Anti-Inflammatory Effects of Alphitolic Acid Isolated from Agrimonia coreana Nakai Extracts Are Mediated via the Inhibition of ICRAC Activity in T Cells.

Agrimonia pilosa Ledeb., an important medicinal herb in traditional East Asian medicine, is primarily used to treat abdominal pain, dysentery, and hemostasis. There are ten other reported species of Agrimonia plants, including Agrimonia coreana Nakai-a naturally growing species in South Korea-and Agrimonia eupatoria Linn. Although recent studies have isolated numerous active constituents and investigated their effects, the medicinal utility of this herb is not yet fully explored. Through patch-clamp recording, a previous study reported that Agrimonia plant extracts inhibit the function of Ca2+ release-activated Ca2+ channels (CRACs). Herein, we aimed to identify and isolate the main compounds in A. coreana responsible for CRAC inhibition while assessing the anti-inflammatory effects mediated by this inhibition. We demonstrated for the first time that alphitolic acid isolated from A. coreana has a dose-dependent inhibitory effect on CRAC activity and, thus, an inhibitory effect on intracellular calcium increase. Furthermore, analysis of human CD4+ T cell proliferation via the carboxyfluorescein diacetate succinimidyl ester method revealed that alphitolic acid inhibited T cell proliferation in a concentration-dependent manner. Our findings provide a theoretical basis for the potential therapeutic use of alphitolic acid in the treatment of inflammatory diseases.

Multi-target modulation of ion channels underlying the analgesic effects of α-mangostin in dorsal root ganglion neurons.

BACKGROUND: α-Mangostin is a xanthone isolated from the pericarps of mangosteen fruit with, and has analgesic properties. Although the effects suggest an interaction of α-mangostin with ion channels in the nociceptive neurons, electrophysiological investigation of the underlying mechanism has not been performed. HYPOTHESIS: We hypothesized that α-Mangostin exerts its analgesic effects by modulating the activity of various ion channels in dorsal root ganglion (DRG) neurons. METHODS: We performed a whole-cell patch clamp study using mouse DRG neurons, HEK293T cells overexpressing targeted ion channels, and ND7/23 cells. Molecular docking (MD) and in silico absorption, distribution, metabolism, and excretion (ADME) analyses were conducted to obtain further insights into the binding sites and pharmacokinetics, respectively. RESULTS: Application of α-mangostin (1-3 µM) hyperpolarized the resting membrane potential (RMP) of small-sized DRG neurons by increasing background K+ conductance and thereby inhibited action potential generation. At micromolar levels, α-mangostin activates TREK-1, TREK-2, or TRAAK, members of the two-pore domain K+ channel (K2P) family known to be involved in RMP formation in DRG neurons. Furthermore, capsaicin-induced TRPV1 currents were potently inhibited by α-mangostin (0.43 ± 0.27 µM), and partly suppressed tetrodotoxin-sensitive voltage-gated Na+ channel (NaV) currents. MD simulation revealed that multiple oxygen atoms in α-mangostin may form stable hydrogen bonds with TREKs, TRAAK, TRPV1, and NaV channels. In silico ADME tests suggested that α-mangostin may satisfy the drug-likeness properties without penetrating the blood-brain barrier. CONCLUSION: The analgesic properties of α-mangostin might be mediated by the multi-target modulation of ion channels, including TREK/TRAAK activation, TRPV1 inhibition, and reduction of the tetrodotoxin-sensitive NaV current. The findings suggest that the phytochemical can be a multi-ion channel-targeting drug and an alternative drug for effective pain management.

Quantification and Validation of an HPLC Method for Low Concentrations of Apigenin-7-O-Glucuronide in Agrimonia pilosa Aqueous Ethanol Extract Topical Cream by Liquid-Liquid Extraction.

In this study, we aimed to develop and validate a pretreatment method for separating and analyzing the small amounts of biomarkers contained in topical cream formulations. Analyzing semisolid formulations that contain low concentrations of active ingredients is difficult. Cream formulations containing an aqueous ethanol extract of 0.1% Agrimonia pilosa is an example. Approximately 0.0013% of apigenin-7-O-glucuronide(A7OG) was contained as a biomarker in the cream. To determine the A7OG content present in the cream formulation, liquid-liquid extraction using dichlormethane was applied. In addition, the volume of the distribution liquid was measured using the peak ratios of the indicator component, A7OG, and an internal standard, baicalin. Subsequently, the A7OG content in the cream formulation was calculated. Using this time-saving method, A7OG can be simply analyzed without additional pretreatment steps, such as evaporation and reconstitution. Moreover, the validation results confirmed that this analytical method met all of the criteria. Consequently, A7OG was successfully isolated from the cream, analyzed, and quantified using the developed method.

