Sixteen New Prenylated Flavonoids from the Fruit of Sinopodophyllum hexandrum

Sixteen new prenylated flavonoids, sinoflavonoids P–Z (1–11) and sinoflavonoids NA–NE (12–16), were isolated from the fruit of Sinopodophyllum hexandrum, along with eight known analogues (17–24). Their structures were elucidated on the basis of extensive spectroscopic data (HR-ESI-MS, 1H-NMR, 13C-NMR, HSQC, HMBC). The cytotoxic activities of compounds 1–18, 20, and 22 were evaluated by MTT assay. Compound 6 showed the most potent cytotoxicity in MCF-7, and HepG2 cell lines, with IC50 values of 6.25 and 3.83 μM, respectively.

Compounds 1-18, 20, 22 were tested for their in vitro cytotoxic activities against MCF-7 and HepG2 cell lines using the MTT assay [11], with 5-fluorouracil as a positive control, and IC 50 values were summarized in Table 3. Among the tested compounds, only compound 6 exhibited the most potent cytotoxic activities against MCF-7 and HepG2 cell lines, with an IC 50 value of 6.25 and 3.83µM, respectively. Compound 6 was more cytotoxic than 5-fluorouracil, whereas compounds 5 displayed no cytotoxicity against MCF-7 and HepG2 cell lines. Compound 5 has the same B and C rings from flavone skeleton as 6, so the variation in cytotoxicity between them indicates 2-(1-hydroxy-1-methylethyl)dihydrofurano group on ring A is structurally required for the cytotoxity against the MCF-7 and HepG2 cells lines. Furthermore, the cytotoxic activity may be affected by the position of furano or dihydrofurano group on the ring A, which needs to be verified with more similar derivatives. With the promising cytotoxicities against two cell lines, compound 6 may be the optimal lead compound for structure optimization studies.

General Experimental Procedures
The UV spectra were measured on a Shimadzu UV-1700 spectrometer (Shimadzu Corporation, Kyoto, Japan). The IR spectra were measured on a Nicolet 10 Microscope Spectrometer (Thermo Scientific, San Jose, CA, USA). The 1D and 2D-NMR spectra were recorded on Bruker-AC (E)-500 spectrometer (Bruker AM 500, Fällanden, Switzerland) using tetramethylsilane (TMS) as an internal standard. The HR-ESI-MS was determined on a Bruker microTOF-Q instrument (Bruker BioSpin, Rheinstetten, Germany). Column chromatography was performed with silica gel (200-300 mesh; Qingdao Marine Chemical Inc., Qingdao, China), sephadex LH-20 (GE Healthcare), and ODS (50 µm; YMC Co. LTD., Kyoto, Japan). Preparative high performance liquid chromatography (HPLC) separations were performed on a SEP system (Beijing Sepuruisi scientific Co., Ltd., China) equipped with a variable-wavelength UV detector, using a YMC-Pack ODS-A column (250 × 20 mm, 5 µm). Chemical reagents for isolation were of analytical grade and purchased from Tianjin Siyou Co., Ltd., China. Biological reagents were from Sigma Company. Human heptocellular (HepG2), and breast (MCF-7) cancer cell lines were from Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, China.

Plant Material
The plant material was collected from Deqin, Yunnan Province, China, in September 2013, and identified by Prof. Chengming Dong as the fruit of S. hexandrum. A voucher specimen (SE 20130929) was deposited at the School of Pharmacy, Henan University of Traditional Chinese Medicine.

Extraction and Isolation
The powered fruit of S. hexandrum (9.1 kg) were refluxed with 95% EtOH three times (each, 2h, 20L). The filtrate was concentrated under reduced pressure to yield a dark brown residue (1.6 kg). The residue was suspended in water and partitioned with petroleum ether (PE), CH 2 Cl 2 , EtOAc, and n-BuOH, successively.

Conclusions
Prenylated flavonoids are characterized by the presence of lipophilic prenylated group on the parent skeleton. Their structure diversity is most attributed to the different position of prenylation, and various length, further cyclization and hydroxylation of prenyl chain. With diverse chemical structure, prenylated flavonoids exhibit extensive pharmacological actions, including antioxidant, anti-inflammatory, anticoagulant, antiviral, antimicrobial anticancer [22], antigenotoxic [23], antiplasmodial [24] and estrogen regulation activities [25]. However, currently, 80% of the approximately 1100 prenylated flavonoids exist in only three plant families (Asteraceae, Cannabinaceae, Leguminosae) [22]. Consequently, their exploitation and use is limited by the narrow distribution in the plant kingdom. Forty-six flavonoids, including 37 prenylated ones, were isolated from S. hexandrum [2,[8][9][10][11]. Most of them were tested for the cytotoxic activity in tumor cell lines [2,9,11]. Further phytochemical studies on S. hexandrum resulted in the isolation of 16 new prenylated flavonoids and eight known analogues. Their cytotoxic activity was evaluated against MCF-7 and HepG2 cell lines. Compound 6 was the most valuable of all tested compounds. Further research is necessary to elucidate the antitumor mechanism. This study also enriches the chemical and pharmacological diversity of prenylated flavonoids.