 min h) Sifted Magnesium Stearate through 40 mesh i) Lubricated the bulk of step (g) with bulk of step (h). Compressed the lubricated blend of step (i) by using 14.50 x 6.50 mm, capsule shaped punches with bisecting line on upper punch and plain 3. EVALUATION TESTS 3.1. Evaluation of tablet: These include the diameter, size, shape, thickness, weight, hardness, disintegration and dissolution characters. The diameters and shape depends on the die and punches selected for the compression of tablets. 3.2. Dissolution Test: The objectives in the development of in vitro dissolution tests are to show that the release of the drug from the dosage form is as close as possible to 100 % and the rate of drug release is uniform from batch to batch. Drug Verapamil is a water insoluble API. 7.4 pH phosphate buffer is taken as dissolution media. Following method was adopted to check dissolution profile. Volume 1 Issue 1 January February 2013 Page 82
S. No
Table 1: Formulation table of Verapamil Hydrochloride SR Tablet 1 2 3 4 5 6 7 8 9 10 11 12 Ingredients Verapamil hyochloride Mannitol SD 200 Croscarmellose sodium (Ac-di Sol) PVP K30 (Povidone K 30) Lactose monohydrate Sodium Alginate (Protanal LF 120M) Microcrystalline Cellulose (Avicel PH101) Hypromellose (Methocel E4M Premium) Hypromellose (Methocel E5 LV) Premium) Purified water Magnesium Stearate Talc Total F2
240 49 35 12 72 17 3.5 1.5 430
F3
240 49 55 12 52 17 3.5 1.5 430
F4
240 15 115 12 29 14 QS 3.5 1.5 430
F5
240 12 115 12 32 14 QS 3.5 1.5 430
F6
240 8 115 16 32 14 QS 3.5 1.5 430
F7
240 8 115 15.5 37 10 QS 4.5 430
F8
240 5.5 100 18 38 24 QS 4.5 430
F9
240 5.5 105 18 38 19 QS 4.5 430
F10
240 5.5 115 18 38 9 QS 4.5 430
F11
240 5.5 115 18 38 9 QS 4.5 430
Table 2 Dissolution method for drug USP Apparatus Type II (Paddle) Speed 50 rpm Medium Phosphate Buffer pH 7.4, 900 ml Sampling times 3,6,9,12,15 Hr Analysis By UV method 278 nm 3.3. Drug release kinetic analysis by using different release model of active molecule: Several theories and kinetic models were described the drug release characteristics of immediate release and modified release dosage forms, by using dissolution data and quantitative interpretation of values obtained in dissolution assay if facilitated by the usage of the generic equation dosage form that mathematically translates the dissolution curve in function of some parameters related with pharmaceutical dosage form. 3.4. Stability Study: Tablets of the final batch were packed in High-Density Polyethylene Containers (HDPE, 60CC) and were subjected to accelerated stability studies (40020C/755%RH 1, 2 & 3 Months). The effects of temperature and humidity with time on the physical and chemical characteristics of the tablet were evaluated for assessing the stability of the prepared formulation. After each time period, the samples were tested for appearance, dissolution, assay and impurities. 4. RESULT AND DISCUSSION 4.1. Solubility: Solubility of the drugs is determined in 5 different media which is given below, Table 3. Solubility of active molecule Table No: 4 Particle size distribution of active molecule Media Solubility (mg/ml) Particle size m Water 0.097 distribution 0.1N HCl 0.1642 D (v, 0.1) 5.40 6.8 Phosphate Buffer 0.1324 D (v, 0.5) 8.94 4.5 Acetate Buffer 0.0485 D (v, 0.9) 16.74 7.4 Phosphate Buffer 0.1398l 4.2. Particle size determination: For many active substances, particle size has an impact on powder flow, content uniformity and drug dissolution. In order to assure consistent product quality, the particle size of the active molecule has been characterized. 4.3. Density and flow properties: From the above data it was concluded that the drug had poor compressibility and poor flow ability. So, during development BD and Flow ability will be improved for better granulometry parameters (Table 5). Table No: 5 Density and flow properties of active molecule Sr. Density (gm/ml) Flow properties No. Bulk Tapped Carrs index Hausners Ratio 1 0.416 0.526 20.91 1.264 4.4. I.R. Spectra of active molecule: Spectrum of Verapamil Hydrochloride in phosphate buffer pH 6.8
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Fig No:1 I.R. Spectra of active molecule
Fig No:2 FTIR Interpretation of IR graphs
The figure shows the IR spectrum of Verapamil Hydrochloride; from the peaks the following functional groups are found. Between 3030 and 2860 cm-1: A broad complex absorption due to superimposing C-H stretching vibrations of the methyl and methylene groups. 2840 cm-1: A band due to C-H stretching vibration of the methoxy groups. Between 2800 and 2300 cm-1: A broad complex absorption due to N-H stretching vibration of the protonated amine. 2236 cm-1: A sharp weak band due to C=N stretching vibration of the saturated alkyl nitrile. 1607, 1591 and 1518 cm-1: Bands due to skeletal stretching vibrations of the benzene ring. 1262 cm-1 : A strong band due to C-O stretching vibrations of the aromatic ethers. 4.5. Drug-excipients compatibility Study: No change appeared in the powder mixer at above all conditions indicate that there are no incompatibilities present between drug and excipients (Table 6) Table No: 6 Drug-excipients compatibility Study
Sample Ratio Observation of total impurity 25C2C / 60%RH 5 % RH one month one month one month one month one month one month one month one month one month 40C2C/ 75%RH 5 % RH Two month Two month Two month Two month Two month Two month Two month Two month Two month
Drug + lactose monohydrate Drug + Mannitol SD 200 Drug + PVP K-30 Drug + Magnesium Sterate Drug + Microcrystalline Cellulose Drug + sodium alginate Drug + Hypermellose E4 Drug + Hypermellose E5 Drug + Talc
1:1 1:1 1: 0.5 1: 0.25 1:1 1:0.5 1:0.5 1:0.5 1: 0.25
4.6. Granule analysis: Granule analysis data such as bulk density, tapped density, carrs index (C I), Hausners ratio (H R) as per table: 7 Table No: 7 Granulometry analysis
Parameter B. D. (gm / ml) T. D. (gm / ml) C. I. (%) H. R. Angle of repose LOD % F2 0.4194 0.5776 28.78 1.456 41.78 2.02 F3 0.4434 0.5976 25.80 1.34 38.66 2.01 F4 0.5214 0.5815 10.34 1.12 34.87 1.99 F5 0.4865 0.5350 9.06 1.10 33.91 2.05 F6 0.5119 0.5811 11.91 1.14 35.34 2.09 F7 0.5434 0.6123 12.67 1.13 34.86 2.15 F8 0.4832 0.5376 10.12 1.11 34.29 2.07 F9 0.4731 0.5423 12.76 1.14 35.75 2.02 F10 0.5162 0.5837 11.56 1.13 34.56 2.12 F11 0.4731 0.5423 12.76 1.14 35.75 2.02
Fig No: 3 In-vitro dissolution INNOVATER VS TRIALprofile F4 TO F9 of F4-F9

120
%DRUG DISSOLVED
100 80 60 40 20 0 3 6 9 12 15 18 TIMES IN HOURS
INNOVATOR TRIAL F4 TRIAL F5 TRIAL F6 TRIAL F7 TRIAL F8 TRIAL F9
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4.7. In-Vitro dissolution study: In-vitro dissolution study of trial batches F4 to F9 was taken and these batches having different concentration with different grade of Hypromellose in F4 and F9, the different concentration of Hypromellose were used. In F4 trial we got 101 % Cumulative Drug Release within 9 hrs and F/05 trial we got 97.8 % Cumulative Drug Release in between 9 to 12 hrs. So this grade cannot be used for extend the dissolution up to 18 hrs and next batch plan with high viscosity grade. In B.No F/08 and F/09, different concentrations of Microcrystalline Cellulose and Sodium Alginate (Protanal LF 120M) were used. In F/08 trial we got up to 80.4% cumulative drug release in 18 Hrs and in F/09 trial we got 76.8 % cumulative drug release in 18 hrs. These batches show very slow drug release compare to target drug release. So this grade was not suitable for further development. In F6 and F7, different concentrations of Sodium Alginate (Protanal LF 120M) and Hypromellose were used. In F6, % drug release was 99.4% in 15 hrs and in F7 trial; % drug release was 98.5% in18 hrs so these F6 and F7 batches were nearly comparable with target drug release so these grade can be used for further development. This may be due to structural reorganization of Hypromellose E5 polymer. Increase in concentration of Hypromellose E5 may result in increase in the tortuosity or gel strength of the polymer. But initial dissolution need to be improved. Assay value of all these batches is acceptable. 4.8. Drug release kinetic analysis by using different release model: To know the mechanism of drug release from these formulations, the data were treated according to first-order (log cumulative percentage of drug remaining vs time), Higuchis (cumulative percentage of drug released vs square root of time), and Korsmeyer Peppas (log cumulative percentage of drug released vs log time) equations along with zero order (cumulative amount of drug released vs time) pattern. (Table 8) Table No: 8.Data analysis by using different model of F/10 Model Zero order First order Higuchi Korsemeyer-Peppas 0.785 0.9994 0.9555 1 Linearity (R2) 4.51 53.05 22.83 0.5 Slope (n) 32.27 66.38 11.66 Intercept (c) The R2 value from plot of zero order and first order shown that the formulation follows first order release pattern. The in vitro release profiles of drug from the formulation could be best expressed by Higuchis equation, as the plots showed high linearity (R 2 = 0.9555). To confirm the diffusion mechanism, the data were fit into Korsmeyer-Peppass equation. The formulations F/12 showed good linearity (R 2 =1), with slope (n) value equal to 0.5, indicating that mechanism of drug release follows non Fickian diffusion. This n value, however, appears to indicate a coupling of diffusion and erosion mechanisms so called anomalous diffusion. The relative complexity of this formulation and its components may indicate that the drug release is controlled by more than one process. 4.9. Stabiity studies: Stability studies were conducted for one month and results were noted in the Table 9. 5. SUMMARY AND CONCLUSION Results of preformulation studies of the active drug indicate that, it has poor flow property and compressibility property, and it was overcome by diluents and excipient compatibility evaluation was carried out, the result indicated that drugs, excipients, and polymers were compatible with each other. Optimized proportion of Hypromellose was decided based on trial and error methods and depending upon the dissolution profile. F10 batch was optimized by employing Hypromellose to check the reproducibility and matching the dissolution profile with the targeted drug release. In Wet granulation, Hypromellose E4 was used as a Controlled Release agent. In batches F04 to F09, different grades and different concentration of Hypromellose were used. The results of these batches conclude that Hypromellose E4 shown fast release compares to targeted release, Hypromellose E5 used as a Binder. In F10, the initial drug release was improved with increased in concentration of Sodium alginate and increased in concentration of Hypromellose E5. In batch F10, obtained with the increase the concentration of Sodium alginate and Hypromellose E5. In F10 obtained with the increase the concentration of SLS and dissolution profile of F10 showed that Percentage cumulative drug release identical with targeted and it were fulfill the criteria and significantly retard the release up to15 hours When compare with the innovator profile. So the batch F10 was considered as the optimized batches for once a day tablet formulation. The F11 was the coated formulation of F10, with opadry blue as coating agent; Coating was carried out till it increased the 2 percentage of weight. The stability study were carried out as per in ICH guideline for the period of one month. The results indicated that the coated formulation (F11) was stable as in period of one. It concluded that the hypromellose e5 could be used as a controlled release excipient in the solid dosage form.
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Table: 10 First month Stability Data of sustained release film coated Tablet at 40C 2C / 75% RH 5 % RH Parameters Description Initial 1 Months Blue, oblong, biconvex film- Blue, oblong, biconvex filmcoated tablets with bisection line coated tablets with bisection on one side. line on one side. 99.40 % 99.13 % 0.29 0.33 (%)
Assay Related Substance (Total Impurities)
3 hr =80.1% 3 hr = 80.1% 6 hr = 74.8% 6 hr = 74.3% 9 hr = 85.3% 9 hr = 85.4% 12 hr = 91.2% 12 hr = 90.8 % 15 hr = 99.5% 15 hr = 99.7% 5.90 5.90 10-18 10-18 Friability 0.017 0.020 100 Revolutions 0.018 0.021 200 Revolutions From the above stability data at 40C 2C / 75% RH 5 % RH, it reveals that the product is stable at 40C/ 75%RH for 1 months. 6. ACKNOWLEDGEMENTS Authors are thankful to Prof. (Dr.) B.Jayakar, Principal, Vinayaka Missions College of Pharmacy, Salem, Tamil Nadu, India, for providing all the facilities for this research work. Dissolution Medium: 1) 750 ml of 0.1 N HCL 2) 7.4 pH Phosphate Buffer , paddle,50 rpm Thickness (mm) Hardness (Kp) REFERENCES Aulton ME, The Science of dosage form design, Churchill living stone, 2002, 2nd edition, 414-418. BellamiWT, P-Glycoprotein and multi drug Resistance, Drug Bank.Com, 36, 1996, 161-183. Fanner DE, Buck JR and Banker GS, Journal of Pharmaceutical Sciences, 1970, 1576-1578. Franz R, Doonan G, Measuring the surface temperature of tablet beds using infrared thermometry, Pharm Technol, 7, 1983, 55-67. Lachman L, Liberman H and Kanig J, The Theory and Practice of Industrial Pharmacy, Third Edition, 1990, 293294, 298, 335, 372, 711, 714. Leon lachman, The theory and practice of industrial pharmacy, 3rd edition, 1987, 336-413. Libermen H, Lachman L, Pharmaceutical Dosage Forms Tablets, Vol. I to III, Marcel Dekker Inc, N.Y, 2003, 85143. Obara S, Mc Ginity J, Influence of processing variables on the properties of free films prepared from aqueous polymeric dispersions by a spray technique, Int J Pharm, 126, 1995, 1-10. Obara S, Mc Ginity J, Influence of processing variables on the properties of free films prepared from aqueous polymeric dispersions by a spray technique, Int J Pharm, 126, 1995, 1-10. Raymond C Rowe, H Kibbe, Handbook of pharmaceutical excipients, 4th edition, publisher- science and practice, Royal pharmaceutical society of great britain, London, 67, 2003, 56-78. Yie W Chain, Novel drug delivery systems, 2nd ed. Madison Avenue (NY), 56, 1992, 198-234.
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FORMULATION AND EVALUATION OF DEXLANSOPRAZOLE DELAYED RELEASE CAPSULES

BS Venkateswarlu*, B Jaykar, Pasupathi A, R Margret Chandira, Palanisamy P Vinayaka missions college of pharmacy, vinayaka missions university, Salem, Tamil Nadu * Corresponding author: Email: palanisamy2907@gmail.com ABSTRACT The present research work is formulation and evaluation of dexlansoprazole delayed release capsules. Dexlansoprazole is proton pump inhibitor (PPI) used in treatment of gastro esophageal reflux disease (G.E.R.D), Erosive esophagitis (E.E) and acid related factors. In this research work various polymers like HPMC P55 S, Eudragit L 100 were used as enteric coating polymers were prepared in a fluidized bed coater (FBC). The pre-formulation studies such as solubility and compatability studies were carried out. In the formulation study of dexlansoprazole different formulation (F1 - F10) have been formulated and evaluated for acid resisitance, dissolution profile and release kinetics. The drug percentage release is more when compared with the marketed product. Stability study was done at various storage conditions and months for description, assay, water content, related substances. Mainly research work is to develop a pharmaceutically equivalent low cost quality improved and stable formulation and evaluation of dexlansoprazole delayed release capsules comparable to innovator product. Key Words: Dexlansoprazole, HPMC P 55 S, Eudragit L 100 55, pH 6.8 Phosphate buffer, Sodium Lauryl Sulphate. 1. INTRODUCTION The present study is to develop a pharmaceutically equivalent, low cost quality improved and stable formulation of Dexlansoprazole delayed release capsules comparable to innovator product. Dexlansoprazole is a proton pump inhibitor used in treating gastric ulcers and gastro esophageal reflux disease (GERD) and also maintaining of all grades of erosive esophagitis (EE). Dexlansoprazole is highly acid labile and presents many formulation challenges and to protect it from acidic environment of the stomach an enteric coated pellets formulation was tried in the present study. The enteric polymers are becoming very popular due to their property of remaining intact in the stomach, but will dissolve and release the contents in the small intestine. The prime intention is to delay the release of drugs which are inactivated by the stomach contents or may cause bleeding or nausea by the irritation of gastric mucosa. The Enteric coated polymers of various different types were tried in the formulation of dexlansoprazole delayed release capsules. 2. MATERIALS AND METHOD Dexlansoprazole was procured from Natco chemical division(India), PVP K30 and HPMC E5 was gifted by Signet chemical corporation (Supp), Dibasic sodium phosphate, Light magnesium carbonate, Sodium lauryl sulphate, HPMC P 55 S, PEG 4000, Tri ethyl citrate and Di ethyl phthalate was gifted by Merck Ltd. 2.1. Drug loading stage: Drug loading was done by following the below mentioned procedure 1. 500 ml of purified water was taken in a beaker and kept for stirring under a mechanical stirrer. 2. Specified quantities of HPMC E5, Light magnesium carbonate and Sodiumlaurylsulphate were added slowly to form a uniform suspension. Specified quantity of dexlansoprazole was added and stirring was continued for 30 mins. 3. Sugar spheres were coated with the prepared drug suspension using fluidized bed coater (FBC). 4. Dried pellets were collected and coating efficiency was calculated. 2.2. Barrier coating stage: This stage was done by following the procedure given below 1. 500 ml of purified water was taken in a beaker and kept for stirring under a mechanical stirrer. 2. Specified quantities of HPMC E5, mannitol and light magnesium carbonate were added slowly to form a uniform suspension. Stirring was continued for 30 mins. 3. Drug loaded pellets were coated with the above suspension using FBC. 4. Dried pellets were collected and coating efficiency was calculated. 2.3. Enteric coating stage: This stage was done by following the procedure given below 1. 300 ml of iso propyl alcohol and 900 ml of Acetone were taken in a beaker and kept for stirring under a mechanical stirrer. 2. Specified quantities of tri ethyl citrate, PEG 4000, Eudragit L 100 55, titanium dioxide and Talc (previously passed through 200#) were added slowly to form a uniform suspension. Stirring was continued for 30 mins. 3. Barrier coated pellets were coated with the above suspension using FBC. 4. Dried pellets were collected and coating efficiency was calculated.
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Table: 1. Formulation of Dexlansoprazole delayed release capsules

S. No 1 2 3 4 5 6 7 8 Ingredients Sugar spheres (18/20#) Dexlansoprazole PVP K-30 HPMC E-5 Dibasic Sodium Phosphate Light Magnesium Carbonate Sodium Lauryl Sulphate Water
*
Formula mg/capsule
F1
171 30 2.8 3.5 0.595 q.s
F2
171 30 5.6 3.5 0.595 q.s
F3
171 30 3.5 0.595 q.s
F4
171 30 5.6 3.5 0.595 q.s
F5
F6
F7
171 30 5.6 3.5 0.945
F8
171 30 5.6 3.5 0.945 q.s
F9
171 30 5.6 3.5 0.945 q.s
F10
171 30 5.6 3.5 0.945 q.s
171 171 Drug loading 30 5.6 5.6 3.5 0.595 30 5.6 3.5 0.945
q.s q.s q.s Barrier coating 10.5 7 10.5 7 10.5 7
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
PVP K-30 HPMC E-5 Dibasic Sodium Phosphate Light Magnesium Carbonate Mannitol Water Eudragit L 100 55 PEG 4000 Tri Ethyl Citrate HPMC P 55 S Di Ethyl Phthalate Cetyl Alcohol Titanium Dioxide Talc Iso Propyl Alcohol Acetone
10.5 3.5 76.40 q.s 42 4.2 1.05 1.4 2.8 q.s q.s
10.5 3.5 73.60 q.s 42 4.2 1.05 1.4 2.8 q.s q.s
10.5 7
10.5 7
10.5 7 76.75 q.s 35 4.2 1.05 1.4 2.8 q.s q.s
10.5 7 69.75 q.s 42 4.2 1.05 1.4 2.8 q.s q.s
10.5 7 62.75 q.s 49 4.2 1.05 1.4 2.8 q.s q.s
70.10 70.10 q.s q.s 42 4.2 1.05 1.4 2.8 q.s q.s 42 4.2 1.05 1.4 2.8 q.s q.s
70.10 69.75 62.75 q.s q.s q.s Enteric coating 42 4.2 1.05 1.4 2.8 q.s q.s 42 4.2 1.05 1.4 2.8 q.s q.s 49 4.2 1.05 1.4 2.8 q.s q.s
* 18 mesh passings and 20 mesh retains 3. RESULTS AND DISCUSSION Formulations from F1 to F10 were tested for acid resistance test, and percentage cumulative drug release. Drug release was compared with innovator product also. F9 has shown better percentage cumulative drug release when compared with other formulations as well as with innovator product also. The results were shown in table 2. Along with F9, F10 also has shown very good drug release profile. Table: 2. In-vitro drug release studies Percentage cumulative drug release F1 F2 F3 F4 F5 F6 F7 0 0 0 0 0 0 0 13.0 61.3 75.9 72.7 76.7 84.1 83.2 85.0 70.1 85.0 82.4 86.0 92.3 89.1 92.0 72.0 86.0 83.9 87.8 93.0 94.3 97.7 69.4 89.0 85.0 89.1 95.9 96.0
S. No. 1 2 3 4 5
Time (min) 0 15 30 45 60
Innovator 0 89.6 95.0 93.0 92.0
F8 0 80.0 89.1 91.0 94.0
F9 0 94.3 96.0 96.6 98.0
F10 0 90.3 93.0 95.0 96.2
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4. SUMMARY AND CONCLUSION The objective of the present study was to formulate and evaluate Dexlansoprazole delayed release capsules comparable to the innovator product (30mg). The Eudragit L 100 55 and HPMC phthalate 55 S were used as enteric polymers. The enteric coated pellets were prepared in a fluidized bed coater. The solubility studies with various PH buffers and compatibility studies for initial analysis, Two weeks (50 0C) were carried out the results found to be satisfactory. The ten formulations of Dexlansoprazole delayed release capsules of Dexlansoprazole were developed by enteric film coating process varying the compositions of drug loading, barrier coating and enteric coating. The Drug Dissolution profiles, Release kinetics, Koresmeyer Peppas plots of Dexlansoprazole delayed release capsule formulations developed were compared with that of innovator product. The process variables for stability studies were standardized and the different batches prepared were evaluated for assay/drug content, content uniformity, and water content. In this the pH 6.8 Phosphate buffer (SLS) shows more solubility and dissolution profile and also stable. Based on the results F9 was selected as the best formulation developed for Dexlansoprazole delayed release capsules. 5. ACKNOWLEDGEMENTS Authors are thankful to Prof. (Dr.) B.Jayakar, Principal, Vinayaka Missions College of pharmacy, Salem, Tamilnadu, India providing all the facilities for this research work. REFERENCES Ansel CH, Poppovich NG, Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed, New Delhi, B.I. Waverly Pvt. Ltd, 1995, 213. Chien WY, Novel Drug Delivery systems, Marcel Dekker, New York, 1992, 156 - 165. Jamie D, Scott, Lesley J, Dexlansoprazole modified release: in erosive oesophagitis and non-erosive reflux disease Croxtall, 70(12), 2010, 1593-1601. Erkoboni DF, Extrusion and Spheronization as a granulation technique, in Parikh DM, Hand book of Pharmaceutical technology, Marcel Dekker, New York, 2002, 333-365. Lee VHL, Robinson JR, Influence of drug properties and routes of drug administration on the design of sustained and controlled release systems, controlled drug delivery, fundamentals and applications, Marcel Dekker, NewYork, 1987, 3 -9. MF Saettone, G Perini, P Rijli, L Rodriguez, M Cini, Effect of different polymer-plasticizer combinations on in-vitro release of Dexlansoprazole from coated pellets, International Journal of Pharmaceutics and research, 1995, 1(2), 83-88. Metha AM, Valazza MJ, Abele SE, Evaluation of fluid bed processes for enteric coating systems, Pharmaceutical technology, 10, 1986, 46-56.
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Pasupathi A et.al.
FORMULATION, OPTIMIZATION AND IN-VITRO EVALUATION OF NIFEDIPINE SUSTAINED RELEASE TABLET

