[Mechanism of Jiaotai Pills in treatment of depression based on quantitative proteomics].

This study aimed to investigate the effect of Jiaotai Pills on protein expression in the hippocampus of the rat model of chronic unpredictable mild stress(CUMS)-induced depression by quantitative proteomics and explore the anti-depression mechanism of Jiaotai Pills. The SD rats were randomized into control, model, Jiaotai Pills, and fluoxetine groups(n=8). Other groups except the control group were subjected to CUMS modeling for 4 weeks. After 4 weeks of continuous administration, the changes of behavior and pathological morphology of the hippocampal tissue were observed. Proteins were extracted from the hippocampal tissue, and bioinformatics analysis was performed for the differentially expressed proteins(DEPs) identified by quantitative proteomics. Western blot was employed to verify the key DEPs. The results showed that Jiaotai Pills significantly alleviated the depression behaviors and hippocampal histopathological changes in the rat model of CUMS-induced depression. A total of 5 412 proteins were identified in the hippocampus of rats, including 65 DEPs between the control group and the model group and 35 DEPs between the Jiaotai Pills group and the model group. There were 16 DEPs with the same trend in the Jiaotai Pills group and the control group, which were mainly involved in sphingolipid, AMPK, and dopaminergic synapse signaling pathways. The Western blot results of Ppp2r2b, Cers1, and Ndufv3 in the hippocampus were consistent with the results of proteomics. In conclusion, Jiaotai Pills may play an anti-depression role by modulating the levels of Ppp2r2b, Cers1, Ndufv3 and other proteins and regulating sphingolipid, AMPK, and dopaminergic synapse signaling pathways.

Comparative pharmacokinetics of three alkaloids in normal and acute hepatitis rats after oral administration of Yanhuanglian total alkaloids extract.

Acute hepatitis is a severe inflammatory disease with high mortality rates. Dehydrocavidine (DC), berberine (BB) and palmatine (PT), the bioactive components of Yanhuanglian total alkaloids extract (YTAE), may play important roles in its protective effect against acute hepatitis. The present study was designed to compare the pharmacokinetic properties of DC, BB and PT after oral administration of YTAE in normal and acute hepatitis rats. The plasma pharmacokinetic profiles of three major bioactive alkaloids from YTAE were analyzed using an LC-MS/MS method. It was found that the pharmacokinetic parameters of DC, BB and PT represented a statistically significant difference (p ˂ 0.05) between the normal rats and the acute hepatitis rats after administration of YTAE. The results indicated that the Cmax , T1/2ß and AUC of DC, BB and PT in acute hepatitis rats were significantly increased, whereas the CL and Vd values were remarkably decreased (p ˂ 0.05) over those of the normal rats. The results suggested that the pharmacokinetic behavior of the active constituents from YTAE could be changed when it was used in acute hepatitis animals, which provide a meaningful basis for developing a clinical dosage regimen in the treatment of acute hepatitis by YTAE.

[Effect of Clopidogrel on Plasma Protein Binding Rates of Ginsenoside Rg1].

Objective To observe the effect of clopidogrel on plasma protein binding rates of gin- senoside Rg1. Methods Concentrations of ginsenoside Rg1 were measured in fetal bovine serum and phosphate buffered solution (PBS). Samples were randomly divided into ginsenoside Rg1 groups (low: 0.4; middle: 1. 0; high 5. 0 mg/L, respectively) and clopidogrel combined ginsenoside Rg1 groups (low: 0. 4 mg/L +2. 0 mg/L clopidogrel; middle: 1. 0 mg/L +2. 0 mg/L clopidogrel; high: 5. 0 mg/L +2. 0 mg/L clo- pidogrel). The effect of clopidogrel on plasma protein binding rates of ginsenoside Rgl was observed u- sing equilibrium dialysis. Then 3-dimensional structure of bovine serum albumin (BSA) was constructed using homology modeling. On this basis, binding effect of small compounds (ginsenoside Rg1 and clopi- dogrel) and BSA was observed using molecular docking method. Results The serum protein binding rate was 11. 2% ±2. 1% in the low dose ginsenoside Rg1 group, 13. 4% ±2. 2% in the middle dose ginsenoside Rgl group, and 14. 6% ±1. 4% in the high dose ginsenoside Rg1 group, respectively. It was 6. 5% ±2. 3% in the clopidogrel combined low dose ginsenoside Rg1 group, 9. 2% ±1. 5% in the clopi- dogrel combined middle dose ginsenoside Rg1 group, 12.1% ± 1. 7% in the clopidogrel combined high dose ginsenoside Rg1 group, respectively. They were lower in clopidogrel combined ginsenoside Rg1 groups than in ginsenoside Rg1 groups with statistical difference (P <0. 05). Results of molecular doc- king showed that competitive binding effect existed between compounds (ginsenoside Rg1 and clopi- dogrel) and BSA. Conclusion Results of equilibrium dialysis and molecular docking comprehensively in- dicated clopidogrel had effect on plasma protein binding rate of ginsenoside Rg1.

