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During senescence, cells express molecules called senescence-associated secretory phenotype (SASP), including growth factors, proinflammatory cytokines, chemokines, and proteases. The SASP induces a chronic low-grade inflammation adjacent to cells and tissues, leading to degenerative diseases. The anti-inflammatory activity of flavonoids was investigated on SASP expression in senescent fibroblasts. Effects of flavonoids on SASP expression such as IL-1α, IL-1β, IL-6, IL-8, GM-CSF, CXCL1, MCP-2 and MMP-3 and signaling molecules were examined in bleomycin-induced senescent BJ cells. In vivo activity of apigenin on SASP suppression was identified in the kidney of aged rats. Among the five naturally-occurring flavonoids initially tested, apigenin and kaempferol strongly inhibited the expression of SASP. These flavonoids inhibited NF-κB p65 activity via the IRAK1/IκBα signaling pathway and expression of IκBζ. Blocking IκBζ expression especially reduced the expression of SASP. A structure-activity relationship study using some synthetic flavones demonstrated that hydroxyl substitutions at C-2′,3′,4′,5 and 7 were important in inhibiting SASP production. Finally, these results were verified by results showing that the oral administration of apigenin significantly reduced elevated levels of SASP and IκBζ mRNA in the kidneys of aged rats. This study is the first to show that certain flavonoids are inhibitors of SASP production, partially related to NF-κB p65 and IκBζ signaling pathway, and may effectively protect or alleviate chronic low-grade inflammation in degenerative diseases such as cardiovascular diseases and late-stage cancer.

Flavonoids from plant origin show anti-inflammatory activity in vitro and in vivo. In addition to inhibition of inflammation-associated enzymes, such as cyclooxygenases (COX) and lipoxygenases, they have been found to regulate the expression of inflammation-associated proteins from in vitro experiments. In order to prove in vivo behavior and the potential for beneficial use against inflammatory skin disorders, the effect of wogonin (5,7-dihydroxy-8-methoxyflavone) on in vivo expression of several inflammation-associated genes was examined in the intact as well as in the inflamed mouse skin by reverse transcriptase-polymerase chain reaction analysis. When applied topically on the intact skin, only a high dose treatment of wogonin (1000 microg/ear/3 days) slightly increased COX-1 and fibronectin mRNA. On the other hand, wogonin at the doses of 250-1000 microg/ear/3 days potently lowered mRNA levels of COX-2 and tumor necrosis factor-alpha with less effect on intercellular adhesion molecule-1 and interleukin-1beta in a sub-chronic skin inflammation model of tetradecanoylphorbol-13-acetate-induced ear edema (multiple treatment). The decrease of prostaglandin E(2) concentration (27.3-34.3%) was concomitantly observed in the wogonin-treated groups. A similar effect was also observed in an acute inflammation model of arachidonic acid-induced ear edema. From the present study, wogonin was proved to differentially regulate the expression of inflammation-associated genes in vivo and to become a useful therapeutic agent for skin inflammatory diseases mainly due to its modulation of the expression of proinflammatory molecules.

Prenylated flavonoids are chemical entities having an isoprenyl, a geranyl, a 1,1-dimethylallyl, and/or a lavandulyl moiety as part of their flavonoid backbone structure. In this study, the effects of 19 naturally occurring prenylated flavonoids, isolated from medicinal plants, on cyclooxygenase (COX)-1 and COX-2 and on 5-lipoxygenase (5-LOX) and 12-LOX were investigated using [14C]arachidonic acid as a substrate. The homogenates of bovine platelets and polymorphonuclear leukocytes were used as COX-1, 12-LOX, and 5-LOX enzyme sources; the homogenate of aspirin-pretreated lipopolysaccharide-induced RAW 264.7 cells was used for the COX-2 enzyme source. Among the 19 prenylated flavonoids, morusin, kuwanon C, sanggenon B, sanggenon D and kazinol B inhibited COX-2 activity (ic(50) = 73-100 microM), but the potencies were far less than that of NS-398 (ic(50) = 2.9 microM). In contrast, many prenylated flavonoids, such as kuraridin, kuwanon C and sophoraisoflavanone A, inhibited COX-1 activity. Of the COX-1 inhibiting prenylated flavonoids, kuraridin, kurarinone, and sophoraflavanone G, all having a C-8 lavandulyl moiety, showed potent activity (ic(50) = 0.1 to 1 microM) comparable to that of indomethacin (ic(50) = 0.7 microM). Most of the prenylated flavonoids tested inhibited 5-LOX activity with ic(50) values ranging from 0.09 to 100 microM. Of these, only kuwanon C, papyriflavonol A and sophoraflavanone G showed inhibitory activity against 12-LOX at low concentration ranges (ic(50) = 19-69 microM) comparable to that of NDGA (ic(50) = 2.6 microM). Our results suggest that the position and the nature of the prenyl substitution greatly influence in vitro biological activities of these molecules.

