The Metabolites and Mechanism Analysis of Genistin against Hyperlipidemia via the UHPLC-Q-Exactive Orbitrap Mass Spectrometer and Metabolomics.

Genistin, an isoflavone, has been reported to have multiple activities. However, its improvement of hyperlipidemia is still unclear, and the same is true with regard to its mechanism. In this study, a high-fat diet (HFD) was used to induce a hyperlipidemic rat model. The metabolites of genistin in normal and hyperlipidemic rats were first identified to cause metabolic differences with Ultra-High-Performance Liquid Chromatography Quadrupole Exactive Orbitrap Mass Spectrometry (UHPLC-Q-Exactive Orbitrap MS). The relevant factors were determined via ELISA, and the pathological changes of liver tissue were examined via H&E staining and Oil red O staining, which evaluated the functions of genistin. The related mechanism was elucidated through metabolomics and Spearman correlation analysis. The results showed that 13 metabolites of genistin were identified in plasma from normal and hyperlipidemic rats. Of those metabolites, seven were found in normal rat, and three existed in two models, with those metabolites being involved in the reactions of decarbonylation, arabinosylation, hydroxylation, and methylation. Three metabolites, including the product of dehydroxymethylation, decarbonylation, and carbonyl hydrogenation, were identified in hyperlipidemic rats for the first time. Accordingly, the pharmacodynamic results first revealed that genistin could significantly reduce the level of lipid factors (p < 0.05), inhibited lipid accumulation in the liver, and reversed the liver function abnormalities caused by lipid peroxidation. For metabolomics results, HFD could significantly alter the levels of 15 endogenous metabolites, and genistin could reverse them. Creatine might be a beneficial biomarker for the activity of genistin against hyperlipidemia, as revealed via multivariate correlation analysis. These results, which have not been reported in the previous literature, may provide the foundation for genistin as a new lipid-lowering agent.

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

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

Experimental and clinical studies on the effects of Portulaca oleracea L. and its constituents on respiratory, allergic, and immunologic disorders, a review.

Various pharmacological effects for Portulaca oleracea were shown in previous studies. Therefore, the effects of P. oleracea and its derivatives on respiratory, allergic, and immunologic diseases according to update experimental and clinical studies are provided in this review article. PubMed/Medline, Scopus, and Google Scholar were searched using appropriate keywords until the end of December 2020. The effects of P. oleracea and its constituents such as quercetin and kaempferol on an animal model of asthma were shown. Portulaca oleracea and its constituents also showed therapeutic effects on chronic obstructive pulmonary disease and chronic bronchitis in both experimental and clinical studies. The possible bronchodilatory effect of P. oleracea and its ingredients was also reported. Portulaca oleracea and its constituents showed the preventive effect on lung cancer and a clinical study showed the effect of P. oleracea on patients with lung adenocarcinoma. In addition, a various constituents of P. oleracea including, quercetin and kaempferol showed therapeutic effects on lung infections. This review indicates the therapeutic effect of P. oleracea and its constituents on various lung and allergic disorders but more clinical studies are required to establish the clinical efficacy of this plant and its constituents on lung and allergic disorders.

Isoflavones Suppress Cyp26b1 Expression in the Murine Colonic Lamina Propria.

Isoflavones have many biological activities and are major bioactive components of kakkonto, a traditional Japanese herbal medicine. We previously reported that the combined therapy of oral immune therapy (OIT) and kakkonto downregulates the mRNA expression of Cyp26b1, a major retinoic acid (RA)-degrading enzyme, in the colon of food allergy mice and thereby ameliorates allergic symptoms. In this study, we evaluated the effects of various isoflavones on Cyp26b1 expression in primary cultured lamina propria (LP) cells isolated from the mouse colon. The mRNA expression of Cyp26b1 was extremely downregulated by all isoflavones tested in the LP cells except for puerarin. In particular, genistein and genistin markedly suppressed Cyp26b1 mRNA expression without affecting RA-synthesizing enzyme expression. Moreover, to evaluate the effects of isoflavones on allergic reactions, genistein and genistin were administered to ovalbumin (OVA)-induced food allergy mice. Oral administration of genistin suppressed the development of allergic symptoms. These results raise the possibility that isoflavones elevated the level of RA in the colon by inhibiting RA degradation and then the high concentration of RA in the colon might exert immunosuppressive and antiallergic effects on food allergy mice.

