1-Carbomethoxy-β-Carboline, Derived from Portulaca oleracea L., Ameliorates LPS-Mediated Inflammatory Response Associated with MAPK Signaling and Nuclear Translocation of NF-κB

Portulaca oleracea is as a medicinal plant known for its neuroprotective, hepatoprotective, antidiabetic, antioxidant, anticancer, antimicrobial, antiulcerogenic, and anti-inflammatory activities. However, the specific active compounds responsible for the individual pharmacological effects of P. oleracea extract (95% EtOH) remain unknown. Here, we hypothesized that alkaloids, the most abundant constituents in P. oleracea extract, are responsible for its anti-inflammatory activity. We investigated the phytochemical substituents (compounds 1–22) using nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS) and screened their effects on NO production in lipopolysaccharide (LPS)-induced macrophages. Compound 20, 1-carbomethoxy-β-carboline, as an alkaloid structure, ameliorated nitric oxide (NO) production, inducible nitric oxide synthase (iNOS), and proinflammatory cytokines associated with the mitogen-activated protein kinase (MAPK) pathways, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Subsequently, we observed that compound 20 suppressed nuclear translocation of nuclear factor κB (NF-κB) using immunocytochemistry. Moreover, we recently reported that compound 8, trans-N-feruloyl-3’, 7’-dimethoxytyramine, was originally purified from P. oleracea extracts. Our results suggest that 1-carbomethoxy-β-carboline, the most effective anti-inflammatory agent among alkaloids in the 95% EtOH extract of P. oleracea, was suppressing the MAPK pathway and nuclear translocation of NF-κB. Therefore, P. oleracea extracts and specifically 1-carbomethoxy-β-carboline may be novel therapeutic candidates for the treatment of inflammatory diseases associated with the activation of MAPKs and NF-κB.

NF-κB acts on a family of inducible transcription factors that regulate a large array of genes engaged in different processes in immune response and inflammation in innate immune cells, including macrophages [13]. The canonical NF-κB pathway responds to several stimuli, including ligands of various cytokine receptors, pattern-recognition receptors (PRRs) [14]. In particular, mammalian cells express five families of PRRs, namely, Toll-like receptors (TLRs), RIG-I-like receptors, nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs), C-type lectin-like receptors, and cytosolic DNA sensors [15]. Among these PRRs, toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) leads to macrophage differentiation toward the M1 phenotype [16]. NF-κB is a novel transcription factor of M1 macrophages and is required for the induction of diverse inflammatory genes, including those encoding TNF-α, IL-6, and IL-1β [17]. Additionally, mitogen-activated protein kinase (MAPK) pathways, associated with extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase, enhance NO production and inducible inducible nitric oxide synthase (iNOS) by the activation of the nuclear translocation of NF-κB [18]. If a natural compound suppresses MAPK pathways and reduces the activation of the nuclear translocation of NF-κB, it may be a potential therapeutic agent for the treatment of inflammatory diseases.
Although P. oleracea extracts exhibit diverse pharmacological effects, including anti-inflammatory activity, individual components of P. oleracea extracts must be separated and the bioactivity of each component should be evaluated prior to the therapeutic application of this material in clinical trials or as a health functional food. In our study, we first elucidated the chemical structures of the phytochemical constituents (compounds 1 to 22) from P. oleracea extracts (95% EtOH) using spectroscopic data, including NMR and ESI-MS. Among these constituents, this is the first report that 1-carbomethoxy-β-carboline, an alkaloid structure, significantly ameliorated NO production and proinflammatory cytokines associated with the MAPK pathway and the nuclear translocation of NF-κB under LPS-mediated inflammatory conditions in RAW 264.7. Additionally, we originally report the identification of a new compound, trans-N-feruloyl-3', 7'-dimethoxytyramine, from the P. oleracea extract.