Plant flavonoids show anti-inflammatory activity both in vitro and in vivo. Some flavonoids, such as flavone derivatives, have been reported previously to inhibit nitric oxide (NO) production by suppressing inducible nitric oxide synthase (iNOS) expression. In this investigation, the effects of wogonin, a potent inhibitor of NO production among the flavonoids tested, on cyclooxygenase-2 (COX-2) induction and activity were elucidated further in connection with iNOS, using a mouse macrophage cell line, RAW 264.7. Wogonin inhibited NO and prostaglandin E 2 (PGE 2 ) production from lipopolysaccharide-induced RAW cells with ic 50 values of 31 and 0.3 μM, respectively. When added after the induction of iNOS and COX-2, wogonin inhibited the formation of PGE 2 ( ic 50 = 0.8 μM), but not the production of NO. Wogonin inhibited COX-2 activity directly ( ic 50 = 46 μM) from the homogenate of aspirin-pretreated RAW cells, as determined by measuring [ 14 C]PGE 2 formation from [ 14 C]arachidonic acid. However, it did not inhibit iNOS or phospholipase A 2 activity. Western blotting showed that wogonin suppressed the induction of both iNOS and COX-2. Prednisolone also suppressed the induction of iNOS and COX-2. Whereas RU-486 (a steroid receptor antagonist) reversed the suppressive activity of prednisolone, it did not affect the suppressive activity of wogonin, suggesting that the suppressive activity of wogonin is not mediated by binding to a steroid receptor. Results from the present study demonstrated that wogonin is a direct COX-2 inhibitor, as well as an inhibitor of iNOS and COX-2 induction. Wogonin may be a potential agent for use in the treatment of inflammatory diseases.

Flavonoids affect the inflammatory process of the mammalian system and possess anti-inflammatory as well as immunomodulatory activities in vitro and in vivo. Since nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is one of the inflammatory mediators, the effects of various naturally occurring flavonoids on NO production in LPS-activated RAW 264.7 cells were evaluated in vitro. Flavonoids such as apigenin, wogonin, luteolin, tectorigenin, and quercetin inhibited NO production, as measured by nitrite formation at 10-100 microM. The most active among 26 flavonoid derivatives tested were apigenin, wogonin, and luteolin, having IC50 values of 23, 17, and 27 microM, respectively, while AMT, a synthetic selective iNOS inhibitor, had an IC50 value of 0.09 microM. In contrast, flavanones, such as naringenin, and flavonoid glycosides, such as apiin, did not demonstrate significant inhibition up to 100 microM. These results clearly indicated that a C-2,3 double bond might be important, and that the potency of inhibition depended upon the substitution patterns of the flavonoid molecules. The inhibitory activity of flavonoids was not due to direct inhibition of iNOS enzyme activity because they did not reasonably inhibit iNOS activity, as measured by [3H]citrulline formation from [3H]arginine, up to 100 microM. In contrast, wogonin and luteolin concentration-dependently reduced iNOS enzyme expression, when measured by western blotting, at 10-100 microM. All these results clearly demonstrated that certain flavonoids inhibit NO production in lipopolysaccharide-activated RAW 264.7 cells, and their inhibitory activity might be due to reduction of iNOS enzyme expression.