*Corresponding Author:
Indu George
Department of Life Sciences, University of Mumbai, Vidyanagari campus, Kalina, Santa Cruz (E), Mumbai-400 098, India
E-mail: [email protected]
Date of Submission 14 February 2005
Date of Revision 23 May 2005
Date of Acceptance 17 March 2006
Indian J Pharm Sci, 2006, 68 (2): 245-247  

Abstract

The in vitro antibacterial activity of different extracts of Cissus quadrangularis Linn (Vitaceae) against some Gram-negative and Gram-positive bacteria, were investigated. The methanol and ethyl acetate extract showed high activity against the bacteria tested.

Cissus quadrangularis Linn (Family: Vitaceae) is a perennial climber, found mostly in the hotter parts of the world such as India, Sri Lanka, Tropical Africa, South Africa, Thailand, Java, and Philippines [1]. The plant is mentioned in the ancient systems of medicine such as Ayurveda, and is useful for treatment of bloody diarrhoea, skin disorders, earache [2], haemorrhoids, irregular menstruation, and accelerates healing of bone fracture [3].

Murthy et al. [4] have previously reported the antimicrobial effects of the plant, along with the values of zones of inhibition. Here, we report the minimum inhibitory concentration (MIC) values of the different extracts of the plant stem using the microtitre plate method [5,6], which is relatively simple, reproducible, and rapid.

The stems of Cissus quadrangularis, collected from the plants maintained in the greenhouse were washed with water, cleaned, cut into small sections, and dried in an oven at 40° for 10-12 d. The dried material was ground to a fine powder in a homogeniser. The powder was stored in a refrigerator.

Extraction was carried out using a Soxhlet apparatus. Ethyl acetate, acetone, petroleum ether (40-60o), methanol (HPLC grade, Sisco Research Laboratories, Mumbai), ethanol, and water were the solvents used for extraction. The extracts were evaporated to obtain dry residues, and then solutions of known concentrations were prepared in respective solvents.

Bacillus subtilis (ATCC No. 6633), Escherichia coli (ATCC No. 25922), Pseudomonas aeruginosa (ATCC No. 27853), and Staphylococcus aureus (ATCC No. 25923), were obtained from National Centre for Industrial Microorganisms, National Chemical Laboratory, Pune. Clinical samples of Salmonella typhi and Streptococcus pyogenes were obtained from the cultures maintained in the department.

The bioassays were performed in 96-well sterile microtitre plates (Nunclon, Denmark). All the 96 wells of the plate were filled with 50 μl of sterile nutrient broth (HiMedia Laboratories). A 50 μl aliquot of the extract was then added to row A, and a 50% serial dilution was achieved (row A to H) by transferring 50 μl at a time to subsequent wells. In all, there were four controls; sterility control: 50 μl nutrient broth only, growth control: 50 μl nutrient broth + 50 μl culture, ampicillin control: 50 μl nutrient broth + 50 μl culture + 250 μg/ml ampicillin, solvent control: 50 μl nutrient broth + 50 μl culture + 50 μl solvent.

An aliquot of a 3 h culture (50 μl) of the test organism in nutrient broth was added in each well, except in the sterility control well. The covered microplates were incubated for 24 h at 37°. Bacterial growth was detected by adding 50 μl of 0.2 mg/ml solution of 2[4-iodophenyl]3[4-nitrophenyl]-5-phenyl-2H-tetrazolium chloride (INT, from SRL) in each test well, and the plate was incubated further for at least 30 min at 37°, to ensure adequate colour development. The lowest concentration in which there was a definite decrease in colour was taken as the MIC of that extract, for that particular organism. All the experiments were performed in triplicates. The results obtained, were subjected to ANOVA and critical difference analysis (CD analysis), at 5% level of significance.

The MIC values of different extracts against all the organisms tested, are detailed in Table 1. The ethyl acetate, acetone, and methanol extracts, showed antimicrobial properties and that B. subtilis, P. aeruginosa,
S. typhi, S. aureus, and S.pyogenes, were susceptible to at least two extracts. Petroleum ether, ethanol, and water extracts, failed to inhibit the bacterial growth of the strains tested. E.coli did not respond to any of the extracts used.

Extract MIC values in mg/ml
E. coli B. subtilis P. aeruginosa S. typhi S. aureus S. pyogenes
Petroleum ether - - - - - -
Ethyl acetate - 0.93 1.87 3.75 0.93 3.75
Acetone - - 3.125 6.25 1.56 -
Methanol - 0.465 3.12 1.24 0.465 0.93
Ethanol - - - - - -
Water - - - - - -

Table 1: Mic values of the test organisms against various extracts

ANOVA was carried out to test the significance of difference between the antimicrobial properties of the extracts (ethyl acetate, acetone and methanol), and difference between the susceptibility of microorganisms
(P. aeruginosa, S. typhi, S. aureus). The analysis showed that there is a significant difference between the extracts (F value=8.84; F critical=3.42) and the microorganisms (F value=15.63; F critical=3.42).

Further, CD analysis was carried out to pinpoint the best extract, and the most susceptible microorganism. The analysis revealed that the methanol extract is significantly better than all the other extracts used (difference of means=2.04 and CD value=1.80), and Staphylococcus aureus is significantly more susceptible as compared to the other tested strains (difference of means=2.77 and CD value=1.80). All the tests were carried out, and the decisions were taken at 5% level. Further studies are needed to isolate and characterize the compound(s) responsible for the inhibitory activity.

Acknowledgements

The authors wish to thank Prof. U. S. Bagde, HOD, Department of Life Sciences, University of Mumbai for providing the necessary facilities and encouragement and Prof. G. K. Rao for his help in the statistical analysis of the results mentioned in the communication.

References