The isolation, characterization and biological activities of the non-glucan polysaccharides from the high-starch-content plant Pueraria mirifica.

The dried root of Pueraria mirifica (P. mirifica) is an edible foodstuff widely used in Asian countries. P. mirifica is known for its high starch content. The isolation of polysaccharides from high-starch plant parts is challenging due to the interference of starch. Therefore, this study aimed to develop a technique for isolating and investigating the structure and activity of non-glucan polysaccharides from P. mirifica (PMP). An effective starch removal process was developed using α-amylase hydrolysis and thorough membrane dialysis. Four non-glucan polysaccharides were isolated, and PMP-2 was subjected to structural elucidation. The results indicated that PMP-2 has a molecular weight of 124.4 kDa and that arabinose and galactose are the main components, accounting for 27.8 % and 58.5 %, respectively. Methylation and NMR analysis suggested that PMP-2 is an Arabinogalactan composed of 1,6-linked Galp and 1,4-linked Galp as the main chain, with arabinan and rhamnose as side chains. Furthermore, PMP-C and PMP-2 exhibited concentration-dependent antioxidant activities against DPPH, ABTS, and hydroxyl radicals and certain immunomodulatory activities related to the release of NO, TNF-α and IL-6. These findings suggest that PMP-2 has potential therapeutically active ingredient in functional foods. The developed method successfully removed starch and isolated non-glucan polysaccharides from the high-starch content plant P. mirifica and can be applied to other high-starch plants.

[Effect and mechanism of Puerariae Lobatae Radix in alleviating insulin resistance in T2DM db/db mice based on intestinal flora].

This study aimed to examine the effect and underlying mechanism of Puerariae Lobatae Radix on insulin resistance in db/db mice with type 2 diabetes mellitus(T2DM) based on the analysis of intestinal flora. Fifty db/db mice were randomly divided into a model group(M group), a metformin group(YX group), a high-dose Puerariae Lobatae Radix group(YGG group), a medium-dose Puerariae Lobatae Radix group(YGZ group), and a low-dose Puerariae Lobatae Radix group(YGD group). Another 10 db/m mice were assigned to the normal group(K group). After continuous administration for eight weeks, body weight and blood sugar of mice were measured. Enzyme linked immunosorbent assay(ELISA) was used to detect glycosylated serum protein(GSP) and fasting serum insulin(FINS), and insulin resistance index(HOMA-IR) was calculated. The histopathological changes in the pancreas were observed by HE staining. Tumor necrosis factor(TNF)-α expression in the pancreas was detected using immunohistochemistry. The structural changes in fecal intestinal flora in the K, M, and YGZ groups were detected by 16S rRNA. Western blot was used to detect the expression of farnesoid X receptor(FXR) and takeda G protein-coupled receptor 5(TGR5) in the ileum, cholesterol 7α-hydroxylase(CYP7A1) and sterol 27α-hydroxylase(CYP27A1) in the liver, and G protein-coupled receptors 41(GPR41) and 43(GPR43) in the colon. Compared with the K group, the M group showed increased body weight, blood sugar, serum GSP, fasting blood glucose(FBG), and FINS, increased HOMA-IR, inflammatory infiltration of islet cells, necrosis and degeneration of massive acinar cells, unclear boundary between islet cells and acinar cells, disturbed intestinal flora, and down-regulated FXR, TGR5, CYP7A1, CYP27A1, GPR41, and GPR43. Compared with the M group, the YX, YGG, YGZ, and YGD groups showed decreased body weight, blood sugar, serum GSP, FBG, and FINS, islet cells with intact and clumpy morphology and clear boundary, necrosis of a few acinar cells, and more visible islet cells. The intestinal flora in the YGZ group changed from phylum to genus levels, and the relative abundance of intestinal flora affecting the metabolites of intestinal flora increased. The protein expression of FXR, TGR5, CYP7A1, CYP27A1, GPR41, and GPR43 increased. The results show that Puerariae Lobatae Radix can improve the inflammatory damage of pancreatic islet cells and reduce insulin resistance in db/db mice with T2DM. The mechanism of action may be related to the increase in the abundance of Actinobacteria, Bifidobacterium, and Bacteroides in the intestinal tract and the protein expression related to metabolites of intestinal flora.

Spectrum-effect relationship study to reveal the pharmacodynamic substances in Flos Puerariae-Semen Hoveniae medicine pair for the treatment of alcohol-induced liver damage.

