Research Article ISSN: 0974-6943

Ravi Kant Upadhyay et al. / Journal of Pharmacy Research 2011,4(1),167-170

Available online through www.jpronline.info Antimicrobial activity of two Indian medicinal plants Tinospora cordifolia (Family: Menispermaceae) and Cassia fistula (Family: Caesalpinaceae) against human pathogenic bacteria
Ravi Kant Upadhyay*, Rajani Tripathi, Shoeb Ahmad Department of Zoology, D D U Gorakhpur University, Gorakhpur, 273009. India

Received on: 12-09-2010; Revised on: 18-10-2010; Accepted on:13-12-2010 ABSTRACT

In the present investigation antimicrobial activity of two Indian medicinal plants i.e. Tinospora cordifolia and Cassia fistula were evaluated against seven human pathogenic bacterial strains. Both were tested by serial micro-dilution method. Antimicrobial susceptibility of aqueous and solvent extracts was assessed. For this purpose, both positive and negative controls were set to determine MIC and MBC values. After bioassays, aqueous and solvent extracts of Tinospora cordifolia and Cassia fistula exhibited significant antimicrobial susceptibility against bacteria both Gram negative and Gram positive i.e. Klebsiella pneumoniae (ATCC 15380), Escherichia coli (ATCC 25922), Micrococcus luteus (ATCC 9341), Streptococcus pneumoniae (ATCC 12755), Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 11778) and Lactobacilus acidophilus (ATCC 53103) at a very low concentration. More specifically, higher percent growth inhibition was obtained in presence of aqueous extracts in comparison to solvent extracts, which was much higher than synthetic antibiotics. Further, different extracts have shown very low MIC value, which was obtained in a range of 0.0078-0.125 mg/ml. It was much lower than the MIC values (0.223-0.892 mg/ml) obtained in presence of standard antibiotics i.e. tetracycline, ampicillin and ciprofloxacin. More specifically, aqueous extracts of both plants have shown lower MIC (0.00975 mg/ml in E. coli and K. pneumoniae) and MBC values (0.078 mg/ml in E. coli and K. pneumoniae) than broad-spectrum antibiotics (0.223-0.892 mg/ml). Further, aqueous extracts of both plant species have shown significantly much higher inhibition zone diameters (20-30mm) against all seven bacterial strains than the synthetic antibiotics (6-18). Certainly, above antimicrobial effects are attributed due to presence of certain chemical substances in the leaves and legumes of above plant species. Key words: MIC, MBC, antibacterial activity, plant extracts, Tinospora cordifolia and Cassia fistula

INTRODUCTION
In the present time multiple drug resistance in microbial pathogens become a serious health problem to humankind worldwide (Peng et al., 2006). It is aroused due to indiscriminate and repetitive use of antimicrobial drugs by inadequate disease treatment (Shariff, 2001). To acquire drug resistance microbes have developed new enzyme system to cleave the drug and make it useless for control of infection (Ritch-Kro et al., 1999). Hence, plant origin herbal medicines are considered as safe alternatives of synthetic drugs. There are varied methods of medicines like Aurveda, Homeopathy and Unani, which utilize plant materials for drug production. Currently, Aurveda considered as a vital system of medicine and governed the worldwide recognition and having non-toxic substances. Hence, in the last few decades, so many plant species were explored for obtaining potential antimicrobials for therapeutic purposes (Rios and Recio, 2005; Liu, 1987), which later on become an integral part of primary health care in many parts of world (Desta, 1993; Anesini and Perez, 1993; Cown, 1999). Hence, plants, which possess strong antimicrobial potential against pathogens are considered as valuable source of medicinal compounds and show lesser side effects. Plants are rich source of wide variety of secondary metabolites viz. tannins, terpenoids, alkaloids, and flavonoids, which possess enormous antimicrobial properties (Suresh et al., 1992). Approximately 25 to 50 % of current pharmaceuticals are derived from plants. Most of them were found effective against many pathogenic bacteria (Bilgrami et al., 1992), fungi (Pacheco et al.,1993), viruses (Nascimento et al., 2000) and even found active against drug-resistant microorganisms (Mohana et al.,2008). Besides this, few antimicrobials such as essential oils (Yang et al., 2010; Nannapaneni et al., 2008), plant extracts (Mohana et al., 2008) and pure compounds have shown broad-spectrum antimicrobial activity against pathogens (Acharya et al., 2009; Serrentino, 1991). Some of the plant products are used as nutraceuticals (Adwan, 2006; Tiwari et al., 2009) and in food preservation (Rege et al., 1999). In the present investigation, two indigenous plant species from India have been screened for their antimicrobial activities. Tinospora cordifolia is a large glabrous ascending shrub belongs to family Menispermaceae and known as Giloy in Hindi. The leaves are membranous and cordate. It is used as a blood purifier and anti-infectious agent. It is also used for the treatment of jaundice, rheumatoid arthritis, diabetes, gout, viral hepatitis, arthopathies and general weakness. Cassia fistula is a tree belongs to family Caesalpiniaceae and is an indigenous medicinal plant, commonly known as Amaltash, Cassia fistula is a medium size tree and having yellow flowers and green leaves. It is an ornamental flowering plant, its seeds and legumes are used for gastric and respiratory problems. Long bunches of flowers are used for decoration purposes. Seeds are used for stomach ailments while bark and leaves for burns. The pulp of the fruit is used as purgative and laxative. EXPERIMENTAL PREPARATION OF EXTRACTS Plant parts of Tinospora cordifolia and Cassia fistula were collected from forest areas of Gorakhpur in eastern U.P. The stem of Tinospora cordifolia and legumes of Cassia fistula were chopped into small pieces, dried, milled and powdered by using pestle and mortar. The acetone, methanol, chloroform and petroleum ether extracts were prepared by using 50g of dried powder of C. fistula legumes and leaves of Tinospora cordifolia in 250 ml of solvent separately. Each solvent extract was fractionated to get different serial fractions in different solvent. The extract was dried; residue was weighed and dissolved in known quantity of fresh solvent. In the preparation of aqueous extract, 50 mg the powdered material was dissolved in 250 ml of distilled water and kept for solubilization overnight at room temperature. For quantization plant extracts were evaporated and re-dissolved in known volume of each solvent separately, MICRO-ORGANISMS Cultures of seven pathogenic bacterial strains each of Escherichia coli (ATCC 25922), Bacillus cereus (ATCC 11778), Lactobacilus acidophilus (ATCC 53103), Micrococcus luteus (ATCC 9341), Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 15380) and Streptococcus pneumoniae (ATCC 12755) were maintained in the laboratory in Luria Broth (2% w/v) regularly for four days at 37 0C before use in experiments. For experiments a portion (100 l) of the overnight culture was mixed in the tests and control for inoculation. For activity testing bacterial cultures were stored at 40 C and sub cultured after every 8th day in solid agar plates.

