Chemical Constituents Of Abies Faxoniana And Their In Vitro Antitumor Activities

The plants of the genus Abies (family Pinaceae) are widely distributed in China, and many of these plants have long been used in folk medicine. China is one of the richest Abies plants countries around the world. Previous studies have demonstrated that this genus is a rich source of terpenoids with high structural diversity:various structure types, diverse substituted positions, multiple chiral centers, and frequent rearrangement. These Abies constituents and their crude extracts exhibit a wide range of biological activities, particularly in cytotoxic, anti-tumoral and anti-inflammatory activities. A. faxoniana is native to China, and is a woody plant distributed mainly in Sichuan and Gansu provinces. However, to date, few reports of related phytochemical and biological investigations toward A. faxoniana exist in the literature. In this study, the chemical constituents of A. faxoniana and their in vitro antitumor activities are investigated.1. chemical constituents from A. faxonianaInvestigations on chemical constituents of the branches and leaves of A. faxoniana led to the isolation and elucidation of 41 new and 60 known compounds, including 60 triterpenoids,23 diterpenoids,8 lignans,2 phenylpropanoids,2 monoterpenoids,1 flavonoid and so on.The major type of triterpenoids that have been found in A. faxoniana is the lanostane type. Six pairs of new epimeric lanostane-type triterpenoids involving spiro-E/F ring (wgw-1/1’-wgw-4/4’, wgw-7/7’, wgw-9/9’), two pairs of new epimeric cycloartane-type triterpenoids bearing spiro-E/F ring (wgw-5/51,6/6’), and a pair of novel epimeric 7(8→9)abeo-spirolanostanes wgw-8/8’with spiro-B/C and E/F ring were isolated from the branches and leaves of A.faxoniana. The spirolactone moiety was elucidated by extensive analysis of NMR spectra and single crystal X-ray diffraction. These triterpenoids were evidenced as inseparable epimer mixtures with a certain proportion, and the C-23 epimerization of the purified isomer for wgw-1/1’ wgw-2/2’ and wgw-9/9’ were observed by HPLC analysis. The interconversion of wgw-9 and wgw-9’ was slower than that of wgw-1 and wgw-1’because of the presence of an olefinic group at C-24 and C-25. The proportions of these epimeric mixtures were determined by HPLC method, and the proportions were 3:1,2:1,3:1, 2:1,4.5:1,4.5:1,8:1,10:1,3:2, respectively. Taking the effect of solvents on epimerization into consideration, we obtained the 13C NMR spectra of wgw-2 in CDCl3 and CD3OD. Close comparison of the two 13C NMR spectrum showed overall similarities. Compounds wgw-19-wgw-21 are usually tautomeric mixtures due to C-23 epimerization of y-lactone moiety with a hemiketal. Compounds wgw-26-wgw-30 are A-seco lanostane-type triterpenoids that can be formed by ring opening at the C-3 and C-4 positions, and wgw-27 contains a characteristic spiro C/D ring that can be formed by 8(14→13) rearrangement. The structures of the isolated compounds were elucidated by the NMR spectra, HR-ESI-MS data, single crystal X-ray diffraction, circular dichroism (CD) data, and PGME method.In the circular dichroism (CD) spectrum of wgw-11, a negative Cotton effect at 216 nm was found, which established a 23R configuration of the lactone side chain in wgw-11.The absolute configuration of 25R for wgw-45 could be elucidated according to the PGME method(△δ=δS-δR).2. in vitro antitumor activities of A. faxonianaThe isolated compounds were tested for cytotoxic activities against human tumor cell lines Huh7, HepG2, SMMC7721,HCT-116, MCF-7, and A549 using MTT assay. Among these isolates, wgw-18 showed cytotoxicities against two hepatoma cell lines HepG2 and SMMC7721 with IC50 values of 9.8 μM and 14.3 μM, respectively, and wgw-6/6’ had IC50 value of 7.5 μM for human hepatoma cell Huh7. Some of the isolated triterpenoids were also evaluated for inhibitory effects on in vitro human DNA topoisomerases Ⅰ and Ⅱ. Compound wgw-23 had inhibition effects against human Topo Ⅱ with an IC50 value of 53.5 μM, which was comparable to a Topo Ⅱ inhibitor, etoposide (IC5o=49.6 μM).Although wgw-18 inhibited the growth of human hepatoma cell lines HepG2 and SMMC7721, the underlying mechanisms of action remain to be elucidated. We further investigated its mechanism of action for inhibition of cell growth using HepG2 and SMMC7721 as representative cell line models.1. HepG2 and SMMC7721 cells were treated with various concentrations of wgw-18 for 24,48,72 h, after which cell growth inhibition assays were performed using the MTT method. The MTT assay showed that wgw-18 inhibited HepG2 and SMMC7721 cells growth in a dose and time dependent manner.2. Cell viability after 5,10,20 μM wgw-18 treatment in normal hepatic cell QSG7701 for 24 were determined by MTT assay. We also tested morphological nuclear changes in QSG7701 cells using Hoechst 33258 staining. These results suggested that wgw-18 had low toxicity to normal hepatic cells.3. Cell apoptosis was revealed by Annexin V-FITC/PI staining and Hoechst 33258 staining in HepG2 and SMMC7721 cells exposed to wgw-18 at different concentrations for 24 h. We examined the accumulation of ROS by staining with DCFH-DA, MMP changes by staining with Rh123, and the expression of apoptosis-related proteins by western blot analysis in HepG2 and SMMC7721 cells. These results indicated that wgw-18 increased cytosolic cytochrome c, Bax, cleaved caspase 3, cleaved caspase 9, and cleaved PARP expressions with a concomitant decrease in the MMP and Bcl-2 expression, leading to cell apoptosis. Additionally, treatment with wgw-18 resulted in a significant increase in intracellular ROS and inhibited Akt expression in HepG2 cells. However, no significant increase in ROS levels was observed in SMMC7721 cells.4. We examined cell cycle distribution using flow cytometry and the expression of cell cycle regulators by western blotting in HepG2 and SMMC7721 cells treated with wgw-18 at different concentrations for 24 h. These results revealed that wgw-18 significantly increased p53 and p21 expression and decreased cyclin Dl and CDK2 expression, leading to cell cycle arrest in G1 phase. In conclusion, our present studies showed that wgw-18 exerted an inhibitory effect on hepatoma cells HepG2 and SMMC7721 by arresting the cell cycle in the G1 phase and inducing apoptosis. Compound wgw-18 induced caspase-dependent apoptosis via both the mitochondrial pathway and the ROS/Akt pathway in HepG2 cells, but the ROS/Akt pathway was not involved in wgw-18-induced SMMC7721 cells apoptosis.