Six New 9,19-Cycloartane Triterpenoids from Cimicifuga foetida L.

Abstract

Six new 9,19-cycloartane triterpene derivatives, as well as 3 known analogues (7-9), were isolated from the roots of Cimicifuga foetida L. Their structures were established on the basis of extensive spectroscopic analyses (IR, UV, ORD, HRESIMS, 1D and 2D NMR).

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Six New 9,19-Cycloartane Triterpenoids from Cimicifuga foetida L.

Guo-Lei Zhu . Di-Fan Zhu . Luo-Sheng Wan . Xing-Rong Peng . Ni-Man Bao . Zhi-Run Zhang . Lin Zhou . Ming-Hua Qiu

Received: 19 February 2016 / Accepted: 27 March 2016 / Published online: 20 May 2016 The Author(s) 2016. This article is published with open access at Springerlink.com

Abstract Six new 9,19-cycloartane triterpene derivatives, as well as 3 known analogues (79), were isolated from the roots of Cimicifuga foetida L. Their structures were established on the basis of extensive spectroscopic analyses (IR, UV, ORD, HRESIMS, 1D and 2D NMR).

Keywords Cimicifuga foetida 9,19-Cycloartane triterpenoids Cycloartane-type

1 Introduction

Cimicifuga foetida L. or actaea foetida also named Shengma, a well known medicinal plant widely distributed in China, has been used for alleviation fever, pain, and inammation since ancient times in China [13].Currently, it is prescribed as one of the source plants for the treatment of headache, sore throat, toothache, and uterine prolapse in the Chinese Pharmacopoeia 2010 [4]. Phyto-chemical investigation have shown that 9,19-cycloartane

triterpenoids and their glycosides are the main constituents of Cimicifuga. Meanwhile, because of their structural diversity and signicant antitumor activity, this kind of components attracted so much attention [514].

In our continuous search for the bioactive triterpenoids, six new cycloartane-type triterpenoids, namely 40-O-acetylcimigenol-3-O-b-D-xylopyranoside (1), 20,12-O-diacetyl-25-anhydrocimicigenol-3-O-b-D-xylopyranoside (2), 12bhydroxy-1,19:9,11-didehydro-9,10-seco-cimigenol-3-O-b-

D-xylopy-ranoside (3), (23S, 24R)-12b-hydroxy-7,8-dihydro-12-deacetyl-acetaeaepoxide-3-one. (4), 16,17-dide-hydro-20,24-O-diacetylhydroshengmanol-3-O-b-D-xylopyranoside (5), and (23S,24S,25S)-16,23:23,- 26-diepoxy-24,25-dihydroxy-9,19-cycloart-1,2-en-3,12-dione (6), together with three known analogues, asiaticoside A (7), 24(S)-O-acetylhydroshengmanol-3-O-b-D-xylopyranoside (8), and cimisterol A (9) were isolated from the roots of C. foetida. All the new compounds were evaluated for their cytotoxicities against ve selected human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW480).

2 Results and Discussion

Compound 1 had the molecular formula of C37H58O10,

which was determined by its HR-EIMS at m/z 662.4414[M]?. The IR spectrum showed absorption for hydroxyl

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G.-L. Zhu L.-S. Wan X.-R. Peng N.-M. Bao

Z.-R. Zhang L. Zhou M.-H. Qiu (&)

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences (CAS), Kunming 650201, Peoples Republic of China e-mail: [email protected]

G.-L. Zhu L.-S. Wan X.-R. Peng N.-M. Bao

Graduate University of Chinese Academy of Sciences, Beijing 100049, Peoples Republic of China

D.-F. ZhuFaculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, Yunnan, Peoples Republic of China

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188 G.-L. Zhu et al.

group at 3425 cm-1. The 1H NMR spectrum (Table 1) showed characteristic cyclopropane methylene signals at dH 0.29 and 0.52 (each 1H, d, J = 3.8 Hz), a secondary

methyl at dH 0.86 (d, J = 6.5 Hz), six tertiary methyls at dH 1.01 to 1.52 (each 3H, s), an acetyl group at dH 1.98, and an anomeric proton at dH 4.89 (d, J = 7.6 Hz). The

Table 1 1H (600 MHz) NMR data of compounds 16 in Pyridine-d5 [d in ppm, J in Hz]

