Inhibitory effects of echinoisoflavanone and sophoraisoflavanone D in Sophora chrysophylla SEEM on lipid peroxidation of mice brain homogenate by interaction of ferrous ion and hydrogen peroxide, in vitro

We present the results of an in vitro investigation of the inhibitory effects of echinoisoflavanone and sophoraisoflavanone D isolated from Sophora chrysophylla SEEM on lipid peroxidation of mice brain homogenate by interaction of ferrous ion and hydrogen peroxide, in vitro. They inhibited lipid peroxidation. The order of inhibitory effects of these isoflavanones and mannitol as a hydroxy radical scavenger was echinoisoflavanone > mannitol > sophoraisoflavanone D. The results suggest that some isoflavanones may be of use in cases where oxidative stress is present.     

Inhibitory effects of sophoraflavanones B and H in Sophora mooracroftiana Beth ex Baker on copper-ion-induced protein oxidative modification of mice brain homogenate in vitro

We present the results of an in vitro investigation of the inhibitory effects of sophoraflavanones B and H isolated from Sophora mooracroftiana Beth ex Baker on copper-induced protein oxidative modification. They inhibited copper-induced protein oxidative modification. Their effects were stronger than those of naringenin as a related flavanone, and of mannitol as a hydroxyl radical scavenger. The results suggest that these natural products may be of use in cases where oxidative stress is present.     

Inhibitory effects of phenylpropanoid metabolites on copper-induced protein oxidative modification of mice brain homogenate, in vitro

We present the results of an in vitro investigation of the inhibitory effects of phenylpropanoid metabolites on copper-induced protein oxidative modification of mice brain homogenate. The effects of caffeic acid, 3-(3, 4-dihydroxyphenyl)-l-alanine, esculetin, ferulic acid, and scopoletin were stronger than that of mannitol as a free-radical scavenger, whereas the effects of other phenylpropanoid metabolites, cinnamic acid, coniferyl alcohol, p-coumaric acid, coumarin, phenylalanine, tyrosine, and umbelliferone, were weak. These results demonstrated that phenolic carboxylic acids with 3,4-dihydroxy or 4-hydroxy-3-methoxy substituents and benzo-α-pyrons with 6,7-dihydroxy or 7-hydroxy-6-methoxy substituents in phenylpropanoid metabolites inhibit metal-induced protein oxidative modification of the brain.     

Novel Approach for Evaluation of Estrogenic and Anti‐Estrogenic Activities of Genistein and Daidzein using B16 Melanoma Cells and Dendricity Assay

The effects of soy isoflavones, genistein and daidzein, which exhibit estrogenic, anti‐estrogenic and/or tyrosine kinase inhibitory activity, on the dendritic morphology of B16 mouse melanoma cells were quantitatively evaluated and compared with those of 17β‐estradiol (Est) and tyrphostin, a tyrosine kinase inhibitor. Dendricity was significantly stimulated in the order of Est >> genistein > daidzein = tyrphostin, but not by glycosides of genistein and daidzein. In competition experiments, Est counteracted the stimulatory activity of genistein and daidzein, but enhanced the activity of tyrphostin additively, suggesting that genistein and daidzein agonized Est. In addition, when the concentration ratios of genistein/Est and daidzein/Est were higher than 5000 and 50 000, respectively, genistein and daidzein agonized Est. In contrast, when the ratio of daidzein/Est was lower than 500, daidzein antagonized Est. Furthermore, genistein and daidzein competed with each other in stimulatory activity. These observations suggest that: 1) dendricity is stimulated by agonists (genistein and daidzein) of Est and tyrosine kinase inhibitors (genistein and tyrphostin), 2) the concentration ratio of isoflavone aglycone/Est is very important as one regulatory factor for estrogenic and/or anti‐estrogenic activity, and 3) daidzein antagonizes not only Est but also genistein. It is concluded that a quantitative and simple dendricity assay using B16 mouse melanoma cells is available to evaluate estrogenic and anti‐estrogenic activity in vitro.     

