Biological Investigation of Macrotyloma uniflorum Linn. Extracts Against Some Pathogens

MATERIALS AND METHODS

Plant material: Macrotyloma uniflorum (Fabaceae) was collected from the village, Susunda of Muradnagar, Comilla, Bangladesh in March, 2002. The botanical identification was made by Prof. Salar Khan (University of Dhaka) and voucher specimen was deposited at the Bangladesh National Herbarium (BNH) (DACB Accession No. 28264). The whole plants were cleaned, air-dried and followed by drying in an oven at 40°C. The dried plants were powdered by grinding in a cyclotec-grinding machine (200 mesh).

Preparation of extracts: The powdered plant was (3.5 kg) was successively extracted with aqueous 80% ethanol (18 Lx3 times, 24 h) at room temperature. The extract was filtered and the filtrate was evaporated to dryness at 40°C under vacuum and finally freeze-dried to obtain crude ethanolic extract of 484 g (13.82%) as solid material. The ethanol extract (484 g) was suspended in water (~2000 mL) and the suspension was transferred into a separating funnel. The aqueous suspension was successively partitioned with dichloromethane (CH₂Cl₂, ca. 2000 mLx3), ethyl acetate (EtOAc, ca.1500 mLx3) and 1-butanol (1-BuOH, ca.1500 mLx3). The CH₂Cl₂, EtOAc, 1-BuOH and aqueous extracts were evaporated separately and lastly freeze-dried. The extractive yield (%) of all the extracts is shown in Table 1. The extracts were tested for their antibacterial and antifungal inhibition activities.

Table 1:	Percentage of different extracts from M. uniflorum plant
aPercentage extract yield (w/w) was estimated as dry extract/dry material weightx100

Microorganisms: The bacterial and fungal strains used in this research were obtained from the Microbiology Laboratory, Department of Microbiology, University of Chittagong, Bangladesh. The bacterial strains comprised: Bacillus subtilis BTCC 17, Bacillus cereus BTCC 19, Bacillus megaterium BTCC 18, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 25922, Salmonella typhi AE 14612, Shigella dysenteriae AE 14396 and Shigella sonnei CRL (ICDDR,B). The fungal pathogens were Fusarim equiseti (corda) Sacc., Macrophomina Phaseolina (Tassi) Goid, Colletotrichum corchori (Ikata Yoshida), Botrydiplodia theobromae pat, Curvularia lunata (wakker becdijin) and Alternaria alternata (Fr.) Kedissler.

Preparation of stock culture and bacterial suspension: Standard Nutrient Agar (NA) medium was used for growing bacterial strains throughout the research. In a hard glass screw cap test tube, sterile slants of NA were prepared. Older cultures were transferred to freshly prepared NA slants separately for each species via sterilized bacterial loop. In such a way, four test tubes were freshly prepared for each bacterial pathogen. These test tubes of inoculated slants were incubated at (35±2)°C in an incubator. After 18-24 h of incubation each culture was used throughout for antibacterial screening studies. For preservation of the stock culture, one set of culture slants were kept in polythene bag, properly tied and preserved as stock culture at 10°C. Subcultures were maintained at 3 to 4 week intervals to ensure culture viability. Bacterial cultures, after 24 h of incubation, were transferred to sterilized distilled water and mixed and these bacterial suspensions were used in the pour plate technique during sensitivity testing.

Antibacterial assay: The antibacterial activities of the different extracts were detected by disc diffusion method (Bauer et al., 1966). Paper discs of 4 mm in diameter and glass petri dish of 90 mm in diameter were used throughout the experiment. Paper discs were sterilized in an autoclave and dried at 100°C in an oven. The discs were then soaked with test extracts at the rate of 500 μg (dry weight) per disc for antibacterial effect analyses. One drop of bacterial suspension was taken in a sterile petri dish and approximately 20 mL of sterilized melted NA (~45°C) was poured into the plate and then mixed thoroughly with the direction of clock wise and anticlock wise motion. After solidification of the seeded NA medium, paper discs, after soaking with test extracts (2% in DMSO; dimethyl sulphoxide) were placed at the centre of the inoculated pour plate. A control plate was also maintained in each case with DMSO. Firstly, the plates were kept for 4 h at low temperature (4°C) and the test extracts diffused from disc to the surrounding medium by this time. The plates were then incubated at (35±2)°C for growth of test organisms and were observed at 24 h intervals for two days. The activity was expressed in terms of inhibition zone diameter in mm. Each experiment was repeated three times and ampicillin (FISONS Bangladesh Ltd.) was used as reference or positive control while DMSO without sample was used as negative control. Antibacterial activity is given in Table 2 and 3.

Medium and cultures: The antifungal activities of the different extracts were investigated against six plant pathogenic fungi. The investigation was based on food poisoning technique (Grover and Moore, 1962). Potato Dextrose Agar (PDA) was used as basal medium for test fungi. DMSO was used as a solvent to prepare the desired solution (2%) of the extracts initially. Glass Petri dishes were cleaned and sterilized in an autoclave after which sterile, molten (~ 45°C) PDA was poured into each plate in 10-12 mL aliqouts. After a few minutes the medium solidified. Then small portions of mycelium of each fungus pathogen were placed at the centre of each PDA plate via sterilized needles. In such a way, each fungus species was transferred into a number of petri dishes. After a few days, the mycelium grows in the entire petri dish and these are then ready for antifungal activity testing of plant extracts.

