Umbelliferose Isolated from Cuminum cyminum L. Seeds Inhibits Antigen-Induced Degranulation in Rat Basophilic Leukemia RBL-2H3 Cells.

Cuminum cyminum L. (cumin) is an annual plant of the Umbelliferae family native to Egypt. We previously showed that the aqueous extract of cumin seeds suppresses degranulation by downregulating the activation of antigen-induced intracellular signaling molecules in rat basophilic leukemia RBL-2H3 cells. However, the active substances in the extract have not yet been identified. Accordingly, herein, we aimed to ascertain the water-soluble substances present in cumin seeds that inhibit degranulation, which led to the identification of umbelliferose, a characteristic trisaccharide present in plants of the Umbelliferae family. Our study is the first to reveal the degranulation-suppressing activity of umbelliferose, and quantification studies suggest that cumin seed powder contains 1.6% umbelliferose. Raffinose, an isomer of umbelliferose, was also found to significantly suppress antigen-induced degranulation, but less so than umbelliferose. Both umbelliferose and raffinose contain sucrose subunits in their structures, with galactose moieties bound at different sites. These differences in structure suggest that the binding of galactose to the sucrose subunit at the α1-2 bond contributes to its strong degranulation-inhibiting properties.

Suppressive effect of ethanol extract from mango (Mangifera indica L.) peel on IgE production in vitro and in vivo.

Immunoglobulin E (IgE) is involved in the onset of allergic reaction, and the suppression of IgE production leads to alleviation of allergic symptoms. We found that mango peel ethanol extract (MPE) significantly suppresses IgE production by human myeloma cell line U266 cells, suggesting that MPE has an anti-allergic effect by inhibiting the production of IgE. Although mangiferin is contained in mango, which suppresses IgE production by U266 cells, it was not contained in MPE. We investigated the suppressive effect of MPE in 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis model mice. The elevation of serum IgE level was significantly suppressed by oral administration of MPE. Intake of MPE also suppressed the expression level of IL-4 in the DNFB-challenged ears, suggesting that MPE suppresses the IL-4-mediated maturation into IgE-producing cells. Our findings indicate that MPE has a potential to alleviate the increase in serum IgE level that is feature of type I allergy.

Immunostimulatory effect of aqueous extract of Coriandrum sativum L. seed on macrophages.

BACKGROUND: Coriandrum sativum L. seed is generally used as a spice and crude drug. Although many functions of the various components in C. sativum L. seed have been reported, the immunostimulatory effect of water-soluble components in C. sativum L. seed has not been studied. In the present study, we focused on the immunostimulatory effect of C. sativum L. seed aqueous extract (CAE) on macrophages as a novel health function of C. sativum L. seed components. RESULTS: CAE significantly enhanced the production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in both RAW264.7 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. CAE also stimulated nitric oxide (NO) production and the phagocytosis activity in RAW264.7 cells. We suggest that the activity of CAE is a result of the upregulation of mitogen-activated protein kinase and nuclear factor-κB cascades via TLR4. In addition, IL-6 production by peritoneal macrophages collected from CAE-administered mice was significantly enhanced, suggesting that CAE could stimulate macrophage activity in vivo. CONCLUSION: The findings of the present study suggest that CAE contains a novel water-soluble component with an immunostimulatory effect on macrophages. CAE would contribute to activating host defense against pathogens by stimulating the innate immunity. © 2017 Society of Chemical Industry.

Immunostimulatory effect of spinach aqueous extract on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages.

We herein report the immunostimulatory effect of spinach aqueous extract (SAE) on mouse macrophage-like J774.1 cells and mouse primary peritoneal macrophages. SAE significantly enhanced the production of interleukin (IL)-6 and tumor necrosis factor-α by both J774.1 cells and peritoneal macrophages by enhancing the expression levels of these cytokine genes. In addition, the phagocytosis activity of J774.1 cells was facilitated by SAE. Immunoblot analysis revealed that SAE activates mitogen-activated protein kinase and nuclear factor-κB cascades. It was found that SAE activates macrophages through not only TLR4, but also other receptors. The production of IL-6 was significantly enhanced by peritoneal macrophages from SAE-administered BALB/c mice, suggesting that SAE has a potential to stimulate macrophage activity in vivo. Taken together, these data indicate that SAE would be a beneficial functional food with immunostimulatory effects on macrophages.

Anti-Inflammatory Activity of the Combination of Nobiletin and Docosahexaenoic Acid in Lipopolysaccharide-Stimulated RAW 264.7 Cells: A Potential Synergistic Anti-Inflammatory Effect.

