Anti-inflammatory effect of flavonoids isolated from Korea Citrus aurantium L. on lipopolysaccharide-induced mouse macrophage RAW 264.7 cells by blocking of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signalling pathways

Citrus fruits are an abundant source of various flavonoids, which have been used as a traditional herbal medicine in Korea and China. Most flavonoids are known to have anti-oxidant, anti-bacterial and analgesic properties. In this study, it was examined whether flavonoids (nobiletin, naringin and hesperidin) isolated from Korea Citrus aurantium L. inhibited the pro-inflammatory mediators including cytokines by blocking nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signalling in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The flavonoids suppressed mRNA and protein expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in LPS-induced macrophages. The molecular mechanism was associated with inhibition of the degradation/phosphorylation of I-κB-α and nuclear translocation of the NF-κB p-65 as well as phosphorylation of MAPK by flavonoids. These results suggest that flavonoids have anti-inflammatory effects by suppressing expression of COX-2, iNOS and cytokines by blocking the NF-κB and MAPK signalling in RAW 264.7 macrophages.     

Comparative effects of tocotrienol-rich fraction, α-tocopherol and α-tocopheryl acetate on inflammatory mediators and nuclear factor kappa B expression in mouse peritoneal macrophages

The effects of tocotrienol-rich fraction (TRF), α-tocopherol (T) and α-tocopheryl acetate (TA) on lipopolysaccharide (LPS)-induced inflammatory responses in mouse peritoneal macrophages were examined. Results showed that at 5–30 μg/ml, all test compounds plus 1 μg/ml LPS exhibited no cytotoxic effects on macrophage cells. Compared with T and TA, TRF showed the strongest anti-inflammatory activity as demonstrated by its potency in inhibiting the LPS-induced nitric oxide (NO), prostaglandin E₂ (PGE₂), and proinflammatory cytokine (TNF-α, IFN-γ, IL-1β and IL-6) production. At 10 μg/ml, it significantly blocked the LPS induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, but has no effect on cyclooxygenase-1 (COX-1). Furthermore, TRF also showed a greater inhibition on the nuclear factor kappa B (NF-κB) expression than T and TA. These results suggest that TRF could be a better agent than T and TA for use in the prevention of chronic inflammatory diseases.     

Suppression of LPS-induced inflammatory activities by Rosmarinus officinalis L.

Rosemary (Rosmarinus officinalis L.) has been used in folk medicine to treat headaches, epilepsy, poor circulation, and many other ailments. It was found that rosemary could act as a stimulant and mild analgesic and could reduce inflammation. However, the mechanisms underlying the anti-inflammatory effects of rosemary need more study to be established. Therefore, in this study, the effects of rosemary on the activation of nuclear factor kappa beta (NF-kB) and mitogen-activated protein kinases (MAPKs), the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and the production of nitric oxide (NO), prostaglandin E₂ (PGE₂), and cytokine in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells were investigated. A methanol extract of rosemary and its hexane fraction reduced NO generation with an IC₅₀ of 2.75 and 2.83 μg/ml, respectively. Also, the methanol extract and the hexane fraction inhibited LPS-induced MAPKs and NF-kB activation associated with the inhibition of iNOS or COX-2 expression. LPS-induced production of PGE₂ and tumour necrosis factor-alpha (TNF-α) were blocked by rosemary. Rosemary extract and its hexane fraction are important for the prevention of phosphorylation of MAPKs, thereby blocking NF-kB activation, which in turn leads to decreased expression of iNOS and COX-2, thus preventing inflammation.     

