Deepak Panwar and Bhatt RP. / Journal of Pharmaceutical Biology, 4(2), 2014, 109-118. Journal of Pharmaceutical Biology www.jpbjournal.com e-ISSN - 2249-7560 Print ISSN - 2249-7579 ANTIBACTERIAL ACTIVITY OF TAGETES MINUTA L. AGAINST STAPHYLOCOCCUS AUREUS AND STREPTOCOCCUS PYOGENES *Deepak Panwar and R.P. Bhatt Department of Botany and Microbiology H.N.B. Garhwal University (A Central University), Srinagar Garhwal, Uttarakhand-246 174, India. ABSTRACT Evaluated the antibacterial effect of aqueous and ethanolic extract of the leaf, bark of Tagetes minuta L. against Staphylococcus aureus and Streptococcus pyogenes was analyzed in this study. These Gram positive strains were isolated and characterized by standard methods. Aqueous and ethanolic extract of the leaf, bark of Tagetes minuta L. were prepared with help of soxhlet unit. Further, evaluated the antimicrobial activity of these extract were analyzed against S. aureus and S. pyogenes. Biochemical test confirmed that isolated strains were Staphylococcus aureus and Streptococcus pyogenes. Aqueous and ethanolic extract of the leaf and bark of Tagetes minuta leaf showed significant antibacterial activity against Staphylococcus aureus and Streptococcus pyogens. 15 µg/ml aqueous extract of T. minuta leaf showed 80, 120% antimicrobial activity against Staphylococcus aureus and Streptococcus pyogens respectively as compared to the control. Similarly, ethanolic extract of leaf of T.s minuta showed significant values against S. aureus and S. pyogens. 15 ethanolic extracts of T. minuta leaf showed more inhibition zones (121.21 and 0 %, respectively) against Staphylococcus aureus and Streptococcus pyogens. 15 µg/ml aqueous extract of bark of T. minuta showed less inhibition zone against Staphylococcus aureus as compared to 15 µg/ml norflox (control drug) whereas 35 µg/ml aqueous extract of bark of T. minuta exhibited more inhibition zone (49.59%) against S. pyogens as compared to the control. Similarly, 20 µg/ml ethanolic extract T. minuta bark marked similar inhibition zone against S. aureus and similarly 20 µg/ml ethanolic extract T. minuta bark showed 50.31% more inhibition zone against S. pyogens as compared to 15 µg/ml norflox (control drug). Aqueous extract of the leaves as well as barks of Tagetes minuta exhibited 5 µg/ml MIC against S. aureus and S. pyogenes. Keywords: MIC, MBC, Tagetes minuta, Staphylococcus aureus, Streptococcus pyogenes. INTRODUCTION Medicinal plants are a source of great economic adults and children, with an estimated 3.5 million deaths value in the Indian subcontinent. It is Ayurveda, the worldwide in 2008 [4]. The human upper respiratory tract foundation of medicinal science of Hindu culture, in its is the reservoir of a diverse community of commensals eight division deals with specific properties of drugs and and potential pathogens (pathobionts), including various aspects of science of life and the art of healing Streptococcus pyogenes, Streptococcus pneumoniae, [1]. In India thousands of species are known to have Haemophilus influenzae, Moraxella catarrhalis, and medicinal value and the use of different parts of several Staphylococcus aureus [5, 6] which occasionally turn into medicinal plants to cure specific ailments has been in pathogens causing infectious diseases. This respiratory vogue since ancient times [2]. infection effects on major population of world wide. Respiratory tract infection are divided into the Rapid division of bacterial cells causes them to upper respiratory tract (nasal passages) prior to evolve resistance to most treatments rather quickly and dissemination to the lower respiratory tract (airways and converted into resistance [7]. Continuous use of drug lungs) [3]. This respiratory infections are common in both makes the micro-organisms into multi drug resistant hospital and community. The acute respiratory infections (MDR). The increasing prevalence of multidrug resistant remain one of the most important causes of death in both strains of bacteria and the recent appearance of strains Corresponding Author:- Deepak Panwar Email:- [email protected] 109 | P a g e Deepak Panwar and Bhatt RP. / Journal of Pharmaceutical Biology, 4(2), 2014, 109-118. with reduced susceptibility to antibiotics raises the spectre 24 hrs and examine the colonies for their size, of untreatable bacterial infections and adds urgency to the morphology and haemolysis. search for new infection-fighting strategies [8]. In addition to this problem, antibiotics are sometimes Characterization of pathogens: Pathogens were associated with adverse effects on the host including characterized on the basis of Biochemical tests [19]. hypersensitivity, immune-suppression and allergic reactions [9]. Preparation of aqueous extraction: Approx. 30 grams Use of antibiotics is not safe so scientists are of dried powder of medicinal plant were transferred into more focus on alternative. There is a continuous and soxhlet unit. Extract was done at 95oC for 24 hours. urgent need to discover new antimicrobial compounds Bottom of soxhlet extraction unit contains plant extract with diverse chemical structures and novel mechanisms of which was filtered through 8 layers of muslin cloth and action because there has been an alarming increase in the then stored at 4 ºC. incidence of new and re-emerging infectious diseases [10]. Plants are the richest resource of drugs of traditional Preparation of ethanol extraction: Approx. 