Cross-linked bromelain inhibits lipopolysaccharide-induced cytokine production involving cellular signaling suppression in rats

Bromelain has been reported to have anti-inflammatory and immunomodulatory effects. It has been cross-linked with organic acids and polysaccharides by gamma irradiation. The cross-linked (CL)-bromelain preparation resisted an acidic environment of pH 3 for 2 h and preserved 80% of its enzyme activity. Pretreatment of rats with CL-bromelain intragastrically for 7 days significantly reduced serum cytokine production induced by injected i.p. with 2.5 mg/kg of lipopolysaccharide (LPS). Bromelain significantly reduced serum glutamate-oxalacetate transaminase induced by LPS. The anti-inflammatory effect of CL-bromelain was correlated with reduced LPS-induced NF-kappaB activity and cyclooxygenase 2 (COX-2) mRNA expression in rat livers. In addition, CL-bromelain dose-dependently inhibited LPS-induced COX-2 mRNA and prostaglandin E2 (PGE2) in BV-2 microglial cells. CL-Bromelain also suppressed the LPS-activated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). In conclusion, the anti-inflammatory effects of the CL-bromelain preparation in vivo and in vitro suggest its therapeutic potentials.     

Anti-inflammatory effects of phenolic compounds isolated from the fruits of Artocarpus heterophyllus

Artocarpus heterophyllus Lam is a large evergreen tree cultivated throughout Southeast Asia for its fruits. Its leaves and roots have been used for medicinal purposes. The aim of this work was to study the in vitro anti-inflammatory effects of phenolic compounds isolated from the ethyl acetate extracts of the fruits of Artocarpus heterophyllus. Three phenolic compounds were characterized as artocarpesin [5,7,2',4'-tetrahydroxy-6-(3-methylbut-3-enyl) flavone] ( 1), norartocarpetin (5,7,2',4'-tetrahydroxyflavone) ( 2), and oxyresveratrol [ trans-2,4,3',5'-tetrahydroxystilbene] ( 3) by spectroscopic methods and through comparison with data reported in the literatures. The anti-inflammatory effects of the isolated compounds ( 1- 3) were evaluated by determining their inhibitory effects on the production of proinflammatory mediators in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophage cells. These three compounds exhibited potent anti-inflammatory activity. The results indicated that artocarpesin ( 1) suppressed the LPS-induced production of nitric oxide (NO) and prostaglandin E 2 (PGE 2) through the down-regulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expressions. Thus, artocarpesin ( 1) may provide a potential therapeutic approach for inflammation-associated disorders.     

Antioxidant and anti-inflammatory potential of hesperetin metabolites obtained from hesperetin-administered rat serum: an ex vivo approach

In recent years much attention has been focused on the pharmaceutical relevance of bioflavonoids, especially hesperidin and its aglycon hesperetin in terms of their antioxidant and anti-inflammatory actions. However, the bioactivity of their metabolites, the real molecules in vivo hesperetin glucuronides/sulfates produced after ingestion, has been poorly understood. Thus, the study using an ex vivo approach is aimed to compare the antioxidant and anti-inflammatory activities of hesperidin/hesperetin or hesperetin metabolites derived from hesperetin-administered rat serum. We found that hesperetin metabolites (2.5-20 μM) showed higher antioxidant activity against various oxidative systems, including superoxide anion scavenging, reducing power, and metal chelating effects, than that of hesperidin or hesperetin. The data also showed that pretreatment of hesperetin metabolites (1-10 μM) within the range of physiological concentrations, compared to hesperetin, significantly inhibited nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production, as evidenced by the inhibition of their precursors, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein levels without appreciable cytotoxicity on LPS-activated RAW264.7 macrophages or A7r5 smooth muscle cells. Concomitantly, hesperetin metabolites dose-dependently inhibited LPS-induced intracellular reactive oxygen species (ROS). Furthermore, hesperetin metabolites significantly downregulate LPS-induced nuclear factor-κB (NF-κB) activation followed by the suppression of inhibitor-κB (I-κB) degradation and phosphorylation of c-Jun N-terminal kinase1/2 (JNK1/2) and p38 MAPKs after challenge with LPS. Hesperetin metabolites ex vivo showed potent antioxidant and anti-inflammatory activity in comparison with hesperidin/hesperetin.     