[Study of change in activity of hepatic drug metabolism enzymes in rat model of chronic unpredictable mild stress].

This study aimed to explore the impact of depression caused by chronic unpredictable mild stress (CUMS) on in vivo activity of six kinds of CYP450 isoforms in rats. According to 'Katz' method, the model of CUMS was established. Tolbutamide, chlorzoxazone, theophylline, midazolam, omeprazole and dextromethorphan were chosen as probe substrates of CYP2C6, CYP2E1, CYP1A2, CYP3A2, CYP2D1 and CYP2D2 of rats. Plasma concentration of six kinds of CYP450 in control group and model group were determined by LC-MS/MS and computed pharmacokinetic parameters. Consequently, metabolism of theophylline and chlorzoxazone accelerated significantly (P < 0.01), but tolbutamide, dextromethorphan, omeprazole and midazolam had no significant difference. The present study proved that depression caused by CUMS had strong induction to CYP1A2 and medium induction to CYP2E1.

[Computational Pharmacological Study on Clopidogrel Metabolism Enzymes Influenced by Fufang Danshen Dripping Pill].

OBJECTIVE: To investigate the effect of Fufang Danshen Dripping Pill on Clopidogrel metabolism enzymes target such as human liver carboxylesterasel (CES1), cytochrome P450 3A4, CYP450 2C19, CYP450 1A2, and CYP450 2B6, and to interpret the interaction effects. METHODS: The CES1, cytochrome P450 3A4, CYP450 2C19, CYP450 1A2 and CYP450 2B6 which involved in Clopidogrel metabolism were selected at first, the chemical ligand database were created then, and finally the interaction effects between the ligand database and Clopidogrel metabolism target were explored. RESULT: 1 MX1 (CES1), 3NXU (CYP450 3A4), 4GQS (CYP450 2C19), 2HI4 (CYP450 1A2) and 3IBD(CYP450 2B6) as well as THA, RIT, OXU, Chlorzoxazone and CPZ were used as receptors and cutoff for each target respectively. The number of hits with potentially positive activities with metabolism enzymes target from the bioactive compounds in the preparation was 29, 8, 31, 51 and 44, respectively. These computational pharmacological docking studies were in accordance with the referenced cocktail experiment results. CONCLUSION: It is suggested that Fufang Danshen Dripping Pill has inhibitory effects on Clopidogrel metabolism enzymes target such as CES1, Cytochrome P450 3A4, CYP450 2C19, CYP450 1A2 and CYP450 2B6.

[Study on biomarker of Tripterygium wilfordii in treatment of rheumatoid arthritis based on PK/PD].

To observe the serum samples and the anti-inflammatory effect of Tripterygium wilfordii in treating RA by using the pharmacokinetic-pharmacodynamic model, make a correlation analysis on concentration-time and effect-time curves, and explore RORγt, IL-17, STAT3, IL-6 mRNA transcriptional levels in rats by PCR. Methotrexate, tripterine and high-dose T. wilfordii could down-regulate RORγt, IL-17, STAT3, IL-6 mRNA transcriptional levels in AA rat lymph nodes. The study on PK-PD model showed correlations between inflammatory factors and blood concentration of T. wilfordii. T. wilfordii and its main active constituent tripterine could show the inflammatory effect and treat RA by inhibiting IL-17 cytokine.

[Effect of Clopidogrel on Pharmacokinetic of Fufang Danshen Dripping Pill (FDDP)].

OBJECTIVE: To investigate the effect of clopidogrel on the pharmacokinetic of Fufang Danshen Dripping Pill (FDDP); METHODS: 20 SD rats were randomly divided into two group,which were intrinsically administrated FDDP(324 mg/kg) alone and combination of FDDP(324 mg/kg) and clopidogrel(30 mg/kg) respectively for 21 days. The ginsenoside Rg, from FDDP was determined by HPLC and its pharmacokinetic parameter were finally compared. RESULTS: The value of Cmax and AUC0-∞ of ginsenoside Rg, were increased from 1.97 ± 0.44 mg/L and 8.44 ± 2.64 mg/L · h to 2.48 ± 0.63 mg/L and 14.38 ± 5.72 mg/L · h respectively. CONCLUSION: Long term with clopidogrel has effect on the pharmacokinetic character of Rg, from FDDP, this research may provides theoretical support for the FDDP-clopidogrel combination treatment in clinical.

