Differential divergences of obligately insect-pathogenic Entomophthora species from fly and aphid hosts

Cecal microbiota of chicken was screened for bacteria involved in the biotransformation of isoflavones. A new facultative anaerobic bacterium, capable of deglycosylation of the isoflavone genistin, was isolated and identified as a Lactobacillus delbrueckii -like strain. The isolate MF-07 was Gram-positive, facultatively anaerobic, catalase negative, non-spore-forming, nonmotile and a straight rod. The polyphasic taxonomic data, along with 16S rRNA gene sequence comparison, demonstrated that the isolate MF-07 was most closely related to L. delbrueckii group of the Lactobacillus genus. Considerable amounts of genistein were accumulated with genistin as a substrate within the first 12 h of fermentation. Formononetin and daidzein were not metabolized. The influence of several carbon sources on the growth of the isolate MF-07 and biotransformation of genistin was also investigated. This is the first study in which an anaerobic Lactobacillus bacterium from the chicken intestinal tract that metabolizes genistin to produce its bioactive metabolite was identified and characterized.     

Oral administration of soy-derived genistin suppresses lipopolysaccharide-induced acute liver inflammation but does not induce thymic atrophy in the rat

Genistein, the principal isoflavone present in soy, has been identified as a protein tyrosine kinase (PTK) inhibitor that has in vitro anti-inflammatory effects. Whether genistein has in vivo anti-inflammatory effects remains unknown yet. Injecting or feeding rats with the unconjugated form of genistein (aglycone) results in decreased thymic weight and lymphocytopenia. However, 95-99% of genistein is present as the conjugated form genistin (genistein glycoside) in soy or soy-derived products. This study was undertaken to reveal whether genistin, as well as genistein, has anti-inflammatory effects in vivo. After oral administration of equimolar genistein (namely 7.4 or 74 micromol/dose) at daily doses of 2.0 or 20 mg/kg, or genistin at daily doses of 3.2 or 32 mg/kg for 3 days to male rats, both aglycone and glycoside suppressed the production of lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, and IL-6 in both from the liver and in the sera. Aglycone induced thymic atrophy while glycoside did not. In vitro preincubation of liver slices from na?ve rat with genistein aglycone or glycoside suppressed LPS-induced TNF-alpha production in a dose-dependent manner. Taken together, both in vivo and in vitro administration of genistin and genistein suppressed LPS-induced liver pro-inflammatory cytokine production. However, equimolar oral administration of genistin did not induce thymus atrophy. Further investigation in long-term isoflavone intake is required especially among neonates. The results suggest that the safety evaluation of the consumption of isoflavone should be based on isoflavone glycoside but not aglycone.     

Anabolic effect of genistein and genistin on bone metabolism in the femoral-metaphyseal tissues of elderly rats: The genistein effect is enhanced by zinc

The effect of genistein and genistin on bone components in the femoral-metaphyseal tissues obtained from elderly female rats was investigated in vitro. The metaphyseal tissues were cultured for 24 h in a medium containing either vehicle, genistein (10^-8-10^-5 M) or genistin (10^-7-10^-5 M). The presence of genistein or genistin caused a significant increase in alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in the metaphyseal tissues. The effect of genistein was greater than that of genistin. The bone components increased by genistein (10^-5 M) or genistin (10^-5 M) were completely blocked by the presence of cycloheximide (10^-6 M). The presence of zinc sulfate (10^-5 M) caused a significant increase in the genistein (10^-5 M)-elevated alkaline phosphatase activity, DNA and calcium contents. The enhancement with zinc was not seen by genistin (10^-5 M). The stimulatory effect of zinc on the genistein-induced increase in bone components of the metaphyseal tissues was completely blocked by the presence of cycloheximide (10^-6 M). The present results suggest that genistein and genistin have an anabolic effect on bone metabolism in the femoral-metaphyseal tissues of elderly rats, and that the genistein effect is enhanced by zinc, an essential trace element.     

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.     

