Rajarajan PN. et al. / Journal of Science / Vol 4 / Issue 6 / 2014 / 371-376. e ISSN 2277 - 3290 Print ISSN 2277 - 3282 Journal of Science Botany www.journalofscience.net ANTIMICROBIAL ACTIVITY AND PHYTOCHEMICAL ANALYSIS OF ANISOMELES MALABARICA (L) R.BR. ON AFLATOXIN PRODUCING FUNGI P.N.Rajarajan*1, K.M. Rajasekaran2, N.K Asha Devi 3 1,2Centre for Botanical Research, The Madura College, Madurai-625011- India. 3Department of Zoology, Thiagarajar College, Madurai-625009, India. ABSTRACT Anisomeles malabarica (Linn.) R.Br.is a traditional medicinal plant of Lamiaceae family, distributed throughout India. The methanolic extracts of Anisomeles malabarica (Linn.) R.Br. were tested for Antimicrobial efficacy against the fungal organisms. The methanolic extract of Anisomeles malabarica (Linn.) R.Br. were exhibited maximum anti fungal activity. Hence it can be concluded that the methanolic extract of Anisomeles malabarica (Linn.) R.Br. possess a significant Antimicrobial activity. This also stands as a scientific support for the usage of this plant for fungi toxic activities, inhibiting the growth of Aspergillus flavus and Aspergillus parasiticus and thus could be considered as an alternative inhibitor of the survival of this fungus in foodstuffs, in addition to offer some protective effect to the production of aflatoxins . Keywords: Anisomeles malabarica, Medicinal plant, Lamiaceae, Antimicrobial. INTRODUCTION Aflatoxins are difuranocoumarin derivatives, There is much difference in quantity of aflatoxin which are produced by a polyketide pathway in production between the different Aspergillus Aspergillus flavus and Aspergillus parasiticus. These flavusstrains; only about half of the Aspergillus toxins were first isolated after the ‘Turkey X disease’, flavusproduce aflatoxin, but those that do can often caused by batches of peanuts in chicken feed, which were produce more than 106 µg/kg. LD50 values for acute contaminated by Aspergillus flavus. There are four major aflatoxin exposure are hard to summarize and extrapolate forms of this toxin, named after the color of light they because of the large differences in susceptibility of the emit when excited by UV light: B1, B2, G1 and G2 (B different animal species; results of animal studies range being blue and the G being green colors); although more from 0.5 – 10.0 mg/kg bodyweight. However, estimated variants exist, these four are the most important. There are LD50 values for humans range from 10-20 mg/kg about a dozen less common variants, such as theP1, Q1, bodyweight for adults; these values have been B2a and G2a, but for the purpose of risk assessment, these extrapolated from animal experiments and a case study. are relatively unimportant. Since Aspergillus flavus is a The case study involved an Indian outbreak of hepatitis in major contaminant in agriculture, contaminating various 1974, in which 100 people died as a result of aflatoxin different foodstuffs such as cereals and rice. Because of intoxication via ingestion of contaminated maize. It was the significance of Aspergillus species as a food later estimated that some adults might have eaten 2-6 mg contaminant, aflatoxins are of great interest in assessing of aflatoxin in a single day [1]. Chronic toxicity data risk to mycotoxin exposure. suggest aflatoxin B1 is the most potent carcinogen known Aspergillus flavusis not the only strain of and chronic exposure has also been shown to cause Aspergillus producing aflatoxins, but it is the most immune suppression [2].Toxicity of aflatoxin is based on common strain and thus of most interest of researchers. the conversion of aflatoxin by cytochrome P450 enzymes Corresponding Author:-P.N.Rajarajan, Email:[email protected] 371 Rajarajan PN. et al. / Journal of Science / Vol 4 / Issue 6 / 2014 / 371-376. into a reactive 8, 9 epoxide form. This form is able to bind on a Perkin Elmer 115 gas chromatograph fitted with a 30 to guanine in DNA and proteins at the N7 position, and m × 0.25 mm ID, 0.25 μm film thickness. Column causing GC-TA transversions in DNA, explaining temperature was initially kept at 70°C for 2 min, and then aflatoxins carcinogenic potential in many studies; they gradually increased to 120°C at 2°C min-1 rate, held for 2 concluded a high correlation between aflatoxin and the min and finally raised to 250°C at 5°C min-1. Diluted number of DNA adducts in vivo[2]. Several studies link samples (1/100 v/v, in hexane) of 1 μl were injected at airborne exposure to aflatoxin B1 to cancer incidence and 250°C. Flame ionization detection (FID) was performed the International Agency for Research on Cancer (IARC) at 280°C. GC-MS analysis was performed on an Agilent has classified aflatoxin B1 as a class I carcinogen [2]. 