[Discovery and confirmation of protein action site AK1 of ginsenosides in brain based on DARTS technology].

This study aims to explore the targets of ginsenosides in brain based on drug affinity responsive target stability(DARTS) technology. Specifically, DARTS technology was combined with label-free liquid chromatography tandem mass spectrometry(LC-MS) to screen out the proteins in the brain that might interact with ginsenosides. Based on the screening results, adenylate kinase 1(AK1) was selected for further confirmation. First, the His-AK1 fusion protein was yielded successively through the construction of recombinant prokaryotic expression vector, expression of target protein, and purification of the fusion protein. Biolayer interferometry(BLI) was employed to detect the direct interaction of Rg_1, Re, Rb_1, Rd, Rh_2, F1, Rh_1, compound K(CK), 25-OH-PPD, protopanaxa-diol(PPD), and protopanaxatriol(PPT) with AK1, thereby screening the ginsenoside monomer or sapogenin that had strong direct interaction with the suspected target protein AK1. Then, the BLI was used to further determine the kinetic parameters for the binding of PPD(strongest interaction with AK1) to His-AK1 fusion protein. Finally, molecular docking technology was applied to analyze the binding properties between the two. With DARTS and LC-MS, multiple differential proteins were screened out, and AK1 was selected based on previous research for target verification. Fusion protein His-AK1 was obtained by prokaryotic expression, and the response(nm) of Re, Rg_1, Rd, Rb_1, Rh_1, Rh_2, F1, PPT, PPD, 25-OH-PPD, and CK with His-AK1 was respectively 0.003 1, 0.001 9, 0.042 8, 0.022 2, 0.013 4, 0.037 3, 0.013 9, 0.030 7, 0.140 2, 0.016 0, and 0.040 8. The K_(on), K_(off), and K_D values of PPD and His-AK1 were determined by the BLI as 1.22×10~2 mol~(-1)·L·s~(-1), 1.04×10~(-2) s~(-1), 8.52×10~(-5) mol·L~(-1). According to the molecular docking result, PPD bound to AK1 with the absolute value of the docking score of 3.438, and hydrogen bonds mainly formed between the two. Thus, AK1 is one of the protein action sites of ginsenosides in the brain. The direct interaction between ginsenoside metabolite PPD and AK1 is the strongest.

[Analysis of anti-fatigue mechanism and potential targets of ginseng].

Ginseng has effects in reinforcing vital energy,invigorating health effectively and relieving fatigue symptoms,and ginsenoside( GS) is the main component of its anti-fatigue effect. Totally 17 active components and 92 drug targets of ginseng compounds were screened from Traditional Chinese Medicine Systems Pharmacology; and 78 intersecting genes of diseases and drug targets were obtained based on R Language Technology. The protein-protein interaction( PPI) network was constructed by STRING 11. 0 software,and Matthews Correlation Coefficient( MCC) algorithm was used to screen core target genes. Gene ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were used to analyze the major genes and their roles in regulatory networks. The results indicated that ginseng could regulate the core target genes,including AKT serine/threonine kinase( AKT1),interleukin-1ß,Toll-like receptor binding molecule 1( ICAM1),mitogen-activated protein kinase 8( MAPK8),AP-1 transcription factor subunit( JUN),transducer and activator of transcription 1( STAT1) and prostaglandin peroxidase synthase 2( PTGS2). It could participate in the functions of cytokine receptor binding,cell adhesion molecule binding and tumor necrosis factor receptor superfamily binding,and also regulate the signal pathways of tumor necrosis factor,interleukin 17 and c-type lectin receptor,so as to exert an anti-fatigue effect. Based on the results of network analysis,32 four-week-old male SPFACR mice were randomly divided into control group,low-dose ginsenoside group,middle-dose ginsenoside group and high-dose ginsenoside group. The corresponding drugs were administrated for 3 weeks. The results showed that GS could significantly up-regulate the expressions of STAT1 and AKT1( P<0. 01,P<0. 05),and downregulate the expressions of PTGS2 and JUN( P<0. 01). However,there was no significant effect on MAPK8,IL-1ß and ICAM1. Ginseng's anti-fatigue regulation network was constructed through network pharmacology,and the results were verified by experiments,in order to reveal the anti-fatigue mechanism of ginseng and provide scientific basis for its clinical application.

