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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Dhanashri et al. World Journal of Pharmacy and Pharmaceutical Sciences Volume 1, Issue 2, 674-692. Research Article ISSN 2278 4357

DEVELOPMENT AND VALIDATION OF HERBAL ANTISEPTIC TOPICAL FORMULATION


Dhanashri B. Nagulwar*1, Pravin K. Bhoyar 2, Jagdish R. Baheti 3, Dinesh M. Biyani 5, Dharmendra R. Mundhada4 , Prasad P. Kathade 2 1. Department of Pharmaceutics, Sharad Pawar College of Pharmacy, Dist: Nagpur, Maharashtra, India. 2. Siddhivinayak College of Pharmacy,Warora, Dist-Chandrapur, Maharashtra, India. 3. Shriman Suresh Dada Jain College of Pharmacy, Chandwad, Nasik, Maharashtra, India 4. Department of Pharmaceutics, Agnihotri College of Pharmacy, Wardha, Maharashtra, India 5. Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, DistRanchi, Jharkhand, India. ABSTRACT
Article Received on 02 June 2012, Revised on 28 June 2012, Accepted on 13 July 2012

For skin infections, topical treatments applied directly to the skin are safest. Present work was decided to carry out antimicrobial activity and antiseptic effect of formulated cream. Oil of J. regia oil of K. galanga, methanolic extract of C. tora seeds and T. arjuna bark were

*Correspondence for Author: * Dhanashri B. Nagulwar, Department of Pharmaceutics, Sharad Pawar College of Pharmacy, Dist: Nagpur, Maharashtra, India. pravinbhoyar007@rediffmail.c om

successfully extracted and phytochemically screened. Thin layer chromatography of above extracts showed that it contains different active constituents. The result of phytochemical screening reveals that the major constituents of extracts were coumarins, flavonoids, steroids and triterpenoids. The in- vitro antimicrobial activity showed that the oil, methanolic extract and formulated cream posses prominent activity compared with that of standard. Zone of inhibition were found to be prominent in concentration 5g / ml of oil and 155ug / ml for C. tora seeds extract. In antimicrobial activity of cream, the formulation containing maximum percent of oils i.e. 5% and 155ug/ml of C. Tora

methanolic extract showed highest zone of inhibition. Validation of cream was done and was found within the limits. The pH of cream was found to be in range of 6.5 7.0 which is good

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for skin pH. The consistency of cream was in the range of 230 to 290 1/10mm. The formulated cream was used for antiseptic action. Patient trial was carried out and successful results were seen. For this purpose cream was applied topically. In human study, trials were conducted on 10 patients suffering from burn infection, tinea pedis and acne. Signs of the infection were healed in the period of 2 week. Key Words: Skin, C. tora, tinea pedis, acne. INTRODUCTION India has an ancient heritage of traditional medicine. Materia medica of India provides lots of information on traditional aspects of therapeutically important natural products. The evaluation of these drugs is mainly based on phytochemical, pharmacological and allied approaches including various techniques like chromatography and microscopy. [1] Herbals are being used, in medicine from time immemorial because they have fitted the immediate personal need, they are accessible and inexpensive. It is widely accepted that the use of herbal medicine is well established and safe. [2] For skin infections, topical treatments applied directly to the skin are safest. Drugs taken orally affect both disease and normal tissues, thus increasing the chance of side effects. Conventional skin disease treatments such as the drugs ketoconazole, ciclopirox, naftifine and tolnaftate can irritate the skin, causing stinging, itching, redness, drying or allergic reactions. Therefore treatment of many herbs can be used safely for superficial skin infections. Herbal medicine, now a days are gaining importance for treating many diseases due to their significant effect and lesser side effects as compared to allopathic medicines.[3] Plants like Clove, Creosote, Garlic, Chamomile, Goldenseal, Tea tree oil, Echinacea, Sassafras has antiseptic and wound healing action. From literature survey, it was revealed that phytochemical analysis of C. tora, j. regia, T. arjuna, k galangal confirmed the presence of flavonoids, steroids, triterpenoids in methanolic extract and these extract exhibited highest antimicrobial activity against S. aureus, B. subtilis, P. bacteria. Plants can be used for antiseptic action therefore decided to use of these plants for further study. From market survey, it was indicated that no cream is available containing combination of these plants therefore decided to formulate antiseptic herbal cream for microbial infection.

