Five New Polyoxypregnane Glycosides from the Vines of Aspidopterysobcordata 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.

1. Introduction

Pregnane glycosides are substances with a basic steroidal skeleton and at least one glycosidic bond structure [1,2,3]. Such glycosides not only have diverse structures but also show various biological activities, such as anti-tobacco mosaic virus [4], anti-inflammatory [5], antiproliferative [6], antioxidant [7], antibacterial [8], antifungal [9] and antitumor activities [10]. In the last few years, many pregnane glycosides have attracted considerable attention from pharmacologists on account of their remarkable cancer inhibitory or anticarcinogenic activities [11,12,13,14,15]. To date, hundreds of different pregnane glycosides have been found in the plants of Malpighiaceae, Asclepiadaceae, Apocynaceae, Ranunculaceaem and Zygophyllaceae [3,16,17,18,19,20].

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.

Figure 1

Structures of compounds 1–5.

2. Results

2.1. Structure Determination

Compound 1 was obtained as a white amorphous powder, and its molecular formula was inferred to be C52H74O20 from the HRESIMS ion peak at m/z 1041.4652 [M + Na]+ (calcd.1041.4666, C52H74O20Na). Its IR spectrum showed absorption bands ascribed to the hydroxyl (3390 cm−1) and carbonyl (1718 cm−1) groups. With the assistance of the HSQC spectrum (Supplementary Figure S4), the 1H- and 13C-APT spectral data of 1 (Table 1 and Table 2) displayed two angular methyl proton signals [δH 1.18 (3H, s, H3-18) and 1.14 (3H, s, H3-19)], one acetyl methyl proton signal [δH 2.06 (3H, s, H3-21)] and one olefic proton signal [δH 5.29 (1H, d, J = 5.4 Hz, H-6)], as well as three downfield carbon signals [δC 138.3 (C-5), 118.4 (C-6) and 211.6 (C-20)], which suggests the presence of a C21 steroidal pregen-5-en-20-one skeleton in the molecular structure [24]. In addition, its 1D-NMR spectral data (Supplementary Figures S1 and S2) revealed the presence of a benzoyl group [δH 7.80 (2H, dd, J = 7.8, 1.2 Hz, Bz-H-3, 7), 7.62 (1H, t, J = 7.4 Hz, Bz-H-5) and 7.49 (2H, t, J = 7.2 Hz, Bz-H-4, 6); δC 164.8 (Bz-C-1), 129.6 (Bz-C-2), 129.2 (Bz-C-3, 7), 128.6 (Bz-C-4, 6) and 133.4 (Bz-C-5)], a tigloyl group [δH 6.49 (1H, q, J = 7.2 Hz, Tig-H-3), 1.53 (3H, d, J = 7.2 Hz, Tig-H3-4) and 1.35 (3H, s, Tig-H3-5); δC 166.8 (Tig-C-1), 127.2 (Tig-C-2), 138.0 (Tig-C-3), 14.1 (Tig-C-4) and 11.3 (Tig-C-5)] and three anomeric methines [δH 4.60 (1H, d, J = 9.6 Hz, Oli-H-1, 4.42 (1H, d, J = 7.8 Hz, Allo-H-1) and 4.21 (1H, d, J = 7.8 Hz, Glc-H-1); δC 96.7 (Oli-C-1), 101.5 (Allo-C-1) and 104.8 (Glc-C-1)].

Table 1

1H-NMR (600 MHz) spectral data of compounds 1–5 in DMSO-d.

