Anti‐inflammatory Potential of Quercetin‐3‐O‐β‐D‐(“2”‐galloyl)‐glucopyranoside and Quercetin Isolated from Diospyros kaki calyx via Suppression of MAP Signaling Molecules in LPS‐induced RAW 264.7 Macrophages

Diospyros kaki (DK) contains an abundance of flavonoids and has been used in folk medicine in Korea for centuries. Here, we report for the first time the anti‐inflammatory activities of Quercetin (QCT) and Quercetin 3‐O‐β‐(“2”‐galloyl)‐glucopyranoside (Q32G) isolated from DK. We have determine the no cytotoxicity of Q32G and QCT against RAW 264.7 cells up to concentration of 50 μM. QCT and Q32G demonstrated potent anti‐inflammatory activities by reducing expression of nitric oxide (NO), tumor necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6 inducible NO synthase (iNOS), cyclooxygenase (COX)‐2, and mitogen‐activated protein kinase (MAPKs) in mouse RAW 264.7 macrophages activated with lipopolysaccharide (LPS). Both QCT or Q32G could decrease cellular protein levels of COX‐2 and iNOS as well as secreted protein levels of NO, PGE₂, and cytokines (TNF‐α, IL‐1β, and IL‐6) in culture medium of LPS‐stimulated RAW 264.7 macrophages. Immunoblot analysis showed that QCT and Q32G suppressed LPS‐induced MAP kinase pathway proteins p‐p38, ERK, and JNK. This study revealed that QCT and Q32G have anti‐inflammatory potential, however Q32G possess comparable activity as that of QCT and could be use as adjuvant to treat inflammatory diseases.     

Phenethyl isothiocyanate decreases thymic stromal lymphopoietin‐induced inflammatory reactions in mast cells

Phenethyl isothiocyanate (PEITC) has been reported to have anti‐inflammatory and anti‐carcinogenic properties. However, the anti‐inflammatory effects of PEITC on thymic stromal lymphopoietin (TSLP)‐induced inflammatory responses are uncertain. This study evaluates pharmacological activities of PEITC on inflammatory reactions in TSLP‐stimulated mast cells. Human mast cell line HMC‐1 was treated with PEITC (0.04, 0.4, and 4 µM) and subjected to inflammation by TSLP. Our results showed that PEITC significantly attenuated IL‐13 and TNF‐α levels increased by TSLP in HMC‐1. PEITC significantly decreased TSLP‐promoted HMC‐1 proliferation and Ki67 mRNA expression. Protein levels of MDM2 and pSTAT6 increased by TSLP were significantly suppressed by PEITC in HMC‐1. In addition, PEITC significantly enhanced protein levels of cleaved poly ADP‐ribose polymerase and p53 decreased by TSLP. Based on the effects of PEITC on inflammation and proliferation in this study, it is possible that PEITC is a potential candidate to treat mast cells‐mediated inflammatory disorders.

Practical applications

This report provides strong evidence that Phenethyl isothiocyanate (PEITC) which is a dietary constituent derived from cruciferous vegetables, may be considered an alternative agent for treatment of mast cells‐mediated inflammatory disorders.     

Bovine casein peptides co‐stimulate naive macrophages with lipopolysaccharide for proinflammatory cytokine production and nitric oxide release

Three bovine casein peptides, ie LLY, PGPIPN and TTMPLW, were used to investigate their effects on cytokine (TNF‐α and IL‐6) production and nitric oxide (NO) release by murine bone marrow macrophages (BMMs). The results showed that these peptides alone were incapable of stimulating cytokine production or NO release in naive or IFN‐γ‐primed BMMs. However, when BMMs were co‐incubated with the peptides at a concentration of 1.0 µM and lipopolysaccharide (LPS; 100 ng ml⁻¹), an augmentative effect on TNF‐α, IL‐6 and NO production was observed. Of the three peptides, TTMPLW had the greatest augmentative effect on NO production by LPS‐stimulated BMMs and induced the highest amount of TNF‐α production at a concentration of 1.0 µM . All the peptides at a concentration of 1.0 µM stimulated IL‐6 production by BMMs. TNF‐α was neutralised by anti‐TNF‐α monoclonal antibody and the release of NO was reduced by about 33.3% (p < 0.01). These results demonstrate that bovine casein peptides can co‐stimulate naive macrophages with LPS for proinflammatory cytokine production and NO release and may play a role in host defence against pathogens. © 2000 Society of Chemical Industry     

