Major isoflavonoid contents of the 1-year-cultivated phytoestrogen-rich herb, Pueraria mirifica

Pueraria mirifica is a tuberous plant enriched with active phytoestrogens. There is no established information about the factors influencing isoflavonoid storage in the tubers. We investigated the tuberous storage of the major isoflavonoids of 1-year-old plants. Four cultivars of P. mirifica were cultivated in the same field trial during the same period to establish a unique plant age and differentiation under the same environment and soil conditions. The tubers collected from the 1-year-old plants in the summer, rainy season and winter were submitted to an HPLC analysis with a gradient system comprising 0.1% acetic acid and acetonitrile. Five major isoflavonoids, puerarin, daidzin, genistin, daidzein and genistein, were adopted as standards. P. mirifica tubers of different cultivars collected in the same season exhibited significant differences in individual and total isoflavonoid contents, showing chemovariety. P. mirifica tubers of the same cultivar collected from different seasons also exhibited significant differences in individual and total isoflavonoid contents, showing the influence of season. In conclusion, the tuberous storage of major isoflavonoids in 1-year-cultivated plants was greatly diverse and was strongly influenced by the season and plant genetics.     

Structure, function, and engineering of enzymes in isoflavonoid biosynthesis

Isoflavonoids are a large group of plant natural products and play important roles in plant defense. They also possess valuable health-promoting activities with significant health benefits for animals and humans. The isoflavonoids are identified primarily in leguminous plants and are synthesized through the central phenylpropanoid pathway and the specific isoflavonoid branch pathways in legumes. Structural studies of some key enzymes in the central phenylpropanoid pathway shed light on the early stages of the (iso)flavonoid biosynthetic process. Significant impact has also been made on structural studies of enzymes in the isoflavonoid branch pathways. Structures of isoflavonoid-specific NADPH-dependent reductases revealed how the (iso)flavonoid backbones are modified by reduction reactions and how enzymes specifically recognize isoflavonoids and catalyze stereo-specific reductions. Structural studies of isoflavonoid methyltransferases and glycosyltransferases revealed how isoflavonoids are further decorated with methyl group and sugars in different methylation and glycosylation patterns that determine their bioactivities and functions. In combination with mutagenesis and biochemical studies, the detailed structural information of these enzymes provides a basis for understanding the complex biosynthetic process, enzyme catalytic mechanisms, and substrate specificities. Structure-based homology modeling facilitates the functional characterization of these large groups of biosynthetic enzymes and their homologs. Structure-based enzyme engineering is becoming a new strategy for synthesis of bioactive isoflavonoids and also facilitates plant metabolic engineering towards improvement of quality and production of crop plants.     

Isoflavonoid exudation from white lupin roots is influenced by phosphate supply, root type and cluster-root stage

The internal concentration of isoflavonoids in white lupin (Lupinus albus) cluster roots and the exudation of isoflavonoids by these roots were investigated with respect to the effects of phosphorus (P) supply, root type and cluster-root developmental stage.To identify and quantify the major isoflavonoids exuded by white lupin roots, we used high-pressure liquid chromatography (HPLC) coupled to electrospray ionization (ESI) in mass spectrometry (MS).The major exuded isoflavonoids were identified as genistein and hydroxygenistein and their corresponding mono- and diglucoside conjugates. Exudation of isoflavonoids during the incubation period used was higher in P-deficient than in P-sufficient plants and higher in cluster roots than in noncluster roots. The peak of exudation occurred in juvenile and immature cluster roots, while exudation decreased in mature cluster roots.Cluster-root exudation activity was characterized by a burst of isoflavonoids at the stage preceding the peak of organic acid exudation. The potential involvement of ATP-citrate lyase in controlling citrate and isoflavonoid exudation is discussed, as well as the possible impact of phenolics in repelling rhizosphere microbial citrate consumers.     

Effect of localized nitrate application on isoflavonoid concentration and nodulation in split-root systems of wild-type and nodulation-mutant soybean plants