ETHNOPHARMACOLOGICAL RELEVANCE: Alcoholic liver disease (ALD) is the most serious and irreversible liver damage associated with alcohol consumption. Flos Puerariae and Semen Hoveniae are traditional Chinese medicines (TCM) for dispelling the effects of alcohol. Many studies have shown that the combination of two medicinal materials has the enhanced effect of treating ALD. AIM OF THE STUDY: The aim of this study is to assess the pharmacological effects of Flos Puerariae-Semen Hoveniae medicine pair, to elucidate its action mechanism in the treatment of alcohol-induced BRL-3A cells, and to reveal the active ingredients in the medicine pair that exerted pharmacological effects by spectrum-effect relationship study. MATERIALS AND METHODS: Firstly, MTT assays, ELISA, fluorescence probe analysis, and Western blot were employed to study the underlying mechanisms of the medicine pair in alcohol-induced BRL-3A cells by examining pharmacodynamic indexes and related protein expression. Secondly, HPLC method was established for chemical chromatograms of the medicine pair with different ratios and the sample extracted by different solvents. Then, principal component analysis, pearson bivariate correlation analysis and grey relational analysis were applied for development of the spectrum-effect correlation between pharmacodynamic indexes and HPLC chromatograms. Moreover, prototype components and their metabolites in vivo were identified by the HPLC-MS method. RESULTS: Flos Puerariae-Semen Hoveniae medicine pair remarkably increased cell viability, decreased the activity of ALT, AST, TC and TG, reduced the generation of TNF-α, IL-1ß, IL-6, MDA and ROS, increased the activity of SOD and GSH-Px, reduced protein expression of CYP2E1, compared with alcohol-induced BRL-3A cells. The medicine pair modulated the PI3K/AKT/mTOR signaling pathways by up-regulating the levels of phospho-PI3K, phospho-AKT and phospho-mTOR. Also, the results of the spectrum-effect relationship study showed that P1 (chlorogenic acid), P3 (daidzin), P4 (6″-O-xylosyl-glycitin), P5 (glycitin), P6 (unknown), P7 (unknown), P9 (unknown), P10 (6″-O-xylosyl-tectoridin), P12 (tectoridin) and P23 (unknown) can be considered as the main components of the medicine pair in the treatment of ALD. Furthermore, 6″-O-xylosyl-tectoridin, tectoridin, daidzin, 6″-O-xylosyl-glycitin and glycitin can be absorbed into the blood and showed clear metabolic and excretion behaviors in rats. CONCLUSION: In this study, the hepatoprotective effects and the pharmacology mechanism of Flos Puerariae-Semen Hoveniae medicine pair in alcohol-induced BRL-3A cells were initially investigated and revealed. Through the spectrum-effect relationship study, the potential pharmacodynamic constituents such as daidzin, 6″-O-xylosyl-glycitin, 6″-O-xylosyl-tectoridin, glycitin, and tectoridin exert pharmacological effects on alcohol-induced oxidative stress and inflammation by modulating the PI3K/AKT/mTOR signaling pathways. This study provided experimental basis and data support for revealing the pharmacodynamic substance basis and pharmacology mechanism in the treatment of ALD. Moreover, it provides a robust mean of exploring the primary effective components responsible for the bioactivity of complicated TCM.

Immunoaffinity separation of miroestrol and deoxymiroestrol from Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham using fragment antigen-binding antibody produced via Escherichia coli.

INTRODUCTION: Miroestrol and deoxymiroestrol are potent phytoestrogens and are oestrogen markers of Pueraria candollei var. mirifica. However, purifying these compounds is difficult because they only exist in trace amounts. OBJECTIVES: Active fragment antigen-binding (Fab) antibodies were produced via Escherichia coli SHuffle® T7 and used to selectively separate these compounds. MATERIALS AND METHODS: Two immunoaffinity separation approaches were developed, namely the immunoaffinity column (IAC) and a cell-based method. Group-specific Fab antibodies against miroestrol and deoxymiroestrol (anti-MD Fab) were used as biological binding reagents for selective separation. RESULTS: The Fab-based IAC effectively separated miroestrol and deoxymiroestrol (0.65 and 2.24 µg per 2 mL of resin, respectively) from P. mirifica root extract. When P. mirifica extract was added to E. coli cultures during Fab expression via a cell-based method, the target compound accumulated in intracellular compartments and, thus, were separated from E. coli cells after the removal of other compounds. A yield of 1.07 µg of miroestrol per gram of cell pellet weight was obtained. Miroestrol and deoxymiroestrol were successfully purified from P. mirifica extract using anti-MD Fab via the IAC and an intracellular cell-based method. CONCLUSION: The proposed methods can simplify the miroestrol and deoxymiroestrol extraction process and provide a basis for applications utilising recombinant antibodies to separate target compounds.

Spatially resolved metabolomics combined with bioactivity analyses to evaluate the pharmacological properties of two Radix Puerariae species.

