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.

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).

All chemicals and solvents used in this experiment were of analytical grade obtained from BDH, Poole, UK.

Microorganisms

Twenty different bacterial strains namely Staphylococcus aureus 29737, Staphylococcus aureus ML 267, Sarcina luteus 9341, Bacillus pumilus 8241, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 10536, Escherichia coli VC Sonawave 3:37 C, Escherichia coli CD/99/1, Escherichia coli RP4, Escherichia coli 18/9, Escherichia coli K88, Shigella dysenteriae 1, Shigella soneii 1, Shigella soneii BCH 217, Shigella flexneri type 6, Shigella boydii 937, Pseudomonas aeruginosa ATCC 25619, Vibrio cholerae 2, Vibrio cholerae 785, Vibrio cholerae 1037 and four different fungal strains namely Candida albicans ATCC 10231, Aspergillus niger ATCC 6275, Penicillium notatum ATCC 11625, Penicillium funiculosum NCTC 287 were collected from institute of microbial technology, Chandigarh, India. The bacterial strains were grown in MacConkey agar plates at 37 ⁰ C and maintained on nutrient agar slants, while fungi were grown at 30 ⁰C 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 10⁶ cfu / ml for bacteria and 2 × 10⁵ 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 ⁰C for 24 hours for bacteria and 30 ⁰C for 3 days for fungal spores ¹⁹. Sensitivity was recorded by measuring the clean zone of growth inhibition on agar surface around the disc.

Determination of Minimum Inhibitory Concentration (MIC), and Minimum Bactericidal Concentration (MBC) and Minimum fungicidal Concentration (MFC) MIC was determined by tube dilution method for each of the test organism in triplicates ²⁰. 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 10⁶ 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 ⁰C for 24 hours for bacteria and 30 ⁰C for 3 days for fungal spores. After incubation the tubes were examined for microbial growth by observing the turbidity.

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 ⁰C for 24 hours and 30 ⁰C 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).

The in vitro antimicrobial activity of MEDP, MECG and MESM were shown in table 1. The MEDP and MESM have shown maximum zone of inhibition against Escherichia coli K88 of 12.56 and 10.67 mm respectively whilst MECG produced maximum zone diameter of 14.10 mm against Pseudomonas aeruginosa ATCC 25619. The activity of MEDP, MECG and MESM among fungi strains was found highest with Candida albicans ATCC 10231 (10.7, 16.5 and 11.2 mm respectively) and lowest with Penicillium funiculosum (7.33, 7.03 and 7.63 mm respectively). In this preliminary antimicrobial assay ciprofloxacin (200 µg/ml), griseofulvin (2000 µg/ml) were taken as standard antibacterial and antifungal agents. The results of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were shown in table 2. The results showed that MEDP is highly sensitive against Escherichia coli strains (MIC and MBC 10 -25 µg/ml), moderately sensitive (MIC 100 µg/ml and MBC 100 - 150 µg/ml) to Staphylococcus aureus and Vibrio cholerae strains, less sensitive (MIC 200 µg/ml) to Shigella spp. and Pseudomonas aeruginosa whilst resistant (MIC and MBC > 200 µg/ml) to Sarcina luteus and Bacillus spp. MECG has shown maximum activity against gram-positive organism including Staphylococcus aureus (MIC 10 µg/ml and MBC 25 µg/ml) and gram negative cultures including Escherichia coli (MIC 10 - 25 µg/ml and MBC 25 - 50 µg/ml), Shigella dysenteriae (MIC and MBC 10 µg/ml), Shigella soneii (MIC 10 µg/ml and MBC 25 µg/ml) and Pseudomonas aeruginosa (MIC and MBC 10 µg/ml); moderately sensitive (MIC 100 µg/ml and MBC 150 µg/ml) to Vibrio cholerae, Sarcina luteus, less sensitive (MIC 200 µg/ml and MBC > 200 µg/ml) to Bacillus spp., whilst resistant (MIC and MBC > 200 µg/ml) to Shigella flexneri and Shigella boydii. MESM was found maximum sensitive (MIC 50 µg/ml and MBC 50 - 75 µg/ml) to Escherichia coli strains; less sensitive (MIC 200 µg/ml) to Vibrio cholerae, Pseudomonas aeruginosa, Staphylococcus aureus, 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.

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.

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.

The authors are thankful to All India Council for Technical Education, New Delhi, India for financial assistance.

REFERENCES