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Research ArticleISSN: 0974-6943 Available online through

www.jpronline.info

*Corresponding author.Ravi Kant UpadhyayDepartment of Zoology,D D U Gorakhpur University, Gorakhpur, India 273009Tel.: +919838448495

E-mail:rkupadhya@yahoo.com

INTRODUCTION

Antimicrobial activity of two Indian medicinal plants Tinospora cordifolia (Family:Menispermaceae) and Cassia fistula (Family: Caesalpinaceae) against human pathogenicbacteriaRavi Kant Upadhyay*, Rajani Tripathi, Shoeb AhmadDepartment 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

ABSTRACTIn the present investigation antimicrobial activity of two Indian medicinal plants i.e. Tinospora cordifolia and Cassia fistula were evaluated against seven humanpathogenic bacterial strains. Both were tested by serial micro-dilution method. Antimicrobial susceptibility of aqueous and solvent extracts was assessed. For thispurpose, both positive and negative controls were set to determine MIC and MBC values. After bioassays, aqueous and solvent extracts of Tinospora cordifolia andCassia 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), Bacilluscereus (ATCC 11778) and Lactobacilus acidophilus (ATCC 53103) at a very low concentration. More specifically, higher percent growth inhibition was obtained inpresence of aqueous extracts in comparison to solvent extracts, which was much higher than synthetic antibiotics. Further, different extracts have shown very low MICvalue, 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 antibioticsi.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 bothplant 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

In the present time multiple drug resistance in microbial pathogens become aserious health problem to humankind worldwide (Peng et al., 2006). It is arouseddue to indiscriminate and repetitive use of antimicrobial drugs by inadequate dis-ease treatment (Shariff, 2001). To acquire drug resistance microbes have devel-oped new enzyme system to cleave the drug and make it useless for control ofinfection (Ritch-Kro et al., 1999). Hence, plant origin herbal medicines are con-sidered as safe alternatives of synthetic drugs. There are varied methods of medi-cines like Aurveda, Homeopathy and Unani, which utilize plant materials for drugproduction. Currently, Aurveda considered as a vital system of medicine and gov-erned the worldwide recognition and having non-toxic substances. Hence, in thelast few decades, so many plant species were explored for obtaining potentialantimicrobials for therapeutic purposes (Rios and Recio, 2005; Liu, 1987), whichlater 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 pos-sess strong antimicrobial potential against pathogens are considered as valuablesource 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 prop-erties (Suresh et al., 1992). Approximately 25 to 50 % of current pharmaceuticalsare derived from plants. Most of them were found effective against many patho-genic bacteria (Bilgrami et al., 1992), fungi (Pacheco et al.,1993), viruses(Nascimento et al., 2000) and even found active against drug-resistant microor-ganisms (Mohana et al.,2008). Besides this, few antimicrobials such as essentialoils (Yang et al., 2010; Nannapaneni et al., 2008), plant extracts (Mohana et al.,2008) and pure compounds have shown broad-spectrum antimicrobial activityagainst pathogens (Acharya et al., 2009; Serrentino, 1991). Some of the plantproducts are used as nutraceuticals (Adwan, 2006; Tiwari et al., 2009) and in foodpreservation (Rege et al., 1999). In the present investigation, two indigenousplant 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 andcordate. It is used as a blood purifier and anti-infectious agent. It is also used for thetreatment of jaundice, rheumatoid arthritis, diabetes, gout, viral hepatitis,arthopathies and general weakness. Cassia fistula is a tree belongs to familyCaesalpiniaceae and is an indigenous medicinal plant, commonly known asAmaltash, Cassia fistula is a medium size tree and having yellow flowers and greenleaves. It is an ornamental flowering plant, it’s seeds and legumes are used forgastric and respiratory problems. Long bunches of flowers are used for decorationpurposes. Seeds are used for stomach ailments while bark and leaves for burns. Thepulp of the fruit is used as purgative and laxative.

