Effect of circulating forms of soy isoflavones on the oxidation of low density lipoprotein

Soy isoflavones are thought to have a cardioprotective effect that is partly mediated by an inhibitory influence on the oxidation of low density lipoprotein (LDL). However, the aglycone forms investigated in many previous studies do not circulate in appreciable quantities because they are metabolised in the gut and liver. We investigated effects of various isoflavone metabolites, including for the first time the sulphated conjugates formed in the liver and the mucosa of the small intestine, on copper-induced LDL oxidation. The parent aglycones inhibited oxidation, although only 5% as well as quercetin. Metabolism increased or decreased their effectiveness. Equol inhibited 2.65-fold better than its parent compound daidzein and 8-hydroxydaidzein, not previously assessed, was 12.5-fold better than daidzein. However, monosulphated conjugates of genistein, daidzein and equol were much less effective and disulphates completely ineffective. Since almost all isoflavones circulate as conjugates, these data suggest that despite the increased potency produced by some metabolic changes, isoflavones may not be effective antioxidants in vivo unless they are deconjugated again.     

Incorporation of esterified soybean isoflavones with antioxidant activity into low density lipoprotein.

We have recently reported that dietary intake of soybean isoflavone phytoestrogens resulted in increased oxidation resistance of isolated low density lipoprotein (LDL). In order to explore the underlying mechanisms we designed two types of in vitro experiments. First, we prepared several different isoflavone fatty acid esters to increase their lipid solubility and studied their incorporation into LDL. Second, the oxidation resistance of the isoflavone-containing LDLs was investigated with Esterbauer's 'conjugated diene' method using Cu2+ as prooxidant. Unesterified daidzein and genistein as well as genistein stearic acid esters were incorporated into LDL to a relatively small extent (0.33 molecules per LDL particle, or less) and they did not significantly influence oxidation resistance. The oleic acid esters of isoflavones were incorporated more effectively, reaching a level of 2.19 molecules per LDL particle or more, and the 4',7-O-dioleates of daidzein and genistein exhibited prolongations of lag times by 46% (P<0.05) and 202% (P<0.01), respectively. A smaller but significant increase in lag time (20.5%, P<0.01) was caused by daidzein 7-mono-oleate. In summary, esterification of soybean isoflavones daidzein and genistein with fatty acids at different hydroxyl groups provided lipophilicity needed for incorporation into LDL. Some isoflavone oleic acid esters increased oxidation resistance of LDL following their incorporation.     

Inhibitory effect of isoflavones on peroxynitrite-mediated low-density lipoprotein oxidation

Peroxynitrite, a potent oxidant formed in vivo from the reaction of nitric oxide with superoxide, can mediate low-density liprotein (LDL) oxidation which is thought to increase the risk of atherosclerosis. This study investigates the inhibitory effect of the isoflavones, genistein and daidzein, together with their glycosidic forms, genistin and daidzin, on the peroxynitrite-mediated LDL oxidation and nitration of tyrosine. Genistein and daidzein were observed to dose-dependently inhibit peroxynitrite-mediated LDL oxidation, while their glucoside conjugates showed less activity. Moreover, all the isoflavones used in this study were found to be potent peroxynitrite scavengers, preventing the nitration of tyrosine. The ability of the isoflavones at 50 microM to decrease the tyrosine nitration induced by peroxynitrite (1 mM) was in the ratios of genistein (49%), daidzein (40%), daidzin (41%) and genistin (42%) when compared to the control (tyrosine incubated only with peroxynitrite). These results suggest that an intake of isoflavones could contribute to protecting against cardiovascular diseases and chronic inflammatory diseases.     