No. 1 2 3 4 5 6

1 1.56 m1.69 m

1.07 m1.52 m

1.47 m2.06 m

1.43 m1.71 m

1.20 m1.50 m

6.65 d (10.0)

6.12 d (10.0)

3 3.51 dd (11.7, 4.3) 3.38 dd (11.7, 4.4) 3.57 dd (4.6, 11.5) 3.38 dd (11.7, 4.4) 5 1.30 m 1.27 m 2.04 m 1.57 dd (12.1, 4.2) 1.33 dd (13.0, 2.6) 1.89 dd (12.7, 5.2) 6 0.72 q (12.4)1.51a

0.72 q (12.7)1.53 m

2 1.95 dt (12.6, 3.7)2.32 dt (12.6, 3.7)

1.26 m

1.82 dt (12.6, 3.7)

1.85 m2.36 m

2.30 m2.63 m

1.89 m2.28 m

1.30 m2.68 m

0.99 m1.37 m

0.76 q (13.0)1.55 m

1.32 m

2.32 d (19.7)

0.85 m1.41 m

8 1.69 m 1.73 dd (12.5, 4.4) 2.56 d (10.9) 1.66 dd (12.0, 5.4) 1.88 m 2.32 m 11 1.16a2.11 m

1.13 m

2.94 dd (16.1, 9.5)

7 1.07a

2.10 m

1.08 m2.23 m

2.19 dd (12.4, 4.5)2.25 dd (18.1, 4.5)

0.96 m1.30 m

1.19 m1.55 m

2.33 s

2.92 d (19.9)

5.92 br s 1.49 m2.64 m

1.05 m1.13 m

12 1.69 m1.56 m

5.27 dd (9.3, 2.50) 4.54 m 4.10 m 1.56 m1.90 m

4.75 d (2.9) 1.88 dd (12.7, 5.2)2.32 m

16 5.09 t (8.2) 4.80 q (7.5 Hz) 17 1.52a 1.64a 1.86 d (12.2) 1.95 m 2.33 m18 1.16 s 1.33 s 1.16 s 1.51 s 1.27 s 1.38 s19 0.29 d (3.8)0.52 d (3.8)

0.26 d (4.2)0.53 d (4.2)

15 4.29 br s 4.47 d (8.8) 4.59 d (6.4) 1.84 m1.96 m

0.87 d (4.8)1.14 d (4.8)

20 1.69 m 1.64 br s 1.79 m 2.41 m 2.56 m 2.33 m21 0.86 d (6.5) 0.94 d (6.0) 1.42 d (6.4) 1.80 d (6.1) 1.00 d (6.7) 1.32 d (6.2) 22 1.03 m2.27 dt (7.1, 2.2)

1.00 s2.24 m

6.11 br s 0.48 d (4.3)0.70 d (4.3)

0.17 d (3.8)0.56 d (3.8)

1.13 m2.39 m

4.02 d (10.6) 1.85 m2.05 m

2.08 m

23 4.79 d (9.0) 4.31 m 4.80 d (8.7) 4.60 d (11.8) 24 3.80 br s 4.19 br s 3.85 s 4.26 br s 5.43 d (2.5) 4.53 d (6.6) 26 1.52 s 5.39 s4.90 s

1.53 s 1.72 s 1.60 s 4.31 d (8.8)4.38 d (8.8)

27 1.32 s 1.86 s 1.54 s 1.80 s 1.63 s 1.97 s 28 1.01 s 1.33 s 1.15 s 0.85 s 0.99 s 0.67 s 29 1.16 s 1.10 s 1.34 s 1.23 s 1.04 s 1.23 s 30 1.06 s 0.96 s 0.95 s 0.97 s 1.10 q 0.97 s AcO-12 2.15 sAcO-24 2.09 sSugar10 4.89 d (7.6) 4.83 d (8.0) 4.85 d (7.6) 4.86 d (8.0 Hz)

20 4.08 t (8.0) 5.59 t (8.0) 4.03 m 5.60 t (8.0 Hz)

30 4.31 t (8.0) 4.19 m 4.19 t (8.8) 4.21 m

40 5.44 dt (11.3, 5.4) 4.21 m 4.25 m 4.25 dd (13.9, 5.1)

50 3.62 t (10.7)4.32 dd (11.3, 5.4)

3.68 t (11.1)4.31 m

3.76 t (8.8)4.39 t (8.8)

