S Dalal, S Rana, K Sastry, S Kataria
antimicrobial activity, eclipta alba, wedelolactone
S Dalal, S Rana, K Sastry, S Kataria. Wedelolactone as an Antibacterial Agent extracted from Eclipta alba . The Internet Journal of Microbiology. 2008 Volume 7 Number 1.
Wedelolactone is a naturally occurring coumestan isolated from aerial parts of
Plants are invaluable sources of pharmaceutical products. India, in particular has yielded an incredible array of plant products that have drawn the attention of ethno pharmacologists from around the world. Medicinal plants are important substances for the study of their traditional uses through the verification of pharmacological effects and can be natural composite sources that act as new anti-infectious agents. In order to find out new sources of drugs, a number of plants have been screened for wide range of biological activities. About 3,000 materials from 2,764 plant species have been screened for their pharmacological and chemotherapeutic properties (Anon, 1988). Traditionally used medicinal plants produce a variety of compounds of known therapeutic properties (Iyengar, 1976; Harborne, 1989; Chopra
The microbes used for the detection of antimicrobial activity were chosen for certain reasons.
However, up to date, research has been done to investigate various pharmacological activities and antimicrobial activity of only crude extracts of this traditionally used herb. We report here our findings on antibacterial effects of wedelolactone (Fig. 1), the principle active compound, extracted from
Materials and Methods
Collection of plant material - Plants of
Plant extraction and fractionation - The three months old 950 gm lyophilized leaves were Soxhlet extracted with methanol for 36 h. The solvent was removed and the residues were suspended in water separately and heated on steam bath below 80 °C for 30 min. After filtration, the aqueous phase was partitioned with ethyl acetate. The organic phase was dried, filtered and the solvents were evaporated to yield 6.8 gm light brown powder. The powder was subjected to fractionation by column chromatography on silica gel, eluted with the solvent of increased polarity i.e. Non-polar - polar - highly polar. The coumestans are polar compounds so the solvent combination found suitable for their elution was Chloroform + Methanol (70 + 30). They were eluted simultaneously in 37 to 48 fractions. The pooled sample was then subjected to TLC, the solvent system (Toluene : Acetone : Formic acid :: 11 : 6 : 1 v/v) showed two spots with Rf values 0.39 and 0.28 which matched with the Rf values of reference wedelolactone and demethylwedelolactone respectively (Courtesy M/s Natural Remedies, Bangalore, India). The purified sample of wedelolactone was put to HPLC for further qualitative analysis using instrument - Thermo Finnigan from Thermo Electron Corp. USA, with quaternary pump and online degasser system with Auto sampler equipped with Photo Diode Array (PDA) detector, ChromQuest Version 5.0 for data interpretation and Supleco C8 Discovery column, 15 cm x 4.6 mm, Lot No. 59353.
Preparation of samples for testing ¬- The study was conducted with purified wedelolactone diluted with 10% dimethylsulfoxide (DMSO). The serial dilutions were performed in a concentration range from 0.005 mg/ml to 50 mg/ml.
Micro organisms - Standardized strains from the American type culture collection (ATCC) were used in bioassays. The Gram-positive bacteria were
Antimicrobial susceptibility testing - MIC of wedelolactone was determined by microdilution technique as described by the National Committee for Clinical Laboratories standards (2000). The MIC was defined as the lowest concentration of the compound to inhibit the growth of microorganism. The bacteria inoculums were prepared in 5 ml nutrient broth and incubated at 37C. The final inoculums were of approximately 5 x 10 6 CFU/ml. Controls with 0.5 ml of culture medium with out the samples and other without microorganisms were used in the tests. Tubes were incubated at 37C for 24 h. The activity was measured as a function of turbidity at 660 nm. Lack of turbidity was further confirmed by pouring suspension aliquot of 0.1 ml into pre-sterilized Petri dishes with nutrient agar medium. The tests were conducted in triplicate.
Agar well diffusion method was carried out by allowing perforation of compound dissolved in DMSO at a concentration of 10 mg/ml. Petriplate containing 30 ml nutrient agar medium were kept for the solidification before inoculating the microorganism, desired numbers of holes of uniform diameter of 8mm were made after solidification, using sterile aluminium borer. 0.2 ml of compound, positive (Gentamycin) and negative (solvent blank) controls were poured into wells. After incubation for 24 h at 37 C the plates were observed and the compound activity was evaluated by measuring zone of inhibition (diameter mm). The tests were conducted in triplicate. Gentamycin (10.0 µg/ml) was used as positive control. The negative control was 10% DMSO.
Wedelolactone exhibited significant antibacterial activity against the four tested strains (Table 1).
In Ayurveda, the traditional Indian system of medicine,
MIC and zone of inhibition were performed to evaluate its antimicrobial potential (Table I). Kumar
The authors wish to thanks Dr. T Velpandian, Associate Professor Ocular Pharmacology Laboratory at All India Institute of Medical Sciences, New Delhi, India for HPLC analysis and the members of laboratory of Department of Biosciences, MD University, Rohtak, Haryana, India.