Effects of naturally occurring dihydroflavonols from Inula viscosa on inflammation and enzymes involved in the arachidonic acid metabolism

The anti-inflammatory properties of three flavanones isolated from Inula viscosa, sakuranetin, 7-O-methylaromadendrin, and 3-acetyl-7-O-methylaromadendrin, have been tested both in vitro and in vivo. Acute inflammation in vivo was induced by means of topical application of 12-O-tetradecanoylphorbol 13-acetate (TPA) to mouse ears or by subcutaneous injection of phospholipase A(2) (PLA(2)) into mouse paws. The test compounds were evaluated in vitro for their effect on both the metabolism of arachidonic acid and on the release and/or activity of enzymes involved in the inflammatory response such as elastase, myeloperoxidase (MPO), and protein kinase C (PKC). The most active compounds in vivo against PLA(2)-induced paw oedema were 7-O-methylaromadendrin (ED(50)=8 mg/kg) and sakuranetin (ED(50)=18 mg/kg). In contrast, the most potent compound against TPA-induced ear oedema was 3-acetyl-7-O-methylaromadendrin (ED(50)=185 microg/ear), followed by sakuranetin (ED(50)=205 microg/ear). In vitro, the latter compound was the most potent inhibitor of leukotriene (LT) B(4) production by peritoneal rat neutrophils (IC(50)=9 microM) and it was also the only compound that directly inhibited the activity of 5-lipoxygenase (5-LOX). 3-Acetyl-7-O-methylaromadendrin also inhibited LTB(4) production (IC(50)=15 microM), but had no effect on 5-LOX activity. The only flavanone that inhibited the secretory PLA(2) activity in vitro was 7-O-methylaromadendrin. This finding may partly explain the anti-inflammatory effect observed in vivo, although other mechanisms such as the inhibition of histamine release by mast cells may also be implicated. Sakuranetin at 100 microM was found to inhibit elastase release, although this result is partly due to direct inhibition of the enzyme itself. At the same concentration, 7-O-methylaromadendrin only affected the enzyme release. Finally, none of the flavanones exhibited any effect on MPO or PKC activities. Taken together, these findings indicate that sakuranetin may be a selective inhibitor of 5-LOX.     

Polymeric black tea polyphenols modulate the localization and activity of 12-O-tetradecanoylphorbol-13-acetate-mediated kinases in mouse skin: Mechanisms of their anti-tumor-promoting action

Polymeric black tea polyphenols (PBPs) have been shown to possess anti-tumor-promoting effects in two-stage skin carcinogenesis. However, their mechanisms of action are not fully elucidated. In this study, mechanisms of PBP-mediated antipromoting effects were investigated in a mouse model employing the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Compared to controls, a single topical application of TPA to mouse skin increased the translocation of protein kinase C (PKC) from cytosol to membrane. Pretreatment with PBPs 1-3 decreased TPA-induced translocation of PKC isozymes (α, β, η, γ, ε) from cytosol to membrane, whereas PBPs 4 and 5 were less effective. The levels of PKCs δ and ζ in cytosol/membrane were similar in all the treatment groups. Complementary confocal microscopic evaluation showed a decrease in TPA-induced PKCα fluorescence in PBP-3-pretreated membranes, whereas pretreatment with PBP-5 did not show a similar decrease. Based on the experiments with specific enzyme inhibitors and phosphospecific antibodies, both PBP-3 and PBP-5 were observed to decrease TPA-induced level and/or activity of phosphatidylinositol 3-kinase (PI3K) and AKT1 (pS473). An additional ability of PBP-3 to inhibit site-specific phosphorylation of PKCα at all three positions responsible for its activation [PKCα (pT497), PKC PAN (βII pS660), PKCα/βII (pT638/641)] and AKT1 at the Thr308 position, along with a decrease in TPA-induced PDK1 protein level, correlated with the inhibition of translocation of PKC, which may impart relatively stronger chemoprotective activity to PBP-3 than to PBP-5. Altogether, PBP-mediated decrease in TPA-induced PKC phosphorylation correlated well with decreased TPA-induced NF-κB phosphorylation and downstream target proteins associated with proliferation, apoptosis, and inflammation in mouse skin. Results suggest that the antipromoting effects of PBPs are due to modulation of TPA-induced PI3K-mediated signal transduction.     

