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.

Protective Effect of Flos Puerariae，Hoveniae Semen， and Their Compatibility on Acute Alcoholic Gastric Mucosal Injury in Mice and Mechanism / 中国实验方剂学杂志

ObjectiveTo explore the improvement effect of Flos Puerariae， Hoveniae Semen， and their compatibility on acute alcoholic gastric mucosal injury， and lay a foundation for further development of Flos Puerariae， Hoveniae Semen， and their compatibility in the prevention and treatment of alcohol-induced multiple organ injury. MethodThe acute alcohol-induced gastric mucosal injury model of mice was established by multiple intragastric administration of 56% Hongxing Erguotou liquor （15 mL·kg-1）. A total of 120 male ICR mice were randomly divided into 8 groups， namely， the blank group， model group， omeprazole group （0.026 g·kg-1）， Flos Puerariae-Hoveniae Semen （compatibility） high， medium， and low-dose groups （29.2，14.6， 7.3 g·kg-1）， Flos Puerariae group （19.5 g·kg-1）， and Hoveniae Semen group （19.5 g·kg-1）， with 15 mice in each group. After one week of adaptive feeding， the animals were pre-administrated with the corresponding drug at the rate of 10 mL·kg-1 for 3 d. From the 4th day， after 1 h of administration， Erguotou liquid was administrated at the rate of 15 mL·kg-1 and the blank group was administrated with the same volume of deionized water to record the drunkenness and sober up time. The administration was lasted for 3 d. One hour after the last administration， the eyeballs were removed and the mice were sacrificed. The concentration of ethanol in serum was determined by gas chromatograph， and the activity of ethanol dehydrogenase （ADH） in gastric mucosa was determined by ultraviolet-vis spectrophotometer. Hematoxylin-eosin （HE） staining was used to observe the pathological changes in gastric mucosa. Serum inflammatory factors were determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA expression of nuclear transcription factor-κB （NF-κB） p65 and NF-κB inhibitory protein α （IκBα） were detected by real-time polymerase chain reaction （Real-time PCR）. ResultAs compared with the normal group， the content of interleukin-6 （IL-6）， interleukin-1β （IL-1β）， and tumor necrosis factor-α （TNF-α） in serum of mice in the model group was increased （P<0.05）， the mRNA expression of NF-κB p65 in gastric mucosa tissues was increased （P<0.01）， and the mRNA expression of IκBα was decreased （P<0.01）. As compared with the model group， the drunkenness time of the omeprazole group， high and medium-dose compatibility groups， and Flos Puerariae group was prolonged （P<0.05）， the sober up time of the high and medium-dose compatibility groups was shortened （P<0.05）， the ethanol concentration in the serum of the high-dose compatibility group was decreased （P<0.05）， the ADH activity in the gastric mucosa of the omeprazole group and high and medium-dose compatibility groups was increased （P<0.05）， the macroscopic injury score of the high， medium， and low-dose compatibility groups and Flos Puerariae group was decreased （P<0.05）， the score of pathological injury in the omeprazole group， high， medium， and low-dose compatibility groups， and Flos Puerariae group was decreased （P<0.01）， the expression of IL-6 in serum of all drug groups was decreased （P<0.05）， the expression of IL-1β in serum of the omeprazole group， high， medium， and low-dose Flos Puerariae groups， and Hoveniae Semen group was decreased （P<0.05）， the expression of TNF-α in serum of high and medium-dose groups was decreased （P<0.05）， the mRNA expression of NF-κB p65 in gastric mucosa tissues of all drug groups was decreased （P<0.05）， and the mRNA expression of IκBα in gastric mucosa tissues of the omeprazole group and high， medium， and low-dose compatibility groups was increased （P<0.05）. As compared with the high-dose compatibility group， the drunkenness time in the low-dose compatibility group and Hoveniae Semen group was shortened （P<0.01）， the sober up time in the Flos Puerariae and Hoveniae Semen groups was prolonged （P<0.01）， the concentration of ethanol in the serum of the medium and low-dose compatibility groups， Flos Puerariae group， and Hoveniae Semen group increased （P<0.05）， the macroscopic injury score of the medium and low-dose compatibility groups and Hoveniae Semen group was increased （P<0.05）， the pathological injury score of the medium and low-dose compatibility groups， Flos Puerariae group， and Hoveniae Semen group was increased （P<0.01）， the content of IL-1β in serum of low-dose compatibility group， Flos Puerariae group， and Hoveniae Semen group was increased （P<0.01）， and the mRNA expression of IκBα in gastric mucosa of the Flos Puerariae group and Hoveniae Semen group was decreased （P<0.05）. As compared with the medium-dose compatibility group， the drunkenness time in the Hoveniae Semen group was shortened （P<0.05）， the sober up time in the Flos Puerariae group was prolonged （P<0.05）， the pathological injury score in the Flos Puerariae group and Hoveniae Semen group was increased （P<0.01）， and the content of IL-1β in serum of the low-dose compatibility group， the Flos Puerariae group， and Hoveniae Semen group was increased （P<0.05）. As compared with the low-dose compatibility group， the pathological injury score of the Hoveniae Semen group was increased （P<0.05）. ConclusionFlos Puerariae， Hoveniae Semen， and their compatibility play a role in preventing and treating acute alcoholic gastric mucosal injury in mice， which may be related to the inhibition of the expression of NF-κB signal pathway in gastric mucosa， and the high-dose compatibility group has the optimal effect.

