Antibacterial Activity of Leaf Extracts of Indian Medicinal Plant ...

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701__________________________________________Research PaperAntibacterial Activity of Leaf Extracts of IndianMedicinal Plant Argyreia involucrataGutal Valyfathulla Shaik* and M. Pavani*** Department of Biotechnology, Acharya nagarjuna university, Guntur, AP, India.** Department of Biotechnology, Rayalaseema University, Kurnool, Andhra Pradesh, India.ABSTRACTAntibacterial activity of aqueous, acetonic, methanolic and ethanolic leaf extracts of Indian medicinal plantArgyreia involucrata was investigated against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus,Escherichia coli , Klebsiella pneumoniae and Pseudomonas spp. The antibacterial activity was performed byagar disc and agar well diffusion methods against 6 bacterial species (3gram positive and 3 gram negative). Allthe leaf extracts has shown their effect on Eschereschia coli (gram negative). Methanolic and ethanolicextracts on Bacillus subtilis and Pseudomonas spp.,. No activity was seen on Bacillus cereus, Staphylococcusaureus and Klebsiella pneumoniae. The significant antibacterial acitivity was compared with standardantibiotics ampicillin and streptomycin. The results obtained in the present study suggest that they can beused in treating diseases caused by the test organisms.INTRODUCTIONIndia is an exquisite example of biodiversity. Fromtime immemorial scholars like Charaka, Barathwaj,Athreyan, Agnivesha, Dhanyandhari, Shushruthan,Wakbadan and Bharathduja etc. have studied andexplored the possibility of such a diversity forhuman welfare and the most conspicuousexploration in this field has lead to the discovery ofso many indigenous medicinal plants that werescripted mainly in Vedas (1500 BC) that containrich materials on herbal lore of that time. Similarto the plant diversity of India the same kind ofdiversity exist in the world of scriptures delineatingthe miracle of medicinal plant 1 . Medicinal plantscan form an excellent source for deviation of leadcompounds or newer drugs 2 .In last three decades number of new antibioticshave produced, but clinical efficacy of theseexisting antibiotics is being threatened by theemergence of multi drug-resistant pathogens 3 .Natural products of higher plants may give a newsource of antimicrobial agents with possibly novelmechanisms of action 4 . Many infectiousmicroorganisms are resistant to synthetic drugs,hence, an alternative therapy is very much neededand attract the attention of many researchers allover the world 5 . Plants were synthesized manycompounds with complex molecular structures by asecondary metabolism. Some of these compounds_______________________________________*Address for correspondence:E-mail: valyfa1979@gmail.comand their derivatives have antimicrobial propertiessuch as alkaloids, flavonoids, isoflavonoids,tannins, cumarins, glycosides, terpenes andphenolic compounds 6 .Plants produce a diverse range of bioactivemolecules, making them a rich source of differenttypes of medicines. A rich heritage of knowledgeto preventive and curative medicines was availablein ancient scholastic works included in the Atharvaveda, Charaka, Sushruta etc. 7 .In recent years, secondary plant metabolites(photochemical), previously with unknownactivities, have been extensively investigated as asource of medicinal agents 8 . A wide range ofmedicinal plant parts is used to extract as raw drugsand they possess varied medicinal properties 9 .Plants with possible antimicrobial activity shouldbe tested against an appropriate microbial model toconfirm the activity and to ascertain the parametersassociated with it. The effects of plant extracts onbacteria have studied by a very large number ofresearchers in different parts of the world 10 - 12 .The present Indian medicinal plant Argyreiainvolucrata belongs to the Family Convolvulaceaewhich is commonly called as morning glory family.Preliminary phytochemical analysis of the leaf andstem extracts indicates the presence ofVol. 2(4) Oct - Dec 2011 www.ijrpbsonline.com 1701

