[Effect of Rehmanniae Radix on depression-like behavior and hippocampal monoamine neurotransmitters of chronic unpredictable mild stress model rats].

To investigate the effect of Rehmanniae Radix on depression-like behavior and monoamine neurotransmitters of chronic unpredictable mild stress(CUMS) model rats. CUMS combined with isolated feeding was used to induce the depression model of rats. The depression-like behavior of rats was evaluated by sucrose preference test, open field test, and forced swim test. Hematoxylin-Eosin(HE) staining was used to investigate the pathological changes of neurons in the CA1 and CA3 area of hippocampus. Ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS) was used to detect the contents of 5-hydroxytryptamine(5-HT), 5-hydroxyindoleacetic acid(5-HIAA), dopamine(DA), 3,4-dihydroxyphenylacetic acid(DOPAC), homovanillic acid(HVA), norepinephrine(NE), and 3-methoxy-4-hydroxyphenyl glycol(MHPG) in rats. Western blot was used to detect the protein expressions of tryptophan hydroxylase 2(TPH2), serotonin transporter(SERT), and monoamine oxidase A(MAO-A) in the hippocampus of rats. Compared with the normal group, depressive-like behavior of rats was obvious in the model group. The arrangements of neurons in the CA1 and CA3 area of hippocampus were loose and disorderly. The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in the hippocampal area were decreased(P<0.01). The protein expression of TPH2 was decreased(P<0.01), but those of SERT and MAO-A were increased(P<0.01). In the Rehmanniae Radix groups with 1.8 g·kg~(-1) and 7.2 g·kg~(-1), the depression-like behavior of CUMS rats and pathological changes of neurons in CA1, CA3 area of hippocampus were improved. The protein expression of TPH2(P<0.05, P<0.01) was increased, and those of SERT and MAO-A were down-regulated(P<0.05, P<0.01). The levels of 5-HT, 5-HIAA, and 5-HT/5-HIAA in hippocampus were increased(P<0.05, P<0.01). The changes in DA, DOPAC, HVA, DA/(DOPAC +HVA), NE, DHPG, and NE/DHPG were not statistically significant. The results suggested that Rehmanniae Radix improved depression-like behavior of CUMS rats, and the mechanism might be related to the regulation of synthesis, transportation, and metabolism of 5-HT neurotransmitter in the hippocampus.

[Formulation optimization of emodin nanostructured lipid carriers by Box-Behnken response surface method and in vitro quality evaluation].

Emodin nanostructured lipid carriers(ED-NLC) were prepared and their quality was evaluated in vitro. Based on the results of single-factor experiments, the ED-NLC formulation was optimized by Box-Behnken response surface method with the dosages of emodin, isopropyl myristate and poloxamer 188 as factors and the nanoparticle size, encapsulation efficiency and drug loading as evaluation indexes. Then the evaluation was performed on the morphology, size and in vitro release of the nanoparticles prepared by emulsification-ultrasonic dispersion method in line with the optimal formulation, i.e., 3.27 mg emodin, 148.68 mg isopropyl myristate and 173.48 mg poloxamer 188. Under a transmission electron microscope(TEM), ED-NLC were spherical and their particle size distribution was uniform. The particle size of ED-NLC was(97.02±1.55) nm, the polymer dispersion index 0.21±0.01, the zeta potential(-38.96±0.65) mV, the encapsulation efficiency 90.41%±0.56% and the drug loading 1.55%±0.01%. The results of differential scanning calorimeter(DSC) indicated that emodin may be encapsulated into the nanostructured lipid carriers in molecular or amorphous form. In vitro drug release had obvious characteristics of slow release, which accorded with the first-order drug release equation. The fitting model of Box-Behnken response surface methodology was proved accurate and reliable. The optimal formulation-based ED-NLC featured concentrated particle size distribution and high encapsulation efficiency, which laid a foundation for the follow-up study of ED-NLC in vivo.

[Anti-depressant components and mechanism of Rehmanniae Radix based on UPLC-Q-Orbitrap HRMS and network pharmacology].

