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 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 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]

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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 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 ) is native to southern . 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 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 [15]. Anti-tick properties of the essential oil of Tagetes minuta L. Sterility Test: The sterility was checked by streaking the (Asteraceae: ) 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

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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. All the strains concentration of medicinal plants such as 1, 2, 3, 4, 5, 10, produced acid from different sugar such as lactose, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 μg/ml maltose, mannitol, fructose, sucrose, trehalose, mannose were prepared. The tubes were inoculated with 20 μL of but did not utilize xylose, cellobiose, raffinose (Table 2). the bacteria suspension per ml of broth, homogenised and Aqueous and ethanolic extract of the leaf and bark of incubated at 37ºC for 24 hours. After incubation, 50 μL Tagetes minuta leaf showed significant antibacterial were taken from each tube and inoculated in a second activity against Staphylococcus aureus and Streptococcus tube containing 1 mL of sterile Mueller-Hinton broth, pyogens. 15 µg/ml aqueous extract of T. minuta leaf homogenised and incubated for another 24 hours at 37ºC. showed 80% antimicrobial activity against The Minimal Inhibitory Concentration (MIC) was Staphylococcus aureus as compared to 15 µg/ml norflox determined as the lowest concentration of medicinal plant (control drug) but highest inhibition zone against for growth was observed in second set of tubes. The Staphylococcus aureus was observed in 35 µg/ml aqueous Minimal Bactericidal Concentration (MBC) was extract of T. minuta leaf which was 180% as compared to determined as the lowest concentration of medicinal plant the control. Similarly, 15 µg/ml aqueous extract of T. for which no growth was observed in the second set of minuta leaf showed 120% more antimicrobial activity tubes [2]. against Streptococcus pyogens as compared to 15 µg/ml norflox (control drug) and 35µg/ml aqueous extract of T. RESULTS minuta leaf showed more inhibition zone by 200%, The pathogenic samples had been collected from respectively against Streptococcus pyogens as compared the hospitals at haridwar, Uttarakhand, with sterilized to the control (Table 3a). cotton swab and transferred on three selective media such Similarly, ethanolic extract of leaf of Tagetes as Phenylethanol Agar medium and Columbia CNA Agar minuta showed significant values against Staphylococcus medium and Sodium azide (NaN3) blood agar media for aureus and Streptococcus pyogens. 15 and 35 µg/ml the cultivation and isolation of gram positive bacteria. ethanolic extracts of T. minuta leaf showed more The isolated Gram positive bacteria had been purified on inhibition zones (121.21 and 202%, respectively) against the same medium by streaking method. Gram staining of Staphylococcus aureus as compared to 15 µg/ml norflox these cultures represent the isolated strains in purple (control drug). Similarly, 15, 20, 25, 30 and 35 µg/ml colour and cocci in cluster or chains. Gram +ve cocci in ethanolic extract of T. minuta leaf showed more inhibition cluster strains had been marked as SAD-1, SAD-2, SAD- zone against Streptococcus pyogens (0, 64.51, 80.64, 3, SAD-4, SAD-5, SAD-6, SAD-7, SAD-8, SAD-9, SAD- 96.77, 120.96%, respectively) as compared to 15 µg/ml 10, SAD-11, SAD-12, SAD-13, SAD-14, SAD-15, SAD- norflox (control drug) (Table 3b). 16, SAD-17, SAD-18, SAD-19 and SAD-20 whereas Aqueous extract of the bark of Tagetes minuta Gram +ve cocci in chain named as SPD-1, SPD-2, SPD-3, represented significant antibacterial activity. 15 µg/ml SPD-4, SPD-5, SPD-6, SPD-7, SPD-8, SPD-9, SPD-10, aqueous extract of bark of T. minuta showed less SPD-11, SPD-12, SPD-13, SPD-14, SPD-15, SPD-16, inhibition zone against Staphylococcus aureus as SPD-17, SPD-18, SPD-19 and SPD-20. compared to 15 µg/ml norflox (control drug) but 20 µg/ml The isolated strains were characterized on the aqueous extract of bark of T. minuta showed 32% more basis of the biochemical tests performed in the laboratory. inhibition zone against Staphylococcus aureus as All the Streptococcus pyogenes strains showed beta- compared to the control. The 15, 20 and 25 µg/ml haemolysis i.e. clear zone around the colony. All aqueous extract of bark of T. minuta did not show more Streptococcus pyogenes strains failed to grow at 45oC, antimicrobial activity against Streptococcus pyogens as 6.5% NaCl broth, 40% bile agar and Bile-aesculine compared to 15 µg/ml norflox (control drug) whereas 35 medium. Further, all Streptococcus pyogenes strains µg/ml aqueous extract of bark of T. minuta exhibited negative results for VP test, pyridoxal or cysteine more inhibition zone (49.59%) against Streptococcus dependence, H2O2 production test, motility and yellow pyogens as compared to the control (Table 4a). pigmentation test. All the strains represent positive tests Similarly, ethanolic extract Tagetes minuta bark in Pyrrolydonylarylamidase, Phosphatase, aesculine showed significant values against Staphylococcus aureus hydrolysis and starch hydrolysis tests. All these strains and Streptococcus pyogens. 20 µg/ml ethanolic extract were sensitive to bacitracin (0.1 unit). None of these Tagetes minuta bark marked similar inhibition zone fermented pyruvate, ribose, arabinose, sorbitol, adonitol, against Staphylococcus aureus as compared to 15 µg/ml

