New Steroidal Saponins from the Rhizomes of Paris vietnamensis and Their Cytotoxicity

Four new spirostanol saponins, named pavitnosides A–D (1–4), with six known steroidal saponins 5–10 were isolated from the rhizomes of Paris vietnamensis. Their chemical structures were determined based on extensive spectroscopic studies and chemical methods. The aglycones of pavitnoside B and pavitnoside C were not reported in previous work. The cytotoxicity of all saponins was evaluated against human glioblastoma U87MG and U251 cell lines. The new spirostanol saponin 1 displayed weak anti-proliferative activity against U87MG cell line and the known saponins 8 and 9 exhibited significant cytotoxicity against the two tumor cell lines, with IC50 values of 2.16 to 3.14 μM, but did not affect the growth of primary cultures of human astrocytes.


Introduction
The genus Paris (Liliaceae) has been used as a traditional Chinese medicine for a long time. In the Chinese Pharmacopoeia, Rhizoma Paridis, recorded as the rhizomes of Paris polyphylla var. yannanensis and Paris polyphylla var. Chinensis [1], are widely used as folk medicine for heat-clearing and detoxifying, relieving swelling and pain, sore throat, snake bites and traumatic injure [2]. By extensive phytochemical and pharmacological studies, steroidal saponins have proven to be the chief active ingredients. Their significant bioactivities, including antitumor, anti-inflammatory, anti-melanogenic, antifungal, antiulcerogenic and hemostatic, have been confirmed in recent years [3][4][5][6][7][8][9][10][11]. As perennial plants, the domestic production of Rhizoma Paridis is insufficient, thus demand exceeds supply [2]. P. vietnamensis was chosen to investigate its chemical characteristics, aimed to replace Rhizoma Paridis and alleviate resource pressure. P. vietnamensis is mainly distributed in Guangxi, Yunnan province, China and North Vietnam. Its ideal growing environment is evergreen broad-leaved forest under 2000 m above sea level [12]. Up to now, one new and ten known steroidal saponins have been identified from the rhizomes of P. vietnamensis, and several compounds exhibit cytotoxic bioactivities [13]. These results prompted to continue further investigation on P. vietnamensis to discover novel structures and bioactive saponins. This paper describes the isolation and structural identification of four new spirostanol saponins and six known saponins (Figure 1), as well as the exploration of their in vitro cytotoxic activities.
GC analysis after acid hydrolysis of Compound 2 demonstrated the same monosaccharides as 1, i.e., presence of D-glucose and L-rhamnose. Compared to Compound 1, Compound 2 also obtained the same linkages of sugar moieties. The only difference was that the signals of 6-acetyl connected with β-D-glucose were not observed in 1 H-NMR and 13  Since steroidal saponins have been reported to possess varying cytotoxic activity against various cancer cell lines [32,33], human glioblastoma U87MG and U251 cells were selected to evaluate the cytotoxicities of the obtained spirostanol glycosides 1-10. The results of their cytotoxicities are shown in Table 2. Compounds 8 and 9 characterized at a branched trisaccharide chain attached to C-3 of the aglycone, exhibited significant cytotoxicities against the two test tumor cells, while saponin 1 displayed weak anti-proliferative activity on U87MG cell line. Saponins 2-7 and 10 possessing a disaccharide chain at C-3 of the aglycone displayed almost no activity (IC 50 > 100 µM). It is worth noting that Compounds 1-10 did not affect the growth of primary cultures of human astrocytes. In fact, exposure of the astrocytes to the highest concentrations of 1-10 (100 µM) for 72 h did not result in any statistically significant change in cell viability, with the inhibition ranging from 3.8% to 9.1% (p > 0.05). Meanwhile, the viability of astrocytes treated with 100 µM ACNU for 72 h decreased to 67.4% (p < 0.05) . Comparing 1 with 2-7, the acetyl group may influence the cytotoxic activity. On the other hand, it seemed that greater number of sugar residues at C-3 of the aglycone showed higher cytotoxic activity. However, the structure-activity relationship (SAR) for the antitumor activity of saponins involved the type of the aglycones; the type, number, sequence and binding sites of the sugar moieties; etc. [1]. For the limitation in the number of the isolated steroidal saponins, further studies on the cytotoxicity of the related saponins from P. vietnamensis are necessary to clarify their SAR. Table 2. Cytotoxicities of saponins 1-10 against two human cancer cell lines and primary cultured astrocytes in vitro (IC 50 , µM) a .