Developmental and nutritional regulation of isoflavone secretion from soybean roots

Isoflavones play important roles in plant–microbe interactions in rhizospheres. Soybean roots secrete daidzein and genistein to attract rhizobia. Despite the importance of isoflavones in plant–microbe interactions, little is known about the developmental and nutritional regulation of isoflavone secretion from soybean roots. In this study, soybeans were grown in hydroponic culture, and isoflavone contents in tissues, isoflavone secretion from the roots, and the expression of isoflavone conjugates hydrolyzing beta-glucosidase (ICHG) were investigated. Isoflavone contents did not show strong growth-dependent changes, while secretion of daidzein from the roots dramatically changed, with higher secretion during vegetative stages. Coordinately, the expression of ICHG also peaked at vegetative stages. Nitrogen deficiency resulted in 8- and 15-fold increases in secretion of daidzein and genistein, respectively, with no induction of ICHG. Taken together, these results suggest that large amounts of isoflavones were secreted during vegetative stages via the hydrolysis of (malonyl)glucosides with ICHG.     

Neutrophil myeloperoxidase chlorinates and nitrates soy isoflavones and enhances their antioxidant properties

Soy isoflavones and other polyphenolics have a number of potentially important beneficial effects on the pro-oxidant aspects of chronic inflammation. The impact of inflammatory cell-specific metabolism of polyphenolics, which can include halogenation and nitration, on the properties of these compounds has not been examined. Using either human neutrophils or differentiated human leukemia cells (HL-60) stimulated with phorbol ester to elicit a respiratory burst, the hypothesis that local generation of reactive oxygen and nitrogen species may metabolize and modify the biological properties of the soy isoflavones was examined. Coincubation of the stimulated cells with genistein or daidzein had no effect on the respiratory burst. Medium from stimulated cells in the presence of the isoflavones and NO(2)(-) increased the inhibition of copper-induced LDL oxidation. Mass spectrometry analysis of this medium revealed that monochlorinated, dichlorinated, and nitrated isoflavones, formed through a myeloperoxidase-dependent mechanism, were present. The consumption of genistein in the presence of cells was both extensive and rapid with > 95% of the genistein converted to either the chlorinated or nitrated metabolites within 30 min. Chemically synthesized 3'-chlorogenistein and 3'-chlorodaidzein increased the inhibition of LDL oxidation by approximately 4-fold and 2-fold over genistein and daidzein, respectively. These results lead to the hypothesis that inflammatory cell-specific metabolism of polyphenolics can modify the properties of these compounds at the local site of inflammation.     

Antiproliferative efficacy of lipophilic soy isoflavone phytoestrogens delivered by low density lipoprotein particles into cultured U937 cells

Some fat-soluble bioactive substances incorporated into low density lipoprotein (LDL) may be delivered into cells via LDL receptor pathway influencing cellular functions. In this study, we synthesized a number of fat-soluble isoflavone esters and investigated their incorporation into LDL as well as their delivery into U937 cells. Using an artificial transfer system (Celite dispersion), genistein and daidzein oleates and daidzein dilinoleate were efficiently incorporated into LDL with concentrations ranging between 2.7 to 16.9 isoflavone molecules/LDL particle, while much smaller amounts of unesterified isoflavones and genistein stearates were transferred into LDL. LDL containing 7-oleates or 4',7-dioleates of genistein and daidzein significantly reduced U937 cell proliferation by 36-43%. The strongest inhibitory effect was shown by daidzein 4',7-dilinoleate with 93% reduction of cell proliferation. Neither of the 4'-oleates of genistein and daidzein contained in LDLs exhibited antiproliferative effects although they were incorporated into LDL. In summary, we demonstrated that isoflavones made fat-soluble by esterification can be incorporated into LDL in vitro and delivered into cultured U937 cells via the LDL-receptor pathway, reducing the cell proliferation.     