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norflox (control drug). Similarly, 15, 20, 25, 30 and 35 leaves as well as barks of Tagetes minuta exhibited µg/ml ethanolic extract T. minuta bark showed more showed variation in the MIC values i.e. ethanolic extracts inhibition zone against Streptococcus pyogens (0, 50.31, of the leaves of Tagetes minuta had 5 µg/ml MIC but 62.89, 88.05 and 125.78%, respectively) as compared to other ethanolic extract showed 10 µg/ml MIC. Further, 15 µg/ml norflox (control drug) (Table 4b). Aqueous aqueous extract of the leaves as well as barks of Tagetes extract of the leaves as well as barks of Tagetes minuta minuta showed variation MBC against Staphylococcus exhibited 5 µg/ml MIC against Staphylococcus aureus aureus and Streptococcus pyogenes. The MBC and Streptococcus pyogenes. But ethanolic extract of the concentrations vary from 10 to 30 µg/ml (Table 5).

Table 1. Biochemical characterization of Streptococcus pyogenes. Haemolysis (β); Growth at 45oC (-ve); Growth in 6.5% NaCl broth (-ve); Growth on 40% bile agar (-ve); Bile-aesculine test (-ve); VP test; Pyrrolydonylarylamidase (+ve) ; Phosphatase (+ve) ; Pyridoxal or cysteine dependence (-ve); Hydrolysis of Aesculine (+ve) and starch (+ve); Sensitive to bacitracin (0.1 unit) (+ve); H2O2 production (-ve); Fermentation of Pyruvate (-ve), Ribose (-ve), Arabinose (-ve), Mannitol (+ve), Sorbitol (-ve), Adonitol (-ve), Sucrose (+ve), Lactose (+ve), Trehalose (+ve), Raffinose (-ve), Inulin (-ve), Starch (+ve), Non-motility, Yellow pigment (No).

Table 2. Biochemical characterization of Staphylococcus aureus strains. Growth anaerobically (+ve); Oxidase (-ve); VP (+ve); Coagulase (+ve); Acid from Lactose (+ve), Maltose (+ve), Mannitol (+ve), Fructose (+ve), Sucrose (+ve), Trehalose (+ve), Xylose (-ve), Cellobiose (-ve), Raffinose (-ve), Mannose (+ve); Phosphatase (+ve); Nitrate (+ve); Arginine (+ve); Urea (+ve) and Protease (+ve).