Nitric Oxide Production Screening Using Twenty-Two Compounds Isolated from Portulaca oleracea
NO is a major proinflammatory mediator, involved in the pathogenesis of inflammation [33]. During an inflammatory response, the large amount of NO formed by the action of iNOS surpasses

Nitric Oxide Production Screening Using Twenty-Two Compounds Isolated from Portulaca oleracea
NO is a major proinflammatory mediator, involved in the pathogenesis of inflammation [33]. During an inflammatory response, the large amount of NO formed by the action of iNOS surpasses the standard physiological amount of NO [34]. A previous study reported that P. oleracea extracts had anti-inflammatory effects and ameliorated inflammatory bowel disease under dextran sulfate sodium-induced colitis by inhibiting NF-κB and MAPK activation [35,36]. However, the specific active compound responsible for the inflammatory effects remained unclear. Then, we evaluated NO production in LPS-induced RAW 264.7, a mouse macrophage cell line, upon treatment with the 22 compounds isolated from the P. oleracea extract ( Figure 3). sodium-induced colitis by inhibiting NF-κB and MAPK activation [35,36]. However, the specific active compound responsible for the inflammatory effects remained unclear. Then, we evaluated NO production in LPS-induced RAW 264.7, a mouse macrophage cell line, upon treatment with the 22 compounds isolated from the P. oleracea extract ( Figure 3).
After screening the impacts of these compounds on NO production, we selected the most potent inhibitor of NO production based on an inhibitory effect of 50% or more as the cut-off. Phenylbutazone (100 μg/mL), one of the nonsteroidal anti-inflammatory drugs, had the best inhibitory effect on nitric oxide of about 50% in RAW 264.7 cells [37]. Of these isolated compounds, compounds that inhibited NO production beyond 50% cut-off under LPS-induced inflammatory conditions were compounds 15 (scinamide C) and 20 (1-carbomethoxy-β-carboline) ( Figure 3A), and their inhibitory rates were 50.92 ± 0.95% (compound 15) and 53.33 ± 2.49% (compound 20).
In NO production screening in Figure. 3, compounds 3, 5, 6, 9, and 16 showed inhibitory effects of NO production similar to compounds 15 and 20. However, compounds 3, 5, 6, 9, and 16 were already reported in previous studies associated with anti-inflammatory effects or cellular toxicity. Compound 3, derived from Arcangelisia gusanlung and Wolfberry, and compound 6, derived from Wolfberry, were previously reported to inhibit NO production [38,39]. Compound 5, derived from Solanum nigrum, had an anti-inflammatory effect by inhibition of leukotriene C4 (LTC4) [40] and compound 16, derived from P. oleracea, had a neuroprotective effect by reducing reactive oxygen species (ROS) and inhibiting ERK 1/2 phosphorylation [41]. Compound 9, derived from Hibiscus tiliaceus, had a cytotoxic effect [42]. Thus, we decided to further evaluate compounds 15 and 20. Additionally, to investigate the cellular toxicology after treatment with compounds 15 and 20, we tested cell viability upon treatment with the test compounds at 1 to 100 μM using the MTT assay ( Figure 3B). Neither compound showed toxicological effects below 100 μM. These results suggested that the major constituents of P. oleracea extract responsible for its ability to inhibit NO production were compounds 15 and 20.  After screening the impacts of these compounds on NO production, we selected the most potent inhibitor of NO production based on an inhibitory effect of 50% or more as the cut-off. Phenylbutazone (100 µg/mL), one of the nonsteroidal anti-inflammatory drugs, had the best inhibitory effect on nitric oxide of about 50% in RAW 264.7 cells [37]. Of these isolated compounds, compounds that inhibited NO production beyond 50% cut-off under LPS-induced inflammatory conditions were compounds 15 (scinamide C) and 20 (1-carbomethoxy-β-carboline) ( Figure 3A), and their inhibitory rates were 50.92 ± 0.95% (compound 15) and 53.33 ± 2.49% (compound 20).
In NO production screening in Figure 3, compounds 3, 5, 6, 9, and 16 showed inhibitory effects of NO production similar to compounds 15 and 20. However, compounds 3, 5, 6, 9, and 16 were already reported in previous studies associated with anti-inflammatory effects or cellular toxicity. Compound 3, derived from Arcangelisia gusanlung and Wolfberry, and compound 6, derived from Wolfberry, were previously reported to inhibit NO production [38,39]. Compound 5, derived from Solanum nigrum, had an anti-inflammatory effect by inhibition of leukotriene C 4 (LTC 4 ) [40] and compound 16, derived from P. oleracea, had a neuroprotective effect by reducing reactive oxygen species (ROS) and inhibiting ERK 1/2 phosphorylation [41]. Compound 9, derived from Hibiscus tiliaceus, had a cytotoxic effect [42]. Thus, we decided to further evaluate compounds 15 and 20. Additionally, to investigate the cellular toxicology after treatment with compounds 15 and 20, we tested cell viability upon treatment with the test compounds at 1 to 100 µM using the MTT assay ( Figure 3B). Neither compound showed toxicological effects below 100 µM. These results suggested that the major constituents of P. oleracea extract responsible for its ability to inhibit NO production were compounds 15 and 20.

