Cytotoxic effects of the dietary flavones chrysin and apigenin in a normal trout liver cell line

Many flavonoids have been shown to possess prooxidant properties, capable of causing oxidative stress, especially at larger doses. Here, we examined the potential cell toxicity caused by exposure to the hydroxylated flavones chrysin, apigenin, luteolin and quercetin in comparison to the methylated flavones 5,7-dimethoxyflavone and 3',4'-dimethoxyflavone in normal Rainbow trout hepatocytes. The hydroxylated flavones, especially chrysin, demonstrated cell toxicity and inhibition of DNA synthesis at very low (2 microM) concentrations. The cytotoxicity of chrysin may partially be due to its metabolism by myeloperoxidase, which was shown to be present in these normal trout liver cells (164pmol/(min mg protein)). In contrast, methylated flavones showed no significant metabolism by myeloperoxidase and no signs of toxicity, even at much higher concentrations. These results may be useful for further investigations of cytotoxicity of dietary flavonoids.     

Effects of flavonoids on vascular smooth muscle of the isolated rat thoracic aorta

The potency, structure-activity relationship, and mechanism of vasorelaxation of a series of flavonoids, representing different subclasses (flavonols: fisetin, rutin, quercetin; flavones: chrysin, flavone, baicalein; flavanones: naringenin, naringin; isoflavones: diadzein and flavanes: epigallo catechin gallate), were examined in the isolated rat aorta. Most of the flavonoids tested showed concentration dependent relaxant effects against K+ (80 mM) and phenylephrine (PE, 0.1 microM)-induced contractions with a greater inhibition of the responses to the alpha1-adrenoceptor agonist. The relaxant effects of most of the flavonoids involve in part the release of nitric oxide and prostaglandins from the endothelium as pretreatment with L-NAME and indomethacin attenuated the responses. In addition, the relaxant action of the flavonoids includes inhibition of Ca+2 influx and release of Ca+2 from intracellular stores. A structure-activity relationship amongst the flavonoids was suggested.     

Flavonoid-induced glutathione depletion: potential implications for cancer treatment

The ability of a number of flavonoids to induce glutathione (GSH) depletion was measured in lung (A549), myeloid (HL-60), and prostate (PC-3) human tumor cells. The hydroxychalcone (2'-HC) and the dihydroxychalcones (2',2-, 2',3-, 2',4-, and 2',5'-DHC) were the most effective in A549 and HL-60 cells, depleting more than 50% of intracellular GSH within 4 h of exposure at 25 microM. In contrast, the flavones chrysin and apigenin were the most effective in PC-3 cells, depleting 50-70% of intracellular GSH within 24 h of exposure at 25 microM. In general, these flavonoids were more effective than three classical substrates of multidrug resistance protein 1 (MK-571, indomethacin, and verapamil). Prototypic flavonoids (2',5'-DHC and chrysin) were subsequently tested for their abilities to potentiate the toxicities of prooxidants (etoposide, rotenone, 2-methoxyestradiol, and curcumin). In A549 cells, 2',5'-DHC potentiated the cytotoxicities of rotenone, 2-methoxyestradiol, and curcumin, but not etoposide. In HL-60 and PC-3 cells, chrysin potentiated the cytotoxicity of curcumin, cytotoxicity that was attenuated by the catalytic antioxidant manganese(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP). Assessments of mitochondrial GSH levels mitochondrial membrane potential and cytochrome c release showed that the potentiation effects induced by 2',5'-DHC and chrysin involve mitochondrial dysfunction.     

Distribution of 8-oxygenated leaf-surface flavones in the genus Ocimum

Nine species of Ocimum (Lamiaceae) were surveyed for leaf-surface flavonoids by means of HPLC with diode array detection and atmospheric pressure chemical ionisation (APCI) mass spectrometry. The analysis revealed the presence of 23 different flavones, most of which were identified by comparing their UV and mass spectra with those of standards. Almost all taxa investigated contained flavones methoxylated in the 6- and 8-positions, such as nevadensin, xanthomicrol and gardenin B. The same taxa also produced flavones methoxylated in the 6-position but hydroxylated in the 8-position, including isothymusin (5,8,4'-trihydroxy-6,7-dimethoxyflavone), pedunculin (5,8-dihydroxy-6,7,4'-trimethoxyflavone) and a new flavone, 5,7,8-trihydroxy-6,4'-dimethoxyflavone, which was given the trivial name pilosin. This compound was isolated from O. americanum var. pilosum and also detected as a minor constituent in O. x citriodorum leaf extracts. Its molecular structure was elucidated by means of NMR spectroscopy. 8-Oxygenated flavones were absent only from O. lamiifolium. APCI mass spectrometry of the flavonoids revealed that the product ions formed by collision induced dissociation of the protonated molecule provided structural information about the substitution pattern of the A-ring. The chemotaxonomic and biogenetic implications of the results are discussed.     

