The Antioxidant and Anti-Inflammatory Activities of 8-Hydroxydaidzein (8-HD) in Activated Macrophage-Like RAW264.7 Cells

8-Hydroxydaidzein (8-HD) is a daidzein metabolite isolated from soybeans. This compound has been studied for its anti-proliferation, depigmentation, and antioxidant activities. However, the anti-inflammatory activities of 8-HD are not well-understood. Through its antioxidant effects in ABTS and DPPH assays, 8-HD reduces the production of sodium nitroprusside (SNP)-induced radical oxygen species (ROS). By triggering various Toll-like receptors (TLRs), 8-HD suppresses the inflammatory mediator nitric oxide (NO) without cytotoxicity. We examined the regulatory mechanism of 8-HD in lipopolysaccharide (LPS)-induced conditions. We found that 8-HD diminishes inflammatory gene expression (e.g., inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and tumor necrosis factor (TNF)-α) by regulating the transcriptional activities of nuclear factor (NF)-κB and activator protein 1 (AP-1). To find the potential targets of 8-HD, signaling pathways were investigated by immunoblotting analyses. These analyses revealed that 8-HD inhibits the activation of TAK1 and that phosphorylated levels of downstream molecules decrease in sequence. Together, our results demonstrate the antioxidant and anti-inflammatory actions of 8-HD and suggest its potential use in cosmetics or anti-inflammatory drugs.

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abstract: ETHNOPHARMACOLOGICAL RELEVANCE
Seed of Torreya nucifera (L.) Siebold & Zucc is used to treat several diseases in Asia. Reports document that T. nucifera has anti-cancer, anti-inflammatory, anti-oxidative activities. In spite of numerous findings on its pharmacological effects, the understanding of the molecular inhibitory mechanisms of the plant remains to be studied. Therefore, we aimed to explore in vitro anti-inflammatory mechanisms of ethyl acetate fraction (Tn-EE-BF) prepared from the seed of T. nucifera in LPS-stimulated macrophage inflammatory responses.



For this purpose, we measured nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated macrophages. Additionally, using RT-PCR, luciferase reporter gene assay, immunoblotting analysis, and kinase assay, the levels of inflammatory genes, transcription factors, and inflammatory signal-regulatory proteins were investigated. Finally, the constituent of Tn-EE-BF was identified using HPLC.



Tn-EE-BF inhibits NO and PGE2 production and also blocks mRNA levels of inducible NO synthase (iNOS), tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 in a dose dependent manner. Tn-EE-BF reduces nuclear levels of the transcriptional factors NF-κB (p65) and AP-1 (c-Jun and FRA-1). Surprisingly, we found that Tn-EE-BF inhibits phosphorylation levels of Src and Syk in the NF-κB pathway, as well as, IRAK1 at the protein level, part of the AP-1 pathway. By kinase assay, we confirmed that Src, Syk, and IRAK1 are suppressed directly. HPLC analysis indicates that arctigenin, amentoflavone, and quercetin may be active components with anti-inflammatory activities.



Tn-EE-BF exhibits anti-inflammatory activities by direct inhibition of Src/Syk/NF-κB and IRAK1/AP-1.

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title: Evaluation of Antioxidant and Anti-Inflammatory Effect of Rhododendron brachycarpum Extract Used in Skin Care Product by in Vitro and in Vivo Test
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title: Moisturizing and Antiinflammatory Properties of Cosmetic Formulations Containing Centella asiatica Extract
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abstract: Covering: up to March 2013. In addition to their prominent role in basic biological and chemical research, natural products are a rich source of commercial products for the pharmaceutical and other industries. Industrial natural product chemistry is of fundamental importance for successful product development, as the vast majority (ca. 80%) of commercial drugs derived from natural products require synthetic efforts, either to enable economical access to bulk material, and/or to optimize drug properties through structural modifications. This review aims to illustrate issues on the pathway from lead to product, and how they have been successfully addressed by modern natural product chemistry. It is focused on natural products of current relevance that are, or are intended to be, used as pharmaceuticals.

title: Natural products and drug discovery: a survey of stakeholders in industry and academia
abstract: In recent decades, natural products have undisputedly played a leading role in the development of novel medicines. Yet, trends in the pharmaceutical industry at the level of research investments indicate that natural product research is neither prioritized nor perceived as fruitful in drug discovery programmes as compared with incremental structural modifications and large volume HTS screening of synthetics.  We seek to understand this phenomenon through insights from highly experienced natural product experts in industry and academia.  We conducted a survey including a series of qualitative and quantitative questions related to current insights and prospective developments in natural product drug development. The survey was completed by a cross-section of 52 respondents in industry and academia.  One recurrent theme is the dissonance between the perceived high potential of NP as drug leads among individuals and the survey participants' assessment of the overall industry and/or company level strategies and their success. The study's industry and academic respondents did not perceive current discovery efforts as more effective as compared with previous decades, yet industry contacts perceived higher hit rates in HTS efforts as compared with academic respondents. Surprisingly, many industry contacts were highly critical to prevalent company and industry-wide drug discovery strategies indicating a high level of dissatisfaction within the industry.  These findings support the notion that there is an increasing gap in perception between the effectiveness of well established, commercially widespread drug discovery strategies between those working in industry and academic experts. This research seeks to shed light on this gap and aid in furthering natural product discovery endeavors through an analysis of current bottlenecks in industry drug discovery programmes.

