Cytotoxic Cycloartane Triterpenes of the Traditional Chinese Medicine “Shengma” (Cimicifuga dahurica)

Abstract

Twelve new 9,19-cycloartane triterpenes (1–12), together with fourteen known compounds (13–26), were isolated from the roots of Cimicifuga dahurica. Their structures were determined by application of spectroscopic analyses and chemical methods. Biological evaluation of the compounds against human HL-60, SMMC-7721, A549, MCF-7, and SW-480 cell lines indicated that cimigenol-type glycosides (1–3, 19, and 20) showed broad-spectrum and moderate cytotoxicities, with IC₅₀ values ranging from 4.2 to 14.5 µM. Meanwhile, cimigenol-type aglycones (6–8, 15, 16, and 18) exhibited broad-spectrum and week cytotoxicities, having IC₅₀ values around 20 µM. In addition, the key points of the structure-activity relationships of aglycones with a cimigenol skeleton were discussed.

Key words

Ranunculaceae - Cimicifuga dahurica - cycloartane triterpenes - cytotoxicity - structure-activity relationships

Introduction

Cancer has become one of the major causes of mortality in humans throughout the world. In 2007, 7.6 million people died from cancer. Significantly, 27 million new cancer cases and 17.5 million cancer deaths are projected to occur in the world by 2050 [1]. Natural products have been a rich source of antitumor agents, and approximately 60 % of currently available drugs are natural compounds or are related to them (from 1940 to 2010) [2].

As one of the three ancient medicinal systems, traditional Chinese medicine (TCM), has gained increasing acceptance and has been recognized by pharmaceutical enterprises as a fountainhead of antitumor drugs [3]. According to the theories of TCM, cancer is caused by imbalances between endogenous physical conditions of the body and exogenous pathogenic factors, including accumulated toxins, heat, and blood stasis [3]. Therefore, the roots of Cimicifuga foetida, belonging to the family Ranunculaceae and officially listed in the Chinese Pharmacopoeia with the name “shengma” as a cooling and detoxicating agent [4], were chosen as the object to investigate potential antitumor constituents. As a result, a number of cytotoxic 9,19-cycloartane triterpenes were successively isolated from this herb medicine [5], [6], [7]. In addition, the preliminary structure-activity relationships (SAR) of the triterpenes with a cimigenol skeleton were proposed [8].

In the Chinese Pharmacopoeia, the roots of C. dahurica are another element of “shengma” [4]. Chemical studies on C. dahurica showed that it also mainly contains 9,19-cyclolanostane triterpenes [9], [10], [11], [12], [13], [14], [15], [16]. Recently, other research groups paid attention to chemical and pharmaceutical properties of the aerial parts of C. dahurica and reported that the ethyl acetate fraction of an 80 % ethanol extract and three isolated cycloartane triterpenoids displayed growth inhibitory activities against several human tumor cell lines [17], [18]. However, little is known about the cytotoxic triterpenes of the roots of C. dahurica [19].

Inspired by the described observations concerning C. foetida, we undertook phytochemical and pharmacological investigations on the roots of C. dahurica. Twelve new 9,19-cyclolanostane triterpenes (1–12), together with fourteen known compounds, cimigenol (13) [20], 7(8)-en-cimigenol (14) [21], 25-O-acetyl-7(8)-en-cimigenol (15) [21], cimigenol-3-one (16) [22], 24-epi-cimigenol-7(8)-en-3-one (17) [21], cimigenol-1(2)-en-3-one (18) [23], 25- O -acetylcimigenol-3-O-[2′-O-acetyl]- α-L-arabinopyranoside (19) [4], 25-anhydrocimigenol-3-O-[2′-O-acetyl]-β-D-xylopyranoside (20) [24], 23-epi-26-deoxyactein (21) [25], actein (22) [26], 24-O-acetyl-7(8)-en-hydroshengmanol (23) [27], 24-O-acetylisodahurinol-3-O-[2′-O-acetyl]-α-L-arabinopyranoside (24) [25], 23-O-acetylshengmanol-3-O-β-D-xylopyranoside (25) [28], and cimiracemonol B (26) [29], were isolated and identified ([Fig. 1]). The isolated compounds were evaluated for their cytotoxicities against human HL-60, SMMC-7721, A549, MCF-7, and SW480 cell lines, using the MTT method. Described herein are the isolation, structure elucidation, and biological activities of the compounds.

Fig. 1 Structures of compounds 1–30.

Materials and Methods

General experimental procedures

Optical rotations were measured in MeOH with a Horiba SEAP-300 polarimeter. ¹H and ¹³C NMR spectra were recorded in pyridine-d ₅ on Bruker DRX-500 and Avance III-600MHz spectrometers (Bruker). Unless otherwise specified, chemical shifts (δ) are expressed in ppm with respect to the solvent signals. ESIMS and HR-TOF-ESIMS data were obtained using a VG Autospec-3000 spectrometer. Infrared spectra were recorded on a Shimadzu IR-450 instrument with KBr pellets. Thin-layer chromatography was performed on precoated TLC plates (200–250 µm thickness, silica gel 60 F₂₅₄; Qingdao Marine Chemical, Inc.), and spots were visualized by heating after spraying with 10 % aq. H₂SO₄ sol. Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph with a YMC-Pack Pro C18 RS 10 mm × 250 mm column. Silica gel (200–300 mesh; Qingdao Marine Chemical, Inc.), Lichroprep RP-18 (40–63 µm; Merck), and Sephadex LH-20 (20–150 µm; Pharmacia) were used for column chromatography (cc).

Plant material

The roots of Cimicifuga dahurica M. (0.9 kg) were collected from Qingyuan County, Liaoning Province, China, in September 2006 and identified by Prof. Shengji Pei, Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (KUN No. 200609004) has been deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, PR China.

Extraction and isolation

The dried and milled roots of Cimicifuga dahurica (0.9 kg) were extracted with MeOH (3 × 3 L × 24 h) at room temperature to give a residue (106 g) after evaporating in vacuum at 50 °C. The extract was subjected to silica gel cc (2 kg, 10 × 150 cm) and eluted with CHCl₃-MeOH [100 : 0 (2 L), 50 : 1 (4 L), 20 : 1 (5 L), 10 : 1 (4 L), 0 : 100 (3 L)] to afford fractions A (21.5 g), B (13.1 g), C (14.5 g), D (16.8 g), and E (16.2 g). Fraction B (13.1 g) was divided into five sub-fractions (B.1–B.5) after performing RP-18 cc (180 g, 5 × 25 cm), eluting with MeOH-H₂O (gradient from 60 : 40 to 100 : 0, 10 L). Fraction B.3 (1.5 g) was subjected to repeated silica gel cc (40 g, 4 × 40 cm) eluted with CHCl₃-Me₂CO (gradient from 20 : 1 to 10 : 1, 4 L) and then to repeated semipreparative HPLC (eluted with CH₃CN-H₂O, gradient from 60 : 40 to 85 : 15) to yield 4 (3.0 mg), 5 (3.0 mg), 6 (2.0 mg), 13 (4.0 mg), 16 (4.7 mg), and 26 (3.6 mg). Compounds 7 (2.8 mg), 14 (3.2 mg), 15 (3.5 mg), 17 (2.3 mg), 18 (2.5 mg), and 23 (2.2 mg) were purified from fraction B.4 (1.8 g) by silica gel cc (30 g, 3.5 × 40 cm) eluted with CHCl₃-Me₂CO (20 : 1, 3 L), followed by repeated semipreparative HPLC (eluted with CH₃CN-H₂O, gradient from 65 : 35 to 85 : 15). Fraction B.5 (0.9 g) was applied to a silica gel column (30 g, 3.5 × 40 cm) eluted with CHCl₃-Me₂CO (20 : 1, 3 L), then purified over semipreparative HPLC (eluted with CH₃CN-H₂O, gradient from 65 : 35 to 90 : 10) to afford 8 (2.7 mg), 9 (1.2 mg), 10 (1.9 mg), and 11 (1.6 mg). Fraction C (14.5 g) was fractionated into four subfractions (C.1–C.4) by performing RP-18 cc (180 g, 5 × 25 cm) eluted with MeOH-H₂O (gradient from 60 : 40 to 90 : 10, 12 L). Fraction C.3 (2.8 g) was subjected to silica gel cc (50 g, 4 × 40 cm) eluted with CHCl₃-Me₂CO (gradient from 10 : 1 to 5 : 1, 4 L), then to repeated semipreparative HPLC (eluted with CH₃CN-H₂O, gradient from 60 : 40 to 75 : 25) to give 1 (5.5 mg), 2 (4.8 mg), 3 (4.3 mg), 12 (5.3 mg), 19 (3.3 mg), and 24 (28 mg). Fractions C.1 and C.2 (4.8 g) were chromatographed on silica gel cc (50 g, 4 × 40 cm), eluting with CHCl₃-Me₂CO (10 : 1, 8 L) to yield 20 (5.2 mg), 21 (6.0 mg), 22 (5.2 mg), and 25 (4.2 mg).

