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ECOTYPE ANALYSIS and BIOACTIVITY ASSAY of RIVINA HUMILIS L. ELSAM JOSEPH * *Department of Botany and Centre for Research, St

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ECOTYPE ANALYSIS and BIOACTIVITY ASSAY of RIVINA HUMILIS L. ELSAM JOSEPH * *Department of Botany and Centre for Research, St Journal of Xi'an University of Architecture & Technology ISSN No : 1006-7930 ECOTYPE ANALYSIS AND BIOACTIVITY ASSAY OF RIVINA HUMILIS L. ELSAM JOSEPH * *Department of Botany and Centre for Research, St. Teresa’s College, Ernakulam, Kerala, India. Abstract - The phenotypic variations among accessions of Rivinahumilis L. were studied for 24 qualitative and 12 quantitative characters and found that ecotypes are not present among accessions. The analysis of antimicrobial activity was done by agar disc diffusion method against 10 bacterial strainsand 4 fungal stains.The plant material was extracted with n-hexane,chloroform and methanol. The studied showed that the plant extracts were more active against gram-negative bacteria than gram-positive bacteria. The most susceptible bacteria were Klebsiellapneumonia (MTCC 109). Pseudomonas. aeruginosa (MTCC 741) also exhibited remarkable antibacterial activity.The fungus Candidaalbicans (MTCC 1637) showed remarkable resistance. All other fungi tested were susceptible and exhibited remarkable antifungal activity.The plant extract wasphytochemicallyassayed for alkaloid, flavonoid, resin, tannin and saponin contents. Key words :Accession,Antibacterial. Antifungal, Disc diffusion method, Medicinal plants,Phyto-compounds . I INTRODUCTION Phytolaccaceae is commonly known as Pokeweed family. The name Phytolaccaceae Lindl. is conserved over Petiveriaceae Link. (Lawrence, 1974 [1]). The genus Rivina (the Pigeon berry) of the family Phytolaccaceae is indigenous to South India from Florida to Texas (Rendle, 1956 [2]).Plants are the exclusive source of drugs for majority of the world population even today. A drug is studied with respect to its constituents, active principles, their isolation, identification and evaluation. This was done by conventional methods earlier, but now it is supplemented by chemical analytical techniques (Daniel, 1991 [3]). Chemical constituents of organisms are taxonomically valuable and have the advantage over classical characters that they can be exactly described. The purposeful search for certain useful compounds in related taxa has been of great interest to the pharmaceutical and food industry.Traditional system of medicine plays a very prominent role in the healthcaresystem of the rural people covering all types of ailments.RigVeda andAtharvaveda have details to cure different diseases. Plants used for traditionalmedicine contain a wide range of substances that can be used to treat chronicas well as infectious diseases. The medicinal value of plants lies in somechemical substances that produce a definite physiological action on the humanbody. The most important of these bioactive compounds of plants are alkaloids,flavanoids, resins, tannins and saponins (Kokate, et al., 2004 [4]).The berries ofthe plant accumulates betalains in its berries (Mohammad et al ., 2015 [5]). The present study deals with the systematic screening of antimicrobial activity and estimation of phyto-compounds of the root and shoot extracts of Rivinahumilis L.It is also aimed to evaluate the phenotypic variations among accessions.The potential of higher plants as source for new drugs is still largely unexplored.Among the estimated 250,000-500,000 plant species, only a smallpercentage has been investigated phytochemically and the fraction submittedto biological or pharmacological screening is even smaller. The antioxidant constituents impart significance to the usage and effectiveness of the plant as they were well-known for the eradication of many degenerative disorders by their free radical quenching capacity (Muhammad et al ., 2013 [6] ). Plants areused medicinally in different countries and are a source of many potent andpowerful drugs. Making antibacterial drug therapy effective, safe and affordable hasbeen the focus of interest during recent years(Bhavnaniand Ballow2000 [7] ; Sharma et al . 2002 [8] ; Janovska et al . 2003 [9] ). There areseveral reports on Volume XIII, Issue 7, 2021 Page No: 51 Journal of Xi'an University of Architecture & Technology ISSN No : 1006-7930 antimicrobial activity of different herbal extracts(Adelakun et al . 2001 [10] ,Camporese et al . 2003 [11],Bonjar 2004 [12] ,de Boer et al . 1966 [13] ,Nair et al . 2005 [14] ).The aim of the present study was to identify the active principles of plant extract which may be useful in developing new lead compounds to combat deadly diseases. II MATERIALS AND METHODS 2.1Collection of plant materials The plant samples for the present study were collected from different localities of Kerala such as Trivandrum, Ernakulam and Muvattupuzha. The collected plant material was identified using standard floras (Hooker 1983 [15] , Gamble, 1967 [16] ; Matthew, 1999 [17] ) and confirmed by comparing with authentic sheets in the herbaria in the Botanical Survey of India, Coimbatore, and Tropical Botanic Garden and Research Institute, Palode, Trivandrum. 