3.75 t (11.1 Hz)4.36 dd (11.1, 5.0)

AcO-20 2.14 s 2.17 s AcO-40 1.98 s

a Signals overlapped

123

Six New 9,19-Cycloartane Triterpenoids from Cimicifuga foetida L. 189

13C NMR and DEPT spectroscopic data (Table 2) of 1 displayed a characteristic cimigenol-type triterpenoid carbon resonances, corresponding to the methylene carbon of

the cyclopropane ring at dC 31.2 (C-19), four oxymethine carbons at dC 89.0 (C-3), 80.6 (C-15), 72.2 (C-23), and90.5 (C-24), and two oxygened quaternary carbons at dC 112.3 and 71.3. Apart from above data, a glycosidic moiety signals [dC 107.7 (d), 76.1 (d), 75.3 (d), 73.5 (d) and 63.5(t)] were also obseverd in its 13C NMR spectrum. These data showed similarities as those of cimigenol-3-O-[20-O-

acetyl]-b-D-xylopyranoside [12]. However, a detailed comparison of their 1D NMR spectra revealed that they had different sugar unit. The upeld shift of H-20 (dH

5.56 ? dH 4.08) and the downeld shift of H-40 (dH4.30 ? dH 5.44) in their 1H NMR spectra, along with the HMBC correlation (Fig. 1) of H-40 (dH 5.44) with the carbonyl group of the acetoxyl group (dC 171.0) and of the anomeric proton with C-3, indicated that a 40-O-acetylxylopyranosyl at C-3 in 1 replaced the 20-O-acetyl-xylopyranosyl in cimigenol-3-O-[20-O-acetyl]-b-D-xylopyranoside. The sugar unit of 1 was further conrmed by comparing its TLC and specic rotation with a standard after acid hydrolysis. Thus, the planar structure of 1 was determined.

In the ROESY spectrum (Fig. 1), correlations of H-3/H-5 and H-15/H3-18 suggested that H-3 and H-15 were a- and b-oriented, respectively. Moreover, the congurations of C-23 and C-24 were assigned as R and S, respectively, by comparing the coupling constants of H-23 (9.0 Hz) and H-24 (0 Hz) of 1 with those of known compounds [15]. Therefore, compound 1 was established to be 40-O-

acetylcimigenol-3-O-b-D-xylopyranoside.

Compound 2 was obtained as a white powder and gave a molecular formula of C39H58O11 by its HR-EIMS (m/z 702.3974 [M]?). The 1H and 13C NMR spectra (Tables 1, 2)

of 2 were very similar to those of 25-anhydrocimicigenol-3-O-[20-O-acetyl]-b-D-xylopyranoside [16], with the exception of an additional acetyl group, which was assigned to

C-12 on the basis of the HMBC correlation of H-12 (dH 5.27) with the acetyl carbonyl carbon at dC 170.9 and the 1H-1H

COSY cross peak of H-12 (dH 1.13) with H-11 (dH 2.94). Signicant ROESY correlation of H-12 with H-17 indicated a b-orientation of the substituent acetyl group at C-12.

Therefore, the structure of 2 was determined as 20,12-O-diacetyl-25-anhydrocimicigenol-3-O-b-D-xylopyranoside.

Compound 3 was isolated as a white powder, showing [M ? Na]? ion at m/z 657.3602 in the HR-ESIMS consistent with the empirical molecular formula C35H54O10

(calcd 657.3615), requiring 9 sites of unsaturation. The IR and UV spectra exhibited absorption bands for hydroxyl group (3431 cm-1) and conjugated double bond (kmax 249 nm; 1631 cm-1), respectively. The

Table 2 13C (150 MHz) NMR data of compounds 16 [d in ppm, J in Hz]

