ANTIMICROBIAL INVESTIGATION OF PIPER NIGRUM L . AGAINST SALMONELLA TYPHI

Medicinal plant is any plant which, in one or more of its organs, contains substances that can be used for therapeutic purposes, or which are precursors for chemopharmaceutical semi-synthesis. Medicinal plants are a source of great medicinal value all over the world. The increasing prevalence of multidrug resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics raises the spectra of untreatable bacterial infections and adds urgency to the search for new infection-fighting and safe strategies .


INTRODUCTION
Medicinal plant is any plant which, in one or more of its organs, contains substances that can be used for therapeutic purposes, or which are precursors for chemopharmaceutical semi-synthesis 1 . Medicinal plants are a source of great medicinal value all over the world 2 . The increasing prevalence of multidrug resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics raises the spectra of untreatable bacterial infections and adds urgency to the search for new infection-fighting and safe strategies 3,4 .
Typhoid fever is caused by Salmonella typhi and is transmitted through the fecal-oral route by the consumption of contaminated water and food. Typhoid fever is an important health problem in many developing countries. The estimated global annual incidence of typhoid in 2000 was 21 million patients, with 217,000 deaths 5 .
Black pepper (Piper nigrum L.) is members of botanical family Piperaceae. Black pepper is a perennial climbing vine grown for its berries extensively used as spice and in medicine. Black pepper is native to Malabar and Travancore coast of India. Other than India, it is mainly cultivated in Vietnam, Brazil, Indonesia, Malaysia, Sri Lanka, China, and Thailand. It is cultivated successfully between 200 North and 200 South of equator and 1500 MSL from sea level 6 . The fruits contain 1.0-2.5% volatile oil, 5-9% alkaloids, of which the major ones are piperine, chavicine, piperidine, and piperetine, and a resin 7 . Pharmacological and clinical studies have revealed that piperine has CNS depressant, antipyretic, analgesic, antiinflammatory 8 , hepatoprotective activity 9 and antioxidant 10 . Black pepper inhibits the growth of certain pathogens 11,12 . Previous finding reveled that Black pipper has good medicinal property and no side effect to human. So aim of this study was to evaluate the antimicrobial activity of Piper nigrum L. against Salmonella. 1. Aqueous extracts: 100g dried finely powdered seeds of Piper nigrum L. were infused in distilled water until completely exhausted. The extract was then filtered using Whatman No. 1 filter paper and the filtrate was evaporated and dried using rotary evaporator at 60°C. The final dried samples were stored at low temperature.

Ethanol extracts:
Dried seeds were grounded and extracted in a percolator with 95% ethanol. About 10ml of ethanol per gram of sample was used. The ethanol extract was dried under a reduced pressure at 40°C. The dried extract was stored in sterile bottles for further use.

Chloroform extracts:
Powdered sample (100g) of seeds were extracted with chloroform using a soxhlet extractor for continuously 10 h or until the used solvent turned pure and colorless. The solvent was removed by evaporation at 40°C to give a concentrated extract, which was then frozen and freeze-dried until further used.

Sterilization and preparation of different concentration of extract:
The dried extracts were exposed to ultra violet light (UV) rays for 24h to sterilize 14 . Liquid extracts were sterilized using a membrane filter (0.45-micron sterile filter). Dry powder extracts were initially dissolved in 1ml of dimethyl sulfoxide (DMSO). Different dilution of extract was prepared. Norfloxacin antibiotic worked as control drug.
Antibacterial activity of Piper nigrum L.: Antibacterial activity was performed according to Deshwal and Vig 15 . The microorganism was activated by inoculating a loopful of the strain in muller hinton broth (30ml) and incubated on a rotary shaker. Then 0.2 ml of inoculum (inoculum size was 10 8 cells/ml as per McFarland standard) was inoculated into the molten Muller Hinton agar media and after proper homogenization it was poured into the sterilized Petri plate. For agar well diffusion method, a well was made in the seeded plates with the help of a sterilized cup-borer. 20μl test compound was introduced into the well and the plates were incubated at 37ºC for 24 h. Microbial growth was determined by measuring the diameter of zone of inhibition. For each bacterial strain, controls were maintained in which pure solvents were used instead of the extract.

RESULTS
10 typhoid patients were selected for present study. Salmonella typhi showed 2-3mm colony, grayish-white, circular, moist, convex colony on blood agar and typical S. typhi surface colonies are black, surrounded by black or brown-black zone with or without a metallic sheen on Bismuth Sulfite Agar medium.   (table 1). Further, three extracts such as aqueous, ethanolic and chloroform extracts of Piper nigrum seeds were prepared. Inhibition zone was increased as increased the concentration of aqueous extract of Piper nigram but 15 mg/ml of ethanolic and chloroform extract showed less inhibition as compared to norfloxacin of same concentration. Maximum inhibition zone was observed in aqueous extract of Piper nigrum as compared to ethanol, chloroform, norfloxacin. Aqueous solution of medicinal plant (30 mg/ml) showed 25.8% more inhibition zone as compared to norfloxacin (30 mg/ml). Similar observation has been shown in ethanol (30 mg/ml), chloroform (30 mg/ml) by 17.2, 9.6% as compared to norfloxacin (30 mg/ml) ( Table 2).  4,12,15,20 . Antibiotics are sometimes associated with adverse effects on the host including hypersensitivity, immune-suppression and allergic reactions 21 . The spices have a unique aroma and flavour which are derived from compounds known as phytochemicals or secondary metabolites 22 . Black pepper (Piper nigrum L.) is used to treat asthma, chronic indigestion, colon toxins, obesity, sinus, congestion, fever 23 .

CONCLUSIONS:
Present study shows that Piper nigrum L. significantly inhibited the growth of Salmonella typhi and it's medicinal value improve its application. Use of antibiotic has side effect to human so it is necessary search for new antimicrobial substance. Medicinal plants are good alternative of chemical antibiotics.