N-Pyridinyl(methyl)-indole-1- or 3-propanamides and propenamides acting as topical and systemic inflammation inhibitors

In this study, the synthetic way to new N-pyridinyl(methyl)indolylpropanamides acting as non acidic NSAIDs has been described. Pharmacomodulation was carried out at N(1) and C(5) of the indole ring and at the level of the propanamide chain. N(3)-pyridinylmethyl-[1(4-chlorobenzyl-5-chloroindol-3-yl)propanamide represents one of the most potent compounds in the TPA-induced mouse ear swelling assay, with a level of activity higher than that of ibuprofen and comparable to that of dexamethasone.     

Phenylpropanoid glycosides from Scrophularia scorodonia: in vitro anti-inflammatory activity

Five phenylpropanoid glycosides isolated from Scrophularia scorodonia L. (Scrophulariaceae), namely angoroside A (1), angoroside C (2), angoroside D (3), acteoside (4) and isoacteoside (5), had been evaluated as potential inhibitors of some macrophage functions involved in the inflammatory process. These compounds have been tested in two experimental systems: ionophore-stimulated mouse peritoneal macrophages and human platelets serve as source of COX-1 and 5-LOX, and mouse peritoneal macrophages stimulated with E. coli LPS are the means of testing for COX-2, NO and TNF-alpha activity. None of compounds assayed had a significant effect on LTC(4)-release from calcium ionophore-stimulated mouse peritoneal macrophages. However, the release of PGE(2) by mouse peritoneal macrophages stimulated with calcium ionophore was inhibited by most of these compounds. In the TXB(2)-release assay, acteoside (4), angoroside A (1) and angoroside C (2) showed a significant effect. These five compounds, except angoroside C (2) significantly inhibited LPS-induced PGE(2), NO and TNF-alpha in a concentration-dependent manner. In LPS-stimulated macrophages, the phenylpropanoid glycoside angoroside C (2) only had activity on NO. These results indicate that the pharmacology of these compounds may participate in the anti-inflammatory effect of Scrophularia scorodonia.     

Hepatoprotective phytocompounds from Cryptomeria japonica are potent modulators of inflammatory mediators

Cryptomeria japonica is an important plantation conifer tree in Asia. This study aimed to characterize the anti-inflammatory and hepatoprotective activities of the phytocompounds from C. japonica wood on LPS- or TPA-induced activation of proinflammatory mediators and CCl(4)-induced acute liver injury in mice. A CJH7-2 fraction was purified from C. japonica extracts following bioactivity-guided fractionation, and it exhibited significant activities on inhibition of NO production and iNOS expression as well as up-regulating HO-1 expression in LPS-stimulated macrophages. CJH7-2 also potently inhibits COX-2 enzymatic activity (IC(50)=5 microg/mL) and TPA-induced COX-2 protein expression in mouse skin (1mg/200 microL/site). CJH7-2 (10 mg/kg BW) can prevent CCl(4)-induced liver injury and aminotransferases activities in mice. Chemical fingerprinting analysis showed that terpenes are the major bioactive compounds in the CJH7-2 fraction. This is the first study to demonstrate that chemical constituents from the wood extract of C. japonica possess anti-inflammatory activities in vitro and in vivo that may play a role in hepatoprotection.     