Cimigenol-3-O-[2′-O-(E)-2-butenoyl]-α-L-arabinopyranoside (1): A white powder; [α]_D ²⁴ = 18.67 (c 0.10, MeOH); IR (KBr): ν _max = 3452, 2931, 2870, 1727, 1632, 1458, 1383, 1044, 977 cm⁻¹; ¹H (C₅D₅ N, 500 MHz) and ¹³C NMR (C₅D₅ N, 150 MHz) spectra, see [Tables 1] and [2]; ESIMS: m/z = 711 [M + Na]⁺; HR-TOF-ESIMS: m/z = 711.4065 (calc. for C₃₉H₆₀O₁₀ Na, 711.4084).

Table 1 ¹H NMR data of compounds 1–12 in pyridine-d ₅ at 500 MHz.

Proton	1	2	3	4	5	6	7	8	9	10	11	12
Chemical shifts are in δ scale with J values in parentheses. a  Signals overlapped
1	1.53 a 1.17 a	1.54 m 1.22 a	1.54 m 1.16 a	1.70 m 1.35 m	2.78 m 1.71 m	2.99 m 1.88 m	1.85 m 1.54 m	6.71 d (10.0)	5.54 brs	1.53 m 1.14 m	1.68 m 1.24 m	1.64 m 1.30 a
2	2.28 m 1.87 m	2.34 m 1.96 a	2.34 dd (4.0, 16.0) 1.95 a	1.92 m 1.86 brs	2.11 m 1.93 m	2.84 m 2.35 m	2.75 ddd (6.5) 2.29 m	6.16 d (10.0)	2.52 m 2.26 m	1.99 m 1.86 dd (3.2, 10.0)	1.95 m 1.89 m	2.36 m 1.95 m
3	3.39 dd (4.0, 11.5)	3.50 dd (5.0, 14.5)	3.47 dd (5.0, 14.2)	3.50 dd (3.5, 11.0)	3.57 dd (5.0, 10.5)				3.78 m	3.48 m	3.51 m	3.49 dd (3.5, 10.5)
4
5	1.28 dd (3.0, 12.3)	1.30 a	1.29 m	1.25 dd (5.0, 12.0)	1.31 dd (5.0, 12.0)	1.64 m	1.54 m	1.84 dd (5.0, 12.5)	2.36 m	1.25 dd (3.2, 10.0)	1.20 a	1.27 a
6	1.48 a 0.66 m	1.52 m 0.70 m	1.49 m 0.69 m	1.94 m 1.68 m	1.92 m 1.77 m	1.82 m 1.64 m	1.70 m 1.54 m	1.75 m 1.60 m	2.64 m 2.36 m	1.51 m 0.65 m	1.93 m 1.56 m	1.76 m 1.43 m
7	2.06 m 1.11 a	2.07 m 1.07 a	2.09 m 1.04 a	6.20 d (7.0)	6.21 d (6.5)	5.23 m	6.05 brd (7.0)	6.11 d (7.0)	5.52 brd (8.0)	2.27 m 1.95 m	6.36 brd (6.5)	5.33 d (7.0)
8	1.65 a	1.66 a	1.66 a							1.72 brd (10.4)
9
10
11	2.06 m 1.01 m	2.07 m 1.15 a	2.09 m 1.15 a	2.96 dd (9.0, 15.0) 1.54 a	4.58 brd (8.5)	4.53 m	2.13 m 1.16 a	2.23 m 1.40 m	5.39 brd (5.0)	2.23 m 1.05 a	1.98 m 1.13 a	2.11 m 1.16 m
12	1.65 a 1.53 a	1.66 a 1.52 m	1.64 a 1.52 m	4.36 t (6.5)	2.74 m 2.11 m	2.73 m 1.99 m	1.81 m 1.68 m	1.81 m 1.70 m	2.21 m 1.94 dd (6.0, 12.5)	1.58 m 1.37 m	1.65 m 1.29 m	1.89 m 1.78 m
13
14
15	4.26 brs	4.27 brs	4.25 brs	4.76 brs	4.62 s	2.46 d (13.0) 2.28 d (13.0)	4.55 brd (8.0)	4.54 d (9.0)	2.36 m 2.09 m			5.92 s
16										3.79 d (9.6)	3.83 d (11.2)
17	1.48 a	1.44 brd (14.0)	1.45 brd (14.0)	1.86 brs	1.48 a	1.59 d (12.0)	1.50 a	1.49 a	1.53 a	1.52 m	1.20 m	2.34 d (8.0)
18	1.13 s	1.13 s	1.13 s	1.51 s	1.27 s	1.26 s	1.16 s	1.10 s	0.81 brs	1.18 s	1.20 s	1.29 s
19	0.45 brs 0.21 brs	0.50 d (4.0) 0.25 d (4.0)	0.52 d (4.0) 0.28 d (4.0)	1.19 a 0.73 d (4.0)	1.97 d (4.0) 1.07 brd (2.5)	2.11 d (4.0) 1.11 d (4.0)	1.19 a 0.67 d (4.0)	1.52 d (4.8) 1.04 d (4.0)	3.24 d (13.5) 3.15 d (13.5)	0.52 d (3.6) 0.29 d (3.6)	1.07 a 0.48 d (4.0)	0.99 a 0.51 d (3.5)
20	1.65 a	1.67 a	1.59 m	1.86 m	1.64 brd (10.5)	1.66 brd (9.5)	1.70 m	1.68 m	1.60 m	1.80 m	1.81 m	2.10 m
21	0.84 d (7.0)	0.83 d (6.4)	0.84 d (6.4)	1.41 d (5.5)	0.82 d (6.0)	0.83 d (6.0)	0.89 d (6.5)	0.89 d (7.0)	0.81 brs	0.91 d (7.2)	0.93 d (6.5)	1.19 d (6.5)
22	2.26 m 1.01 m	2.25 m 0.97 m	2.26 m 0.95 m	2.42 m 1.16 a	2.22 m 0.99 m	2.24 m 0.99 t (11.5)	2.30 m 1.07 m	2.28 m 1.07 m	2.21 m 0.96 a	1.72 brd (10.4) 1.44 m	1.73 m 1.47 m	2.77 t (12.0) 1.68 m
23	4.74 d (9.0)	4.59 d (11.5)	4.59 brd (12.0)	4.80 d (9.0)	4.73 d (9.0)	4.79 d (8.5)	4.78 brd (9.0)	4.77 t (8.0)	4.77 brd (9.0)	4.25 brd (11.2)	4.27 d (11.5)	5.38 brt (9.5)
24	3.76 brs	4.11 brs	4.11 brs	3.87 s	3.77 s	3.71 s	3.80 brs	3.80 s	3.69 brs	5.31 d (1.6)	5.33 d (2.0)	3.03 d (8.0)
25
26	1.48 s	1.68 s	1.68 s	1.52 s	1.47 s	1.51 s	1.48 s	1.49 s	1.52 s	1.61 s	1.62 s	1.25 s
27	1.46 s	1.66 s	1.66 s	1.51 s	1.45 s	1.46 s	1.49 s	1.48 s	1.47 s	1.61 s	1.63 s	1.39 s
28	1.16 s	1.18 s	1.18 s	1.46 s	1.36 s	1.39 s	1.38 s	1.36 s	1.28 s	1.01 s	1.13 s	1.34 s
29	1.08 s	1.31 s	1.22 s	1.17 s	1.19 s	1.16 s	1.04 s	1.15 s	1.31 s	1.19 s	1.21 s	1.31 s
30	0.95 s	1.04 s	1.00 s	1.07 s	1.14 s	1.12 s	1.07 s	0.99 s	0.98 s	1.05 s	1.07 s	1.02 s
3-Ara
1′	4.79 d (7.5)	4.79 d (9.0)	4.88 d (8.0)									4.79 d (7.0)
2′	5.99 t (8.5)	4.38 t (10.0)	4.63 brd (6.0)									4.18 m
3′	4.23 brd (9.5)	4.24 m	5.48 dd (4.5, 11.0)									4.47 t (8.0)
4′	4.30 brs	5.50 brs	4.57 brs									4.33 brs
5′	4.28 m 3.79 brd (12.0)	4.24 m 3.81 d (16.0)	4.30 m 3.85 d (13.5)									4.31 brd (9.5) 3.79 d (12.5)
15-OCOCH 3												2.26 s
23-OCOCH 3												2.02 s
24-OCOCH 3										2.16 s	2.18 s
25-OCOCH 3		1.96 s	1.95 s
3′-OCOCH 3			1.96 s
4′-OCOCH 3		1.96 s
4′-OCOCH=CH-CH3	6.05 d (15.5)
4′-OCOCH=CH-CH3	7.09 m
4′-OCOCH=CH-CH 3	1.63 d (6.5)

