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Biological Activities of Isolated Compounds from Vitex Negundo Leaf

N Y Chowdhury
Institute of Biological Sciences, University of Rajshahi, Rajshahi, Bangladesh

W Islam
Institute of Biological Sciences, University of Rajshahi, Rajshahi, Bangladesh

M Khalequzzaman
Institute of Biological Sciences, University of Rajshahi, Rajshahi, Bangladesh

Abstract/ Executive Summary:

Context: Viyex negundo Linn. (Verbenaceae) is a beautiful tree which is an erect, large aromatic shrub with quadrangular branchlets possess pesticidal, antibacterial and antifungal properties. Objective: To determine the biological activities (antibacterial, antifungal, brine shrimp lethality bioassay) of the two isolated compounds from methanolic leaf extract. Materials and Methods: Powdered leaves of nishinda were extracted with methanol using Soxhlet’s apparatus and subsequent analyses isolated two compounds. Five gram-positive, eight Gram-negative bacterial strains were used for the antibacterial activity using the disc diffusion assay method. The antifungal activities of the isolated compounds were also performed on four pathogenic fungi. Each pure compound was dissolved in 200 μl of methanol to get a concentration 300 μg/10 μl. Minimum Inhibitory Concentrations were determined by serial dilution technique. For brineshrimp bioassay each compound and standard amphicilin trihydrate were dissolved in dimethylsulfoxide to get a five concentrations. Each concentration contained three vials consisting of 10 nauplii in 5 ml of treated sea water. The number of survived nauplii were counted after 24 h and the LD50 values were calculated. Results: The zone of inhibition was prominent for the control (kanamycin) at concentration of 30 μg/disc. At 100 μg/disc Compound 1 exhibited bigger and more prominent clear zone of growth inhibition in all test microorganisms except Shigella shiga. On the contrary, Compound 2 at 100 μg/disc, showed clear zone of inhibition in all bacteria, but inhibition of zones were larger in Compound 1 than Compound 2. Antimicrobial effect of Compound 2 tested on different pathogenic bacteria (MIC 128 μg/ml) and fungi showed that it possesses growth inhibitory effect at various concentrations. MIC of Compound 1 for B. subtilis, S. aureus and S. B-haemolyticus was 64 μg/ml, whereas for P. aeruginosa it was 128 μg/ml. MIC of Compound 2 was 128 μg/ml for B. subtilis, S.-β-haemolyticus and P. aeruginosa whereas it was 64 μg/ml for S. aureus. No fungal activity was observed for Compound 1. Clear inhibition zone was observed for Compound 2 at both concentrations for all of the pathogenic fungi tested. At 100 μg/disc Compound 2 exhibited bigger and prominent clear zone than 50 μg/disc.. Brine shrimp bioassay showed the toxic effect of the both the compounds. Conclusion: The findings indicate promising antibacterial and antifungal activities of V. negundo against life treating pathogens which appears to be an effective material for development of antimicrobial drugs and ecofriendly biopesticides.

Keywords: Vitex negundo, Antimicrobial, MIC, Bacteria, Phytochemical, Medicinal plant activity, Brineshrimp

Location: Department of Botany, University of Rajshahi

Start Date:

End Date:

Program Area: Pest Management

Commodity: Insecticide

Objectives:

To determine the biological activities (antibacterial, antifungal, brine shrimp lethality bioassay) of the two isolated compounds from methanolic leaf extract.

