Int.J.Curr.Microbiol.App.Sci (2014) 3(8) 1069-1076

ISSN: 2319-7706 Volume 3 Number 8 (2014) pp. 1069-1076 http://www.ijcmas.com

Original Research Article Antibacterial Activity, Prelimanary Phytochemical Screening and 1Hnmr Analysis of auriculata and Abuliton indicum

P.Subha Devi, V.Santhi*, Anita Kannagi and J.Jeya Shobana

P.G. and Research Department of Zoology, Jayaraj Annapackiam College For Women (Autonomous), Periyakulum, Theni Dist, Tamil Nadu, . *Corresponding author

A B S T R A C T

The present investigation has been undertaken to find out the antibacterial activity of crude and step gradient solvent of methanol, chloroform and benzene in flower and whole of S. auriculata and A. indicum respectively. The extracts were analyzed for antimicrobial activity using agar well diffusion technique against six bacterial human pathogens viz. S. typhi, S.flexneri E.coli, V.cholerae, M. K e y w o r d s tuberculosis, P. fluorescens. To identify the compound responsible for antibacterial activity the most potent extract was subjected to phytochemical and Antibacterial 1HNMR analysis. The growths of M. tuberculosis and V. cholerae were inhibited activity, by the crude methanol extract of S. auriculata and A. indicum respectively. Among inhibitory the step gradient extract maximum inhibition zone was obtained in Methanol 80% zone, and Benzene 20% and methanol 80% and chloroform 20% in the tested pathogens. medicinal Among the tested pathogens M. tuberculosis, E. coli and V. cholerae were found to 1HNMR be the most susceptible pathogens in both the plants. Phytochemical screening of S. auriculata confirmed the presence of alkaloids, flavonoids, triterpenoids and glycosides and in A. indicum alkaloids, , sterols, flavonoids, terpenoids and saponins. The 1H NMR spectrum of crude methanol extract of A. indicum revealed the presence aliphatic methyl (-CH3) protons, aliphatic methylene (-CH2) protons, CH2 X group, alkenic (C-H=C-H) protons and aromatic C-H protons and S. auriculata confirmed the presence of CH3/aliphatic methyl group, aliphatic methylene (CH2 alkenic C-H or N-H or (HC=C-H) and aromatic C H protons.

Introduction

Medicinal plants are gifts of nature to cure medicines is steadily growing with limitless number of diseases among human approximately 40% of population reporting beings [1]. The abundance of plants on the use of herb to treat medical illness within the earth s surface has led to an increasing past year. Phytochemicals have been interest in the investigation of different recognized as the basis for traditional herbal extracts obtained from traditional medicinal medicine practiced in the past and currently plants as potential sources of new en vogue in parts of the world [3]. The antimicrobial agents [2]. The use of herbal medicinal value of plants lies in some

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Int.J.Curr.Microbiol.App.Sci (2014) 3(8) 1069-1076 chemical substances that produce a definite Materials and Methods physiological action on the human body. Phytochemicals are bioactive chemicals of Collection of Plant Materials plant origin. They are regarded as secondary metabolites because the plants that The fresh S. auriculata flowers and the manufacture them may have little need for whole plant of A. indicum were collected in them. The quantity and quality of the month of December 2013. The plants phytochemicals present in plant parts may were collected from Endappuli at differ from one part to another They are Periyakulam Taluk in Theni District. naturally synthesized in all parts of the plant body; , , stem, , flower, , Preparation of Extract seeds, etc. i.e. any part of the plant body may contain active components [4]. The flowers of S.auriculata and the whole Successful determination of biologically plant of A. indicum were washed with tap active compounds from plant material is water and kept in the room temperature for largely dependent on the type of solvent two weeks. The dried samples were ground used in the extraction procedure [4]. This well to give particle size of 50 150 nm. therefore underscores the need to try as Each 50 grams of S. auriculata and much solvents as possible in screening plant A.indicum fine powder was soaked in parts for phytochemicals. The antimicrobial methanol and Benzene and in Methanol and properties of plants have been investigated Chloroform respectively in various by number of researchers worldwide since proportions M: B and M:C viz.100%, 80:20, past few decades antibiotics from microbial 60:40, 40:60, 20:80 and 100% for one week origin and other chemotherapeutic agents . These extract were filtered by WhatMann have been used for control of bacterial No; 1 filter paper. Crude extracts of both the disease. However due to indiscriminate use plants were extracted by using Soxhlet of these drugs various pathogenic bacteria apparatus. These residues were evaporated have developed resistance to many of the to dryness and stored at 4°C for further currently available antibiotics [5] other studies. drawbacks are their high cost and undesirable side effects [6]. Experimental Microorganism