[Synergistic action of Compound Danshen Dripping Pill (CDDP) on Clopidogrel Bisulfate (CPG) counteracting platelet aggregation].

OBJECTIVE: To study the synergistic action of Compound Danshen Dripping Pill (CDDP) on Clopidogrel Bisulfate (CPG) counteracting platelet aggregation. METHODS: 40 Sprague-Dawley (SD) rats were randomized into four groups: normal control group (CMC-Na), CPG alone group (30 mg/kg), CDDP alone group (324 mg/kg), co-administration group (CPG 30 mg/kg and CDDP 324 mg/kg). The rats received gastric infusion of corresponding drugs for 21 continuous days. Blood sample of SD rats were collected by puncture of the abdominal artery for the determination of coagulation parameters such as prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB) concentration and thrombin time (TT) in each group. Another 40 SD rats were used for the observation of inhibitory effect on the thrombosis in arteriovenous shunt. Finally, homology modeling and molecular docking were employed to simulate their interaction between the P2Y purinoceptor 12 (P2Y12) target and bioactive compounds contained in CDDP. RESULTS: The bleeding time of coagulation parameters was prolonged, the thrombosis in arteriovenous shunt was inhibited in the medication group. The above effect was much better in the combination group. The molecular docking showed that bioactive compounds contained in CDDP was able to inhibit target P2Y12. CONCLUSION: CDDP can enhance the effect of CPG on inhibiting platelet aggregation.

Caffeic Acid Phenethyl Ester Prevents Colitis-Associated Cancer by Inhibiting NLRP3 Inflammasome.

Long-lasting inflammation in the intestinal tract renders individuals susceptible to colitis-associated cancer (CAC). The NOD-like receptor protein 3 (NLRP3) inflammasome plays a key role in the progression of inflammatory bowel disease and CAC. Therefore, identifying effective drugs that prevent CAC by targeting NLRP3 inflammasome is of great interest. Here, we aimed to evaluate the anti-inflammatory effect of caffeic acid phenethyl ester (CAPE) on bone marrow-derived macrophages (BMDMs), THP-1 cells, and azoxymethane/dextran sulfate sodium (AOM/DSS)-induced colon cancer mouse model. We also investigated the anti-tumor mechanism of CAPE. We found that CAPE decreased NLRP3 inflammasome activation in BMDMs and THP-1 cells and protected mice from colorectal cancer induced by AOM/DSS. CAPE regulated NLRP3 at the post-transcriptional level by inhibiting reactive oxygen species (ROS) production. However, CAPE did not affect NLRP3 or IL-1ß transcription, but instead enhanced NLRP3 binding to ubiquitin molecules, promoting NLRP3 ubiquitination, and contributing to the anti-tumor effect in the AOM/DSS mouse model. Moreover, CAPE suppressed the interaction between NLRP3 and CSN5 but enhanced that between NLRP3 and Cullin1 both in vivo and in vitro. Altogether, our findings demonstrate that CAPE prevents CAC by post-transcriptionally inhibiting NLRP3 inflammasome. Thus, CAPE may be an effective candidate for reducing the risk of CAC in patients with inflammatory bowel disease.

Ginsenoside Rb2 inhibits epithelial-mesenchymal transition of colorectal cancer cells by suppressing TGF-ß/Smad signaling.

BACKGROUND: Treating colorectal cancer (CRC) continues to be a clinical challenge. Studies have shown that epithelial-mesenchymal transition (EMT) is a critical step in tumor progression and transforming growth factor-ß1 (TGF-ß1) signaling has been shown to play a crucial role in EMT. Here, we investigate the inhibition effect of Ginsenoside Rb2, main bioactive component of ginseng, in human colorectal cancer cells via TGF-ß1. PURPOSE: The current study aims to study the inhibitory effect of Ginsenoside Rb2 on HCT116 and SW620 cells and its anti-tumor mechanism. METHODS: Histomorphological analysis and western blot analysis were performed to evaluate expression of TGF-ß1 in human cancerous colon samples and the adjacent normal samples. The docking simulation assay were performed to explore the potential mode of binding of Ginsenoside Rb2 to the TGF-ß1 protein. CCK8, adhesion and invasion assay were used to assess the effects of Ginsenoside Rb2 in HCT116 and SW620 cells. RT-PCR, Western blot and Immunohistochemical staining were employed to detect the TGF-ß1-related signaling pathways in the colon cancer cells and/or xenograft mice. RESULTS: The expression of TGF-ß1 in human cancerous colon samples was significantly increased compared with the adjacent normal samples. Ginsenoside Rb2 inhibit the growth, adhesion, EMT and metastasis of human colorectal cancer cells. The docking simulation assay confirmed that Ginsenoside Rb2 bound to the hydrophobic pocket of TGF-ß1, which partially overlaps with the binding sites on TGF-ß1, and thus disrupted TGF-ß1 dimerization. Western Blot analysis further confirmed that Ginsenoside Rb2 could inhibit the expression of TGF-ß1 in vitro and in vivo. Furthermore, Ginsenoside Rb2 could inhibit the expression of Smad4 and phosphorylated Smad2/3. CONCLUSION: Ginsenoside Rb2 could inhibit EMT of colorectal cancer cells through the TGF-ß1/Smad signaling, and might be a potential candidate for the treatment of colorectal cancer.

