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.     

The effect of UV light on isoflavonoid production in G<Emphasis Type="Italic">enista tinctoria</Emphasis> culture in vitro

The effect of UV radiation (254 and 366 nm) on isoflavonoid production in Genista tinctoria callus cultures was investigated. The production of the separated isoflavonoids in callus culture of G. inctoria was changed in the dependence on wavelength of UV treatment. Genistin, genistein, daidzein, biochanin A in higher levels were formed after UV irradiation in callus culture in comparison with untreated callus culture. Maximal genistin content (3.03%) was reached after treatment of UV 254 nm for 300 s (sampled after 48 h). High genistin content (2.06%) was observed also after 120 s of UV 366 nm radiation (sampled after 48 h). Formononetin was detected only after UV treatment in G. tinctoria callus culture.     

Isoflavonoids production in callus culture of Pueraria tuberosa, the Indian kudzu

Isoflavonoid contents of different plant parts and callus tissues of the Indian Kudzu, Pueraria tuberosa (Roxb.ex.Willd.) DC are presented. The initial cultures were slow growing, associated with browning of the tissues. The production of four isoflavonoids (puerarin, genistin, genistein and daidzein) in the callus cultures of P. tuberosa was studied by manipulating the plant growth regulators and sucrose concentration in the medium. Organogenesis was not recorded in callus on any of these treatments. Tuber and stem accumulated puerarin, a glycoside of daidzein, at high amounts, 0.65% and 0.054% respectively. However, the daidzein content of the callus tissues grown on Murashige and Skoog medium containing BA (20.9 microM) and sucrose (60 gl(-1)) was significantly higher (0.056%) than in vivo plant material (0.02%) and other comparable culture systems like Genista and Pueraria lobata.     

Metabolism of daidzein by intestinal bacteria from rhesus monkeys (Macaca mulatta)

PURPOSE: To identify the metabolites produced from an isoflavonoid, daidzein, by colonic bacteria of rhesus monkeys. METHODS: The metabolism of daidzein by the fecal bacteria of nine monkeys was investigated. Daidzein was incubated anaerobically with fecal bacteria, and the metabolites were analyzed by use of liquid chromatography and mass spectrometry. RESULTS: The fecal bacteria of all of the monkeys metabolized daidzein to various extents. Dihydrodaidzein was found in cultures of fecal bacteria from two monkeys; dihydrodaidzein and equol were found in cultures from four monkeys; dihydrodaidzein, equol, and an unknown metabolite (MW = 244) were found in cultures from one monkey; and dihydrodaidzein and the unknown metabolite were found in cultures from two monkeys. CONCLUSIONS: Similar to that in humans, variation was evident in the metabolism of isoflavonoids by fecal bacteria from rhesus monkeys. Some metabolites produced by fecal bacteria from monkeys were the same as those produced by fecal bacteria from humans.     

Growth and isoflavonoid accumulation of Pueraria candollei var. candollei and P. candollei var. mirifica cell suspension cultures

We established cell suspension cultures derived from leaf, stem, and root calli of Pueraria candollei var. candollei and P. candollei var. mirifica using liquid Murashige and Skoog (MS) medium supplemented with 0.56 μM 6-benzyladenine (BA) and 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Growth of the cell suspension cultures progressed to the stationary phase within 15–24 days. Methanolic extracts of cell suspension cultures of both varieties of P. candollei were analyzed using a validated HPLC protocol. All cell lines derived from leaf, stem, and root explants produced four major isoflavonoids: daidzein, daidzin, genistein, and genistin; these isoflavonoids were detected only in the roots of intact plants. Furthermore, the isoflavonoid contents of the cell suspension cultures were higher than those of intact plants. Thus, cell suspension culture of both varieties of P. candollei may be an effective tool for isoflavonoid production.     

