Abstract: Indigenous uses of Walsura trifoliata (A. Juss.) Harms. (Meliaceae) bark in different parts of Eastern Ghats of Southern Peninsular India, curing skin allergies, astringency and diarrhoeia is wide spread. The objective of the present study was to evaluate the antimicrobial and phytochemical activity of bark extract against pathogenic microorganisms. Successive petroleum ether, methanol, benzene and aqueous extracts of Walsura trifoliata bark were tested for their phytochemical constituents, antibacterial and antifungal activity. The methanol and aqueous extracts were found to be most effective against most of the tested organisms. The present findings significantly conform the uses of Walsura trifoliata in the indigenous systems of medicine to treat various diseases like skin allergies, astringency and diarrhoea.
INTRODUCTION
Plants play a vital role in existence and survival of man. Medicinal Plants have been used for centuries as remedies for human diseases because they contain components of therapeutic value (Nostro et al., 2000). It is estimated that there are about 250,000 species of higher plants and the majority of them have not been examined for their pharmacological activities. The antimicrobial properties of certain Indian medicinal plants were reported based on folklore information (Ram et al., 2000; Nagalakshmi et al., 2001; Jayasinghea et al., 2002; Chowdhury et al., 2003; Mishra et al., 2005; Lavanya et al., 2006) and a few attempts were made on inhibitory activity against certain pathogenic bacteria and fungi (Taylor et al., 1995). The plant is well reputed in traditional system of medicine and used by tribal peoples to treat various diseases i.e., skin allergies, astringent and diarrhoeia (Pullaiah and Rani, 1999). This, are common in most of the tribal inhabitants due to lack of sanitation, potable water and awareness of hygienic food habits. Thus there is an increased need for the development of alternative antipathogenic substances. One possible approach is to screen local medicinal plants in search of suitable chemotherapeutic antibacterial and antifungal substances.
MATERIALS AND METHODS
Plant Materials and Extraction
Walsura trifoliata (A. Juss.) Harms. bark were collected from the
evergreen forest of Seshachalam hills ranges of Tirumala hills of Eastern Ghats,
Chittoor district of Andhra Pradesh, in October 2004. The ethnobotanical information
regarding the drug-yielding plant was recorded using the standard methods of
Schultes (1960), Croom (1983) and Hamilton (1995). The information on name,
part used, purpose, mode of administration, etc. was recorded in the field notebooks
as well as audiotapes. The sample specimens were collected in bulk quantities
for analysis. Based on the folk evidences regarding the effective utilization
for different skin allergies, astringent and diarrhoeia, the samples were collected
and screened for antimicrobial properties. The voucher specimens (No. 2089)
were identified with the help of regional floras (Gamble, 1935; Pullaiah and
Rani, 1999) and deposited MMK Herbarium, Vijayawada. The plant material (bark)
were shade dried, powdered (about 60 g) and successively extracted with different
solvents (250 mL) using a soxhlet apparatus for 6 h. The extracts were filtered
and concentrated under reduced pressure, below 40°C to dryness. Phytochemical
analysis for major phytoconstituents of the plant extracts was undertaken using
standard qualitative methods as described by various authors (Kapoor et al.,
1969; Rizk and Bashir, 1980; Fadeyi et al., 1989; Odebiyi and Sofowora,
1990). The plant extracts were screened for the presence of biologically active
compounds like glycosides, phenolics, alkaloids, tannins, flavonoids, saponins
and steroids.
Microorganisms and Media
Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans,
Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis,
Staphylococcus griseus, (gram positive) Escherichia coli, Proteus
vulgaris, Pseudomonas fluorescence, (gram negative) Aspergillus
flavus, Aspergillus niger, Candida albicans, Penicillium
chrysogenum (fungi) were obtained from the Institute of Microbial Technology
(IMTECH), Chandigarh, India. The test organisms were sub-cultured at 37°C
and maintained on nutrient agar media for bacteria and sabouraud agar medium
for Candida albicans.