EXPERIMENTAL

PREPARATION OF EXTRACTSPlant parts of Tinospora cordifolia and Cassia fistula were collected from forestareas of Gorakhpur in eastern U.P. The stem of Tinospora cordifolia and legumesof Cassia fistula were chopped into small pieces, dried, milled and powdered byusing pestle and mortar. The acetone, methanol, chloroform and petroleum etherextracts were prepared by using 50g of dried powder of C. fistula legumes andleaves of Tinospora cordifolia in 250 ml of solvent separately. Each solventextract was fractionated to get different serial fractions in different solvent. Theextract was dried; residue was weighed and dissolved in known quantity of freshsolvent. In the preparation of aqueous extract, 50 mg the powdered material wasdissolved in 250 ml of distilled water and kept for solubilization overnight at roomtemperature. For quantization plant extracts were evaporated and re-dissolved inknown volume of each solvent separately,

MICRO-ORGANISMSCultures of seven pathogenic bacterial strains each of Escherichia coli (ATCC25922), Bacillus cereus (ATCC 11778), Lactobacilus acidophilus (ATCC 53103),Micrococcus luteus (ATCC 9341), Staphylococcus aureus (ATCC 25923), Kleb-siella pneumoniae (ATCC 15380) and Streptococcus pneumoniae (ATCC 12755)were maintained in the laboratory in Luria Broth (2% w/v) regularly for four daysat 370C before use in experiments. For experiments a portion (100 µl) of theovernight culture was mixed in the tests and control for inoculation. For activitytesting bacterial cultures were stored at 40 C and sub cultured after every 8th day insolid agar plates.

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plates. The incubation of test and control cultures was performed at 370C for 24hours. The least concentration at which no visible growth was obtained in agarplates was considered as MBC value. For evaluation of inhibition, both negative(with antibiotics) and positive (without antibiotics and extracts) parallel controlswere set for each and every test. Bacterial growth was obtained in presence andabsence of various concentrations of solvent and aqueous extracts of T. cordifoliaand Cassia fistula separately.

MEASUREMENT OF INHIBITON ZONE DIAMETERAntimicrobial susceptibility of Tinospora cordifolia and Cassia fistula extractswas evaluated by agar disc diffusion method of Kirby et al., (1966). Molten agarwas used as media for bacteria. For this purpose six different concentrations ofeach extract and pure compounds were coated on sterile filter paper discs (WhatmanNo. 1) of 6 mm in sizes. Extract coated discs were dried under laminar flowcabinet. Before experiment inoculum size was determined and adjusted to preparea 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 sterilerubber pad spreader before the coated discs were positioned on the inoculated agarsurface. Each extract and pure compound was assayed in triplicate. For comparisonthree broad spectrum antibiotics i.e. tetracycline, ampicillin and ciprofloxacinwere also used parallel as controls. All treated and untreated plates were incubatedfor 24 hrs at 370C. The antibacterial activity was assessed and size of inhibitionzone diameter surrounding the filter paper discs was measured.

STATISTICAL ANALYSISThe results were interpreted with the standard deviation. Student t- test was

Tinospora cordifoliaPlant extracts K.pneumoniae E.coli B.cereus M.luteus L.acidophilus S.aureus S.pneumoniae

Chloroform 0.0585 0.0146 0.2343 0.0585 0.0292 0.0292 0.0585Acetone 0.0625 0.0312 0.0312 0.0312 0.0625 0.0156 0.125Methanol 0.0625 0.125 0.0312 0.0156 0.0625 0.0625 0.0156Pet.ether 0.0625 0.0312 0.0312 0.0625 0.0312 0.0625 0.0625Aqueous 0.0195 0.00975 0.039 0.0195 0.039 0.039 0.0195Cassia fistulaChloroform 0.0078 0.0156 0.0312 0.0156 0.0312 0.0312 0.0156Acetone 0.0156 0.0156 0.0156 0.0312 0.0312 0.0312 0.0156Methanol 0.0625 0.0312 0.0156 0.0156 0.0156 0.0156 0.0156Pet.ether 0.0312 0.0625 0.0312 0.0156 0.0078 0.125 0.0078Aqueous 0.0975 0.048 0.048 0.048 0.048 0.024 0.024AntibioticsTetracycline* 0.446 0.446 0.446 0.223 0.446 0.446 0.223Ampicillin* 0.223 0.446 0.446 0.892 0.223 0.446 0.223Ciprofloxacin* 0.892 0.446 0.892 0.223 0.223 0.446 0.223

