Characterization of isoflavones and their conjugates in female rat urine using LC/MS/MS

Isoflavone phytoestrogens found in soybeans are the most widely studied phytochemicals in human diets and soy infant formulas. The health benefits of the isoflavones daidzein and genistein have been reported, and concerns about potential adverse effects have also been raised. However, the results of direct analysis of isoflavones and their metabolites in biological fluids after consumption of soy-containing diets are scarce. This study describes an LC/MS/MS method for the analysis of isoflavones and their metabolites in the urine of female rats fed diets made with soy protein isolate. Five isoflavones (daidzein, genistein, glycitein, dihydrodaidzein, and O-desmethylangolensin) were identified by comparison with authentic standards. Seventeen conjugates of isoflavones were characterized in the urine, the most unusual being genistein 5-glucuronide and four glucuronide conjugates of reductive metabolites of daidzein. The application of LC/MS/MS to analyze isoflavone metabolites is simple and sensitive, and appears to be an excellent method for determining the bioavailability and metabolism of food phytochemistry.     

Oxidative metabolism of the soy isoflavones daidzein and genistein in humans in vitro and in vivo.

The soy isoflavones daidzein and genistein are found in high concentrations in human plasma and urine after soy consumption. However, in vitro and in vivo data regarding the oxidative metabolism of isoflavones in humans are scarce. Therefore, we have studied the oxidative metabolites of these compounds formed in human liver microsomes and excreted in urine of male and female humans ingesting soy products for 2 days. Human liver microsomes transformed the soy isoflavone daidzein to three monohydroxylated and three dihydroxylated metabolites according to GC/MS analysis. On the basis of a previous study with rat liver microsomes and with the help of reference substances, these metabolites were identified as 6,7,4'-trihydroxyisoflavone, 7,3',4'-trihydroxyisoflavone, 7,8,4'-trihydroxyisoflavone, 7,8,3',4'-tetrahydroxyisoflavone, 6,7,8,4'-tetrahydroxyisoflavone, and 6,7,3',4'-tetrahydroxyisoflavone. Significant amounts of the same metabolites except 6,7,8,4'-tetrahydroxyisoflavone were also found in urine of female and male volunteers after soy intake. Genistein was metabolized by human liver microsomes to six hydroxylation products. The main metabolites were the three aromatic monohydroxylated products 5,6,7,4'-tetrahydroxyisoflavone, 5,7,8,4'-tetrahydroxyisoflavone and 5,7,3',4'-tetrahydroxyisoflavone. The aliphatic monohydroxylated metabolite 2,5,7,4'-tetrahydroxyisoflavone and two aromatic dihydroxylated metabolites, 5,7,8,3',4'-pentahydroxyisoflavone and 5,6,7,3',4'-pentahydroxyisoflavone, were formed in trace amounts. The same hydroxylated genistein metabolites except the aliphatic hydroxylated one could also be detected in human urine samples. Methylated forms of the catechol metabolites, which were generated by incubations with catechol-O-methyltransferase in vitro could be detected only in trace amounts in the urine samples. This implies that this reaction does not play a major role in the biotransformation of the hydroxylated daidzein and genistein metabolites in vivo. Most of these oxidative metabolites are described as human in vivo metabolites for the first time. Their biological significance remains to be established.     

