Four New Jacaranone Analogs from the Fruits of a Beibu Gulf Mangrove Avicennia marina

Four new jacaranone analogs, marinoids F–I (1–4), were isolated from the fruits of a Beibu Gulf mangrove Avicennia marina. The structures were elucidated based on analysis of spectroscopic data. Marinoids F and G are shown to be diastereoisomers of chlorocornoside, a new halogen containing marine secondary metabolite. The antioxidant activity of the isolates was evaluated using a cellular antioxidant assay, and 4 showed good antioxidant activity (EC50 = 26 μM).


Introduction
Avicennia marina (Forsk.) Vierh. is commonly known as the grey or white mangrove plant resident in the tropical and subtropical regions, it is extremely widespread along the coasts of eastern Africa, islands of the Indian Ocean, tropical Asia, Australia, New Zealand, and islands of the Pacific Ocean to Fiji [1]. The crude extracts are reported to possess antimalarial and cytotoxic activities [1]. Different parts of the plant are used in Egypt as a folk medicine cure for skin diseases [2]. Previous chemical investigation of plants of the genus Avicennina have exhibited the presence of iridoid glucosides, marinoids A-E [1][2][3][4][5], naphthoquinone derivatives [6,7], flavonoids [4,8], and diterpenoids [9]. However, these previous studies did not report any chemical and biological data from the fruits of A. marina. Searching for bioactive secondary metabolites from this specimen afforded four new jacaranone analogs, marinoids F-I (1-4 respectively) ( Figure 1). In this paper, we describe the isolation, structural elucidation, and antioxidant activity of the four new secondary metabolites.

Results and Discussion
Marinoid F (1) was purified as a yellow oil with the molecular formula C 14 -4), in addition to the presence of four olefinic groups [δ C 152.8 (C-6), 148.9 (C-2), 130.5 (C-3) and 125.8 (C-5)], and a quaternary sp 3 carbon (δ C 70.2, C-1) (Table 1), demonstrated that 1 has a para-quinol-type partial structure [10,11]. NMR spectra also indicated the presence of a β-glucosyl group, i.e., one anomeric carbon resonance at δ C 102.6 (C-1ʺ) and one anomeric proton at δ H 4.18 (1H, d, J = 9.2 Hz, H-1ʺ). It was used as a starting point in the homonuclear correlated spectra to determine all glycosidic protons. The J H-1ʺ-H-2ʺ value (9.2 Hz) of compound 1, further confirmed that the sugar was a β-glucosyl group [12]. The gross structure was further established by the aid of COSY and HMBC experiments ( Figure 2). A careful comparison of 1 and cornoside revealed that 1 differs from cornoside by the presence of one chlorine atom attached at C-3 [13].  [13], whose stereochemistry of the aglycone and the β-D-glucosyl residue have been established by enzymatic hydrolysis and other methods [13][14][15]. The reported rotation value after poly-acetylation of cornoside is also negative ([α] 20 D −10.2°) [16]. Moreover, the NMR data of the β-glucosyl residue in compound 1 are in accord with those observed in cornoside [13]. The substitution is simply that of one Cl atom for one H atom, and that many bonds away from the key chiral centre, therefore following those Literature data, we propose that the configuration of C-1 in compound 1 is the same as that found at C-1 in cornoside, namely R. Thus, the structure of 1 is predicted to be as shown in Figure 1.
Marinoid G (2) was obtained as yellow oil. Its molecular formula was determined as C 14 [13], and the observed value for 1 is −14.7°. Although we do not have an explanation for the differences in the absolute values of the optical rotations of cornoside, poly-acetylated of cornoside, and compound 1, and the NMR data of the β-glucosyl residue in compound 2 are greatly similar to those of the β-glucosyl residue in cornoside and compound 1, and also compound 2 showed a positive Cotton effect at 220 nm (Δε +5.97), whereas the observed value for 1 was −4.28, the opposite optical rotation and Cotton effect indicate that 1 and 2 are diastereoisomers. Indeed, we propose that they are enantiomers of the aglycone each with β-glucosyl residues. values [18]. The tentative molecular weight of the compound deduced from NMR analysis suggested compound 3 to be an unsymmetrical dimer.  The connectivity of the two cyclohexanone moieties to C-1′ and C-1ʺʺ were established by the HMBC correlations of H-2 to C-1ʹ and H-6 to C-1ʹ, and of H-2‴ to C-1ʺʺ and H-6‴ to C-1ʺʺ, respectively. The presence of the two β-glucosyl groups in 3 could be proposed on the basis of HMBC correlations from H-4ʺ to C-1′ and H-1ʺ‴ to C-1ʺʺ, respectively. The connection of the two β-glucosyl groups was confirmed by the presence of HMBC correlation from H-4ʺ‴ to C-1ʺ. The configuration of C-1 and C-1‴ were assigned as R because a negative [α] 20 D was observed, which was in accord with that observed in cornoside ([α] 20 D −10.5°), and analysis of the 13 C NMR data of C-1 and C-1‴ in 3 indicated that they were greatly similar to that of C-1 (δ C 68.9) in 4-[2-(β-D-glucopyranosyloxy)ethyl]-4-hydroxy-2-cyclohexen-1-one obtained from Millingtonia hortensis [19]. Consequently, the structure of 3 was determined as showed in Figure 1.
The cellular antioxidant assay (CAA) is a new approach to quantify antioxidants under physiological conditions when compared to chemical antioxidant activity assays [22][23][24]. The CAA assay has been widely used for fruits and vegetables recently, but not yet in marine natural products research. The EC 50 values of compounds 1-3 were weak, 598, 4971, and 1103 μM, respectively. However, the EC 50 value of compound 4 was 26 μM, of the same order of the positive control quercetin (EC 50 = 11 μM).

