Flavanol Derivatives from Rhizophora stylosa and Their DPPH Radical Scavenging Activity

A new acetylated flavanol, 3,7-O-diacetyl (-)-epicatechin (3), and seven known flavanol derivatives, (-)-epicatechin (1), 3-O-acetyl (-)-epicatechin (2), 3,3',4',5,7-O-pentaacetyl (-)-epicatechin (4), (+)-afzelechin (5), (+)-catechin (6), cinchonain Ib (7), and proanthocyanidin B2 (8), were isolated from the stems and twigs of the mangrove plant Rhizophora stylosa and identified. The crude extract, the different fractions and all of the purified compounds were evaluated for DPPH radical scavenging activity.


Introduction
Mangrove plants are well-known as a rich source of tannins.The bark of true mangrove plants contains about 1%~30% tannins [1].The vegetable tannins or plant polyphenols group is comprised of two classes of compounds, the "hydrolysable" and the "condensed" tannins.Flavan-3-ols constitute the latter group [2].Some species of the mangrove plant genus Rhizophora are used by the local people as folk medicines for many diseases.For example, the bark of R. mucronata is used for the treatment of diabetes in India [3], and when boiled in water it is used as an astringent for diarrhea, nausea, and vomiting and as an antiseptic in Thailand [4].The extract of some species of this genus has been reported to have antifungal activity [5], antibacterial activity [6], anti-inflammatory activity [7], gastric antiulcer properties [8], efficacy in wound healing [9], and a protective role in naphthaleneinduced mitochondrial dysfunction [10].Furthermore, the major active principles of these pharmacological and biomedicinal properties are polyphenols that behave as antioxidants.

DPPH Radical Scavenging Activity of isolated compounds
The DPPH radical scavenging activity of compounds 1-8 was presented in Table .1.The results revealed that except for compound 4, compounds 1-8 isolated from R. stylosa displayed DPPH radical scavenging activity comparable to that of the positive control butylated hydroxytoluene (BHT).Compound 8 showed the strongest activity, with IC 50 4.3 µg/mL, four times more active than the positive control, BHT (IC 50 18.0 µg/mL).The DPPH radical scavenging activities of these compounds seemed to be related to the number of phenol groups.Data analysis revealed that compounds having adjacent hydroxyl groups on the ring-B possessed higher activity, a fact confirmed by the literature [18].From the above data, it can be deduced that the main components responsible for the antioxidant activities of R. stylosa extracts were the flavanol derivatives.Further work is in progress to assess whether this species could be used as a source of natural antioxidant for pharmaceutical and food applications.

General
Optical rotations were obtained on JASCO P-1020 digital polarimeter.UV spectra were recorded on a PuXi TU-1810 UV-visible spectrophotometer.IR spectra were taken on a Nicolet 510P FT-IR spectrometer.NMR spectra were recorded on a Bruker Avance 500 MHz NMR spectrometer using TMS as internal standard and chemical shifts were recorded as δ values (500 MHz for 1 H and 125 MHz for 13 C).ESI-MS were measured on a VG AutoSpec 3000 mass spectrometer.Silica gel (200-300 mesh, Qingdao Haiyang Chemical Factory, Qingdao, China) and reversed-phase silica gel C 18 (40-75 µm, Fuji Silysia Chemical Ltd) were used for open column chromatography (CC).Sephadex LH-20 (18-110 µm) was obtained from Pharmacia.Thin-layer chromatography (TLC) was performed on precoated 0.2 mm thick silica gel 60 GF 254 plates (Qingdao Haiyang Chemical Factory, Qingdao, China); spots were detected by spraying with anisaldehyde-H 2 SO 4 reagent, followed by heating.

Plant Material
The stems and twigs of Rhizophora stylosa Griff.were collected on Hainan Island on the South China coastline in July 2004 and authenticated by Prof. H. Peng at Kunming Institute of Botany, Chinese Academy of Sciences.A voucher specimen (0407A) was deposited at the Key Laboratory of Experimental Marine Biology of the Institute of Oceanology, Chinese Academy of Sciences.

DPPH Radical Scavenging Activity
The scavenging effects of samples on DPPH radicals were determined according to the method of literature [11].Synthetic antioxidant BHT was used as positive control.

Figure 1 .
Figure 1.The inhibition ratio (percentage) of extract and different fractions derived from R. stylosa (at 10 µg/mL) in DPPH radical scavenging assay.The results are given as means ± SD (n = 3).