Inhibitory Activities of Proinflammatory Mediators by Compounds 15 and 20
iNOS, which is involved in the production of NO, is a novel signaling molecule associated with the MAPK pathway and NF-κB activity in both microglia and macrophage cells [43]. Subsequently, proinflammatory cytokines such as TNF-α, IL-6, and IL-1β are induced by activating nuclear translocation of NF-κB [44]. To investigate intracellular biological evidence of reduced NO level by compound 15 or 20, we evaluated the effect of compounds 15 and 20 on the proinflammatory mediators such as iNOS, TNF-α, IL-6, and IL-1β in LPS-induced macrophages (Figure 4). In our results, compound 20 was found to be the most effective inhibitor of iNOS under LPS-induced macrophages based on Western blotting compared with compound 15 ( Figure 4A). The inhibitory effect of compound 20 (12.5 µM) was similar to that of dexamethasone (positive control, 10 µM). Moreover, we tested proinflammatory cytokines after treatment with compound 15 or 20. Treatment with compound 15 (25 µM) or 20 (12.5 µM) significantly inhibited the proinflammatory cytokine mRNA of TNF-α, IL-6, and IL-1β. There were significantly inhibitory effects of proinflammatory cytokine mRNA of TNF-α, IL-6, and IL-1β after treatment of compound 15 (25 µM) or 20 (12.5 µM). These results suggested that of the alkaloids in P. oleracea extracts (95% EtOH), the major active compound responsible for its anti-inflammatory activity was compound 20 (1-carbomethoxy-beta-carboline). μM) or 20 (12.5 μM) significantly inhibited the proinflammatory cytokine mRNA of TNF-α, IL-6, and IL-1β. There were significantly inhibitory effects of proinflammatory cytokine mRNA of TNF-α, IL-6, and IL-1β after treatment of compound 15 (25 μM) or 20 (12.5 μM). These results suggested that of the alkaloids in P. oleracea extracts (95% EtOH), the major active compound responsible for its antiinflammatory activity was compound 20 (1-carbomethoxy-beta-carboline). Relative ratio of iNOS versus β-actin was measured using densitometry, and dexamethasone was used as positive control. These graphs represented that compounds 15 and 20 dose-dependently inhibited iNOS using immunoblot analysis. Cells were pretreated with each compound for 2 h and stimulated with LPS (1 μg/mL) for 16 h. Immunoblot analysis was performed in triplicate tests, and results are expressed as means ± SEM. An unpaired Student's t-test was used for statistical analysis. ###p < 0.001 versus Con, *p < 0.05, **p < 0.01, and ***p < 0.001 versus LPS. (C-E) The mRNA expression levels of TNF-α, IL-6, and IL-1β were measured using quantitative real-time PCR experiment, and (B) Relative ratio of iNOS versus β-actin was measured using densitometry, and dexamethasone was used as positive control. These graphs represented that compounds 15 and 20 dose-dependently inhibited iNOS using immunoblot analysis. Cells were pretreated with each compound for 2 h and stimulated with LPS (1 µg/mL) for 16 h. Immunoblot analysis was performed in triplicate tests, and results are expressed as means ± SEM. An unpaired Student's t-test was used for statistical analysis. ### p < 0.001 versus Con, * p < 0.05, ** p < 0.01, and *** p < 0.001 versus LPS. (C-E) The mRNA expression levels of TNF-α, IL-6, and IL-1β were measured using quantitative real-time PCR experiment, and these proinflammatory cytokines were significantly diminished by compounds 15 and 21. Cells were preincubated for 2 h with compounds 1 and 3 at concentrations of 5 and 10 µM, respectively, and activated by LPS (1 µg/mL) for 2 h. Results represented as mean ± SEM, and dexamethasone was used as a positive control. # p < 0.05, ## p < 0.01, ### p < 0.001 versus Con, * p < 0.05, ** p < 0.01, and *** p < 0.001 versus LPS. Con: control, LPS: lipopolysaccharide, Dx: dexamethasone.

