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Previous studies have demonstrated antiestrogenic and antiproliferative effects of these molecules in breast cancer cells. Notably, we have reported that pure synthetic glyceollins I and II act through various pathways, including ERα, FOXM1, AhR, and HIF pathways to inhibit cell proliferation and migration. In this study, the potential antitumor activity of glyceollins enriched in crude soybean extracts, obtained by solid fermentation with Aspergillus sojae, was investigated in vivo on MCF‐7 breast cancer cells implanted in the chorioallantoic membrane of the chick egg and on ovariectomized nude mice. The first trial showed a substantial reduction in the migration of MCF‐7 cells treated with the natural extracts. However, the natural extracts significantly reduced the estrogen‐dependent growth of transplanted tumors in orally fed nude mice. Our results showed that natural soybean extracts slightly but significantly reduced estrogen‐dependent growth of the transplanted tumors in orally fed nude mice. These results were confirmed by immunohistochemistry of Ki‐67 and histone H3S10 phosphorylation (H3S10P), revealing lower expression of these proliferation markers in the transplanted tumors from mice fed with the fermented extracts. Additionally, compared to the control animals, we observed a lower expression of angiogenesis markers such as CD31 and CD34. Surprisingly, transcriptomic analysis of RNA from transplanted MCF‐7 cells revealed no differential gene expression. These results may suggest that orally consumed natural glyceollins exert biological effects throughout the body, acting indirectly to reduce tumor angiogenesis and consequently tumor volume. Overall, our results indicate that glyceollins, elicited components of the soy origin, hold potential therapeutic applications for the prevention and treatment of breast cancer. [ABSTRACT FROM AUTHOR]

Xenohormesis proposes that phytochemicals produced to combat stressors in the host plant exert biochemical effects in animal cells lacking cognate receptors. Xenohormetic phytochemicals such as flavonoids and phytoalexins modulate a range of human cell signaling mechanisms but functional correlations with human pathophysiology are lacking. Here, potent inhibitory effects of grapefruit‐derived Naringenin (Nar) and soybean‐derived Glyceollins (Gly) in human microphysiological models of bulk tissue vasculogenesis and tumor angiogenesis are reported. Despite this interference of vascular morphogenesis, Nar and Gly are not cytotoxic to endothelial cells and do not prevent cell cycle entry. The anti‐vasculogenic effects of Glyceollin are significantly more potent in sex‐matched female (XX) models. Nar and Gly do not decrease viability or expression of proangiogenic genes in triple negative breast cancer (TNBC) cell spheroids, suggesting that inhibition of sprouting angiogenesis by Nar and Gly in a MPS model of the (TNBC) microenvironment are mediated via direct effects in endothelial cells. The study supports further research of Naringenin and Glyceollin as health‐promoting agents with special attention to mechanisms of action in vascular endothelial cells and the role of biological sex, which can improve the understanding of dietary nutrition and the pharmacology of phytochemical preparations. [ABSTRACT FROM AUTHOR]

Soybean products have nutrients, dietary fiber, and phytoalexins beneficial for cardiovascular and overall health. Despite their high consumption in Asian populations, their safety in Western diets is debated. We conducted a dose-escalating clinical trial of the safety and tolerability of soybean products in eight older adults (70–85 years) with obesity. Whole green soybean pods grown under controlled conditions were processed to flour (WGS) at the United States Department of Agriculture using common cooking techniques such as slicing and heat treatment. WGS incorporated into food products was consumed at 10 g, 20 g, and 30 g/day for one week at each dose. The gastrointestinal outcomes, clinical biomarkers, and adverse events were evaluated. We explored the stimulation of phytoalexin (glyceollin) production in live viable soybean seeds (LSS-G). We compared the compositions of WGS and LSS-G with commercial soybean flour and its fermented and enzymatically hydrolyzed forms. We found that although 30 g WSG was well-tolerated, and it made participants feel full. Our processing produced glyceollins (267 µg/g) in LSS-G. Processing soybean flour decreased the iron content, but reduced the oligosaccharides, which could attenuate flatulence. Providing soybean flour at <30 g/day may be prudent for overall health and to prevent the exclusion of other food groups and nutrients in older adults with obesity. [ABSTRACT FROM AUTHOR]

Glyceollins, isoflavonoid-derived antimicrobial metabolites, are the major phytoalexins in soybean (Glycine max). They play essential roles in providing resistance to the soil-borne pathogen Phytophthora sojae and have unconventional anticancer and neuroprotective activities that render them desirable for pharmaceutical development. Our previous studies revealed that the transcription factors GmMYB29A2 and GmNAC42-1 have essential roles in activating glyceollin biosynthesis, yet each cannot activate the transcription of all biosynthesis genes in the absence of a pathogen elicitor treatment. Here, we report that co-overexpressing both transcription factors is also insufficient to activate glyceollin biosynthesis. To understand this insufficiency, we compared the transcriptome profiles of hairy roots overexpressing each transcription factor with glyceollin-synthesizing roots treated with wall glucan elicitor (WGE) from P. sojae. GmMYB29A2 upregulated most of the WGE-regulated genes that encode enzymatic steps spanning from primary metabolism to the last step of glyceollin biosynthesis. By contrast, GmNAC42-1 upregulated glyceollin biosynthesis genes only when overexpressed in the presence of WGE treatment. This is consistent with our recent discovery that, in the absence of WGE, GmNAC42-1 is bound by GmJAZ1 proteins that inhibit its transactivation activity. WGE, and not GmMYB29A2 or GmNAC42-1, upregulated the heat shock family gene GmHSF6-1, the homolog of Arabidopsis HSFB2a that directly activated the transcription of several glyceollin biosynthesis genes. Our results provide important insights into what biosynthesis genes will need to be upregulated to activate the entire glyceollin biosynthetic pathway. [ABSTRACT FROM AUTHOR]