Consumption of lycopene inhibits the growth and progression of colon cancer in a mouse xenograft model

A previous study indicated that lycopene could significantly inhibit the proliferation of human colon cancer cells in vitro. However, the in vivo anticancer effects of lycopene against colon cancer have not been demonstrated yet. Therefore, this study investigated whether consumption of lycopene could prevent the growth and progression of colorectal tumor in a mouse xenograft model. Bioluminescence imaging, histopathological, immunofluorescence (IFC), and immunohistochemical (IHC) staining results indicated that lycopene could effectively suppress the growth and progression of colon cancer in tumor-bearing mice. The results demonstrated that lycopene significantly suppressed the nuclear expression of PCNA and β-catenin proteins in tumor tissues. Consumption of lycopene could also augment the E-cadherin adherent molecule and nuclear levels of cell cycle inhibitor p21(CIP1/WAF1) protein. The chemopreventive effects of lycopene were associated with suppression of COX-2, PGE(2), and phosphorylated ERK1/2 proteins. Furthermore, the inhibitory effects of lycopene were inversely correlated with the plasma levels of matrix metalloproteinase 9 (MMP-9) in tumor-bearing mice. These results suggested that lycopene could act as a chemopreventive agent against the growth and progression of colorectal cancer in a mouse xenograft model.     

Effects of black soybean [Glycine max (L.) Merr.] seed coats and its anthocyanidins on colonic inflammation and cell proliferation in vitro and in vivo

Anti-inflammatory and antiproliferative activities of anthocyanidins and anthocyanin-rich black soybean seed coats were studied in HT-29 human colon adenocarcinoma cells and carcinogen-treated F344 rats, respectively. Cyanidin and delphinidin significantly inhibited cell growth at concentrations of >or=1 microM. Anthocyanidins suppressed cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) mRNAs in TPA-stimulated HT-29 cells. Both yellow and black soybean seed coat supplementation (10%, w/w) did not significantly reduce the number of aberrant crypt foci (ACF), although a modest decrease in the number of ACF was observed in animals fed soybean seed coats. The colonic COX-2 mRNA level was suppressed in rats fed both soybean seed coat diet. The plasma prostaglandin E 2 (PGE 2) level was reduced only in rats fed black soybean seed coats. No difference was observed in either colonic iNOS mRNA or plasma nitric oxide level. These results indicate that anthocyanidins are possible anti-inflammatory agents; however, further studies are required to determine required intake levels in vivo to exert antitumor effect.     

Anti-inflammatory potential of flavonoid contents from dried fruit of Crataegus pinnatifida in vitro and in vivo

The dried fruits of Crataegus pinnatifida, a local soft drink material and medical herb, demonstrated antioxidant effect in a previous study. The present study investigates the anti-inflammatory potential of flavonoid contents from dried fruit of C. pinnatifida (CF-Fs). The preliminary investigation showed that CF-Fs (0.25-0.75 mg/mL) decreased the release of PGE2 and nitric oxide as induced by lipopolysaccharide (LPS, an endotoxin) in macrophage RAW 264.7 cells. The in vivo assay showed that pretreatment of rats with CF-Fs (50-200 mg/kg dosed by gavage) for 5 days significantly decreased the serum levels of the hepatic enzyme markers alanine aminotransferase and aspartate aminotransferase induced by the 6-h treatment with LPS (i.p.; 5 mg/kg). Histopathological evaluation of the rat livers revealed that CF-Fs reduced the incidence of liver lesions such as neutrophil infiltration and necrosis induced by LPS. Furthermore, it was found that pretreatment with CF-Fs decreased the hepatic expression of iNOS and COX-2 induced by LPS in rats. These results demonstrate that CF-Fs present anti-inflammatory potential in vitro and in vivo and that they may play a role in hepatoprotection.     