No. 1 2 3 4 5 6

1 32.7 t 32.5 t 40.6 t 33.7 t 32.0 t 153.5 d

2 30.4 t 30.3 t 32.6 t 37.8 t 30.2 t 127.2 d

3 89.0 d 88.7 d 87.8 d 215.3 s 88.9 d 204.3 s

4 41.7 s 41.3 s 42.7 s 50.4 s 41.3 s 46.6 s

5 47.9 d 47.3 d 51.1 d 48.4 d 47.8 d 47.2 d

6 21.4 t 21.1 t 25.0 t 21.4 t 21.3 t 25.7 t

7 26.7 t 26.3 t 30.6 t 26.1 t 30.3 t 20.3 t

8 49.0 d 47.6 d 50.4 d 46.1 d 47.1 d 45.7 d

9 20.3 s 20.5 s 140.3 s 21.9 s 20.2 s 25.8 s

10 26.9 s 27.0 s 138.7 s 26.6 s 27.4 s 30.6 s

11 26.7 t 37.8 t 135.9 d 41.0 t 26.7 t 46.1 t

12 34.4 t 77.6 d 46.7 s 72.6 d 30.3 t 210.8 s

13 42.2 s 46.3 s 46.7 s 51.5 s 47.5 s 47.5 s

14 47.6 s 48.6 s 49.8 s 48.2 s 49.9 s 60.8 s

15 80.6 d 79.6 d 79.2 d 44.0 t 79.3 d 46.1 t 16 112.3 s 112.6 s 112.7 s 72.8 d 151.3 s 73.0 d

17 59.9 d 59.9 d 58.7 d 53.3 d 121.7 s 48.7 d

18 19.0 q 13.1 q 11.9 q 13.7 q 24.6 q 14.6 q

19 31.2 t 31.2 t 129.6 d 29.6 t 32.4 t 32.6 t

20 24.4 d 24.3 d 24.0 d 35.1 d 27.9 d 25.7 d

21 19.9 q 20.1 q 21.2 q 18.8 q 20.7 q 21.8 q

22 38.5 t 38.8 t 38.9 t 87.9 d 37.2 t 41.3 t

23 72.2 d 74.9 d 72.4 d 106.0 s 77.0 d 106.9 s

24 90.5 d 86.9 d 90.6 d 83.8 d 80.2 d 86.3 d

25 71.3 s 146.1 s 71.4 s 83.9 s 72.3 s 78.2 s

26 24.3 q 113.5 t 27.6 q 25.3 q 27.1 q 78.9 t

27 25.7 q 18.5 q 26.0 q 28.3 q 28.5 q 23.6 q

28 12.2 q 12.4 q 10.4 q 21.1 q 15.6 q 20.3 q

29 26.0 q 25.7 q 25.0 q 22.9 q 25.7 q 22.2 q

30 15.7 q 15.5 q 15.4 q 20.1 q 14.6 q 19.6 q AcO-12 21.6 q

170.9 s

AcO-24 21.3 q

171.4 s

Sugar

10 107.7 d 105.0 d 108.0 d 105.0 d

20 76.1 d 76.0 d 76.1 d 76.0 d

30 75.3 d 76.6 d 79.2 d 76.6 d

40 73.5 d 71.9 d 71.7 d 71.7 d

50 63.5 t 67.4 t 67.7 t 67.5 t

AcO-20 170.4 1 170.4 s22.0 21.6 q

AcO-40 21.2 q 171.0 s

1H NMR

spectrum exhibited a sec-methyl signal at dH 1.42 (3H, d, J = 6.4 Hz), six tert-methyls at dH 0.951.54 (each 3H, s), two olenic methine signals at dH 5.92 (1H, br s) and 6.11 (1H, br s), and an anomeric methine signal at dH 4.85 (1H,

123

190 G.-L. Zhu et al.

Fig. 1 Major correlations in 2D NMR spectra of compound 1

d, J = 7.6 Hz). The 13C NMR showed 35 carbon resonances (Table 2), of which 30 were attributed to a triterpene skeleton and ve to a pentose. A DEPT NMR experiment permitted differentiation of the 30 carbon signals into seven methyls, ve methylenes, eleven methines (including ve oxygenated and two olenic signals), and seven quaternary carbons (including two oxygenated and two olenic signals). The diagnostic signals of two oxygen-bearing methine carbons at dC 90.6 (C-24) and 70.4 (C-23), and a ketal carbon at dC at 112.7 suggested that 3 was a cimigenol-type triterpene compound. Further inspection of the 1D NMR and HSQC spectra of 3, the characteristic cyclopropane methylene resonances H2-19 and two quaternary carbons (C-9 and C-10) were not observed at the characteristic high magnetic eld. Besides, comparison the NMR spectra of 3 with those of 12b-hydroxycimigenol-3-

O-b-D-xylopyranoside [17], the signals due to C-9, C-10, C-11, and C-19 showed a downeld shift from dC 20.5,26.1, 40.6, and 30.8 to 140.3, 138.7, 135.9, and 129.6, respectively, in 3. Such evidences indicated that 3 was a 9,10-seco-9,19-cyclolanostane glycoside with two double bonds. And the location of the double bonds (C10=C19 and

C9=C11) could be further deduced. This was further supported by IR, UV and 2D NMR spectra (Fig. 2). Furthermore, the congurations of C-23 and C-24 were assigned as R and S, respectively, by the same way as 1. Ultimately, the structure of 3 was determined as 12b-hydroxy-10,19:9,11-didehydro-9,10-seco-cimigenol-3-O-b-D-xylopyranoside.

