Two new compounds from Melodinus suaveolens

Abstract Two new compounds, 19-hydroxy-melodinine K (1) and melodiside (2), and 25 known compounds were isolated from leaves and twigs of Melodinus suaveolens. Their structures were elucidated based on 1- and 2-D NMR, FTIR, UV and MS spectroscopic data. 19-hydroxy-melodinine K showed cytotoxic activity against MDA-MB-231 breast, BCG-823 gastric, SW480 colon and Hela cancer cells.


Results and discussion
The MeOH extract of M. suaveolens was partitioned between H 2 O and etOAc combination with alkali treatment, and column chromatography on silica gel and C18 silica gel was used to separate the alkaloidal fraction into 27 compounds.
Compound 2 was isolated as a white powder. The FTIR spectra of 2 exhibited absorption bands for OH (3342 cm −1 ), and ester carboxylic group (1712 cm −1 ) and benzene ring (1616 and 1520 cm −1 ). Its UV spectrum showed the characteristic absorption bands at 246 and 323 nm. The positive HReSI spectrum showed a sodiated molecular ion peak at m/z = 577.1530 [M + Na] + , with a molecular formula of C 25 H 30 O 14 Na. The 1 H NMR spectrum of 2 displayed two pair of aromatic signals at δ H 7.06 (s, 2H) and 6.99 (s, 2H), which demonstrated two 1, 3, 4, 5-tetrasubstituted aromatic rings (Shao et al. 2004). Its 1 H NMR spectrum ( Figure  S6) also exhibited two singlets due to five methoxyl groups at δ H 3.80 (s, 3H) and δ H 3.70 (s, 12H), respectively. The above information, together with the observation of a methyl ester signal (δ C 165.6 (s), 52.2 (q)), indicated the presence of two 4-hydroxy-3,5-dimethoxy benzoic units in the molecule. Furthermore, a glucosyl unit (δ C 101.7, 76.4, 74.2, 74.0, 70.5 and 63.8) was observed in the 13 C NMR spectrum of 2. In addition, HMBC correlations were observed between the anomeric proton signal at δ H 5.09 (1H, d, J = 7.2 Hz) with δ C 137.9 (C-4′), and between δ H 4.43 and 4.19 (H-6) with δ C 165.4 (C-7′′). So 2 was determined as shown in Figure  1 and named as melodiside. The J value (7.2 Hz) of the anomeric proton of the sugar moieties revealed the β-configuration of the glucosyl residue. The identification of the sugar residue was continued by hydrolysis with 10% HCl to afford D-glucose which was confirmed by comparison with authentic samples and determination of their optical rotation values

Plant material
Leaves and twigs of M. suaveolens Champ. ex Benth were collected in July 2012 in Zhongshan, Guangdong Province, P.R. China, and identified by Dr. Xi-Feng Teng. A voucher specimen (Cai120706) was deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.

Cytotoxicity assay
Four human cancer cell lines, MDA-MB-231 breast, BCG-823 gastric, SW480 colon and Hela, were used for cytotoxic assays. Cells were cultured in RPMI-1640 (Sigma-Aldrich, St. Louis, MO, USA) or DMeM medium (Hyclone, Logan, UT, USA), supplemented with 10% foetal bovine serum (Hyclone) in 5% CO 2 at 37 °C. Cytotoxicity assays were performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method in 96-well micro plates. Briefly, 100 μL of adherent cell types were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before the addition of test compounds. Suspended cell types were seeded at an initial density of 1 × 10 5 cells/mL just before drug addition. each tumor cell line was exposed to a test compound at concentrations of 0.04, 0.2, 1.0, 5.0 and 25.0 μM in DMSO in triplicate for 48 h, with cisplatin (Sigma-Aldrich) as the positive control. After treatment, cell viability was assessed, cell growth graphed and IC 50 values calculated by Reed and Muench's method (Reed & Muench 1938).

Conclusions
Alkaloidal fraction of M. suaveolens afforded two new compounds, 19-hydroxy-melodinine K (1) and melodiside (2), and 25 known compounds. 19-hydroxy-melodinine K showed cytotoxic activity against cancer cells, which was consistent with this type skeleton as previously reported in the genus. This type of bisindole could stimulate future phytochemical studies.

Funding
This work was supported by Yunnan province key new product development project [grant number 2015BA006]; and Key project of China National Tobacco Corporation [grant number 110201402042].