A new triterpenoid saponin from Gleditsia sinensis and its antiproliferative activity

Abstract Chemical investigation of the anomalous fruits of Gleditsia sinensis led to the isolation and identification of a new triterpenoid saponin, 3-O-β-D-xylopyranosyl-(1 → 2)-α-L-arabinopyranosyl-(1 → 6)-β-D-glucopyranosyl oleanolic acid 28-O-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyrano--syl-(1 → 4)-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 3)-β-D-glucopyranosyl ester (1), along with other nine known compounds (2–10). All the isolates from this species were reported for the first time. The structure of Compound 1 was determined by a detailed analysis using various analytical techniques, including 1D and 2D NMR. In vitro antiproliferative activities of Compound 1 on MCF-7 and Hep-G2 tumor cell lines were evaluated. IC50 values against the two cell lines were 9.5 and 11.6 μM, respectively.

saponin-rich herbal medicine used for the treatment of apoplexy and as an expectorant and pesticide. In the 2005 edition of Chinese Pharmacopoeia, 'Zhu Ya Zao' is listed as one of the commonly used drugs (National pharmacopoeia committee 2005).
Pharmacological studies suggested that 'Zhu Ya Zao' possessed anti-inflammatory (Dai et al. 2002), antiallergic (Hon et al. 2006) as well as antitumor activities (Teo et al. 2004;Chui et al. 2005;Tang et al. 2007). Previous literature on 'Zhu Ya Zao' identified triterpenoid saponins as the components responsible for the biological activities of the plants. Only a few chemical investigations of this species have so far been reported in Chemical Abstracts (Zhang et al. 1999a(Zhang et al. , 1999b(Zhang et al. and 1999cGao et al. 2008). As a part of our work on biologically active novel secondary metabolites, the whole anomalous fruits of G. sinensis were used to survey chemical composition. As a result, a new triterpenoid saponin along with other nine known compounds was identified from an ethanol extract.
Compound 1 was isolated as a white amorphous powder. HR-ESI-MS showed an [M + Na] + ion peak at m/z 1623.1818 (calc. 1623.7406), which suggested a molecular formula of C 74 H 120 O 37 for Compound 1 combined with the results of 1 H and 13 C NMR. After an extensive 1D and 2D NMR study, the aglycon was identified as oleanolic acid. The chemical shift of δ C 88.10 (C-3) and δ C 175.92 (C-28) indicated that 1 was a bisdesmosidic glycoside (Zhang et al. 1999c). Among the 74 carbon signals in the 13 C NMR spectrum, 30 signals were assigned as aglycone; the remaining 44 signals were indicative of the presence of four hexoses and four pentoses. These were in good agreement with the eight anomeric signals δ H 4.15 (1H, d, J = 7.6 Hz), 4.25 (1H, d, J = 7.2 Hz), 4.38 (1H, d, J = 7.6 Hz), 4.42 (1H, d, J = 7.6 Hz), 4.45 (1H, d, J = 4.8 Hz), 4.48 (1H, br s), 5.13 (1H, br s) and 5.31 (1H, m) in the 1 H NMR spectrum and the eight anomeric carbons observed at δ 93.41, 100.10, 100.57, 101.41, 104.80, 105.08, 105.65 and 105.66 in the 13 C NMR spectrum. The two methyl carbon signals at δ 18.20 and 18.40 and proton signals at δ 1.16 (3H, d, J = 6.0 Hz) and 1.09 (3H, d, J = 6.0 Hz) revealed that 1 included two deoxy sugar moieties. The sugar units were identified as two glucoses, three xyloses, two rhamnoses and one arabinose according to the Thin Layer Chromatography (TLC) analysis following acid hydrolysis together with the data of 1 H and 13 C NMR. All the monosaccharides were in pyranose forms based on the 13 C NMR spectroscopic data for the sugar moieties (Zhang et al. 1999c). The chemical shift and the coupling constant of each of the anomeric protons indicated that two glucoses and three xyloses have β-configuration and one arabinose has α-configuration (Zhang et al. 1999c). The Nuclear Overhauser Enhancement Spectroscopy (NOE) correlation of H-1 with H-2 observed in the ROESY spectrum proved that both the rhamnoses had α-configuration (Zhang et al. 1999c). The identification and full assignments of the proton and carbon signals for the sugar moieties were accomplished by a combination of HSQC, HMBC and ROESY experiments as described previously (Zhang et al. 1999c).
The linkage of sugar units at C-3 was established from the following HMBC correlations: H-1 (δ H 4.15) of Glc with C-3 (δ C 88.10) of the aglycon; H-1 (δ H 4.45) of Ara with C-6 (δ C 68.68) of the Glc; H-1 (δ H 4.25) of Xyl with C-2 (δ C 79.35) of the Ara; while, the attachment of the pentasaccharides chain to C-28 of the aglycon was also based on the HMBC correlations: H-1 (δ H 5. 31) of Glc′ with C-28 (δ C 175.92) of the aglycon; H-1 (δ H 5. 13) of Rha with C-3 (δ C 76.11) of the Glc′; H-1 (δ H 4.42) of Xyl′ with C-4 (δ C 83.67) of the Rha; H-1 (δ H 4.48) of Rha′ with C-4 (δ C 68.50) of the Xyl′; H-1 (δ H 4.38) of Xyl″ with C-4 (δ C 86.26) of the Rha′. The two sugar chains of 1 were also deduced from the following NOE correlations observed in the ROESY spectrum: the proton chemical shift of C-3 in the aglycon (δ H 3.10) with H-   (Zhang et al. 1999c). Many reasons such as temperature, light, precipitation intensity, collection sites, collection date and so on could lead to the difference of secondary metabolites in plants.
Previous pharmacological studies suggested that 'Zhu Ya Zao' possessed antitumor activities (Teo et al. 2004, Chui et al. 2005, Tang et al. 2007, Zhong et al. 2004. To further identify the compounds that play this kind of role in vitro, antiproliferative properties of Compounds 1-4 against Hep-G2 and MCF-7 tumor cell lines were evaluated (Table 1). Compound 1 showed a significant activity against the two cell lines with IC 50 values of 9.5 and 11.6 μΜ, respectively, compared to matrine as a positive control with IC 50 values of 23.6 and 21.2 μΜ, respectively. In addition, Compounds 2-4 exhibited moderate cytotoxicity to the two cell lines with IC 50 values ranging from 42.8 to 71.3 μM. The results are also consistent with the recent reports that a few different structural types of triterpenoids have been confirmed to be antitumor agents (Careaga et al. 2014, Qin et al. 2015).

General
Optical rotations were measured on a WZZ-2B spectropolarimeter. IR spectra were recorded on a NICOLET IR200 FT-IR spectrophotometer. NMR spectra were recorded on a Bruker Avance DRX-400 spectrometer at 400 MHz ( 1 H) and 100 MHz ( 13 C) in DMSO-d 6 solution. Chemical shifts were expressed in δ (ppm) referring to TMS. HR-ESI-MS was carried out on an Agilent Technologies 6224 TOF LC-MS apparatus.

Plant material
The anomalous fruits of G. sinensis were collected from the village of Liuxiayi, Kanzhuang, Zoucheng, Shandong, China in September 2014. The species was identified by Prof. Wei-Ting Wang, Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural Sciences, Jinan, China. A voucher specimen (No. 14-09-16) was deposited in our laboratory.