Genistin: A Novel Potent Anti-Adipogenic and Anti-Lipogenic Agent.

Soy isoflavones are popular ingredients with anti-adipogenic and anti-lipogenic properties. The anti-adipogenic and anti-lipogenic properties of genistein are well-known, but those of genistin and glycitein remain unknown, and those of daidzein are characterized by contrasting data. Therefore, the purpose of our study was to investigate the effects of daidzein, glycitein, genistein, and genistin on adipogenesis and lipogenesis in 3T3-L1 cells. Proliferation of 3T3-L1 preadipocytes was unaffected by genistin and glycitein, but it was affected by 50 and 100 µM genistein and 100 µM daidzein for 48 h. Among the four isoflavones, only 50 and 100 µM genistin and genistein markedly suppressed lipid accumulation during adipogenesis in 3T3-L1 cells through a similar signaling pathway in a dose-dependent manner. Genistin and genistein suppress adipocyte-specific proteins and genes, such as peroxisome proliferator-activated receptor γ (PPARγ), CCAAT-enhancer-binding protein α (C/EBPα), and adipocyte binding protein 2 (aP2)/fatty acid-binding protein 4 (FABP4), and lipogenic enzymes such as ATP citrate lyase (ACL), acetyl-CoA carboxylase 1 (ACC1), and fatty acid synthase (FAS). Both isoflavones also activate AMP-activated protein kinase α (AMPKα), an essential factor in adipocyte differentiation, and inhibited sterol regulatory element-binding transcription factor 1c (SREBP-1c). These results indicate that genistin is a potent anti-adipogenic and anti-lipogenic agent.

[Characteristic fingerprint and multi-components quantitative determination for Fuke Qianjin Capsules by HPLC].

To establish an HPLC characteristic fingerprint method of Fuke Qianjin Capsules,and determine the contents of its main components. The analysis was carried out on a Kromasil 100-5-C18 analytical column(4. 6 mm ×250 mm,5 µm) with gradient elution by acetonitrile(A)-0. 1% phosphoric acid aqueous solution(B),a flow rate at 1. 0 m L·min-1 and the detection wavelength of 254 nm.The column temperature was 30 ℃,and the injection volume was 10 µL. The determination method of genistin,jatrorrhizine,andrographolide and 14-deoxy-11,12-didehydroandrographolide index components were studied methodologically. The common mode of the characteristic fingerprint of Fuke Qianjin Capsules was set up with 8 common peaks,which were identified as genistin,jatrorrhizine,palmatine,berberine,andrographolide,14-deoxy-11,12-didehydroandrographolide,Z-ligustilide,and Z-3-butylidenephthalide,respectively,in comparison with the references. The similarities of 20 batches of Fuke Qianjin Capsules samples were above 0. 95. All of the above-mentioned 4 analytes could be well separated under the optimized chromatographic conditions. RSD of precision and repeatability experiment were both less than 1. 5%,and the sample solution was stable during 72 h. All of the compounds had a good linearity and linear range. The contents of genistin,jatrorrhizine,andrographolide,and 14-deoxy-11,12-didehydroandrographolide in 20 batches of Fuke Qianjin Capsules samples were 28. 66-56. 04,94. 77-197. 92,1 705. 33-4 148. 93 and 462. 16-1 225. 96 µg in each capsule,respectively. The developed HPLC characteristic fingerprint and quantitative analysis methods were reliable,accurate and sensitive,and could be used effectively evaluate the quality of Fuke Qianjin Capsules samples.

A Comprehensive Screening and Identification of Genistin Metabolites in Rats Based on Multiple Metabolite Templates Combined with UHPLC-HRMS Analysis.

