Cytotoxic Cycloartane Triterpenoid Saponins from the Rhizomes of Cimicifuga foetida

Abstract To enrich the bioactive cycloartane triterpenoid glycoside named actein and find out more cytotoxic cycloartane triterpenes, a phytochemical study of Cimicifuga foetida was conducted. 113 g (0.17%) actein was purified by recrystallization while eight cycloartane-type triterpenes (1–8) were isolated from the mother liquid. The chemical structures of new compounds (1–4) were elucidated by 1D and 2D NMR and HRESIMS spectroscopic analyses. Moreover, new compounds showed moderate and broad-spectrum cytotoxicity against 5 human cancer cell lines with IC50 values ranging from 4.02 to 15.80 μM. Graphic Abstract Electronic supplementary material The online version of this article (10.1007/s13659-019-0214-1) contains supplementary material, which is available to authorized users.


Introduction
Cimicifuga foetida, commonly known as "shengma", is a famous traditional chinese medicine native to china. The roots of C. foetida have been widely used to relieve fever and inflammation for thousand years and also have been officially listed in the Chinese Pharmacopoeia [1,2]. During the past decades, the cyclolanostane triterpenoid glycosides of Cimicifuga genus have been noticed for their strong cytotoxic activities on cancer, especially breast cancer. Actein is the representative cyclolanostane triterpene isolated from Cimicifuga, exhibited an anti-angiogenic and anti-cancer activities on human breast cancer models by suppressing the protein expressions JNK/ERK pathways. Further antimetastatic activity study showed that actein could downregulated the protein expression of epidermal growth factor receptor (EGFR) and affect AKT and NF-κB pathways [3][4][5]. In addition, actein also showed cytotoxic activities against the hepatobiliary cancer, glioma growth, liver cancer and cervical cancer [6][7][8][9][10][11][12]. Because the anti-breast cancer activity of actein, the pharmacodynamics, pharmacology and toxicology experiments were tested by us. In order to carry out the preclinical research of actein, a simply and fast method is needed to enrich considerable amount of actein from the roots of C. foetida.
Massive experiments found that triterpenoid saponins which have the similar polar with actein show similar cytotoxic effects [13][14][15]. Meanwhile, analysis of the mother liquid after recrystallization by TLC (thin-layer chromatography) and HPLC (high performance liquid chromatography) showed that a small amount of cycloartane triterpenes still existed. In order to further explore the bioactivities metabolites from C. foetida, the mother liquid was selected for systematically investigated and four new compounds (1)(2)(3)(4) (1)(2)(3)(4) were evaluated for their cytotoxic activities against human HepG2, SMMC-7721, A549, MCF-7, and SW-480 cancer cell lines by using the MTT assay. Herein, the recrystallization process of actein, the isolation, structure's elucidation, and biological activities of new isolates are described.

