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.     

Protective effect of dried safflower petal aqueous extract and its main constituent,carthamus yellow,against lipopolysaccharide‐induced inflammation in RAW264.7 macrophages

BACKGROUND: Safflower, whose botanic name is Carthamus tinctorius L., is a member of the family Compositae or Asteraceae. Carthamus yellow (CY) is the main constituent of safflower and is composed of safflomin A and safflomin B. Dried safflower petals are used in folk medicine and have been shown to invigorate blood circulation, break up blood stasis, and promote menstruation. In addition, dried safflower petals contain yellow dyes that are used to color food and cosmetics. In this study, we investigated the effects of dried safflower petals aqueous extracts (SFA) and CY on lipopolysaccharide (LPS)‐induced inflammation using RAW264.7 macrophages. RESULTS: Our data showed that treatment with SFA (1–1000 µg mL^?1) and CY (1–2000 µg mL^?1) does not cause cytotoxicity in cells. SFA and CY inhibited LPS‐stimulated nitric oxide (NO), prostaglandin E₂ (PGE₂), and interleukin 1β (IL‐1β) release, through attenuation of inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) protein expression. Further, SFA and CY suppressed the LPS‐induced phosphorylation of nuclear factor‐κB, which was associated with the inhibition of IκB‐α degradation. CONCLUSION: These results suggest that SFA and CY provide an anti‐inflammatory response through inhibiting the production of NO and PGE₂ by the downregulation of iNOS and COX‐2 gene expression. Thus safflower petals have the potential to provide a therapeutic approach to inflammation‐associated disorders. 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.     

红松松仁多糖对脂多糖诱导RAW264.7细胞炎症反应的抑制作用

目的:探讨红松松仁多糖PNP40c-1对脂多糖(Lipopolysaccharides,LPS)诱导RAW264.7细胞炎症反应的抑制作用及可能机制。方法:以LPS刺激RAW264.7细胞诱导炎症模型,分别采用MTT法、比色法、酶联免疫法、RT-PCR和Western Blot等方法,比较LPS诱导前后巨噬细胞的细胞活力、吞噬能力、一氧化氮(nitric oxide,NO)/一氧化氮合酶(nitric oxide synthase,iNOS)和细胞因子表达的变化;同时对Nrf2/HO-1信号通路中关键蛋白核因子E2相关因子2(nuclear factor(erythroid-derived 2)-like 2 protein,Nrf2)和血红素氧化酶-1(heme oxygenase-1,HO-1)的mRNA和蛋白表达水平进行研究,以探讨PNP40c-1的具体作用机制。结果:在LPS诱导RAW264.7细胞的炎症反应中,PNP40c-1以剂量依赖性显著提高巨噬细胞的吞噬能力(P<0.05),抑制LPS诱导的NO和iNOS过量表达;PNP40c-1还可通过Nrf2/HO-1信号通路调节炎症因子如肿瘤坏死因子(tumor necrosis factor-α,TNF-α),白介素-1β(interleukin-1β,IL-1β)、白介素-6(IL-6)的分泌,缓解炎症反应。结论:红松松仁多糖PNP40c-1对LPS诱导的炎症反应具有一定的抑制作用,其作用机制与调节Nrf2/HO-1信号通路有关。     

Suppressive effects of extracts from the aerial part of Coriandrum sativum L. on LPS‐induced inflammatory responses in murine RAW 264.7 macrophages

BACKGROUND: Coriandrum sativum is used not only as a spice to aid flavour and taste values in food, but also as a folk medicine in many countries. Since little is known about the anti‐inflammatory ability of the aerial parts (stem and leaf) of C. sativum, the present study investigated the effect of aerial parts of C. sativum on lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophages. We further explored the molecular mechanism underlying these pharmacological properties of C. sativum. RESULTS: Ethanolic extracts from both stem and leaf of C. sativum (CSEE) significantly decreased LPS‐induced nitric oxide and prostaglandin E₂ production as well as inducible nitric oxide synthase, cyclooxygenase‐2, and pro‐interleukin‐1β expression. Moreover, LPS‐induced IκB‐α phosphorylation and nuclear p65 protein expression as well as nuclear factor‐κB (NF‐κB) nuclear protein–DNA binding affinity and reporter gene activity were dramatically inhibited by aerial parts of CSEE. Exogenous addition of CSEE stem and leaf significantly reduced LPS‐induced expression of phosphorylated mitogen‐activated protein kinases (MAPKs). CONCLUSION: Our data demonstrated that aerial parts of CSEE have a strong anti‐inflammatory property which inhibits pro‐inflammatory mediator expression by suppressing NF‐κB activation and MAPK signal transduction pathway in LPS‐induced macrophages. Copyright © 2010 Society of Chemical Industry     

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.     