Soybean products have nutrients, dietary fiber, and phytoalexins beneficial for cardiovascular and overall health. Despite their high consumption in Asian populations, their safety in Western diets is debated. We conducted a dose-escalating clinical trial of the safety and tolerability of soybean products in eight older adults (70–85 years) with obesity. Whole green soybean pods grown under controlled conditions were processed to flour (WGS) at the United States Department of Agriculture using common cooking techniques such as slicing and heat treatment. WGS incorporated into food products was consumed at 10 g, 20 g, and 30 g/day for one week at each dose. The gastrointestinal outcomes, clinical biomarkers, and adverse events were evaluated. We explored the stimulation of phytoalexin (glyceollin) production in live viable soybean seeds (LSS-G). We compared the compositions of WGS and LSS-G with commercial soybean flour and its fermented and enzymatically hydrolyzed forms. We found that although 30 g WSG was well-tolerated, and it made participants feel full. Our processing produced glyceollins (267 µg/g) in LSS-G. Processing soybean flour decreased the iron content, but reduced the oligosaccharides, which could attenuate flatulence. Providing soybean flour at

Background: The high−fat, high−fructose diet (HFFD) provokes overnutrition and inflammation directly, mainly through Toll−like receptors (TLRs). Soybean (Glycine max L.) contains isoflavone that can be transformed into glyceollin by microbial and physical stimuli. Glyceollin possesses many beneficial effects on health. Objective: This study evaluates the beneficial effect of soybean extract elicited by Saccharomyces cerevisiae and light (ESE) on dendritic cells (DCs) profile and naïve T cells in HFFD mice. Materials and methods: Female Balb/C mice were fed with HFFD for 24 weeks then orally administered with simvastatin 2.8 mg/kg BW or ESE 78, 104, and 130 mg/kg BW at the last four weeks. The expression of splenic CD11c+TLR3+, CD11c+TLR4+, NFκB+, CD11c+IL-17+, CD11c+TNF−α+, CD4+CD62L+, and CD8+CD62L+ subsets was measured by flow cytometry. The molecular docking has been measured using Pyrx 0.8, displayed in PyMol and Biovia Discovery Studio. Result: HFFD significantly increased CD11c+TLR3+, CD11c+TLR4+, NFκB+, CD11c+IL-17+, CD11c+TNF−α+ expression and decreased CD4+CD62L+ and CD8+CD62L+ (p

Glyceollins, isoflavonoid-derived antimicrobial metabolites, are the major phytoalexins in soybean (Glycine max). They play essential roles in providing resistance to the soil-borne pathogen Phytophthora sojae and have unconventional anticancer and neuroprotective activities that render them desirable for pharmaceutical development. Our previous studies revealed that the transcription factors GmMYB29A2 and GmNAC42-1 have essential roles in activating glyceollin biosynthesis, yet each cannot activate the transcription of all biosynthesis genes in the absence of a pathogen elicitor treatment. Here, we report that co-overexpressing both transcription factors is also insufficient to activate glyceollin biosynthesis. To understand this insufficiency, we compared the transcriptome profiles of hairy roots overexpressing each transcription factor with glyceollin-synthesizing roots treated with wall glucan elicitor (WGE) from P. sojae. GmMYB29A2 upregulated most of the WGE-regulated genes that encode enzymatic steps spanning from primary metabolism to the last step of glyceollin biosynthesis. By contrast, GmNAC42-1 upregulated glyceollin biosynthesis genes only when overexpressed in the presence of WGE treatment. This is consistent with our recent discovery that, in the absence of WGE, GmNAC42-1 is bound by GmJAZ1 proteins that inhibit its transactivation activity. WGE, and not GmMYB29A2 or GmNAC42-1, upregulated the heat shock family gene GmHSF6-1, the homolog of Arabidopsis HSFB2a that directly activated the transcription of several glyceollin biosynthesis genes. Our results provide important insights into what biosynthesis genes will need to be upregulated to activate the entire glyceollin biosynthetic pathway.

The dynamic changes of glyceollin contents and concentration depend on various conditions. This study aimed to investigate the role of a pure culture of Saccharomyces cerevisiae versus ragi tape as a commercial starter combined with light/no light for boosting glyceollin accumulation in soybeans followed by the evaluation of its antioxidant activity. Soybeans were subjected to five different treatments: untreated, S. cerevisiae (gS), ragi tape (gR), S. cerevisiae with light (gSL), and ragi tape with light (gRL). The isoflavonoids daidzin, glycitin, genistin, daidzein, glycitein, genistein, glyceollin I, glyceollin II, and glyceollin III were identified and measured by High-Performance Liquid Chromatography (HPLC) assay, and the antioxidant activity was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH). Glyceollin I was increased about 2-fold in gR, 3-fold in gS, 4-fold in gSL and 7-fold in gRL compared to untreated soybeans. The gRL also exhibit a potent antioxidant activity compared to gSL. Taken together, ragi tape with light is the most effective elicitor for boosting isoflavonoids contents. Therefore, ragi tape with light deserves as a potential elicitor with valuable insights to increase the isoflavonoids contents to optimize the impact of soybeans for health.