ETHNOPHARMACOLOGICAL RELEVANCE: P. lobata and P. thomsonii are medicinal plants with similar pharmacological functions but different therapeutic effects. A novel method is presented herein to investigate metabolites in terms of their distribution and qualification, quantification is necessary to elucidate the different therapeutic effects of the two Puerariae species. AIM OF THE STUDY: The aim of the present study was to perform spatially resolved metabolomics combined with bioactivity analyses to systematically compare the metabolite differences in P. lobata and P. thomsonii by distribution, qualification, quantification, and biological activity to evaluate their pharmacological properties. MATERIALS AND METHODS: Air flow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADESI-MSI) was performed to characterize the differences in the metabolite distributions of P. lobata and P. thomsonii. Further qualitative and quantitative analyses of the differential metabolites were performed using liquid chromatography-mass spectrometry (LC-MS). Biological activities correlated with the differences in the metabolites were validated by MTT assays. RESULTS: Some metabolites showed complementary distributions of the phloem and xylem in the two species, saccharide, vitamin, and inosine levels were higher in the phloem of P. thomsonii but higher in the xylem of P. lobata. The 3'-hydroxyl puerarin level was higher in the xylem of P. thomsonii but higher in the phloem of P. lobata. Qualitative and quantitative analyses of the metabolites revealed a total of 52 key differential metabolites. MTT assays showed that daidzein, daidzin, puerarin, ononin, genistin, formononetin, 3'-hydroxy puerarin, 3'-methoxy puerarin, mirificin, and 3'-methoxy daidzin exerted protective effects on H9c2 cells against hypoxia/reoxygenation injury. P. lobata extracts exhibited a significantly better protective efficacy than P. thomsonii extracts. CONCLUSIONS: In this study, AFADESI-MSI combined with LC-MS and biological activities comprehensively elucidated metabolite differences in the distribution, qualification, quantification, and pharmacological properties of P. lobata and P. thomsonii. The results of this study could provide a novel strategy for species identification and quality assessment of similar Chinese herbal medicines.

Comparative stability and analytical performance of anti-miroestrol recombinant antibody in different cassettes.

Immunoassays are efficient for the phytochemical analysis of various matrices. However, producing an appropriate recombinant antibody for small molecules is challenging, resulting in costly analyses. In this study, we aimed to develop recombinant fragment antigen-binding (Fab) antibodies against miroestrol, a potent phytoestrogen marker of Pueraria candollei. Two expression cassettes of Fab were established for the production of active Fab antibodies using SHuffle® T7 Escherichia coli cells. The orientation of variable fragment heavy chain (VH) and variable fragment light chain (VL) in the expression vector constructs influences the reactivity, stability, and binding specificity of the resultant Fab. Stability testing of antibodies demonstrated that Fab is a more stable form of recombinant antibody than a single-chain variable fragment (ScFv) antibody in all conditions. Based on the obtained Fab, the ELISA specifically detected miroestrol in the range of 39.06-625.00 ng/mL. The intra- and inter-assay precisions were 0.74-2.98% and 6.57-9.76%, respectively. The recovery of authentic miroestrol spiked into samples was 106.70-110.14%, and the limit of detection was 11.07 ng/mL. The results for P. candollei roots and products determined using our developed ELISA with Fab antibody and an ELISA with anti-miroestrol monoclonal antibody (mAb) were consistent (R2 = 0.9758). The developed ELISA can be applied for the quality control of miroestrol derived from P. candollei. Therefore, the appropriate expression platform of Fab resulted in the stable binding specificity of the recombinant antibody and was applicable for immunoassays.Key points• ELISAs with Fab has higher sensitivity than that with ScFv.• Fab is more stable than ScFv.• Fab-based ELISA can be used for miroestrol determination of Pueraria candollei.

Preparation, Structural Analysis and Antioxidant Activity of Polysaccharides and Their Derivatives from Pueraria lobata.

Pueraria lobata polysaccharides (PLPs) were obtained by hot water extraction using Pueraria lobata as raw material. Structural analysis revealed that PLPs may have a repetitive backbone units of →4) -α-D-Glcp (1→4-α-D-Glcp (1→. Phosphorylated Pueraria lobata polysaccharides (P-PLPs), carboxymethylated Pueraria lobata polysaccharides (CM-PLPs) and acetylated Pueraria lobata polysaccharides (Ac-PLPs) were obtained by chemical modifications of PLPs, respectively. The physicochemical properties and antioxidant activities of these four Pueraria lobata polysaccharides were studied in comparison. In particular, the clearance rate of P-PLPs exceeded 80 %, and was expected to achieve the same effect as Vc . The results showed that the effects of different chemical modifications on the antioxidant activity of PLPs varied greatly.

Combined systematic pharmacology and urine metabonomics to study the therapeutic mechanism of type 2 diabetic treated with the herbal pair of Salvia miltiorrhiza Bunge and Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep.