MIC-Minimum Inhibitory ConcentrationSolubility of each plant extract was determined before treatmentNo turbidity was found visible after 24 hrs incubation*Antibiotics was used for comparison

Tinospora cordifoliaPlant extracts K.pneumoniae E.coli B.cereus M.luteus L.acidophilus S.aureus S.pneumoniae

Chloroform 0.1171 0.0292 0.4687 0.1171 0.0585 0.1171 0.2343Acetone 0.125 0.25 0.125 0.0625 0.125 0.125 0.25Methanol 0.25 0.25 0.0625 0.0312 0.125 0.125 0.0312Pet.ether 0.125 0.25 0.0625 0.25 0.125 0.25 0.125Aqueous 0.078 0.39 0.078 0.078 0.078 0.078 0.078Cassia fistulaChloroform 0.0312 0.0312 0.0625 0.0312 0.0625 0.0625 0.125Acetone 0.0625 0.0625 0.0625 0.0625 0.0625 0.0625 0.0312Methanol 0.25 0.125 0.0312 0.0625 0.0625 0.0625 0.0625Pet.ether 0.0625 0.125 0.0625 0.0312 0.0312 0.25 0.0156Aqueous 0.39 0.39 0.39 0.195 0.195 0.39 0.195AntibioticsTetracycline* 0.892 0.892 1.78 3.57 0.892 0.892 0.892Ampicillin* 1.78 1.78 1.78 7.14 0.892 0.892 0.892Ciprofloxacin* 1.78 1.78 1.78 0.892 3.57 0.892 0.892

MBC- Minimum Bactericidal ConcentrationFor Comparision both negative and positive controls were set*Antibiotics were used for comparison

Plant Conc. K.pneumoniaeE.coli B.cereus M.luteus L.acidophilus S.aureus S.pneumoniaeextracts µg/disc Tc Cf Tc Cf Tc Cf Tc Cf Tc Cf Tc Cf Tc Cf

Chloroform 20 12 7 8 7 12 7 10 7 11 6 13 7 11 8 40 16 8 10 8 13 10 17 9 12 9 16 9 12 13 80 17 10 13 10 15 12 19 12 16 11 17 11 15 14 160 18 13 16 12 17 14 20 14 17 12 21 13 20 20 320 25 15 26 15 21 19 27 20 22 16 26 16 23 21

Acetone 20 9 Nil 10 7 9 7 8 11 8 12 9 13 7 7 40 11 7 12 22 15 13 10 15 14 16 10 17 8 8 80 20 9 18 24 16 16 15 16 15 18 15 19 10 13 160 21 15 20 25 20 18 21 17 17 20 16 22 12 17 320 25 22 22 27 22 20 25 22 25 26 17 26 20 20

Methanol 20 7 Nil 11 7 8 6 9 8 7 7 15 6 15 Nil 40 8 11 15 12 9 10 10 12 8 13 19 17 17 9 80 11 17 16 15 13 12 14 13 10 14 20 18 18 14 160 12 19 20 17 14 16 18 15 17 18 21 22 19 20 320 15 22 24 25 15 20 25 16 25 20 23 32 20 22

Ether 20 9 6 Nil Nil 6 7 10 7 11 7 7 7 11 7 40 10 13 12 10 9 14 11 11 12 11 8 10 13 11 80 12 15 13 11 11 15 13 12 15 16 15 12 15 12 160 14 16 14 16 13 17 15 15 17 16 16 13 19 15 320 25 18 16 17 17 18 20 19 20 19 17 18 27 18

Aqueous 20 8 11 11 12 7 15 7 9 7 8 11 10 10 10 40 14 13 15 13 15 16 11 14 10 11 16 17 15 12 80 16 15 16 14 16 17 15 17 15 13 17 19 17 16 160 17 20 17 17 20 19 16 23 16 17 20 20 20 22 320 22 27 25 25 21 20 20 30 21 20 22 22 25 25