Although isoflavonoids are known to be inducers of nod genes in Bradyrhizobium japonicum, it was recently proposed that internal root levels of isoflavonoids may be important in nodule development on soybean (Glycine max [L.] Merr.). The hypernodulating soybean mutants were shown to accumulate higher root concentrations of isoflavonoid compounds (daidzein, genistein, and coumestrol) and to be more extensively nodulated than was the Williams parent when inoculated with B. japonicum. The hypernodulating mutants and the parent line, Williams, also showed decreased isoflavonoid concentrations and decreased nodule development if N was applied. The current study evaluated the effect of localized NO(3) (-) application on root isoflavonoid concentration and on nodulation in split-root systems of the Williams wild type and a hypernodulating mutant (NOD1-3). Nitrate application markedly decreased isoflavonoid concentrations in non-inoculated soybean roots. When roots were inoculated, nodule number, weight, and nitrogenase activity were markedly suppressed on the root-half receiving 5 millimolar NO(3) (-) compared with the other root-half receiving 0 millimolar NO(3) (-). High performance liquid chromatographic analyses of root extracts showed that the root-half receiving 5 millimolar NO(3) (-) was markedly lower in isoflavonoid concentrations in both soybean lines. This was partially due to the localized stimulatory effect of NO(3) (-) on root growth. The inoculated NOD1-3 mutant had higher isoflavonoid concentrations than did the Williams control in both the presence and absence of NO(3) (-). These results provide evidence that the site of N application primarily controls the site of nodulation inhibition, possibly through decreasing isoflavonoid levels. Although the effect of NO(3) (-) on nodule development and root isoflavonoid concentration was strongly localized, there was evidence that NO(3) (-) also resulted in a systemic effect on root isoflavonoids. The results are consistent with previous speculation that internal levels of root isoflavonoids may affect nodule development.     

In vitro isoflavonoid production in callus from different organs of Pueraria lobata (Wild.) Ohwi

Pueraria lobata (Wild.) Ohwi is a medicinal plant producing large amounts of isoflavonoid glycosides. Here, the ability of in vitro callus cultures to synthesize isoflavonoids was tested. Callus cultures have been initiated from different explants of in vitro germinated plants using modified MS medium. Roots, leaves and stem segments were the best sources of callus tissue. The isoflavonoid profile and content was determined by means of chromatographic methods. Callus from all organs contained isoflavonoid aglycones: genistein and daidzein and daidzein glycosides: daidzin, puerarin and 3'-methoxypuerarin. The differences between each kind of explant were observed in both the total amount of isoflavonoids and in the proportion of individual compounds. The highest content was in root callus, followed by leaf- and stem callus.     

Profiling changes in metabolism of isoflavonoids and their conjugates in Lupinus albus treated with biotic elicitor

Liquid chromatography with ultraviolet and mass spectrometric detection was applied to monitor changes in profiles of isoflavonoid glycosides and free isoflavonoid aglycones in Lupinus albus L. Four isoflavonoid aglycones, fourteen isoflavonoid glycosides, four flavonol glycosides and flavone glycoside were identified in lupin tissue after LC/ESI/MS analyses. An elicitor preparation from purified yeast cell wall was used to inject the shoots of 3-week old seedlings or to infiltrate the cut lupin leaves. Qualitative and quantitative changes of isoflavonoids were measured at different time points after elicitation. In elicited lupin seedlings increased amounts of prenylated isoflavone aglycones were identified. The concentrations of glycosidic conjugates of isoflavones present in plant tissue were less affected.     

Pterocarpans from Bituminaria morisiana and Bituminaria bituminosa

The aerial parts of Mediterranean papilionaceous plants Bituminaria morisiana and B. bituminosa afforded, along with known phenolics, the prenylated pterocarpans bitucarpin A and B, whose structure was elucidated by spectroscopic techniques. A known isoflavonoid (8-prenyldaidzein) was also obtained for the first time as a genuine plant constituent. The accumulation of pterocarpans at the expense of biogenetically more primitive shikimate metabolites like furanocoumarins or isoflavonoids supports the inclusion of this plant, once part of the genus Psoralea, into the distinct genus Bituminaria.     

Effect of inoculation and nitrogen on isoflavonoid concentration in wild-type and nodulation-mutant soybean roots

The isoflavones, daidzein and genistein, have been isolated and identified as the major inducers of nod genes of Bradyrhizobium japonicum. The common nod genes of rhizobia are in turn responsible for stimulating root hair curling and cortical root cell division, the earliest steps in the host response. This study evaluated whether there was a relationship between root isoflavonoid production and the hypernodulation phenotype of selected soybean (Glycine max [L.] Merr.) mutants. Three independently selected hypernodulating soybean mutants (NOD1-3, NOD2-4, and NOD3-7) and a nonnodulating mutant (NN5) were compared with the Williams parent for isoflavonoid concentrations. High performance liquid chromatographic analyses of soybean root extracts showed that all lines increased in daidzein, genistein, and coumestrol concentrations throughout the 12-day growth period after transplanting of both inoculated and noninoculated plants; transplanting and inoculation were done 6 days after planting. No significant differences were detected in the concentration of these compounds among the three noninoculated hypernodulating mutants and the Williams parent. In response to inoculation, the three hypernodulating mutants had higher isoflavonoid concentrations than did the Williams control at 9 to 12 days after inoculation when grown at 0 millimolar N level. However, the inoculated nonnodulating mutant also had higher isoflavonoid concentrations than did Williams. N application [urea, (NH₄)₂SO₄ and NO₃⁻] decreased the concentration of all three isoflavonoid compounds in all soybean lines. Application of NO₃⁻ was most inhibitory to isoflavonoid concentrations, and inhibition by NO₃⁻ was concentration dependent. These results are consistent with a conclusion that differential NO₃⁻ inhibition of nodulation may be partially due to changes in isoflavonoid levels, although the similar response of the nonnodulating mutant brings this conclusion into question. Alternatively, the nodulation control in the NN5 mutant may be due to factors totally unrelated to isoflavonoids, leaving open the possibility that isoflavonoids play a role in differential nodulation of lines genetically competent to nodulate.     