大蒜素对脂多糖诱导腹腔巨噬细胞炎症反应的抑制作用及机制

目的:探讨大蒜素对脂多糖(LPS)诱导小鼠腹腔巨噬细胞炎症反应的抑制作用及机制。方法:建立LPS诱导小鼠腹腔巨噬细胞炎症反应细胞模型,并用地塞米松和不同浓度大蒜素处理,MTT法检测细胞活力,中性红吞噬实验检测吞噬能力,Griess法检测一氧化氮(NO)及ELISA法检测COX-2酶活性和IL-6的分泌,qPCR检测环氧合酶2(COX-2)、一氧化氮合酶(iNOS)和IL-6的mRNA表达水平,Western Blot检测COX-2、iNOS和IL-6的蛋白表达以及核转录因子NF-κB p65及其磷酸化产物的相对表达。结果:大蒜素浓度在40~160 μg/mL范围内对腹腔巨噬细胞均无细胞毒性;与LPS组比较,大蒜素处理组能促进腹腔巨噬细胞的吞噬能力,能显著(P<0.05)抑制炎症因子COX-2酶活性、NO和IL-6的分泌,能显著(P<0.05)抑制基因COX-2、iNOS和IL-6 mRNA和蛋白的相对表达,并极显著(P<0.01)抑制NF-κB p65信号通路的磷酸化。结论:大蒜素能显著抑制LPS诱导的小鼠腹腔巨噬细胞的炎症反应,其机制可能与抑制NF-κB信号通路激活有关。     

Native Australian fruit polyphenols inhibit COX-2 and iNOS expression in LPS-activated murine macrophages

Purified polyphenolic-rich extracts from four native Australian fruits, Illawarra Plum (Podocarpus elatus Endl., Podocarpaceae), Kakadu Plum (Terminalia ferdinandiana Exell, Combretaceae), Muntries (Kunzea pomifera F. Muell., Myrtaceae) and Native Currant (Acrotriche depressa R.Br., Epacridaceae), were screened for their ability to modulate anti-inflammatory activity in LPS-activated murine macrophages (RAW 264.7). The Kakadu Plum extract inhibited the expression of inducible nitric oxide synthase (iNOS), and importantly was the only fruit in this study that displayed differential inhibition of the expression of cyclooxygenase (COX)-2 but not COX-1. Illawarra Plum (COX-2 and iNOS) and Native Currant (iNOS only) also inhibited inflammatory enzymes, while Muntries exhibited none of these activities under the same conditions. All evaluated extracts inhibited the production of prostaglandin E₂ and nitric oxide, suggesting the involvement of alternative pathways in their regulation for the Muntries extract. Further molecular investigations, showed that Kakadu Plum inhibited the NF-??B pathway, but not the p44/42 mitogen activated protein kinase (MAPK) pathway. Collectively, these results demonstrate potential anti-inflammatory activities of native Australian fruits, in particular Kakadu Plum, in LPS-activated murine macrophages, thus confirming the potential biological activities of these fruits.     

Anti-inflammatory activity of ethanolic extract of Sargassum sagamianum in RAW 264.7 cells

Aim of the study is to evaluate the antiinflammatory effects of ethanolic extract of the marine brown alga Sargassum sagamianum collected from Yeonhwari coast of Korea. Ethanolic extract of S. sagamianum (SA-E extract) inhibited expression of nitric oxide (NO) and cytokines (IL-6, IL-1β, and TNF-α) as well as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in lipopolysaccharide (LPS)-induced RAW 264.7 cells without affecting cell viability. In addition, the expression of nuclear factor (NF)-κB p65 was suppressed by SA-E extract. Furthermore, the rate of formation of edema in the mouse ear was reduced by 46% at the highest dose tested (250 mg/kg) compared to that in the control. This study suggests that SA-E extract exerts potent inhibitory effects on LPS-induced expression of inflammatory mediators such as NO, iNOS, COX-2, and cytokines in macrophages through suppression of the NF-κB p65 pathway. SA-E extract might have potential clinical applications as an anti-inflammatory agent.     

