Mastura, Tonel Barus, Lamek Marpaung, & Partomuan Simanjuntak : Isolation and Antioxidant Activity of Phenolic Compounds from Halban Leaves (Vitex pinnata Linn) in Aceh

An isolation and identification of phenolic compounds from Halban Leaves (Vitex pinnata Linn), a traditional medicinal plant in Aceh, has been done. Halban leaves were extracted through methanol maceration followed by isolation. The methanol extract went into n-hexane and ethyl acetate partition consecutively resulting in 3 different fractions of ethyl acetate and n-hexane extract. The ethyl acetate fraction demonstrated the most promising antioxidant and cytotoxic activity. Therefore, purification of ethyl acetate fraction was done by column chromatography [SiO2; (i) nhexane-ethyl acetate 10:1, 1:1 ; (ii) n-hexane-ethyl acetate = 2 : 1); (iii) n-hexaneacetone = 1:1]. The pure isolate as identified by using UV, IR, NMR, and MS spectra afforded 2 phenolic compounds, namely (1) 4-hydroxymethyl benzoate and (2) phydroxymethyl benzoic acid (PHBA). The antioxidant activity of compound 1 and cytotoxicity activity of 2 expressed in IC50 and LD50 was 41.08 ppm and 59.41 ppm, respectively.


Introduction
Vitex pinnata L is known as laban, halban or "Mane" in Acehnese term which means Asian tropical plants with huge potential as a medicinal plant. Almost all parts of the plants can be applied as medicine. As a traditional medicine, the leaves are widely used to treat wounds and fever also to increase appetite. The stem bark is reported to be able to cure stomach ache, wounds and be used as a colouring agent while the roots can be used as stomach pain medicine .  reported that the decoction of V. pinnata bark could cure stomach pain and the leaves can be used as fever and wounds treatment. People in Aceh use Halban leaves as anti-cholesterol, gastric and high blood pressure medications. The fruits are used for carbuncle and fever (Mastura et al., 2018).
The ethnobotany approach allows us to assume that V. pinnata contains active compounds to treat fever, carbuncle, stomach ache and wounds . Generally, Vitex contains chemical compounds like parahydroxymethyl benzoic acid (PBHA) and agnuside (AGN) in which they can be found in genus Vitex negundo and V. trifolia (Bello et al., 2017). Roy et al., 2015 also reported that Vitex negundo Linn exhibited other herbal activities such as anti-inflammatory, antiarthritic, anti-analgesic and hepatoprotective. In Brunei, the young leaves are consumed to treat hypertension and fever. Tea made of the root of the plants is used to treat back pain, body aches and fatigue (Goh et al., 2017). This compound is important to be isolated to determine its chemical structure. The Vitex pinnata L. plant is important for testing its antioxidant activity because the compound to be selected for its chemical structure must be biologically active. The objective of the study is to isolate and identify chemical compounds in Halban leaves (V. pinnata L) from Aceh and the bioactivity test.

Research Methodology Materials
Materials used in this study include leaves of Vitex pinnata L., methanol, water, n-hexane, ethyl acetate.

Extraction
5 kg of Halban leaves is macerated with methanol for 48 hours in triplicate, filtered and evaporated in a rotary evaporator to obtain dense methanol extract. Water was added to the methanol extract, then filtered. The filtrate was partitioned using n-hexane with 4 repetitions. The fraction was evaporated with a rotary evaporator, and it gave 3 different types of extract, which are n-hexane, ethyl acetate and water extract .

Bioactivity: Antioxidant and Toxicity Activity Test
Antioxidant activity was tested based on Molynes procedure in which DPPH concentration has been modified (Molyneux, 2004) whereas toxicity test was done using Brine Shrimp Lethality Test (BSLT). BSLT method is very suitable for the isolated bioactive compounds from plant extracts. Toxicity test was based on Meyer procedure with modifications using live animals (Yang et al., 2015). Extract solutions were made with concentrations of 2000 ppm, 1000 ppm, 200 ppm, 100 ppm, 20 ppm and 10 ppm. The extract used was 40 mg and 5-20 mL of seawater. If the sample is insoluble, 5 drops of 1% DMSO are used. The toxicity test was carried out by inserting 15 larvae of Artemia Salina Leach shrimp for 48 hours into a bottle containing the extract solution and seawater, with the concentration as above. For each concentration carried out 3 times (triple). As a control, seawater was not given a sample extract. The test vials were stored under a TL lamp. Observations were made after 24 hours. The number of shrimp larvae that died was recorded then the percentage of death was calculated. The data obtained were processed using probit analysis.

Identification
Identification was made to both pure VDH 9-3-1 and VDH 8-3-2 compounds by taking spectra data from Ultra Violet (UV), Infrared (IR), Nuclear Magnetic Resonance ( 1 H-NMR and 13 C-NMR) and Mass Spectra. At the characterization stage, the equipment used was the Variant Conc.100 ultraviolet spectrophotometer, the Perkin Elmer Spectrum One FTIR spectrophotometer, the JEOL JNM A5000 NMR spectrometer which operates at 500 MHz NMR (1H) and 125 MHz (13C) frequencies using TMS as the internal standard.

