Anti-inflammatory Lignans from the Fruits of Acanthopanax sessiliflorus

A new lignan, named acanthosessilin A (1), as well as eight known lignan and lignan glycosides 2−9 were isolated from an ethanolic extract of Acanthopanax sessiliflorus fruits. The chemical structures were determined by spectroscopic methods, including HR-EIMS, 1D NMR (1H, 13C, DEPT), 2D NMR (gCOSY, gHSQC, gHMBC, NOESY), and IR spectroscopy. All isolated compounds were tested for the ability to inhibit LPS-induced nitric oxide production in RAW264.7 macrophages.


Introduction
Acanthopanax sessiliflorus (Rupr.et Maxim) Seem, belonging to the Araliaceae family, is widely distributed in Korea, China, and Japan.The bark and twigs of Acanthopanax species are traditionally used in Korea as anti-rheumatoid arthritis, anti-inflammatory, and anti-diabetic drugs and are recognized to have ginseng-like activities [1,2].Previous studies on its phytochemicals resulted in the isolation of lignans from the leaves and roots of Acanthopanax species [3][4][5], and eleutheroside E has been identified as a major compound in the fruits of Acanthopanax species [6].Lignans are thought to OPEN ACCESS be the major active constituents of these plants and are believed to play essential roles in the treatment of diseases [7,8].However, most phytochemical and pharmacological studies have mainly focused on the leaves, bark, and roots of Acanthopanax species, and only a few reports have investigated the fruits.Acanthopanax species are native medicinal plants and the fruits of Acanthopanax species have been used as a remedy to "wipe out evil wind" in traditional medicine [9].To further investigate the bioactive constituents derived in the fruits of these species, the present phytochemical study was initiated.
We report herein on the isolation of a new 3,4-dibenzylfuran lignan (1) from the fruits of A. sessiliflorus, together with eight known compounds 29, and the structural determination of these substances using extensive spectroscopic methods.Several previous studies have provided evidence for the anti-inflammatory effects of extracts and components from Acanthopanax species [10][11][12].Therefore, isolated compounds 19 were evaluated for anti-inflammatory activities through the measurement of nitrite, a soluble oxidation product of nitric oxide (NO), in lipopolysaccharide (LPS)-induced RAW 254.7 macrophage cells.

General
Melting points were obtained using a Fisher-Johns Melting Point Apparatus with a microscope.Ultraviolet spectra were measured on a Shimadzu model UV-1601 spectrophotometer.CD spectra were obtained with a JASCO 715 spectropolarimeter.Optical rotations were measured on a JASCO P-1010 digital polarimeter. 1 H-, 13 C-, and 2D-NMR spectra were recorded on a Varian Unity Inova AS 400 FT-NMR instrument, and chemical shifts were given in δ (ppm) based on tetramethylsilane (TMS) as an internal standard.IR spectra were run on a Perkin Elmer Spectrum One FT-IR spectrometer.EIMS and HR-EIMS spectra were obtained using a JEOL JMS-700 mass spectrometer (Tokyo, Japan).Silica gel 60 (Merck, 230400 mesh), LiChroprep RP-18 (Merck, 4063 μm), and Sephadex LH-20 (Amersham Pharmacia Biotech., Uppsala, Sweden) were used for column chromatography (CC).Pre-coated silica gel plates (Merck, Kieselgel 60 F 254 , 0.25 mm) and pre-coated RP-18 F 254s plates (Merck) were used for analytical thin-layer chromatography analyses.Spots were visualized by spraying with 10% aqueous H 2 SO 4 solution followed by heating.

Plant Material
The fruits of A. sessiliflorus were provided by the Jeongseon Agricultural Extension Center, Jeongseon, Korea in August 2009 and were identified by Prof. Dae-Keun Kim, College of Pharmacy, Woo Suk University, Jeonju, Korea.A voucher specimen (KHU090809) was reserved at the Laboratory of Natural Products Chemistry, Kyung Hee University, Yongin, Korea.

Measurement of NO Production and Cell Viability
Assays for NO production and cell viability were carried out as previously described [23].Briefly, RAW 264.7 macrophages were harvested and seeded in 96-well plates (1 × 10 4 cells/well) for measurement of NO production.The plates were pretreated with various concentrations of samples for 30 min and incubated with LPS (1 μg/mL) for 24 h.The amount of NO was determined by the nitrite concentration in cultured RAW264.7 macrophage supernatants using the Griess reagent.The cell viability was evaluated by MTT reduction.

Conclusions
The new compound 3-(3',4'-dihydroxybenzyl)-4-[(7S),7-hydroxy-3,5-dimethoxybenzyl]tetrahydrofuran, named acanthosessilin A (1), was isolated from Acanthopanax sessiliflorus, together with eight known lignans.According to previous investigations on Acanthopanax species, we have evaluated the inhibitory activities of all compounds against LPS-induced NO production in RAW264.7 macrophages.All compounds moderately inhibited NO production with IC 50 values in the range of 10.34 to 65.07 μM.The results provide a potential explanation for the use of this plant as a herbal medicine in the treatment of inflammatory diseases, and they may be potentially useful in developing new anti-inflammatory agents.
a Assignments were confirmed by DEPT, 1 H 1 H COSY, HSQC, and HMBC.

Table 2 .
Inhibitory effects of compounds 19 against LPS-Induced NO production in RAW 264.7 macrophage cells.IC 50 value of each compound was defined as the concentration (μM) that caused 50% inhibition of NO production in LPS-activated RAW 264.7 macrophage cells.Cells were pretreated for 1 h with compounds before stimulation with LPS (1 μg/mL) for 24 h; b Cell viability indicates mean maximum inhibitory effect, at a concentration of 100 μM, expressed as a percentage inhibition of nitrite production induced by LPS (1 μg/mL) in the presence of vehicle; c Positive control.The results are averages of three independent experiments, and the data are expressed as mean ± SD. a