Toona sinensis and its major bioactive compound gallic acid inhibit LPS-induced inflammation in nuclear factor-κB transgenic mice as evaluated by in vivo bioluminescence imaging

In the present study, we investigated the anti-inflammatory effects of a nutritious vegetable Toona sinensis (leaf extracts, TS) and its major bioactive compound gallic acid (GA) by analysing LPS-induced NF-κB activation in transgenic mice, using bioluminescence imaging. Mice were challenged intraperitoneally with LPS (1 mg/kg) and treated orally with TS or GA (100 or 5 mg/kg, respectively). In vivo and ex vivo imaging showed that LPS increased NF-κB luminescence in the abdominal region, which was significantly inhibited by TS or GA. Immunohistochemical and ELISA analyses confirmed that TS and GA inhibited LPS-induced NF-κB, interleukin-1β, and tumour necrosis factor-α expression. Microarray analysis revealed that biological pathways associated with metabolism and the immune responses were affected by TS or GA. Particularly, LPS-induced thioredoxin-like 4B (TXNL4B) 2 expression in the small intestine, and TXNL4B, iNOS, and COX-2 expression in RAW 264.7 cells were significantly inhibited by TS or GA. Thus, the anti-inflammatory potential of TS was mediated by the downregulation of NF-κB pathway.     

Isolation and synergism of in vitro anti-inflammatory and quinone reductase (QR) inducing agents from the fruits of Morinda citrifolia (noni)

The tropical fruits and fruit products of Morinda citrifolia, commonly known as noni, are consumed as a food or dietary supplement with purported health benefits. The objective of this study was to investigate the potential anti-inflammatory and cancer preventive effects of noni fruit puree extracts. Bioassay-guided fractionation of an ethyl acetate (EtOAc) extract of noni, comprising ~ 2% noni puree solids, led to the isolation of scopoletin (1), rutin (2), and quercetin (3). Quantitative HPLC analysis of the EtOAc extract revealed levels (dry weight basis) of scopoletin at 0.62 ??mol/g, quercetin at 0.26 ??mol/g and rutin at 0.045 ??mol/g. Scopoletin and quercetin inhibited the production of nitric oxide (NO) in a concentration-dependent manner in lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells and exhibited quinone reductase (QR) induction in cultured Hepa 1c1c7 cells. Increases in QR activity in induced cells were associated with increases in QR protein as confirmed by Western blots. Combinations of scopoletin and quercetin at a low (< 10 ??M) concentration resulted in synergistic suppression in nitric oxide (NO) production and down-regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expressions in LPS-induced RAW 264.7 macrophage cells. These results suggest that the combinations of noni compounds with different groups of chemical structures might be useful to efficiently suppress inflammatory and carcinogenic processes related to iNOS and COX-2 gene overexpression. These findings may provide some basis for the purported in vitro anti-inflammatory and anti-cancer effects of noni fruits as functional foods and dietary supplements.     

Antioxidant and antiinflammatory properties of germinated and hydrolysed Brazilian soybean flours

The effect of germination in combination with Alcalase hydrolysis of Brazilian soybean cultivar BRS 133 on the production of soybean flours with bioactive peptides as modulators of oxidative stress and markers of inflammation was monitored. The electrophoretic profile showed a weak protein breakdown during germination. However, a strong breakdown of the proteins can be observed after the first hour of hydrolysis with Alcalase. MALDI-TOF-MS analysis of the protein extracts showed differences in the intensity and profile of peptide mass fingerprint due to germination and hydrolysis. Germinated flour showed higher soluble protein concentration and antioxidant capacity. All soybean protein extracts and protein hydrolysates produced (G0, G18 and G72) showed a significant (p < 0.05) inhibition on inflammatory markers such as nitric oxide (20.5–69.3%), iNOS (22.8–93.6%), PGE₂ (64.0–88.3%), COX-2 (36.2–76.7%), and TNF-α (93.9–99.5%) in LPS-induced RAW 264.7 macrophages. However, protein extracts of flours with 18 h of germination were more potent in inhibiting pro-inflammatory responses when compared to 72 h. It can be concluded that a combination of 72 h of soybean BRS 133 germination and 1 h Alcalase hydrolysis resulted in the formation of bioactive compounds with more potent antioxidant activity, and improvement in the reduction of some of the markers of inflammation.     