Crude extract of Fuzhuan brick tea ameliorates DSS‐induced colitis in mice

Inflammatory bowel disease (IBD) is highly prevalent worldwide and includes ulcerative colitis (UC) and Crohn's disease. It is a high incidence rate disease all over the world and an inducement of colon cancer. The aim of this study was to investigate the potential protective effect of Fuzhuan brick tea (FBT) against colitis induced by dextran sulphate sodium (DSS) in mice. ICR mice were administered FBT orally for 7 days before drinking 3% DSS (w/v). The FBT significantly attenuated the symptoms of colitis including diarrhoea, rectal bleeding and loss of body weight. FBT reduced the shortening of colon length and alleviated the histopathological damages. The myeloperoxidase activity, nitric oxide and malondialdehyde level in colon tissues were also significantly decreased by FBT. Besides, FBT treatment obviously suppressed the expression of the inflammatory cytokines such as TNF‐α, IL‐1β and IFN‐γ. Our results provide a safe and efficient method for preventing and treating colitis.     

Anti‐inflammatory properties of high pressure‐assisted extracts of Grifola frondosa in lipopolysaccharide‐activated RAW 264.7 macrophages

The aim of this study was to determine the in vitro anti‐inflammatory properties of the shake extract (SE) and the high pressure‐assisted extract (PE) of the mycelia of Grifola frondosa in a lipopolysaccharide‐stimulated RAW 264.7 macrophage model. The content of total polysaccharides and β‐glucans of PE at 600 MPa (PE‐600) was 41.2 and 6.2 mg g^?1 dry weight, respectively, which were significantly higher than SE extracts. The results showed that treatment with 500 μg mL^?1 of PE by 600 MPa (PE‐600) did not reduce RAW 264.7 cell viability but did significantly inhibit the production of LPS‐induced NO, PGE₂ and intracellular ROS. The PE‐600 inhibited the activation of NF‐kB and then reduced the production of LPS‐induced TNF‐α, IL‐6 and IL‐1β in a dose‐dependent manner. Thus, the PE could be used as an alternative extraction method for improving the extraction efficacy of G. frondosa and serve as an alternative source of anti‐inflammatory agents.     

Polygonum viviparum L. inhibits the lipopolysaccharide‐induced inflammatory response in RAW264.7 macrophages through haem oxygenase‐1 induction and activation of the Nrf2 pathway

BACKGROUND: Polygonum viviparum L. (PV) is a member of the family Polygonaceae and is widely distributed in high‐elevation areas. It is used as a folk remedy to treat inflammation‐related diseases. This study was focused on the anti‐inflammatory response of PV against lipopolysaccharide (LPS)‐induced inflammation in RAW264.7 macrophages. RESULTS: Treatment with PV did not cause cytotoxicity at 0–50 µg mL^?1 in RAW264.7 macrophages, and the IC₅₀ value was 270 µg mL^?1. PV inhibited LPS‐stimulated nitric oxide (NO), prostaglandin (PG)E₂, interleukin (IL)‐1β and tumour necrosis factor (TNF)‐α release and inducible NO synthase (iNOS) and cyclooxygenase (COX)‐2 protein expression. In addition, PV suppressed the LPS‐induced p65 expression of nuclear factor (NF)‐κB, which is associated with the inhibition of IκB‐α degradation. These results suggest that, among mechanisms of the anti‐inflammatory response, PV inhibits the production of NO and these cytokines by down‐regulating iNOS and COX‐2 gene expression. Furthermore, PV can induce haem oxygenase (HO)‐1 protein expression through nuclear factor E2‐related factor 2 (Nrf2) activation. A specific inhibitor of HO‐1, zinc(II) protoporphyrin IX, inhibited the suppression of iNOS and COX‐2 expression by PV. CONCLUSION: These results suggest that PV possesses anti‐inflammatory actions in macrophages and works through a novel mechanism involving Nrf2 actions and HO‐1. Thus PV could be considered for application as a potential therapeutic approach for inflammation‐associated disorders. © 2012 Society of Chemical Industry     

Anti‐obesity effect of Liupao tea extract by modulating lipid metabolism and oxidative stress in high‐fat‐diet‐induced obese mice

