Evaluation of anti-inflammatory activity of compounds isolated from the rhizome of Ophiopogon japonicas

Background Ophiopogon japonicas (L.f) Ker-Gawl has been used as a traditional Chinese medicine to cure acute and chronic inflammation and cardiovascular diseases including thrombotic diseases for thousands of years. Previous phytochemical studies showed that O. japonicus contained compounds with anti-inflammatory activity. The aim of this study was to identify and isolate compounds with anti-inflammatory activity from the rhizome of O. japonicas. Methods Compounds were isolated by various column chromatography and their structures were identified in terms of nuclear magnetic resonance spectrum (NMR) and mass spectrum (MS). To measure the anti-inflammatory effects of thirteen compounds in LPS-induced RAW 264.7 macrophage cells, we used the following methods: cell viability assay, nitric oxide assay, enzyme-linked immunosorbent assay, quantitative real-time PCR analysis and western blotting analysis. Results One new and twelve known compounds (mainly homoisoflavonoids) were extracted from O. japonicas, in which 4′-O-Demethylophiopogonanone E (10) was considered as a new compound, additionally, compounds 4-O-(2-Hydroxy-1- hydroxymethylethyl)-dihydroconiferyl alcohol (2) and 5,7-dihydroxy-6-methyl-3-(2′, 4′-dihydroxybenzyl) chroman-4-one (12) were isolated from the rhizome of O. japonicas for the first time. The isolated compounds Oleic acid (3), Palmitic acid (4), desmethylisoophiopogonone B [5,7-dihydroxy-3-(4′-hydroxybenzyl)-8- methyl- chromone] (5), 5,7-dihydroxy-6-methyl-3-(4′-hydroxybenzyl) chromone (7) and 10 significantly suppressed the production of NO in LPS-induced RAW 264.7 cells. Especially compound 10 showed the strongest effect against the production of the pro-inflammatory cytokine IL-1β and IL-6 with the IC50 value of 32.5 ± 3.5 μg/mL and 13.4 ± 2.3 μg/mL, respectively. Further analysis elucidated that the anti-inflammatory activity of compound 10 might be exerted through inhibiting the phosphorylation of ERK1/2 and JNK in MAPK signaling pathways to decrease NO and pro-inflammatory cytokines production. Conclusions Our results indicated that 4′-O-Demethylophiopogonanone E can be considered as a potential source of therapeutic medicine for inflammatory diseases.


Background
Inflammation is a biological response of tissue in attempting self-protection against harmful stimuli, caused by a mechanical or biological agent or by an aberrant autoimmune response [1]. Macrophages play a vital role in inflammatory response in the initiation, maintenance and resolution of inflammation [2]. In macrophages, lipopolysaccharide (LPS), a well-known endotoxin, induces the release of numerous pro-inflammatory mediators such as inducible nitric oxide synthase (iNOS) and inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-6 (IL-6), which play a major role in the pathogenesis of various inflammatory disorders and serve as significant biomarkers for the assessment of the inflammatory process [3][4][5][6]. iNOS is a protein whose expression is regulated by activation of NF-κB, which contribute to the production of NO [7,8]. Furthermore, mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERK1/ 2), c-Jun NH 2 -terminal kinases (JNK) and p38, play important roles in regulation of the inflammatory response by mediators. The signaling pathways of MAPKs can lead to the activation of NF-κB and induce expression of pro-inflammatory genes, including IL-1β, IL-6 and iNOS [9][10][11].
The plant Ophiopogon japonicus (L.f ) Ker-Gawl is widely distributed in Southeast Asia, especially in most areas of China. Its rhizome is the primary medical portion and has been used as a traditional Chinese medicine to treat the inflammatory diseases for thousands of years [12]. O. japonicus is an important nourishing-yin drug in the traditional Chinese herbs with various bioactivities, including anti-inflammation, anti-cardiovascular diseases, anti-tumor, anti-aging, immunoregulation [13]. Previous phytochemical studies showed that the rhizome of O. japonicas contained homoisoflavonoids, saponins, amides, monoterpene glycosides, and so on [14].
The study of the biological functions of O. japonicas has been limited largely to demonstration of antioxidant activities in vitro. Few studies have been performed to explore the relationship between the bioactive constituents of O. japonicas and their antiinflammatory properties. Moreover, the molecular mechanisms underlying anti-inflammatory activities of O. japonicus remain unclear in the literature. In this report, we describe the isolation and identification of the compounds extracted from O. japonicus, followed by an evaluation of the anti-inflammatory activities of these compounds. This work was aimed to identify the chemical components with anti-inflammatory activity from O. japonicus and to demonstrate their targeted pathway by the use of a bioactivity guided experimental design.