Flos Puerariae-Semen Hoveniae medicinal pair extract ameliorates DSS-induced inflammatory bowel disease through regulating MAPK signaling and modulating gut microbiota composition.

Background: Inflammatory bowel disease (IBD) is a global gastrointestinal disease characterized by relapsing and remitting inflammatory conditions. Flos Puerariae (the flower of Pueraria lobata [Willd.] Ohwi and P. thomsonii Benth.) and Hovenia dulcis Thunb. (Rhamnaceae) are traditional Chinese medicines. This medicinal pair has been used to treat various diseases due to its excellent anti-oxidant and anti-inflammatory activity. However, the effects of extracts from these plants on dextran sulfate sodium (DSS)-induced colitis have not been investigated; further study is needed to improve the understanding of their mechanisms of action and potential applications. Methods: The chemical constitution of extracts from Flos Puerariae and Semen Hoveniae (PHE) was analyzed using UPLC-LTQ-Orbitrap-MS/MS. The protective effects of PHE on mice with DSS-induced colitis were evaluated through assessment of body weight loss, disease activity index (DAI) score, colon length shortening, and pathological changes. The levels of inflammatory cytokines were determined by ELISA and RT-qPCR. Biomarkers of oxidative stress (ROS, CAT, SOD, MDA, and T-AOC) were analyzed using biochemical kits. The expression of MAPK proteins was determined by Western blotting analysis. Gut microbiota were analyzed via 16S rRNA sequencing. Results: Chemical composition analysis indicated that PHE contains various bioactive compounds, including puerarin, kakkalide, tectoridin, and genistin. The findings from this study suggest that PHE could effectively modulate histopathological score, inflammatory cell infiltration, and inflammatory factor secretion. Notably, PHE ameliorated oxidative stress by inhibiting activation of the MAPK pathway, leading to decreased inflammatory mediators and restored antioxidant enzyme activity. Furthermore, PHE treatment regulated the composition of the gut microbiota by increasing the abundance of benign bacteria, such as Akkermansia, and reducing the abundance of harmful bacteria, such as Proteobacteria. Conclusion: The findings from this study demonstrate the mechanism underlying the amelioration of DSS-induced intestinal oxidative stress by PHE and its positive impact on the restoration of the composition of gut microbiota.

Flos puerariae ameliorates the intestinal inflammation of Drosophila via modulating the Nrf2/Keap1, JAK-STAT and Wnt signaling.

Gut homeostasis is important for human health, and its disruption can lead to inflammatory bowel disease (IBD). Flos Puerariae is a herb with a wide variety of pharmacological activities including antioxidant, antidiabetic, antialcoholismic and anti-inflammatory properties. However, the role of Flos Puerariae on treating IBD remains obscure. Here, we employed Drosophila melanogaster as a model organism to investigate the protective effect of Flos Puerariae extract (FPE) against sodium dodecyl sulfate (SDS)-induced intestinal injury. Our data showed that FPE had no toxic effect in flies, and significantly extended lifespan in SDS-inflamed flies, reduced stem cell proliferation in the midgut, and maintained intestinal morphological integrity. Furthermore, FPE remarkably recused the altered expression level of genes and proteins in Nrf2/Keap1 signaling, JAK-STAT signaling and Wnt signaling pathways in gut of inflammation flies. Thus, FPE has a protective effect against intestinal injury possibly via increasing the Nrf2/keap1 pathway and suppressing the JAK-STAT and Wnt signaling pathways, which would have tremendous potential for treating IBD.

