New Steroidal Saponins Isolated from the Rhizomes of Paris mairei

The genus Paris is an excellent source of steroidal saponins that exhibit various bioactivities. Paris mairei is a unique species and has been widely used as folk medicine in Southwest China for a long time. With the help of chemical methods and modern spectra analysis, five new steroidal saponins, pamaiosides A–E (1–5), along with five known steroidal saponins 6–10, were isolated from the rhizomes of Paris mairei. The cytotoxicity of all the new saponins was evaluated against human pancreatic adenocarcinoma PANC-1 and BxPC3 cell lines.


Introduction
The genus Paris (Liliaceae) includes 33 species around the world, and 27 species and more than 15 varieties have been discovered in China [1]. It has been used as a traditional Chinese medicine for traumatic injuries, heat-clearing and detoxifying, and relief of swelling and long-term pain [2]. Under the development of phytochemistry, steroidal saponins have been proved to be the main chemicals in the genus Paris and present a wide range of pharmacological activities such as anti-tumor [3][4][5], anti-inflammatory [6], anti-fungal [7], hemostasis [8], and immunomodulatory [9]. Moreover, Rhizoma Paridis, documented as rhizomes of Paris polyphylla var. yannanensis and Paris polyphylla var. Chinensis in the 2020 edition of the Chinese Pharmacopoeia, is usually used as adjuvant drugs for postoperative treatment of cancer to improve symptoms and therapeutic effect. However, Paris polyphylla var. yannanensis and Paris polyphylla var. Chinensis as perennial plants need at least 5 years to mature, and the increasing market demand makes wild sources of Rhizoma Paridis seriously scarce [10]. Hence, it is necessary to investigate other species of Paris in order to relieve resource pressure. Paris mairei is mainly distributed in the Guizhou, Sichuan, and Yunnan provinces of China and used as folk medicine for a long time. Herein, this paper reports the isolation and structural identification of five new (1-5) and five known (6-10) saponins ( Figure 1) as well as the cytotoxicity against human pancreatic adenocarcinoma PANC-1 and BxPC3 cell lines.

Results and Discussion
Compound 1, named Pamaiosides A, a white amorphous solid, was positive to Liebermann Burchard and Molisch chemical reactions, which indicates that it might be a steroidal glycoside. The pseudomolecular ion peak was detected in the HR-ESI-MS spectrum at m/z 995.4824 [M + Na] + (calculated for C48H76O20Na, 995.4828), corresponding to

Results and Discussion
Compound 1, named Pamaiosides A, a white amorphous solid, was positive to Liebermann Burchard and Molisch chemical reactions, which indicates that it might be a steroidal glycoside. The pseudomolecular ion peak was detected in the HR-ESI-MS spectrum at m/z 995. 4824 20 . Four methyl groups were tested in the 1 H-NMR Table 1. 13 C-NMR data of aglycone moieties for compounds 1-5 in CD 3 OD.

General
Optical rotations were measured on a Perkin-Elmer 241 MC digital polarimeter (German PerkinElmer Corporation, Boelingen, Germany). 1D and 2D-NMR spectral experiments were measured in CD3OD on a Bruker AVANCE-500 and a Bruksmer AVANCE-800 spectrometer (Bruker Corporation, Karlsruhe, Germany) with TMS as an internal standard. The IR spectra were recorded on a Shimadzu IRPrestige-21 spectrophotometer (Shimadzu Corporation, Tokyo, Japan). The ESI-MS and HR-ESI-MS spectra were carried out on a Waters Micromass Quattro mass spectrometer (Waters, Shanghai, China).

Plant Material
The rhizomes of Paris mairei were collected from Lijiang, Yunnan Province, China, in September 2018 and identified by the corresponding author Haifeng Tang. The voucher sample (No. 20180903) was deposited in the Department of Chinese Materia Medica and Natural Medicines, School of Pharmacy, Air Force Medical University, Xi'an, China.

