New 3′,8′′-Linked Biflavonoids from Selaginella uncinata Displaying Protective Effect against Anoxia

Seven 3′,8′′-linked bioflavonoids, including one new compound, (2′′S)-2′′, 3′′-dihydroamentoflavone-4′-methyl ether (1) and six known compounds: (2S)-2,3- dihydroamentoflavone-4′-methyl ether (2), (2S,2′′S)-2,3,2′′,3′′-tetrahydroamento- flavone-4′-methyl ether (3), (2S,2′′S)-tetrahydroamentoflavone (4), (2S)-2,3-dihydro- amentoflavone (5) and (2′′S)-2′′,3′′-dihydroamentoflavone (6) and amentoflavone (7), were isolated from the 60% ethanolic extract of Selaginella uncinata (Desv.) Spring. The structures of these compounds were elucidated mainly by analysis of their 1D and 2D NMR spectroscopic data, and their absolute configurations were determined by circular-dichroism (CD) spectroscopy. All the seven compounds showed protective effect against anoxia in the anoxic PC12 cells assay, in which compound 6 displayed particularly potent activity.


Introduction
Selaginella uncinata (Desv.) Spring, which is a Chinese herbal medicine known as "Cui Yun Cao", is widely distributed in the southwest China and used to treat jaundice, dysentery, edema and rheumatism diseases. [1] Earlier phytochemical investigation into this plant led to the isolation of several biflavonoids, flavonoids, chromone glycosides and phenolic constituents [2][3][4].
Hypoxia is a common environmental stress in high altitude. It can influence signaling pathways and cell functions, so several cell types including neuroendocrine chromaffin cells have evolved to sense oxygen levels and initiate specific adaptive responses to hypoxia. As a rat pheochromocytoma cell line derived from a tumor of adrenal medulla chromaffin tissue, PC12 is an oxygen-sensitive cell type and a useful system to study the effects of hypoxia [5].

Results and Discussion
The EtOAc soluble fraction was subjected to silica gel, Sephadex LH-20 and ODS column chromatography, and finally purified by preparative reverse-phase HPLC to afford seven compounds. The seven compounds showed positive reaction with Mg/HCl, which indicated that they were flavonoids.
The protective effect against anoxia of compounds 1-7 was evaluated by the anoxic PC12 cells assay ( Table 2). All seven compounds showed protective effect against anoxia, with compound 6 displaying the most potent protective effect, while the other compounds showed moderate effects.

General
UV spectra were obtained on a Shimadzu UV2401PC spectrophotometer and IR spectra were recorded on a Shimadzu FTIR8900 spectrophotometer using KBr disks. Optical rotations were measured on a Jasco P-1020 digital polarimeter and CD spectra were recorded on a JASCO 810 spectropolarimeter. NMR data were obtained on a Bruker AV-400 spectrometer, with TMS as an internal standard. Mass spectra were determined on a Bruker Esquire 2000 spectrometer, and HR-ESI-MS were acquired using a Micromass Q-TOF mass spectrometer. HPLC analyses were carried out on an Agilent 1100 Series instrument equipped with an PDA detector, using an analytical Shim-pack VP-ODS (4.6 × 250 mm, 5 μm, Shimadzu) or a preparative Shim-pack VP-ODS (20 × 250 mm, 5 μm, Shimadzu) column. Column chromatography (CC) was conducted using silica gel (Qing Dao Hai Yang Chemical Group Co., Qing Dao, China), ODS-A120-S150 (YMC Co., Ltd, Komatsu, Japan), and Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden).

Plant material
Herbs of S. unicinata were collected in Guangxi Province, China, in August 2004, and were identified by Professor Sun Qi-shi (Shenyang Pharmaceutical University, Shenyang, China). A voucher specimen (No.Y01156SU) is deposited in the Department of Natural Products Chemistry, Shenyang Pharmaceutical University.

Extraction and isolation
The air-dried whole herbs (4.2 Kg) of S. uncinata were cut into pieces and refluxed with 60% (v/v) EtOH (126 L, 3 times). The dried extract (856 g) was dissolved in water and successively partitioned with EtOAc and water-saturated n-BuOH to give three parts, the EtOAc (160 g), n-BuOH (90.4 g) and H 2 O (600 g) soluble parts. The EtOAc soluble part was subjected to silica gel (200-300 mesh) column chromatography and eluted with a CHCl 3 -MeOH gradient system. Fifteen fractions were obtained.  Table 1; 13 C-NMR (DMSO-d 6 ): Table 1.

The anoxic PC12 cells assay
The PC12 cells were cultured in a medium that consisted of 85% DMEM (Gibco), 10% heat-inactivated (56 °C for 30 min) horse serum (Hyclone), 5% fetal bovine serum (Hyclone) and glutamine 0.10 g/L for 5 generations before used. Then, The cells were dispersed with pipette and seeded in a 35 mm culture dish at a density of 2 × 10 5 cells/mL with 2 mL/per dish and incubated in a gas mixture of 90% air and 10% CO 2 atmosphere at 37 °C for 8 days. The PC12 cells with samples (in 10% DMSO) were divided into groups with three dishes per group (45 μmol/L, 90 μmol/L and 180 μmol/L), respectively. A blank control group without any supplement was carried out and 10% DMSO (DMSO: normal saline = 1:9) was added to the PC12 cells as a negative control. The result showed that the toxicity of 10 % DMSO in the anoxic cells is not significant. Baicalin which displayed potent protective effect against anoxia in the PC12 cells assay was used as a positive control [15]. 24 hours after that, the cells were moved into a sealed container with 90% N 2 and 10% CO 2 gas for 12 hours. Cell viability was assessed by trypan blue exclusion. The viable cells were counted under inverted phase contrast microscope (400×) in 10 visual fields randomly [16]. The promoted survival rate (%) of each sample contrasted to the control was calculated. Each experiment repeats 2 times. The data was presented as mean ± SD, n = 10 visual fields and statistics were performed as Student's T test.