Plant materials
The radix of O. japonicus were purchased from Kangmei Pharmaceutical Co. Ltd., Guangdong, China and were authenticated by Ph. D. Zhi-Hai Huang, department of the Second Institute of Clinical Medicine, Guangzhou University of Chinese medicine, Guangzhou, China. The voucher specimens are deposited at the Guangdong Provincial Academy of Chinese Medical Sciences for future reference, and the voucher specimen number is 110811881.

Instruments
1D-and 2D-NMR spectra data were obtained on a Bruker Avance 300 and 500 NMR spectrometer, with TMS as an internal standard. Electrospray ionization mass spectra (ESI-MS) were measured on a Thermo Scientific Finnigan LTQ mass spectrometer, and Preparative HPLC was conducted using a Waters 2545 Binary gradient module instrument with 2998 Photodiode Array Detector. Column chromatography (CC) separations were performed with D101 resin column (Beijing greenherbs Co. Ltd., China), silica gel (100-200 mesh, Qingdao Haiyang Chemical Co. Ltd., China), Sephadex LH-20 (Pharmcia Biotech AB, Uppsala, Sweden). TLC was carried out on glass precoated silica gel GF 254 plates (Yantai Chemical Industrial Institute, China) and spots were visualized under UV light ((k = 254 nm or 366 nm) or by spraying with 10% (v/v) sulfuric acid in ethanol followed by heating to 105°C. Real-time PCR (Rrism 7500, Applied Biosystems), Nano Drop 2000C Spectrophotometer (Thermo Scientific) and Chemidoc imaging system (BIO-RAD) were for the cell assay.

Cell culture
The mouse RAW264.7 macrophage cell line was purchased from the cell bank of the Chinese Academy of Sciences (CAS, Shanghai, China). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 in Dulbecco′s Modified Eagle Medium (DMEM, Sigma, St. Louis, Mo, USA) supplemented with 10% heatinactivated fetal bovine serum (FBS, Sigma).

Cell viability assay
The cytotoxicity of the isolated compounds toward RAW264.7 macrophage cells were evaluated by a conventional MTT assay as reported previously [15]. RAW 264.7 cells (1.0 × 10 5 cells/well) were given a volume of 100 μl to 96-well plates and incubated for 24 h and then treated with or without different concentrations of compounds (1, 5, 25, 50 and 100 μg/mL). Dimethyl sulphoxide (DMSO) was used to dissolve the dried samples, and its final concentration of DMSO in the culture medium was maintained at less than 0.1% (v/v). The culture plates were kept at 37°C with 5%CO 2 and the assay for each concentration of extracts were performed in triplicates. After additional 24 h incubation, the medium was discarded, and then new medium without PBS was added, and the cells were incubated for 4 h with a solution of 5 mg/mL MTT. The supernatant was removed carefully, and 200 μL DMSO was added to dissolve the formazan crystals. The plate was shaken for 10 mins, and the absorbance was recorded on a Thermo Scientific microplate spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) at the wavelength of 490 nm.

Nitric oxide assay
The accumulation of nitrite, an indicator of NO production in the culture medium, was measured with the Griess reagent [16]. RAW264.7 cells were plated at 4 × 10 5 cells/mL in 24 wells-culture plates. After 24 h incubation, cells were pre-incubated in medium with or without various concentrations of compounds for 2 h. The experimental groups were then stimulated with LPS (final concentration 1 μg/mL) at 37°C for another 24 h, in while the dexamethasone (DXM) group at the concentration of 50 μg/mL was as a positive control. Subsequently, the supernatant was collected. Fifty microlitres of cell culture medium were mixed with 50 μL of Griess reagent (equal volumes of 1% sulphanilamide in 5% phosphoric acid and 0.1% N-1-naphtylethylenediamine dihydrochloride in distilled water), incubated at room temperature for 10 min, and then the absorbance at 540 nm was measured in a microplate reader. Nitrite concentrations in the supernatant were determined by comparison with a sodium nitrite standard curve.

Enzyme-linked immunosorbent assay
The supernatant above-mentioned in the 24 wellsculture plates were collected. Pro-inflammatory cytokines (IL-1β and IL-6) levels in culture medium were determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) according to the manufacturer's instructions.