In-vitro and in-vivo anti-allergic effects of magnolol on allergic rhinitis via inhibition of ORAI1 and ANO1 channels.

ETHNOPHARMACOLOGICAL RELEVANCE: Flos Magnoliae (the dried flower buds of Magnolia biondii Pamp, FM) is a known herbal traditional medicine used for the symptomatic relief of nasal congestion and rhinorrhea caused by rhinitis and sinusitis. Magnolol, a neolignan from the magnolia family, is a secondary metabolite known to have anti-allergic and anti-inflammatory effects. However, the underlying mechanisms and therapeutic effect of magnolol in the treatment of allergic rhinitis (AR) remain elusive. AIMS OF THE STUDY: Anoctamin 1 (ANO1), a calcium-activated anion channel, mediates mucus and electrolyte secretion in nasal airway epithelial cells, whereas calcium release-activated calcium channel protein 1 (ORAI1) participates in the activation of T-lymphocytes and mast cells. The aim of our study is to understand the mechanisms of action of magnolol against AR, i.e., whether it acts through the modulation of ANO1 and ORAI1 channels that are expressed in nasal epithelial cells and T-lymphocytes, respectively. MATERIALS AND METHODS: Whole-cell patch clamp was used to record the activity of ORAI1 and ANO1 ion channels in ORAI1 or ANO1 overexpressed HEK293T cells, while the Ussing chamber apparatus was used to measure electrolyte transport via the epithelium, in Calu-3 cells cultured in an air-liquid interface. Additionally, calcium imaging of Jurkat T-lymphocytes was used to assess changes in the intracellular calcium concentration. Magnolol toxicity was assessed using the CCK-8 assay, and its effect on T-lymphocyte proliferation was measured by labeling human primary T-lymphocytes with carboxyfluorescein succinimidyl ester. Finally, OVA-induced Balb/c mice were employed to evaluate the effect of magnolol on nasal symptoms, as well as cytokine and eosinophil infiltration in AR. RESULTS: Magnolol inhibits ORAI1 and ANO1 channels in a concentration-dependent manner. Magnolol (30 µM) inhibits anti-CD3 induced cellular proliferation and production of IL-2 via ORAI1 channels in T-lymphocytes. Further, ATP-induced electrolyte transport mediated by ANO1 channels is significantly inhibited by magnolol in IL-4 sensitized Calu-3 cells. Notably, 300 µM magnolol significantly attenuates cytokine and eosinophil infiltration, thus alleviating AR symptoms in mice OVA-induced AR. CONCLUSION: Magnolol may be a promising therapeutic agent for the treatment and prevention of AR.

Flos magnoliae constituent fargesin has an anti-allergic effect via ORAI1 channel inhibition.

Flos magnoliae (FM), the dry flower buds of Magnolia officinalis or its related species, is a traditional herbal medicine commonly used in Asia for symptomatic relief of and treating allergic rhinitis, headache, and sinusitis. Although several studies have reported the effects of FM on store-operated calcium entry (SOCE) via the ORAI1 channel, which is essential during intracellular calcium signaling cascade generation for T cell activation and mast cell degranulation, the effects of its isolated constituents on SOCE remain unidentified. Therefore, we investigated which of the five major constituents of 30% ethanoic FM (vanillic acid, tiliroside, eudesmin, magnolin, and fargesin) inhibit SOCE and their physiological effects on immune cells. The conventional whole-cell patch clamp results showed that fargesin, magnolin, and eudesmin significantly inhibited SOCE and thus human primary CD4+ T lymphocyte proliferation, as well as allergen-induced histamine release in mast cells. Among them, fargesin demonstrated the most potent inhibitory effects not only on ORAI1 (IC50 = 12.46 ± 1.300 µM) but also on T-cell proliferation (by 87.74% ± 1.835%) and mast cell degranulation (by 20.11% ± 5.366%) at 100 µM. Our findings suggest that fargesin can be a promising candidate for the development of therapeutic drugs to treat allergic diseases.

Gardenia jasminoides extract and its constituent, genipin, inhibit activation of CD3/CD28 co-stimulated CD4+ T cells via ORAI1 channel.

