Chemical constituents and antitumor activity from Paris polyphylla Smith var. yunnanensis

Abstract Eleven compounds were isolated from the rhizomes of Paris polyphylla Smith var. yunnanensis. Their structures were elucidated on the basis of rigorous 1D and 2D NMR experiments as well as comparison with the literature data. To the best of our knowledge, compound 1 was only predicted by UPLC/Q-TOF MSE and the NMR spectroscopic data was given for the first time. The cytotoxic activities of all compounds on mouse B16 cells were evaluated. Among the tested molecules, compounds 6–9 showed strong cytotoxicities, while compound 1 did not show significant effect.


Introduction
The genus Paris (family: Liliaceae) comprises about 24 species, which are mainly distributed in the tropics and temperate latitudes of Eurasia. Many species in this genus have been historically used for the treatments of fractures, traumatic bleeding, snake bite, mumps and abscess (Editorial Board of Chinese Materia Medica of the State Administration of Traditional Chinese Medicine 1999; Guo et al. 2008). About 70 steroidal saponins have been isolated from 22 species of this genus (Wang et al. 2013). In addition, more than 40 new compounds had been identified by the LC/Q-TOF MS from the Paris polyphylla Ling et al. 2015). However, due to the limitations of the LC/Q-TOF MS in differentiating compounds with geometrically isomeric substituting groups, phytochemical and NMR experiments need to be carried out to get the exact structures of the identified compounds. In our search for bioactive compounds from P. polyphylla Smith var. yunnanensis, we have isolated 11 compounds ( Figure 1) including one polyhydroxylated steroidal saponin (1), which was predicted by UPLC/Q-TOF MS E , and identified the structures by detailed analysis of their NMR spectra in this study. Moreover, the cytotoxicities of compound 1 and other ten compounds on mouse B16 cells were tested.
The sugar moieties were identified by acid hydrolysis of 1 and further GC-MS analysis with authentic samples. Acid hydrolysis of 1 gave d-glucose, d-xylose, l-rhamnose and d-fucose. In addition, the 1 H NMR coupling constants ( 3 J 1, 2 ≥ 7 Hz) were consistent with β-configuration for the glucose, xylose and fucose. The α-configuration for the rhamnose was confirmed by comparing the 13 C NMR spectral data for C-3 and C-5 of rhamnose with the previous report (Kasai et al. 1979). The glycosyl moiety assigned at C-1 of the aglycone was determined by the HMBC correlations of H-1-Glc to C-1 (δ C 84.5) of the aglycone, H-1-Rha to C-2-Glc (δ C 76.7), H-1-Xyl to C-3-Glc (δ C 88.8), which was confirmed by the NOESY correlations of H-1 of aglycone with H-1-Glc, H-2-Glc (δ H 4.12) with H-1-Rha, H-3-Glc (δ H 4.02) with H-1-Xyl. As for another glycoside moiety, the correlation of the anomeric proton of H-1-Fuc with C-24 (δ C 82.6) of the aglycone was observed in the HMBC. Comparison of the NMR and MS data of 1 with those of parisyunnanoside I ) suggested that they have similar structures, but significant difference was that compound 1 lacked the galactose residue. Thus, the structure of the compound 1 was fully determined to be (23S,24S)-spirost-5,25 (27) Compounds 1-11 were evaluated for cytotoxicity against B16 melanoma cells (Table 1). The paridis saponins exhibited a marked structure-dependent growth inhibitory activity against B16 cells. Saponins with a glycosyl group at C-1 of the aglycones (1, 2, 3 and 4), furostanol saponin (5) and diosgenin (10) showed no activities. Sterol having a glycosyl group at C-3 (11) had weak activity. Saponins with a glycosyl group at C-3 of the spirostanol aglycones (6, 7, 8 and 9) exhibited strong cytotoxic activities, which showed statistically 1.83 ± 0.29 a significant difference compared with compound 11 (p = 0.000). It was demonstrated that the 3-O-glycoside moiety and spirostanol structure were essential for the proapoptotic activity with regard to anticancer structural relationship of steroid saponins. These results confirmed the previous inference (Yan et al. 2009). The present results showed that there were no statistically significant differences between compounds 6, 7, 8 and 9 in the cytotoxic activities (p > 0.05). Compared with the cytotoxicity of diosgenin (10), it is supported that the saccharide group increased the cytotoxicity of diosgenin, especially for rhamnose. Active spirostane-type saponins bearing (1→2)-α-L-rha-β-D-glc at C-1 had shown considerable cytotoxic activities (9), further addition of an α-L-rhamnosyl, an α-L-arabinofuranosyl or a β-D-glucosyl, to C-3 or C-4 of the inner glucosyl moiety gave no significant influence on the activity (6, 7 and 8).