Tangeretin reduces ultraviolet B (UVB)-induced cyclooxygenase-2 expression in mouse epidermal cells by blocking mitogen-activated protein kinase (MAPK) activation and reactive oxygen species (ROS) generation

The present study examined the effects of tangeretin, a polymethoxylated flavonone present in citrus fruits, on ultraviolet B (UVB)-induced cyclooxygenase-2 (COX-2) expression in JB6 P+ mouse skin epidermal cells. Tangeretin suppressed UVB-induced COX-2 expression and transactivation of nuclear factor-κB and activator protein-1 in JB6 P+ cells. Moreover, tangeretin blocked UVB-induced phosphorylation of Akt and mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated protein kinase, c-Jun N-terminal kinase, and p38, and attenuated the phosphorylation of MAPK kinases 1/2, 3/6, and 4. Tangeretin also limited the endogenous generation of reactive oxygen species (ROS), thereby protecting the cells against oxidative stress. However, tangeretin did not scavenge the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and influence the nicotinamide adenine dinucleotide phosphate oxidase activity. These results suggest that the anti-inflammatory effects of tangeretin stem from its modulation of cell signaling and suppression of intracellular ROS generation. Tangeretin may have a potent chemopreventive effect in skin cancer.     

Effect of phytocompounds from the heartwood of Acacia confusa on inflammatory mediator production

Acacia confusa Merr. (Leguminosae) is traditionally used as a medicinal plant in Taiwan. In the present study, anti-inflammatory activity of extracts from the heartwood of A. confusa were investigated for the first time. Results demonstrated that ethanolic extracts of A. confusa heartwood strongly suppressed NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Among all fractions derived from ethanolic extracts, the EtOAc fraction exhibited the best inhibitory activity. Following column chromatography and reverse-phase high-performance liquid chromatography, 13 specific phytocompounds including 5 new flavonoids (i.e., 7,8,3',4'-tetrahydroxy-4-methoxyflavan-3-ol, 7,8,3',4'-tetrahydroxyflavone, 7,8,3'-trihydroxy-3,4'-dimethoxyflavone, 7,3',4'-trihydroxyflavone, and 7,3',4'-trihydroxy-3-methoxyflavone) were isolated and identified from the EtOAc fraction. In addition, melanoxetin (3,7,8,3',4'-pentahydroxyflavone), a major compound in the EtOAc fraction, markedly suppressed LPS-induced NO and prostaglandin E 2 (PGE 2) production. Moreover, melanoxetin completely suppressed gene expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) at 50 and 100 microM, respectively. This is the first report to identify the inhibitory bioactivities of melanoxetin on iNOS and COX-2.     

Inhibitory effect of a glycoprotein isolated from golden oyster mushroom ( Pleurotus citrinopileatus ) on the lipopolysaccharide-induced inflammatory reaction in RAW 264.7 macrophage

Mushrooms have become an important source of natural antitumor, antiviral, antibacterial, immunomodulatory, and anti-inflammatory agents. Golden oyster mushroom, Pleurotus citrinopileatus , is a common mushroom in oriental countries for human consumption. The present study investigated the anti-inflammatory reaction of the bioactive nonlectin glycoprotein (PCP-3A) isolated from the fresh fruiting body of this mushroom. Western blot analysis on LPS-induced iNOS, COX-2, and NF-κB expressions in RAW 264.7 cells as affected by PCP3-A was performed to elucidate the mechanism of NO and PGE2 reduction. The results showed that PCP-3A failed to affect RAW 264.7 viability at a concentration up to 6.25 μg/mL, but inhibited LPS (1 μg/mL)-induced expression, and that PCP-3A inhibited the production of NO and PGE2 in LPS-activated macrophages via the down-regulation of certain pro-inflammatory mediators, including iNOS and NF-κB.     

