Cytotoxic polyhydroxylated pregnane glycosides from Cissampelos pareira var. hirsuta

Abstract

Fourteen new polyhydroxylated pregnane glycosides, cissasteroid A–N (1–14), and five known analogues (15–19), were isolated from the dried whole plant of Cissampelos pareira var. hirsuta. Their structures and stereochemistry were elucidated by extensive spectroscopic data, chemical hydrolysis, and ECD measurements. All the compounds were tested for their cytotoxicity against five human cancer cell lines, and inhibitory activity against NO release in LPS-induced RAW 264.7 cells. Compared with cisplatin, compound 7 showed more potent cytotoxicities against the HL-60, A549, SMMC-7721, MCF-7, and SW480 cell lines, with IC₅₀ values of 2.19, 14.38, 2.00, 7.58, and 7.44 μM, respectively. The preliminary study of structure–activity relationship indicated that benzoic acid esterification at C-20 may have a negative effect on the cytotoxic activity of polyhydroxylated pregnane derivatives in these five human cancer cell lines. These results revealed the potential of compound 7 as an ideal antitumor lead compound.

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Introduction

Pregnane glycosides are an important class of secondary metabolites in the plant kingdom. Previous pharmacological investigations have demonstrated various kinds of bioactivities, such as immunosuppressive, anti-oxidant,¹ anti-inflammatory,² anti-epileptic,³ neuroprotective,⁴ anti-diabetic,⁵ anti-proliferative,⁶ anti-obesitic,⁷ and gastroprotective properties.⁸ The genus Cissampelos (Menispermaceae) is composed of 21 species, distributed in the Southwest of China, India, Malaysia, Pakistan, America and East Africa.^9,10 The plants of this genus are used for the treatment of indolent ulcer, asthma, cholera, diarrhea, dysentery, epilepsy, fever, rabies,⁹ malaria,¹⁰ abdominal pain, inflammation, indigestion, wound healing, skin disorders, and snake venom.¹¹ More than 60 natural products have been previously obtained, including alkaloids,¹² flavonoids,¹¹ and terpenes.¹³ Listed in Chinese Pharmacopoeia, the whole plant of C. pareira var. Hirsuta has been used clinically for trauma pain and bleeding as a traditional Dai medicine. In a search for bioactive natural products from traditional Chinese medicines, fourteen new polyhydroxylated pregnane glycosides, cissasteroid A–N (1–14), and five known analogues (15–19), were isolated from the dried whole plant of C. pareira var. hirsuta (Fig. 1). Detailed isolation, structure elucidation and biological assessment of those isolates are reported herein.

Fig. 1 Structures of compounds 1–19.

Results and discussion

Compound 1 was obtained as a white amorphous powder and possessed a molecular formula C₃₇H₅₂O₁₀ with twelve degrees of unsaturation, as revealed from its HR-ESI-MS analysis (m/z 679.3451 [M + Na]⁺, calcd 679.3458). The IR spectrum displayed the presence of aromatic ring (1639, 1451 cm⁻¹), conjugated carbonyl (1703 cm⁻¹), hydroxyl (3398 cm⁻¹), and ether (1029 cm⁻¹) functionalities. The ¹³C-NMR and DEPT spectra showed thirty-seven carbon signals, including eight quaternary carbons [two olefinic/aromatic, δ_C 140.3 (C-5), 136.1 (C-1′), one ester carbonyl δ_C 167.7 (C-9′), three oxygenated δ_C 75.1 (C-8), 89.5 (C-14), 88.9 (C-17)], sixteen methines [eight oxygenated δ_C 79.3 (C-3), 75.1 (C-8), 71.3 (C-12), 76.3 (C-20), 97.2 (C-1′′), 79.2 (C-3′′), 74.5 (C-4′′), 71.4 (C-5′′), eight olefinic/aromatic δ_C 120.1 (C-6), 129.1 (×2) (C-2′, 6′), 130.0 (×2) (C-3′, 5′), 131.3 (C-4′), 120.0 (C-7′), 145.7 (C-8′)], eight methylenes and five methyls (one oxygenated δ_C 58.1, C-7′′). The ¹H and ¹³C-NMR spectra (Tables 1 and 2) revealed the presence of one cinnamoyl group, one O-methylated 2,6-dideoxysugar and one pregnanehexaol skeleton.¹⁴ One monosubstituted benzene ring δ_H 7.59 (2H, m, H-2′, 6′), 7.40 (3H, m, H-3′, 4′, 5′), one set of trans conjugated olefinic protons δ_H 7.74 (1H, d, J = 16.1 Hz, H-7′), 6.52 (1H, d, J = 16.1 Hz, H-8′), one ester carbonyl δ_C 167.7 (C-9′) were observed, suggesting the occurence of trans-cinnamoyl group.¹⁴ The cymaropyranosyl group was based on a series of signals consisting of one methylene group δ_H 2.13 (1H, m, H-2′′), 1.51 (1H, m, H-2′′), δ_C 35.9 (C-2′′), one methoxy group δ_H 3.43 (3H, s, H-7′′), δ_C 58.1 (C-7′′), one secondary methyl group δ_H 1.21 (3H, d, J = 6.3 Hz, H-6′′), δ_C 18.6 (C-6′′), three oxygenated aliphatic carbons δ_C 79.2 (C-3′′), 74.5 (C-4′′), 71.4 (C-5′′), one anomeric carbon δ_C 97.2 (C-1′′), and one monosaccharide anomeric proton δ_H 4.86 (1H, dd, J = 9.9, 1.6 Hz, H-1′′).¹⁴ One cinnamoyl group, two olefinic carbons δ_C 140.3 (C-5), 120.0 (C-7′), and one cymaropyranosyl group accounted for eight out of the twelve degrees of unsaturation, and the remaining four indicated that compound 1 possesses a tetracyclic carbon skeleton. The presence of three methyls δ_H 1.39 (3H, s, H-18), 1.15 (3H, s, H-19), 1.29 (3H, d, J = 6.3 Hz, H-21), δ_C 9.8 (C-18), 18.7 (C-19), 15.2 (C-21), seven methylenes, three oxygenated methines δ_H 3.51 (1H, m, H-3), 3.45 (1H, dd, J = 10.9, 3.9 Hz, H-12), 5.27 (1H, q, J = 6.3 Hz, H-20), δ_C 79.3 (C-3), 71.3 (C-12), 76.3 (C-20); three oxygenated quaternary carbons δ_C 75.1 (C-8), 89.5 (C-14), 88.9 (C-17), one olefinic proton δ_H 5.33 (1H, br. s, H-6), and two olefinic carbons δ_C 140.3 (C-5), 120.1 (C-6), suggested that compound 1 possessed a 3β,8β,12β,14β,17β,20-hexahydroxypregn-5-ene skeleton, and the aglycone was identified as sarcostin.¹⁵ Its ¹H and ¹³C NMR data (Tables 1 and 2) were unambiguously assigned by analysis of the DEPT, HSQC, HMBC, ¹H–¹H COSY spectra. The ¹³C-NMR chemical shift δ_C 35.9 (C-2′′) of methylene group and coupling constant (9.9, 1.6 Hz) of axial anomeric proton allowed the identification of one β-cymaropyranosyl moiety.¹⁶ Acid hydrolysis of 1 yielded D-cymaropyranose, which was identified by its specific dextrorotatory value.¹⁶ The ¹H–¹H COSY spectrum suggested the presence of eight spin-coupling systems, H-1/H-2/H-3/H-4, H-6/H-7, H-9/H-11/H-12, H-15/H-16, H-20/H-21, H-2′/H-3′/H-4′/H-5′/H6′, H-7′/H8′, and H-1′′/H-2′′/H-3′′/H-4′′/H-5′′/H-6′′, as shown in Fig. 2. In the HMBC spectrum, the correlation (Fig. 2) between H-20 (δ_H 5.27) and C-9′ (δ_C 167.7) indicated that 20-OH was esterified by cinnamic acid. The HMBC cross peak of H-1′′ (δ_H 4.86) with C-3 (δ_C 79.3) suggested that the aglycone was glycosylated at C-3 by cymarose.