*Corresponding author.
Ravi Kant Upadhyay Department of Zoology, D D U Gorakhpur University, Gorakhpur, India 273009 Tel.: +919838448495 E-mail:rkupadhya@yahoo.com

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Table 1. MIC values obtained in presence of solvent and aqueous extracts of Tinospora cordifolia and Cassia fistula against seven pathogenic bacterial strains (mg/ml).
Tinospora cordifolia Plant extracts K.pneumoniae E.coli Chloroform Acetone Methanol Pet.ether Aqueous Cassia fistula Chloroform Acetone Methanol Pet.ether Aqueous Antibiotics Tetracycline* Ampicillin* Ciprofloxacin* 0.0585 0.0625 0.0625 0.0625 0.0195 0.0078 0.0156 0.0625 0.0312 0.0975 0.446 0.223 0.892 0.0146 0.0312 0.125 0.0312 0.00975 0.0156 0.0156 0.0312 0.0625 0.048 0.446 0.446 0.446 B.cereus M.luteus 0.2343 0.0312 0.0312 0.0312 0.039 0.0312 0.0156 0.0156 0.0312 0.048 0.446 0.446 0.892 0.0585 0.0312 0.0156 0.0625 0.0195 0.0156 0.0312 0.0156 0.0156 0.048 0.223 0.892 0.223 L.acidophilus S.aureus 0.0292 0.0625 0.0625 0.0312 0.039 0.0312 0.0312 0.0156 0.0078 0.048 0.446 0.223 0.223 0.0292 0.0156 0.0625 0.0625 0.039 0.0312 0.0312 0.0156 0.125 0.024 0.446 0.446 0.446 S.pneumoniae 0.0585 0.125 0.0156 0.0625 0.0195 0.0156 0.0156 0.0156 0.0078 0.024 0.223 0.223 0.223