Table 2 ¹³C NMR data of compounds 1–12 in pyridine-d ₅ at 125 MHz (2–5, 7, 8, 10, 11) and 150 MHz (1, 6, 9, 12).

C	1	2	3	4	5	6	7	8	9	10	11	12
1	32.8 t	32.4 t	32.4 t	30.8 t	27.7 t	29.5 t	31.7 t	152.6 d	121.0 d	32.7 t	31.3 t	30.6 t
2	30.4 t	30.0 t	29.9 t	30.7 t	31.1 t	36.9 t	36.9 t	127.2 d	33.7 t	31.3 t	30.7 t	29.9 t
3	89.2 d	88.9 d	88.7 d	77.8 d	78.1 d	216.1 s	214.9 s	203.8 s	74.2 d	77.9 d	77.6 d	88.5 d
4	41.6 s	41.3 s	41.3 s	40.2 s	40.5 s	49.5 s	48.9 s	45.1 s	39.6 s	41.1 s	40.2 s	40.9 s
5	47.9 d	47.6 d	47.6 d	42.4 d	43.6 d	45.9 d	44.2 d	40.4 d	51.7 d	47.3 d	41.9 d	42.8 d
6	21.5 t	21.1 t	21.1 t	22.0 t	22.2 t	22.7 t	21.9 t	21.8 t	38.5 t	21.0 t	21.9 t	22.2 t
7	26.9 t	26.3 t	26.4 t	114.5 d	115.2 d	114.5 d	114.2 d	115.5 d	125.1 d	26.2 t	115.0 d	115.6 d
8	49.1 d	48.6 d	48.6 d	147.4 s	147.3 s	149.1 s	147.9 s	147.4	144.8 s	43.7 d	141.9	146.6 s
9	20.5 s	20.0 s	20.1 s	21.9 s	28.0 s	28.9 s	22.1 s	26.3 s	138.3 s	20.0 s	21.0 s	21.8 s
10	27.0 s	26.5 s	26.7 s	28.3 s	29.4 s	29.4 s	28.2 s	32.8 s	139.8 s	27.4 s	29.3 s	29.1 s
11	26.8 t	26.4 t	26.5 t	40.3 t	63.5 d	63.7 d	25.6 t	26.5 t	122.1 d	26.0 t	25.3 t	25.3 t
12	34.5 t	34.0 t	34.1 t	72.5 d	49.4 t	48.7 t	33.9 t	33.8 t	38.6 t	31.4 t	30.6 t	33.4 t
13	42.3 s	41.8 s	41.9 s	47.1 s	41.6 s	46.0 s	41.2 s	41.3 s	45.2 s	40.0 s	40.2 s	41.9 s
14	47.7 s	47.2 s	47.3 s	51.3 s	50.7 s	48.6 s	50.6 s	50.9 s	48.3 s	55.1 s	56.4 s	48.9 s
15	80.7 d	80.2 d	80.2 d	78.1 d	78.2 d	45.8 t	78.1 d	77.9 d	44.3 t	213.9 s	211.2 s	82.1 d
16	112.5 s	112.4 s	112.5 s	112.5 s	112.4 s	114.9 s	112.2 s	112.1 s	114.5 s	84.3 d	84.3 d	214.2 s
17	60.0 d	59.4 d	59.4 d	59.8 d	58.9 d	61.6 d	59.4 d	59.4 s	60.4 d	52.4 d	52.3 d	60.3 d
18	20.0 q	19.5 q	19.5 q	13.2 q	21.0 q	21.4 q	21.7 q	21.5 q	17.8 q	20.3 q	25.4 q	22.3 q
19	31.2 t	30.9 t	30.9 t	28.8 t	18.7 t	18.7 t	27.7 t	30.3 t	44.3 t	31.3 t	28.5 t	30.4 t
20	24.5 d	23.9 d	23.9 d	23.8 d	24.0 d	24.3 d	24.0 d	24.0 d	24.4 d	33.3 d	33.0 d	28.9 d
21	20.1 q	19.5 q	19.5 q	21.3 q	19.6 q	20.2 q	19.7 q	19.7 q	20.5 q	20.0 q	20.0 q	20.3 q
22	38.6 t	37.9 t	37.9 t	38.5 t	38.0 t	38.5 t	38.0 t	38.6 t	25.9 t	38.8 t	38.7 t	37.5 t
23	72.3 d	71.7 d	71.7 d	72.0 d	72.0 d	72.4 d	72.1 d	72.1 d	72.3 d	79.1 d	79.0 d	72.3 d
24	90.7 d	86.8 d	86.8 d	90.1 d	90.3 d	91.1 d	90.3 d	90.3 d	91.0 d	79.8 d	79.8 d	65.6 d
25	71.4 s	83.1 s	83.1 s	71.0 s	71.9 s	71.4 s	70.9 s	70.9 s	71.5 s	72.1 s	72.0 s	59.1 s
26	27.6 q	23.4 q	23.4 q	27.0 q	27.1 q	28.4 q	27.1 q	27.1 q	28.4 q	26.8 q	26.8 q	25.2 q
27	25.9 q	21.5 q	21.5 q	25.6 q	25.4 q	25.2 q	25.4 q	25.4 q	25.1 q	28.4 q	28.4 q	19.8 q
28	12.3 q	11.8 q	11.8 q	18.3 q	19.5 q	27.9 q	18.4 q	18.2 q	25.3 q	17.6 q	21.6 q	20.0 q
29	26.0 q	25.7 q	25.7 q	26.1 q	26.3 q	23.2 q	22.6 q	21.7 q	25.7 q	26.1 q	26.1 q	26.3 q
30	15.7 q	15.4 q	15.4 q	13.6 q	13.9 q	20.8 q	20.2 q	18.9 q	15.1 q	14.9 q	13.6 q	14.7 q
3-Ara
1′	105.0 d	107.3 d	106.8 d									108.1 d
2′	74.5 d	73.2 d	69.7 d									73.4 d
3′	73.0 d	72.5 d	76.9 d									75.2 d
4′	70.3 d	72.3 d	66.7 d									70.2 d
5′	67.6 t	63.2 t	66.2 t									66.5 t
15-OCOCH3												170.9 s
15-OCOCH3												21.4 q
23-OCOCH3												171.2 s
23-OCOCH3												21.5 q
24-OCOCH3										171.1 s	171.1 s
24-OCOCH3										21.0 s	20.9 s
25-OCOCH3		170.2 s	170.2 s
25-OCOCH3		21.2 q	22.3 q
3′-OCOCH3			170.8 s
3′-OCOCH3			21.6 q
4′-OCOCH3		170.8 s
4′-OCOCH3		22.2 q
4′-OCOCH=CH-CH3	166.3 s
4′-OCOCH=CH-CH3	122.3 d
4′-OCOCH=CH-CH3	145.2 d
4′-OCOCH=CH-CH3	18.2 q