Table 3a. Effect of aqueous extract of leaf of Tagetes minuta against Staphylococcus aureus and Streptococcus pyogens Inhibition zone (mm) Pathogen Medicinal plant control 15µg/ml 20µg/ml 25µg/ml 30µg/ml 35 µg/ml 15µg/ml SAD-1 17 21 23 25 27 9 SAD-2 18 22 24 26 28 10 SAD-3 18 22 24 26 28 10 SAD-4 18 22 24 26 28 10 SAD-5 19 23 25 27 29 11 SAD-6 18 22 24 26 28 10 SAD-7 18 22 24 26 28 10 SAD-8 18 22 24 26 28 10 SAD-9 19 23 25 27 29 11 SAD-10 17 21 23 25 27 9 SAD-11 17 21 23 25 27 9 SAD-12 18 22 24 26 28 10 SAD-13 18 22 24 26 28 10 SAD-14 18 22 24 26 28 10 SAD-15 19 23 25 27 29 11 SAD-16 18 22 24 26 28 10 SAD-17 18 22 24 26 28 10 SAD-18 18 22 24 26 28 10 SAD-19 19 23 25 27 29 11 SAD-20 17 21 23 25 27 9 Average 18 22 24 26 28 10 SD 0.632456 0.632456 0.632456 0.632456 0.632456 0.632456 SPD-1 22 24 26 28 30 10 SPD-2 22 24 27 28 30 10 SPD-3 22 24 26 28 30 10 SPD-4 22 24 26 28 30 10 SPD-5 23 25 27 29 31 11 SPD-6 21 23 25 27 29 9

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SPD-7 22 24 26 28 30 10 SPD-8 21 23 25 27 29 9 SPD-9 22 24 26 28 30 10 SPD-10 23 25 27 29 31 11 SPD-11 22 24 26 28 30 10 SPD-12 22 24 26 28 30 10 SPD-13 22 24 26 28 30 10 SPD-14 22 24 26 28 30 10 SPD-15 22 24 26 28 30 10 SPD-16 23 25 27 29 31 11 SPD-17 22 24 26 28 30 10 SPD-18 22 24 26 28 30 10 SPD-19 22 24 26 28 30 10 SPD-20 21 23 25 27 29 9 Average 22 24 26.05 28 30 10 SD 0.547723 0.547723 0.589491 0.547723 0.547723 0.547723 SAD = Staphylococcus aureus; SPD= Streptococcus pyogens.

Table 3b. Effect of ethanolic extract of leaf of Tagetes minuta against Staphylococcus aureus and Streptococcus pyogens Inhibition zone (mm) Pathogen Medicinal plant control 15µg/ml 20µg/ml 25µg/ml 30µg/ml 35 µg/ml 15µg/ml SAD-1 22 24 26 28 30 10 SAD-2 22 24 26 28 30 10 SAD-3 23 25 27 29 31 11 SAD-4 21 23 25 27 29 9 SAD-5 22 24 26 28 30 10 SAD-6 22 24 26 28 30 10 SAD-7 22 24 26 28 30 10 SAD-8 22 24 26 28 30 10 SAD-9 21 23 25 27 29 9 SAD-10 21 23 25 27 29 9 SAD-11 22 24 26 28 30 10 SAD-12 22 24 26 28 30 10 SAD-13 22 24 26 28 30 10 SAD-14 23 25 27 29 31 11 SAD-15 23 25 27 29 31 11 SAD-16 22 24 26 28 30 10 SAD-17 21 23 25 27 29 9 SAD-18 22 24 26 28 30 10 SAD-19 22 24 26 28 30 10 SAD-20 21 23 25 27 29 9 Average 21.9 23.9 25.9 27.9 29.9 9.9 SD 0.6245 0.6245 0.6245 0.6245 0.6245 0.6245 SPD-1 14 22 24 26 29 14 SPD-2 12 20 22 24 27 12 SPD-3 13 21 23 25 28 13 SPD-4 12 20 22 24 27 12 SPD-5 12 20 22 24 27 12 SPD-6 12 20 22 24 27 12 SPD-7 13 21 23 25 28 13 SPD-8 12 20 22 24 27 12 SPD-9 12 20 22 24 27 12