This study aimed to investigate a synergistic anti-inflammatory effect of a citrus flavonoid nobiletin and docosahexaenoic acid (DHA), one of n-3 long-chain polyunsaturated fatty acids, in combination. Simultaneous treatment with nobiletin and DHA synergistically inhibited nitric oxide production (combination index < 0.9) by mouse macrophage-like RAW 264.7 cells stimulated with lipopolysaccharide (LPS) without cytotoxicity. On the other hand, the inhibitory effect of nobiletin and DHA in combination on proinflammatory cytokine production was not synergistic. Neither nobiletin nor DHA affected the phagocytotic activity of RAW 264.7 cells stimulated with LPS. Immunoblot analysis revealed that the inhibition potency of DHA on the phosphorylation of ERK and p38 and nuclear translocation of NF-κB is markedly enhanced by simultaneously treating with nobiletin, which may lead to the synergistic anti-inflammatory effect. Overall, our findings show the potential of the synergistic anti-inflammatory effect of nobiletin and DHA in combination.

Aqueous Extract from Cuminum cyminum L. Seed Alleviates Ovalbumin-Induced Allergic Rhinitis in Mouse via Balancing of Helper T Cells.

Cuminum cyminum L. (cumin) seeds are widely used as a spice. Although we previously reported that the aqueous extract of cumin seeds suppresses the degranulation of rat basophilic RBL-2H3 cells, it has not been clarified whether the extract alleviates actual allergy symptoms in vivo. Therefore, in this study, we investigated the effect of oral administration of cumin seed aqueous extract (CAE) in ovalbumin (OVA)-induced allergic rhinitis. BALB/c mice were randomly divided into the following three groups: control group (five mice), OVA group (five mice), and OVA + CAE group (five mice). Allergic rhinitis was induced by sensitization (intraperitoneal, 25 µg OVA and 1.98 mg aluminum hydroxide gel) followed by challenge (intranasal, 400 µg OVA). The oral administration of CAE (25 mg/kg) reduced the sneezing frequency of OVA-induced allergic rhinitis model mice. In addition to reducing the serum immunoglobulin E and IL-4 levels, the oral administration of CAE reduced the production of T-helper type-2 (Th2) cytokines (IL-4, IL-5, IL-10, and IL-13) in the splenocytes of the model mice. Furthermore, a significant increase in the ratio of Th1 to Th2 cells was observed in the CAE-administered group. Our findings suggest that the ingestion of CAE improves T cell balance, the dominant state of Th2, and alleviates allergic rhinitis symptoms.

p-Synephrine suppresses inflammatory responses in lipopolysaccharide-stimulated RAW264.7 cells and alleviates systemic inflammatory response syndrome in mice.

p-Synephrine is the primary protoalkaloid found in Citrus species such as Citrus aurantium (bitter orange) and is widely used as a dietary supplement. Although studies have shown the anti-inflammatory effect of p-synephrine, the cells targeted and detailed mechanism(s) of action are not established. Therefore, we investigated the anti-inflammatory effects of p-synephrine and elucidated its underlying mechanisms in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, peritoneal macrophages, and an LPS-induced systemic inflammatory response syndrome (SIRS) mouse model. We found that p-synephrine inhibits the production of proinflammatory cytokines and nitric oxide in LPS-stimulated RAW264.7 cells, and proinflammatory cytokines in primary peritoneal macrophages. This effect of p-synephrine is due to downregulation of the p38 MAPK and NF-κB signaling pathway and is mediated by ß-adrenergic receptors. Oral administration of p-synephrine to SIRS mice inhibited the serum levels of proinflammatory cytokines and improved their survival rate. Thus, our findings show that p-synephrine alleviates the hyperinflammatory response in macrophages and a SIRS mouse model.

Identification of components in coriander (Coriandrum sativum L.) inhibiting degranulation of RBL-2H3 cells.

We found that a water-soluble extract of coriander (Coriandrum sativum L.) (leaves, petioles and stems) inhibits antigen-induced degranulation of RBL-2H3 cells, a rat basophil leukemia cell line. The aim of this study was to elucidate the anti-degranulation active components in the extract. The methanol-eluate fraction obtained by fractionation of the water-soluble extract using MCI gel column chromatography had strong activity, and eight components were isolated and identified. Two of them were identified as new compounds, (3S)-3-methyl-6-hydroxyisocoumarin 8-O-ß-D-glucopyranoside (compound 1) and (7S,8R)-7,8-dihydro-8-ß-D-glucopyranosyloxy-4-methoxy-7-methyl-5H-fro[2,3-g][2]benzopyran-5-one (compound 2). As a result of evaluation of anti-degranulation activity of eight components, seven of them, such as tryptophan, phenylalanine, dihydroxycoumarin glucoside, quercetin glycoside, rutin, compound 1, and compound 2, had the activity. These results indicated that the water-soluble extract of coriander contains several anti-degranulation substances.