General Experimental Procedures
UV spectra were recorded in MeOH on a Perkin-Elmer Lambda 35 UV-Vis spectrophotometer (Wellesley, MA, USA). The IR spectra were measured in KBr on a WQF-410 FT-IR spectrophotometer (Beifen-Ruili, Beijing, China). NMR spectra were recorded on a Bruker AV 600 NMR spectrometer (Bruker, Bremen, Germany) with TMS as an internal standard. HR-ESI-MS data were obtained from Bruker Maxis mass spectrometer (Bruker, Bremen, Germany). Waters-2695 HPLC system (Waters, Milford, MA, USA), using a Sunfire™ C 18 column (150 × 10 mm i.d., 10 μm, Waters, Milford, MA, USA) coupled to a Waters 2998 photodiode array detector (Waters, Milford, MA, USA). Optical rotation data were measured by Perkin-Elmer Model 341 polarimeter (Wellesley, MA, USA). CD spectra were recorded on a spectropolarimeter (MODEL J-810-150S, Tokyo, Japan). The silica gel GF 254 used for TLC were supplied by the Qingdao Marine Chemical Factory, Qingdao, China. Spots were detected on TLC under UV light or by heating after spraying with 5% H 2 SO 4 in EtOH. All solvent ratios are measured v/v.

Plant Material
The fruits of A. marina were collected from Beihai city, Guangxi province, China, in September, 2011. The specimen was identified by Professor Hangqing Fan who is from Guangxi Mangrove Research Center, Guangxi Academy of Sciences. A voucher specimen (2011-GXAS-008) was deposited in Guangxi Key Laboratory of Marine Environmental Science, Guangxi Academy of Sciences, China.

Extraction and Isolation
The fruits of A. marina (35.4 kg, wet weight) were exhaustively extracted with EtOH-CH 2 Cl 2 (2:1, v/v). The solvent was evaporated in vacuo to afford a syrupy residue that was suspended in distilled water and fractionated successively with petroleum ether, ethyl acetate, and n-butanol. The n-butanol soluble portion (269 g) was subjected to column chromatography (CC) on silica gel, using

Conclusions
In conclusion, four new jacaranone analogs, marinoids F-I (1-4 respectively), were isolated from a Beibu Gulf mangrove A. marina and identified. Marinoids F and G are shown to be diastereoisomers of chlorocornoside, a new halogen containing marine secondary metabolite. The CAA assay is considered to be a more physiologically relevant assay in the measurement of antioxidant activity of food when compared to the common chemistry antioxidant activity assays [25,26]. Until today, there have been no reports of the use of this CAA assay in the marine research area. Using the assay, the antioxidant activity of the isolates was therefore determined. This is the first report of chlorocornoside and of the dimeric disaccharide 4 which showed good antioxidant activity (EC 50 = 26 μM), comparable with the positive control quercetin.