Oxidative metabolism and genotoxic potential of major isoflavone phytoestrogens

The soy isoflavones daidzein, genistein and glycitein are extensively metabolized by rat liver microsomes to a variety of catechol metabolites. Hydroxylated metabolites of daidzein and genistein have also been demonstrated in incubations with human hepatic microsomes and in the urine of humans after ingestion of soy food. Although the microsomal metabolism of formononetin and biochanin A is dominated by demethylation to daidzein and genistein, respectively, catechols of the parent isoflavones and of the demethylation products are also formed. Thus, oxidative metabolism appears to be common among isoflavones and may have implications for their biological activities. As genistein but not daidzein exhibits clastogenic activity in cultured mammalian cells, the role of oxidative metabolism for the genotoxicity of isoflavones is of particular interest.     

Genistein and daidzein repress adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells via Wnt/β-catenin signalling or lipolysis

Objectives: One aspect of the effects of isoflavones against fat deposition might be at least associated with the mechanism by which Wnt/β‐catenin signalling inhibits adipocyte differentiation. However, it remains completely unknown as to whether isoflavones might influence Wnt signalling during commitment of pluripotent mesenchymal stem cells (MSCs) to adipose lineages. In the present study, we have investigated the mechanisms underlying effects of genistein and daidzein, the major soy isoflavones, on anti‐adipogenic Wnt/β‐catenin signalling. Materials and methods: Adipose tissue‐derived (AD) MSCs were exposed continuously to genistein and daidzein (0.01–100 μm ) during adipogenic differentiation (21 days). An oestrogen antagonist, ICI 182,780, was used to determine whether or not the isoflavones activated Wnt signalling via oestrogen receptors (ERs). Results: Genistein and daidzein suppressed adipogenic differentiation of AD‐MSCs in a dose‐dependent manner and inhibited expression of adipogenic markers, PPARγ, SREBP‐1c and Glut 4, from mid‐phase differentiation. Microarrays showed that anti‐adipogenic effects of genistein were principally attributable to activation of Wnt signalling via ERs‐dependent pathway, such as Erk/JNK signalling and LEF/TCF4 co‐activators. These findings were supported by evidence that the effects of genistein were offset by ICI182,780. Unlike genistein, daidzein inhibited adipogenesis through stimulation of lipolysis, with for example, PKA‐mediated hormone sensitive lipase. This is consistent with the increase in glycerol released from AD‐MSCs. In conclusion, understanding that different sets of mechanisms of the two isoflavones on adipogenesis will help the design of novel strategies to prevent observed current epidemic levels of obesity, using isoflavones.     

Genistein as a potential inducer of the anti-atherogenic enzyme paraoxonase-1: studies in cultured hepatocytes in vitro and in rat liver in vivo

A number of cardioprotective effects, including the reduced oxidation of the low‐density lipoprotein (LDL) particles, have been attributed to dietary soy isoflavones. Paraoxonase 1 (PON1), an enzyme mainly synthesized in the liver, may exhibit anti‐atherogenic activity by protecting LDL from oxidation. Thus, dietary and pharmacological inducers of PON1 may decrease cardiovascular disease risk. Using a luciferase reporter gene assay we screened different flavonoids for their ability to induce PON1 in Huh7 hepatocytes in culture. Genistein was the most potent flavonoid with regard to its PON1‐inducing activity, followed by daidzein, luteolin, isorhamnetin and quercetin. Other flavonoids such as naringenin, cyanidin, malvidin and catechin showed only little or no PON1‐inducing activity. Genistein‐mediated PON1 transactivation was partly inhibited by the oestrogen‐receptor antagonist fulvestrant as well as by the aryl hydrocarbon receptor antagonist 7‐ketocholesterol. In contrast to genistein, the conjugated genistein metabolites genistein‐7‐glucuronide, genistein‐7‐sulfate and genistein‐7,4′‐disulfate were only weak inducers of PON1 transactivation. Accordingly, dietary genistein supplementation (2 g/kg diet over three weeks) in growing rats did not increase hepatic PON1 mRNA and protein levels as well as plasma PON1 activity. Thus, genistein may be a PON1 inducer in cultured hepatocytes in vitro, but not in rats in vivo.     