Investigation of the membrane localization and distribution of flavonoids by high-resolution magic angle spinning NMR spectroscopy

To investigate the structural basis for the antioxidative effects of plant flavonoids on the lipid molecules of cellular membranes, we have studied the location and distribution of five different flavonoid molecules (flavone, chrysin, luteolin, myricetin, and luteolin-7-glucoside) with varying polarity in monounsaturated model membranes. The investigated molecules differed in the number of hydroxyl groups attached to the polyphenolic benzo-gamma-pyrone compounds. To investigate the relation between hydrophobicity and membrane localization/orientation, we have applied (1)H magic angle spinning NMR techniques measuring ring current induced chemical shift changes, nuclear Overhauser enhancement cross-relaxation rates, and lateral diffusion coefficients. All investigated flavonoids show a broad distribution along the membrane normal with a maximum in the lipid/water interface. With increasing number of hydroxyl groups, the maximum of this distribution is biased towards the lipid headgroups. These results are confirmed by pulsed field gradient NMR measurements of the lateral diffusion coefficients of phospholipids and flavonoids, respectively. From the localization of different flavonoid protons in the membrane, a model for the orientation of the molecules in a lipid bilayer can be deduced. This orientation depends on the position of the polar center of the flavonoid molecule.     

Dietary flavonoids attenuate tumor necrosis factor alpha-induced adhesion molecule expression in human aortic endothelial cells. Structure-function relationships and activity after first pass metabolism

Flavonoids have been suggested to exert human health benefits by anti-oxidant and anti-inflammatory mechanisms. In this study, we investigated whether and by what mechanisms dietary flavonoids inhibit expression of cellular adhesion molecules, which is relevant to inflammation and atherosclerosis. We found that the capacity of flavonoids to inhibit tumor necrosis factor alpha-induced adhesion molecule expression in human aortic endothelial cells was dependent on specific structural features of the flavonoids. The 5,7-dihydroxyl substitution of a flavonoid A-ring and 2,3-double bond and 4-keto group of the C-ring were the main structural requirements for inhibition of adhesion molecule expression. In striking contrast, hydroxyl substitutions of the B- and C-rings but not the A-ring were essential for antioxidant activity. Hence, only hydroxyl flavones, such as apigenin and chrysin, and flavonols, such as galangin, kaempferol, and quercetin, were able to inhibit endothelial adhesion molecule expression, whereas flavone, chromone, the flavanone, naringenin, and the flavanol, (-)-epicatechin, were ineffectual. At low concentrations, the active flavonoids significantly attenuated expression of E-selectin and intercellular adhesion molecule 1 but not vascular cell adhesion molecule 1. In addition, exposure of apigenin and kaempferol to cultured hepatocytes, mimicking first pass metabolism, greatly diminished the inhibitory effect of flavonoids on endothelial intercellular adhesion molecule 1 expression. We conclude that the effect of dietary flavonoids on endothelial adhesion molecule expression depends on their molecular structure, concentration, and metabolic transformation but not their antioxidant activity.     

Mutagenicity of flavones, chromones and acetophenones in Salmonella typhimurium. New structure-activity relationships