Oxidative in vitro metabolism of the soy phytoestrogens daidzein and genistein

The oxidative metabolism of the major soy isoflavones daidzein and genistein was investigated using liver microsomes from Aroclor-treated male Wistar rats. Both daidzein and genistein were extensively metabolized and are therefore excellent substrates for cytochrome P450 enzymes. The identity of the metabolites was elucidated using high-performance liquid chromatography (HPLC) with diode array detection, gas chromatography-mass spectrometry (GC/MS), and HPLC/atmospheric pressure ionization electrospray mass spectrometry (API-ES MS) as well as reference substances. Daidzein was converted to nine metabolites, comprising four monohydroxylated, four dihydroxylated, and one trihydroxylated metabolite. Genistein was metabolized to four monohydroxylated and two dihydroxylated products. With both isoflavones the additional hydroxy groups are exclusively introduced into the ortho positions of existing phenolic hydroxy groups. The major metabolites of daidzein were identified as 6,7,4'-trihydroxyisoflavone, 6,7,3',4'-tetrahydroxyisoflavone, 7,8, 4'-trihydroxyisoflavone, and 5,6,7,4'-tetrahydroxyisoflavone. The main microsomal metabolites of genistein were 5,6,7, 4'-tetrahydroxyisoflavone and 5,7,8,4'-tetrahydroxyisoflavone. Furthermore, the GC/MS and HPLC/API-ES MS analysis support the conclusion that one monohydroxylated metabolite of daidzein and genistein is hydroxylated at the aliphatic position C-2 of the C-ring. The UV-vis, GC/MS, and HPLC/MS data of all detected metabolites as well as the derived chemical structure of the metabolites are presented. Most metabolites are reported in this paper for the first time. On the basis of these findings it is suggested that hydroxylation reactions may also play an important role in the in vivo metabolism of the soy isoflavones daidzein and genistein.     

Soy isoflavones protect the intestine from lipid hydroperoxide mediated oxidative damage

The effects of 24 h supplementation of human colon carcinoma cells (Caco-2) with isoflavones, genistein, and daidzein and their activities against oleic acid hydroperoxide mediated oxidative stress were investigated. Genistein, at 25, 50, and 100 microM, and daidzein, at 25 and 50 microM, did not induce cell injury to Caco-2 cells. Both compounds reduced cell injury and DNA damage mediated by 5 microM oleic acid hydroperoxides in Caco-2 cells. The effects of genistein and daidzein on antioxidant enzymes were dependent upon the compound and its concentration.     

Thermal stability of genistein and daidzein and its effect on their antioxidant activity

Soy isoflavones, present in many processed soy foods, are known for their phytoestrogenic and antioxidant activities. The aim of this work was to study the kinetics of genistein and daidzein degradation at elevated temperatures and to follow changes in their antioxidant activity. Daidzein and genistein in model solutions (pH 7 and 9) were thermally treated at 120 degrees C or incubated at 70, 80, and 90 degrees C. Isoflavone degradation was observed at all temperatures, with apparent first-order kinetics at 70-90 degrees C, and E(a) = 8.4 and 11.6 kcal/mol at pH 9, respectively. Microcalorimetric stability tests showed a similar pattern of degradation, however, with higher E(a) (genistein, 73.7 kcal/mol; daidzein, 34.1 kcal/mol) that may be attributed to the anaerobic conditions. The antioxidant activity of incubated isoflavone solutions, followed by the ABTS test, decreased rapidly at pH 9 for genistein, whereas only moderate reduction was observed for daidzein (pH 7 and 9) or genistein at pH 7. This may indicate different degradation mechanisms for genistein and daidzein.     

Identification of urinary metabolites of the red clover isoflavones formononetin and biochanin A in human subjects

Dietary supplements manufactured from red clover are widely marketed to provide the beneficial health effects of isoflavones without changing the original diet. In this study the metabolism of formononetin and biochanin A, the principal isoflavones of red clover, was studied in human subjects. Seven women ingested four red clover dietary supplements, and the metabolites of the isoflavones were identified in their urine samples. The structures of trimethylsilyl derivatives of the metabolites were established by GC-MS. New reduced metabolites of formononetin (dihydroformononetin and angolensin) and biochanin A (dihydrobiochanin A and 6'-hydroxyangolensin) were identified in urine samples using authentic reference compounds. Possible metabolic pathways are presented for the red clover isoflavones formononetin and biochanin A.     

