2-Benzyl-benzofurans from the tubers of Ophiopogon japonicus

Background The overproduction of nitric oxide (NO) is known to involve in various inflammatory processes. A methanol extract of the tubers of Ophiopogon japonicus was found to strongly inhibit NO production. The present paper deals with the isolation, structural identification and NO inhibitory effect of five compounds isolated from the MeOH extract of O. japonicus tubers. Results Three new compounds were elucidated to be (2R)-(4-methoxybenzyl)-5,7-dimethyl-6-hydroxyl-2,3-dihydrobenzofuran (1), 2-(2-hydroxyl-4-methoxy-benzyl)-5-methyl-6-methoxyl-2,3-dihydrobenzofuran (2), and 2-(4-hydroxy-benzyl)-5,6-dihydroxybenzofuran (3). In addition, two known compounds were isolated from a natural source for the first time including 2-(4-methoxy-benzyl)-6,7-dimethoxyl-2,3-dihydrobenzofuran (4), and 2-(4-methoxy-benzyl)-6,7-methylenedioxy-2,3-dihydrobenzofuran (5). The absolute configuration of compound 1 was determined by experimental and calculated circular dichroism spectra. The effects of the isolated compounds on LPS-induced NO production in RAW264.7 cells were evaluated. Compound 1 and 2 showed the inhibitory activity with IC50 values of 11.4 and 29.1 μM, respectively. Conclusions The class of 2-benzyl-2,3-dihydrobenzofuran is uncommon in nature. In this work, three such compounds were isolated from O. japonicus. Two of them showed promising anti-inflammatory activity by inhibition of NO production. Electronic supplementary material The online version of this article (doi:10.1186/s13065-017-0242-z) contains supplementary material, which is available to authorized users.

Nitric oxide (NO) is produced by inducible nitric oxide synthase (iNOS) in macrophages, hepatocytes, and renal cells. When produced in excess, NO directly damages normal tissues and triggers inflammation. Therefore, inhibitors of NO production have potential therapeutic value as anti-inflammatory agents [11]. In our search for anti-inflammatory compounds from natural sources, a methanol (MeOH) extract of the tubers of O. japonicus was found to strongly inhibit NO Open Access  Phytochemical fractionation of the CHCl 3soluble fraction of the MeOH extract led to the isolation  of five 2-benzyl benzofurans, including three new (1-3) and two known (4, 5) compounds (Fig. 1). Compound 1 strongly inhibited NO production in lipopolysaccharide (LPS)-induced RAW264.7 cells.

Table 1 1 H NMR data of compounds 1-5 (δ H in ppm, J in Hz)
The isolated compounds were tested for their ability to inhibit NO production in LPS-stimulated RAW264.7 cells. NO is an important signalling molecule in various physiological and pathophysiological responses, including the circulation, blood pressure, platelet function, and host defence. The overproduction of NO is also important in inflammation and related processes [16]. High NO levels are used as a marker for the treatment of inflammatory disorders. According to its specific functions and characteristics, inhibition of NO production by immune cells, typically macrophages, is suggested as one strategy for the development of antiinflammatory agents. Our test revealed that compounds 1 and 2 reduced NO levels in LPS-stimulated RAW264.7 cells. Compound 1 was the most active compound with an IC 50 of 11.4 μM, while compound 2 had a moderate effect (IC 50 = 29.1 μM). Compounds 3-5 were inactive up to the highest concentration tested (30 μM). The cell viability test showed that neither compound had significant toxicity at its effective dose for NO inhibition (data not shown). 2,3-Dihydrobenzofurans have been known as potent anti-inflammatory compounds. Closse et al. [17] demonstrated that the synthetic analogues of 2,3-dihydrobenzofuran-2-one had powerful anti-inflammatory activity in vivo, and 5-chloro-6-cyclohexyl-2,3-dihydrobenzofuran-2-one was significantly more potent than the reference compound, diclofenac, in all testing models. More recently, a series of dihydrobenzofurans was isolated from the seeds of Prunus tomentosa, some of which strongly inhibited NO production in LPS-stimulated BV-2 cells [18]. Consistently, our results suggest that O. japonicus is a potential natural source of antiinflammatory dihydrobenzofurans.

General experimental procedures
Optical rotation values were recorded on a JASCO P-2000 digital polarimeter (JASCO, Tokyo, Japan). The IR spectra were obtained from a Tensor 37 FT-IR spectrometer (Bruker, Ettlingen, Germany). CD spectra were obtained with a JASCO J-1100 spectropolarimeter. NMR experiments were carried out on a Bruker AM500 FT-NMR spectrometer (Bruker, Rheinstetten, Germany) using residual solvent peak as a reference or tetramethylsilane (TMS) as internal standard. The HR-ESI-MS were recorded on a Waters Q-TOF micromass spectrometer Waters Q-TOF micromass spectrometer and an LTQ Orbitrap XL ™ Mass spectrometer. Absorbance of bioassay solutions was read on an xMark microplate spectrophotometer.

Plant materials
The tubers of O. japonicus were collected in Feb. 2014 at Me Linh, Hanoi and identified by Prof. Tran Huy Thai, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology. The voucher specimens were deposited at the Department of Bioactive Products, Institute of Marine Biochemistry, Vietnam Academy of Science and Technology.

Assay for inhibition of NO production
The effects of compounds on the NO production in LPS-stimulated RAW264.7 cells were evaluated as previously described [19]. The cells were seeded in 96-well plate at 2 × 10 5 cells/well and incubated for 12 h. The plate were pretreated with compounds in various concentrations (from 1 to 30 µM) for 30 min and then incubated for another 24 h with or without 1 μg/ml LPS. 100 μl of the culture supernatant were transferred to other 96-well plate and 100 μl of Griess reagent were added. The absorbance of the reaction solution was read at 570 nm with a microplate reader (XMark microplate reader, Biorad, USA). The remaining cell solutions in cultured 96-well plate were used to evaluate cell viability by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cardamonin was used as a positive control (IC 50 = 2.80 μM).