Pasupathi A*, Palanisamy P, B Jaykar, R Margret Chandira, BSVenkateswarlu Vinayaka Missions College of Pharmacy, Vinayaka Missions University, Salem, Tamil Nadu. *corresponding author; Email: palanisamy2907@gmail.com
ABSTRACT The objective of the present study was to develop sustained release tablet of Nifedipine. The drug is a dihydropyridine derivative mainly indicated for the treatment of Hypertension. The drug belongs to the therapeutic classification as calcium channel blocker and antianginal. The tablets were prepared by wet granulation method.The granules were evaluated for angle of repose, bulk density, tapped density, compressibility index, and Hausner ratio. The tablets were subjected to thickness, hardness, friability, weight variations, and drug content by assay and in vitro dissolution studies. The granules showed satisfactory flow properties, compressibility index and drug content. All the tablet formulations showed acceptable pharmaceutical properties.The in-vitro drug release from Nifedipine sustained release tablet was carried out in 1.2 N HCl, and 6.8 pH phosphate buffer for 24hrs. The optimized formulation F3 showed the highest f2 (similarity factor) (f2 = 75.5) value. The drug release from the developed formulation was independent of agitational intensity. The release rate was influenced by medium, the amount of rate controlling polymer. The similarity factor, f2 was applied between the optimized formulation and the theoretical dissolution profile. The comparison of optimized formulation with marketed product gave a satisfactory release profile. The drug release data were plotted using various kinetic equations (Zero order, First order, Higuchis kinetics, Korsmeyer and Peppas kinetics and Hixson and Crowell kinetics) to evaluate the drug release mechanism and kinetics. The formulations were found to be stable for after 2 months of accelerated stability studies. Key words: sustained release tablet, Nifedipine, calcium channel blocker, rate controlling polymer, in-vitro drug release 1. INTRODUCTION The aim of the work is to investigate the possibility of obtaining a prolonged, relatively constant level of Nifedipine. Nifedipine which is an antihypertensive drug has poor aqueous solubility, short half life and undergo an excessive first pass metabolism. So it is prescribed 2-3 times/day for the treatment of hypertension which leads to poor patient complaints and development of tolerance. Present studies investigate the possibility for the development of sustained release tablet of Nifedipine, to reduce the side effect, dosing frequency and improve patient compliance. Keeping these factors in view it was aimed to formulate and evaluate SR Nifedipine tablets 30 mg, to provide a controlled and predictable release of Nifedipine for once daily administration. 2. MATERIALS AND METHODS Nifedipine was procured by Merck (Germany), HPMC K15, HPMC K100 and PVP was gifted by FMC Biopolymer, Eudragit was gifted by Merck Limited (India), Magnesium stearate was gifted by Amishi drugs and Chemicals (India), Lactose was gifted by HMS (Kerala), Isopropyl alcohol was gifted by Cabot Sanmar Ltd (Kerala), Potassium chloride, Hydrochloric acid, Potassium phosphate (monobasic), Sodium hydroxide and Methanol was gifted by Nice Chemicals. Cochin (Kerala). Table : 1 Development of Various Tablet Formulations INGREDIENTS F1(mg) F2(mg) F3(mg) F4(mg) F5(mg) F6(mg) Nifedipine 30 30 30 30 30 30 HPMC K15 45 60 ------------HPMC K100 ------45 60 -------PVP ------------45 60 MCC 40 40 40 40 40 40 Lactose 35 20 35 20 35 20 Sodium CMC 8 8 8 8 8 8 Eudragit 5 5 5 5 5 5 Magnesium stearate 2 2 2 2 2 2 Isopropyl alcohol Qs Qs Qs Qs Qs Qs Total 165 165 165 165 165 165 2.1. Evaluation of the sustained release developed formulations : Weight variation, friability, hardness, thickness, diameter and drug content were measured and tabulated in Table 2. 2.2. Dissolution Study: Dissolution test was conducted by using medium 900ml of 1.2N HCl for the first two hours and then replaced with 6.8 PH phosphate buffer using USP-1 (Basket) apparatus, at 100rpm speed. Samples were collected at the time intervals 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours. Temperature of the Volume 1 Issue 1 January February 2013 Page 90
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medium was maintained at 37 0.5C. Samples were withdrawn at specified time required amount of sample into amber colored volumetric flask and made up to the volume and absorbance was measured at wavelength 350nm and calculated the percentage release. 2.3. Performance evaluation of optimized formulation: The optimized formulation was selected by comparing the results of various evaluation techniques including % drug release and a formulation which gives the most satisfactory results was considered to be the optimized formulation. Above all the formulation F3 showed maximum and comparable % drug release after 24hrs of dissolution studies. Therefore, F3 was taken as the optimized formulation. The optimized formulation was evaluated based on various pharmacopoeial and nonpharmacopoeial tests. 2.4. Effect of agitational intensity: To study the effect of agitational intensity of the release media, release studies of the optimized formulations were carried out in dissolution apparatus at variable rotational speed. Dissolution apparatus used was USP-1 apparatus, at 50rpm, 100rpm.The result showed that there was no significant difference in release profile under different agitation rates. Therefore, it might be expected that the mobility of the gastrointestinal tract hardly affects the drug release of sustained release tablet system. The samples were withdrawn at predetermined intervals and analyzed at 350nm spectrophotometrically. The results are given in Table 5 and graphically represented in Fig no 3 2.5. Effect of dissolution media: To study the effect of release media on drug release, the release was conducted in deionised water (DIW), Simulated gastric fluid(SGF) and simulated intestinal fluid(SIF), respectively. No significant difference in release profile could be found. In other words, the sustained release tablet system exhibited a media independent release. Thus it may be expected that the fluid in different parts of gastrointestinal tract scarcely affect drug release of sustained release tablet system. 2.6. Comparison of optimized formulation with marketed formulation: The in-vitro dissolution profile of the optimized formulation was compared against in-vitro dissolution profile of a marketed formulation under similar experimental conditions. The results of this comparison study is given in Table 6 Table. 2 Evaluation of physical parameters of formulations B. Weight Diameter Thickness Hardness Friability Drug No. variation(mg)* (mm)* (mm)* (Kg/cm) 2 (%) content (%) 1652.23 4.40.03 3.20.03 5.31 0.35 99.01 F1 1644.53 4.40.01 3.20.04 5.26 0.36 98.9 F2 99.55 1650.98 4.40.04 3.20.06 5.51 0.35 F3 1642.78 4.40.03 3.20.08 5.47 0.38 98.8 F4 1661.57 4.40.02 3.20.07 5.15 0.41 99.27 F5 1651.34 4.40.01 3.20.02 5.34 0.36 98.69 F6 *Each value represents the mean standard deviation (n = 10) 3. RESULT AND DISCUSSION 3.1. In-vitro dissolution studies: In-vitro drug release study of Nifedipine was carried out in 1.2 N HCl and 6.8 pH phosphate buffer for 24 hours. In order to find out the order of release and the mechanism, which predominantly influences the drug release from the tablet, the in-vitro dissolution data was subjected to graphical treatment i.e. percentage cumulative drug release Vs time. The results are shown in Table 3. The in-vitro dissolution profile is graphically represented in Figure.1 for different formulations. The releases of Nifedipine from sustained release tablet of the various formulations varied according to the ratio of different polymers. In the case of F1 the drug-HPMC K15 ratio was 1:1.5 and the drug release rate at the end of 24th hour was found to be 50.34%. F2 formulation was taken with the 60mg of HPMC K15 (1:2) to control the release of Nifedipine from the tablet. The drug release rate was found to be 82.99% at the end of 24th hour. In batch F3 was taken with 45mg of HPMC K100 to control the release of Nifedipine from the tablet. The composition and processing technique were same as that of other batches. It releases 95.67% of drug release at the end of 24th hour, so it is better than the other formulation. In batch F4, 60 mg of HPMC K100 was taken as polymer. The drug release rate was found to be 85.98% at the end of 24th hours. In batch F5, 1:1.5 ratio of drug and PVP used to control the release of Nifedipine from the tablet. The drug release at the end of 24th hour was found to be 58.77%. In formulation F6, 60 mg PVP which is 2:1 ratio with the drug was taken as polymer concentration. In this batch the release is controlled for 24th hour and was found to be 48.14%.
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Table 3. In vitro percentage drug release of different formulations Time in hrs F1 F2 F3 F4 F5 0.5 1.006 1.331 1.956 1.617 1.094 1 2.376 4.69 6.086 5.187 3.452 2 5.85 12.106 10.321 12.184 7.717 4 9.324 17.38 19.176 16.965 12.876 6 12.94 23.178 28.656 24.345 17.783 8 18.467 31.876 37.633 35.171 23.965 10 26.99 44.976 47.874 55.793 33.849 12 35.678 61.003 55.746 73.179 42.176 16 42.789 73.543 83.753 78.789 49.134 20 48.569 79.156 92.718 83.759 54.768 24 50.34 82.987 95.672 85.987 58.767 Figure1. In-vitro dissolution profile of different formulations
P erc entag e drug releas e
120 100 80 60 40 20 0 0 4 8 12 T im e in hours 16 20 24
F6 0.874 1.405 3.194 6.118 10.276 16.984 21.561 28.789 39.345 44.478 48.143
Dissolution profile F1-F6
F1 F2 F3 F4 F5 F6
3.2. Effect of Different Media on the Dissolution of the Optimized Formulation (F3): The dissolution profile of optimized formulation at different release media was evaluated and results were shown in Table 4. Table 4. Effect of different media on the dissolution of the optimized formulation (F3) Figure 2. EffectOptimiz of different media 3) on the dissolution ed F ormulation(F of the optimized formulation (F3)
P erc en tag e releas e
E ffec t of Different Media on the Dis s olution of the
Time (Hrs) DIW* SGF** SIF*** 100 0.5 1.334% 0.658% 1.756% 80 DIW 1 5.737% 1.123% 4.187% 60 S GF 2 10.679% 3.546% 10.631% 40 S IF 4 19.103% 5.89% 16.455% 20 6 27.157% 10.145% 23.067% 0 8 37.756% 16.958% 30.123% 0 4 8 12 16 20 24 10 48.535% 22.715% 37.935% Time in hours 12 61.278% 30.374% 50.821% *DIW-Deionised Water 16 75.125% 39.932% 64.083% **SGF-Simulated Gastric Fluid 20 85.149% 44.918% 76.713% ***SIF-Simulated Intestinal Fluid 24 91.347% 46.119% 80.554% To study the effect of release media on drug release, the release was conducted in deionised water (DIW), Simulated gastric fluid(SGF) and simulated intestinal fluid(SIF), respectively. A significant difference in release profile could be found. In other words, the sustained release tablet system exhibited a media dependent release. Thus it may be expected that the fluid in different parts of gastrointestinal tract affect drug release of sustained release tablet system. 3.3. Effect of Speed of Rotation on Dissolution Profile of Optimized Formulation (F3): The optimized formulation F3 was evaluated for dissolution profile at different speed of rotation and results were shown in Table 5. The result showed that there was no significant difference in release profile under different agitation rates. Therefore, it might be expected that the mobility of the gastrointestinal tract hardly affects the drug release of sustained release tablet system.
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Table 5. Effect of speed of rotation on dissolution profile of optimized formulation (F3) Time (Hrs) 50 rpm 100 rpm 0.5 1.472% 1.956% 1 6.501% 6.086% 2 10.122% 10.321% 4 18.934% 19.176% 6 27.347% 28.656% 8 35.825% 37.633% Figure.3. EEffect ofofSpeed of on Dissolution ffec t of S peed R otation on DisRotation s olution P rofile of Optimiz ed F ormulation (F 3) Profile of Optimized Formulation (F3)
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P erc en tag e d ru g releas ed
P erc en tag e releas e
Time (Hrs) 10 12 16 20 24
50 rpm 44.721% 49.868% 79.438% 86.768% 91.513%
100 rpm 47.874% 55.746% 83.753% 92.718% 95.672%
Figure.4. Comparison Optimized Formulation C omparis on of Optimiz edof F ormulation (F 3) with Marketed (F3) with Marketed Formulation F ormulation.
120 100 80 60 40 20 0 0 0.5 1 2 4 6 8 10 12 16 20 24 Optimiz ed Marketed
100 80 60 40 20 0 0 4 8 12 Time in hours 16 20 24 50 rpm 100rpm
Time in hours
3.4. Comparison of Optimized Formulation (F3) with Marketed Formulation: The optimized formulation F3 was compared with marketed formulation for cum. % drug release. The results are shown in Table 6. The comparison of the optimized formulation (F3) with the marketed formulation showed a satisfactory result and is found to be acceptable. Table 6. Comparison of Optimized Formulation (F3) with Marketed Formulation Time Optimized Marketed Time (Hrs) Optimized Marketed (Hrs) Formulation Formulation Formulation Formulation 0.5 1.956 3.396 10 47.874% 48.777% 1 6.086% 7.415% 12 55.746% 66.383% 2 10.321% 11.354% 16 83.753% 89.445% 4 19.176% 19.906% 20 92.718% 96.208% 6 28.656% 30.637% 24 95.672% 99.908 8 37.633% 38.213% 3.5. Stability studies: The batch F3 was selected as an optimum batch and the stability study was carried out at room temperature and at accelerated condition of 40C/75 % RH condition for a period of two month. The dissolution profile at accelerated temperature did not show any significant reduction in the release rate of drug from formulation which is found to be acceptable and the results were shown in Table 7. Table 7. Dissolution Profile of Optimized Formulation F3 at Room Temp and at Accelerated Temp Time Cumulative Percentage Release Room Temperature Accelerated Temperature Initial 30 Days 60 days Initial 30 Days 60 Days 0 0 0 0 0 0 0 0.5 1.956 1.652 1.033 1.956 1.652 0.933 1 6.086 5.897 5.663 6.086 5.897 3.663 2 10.321 10.136 9.813 10.321 9.875 8.713 4 19.176 18.984 18.443 19.176 18.367 17.243 6 28.656 27.896 27.017 28.656 27.677 25.077 8 37.633 36.576 35.574 37.633 35.976 32.774 10 47.874 46.302 45.056 47.874 44.902 40.756 12 55.746 53.794 52.614 55.746 52.294 49.014 16 83.753 82.645 80.943 83.753 80.945 77.443 20 92.718 90.856 88.996 92.718 89.556 85.996 24 95.672 93.834 92.486 95.672 92.744 89.486
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4. SUMMARY AND CONCLUSION Under preformulation study, the organoleptic properties were complied with the BP specification. Physical properties such as bulk density and tapped density were more in case of granules ready for compression than that of Nifedipine raw powder. In vitro studies showed formulation F3 followed the sustained drug release. The coating solution prepared by using polymethacrylate polymer was the ideal to formulate a sustained release drug delivery system.
Dis s olution P rofile of Optimiz ed F ormulation F 3 at Figure.5. Dissolution Profile of Optimized R oom T emp Formulation F3 at Room Temp
Figure.6. Dissolution Profile of Optimized D is s olution P rofile of Optimiz ed F ormulation F 3 at FormulationAc F3 at Accelerated Temp c elerated T emp.
120
D is s olution profile at ac c elerated temperature
120 100 80 60 40 20 0 0 4 8 12 Time in hrs 16 20 24 Initial 30 Days 60 Days
100 80 60 40 20 0 0 4 8 12 T ime in hours 16 20 24 Initial 30 Days 60 Days
The compatibility evaluation was carried out by FT-IR spectroscopy analysis. The study revealed that there were no interactions between the drug and the polymers. Hence the drug and polymers were compatible. The optimized formulation F3 was evaluated on the basis of Pharmacopoeial specifications. The physical parameters like thickness, diameter, hardness, friability, weight variations were carried out. The assay was carried out for optimized formulation and the result was found to be 99.55%. The optimum release was shown with HPMC K 100 in ratio 1.5:1 with Nifedipine. The optimized formulation showed a satisfactory release profile on comparison with the marketed formulation. The drug release was found to decrease with the increase in the weight of PVP. The drug release was found to be more with HPMC than with PVP. The similarity factor f2 was applied between the dissolution profile of optimized batch and the theoretical dissolution profile, which also indicate a decent similarity between both dissolution profiles. The optimized batch was kept for stability studies at room temperature (25C 2C/ 60% 5% RH) and at accelerated temperature (40C 2C/ 75% 5% RH) in stability chamber for 60 days. The result of stability studies revealed no change in physical appearance, drug content and in vitro dissolution profile, hence F3 formulation was found to be stable. The drug release from the selected formulation (F3) was plotted for the zero order kinetics. From the results obtained, it can be concluded that formulation F3 has achieved the objectives of sustained drug release, patient compliance and cost effectiveness as a single daily dose of the drug. 5. ACKNOWLEDGEMENTS Authors are thankful to Prof.(Dr.) B.Jayakar, Principal, Vinayaka missions college of pharmacy, Salem, Tamilnadu, India providing all the facilities for this research work. REFERENCE Kanvide SA, Kulkarni MS, Stability of oral solid dosage form-a global perspective, Pharma Times, 37(5), 2005, 9-15 Rong Kun Chang, Joseph R Robinson, Sustained Drug Release from tablets and particles through coating, Pharmaceutical Dosage forms, Leon Lachman, H.A Librman, Tablet Vol-3, 1990, 374-412. Shargel L, Yu ABC, Modified release drug products in: Applied Biopharmaceutics and Pharmacokinetics, 4th ed. McGraw Hill, 1999, 169-171. United States Pharmacopia, XXIV NF 18, United State Pharmacopeia Convention, Rockville, 1995, 17911806. Vyas SP, Khar RK, Controlled Drug Delivery, Concepts and Advances, Ist Ed, vallabh prakashan, 2002, 156189. Wolfgang A Ritschel, Gregory L Kearns, Hand Book of Basic Pharmacokinetics, 2004, 5 American Pharmaceutical Assosiation, 484-485. Xuan Ding Adam, W G Alani, Joseph R Robinson, Sustained Release Dosage forms in Remingtons Phramaceutical sciences. (Pub: Lippincot Williams & Wilkins) Gennaro A R et.al (2005), 21 st edn, Vol-I, 1600-1610.
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FORMULATION, DEVELOPMENT, EVALUATION OF CALCITRIOL AND CLOBETASOL PROPIONATE OINTMENT

Pasupathi A*, Palanisamy P, B Jaykar, R Margret Chandira, B S Venkateswarlu Vinayaka Missions College of Pharmacy, vinayaka missions university, Salem, India *Corresponding author: Email: palanisamy2907@gmail.com ABSTRACT Psoriasis vulgaris is a common skin disorder characterized by focal formation of inflamed, raised plaques that constantly shed scales derived from excessive growth of skin epithelial cells. Prevalence of Psoriasis is 2-3 % in general population. Currently, Topical Corticosteroids remain a pivotal treatment due to their effective anti-inflammatory properties; however potential adverse effects associated with chronic application limit long-term continuous therapy. Vitamin D analogues provide another mechanism of action reducing lesions through effects on keratinocytes and on cytokine environment. A topical combination of corticosteroid & Vitamin D derivative appears to provide a balanced approach to psoriasis treatment. When Calcitriol is used in combination with topical steroids, some improvement in psoriasis treatment was observed. The main side effect of Calcitriol is skin irritation. Topical steroids Clobetasol used in conjunction with Calcitriol may lessen skin irritation. Combination reduces hazards associated with long term use of topical Corticosteroids (atrophy and rebound) as well as irritation associated with Calcipotriol. Various formulations of Calcitriol (0.0003%) and Clobetasol Propionate (0.05%) were taken for optimization in relation to ointment base, consistency, ointment stability, stability with antioxidant, stability with different preservatives, and effect of temperature of Calcitriol phase addition to bulk white petrolatum base of ointment. Observations of all formulations for physical characterization and drug release profile had shown that, all comply with the specifications of official pharmacopeias and or standard reference. It was observed that, formulation batch- F12 selected as optimized formulation of Ointment, as it fulfills all requirements of Topical ointment. KEY WORDS: Psoriasis vulgaris, Calcitriol (0.0003%) and Clobetasol Propionate (0.05%). 1. INTRODUCTION Psoriasis vulgaris (Dr. Peter Pugliese, 2001) is a common skin disorder characterized by focal formation of inflamed, raised plaques that constantly shed scales derived from excessive growth of skin epithelial cells. Prevalence of Psoriasis is 2-3%in general population. The disease is defined by a series of linked cellular changes in the skin, Hyperplasia of epidermal keratinocytes, vascular hyperplasia and ectasia and infiltration of T lymphocytes, neutrophils, and other types of leucocyte in affected skin. Psoriasis (Dr. Robert E. Connolly, 2009) is T-cell-mediated autoimmune disorder. The process begins with an environmental factor, perhaps a viral antigen, which induces T cells to produce cytokines. The cytokines stimulate keratinocyte proliferation and production of antigenic adhesion molecules in the dermal blood vessels. These adhesion molecules further stimulate T cells to produce cytokines, thus perpetuating the response. 1.1. Rationale for combination of Calcitriol and Clobetasol propionate: Currently, topical corticosteroids remain a pivotal treatment due to their effective anti-inflammatory properties; however, potential adverse effects associated with chronic application limit long therapy. Vitamin D analogues provide another mechanism of action, reducing lesions through effects on both keratinocytes and on cytokine environment. A topical combination of corticosteroid and vitamin D derivative appears to provide a balanced approach to psoriasis treatment. When Calcitriol is used in combination with topical steroids, psoriasis improves more than with either agent alone. The main side effect of Calcitriol is skin irritation. Topical steroids Clobetasol propionate used in conjunction with Calcitriol may lessen skin irritation. Combination reduces the hazards associated with the long-term use of topical corticosteroids. Aim of the present work was to formulate efficacious and safe and stable semisolid dosage form with combination therapy for a super-potent topical Steroid and a topical Vitamin. In order to Reduce dosing frequency. To provide synergistic effect. To provide Ease of application. To increase bioavailability of drug. 1.2. Objective of work: The objectives of the work were to formulate topical preparation of Calcitriol and Clobetasol propionate combination ointment. The combination in single pharmaceutical composition of Calcitriol with Clobetasol propionate is not without certain problems. The reason for this is that the Calcitriol is unstable in aqueous media and more particularly at acidic pH values, where as Clobetasol propionate is unstable in basic environment. The critical role of vehicle is to enhance therapeutic efficacy and patient compliance. So Attempts have been made to develop Calcitriol and Clobetasol propionate combination ointment. 2. MATERIALS AND METHODS Clobetasol Propionate (Micronized) was procured from Sumit Lab and Calcitriol was procured from Biocon (India), White petrolatum (W.S.P.) was gifted by Unicorn / Wax oils (India), Liquid paraffin was gifted by Wax oils (India), Cetosteryl alcohol, Sorbiton sesquioleate was gifted by Croda (India), Shea Butter was gifted by Dow Corning (India), PEG-4000 was gifted by Clariant (India), White bees Wax was gifted by Manali Petro Volume 1 Issue 1 January February 2013 Page 95
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(India), Propylene glycol was gifted by Wax oils LTD(India), Chlorocresol, Vitamin E Acetate was gifted by Merck Ltd(India), Caprilic capric tri-glyceride (Fractionated coconut oil) was gifted by Subhash chemicals (Bangalore), Citric Acid, Monohydrate, Sodium Citrate, Dihydrate was gifted by RFCL LTD (India), Butylated Hydroxytoluene (BHT) was gifted by Loba chem.(India). 3. FORMULATION OF BATCHES Table No. 1 Formulation of trial batches from F1 to F6 Ingredient (%w/w) Batches (Quantities in % w/w) F1 F2 F3 F4 F5 F6 White Petrolatum 64.909 --64.90 84.90 -Bees Wax 5.000 -1.25 5.00 -1.25 Shea Butter 5.000 --5.00 --Butylated Hydroxytoluene 0.04 0.04 ----Propylene Glycol 10 -47.45 10.00 10.00 47.45 Clobetasol propionate 0.05020 0.05020 0.05020 0.05020 0.05070 0.05020 Caprylic capric Triglyceride 15.00 --15.00 --Calcitriol 0.000307 0.000307 0.000307 0.000307 0.000307 0.000307 Vitamin E Acetate ----0.05 0.05 Liquid Paraffin ------Sorbiton Sesquioleate ------Gelucire 44/14 ---0.05 --PEG-400 -64.910 ----PEG-4000 -35.00 ----Cetostearyl Alcohol --8.40 --8.40 Glyceryl Monostearate --11.00 --11.00 Stearoyl Macrogol --1.50 --1.50 Chlorocresol --0.08 --0.08 Citric Acid, monohydrate --0.05 --0.05 Sodium Citrate, dehydrate --0.05 --0.05 Purified Water --30.18 --30.18 3.1. Evaluation of formulation: 3.1.1.Measurements of pH: (Amra Osmancevic and Kerstin Landin-Wilhelmsen, 2010) (By Extraction methodology): 2.5gm Ointment sample was taken in 100 ml dry beaker, then 50 ml water was added to it. Beaker was heated on water bath maintained at about 60C to 70C for 10 minutes, cooled to room temperature, and then centrifuged at 3000 rpm for 10 minutes. The pH of supernant water extract was measured by using Labindia 200 + pH meter. The pH measurements were done by using a digital type pH meter by dipping the glass electrode into the ointment formulation. 3.1.2. Determination of viscosity: ( Zaira Kharaeva, 2009) The measurement of viscosity of the formulated Ointment was done by using Brookfield Viscometer (CAP 2000+ Viscometer). Spindle No. 1 was used for the determination of viscosity of ointment. Spindle was rotated at 80 rpm for 30 second for each measurement. The results are shown in Table No.5. 3.1.2. Determination of extrudability: (Christina MP, 1994) (Smith, Jan G, 2010) Extrudability test is the measure of the force required to extrude the material from a collapsible tube when certain amount of force has been applied on it in the form of weight. In the present study the quantity in percentage of cream extruded from the tube on application of certain load was determined. More the quantity extruded, better was the extrudability of ointment. 3.1.3. Determination of spreadability: (Christina MP, 1994) (Smith, Jan G, 2010) One of the criteria for a cream, ointment or gel is that it should possess good spreadability. Spreadability is a term expressed to denote the extent of area to which the cream readily spreads on application to skin or affected part. The therapeutic efficacy of a formulation also depends on its spreading value. Hence determination of spreadability is very important in evaluating ointment characteristics. Special apparatus was designed to study the spreadability of ointment formulations. The spreadability is expressed in terms of time in seconds taken by two slides to slip off from ointment, placed in between the slides under the direction of certain load. Lesser the time taken for separation of twoslides, better the spreadability of ointment. 3.1.4. Microscopic evaluation: Microscopic evaluation of all formulated batches was done by using Compound microscope with the help of MOTIC software. Evaluation of the sample was carried initially and after each stability condition. Procedure: Clean and dry glass slide and cover slip were taken Volume 1 Issue 1 January February 2013 Page 96
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Very small quantity of sample was taken on glass slide Sample was covered by placing cover slip on it. Precaution was taken that there was no entrapment of air Cover slip was slightly pressed to spread the sample uniformly to prepare the thin film It was observed under microscope with different power of lenses like 10x, 40x and 100x.
Table No. 2. Formulation of trial batches from F7 to F12 FIngredient (%w/w) Batches (Quantities in % w/w) F7 F8 F9 F10 F11 F12 White Petrolactum 89.41 84.90 89.90 89.399 84.45 89.399 Bees Wax -----Shea Butter ------Butylated Hydroxytoluene -----0.05 Propylene Glycol 5.000 10.00 5.00 5.00 10.00 5.00 Clobetasol propionate 0.05020 0.05070 0.05020 0.05070 0.05070 0.05070 Caprylic capric Triglyceride 5.000 -----Calcitriol 0.000307 0.000307 0.000307 0.000307 0.000307 0.000307 Vitamin E Acetate 0.05 0.05 0.05 0.05 0.05 -Liquid Paraffin -5.00 5.00 5.00 5.00 5.00 Sorbiton Sesquioleate 0.50 ---0.50 0.50 Gelucire 44/14 ---0.50 --3.1.5. Microbiological enumeration test: 10 gm of product was transferred aseptically to make 100ml of buffered phosphate buffer solution pH 7.2 or fluid soyabean casein digest medium containing 4% polysorbate 20 (solutionA) 3.1.6. Pour-plate method: 1ml of prepared sample was taken and transferred in to sterile Petri dishes. 15 to 20ml of liquefied soyabean-casein digest agar was poured in to sterile petri dishes, suitable for cultivation of bacteria and liquefied sabouraud dextrose agar suitable for cultivation of fungi at not more than 45C then covered them. The sample was mixed with media by tintling or rotating the dishes and contents were allowed to solidify at room temperature. The plates of soyabean-casein digest agar were incubated at 30 to 35C for 3 to 5 days and the plates of sabouraud dextrose agar at 20 to 25C for 5 to 7 days. Plates were selected corresponding to given dilution and showing the highest number of colonies less than 250 for total aerobic microbial count and 50 for total yeast and mould count (TYMC). The arithmetic mean per culture medium of counts was taken and the number of colony forming units (cfu) per gram of sample was calculated. 3.1.7. Interpretation of the results: The total aerobic microbial count (TAMC) was considered equal to the number of cfu found using soyabean-casein digest agar. If colonies of fungi were detected on this medium, they were counted as part of TAMC. The total yeast and mould count (TYMC) was considered equal to the number of cfu found using sabouraud dextrose agar. If colonies of bacteria were detected on this medium, they were counted as a part of TYMC. When the TYMC was expected to exceed the acceptance criteria due to bacterial growth, sabouraud dextrose agar containing antibiotics needed to be use. 3.2. Test for specified micro-organisms 3.2.1. Sample preparation and pre-incubation: A sample was prepared using a 1 in 10 dilution of quantity corresponding not less than 1 gm of product to be examined or 10ml of solution A was transferred aseptically to make 100ml soyabean-casein digest broth. If the product was known to have antimicrobial activity, an inactivating agent such as polysorbate 80, soyalecithin needed to be adding. It was incubated at 30 to 35C for 18 to 24 hours. After 24 hours incubation, the test for Pseudomonas aeruginosa and Staphylococcus aureus were carry out. 3.2.2. Test for Pseudomonas aeruginosa Selection and subculture: After 24 hours of incubation of soyabean-casein digest broth, a loopful of enriched culture was streaked on a plate of centrimide agar. The plates were incubated at 30-35C for 18 to 72 hours. Interpretation: The presence of Pseudomonas aeruginosa was indicated by growth of colonies. This was confirmed by identification tests. The product comply the test if the colonies were not present. Identification tests: Any colony showing greenish colour was subcultured on the surface of pseudomonas agar medium for detection of pyocyanin and pseudomonas agar medium for detection of fluroresin, plates were covered and inverted and incubated at 35-37C for not less than three days. Observation and result: The streaked surface was examined under UV light to determine whether colonies are having characteristics given below. Morphological characteristics: Addition of fluroresin to pseudomonas agar medium shows generally colourless to yellowish colonies and yellowish fluorescence in UV light. Morphological characteristics pseudomonas agar medium for detection of pyocyanin shows generally greenish colonies and bluish fluorescence pseudomonas agar medium in UV light.
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Table No. 2 Morphological characteristics Characteristic colonial fluorescence in UV light Morphology Pseudomonas Agar medium for Generally colourless to Yellowish detection of Pyocyanin yellowish colonies Pseudomonas Agar medium for Generally greenish Blue detection of Pyocyanin colonies Any suspect colonial growth was confirmed by oxidase test. Oxidase test: if growth of suspect colonies occurs, smear the suspected colony on oxidase disc. If there is no development of purple colour within 30 seconds, sample meets the requirement for the test of absence of pseudomonas aeruginosa. 3.2.3. Test for Staphylococcus aureus Selection and subculture: After 24 hours of incubation of Soyabean-Casein Digest Broth, a loopful of enriched culture was streaked on a plate of mannitol salt agar. The plates were incubated at 30-35C for 18 to 72 hours. Interpretation:The presence of staphylococcus aureus was indicated by growth of yellow or white colonies surrounded by yellow zone. This was confirmed by identification tests. The product comply the test if the colonies were not present. Identification tests: In case the colonies characteristics matches with the description as mentioned above, then presence of positive cocci was confirmed by gram staining. Coagulase Test (Staphylococcus aureus): With the aid of an inoculating loop, transfer representative suspect colonies from the agar surfaces of the mannitol salt agar medium to individual tubes, each containing 0.5ml of mammalian, preferably rabbit or horse plasma with or without suitable additives. Incubate in water bath at 37C, examining the tubes at 3 hours and subsequently at suitable intervals up to 24 hours. Test positive and negative sample meets the requirements of the test for absence of Staphylococcus aureus. Table No. 3 Culture Conditions for Inoculum Preparation Medium
Organism Pseudomonas aeruginosa (ATCC No. 9027) Staphylococcus aureus (ATCC No. 6538) Suitable Medium SoybeanCasein Digest Agar SoybeanCasein Digest Agar Incubation Temperature 32.5 2.50C 32.5 2.50C Inoculum Incubation Time 18 to 24 hours 18 to 24 hours Microbial Recovery Incubation Time 3 to 5 days 3 to 5 days
Interpretation criteria: Table No. 4 Acceptance criteria for microbiology interpreted as follows. Limit Maximum acceptable count 101CFU 20 102CFU 200 103CFU 2000 3.2.4. Stability Study:Stability study was performed as per ICH guideline. The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity and light. Therefore, stability studies provide data to justify the storage condition and shelf-life of the drug product. For drug substance, such studies establish the retest date in addition to the storage condition of raw material. 4. RESULT AND DISCUSSION 4.1. Related Substances calculation:
Figure No. 1: Chromatogram for RS of mixed Standard
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Table No. 5 Peak results for RS of mixed standard Name RT Area % Area 10.7 90.3 USP Tailing 1.13 1.093 USP Plate Count 6746 12416
12.85 25468 Clobetasol propionate 17.4 255216 Calcitriol Chromatogram for RS of sample (Optimized batch F12):
Figure No. 2 Chromatogram for RS of Sample for Clobetasol propionate (Optimized batch F12) Table No. 6 Peak results for RS of sample (optimized batch F12) Peak No. 1 2 3 4 5 Name Unknown-1 Unknown-2 Unknown-3 Clobetasol propionate Unknown-4 RT 4.7 5.3 13.9 12.9 5.7 Area 6052 7241 20389 253981 28706 % Area 0.41 0.49 13.93 83.20 1.96 % Impurity 0.0297 0.031 0.100 -0.126
Single maximum impurity- 0.12 Total impurities- 0.2867 RS study of sample (optimized batch, F12) after accelerated stability study at the condition 40 0C/75 %RH showed that related substances formed due to degradation of active ingredients were below the limits prescribed. 4.2. Evaluation of Ointment: The evaluated parameters were pH, viscosity, spreadability, extrudability and assay. Table No.7 Initial evaluation of formulation batches pH * Viscosity* % Drug content* %Drug content* (Poise) (Clobetasol Propionate) (Calcitriol) 6.15 0.01 6.46 0.03 1.29 0.22 6.43 0.04 3.57 0.26 6.21 0.12 5.05 0.12 6.63 0.15 3.58 0.25 98.28 0.17 98.37 1.08 6.75 0.15 4.10 0.14 97.26 0.13 98.46 1.15 6.10 0.15 3.56 0.17 98.41 0.08 97.89 1.23 5.70 0.15 3.55 0.27 98.29 0.05 99.19 0.94 5.25 0.26 3.50 0.37 98.17 0.35 99.42 0.29 5.45 0.26 3.55 0.52 99.40 0.23 98.94 0.54 5.10 0.26 3.49 0.45 99.27 0.25 99.94 0.19 5.32 0.26 3.61 0.57 99.83 0.23 99.62 0.86 Note: * = Mean S.D., CP=Clobetasol propionate, C=Calcitriol
Batch N0. F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12
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Figure No. 3 Diffusion Profile for Calcitriol (Trial Batch F7, F8, F9)
Diffusion Drug Release Profile of Calcitriol
4.2.1.In-vitro Drug release profile of Calcitriol:

