Hyun Pyo Kim

Effects of naturally occurring flavonoids on nitric oxide production in the macrophage cell line RAW 264.7 and their structure–activity relationships

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.

Effect of wogonin, a plant flavone from Scutellaria radix, on the suppression of cyclooxygenase-2 and the induction of inducible nitric oxide synthase in lipopolysaccharide-treated RAW 264.7 cells.

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 microM, respectively. When added after the induction of iNOS and COX-2, wogonin inhibited the formation of PGE(2) (IC(50) = 0.8 microM), but not the production of NO. Wogonin inhibited COX-2 activity directly (IC(50) = 46 microM) 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.

Effects of naturally occurring prenylated flavonoids on enzymes metabolizing arachidonic acid: Cyclooxygenases and lipoxygenases

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.

Effects of wogonin, a plant flavone from Scutellaria radix, on skin inflammation: in vivo regulation of inflammation-associated gene expression

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.

Anti-inflammatory effects of schisandrin isolated from the fruit of Schisandra chinensis Baill

Schisandrin is the main active ingredient isolated from the fruit of Schisandra chinensis Baill. Recent studies have demonstrated that schisandrin exhibits anti-oxidative effects in vivo. In the present study, the effect of schisandrin on plasma nitrite concentration in lipopolysaccharide (LPS)-treated mice was evaluated. It also significantly inhibited carrageenan-induced paw edema and acetic acid-induced vascular permeability in mice. Furthermore, schisandrin had a protective effect on lipopolysaccharide (LPS)-induced sepsis. In vitro, our results are the first that show that the anti-inflammatory properties of schisandrin result from the inhibition of nitric oxide (NO) production, prostaglandin E(2) (PGE(2)) release, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, which in turn results from the inhibition of nuclear factor-kappaB (NF-kappaB), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) activities in a RAW 264.7 macrophage cell line.

Inhibition of cyclooxygenase/lipoxygenase from human platelets by polyhydroxylated/methoxylated flavonoids isolated from medicinal plants.

Various flavonoid derivatives were previously reported to possess the inhibitory activity on cyclooxygenase/lipoxygenase. And these properties of flavonoids might contribute to their antiinflammatory activityin vivo. In this study, several polyhydroxylated/methoxylated flavonoid derivatives such as oroxylin A, wogonin, skullcapflavone II, tectorigenin and iristectorigenin A were isolated from the medicinal plants. These compounds were evaluated for their inhibitory effects on cyclooxygenase/lipoxygenase from the homogenate of human plateletsin vitro. It was found that isoflavones including daidzein and tectorigenin possessed the inhibitory activity on cyclooxygenase, although the potency of inhibition was far less than that of indomethacin. In addition, oroxylin A, baicalein and wogonin inhibited 12-lipoxygenase activity without affecting cyclooxygenase, which suggested that 5,6,7- or 5,7,8-trisubstitutions of A-ring of flavone gave favorable results. The IC50 values of oroxylin A and NDGA aginst 12-lipoxygenase were found to be 100 and 1.5 uM, respectively.

Anti-inflammatory and anti-arthritic activity of total flavonoids of the roots of Sophora flavescens

ETHNOPHARMACOLOGICAL RELEVANCE The roots of Sophora flavescens have long been used in Chinese medicine for the treatment of fever, inflammatory disorders, ulcers and skin burns. Sophora flavescens contains flavonoids and alkaloids. AIM OF THE STUDY This study was conducted to develop a plant-based anti-inflammatory agent focused on chronic inflammatory disorders. To accomplish this, the alkaloid-free prenylated flavonoid-enriched fraction (PFS) of rhizomes of Sophora flavescens was prepared and its in vitro and in vivo anti-inflammatory activities were then evaluated for the first time. MATERIALS AND METHODS The inhibitory activity of PFS on PGE(2), NO, IL-6 and TNF-alpha production of lipopolysaccharide (LPS)-treated RAW 264.7 cells was measured. Additionally, adjuvant-induced arthritis in rats was used as an animal model of chronic inflammation to establish the in vivo anti-inflammatory effects of PFS. RESULT PFS inhibited cyclooxygenase-2 (COX-2)-catalyzed PGE(2) and inducible nitric oxide synthase (iNOS)-catalyzed NO production by lipopolysaccharide (LPS)-treated RAW 264.7 cells at 10-50 microg/ml, and these effects primarily occurred via COX-2 inhibition and iNOS down-regulation, respectively. PFS also inhibited IL-6 and TNF-alpha production. When tested against adjuvant-induced arthritis in rats (chronic inflammation), PFS strongly inhibited arthritic inflammation when administered orally at doses of 10-100mg/kg/day. In addition, PFS administered orally potently inhibited acetic acid-induced writhing in mice. CONCLUSIONS Our results suggest that PFS inhibits chronic inflammatory response and the inhibition of proinflammatory molecules such as COX-2, iNOS and IL-6 may contribute, at least in part, to the anti-inflammatory activity in vivo. Overall, these results indicate that PFS from Sophora flavescens may have the potential for treatment of chronic inflammatory disorders such as rheumatoid arthritis.

