Sitosterol and Betulonic Acid from n-Hexane Extract the Stem Bark of Tetracera indica

The Tetracera indica has been used traditionally to treat various diseases including skin medications, lower blood pressure, fever, headache, wound and gout medicine. The objective of the research was isolated and characterized the β-sitosterol (1) and betulonic acid (2) from n-hexane extract of the stem bark of Tetracera indica. The dry powder the stem bark (550 g) was maceration with n-hexane for 24 hrs. This extraction process was repeated three times. The nhexane extract was separated and purified by chromatography technique. The structure of isolated compound was determined by spectroscopy data UV, IR, NMR and compared with reported data. Two compounds have been isolated and base on spectroscopy data the compounds is β-sitosterol (1) and betulonic acid (2). Betulonic acid is being reported for the first time from T. indica, mean while the β-sitosterol previously reported from MeOH extract of leaves of T. indica but is being reported for the first time from the stem bark of T. indica.


INTRODUCTION
Indonesia is one country of the largest users of medicinal plants in the world along with other countries in Asia, such as China and India (Widjaja et al., 2014). The used of medicinal plants traditionally has been done by Indonesian people for generations. In previous studies, exploration and inventory of medicinal plants and thereused in people local community have been done. One of the medicinal plants is Tetracera indica (Yustian, Muharni, Sukarmi, Zulaicha, & Arbi, 2012). This species also known by the name Tetracera assay (Hoogland, 1953). The stem bark of T indica was used as a rope and the fruit used as vinegar in cooking. T. Indica in Malaysia is known locally name mempelas oil or mempelas rich and in Cina is know locally name caygiaychieu. Malay communities in Malaysia used dry powder of leaves to cure inflammation, while the Temuan Tribe in Selangor used the roots of T. indica to treatment of hypertensive patients and high fever. The Machang tribe in Kelantang used mixture the powdered of roots and leaves treat skin diseases (Faridah and Nurul huda, 1999). In Indonesia, especially the people of South Sumatra using the stem bark of the T. indica for the treatment of kidney stones (Muharni, Fitriya & Farida, 2016;Ong & Nordiana, 1999) and the leaves used for hypercholesterolemia (Samitra & Rozi, 2017). In India was used for the treatment of dysentery, hepatitis, diuretics, fever and fatigue (Lima, Lemos & Conserva, 2014).

EXPERIMENTAL SECTION Materials
The materials : The stem bark simplicia of T. indica.The fresh stem bark of T. indica was collected in January 2018 from Musi Banyuasin South Sumatera Indonesia and identified by Dr. Laila Hanum (number specimen VIC 2702), Head of the Botany Laboratory, University of Sriwijaya. The stem bark (2 kg) is cut into pieces and then dried at room temperature after that the stem bark was ground until fine so that obtained the dried powder. Material for isolation: n-hexane, ethyl acetate, methanol, acetone pa, chlotoform p.a, cerium sulphate, silica gel G 60 (70-230 mesh), Thin Layer Chromatography (TLC) plate 60G F254 Merck (Art 5554). All technical quality solvents were distilled before used.

Instrumentation
Melting point was determined by Gallen Camp melting point apparatus. UV spectrum was recorded on spectrophotometer ultraviolet Beck DU-7500. The IR (KBr) spectrum was recorded on Perkin Elmer-FTIR spectrometer, Shimadzu UV-168A. The 1 H-NMR and 13 C-NMR spectra were recorded on Agilent DD2 ( 1 H-NMR500 MHz, 13 C-NMR125 MHz),ultra-violet lamps used CAMAG 254 nm, Thin Layer Chromatography (TLC) was performed with silica gel 60G F254 using Merck (Art.5554) and spots were visualized by cerium sulfate vapors and ultra violet light.

Extraction and Isolation
The dried powdered plant material about 550 g was macerated with n-hexane for 24 hours The extraction process were repeated three times.The nhexane extract was concentrated by Rotary evaporator at about 50C and obtained crude nhexane extract (250 mg). The crude n-hexane (250 mg) was dissolved in the of acetone and adsorbed on 500 mg silica gel (70-230 mesh), stirred evenly and the slurry formed was allowed to dry. Crude nhexane extract was separated by column chromatography technique using silica gel G60 (70 -230 mesh) 60 g as stationary phase. The column (60 cm x 2 cm) was prepared by place cotton at the bottom of the column. The slurry of silica in nhexane was poured into the column. The silica slurry were compacted by flowing n-hexane solvent repeatedly. The Extract has been preparate was charged into the column. The column was first eluted with n-hexane and then with the solvent n-hexane: ethyl acetate (9:1 to 3:7). The fraction of 10 mL were collected in bottles vial and evaporated.TLC analysis toidentify the component with UV lamp and cerium sulfate to the component viewer.
Characterization of the compounds The structure of compounds was determined the structure using UV, IR, 1 H-NMR, 13 C-NMR and DEPT spectroscopy. .