BACKGROUND: The herbal pair of Salvia miltiorrhiza Bunge and Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep (DG) is commonly used in the treatment of type 2 diabetes (T2DM) in traditional Chinese medicine (TCM). The drug pair DG was designed by Dr. Zhu chenyu to improve the treatment of T2DM. AIM: This study combined with systematic pharmacology and urine metabonomics to explore the mechanism of DG in the treatment of T2DM. METHODS: The therapeutic effect of DG on T2DM was evaluated by fasting blood glucose (FBG) and biochemical indexes. Systematic pharmacology was used to screen the active components and targets that may be related to DG. Metabonomics was established to find urinary metabolites and pathways that may be induced by DG. Finally, integrate the results of these two parts for mutual verification. RESULTS: FBG and biochemical indexes showed that DG could reduce FBG and adjust the related biochemical indexes. Metabolomics analysis indicated that 39 metabolites were related to DG for T2DM treatment. In addition, systematic pharmacology showed compounds and potential targets which were associated with DG. Finally, 12 promising targets were selected as targets for T2DM therapy by integrating the results. CONCLUSION: The combination of metabonomics and systematic pharmacology based on LC-MS is feasible and effective, which provides strong support for exploring the effective components and pharmacological mechanism of TCM.

Identification of yeast α-glucosidase inhibitors from Pueraria lobata by ligand fishing based on magnetic mesoporous silicon combined with knock-out/knock-in technology.

In this study, a ligand fishing technique based on magnetic mesoporous silicon was established and used to screen α-glucosidase inhibitors from Pueraria lobata. To clarify quantity-activity relationships in a holistic view, the knock-out/knock-in technology was used to analyse the interactions of several active constituents in P. lobata. Magnetic mesoporous silicon with a large specific surface area and better biocompatibility was synthesised. Subsequently, α-glucosidase was immobilised on -NH2-modified magnetic mesoporous silicon, and the compounds in the crude extract of P. lobata were screened across enzyme binding. The structures of the ligands were elucidated using UPLC-Q-TOF-MS/MS, and their activities were verified by knock-out/knock-in experiments and molecular docking. Daidzein and puerarin showed α-glucosidase inhibitory activities with an IC50 of 0.088 ± 0.003 mg mL-1 and 0.414 ± 0.005 mg mL-1, respectively. Among them, puerarin, which accounted for more than 40% of the total content, showed synergistic effects with other components and was the main contributor to the α-glucosidase inhibitory activity of P. lobata.

Oriental traditional herbal Medicine--Puerariae Flos: A systematic review.

ETHNOPHARMACOLOGICAL RELEVANCE: Pueraria Flos (PF), a traditional herbal medicine, is botanically from the dried flowers of Pueraria lobate (Willd.) Ohwi. (Chinese: ) or Pueraria thomsonii Benth. (Chinese: ). It has a long history of thousands of years in China for awakening the spleen, clearing the lungs, relieving alcohol. AIM OF THE REVIEW: This review aims to report the up-to-date research progress in ethnopharmacology, phytochemistry, pharmacology and toxicology, metabolism and therapeutic application of PF, so as to provide a strong basis for future clinical treatment and scientific research. MATERIALS AND METHODS: Relevant information on PF was collected from scientific literature databases including PubMed, CNKI and other literature sources (Ph.D. and M.Sc. dissertations and Chinese herbal classic books) by using the keyword "Puerariae". RESULTS: Briefly, phytochemical research report has isolated 39 flavonoids, 19 saponins and 25 volatile oils from PF. Flavonoids and saponins are the most important bioactive compounds, and most of the quality control studies focus on these two types of compounds. Modern pharmacological studies have revealed their significant biological activities in relieving alcoholism, hepatoprotective, anti-tumor, anti-inflammatory, and anti-oxidation, which provides theoretical support for the traditional use. CONCLUSIONS: Comprehensive analysis showed that pharmacological activity of most purified compounds from PF had not been reported. Kakkalide, tectoridin and their deglycosylated metabolites (irisolidone and tectorigenin) has been focused on excessively due to their higher content and better activities. This leads to low development and resources waste. Interestingly, PF made a breakthrough in the field of food. Many kinds of fat-lowering foods such as PILLBOX Onaka have been popular in Japan market, which received extensive attention. Therefore, we suggest that future research can be paid attention on the development of the plant's function in the field of food and medicine, as well as the transformation from experimental to clinical.