Extraction and purification of isoflavones from soybeans and characterization of their estrogenic activities

Soybean isoflavones have multiple beneficial health effects especially on estrogen-deficient diseases such as menopausal symptoms. In this study, isoflavones were produced from soybean flour, and the extraction and purification parameters were optimized to give a high yield of total isoflavones, about 0.62 mg of aglycones/g of soybean flour, which is >2 times the initial yield. HPLC analysis and MTT cell proliferation assay using MCF-7 cells revealed that the product thus obtained not only contained a high content of isoflavone aglycones but also had estrogenic activity. MTT data also revealed that both genistein and daidzein exhibited estrogenic effects at lower concentrations and antiproliferative effects at higher concentrations, and 1 microM genistein and 10 microM daidzein exerted significant estrogenic activities, which were not more than that of the endogenous level of 17beta-estradiol (E2). The production method developed can be used as a guideline for manufacturing soy isoflavones, and the MTT assay was demonstrated to be suitable for quality control on isoflavone products. The results on the estrogenic properties of isoflavones can be used as reference data for their effective and safe usages in estrogenic therapy.     

合成大豆异黄酮的植物基因工程研究

异黄酮是主要存在于豆科植物中的一种具有广谱抗菌活性的黄酮类物质。经过多年生理生化研究发现，异黄酮，特别是作为大豆异黄酮代表的染料木素和大豆黄素，具有良好的预防骨质疏松、更年期综合症、心脑血管疾病和多种癌症的保健效果，是大豆及豆制品中重要的营养成分之一，已有多年被用作保健品功能成分的历史。异黄酮是植物苯丙烷类次生代谢途径的一类产物，随着近年来对植物次生代谢途径研究的不断深入，特别是合成异黄酮的关键酶——异黄酮合酶（isoflavone synthase, IFS）基因的成功克隆，异黄酮合成途径已经得到完全解析。研究者随即展开了合成异黄酮的基因工程研究，在研究过程中逐渐了解到这条代谢途径受到了复杂的分子调控。本文将结合本课题组的工作，综述基因工程合成异黄酮的研究历史与成果，重点深入分析和讨论异黄酮生物合成所受到的分子调控。     

Studies of the in vitro intestinal metabolism of isoflavones aid in the identification of their urinary metabolites

Soy isoflavones have recently gained considerable interest due to their possible health benefits. However, detailed studies on the metabolism of isoflavones are lacking. The aims of the investigation presented here were (1) to study the in vitro intestinal metabolism of isoflavones and their hydroxylated analogues 3'-OH-daidzein, 6-OH-daidzein, 8-OH-daidzein, and 3'-OH-genistein and (2) to characterize the structures of some earlier identified urinary metabolites of soy isoflavones, for which no authentic reference compounds have been available. Isoflavone standards (1-2 mg) were fermented with human fecal flora (16.7%) for 24 h. Metabolites formed during the fermentation were tentatively identified by interpretation of the mass spectra of trimethylsilylated compounds obtained by GC-MS. Compounds having hydroxyl groups at 5-position (i.e., genistein and 3'-OH-genistein) were completely converted to metabolites that could not be detected by the methods used in this study. The metabolism of daidzein and its hydroxylated analogues, 3'-OH-daidzein, 6-OH-daidzein, and 8-OH-daidzein, occurred to a much lesser extent. Minor amounts of reduced metabolites (i.e., isoflavanones and alpha-methyldeoxybenzoins) of these compounds were tentatively identified in fermentation extracts. The retention times and the mass spectra of reduced isoflavone metabolites, obtained from in vitro fermentations of pure compounds, were utilized to identify unknown urinary metabolites of soy isoflavones. Four novel isoflavone metabolites were identified in human urine collected after soy supplementation: 3' '-OH-O-desmethylangolensin, 3',4',7-trihydroxyisoflavanone, 4',7,8-trihydroxyisoflavanone, and 4',6,7-trihydroxyisoflavanone.     

