Three new triterpenoid saponins from the roots of Ardisia crenata and their cytotoxic activities

Abstract Three new triterpenoid saponins, ardisicrenoside O (1), ardisicrenoside P (2) and ardisicrenoside Q (3) together with three known compounds, 3β,16α-dihydroxy-30-methoxy-28, 30-epoxy-olean-12-en, cyclamiretin A 3-O-β-d-glucopyranosyl-(1→2) -α-l-arabinopyranoside and cyclamiretin A 3-O-β-d-glucopyranosyl-(1→4) -α-l-arabinopyranoside were isolated from the roots of Ardisia crenata Sims. Their structures were determined by one- and two-dimensional NMR techniques, including HSQC, HMBC and TOCSY experiments, as well as acid hydrolysis and GC analysis. All isolates were evaluated for the cytotoxic activities on two human cancer cell lines and compounds 3, 5 and 6 showed significant cytotoxicity.


Introduction
Saponins have a wide variety of biological activities and get more and more attention by chemists (Abbas et al. 2015;Almutairi & Ali 2015). Every year, many new saponins are discovered and reported (Silchenko et al. 2013;Zhang et al. 2013;Gedara & Galala 2014). Over the past 10 years, much attention has been paid to saponins from the genus of Ardisia. Up to now, 66 triterpenoid saponins, belonging to 10 different aglycones, were identified from this genus, which showed the strong pharmacological activities of antitumour and enhance the immunological function of body (Liu et al. 2004;Han et al. 2012). So it was revealed that triterpenoid saponins were the main bioactive constituents of this genus.
Ardisia crenata Sims (Chinese name "Zhushagen"), belongs to the genus Ardisia of the family Myrsinaceae, is a widely occurring shrub in southern China. Its roots have been used as a traditional medicine for the treatment of respiratory tract infections and menstrual disorders in China (Jiangsu New Medical College 1985). Previous chemical studies of Ardisia crenata extract have led to the isolation of 20 triterpenoid saponins, ardisiacrispin A-B, ardisicrenoside A-M and five kinds of two glycosides (Jia et al. 1994;Koike et al. 1999;Liu et al. 2007;Zhang et al. 2011) with significant effects of antitumour, inhibiting platelet aggregation and protecting the liver (Song 2014;Yao et al. 2015). At present, the skeletons of triterpenoid saponin are mainly 12-alkene and 13, 28-epoxy ether. Our continuing phytochemical studies on this extract led to the isolation of three new oleanane-type triterpenoid saponins, ardisicrenoside O (1), ardisicrenoside P (2) and ardisicrenoside Q (3), together with three known compounds, 3β,16α-dihydroxy-30-methoxy-28, 30-epoxy-olean-12-en, cyclamiretin A 3-O-β-d-glucopyranosyl-(1→2)-α-l-arabinopyranoside and cyclamiretin A 3-O-β-d-glucopyranosyl-(1→4)-α-l-arabinopyranoside. And the aglycone of three new saponins was not reported from this genus before. In this paper, we reported the isolation and structural elucidation of three new triterpenoid saponins as well as their cytotoxicity against two cell lines of MCF-7 and NCI-H460.