28 flavones and 11 structurally-related flavonoids, chromones, and acetophenones, were tested for mutagenicity in the Salmonella typhimurium his reversion assay. 7 flavones, all of which were hydroxy- or methoxy-substituted at position 8, were moderate to strong mutagens in strain TA100 in the presence of rat liver S9 mix. In each case, the response of strain TA98 was either not significant or was very much weaker than that observed in strain TA100. The activation by S9 is not mediated by the microsomal cytochrome P450 system, since activation was not diminished when microsomes were removed by centrifugation at 100 000 X g. The observed strain specificity and structural requirements for activity indicate a mutagenic mechanism different from that associated with previously reported mutagenic flavonols (3-hydroxy-flavones) which are most active in strain TA98. The most mutagenic flavone investigated, 5,7,8-trihydroxy-flavone (norwogonin), had a potency of 17 revertants/nmole. Simplification of the chemical structures to hydroxy-substituted chromone and acetophenone systems revealed similar strain specificity, hydroxylation requirements, and S9 dependence within these structural classes, suggesting a similar activation pathway and mutagenic mechanism. The greatest mutagenic potency was observed within the flavone series, but significant potency was retained by similarly hydroxylated chromones and acetophenones. No mutagenic activity was observed in the absence of the aryl ketone moiety.     

Accumulation and metabolism of the anticancer flavonoid 5,7-dimethoxyflavone compared to its unmethylated analog chrysin in the Atlantic killifish

The use of dietary flavonoids as potential chemopreventive agents is a concept of increasing interest. Recent findings indicate that methylated flavones have the advantage of increased metabolic stability. One such compound, the naturally-occurring 5,7-dimethoxyflavone (5,7-DMF), has been shown to be a potential chemopreventive agent in human cancer originating from the liver, mouth, esophagus and lung. As bioavailability is a key issue for potential in vivo effects, the tissue accumulation and biliary elimination of 5,7-DMF and its non-methylated analog chrysin were examined in a small fish model (Fundulus heteroclitus). The fish were exposed to 5,7-DMF, chrysin or vehicle control (DMSO<0.01%) in seawater for 8h. Toxicity was not observed at the 5microM exposure level. Tissues and bile were harvested and analyzed by HPLC and LC/MS for quantitation and identification of parent compound and metabolites. 5,7-DMF accumulated 20-fold to 100-fold in all tissues examined, with the highest accumulation in liver and brain, whereas chrysin was barely detectable in any tissues except the liver. The bile of chrysin-exposed fish contained very low concentrations of unchanged chrysin but high concentrations of two glucuronic acid conjugates. In the bile of 5,7-DMF-exposed fish, the parent compound was detectable in significant amounts along with glucuronic acid conjugates of O-demethylated 5,7-DMF. In conclusion, our study demonstrated high tissue accumulation and limited metabolism of 5,7-DMF compared to chrysin in vivo, making this flavone a promising chemopreventive molecule.     

Comparative CYP1A1 and CYP1B1 substrate and inhibitor profile of dietary flavonoids

CYP1A1 and CYP1B1 are two extrahepatic enzymes that have been implicated in carcinogenesis and cancer progression. Selective inhibition of CYP1A1 and CYP1B1 by dietary constituents, notably the class of flavonoids, is a widely accepted paradigm that supports the concept of dietary chemoprevention. In parallel, recent studies have documented the ability of CYP1 enzymes to selectively metabolize dietary flavonoids to conversion products that inhibit cancer cell proliferation. In the present study we have examined the inhibition of CYP1A1 and CYP1B1-catalyzed EROD activity by 14 different flavonoids containing methoxy- and hydroxyl-group substitutions as well as the metabolism of the monomethoxylated CYP1-flavonoid inhibitor acacetin and the poly-methoxylated flavone eupatorin-5-methyl ether by recombinant CYP1A1 and CYP1B1. The most potent inhibitors of CYP1-EROD activity were the methoxylated flavones acacetin, diosmetin, eupatorin and the di-hydroxylated flavone chrysin, indicating that the 4'-OCH(3) group at the B ring and the 5,7-dihydroxy motif at the A ring play a prominent role in EROD inhibition. Potent inhibition of CYP1B1 EROD activity was also obtained for the poly-hydroxylated flavonols quercetin and myricetin. HPLC metabolism of acacetin by CYP1A1 and CYP1B1 revealed the formation of the structurally similar flavone apigenin by demethylation at the 4'-position of the B ring, whereas the flavone eupatorin-5-methyl ether was metabolized to an as yet unidentified metabolite assigned E(5)M1. Eupatorin-5-methyl ether demonstrated a submicromolar IC(50) in the CYP1-expressing cancer cell line MDA-MB 468, while it was considerably inactive in the normal cell line MCF-10A. Homology modeling in conjunction with molecular docking calculations were employed in an effort to rationalize the activity of these flavonoids based on their CYP1-binding mode. Taken together the data suggest that dietary flavonoids exhibit three distinct modes of action with regard to cancer prevention, based on their hydroxyl and methoxy decoration: (1) inhibitors of CYP1 enzymatic activity, (2) CYP1 substrates and (3) substrates and inhibitors of CYP1 enzymes.     