This study aimed to explore the anti-depressant components of Rehmanniae Radix and its action mechanism based on network pharmacology combined with molecular docking. The main components of Rehmanniae Radix were identified by ultra-high performance liquid chromatography-quadrupole/Orbitrap high resolution mass spectrometry(UPLC-Q-Orbitrap HRMS), and the related targets were predicted using SwissTargetPrediction. Following the collection of depression-related targets from GeneCards, OMIM and TTD, a protein-protein interaction(PPI) network was constructed using STRING. GO and KEGG pathway enrichment analysis was performed by Metascape. Cytoscape 3.7.2 was used to construct the networks of "components-targets-disease" and "components-targets-pathways", based on which the key targets and their corresponding components were obtained and then preliminarily verified by molecular docking. Rehmanniae Radix contained 85 components including iridoids, ionones, and phenylethanoid glycosides. The results of network analysis showed that the main anti-depressant components of Rehmanniae Radix were catalpol, melittoside, genameside C, gardoside, 6-O-p-coumaroyl ajugol, genipin-1-gentiobioside, jiocarotenoside A1, neo-rehmannioside, rehmannioside C, jionoside C, jionoside D, verbascoside, rehmannioside, cistanoside F, and leucosceptoside A, corresponding to the following 16 core anti-depression targets: AKT1, ALB, IL6, APP, MAPK1, CXCL8, VEGFA, TNF, HSP90 AA1, SIRT1, CNR1, CTNNB1, OPRM1, DRD2, ESR1, and SLC6 A4. As revealed by molecular docking, hydrogen bonding and hydrophobicity might be the main action forms. The key anti-depression targets of Rehmanniae Radix were concentrated in 24 signaling pathways, including neuroactive ligand-receptor interaction, neurodegenerative disease-multiple diseases pathway, phosphatidylinositol 3-kinase/protein kinase B pathway, serotonergic synapse, and Alzheimer's disease.

Development of an ultra-performance liquid chromatography-electrospray ionization-Orbitrap mass spectrometry method for determination of xanthopurpurin in rat plasma and its application to pharmacokinetic study.

A rapid and sensitive method was developed and validated for the quantitative determination of xanthopurpurin (XPP) in rat plasma using ultra-performance liquid chromatography-electrospray ionization-Orbitrap mass spectrometry. XPP inhibits IgE production and prevents peanut-induced anaphylaxis. The XPP and emodin (internal standard) were determined in negative ion mode with m/z 239.0350 → 211.0400 and 269.0455 → 241.0507, respectively. The separation process was achieved using an ACQUITY UPLC HSS T3 column with acetonitrile and 0.1% formic acid in water (85:15). The linear range was 0.5-100 ng/mL, and the correlation coefficient (r2 ) was > 0.993. The inter-day and intra-day precision was within an acceptable range of 15%. The extraction recovery and matrix effect were 78.9-87.2% and 94.3-98.5%, respectively. Under different conditions, the XPP was stable in the range of 5.6-10.6%. This method was successfully applied to study the pharmacokinetics of XPP with an oral dose of 10.0 mg/kg and intravenous dose of 2.0 mg/kg in rats. The absolute oral bioavailability of XPP was 4.6%.

[Quantitative analysis of 16 components in Cyclocarya paliurus leaf by UPLC-QE-Orbitrap-MS].

Modern research showed that components in the dried leaf of Cyclocarya paliurus. had various biological activities. The current quality control research was focused on content determination of polysaccharides and flavonoids, while there were less research on quantitative analysis of terpenes and phenolic acids. In this paper, the contents of 16 components of 3 kinds in C. paliurus leaf were determined by UPLC-QE-Orbitrap-MS. The results were as following: good linear relationship of 16 analytes existed within the studied concentration rages (R²>0.996), and RSDs were of <3.0% in the precision test and replicate test, with the average recovery rates 95.20%-104.4%, respectively. The results indicatod that the method is simple and accurate, which can be used for the comprehensive quality evaluation of C. paliurus leaf. The established method was applied to determine the contents of 12 batches of C. paliurus leaf from different areas, and the 16 analvtes contents in the samples could be different from several times to dozens times, which indicated that there might be significant quality difference in C. paliurus leaf from different areas.

Comprehensive study of cationic liposomes composed of DC-Chol and cholesterol with different mole ratios for gene transfection.

The ability of cationic liposomes composed of DC-Chol and cholesterol to carry pDNA into 293 T cells was investigated. Lipoplexes formed between DC-Chol/cholesterol liposomes and pDNA (encoding green fluorescent protein, GFP) were analyzed in terms of morphology observation, turbidity determination, particle size and zeta potential measurement, differential scanning calorimetry (DSC), gel retardation assay, cytotoxicity analysis in 293 T cells and transfection efficiency. The results showed that liposome preparation at or above 66.7 mol% cholesterol in formulation exhibited a calorimetric transition caused by anhydrous cholesterol domain at about 41°C. In comparison with the control, DOPE-containing liposomes, DC-Chol/cholesterol carriers showed more stable particle size, lower turbidity, higher activity for transfecting cells in the presence of high concentration serum (50% FBS), primarily due to the neutral domain formation by increasing mole ratios of cholesterol in formulation, as well as relatively lower cytotoxicity. Based on the results, it is suggested that incorporating high contents of cholesterol might be a potentially applicable method for various kinds of cationic lipids to obtain the gene carriers with high capability for in vivo transfection.

