ANTIBACTERIAL, INSECTICIDAL AND IN VIVO CYTOTOXICITY ACTIVITIES OF SALIX TETRASPERMA

ANTIBACTERIAL, INSECTICIDAL AND IN VIVO CYTOTOXICITY ACTIVITIES OF SALIX TETRASPERMA

1. Saiful Islam ¹, Ronok Zahan ², Laizuman Nahar ³, M. Badrul Alam ², Marufa Naznin ⁴, Gopal C. Sarkar ⁵, M. A. Mosaddik ⁶ and M. Ekramul Haque*⁶

Department of Pharmacy, International Islamic University ¹, Chittagong, Bangladesh

Department of Pharmacy, Atish Dipankar Unversity of Science & Technology ², Dhaka, Bangladesh

Department of Pharmacy, Southeast University ³, Dhaka, Bangladesh

Department of Environmental Science, Independent University ⁴, Bangladesh

Quality Compliance Department, Square Pharmaceuticals Ltd., ⁵ Pabna, Bangladesh

Department of Pharmacy, University of Rajshahi ⁶, Rajshahi-6205, Bangladesh

ABSTRACT

The present study was designed to investigate antibacterial, insecticidal and in vivo cytotoxic activities of the methanol: ethylacetate (1:9) extract of leaves, barks and roots of Salix tetrasperma. Antibacterial test were done against five Gram-positive and four Gram-negative bacteria. Bark extract was active against all tested microbial species and the highest activity was shown against Escherichia coli with a zone of inhibition 12 ± 0.02 mm. The leaves extract was active against all the Gram positive bacterial strain whereas inactive against all the Gram negative bacterial strain except Shegella flexneri while root extract showed insignificant activity. In insecticidal study, the root and leaves extract of Salix tetrasperma showed moderate activity with 26% and 20% mortality rate of Tribolium castaneum at a dose of 50mg/ml in 48 hours, respectively and bark extract had no activity. In respect of in vivo cytotoxicity, Root and bark extract showed moderate cell growth inhibition, whereas leaves extract had no activity at all.

Salix tetrasperma,

Antibacterial,

Insecticidal,

Ehrlich ascites carcinoma (eac) cells

INTRODUCTION: The history of plants being used for medicinal purpose is probably as old as the history of mankind. Simultaneous with population explosion, virulent strains of microorganisms become more common and their increased attack accounts for increased mortality ¹.

Bangladesh, being a country with high density of population, infectious diseases becomes a great challenge in the health and economic sector. Despite the large numbers of synthetic antibiotics having different chemical nature have been developed in the last few decades, inappropriate and injudicious uses of antibiotics or self treatment practices developed resistance to microbes ². So the developments of new antibacterial agents are necessary to combat the problem of microbial resistance and for substitution with ineffective ones. Moreover, higher plants are rich source of novel natural substances that can be used to develop new antibacterial agent as well as environmental safe methods for insect control ³.

Not only the world wide annual losses of food grains storage caused by insects have been estimated to be about 10% of the world’s production, but losses of 25% or more may also occur in tropical countries through insect attack after harvest ⁴. Control of these insects relies heavily on the use of synthetic insecticides and fumigants, which has led to problem such as disturbances of the environment, increasing cost of application, pest resurgence, resistant to pesticides and lethal effects on non-target organism in addition to direct toxicity to users ⁵. So there is an immediate need to develop safe alternatives with low cost, easy to use and friendly to the environment.

Cancer, the second leading cause of death worldwide next to cardiovascular diseases, is a group of more than 100 different diseases, characterized by uncontrolled cellular growth, local tissue invasion, and distant metastases ⁶ and can be treated with surgery, radiation, chemotherapy, hormone therapy and biological therapy. Chemotherapy is still a major challenge to the cancer patients because less than 1% of injected drug molecules can reach their target cells, whereas the rest may damage healthy cells and tissue ⁷.

Plants have a long history of use in the treatment of cancer. It is noteworthy that a number of plants are known to be the source of useful drugs in modern medicine⁸ and have been accepted currently as one of the main source of cancer chemoprevention drug discovery and development ⁹ due to their diverse pharmacological properties including cytotoxic and cancer chemopreventive effects ¹⁰. Salix tetrasperma Roxburgh. (Family: Salicaceae), generally called Indian Willow ¹¹. Phytochemical studies of this plant resulted in the isolation of several tannins, triterpenes, viz. -amyrin, lupeol ¹² and chalcinasterol ¹³, steroids viz. -sitosterol and stigmasterol ¹⁴.

Various types of sapogenins such as quinovic acid, salicortin, saligenin, phenolic glycosides and pyrocatechol were isolated from the barks and leaves ¹⁵. The active extract of the bark, called salicin ¹⁶ was isolated to its crystalline form. Aqueous extract of dried leaf reported to possess cardiotonic activity and the methanol extract of the dried leaf possess reverse transcriptase inhibition effect ¹⁷. The aqueous extract of the stem bark has been reported to increase testosterone level in rats at 500.0 mg/kg, p.o. ¹⁸ and also accelerates semen coagulation in rats at a concentration of 2%w/v ¹⁹. A dose of 0.094 mg/kg of aerial parts shows hypothermic activity in mice ²⁰. The paste of both leaf and root is applied externally in scorpion stings, bug bites, for sores and warts and the decoction of the dried root is taken orally for the treatment of hepatitis ²¹ and whooping cough in children ²². Based on these reports our studies have been designed to examine whether the methanol: ethyl acetate (1:9) extract of Salix tetrasperma exerts antibacterial, insecticidal and in vivo cytotoxicity activity.

MATERIALS AND METHODS:

Plant materials: Different parts (roots, leaves and barks) of S. tetrasperma were collected from the adjoining area of Rajshahi University Campus, Bangladesh during the month of April 2009 and were identified by Taxonomist, Department of Botany and University of Rajshahi, Bangladesh where a voucher specimen (Voucher No # 35) has been deposited for future references.

Preparation of extracts: The roots, leaves and barks of S. tetrasperma were dried in an oven at 37⁰C and then pulverized into course powder with a mechanical grinder, passing through sieve #40, and stored separately in an air tight container. The dried powdered materials (1.0 kg) each, extracted three times by sonication for 30 min with MeOH: EtOAc (1:9) mixture (1000ml) and then filtered. Each of the filtrate was concentrated to dryness, in vacuum at 40⁰C to render the crude extracts 300 g, 240 g and 320 g respectively.

Antibacterial assay: Sterile 6.0 mm diameter blank discs (BBL, Cocksville, USA) were impregnated with test substances at the dose of 200 µg/disc. This disc, along with standard discs (Ciprofloxacin, Oxoid Ltd, UK) and control discs were placed in petri dishes containing a suitable agar medium seeded with the test organisms using sterile transfer loop and kept at 4⁰C to facilitate maximum diffusion. The plates then kept in an incubator (37^oC) to allow the growth of the bacteria. The antibacterial activities of the test agents were determined by measuring the diameter of the zone of inhibition in terms of millimeter. Antimicrobial activity was tested against Bacillus anthracis, Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonus aeruginosa, Shigella flexneri, and Shigella boydii were obtained from International Centre for Diarrhoeal