Qualitative and quantitative evaluation of Flos Puerariae by using chemical fingerprint in combination with chemometrics method.

In order to better control the quality of Flos Puerariae (FP), qualitative and quantitative analyses were initially performed by using chemical fingerprint and chemometrics methods in this study. First, the fingerprint of FP was developed by HPLC and the chemical markers were screened out by similarity analysis (SA), hierarchical clustering analysis (HCA), principal components analysis (PCA), and orthogonal partial least squares discriminant analysis (OPLS-DA). Next, the chemical constituents in FP were profiled and identified by HPLC coupled to Fourier transform ion cyclotron resonance mass spectrometry (HPLC-FT-ICR MS). Then, the characteristic constituents in FP were quantitatively analyzed by HPLC. As a result, 31 common peaks were assigned in the fingerprint and 6 of them were considered as qualitative markers. A total of 35 chemical constituents were detected by HPLC-FT-ICR MS and 16 of them were unambiguously identified by comparing retention time, UV absorption wavelength, accurate mass, and MS/MS data with those of reference standards. Subsequently, the contents of glycitin, genistin, tectoridin, glycitein, genistein, and tectorigenin in 13 batches of FP were detected, ranging from 0.4438 to 11.06 mg/g, 0.955 to 1.726 mg/g, 9.81 to 57.22 mg/g, 3.349 to 41.60 mg/g, 0.3576 to 0.989 mg/g, and 2.126 to 9.99 mg/g, respectively. In conclusion, fingerprint analysis in combination with chemometrics methods could discover chemical markers for improving the quality control standard of FP. It is expected that the strategy applied in this study will be valuable for further quality control of other traditional Chinese medicines.

Studies on chemical constituents of Flos Puerariae-Semen Hoveniae medicine pair by HPLC and Fourier transform ion cyclotron resonance mass spectrometry.

Alcoholic liver disease is currently the most clinically concerning liver disease, which occurs from chronic alcohol abuse. Flos Puerariae and Semen Hoveniae have been used to treat alcohol drinking excessively for thousands of years in China. In this study, the ethanol extract of the medicine pair was qualitatively and quantitatively analyzed by high-performance liquid chromatography and Fourier transform ion cyclotron resonance mass spectrometry. First, the high-performance liquid chromatography fingerprint was established to obtain the overall chromatographic data of its chemical constituents. Next, high-performance liquid chromatography-mass spectrometry was applied to identify its chemical constituents. Then, the characteristic constituents were simultaneously quantified by high-performance liquid chromatography. In addition, the chemical constituents that were absorbed into rat plasma were identified by high-performance liquid chromatography-mass spectrometry. As a result, a total of 48 chemical constituents in the medicine pair were detected and identified in vitro. Meanwhile, the content of seven representative constituents, including dihydromyricetin, glycitin, genistin, tectoridin, glycitein, genistein, and tectorigenin were simultaneously determined. Furthermore, a total of 19 chemical constituents were detected in rat plasma after oral administration. In short, the chemical constituents of the medicine pair were initially investigated in this study, which will lay the foundation for the discovery of its pharmacodynamic substances in further works.

Qualitative and quantitative evaluation of Flos Puerariae by using chemical fingerprint in combination with chemometrics method / 药物分析学报

In order to better control the quality of Flos Puerariae(FP),qualitative and quantitative analyses were initially performed by using chemical fingerprint and chemometrics methods in this study.First,the fingerprint of FP was developed by HPLC and the chemical markers were screened out by similarity analysis(SA),hierarchical clustering analysis(HCA),principal components analysis(PCA),and orthogonal partial least squares discriminant analysis(OPLS-DA).Next,the chemical constituents in FP were profiled and identified by HPLC coupled to Fourier transform ion cyclotron resonance mass spectrometry(HPLC-FT-ICR MS).Then,the characteristic constituents in FP were quantitatively analyzed by HPLC.As a result,31 common peaks were assigned in the fingerprint and 6 of them were considered as qualitative markers.A total of 35 chemical constituents were detected by HPLC-FT-ICR MS and 16 of them were unambiguously identified by comparing retention time,UV absorption wavelength,accurate mass,and MS/MS data with those of reference standards.Subsequently,the contents of glycitin,genistin,tectoridin,glycitein,genistein,and tectorigenin in 13 batches of FP were detected,ranging from 0.4438 to 11.06 mg/g,0.955 to 1.726 mg/g,9.81 to 57.22 mg/g,3.349 to 41.60 mg/g,0.3576 to 0.989 mg/g,and 2.126 to 9.99 mg/g,respectively.In conclusion,fingerprint analysis in combination with chemometrics methods could discover chemical markers for improving the quality control standard of FP.It is expected that the strategy applied in this study will be valuable for further quality control of other traditional Chinese medicines.