Genistin, an isoflavone belonging to the phytoestrogen family, has been reported to possess various therapeutic effects. In the present study, the genistin metabolites in rats were investigated by UHPLC-LTQ-Orbitrap mass spectrometer in both positive and negative ion modes. Firstly, the data sets were obtained based on data-dependent acquisition method and then 10 metabolite templates were established based on the previous reports. Then diagnostic product ions (DPIs) and neutral loss fragments (NLFs) were proposed to efficiently screen and ascertain the major-to-trace genistin metabolites. Meanwhile, the calculated Clog P values were used to identify the positional isomers with different retention times. Consequently, a total of 64 metabolites, including prototype drug, were positively or putatively characterized. Among them, 40 metabolites were found according to the templates of genistin and genistein, which was the same as the previous research method. After using other metabolite templates, 24 metabolites were added. The results demonstrated that genistin mainly underwent methylation, hydrogenation, hydroxylation, glucosylation, glucuronidation, sulfonation, acetylation, ring-cleavage and their composite reactions in vivo biotransformation. In conclusion, the research not only revealed the genistein metabolites and metabolic pathways in vivo comprehensively, but also proposed a method based on multiple metabolite templates to screen and identify metabolites of other natural compounds.

An Enzyme-linked Immunosorbent Assay for Genistein 7-O-[α-rhamnopyranosyl-(1→6)]-ß-glucopyranoside Determination in Derris scandens using a Polyclonal Antibody.

INTRODUCTION: Genistein 7-O-[α-rhamnopyranosyl-(1→6)]-ß-glucopyranoside (GTG) is a major bioactive compound in Derris scandens. It is responsible for anti-inflammatory activity by inhibition of cyclooxygenase and lipoxygenase. There are many commercial products of D. scandens available in Thailand. OBJECTIVE: To develop an enzyme-linked immunosorbent assay (ELISA) for the quantitative analysis of GTG in plant material and derived products using a polyclonal antibody. METHODS: An immunogen was synthesised by conjugating GTG with a carrier protein. The polyclonal antibody against GTG (GTG-PAb) was produced in New Zealand white rabbits. The ELISA method was validated for specificity, sensitivity, accuracy, precision and correlation with HPLC. RESULTS: The polyclonal antibody was specific to GTG and genistin within the range of compounds tested. The GTG ELISA was applied in the range 0.04-10.00 µg/mL with a limit of detection of 0.03 µg/mL. The recovery of GTG in spiked Derris scandens extracts ranged from 100.7 to 107.0%, with a coefficient of variation less than 7.0%. The intra- and inter-assay variations were less than 5.0%. The ELISA showed a good correlation with HPLC-UV analysis for GTG determination in samples, with a coefficient of determination (r2 ) of 0.9880. CONCLUSION: An ELISA was established for GTG determination in Derris scandens. The GTG-PAb can react with GTG and genistin, but genistin has not been found in the plant. Therefore, the ELISA can be used for high throughput quality control of GTG content in D. scandens and its products. Copyright © 2016 John Wiley & Sons, Ltd.

Improving Free Radical Scavenging Activity of Soy Isoflavone Glycosides Daidzin and Genistin by 3'-Hydroxylation Using Recombinant Escherichia coli.

The present study describes the biotransformation of a commercially available crude extract of soy isoflavones, which contained significant amounts of the soy isoflavone glycosides daidzin and genistin, by recombinant Escherichia coli expressing tyrosinase from Bacillus megaterium. Two major products were isolated from the biotransformation and identified as 3'-hydroxydaidzin and 3'-hydroxygenistin, respectively, based on their mass and nuclear magnetic resonance spectral data. The two 3'-hydroxyisoflavone glycosides showed potent 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity with IC50 values of 7.4 and 9.8 µM for 3'-hydroxydaidzin and 3'-hydroxygenistin, respectively. The free radical scavenging activities of the two 3'-hydroxyisoflavone glycosides were, respectively, 120 and 72 times higher than the activity of their precursors, daidzin and genistin, and were also stronger than the activity of ascorbic acid, which showed an IC50 value of 15.1 µM. This is the first report of the bio-production and potential antioxidant applications of both 3'-hydroxydaidzin and 3'-hydroxygenistin.

Soybean resistance to stink bugs (Nezara viridula and Piezodorus guildinii) increases with exposure to solar UV-B radiation and correlates with isoflavonoid content in pods under field conditions.

