Anti-Inflammatory Activity of Heterocarpin from the Salt Marsh Plant Corydalis heterocarpa in LPS-Induced RAW 264.7 Macrophage Cells

The inhibitory effect of three chromones 1–3 and two coumarins 4–5 on the production of nitric oxide (NO) was evaluated in LPS-induced RAW 264.7 macrophage cells. Among the compounds tested heterocarpin (1), a furochromone, significantly inhibited its production in a dose-dependent manner. In addition, heterocarpin suppressed prostaglandin E2 (PGE2) production and expression of cytokines such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6).


Introduction
Halophytes are naturally salt-tolerant plants that may be potentially useful as new sources of biologically active substances. These plants are exposed in their habitats to various abiotic constraints (salinity, drought, heat/cold, luminosity and other harsh environmental conditions), and as a result, they have had to adapt to their environment by developing specific responses which often include the synthesis of unusual secondary metabolites. Corydalis heterocarpa is a salt-tolerant plant species that grows on sandy seashores throughout the western coastal area of South Korea. The genus Corydalis is composed of more than 400 species in Eurasia and North America. For centuries some Corydalis species have been long used for the treatment of ailments in Asian countries including China, Korea, and Japan. C. heterocarpa has been also used in Korean folk medicine to treat labor pains, spasms, boils, and dysentery. Nevertheless, only a few studies of this species have been reported. To date C. heterocarpa has been shown to possess UVB-protection, anti-tumor, anti-oxidant and anti-inflammatory effects. These effects seem to be due to coumarins, promoting them as a source of biofunctional products [1][2][3][4][5]. Like other salt marsh plants, a high content of phytochemicals which are crucially needed to endure the highly salinic environmental conditions is a characteristic of C. heterocarpa. Coupled with the high potential of phytochemicals and high rates of nutritional consumption, the utilization of C. heterocarpa as a functional food has been suggested. In this regard, research on the secondary metabolites and bioactivities of the C. heterocarpa has not been done intensely enough.

Isolation and Structure Determination of Compounds 1-5
Shade-dried whole plants of Corydalis heterocarpa were extracted twice overnight with CH2Cl2 and MeOH at room temperature. The combined crude extracts were concentrated in vacuo at 40 °C to leave a dark brown gum and then partitioned between CH2Cl2 and H2O. Each layer was further partitioned with n-hexane/85% aq. MeOH and n-BuOH/H2O, respectively. The n-BuOH and 85% aq. MeOH fractions were purified by various column chromatographies, TLC and HPLC methods using different solvent combinations to yield three chromones and two coumarins ( Figure 1A).

Cell Viability
We first measured the cytotoxicity of compounds 1-5 in RAW 264.7 cells by using the MTT assay. The result showed compounds 1-5 at the concentrations used (50 and 100 μM) had no cytotoxic effect, except compound 4 at a concentration of 100 μM ( Figure 1B).

Effect of Compounds 1-5 on LPS-induced NO Production and iNOS Expression
To investigate the inhibitory effect of compounds 1-5 on NO production, RAW 264.7 cells were pretreated with each compound for 1 h prior to stimulation with LPS. Following 24 h of LPS stimulation, the levels of NO production in the culture media were measured. As shown in Figure 2A, LPS stimulation resulted in a marked induction of NO production when compared to the untreated cells and pretreatment with compound 1 suppressed the NO production with an IC50 value of 66.6 μM ( Figure 2B), whereas other compounds did not reduce nitrite levels ( Figure 2A). To determine whether suppression of NO production by compound 1 was due to reduced iNOS protein expression, cells were pretreated with different concentrations of compound 1 and stimulated with LPS for 24 h. The significant induction of iNOS protein by LPS stimulation was inhibited by compound 1 in a dose-dependent manner ( Figure 2C). These results showed that compound 1 inhibited NO production by the decreased iNOS protein expression.

Effect of Compound 1 on LPS-Induced PGE2 Production and COX-2 Expression
To further elucidate the anti-inflammatory response of compound 1, we examined the inhibitory effect of compounds 1 on PGE2 production following LPS stimulation in RAW 264.7 cells. Briefly, the cells were pretreated with various concentrations of compound 1 for 1 h and then stimulated with LPS. After incubation for 24 h, the production of PGE2 from the culture supernatant was measured using ELISA. As shown in Figure 3A, the amount of PGE2 in the culture supernatant increased with LPS stimulation, and then this increase was reduced by treatment with compound 1 in a dose-dependent manner (p < 0.05). To investigate whether the inhibitory effects of compound 1 on PGE2 production are related to the modulation of the COX-2 expression, we evaluated the COX-2 protein levels using Western blot analysis. The COX-2 protein was not detected in the absence of LPS stimulation, but the levels of COX-2 were induced after LPS exposure. As shown in Figure 3B, compound 1 reduced LPS-stimulated COX-2 protein expression. Control values were obtained in the absence of LPS or compound 1. The values are the mean ± SD of three independent experiments (significant as compared to control. * p < 0.05 and ** p < 0.01).