Inhibition of microsome-mediated binding of benzo[a]pyrene to DNA by flavonoids either in vitro or after dietary administration to rats.

The in vitro and in vivo effects of selected natural flavonoids (flavone, flavanone, tangeretin, quercetin, chrysin) on the microsome-catalysed binding of [3H]benzo[a]pyrene to calf thymus DNA were investigated and compared with those of two synthetic flavonoids, 7,8-benzoflavone and 5,6-benzoflavone. In vitro addition of these flavonoids (0.1 mM) to an incubation system containing hepatic microsomes prepared from Aroclor 1254-pretreated rats strongly inhibited BaP-DNA adduct formation (72-89%). The incubation of BaP with hepatic microsomes prepared from animals fed 0.3% quercetin, tangeretin and 7,8-benzoflavone for 2 weeks also resulted in less effective binding of BaP metabolites to added DNA, than with microsomes from untreated rats. Other tested compounds, chrysin, flavone, flavanone and 5,6-benzoflavone showed no or little effect. The influence of flavonoid pretreatment on hepatic microsomal enzymes involved in BaP metabolism has also been examined. Aryl hydrocarbon hydroxylase activity was moderately increased (1.5-1.8-fold) in microsomes prepared from rats fed flavone, tangeretin, 7,8-benzoflavone and 5,6-benzo-flavone. Epoxide hydrolase activity was enhanced by 7,8-benzoflavone (1,6-fold), and by flavone and flavanone (5-fold). These results confirm that flavonoids, in vitro, are potent inhibitors of carcinogen-DNA binding. Oral administration of 0.3% flavonoids alters the properties of liver microsomes, resulting in the decreased ability of BaP metabolites to bind DNA.     

Aromatase inhibition by bioavailable methylated flavones

Previous studies have shown chrysin, 7-hydroxyflavone and 7,4'-dihydroxyflavone to be the most potent flavonoid inhibitors of aromatase. However, very poor oral bioavailability is a major limitation for the successful use of dietary flavonoids as chemopreventive agents. We have recently shown that methylated flavones, including 5,7-dimethoxyflavone, 7-methoxyflavone and 7,4'-dimethoxyflavone, are much more resistant to metabolism than their unmethylated analogs and have much higher intestinal absorption. In this study, we examined these fully methylated flavones as potential aromatase inhibitors for the prevention and/or treatment of hormone-dependent cancers. Whereas 5,7-dimethoxyflavone had poor effect compared to its unmethylated analog chrysin, 7-methoxyflavone and 7,4'-dimethoxyflavone were almost equipotent to their unmethylated analogs with IC(50) values of 2-9 microM. Thus, some fully methylated flavones appear to have great potential as cancer chemopreventive/chemotherapeutic agents.     

Effect of flavonoids on mycelial growth of Verticillium albo-atrum

Twenty-five flavonoids were examined for their effect on the mycelial growth of a crop pathogen, Verticillium albo-atrum. The minimum inhibitory concentrations (MIC) for the two most active compounds, flavone and flavanone, were 1 and 5 ppm, respectively. Other flavonoids inhibited hyphal growth at concentrations above 5 ppm and some compounds were ineffective at 200 ppm, the highest concentration used. Active compounds did not share a common pattern of substitutions. The unsubstituted flavonoids were stronger growth inhibitors and, in most cases, increasing the number of substitutions (hydroxylation, methoxylation, and glycosylation) resulted in the loss of antifungal activity.     