Abstract Terpenes produced by plants comprise a diverse range of secondary metabolites, including volatile organic compounds (VOCs). Terpene VOC production may be altered after damage or by biological stimuli such as bacterial, fungal and insects, and subsequent triggering of plant defense responses. These VOCs originate in plants from two independent pathways: the mevalonate and the methylerythritol phosphate pathways, which utilize dimethylallyl and isopentenyl diphosphates to form the terpenoidal precursors. Phakopsora pachyrhizi fungi causes Asian soybean rust, limiting soybean production and resulting in losses of up to 80% if no control strategies are applied. By using a transcriptome datasets, we investigated the regulation of genes of the mevalonate pathway under different biotic stresses. We studied the impact of P. pachyrhizi infection in vivo expression profile of genes involved in terpenoid and glyceollin biosynthesis in genotypes harboring different resistance genes (Rpp), and across the infection cycle. In addition, we used UPLC and UPGC analysis to evaluate glyceollin and VOC production, respectively, to identify metabolites associated with soybean responses to pathogen infection. The regulation of soybean genes involved in terpene production was influenced by genotypes, depending on the Rpp gene, while glyceollin was induced in all genotypes. Furthermore, a sesquiterpene was identified as a potential marker associated with rust symptoms on soybean.

Cytochrome P450, CYP93A1, is involved in the synthesis of the phytoalexin glyceollin in soybean (Glycine max L. Merr). The gene encoding CYP93A1 has been used as defense marker in soybean cell cultures, however, little is known regarding how this gene is expressed in the intact plant. To further understand the tissue-specific role of CYP93A1 in soybean defense, we analyzed the expression of this gene in mechanically damaged leaves and stems. In leaves, CYP93A1 was constitutively expressed; its expression did not change in response to mechanical damage. In stems, however, expression of CYP93A1 was induced as quickly as 4 h after mechanical damage and remained upregulated for at least 48 h. The induction of CYP93A1 was associated with the synthesis of glyceollins. In comparison to several other defense-related genes encoding cysteine protease inhibitors L1 and R1 and storage proteins vspA and vspB, CYP93A1 was the most strongly induced by stem wounding. The induction of CYP93A1 was observed only locally, not systemically. Similar stem expression patterns were consistently observed among three different soybean genotypes. The strong induction of CYP93A1 in mechanically damaged stems suggests an important role in the soybean stem defense response; therefore, this study expands the use of CYP93A1 as a defense response marker to stems, not just soybean cell cultures.

The WRKYs are a group of plant-specific transcription factors that play important roles in defense responses. In this study, we silenced 2 GmWRKY33B homologous genes using a bean pod mosaic virus (BPMV) vector carrying a single fragment from the conserved region of the GmWRKY33B genes. Silencing GmWRKY33B did not result in morphological changes. However, significantly reduced resistances to Pseudomonas syringae pv. glycinea ( Psg ) and soybean mosaic virus (SMV) were observed in the GmWRKY33B -silenced plants, indicating a positive role of the GmWRKY33B genes in disease resistance. Kinase assay showed that silencing the GmWRKY33B genes significantly reduced the activation of Gm MPK6, but not Gm MPK3, in response to flg22 treatment. Reverse transcriptase PCR (RT-PCR) analysis of the genes encoding prenyltransferases (PTs), which are the key enzymes in the biosynthesis of glyceollin, showed that the Psg -induced expression of these genes was significantly reduced in the GmWRKY33B -silenced plants compared with the BPMV-0 empty vector plants, which correlated with the presence of the W-boxes in the promoter regions of these genes. Taken together, our results suggest that Gm WRKY33Bs are involved in soybean immunity through regulating the activation of the kinase activity of Gm MPK6 as well as through regulating the expression of the key genes encoding the biosynthesis of glyceollins.