Effects of Compound 1 on Pro-Inflammatory Cytokine Production
To further determine the anti-inflammatory effect of compound 1, the secreted pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6 were measured by ELISA. Treatment of RAW 264.7 cells with LPS alone resulted in a marked induction of cytokine production when compared to the untreated cells (p < 0.001) ( Figure 4). However, the pretreatment of compound 1 at 100 μM significantly decreased TNF-α levels compared to the supernatant of the LPS-stimulated cells (p < 0.05). In addition, the levels of IL-1β and IL-6 were increased in the culture supernatant of the LPS-stimulated cells whereas pretreatment with compound 1 at 50 and 100 μM resulted in a decrease in cytokine production (p < 0.05 or 0.01).

Discussion
In recent years, there has been much interest in and research on the influence of natural products on several diseases, including aging-related illness, cancer, neurodegenerative disease, and cardiovascular disease. Corydalis species have been extensively investigated for this purpose, and have yielded alkaloids, isoquinoline alkaloids, and cyanidin glycosides as their secondary metabolites [10][11][12]. Corydalis heterocarpa has been widely used as a folk medicine for treatment of labor pains, spasms, boils and dysentery [2][3][4][5]. Macrophages play an important role in inflammatory disorders through the release of factors such as NO, cytokines, and prostaglandin mediators in the immune system [13][14][15][16]. The inducible form of NO is generated from L-arginine by iNOS. NO, a free radical, plays important roles in a variety of physiological functions such as host defense, vasodilation, and regulation of cellular functions [17,18]. However, overproduction of NO by iNOS further enhances cyclooxygenase 2 (COX-2) activities, and can be deleterious to the host [19][20][21]. COX-2 is a highly inducible enzyme that catalyzes the conversion of arachidonic acid into prostaglandins (PGs) during inflammation. One of the PGs produced at high levels in inflammation is prostaglandin E2 (PGE2), a major COX-2 product at inflammatory sites, which contributes to local blood flow increases, edema formation, and pain sensitization [22,23]. Data represent the mean ± SD with three separate experiments (significant as compared to control. * p < 0.05, ** p < 0.01).
Five compounds from Corydalis heterocarpa were screened for their ability to inhibit LPS-induced NO production in RAW 264.7 macrophages at a concentration of 100 μM. This activity was not attributable to cytotoxicity of 1-5 as confirmed by the MTT assay, which showed little cytotoxicity for any of these compounds at the screened concentrations. Among the compounds tested, heterocarpin (1) most strongly inhibited NO production, so heterocarpin was further investigated for its inhibitory effect on production of PGE2, the major metabolite of the COX-2 pathway, which plays a critical role in the pathogenesis of acute and chronic inflammatory diseases [24,25]. PGE2 production was prominently decreased by heterocarpin, showing inhibition rate of 85.5% at a concentration of 100 μM. To confirm the underlying mechanism of this potent effect, we examined its effect on protein expression of both iNOS and COX-2 enzymes, which are known to be responsible for NO and PGE2 production, respectively. We found that heterocarpin suppressed LPS-induced iNOS and COX-2 protein expression. This means that the inhibition of NO and PGE2 production by heterocarpin is a result of the inhibition of the iNOS and COX-2 expression.
NO and PGE2 are known to act as secondary mediators of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6, which are considered to be important initiators of the inflammatory response and mediators of the development of various inflammatory diseases [26,27]. Among these, TNF-α is implicated as a key cytokine playing an important role in the immune response such as autoimmune reactions, and its production is crucially required for the synergistic induction of NO synthesis in LPS-stimulated macrophages [28]. IL-1β is also an important mediator of the inflammatory response produced by activated macrophages, and is involved in a variety of cellular activities such as cell proliferation, differentiation, and apoptosis. Likewise, IL-6 plays a role as a crucial pro-inflammatory cytokine, regarded as an endogenous mediator of LPS-induced fever [15,16,[29][30][31]. In this study, heterocarpin inhibited production of TNF-α, IL-1β, and IL-6 in a dose dependent manner in LPS-stimulated RAW 264.7 cells. In particular, IL-1β production was greatly decreased by heterocarpin, with an inhibition ratio of 95.7% at a concentration of 100 μM.
In conclusion, these results suggest that heterocarpin suppresses the production of NO, PGE2, and pro-inflammatory cytokines TNF-α, IL-1β and IL-6 by reducing their protein expression levels as measured by western blot analysis. Therefore, heterocarpin may be useful for the treatment of inflammatory diseases, although further studies are needed to elucidate the mechanism of its in vivo anti-inflammatory activity.