6850 Series II apparatus, fitted with a fused silica HP- Plants are the main source of food. They are rich 5MS non-polar capillary column (30 m × 0.25 mm ID, in nutrients .They are also rich in compounds which have film thickness 0.25 μm). Carrier gas was helium at a flow pain relieving and healing abilities. From earliest times rate of 1 ml/min. Injector and MStransfer line itself plants were used for treatment of disease without temperatures were set at 230 and 270°C, respectively. Ion knowledge about the compounds present and their mode source temperature was 200°C. The injection volume was of action. Over the centuries 0.1 μl with a split ratio of 1:50. Mass range was from m/z Societies around the world have developed their 40 to 550 amu. Identification of components of the own tradition to make sense of medicinal plants and their essential extracts was based on GC retention indices and use. Many efforts have been made to discover new computer matching with the Wiley Library as well as by antimicrobial compounds from various kinds of sources comparison of the fragmentation patterns of the mass such as micro-organisms, animals, and plants. One of spectra with those reported in the literature [6]. such resources is folk medicines. Systematic screening of them may result in the Test Fungi: discovery of novel effective compounds [3]. Anisomeles Two species of Aspergillus viz Aspergillus malabarica (L.) (Malabar catmint) Cogn.Syn. Nepeta flavus and Aspergillus parasiticus which were frequently malabarica L., (Family: Lamiaceae) is a medicinal plant associated with Peanut seeds with higher percentage were has been used as a folkloric medicine to treat amentia, selected for antifungal activity assay of methanolic extract anorexia, fevers, swellings, rheumatism [4]. The herb is of test plant. reported to possess anticancer, allergenic, anthelmintic, anti-allergic, anti-anaphylactic, anti-bacterial, anti-fungal, Antifungal activity test anti-carcinomic, anti-edemic, anti-histaminic, anti- Antifungal activity was screened by agar well inflammatory, anti-leukemic, anti-nociceptive, anti- diffusion method [7]. The methanol extracts of plasmodial, anti-septicandantiperotic properties [5]. Anisomeles malabarica plants were tested against Aspergillus flavus and Aspergillus parasiticus. The PDA MATERIALS AND METHODS medium was poured in to the sterile petriplates and Collection of Plant Material allowed to solidify. The test fungal culture was evenly Fresh, healthy leaves of Anisomeles malabarica spread over the media by sterile cotton swabs. Then wells were collected from Pasumalai region, Madurai, (6 mm) were made in the medium using sterile cork borer. Tamilnadu (Fig 1). The plants were identified and 200μl of each extracts were transferred into the separate authenticated at Centre for Botanical Research, The wells. The plates were incubated at 27°C for 48 – 72 hrs. Madura College Madurai. The leaves were washed After the incubation the plates were observed for thoroughly 2-3 times with running water and once with formation of clear incubation zone around the well sterile distilled water then air-dried on sterile blotter under indicated the presence of antifungal activity. The zone of shade. inhibition was recorded. Solvent Extraction RESULTS AND DISCUSSION Thoroughly washed dried leaves of five plants The GC-MS analysis carried out on Anisomeles Anisomeles malabarica were dried in shade for ten days malabarica revealed the presence of many compounds in and then powdered with the help of Waring blender. 25 g the retention time range of 14.291-49.067min (Table1and of shade-dried powder was filled in the thimble and 2).The major constituents were 3 –Oxo-10(14)- extracted successively with methanol as solvent in epoxyguai-11(13)-en-6,12-olid (RT=44.354, MW=262, Soxhlet extractor for 48h. The solvent extracts were molecular formula= C15 H18O4 , %Area= 13.57%), concentrated under reduced pressure and preserved at 5°C Caryophellene (RT=14.774,MW=204, molecular formula in airtight bottle until further use. = C15 H18O4, %Area = 0.17%), 1 Phenenthrene methanol (RT=40.031 ,MW=286, molecular formula= C20 H30O , GCMS Analysis %Area=0.39%) and Thunbergol (RT=40.031,MW=60, Analytical gas chromatography was carried out molecular formula= C20 H34O ,%Area=0.53%). The minor 372 Rajarajan PN. et al. / Journal of Science / Vol 4 / Issue 6 / 2014 / 371-376. constituents present were Octadecane (RT=19.127, Antimicrobial Sensitivity Test MW=258, molecular formula C18H38 , %Area=0.38%), Antifungal activity of field grown Anisomeles 2,6,10-Trimethyl,14-ethylene 14 pentadecane malabarica was screened against aflatoxigenic fungi (RT=24.292, MW=278, molecular formula = C20 H38 , Aspergillus flavus and Aspergillus parasiticus. The %Area =1.40 %), 3,7,11,15-tetramethyl -2 hexadecane extracts showed varying of inhibitory effects (Fig 3). The (RT=24.80, MW=296, molecular formula = C20 H40 O , inhibitory effects of extracts were directly proportional to %Area = 0.39%), 3,5,9 Trimethyl-deca 2,4,8-trien-1-ol the increasing concentration of field grown leaf. The (RT=28.106, MW=194, molecular formula= C13H22O, Methanolic extract produced good antimicrobial activity %Area=0.53%).