Triterpenoids from Abies faxoniana and their cytotoxic activities

Abstract Two previously unreported triterpenoids (1 and 2) and four known compounds were isolated from Abies faxoniana. Compound 1 has a α,β-unsaturated-γ-lactone ring conjugated with the C-22/23 olefin in the C-17 side chain. The structures of the new compounds were established on the basis of spectroscopic data analysis. These compounds were tested for their cytotoxicities against six human tumour cell lines. Compound 1 showed cytotoxic activities against MCF-7 and A549 cells with IC50 values of 7.5 and 8.7 μM, respectively.


Introduction
There are approximately 50 species in the genus Abies (Pinaceae), 20 of which are indigenous to China (Zheng & Fu 1978). Phytochemical studies have indicated that terpenoids are characteristic constituents of the Abies species (Yang et al. 2010(Yang et al. , 2014Lavoie et al. 2012;Li et al. 2012Li et al. , 2015Handa et al. 2013;Wang et al. 2015aWang et al. , 2015bBelhadj Mostefa et al. 2016;Kim et al. 2016;Wu et al. 2016). These Abies terpenoids have been demonstrated to exert diverse biological activities, particularly in vitro antitumor activities (Yang et al. 2010;Lavoie et al. 2012;Li et al. 2012;Wang et al. 2015a;Wu et al. 2016). Abies faxoniana is a woody plant in China distributed exclusively in the mountains of Sichuan and Gansu provinces (Zheng & Fu 1978). Previously, we reported the isolation of eight pairs of epimeric triterpenoids involving a characteristic spiro-e/F ring from A. faxoniana (Wang et al. 2015a). These triterpenoids were obtained as epimeric mixtures arising from tautomerism at C-23 of the spirolactone structure. Herein, the branches and leaves of A. faxoniana were collected for further phytochemical investigation, which led to the isolation of two new triterpenoids (1 and 2) and four known compounds (3-6) ( Figure 1). The cytotoxic activities of the isolated compounds against six human tumour cell lines are also described.
The molecular formula of compound 2 was established to be C 30 H 48 O 2 on the basis of its [M + H] + ion at a m/z of 441.3721 in positive HReSIMS, indicating a hydrogen deficiency index of seven. The IR spectrum suggested the presence of hydroxy (3450 cm −1 ), aldehyde (1725, 2820, 2720 cm −1 ) and olefinic (1646 cm −1 ) groups. The 1 H, 13 C and DePT nMR spectroscopic data of 2 indicated 30 carbon signals including six methyl singlets, one methyl doublet, nine methylenes, eight methines and six quaternary carbons with the aid of HSQC data, including one aldehyde group at δ C 195.4; δ H 9.37 (s, 1H), an oxygenated secondary carbon at δ C 79.2; and two pairs of double bonds at δ C 121.5/148.6, 139.0/155.5. By combining the above evidence with comparison of nMR data with those of lanostane triterpenoids from the Abies plants, 2 was found to be similar to the known compound abiesatrine K (Wang et al. 2015b), except for the presence of an aldehyde group at δ C 195.4; δ H 9.37 (s, 1H) in 2 instead of a carboxylic moiety. This aldehyde group attached to C-26 was established based on the HMBC correlations from the aldehydic hydrogen at δ H 9.37 to C-24, C-25 and C-27, and from H-24 and H 3 -27 to the aldehydic carbonyl at δ C 195.4 (( Figure S18, Supplementary material)). The hydroxy group in 2 could be located at C-3 because of the HMBC correlations from H 3 -28 and H 3 -29 to the oxymethine at δ C 79.2. The nOeSY correlations of H-3 with H 3 -19, H-3 with H 3 -29, and H 3 -19 with H-9 revealed a co-facial relationship of H-3, H 3 -19 and H-9 and were assigned as β-oriented. no nOeSY correlations could be detected between H-9 and H-5, and H-9 and H 3 -30, indicating that H-5 and H 3 -30 resided on the opposite side of the plane, and were deduced to be α-orientation. The α-orientation of H-17 was established on the basis of the nOeSY correlation of H-17 with H 3 -30. Consequently, the structure of compound 2, named abiesatrine Q, was determined to be 3α-hydroxy-9β-lanosta-7,24E -diene-26-al.
All the isolated compounds (1-6) were tested for their cytotoxic activities against the human tumour cells Huh7, HepG2, SMMC7721, HCT-116, MCF-7 and A549 by MTT method (Table 1). Doxorubicin was used as a positive control. Among these compounds, compound 1 showed the strongest cytotoxicity against MCF-7 and A549 cells with IC 50 values of 7.5 and 8.7 μM, respectively; compounds 3 and 4 demonstrated inhibitory activity against HepG2 cells with IC 50 values of 17.9 and 15.0 μM, respectively.

General experimental procedures
nMR spectra were determined with a Bruker Avance 500 spectrometer with TMS as an internal standard. Optical rotations were measured with a JASCO P-1020 digital polarimeter in MeOH. HR-eSI-MS were performed on an Agilent 6520 Accurate-Mass Q-TOF mass spectrometer. IR spectra were recorded in KBr disk by a Bruker FTIR Vector 22 spectrometer. uV spectra were recorded with a Shimadzu uV-2500 spectrometer. Column chromatography (CC) was carried out on silica gel (Qingdao Haiyang Chemical Co., Ltd, China), YMC 50 μm ODS-A (Milford, uSA) and Sephadex LH-20 (Ge Healthcare Bio-Sciences AB, uppsala, Sweden), respectively. TLC was conducted on silica gel plates GF254 (Yan Tai Heng nuo Chemical Technology Co. Ltd, China). HPLC purification (Shimadzu LC-2010A HT, Shimadzu Crop., Kyoto, Japan) was conducted on a semi-preparative RP-C 18 silica column (Agilent ZORBAX SB-C18, 5 μm, 9.4 × 250 mm).

Cytotoxic MTT assay
HepG2, Huh7, SMMC7721, HCT-116, MCF-7 and A549 cell lines were obtained from Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Cytotoxicity assay was determined by the MTT method. The test compounds were dissolved in 0.1% DMSO. Cells were seeded in a 96 well plate at 6 × 10 3 cells/well and exposed to the test compounds for 24 h. The cultures were treated with 0.1% DMSO as the vehicle control. After 24-h incubation, 10-μL MTT solution (5 mg/mL) in PBS was added into 100-μL medium, and the plates were incubated for 4 h at 37°C in the incubator chamber. The supernatant was then removed from the formazan crystals, and 100-μL DMSO was added to each well. The absorbance was measured by a microplate reader using a wavelength of 570 nm.

Conclusion
In our continuing search for bioactive compounds from Abies species, two new triterpenoids (1 and 2) and four known compounds (3-6) were isolated from the branches and leaves of A. faxoniana. Since triterpenoids from Abies plants displayed various interesting biological activities, particularly in cytotoxic and antitumor activities, the cytotoxicities of these isolated compounds (1-6) were evaluated against six human tumour cell lines. Among them, the most potent cytotoxicity against MCF-7 and A549 cells was found for 1 with IC 50 values of 7.5 and 8.7 μM, respectively. It is therefore interesting to note that an α,β-unsaturated-γ-lactone conjugated with a double bond in C-17 side chain seemed to enhance the cytotoxic activity. However, further investigation on the mechanism of action underlying the in vitro antitumor effect of 1 is necessary.

Supplementary material
Supplementary material relating to this article is available online, including the HR-eSI-MS and 1D/2D nMR spectra for compounds 1and 2.

Disclosure statement
no potential conflict of interest was reported by the authors.