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In present project, methnolic extract of C. tora seeds and n-hexane extract (oil) of j. regia and k. galanga will be used for antiseptic action of cream. extracted oil are soluble in oily phase and C. tora methanolic extract is soluble in aq. phase therefore it is expected that extract should be compatible with cream base therefore decided to use o/w cream base for development of herbal antiseptic topical formulation. o/w creams also cause less dryness and burning, nonstaining and water miscible. Creams are well absorbed and tolerated by many people. [4] In addition to this combination of plant extracts formulation will be enriched with Aloe-vera extract as emollient for the skin. Therefore these formulations will be expected to exhibit better efficacy as compared in the marketed antiseptic formulation. In present work main objective is development and validation of Herbal antiseptic cream, in vitro evaluation of antimicrobial activity, and in vivo study, assessment of antiseptic activity of developed cream on patients through various skin infections will be expected to exhibit better antiseptic effect as compared in the marketed antiseptic formulation. MATERIAL AND METHODS Collection and authentication of plant material The T. arjuna bark, J. rgia seeds, K. galanga rhizome, C. tora seeds were collected from the Shri Shail Medi Pharma of Nagpur district. The plant specimen was dried and its herbarium sheet was prepared and it is available in Department of Botany, Nagpur University, Nagpur. Procurement of Material and Extraction These parts of the above mentioned plants subjected to size reduction to get coarse powder. Such powdered material was charged into the Soxhlet apparatus and extraction was carried out using n-hexane as solvent for J. regia seeds and for K. galanga rhizomes whereas petroleum ether (60-80o) and methanol (GR) for T.arjuna bark and C. tora seeds. Various Phytochemical screening tests are carried out such as tests for sterol like salkowaski test, liebermanns test, liebermann-burchard test.[5] Tests for alkaloids like dragendorffs reagent, mayers reagent, wagners reagent, hagers reagent. Test for saponins and test for tannins like ferric chloride test, lead acetate test, potassium dichromate test, gelatin solution test. Test for flavonoids (Shinoda test) and Test for proteins like biuret test, xanthoproteic test, millon's test (Mercury nitrate solution) and test for fixed oils are carried out.

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Thin Layer Chromatography of different extracts [6-7] Chromatographic pattern of active extract of T. arjuna bark, C. tora seeds j. regia seeds and K. galanga rhizome was studied by thin layer chromatography to find out the number of phytoconstituents present in them. The adsorbent material used for the thin layer chromatography was silica gel G. Analysis of oil [8] The various physical constants like acid value, saponification value and ester value were determined by using standard methods of Indian Pharmacopoeia (1996). Acid value Oil sample (2 g) was accurately weighed and dissolved in 50ml of mixture of ethanol (95%) and ether (1:1), which was previously neutralized with 0.1M potassium hydroxide solution. The flask was connected with a reflux condenser and warmed slowly with frequent shaking until the sample was dissolved; 1ml of phenolphthalein solution was added and titrated with 0.1M potassium hydroxide until the solution remained faint pink after shaking for 30sec. The acid value was calculated from the expression. Acid Value = 5.61 n / m Where, n = ml of KOH required. w = weight in gram of sample. Saponification value Accurately weighed 2g of the oil was added in 250ml flask of borosilicate glass fitted with reflux condenser. 25ml of 0.5M ethanolic potassium hydroxide was added and boiled under reflux on water bath for 30min, Phenolphthalein solution (1 ml) was added and titrated with 0.5M hydrochloric acid (ml) and repeated the same procedure for blank (bml).

