Two new triterpenoid saponins from Bupleurum marginatum Wall. ex DC

Abstract Two new triterpenoid saponins (1 and 2), together with two known saponins (3 and 4) were isolated from Bupleurum marginatum Wall. ex DC. Their structures were elucidated by the comprehensive spectroscopic analysis. Compounds 1 and 2 represented the rare example of an oleanane-type triterpenoid with two sugar moieties at C-3 and C-28. The cytotoxic activity of four compounds was evaluated against normal heptocell BRL-3A in vitro. Graphical abstract


Introduction
The herb Bupleurum marginatum Wall. ex DC., widely distributed in the southwest of China, has been used to treat liver depression and qi stagnation in Traditional Chinese Medicine (Yao et al. 2019). It is one of three important sources of Bupleurum medicinal materials, including Bupleurum chinense and Bupleurum scorzonerifolium (Ding et al. 2016). Phytochemical investigations have revealed a number of structurally diverse metabolites, such as triterpenoid saponins (Lu et al. 2012;Lei et al. 2020), flavonoids (Wang et al. 2007;Lei et al. 2020;Ma et al. 2021), lignins (Liu et al. 2008;Ashour et al. 2012;Zhou et al. 2021), volatile oils (Ashour et al. 2009), and polysaccharides (Lu et al. 1997). Most of the compounds were tested to exhibit extensive biological activities, main including anticancer (Ashour et al. 2012;Burke et al. 1997), antiviral (Fang et al. 2017), antibacterial (Qiao et al. 2016), analgesia and sedation (Ashour et al. 2014), anti-inflammatory (Lu et al. 2012), and antioxidant (Ashour et al. 2018;Zhou et al. 2021) activities. In the previous investigation based on dimethylnitrosamine (DMN)-induced liver fibrosis in rat and proteomic analysis, triterpenoid saponins from B. marginatum Wall. ex DC. displayed potential anti-fibrotic activity (Liu et al. 2018). Further investigation for new natural cytotoxic components, the isolation of water fraction of the root of B. marginatum Wall.ex DC. was carried out. Two new triterpenoid saponins (1 and 2) with two known saponins, Saikosaponin Q (3) (Liang et al. 2001) and Saikosaponin D (4) (Liang et al. 2003) were identified ( Figure 1). Compounds 1 and 2 represent the rare example of oleanane-type triterpenoid with sugar groups at C-3 and C-28 of the aglycone in B. marginatum. In addition, The cytotoxicity of these compounds was evaluated against BRL-3A cell lines. Compound 4 showed obvious cytotoxicity (IC 50 15.58 ± 0.94 lM), and compounds 1-3 exhibited no cytotoxicity (IC 50 > 50 lM) in vitro.

Results and discussion
Compounds 1 and 2 were isolated as white amorphous powder. These 1 H and 13 C NMR spectra displayed characteristic signals of oleanane-type triterpene backbone (Wandji et al. 2003). The monosaccharides obtained by acid hydrolysis of two compounds were identified by comparison on TLC with authentic samples as glucose, rhamnose and xylose. The absolute configuration was determined by specific optical rotations to be D-form for glucose and xylose, and L-form for rhamnose.
Compound 1 has the molecular formula C 54 H 88 O 23 determined by HR-ESIMS analysis (m/z 1127.5637 [M þ Na] þ ). Thirty of the 54 carbons were assigned to the triterpenoid skeleton and 24 to saccharide moiety. Singlets corresponding to six methyl groups at d H 0.76, 0.85, 0.87, 0.94, 1.08, and 1.26 were correlated with the signals at d C 15. 1, 16.2, 17.3, 19.4, 20.7, and 26.8 in HSQC spectrum. Two olefinic protons at d H 5.62 (1H, d, J ¼ 10.5 Hz) and 6.45 (1H, d, J ¼ 10.5 Hz) and four olefinic signals at d C 136.5, 130.2, 126.0 and 125.3 corresponding to two conjugated double bonds (D 11,12 and D 13,18 ). 13 C NMR data of the aglycone moiety of compound 1 were in good agreement with those of BK2 (Luo et al. 1987), which indicated that compound 1 had the skeleton of 3b, 16a, 28, 30-tetrahydroxyolean-11, 13(18)-dien. Four anomeric proton signals at d H 4.92 (1H, d, J ¼ 7.7 Hz), 4.38 (1H, d, J ¼ 7.9 Hz), 4.20 (1H, d, J ¼ 7.8 Hz) and 4.34 (1H, d, J ¼ 7.8 Hz) were observed in NMR spectrum of its sugar moiety. The splitting patterns of anomeric proton signals indicated that compound 1 had three glucose with b-linkages (Badaoui et al. 2020) and one rhamnose with a-linkage (Escobedo-Mart ıneza et al. 2010). The correlations of three sugar units established by analysis of HMBC spectrum between H-1 of Glc (d H 4.38) and C-3 of the aglycone (d C 89.5), H-1 of Glc' (d H 4.34) and C-6 of Glc (d C 68.0), H-1 of Rha (d H 4.92) and C-4 of Glc (d C 78.9) confirmed the existence of a trisaccharide at C-3 of compound 1. The linkage position of another sugar unit was found to be at C-28 of compound 1 based on the long-range correlation between the anomeric proton of Glc" (d H 4.20) and C-28 of the aglycone (d C 71.6). All 1 H and 13 C NMR signals were assigned well by HSQC, HMBC and 1 H-1 H COSY analyses. The key correlations are shown in Figure  S1. Therefore, the structure of compound 1 was elucidated as 28- Compound 2 has the molecular formula C 53 H 86 O 23 determined by HR-ESIMS analysis (m/z 1113.5444 [M þ Na] þ ). 1 H and 13 C NMR data of the aglycone of 2 were similar to those of 1 and the difference was that one of angular methyl groups was substituted by the hydroxymethyl group. The position of the hydroxymethyl group was ascertained to be C-4 on the base of HMBC correlations between H 2 -23 (d H 3.70) to C-4 of the aglycone (d C 42.6). Four anomeric proton signals were observed in its sugar moiety. The J H-1,H-2 values (7.0-8.0 Hz) and TLC behaviors suggested that four sugar units were two b-glucose, one a-rhamnose, and one b-xylose The key correlations are shown in Figure S2. Therefore, the structure of compound 2 was elucidated as 28-(b-D-glucopyranosyloxy)-16a,23,30-trihydroxyoleana-11,13 All compounds were tested for cytotoxic activities against BRL-3A. Compounds 1-3 showed no cytotoxicity effect, and IC 50 values were 147.24 ± 2.69 lM, 125.77 ± 1.95 lM, and 66.42 ± 1.72 lM, respectively. Compound 4 showed obvious cytoxicity with IC 50 value of 15.58 ± 0.94 lM.

