Antimicrobial Activity of Bauhinia variegata Leaves (Ethanolic) Extract.

 

S.M. Bairagi1*, R.C. Agrawal 2 and Nitin Nema3

1Department of Pharmacology, MES College of Pharmacy Sonai, Ahmednagar, (M.S.) India

2Jawaharlal Nehru Cancer Hospital and Research Center, Bhopal, (M.P.) India.

 

ABSTRACT:

Purpose: The antimicrobial activity of the 50 % methanolic extract of the leaves of Bauhinia variegata. (Leguminosae) was investigated in other to verify its claimed ethno medicinal use in the treatment of microbial infections.

Method: The antimicrobial activity of the extract was tested against standard strains and clinical isolates of some aerobic bacteria using the Agar well diffusion method. Commercial antibiotics were used as positive reference standards to determine the sensitivity of the strains.

Results: The extracts showed significant inhibitory activity against standard strains and clinical isolates of Staphylococcus aureus, clinical isolates of Bacillus subtilis and Streptococcus epidemidis. The MIC values obtained using the Agar-dilution test ranged from 5.0 mg/ml. 10.0 mg/ml. Neither the concentrated extract nor its dilutions inhibited Esherichia coli, Pseudomonas aeruginosa and Shigella floxineri

Conclusion: The results demonstrate that the crude extract of the leaves of Bauhinia Variegata has a narrow spectrum of activity and suggest that it may be useful in the treatment of infections caused by Gram positive aerobic bacteria.

 

KEYWORDS: Antimicrobial activity, Bauhinia variegata, Methanol extract.

 

 

INTRODUCTION:

Various plants are identified as rasayanas in the Indian ayurvedic system of medicine having various pharmacological properties such as immunostimulant, tonic, neurostimulant, antibacterial, antirheumatic, adaptogenic and antistress1. According to the ayurveda, Bauhinia variegata. (Leguminosae) is an herbaceous plant, leaves are shaped little like cows hoof, flowers are hermaphrodite which grows abundantly on cultivated fields, waste areas, roadsides and open clearing in India1. The plant has a variety of traditional uses. The juice of the bark is used in the treatment of amoebic dysentery, diarrhoea and other stomach disorders.2. The bark powdered is traditionally used as for tonic, ulcers. The root is used as antitode to snake poison3. The insulin-like proteins isolated from the leaves of the Bauhinia variegata4. The bark of the Bauhinia variegata showed the hepatoprotective activity. The hepatoprotective activity was investigated in carbon tetrachloride (CCl4) intoxicated Sprague-Dawley rats5-7.This present study examined the activity of the 50% methanol extract of the leaves of Bauhinia Variegata against some bacteria.

 

 


MATERIALS AND METHODS:

Plant material:

The leaves of Bauhinia variegata were collected locally from herbal garden Bhopal.(M.P.) India. The fresh plat material was washed under running tab water and air-dried on the laboratory bench for 5 days and then ground to powder using an electric mill. 500 g of the powdered leaves of the plant material was macerated with 2800 ml of 50 % ethanol for 72 hours. The extract was filtered and concentrated to dryness by evaporation in a vacuum to yield 12.60 g of the crude methanol extract, which was used for the antimicrobial assay.

 

Test organisms:

The following microorganisms were used for the study. Standard strain of Staphylococcus aureus, Bacillus subtilis, Streptococcus epidermidis, Esherichia coli,Pseudomonas aeruginosa and Shigella floxineri. These microorganisms were obtained from the laboratory stock of the Jawaharlal Nehru Cancer Research and Hospital, Bhopal (M.P.) India They were maintained on agar slants at 4oC in the refrigerator.

 

Drugs and Microbiological Media:

The antimicrobial agents used were: amoxicillin, ciprofloxacin, fluconazole, nutrient broth and nutrient Agar (Himedia Lab Ltd., Mumbai)

 

Preparation of plates for susceptibility tests:

The agar-well diffusion method, suitably modified was adopted for the susceptibility studies8-9. Inoculate of the test organisms were prepared by growing each pure isolate in nutrient broth overnight 37o C .The overnight broth culture was, subcultured in fresh nutrient broth and grown for 3 hours, to obtain log phase culture. Aliquots of 0.2 ml were used to seed a molten nutrient agar medium, which was cooled to 450C. This was poured into the sterile petri dishes and used for assay. The crude extract was reconstituted with sterile distilled water and stock concentration of 1 g/ml or 1000mg/ml was made. The extract was tested at a concentration of 25 mg/ml. 200 μl of this concentration was delivered into wells (8 mm in diameter) bored into the already seeded nutrient agar plates. Equal volume of distilled water was assayed as control. Ciprofloxacin (5 μg/ml), amoxycillin (25 μg/ml) and fluconazole (20 μg/ml) were included as standard antimicrobial agents and tested along with the extract. The nutrient agar plates were incubated at 37o C for 24 hours. The diameters of zones of inhibition were measured in millimeter with a ruler and recorded. This was repeated three times and average diameters were recorded.