Liupao tea (LPT) is traditional dark Chinese tea. The effect of LPT extract on high‐fat‐diet‐induced obese mice was investigated systematically. The results showed that LPT extract could reduce body weight and significantly alleviate liver damage and fat accumulation. LPT could also decrease the levels of alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (AKP), total cholesterol (TC), triglycerides (TG), and low‐density lipoprotein cholesterol (LDL‐C) and increase the level of high‐density lipoprotein cholesterol (HDL‐C) in the liver. It also decreased the serum levels of inflammatory cytokines, including tumor necrosis factor alpha (TNF‐α), interferon gamma (IFN‐γ), interleukin (IL)‐1β, and IL‐6 and increased the serum levels of anti‐inflammatory cytokines, including IL‐10 and IL‐4. Moreover, LPT improved the levels of total superoxide dismutase (T‐SOD), glutathione peroxidase (GSH‐Px), and catalase (CAT) and reduced the level of malondialdehyde (MDA) in the liver. Moreover, LPT could upregulate the mRNA and protein expressions of peroxisome proliferator‐activated receptor alpha (PPAR‐α), lipoprotein lipase (LPL), carnitine palmitoyltransferase 1(CPT1), and cholesterol 7 alpha‐hydroxylase (CYP7A1) and downregulate those of PPAR‐γ and CCAAT/enhancer‐binding protein alpha (C/EBP‐α) in the liver. It also increased the mRNA expression of copper/zinc superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2), CAT, gamma‐glutamylcysteine synthetase 1 (GSH1), and GSH‐Px. The components of LPT extract include catechin, rutin, taxifolin, and astragalin, which possibly have a wide range of biological activities. In conclusion, our work verified that LPT extract possessed an anti‐obesity effect and alleviated obesity‐related symptoms, including lipid metabolism disorder, chronic low‐grade inflammation, and liver damage, by modulating lipid metabolism and oxidative stress.     

Preventive effects of black tea theaflavins against mouse type IV allergy

BACKGROUND: Tea (Camellia sinensis L.), one of the most popular beverages, contains various beneficial constituents. We investigated the preventive effects of black tea theaflavins, theaflavin‐3‐gallate (3‐TF) and theaflavin‐3,3′‐digallate (TFDG), on oxazolone‐induced type IV allergy in male ICR mice. RESULTS: Percutaneous administration of both 3‐TF and TFDG at 0.2 mg ear^?1 showed significant preventive effects against mouse type IV allergy. Oral administration of these agents at 50 mg kg^?1 body weight also showed significant preventive effects against mouse type IV allergy. Oral administration of 3‐TF and TFDG at a dose of 50 mg kg^?1 body weight prevented the increases in levels of some proinflammatory cytokines, interleukin‐12 (IL‐12), interferon‐gamma (IFN‐γ), and tumour necrosis factor‐alpha (TNF‐α), in the sera and/or ears of mice with type IV allergy. Lowering of serum antioxidant activity in mice with allergic symptoms was also prevented by oral administration of these theaflavins at a dose of 50 mg kg^?1 body weight. The anti‐allergic mechanisms of action of theaflavins involve inhibition of the fluctuations of cytokines and maintenance of antioxidant status in allergic mice. CONCLUSION: These results suggest that the theaflavins as well as catechins contribute to the anti‐allergic effects of black tea. Copyright © 2010 Society of Chemical Industry     

Enhanced Anti‐Inflammatory Activities by the Combination of Luteolin and Tangeretin

Dietary components in combination may act synergistically and produce enhanced biological activities. Herein, we investigated the anti‐inflammatory effects of 2 flavonoids, that is luteolin (LUT) and tangeretin (TAN) in combination. Lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophages were treated with noncytotoxic concentrations of LUT, TAN, and their combinations. The results showed that LUT/TAN in combination produced synergistic inhibitory effects on LPS‐stimulated production of nitric oxide (NO). ELISA results demonstrated that LUT/TAN in combination caused stronger suppression on the LPS‐induced overexpression of proinflammatory mediators, such as prostaglandin E₂ (PGE₂), interleukin (IL)‐1β, and IL‐6 than LUT or TAN alone. Immunoblotting and Real‐Time PCR analyses showed that LUT/TAN combination significantly decreased LPS‐induced protein and mRNA expression of inducible nitric oxide synthase and cyclooxygenase‐2. These inhibitory effects of the combination treatment were stronger than those produced by LUT or TAN alone. Overall, our results demonstrated for the first time that combination of LUT and TAN produced synergistic anti‐inflammatory effects in LPS‐stimulated RAW 264.7 macrophages.     