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SPD-10 12 20 22 24 27 12 SPD-11 12 20 22 24 27 12 SPD-12 12 20 22 24 27 12 SPD-13 12 20 22 24 27 12 SPD-14 13 21 23 25 28 13 SPD-15 14 22 24 26 29 14 SPD-16 12 20 22 24 27 12 SPD-17 13 21 23 25 28 13 SPD-18 12 20 22 24 27 12 SPD-19 12 20 22 24 27 12 SPD-20 12 20 22 24 27 12 Average 12.4 20.4 22.4 24.4 27.4 12.4 SD 0.663325 0.663325 0.663325 0.663325 0.663325 0.663325 SAD = Staphylococcus aureus; SPD= Streptococcus pyogens.

Table 4a. Effect of aqueous extract of bark of Tagetes minuta against Staphylococcus aureus and Streptococcus pyogens Inhibition zone (mm) Pathogen Medicinal plant control 15µg/ml 20µg/ml 25µg/ml 30µg/ml 35 µg/ml 15µg/ml SAD-1 3 8 11 12 14 6 SAD-2 3 8 11 12 14 6 SAD-3 3 8 11 12 14 6 SAD-4 3 8 11 12 14 6 SAD-5 4 9 12 13 15 7 SAD-6 3 8 11 12 14 6 SAD-7 3 8 11 12 14 6 SAD-8 3 8 11 12 14 6 SAD-9 3 8 11 12 14 6 SAD-10 4 9 12 13 15 7 SAD-11 3 8 11 12 14 6 SAD-12 3 8 11 12 14 6 SAD-13 3 8 11 12 14 6 SAD-14 3 8 11 12 14 6 SAD-15 3 8 11 12 14 6 SAD-16 3 8 11 12 14 6 SAD-17 3 8 11 12 14 6 SAD-18 4 9 12 13 15 7 SAD-19 4 9 12 13 15 7 SAD-20 4 9 12 13 15 7 Average 3.25 8.25 11.25 12.25 14.25 6.25 SD 0.433013 0.433013 0.433013 0.433013 0.433013 0.433013 SPD-1 7 10 12 16 19 13 SPD-2 8 9 12 15 18 12 SPD-3 7 10 12 16 19 13 SPD-4 8 9 12 15 18 12 SPD-5 8 9 12 15 18 12 SPD-6 9 11 13 17 20 13 SPD-7 8 9 12 15 18 12 SPD-8 8 9 12 15 18 12 SPD-9 7 10 12 16 19 13 SPD-10 8 9 12 15 18 12 SPD-11 9 11 13 17 20 13 SPD-12 8 9 12 15 18 12

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SPD-13 8 9 12 15 18 12 SPD-14 8 9 12 15 18 12 SPD-15 8 9 12 15 18 12 SPD-16 8 9 12 15 18 12 SPD-17 8 9 12 15 18 12 SPD-18 8 9 12 15 18 12 SPD-19 8 9 12 15 18 12 SPD-20 7 10 12 16 19 13 Average 7.9 9.4 12.1 15.4 18.4 12.3 SD 0.538516 0.663325 0.3 0.663325 0.663325 0.458258 SAD = Staphylococcus aureus; SPD= Streptococcus pyogens