Summary: Phytoalexin glyceollins are soybean‐specific antimicrobial compounds that are derived from the isoflavonoid pathway. They are synthesized by soybean in response to extrinsic stress such as pathogen attack or injury, thereby conferring partial resistance if synthesized rapidly at the site of infection and at the required concentration. Soybean produces multiple forms of glyceollins that result from the differential prenylation reaction catalyzed by prenyltransferases (PTs) on either the C‐2 or C‐4 carbon of a pterocarpan glycinol. The soybean genome contains 77 PT‐encoding genes (GmPTs) where at least 11 are (iso)flavonoid‐specific. Transcript accumulation of five candidates GmPTs was increased in response to Phytophthora sojae infection, suggesting their role in phytoalexin synthesis. The induced GmPTs localize to plastids and display tissue‐specific expression. We have in this study identified two additional GmPTs: an isoflavone dimethylallyltransferase 3 (IDT3); and a glycinol 2‐dimethylallyl transferase GmPT01. GmPT01 prenylates (−)‐glycinol at the C‐2 position, localizes in the plastid, and exhibits root‐specific gene expression. Furthermore, its expression is induced rapidly in response to stress, and is associated with a quantitative trait loci linked with resistance to P. sojae. Based on these results, we conclude that GmPT01 are possibly one of the loci involved in conferring partial resistance against stem and root rot disease in soybean. Significance Statement: GmPT01 is a pterocarpan 2‐dimethylallyltransferase that catalyzes the key prenylation reaction in phytoalexin glyceollin biosynthesis in soybean. The rapidly induced gene expression in response to stress and its location near a QTL linked to P. sojae resistance suggest a role of GmPT01 in partial resistance that can be utilized in resistance breeding. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Glyceollin produced by soybean callus suspension cultures upon AOS was first found. • Compound content and gene expression changed during glyceollin synthesis under AOS. • Glyceollin I was prominent among isomers in AOS-treated callus suspension cultures. • Glyceollin II and III were dominant among isomers in AOS-induced soybean cotyledon tissues. Abstract Glyceollins are a group of inducible soybean phytoalexins. The aim of the present study was to investigate the mechanism of glyceollin production in callus suspension cultures (CS) and cotyledon tissues elicited with alginate oligosaccharides (AOS) at 1%, 2%, and 3% concentrations that were applied for three days. HPLC analysis of the extracts from 2% AOS-elicited callus suspension cultures (ECS) revealed that glyceollins content was 85.3-fold and 8.2-fold higher than that in unelicited controls and elicited cotyledon tissues (ECT), respectively. UPLC-Q-Exactive Orbitrap MS analysis showed that the 2% AOS induction resulted in elevated levels of metabolites, the intermediary products of glyceollin biosynthesis, such as daidzein, glycitein, genistein, naringenin, glycinol, glyceollidin I/II, and glyceollin I, II, and III in ECS and ECT. Glyceollin I was prominent among the three glyceollin isomers in ECS, whereas isomer II and III were dominant in the synthetic route for glyceollins in ECT. qRT-PCR analysis of related genes showed that UGT , CYP93A1 , G2DT , and G4DT displayed a direct effect on glyceollin synthesis. Our results suggested that 2% AOS-induced CS produced glyceollins in higher content compared to ECT by altering metabolite content and gene expression. Elicitation of CS is thus a promising technology for glyceollin synthesis extensively. [ABSTRACT FROM AUTHOR]

Alginate oligosaccharides (AOs) are linear oligosaccharides with alternating sequences of mannuronic acid (M) and guluronic acid (G) residues. AOs can be used as a safe elicitor to induce glyceollins, which have many human health benefits, in soybean seeds. In this research, four AO fractions with different chemical structures and molecular weights were separated, purified, and then characterized by NMR spectroscopy and ESI-MS. With a 4,5-unsaturated hexuronic acid residue (△) at the non-reducing terminus, the structures of these four AO fractions were △G, △MG, △GMG and △MGGG, which exhibited glyceollin-inducing activities of 1.2339, 0.3472, 0.6494 and 1.0611 (mg/g dry weight) in soybean seeds, respectively. The results demonstrated that a larger molecular weight or a higher G/M ratio might correlate with a higher glyceollin-inducing activity. Moreover, the alginate disaccharide △G could be introduced as relatively safe and efficient elicitor of high glyceollin content in soybeans. [ABSTRACT FROM AUTHOR]

Phytoalexins are metabolites biosynthesized in plants in response to pathogen, environmental, and chemical stresses that often have potent bioactivities, rendering them promising for use as therapeutics or scaffolds for pharmaceutical development. Glyceollin I is an isoflavonoid phytoalexin from soybean that exhibits potent anticancer activities and is not economical to synthesize. Here, we tested a range of source tissues from soybean, in addition to chemical and biotic elicitors, to understand how to enhance the bioproduction of glyceollin I. Combining the inorganic chemical silver nitrate (AgNO3) with the wall glucan elicitor (WGE) from the soybean pathogen Phytophthora sojae had an additive effect on the elicitation of soybean seeds, resulting in a yield of up to 745.1 μg gt-1 glyceollin I. The additive elicitation suggested that the biotic and chemical elicitors acted largely by separate mechanisms. WGE caused a major accumulation of phytoalexin gene transcripts, whereas AgNO3 inhibited and enhanced the degradation of glyceollin I and 6"-O-malonyldaidzin, respectively. [ABSTRACT FROM AUTHOR]