Flavones and flavone synthases

Within the secondary metabolite class of flavonoids which consist of more than 9000 known structures, flavones define one of the largest subgroups. Their natural distribution is demonstrated for almost all plant tissues. Various flavone aglyca and their O- or C-glycosides have been described in the literature. The diverse functions of flavones in plants as well as their various roles in the interaction with other organisms offer many potential applications, not only in plant breeding but also in ecology, agriculture and human nutrition and pharmacology. In this context, the antioxidative activity of flavones, their use in cancer prevention and treatment as well as the prevention of coronary heart disease should be emphasized. The therapeutic potential of flavones makes these compounds valuable targets for drug design, including recombinant DNA approaches. The biosynthesis of flavones in plants was found to be catalyzed by two completely different flavone synthase proteins (FNS), a unique feature within the flavonoids. The first, FNS I, a soluble dioxygenase, was only described for members of the Apiaceae family so far. The second, FNS II, a membrane bound cytochrome P450 enzyme, has been found in all other flavone accumulating tissues. This phenomenon is particularly of interest from the evolutionary point of view concerning the flavone biosynthesis and functions in plants. Recently, FNS I and FNS II genes have been cloned from a number of plant species. This now enables detailed biochemical and molecular characterizations and also the development of direct metabolic engineering strategies for modifications of flavone synthesis in plants to improve their nutritional and/or biopharmaceutical value.     

Flavonoid systematics of the genus Perideridia (Umbelliferae)

A survey of flavonoids in sixteen of the seventeen taxa in the genusPerideridia (Umbelliferae) showed the presence of thirteen glycosides of the flavonols kaempferol, quercetin, and isorhamnetin, and seven glycosides of the flavones apigenin, luteolin and chrysoeriol. An anthocyanin and four other flavonoids also occur, but remain unidentified dueto their low concentration. Several species characteristically produce speciesspecific compounds. The majority of species, however, produce flavonoids common to one or more taxa, but each taxon can be distinguished by its own specific complement of these flavonoids. Based on classes of flavonoids the genus can be divided into three groups: (1) those species which produce only flavonols; (2) those which produce mainly flavonols and a few flavones; and (3) those which produce predominantly flavones with flavonols absent or present only in trace amounts. Geographically, the flavonol-producing species are centered in California, extending northeastward to Idaho and eastward into Arizona. The flavonol/flavone producers are concentrated more towards the Pacific Northwest and eastward through the Rocky Mountains to the midwestern United States.     

Aromatase inhibition by flavonoids

Several synthetic flavones were found to inhibit the aromatization of androstenedione to estrone catalyzed by human placental microsomes. Twenty-one compounds were tested and the IC50 of the most active were: flavone, 10 microM; 7-hydroxyflavone, 0.5 microM; 7,4'-dihydroxyflavone, 2.0 microM; flavanone, 8.0 microM; and 4'-hydroxyflavanone, 10 microM. Most of the others had IC50 values ranging from 80 to greater than 200 microM. These findings show that 4'-hydroxylation results in either no change or very little change in IC50 for flavanone, isoflavone and isoflavanone as well as other ring A hydroxylated flavones. Derivatives of flavone with a hydroxyl substituent at position 5, 6 and 7 were also screened. 7-Hydroxyflavone (11) was the most effective competitive inhibitor (IC50 = 0.5 microM) with an apparent Ki value of 0.25 microM. Compound 11 also induced a change in the absorption spectrum of the aromatase cytochrome P-450 which is indicative of substrate displacement. The relative binding affinities of the flavonoid analogs were determined and only ring A adn ring B dihydroxylated analogs were found to bind to the estrogen receptor.     

Correlation between the affinity of flavonoids binding to the cytosolic site of Leishmania tropica multidrug transporter and their efficiency to revert parasite resistance to daunomycin

The C-terminal nucleotide-binding domain (NBD2) of a P-glycoprotein-like transporter, encoded by the ltrmdr1 gene in Leishmania tropica and involved in parasite multidrug resistance (MDR), was overexpressed in Escherichia coli as a hexahistidine tagged protein and purified. The L. tropica recombinant domain efficiently bound fluorescent derivatives of ATP, the hydrophobic steroid analogue RU 486, and different classes of flavonoids with the following efficiency: flavone > flavanone > isoflavone > glucorhamnosyl-flavone > chromone. The affinity for flavones was dependent on the presence of hydroxyl groups at positions 5 and 3 and was further increased by a hydrophobic 1,1-dimethylallyl substituent at position 8. When flow cytometry was used to measure daunomycin accumulation in a MDR L. tropica line, a reversing effect was observed with flavones such as dimethylallyl-kaempferide at low concentration or apigenin at higher concentration, but neither with the glucorhamnosyl derivative rutin nor with the isoflavone genistein. The in vivo reversing effect of dimethylallyl-kaempferide was correlated to a high inhibition of MDR cell growth in the presence of daunomycin. The results suggest that flavone inhibition of both daunomycin efflux and parasite growth in the presence of the drug correlates to direct binding of the compound to cytosolic domain of the P-glycoprotein-like transporter.     