Table 1 ¹H NMR data of compounds 1–14^a

No.	1^b	2^b	3^b	4^b	5^b	7^c	8^b
a Recorded at 500 MHz. δH in ppm, J in Hz.b Recorded in methanol-d4.c Recorded in chloroform-d1.
1	1.86, m; 1.09, m	1.89, m; 1.16, m	1.86, m; 1.10, m	1.87, m; 1.08, m	1.84, m; 1.11, m	1.85, m; 1.08, m	1.90, m; 1.10, m
2	1.53, m; 1.84, m	1.61, m; 1.90, m	1.57, m; 1.84, m	1.59, m; 1.84, m	1.59, m; 1.84, m	1.58, m; 1.89, m	1.87, m; 1.63, m
3	3.51, m	3.56, m	3.52, m	3.58, m	3.59, m	3.53, m	3.54, m
4	2.36, m; 2.22, m	2.39, m; 2.26, m	2.34, m; 1.99, m	2.34, m; 2.20, m	2.39, dd (12.6, 3.9); 2.20, m	2.37, m; 2.27, m	2.36, m; 2.23, m
6	5.33, br s	5.38, br s	5.34, br s	5.32, br s	5.37, br s	5.37, br s	5.34, br s
7	2.13, m	2.18, m	2.17, m	2.13, m	2.14, m	2.17, m	2.12, m
9	1.44, m	1.53, m	1.55, m	1.44, m	1.55, m	1.51, m	1.52, m
11	1.60, m; 1.32, m	2.15, m; 1.83, m	1.96, m; 1.65, m	1.87, m; 1.51, m	2.07, m; 1.54, m	2.07, m; 1.55, m	2.06, m; 1.68, m
12	3.45, dd (10.9, 3.9)	4.93, dd (11.2, 4.3)	4.86, dd (11.5, 4.2)	3.47, dd (110, 4.1)	4.77, dd (11.5, 4.3)	4.87, dd (11.4, 4.3)	4.70, dd (11.5, 4.2)
15	1.89, m	2.01, m; 1.90, m	2.16, m	1.88, m; 1.82, m	1.86, m; 1.77, m	1.88, m	1.91, m; 1.72, m
16	1.85, m; 1.52, m	1.85, m	2.01, m; 1.87, m	1.85, m	1.78, m	1.80, m	1.82, m; 1.78, m
18	1.39, s	1.70, s	1.63, s	1.38, s	1.60, s	1.62, s	1.55, s
19	1.15, s	1.18, s	1.11, s	1.14, s	1.19, s	1.13, s	1.15, s
20	5.27, q (6.3)	3.61, q (6.3)	4.79, q (6.2)	5.28, q (6.2)	3.54, q (6.2)	3.55, q (6.2)	3.48, q (6.3)
21	1.29, d (6.3)	1.06, d (6.3)	1.32, d (6.4)	1.28, d (6.2)	1.25, d (6.2)	1.24, d (6.2)	1.07, d (6.3)
2′	7.59, m	8.16, d (8.4)	7.24, d (7.3)	7.59, m	7.65, m	8.07, d (7.2)
3′	7.40, m	7.50, t (7.9)	7.32, m	7.38, m	7.43, m	7.45, t (7.7)	7.00, qd (7.1, 1.3)
4′	7.40, m	7.62, t (7.5)	7.32, m	7.38, m	7.43, m	7.57, t (7.4)	1.82, d (7.1)
5′	7.40, m	7.50, t (7.9)	7.32, m	7.38, m	7.43, m	7.45, t (7.7)	1.87, s
6′	7.59, m	8.16, d (8.4)	7.24, d (7.3)	7.59, m	7.65, m	8.07, d (7.2)
7′	7.74, d (16.1)		7.35, d (16.0)	7.73, d (16.0)	7.81, d (16.0)
8′	6.52, d (16.1)		6.05, d (16.0)	6.50, d (16.0)	6.66, d (16.0)
1′′	4.86, dd (9.9, 1.6)	4.88, dd (9.6, 1.8)		4.85, dd (9.7, 1.4)	4.85, dd (9.7, 1.4)	4.82, dd (9.7, 1.6)	4.82, dd (10.0, 1.3)
2′′	2.13, m; 1.51, m	2.15, m; 1.54, m	7.94, d (7.2)	2.22, m; 1.53, m	2.23, m; 1.56, m	2.17, m; 1.59, m	2.12, m; 1.49, m
3′′	3.59, m	3.62, m	7.32, m	3.49, m	3.52, m	3.58, m	3.59, m
4′′	3.16, dd (9.5, 3.0)	3.19, dd (9.4, 3.0)	7.54, t (7.5)	3.22, dd (9.7, 2.8)	3.24, dd (9.6, 2.9)	3.19, dd (9.6, 2.8)	3.16, dd (9.5, 3.1)
5′′	3.71, m	3.74, m	7.32, m	3.79, m	3.81, m	3.83, m	3.71, m
6′′	1.21, d (6.3)	1.25, d (6.2)	7.94, d (7.2)	1.18, d (6.3)	1.22, d (6.2)	1.03, d (6.2)	1.22, d (6.3)
7′′	3.43, s	3.46, s		3.42, s	3.42, s	3.40, s	3.43, s
1′′′			4.84, dd (9.8, 2.0)	4.77, dd (9.6, 1.7)	4.79, dd (9.8, 1.6)	4.65, dd (9.7, 1.6)
2′′′			2.14, m; 1.50, m	2.06, m; 1.52, m	2.07, m; 1.53, m	2.07, m; 1.53, m
3′′′			3.58, m	3.84, m	3.84, m	3.77, m
4′′′			3.15, dd (9.6, 3.2)	3.16, dd (9.6, 3.2)	3.19, dd (9.6, 3.2)	3.17, dd (9.6, 2.8)
5′′′			3.71, m	3.72, m	3.72, m	3.53, m
6′′′			1.20, d (6.2)	1.22, d (6.3)	1.11, d (6.3)	1.20, d (6.2)
7′′′			3.43, s	3.42, s	3.42, s	3.42, s

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No.	6^b	9^b	10^b	11^b	12^b	13^b	14^b
1	1.76, m; 1.09, m	1.84, m; 1.10, m	1.85, m; 1.13, m	1.90, m; 1.10, m	1.90, m; 1.10, m	1.79, m; 1.10, m	1.79, m; 1.10, m
2	1.54, m; 1.82, m	1.60, m; 1.87, m	1.63, m; 1.89, m	1.87, m; 1.61, m	1.60, m; 1.87, m	1.59, m; 1.85, m	1.59, m; 1.86, m
3	3.58, m	3.53, m	3.54, m	3.42, m	3.52, m	3.50, m	3.51, m
4	2.34, m; 2.19, m	2.37, m; 2.23, m	2.39, m; 2.26, m	2.36, m; 2.21, m	2.38, m; 2.21, m	2.35, m; 2.21, m	2.33, m; 2.21, m
6	5.35, br s	5.34, br s	5.36, br s	5.33, br s	5.34, br s	5.33, br s	5.33, br s
7	2.19, m	2.13, m	2.17, m	2.13, m	2.14, m	2.12, m	2.13, m
9	1.58, m	1.54, m	1.53, m	1.49, m	1.49, m	1.49, m	1.49, m
11	1.58, m; 2.04, m	1.64, m; 2.03, m	1.66, m; 2.04, m	1.95, m; 1.61, m	1.62, m; 1.93, m	1.62, m; 1.93, m	1.61, m; 1.92, m
12	5.03, dd (11.4, 4.3)	4.71, dd (11.6, 4.3)	4.73, dd (11.5, 4.2)	4.67, dd (11.5, 4.1)	4.68, dd (11.5, 4.1)	4.65, dd (11.5, 4.2)	4.66, dd (11.4, 4.1)
15	1.95, m; 2.06, m	1.92, m; 1.85, m	1.95, m	1.89, m	1.89, m	1.95, m; 1.88, m	1.95, m; 1.87, m
16	1.56, m	1.77, m; 1.82, m	1.79, m	1.95, m; 1.89, m	1.94, m; 1.89, m	1.88, m	1.87, m
18	1.68, s	1.55, s	1.57, s	1.45, s	1.46, s	1.45, s	1.45, s
19	1.10, s	1.15, s	1.17, s	1.13, s	1.13, s	1.12, s	1.12, s
20	4.82, q (6.1)	3.48, q (6.3)	3.52, q (6.2)	4.56, q (6.2)	4.58, q (6.2)	4.56, q (6.0)	4.56, q (6.5)
21	1.27, d (6.1)	1.07, d (6.3)	1.21, d (6.2)	1.20, d (6.2)	1.21, d (6.2)	1.20, d (6.0)	1.20, d (6.5)
2′	7.62, d (8.1)			1.88, s	1.89, s	1.88, s	1.88, s
3′	7.09, t (7.8)	7.01, q (7.1)	7.02, qd (7.1, 1.3)
4′	7.43, t (7.4)	1.82, d (7.1)	1.85, d (7.1)
5′	7.09, t (7.8)	1.88, s	1.90, s
6′	7.62, d (8.1)
1′′		4.88, dd (9.6, 1.9)	4.84, dd (9.6, 1.6)
2′′	7.62, d (8.1)	2.09, m; 1.54, m	2.07, m; 1.57, m
3′′	7.33, t (7.8)	3.84, m	3.84, m	7.01, q (6.2)	7.01, q (6.2)	7.00, qd (7.0, 1.0)	7.00, qd (7.0, 1.0)
4′′	7.54, t (7.4)	3.29, dd (9.1, 4.1)	3.27, dd (9.3, 3.0)	1.82, d (7.1)	1.84, d (7.0)	1.83, d (7.1)	1.82, d (7.1)
5′′	7.33, t (7.8)	3.84, m	3.82, m	1.86, s	1.86, s	1.85, s	1.85, s
6′′	7.62, d (8.1)	1.21, d (6.3)	1.21, d (6.1)
7′′		3.43, s	3.45, s
1′′′	4.85, dd (9.7, 1.6)	4.61, dd (9.0, 1.8)	4.60, dd (9.7, 1.5)	4.86, dd (9.6, 1.7)	4.87, dd (9.6, 1.7)	4.86, dd (9.6, 1.8)	4.86, dd (9.6, 1.7)
2′′′	2.04, m; 1.55, m	2.34, m; 1.37, m	2.10, m; 1.57, m	2.13, m; 1.52, m	2.07, m; 1.55, m	2.03, m; 1.55, m	2.05, m; 1.58, m
3′′′	3.50, m	3.21, m	3.84, m	3.59, m	3.85, m	3.60, m	3.81, m
4′′′	3.21, dd (9.6, 2.9)	2.97, t (9.0)	3.26, dd (9.4, 3.0)	3.16, dd (9.6, 3.1)	3.26, dd (9.1, 2.5)	3.22, dd (9.6, 2.9)	3.30, dd (9.6, 3.3)
5′′′	3.80, m	3.27, m	3.82, m	3.72, m	3.82, m	3.81, m	3.81, m
6′′′	1.22, d (6.2)	1.28, d (6.2)	1.08, d (6.2)	1.22, d (6.3)	1.21, d (6.2)	1.18, d (6.4)	1.21, d (6.1)
7′′′	3.42, s	3.44, s	3.44, s	3.43, s	3.44, s	3.42, s	3.42, s
1′′′′	4.79, dd (9.6, 3.2)		4.73, dd (9.6, 1.6)		4.60, dd (9.7, 1.5)	4.76, dd (9.8, 1.8)	4.78, dd (9.7, 1.8)
2′′′′	2.05, m; 1.53, m		2.34, m; 1.36, m		2.33, m; 1.37, m	2.21, m; 1.56, m	2.02, m; 1.58, m
3′′′′	3.83, m		3.20, m		3.42, m	3.84, m	3.81, m
4′′′′	3.15, dd (9.6, 3.2)		2.97, t (9.0)		2.97, t (9.0)	3.16, dd (9.7, 3.2)	3.24, dd (9.7, 3.0)
5′′′′	3.70, m		3.26, m		3.26, m	3.72, m	3.80, m
6′′′′	1.17, d (6.2)		1.30, d (6.2)		1.28, d (6.2)	1.22, d (6.5)	1.18, d (6.3)
7′′′′	3.42, s		3.45, s		3.42, s	3.41, s	3.42, s
1′′′′′							4.59, dd (9.8, 1.8)
2′′′′′							2.32, m; 1.37, m
3′′′′′							3.20, m
4′′′′′							2.97, t (9.0)
5′′′′′							3.27, m
6′′′′′							1.27, d (6.2)
7′′′′′							3.41, s

