Inhibitory Effect of 1,5-Dimethyl Citrate from Sea Buckthorn (Hippophae rhamnoides) on Lipopolysaccharide-Induced Inflammatory Response in RAW 264.7 Mouse Macrophages

Hippophae rhamnoides L. (Elaeagnaceae; commonly known as “sea buckthorn” and “vitamin tree”), is a spiny deciduous shrub whose fruit is used in foods and traditional medicines. The H. rhamnoides fruit (berry) is rich in vitamin C, with a level exceeding that found in lemons and oranges. H. rhamnoides berries are usually washed and pressed to create pomace and juice. Today, the powder of the aqueous extract of H. rhamnoides berries are sold as a functional food in many countries. As part of our ongoing effort to identify bioactive constituents from natural resources, we aimed to isolate and identify those from the fruits of H. rhamnoides. Phytochemical analysis of the extract of H. rhamnoides fruits led to the isolation and identification of six compounds, namely, a citric acid derivative (1), a phenolic (2), flavonoids (3 and 4), and megastigmane compounds (5 and 6). Treatment with compounds 1–6 did not have any impact on the cell viability of RAW 264.7 mouse macrophages. However, pretreatment with these compounds suppressed lipopolysaccharide (LPS)-induced NO production in RAW 264.7 mouse macrophages in a concentration-dependent manner. Among the isolated compounds, compound 1 was identified as the most active, with an IC50 of 39.76 ± 0.16 μM. This value was comparable to that of the NG-methyl-L-arginine acetate salt, a nitric oxide synthase inhibitor with an IC50 of 28.48 ± 0.05 μM. Western blot analysis demonstrated that compound 1 inhibited the LPS-induced expression of IKKα/β (IκB kinase alpha/beta), I-κBα (inhibitor of kappa B alpha), nuclear factor kappa-B (NF-κB) p65, iNOS (inducible nitric oxide synthase), and COX-2 (cyclooxygenase-2) in RAW 264.7 cells. Furthermore, LPS-stimulated cytokine production was detected using a sandwich enzyme-linked immunosorbent assay. Compound 1 decreased interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) production in LPS-stimulated RAW 264.7 cells. In summary, the mechanism of action of 1 included the suppression of LPS-induced NO production in RAW 264.7 cells by inhibiting IKKα/β, I-κBα, NF-κB p65, iNOS, and COX-2, and the activities of IL-6 and TNF-α.


Plant Material
The powder of H. rhamnoides fruits was purchased in October of 2018 from Korea Beauty and Healthcare Co., Ltd. Thereafter, the material was identified by one of the authors (K.H.K). A voucher specimen of the material (VT-2018) was deposited in the herbarium at the School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea.

Measurement of RAW 264.7 Cell Viability
RAW 264.7 cell viability was determined using an Ez-Cytox cell viability detection kit (Daeil Lab Service Co., Seoul, Korea). Cells were plated in 96-well plates at a density of 3 × 10 4 cells/well and incubated for 24 h at 37 • C. Cells were exposed to compounds at concentrations of 5, 10, 25, 50, and 100 µM for 1 h at 37 • C. Thereafter, they were incubated with vehicle (medium containing 0.5% DMSO) for 24 h at 37 • C. After that, they were incubated with the Ez-Cytox solution for 40 min at 37 • C. Absorbance was determined at 450 nm using a PowerWave XS microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

Measurement of NO Production in RAW 264.7 Cells
RAW 264.7 cells were plated in 96-well plates at a density of 3 × 10 4 cells/well for a 24 h incubation at 37 • C. Cells were either exposed to the compounds or N G -methyl-L-arginine acetate salt (L-NMMA) at concentrations of 5, 10, 25, 50, and 100 µM for 1 h at 37 • C. Thereafter, they were incubated with 1 µg/mL LPS for 24 h at 37 • C. The supernatant was mixed with an equal volume of Griess reagent containing 0.2% naphthylethylenediamine dihydrochloride (Sigma-Aldrich, St. Louis, MO, USA) and 2% sulfanilamide (Sigma-Aldrich) in 5% phosphoric acid (Sigma-Aldrich) and incubated for 40 min at 37 • C. The absorbance was then measured at 540 nm using a PowerWave XS microplate reader.