Gardenia jasminoides (GJ) is a widely used herbal medicine with antiinflammatory properties, but its effects on the ORAI1 channel, which is important in generating intracellular calcium signaling for T cell activation, remain unknown. In this study, we investigated whether 70% ethanolic GJ extract (GJEtOH) and its subsequent fractions inhibit ORAI1 and determined which constituents contributed to this effect. Whole-cell patch clamp analysis revealed that GJEtOH (64.7% ± 3.83% inhibition at 0.1 mg/ml) and all its fractions showed inhibitory effects on the ORAI1 channel. Among the GJ fractions, the hexane fraction (GJHEX, 66.8% ± 9.95% at 0.1 mg/ml) had the most potent inhibitory effects in hORAI1-hSTIM1 co-transfected HEK293T cells. Chemical constituent analysis revealed that the strong ORAI1 inhibitory effect of GJHEX was due to linoleic acid, and in other fractions, we found that genipin inhibited ORAI1. Genipin significantly inhibited IORAI1 and interleukin-2 production in CD3/ CD28-stimulated Jurkat T lymphocytes by 35.9% ± 3.02% and 54.7% ± 1.32% at 30 µM, respectively. Furthermore, the same genipin concentration inhibited the proliferation of human primary CD4+ T lymphocytes stimulated with CD3/CD28 antibodies by 54.9% ± 8.22%, as evaluated by carboxyfluorescein succinimidyl ester assay. Our findings suggest that genipin may be one of the active components of GJ responsible for T cell suppression, which is partially mediated by activation of the ORAI1 channel. This study helps us understand the mechanisms of GJ in the treatment of inflammatory diseases.

Humulus japonicus extract ameliorates collagen­induced arthritis in mice through regulation of overall articular inflammation.

Humulus japonicus (HJ) is a widely used herbal medicine in Asia with anti­oxidative, anti­microbial, and anti­inflammatory effects. We investigated the potential therapeutic effects of HJ in rheumatoid arthritis (RA) using a mouse model of collagen­induced arthritis (CIA) and a lipopolysaccharide­stimulated murine macrophage cell line (RAW 264.7). The CIA mice were administered 300 mg/kg HJ orally starting 3 days prior to second immunization. The clinical and histopathological findings were assessed in the paw of CIA mice. The levels of autoantibodies and inflammatory markers were determined in the plasma and cell culture supernatant, respectively. The expression at mRNA and protein levels was analyzed by reverse transcription quantitative­PCR and western blot analysis, respectively. HJ significantly decreased the gross arthritic scores and paw swelling in CIA mice. Furthermore, synovial inflammation, cartilage destruction, and bone erosion were markedly reduced by HJ. It also decreased the expression of inflammatory enzymes in both the paw of mice and RAW 264.7 cells. Moreover, the expression of genes related to all macrophages and pro­inflammatory M1 macrophage were significantly decreased, whereas the expression of anti­inflammatory M2 macrophage marker was markedly increased in the paw of HJ­treated CIA mice. In addition, HJ suppressed the levels of plasma anti­type II collagen antibody following the decreased expression of T helper type 1 (Th1) and Th2 cell­associated surface markers and cytokines in the paw. HJ also significantly inhibited the expression of IL­6 both in vitro and in vivo, followed by reduced STAT3 phosphorylation and expression in the paw of CIA mice. Finally, the expression of osteoclast­related genes was decreased in the paw of HJ­treated CIA mice. These findings suggest that HJ can play a role in suppressing the development of CIA by overall regulation of articular inflammation. This study should provide new insights into the use of HJ as a therapeutically effective natural product against RA.

Flos Magnoliae and its Constituent Linoleic Acid Suppress T Lymphocyte Activation via Store-Operated Calcium Entry.

Intracellular calcium signaling is crucial for type 2 helper T cell and mast cell activation, which is essential for allergic inflammation. It is initiated by antigen-mediated receptor stimulation that triggers store-operated calcium entry (SOCE) via ORAI1 calcium channel. Flos Magnoliae (FM) is widely used to treat allergic diseases such as allergic rhinitis and asthma. Although many studies have reported that FM regulates intracellular calcium signaling, research on the exact type of calcium channel modulated by FM is scarce. Therefore, we hypothesized that the anti-allergic effects of FM might result from ORAI1 inhibition in T cells. We investigated whether a 70% ethanolic extract of FM (FMEtOH) and its constituents inhibit ORAI1 channel activity and subsequent T cell activation. We performed conventional whole-cell patch clamp studies in hSTIM1 and hORAI1-overexpressing HEK293T cells (HEKORAI1). Intracellular calcium concentration was determined using Fura-2 dye and cytokine production measurement in Jurkat T lymphocytes. FMEtOH (0.03 mg/mL) and its fractions, especially hexane fraction (FMHex, 0.01 mg/mL), significantly inhibited SOCE and IL-2 cytokine production in Jurkat T lymphocytes. GC/MS analysis showed linoleic acid (LA) as the major component of FMHex. FMHex at 0.01 mg/mL (equivalent to 10 µM LA) inhibited not only SOCE but also IL-2 production, as well as CD3/CD28 receptor co-stimulation induced calcium signaling in Jurkat T lymphocytes. FMEtOH and LA suppressed CD4+ T lymphocyte activation, at least in part, by inhibiting ISOCE. Thus, ISOCE inhibition may be a potential strategy to inhibit immune responses in inflammation.

