Two new ent-atisanes from the root of Euphorbia fischeriana Steud.

Two new ent-atisanes ent-1β,3β,16β,17-tetrahydroxyatisane (1), ent-1β,3α,16β,17-tetrahydroxyatisane (2) together with 11 known diterpenes were isolated from the anti-tumour activity fraction of Euphorbia fischeriana Steud. The compounds were identified by detailed spectroscopic analysis, including extensive 2D-NMR experiments. X-ray analysis was applied to determine the structure of compound 2. All 13 compounds were screened for cytotoxicity in vitro against human tumour MCF-7, HepG-2 and SGC-7901 cell lines. Compounds 1 and 2 showed the inhibitory effects against MCF-7 with IC50 levels of 23.21 and 15.42 μM; simultaneously, compounds 4, 6, 8 and 11 also had definite inhibitory effect against different cell lines.

The stereochemistry of compound 2 was analysed by X-ray. Suitable crystals were mounted on a glass fibre on a single-crystal X-ray diffractometer (German Bruker SMART 1000 CCD) operating at 50 kV and 40 mA using Mo Ka radiation (0.71073 Å ). Data collection and reduction were performed using the SMART and SAINT software. The structure was solved by direct methods, and the non-hydrogen atoms were subjected to anisotropic refinement by full-matrix least squares on F 2 using SHELXTL package. All hydrogen atoms were located in their calculated positions and treated using a riding model. Compound 2: empirical formula C 20 H 34 O 4 , formula weight 338.47, orthorhombic, space group, P 21 21 21, According to the data of X-ray and NOESY correlations, simultaneously compared with the literature (Allick et al. 1990;Zhan et al. 2013), the orientations of H-5 and H-20 have been determined as b and a, respectively, in atisane skeleton. Therefore, in compound 2, the orientations of H-1, H-5, H-9 and CH 3 -19 have been confirmed as b while H-3, CH 3 -18 and CH 3 -20 have been determined as a. Compound 2 was elucidated as ent-1b,3a,16b,17tetrahydroxyatisane.

Plant material
The root of Euphorbia fishcheriana Steud. was collected in Heilongjiang Province of China in September 2011, and identified by Prof. Zhenyue Wang (Heilongjiang University of Chinese Medicine). The voucher specimen (20110047) was deposited at the Herbarium of Heilongjiang University of Chinese Medicine, Harbin, China.

Cytotoxicity
The cytotoxicity of all 13 compounds was tested by MTT method (Wang et al. 2009(Wang et al. , 2011. The result showed that compounds 1 and 2 had an intensive effect with IC 50 levels of 23.

Conclusion
In our study, 13 diterpenes compounds were isolated from E. fischeriana Steud. and identified. Compounds 1 and 2 are two new ent-atisane compounds and stereoisomers for each other whose configuration was further determined by X-ray. Compounds 3, 4 and 6 were isolated from E. fischeriana Steud. for the first time. According to the result of MTT, different degrees of inhibition on three different human cell lines MCF-7, HepG-2 and SGC-7901 were indicated by compounds 1, 2, 4, 8 and 13, respectively.

Supplementary material
Experimental details relating to this article are available online, alongside Tables S1and S2 and Figures S1 -S17.

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