Western Blot Analysis
RAW 264.7 cells were plated in 6-well plates at a density of 2 × 10 5 cells/well and incubated for 24 h at 37 • C. Cells were exposed to 50 and 100 µM of compound 1 for 1 h at 37 • C prior to incubation with 1 µg/mL LPS for 24 h at 37 • C. Whole-cell extracts were then prepared using radio immunoprecipitation assay buffer (RIPA buffer; Cell Signaling Technology, Inc., Beverly, MA, USA) containing 1× ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF), according to the manufacturer's instructions. Protein concentration of the whole-cell extracts was determined with a bicinchoninic acid (BCA) protein assay kit. Proteins (20 µg/lane) were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for 90 min at 110 V, transferred to a polyvinylidene fluoride (PVDF) membrane, and analyzed with epitope-specific primary antibodies to phospho-IκB kinase (IKK)α/β, IKKα (IκB kinase alpha), IKKβ (IκB kinase beta), I-κBα (inhibitor of kappa B alpha), phospho-I-κBα, phospho-NF-κB p65, NF-κB p65, and iNOS, followed by cyclooxygenase-2 (COX-2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies (Cell Signaling, MA, USA). The bound antibodies were then visualized with ECL Advance Western Blotting Detection Reagents (GE Healthcare, Cambridge, United Kingdom) and a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany). The optical densities of the immunoreactive bands were obtained using ImageJ software (Version 1.51J; National Institutes of Health, Bethesda, MD, USA) and normalized with those of the control. Data represented by fold-increases were compared to the control. The GAPDH was used for loading control.

Determination of IL-6 and TNF-α Production
RAW 264.7 cells were plated in 24-well plates at a density of 4 × 10 5 cells/well and incubated for 24 h at 37 • C. Cells were then exposed to compound 1 at concentrations of 50 and 100 µM for 1 h at 37 • C prior to incubation with 1 µg/mL LPS for 24 h at 37 • C. To determine IL-6 and tumor necrosis factor alpha (TNF-α) production, the supernatant was obtained and assayed to quantify the levels of these cytokines using an IL-6 sandwich enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, CA, USA) and a TNF-α ELISA kit (eBiosciences, CA, USA), according to the manufacturer's instructions.

Statistical Analysis
At least three independent determinations were carried out for each experiment. All data are presented as average value and standard deviation (SD). Statistical significance was determined using one-way analysis of variance (ANOVA), and multiple comparisons were carried out with a Bonferroni correction. A p-value less than 0.05 indicated statistical significance. All statistical analyses were performed using SPSS Statistics version 19.0 (SPSS Inc., Chicago, IL, USA).

Isolation and Identification of Compounds
The aqueous extract powder of H. rhamnoides fruits was first suspended in distilled water, and then subjected to solvent-partitioning with n-hexane, CH 2 Cl 2 , ethyl acetate, and n-BuOH to obtain each solvent fraction. Phytochemical analysis of the CH 2 Cl 2 -soluble and ethyl acetate-soluble fractions was performed using repeated column chromatography, HPLC, and LC/MS. Through chemical analysis, six compounds (1-6), including a citric acid derivative (1), a phenolic (2), two flavonoids (3 and 4), and two megastigmane compounds (5 and 6) ( Figure 1) were isolated; compounds 1-4 were isolated from the ethyl acetate-soluble fraction whereas compounds 5-6 were isolated from the CH 2 Cl 2 -soluble fraction. By comparing the NMR spectroscopic data of the compounds to their reported values and the LC/MS results, we identified that the compounds were 1,5-dimethyl citrate (1) [23], 5-methoxysalicylic acid (2) [24], syringetin-3-O-glucoside (3) [25], isorhamnetin-3-O-glucoside (4) [9], (+)-dehydrovomifoliol (5) [26], and (+)-vomifoliol (6) [27]. In particular, the absolute configurations for compounds 5 and 6 were established by their positive specific rotation values and comparison of their NMR spectroscopic data with those reported earlier and electronic circular dichroism (ECD) data ( Figure S1) [26,27]. RAW 264.7 cells were plated in 24-well plates at a density of 4 × 10 5 cells/well and incubated for 24 h at 37 °C. Cells were then exposed to compound 1 at concentrations of 50 and 100 μM for 1 h at 37 °C prior to incubation with 1 μg/mL LPS for 24 h at 37 °C. To determine IL-6 and tumor necrosis factor alpha (TNF-α) production, the supernatant was obtained and assayed to quantify the levels of these cytokines using an IL-6 sandwich enzyme-linked immunosorbent assay (ELISA) kit (BD Biosciences, CA, USA) and a TNF-α ELISA kit (eBiosciences, CA, USA), according to the manufacturer's instructions.

