A new isocoumarin from the aerial parts of Aconitum gymnandrum

Abstract A new isocoumarin, along with 10 known compounds, was isolated from the aerial parts of Aconitum gymnandrum. Their structures were elucidated by spectroscopic methods including extensive 1D and 2D NMR techniques. Among the known compounds, compound 11 was obtained as a natural product for the first time, which was previously reported as a synthetic product. In addition, compounds 1–5, 7 and 9 were tested for their cytotoxicity against four human cancer cell lines. The results showed that compounds 3, 4 and 7 displayed cytotoxicity against lung cancer A549 and gastric cancer MGC80, respectively, whereas 5 and 9 showed selective cytotoxicity against hepatocellular carcinoma HepG2.


Introduction
Aconitum (Ranunculaceae) is a large resource of medicinal plants. About 36 kinds of Aconitum species have been used for treating rheumatism and analgesia in China (Fu 2004). Aconitum gymnandrum is the sole representative of the monotypic subgenus Gymnaconitum (Stapf.) Rapaics (Wang et al. 2009). This species is widely distributed in alpine meadows and hillsides ABSTRACT A new isocoumarin, along with 10 known compounds, was isolated from the aerial parts of Aconitum gymnandrum. Their structures were elucidated by spectroscopic methods including extensive 1D and 2D NMR techniques. Among the known compounds, compound 11 was obtained as a natural product for the first time, which was previously reported as a synthetic product. In addition, compounds 1-5, 7 and 9 were tested for their cytotoxicity against four human cancer cell lines. The results showed that compounds 3, 4 and 7 displayed cytotoxicity against lung cancer A549 and gastric cancer MGC80, respectively, whereas 5 and 9 showed selective cytotoxicity against hepatocellular carcinoma HepG2. along the edges of the Qinghai-Tibetan Plateau as well as on the Qinghai-Tibetan platform itself. It differs from other congeneric species by being pollinated by both insects and wind to adapt to the arid habitats (Duan et al. 2010). The aerial parts of A. gymnandrum are used as a traditional Tibetan medicine to treat rheumatic numbness and joint pain. The water extracts of stems and leaves of A. gymnandrum exhibited anti-inflammatory activities (Fu et al. 2005). A. gymnandrum is also a kind of poisonous plant in natural grassland, and it's reported that the methanol extracts of A. gymnandrum had strong anti-feeding effects against larva of Pieris rapae (Hu et al. 2011). Phytochemical investigation of this species was rare and previous research revealed that diterpenoid alkaloids were the major components of Aconitum plants (Wu ＆ Zhu 1984;Jiang et al. 1986;Ding et al. 1993;Atta-ur-Rahman & Choudhary 1999;Guo et al. 2014;Yin et al. 2014). As a part of our research for new bioactive secondary metabolites from the Tibetan medicine, our group conducted the phytochemical research of this species. This led to the isolation of a new isocourmain, 6,7-dimethoxy-8-hydroxy-3-[β-(p-hydroxyphenyl)ethyl]-3,4-dihydroiso-coumarin (1), together with 10 known compounds (2-11) ( Figure 1). In addition, the cytotoxicity of the compounds 1-5, 7 and 9 against four human cancer cell lines was evaluated. Herein, we report the isolation, structural elucidation and cytotoxicity of these compounds.
Compounds 1-5, 7 and 9 were evaluated for their cytotoxicity against four human cancer cell lines (gastric cancer MGC80, liver cancer HepG2, lung cancer A549 cells and colon cancer Hct116). The results showed that compounds 3 displayed selective cytotoxicity against lung cancer A549 with IC 50 values of 35.31 μg mL −1 , and compounds 4 and 7 displayed selective cytotoxicity against gastric cancer MGC80 with IC 50 values of 24.7 and 38.1 μg mL −1 , respectively, while 5 and 9 showed selective cytotoxicity against liver cancer HepG2 with IC 50 values of 48.70 and 27.40 μg mL −1 , respectively. Compounds 1 and 2 showed no cytotoxicity against any of the four human cancer cell lines at a concentration of 50 μg mL −1 .

General
optical rotations were measured with a HoRIBA SePA300 high sensitive polarimeter. IR spectra were measured on a Bio Rad FTS 135 series spectrometer in dry film. uV spectra were recorded on a Shimadzu uV2401A uV-Vis spectrophotometer. eSIMS and HReSI-MS were run on an API QSTAR Pular-1 spectrometer. NMR spectra measured in CDCl 3 or CD 3 oD solution and recorded on a Bruker Avance III-600 spectrometer at 25 °C, using TMS as an internal standard. Chemical shifts were reported in units of δ (ppm) and coupling constants (J) were expressed in Hz. CC were carried out over silica gel (200-300 mesh, Qingdao Haiyang Chemical Co.) and Rp-18 (40-63 μm, Merck). Pre-coated silica gel plates (Qingdao Haiyang Chemical Co.) were used for TLC. Detection was done under uV light (254 nm and 365 nm) and by spraying the plates with 10% sulphuric acid followed by heating. An Agilent series 1200 (Agilent Technologies) were used for HPLC. A Hanbon preparative HPLC (Hanbon Sci & Tech, Jiangsu) and C18-HC column 10 μm 100 Å (250 mm × 20 mm, Acchrom, Beijing, China) was used for semi-preparative HPLC separations.

Plant material
The aerial parts of A. gymnandrum were collected in Qi-lian, Qing-hai Province, People's Republic of China, in August 2013. The identification of the plant material was verified by professor Li-juan Mei (Northwest Institute of Plateau Biology, Chinese Academy of Science). A voucher specimen (No. 0295343) has been deposited in the Key Laboratory of Tibetan Medicine Research.

In vitro cytotoxic activity
Four human cancer cell lines, gastric cancer MGC80, liver cancer HepG2, lung cancer A549 and colon cancer Hct116 were used in the cytotoxic assay. All the cells were cultured in RPMI-1640 or DMeM medium (Hyclone, uSA), supplemented with 10% fetal bovine serum (Hyclone, uSA). The cytotoxicity assay was performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method in 96-well microplates. (Mosmann 1983) Briefly, adherent cells (100 μL) were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition with an initial density of 0.5 × 105-1 × 10 5 cells/mL. each tumour cell line was exposed to the test compound dissolved in DMSo in triplicates for 48 h at 37 °C with doxorubicin as positive control. Then, MTT (50 μL) was added to each well, and the tumour cells were incubated for another 4 h at 37 °C. After the supernatant liquor was removed, SDS (200 μL) was added to each well. The optical density was measured at 595 nm on a microplate reader. Cell viability was detected and a cell growth curve was graphed. IC 50 values were calculated by Reed and Muench's method. (Reed & Muench 1938).

Conclusions
In this study, a new isocoumarin together with 10 known compounds was isolated from the aerial parts of A. gymnandrum. Among the known compounds, compound 11 was firstly reported as a natural product, which was previously reported as a synthetic product. Herein, the structure elucidation of the new compound was reported. Furthermore, the cytotoxic activity of compounds 1-5, 7 and 9 was tested for their cytotoxicity against four human cancer cell lines (MGC80, HCT116, A549 and HepG2) by the MTT assay. The results showed that compounds 3, 4 and 7 displayed cytotoxicity against lung cancer A549 and gastric cancer MGC80, respectively, whereas 5 and 9 showed cytotoxicity against hepatocellular carcinoma HepG2.

Supplementary material
experimental details relating to this article are available online, alongside Table S1 and Figures S1-S7.

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