Potential Anti-Diabetic Activity of Pueraria lobata Flower (Flos Puerariae) Extracts.

Pueraria lobata (Wild.) Ohwi. (P. lobata) flowers known as 'Kudzu flower' contain isoflavonoids and essential oil components. They have a wide range of biological and pharmacological activities, including protective effects against non-alcoholic fatty liver disease, hyperglycemia, and hypolipidemia, anti-mutagenic effects, and benefits for weight loss. However, the molecular mechanism of these effects remains unclear. Our study aimed to systematically examine the effects of flos puerariae crude extract (FPE) as an anti-diabetic agent using in vitro assays. The cytotoxicity of FPE was evaluated using MTS assay in L6 rat myocyte and 3T3-L1 murine fibroblast cell lines. PPARγ binding activity and adipogenesis were examined using dual-luciferase and differentiation assays, respectively. For investigating the anti-diabetic activity, glucose utilization, including GLUT4 protein expression, glucose uptake assay, and GLUT4 translocation using immunofluorescence microscopy were conducted in L6 cells. Furthermore, we assessed the antioxidant and anti-inflammatory activities of FPE. Our results demonstrated the ability to augment glucose uptake in L6 cells and enhance glucose utilization activity by increasing the expression of glucose transporter type 4 (GLUT4). In summary, our findings suggest that FPE may be a potential anti-diabetic substance for the treatment of diabetic patients and can prevent inflammatory or oxidation-related diseases.

[Study on the mechanism of Flos Puerariae and Semen Hoveniae in treatment of alcoholic liver injury based on network pharmacology and molecular docking].

OBJECTIVE: To explore the mechanism of Flos Puerariae and Semen Hoveniae in treatment of alcoholic liver injury (ALI) based on network pharmacology and molecular docking. METHODS: The information of chemical constituents and targets of Flos Puerariae and Semen Hoveniae was collected from TCMSP and Swiss databases, and the threshold values of oral bioavailability (OB) ≥ 30%, drug likeness (DL) ≥0.18 were used to screen the potential active compounds. The GeneCard and DrugBank databases were used to obtain the targets corresponding to ALI. The common targets were queried using Venn Diagram, and the network of PPI and Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed through DAVID and Reactome database. Autodock Vina software was used for molecular docking of potential ingredients and key targets. RESULTS: A total of 21 potential active compounds and 431 therapeutic targets were gathered in Flos Puerariae and Semen Hoveniae, which involved 273 biological functions, 90 KEGG pathways and 362 Reactome pathways. The GO functions involved protein binding, ATP binding, etc.; the KEGG pathways mainly included PI3K-Akt signaling pathway and TNF signaling pathway; the Reactome pathways contained signal transduction and immune system, etc. The results of molecular docking showed that 21 potential active ingredients had good affinity with the core targets Akt1, TP53 and IL-6. CONCLUSIONS: The network pharmacology and molecular docking analysis demonstrate the synergetic effect of Flos Puerariae and Semen Hoveniae with multi-compounds, multi-targets and multi-pathways in the treatment of ALI; and also predict the possible medicinal substance, key targets and pathways, which provides clues for the new drug development and mechanism research.

