Actaticas A−G, Cycloartane Triterpenes From Actaea asiatica With Their Antiproliferative Activity

Phytochemical studies on the rhizomes of Actaea asiatica led to the isolation of seven new cycloartane triterpenes, actaticas A−G (1−7). Their structures were determined by NMR, HRESIMS, and chemical analysis. All the isolates were evaluated for their antiproliferative activity against HT-29 and McF-7 cell lines. The results showed that all the compounds displayed cytotoxicity. All compounds showed significant inhibitory effects with IC50 values of 9.2–26.4 μM.


INTRODUCTION
Actaea asiatica H. Hara, a perennial herb belonging to the family Ranunculaceae, is mainly distributed in the southwest and northwest of China. Its roots have been traditionally used among the Tujia folk in Hubei Province for treating headache, sore throat, rheumatic pain, rubella, measles, pertussis, uterine prolapse, and dog bites (Gao et al., 2006a;Gao et al., 2006b;Fan et al., 2007;Gao et al., 2007). Phytochemical studies indicated that the genus Actaea contained cycloartane triterpene glycosides with cytotoxic activities (Kusano et al., 1998;Kusano et al., 1999;Gao et al., 2006b). However, little systematic chemical work on A. asiatica has been carried out so far. In order to find the bioactive constituents from A. asiatica, chemical research were carried out, resulting in the isolation of seven new cycloartane triterpene glycosides, namely, actaticas A-G (1-7) ( Figure 1). Their structures were determined by spectroscopic analysis and chemical methods. Herein, structural elucidation of compounds 1-7 was reported as well as their cytotoxic activities.

General Experimental Procedures
Optical rotations were obtained on a PerkinElmer 341 digital polarimeter. IR spectra were recorded on Shimadzu FTIR-8400S spectrometers. NMR spectra were obtained with a Bruker AV III 600 NMR spectrometer (chemical shift values are presented as δ values with TMS as the internal standard). HR-ESIMS spectra were performed on a LTQ-Obitrap XL spectrometer. Preparative HPLC was performed on a Lumtech K-1001 analytic LC equipped with two pumps of K-501, a UV detector of K-2600, and an YMC Pack C 18 column (250 × 10 mm, i.d., 5 μm, YMC Co. Ltd., Japan) eluted with CH 3 OH-H 2 O at a flow rate of 2 ml/min. C 18 reversed-phase silica gel (40-63 μm, Merk, Darmstadt, Germany), MCI gel (CHP 20P, 75-150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), and silica gel (100-200 mesh, Qingdao Marine Chemical plant, Qingdao, the People's Republic of China) were used for column chromatography. Pre-coated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, the People's Republic of China) were used for TLC. All solvents used were of analytical grade (Beijing Chemical Works).

Plant Material
The plants of A. asiatica were collected at Jinfuo Mountain in Chongqing province, the People's Republic of China, in November 2016, and were authenticated by Professor Sirong Yi. The voucher specimen (CS161108) has been deposited at the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences.

Hydrolysis of Compounds
Acid hydrolysis of 1 and 4-7: A solution of compounds 1, 4, 5, 6, and 7 (5 mg) in 2 M HCl (1 ml) was heated at reflux for 24 h. The reaction mixture was neutralized with 2 M NaOH and extracted by partition with EtOAc (5 × 1 ml). 10 ml of water was added to the residue and extracted with CH 2 Cl 2 three times. Sugars were analyzed by TLC and GC analysis and compared with authentic sample of D-sugars. The spots were visualized by spraying with EtOH-H 2 SO 4 -anisaldehyde (9:0.5:0.5, v/v), then heated at 150°C. Furthermore, the absolute configurations of the sugars were determined by gas chromatography according to a method previously described (Ma et al., 2016;Li et al., 2017). Compound 3 was dissolved in MeOH (15 ml), then 4% K 2 CO 3 (15 ml) was added and each solution was stirred at room temperature overnight. Each solution was neutralized by 10% AcOH, and extracted with EtOAc (2 × 20 ml). EtOAc extract after removal of solvent, was dissolved in MeOH (10 ml) and refluxed with 0.5 N HCl (3 ml) for 4 h (Yin et al., 2010).

