Phenolic Glucosides from Dendrobium aurantiacum var. denneanum and Their Bioactivities

A new 8,4'-oxyneolignane glucoside 1 has been isolated from the stems of Dendrobium aurantiacum var. denneanum together with six known phenolic glucosides 2−7. The structure of the new compound, including its absolute configuration, was determined by spectroscopic and chemical methods as (−)-(7S,8R,7'E)-4-hydroxy-3,3',5,5'-tetramethoxy-8,4'-oxyneolign-7'-ene-7,9,9'-triol 7,9'-bis-O-β-D-glucopyranoside (1). In the in vitro assays, compound 1 and (−)-syringaresinol-4,4'-bis-O-β-D-glucopyranoside (2) showed evident activity against glutamate-induced neurotoxicity in PC12 cells. Shashenoside I (4) showed a selective cytotoxic activity with the IC50 value of 4.17 μM against the acute myeloid leukemia cell line MV4-11, while it was inactive against 10 other human tumor cell lines.


General
NMR spectra were recorded on a INOVA-500 spectrometer. HRESIMS were measured with Waters Synapt G 2 HDMS. IR were recorded on a Vector 22 FT-IR spectrometer. UV spectra were obtained on a Shimadzu UV-260 spectrophotometer. Optical rotations were measured with a Perkin-Elmer 341 plus. CD spectra were recorded on a JASCO J-815 CD spectrometer. Column chromatography was performed with silica gel (200-300 mesh, Yantai Institute of Chemical Technology, Yantai, China) and Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden). HPLC separation was performed on an instrument consisting of a Cometro 6000LDS pump and a Cometro 6000PVW UV/VIS detector with an Ultimate (250 × 10 mm) preparative column packed with C 18 (5 μm).

Plant Material
The stem of Dendrobium aurantiacum var. denneanum was collected in April of 2011 from a culture field in Shuangliu, Sichuan Province, China. Plant identity was verified by Prof. Min Li (Chengdu University of TCM, Sichuan, China). A voucher specimen (SSF-20110410) was deposited at the School of Pharmacy, Chengdu University of TCM, Chengdu, China.

Extraction and Isolation
The air-dried stem of D. aurantiacum var. denneanum (10 kg) was powdered and extracted three times with 95% EtOH (30 L) for 3 h under reflux. The EtOH extract was evaporated under reduced pressure to yield a dark brown residue (530 g), which was suspended in H 2 O (2.5 L) and then successively partitioned with EtOAc and n-BuOH (6 × 2.5 L). The n-BuOH extract (110 g) was applied to a D-101 macroporous adsorbent resin (1.5 Kg) column. Successive elution of the column with H 2 O, 10% EtOH, 30% EtOH, 50% EtOH, and 95% EtOH (4 L each) yielded five portions. The portion (48 g) eluted by 30% EtOH was separated by MPLC over reversed-phase silica gel eluting with a gradient of increasing MeOH   Table 1.

Enzymatic Hydrolysis of 1
A solution of compound 1 (2 mg) in H 2 O (10 mL) was hydrolyzed with β-glucosidase (15 mg) at 37 °C for 96 h. The reaction mixture was extracted with EtOAc (3 × 10 mL) to yield the individual EtOAc extract and H 2 O phase after removing the solvents. The aqueous phases were subjected to preparative TLC eluted with MeCN−H 2 O (8:1) to yield the sole sugar, which could be identified as D-glucose by the sign of its positive optical rotation. The EtOAc extracts were purified by preparative TLC using CHCl 3 -MeOH (12:1) to afford 1a (0.

Cell Culture and Assessment of Cytotoxic Activity against Human Tumor Cells
The human lung cancer cell lines H1975, H358 and A549, human hepatocellular carcinoma cell lines HepG-2 and SMMC7721, human colorectal carcinoma cell line HCT116, human mammary carcinoma cell lines MDA-MB-231 and MCF-7, human melanoma cell line A2058, human pancreatic cancer cell line PANC-1, human acute myeloid leukemia cell line MV4-11 were obtained from the American Type Culture Collection (ATCC) and grown in RPMI1640, DMEM or IMDM containing 10% fetal bovine serum (v/v) in 5% CO 2 at 37 °C. Cells (2 × 10 3 -10 × 10 3 ) were seeded in 96-well plates and cultured for 24 h, followed by the test compounds treatment at concentrations of 0.625-20 µM for 72 h. After the culture period, 20 µL of MTT (5 mg/mL) was added per well and incubated for 4 h at 37 °C, then 50 μL of 20% acidified SDS was used to lyse the cells. Finally, absorbance was measured at 570 nm using a microplate reader. Each assay was replicated three times. The effect of the compounds on tumor cells viability was calculated and expressed by IC 50 of each cell line.

Cell Culture and Assessment of Neuroprotective Activity
PC12 cells at a density of 5 × 10 3 cells per well in 96-well plates were suspended in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) media supplemented with 5% fetal bovine serum (FBS, Hyclone) and 5% horse serum, penicillin (100 IU/mL), streptomycin (100 μg/mL), and L-glutamine (2 μM) and incubated in a CO 2 incubator (5%) at 37 °C for 24 h. The cells were pre-treated with test compounds and MK-801 for 1 h, respectively, and then exposed to glutamate (50 μM). After incubation for an additional 24 h, MTT (0.5 mg/mL) was added to the medium and incubated for 4 h. Absorbance was measured at 570 nm using a microplate reader, the cell viability was evaluated by relative protection, which was calculated as 100 × [optical density (OD) of test compound + glutamate-treated culture − OD of glutamate-treated culture]/[OD of control culture − OD of glutamate-treated culture].