Plant Material
The dried rhizomes of Paris vietnamensis were collected from Ya'an, Sichuan Province, China in May 2016, and were identified by one of the authors, Haifeng Tang. The voucher sample (No. 20160520) was deposited in the Herbarium of Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University, Xi'an, China.

Extraction and Isolation
The dried rhizomes of Paris vietnamensis (2 kg) were cut into pieces and refluxed with 70% ethanol (10 L) thrice (each 1.5 h). The solution was merged and condensed with a vacuum rotary evaporator to receive a syrupy residue (630 g). The extraction was suspended in water (3 L) and successively extracted with petroleum ether and water saturated n-BuOH. The water saturated n-BuOH layer was evaporated under reduced pressure to afford an extraction (200 g). The crude extract was offered to silica gel column chromatography (CC) and eluted by gradient eluent of The purity of all compounds was assessed by HPLC as more than 95%.

Acid Hydrolysis and GC Analysis of the Sugar Moieties in 1-4
Compounds 1-4 (each 2 mg) were heated in an ampule with 2 mol/L CF 3 COOH (2 mL) at 120 • C for 2 h. The reaction mixture was extracted with CHCl 3 (3 × 5 mL). The aqueous layer was concentrated in vacuo; 1 mL pyridine and 2 mg NH 2 OH·HCl were added to the dried residue; and the mixture was stirred at 90 • C for 1 h. After the reaction mixtures were cooled, 1.5 mL of Ac 2 O was added and the mixtures were heated at 90 • C for 1 h. The reaction mixtures were concentrated under reduced pressure, and the resulting aldononitrile peracetates were analyzed by GC. The carbohydrates were determined by comparing the retention times with standard aldononitrile peracetates prepared from authentic sugars by the same procedure performed for the sample [1,34]. Retention times for authentic sugars after being derivatized were 5.61 min (L-Rha) and 11.56 min (D-Glc), respectively. L-Rha and D-Glc were identified in a ratio of 1:1 for Compounds 1-4.

Cytotoxicity Assay for Compounds 1-10
Human glioma cell lines U251 and U87MG were obtained from the Cell Bank of Chinese Academy of Science (Shanghai, China). Cultured primary astrocytes were obtained from a slightly impaired brain tissue fragment of a volunteer with cerebral trauma who consented to the procedure as described previously [35]. Acquisition of the tissue was approved by the local medical research ethics committee at Xijing Hospital, Fourth Military Medical University. The cell lines were cultured in DMEM (Corning, Beijing, China) supplemented with 10% FBS (Ausbian, Harbin, China) at 37 • C with 5% CO 2 . The logarithmic phase cells were seeded on 96-well plates at the concentration of 5000 cells/well and incubated with various concentrations (100, 10, 1, 0.1, and 0.01 µM in medium containing less than 0.1% DMSO) of Compounds 1-10 in triple wells for 72 h. Nimustine hydrochloride (ACNU, Sigma, ≥ 99%, Shanghai, China) was used as the positive control. Cell viability was determined according to reported assay methods using commercial CCK8 kit (7sea biotech, Shanghai, China) [36,37]. The optical density of each well was measured with a Bio-Rad 680 microplate reader at 450 nm (Bio-Rad Corporation, Hercules, CA, USA). Cytotoxicity was expressed as the concentration of drug inhibiting cell growth by 50% (IC 50 ).

Statistical Analysis
All data are presented as the mean ± standard deviation (SD) and analyzed using SPSS version 19.0 (SPSS Inc., Chicago, IL, USA). Unpaired test and one-way Analysis of Variance (ANOVA) followed by LSD test were performed for the differences between different groups. A value of p < 0.05 was considered as statistically significant difference.

Conclusions
Phytochemical investigation of P. vietnamensis afforded 10 compounds, including four new compounds. The aglycones of Compounds 2 and 3 had never been isolated in the phytochemical studies and the 1 H-NMR and 13 C-NMR spectroscopic data were given for the first time in this study.
The experiments of cytotoxicity of all compounds showed that 8 and 9 exhibited notable cytotoxicity against the U87MG and U251 cell lines. However, Compounds 1-10 did not affect the growth of primary cultures of human astrocytes. It is suggested that saponins 8 and 9 are reliable candidates for chemotherapeutic treatment of human glioma.
Supplementary Materials: The physical and spectroscopic data of saponins 5-10, and NMR and MS spectra of saponins 1-4 are available online.