title: Antioxidant activity applying an improved ABTS radical cation decolorization assay.
abstract: A method for the screening of antioxidant activity is reported as a decolorization assay applicable to both lipophilic and hydrophilic antioxidants, including flavonoids, hydroxycinnamates, carotenoids, and plasma antioxidants. The pre-formed radical monocation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS*+) is generated by oxidation of ABTS with potassium persulfate and is reduced in the presence of such hydrogen-donating antioxidants. The influences of both the concentration of antioxidant and duration of reaction on the inhibition of the radical cation absorption are taken into account when determining the antioxidant activity. This assay clearly improves the original TEAC assay (the ferryl myoglobin/ABTS assay) for the determination of antioxidant activity in a number of ways. First, the chemistry involves the direct generation of the ABTS radical monocation with no involvement of an intermediary radical. Second, it is a decolorization assay; thus the radical cation is pre-formed prior to addition of antioxidant test systems, rather than the generation of the radical taking place continually in the presence of the antioxidant. Hence the results obtained with the improved system may not always be directly comparable with those obtained using the original TEAC assay. Third, it is applicable to both aqueous and lipophilic systems.

title: Effect of Ixeris dentata Nakai Extract on Nitric Oxide Production and Prostaglandin E2 Generation in LPS-stimulated RAW264.7 Cells
abstract: Inflammation is the basis of severe acute and chronic diseases. This study investigated the anti-inflammatory property of a crude methanol extract (MeOH-ex) and the solvent fractions of Ixeris dentata Nakai (IDN) in LPS-stimulated murine macrophage-like cell line RAW264.7. Here, we showed that the ethyl acetate fraction (EtOAc-fr) had the most potent inhibitory activity on LPS-induced nitric oxide (NO) production among the tested samples, i.e., IDN MeOH-ex and the three different solvent fractions (chloroform, n-hexane, and EtOAc). We further found that the EtOAc-fr significantly inhibited LPS-induced prostaglandin PGE2 (PGE2) generation in RAW264.7 cells. Furthermore, the treatment with EtOAc-fr effectively suppressed the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). These results suggest that the EtOAc-fr of IDN MeOH-ex exhibits an anti-inflammatory activity in vitro by inhibiting LPS-induced NO production and PGE2 generation via suppression of iNOS and COX-2 expression.

title: In vitro and in vivo anti-inflammatory activities of Korean Red Ginseng-derived components
abstract: Although Korean Red Ginseng (KRG) has been traditionally used for a long time, its anti-inflammatory role and underlying molecular and cellular mechanisms have been poorly understood. In this study, the anti-inflammatory roles of KRG-derived components, namely, water extract (KRG-WE), saponin fraction (KRG-SF), and nonsaponin fraction (KRG-NSF), were investigated.  To check saponin levels in the test fractions, KRG-WE, KRG-NSF, and KRG-SF were analyzed using high-performance liquid chromatography. The anti-inflammatory roles and underlying cellular and molecular mechanisms of these components were investigated using a macrophage-like cell line (RAW264.7 cells) and an acute gastritis model in mice.  Of the tested fractions, KGR-SF (but not KRG-NSF and KRG-WE) markedly inhibited the viability of RAW264.7 cells, and splenocytes at more than 500 μg/mL significantly suppressed NO production at 100 μg/mL, diminished mRNA expression of inflammatory genes such as inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α, and interferon-β at 200 μg/mL, and completely blocked phagocytic uptake by RAW264.7 cells. All three fractions suppressed luciferase activity triggered by interferon regulatory factor 3 (IRF3), but not that triggered by activator protein-1 and nuclear factor-kappa B. Phospho-IRF3 and phospho-TBK1 were simultaneously decreased in KRG-SF. Interestingly, all these fractions, when orally administered, clearly ameliorated the symptoms of gastric ulcer in HCl/ethanol-induced gastritis mice.  These results suggest that KRG-WE, KRG-NSF, and KRG-SF might have anti-inflammatory properties, mostly because of the suppression of the IRF3 pathway.

title: "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.
abstract: A simple and efficient procedure was employed for the electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to sheets of pure, unmodified nitrocellulose. Immobilized proteins could then be radiographically visualized in situ by reaction with specific antibody and the subsequent binding of radioiodinated Staphylococcus protein A to the immune complexes. The detection of murine leukemia virus antigens in complex cellular lysates was used to demonstrate the efficacy of this technique.