Table 3. Inhibition zone diameters obtained in presence of different solvent and aqueous extracts of Tinospora cordifolia and Cassia fistula were measured by agar disc diffusion method (in mm) and compared with broad spectrum antibiotic drugs.
Plant extracts Conc. g/disc K.pneumoniae E.coli B.cereus M.luteus L.acidophilus S.aureus S.pneumoniae Tc Cf Tc Cf Tc Cf Tc Cf Tc Cf Tc Cf Tc Cf 12 16 17 18 25 9 11 20 21 25 7 8 11 12 15 9 10 12 14 25 8 14 16 17 22 6 8 13 16 17 7 8 14 16 17 20 8 10 13 17 7 8 10 13 15 Nil 7 9 15 22 Nil 11 17 19 22 6 13 15 16 18 11 13 15 20 27 8 10 13 16 26 10 12 18 20 22 11 15 16 20 24 Nil 12 13 14 16 11 15 16 17 25 7 9 12 14 17 6 9 13 16 17 6 9 12 15 16 7 8 10 12 15 7 22 24 25 27 7 12 15 17 25 Nil 10 11 16 17 12 13 14 17 25 12 13 15 17 21 9 15 16 20 22 8 9 13 14 15 6 9 11 13 17 7 15 16 20 21 6 9 11 13 15 6 9 12 15 16 6 9 12 16 17 7 10 12 14 19 7 13 16 18 20 6 10 12 16 20 7 14 15 17 18 15 16 17 19 20 10 17 19 20 27 8 10 15 21 25 9 10 14 18 25 10 11 13 15 20 7 11 15 16 20 7 10 12 14 16 7 8 14 15 17 7 8 14 17 18 7 9 12 14 20 11 15 16 17 22 8 12 13 15 16 7 11 12 15 19 9 14 17 23 30 11 12 16 17 22 8 14 15 17 25 7 8 10 17 25 11 12 15 17 20 7 10 15 16 21 6 8 12 15 16 7 10 15 16 17 7 9 15 16 17 6 9 11 12 16 12 16 18 20 26 7 13 14 18 20 7 11 16 16 19 8 11 13 17 20 13 16 17 21 26 9 10 15 16 17 15 19 20 21 23 7 8 15 16 17 11 16 17 20 22 6 8 10 14 16 6 8 13 16 17 7 9 14 15 17 7 9 11 13 16 13 17 19 22 26 6 17 18 22 32 7 10 12 13 18 10 17 19 20 22 11 12 15 20 23 7 8 10 12 20 15 17 18 19 20 11 13 15 19 27 10 15 17 20 25 7 10 13 16 18 6 9 16 17 18 7 8 10 13 15 8 13 14 20 21 7 8 13 17 20 Nil 9 14 20 22 7 11 12 15 18 10 12 16 22 25

MIC-Minimum Inhibitory Concentration Solubility of each plant extract was determined before treatment No turbidity was found visible after 24 hrs incubation *Antibiotics was used for comparison Table 2 . MBC values obtained in presence of solvent and aqueous extracts of Tinospora cordifolia and Cassia fistula against seven pathogenic bacterial strains (mg/ml).
Tinospora cordifolia Plant extracts K.pneumoniae E.coli Chloroform Acetone Methanol Pet.ether Aqueous Cassia fistula Chloroform Acetone Methanol Pet.ether Aqueous Antibiotics Tetracycline* Ampicillin* Ciprofloxacin* 0.1171 0.125 0.25 0.125 0.078 0.0312 0.0625 0.25 0.0625 0.39 0.892 1.78 1.78 0.0292 0.25 0.25 0.25 0.39 0.0312 0.0625 0.125 0.125 0.39 0.892 1.78 1.78 B.cereus M.luteus L.acidophilus S.aureus S.pneumoniae 0.4687 0.125 0.0625 0.0625 0.078 0.0625 0.0625 0.0312 0.0625 0.39 1.78 1.78 1.78 0.1171 0.0625 0.0312 0.25 0.078 0.0312 0.0625 0.0625 0.0312 0.195 3.57 7.14 0.892 0.0585 0.125 0.125 0.125 0.078 0.0625 0.0625 0.0625 0.0312 0.195 0.892 0.892 3.57 0.1171 0.125 0.125 0.25 0.078 0.0625 0.0625 0.0625 0.25 0.39 0.892 0.892 0.892 0.2343 0.25 0.0312 0.125 0.078 0.125 0.0312 0.0625 0.0156 0.195 0.892 0.892 0.892

Chloroform 20 40 80 160 320 Acetone 20 40 80 160 320 Methanol 20 40 80 160 320 Ether 20 40 80 160 320 Aqueous 20 40 80 160 320 Tetracycline* 20 40 80 160 320 Ampicillin* 20 40 80 160 320 Ciprofloxacin* 40 80 160 320