Table No.8 Diffusion Profile for Calcitriol (Trial Batch F7, F8, F9)
Time (min) 0 30 60 120 180 240 300
Galderma (innovator) 0 4.3 12.4 37.2 53.9 70.9 86.3
F7 0 2.6 7.4 19.9 32.7 45.3 55.6
F8 0 3.1 8.7 25.3 44.5 59.4 69.8
F9 0 3.4 9.9 28.2 46.7 60.7 75.9
100 90 80 70 60 50 40 30 20 10 0 0 100 200 300 400
% Drug Release
Innovator F7 F8 F9
Time
Table No.9 Diffusion Profile for Calcitriol (Trial Batch F10, F11, F12) Time (min) 0 30 60 120 180 240 300 Galderma (Innovator) 0 4.3 12.4 37.2 53.9 70.9 86.3 F10 0 3.5 9.9 28.7 47.9 62.7 77.9 F11 0 3.9 11.1 32.4 50.2 66.6 84.8 F12 0 4.1 12.6 37.1 54.3 70.5 87.3
Figure No. 4 Diffusion Profile for Calcitriol (Trial Batch F10, F11, F12)
Diffusion drug release profile of Calcitriol
100 90 80 70 60 50 40 30 20 10 0 0 100 200 300 400
%Drug Release
INNOVATOR F10 F11 F12
Time(Min)
Table No.10 Diffusion Profile for Calcitriol (Trial Batch F12)
Figure No. 5 Diffusion Profile for Calcitriol (Trial Batch F12 & Innovator)
Diffusion drug release profile of Calcitriol
100 90 80 70 60 50 40 30 20 10 0 0 100 200 300 400
Time (min) 0 30 60 120 180 240 300
Galderma (Innovator) 0 4.3 12.4 37.2 53.9 70.9 86.3
F12 0 4.1 12.6 37.1 54.3 70.5 87.3
%Drug Release
INNOVATOR F12
Time(Min)
4.2.2. In-vitro Drug release profile of Clobetasol propionate: Table No.11 Diffusion Profile for Clobetasol propionate (Trial Batch F7, F8, F9) Time (min) 0 30 60 120 180 240 300 Galderma (Innovator) 0 9.1 18.4 42.4 60.2 76.6 91.1 F7 0 7.0 12.8 37.1 53.2 69.4 81.4 F8 0 6.4 11.3 30.7 47.5 63.7 78.2 F9 0 7.3 13.4 38.7 55.8 70.1 83.8 Figure No. 6 Diffusion Profile for Clobetasol propionate (Trial Batch F7, F8, F9)
Diffusion Drug Release Profile of Clobetasol propionate
100 90 80 70 60 50 40 30 20 10 0 0 100 200 300 400
% Drug Release
Innovator F7 F8 F9
Time
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In-vitro Drug release profile of Clobetasol propionate: Table No.12 Diffusion Profile for Clobetasol propionate (Trial Batch F10, F11, F12) Time Galderma F10 F11 F12 (min) (Innovator) 0 0 0 0 0 30 9.1 7.4 8.5 9.4 60 18.4 13.5 15.6 18.9 120 42.4 38.4 39.8 42.7 180 60.2 55.9 58.2 61.1 240 76.6 70.3 73.5 77.0 300 91.1 83.5 87.4 92.1 In-vitro Drug release profile of Clobetasol propionate: Table No.13 Diffusion Profile for Clobetasol propionate (Trial Batch F12) Time(min) 0 30 60 120 180 240 300 Galderma(Innovator) 0 9.1 18.4 42.4 60.2 76.6 91.1 F12 0 9.4 18.9 42.7 61.1 77.0 92.1
Figure No. 7 Diffusion Profile for Clobetasol propionate (Trial Batch F10, F11, F12)
120 110 100 90 80 70 60 50 40 30 20 10 0 0 100 200 300 400
% Drug Release
Innovator F10 F11 F12
Time(Min)
Figure No. 8 Diffusion Profile for Calcitriol (Trial Batch F12 & Innovator)
100 90 80 70 60 50 40 30 20 10 0 0 100 200 300 400
% Drug Release
Innovator F12
Time(min)
4.3. Optimization of Ointment base for consistency: The base selection was carried out in F3, F4 and F5. First batch was formulated as per the Galderma ointment and Calcitriol was incorporated in Caprylic capric triglyceride, consistency suddenly found to be dropped and drug phase was not dispersed uniformly. In next batch F2, micronized form of both the drugs was used and Calcitriol phase was incorporated in PEG base after emulsification at 700C. Micronized form of drug gave smooth texture to ointment but consistency again found to be dropped. After that to improve the consistency, in trial batch F3 Dermovat Cream base with 8.4% w/w Cetostearyl alcohol was incorporated in water phase. Viscosity was found good but after the stability testing (e.g. 25C/60%RH, 30C/65%RH, 40C/75%RH) assay results indicated that concentration of both drugs were decreasing day by day. F1 & F3 has better consistency than PEG base. In next trial F4 white petrolatum base Gelucire 44/14 as emulsifier was used, but consistency was increased too high due to Shea butter and bees wax. After evaluation it was found that consistency was needed to reduce slightly and spreadability needed to improve as it showed tackiness. Results showed that F5 exhibited good spreadability property as compared to previous batches. 4.4. Batches with the antioxidant: In the Innovator formula, BHT was used as antioxidant. The antioxidant was used to find out the more stable formulation as both the drugs are prone to oxidative degradation. The continued degradation of drugs results in a total loss of its therapeutic activity. Previous batch F2 was prepared with single antioxidant i.e., BHT. And next trial batch F5 was taken with antioxidant Vitamin E Acetate were evaluated for the assay initially and after the stability testing (e.g. 25C/60%RH, 30C/65%RH, 40C/75%RH). The antioxidant vitamin E was added to WSP base, PEG base, & Cream base formulation batches. (F2, F5, F6). But according to microbiological enumeration test, it was observed that batch F5 with white petrolatum and Vitamin E Acetate having better stability than PEG base or Cream base. Finally Vitamin E Acetate was selected as antioxidant for next formulation trial batches.
F2
F5 Figure No. 9: Microscopic evaluation of F2, F5 and F6
F6
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4.5. Batches with different Solubilizer: To evaluate the effect of Solubilizer on stability of formulation, batches with the different Solubilizer concentrations were evaluated for assay to find out the effect of Solubilizer on % drug content after stability study. Initial batches showed that variation in Solubilizer effects on assay and variation of drug content at different points of the SS container as such top, middle and bottom. In trial batch F7 Fractionated coconut oil (5%) was used as Solubilizer for Calcitriol with propylene glycol (5%) for Clobetasol propionate as Solubilizer. In trial batch F8 Liquid paraffin (5%) used as Solubilizer for Calcitriol with propylene glycol (10%) for Clobetasol propionate as Solubilizer. In trial batch F9 Liquid paraffin (5%) was used as Solubilizer for Calcitriol with propylene glycol (5%) for Clobetasol propionate as Solubilizer. It provides uniformity of both drugs dispersion in ointment base. The trial batches were evaluated initially and after the stability period of 3 month at different stability conditions. 4.6. Selection of Emulsifier for stabilization of Ointment (F10): As in trial batch F7, F8 & F9 oily appearance was observed on the top surface of the ointment, due to separation of Solubilizer to the some extent, after keeping over night. This was due to separation of propylene glycol or liquid paraffin from white petrolatum base. So it was needed to add an Emulsifying agent. The two emulsifiers were selected as Gelucire 44/14 & Sorbiton sesquioleate. HLB of Sorbiton sesquioleate = 03.7 HLB of Gelucire 44/14= 14 As the formulation to be prepared was consist of a combination of more than one oleaginous material such as water-insoluble hydrophobic oils and fats. Most of the early ointment bases used to be exclusively oleaginous in nature. All the previous batches were formulated without the surfactant. Except trial batch F4. Consistency was found good but separation of propylene glycol or liquid paraffin from white petrolatum base. To overcome the non uniformity of Solubilizer with white petrolatum base, trial batch F10 was not found good. But Trial batch F11 given better uniformity and homogenousity of Solubilizer with white petrolatum base. Fraction of surfactant was added in both the drug phases (i.e., Clobetasol propionate and Calcitriol). 4.7. Batches with different Temp. of addition of Calcitriol phase to the bulk white petrolatum base: In, Trial batch F11 & F12 the Calcitriol in liquid paraffin phase was added to the bulk White petrolatum base at 70oC to 72oC & 35oC to 37oC. F12 batch was observed more consistent than F11. Hence effect of Temp. On formulation was optimized. Depending on the antimicrobial stability, assay and In-vitro Drug release profile, final optimized batch F12 was selected. 4.8. Stability study of different batches: Stability study was carried out according to the ICH guidelines at 250C/60% RH, 300C/75% RH, 400C/75% RH for three months. The formulation was packed in aluminum lacquered tube and kept in stability chamber. All the parameters were evaluated after stability study. Table No. 14 Stability results after 1 month
Parameters pH* Viscosity* (Poise) Spreadability.* (gm cm/sec) Extrudability* (%) %Assay (CP)* %Assay(C)* pH* Viscosity* (Poise) Spreadability.* (gm cm/sec) Extrudability* (%) %Assay (CP)* %Assay(C)* pH* Viscosity* (Poise) Spreadability.* (gm cm/sec) Extrudability* (%) %Assay (CP)* %Assay(C)* F7 7.110.04 3.700.11 4.110.16 760.94 99.461.25 98.831.05 7.210.04 3.660.15 4.160.14 780.87 98.981.43 98.781.21 7.920.28 3.600.12 4.250.11 800.87 97.341.12 97.111.01 Stability result at 250C/60 %RH F8 F9 F10 F11 7.720.05 5.160.10 5.220.25 5.620.12 3.620.08 3.640.05 3.570.04 3.620.04 4.200.08 4.120.07 4.200.23 4.270.11 800.54 820.16 810.26 160.33 99.470.72 99.621.01 98.210.72 99.740.22 99.530.89 99.630.96 98.100.49 99.000.31 Stability result at 300C/75 %RH 7.760.12 5.180.02 5.190.25 5.330.17 3.600.27 3.590.04 3.580.71 3.620.26 4.240.26 4.180.08 4.180.21 4.200.18 801.01 900.84 810.42 840.06 99.450.14 99.541.03 99.140.04 98.700.17 99.530.04 99.620.78 99.050.26 99.000.42 Stability result at 400C/75 %RH 8.300.18 5.160.15 5.190.25 5.310.54 3.550.34 4.180.12 811.12 99.191.53 99.231.19 3.620.54 4.120.65 910.33 99.541.34 99.520.23 3.580.06 4.180.21 810.87 99.140.06 99.050.26 3.540.06 4.160.27 840.06 98.640.42 99.040.42 F12 5.620.05 3.610.18 4. 500.08 870.16 98.780.62 99.200.43 5.600.23 3.610.08 4.210.26 860.05 99.710.42 99.660.19 5.580.52 3.610.87 4.210.06 860.05 99.700.27 99.580.06
Note: * = Mean Standard deviation, CP=Clobetasol propionate, C=Calcitriol Once the ointment base was optimized, trials with different antioxidants, with different Solubilizer and temperature of addition of Calcitriol phase to bulk phase (F7-F12) were formulated and kept for the stability study. Results of the stability studies are shown in Tables 14, 15 and 16. Assay of F3 and F4 was found to be dropped at the accelerated stability conditions of 400C/75 %RH. This drop in assay may be due to ineffective Volume 1 Issue 1 January February 2013 Page 102
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protection against oxidative degradation. Use of antioxidants in F5 and F6 gave better results. Also the dispersion of both drugs in ointment base was not uniform and homogenous so in F7, F8 and F9 different Solubilizer with different concentration were used. 5% propylene glycol and 5% liquid paraffin for Clobetasol propionate and Calcitriol found better results. In F10 to overcome the non uniformity of solubilizer with white petrolatum base was optimized by using Emulsifying agent Gelucire 44/14 and Sorbiton sesquioleate. Result of Sorbiton sesquioleate was found better. In trial F11, F12 Temp. of addition of Calcitriol phase to the bulk white petrolatum base was optimized. Also % drug content was affected to minor extent was considerable. Final optimized formulation was selected on the basis of Microbiological enumeration testing of F9-F12. 4.9. Related Substances: Determination of related substances of Clobetasol propionate (active ingredient) was carried out for the batches kept for stability study at different conditions (i.e., F7-F12). Determination of related substances of Calcitriol (active ingredient) was not carried out due to conc. was too much low 0.0003% w/w. All the stability batches showed the total impurities for Clobetasol propionate below the prescribed limit 4.10. Microbiological Enumeration Test: The preservative efficacy test was designed to perform the quantitative and qualitative estimation of specific viable microorganisms present in formulation by using various cultures of bacteria and fungi (Staphylococcus aureus, Pseudomonas aeruginosa) for 28 days. Cfu/gm of sample was counted at the interval of 7 days. Results are tabulated in Table No. 17. Table No. 15 Stability results after 2 months
Parameters pH* Viscosity* (Poise) Spreadability.* (gm cm/sec) Extrudability* (%) %Assay (CP)* %Assay(C)* pH* Viscosity* (Poise) Spreadability.* (gm cm/sec) Extrudability* (%) %Assay (CP)* %Assay(C)* pH* Viscosity* (Poise) Spreadability.* (gm cm/sec) Extrudability* (%) %Assay (CP)* %Assay(C)* F7 7.250.54 3.650.02 4.410.17 760.27 99.350.65 98.640.21 7.210.81 3.660.11 4.160.31 780.35 98.981.02 98.780.22 7.920.28 3.600.12 4.250.11 800.12 97.040.06 97.050.01 F8 8.130.18 3.510.96 4.180.08 800.04 99.310.72 99.370.21 7.760.12 3.600.07 4.240.13 801.01 99.011.03 99.140.41 8.300.31 3.550.34 4.180.12 801.09 98.860.42 98.940.13 Stability result at 250C/60 %RH F9 5.160.09 3.640.04 4.120.41 F10 5.220.19 3.570.22 4.200.33 F11 5.280.19 3.600.18 4.250.08 820.49 98.70.62 99.000.89 5.330.23 3.620.53 4.200.12 840.06 98.500.06 99.000.23 F12 5.580.12 3.620.04 4.501.21 860.32 99.700.07 99.200.31 5.560.12 3.610.05 4.210.21 860.08 99.600.37 99.150.12 5.550.02 3.610.15 4.210.12 860.65 99.430.27 99.100.02
820.12 810.21 99.640.15 98.210.22 99.630.31 98.100.16 Stability result at 300C/75 %RH 5.180.02 5.190.78 3.590.04 3.58 0.11 4.180.18 4.180.25 900.71 99.541.03 99.621.05 810.42 99.140.24 99.050.41
Stability result at 400C/75 %RH 5.200.15 5.270.25 5.310.54 3.610.54 3.340.06 3.540.06 4.020.42 4.110.21 4.160.27 910.33 99.451.04 99.020.21 810.87 99.140.06 99.050.46 840.23 98.640.17 99.040.65
All batches with different preservative combinations showed decrease in Cfu count but F12 containing Vitamin E Acetate (0.5% w/w) was found to be most effective in inhibiting the microbial growth. Preservative in F12 was found to be effective as, The concentration of viable bacteria was not more than 0.1% of initial concentration by the 14 th day. The concentration of each test microorganism remained at or below the designated levels during the remainder of 28 day test period. On the basis all the evaluation parameters of formulation including assay, RS, viscosity, spreadability, extrudability and emulsion stability initially and after exposure to accelerated stability conditions and finally on the basis of antimicrobial preservative efficacy testing, F12 was selected as optimized formula for topical delivery of Clobetasol propionate and Calcitriol. 4.11. Microscopic evaluation of optimized batch: From microscopy it was observed that the uniform sized globules were distributed throughout White petrolatum base (Figure 10). Microscopic evaluation after accelerated stability studies showed that ointment was stable as retaining its integrity and globule size did not increase to considerable extent.
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Pasupathi A et.al. Parameters pH* Viscosity* (Poise) Spreadability.* (gm cm/sec) Extrudability* (%) %Assay (CP)* %Assay(C)* pH* Viscosity* (Poise) Spreadability.* (gm cm/sec) Extrudability* (%) %Assay (CP)* %Assay(C)* pH* Viscosity* (Poise) Spreadability.* (gm cm/sec) Extrudability* (%) %Assay (CP)* %Assay(C)* F7 7.300.26 3.650.14 4.501.21 760.27 99.040.65 98.130.21 7.210.81 3.660.11 4.160.31 780.32 98.79.17 98.720.05 7.920.06 3.600.12 4.250.11 800.13 97.000.06 96.870.15
Table No. 16 Stability results after 3 months

Stability result at 250C/60 %RH F8 F9 F10 F11 8.130.18 5.460.09 5.320.19 5.280.19 3.510.96 3.640.04 3.570.22 3.600.18 4.060.08 4.120.41 4.200.33 4.250.08 800.04 99.120.72 820.12 99.640.15 810.21 98.210.22 820.49 98.70.62 F12 5.510.12 3.620.04 4.410.81 860.32 99.700.07 99.200.31 5.500.12 3.610.05 4.210.21 860.08 99.610.37 99.150.12 5.510.02 3.610.21 4.220.12 860.65 99.310.31 99.140.32
99.190.21 99.630.31 98.100.16 99.000.89 Stability result at 300C/75 %RH 7.760.12 5.380.02 5.280.78 5.330.23 3.600.07 4.240.13 801.01 98.450.23 98.710.37 3.590.02 4.180.18 900.71 99.541.03 99.621.05 3.58 0.11 4.180.25 810.42 99.140.24 99.050.41
0
3.620.53 4.200.12 840.06 98.700.06 99.000.23
Stability result at 40 C/75 %RH 8.300.31 5.200.36 5.270.25 5.390.54 3.550.34 4.180.23 801.09 98.290.23 98.580.37 3.610.54 4.020.42 910.15 99.391.04 99.120.26 3.340.06 4.110.21 810.11 99.140.16 99.020.13 3.540.18 4.360.27 840.31 98.520.06 99.000.28
Table No.17 Results of Microbiological Enumeration Test Evaluation time Trial Culture used Staph. aureus Pseudomonas aeruginosa Batch
ATCC 6538 ATCC 9027
0 hour count Cfu/gm of sample Cfu x 103
7 days count Cfu/gm of sample Cfu x 103
F9 F10 F11 F12 F9 F10 F11 F12 F9 F10 F11 F12 F9 F10 F11 F12 F9 F10 F11 F12
213 215 243 209 212 189 189 183 132 45 49 39 59 9 7 3 21 0 1 0
210 208 234 201 221 203 196 195 113 39 41 45 54 5 5 2 12 1 0 0
14 days count Cfu/gm of sample
Figure No. 10: Microscopy of optimized batch (F12) Cfu = Colony forming unit
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5. SUMMARY AND CONCLUSSION A topical combination of corticosteroid & Vitamin D derivative appears to provide a balanced approach to psoriasis treatment. When Calcitriol is used in combination with topical steroids, Psoriasis improves more than one agent alone. The main side effect of Calcitriol is skin irritation. Topical steroids Clobetasol used in conjunction with Calcitriol may lessen skin irritation. Combination reduces hazards associated with long term use of topical Corticosteroids. Various formulations of Calcitriol (0.0003%) and Clobetasol Propionate (0.05%) were taken for optimization in relation to ointment base, consistency, ointment stability, stability with antioxidant, stability with different preservatives, and effect of temperature of Calcitriol phase addition to bulk white petrolatum base of ointment. Initial batch based on innovator Galderma showed sudden drop in consistency and also the drugs were not dispersed uniformly in formulation as it showed grittiness. From trial batch F2 micronized form of both the drugs was used to prevent grittiness. In F2, F3, F4 and F5 selection of base was carried out to improve the consistency. Homogeneity and consistency were improved in F5 than F1, F2, F3 and F4 but it showed tackiness as previous batches. To improve spreadability, Bees wax, Shea butter was not added in F5. Homogeneity of F5 & F6 was satisfactory with uniform globule size and drug dispersion. In trial batch F7 Fractionated coconut oil (5%) was used as Solubilizer for Calcitriol with propylene glycol (5%) for Clobetasol propionate as Solubilizer. In trial batch F8 Liquid paraffin (5%) used as Solubilizer for Calcitriol with propylene glycol (10%) for Clobetasol propionate as Solubilizer. In trial batch F9 Liquid paraffin (5%) was used as Solubilizer for Calcitriol with propylene glycol (5%) for Clobetasol propionate as Solubilizer. Trial batch F11 & F12 the Calcitriol in liquid paraffin phase was added to the bulk White petrolatum base at 70oC to 72oC & 35oC to 37oC. F12 batch was observed more consistent than F11. Hence effect of Temperature on formulation was optimized. Depending on the antimicrobial stability, assay and In-vitro Drug release profile, final optimized batch F12 was selected. Observations of all formulations for physical characterization, assay & in-vitro drug release had shown that, all comply with the specifications of official pharmacopeias and or standard reference. It was observed that, formulation batch- F12 selected as optimized formulation of Ointment, as it fulfills all requirements of Topical ointment identical with innovator product ( GALDERMA). It was concluded that ointment of Calcitriol & Clobetasol propionate could be formulated components and processing aspects. 6. ACKNOWLEDGEMENTS Authors are thankful to Prof.(Dr.) B.Jayakar, Principal, Vinayaka missions college of pharmacy, Salem, Tamilnadu, India providing all the facilities for this research work. REFERENCES Amra Osmancevic and Kerstin Landin-Wilhelmsen, Vitamin D status in psoriasis patients during different treatments with phototherapy, Journal of Photochemistry and Photobiology, B Biology, 101(2), 2010, 117-121. Christina MP, Mouse Model of psoriasis, Research in Immunology, 1994, 145(5), 357-362. Dr. Peter Pugliese, Physiology of the Skin, Workbook and Study Guide, Allured Pub Corp, 1st edition, 2001, 35-41. Dr. Robert E. Connolly, Psoriasis Can Be Cured, 2009, 350-353. Smith Jan G, United States Application US2010/0249060 A1, Topical formulation of low level Clobetasol propionate for treating disorders of the skin and mucous membranes, 2010. The United State Pharmacopoeia 2007, Volume 32(I) The official compendia of standard, USP-30, NF-25, 1787. Zaira Kharaeva, Elena Gostova, Chiara De Luca, Desanka Raskovic, Liudmila Korkina, Clinical and biochemical effects of coenzyme Q10, vitamin E, and selenium supplementation to psoriasis patients, Journal of Nutrition, 25(3), 2009, 295-302.
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HEPATOPROTECTIVE EFFECT OF LEAVES OF MADHUCA LONGIFOLIA (KOENIG) AGAINST PARACETAMOL INDUCED ACUTE LIVER DAMAGE IN RATS
Arun Kumar1, Kaushik Biswas1, Bhavin A1, Babaria2, S Ramachandra Setty2* 1. S.C.S College of Pharmacy, Harapanahalli, Karnataka 2. Government College of Pharmacy, Bengaluru, Karnataka * Corresponding author: Email: arun_pharma44@yahoo.co.in ABSTRACT The present study was desined to evaluated the hepatoprotective activity of 70% ethanolic extract of leaves of Madhuca longifolia in paracetamol (2gm/kg) induced liver toxicity in albino wistar rats. The paracetamol injected rats were found to be with significantly increased level of biochemical markers like SGPT (Serum glutamate pyruvate transaminase), SGOT, (Serum glutamate oxaloacetate transaminase) ALP (alkaline phosphatases), serum bilirubin (total and direct), total cholesterol and serum triglycerides. The depleted GSH (glutathione) leves and increased lipid peroxidation indicated the involvement of free radicals in the liver toxicity. 70% ethanolic extract at the dose of 20mg/kg and 40mg/kg produced significant reduction in the serum levels of biochemical markers mentioned above. The extract also increased the levels of GSH and significantly decreased the lipid peroxidation. The results of 70% ethanolic extract were comparable with the standard silymarin. These results suggest that 70% ethanolic extract of Madhuca longifolia (Koenig) may possess the potential therapeutic value in the treatment of some liver diseases, which can be attributed to its anti-oxidant action. Key words: 70% extract, Madhuca longifolia, hepatoprotectve, GSH, lipid peroxidation, biochemical markers. INTRODUCTION Herbal medicines derived from plant extracts are being increasingly utilized to treat a wide variety of clinical disease, though relatively little knowledge about their mode of action is available. So there is a growing interest in the pharmacological evaluation of various plants used in traditional system of medicine. India is also a prosperous example of the same trend. Free radical and reactive oxygen species are often generated as by product of biological reaction. The involvement of these species in the pathogenesis of large number of disease including atherosclerosis, diabetis mellitus, asthama, nephritis, liver disease is well documented (Maxwell A G, 1999). So a potent scavenger of these species may serve as a possible preventive intervention for free radicals mediated disease. Natural anti-oxidant may scavenge the free radical and can protect the organ from damage (Susanta K M, 2006). Liver is often abused by environmental toxins, poor eating habits, alcohol consumption and over the counter drug use that damage and weaken the liver leading to important public health problems. There are no satisfactory remedies available from synthetic and herbal world of medicine to treat the liver disease. Paracetamol is widely used analgesic and anti-pyretic, produces acute liver damage if over doses are consumed. The hepatotoxicity of paracetamol has been attributed to the formation of toxic highly reactive metabolite n-acetyl para benzoquineimine (NAPQI). Parcetamol also causes nephro toxicity (Diadelis Remirez, 1995). MATERIALS AND METHODS Animals: Albino rats (Wistar) weighing 150-200g and albino mice weighing 20-25g of either sex were used in this study. They were procured from Sri Venkateshwara Enterprises, Bangalore. The animals were acclimatized for one week under laboratory conditions. They were housed in polypropylene cages and maintained at 27 C 2C under 12 hours dark / light cycle. They were fed with standard rat feed (Gold Mohur Lipton India Ltd.) and water ad libitum was provided. Ethical clearance for handling the animals was obtained from the Institutional animals ethical committee prior to the beginning of the project work. Since there are no reports on hepatoprotective and nephroprotective properties of Madhuca longifolia (Koenig) leaves and the pharmacological profile is also incomplete, the present study is planned to study the hepatoprotective effect of leaves of Madhuca longifolia. Plant Material: Madhuca longifolia (Koenig) leaves (both tender and mature) were collected from the herbal garden of S.C.S. College of Pharmacy, Harapanahalli. The plant was identified and authenticated by Prof. K. Prabhu, Department of Pharmacognosy, S.C.S. College of Pharmacy, Harapanahalli. A herbarium specimen is deposited in our college museum. The leaves were shade dried separately at room temperature and pulverized. The powder obtained was subjected to successive soxhlet extraction with the solvents with increasing order of polarity i.e petroleum ether (60-80%), chloroform (59.5-61.5%), ethanol (64.5-65.5%) and water. In addition the shade-dried powder was extracted directly with 70% ethanol (hydro-alcoholic extract), which was used for biological investigations and in vitro and in vivo antioxidant studies, after subjecting it to preliminary qualitative phytochemical studies. The extracts were concentrated under reduced pressure and stored in a desicator until further use and the percentage yield of corresponding extracts were calculated. Preparation of extract: The powder obtained was subjected to successive soxhlet extraction with the solvents with increasing order of polarity i.e. pet. ether (60 80), chloroform (59.561.5), ethanol (64.5-65.5) and water. In addition the shade-dried powder was extracted directly with 70% ethanol (hydro-alcoholic extract), which was used for biological investigations and in-vitro and in-vivo antioxidant studies, after subjecting it to preliminary Volume 1 Issue 1 January February 2013 Page 106
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qualitative phytochemical studies. The extracts were concentrated under reduced pressure and stored in a desicator until further use and the percentage yield of corresponding extracts were calculated. Preliminary phytochemical investigation: The preliminary phytochemical screening was carried out on petroleum ether, chloroform, 95% ethanolic, aqueous and 70% ethanolic extract of Peltophorum pterocarpum leaves for qualitative identification of type of phytoconstituents present (Kokate CK, 1999) (Khandelwal KR, 2000). Aute toxicity (LD 50) study: The acute toxicity for 70% ethanolic extracts of Peltophorum pterocarpum leaves were determined on albino mice, maintained under standard conditions. The animals were fasted overnight prior to the experiment. Fixed dose method of OCED (organization for economic cooperation and development) Guideline No. 420 given by CPCSEA was adopted for toxicity studies. Animal treatment (Chattopadhyay RR, 2003): In dose response experiment albino rats were randomly assigned into 5 groups of 6 animal each. Group- I (Negaitve control) received 1ml/kg normal saline for 7 days, p.o. GroupII(positive control), Group-III(standard silymarin ), Group- IV and V(test extract) on 5 th day 30 minutes after aministrstion of normal saline(1ml/kg), 100 mg/kg silymarin,70% ehanolic extract 100mg/kg and 70% ehanolic extract 200mg/kg of Peltophorum pterocarpum leaves, received paracetamol 2g/kg orally. After 48 hours of paracetamol feeding rats were sacrificed under mild ether anesthesia. Biochemical studies (Tietz MN, 1983): The blood was obtained from all the animals by puncturing retro-orbital plexus. The blood sample was centrifuged immediately to get clear serum and subjected for estimation of various biochemical parametears namely SGPT, SGOT, ALP, serum bilirubin (total and direct), total cholesterol, serum triglcerieds. IN-VIVO ANTI-OXIDANT GSH estimation (Aykae G, 1985): Tissue samples were homogenized in ice cold Trichloroacetic acid (TCA) (1 gm tissue plus 10 ml 10% TCA) in a ultra turrax tissue homogenizer. Glutathione measurements were performed using a modification of the Ellamn procedure Briefly, after centrifugation at 3000 rpm for 10 minutes, 0.5 ml supernatant was added to 2 ml of 0.3 M disodium hydrogen phosphate solution. A 0.2 ml solution of dithiobisnitrobenzoate (0.4 mg/ml in 1% sodium citrate) was added and the absorbance at 412 nm was measured immediately after mixing. % increase in OD is directly proportional to the increase in the levels of Glutathione. Hence, % increase in OD is calculated. Lipid peroxidation estimstion (John Buege A, 1978): The degree of lipid peroxide formation was assayed by monitoring thiobarbituric reactive substance formation. Combine 1.0 ml of biological sample (0.12.0 mg of membrane protein or 0.1-0.2 mol of lipid phosphate) with 2.0 ml of TCA-TBA-HCl and mix thoroughly. The solution is heated for 15 min in a boiling water bath. After cooling, the flocculent precipitate was removed by centrifugation at 1000 rpm for 10 min. The absorbance of the sample is determined at 535 nm against a blank that contains all the reagents minus the lipid. Statistical analysis: Results were expressed as mean SEM, (n=6). Statistical analysis was performed with one way analysis of variance (ANOVA) followed by Tukey-Kramer Multiple Comparisons Test by using Graph Pad Instat Software. P value less than 0.05 was considered to be statistically significant. *P<0.05, **<0.01 and ***<0.001, when compared with control and toxicant group as applicable Table-1 Effects of 70% ethanolic extract of Madhuca longifolia (Koenig) tender leaves in paracetamol induced hepatotoxicity
Treatment SGPT U/L SGOT U/L Biochemical parameters Mean SEM Total Direct Total ALP Bilirubin Bilirubin Cholesterol IU/L mg/dl mg/dl mg/dl 0.89 0.213 115.63 131.68 0.077 0.006 5.406 3.143 4.217 1.45 185.36 425.36 0.058 0.056 4.106 8.282 1.092 0.296 118.67 170.43 0.064*** 0.006*** 6.235*** 4.325*** 335.71 5.594*** 219.16 7.827*** Triglycerides mg/dl 175.04 5.527 210.56 7.688 178.82 8.823** 199.15 2.734 184.93 1.854*
Negative control 64.741 67.16 (1ml vehicle p.o.) 5.061 3.753 Paracetamol (positive control) 263.17 323.51 (1ml vehicle p.o.+ 2 g/kg p.o.) 6.908 8.251 Paracetamol +Silymarin 78.567 93.47 (2 g/kg p.o. + 100mg/kg, p.o.) 6.230*** 4.525*** Paracetamol +70% ethanolic 195.23 254.17 2.650 0.926 159.51 extract 4.151*** 5.395*** 0.057*** 0.009*** 5.287* (2 g/kg p.o.+ 20 mg/kg p.o.) Paracetamol +70% ethanolic 112.025 149.78 1.48 0.482 125.27 extract 6.252*** 4.866*** 0.077*** 0.009*** 5.009*** (2 g/kg p.o. + 40 mg/kg p.o.) Values are the mean S.E.M. of six rats/treatment, Significance *P<0.05, **P <0.01 and *** P<0.001, compared to paracetamol treatment
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Fig. No. 1