Study on the mechanism of Flos Puerariae and Semen Hoveniae in treatment of alcoholic liver injury based on network pharmacology and molecular docking / 浙江大学学报·医学版

OBJECTIVE@#To explore the mechanism of Flos Puerariae and Semen Hoveniae in treatment of alcoholic liver injury (ALI) based on network pharmacology and molecular docking.@*METHODS@#The information of chemical constituents and targets of Flos Puerariae and Semen Hoveniae was collected from TCMSP and Swiss databases, and the threshold values of oral bioavailability (OB) ≥ 30%, drug likeness (DL) ≥0.18 were used to screen the potential active compounds. The GeneCard and DrugBank databases were used to obtain the targets corresponding to ALI. The common targets were queried using Venn Diagram, and the network of PPI and Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed through DAVID and Reactome database. Autodock Vina software was used for molecular docking of potential ingredients and key targets.@*RESULTS@#A total of 21 potential active compounds and 431 therapeutic targets were gathered in Flos Puerariae and Semen Hoveniae, which involved 273 biological functions, 90 KEGG pathways and 362 Reactome pathways. The GO functions involved protein binding, ATP binding, etc.; the KEGG pathways mainly included PI3K-Akt signaling pathway and TNF signaling pathway; the Reactome pathways contained signal transduction and immune system, etc. The results of molecular docking showed that 21 potential active ingredients had good affinity with the core targets Akt1, TP53 and IL-6.@*CONCLUSIONS@#The network pharmacology and molecular docking analysis demonstrate the synergetic effect of Flos Puerariae and Semen Hoveniae with multi-compounds, multi-targets and multi-pathways in the treatment of ALI; and also predict the possible medicinal substance, key targets and pathways, which provides clues for the new drug development and mechanism research.

Optimized extraction technology of flos Puerariae lobata isoflavone using orthog-onal test / 药学实践杂志

Objective To optimize the extraction technology of flos Puerariae lobata isoflavone .Methods The flos Pu-erariae lobata isoflavone was distilled by ethanol circumfluence .Total flavonoids ,tectoridin and tectorigenin extracted from Puerariae using the UV and HPLC spectromertry methods were taken as evaluation indexes .Extraction technology was opti-mized with L9 (34 ) orthogonal test on the base of single observation of ethanol concentration ,solvent dosage and distilling time . Results The best extraction technology of flos Puerariae lobata isoflavone was :to add 12 times the amount of 70% ethanol for 90 minutes for the first time ,and 10 times the amount of 70% ethanol for 60 minutes for the second time .Conclusion The op-timized extraction process of flos Puerariae lobata isoflavone is reasonable and feasible ,and it can offer reference to actual pro-duction .

Protective effect of Flos puerariae extract following acute alcohol intoxication in mice.

BACKGROUND: The effect of Flos Puerariae extract (FPE) on alcohol metabolism, hepatic injury, and memory impairment was assessed following acute ethanol (EtOH) intoxication in mice. METHODS: The model of acute EtOH intoxication was established by intragastric administration with 8 g/kg EtOH in mice. FPE was orally administrated (gavage) once a day for 7 consecutive days. Mice were randomly divided into 4 groups: control group, model group, and FPE groups (100, 200 mg/kg). Alcohol tolerance and intoxication time, blood alcohol concentration, the activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in liver, aspartate amino transferase (AST) and alanine amino transferase (ALT) in serum, superoxide dismutase (SOD), glutathione peroxidase (GSH-px), catalase and the formation of malondialdehyde (MDA) in both liver and brain, as well as memory ability were determined after acute alcohol exposure. RESULTS: Compared with model group, pretreatment with FPE significantly prolonged alcohol tolerance time and shortened intoxication time, which is accompanied by decreased blood alcohol concentration and elevated activities of ADH and ALDH in liver. Moreover, the index of hepatic injury, ALT, and AST activities in serum was markedly decreased by pretreatment with FPE. Additionally, decreased MDA level, enhanced GSH-px and catalase activities in liver, as well as enhanced SOD and catalase activities in brain were found in FPE pretreated mice after acute exposure to EtOH. Furthermore, FPE pretreated mice showed markedly relieved memory disruption following acute EtOH intoxication. CONCLUSIONS: This study suggests that FPE pretreatment could enhance alcohol metabolism, prevent hepatic injury, and relieve memory impairment after acute alcohol intoxication and that this effect is likely related to its modulation on the alcohol metabolizing and antioxidant enzymes.