Statistical Analysis
At least three independent determinations were carried out for each experiment. All data are presented as average value and standard deviation (SD). Statistical significance was determined using one-way analysis of variance (ANOVA), and multiple comparisons were carried out with a Bonferroni correction. A p-value less than 0.05 indicated statistical significance. All statistical analyses were performed using SPSS Statistics version 19.0 (SPSS Inc., Chicago, IL, USA).

Effects of Compounds 1-6 on Cell Viability
Prior to evaluating the effect of the compounds on NO production, we determined their cytotoxicity in RAW 264.7 mouse macrophages using an Ez-Cytox cell viability assay. As a result, the

Effects of Compounds 1-6 on Cell Viability
Prior to evaluating the effect of the compounds on NO production, we determined their cytotoxicity in RAW 264.7 mouse macrophages using an Ez-Cytox cell viability assay. As a result, the compounds were considered to be cytotoxic when the cell viability in the compound-treated group was less than 80% of that in the untreated group. At all tested concentrations, all concentrations of compounds 1-6 had no cytotoxicity on RAW 264.7 mouse macrophages ( Figure 2). Therefore, all concentrations were employed for the subsequent experiments.
Foods 2020, 9, x FOR PEER REVIEW 6 of 13 compounds were considered to be cytotoxic when the cell viability in the compound-treated group was less than 80% of that in the untreated group. At all tested concentrations, all concentrations of compounds 1-6 had no cytotoxicity on RAW 264.7 mouse macrophages ( Figure 2). Therefore, all concentrations were employed for the subsequent experiments.

Effects of Compounds 1-6 on NO Production
The production of NO in LPS-induced RAW 264.7 macrophages has been used as an indicator to evaluate the anti-inflammatory effects of natural products. In the present study, we evaluated the effect of compounds 1-6 on LPS-induced NO production in RAW 264.7 cells. In this assay, L-NMMA, a nitric oxide synthase inhibitor, was used as the positive control. As shown in Figure 3, exposure to 1 μg/mL LPS induced a significant increase in NO production; however, this was suppressed by pretreatment with compounds 1-6, which had 50% inhibitory concentration (IC50) values of 39.76 ± 0.16 μM, 79.39 ± 0.29 μM, 86.33 ± 0.54 μM, 87.01 ± 0.30 μM, 74.71 ± 0.24 μM, and 76.12 ± 0.14 μM, respectively. Although the IC50 values of most compounds were slightly weaker than that of L-NMMA (IC50, 28.48 ± 0.05 μM), compound 1 displayed a similar IC50 value, which indicates that it might inhibit the increase of LPS-induced NO production in RAW 264.7 macrophages. This finding suggests that the inflammatory responses induced by extracellular stimuli might be suppressed via treatment with compound 1. Thus, we proceeded to investigate the potential benefit of compound 1 as an NO production inhibitor by carrying out a mechanism study.

Effects of Compounds 1-6 on NO Production
The production of NO in LPS-induced RAW 264.7 macrophages has been used as an indicator to evaluate the anti-inflammatory effects of natural products. In the present study, we evaluated the effect of compounds 1-6 on LPS-induced NO production in RAW 264.7 cells. In this assay, L-NMMA, a nitric oxide synthase inhibitor, was used as the positive control. As shown in Figure 3, exposure to 1 µg/mL LPS induced a significant increase in NO production; however, this was suppressed by pretreatment with compounds 1-6, which had 50% inhibitory concentration (IC 50 ) values of 39.76 ± 0.16 µM, 79.39 ± 0.29 µM, 86.33 ± 0.54 µM, 87.01 ± 0.30 µM, 74.71 ± 0.24 µM, and 76.12 ± 0.14 µM, respectively. Although the IC 50 values of most compounds were slightly weaker than that of L-NMMA (IC 50 , 28.48 ± 0.05 µM), compound 1 displayed a similar IC 50 value, which indicates that it might inhibit the increase of LPS-induced NO production in RAW 264.7 macrophages. This finding suggests that the inflammatory responses induced by extracellular stimuli might be suppressed via treatment with compound 1. Thus, we proceeded to investigate the potential benefit of compound 1 as an NO production inhibitor by carrying out a mechanism study.