25-O-Acetylcimigenol-3-O-[4′-O-acetyl]-α-L-arabinopyranoside (2): A white powder; [α]_D ²⁴ = 6.67 (c 0.06, MeOH); IR (KBr): ν _max = 3472, 2968, 2870, 1739, 1458, 1374, 1072, 972 cm⁻¹; ¹H (C₅D₅ N, 500 MHz) and ¹³C NMR (C₅D₅ N, 125 MHz) spectra, see [Tables 1] and [2]; ESIMS: m/z = 727 [M + Na]⁺; HR-TOF-ESIMS: m/z = 727.4029 (calc. for C₃₉H₆₀O₁₁Na, 727.4033).

25-O-Acetylcimigenol-3-O-[3′-O-acetyl]-α-L-arabinopyranoside (3): A white powder; [α]_D ²⁴ = − 6.80 (c 0.05, MeOH); IR (KBr): ν _max = 3467, 2935, 2870, 1741, 1458, 1375, 1067, 972 cm⁻¹; ¹H (C₅D₅ N, 500 MHz) and ¹³C NMR (C₅D₅ N, 125 MHz) spectra, see [Tables 1] and [2]; ESIMS: m/z = 703 [M – H]⁻; HR-TOF-ESIMS: m/z = 703.4055 (calc. for C₃₉H₅₉O₁₁, 703.4057).

12β-Hydroxy-7(8)-en-cimigenol (4): A white powder; [α]_D ²⁴ = 3.20 (c 0.10, MeOH); IR (KBr): ν _max = 3423, 2931, 2872, 1638, 1447, 1381, 1025, 979 cm⁻¹; ¹H (C₅D₅ N, 500 MHz) and ¹³C NMR (C₅D₅ N, 125 MHz) spectra, see [Tables 1] and [2]; ESIMS: m/z = 525 [M + Na]⁺; HR-TOF-ESIMS: m/z = 525.3192 (calc. for C₃₀H₄₆O₆Na, 525.3192).

11β-Hydroxy-7(8)-en-cimigenol (5): A white powder; [α]_D ²⁴ = 0.47 (c 0.10, MeOH); IR (KBr): ν _max 3441, 2997, 2882, 1634, 1447, 1380, 1027, 973 cm⁻¹; ¹H (C₅D₅ N, 500 MHz) and ¹³C NMR (C₅D₅ N, 125 MHz) spectra, see [Tables 1] and [2]; ESIMS: m/z = 525 [M + Na]⁺; HR-TOF-ESIMS: m/z = 525.3199 (calc. for C₃₀H₄₆O₆Na, 525.3192).

11β-Hydroxy-15-deoxycimigenol-7(8)-en-3-one (6): A white powder; [α]_D ²⁴ = − 23.33 (MeOH, c 0.05); IR (KBr): ν _max = 3441, 2969, 2869, 1711, 1633, 1382, 975 cm⁻¹; ¹H (C₅D₅ N, 500 MHz) and ¹³C NMR (C₅D₅ N, 150 MHz) spectra, see [Tables 1] and [2]; ESIMS: m/z = 507 [M + Na]⁺; HR-TOF-ESIMS: m/z = 507.3090 (calc. for C₃₀H₄₄O₅Na, 507.3086).

Cimigenol-7(8)-en-3-one (7): A white powder; [α]_D ²⁴ = − 21.67 (c 0.10, MeOH); IR (KBr): ν _max = 3492, 2984, 2839, 1709, 1637, 1452, 1382, 1024, 987 cm⁻¹; ¹H (C₅D₅ N, 500 MHz) and ¹³C NMR (C₅D₅ N, 125 MHz) spectra, see [Tables 1] and [2]; ESIMS: m/z = 507 [M + Na]⁺; HR-TOF-ESIMS: m/z = 507.3086 (calc. for C₃₀H₄₄O₅Na, 507.3086).

Cimigenol-1(2),7(8)-dien-3-one (8): A white powder; [α]_D ²⁴ = − 60.60 (c 0.10, MeOH); IR (KBr): ν _max = 3432, 2947, 2852, 1693, 1637, 1460, 1382, 1070, 978 cm⁻¹; ¹H (C₅D₅ N, 500 MHz) and ¹³C NMR (C₅D₅ N, 125 MHz) spectra, see [Tables 1] and [2]; ESIMS: m/z = 505 [M + Na]⁺; HR-TOF-ESIMS: m/z = 505.2944 (calc. for C₃₀H₄₂O₅Na, 505.2929).

9,10-Seco-1(10),7(8),9(11)-trien-cimigenol (9): A white powder; [α]_D ²⁴ = 5.73 (c 0.05, MeOH); IR (KBr): ν _max = 3432, 2947, 2852, 1624, 1460, 1383, 1070, 978 cm⁻¹; ¹H (C₅D₅ N, 500 MHz) and ¹³C NMR (C₅D₅ N, 150 MHz) spectra, see [Tables 1] and [2]; ESIMS: m/z = 469 [M + H]⁺; HR-TOF-ESIMS: m/z = 469.3311 (calc. for C₃₀H₄₅O₄, 469.3317).

24-O-Acetylisodahurinol (10): A white powder; [α]_D ²⁴ = 2.09 (c 0.07, MeOH); IR (KBr): ν _max = 3473, 2946, 2862, 1738, 1471, 1377, 1024, 994 cm⁻¹; ¹H (C₅D₅ N, 500 MHz) and ¹³C NMR (C₅D₅ N, 125 MHz) spectra, see [Tables 1] and [2]; ESIMS: m/z = 553 [M + Na]⁺; HR-TOF-ESIMS: m/z = 553.3494 (calc. for C₃₂H₅₀O₆Na, 553.3505).

24-O-Acetyl-7(8)-en-isodahurinol (11): A white powder; [α]_D ²⁴ = − 4.80 (c 0.05, MeOH); IR (KBr): ν _max = 3437, 2920, 2875, 1736, 1640, 1472, 1375, 1026, 996 cm⁻¹; ¹H (C₅D₅ N, 500 MHz) and ¹³C NMR (C₅D₅ N, 125 MHz) spectra, see [Tables 1] and [2]; ESIMS: m/z = 551 [M + Na]⁺; HR-TOF-ESIMS: m/z = 551.3346 (calc. for C₃₂H₄₈O₆Na, 551.3348).