Estrogenic activity of glycitein, a soy isoflavone

Glycitein (4',7-dihydroxy-6-methoxyisoflavone) accounts for 5-10% of the total isoflavones in soy food products. The biological activity of this compound has not been reported to date, although numerous studies have been performed with the other soy isoflavones, daidzein and genistein. Glycitein was isolated from soy germ to 99% purity. Weaning female B6D2F1 mice were dosed with glycitein (3 mg/day), genistein (3 mg/day), and diethylstilbestrol (DES) (0.03 microg/day) in 5% Tween 80 by gavage for 4 days. A control group received an equal volume of 5% Tween 80 solution daily. The uterine weight increased 150% with glycitein (p < 0.001), 50% with genistein (p < 0. 001), and 60% with DES (p < 0.001) compared with the control group. DES, 17beta-estradiol, and three isoflavones (daidzein, genistein, and glycitein) were examined for their competitive binding abilities with 17beta-((3)H)estradiol to the estrogen receptor proteins of the B6D2F1 mouse uterine cytosol. The concentrations of each compound required to displace 50% of the ((3)H)estradiol at 5 nM in the competitive binding assay were 1.15 nM DES, 1.09 nM 17beta-estradiol, 0.22 microM genistein, 4.00 microM daidzein, and 3.94 microM glycitein. These data indicated that glycitein has weak estrogenic activity, comparable to that of the other soy isoflavones but much lower than that of DES and 17beta-estradiol.     

LC/UV/ESI-MS analysis of isoflavones in Edamame and Tofu soybeans

High-performance liquid chromatography coupled with ultraviolet and electrospray ionization mass spectrometry (HPLC/UV/ESI-MSD) was applied to the study of isoflavones in both Edamame and Tofu soy varieties, from which the immature fresh soybeans or the mature soybean seeds are consumed, respectively. Positive atmospheric pressure interface (API) MS and MS/MS were used to provide molecular mass information and led to the identification of a total 16 isoflavones, including three aglycones, three glycosides, two glycoside acetates, and eight glycoside malonates. The major isoflavones in soybean seeds were daidzein and genistein glycoside and their malonate conjugates. Trace levels of daidzein and genistein acetyl glycosides were found only in the mature dry soybean seeds. To facilitate quantitative analysis, acid hydrolysis during extraction of soy samples was selected to convert the various phytoestrogen conjugates into their respective isoflavone aglycones, allowing accurate quantitation of total phytoestrogens as aglycones. On the basis of HPLC combined with UV and MS detection, all three targeted soy isoflavone aglycones, daidzein, genistein and glycitein in hydrolyzed extracts were successfully quantified within 25 min with formononetin used as the internal standard. The standard curves of UV detection were fitted in the range of 14.16-29000 ng/mL for daidzein, 15.38-31500 ng/mL for genistein, and 11.72-24000 ng/mL for glycitein. For MS detection, the standard curves were established in the range of 3.54-1812.5 ng/mL for daidzein, 3.85-1968.75 ng/mL for genistein, and 2.93-1500 ng/mL for glycitein. Good linearities (r(2) > 0.999 for UV and r(2) > 0.99 for MS) for standard curves were achieved for each isoflavone. The accuracy and precision (RSD) were within 10% for UV detection and 15% for MS detection (n = 10). Using this method, the phytoestrogen levels of total isoflavone aglycones from 30 soybean seed varieties were then evaluated for confirmation of the technique. Total isoflavones ranged across the varieties from 0.02 to 0.12% in the Edamame varieties, which are harvested while the seeds are still immature, and from 0.16 to 0.25% in Tofu varieties, harvested when the seeds are physiologically mature. While the literature has focused on the isoflavone content of soy products and processing soy, this report provides a reliable analytical technique for screening of authenticated fresh immature Edamame soybeans and Tofu soybeans.     