Enhanced anti-inflammatory activities of Monascus pilosus fermented products by addition of ginger to the medium

Hypercholesterolemia initiates the atherogenic process; however, chronic inflammation promotes atherogenesis. Monascus spp. fermented products are recognized for their anti-hypercholesterolemic effect, but their anti-inflammatory activity is not as significant as that of many plant-derived foods. To enhance the anti-inflammatory function of Monascus pilosus fermented products, ginger was added to the PDB medium at a ratio of 20% (v/v). The mycelia and broth were collected, freeze-dried, and extracted by ethanol for assays. Macrophage RAW264.7 was challenged with lipopolysaccharide (LPS) and coincubated with the extracts of fermented product cultured in ginger-supplemented medium (MPG) or extracts of fermented product cultured in regular PDB medium (MP) for 18 h. Human umbilical vein endothelial cell HUVEC was challenged with tumor necrosis factor (TNF)-α and coincubated with the extracts of either MPG or MP for 6 h. The results showed that MPG significantly (p<0.05) lowered the production of macrophage pro-inflammatory cytokines TNF-α, nitric oxide (NO), interleukin (IL)-1, IL-6, and prostaglandin E2 (PGE2) by 68.53%, 84.29%, 32.55%, 84.49%, and 69.49%, respectively; however, MP had no inhibitory effect. MPG significantly downregulated the expression of p-IκB, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) in macrophage by 42.16%, 50.87%, and 51.35%, respectively, while MP had no inhibition on COX-2 expression and only 16.64% and 19.22% downregulatory effect on iNOS and phosphorylated-IκB (p-IκB), respectively. Moreover, MPG significantly suppressed the expression of vessel cell adhesion molecule-1 (VCAM-1) and p-IκB in endothelial cell by 63.48% and 63.41%, respectively. LC/MS/MS analysis indicated that 6-gingerdiol was formed in the ginger-modified medium during fermentation. The results of this study will facilitate the development of Monascus spp. fermented products as antiatherosclerotic nutraceuticals.     

Anti-inflammatory activities of mogrosides from Momordica grosvenori in murine macrophages and a murine ear edema model

Momordica grosvenori (Luo Han Guo), grown primarily in Guangxi province in China, has been traditionally used for thousands of years by the Chinese to make hot drinks for the treatment of sore throat and the removal of phlegm. The natural noncaloric sweetening triterpenoid glycosides (mogrosides) contained in the M. grosvenori fruits are also antioxidative, anticarcinogenic, and helpful in preventing diabetic complications. The aim of this study was to assess the anti-inflammatory properties of mogrosides in both murine macrophage RAW 264.7 cells and a murine ear edema model. The results indicate that mogrosides can inhibit inflammation induced by lipopolysaccharides (LPS) in RAW 264.7 cells by down-regulating the expression of key inflammatory genes iNOS, COX-2, and IL-6 and up-regulating some inflammation protective genes such as PARP1, BCL2l1, TRP53, and MAPK9. Similarly, in the murine ear edema model, 12-O-tetradecanoylphorbol-13-acetate-induced inflammation was inhibited by mogrosides by down-regulating COX-2 and IL-6 and up-regulating PARP1, BCL2l1, TRP53, MAPK9, and PPARδ gene expression. This study shows that the anticancer and antidiabetic effects of M. grosvenori may result in part from its anti-inflammatory activity.     

