"In vitro evaluation of antimicrobial activity of crude extracts"
Biyoloji Bilimleri Araştırma Dergisi 2 (2): 97-101, 2009 ISSN:1308-3961, www.nobel.gen.tr 97 In vitro evaluation of antimicrobial activity of crude extracts of medicinal plants against multi drug resistant pathogens Nanda Kumar RAJENDRAN¹, Jayapradha RAMAKRISHNAN² ¹School of Chemical and Biotechnology, SASTRA University, Thanjavur, India Corresponding author Geliş Tarihi : 10 Ekim 2008 e-mail: email@example.com Kabul Tarihi : 15 Kasım 2008 Abstract In vitro antibacterial activity of aqueous and methanol extracts of some medicinal plants were screened against multi-drug resistant bacteria including Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis and Streptococcus pyogenes isolated from clinical specimen. The selection of medicinal plants was based on the fact that most of these plants were not previously screened against multi-drug resistant pathogenic organisms. Methanol extracts of W.somnifera showed higher antibacterial activity compared to other plant extracts tested. The minimum inhibitory concentration (MIC) of methanol extracts of W.somnifera was in the range of 50 to 100 mg/ml. The antibacterial activity of the extracts was not signiﬁcantly affected by heating at 100oC for 1 h but the activity was lost at pH 8. Phytochemical studies on the leaf extracts of medicinal plants revealed the presence of alkaloids, saponins, Cardiac glycosides, steroids, tannins and flavonoids. Keywords: Antimicrobial activity, W.somnifera, multidrug resistant pathogens, MIC, Phytochemical studies INTRODUCTION MATERIALS AND METHODS There is a continuous and urgent need to discover new antimicrobi- Plant materials al compounds with diverse chemical structures and novel mechanisms The plant leaves were collected locally and were further of action because there has been an alarming increase in the incidence identiﬁed and the respective voucher specimens have been de- of new and re-emerging infectious diseases. Another big concern is the posited in the Department of Environment and herbal science, development of resistance to the antibiotics in current clinical use. In re- Tamil University, Thanjavur, India. cent years, drug resistance to human pathogenic bacteria has been com- Extraction of Plant Materials and their extractive value monly reported from all over the world. The drug-resistant bacteria and fungal pathogens have further complicated the treatment of infectious Aqueous Extraction diseases in AIDS and cancer patients [3, 14]. In the present scenario For aqueous extraction, 10 g of air dried powder was placed of emergence of multiple drug resistance to human pathogenic organ- in 100 ml of distilled water (1:10 ratio) and boiled for 6 hours and isms, this has necessitated a search for new antimicrobial substances then ﬁltered. The ﬁltrate was condensed in boiling water bath and from other sources including plants. Higher plants produce hundreds used for further analysis . to thousands of diverse chemical compounds with different biological Methanol extraction activities . The antimicrobial compounds produced by plants are 10 g of air dried powder was placed in 100 ml of methanol active against plant and human pathogenic microorganisms . The in a conical flask and kept in rotary shaker at 150 rpm for 24 h. substances that can either inhibit the growth of pathogens or kill them After 24 h, it was ﬁltered and the solvent was evaporated to make and have no or least toxicity to host cells are considered candidates for the ﬁnal volume one-fourth of the original volume. It was stored developing new antimicrobial drugs. It is expected that plant extracts at 4oC for further studies. The extractive values of aqueous and showing target sites other than those used by antibiotics will be active methanolic plant extracts were analysed . against drug-resistant microbial pathogens. However, very little infor- Preliminary Phytochemical Analysis mation is available on such activity of medicinal plants [6, 8]. Qualitative phytochemical analysis of the of the plants col- In the present study, we have selected some medicinal plants to be lected was determined as follows: Phytochemical analysis for screened against multi-drug resistant bacteria including Staphylococ- major phyto constituents of the plant extracts was undertaken us- cus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escher- ing standard qualitative methods as described by various authors ichia coli, Proteus mirabilis and Streptococcus pyogenes. The selection [4, 7, 12, 15, 17]. The plant extracts were screened for the pres- of medicinal plants is based on the fact that most of these plants were ence of biologically active compounds like glycosides, alkaloids, not previously screened against multi-drug resistant pathogenic organ- tannins, flavonoids, saponins and steroids. isms. Phytochemical analysis of active plant extracts for their major group of phytoconstituents and the active group of certain extracts is Microrganisms used also reported here. Clinical isolates of Escherichia coli, Klebsiella Pneumoniae, 98 N. K. Rajendran ve J. Ramakrishnan / Bibad, 2 (2): 97-101, 2009 Proteus mirabilis, Pseudomonas aeruginosa, Staphy- ously adjusted to a concentration of 105 cells/ml was then lococcus aureus, and Streptococcus pyogenes obtained introduced. A set of test tubes containing broth alone were from the Department of Microbiology, Thanjavur Gov- used as control. All the test tubes and control were then ernment Medical College were used in this study. All incubated at 37oC for 18 h. After the period of incuba- cultures were bio chemically tested for purity. tion, the tube containing the least concentration of extract Culture media and inoculum preparation showing no visible sign of growth was considered as the minimum inhibitory concentration  Clinical isolates were frequently sub cultured and maintained in Nutrient Agar plates. For Antimicrobial as- Stability tests say, microbial cultures freshly grown at 37°C were appro- pH and temperature stability of the extracts were de- priately diluted in sterile normal saline solution to obtain termined using the method of Sammuelson . 