Pro-apoptotic effect and cytotoxicity of genistein and genistin in human ovarian cancer SK-OV-3 cells

We investigated the effects of genistein and genistin on proliferation and apoptosis of human ovarian SK-OV-3 cells and explored the mechanism for these effects. SK-OV-3 cells were treated with genistein and genistin at various concentrations (ranging from 1 to 100 muM) either alone or in combination for 24 and 48 h. Cell proliferation was estimated using an MTT assay, and cell cycle arrest was evaluated using FACS. Caspase-3 activity and annexin-based cell cycle analysis were used as measures of apoptosis. In addition, genistein- and genistin-induced cytotoxicity was determined by measuring release of LDH. Genistein treatment for 24 or 48 h substantially inhibited SK-OV-3 cell proliferation in a dose-dependent manner, and genistin treatment for 48 h also inhibited cell proliferation. Genistein caused cell cycle arrest at G2/M phase in dose- and time-dependent manner, and genistin caused cell cycle arrest not only at G2/M phase but also at G1 phase. Genistein markedly induced apoptosis and significantly increased LDH release, whereas genistin did not affect LDH release. Moreover, exposure to both genistein and genistin in combination for 48 h induced apoptosis without increasing LDH release. Genistein and genistin inhibit cell proliferation by disrupting the cell cycle, which is strongly associated with the arrest induction of either G1 or G2/M phase and may induce apoptosis. Based on our findings, we speculate that both genistein and genistin may prove useful as anticancer drugs and that the combination of genistein and genistin may have further anticancer activity.     

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.     

Effect of protein tyrosine kinase inhibitors on the current through the Ca(V)3.1 channel

In the present study, we have investigated the effects of protein tyrosine kinase (PTK) inhibitors on the Ca(V)3.1 calcium channel stably transfected in HEK293 cells using the whole-cell configuration of the patch-clamp technique. We have tested two different tyrosine kinase inhibitors, genistein and tyrphostin AG213, and their inactive analogs, genistin and tyrphostin AG9. Bath application of genistein, but not genistin, decreased the T-type calcium current amplitude in a concentration-dependent manner with an IC(50) of 24.7+/-2.0 microM. This effect of genistein was accompanied by deceleration of channel activation and acceleration of channel inactivation. Intracellular application of neither genistein nor genistin had a significant effect on the calcium current. Extracellular application of 50 microM tyrphostin AG213 and its inactive analogue, tyrphostin AG9, did not affect the current through the Ca(V)3.1 channel. The effect of genistein on the channel was also not affected by the presence of catalytically active PTK, p60(c-src) inside the cell. We have concluded that genistein directly inhibited the channel. This mechanism does not involve a PTK-dependent pathway. The alteration of the channel kinetics by genistein suggests an interaction with the voltage sensor of the channel together with the channel pore occlusion.     

A process for high-efficiency isoflavone deglycosylation using Bacillus subtilis natto NTU-18

In order to produce isoflavone aglycosides effectively, a process of isoflavone hydrolysis by Bacillus subtilis natto NTU-18 (BCRC 80390) was established. This process integrates the three stages for the production of isoflavone aglycosides in one single fermenter, including the growth of B. subtilis natto, production of β-glucosidase, deglycosylation of fed isoflavone glycosides. After 8 h of batch culture of B. subtilis natto NTU-18 in 2 L of soy medium, a total of 3 L of soy isoflavone glucoside solution containing 3.0 mg/mL of daidzin and 1.0 mg/mL of genistin was fed continuously over 34 h. The percentage deglycosylation of daidzin and genistin was 97.7% and 94.6%, respectively. The concentration of daidzein and genistein in the broth reached 1,066.8 μg/mL (4.2 mM) and 351 μg/mL (1.3 mM), respectively, and no residual daidzin or genistin was detected. The productivity of the bioconversion of daidzein and genistein over the 42 h of culture was 25.6 mg/L/h and 8.5 mg/L/h, respectively. This showed that this is an efficient bioconversion process for selective estrogen receptor modulator production.     

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.     

Sensitive high-performance liquid chromatographic method for profiling phytoestrogens using coulometric electrode array detection: application to plasma analysis.