Background: While current therapies for osteoporosis focus on reducing bone resorption, the development of therapies to regenerate bone may also be beneficial. Promising anabolic therapy candidates include phytoestrogens, such as daidzein, which effectively induce osteogenesis of adipose-derived stromal cells (ASCs) and bone marrow stromal cells (BMSCs).Purpose: To investigate the effects of glyceollins, structural derivatives of daidzein, on osteogenesis of ASCs and BMSCs.Study Design: Herein, the osteoinductive effects of glyceollin I and glyceollin II were assessed and compared to estradiol in ASCs and BMSCs. The mechanism by which glyceollin II induces osteogenesis was further examined.Methods: The ability of glyceollins to promote osteogenesis of ASCs and BMSCs was evaluated in adherent and scaffold cultures. Relative deposition of calcium was analyzed using Alizarin Red staining, Bichinchoninic acid Protein Assay, and Alamar Blue Assay. To further explore the mechanism by which glyceollin II exerts its osteoinductive effects, docking studies of glyceollin II, RNA isolation, cDNA synthesis, and quantitative RT-PCR (qPCR) were performed.Results: In adherent cultures, ASCs and BMSCs treated with estradiol, glyceollin I, or glyceollin II demonstrated increased calcium deposition relative to vehicle-treated cells. During evaluation on PLGA scaffolds seeded with ASCs and BMSCs, glyceollin II was the most efficacious in inducing ASC and BMSC osteogenesis compared to estradiol and glyceollin I. Dose-response analysis in ASCs and BMSCs revealed that glyceollin II has the highest potency at 10nM in adherent cultures and 1µM in tissue scaffold cultures. At all doses, osteoinductive effects were attenuated by fulvestrant, suggesting that glyceollin II acts at least in part through estrogen receptor-mediated pathways to induce osteogenesis. Analysis of gene expression demonstrated that, similar to estradiol, glyceollin II induces upregulation of genes involved in osteogenic differentiation.Conclusion: The ability of glyceollin II to induce osteogenic differentiation in ASCs and BMSCs indicates that glyceollins hold the potential for the development of pharmacological interventions to improve clinical outcomes of patients with osteoporosis. [ABSTRACT FROM AUTHOR]

The rampant use of antibiotics in the swine industry has caused an increase in antibiotic-resistant microorganisms. Thus, investigating novel natural antimicrobial compounds that can be supplemented into swine feed is of great importance. Glyceollins are members of a class of inducible phytochemicals that are naturally produced by soybeans and have antimicrobial and antifungal properties. Previous research has concluded that 3 mg glyceollins per g soybean is the target production that would enable glyceollins to be used as an antimicrobial in swine feed. In this study, the optimal soybean germination conditions for glyceollin production were elucidated and utilized. Also, 8 fungal strains were evaluated for their ability to stimulate glyceollin production in soybeans. The fungal strains were inoculated on soybeans that were either manually de-hulled and halved or still encased by the hull. It was determined that Trichoderma reesei NRRL 3653 inoculated on de-hulled soybeans yielded significantly higher glyceollin production (3.763 mg/g) than all other treatments investigated in this study, which was a ∼3-fold increase in production compared to a previously published study. This study strongly suggests that T. reesei can stimulate glyceollin production from soybeans at a high enough concentration to become a novel natural antimicrobial in swine feed. [ABSTRACT FROM AUTHOR]

Cytochrome P, CYP93A1, is involved in the synthesis of the phytoalexin glyceollin in soybean ( Glycine max L. Merr). The gene encoding CYP93A1 has been used as defense marker in soybean cell cultures, however, little is known regarding how this gene is expressed in the intact plant. To further understand the tissue-specific role of CYP93A1 in soybean defense, we analyzed the expression of this gene in mechanically damaged leaves and stems. In leaves, CYP93A1 was constitutively expressed; its expression did not change in response to mechanical damage. In stems, however, expression of CYP93A1 was induced as quickly as 4 h after mechanical damage and remained upregulated for at least 48 h. The induction of CYP93A1 was associated with the synthesis of glyceollins. In comparison to several other defense-related genes encoding cysteine protease inhibitors L1 and R1 and storage proteins vspA and vspB, CYP93A1 was the most strongly induced by stem wounding. The induction of CYP93A1 was observed only locally, not systemically. Similar stem expression patterns were consistently observed among three different soybean genotypes. The strong induction of CYP93A1 in mechanically damaged stems suggests an important role in the soybean stem defense response; therefore, this study expands the use of CYP93A1 as a defense response marker to stems, not just soybean cell cultures. [ABSTRACT FROM AUTHOR]

Foram desenvolvidos três experimentos com o objetivo de avaliar o potencial de preparados a base de calêndula (Calendula officinalis L.) na indução de fitoalexinas em cotilédones de soja, na indução de mecanismos de resistência em frutos de morango, e o efeito fungistático sobre Botrytis cinerea in vitro. O delineamento experimental foi inteiramente casualizado com quatro repetições, para 15 tratamentos resultantes da combinação de três formas de extração (extrato alcoólico, infusão, e maceração) em cinco concentrações (zero; 1,25; 2,5; 5; e 10%). Os resultados demonstram que os preparados de C. officinalis apresentaram capacidade de indução das fitoalexinas gliceolinas em cotilédones de soja. Na aplicação dos preparados em pós-colheita de morangos ocorreu alteração no teor de flavonóides, bem como a atividade da enzima FAL foi estimulada pela aplicação dos extratos; no entanto, não foi constatado o controle de podridão dos frutos. O efeito fungistático foi observado na extração por maceração em todas as suas concentrações reduzindo o crescimento do fungo B. cinerea in vitro sendo que, a partir de 2,5%, observou-se inibição total. A extração por infusão também apresentou resposta positiva na redução do crescimento de B. cinerea, com melhor resposta na concentração de 10% do preparado.Three experiments were carried out to evaluate the potential of calendula (Calendula officinalis L.) extracts for phytoalexin induction in soybean cotyledons, resistance mechanism induction in strawberry fruits and fungistatic effect on Botrytis cinerea in vitro. Experimental design was completely randomized with 15 treatments resulting from the combination of three forms of extraction (alcohol extract, infusion and maceration) at five concentrations (zero, 1.25, 2.5, 5 and 10%). Results showed that C. officinalis extracts could induce the phytoalexins glyceollins in soybean cotyledons. In the application of extracts during the strawberry postharvest, there was a change in flavonoid content, as well as in the activity of the enzyme PAL, which was stimulated by the application of extracts; however, fruit rot control was not noted. Fungistatic effect was observed for the extract obtained by maceration at all used concentrations, reducing in vitro the growth of the fungus B. cinerea, and from 2.5% there was total inhibition. The extract obtained by infusion also showed a positive response in reducing B. cinerea growth, with better response at the concentration of 10% extract.