Western blotting analysis
Western blotting was performed according to a standard method [18]. The cells were seeded in 6-well plates (1.0 × 10 6 cells/well) and the groups were the same as above. Proteins were extracted from cells in the ice-cold RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing with PMSF, and incubated with 40 min on the ice. After centrifugation at 12,000 rpm for 20 min, the supernatant was collected. The protein concentration was determined using the BCA kit (Sigma-Aldrich) according to the manufacturer's instruction. The protein from each sample was boiled with loading buffer for 10 min, forty microgram of protein per lane was electrophoresed through 12% SDS-PAGE gel, and followed by transferring to a PVDF membranes, which were activated in methanol. The membrane was blocked with 5% skim milk for 1 h at room temperature, and then incubated with primary antibodies (Cell Signaling Technology Inc., Beverly, MA, USA) at 1:1000 (v/v) dilution in 5% BSA at 4°C overnight, and washed three times with TBST buffer. The membrane was followed by incubation for 1 h at room temperature with horseradish peroxidaseconjugated anti-rabbit IgG secondary antibody (CST). Then the blots were washed with TBST buffer three times and visualized by an enhanced chemiluminescent (ECL) detection solution.

Statistical analysis
Statistical comparisons were performed between the control and treated groups. The data obtained from three independent experiments were reported as mean values ± standard deviations. Statistical analysis was carried out using SPSS 19.0 software. The data were subjected to analysis of variance (ANOVA) for comparing three or more groups, a significant difference was assumed at a level of p < 0.05.
Ophiopogonanone E (1). Yellow amorphous powder;   1H, m, H-3) were observed, which indicated the γdihydropyrone moiety. Combined with the two benzylmethylene protons appeared at δ H 3.14 (1H, dd, J = 13.7, 4.9 Hz, H-9a) and 2.57 (1H, dd, J = 13.7, 10.0 Hz, H-9b), compound 10 was presumed to be a homoisoflavonoid derivative [19]. After carefully analysis of the NMR spectra of 10, the spectral properties were found very similar to those of ophiopogonanone E [19]. The notable difference was the presence of a hydroxy group instead of a methoxy group at C-4′ in 10, compared with ophiopogonanone E. The change and the structure of 10 were further confirmed by HMBC correlations from δ H 3.71 (3H, s, OCH 3

Effects of isolated compounds (1-13) on Cell Viability of RAW 264.7Macrophages
The cytotoxicity of these compounds on the proliferation of RAW264.7 cells was measured using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay [27]. The results were shown as relative cell viability referred to control (equal to 100%). In this study, 13 compounds of O. japonicas were shown different cytotoxicity effect to RAW 264.7 cells. The safe concentration of compound 2, 4 and 13 is 100 μg/mL, while compound 3, 5 and 10 is 50 μg/mL, which is the cell survival probability more than 80%, the other compounds have some cytotoxicity at the concentrations (1-100 μg/mL) (Fig. 3).