Solar UV-B radiation (280-315 nm) has a significant influence on trophic relationships in natural and managed ecosystems, affecting plant-insect interactions. We explored the effects of ambient UV-B radiation on the levels of herbivory by stink bugs (Nezara viridula and Piezodorus guildinii) in field-grown soybean crops. The experiments included two levels of UV-B radiation (ambient and attenuated UV-B) and four soybean cultivars known to differ in their content of soluble leaf phenolics. Ambient UV-B radiation increased the accumulation of the isoflavonoids daidzin and genistin in the pods of all cultivars. Soybean crops grown under attenuated UV-B had higher numbers of unfilled pods and damaged seeds than crops grown under ambient UV-B radiation. Binary choice experiments with soybean branches demonstrated that stink bugs preferred branches of the attenuated UV-B treatment. We found a positive correlation between percentage of undamaged seeds and the contents of daidzin and genistin in pods. Our results suggest that constitutive and UV-B-induced isoflavonoids increase plant resistance to stink bugs under field conditions.

Pharmaco- and toxicokinetics of selected exogenous and endogenous estrogens: a review of the data and identification of knowledge gaps.

Chemicals with estrogenic activity are derived from many different natural and synthetic processes and products, including endogenous production (e.g., estradiol, conjugated estrogens), drugs (e.g., ethinyl estradiol, conjugated estrogens), plants used as foods (phytoestrogens such as genistein, daidzein, S-equol), and man-made chemicals (xenoestrogens such as bisphenol A). Human exposure to low doses of endogenous estrogens, estrogenic drugs, phytoestrogens, and xenoestrogens has the potential to improve health or disrupt normal endocrine activity, as well as impact the diverse systems with which estrogens interact, including the cardiovascular system, and lipid and carbohydrate metabolism. Mechanisms of action and diversity of adverse and non-adverse effects following human exposure to low doses of estrogen active chemicals (EACs, defined as chemicals which interact with an estrogen receptor [ER]) are poorly understood. This review summarizes our current understanding of the pharmacological action with a focus on pharmacokinetics (PK) and toxicokinetics (TK) of several representative EACs in both physiological and pathological processes. The goal of this review is to assess the current state-of-the-science on: (i) the potential for EACs to interfere with endocrine activity, (ii) factors which contribute to endocrine-related clinical outcomes, and (iii) existing knowledge gaps. While classical PK approaches (compartmental or non-compartmental) can be used to characterize absorption, distribution, metabolism, and elimination of EACs, many of the detailed pharmacological characteristics necessary to understand benefit-risk balance have not yet been clarified. Pharmacological complexities mirror the complexity of determining whether and under what conditions exposure to estrogens in drugs, foods or to xenoestrogenic chemicals are beneficial or harmful to human health.

Effect of ultrasound assisted extraction upon the Genistin and Daidzin contents of resultant soymilk.

The purpose of this study was to determine the effect of ultrasound treatment on the contents of daidzin, genistin, and their respective aglycones, daidzein and genistein, in resultant soymilk. Soybean slurry was exposed to ultrasound treatment, filtered, and placed in an ultrasound cleaning bath set with different frequencies (35and 130 KHz), treatment temperatures (20 and 40 °C), and times (20, 40, and 60 min). Concentrations for these isoflavones were determined using reverse-phase high-performance liquid chromatography. Results indicated that both frequencies significantly (p < 0.05) increased isoflavone content (IC), glycosides, and aglycones in extracted soymilk. These results were attributed to induced cavitation, which increases the permeability of plant tissues. However, the frequency of 35 kHz caused a noticeably higher increase in IC than 130 kHz. Results also revealed significant increases in IC with increased sonication time (from 20 to 60 min) and with increased temperature (from 20 to 40 °C).

Flavonoids and their relationship with the physiological quality of seeds from different soybean genotypes.

