Isolation of Chemical Compounds Fraction 8 in Ethyl Acetate Phase from Metanol Extract in Binahong Leaves (Anredera cordifolia (Ten) Steenis)

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1 Isolation of Chemical Compounds Fraction 8 in Ethyl Acetate Phase from Metanol Extract in Binahong Leaves (Anredera cordifolia (Ten) Steenis) Ratna Djamil, Henri Marjuandi Faculty of Pharmacy Pancasila University, Jakarta 12640, Indonesia ratnadj_ffup@yahoo.co.id ABSTRACT Binahong is one type of plants whose leaves are used as medicine. From previous research, it is reported that ethyl acetate phase from methanol extract in binahong leaves contain secondary metabolites such as saponin, steroids, triterpenoids, and flavonoids. Ethyl acetate phase from methanol extract of binahong leaves also have bioactivity toward the larvae of Artemia Salina Leach. This research aims to determine the content of chemical compounds fraction 8 in ethyl acetate phase from methanol extract in binahong leaves that is obtained through vacuum liquid chromatography using dichloromethane-isopropanol and methanol as the eluent. It is obtained nine fractions (fraction 1 fraction 9) which then fraction 8 is refractionated by column chromatography using methanol as eluent that acquires five fractions (fraction 8.1 fraction 8.5). Later on, through preparative high performance liquid chromatography using methanol-water as eluent, fraction 8 obtains three peaks. The first peak is identified by UV-VIS, FTIR, and GC-MS. The result exhibits the maximum isolate absorption at 264 nm from UV-VIS, bears the group of C=C and NH in FTIR, and according to GC-MS analysis the isolate of fraction contains adenine with 97% similarity. Keywords: Binahong (Anredera Cordifolia (Ten.) Steenis), Amino acid, Adenine. INTRODUCTION Anredera is one species of the Basellaceae has been recognized to have many benefits for treating various diseases. Familia Basellaceae have various species such as Anredera baselloides (Kunth) Baill, Anredera cordifolia (Ten.) Steenis, Anredera diffusa (Moq.), Anredera

2 leptostachys (Moq.) Steenis, Anredera spicata, Anredera vesicaria, Anredera cumingii, and others. Binahong is the Indonesian names for Anredera cordifolia, From previous researches, binahong was identified to have several secondary metabolites such as flavonoids, saponins, steroids, triterpenoids, and coumarins that have various bioactivity for human benefits. This research will be delivered as a discovery of fraction 8 in ethyl acetate phase from methanol extract in binahong leaves. The molecular structure of compounds is determined based on UV-VIS, FTIR, and GC-MS. METHODS General Experimental Procedure Ethyl acetate crude extract from the binahong leaves is fractionated by vacuum liquid chromatography, column chromatography, and thin layer chromatography. The result is isolated furthermore by preparative high performance liquid chromatography. The obtained isolate is identified by UV-VIS, Fourier Transform Infra-Red (FTIR), and Gas Chromatography Mass Spectrophotometer. Extraction and Isolation Prior to fractionation of ethyl acetate phase from methanol extract of the leaves, thin layer chromatography analysis is conducted with dichloromethane-isopropanol and methanol as eluent. Ethyl acetate fraction is refractionated by Vacuum Liquid Chromatography (VLC) using silica gel H60 and dichloromethane-isopropanol and methanol as gradient. The produced fractions are thickened by rotary evaporator vacuum until it forms crude. Fraction 8 from VLC procedure is refractionated by sephadex column chromatography to obtained a more simplified fraction. The result with similar chromatogram is combined together. The purification of isolate is performed by High Performance Liquid Chromatography (HPLC) at 254 nm. Sample is diluted in methanol, centrifuged and injected to HPLC until certain peaks are acquired. The final isolate is evaporated from methanol by rotary evaporator vacuum and is frozen by freeze dryer for 48 hour. The dried powder of isolate from fraction 8 is identified with UV-VIS, FTIR, and GC-MS to determine the chemical compounds molecular structure.