23-O-Diacetyl-7(8)-en-shengmanol-3-O-α-L-arabinopyranoside (12): A white powder; [α]_D ²⁴ = − 7.69 (MeOH, c 0.07); IR (KBr): ν _max = 3473, 2942, 2871, 1736, 1643, 1458, 1376, 1024, 972 cm⁻¹; ¹H (C₅D₅ N, 500 MHz) and ¹³C NMR (C₅D₅ N, 150 MHz) spectra, see [Tables 1] and [2]; ESIMS: m/z = 725 [M + Na]⁺; HR-TOF-ESIMS: m/z = 725.3895 [M + Na]⁺ (calc. for C₃₉H₅₈O₁₁Na, 725.3876).

Hydrolysis and identification of the sugar moieties in compounds 1, 2, 3, and 12

Compound 1 (4.0 mg) together with 2 and 3 (3.0 mg of each) were individually dissolved in MeOH (5 mL), then 4 % K₂CO₃ (5 mL) was added, and each solution was stirred at rt overnight. Each solution was neutralized by 10 % HOAc and extracted with EtOAc (3 × 15 mL). Each EtOAc extract, after removal of solvent, was dissolved in MeOH (5 mL) and refluxed with 0.5 N HCl (3 mL) for 4 h [7]. Compound 12 (4.0 mg), by contrast, was directly dissolved in MeOH (5 mL) and refluxed with 0.5 N HCl (3 mL) for 4 h. Each reaction mixture was diluted with H₂O and extracted with CHCl₃ (3 × 10 mL). Each aqueous layer was then neutralized by Ag₂CO₃, and the formed precipitation was filtered to give a monosaccharide, which had an Rf (EtOAc-CHCl₃-MeOH-H₂O, 3 : 2 : 2 : 1) and specific rotation [α]_D ²⁰ + 82.78 (c 0.05, MeOH) corresponding to those of L-arabinose (Sigma-Aldrich).

Cytotoxicity bioassay

Five human cancer cell lines, human myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, lung cancer A-549, breast cancer MCF-7, and colon cancer SW480, were used in the cytotoxic assay. Cells were cultured in DMEM medium (Hyclone) supplemented with 10 % fetal bovine serum (Hyclone), in 5 % CO₂ at 37 °C. The cytotoxicity assay was performed according to the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] method in 96-well microplates [32], [33]. Briefly, 100 µL of adherent cells were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before addition of test compounds, while suspended cells were seeded just before drug addition with an initial density of 1 × 10⁵ cells/mL. Each tumor cell line was exposed to the test compounds (dissolved in DMSO, then diluted by DMEM medium) at concentrations of 0.064, 0.32, 1.6, 8, and 40 µM in triplicate for 48 h, with cisplatin (Sigma) and culture solution as positive and negative controls, respectively. After compound treatment, cell viability was detected, and a cell growth curve was graphed. IC₅₀ values were calculated by Reed and Muenchʼs method [34].

Supporting information

The NMR spectra of compounds 1–12, as well as ESI and HR-TOF-ESI data of 1–12 are available as Supporting Information.

Results and Discussion

Compound 1 was obtained as white powder. Its molecular formula (C₃₉H₆₀O₁₀) was deduced from the analysis of ¹³C NMR and HR-TOF-ESIMS data {m/z: 711.4065 [M + Na]⁺ (calc. for C₃₉H₆₀O₁₀Na, 711.4084)}. The IR spectrum showed absorptions for hydroxy groups at 3452 cm⁻¹, carbonyl groups at 1727 cm⁻¹, and double bonds at 1632 cm⁻¹, respectively. The ¹H-NMR spectrum ([Table 1]) showed the presence of the characteristic cyclopropane methylene signals at δ _H 0.21 and 0.45 (each 1H, brs), an anomeric proton at δ _H 4.79 (d, J = 7.5 Hz), two olefinic protons at δ _H 6.05 (1H, d, J = 15.5 Hz) and 7.09 (1H, m), two secondary methyl signals at δ _H 0.84 (d, J = 7.0 Hz) and 1.63 (d, J = 6.5 Hz), and six tertiary methyl groups at δ _H 0.95–1.48. In the ¹³C and DEPT NMR spectra of 1 ([Table 2]), the signals ascribable to an α,β-unsaturated ketone moiety at δ _C 166.3, 122.3, and 145.2 were observed. A comparison of the spectroscopic data of 1 with those of cimigenol-3-O-α-L-arabinopyranoside (27) [15] showed that, structurally, 1 closely resembles 27, with the main differences of the sugar unit and the presence of another tetra-carbon unit, including the α,β-unsaturated ketone resonances. In the ¹H-¹H COSY spectrum ([Fig. 2]), a correlation was observed between the secondary methyl signal at δ _H 1.63 (d, J = 6.5 Hz) and the olefinic proton at δ _H 7.09 (m), which indicated the tetra-carbon unit to be a 2-butenoyl. In addition, the coupling constant (J = 15.6 Hz) of the two olefinic protons at δ _H 6.05 and 7.09 confirmed the E-geometry of a double bond in the 2-butenoyl moiety. In the HMBC spectrum ([Fig. 2]), a correlation was observed between the anomeric proton at δ _H 4.79 (H-1′, 1H, d, J = 7.5 Hz) and the methine signal at δ _C 89.2 (C-3), suggesting that a sugar unit was attached at the C-3. The sugar obtained after acid hydrolysis was identified as α-L-arabinose by comparing its TLC and specific rotation with the standard. In the ¹H NMR spectrum, a downfield resonance was observed at δ _H 5.99 (t, J = 8.5 Hz), which showed correlations with the methine resonance at δ _H 4.30 (H-3′) and the anomeric proton at δ _H 4.79 in the ¹H-¹H COSY spectrum ([Fig. 2]). Furthermore, the HMBC ([Fig. 2]) correlation between the carbonyl group (δ _C 166.3) and the proton resonance (δ _H 5.99, t, J = 8.5 Hz) indicated the (E)-2-butenoyl unit was attached at C-2′. In the ROESY spectrum ([Fig. 3]), H-3 showed a correlation with H-5 suggesting an α-orientation of the H-3, while H-15 showed a correlation with Me-18, indicating an α-orientation of the hydroxyl group at C-15. The configuration of C-23 and C-24 was deduced as R and S, respectively, by comparison of the coupling constant of H-23 and H-24 with those of cimigenol-type compounds [6], [22], [26]. Therefore, the structure of 1 was determined as cimigenol-3-O-[2′-O-(E)-2-butenoyl]-α-L-arabinopyranoside.

Fig. 2 Major HMBC (→) and ¹H-¹H COSY (—) correlations of compound 1.

Fig. 3 Key NOESY correlations of compound 1.