Differential mechanisms for the inhibition of human cytochrome P450 1A2 by apigenin and genistein

The inhibitory effects of flavonoids on the human cytochrome P450 1A2 (CYP1A2) were examined. Among flavonoids tested, galangin, kaempferol, chrysin, and apigenin were potent inhibitors. Although apigenin belonging to flavones and genistein belonging to isoflavones are similar in the chemical structures, the inhibitory potencies for CYP1A2 were distinguished markedly between these two flavonoids. In computer‐docking simulation, apigenin interacted with the same mode of cocrystallized α‐naphthoflavone in the active site of CYP1A2, and then the B ring of apigenin was placed close to the heme iron of the enzyme with a single orientation. In contrast, the docked genistein conformation showed two different binding modes, and the A ring of genistein was oriented to the heme iron of CYP1A2. Furthermore, the binding free energy of apigenin was lower than that of genistein. These results demonstrate a possible mechanism that causes the differential inhibitory potencies of apigenin and genistein for CYP1A2. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:230–234, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20328     

Variable effects of tyrosine kinase inhibitors on avian osteoclastic activity and reduction of bone loss in ovariectomized rats

We compared the effects of the tyrosine kinase inhibitor genistein, a naturally occurring isoflavone, to those of tyrphostin A25, tyrphostin A47, and herbimycin on avian osteoclasts in vitro. Inactive analogs daidzein and tyrphostin A1 were used to control for nonspecific effects. None of the tyrosine kinase inhibitors inhibited bone attachment. However, bone resorption was inhibited by genistein and herbimycin with ID₅₀s of 3 μM and 0.1 μM, respectively; tyrphostins and daidzein were inactive at concentrations below 30 μM, where nonspecific effects were noted. Genistein and herbimycin thus inhibit osteoclastic activity via a mechanism independent of cellular attachment, and at doses approximating those inhibiting tyrosine kinase autophosphorylation in vitro; the tyrphostins were inactive at meaningful doses. Because tyrosine kinase inhibitors vary widely in activity spectrum, effects of genistein on cellular metabolic processes were compared to herbimycin. Unlike previously reported osteoclast metabolic inhibitors which achieve a measure of selectivity by concentrating on bone, neither genistein nor herbimycin bound significantly to bone. Osteoclastic protein synthesis, measured as incorporation of ³H-leucine, was significantly inhibited at 10 μM genistein, a concentration greater than that inhibiting bone degradation, while herbimycin reduced protein synthesis at 10 nM. These data suggested that genistein may reduce osteoclastic activity at pharmacologically attainable levels, and that toxic potential was lower than that of herbimycin. To test this hypothesis in a mammalian system, bone mass was measured in 200 g ovariectomized rats treated with 44 μmol/day genistein, relative to untreated controls. During 30 d of treatment, weights of treated and control group animals were indistinguishable, indicating no toxicity, but femoral weight in the treated group was 12% greater than controls (P < 0.05). Our data indicate that the isoflavone inhibitor genistein suppresses osteoclastic activity in vitro and in vivo at concentrations consistent with its ID₅₀s on tyrosine kinases, with a low potential for toxicity. © 1996 Wiley-Liss, Inc.     

In Vitro and in Vivo Melanogenesis Inhibition by Biochanin A from Trifolium pratense

Our previous study showed that a methanol extract from Trifolium pratense exerted potent inhibitory activity on melanogenesis in mouse B16 melanoma cells. In the present study, the active compound in this Chinese herb extract was isolated and identified as biochanin A by mass spectrum, ¹H-NMR, and ¹³C-NMR analysis. The inhibitory effects of biochanin A on melanogenesis were investigated in vitro in cultured melanoma cells and in vivo in zebrafish and mice. Biochanin A dose-dependently inhibited both melanogenesis and cellular tyrosinase activity in B16 cells and in zebrafish embryos. Application of a cream containing 2% biochanin A twice daily to the skin of mice also increased the skin-whitening index value after 1 week of treatment, and the increase continued for another 2 weeks. Biochanin A was confirmed as a good candidate for use as a skin-whitening agent in the treatment of skin hyperpigmentation disorders.     

Pre-incubation of Bradyrhizobium japonicum with jasmonates accelerates nodulation and nitrogen fixation in soybean (Glycine max) at optimal and suboptimal root zone temperatures