Metabolomics of the anti-inflammatory effect of Pueraria lobata and Pueraria lobata var. Thomsonii in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Pueraria lobata (Willd.) Ohwi and Pueraria lobata var. Thomsonii (Benth.) Maesen are essential medicinal and edible homologous plants widely cultivated in Asian countries. Therefore, P. lobata and P. thomsonii are widely used in the food, health products and pharmaceutical industries and have significant domestic and international market potential and research value. P. lobata and P. thomsonii have pharmacological effects in the clinic, such as antipyretic, analgesic, anti-inflammatory and antioxidant effects. These plants are commonly used in the treatment of inflammatory diseases and other related diseases. However, the potential mechanisms of the anti-inflammatory effects of P. lobata and P. thomsonii have not been elucidated. AIM OF THE STUDY: This study aimed to confirm the anti-inflammatory effects of P. lobata and P. thomsonii on inflammatory model diseases and to investigate the mechanism of their anti-inflammatory effects from the perspective of plasma metabolomics. MATERIALS AND METHODS: First, P. lobata and P. thomsonii were identified by high‒performance liquid chromatography (HPLC). Second, we established the following three inflammation models: an acute inflammation model of auricular swelling in mice induced by xylene, an acute inflammation model of foot swelling in rats induced by carrageenan gum, and a chronic inflammation model of cotton ball granuloma in rats. Then we examined the weight and swelling rate of auricular swelling in mice; the residence time, contact area, and mean contact pressure in rats on the gait meter; and the weight of granulomas in rats and the content of IL-1ß and TNF-α in plasma to investigate the anti-inflammatory pharmacodynamics of P. lobata and P. thomsonii. Third, we used LC‒MS‒based plasma metabolomics techniques to obtain potential biomarkers of P. lobata and P. thomsonii related to inflammation. Then, the potential biomarkers were enriched by MetaboAnalyst and KEGG metabolomics analysis tools to obtain metabolic pathways related to inflammation. Finally, we tested the indicators of COX-2, 5-LOX, GSH, GSSG and γ⁃GCL in rat plasma from the granuloma model by enzyme-linked immunosorbent assays (ELISAs) to verify the inflammation-related metabolic pathway. RESULTS: The experimental results showed that P. lobata and P. thomsonii could reduce the swollen weight and swelling rate of the auricle in mice, and could increase the residence time, contact area and mean contact pressure in rats on the gait meter. Moreover, P. lobata and P. thomsonii could inhibit the growth of granulomas and reduce the content of IL-1ß and TNF-α in plasma in rats. The above results preliminarily verified that P. lobata and P. thomsonii have different anti-inflammatory effects. We identified eighteen plasma biomarkers associated with P. lobata and sixteen plasma biomarkers related to P. thomsonii in regulating inflammation by a plasma metabolomics analysis. The following two major metabolic pathways were further screened and enriched: arachidonic acid metabolism and glutathione metabolism. Then we noted that P. lobata and P. thomsonii could reduce the COX-2, 5-LOX and GSSG levels and increase the GSH, GSH/GSSG and γ⁃GCL levels based on the ELISA results, which demonstrated that P. lobata and P. thomsonii affect the anti-inflammatory mechanism through arachidonic acid metabolism and glutathione metabolism. CONCLUSIONS: The results of this study further elucidate the anti-inflammatory mechanism of action of P. lobata and P. thomsonii, providing a scientific basis for developing new drugs for the treatment of inflammation-related diseases and laying a foundation for the development of herbal resources, such as P. lobata and P. thomsonii.

Exploring the mechanism of the antithrombotic effects of Pueraria lobata and Pueraria lobata var. thomsonii based on network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Pueraria lobata (Willd.) Ohwi and Pueraria lobata var. thomsonii (Benth.) Maesen are nutritious medicine food homology plants that are widely used in the food and health products industry and are excellent natural materials for the development of new health foods, with great potential for domestic and foreign markets. Clinically, P. lobata and P. thomsonii are used to treat coronary heart disease, atherosclerosis, cerebral infarction and other cardiovascular diseases, and antithrombotic actions may be their core effect in the treatment of thrombotic diseases. However, the underlying mechanisms of the antithrombotic properties of P. lobata and P. thomsonii have not been clarified. METHODS: First, P. lobata and P. thomsonii were identified by high-performance liquid chromatography (HPLC). An arteriovenous bypass thrombosis rat model was established. Thrombus dry‒wet weight, platelet accumulation rate and the four coagulation indices, including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB), were detected in plasma to manifest the P. lobata and P. thomsonii antithrombotic function. Network pharmacology and molecular docking methods were used to obtain key targets and verify reliability. David 6.8 was used for GO and KEGG analyses to explore pathways and potential targets for P. lobata and P. thomsonii antithrombotic functions. Prostaglandin I2 (PGI2), thromboxane A2 (TXA2), cyclooxygenase 2 (COX-2), myeloperoxidase (MPO) and endothelial nitric oxide synthase (eNOS) were tested by enzyme-linked immunosorbent assay (ELISA). RESULTS: The results indicated that P. lobata and P. thomsonii can reduce thrombus dry‒wet weight and platelet accumulation in rats and inhibit TT, APTT, FIB, and PT. A comprehensive network pharmacology approach successfully identified 9 active ingredients in P. lobata and P. thomsonii. The main active ingredients include polyphenols, amino acids and flavonoids. A total of 15 antithrombotic function targets were obtained, including 3 key targets (PTGS2, NOS3, MPO). Pathway analysis showed 10 significant related pathways and 29 biological processes. P. lobata and P. thomsonii inhibited platelet aggregation by upregulating PGI2 and downregulating TXA2, inhibited PTGS2 to reduce inflammation, and increased the level of eNOS to promote vasodilation. In addition, P. lobata and P. thomsonii alleviated oxidative stress by increasing SOD levels and significantly decreasing MDA contents. CONCLUSION: The results of the study further clarify the antithrombotic mechanism of action of P. lobata and P. thomsonii, which provides a scientific basis for the development of new drugs for thrombogenic diseases and lays the foundation for the development of P. lobata and P. thomsonii herbal resources and P. lobata and P. thomsonii health products.