Effect of water-to-bean ratio on the contents and compositions of isoflavones in tofu

The present study investigated the changes of the bioavailable isoflavones, including daidzin, genistin, daidzein, and genistein, during the making of tofu. The amount of extracted daidzin and genistin in soy milk increased with increasing water-to-bean ratios from 5 to 9 and reached the maximum level at the ratios of 9-11. On the other hand, the amount of extracted free isoflavones (daidzein and genistein) was not affected by the water-to-bean ratio at the range of 5-11, and their extracted amounts in soy milk were 2-4-fold those in raw soybean. It is suggested that these free isoflavones are mainly derived from daidzin, genistin, malonyldaidzin, and malonylgenistin through enzymatic hydrolysis during the making of soy milk. Tofu made with water-to-bean ratios of 9:1 and 10:1 had the maximal retentions of daidzin and genistin, which were due to the fine homogeneous network microstructure that is supposed to be more effectively retained through hydrophilic interaction with protein. On the contrary, the retained amount of free isoflavones decreased significantly as the water-to-bean ratio increased from 7 to 11, due to their weakening hydrophobic interaction with protein. In this study it was found that the homogeneous microstructure of tofu improved the retention of hydrophilic daidzin and genistin and that the increased amount of drained water does not significantly reduce their retention in the final tofu products as generally imagined.     

Seasonal variation of red clover (Trifolium pratense L., Fabaceae) isoflavones and estrogenic activity

Red clover (Trifolium pratense L., Fabaceae) dietary supplements are currently used to treat menopausal symptoms because of their high content of the mildly estrogenic isoflavones daidzein, genistein, formononetin, and biochanin A. These compounds are estrogenic in vitro and in vivo, but little information exists on the best time to harvest red clover fields to maximize content of the isoflavones and thus make an optimal product. Samples of cultivated red clover above-ground parts and flower heads were collected in parallel over one growing season in northeastern Illinois. Generally, autohydrolytic extracts of above-ground parts contained more isoflavones and had more estrogenic activity in Ishikawa endometrial cells as compared with extracts of flower heads. Daidzein and genistein contents peaked around June to July, while formononetin and biochanin A contents peaked in early September. Flower head and total above-ground parts extracts exhibited differential estrogenic activity in an Ishikawa (endometrial) cell-based alkaline phosphatase induction assay, whereas nondifferential activity was observed for most extracts tested in an MCF-7 (breast) cell proliferation assay when tested at the same final concentrations. Ishikawa assay results could be mapped onto the extracts' content of individual isoflavones, but MCF-7 results did not show such a pattern. These results suggest that significant metabolism of isoflavones may occur in MCF-7 cells but not in Ishikawa cells; therefore, caution is advised in the choice of bioassay used for the biological standardization of botanical dietary supplements.     

Antioxidant isoflavones in Osage orange, Maclura pomifera (Raf.) Schneid

Recent findings that many human chronic diseases are associated with oxidative stresses have instigated the search for dietary antioxidants. Many phytochemicals, particularly phenolic compounds, have been found to possess strong antioxidant activity and reduce the risks of those diseases. Isoflavones, a special phenolic group found in soybean, have been found to act as antioxidants in some model systems. This study investigated the isoflavone content in a unique nonedible tree fruit, Osage orange [Maclura pomifera (Raf.) Schneid], and methods for the extraction, identification, and quantification of the two major isoflavones, osajin and pomiferin, were developed. The ethyl acetate extract contained 25.7% osajin and 36.2% pomiferin, and the two isoflavones were at 9.5 g kg(-1) of fresh Osage orange. Two model systems, FRAP and beta-CLAMS, were used to measure the antioxidant activity of these two isoflavones. Pomiferin was found to be a strong antioxidant in both systems, comparable to the antioxidant vitamins C and E and the synthetic antioxidant BHT. Osajin and the two soybean isoflavones (genistein and daidzein) showed no antioxidant activity. Although the Osage orange fruit is not a food source, it is considered to be safe and, therefore, a potentially good source of an antioxidant nutraceutical and functional food ingredient.     

