Cycloartane Glycosides from the Roots of Cimicifuga foetida with Wnt Signaling Pathway Inhibitory Activity

Four new 9,19-cycloartane triterpenoids, cimilactone E (1), cimilactone F (2), 2′-O-(E)-butenoyl-23-epi-26-deoxyactein (3), and 2′,12β-O-diacetylcimiracemonol-3-O-β-d-xylopyranoside (4), together with four known constituents (5–8) were isolated from the roots of Cimicifuga foetida. The new structures were elucidated by extensive spectroscopic analysis. In addition, compounds 7 and 8 showed significant Wnt signaling pathway inhibitory activity, with IC50 values of 3.33 and 13.34 μM, respectively, using the luciferase reporter gene assay.


Introduction
Wnt signaling pathway plays an important role in numerous biological processes, including axis formation, cell fate specification, cell polarity determination, and cell migration [1]. Pathologically, Wnt signaling pathway is frequently aberrant in wide spectrum of malignancies, such as colon cancer, liver cancer, leukemia, melanoma, pancreatic cancer, and breast cancer [2]. Thus, screening inhibitors of Wnt signaling pathway has been considered as effective therapeutic strategies to combat cancer [3].

Results and Discussion
Compound 1 was obtained as a white powder. The IR spectrum showed absorptions for hydroxyl groups at 3438 cm -1 and carbonyl groups at 1735 cm -1 , respectively. Its 13 C NMR and DEPT spectra of 1 exhibited 35 signals, of which 26 were attributed to the aglycon, five to a pentose residue, and four to two acetyl groups. The 13 C NMR and DEPT spectrum of the aglycon of 1 also showed an ester carbonyl carbon at d C 174.2 and two acetoxy carbonyl groups at d C 171.1 and 170.5. The aforementioned data suggested that 1 was a 9,19-cycloartane tetranortriterpene glycoside with three carbonyl groups. The NMR spectroscopic data of 1 ( Table 1) closely resembled that of cimilactone A (5), except for the presence of an additional acetoxy group. In the HMBC spectrum (Fig. 3), a correlation was observed between the proton at d H 4.87 (1H, d, J = 8.0 Hz, H-1 0 ) and the methine carbon at d C 88.8 (C-3), suggesting that the sugar moiety was located at C-3. In 1 H-1 H COSY spectrum, the     H-17 were proposed as a-orientation, respectively, by the same way as that of 1. Therefore, the structure of 2 was identified as (3b,12b,16b)-12-acetoxy-3-hydroxy-24,25,26,27tetranor-cycloartan-23, 16 Table 1). The 13 C NMR and DEPT spectrum (Table 1) of 3 exhibited 41 signals, of which 30 were attributed to the aglycon, five to a pentose residue, two to an acetyl group, and four to an 2-butenoyl group. Additionally, the aglycon of 3 also showed a hemiketal moiety at d C 74.9 (d), 106.3 (s) and 68.5 (t), and an epoxyethane signals at d C 62.6 (d) and 62.9 (s), which indicated that the aglycon of 3 was similar with 23-epideoxyacteol. A comparison of the spectroscopic data of 3 with those of 23-epi-26-deoxyactein showed that 3 closely resembles of it except for the presence of another tetracarbon unit (2-butenoyl group) [19]. Additionally, the Egeometry of a double bond in the 2-butenoyl was confirmed in the same way with that of 2. Compound 4 was obtained as a white powder. The IR spectrum showed absorptions for hydroxyl (3439 cm -1 ), carbonyl groups (1730 cm -1 ), respectively. Its molecular formula (C 39 H 60 O 12 ) with ten degrees of unsaturation was deduced from the analyses of 13  exhibited 39 signals, of which 30 were attributed to the aglycon, five to a pentose residue, and four to two acetyl groups. All above showed 4 was similar to cimiracemoside H, except for the presence of an additional acetoxy group. In 1 H- 1  As noted in the introduction, the roots of C. foetida have been used as cooling and detoxification agents by Chinese people since ancient time. Previous reports have shown that many pure 9,19 cycloartane triterpenoids isolated from this species exhibited cytotoxic activity against 11 tumor cell lines (including HepG2, MDA-MB-A231, HL-60, SMMC-7721, A549, SK-BR-3, PANC-1, K562, U933, HEG-2, and SGC-7091) in vitro, respectively [5-7, 17, 20, 21]. But there is no report on inhibition of Wnt signaling pathway. The pure components isolated in the present paper were screened against Wnt signaling pathway using the luciferase reporter gene assay. The known compounds 7 and 8 showed notable activity with the IC 50 values of 3.33 and 13.34 lM, respectively. To the best of our knowledge, this is the first time to report the inhibitory activity against Wnt signaling pathway of 9,19-cycloartane triterpenes. These data suggested that some chemical constituents from C. foetida might be valuable to against tumorigenesis through inhibition to Wnt signaling pathway.

General Experimental Procedures
UV spectra were recorded in MeOH on a shimadizu UV-210A spectrometer. IR spectra were recorded on Shimadzu IR-450 spectrometer with KBr disc. Optical rotations were measured a Horiba SEAP-300 polarimeter. 1 H NMR and 13 C NMR spectra were recorded using a Bruker AM-600 spectrometer with TMS as internal standard, operating at 600 and 150 MHz, respectively. All compounds were measured in solvents pyridine-d 5 . ESIMS and HRESIMS were carried out on a Waters Autospec Premier-P776 spectrometer. TLC analysis was performed on silica gel GF 254 plate (Qingdao Marine Chemical, Inc.). Lichroprep RP-18 (40-63 lm, Merck) and silica gel (200-300 mesh) was used for column chromatography. Semipreparative HPLC was carried out on an Agilent 1260 liquid chromatograph with a ZORBAX SB C-18 column (9.4 9 250 mm,5 lm,) and a ZORBAX XDB C-18 column (9.4 9 250 mm, 5 lm).

Plant Materials
The roots of C. foetida (82 kg) were collected from Yulong County of Yunnan province of China in September 2010 and authenticated by Prof. Shen-Ji Pei of Kunming Institute of Botany, where a voucher specimen (KUN No. 20100906) is deposited.

Luciferase Activity
The Wnt signaling inhibitory activity of the eight 9, 19-cycloartane triterpenes (1-8) using the luciferase reporter gene assay as previously described [2]. Briefly, HEK293W cells were seeded in 96 well plate, and the luciferase activities were measured after incubation with the triterpenes for 24 h, using the Dual-Lucy Assay Kit (Promega) according to the manufacturer's instructions.