MBC- Minimum Bactericidal Concentration For Comparision both negative and positive controls were set *Antibiotics were used for comparison MEDIA PREAPRATION AND ITS STERILIZATION For antimicrobial susceptibility testing both solid (Agar-agar) and liquid agar (Luria broth) media were used. It has shown a good bacterial growth and reproducibility. For suspension culture of bacterial cells 1% Lauria Broth (w/v) was prepared by dissolving 1 gm of broth media in 100 ml of distilled water, while 2% agar solid was used for developing surface colony growth. DETERMINATION OF MIC AND MBC VALUES For determination of antimicrobial activity, bacterial growth inhibition was accessed in the presence of different increasing concentrations of Tinospora cordifolia and Cassia fistula extracts. For this purpose both crude extracts were separately diluted by using serial micro dilution method with Luria Broth culture medium at a final concentration range from 8.0 to 0.00975 mg/ml. The tested crude extracts and pure compounds were added to fresh suspension after following the serial dilutions up to 10-10. Each extract was assayed in triplicate. Before conducting experiments all the conditions for in vitro anti-microbial activity were standardized to determine MIC and MBC values. The MIC values considered as the lowest concentration of crude extract and pure compounds, which have shown no turbidity in the culture flask after 24 hrs of incubation at 370C. The turbidity in the culture flasks was considered as visible growth of microorganisms. Further, it was standardized in terms of absorbance at 600 nm in a visible spectrophotometer. For determination of minimum bactericidal concentration (MBC) growth inhibitory assays were performed. For this purpose inoculum size was adjusted to prepare a final colony number as 108 colony forming units (CFU/ml in sterile agar

* The effectiveness of different extracts demonstrated by the size of growth inhibition diameter obtained in filter paper disc diffusion assay in mm.*Tc: Tinospora cordifolia*Cf: Cassia fistula * The strength of activity is presented as resistant (>7mm), Intermediate (>12mm), and Susceptible (>18mm). In negative control no antibiotic was used. In positive control antibiotic was used for comparison . plates. The incubation of test and control cultures was performed at 370C for 24 hours. The least concentration at which no visible growth was obtained in agar plates was considered as MBC value. For evaluation of inhibition, both negative (with antibiotics) and positive (without antibiotics and extracts) parallel controls were set for each and every test. Bacterial growth was obtained in presence and absence of various concentrations of solvent and aqueous extracts of T. cordifolia and Cassia fistula separately. MEASUREMENT OF INHIBITON ZONE DIAMETER Antimicrobial susceptibility of Tinospora cordifolia and Cassia fistula extracts was evaluated by agar disc diffusion method of Kirby et al., (1966). Molten agar was used as media for bacteria. For this purpose six different concentrations of each extract and pure compounds were coated on sterile filter paper discs (Whatman No. 1) of 6 mm in sizes. Extract coated discs were dried under laminar flow cabinet. Before experiment inoculum size was determined and adjusted to prepare a final colony number as 108 CFU/ml (Colony Forming Unit) in sterile agar plates. Bacterial inoculum was spread evenly on to the surface of agar plate with sterile rubber pad spreader before the coated discs were positioned on the inoculated agar surface. Each extract and pure compound was assayed in triplicate. For comparison three broad spectrum antibiotics i.e. tetracycline, ampicillin and ciprofloxacin were also used parallel as controls. All treated and untreated plates were incubated for 24 hrs at 370C. The antibacterial activity was assessed and size of inhibition zone diameter surrounding the filter paper discs was measured. STATISTICAL ANALYSIS The results were interpreted with the standard deviation. Student t- test was