Anti-inflammatory activities of aqueous extract of Mesona procumbens in experimental mice

BACKGROUND: Mesona procumbens is consumed as a herbal drink and jelly‐type dessert in Taiwan. The aim of this study was to determine the mechanism of anti‐inflammatory activities of the aqueous extract of M. procumbens (AMP) using the λ‐carrageenin (Carr)‐induced mouse paw oedema model. The fingerprint chromatogram of AMP was obtained by high‐performance liquid chromatography (HPLC) analysis. To investigate the anti‐inflammatory mechanism of AMP, the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) and the level of malondialdehyde (MDA) in paw oedema were monitored. Serum nitric oxide (NO), tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) were also evaluated. RESULTS: The fingerprint chromatogram from HPLC indicated that AMP contained protocatechuic acid, chlorogenic acid, vanillic acid and caffeic acid. In the anti‐inflammatory test, AMP decreased paw oedema after Carr administration and increased the CAT, SOD and GPx activities and decreased the MDA level in paw oedema at 5 h after Carr injection. AMP also affected the serum NO, TNF‐α and IL‐1β levels at 5 h after Carr injection. Western blotting revealed that AMP decreased the expression of Carr‐induced inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2). CONCLUSION: Mesona procumbens has the potential to provide a therapeutic approach to inflammation‐associated disorders. Copyright © 2011 Society of Chemical Industry     

Anti‐inflammatory and anticancer activities of water‐soluble peptide extracts of buffalo and cow milk Cheddar cheeses

This article aimed to assess the anti‐inflammatory and anticancer potential of water‐soluble peptide (WSP) extracts from buffalo and cow milk Cheddar cheeses. Anti‐inflammatory activity was evaluated on the basis of nitric oxide (NO) production in lipopolysaccharide‐stimulated macrophage (RAW‐264.7) cells. A cell viability assay, cell cycle arrest and apoptosis were performed to explore anticancer activity in a colon cancer model (HT‐29). The WSP extracts of both Cheddar cheeses effectively inhibited NO production in activated macrophages. Maximum growth inhibition was observed in the HT‐29 cells at concentrations of 400 and 500 μg/mL. A significant increase in cell population at G₀/G₁ phase of the cell cycle was observed. Moreover, the WSP extracts also induced extensive apoptosis in colon cancer cells.     

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.     

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     

桑葚提取物体外抗炎作用及机制的研究

本文采用脂多糖(LPS)刺激小鼠腹腔巨噬细胞RAW264.7,建立细胞体外的炎症模型,研究桑葚提取物对LPS诱导巨噬细胞RAW264.7分泌功能的影响及其作用机制。实验用1μg/mL LPS刺激RAW264.7细胞,在不同浓度样品的干预下,用MTT法检测不同浓度的样品对RAW264.7细胞的作用;用Griess法检测细胞液中NO的含量;用酶联免疫吸附法(ELISA)检测细胞液中PGE2含量;用免疫印迹法(Western Blot)和RT-PCR法检测桑葚提取物对细胞iNOS和COX-2表达的影响;用HPLC法检测桑葚提取物中白藜芦醇的含量。结果表明桑葚提取物浓度在0.5~2 mg/mL范围内对细胞生长无明显影响;在1~2 mg/mL范围内能有效抑制NO和PGE2的分泌并能有效抑制iNOS和COX-2的表达;桑葚提取物中白藜芦醇的含量为107.44±0.48μg/g。这表明桑葚提取物抑制炎症相关因子表达量,从而减弱促炎症反应,发挥抗炎功效,其抗炎活性可能与桑葚中含有较高的白藜芦醇相关。     

Evaluation of the anti-inflammatory effects of phloretin and phlorizin in lipopolysaccharide-stimulated mouse macrophages