Regulation of isoflavone production in hydroponically grown Pueraria montana (kudzu) by cork pieces, XAD-4, and methyl jasmonate

A mini-hydroponic growing system was employed for seedlings of kudzu vine (Pueraria montana) and contents of isoflavones (daidzein, genistein, daidzin, genistin, and puerarin) from shoot and root parts of seedlings were analyzed quantitatively. In addition, exogenous cork pieces, polymeric adsorbent, XAD-4, and universal elicitor, methyl jasmonate (MeJA), were used to regulate the production of these isoflavones. It was shown that cork pieces up-regulate the production of daidzein and genistein up to seven- and eight-fold greater than the levels obtained for control roots. In contrast, levels of glucosyl conjugates, daidzin and genistin, decrease up to five- and eight-fold, respectively. Cork treatment also induces the excretion of the root isoflavone constituents into the growth medium. Minimal levels of isoflavones are absorbed by the cork pieces. XAD-4 stimulates the production of glucosyl conjugates, daidzin and genistin, in root parts about 1.5-fold greater than that obtained in control roots. These are the highest amounts of daidzin and genistin that are observed (5.101 and 6.759 mg g⁻¹ dry weight, respectively). In contrast to these two adsorbents, MeJA increases the accumulation of isoflavones in shoot rather than in root parts of seedlings, about three- to four-fold over control levels, with the exception of genistein. These studies reveal new observations on the regulation of isoflavone production in hydroponically grown Pueraria montana plants by two adsorbents (cork pieces and XAD-4) and MeJA elicitor.     

Genistein and daidzein induce neurotoxicity at high concentrations in primary rat neuronal cultures

Summary It is known that estrogen can protect neurons from excitotoxicity. Since isoflavones possess estrogen-like activity, it is of interest to determine whether isoflavones can also protect neurons from glutamate-induced neuronal injury. Morphological observation and lactate dehydrogenase (LDH) release assay were used to estimate the cellular damage. It is surprising that, contrary to estrogen, isoflavones, specifically genistein and daidzein, are toxic to primary neuronal culture at high concentration. Treatment of neurons with 50 μM genistein and daidzein for 24 h increased LDH release by 90% and 67%, respectively, indicating a significant cellular damage. Under the same conditions, estrogen such as 17β-estradiol did not show any effect on primary culture of brain cells. At 100 μM, both genistein and daidzein increased LDH release by 2.6- and 3-fold, respectively with a 30-min incubation. Furthermore, both genistein and daidzein at 50 μM increased the intracellular calcium level, [Ca²⁺]_i, significantly. To determine their mode of action, genistein and daidzein were tested on glutamate and GABA_A receptor binding. Both genistein and daidzein were found to have little effect on glutamate receptor binding, while the binding of [³H]muscimol to GABA_A receptors was markedly inhibited. However, 17β-estradiol did not affect GABA_A receptor binding suggesting that the toxic effect of genistein and daidzein could be due to their inhibition of the GABA_A receptor resulting in further enhancement of excitation by glutamate and leading to cellular damage. Ying Jin, Heng Wu contributed equally to this article.     

Flavonoid and isoflavonoid distribution in developing soybean seedling tissues and in seed and root exudates