Amino acids stimulate Akt phosphorylation,and reduce IL‐8 production and NF‐κB activity in HepG2 liver cells

Hepatic insulin resistance and inflammatory cytokine production contribute to the manifestation of the metabolic syndrome. As amino acids have been implicated in modulating insulin signaling and inflammation, we have investigated the effects of glutamine, leucine and proline on markers of inflammation and insulin sensitivity in HepG2 liver cells. Cells were incubated with IL‐1β (5 ng/mL) to stimulate IL‐8 production. After 24 h, glutamine inhibited IL‐8 production significantly (p<0.05) at 2, 5 and 10 mM (to 82, 73 and 72% of control), whereas leucine reduced IL‐8 production significantly only at 10 mM (66%) and proline at 5 and 10 mM (71 and 52%). Glutamine, leucine and proline all reduced NF‐κB activity after 3 h of IL‐1β stimulation at 2, 5 and 10 mM (p<0.001). Insulin‐induced (1 nM) Akt phosphorylation was reduced in cells treated with tumour necrosis factor‐α (10 ng/mL) for 24 h, but was partly restored by simultaneous incubation with glutamine, leucine and proline (25 mM). Phosphorylation of glycogen synthase kinase‐3β was unaffected by insulin stimulation and amino acid treatment. Our results indicate that glutamine, leucine and proline attenuate IL‐8 production, probably through inhibition of NF‐κB, and that they increase Akt phosphorylation in HepG2 cells.     

Chinese Yam Extracts Containing β‐Sitosterol and Ethyl Linoleate Protect against Atherosclerosis in Apolipoprotein E‐Deficient Mice and Inhibit Muscular Expression of VCAM‐1 In Vitro

Atherosclerosis is a chronic inflammatory disease, which is associated with increased expression of adhesion molecules and monocyte recruitment into the arterial wall. This study evaluated whether hexane extracts from the edible part (DB‐H1) or bark region (DB‐H2) of Dioscorea. batatas Decne have anti‐atherosclerotic properties in vivo and in vitro experiments. We also identified bioactive components in the hexane extracts. Thirty‐six apolipoprotein E (ApoE^?/?) mice and 12 control (C57BL/6J) mice were given a Western‐type diet for 11 or 21 wk. To examine the effects of yam extracts on lesion development, ApoE^?/? mice were orally administered DB‐H1 or DB‐H2 for the duration of the study (200 mg/kg b.w./day, 3 times per wk). Both DB‐H1 and DB‐H2 significantly reduced the total atherosclerotic lesion area in the aortic root. In addition, plasma concentrations of total cholesterol, oxidized‐low‐density lipoprotein, and c‐reactive protein were decreased by administration of DB‐H1 and DB‐H2. Consistent with the in vivo observations, DB‐H1 and DB‐H2 inhibited tumor necrosis factor (TNF)‐α–induced vascular cell adhesion molecule‐1 expression and adhesion of THP‐1 monocytes to TNF‐α–activated vascular smooth muscle cells. It was also found that treatment with DB‐H1 or DB‐H2 resulted in the inhibition nitric oxide (NO) and reactive oxygen species production and iNOS expression in macrophages. Thus, DB‐H1 and DB‐H2 seem to influence atherosclerosis by affecting the production of inflammatory mediators in vivo. Our results suggest that yam extracts have the potential to be used in the prevention of atherosclerosis.     

Phenethyl isothiocyanate inhibits STAT3 activation in prostate cancer cells

This study was undertaken to investigate the mechanism by which phenethyl isothiocyanate (PEITC), a natural compound from cruciferous vegetables, exhibits antitumor effect on prostate cancer cells. Cell proliferation, cell cycle, Western blot, gene transfer, and reporter assays were used to test the effects of PEITC on the growth and IL6/JAK/STAT3 pathway in prostate cancer. The result showed that PEITC significantly inhibited DU145 cell proliferation in a dose‐dependent manner and induced the cell arrest at G2‐M phase. PEITC inhibited both constitutive and IL‐6‐induced STAT3 activity in DU145 cells. IL‐6‐stimulated phosphorylation of JAK2, an STAT3 upstream kinase, was also attenuated by PEITC. Moreover, an antioxidant reagent, N‐acetyl‐L ‐cysteine (NAC) which suppresses reactive oxygen species (ROS) generation, reversed the early inhibitory effects of PEITC on cell proliferation, constitutive or IL‐6‐mediated JAK‐STAT3 phosphorylation in PCa cells. Taken together, our data demonstrated that PEITC can inhibit the activation of the JAK‐STAT3 signal‐cascade in prostate cancer cells and the underlying mechanism may be partially involved with blocking cellular ROS production during the early stage of the signaling activation by IL‐6.     