Fig. No. 2 Effect of 70% alcoholic extract of Madhuca longifolia (Koenig) leaves on biochemical parameters in paracetamol induced hepatotixicity
Serum SGPT levles (U/L)
300 250
Serum SGPT levles (U/L)
Effect of 70% alcoholic extract of Madhuca longifolia (Koenig) leaves on biochemical parameters in paracetamol induced hepatotixicity Normal Control
PCM Trtd.
PCM + 100mg/kg Silymarin PCM + 20mg/kg 70% Et. Ext. PCM + 40mg/kg 70% Et. Ext.
300 200 100 0
Normal Control PCM Trtd. PCM + 100mg/kg Silymarin PCM + 20mg/kg 70% Et. Ext. PCM + 40mg/kg 70% Et. Ext.
200 150 100 50
Groups
0 Groups
Total Bilirubin levels (mg/dl)
Fig. No. 3 Effect of 70% alcoholic extract of Madhuca longifolia (Koenig) leaves on biochemical parameters in paracetamol induced hepatotixicity 5 4 3 2 1 0
Normal Control PCM Trtd. PCM + 100mg/kg Silymarin PCM + 20mg/kg 70% Et. Ext. PCM + 40mg/kg 70% Et. Ext.
Groups
RESULTS There is an increase in SGPT U/L levels observed in paracetamol treated group (263.170). The extract has shown a dose dependent effect. SGPT levels were restored to 112.025 by 40 mg/kg of 70% ethanolic extract of the leaves, whereas standard 100 mg/kg silymarin has also shown a statistically significant effect i.e. 78.567. SGOT U/L levels have been increased significantly in paracetamol treated group i.e 323.51. 40 mg/kg of 70% ethanolic extract of the leaves reduced the elevated levels of SGOT to 149.78, which is statistically significant when compared to PCM (paracetamol) treatment. Standard silymarin 100 mg/kg has reduced the SGOT levels significantly i.e. 93.47. In case of total and direct bilirubin, a dose dependent effect of the extract is observed. 40 mg/kg 70% of ethanolic extract has reduced the elevated levels of total and direct bilirubin levels by paracetamol from 4.217mg/dl and 1.45 mg/dl to 1.480 mg/dl and 0.482 mg/dl respectively. The results of 40 mg/kg 70% ethanolic extract was found to be comparable with the results of 100 mg/kg silymarin on the same marker enzymes. There was a significant rise in total cholesterol took place in paracetamol treated group. Dose dependent effect was observed with the 70% ethanolic extract and result of 40 mg/kg 70% ethanolic extract is comparable with 100 mg/kg silymarin. There is an increase in ALP levels observed in paracetamol treated group (425.360 U/L). The extract has shown a dose dependent effect. ALP levels were restored to 219.16 U/L by 40 mg/kg 70% ethanolic extract of the leaves which is statistically significant when compared with PCM treated group. The restoration by standard silymarin 100 mg/kg was also significant i.e. 170.43. CONCLUSION The Madhuca longifolia (Koenig) leaves contain steroids, flavonoids, carbohydrates, saponins and tannins. 70% Ethanolic extract of Madhuca longifolia (Koenig) leaves demonstrated dose dependant reducing power, superoxide anion scavening, hydroxyl radical scavening and nitric oxide scavenging activities. The 70% Ethanolic extract of Madhuca longifolia (Koenig) leaves possess hepatoprotective and nephroprotective activity and this may be due to the presence of flavonoids and others antioxidant principles. REFERENCES Aykae G, Vysal M, Yalein AS, Kocak-Toker N, Sivas A, Oz H, The effect of chronic ethanol ingestion on hepatic lipid peroxide, Glutathione, glutathione peroxidase and glutathione transferase in rats, Toxicology, 36, 1985, 71-76. Burtis CA, Ashwood ER, Eds Tietz, Textbook of Clin Chem, Philadelphia, WB Saunders Co, 3, 1999; 1829. Chattopadhyay RR, Possible mechanism of hepatoprotective activity of Azadirachta indica leaf extract: Part II. J. Ethnopharmacol, 89, 2003; 217-219
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Diadelis Remirez, Jan NM, Commandeur, Groot Ed, Nico PE, Vermeulen, Mechanism of protection of lobenzarit against paracetamol-induced toxicity in rat hepatocytes, Eur J Pharmacol, 293, 1995, 301-308. Dipak V Parmar, Gazala Ahmed, Milind A, Khandkar A, Surendra S Katyara, Mitochondrial ATPase, a target for paracetamol-induced hepatotoxocity, Eur J Pharmacol, 293, 1995, 225-229. Dortman RB, Lawhorn GT, Serum enzymes as indicators of chemical induced liver damage, Drug and chemical toxicology, 1, 1978, 163-171. John Buege A, Steven Aust D, Microsomal lipid peroxidation. London : Moury Kleiman Co., 1978 pp 302. Khandelwal KR, Practical Pharmacognosy techniques and experiments, 2nd ed. Pune, Nirali Prakashan, 2000, 149-156 Kokate CK, Practical Pharmacognosy 4th ed, New Delhi, Vallabha prakashan, 1999: 149-156. Maxwell A G, Masato Y, Yoko A. Free radical scavenging action of medical herbs from Ghana Thonningia sanginea on experimentally induced liver injuries, General Pharmacology, 1999, 32, 661-667. Rajesh MG, Latha MS, Preliminary evaluation of the antihepatotoxic activity of Kamilari, a polyherbal formulation. Journal of Ethnopharmacology, 91, 2004, 99-104. Sing J, Reen RK, In vitro assessment of paracetamol-induced toxicity in the reuber hepatoma H4IIEC3/G cell line competent of xenobiotics metabolism, Toxicology in vitro, 13, 1999, 897-903. Susanta K M, Goutam C, Gupta M, Majumder U K, In vitro antioxidant activity of Diospyros malabarica Kostel bark. Ind J Exp Bio, 2006, 4, 39-44. Tietz MN, Rinker D, Show LM, IFCC method for alkaline phosphatase, J Clin Chem Clin Biochem, 21, 1983, 731-748.
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FORMULATION AND EVALUATION OF ORAL CONTROLLED FLOATING TABLETS OF ANTI-ASTHAMATIC DRUG

Sony Yellapu1, Debjith Bhowmick2, Harish Gopinath 2, Arvind Gurram2, Anusha P2 1. Indira Gandhi Institute of Pharmaceutical Sciences, IRC Village, Bhubaneswar, Odisha 2. Nimra College of Pharmacy, Jupudi, Vijayawada, Andhra Pradesh Mail Id: sony.pharma56@gmail.com
Abstract
The objective of the present research work is to formulate and evaluate oral controlled floating tablet of anti-asthmatic drug Terbutaline sulphate. The pre and post compression parameters have been studied and the results were found to be within the IP specification. The formulations F3 and F6 showed higher swelling index compared to others. In-vitro release rate studies showed that the maximum drug release was observed in F2 and F9 formulations up to 12 hrs. Optimized formula found to be stable at 45oC and 75% RH for a period of 3 month. FT-IR studies revealed that there was no interaction between drug and the polymers used. From the study it is evident that a promising controlled release floating tablets of Terbutaline sulfate can be developed to increase gastric residence time. Further detailed investigations are required to establish efficacy of these formulations and fix the required dose. Key words: Terbutaline sulphate, Conventional tablet, Anti-Asthmatic 1. INTRODUCTION The controlled gastric retention of solid dosage forms may be achieved by the mechanisms of mucoadhesion, floatation, sedimentation, expansion, modified shape systems or by the simultaneous administration of pharmacological agents that delay gastric emptying. Drugs which are locally active in the stomach e.g. misroprostol, antacids etc, drugs that have narrow absorption window in gastrointestinal tract (GIT) e.g. L-DOPA, para aminobenzoic acid, furosemide, riboflavin etc, drugs those are unstable in the intestinal or colonic environment e.g. captopril, ranitidine HCl, metronidazole are suitable to formulate as floating or gastro retentive drug delivery systems. To formulate GRDDS, It must have sufficient drug loading capacity, ability to control the drug release profile, fully degraded and evacuated from the system once the drug release is over, should not have effect on gastric motility including emptying pattern and It should not have other local adverse effects. Various approaches have been worked out to improve the retention of oral dosage form in the stomach as floating systems, swelling or expanding systems, bio-adhesive systems and high density systems. Floating systems are low density systems that have sufficient buoyancy to float over the gastric contents and remain in the stomach for a prolonged period. While the system floats over the gastric contents, the drug is released slowly at the desired rate, which results in the increased gastro retention time and reduces fluctuations in plasma drug concentration. After release of drug the residual system is emptied from the stomach. This results in an increased gastric retention time and a better control of the fluctuations in the plasma drug concentration. However, besides a minimal gastric content is needed to allow the proper achievement of the buoyancy. To formulate a successful stomach specific or gastro retentive or floating drug delivery system (GRDDS) several techniques are currently used such as non-effervescent systems, effervescent floating dosage forms, floating system based on ion exchange resin, volatile liquid containing system, multiple unit floating systems and raft forming systems. 2. MATERIALS AND METHODS 2.1 materials: Terbutaline sulfate was obtained as a gift sample from Karnataka antibiotics .pvt.ltd, HPMC k15M was obtained as a gift sample from yarrow chem products, Mumbai, carbopol 934P, sodium bicarbonate, and magnesium stearate was procured from SD fine chem.Limited, Mumbai. 2.2. Formulation of hydro-dynamically balanced tablets: Floating matrix tablets containing Terbutaline sulfate were prepared by direct compression technique using varying concentrations of different grades of polymers with sodium bicarbonate. All the ingredients except magnesium stearate were blended in glass mortar uniformly. After sufficient mixing of drug as well as other components, magnesium stearate was added and further mixed for additional 2-3 minutes. The tablets were compressed with 8mm punch using hydraulic press. The weight of the tablets was kept constant for formulations F1 to F13. The composition of all formulations was given in Table no 1 2.3. Evaluation of hydrodynamically balanced tablets: Pre-formulation studies were performed on the drug which included melting point determination, solubility and compatibility studies. The formulated hydrodynamically balanced tablets were evaluated for its shape, Thickness, hardness, friability, tablet density, Weight Variation, Test for Content, Buoyancy / Floating, Swelling, In-vitro dissolution studies. 3. RESULTS AND DISCUSSION The values obtained for angle of repose, compressibility index for all formulations are tabulated in Table 2 . The values were found to be in the range from 22.3o + 0.9 to 29.5o + 0.8, 15.15 0.56 % to 22.09 0.32 % for angle of repose and compressibility index respectively. This indicates good flow property of the powder Volume 1 Issue 1
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blend for direct compression. Tablet dimensions determined for formulated tablets were tabulated in Table 3. The %friability was less than 1% in all the formulations ensuring that the tablets were mechanically stable. The percentage weight variation for all formulations was shown in Table 3. All the tablets passed weight variation test as the % weight variation was within the pharmacopoeial limits of 5% of the weight. Results of Buoyancy study were tabulated in table no 3, and that the batch containing HPMC K15 and carbopol 934 p polymers showed good Buoyancy lag time (BLT) and Total floating time (TFT) for 24 hrs. Swelling study was performed on all the batches for 8 hr. The results of swelling index are given in Table 4. While the plot of swelling index against time (hr) is depicted in Figure 5. From the results it was concluded that swelling increases as the time passes because the polymer gradually absorbs water due to hydrophilicity of polymer. In the present study, the higher swelling index was found for tablets batch F8 batch containing HPMC K15M having nominal viscosity of 15,000 cps. Thus, the viscosity of the polymer had major influence on swelling process, matrix integrity, as well as floating capability, hence from the above results it can be concluded that linear relationship exists between swelling process and viscosity of polymer. The percentage of drug content was found to be between 96.48% to 98.13% of Terbutaline sulfate, which was within acceptable limits. Table 3 shows the results of drug content uniformity in each batch. The Invitro drug release profile of tablet from each batch (F1 to F8) was shown in Table 5. The plot of % cumulative drug release V/s time (hr) was plotted and depicted as shown in Fig. 6 & 7. From the In-vitro dissolution data it was found that, Tablet of batch F1, F2, F3, F4,F5,F6,F7,F8,and F9 contained different concentrations of polymers HPMC K15M with Carbopol 934p showed drug release at the end of 1 st hr were 18.57,21.42,15.71,12.81.22.85,15.71,27.14,30.00,and 21.42. Drug release at 8th Hr ranges from 51.14 % to 70.22 %. Decreased rate of drug release was observed with increased concentration of polymers. When the tablets contact with water occurs which acts as rate controlling matrix for the release of drug molecules. Drug Release at 12th hr ranges from 77.03 % to 93.04 %. Decreased rate of drug release was observed with increased concentration of polymers. When the tablets contact with water occurs which acts as rate controlling matrix for the release of drug molecules Curve fitting analysis results of dissolution data fitted to various drug release kinetic equations. Peppas model was found to be best fitted in all dissolution profile having higher correlation coefficient (r value) followed by Higuchi model and zero Order Release equation. Mechanism of drug release was found to be non-fickian diffusion. Stability studies were carried out on optimized formulation as per ICH Guidelines Q1C. Table 1. Formulation chart Formulation Drug in HPMC Carbopol NaHCO3 Mag.Stearate Lactose Code (mg) K15M (mg) (mg) (mg) (mg) (mg) F1 15 25 22.07 40 10 157.30 F2 15 25 15 40 10 165.00 F3 15 46.21 15 40 10 143.79 F4 15 3.79 15 40 10 186.21 F5 15 40 10 40 10 155.00 F6 15 40 20 40 10 145.00 F7 15 10 10 40 10 185.00 F8 15 25 7.93 40 10 172.07 F9 15 10 20 40 10 175.00 Table 2. Evaluation of micromeritic properties of the granules Formulat Bulk Tapped Angle of Carrs ion code density (g/cc) density repose index o F1 0.337+0.005 0.427+0.041 27.7 +1.0 21.26+0.74 F2 0.331+0.113 0.413+0.013 25.9o +0.5 19.85+0.32 F3 0.329+0.014 0.423+0.018 26.9o +0.4 20.81+0.79 F4 0.324+0.012 0.385+0.016 23.2o +1.2 16.84+0.13 o F5 0.335+0.011 0.406+0.041 24.1 +0.3 17.48+0.52 F6 0.342+0.147 0.439+0.038 29.5o +0.8 22.09+0.32 F7 0.328+0.034 0.392+0.013 22.7o +1.1 15.23+0.32 o F8 0.327+0.147 0.390+0.052 22.3 +0.9 15.15+0.56 F9 0.342+0.013 0.410+0.041 25.2o +0.6 18.31+0.14
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Sony Yellapu et.al. Batch es F1 F2 F3 F4 F5 F6 F7 F8 F9 Diameter (mm) 8.22 0.010 8.39 0.006 8.57 0.031 8.74 0.053 8.03 0.013 8.09 0.010 8.14 0.019 8.17 0.052 8.28 + 0.041 Thickness (mm) 4.2 0.012 4.29 0.021 4.4 0.015 4.5 0.006 4.3 0.011 4.2 0.016 4.41 0.002 4.18 0.025 4.32 + 0.016 Hardness (Kg/cm2) 4.2 0.011 4.3 0.018 4.4 0.005 4.4 0.034 4.7 0.003 4.1 0.025 4.1 0.032 4.3 0.001 4.2 + 0.005
ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology Friability (%) 0.76 0.71 0.65 0.69 0.84 0.88 0.82 0.79 0.85 Weight variation (mg) 274.8 278.2 273.5 276.8 272.6 278.4 274.5 277.3 276.4 Drug content uniformity (mg) 97.06 96.14 97.32 96.48 98.16 97.36 96.43 98.13 96.48 Buoyancy Lag time (Sec) 38.32 45.19 49.43 57.54 14.51 17.32 18.17 22.47 27.32 Total floating time (hrs) 16 14 12 12 24 24 24 24 24
Table 3. Physical properties of tablets, Buoyancy lag time, Total floating time of Batch F1-F9
Time 1 2 3 4 5 6 7 8
F1 24.07 40.74 58.88 78.51 91.48 107.77 116.29 137.40
Table 4. Swelling Index of Tablets of Batch F1 to F9 F2 F3 F4 F5 F6 F7 21.11 30 8.51 22.96 25.9 19.62 30.74 45.18 17.40 37.40 44.44 37.40 47.03 52.59 35.92 48.88 61.11 57.03 60.74 70 47.40 73.33 74.44 73.33 82.96 85.55 55.18 88.14 93.70 87.77 95.92 102.22 67.03 104.07 105.18 100 104.81 121.85 78.8 112.22 118.51 116.66 122.59 143.70 87.77 130 142.59 137.40 Table 5. In-vitro dissolution study of F1-F9 Formulations % cumulative drug release F2 F3 F4 F5 F6 F7 21.42 27.26 34.55 41.88 46.40 52.37 59.80 64.41 71.90 78.00 82.71 87.44 15.71 21.51 25.92 27.49 33.35 36.39 45.19 53.98 57.13 64.58 74.93 83.91 12.85 18.64 25.88 30.31 33.34 42.09 45.18 51.14 55.70 66.00 69.22 81.02 22.85 32.98 38.88 41.95 45.03 55.28 64.15 70.22 76.31 81.01 82.88 86.18 15.71 24.37 30.22 31.81 37.70 47.91 56.74 59.91 63.09 64.86 73.78 77.03 27.14 40.15 43.23 47.75 49.44 54 57.15 58.88 69.20 73.86 77.11 80.38
F8 22.59 36.66 54.44 72.22 85.55 100.74 117.40 133.70
F9 22.22 41.11 57.40 74.07 87.03 99.62 116.66 134.44
Time in hrs 1 2 3 4 5 6 7 8 9 10 11 12
F1 18.57 24.38 30.23 36.11 46.31 49.42 59.69 68.59 71.82 79.35 82.64 85.94
F8 30 40.16 47.53 49.22 53.77 56.92 60.09 61.84 65.03 72.53 78.63 87.62
F9 21.42 30.11 37.42 40.49 45 50.96 55.92 61.53 66.15 69.37 82.60 93.04
Table 6. Comparative data of percentage drug release from the formulations F1 to F9 Time In F1 F2 F3 F4 F5 F6 F7 F8 F9 Hours 1 18.57 21.42 15.71 12.85 22.85 15.71 27.14 30 21.42 2 24.38 27.26 21.51 18.64 32.98 24.37 40.15 40.16 30.11 3 30.23 34.55 25.92 25.88 38.88 30.22 43.23 47.53 37.42 4 36.11 41.88 27.49 30.31 41.95 31.81 47.75 49.22 40.49 5 46.31 46.40 33.35 33.34 45.03 37.70 49.44 53.77 45 6 49.42 52.37 36.39 42.09 55.28 47.91 54 56.92 50.96 7 59.69 59.80 45.16 45.18 64.12 56.74 57.15 60.09 55.52 8 68.59 64.41 53.98 51.14 70.22 59.91 58.88 61.84 61.53 9 71.82 71.90 57.13 55.70 76.31 63.09 69.20 65.03 66.15 10 79.35 78.00 64.58 66.00 81.01 64.86 73.86 72.53 69.37 11 82.64 82.71 74.93 69.22 82.88 73.78 77.11 78.63 82.60 12 85.94 87.44 83.91 81.02 86.18 77.03 80.38 87.62 93.04
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Time In Hours 1 2 3 4 5 6 7 8 9 10 11 12
Table 7. Comparison of zero order of in vitro drug release F1-F9 F1 F2 F3 F4 F5 F6 F7 18.57 24.38 30.23 36.11 46.31 49.42 59.69 68.59 71.82 79.35 82.64 85.94 21.42 27.26 34.55 41.88 46.40 52.37 59.80 64.41 71.90 78.00 82.71 87.44 15.71 21.51 25.92 27.49 33.35 36.39 45.16 53.98 57.13 64.58 74.93 83.91 12.85 18.64 25.88 30.31 33.34 42.09 45.18 51.14 55.70 66.00 69.22 81.02 22.85 32.98 38.88 41.95 45.03 55.28 64.12 70.22 76.31 81.01 82.88 86.18 15.71 24.37 30.22 31.81 37.70 47.91 56.74 59.91 63.09 64.86 73.78 77.03 27.14 40.15 43.23 47.75 49.44 54 57.15 58.88 69.20 73.86 77.11 80.38
F8 30 40.16 47.53 49.22 53.77 56.92 60.09 61.84 65.03 72.53 78.63 87.62
F9 21.42 30.11 37.42 40.49 45 50.96 55.52 61.53 66.15 69.37 82.60 93.04
Table 8. Comparison of First order of in vitro release F1-F9