3,5-Di-C-ß-D-glucopyranosyl phloroacetophenone, a major component of Melicope ptelefolia, suppresses fibroblast activation and alleviates arthritis in a mouse model: Potential therapeutics for rheumatoid arthritis.

Melicope ptelefolia has been traditionally used to treat rheumatism and fever. The present study aimed to investigate the therapeutic effect of 3,5­di­C­ß­D­glucopyranosyl phloroacetophenone (ßGP), a main component of M. ptelefolia, on rheumatoid arthritis (RA). A model of collagen­induced arthritis (CIA) was established in mice using the RAW 264.7 murine macrophage cell line and mouse embryonic fibroblasts (MEFs). The clinical scores of arthritis, swelling, histopathological findings, and micro­computed tomography in CIA mouse paws were assessed. The levels of anti­type II collagen antibody and cytokines were determined in the plasma and cell culture supernatant, respectively. Protein and gene expression levels were analyzed by western blot and reverse transcription­quantitative polymerase chain reaction analyses. ßGP significantly decreased the gross arthritic scores of CIA mice and joint swelling, and decreased articular inflammation, cartilage degradation and bone erosion. However, ßGP did not exert any effect on anti­type II collagen immunoglobulin G plasma levels or inflammatory cytokine expression in macrophages. ßGP significantly suppressed the expression of interleukin­6 and leukemia inhibitory factor and decreased the phosphorylation of signal transducer and activator of transcription 3, and expression of receptor activator of nuclear factor­κB ligand in tumor necrosis factor­α­stimulated MEFs and in CIA mouse paws. Osteoclast­related gene expression was significantly reduced in CIA mouse paws. Taken together, ßGP suppressed the development of RA by regulating the activation of synovial fibroblasts.

Spirodela polyrhiza and its Chemical Constituent Vitexin Exert Anti-Allergic Effect via ORAI1 Channel Inhibition.

Intracellular calcium signaling cascades are integral to early and late allergic responses involving mast cell degranulation and type 2 helper T cell activation, respectively. Both the responses are accompanied by the movement of calcium through the calcium release-activated calcium (CRAC) channel, encoded by the ORAI1 gene. Spirodela polyrhiza (L.) Schleid (SP) has anti-inflammatory and anti-allergic effects, but its effect on calcium signaling has not been reported. This study investigated whether a 30% ethanolic SP extract (SPEtOH) and its constituents can reduce CRAC currents ([Formula: see text]), and thus inhibit mast cell degranulation and T cell activation. In Jurkat T lymphocytes, we found that 3[Formula: see text]mg/mL SPEtOH inhibited the [Formula: see text] by [Formula: see text]%, whereas one of its constituents vitexin (100[Formula: see text][Formula: see text]M) inhibited the [Formula: see text] by [Formula: see text]%. Furthermore, in the RBL-2H3 mast cell, the [Formula: see text] was inhibited by 3[Formula: see text]mg/mL SPEtOH ([Formula: see text]%) and 100[Formula: see text][Formula: see text]M vitexin ([Formula: see text]%). Investigation of human primary T cell proliferation induced by co-stimulation with antibodies to cluster of differentiation 3 and 28, and of RBL-2H3 mast cell degranulation following IgE-antigen complex stimulation revealed that 100[Formula: see text][Formula: see text]M vitexin inhibited both T-cell proliferation (by [Formula: see text]%) and mast cell degranulation (by [Formula: see text]%). These effects were concentration-dependent, and no cytotoxicity was observed. Our findings suggest that vitexin is a promising candidate compound for the development of therapeutic agents to prevent and treat allergic diseases.

Flos Magnoliae Inhibits Chloride Secretion via ANO1 Inhibition in Calu-3 Cells.