Glyceollins are phytoalexins produced in soybeans under stressful growth conditions. On the basis of prior evaluations, they show potential to treat multiple diseases, including certain cancers, Type 2 diabetes, and cardiovascular conditions. The aim of the present study was to expand on recent studies designed to initially characterize the intestinal disposition of glyceollins. Specifically, studies were undertaken in Caco-2 cells to evaluate glyceollins' effects on apical efflux transporters, namely, MRP2 and BCRP, which are the locus of several intestinal drug-drug and drug-food interactions. 5- (and 6)-carboxy-2',7'-dichloroflourescein (CDF) was used to provide a readout on MRP2 activity, whereas BODIPYprazosin provided an indication of BCRP alteration. Glyceollins were shown to reverse MRP2-mediated CDF transport asymmetry in a concentration-dependent manner, with activity similar to the MRP2 inhibitor, MK-571. Likewise, they demonstrated concentration-dependent inhibition of BCRP-mediated efflux of BODIPY-prazosin with a potency similar to that of Ko143. Glyceollin did not appreciably alter MRP2 or BCRP expression following 24 h of continuous exposure. The possibility that glyceollin mediated inhibition of genistein metabolite efflux by either transporter was evaluated. However, results demonstrated an interaction at the level of glyceollin inhibition of genistein metabolism rather than inhibition of metabolite transport. [ABSTRACT FROM AUTHOR]

Phytoalexins are metabolites biosynthesized in plants in response to pathogen, environmental, and chemical stresses that often have potent bioactivities, rendering them promising for use as therapeutics or scaffolds for pharmaceutical development. Glyceollin I is an isoflavonoid phytoalexin from soybean that exhibits potent anticancer activities and is not economical to synthesize. Here, we tested a range of source tissues from soybean, in addition to chemical and biotic elicitors, to understand how to enhance the bioproduction of glyceollin I. Combining the inorganic chemical silver nitrate (AgNO3) with the wall glucan elicitor (WGE) from the soybean pathogen Phytophthora sojae had an additive effect on the elicitation of soybean seeds, resulting in a yield of up to 745.1 µg gt−1 glyceollin I. The additive elicitation suggested that the biotic and chemical elicitors acted largely by separate mechanisms. WGE caused a major accumulation of phytoalexin gene transcripts, whereas AgNO3 inhibited and enhanced the degradation of glyceollin I and 6″-O-malonyldaidzin, respectively.

An estimated 70% of breast cancer tumors utilize estrogen receptor (ER) signaling to maintain tumorigenesis and targeting of the estrogen receptor is a common method of treatment for these tumor types. However, ER-positive (+) breast cancers often acquire drug resistant or altered ER activity in response to anti-estrogens. Here we demonstrate glyceollin, an activated soy compound, has anti-estrogen effects in breast cancers. We demonstrate through estrogen response element luciferase and phosphorylation-ER mutants that the effects of glyceollin arise from mechanisms distinct from conventional endocrine therapies. We show that glyceollin suppresses estrogen response element activity; however, it does not affect ER-alpha (α) phosphorylation levels. Additionally we show that glyceollin suppresses the phosphorylation of proteins known to crosstalk with ER signaling, specifically we demonstrate an inhibition of ribosomal protein S6 kinase, 70 kDa (p70S6) phosphorylation following glyceollin treatment. Our data suggests a mechanism for glyceollin inhibition of ERα through the induced suppression of p70S6 and demonstrates novel mechanisms for ER inhibition. [ABSTRACT FROM AUTHOR]

Glyceollins are phytoalexins produced in soybeans from their isoflavone precursor daidzein. Their impressive anticancer and glucose normalization effects in rodents have generated interest in their therapeutic potential. The aim of the present studies was to begin to understand glyceollin intestinal transport and metabolism, and their potential effects on P-glycoprotein (Pgp) in Caco-2 cells. At 10 and 25 μM, glyceollin permeability was 2.4±0.16×10−4 cm/sec and 2.1±0.15×10−4 cm/sec, respectively, in the absorptive direction. Basolateral to apical permeability at 25 μM was 1.6±0.10×10−4 cm/sec. Results suggest high absorption potential of glyceollin by a passive-diffusion-dominated mechanism. A sulfate conjugate at the phenolic hydroxyl position was observed following exposure to Caco-2 cells. In contrast to verapamil inhibition of the net secretory permeability of rhodamine 123 (R123) and its enhancement of calcein AM uptake into Caco-2 cells, neither glyceollin nor genistein inhibited Pgp (MDR1; ABCB1) up to 300 μM. There was no significant change in MDR1 mRNA expression, Pgp protein expression, or R123 transport in cells exposed to glyceollin or genistein for 24 h up to 100 μM. Collectively, these results suggest that glyceollin has the potential to be well absorbed, but that, similar to the isoflavone genistein, its absorption may be reduced substantially by intestinal metabolism; further, they indicate that glyceollin does not appear to alter Pgp function in Caco-2 cells. [ABSTRACT FROM AUTHOR]