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applied to know significant differences between the antimicrobial effectiveness of each extract and antibiotics. The data was also statistically analyzed by applying ANOVA to know the significant difference in antimicrobial susceptibility of broadspectrum antibiotics and various plant extracts. The related effectiveness was also tested by applying linear correlation between control and tests. RESULTS DETERMINATION OF MIC AND MBC VALUES Chloroform extract of T. cordifolia showed least MIC value i. e. o.146 mg/ml against E. coli while acetone extract 0.0156 mg/ml against S. pneumoniae . Aqueous extract of T. cordifolia showed lowest MIC values i.e. 0.00975 mg/ml against E. coli, while methanol extract has shown highest activity against M. luteus and S. pneumoniae at 0.0156 mg/ml concentration (Table 1). Petroleum ether extract of T. cordifolia showed high activity against E. coli, B. cereus, and L. acidophillus at 0.312 mg/ml (Table 1) Similarly, Cassia fistula chloroform extract was found to be highly susceptible, as it has shown very low MIC value i.e. 0.0078 mg/ml against K. pneumoniae. Acetone extract has shown 0.0156 mg/ml MIC value against K. pneumoniae, E. coli, B. cereus and S. pneumoniae . Cassia fistula aqueous extract has shown MIC value i.e. 0.0975 mg/ml in K. pneumoniae and 0.048 mg/ml in E. coli, B. cereus, M. luteus and L. acidophilus. Besides this, antibiotics such as tetracycline, amphicillin and ciprofloxacin has shown higher MIC values which were obtained in a range of 0.892-3.57mg/ml (Table 1). Lowest MBC value was found in Tinospora cordifolia methanol extract i.e. 0.0312 mg/ml in M. luteus and S. pneumoniae (Table 2). Similarly, Cassia fistula ether extract has shown very low MBC values i.e. 0.0156 mg/ml in S. pneumoniae (Table 2), while chloroform extract has shown MBC values i.e. 0.0312 mg/ml MBC value against K. pneumoniae, E. coli and M. luteus, while its aqueous extract has shown very low MBC values i.e. 0.39 mg/ml in K. pneumoniae, E. coli, S. aureus and B. cereus also than antibiotics (Table 2). Antibiotics tetracycline and ampicillin have shown very high MBC values i.e. 3.57 mg/ml and 7.14 mg/ml in M. luteus, while ciprofloxacin has shown very high MBC value i.e.3.57 mg/ml in L. acidophilus (Table 2). MEASUREMENT OF INHIBITION ZONE DIAMETER Antimicrobial activity of crude extract was also confirmed by agar disc diffusion method and inhibition zone diameters were measured in presence and absence of each solvent and aqueous extract of Tinospora cordifolia and Cassia fistula . Results are presented in table 3. In presence of Tinospora cordifolia chloroform extract maximum inhibition zone diameter was obtained i.e. 25 mm in K. pneumoniae, 26 mm in E. coli and S. aureus, 27 mm in M. luteus at 320g concentration. In Tinospora cordifolia acetone extract maximum inhibition zone diameter was obtained i.e. 25 mm in K. pneumoniae, M. luteus and L. acidophilus. Similarly, Tinospora cordifolia methanol, ether and aqueous extracts have shown maximum inhibition zone diameter at 320g concentration (Table 3). In presence of Cassia fistula chloroform extract inhibition zone diameter was obtained 15mm in K. pneumoniae and E. coli, 16mm in S. aureus and L. acidophilus, 20mm in M. luteus and 21mm in S. pneumoniae at 320g concentration (Table 3). Cassia fistula acetone extract has shown highest inhibition zone diameter i.e. 27mm in E. coli, 26 mm in S. aureus and L. acidophilus, while rest of the bacterial strains have shown inhibition zone diameter between 20-22mm. Cassia fistula methanol extract has shown higher inhibition zone diameter in S. aureus 32mm and 25mm in E. coli. It has shown 16-22mm inhibition zone diameter in other bacterial strains. In presence of Cassia fistula ether extract, lowest inhibition zone diameter was obtained between 17-19 mm against all pathogenic bacterial strains at 20g concentration. Similarly, Cassia fistula aqueous extract has shown highest inhibition zone diameter i.e. 30 mm in M. luteus, 27 mm in K. pneumoniae , 25 mm in E. coli and S. pneumoniae , 22 mm in S. aureus. Morespecifically, aqueous extract represented higher susceptibility to bacterial strains (Table 3). Antibiotics tetracycline, ampicillin and ciprofloxacin have shown significantly smaller inhibition zone diameter than that of plant extracts. It was obtained in a range of 7-17 mm (Table 3). DISCUSSION Both herbs and herbal products are known to have antibacterial potential (De Boer et al., 2005; Adwan et al., 2006). Various ethnic groups and local population use these, as medicine worldwide. Herbal treatments become very popular because it is easily available, cheaper and less toxic than synthetic drugs. In the present investigation, solvent and aqueous extracts of Tinpospora cordifolia and Cassia fistula were evaluated for exploration of their antimicrobial activity against certain Gram negative and Gram positive human pathogenic bacteria. Susceptibility of each plant extract was tested by serial microdilution method and MIC and MBC values were determined. Aqueous and solvent extracts of both plant species have shown very high antimicrobial susceptibility against all seven bacterial strains at a