Position	1	2	3	4	5
1	1.17 (m), 1.81 (m)	1.17 (m), 1.81 (m)	1.17 (m), 1.81 (m)	1.20 (m), 1.71 (m)	0.92 (m), 1.90 (m)
2	1.71 (m), 1.99 (m)	1.71 (m), 2.00 (m)	1.61 (m), 2.01 (m)	1.75 (m), 1.99 (m)	1.59 (m), 2.03 (m)
3	3.01 (m)	3.06 (m)	3.06 (m)	3.01 (m)	3.26 (m)
4	2.21 (m), 2.43 (m)	2.21 (m), 2.42 (m)	2.21 (m), 2.31 (m)	1.59 (m), 1.62 (m)	1.44 (m), 2.09 (m)
5	-	-	-	1.18 (m)	1.90 (m)
6	5.29 (d, J = 5.4 Hz)	5.17 (d, J = 5.4 Hz)	5.45 (d, J = 5.4 Hz)	1.07 (m), 1.61 (m)	1.04 (m), 1.66 (m)
7	1.81 (m), 2.24 (m)	1.88 (m), 2.21 (m)	1.17 (m), 2.11 (m)	1.42 (m), 1.60 (m)	1.36 (m), 1.37 (m)
8	-	-	1.80 (m)	2.06 (m)	2.06 (m)
9	2.01 (d, J = 10.8 Hz)	1.96 (d, J = 10.2 Hz)	1.63(d, J = 10.2 Hz)	1.65 (d, J = 10.8 Hz)	1.66 (d, J = 10.2 Hz)
10	-	-	-	-	-
11	5.89 (t, J = 10.8 Hz)	5.70 (t, J = 10.2 Hz)	5.25 (t, J = 10.2 Hz)	5.16 (t, J = 10.2 Hz)	5.21 (t, J = 10.8 Hz)
12	5.02 (d, J = 10.8 Hz)	4.89 (d, J = 10.2 Hz)	4.78 (d, J = 10.2 Hz)	4.97 (d, J = 10.2 Hz)	4.75 (d, J = 10.8 Hz)
13	-	-	-	-	-
14	-	-	-	-	-
15	1.81 (m), 2.21 (m)	1.85 (m), 2.21 (m)	1.21 (m), 1.31 (m)	1.41 (m), 1.53 (m)	1.49 (m), 1.56 (m)
16	1.80 (m), 2.11 (m)	1.79 (m), 2.11 (m)	1.39 (m), 2.41 (m)	1.81 (m), 2.19 (m)	1.81 (m), 2.12 (m)
17	2.70 (m)	2.89 (m)	3.34 (m)	3.35 (m)	3.34 (m)
18	1.14 (s)	1.22 (s)	0.96 (s)	0.81(s)	0.82 (s)
19	1.35 (s)	1.43 (s)	1.01 (s)	0.91 (s)	0.92 (s)
20	-	-	-	-	-
21	2.13 (s)	2.15 (s)	2.06 (s)	2.10 (s)	2.07 (s)
11-O	Bz	Tig	Ac	Ac	Tig
2	-	-	1.80 (s)	1.78 (s)	-
3	7.80 (dd, J = 7.2, 1.2 Hz)	6.61 (q, J = 7.2 Hz)	-	-	6.58 (q, J = 7.2 Hz)
4	7.49 (t, J = 7.2 Hz)	1.73 (d, J = 7.2 Hz)	-	-	1.69 (d, J = 7.2 Hz)
5	7.62 (t, J = 7.2 Hz)	1.61 (s)	-	-	1.60 (s)
6	7.49 (t, J = 7.2 Hz)	-	-	-	-
7	7.80 (dd, J = 7.2, 1.2 Hz)	-	-	-	-
12-O	Tig	Tig	Tig	Tig	Tig
2	-	-	-	-	-
3	6.49 (q, J = 7.2 Hz)	6.69 (q, J = 7.2 Hz)	6.83 (q, J = 7.2 Hz)	6.81 (q, J = 7.2 Hz)	6.68 (q, J = 7.2 Hz)
4	1.53 (d, J = 7.2 Hz)	1.74 (d, J = 7.2 Hz)	1.80 (d, J = 7.2 Hz)	1.79 (d, J = 7.2 Hz)	1.73 (d, J = 7.2 Hz)
5	1.35 (s)	1.69 (s)	1.78 (s)	1.76 (s)	1.60 (s)
Oli/Ole-1	4.60 (d, J = 9.6 Hz)	4.62 (d, J = 9.6 Hz)	4.78 (d, J = 9.6 Hz)	4.65 (t, J = 9.6 Hz)	4.56 (t, J = 9.6 Hz)
2	1.82 (m), 2.12 (m)	1.82 (m), 2.23 (m)	1.82 (m), 2.21 (m)	1.42 (m), 2.21 (m)	1.91 (m), 2.25 (m)
3	3.01 (m)	3.42 (m)	3.52 (m)	3.02 (m)	3.47 (m)
4	3.85 (m)	3.86 (m)	3.21 (m)	3.82 (m)	3.37 (m)
5	3.14 (m)	3.01 (m)	3.01 (m)	3.00 (m)	3.45 (m)
6	1.22 (d, J = 6.0 Hz)	1.19 (d, J = 6.0 Hz)	1.24 (d, J = 6.0 Hz)	1.19 (d, J = 6.0 Hz)	1.09 (d, J = 6.0 Hz)
-OCH3	-	-	-	-	3.26 (s)
Allo-1	4.42 (d, J = 7.8 Hz)	4.45 (d, J = 8.4 Hz)	4.48 (d, J = 7.8 Hz)	4.46 (d, J = 7.8 Hz)	4.55 (d, J = 7.8 Hz)
2	3.21 (m)	3.22 (m)	3.01 (m)	3.24 (m)	3.15 (m)
3	3.82 (m)	3.81 (m)	3.81 (m)	3.85 (m)	3.04 (m)
4	3.38 (m)	3.61 (m)	3.33 (m)	3.25 (m)	3.34 (m)
5	3.61 (m)	3.61 (m)	3.61 (m)	3.27 (m)	3.26 (m)
6	1.21 (d, J = 6.0 Hz)	1.23 (d, J = 6.0 Hz)	1.20 (d, J = 6.0 Hz)	1.23 (d, J = 6.0 Hz)	1.23 (d, J = 6.6 Hz)
3-OCH3	3.74 (s)	3.74 (s)	3.46 (s)	3.46 (s)	3.47 (s)
Glc-1	4.21(d, J = 7.8 Hz)	4.22(d, J = 7.8 Hz)	4.22(d, J = 7.8 Hz)	4.22(d, J = 7.8 Hz)	-
2	2.89 (m)	2.89 (m)	2.99 (m)	3.24 (m)	-
3	3.05 (m)	3.02 (m)	3.15 (m)	3.05 (m)	-
4	3.40 (m)	3.41 (m)	3.01 (m)	3.04 (m)	-
5	3.31 (m)	3.24 (m)	3.71 (m)	3.19 (m)	-
6	3.51 (m)	3.74 (m)	3.50 (m)	3.46 (m)	-
8-OH/14-OH	3.98 (s)/4.75 (s)	3.90 (s)/4.71 (s)	-/4.48 (s)	-/4.42 (s)	-/4.40 (s)