Aqueous Extract from Leaves of Citrus unshiu Attenuates Lipopolysaccharide-Induced Inflammatory Responses in a Mouse Model of Systemic Inflammation.

Inflammation is related to various life-threatening diseases including cancer, neurodegenerative diseases, and metabolic syndrome. Because macrophages are prominent inflammatory cells, regulation of macrophage activation is a key issue to control the onset of inflammation-associated diseases. In this study, we aimed to evaluate the potential anti-inflammatory activity of Citrus unshiu leaf extract (CLE) and to elucidate the mechanism underlying its anti-inflammatory effect. We found the inhibitory activity of CLE on the secretion of proinflammatory cytokines and a chemokine from mouse macrophage-like RAW 264.7 cells and mouse peritoneal macrophages. The inhibitory activity of CLE was attributed to downregulated JNK, p38 MAPK, and NF-κB signaling pathways, leading to suppressed gene expression of inflammation-associated proteins. Oral administration of CLE significantly decreased the serum level of proinflammatory cytokines IL-6 and TNFα and increased that of anti-inflammatory cytokine IL-10 in lipopolysaccharide-induced systemic inflammation mice. In addition, oral administration of CLE decreased secretion and gene expression of several proinflammatory proteins in the liver and spleen of the model mice. Overall results revealed that C. unshiu leaf is effective to attenuate inflammatory responses in vitro and in vivo.

Trigonelline: An alkaloid with anti-degranulation properties.

Trigonelline, one of the alkaloids contained in coffee, is important not only as one of the constituents of aroma and flavor in coffee but also as a useful source of nutrition. Its anti-microbial, anti-carcinogenic, and anti-hyperglycemic effects have been investigated in previous studies. However, there have not been any studies examining the anti-degranulation effect of trigonelline. In this study, the anti-degranulation effect of trigonelline was evaluated in in vitro and in vivo models using a rat basophilic leukemia cell line, RBL-2H3 cells, and a passive cutaneous anaphylaxis (PCA) reaction in mice, respectively. In the ß-hexosaminidase release assay, trigonelline effectively suppressed antigen-induced degranulation of RBL-2H3 cells in a dose-dependent manner without cytotoxicity. Trigonelline also inhibited FcεRI-mediated intracellular signaling pathways, such as phosphorylation of PLCγ1, PI3 K, and Akt, in antigen-stimulated RBL-2H3 cells and suppressed the PCA response in mice. Moreover, trigonelline also inhibited the microtubule formation in RBL-2H3 cells, indicating that trigonelline could inhibit IgE-sensitized mast cell degranulation by attenuating both the intracellular calcium-dependent and independent pathways. These results revealed that trigonelline possesses the anti-degranulation effect against the development of allergic diseases.

Anti-inflammatory effect of aqueous extract from Kawachi-bankan (Citrus maxima) peel in vitro and in vivo.

Kawachi-bankan (Citrus maxima) is one of the citruses produced in Ehime, Japan. Although health functions of flavonoids and carotenoids in citrus peel have been studied very well, those of water-soluble substances in the peel have not been focused. We herein indicated the anti-inflammatory effect of Kawachi-bankan peel aqueous extract (KPE) in vitro and in vivo. KPE significantly inhibited the production of inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α by LPS-stimulated RAW264.7 cells without cytotoxicity. KPE also significantly inhibited the mRNA expression levels of IL-6 and TNF-α in the cells, suggesting that KPE inhibits the production of inflammatory cytokines by suppressing the gene expression levels. Immunoblot analysis revealed that KPE shows an anti-inflammatory effect on macrophages through the suppression of the phosphorylation of p38 and the translocation of NF-κB into nucleus. The oral administration of KPE inhibited the serum levels of inflammatory cytokines and improved the survival rate in systemic inflammatory response syndrome (SIRS) model mice. Our experiments using a cell line suggested that KPE inhibits the production of inflammatory cytokines by macrophages in hyperinflammatory state. In addition, experiments in vivo showed that the oral administration of KPE inhibited the serum levels of inflammatory cytokines and improved the survival rate in SIRS model mice. Our findings indicated that KPE contributes to alleviating of a hyperinflammatory response.

Inhibitory effect of aqueous extract of Cuminum cyminum L. seed on degranulation of RBL-2H3 cells and passive cutaneous anaphylaxis reaction in mice.