Saponification value = 28.05 Where, w = weight in g of the sample Ester value Acid value and saponification value were determined as per the Indian pharmacopoeia procedures and the ester value was calculated from the expression,

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Ester Value = Saponification Value Acid Value Anti-microbial activity


[9-11]

The antimicrobial activity of K. galanga oil, J. regia oil , methanolic exrtract of C. tora seeds and T. arjuna bark were studied against Gram-positive S. aureus and B. substilis, P. bacteria and C. albicans and A. niger fungi by agar diffusion method. For antibacterial activity streptomycin while for antifungal activity griseofulvin was used as the standard. Screening of antimicrobial activity was carried out using the cup plate agar diffusion method. This method depends on the diffusion of drug through the solidified agar layer of a petridish to an extent such that growth of the inoculated microorganism prevented entirely in a circular area zone around the cup containing the solution of the compound under test. One loopful of the stock culture was inoculated at 10 ml of agar slant previously in sterilized test tubes, and incubated at 37oC for 24 h and 20oC for 48 h respectively for bacteria and fungi. About 3ml of distilled water was added to the test tube and a suspension of the culture was obtained by shaking for few minutes. The solutions were made by dissolving the test oil compound in minimum amount of DMSO (dimethyl sulphooxide), and solid compound in minimum amount distilled water, volume was made with sterilized water to produce a concentration of 100g/ml. Respective sterile medium was melted on water bath and kept at 45oC. In each sterile petridish 25ml of molten medium was added and 107/ml of sub cultured organism under study was inoculated. Formulation of cream [12-13] For the convenient application of oil and other methanolic extract and by considering the antimicrobial activity, cream was selected as a suitable dosage for topical application. Creams are nonstaining and water miscible. Creams are well absorbed and tolerated by many people. Cream base formula was selected for the development of herbal antiseptic cream as shown in table 1.

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Table 1: formula for development of cream base

Sr. No

Ingredients

% w/ w

Oil phase 1. 2. 3. 4. Stearic acid Cetyl alcohol Propyl paraben Butylated hydroxyl toluene 26.56% 1.0% 0.01% 0.02%

Water phase 1. 2. 3. 5. 6. Propylene glycol Glycerine Triethanol amine Methyl paraben Water 5.0 % 5.0 % 1. 0 % 0.01% 61.42%

Method of preparation Oil soluble components were dissolved in oil phase and heated to 75C. Other water soluble components were dissolved in aqueous phase and heated to 75C. After heating, oil phase was added in aqueous phase with continuous stirring till a thick viscous cream was obtained. Formulation development of herbal creams of botanical extract. Topical herbal creams of mentioned botanical extracts were prepared as per the following way on trial and error basis. Batch I- In this batch, a single botanical extract (C. tora seeds methanolic extract ) was added which was compatible with above base. Batch II- In this batch, cassia tora seeds methanolic extract and Juglans regia oil were added which were also compatible and more emollient than the pervious. Batch III- In this batch, all mentioned extracts were added, it was noted that all extracts were compatible with the base formula and cream texture and color was more acceptable than the

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previous batch. Cream formulated in trial batch III was taken as the master formula and subjected for further evaluation study. Formulas for each trial batch cream as shown in table 2. Table 2: Formula for each batch Ingredients Stearic acid Cetyl alcohol Batch I Batch II Batch III 26.56% 1% 0.02%

26.56% 26.56% 1% 1% 0.02%

Butylated hydroxy 0.02% toluene Propyl paraben J. regia oil K. galangal oil Glycerine Propyleneglycol Triethanol amine Methyl paraben 0.01% 5% 5% 1% 0.01%

0.01% 5.0% 5% 5% 1% 0.01% 1.55%

0.01% 5.0% 5.0% 5% 5% 1% 0.01% 1.55%

C. tora seeds 1.55% methanol extract Aloe vera extract Water Evaluation of cream -

2.0% 54.15%

59.85% 54.15%

As per the requirements for skin creams specified in Indian standards (6608: 2004), following parameters were used for evaluation of cream. Thermal stability For thermal stability testing, a humidity chamber and clear glass container of around 30ml capacities with screw cap were used. With the help of spatula cream was inserted in the glass containers and tapped to settle to the bottom and plug was inserted and tightens the cap. The filled bottle was kept inside the incubator at 45 10C for 48h. On removal from the