Table 2

13C-NMR (150 MHz) spectral data of compounds 1–5 in DMSO-d.

Position	1	2	3	4	5	Position	1	2	3	4	5
1	38.0	38.0	37.8	37.0	37.0	7	129.2	-	-	-	-
2	29.0	29.1	29.4	27.5	27.5	12-O	Tig	Tig	Tig	Tig	Tig
3	76.8	76.6	76.6	76.6	75.2	1	166.8	166.9	166.9	166.8	166.8
4	38.6	38.1	38.4	33.6	32.7	2	127.2	127.5	127.4	127.5	127.5
5	138.4	138.4	139.0	43.6	43.6	3	138.0	138.0	138.7	138.5	138.0
6	118.4	118.3	121.9	29.6	29.4	4	14.1	14.3	14.4	14.4	14.2
7	26.0	26.2	27.1	28.6	28.6	5	11.3	11.6	11.9	11.8	11.7
8	75.1	75.0	36.8	36.5	36.6	Oli/Ole-1	96.7	96.8	96.8	96.5	96.4
9	47.6	47.6	46.5	48.7	48.9	2	38.9	38.9	38.9	38.7	36.6
10	39.2	39.3	39.0	37.1	37.1	3	68.8	68.8	68.8	68.8	78.6
11	71.4	70.5	70.5	70.8	70.8	4	87.2	87.2	87.2	87.3	82.3
12	77.6	77.6	77.0	77.4	77.5	5	69.9	69.9	70.0	69.9	70.5
13	54.6	54.6	54.0	54.0	53.9	6	17.2	17.2	17.2	17.2	18.0
14	84.5	84.5	82.9	82.8	82.9	-OCH3	-	-	-	-	56.3
15	35.5	35.5	33.3	34.6	34.6	Allo-1	101.5	101.6	101.6	101.6	100.8
16	23.2	22.8	22.8	23.0	23.1	2	70.5	70.5	70.5	70.5	73.1
17	58.6	58.6	57.8	57.8	57.8	3	81.4	81.4	81.4	81.4	82.8
18	12.9	12.9	11.4	11.5	11.5	4	81.6	81.6	81.6	81.6	71.6
19	17.6	17.2	18.6	13.6	11.7	5	68.6	68.6	68.6	68.6	69.4
20	211.6	211.5	211.1	211.1	211.1	6	17.3	17.7	17.7	17.7	18.4
21	30.6	30.6	30.7	30.7	30.7	3-OCH3	60.9	61.0	61.0	61.0	61.4
11-O	Bz	Tig	Ac	Ac	Tig	Glc-1	104.8	104.9	104.9	104.9	-
1	164.8	165.9	169.6	169.8	166.4	2	76.6	76.6	76.6	76.6	-
2	129.6	128.0	21.1	21.2	127.9	3	73.7	73.7	73.7	73.7	-
3	129.2	138.0	-	-	138.0	4	70.1	70.2	70.2	70.2	-
4	128.6	14.3	-	-	14.2	5	76.9	76.9	77.0	76.9	-
5	133.4	11.7	-	-	11.6	6	61.4	61.4	61.4	61.4	-
6	128.6	-	-	-	-