Qualitative detection of ginsenosides in brain tissues after oral administration of high-purity ginseng total saponins by using polyclonal antibody against ginsenosides.

Given the limited studies and conflicting findings, the transport character of ginsenosides crossing the blood-brain barrier (BBB) remains unclear. The present study was designed to qualitatively determine the distribution of ginsenosides in brain tissues after oral administration of ginseng total saponins, using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) combined with immunohistochemistry. In brain tissue homogenates, ginsenoside Rg1 was detectable and no other ginsenosides or their metabolites were found. No ginsenosides were detected in cerebrospinal fluid. Immunohistochemistry staining of brain tissue sections by using anti-ginsenoside polyclonal antibodies revealed the localization of ginsenosides in brain tissues. Furthermore, immunofluorescence double staining revealed that ginsenosides widely existed in vascular endotheliocytes and astrocytes, and in few neurons. These results indicated that Rg1 was the main component that entered the brain after oral administration of ginseng total saponins and that ginsenosides could cross the BBB, although the transport capability of ginsenosides through the BBB may be poor.

Preventive Effects of Ginseng Total Saponins on Chronic Corticosterone-Induced Impairment in Astrocyte Structural Plasticity and Hippocampal Atrophy.

To further explore the underlying antidepressant mechanism of ginseng total saponins (GTS), this study observed the effects on hippocampal astrocyte structural plasticity and hippocampal volume in the corticosterone-induced mouse depression model. Corticosterone (20 mg/kg/day) was administered subcutaneously for 5 weeks, and GTS (12.5, 25, and 50 mg/kg/day; namely GTSL, GTSM, and GTSH) or fluoxetine (10 mg/kg/day) were given intragastrically during the last 3 weeks. On day 33 and day 34, depression-like behavior was observed via a forced swimming test and a tail suspension test, respectively. At 6 h after the last dose of corticosterone (day 35), all mice were sacrificed followed by serum corticosterone assays, stereological analysis of hippocampal glial fibrillary acidic protein-positive (GFAP+ ) astroctyes and hippocampal volume, and hippocampal glycogen tests. Results showed that all doses of GTS ameliorated depression-like behavior and the decrease in hippocampal glycogen without normalizing hypercortisolism. Moreover, GTSH and GTSM reversed the corticosterone-induced reduction in the total number of hippocampal GFAP+ astrocytes and hippocampal volume. Additionally, GTSH alleviated the diminished protrusion length and somal volume of GFAP+ astrocytes induced by corticosterone. These findings imply that the effects of GTS on corticosterone-induced depression-like behavior may be mediated partly through the protection to hippocampal astrocyte structural plasticity. Copyright © 2017 John Wiley & Sons, Ltd.

Determination of total ginsenosides in ginseng extracts using charged aerosol detection with post-column compensation of the gradient.

AIM: Variation in structure-related components in plant products prompted the trend to establish methods, using multiple or total analog analysis, for their effective quality control. However, the general use of routine quality control is restricted by the limited availability of reference substances. Using an easily available single marker as a reference standard to determine multiple or total analogs should be a practical option. METHOD: In this study, the Ultra-HPLC method was used for the baseline separation of the main components in ginseng extracts. Using a plant chemical component database, ginsenosides in ginseng extracts were identified by Ultra-HPLC-MS analysis. The charged aerosol detection (CAD) system with post-column compensation of the gradient generates a similar response for identical amounts of different analytes, and thus, the content of each ginsenoside in ginseng extracts was determined by comparing the analyte peak area with the reference standard (determination of total analogs by single marker, DTSM). The total ginsenoside content was determined by the summation of reference standard and other ginsenoside components. RESULTS: The results showed that DTSM approaches were available for the determination of total ginsenosides in a high purity ginseng extract because of the removal of impurities. In contrast, DTSM approaches might be suitable for determination of multiple ginsenosides without interference from impurities in the crude ginseng extract. CONCLUSION: Future practical studies similar to the present study should be conducted to verify that DTSM approaches based on CAD with post-column inverse gradient for uniform response are ideal for the quality control of plant products.