Tetracycline* 20 6 7 6 7 6 6 7 40 8 9 9 10 8 8 10 80 13 12 11 12 12 10 13 160 16 14 13 14 15 14 16 320 17 17 15 16 16 16 18

Ampicillin* 20 7 6 6 7 7 6 6 40 8 9 9 8 10 8 9 80 14 13 12 14 15 13 16 160 16 16 15 15 16 16 17 320 17 17 16 17 17 17 18

Ciprofloxacin* 20 6 6 7 7 7 7 6 40 8 9 9 8 9 9 8 80 10 12 12 14 15 14 10 160 13 15 16 17 16 15 13 320 17 16 17 18 17 17 15

Table 3. Inhibition zone diameters obtained in presence of differentsolvent and aqueous extracts of Tinospora cordifolia and Cassia fistula weremeasured by agar disc diffusion method (in mm) and compared withbroad spectrum antibiotic drugs.

* The effectiveness of different extracts demonstrated by the size of growth inhibi-tion diameter obtained in filter paper disc diffusion assay in mm.*Tc: Tinosporacordifolia*Cf: Cassia fistula * The strength of activity is presented as resistant(>7mm), Intermediate (>12mm), and Susceptible (>18mm). In negative controlno antibiotic was used. In positive control antibiotic was used for comparison.

MEDIA PREAPRATION AND ITS STERILIZATIONFor antimicrobial susceptibility testing both solid (Agar-agar) and liquid agar (Luriabroth) 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 bydissolving 1 gm of broth media in 100 ml of distilled water, while 2% agar solid wasused for developing surface colony growth.

DETERMINATION OF MIC AND MBC VALUESFor determination of antimicrobial activity, bacterial growth inhibition was ac-cessed in the presence of different increasing concentrations of Tinospora cordifoliaand Cassia fistula extracts. For this purpose both crude extracts were separatelydiluted by using serial micro dilution method with Luria Broth culture medium at afinal concentration range from 8.0 to 0.00975 mg/ml. The tested crude extractsand pure compounds were added to fresh suspension after following the serialdilutions up to 10-10. Each extract was assayed in triplicate. Before conductingexperiments all the conditions for in vitro anti-microbial activity were standard-ized to determine MIC and MBC values. The MIC values considered as the lowestconcentration of crude extract and pure compounds, which have shown no turbid-ity in the culture flask after 24 hrs of incubation at 370C. The turbidity in theculture flasks was considered as visible growth of microorganisms. Further, it wasstandardized in terms of absorbance at 600 nm in a visible spectrophotometer.For determination of minimum bactericidal concentration (MBC) growth inhibi-tory assays were performed. For this purpose inoculum size was adjusted to pre-pare a final colony number as 108 colony forming units (CFU/ml in sterile agar

Table 2 . MBC values obtained in presence of solvent and aqueous ex-tracts of Tinospora cordifolia and Cassia fistula against seven pathogenicbacterial strains (mg/ml).

Table 1. MIC values obtained in presence of solvent and aqueous extractsof Tinospora cordifolia and Cassia fistula against seven pathogenic bacte-rial strains (mg/ml).

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applied to know significant differences between the antimicrobial effectiveness ofeach extract and antibiotics. The data was also statistically analyzed by applyingANOVA to know the significant difference in antimicrobial susceptibility of broad-spectrum antibiotics and various plant extracts. The related effectiveness was alsotested by applying linear correlation between control and tests.

RESULTSDETERMINATION OF MIC AND MBC VALUESChloroform extract of T. cordifolia showed least MIC value i. e. o.146 mg/mlagainst E. coli while acetone extract 0.0156 mg/ml against S. pneumoniae. Aque-ous extract of T. cordifolia showed lowest MIC values i.e. 0.00975 mg/ml againstE. coli, while methanol extract has shown highest activity against M. luteus andS. pneumoniae at 0.0156 mg/ml concentration (Table 1). Petroleum etherextract 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, asit 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 MICvalue 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 obtainedin a range of 0.892-3.57mg/ml (Table 1).

