Scholarly article on topic 'Six New 9,19-Cycloartane Triterpenoids from Cimicifuga foetida L.'

Six New 9,19-Cycloartane Triterpenoids from Cimicifuga foetida L. Academic research paper on "Chemical sciences"

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Academic research paper on topic "Six New 9,19-Cycloartane Triterpenoids from Cimicifuga foetida L."

Nat. Prod. Bioprospect. CrossMark

DOI 10.1007/sl3659-016-0097-3

ORIGINAL ARTICLE

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

© 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 (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).

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 inflammation since ancient times in China [1-3]. 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

Electronic supplementary material The online version of this article (doi: 10.1007/s13659-016-0097-3) contains supplementary material, which is available to authorized users.

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, People's Republic of China e-mail: mhchiu@mail.kib.ac.cn

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

Graduate University of Chinese Academy of Sciences, Beijing

100049, People's Republic of China

D.-F. Zhu

Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, Yunnan, People's Republic of China

triterpenoids and their glycosides are the main constituents of Cimicifuga. Meanwhile, because of their structural diversity and significant antitumor activity, this kind of components attracted so much attention [5-14].

In our continuous search for the bioactive triterpenoids, six new cycloartane-type triterpenoids, namely 4-O-acetyl-cimigenol-3-O-b-D-xylopyranoside (1), 20,12-O-diacetyl-25-anhydrocimicigenol-3-O-b-D-xylopyranoside (2), 12b-hydroxy-1,19:9,11 -didehydro-9,10-seco-cimigenol- 3-O-ft-D-xylopy-ranoside (3), (23S, 24R)-12b-hydroxy-7,8-dihy-dro-12-deacetyl-acetaeaepoxide-3-one. (4), 16,17-dide-hydro-20,24- O-diacetylhydroshengmanol- 3- O-b-D-xylopy-ranoside (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 five 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

Published online: 20 May 2016

1 Springer

group at 3425 cm- _1. The NMR spectrum (Table 1) methyl at dH 0.86 (d, J = 6.5 Hz), six tertiary methyls at showed characteristic cyclopropane methylene signals at dH 1.01 to 1.52 (each 3H, s), an acetyl group at dH 1.98, dH 0.29 and 0.52 (each 1H, d, J = 3.8 Hz), a secondary and an anomeric proton at dH 4.89 (d, J = 7.6 Hz). The

Table 1 1H (600 MHz) NMR data of compounds 1-6 in Pyridine-d5 [d in ppm, J in Hz]

No. 1 2 3 4 5 6

1 1.56 m 1.07 m 1.47 m 1.43 m 1.20 m 6.65 d (10.0)

1.69 m 1.52 m 2.06 m 1.71 m 1.50 m

2 1.95 dt (12.6, 3.7) 1.26 m 1.85 m 2.30 m 1.89 m 6.12 d (10.0)

2.32 dt (12.6, 3.7) 1.82 dt (12.6, 3.7) 2.36 m 2.63 m 2.28 m

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) 0.72 q (12.7) 1.30m 0.99 m 0.76 q (13.0) 1.32 m

1.51a 1.53 m 2.68 m 1.37 m 1.55 m 2.32 d (19.7)

7 1.07a 1.08 m 2.19 dd (12.4, 4.5) 0.96 m 1.19 m 0.85 m

2.10 m 2.23 m 2.25 dd (18.1, 4.5) 1.30 m 1.55 m 1.41 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 1.16a 1.13 m 5.92 br s 1.49 m 1.05 m 2.33 s

2.11 m 2.94 dd (16.1, 9.5) 2.64 m 1.13 m 2.92 d (19.9)

12 1.69 m 5.27 dd (9.3, 2.50) 4.54 m 4.10 m 1.56 m -

1.56 m 1.90 m

15 4.29 br s 4.47 d (8.8) 4.59 d (6.4) 1.84 m 4.75 d (2.9) 1.88 dd (12.7, 5.2)

1.96 m 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 m

18 1.16 s 1.33 s 1.16 s 1.51 s 1.27 s 1.38 s

19 0.29 d (3.8) 0.26 d (4.2) 6.11 br s 0.48 d (4.3) 0.17 d (3.8) 0.87 d (4.8)

0.52 d (3.8) 0.53 d (4.2) 0.70 d (4.3) 0.56 d (3.8) 1.14 d (4.8)

20 1.69 m 1.64 br s 1.79 m 2.41 m 2.56 m 2.33 m

21 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 m 1.00 s 1.13 m 4.02 d (10.6) 1.85 m 2.08 m

2.27 dt (7.1, 2.2) 2.24 m 2.39 m 2.05 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 s 1.53 s 1.72 s 1.60 s 4.31 d (8.8)

