Five New Polyoxypregnane Glycosides from the Vines of Aspidopterys obcordata and Their Antinephrolithiasis Activity

From the dried vines of Aspidopterys obcordata Hemsl, five new polyoxypregnane glycosides, named obcordatas J–N (1–5), were obtained. Their structures were fully elucidated and characterized by HRESIMS and extensive spectroscopic data. In addition, all of the new compounds were screened for their antinephrolithiasis activity in vitro. The results showed that compounds 1–3 have prominent protective effects on calcium oxalate crystal-induced human kidney 2 (HK-2) cells, with EC50 values ranging from 6.72 to 14.00 μM, which is consistent with the application value of A. obcordata in folk medicine for kidney stones.

A. obcordata, a wood liana of the family Malpighiaceae, is distributed mainly in Xishuangbanna, Yunnan Province, China. The vines of this plant have a long history as a "Dai Medicine" for the treatment of urinary tract infections, chronic nephritis, rheumatic bone pain, cystitis and kidney stones [21,22]. In our previous research, the antinephrolithiasis effects of a distinct polar extract of A. obcordata were investigated. The results showed that the 95% ethanol extract of this plant could reduce the volume of kidney stones and decrease urea nitrogen levels and serum creatinine in rats with nephrolithiasis [23]. Although A. obcordata has been indicated as safe and effective in the treatment of kidney stones, the material basis of this plant's antinephrolithiasis effect is still unclear. In order to further define the active ingredients, an investigation of the 95% ethanol extracts of the dried vines of A. obcordata was carried out. Finally, five new polyoxypregnane glycosides, obcordatas J-N (1-5) (Figure 1), were obtained in the experiment. Thus, this article reports the isolation process and full structural elucidation of these glycosides, as well as their antinephrolithiasis activity in vitro.
article reports the isolation process and full structural elucidation of these glycosides, as well as their antinephrolithiasis activity in vitro.
Compound 4 was isolated and purified as a white amorphous powder. The HRESIMS displayed an ion peak at m/z 965.4703 [M + Na] + (calcd. 965.4717, C 47 H 74 O 19 Na), which showed the molecular formula C 47 H 74 O 19 and two more mass units than obcordata L (3). By detailed comparison of the 1D-NMR spectral data (Tables 1 and 2, Supplementary Figures S19 and S20) with those of 3, significant differences were the disappearance of one double bond and the presence of an extra methine δ C 43.6 (C-5) and one additional methylene δ C 29.6 (C-6), which indicates that 4 is the reduction product of 3. In the NOESY spectrum, correlations between H-3 and H-5, H-5 and H-9 suggest that H-5 is α-oriented. Finally, compound 4 was illustrated and given the name obcordata M.

Antinephrolithiasis Activity
The antinephrolithiasis activity of the obtained compounds 1-5 was evaluated in HK-2 cells injured by calcium oxalate crystals via the MTT assay [23,25,26]. In view of the potential cytotoxicity of compounds 1-5 in mammalian cells, normal HK-2 cells were treated with 100 µM of all compounds for 24 h, and the cell viabilities were not significantly affected. Afterwards, the protective effects of compounds (1-5) against calcium oxalate crystal-induced HK-2 cells were determined in vitro. The results of the viabilities of injured HK-2 cells (Figure 4) showed that the EC 50 of all isolated compounds ranged from 6.72 to 50.69 µM. Among them, compounds 1-3 displayed better protection in injured HK-2 cells, with EC 50 values of 6.72, 11.85 and 14.00 µM, respectively. It is worth noting that compound 1 exhibited the most potent antinephrolithiasis activity, with an EC 50 value of 6.72 µM, compared with the positive control apocynin (Apo.), with an EC 50 value of 6.88 µM. Therefore, the protective mechanism of obcordata J (1) against nephrolithiasis activity deserves significant further exploration.

General Experimental Materials
UV spectra and optical rotations were measured with a UV2550 (Shimadzu, Kyoto, Japan) and a 341 digital polarimeter (PerkinElmer, Norwalk, CT, USA), respectively. IR spectral data were determined with FTIR-8400 spectrometers (Shimadzu, Japan). NMR spectral data were recorded on a Bruker AV III 600 NMR spectrometer (Bruker, Billerica, Germany). Mass spectra were performed by using the Waters micromass Q-TOF system (Waters, Bremen, GA, USA). Silica gels (200-300 mesh, Qingdao Marine Chemical Plant, Qingdao, China) were used for column chromatography (CC). TLC analyses were measured by spraying with 5% H2SO4 and heating at 100 °C (silica gel GF 254, Qingdao Haiyang Chemical Co., Qingdao, China). All solvents (Beijing Chemical Works, Beijing, China) used were analytical grade.

Plant Material
The vines of A. obcordata were collected from Jinghong (Yunnan Province, China) and were authenticated by Professor Rongtao Li (Yunnan Branch, Institute of Medicinal Plant (IMPLAD)). The voucher specimen (CS-16368) was deposited at IMPLAD.

Extraction and Isolation
The vines of A. obcordata (5.0 kg) were air-dried, powdered and then repeatedly extracted with 95% ethanol (25 L) four times, and each extraction lasted for 3 h. The extracted solution was evaporated under vacuum to provide the crude ethyl alcohol extract (280.0

General Experimental Materials
UV spectra and optical rotations were measured with a UV2550 (Shimadzu, Kyoto, Japan) and a 341 digital polarimeter (PerkinElmer, Norwalk, CT, USA), respectively. IR spectral data were determined with FTIR-8400 spectrometers (Shimadzu, Japan). NMR spectral data were recorded on a Bruker AV III 600 NMR spectrometer (Bruker, Billerica, Germany). Mass spectra were performed by using the Waters micromass Q-TOF system (Waters, Bremen, GA, USA). Silica gels (200-300 mesh, Qingdao Marine Chemical Plant, Qingdao, China) were used for column chromatography (CC). TLC analyses were measured by spraying with 5% H 2 SO 4 and heating at 100 • C (silica gel GF 254, Qingdao Haiyang Chemical Co., Qingdao, China). All solvents (Beijing Chemical Works, Beijing, China) used were analytical grade.

Plant Material
The vines of A. obcordata were collected from Jinghong (Yunnan Province, China) and were authenticated by Professor Rongtao Li (Yunnan Branch, Institute of Medicinal Plant (IMPLAD)). The voucher specimen (CS-16368) was deposited at IMPLAD.

Compound Hydrolysis
The hydrolysis method of compounds 1-5 was the same as reported in our previous study [24].

Cytotoxicity Assay
In 96-well microplates, HK-2 cells were seeded (2 × 10 4 cells/mL) and treated with compounds 1-5 in different concentrations (6.25, 12.5, 25, 50 and 100 µM) for 24 h. After that, the cytotoxicity of all compounds against HK-2 cells was determined via the MTT assay. Furthermore, an automatic multifunctional microplate reader was used to measure the OD values at 570 nm.