Flavonoid metabolites reduce tumor necrosis factor-α secretion to a greater extent than their precursor compounds in human THP-1 monocytes.

Scope - Flavonoids are generally studied in vitro, in isolation, and as unmetabolized precursor structures. However, in the habitual diet, multiple flavonoids are consumed together and found present in the circulation as complex mixtures of metabolites. Using a unique study design, we investigated the potential for singular or additive anti-inflammatory effects of flavonoid metabolites relative to their precursor structures. Methods and results - Six flavonoids, 14 flavonoid metabolites, and 29 combinations of flavonoids and their metabolites (0.1–10 μM) were screened for their ability to reduce LPS-induced tumor necrosis factor-α (TNF-α) secretion in THP-1 monocytes. One micromolar peonidin-3-glucoside, cyanidin-3-glucoside, and the metabolites isovanillic acid (IVA), IVA-glucuronide, vanillic acid-glucuronide, protocatechuic acid-3-sulfate, and benzoic acid-sulfate significantly reduced TNF-α secretion when in isolation, while there was no effect on TNF-α mRNA expression. Four combinations of metabolites that included 4-hydroxybenzoic acid (4HBA) and/or protocatechuic acid also significantly reduced TNF-α secretion to a greater extent than the precursors or metabolites alone. The effects on LPS-induced IL-1β and IL-10 secretion and mRNA expression were also examined. 4HBA significantly reduced IL-1β secretion but none of the flavonoids or metabolites significantly modified IL-10 secretion. Conclusion - This study provides novel evidence suggesting flavonoid bioactivity results from cumulative or additive effects of circulating metabolites

Flavonoid combinations cause synergistic inhibition of proinflammatory mediator secretion from lipopolysaccharide-induced RAW 264.7 cells.

Objectives: We evaluated several flavonoid combinations for synergy in the inhibition of proinflammatory mediator synthesis in the RAW 264.7 cellular model of inflammation. Methods: The inhibitory effect of chrysin, kaempferol, morin, silibinin, quercetin, diosmin and hesperidin upon nitric oxide (NO), prostaglandin E2 (PGE2) and tumour necrosis factor-α (TNF-α) secretion from the LPS-induced RAW 264.7 monocytic macrophage was assessed and IC50 values obtained. Flavonoids that showed reasonable inhibitory effects in at least two out of the three assays were combined in a series of fixed IC50 ratios and reassessed for inhibition of NO, PGE2 and TNF-α. Dose-response curves were generated and interactions were analysed using isobolographic analysis. Results: The experiments showed that only chrysin, kaempferol, morin, and silibinin were potent enough to produce dose-response effects upon at least two out of the three mediators assayed. Combinations of these four flavonoids showed that several combinations afforded highly significant synergistic effects. Conclusions: Some flavonoids are synergistic in their anti-inflammatory effects when combined. In particular chrysin and kaempferol significantly synergised in their inhibitory effect upon NO, PGE2 and TNF-α secretion. These findings open further avenues of research into combinatorial therapeutics of inflammatory-related diseases and the pharmacology of flavonoid synergy

Common Phenolic Metabolites of Flavonoids, but Not Their Unmetabolized Precursors, Reduce the Secretion of Vascular Cellular Adhesion Molecules by Human Endothelial Cells

Background: Flavonoids have been implicated in the prevention of cardiovascular disease; however, their mechanisms of action have yet to be elucidated, possibly because most previous in vitro studies have used supraphysiological concentrations of unmetabolized flavonoids, overlooking their more bioavailable phenolic metabolites. Objective: We aimed to explore the effects of phenolic metabolites and their precursor flavonoids at physiologically achievable concentrations, in isolation and combination, on soluble vascular cellular adhesion molecule-1 (sVCAM-1). Method: Fourteen phenolic acid metabolites and 6 flavonoids were screened at 1 μM for their relative effects on sVCAM-1 secretion by human umbilical vein endothelial cells stimulated with tumor necrosis factor alpha (TNF-α). The active metabolites were further studied for their response at different concentrations (0.01 μM–100 μM), structure-activity relationships, and effect on vascular cellular adhesion molecule (VCAM)-1 mRNA expression. In addition, the additive activity of the metabolites and flavonoids was investigated by screening 25 unique mixtures at cumulative equimolar concentrations of 1 μM. Results: Of the 20 compounds screened at 1 μM, inhibition of sVCAM-1 secretion was elicited by 4 phenolic metabolites, of which protocatechuic acid (PCA) was the most active (−17.2%, P = 0.05). Investigations into their responses at different concentrations showed that PCA significantly reduced sVCAM-1 15.2–36.5% between 1 and 100 μM, protocatechuic acid-3-sulfate and isovanillic acid reduced sVCAM-1 levels 12.2–54.7% between 10 and 100 μM, and protocatechuic acid-4-sulfate and isovanillic acid-3-glucuronide reduced sVCAM-1 secretion 27.6% and 42.8%, respectively, only at 100 μM. PCA demonstrated the strongest protein response and was therefore explored for its effect on VCAM-1 mRNA, where 78.4% inhibition was observed only after treatment with 100 μM PCA. Mixtures of the metabolites showed no activity toward sVCAM-1, suggesting no additive activity at 1 μM. Conclusions: The present findings suggest that metabolism of flavonoids increases their vascular efficacy, resulting in a diversity of structures of varying bioactivity in human endothelial cells

