The azalea (Rhododendron simsii Planch.) is an important ornamental woody plant with various medicinal properties due to its phytochemical compositions and components. However little information on the metabolite variation during flower development in Rhododendron has been provided. In our study, a comparative analysis of the flavonoid profile was performed in Rhododendron pulchrum sweet at three stages of flower development, bud (stage 1), partially open flower (stage 2), and full bloom (stage 3). A total of 199 flavonoids, including flavone, flavonol, flavone C-glycosides, flavanone, anthocyanin, and isoflavone were identified. In hierarchical clustering analysis (HCA) and principal component analysis (PCA), the accumulation of flavonoids displayed a clear development stage variation. During flower development, 78 differential accumulated metabolites (DAMs) were identified, and most were enriched to higher levels at the full bloom stage. A total of 11 DAMs including flavone (chrysin, chrysoeriol O-glucuronic acid, and chrysoeriol O-hexosyl-O-pentoside), isoflavone (biochanin A), and flavonol (3,7-di-O-methyl quercetin and isorhamnetin) were significantly altered at three stages. In particular, 3,7-di-O-methyl quercetin was the top increased metabolite during flower development. Furthermore, integrative analyses of metabolomic and transcriptomic were conducted, revealing that the contents of isoflavone, biochanin A, glycitin, and prunetin were correlated with the expression of 2-hydroxyisoflavanone dehydratase (HIDH), which provide insight into the regulatory mechanism that controls isoflavone biosynthesis in R. pulchrum. This study will provide a new reference for increasing desired metabolites effectively by more accurate or appropriate genetic engineering strategies.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) accounts as a crucial health concern with a huge burden on health and economic systems. The aim of this study is to evaluate the effect of soy isoflavones supplementation on metabolic status in patients with NAFLD.
METHODS: In this randomized clinical trial, 50 patients with NAFLD were randomly allocated to either soy isoflavone or placebo groups for 12 weeks. The soy isoflavone group took 100 mg/d soy isoflavone and the placebo group took the similar tablets containing starch. Anthropometric indices, blood lipids, glycemic parameters and blood pressure were measured at the beginning and at the end of the study.
RESULTS: At the end of week 12 the level of serum triglyceride (TG), low density lipoprotein (LDL) and total cholesterol (TC) was significantly decreased only in soy isoflavone group compared to baseline (P < 0.05). Although waist circumference (WC) decreased significantly in both groups after 12 weeks of intervention (P < 0.05), hip circumference (HC) decreased significantly only in soy isoflavone group (P = 0.001). No significant changes observed regarding high density lipoprotein (HDL) and blood pressure in both groups. At the end of the study, serum glucose level was significantly decreased in the placebo group compared to baseline (P = 0.047). No significant changes demonstrated in the soy isoflavone group in regard to glycemic parameters (P > 0.05).
CONCLUSIONS: This study revealed that soy isoflavones could significantly reduce TG, LDL TC, WC and HC in NAFLD patients.
BACKGROUND: The Ethics committee of Ahvaz Jundishapur University of Medical Sciences approved the protocol of the present clinical research (IR.AJUMS.REC.1401.155). The study was in accordance with the Declaration of Helsinki. This study\'s registered number and date are IRCT20220801055597N1 and 20.09.2022, respectively at https://fa.irct.ir .

Plant roots secrete various metabolites, including plant specialized metabolites, into the rhizosphere, and shape the rhizosphere microbiome, which is crucial for the plant health and growth. Isoflavones are major plant specialized metabolites found in legume plants, and are involved in interactions with soil microorganisms as initiation signals in rhizobial symbiosis and as modulators of the legume root microbiota. However, it remains largely unknown the molecular basis underlying the isoflavone-mediated interkingdom interactions in the legume rhizosphere. Here, we isolated Variovorax sp. strain V35, a member of the Comamonadaceae that harbors isoflavone-degrading activity, from soybean roots and discovered a gene cluster responsible for isoflavone degradation named ifc. The characterization of ifc mutants and heterologously expressed Ifc enzymes revealed that isoflavones undergo oxidative catabolism, which is different from the reductive metabolic pathways observed in gut microbiota. We further demonstrated that the ifc genes are frequently found in bacterial strains isolated from legume plants, including mutualistic rhizobia, and contribute to the detoxification of the antibacterial activity of isoflavones. Taken together, our findings reveal an isoflavone catabolism gene cluster in the soybean root microbiota, providing molecular insights into isoflavone-mediated legume-microbiota interactions.