Compound 1 Downregulated IKKα/β, I-κBα, and NF-κB p65 in LPS-Stimulated RAW 264.7 Mouse Macrophages
When macrophages are stimulated with LPS, IKKα/β is phosphorylated, thereby leading to the subsequent phosphorylation and degradation of IκBα, which enables the nuclear translocation of NF-κB [28]. NF-κB p65 is a core mediator of the innate immune response that leads to the transcription of genes encoding pro-inflammatory mediators, such as iNOS and COX-2, and pro-inflammatory cytokines, such as TNF-α and IL-6 [29,30]. Therefore, to confirm whether pre-treatment with compound 1 could suppress LPS-stimulated NO production through the NF-κB p65 signaling pathway, we determined the effect of compound 1 on the expression of IKKα/β, I-κBα, and NF-κB p65 in LPS-stimulated RAW 264.7 mouse macrophages. As shown in Figure 4, exposure to 1 μg/mL LPS significantly upregulated IKKα/β, I-κBα, and NF-κB p65; however, pretreatment with 50 and 100 μM of compound 1 reversed these changes induced by LPS. Such findings indicate that compound 1 inhibits NO production by inhibiting the phosphorylation of IKKα/β, I-κBα, and NF-κB p65 in RAW 264.7 macrophages. On the basis of these results, it appears that the action site of compound 1 is upstream of the IKK site, thus modulating inflammatory mediator expression.

Compound 1 Downregulated IKKα/β, I-κBα, and NF-κB p65 in LPS-Stimulated RAW 264.7 Mouse Macrophages
When macrophages are stimulated with LPS, IKKα/β is phosphorylated, thereby leading to the subsequent phosphorylation and degradation of IκBα, which enables the nuclear translocation of NF-κB [28]. NF-κB p65 is a core mediator of the innate immune response that leads to the transcription of genes encoding pro-inflammatory mediators, such as iNOS and COX-2, and pro-inflammatory cytokines, such as TNF-α and IL-6 [29,30]. Therefore, to confirm whether pre-treatment with compound 1 could suppress LPS-stimulated NO production through the NF-κB p65 signaling pathway, we determined the effect of compound 1 on the expression of IKKα/β, I-κBα, and NF-κB p65 in LPS-stimulated RAW 264.7 mouse macrophages. As shown in Figure 4, exposure to 1 µg/mL LPS significantly upregulated IKKα/β, I-κBα, and NF-κB p65; however, pretreatment with 50 and 100 µM of compound 1 reversed these changes induced by LPS. Such findings indicate that compound 1 inhibits NO production by inhibiting the phosphorylation of IKKα/β, I-κBα, and NF-κB p65 in RAW 264.7 macrophages. On the basis of these results, it appears that the action site of compound 1 is upstream of the IKK site, thus modulating inflammatory mediator expression.

Compound 1 downregulated iNOS and COX-2 expression in LPS-stimulated RAW 264.7 mouse macrophages
The induction of iNOS and COX-2 during inflammation directly contributes to NO synthesis [31]. Therefore, we proceeded to verify whether compound 1 (1,5-dimethyl citrate) affects LPSstimulated iNOS and COX-2. As shown in Figure 5, exposure to 1 μg/mL LPS significantly upregulated iNOS and COX-2; however, pretreatment with 50 and 100 μM of compound 1 could reverse the changes induced by LPS. Compound 1 could therefore inhibit the increase in iNOS and COX-2 induced via the NF-κB signaling pathway. On the basis of these results, the suppression in NO production of compound 1 might be associated with downregulating the expression of iNOS and COX-2 by LPS stimulation in RAW 264.7 macrophages.

Compound 1 downregulated iNOS and COX-2 expression in LPS-stimulated RAW 264.7 mouse macrophages
The induction of iNOS and COX-2 during inflammation directly contributes to NO synthesis [31]. Therefore, we proceeded to verify whether compound 1 (1,5-dimethyl citrate) affects LPS-stimulated iNOS and COX-2. As shown in Figure 5, exposure to 1 µg/mL LPS significantly upregulated iNOS and COX-2; however, pretreatment with 50 and 100 µM of compound 1 could reverse the changes induced by LPS. Compound 1 could therefore inhibit the increase in iNOS and COX-2 induced via the NF-κB signaling pathway. On the basis of these results, the suppression in NO production of compound 1 might be associated with downregulating the expression of iNOS and COX-2 by LPS stimulation in RAW 264.7 macrophages.