very low concentration. More specifically, higher percent growth inhibition was obtained in presence of plant extracts in comparison to synthetic antibiotics.. All different extracts have shown very low MIC value, which was found to be lower (0.0078-0.125 mg/ml) than the MIC values (0.223-0.892 mg/ml) obtained in presence of standard antibiotic drugs i.e. tetracycline, ampicillin and ciprofloxacin. It proves antimicrobial susceptibility of plant extracts. Tinospora cordifolia aqueous extract has shown lowest MIC value i.e. 0.00975 mg/ml in E. coli. Similar antimicrobial activity was reported by (Shashidharan et al.,2007) in leaves of Stachytapheta jamaicensis against E coli, S. epideremidis and Pseudomonas aeurginosa with an MIC value of 5.00 mg/ml. Bixa orellana (L) exhibited an MIC of 0.2 g/ml against B cereus (Rastogi and Mehrotra, 1999). Similarly Cassia fistula aqueous extract exhibited a significant antimicrobial activity against Gramve and Gram+ve bacteria. The results are summarized in Table 1-3. Such inhibitory effects of in C. fistula can be attributed to the chemical substances present in the fruits/legumes (Rojas et al., 2006). It might be tannins and propyl gallate which are formed in ripening fruits and inhibit the growth of infectious microorganisms (Chung et al., 1998). Further, antimicrobial activity of plant extracts is strengthened by low MBC values obtained in plant extracts against all seven bacterial strains. However, MBC values obtained in solvent and aqueous extracts of Tinospora cordifolia were in range of 0.0292-0.39 mg/ml while these were 0.0156-0.39 mg/ ml in Cassia fistula in same extracts. Further MBC values obtained in plant extracts were significantly much lower than broad spectrum antibiotics 0.8927.17 mg/ml Again it proves antimicrobial potential of plant extracts. Further, effectiveness of each plant extract was determined by agar disc diffusion method and inhibition zone diameters obtained in presence and absence of each extract. Based on growth inhibition zone diameter obtained bacterial strains were divided into three categories i.e. resistant (> 7 mm), intermediate (> 12 mm) and susceptible (> 18 mm). However, at a very low concentration (20-320 g/disc) 1532 mm inhibition zone diameters were obtained in chloroform, acetone, methanol, petroleum ether and aqueous extracts of both plant species (Table 3). These were significantly much larger than the antibiotic drugs i.e. tetracycline, ampicillin and ciprofloxacin which have shown inhibition zone diameters in a range of 618 mm against K. pneumoniae, E. coli, B. cereus, M. luteus, L. acidophilus, S. aureus and S. pneumoniae at the same concentration. Highest inhibition zone diameter 30 mm was obtained in aqueous extract of Cassia fistula against M. luteus (Table 3) at 320 g/disc. Similar inhibition zone diameter (22 mm) was obtained in methanolic extract of R. coraria (Abdelrahim et al., 2002). Results clearly indicate that aqueous and solvent extracts of both plant species were found to be more effective against both Gram+ve and Gram-ve bacteria. Similarly Psidium guajava plant showed very strong antimicrobial activity against Staphylococcus, Shigella, Salmonella, Bacillus, E. coli, Clostridium and Pseudomonas (Abere et al., 2007; Kamath et al., 2008) due to presence of phyto-chemicals, like diterpenes, terepenes, phenols, lectins, saponins and flavonoides (Adeniyi et al., 2008; Aijyegoro et al., 2008). Similar antimicrobial susceptibility was reported in leaf extract of Mitracarpus scaber (Azu and Onyeagba, 2007), Capparis decidua (Upadhyay et al.,2010a), bark extract of Distemonanthus benthamianus (Baill) (Zhao et al.,1991), Allium cepa and Zingiber officinalis (Kumar et al., 2000) Parthenium hysterophorous Linn (Serrentino, 1991) and volatile components of Phyllanthus emblica (Yang et al., 2010), Fortunella japonica and Citrus sunki essential oils against skin pathogens (Nannapaneni et al., 2008). From the present work, it can be stated that aqueous extracts of above plant species were found to be more potent which can efficiently reduce the contamination of pathogenic bacteria. There are two possible explanations for the observed antimicrobial activity. First, the components are water soluble and bioactive, second these are less soluble in organic solvents. It is the main reason behind their antimicrobial activity. In the present study, both plant species have shown nearly equal antimicrobial effects on both gram positive and gram-negative bacteria in suspension culture. It is further proved by inhibition zone diameters obtained in filter paper disc diffusion assays, which have shown better effectiveness of aqueous extracts against Gram-positive bacteria. It shows the presence of some strong antimicrobial constituents belonging to the different groups (Batista et al., 1994; Scazzocchino et al., 2001; Lambert et al., 2001), which might have very high permeability across the bacterial cell wall (Walsh et al., 2003; Innouye et al., 2001) due to ion leakage (Delequis et al., 2002) There might be another possibility that plant extract may successfully inhibit microbial respiration and increase the plasma membrane permeability, which results in to death of bacterial cells