Compounds 2 and 3 were determined to have the same molecular formula C₃₉H₆₀O₁₁ by HR-TOF-ESIMS (m/z 727.4029 [M + Na]⁺ and 703.4055 [M – H]⁻, respectively). The NMR spectroscopic data ([Tables 1] and [2]) of 2 and 3 were similar to those of 25-O-acetylcimigenol-3-O-[2′-O-acetyl]-α-L-arabinopyranoside (19), except for the sugar moiety. In the ¹H-NMR spectrum ([Table 1]) of 2, the signal due to H-4′ showed a downfield shift from δ _H 4.16 to 5.50. Meanwhile, the signal of H-2′ was shifted from δ _H 5.89 to δ _H 4.38. In addition, the signal due to C-4′ exhibited a downfield shift from δ _C 69.8 to 72.3 in the ¹³C NMR spectrum ([Table 2]). The changes of these chemical shifts may be explained by the O-acetyl group being attached to C-4 of the sugar unit, which was further confirmed by the presence of the HMBC correlation between the H-4′ signal at δ _H 5.50 and the carbonyl group signal at δ _C 170.8. The sugar obtained after acid hydrolysis was confirmed as α-L-arabinopyranose by comparing its TLC and specific rotation with the standard. The configurations of C-23 and C-24 are proposed as R and S, respectively, by the same way as that of 1. Thus, the structure of 2 was assigned as 25-O-acetylcimigenol-3-O-[4′-O-acetyl]-α-L-arabinopyranoside. In the same way, an acetoxy group was determined to be at C-3′ for 3, which was further confirmed by the presence of the HMBC correlation between the H-3′ signal at δ _H 5.48 and the carbonyl group signal at δ _C 170.8. Therefore, 3 was identified as 25-O-acetylcimigenol-3-O-[3′-O-acetyl]-α-L-arabinopyranoside.

Compound 4 gave a molecular formula of C₃₀H₄₆O₆ as established by HR-TOF-ESIMS (m/z 525.3192 [M + Na]⁺), which is 16 Da more than those of 7(8)-en-cimigenol (14). The ¹H NMR spectrum ([Table 1]) displayed downfield cyclopropane methylene signals at δ _H 0.73 (1H, d, J = 4.0 Hz) and 1.19 (overlapped), seven methyl groups at δ _H 1.07–1.52, and an olefinic proton at δ _H 6.20 (1H, d, J = 7.0 Hz), respectively, suggesting 4 to be a 9,19-cyclolanostane aglycone with a pair of olefinic carbons close to the cyclopropane methylene at C-19 [20]. The NMR data of 4 showed a close resemblance with those of 14, except that the methylene signal at δ _C 33.7 (C-12) was absent, showing instead a hydroxymethine at δ _C 72.5. On the basis of the above observations, it was reasonable to deduce that 4 was a 12-hydroxy derivative of 14, which was also supported by the HMBC correlations of H-12 at δ _H 4.36 with C-11 (δ _C 40.3), C-13 (δ _C 47.1), and CH₃-18 (δ _C 13.2), as well as by the downfield shift of C-11 about 14.9 ppm in the ¹³C spectrum ([Table 2]). Significant ROESY correlations of H-12 with H-5, and CH₃-28 suggested a β-orientation of the substituent at C-12. Therefore, 4 was elucidated as 12β-hydroxy-7(8)-en-cimigenol.

Compound 5 was assigned as C₃₀H₄₆O₆, as deduced from the HR-TOF-ESIMS (m/z 525.3199 [M + Na]⁺), which is identical to that of compound 4. The NMR data of 5 were similar to that of 4 with the major difference being that a hydroxyl group was shifted from C-12 to C-11. In the ¹³C NMR spectrum ([Table 2]) of 5, the signal due to C-12 exhibited an upfield shift from δ _C 72.5 to 49.4, while C-11 showed a downfield shift from δ _C 40.3 to 63.5, further confirming the deduction. The relative configuration of the hydroxyl group at C-11 was proposed as β-orientated by analyses of the ROESY spectrum. Accordingly, compound 5 was characterized as 11β-hydroxy-7(8)-en-cimigenol.

Compound 6 had the molecular formula C₃₀H₄₄O₅ from its positive mode HR-TOF-ESIMS (507.3090 [M + Na]⁺). In the ¹³C and DEPT spectra ([Table 2]), 6 exhibited signals very similar to those of 5 except that hydroxy methine signals due to C-3 and C-15 were absent, whereas a carbonyl carbon at δ _C 216.1 and a downfield methylene at δ _C 45.8 were observed. In addition, HMBC correlations of H-2, CH₃-29, and CH₃-30 with the carbonyl group at δ _C 216.1 and protons (δ _H 2.46 and 2.28, each 1H) due to the methylene at δ _C 45.8 with C-14, C-16, and CH₃-28 indicated that a carbonyl carbon replaced a hydroxyl group at C-3, and the methine at C-15 was transformed to a methylene in 6. Ultimately, 6 was elucidated as 11β -hydroxy-15-deoxycimigenol-7(8)-en-3-one.

The HR-TOF-ESIMS (m/z 507.3086 [M + Na]⁺) of compound 7 determined its molecular formula as C₃₀H₄₄O₅, which is identical with 24-epi-cimigenol-7(8)-en-3-one (17). The NMR data ([Tables 1] and [2]) of 7 resembled those of 17 with major differences at C-22 (δ _C 38.0), C-23 (δ _C 72.1), C-24 (δ _C 90.3), and C-25 (δ _C 70.9), which are similar to the key structural differences between cimigenol-type and 24-epi-cimigenol-type triterpenes [21]. By comparing the coupling constant of H-24 (0 Hz) with those of cimigenol-type compounds (0 Hz) and 24-epi-cimigenol-type constituents (4 Hz), the configuration of C-24 of 7 was determined to be S [6], [21], [25]. Thus, 7 was elucidated as cimigenol-7(8)-en-3-one.

The molecular formula of compound 8 was determined as C₃₀H₄₂O₅ from the HR-TOF-ESIMS (m/z 505.2944 [M + Na]⁺). Its NMR data ([Tables 1] and [2]) were similar to those of 7 except for the signals of ring A. Unsaturated carbon signals at δ _C 152.6 and 127.2 were observed in 8, whereas signals of two methylenes due to C-1 and C-2 were absent. Significant HMBC correlations were observed between the carbonyl C-atom at δ _C 203.8 and the olefinic protons at δ _H 6.16 and 6.71 (each 1H, d, J = 10.0 Hz). The above evidence suggested that compound 8 is transformed from 7 through dehydrogenation between C-1 and C-2. Therefore, compound 8 was characterized as cimigenol-1(2),7(8)-dien-3-one.

Compound 9 was assigned a molecular formula of C₃₀H₄₆O₄ from its HR-TOF-ESIMS (m/z 469.3311 [M + H]⁺). In the ¹H NMR spectrum, signals for the significant downfield cyclopropane methylene at δ _H 3.15 and 3.24 (1H each, d, J = 14.0 Hz), seven methyl groups at δ _H 0.81–1.52, and three olefinic protons at δ _H 5.39, 5.52, and 5.54 were observed, suggesting 9 is a 9,10-seco-9,19-cycloartane triterpene [30]. The ¹³C NMR and DEPT spectroscopic data of 9 were identical with the aglycone resonances of cimicinol (28) [31], except for the upfield shift of the C-3 by 9.7 ppm, which could be explained by the absence of a sugar unit at C-3. Therefore, 9 was elucidated as 9,10-seco-1(10),7(8),9(11)-trien-cimigenol.

The molecular formula of compound 10 was established as C₃₂H₅₀O₆ on the basis of HR-TOF-ESIMS (m/z 553.3494 [M + Na]⁺). In the IR spectrum, absorption bands at 3473 and 1733 cm⁻¹ for hydroxyl and carbonyl groups were observed. In the ¹³C and DEPT NMR spectra, the signals ascribable to the methylene carbon of the cyclopropane ring at δ _C 31.3 (C-19), four oxygen-bearing methine carbons at δ _C 84.3 (C-16), 79.8 (C-24), 79.1 (C-23), and 77.9 (C-3), as well as two carbonyl carbons at δ _C 213.9 (C-15) and 171.1 (C₂₄-acetoxy) were observed, suggesting that 10 was a highly oxygenated 9,19-cycloartane triterpene aglycon with an O-acetyl group. By comparison of NMR spectroscopic data, 10 was determined to be the aglycon of 24-O-acetylisodahurinol-3-O-α-L-arabinopyranoside (29) [7]. The configuration of C-24 was deduced as S by comparison of the coupling constants of H-24 (1.6 Hz) with those of dahurinyl diacetate (9 Hz) and isodahurinyl diacetate (2 Hz) [29]. Accordingly, compound 10 was characterized as 24-O-acetylisodahurinol.