An HPLC method for profiling 13 phytoestrogens and their metabolites using coulometric electrode array detection was developed. Sensitivity of the method was slightly less than that of our GC-MS method, but significantly higher compared to the HPLC methods using diode-array or UV detection. Detection limits varied from 3.4 (secoisolariciresinol) to 40.3 (genistin) pg on column. Signal linearities ranged from the detection limits to 61 ng on column. Resolution values for the peak pairs varied from 1.1 (O-desmethylangolensin-anhydrosecoisolariciresinol) to 16 (daidzin-genistin). Intra- and interassay retention time variations were negligible and detector response variation was eliminated by frequent calibration. Chromatographic method was applied to plasma analyses and 6 of the 13 compounds were detected. Method accuracy for those six analytes varied from 69% (enterodiol) to 118% (genistein). Intraassay precision CVs ranged from 1.5% (enterolactone, 12.4 nmol/liter) to 14% (genistein, 245 nmol/liter) and interassay precision CVs ranged from 9.9% (daidzein, 67.4 nmol/liter) to 44% (enterodiol, 1.20 nmol/liter).     

Isoflavonoids in root and hypocotyl of soybean seedlings (Glycine max,Fabaceae)

Isoflavonoids, some of which are highly fluorescent, are produced by soybean [Glycine max (L.) Merr.] and serve as chemical signals for certain aspects of nitrogen fixation and microbial resistance. This study was conducted to determine whether soybean mutants with nonfluorescent roots contained abnormal concentrations of isoflavonoids. Isoflavonoids were extracted from the root and hypocotyl of 4-d-old wild-type soybean seedlings (cv. Hark) having fluorescent roots and from four nonallelic mutant, near isogenic lines of Hark having nonfluorescent roots. In addition, isoflavonoids were extracted from the root and hypocotyl of 4-d-old seedlings of near isogenic lines of Hark harboring two pairs of the mutant alleles for nonfluorescent roots. Malonyl daidzin, daidzin, malonyl genistin, and genistein were the most abundant isoflavonoids extracted from either the root or hypocotyl of seedlings with either fluorescent or nonfluorescent roots. Extracted malonyl daidzin, malonyl genistin, and malonyl glycitin decomposed readily, yielding daidzin, genistin, and glycitin, respectively. The concentrations of malonyl genistin and genistein, two highly fluorescent compounds, were similar in both fluorescent and nonfluorescent roots. Thus, root fluorescence was not correlated with abundance per se of fluorescent isoflavonoids in roots. In addition, the abundance of isoflavonoids extractable from the hypocotyl did not correlate with root fluorescence.     

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.     

Genistin inhibits UV light-induced plasmid DNA damage and cell growth in human melanoma cells

In recent years, genistein has received considerable attention because epidemiologic studies showed that consumption of soybean-containing diets was associated with a lower incidence of certain human cancers in Asian populations. In vitro studies further showed that such chemopreventive and antineoplastic effects were associated with the antioxidant activity of genistein and inhibitor activities on cell proliferation and angiogenesis. Genistein was shown to arrest the growth of malignant melanoma in vitro and to inhibit ultraviolet (UV) light-induced oxidative DNA damage. Recently, it has been demonstrated that genistin, as other flavonoid glycosides, is partly absorbed without previous cleavage and does not have to be hydrolyzed to be biologically active. Therefore, not only isoflavone aglycons, but also glycosides can be of physiological relevance. In the present study, we evaluated in cell-free systems the effect of genistin and daidzin on pBR322 DNA cleavage induced by hydroxyl radicals, generated from UV photolysis of hydrogen peroxide, and their superoxide anion scavenging capacity. In addition, we investigated the growth inhibitory activity of these isoflavones against human melanoma cell line (M14). Under our experimental conditions, genistin and daidzin showed a protective effect on DNA damage and exhibited a superoxide dismutase-like effect, but only genistin was able to reduce significantly the vitality of M14 cells, confirming the importance of the 5,7-dihydroxy structure in the A ring. These results suggest that also genistin, due to its antioxidant and anticarcinogenic properties, contributes to the overall biological activity of soy and could have promising applications in the field of dermatology.     