Identification and characterization of human UDP-glucuronosyltransferases responsible for the in vitro glucuronidation of salvianolic acid A.

Glucuronidation is an important pathway in the elimination of salvianolic acid A (Sal A); however the mechanism of UDP-glucuronosyltransferases (UGTs) in this process remains to be investigated. In this study, the kinetics of Sal A glucuronidation by pooled human liver microsomes (HLMs), pooled human intestinal microsomes (HIMs) and 12 recombinant UGT isozymes were investigated. The glucuronidation of Sal A can be shown both in HLMs and HIMs with K(m) values of 39.84 ± 3.76 and 54.04 ± 4.36 µM, respectively. Among the 12 human UGTs investigated, UGT1A1 and UGT1A9 were the major isoforms that catalyzed the glucuronidation of Sal A (K(m) values of 29.72 ± 2.20 and 24.40 ± 2.60 µM). UGT1A9 showed the highest affinity of Sal A glucuronidation. Furthermore, a significant correlation between Sal A glucuronidation and propofol glucuronidation (a typical UGT1A9 substrate) was observed. The chemical inhibition study showed that the IC(50) for phenylbutazone inhibition of Sal A glucuronidation was 50.3 ± 4.3 and 39.4 ± 2.9 µM by HLMs and UGT1A9, respectively. Mefenamic acid inhibited Sal A glucuronidation in UGT1A1 and HLMs with IC(50) values of >200 and 12.4 ± 2.2 µM, respectively.

Influence of rifampicin on the pharmacokinetics of salvianolic acid B may involve inhibition of organic anion transporting polypeptide (Oatp) mediated influx.

This article studied the possible effect of rifampicin (RIF), an inhibitor of organic anion transporting polypeptide (Oatp), on the pharmacokinetics of salvianolic acid B (SAB) in rats. Rifampicin was administered intravenously 15 min prior to SAB (5 mg/kg) in rats at doses of 0, 5.0, 10.0 and 20.0 mg/kg, respectively. The concentrations of SAB in plasma and bile were determined using a Shimadzu HPLC system coupled to a LC-MS-2010EV mass spectrometer. Compared with the control group, the AUC(0-t) and C(max) values of SAB were increased significantly, while the CL(total) and CL(bile) were decreased significantly. These results suggested that pretreatment with rifampicin prior to SAB administration could decrease significantly the total and bile elimination of SAB and alter its pharmacokinetic profiles. The influence of rifampicin on the pharmacokinetics of SAB may be attributed to the inhibition of Oatp-mediated influx.

Simultaneous determination of six phenolic constituents of Danshen injection in rat plasma by LC-ESI-MS and its application to a pharmacokinetic study.

Salvianolic acid A, salvianolic acid B, danshensu, protocatechuic aldehyde, rosmarinic acid and lithospermic acid are the six major active constituents in Danshen injection. In this study, a rapid, sensitive and specific liquid chromatographic-electrospray ionization-mass spectrometry method for the simultaneous quantitative determination of these compounds in rat plasma was developed. After a single step of liquid-liquid extraction with ethyl acetate, they were eluted by a Hypersil C18 column (5 µm, i.d. 4.6 × 200 mm) within 4 min with a mobile phase consisting of acetonitrile and 0.1% formic acid water solution (35:65, v/v). The assay was linear in the concentration range of 0.05-10 µg mL(-1). Absolute recoveries were above 60%. The precisions and accuracies determined within three consecutive days were within acceptable limits. The method was successfully applied to a pharmacokinetic study in rats after an intravenous administration of Danshen injection.

Determination of nimodipine in human plasma by HPLC-ESI-MS and its application to a bioequivalence study.