Flos Magnoliae (FM, Chinese name: Xin-yi) is an oriental medicinal herb commonly used for symptomatic relief from allergic rhinitis, sinusitis, and headache, including in traditional Chinese and Korean medicine formulations. FM inhibits histamine release from mast cells and cytokine secretion from T cells. However, the mechanism of action of FM on the anoctamin-1 (ANO1) ion channel, which is responsible for nasal hypersecretion in allergic rhinitis, has not been elucidated. Therefore, in this study, we investigated the effect of a 30% ethanolic extract of FM (FMEtOH) and its chemical constituents on ANO1 activity. We used high-performance liquid chromatography analysis to identify five major chemical constituents of FMEtOH: vanillic acid, tiliroside, eudesmin, magnolin, and fargesin. Using a conventional whole-cell patch clamp method, we found that FMEtOH (30, 100, and 300[Formula: see text][Formula: see text]g/mL) and its chemical constituent tiliroside inhibited ANO1 activity in ANO1-overexpressing HEK293T cells. In addition, we found that the treatment of the airway epithelial cell line Calu-3 with interleukin 4 significantly increased Ca[Formula: see text] activated Cl[Formula: see text] current (ICaCC), but not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride current (ICFTR). FMEtOH and tiliroside specifically inhibited ICaCC. Thus, in this study, we identified a novel mechanism underlying the alleviation of allergic rhinitis by FMEtOH. Our results indicate that FMEtOH and its chemical constituent tiliroside are promising and potent agents for the prevention and treatment of allergic rhinitis.

Activation of transient receptor potential vanilloid 3 by the methanolic extract of Schisandra chinensis fruit and its chemical constituent γ-schisandrin.

Transient receptor potential vanilloid 3 (TRPV3) is a non-selective cation channel with modest permeability to calcium ions. It is involved in intracellular calcium signaling and is therefore important in processes such as thermal sensation, skin barrier formation, and wound healing. TRPV3 was initially proposed as a warm temperature sensor. It is activated by synthetic small-molecule chemicals and plant-derived natural compounds such as camphor and eugenol. Schisandra chinensis (Turcz.) Baill (SC) has diverse pharmacological properties including antiallergic, anti-inflammatory, and wound healing activities. It is extensively used as an oriental herbal medicine for the treatment of various diseases. In this study, we investigated whether SC fruit extracts and seed oil, as well as four compounds isolated from the fruit can activate the TRPV3 channel. By performing whole-cell patch clamp recording in HEK293T cells overexpressing TRPV3, we found that the methanolic extract of SC fruit has an agonistic effect on the TRPV3 channel. Furthermore, electrophysiological analysis revealed that γ-schisandrin, one of the isolated compounds, activated TRPV3 at a concentration of 30 µM. In addition, γ-schisandrin (~100 µM) increased cytoplasmic Ca2+ concentrations by approximately 20% in response to TRPV3 activation. This is the first report to indicate that SC extract and γ-schisandrin can modulate the TRPV3 channel. This report also suggests a mechanism by which γ-schisandrin acts as a therapeutic agent against TRPV3-related diseases.

Synergistic effects of glutathione and ß-mercaptoethanol treatment during in vitro maturation of porcine oocytes on early embryonic development in a culture system supplemented with L-cysteine.

Various methods have been used to remove reactive oxygen species (ROS) generated from in vitro culture (IVC) conditions that can cause cell injury or death, including the application of low oxygen (O(2)) tension and the addition of antioxidants. The beneficial effects of antioxidants and O(2) tension on IVC of porcine embryos, however, are controversial among researchers. In this study, we sought to determine the effects and optimal concentrations of antioxidants for the development of porcine embryos in an IVC system. Specifically, we examined the synergistic effects of antioxidants on development to the blastocyst stage in a culture system supplemented with L-cysteine during IVM. Of the antioxidants tested (melatonin, glutathione (GSH), ß-mercaptoethanol (ß-ME), N-acetylcysteine (NAC) and dithiothreitol (DTT)), addition of GSH (1 mM) or ß-ME (25 µM) significantly increased development to the blastocyst stage compared with the controls without antioxidant treatment (22.2 ± 4.2% for 1 mM GSH, 25.9 ± 2.2% for 25 µM ß-ME and 12-13% for the control, P<0.05). In addition, the mean cell number per blastocyst was increased by approximately 1.7-fold in the presence of GSH or ß -ME. These GSH- and ß-ME-induced increases in development to the blastocyst stage and total cell number, however, were not mimicked by melatonin, NAC or DTT, all of which are ROS scavengers. The combination of GSH or ß-ME with L-cysteine significantly reduced high O(2) tension-induced ROS production (P<0.05). These results suggest that a combination of 1 mM GSH or 25 µM ß-ME with 1 mM L-cysteine could be used for production of high quality porcine blastocysts in IVC systems.