Table 4b. Effect of ethanolic extract of bark of Tagetes minuta against Staphylococcus aureus and Streptococcus pyogens Inhibition zone (mm) Pathogen Medicinal plant control 15µg/ml 20µg/ml 25µg/ml 30µg/ml 35 µg/ml 15µg/ml SAD-1 4 5 8 10 12 5 SAD-2 4 5 8 10 12 5 SAD-3 4 5 8 10 12 5 SAD-4 5 6 9 11 13 6 SAD-5 4 5 8 10 12 5 SAD-6 4 5 8 10 12 5 SAD-7 5 6 9 11 13 6 SAD-8 4 5 8 10 12 5 SAD-9 4 5 8 10 12 5 SAD-10 5 6 9 11 13 6 SAD-11 4 5 8 10 12 5 SAD-12 4 5 8 10 12 5 SAD-13 4 5 8 10 12 5 SAD-14 4 5 8 10 12 5 SAD-15 5 6 9 11 13 6 SAD-16 4 5 8 10 12 5 SAD-17 5 6 9 11 13 6 SAD-18 4 5 8 10 12 5 SAD-19 4 5 8 10 12 5 SAD-20 4 5 8 10 12 5 Average 4.25 5.25 8.25 10.25 12.25 5.25 SD 0.433013 0.433013 0.433013 0.433013 0.433013 0.433013 SPD-1 7 11 12 14 17 7 SPD-2 8 12 13 15 18 8 SPD-3 8 12 13 15 18 8 SPD-4 7 11 12 14 17 7 SPD-5 8 12 13 15 18 8 SPD-6 9 13 14 16 19 9 SPD-7 9 13 14 16 19 9 SPD-8 9 13 14 16 19 9 SPD-9 8 12 13 15 18 8 SPD-10 8 12 13 15 18 8 SPD-11 8 12 13 15 18 8 SPD-12 8 12 13 15 18 8 SPD-13 8 12 13 15 18 8 SPD-14 7 11 12 14 17 7 SPD-15 8 12 13 15 18 8

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SPD-16 8 12 13 15 18 8 SPD-17 7 11 12 14 17 7 SPD-18 8 12 13 15 18 8 SPD-19 8 12 13 15 18 8 SPD-20 8 12 13 15 18 8 Average 7.95 11.95 12.95 14.95 17.95 7.95 SD 0.589491 0.589491 0.589491 0.589491 0.589491 0.589491 SAD = Staphylococcus aureus; SPD= Streptococcus pyogens

Table 5. MIC and MBC of various medicinal against Staphylococcus aureus and streptococcus aureus Antimicrobial activity of plant extract S.No. Plant part Pathogen MIC (µg/ml) MBC (µg/ml) Aqueous Ethanolic Aqueous Ethanolic 1 Leaf Staphylococcus aureus 5 5 10 10 2 Leaf Streptococcus pyogens 5 10 15 30 3 Bark Staphylococcus aureus 5 10 10 20 4 Bark Streptococcus pyogens 5 10 15 25