Suppressive effects of citrus fruits on free radical generation and nobiletin, an anti-inflammatory polymethoxyflavonoid

Citrus fruit intake is known to be associated with a reduction of cancer incidence. Free radicals, including superoxide (O2-) and nitric oxide (NO), are involved in some epithelial carcinogenesis processes. In the present study, we screened thirty-one citrus fruits for their suppressive activities toward three lines of free radical generating systems: 1) O2- generation by the xanthine (XA)-xanthine oxidase (XOD) system; 2) O2- generation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in differentiated human promyelocytic HL-60 cells; and 3) NO generation in murine macrophage RAW264.7 cells stimulated with lipopolysaccharide (LPS) and interferon (IFN)-gamma. As a result, the inhibitory activities of peel parts were largely found to be higher than those of the corresponding juice sac parts. In particular, the peel portion of Dancy tangerine (Citrus tangerinia) showed marked anti-oxidative activities in these systems. In addition, nobiletin, a polymethoxyflavonoid isolated from C. nobilis, showed a higher anti-inflammatory activity than indomethacin in a TPA-induced edema formation test in mouse ears. These results indicate that citrus fruits could be notable sources of anti-oxidative, anti-inflammatory, and cancer preventive compounds.     

Anti-tumor promoting potential of naturally occurring diarylheptanoids structurally related to curcumin.

In recent years, there have been considerable efforts to search for naturally occurring substances for intervention of carcinogenesis. Many components from medicinal or dietary plants have been identified to possess potential chemopreventive properties. For instance, curcumin, a yellow colouring agent from turmeric (Curcuma longa Linn., Zingiberaceae) has been shown to inhibit tumor formation in diverse animal models. Alpinia oxyphylla Miquel that also belongs to ginger family has been used in oriental herbal medicine. In the present work, we have evaluated the anti-tumor promoting potential of yakuchinone A (1-[4'-hydroxy-3'-methoxyphenyl]-7-phenyl-3-heptanone) and yakuchinone B (1-[4'-hydroxy-3'-methoxyphenyl]-7-phenylhept-1-en-3-one), major pungent ingredients of A. oxyphylla. Thus, topical application of yakuchinone A or B significantly suppressed TPA-induced epidermal ornithine decarboxylase activity. They also reduced TPA-stimulated production of tumor necrosis factor-alpha in cultured human promyelocytic leukemia (HL-60) cells. Both compounds blunted the TPA-induced superoxide generation in differentiated HL-60 cells in a concentration-related manner and also inhibited lipid peroxidation in rat brain homogenates. Furthermore, yakuchinone A and yakuchinone B nullified the activation of the activator protein-1 (AP-1) in immortalized mouse fibroblast cells in culture. These findings indicate that pungent diarylheptanoids from A. oxyphylla have anti-tumor promotional properties that can contribute to their chemopreventive potential.     

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine(H-7), a potent inhibitor of protein kinases, inhibits the differentiation of HL-60 cells induced by phorbol diester

Treatment of the human promyelocytic leukemia cell line HL-60, with 12-o-tetradecanoylphorbol acetate (TPA) results in the differentiation into macrophage-like cell. A potent inhibitor of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine(H-7), suppressed the proliferation of HL-60 cells and also inhibited TPA-induced cell differentiation of these cells. N-(2-guanidinoethyl)-5-isoquinolinesulfonamide(HA-1004), a weaker analog of H-7, failed to inhibit this TPA-induced cell differentiation. H-7 also inhibited TPA-induced protein phosphorylation in these cells. Thus, protein kinase C-mediated phosphorylation may be involved in the process of TPA-induced HL-60 cell differentiation.     

New N-pyridinyl(methyl)-indolalkanamides acting as topical inflammation inhibitors

The authors have described the synthetic way to new N-pyridinyl(methyl)indolylpropanamides acting as non acidic NSAIDs. Pharmacomodulation was carried out at N-1 and C-5 of the indole ring and at the level of the propanamide chain. N-(pyridin-3-ylmethyl)-3-[5-chloro-1-(4-chlorobenzyl)-indol-3-yl]propanamide 32 represents one of the most potent compounds evaluated in the TPA-induced mouse ear swelling assay, with a level of activity higher than that of ibuprofen and comparable to that of dexamethasone.     

3-Geranyl-4-hydroxy-5-(3'-methyl-2'-butenyl)benzoic acid as an anti-inflammatory compound from Myrsine seguinii.

