Flavon Compound from The Ethyl Acetate Extract of The Stem of Supit ( Tetracera indica Merr

Tetracera indica Merr. in Musi Banyuasin, is one of the traditional medicine used by the community for the treatment of kidney stone disease and gout, but this claim is not recorded in the treatment of kidney stones and gout in Indonesia. In this study, isolation of antioxidant compound from ethyl acetate extracts of supit (Tetracera indica) was done. The isolation was carried out through step gradient polarity extraction, and separated and purified by chromatography technique. The determination of the structure of the isolated compound was performed by spectroscopy method including UV, IR, and NMR 1D and 2D, and antoxidant activity was determined by DPPH method. An active antioxidant compound was isolated from ethyl acetate extract in form of yellow solid (15 mg). Based on spectroscopic analysis the isolated compound was 5,8dihydroxy-7-methoxyflavone. The compound showed strong antioxidant activity (IC50 8.25 μg/mL) higher than standard ascorbic acid (IC50 11.3 μg/mL). This data concluded that efficacy of supit (Tetracera indica) for the treatment related to antioxidant activity (uric acid) is proven by the identification of one antioxidant compound of this plant. Key word: Flavon, ethyl acetate extract, Tetracera indica, antioxidant


INTRODUCTION
Indonesia is a country with the second largest diversity of plants in the world, but there are still many medicinal plants that have not been fully utilized (Hanafi, Nina, Zorni, and Nurbaiti, 2005).The use of medicinal plants has been practiced by the Indonesian people for generations.Indonesian herbal medicines are used based on empirical practice: diseases preventive (48.9%) (Hanafi et al, 2005).Although modern medicine has grown tremendously in Indonesia, but traditional medicine is still very popular in rural as well as in urban areas (Elfahmi, Herman, Woerdenbag, and Oliver, 2014).Moreover to having a high diversity of plants, Indonesia is also rich with tribal and cultural diversity (Ernie, 2013).In some tribes, endemic plants was found to be used as traditional treatment.Some times in some tribes are found endemic plants used for traditional treatment.
In previous research has been done exploration and inventory of medicinal plants and their utilization in society based on local resources.One of them is the ethnic Musi in Musi Banyuasin district of South Sumatra.In research of medicinal plants and herbal medicine has been identified 95 species of medicinal plants (Yustian, Muharni, Sukarmi, Zulaicha, and Arbi, 2012).From the 20 selected plant, 9 species of them are traditionally used for the treatment of diseases atherosclerosis, diabetes , prostate, gout, and kidney stones.These types of diseases are related to free radical activity (Paravicini and Touyz, 2008).
In vitro antioxidant activity test of 9 selected plant extracts is one of them is supit stems, showed high antioxidant activity with value of % inhibition 69.6% at test concentration 1000 ppm.Based on literature study of extracts that showed antioxidant activity> 50% categorized as active source of antioxidant compound (Chaudhary, Bhandari, and Pandurangan, 2011;Muharni, Fitrya, and Nurmaliana, 2016).
Plants produce a wide diversity of secondary metabolites exhibit and a wide array of biological and pharmacological properties (Michael, 2015).Secondary metabolites from plants are the basis for many drugs currently used to treat various diseases, including diseases related to radical activity (Rogerio, Sá-Nunes, and Faccioli, 2010).Phytochemical tests of ethanol ektract of supit (Tetracera indica) have been performed and exhibited positive phenolic compounds.Phenolic compounds are generally active as antioxidant (Kenari, Mohsenzades, and Amiri, 2014;Brewer, 2011;Kumar, Mishra, and Pandey, 2013).The limited ingredients available medicines has prompted researchers to explore the potential of nature to find new bioactive compounds.
In another study of Tetracera indica leaf extract containing flavonoid wagonin compound was active as an antibacterial (Lima, Lemos, and Conserva, 2014).Abdullah, Ismail, Jamaludin, and Mashim (2013) reported that the stems of supit show activity as antihiperuricemia, antidiabetic and anti-inflammatory and two compound has been reported of stem supit that is betulinic acid and 5,7-dihydroxyl-8-methoxyflavone.In this paper will be reported antioxidant compound active by DPPH method from ethyl acetate extract stem of supit.

Preparation of Extracts
The stem of Tetracera indica were dried at 30 °C for 20 days with continuous moisture monitoring.After the material was completely dry, it was pulverized in a knife grinder, obtained 550 g of sample.The dried stem bark were subjected to exhaustive extraction in maceration apparatus using an increasing polarity solvent system, with nhexane, ethyl acetate and ethanol as solvents each for 24 h.The extracts were then concentrated at reduced pressure used Rotary evaporator.Each of the extracts was tested antioxidant activity.