Time 1 2 3 4 5 6 7 8 9 10 11 12 F1 1.91 1.87 1.84 1.81 1.73 1.71 1.61 1.50 1.45 1.31 1.24 1.14 F2 1.89 1.86 1.815873 1.764259 1.729126 1.677853 1.604209 1.551295 1.448633 1.342266 1.237687 1.098836 F3 1.925754 1.894782 1.869697 1.860386 1.823754 1.803479 1.739045 1.662908 1.632104 1.549159 1.399042 1.206483 F4 1.940232 1.910396 1.869883 1.843124 1.823857 1.762714 1.738919 1.688928 1.646341 1.531378 1.488237 1.278209 F5 1.887296 1.826178 1.786177 1.763784 1.740049 1.650446 1.554383 1.473892 1.374428 1.278391 1.23348 1.140428 F6 1.925754 1.878677 1.843717 1.833673 1.794444 1.712732 1.636026 1.603007 1.567082 1.545789 1.418538 1.360978 F7 1.862472 1.777058 1.754118 1.718053 1.703769 1.662758 1.631943 1.613959 1.488461 1.417221 1.359474 1.292502 F8 1.845098 1.776943 1.719897 1.705674 1.664851 1.634189 1.601025 1.581504 1.543575 1.438831 1.329705 1.092476 F9 1.895265 1.844359 1.796376 1.774575 1.740363 1.690548 1.648128 1.585013 1.529447 1.486104 1.24047 0.842134
Table 9. optimized formula *Ingredients R Terbutaline sulfate 15 Hpmc K15M 40 Carbopol 934p 10.33 NaHCO3 40 Mg. stearte 10
Table 10. Response variables of optimized formula Formulation hardness Buoyancy Swelling Drug code index content (%) Optimized 4.85 + 0.79 28+ 0.29 133+ 0.33 97.48 + 0.26 formula
Table 11. Drug release studies of optimized formula: Time (hr) CDR %CDR Time (hr) CDR %CDR 0 0 0 7 9.01 60.09 1 4.5 30 8 9.27 61.84 2 6.02 40.16 9 9.75 65.03 3 7.12 47.53 10 10.87 72.53 4 7.38 49.22 11 11.79 78.63 5 8.06 53.77 12 13.14 87.62 6 8.53 56.92 3.1. Stability studies: Stability studies were conducted as per ICH guidelines and the data was presented in Table 12 and Table 13. Table 12. Response variables of optimized formulation (after stability) Time Hardness Buoyancy Swelling index Drug content (%) (kg/cm2 ) (%) 4.85 + 0.79 133+ 0.33 97.48 + 0.26 Initial 28 + 0.29 After one Month Volume 1 Issue 1 4.91 + 0.32 28 + 0.87 136 + 0.13 97. 98 + 0.62
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Table 13. Drug release studies (after stability) Time (hr) CDR %CDR Time (hr) CDR %CDR 0 0 0 7 9.28 61.86 1 4.91 32.73 8 9.65 64.33 2 6.01 40.06 9 9.99 66.60 3 7.22 48.13 10 10.87 72.46 4 8.01 53.40 11 11.68 77.86 5 8.66 57.73 12 13.14 87.60 6 8.99 59.93
Figure: 1 FTIR of Terbutaline Sulphate
Figure: 2 FT-IR of TerbutalineSulphate +Carbopol 934
Figure: 3 FT-IR of Terbutaline sulphate + HPMC K 15 M

200 150 100 50
Figure: 4 FT-IR of Terbutaline sulphate Carbopol 934 + HPMC K 15 M

100 80 %cdr 60
%swelling index
40
20 0 0 f1 5 Time 10 f2 f3 15 f4
0
Time f1 f2 f3 f4 f5 f6 f7 f8 Formulation f9
Figure: 5 Swelling index of F1-F9
Figure: 6 Comparative data of % drug release from the formulations F1 to F4
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ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology 100 80 60 %cdr 40 20 0 0 f5 f6 5 Time 10 f7 f8 15 f9 f1 0 f2 5 Time 10 f3 f4 15
%cdr
40 20 0
Figure :7 Comparative data of % drug release from the formulations F5 to F9 Kinetics of drug release
Figure: 8 Comparison of zero order In-vitro drug release from F4-F9
4. CONCLUSION From the above experimental results it can be concluded that, formulated tablets gave satisfactory results for various physicochemical parameters like hardness, friability, thickness, weight variation and content uniformity. Sodium bicarbonate has predominant effect on the buoyancy lag time, while HPMC K15M and Carbopol 934p has predominant effect on total floating time and drug release carbopol shows significant effect on drug release. Swelling index has a significant effect on the drug release. The formulations F3 and F6 showed higher swelling index compared to others. In-vitro release rate studies showed that the maximum drug release was observed in F2 and F9 formulations up to 12 hrs. Optimized formula found to be stable at 45oC and 75% RH for a period of 3 month. FT-IR studies revealed that there was no interaction between drug and the polymers used. From the study it is evident that a promising controlled release floating tablets of Terbutaline sulfate can be developed to increase gastric residence time. Further detailed investigations are required to establish efficacy of these formulations and fix the required dose. REFERENCES Amit KN, Ruma M, Biswarup D, Gastroretentive drug delivery systems: a review, Asian J Pharm Clin Res 2010; 3(1), 2-10. Arkash V, Vikas J, Saurabh M, Vidushi S, A review on gastroretentive drug delivery system, Int J Pharm Life Sci 2(5), 2011, 773-781. Azhar DK, Meenakshi B, Floating Drug Delivery System: An Overview, Int J Pharm Tech Res, 2(4), 2010, 24972505. Hetangi R, Vishnu P, Moin M, Floating drug delivery system: innovative approach of gastroretention, Int J Pharm Sci Rev Res, 4(3), 2010, 183-92. Natasha S, Dilip A, Gupta MK, Mahaveer KPR, A comprehensive review on floating drug delivery system, Int J Res Pharm Biomed Sci, 2(2), 2011, 428-441. Neha N, An updated review on: floating drug delivery system, Int J Appl Pharm, 3(1), 2011, 1-7. Pooja M, Kamal S, Navneet S, Surender V, Sanju N, Vinay V, An overview on recent advancements and developments in gastroretentive buoyant drug delivery system, Der Pharmacia Sinica, 2 (1), 2011,161-169. Ravi SP, Ashish PV, Rahul PB, Patel MR, Patel KR, Patel NM, Gastroretentive drug delivery systems: a review. Int J Pharm World Res, 2(1), 2011, 1-24. Shweta A, Javed A, Alka A, Roop K, Khar, Sanjula B, Floating drug delivery systems: a review, AAPS Pharm Sci Tech, 6(3), 2005, 372-390. Vinod KR, Santhosh V, Anbuazaghan S, David B, Padmasri A, Sandhya S, Approaches for gastrotentive drug delivery systems, Int J Appl Biol Pharm Technol, 1(2), 2010, 589-601.
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FORMULATION DEVELOPMENT AND EVALUATION OF LOPERAMIDE HYDROCHLORIDE ORALLY DISINTEGRATING TABLETS

A Bharathi*, K Mohan Guptha, Y Uma Jagannadha Rao KVSR Siddhartha College of Pharmaceutical Sciences, Vijayawada-520010, AP, India *Corresponding author:bharathi_arigela@yahoo.com ABSTRACT There is an increasing demand for more patient compliant dosage form and a novel method is the development of orally disintegrating tablets which dissolve or disintegrates instantly on the patient tongue or buccal mucosa. It is suited for tablets undergoing high first pass metabolism and is used for improving bioavailability with reducing dosing frequency to minimize side effect and make it more cost effective. Loperamide hydrochloride is a drug of choice for diarrhoea. Loperamide hydrochloride has systemic bioavailability too low due to extensive hepatic first pass metabolism. Hence the main objective of the study was to formulate orally disintegrating tablets of Loperamide hydrochloride to achieve a better dissolution rate and further improving the bioavailability of the drug and to get relief from diarrhoea very quickly. Orally disintegrating tablets prepared by direct compression and using super disintegrant crosspovidone were prepared and evaluated for the precompression parameters such as bulk density, compressibility, angle of repose etc. The prepared batches of tablets were evaluated for hardness, weight variation, friability,drug content, disintegration time and in-vitro dissolution profile and found satisfactory. Among the 6 groups (f1, f2, f3, f4, f5, f6),formulation f4 emerged as the best formulation and showed rapid dissolution rate i.e. it releases 95% drug in 5 min. Key Words: Loperamide hydrochloride, orally disintegrating tablets, Aspartame 1. INTRODUCTION Recent advance in novel drug delivery system aims to enhance the safety and efficacy of the drug molecule b y formulating a dosage form being for the administration (Kuchekar BS, 2003) Difficulty in swallowing is experienced by patient such as paediatric, geriatric, bedridden, disabled and mentally ill, including motion sickness and sudden episodes of allergic attacks, hence resulting in higher incidence of non-compliance and ineffective therapy2. To improve the quality of life and treatment compliances of such patients fast disintegrating or orally disintegrating tablets dosage form is a better alternative for oral medication (Yutaka M, 2002). Orally disintegrating tablets are solid dosage form containing medical substances which disintegrate rapidly, usually within few seconds when placed upon tongue requiring no additional water to facilate swallowing (Shu T, 2002) It is suited for tablets undergoing high first pass metabolism and is improving bioavailability with reducing dosing frequency to minimize side effect. Fast disintegrating tablets are gaining prominence as new drug delivery systems. These dosage forms disintegrate within a minute with very less quantity of water. This can be achieved by addition of various superdisintegrants like croscaramellose sodium, crosspovidone, sodium starch glycolate alone or in various combinations. Due to the fast disintegration of dosage form, patients o b t a i n q u i c k p h a r m a c o l o g i c a l effect of active pharmaceutical ingredient (Kharwade RS, 2011). Loperamide hydrochloride is the drug of choice for diarrhoea. It undergoes first pass metabolism and the resulting bioavailability is too low. Hence, there is a need to develop rapidly disintegrating tablets, which disintegrate in matter of seconds in the oral cavity, thereby reducing first pass metabolism, increasing the bioavailability and also to reduce the onset of pharmacological action. Direct compression is one of the technique requires the incorporation of superdisintegrants into the formulation and use of highly water soluble excipients achieve faster tablet disintegration. Direct compression does not require the use of water or heat during the formulation procedure and is the ideal method for moisture and heat-labile medications. In this study, an effort has been made to formulate orally d i s i n t e g r a t i n g tablets of Loperamide hydrochloride using crospovidone as superdisintegrant. Objective of study was to enhance dissolution and absorption of drug, which may produce the rapid onset of action in the treatment of Diarrhoea. 2. MATERIALS AND METHODS 2.1. Materials: Loperamide hydrochloride was obtained as a gift sample from Vasudha phar machem, Hyderabad, crosspovidone from ISP, USA, Manitol obtained from Roquette, France, Microcrystalline cellulose obtained from FMC Ireland. All chemicals and reagents used were of analytical grade. 2.2. Preparation of orally disintegrating tablets: Loperamide hydrochloride orally disintegrating tablets were prepared by direct compression method. Different concentration of excipients was used to prepare different groups of orally disintegrating tablets. Compositions of various formulations are shown in Table 1. All the ingredients of the orally disintegrating tablets of Loperamide hydrochloride were weighed, sieved and mixed, and then finally to the blend Magnesium Stearate and Aerosil were added and compressed on the 8mm standard concave punch using an 8 station Kambert tablet machine. The total weight of the formulation was maintained 100mg Table: 1 Formulation design of directly compressible loperamide hydrochloride tablets Volume 1 Issue 1 January February 2013 Page 116
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INGREDIENTS (mg) Loperamide HCL Crospovidone Microcrystalline cellulose Mannitol Aspartame Magnesium stearate Aerosil Pepperment
F1 2 5 5 81.5 4 1 1 0.5
F2 2 5 15 71.5 4 1 1 0.5
F3 2 7.5 5 79 4 1 1 0.5
F4 2 7.5 15 69 4 1 1 0.5
F5 2 10 5 76.5 4 1 1 0.5
F6 2 10 15 66.5 4 1 1 0.5
Table: 2 Evaluation parameters of tablets Formulae Hardness (kg/ cm2) FI FII FIII FIV FV FVI 5.0 5.0 5.0 5.0 5.0 5.0 Friability Weight Thickness(mm) Disintegration Wetting Variation time (sec) time(sec) (mg) complies 3.50 14 25.34 complies 3.50 14 25.46 complies 3.50 13 18.38 complies 3.50 12 18.47 complies 3.50 13 20.71 complies 3.50 13 22.92
0.145 0.150 0.140 0.135 0.170 0.172
2.3. Evaluation of orally disintegrating tablets of Loperamide hydrochloride: All the batches of tablets were evaluated for various parameters like weight variation, friability, hardness, drug content, disintegration and dissolution and results reported in Table 2. Table 3: Dissolution profiles of formulations Time (hr) 5 10 20 30 % Cumulative drug release F1 F2 F3 F4 F5 F6 INNOVATOR 863. 851. 920 951. 891.1 922.2 990.6 873. 871. 920 972. 932.2 963.2 990.6 1 4 .6 6 3 3 .6 7 922. 900. 931 972. 932.2 963.2 990.6 8 6 .7 7 922. 920. 931 972. 932.2 963.2 990.6 8 6 .7 7 Figure :1 Comparitive dissolution profiles of F1-F6 with innovator
Cummulative % Drug release 120 100 80 60 40 20 0 0 10 20 Time(min) 30 40 F1 F2 F3 F4 F5 F6 Innovator
2.3.1. Weight variation test (Ansel HC, 1995): Twenty tablets were taken and their weight was determined individually and collectively on a digital weighting balance. The average weight of one tablet was determined from the collective weight. 2.3.2. Hardness test (Banker GS, 1987) (Schiermeier S, 2002): The hardness of the tablet was determined using Dr. Schleuniger Hardness tester. 2.3.3. Friability test: Six tablets from each batch were examined for friability using Roche Friabalator (Tropical Equipment Pvt. Ltd. Mumbai, India) and the equipment was run for 4min at 25 revolutions per minute. The Volume 1 Issue 1 January February 2013 Page 117
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tablets were taken out, dedusted and reweighted and % friability was calculated. %Friability = (Loss in weight/Initial weight) x 100 2.3.4. Wetting time (Sunada H, 2002) (Schiermeier S, 2002): A piece of tissue paper folded twice was kept in a Petri dish (inter diameter 5.5cm) containing 6ml of purified water. The tablet was placed on the tissue paper and allowed to wet completely. The time required for complete wetting of the tablet was then recorded 2.3.5. Tablet thickness: Ten tablets were taken and their thickness is recorded using vernier callipers. Disintegration test: The disintegration test was performed using an USP disintegration apparatus, with distilled water at 37 2 C. The time reported to obtain complete disintegration of six tablets were recorded and average was reported. 2.3.6. In vitro Dissolution testing: Dissolution study was conducted for all the formulation using USP Type-II apparatus (paddle type). The dissolution test was performed using 900 ml of 0.01 M HCl taken as dissolution medium at 50 rpm and 37 C 0.5 C. Five millilitres of aliquots were periodically withdrawn and the sample volume was replaced with an equal volume of fresh dissolution medium. The samples were suitably diluted and analysed by HPLC. For comparison, dissolution studies of commercial tablets were also conducted. 3. RESULTS Loperamide hydrochloride orally disintegrating tablets were prepared by direct compression method. The compositions of the formulations are shown in the Table1. Table 2 shows the data obtained from the evaluation of tablets. All batches of the tablets were preliminarily evaluated for various physical parameters such as hardness, friability, drug content, wetting time, disintegration and dissolution which were reported in Table no 2. All above properties and value were near to boundary of standard limit. All the tablets maintained hardness in the range 5.0 kg/cm2. The loss in total weight of the tablets due to friability was in the range of 0.14 - 0.17%. The drug content in different formulation was highly uniform and in the range of 97 - 99%. Wetting time is used as an indicator of the ease of tablet disintegration and found to be 18.38 - 25.46 sec. The result of In-vitro disintegration were within the prescribed limits and comply with the criteria for orally disintegrating tablets, the value were with 12-14sec. In-vitro dissolution studies are shown in Table 3 and Figure 1. The concept of super disintegrant addition method proved to be beneficial in order to lower the disintegration time. The quicker disintegration time may be attributed to faster water uptake by the tablets. Dissolution profiles revealed that, after 5 minutes, formulations F1-F6 showed % drug release of 86,85,92,95,89,92% respectively. Among all the formulations, F4 formulation shows better dissolution efficiency and rapid disintegration with release of 95% within 5min. 4. CONCLUSION The Fast disintegrating tablets of Loperamide hydrochloride were formulated by using the superdisintegrant Crosspovidone. The use of super disintegrant crosspovidone at concentration of 7.5% has given the better release of drug when compared to other concentrations. The proposed Fast disintegrating formulation possessed ideal and reproducible characteristics of disintegration time and drug release profile. REFERENCES
0 0 0
Kuchekar BS, Badhan AC, Mahajan HS, Mouth Dissolving Tablets: A Novel Drug Delivery System, Pharma Times, 35, 2003, 7-9. Seager H, Drug-delivery product and the zydis fast dissolving form, J Pharm Pharmacol, 50(4), 1998, 375382. Yutaka M, Yuki T, Masanobu Y, Ryoji T, Junko A, Kozo T, Evalution of the disintegration time of rapidly disintegrating tablets via a novel method utilizing a CCD camera, Chem Pharm Bull, 50(9), 2002, 1181-1186. Shu T, Suzuki H, Hironaka K, Ito K, Studies of rapidly disintegrating tablets in the oral cavity using cogrinding mixtures of Mannitol with Crosspovidone, Chem. Pharm. Bull., 50, 2002, 193-198. Kharwade RS, Vyavhare NS, More SM, Formulation of mucoadhesive tablet by u sing Aegle marmelos gum, I nternational j ournal o f applied biology and pharmaceutical t echnology, 2(1), 2011, 154161. Ansel HC, Popovich NG, Allen LV, Pharmaceutical dosage forms and drug delivery system. 8th ed, New Delhi, B.I. Waverly Pvt. Ltd, 1995, 305-312. Banker GS, Anderson NR, Tablets. In: Lachman L, Lieberman HA, Kanig JL, editors, The theory and practice of industrial pharmacy, 3rd ed. Mumbai: Varghese Publishing House, 1987, 296303, 316317. Sunada H, Bi YX, Yonezawa Y, Danjo K. Preparation, evaluation and optimization of rapidly disintegrating tablets, Powder Tech, 122, 2002, 188198. Schiermeier S, Schmidt PC, Fast dispersible ibuprofen tablets, Eur J Pharm Sci, 15, 2002; 295305. Volume 1 Issue 1 January February 2013 Page 118
Haritha M et.al.
DRY EMULSION: A PROMISING DOSAGE FORM TO DELIVER LIPOPHILIC DRUG MOLECULES WITH IMPROVED STABILITY AND EFFECTIVENESS
Haritha M*, Priyanka M, Abeda Aqther, Neeharika R, Pragati Kumar B Nimra College of Pharmacy, Vijayawada, AP, India *Corresponding author: Email: haritham29@yahoo.com ABSTRACT Liquid emulsions have distinct advantages over the other oral dosage forms by improving the bio availability and by reducing the side effects, but the number of emulsion formulations currently in use are few compared with other oral dosage forms due to lack of physical - chemical and compliance problems. To overcome these problems dry emulsions are prepared. Dry emulsions are prepared by drying liquid o/w emulsions containing a solid carrier in the aqueous phase. The solid carrier provides the dry emulsions with bulk and mass. Dry emulsions are lipid based powder formulations from which an O/W emulsion can be reconstituted in-vivo or in-vitro. Dry emulsions can be prepared by spray drying, lyophilization and rotary evaporation. Unfortunately the dry emulsions were cohesive powders. The cohesiveness was reduced by addition of sucrose. 1. INTRODUCTION Dry emulsions are of interest because of their stability and sustained release effect. Dry emulsions present a potential oral drug delivery system for lipophilic and low soluble drug substances and for drug substances needing protection against light (Corveleyn S, Remon JP, 1998a) or oxidation (Corveleyn S, Remon JP, 1998b). For the preparation of dry emulsions we use drug, solid carrier, aqueous phase and lipophilic solvent. The solid carriers used to prepare dry emulsions are gelatin, lactose, maltodextrin, mannitol, povidone, sucrose etc. Insoluble carrier like colloidal silica (Corveleyn S, Remon JP, 1999) can also be used. Dry emulsions can be prepared by spray drying, (Ford JL, 1999) Lyophilization (Faldt P, Bergenstahl B, 1995) and rotary evaporation. The solid carrier may undergo partial or complete transformation into an amorphous state. Since the amorphous carrier exhibits a strong tendency to crystallize at a particular elevated temperature and relative humidity, physical stability problems may arise. Stability tests for amorphous solid carriers like lactose, maltodextrin, mannitol and sucrose will be carried out. To avoid stability problems water soluble polymers like hydroxyl propyl methyl cellulose, methyl cellulose and povidone are used as solid carriers. After conducting preliminary studies on these water soluble polymers, it was concluded that dry emulsions with hydroxyl propyl methyl cellulose (HPMC) as solid carrier is most promising. For preparing dry emulsions lipidic solvents like fractionated coconut oil, Miglyol 812, Capmul MCML 8, Phosal 53 MCT, sesame oil, Lecithin, almond oil etc., can be used. Water soluble polymer HPMC facilitated the emulsification of liquid o/w emulsions due to its ability to reduce the surface tension. As the concentration of HPMC in liquid O/W emulsions is increased a reduced droplet size distribution can be obtained (Faldt P, Bergenstahl B, 1996a). Problems by spray drying liquid O/W emulsions with high concentration of HPMC may arise, Such as a blocked atomizer, because aqueous solutions of HPMC may exhibit a thermoreversible gelation. Thus optimization of spray drying process can be obtained by reducing the temperature of the atomizer by water cooling (Faldt P, Bergenstahl B, 1996b). Among lipid solvents fractionated coconut oil is preferred, as it is made up of short or middle chain triglycerides containing only saturated fatty acids which make it stable to oxidation. 2. PREPARATION OF DRY EMULSION General method of preparation of dry emulsion follows the steps given below 1. Dissolve drug in lipophilic solvent. 2. Add aqueous phase containing bulking agents 3. Form an emulsion 4. Remove water (Lyophilization, spray drying) 5. Fill powder into capsules or compress into tablets Dry emulsions are prepared by using Spray drying Lyophilization Rotary evaporation 2.1. Method: Liquid O/w emulsions are prepared with desired % of dry powder mass. The aqueous solution containing dissolved solid carrier and lipophilic vehicle is homogenized (Faldt P, Bergenstahl B, 1996c) in a high speed colloid mill. Then the liquid O/W emulsions were spray dried in a laboratory spray dryer.
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2.2. Reconstitution of liquid emulsions: A total of 1.0 g of dry emulsion was suspended in 4.0 ml. of distilled water in a 17 ml. container. After 1 hour of rotation at approximately 20 rpm, samples are withdrawn for further characterization. 2.3. Storage of dry emulsion: The dry emulsions were stored in a well closed containers protected from light at ambient temperature and 400 C in 75% relative humidity in a humidity chamber created by a saturated NaCl solution. 3. CHARACTERISATION OF DRY EMULSIONS 3.1. Drug content: Dry emulsion equivalent to 10 mg of drug is weighed accurately and dissolved in suitable solvent. Filter through Whatman filter paper no. 41. The stock solutions are diluted suitably. The drug content is analyzed by U V spectrophotometer. 3.2. Scanning electron microscopy: SEM photomicrographs are taken by analytical scanning electron microscope for studying surface morphology (Kiekens F, 1997). Globule size determination: Microscopic examination of the emulsion before and after reconstitution is observed. 3.3. Density: The density of the dry emulsions was determined by helium pycnometry. For one determination each sample was measured seven times. A Pascal 140 equipped with a dilatometer type CD3P was applied to determine the density of the dry emulsions by mercury porosimetry. 3.4. Moisture content: Moisture content was determined by Thermo Gravimetric Analysis ( Appro Adley Lima AN, 2008) approximately 15.00 20.00 mg. samples were placed in the sample pan and the effluent gas was dry nitrogen. The scanning rate was 100 C / min in the scan range 50-2000 C. The moisture content can be determined as the weight loss between 50 and 1200 C. 3.5. Surface characterization: Scanning Electron Microscopy (SEM) is used to examine the outer macroscopic structure of the dry emulsion. Prior to microscopy samples were coated with gold/palladium by sputtering for 300 seconds in a Bio Rad, E5200 Auto Sputter Coater. The samples were scanned at a voltage of 15 KV. 4. NECISSITY OF DRY EMULSIONS They improve the bio availability of drug substances Side effects are reduced Dry emulsions are attractive because they are physically and microbiologically stable formulations. They represent a potential oral drug delivery system for lipophilic and low soluble drug substances. Used for drug substances needing protection against light or oxidation. Dry emulsions provide most constant possible effective blood levels over prolonged durations of therapy. 5. APPLICATIONS OF DRY EMULSIONS The dried emulsion may be used in plan protection formulations (Chiou WL and Riegelman S, 1971) In the formulation of antifoams (Chiou WL and Riegelman S, 1971) In cosmetic formulations In household care wipes, in skin care wipes, in baby care wipes In makeup removing wipes In bath salt formulations In surface coating formulations for e.g: in paints (Lladser M, 1968). 6. TECHNICAL PROPERTIES OF THE DRY EMULSIONS The type of rotary atomizer and the rotation rate had no noticeable effect on the technical properties of the dry emulsions containing 40% lipid. The reconstitution properties of the dry emulsions were affected by both the type of rotary atomizer and by the rotation rate of the atomizer. The porosimeter density of the dry emulsions was affected by the rotation rate of the atomizer. This is probably due to a particle size effect caused by the reduction of particle size with increased rotation rate of atomizer. Dry emulsions with small particles become more cohesive, having properties of low flow ability, and poor packing, explaining the lower porosimeter density seen at the higher rotation rate. The dry emulsions are cohesive powders having poor flow ability due to low density, the size and shape of the particles. The technical properties have to be improved for example by melt our wet granulation. The droplet size distribution of the liquid O/W emulsions before spray drying and after reconstitution increased with increasing lipid content. Dry emulsions having lipid content below 50% reformed the original emulsion. With increasing the lipid content from 30 to 80% the droplet size of dry emulsions is reduced. It is possible to encapsulate up to 80% lipid with pharmacoat 603 as a solid carrier. Dry emulsions having lipid content up to 40% dry powder mass reformed the original O/W emulsion upon reconstitution. Higher the liquid viscosity, the atomized droplet size increases resulting in a bigger particle size of the powder. Volume 1 Issue 1 January February 2013 Page 120
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Since solid carriers like HPMC undergo thermo reversible gelation, it was confirmed by experimentation that by applying water cooling at the atomizer a smaller particle size and a better emulsion were obtained. 7. STABILITY OF THE DRY EMULSIONS UPON AGEING In the storage period dry emulsions are physically stable. After 6 months of storage the outer structure of the dry emulsions had not changed and the dry emulsions were able to reform the original O/W emulsion after reconstitution in water. Dry emulsions having a lipid content of 40% dry powder mass present a potential far an oral drug delivery system. Table.1. Drugs formulated in the form of dry emulsion DRUG LOADED METHOD OF LOADING RESULT Spray drying Improved absorption Lovastatin 1% W/O Emulsion, free flowing Improved absorption Theophylline 7% reconstitutable powder. Adsorption on carrier Improved intestinal absorption Indomethacine/5Fluorouracil ester 7.5% Spray drying Improved intestinal absorption Amlodipine 1.1% Spray drying Improved in-vitro release Vitamin E 40% REFERENCES Adley Lima AN, Jose Sobrinho LS, Roberto AC, Correa JR, & PedroRolim Neto J, Alternative technologies to improve solubility of poorly water soluble drugs, Lat Am J Pharm, 27 (5), 2008, 789- 797. Chiou WL, Riegelman S, Pharmaceutical applications of Dry emulsion systems, J Pharm Sci, 60(9), 1971, 12811302. Corveleyn S, Remon JP, 1998a, Formulation of a lyophilized dry emulsion tablet for the delivery of poorly soluble drugs, Int J Pharm, 166, 1998a, 6574. Corveleyn S, Remon JP, Bioavailability of hydrochlorothiazide: conventional versus freeze-dried tablets, Int J Pharm, 173, 1998b, 149155. Corveleyn S, Remon JP, Stability of freeze-dried tablets at different relative humidities, Drug Dev Ind Pharm, 25, 1999, 10051013. Faldt P, Bergenstahl B, Fat encapsulation in spraydried food powders, J Am Oil Chem Soc, 72, 1995, 171176. Faldt P, Bergenstahl B, Changes in surface composition of spray-dried food powders due to lactose crystallisation, L.W.T. 29, 1996a, 438446. Faldt P, Bergenstahl B, Spray-dried whey protein: lactose:soybean oil emulsions; Surface composition and particle structure. Food Hydrocoll, 10, 1996b, 421429. Faldt P, Bergenstahl B, Spray-dried whey protein: lactose:soybean oil emulsions; redispersability, wettability and particle structure, Food Hydrocoll, 10, 1996c, 431439. Ford JL, Thermal analysis of hydroxypropylmethylcellulose and methylcellulose: powders, gels and matrix tablets. Int. J. Pharm. 179, 1999, 209228. Kiekens F, Vermeire A, Samyn N, Demeester J, Remon JP, Optimisation of electricalconductance measurements for the quantification and prediction of phase separation in o:w-emulsions, containing hydroxypropylmethylcellulose as emulsifying agents, Int J Pharm, 146, 1997, 239245. Lladser M, Medrano C, Arancibia A, The use of supports in the lyophilization of oil-in-water emulsions, J Pharm Pharmacol, 20, 1968, 450455.
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AN ICE-CREAM TYPE PHARMACEUTICAL FORMULATION OF PIPERAZINE HYDRATE AND ITS EVALUATION

