In Vitro Evaluation of Antimicrobial Activity of Crude Extract from Plants Diospyros peregrina , Coccinia grandis and Swietenia macrophylla

Purpose: The aim of the present study was to investigate antimicrobial activity of methanol extract of Diospyros peregrina fruits (MEDP), Coccinia grandis leaves (MECG) and Swietenia macrophylla barks (MESM). Methods: MEDP, MECG and MESM were examined against some selective gram positive and gram negative bacterial (20) and fungal (4) strains. Preliminary antimicrobial activity was evaluated by agar disc diffusion method. Minimum inhibitory concentration was determined by tube dilution (MIC) whilst minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were determined by agar diffusion method. Results: MEDP and MESM both have shown highest sensitivity against Escherichia coli strains. MEDP was found resistant to Sarcina luteus and Bacillus spp whereas MESM was resistant to all Shigella strains. MECG has shown major activity against Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, Shigella soneii and Pseudomonas aeruginosa; whilst resistant to Shigella flexneri and Shigella boydii. Against fungi strains extracts were found effective at higher concentrations. Candida albicans has shown highest sensitivity whilst Penicillium spp. was least effective to all three extracts. Conclusion: The study confirms that MEDP, MECG, MESM all possess antimicrobial activity with different potency against variety of selected microorganisms. The differentiating activities of these three extracts encourage developing a novel broad spectrum antimicrobial herbal formulation in future.


INTRODUCTION
In recent times, the rapid development of multiresistant bacterial and fungal strains of clinically important pathogens fetches the interest of scientist to develop newer broad spectrum antimicrobial agents 1 .The less availability and high cost of new generation antibiotics necessitates looking for the substances from alternative medicines with claimed antimicrobial activity.A number of herbs with significant antimicrobial activity have been reported in different traditional literatures 2,3,4 .Now it is aimed to explore scientifically the antimicrobial potential of three traditional plants and substantiate the folklore claims.Diospyros peregrina Gurke.(Ebenaceae) is a small middle sized tree of costal West Bengal.The fruits have ethnomedicinal significance for the treatment of diarrhoea, dysentery, cholera, ulcer of mouth and in wounds 5,6 .The fruits contain triterpenes, alkanes, flavonoids and tannins 7,8,9,10 .Coccinia grandis (L.) Voigt.(Family: Cucurbitaceae) is a climbing perennial herb distributed almost all over the world.The leaves of the plant possess antidiabetic, antiinflammatory, antipyretic, analgesic, antispasmodic, antimicrobial, cathartic, expectorant activities 11,12 .The leaves contain triterpenoids, alkaloids and tannins 13 .The plant Swietenia macrophylla (Family: Meliaceae) is a large evergreen tree native to tropical America distributed almost all over the world.The barks of this plant possess anti-HIV, antimicrobial, antimalarial, and antitumor activities 14 .The barks contain triterpenoids, limonoids, flavonoids and tannins 15,16 .The objective of this research was to authenticate the antimicrobial sensitivity of the methanol extract of unripe matured fruits of Diospyros peregrina, Coccinia grandis leaves and Swietenia macrophylla bark and against some selected bacterial and fungal strains to lengthen the queue of antimicrobial herbs.

Plant material
Matured unripe fruits of Diospyros peregrina (Family: Ebenaceae) were collected in the month of June from the villages of South 24 Parganas, West-Bengal, India; the leaves of Coccinia grandis (L) Voigt.(Family: Cucurbitaceae) and barks of Swietenia macrophylla King.(Family: Meliaceae) were collected in the month of April, from the villages of Midnapore (E), West Bengal, India.The plants were authenticated by the Botanical Survey of India.Voucher specimens number entitled CHN/1-1(69), CNH/1-1 (44) and CNH/1-1(64) were deposited at our institute for future reference.

Preparation of methanol extract
The powdered plant materials (matured unripe fruits of Diospyros peregrina, leaves of Coccinia grandis and barks of Swietenia macrophylla) of 600 g each were extracted separately with methanol using Soxhlet apparatus.The resulting extracts were evaporated in vacuum and finally lyophilized into solid mass devoid of solvent (Yield = 8.75, 13.02 and 13.62 % respectively) and stored in desiccators for future use.Preparation of sample In the study of antimicrobial activity, extracts were dissolved in Dimethyl sulphoxide (DMSO).The corresponding concentration was expressed in term of µg of extract per ml of solvent (µg/ml).

