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.

Multi-Index Comprehensive Assessment Optimized Critical Flavonoids Extraction from Semen Hoveniae and Their In Vitro Digestive Behavior Evaluation.

Critical flavonoids from Semen Hoveniae have huge potential bioactivities on hypoglycemic. A multi-index comprehensive assessment based on Analytic Hierarchy Process (AHP) method was performed to optimize the extraction process of flavonoids from Semen Hoveniae, which taking dihydromyricetin, taxifolin, myricetin and quercetin as indexes, and, then, an in vitro simulated gastrointestinal digestion model was established to investigate the changes of flavonoids contents and their antioxidant capacity before and after digestion. The results showed that three influence factors acted significantly with the order of ethanol concentration > solid-liquid ratio > ultrasound time. The optimized extraction parameters were as follows: 1:37 w/v of solid-liquid ratio, 68% of ethanol concentration and 45 min for ultrasonic time. During in vitro digestion, the order of remaining ratio of four flavonoids in the extract was dihydromyricetin > taxifolin > myricetin > quercetin in gastric digestion, and remaining ratio of taxifolin was 34.87% while others were restructured in intestinal digestion. Furthermore, the 1,1-dipheny-2-picryhydrazyl free radical (DPPH ·) scavenging ability and oxygen radical absorption capacity (ORAC) of extract were more stable in gastric digestion. After an hour's intestinal digestion, the extract had no DPPH antioxidant capacity, but amazingly, its ORAC antioxidant capacity was retained or increased, which implied that substances were transformed and more hydrogen donors were produced. This study has carried out a preliminary discussion from the perspective of extraction and put forward a new research idea, to improve the in vivo bioavailability of the critical flavonoids from Semen Hoveniae.

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.

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.

Quality control of Semen Hoveniae by high-performance liquid chromatography coupled to Fourier transform-ion cyclotron resonance mass spectrometry.

A method based on high-performance liquid chromatography and Fourier transform-ion cyclotron resonance mass spectrometry was developed to control the quality of Semen Hoveniae. First, the chromatographic fingerprint was established in combination with the chemometrics methods such as similarity analysis, cluster analysis, principal component analysis, and orthogonal partial least squares discriminant analysis to discover the qualitative markers. Then, an high-performance liquid chromatography mass spectrometry method was developed to identify the chemical constituents in Semen Hoveniae. Moreover, the content of dihydromyricetin and dihydroquercetin in Semen Hoveniae were determined by high-performance liquid chromatography. As a result, nine common peaks were assigned in the fingerprints and the similarity of the 13 batch samples varied from 0.425 to 0.993, indicating an obviously different quality. Dihydromyricetin and dihydroquercetin were the main qualitative markers to differ the quality of Semen Hoveniae. Meanwhile, a total of 21 chemical compounds were characterized by high-performance liquid chromatography mass spectrometry and six of them were identified by comparing with information of reference standards. Finally, the content of dihydromyricetin and dihydroquercetin in 13 batch samples varied from 0.824  to 7.499 mg/g and from 0.05941  to 4.258 mg/g , respectively. In conclusion, the methods developed here will provide sufficient qualitative and quantitative information for the quality control of Semen Hoveniae.

[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.

Semen hoveniae extract ameliorates alcohol-induced chronic liver damage in rats via modulation of the abnormalities of gut-liver axis.

BACKGROUND: Hovenia dulcis Thunb. is considered as a traditional herbal medicine that has been used in the treatment for ethanol-induced liver disease for centuries. Recently, substantial studies demonstrated that Semen hoveniae extract (SHE) not only suppressed the hepatic steatosis caused by chronic ethanol exposure, but also inhibited lipopolysaccharide-stimulated inflammatory responses. Nevertheless, the underlying molecular mechanisms largely remained elusive. AIM: To determine the hepatoprotective effects of SHE on ethanol-triggered liver damage and further elucidate its potential mechanisms. METHODS: In the present study, the Sprague-Dawley rats were fed with the Lieber-DeCarli diet containing alcohol or isocaloric maltose dextrin as control diet with or without SHE (300 and 600 mg/kg/d bw) for 8 weeks. The levels of serum biomarkers (ALT, AST and LDH) and LPS were detected by biochemical assay kits and endotoxin detection LAL kit, respectively. The histopathological changes of liver and intestinal tissues were observed by hematoxylin and eosin (H&E) staining and Transmission electron microscope (TEM). The expressions of CD14, TLR4, MyD88, NF-κB, Iκ-B, P-Iκ-B and TNF-α in liver, and ZO-1 and occludin in intestine were determined by western blot. The faecal microbial composition was determined by16S rRNA Gene Sequencing Analysis. RESULTS: Biochemical and histopathological analysis revealed that SHE significantly alleviated the lipid deposition and inflammation response in liver induced by ethanol. SHE remarkably inhibited the TLR4 pathway and its downstream inflammatory mediators, and up-regulated the expressions of ZO-1 and occludin in the intestine. The further investigations suggested SHE dramatically reversed ethanol-induced alterations in the intestinal microbial flora and decreased the generation of gut-derived endotoxin. CONCLUSION: In summary, SHE probably modulated abnormalities of gut-liver axis and inhibited TLR4-associated inflammatory mediators activation to exert its hepatoprotective properties. These findings suggested that SHE as a traditional therapeutic options which may play an essential role in protecting against the chronic ethanol-triggered liver injury.

Analysis of Flavonoids in Semen Hoveniae by HPLC-ESI-MS / 中药新药与临床药理

Objective To establish a HPLC-ESI-MS method for simultaneous analysis and determination of flavonoids in Semen Hoveniae. Methods The separation was performed on Acquity UPLCTM BEH C18 column (50 mm× 2. 1 mm,1.7 μm) with a mobile phase of formic acid aqueous solution (B) and MeOH (A). The flow rate was at 1.0 mL/min and detection wavelength was at 290 nm. Samples were analyzed in positive-ion detection model. Results Five kinds of flavonoids in Semen Hoveniae were separated well and were tentatively identified by ESI-MS and by reference to litera-ture data. Conclusion The method is accurate and rapid, and can be used for the identification of flavonoids in Semen Hoveniae.

Determination of Quercetin in Semen Hoveniae by RP-HPLC / 中药新药与临床药理

Objective To determine the content of quercetin in Semen Hoveniae.Methods A RP-HPLC method was established.The chromatographic column was Synergi 4u Fusion-RP 80.The mobile phase was acetonitrile-0.5 %phosphoric acid(33 ∶67).The flow rate was 1.0 mL?min-1,the detection wavelength was at 360 nm,and the column temperature was 30 ℃.Results The average recovery of quercetin was 98.6 %,and RSD was 2.39 %(n=6).Conclusion The method is effective,simple and accurate,and can be used to determine the content of quercetin in Semen Hoveniae.