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.

This study aims to investigate the quality changes of germinated soybeans during refrigerated storage (4 °C), with an emphasis on the stimulatory effect of refrigeration on their special functional compounds. After germinating for two days, germinated soybeans were stored at 4 °C for seven days, while the germinated soybeans stored at 25 °C served as control group. The results showed that refrigerated storage significantly affected the physiological changes in germinated soybeans. The weight loss rate, browning rate, malondialdehyde (MDA) content and H2O2 content all decreased dramatically during refrigerated storage compared to the control group. The total phenolic and total flavonoid contents of germinated soybeans under refrigeration exhibited a trend of increasing and then decreasing over time. Additionally, during refrigerated storage, the total isoflavone content reached a peak of 8.72 g/kg on the fifth day, in which the content of daidzein and glycitin increased by 45% and 49% respectively, when compared with the control group. Moreover, the content of γ-aminobutyric acid (GABA) peaked on the first day, and kept a high level during storage. In which, the refrigerated group was 2.35-, 2.88-, 1.67-fold respectively after storage for three to seven days. These results indicated that refrigeration stimulated the biosynthesis of isoflavones and GABA in germinated soybeans during storage. More importantly, there was a sequential difference in the timing of the stimulation of the two functional components under refrigeration.

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.

Auricularia auricula fermentation was performed to reduce anti-nutritional factors, improve nutritional components, and enhance biological activity of soybean. Results showed that the contents of raffinose, stachyose, and trypsin inhibitor were significantly decreased from initial 1.65 g L-1, 1.60 g L-1, and 284.67 μg g-1 to 0.14 g L-1, 0.35 g L-1, and 4.52 μg g-1 after 144 h of fermentation, respectively. Simultaneously, the contents of polysaccharide, total phenolics, and total flavonoids were increased, and melanin was secreted. The isoflavone glycosides were converted to their aglycones, and the contents of glyctin and genistin were decreased from initial 1107.99 μg g-1 and 2852.26 μg g-1 to non-detection after 72 h of fermentation, respectively. After 96 h of fermentation, the IC50 values of samples against DPPH and ABTS radicals scavenging were decreased from 17.61 mg mL-1 and 3.43 mg mL-1 to 4.63 mg mL-1 and 0.89 mg mL-1, and those of samples inhibiting α-glucosidase and angiotensin I-converting enzyme were decreased from 53.89 mg mL-1 and 11.27 mg mL-1 to 18.24 mg mL-1 and 6.78 mg mL-1, respectively, indicating the significant increase in these bioactivities. These results suggested A. auricula fermentation can enhance the nutritional quality and biological activity of soybean, and the fermented soybean products have the potential to be processed into health foods/food additives.

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.

Objective: Metabolic syndrome (MetS) is a global disease burden that has resulted in 10 million people being affected by it, yet no new drugs have been approved for clinical treatment. Isoflavone may be able to stop the development of MetS or enhance its treatment. Therefore, we investigated the relationship between dietary intake of isoflavone and prevalence of MetS to find potentially effective treatments. Methods: We conducted a cross-sectional study using data from 8512 National Health and Nutrition Examination Survey (NHANES) participants from 2007 to 2010 and 2017 to 2018 and their associated isoflavone intake from the flavonoid database in the USDA Food and Nutrient Database for Dietary Studies (FNDDS). We investigated the relationship between MetS status and isoflavone intake by adjusting for confounding variables using multivariable logistic regression models. Results: In a multivariable-adjusted model, there was a negative association between isoflavone intake and the incidence of MetS (odds ratio for Q4 vs. Q1 was 0.66, 95% confidence interval = 0.51-0.86, P = 0.003, p for trend was <0.001). This inverse association remained robust across most subgroups, while nonsignificant interactions were tested between isoflavone intake and age, sex, ethnicity, economic status, body mass index, smoking status, alcohol consumption, and physical activity level (P values for interaction >0.05). Conclusions: We found that MetS prevalence decreased with increased isoflavone intake, suggesting that dietary patterns of soy food or supplement consumption may be a valuable strategy to reduce the disease burden and the prevalence of MetS.

A simple and rapid instantaneous nebulization dispersive liquid-phase microextraction method was developed, and combined with high-performance liquid chromatography for determination of the contents of seven analytes in traditional Chinese medicines. In this study, using the sprinkler device to achieve instantaneous synchronous dispersion and extraction, only one spray can rapidly achieve the concentration and enrichment of seven kinds of chalcone and isoflavones. The key factors affecting the extraction efficiency were optimized including the type and volume of extractant, the pH and salt concentration of the sample phase, and the number of dispersion. Under the optimal conditions, the enrichment factor of the target analytes ranged from 103.1 to 180.9, with good linearity and correlation coefficients above 0.9970. The limits of detection ranged from 0.02 to 0.15 ng/mL, with good accuracy (recoveries 91.1 to 108.9%) and precision (relative standard deviations 1.5-7.1%). This method has short extraction time (2 s), low organic solvent consumption and high enrichment effect, so it has a wide application prospects.