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after massive ion leakage (Knobloch et al.,1986). It may also happen due to hydrophilic nature of bacterial cell wall (Upadhyay et al., 2010b) This explained that there was a natural and efficient substance (Upadhyay et al., 2010c), which is capable to inhibit the growth of bacteria, which can resist the antibiotics. Above findings indicate that further studies on chemical and biological properties of their active components should be preformed. It is also suggested based on above study that pure chemical components of above plant species can be used for development of herbal medicine which might have stronger application against disease pathogens. ACKNOWLEDGMENTS The authors wish to thanks University Grants Commission, New Delhi, for financial assistance REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Abdelrahim SI, Almagboul AZ, Omer ME, Elegami A. Antimicrobial activity of Psidium guajava L., Fitoterapia, 73(7-8), 2002, 713-715. Abere TA, Onyekweli AO, Ukoh GC. In vitro antimicrobial activity of the extract of Mitracarpus scaber leaves formulated as syrup., Trop. J. Pharmaceutical Res., 6, 2007, 679-682. Acharyya P, Barua NC, Sarma A. Anti microbial activity of a pseudoguaianolids isolated from Parthenium hysterophorus Linn., Asian Journal of Microbiology, Biotechnology and Environmental Sciences, 10(2), 2009, 281-282. Adeniyi BA, Ayepola OO. The phytochemical screening and antimicrobial activity of leaf extracts of Eucalyptus camaldulensis and Eucalyptus torelliana, Research Journal of Medical Plant, 2(1),2008, 34-38. Adwan G, Abu-Shanab B, Adwan K, Abu-Shanab F. Antibacterial effects of nutraceutical plants growing in palestine on Pseudomonas aeruginosa, Turk. J. Biol., 30, 2006, 239-242. Adwan S, Abu-Shanab B, Adwan K, Abu-Saneb F. Antibacterial effects of nutraceutical plants growing in palestine on Pseudomonas aeruginosa, Turk J. Biol., 30,2006, 239-242. Aijyegoro OA, Akinpelu DA, Afolayan AJ, Okoh AI. Antibacterial activities of crude stem bark extracts of Distemonanthus benthamianus Baill, Journal of BiologicalSciences, 8(2), 2008, 356361. Anesini E, Perez C. Screening of plants used in Argentine folk medicine for antimicrobial activity, J. Ethnophormacol., 39, 1993, 119-128. Azu NC, Onyeagba RA. Antimicrobial properties of extracts of Allium cepa and Zingiberofficinale on Escherichia coli, Salmonella typhi and Bacillus subtilis, Internet. Journal of Tropical Medicine, 3, 2007, 2. Batista O, Duarte J, Nascimento, Simones MF. Structure and antimicrobial activity of diterpenes from the roots of Plectranthus hereroensis, J. Nat. Prod., 57, 1994, 858-861. Bilgrami KS, Sinha KK, Singh AK. Inhibition of Aflatoxin production and growth of Aspergillus flavus by eugenol and onion and garlic extracts, Indian J. Med. Res., 96, 1992, 171-175. Chung KT, Wong TY, Wel CI, Huang YW, Lin Y. Tannins and human health: a review, Crit. Rev. Food Sci. Nutr., 38, 1998, 421-464. Cowan MM. Plant product as antimicrobial agents, Clinical Microbiology , 12(4), 1999, 564-582. De Boer HJ, Kool A, Broberg A, Mziray WR, Hedberg I, Levenfors, JJ. Antifungal and antibacterial activity of some herbal remedies from Tanzania, J. Ethnophamacol., 96, 2005, 461-469. Delaquis PJ, Stanich K, Girard B, Mazza G. Antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils, International Journal of Food Microbiology, 74, 2002, 101109. Desta B. Ethiopian traditional herbal drugs. Part II:Antimicrobial activity of 63 medicinal plants, J. Ethnopharmacol., 39(2),1993, 129-139. Dulger B, Gonuz A. Antimicrobial activity of some endemic verbascum, salvia and Stachys species, Pharm. Biol., 42, 2004, 301-304. Inouye S, Takizawa T, Yamaguchi H. Antibacterial activity of essential oils and their major constituents against respiratory tract pathogen by gaseous contact, Journal of. Antimicrobial Chemotherapy, 47,2001, 565-573. Kamath JV, Nair R, Kumar ACK, Mohana SL. Psidium guajava L. A review, I.J.G.P., 2008, 912. Kirby WM, Bauer AW, Sherris JC, Turck M. Antibiotic susceptibility testing by a standard single disc method, Am. J. Clin. Pathol., 45, 1966, 493-496. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 21. 22. 23. 24. 25. 26. 27. Knobloch KH, Weigand N, Weis HM, Schwa H, Vigenschow. Action of terpenoids on energy metabolism, In: De Gruyter ed. Progress in Essential oil Research, 16th International Symposium on Essential Oils, Germany, Berlin. 