The 1H-1H COSY spectrum (Supplementary Figure S3) showed four spin systems, H2-1/H2-2/H-3/H2-4, H-6/H2-7, H-9/H-11/H-12 and H2-15/H2-16/H-17, in the aglycone moiety (highlighted in red in Figure 2). The positions of benzoyl and tigloyl groups were located at C-11 and C-12, respectively, based on HMBC correlations (highlighted in blue in Figure 2) between H-11 (δH 5.88) and Bz-C-1 (δC 164.8), H-12 (δH 5.01) and Tig-C-1 (δC 166.8). Meanwhile, the key HMBC correlations between 8-OH (δH 3.98) and C-8 (δC 75.1) and between 14-OH (δH 4.75) and C-8 (δC 75.1), C-13 (δC 54.6) and C-14(δC 84.5) indicate that two hydroxyl groups are substituted at C-8 and C-14, respectively. Afterwards, according to careful analysis of the 2D NMR spectral data (Supplementary Figures S3–S6), three sugars were proposed to be D-olivopyranose (Oli), 6-deoxy-3-O-methyl-D-allopyranose (Allo) and D-glucose (Glc), further confirmed by TLC and gas chromatography (GC) in comparison with authentic monosaccharides. The connectivity and linkage position of these sugars were identified by their crucial HMBC correlations between Glc-H-1 (δH 4.21) and Allo-C-4 (δC 81.6), between Allo-H-1 (δH 4.42) and Oli-C-4 (δC 87.2), and between Oli-H-1 (δH 4.60) and C-3 (δC 76.8). Based on the above analysis, the planar construction of 1 was determined.

Figure 2

Key 1H-1H COSY and HMBC correlations for compound 1.

The coupling constants and NOESY spectral data (Table 1 and Supplementary Figure S6) were used to clarify the relative configuration of 1. The β configurations of the three sugars were each confirmed by their large coupling constants (3J1,2 > 7 Hz). Moreover, the coupling constant (J = 10.8 Hz) between H-11 and H-12 suggests that both protons are in different directions, which was further verified in the NOESY experiment. Subsequently, the NOE correlations (Figure 3) between H-1α and H-3 and H-9, between H-12 and H-17 and H-9, between H3-19 and H-11 and H-1β, between H3-18 and 8-OH, and between H3-21 and H3-18 and 14-OH indicate that H-3/H-9/H-12/H-17 are α-oriented, and H-11/8-OH/14-OH/H3-18/H3-19 are β-oriented. Considering the polyoxypregnane glycosides previously reported for A. obcordata [18,24], the absolute configuration of 1 was established. Finally, the whole structure of 1 was identified and named obcordata J.

Figure 3

Key nuclear Overhauser effect (NOE) correlations for compound 1.

Compound 2 was suggested to have the molecular formula C50H76O20, as determined by the HRESIMS ion at m/z 1019.4803 [M + Na]+ (calcd. 1019.4822, C50H76O20Na). The 1H-NMR and 13C-APT spectral data (Table 1 and Table 2, Supplementary Figures S7 and S8) of 2 were structurally similar to those of 1, except for the absence of the benzoyl group and the presence of an additional tigloyl group in 2. In the 13C-APT spectrum (Supplementary Figure S8) of 2, the carbon signals at δC 166.9, 165.9, 138.0, 138.0, 128.0, 127.5, 14.3, 14.3, 11.7 and 11.6 suggest the existence of two tigloyl groups in 2. At the same time, the key HMBC correlations between H-11 (δH 5.69) and 11-O-Tig-C-1 (δC 165.9), H-12 and 12-O-Tig-C-1 (δC 166.9) illustrate that two tigloyl groups are located at C-11 and C-12. Thus, the complete structure of 2 was established and named obcordata K.