Cytotoxic Assay
The cytotoxicity of compounds 1−7 was evaluated using the MTT procedure with HT-29 and McF-7 cancer cell lines. The cells were incubated in DMEM supplemented with 10% fetal bovine serum and cultured at a density of 1.2 × 10 4 cells/ml in a 96-well microtiter plate. Five various concentrations of each agent dissolved in dimethyl sulfoxide (DMSO) were then put in the wells. Each concentration was evaluated three times. After incubation under 5% CO 2 at 37°C for 48 h, 10 ml of MTT (4 mg/ml) was placed into each well, and the cells were incubated for an additional 4 h. Then, the liquid was taken out, and DMSO (200 ml) was put into the wells. The absorbance was documented with a microplate reader at wave length of 570 nm.

RESULTS AND DISCUSSION
Compound 1 was obtained as white amorphous powder. Its IR spectrum showed absorptions of hydroxyl group at 3,440 and 3,397 cm −1 and carbonyl at 1,733 cm −1 . The HRESIMS spectrum showed a pseudo-molecular ion at m/z 729.4233 [M + Na] + in the positive ion mode from which in conjunction with NMR data the molecular formula was established as C 39 H 62 O 11 , consistent with nine degrees of unsaturation. In the 1 H NMR spectrum (Table 1) two cyclopropane-methylene protons as an AX system at δ H 0.18 and 0.47 (each 1H, d, J 4.0 Hz, H 2 -19) together with seven tertiary methyl groups at δ H 1.49, 1.47, 1.32, 1.36, 1.35, 1.02, and 1.09, indicated a cycloartane triterpenoid structure (Ju et al., 2002a;Mohamed, 2014;Gan et al., 2015;Wu et al., 2017). The 1 H NMR  -20), and an anomeric carbon at δ C 108.1, together with acetyl signals at δ C 170.9, 171.2, 21.2, and 22.0. The 1 H and 13 C NMR spectroscopic data of 1 confirmed that the compound was a cycloartane triterpene glycoside (Jung et al., 2002;Wu et al., 2017;Wua et al., 2017). All proton signals were assigned to the corresponding carbons through direct 1 H and 13 C correlations in the HSQC spectrum. Inspection of the 1 H-1 H COSY spectrum showed fragments of C-1/ C-2/C-3, C-5/C-6/C-7/C-8, C-11/C-12, C-15/C-16/C-17, and C-22/C-23/C-24. In the HMBC spectrum (Figure 2), the correlations were observed from H-28/29 to C-3 and C-5, H-19 to C-1, C-5, C-6, C-9, and C-11, and H-18 to C-12 and C-17, H-30 to C-8, C-14, C-16 and C-18, H-21 to C-22, H-22 to C-24, and H-24 to C-26 and C-2 fully confirmed the basic skeleton cycloartane triterpene of compound 1, which was consistent with the above deduction. The acetyl groups were connected with C-15 and C-16 supported by the correlations from H-15 to δ C 170.9 (the carbonyl carbon of OAc) and H-16 to δ C 171.2 (the carbonyl carbon of OAc). The sugar was connected with C-3 based on the key HMBC correlation between H-1′ (δ H 4.86, d, J 7.2 Hz) and C-3 (δ C 88.8), which was identified as D-xylose by TLC in comparison with authentic monosaccharides (visualization with ethanol-5% H 2 SO 4 spraying) followed by gas chromatography.
The NOESY experiment and coupling constants established the relative configuration of compound 1 (Figure 3), in which correlation of H-3/H-5 showed α-orientation of H-3. The larger coupling constants ( 3 J 1,2 > 7.0 Hz) of the anomeric protons indicated the β configuration of the sugar unit. The significant cross peaks from H-15 to H 3 -18, H-17α to  to Me-21 were observed, which enabled the establishment of OAc-15α and OAc-16β. Until now, all the isolated cycloartane triterpene share the identical absolute configuration with trans A/B, B/C, C/D rings. Considering the same cycloartane triterpene skeleton and identical carbon signals at C-20/C-24, compound 1 was established as 20S and 24R configurations (Ju et al., 2002a). Therefore, the structure of the compound was identified as shown and given the trivial name actatica A.
