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Int. J. Curr. Res. Biosci. Plant Biol. (2020) 7(5), 40-45 International Journal of Current Research in Biosciences and Plant Biology Volume 7 ● Number 5 (May-2020) ● ISSN: 2349-8080 (Online) Journal homepage: www.ijcrbp.com Original Research Article doi: https://doi.org/10.20546/ijcrbp.2020.705.006 Contemplating Macrotyloma uniflorum in traditional snake envenomation management practices through analysis on various solvents by a scheme of spectroscopic, phytochemical and chromatographical analysis Haritha Venugopal1, K. G. Sasikumar2, R. Prathapachandran3 and M. Anoop1* 1, 3K. V. M. College of Science & Technology, Kokkothamangalam PO, Cherthala- 688527, Kerala, India 2Shanthinikethan, Pattanakad PO, Cherthala, Kerala, India *Corresponding author; e-mail: [email protected];. +91-95675-20201. Article Info ABSTRACT Date of Acceptance: The idea of extrapolating the active principles in snake envenomation research in 18 April 2020 Macrotyloma uniflorum is presented in this work on a preliminary analysis through spectroscopic, chromatographic and phytochemical analysis. Similarity in the Rf values of Date of Publication: 0.9 ranges clearly indicated the presence of a common component .The UV-VIS 06 May 2020 spectroscopic analysis have indicated the presence of unique peaks in 200-320 ranges which predicted the presence of terpenoid compounds as it was discussed through the Keywords presence of chromophores. The phytochemical analyses have confirmed the presence of Active principles terpenoid through the presence of a brown color ring. The preliminary analysis floors a Chromatography base for the future drug formulation extending through the strategy of structural Envenomation interpretation with NMR and mass spectrometer assays. With the climatic change and Phytochemicals poor waste management there occurs an increasing overlap between humans and Spectroscopy poisonous snakes. The risk factors are assessed and which leads to the formulation of novel drug therapeutics. The work presents novel strategies in snake envenomation research through a systems approach of research with Macrotyloma uniflorum alone and in combination and was found to be effective in traditional snake envenomation practices. Introduction insecticide or pesticide in order to protect themselves and humans make use of these special Most of the plants we see around us in our day components produced by plants for making natural today life have their own medicinal properties due insecticide, pesticide herbicide etc. to the presence of certain special compounds called secondary metabolites or in other words the A wide variety of plants and their active principles presence of these special secondary metabolites have been evaluated for pharmacological gives certain plants their medicinal properties. properties (Gupta and Peshin, 2012). The plants These secondary metabolites are organic used in experiments were Macrotyloma uniflorum compounds which do not directly involve in the have medicinal properties and in Ayurveda and growth of the plants. Usually these secondary was being used as part of different medicinal metabolites act as a defense mechanism for the self combination over several years. Macrotyloma protection of the plants that is, they could act as an uniflorum was considered as the most extensive V. Haritha et al. (2020) / Contemplating Macrotyloma uniflorum in traditional snake envenomation management practices 40 through analysis on various solvents by a scheme of spectroscopic, phytochemical and chromatographical analysis Int. J. Curr. Res. Biosci. Plant Biol. (2020) 7(5), 40-45 crop grown in south India, the maximum in ether, acetone, ethanol-acetone, chloroform, ethyl Andhra Pradesh, Karnataka and Tamil Nadu acetate, methanol and water were spotted on (Parvathiraj et al., 2015). These medicinal standard TLC silica plates and silica plates made of properties are due to the presence of those special silica gel and calcium carbonate. The spots were components in it. analyzed and RF values were calculated. The spots were scrapped and the sample kept in refrigerator Secondary metabolites can be simply classified into for FTIR and LCMS analysis. three main groups: terpenes (such as cardiac glycosides, sterols, and carotenoids), phenolics UV-Visible spectroscopy (such as flavonoids, coumarins, tannins, and lignin) and nitrogen containing compounds (such The Optical Density values were recorded with the as alkaloids and glucisinolates) (Agostini-Costa help of an UV-Vis spectrophotometer in varying et al., 2012). The secondary metabolites are wavelengths from 280 nm - 750 nm in 50 nm considered as the bioactive components and were intervals for Macrotyloma uniflorum macerated in elucidated with phytochemical analysis. The