Jasmonic acid (JA) and methyl jasmonate, collectively known as jasmonates, are naturally occurring in plants; they are important signal molecules involved in induced disease resistance and mediate many physiological activities in plants. We studied the effect of JA and its methyl ester, methyl jasmonate (MeJA), on the induction of nod genes in Bradyrhizobium japonicum GG4 (USDA3) carrying a plasmid with a translational fusion between B. japonicum nodY and lacZ of Escherichia coli, and the expression activity was measured by β-galactosidase activity. Both JA and MeJA strongly induced the expression of nod genes. They have little or no deleterious effects on the growth of B. japonicum cells, while genistein (Gen) showed inhibitory effects. We further studied the effect of JA- and MeJA-induced B. japonicum on soybean nodulation and nitrogen fixation under optimal (25°C) and suboptimal (17°C) root zone temperature (RZT) conditions. B. japonicum cells were grown in liquid yeast extract mannitol media and induced with a range of Gen, JA, and MeJA concentrations, including a treatment control with no inducer added. Soybean seedlings were grown at 25 or 17°C RZT with a constant air temperature (25°C) and inoculated, at the vegetative cotyledonary stage, with various B. japonicum induction treatments. Addition of Gen or jasmonates to B. japonicum, prior to inoculation, enhanced nodulation, nitrogen fixation, and plant growth at suboptimal RZT conditions. A higher concentration of Gen was inhibitory at 25°C, while this same concentration was stimulatory at 17°C. Interestingly, pre-incubation of B. japonicum with JA and MeJA enhanced soybean nodulation and nitrogen fixation under both optimal and suboptimal RZTs. We show that jasmonates are thus a new class of signaling molecules in the B. japonicum-soybean symbiosis and that pre-induction of B. japonicum with jasmonates can be used to enhance soybean nodulation, nitrogen fixation, and early plant growth.     

Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A

The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.     

New Isoprenylated 2‐Arylbenzofurans and Pancreatic Lipase Inhibitory Constituents from Artocarpus nitidus

Two new isoprenylated 2‐arylbenzofurans, artonitidin A (=(2′R)‐2′,3′‐dihydro‐2′‐(1‐hydroxy‐1‐methylethyl)‐5′,7‐bis(3‐methylbut‐2‐en‐1‐yl)‐2,4′‐bi‐1‐benzofuran‐6,6′‐diol; 1 ) and artonitidin B (=5‐[6‐hydroxy‐7‐(3‐methylbut‐2‐en‐1‐yl)‐1‐benzofuran‐2‐yl]‐4‐(3‐methylbut‐2‐en‐1‐yl)benzene‐1,3‐diol; 2 ), together with 14 known compounds, 3 – 16 , were isolated from the stems of Artocarpus nitidus Trec. The structures were elucidated by spectroscopic methods. Norartocarpin ( 3 ), cudraflavone C ( 5 ), brosimone I ( 8 ), artotonkin ( 11 ), albanin A ( 13 ), and artopetelin M ( 14 ) showed inhibitory effects on pancreatic lipase with IC₅₀ values ranging from 1.8±0.1 to 63.8±3.6 μM .     

Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis

Oxidative stress may be an important factor in the development of diabetic complications. Advanced glycation end-products have drown attention as potential sources of oxidative stress in diabetes. We investigated the protective effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on oxidative DNA damage from reactive oxygen species or advanced glycation end-products in vitro, as well as effects of main fluvastatin metabolites and other inhibitors of the same enzyme, pravastatin and simvastatin. Protective effects were assessed in terms of the DNA breakage rate in a single-stranded phage DNA system in vitro. DNA was exposed to either reactive oxygen species or advanced glycation end-products. Fluvastatin and its metabolites showed a strong protective effect comparable to those seen with thiourea and mannitol, though pravastatin and simvastatin did not exert clear protective effects. Furthermore, fluvastatin reduced the mutagenesis by reactive oxygen species or advanced glycation end-products in Salmonella typhimurium test strains. Both pravastatin and simvastatin still lacked protective activity. Fluvastatin and its metabolites protect against oxidative DNA damage and may reduce risk of consequent diabetic complications.     

Novel approach for evaluation of estrogenic and anti-estrogenic activities of genistein and daidzein using B16 melanoma cells and dendricity assay