2.2 . Phenotypic variation among accessions The phenotypic variations among accessions were studied in the plants collected. The accessions of Rivinahumilis L. were collected from Ernakulam, Trivandrum and Muvattupuzha. A total of 30 specimens were collected from 3 localities of South India. Each specimen was scored for 12 morphometric characters and 24 qualitative morphological characters. Karl Pearson’s correlation coefficient was calculated to test the association between pairs of morphometric characters of the accessions.The variations in phenotypic characters of accessions of the three samples were studied by breaking down the total variances of a variable into additive components which may be attributed to regional factors and genus respectively, by applying the statistical method of analysis of variance (ANOVA). For the analysis of variance and estimation of correlations, SPSS Windows Version 17 was used. ∑ʚ͒ − ͒Ŭʛʚ͓ − ͓Ŭʛ ͦ = ǭ∑ʚ͒ − ͒Ŭʛͦ ∑ʚ͓ − ͓Ŭʛͦ ͫℎ͙͙ͦ ͒ ͕͘͢ ͓ ͕͙ͦ ͨℎ͙ ͨͫͣ ͕͕͖͙ͪͦͧ͝͠ ͙ͩͦ͘͢ ͙͕͗ͣͧͦͨͣ͘͢͢͝͝ 2.3. Screening of phyto-compounds Extracts of the powdered samples of Phytolaccaoctandra L. , Petiveriaalliacea L., and Rivinahumilis L.were subjected to treatments with various chemical reagents for quantitative estimation of alkaloids, flavonoids, resins, tannins and saponins (Harborne 1973 [18] ). 2.4.Bioactivity assay of plant extracts Roots and shoots were collected separately and washed thoroughly in running tap water and air dried in shades for one week. The dried samples were powdered and extracted the active principles by solvent extraction method.The powdered plant materials were loaded in the soxhlet apparatus and exhaustively extracted for 7 hours with n-hexane, chloroform and methanol. The extracts were concentrated using rotary vacuum evaporator. The concentrated residue was carefully packed in small clean bottles and stored at room temperature. A known volume of the extract was taken in a pre-weighed petridish. The weight of the petridish along with the extract was taken. The extract was allowed to dry in reduced pressure and ambient temperature. After complete drying, the weight was measured and the yield (concentration) of the content was calculated. Volume XIII, Issue 7, 2021 Page No: 52 Journal of Xi'an University of Architecture & Technology ISSN No : 1006-7930 2.4.1. Bacterial and fungal strains used for the analysis The microbial strains for the antimicrobial screening were obtained as the Microbial Type Culture Collection (MTCC) from the Institute of Microbial Technology, Chandigarh (India). The following strains were used for the screening of antibacterial and antifungal activities. Gram-positive bacteria:Staphylococcus aureus (MTCC 96), Bacillus cereus (MTCC 430), B. subtilis (MTCC 441) Gram-negative bacteria:Serratiamarcescens (MTCC 97), Pseudomonas fluorescens (MTCC 103), P. aerugin osa (MTCC 741), Klebsiellapneumoniae (MTCC 109), Proteus vulgaris (MTCC 426), Escherichia coli (MTCC 443), Salmonella typhi (MTCC 733) Fungi:Candida albicans (MTCC 227), C. albicans (MTCC 1637), C. albicans (MTCC 3017), C. glabrata (MTCC 3019) 2.4.2.Evaluation of antibacterial activity Antibacterial activity of the plant extractwas tested against the Gram-positive and Gram-negative bacteria by the agar disc diffusion method. The bacteria were grown on Mueller-Hinton agar media (pH 7.3). Agar media were poured into the plates to a uniform depth of 5 mm and allowed to solidify. The microbial suspensions at 5x10 6cfu ml –1 were streaked over the surface of media using a sterile cotton swab to ensure confluent growth of the organism. The discs (6 mm in diameter) used were Whatmann No. 1 paper discs. 10µl aliquots of the extracts in DMSO were spotted on filter paper discs. The discs were then aseptically applied to the surface of agar plates at well-spaced intervals. The plates were incubated at 37 0C for 24 hours and the growth inhibition zones, including disc diameters were measured. Control discs, impregnated with 10 µl of the solvent DMSO and streptomycin (2 µg per disc) were used alongside the test discs in each experiment. 2.4.3.Evaluation of antifungal activity For the screening of antifungal properties, the fungi were obtained from MTCC, cultured in modified Sabouraud’s agar. Suspensions at 5x10 6cfu ml –1 were used. 10 µl of extract (33.3% v/v) in DMSO was impregnated on discs. 10 µl of the solvent DMSO and fluconazole (2µg per disc) were used as controls. The growth inhibition zone including disc diameters were measured. III RESULTS AND DISCUSSION 3.1. Phenotypic variation among accessions All the observations were mean of 10 samples. The statistical analysis of correlation
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