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701carbohydrates, cardiac glycosides, phytosterols,phenols, falvonoids, diterpenes and saponins. Theaim of this present study was to evaluate theactivity of different extracts against Gram positiveand Gram negative test bacterial strains.MATERIALS AND METHODSBACTERIAL STRAINSThe microbial strains are identified strains andwere obtained from the National ChemicalLaboratory (NCL), Pune, India. The bacterialstrains studied are Bacillus cereus NCIM 2157,Bacillus subtilis NCIM 2063, Staphylococcusaureus NCIM 2901, Escherichia coli NCIM 2065,Klebsiella pneumoniae NCIM 2883 andPseudomonas sp. NCIM 2207.PLANT MATERIALThe plant Argyreia involucrata was collected fromMadhukarai hills, Coimbatore District, TamilNadu. The plant was taxonomically identified.The fresh plant parts were collected. The leaveswere detached and washed with clean water.Leaves were air dried on a clean sheet for one weekat room temperature. Dried leaf material ofArgyreia involuncrata was powdered and passedthrough sieve #10.SOLVENT EXTRACTIONTen grams of air dried leaf powder was placed in100 ml of organic solvents (Acetone, Methanol andEthanol) in a conical flask, plugged with cotton andthen kept on a rotary shaker at 175 – 200 rpm for24 h. After 24 h, it was filtered through 8 layers ofmuslin cloth and centrifuged at 5000 x g for 15min. The supernatant was collected and the solventwas evaporated to make the final volume onefourthof the original volume. It was stored at 4º Cin airtight bottles for further studies.AQUEOUS EXTRACTIONFor aqueous extraction, 10 g of air – dried leafpowder was placed in distilled water and boiled for6 h. At intervals of 2 h it was filtered through 8layers of muslin cloth and centrifuged at 5000 x gfor 15 min. The supernatant was collected. After 6h, the supernatant was concentrated to make thefinal volume one – fourth of the original volume. Itwas then autoclaved at 121º C and 15 lbs pressureand stored at 4º C.MEDIA PREPERATION ANDANTIBACTERIAL ACTIVITYThe antimicrobial assay of leaf was performed bytwo methods viz., agar disc diffusion method 13 foraqueous extract and agar well diffusion method 14for solvent extract.A loop full of the strain was inoculated in 30 ml ofnutrient broth in a conical flask and incubated on arotary shaker for 24 h to activate the strain.Muller Hinton Agar (MHA) (3.8g/100ml) wasweighed and dissolved in 100 ml of distilled waterin a sterile conical flask. The medium wassterilized by autoclaving and was allowed to cool atroom temperature. The molten Muller Hinton Agarwas inoculated with 200 µl of the inoculum andpoured into the Petri plate.For Agar disc diffusion method, the disc (0.6 cm)was saturated with 50 µl of the compound, allowedto dry and was introduced on to the upper layer ofthe medium with bacteria. Antibiotic paper(streptomycin, ampicillin) discs were used aspositive control. These test discs was placed onMHA plate swabbed with the culture ofmicroorganisms. The plates were incubated at 37°C for overnight.For agar well diffusion method, seven wells wereprepared in the plates with the help of a cork-borer(0.6 cm). 