The active components of Erzhi wan and their anti-Alzheimer's disease mechanisms determined by an integrative approach of network pharmacology, bioinformatics, molecular docking, and molecular dynamics simulation.

Erzhi Wan (EZW), a classic Traditional Chinese Medicine formula, has shown promise as a potential therapeutic option for Alzheimer's disease (AD), yet its mechanism remains elusive. Herein, we employed an integrative in-silico approach to investigate the active components and their mechanisms against AD. We screened four active components with blood-brain barrier permeabilities from TCMSP, along with 307 corresponding targets predicted by SwissTargetPrediction, PharmMapper, and TCMbank websites. Then, we retrieved 2260 AD-related targets from Genecards, OMIM, and NCBI databases. Furthermore, we constructed the protein-protein interaction (PPI) network of the intersected targets via the STRING database and performed the GO and KEGG enrichment analyses using the "clusterProfiler" R package. The results showed that the intersected targets were intimately related to the p53/PI3K/Akt signaling pathway, serotonergic synapse, and response to oxygen level. Subsequently, 25 core targets were found differentially expressed in brain regions by bioinformatics analyses of GEO datasets of clinical samples from the Alzdata database. The binding sites and stabilities between the active components and the core targets were investigated by the molecular docking approach using Autodock 4.2.6 software, followed by pocket detection and druggability assessment via the DoGSiteScorer server. The results showed that acacetin, ß-sitosterol, and 3-O-acetyldammarenediol-II strongly interacted with the druggable pockets of AR, CASP8, POLB, and PREP. Eventually, the docking results were further cross-referenced with the literature research and validated by 100 ns of molecular dynamics simulations using GROMACS software. Binding free energies were calculated via MM/PBSA strategy combined with interaction entropy. The simulation results indicated stable bindings between four docking pairs including acacetin-AR, acacetin-CASP8, ß-sitosterol-POLB, and 3-O-acetyldammarenediol-II-PREP. Overall, our study demonstrated a theoretical basis for how three active components of EZW confer efficacy against AD. It provides a promising reference for subsequent research regarding drug discoveries and clinical applications.

Multi-omics analysis of kidney, bone and bone marrow explored potential mechanisms of Erzhi Wan against osteoporosis with kidney-Yin deficiency.

Osteoporosis (OP) is a metabolic bone disease that can lead to major health challenges. The theory of Traditional Chinese medicine believes that kidney-Yin deficiency (KYD) is the main cause of postmenopausal osteoporosis. This study was aimed to investigate the effect of EZW on anti-osteoporosis with KYD, and explore potential mechanisms from the perspective of the kidney, bone and bone marrow through analysis of metabolomics and proteomics. The model of OP with KYD was established by rats treated with bilateral ovariectomy (OVX), and then given intragastric administration of thyroid and reserpine to induce. Micro-CT was applied to determine the microstructures of bone. Serum levels associated with bone turnover markers and kidney-Yin deficiency were detected by enzyme-linked immunosorbent (ELISA) assay. The differential metabolites in the kidney, bone and bone marrow were analyzed by metabolomics. The differentially expressed proteins in these three tissues were detected via proteomics. The findings suggested that EZW could alleviate a variety of metabolites and proteins among the kidney, bone and bone marrow, primarily in amino acid metabolism, carbohydrate metabolism, nucleotide metabolism and lipid metabolism, thus leading to improvements of OP with KYD, which provided theoretical basis for clinical treatment of EZW on OP with KYD.