Abstract: The glyceollins are soybean-derived phytoalexins that accumulate in the seeds in response to various extrinsic factors. Several elicitors such as fungi, UV irradiation and physical injury have been demonstrated to alter the secondary metabolome of soybeans via induction of glyceollin synthesis and accumulation. The pterocarpan phytoalexins have exhibited antiestrogenic activity against estrogen receptor-mediated cellular processes. As a result, the glyceollins display anticancer activities against breast and ovarian cancers through inhibition of tumor growth and progression, and also by inducing cell cycle arrest in the cancer cells. Moreover, glyceollins were found to exhibit antimicrobial activity against pathogenic fungi, cellular antioxidant activities against endotoxin-induced oxidative stress, and regulatory effects on glucose metabolism and endotoxin-induced inflammatory response. The soybean-derived compounds warrant further studies to evaluate detailed molecular mechanisms during aberrant cellular processes, safety and efficacy in animal disease models and human subjects, and potential use for food preservation. The bioactivity of glyceollins can result in the enrichment of soybean with these phytoalexins for improved health functions and other novel applications. [Copyright &y& Elsevier]

Abstract: Glyceollin has been shown to have antidiabetic properties, although its molecular mechanism is not known. Here, we have investigated the metabolic effects of glyceollin in animal models of insulin resistance and in endoplasmic reticulum (ER) stress-responsive muscle cells. db/db mice were treated with glyceollin for 4weeks and triglycerides, total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) levels were measured. Glyceollin reduced serum insulin and triglycerides and increased HDL levels in db/db mice. Furthermore, glyceollin caused a significant improvement in glucose homeostasis without altering body weight and food intake in db/db mice. In muscle cells, glyceollin increased the activity of AMP-activated protein kinase (AMPK) as well as cellular glucose uptake. Fatty acid oxidation was also increased. In parallel, phosphorylation of acetyl-CoA carboxylase (ACC) at Ser-79 was increased, consistent with decreased ACC activity. An insulin-resistant state was induced by exposing cells to 5μg/ml of tunicamycin as indicated by decreased insulin-mediated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and glucose uptake. Inhibition of insulin-mediated tyrosine phosphorylation of IRS-1 and glucose uptake under ER stress was prevented by glyceollin. Strikingly, glyceollin reduced ER stress-induced, c-Jun NH2-terminal kinase activation and subsequently increased insulin signaling via stimulation of AMPK activity in L6 myotubes. Pharmacologic inhibition or knockdown of Ca2+/calmodulin-dependent protein kinase kinase blocked glyceollin-increased AMPK phosphorylation and insulin sensitivity under ER stress conditions. Taken together, these results indicate that glyceollin-mediated enhancement of insulin sensitivity under ER stress conditions is predominantly accomplished by activating AMPK, thereby having beneficial effects on hyperglycemia and insulin resistance. [Copyright &y& Elsevier]

Soy milk was prepared from regular soybean (M1), soybean germinated for 3 days at 25°C (M2), and soybean germinated under fungal infection (M3). Rhizopus microsporus var. oligosporus ATCC 22959 was used as the elicitor for glyceollin production. Each soy milk was fermented with Streptococcus infantarius 12 and Weissella sp. 4 (1:5, v/v) for 12 h at 37°C. Significant induction of glyceollins was confirmed only in M3 soy milk and glyceollins maintained stably during 12 h fermentation period. The concentration of glyceollins in M3 yogurt was 2,400.4±83 and 2,525.2±158 μg/g dry matter (d.m.) at 0 and 12 h, respectively. The amount of daidzein was significantly higher in M3 yogurt (635.1±21) than that of M1 (417±11) and M2 (545±17 μg/g d.m.) yogurt in 12 h ( p<0.05). M2 yogurt had the highest amount of genistein (695.3±17) followed by M3 (634.5±26) and M1 (612.5±14 μg/g d.m.) yogurt. M3 soy yogurt also showed the highest content of total phenolic compounds (5.37 mg/g) and antioxidant activity. The results indicated that functional soy yogurt can be prepared from soybean enriched with glyceollins. [ABSTRACT FROM AUTHOR]

This review covers the biosynthesis of glyceollin and its biological activities including antiproliferative/antitumor action (toward B16 melanoma cells, LNCaP prostate cancer cells, and BG-1 ovarian cancer cells), anti-estrogenic action (through estrogen receptors α- and β-), antibacterial action (toward Erwinia carotovora, Escherichia coli, Bradyrhizobium japonicum, Sinorhizobium fredii ), antinematode activity, and antifungal activity (toward Fusarium solani, Phakospora pachyrhizi, Diaporthe phaseolorum, Macrophomina phaseolina, Sclerotina sclerotiorum, Phytophthora sojae, Cercospora sojina, Phialophora gregata, and Rhizoctonia solani). Other activities include insulinotropic action and attenuation of vascular contractions in rat aorta. [ABSTRACT FROM AUTHOR]