Portulaca oleracea Inhibits Proinflammatory and Inflammatory Signaling
If compound 20 decreased proinflammatory mediators associated with MAPKs and nuclear translocation of NF-κB with a potency similar to dexamethasone and nonsteroidal anti-inflammatory drugs (NSAIDs) [45], compound 20 may be a useful therapeutic agent for treating inflammatory diseases with minimal toxic side effects. Then, to further investigate the anti-inflammatory effects associated with the inhibition of NO production and iNOS, we evaluated the major inflammatory signaling pathways, MAPKs, associated with p38, ERK, and JNK and nuclear translocation of NF-κB in LPS-induced murine macrophages, RAW 264.7 cells upon treatment with or without compound 20. Western blotting indicated that pretreatment with compound 20 remarkably disturbed the MAPK signaling pathways associated with JNK, ERK, and p38 under LPS-induced inflammatory conditions in RAW 264.7 cells ( Figure 5A). The protein expression ratios of p38, ERK, and JNK are shown in Figure 5B-D.

Portulaca oleracea Inhibits Proinflammatory and Inflammatory Signaling
If compound 20 decreased proinflammatory mediators associated with MAPKs and nuclear translocation of NF-κB with a potency similar to dexamethasone and nonsteroidal anti-inflammatory drugs (NSAIDs) [45], compound 20 may be a useful therapeutic agent for treating inflammatory diseases with minimal toxic side effects. Then, to further investigate the anti-inflammatory effects associated with the inhibition of NO production and iNOS, we evaluated the major inflammatory signaling pathways, MAPKs, associated with p38, ERK, and JNK and nuclear translocation of NF-κB in LPS-induced murine macrophages, RAW 264.7 cells upon treatment with or without compound 20. Western blotting indicated that pretreatment with compound 20 remarkably disturbed the MAPK signaling pathways associated with JNK, ERK, and p38 under LPS-induced inflammatory conditions in RAW 264.7 cells ( Figure 5A). The protein expression ratios of p38, ERK, and JNK are shown in Figure 5B-D. Subsequently, we tested prevention by pretreatment with compound 20 and qualitatively and quantitatively analyzed NF-κB ( Figure 6). First, we determined whether compound 20 suppressed the nuclear translocation of NF-κB. Immunocytochemistry clearly demonstrated that compound 20 reduced the translocation of NF-κB into the nucleus in RAW 264.7 cells ( Figure 6A). Furthermore, we evaluated nuclear NF-κB and cytosolic IκBα using Western blotting ( Figure 6B). Compound 20 remarkably ameliorated translocation of nuclear NF-κB by suppressing the degradation of IκBα, similar to dexamethasone ( Figure 6C,D). A schematic of the mechanism of compound 20 is shown in Figure 6E. These results suggest that compound 20 significantly inhibited the MAPK pathway and Subsequently, we tested prevention by pretreatment with compound 20 and qualitatively and quantitatively analyzed NF-κB ( Figure 6). First, we determined whether compound 20 suppressed the nuclear translocation of NF-κB. Immunocytochemistry clearly demonstrated that compound 20 reduced the translocation of NF-κB into the nucleus in RAW 264.7 cells ( Figure 6A). Furthermore, we evaluated nuclear NF-κB and cytosolic IκBα using Western blotting ( Figure 6B). Compound 20 remarkably ameliorated translocation of nuclear NF-κB by suppressing the degradation of IκBα, similar to dexamethasone ( Figure 6C,D). A schematic of the mechanism of compound 20 is shown in Figure 6E. These results suggest that compound 20 significantly inhibited the MAPK pathway and suppressed the degradation of IκBα along with the nuclear localization of NF-κB under LPS-induced inflammation in RAW 264.7 cells.  Values are means ± SEM, and an unpaired Student's t-test was used for statistical analysis. #p < 0.05, and ###p < 0.001 versus Con, *p < 0.05, **p < 0.01, and ***p < 0.001 represented significant differences from the LPS-treated group. Con: control, LPS: lipopolysaccharide, Dx: dexamethasone.