General Procedures
Optical rotation was determined on a Perkin-Elmer polarimeter 341 (The Perkin-Elmer Co., Norwalk, CT, USA) using a 1 cm cell. NMR spectra were recorded in CD3OD and CDCl3 on a Varian Mercury 300 instrument (Varian, Palo Alto, CA, USA) at 300 MHz for 1 H and 75 MHz for 13 C using standard pulse sequence programs. All chemical shifts were recorded with respect to TMS as an internal standard. Mass spectroscopic data were obtained at the Korean Basic Science Institute, Seoul, Korea. High performance liquid chromatography (HPLC) was performed with a Dionex P580 (Dionex Corp., Sunnyvale, CA, USA) with a Varian 350 RI detector (Varian, Walnut Creek, CA, USA). All solvents used were spectroscopic grade or were distilled from glass prior to use.

Extraction and Isolation
The collected samples (300 g) were air-dried, chopped into small pieces, and extracted for 2 days with MeOH (3 L × 2) and CH2Cl2 (3 L × 2), respectively. The combined crude extracts (41.1 g) were evaporated under reduced pressure and partitioned between CH2Cl2 and water. The organic layer was further partitioned between 85% aq. MeOH and n-hexane, and the aqueous layer was fractionated with n-BuOH and H2O, successively, to afford n-hexane (7.3 g), 85% aqueous MeOH (12.0 g), n-BuOH (4.3 g), and water (20.0 g).

Cell Culture
A RAW 264.7 murine macrophage cell line was obtained from the Korean Cell Line Bank (Seoul, Korea) and then cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 100 μM MEM non-essential amino acid solution at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.

Measurement of Cell Viability
Cell viability was assessed using the CellTiter 96 Aqueous One kit (Promega). Briefly, RAW 264.7 cells were seeded onto a 96 well plate at 5 × 10 4 cells/well. After incubation with various concentration of compounds, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2Htetrazolium (MTS), which is converted to a formazan product by metabolically active cells, was added to each well. After 2 h of incubation, the optical densities at 490 nm were measured using a microplate reader (Tecan Systems, San Jose, CA, USA). The compounds were considered to be cytotoxic when the optical density of the sample-treated group was less than 90% of that in the control group.

Measurement of Nitrite and PGE2
RAW 264.7 cells (2 × 10 5 cells/well) were seeded onto a 24-well culture plate at 37 °C for overnight in medium. The cells were preincubated with different concentrations of the compounds for 1 h and then incubated for 24 h with or without LPS. NO production was monitored by measuring nitrite levels in the culture media using Griess reagent (1% sulfanilamide, 0.1% N-1-naphthylenediamine dihydrochloride and 2.5% phosphoric acid). The absorbance was measured at 570 nm after incubation for 10 min. The nitrite levels in the samples were calculated from a standard curve generated using known concentrations of sodium nitrite. The PGE2 concentration in the culture supernatant was also measured to determine the inhibitory activities of compound 1 using an enzyme immunoassay kit (Cayman Chemical) according to the manufacturer's instructions.

Enzyme-Linked Immunosorbent Assay (ELISA)
RAW 264.7 cells (2 × 10 5 cells/well) were plated in a 24-well plate and then incubated with compound 1 for 1 h in the present or absence of LPS (100 ng/mL) for 24 h. The supernatants of cell cultures were used to measure the TNF-α, IL-1β and IL-6 levels using ELISA kits (R&D Systems) according to the manufacturer's instructions. The cytokine concentration in the samples was calculated from a standard curve developed using a known concentration of recombinant TNF-α, IL-1β and IL-6.

Western Blot Analysis
After activation with LPS, RAW 264.7 cells (2 × 10 6 cells/well) were washed once with 10 mM PBS (pH 7.4) containing 150 mM NaCl and then lysed with PRO-PREP™ Protein extraction solution (iNtRON Biotechnology). The protein concentration was determined using the Pierce ® 660 nm protein assay reagent (Rockford, IL, USA) according to the manufacturer's instructions. Lysates (20 μg/lane) were separated by 10% SDS-PAGE on polyacrylamide gels and then transferred to polyvinylidene difluoride membranes. Next, the membranes were blocked with 5% non-fat dry milk or BSA in PBS-T solution for 1 h and then probed with iNOS (BD Biosciences) and COX-2 antibodies (Cayman Chemical) overnight at 4 °C. The membranes were washed with PBS-T three times and incubated with a secondary HRP-conjugated antibody for 2 h at room temperature. Following three further washings in PBS-T, the protein bands were visualized using an ECL detection reagent (Pierce).

Statistical Analysis
The data were presented as mean ± SD. The statistical significance of the difference between mean values was determined by the pair t-test. p-values less than 0.05 or 0.01 were considered statistically significant.