Dietary soy protein and soy isoflavones have anti-inflammatory properties. Previously, we reported that feeding soy protein concentrate diet (SPC) with low or high isoflavone (LIF or HIF) to young (seven-week-old) obese (fa/fa) Zucker rats inhibits lipopolysaccharide (LPS) translocation and decreases liver inflammation compared to a casein control (CAS) diet. The current study investigated whether SPC-LIF and SPC-HIF diets would reduce liver inflammation in adult obese Zucker rats fed a CAS diet. A total of 21 six-week-old male obese (fa/fa) Zucker rats were given CAS diet for 8 weeks to develop obesity then randomly assigned to CAS, SPC-LIF, or SPC-HIF (seven rats/group) diet for an additional 10 weeks. The expression of LPS-translocation, inflammation, and intestinal permeability markers were quantified by qPCR in liver, visceral adipose tissue (VAT), and colon. LPS concentration was determined in both the colon content and fecal samples by a Limulus amebocyte lysate (LAL) test. SPC-LIF and SPC-HIF diets significantly decreased liver LPS-binding protein (LBP) expression compared to CAS diet (p < 0.01 and p < 0.05, respectively). SPC-HIF diet also significantly decreased liver MCP-1 and TNF-α expression (p < 0.05) and had a trend to decrease liver iNOS expression (p = 0.06). In the colon, SPC-HIF diet significantly increased LBP expression compared to CAS diet (p < 0.05). When samples from all three groups were combined, there was a negative correlation between colon LBP expression and liver LBP expression (p = 0.046). SPC diets did not alter the expression of intestinal permeability markers (i.e., occludin, claudin 3, and zonula occludens-1) in the colon or inflammation markers (i.e., TNF-α and iNOS) in VAT or the colon. LPS levels in the colon content did not differ between any groups. Fecal LPS levels were significantly higher in the SPC-LIF and SPC-HIF groups compared to the CAS group (p < 0.01). In conclusion, SPC, particularly SPC with HIF, reduces liver LBP expression and inflammation makers (i.e., TNF-α and MCP-1 expression) in adult obese Zucker rats, likely by reducing LPS translocation.

Polycystic ovary syndrome (PCOS), a hormonal and metabolic disorder manifested in women of reproductive age, is still being treated using drugs with side effects. As an alternative to these drugs, isoflavone, also identified as phytoestrogen, has anti-PCOS activity. Isoflavone can help relieve PCOS symptoms by lowering the level of testosterone, which causes hyperandrogenism, thereby normalizing the menstrual cycle and restoring normal ovarian morphology. Furthermore, isoflavone influences the improvement of the metabolic profile, which changes because of PCOS, as well as the reduction of inflammatory markers and oxidative stress. However, both significant and non-significant results have been generated on the activity of isoflavones in PCOS. The present review aims to discuss the existing literature on the effect of isoflavone on PCOS symptoms based on in vivo and clinical trial studies.

The active metabolite (S)-equol, derived from daidzein by gut microbiota, exhibits superior antioxidative activity compared with its precursor and plays a vital role in human health. As only 25% to 50% of individuals can naturally produce equol when supplied with isoflavone, we engineered probiotic E. coli Nissle 1917 (EcN) to convert dietary isoflavones into (S)-equol, thus offering a strategy to mimic the gut phenotype of natural (S)-equol producers. However, co-fermentation of EcN-eq with fecal bacteria revealed that gut microbial metabolites decreased NADPH levels, hindering (S)-equol production. Transcriptome analysis showed that the quorum-sensing (QS) transcription factor SdiA negatively regulates NADPH levels and (S)-equol biosynthesis in EcN-eq. Screening AHLs showed that SdiA binding to C10-HSL negatively regulates the pentose phosphate pathway, reducing intracellular NADPH levels in EcN-eq. Molecular docking and dynamics simulations investigated the structural disparities in complexes formed by C10-HSL with SdiA from EcN or E. coli K12. Substituting sdiA_EcN in EcN-eq with sdiA_K12 increased the intracellular NADPH/NADP+ ratio, enhancing (S)-equol production by 47%. These findings elucidate the impact of AHL-QS in the gut microbiota on EcN NADPH metabolism, offering insights for developing (S)-equol-producing EcN probiotics tailored to the gut environment.