Distribution of isoflavonoids in non-leguminous taxa - an update

Common emphasis of the fact that isoflavonoids are characteristic metabolites of leguminous plants sometimes leads to overlooking that the presence of isoflavonoids has been reported in several dozen other families. The spectrum of isoflavonoid producing taxa includes the representatives of four classes of multicellular plants, namely the Bryopsida, the Pinopsida, the Magnoliopsida and the Liliopsida. A review, recently published by Reynaud et al. [Reynaud, J., Guilet D., Terreux R., Lussignol M., Walchshofer N., 2005. Isoflavonoids in non-leguminous families: an update. Nat. Prod. Rep. 22, 504-515], provided listing of 164 isoflavonoids altogether reported in 31 non-leguminous angiosperm families. In this contribution we complement the abovementioned inventory bringing the references on further 17 isoflavonoid producing families and on additional 49 isoflavonoids reported to occur in non-leguminous plants.     

Effects of high-dose soy isoflavones and equol on reproductive tissues in female cynomolgus monkeys

Soy isoflavonoids have well-established estrogenic properties in cell culture and rodent models, raising concerns that high isoflavonoid intake may promote development of uterine and breast cancers. To address this concern we evaluated the effects of high-dose isoflavonoid supplements on reproductive tissues in a postmenopausal primate model. Thirty adult female ovariectomized monkeys (Macaca fascicularis) were randomized to receive a control diet 1) alone, 2) with 509 mg/day of the soy isoflavones genistein and daidzein (IF), or 3) with 1020 mg/day of racemic equol (EQ), an isoflavan, for approximately 1 mo. Doses are expressed in aglycone units as calorically scaled human equivalents. Total serum isoflavonoid levels 4 h postfeeding were <20 nmol/L, 2570.7 nmol/L, and 6944.8 nmol/L for control, IF, and EQ groups, respectively. Equol was the predominant serum isoflavonoid in both IF (72.5%) and EQ (99.7%) groups. Aglycones represented 0.9% (IF) and 0.5% (EQ) of total serum isoflavonoids. Histologically, uteri and mammary glands were diffusely atrophic in all groups. Uterine weight, endometrial thickness, glandular area, and epithelial proliferation in the uterus were not significantly different among treatment groups (ANOVA P > 0.1 for all). Endometrial progesterone receptor gene expression was significantly increased in the IF group (P = 0.02), while protein expression was not altered (ANOVA P > 0.1). Within the mammary gland, proliferation and indicators of estrogen exposure did not differ among treatment groups (ANOVA P > 0.1 for all). These findings indicate that high doses of dietary soy isoflavonoids have minimal uterotrophic or mammotrophic effects in an established primate model.     

Isoflavonoids in the Rutaceae family: 1. Fortunella obovata, Murraya paniculata and four Citrus species

Several types of compounds with immunoreactivity similar to isoflavonoids were detected in water: ethanol extracts of leaves of Fortunella obovata Hort. ex Tanaka, Murraya paniculata Jack. and four Citrus species, namely C. aurantium L, C. grandis Osbeck, C. limonia Osbeck., and C. sinensis Osbeck (Rutaceae). The chromatographic mobilities of the immunoreactive substances were compared with those of authentic standards, revealing a spectrum of isoflavonoid metabolites in all plants studied. Aglycones as well as glycosides were recognized, namely daidzin, genistin, daidzein, genistein, formononetin, biochanin A, prunetin, and several incompletely characterized isoflavonoids. A subsequent HPLC-MS study verified the identities of the main immunoreactive isoflavonoids and established the identities of several others, viz. glycitein, glycitin, ononin and sissotrin, including the malonylated and acetylated isoflavonoid glucosides. The estimated content of the individual immunoreactive entities ranged from a few microg to about 2 mg/kg (dry weight). It is concluded that the isoflavonoid metabolic pathway is present throughout the Rutaceae family.     

An arylbenzofuran and four isoflavonoids from the roots of Erythrina poeppigiana

An arylbenzofuran, erypoegin F and four isoflavonoids, erypoegins G-J, together with six known compounds were isolated from the roots of Erythrina poeppigiana, and their structures were elucidated on the basis of spectroscopic evidence. Erypoegin F is a rare 2-arylbenzofuran possessing a formyl group from a natural source, and erypoegin I is the first naturally occurring isoflavonoid with a 2-oxo-3-methylbutyl group.     