3 voltage RESULTS AND DISCUSSION From the extracted binahong leaves (Anredera cordifolia (Ten.) Steenis) with ethyl acetate adenine is found in the compound. This compound is obtained through several stages ofisolation include partitioning,fractionationand variouschromatographic techniques. Fractination by VLC obtains nine fractions and fraction 8 is chosen to be isolated furthermore by column chromatography. In column chromatography, 350 fractions are earned and then combined based on their same chromatogram. Afterwards, the simplified fractions go through refractination by HPLC process. In HPLC process fraction 8.4 is indicated to have the dominant absorbent peaks. Fraction 8.4 in preparative HPLC obtains 3 peaks. Fraction at Rt 4.8 minutes, fraction at Rt 8.9 minutes, and fraction at Rt 15.8 minutes (figure 1). [Au] 1.4 C:\Clarity\D.Binahong Ratna\Data\FRAKSI _31 26-Jan Time [min.] Figure 2 Fraction is identified by UV-VIS spectrophotometer at wave length 600nm-200nm and exhibits spectrum with maximum absorbent at nm wave length (figure 2). According to FTIR identification indicates functional groups stretched C=C aliphatic, aromatic, and C=N at wave 1660 cm -1, also the presence of amine group (NH) at wave 3640 cm - 1 (figure 3). Figure 3

4 Abundance TIC: BINAHONG F1.D Time--> A b u n d a n c e Figure 4 m / z --> A b u n d a n c e S c a n ( m in ): B I N A H O N G F 1. D m / z --> # : A d e n in e Figure 5 Identification with gas chromatography is aimed to determine molar mass from chemical compound and its similarity by Willey database. The chromatogram result from fraction exhibits that the isolate is pure. The indicated chemical compounds content in fraction can be seen in (figure 4). Adenine has the molecular formula C 5 H 5 N 5 with 135 as its molar mass. It is identified as adenine because it has similar Rt with adenine which are 5.77 minutes, 5.81 minutes, 5.92 minutes, 5.98 minutes and 6.23 minutes, also it s calculated that fraction has 97% similarity with adenine. Fraction isolate at 5.7 minutes compared with adenine with library Willey is identified with mass spectrum which result can be seen on figure 5. structure of adenine

5 CONCLUSION Ina study ofleafbinahong(anredera cordifolia) have successfullyisolatedanadenincompound from fraction and exhibits molecular structure through various spectrophotometric and chromatography procedure of UV-VIS, FTIR, GCMS, and preparative HPLC. REFERENCES [1] Murakami, T., Hirano, K., Yoshikawa, M., (2001). Structures of New Oleanane-Type Triterpene Oligoglycosides, Basellasaponins A, B, C, and D, from the Fresh Aerial Parts of Basella rubra L, Chem.Pharm.Bull, 49, (6), [2] Chuang, M.T., Lin, Y., Hou, W., (2007), Ancordin, the major rhizome protein of madeira-vine, with trypsin inhibitory and stimulatory activities in nitric oxide productions. Elsevier Inc,28, [3] Abou Zeid, A.H.S., Soliman, F.M., Sleem, A.A., Mitry, M.N.R. (2007). Phytochemical and bio-activity investigations of the aerial parts of Anredera cordifolia (Ten.) Steenis. Bulletin of the National Research Centre Cairo, 32,(1); [4] Kochhar,S.P., J.B.Rossell. (1990).Detection, Estimation and Evaluation of antioxidant in Food Systems. London: Elsevier Applied Science [5] Paul, S. B., Singha, S. (2012). Isolation and Identification of physiologically important Sterols and sterol glucoside from Basella rubra Linn, Assam University Journal of Science & Technology: Biological and Enviromental Sciences, 5, (1), [6] Lemmens, R. H. M. J., Bunyapraphatsara, N. (2003). Medicinal and poisonous plants Plant Resources of South-East Asia,12,(3), [7] Moura-Letts, G., Leon, F.V., Ana,Marcolo., Abraham, J.V., Gerald, B.H., (2006).In Vivo Wound-Healing Activity of Oleanolic Acid Derived from the Acid Hydrolysis of Anredera diffusa, Journal Natural Product, 69, (6),