Methodology:

Plant: The leaves of V. negundo were collected from the Rajshahi University and authenticated by the authority of the Department of Botany, University of Rajshahi where a voucher specimen (# 528) has been deposited. Preparation of Extracts and isolation of two compound: Powdered leave (500 g) was extracted with methanol (MeOH) (BDH, England) using a Soxhlet’s apparatus. The crude extract were stored in a refrigerator at -20°C. The subsequent analyses led to the isolation, identification and structural elucidations of two compounds 22, 23-dihydro α-spinasterol-β -D-glucoside (Compound-1) and 2-hydroxy benzoic acid (salicylic acid) (Compound 2). Both compounds were first time isolated from this plant. Antibacterial activity: Five gram-positive (Bacillus subtilis, B. megaterium, Sarcina lutea, Staphylococcus aureus, St. β-haemolyticus) and eight gram-negative (Shigella dysenteriae, Sh. shiga, Sh. boydii, Sh. sonnei, Escherichia coli, Pseudomonus aeruginosa, Klebsiella sp., Salmonella typhi) strains were used for the anti-bacterial activity using the disc diffusion assay method (Bauer et al. 1966). The bacteria were collected from the cultures maintained in the microbiology laboratory of the Department of Pharmacy, University of Rajshahi. The test organism was transferred from the subculture to the test tube containing 20 ml sterile media. The bacterial suspensions were aseptically transferred to the sterile petri dish giving a uniform depth of media (4 mm). Kanamycin 30 μg/disc was used as control. Standard disc was prepared by pouring 10 μl of kanamycin stock solution (3 μg/μl). The sample disc, standard antibiotic disc and control disc were placed gently on the solidified agar plates. The plates were then inverted and kept in a refrigerator for about 24 h at 4°C to obtain maximum diffusion. Finally, the plates were incubated at 37.5°C for 18-24 h. The antibacterial activities of the test sample were determined by measuring the diameter of inhibitory zones in mm. Brine shrimp bioassay: Brine shrimp bioassay (Mayer et al. 1982) of both the compounds was done. One milligram of each sample compound and standard antibiotic amphicilin trihydrate were dissolved in 200 μ1 of dimethylsulfoxide (DMSO) to get a concentration of 5 μg/μl. The experiment was conducted into five groups. Each group contained three vials consisting of 10 nauplii in 5 ml of sea water. The concentrations of the sample were 5, 10, 20, 40 and 80 μg/ml, respectively. For control, three vials containing 10 brine shrimp nauplii in 5 ml seawater were taken and 20 μ1 DMSO was added in each vial. After 24 h, the vials were observed and the number of survived nauplii in each vial was counted. The LD50 was calculated by Probit analysis and regression lines were drawn according to Goldstein et al. (1974). Determination of minimum inhibitory concentration: The minimum inhibitory concentration (MIC) of pure compounds was determined by agar dilution method (Vander-Berghe and Vlietinck 1991). The test organisms were Bacillus subtilis, Staphylococcus aureus, St.-β-haemolyticus and Pseudomonus aerugonosa. The compounds (Compound 1 and Compound 2) in various concentrations (2-512 μg/ml) and 10μl of bacterial culture (107cells/ml) were added in culture tubes containing 1 ml sterile nutrient broth medium. The cultures were mixed well and incubated at 37.5°C for 24 h and observed for growth of the bacteria. Antifungal activity: The same procedure was followed as did for that of antibacterial activity. Nystatin was used as standard at and 50 μg/disc from the stock solution (5 μg/μl). The period of incubation was 48 h. The fungi (Aspergillus niger, A. flavus, Candida albicans and Trichoderma sp.) were collected from the Department of Botany, University of Rajshahi.

Source of Information: J. bio-sci. 18: 53-59, 2010 ISSN 1023-8654

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The present results indicate that Compound 1 is highly active against Gram-positive bacteria (Sarcina lutea) and Gram-negative bacteria (S. boydii and Klebsiella sp.). The MIC results indicate that both the compounds are effective to inhibit bacterial growth at low concentration (64 μg mL-1). Compound 2 showed strong fungicidal activities at 50 μg/disc. Compound 1 was more cytotoxic in brineshrimp bioassay (LD50 25.153) than Compound 2. These findings lead to further in vivo studies, using animal model, to explore the potential application of this protocol for bacterial pathogen treatment in immune compromised patients and also in preservation of food, pharmaceutical and cosmetic formulations to protect product from microbial activity.

Knowledge Management: Journal