Extraction and Isolation
The dried rhizomes of Paris mairei (1.0 kg) were chopped and refluxed with 70% ethanol (10.0 L) thrice (each 2 h). The ethanol solution was mixed and condensed with a vacuum rotary evaporator to receive a syrupy residue (584.0 g). The extraction was suspended in water (3.0 L) and extracted with same volume of petroleum ether and water saturated n-BuOH 3 times, successively. The water saturated in the n-BuOH layer was vacuum evaporated to give a gummy residue (132.0 g). The crude extraction was separated by silica gel column chromatography and eluted by gradient eluent of  1 g) and Fr.13-2 (830 mg). Fr.13-1 was eluted by MeOH on a Sephadex LH-20 to get rid of pigmentum and separated to Fr.13-1-1 (64 mg), Fr.13-1-2 (57 mg), and Fr-13-1-3 (145 mg) on ODS silica gel. Then, Fr.13-1-1 and Fr.13-1-3 were isolated by semi-preparative HPLC using MeCN-H 2 O (35:65, 40:60) as the mobile phase at a flow rate of 8.0 mL/min to afford compound 1 (9.1 mg, t R = 24.3 min) and 4 (8.8 mg, t R = 48.6 min), respectively. Fr.11 was eluted by MeOH on a Sephadex LH-20 to remove pigmentum to receive Fr.11-1 (4.2 g), Fr.11-2 (5.0 g), and Fr.11-3 (430 mg). Fr.11-2 was N 2 protection. The reaction products were dissolved in the mixed solution of 0.2 mL N-(trimethylsilyl)imidazole and 2 mL anhydrous pyridine, and the mixture was warmed at 60 • C for another 1 h. Then, the solvent was evaporated under N 2 protection. The residue was suspended in cyclohexane and water, the cyclohexane layer was the trimethylsilyl ether derivatives of monosaccharide. The mixture was filtered through a 0.45 µm membrane to remove the precipitate and analyzed by GC. Separations were carried out on an HP-5 capillary column (30 m × 0.32 mm, 0.5 µm). Highly pure N 2 was used as a carrier gas (1.0 mL/min flow rate), and the FID detector operated at 250 • C (column temperature 250 • C). The carbohydrates were determined by comparing the retention times with standard trimethylsilyl ether derivatives prepared from authentic sugars using the same procedure performed for the sample. Retention times for authentic sugars after being derivatized were 11.23 min (D-Api), 12.20 min (L-Ara), 13.34 min (D-Xyl), and 14.48 min (L-Rha), respectively.

Cytotoxicity Assay for Compounds 1-5
The human pancreatic adenocarcinoma PANC-1 and BxPC3 cell lines were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China) and cultured in DMEM (Corning, Beijing, China) supplemented with 10% FBS (Sigma, Shanghai, China) and 1% Penicillin-Streptomycin (Sigma, Shanghai, China) at 37 • C with 5% CO 2 . In the exponential phase of the growth, cells were plated onto 96-well plates at a concentration of 8000 cells/well for 24 h. Compounds 1-5 were prepared to various concentrations (80, 40, 20, 10, 5, 2.5, 1.25, and 0.625 µM in medium containing less than 0.1% DMSO) and incubated in 96-well plates (each concentration in six-fold wells) for 72 h. Gemcitabine (Gem, Meilunbio, ≥98%, Dalian, China) was offered as the positive control. Cell viability was determined according to reported assay methods using the commercial CCK8 kit (Elabscience, Wuhan, China) [29]. The optical density (OD) of each well was measured with an AMR-100 microplate reader at 450 nm (Allsheng Corporation, Hangzhou, China). Cytotoxicity emerged as the value of the drug concentration at the inhibition of cell growth by 50% (IC 50 ).

Conclusions
This study afforded 10 compounds from the rhizomes of Paris mairei, including five new spirostane saponins. None of the new compounds exhibited cytotoxicity against PANC-1 and BxPC3 pancreatic cell lines, implying that the polyglucosides at 1-hydroxy in spirostane saponins may significantly decreased the activities of antitumor.

Data Availability Statement:
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds 6-10 are available from the authors.