Abstract: The petroleum ether, ethyl acetate and methanol extracts of Vanda roxburghii as well as glycoside, melianin (VR-1) isolated from it were screened for antimicrobial activity against a wide variety of bacteria and fungi. The ethyl acetate and methanol extracts showed moderate antibacterial activity against almost all the tested organisms. The compound melianin (VR-1) exhibited strong activity against all the tested organisms and produced zone of inhibition between 17 and 27 mm. The petroleum ether extract was found comparatively less active against the organisms. All the tested materials showed antifungal activity against Aspergillus fumigatus, Candida albicans, Hensinela californica and Rhizopus arijae. The minimum inhibitory concentrations (MIC) of melianin against Bacillus cereus, Bacillus subtilis, Escherichia coli and Shigella dysenteriae were 32, 64, 64 & 128 μg ml–1 respectively. The findings may provide the basis for traditional use of this plant in the treatment of infectious diseases.
Introduction
Vanda roxburghii Br., an epiphytic herb belonging to the family Orchidaceae is widely distributed in tropical Asiatic & Australian Zones (Kirtikar and Basu, 1994). It is found in Bengal, Chosta, Nagpur, Bihar, Central Provinces, W. Penisula, Travancore, Ceylon (Hooker, 1985). In Bangladesh it grows as an epiphyte in Mango, Black berry and Guava trees. The plant has a folkloric reputation as a cure of various infectious diseases (Biswas, 1973). The juice of leaves is used by the native physicians of Bangladesh as a wonderful painkiller of ears. The root is used in the treatment of diarrhoea, dysentery, dyspepsia, bronchitis, rheumatic pains, diseases of the abdomen, hiccough and tremor. The root of this plant is also used as laxative and tonic to the liver & brain. It is also effective against piles, lumbago toothache, boils on the scalp, inflammation, fracture (Kirtikar and Basu, 1994; Biswas, 1973).
Although V. roxburghii is locally credited with medicinal properties, no antimicrobial study of this plant has been reported. As a part of our continuing search for novel antimicrobial principles from the medicinal plants of Bangladesh, we studied V. roxburghii and herein reported the results of in vitro antimicrobial investigation.
Materials and Methods
Collection of the plant: The roots of V. roxburghii Br. were collected from Rajshahi University campus and were identified by Bangladesh National Herbarium, Dhaka.
Extraction, isolation and characterization: The roots were dried in an oven at 45°C, crushed and then extracted successively with petroleum ether (C2H5–O-C2H5) (40-60°C), ethyl acetate (CH3COO C2H5) and methanol in a soxhlet apparatus (Morrison and Boyd, 1994). The extracts were concentrated by a vacuum rotary evaporator under reduced pressure and then were used for antimicrobial screening.
Compound VR-1 was isolated from the ethyl acetate extract by column chromatography (Beckett and Stenlake, 1986) followed by TLC and PTLC (Stahl, 1969). The compound was identified as melanin on the basis of their 1H and 13C-NMR, and mass spectral data (Ahmed, 1996) and comparison with previously reported data for melianin from Melia volkensii (Lingling et al., 1998). It was then subjected to antimicrobial screening.
Antibacterial screening: The in vitro antibacterial activity of the extracts and isolated compound VR-1 were studied against 5 gram-positive and 9 gram-negative bacterial strains by the standardized disc-diffusion method (Barry, 1980; Berghe and Vlietnck, 1991) against selected test organisms. Nutrient agar media was used as the bacteriological media. The petroleum ether, ethyl acetate and methanol extracts were dissolved separately in sufficient amount of methanol to get a concentration of 400μg 10μl–1. Compound VR-1 was dissolved in sufficient volume of methanol to get a concentration of 200μg 10μl–1. The diameters of zone of inhibition produced by the agent were compared with those produced by the standard antibiotic (Kanamycin, 30μg disc–1). The experiment was performed in duplicate to minimize the errors.
Minimum inhibitory concentration (MIC): The MIC value of the compound VR-1 was determined against one gram-positive (Bacillus cereus) and one gram-negative (Escherichia coli) bacteria. The test was carried out by serial dilution technique (Reiner, 1982). Nutrient agar and nutrient broth were used as bacteriological media.
Antifungal screening: Seven pathogenic fungi were used for the test. Potato Dextrose Agar (PDA) was used as fungicidal media. The in vitro antifungal activities of the samples were performed by disc diffusion method. Clotrimazole was used as a standard one.
Results and Discussion
In antibacterial screening, it was found that the ethyl acetate and methanol extracts showed moderate activity against almost all gram-positive and gram-negative bacteria (Table 1). Whereas the petroleum ether extract exhibited mild antibacterial activity against the test organisms. The zone of inhibition of ethyl acetate and methanol extracts was found in between 11-17 mm and 9-14 mm respectively. On the other hand, compound melianin (VR-1), isolated from the ethyl acetate extract showed strong activity against all the tested organisms and produced zone of inhibition in between 17 and 29 mm. Compound VR-1 showed intense activity against Bacillus cereus, Bacillus subtilis, Escherichia coli and Shigella dysenteriae.
Antifungal screening of the extracts and compound revealed that all the tested materials were active against Aspergillus fumigatus, Candida albicans, Hensinela californica and Rhizopus arijae. The compound did not show any activity against Saccharomyces cerevisae, Pigmented yeast and Rhizopus digasporum (Table 2).
The minimum inhibitory concentration (MIC) of the compound VR-1 against Bacillus cereus, Bacillus subtilis, Escherichia coli and Shigella dysenteriae was determined and the values were 32, 64, 64 & 128μg ml–1 respectively (Table 3).
| Table 1: | Antibacterial activity of extracts and compound VR-1 isolated form Vanda roxburghii Br. |
| A= Petroleum ether extract, 400μg disc–1 B= Ethyl acetate extract, 400μg disc–1 C= Methanol extract, 400μg disc–1 D= VR-1, 200μg disc–1 E= Standard Kanamycin, 30μg disc–1 ‘-’ = No sensitivity | |
| Table 2: | Antifungal activity of extracts and compound VR-1 isolated form Vanda roxburghii Br. |
| A= Petroleum ether extract, 400μg disc–1 B= Ethyl acetate extract, 400μg disc–1 C= Methanol extract, 400μg disc–1 D= VR-1, 200μg disc–1 E= Standard Clotrimazole, 30μg disc–1 ‘-’ = No sensitivity | |
| Table 3: | The minimum inhibitory concentration (MIC) values of the compound VR-1 against test organisms. |
| ‘+’ = Growth; ‘-’ = No Growth, | |
In conclusion, the present study reports for the first time the antibacterial and antifungal activity of the compound and extracts of V. roxburghii Br. The findings may provide the basis for traditional use of this plant in the treatment of infectious diseases.
Acknowledgment
The authors are thankful to Dr. Abdur Rashid, the NCI Frederick, Cancer Research and Development Center, Frederick MD, 21702-1201 USA, for spectroscopic analyses of this research samples.