Flavonoids are compounds that result from the secondary metabolism of plants and play a crucial role in plant development and mitigating biotic and abiotic stresses. The highest levels of flavonoids are found in legumes such as soybean. Breeding programs aim to increase desirable traits, such as higher flavonoid contents and vigorous seeds. Soybeans are one of the richest sources of protein in the plant kingdom and the main source of flavonoid derivatives for human health. In view of this, the hypothesis of this study is based on the possibility that the concentration of isoflavones in soybean seeds contributes to the physiological quality of the seeds. The aim of this study was to analyze the content of flavonoids in soybean genotypes and their influence on the physiological quality of the seeds. Seeds from thirty-two soybean genotypes were obtained by carrying out a field experiment during the 2021/22 crop season. The experimental design was randomized blocks with four replications and thirty-two F3 soybean populations. The seeds obtained were subjected to germination, first germination counting, electrical conductivity and tetrazolium vigor and viability tests. After drying and milling the material from each genotype, liquid chromatography analysis was carried out to obtain flavonoids, performed at UPLC level. Data were submitted to analysis of variance and, when significant, the means were compared using the Scott-Knott test at 5% probability. The results found here show the occurrence of genotypes with higher amounts of flavonoids when compared to their peers. The flavonoid FLVD_G2 had the highest concentration and differed from the others. Thus, we can assume that the type and concentration of flavonoids does not influence the physiological quality of seeds from different soybean genotypes, but it does indirectly contribute to viability and vigor, since the genotypes with the highest FLVD_G2 levels had better FGC values. The findings indicate that there is a difference between the content of flavonoids in soybean genotypes, with a higher content of genistein. The content of flavonoids does not influence the physiological quality of seeds, but contributes to increasing viability and vigor.

Genistin: A Novel Estrogen Analogue Targeting ERß to Alleviate Thrombocytopenia.

Thrombocytopenia, a prevalent hematologic challenge, correlates directly with the mortality of numerous ailments. Current therapeutic avenues for thrombocytopenia are not without limitations. Here, we identify genistin, an estrogen analogue, as a promising candidate for thrombocytopenia intervention, discovered through AI-driven compound library screening. While estrogen's involvement in diverse biological processes is recognized, its role in thrombopoiesis remains underexplored. Our findings elucidate genistin's ability to enhance megakaryocyte differentiation, thereby augmenting platelet formation and production. In vivo assessments further underscore genistin's remedial potential against radiation-induced thrombocytopenia. Mechanistically, genistin's efficacy is attributed to its direct interaction with estrogen receptor ß (ERß), with subsequent activation of both ERK1/2 and the Akt signaling pathways membrane ERß. Collectively, our study positions genistin as a prospective therapeutic strategy for thrombocytopenia, shedding light on novel interplays between platelet production and ERß.

Biosynthesis and Anticancer Activity of Genistein Glycoside Derivatives.

As a beneficial natural flavonoid, genistein has demonstrated a wide range of biological functions via regulating a number of targets and signaling pathways, such as anti-cancer, antioxidant, antibacterial, antiinflammatory, antifungal, antiviral, iron chelation, anti-obesity, anti-diabetes, and anti-hypertension. Pub- Med/Medline and Web of Science were searched using appropriate keywords until the end of December 2023. Despite its many potential benefits, genistein's clinical application is limited by low hydrophilicity, poor solubility, and suboptimal bioavailability due to its structure. These challenges can be addressed through the conversion of genistein into glycosides. Glycosylation of active small molecules may enhance their solubility, stability, and biological activity. In recent years, extensive research has been conducted on the synthesis, properties, and anticancer activity of glycoconjugates. Previous reviews were devoted to discussing the biological activities of genistin, with a little summary of the biosynthesis and the structure-activity relationship for their anticancer activity of genistein glycoside derivatives. Therefore, we summarized recent advances in the biosynthesis of genistein glycosylation and discussed the antitumor activities of genistein glycoside derivatives in a structure-activity relationship, which may provide important information for further development of genistein derivatives.

Genistin Represses the Proliferation and Angiogenesis While Accelerating the Apoptosis of Glioma Cells by Modulating the FOXC1-Mediated Wnt Signaling Pathway.