The molecular of compound 4 was assigned as C30H46O6 by HR-EIMS at m/z 502.3294 [M]?. The 1D

NMR data of 4 (Tables 1, 2) showed that 4 was a highly oxygenated 9,19-cycloartane triterpene and resembled that of the aglycone of 7,8-dihydroactaeaepoxide-3-O-b-D-xylopyranoside [18]. However, the signals for the oxymethine at C-3 and the acetoxyl group at C-12 were absent. Instead a carbonyl group signal at dC 215.3 and an upeld oxymethine at dC 72.6 were observed, which indicated that the oxymethine (C-3) and the acetoxyl group (C-12) were replaced by a carbonyl group and a hydroxyl group,

respectively. The evidence was established from HMBC correlations (Fig. 2) of H3-29 (dH 1.23) and H3-30 (dH0.94) with C-3 (dC 215.3), of H3-18 (dH 1.51) with C-12 (dC 72.6), together with the

1H-1H COSY correlations (Fig. 2) of H-1 (dH 1.43, 1.71)/H-2 (dH 2.30, 2.63), H-5 (dH1.57)/H-6 (dH 0.99, 1.37)/H-7 (dH 0.96, 1.30)/H-8 (dH1.66), H-11 (dH 1.49, 2.64)/H-12 (dH 4.10), H-15 (dH 1.84,1.96)/H-16(dH 5.09)/H-17 (dH 1.53). Thus, the planar structure of 4 was elucidated. Additionally, the substituent hydroxyl group at C-12 was assigned as b orientation by the ROESY correlation of H3-28 with H-12. Further inspection of the ROESY spectrum, the correlations of H-16 with H-22 and H-24, of H-22 with H-12, H-21, and H-28, and of H-24 with H-16, suggested that an a orientation of H-16, H-22, H-12, and H-24, respectively. So the conguration of C-23 was elucidated as S since C-23 and C-24 formed an oxirane. Ultimately, compound 4 was elucidated as (23S, 24R)-12b-hydroxy-7,8-dihydro-12-deacetylacetaeaepoxide-3-one.

Compound 5 was determined to have the molecular formula of C39H60O11 by its HR-EIMS (m/z 704.4138 [M]?).

The 1H NMR spectrum (Table 1) displayed characteristic cyclopropane methylene signals at dH 0.17 and 0.56 (each 1H, d, J = 3.8 Hz), a sec-methyl at dH 1.00 (3H, d,

Fig. 2 Key HMBC ( ) and 1H-1H COSY ( ) correlations of compounds 3, 4 and 6

123

Six New 9,19-Cycloartane Triterpenoids from Cimicifuga foetida L. 191

J = 6.7 Hz), six tert-methyls at dH 0.991.63 (each 3H, s), an acetyl methyl group at dH 2.09 (3H, s), and an anomeric proton signal at dH 4.86 (1H, d, J = 8.0 Hz), respectively, suggersting 5 to be a 9,19-cycloartane triterpene glycoside with a substituent acetyl group. The NMR data (Tables 1, 2) of 5 resembled those of 25-O-methyl-24-O-acetylhydroshengmanol-3-O-b-D-xylopyranoside [19], except for one more acetyl group for the sugar unit, one less substituent methoxy group at C-25, and the presence of two downeld signals at dC 151.3 and 121.7 while the absence of an oxygen-bearing quaternary carbon and a methine resonance due to

C-16 and C-17, respectively. On the basis of these observations, it was reasonable to deduce that 5 was a 16,17-dehydrated derivative with an additional acetyl group and a skimpy methoxy group of 25-O-methyl-24-O-acetylhydroshengmanol-3-O-b-D-xylopyranoside. As for 2, an acetyl group was determined to be at C-20 for 5, which was further conrmed by the HMBC correlation between the H-20 signal at dH 5.20 and the carbonyl group signal at dC 170.4. In the NMR spectra, the signals of the methoxy appeared in 25-

O-methyl-24-O-acetylhydro-shengmanol-3-O-b-D-xylopyranoside couldnt be observed in 5, while the chemical shift of C-25 shifted upeld from dC 76.0 to 72.3. This suggested the methoxy group located at C-25 wasnt appeared in 5.