Intestinal ellagitannin metabolites ameliorate cytokine-induced inflammation and associated molecular markers in human colon fibroblasts

Pomegranate ellagitannins (ETs) are transformed in the gut to ellagic acid (EA) and its microbiota metabolites, urolithin A (Uro-A) and urolithin B (Uro-B). These compounds exert anti-inflammatory effects in vitro and in vivo. The aim of this study was to investigate the effects of Uro-A, Uro-B, and EA on colon fibroblasts, cells that play a key role in intestinal inflammation. CCD18-Co colon fibroblasts were exposed to a mixture of Uro-A, Uro-B, and EA, at concentrations comparable to those found in the colon (40 μM Uro-A, 5 μM Uro-B, 1 μM EA), both in the presence or in the absence of IL-1β (1 ng/mL) or TNF-α (50 ng/mL), and the effects on fibroblast migration and monocyte adhesion were determined. The levels of several growth factors and adhesion cytokines were also measured. The mixture of metabolites significantly inhibited colon fibroblast migration (～70%) and monocyte adhesion to fibroblasts (～50%). These effects were concomitant with a significant down-regulation of the levels of PGE(2), PAI-1, and IL-8, as well as other key regulators of cell migration and adhesion. Of the three metabolites tested, Uro-A exhibited the most significant anti-inflammatory effects. The results show that a combination of the ET metabolites found in colon, urolithins and EA, at concentrations achievable in the intestine after the consumption of pomegranate, was able to moderately improve the inflammatory response of colon fibroblasts and suggest that consumption of ET-containing foods has potential beneficial effects on gut inflammatory diseases.     

Lipoperoxidation and cyclooxygenases 1 and 2 inhibitory compounds from Iryanthera juruensis

Plants from Iryanthera genus have been traditionally used as food supplements by South American Indians. The MeOH extract of leaves of Iryanthera juruensis, one of the plants endemic to the Amazon region and consumed in Brazil, and the hexane extract from its seeds inhibited lipid peroxidation (LPO) and cyclooxygenase (COX-1 and -2)) enzymes in in vitro assays. Further analyses of these extracts yielded 5-deoxyflavones (1-5) from the leaf extract and sargachromenol (6), sargaquinoic acid (7), a novel juruenolic acid (8), omega-arylalkanoic acids (9a-c), and the lignan guaiacin (10) from the seed extract. Compounds 3-5 inhibited LPO by 86%, 77%, and 88% at 10 ppm, respectively, and compounds 6 and 9a-c showed inhibition at 76% and 78% at 100 ppm, respectively. However, compounds 7 and 8 were inactive and lignan 10 exhibited LPO inhibitory activity by 99% at 100 ppm compared to commercial antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and vitamin E. The flavones 1-5 also inhibited COX-1 and -2 enzymes by 50-65% at 100 ppm. Compound 6 showed high but nonselective inhibition of COX-1 and COX-2 enzymes, when compared to aspirin and Celebrex, a nonsteroidal anti-inflammatory drug (NSAID). Compounds 7 and 10 inhibited COX-1 by 60% and 65% and COX-2 by 37% and 18%, respectively, whereas compounds 8 and 9a-c showed little or no activity against these enzymes.     

Anti-inflammatory activities of 6β-acetoxy-7α-hydroxyroyleanone from Taiwania cryptomerioides Hayata ex vivo and in vivo

Excess production of nitric oxide (NO) by inducible NO synthase (iNOS) in activated macrophages is linked to acute and chronic inflammation. Thus, it would be valuable to develop inhibitors of NO production and/or iNOS for potential therapeutic use. This study investigated the anti-inflammatory effects of 6β-acetoxy-7α-hydroxyroyleanone (AHR), a compound isolated from the bark of Taiwania cryptomerioides Hayata, using lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW 264.7) ex vivo and carrageenan (Carr)-induced mouse paw edema model in vivo. When RAW 264.7 macrophages were treated with different concentrations of AHR (0, 0.31, 0.62, 1.25, and 2.50 μg/mL) together with LPS (100 ng/mL), a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that AHR blocked protein expression of iNOS and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 macrophages, significantly. In the anti-inflammatory test, AHR (1.25 and 2.50 mg/kg) decreased paw edema at 4 and 5 h after λ-carrageenan (Carr) administration and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver tissue. It was also demonstrated that AHR significantly attenuated the malondialdehyde (MDA) level in the edema paw at 5 h after Carr injection. AHR (0.62, 1.25, and 2.50 mg/kg) decreased the NO levels on both edema paw and serum at 5 h after Carr injection. Also, AHR diminished the serum tumor necrosis factor (TNF-α) at 5 h after Carr injection. Western blotting revealed that AHR (2.50 mg/kg) decreased Carr-induced iNOS and COX-2 expressions at 5 h in the edema paw. An intraperitoneal (ip) injection treatment with AHR also diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory activities of AHR might be related to the decrease in the levels of MDA, iNOS, and COX-2 in the edema paw and to the increase in the activities of CAT, SOD, and GPx in the liver through the suppression of TNF-α and NO.     