Production of daidzein by callus cultures of Psoralea species and comparison with plants

Callus cultures were established from five Psoralea species (Leguminosae) with the objective of producing daidzein (isoflavone). The biomass doubling times ranged from 7 to 16 days according to the species and a 48 weeks period was necessary to obtain lines with stable growth characteristics. All the 217 callus lines were analyzed for their daidzein content using HPLC. Our callus collection showed a large interspecific variation and the highest concentrations were recovered in P. obtusifolia callus lines (maximum of 0.9680% DW). Intraspecific variation was also important and allowed the recovery of high-producing lines (production exceeding 0.3000% DW) with four out of the five Psoralea species studied. The daidzein repartition was investigated in planta with P. cinerea in order to evaluate the potential of in vivo production. Mature fruits were the richest organs for daidzein concentration in P. cinerea and were used as indicators to evaluate the possible production with the other four plant species. In vitro concentrations were always much higher than in planta, and no correlation could be established between the calluses and plants for the five species. Our callus lines contained concentrations comparable to Psoralea hairy root lines. They can be considered as an interesting material to further study the production of daidzein. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tissue culture in synthetic atmospheres: diffusion rate effects on cytokinin-induced callus growth and isoflavonoid production in soybean [Glycine max (L.) Merr. cv. Acme]

Concentration is one factor that is known to determine how metabolic gases influence the growth and secondary metabolism of plant tissues in culture. How actual gas bioavailability influences these processes has not been studied despite its potential importance in specialized applications. A simple model system, soybean [Glycine max (L.) Merr. cv. Acme] callus culture, was selected for experiments because exogenous cytokinin (6-benzylaminopurine; BAP) elicits two types of responses: (1) enhanced callus proliferation, and (2) rapid induction of the isoflavonoid daidzein (7,4′-dihydroxyisoflavone). Synthetic atmospheres supplying metabolic gases with higher or lower bioavailability than in air were created by replacing the nitrogen moiety in standard air with either helium or argon, respectively. Callus was cultured on agar or in liquid shake cultures according to standard procedures. At an optimal cytokinin concentration for stimulation of callus proliferation, 4.4 × 10⁻⁷ M BAP, increased diffusion rates for the metabolic gases resulted in greater weight gain in agar cultures. Weight gain was 11% higher for He-treated and 39% lower for Ar-treated cultures than for the nitrogen control. In contrast, there was no significant effect of metabolic gas diffusion rate on daidzein production in either agar or liquid cultures. Apart from the potential application of these synthetic atmospheres for enhancing plant tissue culture growth, they may have unique value for the space program as an effective way of replicating the gas exchange limitations posed for plants by microgravity (Ar atmosphere), and as a countermeasure for this limitation (He atmosphere).     

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.     

Variation in nicotine content of tobacco callus cultures

Plants ofNicotiana tabacum L. cv. Burley 21 which showed no difference in nicotine content were used to establish callus cultures. Cultures were initiated from different plants and from different leaves within each plant. The nicotine content of the calli was determined, and the results subjected to an analysis of variance. Differences between plants and differences within plants significantly affected the nicotine content of the cultures. The differences between plants were transmitted sexually and asexually, providing evidence that they are genetically determined. No such differences in nicotine content were found between the plants from which the cultures were established, indicating that nicotine production in vitro involves additional genes to those which are needed for nicotine production in the plant. The differences within plants were further investigated by establishing callus cultures from pith explants taken from different parts of the stem. Explants from apical pith tissue gave calli having far more nicotine and more roots than cultures derived from basal pith explants. This results may reflect the proximity of the apical pith explants to the site of auxin synthesis in the stem apex. Callus cultures derived from pith explants showed greater growth and nicotine production than those derived from leaf explants when the calli were induced on Murashige-Skoog medium containing -naphthalene acetic acid. This result is in conflict with the widely held belief that explants from different parts of the plant give cultures with similar yields of species-specific compounds.Abbreviations HN High nicotine - LN low nicotine     

Effects of Agrobacterium rhizogenes strains and other parameters on production of isoflavonoids in hairy roots of Pueraria candollei Grah. ex Benth. var. candollei

Using several explants of Pueraria candollei Grah. ex Benth. var. candollei and two strains of Agrobacterium rhizogenes (ATCC 15834 and 43057), hairy root cultures were established. Including 100???M acetosyringone in the culture medium enhanced frequency of hairy root induction by up to 58?%. Subsequently, effects of inoculum size (IS) and temperature on growth and production of isoflavonoids in hairy roots were determined. Conditions of 1?%?IS and 32?°C promoted the highest accumulation of total isoflavonoid content, up to 31.0?±?22.6?mg/g dry weight (DW), in hairy roots. Moreover, culture of hairy roots at 32?°C decreased browning of hairy roots. Furthermore, this temperature promoted accumulation of the secondary metabolite daidzein; whereas, hairy root cultures at the stationary phase accumulated higher amounts of the isoflavonoid puerarin rather than daidzein.     