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incubator, it was noted that no oil separation or any other phase separation was not observed, formulated cream was stable at 450C. Determination of pH of formulated Cream The digital pH meter was calibrated using buffer solution of pH 4.01, 7.0 and 9.2. Cream was taken in a beaker and the pH of the cream was determined. Determination of total fatty substance content About 2g of the accurately weighed formulated cream was taken into a conical flask, Dilute Hydrochloric acid (25ml) was added and a reflux condenser was fitted into the flask and the solution was boiled until it perfectly cleared. Then contents of the flask were poured into a 300ml-separating funnel and was cooled to room temperature. In portion of 10ml the conical flask was rinsed with 50 ml of petroleum ether and poured into the separating funnel, separating funnel was then shaked well and left until the layers were separated. An aqueous phase was separated and all ether extract was then washed with water. This petroleum ether extract was then filtered through a filter paper and dried the material in the flask at a temperature 90 20C. of to constant mass. Formula: Total fatty substance = 100 M1/ M2 Where, M1= mass (g) of the residue M2 = mass (g) of the cream Determination of residue About 5g of the cream was taken in a weighed, clean and dry squat form weighing bottle and dried to constant mass at 105 10 C. Cooled in a desiccator and weighted. Formula: Residue = 100 M1/ M2 Where, M1 = mass (gm) of the residue M2 = mass (gm) of the cream Test for lead Standard lead solution was prepared by using 1.600gm of the lead nitrate taken in water and the solution was made to 1000ml. solution (10ml) was Pipette out and diluted to 1000ml with water. One mm of this solution contains 0.01mg of lead (Pb). About 2.00gm of cream was 681

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taken in a crucible and heated on a hot plate and then taken in a muffle furnace to ignite it at 6000 C to constant mass. Dilute hydrochloric acid 3ml (5N) was added and warmed and volume was made to 100ml. solution was then filtered. In the second Nessler' s cylinder, 2 ml of dilute acetic acid (1N) was added, volume was made with water to 25ml. standard hydrogen sulphide(10ml) solution was added to each Nesslers cylinder and volume was made with water (50ml) mixed and allowed to stand for 10min and the colour produced in two Nesslers cylinders was compared. The colored produced with hydrogen sulphide was matched against that obtained with standard lead solution. Spreadability Spreadability of cream was measured with the glass slide apparatus, excess of cream was

placed between two slides and 1 kg weight was placed on slide for 5 min. to compress the sample to uniform thickness, time in seconds to separate two slides was taken as measure of spreadability. S=wl/t where, S = spreadability (g cm/sec) w = weight on upper slide (g) l = length of Slide (cm) t = time taken in sec (sec) Homogeneity The developed cream was tested for homogeneity by visual inspection, after the cream have been set in the container, spread on the glass slide for the appearance, tested for the presence of any lumps, flocculates or aggregates. Skin irritation test The skin irritation was carried out on human volunteers. For formulated cream, five volunteers were selected and 1.0g of formulated cream was applied on an area of two square inch to the back of the hand. The volunteers were observed for lesions or irritation. Consistency Consistency of the formulation was determined by penetrometer model no. 2. Associated instrument Pvt. Ltd. Culcutta.