Effects of sophoraflavanone G, a prenylated flavonoid from Sophora flavescens, on cyclooxygenase-2 and in vivo inflammatory response.

Previously, several prenylated flavonoids having a C-8 lavandulyl moiety were found to inhibit cyclooxygenase-1 (COX-1) as well as 5-lipoxygenase (5-LOX), and sophoraflavanone G was the most potent inhibitor against these eicosanoid generating enzymes among 19 prenylated flavonoids tested. In this investigation, effects of sophoraflavanone G on COX-2 induction from RAW 264.7 cells andin vivo inflammatory response were studied. Sophoraflavanone G inhibited prostaglandin E2 (PGE2) production from lipopolysaccharide (LPS)-treated RAW cells by COX-2 down-regulation at 1-50 uM. Other prenylated flavonoids including kuraridin and sanggenon D also down-regulated COX-2 induction at 10-25 uM, while kurarinone and echinoisoflavanone did not. In addition, sophoraflavanone G showedin vivo anti-inflammatory activity against mouse croton oil-induced ear edema and rat carrageenan paw edema via oral (2-250 mg/kg) or topical administration (10-250 ug/ear). Although the potencies of inhibition were far less than that of a reference drug, prednisolone, this compound showed higher antiinflammatory activity when applied topically, suggesting a potential use for several eicosanoidrelated skin inflammation such as atopic dermatitis.

Amentoflavone, a plant biflavone: A new potential anti-inflammatory agent

Biflavonoid is one of unique classes of naturally-occurring bioflavonoids. Certain biflavonoids including amentoflavone were previously reported to have inhibitory effect on the group II phospholipase A2 activity. Amentoflavone was also found to inhibit cyclooxygenase from guinea-pig epidermis without affecting lipoxygenase. In this study, anti-inflammatory and analgesic activities of amentoflavone were evaluated. When amentoflavone was administered intraperitoneally, it showed a potent anti-inflammatory activity as determined by amelioration of croton-oil induced mouse ear edema. It also showed a potent anti-inflammatory activity in the rat carrageenan paw edema model (ED50=42 mg/kg) compared to the activity of prednisolone (35 mg/kg) and indomethacin (10 mg/kg). However, amentoflavone did not show a significant inhibitory activity against rat adjuvant-induced arthritis, a chronic inflammatory model. In addition, amentoflavone was found to possess a potent analgesic activity in the acetic acid writhing test (ED50=9.6 mg/kg) compared to the activity of indomethacin (3.8 mg/kg). These results suggest that amentoflavone may be a potential lead for a new type of anti-inflammatory agents having dual inhibitory activity of group II phospholipase A2 and cyclooxygenase.

Chemical constituents of the root of Dystaenia takeshimana and their anti-inflammatory activity.

In our ongoing search for bioactive compounds originating from the endemic species in Korea, we found that the hexane and EtOAc fractions of the MeOH extract from the root ofDystaenia takeshimana (Nakai) Kitagawa (Umbelliferae) showed cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) dual inhibitory activity by assessing their effects on the production of prostaglandin D2 (PGD2) and leukotriene C4 (LTC4) in mouse bone marrow-derived mast cells. By activity-guided fractionation, five coumarins, viz. psoralen (2), xanthotoxin (3), scopoletin (4), umbelliferone (5), and (+)-marmesin (6), together with β-sitosterol (1), were isolated from the hexane fraction, and two phenethyl alcohol derivatives, viz. 2-methoxy-2-(4′-hydroxyphenyl)ethanol (7) and 2-hydroxy-2-(4′-hydroxyphenyl)ethanol (8), three flavonoids, viz. apigenin (9), luteolin (10), and cynaroside (11), as well as daucosterol (12) were isolated from the EtOAc fraction using silica gel column chromatography. In addition, D-mannitol (13) was isolated from the BuOH fraction by recrystallization. Two of the coumarins, scopoletin (4) and (+)-marmesin (6), the two phenethyl alcohol derivatives (7, 8) and the three flavonoids (9–11) were isolated for the first time from this plant. Among the compounds isolated from this plant, the five coumarins as well as the three flavonoids showed COX-2/5-LOX dual inhibitory activity. These results suggest that the anti-inflammatory activity ofD. takeshimana might in part occurvia the inhibition of the generation of eicosanoids.