The distribution of flavonoids, isoflavonoids, and their conjugates in developing soybean (Glycine max L.) seedling organs and in root and seed exudates has been examined. Conjugates of the isoflavones daidzein and genistein are major metabolites in all embryonic organs within the dry seed and in seedling roots, hypocotyl, and cotyledon tissues at all times after germination. Primary leaf tissues undergo a programmed shift from isoflavonoid to flavonoid metabolism 3 days after germination and become largely predominated by glycosides of the flavonols kampferol, quercetin, and isorhamnetin by 5 days. Cotyledons contain relatively constant and very high levels of conjugates of both daidzein and genistein. Hypocotyl tissues contain a third unidentified compound, P19.3, also present in multiple conjugated forms. Conjugates of daidzein, genistein, and P19.3 are at their highest levels in the hypocotyl hook and fall off progressively down the hypocotyl. These isoflavones also undergo a programmed and dramatic decrease between 2 and 4 days in the hypocotyl hook. All root sections are predominated by daidzein and its conjugates, particularly in the root tip, where they reach the highest levels in the seedling. Light has a pronounced effect on the distribution of the isoflavones; in the dark, isoflavone levels in the root tips are greatly reduced, while those in the cotyledons are higher. Finally, the conjugates of daidzein and genistein and several unidentified aromatic metabolites are selectively excreted into root and seed exudates. Analysis of seed exudates suggests that this is a continuous, but saturable event.     

Measurement of intact sulfate and glucuronide phytoestrogen conjugates in human urine using isotope dilution liquid chromatography-tandem mass spectrometry with [13C(3)]isoflavone internal standards

A method has been developed for the analysis of phytoestrogens and their conjugates in human urine using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Stable isotopically labeled [13C(3)]daidzein and [13C(3)]genistein were synthesized and used as internal standards for isotope dilution mass spectrometry. Free aglycons and intact glucuronide, sulfate, diglucuronide, disulfate, and mixed sulfoglucuronide conjugates of isoflavones and lignans were observed in naturally incurred urine samples. Sample pretreatment was not necessary, other than addition of internal standards and pH adjustment. Urine was injected directly onto the analytical column. The limits of detection were generally <50ng/ml, precision was generally <10% CV for conjugates. Total hydrolyzed daidzein and genistein were measured against quality assurance urine sample and were accurate to within 12%. The accuracy of conjugate measurement can not be ascertained, as no reference samples are available. The mean sum of daidzein and its conjugates was within 20% of the hydrolyzed value. Concentrations of the free aglycons of up to 22% of genistein and 18% of daidzein were observed. The average pattern was ca. 54% 7-glucuronide, 25% 4(')-glucuronide, 13% monosulfates, 7% free daidzein, 0.9% sulfoglucuronides, 0.4% diglucuronide, and <0.1% disulfate. Selective enzymatic deconjugation with glucuronidase and mixed glucuronidase/sulfatase were used to validate the accuracy of the quantitation of the intact daidzein conjugates. There were no apparent sex differences, or conditioning effects on the conjugation profile of isoflavones after chronic dosing.     

Antioxidant mechanisms of isoflavones in lipid systems: paradoxical effects of peroxyl radical scavenging

Oxidation of lipids has been implicated in the pathophysiology of atherosclerosis. It has been suggested that scavenging of lipid peroxyl radicals contribute to the antiatherosclerotic effects of naturally occurring compounds such as the isoflavones. This group of polyphenolics includes genistein and is present in relatively high concentrations in food products containing soy. Soy isoflavones are capable of inhibiting lipoprotein oxidation in vitro and suppressing formation of plasma lipid oxidation products in vivo. However, key aspects of the antioxidant mechanisms remain unknown. In this study the antioxidant effects of genistein and other soy isoflavones on lipid peroxidation initiated by mechanistically diverse oxidants was investigated. Although isoflavones inhibited lipid peroxidation stimulated by both metal-dependent and independent processes, the concentration required for these effects were relatively high compared to those found in vivo. Interestingly, however, isoflavones were not consumed and remained in the native state over the time during which inhibition of lipid peroxidation was observed. This was also the case under conditions where synergistic inhibition of LDL oxidation was observed with ascorbate. Furthermore, in an oxidation system driven solely by peroxyl radicals, isoflavones were found to be relatively poor peroxyl radical scavengers. Consistent with the apparent lack of reactivity with lipid-derived oxidants, isoflavones were also relatively resistant to oxidation mediated by the potent oxidant peroxynitrite. The potential antioxidant mechanisms of isoflavones are discussed in the context of possible reactivities of isoflavone-derived phenoxyl radicals.     