Elicitor-Induced Association of Isoflavone O-Methyltransferase with Endomembranes Prevents the Formation and 7-O-Methylation of Daidzein during Isoflavonoid Phytoalexin Biosynthesis

The bioactive isoflavonoids of the Leguminosae often are methylated on the 4'-position of their B-rings. Paradoxically, reverse genetic evidence implicates alfalfa isoflavone O-methyltransferase (IOMT) in the biosynthesis of 4'-O-methylated isoflavonoids such as the phytoalexin medicarpin in vivo, whereas biochemical studies indicate that IOMT has strict specificity for methylation of the A-ring 7-hydroxyl of daidzein, the presumed substrate for O-methylation, in vitro. Radiolabeling and isotope dilution studies now confirm that daidzein is not an intermediate in isoflavonoid phytoalexin biosynthesis in alfalfa. Furthermore, protein gel blot analysis and confocal microscopy of a transiently expressed IOMT-green fluorescent protein fusion in alfalfa leaves show that the operationally soluble IOMT localizes to endomembranes after elicitation of the isoflavonoid pathway. We propose that IOMT colocalizes with the endoplasmic reticulum-associated isoflavone synthase cytochrome P450 to ensure rapid B-ring methylation of the unstable 2,4',7-trihydroxyisoflavanone product of isoflavone synthase, thereby preventing its dehydration to daidzein and subsequent A-ring methylation by free IOMT. In this way, metabolic channeling at the entry point into isoflavonoid phytoalexin biosynthesis protects an unstable intermediate from an unproductive metabolic conversion.     

Partial reconstruction of flavonoid and isoflavonoid biosynthesis in yeast using soybean type I and type II chalcone isomerases

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4′-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3β-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.     

Improved isoflavonoid production in Pueraria candollei hairy root cultures using elicitation

The effect of abiotic and biotic elicitors (methyl jasmonate, chitosan, salicylic acid, Agrobacterium, and yeast extract) at various concentrations on total isoflavonoid accumulation was studied in the hairy root cultures of Pueraria candollei. All elicitors stimulated isoflavonoid production. Yeast extract (0.5 mg/ml) was the most efficient giving total isoflavonoids at 60 ± 1 mg/g dry wt, which was 4.5-fold higher than control hairy roots on day 3 of elicitation.     

HPLC analysis of white lupin isoflavonoids

An investigation of the HPLC analytical conditions for simple isoflavones, prenylated isoflavones and some of their glucosyl derivatives resulted in reasonable separation and total elution in 35 min when using a reversed-phase C18 Lichrospher column and a gradient elution system of MeCN-THF-H2O. This method was successfully applied to quantify the changes in isoflavonoid constituents in white lupin (Lupinus albus L.) tissues: (a) young legumes (pods and seeds) during maturation, and (b) soaked, germinating seeds. In developing legumes, genistein and 2'-hydroxygenistein, as well as their prenylated derivatives, were present in the pods as the major components, together with minor amounts of glucosides, whereas only minute amounts of isoflavonoids were detectable in the ripening seeds. When soaked with water, mature lupin seeds which normally contain trace amounts of isoflavonoids, started rapidly to biosynthesize simple isoflavones and accumulate large amounts of genistein 7-O-glucoside and its 6"-O-malonyl derivative. These dynamic changes are discussed in relation to the role of isoflavonoids in the lupin defense system.     

Antibacterial properties of a new isoflavonoid from Erythrina poeppigiana against methicillin-resistant Staphylococcus aureus.

A new isoflavonoid, together with four known isoflavonoids, was isolated from the roots of Erythrina poeppigiana. The chemical structure was determined by extensive spectroscopic studies, and then its antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) was investigated. The new isoflavonoid was identified as 3,9-dihyroxy-10-gamma,gamma-dimethylallyl-6a,11a-dehydropterocarpan (compound 1). Compound 1 inhibited bacterial growth most potently of the five isolates, and had a minimum inhibitory concentration (MIC) of 125 microg/ml against thirteen MRSA strains. Inhibitory activity was based on bactericidal action and viable cell number reduced by approximately 1/10,000 after 4 h incubation with compound 1. Despite intense bactericidal action against MRSA, compound 1 never resulted in leakage of 260 nm-absorbing substances from bacterial cells. Compound 1 (12.5 microg/ml) completely inhibited incorporation of radio-labeled thymidine, uridine and leucine into MRSA cells. Although glucose incorporation was also markedly inhibited by the compound, the amount of glucose incorporated by bacterial cells increased gradually with incubation time. These findings suggest that compound 1 exhibits anti-MRSA activity by interfering with incorporation of metabolites and nutrients into bacterial cells or by affecting the nucleic acids of MRSA cells. Furthermore, this new compound could be a potent phytotherapeutic agent for treating MRSA infections.