Preparation and quality assessment of high-purity ginseng total saponins by ion exchange resin combined with macroporous adsorption resin separation.

AIM: To prepare high-purity ginseng total saponins from a water decoction of Chinese ginseng root. METHOD: Total saponins were efficiently purified by dynamic anion-cation exchange following the removal of hydrophilic impurities by macroporous resin D101. For quality control, ultrahigh-performance liquid chromatography with a charged aerosol detector (CAD) was applied to quantify marker components. The total saponin content was estimated by a colorimetric method using a vanillin-vitriol system and CAD response. RESULTS: D201, which consisted of a cross-linked polystyrene matrix and -N(+)(CH3)3 functional groups, was the best of the four anion exchange resins tested. However, no significant difference in cation exchange ability was observed between D001 (strong acid) and D113 (weak acid), although they have different functional groups and matrices. After purification in combination with D101, D201, and D113, the estimated contents of total saponins were 107% and 90% according to the colorimetric method and CAD response, respectively. The total amount of representative ginsenosides Re, Rd, Rg1, and compound K was approximately 22% based on ultrahigh-performance liquid chromatography-CAD quantitative analysis. CONCLUSION: These findings suggest that an ion exchange resin, combined with macroporous adsorption resin separation, is a promising and feasible purification procedure for neutral natural polar components.

[Advance of studies on application of hapten antibodies].

Hapten antibodies are active components of traditional Chinese medicines, have been widely applied in all of study fields of traditional Chinese medicine. First, hapten monoclonal antibodies could be designed into ELISA kits for quantitative analysis on the content of effective components in plant crude extracts or biological samples, which be applied for quality control and studies on pharmacokenetics of traditional Chinese medicines. Second, hapten monoclonal antibodies could be coupled with solid-phase carriers to generate immunoaffinity chromatography column, which could be used for knock-out extract preparation or pre-treatment of complicated sampless. Finally, a single-chain variable fragment antibody (scFV) gene segment of effective components of hapten monoclonal antibodies could be transformed into relative plant cells to gain new varieties with high-enrichment effective components, and thus achieve the molecular breeding of medicinal plants.

Brazilein, an important immunosuppressive component from Caesalpinia sappan L.

Caesalpinia sappan has been shown to have interesting immunosuppressive properties. Its heartwood has long been used in Chinese medicines for treating a variety of immune-mediated pathology and inflammatory disease. The purpose of this work was to evaluate the immunocompetence effects of brazilein on mice lymphocytes in vitro and in vivo. The results showed that brazilein and Caesalpinia sappan ethanol extract (SME) could distinctly inhibit the proliferation of T lymphocyte stimulated by Concanavalin A (Con A) and the proliferation of B lymphocyte stimulated by lipopolysaccharides (LPS), and brazilein could suppress mice humoral immune response by plaque forming cell (PFC) test. In addition, immune organs (thymus and spleen) in mice treated with brazilein were notably atrophied and weight loss in vivo (intraperitoneal injection, i.p.). In attempting to investigate the mechanisms of the immunosuppressive activity of brazilein, we discovered that brazilein can induce apoptosis in mice spleen lymphocytes by flow cytometry analysis and DNA fragmentation assay, which may be one of the pathways that brazilein inhibited immunocompetence of mice lymphocytes.

Study on cardioactive effects of brazilein.

