Cytotoxic monoterpenoid indole alkaloids isolated from the barks of Voacanga africana Staph.

A new monoterpenoid indole alkaloid compound (1) and six known monoterpenoid indole alkaloids compounds (2–7) were isolated from the barks of Voacanga africana Staph. The structures were established by spectral analysis as ibogamine-16-carboxylic acid,17,20-didehydro-5,6-dioxo-10-methoxy-methyl ester (1), voacamine (2), vobasine (3), voacangine (4), voacristine (5), 19-epi-voacristine (6) and 19-epi-heyneanine (7). Compound 1 was confirmed by X-ray crystallographic analysis. All of the isolated compounds were evaluated for cytotoxicity against five cell lines (HEPG-2, A375, MDA-MB-231, SH-SY5Y, CT26). Among them, compounds 2 and 6 displayed significant inhibitory activities, compounds 3, 4 and 5 showed moderate inhibitory activities, while compounds 1 and 7 showed no inhibitory activities against the five cell lines.


Introduction
Voacanga africana Staph. from the family Apocynaceae is widespread in West Africa, Congo and Tanzania. The plant is widely distributed over secondary forests and transitional zones (Adolfina et al. 2009). It has been traditionally used for the treatment of leprosy, diarrhoea, generalised oedema, convulsions in children, madness, diuretic and infant tonic (Iwu 1993;Neuwinger 2000;Tan et al. 2000). V. africana Staph. is a very rich source of a number of indole alkaloids with intriguing carbon skeletons and biological activities (Kang et al. 2012;Mei et al. 2012). In our previous investigation on this plant, 11 alkaloids with cancer toxicity were obtained (Mei 2012;Dan 2014). Now, further investigation on the ethyl acetate extract of V. africana Staph. led to the isolation of a new monoterpenoid indole alkaloid 1 and six known monoterpenoid indole alkaloid compounds 2 -7 ( Figure 1). Furthermore, these seven compounds were evaluated in vitro against the HEPG-2, A375, MDA-MB-231, SH-SY5Y and CT26 tumour cell lines. We herein report the isolation, structural elucidation and bioactivity of these compounds.
3.4. Crystal data for 1 X-ray data were collected at room temperature with Mo Ka radiation (graphite monochromator l ¼ 0.71073 A ) on a Huber four circle diffractometer equipped with a Bruker APEX-II CCD area detector. Data reduction was carried out using Bruker SAIN.

MTT assay
The cancer cell lines HEPG-2, A375, MDA-MB-231, SH-SY5Y and CT26 were obtained from the American Type Culture Collection, USA. Cultures were incubated in a humidified atmosphere of 5% CO 2 at 378C. When the cells enter the logarithmic growth phase, cells (3 £ 10 3 /well) were seeded in supplemented culture medium (100 mL/well) in a 96-well plate and incubated for cells adherent. The medium was then replaced with a test compound-containing medium, and the cells were further incubated for 72 h. DMSO 10% was used as blank sample while taxol was used as positive control. The cell viabilities were evaluated by MTT assays. The plate was then read on a microplate reader at 570 nm to evaluate the effects of the test compounds on cell growth. Experiments were conducted in triplicate.

Conclusions
A new compound and six known compounds were isolated from the barks of V. africana Staph. They were bis monoterpenes indole alkaloid (compound 2) and monoterpenes indole alkaloid (compounds 1, 3-7). All the compounds were tested for their in vitro cytotoxic activities against the HEPG-2, A375, MDA-MB-231, SH-SY5Y and CT26 cancer cell lines. Ibogamine monoterpenoid indole alkaloids showed important cytotoxicities against tumour cells, the presence methyl ester of C-16 was necessary to maintain the activities (Wang 2008). Compound 2 was bis monoterpenes indole alkaloid, which showed stronger activity than monoterpenes indole alkaloids.

Supplementary material
The UV, IR, HR-ESI-MS, 1 H NMR, 13 C NMR, 13 C DEPT 908, 13 C DEPT 1358, HSQC and HMBC spectra of 1 can be found in the online-only supplementary material, Figures S1 -S10. Crystallographic data in CIF format are available free of charge via the Internet at CCDC 1016804. These data can be obtained free of charge via https://www.ccdc.cam.ac.uk/services/ structure_deposit/ (or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: þ 44 1223 336033; or deposit_reply@ccdc.cam.ac.uk).