Evaluation of Antioxidant Activity of Each Extract
The antioxidant activity test was performed using DPPH radical-scavenging activity (Selvi, Joseph, and Jayaprakasha, 2003;Tuanjai, Supalax, Thawatchai, and Wittaya, 2011).The concentration series for extract :1000, 500, 250, 125, 62.5 and 31.25 1000 µg/mL (in DMSO) was prepared.0.2 mL aliquot of sample solution was mixed with 3.8 mL of DPPH (0.5 mM in methanol) where 1.98 mg DPPH was placed on a 100 mL volumetric flask then add methanol until the volume 100 mL.This mixture was shaken at room temperature for 30 min.
The absorbance of the mixture was then measured by UV-Vis spectrophotometer at λmax 517 nm (Selvi et al., 2003).For positive control (standard) used ascorbic acid and negative control (blank) used methanol.Isolated compound solution was analyzed in triplicate, The radical-scavenging activity was evaluated as the percentage of inhibition according to the following equation: %inhibition= [(absorbance of controlabsorbance of sample)/ absorbance of control)]x 100 (Delwar, Shahid, Masudur, Shohel, Shahid, and Mohammad, 2014) and the average values were plotted to obtain the IC50 against DPPH by linear regression.

Elucidation Structure
The structure of the isolated compound was elucidated using UV, IR, NMR 1D( 1 H and 13 C NMR, DEPT), and NMR 2D (HMQC, and HMBC) spectroscopy and by comparison with data from the literature.

Evaluation Antioxidant Activity of Isolated Compound
The pure isolated compound was determined antioxidant activity by the same procedure as the antioxidant activity assay of the extract but using the concentration 100, 50, 25, 12.5 and 6.75 µg/mL.

RESULTS AND DISCUSSION
The stem of supit ( 2 kg ) after dried obtained 550 g of powder.The powder Tetracera indica of 550 g was extracted with n-hexane solvent, ethyl acetate and ethanol respectively and after concentrated was obtained n-hexane extract 250 mg, ethyl acetate 3,50 g and ethanol extract 3.7 g.Each extract, has been done antioxidant activity test with varying concentration (Table 1).Mariod, Matthaus and Hussein, (2008) state that the value of antioxidant activity is determined based on the value of percentage inhibition, the higher the value of the inhibition, the higher the antioxidant activity.
Table 1 showed that at the same concentration 1000 μg / mL, the ethanol extract showed the highest activity with inhibition 94.27% followed by ethyl acetate extract with inhibition 90.12%.Meanwhile, nhexane extract only gives an inhibition of 1 2 3 29.63%.Extracts are categorized potent containing antioxidant compounds when its have percent inhibition > 50% at a concentration of 1000 μg/mL (Chaudhary et al., 2012).Based on this data it is stated that the extract of ethyl acetate and ethanol extract from stems of supit (Tetracera indica) potentially contain antioxidant compounds.The Compounds that exhibit antioxidant activity generally provide fluorescent under UV light at a wavelength of λ 365 nm.Ethyl acetate extract after separation and purification a compound was isolated from the ethyl acetate extract of steam T. indica.The isolated compound was as a yellow solid (15 mg).The purity test of the isolated compound was carried out by TLC analysis using various eluents : n-hexane : ethyl acetate (9 : 1 ; 5 : 5) and EtOAC : MeOH (9.5 : 1.5) showed a single spot on the UV lamp at λ 365 nm (Figure 1).The 1 H NMR spectrum of the isolated compound (Figure 4) show the proton signal of methoxy at δH 4.04 (3H, s) and the signal of the aromatic proton at the δH 6.0-8.0 ppm .Signals at δH 6.43 (1H, brs) with widened peaks are signals for phenolic OH.Signals at δH 6.45 ppm (1H, s) and 6.69 (1H, s) are 2 non-split aromatic protons, 7,54 -7,56 (3H, m) are 3 proton aromatics that split with protons at δH 7.91 (2H, dd J = 3.85) and signal at δH 12.49 (1H, s) are signal for proton OH chelate.The 13 C NMR spectrum of the isolated compound showed 14 signals.Signal δC 62.2 ppm is characteristic signal for methoxy carbon.Signals at δC 99.00 -165 ppm are signals for C SP 2 from aromatic carbon.The signal at δC 182.4 ppm is a signal for C carbonyl in the form of a ketone.In HMQC spectrum (Figure 6) showed proton methoxy at δH 4.04 ppm bound to carbon δC 62.2 ppm and on spectrum of HMBC showed correlation with carbon at δC 127.0 ppm.The HMQC spectrum also showed proton at δH 6.45 ppm (1H, s) and δH 6.69 (1H, s) respectively bound to carbon at δC 99.0 ppm and δC 105.4 ppm.In the HMBC spectrum (Figure 6) showed protons at δH 6.45 ppm (1H, s) correlated with carbon at δC 106.1(C10), 127.0 (H8) 155.4 (C7), 157.9 ppm (C9).Meanwhile the proton at δ H 6.69 (1H, s) correlated with carbon at δC 106.1 (C10), 131.4 (1') 163.5 (C2), 182.4 ppm (C4).This indicates the carbon at δC 106.1 ppm was located between the carbon at δC 99.0 and 105.4 ppm.
The HMQC spectrum also showed proton at δH 7.56 (2H) ppm, 7.57 (1H, s) and Furthermore, it was also seen in the HMQC spectrum where proton at δH 12.49 ppm bound to carbon at δC 163.76 ppm and on