Antimicrobial Activity
The antimicrobial activity of the ethanol extracts of each sample was evaluated
by using disc diffusion method (Bauer et al., 1966). Petriplates containing
20 mL of respective medium was seeded with selected microbial strains. Five
milliliters of nutrient broth was inoculated with a loop (6 mm) of bacteria/yeast
and incubated at 35°C for 6 h. One milliliter of broth was taken at 0.6
optical density (at log phase, ca.108 cells mL-1) and inoculated
the nutrient agar (sterile) and transferred to 180x20 mm petri dishes. The sterile
Whatmann No. 1 filter paper discs of 6 mm diameter were impregnated with 1000-5000
μg of concentrated plant extracts and placed on the surface of the freshly
inoculated medium. Standard antibiotic discs viz., Ampicillin, Tetracy-cline,
Gentamycine and Clotrimazole were obtained from Hi-Media, Mumbai, were used
as positive controls. Ethanol and water alone served as negative controls. The
assessment of antimicrobial activity was based on measurement of inhibition
zones formed around the discs. The media were incubated for 24 h at 37°C
and the diameters of the inhibition zones were recorded. Three independent trials
were conducted for each concentration.
RESULTS AND DISCUSSION
The methanol, aqueous, petroleum and benzene extract of bark of Walsura trifoliata were tested and results shown in Table 1. Different extracts inhibited the growth of 12, 10, 3 and 3 of the 14 used microorganisms, respectively. Methanol extracts of Walsura trifoliata exhibits significant activity against Staphylococcus aureus, Bacillus cereus and moderate inhibition on Bacillus coagulans and Candida albicans, less activity against Aspergillus flavus. However, aqueous extracts also exhibited significant activity against Bacillus cereus, Bacillus coagulaus and Escherchia coli, moderate inhibition on Bacillus subtilis, over all when aqueous extract showed more significant activity. The petroleum ether and benzene extracts showed less inhibitory activity when compare with the above two extracts. The petroleum ether showed activity against Escherchia coli, Aspergillus niger and Pseudomonas fluorescence and where as Benzene extracts were against Bacillus lichenigormis, Bacillus cereus and Staphylococcus epidermidis.
| Table 1: | Antimicrobial activity of Walsura trifoliate bark extractsa |
| a: Values (zone of inhibition in mm) are the mean of three replicates; minimum inhibitory concentration (mg mL-1) are reported in parentheses, when determined. P: Petroleum ether extract; M: Methanol extract B, benzene extract; A: Aqueous extract; (all tested at 25 mg mL-1); -: No inhibition; NT: Not Tested; b: Ampicillin (10 mg mL-1) for Bacillus, Staphylococcus species, Tetracycline (30 mg mL-1) for E. coli, Gentamycine (10 mg mL-1) for Proteus and Pseudomonas species and Clotrimazole (25 mg mL-1) for fungal species | |
| Table 2: | Phytochemical screening of petroleum ether (P), methanol (M), benzene (B), aqueous (A) extracts of Walsura trifoliata bark extractsa |
| - : Not detected; +: Low concentration; ++: High concentration; +++: Very high concentration | |
Phytochemical screening of these extracts showed the presence of alkaloids, carboxylic acids, fatty acids, phenols, saponins and steroids (Table 2). Catechols, coumarins, proteins, tannins, volatile oils were observed in low concentrations. We suspect that tannins found in the in the Phytochemical analysis of the extracts could be responsible for the antibacterial activity. Tannins have astringent actions, which form the basis for their therapeutic applications (Edward et al., 1970). If was found that plants which contain tannins posses antimicrobial activity (Trease and Evans, 1989).
The results showed antimicrobial activates agreeing with comparable results of previous researches using extracts other plants species like Azadirachta indica (Ram et al., 2000) Chukrasia tabularis (Nagalakshmi et al., 2001), Toona ciliata and Amoora rohituka (Chowdhury et al., 2003) Aglaia spectabilis (Lavanya et al., 2006) etc. The effectiveness may be due to the cumulative action of different compounds present in the plant parts (Bai, 1990). They include alkaloids, flavonoids, triterpenoids and other compounds of phenolic nature and are classified as active antimicrobial compounds (Rojas et al., 1992). This study revealed that the bark extracts of
Walsura trifoliata had antimicrobial activities to certain pathogens with a broad spectrum result to standard antibiotics. The results obtained also provide support to the uses of the plants in traditional medicine. However the toxicological analysis of the active compounds is necessary in order to assess its tolerance in the human body when administered.
CONCLUSION
Based on the results of the present study it can be concluded that the methanol and aqueous extract of Walsura trifoliata bark has exhibited significant activity. The above results indicate that the bark extracts may be used in the treatment of skin allergies, astringency and diarrhoeia, caused by microorganisms tested. Further studies with purified active compounds may be useful to evaluate the actual antibacterial properties of this plant. In addition toxicological experiments must also be undertaken to ensure the safe use.
ACKNOWLEDGMENT
KSM sincerely thanks the Department of Science and Technology, New Delhi for financial support under SERC-Young Scientist.