Effects of isolated compounds (1-13) on the Production of NO in LPS-induced RAW264.7 Macrophages
To evaluate the effect of compounds (1-13) on NO production in LPS-stimulated RAW 264.7 cells, we measured the nitrite concentration in the cultured media using Griess reagent [28]. Compared with the LPS group, the anti-inflammatory activities of compounds 3, 4, 5, 7 and 10 increased in accordance with the increase in concentration. As shown in Fig. 4, compounds 3, 4, 5, 7 and 10 displayed an inhibitory potency, with IC 50 values of 80.2 ± 2.3, 33.4 ± 2.9, 14.1 ± 1.5, 10.9 ± 0.8 and 66.4 ± 3.5 μg/mL, respectively. Moreover, compounds 4, 5 and 7 showed stronger anti-inflammatory abilities than the positive control DXM at the concentration of 50 μg/ mL (p < 0.01), in which compounds 5 and 7 belonged to homoisoflavonoids. Thus, it is possible to demonstrate that the anti-inflammatory activity of O. japonicas might be derived from its rich in homoisoflavonoids compounds, however, there are few literatures reported on its anti-inflammatory mechanism.
Effects of the compounds 3-5, 7 and 10 on the Production of Pro-Inflammatory Cytokines (IL-1β and IL-6) in LPS-induced RAW264.7 Macrophages Because IL-1β and IL-6 are early secreted proinflammatory cytokines and their elevated levels can be detected in a variety of acute and chronic inflammatory diseases [29], we measured IL-1β and IL-6 production in the supernatant of RAW 264.7 cells by ELISA. The LPS group significantly increased IL-1β and IL-6 production compared with the control group. The release of both cytokines were remarkably decreased in a dose-  Of three independent experiments. ## p < 0.01 as compared with the control group, * p < 0.05 and ** p < 0.01 versus the LPS group, △p < 0.05 and △△p < 0.01 versus the DXM group dependent manner (p < 0.05) in the groups pretreated with compounds 3, 4, 5, 7 and 10 ( Fig. 5a and b). Moreover, the IL-1β IC 50 values of compounds 4, 5 and 10 were 65.3 ± 6.8, 64.3 ± 7.9 and 32.5 ± 3.5 μg/mL, respectively. In addition to IL-6, compounds 3, 4, 5, 7 and 10 displayed an inhibitory potency, with IC 50 values of 58.9 ± 6.8, 71.6 ± 11.7, 32.4 ± 3.6, 11.5 ± 2.8 and 13.4 ± 2.3 μg/ mL, respectively. This result indicated that 4'-O-Demethylophiopogonanone E (10) efficiently suppressed LPS-induced IL-1β and IL-6 production, especially, it showed stronger anti-inflammatory ability than DXM at the concentration of 50 μg/mL (p < 0.05). Its effect on the LPS-induced IL-1β, IL-6 and iNOS mRNA expressions needs further investigation. Previous studies have indicated that IL-1β and IL-6 are crucial factors involved in all kinds of inflammatory processes that can be regulated by activation of NF-κB [30][31][32]. In addition, MAPKs are also known to be important for the transcriptional NF-κB pathway activation and are related to iNOS expression [33]. To investigate whether the suppression of inflammatory responses by 4'-O-Demethylophiopogonanone E was mediated through the MAPKs pathways, we estimated the effects of compound 10 on the LPS-stimulated phosphorylation of ERK1/2 and JNK in RAW 264.7 cells by Western blotting. As shown in Fig. 7, this research demonstrated that LPS significantly increased the phosphorylation of MAPKs signaling proteins, while pre-treatment with compound 10 considerably Fig. 6 Effects of 4'-O-demethylophiopogonanone E (10) on the mRNA expression of IL-1β (a), IL-6 (b) and iNOS (c) in LPS-induced RAW264.7 macrophages. Cells were pre-treated for 2 h with indicated concentrations of 10 and then stimulated with LPS (final concentration 1 μg/mL) for another 12 h. Data are expressed as mean ± S.D. Of three independent experiments. ## p < 0.01 as compared with the control group, * p < 0.05 and ** p < 0.01 versus the LPS group, △ p < 0.05 and △△ p < 0.01 versus the DXM group inhibited the phosphorylation of ERK1/2 and JNK, however it did not have a distinct dose-dependent pattern. In conclusion, our findings suggested that 4'-O-Demethylophiopogonanone E might decrease NO, IL-1β and IL-6 production via inhibitions of the MAPKs signaling pathways.

Conclusion
In this study, a new compound 4'-O-Demethylophiopogonanone E was isolated and identified from the rhizome of O. japonicas. Moreover, we firstly investigated the effect and mechanism of 4'-O-Demethylophiopogonanone E on LPS-stimulated NO and pro-inflammatory cytokines including IL-1β and IL-6. NO is synthesized from L-arginine by iNOS, and its overproduction is involved in cytotoxicity and tissue damage in the inflammatory process [34,35]. Overexpression of iNOS by inflammatory agents accompanied with inflammatory disorder including IL-1β and IL-6, which are resulted in acute and chronic response to inflammatory diseases [36]. In the present study, we clearly demonstrated that 4'-O-Demethylophiopogonanone E significantly inhibited LPS-stimulated NO production and pro-inflammatory cytokines (IL-1β and IL-6) in macrophages via downregulation of the mRNA expressions of iNOS, IL-1β and IL-6 ( Fig. 6).
Moreover, MAPKs have been involved in pro-inflammatory signaling cascades and large numbers of evidence has demonstrated that the activation of ERK1/2 and JNK is involved in up-regulation of nitric oxide and pro-inflammatory cytokines in LPS-induced macrophages [37,38]. Thus, we assessed that 4'-O-Demethylophiopogonanone E inhibited the inflammatory response via blocking the phosphorylation of MAPKs in LPSstimulated macrophages. In the results of 4'-O-Demethylophiopogonanone E pretreatment, we found that 4'-O-Demethylophiopogonanone E suppressed LPS-stimulated ERK1/2 and JNK MAPK phosphorylation (Fig. 7). This result suggests that 4'-O-Demethylophiopogonanone E exerted anti-inflammatory actions via inhibition of iNOS, IL-1β and IL-6 gene expressions through suppression of the MAPKs signaling pathways.
Consequently, based on these findings, we provide clear evidence and molecular basis for the antiinflammatory mechanism of 4'-O-Demethylophiopogonanone E and show great potential as a novel herbal ingredient for the treatment of inflammatory diseases.