Sustainable production of genistin from glycerol by constructing and optimizing Escherichia coli

Genistin is one of the bioactive isoflavone glucosides found in legumes, which have great nutraceutical and pharmaceutical significance. The market available isoflavones are currently produced by direct plant extraction. However, its low abundance in plant and structural complexity hinders access to this phytopharmaceutical via plant extraction or chemical synthesis. Here, the E. coli cell factory for sustainable production of genistin from glycerol was constructed. First, we rebuilt the precursor genistein biosynthesis pathway in E. coli, and its titer was then increased by 668% by identifying rate-limiting steps and applying an artificial protein scaffold system. Then de novo production of genistin from glycerol was achieved by functional screening and introduction of glycosyl-transferases, UDP-glucose pathway and specific genistin efflux pumps, and 48.1 mg/L of genistin was obtained. A further engineered E. coli strain equipped with an improved malonyl-CoA pathway, alternative glycerol-utilization pathways, acetyl-CoA carboxylase (ACC), and CRISPR interference (CRISPRi) mediated regulation produced up to 137.8 mg/L of genistin in shake flask cultures. Finally, 202.7 mg/L genistin was achieved through fed-batch fermentation in a 3-L bioreactor. This study represents the de novo genistin production from glycerol for the first time and will lay the foundation for low-cost microbial production of glucoside isoflavones. In addition, the multiphase workflow may provide a reference for engineering the biosynthetic pathways in other microbial hosts as well, for green manufacturing of complex natural products.     

In vivo response of neutrophils and epithelial cells to lipopolysaccharide injected into the monkey ileum

Since few previous studies have investigated the in vivo response of intestinal mucosa to the luminally administered lipopolysaccharide (LPS), we examined the cellular localization of exogenously applied LPS in the intestinal mucosa and the expression of Toll-like receptor (TLR) and IL-1 receptor-associated kinase (IRAK) in the epithelial cells of monkey ileum. FITC-labeled LPS was injected into the lumen of monkey ileum. Thirty minutes after the LPS injection, the ileal tissue was fixed and localization of FITC fluorescence in the ileal mucosa was examined. We applied Factor C immunohistochemistry to demonstrate the bioactivity of LPS taken up by the mucosal tissue. The expression of TLR4 and IRAK-1 in the epithelial cells was also examined by immunohistochemistry. FITC fluorescence was detected in the cells migrated into the epithelium and those in the lamina propria. The FITC-labeling cells were completely overlapped with the Factor C immunoreactive cells. These FITC-labeling/Factor C-positive cells were identified as neutrophils by the immunoelectron microscopic analysis. TLR4 and IRAK-1 were expressed at the apical membrane of the epithelial cells in the ileum of both control and FITC-LPS injected animals. These results suggest that intraluminal injection of LPS stimulates the transmigration of neutrophils into the epithelium and these neutrophils may uptake luminally applied LPS and possibly inactivate the enterotoxin. Expression of TLR4 and IRAK-1 in the epithelial cells suggests that epithelial cells may react to LPS and produce chemoattractant mediator to induce the neutrophil chemotaxis.     

Enhanced bioavailability of soy isoflavones by complexation with beta-cyclodextrin in rats

In order to improve the solubility and bioavailability of a soy isoflavone extract (IFE), inclusion complexes (IFE-beta-CD) of the isoflavone extract with beta-cyclodextrin (beta-CD) were prepared and studied for their solubility and bioavailability. The aqueous solubility of the complexes of IFE with beta-CD (2.0 mg/ml) was about 26 times that of IFE itself (0.076 mg/ml). The same dosages of IFE and IFE-beta-CD were orally administered to SD rats (Sprague-Dawley) on an isoflavone glycoside (IFG) basis (daidzin, genistin and glycitin), and the plasma concentrations of daidzein, genistein and glycitein were measured over time to estimate the average AUC (area under the plasma concentration versus time curve) of the isoflavones. After the oral administration, the AUC values for daidzein, genistein and glycitein were 340, 11 and 28 microg x min/ml, respectively. In contrast, the respective AUC values after the administration of IFE-beta-CD were 430, 20 and 48 microg x min/ml. The bioavailability of daidzein in IFE-beta-CD was increased to 126% by the formation of inclusion complexes with beta-CD, compared with that in IFE. Furthermore, the bioavailability of genistein and glycitein in IFE-beta-CD formulation was significantly higher by up to 180% and 170%, respectively, compared with that of IFE p=0.008 and p=0.028, respectively). These results show that the absorption of IFE could be improved by the complexation of IFE with beta-CD (IFE-beta-CD).     

