Phytochemical analysis of Daphne pontica L. stems with their pro-apoptotic properties against DU-145 and LNCaP prostate cancer cells.

Background: Daphne pontica is an endemic plant grown wild in the North part of Iran, with anticancer activities.

Objectives: This study aims to analyze the phytochemistry and screen the cytotoxic activity of new bioactive compounds against a panel of cancer cells, in addition to proapototic properties against prostate cancer cells.

Method: Purification procedure was done using repeated column chromatographies by MPLC and HPLC systems. The structures were elucidated by the NMR and exact mass spectroscopy, stereochemistry by NOESY, and absolute configuration by electronic circular dichroism (ECD) spectra. Cytotoxicity was done against DU 145, LNCaP, HeLa, MCF-7, and MDA-MB 231 cells by standard MTT assay. An annexin V/PI assay was performed to measure the type of death following treatment with these compounds for 48 h, followed by the caspase-3 activity test.

Results: In this study, one new dilignan named lignopontin A (9), in addition to 13 known compounds including two phenolic acids (3, 5), one flavanone (6), one bis flavonoid (1), one cumarin glycoside (2), one mono (4) and two dicumarins (10, 11), two lignans (7, 8), and three daphnane diterpenoids (12-14) were isolated for the first time from D. pontica stems. Complete spectral data of compound 12, named as 6,7α-epoxy-5β-hydroxy-9,13,14-ortho-(4,2E)-pentadeca-2,4-diene-1-yl)-resiniferonol, and compound 14, named as 6,7α-epoxy-5β-hydroxy-9,3,14-ortho-(2,4E)-pentadeca-2,4-di-1-yl)-resiniferonol-12β-yl-acetate are reported for the first time. In the MTT assay of newly described compounds against a panel of cancer cells, compounds 9, 12, and 14 possessed moderate to potent cytotoxicity against prostate, breast, and cervical cancer cells in a dose-dependent manner. Flow cytometry analysis against prostate cancer cells indicated that the cytotoxicity of compounds 12 and 14 was due to their ability to induce apoptosis. In the case of compound 9, in Du 145 cells, cell death was mainly through apoptosis. In contrast, LNCaP cells showed both apoptosis and necrotic cell death, predominated by necrosis at the higher concentrations. Caspase-3 activity confirmed apoptosis observed in these compounds through the caspase pathway in prostate cancer cells.

Conclusion: D. pontica is a new source of dimeric phenolic compounds, including bisflavonoids, phenylpropanoid-cumarin adduct, and dilignans, as well as daphnane diterpenoids with resiniferonol core with long-chain orthoester moieties. In cytotoxicity screening, compounds 9, 12, and 14 inhibited the growth of DU-145 and LNCaP cells in a dose-dependent manner with IC₅₀ varied from 0.9 - 27.3 and 25.2 - 87.4 μM, respectively. Among them, 9 exhibited selective growth inhibition against DU 145 treated cells. LNCaP cells demonstrated the highest sensitivity to treatment with compound 12.