Dibenzocyclooctadiene lignans from the fruits of Schisandra chinensis and their cytotoxicity on human cancer cell lines

Repeated chromatographic separations of the EtOAc fraction of Schisandra chinensis fruits on silica gel, octadecyl silica gel, and Sephadex LH-20 led to the isolation and identification of seven dibenzocyclooctadiene lignans (1–7). The NMR data reported in the literature for angeloyl gomisin H (5) were shown to be incorrect. We unambiguously identified the compounds based on detailed analysis of the 1D and 2D NMR data, especially from HMBC and NOESY experiments. In addition, MTT assays and cell viability experiments verified the cytotoxicity of the isolated dibenzocyclooctadiene lignans against the human cancer cell lines AGS, HeLa, and HT-29.


Introduction
Schisandra chinensis is a deciduous woody vine native to Far East Asia. The fruits, red berries called "Omija" in South Korea, have been used as food as well as a traditional medicine with hepaprotective, cardiovascular, and antibacterial benefits [1,2]. The Korean word Omija, meaning "five flavors" (sweet, sour, bitter, salty, and spicy), indicates that the fruit has a variety of components that exhibit pharmacological effects. To date, phytochemical studies of S. chinensis fruits have led to the isolation of lignans, triterpenoids, monoterpenes, sesquiterpenes, organic acids, and sterols, [3,4] among which dibenzocyclooctadiene lignans are overwhelmingly the major components [5]. Among its major components, the NMR data reported in the literature for angeloyl gomisin H (5) were identified to be a little bit incorrect. Therefore, we unambiguously identified the compound 5 to correct its NMR value based on detail NMR analysis technics especially gHMBC and NOESY. Also, S. chinensis extracts and some dibenzocyclooctadiene lignans from this plant have been reported to be cytotoxic to certain cancer cell lines [6][7][8][9]. Despites their significant anti-cancer effects, there has been no report concerning their chemical structures relationship between their cytotoxic activity against various cancer cells.
This paper describes the isolation for seven dibenzocyclooctadiene lignans from the fruits of S. chinensis, structure determination of the isolation ones, especially angeloyl gomisin H (5). In addition, their cytotoxicities were evaluated against several human cancer cell lines (AGS, HeLa, and HT-29), and the relationship of their structure to their activity.

Cell viability assay
Cell reasonability was dictated by MTT measure as recently portrayed [13]. Cells were seeded at a thickness of 1 × 10 3 cells/well in a 96-well plate and refined with sans serum DMEM or RPMI-1640 for 16 h. At that point, the cells were treated with sequential groupings of Angeloyl gomisin H, Gomisin A, Gomisin C, Gomisin J, (-)-Gomisin K1, Schisandrin, in different concentration (10, 25, 50 μg/mL) for 24 h. Treatment at every fixation was acted in triplicate. After medicines, the medium was suctioned and cells were washed with PBS. Cells were in this manner hatched with MTT arrangement (5 mg/mL) for 6 h. The supernatant was expelled, and formazan was solubilized in isopropanol and estimated spectrophotometrically at 570 nm. The level of practical cells was assessed in examination with untreated cells. The information shows the mean ± SD of at least three free trials.

Hoechst 33258 and propidium iodide staining
All the cells were seeded onto amplifying instrument coverslip in a 6-well plate until further notice and were treated with IC 50 union of ginger blends. In the wake of washing twice with PBS, cells were fixed with 4% paraformaldehyde for 15 min. The joined cells were recolored with 500 μL Hoechst 33258 (5 μg/mL) plan and 500 μL propidium iodide (PI, 5 μg/mL) course of action at room temperature for 30 min, exclusively. Apoptotic cells with combined and partitioned centers were overviewed using  a Leica DMLB fluorescence amplifying focal point (Wetzlar, Germany) [14,15].
Schisandra chinensis extract and major components from this plant are previously reported to have an anticancer activity including cytotoxicity on human cancer cell lines [22]. Especially almost compounds which have dibenzocylooctadiene-type lignans structure, deoxyschisandrin, gomisin A, and γ-schisandrin, have significant anti-cancer effect [23][24][25][26]. However, there are no reports for the cytotoxicity of dibenzocylooctadienetype lignans on gastric (AGS), cervical (HeLa), and colon (HT-29) human cancer cells. Therefore, we evaluated the dibenzocylooctadiene lignans (1-7) from S. chinensis fruits for the cytotoxicity of the human cancer cells using MTT assay.
Compounds 4-6, which have a hydroxy group at C-7, showed relatively weak toxicity on RAW 264.7 cells compared with lignans without a hydroxyl group at C-7. In comparison, compounds 2, 3, and 7, which have S-biphenyl positions, and compound 1, in which all the hydroxy groups in the benzene ring are substituted by methoxy groups, showed relatively strong toxicity toward RAW 264.7 cells. Additionally, compounds 2 and 3, which have S-biphenyl positions and one or two hydroxyl groups on the benzene ring, showed significant inhibition of HeLa cells. Compounds 4 and 5, which have relatively low toxicity toward normal cells, suppressed the proliferation and viability of AGS and HeLa cells. In particular, compound 5, with its angeloyl moiety, exhibited a slightly stronger effect on AGS, HeLa, and HT29 cells than compound 4. These results indicate that the stereochemistry, the presence of an angeloyl or a hydroxy group at C-7, and the benzene ring could be key factors of dibenzocyclootadiene-type lignans affecting the cytotoxicity against AGS, HeLa, and HT29 cells.
After treatment of the compounds, the cell nuclei were stained with Hoechst 33258 and PI were observed by fluorescence microscopy, respectively. The treated cells exhibited apoptotic morphology, such as cell shrinkage with DNA condensation, high fluorescence, and formation of the apoptotic body. And the IC 50 value represents the concentration of each compound that inhibits cell activity by 50% (Table 2).
In conclusion, our data reveal that dibenzocyclooctadiene lignans 2-5 from S. chinensis fruits can be effective candidates as anticancer materials for stomach, cervical, and colon cancers.