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Table 2 ¹³C NMR data of compounds 1–14^a

No.	1^b	2^b	3^b	4^b	5^b	6^b	7^c	8^b	9^b	10^b	11^b	12^b	13^b	14^b
a Recorded at 125 MHz.b Recorded in methanol-d4.c Recorded in chloroform-d1.
1	39.8 t	39.8 t	39.7 t	39.8 t	39.8 t	39.7 t	38.9 t	39.8 t	39.8 t	39.8 t	39.7 t	39.7 t	39.7 t	39.7 t
2	30.2 t	30.2 t	30.1 t	30.2 t	30.2 t	30.1 t	29.0 t	30.2 t	30.2 t	30.2 t	30.1 t	30.1 t	30.1 t	30.1 t
3	79.3 d	79.3 d	79.22 d	79.4 d	79.3 d	79.3 d	78.0 d	79.3 d	79.3 d	79.3 d	79.3 d	79.3 d	79.2 d	79.2 d
4	39.9 t	39.8 t	39.8 t	39.9 t	39.8 t	39.8 t	38.8 t	39.8 t	39.8 t	39.8 t	39.8 t	39.8 t	39.7 t	39.8 t
5	140.3 s	140.1 s	140.1 s	140.3 s	140.1 s	140.1 s	139.7 s	140.0 s	140.1 s	140.1 s	140.0 s	140.1 s	140.0 s	140.1 s
6	120.1 d	120.0 d	120.1 d	120.0 d	120.0 d	119.8 d	118.4 d	120.0 d	120.0 d	120.0 d	119.8 d	119.8 d	120.0 d	119.8 d
7	35.3 t	35.3 t	35.2 t	35.3 t	35.2 t	35.2 t	33.7 t	35.2 t	35.2 t	35.2 t	35.1 t	35.1 t	35.1 t	35.1 t
8	75.1 s	75.0 s	75.0 s	75.1 s	75.5 s	75.0 s	72.4 s	75.0 s	75.2 s	75.2 s	75.3 s	75.4 s	74.9 s	75.5 s
9	45.3 d	44.8 d	44.7 d	45.4 d	44.8 d	44.6 d	43.4 d	44.7 d	44.7 d	44.7 d	44.7 d	44.7 d	44.6 d	44.7 d
10	38.0 s	38.1 s	38.0 s	38.0 s	38.0 s	38.0 s	37.0 s	38.0 s	38.0 s	38.0 s	38.0 s	38.0 s	37.9 s	38.0 s
11	29.8 t	26.0 t	26.1 t	29.8 t	26.0 t	26.3 t	24.7 t	26.0 t	26.0 t	26.0 t	26.0 t	26.0 t	26.0 t	26.0 t
12	71.3 d	75.5 d	75.2 d	71.3 d	75.0 d	75.5 d	74.0 d	75.1 d	75.0 d	75.0 d	75.0 d	75.0 d	75.4 d	75.0 d
13	59.2 s	57.7 s	57.7 s	58.1 s	57.5 s	57.8 s	56.3 s	57.6 s	57.7 s	57.4 s	57.4 s	54.8 s	57.3 s	57.4 s
14	89.5 s	89.2 s	88.5 s	88.9 s	89.1 s	88.6 s	87.9 s	89.2 s	89.2 s	89.2 s	88.5 s	88.5 s	88.4 s	88.5 s
15	34.2 t	34.4 t	34.5 t	34.2 t	34.3 t	34.5 t	33.3 t	34.3 t	34.3 t	34.3 t	34.2 t	34.2 t	34.1 t	34.2 t
16	34.1 t	33.5 t	34.1 t	34.1 t	33.6 t	34.1 t	31.7 t	33.5 t	33.5 t	33.5 t	33.0 t	34.0 t	34.0 t	34.0 t
17	88.9 s	89.4 s	89.6 s	89.5 s	89.3 s	89.6 s	87.9 s	89.3 s	89.3 s	89.3 s	89.5 s	89.5 s	89.4 s	89.4 s
18	9.8 q	11.3 q	11.3 q	9.8 q	11.2 q	11.3 q	11.1 q	11.2 q	11.2 q	11.2 q	10.9 q	10.9 q	10.9 q	10.9 q
19	18.7 q	18.5 q	18.5 q	18.6 q	18.5 q	18.5 q	18.2 q	18.5 q	18.9 q	18.4 q	18.7 q	18.5 q	18.5 q	18.6 q
20	76.3 d	71.6 d	76.4 d	76.3 d	71.7 d	76.0 d	74.6 d	71.4 d	71.6 d	71.6 d	75.5 d	75.5 d	75.3 d	75.3 d
21	15.2 q	18.9 q	15.2 q	15.2 q	18.7 q	15.2 q	15.5 q	18.9 q	18.5 q	15.1 q	15.2 q	15.2 q	15.2 q	15.2 q
1′	136.1 s	131.9 s	135.6 s	136.1 s	135.9 s	132.2 s	130.1 s	169.1 s	169.2 s	169.2 s	173.0 s	173.0 s	172.9 s	172.9 s
2′	129.1 d	131.0 d	129.3 d	129.1 d	129.4 d	130.5 d	129.6 d	139.6 s	139.4 s	139.6 s	22.1 q	22.1 q	22.1 q	22.1 q
3′	130.0 d	129.5 d	129.8 d	130.0 d	130.0 d	129.2 d	128.7 d	130.1 d	130.0 d	130.1 d
4′	131.3 d	134.3 d	131.3 d	131.3 d	131.5 d	133.7 d	133.4 d	14.5 q	14.5 q	14.5 q
5′	130.0 d	129.5 d	129.8 d	130.0 d	130.0 d	129.2 d	128.7 d	12.1 q	12.1 q	12.1 q
6′	129.1 d	131.0 d	129.3 d	129.1 d	129.4 d	130.5 d	129.6 d
7′	120.0 d	167.8 s	119.8 d	119.9 d	119.3 d	167.8 s	165.8 s
8′	145.7 d		145.3 d	145.7 d	146.8 d
9′	167.7 s		168.0 s	167.7 s	168.4 s
1′′	97.2 d	97.2 d	131.7 s	97.2 d	97.2 d	131.6 s	96.1 d	97.2 d	97.2 d	97.2 d	168.5 s	168.6 s	168.5 s	168.5 s
2′′	35.9 t	35.9 t	131.0 d	35.6 t	35.6 t	130.8 d	34.5 t	35.9 t	36.7 t	36.6 t	139.5 s	139.5 s	139.4 s	139.5 s
3′′	79.2 d	79.2 d	129.5 d	79.2 d	79.2 d	129.2 d	77.4 d	79.2 d	78.5 d	78.5 d	130.0 d	130.0 d	129.9 d	130.0 d
4′′	74.5 d	74.5 d	134.2 d	83.8 d	83.8 d	134.0 d	82.5 d	74.5 d	83.9 d	83.9 d	14.5 q	14.5 q	14.5 q	14.5 q
5′′	71.4 d	71.4 d	129.5 d	70.0 d	70.0 d	129.2 d	68.5 d	71.5 d	70.0 d	69.8 d	12.2 q	12.2 q	12.2 q	12.2 q
6′′	18.6 q	18.7 q	131.0 d	18.5 q	18.5 q	130.8 d	18.2 q	18.7 q	18.5 q	18.5 q
7′′	58.1 q	58.1 q	166.9 s	58.4 q	58.1 q	166.6 s	57.2 q	58.1 q	57.4 q	57.6 q
1′′′			97.2 d	101.2 d	101.2 d	97.2 d	99.4 d		102.8 d	101.2 d	97.2 d	97.2 d	97.2 d	97.2 d
2′′′			35.9 t	36.7 t	36.6 t	35.6 t	35.5 t		37.4 t	36.4 t	35.9 t	36.7 t	36.6 t	36.6 t
3′′′			79.17 d	78.6 d	78.6 d	79.2 d	77.3 d		81.6 d	78.6 d	79.2 d	78.5 d	79.1 d	78.5 d
4′′′			74.4 d	74.5 d	74.4 d	83.8 d	71.0 d		77.0 d	83.9 d	74.7 d	83.8 d	83.8 d	83.8 d
5′′′			71.4 d	71.3 d	71.3 d	70.0 d	70.7 d		73.3 d	70.0 d	71.4 d	69.9 d	69.9 d	69.8 d
6′′′			18.7 q	18.7 q	18.8 q	18.5 q	18.3 q		18.4 q	18.9 q	18.4 q	18.4 q	18.5 q	18.5 q
7′′′			58.1 q	59.3 q	58.5 q	58.1 q	58.0 q		58.5 q	58.5 q	58.1 q	58.5 q	58.1 q	57.4 q
1′′′′						101.2 d				102.8 d		102.8 d	101.1 d	101.2 d
2′′′′						36.6 t				37.4 t		37.4 t	35.6 t	36.4 t
3′′′′						78.6 d				81.6 d		81.6 d	78.5 d	78.54 d
4′′′′						74.4 d				77.0 d		77.0 d	74.4 d	83.8 d
5′′′′						71.3 d				73.3 d		73.3 d	71.2 d	69.9 d
6′′′′						18.7 q				18.6 q		18.4 q	18.8 q	18.5 q
7′′′′						58.4 q				58.4 q		57.4 q	58.4 q	58.5 q
1′′′′′														102.8 d
2′′′′′														37.4 t
3′′′′′														81.6 d
4′′′′′														76.9 d
5′′′′′														73.2 d
6′′′′′														18.4 q
7′′′′′														58.4 q

