*Corresponding Author:
T. Ghosh
Institute of Pharmacy and Technology, Salipur - 754 202, India
E-mail: [email protected]
Date of Submission 16 January 2006
Date of Revision 28 August 2006
Date of Acceptance 16 April 2007
Indian J Pharm Sci,2007, 69 (2): 312-314  

Abstract

The ethyl acetate and n-butanol fractions of ethanol extract of Bacopa monnieri Linn. aerial parts were screened for antibacterial and antifungal activities by both zone of inhibition study and determination of minimum inhibitory concentration (MIC). The ethyl acetate fraction was found to be more potent than the n-butanol fraction, though both of them were endowed with antimicrobial activity. The present study reveals the potential usefulness of B. monnieri aerial parts in the treatment of various pathogenic diseases as mentioned in the Ayurvedic literature.

Introduction

Bacopa monnieri Linn. (Family: Scrophulariaceae), is a creeping, glabrous, succulent herb, rooting at nodes. It is distributed throughout India in all plain districts, ascending to an altitude of 1,320m [1]. In Ayurveda, B. monnieri have been used in the treatment of insanity, epilepsy, hysteria and skin diseases [2]. The alcoholic extract is reported to increase the learning performance of rats and the activity is attributed to saponin mixtures consisting mainly of bacosides A and B [3,4]. The plant is also reported to show sedative [5], antiepileptic [6], vasoconstrictor [7], antiinflammatory [8] and anthelmintic [9] activities. The plant is reported to contain tetracyclic triterpenoid saponins, bacosides A and B [10,11], hersaponin [12] and some flavonoids [13,14]. The antimicrobial activity of the ethanol extract of the plant has already been reported by the authors [15]. In the present investigation, the antimicrobial activity of ethyl acetate and n-butanol fractions of the ethanolic extract of B. monnieri aerial parts were studied in a scientific manner.

The plant material was identified by the taxonomists of the Botanical Survey of India, Shibpur, Howrah. After authentication, fresh aerial parts were collected in bulk from young matured plants from the rural belts of Salipur during early summer, washed, shade dried and then milled into coarse powder by a mechanical grinder.

The powdered plant material (500 g) was defatted with petroleum ether (60-80°) and then extracted with 1.5 l of ethanol (95%) in a Soxhlet apparatus. The solvent was then removed under reduced pressure, to yield a greenish-black sticky residue (yield: 11.6% w/w with respect to dried plant material). A part of the ethanol extract was kept as such and the remaining part was partitioned successively between ethyl acetate-water system and then between n-butanol-water system. The ethyl acetate (yield: 48.28% w/w with respect to ethanol extract) and n-butanol (yield: 24.74% w/w with respect to ethanol extract) fractions of the ethanolic extract were used in the present study.

The microorganisms used in the present study include Staphylococcus aureus, Bacillus subtilis, Bacillus polymexia, Streptococcus faecalis, Pseudomonas aeruginosa, Salmonella typhi, Vibrio cholerae, Shigella dysenteriae, Escherichia coli, Penicillium notatum, Aspergillus niger and Candida albicans. Suitable strains of these microorganisms were procured from the microbiology laboratory of our institute, which were originally obtained from Department of Microbiology, Orissa University of Agricultural University, Bhubaneswar.

Antibacterial and antifungal activities were studied by agar disc diffusion method [16] and determination of minimum inhibitory concentration (MIC) by broth dilution method [17].The zone of inhibition of the fractions was performed at concentrations of 2, 5 and 10 mg/ml of the fractions in dimethyl sulphoxide (DMSO). Ciprofloxacin (5 µg/ml) and clotrimazole (25 µg/ml) were used as reference controls for the antibacterial and antifungal studies, respectively. Solvent control (only DMSO) was also maintained throughout the experiment. MIC was performed at concentrations of the fractions ranging from 25 to 800 µg/ml in DMSO against all the test microorganisms.

The ethyl acetate fraction was found to be more potent than the n-butanol fraction. Zone of inhibition study reveals that both the fractions showed antibacterial and antifungal activity in a dose dependant manner and was comparable with the standard drugs (Table 1). The MIC results showed that both the fractions have antibacterial and antifungal activity against the tested strains. MIC value ranges from 25 to 200 µg/ml for ethyl acetate fraction and 100 to 600 µg/ml for n-butanol fraction.The results also reveal that both the fractions were more active against B. subtilis and less active against P aeruginosa, which is naturally resistant to antibacterial agents [18] (Table 2).

Micro-organisms EAF (mg/ml) NBF (mg/ml) Standardsa
Gram-positive bacteria 2 5 10 2 5 10  
S. aureus ATCC 25923 10.7 15 20.7 8.3 15.3 17.7 27.3
B. subtilis UC 564 11.7 18.7 22 8.7 14.3 15.7 25
B. polymexia 474 8.7 14 17.7 10.3 11.7 15 22.3
Streptococcus faecalis 9.3 12.7 16.7 7 11.3 14.3 26.7
Gram-negative bacteria            
P. aeruginosa 25619 8 11.3 15.3 7.3 9.7 12.3 24.3
S. typhi 57 10.7 18 23.3 8.7 13 17.3 23.3
V. cholerae 824 10.3 12.7 17 7.3 10.7 14.3 22.3
S. dysenteriae ATCC C 3 9.3 14 17.7 7 11.3 13 25.3
E. coli NCTC 8196 9 13.7 16.3 7.3 10.7 13 21
Fungi              
P. notatum ATCC 11625 8.3 12.3 15.7 7.3 11 14.7 20.3
A. niger AB 41 8.3 13 18 7.7 11.7 13.7 23.7
C. albicans ATCC 18804 12.3 18 22.3 7.7 12.3 15.7 28.3

Table 1: Zone of inhibition of various fractions of ethanol extract of b. Monnieri aerial parts

micro-Organisms EAF NBF
Gram-positive bacteria    
S. aureus ATCC 25923 50 100
B. subtilis UC 564 25 200
B. polymexia 474 50 100
Streptococcus faecalis 100 400
Gram-negative bacteria    
P. aeruginosa 25619 200 600
S. typhi 57 25 100
V. cholerae 824 100 200
S. dysenteriae ATCC C 3 50 400
E. coli NCTC 8196 100 400
Fungi    
P. notatum ATCC 11625 200 600
A. niger AB 41 100 400
C. albicans ATCC 18804 50 200

Table 2: Zone of inhibition of various fractions of ethanol extract of b. Monnieri aerial parts

Acknowledgements

Authors are thankful to the authority of Botanical Survey of India, Shibpur, Howrah for identification of the plant specimen. The facilities provided by the authorities of Institute of Pharmacy and Technology, Salipur and the authorities of Jadavpur University during the course of study are gratefully acknowledged.

References