Bioassay-guided isolation of anti-inflammatory compounds from the methanol extract of Myrsine seguinii yielded an anti-inflammatory compound (1). The structure of compound 1 was elucidated to be 3-geranyl-4-hydroxy-5-(3'-methyl-2'-butenyl)benzoic acid on the basis of its spectroscopic data. Compound 1 strongly suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation on mouse ears at a dose of 500 micrograms (inhibitory effect (IE): 65%). The acetate and the methyl ether of 1 showed moderate activity at a 500-microgram application, with IE 38% and 27%, respectively. However, the methyl ester and the dimethyl derivative of 1 did not show activity at the same dose. The related compounds of 1, o-, m- and p-hydroxybenzoic acids also did not exhibit notable activity. These results indicate that the carboxylic acid and lipophilic terpene moieties of 1 were significant structural features for anti-inflammatory activity.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin ornithine decarboxylase and protein kinase C by polyphenolics from grapes

Ornithine decarboxylase is the rate-limiting enzyme in the biosynthesis of polyamines, which are believed to play an essential role in diverse biological processes including cell proliferation and differentiation. We have previously reported [J. Bomser, K. Singletary, M. Wallig, M. Smith, Inhibition of TPA-induced tumor promotion in CD-1 mouse epidermis by a polyphenolic fraction from grape seeds, Cancer Letters 135 (1999) 151-157] that pre-application of a grape polyphenolic fraction (GPF) to mouse skin epidermis inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity, as well as 7, 12-dimethylbenz[a]anthracene (DMBA)-initiated, TPA-promoted mouse skin tumorigenesis. The present studies were designed to further characterize the effect of time and dose of application of GPF on TPA-induced ODC activity and protein expression, and on protein kinase C activity in mouse skin epidermis. In addition, the effect of GPF on ODC kinetics in vitro was examined. Application of 5, 10, and 20 mg of GPF 20 min prior to treatment with TPA resulted in a significant decrease in epidermal ODC activity of 54, 53, 90%, respectively, compared with controls. Yet, ODC protein levels (Western blot) in the 10 and 20 mg GPF groups were significantly increased by 1.8 and 1.9-fold, respectively, compared with controls. A similar response was observed with the ODC inhibitor 2-difluoromethylornithine (DFMO), which served as a positive control. Application of grape polyphenolics (20 mg) at 60 and 30 min prior to treatment with TPA inhibited ODC activity by 62 and 68%, respectively, compared with controls (P<0.05). In contrast, application of grape polyphenolics (20 mg) at 60, 120 and 240 min after treatment with TPA resulted in no significant changes in ODC activity. A similar increase in epidermal ODC protein was observed in these GPF-treated animals, similar to that observed when GPF application preceded TPA. When applied to mouse skin prior to TPA, GPF was associated with a decrease in subsequent PKC activity compared with controls at 10 and 30 min following TPA treatment. The GPF-associated decrease in PKC activity preceded the decrease in ODC activity. In a separate in vitro study, kinetic analyses indicated that GPF is a competitive inhibitor of ODC activity. Collectively these data suggest that the grape polyphenolic fraction is effective as an inhibitor of ODC activity when applied before TPA, and that the magnitude of inhibition is independent of epidermal ODC protein content. In addition, GPF is a competitive inhibitor of ODC activity in vitro. The decrease in TPA-induced ODC activity due to GPF treatment is preceded by an inhibition of TPA-induced PKC activity. Thus, the polyphenolic fraction from grapes warrants further examination as a skin cancer chemopreventive agent that interferes with cellular events associated with TPA promotion.     

In vivo activity of pseudoguaianolide sesquiterpene lactones in acute and chronic inflammation