The effects of soy isoflavones, genistein and daidzein, which exhibit estrogenic, anti-estrogenic and/or tyrosine kinase inhibitory activity, on the dendritic morphology of B16 mouse melanoma cells were quantitatively evaluated and compared with those of 17 beta-estradiol (Est) and tyrphostin, a tyrosine kinase inhibitor. Dendricity was significantly stimulated in the order of Est > genistein > daidzein = tyrphostin, but not by glycosides of genistein and daidzein. In competition experiments, Est counteracted the stimulatory activity of genistein and daidzein, but enhanced the activity of tyrphostin additively, suggesting that genistein and daidzein agonized Est. In addition, when the concentration ratios of genistein/Est and daidzein/Est were higher than 5000 and 50,000, respectively, genistein and daidzein agonized Est. In contrast, when the ratio of daidzein/Est was lower than 500, daidzein antagonized Est. Furthermore, genistein and daidzein competed with each other in stimulatory activity. These observations suggest that: 1) dendricity is stimulated by agonists (genistein and daidzein) of Est and tyrosine kinase inhibitors (genistein and tyrphostin), 2) the concentration ratio of isoflavone aglycone/Est is very important as one regulatory factor for estrogenic and/or anti-estrogenic activity, and 3) daidzein antagonizes not only Est but also genistein. It is concluded that a quantitative and simple dendricity assay using B16 mouse melanoma cells is available to evaluate estrogenic and anti-estrogenic activity in vitro.     

Five New Glycoside Constituents from the Roots of Gentiana crassicaulis Duthie ex Burk.

Three undescribed glycoside constituents, macrophyllosides E-G and a pair of iridoid glycosides genticrasides A/B, together with eleven known glycoside compounds were isolated from the roots of Gentiana crassicaulis Duthie ex Burk. Their structures were identified by means of spectra analysis and data comparison with previous literatures. Interestingly, the glucose moieties in macrophylloside E and F possess free anomeric hydroxy groups. Genticrasides A/B, identified as a pair of iridoid originated lactones, have not been reported from Gentianaceae family up to now. The anti-inflammatory effects of selected compounds were also evaluated through the nitric oxide (NO) production inhibition in lipopolysaccharides (LPS)-induced RAW264.7 macrophage cells. In which, macrophyllosides G and D showed NO inhibitory activities with rates of 76.14±4.02 % and 52.44±8.29 % at 100 μg/mL.     

Genistein attenuates D-galactose-induced oxidative damage through decreased reactive oxygen species and NF-κB binding activity in neuronal PC12 cells

AimsWe investigated the mechanism of D-galactose (DG)-induced oxidative damage and the neuroprotective action of genistein in PC12 cells.Main methodsPC12 cells were treated with 40 mM DG dissolved in medium containing 85% RPMI1640, 10% HBS and 5% FBS with or without genistein. We measured the protein expression of β-amyloid (Aβ), advanced glycation end products (AGEs), IκB-α and manganese-superoxide dismutase (MnSOD) by western blotting, intracellular reactive oxygen species (ROS) by 2, 7-dichlorofluorescin-diacetate, and the binding activity of nuclear factor kappa B (NF-κB) by electrophortic mobility shift assay.Key findingsDG (40 mM) completely retarded cell growth after incubation for 72 h, and this effect was not due to osmotic changes, as 40 mM mannitol had no effect. Mechanistically, we found that DG increased intracellular ROS starting at 4 h and increased Aβ and AGEs at 24 h. DG treatment for 24 h also increased the binding activity of NF-κB but strongly decreased the expression of IκB-α protein. Furthermore, DG treatment for 48 h increased MnSOD protein expression. All these effects of DG were effectively inhibited by genistein (0.5–10 μM).SignificanceThe present study indicates that the protection of genistein against DG-induced oxidative stress in PC12 cells, and the effect is likely mediated by decreased intracellular ROS and binding activity of NF-κB.     

Genistein preincubation of Bradyrhizobium japonicum cells improves strain competitiveness under greenhouse,but not field conditions

In the soybean-B. japonicum symbiosis, genistein has been identified as one of the major compounds in soybean seed and root exudates responsible for inducing expression of the B. japonicum nodYABC operon. In this study, we have tested the possibility that genistein treatment prior to inoculation can increase the competitiveness of the treated B. japonicum strain under both greenhouse and field conditions. Two mutants of the two B. japonicum strains each with a different antibiotic resistant marker were selected. They were tested with one or the other treated with genistein. The results showed genistein treated mutants had higher levels of nodule occupancy than the untreated mutant or parent strain under greenhouse conditions. Mutants from 532C had higher nodule occupancies than mutants from USDA110, especially at 15 °C. In the more complex field environment, genistein treated mutants formed fewer nodules than the untreated mutants. The contradictory results of strain competitiveness for greenhouse and field experiments are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Preincubation of B. japonicum cells with genistein reduces the inhibitory effects of mineral nitrogen on soybean nodulation and nitrogen fixation under field conditions