Compound 3, obtained as a white amorphous powder, was established to have the molecular formula C47H72O19 by the HRESIMS ion peak at m/z 963.4518 [M + Na]+ (calcd. 963.4560, C47H72O19Na). Its 1D-NMR data (Supplementary Figures S13 and S14, Table 1 and Table 2) were quite similar to those of 1, except for the absence of the benzoyl and hydroxy groups and the presence of an extra acetoxyl group [δH 1.80 (3H, s, Ac-H3-2); δC 169.6 (Ac-C-1), 21.1 (Ac-C-2)] in 3. The HMBC correlations between H-11 (δH 5.25) and Ac-C-1 (δC 169.6) indicate that the acetoxyl group is replaced at C-11. Based on the HMBC correlations between 14-OH (δH 4.48) and C-8 (δC 36.8), C-13 (δC 54.0), C-14 (δC 82.9) and C-15 (δC 33.3), the remaining hydroxyl group was identified to be substituted at C-14. Together with its NOESY spectral data (Supplementary Figure S18), the structure of 3 was finally confirmed and named obcordata L.

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, C47H74O19Na), which showed the molecular formula C47H74O19 and two more mass units than obcordata L (3). By detailed comparison of the 1D-NMR spectral data (Table 1 and Table 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 δC 43.6 (C-5) and one additional methylene δC 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.

Compound 5 was suggested to be C45H70O14 based on the HRESIMS pseudomolecular ion peak at m/z 857.4640 [M + Na]+ (calcd. 857.4658, C45H70O14Na). The overall consideration of 1D- (Table 1 and Table 2) and 2D-NMR spectral data (Supplementary Figures S25–S30) suggests the presence of a (5α,8β,9α,17α)-20-one-3β,11α,12β,14β-tetradroxypregnane aglycone moiety in 5. With the aid of the HSQC spectrum (Supplementary Figure S28), the 13C-APT spectrum (Table 2 and Supplementary Figure S26) of 5 revealed the appearance of two tigloyl groups (δC 166.8, 166.4, 138.0, 138.0, 127.9, 127.5, 14.2, 14.2, 11.7 and 11.6) and two sugar units (two anomeric carbons at δC 100.8 (Allo-C-1) and 96.4 (Ole-C-1)). Two tigloyl groups were confirmed to be located at C-11 and C-12 by the HMBC correlations between H-11 (δH 5.21) and 11-O-Tig-C-1 (δC 166.4) and between H-12 (δH 4.75) and 12-O-Tig-C-1 (δC 166.8). With the assistance of 2D-NMR spectral data, two sugar units were fully assigned as D-oleandrose (Ole) and 6-deoxy-3-O-methyl-D-allopyranose (Allo) after hydrolysis, which are consistent with the sugars of obcordata A previously reported from A. obcordata [23]. Based on the NOESY spectral data, compound 5 was finally identified and named obcordata N.

2.2. 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 EC50 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 EC50 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 EC50 value of 6.72 μM, compared with the positive control apocynin (Apo.), with an EC50 value of 6.88 μM. Therefore, the protective mechanism of obcordata J (1) against nephrolithiasis activity deserves significant further exploration.

Figure 4

Protective effects of compounds 1–5 in HK-2 cells injured by calcium oxalate crystals.

3. Materials and Methods

3.1. 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.

3.2. 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.

3.3. 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 g). The concentrated 95% ethanol extract was dissolved in water and partitioned successively using different solvents (petroleum ether, dichloromethane, ethyl acetate and n-butanol, 2 L, 3 times) to obtain different extracts. The ethyl acetate fraction (80 g) was selected for further separation on account of its moderate antinephrolithiasis effect on HK-2 cells exposed to calcium oxalate crystals [27]. The crude ethyl acetate extracts were chromatographed over silica gel CC (200–300 mesh, 10 cm × 100 cm) using dichloromethane–methanol (1:0→0:1, 5 L), which yielded 12 fractions. Semi-preparative HPLC (S-HPLC) with a Lumtech K-1001 analytic HPLC system (a K-2600 UV detector and two K-501 pumps) and a YMC Pack C18 column (250 mm × 10 mm, 5 μM, YMC Co. Ltd., Kyoto, Japan) was used to purify compounds (methanol–water system, 2 mL/min). Fr.8 (5.6 g) was purified by S-HPLC (methanol–water system, 72:28, v/v) to yield obcordata M (4)(1.5 mg, tR = 18.5 min) and obcordata N (5) (1.6 mg, tR = 25.2 min). Fr.9 (6 g) was purified by S-HPLC (methanol–water system, 52:48, v/v) to yield obcordata J (1) (4 mg, tR = 13.4 min), obcordata K (2) (1.9 mg, tR = 16.2 min) and obcordata L (3) (1.8 mg, tR = 19.3 min).