Many reports suggest that phloretin and phlorizin have antioxidant properties and can inhibit glucose transportation, the anti-inflammatory effects and mechanism of phloretin and phlorizin remain unclear. This study aims to evaluate the anti-inflammatory effects of phloretin and phlorizin in LPS-stimulated murine RAW264.7 macrophages. RAW264.7 cells were pretreated with various concentrations of phloretin or phlorizin (3–100 μM) and cell inflammatory responses were induced with LPS. Pretreated with 10 μM phloretin significantly inhibited the levels of NO, PGE₂, IL-6, TNF-α, iNOS and COX-2. Furthermore, it was demonstrated that phloretin suppressed the nuclear translocation of NF-κB subunit p65 proteins, and decreased phosphorylation in MAPK pathways. Surprisingly, phlorizin did not suppress the inflammatory response in LPS-stimulated RAW264.7 cells. These results suggest that phloretin has an anti-inflammatory effect that reduces levels of proinflammatory cytokines and mediators in RAW264.7 cells.     

Phosphorylation of peptidoglycan from Lactobacillus acidophilus and its immunoregulatory function

Peptidoglycan (PG) is available from a wide variety of lactic acid bacteria (LAB) and is the main structure of cell wall components. Phosphorylated modification would bring new properties such as the potential antioxidant activities and antiviral capability to an organic molecule. In the present work, small molecular fragments of PG (derived from Lactobacillus acidophilus) hydrolysed by mutanolysin were phosphorylated under optimal conditions. P‐PG had a monomer molecular structure of GlcNAC[PO₃]–MurNAC–Ala–Glu–Lys–Ala, with a molecular mass of 884 Da and a phosphorus content of 8.9%. P‐PG displayed some immunoregulatory activity in lipopolysaccharides (LPS) stimulated RAW 264.7 macrophages. Compared with the LPS‐stimulated group, the addition of P‐PG inhibited the secretion of GM‐CSF, TNF‐α and IL‐1 in a dose‐dependent manner. The effect of 50 μg mL^?1 of P‐PG was more significant than 50 μg mL^?1 of PG. Lower fluorescence of lysosomes was observed in P‐PG‐treated RAW 264.7 cells may also reveal some immune defence function in the LPS‐induced macrophages.     

γ-谷维素对脂多糖诱导巨噬细胞RAW264.7炎症因子表达的影响

利用细菌脂多糖(lipopolysaccharide,LPS)刺激巨噬细胞RAW264.7形成炎症模型,评价γ-谷维素对巨噬细胞炎症因子表达的影响。在LPS刺激的RAW264.7细胞培养基中,添加不同浓度的γ-谷维素,分析培养基中炎症因子白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)的含量,以及NO_2~-/NO_3~-含量,发现γ-谷维素能抑制炎症因子的分泌;利用实时荧光定量聚合酶链式反应(real-time quantitative polymerase chain reaction,qPCR)分析mRNA表达水平,确定γ-谷维素能抑制炎症因子的基因表达;采用Western blotting分析进一步确定γ-谷维素能抑制炎症因子的蛋白表达。综上所述,γ-谷维素能明显抑制炎症因子IL-1β、TNF-α、IL-6、NO等分泌和表达。     

Dimethoxycurcumin, a synthetic curcumin analogue with higher metabolic stability, inhibits NO production, inducible NO synthase expression and NF-kappaB activation in RAW264.7 macrophages activated with LPS

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 and/or iNOS for potential therapeutic use. We investigated whether dimethoxycurcumin (DiMC), a synthetic curcumin analogue with higher metabolic stability over curcumin, could inhibit NO production and iNOS expression in activated macrophages. RAW264.7 macrophages were activated with lipopolysaccharide (LPS) in the absence or presence of DiMC, which contains four methoxy groups at two aromatic rings, curcumin containing two, bis-demethoxycurcumin (BDMC) containing none, or tetrahydrocurcumin (THC) containing two but lacking conjugated double bonds in the central seven-carbon chain. NO production, iNOS expression and NF-kappaB activity were examined. DiMC, curcumin and BDMC inhibited NO production, iNOS expression and NF-kappaB activation, with DiMC being the most effective, followed by curcumin and BDMC. THC failed to inhibit NO production, iNOS expression and NF-kappaB activation. Our results suggest that DiMC inhibits NO production, iNOS expression and NF-kappaB activation in LPS-activated macrophages, which may be due not only to the conjugated double bonds but also the increased number of methoxy groups.