Compound 1 downregulated iNOS and COX-2 expression in LPS-stimulated RAW 264.7 mouse macrophages
The induction of iNOS and COX-2 during inflammation directly contributes to NO synthesis [31]. Therefore, we proceeded to verify whether compound 1 (1,5-dimethyl citrate) affects LPSstimulated iNOS and COX-2. As shown in Figure 5, exposure to 1 μg/mL LPS significantly upregulated iNOS and COX-2; however, pretreatment with 50 and 100 μM of compound 1 could reverse the changes induced by LPS. Compound 1 could therefore inhibit the increase in iNOS and COX-2 induced via the NF-κB signaling pathway. On the basis of these results, the suppression in NO production of compound 1 might be associated with downregulating the expression of iNOS and COX-2 by LPS stimulation in RAW 264.7 macrophages.

Compound 1 downregulated IL-6 and TNF-α production in LPS-stimulated RAW 264.7 mouse macrophages
Elevated expression levels of iNOS and COX-2 followed by an increase in the production of NO have been found to be evoked by TNF-α and IL-6 in RAW 264.7 macrophages [32]. Lastly, we proceeded to examine whether compound 1 affected the production of pro-inflammatory cytokines, including IL-6 and TNF-α, in LPS-stimulated RAW 264.7 mouse macrophages. As shown in Figure 6, treating RAW 264.7 macrophages with LPS markedly increased IL-6 and TNF-α production, which were ameliorated by 50 and 100 µM of compound 1 in a concentration-dependent manner. These results suggest that compound 1 inhibits IL-6 and TNF-α production, ultimately leading to a reduction in inflammatory response. Altogether, we found that the inhibition of NO production in LPS-activated RAW 264.7 macrophages by compound 1 was mediated by the inhibition of IKKα/β, I-κBα, NF-κB p65, iNOS, and COX-2, as well as the activities of IL-6 and TNF-α (Figure 7). Foods 2020, 9, x FOR PEER REVIEW 9 of 13 expression. Quantitative graph of (B) iNOS and (C) COX-2 (mean ± SD, * p < 0.05 compared to the LPS-treated group). C: control group treated with 0.5% DMSO. 1,5-dimethyl citrate (1).

Compound 1 downregulated IL-6 and TNF-α production in LPS-stimulated RAW 264.7 mouse macrophages
Elevated expression levels of iNOS and COX-2 followed by an increase in the production of NO have been found to be evoked by TNF-α and IL-6 in RAW 264.7 macrophages [32]. Lastly, we proceeded to examine whether compound 1 affected the production of pro-inflammatory cytokines, including IL-6 and TNF-α, in LPS-stimulated RAW 264.7 mouse macrophages. As shown in Figure 6, treating RAW 264.7 macrophages with LPS markedly increased IL-6 and TNF-α production, which were ameliorated by 50 and 100 μM of compound 1 in a concentration-dependent manner. These results suggest that compound 1 inhibits IL-6 and TNF-α production, ultimately leading to a reduction in inflammatory response. Altogether, we found that the inhibition of NO production in LPS-activated RAW 264.7 macrophages by compound 1 was mediated by the inhibition of IKKα/β, I-κBα, NF-κB p65, iNOS, and COX-2, as well as the activities of IL-6 and TNF-α (Figure 7).  expression. Quantitative graph of (B) iNOS and (C) COX-2 (mean ± SD, * p < 0.05 compared to the LPS-treated group). C: control group treated with 0.5% DMSO. 1,5-dimethyl citrate (1).