Compound 11 gave a molecular formula of C₃₂H₄₈O₆ by HR-TOF-ESIMS at m/z 551.3346 [M + Na]⁺. The NMR spectroscopic data ([Tables 1] and [2]) of 11 resembled those of 10 except for major differences at C-7 (δ _C 115.0) and C-8 (δ _C 141.9) due to dehydrogenation at these positions. This deduction was confirmed by the correlations of δ _H 6.36 (H-7) with δ _H 1.56 and 1.93 (H-6) in the ¹H-¹H COSY spectrum. Accordingly, compound 11 was characterized as 24-O-acetyl-7(8)-en-isodahurinol.

Compound 12 exhibited the molecular formula C₃₉H₅₈O₁₁, as established by HR-TOF-ESIMS at m/z 725.3895 [M + Na]⁺. The molecular weight of 12 is 42 Da more than that of 23-O-acetyl-7(8)-en-shengmanol-3-O-α-L-arabinopyranoside (30) [27], which may be due to an acetyl group. When its spectroscopic data ([Tables 1] and [2]) were compared with those of 30 [27], an additional O-acetyl group was assigned to C-15 on the basis of the upfield shift of the carbonyl carbon (C-16) from δ _C 220.3 to 214.2, the downfield shift of H-15 from δ _H 4.56 to 5.92, as well as the HMBC correlation of H-15 and the carbonyl group signal at δ _C 170.9. Significant ROESY correlations of H-15 with CH₃-18 suggested an α-orientation of the substituent at C-15. The configurations of C-23 and C-24 were considered to be R and S, respectively, by comparing coupling constants of H-23 and H-24 with those of known 9,19-cyclolanostane triterpene glycosides [25]. In the ¹³C NMR spectrum ([Table 2]), compound 12 showed resonances corresponding to an α-L-arabinose moiety at δ _C 108.1 (d), 73.4 (d), 75.2 (d), 70.2 (d), and 66.5 (t) [8], [29], which was further confirmed by comparing its TLC and specific rotation with the standard after hydrolysis. Ultimately, 12 was elucidated as 15,23-O-diacetyl-7(8)-en-shengmanol-3-O-α-L-arabinopyranoside.

All isolated compounds were screened for their in vitro antitumor activities. As summarized in [Table 3], the new compounds 1–3 and the known compounds 19 and 20 showed broad-spectrum and moderate cytotoxicities against human HL-60, SMMC-7721, A549, MCF-7, and SW480 cell lines, with IC₅₀ values ranging from 4.2 to 14.5 µM. In addition, the new compounds 6–8 and the known compounds 15, 16, and 18 exhibited broad-spectrum and week cytotoxicities, having IC₅₀ values around 20 µM. Based on the above results, we suggest that the roots of C. dahurica may be another potential resource for promising antitumor agents.

Table 3 Cytotoxicity ^a (IC₅₀, µM ± SD) of compounds isolated from the roots of C. dahurica.

Compounds	HL-60	SMMC-7721	A-549	MCF-7	SW480
a Cytotoxicity is the average (n = 3) of calculated IC50s; the purity of compounds 1–26 is greater than 95 % and of cisplatin greater than 99 %
1	4.2 ± 0.4	13.4 ± 1.1	13.8 ± 0.8	14.2 ± 0.7	11.8 ± 1.3
2	8.1 ± 0.5	12.2 ± 1.2	14.5 ± 0.7	12.7 ± 1.4	13.1 ± 0.9
3	5.8 ± 0.2	8.7 ± 0.3	10.3 ± 1.1	12.6 ± 0.5	11.3 ± 1.2
4	> 40	> 40	> 40	> 40	> 40
5	> 40	> 40	> 40	> 40	> 40
6	18.9 ± 1.6	19.5 ± 0.7	21.1 ± 1.8	23.7 ± 1.5	20.3 ± 1.7
7	21.1 ± 1.5	18.3 ± 1.7	20.5 ± 1.0	19.2 ± 0.9	> 40
8	21.9 ± 2.2	17.4 ± 1.8	19.5 ± 0.8	17.5 ± 1.3	22.5 ± 1.2
9	> 40	> 40	> 40	> 40	> 40
10	> 40	> 40	> 40	> 40	> 40
11	> 40	> 40	> 40	> 40	> 40
12	> 40	> 40	> 40	> 40	> 40
13	> 40	> 40	> 40	> 40	> 40
14	> 40	> 40	> 40	> 40	> 40
15	10.5 ± 0.9	22.5 ± 1.3	19.1 ± 2.1	20.6 ± 1.4	13.8 ± 0.8
16	20.2 ± 0.7	22.9 ± 1.5	20.4 ± 1.7	21.1 ± 1.2	21.3 ± 1.4
17	> 0	> 40	> 40	> 40	> 0
18	20.2 ± 0.8	22.9 ± 1.7	20.4 ± 1.3	15.1 ± 1.2	> 40
19	5.8 ± 0.7	8.7 ± 0.8	10.3 ± 2.1	12.6 ± 1.7	11.3 ± 0.7
20	8.1 ± 0.6	12.2 ± 1.4	14.5 ± 1.2	12.7 ± 2.3	13.1 ± 1.5
21	> 40	> 40	> 40	> 40	> 40
22	> 40	> 40	> 40	> 40	> 40
23	> 40	> 40	> 40	> 40	> 40
24	> 40	> 40	> 40	> 40	> 40
25	> 40	> 40	> 40	> 40	> 40
26	> 40	> 40	> 40	> 40	> 40
Cisplatin	0.52 ± 0.05	13.4 ± 0.6	12.4 ± 0.7	15.0 ± 1.4	14.4 ± 1.1

In the present study, structural and bioactive properties of five cimigenol-type glycosides (1–3, 19, 20) are completely in accordance with the SAR we proposed before. Thus, the SAR proposed in our previous studies may be used for the design of more potent lead compounds. Furthermore, six cimigenol-type aglycons (6–8, 15, 16, 18) exhibited broad-spectrum and week cytotoxicities. The main structural characters of these compounds are: (1) the configurations of C-23 and C-24 are R and S, respectively; (2) carbonyl and acetoxy groups instead of a hydroxyl group at C-3 or C-25. Previously, we reported that cimigenol-type aglycone actrin-3-one has potent and moderate activities against human HepG-2 and HT 29 cell lines, respectively. Meanwhile, 25-O-acetylcimigenol exhibited moderate activity against the human HepG-2 cell line [7]. Based on the analyses of these data, we may propose that for cimigenol-type aglycones, hydrophobic groups, such as carbonyl and acetoxy, instead of a hydroxyl group at C-3 or C-25 are essential for cytotoxicity.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (No. U1132604), Knowledge Innovation Program of the CAS (Grant No. KZCX2-XB2-15-03, KSCX2-EW-R-15), as well as Top Talents Program of Yunnan province (2009C1120), and the Foundation of State Key Laboratory of Phytochemistry and Plant Resources in West China (P2010-ZZ14).

Conflict of Interest

All authors declare that there are no conflicts of interest. The isolation and structural elucidations were accomplished by Yin Nian, Hai-Yan Wang, Lin Zhou, and Ming-Hua Qiu, and the cell culture and cytotoxicity assay were performed by Jia Su and Yan Li. All the authors knew about this manuscript and had no objection to submitting it.