Study on material basis of cathepsin K targeted inhibitor in Erzhi Wan / 药学实践杂志

Objective To investigate the active ingredients and components that inhibiting cathepsin K activity in Erzhi Wan, a classic kidney-tonifying formula. Methods Then-butanol, dichloromethane, ethyl acetate and petroleum ether parts and 30 active components in Erzhi Wan were screened by established high throughput fluorescence methods of inhibit the binding activity of CTSK with Z-FR-MCA substrate, the formation of CTSK and chondroitin sulfate A (CSA) complex activity, and the activity of substrate type I collagen degradation by CTSK. Molecular docking and insoluble collagen substrate binding assays were applied to verify the potential CTSK inhibitors. Results The n-butanol and petroleum ether parts of Erzhi Wan inhibited the formation of CTSK and CSA* complex by more than 90%, the petroleum ether part inhibited the binding of CTSK to substrate Z-FR-MCA by more than 90%, the collagen degradation inhibition rate of CTSK in n-butanol part was more than 95% and that in petroleum ether part was 58.6%. Among the 30 active components, 11 showed that the inhibition rate of CTSK and CSA* complex formation was more than 50%, and 5 components with the inhibition rate of Z-FR-MCA binding activity more than 50%. Finally, there were four components including eclalbasaponin Ⅸ, (-)-epicatechin gallate, nuezhenoside and wedelolactone. The inhibition rate of collagen degradation was more than 50%. Eclipta saponin IX inhibited the binding rate between collagen fibers and CTSK, up to 60%, but all of them failed to dock with CTSK active site. Conclusion There are active components that inhibiting cathepsin K in Erzhi Wan, which mainly exists in the n-butanol ingredients, but the active components is not an active-site inhibitor. It might inhibit the binding of CTSK with oligosaccharides by binding to other sites of CTSK, and then reduce the collagen degradation activity of CTSK.

The protective effect of drug-containing serums and polar extracts of Erzhi Wan on H2O2-induced hepatocyte senescence / 药学学报

Using a H2O2-induced BRL cell senescence model, we investigated the anti-aging effects of drug-containing serums of Erzhi Wan (EZW) and various polar extracts (petroleum ether, ethyl acetate, n-butanol, water, and iridoid glycoside-enriched fractions). Cell viability was detected by MTT assay. Cell senescence was evaluated with β-galactosidase staining assay. Intracellular reactive oxygen species (ROS) were measured by flow cytometry. UFLC-Q-TOF-MS/MS was used to identify chemical components in EZW and the extracts, and molecular docking technology was used to predict the anti-aging components of EZW. Results showed that treatment of cells with 600 μmol·L-1 H2O2 for 72 h markedly induced cell senescence, inhibited cell proliferation and increased intracellular β-galactosidase activity and ROS levels. If cells were pretreated with drug-containing serum of EZW this induction of senescence was decreased. A total of 49 chemical compounds were identified in EZW by liquid chromatography-mass spectrometry, 14 of these were identified by molecular docking as potential active ingredients. Based on these analyses, and the in vitro experiments with polar extracts, we conclude that the anti-aging components of EZW are triterpenes, flavonoids and phenyl alcohols, providing a basis for further elucidation of the active agents and mechanism of the anti-aging effect of EZW.

[Molecular mechanism analysis of Erzhi Wan in treatment of aging based on network pharmacology].

This present study aimed to explore the molecular mechanism of Erzhi Wan(a prescription of nourishing Yin and toni-fying liver and kidney) in treatment of aging by network pharmacology. The active constituents and target proteins of Erzhi Wan were searched from Traditional Chinese Medicine Systems Pharmacology Database(TCMSP) and PubChem databases respectively. Aging-related genes were searched from Gene and HAGR databases. Based on the Ingenuity Pathway Analysis(IPA), we analyzed the common molecular network, biological pathway and interaction sites between these two parts, and verified some of them by Western blot. Twelve active constituents of Erzhi Wan were screened by TCMSP databases, 69 protein targets were predicted through PubChem, and 148 aging-related genes were found in Gene and HAGR databases. IPA comparison showed that the molecular networks of these two were complex, with diversity of biological functions. The common pathways involved 292 pathways, mainly related to tumors. They acted on hypoxia inducible factor-1α gene(HIF1α), nuclear factor-E2 related factor(Nrf2/NFE2 L2), tumor necrosis factor(TNF) and other sites. Western blot results suggested that Erzhi Wan could down-regulate the expression of HIF1α, with statistical difference(P<0.05). It was concluded that, Erzhi Wan could intervene aging through improving pseudo-hypoxic microenvironment and inflammation. The molecular mechanism of Erzhi Wan in delaying aging was preliminarily revealed, which laid a foundation for further stu-dying the anti-aging mechanism of Erzhi Wan, and also provided a reference for the compatibility mechanism and extended application of Chinese medicine compounds.