General experimental procedures
HPLC analyses were performed on Shimadzu LC-20AD (Shimadzu, Kyoto, Japan) equipped with a ZORBAX Eclipse XDB-C 18 column (250 Â 4.6 mm, 5 lm) and a SPD-20A detector. Preparative HPLC separations were conducted on a Shimadzu LC-6AD system with a preparative reversed-phase C 18 column (250 Â 20 mm, 5 lm) and a SPD-6A detector. Mobile phase were purified water and methanol with chromatographic grade, which were bought from Merck. Organic reagents were analytical grade. NMR spectra were recorded on Bruker 700 MHz AVANCE III spectrometer (700 MHz for 1 H and 176 MHz for 13 C) in deuterated Methanol-D4 solutions using TMS as an internal standard. HR-ESI-MS were obtained from a 1100 Agilent Series coupled to an Agilent 6520 Accurate Mass Q-TOF LC/MS.

Plant material
Bupleurum marginatum Wall. ex DC. was bought from Dai hospital of Xishuangbanna, Yunnan Province, China, and identified by the pharmacist Yinghong Zhao from Dai hospital of Xishuanbanna. A voucher specimen (batch number: YN20130561) was stored at Key Laboratory for Separation and Analysis in Biomedicine and Pharmaceuticals, School of Life Science, Beijing Institute of Technology.

Extraction and isolation
Air dried powder of the root of Bupleurum marginatum Wall. ex DC. (17 kg) was decocted with 75% ethanol with 5% ammonia for 1 h (three times) and extracting solution was merged and filtered. The filtrate was evaporated under reduced pressure to acquire crude extraction (2.7 kg), which was extracted with petroleum ether, ethyl acetate, n-butanol and water to obtain the corresponding fractions.
Compound 1 3.4. Determination of the absolute configurations of sugars in compounds 1 and 2 CH 3 OH (15 mL) and 0.6 M H 2 SO 4 (5 mL) were added to compound 1 (5 mg), and the mixture was kept at 90 C for 5 hours. Hydrolysate was extracted by CHCl 3 to yield the aglycone. The hydrolysate left was neutralized with 5% Ba(OH) 2 aqueous solution. The sediment was filtered off and the aqueous phase was freeze-dried to provide the product, which was subjected to silica gel column eluting with the upper layer of n-BuOH-HOAc-H 2 O (4:1:5, v/v) to yield glucose (R f 0.33) and rhamnose (R f

Cytotoxicity assay of BRL-3A
BRL-3A cells were maintained in DMEM medium supplemented with 10% fetal bovine serum in an incubator with constant temperature at 37 C and a humidified atmosphere of 5% CO 2 . The cells were trypsinized and passaged to new plates every two or three days. BRL-3A cells were seeded in 96-well plates (3 Â 10 4 /mL) for 24 hours, followed by cells incubation with the different concentration of compounds (5, 10, 50, 100, 150, 200, 500 lg/mL) for 4 hours. Each group was provided with 6 independent duplicates. Cell viability was determined using MTS/PMS assay. Absorbance values were read at 490 nm on an ELISA reader. 0.1% DMSO was considered as blank control. IC 50 values were calculated using Microsoft Excel software.

Conclusions
Two previously unreported triterpenoid saponins (1and 2), combined two known saponins (3 and 4) were identified from B. marginatum Wall. ex DC. and evaluated for cytotoxicity against BRL-3A in vitro. The result further proved that the triterpenoid saponins were important characteristic components of this plant. Although 117 triterpenoid saponins were identified from 15 species of Bupleurum (Luo et al. 2019), the saponin with two sugar chains at C-3 and C-28 of the aglycone was rare, and it was the first time found in B. marginatum Wall. ex DC. According to the structural features of the isolates, compound 4 displayed cytotoxicity activity against BRL-3A while compounds 1-3 exhibited no cytotoxicity (IC 50 > 50 lM), suggesting that the 13,28-epoxy bridge in the aglycone was necessary for compounds to exhibit cytotoxic activity (Li et al. 2015). However, as the herb B. marginatum Wall. ex DC. has been used to liver depression and qi stagnation, the protection criteria to liver by compounds from this herb should be no cytotoxicity to normal heptocells (e.g., BRL-3A). The isolates 1 and 2 are of great significance to further explore the structure-activity relationship, as well as enrich the types of chemical structures of this plant.

Disclosure statement
No potential conflict of interest was reported by the authors.