Additionally, the location of the double bond was further conrmed by the HMBC correlations of H-18 (dH 1.27) and

H-21 (dH 1.00) with dC 121.7, of H-15 (dH 4.75) with dC 151.3 and 121.7, and of H-23 (dH 4.60) with dC 151.3, respectively. The conguration of C-23 could be determined as b by ROESY correlation of H-23 with H-20. And the conguration of C-24 was assigned as S by comparison the coupling constant of H-24 (J = 2.5 Hz) with those of dahurinyl deacetate (J = 9 Hz, 24R) and isodahurinyl deacetate (J = 2.0 Hz, 24S) [20]. Therefore, 5 was elucidated as 16,17-didehydro-20,24-O-diacetyl- hydroshengmanol-3-O-b-D-xylopyranoside.

Compound 6 was isolated as a white powder. Its molecular formula (C30H42O6) was deduced from HR

EIMS (m/z 498.2991 [M]?), corresponding to nine degrees of unsaturation. The 1H and 13C NMR spectroscopic data (Tables 1, 2) of 6 showed similarities with those of yunnanterpene A [21], except for the differences of rings A and C, and the chemical shifts of C-22, C-23, and C-24. Two methylene signals due to C-1 at dC 33.3 and C-2 at dC37.4 appeared in the ring A of yunnanterpene A were absent from the 13C-DEPT spectrum of 6, respectively. Instead, two olenic carbon signals at dC 153.5 and 127.2 were observed. Besides, the signal due to C-3 showed an upeld shift from dC 215.0 to 203.4. These evidences suggested the double bond was located at C-1 and C-2, which was further conrmed by the UV, IR (kmax 262 nm;

1669 cm-1), and the HMBC correlations (Fig. 2) of the olenic protons at dH 6.65 and 6.12 with the carbonyl

carbon signals at dC 204.3 (C-3), The other changes of the ring C was that the methine signal at dC 72.1 (C-12)

appeared in yunnanterpene A was absent instead of a quarternary carbonyl carbon signal at dC 210.8 (C-12) in 6.

Meanwhile, the 13C NMR signal due to C-11 showed a downeld shift from dC 40.6 in yunnanterpene A to 46.1 in 6, and the signal due to C-13 exhibited an uneld from dC50.5 to 46.6, respectively. These observations indicated that the hydroxyl group was attached to C-12 was replaced by the carbonyl group, which was further conrmed by the HMBC correlations (Fig. 2) from H-11 (dH 2.33 and 2.92)

and H3-18 (dH 1.38) to C-12 (dC 210.8), respectively. All of the above observations were consistent with the HSQC, HMBC, and 1H-1H COSY correlations (Fig. 2). Besides, the downeld shift of C-22 and C-24 from dC 37.5 and 83.7 to 41.3 and 86.3 and the upeld of C-23 from dC 110.9 to 106.9 suggested the congurations of two compounds may be different. Furthermore, the diagnostic ROESY congurations of H-16 with H-16, H3-28 and H3-27, of H-24 with

H-26a, of H3-27 with H-16 and H-26a, indicated that the a-orientation of H-16, H3-27, and H-24. And the conguration of C-23 was identied as S by comparision the chemical shifts of C-16 (dC 73.0) and C-20 (dC 25.7) with the 26-deoxyactein compound [C-16 (dC 73.0) and C-20 (dC 26.0)] [16] and 23-epi-26-deoxyactein [C-16 (dC 74.5)

and C-20 (dC 23.0)] [22]. Hence, compound 6 was determined as (23S,24S,25S)-16,23:23,26-diepoxy-24,25-dihydroxy-9,19-cycloart-1,2-en-3,12-dione.

Three known compounds asiaticoside A (7) [23], 24(S)-O-acetylhydroshengmanol-3-O-b-D-xylopyranoside (8)

[19], and cimisterol A (9) [13] were also isolated from this species. Their structures were identied by its 1D NMR spectra as well as comparison with reported data.