Chemicals
All chemicals and solvents used in this experiment were of analytical grade obtained from BDH, Poole, UK. grown in MacConkey agar plates at 37 0 C and maintained on nutrient agar slants, while fungi were grown at 30 0 C and maintained in Saboraud glucose agar slants.Preliminary screening for antimicrobial activity The test was performed by disc diffusion assay as per NCCLS, 1993 17 .The nutrient agar plates containing an inoculum size of 10 6 cfu / ml for bacteria and 2 × 10 5 spores for fungi on Saboraud glucose agar plates, were used 18 .Previously prepared extract impregnated disc (6 mm in diameter) at the concentrations of 200 µg/ml for bacterial and 2000 µg/ml for fungal strains were placed aseptically on sensitivity plates with appropriate controls.Ciprofloxacin (200 µg/ml) and griseofulvin (2000 µg/ml) were used as standard antibacterial and antifungal antibiotics respectively.Plates were incubated at 37 0 C for 24 hours for bacteria and 30 0 C for 3 days for fungal spores 19 ..To 0.5 ml of varying concentrations of the extracts (0 -200 µg/ml for bacterial strains and 0 -2000 µg/ml for fungal strains), 2ml of nutrient broth was added and then a loopful of test organism previously diluted to 0.5 McFarland turbidity standard for (Bacterial isolates) and 10 6 cfu/ml (for fungal strains) was introduced to the tubes.The procedures were repeated on the test organisms using standard antibiotics ciprofloxacin (for bacteria) and griseofulvin (for fungi).A tube containing nutrient broth only seeded with the test organisms was served as control.Tubes containing bacterial cultures were then incubated at 37 0 C for 24 hours for bacteria and 30 0 C for 3 days for fungal spores.After incubation the tubes were examined for microbial growth by observing the turbidity.

Microorganisms
To determine the MBC and MFC, for each set of test tubes in the MIC determination, a loopful of broth was collected from those tubes which did not show any growth and inoculated on sterile nutrient agar (for bacteria) and Saboraud glucose agar (for fungi) by streaking.
Plates inoculated with bacteria and fungi were then incubated at 37 0 C for 24 hours and 30 0 C for 3 days respectively.After incubation the concentration at which no visible growth was seen was noted as MBC (for bacteria) and MFC (For fungi).

RESULTS
The in vitro antimicrobial activity of MEDP, MECG and MESM were shown in

DISCUSSION
The antimicrobial activities of various plants have been reported by many researchers 21,22 .Phytoconstituents present in plants namely flavonoids, alkaloids, tannins and triterpenoids are producing exciting opportunity for the expansion of modern chemotherapies against wide range of microorganisms 23,24 .In present study a variety of gram positive, gram negative bacteria and fungal stains were selected for the screening of antimicrobial effect of three selected plant extracts to perceive the antimicrobial spectrum as well to authenticate ethnomedicinal claims.The results of this study showed that the MEDP, MECG and MESM have varied antimicrobial activities against the tested organisms.Among these three extracts MECG was found most effective against selected strains followed by MEDP and MESM in order effectiveness.Thus in search of novel broad spectrum antimicrobial agent, the formulation comprising different proportions of these extracts may be proven good.This study has not only shown the scientific basis for some of the therapeutic uses of traditional plants, but also confirmed the ethnomedicinal claims for the selected plants.

CONCLUSION
In conclusion, the results of this investigation revealed that methanol extracts of all three plants possess differentiating antimicrobial activity against selected bacterial and fungal strains.The differentiating activities against variety of microorganisms of these three extracts encourage developing a novel broad spectrum antimicrobial formulation in future.Now our research will be directed to develop a broad spectrum antimicrobial herbal formulation with these plants.

Table 1 :
Preliminary antimicrobial activity of MEDP, MECG and MESM Key: '-' no measurable zone.Values are mean ± S.E.M. of 3 replications.MEDP -methanol extract of mature fruits of Diospyros peregrina, MECG -methanol extract of the leaves of Coccinia grandis, MESM -methanol extract of the bark of Swietenia macrophylla.

Table 2 :
Minimum inhibitory concentration of the three methanol extracts

table 1
Sarcina luteus and Bacillus spp.and resistant (MIC and MBC > 200 µg/ml) to all Shigella spp.Against fungi strains all extracts were found effective at higher concentrations.Candida albicans has shown highest sensitivity with MIC values of 800, 200, 800 µg/ml and MFC values of 900, 300, 1000 µg/ml with MEDP, MECG and MESM respectively whilst Penicillium spp.were found least effective with MIC and MFC values of 1500 µg/ml and 2000 µg/ml respectively with all three extracts.