Activation of glutathione peroxidase via Nrf1 mediates genistein's protection against oxidative endothelial cell injury

Cellular actions of isoflavones may mediate the beneficial health effects associated with high soy consumption. We have investigated protection by genistein and daidzein against oxidative stress-induced endothelial injury. Genistein but not daidzein protected endothelial cells from damage induced by oxidative stress. This protection was accompanied by decreases in intracellular glutathione levels that could be explained by the generation of glutathionyl conjugates of the oxidised genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone. Both isoflavones evoked increased protein expression of gamma-glutamylcysteine synthetase-heavy subunit (gamma-GCS-HS) and increased cytosolic accumulation and nuclear translocation of Nrf2. However, only genistein led to increases in the cytosolic accumulation and nuclear translocation of Nrf1 and the increased expression of and activity of glutathione peroxidase. These results suggest that genistein-induced protective effects depend primarily on the activation of glutathione peroxidase mediated by Nrf1 activation, and not on Nrf2 activation or increases in glutathione synthesis.     

Proteome analysis suggests that mitochondrial dysfunction in stressed endothelial cells is reversed by a soy extract and isolated isoflavones

Apoptosis is a driving force in atherosclerosis development. A soy extract or a combination of the soy isoflavones genistein and daidzein inhibited apoptosis induced by oxidized LDL in endothelial cells. Proteome analysis revealed that the LDL-induced alterations of numerous proteins were reversed by the extract and the genistein/daidzein mixture but only three protein entities, all functionally linked to mitochondrial dysfunction, were regulated in common by both treatments.     

Properties of Quercetin Conjugates: Modulation of LDL Oxidation and Binding to Human Serum Albumin

Quercetin is an important dietary flavonoid with in vitro antioxidant activity. However, it is found in human plasma as conjugates with glucuronic acid, sulfate or methyl groups, with no significant amounts of free quercetin present. The antioxidant properties of the conjugates found in vivo and their binding to serum albumin are unknown, but essential for understanding possible actions of quercetin in vivo. We, therefore, tested the most abundant human plasma quercetin conjugates, quercetin-3-glucuronide, quercetin-3′-sulfate and isorhamnetin-3-glucuronide, for their ability to inhibit Cu(II)-induced oxidation of human low density lipoprotein and to bind to human albumin, in comparison to free flavonoids and other quercetin conjugates. LDL oxidation lag time was increased by up to four times by low (<2?μM) concentrations of quercetin-3-glucuronide, but was unaffected by equivalent concentrations of quercetin-3′-sulfate and isorhamnetin-3-glucuronide. In general, the compounds under study prolonged the lag time of copper-induced LDL oxidation in the order: quercetin-7-glucuronide>quercetin>quercetin-3-glucuronide=quercetin-3-glucoside>catechin>quercetin-4′-glucuronide>isorhamnetin-3-glucuronide>quercetin-3′-sulfate. Thus the proposed products of small intestine metabolism (quercetin-7-glucuronide, quercetin-3-glucuronide) are more efficient antioxidants than subsequent liver metabolites (isorhamnetin-3-glucuronide, quercetin-3′-sulfate). Albumin-bound conjugates retained their property of protecting LDL from oxidation, although the order of efficacy was altered (quercetin-3′-sulfate>quercetin-7-glucuronide>quercetin-3-glucuronide>quercetin-4′-glucuronide=isorahmnetin-3-glucuronide). K_q values (concentration required to achieve 50% quenching) for albumin binding, as assessed by fluorescence quenching of Trp214, were as follows: quercetin-3′-sulfate (～4?μM)=quercetin≥quercetin-7-glucuronide>quercetin-3-glucuronide=quercetin-3-glucoside>isorhamnetin-3-glucuronide>quercetin-4′-glucuronide (～20?μM). The data show that flavonoid intestinal and hepatic metabolism have profound effects on ability to inhibit LDL oxidation and a lesser but significant effect on binding to serum albumin.     