Genistein is the major root produced isoflavonoid inducer of nod genes in the symbiosis between B. japonicum and soybean plants. Reduction in the isoflavonoid content of the host plants has recently been suggested as a possible explanation for the inhibition of mineral nitrogen (N) on the establishment of the symbiosis. In order to determine whether genistein addition could overcome this inhibition, we incubated B. japonicum cells (strain 532C) with genistein. Mineral N (in the form of NH₄NO₃) was applied at 0, 20 and 100 kg ha^-1. The experiments were conducted on both a sandy-loam soil and a clay-loam soil. Preincubation of B. japonicum cells with genistein increased soybean nodule number and nodule weight, especially in the low-N-containing sandy-loam soil and the low N fertilizer treatment. Plant growth and yield were less affected by genistein preincubation treatments than nitrogen assimilation. Total plant nitrogen content was increased by the two genistein preincubation treatments at the early flowering stage. At maturity, shoot and total plant nitrogen contents were increased by the 40 μM genistein preincubation treatment at the sandy-loam soil site. Total nitrogen contents were increased by the 20 μM genistein preincubation treatment only at the 0 and 20 kg ha^-1 nitrate levels in clay-loam soil. Forty μM genistein preincubation treatment increased soybean yield on the sandy-loam soil. There was no difference among treatments for 100-seed weight. The results suggest that preincubation of B. japonicum cells with genistein could improve soybean nodulation and nitrogen fixation, and at least partially overcome the inhibition of mineral nitrogen on soybean nodulation and nitrogen fixation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Different effects of genistein and resveratrol on oxidative DNA damage in vitro

Previous studies have demonstrated that phenolic compounds, including genistein (4′,5,7-trihydroxyisoflavone) and resveratrol (3,4′,5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in φX-174 plasmid DNA. H₂O₂/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H₂O₂/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H₂O₂ suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H₂O₂/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H₂O₂ were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro.     

Influence of genistein (4′,5,7-trihydroxyisoflavone) on the growth and proliferation of testicular cell lines

The effects of genistein on testicular cells, TM3, TM4, and GC-1 spg, were studied in vitro. First, each cell line was cultured with pre-determined concentrations of genistein for a maximum of 72 h to assess the effects of genistein on in vitro growth of the test cells. A second series of experiments were performed to determine the degree of genistein-induced apoptosis in these cells, using Apop-Tag^R kit reagents, to detect apoptotic cells in situ by specific end labeling, and detection of DNA fragments produced by the apoptotic process. The results obtained indicate that: i) genistein inhibits the growth and proliferation of testicular cells; ii) growth inhibition and proliferation is dose- and exposure-time dependent; iii) there is significant difference in sensitivity of the different testicular cells to genistein; iv) genistein induces apoptosis in testicular cells in a concentration-dependent manner. Genistein-induced apoptosis identifies genistein as a potential diagnostic and therapeutic tool in testicular pathophysiological research.     

Variability in yield and yield component responses to genistein pre-incubated Bradyrhizobium japonicum by soybean [Glycine max (L.) Merr] cultivars

The sub-tropical legume, soybean [Glycine max (L.) Merr.], has lower grain yields at low temperatures, mainly due to reduced nitrogen fixation. The isoflavone genistein has been identified as one of the major compounds in soybean seed and root extracts responsible for inducing the expression of the B. japonicum nod genes. A 2-year field study was conducted in 1997 and 1998 with 11 soybean cultivars recommended for Québec, and representing a range of yield potentials and maturity groups. The objective of this study was to assess the variability among soybean cultivar maturity groups in terms of response to genistein application under Canadian short season and cool-spring conditions. The experiments were organized in a randomized complete block design with three replications. The two genistein treatments included B. japonicum inoculant pre-incubated with 20 m genistein and B. japonicum inoculant only. The inoculants were applied into the furrow at the time of planting. The results of this study showed that genistein pre-incubated B. japonicum increased soybean grain yield and protein content over two years. In 1998, pod number per plant^–1 and seed number plant^–1 were also clearly increased. When 20 m genistein was applied in 1998, cultivars in the late maturity group had 28 and 70% more shoot and total protein content, respectively, than the early maturity groups with or without genistein, or the late maturity without genistein, in 1998. There was no interaction between genistein application and soybean cultivar in this study, indicating that both early and late maturing cultivars responded similarly to genistein pretreated inocula. Pre-incubation of B. japonicum with genistein can increase N₂ fixation potential in short season areas. Key words: Soybean, cultivars, genistein, yield, and yield components