M Komala1*, K Hima Bindu1 , P Shanmugapandiyan1, P Veera Swamy2, R Sivakumar2, Debjit Bhowmik3 1. Mohamed Sathak A.J. College of pharmacy, Chennai, India 2. The Erode College of Pharmacy, Erode, Tamil Nadu, India 3. Karpagam university, Coimbatore, Tamil Nadu, India *Corresponding author: E.mail:komala_sivakumar@rediffmail.com ABSTRACT The main objective of this work is to develop an ice cream type pharmaceutical formulation of piperazine hydrate and a process for preparing the same to substitute for tablets and syrups. Ice cream type formulations have merits over conventional formulae in-terms of compliance, so it can be used as an improved pharmaceutical formula for children affected by helminthic infection. The in-vitro diffusion profile of the formulation was studied in pH 1.5 buffer (acidic medium) and in pH 7.8 buffer (alkaline medium) for 2 hours in each buffer medium. The release in acid medium was 23.5% maximum at the end of 2 hours. This showed that only negligible amount of drug was released in acid medium. The release in pH 7.8 buffer was found to be 80% at the first 15mins which showed an immediate on-setting action of the drug in the body system. The drug release is prolonged up to 2 hours and absorption of up to 95% of the drug within 2 hours in the intestinal tract is possible. The diffusion study results concluded that major portion of the drug was available for the body system to cure the infection of Ascariasis. Keywords- Ice creams, In-vitro diffusion profile, helminthic infection, piperazine hydrate. 1. INTRODUCTION Ascariasis is an infection of the intestinal tract caused by the adult Ascaris lumbricoides is clinically manifested by vague symptoms of nausea, abdominal pain and cough. Live worms are passed in the stool or vomited. Occasionally, they may produce intestinal obstruction or may migrate into the peritoneal cavity. Ascaris lumbricoides lives in the lumen of small intestine, where it moves freely. Life span of adult is between 6-12 months. Maximum reported being 1.5 years. Ascaris is a soil-transmitted helminth. The eggs remain viable in the soil for months or years under favorable conditions of temperature, moisture, oxygen, pressure and ultra violet radiation from the sunlight. Foods that are eaten raw such as salads and vegetables readily convey the infection and so is polluted water. Piperazine hexahydrate belongs to Anti-helminthic which is freely soluble in water & alcohol, insoluble in solvent ether. The most widely used preparations are Piperazine citrate as a flavored syrup and Piperazine phosphate as tablets. An accepted safe schedule is an oral dose of 75 mg/kg of body weight with a maximum individual dose of 4gm. Piperazine hexahydrate blocks the neuromuscular transmittance in round worm by antagonizing acetyl choline action and causing hyper polarization and causing flaccid paralysis of the worm and ultimate peristaltic movement, no fasting or patient preparation is required. It has effective against Ascaris lumbricoides and E. vermicularis. 2. MATERIALS AND METHODS 2.1. Commercial preparation of ice cream type formulation: Milk ,Skimmed milk powder (SMP) ,Solid not fat (SNF), Sugar, Sodium alginate (stabilizer), Mono tri glycerides (GMS) (Smoothening agent), Emulsion cream (or) Butter, Suitable flavours, Water. 2.2. Preparation of ice cream: There are three distinct stages in the preparation of ice creams. 1) Preparation of basic mixture 2) Freezing 3) Molding 2.2.1. Preparation of the basic mixture: Milk and water required for the formulation was placed in a stainless steel vat providedwith power stirrer and heating was started.Slowly dry ingredients like milk powder, sugar, etc.., were added little before the temperature reaches to 500C.The stabilizer sodium alginate, should not be added before the temperature reaches to 650C.The mix homogenized at 600C to 770C using pressure of about 2500 3000 PSI using homogenizer. The mix was pasteurized to 700C for 30 mins and allowed to cool to room temperature.Three concentrations of drug in ice cream were prepared as follows: i) 10gm of Piperazine hydrate was dissolved in 100 ml of ice cream mix (1:9). ii) 20gm of Piperazine hydrate was dissolved in 90ml of ice cream mix (2:8). iii) 30gm of Piperazine hydrate was dissolved in 80ml of ice cream mix (3:7). Cool the above (1:9) (2:8) (3:7) mixes to 00 to 40C separately. 2.2.2. Freezing of ice cream: The mix was run into the freezer. Required amount of flavor Vanilla was added. The dasher and then the refrigerant in the freezing chamber were started. When the ice cream has been frozen to the proper stiffness, the refrigerant was shut off but whipping was allowed to continue for few minutes. Whipping was continued and the over-run test was made. When the desired over run has been reached, the product was drawn out from freezer. 2.2.3. Over run test: Over run test is the increase in volume of ice cream over the volume of the mix. Volume 1 Issue 1 January February 2013 Page 122
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. . 100 . 2.2.4. Storage: The formulation should be stored freezing temperature at about 18 0C to 240C. 2.3. Comparison of three formulations: When the three formulations were compared, the formulation of 1:9 concentration has good texture and its color and odor was unchanged as that of standard ice cream. So formulation: I was selected for evaluation. Table.1. Comparison of three formulations FORMULATION PHYSICAL CHARACTERISTICS OF FORMULATION RATIO COLOUR ODOUR TEXTURE Standard Creamy white Vanilla Smooth & fluffy % = 1:9 (Formulation I) 2:8 (Formulation II) 3:7 (Formulation III) Similar to standard ice cream Intense than 1:9 ratio formulation More Intense than 1:9 ratio formulation Similar to standard ice cream Intense than 1:9 ratio formulation More Intense than 1:9 ratio formulation Similar to standard ice cream Intense than 1:9 ratio formulation More Intense than 1:9 ratio formulation
2.4. Evaluation of formulation: 2.4.1. Specific Gravity: Specific gravity of the formulation was determined by using pycnometer 2.4.2. Determination of pH: pH of the formulation was determined by using pH meter 2.4.3. Determination of fat content: Fat content was determined by using centrifuge (Gerber test) 2.4.4. Gerber test: 1 ml of ice cream was diluted with 5ml of water. To 1m of the above solution 10 ml of 80% Sulfuric acid and 1 ml of amyl alcohol was added and centrifuged. The fat content can be directly measured by the amount of precipitate obtained. 2.4.5. Identification tests for type of emulsion: Basically ice cream is an emulsion. The type of emulsion is determined by the following tests. a) Dilution test b) Dye solubility test a) Dilution test: Table.2. Dilution test Experiment Observation Inference Water is added and shaken well. Water is distributed uniformly o/w type of emulsion b) Dye solubility test: Table.3. Dye solubility test Experiment Observation Inference To 1ml of ice cream few drops of water Continuous phase appears pink. o/w type of emulsion. soluble dye (Amaranth) is added. Thin Disperse phase appears smear was made on the glass slide and colorless. examined under microscope. 2.5. Accelerated stability studies: The emulsion was subjected to different centrifugal speeds (200 300 rpm) at room temperature and the separation of phase was observed at different time periods is as follows. Table.4. accelerated stability studies Time in Minutes Speed (RPM) Observation 1 200 No phase separation 2 250 No phase separation 3 300 No phase separation 2.6. Particle size analysis: The measurement of the size of the globules of an emulsion is one of the methods to determine the stability of the emulsion. As the size of the globule increases, stability of emulsion decreases. Hence, size of globules and their size distribution is generally ascertained in an emulsion over a certain time span. The globule size is measure by microscopic methods. Table.5. Lists of standard particle size S.NO. Size in m Type of Emulsion 1 0.5 10 Fine emulsion 2 10 50 Coarse emulsion 2.7. Microscopic method: Measurement of globule size: Sample is diluted sufficiently. A drop of the diluted sample is mounted on the slide and covered with cover slip without allowing any air bubble. The slide is placed Volume 1 Issue 1 January February 2013 Page 123
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under the microscope and the size of the globule is measured. 300 globules are measured and the average size of the globules is calculated. The average particle size of the formulation was 18.33. Frequency distribution curve and a histogram (graph - I) was developed using the observed values from the above particle size distribution studies. Table.6.The tabulation for the report is as follows
Size of particles() Mean (d) Frequency (n) nd
Figure.1. Particle size distribution

FREQUENCY 200 100 0 5 15 25 35 45 55
0-10 10-20 20-30 30-40 40-50 50-60
5 15 25 35 45 55
35 178 56 17 11 3
175 2670 1400 595 495 165
SIZE OF PARTICLES IN MICRONS
2.8. Assay: To 10ml each of sample and standard solutions 1ml of 2, 6 dichloroquinine chloramide was added (chloramide reagent). Heat gently on water bath for 30 min. Cool to room temperature and added 1ml of Hydrochloric acid (concentrated). Adjust the volume to 25ml with water and measure the extinction at about 525 nm against reagent blank. 2.9. Diffusion study: The diffusion is studied at constant time intervals in the following medium. 1. Acid medium. 2. Alkaline medium. 2.9.1. Diffusion study in acid medium: Acid medium of pH 1.5 was prepared using hydrochloric acid. 750 mg of formulation was taken in cellophane bag dipped intothe 1000ml of acid medium in dissolution jar and maintained at 370C. Paddle was set for 50 rpm. The dissolution was carried out for 2 hours. Sample of the medium 10 ml was taken at regular intervals of 15mins, 30mins, 45mins, 60mins, 90mins and 120 minutes. To each of the 10 ml sample, 1ml of2,6 dichloroquinine chloramide was added, heated for half an hour cooled to room temperature to it was added 1ml of concentrated hydrochloric acid. The solution was made upto25ml with distilled water and absorbance was measured at 525 nm. The results were presented in Table 7. Table.7. Diffusion study in acid medium Time in Absorbance in Percentage of drug diffused minutes acid medium (label claim 10% W/w) 15 0.015 0.560 30 0.015 0.560 45 0.018 0.672 60 0.042 1.568 90 0.048 1.792 120 0.063 2.350 2.9.2. In alkaline medium: The drug is readily absorbed in the intestine. So, the diffusion of drug was studied in intestinal pH (7.8) for diffusion study in alkaline medium phosphate buffer of pH 7.8 is used. The sample is packed in cellophane sheet same as that of the acid medium is made dipped in alkaline medium of pH 7.8. The dissolution test was carried out for 15 min, 30mins, 45mins, 60mins, 90mins, 120mins with-drawing 10ml of sample. To each of 10 ml sample, 1ml of 2,6 dichloro quinine chloramide was added, heated for half an hour, cooled to room temperature and 1ml of concentrated hydrochloric acid added. The solution was made upto 25ml with de -mineralized water and the absorbance measured at 525 nm. The results were presented in Table 8. Table.8. Diffusion study in alkaline medium Time in Absorbance Percentage of (10%) minutes Drug diffused (w/w) 15 0.216 8.06% 30 0.219 8.17% 45 0.225 8.40% 60 0.234 8.73% 90 0.255 9.52% 120 0.258 9.63%
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2.10. Stability study: The ice cream type formulation of Piperazine was stored in a frozen condition under refrigeration and the sample was checked every fortnight for changes in color, odour and texture. After 3 weeks the formulation was assayed. 3. RESULTS AND DISCUSSION Ice cream type formulation of Piperazine hexahydrate was developed and evaluated for the efficacy of the formulation and suitability for dispensing. The pH of the Piperazize hexahydrate was found to be 10.85. The percentage purity of Piperazine hexahydrate was found to be 99.71% by Gravimetric method. The formulation was made in three concentrations of 1:9, 2:8, 3:7. When the three formulations were compared (Table. 1) 1:9 concentration formulation had good texture, consistency and its colour and odour was unchanged. Thus 1:9 concentration was selected for evaluation. The specific gravity of the formulation was found to be 1.1252. pH was determined to be 10.2.The fat content of the formulation was found to be 12% mentioned by preventive food adulteration act and Indian Standard Association. Identification tests (Table.2 & 3) for determining the type of emulsion confirmed that the formulation belongs to O/W type of emulsion. The accelerated stability study (Table.4) indicated that the formulation is a good emulsion as no phase separation took place at 300 rpm. The particle size distribution of the formulation was found to be 18.33m which concluded that the ice cream was a coarse emulsion (Table.5) from Graph I. The particle size distribution was in the range of 10 20 m. The formulated product was analysed for its purity and was found to have a purity of 96.3%.The in-vitro diffusion profile of the formulation was studied in pH 1.5 buffer (acidic medium) and in pH 7.8 buffer (alkaline medium) for 2 hours in each buffer medium. The release in acid medium was 23.5% maximum at the end of 2 hours. (Table.7). this showed that only negligible amount of drug was released in acid medium. The release in pH 7.8 buffer was found to be 80% at the first 15mins which showed an immediate on-setting action of the drug in the body system. The drug release is prolonged up to 2 hours and absorption of up to 95% of the drug within 2 hours in the intestinal tract is possible. The diffusion study results concluded that major portion of the drug was available for the body system to cure the infection of Ascariasis. The above result was found to be within the limits of preventive food adulteration act an Indian Standard Association. The amount of active drug in the formulation after 3 weeks of storage under refrigeration was 93.4% w/w which is almost equivalent to that of initial assay value. The stability of the product could further be improved by storing below freezing temperature (-180C to -240C) in deep freezing units. 4. CONCLUSION Ice cream type pharmaceutical formulation of Piperazine hexahydrate prepared on large scale was found to be suitable to substitute for conventional dosage form of tablets and syrups. The formulation has good absorption of drug in the intestinal tract. Ice cream type formulation would be a favorable dosage form for children affected by helmintic infection in terms of taking preference. The formulation can be marketed commercially if proper storage facilities are provided during manufacture, transportation and in community pharmacy. REFERENCES Akalin AS and Erisir D, Effects of inulin and oligofructose on the rheological characteristics and probiotic culture survival in low-fat probiotic ice cream, J of Food Sci, 73, 2002, 184-188. Cummings JH and Roberfroid MB, A new look at dietary carbohydrate: chemistry, physiology and health, Eur J Clin Nutr, 51, 1997, 417 442. Duncan C, Gannon J and Walker WA, Protective nutrients and functional foods for the gastrointestinal tract, Am J Clin Nutr, 75(5), 2002, 789-808. Food and Drug Administration. (2003) Agency Response Letter GRAS Notice No. GRN 000118, Available at http://www.cfsan.fda.gov/~rdb/opa-g118.html, Accessed August 18, 2008. Gibson GR and Roberfroid MB, Dietary modulation of the human colonic micro flora: introducing the concept of prebiotics, J Nutr, 125, 1995, 140112. Indian pharmacopoeia, 1996, Vol II, 299. K Park, Parks Text book of preventive and social medicine, Banarsidas Bhanot Publishers located in Jabalpur, Madhya Pradesh, India, 21th edn, 2011, 181. Niness KR, Inulin and oligofructose: what are they? J Nutr, 129 (7), 1999, 1402-1406. Pereira DI and Gibson GR, Effects of consumption of probiotics and prebiotics on serum lipid levels in humans, Crit Rev Biochem Mol Biol, 37, 2002, 259-281.
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ISSN: 2320 3471(Online) Nalla Chandana et.al. Indian Journal of Research in Pharmacy and Biotechnology RECENT TRENDS IN POLYMER USE IN BIOADHESIVE DRUG DELIVERY SYSTEM Nalla Chanda1*, Thirupathi Reddy A1, B Appanna2, Ch Papinaidu2, D Suvarchala2 1. Nimra College of Pharmacy, Vijayawada, India 2. Bellammkonda College of Pharmacy, Prakasam District, India *Corresponding author: Email: chandananalla@gmail.com ABSTRACT Bioadhesion can be defined as the process by which a natural or a synthetic polymer can adhere to a biological substrate. When the biological substrate is a mucosal layer then the phenomena is known as mucoadhesion. The substrate possessing bioadhesive property can help in devising a delivery system capable of delivering a bioactive agent for a prolonged period of time at a specific delivery site. The current review provides a good insight on mucoadhesive polymers, the phenomenon of mucoadhesion and the factors which have the ability to affect the mucoadhesive properties of a polymer. The drugs which have local action or those which have maximum absorption in gastrointestinal tract require increased duration of stay in GIT. Thus, mucoadhesive dosage forms are advantageous in increasing the drug plasma concentrations and also therapeutic activity. Key words: Mucoadhesion, mucosa, mucoadhesive polymers, drug delivery. 1. INTRODUCTION Bioadhesion may be defined as the state in which two materials, at least one of which is biological in nature, are held together for extended period of time by interfacial forces. In pharmaceutical sciences, when the adhesive attachment is to mucus or a mucous membrane, the phenomenon is referred to as mucoadhesion. In the early 1980s; academic research groups working in the ophthalmic field pioneered the concept of mucoadhesion as a new strategy to improve the efficacy of various drug delivery systems. Since then, the potential of mucoadhesive polymers were shown in ocular, nasal, vagina and buccal drug delivery systems leading to a significantly prolonged residence time of sustained release delivery systems on these mucosal membranes. In addition, the development of oral mucoadhesive delivery systems was always of great interest as delivery systems capable of adhering to certain gastrointestinal (GI) segments would offer various advantages. With few exceptions, however, mucoadhesive drug delivery systems have so far not reached their full potential in oral drug delivery, because the adhesion of drug delivery systems in the GI tract is in most cases insufficient to provide a prolonged residence time of delivery systems in the stomach or small intestine. This overview about the mucoadhesive dosage forms might be a useful tool for the efficient design of novel mucoadhesive drug delivery systems. Mucoadhesive drug delivery systems have applications from different angles, including development of novel mucoadhesives, design of the device, mechanisms of mucoadhesion and permeation enhancement. With the influx of a large number of new drug molecules due to drug discovery, mucoadhesive drug delivery will play an even more important role in delivering these molecules. 2. THEORY 2.1. Electronic theory: Electronic theory is based on the premise that both mucoadhesive and biological materials possess opposing electrical charges. Thus, when both materials come into contact, they transfer electrons leading to the building of a double electronic layer at the interface, where the attractive forces within this electronic double layer determines the mucoadhesive strength. 2.2. Adsorption theory: According to the adsorption theory, the mucoadhesive device adheres to the mucus by secondary chemical interactions, such as in Van der Waals and hydrogen bonds, electrostatic attraction or hydrophobic interactions. For example, hydrogen bonds are the prevalent interfacial forces in polymers containing carboxyl groups. Such forces have been considered the most important in the adhesive interaction phenomenon because, although they are individually weak, a great number of interactions can result in an intense global adhesion. 2.3. Wetting theory: The wetting theory applies to liquid systems which present affinity to the surface in order to spread over it. This affinity can be found by using measuring techniques such as the contact angle. The general rule states that the lower the contact angle then the greater the affinity. The contact angle should be equal or close to zero to provide adequate spread ability. 2.4. Diffusion theory: Diffusion theory describes the interpenetration of both polymer and mucin chains to a sufficient depth to create a semi-permanent adhesive bond. It is believed that the adhesion force increases with the degree of Penetration of the polymer chains. This penetration rate depends on the diffusion coefficient, flexibility and nature of the mucoadhesive chains, mobility and contact time. The adhesion strength for a polymer is reached when the depth of penetration is approximately equivalent to the polymer chain size. In order for diffusion to occur, it is important that the components involved have good mutual solubility, that is, both the bioadhesive and the mucus have similar chemical structures. The greater the structural similarity, the better is the mucoadhesive bond. 2.5. Fracture theory: This is perhaps the most-used theory in studies on the mechanical measurement of mucoadhesion. It analyses the force required to separate two surfaces after adhesion is established. This force, Sm, Volume 1 Issue 1 January February 2013 Page 134
ISSN: 2320 3471(Online) Nalla Chandana et.al. Indian Journal of Research in Pharmacy and Biotechnology is frequently calculated in tests of resistance to rupture by the ratio of the maximal detachment force, Fm, and the total surface area, A0, involved in the adhesive interaction, = 0 . Since the fracture theory is concerned only with the force required to separate the parts, it does not take into account the interpenetration or diffusion of polymer chains. Consequently, it is appropriate for use in the calculations for rigid or semi-rigid bio adhesive materials, in which the polymer chains do not penetrate into the mucus layer. 2.6. Mechanical theory: Mechanical theory considers adhesion to be due to the filling of the irregularities on a rough surface by a mucoadhesive liquid. Moreover, such roughness increases the interfacial area available to interactions thereby aiding dissipating energy and can be considered the most important phenomenon of the process. The mechanisms governing mucoadhesion are also determined by the intrinsic properties of the formulation and by the environment in which it is applied. Intrinsic factors of the polymer are related to its molecular weight, concentration and chain flexibility. For linear polymers, mucoadhesion increases with molecular weight, but the same relationship does not hold for non-linear polymers. It has been shown that more concentrated mucoadhesive dispersions are retained on the mucous membrane for longer periods, as in the case of systems formed by in-situ gelification. After application, such systems spread easily, since they present rheological properties of a liquid, but gelify as they come into contact the absorption site, thus preventing their rapid removal. Chain flexibility is critical to consolidate the interpenetration between formulation and mucus. Environment-related factors include pH, initial contact time, swelling and physiological variations. The pH can influence the formation of ionizable groups in polymers as well as the formation of charges on the mucus surface. Contact time between mucoadhesive and mucus layer determines the extent of chain interpenetration. Super-hydration of the system can lead to build up of mucilage without adhesion. The thickness of the mucus layer can vary from 50 to 450 m in the stomach to less than 1m in the oral cavity. Other physiological variations can also occur with diseases. 3. FACTORS AFFECTING MUCOADHESION 3.1. Molecular weight: The mucoadhesive strength of a polymer increases with molecular weights above 100,000. Direct correlation between the mucoadhesive strength of polyoxyethylene polymers and their molecular weights lies in the range of 200,0007,000,000. 3.2. Flexibility: Muco adhesion starts with the diffusion of the polymer chains in the interfacial region. Therefore, it is important that the polymer chains contain a substantial degree of flexibility in order to achieve the desired entanglement with the mucus. The increased chain interpenetration was attributed to the increased structural flexibility of the polymer upon incorporation of polyethylene glycol. In general, mobility and flexibility of polymers can be related to their viscosities and diffusion coefficients, as higher flexibility of a polymer causes greater diffusion into the mucus network. 3.3. Cross-linking density: The average pore size, the number and average molecular weight of the cross-linked polymers, and the density of cross-linking are three important and inter-related structural parameters of a polymer network. Therefore, it seems reasonable that with increasing density of cross-linking, diffusion of water into the polymer network occurs at a lower rate which, in turn, causes an insufficient swelling of the polymer and a decreased rate of interpenetration between polymer and mucin. One of the studies addressing this factor demonstrated that high concentrations of flexible polymeric films based on polyvinyl pyrrolidone or poly (vinyl alcohol) as film-forming polymers did not further enhance the mucoadhesive properties of the polymer. 3.4. Hydrogen bonding capacity: Hydrogen bonding is another important factor in mucoadhesion of a polymer. Desired polymers must have functional groups that are able to form hydrogen bonds, and flexibility of the polymer is important to improve this hydrogen bonding potential. Polymers such as poly (vinyl alcohol), hydroxylated methacrylate, and poly (methacrylic acid), as well as all their copolymers, have good hydrogen bonding capacity. 3.5. Hydration: Hydration is required for a mucoadhesive polymer to expand and create a proper macromolecular mesh of sufficient size, and also to induce mobility in the polymer chains in order to enhance the interpenetration process between polymer and mucin. Polymer swelling permits a mechanical entanglement by exposing the bioadhesive sites for hydrogen bonding and/or electrostatic interaction between the polymer and the mucus network. However, a critical degree of hydration of the mucoadhesive polymer exists where optimum swelling and mucoadhesion occurs. 3.6. Charge: Some generalizations about the charge of bioadhesive polymers have been made previously, where non-ionic polymers appear to undergo a smaller degree of adhesion compared to anionic polymers. Strong anionic charge on the polymer is one of the required characteristics for mucoadhesion. Some cationic polymers are likely to demonstrate superior mucoadhesive properties, especially in a neutral or slightly alkaline medium. Additionally, some cationic high molecular-weight polymers, such as chitosan, have shown to possess good adhesive properties. There is no significant literature about the influence of the charge of the membrane on the mucoadhesion but the pH of the membrane affects the mucoadhesion as it can influence the ionized or un-ionized forms of the polymers. 3.7. Concentration: The importance of this factor lies in the development of a strong adhesive bond with the Volume 1 Issue 1 January February 2013 Page 135
ISSN: 2320 3471(Online) Nalla Chandana et.al. Indian Journal of Research in Pharmacy and Biotechnology mucus, and can be explained by the polymer chain length available for penetration into the mucus layer. When the concentration of the polymer is too low, the number of penetrating polymer chains per unit volume of the mucus is small and the interaction between polymer and mucus is unstable. In general, the more concentrated polymer would result in a longer penetrating chain length and better adhesion. However, for each polymer, there is a critical concentration, above which the polymer produces an unperturbed state due to a significantly coiled structure. 4. MUCOADHESIVE POLYMERS Mucoadhesive polymers are water-soluble and water insoluble polymers, which are swellable networks, jointed by cross-linking agents. These polymers possess optimal polarity to make sure that they permit sufficient wetting by the mucus and optimal fluidity that permits the mutual adsorption and interpenetration of polymer and mucus to take place. Mucoadhesive polymers that adhere to the mucin-epithelial surface can be conveniently divided into three broad classes: Polymers that become sticky when placed in water and owe their mucoadhesion to stickiness. Polymers that adhere through nonspecific, non-covalent interactions that is primarily electrostatic in nature (although hydrogen and hydrophobic bonding may be significant). Polymers that bind to specific receptor site on tile self surface. 4.1. Characteristics of an ideal mucoadhesive polymer: An ideal mucoadhesive polymer has the following characteristics: The polymer and its degradation products should be nontoxic and should be non-absorbable from the gastrointestinal tract. 1. It should be nonirritant to the mucous membrane. 2. It should preferably form a strong non-covalent bond with the mucin-epithelial cell surfaces. 3. It should adhere quickly to most tissue and should possess some site-specificity. 4. It should allow daily incorporation to the drug and offer no hindrance to its release. 5. The polymer must not decompose on storage or during the shelf life of the dosage form. 6. The cost of polymer should not be high so that the prepared dosage form remains competitive. 4.2. Molecular characteristics: The properties exhibited by a good mucoadhesive may be summarized as follows 1. Strong hydrogen bonding groups (-OH, -COOH). 2. Strong anionic charges. 3. Sufficient flexibility to penetrate the mucus network or tissue crevices. 4. Surface tension characteristics suitable for wetting mucus/mucosal tissue surface. 5. High molecular weight. 4.3. There are two broad classes of mucoadhesive polymers: 4.3.1. Hydrophilic polymers: In the large classes ofhydrophilic polymers those containing carboxylic group exhibit the best mucoadhesive properties, poly vinylpyrrolidone (PVP), Methyl cellulose (MC), Sodium carboxy methylcellulose (SCMC) Hydroxy propyl cellulose (HPC) and other cellulose derivative. 4.3.2. Hyrogels: These are the class of polymeric biomaterial that exhibit the basiccharacteristics of an hydrogels to swell by absorbing water interacting bymeans of adhesion with the mucus that covers epithelia i.e. Anionic group : Carbopol, Polyacrylates and their crosslinked modifications Cationic group : Chitosan and its derivatives Neutral group : Eudragit- NE30D etc. 4.3.3. Natural polymers: Tragacanth, Sodium alginate, Karaya gum, Guar gum, Xanthan gum, Lectin, Soluble starch, Gelatin, Pectin, Chitosan. 4.3.4. Synthetic polymers: Cellulose derivatives (methylcellulose, ethyl cellulose, hydroxy-ethylcellulose, Hydroxyl propyl cellulose, hydroxyl propyl methylcellulose, sodium carboxy methylcellulose, Poly (acrylic acid) polymers (carbomers, polycarbophil), Poly (hydroxyethyl methylacrylate), Poly (ethylene oxide), Poly (vinyl pyrrolidone), Poly (vinyl alcohol). 5. CONCLUSION Mucoadhesive polymers may provide an important tool to improve the bioavailability of the active agent by improving the residence time at the delivery site. The various sites where mucoadhesive polymers have played an important role include buccal cavity, nasal cavity, rectal lumen, vaginal lumen and gastrointestinal tract. Development of novel mucoadhesive delivery systems are being undertaken so as to understand the various mechanism of mucoadhesion and improved permeation of active agents. Mucoadhesive dosage forms have a high potential of being useful means of delivering drugs to the body, perhaps particularly for topical or local administration where the mechanical trauma experienced by the dosage form may be minimized.
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ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology Table-1 Polymer and its bioadhesive property Polymer Bioadhesive Polymer Bioadhesive property property CMC Sodium +++ Guar Gum ++ Carbopol 934 +++ Thermally modified starch + Polycarbophil +++ Pectin + Tragacanth +++ PVP + Poly acrylic acid +++ Acacia + Sodium Alginate +++ Psyllium + Hydroxy Ethyl Cellulose +++ Amberlite-200 resin + HPMC +++ Hydroxy Propyl Cellulose + Gum Karaya ++ Chitosan + Gelatin ++ + + + Excellent, + + Fair, + Poor REFERENCES Nalla Chandana et.al. Devarajan PV and Adani MH, In, Jain NK, Eds , Controlled and Novel Drug Delivery, CBS Publishers & Distributors, New Delhi, 2002, 52-81. Gandhi RB and Robinson JR, Oral cavity as a Site for Bioadhesive Drug Delivery, Adv Drug Del Rev, 1994, 13, 1994, 43-74. Harris D and Robinson JR, Drug Delivery via the Mucous Membranes of The Oral Cavity, J Pharm Sci, 81, 1992, 1-10. Ilango R, Jaykar B and Kavimani S, Chitosan as a New Pharmaceutical Excipient, East Pharm, 1998, March, 47- 49. Junginger HE, Thanou M and Verhoef JC, In, Swarbrick J and Boylan JC, Eds, Encyclopedia of Pharmaceutical Technology, Marcel Dekker, 20 (3), 1990, 169-192. Khar RK, Ahuja A and Ali J, In, Jain NK, Eds , Controlled and Novel Drug Delivery, CBS Publishers & Distributors, New Delhi, 2002, 353-55. Mandal SC and Morgan TM, Polymer Used in Transdermal Drug Delivery System, East Pharm, 1994, 56-64. Mathiowitz E, Chickering D, Jacob J S and Santos C, In, Mathiowitz E, Eds, Encyclopedia of Controlled Drug Delivery, Vol 1, John Willey & Sons, New York, 1999, 9- 44. Prakash SR, Gothoskar AV and Karad MT, A Novel Method for the Study of Water Absorption Rates by Swellable Matrices, Pharm. Tech, 2003, 27 (5), 40-48. Semalty A, Bhojwani Mona, Bhatt GK, Gupta GD, Shrivastav AK, Design and Evaluation of Mucoadhesive Buccal Films of Diltiazem Hydrochloride, Indian J Pharm Sci, 2005, 67(5), 548-552. Squier CA, The permeability of oral mucosa, Crit Rev Oral Biol Med, 2, 1991, 13-32. Squier CA and Wertz PW, Structure and Function of the Oral Mucosa and Implicationsfor Drug Delivery, in, MJ Rathbone, Eds; Oral Mucosal Drug Delivery, 1996, Marcel Dekker, Inc, New York, 1-26. The Indian Pharmacopoeia, Controller of Publications, Ministry of Health and Family Welfare, 1, 1996, 381382.
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Arihara Siva Kumar G et.al.
EFFECT OF METHANOLIC EXTRACT OF ADENANTHERA PAVONINA LINN ON DALTON'S ASCITIC LYMPHOMA