Japanese bog orchid (Eupatorium japonicum) extract suppresses expression of inducible nitric oxide synthase and cyclooxygenase-2 induced by toll-like receptor agonists

Toll-like receptors (TLR) play an important role in the recognition of many pathogen-associated molecular patterns and the induction of innate immunity. Dysregulated activation of TLR signaling pathways is associated with certain inflammatory diseases. Japanese bog orchid (Eupatorium japonicum), which belongs to a family of Asteraceae plants, is consumed as a tea. The present study investigated the effect of the ethanol extracts of flowers of Japanese bog orchid (EJE) on nuclear factor (NF)-κB activation and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by TLR agonists in murine macrophages. EJE suppressed NF-κB activation and iNOS and COX-2 expression induced by lipopolysaccharide (TLR4 agonist), polyriboinosinic polyribocytidylic acid (TLR3), and macrophage-activating 2 kDa lipopeptide (TLR2 and TLR6). These results suggest that EJE can regulate TLR signaling pathways and indicated its potential as a potent anti-inflammatory drug.     

铁观音茶提取物对脂多糖诱导RAW264.7细胞炎症反应的抑制作用及机制

研究铁观音茶提取物对脂多糖(LPS)诱导的RAW264.7细胞炎症反应的抑制作用及机制。用脂多糖作用于RAW264.7细胞,建立炎症模型,并用吲哚美辛和不同浓度铁观音提取物处理,检测NO和IL-6的分泌情况,qPCR检测一氧化氮合酶(iNOS)、环氧合酶2(COX-2)、肿瘤坏死因子α(TNF-α)、单核细胞趋化蛋白1(MCP-1)、白细胞介素6(IL-6)mRNA相对表达,Western Blot检测炎症相关蛋白激酶(IKKβ),核转录因子κB抑制因子(IκB)、核转录因子κB p65(NF κB p65)及其磷酸化产物的相对表达。结果显示,铁观音茶提取物能显著抑制炎症介质NO分泌和IL-6蛋白表达量(p<0.05),抑制炎症相关基因iNOS、COX-2、TNF-α和MCP-1等表达,并极显著抑制NF-κB信号通路相关蛋白IKKβ、IkB和p65的磷酸化(p<0.01)。以上结果表明,铁观音茶提取物可明显抑制LPS诱导的RAW264.7细胞炎症反应,其机制可能与抑制NF-κB信号通路激活有关。     

Common bean (Phaseolus vulgaris L.) hydrolysates inhibit inflammation in LPS-induced macrophages through suppression of NF-κB pathways

The objectives of this study were to evaluate the antioxidant capacity of protein hydrolysates of the common bean (Phaseolus vulgaris L.) varieties Negro 8025 and Pinto Durango and determine their effect on the markers of inflammation in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Cell viability was determined and the percentage of viable cells was calculated and concentrations that allowed >80% cell viability were used to determine the markers of inflammation. Alcalase hydrolysates and pepsin–pancreatin hydrolysates showed the highest antioxidant capacity after 80 and 120 min of hydrolysis, respectively. Alcalase hydrolysates of the common bean Pinto Durango at 120 min inhibited inflammation, with IC₅₀ values of 34.9 ± 0.3, 13.9 ± 0.3, 5.0 ± 0.1 and 3.7 ± 0.2 μM, while var. Negro needed 43.6 ± 0.2, 61.3 ± 0.3, 14.2 ± 0.3 and 48.2 ± 0.1 μM for the inhibition of cyclooxygenase-2 expression, prostaglandin E₂ production, inducible nitric oxide synthase expression and nitric oxide production, respectively. Also, hydrolysates significantly inhibited the transactivation of NF-κB and the nuclear translocation of the NF-κB p65 subunit. In conclusion, hydrolysates from the common bean can be used to combat inflammatory and oxidative-associated diseases.