Triterpenoid Saponins from Anemone rivularis var. Flore-Minore and Their Anti-Proliferative Activity on HSC-T6 Cells

Five previously undescribed triterpenoid saponins (1–5), along with eight known ones (6–13), were isolated from the whole plants of Anemone rivularis var. flore-minore. Their structures were clarified by extensive spectroscopic data and chemical evidence. For the first time, the lupane-type saponins (3 and 12) were reported from the Anemone genus. The anti-proliferative activity of all isolated saponins was evaluated on hepatic stellate cells (HSC-T6). Saponins 12 and 13, which possess more monosaccharides than the others, displayed potent anti-proliferative activity, with IC50 values of 18.21 and 15.56 μM, respectively.

The monosaccharides of 1 were determined as L-arabinose (Ara), L-rhamnose (Rha), and D-glucose (Glc), in a ratio of 1:1:1 by acidic hydrolysis followed by gas chromatography (GC) analysis [20]. The 1 H-NMR spectrum of compound 1 exhibited three anomeric protons at δ H = 6.32 (d, J = 8.2 Hz), 6.17 (s) and 5.03 (d, J = 7.3 Hz), and one methyl group of 6-deoxy-hexopyranosyl moiety at δ H 1.62 (d, J = 6.2 Hz). The α anomeric configuration of the Ara unit was deduced from the 3 J H-1/H-2 (7.3 Hz) value observed in the 4 C 1 form. The Glc unit was determined to have a β anomeric configuration on the basis of its 3 J H-1/H-2 coupling constant (8.2 Hz). Although the anomeric proton of the Rha moiety was observed as a singlet in the 1 H-NMR spectrum, the 13 C-NMR shift of Rha C-5 at δ C = 69.6 indicated the α anomeric configuration [21,22]. The complete assignments of proton signals belonging to sugars were based on 2D NMR of 1 H-1 H COSY, TOCSY, and NOESY, and the carbon signals were assigned by HSQC and further confirmed by the HMBC spectrum ( Table 2). The above NMR data indicated that all the monosaccharides were in their pyranose forms. The sequence and binding sites of the oligosaccharide chains were deduced from the HMBC spectrum ( Figure 2). A cross peak between C-3 of the aglycone and H-1 of Ara revealed that Ara was connected to C-3 of the aglycone. Similarly, the linkage of Glc at C-28 of the aglycone was indicated by the cross peak Glc H-1/C-28, and the linkage of Rha at C-2 of Ara was indicated by the cross peak Rha H-1/Ara C-2. This conclusion was also supported by the NOESY correlations ( Figure 2). On the basis of the above analysis, the structure of 1 was elucidated as 3β-O-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl gypsogenin 28-O-β-D-glucopyranosyl ester. Table 1. 1 H-(500 MHz) and 13 C-NMR (125 MHz) data for the aglycone moieties of 1-5 in pyridine-d 5 .  The monosaccharides of 1 were determined as L-arabinose (Ara), L-rhamnose (Rha), and Dglucose (Glc), in a ratio of 1:1:1 by acidic hydrolysis followed by gas chromatography (GC) analysis [20].   13 C-NMR (125 MHz) data for the sugar moieties of 1-5 in pyridine-d 5 .  21 . By comparing with the 1D NMR data of 1, the aglycone moiety of 2 was identical to 1 ( Table 1) .85 with two olefinic carbon signals at δ C 110.1 and 150.8, and one carbonyl signal at δ C 174.9, which were characteristic of the ∆ 20(29) -lupane-type aglycone. The full assignments of the aglycone NMR signals were derived from 2D NMR data ( Table 1), suggesting that the aglycone of 3 was betulinic acid [23,24]. The sugar moieties of 3 were determined as L-arabinose, L-rhamnose, and D-glucose in a ratio of 1:1:2 by acid hydrolysis, followed by GC analysis. Meanwhile, the 1D NMR spectra of 3 exhibited four anomeric protons at δ  Figure S3 in Supplementary data). The assignments of the NMR signals associated with the aglycone moiety were derived from 2D NMR spectra (Table 1). These data revealed that the aglycone of 4 was 21α-hydroxy-oleanolic acid, which was in a good agreement when comparing the literature data [26,27]. Further comparison of the 1D NMR data assignable to the sugar part between 4 and 3 led to the determination of the same monosaccharide units and glycosylation sequence observed for both at C-3 and C-28 ( Table 2). The conclusion was confirmed by the HMBC and NOESY spectra data ( Figure S3 in Supplementary data). Therefore, the structure of saponin 4 was elucidated as 3β-O-α-L-arabinopyranosyl 21α-hydroxy-oleanolic acid 28-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl ester.
For a long time, A. rivularis var. flore-minore was used as a folk medicine in Shaanxi Province for the treatment of hepatitis [9]. Inhibition of hepatic stellate cell (HSC) proliferation plays a key role in the pathogenesis of liver fibrosis caused by chronic hepatocellular damage [36]. HSC-T6, an immortalized rat hepatic stellate cell line, has generally been applied as a screening tool to evaluate the potential antifibrotic activity [37].