Discussions
The extract of Halban leaves (Vitex pinnata Linn) from Aceh gives more water fraction (%) compared to the others (n-hexane and ethyl acetate fraction). Halban leaves are assumed to contain more polar compounds than non-polar or semipolar compounds (Breda et al., 2016). The yield of Halban leaves extract can be seen in Table 1.  -Kang et al., 2016). The results of the toxicity test can be seen in Table 2. An extract is called active and has toxic properties if it causes 50% death of test animal at the concentration below 1,000 ppm and does not have toxic properties if it is found at above 1,000 ppm (Sobolewska et al., 2016;Molyneux, 2004).
Fraction VDH 9 -3-13 obtained is a transparent white crystal. The result of the antioxidant activity test from free radical scavenging method using DPPH (1,1-diphenyl-2-picrylhydrazyl) reagent. The results of antioxidant activity test for VDH 9-3-13 in resistance percentage and IC 50 values can be seen in Table 3 (Iqbal et al., 2015). Based on antioxidant activity test shown in Table 3 above, VDH 9-3-13 has IC 50 (Inhibition Concentration) value of 41.087. This indicates that the compound has a very strong antioxidant activity.

Identification of VDH 8-3-2 and VDH 9-3-13 Compounds based on Spectra Data
Based on UV spectra, VDH 8-3-2 gives a peak at 251.5 nm wavelength. According to (Shah, 2016), the peak at 230 nm wavelength is a carboxylic acid which binds with benzene functional groups (λ230 nm) and OH attached at para position (25 nm). It is predicted that the functional group appears at 255 nm wavelength. While VDH 9-3-13 gives a peak at λ255.5 nm. According to (Shah, 2016), the peak at 230 nm is methyl functional group that is attached with carboxylic acid and benzene functional group (λ230 nm), and OH is binded at para position (25 nm). Infrared spectra show VDH 8-3-2 contains OH functional groups at 3429.43 cm -1 wavenumbers, carboxylic acid carbonyl group at 1726.29 cm -1 and C=C aromatic benzene group at 1600.02 cm -1 wavenumber. Hydroxyl group in Infrared spectra for pure VDH 9-3-13 is shown in 3350 cm -1 wavelength, carboxyl group at 1787 cm -1 and benzene aromatic is shown at 1606 cm -1 (da Silva et al., 2016).
The analysis on NMR from 13 C-NMR for VDH 8-3-2 gives 5 signal of Csp 2, which represents 7 carbon atoms. The 5 signals consist of one conjugated carbonyl (carboxylate) (C-7), which is very specific at carbon shifting (δ C 168.10 ppm). The other 4 signals (C-2, C-6) and (C-3, C-5) have carbon shifting (δ C 137.2 ppm) and (δ C 116.00 ppm), respectively. Benzene at C4 carbon shifts (δ C 162.7 ppm) is oxi aryl carbon. Whereas for C-1 carbon shift is (δ C 122.4 ppm). Spectra data 1 H-NMR for VDH 8-3-2 shows that there are two signals for the aromatic area with 2H integration each. Both signals, H-2 and H-6, are found in the shifts, (δ H 7.93 ppm) and they have doublet multiplicity while H-3 and H-5 have proton shift (δ H 6.92 ppm). The signals with those characteristics are specific for 1,4disubstituted benzene. The chemical shift values of 1 H-NMR and 13 C-NMR from VDH 8-3-2 compound is concluded in Table 4 (Juvik et al., 2016). Because the chemical structure of compounds is very simple, only 1-D NMR was used. Based on UV, IR, proton and carbon NMR spectra data, the chemical structure of the pure VDH 8-3-2 compound can be determined as para hydroxybenzoic acid (PHBA). The chemical structure of the compound in Halban leaves from Aceh (V. pinnata L) is shown in Figure 1  . Proton NMR spectra analysis of VDH 9-3-13 shows proton shift at δ H 3.81 (OCH 3 ), δ H 7.89 (2 H) dan δ H 6.91 (2H) (aromatic functional groups). Carbon NMR gives 8 carbon atoms which consist of five -CH= (d); one (s) -C, one C=O and one OCH 3 . The shift value of 1 H-NMR 13 C-NMR of VDH 9-3-13 is concluded in Table 5. Based on Ultra Violet (UV) data, there is benzene chromophore functional groups. From infrared (IR), there is OH, C=O and aromatic functional groups. Proton NMR shows OCH 3 functional groups and CH aromatic while carbon NMR gives OCH 3 , carbonyl and aromatic. Therefore the chemical structure of VDH 9-3-13 isolated from Halban leaves (V. pinnata L) from Aceh can be determined as 4-hydroxy methyl benzoate compound   (Figure 2).

Conclusions
From the research, it can be concluded that Halban leaves (V. pinnata Linn) from Aceh. Ethyl acetate fraction contains phenolic compounds such as para hydroxybenzoic acid (PHBA) and 4-hydroxy methyl benzoate. Based on the test results of all fractions, only PHBA has antioxidant activity, and only 4-hydroxy methyl benzoate has cytotoxic activity. Bioactivity test for both compounds shows that PHBA has toxicity with LC 50 = 59.41 and 4-hydroxy methyl benzoate has antioxidant activity with IC 50 = 41.807.