---

Fig. 2 Key ¹H–¹H COSY and HMBC correlations of compounds 1–14.

The absolute configuration of the ring substituents of aglycone skeleton was determined by analysis of the ECD and NOESY spectrum (Fig. 3). The ECD curve of C21 steroid with a (2E,4E)-5-phenyl-2,4-pentadienoate group at C-20 (lyciumsterol A) showed that 20S derivative gave positive Cotton effect at around 300 nm, while 20R derivative showed the negative Cotton effect.¹⁶ The ECD spectrum of compound 1 exhibited the positive Cotton effect at 279 nm. Consequently, the absolute configuration of C-20 was determined to be S.¹⁶ The NOESY correlations (Fig. 3) from H-1α to H-3 and H-9, from H-9 to H-12, from H-16α to H-20, from H-12 to H-20, from H-19 to H-18, and from H-1β to H-19, indicated the α-orientation for H-3, H-9, H-12, and H-20, and the β-orientation for Me-19 and Me-18. All pregnanes from natural sources possess the trans/trans/cis connection modes for the A, B, C, and D rings, so 8-OH and 14-OH were β-oriented.¹⁶ In combination with the same biosynthetic relationship, the absolute configurations of the chiral carbons in the pregnane skeleton were defined as 3S, 8S, 9S, 10R, 12S, 13S, 14R, and 17S in 1.^15–18 Based on these data, compound 1 was established as 20-O-trans-cinnamoylsarcostin 3-O-β-D-cymaropyranoside, and named cissasteroid A.

Fig. 3 Selected NOE correlations of compound 1.

Compound 2 was obtained as a white amorphous powder. Its ¹H and ¹³C NMR spectra (Tables 1 and 2) were analogous to those of 1, except that benzoyl group [one monosubstituted benzene ring δ_H 8.16 (2H, d, J = 8.4 Hz, H-2′, 6′), 7.50 (2H, t, J = 7.9 Hz, H-3′, 5′), 7.62 (1H, t, J = 7.5 Hz, H-4′), on ester carbonyl δ_C 167.8 (C-7′)] was observed in 2 instead of the cinnamoyl group in 1. This was further supported by their HR-ESI-MS, which gave a sodium adduct ion m/z 653.3304 (calcd 653.3302) in 2, with 26 mass-units less than that of 1. The HMBC correlation (Fig. 2) between H-12 (δ_H 4.93) and C-7′ (δ_C 167.8), indicated that 12-OH was esterified by benzoic acid. Hence, compound 2 was assigned as 12-O-benzoylsarcostin 3-O-β-D-cymaropyranoide, and named cissasteroid B.

Compound 3 was obtained as a white amorphous powder. Its ¹H and ¹³C NMR data (Tables 1 and 2) were almost superimposable on those of 2, except that one additional cinnamoyl group [one monosubstituted benzene ring δ_H 7.24 (2H, d, J = 7.3 Hz, H-2′, 6′), 7.32 (3H, m, H-3′, 4′, 5′), one set of trans conjugated olefinic protons δ_H 7.35 (1H, d, J = 16.0 Hz, H-7′), 6.05 (1H, d, J = 16.0 Hz, H-8′), one ester carbonyl δ_C 168.0 (C-9′)] was observed in 3. This was further supported by their HR-ESI-MS, which gave a sodium adduct ion m/z 783.3720 (calcd 783.3720) in 3, with 130 mass-units more than that of 2. The HMBC correlations (Fig. 2) from H-12 (δ_H 4.86) to C-9′ (δ_C 168.0), from H-20 (δ_H 4.79) to C-7′′ (δ_C 166.9), indicated that 12-OH and 20-OH were esterified by cinnamic acid and benzoic acid, respectively. Thus, compound 3 was identified as 12-O-trans-cinnamoyl-20-O-benzoylsarcostin 3-O-β-D-cymaropyranoside, and named cissasteroid C.

Compound 4 was obtained as a white amorphous powder. Its ¹H and ¹³C NMR spectra (Tables 1 and 2) bore a resemblance to those of 1, with the notable difference given by the presence of one additional β-cymaropyranosyl group [the anomeric proton δ_H 4.77 (1H, dd, J = 9.6, 1.7 Hz, H-1′′′), seven carbon signals δ_C 101.2 (C-1′′′), 36.7 (C-2′′′), 78.6 (C-3′′′), 74.5 (C-4′′′), 71.3 (C-5′′′), 18.7 (C-6′′′), 59.3 (C-7′′′)] in 4. Acid hydrolysis of 4 yielded only D-cymaropyranose. The HMBC cross peaks of H-1′′ (δ_H 4.85) with C-3 (δ_C 79.4), and H-1′′′ (δ_H 4.77) with C-4′′ (δ_C 83.8), indicated that one cymaropyranosyl was at C-3 of the aglycone and the other was substituted at C-4′′ of the inner cymarose. Therefore, compound 4 was identified as 20-O-trans-cinnamoylsarcostin 3-O-β-D-cymaropyransyl-(1→4)-β-D-cymaropyranoside, and named cissasteroid D.

Compound 5 was obtained as a white amorphous powder. It gave the same molecular formula C₄₄H₆₄O₁₃ as that of 4, based on a sodium adduct ion m/z 823.4244 (calcd 823.4245). A comparison of the NMR spectroscopic data demonstrated that the difference between these two compounds was in the linkage position of the cinnamoyl group. The HMBC correlation from H-12 (δ_H 4.77) to C-9′ (δ_C 168.4) confirmed that the cinnamoyl group was located at C-12. From the above analysis, compound 5 was characterized as 12-O-trans-cinnamoylsarcostin 3-O-β-D-cymaropyransyl-(1→4)-β-D-cymaropyranoside, and named cissasteroid E.