Compounds 16 isolated in the present study were evaluated for their cytotoxicities against ve human cancer cell lines using MTT method, with cisplatin and taxol as the positive control. Unfortunately, none of them showed signicant activity [24].

3 Experiments Section

3.1 General Experimental Procedures

Optical rotations were measured in MeOH with a Horiba SEAP-300 polarimeter. 1H and 13C NMR spectra were recorded in pyridine-d5 on Bruker Avance III-600 MHz spectrometers (Bruker, Zrich, Switzerland), using TMS as internal standard for chemical shifts. Chemical shifts (d)

were expressed in ppm with reference to the TMS resonance. ESIMS, HRTOF-ESIMS and EIMS, HR-EIMS data were obtained using a VG Autospec-3000 and API QSTAR TOF spectrometer, respectively. Infrared spectra were

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192 G.-L. Zhu et al.

recorded on a Shimadzu IR-450 instrument with KBr pel-lets. CD was detected with a Chirascan circular dichroism spectrograph (Applied Photophysis, England). Thin-layer chromatography was performed on precoated TLC plates (Silica gel GF254, Qingdao Marine Chemical, Inc.) and spots were visualized by heating after spraying with 10 % H2SO4 in EtOH. Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph with a YMC-Pack Pro C18 RS 10 mm 9 250 mm column. Silica gel (mesh 200300, Qingdao Marine Chemical, Inc.), Lichroprep RP-18 (4063 lm, Merck), Amberlite IR-35 (10 mL) column and Sephadex LH-20 (Pharmacia) were used for column chromatography.

3.2 Plant Materials

The roots of Cimicifuga foetida (82 kg) were collected from Yulong County, Yunnan Province, in September 2010 and identied by Professor Pei shengji, Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (KUN No. 20100906) 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.

3.3 Extraction and Isolation

The air-dried roots of C. foetida (82 kg) were crushed with a blender and reuxed with 95 % EtOH at 70 C for three times (5 h, each). The residue was yielded by removal of the solvent was dissolved in water to form a suspension. The aqueous suspension was successively partitioned with EtOAc and n-BuOH. The EtOAc (5.6 kg) fraction was absorbed on 12 kg silica gel and chromatographed on a prepacked (120 kg) silica gel column, eluting stepwise with CHCl3MeOH (1:0, 100:1, 50:1, 20:1, 5:1) to give ve fractions (AE) Fraction C (230 g) was subjected to column chromatograph (CC) on silica gel (1.5 kg) and eluted with PE-Me2CO (5:1, 2:1, 0:1) to obtain C-1 (60 g),

C-2 (40 g), and C-3 (105 g) as in the previous report [25]. Fraction C-2 (40 g) was puried using an ODS silica gel column with MeOH-H2O (60:40, 80:20, 100:0), followed by purication using preparative HPLC eluted with CH3CN-H2O (65:35), furnished compound 4 (2.3 mg).

Similarly, using CH3CN-H2O (70:30) as eluent with a ow rate of 3 ml/min, compound 6 (9.6 mg) was puried from C-3 (105 g). Fraction D (200 g) was separated on silica gel eluted with CHCl3Me2CO (gradient polarity from 15:1 to 5:1) to give ten subfractions (D-1-D-10). Fraction D-1 (10 g) was separated by CC (ODS silica gel) with MeOH H2O (gradient polarity from 60:40 to 90:10) and puried by

HPLC eluting with CH3CNH2O (60:40, ow rate of 3 ml/min) to obtain 3 (6.0 mg) and 9 (2.5 mg) with retention times of 9.30 and 18.50 min, respectively. Sub-fraction D-6 (15 g) was subjected to silica gel (CH3Cl3

MeOH, gradient from 25:1 to 15:1) and then puried by an ODS silica gel column (MeOHH2O, 70:30 to 100:0)

and HPLC spectrum eluting with (CH3CNH2O, 50:50, ow rate of 3 ml/min) to obtain 1 (6.2 mg) and 5(5.1 mg) with retention times of 10.80 and 13.40 min, respectively. Sub-fraction D-8 (20 g) was chromatographed on a silica gel (CH3Cl3MeOH, 30:1, 20:1, 15:1) and ODS (MeOHH2O, 70:30 to 100:1), followed puried on HPLC CH3CNH2O (67:33, ow rate of 3 ml/

min) to yield 2 (5.3 mg), 7 (5.5 mg), and 8 (6.0 mg) with the retention times of 19.20, 26.70, and 12.40 min, respectively.