A simple, specific, and precise liquid chromatographic-electrospray ionization mass spectrometric (LC-ESI-MS) method has been developed for determination of nimodipine concentration in human plasma. The method involves the addition of 200 microL of saturated sodium bicarbonate (NaHCO(3)) solution to plasma to improve the extraction recovery, liquid-liquid extraction of nimodipine from plasma samples with anhydrous diethyl ether, simple reversed-phase chromatography, and ESI mass spectrometric detection in negative ion selected ion monitoring mode (SIM) using target [M-] ions at m/z 417 and m/z 359 for nimodipine and nitrendipine (internal standard, IS), respectively. A complete analytical run was achieved within 3.5 min. The limit of quantification was 0.5 ng/mL. The method was validated within a linear range of 0.5-100 ng/mL. The correlation coefficient for the calibration regression line was 0.9995 or better. Intra- and inter-batch accuracy and precision were acceptable. Analyte was stable in a battery of stability studies. The method has been successfully used in a bioequivalence study.

[Effect of cyclosporine A on the pharmacokinetics of ginkgolide B in rats].

The paper is aimed to investigate the effect of cyclosporine A (CyA) on the pharmacokinetics of ginkgolide B (GB) in rats, and to look for the mechanism of the changes in pharmacokinetic behaviors of GB. GB concentration in plasma, brain homogenate and urine samples of rats was determined by LC-MS. Effects of CyA on plasma levels, brain distributions as well as urinary excretions after intravenous administration of GB were evaluated. CyA co administrated intravenously at 10 mg kg(-1) or 20 mg kg(-1) significantly increased AUC(0-360 min) (P < 0.01) and decreased total CL of GB in rats. While co administrated CYP3A inhibitor itraconazole (ICZ) has no appreciable effect on the pharmacokinetic behavior of GB. CyA increased the brain uptake of GB in a dose-dependent manner. The brain distribution of GB was significantly increased at 5 min by different doses of CyA (P < 0.001), while at 20 and 60 min only high dose of CyA could significantly increase the levels of GB in the brain (P < 0.01 and P < 0.001). Different P-gp inhibitors CyA or verapamil (VER) or digoxin (DGX) decreased the urinary GB excretion, the urinary excretion of GB in 0-8 h were about 34.8% (P < 0.001), 59.4% (P < 0.001) and 79.7% (P < 0.05) of the control, separately. No appreciable effect of ICZ was observed on urinary excretion of GB. Coadministration of P-gp inhibitors CyA could significantly increase the plasma level, accelerate the brain distribution and decrease the urinary excretion of GB.

Liquid chromatography mass spectrometry for the determination of salvianolic acid B, a natural compound from the herb Danshen in rat plasma and application to pharmacokinetic study.

A sensitive and specific liquid chromatographic-electrospray ionization mass spectrometric method was developed for quantification of salvianolic acid B in rat plasma with resveratrol as the internal standard. The analytes were separated on a reversed-phase column with acetonitrile (40%) and water (60%) containing 0.75% formic acid as mobile phase at a flow rate of 1 mL/min. Liquid-liquid extraction was adopted for the sample preparation, and the analytes were determined using electrospray negative ionization mass spectrometry in the selective monitoring mode. The method was validated over the concentration range 0.1-40 microg/mL using 0.1 mL of plasma with coefficients of correlation >0.999. The intra- and inter-day precisions of analysis were <10%, and accuracy ranged from 94 to 101%. This method was successfully applied to a pharmacokinetics of salvianolic acid B in rats.

[Effect of metastasis suppressor gene KAI1 on adhesion of hepatocellular carcinoma cell line MHCC97-H].

BACKGROUND & OBJECTIVE: Metastasis suppressor gene KAI1 can inhibit the metastasis potential of many malignant tumors. This study was to explore the effect of KAI1 gene on the adhesion of hepatocellular carcinoma cell line MHCC97-H with high metastatic potential, and to find the possible mechanism. METHODS: The plasmid containing KAI1 gene was transfected into MHCC97-H cells. The growth status of the cells was observed. The changes of adhesion factors, soluble intercellular adhesive molecule-1 (sICAM-1) and E-cadherin, in the cells were detected by ELASA and Western blot. The character of cell adhesion was determined through plate colony formation test and cell adhesion test. RESULTS: The morphology of MHCC97-H cells did not change markedly after transfection of KAI1 gene, but black granules in cytoplasm were increased. Four days after transfection, the expression of sICAM-1 and E-cadherin were decreased by 24.28% and 26.02%. The adhesion rate was decreased by 11.34% at 3 h after transfection, and by 24.00% at 4 h after transfection, but the cloning efficiency did not change much. CONCLUSION: KAI1 gene could affect the growth pattern and proliferation of MHCC97-H cells, suppress sICAM-1 secretion and E-cadherin production, and inhibit adhesion of MHCC97-H cells.