Cuminum cyminum L. (cumin) seed is used as a spice in various countries. Although several functions of the components in cumin seed have been reported, the anti-allergic effect of the water-soluble component in cumin seed has not been reported yet. In this study, we focused on the suppressive effect of cumin seed aqueous extract on degranulation in order to reveal the anti-allergic effect of cumin. Cumin seed aqueous extract significantly suppressed the antigen-induced degranulation of rat basophilic leukemia cell line RBL-2H3 cells in a dose-dependent manner without cytotoxicity. The extract also inhibited the elevation of the intracellular calcium ion concentration induced by antigen. Immunoblot analysis revealed that the extract suppresses phosphorylation of phosphatidylinositol 3-kinase, Bruton's tyrosine kinase, phospholipase C-γ1/2, and Akt in the signaling pathways activated by antigen induction via FcεRI. Furthermore, the extract suppressed microtubule formation induced by antigen. In addition, oral administration of cumin seed aqueous extract significantly suppressed the passive cutaneous anaphylaxis reaction in BALB/c mice. Our findings suggest that cumin seed contains water-soluble components with the anti-allergic effect. Therefore, cumin seed has potential as anti-allergic functional food.

Inhibitory effect of Japanese black vinegar on IgE-mediated degranulation of RBL-2H3 cells and a murine model of Japanese cedar pollinosis.

Japanese black vinegar (JBV) is a traditional vinegar manufactured with steamed unpolished rice. After screening, beneficial effects of JBV on IgE-mediated allergic responses were found. In this study, acetic acid-free JBV was used to evaluate its antiallergic effects. JBV suppressed degranulation of rat basophilic leukemia RBL-2H3 cells in a dose-dependent manner without cytotoxicity. The inhibitory effect of JBV on the degranulation seemed to be caused by the bioactive ingredients other than proteins, because the activity was not affected by heat treatment or protease digestion. JBV inhibited the elevation in the intracellular Ca2+ concentration induced by antigen. Immunoblot analysis revealed that JBV suppresses degranulation of RBL-2H3 cells by downregulated phosphorylation of PI3K, Akt, and PLCγ1. In addition, oral administration of JBV significantly suppressed passive cutaneous anaphylaxis reaction in mice and an allergic symptom in Cry j1-induced pollinosis model mice. Thus, JBV has a potential as a health-promoting food with the antiallergy effect.

Immunostimulatory effect of dried bonito extract on mouse macrophage cell lines and mouse primary peritoneal macrophages.

Dried bonito is a preserved food used in Japan, which contains abundant flavor ingredients and functional substances. We focused on the immunostimulatory effect of dried bonito extract (DBE) on mouse macrophage-like J774.1 cells, RAW264.7 cells, and mouse primary peritoneal macrophages. DBE significantly stimulated the production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by both J774.1 cells and peritoneal macrophages by enhancing the cytokine gene expression levels. In addition, DBE stimulated nitric oxide production by enhancing the expression of inducible nitric oxide synthase in RAW264.7 cells. DBE also increased the phagocytosis activity of J774.1 cells. Immunoblot analysis revealed that DBE has an immunostimulatory effect on macrophages through activation of mitogen-activated protein kinase and nuclear factor-κB cascades. TNF-α production enhanced by DBE was partially inhibited by treatment with TLR4 inhibitor TAK-242, whereas IL-6 production enhanced by DBE was almost inhibited. These results suggested that DBE is thought to strongly stimulate the TLR4 signaling pathway for macrophage activation, and its activation is also involved in other signaling. Finally, the phagocytosis activity of peritoneal macrophages from DBE-administered BALB/c mice increased significantly, suggesting that DBE has the potential to stimulate macrophage activity in vivo. In conclusion, these data indicated that DBE contributes to activating host defense against pathogens by activating innate immunity.

Inhibitory effect of aqueous spinach extract on degranulation of RBL-2H3 cells.

The inhibitory effect of an aqueous extract from spinach on degranulation of RBL-2H3 cells is herein reported. The extract significantly suppressed antigen-induced degranulation in a dose-dependent manner without affecting cell viability. Active substances in the extract were heat-stable and trypsin-resistant with molecular weights ranging from 500 Da to 14 kDa. The extract inhibited elevation of the intracellular Ca(2+) concentration caused by stimulation by antigen, while not suppressing degranulation induced by a calcium ionophore A23187. Immunoblot analysis revealed that the inhibitory effect results from downregulation of phosphorylation of both Syk kinase and phosphatidylinositol 3-kinase in the signalling pathways involved in degranulation caused by the antigen-antibody interaction. Taken together, these findings suggest that aqueous spinach extract has an anti-allergic activity that controls degranulation.