Results and discussion
The n-butanol soluble fraction of the 60% EtOH extract of the roots of A.crenata was subjected to repeated column chromatography over silica gel, ODS and reversed-phase PHPLC, eluting with various solvent systems, to afford three new triterpenoid saponins.
Compound 1 Compound 1 displayed 53 carbon signals in its 13 C NMR spectrum, of which 31 carbon signals could be assigned to the signals of the aglycone. It was evident that 1 was a triterpenoid saponin related to oleanane skeleton based on the 1 H NMR spectral signals (Table 1) assigned to six tertiary methyl groups at δ H 1.38(Me-27), 1.21(Me-23), 1.08 (Me-24), 0.98 (Me-29), 0.91(Me-26) and 0.82(Me-25), together with six corresponding sp3 carbon signals in the 13 C NMR spectrum (Table 1). 13 C NMR spectral data of compound 1 are similar to those of the known saponin ardisicrenoside H . As shown in Table 1, there is a signal at δ C 110.1 (CH, by DEPT) instead of a signal at δ C 180.6 due to the 30-COOH group of ardisimamilloside H. Furthermore, in the HMBC spectrum of compound 1 (Figure 2), the carbon signal at δ C 110.1 not only correlates with Me-29 and H-28 (δ H 4.68), but also with a methoxyl signal at δ H 3.44 (3H, s), confirming that δ C 110.1 is the signal of C-30 which connects with C-28 through the epoxy ether bond and the methoxyl group is at C-30 ( Figure 1). The presence of one 28, 30-epoxy group was also deduced from the 1 H NMR resonance at δ H 4.43 (CH, c NMr (100 Mhz) data of compound 1, 2 and 3 in c5d5 N (δ in ppm, J in hz).   distinguished by HSQC) as well as the long-range coupling with C-21(δ C 31.6) and C-28(δ C 78.6) in the HMBC experiment. 13 C NMR spectral data showed the presence of two olefinic carbon signals at δ C 121.7 (C-12) and δ C 143.3 (C-13) in a low magnetic field. Further, a hydroxyl group at C-16 could be explained by the 13 C NMR resonance at δ C 74.9 (C-16). The α configuration for 16-OH was determined by comparing the chemical shift of C-16 with that of triterpenoid analogues (for 16α-OH: ca. δ C 77.0, for 16β-OH: ca. δ C 64.0) . In addition, the oligosaccharide moieties at C-3 (δ C 88.9) could be identified by the analysis of HMBC correlations. In the NOESY spectrum, the correlations of H-3 (δ H 3.14) with Me-23 (δ H 1.21) and H-5 (δ H 0.70) indicated the β configuration for 3-O-sugar moiety. The assignments of the NMR signals associated with the aglycone moiety (Table 1) were derived from 1 H-1 HCOSY, TOCSY, HSQC, HMBC and NOESY experiments. Thus, the sapogenin of 1 was determined as 3β, 16α-dihydroxy-30-methoxy-28, 30-epoxy-olean-12-en. The presence of l-arabinose, d-glucose and d-xylose in a ratio of 1:2:1 in 1 was established by acid hydrolysis with 2 mol/L HCl followed by GC analysis of the corresponding trimethylsilylated derivatives using an l-Chirasil-Val column. The 1 H NMR spectrum of 1 displayed four signals ascribable to the anomeric protons (δ H 4.76, 4.90, 4.99 and 5.48) that correlated in the HSQC experiment with carbon signals at δ C 104.6, 107.6, 104.2 and 104.9, respectively. The β anomeric configurations for two glucose units and one xylose unit were determined from their 3 J H-1/H-2 coupling constants (7.6, 7.8 and 7.3 Hz). The α anomeric configuration for arabinose residue was judged from the correlation between H-1 and H-3 and between H-1 and H-5 in the NOESY experiment observed for the 4 C 1 form, although its H-1 coupling constant (5.9 Hz) was smaller than methyl-α-l-arabinopyranoside (8 Hz) (Agrawal 1992). This could be explained by the quick conformational equilibrium between its 4 C 1 and 1 C 4 conformers (De Tommasi et al. 1993). All proton signals due to sugars were identified by careful analysis of the 1 H-1 H COSY, TOCSY and NOESY spectra, and the carbon signals were assigned by HSQC and further confirmed by HMBC spectrum. Data from the above experiments (Table 1) indicated that four sugar residues are in the pyranose forms. Detail inspection of HMBC and NOESY spectra led to the determination of the sequence and binding sites of the sugar chain ( Figure 2). In the HMBC spectrum, a cross-peak between C-3 of the aglycone and H-1 of arabinose (Ara) indicated that Ara was connected to C-3 of the aglycone. The linkages of the two glucose, (terminal Glc 1) at C-2 of Ara and (inner Glc 1) at C-4 of Ara were indicated by the cross-peak of Glc I H-1/Ara C-2 and Glc II H-1/Ara C-4, respectively. Similarly, the linkage of the xylose (Xyl 1) at the inner Glc-2 was indicated by cross-peak of Xyl I H-1/ inner Glc-2. The conclusion was confirmed by NOESY experiment. Hence, the structure of 1 was elucidated as 3β-O-{β-d-xylopyranosyl-(1→2)-β-d-glucopyranosyl-(1→4)-[β-dglucopyranosyl-(1→2)]-α-l-arabinopyranosyl}-16α-hydroxy-30-methoxy-28, 30-epoxyolean-12en and named as ardisicrenoside O.
Compounds 1, 2 and 3 are new oleanane triterpenoid saponins. To the best of our knowledge, the saponin containing monosaccharide group at the C-3 hydroxy group of the aglycone of 3β, 16α-dihydroxy-30-methoxy-28, 30-epoxy-olean-12-en, has not been reported from natural sources.
The MTT method was used to test the cytotoxicity of these compounds on MCF-7 and NCI-H460 cancer cell lines. The bioassay results of all isolated compounds are collected in Table 2. From these results, it was concluded that compounds 3, 5 and 6 exhibited strong cytotoxicities against cancer cell lines, compounds 1 and 2 had relatively weak activities and compound 4 showed no activity. It is remarkable that compounds 3, 5 and 6 had stronger activity against NCI-H460 than that of MCF-7.