The phytoestrogen equol increases nitric oxide availability by inhibiting superoxide production: an antioxidant mechanism for cell-mediated LDL modification

Estrogen replacement therapy (ERT) is reported to lower the incidence of cardiovascular disease in postmenopausal women. ERT also lowers the levels of oxidatively modified low-density lipoprotein (LDL). Because modified LDL can mediate the development of atherosclerosis by inflammatory processes, ERT may exert its LDL protective effect through enhanced antioxidant activity in vascular tissues. Plant sources of estrogenic compounds have been used as alternatives for ERT because they avoid a number of negative health effects produced by estrogen. In this study, the antioxidant properties of the soy isoflavone metabolite, equol (an estrogenic metabolite of daidzein) were studied. Equol has a greater antioxidant activity than the parent isoflavone compounds genistein and daidzein, found in high concentration in soy. Equol inhibits LDL oxidation in vitro and LDL oxidative modification by J774 monocyte/macrophages to LDL(-), an electronegative modified LDL found in human plasma. An antioxidant effect of equol was found to be mediated by inhibition of superoxide radical (O(2)(-*)) production and manifested through enhanced levels of free nitric oxide (NO) that prevents LDL modification. Thus, when NO levels were increased by donor agents, generators, or compounds that facilitate nitric oxide synthase activity, LDL(-) formation by J774 cells was strongly inhibited. Conversely, inhibition of NO production enhanced LDL(-) formation, and the combination of reduced NO and increased O(2)(-*) production yielded maximum LDL(-) formation. Pretreatment of cells with equol inhibited production of O(2)(-*) by J774 cells apparently via the inactivation of the reduced nicotinamide adenine dinucleotide phosphate oxidase complex. Decreased O(2)(-*) production resulted in increased free NO levels (but not total NO production) indicating that decreased reactions between O(2)(-*) and NO are an outcome of equol's antioxidant activity in cell culture.     

Chlorination and nitration of soy isoflavones.

Diets enriched in soy foods containing a high concentration of isoflavonoids are associated with a decrease in the incidence of several chronic inflammatory diseases. Studies with experimental models of diseases, such as atherosclerosis, suggest that these effects can be ascribed to the biological properties of the isoflavones. Since the isoflavones and tyrosine have structural similarities and modifications to tyrosine by inflammatory oxidants such as hypochlorous acid (HOCl) and peroxynitrite (ONOO(-)) have been recently recognized, we hypothesized that the isoflavones also react with HOCl and ONOO(-). Using an in vitro approach, we demonstrate in the present study that the isoflavones genistein, daidzein, and biochanin-A can be chlorinated and nitrated by these oxidants. These reactions were investigated using high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance. In the reaction with HOCl, both mono- and dichlorinated derivatives of genistein and biochanin-A are formed, whereas with daidzein only a monochlorinated derivative was detected. The reaction between genistein or daidzein and ONOO(-) yielded a mononitrated product. However, no nitrated product was detected with biochanin-A. Furthermore, the reaction between genistein and sodium nitrite and HOCl yielded a chloronitrogenistein derivative, as well as a dichloronitrogenistein derivative. These results indicate that the ability of the isoflavones to react with these oxidant species depends on their structure and suggest that they could be formed under conditions where these reactive species are generated under pathological conditions.     

Comparison of antioxidative and chelating effects of daidzein and daidzin on protein oxidative modification by copper in vitro

Daidzein and its glycoside daidzin are isoflavones. Their antioxidative effects were compared in vitro. Although both compounds inhibited protein oxidative modification by copper, the inhibitory effect of daidzein was stronger than that of daidzin. Because daidzein showed a greater affinity for Cu²⁺, the antioxidant effect of these isoflavones may be dependent on their respective copper-chelating abilities.