Chitosan/xanthan gum-based (Hydroxypropyl methylcellulose-co-2-Acrylamido-2-methylpropane sulfonic acid) interpenetrating hydrogels for controlled release of amorphous solid dispersion of bioactive constituents of Pueraria lobatae.

Pueraria lobatae (Willd) Ohwi is a traditional Chinese medicine used to treat alcohol intoxication, diabetes, cerebrovascular and cardiovascular diseases. Some of its active components include the flavonoids puerarin, daidzin, daidzein, and genistin. The therapeutic efficacy of these agents is hampered by their poor pharmacokinetic profiles (rapid systemic clearance, low oral bioavailability, short half-life) and physicochemical properties (such as poor aqueous solubility and stability). In the current study, chitosan/xanthan gum-based (hydroxypropyl methylcellulose-co-2-acrylamido-2-methylpropane sulfonic acid) hydrogels for the controlled release of Pueraria lobata-solid dispersion (SD) were successfully prepared and characterized. A total of 61 compounds were identified in the Pueraria lobatae-SD using UHPLC-Q-TOF-MS analysis. Hydrogel structure was confirmed by FTIR, XRD, TGA, DSC, and SEM showed a porous structure. Correlations between hydrogels structural properties was also investigated. The hydrogels showed higher swelling after 48 h at pH 1.2 (21.15 %) than pH 7.4 (15.91 %). In vitro drug release study demonstrated that drug release was maximum at pH 1.2 (63 %) compared to pH 7.4 (49 %) after 48 h. The gel fraction of the synthesized hydrogel was increased with the increase in the polymer and crosslinker concentrations. Furthermore, in vitro studies demonstrated that the developed hydrogels possess good antioxidant and antimicrobial properties.

Daidzein Hydroxylation by CYP81E63 Is Involved in the Biosynthesis of Miroestrol in Pueraria mirifica.

White Kwao Krua (Pueraria candollei var. mirifica), a Thai medicinal plant, is a rich source of phytoestrogens, especially isoflavonoids and chromenes. These phytoestrogens are well known; however, their biosynthetic genes remain largely uncharacterized. Cytochrome P450 (P450) is a large protein family that plays a crucial role in the biosynthesis of various compounds in plants, including phytoestrogens. Thus, we focused on P450s involved in the isoflavone hydroxylation that potentially participates in the biosynthesis of miroestrol. Three candidate P450s were isolated from the transcriptome libraries by considering the phylogenetic and expression data of each tissue of P. mirifica. The candidate P450s were functionally characterized both in vitro and in planta. Accordingly, the yeast microsome harboring PmCYP81E63 regiospecifically exhibited either 2' or 3' daidzein hydroxylation and genistein hydroxylation. Based on in silico calculation, PmCYP81E63 had higher binding energy with daidzein than with genistein, which supported the in vitro result of the isoflavone specificity. To confirm in planta function, the candidate P450s were then transiently co-expressed with isoflavone-related genes in Nicotiana benthamiana. Despite no daidzein in the infiltrated N. benthamiana leaves, genistein and hydroxygenistein biosynthesis were detectable by liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Additionally, we demonstrated that PmCYP81E63 interacted with several enzymes related to isoflavone biosynthesis using bimolecular fluorescence complementation studies and a yeast two-hybrid analysis, suggesting a scheme of metabolon formation in the pathway. Our findings provide compelling evidence regarding the involvement of PmCYP81E63 in the early step of the proposed miroestrol biosynthesis in P. mirifica.

Research on the Material Basis and Mechanism of Kudzu Root in Preventing and Treating Cerebral Ischemia based on Network Pharmacology.

BACKGROUND: It has been shown that Kudzu root has significant pharmacological effects such as improving microcirculation, dilating coronary arteries, and increasing cerebral and coronary blood flow, but its material basis and mechanism of action are not clear. OBJECTIVE: The aim of this study was to investigate the mechanism of action of Kudzu root in the prevention and treatment of cerebral ischemia (CI) through network pharmacology combined with animal experiments. METHODS: The components of kudzu root were screened by using the Chemistry Database, Chinese Academy of Science. Linpinski's five rules were used to perform pharmacophore-like analysis to obtain the active ingredients of Kudzu root. The Swiss Target Prediction Service database was used to predict the potential protein targets of kudzu root components associated with CI. An active ingredient-target network was constructed by using Cytoscape 3.6.0. A rat model of middle cerebral artery occlusion (MCAO) was established, then the main targets and signaling pathways predicted were verified by observing the area of cerebral infarction and Western blot experiments. RESULTS: In total, 84 major active compounds and 34 targets included gerberoside, belonging to the isoflavone class, gallic acid, amino acid class, 4-Methylphenol, phenolic class, and quercetin, and flavonoid class (Flavonoids). The targets covered were proteins related to excitatory amino acids and calcium overload, including Excitatory amino acid transporter 2 (SLC1A2), Glutamate receptor ionotropic, kainate 1 (GRIK1), Glutamate receptor ionotropic, NMDA 1 (GRIN1), Glutamate receptor 2(GRIA2), Calcium/calmodulin-dependent protein kinase II (CaMKII), Neuronal nitric oxide synthase(nNOS). Glutamatergic energy is prominent, and calcium transport across the membrane is central to the network and occupies an important position. CONCLUSION: Kudzu root can significantly reduce neurological damage in rats with CI, and also significantly reduce the rate of cerebral infarction. It is worth noting that Kudzu root can prevent and treat CI by reducing excitatory amino acid toxicity and improving calcium overload.