Molecular mechanism analysis of Erzhi Wan in treatment of aging based on network pharmacology / 中国中药杂志

This present study aimed to explore the molecular mechanism of Erzhi Wan(a prescription of nourishing Yin and toni-fying liver and kidney) in treatment of aging by network pharmacology. The active constituents and target proteins of Erzhi Wan were searched from Traditional Chinese Medicine Systems Pharmacology Database(TCMSP) and PubChem databases respectively. Aging-related genes were searched from Gene and HAGR databases. Based on the Ingenuity Pathway Analysis(IPA), we analyzed the common molecular network, biological pathway and interaction sites between these two parts, and verified some of them by Western blot. Twelve active constituents of Erzhi Wan were screened by TCMSP databases, 69 protein targets were predicted through PubChem, and 148 aging-related genes were found in Gene and HAGR databases. IPA comparison showed that the molecular networks of these two were complex, with diversity of biological functions. The common pathways involved 292 pathways, mainly related to tumors. They acted on hypoxia inducible factor-1α gene(HIF1α), nuclear factor-E2 related factor(Nrf2/NFE2 L2), tumor necrosis factor(TNF) and other sites. Western blot results suggested that Erzhi Wan could down-regulate the expression of HIF1α, with statistical difference(P<0.05). It was concluded that, Erzhi Wan could intervene aging through improving pseudo-hypoxic microenvironment and inflammation. The molecular mechanism of Erzhi Wan in delaying aging was preliminarily revealed, which laid a foundation for further stu-dying the anti-aging mechanism of Erzhi Wan, and also provided a reference for the compatibility mechanism and extended application of Chinese medicine compounds.

[Study on Material Basis and Mechanism of Erzhi Wan prevent Alzheimer's disease by network pharmacology].

The present study is to explore the material basis and mechanism of Erzhi Wan the prevented Alzheimer's disease by using network pharmacology. The key target of Alzheimer's disease was docked with the Erzhi Wan compounds, and the drugs-target combined with target-signal pathway network model was established by Cytoscape 3.2.1 software. Thirty compounds have a strong interaction with key target of Alzheimer's disease and three key pathways related with Wnt, MAPK and PI3K-Akt-mTOR. There are 5 ingredients such as quercetin,geraniol,beta-sitosterol,nerol,eriodictyol that could be verified from literature.This result initially revealed the material basis for Erzhi Wan for Alzheimer's disease and the mechanism in terms of three signaling pathways. The network pharmacology method found that the active ingredients of Erzhi Wan for Alzheimer's disease may be quercetin,geraniol,beta-sitosterol,nerol,and eriodictyol, and the mechanism may be related to three signal pathways including Wnt, MAPK, and PI3K-Akt-mTOR.

Study on Material Basis and Mechanism of Erzhi Wan prevent Alzheimer's disease by network pharmacology / 中国中药杂志

The present study is to explore the material basis and mechanism of Erzhi Wan the prevented Alzheimer's disease by using network pharmacology. The key target of Alzheimer's disease was docked with the Erzhi Wan compounds, and the drugs-target combined with target-signal pathway network model was established by Cytoscape 3.2.1 software. Thirty compounds have a strong interaction with key target of Alzheimer's disease and three key pathways related with Wnt, MAPK and PI3K-Akt-mTOR. There are 5 ingredients such as quercetin,geraniol,beta-sitosterol,nerol,eriodictyol that could be verified from literature.This result initially revealed the material basis for Erzhi Wan for Alzheimer's disease and the mechanism in terms of three signaling pathways. The network pharmacology method found that the active ingredients of Erzhi Wan for Alzheimer's disease may be quercetin,geraniol,beta-sitosterol,nerol,and eriodictyol, and the mechanism may be related to three signal pathways including Wnt, MAPK, and PI3K-Akt-mTOR.