BACKGROUND: Triple-negative breast cancer (TNBC) is a heterogeneous carcinoma characterized by the most aggressive phenotype among all breast cancer subtypes. However, therapeutic options for TNBC patients have limited clinical efficacy due to lack of specific target and efficient targeted therapeutics.
OBJECTIVE: To investigate the biological characteristics of a novel estrogen receptor (ER)-α splice variant ER-α30 in breast cancer cells, and its possible role in the anticancer effects of calycosin, a typical phytoestrogen derived from the herbal plant Astragalus membranaceus, against TNBC. This may also provide a better understanding of the inhibitory activity of calycosin on TNBC progression.
METHODS: Breast cancer tissues and para-cancer tissues were collected and analyzed for the expression levels of ER-α30 using immunohistochemistry (IHC), and its expression in two TNBC cell lines (MDA-MB-231 and BT-549) was detected by western blot and qRT-PCR assays. Then the alteration of cell viability, apoptosis, migration, invasion and epithelial-mesenchymal transition (EMT) in response to overexpression or knockdown of ER-α30 was separately determined by CCK-8, Hoechst 33258, wound healing, transwell and western blot assays in two TNBC cell lines. Next, the anticancer effects of calycosin on MDA-MB-231 cells were evaluated through CCK-8, colony formation, flow cytometry, Hoechst 33258 and western blot assays, along with the role of ER-α30 in these effects and the possible downstream targets of ER-α30. In addition, the in vivo experiments were carried out using MDA-MB-231 xenograft model intraperitoneally treated with calycosin. The volume and weight of xenograft tumor were measured to evaluate the in vivo anticancer activities of calycosin, while the corresponding changes of ER-α30 expression in tumor tissues were detected by IHC.
RESULTS: It was demonstrated that the novel ER-α splice variant ER-α30 was primarily distributed in the nucleus of TNBC cells. Compared with normal breast tissues, ER-α30 expression was found in significantly higher levels in breast cancer tissues of ER- and progesterone receptor (PR)-negative subtype, so did in TNBC cell lines (MDA-MB-231 and BT-549) when compared to normal breast cell line MCF10A. Moreover, ER-α30 overexpression strikingly enhanced cell viability, migration, invasion and EMT progression and reduced apoptosis in TNBC cells, whereas shRNA-mediated knockdown of ER-α30 revealed the opposite results. Notably, calycosin suppressed the expression of ER-α30 in a dose-dependent manner, accompanied with the inhibition of TNBC growth and metastasis. A similar finding was observed for the xenografts generated from MDA-MB-231 cells. The treatment with calycosin suppressed the tumor growth and decreased ER-α30 expression in tumor tissues. Furthermore, this inhibition by calycosin was more pronounced in ER-α30 knockdown cells. Meanwhile, we found a positive relationship between ER-α30 and the activity of PI3K and AKT, which could also be inactivated by calycosin treatment.
CONCLUSIONS: For the first time, it is demonstrated that the novel estrogen receptor-α splice variant ER-α30 could function as pro-tumorigenic factor in the context of TNBC by participating in cell proliferation, apoptosis, invasion and metastasis, thus it may serve as a potential therapeutic target for TNBC therapy. Calycosin could reduce the activation of ER-α30-mediated PI3K/AKT pathway, thereby inhibited TNBC development and progression, suggesting that calycosin may be a potential therapeutic option for TNBC.

Soybeans have medicinal value and are an oil crop with medicinal and food properties. The present work investigated two aspects of isoflavone accumulation in soybean. First, germination conditions for exogenous-ethephon-mediated accumulation of isoflavone were optimised through response surface methodology. Second, various influences of ethephon on the growth of germinating soybeans and isoflavone metabolism were investigated. The findings of the research led to the conclusion that exogenous ethephon treatment effectively facilitated the enrichment of isoflavones in soybeans during germination. Optimal germination conditions were obtained through a response surface optimization test, which yielded the following criteria: a germination time of 4.2 d, an ethephon concentration of 102.6 μM, and a germination temperature of 30.2 °C. The maximum isoflavone content was 544.53 μg/sprout FW. Relative to the control, the addition of ethephon significantly inhibited sprout growth. Exogenous ethephon treatment led to the phenomenon that peroxidase, superoxide dismutase, and catalase activities and their gene expression increased significantly in germinating soybeans. Meanwhile, the expression of genes related to ethylene synthetase increase under the effect of ethephon promoting ethylene synthesis. Ethylene multiplied the total flavonoid content of soybean sprouts relying on the increase in activity and gene expression of crucial isoflavone biosynthesis-related enzymes (phenylalanine ammonia-lyase and 4-coumarate coenzyme A ligase) during germination.

BACKGROUND: Epidemiological studies that focus on the relationship between dietary isoflavone intake and the risk of breast cancer still lead to inconsistent conclusions. Herein, we conducted a meta-analysis of the latest studies to explore this issue.
METHODS: We performed a systematic search using Web of Science, PubMed, and Embase from inception to August 2021. The robust error meta-regression (REMR) model and generalized least squares trend (GLST) model were used to establish dose-response relationships between isoflavones and breast cancer risk.
RESULTS: Seven cohort studies and 17 case-control studies were included in the meta-analysis, and the summary OR for breast cancer was 0.71 (95% CI 0.72-0.81) when comparing the highest to the lowest isoflavone intake. A subgroup analysis further showed that neither menopausal status nor ER status has a significant influence on the association between isoflavone intake and breast cancer risk, while the isoflavone intake doses and study design does. When the isoflavones exposure was less than 10 mg/day, no effects on breast cancer risk were detected. The inverse association was significant in the case-control studies but not in the cohort studies. In the dose-response meta-analysis of the cohort studies, we observed an inverse association between isoflavone intake and breast cancer: a 10 mg/day increase in isoflavone intake was related to reductions of 6.8% (OR = 0.932, 95% CI 0.90-0.96) and 3.2% (OR = 0.968, 95% CI 0.94-0.99) in breast cancer risk when using REMR and GLST, respectively. In the dose-response meta-analysis of the case-control studies, the inverse association for every 10 mg/day isoflavone intake was associated with breast cancer risk reductions by 11.7%.
CONCLUSIONS: present evidence demonstrated that taking in dietary isoflavone is helpful in reducing the breast cancer risk.