4.90 s 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 s

AcO-24 2.09 s

10 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) 3.68 t (11.1) 3.76 t (8.8) 3.75 t (11.1 Hz)

4.32 dd (11.3, 5.4) 4.31 m 4.39 t (8.8) 4.36 dd (11.1, 5.0)

AcO-20 2.14 s 2.17 s

AcO-40 1.98 s

a Signals overlapped

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

Table 2 13C (150 MHz) NMR data of compounds 1-6 [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

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-2' 170.4 1 22.0 170.4 s 21.6 q

AcO-4' 21.2 q 171.0 s

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), and 90.5 (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-[2'-O-acetyl]-b-D-xylopyranoside [12]. However, a detailed comparison of their 1D NMR spectra revealed that they had different sugar unit. The upfield shift of H-2' (dH 5.56 ? dH 4.08) and the downfield shift of H-4' (dH 4.30 ? dH 5.44) in their :H NMR spectra, along with the HMBC correlation (Fig. 1) of H-4' (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 4'-O-acetyl-xylopyranosyl at C-3 in 1 replaced the 2'-O-acetyl-xy-lopyranosyl in cimigenol-3-O-[2'-O-acetyl]-b-D-xylopyra-noside. The sugar unit of 1 was further confirmed by comparing its TLC and specific 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 configurations 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 4'-O-acetylcimigenol-3-O-b-D-xylopyranoside.

Compound 2 was obtained as a white powder and gave a molecular formula of C39H58On by its HR-EIMS (m/z 702.3974 [M]?). The :H and 13C NMR spectra (Tables 1, 2) of 2 were very similar to those of 25-anhydrocimicigenol-3-O-[2'-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 :H-:H COSY cross peak of H-12 (dH 1.13) with H-11 (dH 2.94). Significant 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 2',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- ) and conjugated double bond (kmax 249 nm; 1631cm-1), respectively. The :H NMR spectrum exhibited a sec-methyl signal at dH 1.42 (3H, d, J = 6.4 Hz), six tert-methyls at dH 0.95-1.54 (each 3H, s), two olefinic 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,

'H-'H COSY:

ROESY: A"*

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 triter-pene skeleton and five to a pentose. A DEPT NMR experiment permitted differentiation of the 30 carbon signals into seven methyls, five methylenes, eleven methines (including five oxygenated and two olefinic signals), and seven quaternary carbons (including two oxygenated and two olefinic 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 field. 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 downfield 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 (Ci0=C19 and C9=C11) could be further deduced. This was further supported by IR, UV and 2D NMR spectra (Fig. 2). Furthermore, the configurations 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-3-0-b-D-xylopy-ranoside.

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-xy-lopyranoside [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 upfield 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,

Fig. 2 Key HMBC (-»-) and 1H-1H COSY (

of compounds 3, 4 and 6

) correlations

respectively. The evidence was established from HMBC correlations (Fig. 2) of H3-29 (dH 1.23) and H3-30 (dH 0.94) with C-3 (dC 215.3), of H3-18 (dH 1.51) with C-12 (dC 72.6), together with the :H-:H COSY correlations (Fig. 2) of H-1 (dH 1.43, 1.71)/H-2 (dH 2.30, 2.63), H-5 (dH 1.57)/H-6 (dH 0.99, 1.37)/H-7 (dH 0.96, 1.30)/H-8 (dH 1.66), 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 configuration 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 C39H60On 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,

J = 6.7 Hz), six tert-methyls at dH 0.99-1.63 (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-acetylhy-droshengmanol-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 downfield 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-dehy-drated derivative with an additional acetyl group and a skimpy methoxy group of 25-O-methyl-24-O-acetylhy-droshengmanol-3-O-b-D-xylopyranoside. As for 2, an acetyl group was determined to be at C-20 for 5, which was further confirmed by the HMBC correlation between the H-2' 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-xylopy-ranoside couldn't be observed in 5, while the chemical shift of C-25 shifted upfield from dC 76.0 to 72.3. This suggested the methoxy group located at C-25 wasn't appeared in 5. Additionally, the location of the double bond was further confirmed 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 configuration of C-23 could be determined as b by ROESY correlation of H-23 with H-20. And the configuration 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-2',24-O-diacetyl- hydrosheng-manol-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 yun-nanterpene 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 dC 37.4 appeared in the ring A of yunnanterpene A were absent from the 13C-DEPT spectrum of 6, respectively. Instead, two olefinic carbon signals at dC 153.5 and 127.2 were observed. Besides, the signal due to C-3 showed an upfield 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 confirmed by the UV, IR (kmax 262 nm; 1669 cm-1), and the HMBC correlations (Fig. 2) of the olefinic 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 downfield shift from dC 40.6 in yunnanterpene A to 46.1 in 6, and the signal due to C-13 exhibited an unfield from dC 50.5 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 confirmed 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 downfield shift of C-22 and C-24 from dC 37.5 and 83.7 to 41.3 and 86.3 and the upfield of C-23 from dC 110.9 to 106.9 suggested the configurations of two compounds may be different. Furthermore, the diagnostic ROESY configurations 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 configuration of C-23 was identified 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-dihy-droxy-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 identified by its 1D NMR spectra as well as comparison with reported data.