The pseudoguaianolide sesquiterpene lactones 4-alpha-O-acetyl-pseudoguaian-6beta-olide (1), hymenin (2), ambrosanolide (3), tetraneurin A (4), parthenin (5), hysterin (6) and confertdiolide (7) were evaluated for their ability to affect the inflammation responses induced by different agents. All the compounds showed activity against the 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced mouse ear edema. The ethyl phenylpropiolate (EPP)-induced mouse ear edema was inhibited by compounds 3, 5 and 7. However, when sesquiterpene-lactones were assayed on the arachidonic acid (AA)-induced mouse ear edema, none of them were active. The only sesquiterpene lactone orally active against the paw mouse edema induced by carrageenan was 7, which gave a 46% edema inhibition after 3 h. On the other hand, compounds 3, 5 and 7 reduced the serotonin-induced paw edema in mice, although compound 7 was inactive in presence of cycloheximide. In addition, the sesquiterpene lactones were assayed against the chronic inflammation induced by repeated application of TPA on mouse ear. Confertdiolide was the most active compound; it reduced the edema by 87% and had a more moderate effect against the leukocyte recruitment (36% reduction in myeloperoxidase (MPO) levels). A histological study of ear the samples treated with 7 presented no detectable morphological lesions such as those treated with dexamethasone. On the oxazolone-induced delayed type hypersensitivity (DTH) only compounds 4 and 5 were active 24 h after the challenge. Compound 5 affected polymorphonuclear leukocyte infiltration (69% reduction in MPO levels). The results suggest that the especial chemical structure and spatial conformation of confertdiolide may facilitate its anti-inflammatory effect.     

广东紫珠中的萜类化合物及其抗炎活性

对岭南药材广东紫珠(Callicarpa kwangtungensis)地上部分进行化学成分研究,得到11个萜类化合物,分别鉴定为sambucunlin A(1)、2α-羟基羽扇豆醇(2)、swinhoeic acid(3)、3β-羟基-乌苏烷-11-烯-13β,28-内酯(4)、蔷薇酸(5)、2α,3β,6β,18β,23-pentahydroxy-olean-12-en-28-oic acid(6)、rel-5-(3S,8S-dihydroxy-1R,5S-dimethyl-7-oxa-6-oxobicyclo-oct-8-yl)-3-methyl-2Z,4E-pentadienoic acid(7)、salvionoside B(8)、齐墩果酸(9)、白桦脂酸(10)和α-香树脂醇(11)。其中,化合物1～4和6～8为首次从该属植物中分离得到。在化学成分分离基础上,进一步选择脂多糖(LPS)诱导的RAW 264.7小鼠巨噬细胞炎症模型进行萜类化合物抗炎活性测试。结果表明:化合物3和9具有显著的抗炎活性,对比结构发现,三萜类化合物(3～6、9和11)抗炎活性优于倍半萜类化合物(7和...     

Modulation of phorbol ester-elicited events in mouse epidermis by dietary n-3 and n-6 fatty acids

Because arachidonic acid-derived eicosanoids are potent modulators of hyperproliferation and inflammation during skin tumor promotion with the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate (TPA) (17, 18), it was hypothesized that dietary modification of epidermal fatty acids might modulate TPA-induced biochemical events in mouse skin. Semipurified diets containing 10% total fat composed of corn oil (CO) or a combination of CO and menhaden oil (MO) or coconut oil (CT) were fed to SENCAR mice for 4 weeks. Fatty acid composition of epidermal phospholipids generally reflected fatty acid composition of dietary oils fed to the mice. Since fatty acid-derived eicosanoids are thought to be essential in tumorigenesis, we compared the effects of dietary fats on prostaglandin E (PGE) production in epidermis treated with a single dose of TPA. TPA-induced PGE production in mouse epidermis from mice fed the MO diet was significantly reduced compared to PGE production in epidermal homogenates from mice fed the CO or CT diets. Type of dietary fats did not appear to modulate TPA-induced vascular permeability, however hyperplasia was slightly elevated in skins of mice fed MO. The subcellular distribution of protein kinase C, the plasma membrane receptor for TPA predominantly located in the cytosol (80%), was altered in epidermis from mice fed the MO diet compared to preparations from mice fed CO or CT diets which exhibited normal protein kinase C distribution. Our results suggest that n-3 rich dietary lipids modulate TPA-elicited events in mouse skin to a greater extent than diets containing higher proportions of saturated or n-6 fatty acids.     

Phorbol ester induced prostaglandin synthesis and [3H]-TPA metabolism by TPA-sensitive and TPA-resistant Friend erythroleukemia cells.