1986, 429-445. Kumar S, Kumar S, Verma NS, Pande D, Srivastava PS. In vitro regeneration and screening of berberine in T. cordifolia , J. Med. Arom. Plant Sci., 22, 2000, 61. Lambert RJ, Skandamis PN, Coote PJ, Nycas GJ. A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol, Journal of Applied. Microbiology , 2001, 91, 453 462. Liu CX. Development of Chinese medicine based on pharmacology and therapeutics, J. Ethnopharmacol., 19, 1987, 119-123. Liu X, Zhao M, Luo W, Yang B, Jiang Y. Identification of volatile components in Phyllanthus emblica L. and their antimicrobial activity., J. Med. Food, 12(2), 2009, 423-428. Mohana DC, Satish S, Raveesha KA. Antibacterial evaluation of some plant extracts against some human pathogenic bacteria, Biological Research., 2(3-4), 2008, 49-55. Nannapaneni R, Muthaiyan A, Crandall PG, Johnson MG, OBryan CA, Chalova VI, Callaway TR, Carroll JA, Arthington JD, Nisbet DJ, Ricke SC. Antimicrobial activity of commercial citrusbased natural extracts against Escherichia coli O157:H7 isolates and mutant strains, Foodborne Pathog Dis., 5(5), 2008, 695-699. Nascimento GGF, Locatelli J, Paulo C, Freitas-Giuliana, L, Silva S. Antibacterial activity of plant extracts and Phytochemical on antibiotic resistant bacteria, Brazilian Journal of Microbiology, 31, 2000, 247-256. Pacheco P, Sierra J, Schmeda-Hirschmann G, Potter CW, Jones, BM, Moshref, M. Antiviral activities of Chilean medicinal plant extracts, Phytother Res., 7, 1993, 415-418. Peng Y, Rakowski SA, Filutowiez M. Small deletion variants of the replication protein Pi and their potential for over-replication-based antimicrobial activity, FEBS Microbiol Lett., 261(2), 2006, 245-252. Rastogi P, Mehrotra BN. Compendium in Indian medicinal plants, Drug research perspective, CDRI Lucknow and NISCOM, New Delhi. 2, 1999, 2-859. Rege NN, Thatte UM, Dahanukar SA. Adaptogenic properties of six rasayana herbs used in ayruvedic medicine, Phytother. Res., 13, 1999, 275-291. Rios JL, Recio MC. Medicinal plants and antimicrobial activity, J. Ethanopharmacol., 100, 2005, 80-84. Ritch-Kro EM, Turner NJ, Towers GH. Carrier herbal medicine: an evoluation of the antimicrobial and anti-cancer activity in some frequently used remedies, J. Ethno. Pharmacol., 5, 1996, 151-156. Rojas JJ, Ochoa VJ, Ocampo SA, Munoz JF. Screening for antimicrobial activity of ten medicinal plants Colombian folkloric medicine: A possible alternative in the treatment of non-nasocomial infection, BMC. Comelmentary and Alternative Medicine, 2006, 6, 2. Scazzocchino, F., Cometa, M. F., Tomassini, L. and Palmery, M. Antibacterial activity of Hydrastis canadensis extract and its major isolated alkaloids, Planta Med., 67, 2001, 561-563. Serrentino J. How Natural Remedies work, Point Robert, W.A. ed., Harley and Marks publishers, 1991, 20-22. Shariff ZU. Modern Herbal Therapy for common Ailments, United Kingdom, 2001, 9-84. Shashidharan S, Yoga Latha L, Zuraini Z, Suryani S, Sangetha S, Shirley L. Antidiarrheal and antimicrobial activities of Stachytarpheta jamaicensis leaves, Applied Sciences, 39, 2007, 245248. Suresh P, Ingle VK, Vijaya Lakshmi V. Antibacterial activity of Eugenol in Comparision with other antibiotics, J. Food. Sci. Technol., 29,1992, 254-256. Tiwari BK, Valdramidis VP, ODonnell CP, Muthukumarappan K, Bourke P, Cullen PJ. Application of natural antimicrobials for food preservation, J. Agric. Food Chem., 57(14), 2009, 59876000. Upadhyay RK, Ahmad S, Tripathi R, Rohtagi L, Jain SC. Screeining of antimicrobial potential of extracts and pure compounds isolated from Capparis deciduas, Journal of Medicinal Plants Research, 4(6), 2010a, 439-445. Upadhyay RK, Dwivedi P, Ahmad S. Antimicrobial activity of photoactivated cow urine against certain pathogenic bacterial strains African Journal of Biotechnology, 9(4), 2010b, 518-522. Upadhyay RK, Dwivedi P, Ahmad S. Screening of antibacterial activity of six plant essential oils against pathogenic bacterial strains, Asian Journal of Medical Sciences, 2(3), 2010, 152-158. Walsh SE, Maillard JY,Russell AD, Catrenich CE, Charbonneau DL Bartolo RJ. Activity and mechanism of action of selected biocidal agents on Gram-positive and negative bacteria. Journal of Applied Microbiology, 94, 2003, 240-247. Yang EJ, Kim SS, Moon JY, Oh TH, Baik JS, Lee NH, Hyun CG. Inhibitory effects of Fortunella japonica var. margarita and Citrus sunki essential oils on nitric oxide production and skin pathogens , Acta Microbiol Immunol Hung., 57(1), 2010, 15-27. Zhao TF, Wang X. Rimando AM, Che C. Folkloric medicinal plants: Tinospora sagittata var. cravoniana and Mahonia bealei, Planta Medica, 57, 1991, 505-505.

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Source of support: University Grants Commission, New Delhi, ; Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 1. January 2011

167-170

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