Results and Discussion
Actein obtained by simply repeated recrystallization. And eight triterpenoid saponins (1-8) were isolated from the mother liquid. The structure elucidation of new compounds (1-4) are as followed.
Actein was obtained as white needles. The details of recrystallization process can be found in experiments section.
Compound 1 was obtained as white, amorphous powder. And the molecular formula C 41 H 60 O 12 -24)] indicated that 1 was acteol-type triterpenoid. In the 13 C NMR (DEPT) spectra, the signals ascribable to an α, β-unsaturated ketone moiety at δ C 165.9 (s), 122.1 (d), 149.7 (d), 31.6 (q) were observed. A comparison of the spectroscopic data of 1 with actein [16] showed that, structurally, 1 closely resembles actein, with the main differences of the sugar moiety, including the α,β-unsaturated ketone resonances. And in the 1 H-1 H COSY spectrum (Fig. 3) an ester carbonyl carbon at [δ C 170.5 (-OAc)], therefore, a acetyl group is located at C-12. Finally, in the HMBC spectrum ( Fig. 1), the correlation of the anomeric proton [δ H 4.88 (d, J = 7.9 Hz, H-1′)] with the methine carbon [δ C 88.2 (C-3)] illustrated that the sugar moiety was located at C-3. After acid hydrolysis, the sugar was identified as D-xylose by comparing its TLC and specific rotation with an authentic sample (Fig. 2).
In the ROESY spectrum (Fig. 3), the d-xylose moiety was suggested to be in the β-configuration by the large coupling constant of the anomeric proton (J 1,2 = 7.9 Hz) in the 1 H NMR spectrum. Additionally, when combined with the key correlations observed in the ROESY spectrum of H-1′ (axial), H-2′ (equatorial), H-3′ (axial), and H-4′ (equatorial), it indicated that C-2′, C-3′, and C-4′ are α-, β-, and α-configurations, respectively. These findings confirmed that the monoglycoside unit of 1 is a xylopyranoside moiety. Moreover, correlations between H-3/H-5, The absolute configuration of C-23 was established by comparing to the 13 C NMR spectroscopic data of 23-epi-26-deoxyActein (23S) and actein (23R). When the chemical shifts of C-16 and C-20 were δ C 73.0 and 26.0, the absolute configuration of C-23 was assigned as R, similar to actein, while signals at δ C 74.5 and δ C 23.3 were corresponding to S-configuration, similar to 23-epi-26-deoxyActein [17]. Thus, chemical shifts of C-16 and C-20 were δ C 25.9 and 72.9 in compound 1, respectively, indicating that the absolute configuration of C-23 was R. Therefore, the structure of 1 was elucidated as Compound 2 was obtained as a white, amorphous powder, showing an [M−H] − ion at m/z 761.4119 in the HR-ESIMS consistent with the empirical molecular formula C 41 H 62 O 13 (calc. 761.4118), requiring 11 sites of unsaturation. The IR spectrum showed absorptions of hydroxy and carbonyl groups at 3436 and 1739 cm −1 , respectively. In the 13 C NMR (DEPT) spectrum, 41 carbon signals could be resolved as nine methyls, ten methylenes, twelve methines, and ten quaternary carbons. In the 1 H NMR spectrum of 2, the characteristic cyclopropane methylene resonances at δ H 0.23 and 0.46 (each 1H, d, J = 3.8 Hz), an anomeric proton at  The above evidences, together with the diagnostic signals for two oxygen-bearing methine carbons [δ C 71.5 (C-23) and 86.6 (C-24)] and a ketal carbon [δ C 112.3 (C-16)] in the 13 C NMR spectrum, confirmed that 2 is a 9,19-cimigenoltype monoglycoside with one acetoxy group. The 13 C NMR spectrum also revealed carbons assignable to a 3-methoxy-3-oxo-propionyl moiety In the HMBC spectrum (Fig. 4), the correlation of the proton at δ H 4.81 (d, J = 7.9 Hz, H-1′) with the methine carbon at δ C 88.5 (d, C-3) indicated that the sugar moiety was located at C-3. Aforementioned data indicated that the structure of 2 was similar to 25-O-acetylcimigenol-3-O-β-dxylopyranoside (8) [18], with the only difference in the sugar moiety. In 8 the H-2′ resonance was observed at δ H 4.02, whereas in 2 it shifted downfield to δ H 5.52. In addition, the C-1′ resonances at δ C 107.5 and C-3′ at δ C 78.6 in 8 shifted upfield to δ C 104.0 and 76.6, respectively, in 2. Which may be explained by the presence of the C-2′ 3-methoxyl-3-oxopropionyl moiety group of the xylose unit. This deduction was confirmed by the HMBC correlation observed between the correlation of the proton at δ H 5.52 (1H, t, J = 8.6 Hz, H-2′) with the ester carbonyl carbon at δ C 167.0 (s, C-1′′).
In the ROESY spectra (Fig. 4), the d-xylose moiety was suggested to be in the β-configuration by the large coupling constant of the anomeric proton (J 1,2 = 7.9 Hz) in the 1 [19]. The configuration at C-23, C-24 were assigned as R and S, respectively. Therefore, the structure of 2 was elucidated as 25 Compound 3 gave the same molecular formula as compound 2 by HR-ESIMS. The IR and 1D NMR spectra of compound 3 showed close resemblances to those of 2 except for slight differences resonances of the sugar moiety. In 2, the H-3′ resonance was observed at δ H 4.12 m, whereas in 3 it shifted downfield to δ H 5.73 (1H, t, J = 9.2 Hz). In addition, the C-2′ resonances at δ C 75.9 and C-4′ at δ C 71.1 in 2 shifted upfield to δ C 72.8 and 68.9, respectively, in 3, which may be explained by the presence of the C-3′ 3-methoxy-3-oxo-propionyl moiety of the xylose unit. Which was further proved by the HMBC correlation. And used the same method as compound 2, the configuration at C-23, C-24 were assigned as R and S, respectively. Thus  (Tables 1, 2). The 1D NMR spectra (Tables 1, 2) showed 4 were similar to 4′-O-acetylcimigenol-3-O-β-dxylopyranoside [20], except for the presence of an additional methoxy group. Furthermore, the position of the attached methoxy group was supported by a downfield chemical shift of δ C 76.2 (C-25) in the 13 C NMR spectra (   Especially, all the new compounds exhibited stronger cytotoxicities against A-549, MCF-7 and SW-480 cell lines than SMMC-7721 and HL-60 cell lines, even stronger than positive control (Cisplatin). Based on the above results, we also found that the cytotoxic activities of new compounds, which means these substituents (like acetyl, butenoyl or methoxyloxo-propionyl) attached to sugar unit may contribute to inhibition activity.
In this present study, a great amount of high-purity actein were obtained by recrystallization, which laid a material foundation for the pre-clinical research. Compounds 1-4 isolated from the mother liquid also showed inhibition activity to different cancer cell lines, which provide not only chemical model for discovering potential anticancer agents, but also a proof for further development and utilization of Cimiciguga genus.