Arihara Siva Kumar G1, Rajesh Kumar Javvadi1*, Vinay Kumar K2, Manohar Reddy E1, Veera Reddy Y1, Harshavardhan G1, Akbar MD3 1. K M C H College of Pharmacy, Coimbatore, Tamil Nadu 2. C R Reddy College of Pharmacy, Eluru, A.P 3. Nimra College of Pharmacy, Vijayawada, A.P *Corresponding author: Email: rajesh.vbcops@gmail.com. Abstract The present study was undertaken to examine anticancer activity of Adenanthera pavonina L stem bark extract on Dalton's ascitic lymphoma (DAL) in male Swiss albino mice. Tumour was induced in mice by intraperitoneal injection of DAL cells (1 x 1000000 cells / mouse). Methanol extract of Adenanthera pavonina L (MAP) was administrated to the mice at the dose of 125 and 250 mg/kg/day, p.o. The antitumor effect of the extract was evaluated by using In-vitro cytotoxic assay; Mean survival time (MST), Tumour volume (TV), Percentage Increase in Life Span (ILS), viable and non-viable tumour cells count. The findings of this study indicate that the MAP possesses significant antitumor activity. Key words: Dalton's ascitic lymphoma, Tumor volume, Percentage Increase in Life Span, antitumor activity 1. INTRODUCTION According to WHO (Shin Young., 2009) Adenanthera pavonina Linn is a weak cyto toxic. In Ayurveda is widely used in various ayurvedic herbal preparations for treating cancer, it is good (Rodrigo et al., 2007) cytotoxic and antioxidant. The present study is focused on the evaluation of antitumor activity of methanolic extract of Adenanthera pavonina L (MAP) against Daltons ascetic lymphoma. 2. MATERIALS AND METHODS 2.1. Collection and Extraction: The steam bark of Adenanthera pavonina Linn were collected in Coimbatore in the month of December and authenticated by P.Satyanarayana Scientist, Botanical Survey of India (BSI), Coimbatore,Tamil Nadu. The authentication certificate number was BSI/SRC/5/23/ Tech-244. The steam bark was chopped, air dried for a week and powered. The powder was then extracted in petroleum ether to de waxing using soxhlet extractor. Then the material was air dried and again subjected to extraction with methanol by using soxhlet continuous extraction until the colour of the material disappears. The obtained extract was concentrated under vacuum and dried in a vacuum desiccator. 2.2. Animals used: Male Swiss albino mice (2025 g) were used throughout the study. They were housed in standard microlon boxes and were given standard laboratory diet and water ad libitum. The experiments were conducted in accordance with protocol duly approved (KMCRET/M.Pharm/05/2010-11) by the institutional animal ethical committee (IAEC /18/2009-10) by the institutional animal ethical committee (IAEC) of KMCH College of Pharmacy, Coimbatore-48. 2.3. Tumor cell line: Daltons ascitic lymphoma (DAL) cells were obtained through the courtesy of Amala Cancer Institute, Thrissur, Kerala; DAL cells were maintained by weekly intraperitoneal (i.p.) inoculation of 110 6 cells/mouse. 2.4. In-vitro short term cytotoxicity assay: Trypan blue exclusion method: Dalton lymphoma ascites (DAL) cells were aspirated from the peritoneal cavity of mice and washed three times in phosphate buffer saline. One million cells were incubated with different concentrations of the extract (10, 25, 50, 100 and 200 g/ml) and the volume was made up to 1.0 ml using Phosphate buffer saline & Control tubes contained only cell suspensions. The assay mixture was incubated for 3 hr at 37 C and their graphical representation in figure I. The viability of the cells was then determined using the following formula. % viability = [(total cells-dead cells) 100]/total cells % cytotoxicity =100-(%viability) 2.5. Effect of MAP on DAL-induced ascitic antitumor studies: The animals were inoculated with DAL (1 106 cells /mouse) and divided into 4 groups containing 12 mice in each group. Group I, served as the tumor control and received normal saline 5ml/kg. Group II, which served as a positive control, was treated with 5-FU at the dose of 20 mg/kg body weight. Groups III and IV were treated with MAP at125 and 250 mg/ kg body weight, respectively. All the treatments were given orally at 24 h after tumor inoculation and continued once daily for 13 days. On the 13th day, Six animals from each group were anaesthetized slightly with anesthetic ether, blood was collected from retro-orbital sinus used for hematological parameters and intraperitonial fluid is used for evaluate the tumor volume (TV), viable and non-viable tumor cells count and the remaining six animals from each group is used to evaluate the Body Weight Analysis, Mean survival time (MST), Percentage increase in life span (ILS). 2.6. Determination of tumor cell count: 0.5ml of 0.4% typhan blue, 0.3ml of normal saline and 0.2ml of cell suspension were mixed and kept aside for 5min and not more than 15min. From this one drop of solution was taken Volume 1 Issue 1 January February 2013 Page 138
on a neubar chamber and a cover slip is placed. This is placed on Haemocytometer and the viable and non-viable cells were counted under 10X power. Viable cells dont take color and these cells appear in white colour on blue background. Non-viable cells (dead cells) take blue color and give dark blue shading to the cells. Serial dilutions were made with phosphate buffer saline and adjusted to the required concentrations. Tumor cell count for respective groups was shown in the figure2. Cell count = (No. of cells x Dilution) / (Area x Thickness of liquid film). 2.7. Body weight analysis: All the mice were weighed for every ten days, after tumor inoculation. Average gain in body weight was determined and recorded in table3. 2.8. Mean survival time (MST): The Mean survival time of all groups was calculated by giving respective doses for 13 days. Noted every death and take the average. 2.9. Percentage increase in life span: Recording the mortality monitored the effect of the Adenanthera povanina linn on tumor growth and percentage increase in life span (ILS %) was calculated. ILS (%) = [(Mean survival of treated group/ Mean survival of control group)-1] x100 2.10. Determination of tumor volume: The mice were dissected and the ascitic fluid was collected from the peritoneal cavity. The volume was measured by taking it in a graduated centrifuge tube and packed cell volume (tumor volume) determined by centrifuging at 1000 g for 5 min. 3. RESULTS Table: 1 In-vitro short term cytotoxicity assay by using DAL cells against MAP Concentration (g/ml) % cytotoxicity Control(blank) 6.667 10 17.241 25 21.127 50 26.667 100 37.5 200 67.308 Ctc50 138.3
Figure 1 Percentage cytotoxicity of MAP

80
%Cytotoxicity
60 40 20 0 0 50 100 150 200 concentration(g/ml)
CTC50 = 138.3 g/ml
250
Table: 2 Shows Hematological and tumor cell parameters

Treatme nt group DAL control 5-Fu, 20 mg/kg MAP, 125 mg/kg MAP, 250 mg/kg Hb (g/dl) RBC (million/mm3) 4.3160.23 12.1000.47 b 7.5670.41b 10.9160.44 b Total WBC (1103/mm3) 14.660.21 7.800.19 6.6170.17 9.5670.16 Tumor cell volume (ml) 4.9500.10 1.1170.09a 2.4830.14a 3.2830.09b Viable tumor cell count106 cells/ml 9.770.17 2.280.07a 2.900.10a 4.500.22a Non-viable tumor cell count106 cells/ml 0.840.10 5.750.07a 5.230.08a 4.600.26a
9.9670.24 14.4170.3 a 11.6840.23 ns 13.2500.28 a
Values are expressed as the mean S.E.M., (n = 6); Statistical sigmificance (p) calculated by one way ANOVA followed by dunnetts a-P<0.001, b- P<0.01, ns-non significant, calculated by comparing treated group with DAL control group. Table: 3 Effect of Methanolic extract of Adenanthera pavonina Linn on body weight of mice challenged with Daltons lymphoma ascetic cells for every 10 days EXPERIMENTAL Body weight (gm) Decrease in body GROUPS weight from 11th day 0 day 11th day 20th day to 20th day(gm) DAL-CONTROL 23.50.42 27.830.3 29.50.43 DAL+5- Fu 23.50.42 27.170.48 210.37 6.17 DAL+MAP, 125mg/kg 23.170.6 26.830.3 22.170.31 4.66 DAL+MAP, 250mg/kg 23.830.48 26.830.3 21.330.49 5.5 Values are expressed as the mean S.E.M., (n = 6) Figure 2 Shows cell count of all groups
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Figure 3 shows animals which were used for body weight analysis and survival time
150
Figure 4 Effect of MAP on survival time
survival time(days)
% increase in lifespan
100
50
0
m g/ kg O N m g/ kg 5 20 12 C Fu 25 D A L+ M A P 0 m g/ kg TR O L
A L
A L+ 5-
Table: 4 Shows mean survival time and percentage increase in life span of various groups Experimental groups Mean survival time in days %ILS DAL-CONTROL 21.500.43 5-FU, 20mg/kg 42.500.76b 97.674 MAP, 125 mg/kg 38.330.67b 78.279 b MAP, 250 mg/kg 30.331.02 41.069 Values are expressed as the mean S.E.M.; Statistical significance (p) calculated by one way ANOVA followed by dunnetts aP< 0.001, bP< 0.01, cP< 0.05 , NS non significant calculated by comparing treated group with DAL control group 4. DISCUSSION The results of the present study clearly demonstrate the tumour inhibitory activity of MAP against DAL. The reliable criteria for evaluating an anticancer drug are prolongation of lifespan of the animal and decrease in WBC count of blood. Our results show an increase in lifespan accompanied by a reduction in WBC count in MAP treated mice. It had significant effect in increasing the life span of ascities tumour bearing. These results clearly demonstrate the anti-tumour effect of MAP against DAL. In cancer chemotherapy the major problems are of myelosuppression and anaemia the anaemia encountered in tumour bearing mice is mainly due to reduction in RBC and HB% and this may occur either due to iron deficiency or due to hemolytic or myelopathic conditions. Treatment with MPA restored the hemoglobin content, RBC and WBC cell count to Volume 1 Issue 1 January February 2013 Page 140
A L+ M
normal near values. This indicates a regular and rapid increase in ascetic fluid volume was observed in DAL bearing mice. Ascetic fluid is the direct nutritional source for tumour growth; it meets the nutritional requirements of tumour cells. MAP treatment decrease viable cancer cell count, and increased the lifespan. It may be suggested that MAP decreases the nutritional fluid volume and thereby arrests tumour growth and increases the life span. Preliminary phytochemical screening indicated the presence of triterpenoids, flavonoids and steroids in MAP. Flavonoids have been shown to possess anti-mutagenic and anti-malignant effect. The cytotoxic and antitumor properties of the extracts may be due to these compounds. REFERENCES L. Sathiyanarayanan, Sinnathambi Arulmozhi and N. Chidambaranathan, Anticarcinogenic activity of leptadenia reticulate against Daltons ascitic lymphoma, Iranian journal of pharmacology & therapeutics 62, 2007, 133-135. NV Rajesh Kumar, KL Joy, Girija Kuttan, RS Ramsewak, Muraleedharan, G Nair, Ramadasan Kuttan, Antitumour and anticarcinogenic activity of Phyllanthus amarus extract, Journal of Ethnopharmacology 2002; 81 (2002) 1722. P Sivakumar, Antitumor and antioxidant activities of Triumfetta rhomboidea against Daltons ascites lymphoma bearing swiss albino mice, Research journal of medical sciences, 2 (4), 2008, 203-208. Ramalingam Radha, Subramaniyam Kavimani, Velayudham Ravichandran, Antitumor activity of methanolic extract of Plumeria alba leaves against Dalton lymphoma ascites in mice, International Journal of Health Research, 1(2), 2008, 79-85. Roberta R, Luciana GM, Luciana CC and Glucia P, Evaluation of the antioxidant Properties of the Brazilian Cerrado fruit Annona crassiflora (Araticum), Journal of Food Science, 71, 2006, 102-107. S. Kavimani & KT Manisenthl Kumar, Effect of methanolic extract of Enicostemma littorale on Daltons ascitic lymphoma, Journal of Ethnopharmacology, 71, 2000, 349352.
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Dipankar Das et.al.
DEVELOPMENT AND EVALUATION OF HIGH POROUS MOUTH DISSOLVING TABLETS CONTAINING LAMOTRIGINE COMPLEXATION
Dipankar Das*, B. K. Sridhar National College of Pharmacy, Shimoga, Karnataka * Corresponding author: Email: dipankar1986@yahoo.com ABSTRACT Lamotrigine (LMG) is an Antiepileptic drug which is widely used in epilepsy. It is also used in simple and complex partial seizures and secondary generalized tonic-clonic seizures. It is poorly water soluble drug (0.17mg/ml at 250C). Thus, in the work under taken, an attempt was made to enhance the water solubility by complexation with -cyclodextrin (1:5 molar ratios). The mouth dissolving tablet of LMG was prepared by direct compression method using different concentration of subliming agent like menthol and superdisintegrant like sodium starch glycolate. The formulations were evaluated for weight variation, hardness, friability, drug content, wetting time, water absorption ratio, in vitro disintegration time and in vitro dissolution studies etc. The prepared tablets were characterized by Fourier transform infra red spectroscopy, differential scanning calorimetry, powder X-ray diffraction studies, scanning electron microscopy. The disintegration time for the complexed tablets prepared by different concentration of menthol are found to be in range of 252.52 to 913.05 seconds and the formulation which are prepared by different concentration of sodium starch glycolate are found to be in range of 301.00 to 952.52 sec. All the formulation showed almost 100 percent of drug release within 40 min. Among all the formulation, F5 prepared with 40 mg menthol and F13 prepared with 35 mg sodium starch glycolate shows faster drug release, respectively 12 min and 15 min. Further formulations were subjected to stability testing for 2 months at temperature of 405C/755%RH. Tablets have shown no appreciable changes with respect to physical appearance, drug content, disintegration time, and dissolution profiles. Keywords: Lamotrigine, -cyclodextrin, Sublimation, Super disintegrants, Mouth dissolving tablets. INTRODUCTION Mouth-dissolving/disintegrating tablets (MDDDS) have the unique property of rapidly disintegrating and/or dissolving and releasing the drug as soon as they come in contact with saliva, thus obviating the requirement of water during administration. Therefore, these dosage forms have lured the market for a certain section of the patient population which includes dysphagic, bed ridden, psychic, and geriatric and pediatric patients. Mouthdissolving/disintegrating tablets (MDDTs) are also called as fast -dissolve, fast-melt, rapidly disintegrating, quick-melt, quick-dissolve, crunch-melt, bite-dispersible, orally disintegrating, and orodispersible tablets. Drugs without any bitter taste, having dose lower than 20mg, Small to moderate molecular weight, good stability in water and saliva, partially non ionized at the oral cavities pH, and having ability to diffuse and partition into the epithelium of the upper GIT (log p>1, or preferably>2) are ideal candidates to formulate as MDTS. Various approaches used for preparation of MDT include Freeze-drying, Moulding, Compression moulding, Heat moulding, No vacuum lyophilization, Sublimation Phase transition process, Mass extrusion Three-dimensional Printing (3DP), Spray drying Cotton Candy Process, Direct compression. Use of disintegrants is the basic approach in development of MDTs. Disintegrants play a major role in the disintegration and dissolution of MDT. It is essential to choose a suitable disintegrant, in an optimum concentration so as to ensure quick disintegration and high dissolution rates. 2. MATERIALS AND METHODS 2.1 Materials Lamotrigine was obtained as a gift sample from Torrent Pharmaceuticals Ltd., Indrad, India, Pearlitol SD 200 was obtained as a gift sample from Strides Arcolab Ltd., Bangalore, India, -cyclodextrin, and Magnesium stearate was procured from S.D. Fine Chem. Ltd, Mumbai, India, Menthol, Aerosil 200 was obtained from Himedia Laboratories Ltd., Mumbai, and Sodium Starch Glycolate from Wockhardt Research center, Aurangabad. 2.2 Methods 2.2.1 Inclusion Complexation of Lamotrigine with -Cyclodextrin Methods of Preparation of inclusion complex: Inclusion complexes were prepared by different methods like physical mixture, kneading and co-evaporation method. In physical mixture method Lamotrigine and Cyclodextrin were accurately weighed in different molar ratios viz. 1:1, 1:2, 1:3 and 1:5 separately and blended thoroughly by triturating in a mortar at 20C for about 10 minutes. The powder mixtures were then pulverized through sieve no.80 and stored in desiccator till further use. In kneading method, physical mixture of LMG: -CD in the different molar ratios viz. 1:1, 1:2, 1:3 and 1:5 was wetted with methanol and water mixture (1:1, by volume) with a pestle to obtain a paste like consistency. The paste was then dried under vacuum at room temperature, pulverized by passing through sieve no. 80 and stored in a dessicator till further use. In co-evoparation method, Lamotrigine was dissolved in methanol and -cyclodextrin was dissolved in water. The two solutions were mixed, stirred for 6 hours at 40C and finally were dried at 40C for 24 hours. The dried complex was then pulverized by passing through sieve no. 80 and stored in a dessicator till further use. Volume 1 Issue 1 January February 2013 Page 142
Dipankar Das et.al.
2.2.2 Characterization of LMG Inclusion complexes: LMG Inclusion complexes were characterized by Differential Scanning Calorimetry (DSC), Fourier Transform Infrared spectroscopy (FT-IR), Powder X-Ray Diffraction study (XRD), and Scanning Electron Microscopy (SEM). It was analyzed for its drug content, In vitro dissolution studies by using dissolution testing apparatus II (paddle method) in 900 ml of 0.1 N hydrochloric acid (HCl) solution of pH 1.2 at 370.5C and at a speed of 50 rpm and samples were analyzed spectrophotometrically at 267 nm against suitable blank. 2.2.3 Preparation of high porous mouth dissolving/disintegrating tablets: Inclusion complex of LMG: -CD (1:5 molar ratios) equivalent to 25 mg of drug prepared by kneading method were taken and mixed with directly compressible diluent, subliming agent, super disintegrant and other excipients in a plastic container. Table1 gives composition of the tablet formulation. Powder blend were directly compressed using 10.05 mm, round-shaped flat punch in a single station tablet compression machine (Cadmach, Ahmedabad, India). The compressed tablets were subjected to drying in hot air oven at 60C for 6 hours to facilitate the sublimation of menthol during drying. 3. EVALUATION PARAMETERS FOR MOUTH DISSOLVING TABLETS OF INCLUSION COMPLEXES OF LAMOTRIGINE: Precompresion parameters Angle of repose Bulk Density (Db), Tapped Density (Dt), Carrs Index (I), Hausners ratio (H) were studied. The prepared tablets were characterized for Weight uniformity, Hardness, Friability, Wetting time & Water Absorption Ratio, Drug content uniformity, In vitro disintegration time, In vitro dissolution studies, and Stability studies. 4. DISCUSSION The percentage drug content for all the prepared inclusion complexes of LMG with -CD were found to be in the range of 96.900.50% to 99.140.44%, indicating uniform drug distribution. In-vitro dissolution studies data of pure drug and its complex method are shown in table 4 and 5. Here dissolution study of pure drug data showed that 19.950.45 percent of drug was released within 30 min. In Physical mixture complete dissolution of inclusion complexes occurred within 30 min. Complex P5 containing 1:5 molar ratios of drug and -CD showed faster dissolution rate, about 100% drug release was observed within 12min. In case of kneading method complete dissolution of drug complexes occurred within 25 min. Complex K5 containing 1:5 molar ratios of drug and -CD showed faster dissolution rate, about 100% drug was released within 10min and in co-evaporation method complete dissolution of inclusion complexes occurred within 25 min. Complex C5 containing 1:5 molar ratios of drug and -CD, showed faster dissolution rate, i.e. about 100% drug release within 12min. So, among the different methods of preparation of inclusion complexes, kneading method was found to be most effective. The formulation K5 showed faster dissolution rate (10min). The values for angle of repose were found to be in the range of 30 to 34. Bulk densities and tapped densities of various formulations were found to be in the range of 0.430.021 to 0.530.006 (g/cc) and 0.510.017 to 0.580.005 (g/cc) respectively. Carrs index of the prepared blends fall in the range of 7.54% to 16.98% and Hausners Ratio of the powdered blends was in the range of 1.08 to 1.20. From the result it was concluded that the powder blends had good flow properties and compressibility, so these can be used for tablet manufacture. Thickness and Diameter results are shown in Table 6. The results showed the thickness and diameter of the tablets prepared by different concentration of menthol was in the range of 3.110.02 mm to 3.280.021 mm and 10.060.01 mm to 10.140.015 mm respectively. Hardness results are shown in Table 6 and it was found to be in the range of 3.80.20 to 4.30.12 Kg/cm2. Difference in weight and percent deviation was calculated for each tablet, shown in the Table 6. The results of the test showed that, the tablet weights were within pharmacoepial limit. Drug content uniformity study data is shown in the Table 6 and was found to be in the range of 98.041.49 % to 100.340.94 % which showed that there was uniform distribution of the drug in tablets of all formulations. Tablets evaluated for percentage friability and the data is shown in the Table 7. The test for friability of all the tablets formulation (except F6) lies in the range of 0.53 % to 0.93 % (less than 1 %) indicating good mechanical strength of tablets. But the friability of F6 prepared with higher concentration of menthol was found to be 1.131%, which is more than 1 %. So it indicates poor mechanical strength. The formulation F0 prepared without disintegrating agent or subliming agent had shown maximum wetting time of 2072.00 seconds. The average wetting time of all the formulations was obtained in the range of 20 to 96 seconds as shown in the Table 7. The formulation FM prepared without menthol showed maximum wetting time of 963.00 seconds and F6 containing 50 mg of menthol showed minimum wetting time of 202.51 seconds. It was observed that with the increase concentration of subliming agent in the tablet formulations wetting time was minimized. The formulation F0 prepared without disintegrating agent or subliming agent had shown minimum water absorption ratio of 211.01 %. The formulation FM prepared without menthol had shown minimum water absorption ratio of 241.53 % and F6 prepared using 50 mg menthol had shown maximum water absorption ratio of 621.51 %. So the result indicates that due to pore formation by subliming agent, water absorption ratio maximizes. Tablets of each batch were evaluated for in vitro disintegration time. The formulation F0 containing neither disintegrating agent nor subliming agent had shown maximum disintegration time of 2184.04 seconds. Volume 1 Issue 1 January February 2013 Page 143
Dipankar Das et.al.
The average disintegration time of all the formulations prepared by using different concentration of menthol are shown in the Table 7. The results showed that the disintegration time of prepared tablets are in the range of 252.52 to 913.05 seconds. The tablets of formulation FM prepared without menthol showed slower disintegration time of 913.05 seconds and formulation F6 prepared using 50 mg of menthol showed the faster disintegration time of 252.52 seconds. Finally, the tablets were evaluated for in-vitro dissolution studies in simulated gastric fluid. The formulation F0 prepared without super disintegrant and subliming agent showed about 100 % of drug release within 60 min. The average dissolution study data of all the formulations prepared by using different concentration of menthol are shown in the Table 7. Among all the formulations, FM prepared without menthol showed about 100 % drug release within 35 min and F6 containing 50 mg of menthol showed faster drug release of about 100% within 9 min. This result suggests a direct relationship of concentration of subliming agent with drug release. As the amount of subliming agent increases in the acceptable range, the drug release also increases. Stability studies were carried out for the selected formulations F5 and F13, the results are shown in Table 10. The result showed that there was no significant difference in the hardness, friability, drug content and disintegration time, at various sampling intervals. The in vitro dissolution profiles were super imposable which confirms the stability of the product. 5. CONCLUSION From the DSC, FT-IR, XRD, SEM, drug content and in vitro dissolution studies of LMG inclusion complexes it was concluded that the formulation K5 i.e., the inclusion complex of LMG with -CD (1:5 molar ratio) prepared by kneading method is the best formulation. The present study is an attempt to select the best possible inclusion complex formulation to formulate high porous mouth dissolving tablets of LMG using super disintegrant such as sodium starch glycolate by sublimation technique. The precompression parameters of all formulations showed good flow properties and compressibility, so these can be used for tablet manufacture. The postcompression parameters of all formulations were determined and the values were found to be within IP limits. The various formulation of mouth dissolving tablet of high porous LMG inclusion complex were prepared using various concentration of subliming agent like as menthol and various concentration of super disintegrant like as sodium starch glycolate. Among the formulations prepared with various concentration of menthol, formulation F6 containing 50 mg of menthol showed complete release of drug in 9 min. But because of higher concentration of menthol the friability is beyond the limit. So formulation F5 containing 40 mg menthol showed complete release of drug in 12 min, was considered as best formulation. And among the formulations prepared with various concentration of sodium starch glycolate, formulation F13 containing 35 mg of SSG showed complete release of drug in 15 min, was considered as best formulation. Accelerated stability study was carried out for selected formulations F5 and F13 which showed no significant difference in the drug content, disintegration time, hardness, friability and in vitro dissolution studies which confirms the stability of the product. As a result of this study, it may be concluded that the inclusion complexation technique may be useful to improve solubility, dissolution rate and subsequently bioavailability of poorly soluble drug. The concept of formulating high porous mouth dissolving tablets of LMG inclusion complexes using super disintegrants by sublimation technique offers a suitable and practical approach in serving desired objectives of faster disintegration and dissolution characteristics. Table 1: Composition of LMG inclusion complexes Drug--CD ratio Inclusion complex Method Formulation Code Composition
Lamotrigine: -cyclodextrin Physical mixture 1:1 1:2 1:3 1:5 1:1 1:2 1:3 1:5 1:1 1:2 1:3 1:5 P1 P2 P3 P5 K1 K2 K3 K5 C1 C2 C3 C5
Kneading method
Co-evaporation Method
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Table 2: Composition of mouth dissolving tablets of LMG inclusion complex Formulation code Ingredients (mg/tablet) Inclusion Pearlitol Sodium Menthol Aerosil 200 Magnesium complex SD 200 starch Stearate glycolate F1 150 124 15 5 3 3 F2 150 119 15 10 3 3 F3 150 109 15 20 3 3 F4 150 99 15 30 3 3 F5 150 89 15 40 3 3 F6 150 79 15 50 3 3 FM 150 129 15 0 3 3 F0 150 144 0 0 3 3 F7 150 119 5 20 3 3 F8 150 114 10 20 3 3 F9 150 109 15 20 3 3 F10 150 104 20 20 3 3 F11 150 99 25 20 3 3 F12 150 94 30 20 3 3 F13 150 89 35 20 3 3 FS 150 124 0 20 3 3 Table 3: Percentage drug contents of LMG in inclusion complexes Formulation code Drug content (%) P1 P2 P3 P5 K1 K2 K3 K5 C1 C2 C3 C5 97.760.43 98.341.86 96.900.50 98.130.86 98.200.82 97.661.24 99.020.36 98.340.45 97.050.81 96.940.39 97.860.42 99.140.44
Time (min)
Table 4: Comparisons of in vitro dissolution profile data of LMG in pure form and its complexes with -CD Cumulative % drug released
Drug 0 0.92 1.54 2.66 4.68 6.88 8.79 9.63 11.60 12.78 13.82 16.04 19.95 P1 0 14.67 25.38 31.06 39.93 51.99 59.48 66.69 73.90 83.25 89.80 98.31 100.03 P2 0 15.29 27.70 41.14 53.26 65.74 72.62 87.89 96.79 101.04 P3 0 13.62 30.45 49.77 64.88 78.22 95.34 99.75 P5 0 21.15 36.43 55.18 72.77 85.25 98.88 K1 0 13.15 25.95 34.08 44.01 55.63 69.06 82.76 88.87 91.36 95.66 100.2 K2 0 12.92 26.22 30.86 45.28 55.94 70.60 87.63 99.41 K3 0 16.70 33.38 50.25 66.091 84.66 100.08 K5 0 18.33 38.49 53.19 78.34 98.96 C1 0 16.13 20.79 32.81 43.98 54.83 60.64 71.36 78.84 84.93 92.66 100.0 C2 0 16.27 25.89 38.70 45.13 57.27 78.93 84.18 99.83 C3 0 17.3 33.06 48.64 60.93 77.69 93.89 100.05 C5 0 21.14 38.26 60.26 74.28 87.98 99.04
0 2 4 6 8 10 12 14 16 18 20 25 30
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Table 5: Comparison of in vitro dissolution profile data of optimized complexes of different method Time (min) Cumulative % drug released Drug P5 K5 C5 0 0 0 0 0 2 0.920.06 21.150.58 18.330.86 21.140.67 4 1.540.27 36.430.72 38.490.68 38.260.55 6 2.660.53 55.180.70 53.190.75 60.260.74 8 4.680.44 72.770.76 78.340.69 74.280.71 10 6.880.63 85.251.15 98.960.39 87.980.39 12 8.790.68 98.880.61 99.040.60 Table 6: Evaluation Parameters for Mouth Dissolving Tablets of LMG: -CD Pre-compression and post compression Parameters of LMG inclusion complex
Formul ation F0 F1 F2 F3 F4 F5 F6 FM F7 F8 F9 F10 F11 F12 F13 FS Bulk density (g/cc) 0.53 0.46 0.44 0.46 0.44 0.43 0.44 0.49 0.46 0.46 0.44 0.47 0.45 0.46 0.44 0.46 Tapped density (g/cc) 0.58 0.52 0.51 0.53 0.52 0.51 0.53 0.53 0.51 0.52 0.51 0.53 0.53 0.54 0.52 0.51 Angle of repose () 34.2 32.21 32.89 31.31 31.89 32.71 33.37 33.86 31.82 32.67 30.91 33.19 33.83 31.81 34.21 34.93 Carrs index (%) 08.62 11.53 13.72 13.20 15.38 15.68 16.98 07.54 09.80 11.53 13.72 11.32 15.09 14.81 15.38 09.80 Hausner s Ratio 1.09 1.13 1.15 1.15 1.18 1.18 1.20 1.08 1.10 1.13 1.15 1.12 1.17 1.17 1.18 1.10 Thickness (mm) 3.15 3.28 3.28 3.24 3.23 3.18 3.16 3.11 3.14 3.12 3.17 3.23 3.22 3.14 3.20 3.24 Diameter (mm) 10.06 10.08 10.09 10.08 10.11 10.10 10.12 10.14 10.09 10.08 10.09 10.07 10.10 10.12 10.13 10.08 Hardness (Kg/cm2) 4.3 4.0 3.8 4.0 4.1 3.9 4.0 4.3 4.2 3.9 4.3 4.0 4.1 3.8 4.1 3.7 Weight variation (mg) 299.6 298.3 298.0 300.3 300.6 299.0 297.0 300.3 298.6 299.0 298.6 300.0 299.3 301.0 301.0 298.3 % Drug Content 99.6 99.0 98.6 100.3 99.8 98.9 98.0 99.7 99.2 99.3 97.8 100.2 98.0 99.5 98.5 100.0
Formulation F0 F1 F2 F3 F4 F5 F6 FM F7 F8 F9 F10 F11 F12 F13 FS
Table 7: evaluation parameters for prepared tablets Percentage Disintegration time Wetting time Friability (%) (sec) (sec) 0.535 2184.04 2072.00 0.714 762.08 692.08 0.792 682.00 581.53 0.822 492.52 421.53 0.882 331.15 282.08 0.937 282.08 231.52 1.131 252.52 202.51 0.627 913.05 963.00 0.732 721.53 692.52 0.895 632.08 542.00 0.745 511.00 443.00 0.886 441.00 401.53 0.824 381.53 331.53 0.905 332.52 292.00 0.867 301.00 262.08 0.923 952.52 812.52
Water Absorption Ratio (%) 211.01 252.00 281.53 361.00 442.52 511.10 621.51 241.53 241.00 290.56 372.08 461.53 591.72 681.04 701.53 231.15
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Table 8: In vitro dissolution profile data of mouth dissolving tablets of LMG inclusion complex using different concentration of subliming agent Time F0 FM F1 F2 F3 F4 F5 F6 (min) 0 0 0 0 0 0 0 0 0 3 12.08 17.27 19.59 25.2 28.19 31.68 35.01 40.54 6 18.41 26.47 27.9 33.94 41.06 45.8 56.75 76.62 9 26.15 36.97 36.71 49.34 55.42 61.34 81.68 99.51 12 32.07 49.41 51.43 56.79 66.4 79.8 99.63 15 39.8 58.19 60.34 65.36 77.85 93.83 18 47.16 65.88 69.13 76.81 89.65 100.28 21 55.25 76.61 79.79 87.18 95.96 24 64.31 83.98 88.57 94.17 99.83 27 71.83 90.87 94.71 99.19 30 80.65 95.8 100.16 35 86.62 99.71 40 93.25 50 98.41 60 100.14 Table 9: In vitro dissolution profile data of mouth dissolving tablets of LMG inclusion complex using different concentration of super disintegrants Time Cumulative % drug released (min) F0 FS F7 F8 F9 F10 F11 F12 F13 0 0 0 0 0 0 0 0 0 0 3 12.08 14.55 17.98 22.09 27.74 32.15 34.66 40.12 44.45 6 18.41 21.13 25.43 29.44 35.6 40.52 47.68 60.39 60.39 9 26.15 33.23 34.14 40.94 46.04 55.76 67.05 74.67 78.3 12 32.07 48.30 48.73 50.13 56.99 68.03 81.55 93.88 94.25 15 39.8 55.5 56.84 60.95 67.82 83.69 92.28 99.17 100.73 18 47.16 63.35 66.93 69.75 84.23 94.98 100.08 21 55.25 70.72 72.82 79.06 93.81 99.78 24 64.31 79.81 81.90 86.74 99.34 27 71.83 86.14 87.85 93.68 30 80.65 90.96 95.22 100.13 35 86.62 96.33 99.68 40 93.25 101.06 50 98.41 60 100.14 Table 10: Stability study of formulation F5 and F13 Hardness Friability Drug Disintegration (Kg/cm2 (%) content (%) Time (sec) F5 F13 F5 F13 F5 F13 F5 F13 3.9 3.8 3.6 4.1 4.0 4.0 0.937 0.941 0.956 0.867 0.873 0.895 98.92 98.76 98.24 98.56 98.4 98.02 28 26 26 30 29 28
Time
Cumulative % drug released F5 F13 99.63 98.46 98.03 100.73 99.47 98.83
Zero month First Month Second Month
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Dipankar Das et.al.
Figure 1: DSC thermogram of pure LMG: -CD (1:5 molar ratios)