petroleum ether, acetone, ethanol-acetone, water present study aims in extrapolating the active and Optical Density values were recorded with the principles in Macrotyloma uniflorum help of an UV-Vis spectrophotometer in varying contemplating the idea of traditional snake wavelengths from 300 nm - 500 nm in 30 nm envenomation practice. intervals for Macrotyloma uniflorum macerated in chloroform, ethyl acetate, methanol and water. Materials and methods Phytochemical analysis Collection of samples Acetone extracts of Macrotyloma uniflorum were Seeds of Macrotyloma uniflorum, were collected assayed for selected phytochemical tests like from traditional Ayurvedic medical shop at flavonoid, phenol, alkaloid and terpenoid tests Alappuzha, Kerala. The plant was identified at under standard protocol to trace out the bioactive biotechnology department of KVM College of compounds which might be showing the potential Science and Technology, Kokothamangalam, in snake venom treatments (Rufus Auxila et al., Cherthala. The sample collected and was powdered 2013). finely and dried under direct sunlight for 3 days and was stored in dry conditions. Test for flavonoids: Alkaline reagent test: 1ml of extract was mixed with 2ml of 2% of sodium Cold extraction hydroxide solution. An intense yellow colour were formed which turn colorless on addition of few 2.5g of the seed powder of Macrotyloma uniflorum drops dilute acid which indicate the presence of were macerated separately in petroleum ether, flavonoid. acetone, ethanol-acetone, chloroform, ethyl acetate, methanol and water for 24 hours in the Test for phenol: 1ml of extract was mixed with 2 increasing order of their polarity. The collected ml of 2% ferric chloride solution. A Blue, Green or samples were evaporated and filtered using black coloration indicate the presence of phenols, Whatman filter paper No. 1. tannins. Thin layer chromatography Test for alkaloids: 1 ml of plant extract was mixed with 2 ml of Wagner’s reagent (dissolve 2 g Thin layer chromatography is a technique used for of iodine and 6 g of potassium iodide in 100 ml of the separation of non-volatile mixtures. Here we use water) and shaken well. Reddish brown precipitate this technique to separate different components of indicates the presence of alkaloids. the extracts. The solvent system used as the mobile phase is chloroform: ethyl acetate: acetic acid in the Test for terpenoids: 0.1 ml of plant extract was ratio 6.5:3.5:0.5. The plant extract in Petroleum mixed with 0.5 ml chloroform and 1.25 ml of V. Haritha et al. (2020) / Contemplating Macrotyloma uniflorum in traditional snake envenomation management practices 41 through analysis on various solvents by a scheme of spectroscopic, phytochemical and chromatographical analysis Int. J. Curr. Res. Biosci. Plant Biol. (2020) 7(5), 40-45 concentrated sulfuric acid were carefully added to UV-Visible Spectroscopy form a layer. Presence of reddish brown color at the interface gives the positive result. The UV-Vis spectrophotometer presented maximum absorbance in aqueous extracts and Terpenoid assay for different solvent minimum in petroleum ether. The maximum peak extracts of Macrotyloma uniflorum: 0.5 ml was found in 380nm in aqueous extract 230-320 of plant extract was mixed with 2 ml chloroform nm in acetone. The UV-Vis absorbance different and 5 ml of conc. H2SO4 carefully added to form a extracts are shown in Fig. 1 and Fig. 2. layer. Presence of reddish brown color at the interface gives the positive results. Terpenoid test was carried out for Macrotyloma uniflorum in Most of the unique peaks were distributed between petroleum ether, acetone, ethanol-acetone, 300nm-500nm for various solvent extracts as they chloroform, ethyl acetate, methanol and water. have shown minimum absorption. This was further assessed in 30nm intervals for Macrotyloma Results and discussion uniflorum in other solvents like chloroform, ethyl acetate, methanol and water. The maximum Thin layer chromatography absorbance was for chloroform and water at 500 nm. Petroleum ether extract showed a maximum Thin layer chromatography of plant extract in seven peak at 200nm but was not showing any further different solvents was spotted on TLC plate and the activities upon higher peak ranges from 230 to RF values calculated are shown in Table 1. 750. Table 1. RF values depicting different solvent extracts of Macrotyloma uniflorum. Solvents Petroleum Ethanol- Ethyl Acetone Chloroform Methanol water used ether acetone acetate RF value 0.9 0.9 0.9 0.9 0.975 0.9574 0.9 3.5 3 Petroleum ether Acetone 2.5 Ethanol-acetone 2 Aqueous 1.5 Absorbance 1 0.5 0 200 230 260 290 320 380 430 490 530 590 630 690 750 Wavelength in nm Fig. 1: UV-Vis spectrum of Macrotyloma uniflorum in Pet-ether, acetone, ethanol-acetone and aqueous solvents.