[Mechanism of Puerariae Lobatae Radix against lung cancer by inhibiting histone demethylase LSD1].

Histone lysine-specific demethylase 1(LSD1) has become a promising molecular target for lung cancer therapy. Upon the screening platform for LSD1 activity, some Chinese herbal extracts were screened for LSD1 activity inhibition, and the underlying mechanism was preliminarily investigated at both molecular and cellular levels. The results of LSD1 inhibition showed that Puerariae Lobatae Radix extract can effectively reduce LSD1 expression to elevate the expression of H3 K4 me2 and H3 K9 me2 substrates in H1975 and H1299 cells. Furthermore, Puerariae Lobatae Radix was evaluated for its anti-lung cancer activity. It had a potent inhibitory ability against the proliferation and colony formation of both H1975 and H1299 cells. Flow cytometry and DAPI staining assays indicated that Puerariae Lobatae Radix can induce the apoptosis of lung cancer cells. In addition, it can significantly suppress the migration and reverse the epithelial-mesenchymal transition(EMT) process of lung cancer cells by activating E-cadherin and suppressing the expression of N-cadherin, slug and vimentin. To sum up, Puerariae Lobatae Radix displayed a robust inhibitory activity against lung cancer, and the mechanism may be related to the down-regulation of LSD1 expression to induce the cell apoptosis and suppress the cell migration and EMT process. These findings will provide new insights into the action of Puerariae Lobatae Radix as an anti-lung cancer agent and offer new ideas for the study on the anti-cancer action of Chinese medicine based on the epigenetic modification.

Chromosome-Level Genome Assembly and Multi-Omics Dataset Provide Insights into Isoflavone and Puerarin Biosynthesis in Pueraria lobata (Wild.) Ohwi.

Pueraria lobata (wild.) Ohwi is a leguminous plant and one of the traditional Chinese herbal medicines. Its puerarin extract is widely used in the pharmaceutical industry. This study reported a chromosome-level genome assembly for P. lobata and its characteristics. The genome size was ~939.2 Mb, with a contig N50 of 29.51 Mbp. Approximately 97.82% of the assembled sequences were represented by 11 pseudochromosomes. We identified that the repetitive sequences accounted for 63.50% of the P. lobata genome. A total of 33,171 coding genes were predicted, of which 97.34% could predict the function. Compared with other species, P. lobata had 757 species-specific gene families, including 1874 genes. The genome evolution analysis revealed that P. lobata was most closely related to Glycine max and underwent two whole-genome duplication (WGD) events. One was in a gamma event shared by the core dicotyledons at around 65 million years ago, and another was in the common ancestor shared by legume species at around 25 million years ago. The collinearity analysis showed that 61.45% of the genes (54,579 gene pairs) in G. max and P. lobata had collinearity. In this study, six unique PlUGT43 homologous genes were retrieved from the genome of P. lobata, and no 2-hydroxyisoflavanone 8-C-glucoside was found in the metabolites. This also revealed that the puerarin synthesis was mainly from the glycation of daidzein. The combined transcriptome and metabolome analysis suggested that two bHLHs, six MYBs and four WRKYs were involved in the expression regulation of puerarin synthesis structural genes. The genetic information obtained in this study provided novel insights into the biological evolution of P. lobata and leguminous species, and it laid the foundation for further exploring the regulatory mechanism of puerarin synthesis.

Isolation of Mirificin and Other Bioactive Isoflavone Glycosides from the Kudzu Root Lyophilisate Using Centrifugal Partition and Flash Chromatographic Techniques.

Pueraria lobata (Willd.) Ohwi is a legume taxon native to Southeast Asia and widely used in traditional medicine systems of that region. The therapeutic applications of the underground parts of this species (known as kudzu root) are related to its high content of isoflavones, mainly the characteristic C-glycoside derivatives. Within this group, the most explored compound both phytochemically and pharmacologically is puerarin. However, current scientific findings document important anti-biodegenerative effects for some of the minor isoflavones from kudzu roots. Therefore, the main objective of the study was to develop an original preparative method that allowed the efficient isolation of closely related hydrophilic daidzein C-glycosides, including mirificin, from vacuum-dried aqueous-ethanolic extracts of kudzu roots. For this purpose, the combined centrifugal partition (CPC) and flash chromatographic (FC) techniques were elaborated and used. The optimized biphasic solvent system in CPC, with ethyl acetate, ethanol, water, and 0.5% (V/V) acetic acid as a mobile phase modifier, enabled the purification and separation of the polar fraction containing bioactive isoflavones and ultimately the isolation of mirificin, 3'-hydroxy- and 3'-methoxypuerarin, puerarin, and daidzin using FC. The identity of isoflavones was confirmed using spectroscopic (UV absorption and nuclear magnetic resonance) and mass spectrometric methods. The determined purity of isolated mirificin was 63%.