General Experimental Procedures
The structures of isolated compounds were identified by spectroscopic data including 1 H NMR, 13 C NMR, COSY, HMBC, HMQC, HRESIMS, IR, and optical rotation. 1 H, 13 C, and 2D NMR spectra were recorded on a JNM-ECA600 (Jeol, Tokyo, Japan) instrument using TMS as references. High-resolution electrospray ionization mass spectrometry (HRESIMS) was carried out using a Waters SYNAPT G2-Si HDMS spectrometer (Waters, Milford, MA, USA). IR spectra were obtained on a FT/IR-4600 (Jasco, Tokyo, Japan) spectrometer. UV spectra were recorded on a spectraMax M2 ϴ (Molecular Devices, Sunnyvale, CA, USA) spectrophotometer. Optical rotations were measured on a Jasco P-2000 (C) The graph is described as relative ratio of NF-κB to lamin B. (D) The graph shows the relative ratio of IκBα to β-actin using densitometry of ImageJ. (E) The schematic pathway of compound 20 is exhibited. Cells were pretreated for 2 h with compound 20 at concentration of 12.5 µM, and stimulated with LPS (1 µg/mL) for 1 h. Dexamethasone was used as positive control, and immunoblot analysis was performed as triplicate experiments. Values are means ± SEM, and an unpaired Student's t-test was used for statistical analysis. # p < 0.05, and ### p < 0.001 versus Con, * p < 0.05, ** p < 0.01, and *** p < 0.001 represented significant differences from the LPS-treated group. Con: control, LPS: lipopolysaccharide, Dx: dexamethasone.

General Experimental Procedures
The structures of isolated compounds were identified by spectroscopic data including 1 H NMR, 13 C NMR, COSY, HMBC, HMQC, HRESIMS, IR, and optical rotation. 1 H, 13 C, and 2D NMR spectra were recorded on a JNM-ECA600 (Jeol, Tokyo, Japan) instrument using TMS as references. High-resolution electrospray ionization mass spectrometry (HRESIMS) was carried out using a Waters SYNAPT G2-Si HDMS spectrometer (Waters, Milford, MA, USA). IR spectra were obtained on a FT/IR-4600 (Jasco, Tokyo, Japan) spectrometer. UV spectra were recorded on a spectraMax M 2 θ (Molecular Devices, Sunnyvale, CA, USA) spectrophotometer. Optical rotations were measured on a Jasco P-2000 polarimeter (Jasco). The HPLC analysis was performed using an Agilent 1220 infinity HPLC system (Agilent Technologies, CA, USA) equipped with a quaternary pump.