Perturbation of the lipid phase of a membrane is not involved in the modulation of MRP1 transport activity by flavonoids

The expression of transmembrane transporter multidrug resistance-associated protein 1 (MRP1) confers the multidrug-resistant phenotype (MDR) on cancer cells. Since the activity of the other MDR transporter, P-glycoprotein, is sensitive to membrane perturbation, we aimed to check whether the changes in lipid bilayer properties induced by flavones (apigenin, acacetin) and flavonols (morin, myricetin) were related to their MRP1 inhibitory activity. All the flavonoids inhibited the efflux of MRP1 fluorescent substrate from human erythrocytes and breast cancer cells. Morin was also found to stimulate the ATPase activity of erythrocyte ghosts. All flavonoids intercalated into phosphatidylcholine bilayers as judged by differential scanning calorimetry and fluorescence spectroscopy with the use of two carbocyanine dyes. The model of an intramembrane localization for flavones and flavonols was proposed. No clear relationship was found between the membrane-perturbing activity of flavonoids and their potency to inhibit MRP1. We concluded that mechanisms other than perturbation of the lipid phase of membranes were responsible for inhibition of MRP1 by the flavonoids.     

Phytoestrogens as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase

Different phytoestrogens were tested as inhibitors of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a member of the short-chain dehydrogenase/reductase superfamily. Phytoestrogens inhibited the oxidation of 100 microM 17beta-hydroxyestra-4-en-3-one and the reduction of 100 microM estra-4-en-3,17-dione, the best substrate pair known. The best inhibitors of oxidation, with IC(50) below 1 microM, were flavones hydroxylated at positions 3, 5 and 7: 3-hydroxyflavone, 3,7-dihydroxyflavone, 5,7-dihydroxyflavone (chrysin) and 5-hydroxyflavone, together with 5-methoxyflavone. The best inhibitors of reduction were less potent; 3-hydroxyflavone, 5-methoxyflavone, coumestrol, 3,5,7,4'-tetrahydroxyflavone (kaempferol) and 5-hydroxyflavone all had IC(50) values between 1 and 5 microM. Docking the representative inhibitors chrysin and kaempferol into the active site of 17beta-HSDcl revealed the possible binding mode, in which they are sandwiched between the nicotinamide moiety and Tyr212. The structural features of phytoestrogens, inhibitors of both oxidation and reduction catalyzed by the fungal 17beta-HSD, are similar to the reported structural features of phytoestrogen inhibitors of human 17beta-HSD types 1 and 2.     

Flavonoid inhibition of overexpressed human 3beta-hydroxysteroid dehydrogenase type II

The inhibitory effects of various flavonoids on human 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase type II (3beta-HSD type II), overexpressed in baculovirus, were investigated, and the structure-inhibition relationship was examined. The isoflavone derivatives daidzein, genistein, formononetin and biochanin A inhibited 3beta-HSD type II activity at a concentration of 10 microM and of these, genistein was the most potent inhibitor. 6-Hydroxyflavone (6-HF), a synthetic flavone, also strongly inhibited 3beta-HSD activity but 5-HF, 7-HF and other natural flavones were less potent. Energy minimization structures of the flavonoids, as produced using MOE software, showed that isoflavones and flavones have an almost flat A-C ring structure, and that flavonoids that acted as inhibitors had similar steric structures to DHEA. Genistein, 6-HF and cyanoketone, which is known as a typical 3beta-HSD inhibitor, were found to act as competitive inhibitors with K(i) values of 0.12 microM, 0.19 microM and 0.67 nM, respectively. Furthermore, the LUMO (lowest unoccupied molecular orbital (LUMO)) values, as calculated using WinMOPAC (Fujitsu, Japan), of the inhibitors were correlated with the IC(50) values (r2 = 0.84). From these results, it appears that inhibitory effects of flavonoids are due to the combination of steric structure and electron affinity between the active center of 3beta-HSD type II and the flavonoid molecule.