BACKGROUND: Glioma is a tumor originating from glial cells and is the most common primary brain tumor. At present, the main treatment methods for glioma include surgical resection and radiotherapy and chemotherapy, but the treatment effect is not very ideal. Genistin (GS) inhibits breast cancer cell growth while promoting apoptosis, but its effect and detailed molecular mechanism on glioma are yet to be defined. In addition, forkhead box C1 (FOXC1) has been found to be involved in the growth, invasion, and angiogenesis processes of glioma cells. METHODS: Human glioma cells in the Control, GS-6.25, GS-12.5, and GS25 (GS) groups were treated with 0, 6.25, 12.5, and 25 µM of Genistin, respectively, for 72 hours, and cells in the GS + NC (negative control) and GS + FOXC1 groups were transfected with negative control or forkhead box C1 (FOXC1) overexpression plasmids, respectively, prior to Genistin (25 µM) treatment for 72 hours. Next, the viability, proliferation, apoptosis, and angiogenesis of treated glioma cells were detected using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'deoxyuridine (EdU) proliferation, flow cytometry, and tube formation assays. Meanwhile, the half-maximal inhibitory concentration (IC50) of Genistin in the treated glioma cells was calculated. Afterwards, quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot quantified the levels of FOXC1, Wnt1, Wnt3a, glycogen synthase kinase-3ß (GSK3ß), and phosphorylated GSK3ß (p-GSK3ß). RESULTS: Genistin inhibited viability, proliferation, and angiogenesis while promoting the apoptosis of glioma cells (p < 0.05, p < 0.001). Also, Genistin decreased the levels of FOXC1, Wnt1, and Wnt3a while increasing p-GSK3ß levels in glioma cells (p < 0.05, p < 0.01, p < 0.001). FOXC1 was up-regulated in glioma cells and tissues, and overexpressed FOXC1 overturned the effects of Genistin on the abovementioned factors in glioma cells (p < 0.05, p < 0.001). CONCLUSIONS: Genistin inhibits viability, proliferation, and angiogenesis while accelerating glioma cell apoptosis by modulating the FOXC1-mediated Wnt signaling pathway.

Sprouting facilitates the antiglycative effect of black soybean (Glycine max (L.) Merr.) by promoting the accumulation of isoflavones.

The exposure of advanced glycation end products (AGEs) can induce chronic inflammation, oxidative stress, and accelerated aging, contributing the onset and progression of many diseases especially diabetic complications. Therefore, the searching of antiglycative foods is of practical significance, which may serve as a strategy in the attenuation of AGEs-associated diseases. In this study, we evaluated the antiglycative potential of some beans and bean sprouts that were common in our daily life. The results revealed that sprouting enhanced the antiglycative activity of beans, with black soybean sprouts demonstrating the highest efficacy (4.92-fold higher than the unsprouted beans). To assess practical implications, we examined the antiglycative activity of black soybean sprouts in pork soup, a popular food model that incorporates sprouts. Our findings confirmed the inhibitory effect on a dose-dependent manner. Through open column fractionation, we identified isoflavones and soyasaponin Bb as the candidates responsible for these effects. Additionally, compare to the unsprouted black soybeans, we found significant increases in the levels of antioxidative properties (2.51-fold), total phenolics (7.28-fold), isoflavones, and soyasaponin Bb during the sprouting process. Further studies determined that genistein, genistin, and daidzin were the major antiglycative compounds in black soybean sprouts. Collectively, this study emphasizes the benefits of sprouted beans and offers foundation for the development of functional sprouting foods.

Study on biotransformation and absorption of genistin based on fecal microbiota and Caco-2 cell.

Introduction: Genistin, as a kind of natural isoflavone glycoside, has good biological activity, and its weak absorption makes it closely related to intestinal flora. However, the role of the intestinal flora is still unclear and whether the metabolites produced by the intestinal flora are absorbed systemically is also variable. Methods: Genistin was fermented for 24 h based on fecal bacteria fermentation technology. The components were qualitatively and quantitatively analyzed by HPLC and UHPLC-Q-Exactive Orbitrap Mass spectrometry. The composition of intestinal flora in fermentation samples from fecal bacteria was detected by 16S rRNA sequencing. Five representative probiotics were cultured in vitro and fermented with genistin to determine similarities and differences in genistin metabolites by different bacteria at different times. Finally, the absorption results of metabolites by fermentation were verified by a Caco-2 cell monolayer. Results: The HPLC results of fecal fermentation showed that genistein levels increased from 0.0139 ± 0.0057 mg/mL to 0.0426 ± 0.0251 mg/mL and two new metabolites were produced. A total of 46 metabolites following fecal fermentation were identified, resulting from various biotransformation reaction products, such as decarbonylation, hydroxylation, and methylation. Simultaneously, the 16S rRNA results showed that the intestinal flora changed significantly before and after fermentation and that the intestinal microorganisms in the control (Con) group and the fermentation (Fer) group showed a significant separation trend. Five genera, Lactobacillus, Bifidobacterium, Parabacteroides, Sutterella, and Dorea, were considered the dominant flora for genistin fermentation. The qualitative results of fermentation of genistin by five probiotics at different times showed that there were significant differences in small molecule metabolites by fermentation of different bacteria. Meanwhile, most metabolites could be identified following fecal bacteria fermentation, which verified the importance of the dominant bacteria in the feces for the biotransformation of components. Finally, the absorption results of the metabolites based on the Caco-2 cell monolayer showed that 14 metabolites could be absorbed into the circulation in vivo through the mesentery. Discussion: The small molecule metabolites of genistin by fermentation of fecal bacteria can be well absorbed systemically by the body. These studies provide a reference value for explaining the transformation and absorption of flavonoid glycosides in the intestine.