Antioxidant properties of soybean isoflavone extract and tofu in vitro and in vivo

Isoflavones in soybean were extracted in the crude form using 80% food-grade ethanol at 80 degrees C for 6 h and followed by concentration and dehydration. The soy extract contained isoflavones primarily in the forms of glucosides. In vitro antioxidant activities of the soy extract containing 20-500 ppm isoflavones were conducted using a Rancimat method. The results showed that soy isoflavone extract had strong in vitro antioxidant activity. There was a dose-dependent response for the in vitro antioxidant activity at the lower concentrations but not at the higher concentrations. In vivo antioxidant property was determined by measuring the antioxidant enzymes, superoxide dismutase, and catalase in various organs of rats that were fed with diets containing partially oxidized oil and various levels of isoflavones for up to 24 weeks. Neither short-term (8 weeks) feeding nor low isoflavone content (50 ppm) induced changes in superoxide dismutase or catalase activities in rats. Only diets containing high isoflavone contents (150 and 250 ppm) showed obvious elevated enzymatic levels in various organs. In addition, a laboratory-prepared tofu containing approximately 50 ppm isoflavones had better effects than the soy extract with the 250 ppm isoflavone group, which indicated that molecules other than isoflavones may have a synergistic effect on in vivo antioxidant enzyme inductions of tofu.     

Polyphenolic content in different plant parts of soy cultivars grown under natural conditions

Roots, cotyledons, leaves, stems, pods, and seeds of three soy cultivars were analyzed for their content of isoflavones, flavonols, coumarins, and phenolic acid derivatives with three samplings during a three-month period. The extracts were analyzed by HPLC/DAD and HPLC/MS, allowing us to confirm the presence of daidzein and genistein derivatives as the major isoflavones and to characterize coumarins, most flavonols and phenolic acid derivatives. Seeds exhibited the highest content of isoflavones: 12.61 g/kg of dry weight (DW) in cv. Emiliana; 8.97 g/kg of DW in cv. Elvir; 4.49 g/kg of DW in cv. Kure, and roots are the only part with coumarins, ranging from 4.08 g/kg of DW (cv. Emiliana) to 1.29 g/kg of DW (cv. Elvir) for the longest sampling period. Leaves, pods, and stems have flavonols, and in particular leaves showed: 7.28 g/kg of DW in cv. Emiliana; 6.57 g/kg of DW in cv. Elvir; 7.08 g/kg of DW in cv. Kure. The high content of isoflavones found in the seeds could be ascribed to the natural conditions under which the soy plants were grown.     

Suppression effect of soy isoflavones on nitric oxide production in RAW 264.7 macrophages

Genistein, daidzein, and glycitein, as primary isoflavones in soybeans, are reported to have beneficial effects on atherosclerosis, chronic inflammatory diseases, and cancers that are conducted by nitric oxide (NO) injury. The objectives of this study were to investigate the effects and mechanisms of these soy isoflavones on the inducible nitric oxide synthase (iNOS) system in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Genistein, daidzein, and glycitein dose-dependently suppress NO production (IC(50) = 50 microM) in supernatants of LPS-activated macrophages as measured on the basis of nitrite accumulation. In addition, direct inhibition of iNOS activity, determined by means of the conversion of L-[(3)H]arginine to L-[(3)H]citrulline, and markedly reduced iNOS protein and mRNA levels, evaluated by means of Western blot and RT-PCR, respectively, were found in homogenates of LPS-activated cells treated with each isoflavone. Moreover, genistein was found to have a greater inhibitory effect on NO production but no significant effect on iNOS activity or protein and gene expression to daidzein and glycitein. These observations reveal that the suppression of NO production by genistein, daidzein, and glycitein might be due to the inhibition of both the activity and expression of iNOS in LPS-activated macrophages. The result suggests that soy isoflavones might attenuate excessive NO generation at inflammatory sites.     

Antioxidant capacity of seed coat, dehulled bean, and whole black soybeans in relation to their distributions of total phenolics, phenolic acids, anthocyanins, and isoflavones