Compound 6 was obtained as a white amorphous powder. Its ¹H and ¹³C NMR spectra (Tables 1 and 2) were almost consistent with those of 5, except that two benzoyl groups [two monosubstituted benzene rings δ_H 7.62 (4H, t, J = 8.1 Hz, H-2′, 6′, 2′′, 6′′), 7.09 (2H, t, J = 7.8 Hz, H-3′, 5′), 7.43 (1H, t, J = 7.4 Hz, H-4′), 7.33 (2H, t, J = 7.8 Hz, H-3′′, 5′′), 7.54 (1H, t, J = 7.4 Hz, H-4′′), two ester carbonyls δ_C 167.8 (C-7′), 166.6 (C-7′′)] were observed in 6 instead of one cinnamoyl group in 5. This was further confirmed by their HR-ESI-MS, which gave a sodium adduct ion m/z 901.4350 (calcd 901.4350) in 6, with 78 mass-units more than that of 5. Moreover, the HMBC correlations of H-12 (δ_H 5.03) with C-7′ (δ_C 167.8), and of H-20 (δ_H 4.82) with C-7′′ (δ_C 166.6), suggested that 12-OH and 20-OH were esterified by benzoic acids. Consequently, compound 6 was designated as 12,20-O-dibenzoylsarcostin 3-O-β-D-cymaropyranoide-(1→4)-β-D-cymaropyranoside, and named cissasteroid F.

Compound 7 was obtained as a white amorphous powder. Its ¹H and ¹³C NMR spectra (†) were analogous to those of 6, except that the absence of one benzoyl group in 7. This was further supported by their HR-ESI-MS, which gave a sodium adduct ion m/z 797.4087 (calcd 797.4088) in 7, being 104 mass-units less than that of 6. The HMBC correlation (Fig. 2) between H-12 (δ_H 4.87) and C-7′ (δ_C 165.8), indicated that 12-OH was esterified by benzoic acid. Thus, compound 7 was designated as 12-O-benzoylsarcostin 3-O-β-D-cymaropyranoide-(1→4)-β-D-cymaropyranoside, and named cissasteroid G.

Compound 8 was obtained as a white amorphous powder. Its ¹H and ¹³C NMR spectra (Tables 1 and 2) were closely related to those of 1, except that one tigloyl group [one olefinic proton δ_H 7.00 (1H, qd, J = 7.1, 1.3 Hz, H-3′), one tertiary methyl δ_H 1.87 (3H, s, H-5′), δ_C 12.1 (C-5′), one secondary methyl δ_H 1.82 (3H, d, J = 7.1 Hz, H-4′), δ_C 14.5 (C-4′), two olefinic carbons δ_C 139.6 (C-2′), 130.1 (C-3′), and one ester carbonyl δ_C 169.1 (C-1′)] was observed in 8 instead of the cinnamoyl group in 1. This was further supported by their HR-ESI-MS, which gave a sodium adduct ion m/z 631.3458 (calcd 631.3458) in 8, with 48 mass-units less than that of 1. The HMBC correlation (Fig. 2) between H-12 (δ_H 4.70) and C-1′ (δ_C 169.1), indicated that 12-OH was esterified by tiglic acid. Thus, compound 8 was established as 12-O-tigloylsarcostin 3-O-β-D-cymaropyranoside, and named cissasteroid H.

Compound 9 was obtained as a white amorphous powder. Its ¹H and ¹³C NMR spectra (Tables 1 and 2) were quite similar to those of 8, except that one oleandropyranosyl group [δ_C 102.8 (C-1′′′), 37.4 (C-2′′′), 81.6 (C-3′′′), 77.0 (C-4′′′), 73.3 (C-5′′′), 18.4 (C-6′′′), 58.5 (C-7′′′) ]was observed in 9. This was further supported by their HR-ESI-MS, which gave a sodium adduct ion m/z 775.4244 (calcd 775.4245) in 9, with 144 mass-units more than that of 8. D-Oleandropyranose was identified by acid hydrolysis and specific rotation value.¹⁶ The β-configuration of D-oleandropyranose was determined by the large coupling constants (J = 9.0, 1.8 Hz) of the anomeric proton and the chemical shifts (δ_C 37.4, C-2′′′) of the methylene carbon.¹⁶ The HMBC cross peaks of H-1′′ (δ_H 4.88) with C-3 (δ_C 79.3), and H-1′′′ (δ_H 4.61) with C-4′′ (δ_C 83.9), indicated that the cymaropyranosyl group was at C-3 of the aglycone and the oleandrose was substituted at C-4′′ of the inner cymarose. Thus, compound 9 was identified as 12-O-tigloylsarcostin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranoside, and named cissasteroid I.

Compound 10 was isolated as a white amorphous powder. Its ¹H and ¹³C NMR (Tables 1 and 2) bore a resemblance to those of 9, with the obvious difference being the resonances of seven carbon signals [δ_C 101.2 (C-1′′′), 36.4 (C-2′′′), 78.6 (C-3′′′), 83.9 (C-4′′′), 70.0 (C-5′′′), 18.9 (C-6′′′), and 58.5 (C-7′′′)] and one anomeric proton δ_H 4.60 (1H, dd, J = 9.7, 1.5 Hz, H-1′′′), which indicated the occurrence of one additional β-cymaropyranosyl moiety. Furthermore, the absolute configurations of the three deoxysugars were confirmed as D-series by the same method as 9. The sequence of this trisaccharide moiety was established as β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside, based on the HMBC correlations from H-1′′′′ (δ_H 4.73) to C-4′′′ (δ_C 83.9), and from H-1′′′ (δ_H 4.60) to C-4′′ (δ_C 83.9). In addition, the HMBC correlation from H-1′′ (δ_H 4.84) to C-3 (δ_C 79.3) suggested that the trisaccharide moiety is attached at C-3. Thus, compound 10 was defined as 12-O-tigloylsarcostin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside, and named cissasteroid J.

Compound 11 was obtained as a white amorphous powder. Its ¹H and ¹³C NMR (Tables 1 and 2) data showed a distinct similarity with those of 8, except that an acetyl group δ_H 1.88 (3H, s, H-2′), δ_C 22.1 (C-2′), 173.0 (C-1′) was observed in 11. This was further supported by their HR-ESI-MS, which gave a sodium adduct ion m/z 673.3565 (calcd 673.3564) in 11, with 42 mass-units more than that of 8. The HMBC correlations (Fig. 2) between H-12 (δ_H 4.67) and C-1′ (δ_C 173.0), between H-20 (δ_H 4.56) and C-1′′ (δ_C 168.5), indicated that 12-OH and 20-OH were esterified by acetic acid and tiglic acid, respectively. Thus, compound 11 was deduced as 12-O-acetyl-20-O-tigloylsarcostin 3-O-β-D-cymaropyranoide, and named cissasteroid K.

Compounds 12 and 13 were obtained as white amorphous powders. Their HR-ESI-MS showed the same molecular formula of C₄₂H₆₆O₁₄, according to a sodium adduct ion [m/z 817.4350 in 12; m/z 817.4360 in 13 (calcd 817.4350)]. Analysis of the UV, IR, and NMR data suggested that compounds 5 and 6 possess the same planar structure. Their ¹H and ¹³C NMR spectra (Tables 1 and 2) bore a resemblance to those of 11, except that another 2,6-deoxysugar moiety [one anomeric proton and seven carbon signals, δ_H 4.60 (1H, dd, J = 9.7, 1.5 Hz, H-1′′′′), δ_C 102.8 (C-1′′′′), 37.4 (C-2′′′′), 81.6 (C-3′′′′), 77.0 (C-4′′′′), 73.3 (C-5′′′′), 18.4 (C-6′′′′), 57.4 (C-7′′′′) in 12; δ_H 4.76 (1H, dd, J = 9.8, 1.8 Hz, H-1′′′′), δ_C 101.1 (C-1′′′′), 35.6 (C-2′′′′), 78.5 (C-3′′′′), 74.4 (C-4′′′′), 71.2 (C-5′′′′), 18.8 (C-6′′′′), 58.4 (C-7′′′′) in 13] were observed. Acid hydrolysis of 12 and 13 yielded D-oleandropyranose and D-cymaropyranose, and D-cymaropyranose, respectively. Their β-configurations were established by the large coupling constants (cymarose, J = 9.6, 1.7 Hz, oleandrose, J = 9.7, 1.5 Hz in 12; cymarose, J = 9.6, 1.8 Hz, cymarose, J = 9.8, 1.8 Hz in 13) of the anomeric protons and the chemical shifts (δ_C 36.7 (C-2′′′), 37.4 (C-2′′′′) in 12; δ_C 36.6 (C-2′′′), 35.6 (C-2′′′′) in 13) of the methylene carbons.¹⁶ The disaccharide moieties at C-3 in 12 and 13 were determined as β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl and β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl sugar sequences, respectively, based on the HMBC correlations from H-1′′′′ (δ_H 4.60) to C-4′′′ (δ_C 83.8) in 12, from H-1′′′′ (δ_H 4.76) to C-4′′′ (δ_C 83.8) in 13, respectively. Thus, compounds 12 and 13 were identified as 12-O-acetyl-20-O-tigloylsarcostin 3-O-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranoside and 12-O-acetyl-20-O-tigloylsarcostin 3-O-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside, and named cissasteroid L (12) and M (13), respectively.