DSC mW
Figure 2: DSC thermogram of LMG, inclusion Complex prepared by kneading method

DSC mW 40.00
S-II
S-III
15.00
30.00
10.00
P eak Onset E ndset Heat 281.80 0 C x10
0 280.47 x10 C 0 282.57 x10 C
8.41 0 mJ x10
20.00
5.00
P eak Onset E ndset Heat
0 116.60 x10 C 0 109.56 x10 C 0 127.29 x10 C 0 -79.92 x10 mJ
10.00
0.00 100.00 200.00 Temp [C] 300.00
0.00
100.00 Temp [C]
200.00
300.00
Figure 3: FT-IR spectra of pure LMG
Figure 4: FT-IR spectra of -CD
Figure 5: FT-IR spectra of LMG: -CD (1:5 molar ratios) inclusion complex prepared by kneading method
Figure 6: SEM images of pure LMG
Figure 7 : SEM images of LMG: -CD (1:5 molar ratios) inclusion Complex prepared by kneading method
REFERENCES Volume 1 Issue 1 January February 2013 Page 148
Dipankar Das et.al.
Ammara HO, Salamaa HA, Ghorabb M, Mahmouda AA, Inclusion complexation of glimepiride in dimethyl-cyclodextrin, Asian J Pharm Sci, 2(2), 2007, 44-55. Banker GS, Anderson NR, Tablets in Lachman L, Lieberman HA, Kanig JL, The theory and practice of industrial pharmacy, 3rd ed, Philadelphia PA: Lea & Febiger, 2, 1986, 293-345. Bhowmik D, Chiranjib B, Krishnakanth, Pankaj, Chandira RM, Fast dissolving tablet: an overview, J Chem and Pharm Res, 1(1), 2009, 163-177. Brahmanker DM, Sunil B Jaiswal. Bioavailability and bioequivalence, Biopharmaceutics & Pharmacokinetics -A Treatise, 1st ed, Vallabh Prakashan, New Delhi, India, 1995, 296-302. Brewster ME, Loftsson T, Cyclodextrins as pharmaceutical solubizers, Adv Drug Deliv Rev, 59, 2007, 645-666. Del Valle EMM, Cyclodextrins and their uses: a review, Process biochemistry, 2003, 1-14. Gohel MC, Jogani PD, A review of co-processed directly compressible excipients, J Pharm Sci, 8, 2005, 76-93. Gowthamarajan K, Singh SK, Prakash D, Somashekhar CN, Raju KNS, Dissolution enhancement of poorly soluble aceclofenac by solid dispersion technique and its comparison with marketed formulations, Int J Pharm Tech Res 2(4), 2010, 2347-2356. http://www.pharmainfo.net/reviews/formulation-strategies-improving-drug-solubility-using-solid-dispersions.htm Loftsson T, Brewster ME, Pharmaceutical applications of cyclodextrins, J Pharm Sci, 85, 1996, 1017-1025. Loftsson T, Jarho P, Masson M & Jarvinen T, Cyclodextrins in drug delivery, Expert Opin Drug Deliv, 2(2), 2005, 335-351. Mahapatra AK, Murthy PN, Sahoo J, Biswal S, Sahoo SK, Formulation design and optimization of mouth dissolving tablets of levocetirizine hydrochloride using sublimation technique, Ind J Pharm Educ Res 2009, 43(1), 39-45. Martin A, Drug product design in physical pharmacy, 4th ed, Philadelphia PA: Lippincott Williams & Wilkins 2001, 512-555. Mishra PR, Mishra M, Namdeo A, Jain NK, Review article: Pharmaceutical potential of cyclodextrin, Indian J Pharm Sci, 61, 1999, 193-198. Mohire NC, Yadav AV, Gaikwad VK, Novel approaches in development of metronidazole orodispersible tablets, Res J Pharm and Tech, 2009, 2(2), 283-286. Mosher G, Thompson D. Complexation and cyclodextrin, Encyclopedia of pharmaceutical technology, 2nd ed: edited by Swarbrick J, Boylon JC by Marcel Dekker, 2002, 531-558. Patel HM, Suhagia BN, Shah SA, Rathod IS, Parmar VK, Preparation and characterization of etoricoxib-cyclodextrin complexes prepared by the kneading method, Acta Pharm, 57, 2007, 351-359. Pokharkar V, Khanna A, Venkatpurwar V, Dhar S, Mandpe L, Ternary complexation of carvedilol -cyclodextrin and citric acid for mouth-dissolving tablet formulation, Acta Pharm, 59, 2009, 121-132. Prajapati BG, Ratnakar N, A review on recent patents on fast dissolving drug delivery system, Int J Pharm Tech Res, 1(3), 2009, 790-798. Sastry S, Nyshdham J, Fix J, Recent technological advances in oral drug delivery: A review, AAPS Pharm Sci Tech, 13, 2000, 138-44. Satish NK, Satish KN, Design and optimization of fast dissolving tablets containing Metoprolol by sublimation method, Int Res J Pharm, 1(1), 2010, 346-357. Shukla D, Chakraborty S, Singh S, Mishra B, Mouth Dissolving Tablets , An Over view of Formulation Technology, Sci Pharm, 76, 2009, 309-326. Vemula VR, Lagishetty V, Lingala S, Solubility enhancement techniques, Int J of Pharm Sci Rev and Res, 5(1) 2010, 41-51. Yeola B, Pisal S, Paradkar A, Mahadik K, New drug delivery system for elderly, Ind Drugs, 13, 2000, 312-318.
Volume 1 Issue 1
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Subhasish Chaudhuri and Biswajit Chandra Das
POISONOUS HOUSEHOLD ITEMS AND THEIR SOCIAL IMPACT

Subhasish Chaudhuri1*, Biswajit Chandra Das2 1. Tripura State Forensic Science Laboratory, Agartala, Tripura, India 2. Tripura State AIDS Control Society, Agartala, Tripura, India *Corresponding author: Email:bisu_25d@yahoo.com ABSTRUCT Many of the products we use for housework, gardening and home improvement contain hazardous chemicals that endanger our health as well as pollute the environment. These poisons affect our body either immediately (acutely toxic) or over a long period of time (chronically toxic) can damage and destroy cells and chromosomal material (known to cause cancer, mutations and fetal harm). People expose those harmful chemicals in different ways- by eating, breathing, drinking and the most important exposure rotate is through skin. The person involves applying those products and farm workers are most likely to be affected. In other hand those items are uses as sniffer drugs especially by the teenagers. A quite good numbers of common products are presently abused by the young generation in the society just to enjoy the feeling of well being and also to get a quick high for a short period. Identification of those poisonous household products is necessary for awareness to handle the same. An overview of common health and safety hazards is presented. KEY WORDS: Poisonous household items, hazardous chemicals. INTRODUCTION The household items like floor cleaner, toilet cleaner, mosquito coil, spot remover, paint, flea powder, cockroaches, correction fluid, hair colour, liquid soap, shoes polish & perfumes, air fresheners etc. are found every stationery shops and due to low cost and availability of those are used almost all families. Use of those items is inevitable and potential for harm or injury could be significant if they are misused or mishandled specially for children. The bathroom is one of the most toxic rooms in the house for most families. People use deodorants containing aluminum (Alzheimer s disease), Shampoos containing harsh solvents (liver toxicity), toothpaste containing non-organic fluoride (osteoporosis), mouthwash with aspartame (brain tumors) or saccharin (cancer), and to top it off, most people slap on a dab of perfume or cologne containing highly toxic cancer-causing chemicals. In a laboratory analysis, one popular perfume was found to contain more than forty chemicals classified as hazardous to the liver, and yet the FDA still does not require perfume manufacturers to warn consumers about the toxic chemicals found in their products. The laundry room is also highly toxic, containing the same chemical perfumes in both the laundry detergent and especially the dryer sheets. Dryer sheets coat all your clothes with a layer of toxic chemicals. When you wear those clothes, your body moisture causes those chemicals to come into contact with your skin and be absorbed directly into your bloodstream. Its an easy way to poison your system with cancer-causing chemicals. The kitchen is also highly toxic: consumers purchase antibacterial soap products made with potent nerve chemicals similar to Agent Orange thats what kills the bacteria. They also use automatic dishwashing detergent containing yet more chemicals and toxic fragrance compounds that coat the plates, glasses and silverware with a thin layer of cancer-causing chemicals. Subsequently, families then eat off those dishes and ingest the chemicals. Gardening Chemicals pesticides are one of the most hazardous chemicals used around the home. There are approximately 1,400 pesticides, herbicides, and fungicides that are common ingredients in household products. Often, these chemicals are combined with other toxic substances like solvents and these combinations create over 34,000 different product formulations. ABUSES OF CHEMICALS AND SOCIAL IMPECT It is the cheap availability in most households or shops, easy to carry and conceal simplicity in mode of ingestion and above all legal to posses that prompts the abusers to get involved to it. The inhalants produce a quick high that wanes in a few hours and also the users can evade detection or punishment. The adolescents usually gather in a group to inhalant abuse or as a user gains entry to a group. At the final stage the users are left to leave in a impoverished isolated community, only with few others available users. This situation may increase the attractiveness of inhalants to them as a exciting rebellious and novel experience. This is the extrinsic factor to this addiction and the other intrinsic factor to this addiction is the risk taking tendency among some individuals. The no cost availability of some inhalants like the empty containers of erase ink, correction fluid, aerosols, shoe polish, hair spray and adhesives amongst the pavement dwellers have provided the teenagers of this society to get more and more involved to the list of sniffer and solvent abusers. This has posed a more serious threat to the society as there is least morality remains in them, of course not to all, they get more and more involved to crime and continue with their addiction and gradually move towards higher drug abusers. PHYSIOLOGICAL ACTION In course of inhalation, central nervous system is first stimulated and then depressed. The inhalant pharmacology reinforces repeated self administration in animals. Intoxication stages are somewhat similar to that Volume 1 Issue 1 January February 2013 Page 150
of alcohol; euphoria, blurring of vision, slurring of speech, staggering, in coordination, nausea, vomiting, coma even up to death. However compared to alcohol intoxication, solvent intoxication develops and wanes rapidly (within few minutes to hours). Seldom there may develop hallucination. The most serious threat is the total combination of these effects which may lead to severe accidents. Solvent inhalation no doubt has fatal effects. Many cases are there where death is due to direct toxic effect of the solvent, which result from cardiac arrhythmias, respiratory depression, asphyxiation, accident or injury during intoxicated condition. Among other severe irreversible effects are the hepatic or renal damage and permanent muscle damage associated with rhabdomyolysis. Certain solvents have neurotoxin effect, e.g., hexane is metabolized to 2, 5-hexanedione, which causes neuropathy. Table 1. List of commonly used hazardous household items
Hazardous chemicals Acetone Use in household items Nail polish, spot remover/glues & as solvent. Correction fluid, Present in wood putty & as solvent. Floor cleaner, Antifreeze Air freshener and as preservative Petrol/gasoline Harmful affect Slow decomposition; liver and kidney damage; Corrosive; vapor extremely irritable to skin, eyes and respiratory passages; ingestion causes tissue burns. Highly toxic and may cause skin, kidney, liver, and central nervous system damage. In addition, may damage the reproductive system. Toxic, causes central nervous system depression and kidney, liver lesions. Toxic; carcinogen; irritant to eyes, nose, throat and skin; may cause nausea, headaches, nose bleeds, dizziness, memory loss, and shortness of breath. Highly flammable; Benzene is a carcinogen, Damage to digestive, genitourinary, neuromuscular and central nervous system; anemia and brain damage Flammable; skin irritant; possible liver and kidney damage, eye, nose, throat, lung irritant; air concentrations may cause unconsciousness/death. Caustic; irritant; inhibits reflexes; burns to skin, poisonous if swallowed. Short-term exposure may cause mild asthmatic symptoms. Long term exposure may result in more serious respiratory problems. Ammonia is an eye irritant that may cause headaches and lung irritation. This may cause skin discoloration, shallow breathing, vomiting, and even death. This chemical is associated with both cancer and birth defects. Naphthalene is a suspected carcinogen that may harm eyes, blood, liver, kidneys, skin, as well as the central nervous system. Burns the skin on contact or will cause vomiting diarrhea and stomach burns if swallowed. May also cause blindness if contacts the eyes. Flammable; very toxic; respiratory, circulatory or cardiac damage
Toluene
Di-ethylene glycol & Ethylene glycol Formaldehyde
Petroleum hydrocarbon(Benzene) Aromatic hydrocarbons
Paint, perfumes
Sodium or Potassium hydroxide. Sodium hypochlorite
Drain Cleaner, Over Cleaner Disinfectants, Bleach, Spot Removers. Contained in glass cleaners. floor and furniture polishes
Ammonia Nitrobenzene
Naphthalene or dichlorobenzene Hydrochloric Acid Sodium Acid Sulfate Glycols and Phenol
Para
Uses as insecticide.
or
Common ingredients toilet bowl cleaners
in
Petroleum Distillates
Common chemicals giving fragrances and colors to lotions, creams, and moisturizers. Contained in metal polishes. Mosquito coil, Cockroaches, Flea powder etc.
Pesticides
Short-term exposure may result in temporary eye clouding; long term exposure may result in damage to the nervous system, skin, kidneys, and eyes. Very toxic, interferes with human nervous system, may cause Skin irritation, may damage liver, kidney, spleen and central nervous system.
DISCUSSION Many of the products we use for housework, gardening, home improvement, or car maintenance contain hazardous materials that endanger our health as well as pollute the environment. The average house has an estimated three to 10 gallons of hazardous products. Collectively, these materials can contaminate our drinking water if they are not stored carefully and disposed of properly. In addition to poisoning our water, inappropriate use and disposal of hazardous household products can cause injuries, poisoning and air pollution. In the day to day bustle of identification and analyses health and safety can be easily overlooked. However with proper guidance we can find and correct common mistakes and prevent illness or injury. It may be impossible to totally eliminate Volume 1 Issue 1 January February 2013 Page 151
hazardous products in your home. The following guidelines will help you when using hazardous products to keep your home and environment safe. Read the directions on the label and follow them. Use the product only for the tasks listed on the label. Wear protective equipment recommended by the manufacturer. Handle the product carefully to avoid spills and splashing. Close the lid as soon as the product is used. This will control vapors and reduce chances of spills. Secure lids tightly. Use products in well-ventilated areas to avoid inhaling fumes. Work outdoors if possible. When working indoors, open windows. Use a fan to circulate the air toward the outside. Take plenty of fresh-air breaks. If you feel dizzy, headachy or nauseous take a break and go outside. Do not eat, drink or smoke while using hazardous products. Traces of hazardous chemicals can be carried from hand to mouth. Smoking can start a fire if the product is flammable. Do not mix products unless directions indicate that you can safely do so. This can cause explosive or poisonous chemical reactions. Even different brands of the same product may contain incompatible ingredients. If pregnant, avoid toxic chemical exposure as much as possible. Many toxic products have not been tested for their effect on unborn infants. Avoid wearing soft contact lenses when working with solvents and pesticides. They can absorb vapors and hold the chemical near your eyes. Carefully and tightly seal products when you have finished. Escaping fumes can be harmful and spills can occur. Follow label directions for proper storage conditions. Leave the product in its original container with original label attached. Never store hazardous products in food or beverage containers. Make sure lids and caps are tightly sealed. Store hazardous products on high shelves or in locked cabinets out of reach of children and animals. Store incompatibles separately keep flammables away from corrosives. Store volatile productsthose that warn of vapors and fumes in a well-ventilated area, out of reach of children and pets. Keep containers dry to prevent corrosion. Store rags used with flammable products (furniture stripper, paint remover, etc.) in a sealed marked container. Keep flammable products away from heat, sparks or sources of anything that could ignite them. Know where flammable materials in your home are located and know how to extinguish them. Consider the sociological effects of solvent addiction. An addict looses the normalcy of life and day by day moves towards a un aimed destiny. Besides this the side effects like muscle weakness, renal dysfunction, acute respiratory distress etc. arising out of continuous exposure to solvents disable the addict to lead a normal life, thus making them a burden to the society. Fragrance actually dulls the mind and the senses, by the way. That's a completely different topic, but the short version is that if you wear perfume and use fragrance in your laundry, your mind is dulled. By using only fragrance-free products, you will literally become more intelligent. No kidding. REFERENCES Toxic Household Chemicals, by: Jo Hartley, 2009, http://www.naturalnews.com/025507_chemicals_toxic_products.html Edit by Natural News.com
Hazardous Household Products, Prepared by: Wilma Hemet Published by: North Carolina Cooperative Extension Service Publication Number: HE-368-1 Gelder M, Gath D, Mayou R, Oxford Textbook of Psychiatry, ELBS, 2nd ed, 554-555. Ed Kaplan HI, Shaddock BJ, Comprehensive Textbook of Psychiatry-VI, Volume-I, 6th Ed, Williams & Wilkins, 838-842. Saferstein R, Criminilastics, An introduction to Forensic Sciences, Prentice hall, 6th ed, 1998. Varghese L, Mathew NM, How to develop better NR solution adhesives, Rubber Asia, May-June, 1997, 27-28. Ed Moffet, AC, Osselton ND, Widdop B, Clarkes analysis of Drugs and Poisons, Vol-I & II, Pharmaceutical Press, 3rd edition. Volume 1 Issue 1 January February 2013 Page 152

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QUANTITATIVE ANALYSIS OF ACETAMINOPHEN IN NUROMOL TABLETS BY HIGH PRESSURE LIQUID CHROMATOGRAPHY

Series1 300000 Series2 Linear (Series1) 200000 100000

Figure: 1 Chromatogram of Acetaminophen Volume 1 Issue1

Figure: 2 Calibration curve for Acetaminophen Page 1

January February 2013

Krupa Rani Gorthi and Aravind G

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

Krupa Rani Gorthi and Aravind G

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

January February 2013

Harish Gopinath et. al

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

January February 2013

Harish Gopinath et. al

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

January February 2013

Harish Gopinath et. al

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

Table.1.Standard graph of captopril at 226.0nm

Table.2.Evaluation of formulation and micromeritic properties

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

C1 C2 C3 C4 E1 E2 E3 E4 S1 S2 S3 S4 SH1 SH2 SH3 SH4

60 64 68 66 68 72.8 77.6 72 69.2 73.6 77.6 78.7 72 77.6 77.9 79.4

January February 2013

Harish Gopinath et. al

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

Figure 1. SEM of the Captopril microcapsules

Table.3. In-vitro Dissolution Studies

Figure 2(a) FTIR studies of pure drug Captopril

Figure 2(b) FTIR studies of Chitosan

January February 2013

Harish Gopinath et. al

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

Figure 2(c) FTIR studies of Ethyl cellulose

Figure 2(d) FTIR studies of Sodium alginate

Figure 2(e) FTIR studies of HPMC

Figure 2(f) FTIR studies of HPMC+ Sod.Alginate

Figure 2(g) FTIR studies of Captopril + chitosan

Figure 2(h) FTIR studies of Captopril + Ethyl cellulose

Figure 2(i) FTIR studies of Captopril + Sod.alginate

Figure 2(j) FTIR studies of Captopril + Sod.alginate+ HPMC

January February 2013

Harish Gopinath et. al

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

January February 2013

Harish Gopinath et.al.,

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

Harish Gopinath et.al.,

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

Diclofenac sodium PEG-4000

Diclofenac sodium PEG-6000

Diclofenac sodium (PEG-4000 & PEG6000)

Harish Gopinath et.al.,

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology

Percentage drug release

Table.5. Percentage of Diclofenac Sodium Dissolved From Tablets

1.35 4.50 10.35 14.85 25.65 28.35

5.025 22.50 45.00 73.50 84.37 95.62

January February 2013

January February 2013

Dayananda Bhowmik et.al.,

ISSN: 2320 3471(Online) Indian Journal of Research in Pharmacy and Biotechnology