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.     

Occurrence of iridoid glycosides in in vitro cultures and intact plants of <Emphasis Type="Italic">Scrophularia nodosa</Emphasis> L.

Shoot, root, and callus cultures of Scrophularia nodosa L. (Scrophulariaceae) were established and cultivated in vitro. Iridoid glycosides, such as harpagoside, aucubin, and catalpol were identified by LC-ESI-MS and their contents determined by HPLC. For comparison intact plants of S. nodosa were analysed. In shoot cultures slightly lower amounts of detectable iridoid glycosides (4.36% dry weight) were determined than in the field grown plants (4.88%). Concentration of harpagoside was highest in leaves of field plants (1.05%) and in flowers of in vitro plantlets (1.10%). For aucubin the highest amount was found in the leaves of in vitro plantlets (1.67%) whereas the levels of aucubin in the leaves of field plants were remarkably lower. Catalpol was produced as a trace compound in intact plants and shoot cultures. Callus and root cultures were apparently not able to synthesise iridoid glycosides.     

Isoflavones in the Rutaceae family: twenty selected representatives of the genera Citrus, Fortunella, Poncirus, Ruta and Severinia

Enzyme-linked immunosorbent assays in combination with semi-preparative high-performance liquid chromatography (HPLC) and analytical HPLC with mass spectroscopy in the selective ion monitoring mode were used for the determination of selected isoflavones, daidzein, genistein, biochanin A and their homologues, in 20 representatives of the Rutaceae family. Species belonging to five genera were studied, namely Citrus, Fortunella, Poncirus, Ruta and Severinia. The enzyme immunoassays used were based on polyclonal antibodies raised against isoflavonoid conjugates with bovine serum albumin (BSA), namely biochanin A-7-BSA, daidzein-7-BSA, daidzein-4'-BSA, genistein-7-BSA and genistein-4'-BSA. Aglycones as well as glycosides were detected, and methoxyisoflavones appeared to be more abundant than hydoxyisoflavones. The content of individual isoflavonoids ranged from 0 to 2.6 mg/kg (dry weight); the sum of all measured substances reached up to 5.9 mg.     

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.     

Effect of spaceflight on isoflavonoid accumulation in etiolated soybean seedlings

In order to explore the potential impact of microgravity on flavonoid biosynthesis, we examined isoflavonoid levels in soybean (Glycine max) tissues generated under both spaceflight and clinorotation conditions. A 6-day Space Shuttle-based microgravity exposure resulted in enhanced accumulation of isoflavone glycosides (daidzin, 6"-O-malonyl-7-O-glucosyl daidzein, genistin, 6"-O-malonyl-7-O-glucosyl genistein) in hypocotyl and root tissues, but reduced levels in cotyledons (relative to 1g controls on Earth). Soybean seedlings grown on a horizontally rotating clinostat for 3, 4 and 5 days exhibited (relative to a vertical clinorotation control) an isoflavonoid accumulation pattern similar to the space-grown tissues. Elevated isoflavonoid levels attributable to the clinorotation treatment were transient, with the greatest increase observed in the three-day-treated tissues and smaller increases in the four- and five-day-treated tissues. Differences between stresses presented by spaceflight and clinorotation and the resulting biochemical adaptations are discussed, as is whether the increase in isoflavonoid concentrations were due to differential rates of development under the "gravity" treatments employed. Results suggest that spaceflight exposure does not impair isoflavonoid accumulation in developing soybean tissues and that isoflavonoids respond positively to microgravity as a biochemical strategy of adaptation.     

Flavonol glycoside production in callus cultures of Epimedium diphyllum.

Callus cultures of Epimedium diphyllum produced a large amount of epimedoside A in addition to a small amount of diphylloside B, ikarisoside C, epimedoside E, diglycosides of des-O-methylanhydroicaritin (8-gamma, gamma-dimethylallylkaempfero). Icariin, epimedins A-C, which are glycosides of anhydroicaritin, were also produced in the callus cultures. Contents of the flavonol glycosides in callus tissue were higher than those of mother plants, but the composition of each flavonol glycoside mixture in the callus cultures was different from that of the original plants. The time-course experiments showed that an inverse relationship existed between cell growth and flavonol glycoside production. Effects of hormonal factors on cell growth and flavonol glycoside production indicated that 2,4-dichlorophenoxyacetic acid was needed for the production of flavonol glycosides.     