DISCUSSION AND CONCLUSION Through a series of experiments carried out uniformly positive [26]. In other report, purified S. aureus during the present investigation, Staphylococcus aureus isolates through different biochemical tests such as and Streptococcus pyogenes have been established that catalase, coagulase (tube test), oxidation- fermentation, pathogenic gram positive cocci. These were isolated detection of haemolysin and mannitol fermentation tests forms as pathogen in the respiratory tract of human [27]. Further, isolated Streptococcus pyogenes strains beings. Phenylethanol, Columbia CNA and Sodium azide represent positive tests in Pyrrolydonylarylamidase, (NaN3) blood agar mediums had been used the isolation Phosphatase, aesculine hydrolysis and starch hydrolysis of gram positive bacteria. All isolated Gram positive tests but negative results for VP test, pyridoxal or cysteine strains were cocci and arrangement confirms the dependence, H2O2 production test, motility and yellow i.e. cocci in cluster form suggested that isolated strains pigmentation test. Streptococcus pyogenes has been belongs to genus Staphylococcus and chain arrangement reported as an another important human pathogen. On the confirm that genus was Streptococcus. Our isolated basis of biochemical tests performed on 172 clinical Staphylococcus aureus respond positively for VP test, isolates of group A, C, F, or G "nongroupable" beta- coagulase test, phosphatase test, nitrate test, arginine test, hemolytic streptococci [28]. Further, previously, isolated urea test, protease test but not produce oxidase. Similar Streptococcus pyogenes and confirmed by biochemical studies have confirmed that genus, Staphylococcus is tests [29]. Similar experiment had been conducted and gram positive [23]. For this purpose they had been isolated the Streptococcus pyogenes and characterized on performed Gram staining as well as other various the basis of the biochemical tests such as production of biochemical tests including Catalase (+ve), Oxidase (-ve), pyrrolidonyl arylamidase (+ve); voges proskauer reaction Indole (-ve), Methyl red (+ve), Voges-Proskauer (+ve), (-ve), hydrolysis of starch (-ve) and acid formation tests Nitrate reduction (+ve), and the test of acid from different [30]. Further, gram staining, morphological and sugars (glucose, mannitol, malyose, lactose, sucrose), biochemical tests have confirmed that the isolated strains including haemolysis on Sheep Blood Agar (SBA) (+ve) comparise Staphylococcus aureus and Streptococcus and Coagulase tests (+ve) [23]. Similarly, isolated clinical pyogenes. The studies performed by all the above authors S. aureus strains characterized by the standard also suggest and supports our finding that the isolated biochemical tests whereas all S. aureus strains represented strains were Staphylococcus aureus and Streptococcus urea hydrolysis activity and fermentation tests, out of pyogens. these only 35% isolates confirm protease activity for Available literatures and their studies also identification of Staphylococcus aureus [24]. confirm that these two gram positive bacteria cause Microbiological methods like colonial morphology, Gram various types of diseases in human beings. It is also staining as well as catalase and coagulase tests has evident from various studies that the use of antibiotics is applied [25]. not safe for human beings because it develops Multi Drug Previously, 78 strains of S. aureus had been Resistant pathogens. Therefore, scientists have been more reported and the strains had also been tested for focused on the use of alternative medicines. The plants coauglase, acetoin production from glucose, novobiocin are the richest resource of the drugs of traditional systems sensitivity and mannitol fermentation and fount to be of medicine, modern medicines, nutraceuticals, food

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supplements, folk medicines, pharmaceutical concentration (MIC) and Minimum Bactericidal intermediates and chemical entities for synthetic drugs concentration of aqueous and ethanolic extract of leaf [11]. The medicinal plants exhibit antimicrobial activity as well as bark of Tagetes minuta. Similarly, MIC values against pathogenic micro-organisms [2, 21, 30, 31]. of leaf methanol extract of A. indica against K. Aqueous extract of Tagetes minuta inhibited the pneumonia (22.6 µg/ml), flower extract against E. coli growth and reproduction of Staphylococcus aureus and (MIC: 24.2 µg /ml) and leaf ethyl acetate extract of C. Streptococcus pyogens. 15µg/ml ethanolic extract of the angustifolia against K. pneumoniae (28.4 µg/ml) has been bark of Tagetes minuta did not inhibit the growth of reported [32]. The extract of ethnomedicinal plants such Streptococcus pyogens but same concentration of extract as Selaginella bryopteris (Amarbatooti Sanjivini), retard the growth of Staphylococcusaureus by 121% as Lycopodium flexuousm (Kalijar), Adiantum philippense compared to control (norflox 15µg/ml). Similarly, other (Kalijhant), Drypteris eochleata (Jatashankari) and reports state that the leaves and flowers of many plants Tectacria coadunat(Jatamasi) exhibited antimicrobial such as Aristolochiaindica, Cassia angustifolia, leaves of activities against one or more of the tested Catharanthus roseus, Diospyros melanoxylon, Dolichos microorganisms and also mentioned that the extract biflorus, Gymnema sylvestre and Justicia procumbens concentrations ranges from 25 μg/ml to 2 mg/ml or even represent antimicrobial activity against many bacteria 40 mg/ml [33]. The investigator had isolated two gram such as Bacillus cereus, Aeromonas hydrophila, positive bacterial strains i.e. Staphylococcus aureus and Enterobacter aerogenes, Escherichia coli and Streptococcus pyogens. During the tenure of the present Klebsiellapneumoniae [32]. research, angiospermic plant - Tagetes minuta had been Further, we evaluated that Minimum inhibitory found to inhibit the growth of the isolated pathogens.

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