Isolation and identification of phytochemical constituents from the fruits of Acanthopanax senticosus

Phytochemical constituents were isolated from the fruits of Acanthopanax senticosus by repeated chromatography and prep-HPLC. Their structures were identified as β-sitosterol (1), daucosterol (2), phydroxybenzoic acid (3), vanillic acid (4), uracil (5), eleutheroside K (6), songoroside A (7), copteroside B (8) and myo-inositol (9) by spectroscopic analysis. Among them, p-hydroxybenzoic acid (3), eleutheroside K (6) and songoroside A (7) were isolated for the first time from the fruits of A. senticosus, and songoroside A (7) was isolated for the first time from Acanthopanax species.

There are many reports on the analysis of phytochemicals in A. senticosus (Row and Song, 2004;Lee et al., 2004b;Apers et al., 2005;Kim et al., 2006;Li et al., 2006;Ma et al., 2011).There have been many reports on the medicinal effects, isolation, and identification of compounds from the roots, stems and leaves of A. senticosus.However, there have been few investigations of the fruits of A. senticosus.Therefore, this research is focused on the isolation and identification of compounds from A. senticosus fruits by repeated chromatography and recycling preparative high performance liquid chromatography (prep-HPLC).

Plant materials
The fruits of A. senticosus (Araliaceae) were collected at Gongju and verified by Prof. Seon Haeng Cho, Gongju National University of Education, Republic of Korea.A voucher specimen (No.LEE 2008-01) was deposited at the Herbarium of Department of Integrative Plant Science, Chung-Ang University, Republic of Korea.

General experimental procedures
Electron ionization mass spectrometry (EI-MS) was measured with a Jeol JMS-600 W (Tokyo, Japan) mass spectrometer. 1H-and 13 Cnuclear magnetic resonance (NMR) spectra were recorded with a Bruker Avance 300 or 500 NMR (Rheinstetten, Germany) spectrometers in CDCl 3 , C 5 D 5 N, DMSO or CD 3 OD using tetra methyl silane (TMS) as an internal standard.Chemical shifts were reported in parts per million () and coupling constants (J) were expressed in Hertz (Hz).Thin layer chromatography (TLC) analysis was conducted with Kiesel gel 60 F254 (Art.5715, Merck Co., Germany) plates (silica gel, 0.25 mm layer thickness), with compounds visualized by spraying with 10% H 2 SO 4 in MeOH.Repeated chromatography was conducted with a silica gel (200 to 400 mesh ASTM; Merck Co., Germany).All other chemicals and reagents were analytical grade.Prep-HPLC was conducted by a JAI LC-9104 (Tokyo, Japan) system equipped with an L-6050 pump and UV-3702 UV/VIS detector.

RESULTS AND DISCUSSION
A chromatographic separation of the MeOH extract of A. senticosus led to the isolation of compounds 1 to 9 (Figure 1).Compounds 1 and 2 were obtained as white powders from the CHCl 3 fractions. 1H-NMR spectra of 1 and 2 showed the existence of a sterol skeleton and a molecular ion peak at m/z 414 [M] + in the EI-MS and 577 [M + H] + in the FAB-MS.Two angular methyl singlets of H-18 and -19 at δ 0.67 to 0.68 and 0.94 to 1.01 and three doublets of H-21, -26 and -27 at δ 0.92 to 1.00, 0.81 to 0.94 and 0.86 to 0.89 were observed, respectively.An olefinic proton signal of H-6 was observed at δ 5.35. 13C-NMR spectra of compounds 1 and 2 showed 29 and 35 resonances, respectively.C-5 and -6 signals of compounds 1 and 2 were observed at δ 141.0 to 141.3 and 121.9 to 122.3, respectively.Compounds 1 and 2 had similar structural signals.The typical pattern of a glucose moiety was observed in the 1 H-and 13 C-NMR spectra in compound 2. The anomeric proton of compound 2 produced a peak at δ 5.09 (d, J = 6.9 Hz), and the glucose position was at C-3 (β-linkage) of the aglycone according to HMBC analysis.
Accordingly, the structures of compounds 3 and 4 were identified as p-hydroxybenzoic acid and vanillic acid,  respectively, by comparison of the spectral data, as described in the literature (Shimizu et al., 1983;Pyo et al., 2002;González-Baró et al., 2008;Zhang et al., 2011;Yuan et al., 2012).p-Hydroxybenzoic acid shows antioxidant activity on DPPH radical assay and the inhibition of lipoperoxidation (Yamaguchi et al., 2006).Vanillic acid is a phenolic derivative of edible plants and fruits and has antibacterial and antimicrobial properties against Listeria monocytogenes, Listeria innocua, Listeria grayi and Listeria seeligeri (Rai and Maurya, 1966;Delaquis et al., 2005).Compound 5 was obtained as white powder from the EtOAc fraction and showed a molecular ion peak at m/z 112 [M] + in the EI-MS.In the 1 H-NMR spectrum of compound 5, two doublets of typical olefinic proton signals at δ 7.41 (J = 7.8 Hz) and 5.63 (J = 7.8 Hz) were observed.Accordingly, the structure of compound 5 was elucidated as uracil by comparison of the spectral data, as described in the literature (Lee et al., 2002).Uracil can be used to determine microbial contamination of tomatoes as its presence is an indication of lactic acid bacteria contamination in the fruit (Hidalgo et al., 2005).Compounds 6 to 8 were obtained as amorphous powders from the EtOAc fraction and showed molecular ion peaks at m/z 735 [M+H] + , 589 [M+H] + and 649 [M+H] + in the FAB-MS, respectively.The aglycone of compounds 6 and 7 was oleanolic acid, while that of compound 8 was hederagenin.In the 1 H-NMR spectra of compounds 6 to 8, one olefinic proton signal at δ 5. 47-5.50 (H-12) and one oxygen-bearing methine proton signal at δ 3.27-3.31(H-3) were observed.Seven tertiary methyl group signals at δ 0.78 to 1.33 (each s, H-23, 24, 25, 26, 27, 29 and 30) were observed in compounds 6 and 7.In addition, six tertiary methyl groups signals at δ 0.91 to 1.27 (each s, H-24, 25, 26, 27, 29 and 30) were observed in compound8.
Compound 9 was obtained as a brown powder from the n-BuOH fraction.Three hydroxyl proton signals at δ 4.58, 4.52, and 4.38 and two multiplets of CH proton signals at δ 3.11 and 3.00 were observed.The 13 C-NMR spectrum of compound 9 showed four ring carbon signals at δ 75.6, 74.6, 73.1 and 72.2.Accordingly, the structure of compound 9 was elucidated as myo-inositol by comparison of the spectral data, as described in the literature (Yasue et al., 1968).A previous placebo-controlled study has demonstrated that myo-inositol supplementation improves features of dysmetabolic syndrome in postmenopausal women, including triglycerides, HDL cholesterol and diastolic blood pressure (Giordano et al., 2011).
Chemical shifts are reported in parts per million (), and coupling constants (J) are expressed in Hertz.

Table 3 .
1 H and 13 C-NMR spectral data of compounds 6 to 8.
Chemical shifts are reported in parts per million (), and coupling constants (J) are expressed in Hertz.