Efficacy and Mechanism of Pueraria lobata and Pueraria thomsonii Polysaccharides in the Treatment of Type 2 Diabetes.

Diabetes is called a "wasting and thirsting disorder" in Chinese traditional medicine because there is a depletion of vital substances in the body independent of the intake of food or water and an inability to reintroduce fluids through drinking. Pueraria lobata (Willd.) Ohwi (GG) and Pueraria thomsonii Benth. (FG) are traditional Chinese herbal medicines used in the treatment of wasting-thirst that reduce blood glucose levels. Flavonoids are the main pharmacodynamic components of GG and FG, and they are also the most studied components at present, but polysaccharides are also active components of GG and FG, which, however, are less studied. Therefore, this study aimed to investigate the effect of Pueraria polysaccharides (GG and FG polysaccharides) on type 2 diabetes (T2D), as well as their related mechanisms of action in terms of both intestinal flora and metabolomics. The C57BL/KsJ-db/db mouse model, a well-established model of obesity-induced T2D, was used in this study. The metabolomic analysis showed that Pueraria polysaccharides improved the metabolic profile of diabetic mice and significantly regulated metabolites and metabolic pathways. Both GG and FG polysaccharides regulated insulin resistance in mice by regulating PPAR signaling pathway so as to treat T2D. Additionally, Pueraria polysaccharides regulated the structure of gut microbiota and improved the diabetes-related metabolic pathway. Therefore, this study discovered the antidiabetic effects and potential mechanisms of Pueraria polysaccharides through multiple pathways involving gut microbiota and metabolites, providing a theoretical basis for further studies on their effects in the treatment of T2D.

Puerarin: A review of its mechanisms of action and clinical studies in ophthalmology.

BACKGROUND: Pueraria is the common name of the dried root of either Pueraria montana var. lobata (Willd.) Maesen & S.M.Almeida ex Sanjappa & Predeep (syn. Pueraria lobata (Willd.) Ohwi) or Pueraria montana var. thomsonii (Benth.) M.R.Almeida (syn. Pueraria thomsonii Benth.). Puerarin is a C-glucoside of the isoflavone daidzein extracted from Pueraria. It has been widely investigated to explore its therapeutic role in eye diseases and the molecular mechanisms. PURPOSE: To collect the available literature from 2000 to 2022 on puerarin in the treatment of ocular diseases and suggest the future required directions to improve its medicinal value. METHOD: The content of this review was obtained from databases such as Web of Science, PubMed, Google Scholar, China National Knowledge Infrastructure (CNKI), and the Wanfang Database. RESULTS: The search yielded 428 articles, of which 159 articles were included after excluding duplicate articles and articles related to puerarin but less relevant to the topic of the review. In eleven articles, the bioavailability of puerarin was discussed. Despite puerarin possesses diverse biological activities, its bioavailability on its own is poor. There are 95 articles in which the therapeutic mechanisms of puerarin in ocular diseases was reported. Of these, 54 articles discussed the various signalling pathways related to occular diseases affected by puerarin. The other 41 articles discussed specific biological activities of puerarin. It plays a therapeutic role in ophthalmopathy via regulating nuclear factor kappa-B (NF-ĸB), mitogen-activated protein kinases (MAPKs), PI3K/AKT, JAK/STAT, protein kinase C (PKC) and other related pathways, affecting the expression of tumour necrosis factor α (TNF-α), interleukin-1ß (IL-1ß), intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), superoxide dismutase (SOD), B-cell lymphoma-2 (Bcl-2) and other cytokines resulting in anti-inflammatory, antioxidant and anti-apoptotic effects. The clinical applications of puerarin in ophthalmology were discussed in 25 articles. Eleven articles discussed the toxicity of puerarin. The literature suggests that puerarin has a good curative effect and can be used safely in clinical practice. CONCLUSION: This review has illustrated the diverse applications of puerarin acting on ocular diseases and suggested that puerarin can be used for treating diabetic retinopathy, retinal vascular occlusion, glaucoma and other ocular diseases in the clinic. Some ocular diseases are the result of the combined action of multiple factors, and the effect of puerarin on different factors needs to be further studied to improve a more complete mechanism of action of puerarin. In addition, it is necessary to increase the number of subjects in clinical trials and conduct clinical trials for other ocular diseases. The information presented here will guide future research studies.