Plant Material
Rhizomes of C. foetida (67 kg) were collected from Yulong County, Yunnan Province, China, in August 2014. The material was identified by Prof. Shengji Pei at Kunming Institute

Extraction and Isolation
The air-dried and powdered rhizomes of C. foetida (67 kg) were extracted three times with 90% aqueous methanol (50 L × 3) at 60 °C to give a residue after evaporating under vacuum at 50 °C. The residue was suspended in water and then partitioned with petroleum ether (PE), EtOAc and n-BuOH. The EtOAc portion (8 kg) was subjected to the D101 macroporous absorption resin and eluted with a gradient of (MeOH/H 2 O = 50:50, 70:30, 90:10) to afford three fractions. The fraction (MeOH/H 2 O = 90:10, 4 kg) was fragmented by a silica gel column (CHCl 3 /MeOH = 100:1, 50:1, 20:1) and yields three subfractions. Further analysis of TLC and HPLC showed that actein existed in the (CHCl 3 / MeOH = 50:1, 160 g) fraction. A multi-solvent (MeOH/ MeCO 2 = 1:3) recrystallization was used to purify the actein. The mixture (160 g) was dissolved in the MeOH (300 ml), which was a saturated solution at 45 °C. Then filter the solution to remove the insoluble impurities. Later, MeCO 2 (600 ml) was added to above solution to obtain a mixed solvent system. The mixed solvent system allowed to cool over time to give crystals. The next step, separated the crystals from the solvent system by filter. Next, the mother liquid was evaporated again under the vacuum at 50 °C to give a residue, which was treated on the basis of aforementioned process until no actein monitored by TLC. Finally, 113 g of actein was obtained.
The mother liquid was evaporated under vacuum at 50 °C, and a residue (40 g) was obtained. Then, the residue was separated by a silica gel column (petroleum ether/ MeCO 2 = 10:1, 2:1, 1:1) to obtain three parts. Fraction

Hydrolysis and Identification of the Sugar Moieties in Compounds 1-4
The new compounds 1-4 (3 mg of each) were separately dissolved in MeOH (5 ml); 4% K 2 CO 3 (5 ml) was added, and the solution was stirred at room temperature overnight. The solution was neutralized with 10% HOAc and extracted with EtOAc (3 × 10 ml). After removal of the solvent, the EtOAc extract was dissolved in MeOH (5 ml) and refluxed with 0.5 N HCl (1 ml) for 4 h. Each reaction mixture was diluted with H 2 O and extracted with CHCl 3 . The water layer was applied on an Amberlite IR-35 (5 ml) column, and the resultant fraction was concentrated in vacuo to give a monosaccharide, which had an R f 0.4 (EtOAc-CHCl 3 -MeOH-