30 grams of systems of medicine, modern medicines, nutraceuticals, dried powder of medicinal plant were transferred into food supplements, folk medicines, pharmaceutical soxhlet unit. Extract was done at 45oC for 72 hours. intermediates and chemical entities for synthetic drugs Bottom of soxhlet extraction unit contains plant extract [11]. which was filtered through 8 layers of muslin cloth and Wild marigold (Tagetes minuta L., syn. T. then stored at 4 ºC. glandulifera, familia Asteraceae) is native to southern South America. Introduced to Europe, Asia, Africa, Preparation of different concentration: The extracts Madagascar, India, Australia, Hawaii [12, 13]. Wild were sieved through a fine mesh cloth and sterilized using marigold - Tagetes minuta L. is an annual, strongly a membrane filter (0.45-micron sterile filter). This extract aromatic herb with height of 1-2 meter and stem of was considered as the 100% concentration of the extract Tagetes minuta is glabrous erect, branched and furrowed [20]. The concentrations such as 15, 20, 25, 30, 35 µg/ml [13]. The Leaves of Tagetes minuta are opposite, were prepared and norflox 15 µg/ml worked as control pinnately parted but the upper leaves are alternate and drug. length of leaf varies from 4 to 8 cm as well as width is varies from 3 to 4.5 cm [14]. The essential oil of this Sterilization of extract: The dried extracts were exposed plant, known commercially as “Tagetes oil”, has to ultra violet light (UV rays for 24 h to sterilize [21]. applications in food production, including the preparation Liquid extracts were sterilized using a membrane filter of alcoholic beverages, cola and frozen dairy desserts, as (0.45-micron sterile filter). well as sweets, jellies, puddings and spices [15]. Anti-tick properties of the essential oil of Tagetes minuta L. Sterility Test: The sterility was checked by streaking the (Asteraceae: Asterales) has been examined against extracts on nutrient agar plate and incubated at 37° C for Hyalomma rufipes ticks [16]. T. minuta showed 24 h. It was confirmed that there were no artifacts to antifungal activity against Fusarium oxysporum [17] and contaminate the sensitivity testing [21]. 700 and 800 ppm of the Tagetes oil inhibit 67% of growth of fungus Ascosphaera apis [18]. Therefore, this study Antibacterial Activity by disc diffusion method and was carried out to evaluate antibacterial activity of agar well diffusion method: The microorganism was aqueous and ethanolic extract of leaf as well as bark of activated by inoculating a loopful of the strain in the the Tagetes minuta L. against Staphylococcus aureus and nutrient broth (30 ml) and incubated on a rotary shaker. Streptococcus pyogenes. Then 0.2 ml of inoculum (inoculum size was 108 cells/ml as per McFarland standard) was inoculated into the MATERIALS AND METHODS molten Muller Hinton agar media and after proper Isolation of microorganisms: Sometimes the sample homogenization it was poured into the Petri plate. contains Gram-negative bacteria in the sample or the population of Gram-positive bacteria is low. In that case, For agar disc diffusion method, the test compound (0.1 it is very difficult to isolate the Gram-positive bacteria. ml) was introduced on the disc (0.7 cm) and then allowed Different types of selective media are used to isolate the to dry. Then the disc was impregnated on the seeded agar Gram-positive bacteria. Different types of media such as plate. The plates were incubated at 37 ºC for 24 h. Phenylethanol Agar medium and Columbia CNA Agar Microbial growth was determined by measuring the medium and Sodium azide (NaN3) blood agar medium are diameter of zone of inhibition. For each bacterial strain, preferred for isolation of gram positive bacteria. Swab controls were maintained in which pure solvents were samples were collected from the mouth of Patients and used instead of the extract. The control zones were streaked on the agar medium and incubated at 35±1oC for subtracted from the test zones and the resulting zone 110 | P a g e Deepak Panwar and Bhatt RP. / Journal of Pharmaceutical Biology, 4(2), 2014, 109-118. diameter is shown in the graph. The experiment was done raffinose, inulin but showed fermentation mannitol, three times and the mean values are presented [22]. sucrose, lactose, trehalose, starch (Table 1). All the Staphylococcus aureus strains had been tried to grow Antibacterial Activity by serial dilution in tubes: Dry under aerobic as well as anaerobic condition. They the extract of medicinal plant. This powder of medicinal respond positively for VP test, coagulase test, plant was dissolved in sterilized Mueller-Hinton broth and phosphatase test, nitrate test, arginine test, urea test, sterilized by membrane filter method. Various protease test but not produce oxidase.