Lowest MBC value was found in Tinospora cordifolia methanol extract i.e. 0.0312mg/ml in M. luteus and S. pneumoniae (Table 2). Similarly, Cassia fistula etherextract 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/mlMBC value against K. pneumoniae, E. coli and M. luteus, while it’s aqueous extracthas 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 andampicillin have shown very high MBC values i.e. 3.57 mg/ml and 7.14 mg/ml inM. luteus, while ciprofloxacin has shown very high MBC value i.e.3.57 mg/ml inL. acidophilus (Table 2).

MEASUREMENT OF INHIBITION ZONE DIAMETERAntimicrobial activity of crude extract was also confirmed by agar disc diffusionmethod and inhibition zone diameters were measured in presence and absence ofeach solvent and aqueous extract of Tinospora cordifolia and Cassia fistula .Results are presented in table 3. In presence of Tinospora cordifolia chloroformextract 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 320µgconcentration. In Tinospora cordifolia acetone extract maximum inhibitionzone diameter was obtained i.e. 25 mm in K. pneumoniae, M. luteus and L.acidophilus. Similarly, Tinospora cordifolia methanol, ether and aqueous extractshave shown maximum inhibition zone diameter at 320µg concentration (Table3).

In presence of Cassia fistula chloroform extract inhibition zone diameter wasobtained 15mm in K. pneumoniae and E. coli, 16mm in S. aureus and L. acido-philus, 20mm in M. luteus and 21mm in S. pneumoniae at 320µg concentration(Table 3). Cassia fistula acetone extract has shown highest inhibition zone diam-eter i.e. 27mm in E. coli, 26 mm in S. aureus and L. acidophilus, while rest of thebacterial strains have shown inhibition zone diameter between 20-22mm. Cassiafistula methanol extract has shown higher inhibition zone diameter in S. aureus32mm and 25mm in E. coli. It has shown 16-22mm inhibition zone diameter inother bacterial strains. In presence of Cassia fistula ether extract, lowest inhibi-tion zone diameter was obtained between 17-19 mm against all pathogenic bacte-rial strains at 20µg concentration. Similarly, Cassia fistula aqueous extract hasshown 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 bacterialstrains (Table 3). Antibiotics tetracycline, ampicillin and ciprofloxacin have shownsignificantly smaller inhibition zone diameter than that of plant extracts. It wasobtained in a range of 7-17 mm (Table 3).

DISCUSSIONBoth herbs and herbal products are known to have antibacterial potential (DeBoer et al., 2005; Adwan et al., 2006). Various ethnic groups and local populationuse these, as medicine worldwide. Herbal treatments become very popular becauseit is easily available, cheaper and less toxic than synthetic drugs. In the presentinvestigation, solvent and aqueous extracts of Tinpospora cordifolia and Cassia

fistula were evaluated for exploration of their antimicrobial activity against cer-tain Gram negative and Gram positive human pathogenic bacteria. Susceptibilityof each plant extract was tested by serial microdilution method and MIC and MBCvalues were determined. Aqueous and solvent extracts of both plant species haveshown very high antimicrobial susceptibility against all seven bacterial strains at avery low concentration. More specifically, higher percent growth inhibition wasobtained in presence of plant extracts in comparison to synthetic antibiotics.. Alldifferent 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 inpresence of standard antibiotic drugs i.e. tetracycline, ampicillin and ciprofloxacin.It proves antimicrobial susceptibility of plant extracts. Tinospora cordifolia aque-ous extract has shown lowest MIC value i.e. 0.00975 mg/ml in E. coli. Similarantimicrobial activity was reported by (Shashidharan et al.,2007) in leaves ofStachytapheta jamaicensis against E coli, S. epideremidis and Pseudomonasaeurginosa with an MIC value of 5.00 mg/ml. Bixa orellana (L) exhibited an MICof 0.2 µg/ml against B cereus (Rastogi and Mehrotra, 1999). Similarly Cassiafistula aqueous extract exhibited a significant antimicrobial activity against Gram-ve and Gram+ve bacteria. The results are summarized in Table 1-3. Such inhibitoryeffects of in C. fistula can be attributed to the chemical substances present in thefruits/legumes (Rojas et al., 2006). It might be tannins and propyl gallate whichare formed in ripening fruits and inhibit the growth of infectious microorganisms(Chung et al., 1998). Further, antimicrobial activity of plant extracts is strength-ened by low MBC values obtained in plant extracts against all seven bacterialstrains. However, MBC values obtained in solvent and aqueous extracts of Tinosporacordifolia 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 plantextracts were significantly much lower than broad spectrum antibiotics 0.892-7.17 mg/ml Again it proves antimicrobial potential of plant extracts.