Compounds 1-6 isolated in the present study were evaluated for their cytotoxicities against five human cancer cell lines using MTT method, with cisplatin and taxol as the positive control. Unfortunately, none of them showed significant activity [24].

3 Experiments Section

3.1 General Experimental Procedures

Optical rotations were measured in MeOH with a Horiba SEAP-300 polarimeter. :H and 13C NMR spectra were recorded in pyridine-d5 on Bruker Avance III-600 MHz spectrometers (Bruker, Zurich, 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

recorded on a Shimadzu IR-450 instrument with KBr pellets. 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 x 250 mm column. Silica gel (mesh 200-300, Qingdao Marine Chemical, Inc.), Lichroprep RP-18 (40-63 im, 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 identified 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 refluxed 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 CHCl3-MeOH (1:0, 100:1, 50:1, 20:1, 5:1) to give five fractions (A-E) 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 purified using an ODS silica gel column with MeOH-H2O (60:40, 80:20, 100:0), followed by purification 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 flow rate of 3 ml/min, compound 6 (9.6 mg) was purified from C-3 (105 g). Fraction D (200 g) was separated on silica gel eluted with CHCl3-Me2CO (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 purified by

HPLC eluting with CH3CN-H2O (60:40, flow 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. Subfraction D-6 (15 g) was subjected to silica gel (CH3Cl3-MeOH, gradient from 25:1 to 15:1) and then purified by an ODS silica gel column (MeOH-H2O, 70:30 to 100:0) and HPLC spectrum eluting with (CH3CN-H2O, 50:50, flow 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 chro-matographed on a silica gel (CH3Cl3-MeOH, 30:1, 20:1, 15:1) and ODS (MeOH-H2O, 70:30 to 100:1), followed purified on HPLC CH3CN-H2O (67:33, flow 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 4'-O-Acetylcimigenol-3-O-b-D-xylopyranoside (1)

White powder; [a]D -3.59 (c 0.2, MeOH); IR (KBr) vmax 3425, 2964, 2936, 2870, 1742, 1626, 1458, 1413, 1379, 1308, 1252, 1170, 1047, 979 cm-1; :H and 13C NMR data see Tables 1, 2; HREIMS m/z 662.4414 [M]? (calcd for 662.4394).

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

White powder; [a]D -1.63 (c 1.8, MeOH); IR (KBr) vmax 3442, 2935, 2871, 1736, 1629, 1455, 1413, 1239, 1159, 1071, 1044, 982 cm-1; :H 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-seco-cimigenol-3-O-b-D-xylopyranoside (3)

White powder; [a]D° -1.63 (c 0.01, MeOH); IR (KBr) vmax 3431, 2931, 2873, 1631, 1456, 1384, 1238, 1161, 1041, 975 cm-1; :H (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-deacetyl-acetaeaepoxide-3-one. (4)

White powder; [a]D -18.17 (c 1.1, MeOH); IR (KBr) vmax 3441, 2966, 2932, 2875, 1705, 1628, 1465, 1383, 1248, 1061 cm-1; :H and 13C NMR data see Tables 1, 2; HREIMS m/z 502.3294 [M]? (calcd for 502.3294).

3.8 16,17-Didehydro-2',24-O-diacetylhydrosheng-manol-3-O-ß-D-xylopyranoside (5)

White powder; [a]DO -10.28 (c 0.01, MeOH); IR (KBr) vmax 3431, 2929, 2870, 2853, 1739, 1629, 1459, 1375, 1237, 1240, 1167, 1125, 1072, 1046, 980 cm-1; :H 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]D° -24.21 (c 0.5, MeOH); IR (KBr) vmax 3440, 2931, 2871, 1712, 1669, 1457, 1380, 1278, 1167, 1019, 940 cm-1; :H 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 refluxed with 0.5 N HCl (3 mL) for 4 h. Each reaction mixture was diluted with H2O and extracted with CHCl3 (3 x 10 mL). The water layer was then neutralized by Ag2CO3, and the precipitate filtered to give a monosaccharide. Each monosaccharide of those compounds had an Rf (EtOAc/CHCl3/MeOH/H2O, 3:2:2:1) and specific rotation [a]D° ?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

Conflict of Interest All authors declare no conflict 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/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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