3,3’-di-O-methylellagic acid, an Antioxidant Phenolics Compound from Sonneratia alba Bark

Free radicals play an important role in some pathogenesis of serious diseases, such as neurodegenerative disorders, cancer, liver cirrhosis, cardiovascular diseases, atherosclerosis, cataracts, diabetes and inflammation. Compounds that can scavenge free radicals have great potential in ameliorating these diseases. It is reported that phenolic compounds in plants possess strong antioxidant activity and may help to protect cells against the oxidative damage caused by free radicals. Previous study revealed that mangrove trees, Sonneratia alba Bark showed strong antioxidant activity. Ethyl acetate fraction exhibited the best antioxidant performance. The antioxidant activity of this fraction was attributed to the presence of compounds of different polarities such as phenolics. Furthermore, the phenolic antioxidant of ethyl acetate fraction were purified and identified with UV, IR, MS, and 2D NMR spectrometry. 3,3’-di-O-methylellagic acid was found in brown amourphous powder. Antioxidant activity was evaluated and compared with L-(+)-ascorbic acid as standard by using DPPH assay. The compound has strong antioxidant activity than ascorbic acid standar with the value of IC 50 11.35 and 17.64 μg/mL, respectively. The high value of antioxidant activity of compound indicate that S. alba is a potential source of natural antioxidant agent.


INTRODUCTION
There is an increasing interest in the role of free radicalmediated damage in the aetiology of human diseases.Free radicals formed during oxidation process occurring in various products and biological systems are known to be responsible for oxidative deterioration, health damage and accelerated aging (Aruoma 2003).Consequently, antioxidants have became an essential part of preservation technology and contemporary health care.Synthetic antioxidants have been widely used in food and medicine.The most commonly used synthetic antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and tert-butylated hydroxyquinone (TBHQ) (Lo liger 1991).However, BHA, and BHT were found to be anticarcinogenic as well as carcinogenic in experimental animals.Originally, BHA appeared to have tumor-initiating as well as tumor-promoting action.Recently, it has been established that tumor formation appears to involve only tumor promotion caused by BHA and BHT (Botterweck et al. 2000).The potential toxicity of some synthetic antioxidants, however, has intensified efforts to discover and utilise antioxidants from natural sources (Madsen & Bertelsen 1995;Nieto et al. 1993;Shahidi & Wanasundara 1992;Thamavit et al. 1985).
Sonneratia alba, one of mangrove species, has been used as preservative in production of alcoholic traditional beverage from palm trees.It has been reported that S. alba bark extract played an important role to prevent the formation of acetate acid generated from oxidation of ethanol (Firdaus & Sinda 2003).Furthermore, extract of this plant has been used as astringent, antiseptic, and to treat internal bleeding and disentry.We suggest that preservative property of this plant due to the presence of antioxidant or antibacterial compound (Bandarnayake 2002;Kumar et al. 2008).
Therefore, this plant might be a good candidate for further development as a nutraceutical or for its antioxidant remedies.
However, the potential compound of the bark extracts of S. alba as a source of natural antioxidant have not been studied in detail to date.Therefore, it is important to find out the compound that responsible for the extract activity.
The stable radical species 1,1-diphenyl-2-picrylhydrazyl (DPPH), has been widely used for antioxidant capacity screening and estimation due to its clear reaction mechanism, solvent compatibility and the technical simplicity of its assays which requires no special equipment (Chen et al. 1999;Cheng et al. 2006).The purple coloured of DPPH has a strong characteristic absorption at 515 nm and can undergo reactions with hydrogen donating antioxidant compounds to yield the stable yellow DPPH-H molecule easily monitored with Ultra Violet (UV) spectroscopy (Prior et al. 2005;Roginsky & Lissi 2005).In this study, several column chromatography (CC) were applied separate and purify the S. alba bark extracts; and their antioxidant activities were further evaluated by 1, 1-dipheny1-2-picrylhydrazyl (DPPH) free radical scavenging assay.EtAOc showed the highest antioxidant activity, so only this fraction would be continued for further analysis (Figure 1).2).Thus, compound 1 was identified as 3,3'-di-O-methylellagic acid (Figure 2).

Materials
IR spectrum of compound 1 showed an absorption in the region above 3000 cm -1 (3232 cm -1 ) derived from The DPPH assay, which measures the ability of compounds to transfer labile H-atoms to radicals, is the commonest method of antioxidant activity evaluations (Brand-Williams et al. 1995).The abstraction of hydrogen by this stable free radical is known to lead to the bleaching of the maximum absorption 517 nm and can easily be monitored spectrophotometrically.The synthetic nitrogencentered DPPH radical is not biologically relevant but DPPH assay is often used to evaluate the ability of antioxidant to scavenge free radicals which are known to be a major factor

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All chemical and reagent were analytical grade purchased from Merck (Darmstard-Germany). 1, 1diphenyl-2-picryl hydrazyl (DPPH), L-(+)-ascorbic acid, hexane, chloroform, ethyl acetate, methanol, column Chromatography was carried out on Merck silica gel (200-400 mesh).Sample preparing was carried out at Organic Chemistry laboratory of Hasanuddin University.The measuring of 1H, 13C, EI-MS, UV, and IR spectra was done at LIPI Serpong Banten.Antioxidant activity was determined at Uji Biofarmaka Laboratory of LPPM IPB Bogor.Extraction of Bark.Dried and powdered bark of S. alba (10 kg) were extracted with methanol.After filtration, the combined extracts were evaporated to dryness to produce the methanolic extract (183 g).The methanolic extract was dissolved in a mixture of methanol:water (2:3, v/v) at room temperature and partitioned successively with hexane, chloroform and ethyl acetate, produced hexane (23 g), chloroform (49 g) and ethyl acetate (65 g) extracts, respectively.The extracts were concentrated under vacuum.

Figure 2
Figure 1 Extraction, fractionation and column chromatography separation of S. alba Bark unsaturated CH bond.The presence of absorption at 1612 cm -1 support that CH is derived from unsaturated aromatic group.Strong absorptions at 1730 cm -1 indicates that the compound contains a carbonyl group (C = O), and the absorption is specific for lactone (cyclic ester).Broader absorption at 3277 cm -1 indicates that the compound contained an OH group, this absorption is too low for the range of NH groups.The presence of the methyl group is characterized by the presence of absorption in the region near 1385 cm -1 and also at 1354 cm -1 .The existence of more than one absorption in the region 1000-1300 cm -1 states that there are many type C-O bonding.UV spectrum (methanol) shows a maximum absorption at 249 nm (methanol).This data is consistent with the molecular structure of obtained compounds that indicate the presence of substituted aromatic systems.3,3'-di-O-methylellagic acid is not a new compound.Tian et al. (2009) has reported several compounds from S. caseolaries involve 3,3'-di-O-methylellagic acid.It leads that this compound may attributed compound to the family of mangrove trees, Sonneraceae.IC 50 values of extract, fractions, compound from S. alba Bark, and control were determined from linier regression as showen in Tabel 1. 3,3'-di-O-methylellagate acid, and L-(+)ascorbic acid showed concentration dependence-activity until reach the maximum (Figure 3).Maximum activity is reached at concentration 34 µg/mL for 3,3'-di-O-methylellagic acid.