50 µl of each test extract was introducedinto well. Antibiotics were added to the wells. Theplates were incubated overnight at 37° C.Microbial growth was determined by measuring thediameter of zone of inhibition. For each bacterialstrain, controls were maintained where puresolvents were used instead of the extract. Theresult was obtained by measuring the zonediameter. The experiment was done three timesand the mean values are presented.MINIMUM INHIBITORY CONCENTRATION(MIC)MIC is defined as the lowest concentration whereno visible turbidity is observed in the test tube(bacteriostatic concentration). The Vollekova etal., 15 (2001) method modified by Usman et al., 16(2007) was employed. In this method, the brothdilution technique was utilized where the plantextract was prepared to the highest concentration insterile distilled water and serially diluted (two-fold)to a working concentration using nutrient broth andlater inoculated with 0.2 ml suspension of bacterialstrains. After 18 hours of incubation at 37º C, thetest tubes were observed for turbidity. The leastwhere no turbidity was observed was determinedand noted as the minimum inhibitory concentration(MIC) value.MINIMUM BACTERIAL CONCENTRATION(MBC)The MBC is defined as the lowest concentrationwhere no bacterial growth is observed(bacteriocidal concentration). This was determinedVol. 2(4) Oct - Dec 2011 www.ijrpbsonline.com 1702

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701by the broth dilution resulting from the MIC tubesby sub-culturing to antimicrobial free agar asdescribed by Vollekova et al., 15 and Usman et al.,16. In this technique, the contents of the test tubesresulting from MIC was streaked using a sterilewire loop on agar plate free of bacteria andincubated as 37º C for 18 hours. The lowestconcentration of the extract which showed nobacterial growth was noted and recorded as theMBC.RESULTS AND DISCUSSIONThe antibacterial activity of Argyreia involucrataplant extracts was assayed against 6 bacterialspecies (3 gram positive and 3 gram negative).Table 1 & 2 shows the microbial growth inhibitionof aqueous, acetonic, methanolic and ethanolicextracts of the plant species. The methanolic andethanolic extracts has showed the effect on grampositive Bacillus subtilis. All the extracts hasshown their effect on Eschereschia coli (gramnegative). Whereas ethanolic extract on Klebsiellapneumoniae (gram negative) and methanolic andethanolic extracts on Pseudomonas spp., has showntheir effect. The rest were resistant to the extracts.The maximum antibacterial activity was shown byall the extracts on gram negative bacterial speciesparticularly on Eschereschia coli. The significantantibacterial activity has compared with thestandard antimicrobial antibiotics ampicillin andstreptomycin (50 µL). MIC values were alsorepresented in the table 3 & 4. No profound effectwas shown in MBC.Phytomedicine is a great relief to the presentsituation of medical treatments. The developmentof antimicrobials from the plant species will lead tothe development of newer green medicines againstmicrobes. Green medicines can serve the purposewith lesser side effects than the syntheticantimicrobials.ACKNOWLEDGEMENTI am thankful to all my Gutal family members fortheir support in the present work. I am thankful toVice-chancellor, Registrar, Principal ofRayalaseema University, Kurnool for encouragingand providing the lab facilities in Department ofBiotechnology.Table 1: Antimicrobial activity of leaf extracts of Argyreia involuncrataagainst various Gram positive bacteriaGram positive bacteriaZone of Inhibition (cm)Aqueous extract Acetonic Methanolic Ethanolic Streptomycin AmpicillinextractextractextractBacillus cereus - - - - 3.0 1.7Bacillus subtilis - - 1.0 0.8 3.0 1.7Staphylococcus aureus - - - - 3.0 1.7“ – “ = no activityGram negativebacteria“ – “ = no activityTable 2: Antimicrobial activity of leaf extracts of Argyreia involuncrataagainst various Gram negative bacteriaZone of Inhibition (cm)Aqueous extract Acetonic extract Methanolic extract EthanolicextractStreptomycinAmpicillinEschereschia coli 1.0 1.0 1.0 1.0 2.5 1.7Klebsiella- - - 1.0 2.5 1.7pneumoniaePseudomonas spp., - - 1.0 1.0 2.5 1.7Vol. 2(4) Oct - Dec 2011 www.ijrpbsonline.com 1703