Callus cultures and bark from Norway spruce clones show similar cellular features and relative resistance to fungal pathogens

In field experiments, clones of Norway spruce [Picea abies (L.) Karst.] showed different degrees of resistance against pathogenic fungi inoculated into the bark that correlate with differences in polyphenolic parenchyma (PP) cells of the bark. Cells of spruce callus cultures, particularly towards the callus surface, resemble PP cells and this study looks at changes in callus cells during infection and the relative resistance of cultures from clones of low (weak) or high (strong) resistance to fungal infection. Callus cultures, initiated from trees with different resistance, were co-inoculated with Ceratocystis polonica (Siem.) C. Moreau and Heterobasidion annosum (Fr.) Bref. Callus cells from strong clones resemble PP cells of bark tissue from strong clones, having more polyphenolic bodies, while callus cells from weak clones are more similar to PP cells from those clones, which have less extensive phenolic bodies. Callus cultures from trees with weak resistance were more quickly overgrown by both species of pathogenic fungi than cultures from trees with strong resistance. Callus cells of infected cultures showed changes similar to activated PP cells of bark, including enhanced accumulation of polyphenolics. Phenolic bodies were more numerous and more extensive (larger and denser) in callus cells of strong versus weak clones under all conditions. Thus, callus cells may perform similar functions in defense as PP cells in the bark. Callus from trees of varying resistance seem to reflect the relative resistance of the trees from which they are derived, and this study indicates that some mechanisms of resistance can be studied using callus from trees of different resistance.     

Morphogenesis and tissue cultures of three citrus species

Studies were performed to define tissue culture techniques and culture conditions for morphogenesis, callus culture and plantlet culture of sweet orange (Citrus sinensis (L.) Osb.), citron (C. medica L.) and lime (C. aurantifolia) (Christm. Swing). The optimal concentrations of NAA to induce root formation on stem segments were 10 mg l^-1 for sweet orange and lime, and 3 mg l^-1 for citron. The optimal BA concentration for shoot and bud proliferation was 3 mg l^-1 for sweet orange and citron, and 1 mg l^-1 for lime. Callus initiation was accomplished in a culture medium containing 10 mg l^-1 NAA and 0.25 mg l^-1 BA. Callus was maintained by periodical subculture into the same medium supplemented with 10% (v:v) organge juice. In vitro plantlets of the three species were obtained by rooting of shoots developed from bud cultures, and of citron and lime by development of shoots from root cultures. The plants were successfully established on soil.     

Ultraviolet irradiation induces accumulation of isoflavonoids and transcription of genes of enzymes involved in the calycosin-7-O-β-d-glucoside pathway in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao

Isoflavonoids are a group of phenolic secondary metabolites found almost exclusively in leguminous plants. Formononetin, calycosin and calycosin-7-O-β-d-glucoside (CG) are isoflavonoid products in the CG pathway. Accumulation of the three isoflavonoids plus daidzein and expression of six genes of enzymes involved in the CG pathway were studied in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao with ultraviolet (UV) irradiation. Our results showed that (1) main isoflavonoids in roots, stems and leaves were CG, daidzein and calycosin, respectively; they accumulated significantly under the induction of UV irradiation during 8 days but their content declined later; (2) expression of six genes of enzymes involved in the CG pathway was inhibited slightly at early stage but the expression was increased greatly afterward; (3) chalcone synthase, chalcone reductase and chalcone isomerase were expressed to their individual maximum level within shorter hours than were cinnamate 4-hydroxylase, isoflavone synthase (IFS) and isoflavone 3'-hydroxylase and (4) more calycosin but less daidzein accumulated in leaves. IFS was highly expressed in leaves, which might lead to high accumulation of the common precursor of daidzein and 2,7-dihydroxy-4'-O-methoxy-isoflavanone, the latter of which would be converted to formononetin, calycosin and CG via a series of reactions. Little daidzein accumulated in leaves, which suggested that rather than be converted to daidzein, the 2,7,4'-trihydroxyisoflavanone was probably more easily caught by 2-hydroxyisoflavanone 4'-O-methyltransferase and hence provided more precursors for formononetin. The findings were discussed in terms of the influence of UV irradiation in the accumulation of isoflavonoids.