Biotransformation and Metabolic Profile of Limonin in Rat Liver Microsomes, Bile, and Urine by High-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry.

Limonin is a triterpenoid in citrus seeds, which has significant biological activities. However, the metabolic profile of limonin has not been fully understood. To expound its metabolism in vivo and in vitro, the metabolites of limonin was studied by rat liver microsomes, urine, and bile. High-performance liquid chromatography/quadrupole time-of-flight mass spectrometry was used for identification. Among the metabolites, the structures of M1 and M3 were confirmed by chemical synthesis and nuclear magnetic resonance spectra analysis. Our results indicated that reduction and hydrolysis were the two major pathways during limonin metabolism in vivo and in vitro. The results from this work are valuable and important for understanding the metabolic process of limonin.

Determination of Paeoniflorin in Rat Plasma by Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry and its Application to a Pharmacokinetic Study.

In this study, a rapid and highly sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method was established for the quantification of paeoniflorin in rat plasma. The analyte and the internal standard (IS), hyperoside, were extracted from rat plasma via liquid-liquid extraction with ethyl acetate. LC separation was carried on a Phenomenex Luna C18 column within 2.5 min. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization and operated in the multiple reaction monitoring and negative ion modes. The precursor to product ion transitions monitored for paeoniflorin and the IS were m/z 524.8→449.0 and 463.1→300.0, respectively. The assay was validated with a linear range of 0.02~25.6 µg/mL for paeoniflorin. The intra- and inter-day precisions (relative standard deviation%) were within 9.7%, and the recoveries were greater than 65.2%. Paeoniflorin was proven to be stable during all sample storage, preparation and analytical procedures. The method was successfully applied to a pharmacokinetic study of the Baixiangdan Capsule in eight female rats.

Pharmacokinetic and pharmacodynamic modeling of oral mitiglinide on glucose lowering in healthy Chinese volunteers.

BACKGROUND: Mitiglinide is a widely used agent for diabetic treatment. We established a pharmacokinetic-pharmacodynamic (PK-PD) model to illustrate the relationship between mitiglinide plasma concentration and its glucose lowering effects in healthy volunteers. METHODS: The volunteers participated in the test after the administration of a single dose of 10 mg mitiglinide. The drug concentration in Plasma and the values of glucose levels were determined by LC-MS/MS assay and hexokinase method. A PK-PD model was established with a series of equations to describe the relationship between plasma medicine and glucose, and the equations were solved numerically and fitted to the data with the Phoenix NLME software. RESULTS: The results of the two-compartment model analysis were based on the maximum likelihood criterion and visual inspection of the fittings. The terminal elimination half-life (t 1/2) was 1.69 ± 0.16 h and the CL/F was 7.80 ± 1.84 L/h. The plasma glucose levels began to decline by 0.2 h, and hit its bottom decreasing values of 2.6 mg/L at 0.5 h after administration. The calculated parameter and fitting curve indicated that the model established in our experiment fitted well. CONCLUSIONS: A PK/PD model illustrates that the relationship between mitiglinide concentration in plasma and glucose lowering effect in healthy volunteers was established. The results of our experiment suggested that the model can be used reasonably to predict the relationship between PK and PD in mitiglinide, which could be used in diabetes mellitus dosage control in clinical trials and other fields.

Measurement of hydroxysafflor yellow A in human urine by liquid chromatography-tandem mass spectrometry.