General experimental procedures
HR-ESI-MS spectra were performed on a Bruker Micro TOF-Q mass spectrometer. 1D and 2D NMR spectra were obtained on a Bruker AVANCE-600 (600 MHz

Plant materials
The rhizomes of P. polyphylla Smith var. yunnanensis were collected from Lijiang, Yunnan province, China and authenticated by Professor Wenyuan Gao (School of Pharmaceutical Science and Technology, Tianjin University). A voucher specimen (No.2010910) has been deposited in the School of Pharmaceutical Science and Technology, Tianjin University, China.

Acidic hydrolysis of 1
Compound 1 (2 mg) was refluxed with 2 M HCl on water bath for 2 h. After cooling, the reaction mixture was extracted with CHCl 3 (3 × 5 mL). The aqueous layer was evaporated to dryness with MeOH until neutral. The dried residue was dissolved in 1 mL of anhydrous pyridine and treated with l-cysteine methyl ester hydrochloride (1.5 mg) stirred at 60 °C for 1 h. Trimethylsilyl imidazole (1.0 mL) was added to the reaction mixtures, and they were kept at 60 °C for 30 min. The supernatants (4 μL) were analysed by GC, quartz capillary column (30 m × 0.32 mm), column temperature: 180-280 °C and the carrier gas was N 2 (1 mL/min); injector temperature: 230 °C; compound 1 was determined by comparison of the retention times of the corresponding derivatives with those of standard l-rhamnose, d-fucose, d-xylose and d-glucose giving a single peak at 12.94, 15.27, 16.33 and 25.67 min, respectively.

Determination of cell viability (MTT assay)
B16 cells were seeded in 96-well plates at a density of 3.5 × 10 4 cells per well (200 μL of medium per well). After overnight incubation, cells were synchronised by changing to a medium free of FCS. After another 24 h, serial concentrations of the test compounds in DMSO solutions (0.1% DMSO) were added and further incubated for 24 h. The supernatant was discarded and 100 μL of 0.5 mg/mL MTT medium (Sigma, USA) was added to all wells for another 4 h of culture. The supernatant was then removed, 100 μL per well of DMSO was added with samples being shaken for 15 min. The control group was cells without any addition and was referred to as 100% of viable cells. Absorbance at 570 nm was measured with an enzyme-linked immunosorbent assay plate reader (TECAN A-5002 Spectra, Austria). This experiment was carried out three times independently. IC 50 values were calculated using a linear regression of dose-dependent curves plotted from at least six points. All statistical comparisons were carried out by using SPSS software (SPSS 19.0, SPSS Inc., Chicago, USA). The analyses were run in triplicate, and the results are expressed as means and standard deviations. Statistical differences were assessed by one-way ANOVA tests. P-value less than or equal to 0.05 was considered to be statistically significant.

Conclusion
Phytochemical investigation of P. polyphylla Smith var. yunnanensis afforded 11 compounds including compound 1, a polyhydroxylated steroidal saponin, which was predicted by UPLC/Q-TOF MS E and never been isolated in the phytochemical studies. The 1 H NMR and 13 C NMR spectroscopic data of it were given for the first time in this study. The tests of cytotoxic activities of all the 11 compounds showed the cytotoxic activities of steroidal saponins were influenced by both the variety of aglycone and the sugar chain.

Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1-S7 and Table S1.

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

Funding
This work was supported by National Natural Science Foundation of China [grant number 30873378].