 

Determination of Minimum Inhibitory Concentration (MIC):

The standard agar dilution protocol with doubling dilution was used. The extract was incorporated into nutrient agar at concentrations of 2.5 mg/ml, to 20 mg/ml. A control without the extract was also set up. 10μl each of the test organisms, previously diluted to give 10 6 cfu/ml was used to inoculate the plates. These were incubated at 37o C for 24 hours in the first instance, and for another 24 hours, before the results were recorded after observing for growth. The minimum inhibitory concentrations (MICs) of the extract for each test microorganism were regarded as the agar plate with the lowest concentrations without growth.

 

Table.1. Result of antibacterial activity of B.Variegata

Organism

Mean diameter of zone of inhibition(mm)

B. variegata (25 mg/ml)

OF 5g/ml

Amox 25g/ml

AZ 20g/ml

Staphylococcus aureus

21.6

26.3

15.4

18

Streptococcus epidermidis

16.5

32.2

23.2

20.4

Bacillus

subtilis

15

19

16.4

21

Shigella floxineri

16

19.8

18.2

20.2

Pseudomonas aeruginosae

-

27.2

-

30

Esherichia

coli

-

26.4

16.2

21

OF-Ofloxacin, Amox-Amoxycilin, Az-Azithromycin

 

RESULTS:

The results of the antibacterial activity of the ethanol extract of the leaves of Bauhinia variegata (the extract) and some Standard strains of Staphylococcus aureus, Bacillus subtilis and Streptococcus epidermidis, are shown in Table 1 .The extract did not show any activity against Esherichia coli, Pseudomonas aeruginosa (Table 1). The minimum inhibitory concentrations (MICs) of the extract against the test organisms are shown in Table 1. The MIC was 5.0 mg/ml against both the clinical isolates of Staphylococcus aureus, 10 mg/ml against Streptococcus epidermidis, and 10 mg/ml against Bacillus subtilis. The control did not produce any inhibitory activity against the organisms. The Gram negative organisms were not inhibited within the concentration range used in this study.

 

The MIC of the crude extract for Candida albicans was not determined, since there was no inhibitory activity. The zone of inhibition produced by 25 mg/ml of the extract was 21.4 mm as against 15.3 mm produced by 25 μg/ml of amoxycillin against the clinical isolates of Staphylococcus aureus. The zone of inhibition produced by 25 mg/ml of the extract against Standard strains of Staphylococcus aureus was much lower (16 mm) when compared with that of the clinical isolates of Staphylococcus aureus (21.3 mm).

 

DISCUSSION:

The methanolic total crude extract of Bauhinia Variegata showed reasonable, comparable inhibitory activity against the Gram positive organisms; whereas there was no activity against any Gram negative bacteria. The bioactives of the extract which elicited antibacterial activity appeared to have preferential and specific activity against Gram positive bacteria. This observation could possibly justify the usefulness of the plant as earlier reported5-7. The extract presents narrow spectrum antibacterial activity since there was no activity against Gram negative bacteria, like Esherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and even Candidaalbicans - a fungus.

 

CONCLUSION

The methanol extract of the leaves of Bauhinia variegata (Leguminosae) has activity against Gram positive bacteria. However, the activity shown against susceptible organisms, as observed in this study, would appear to justify the ethnomedicinal use in recipes for infections. Further studies would focus on isolation of the bioactives, biological and chemical characterization.

 

ACKNOWLEDGEMENTS

The authors are thankful to Dr. R. C. Agrawal for his great support.

 

REFERENCES:

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4.       Azevedo, C. R., Maciel, F. M.. Silva, L. B., Brazilian Journal of Medical Biological Research, 2006, 39, 1435-1444.

5.       Kirtikar, K.R., Basu, B.D., Dehradun,International book distributors and publishers, Indian Medicinal Plants. 2001, 3.1257-1260

6.       Sharma, R. N., Saxsena, V.K., Asian J. Chem., 1996, 4, 811-812.

7.       Dubey, G.P., Agrawal A., Dixit, S.P., Antiseptic, 1994, 91, 205-208.

8.       Perez, C., Paul, M., Bazerque, P., An antibiotic assay by the agar well diffusion method. Acta Bio Med Exp 1990, 15,113-115,

9.       Nair, R., Chanda, S, Antibacterial activity of Punica granatum in different solvents, Indian J Pharm Sci 2005,67,239-243.

 

 

Received on 29.04.2011

Accepted on 10.06.2011

A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 3(5): Sept.- Oct. 2011, 244-246

 

 

 

 

 

 

 

 

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