Cytotoxic phenyl polyketides from Hypericum curvisepalum N. Robson

Abstract (±)-Hypecurvone A (1) and B (2), two new undescribed phenyl polyketides, along with seven known analogues (3–9) were isolated from the whole plant of Hypericum curvisepalum. Chiral separation of 1 and 2 yielded two pairs of enantiomers 1a/1b and 2a/2b, respectively. The structures of these compounds were elucidated by extensive spectroscopic analyses and ECD spectra simulations. All isolates exhibited moderate cytotoxicity against human hepatocellular carcinoma SMMC-7721 cells, and compound 3 also showed weak cytotoxicity toward MGC-803 cells. The cytotoxicity of these compounds was found to be related to enhanced mitochondria-mediated apoptosis and inhibition of the G2/M phase of the cell cycle. Graphical abstract


Results and discussion
Because the ECD spectrum of 1 displayed no obvious Cotton effect, and its specific rotation value was approximately zero, 1 was considered a racemic mixture.Subsequent chiral separation of 1 yielded a pair of enantiomers 1a and 1b in an approximate ratio of 1:1 (Figure S2, in Supplementary material) exhibiting mirror image-like ECD curves (Figure S3, in Supplementary material).The absolute configurations of 1a and 1b were assigned as (1 00 S) and (1 00 R), respectively, based on ECD calculations.
Compound 2 was obtained as a pale yellow oil.The HRESIMS ion at m/z 337.1048 337.1046) revealed a molecular formula of C 18 H 18 O 5 .The 1 D and 2 D NMR data of compound 2 closely resembled those of known compound peplidiforone B (4) (Fobofou et al. 2016).The only difference was that the methyl group at C-2 00 in 4 was replaced by a acetoxy methyl moiety in 2, as suggested by the HMBC (Figure S1, in Supplementary material) correlations from H-3 00 to C-1 00 , C-2 00 , and C-6 00 , from H-7 00 to C-3 00 and C-6 00 .The specific rotation value (near zero) and the ECD spectrum indicated that 2, like compound 1, was also a racemic mixture.Enantioseparation of 2 into 2a and 2b was achieved by employing chiral HPLC (Figure S2, in Supplementary material).The absolute configurations of 2a and 2b were determined as (2 00 S) and (2 00 R), respectively, by comparison of their experimental and calculated ECD spectra (Figure S3, in Supplementary material).

Cytotoxic activities assay
The cytotoxic effects of all isolated compounds against two human cancer cell lines, SMMC-7721 and MGC-803, were evaluated.As showed in Table S2 (in Supplementary material), all tested compounds exhibited cytotoxicities against SMMC-7721 cells; the IC 50 values ranged from 43.8 to 120.8 lM.In addition, compound 3 exhibited cytotoxicity against MGC-803 cells (IC 50 152.9lM).
To investigate their possible cytotoxic action mechanism of the four compounds with high cytotoxic activities, the effects of compounds 5-8 on the mitochondrial membrane potential (MMP) of SMMC-7721 cells were evaluated.The results (Figure S4a, in Supplementary material) indicated that MMP in SMMC-7721 cells was hyperpolarized, it might be caused by a stimulus of ROS, which play important roles as an early initiator of apoptosis in SMMC-7721 cell.To verify this, the ROS in SMMC-7721 cells were then determined.Results showed that the percentage of ROS-positive cells was 21.42 ± 0.13%, 23.51 ± 0.12%, 13.51 ± 1.41% and 53.43 ± 1.01%, respectively (Figure S4b, in Supplementary material), which demonstrating a significant increase of ROS caused by compounds 5-8.Annexin V-FITC/PI double staining experiment was employed to corroborate the percentage of apoptotic cells increased from 14% in the control to 26% in SMMC-7721 cells line (Figure S4c, in Supplementary material), suggesting that compounds 5-8 could induce apoptosis of SMMC-7721.Compared with untreated cells, DAPI stained results manifested a more concentrated fluorescence intensity in single cells (Figure S4d, in Supplementary material).Above analyses supported the nuclear morphologic change in SMMC-7721 cells.The effects of compounds 5-8 on SMMC-7721 cell cycle distribution was monitored by flow cytometry analysis after PI staining of the cellular DNA.As indicated in Figure S5 (in Supplementary material), compounds 5-8 induced accumulation of cells in the G2/M phase in contrast to untreated control cells.

Conclusions
As part of our ongoing investigation on novel bioactive constituents from plants of the genus Hypericum, two pairs of new phenyl polyketides (1 and 2), and seven previous reported compounds (3-9) were isolated from the whole plant of H. curvisepalum.
All compounds exhibited moderate cytotoxicity against SMMC-7721 cells with IC 50 values of 43.8-120.8lM, and compound 3 also showed cytotoxicity against MGC-803 cells with IC 50 values of 152.9 lM.The cytotoxicity of these compounds was found to be related to enhanced mitochondria-mediated apoptosis and inhibition of the G2/M phase of the cell cycle.
Compound 1 was isolated as a pale brown solid with a molecular formula of C 17 H 20 O 4 deduced from HRESIMS m/z 287.1277 [M -H] -(calcd for C 17 H 19 O 4 -, 287.1288].The UV spectrum showed absorption maxima at 235 and 325 nm.The absorption bands of IR spectrum indicated the coexistence of carbonyl (1 692 cm À1