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Microbial evaluation of herbal formulations is essential to check the limits of microbial contamination and extents of pathogencity. This evaluation has direct correlation with the quality of products. For the evaluation of total microbial count details of different count media were used, nutrient agar medium used for the growth of bacteria and Potato dextrose agar medium was used for the growth of fungi. [15] In aseptic conditions cream equivalent to one gram was dissolved in 10ml of sterile water and was serially diluted. The medium and apparatus required for experimental were sterilized in an autoclave at 1210C for 15min. In aseptic conditions, 1ml of test sample was transferred to petridish containing melted agar medium at about 420C and mixed well by rotating the petridish. It was allowed to solidify and then incubated at 370C for 24h for detection of bacteria. After incubation period, colonies were counted. Formula: No. of colonies No. of microorganisms = Volume of sample Stability testing of formulated cream. For assessing the stability of formulated creams following parameters were taken into consideration like Thermal stability testing, pH, Total fatty substance content, Total residue, General test for lead, Consistency, Spreadability. [16] These studies are essential to ensure that product is stable over its designated shelf life. The stability study was carried out for three months as per ICH norms, at three different temperatures such as at room temperature, 450C and 8 to 100C. Results of stability testing of formulated cream are given in table. Patients study Realising the importance of patient acceptability the formulation was filled into 20gm capacity container and dispensed to the patients suffering from acne, Tinea pedis, burn, in the dermatology department of government Ayurvedic hospital Nagpur. The study was supervised under the Dr. Kabra and Dr. Deepak Madavi and others doctors. Each Polyherbal cream (containing combination of methanolic extract of Cassia tora seeds, Juglans regia oil, Kaemferia galanga oil) was prescribed to the patients suffering from skin diseases. A retrospective analysis of their record revels that the cream was well tolerated by 683 dilution factor

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the patients. Consent form was signed by the patients before prescribing cream. The treatment was given for two weeks to the patients suffering from burn infection, Tinea pedis and acne. Patient study for Burn infection Three patients suffering from burn infection were taken for patients study. Damage in superficial burns was not deeper than papillary epidermis.[17] Clinical features were oedema redness, bleb formation and pain due to preserved nerve endings. Compliance of patients was good against formulated cream than the marketed preparation. Patient study for acne Acne is an inflammatory skin disorder of the skins sebaceous glands and hair follicles and occurs when the cells lining the sebum canals are injured or when follicles or pores are blocked or clogged by sebum (oil) and dead cells mixed together. Patient of either sex, age between 21- 45 yrs. were selected, two types of pigmentation were observed having black coloured and white coloured pigmentation, while giving the treatment it was observed that pimple infection did not spread further, cream act effectively on P. bacteria, sebum irritation decreases, pores did not clog further. Patients study for Tinea pedis Tinea pedis is the fungal infection of fact mostly involving in between the toes. It is usually associated with prolonged exposure to moisture. It involved, skin appears blanched with cracks and associated itching. Treatment involves the application of cream base, formulated cream and marketed preparation (skina cream). RESULT AND DISCUSSION All the extracts of plant material were subjected to preliminary phytochemical screening for the detection of various plant constituents. Phytochemical screening of methanolic extract of T. arjuna bark, C. tora seeds showed presence of sterols, saponins, tannins, flavonoids, sugar, proteins and coumarin. Hexane extracts of J. regia seeds and K. galanga rhizome showed positive test for sterols, flavonoid and coumarin hence both the extracts were used for further study. Chromatographic pattern of above mentioned plant extracts were studied by thin layer chromatography to find out the number of constituents present in them. Stationary phase used www.wjpps.com 684

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was Silica gel G and for mobile phase different combinations of the solvents were tried for the development of the solvent system for different extracts. Mobile phase with Benzene: Ethyl acetate (8:2) showed maximum resolution and reproductive results. After spraying with 50 % H2SO4 and in iodine vapour gives 3 spots with Rf values 0.34, 0.61, 0.87. of petrolium ether extract T. arjuna bark and gives 3 spots with Rf values 0.34, 0.54, 0.74 C. of petrolium ether extract of C. tora seeds. Mobile phase with Butanol; Acetic acid; Water (6:2:2) showed maximum resolution and reproductive results. After spraying with 50 % H2SO4 and in iodine vapour gives 2 spots with Rf values 0.48, 0.76 methanol extract of. T. arjuna bark and 6 spots with Rf values 0.32, 0.45, 0.57, 0.74, 0.81, 0.96 of methanol extract of C. tora seeds. Mobile phase with Benzene: Ethyl acetate: Acetone (8:1:0.5) showed maximum resolution. After spraying with 10 % vanniline sulphuric acid and in iodine vapour gives 4 spots with Rf values 0.32, 0.68, 0.82.0.94 of J. Regia oil and 6 spots with Rf values 0.38, 0.46, 0.52, 0.64, 0.77, 0.84 of K. galanga oil. As shown in Table 11, acid value, saponification value and ester value of J. regia seeds oil were found to be 15.54, 134.5, and 118.7 and of K. galanga oil were found to be 1.2, 105.4 and 121.7 respectively as shown in table 3. Table 3: Evaluation parameters of J. regia and K. galanga oils Parameter Acid value Saponification Value Ester value J. regia Oil 15.54 134.5 118.7 K. galanga oil 1.2 105.4 121.7

