New Triterpene Diglycosides from the Rhizome of Cimifuga foetida

Five new 9,19-cycloartane triterpene diglycosides, which have been named cimiaceroside C (1), and cimifosides A-D (2-5) together with the known compounds cimiracemoside D (6), cimidahurine (7) and α-d-glucopyranosyl-l-β-d-fructofuranoside (8) were isolated from the rhizome of Cimicifuga foetida. The new triterpene diglycosides 1-5 were identified as cimiacerol-3-O-β-d-xylopyranosyl-(1''→3')-β-d-xylopyranoside, 12β-hydroxycimigenol-3-O-β-d-xylopyranosyl-(1''→3')-β-d-xylopyranoside, 25-O-acetylcimig- enol-3-O-β-d-xylopyranosyl-(1''→3')-β-d-xylopyranoside, 24-acetylhydroshengmanol-3-O-β-d-xylopyranosyl-(1''→3')-β-d-xylopyranoside and 26-deoxyacetylacteol-3-O-β-d-xylo- pyranosyl-(1''→3')-β-d-xylopyranoside, respectively, based on analysis of their spectral data and chemical reactions.


Introduction
The rhizome of Cimicifuga foetida (family Ranunculaceae) is a popular Chinese Traditional Medicine. Under the trivial name of "Shengma", it has been used as an antipyretic and analgesic agent since ancient times [1]. Recently, all Cimicifuga species were returned to the genus Actaea L based on evidence from DNA sequence data [2][3][4]. In the United States and the European Union, Actaea racemosa (L.) Nutt [2][3][4], commonly known as black cohosh, has been reputed to reduce the frequency and intensity of hot flashes and other menopause symptoms [5,6]. Chemical constituents of Cimicifuga species have been extensively studied by several groups [7][8][9]. Previous phytochemical studies have revealed that Cimicifuga species mainly contain constituents such as chromones, cinnamic acid derivatives, and 9,19-cyclolanostane triterpenes. To date, more than 200 triterpenes have been isolated from the genus [10,11] and triterpenoid glycosides are considered to be the main active components, which have been used as marker compounds to standardize Cimicifuga extracts [12].
The relative stereochemistry of 1 was determined on the basis of the ROESY experiments (   Table 1 The 13 C-NMR data of compounds 1-5 in C 5 D 5 N (δ in ppm).    , H-1''). The 13 C-NMR spectrum exhibited 40 resonances, of which 30 were attributed to a triterpene skeleton, ten to two sugar units. A comparison of the 1 H-and 13 C-NMR spectra of 2 with those of cimiside A [17] revealed that 2 has an additional sugar unit. Only xylose was detected in the aqueous fraction of the acid hydrolysis products of 2. In the HMBC spectrum, long-range correlations between δ H 5.29 (H-1') and 88.7 (C-3), and between δ H 4.81 (H-1'') and 87.4 (C-3') were observed for 2, which assigned the inner xylose at C-3, and the terminal xylose at C-3' of the inner xylose. The configurations of C(23) and C(24) were ascribed as R and S respectively, by comparing the spectral data of C-23 and coupling constants of H-24 signals of 2 with those of known 9,19-cyclolanostane triterpene glycosides [18]. Therefore, cimifoside A (2) was determined to be 12β-hydroxycimigenol and two for a carbonyl group. All this evidence suggested that 3 was a 9,19-cycloartane triterpenoid diglycoside. The structure of 3 resembled that of the known compound cimiside B [17]. It differs from cimiside B only by the presence of an acetyl group, which was assigned to C-25 due to dramatic chemical shift changes of C-25 (+Δ12.2 ppm), C-24 (-Δ3.4 ppm), C-26 (-Δ1.4 ppm) and C-27 (-Δ3.9 ppm) ( Table 1). The relative configuration of 3 was determined to be the same as that of 2 by comparison with literature data [19]. Thus, the chemical structure of 3 was identified to be 12βhydroxycimigenol . The 1 H-and 13 C-NMR spectral data of 5 were very similar to those of 23-epi-26-deoxyactin [8], except for an additional xylose. The second xylose was connected to C-3' of the first xylose due to an HMBC correlation between δ H 4.77 (H-1'') and 87.4 (C-3'), and the disaccharide unit was further assigned to C-3 by an HMBC correlation of δ H 5.27 (H-1') and 88.3 (C-3). Hence, 5 was determined to be 23-epi-26-deoxyacetylacteol-3-O-β-D-xylopyranosyl-(1''→3')-β-Dxylopyranoside.

Conclusions
Although the rhizome of C. foetida is a very famous Chinese Traditional Medicine and it has been the subject of extensive phytochemical investigations, its chemical components have not been completely identified yet. Triterpene monoglycosides are considered to be the main components of Cimicifugu foetida, but in our present study a series of new cycloartane diglycosides 1-5 have been isolated and identified, which further clarified the triterpene glycoside components from C. foetida. Moreover this finding will be helpful for identifying extracts of A. racemosa and C. foetida, as some black cohosh products in America and European market are contaminated with related Asian Cimicifuga species, such as C. foetida, C. simplex, and C. dahurica.

Experimental
General IR spectra were recorded on a Shimadzu IR-450 instrument, and are reported in cm -1 . 1 H (400 and 500 MHz) and 13 C-NMR (100 and 125 MHz) spectra (all in C 5 D 5 N) were recorded with Bruker AV 400 and DRX500 instruments, using TMS as an internal standard. Silica gel (200-300 mesh, Qingdao Marine Chemical, P.R. China), Lichroprep RP-18 (40-63um, Merck, Darmstadt, Germany) were used for column chromatography (CC). Fractions were monitored by TLC, and spots were visualized by heating TLC sprayed with 10% H 2 SO 4 . Mass spectral data were recorded on a VG Autospec 3000 spectrometer.

Plant material
The rhizomes of Cimicifuga foetida were collected in Lijiang, Yunnan Province, China, in July