This situation forced scientists to search for Six different bacterial strains used for the new antimicrobial substances. Therefore study were S. typhi, S.flexneri E.coli, there is a need to develop alternative V.cholerae, M. tuberculosis, P. fluorescens. antimicrobial drugs for the treatment of The standard strains were obtained from infectious diseases from medicinal plants [7 Basic Biomedical Science, Bharathidasan and 8]. Keeping the importance of plants in University, Trichy. The stock culture was terms of bioactive compounds with maintained on Hi media agar medium antibacterial properties the present study has incubated at 4°C. been undertaken to ascertain the antibacterial activity of extracts from A. Preparation of Inoculam indicum and S. auriculata against some human pathogens and to identify the most Stock cultures of V.cholera, S.typhi, potent probable antibacterial bioactive S.flexneri, E.coli and P.fluorescens were compound by phytochemical analysis and maintained at 4°C in slopes of nutrient agar. 1HNMR study. 1070

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Active cultures for experiment were 1HNMR prepared by transferring a loop full of microorganisms from stock cultures to test 1HNMR spectra of methanol extract of tubes of nutrient broth and incubated for 24 S.auriculata and A. indicum were recorded ° hours at 37 C. on a Burker (Avance) 300 MH2, NMR instrument using TMS as internal standard Antibacterial susceptibility assay and chloroform as solvent. Standard Burker software was used throughout, chemical Antibacterial activity was assayed by the shifts are recorded as ( - scale) parts per disc diffusion method [9]. A known mg of million and the coupling constants are given crude extract was dissolved in 0.6 ml of in Hertz. The result graph was compared solvent. 6 mm of sterile disc was soaked in with the reference chart and the possible the solvent as control. Both the discs were functional groups present in the test plants allowed to dry at room temperature. were determined. Pathogenic bacterial strains were inoculated in sterile broth and incubated at 37°C for 24 Results and Discussion hours. Pathogens were swapped on the surface of sterile petridishes in 20 ml of The results of antibacterial activity of crude solidified nutrient agar. The control and the and step gradient extract of Senna ariculata experimental discs were placed in the sterile and Abutilon indicum are given in Tables 1 solidified nutrient agar petriplates to assess & 2 and Figures 1- 14 the effect of solvent and extracts on pathogens. These agar plates were Antibacterial activity of S. aruiculata incubated at 37°C for 24 hours and the antibacterial activity was measured Antibacterial activity of S. auriculata the accordingly based on the inhibition zone result revealed that the crude methanol around the disc impregnated with plant extract showed the highest activity (9mm) extract. Antibacterial activity was expressed against M. tuberculosis and the least against in diameter zone of inhibition, which was S.typhi. It also showed activity against S. with the outer side of the disc to inner side flexneri, V. cholerae, P. fluorescens (8mm) of the inhibition zone. Each active extract (Figure-1). Similar result was reported by was tested for thrice for confirmation of Sumathi and Pushpa [11] that, methanol activity. The most potent extract was extract of Datura metal was tested against subjected to Phytochemical analysis and different bacterial pathogens. The methanol 1 HNMR study to find out the potential extract of Datura matel showed (9mm) bioactive compound. inhibition zone against to E. coli. Among the step gradient extracts, of the six bacterial Characterization of antibacterial pathogens tested maximum numbers of compounds Phytochemical screenings pathogens were inhibited in 80:20% Methanol: Benzene (Figure-2). In Methanol The most potent extract was subjected to 60% and Benzene 40% extract the highest preliminary phytochemical testing activity was renowned against E. coli (7mm) qualitatively to detect for the presence of (Figure-3) and same mm was exhibited alkaloids, carbohydrates, cardiac glycosides, against E. coli in Methanol 40% and saponins, steroids, tannins and terpenoids by Benzene 60% extract (Figure-4). Very using the standard method [11]. meager activity was exhibited by methanol