It was indicated that methanolic extract of C. tora seeds, K. galanga oil and J. regia oil had maximum antimicrobial activity whereas methanolic extract of T. arjuna bark had not maximum antimicrobial activity as compared to that of other plants extract so T. arjuna bark extract was not used in formulation of cream as shown in table 4, 5, 6 and 7.

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Table 4: Antibacterial activity of K. galanga oil. Microorganism 1g/ml 4 7 7 2g/ml 7 10 9 Zone of Inhibition in mm K. galangal 3g/ml 4g/ml 10 13 14 13 15 17 5g/ml 14 17 19 Streptomycin (5g/ml) 18.2 21.1 23.2

P. bacteria B. subtilis S. aureus

Table 5: Antibacterial activity of J. regia oil. Zone of Inhibition in mm J. regia oil 2g/ml 3g/ml 4g/ml 5g/ml 11.2 11.1 9 12.5 12.2 13 13.3 13.2 15 14.1 16.7 18.7

Microorganism P. bacteria B. subtilis S. aureus

Streptomycin (5g/ml) 18.2 21.1 23.2

1g/ml 9 10.2 7

Table 6: Antibacterial activity of methanolic extract of C. tora seeds and T. arjuna bark extract
Zone Of Inhibition in mm Extracts

B. Subtilis 14.3

S. aureus 16.4

P. bacteria 13.2

C. tora seeds methanolic extract (155ug/ml) T. arjuna methanolic extract (170ug/ml) Streptomycin 5ug/ml

3 18.2

2 21.1

23.2

Table 7: Antimicrobial activity of all combined extracts Zone of Inhibition in mm J. regia C. tora seeds oil + methanolic + extract Streptomycin 5g/ml 155 ug/ml (5g/ml) 16.7 18.2 19.5 21.9 21.1 23.2

Microorganism P. bacteria B. subtilis S. aureus

K. galanga oil + 5 g/ml

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Cream formulated in trial batch III was taken as the master formula and subjected for further evaluation study. It was indicated that evaluation parameter of formulated cream are within the limits as per the requirements for skin creams specified in Indian standards as shown in table 8. Table 8: Evaluation of Cream Cream base + C. tora methanolic extract + J. regia oil + K. galanga oil No phase separation 6.8 37.78%/gm Passes the test 46.94%/gm No lumps

Sr. No

Parameter

Cream base

Cream base + C. tora methanolic extract

Cream base + C. tora methanolic extract + J. regia oil

1 2 3 4 5 6 7 8

Thermal stability testing pH Total fatty substance content, (%/gm) General test for lead Total residue, (%/gm) Homogeneity Spreadability Skin irritation test Consistency at room temperature

No phase separation 6.4 26.32%/gm Passes the test 37.26%/gm No lumps

No phase separation 6.6 27.56%/gm Passes the test 38.81%/gm No lumps

No phase separation 6.7 32.97%/gm Passes the test 42.57%/gm No lumps

10.32gm.cm/se 10.30gm.cm/se 12.59gm.cm/sec 13.01gm.cm/sec c c No irritation 237 276 151 No irritation 233 273 149 No irritation 239 179 159 No irritation 243 283 165

9 At 450C 1/10 mm At 8-10 0C 1/10 mm

The results for determination of total plate count of bacteria and fungi are shown in following table 9 and 10.