Saponins
For a long time, A. rivularis var. flore-minore was used as a folk medicine in Shaanxi Province for the treatment of hepatitis [9]. Inhibition of hepatic stellate cell (HSC) proliferation plays a key role in the pathogenesis of liver fibrosis caused by chronic hepatocellular damage [36]. HSC-T6, an immortalized rat hepatic stellate cell line, has generally been applied as a screening tool to evaluate the potential antifibrotic activity [37].
In this study, the anti-proliferative activity of isolated saponins on HSC-T6 cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT colorimetric assay. Colchicine was used as a positive control. As shown in Table 3, saponins 12 and 13 displayed potent antiproliferative activity with IC50 values of 18.21 and 15.56 μM, respectively. Saponins 3-5 and 8-11 showed moderate antiproliferative activity with IC50 values ranging from 22.85 to 52.65 μM. These results revealed that the number of monosaccharides in sugar chains (both at C-3 and C-28) increased the anti-proliferative activity (Figure 3). The gypsogenin aglycone saponins (1 and 2) and monodesmosidic ones (6 and 7) were inactive, which suggested that the aldehyde functional group at C-23 and the free carboxyl functional group at C-28 had negative effects on anti-proliferative activity (Figure 3). It is worth mentioning that the monodesmosidic saponins (the sugar chain attached at C-3 and a free carboxylic acid at C-28) were cytotoxic against tumor cells in previous studies [10,14,15,17]. These opposing effects may be due to their different mechanisms against different types of cells. Nevertheless, the anti-proliferative activity of these saponins was very sensitive to their precise functionalization, especially for the sugar moieties. Further research is needed to approach a significative structureactivity relationship.

Plant Material
The plant material was collected on Tsinling Mountains, Shaanxi Province, China, in October 2016. The plant was identified by Prof. Ji-Tao Wang at the Department of Pharmacognosy, School of Pharmacy, Shaanxi University of Chinese Medicine. A voucher specimen (No. 161018) was deposited in the Herbarium of Shaanxi University of Chinese Medicine.

Extraction and Isolation
The air-dried whole plants of A. rivularis var. flore-minore (5 kg) were powdered and extracted with 70% EtOH (3 × 8 L × 2 h) under reflux to give a crude extract (800 g), which was further suspended in water (8 L) and partitioned successively with petroleum ether (8 L × 2) and n-BuOH in triplex wells for 48 h at 37 • C in a humidified 5% CO 2 atmosphere. An amount of 20 µL MTT (Sigma-Aldrich) reagent solved in PBS was added to each well (final concentration = 5 mg/mL), and further incubated for 4 h. After removing the supernatant, DMSO was added to solubilize the formazan crystals. The optical density of each well was measured with a Bio-Rad 680 microplate reader at 560 nm. Anti-proliferative activity was expressed as the concentration of compound producing 50% of cell inhibitory rate (IC 50 ).

Conclusions
In this study, thirteen triterpenoid saponins, including five new ones, were isolated from A. rivularis var. flore-minore. All the structures were established on the basis of extensive spectroscopic studies along with MS analyses and acid hydrolysis. Five kinds of aglycones were identified, i.e., gypsogenin, betulinic acid, 21-hydroxy-oleanolic acid, hederagenin, and oleanolic acid. The lupane-type saponins (3 and 12) were reported from the Anemone genus for the first time. The anti-proliferative activity of all isolated saponins was evaluated on hepatic stellate cells (HSC-T6). The preliminary structure-activity relationship analyses revealed that the more monosaccharides the saponins possessed, the stronger the anti-proliferative activity exhibited. This work will not only enrich the diversity of triterpenoid saponins of this genus, but will also provide a reference for the discovery of potential lead compounds for liver disease drug development.