50 mg/ the cell suspension at 105CFU/ml. ml concentration of the constituted extracts was treated Antibiotic resistance of test strains at 60oC and 100oC separately in a water bath for 60 min. The antimicrobial activity of each of the treated Antibiotic sensitivity of test strains was determined by and non-treated samples was determined as described the standard Disc diffusion against a number of antibiotics earlier. For determination of effect of pH, 50 mg/ml dilu- . The potency of antibiotics per disc was as follows, tions of the samples were adjusted to pH ranges between Amoxyclav (Ac), Clindamycin (Cd) (10 µg/ disc each); 6.0 and 8.0 using 0.1 N HCl and 0.1N NaOH. The antimi- Clarithromycin (Cw) (15 µg/ disc each); Cefaclor (Cj), crobial activity of each of treated and non-treated extracts Cloxacillin (cx) (30 µg/ disc). All antibiotic discs were were determined. purchased from the Hi-Media Pvt.Ltd. (Bombay, India). Antimicrobial susceptibility testing The agar well diffusion method  as adopted ear- RESULTS AND DISCUSSION lier  was used; 0.1 ml of diluted inoculum (105CFU/ ml) of test organism was spread on MHA plates. Wells of Phytochemical Analysis 8 mm diameter were punched into the agar medium and The Phytochemical screening of the plant extracts re- ﬁlled with 100 µl (150 mg/ml) of plant extract and solvent vealed the presence of tannins, alkaloids and flavanoids blanks. The plates were incubated for 18 h at 37°C. Anti- in all the plant extracts studied as shown in Table 1. The microbial activity was evaluated by measuring the zone of presence of saponins, tannins, alkaloids, flavanoids, ste- inhibition against the test organism. roids and cardiac glycosides provoked for an in-depth Determination of the minimum inhibitory concen- study on the plant. The metabolites are of various phar- tration (MIC) macological importance. Many triterpene saponins and The methanol extract of W.somnifera which showed their aglycones have been reported to have varied uses as signiﬁcant antimicrobial activity were selected for deter- anti-ulcerogenic, anti-inflamatory, ﬁbrinolytic, antipyret- mination of MIC. A stock solution of 100 mg/ml was pre- ic, analgesic and anti-edematous in action. The presence pared. This was serially diluted to obtain various ranges of tannin in most of plant extract could be responsible for of concentrations between 5mg/ml to 100mg/ml. 0.5 ml possible antitumor and anti oxidant activities [7, 11]. of each of the dilutions of different concentrations was Antibiotic resistance of test strains transferred into sterile test tube containing 2.0 ml of nutri- Results in Table 2 showed that test organisms exhib- ent broth. To the test tubes, 0.5 ml of test organism previ- ited varying degrees of multi drug resistance to standard Table 1. Phytochemical screening of the plant extracts + indicates Presence and - indicates Absence N. K. Rajendran ve J. Ramakrishnan / Bibad, 2 (2): 97-101, 2009 99 antibiotics disc used in this study. The test organisms pneumoniae, Proteus mirabilis, P. aeruginosa, S. aureus used in this study were resistant to Clindamycin (Cd) and Streptococcus pyogenes) were studied. The results , Cloxacillin (cx) and Clarithromycin (Cw) at 10 µg/disc, presented in the table 3, showed that MeOH extract in- 30 µg/disc and 15 µg/disc respectively ,but susceptible to hibited the growth of most of the organisms used. But Amoxyclav (Ac) at 10 µg/disc and Cefaclor (Cj) 30 µg/ aqueous extract showed least activity against the micro disc . When compared to the antibacterial activity of organisms. the extracts it was observed that the inhibitory activity Minimum inhibitory concentration of the active ex- of the methanol extracts of W.somnifera at 50 mg/ml tract against the test organisms was greater than those of the standard antibiotics. Most The MIC results are presented in Table 4. The crude interestingly these extracts exerted inhibitory properties extract of the Withania somnifera inhibited and fully on β-lactamase positive isolates. prevented the growths of Proteus mirabilis at a concen- Antimicrobial susceptibility testing tration of 50 mg/ml, E.coli and P.aeruginosa at a concen- In the present investigation crude extracts were de- tration of 60 mg/ml, K.pnemoniae at a concentration of 80 rived from the plant leaves and their efﬁcacy to inhibit mg/ml, S.aureus and Streptococcus pyogenes at a concen- the growth of Multi drug resistant organisms (E. coli, K. Table 2. Antibiogram of clinical isolates against various standard drugs Conc. = Concentration Table 3. Effect of aqueous and methanolic extract on microbicidal activity -- indicates no zone of inhibition Aq = aqueous extracts MeOH = Methanolic extracts 100 N. K. Rajendran ve J. Ramakrishnan / Bibad, 2 (2): 97-101, 2009 tration of 100 mg/ml as recorded in Table. Hence the crude ing as reported that many natural antimicrobials including leaf extract contains maximum active phyto compounds. honey are active against animal pathogens at acidic pH . Stability tests The stability of the antimicrobial activity of the extracts REFERENCE of Withania somnifera was analysed by heating at 60oC and 100oC for one hour and the results from the table 5 and  Ahmad I, Mehmood Z, Mohammad F. 1998. Screening 6 depicted that the stability of crude extract showed slight de- of some Indian medicinal plants for their antimicrobial crease with increased temperature treatment. Also, the stabil- properties. Journal of Ethnopharmacology. 62: 183–193. ity of the antimicrobial property at acidic pH is not surpris- Table 4. MIC of methanolic extract of W. somnifera against human drug resistant pathogens *= MIC concentration, --= No growth, += cloudy solution (slight growth), ++= Turbid solution (strong growth), +++= Highly turbid solution (dense growth) Table 5. 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