LC-MS determination and pharmacokinetic study of salidroside in rat plasma after oral administration of suspensions of traditional Chinese medicine Erzhi Wan and Fructus Ligustri lucidi.

A simple, rapid and sensitive liquid chromatography-mass spectrometry (LC-MS) method was developed for the determination of salidroside in rat plasma and study of its pharmacokinetics after oral administration of suspension of Erzhi Wan and Fructus Ligustri lucidi into Wistar rats. Plasma sample of 200 µL was extracted with acetic ether-isopropanol (2:1) and the extraction was performed on a Kromasil C18 column (150 mm × 4. 6 mm, 5 µm) with the mobile phase of methanol-water (41:59, v/v) within a run time of 6.0 min. The analyte was monitored with positive electrospray ionization (ESI) by selected ion monitoring (SIM) mode. The target ions were m/z 323.05 for salidroside and m/z 411.05 for internal Standard (IS) geniposide. A good linear relationship was obtained over the range of 5.0-500.0 ng/mL and the lower limit of quantification was 5.0 ng/mL. The validated method was successfully applied to the pharmacokinetic study of salidroside in rat plasma after oral administration of suspension of Erzhi Wan and Fructus Ligustri lucidi.

LC-MS determination and pharmacokinetic study of salidroside in rat plasma after oral administration of suspensions of traditional Chinese medicine Erzhi Wan and Fructus Ligustri lucidi / 药物分析学报

A simple,rapid and sensitive liquid chromatography-mass spectrometry (LC-MS) method was developed for the determination of salidroside in rat plasma and study of its pharmacokinetics after oral administration of suspension of Erzhi Wan and Fructus Ligustri lucidi into Wistar rats.Plasma sample of 200 μL was extracted with acetic etherisopropanol (2:1) and the extraction was performed on a Kromasil C18 column (150 mm×4.6mm,5 μm) with the mobile phase of methanol-water (41:59,v/v) within a run time of 6.0min.The analyte was monitored with positive electrospray ionization (ESI) by selected ion monitoring (SIM) mode.The target ions were m/z 323.05 for salidroside and m/z 411.05 for internal standard (IS) geniposide.A good linear relationship was obtained over the range of 5.0-500.0 ng/mL and the lower limit of quantification was 5.0 ng/mL.The validated method was successfully applied to the pharmacokinetic study of salidroside in rat plasma after oral administration of suspension of Erzhi Wan and Fructus Ligustri lucidi.

LC-MS determination and pharmacokinetic study of salidroside in rat plasma after oral administration of suspensions of traditional Chinese medicine Erzhi Wan and Fructus Ligustri lucidi / 药物分析学报

A simple,rapid and sensitive liquid chromatography-mass spectrometry（LC-MS）method was developed for the determination of salidroside in rat plasma and study of its pharmacokinetics after oral administration of suspension of Erzhi Wan and Fructus Ligustri lucidi into Wistar rats.Plasma sample of 200 μL was extracted with acetic ether-isopropanol（2∶1）and the extraction was performed on a Kromasil C18 column（150 mm×4.6 mm,5 μm）with the mobile phase of methanol-water（41∶59,v/v）within a run time of 6.0 min.The analyte was monitored with positive electrospray ionization（ESI）by selected ion monitoring（SIM）mode.The target ions were m/z 323.05 for salidroside and m/z 411.05 for internal standard（IS）geniposide.A good linear relationship was obtained over the range of 5.0-500.0 ng/mL and the lower limit of quantification was 5.0 ng/mL.The validated method was successfully applied to the pharmacokinetic study of salidroside in rat plasma after oral administration of suspension of Erzhi Wan and Fructus Ligustri lucidi.