A rapid and specific high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the quantification of hydroxysafflor yellow A (HSYA) in human urine with isorhamnetin-3-O-neohespeidoside as internal standard (IS). HSYA and IS were extracted from urine samples by simple solid-phase extraction and separated on an Agilent Zorbax SB C18 column (4.6 mm × 150 mm, 5 µm) with the mobile phase of 0.2 mM ammonium acetate: methanol (30/70, v/v) at a flow rate of 0.4 mL/min. Polar endogenous interferences eluted in 0.1-2.5 min were switched into waste channel by the Valve Valco, to reduce the possible matrix effect for MS detection in each run. The MS detection of analytes was performed on a tandem mass spectrometer equipped with an electrospray ionization source in negative mode using multiple-reaction monitoring. The MS/MS ion transitions monitored were m/z 611.3→491.2 for HSYA and m/z 623.2→299.2 for IS. The method was fully validated for selectivity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability, and then was applied to the urinary excretion study of injectable powder of pure HSYA in healthy Chinese volunteers for the first time. The results suggested that urine was the main excretion way of HSYA in healthy volunteers, further demonstrating the feasibility and necessity of our current method.

In vitro inhibitory effects of ethanol extract of Danshen (Salvia miltiorrhiza) and its components on the catalytic activity of soluble epoxide hydrolase.

BACKGROUND: Soluble epoxide hydrolase (sEH) has been demonstrated to be a key enzyme involved in the pathologic development of several cardiovascular diseases and inflammation, and inhibition of sEH is therefore very helpful or crucial for the treatment of ischemia-reperfusion injury, cardiac hypertrophy, hypertension and inflammation. Danshen, the dried root of Salvia miltiorrhiza (Fam. Labiatae), has been used for the treatment of cardiovascular and cerebrovascular diseases in China and other countries for hundreds of years. Recent studies indicated that Danshen and its preparations also have potential for the management of inflammation. However, little information is available about the possibility of Danshen and its components on sEH inhibition. PURPOSE AND METHODS: Danshen extracts and its constituents were tested for sEH inhibition using its physiological substrate, 8,9-EET, based on a LC-MS/MS assay in this study. RESULTS: Among the tested 15 compounds, tanshinone IIA and cryptotanshinone were found to be the potent (Ki = 0.87 µM) and medium (Ki = 6.7 µM) mixed-type inhibitors of sEH, respectively. Salvianolic acid C (Ki = 8.6 µM) was proved to be a moderate noncompetitive sEH inhibitor. In consistent with the inhibition results of the pure compounds, the 75% ethanol extract of Danshen (EE, IC50 = 86.5 µg/ml) which contained more tanshinone IIA and cryptotanshinone exhibited more potent inhibition on sEH than the water extract (WE, IC50 > 200 µg/ml) or 1 M NaHCO3 (BE, IC50 > 200 µg/ml) extract. CONCLUSION: These data indicated that using the ethanol fraction of Danshen and increasing the amounts of tanshinone IIA, cryptotanshinone and salvianolic acid C, especially the contents of tanshinone IIA in Danshen extract or preparations to enhance the inhibitory effects on sEH might be efficient ways to improve its cardiovascular protective and anti-inflammatory effects, and that herbal medicines could be an untapped reservoir for sEH-inhibition agents and developing sEH inhibitors from the cardiovascular protective and anti-inflammatory herbs is a promising approach.

[Effect of clopidogrel on plasma protein binding rate of ginsenosides: a liquid chromatography-mass spectrometry-based study].

OBJECTIVE: To investigate the effect of clopidogrel on the binding rate of ginsenosides with rat serum proteins (RSA). METHODS: Equilibrium dialysis and liquid chromatography-mass spectrometry were employed to quantify the concentration of ginsenoside Rg1 and Rb1. The protein-binding rates of Rg1 and Rb1 in the presence or absence of clopidogrel (1.0 mg/L) were determined. A molecular simulation model (consisting of homology modeling and molecular docking interaction) was used to reveal the target protein-compound interactions. RESULTS: The binding rates of ginsenosides Rg1 (0.4, 1.0, and 2.0 mg/L) with RSA were (30.16∓2.82)%, (33.42∓4.21)%, and (34.61∓3.42)%, and those of and Rb1 were (50.13∓2.34)%, (51.23∓3.23)%, and (53.11∓3.26)%, respectively. In the presence of clopidogrel, the binding rates of Rg1 decreased to (22.13∓2.72)%, (21.42∓3.22)%, and (25.45∓3.52)%, and those of Rb1 to (40.13∓3.24)%, (41.25∓4.15)%, and (43.11∓3.31)%, receptively. The molecular docking suggested that these compounds competed to bind with RSA. CONCLUSION: Clopidogrel can competitively bind to RSA with ginsenosides to lower the plasma protein binding rates of ginsenosides.