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20% Benzene 80% and also in crude reported that the Phytochemical analysis of benzene in all the tested pathogens (2mm) methanol extract of , stem, seed, and root (Figure-5 & 6). sample of Cassia auriculata showed the presence of alkaloids, tannins, flavonoids Antibacterial activity of A. indicum and anthroquinones while saponins was not detected. Sarayu et al. [14] also reported the The crude extract inhibitory zone of A. similar result in the methanolic extract of the indicum range varied from 2mm (S. typhi) to leaf sample in Coriander sativum. 8mm (V. cholerae). Antibacterial activity of A. indicum the result revealed that the crude The bioactive compounds obtained from methanol extract showed the highest activity medicinal plants have been used to treat (8mm) against V. cholerae and the least various ailments caused by microorganisms. activity (2mm) in S. typhi (Figure-7). The most important of their bioactive Comparable result was obtained by Mahesh principles are alkaloids, phenolic and Satish[12] in Acacia nilotica, Sida compounds, flavonoids and tannins that may cordifolia, Tinospora cordifolia, withania be evolved in plants as self defense against somnifer and Ziziphus mauritiana in pest and pathogens [15]. methanol leaf extracts against Bacillus subtilis, E. coli and P. fluorescens. Of the 1HNMR spectrum of crude methanol four step gradient extract Methanol 80% and extract of S.auriculata and A.indicum chloroform 20% showed the highest activity (7mm) against P. fluorescens (Figure-8) and The 1HNMR spectra (Figure - 13) of crude in Methanol 60% and chloroform 40% the methanol extract of A. indicum showed the same inhibitory zone was obtained in E. coli presence of an aliphatic methyl (-CH3) (7mm) (Figure-9). In 40:60 Methanol: ,aliphatic methylene (-CH2), CH2 X group, Chloroform step gradient extract, the upper alkenic (C-H=C-H) and aromatic C-H most activity was exhibited by E. coli (5mm) protons) whereas the 1HNMR spectra and trace activity (1mm) were observed (Figure -14) of crude methanol extract of S. against S. typhi, V. cholera and M. auriculata showed four major signals which tuberculosis (Figure -10) and very minimum revealed the presence of (CH3/aliphatic activity was exhibited in Methanol 20% and methyl proton, aliphatic methylene (CH2) chloroform 80%as well as in crude proton, alkenic C-H or N-H or (HC=C-H) chloroform extract (Figure-11& 12) in all proton and aromatic C H protons. the tested pathogens. Silverstein et al., [16] reported that the 1H Phytochemical analysis of S. auriculata NMR analysis of methanol extract of E. and A. indicum agallocha showed the presence of aliphatic group of compounds which might be The results of phytochemical analysis responsible for the biological effects of the carried out on the sample S. auriculata and plant extract including antimicrobial A. indicum were shown in (Table 1 &2). The properties. The result showed that methanol results revealed the presence of alkaloids, was the best solvent for extracting the flavonoids, triterpenoids, and glycosides in effective antimicrobial substances from the S. aruiculata and the presence of alkaloids, medicinal plant S. aruiculata and A. indicum tannins, sterols, flavonoids, terpenoids and than the rest of the solvents tested. saponins in A. indicum. Sanjivani et al. [13]

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Fig.1 Antibacterial activity of Fig. 2 Antibacterial activity of methanol 80%, crude methanol extract of S. auriculata benzene 20% extracts of S.auriculata

Fig. 3 Antibacterial activity of methanol 60%, Fig.4 Antibacterial activity methanol 40%, benzene 40% extract of S.auriculata benzene 60% extract of S.auriculata

Fig.5 Antibacterial activity of methanol 20%, Fig.6 Antibacterial activity of crude benzene 80% of S.auriculata Benzene extract of S. auriculata

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Fig.7 Antibacterial activity of crude Fig.8 Antibacterial activity of methanol 80%, methanol extract of A. indicum chloroform 20% of A.indicum

Fig.9 Antibacterial activity of methanol 60%, Fig.10 Antibacterial activity of methanol 40% chloroform 40% of A.indicum chloroform 60% of A.indicum

Fig.11 Antibacterial activity of methanol 20%, Fig.12 Antibacterial activity of crude chloroform 80% of A.indicum chloroform extract of A. indicum

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Fig.13 1HNMR spectrum of crude Fig.14 1HNMR spectrum of crude methanol extract of S. auriculata methanol extract of A.indicum