Determination of 5-aminoimidazole-4-carboxamide in human plasma by ion-pair extraction and LC-MS/MS.

A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was established for the determination of 5-aminoimidazole-4-carboxamide (AICA) in human plasma. The method included a solvent extraction of AICA as an ion pair with 1-pentanesulfonate ion and a separation on a Hypersil ODS2 column with the mobile phase of methanol-water (68:32, v/v). Determination was performed using an electrospray ionization source in positive ion mode (ESI(+)). Multiple reaction monitoring (MRM) was utilized for the detection monitoring m/z at 127-->110 for AICA, and 172-->128 for IS. The calibration curve was linear within a range from 20 to 2000 ng/mL and the limit of quantity for AICA in plasma was 20 ng/mL. RSD of intra-assay and inter-assay were no more than 5.90% and 5.65%.

Prevention of central cell damage to isolated islets of Langerhans in hamsters by low temperature preconditioning.

BACKGROUND: The efficacy of clinical islet transplantation has been demonstrated with autografts, and although islet allografts have established insulin independence in a small number of IDDM patients, the treatment is confounded by the necessity of central cell damage immunosuppression, the lack of donor tissue, and recurring islet immunogenicity. These limitations underscore a need to develop therapies to serve the large population of diabetic patients. This study was designed to document central cell damage to isolated islets of Langerhans in hamsters and its prevention. METHODS: Islets were cultured at 37 degree centigrade for 7-14 days after isolation, and then at 26 degree centigrade for 2,4 and 7 days before additional culture at 37 degree centigrade for an additional 7 days. Central cell damage in the isolated islets was monitored by video-microscopy and analyzed quantitatively by a computer-assisted image analysis system. The analysis included daily measurement of the diameter and the area of the isolated islets and the area of the central cell damage that developed in those islets over time during culture. Histological examination and TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay were used to characterize cell damage and to monitor islet function. RESULTS: Microscopic analysis showed that during the 7 to 14 days of culture at 37 degree centigrade, central cell damage appeared in the larger islets with diameters greater than 200 microm, which included both necrotic and apoptotic cell death. Low temperature (26 degree centigrade) culture prevented central cell damage of isolated islets. The 7-day culture procedure at 26 degree centigrade could inhibit most of the central cell (excluding diameters greater than 300 microm) damage when the islets were rewarmed to 37 degree centigrade. CONCLUSIONS: Our results indicate that central cell damage to isolated islets of Langerhans correlates with the size of the islets. Low temperature (26 degree centigrade) culture can prevent central cell damage to the isolated islets, and is capable to successfully precondition these islets for 37 degree centigrade culture. These novel findings may help to understand the pathophysiology of early loss of islet tissue after transplantation, and may provide a new strategy to improve graft function in the clinical setting of islet transplantation.

Prevention of core cell damage in isolated islets of Langerhans by low temperature preconditioning.

AIM: To study the core cell damage in isolated islets of Langerhans and its prevention by low temperature preconditioning (26 degrees). METHODS: Islets were cultured at 37 degrees for 7-14 d after isolation, and then at 26 degrees for 2, 4 and 7 d before additional culture at 37 degrees for another 7 d. Core cell damage in the isolated islets was monitored by video-microscopy and analyzed quantitatively by use of a computer-assisted image analysis system. The analysis included daily measurement of the diameter and the area of the isolated islets and the area of the core cell damage that developed in those islets over time during culture. Histology and TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay were used to characterize the cell damage and to monitor islet function. RESULTS: Microscopic analysis showed that during the 7 to 14 d of culture at 37 degrees, core cell damage occurred in the larger islets with diameters >200 microm, which included both necrotic and apoptotic cell death. Low temperature (26 degrees) culture could prevent core cell damage of isolated islets. The 7-d culture procedure at 26 degrees could inhibit most of the core cell (excluding diameters >300 microm) damages when the islets were re-warmed at 37 degrees. CONCLUSION: Our results indicate that core cell damage within isolated islets of Langerhans correlates with the size of islets. Low temperature (26 degrees) culture can prevent core cell damage in isolated islets, and successfully precondition these islets for incubation at 37 degrees. These novel findings may help to understand the pathophysiology of early loss of islet tissue after transplantation, and may provide a new strategy to improve graft function in the clinical setting of islet transplantation.