Soybean [Glycine max (L.) Merr.] isoflavones, which are secondary metabolites with various functions, are included in food, cosmetics, and medicine. However, the molecular mechanisms regulating the glycosylation and malonylation of isoflavone glycoconjugates remain unclear. In this study, we conducted an RNA-seq analysis to compare soybean genotypes with different isoflavone contents, including Danbaek and Hwanggeum (low-isoflavone cultivars) as well as DB-088 (high-isoflavone mutant). The transcriptome analysis yielded over 278 million clean reads, representing 39,156 transcripts. The analysis of differentially expressed genes (DEGs) detected 2654 up-regulated and 1805 down-regulated genes between the low- and high-isoflavone genotypes. The putative functions of these 4459 DEGs were annotated on the basis of GO and KEGG pathway enrichment analyses. These DEGs were further analyzed to compare the expression patterns of the genes involved in the biosynthesis of secondary metabolites and the genes encoding transcription factors. The examination of the relative expression levels of 70 isoflavone biosynthetic genes revealed the HID, IFS, UGT, and MAT expression levels were significantly up/down-regulated depending on the genotype and seed developmental stage. These expression patterns were confirmed by quantitative real-time PCR. Moreover, a gene co-expression analysis detected potential protein-protein interactions, suggestive of common functions. The study findings provide valuable insights into the structural genes responsible for isoflavone biosynthesis and accumulation in soybean seeds.

Phytoestrogens, such as isoflavones, are known for their capacity to simulate various physiological impacts of estrogen in the human body. Our research evaluated the effects of isoflavone-enriched soybean leaves (IESL) on collagen fiber loss prompted by ovariectomy in Sprague Dawley (SD) rats, thereby simulating menopausal changes in women. IESL, bolstered with an increased concentration of isoflavones through a metabolite farming process, contained a significantly higher amount of isoflavones than regular soybean leaves. Our results indicate that the administration of IESL can counteract the decrease in relative optical density and dermal thickness of collagen fibers caused by ovariectomy in SD rats, with more pronounced effects observed at higher isoflavone dosages. These outcomes suggest that soybean leaves rich in isoflavones may hold potential benefits in combating collagen degradation and skin aging symptoms related to menopause. Further research is needed to fully understand the exact molecular pathways at play and the potential clinical relevance of these findings.

Soybean (Glycine max (L.) Merrill) isoflavones are among the most important secondary metabolites, with functional benefits for human health. Soybeans accumulate three aglycone forms of isoflavones: genistein, daidzein, and glycitein. Soybean landrace Kumachi-1 does not accumulate malonylglycitin at all. Gene structure analysis indicated that Glyma.11G108300 (F6H4) of Kumachi-1 has a 3.8-kbp insertion, resulting in a truncated flavonoid 6-hydroxylase (F6H) sequence compared to the wild-type sequence in Fukuyutaka. Mapping experiments using a mutant line (MUT1246) with a phenotype similar to that of Kumachi-1, with a single-nucleotide polymorphism (SNP) in F6H4, revealed co-segregation of this mutation and the absence of glycitein isoflavones. We also identified a mutant line (K01) that exhibited a change in the HPLC retention time of glycitein isoflavones, accumulating glycoside and malonylglycoside forms of 6-hydroxydaidzein. K01 contains an SNP that produces a premature stop codon in Glyma.01G004200 (IOMT3), a novel soybean isoflavone O-methyltransferase (IOMT) gene. We further analyzed transgenic hairy roots of soybeans expressing Glyma.11G108300 (F6H4) and Glyma.01G004200 (IOMT3). Those overexpressing F6H4 accumulated malonylglycoside forms of 6-hydroxydaidzein (M_6HD), and co-expression of F6H4 and IOMT3 increased the level of malonylglycitin but not of M_6HD. These results indicate that F6H4 and IOMT3 are responsible for glycitein biosynthesis in soybean seed hypocotyl.

Lipopolysaccharide (LPS) translocation and inflammation contribute to the increased risk of chronic diseases, including non-alcoholic fatty liver disease (NAFLD), associated with obesity. Previously, we reported that feeding soy protein with high or low (negligible) isoflavone reduces liver steatosis in obese Zucker rats, and the reduced steatosis is accompanied by decreased serum C-reactive protein levels. The current study investigated the effect of feeding soy protein concentrate (SPC) with high or low isoflavone (HIF or LIF) on liver inflammation and LPS translocation in obese Zucker rats. Six-week-old male lean (L, n = 21) and obese (O, n = 21) Zucker rats were fed casein control, SPC-LIF, or SPC-HIF diets for 18 weeks. At the end of 18 weeks, the expression levels of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), inducible nitric oxide synthase (iNOS), arginase 1 (ARG1), lipopolysaccharide binding protein (LBP), myeloperoxidase (MPO), and sterol regulatory element-binding protein 1 (SREBP-1) were significantly higher in obese rats compared to lean rats. Compared to the casein control diet, both the SPC-LIF and SPC-HIF diets significantly decreased TNF-α, MCP-1, iNOS, and LBP expression in obese rats, which is accompanied by significantly less LPS staining in liver slides from SPC-LIF-and SPC-HIF-fed obese rats compared to the casein control diet-fed obese rats. Taken together, the SPC-LIF and SPC-HIF diets attenuated liver inflammation in obese Zucker rats, likely by decreasing LPS translocation.