Table.1 Phytochemical screening of crude methanol extract of A.indicum

Phytochemicals Results Alkaloids + Glycosides - Steroids + Flavanoids + Reducing Sugars - Triterrpenes + Phenolic Compounds - Tannins +

Table.2 Phytochemical screening of crude methanol extract of S.auriculata

Phytochemicals Result Alkaloids + Glycosides + Steroids - Flavanoids + Reducing Sugars - Triterrpenes + Phenolic Compounds - Tannins - alkali proton in S. auriculata. The data This might be due to the effective generated from the experiment have bioactive compounds as confirmed by provided the chemical basis for the phytochemical as alkaloid, flavonoid, 1 bioactivity and the therapeutic use of S. triterpenoids and glycosides and the H auriculata and A. indicum for various NMR as aliphatic methyl protons and ailments. Our study demonstrated that alkanic protons in A. indicum and methanol, methanol: chloroform (80:20) aliphatic proton, aromatic proton and and methanol: benzene (80:20) are found

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to be the effective solvent system for [7] Clark. A.,M, Phar.Res.,(1996). 13, separation of bioactive compounds of the 1133 test plants however further study is [8] Cordell. G.A., Phytochemistry., essential to purify, identify and (2000). 55, 463 characterize the biomolecules. [9] Bauer,,A.W., W.M.M. Kirby, J.C. Sherris and M. Turck, 1996. Antibiotic susceptibility testing by a standard single disc method. Am. J.Clin. Patol., 45: 493 [10] Harborne,J.B. (1998). Phytochemical methods. A guide to modern The crude methanol extracts of S. nd auriculata and A. indicum showed techniques of plant analysis.2 maximum activities when compared to edition. Pages 1-32. J. B. Harbone that of step gradient extracts. This study Publishers, London, Chappman & Hall. confirms the antibacterial properties [11] Sumathi and Puspha, (2007). obtained by a plant and it may have fewer Evaluation of antibacterial activity of side effects as it falls in the category of some Indian medicinal plants. Asian natural medicine. The present study Journal of Microbiology, confirms the antibacterial effect of Biotechnology and Environmental methanol extract and justified the Science, 9: 201-205. medicinal use of S. auriculata and A. [12] Mahesh, B., Department of Studies in indicum and further study is required to Applied Botany and Biotechnoloty, find out the active component of medicinal University of Mysore, value. Manasagangotri, Mysore-570 006. [13] Sanjivani, R. Bhalsing school of life References sciences. North Maharashtra University, Jalgaon 425 002. [1] Bushra Begum,.N.R. and T.Ganga [14] Sarayu,L., Rajendran S., Prabhakar S., devi,Asian Jr.of and Uma B., (2009). Antibacterial Microbiol.Biotech.Env. Sc., (2003), Activity and Phytochemical analysis 5(3), 319 of Corianter sativum against [2] Bonjar Glls and P.R.Farrokhi,J. Nat infectious diarrhea. Ethnobacterial Prod Med.,(2004),8,34 leaflets, 13: 590-594. [3] Lalitha. T.P,, P.Jayanthi. Asian [15] Sukumaran,S., S. Kiruba., M. J.Plant Sci.Res., (2012) 2(2): 115-122 Mahesh., S.R.Nisha., P.Z. Millar., [4] Tiwari. P., B.Kumar, M.Kaur, C.P. Ben., S.Jeeva., (2011) H.Kaur. (2011). Int.pharm.science, 98 Phytochemical constituents and - 108 antibacterial efficacy of the flowers of [5] Arumugam, M., Karthikeyan,S., and Peltophorum pterocarpum(DC) Baker AhamedJohn, S., (2009). ex Heyne, Asia Pacific Journal of Antibacterial activity of Indonessiella. Tropical medicine. Vol.4 (9) 735- Echinoids Research Journal of 738. Biological science, 1(3):157-161. [16] Silverstein R.M., Webster FX, [6] Rajasekara Pandiyan, M., Sharmila Kiemle DJ . Proton NMR Banu, G., and Kumar,G., (2007). spectrometry.Spectrometric Antibacterial activities of natural identification of organic Compounds. honey from medicinal plants or USA:John Wiley and Sons,Inc; antibiotic resistant strains of bacteria. (2005) 127-204. Asian Journal of Microbiology Biotechnology and Environmental Science 9:219-224.

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