Protective effect of flos puerariae flavonoid on adriamycin-induced toxic myocarditis / 中国生化药物杂志

Objective To investigate the protective effect of flos puerariae flavonoid on adriamycin (ADR)-induced toxic myocarditis and its mechanisms from morphological,biochemical and molecular levels.Methods 96 healthy Kunming male mice were randomly divided into 6 groups:normal control group,ADR model control group,ADR+low dose of flos puerariae flavonoid group(50 mg/kg),ADR +middle dose of flos puerariae flavonoid group(100 mg/kg),ADR +high dose of flos puerariae flavonoid group(200 mg/kg),and Vit E positive control group(40 mg/kg),16 in each group.The drugs were orally administered for consecutive 15 d and the model of toxic myocarditis was induced by intraperitoneal injection of ADR(3 mg/kg)in mice from day 2,one time every other day,for 7 times Colorimetry was used to measure the changes of marker enzymes about myocardial injury and inducible nitric oxide synthase(iNOS)activity in serum and tissue;immunohistochemical method was adopted to detecte the expression of myocardial apoptosis related proteins Bax and bcl-2;HE staining was conducted to observe the pathological changes of cardiac structure.Results Compared with normal control group,ADR(3 mg/kg,ip,7 times)induced the elevation of serum creatine kinase (CK),lactate dehydrogenase (LDH),aspartate transaminase(GOT)and iNOS activity increased significantly in mice(P<0.01).Meanwhile myocardial superoxide dismutase(SOD)activity decreased, and the malondialdehyde(MDA)content increased(P<0.01).Myocardial cell apoptosis in mice increased significantly,and the apoptosis rate was(40.5 ± 5.2)%;the expressions of Bax and Bcl-2 were significantly increased(P<0.01),However,the Bcl-2/Bax ratio decreased.The flos puerariae flavonoid (50,100,200 mg/kg,ig,15 d)and Vit E positive control group could reverse the changes induced by ADR,decrease serum CK,LDH,GOT and iNOS activities,increased myocardial SOD activity,lower MDA content and the expression of bax protein,and elevated Bcl-2/Bax ratio,in a dose-dependent manner.Light microscopy confirmed that flos puerariae flavonoid significantly alleviated the changes of myocardial microstructure.Conclusion ADR could induce myocardial cell apoptosis and lead toxic myocarditis in experimental mice.The flos puerariae flavonoid has protective effect on ADR-induced myocardial injury and the mechanism may be related to elevating myocardial SOD activity and anti-lipid peroxidation,inhibiting the expression of Bax protein and adriamycin-induced cardiomyocyte apoptosis.

The characteristic analysis on intestinal metabolism of Pueraria lobata (Willd.)Ohwi / 国际中医中药杂志

Objective To establish a method to compare the difference of exposure component of oral taking different part ofPueraria Lobata (Willd.) Ohwi in intestine.Methods After the incubation of puerarin,extraction Radix Puerariae and Flos Puerariae with S9,the mixtures were centrifuged to get the supernatant for analysis with HPLC.Results The research showed that the metabolic rate of puerarin was higher than that of extractions because of the concentration of enzyme.The difference between the fingerprints of Radix Puerariae and Flos Puerariae Lobata indicated that there was a difference of chemical components in such two parts of Kudzuvine Root.Conclusion The profile of intestinal metabolism can be revealed in the research of gut wall metabolism in the course of intestinal absorption by S9 incubation.

Quick Detection of Four Isoflavone Compounds from Extracts of Pueraria Lobata Flowers by HPLC / 中国药师

Objective:To establish an HPLC method for the determination of four kinds of isoflavone compounds including daid-zin, tectoridin, daidzein and tectorigenin in the extracts of Pueraria lobata flowers. Methods:The separation was performed on an Agi-lent C18 column(250 mm × 4. 6 mm, 5 μm) using a mobile phase of methanol-acetonitrile-water(2∶1∶2). The flow rate was 1. 0 ml· min-1 ,and the detection wavelength was 264nm. The column temperature was 25℃ and the sample size was 20 μl. Results:The de-tection could be accomplished within 10 minutes with good separation and specificity of four isoflavone compounds with the retention timeof3.4,3.8,5.7and7.2min,respectively. Thelinearrangewas0.784-78.440 μg·ml-1(daidzin),2.000-200.000 μg· ml-1(tectoridin) and 0.800-80.020 μg·ml-1 (daidzein and tectorigenin),and the relative coefficient was 0.999 9, 0.999 8, 0. 999 7 and 0. 999 9, respectively. The average recovery was 100. 54%(RSD=1. 66%,n=6),100. 03%(RSD=1. 00%, n=6), 99. 48%(RSD=1. 76%, n=6) and 100. 92%(RSD=2. 26%, n=6), respectively. Conclusion: The method is simple, rapid and accurate with good repeatability, which can be used in the rapid determination of isoflavone compounds in the flowers of Pueraria loba-ta.