General experimental procedures
Melting points were determined on a Yanaco MP-S3 micromelting point apparatus, and were uncorrected. Optical rotations were measured using a P-1020 digital polarimeter (JASCO Corporation). ESI-MS was measured using Bruker esquire 2000 mass spectrometer. IR spectra were recorded on SHIMADZU FT/IR-8400 spectrometer. 1 H and 13 C-NMR, along with 2D NMR spectra were determined on a Bruker AV-400 (400 MHz for 1 H, 100 MHz for 13 C) NMR spectrometer, using TMS as an internal standard. Chemical shifts were expressed in δ (ppm) and coupling constants (J) were reported in Hertz (Hz). TLC was carried out on silica gel 60F 254 and the spots were visualised by spraying with 10% H 2 SO 4 and heating. Diaion HP-20 (Mitsubishi Kasei) and ODS (Lobar, 40-63 μm, Merck) were used for column chromatography. Preparative HPLC was performed using an ODS column (C-18, 250 × 20 mm, SHIMADZU Pak; detector: RID).

Plant materials
The root of Ardisia crenata Sims was collected from Guangxi province of China in 2000, and identified by Professor Qishi Sun. A voucher specimen (YL-2001-113) has been deposited in the Shenyang Pharmaceutical University, Liaoning province of China.

Acid hydrolysis for sugar analysis
A solution of 1 (2.0 mg) was heated with 2 M HCl at 80 °C for 8 h. The mixture was neutralised by addition of Amberlite IRA 400 (OHform) and filtered. The filtrate was dried in vacuo, dissolved in 0.2 ml of pyridine containing l-cysteine methyl ester (10 mg/ml) and reacted at 60 °C for 1 h. To this mixture, a solution (0.2 ml) of trimethylsilylimidazole in pyridine (10 mg/ml) was added, and it was heated at 60 °C for 1 h. The sugars were first analysed by TLC over silica gel (CHCl 3 -MeOH-H 2 O, 8:5:1) by comparison with standard samples. The absolute configuration of each monosaccharide was determined from GC-MS analysis of their trimethylsilylated derivatives by comparison with authentic samples using the method previously described (Chaabi et al. 2010). The following sugars were detected: d-glucose, l-rhamnose and l-arabinose.

Cytotoxicity assay
Cancer cells were cultured in PRMI-1640 medium supplemented with 5% foetal bovine serum and incubated at 37 °C in a 5% CO 2 humidified incubator and subcultured every two days to maintain them in a state of logarithmic growth. Then, the cells were seeded into 96-well microtiter plates (1 × 10 4 cells per well). Compounds were dissolved in DMSO and added to the 96-well microtiter plates 24 h after seeding. The cells were incubated for two days in the presence of sample. For the evaluation of in vitro cytotoxicity, MTT assay was used. The anticancer drug cis-dichlorodiamine platinum (cis-DDP) was used as the positive control.

Conclusion
The phytochemical studies of A. crenata have led to the isolation of three new triterpenoid saponins, along with two known saponins and an aglycone. The aglycone of these three new saponins was different with other aglycones reported in this genus before, which has been rarely reported in the plant kingdom. The observed inhibition of cancer cell growth by compounds 3, 5 and 6 suggests that they are potential anticancer active ingredients.

Funding
This work was supported by the Tianjin Natural Science Foundation [number YFJMJC08300].