Compound 14 was obtained as a white amorphous powder. Comparison of its NMR spectra with those of 13 revealed these two compounds differ by the presence of an additional oleandropyranosyl group in 14. The β-oleandropyranose was confirmed by the large coupling constant (J = 9.8, 1.8 Hz) of the anomeric proton and the chemical shifts δ_C 37.4 (C-2′′′′′) of the methylene carbon. Acid hydrolysis gave D-oleandrose and D-cymarose. The sugar sequence of β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside and its linkage at C-3 were determined, based on the HMBC correlations from H-1′′′′′ (δ_H 4.59) to C-4′′′′ (δ_C 83.8), from H-1′′′′ (δ_H 4.78) to C-4′′′ (δ_C 83.8) from H-1′′′ (δ_H 4.86) to C-3 (δ_C 79.2), respectively. Consequently, compound 14 was characterized as 12-O-acetyl-20-O-tigloylsarcostin 3-O-β-D -oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside, and named cissasteroid G (14).

Five known compounds were obtained and identified as isokidjoladinin (15),¹⁷ deacetylkidjoladinin (16),¹⁸ 12,20-O-dibenzoylsarcostin (17),¹⁹ 12-O-cinnamoyl-3β,5α,8β,12β,14β,17β,20-heptahydroxy-(20S)-pregn-6-ene (18),²⁰ 12,20-O-dibenzoylsarcostin-3-O-β-D-cymaropyranoide (19),²¹ by comparison of their spectroscopic data with values reported in the literature.

Polyoxypregnane glycosides have been reported to show various cytotoxic or anti-proliferative activities against MCF-7, H1299, HeLa, HepG2,²² PC-3, HT-29,²³ and A-549 (ref. 24) cell lines. All the isolates (1–19) were evaluated for their cytotoxicity against five human cancer cell lines: HL-60, A549, SMMC-7721, MCF-7, and SW480 (Table 3), and against NO release in LPS-induced RAW 264.7 cells. Unfortunately, they were devoid of any NO production inhibitory activity. Compared with cisplatin, compound 7 showed more potent cytotoxicities against the HL-60, A549, SMMC-7721, MCF-7, and SW480 cell lines, with IC₅₀ values of 2.19, 14.38, 2.00, 7.58, and 7.44 μM, respectively. However, all the tested compounds were less active than paclitaxel. Compound 7 was more cytotoxic than compound 6, suggesting that benzoic acid esterification at C-20 may have a negative effect on the cytotoxic activity of polyhydroxylated pregnane derivatives against these five cell lines.

Table 3 Cytotoxicities of compounds 1–19 against HL-60, A549, SMMC-7721, MCF-7 and SW480 cell lines (IC₅₀, μM)

No.	HL-60	A549	SMMC-7721	MCF-7	SW480	No.	HL-60	A549	SMMC-7721	MCF-7	SW480
3	14.85 ± 0.55	>40	24.05 ± 0.61	30.97 ± 0.48	>40	1, 2, 4, 5, 8–16, 18, 19	>40	>40	>40	>40	>40
6	36.74 ± 0.72	>40	>40	>40	>40	Cisplatin	3.38 ± 0.23	24.58 ± 1.30	18.25 ± 0.57	20.37 ± 0.71	11.79 ± 1.08
7	2.19 ± 0.07	14.38 ± 0.65	2.00 ± 0.08	7.58 ± 0.25	7.44 ± 0.22	Paclitaxel	<0.008	<0.008	1.68 ± 0.21	<0.008	<0.008
17	>40	>40	32.81 ± 1.47	>40	>40

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Conclusions

Compounds 1–19 represent the first report of polyhydroxylated pregnane glycosides from the genus Cissampelos. This also lays a solid chemical foundation for pharmacological research of C. pareira var. hirsuta. Compound 7 was the most promising of all isolated compounds based upon their IC₅₀ values. Further studies are necessary to explore antitumor mechanism, cytotoxicities in normal cells, and structure optimization.

Experimental method

General experimental procedures

Optical rotations and ECD spectra were measured by a Rudolph AP-IV polarimeter (Rudolph, Hackettstown, NJ, USA) and an Applied Photophysics Chirascanq CD spectropolarimeter (Applied Photophysics, Leatherhead, Surrey, UK), respectively. UV and IR spectra were acquired using a ThermoEVO 300 spectrometer (Thermo, Waltham, MA, USA) and a ThermoNicolet IS 10 spectrometer (Thermo, Waltham, MA, USA), respectively. NMR and mass spectra were recorded on a Bruker Avance III 500 spectrometer (Bruker, Germany) and a Bruker maXisHD mass spectrometer (Bruker, Germany), respectively. Preparative HPLC separations were performed on a SEP system (Beijing Sepuruisi scientific Co., Ltd, China) equipped with a variable-wavelength UV detector, using a YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Monosaccharide isolation was conducted on a Waters 2695 separation module with an evaporative light scattering detector (ELSD) (Waters, Milford, MA, USA). MCI gel CHP-20, ODS gel (50 μm), sephadex LH-20 (40–70 μm), and silica gel (160–200 mesh) were acquired from TOSOH Corp., Tokyo, Japan, YMC Group, Kyoto, Japan, Amersham Pharmacia Biotech AB, Uppsala, Sweden, and Marine Chemical Industry, Qingdao, China, respectively. Chemical reagents for isolation were of analytical grade and purchased from Tianjin Siyou Co., Ltd, China. Biological reagents were from Sigma Company. Human hepatocellular carcinoma (SMMC-7721) cell line was bought from China Infrastructure of Cell Line Resources (Beijing, China), from Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, China. Human myeloid leukemia cell line (HL-60), lung cancer (A549), breast cancer (MCF-7), and colon cancer (SW-480) were from American Type Culture Collection (ATCC, Manassas, VA, USA).

Plant material

The dried whole plants of C. pareira var. hirsuta were collected in Yunnan province, China, in July 2018, and authenticated by Prof. Cheng-Ming Dong at School of Pharmacy, Henan University of Chinese Medicine, where a voucher specimen (SE 20180705) was deposited.

Extraction and isolation

The dried and powdered whole plants of C. pareira var. hirsuta (50.7 kg) were refluxed with 95% EtOH (3 × 300 L) to yield a crude extract (2.7 kg). The extract was dispersed in water (9 L) and sequentially partitioned with petroleum ether (PE, 9 L × 3), CH₂Cl₂ (9L × 3), and n-BuOH (3.2 L × 3) to afford the PE (351.1 g), the CH₂Cl₂ (740.1 g), and n-BuOH fractions (594.5 g). The CH₂Cl₂ fraction was separated into five fractions (A1–A5) by silica gel column chromatography (CC, 125 × 15 cm) with a gradient of PE (60–90 °C)–acetone (v/v 100 : 0, 100 : 1, 100 : 3, 100 : 5, 100 : 10, 100 : 30, 100 : 50, 1 : 1, 1 : 2). Fraction A4 (10.32 g) was chromatographed over open MCI gel CHP-20 CC (23 × 4 cm) eluted with a gradient of methanol–H₂O (v/v 10 : 90, 30 : 70, 40 : 60, 70 : 30, 80 : 20) to yield five subfractions (A4-1–A4-5). Subfraction A4-2 (3.21 g) was passed through sephadex LH-20 CC (4.4 × 120 cm) eluted by MeOH to obtain six subfractions (A4-2-1–A4-2-6). The subfraction A4-2-2 (864.3 mg) was subjected to silica gel CC (35 × 2.5 cm) with a CH₂Cl₂ : MeOH (80 : 1, 50 : 1, 30 : 1, 20 : 1, 10 : 1, 0 : 1) gradient to give five subfractions (A4-2-2-1–A4-2-2-5). Subfraction A4-2-2-1 (350.0 mg) was purified by preparative HPLC (MeOH : H₂O 65 : 35) to afford compounds 15 (18.8 mg, t_R 23.0 min), and 16 (14.9 mg, t_R 17.4 min). Subfraction A4-3 (2.17 g) was submitted to silica gel CC (45 × 5 cm) eluted by PE–EtOAC (10 : 1, 8 : 1, 5 : 1, 3 : 1, 2 : 1, 1 : 1, 1 : 2) to afford five subfractions (A4-3-1–A4-3-5). Subfraction A4-3-3 (936.2 mg) was further purified by preparative HPLC (CH₃CN–H₂O 50 : 50) to produce compounds 1 (10.0 mg, t_R 51.0 min), 8 (15.2 mg, t_R 45.3 min), and 11 (9.8 mg, t_R 71.2 min). Subfraction A4-4 (1.36 g) was subjected to silica gel CC (45 × 5 cm) eluted by PE–EtOAC (10 : 1, 8 : 1, 5 : 1, 3 : 1, 2 : 1, 1 : 1, 1 : 2) to obtain four subfractions (A4-4-1–A4-4-4). Subfraction A4-4-4 (686.6 mg) was rechromatographed by sephadex LH-20 CC (100 × 2.5 cm) eluted by MeOH to provide three subfractions (A4-4-4-1–A4-4-4-3). Compounds 4 (3.9 mg, t_R 31.9 min), 7 (12.5 mg, t_R 48.4 min), 9 (4.3 mg, t_R31.2 min), 10 (8.9 mg, t_R 55.0 min), 12 (5.4 mg, t_R 51.8 min), and 13 (7.0 mg, t_R 63.1 min) were obtained from subfraction A4-4-4-2 (280.3 mg) using preparative HPLC (CH₃CN–H₂O, 60 : 40) at a flow rate of 6 mL min⁻¹. Compounds 2 (18.9 mg, t_R 27.2 min), 17 (4.5 mg, t_R 42.3 min), and 18 (38.0 mg, t_R 38.4 min) were isolated from sub-fraction A4-4-4-3 (265.8 mg) using preparative HPLC (CH₃CN–H₂O 60 : 40) at a flow rate of 6 mL min⁻¹. Subfraction A4-5 (3.32 g) was subjeced to silica gel CC (45 × 5 cm) eluted by PE–EtOAC (10 : 1, 8 : 1, 5 : 1, 3 : 1, 2 : 1, 1 : 1, and 1 : 2) to obtain three subfractions (A4-5-1–A4-5-3). Further separation of subfraction A4-5-2 (988.5 mg) using sephadex LH-20 CC (91 × 2.4 cm) eluted by MeOH resulted in five subfractions A4-5-2-1–A4-5-2-5. Subfraction A4-5-2-2 (334.2 mg) was purified by preparative HPLC eluted with MeOH–H₂O (75 : 25) at a flow rate of 6 mL min⁻¹ to give compounds 5 (23.0 mg, t_R 75.1 min), 6 (23.3 mg, t_R108.2 min), and 14 (20.0 mg, t_R 88.4 min). Compounds 3 (50.0 mg, t_R 102.0 min) and 19 (19.8 mg, t_R 57.3 min) were isolated from subfraction A4-5-2-3 (349.8 mg) by preparative HPLC (MeOH–H₂O 75 : 25) at a flow rate of 6 mL min⁻¹.