The potent mouse skin tumour promoter, 12-O-tetra-decanoyl phorbol-13-acetate (TPA), causes a release of arachidonic acid and prostaglandins E₂ and F_2α from Friend erythroleukemia cells (FELC). This effect is not seen with a subclone which is resistant to both TPA-induced inhibition of differentiation and TPA-induced adhesion. TPA-sensitive and TPA-resistant clones metabolize [³H]-TPA at the same rate to phorbol-13-monoacetate (P(13)A). P(13)A has no effect on differentiation or adhesion of FELC. Together with previous findings, these results suggest that TPA-induced changes in membrane lipid metabolism are closely linked to its effects on growth, differentiation and adhesion. The resistance of a subclone of FELC to TPA appears to be at the level of membrane action rather than the metabolism of TPA.     

New anti-inflammatory ergostane-type ecdysteroids from the sclerotium of Polyporus umbellatus

Bioassay-guided fractionation of the ethyl acetate extract from the sclerotium of Polyporus umbellatus resulted in the isolation of three new ergostane-type ecdysteroids, named polyporoid A (1), B (2), and C (3), together with five known ecdysteroids. The structures of the new compounds were determined on the basis of extensive spectroscopic data (IR, MS, (1)H and (13)C NMR, and 2D NMR) analyses. All compounds (1-8) exhibited potent anti-inflammatory activity in the test of TPA-induced inflammation (1 microg/ear) in mice, with ID(50) values in the range of 0.117-0.682 microM/ear.     

Cyanidin-3-glucoside, a natural product derived from blackberry, exhibits chemopreventive and chemotherapeutic activity

Epidemiological data suggest that consumption of fruits and vegetables has been associated with a lower incidence of cancer. Cyanidin-3-glucoside (C3G), a compound found in blackberry and other food products, was shown to possess chemopreventive and chemotherapeutic activity in the present study. In cultured JB6 cells, C3G was able to scavenge ultraviolet B-induced *OH and O2-* radicals. In vivo studies indicated that C3G treatment decreased the number of non-malignant and malignant skin tumors per mouse induced by 12-O-tetradecanolyphorbol-13-acetate (TPA) in 7,12-dimethylbenz[a]anthracene-initiated mouse skin. Pretreatment of JB6 cells with C3G inhibited UVB- and TPA-induced transactivation of NF-kappaB and AP-1 and expression of cyclooxygenase-2 and tumor necrosis factor-alpha. These inhibitory effects appear to be mediated through the inhibition of MAPK activity. C3G also blocked TPA-induced neoplastic transformation in JB6 cells. In addition, C3G inhibited proliferation of a human lung carcinoma cell line, A549. Animal studies showed that C3G reduced the size of A549 tumor xenograft growth and significantly inhibited metastasis in nude mice. Mechanistic studies indicated that C3G inhibited migration and invasion of A549 tumor cells. These finding demonstrate for the first time that a purified compound of anthocyanin inhibits tumor promoter-induced carcinogenesis and tumor metastasis in vivo.     

N-Pyridinyl(methyl)-indole-1- or 3-propanamides and propenamides acting as topical and systemic inflammation inhibitors

In this study, the synthetic way to new N-pyridinyl(methyl)indolylpropanamides acting as non acidic NSAIDs has been described. Pharmacomodulation was carried out at N¹ and C⁵ of the indole ring and at the level of the propanamide chain. N³-pyridinylmethyl-[1(4-chlorobenzyl-5-chloroindol-3-yl)propanamide represents one of the most potent compounds in the TPA-induced mouse ear swelling assay, with a level of activity higher than that of ibuprofen and comparable to that of dexamethasone.     

Virginols A–C,three new withanolides from Physalis virginiana

Virginols A–C are three new withanolides containing an epoxylactol at the side chain. They were isolated from the aerial parts of Physalis virginiana Miller. Structures of these compounds were determined by analyses of their spectroscopic data, including 1D and 2D NMR. The structure of virginol A was confirmed by X-ray diffraction analysis. The anti-inflammatory activity of virginols was evaluated in the TPA-induced ear mouse edema model.