Inducible nitric oxide synthase and cyclooxygenase-2 participate in anti-inflammatory activity of imperatorin from Glehnia littoralis

In this study, we have investigated the anti-inflammatory effects of imperatorin, a compound isolated from the roots of Glehnia littoralis, using a lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7) in vitro and a carrageenan (Carr)-induced mouse paw edema model in vivo. When RAW264.7 macrophages were treated with imperatorin together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that imperatorin blocked the protein expression of iNOS and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW264.7 macrophages significantly. In the anti-inflammatory test, imperatorin decreased the paw edema at 4 and 5 h after Carr administration and increased the activities of catalase, superoxide dismutase, and glutathione peroxidase in paw edema. We also demonstrated that imperatorin significantly attenuated the malondialdehyde level in the edema paw at the fifth hour after Carr injection. Imperatorin decreased the NO and tumor necrosis factor and prostaglandin E2 levels on serum at 5 h after Carr injection. Western blotting revealed that imperatorin decreased Carr-induced iNOS and COX-2 expressions at 5 h in edema paw. An intraperitoneal injection treatment with imperatorin also diminished neutrophil infiltration into sites of inflammation as did indomethacin. The results suggested that imperatorin had anti-inflammatory effects in LPS-stimulated RAW 264.7 cells and Carr-injected mice, respectively. In addition, inhibition of elevated iNOS and COX-2 protein expression as well as neutrophil infiltration of Carr-injected paws may be involved in the beneficial effects of imperatorin.     

Composition of liquid rice hull smoke and anti-inflammatory effects in mice

A new liquid rice hull smoke extract with a smoky aroma and sugar-like odor prepared by pyrolysis of rice hulls followed by liquefaction of the resulting smoke contained 161 compounds characterized by GC/MS. Antioxidative, antiallergic, and anti-inflammatory activities of the extract were assessed in vitro and in vivo. At pH 5, the extract inhibited 1-diphenyl-2-picrylhydrazyl (DPPH) free radicals and suppressed nitric oxide (NO) and β-hexosaminidase releases from lipopolysaccharide (LPS)-induced RAW264.7 mouse macrophage leukemia cells and ionophore A23187-stimulated RBL-2H3 rat basophilic cells without significant cytotoxicity. 12-O-Tetradecanolylphorbol-13-acetate (TPA) was applied to the ears of CD-1 mice to induce inflammation (edema), which was accompanied by increases in a series of biomarkers. Topical application of 1% of the extract as well as feeding mice a standard diet with 1% extract for two weeks significantly reduced the expression of biomarkers associated with the TPA-induced inflammation. These include tumor necrosis factor-α (TNF-α), IL-1β, interleukin-1β (IL-1β), interleukin-6 (IL-6), leukotriene B(4) (LTB(4)), prostaglandin E(2) (PGE(2)), myeloperoxidase (MPO). These in vitro and in vivo findings demonstrate the potential value of rice hull smoke extract derived from a major agricultural byproduct to serve as a new biomaterial for the improvement of food quality and safety and the environment.