Black soybeans have been used as an excellent dietary source for disease prevention and health promotion in China for hundreds of years. However, information about the distribution of health-promoting phenolic compositions in different physical parts of black soybean and the contribution of phenolic compositions to overall antioxidant capacity is limited. To elucidate the distribution of phenolic composition and their contribution to antioxidant activities in black soybean, the total and individual phenolic profiles, and antioxidant capacities of seed coat, dehulled and whole black soybean were systematically investigated. The seed coat exhibited much higher total phenolic indexes and antioxidant activities than whole and dehulled black soybean. Dehulled black soybean possessed similar levels of total phenolic content, total flavonoid content, 2-diphenyl-1-picryhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP), and oxygen radical absorbance capacity (ORAC) activities as compared to whole yellow soybean. Cyanidin-3-glucoside, petunidin-3-glucoside, and peonidin-3-glucoside were detected in the seed coat but not in dehulled black soybean and yellow soybean. Among benzoic acid detected, caffeic and chlorogenic acid were the predominant phenolic acids. Whole black soybean and dehulled black soybean exhibited similar isoflavone contents in 7- O-beta-glucosides and malonylglucosides of daidzein and genistein. The seed coat possessed significantly ( p < 0.05) lower 7- O-beta-glucosides and malonylglucosides of daidzein and genistein, acetylglycitin, and total isoflavones than whole and dehulled black soybean. The contribution of phenolics in the seed coat to the antioxidant activity of black soybean parts depends on the assay methods. When measured with the DPPH and FRAP methods, the seed coat contributed 90% of the total antioxidant capacity of black soybean. However, when measured with the ORAC method, the seed coat and dehulled portion contributed approximately equally the total antioxidant capacity of black soybeans. The information generated from this study on the distribution and content of their active components is useful for the effective use of black soybeans as an ingredient for promoting health.     

Inhibition of extrahepatic human cytochromes P450 1A1 and 1B1 by metabolism of isoflavones found in Trifolium pratense (red clover)

Biochanin A and formononetin are the predominant isoflavones in red clover. In a previous study (J. Agric. Food Chem. 2002, 50, 4783-4790), it was demonstrated that human liver microsomes converted biochanin A and formononetin to genistein and daidzein. This paper now shows CYP1B1-catalyzed O-demethylation of biochanin A and formononetin to produce genistein and daidzein, respectively, which inhibit CYP1B1. Recombinant human CYP1A1 or CYP1B1 was incubated with biochanin A or formononetin. CYP1A1 catalyzed isoflavone 4'-O-demethylation and hydroxylations with similar efficiency, whereas CYP1B1 favored 4'-O-demethylation over hydroxylations. Three of the biochanin A metabolites (5,7,3'-trihydroxy-4'-methoxyisoflavone, 5,7,8-trihydroxy-4'-methoxyisoflavone, and 5,6,7-trihydroxy-4'-methoxyisoflavone) were characterized by 1H NMR spectroscopy and mass spectrometry. Daidzein (Ki = 3.7 microM) exhibited competitive inhibition of CYP1B1 7-ethoxyresorufin O-deethylase activity, and genistein (Ki = 1.9 microM) exhibited mixed inhibition. Biochanin A and/or formononetin may exert anticarcinogenic effects directly by acting as competitive substrates for CYP1B1 or indirectly through their metabolites daidzein and genistein, which inhibit CYP1B1.     

Separation of quercetin's biological activity from its oxidative property through bioisosteric replacement of the catecholic hydroxyl groups with fluorine atoms

An oxidative property has endowed quercetin with numerous biological benefits, and some of quercetin's biological activities may be related, at least partly, to its antioxidant activity. On the other hand, the oxidative property and associated susceptibility to oxidative decomposition has hampered in-depth investigation of the biological targets as well as underlying mechanisms for quercetin's biological activity. This study was undertaken to separate quercetin's biological activities from its antioxidant properties through bioisosteric replacement of the phenolic hydroxyl groups. The novel quercetin derivative 3',4'-difluoroquercetin (2), thus prepared, showed nonoxidizable property with no attenuation of biological activity. Rather, 2 showed a subtle but significant increase in biological activity compared with quercetin, which might be attributed to its lack of oxidative property. The nonoxidizable nature along with the potent biological activity of the quercetin mimic 2 suggests possible oxidation-independent mechanisms for the biological activities of the quercetin that do not require oxidative formation of the highly electrophilic metabolites.