Cissasteroid A (1). White, amorphous powder; [α]^D₂₀ +56.1 (c 0.02, MeOH); UV (MeOH) λ_max (log ε) 207 (3.88), 216 (3.81), 276 (3.92), 377 (1.42) nm; IR (iTR)ν_max 3398, 2933, 1703, 1639, 1451, 1376, 1311, 1278, 1187, 1082, 1029 cm⁻¹; HR-ESI-MS (positive): m/z 679.3451 [M + Na]⁺ (calcd for C₃₇H₅₂O₁₀Na, 679.3458); NMR data (CD₃OD), see Tables 1 and 2.

Cissasteroid B (2). White, amorphous powder; [α]^D₂₀ +18.2 (c 0.08, MeOH); UV (MeOH) λ_max (log ε) 205 (3.64), 231 (3.75), 274 (2.93) nm; IR (iTR)ν_max 3395, 2934, 1708, 1452, 1382, 1316, 1277, 1163, 1074, 1027 cm⁻¹; HR-ESI-MS (positive): m/z 653.3304 [M + Na]⁺ (calcd for C₃₅H₅₀O₁₀Na, 653.3302); NMR data (CD₃OD), see Tables 1 and 2.

Cissasteroid C (3). White, amorphous powder; [α]^D₂₀ +77.0 (c 0.02, MeOH); UV (MeOH) λ_max (log ε) 224 (4.09), 281 (4.04) nm; IR (iTR)ν_max 3365, 2939, 1705, 1638, 1451, 1311, 1278, 1165, 1075, 1025 cm⁻¹; HR-ESI-MS (positive): m/z 783.3720 [M + Na]⁺ (calcd for C₄₄H₅₆O₁₁Na, 783.3720); NMR data (CD₃OD), see Table 1 and 2.

Cissasteroid D (4). White, amorphous powder; [α]^D₂₀ +46.5 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 207 (3.84), 216 (3.83), 276 (3.86) nm; IR (iTR)ν_max 3418, 2931, 1704, 1638, 1451, 1368, 1311, 1280, 1165, 1084, 1061 cm⁻¹; HR-ESI-MS (positive): m/z 823.4244 [M + Na]⁺ (calcd for C₄₄H₆₄O₁₃Na, 823.4245); NMR data (CD₃OD), see Tables 1 and 2.

Cissasteroid E (5). White, amorphous powder; [α]^D₂₀ +12.4 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 207 (3.84), 218 (3.86), 280 (3.99), 375 (1.35) nm; IR (iTR)ν_max 3384, 2935, 1702, 1636, 1578, 1451, 1368, 1312, 1280, 1165, 1081, 1059, 1027 cm⁻¹; HR-ESI-MS (positive): m/z 823.4244 [M + Na]⁺ (calcd for C₄₄H₆₄O₁₃Na, 823.4245); NMR data (CD₃OD), see Tables 1 and 2.

Cissasteroid F (6). White, amorphous powder; [α]^D₂₀ +58.1 (c 0.12, MeOH); UV (MeOH) λ_max (log ε) 206 (4.02), 230 (4.18), 275 (3.21), 376 (1.78) nm; IR (iTR)ν_max 3405, 2933, 1711, 1602, 1451, 1307, 1316, 1277, 1078, 1026 cm⁻¹; HR-ESI-MS (positive): m/z 901.4350 [M + Na]⁺ (calcd for C₄₉H₆₆O₁₄Na, 901.4350); NMR data (CD₃OD), see Tables 1 and 2.

Cissasteroid G (7). White, amorphous powder; [α]^D₂₀ −10.9 (c 0.03, MeOH); UV (MeOH) λ_max (log ε) 207 (2.80), 228 (3.95), 274 (3.05) nm; IR (iTR)ν_max 3379, 2934, 1708, 1602, 1451, 1369, 1316, 1277, 1194, 1164, 1146, 1081, 1027 cm⁻¹; HR-ESI-MS (positive): m/z 797.4087 [M + Na]⁺ (calcd for C₄₂H₆₂O₁₃Na, 797.4088); NMR data (CD₃OD), see Tables 1 and 2.

Cissasteroid H (8). White, amorphous powder; [α]^D₂₀ +9.8 (c 0.04, MeOH); UV (MeOH) λ_max (log ε) 219 (2.89) nm; IR (iTR)ν_max 3409, 2935, 1690, 1648, 1454, 1380, 1267, 1194, 1161, 1142, 1078, 1030 cm⁻¹; HR-ESI-MS (positive): m/z 631.3458 [M + Na]⁺ (calcd for C₃₃H₅₂O₁₀Na, 631.3458); NMR data (CD₃OD), see Tables 1 and 2.

Cissasteroid I (9). White, amorphous powder; [α]^D₂₀–10.9 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 207 (3.86) nm; IR (iTR)ν_max 3395, 2932, 1703, 1649, 1451, 1377, 1268, 1194, 1149, 1060, 1029 cm⁻¹; HR-ESI-MS (positive): m/z 775.4244 [M + Na]⁺ (calcd for C₄₀H₆₄O₁₃Na, 775.4245); NMR data (CD₃OD), see Tables 1 and 2.

Cissasteroid J (10). White, amorphous powder; [α]^D₂₀ +8.9 (c 0.26, MeOH); UV (MeOH) λ_max (log ε) 216 (3.93) nm; IR (iTR)ν_max 3432, 2933, 1702, 1650, 1453, 1369, 1318, 1266, 1195, 1149, 1082, 1058, 1031 cm⁻¹; HR-ESI-MS (positive): m/z 919.5031 [M + Na]⁺ (calcd for C₄₇H₇₆O₁₆Na, 919.5031); NMR data (CD₃OD), see Tables 1 and 2.

Cissasteroid K (11). White, amorphous powder; [α]^D₂₀ +3.6 (c 0.07, MeOH); UV (MeOH) λ_max (log ε) 215 (4.25) nm; IR (iTR)ν_max 3409, 2933, 1730, 1701, 1650, 1452, 1374, 1265, 1237, 1146, 1076, 1027 cm⁻¹; HR-ESI-MS (positive): m/z 673.3565 [M + Na]⁺ (calcd for C₃₅H₅₄O₁₁Na, 673.3564); NMR data (CD₃OD), see Tables 1 and 2.

Cissasteroid L (12). White, amorphous powder; [α]^D₂₀ +13.9 (c 0.05, MeOH); UV (MeOH) λ_max (log ε) 209 (3.92) nm; IR (iTR)ν_max 3378, 2935, 1732, 1703, 1650, 1451, 1373, 1267, 1238, 1194, 1149, 1065, 1028 cm⁻¹; HR-ESI-MS (positive): m/z 817.4350 [M + Na]⁺ (calcd for C₄₂H₆₆O₁₄Na, 817.4350); NMR data (CD₃OD), see Tables 1 and 2.