Compound 4 has a molecular formula of C 39 H 60 O 13 according to the HRESIMS (m/z 759.3974 [M + Na] + , calcd for C 39 H 60 O 13 Na, 759.3926). Its IR spectrum showed strong hydroxyl (3,439, 1,044 cm −1 ) and carboxyl (1730 cm −1 ) absorptions. The 1 H and 13 CNMR spectra indicated that 4 had two acetoxyl groups. Detailed NMR spectral analysis revealed that 4 possessed a cyclopropane ring, six methyl groups, a hydroxymethyl group at C-18, and a D-xylosyl unit at C-3. The 1 H and 13 C NMR spectra of 4 were similar to those of beesioside J (Ju et al., 2002b), except for a carbonyl group (C O) connected to C-12 of 4, which causes the downfield chemical shift of C-12 (δ C 216.6). The correlation from δ H 4.54 (H-11) to δ C 216.6 (C O) according to the HMBC supported the above result. Therefore, compound 4 was tentatively determined and named actatica D.
Compound 5 Table 1) two cyclopropane-methylene protons as an AX system at δ H 0.21 and 0.59 (each 1H,d,J 4.0 Hz, together with nine tertiary methyl groups indicated a cycloartane triterpenoid structure. The 1 H NMR and 13 C APT data for this compound were analogous to 1, except for the additional NMR signals at δ C 30.3 and 27.4, and δ H 1.31 (3H, s), and 1.45 (3H, s). The differences showed that 5 had one more hydroxyisopropyl group connected at C-24. In the HMBC spectrum, the correlations from H-24 to C-26, C-26′, C-27, and C-27′ confirmed the above deduction. Taken together with the NOESY spectra data, compound 5 was established as 24R configurations. Thus, compound 5 was established and named actatica E.
Compound 6 was determined to have the molecular formula of C 37 H 58 O 11 based on the 13 C APT data and by the HRESIMS ion peak at m/z 701.3904 ([M + Na] + , calcd for C 37 H 58 O 11 Na, 701.3926). The 1 H-NMR spectrum ( Table 1) displayed signals for seven tertiary methyls (δ 1.02, 1.22, 1.23, 1.30, 1.53, and 1.55), two typical signals at δ 0.12 (1H, d, J 4.2 Hz) and 0.44 (1H, d, J 4.2 Hz) ascribable to a cyclopropane methylene group, indicating that 6 might be a cycloartane-type triterpenoid. Examination of the 1 H and 13 C APT data (Tables 1, 2) showed the structure of 6 to be similar to 4. The NMR spectrum showed that compound 6 has only one set of acetyl group data. On the basis of 1 H-1 H COSY and HSQC and comparison with related 4, all signals were assigned as shown in Tables 1, 2. The correlation from H-16 (δ H 5.47) to acetyl carbon (δ C 171.8) was observed in HMBC spectrum, which means the acetyl group was connected to C-16. Therefore, compound 6 was clearly determined and named actatica F.

Bioactive Activity
The cytotoxic of all compounds 1-7 were tested for their inhibitory activity against human HT-29 and McF-7 cancer cell lines using MTT assay. All compounds showed significant inhibitory effects with IC 50 values of 9.2-26.4 μM ( Table 3). Compound 7, with an oxygen bridge between C-18 and C-24, showed the best potency among the isolated constituents. With a tetrahydrofuran fragment connected by C-20 and C-24, compounds 1 and 4-7 showed better activity than 2 and 3.
Seven new 9,19-cycloartane glycosides were isolated from the rhizomes of A. asiatica H. Hara. Until now, nearly 200 naturally occurring triterpenes with a 9,19-cycloartane have been reported (Su et al., 2016;Hassan et al., 2020). However, compound 5 with one more hydroxy isopropyl group was first isolated from the genus Actaea. All compounds displayed inhibitory activity against human HT-29 and McF-7 cancer cell lines. Further analysis of the data showed that compounds 1 and 4-7 exhibited better protective effect than other compounds, which indicated that the tetrahydrofuran fragment connected by C-20 and C-24 may affect the inhibitory activity regarding HT-29 and McF-7.