Plant Material
Dried P. oleracea (20 kg) was purchased from the Kyung-dong market in Seoul, Korea in May 2013. One of the authors (M.-C.R.) performed botanical identification, and a voucher specimen (KRIB-KR2013-003) was deposited at the laboratory of the Immunoregulatory Materials Research Center, Jeonbuk Branch of the Korea Research Institute of Bioscience and Biotechnology.

Real-Time PCR Using TaqMan Probe
Total RNA was extracted from RAW 264.7 cells using the TaKaRa MiniBEST Universal RNA Extraction Kit following the manufacturer's instructions (TaKaRa Bio Inc., Shiga, Japan). The complementary DNA (cDNA) was synthesized from 1 µg of the total RNA using a PrimeScript 1st strand cDNA synthesis kit (Takara Bio Inc. Japan). Quantitative real-time PCR (qPCR) of Il1bβ (Mm00434228_m1), Il6 (Mm00446190_m1), and Tnf (Mm00443258_m1) was performed with a TaqMan Gene Expression Assay Kit (Thermo Fisher Scientific, San Jose, CA, USA). To normalize the gene expression, an 18S rRNA endogenous control (Applied Biosystems, Foster City, CA, USA) was used. The qPCR was employed to verify the mRNA expression using a Step-One Plus Real-Time PCR system. To quantify mRNA expression, TaqMan mRNA assay was performed according to the manufacturer's protocol (Applied Biosystems) [49]. PCR amplification was analyzed using the comparative ∆∆CT method.

Immunocytochemistry
RAW 264.7 (1 × 10 6 per well) cells were seeded on collagen-coated coverslips in six-well plates and incubated overnight. Then, the cells were pretreated with compound 20 (12.5 µM) or dexamethasone (10 µM) for 2 h. Then, the coverslips were washed with phosphate-buffered saline (PBS) and fixed with ice-cold methanol for 10 min at room temperature. After blocking with 1% bovine serum albumin (BSA) in PBS containing 0.1% Tween 20 (PBST) for 30 min, the coverslips were incubated overnight with an anti-p65 NF-κB antibody in a humidified chamber at 4 • C, followed by coincubation with an FITC-labeled anti-rabbit IgG antibody in a 37 • C humidified chamber for 60 min in the dark. The coverslips were sealed and stained with DAPI by using ProLong Gold antifade reagent with DAPI (Thermo Fisher Scientific, Waltham, MA, USA). Immunofluorescence images were obtained using an Olympus IX73 microscope with cellSens software (Olympus, Center Valley, PA, USA) [47].

Statistical Analysis
GraphPad Prism 5 software (GraphPad software, San Diego, CA, USA) was used for the statistical analysis of the experimental results. Each experiment, including NO assay, MTT assay, immunoblot, and real-time PCR, was performed independently three times, and these data represent the mean ± standard error of the mean (SEM). For comparisons between the control and LPS-treated groups, the unpaired t-test or Mann-Whitney U-test was used according to the data distribution. The statistical significance of each value was measured by the unpaired Student's t-test or Mann-Whitney U-test. * p < 0.05, ** p < 0.01, and *** p < 0.001 were considered significant.

Conclusions
The 1-carbomethoxy-β-carboline (compound 20), which has an alkaloid structure as an active compound responsible for the anti-inflammatory activity of P. oleracea extract (95%), disturbed one of the major intracellular inflammatory signaling pathways associated with MAPKs and suppressed the nuclear translocation of NF-κB, decreasing proinflammatory mediators such as iNOS, TNF-α, IL-6, and IL-1β. These results indicate that P. oleracea extract and its components, specifically alkaloid compound 20, may be useful and safe therapeutic agents for treating the painful symptoms of inflammatory diseases such as rheumatoid arthritis, allergic asthma, and atopic dermatitis as an alternative to NSAIDs and dexamethasone. Further studies are necessary to improve the extraction efficiency of compound 20 and determine the structural characteristics responsible for its bioactivity and to evaluate better clinical mimic experiments such as using human monocytes rather than the single mouse cell line, RAW 264.7.

Conflicts of Interest:
The authors have declared that there is no conflict of interest.