3.4. Characterization of Compounds 1–5

Obcordata J (1): White amorphous powder; [α]25D +37 (c 0.15, methanol); UV λmax (methanol) (log ε): 275 (3.62) nm; IR (KBr) νmax: 3,423, 1718, 1660, 1512, 1420 and 1210 cm−1; HRESIMS m/z 1041.4652 [M + Na]+ (calculated for C52H74O20Na, 1041.4666,); 1H- and 13C-NMR spectral (600, 150 MHz, DMSO-d) data: see Table 1 and Table 2.

Obcordata K (2): White amorphous powder; [α]25D +40.5 (c 0.10, methanol); UV λmax (methanol) (log ε): 275 (3.35) nm; IR (KBr) νmax: 3390, 1715, 1658, 1503, 1427 and 1215 cm−1; HRESIMS m/z 1019.4803 [M + Na]+ (calculated for C50H76O20Na, 1019.4822); 1H- and 13C-NMR spectral (600, 150 MHz, DMSO-d) data: see Table 1 and Table 2.

Obcordata L (3): White amorphous powder; [α]25D +48.2 (c 0.11, methanol); UV λmax (methanol) (log ε): 272 (3.04) nm; IR (KBr) νmax: 3403 and 1720 cm−1; HRESIMS m/z 963.4518 [M + Na]+ (calculated for C47H72O19Na, 963.4560); 1H- and 13C-NMR spectral (600, 150 MHz, DMSO-d) data: see Table 1 and Table 2.

Obcordata M (4): White amorphous powder; [α]25D +52.2 (c 0.13, methanol); UV λmax (MeOH) (log ε): 225 (2.84) nm; IR (KBr) νmax: 3410, 1718; 1636 and 1524 cm−1; HRESIMS m/z 965.4703 [M + Na]+ (calculated for C47H74O19Na, 965.4717); 1H- and 13C-NMR spectral (600, 150 MHz, DMSO-d) data: see Table 1 and Table 2.

Obcordata N (5): White amorphous powder; [α]25D +44.6 (c 0.16, methanol); UV λmax (methanol) (log ε): 230 (2.90) nm; IR (KBr) νmax: 3385, 1690, 1620 and 1542 cm−1; HRESIMS m/z 857.4640 [M + Na]+ (calculated for C45H70O14Na, 857.4658); 1H- and 13C-NMR spectral (600, 150 MHz, DMSO-d) data: see Table 1 and Table 2.

3.5. Compound Hydrolysis

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

3.6. Cytotoxicity Assay

In 96-well microplates, HK-2 cells were seeded (2 × 104 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.

3.7. Antinephrolithiasis Screening

The method of preparing COM (calcium oxalate monohydrate) crystals was used with little modification, as described previously [23,25,26]. Different concentrations (1.56, 3.12, 6.25, 12.5, 25 and 50 µM) of compounds 1–5 were used in the dose-dependent study using this method. Cells without any treatment were used as a control. Furthermore, various concentrations of Apo. were used as a positive control. After 24 h, the viability of the injured HK-2 cells was measured by the MTT assay.

4. Conclusions

In this study, five new polyoxypregnane glycosides, obcordatas J–N (1–5), were obtained from 95% EtOH extracts of the dried vines of A. obcordata. The complete structures of all of these new compounds were eventually elucidated by extensive spectral analysis. Their antinephrolithiasis activities were measured based on the viability of HK-2 cells exposed to COM crystals in vitro. Among all of the tested compounds, obcordata J (1) was revealed to have the most potent antinephrolithiasis activity, with an EC50 value of 6.72 μM. To date, about 60 compounds, including 17 polyoxypregnane glycosides, have been isolated from A. obcordata. Interestingly, the glycoside obcordata A was reported to exert its antinephrolithiasis activity through the NOX4/ROS/P38 MAPK pathway [18,22,23,24,28]. In sum, polyoxypregnane glycosides might be the material basis of the A. obcordata antinephrolithiasis effect, which deserves in-depth study.