PACAP/VIP in the prefrontal cortex mediates the rapid antidepressant effects of zhizichi decoction.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhizichi decoction (ZZCD) is a traditional Chinese medicine formula that consists of Gardenia jasminoides J.Ellis (GJ) and Semen Sojae Praeparatum. It is used to treat insomnia and emotion-related disorders, such as irritability. Previous studies have found that GJ has a rapid antidepressant effect. The study found that ZZCD is safer than GJ at the same dosage. Consequently, ZZCD is a superior drug with quicker antidepressant effects than GJ. The rapid antidepressant effects of ZZCD were examined in this study, along with the components that make up this effect. It was determined that the activation of prefrontal Pituitary Adenylate Cyclase Activating Polypeptide (PACAP)/Vasoactive Intestinal Polypeptide (VIP) is essential for ZZCD's rapid antidepressant effects. AIM: This study identified and discussed the rapid antidepressant effects and biological mechanisms of ZZCD. MATERIALS AND METHODS: The tail suspension test (TST) and the forced swimming test (FST) were used to screen the effective dosage of ZZCD (0.67 g/kg, 1 g/kg, 4 g/kg). The effective dosage of ZZCD (1 g/kg) was tested in the TST conducted on Institute of Cancer Research (ICR) mice that were treated with lipopolysaccharide (LPS) at a concentration of 0.1 mg/mL. To confirm the expression of c-Fos, PACAP, and VIP in the prefrontal cortex (PFC), immunohistochemistry tests were conducted on mice following intragastric injection of ZZCD. Chemical characterization analysis and HPLC quality control analysis were conducted using UHPLC-Q-Obitrap-HRMS and chromatographic analysis. RESULTS: The results showed that an acute administration of ZZCD (1 g/kg) decreased the immobility time of Kunming (KM) mice in TST and FST. Depressive behaviors in TST-induced ICR mice treated with LPS (0.1 mg/mL) were reversed by ZZCD (1 g/kg). The results of immunohistochemical experiments showed that ZZCD (1 g/kg) activated neurons in the PFC and PACAP/VIP in the PFC. In this study, 22 substances in ZZCD were identified. Five primary distinctive fingerprint peaks-geniposide, genistin, genipin-1-ß-D-gentiobioside, glycitin, and daidzin-were found among the ten common peaks. CONCLUSION: ZZCD (1 g/kg) had significant rapid antidepressant effects. PACAP/VIP in the PFC was found to mediate the rapid antidepressant effects of ZZCD.

Transformation profiles of the isoflavones in germinated soybean based on UPLC-DAD quantification and LC-QTOF-MS/MS confirmation.

Germinated soybean is one kind of food and a medicine. In the actual process of producing a large amount of naturally germinated soybean, it is difficult to strictly control the germination process conditions. However, sprout length may be more suitable as the terminal judgment indicator for naturally germinated soybean. An UPLC-DAD method was developed and validated to explore the transformation profiles of soybean isoflavones in germinated yellow or black soybean with different sprout lengths. Moreover, an LC - QTOF-MS/MS method was used to avoid false positive results. The contents of daidzein, glycitein, and genistein almost reached their corresponding maximum values when the sprout length ranged from 1.0 cm to 1.5 cm (P < 0.05). Therefore, yellow soybean is suggested to be the processing raw material with higher contents of those isoflavones, and the optimal sprout length for germinated soybean may be in the range of 1.0-1.5 cm.