3.4 40-O-Acetylcimigenol-3-O-b-D-xylopyranoside (1)

White powder; a 20D -3.59 (c 0.2, MeOH); IR (KBr) mmax

3425, 2964, 2936, 2870, 1742, 1626, 1458, 1413, 1379, 1308, 1252, 1170, 1047, 979 cm-1; 1H and 13C NMR data see Tables 1, 2; HREIMS m/z 662.4414 [M]? (calcd for 662.4394).

3.5 20,12-O-Diacetyl-25-anhydrocimicigenol-3-O-b-D-xylopyranoside (2)

White powder; a 20D -1.63 (c 1.8, MeOH); IR (KBr) mmax

3442, 2935, 2871, 1736, 1629, 1455, 1413, 1239, 1159, 1071, 1044, 982 cm-1; 1H and 13C NMR data see Tables 1, 2; HREIMS m/z 702.3974 [M]? (calcd for 702.3979).

3.6 12b-Hydroxy-1,19:9,11-didehydro-9,10-secocimigenol-3-O-b-D-xylopyranoside (3)

White powder; a 20D -1.63 (c 0.01, MeOH); IR (KBr) mmax

3431, 2931, 2873, 1631, 1456, 1384, 1238, 1161, 1041, 975 cm-1; 1H (C5D5N, 600 MHz) and 13C NMR (C5D5N, 150 MHz) data see Tables 1, 2; ESIMS m/z 657 [M ? Na]?; HRESIMS m/z 657.3602 (calcd for 657.3615).

3.7 (23S,24R)-12b-Hydroxy-7,8-dihydro-12-deacetylacetaeaepoxide-3-one. (4)

White powder; a 20D -18.17 (c 1.1, MeOH); IR (KBr) mmax

3441, 2966, 2932, 2875, 1705, 1628, 1465, 1383, 1248, 1061 cm-1; 1H and 13C NMR data see Tables 1, 2; HREIMS m/z 502.3294 [M]? (calcd for 502.3294).

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Six New 9,19-Cycloartane Triterpenoids from Cimicifuga foetida L. 193

3.8 16,17-Didehydro-20,24-O-diacetylhydroshengmanol-3-O-b-D-xylopyranoside (5)

White powder; a 20D -10.28 (c 0.01, MeOH); IR (KBr)

mmax 3431, 2929, 2870, 2853, 1739, 1629, 1459, 1375, 1237, 1240, 1167, 1125, 1072, 1046, 980 cm-1; 1H and 13C

NMR data see Tables 1, 2; HREIMS m/z 704.4138 [M]? (calcd for 704.4136).

3.9 (23S,24S,25S)-16,23:23,26-Diepoxy-24,25-diihydroxy-9,19-cycloart-1,2-en-3,12-dione (6)

White powder; a 20D -24.21 (c 0.5, MeOH); IR (KBr) mmax

3440, 2931, 2871, 1712, 1669, 1457, 1380, 1278, 1167, 1019, 940 cm-1; 1H and 13C NMR data see Tables 1, 2; HRESIMS m/z 498.2991 [M]? (calcd for 498.2981).

3.10 Acidic Hydrolysis of 1, 2 and 5

The new compounds 1, 2 and 5 (4 mg of each) were dissolved in MeOH (5 mL) and reuxed with 0.5 N HCl (3 mL) for 4 h. Each reaction mixture was diluted with H2O and extracted with CHCl3 (3 9 10 mL). The water layer was then neutralized by Ag2CO3, and the precipitate ltered to give a monosaccharide. Each monosaccharide of those compounds had an Rf (EtOAc/CHCl3/MeOH/H2O, 3:2:2:1) and specic rotation a 20D ?24.3 (c 0.10, H2O)

corresponding to those of D-xylose (Sigma-Aldrich).

Acknowledgments This project was supported by Program for National Natural Science Foundation of China (Nos. U1132604 and 81302670), The Major Deployment Program of the CAS (No. KSZDEW-Z-004-03), and Foundation of State Key Laboratory of Phytochemistry and Plant Resources in West China (P2008-ZZ05).

Compliance with Ethical Standards

Conict of Interest All authors declare no conict of interest.

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/

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