A B
the HMBC spectrum this proton visible correlated with carbon at δC 99.0; 105.4 and 157.9 ppm δC 99.0; 106.1 ppm.This indicates that this hydroxyl proton is a chelated proton adjacent to a proton δH 6.45 ppm.The correlation between proton and carbon is shown in Figure 8.Based on UV IR, NMR 1d and 2 D spectroscopy data, it was concluded that isolation compound was flavonoid 5,8dihydroxy-7-methoxyflavone with molecular formula C16H12O5 with the structure shown in Figure 8.

Antioxidant activity of isolated compound
Antioxidant activity test was done by DPPH method with concentration variation 100, 50, 25, 12.5 and 6.75 ppm.Antioxidant activity is determined based on the value of% inhibition (Table 3).
The higher the% inhibition, the stronger the antioxidant activity Table 3 showed the higher the concentration of the test the smaller the absorbance and the percent inhibition value will also be higher.Based on the percentage of inhibition seen at a concentration of 6.25 μg / mL, the isolation compound results showed higher activity than the standard ascorbic acid.To determine the value of IC50, the plot curve between concentration and the value of % inhibition based on the linear regression.Base on regression linear obtained equation shows that IC50 value of the isolated compound is 8.25 μg / mL, while the ascorbit acid gives IC50 value of 11.3 μg / mL.Based on this data concluded the efficacy of supit plants (Tetracera indica) for the treatment related to antioxidant activity (uric acid) is proven by the identification of one antioxidant compound of this plant.Isolatet compound is flavonoid group.Many flavonoids are shown to have antioxidant activity, such as for the prevention of coronary heart disease, hepatoprotective activity, anti-inflammatory, and anticancer (Kumar and Pandey, 2013).
The isolated compound 5,8-dihydroxy-7-methoxyplavone was known by another name wogonin and has been previously found on the leaf extract (Dogarai et al., 2011) and stem extract of Ttetracera indica (Abdullah et al., 2013), but is first reported as active antioxidant against DPPH radicals.

CONCLUSIONS
A flavon compound has been isolated from ethyl acetate extract of the stem of supit (Tetracera indica) and identified as 5,8dihydroxy-7-methoxyplavone.This compound showed strong antioxidant activity by DPPH method with IC50 value 8.25 μg / mL.

Figure 4 .
Figure 4. Spectra 1 H NMR of isolated compound

Figure 7 .
Figure 7. Spectrum HMBC at δH 6.2 -8.1 ppm and δC 12.2 -12.9 ppm and δH 100 -165 ppm.Based on this HMQC data the number of carbon signals is 16 signals and the compound has an aromatic group in the form of mono substitution.In the HMBC spectrum the proton signals at δ H 7.56 ppm correlate with the carbon at 131.4(1'), 129.4 ppm (C3',5'), meanwhile the signal at δH 7.91ppm Showed

Table 1 .
Antioxidant activity of each extract

Table 2 .
Chemical shift data of proton and carbon of the 1H and 13CNMR spectra of isolation compound at 500 MHz for 1 H and 125 MHz for 13 C in CDCl3.
A B Figure 8. HMBC correlation (A) and structure isolation compound (B)

Table 3 .
Antioxidant activity of isolated compound