Effects of Catechol O‐Methyl Transferase Inhibition on Anti‐Inflammatory Activity of Luteolin Metabolites

Although luteolin is known to have potent anti‐inflammatory activities, much less information has been provided on such activities of its hepatic metabolites. Luteolin was subjected to hepatic metabolism in HepG2 cells either without or with catechol O‐methyl transferase (COMT) inhibitor. To identify hepatic metabolites of luteolin without (luteolin metabolites, LMs) or with COMT inhibitor (LMs+CI), metabolites were treated by β‐glucuronidase and sulfatase, and found that they were composed of glucuronide and sulfate conjugates of diosmetin in LMs or these conjugates of luteolin in LMs+CI. LMs and LMs+CI were examined for their anti‐inflammatory activities on LPS stimulated Raw 264.7 cells. Expression of iNOS and production of nitric oxide and pro‐inflammatory cytokines such as TNF‐α, IL‐1β, and IL‐6 were suppressed more effectively by the treatment with LMs+CI than LMs. Our data provide a new insight on possible improvement in functional properties of luteolin on target cells by modifying their metabolic pathway in hepatocytes.     

Sarcodon aspratus Extract Ameliorates Dextran Sulfate Sodium‐Induced Colitis in Mouse Colon and Mesenteric Lymph Nodes

Mushrooms have been previously investigated for their immune‐modulating and anti‐inflammatory properties. We examined whether the anti‐inflammatory properties of Sarcodon aspratus ethanol extract (SAE) could elicit protective effects against dextran sulfate sodium (DSS)‐induced colitis in vivo. Male C57/BL6 mice were randomly assigned to 1 of 4 treatment groups: control (CON; n = 8), DSS‐treated (DSS; n = 9), DSS+SAE at 50 mg/kg BW (SAE50; n = 8), and DSS+SAE at 200 mg/kg BW groups (SAE200; n = 9). DSS treatment induced significant weight loss, which was significantly recovered by SAE200. Although SAE did not affect DSS‐mediated reductions in colon length, it improved diarrhea and rectal bleeding induced by DSS. SAE at 200 mg/kg BW significantly attenuated IL‐6 and enhanced IL‐10 expression in mesenteric lymph nodes (MLN), and significantly reduced IL‐6 levels in splenocytes. SAE200 also significantly attenuated DSS‐induced increase in IL‐6 and IL‐1β, and reductions in IL‐10 in colon tissue. High levels of SAE were also observed to significantly decrease inflammatory COX‐2 expression that was upregulated by DSS in mice colon. These findings may have relevance for novel therapeutic strategies to mitigate inflammatory bowel disease‐relevant inflammatory responses, via the direct and indirect anti‐inflammatory activity of SAE. We also found that SAE harbors significant quantities of total fiber and β‐glucan, suggesting a possible role for these components in protection against DSS‐mediated colitis.     

Dietary grape seed extract ameliorates symptoms of inflammatory bowel disease in IL10‐deficient mice

Grape seed extract (GSE) is a by‐product of the wine industry, with abundant polyphenolic compounds known for their anti‐inflammatory and anti‐oxidative effects. Using IL10‐deficient mice (IL10KO), here we showed that GSE (1% of dry feed weight) ameliorated inflammatory bowel disease indices, increased colonic goblet cell numbers and decreased myeloperoxidase levels in the large intestine. Concomitantly, GSE supplementation attenuated inflammation, decreased the expression of pore forming tight junction protein claudin2, and increased levels of Lactobacilli and Bacteroides in the gut microbiota of IL10KO mice. In summary, our study shows that GSE has protective roles on inflammatory bowel disease through altering gut inflammation, tight junction protein expression, and gut microbiota composition.