Brazilein (6a,7-dihydro-3,6a,10-trihydroxy-benz[b]indeno[1,2-d]pyran-9(6H)-one) is a compound obtained in a large amount from Caesalpinia sappan ethanol extracts with a high purity of about 98%. In isolated cardiac tissues, we found that brazilein exhibited a positive inotropic action in a concentration-dependent manner with little effect on heart rate and coronary perfusion. To study its possible mode of action, isolated rat hearts were treated with propranolol. This treatment did not alter the cardiotonic effect of brazilein, suggesting that this effect does not involve stimulation of beta-adrenoceptors. On the other hand, an analysis of the interaction between Na(+),K(+)-ATPase and brazilein was carried out. Albino guinea pig erythrocytes (mainly alpha1-Na(+),K(+)-ATPase isoforms) enriched with Na(+),K(+)-ATPase isoforms were utilized to compare the inhibition promoted by brazilein with that of classical inhibitors such as the cardiac glycoside deslanoside. Analysis of inhibition curves revealed that unlike deslanoside, brazilein had a relatively low affinity for erythrocyte isoforms and failed to completely inhibit the Na(+),K(+)-ATPase activity. The extent of the maximum inhibition rate was about 50%. The inhibitory effect of brazilein was not antagonized by 10 mmol/l K(+), as observed with deslanoside. Electrocardiogram research in vivo showed that brazilein did not induce the ventricular arrhythmias observed with deslanoside, suggesting that brazilein might have a less adverse effect and higher therapeutic index than cardiac glycosides. In light of all the above-mentioned observations, it can be concluded that brazilein, a molecule with a non-steroidal skeleton, produced its positive inotropic effect through inhibiting Na(+),K(+)-ATPase and could thus serve as a structural paradigm to develop new inotropic drugs.

A new approach to investigate the pharmacokinetics of traditional chinese medicine YL2000.

Much progress has been made in the pharmacology of Traditional Chinese Medicine (TCM). However, the question on how to investigate pharmacokinetics of TCM extract remains. In this study, we selected a new TCM extract YL2000 developed in our laboratory as the research object and investigated both the pharmacokinetics of baicalin and berberine in YL2000 and the pharmacodynamics of YL2000 in febrile rats. The correlation analysis between the time-concentration curves of baicalin and berberine and the time-effect curve of YL2000 was conducted in plasma by statistical methods. The results showed that the time-effect data of anti-pyretic effect of YL2000 had a negative correlation (r = -0.8312, P < 0.1) with the time-concentration data of baicalin in plasma, but had no correlation (r = 0.01368, P > 0.5) with berberine. These data suggested that baicalin could be selected as a marker of anti-pyretic effect, and that YL2000 could be used to treat fevers according to the disposition of baicalin in vivo. In this study, we also proposed that one or more active elements in TCM extracts could be selected to represent the pharmacokinetics of TCM extracts in vivo, combined with the pharmacodynamics of TCM extract.

Pharmacokinetic study of ellagic acid in rat after oral administration of pomegranate leaf extract.

Quantification of ellagic acid, the principal bioactive component of pomegranate leaf extract, in rats plasma following oral administration of pomegranate leaf extract was achieved by using a high-performance liquid chromatographic method. The calibration curve for ellagic acid was linear (r2=0.9998) ver the concentration range 0.026-1.3 microg/ml. The intra- and inter-day assays of ellagic acid from rat plasma were less than 6.52% at concentration range from 26 to 1300 ng/ml and good overall recoveries (94.5-102.4%) were found on same concentrations. The concentration-time profile was fitted with an open two-compartment system with lag time and its max concentration of ellagic acid in plasma was 213 ng/ml only 0.55 h after oral administration extract 0.8 g/kg. The pharmacokinetic profile indicates that ellagic acid has poor absorption and rapid elimination after oral administration pomegranate leaf extract, and part of it was absorbed from stomach.