Cissasteroid M (13). White, amorphous powder; [α]^D₂₀ +23.6 (c 0.40, MeOH); UV (MeOH) λ_max (log ε) 215 (3.72) nm; IR (iTR)ν_max 3387, 2934, 1731, 1702, 1650, 1451, 1371, 1319, 1266, 1237, 1194, 1162, 1147, 1079, 1027 cm⁻¹; HR-ESI-MS (positive): m/z 817.4360 [M + Na]⁺ (calcd for C₄₂H₆₆O₁₄Na, 817.4350); NMR data (CD₃OD), see Tables 1 and 2.

Cissasteroid N (14). White, amorphous powder; [α]^D₂₀ +20.5 (c 0.26, MeOH); UV (MeOH) λ_max (log ε) 212 (3.86) nm; IR (iTR)ν_max 3382, 2935, 1732, 1703, 1649, 1451, 1371, 1318, 1267, 1238, 1194, 1150, 1058, 1027 cm⁻¹; HR-ESI-MS (positive): m/z 961.5137 [M + Na]⁺ (calcd for C₄₉H₇₈O₁₇Na, 961.5137); NMR data (CD₃OD), see Tables 1 and 2.

Absolute configuration determination of sugar moieties

A solution of 10 (2.2 mg) in 1 mL of MeOH was hydrolyzed with 100 μL of 0.05 N H₂SO₄. The solution was stirred at 60 °C for 2 h. After cooling, the reation mixture was diluted with 10 mL of H₂O and extracted with 10 mL of CH₂Cl₂. The H₂O phase was neutralized with saturated aqueous Ba(OH)₂ solution. The precipitate was filtered off, and then the filtrate was evaporated under reduced pressure to give the sugar fraction. The residue was isolated by preparative HPLC-ELSD (a TSKgel G3000PWXL column, 300 mm × 7.8 mm, 5 μm) and eluted with H₂O at a flow of 0.2 mL min⁻¹ to obtain oleandropyranose (t_R 53.9 min) and cymaropyranose (t_R 55.3 min), respectively. Compounds 1–9, and 11–14 were hydrolyzed by the above procedure. D-Oleandrose and D-cymarose were identified by comparing their experimental and reported rotation values.¹⁶

Cytotoxicity assay in vitro

Cytotoxicity was tested by the MTS method previously described.²⁵ Cisplatin and paclitaxel were used as positive control. The cytotoxicity of compounds 1–19 was evaluated against HL-60, A-549, SMMC-7721, MCF-7, and SW-480 cell lines. All the cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere with 5% CO₂. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS). To be brief, 100 μL of adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 24 h before drug addition, each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h. After the incubation, MTS (20 μL) was added to each well, and the incubation continued for 4 h at 37 °C. The optical density of each well was measured at 492 nm in a 96-well microtiter plate reader. The IC₅₀ value of each compound was calculated by the Reed–Muench's method.

NO inhibitory activity

The NO inhibitory activity was evaluated by the previously reported protocol.²⁵ L-N^G-Monomethyl arginine (L-NMMA) was used as a positive control. RAW 264.7 macrophages cells (2 × 10⁵ cells per well) were precultured in 96-well microplates for 24 h. The test compounds (50 μM) and L-NMMA with 1 μg mL⁻¹ LPS were added and incubated for another 18 h at 37 °C. Nitric oxide production was assessed by the Griess Reagent System.

Author contributions

H. J. Chen and H. Chen performed the experiments, data analysis, and experimental planning. Y. Y. Si, M. Li, and K. Du screened the biological activities. The project was conceived and supervised by Y. J. Sun and W. S. Feng. The manuscript was written by Y. J. Sun, R. J. Han, and C. Zhao. All authors reviewed the manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by Basic Science Foundation of Henan University of Chinese Medicine (No. 2014KYYWF-QN26), Science and Technology Innovation Talent Support Scheme of Henan University of Chinese Medicine (No. 2016XCXRC01), Scientific and Technological Key Project in Henan Province (No. 192102310438), and Research Project on Chinese Medicine Science in Henan Province (No. 20-21ZY1039).

Notes and references

1. Z. Y. Wang, M. J. Jiang, A. F. Khan, S. B. Cai, X. N. Li, J. Q. Liu, G. Y. Kai, T. R. Zhao, G. G. Cheng and J. X. Cao, Phytochemistry, 2019, 168, 112127.
2. Y. Liu, W. Guan, C. L. Yang, Y. M. Luo, Y. Liu, Y. Y. Zhou, L. N. Liu, B. Y. Yang and H. X. Kuang, Can. J. Chem., 2020, 98, 74.
3. J. L. Li, Z. B. Gao and W. M. Zhao, J. Nat. Prod., 2015, 79, 89.
4. Z. M. Zhao, Z. H. Sun, M. H. Chen, Q. Liao, M. Tan, X. W. Zhang, H. D. Zhu, R. B. Pi and S. Yin, Steroids, 2013, 78, 1015.
5. P. V. Kiem, D. T. H. Yen, N. V. Hung, N. X. Nhiem, B. H. Tai, D. T. Trang, P. H. Yen, T. M. Ngoc, C. V. Minh, S. Park, J. H. Lee, S. Y. Kim and S. H. Kim, Molecules, 2020, 25, 2525.
6. C. Liu, Z. X. Liao, S. J. Liu, J. Y. Sun, G. Y. Yao and H. S. Wang, Nat. Prod. Res., 2014, 28, 1843.
7. S. Z. Liu, Z. H. Chen, J. Wu, L. Y. Wang, H. M. Wang and W. M. Zhao, Phytochemistry, 2013, 93, 144.
8. R. A. El-Shiekh, A. Salama, A. K. Al-Mokaddem, A. Bader and E. A. Abdel-Sattar, Steroids, 2021, 165, 108759.
9. D. K. Semwal, R. B. Semwal, I. Vermaak and A. Viljoen, J. Ethnopharmacol., 2014, 155, 1011.
10. H. Singh, P. A. Dhole, G. Krishna, R. Saravanan and P. K. Baske, Indian J. Nat. Prod. Resour., 2018, 9, 160.
11. B. R. Verrastro, A. M. Torres, G. Ricciardi, P. Teibler, S. Marunak, C. Barnaba, R. Larcher, G. Nicolini and E. Dellacassa, J. Ethnopharmacol., 2018, 212, 36.
12. M. Bala, S. Kumar, K. Pratap, P. K. Verma, Y. Padwad and B. Singh, Nat. Prod. Res., 2019, 33, 622.
13. M. Bala, K. Pratap, P. K. Verma, Y. Padwad and B. Singh, Nat. Prod. Res., 2015, 29, 686.
14. J. R. Dong, X. R. Peng, L. Li, S. Y. Lu, L. Zhou and M. H. Qiu, Bioorg. Med. Chem. Lett., 2018, 28, 1520.
15. X. Y. Li, H. X. Sun, Y. P. Ye, F. Y. Chen, J. Tu and Y. J. Pan, Steroids, 2006, 71, 683.
16. Y. W. An, Z. L. Zhan, J. Xie, Y. N. Yang, J. S. Jiang, Z. M. Feng, F. Ye and P. C. Zhang, J. Nat. Prod., 2016, 79, 1024.
17. Y. P. Ye, H. X. Sun, X. Y. Li, F. Y. Chen, F. Qin and Y. J. Pan, Steroids, 2005, 70, 791.
18. X. Y. Li, S. L. Zong, F. Y. Chen, S. F. Xu and Y. P. Ye, Nat. Prod. Commun., 2012, 7, 1269.
19. R. Aquino, C. Pizza, N. De Tommasi and F. De Simone, J. Nat. Prod., 1995, 58, 672.
20. Y. L. Lin, Y. M. Wu, Y. H. Kuo and C. F. Chen, J. Chin. Chem. Soc., 1999, 46, 841.
21. Y. J. Hu, X. L. Shen, Y. M. Shen and Q. Z. Mu, Acta Chim. Sin., 1998, 56, 507.
22. X. S. Li, X. M. Yang, W. J. Ding, Z. P. Xu, C. M. Zhang, J. Long, L. Liu, C. Y. Lu and J. S. Tang, Fitoterapia, 2021, 149, 104833.
23. Y. S. Makong, G. W. Fotso, G. H. Mouthe, B. Lenta, R. Rennert, N. Sewald, N. Arnold, J. D. Wansi and B. T. Ngadjui, Nat. Prod. Res., 2021, 35, 2037.
24. F. Y. Yuan, X. L. Wang, T. Wang, T. Shen, D. M. Ren, H. X. Lou and X. N. Wang, J. Nat. Prod., 2019, 82, 1542.
25. Z. Z. Zhao, Q. L. Zhao, W. S. Feng, H. R. He, M. Li, G. M. Xue, H. P. Chen and J. K. Liu, RSC Adv., 2021, 11, 18693.

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Footnote

† Electronic supplementary information (ESI) available: 1D and 2D NMR spectra for compounds 1–14. See DOI: 10.1039/d1ra07498a

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