Further, effectiveness of each plant extract was determined by agar disc diffusionmethod and inhibition zone diameters obtained in presence and absence of eachextract. Based on growth inhibition zone diameter obtained bacterial strains weredivided into three categories i.e. resistant (> 7 mm), intermediate (> 12 mm) andsusceptible (> 18 mm). However, at a very low concentration (20-320 µg/disc) 15-32 mm inhibition zone diameters were obtained in chloroform, acetone, metha-nol, petroleum ether and aqueous extracts of both plant species (Table 3). Thesewere significantly much larger than the antibiotic drugs i.e. tetracycline, ampicil-lin and ciprofloxacin which have shown inhibition zone diameters in a range of 6-18 mm against K. pneumoniae, E. coli, B. cereus, M. luteus, L. acidophilus, S.aureus and S. pneumoniae at the same concentration. Highest inhibition zonediameter 30 mm was obtained in aqueous extract of Cassia fistula against M. luteus(Table 3) at 320 µg/disc. Similar inhibition zone diameter (22 mm) was obtained inmethanolic extract of R. coraria (Abdelrahim et al., 2002). Results clearly indi-cate that aqueous and solvent extracts of both plant species were found to be moreeffective against both Gram+ve and Gram-ve bacteria. Similarly Psidium guajavaplant 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 ofMitracarpus scaber (Azu and Onyeagba, 2007), Capparis decidua (Upadhyay etal.,2010a), bark extract of Distemonanthus benthamianus (Baill) (Zhao etal.,1991), Allium cepa and Zingiber officinalis (Kumar et al., 2000) Partheniumhysterophorous Linn (Serrentino, 1991) and volatile components of Phyllanthusemblica (Yang et al., 2010), Fortunella japonica and Citrus sunki essential oilsagainst skin pathogens (Nannapaneni et al., 2008).

From the present work, it can be stated that aqueous extracts of above plantspecies were found to be more potent which can efficiently reduce the contamina-tion of pathogenic bacteria. There are two possible explanations for the observedantimicrobial activity. First, the components are water soluble and bioactive,second these are less soluble in organic solvents. It is the main reason behind theirantimicrobial activity. In the present study, both plant species have shown nearlyequal antimicrobial effects on both gram positive and gram-negative bacteria insuspension culture. It is further proved by inhibition zone diameters obtained infilter paper disc diffusion assays, which have shown better effectiveness of aque-ous extracts against Gram-positive bacteria. It shows the presence of some strongantimicrobial constituents belonging to the different groups (Batista et al., 1994;Scazzocchino et al., 2001; Lambert et al., 2001), which might have very highpermeability 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 possibil-ity that plant extract may successfully inhibit microbial respiration and increasethe plasma membrane permeability, which results in to death of bacterial cells

Shashidharan et al., (2007)

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after massive ion leakage (Knobloch et al.,1986). It may also happen due tohydrophilic nature of bacterial cell wall (Upadhyay et al., 2010b) This explainedthat there was a natural and efficient substance (Upadhyay et al., 2010c), which iscapable to inhibit the growth of bacteria, which can resist the antibiotics. Abovefindings indicate that further studies on chemical and biological properties of theiractive components should be preformed. It is also suggested based on above studythat pure chemical components of above plant species can be used for develop-ment of herbal medicine which might have stronger application against diseasepathogens.

ACKNOWLEDGMENTSThe authors wish to thanks University Grants Commission, New Delhi, for finan-cial assistance

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

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