Test for antifungal activity: A required amount of medium (PDA) was taken in conical flasks separately and was sterilized by autoclave (121°C, 15 psi) for 15 min. After autoclaving, calculated amounts of test extracts (2%) was added to the sterilized medium in conical flask and the flasks were shaken thoroughly to mix the extract with the medium before pouring. The medium with (2%) of extract was then transferred in 10 μL aliquots into sterilized glass petri dishes individually. Proper control was maintained separately with sterilized PDA medium without extract and three replications were prepared for each treatment. After solidification of medium, the fungal inoculum (5 mm mycelial block) was placed on the centre of the petri dishes in the inverted position.

All the plates were incubated at room temperature on a laboratory bench top for three days at (25±2)°C in triplicate. After three days of incubation, the diameter of fungal mycelial growth was measured. The average of two measurements was taken as mycelial colony diameter of the fungus in mm. Nystatin was used as positive control. Antifungal activity of the extracts is presented in the Table 3. The percentage inhibition of mycelial growth of test fungus was calculated by following equation:

where, C was the diameter of the fungal colony in control and T was the diameter of the fungal colony in treatment.

RESULTS AND DISCUSSION

The extractives of M. uniflorum demonstrated varying degrees of inhibition to growth of microorganisms. The dichloromethane, ethyl acetate, 1-butanol and aqueous extracts were subjected to screening for inhibition of microbial growth against 8 bacteria and 6 fungal strains. The in vitro growth inhibition of all gram-positive bacterial strains are mentioned in Table 2 and it was found that the ethyl acetate extract showed significant antibacterial activity against Bacillus subtilis, Bacillus cereus and Staphylococcus aureus by showing zones of inhibition of 22, 17 and 16 mm, respectively. The ethyl acetate extract also showed more activity (22 mm) against Bacillus subtilis than the standard antibiotic, ampicillin (18 mm) and the dichloromethane extract was active against only one bacterium, Staphylococcus aureus (16 mm). The 1-butanol and aqueous extracts were less active against Bacillus subtilis and Staphylococcus aureus than standard antibiotic. However, Bacillus megaterium bacteria did not show any signs of inhibition against any extracts.

Table 2:	Zone of inhibition observed against Gram-positive bacteria by the extracts
aValues are mean of three replicates, -no. inhibition zone, 10-15: moderately active, >16: significantly active, bStandard antibiotic

Table 3:	Zone of inhibition observed against Gram-negative bacteria by the extracts
aValues are mean of three replicates, -no. inhibition zone, 10-15: moderately active, >16: significantly active, bstandard antibiotic

Table 4:	Antifungal activities of the different extracts
aValues are mean of three replicates, -no. inhibition zone, bstandard antibiotic

The screening data presented in Table 3 against gram-negative bacteria suggests that the ethyl acetate extract showed significant inhibition against Salmonella typhi (24 mm) but good inhibition against Shigella dysenteriae (17 mm) and Escherichia coli (15 mm). Contrastively, the dichloromethane extract showed little growth inhibition for any bacteria. 1-butanol and aqueous extracts also showed good activity against a few bacteria but none of them was stronger than the standard antibiotic. The antibacterial activity increased as the polarity of the solvents increased and the maximum antibacterial activity was found to be caused by the ethyl acetate extract.

The dichloromethane extract gave significant (32%) antifungal activity against Fusarim equiseti which was higher inhibition than the standard antibiotic, nystatin (30%). The same extract showed moderate to good inhibition of mycelial growth against Alternaria alternate (22%), Colletotricum corchori (20%) and Curvularia iunata (18%) as well. The ethyl acetate extract showed significant antifungal activity against Colletotricum corchori (21%). 1-butanol and aqueous extracts showed good antifungal activity against Alternaria alternata (23%) and Botrydiplodia theobromae (18%), respectively (Table 4). The overall results indicated that out of six fungi tested against M. uniflorum plant extracts, minimum average inhibition was observed in the case of Colletotricum corchori while Fusarim equiseti showed maximum average inhibition.

The antibacterial activities of the plant extracted in different solvents varied greatly because there were many factors that influence the active compounds present in the plant. Here the polarity of the extracting solvents was different and it greatly influenced the antimicrobial properties (Table 2, 3). This may be due to the better solubility of the active compounds in organic solvent (De Boer et al., 2005; Parekh et al., 2006). The extractive yield of different solvents is given Table 1. Maximum yield obtained was with 1-butanol (3.14%) while the minimum was with dichloromethane (1.14%) (Table 1).

Traditional healers use primarily water as solvents but in this study showed that the plant extracts, extracted in different organic solvents, showed profoundly distinct antibacterial activities compared to aqueous extracts. These observations can be rationalized in terms of the polarity of the compound being extracted by each solvent and, in addition to their intrinsic bioactivity by their ability to dissolve or diffuse in the media used in the assay. The growth media also seem to play an important role in the determination of the antibacterial activity (Lin et al., 1999). Amongst the gram-positive and gram-negative bacteria, gram-negative bacterial strains were more susceptible to the extracts as compared to gram-positive bacteria. Generally gram-negative bacteria are more resistant than gram-positive bacteria (Kelmanson et al., 2000; Rabe and Staden, 1997) but the result of this present work is different. Here the maximum antibacterial activity was seen against gram-negative bacteria (Table 3). The biological activities exhibited by the different extracts of M. uniflorum is most likely due to the presence of different chemical components in the extracts.

CONCLUSION

Extracts of M. uniflorum Linn. in this study demonstrated a broad-spectrum of activity against both gram-positive and gram-negative bacteria and fungi. The results of present study support the traditional usage of the studied plant and suggest that the plant extracts possess compounds with antimicrobial properties that can be used as antimicrobial agents after further pharmacological study.

ACKNOWLEDGMENTS

The authors are indebted to the Chairman of the Department of Microbiology, University of Chittagong to give the opportunity to carry out this present research. We are also indebted to Dr. Robert Kanaly for critical reading of this manuscript.