Abstract: Glyceollins are pterocarpan phytoalexins elicited in high concentrations when soybeans are stressed. We have previously reported that the three glyceollin isomers (GLY I–III) exhibit antiestrogenic properties, which may have significant biological effects upon human exposure. Of the three isomers, we have recently shown that glyceollin I is the most potent antiestrogen. Natural (−)-glyceollin I recently was synthesized along with its racemate and unnatural (+) enantiomer. In this study, we compared the glyceollin I enantiomers’ ER binding affinity, ability to inhibit estrogen responsive element transcriptional (ERE) activity and endogenous gene expression in MCF-7 cells. The results demonstrated similar binding affinities for both ERα and ERβ. Reporter gene assays in MCF-7 cells revealed that while (+)-glyceollin I slightly stimulated ERE transcriptional activity, (−)-glyceollin I decreased activity induced by estrogen. Co-transfection reporter assays performed in HEK 293 cells demonstrated that (+)-glyceollin I increased ERE transcriptional activity of ERα and ERβ with and without estrogen with no antiestrogenic activity observed. Conversely, (−)-glyceollin I decreased the activity of both ER subtypes stimulated by estradiol demonstrating potent antiestrogenic properties. Additionally, each Gly I enantiomer induced unique gene expression profiles in a PCR array panel of genes commonly altered in breast cancer. [ABSTRACT FROM AUTHOR]

The present study was undertaken to investigate the molecular mechanisms by which glyceollin I inhibits vascular contraction in rat aorta. Rat aortic rings were treated with either glyceollin I or vehicle when vascular contraction reached plateaus. We measured the activity of GTP-RhoA and Rho GTPase-activating protein (RhoGAP) and the phosphorylation level of the myosin light chain (MLC20), myosin phosphatase targeting subunit 1 (MYPT1), and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI17). Glyceollin I reduced vascular contraction whether endothelium is present or denuded. Glyceollin I reduced vascular contraction induced by NaF, U46619, phenylephrine, or PDBu. Blockers of K+ channels did not affect the vasorelaxation induced by glyceollin I. Glyceollin I reduced activation of RhoA as well as phosphorylation level of MLC20. Glyceollin I also reduced phosphorylation of MYPT1 and CPI17 induced by NaF but not PDBu. However, glyceollin I had no direct effect on RhoGAP activation in vitro. Glyceollin I reduced vascular contraction, at least in part, through inhibition of the RhoA/Rho-kinase signaling pathway. [ABSTRACT FROM AUTHOR]

Hairy roots were initiated from two soybean [Glycine max (L.) Merr.] genotypes with different susceptibility (susceptible ‘Spencer’ and partially resistant ‘PI567.374’) to the disease sudden death syndrome (SDS) caused by the soil-borne fungal pathogen Fusarium solani f. sp. glycines (FSG) to study the role of isoflavonoids in the plant response to FSG infection. Hairy root cultures obtained by transformation with Agrobacterium rhizogenes allows normal root growth that can be visually monitored. The principal isoflavones (genistin, daidzin, glycitin and their malonyl conjugates and aglycones) and also isoflavonoid phytoalexins (coumestrol and glyceollin) were measured by HPLC in extracts of the FSG-inoculated and non-inoculated hairy roots. FSG mycelia grew more slowly on inoculated PI567.374 hairy roots than on Spencer hairy roots. The glyceollin content was higher in FSG-inoculated PI567.374 hairy roots than in Spencer hairy roots even though the glyceollin precursor, the isoflavone daidzein, was higher in Spencer. The de novo synthesis of isoflavones and glyceollin was confirmed by [14C]Phe incorporation into glyceollin, which was higher both in the FSG-inoculated roots and surrounding medium of the cv. PI567.374 than that of Spencer. Glyceollin was the most inhibitory to FSG growth among eight isoflavonoids tested. The levels of coumestrol, a putative phytoalexin, did not change upon FSG inoculation. The defense response was also elicited by FSG culture filtrates in hairy roots grown in liquid culture. The data obtained indicate that the ability of soybean roots to rapidly produce sufficient amounts of glyceollin in response to FSG infection might be important in providing partial resistance to this fungus. [Copyright &y& Elsevier]

Fecundity, mortality, and food consumption of the Formosan subterranean termite, Coptotermes formosanus Shiraki, were evaluated in response to five plant ifavonoids (genistein, biochanin A, apigenin, quercetin, and glyceohm). Apigenin fed at 50 μg/primary reproductive pair proved to be the most toxic fiavonoid. Biochanin A was most effective in reducing fecundity. Subsequently, these two flavonoids were tested through oral feeding and topical application at 100-μg dose. Significant reduction in the numbers of progeny was evident for biochanin A in both treatment methods. Choice feeding tests with termite workers showed that initially termites were attracted to filter paper treated with biochanin A, but over a period of 72 hr, consumed significantly less material when compared to controls. Biochanin A is a promising phytochemical with ability to reduce fecundity in primary reproductives of the Formosan subterranean termite, but it does not elicit phagostimulant activity. [ABSTRACT FROM AUTHOR]