Identification of tentative antioxidant constituents from stem and leaves of leea indica species using spectroscopic analysis

The tentative antioxidative constituents from stem and leaves extracts of Leea indica which traditionally known as Mali-mali had been investigated. Three different polarities of crude extracts of each part was prepared using consecutive soaking with petroleum ether, dichloromethane and methanol. Screening of antioxidants of each extract on TLC was performed with the use of 2,2 diphenyl-1-picrylhydrazyl (DPPH) as spraying reagent and have been previously reported. The isolation of antioxidative compounds was performed through preparative thin layer chromatography(pTLC) with the use of certain binary solvents system. The structure determination was carried out using Fourier Transform Infra-Red (FTIR), Gas Chromatography Mass Spectrometer (GCMS) and Proton Nuclear Magnetic Resonance (NMR) along with comparison with literature. FTIR analysis depicted some important functional groups such as OH, C-H, C=C and C-O stretching in both parts. The C=O and C-N stretching only appeared in leaves extract of L. indica. According to proton NMR spectrum of leaves part, the chemical shifts belonged to aliphatic protons, vinylic protons, hydroxyl proton and aromatic protons. The proton NMR spectrum of stem part revealed the existence of proton aliphatic, vinylic proton and hydroxyl proton. Based on GCMS analysis with the assistance of Wiley 275.L database matching individually as well as with proton NMR data, the antioxidative compounds isolated from stem part were proposed as 1-(hydroxymethyl)-1,2ethanediyl ester (LI-1). 9-Oxononanoic acid methyl ester (LI-2), 9,12-Octadecadienoic acid (LI-3), 3’,8,8’-Trimethoxy-3-piperidyl-2,2’-binaphthalene1,1’,4,4’-tetrone (LI-4). Keyword: Leea indica, Antioxidant, Secondary Metabolites, proton NMR Introduction Leea indica species from Leeaceae family has a vast application in ethnomedicinal purpose and lately the search of various active components is crucial and gaining popularity. Previous research found some important secondary metabolites as an anti-cancer, anti-hyperglycemic activity, hypolipidemic activity and antioxidant agent (Dalu et al., 2014). The leaf part has been used traditionally to treat vertigo and diabetes while the root was used as antidysentric, anthelmintic, spasmolytic and sudorific and to treat cardiac and skin diseases (Chatterjee and Prakashi, 1997; Khare, 2007; Rahman et al., 2007). According to Srinivasan et al. (2008), the presence of gallic acid, ursolic acid (triterpenes), β-sitosterol (steroids) and lupeol (triterpenes) was identified from phytochemical screening test done on methanol extract of leaves extract of L. Indica by utilizing co-TLC technique. The flower contains ester of GADING Journal of Science and Technology Vol 3 No (1) (2020) – eISSN: 2637-0018 Published by Universiti Teknologi MARA (UiTM) Cawangan Pahang March 2020 | 130 phthalic acid, di-isobutylphthalate, di-n-butylphthalate, n-butylisophthalate, and 3,5 butylisohexylphthalate (Srinivasan et al., 2009). Rahman et al. (2013) reported the presence alkaloids, flavanoids, glycosides, terpenoids, and tannin in L. indica leaf extract. Studies conducted by Emran et al. revealed methanol leaf extract exhibited significant DPPH free radical scavenging activity compared with standard antioxidant ascorbic acid. IC50 value of ascorbic acid and leaf extract was found 1.468μg/ml and 139.837μg/ml, respectively. Phytochemical analyses have screened the presence of alkaloid, flavonoid, terpenoids and steroid which responsible for antioxidant potency of methanol leaf extract of L. indica. This herb shows seasonal and varietal variation in the quantity of secondary metabolites (Emran et al., 2012). Currently, the investigation of total phenolic content, flavonoid and its antioxidant properties of L. indica leaves extracts from n-hexane, ethyl acetate, and methanol was successfully conducted (Sulistyaningsih et al., 2017) and the results revealed that the highest fraction of total phenolic as well as total flavonoid was resulted from methanol extract. It was also demonstrated the strongest antioxidant activity with the IC50 of DPPH scavenging effect 1.62 + 0.02 g GAE/ml and the highest percentage of inhibition for hydroxyl radical 57.60 + 2.52 %. This study was conducted by means to confirm and establish our previous work (Harun et al., 2018) through proton NMR analysis of four proposed structures of antioxidative constituents from stem and leaves of L. indica. Materials and Methods Sample Preparation and Plant Extraction About 500 g of L. indica leaves and stem were collected from Hutan Simpan UiTM Pahang. They were washed and cut into small pieces to assist the drying process. The leaves and stem were dried for several days at room temperature and were grinded into fine powder. Extraction Process The extraction process was performed by utilizing three types of solvents with different polarities and they were petroleum ether (PE), dichloromethane (DCM) and methanol (MeOH). The soaking process and extract preparation of both parts were followed the same method as reported by Harun et al., 2018. Fourier Transform Infra-Red (FTIR) analysis The extract was placed on the sampling plate using dropper. Since the instrument is an ATRFTIR, the sample can be analysed directly in liquid form. The wavenumber was set between 400 – 4000 cm. A background spectrum was firstly obtained before the sample was analysed. The functional groups were detected and were recognized as absorption peaks at different wave number. Isolation of Selected Antioxidative Components of LI-1, LI-2, LI-3 and LI-4 from Preparative Thin layer Chromatography(pTLC) The TLC of each extracts were developed in suitable developing solvents such as combinatio n of hexane with dichloromethane, dichloromethane with methanol until all components were well separated on TLC (Harun et al., 2018). Further work on isolation of antioxidative constit uents was conducted through pTLC development. The target constituent at certain Rf value w as carefully scratched and collected. Based on our previous investigation (Harun et al., 2018) the antioxidative constituents were observed at Rf values of 0.14, 0.66 and 0.85 for leaves part and at Rf 0.17 for stem part. The constituent was separated the from silica gel and was kept in the vial prior to analysis. GADING Journal of Science and Technology Vol 3 No (1) (2020) – eISSN: 2637-0018 Published by Universiti Teknologi MARA (UiTM) Cawangan Pahang March 2020 | 131 Gas Chromatography (GCMS) Analysis of LI-1, LI-2, LI-3 and LI-4 The procedure of GCMS analysis of four antioxidative constituents was followed the same m ethod as previously reported (Harun et al., 2018) One Dimensional Nuclear Magnetic Resonance (NMR) Analysis of LI-1, LI-2, LI-3 and LI-4 The isolated sample was subjected to one dimensional 500 MHz H-NMR (JOEL) for structure determination. The sample was mixed with standard tetramethylsilane (TMS) in CDCl3 prior to analyze. The resolve peaks at different chemical shifts indicated different type of protons present in the isolated compounds. The proton NMR data was used to confirm the types of protons present in the structure which was previously identified from GCMS (Harun et al., 2018) Results and Discussion Fourier Transform Infra-Red (FTIR) Fourier Transform Infrared Spectrometer (FTIR) is a common device used to determine functional group of a compound based on absorption of ultraviolet light at certain wavelength. Since methanol extract was used for analysing purpose, all of the functional groups present in this extract represented the functional groups of L. indica. The FTIR analysis was started by obtaining the background spectrum to eliminate the noises which can interfere the spectrum. The sample was directly analysed on the Spectrum 100 Spectrometer’s crystal plate since we used Attenuated Total Reflectance (ATR) sampling. The FTIR spectrum of both stem and leaf revealed the presence of hydroxyl group, OH absorption peak at range 3208-3325 cm, C-H stretching at 2947-2950 cm, C=O stretching at 1611 cm, C=C stretching at 1646-1539 cm, C-N stretching at 1340 cm and C-O stretching at 1014-1016 cm. The result of FTIR revealed some basic information to aid structure determination since it depicted some important functional groups present in the compound as well as covalent bond between atoms. In normal cases of the determination of structure of unknown compound, FTIR data was used along with Rule 13 of molecular weight to predict actual molecular formula of unknown compound. Preparative Thin Layer Chromatographic (pTLC) Preparative TLC method is the most convenient method for isolating matters especially in isolating active components such as antioxidative constituents. Figure 1 shows an example of the target antioxidative constituent of dichloromethane extract of L. indica stem at Rf 0.17 whereby this Rf value was obtained from developed TLC sheet in 100% dichloromethane. The greenish yellow colour of antioxidative compound was successfully isolated after careful scrapping process at targeted Rf value, dissolving and filtering to get rid of gel silica. The pTLC analysis of petroleum ether extract of L.indica leaves revealed two antioxidative marks at Rf 0.14 and 0.85 and one antioxidative marks at Rf 0.66 for DCM extract of L. indica leaves. The isolation of respective antioxidative marks was done in the same manner as depicted for compound at Rf 0.17 in Figure 1. Previously, all marks constituents revealed yellow color against purple background when sprayed with DPPH reagent (Harun et al., 2018). GADING Journal of Science and Technology Vol 3 No (1) (2020) – eISSN: 2637-0018 Published by Universiti Teknologi MARA (UiTM) Cawangan Pahang March 2020 | 132 Figure 1 pTLC of Antioxidative compound (Rf 0.17) of DCM extract of L.indica stem marked on pTLC Proposed Structure from Gas Chromatography (GCMS) All four isolated compounds were subjected to GCMS analysis by means of determination of their structural formula