RESULTS AND SISCUSSION
Compound 1 is white crystalline, m.p 135 -137C. Spektrum UV (MeOH) indicates maximum absorption at λmax 206 nm. The UV spectrum indicates that the isolated compound is a steroid group because there is no visible absorption for the chromophore group. IR absorptions bands appeared at IR Spectroscopic analysis, the observed absorption bands are bands appeared at (λmax cm -1 ) 3423 cm -1 (O-H stretching), 2935 and 2886 cm -1 (aliphatic C-H stretching asymmetric and symmetric), 1463 and 1377 cm -1 (cyclic(CH2)n bending), and the absorption frequency at 1056 cm -1 (C-O). The absorption frequencies in IR spectrum show similarity the absorption frequencies for β-sitosterol.
1 H-NMR ((CD3)2CO, 500 MHz) δH ppm and 13 C-NMR ((CD3)2CO, 125 MHz) δC ppm for compound 1 can see Table 1. The proton 1 H-NMR spectrum showed the proton of H-3 appeared as δH 3.30 ppm (1H,m), and signal for vinylic proton appeared at δH 5.20 (1H,m) indicating the presence of one methine group that is characteristic of β-sitosterol. 1 H-NMR spectrum of compound 1 showed the presence of 6 high intensity peaks at δH 0.71; 0.81; 0.83; 0.86; 0.96; and 1.02 ppm, indicating the presence of six methyl groups (Figure 1). From the 6 methyl proton signals, two between shown as double signal supported by 1 H-NMR spectrum and the DEPT spectrum which is only shows 6 methyl carbon signals. The 13 C-NMR spectra shown signals at δC 141.5 and 120.7 ppm recognizable as signal which assigned at C-5 and C-6 double bonds respectively in β-sitosterol. Spectra 13 C-NMR compound 1 show twenty nine carbon signal. To determined the type of carbon, methyl methylene, methine or quaternary carbon can be used DEPT spectrum (Figure 2 (2) The UV, IR, 1 H NMR, and 13 C NMR spectra data of compound 1 has been comparison with literature data of β-sitosterol and show the structure of compound 1 is β-sitosterol. (Figure 3). The βsitosterol has also been previously reported from MeOH extract leaves of T.indica but for the first time reported from the extract of stem bark of T. Indica.
The 1 H-NMR spectrum of the isolated compound 2 (Figure 4) shows signal peaks that accumulate below the δH 3 ppm area and also the peak for the dominant methyl signals. This indicates that the isolated compounds are triterpenoid or steroid groups. The widening of the 1 H-NMR spectrum in the area of δH 0.85-1.80 ppm showed six signal methyl groups at δH 0.95 ppm (3H,s); 1.00 (6H,s); 1.04 (6H,brs); δH 1.70 ppm (3H,s). The signal at 4.59 (1H, brs) and 4.72 (1H,brs) for two signal vinylic protons. The 13 C-NMR spectrum ( Figure 5) shows a characteristic spectrum for triterpenoids where the signals appear to accumulate below δC 60 ppm which is a signal for C, CH, CH2 and CH3 from cyclo aliphatic from triterpenoid.
The 13 C-NMR spectrum shows 30 signal (Table 2), 4 signal appeared on δC 100 ppm at δC 110.0; δC 151.6 ppm for C=C and two signal at δC 177.5 ppm (C=O acid) and δC 216.4 ppm (C=O ketone). In the 13 C-NMR spectrum no signal appears at δC 73−75 ppm which the characteristics for signal C-3 which bind OH which is commonly found in triterpenoid compounds.To determine the type of carbon determined from the DEPT 135 spectrum so that it can be determined the amount of C quaternary, CH (methine), CH2 (methylene), and CH3 (methyl). In the DEPT 135 spectrum, the CH3 and CH signals appear upward, the CH2 signal downwards and the C quartener signal does not appear. DEPT Spectrum show 6 signal methyl at 14.9; 16.3; 16.4; 19.5; 21.3 and 30.1 ppm), 11 methylene signal at δC (20.4; 22.2; 26.4; 30.5; 31.3; 32.7; 34.4; 34.5; 37.5; 40.2 and 110.0 ppm), 5 methine signal at δC (39.1; 47.9; 49.8; 50.6; and 55.5 ppm) and 8 signal C quaternary at δC (37.6; 41.5; 43.3; 47.7; 56.8; 15.6; 177.5; and 216.4 ppm ). The UV, IR, 1 H-NMR, and 13 C-NMR spectra data and their comparison with those described in literature (Liu et al., 2012) showed in Table 2. Base on data in Table 2 the structure of compound 2 is betulonic acid (Figure 3). Comparison of 13 C-NMR data in Table 2 shows that the isolated compound show a chemical shift value that is almost the same as the comparison data, while there is a slight difference in chemical shift value because the solvent used to isolated compound of solvent (CD3)2CO which is different from the literature data that used solvents CDCl3. Liu et al., (2012) reported that betulonic acid compound from root extracts of Belamcanda chinensis (L.) which had biological activity as antitumor. According to Shabana, El Sayed, Yousif & Sleem, (2011) in Harpephyllum caffrum leaves also contain betulonic acid which is active as an antioxidant and antitumor. Betulonic acid alsowas found by Zhang, Li, Fong, & Soejarto, (2016) in leaves of Vatica cinerea King (Dipterocarpaceae) which showed anti-HIV activity. Betulonic acid has been widely reported from other species but for the first time reported from the extract of stem bark of T. indica.  (2) Table2. 1 H and 13 C NMR chemical shift values for compound 2 a ( 1 H-500 MHz, 13 C-125 MHz, in (CD3)2CO ) and betulonic acid b ( 1 H-400 MHz, 13 C-100 MHz, in CDCl3, ppm) (Liu et al., 2012) C No.
δC ( CONCLUSIONS Two compounds β-sitosterol (1) and betulonic acid (2) were successfully isolated and identified from the n-hexane extracts of stem bark of Tetracera indica. betulonic acid was first reported from T. indica.