• References

• 1 Pan L, Chai HY, Kinghorn AD. The continuing search for antitumor agents from higher plants. Phytochem Lett 2010; 3: 1-8
• 2 Newman DJ, Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod 2012; 75: 311-335
• 3 Hsiao WLW, Liu L. The role of traditional Chinese herbal medicines in cancer therapy from TCM theory to mechanistic insights. Planta Med 2010; 76: 1118-1131
• 4 Chinese Pharmacopoeia Commission. The Pharmacopoeia of Chinese Peopleʼs Republic. 2010 edition. Beijing: The Chemical Industry Publishing House; 2010: 68-69
• 5 Sun LR, Qing C, Zhang YL, Ji SY, Li ZR, Pei SJ, Qiu MH, Gross ML, Qiu SX. Cimicifoetisides A and B, two cytotoxic cycloartane triterpenoid glycosides from the rhizomes of Cimicifuga foetida, inhibit proliferation of cancer cells. Beilstein J Org Chem 2007; 3: 1-6
• 6 Lu L, Chen JC, Song HJ, Li Y, Nian Y, Qiu MH. Five new triterpene bisglycosides with acyclic side chains from the rhizomes of Cimicifuga foetida L. Chem Pharm Bull 2009; 58: 729-733
• 7 Nian Y, Zhang YL, Chen JC, Lu L, Qing C, Qiu MH. Cytotoxic chemical constituents from the roots of Cimicifuga foetida . J Nat Prod 2010; 73: 93-98
• 8 Nian Y, Zhang XM, Li Y, Wang YY, Chen JC, Lu L, Zhou L, Qiu MH. Cycloartane triterpenoids from the aerial parts of Cimicifuga foetida Linnaeus. Phytochemistry 2011; 72: 1473-1481
• 9 Sakurai N, Inoue T, Nagai M. Studies on the Chinese crude drug “Shoma.” II. Triterpenes of Cimicifuga dahurica MAXIM. Yakugaku Zasshi 1972; 92: 724-728
• 10 Kimura O, Sakurai N, Inoue T. Studies on the Chinese crude drug “Shoma.” VII. Isolation and determination of genuine natural products, acetyl shengmanol xyloside, 24-O-acetylhydroshengmanol xyloside, and shengmanol xyloside, in Cimicifuga dahurica and the other Cimicifuga plants. Yakugaku Zasshi 1983; 103: 293-299
• 11 Li CJ, Chen DH, Xiao PG. Studies on the chemical constituents from the aerial parts of Cimicifuga dahurica . Acta Pharm Sin 1993; 28: 777-781
• 12 Li CJ, Chen DH, Xiao PG. Chemical constituents of traditional Chinese drug “sheng-ma” (Cimicifuga dahurica). II. chemical structure of cimicifugamide. Acta Chim Sin 1994; 52: 296-300
• 13 Li CJ, Chen DH, Xiao PG. Chemical constituents of traditional Chinese drug “sheng-ma” (Cimicifuga dahurica) III structures of cimiside C and cimiside D. Acta Chim Sin 1994; 52: 722-726
• 14 Zhang QW, Ye WC, Hsiao W, Zhao SX, Che CT. Cycloartane glycosides from Cimicifuga dahurica . Chem Pharm Bull 2001; 49: 1468-1470
• 15 Ye WC, Zhang JW, Che CT, Ye T, Zhao SX. Cycloartane glycosides from Cimicifuga dahurica . Planta Med 1999; 65: 770-772
• 16 Liu Y, Chen DH, Si JY, Tu GZ, An DG. Two new cyclolanostanol xylosides from the aerial parts of Cimicifuga dahurica . J Nat Prod 2002; 65: 1486-1488
• 17 Tian Z, Yang MS, Huang F, Li KG, Si JY, Shi L, Chen SB, Xiao PG. Cytotoxicity of three cycloartane triterpenoids from Cimicifuga dahurica . Cancer Lett 2005; 226: 65-75
• 18 Tian Z, Si JY, Chang Q, Zhou L, Chen SL, Xiao PG, Wu E. Antitumor activity and mechanisms of action of total glycosides from aerial part of Cimicifuga dahurica targeted against hepatoma. BMC Cancer 2007; 7: 237-246
• 19 Einbonda LS, Ye WC, Hec K, Wu HL, Cruz E, Rollerc M, Kronenberg M. Growth inhibitory activity of extracts and compounds from Cimicifuga species on human breast cancer cells. Phytomedicine 2007; 15: 504-511
• 20 Sun LR, Yan J, Pei SJ, Qiu MH. A new cycloartane triterpenoid from the rhizome of Cimicifuga foetida collected in Dali. Acta Bot Yunn 2005; 27: 331-336
• 21 Li JX, Kadota S, Hattori M, Yoshimachi S, Shiro M, Oogami N, Mizuno H, Namba T. Isolation and characterization of ten new cycloartenol triterpenes from Cimicifuga heracleifolia Komarov. Chem Pharm Bull 1993; 41: 832-841
• 22 Gao JC, Zhang JC, Lu ZJ, Zhu GY, Yang MS, Xiao PG. Chemical constituents of Actaea asiatica Hara and their anti-osteoporosis activities. Biochem Syst Ecol 2006; 34: 710-713
• 23 Kusano A, Takahira M, Shibano M, Miyase T, Okuyama T, Kusano G. Studies on the constituents of Cimicifuga species. XXII. Structure of two new cyclolanostanol xylosides, cimiacerosides A and B. Heterocycles 1998; 48: 1003-1013
• 24 Zhou L, Yang JS, Tu GZ, Zou JH. Cyclolanostane triterpene glycosides from Souliea vaginata . Chem Pharm Bull 2006; 54: 823-826
• 25 Chen SN, Fabricant DS, Lu ZZ, Fong HHS, Farnsworth NR. Cimiracemosides I–P, new 9,19-cyclolanostane triterpene glycosides from Cimicifuga racemosa . J Nat Prod 2002; 65: 1391-1397
• 26 Kusano A, Takahira M, Shibano M, In Y, Ishida T, Miyase T, Kusano G. Studies on the constituents of Cimicifuga species. XX. Absolute stereostructures of cimicifugoside and actein from Cimicifuga simplex WORMSK. Chem Pharm Bull 1998; 46: 467-472
• 27 Kusano A, Shibano M, Kusano G, Miyase T. Studies on the constituents of Cimicifuga species. XIX. Eight new glycosides from Cimicifuga simplex WORMSK. Chem Pharm Bull 1996; 44: 2078-2085
• 28 Kusano A, Shibano M, Kitagawa S, Kusano G. Studies on the constituents of Cimicifuga species. XV. Two new diglycosides from the aerial parts of Cimicifuga simplex WORMSK. Chem Pharm Bull 1994; 42: 1940-1943
• 29 Shao Y, Harris A, Wang M, Zhang H, Cordell GA, Bowman M, Lemmo E. Triterpene glycosides from Cimicifuga racemosa . J Nat Prod 2000; 63: 905-910
• 30 Ali Z, Khan SI, Fronczek FR, Khan IA. 9,10-seco-9,19-cyclolanostane arabinosides from the roots of Actaea podocarpa . Phytochemistry 2007; 68: 373-382
• 31 Kadota S, Li JX, Tanaka K, Namba T. Constituents of Cimicifugae rhizoma II. Isolation and structures of new cycloartenol triterpenoids and related compounds from Cimicifuga foetida L. Tetrahedron 1995; 51: 1143-1166
• 32 Mosmman T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Immunol Methods 1983; 65: 55-63
• 33 Alley MC, Scudiero DA, Monks A. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res 1988; 48: 589-601
• 34 Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. Am J Hyg 1938; 27: 493-497