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701Table 3 : Minimum Inhibitory concentration (MIC) of leaf extracts ofArgyreia involuncrata against various Gram positive bacteriaGram positivebacteria/extractsBacillus cereusAqueousAcetonicMethanolicEthanolicBacillus subtilisAqueousAcetonicMethanolicEthanolicS. aureusAqueousAcetonicMethanolicEthanolicExtract dilution2 4 8 16 32 64 128 256––––––––––––––––––––––––––––––––––––––––––––––––––––––++––––––––––––––––––––––––––––––––––––––––S. aureus = Staphylococcus aureus– = turbidity+ = least concentration showing no turbidityTable 4 : Minimum Inhibitory concentration (MIC) of leaf extracts ofArgyreia involuncrata against various Gram negative bacteriaGram negativebacteria/extractsEschereschia coliAqueousAcetonicMethanolicEthanolicKlebsiellapneumoniaeAqueousAcetonicMethanolicEthanolicPseudomonas spsAqueousAcetonicMethanolicEthanolicExtract dilution2 4 8 16 32 64 128 256––––––––––––––––––––––––––––––––––––+––––––––––––+++––––––++–––––––+––––––––––––––––––––––––––––– = turbidity+ = least concentration showing no turbidityREFERENCES1. Gayatri, R., Nambiar, and Raveendran. (2008).Indigenous Medicinal Plants Scripted inAmarakosam. Am-Euras. J.. Bot., Vol 1 (3): 68– 72.2. Balick J.M. and P.A. Cox, (1996). Plants,People and Culture: the science ofEthnobotany, Scientific American Library,New York, pp; 228.3. Cohen, M.L., (1992). Epidemiology of DrugResistance: Implications for a Post-Antimicrobial Era. Sci., 257: 1050 – 1055.4. Barbour, E.K.; Al Sharif, M,; Sagherian, V.K.;Habre, A.N.; Talhouk, R.S. and Talhouk, S.N.(2004). Screening of selected indigeneousplants of Lebanon for antimicrobial activity. J.Ethnopharmacol., 93: 1 – 7.5. Mohanan, P.V.; Rao, J.M,; Kutti, M.A.S. andDevi, K.S.(1998). Cytotoxicity of extracts ofSolanum trilobatum and anticarcinogenicactivity of sobatum. Biomedicine, 18: 106 –111.6. Simoes, C.M.O.; Schenkel, E.P.; Gosman, G.;Mello, J.C.P.; Mentz, L.A. and Perovick, P.R.Vol. 2(4) Oct - Dec 2011 www.ijrpbsonline.com 1704

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701(1999). Pharmacognosia: da planta aomedicamento. Santa Catarina: UFSC eUFRGS.7. Farhana, A.R.; Mahmuda, H.; and Imran-Ul-Haque, Md. (2009). In vitro antimicrobial,cytotoxic and antioxidant activity of flowerextract of Saccharum Spontaneum Linn.,European J. of Scientific Research Vol.30No.3: 478 – 483.8. Krishnaraju, A.V., Rao, T.V.N. andSundararaju D et al., (2005). Assessment ofbioactivity of Indian medicinal plants usingBrine shrimp (Artemia salina) lethality assay.Int. J. Appl. Sci. Eng 2 : 125 – 134.9. Uniyal, S.K., Singh, K.N., Jamwal. P. and Lal,B. (2006). Traditional use of medicinal plantsamong the tribal communities of UnhotaBhangal, Western Himalayan. J. Ethnobiol.Ethnomed., 2 : 1 – 14.10. Reddy, P.S., Jamil, K. and Madhusudhan P etal., (2001). Antibacterial activity of isolatesfrom Piper longum and Taxus baccata.Pharmaceutical Biol 39: 236 – 238.11. Erdogul O.T., (2002). Antibacterial activitiesof some plant extracts used in folk medicine.Pharmaceutical Biol 40: 269-273.12. Ates D.A, and Erdogul O.T., (2003).Antimicrobial activities of various medicinaland commercial plant extracts. Turk. J. Biol27: 157–162.13. Parekh. J, Nair. R, and Chanda. S. (2005).Preliminary screening of some folkloremedicinal plants from western India forpotential antimicrobial activity. Indian JPharmacol 37 : 408 – 409.14. Bauer, A.W. Kirby W.M.M, and Sherris J.C. etal., (1966). Antibiotic susceptibility testing bya standardized single disk method. Am J ClinPathol 45 : 493 – 496.15. Vollekova, A.D., Kostalova and R. Sochorova(2001). Isoquinoline Alkaloids from Mahoniaaquifolium stem bark as active againstMalassezia Sp. Folia Microbiol., 46: 107 –111.16. Usman, H., Abdulrahman, F.I. and A.H. Ladan(2007). Phytochemical and AntimicrobialEvaluation of Tribulus terrestris L. (zygophylaceae). Growing in Nigeria. Res. J.Bio. Sci. Medwell Journals, 2 (3): 244 – 247.Vol. 2(4) Oct - Dec 2011 www.ijrpbsonline.com 1705