The formation of glucosides of isoflavones and of some other phenols by rabbit liver microsomal fractions

1. Rabbit liver microsomal fractions in vitro effected the transfer of glucuronic acid from UDP-glucuronic acid to biochanin A, formononetin, daidzein, genistein and equol. Only monoglucuronides were formed. 2. The same isoflavones were converted into monoglucosides when UDP-[6-(3)H]glucose was substituted for UDP-glucuronic acid in the incubation medium in vitro. The glucosides were formed in much lesser yield than were the glucuronides. 3. The glucoside of genistein was identified as genistin (genistein 7-glucoside) by Sephadex chromatography and reverse isotope dilution. 4. The specificity of the glucuronyl- and glucosyl-transfer mechanisms was compared for a series of steroids and other phenols in addition to the isoflavones. It was concluded that separate transferases were responsible for the formation of the two types of glycosides.     

Biological aspects of Bemisia tabaci biotype B and the chemical causes of resistance in soybean genotypes

In Brazil, the silverleaf whitefly (SLW), Bemisia tabaci biotype B, is a serious soybean pest. SLW management is difficult and new control strategies, such as host–plant resistance, are required. Herein, we aimed to evaluate the biology of SLW, from eggs to adults, on seven soybean cultivars. The emergence of adult insects was monitored daily. Defense-related compounds were identified and quantified from the V3 to the V8 stages in SLW-infested and non-infested plants. The rates of emergence of SLW adults were lower in cultivars ‘IAC 17,’ ‘IAC 19’, and ‘IAC 24’ compared with the susceptible cultivar ‘IAC Holambra Stwart’. Rutin, genistin, genistein, and salicylic acid were identified and quantified in plant extracts. The rutin, genistin, and genistein levels decreased after SLW infestation. Rutin concentrations increased in infested plants of ‘IAC 17’ (V6) and ‘IAC 19’ (from V5 to V8). ‘Barreiras’ showed the highest genistein content in non-infested plants; from V5 growth stage, it was not detected in cultivars Doko (infested), Vencedora, ‘IAC 17’, and ‘IAC 24’ (non-infested). High levels of salicylic acid were observed in ‘IAC ‘19’-infested plants (at V3 and V5). The results suggest that rutin can be related to SLW adults’ emergence only when the feeding source was ‘IAC 19’. Consequently, further studies are needed to access the associated gene expressions and the effect of other secondary metabolites, mainly volatile compounds from SA pathway, including its consequences on feeding preference and mostly in relation to IAC cultivars.     

Protein glycosylation in Haloferax volcanii: partial characterization of a 98-kDa glycoprotein

Eubacterium ramulus, a flavonoid-degrading anaerobic bacterium from the human gastrointestinal tract, was tested for its ability to transform the isoflavonoids genistein-7-O-glucoside (genistin), genistein and daidzein. Genistein was completely degraded by E. ramulus via 6'-hydroxy-O-desmethylangolensin to 2-(4-hydroxyphenyl)-propionic acid. Dihydrogenistein was neither observed as an intermediate in this transformation nor converted itself by growing cells or cell-free extracts of E. ramulus. Genistein-7-O-glucoside was partially transformed by way of genistein to the product 2-(4-hydroxyphenyl)-propionic acid. Daidzein was in part degraded to O-desmethylangolensin, the corresponding metabolite to 6'-hydroxy-O-desmethylangolensin. The hydroxyl group in position 6' of O-desmethylangolensin is crucial for further degradation.