Synergism of selected flavonoids in inflammation

Inflammation is characterized by the release of various pro-inflammatory mediators, including the free radical nitric oxide (NO), prostaglandin E2 (PGE2) and tumor necrosis factor alpha (TNF-a) from stimulated macrophage cells. Sustained release of these mediators can lead to chronic diseases, tissue injury and multiple organ dysfunction syndrome (MODS). Thus, suppression of these mediators is a useful strategy for the treatment of chronic inflammatory diseases. The use of naturally abundant flavonoid compounds is widely reported to have anti-inflammatory, anticancer, anti-oxidant and estrogenic effects. In particular, chrysin, kaempferol, morin, silibinin, quercetin, diosmin and hesperidin are known to alleviate the generation of key pro-inflammatory mediators. Several of the above mentioned flavonoids have shown biological benefits on models of inflammation. Nevertheless, the combinatorial effects of these flavonoids have not been reported. In this study, the synergistic effects of several flavonoid combinations on secretion of major pro-inflammatory mediator from lipopolysaccharide (LPS)-stimulated RAW264.7 cells as a cellular model of inflammation, were investigated. To further assess the therapeutic efficacy of flavonoid combination during the progression of sepsis, survival studies against polymicrobial sepsis in ICR mice were done as an animal model of inflammation. Prior to in vivo experiments, the effects of all compounds on NO, PGE2 and TNF-Ct secretion from LPS-stimulated RAW 264.7 cells were determined by ELISA and Griess assay; as well as cellular viability by MTT assay. After assessing and obtaining the IC50 values, flavonoids that expressed inhibitory effects, in at least two out of the three mediators, were combined in a series of fixed IC50 ratios and reassessed to generate dose response curves. Flavonoid combination that exhibited highest synergistic potency as detected by isobolographic analyses, were employed to further investigate its effects in an animal model of sepsis by cecal ligation and puncture (CLP)-induced septic shock in ICR mice. Key inflammatory 'mediators secreted from septic mice were measured through ELISA and fluorometric determinations; and pharmacological effects upon vital organs were investigated. Chrysin, kaempferol, morin and silibinin were found to have an adequate potency to produce dose-response effects upon at least two out of the three mediators assayed. Significant synergistic effects have been observed among combinations of the flavonoids mentioned above. In particular, the chrysin / kaempferol combination significantly synergized to increase the potency of inhibiting the mediators NO, PGE2 and TNF-Ct secreted from LPS-stimulated RAW 264.7 cells with IC50 = 2.27IlM, 2.281lM and 20.531lM respectively, as well as a 29% significant increase in survival rate in CLP-induced septic shock in ICR mice. Conclusively, this study demonstrated that chrysin / kaempferol combination significantly synergized to increase the anti-inflammatory activity through inhibition of several mediators which contributed to improved survival rate. These fmdings suggest that chrysin / kaempferol combination has reasonable potential as a natural approach in treating inflammation. Further studies are required to investigate the underlying mechanisms involved during inflammation

Kaempferol and chrysin synergies to improve septic mice survival

Previously, we reported the role of synergy between two flavonoids—namely, chrysin and kaempferol—in inhibiting the secretion of a few major proinflammatory mediators such as tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2), and nitric oxide (NO) from lipopolysaccharide (LPS)-induced RAW 264.7 cells. The present study aims to evaluate the effects of this combination on a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Severe sepsis was induced in male ICR mice (n = 7) via the CLP procedure. The effects of chrysin and kaempferol combination treatment on septic mice were investigated using a 7-day survival study. The levels of key proinflammatory mediators and markers—such as aspartate aminotransferase (AST), TNF-α, and NO—in the sera samples of the septic mice were determined via ELISA and fluorescence determination at different time point intervals post-CLP challenge. Liver tissue samples from septic mice were harvested to measure myeloperoxidase (MPO) levels using a spectrophotometer. Moreover, intraperitoneal fluid (IPF) bacterial clearance and total leukocyte count were also assessed to detect any antibacterial effects exerted by chrysin and kaempferol, individually and in combination. Kaempferol treatment improved the survival rate of CLP-challenged mice by up to 16%. During this treatment, kaempferol expressed antibacterial, antiapoptotic and antioxidant activities through the attenuation of bacterial forming units, AST and NO levels, and increased polymorphonuclear leukocyte (PMN) count in the IPF. On the other hand, the chrysin treatment significantly reduced serum TNF-α levels. However, it failed to significantly improve the survival rate of the CLP-challenged mice. Subsequently, the kaempferol/chrysin combination treatment significantly improved the overall 7-day survival rate by 2-fold—up to 29%. Kaempferol and chrysin revealed some synergistic effects by acting individually upon multiple pathophysiological factors involved during sepsis. Although the kaempferol/chrysin combination did not exhibit significant antibacterial effects, it did exhibit anti-inflammatory and antioxidant activities, which translate to significant improvement in the survival rate of septic animals. These findings suggest the potential application of this combination treatment as a beneficial adjuvant supplement strategy in sepsis control