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Table 9: Total number of viable bacteria present in formulated cream. Types of microorganism Bacteria (C.F.U./ml) Cream base + C. tora methanolic extract + J. regia oil Cream base + C. tora methanolic extract + J. regia oil + K. galanga oil 1.21102

Formulat ed cream

Cream base

Cream base + C. tora methanolic extract

3.12 102

2.57 102

2.39 102

Table 10: Total number of viable fungi present in formulated cream. Formulated cream Cream base Types of microorganism Fungi (C.F.U./ml) Cream base + C. tora methanolic extract 2.42 102 Cream base + C. tora methanolic extract + J. regia oil 2.21 102 Cream base + C. tora methanolic extract + J. regia oil + K. galanga oil 1.15 102

3.29 102

From stability study of cream, it was indicated that cream was stable at three different temperatures such as at room temperature 450C and 8 to 100C. From ANOVA, it was indicated that there were no significant difference between the consistency, pH, spreadability, total fatty substance content, total residue content values of the different batches of formulated cream. Calculated F ratio were less than table F value, therefore accepted hypothesis that there were no difference between the batches at 5% level of significance. Three batches of cream were formulated, F1, F2 and F3 and their antimicrobial activity was carried out and the results of zone of inhibition are as shown in table 11, 12 and figure 1 and 2.

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Table11: Antibacterial activity of cream Microorgani sm P. bacteria B. subtilis S. aurbeus Zone of Inhibition in mm Formulated cream Streptomycin (5g/ml) F1 F2 F3 17.2 17.4 17.4 18.2 19.7 19.7 19.7 21.1 22.2 22.3 22.3 23.2
Antibacterial activit
25 20 m
m n i 15 n o i t 10 i b i h n i 5 f o e0 n o z

F1 F2 F3 Sreptomycin
1 . B. subtilis 2. S. aureus 3. P. bacteria 1 2 3

Figure 1: Antibacterial activity of cream Table 11: Antifungal activity of cream

Microorg anism

C. albicans A. niger

Zone of Inhibition in mm Formulated cream 5ug/ml grisevofulvi n F1 F2 F3 (5g/ml) 23.4 23.5 23.7 25.2 20.6 20.9 20.9 21.7

Thus, maximum zone of inhibition is found in cream containing all polyherbal extract which is more or less equal to that of standard.
Antifungal activity
30 n o i t i b 20 i hm n i f m on i 10 e n o z 0 1 2 Bacterial
F1 F2 F3 grisevofulvin

1. B . su b tilis
2. S . au re u s 3. P .b acte ria

Figure 2: Antifungal activity of of formulated cream

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From patients study it was indicated that all the parameters and signs of the disease infection were healed in the period of 2 week. Following photo shows the effect of base, cream and marketed preparation on patients suffering from Tinea pedis, burn injury and acne as shown in figure 3, 4, 5, 6 and 7. Figure 3: Effect of marketed cream on tinea pedis patients.

Before treatment

after one week

after two week

Figure 4: Effect of formulated cream on tinea pedis patients

Before treatment

after one week

after two week

Figure 5: Effect of marketed cream on acne patients

Before treatment

after one week

after two week

Figure 6: Effect of formulated cream on acne patients

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Before treatment

after one week

after two week

Figure 7: Effect of formulated cream on burn patients.

Before treatment After two week

After one week

After two week

Before treatment After one week

After two week

Before treatment After one week

After two week

REFERENECES 1. Kumar V, Parmar NS. Herbs: A Potential Source for the Development of New Phytomedicinals. The Pharma Review. 2003; 1: 56. 2. Mukherjee PK. Quality Control of Herbal Drugs. 1st ed., New Delhi; Business Horizon Pharmaceutical Publishers: 2002. 3. World Health Organization. Research Guidelines for Evaluating the Safety and Efficacy of Herbal Medicines; 1993.p. 397. 4. Khan MR, Khihana M, Omoloso AD. Antimicrobial activity of Michelia champaca. Fitoteripia, 2003; 73: 744. 5. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 1st ed., Mumbai; Nirali Prakashan: 1990. 6. Stalhl E. Thin Layer Chromatography. 2nd ed., Berlin; Springer- Verlay Berlin

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