Compound 1 downregulated IL-6 and TNF-α production in LPS-stimulated RAW 264.7 mouse macrophages
Elevated expression levels of iNOS and COX-2 followed by an increase in the production of NO have been found to be evoked by TNF-α and IL-6 in RAW 264.7 macrophages [32]. Lastly, we proceeded to examine whether compound 1 affected the production of pro-inflammatory cytokines, including IL-6 and TNF-α, in LPS-stimulated RAW 264.7 mouse macrophages. As shown in Figure 6, treating RAW 264.7 macrophages with LPS markedly increased IL-6 and TNF-α production, which were ameliorated by 50 and 100 μM of compound 1 in a concentration-dependent manner. These results suggest that compound 1 inhibits IL-6 and TNF-α production, ultimately leading to a reduction in inflammatory response. Altogether, we found that the inhibition of NO production in LPS-activated RAW 264.7 macrophages by compound 1 was mediated by the inhibition of IKKα/β, I-κBα, NF-κB p65, iNOS, and COX-2, as well as the activities of IL-6 and TNF-α (Figure 7).  In previous studies, flavonoids as main components of sea buckthorn (H. rhamnoides) including isorhamnetin, quercetin, and kaempferol, inhibited LPS-induced NO production in RAW 264.7 cells through the inhibition of expressions of iNOS, COX-2, proteins in the mitogen-activated protein kinase (MAPK) pathway (c-Jun N-terminal kinase and p38), I-κBα, NF-κB p65, and production of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) [33]. The mechanism of action of flavonoids from sea buckthorn was similar to that of 1,5-dimethyl citrate (1) that we identified in this study. However, it has not been known exactly which flavonoid of sea buckthorn exhibits the anti-inflammatory effect. To the best of our knowledge, this study is the first to evaluate the anti-inflammatory activity of citric acid derivative isolated from sea buckthorn on LPS-induced inflammatory response in RAW 264.7 mouse macrophages. The active compound, 1,5-dimethyl citrate (1), was a simple derivative of citric acid present in many plants and fruits [34][35][36][37]. An anti-inflammatory effect of citric acid derivatives has been reported in previous studies [34][35][36][37]. In LPS-treated mice, increased TNF-α in mice brain tissue and iNOS expression in the cytoplasm of hepatocytes were decreased after treatment of citric acid [34]. In a recent study, wheat germ extract with citric acid was reported to inhibit secretion of the pro-inflammatory cytokines, TNF-α, IL-6, and IL-12 and the synthesis of COX-2, as well as phosphorylation of NF-κB p65 and p38 kinase in LPS-activated macrophages [35]. In addition, calcium citrate showed antioxidant enzyme activities, and inhibited NO production by suppression of the expression of pro-inflammatory mediators (NF-κB, iNOS, and COX-2) and cytokines (TNF-α, IL-1β, and IL-6) in LPS-stimulated RAW 264.7 macrophages [36]. Another recent study reported that citric acid displayed anti-inflammatory function via the toll-like receptor (TLR)-mediated activation of NF-κB and interferon regulatory factor-3 signaling pathways, and that citric acid contributed to the maintenance of tight junction proteins via the TLR-mediated p38 and c-Jun N-terminal kinase pathways [37]. Interestingly, a new oligosaccharide citric acid derivative isolated from Vigna angularis (ohwi et ohashi. var. Dainagon) seeds displayed promising anti-influenza A virus activity [38]. Additionally, a recent paper reported that citric acid showed protective effect against hepatic ischemia reperfusion injury in Sprague-Dawley rats, which suggests significant therapeutic potential of citric acid in ischemic liver injury [39]. Although future in vivo studies should support the anti-inflammatory effects of 1,5-dimethyl citrate (1), identified as an active compound in this study, our findings suggest a potential application of 1,5-dimethyl citrate (1) for the treatment of inflammatory diseases.

Conclusions
Herein, we provided experimental evidence of the potential role of 1,5-dimethyl citrate from H. rhamnoides fruits in the management and treatment of inflammatory diseases. By performing a phytochemical analysis of the extracts of H. rhamnoides fruits using repeated column chromatography, HPLC, and LC/MS, we isolated and identified six compounds (1-6), namely, a citric acid derivative (1), a phenolic (2), two flavonoids (3 and 4), and two megastigmane compounds (5 and 6). Among them, 1,5-dimethyl citrate (1) was demonstrated as effectively preventing LPS-induced NO production and markedly inhibited the expression of IKKα/β, I-κBα, NF-κB p65, iNOS, and COX-2, and the activities of IL-6 and TNF-α. On the basis of our findings, we have provided experimental evidence that the 1,5-dimethyl citrate (1) obtained from H. rhamnoides fruits could function as an effective agent for the treatment of inflammatory diseases.
Supplementary Materials: The following are available online at http://www.mdpi.com/2304-8158/9/3/269/s1: Figure S1: ECD data of compounds 5 (A) and 6 (B), Figure S2: LC/MS-based analysis (detection wavelength was set as 210 nm) of the ethyl acetate-soluble fraction as well as the chemical structures of compounds 1-4, Figure S3: LC/MS-based analysis (detection wavelength was set as 210 nm) of the CH2Cl2-soluble fraction as well as the chemical structures of compounds 5-6, Figure S4: LC/MS analysis for purity of compounds 1-6.