Suitable pretreatment of biological samples can truly reflect the role of law of the measured components played in the body and will provide experimental evidence for the studies on metabolic process, material basis of efficacy, mechanism of action, pharmacology, toxicology and the others. Biological samples include blood, urine, hair, tears, etc. There are also many samples processing methods, such as the direct protein precipitation, liquid-liquid extraction and solid phase extraction and so on. These methods could be used alone or combined.
Animals ; Body Fluids ; chemistry ; Chemical Precipitation ; Chemistry Techniques, Analytical ; methods ; Humans ; Liquid-Liquid Extraction ; Proteins ; isolation & purification ; Solid Phase Extraction

To establish a UPLC-MS/MS method for the simultaneous determination of geniposide, genipin 1-O-beta-D-gentiobioside and geniposidic acid in rat brains and study the brain pharmacokinetics of the three iridoid glycosides in stroke rat after the oral administration of Xingnaojing. In this experiment, brain samples were precipitated with protein for twice. Acquity BEH C18 column was adopted, with acetonitrile-0.1% formic acid-water as the mobile phase for gradient elution. ESI source was adopted for mass spectra; multiple reaction monitoring (MRM) was conducted to detect negative ions. The time for sample analysis was 3.5 min. the results showed good linear relations among the three iridoid glycosides, with the extraction recovery between 99.6% and 114.3%, good intra- and inter-day precisions and accuracies and stability in line with the requirements. The t1/2 and MRT in the three components were similar in brains of stroke rats. Geniposide and genipin 1-O-beta-D-gentiobioside showed double peaks; where as geniposidic acid showed a single peak. In conclusion, the method is so specific, sensitive, accurate and reliable that it can be used to study the brain pharmacokinetics of Xingnaojing oral preparation.
Animals ; Brain ; metabolism ; Brain Chemistry ; Chromatography, High Pressure Liquid ; methods ; Drug Stability ; Drugs, Chinese Herbal ; chemistry ; pharmacokinetics ; Iridoids ; chemistry ; pharmacokinetics ; Male ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry ; methods

OBJECTIVETo investigate the association of C-erb B-2 expression with angiogenesis in nasopharygeal carcinoma.

METHODSSeventy-seven specimens of nasopharygeal carcinoma were examined immunohistochemically for protein expressions of C-erb B-2 and vascular endothelial growth factor (VEGF), and the microvessel density (MVD) was determined by immunostaining of the endothelial cells for factor VIII-related antigen (F8).

RESULTSPositive C-erb B-2 immunostaining was observed in 36.36% (28/77) of the nasopharygeal carcinoma tissues, which had a VEGF positivity rate of 32.48% (25/77). High positivity rate of C-erg B-2 was associated with high positivity rate of VEGF and high MVD (P<0.05).

CONCLUSIONThe expression of C-erb B-2 may contribute to angiogenesis in nasopharygeal carcinoma.

Adult ; Aged ; Carcinoma ; blood supply ; genetics ; metabolism ; pathology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; blood supply ; genetics ; metabolism ; pathology ; Neovascularization, Pathologic ; Receptor, ErbB-2 ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; Young Adult

The aim of the present research was to study the transdermal absorbability of ferulic acid and imperatorin in Zhitong cataplasm. Using methanol to extract the effective components, HPLC was established to determine drug content in Zhitong cataplasm. The excised abdominal skin of nude rats was used as the permeation model Utilizing the modified Franz diffusion cell, the suitable receiving solution was elected. The contents of ferulic acid and imperatorin in Zhitong cataplasm were 455.10, 371.66 microg x g(-1), respectively. Taken 20% ethanol- PBS (pH 8) as receiving solution, ferulic acid and imperatorin could be detected maximize, with the steady-state infiltration rates of 1.29, 0.15 microg x h(-1) x cm(-2), respectively. After 24 h, their penetration quantities were 30.03, 3.31 microg x cm(-2), with the penetration rates of 41.45%, 5.60%, respectively. The residual quantities of ferulic acid and imperatorin in skin were 0.69, 2.60 microg x cm(-2), with the retention rates of 0.95%, 4.40%, respectively. The in vitro transdermal behavior of ferulic acid in Zhitong cataplasm was close to a zero-order process, with the stratum corneum playing a rate-limiting role; whereas the percutaneous behavior of imperatorin was closer to Higuchi model, where diffusion played an important role in drugs permeation.
Analgesics ; chemistry ; metabolism ; therapeutic use ; Animals ; Coumaric Acids ; metabolism ; therapeutic use ; Drugs, Chinese Herbal ; chemistry ; metabolism ; therapeutic use ; Furocoumarins ; metabolism ; therapeutic use ; Kinetics ; Pain ; drug therapy ; Rats ; Skin Absorption

The object of this paper was to study the in vitro percutaneous factors that affect permeability of imperatorin. Excised abdominal skin of SD rats was used as the permeation model. Modified Franz diffusion cell was used to evaluate receiving solution, skin treatment and drug concentration on the effect of permeation. HPLC was established to determine drug content in recetpor at specified time points. The result showed that 20% ethanol and 0.5% Tween-80 could significantly increase the steady-state infiltration rate (Jass) of imperatorin. Skin treated of 20% ethanol, 20% ethanol-1% Tween-80 before experiments got an increased transdermal rate, whereas, those only accepted the treatment of 1% Tween-80 showed a decreased transdermal rate. The steady-state infiltration rate of imperatorin had a relationship with drug concentration, with the concentration increase, a bigger steady-state infiltration rate was obtained. When the drug concentration reached a certain concentration, the value was no longer changed. From the above research it could be found that ethanol could change the structure of the skin stratum corneum, which resulted in the increase of steady-state infiltration rate. The effect of Tween-80 on the steady-state permeation rate had nothing to do with skin structure. The concentration of drug in the diffusing cell had an effect on the penetration of imperatorin, which was the combined action of different diffusion route.
Animals ; Chemistry, Pharmaceutical ; Furocoumarins ; chemistry ; metabolism ; Hydrophobic and Hydrophilic Interactions ; Permeability ; Rats ; Rats, Sprague-Dawley ; Skin ; metabolism

OBJECTIVETo study the transdermal permeation and in vivo pharmacokinetics of effective constituent ferulic acid from Ligusticum chuanxiong, in order to establish the in vitro/in vivo correlation in transdermal permeation.

METHODFranz diffusion cell was adopted in the in vitro transdermal permeation, with CD-1 nude rat abdominal skin as the permeation medium. Linear probes were implanted in CD-1 nude rats. With PBS as perfusate, microdialysis was employed to study the pharmacokinetics. Ferulic acid concentrations in the receptor solution and dialysate were assessed by high performance liquid chromatography (HPLC). The study on correlation between in vitro and in vivo data was conducted by deconvolution methods.

RESULTThe transdermal permeation rate of ferulic acid from Ligusticum chuanxiong was (0.094 4 +/- 0.049 4) microg x cm2 x min, with Cmax of ferulic acid being 808.91 microg x L(-1), and Tmax being 183 min after dermal administration. The in vitro/in vivo correlation was 93.61.

CONCLUSIONFerulic acid in extracts from L. chuanxiong can quickly penetrate skins. By using the in vitro/in vivo correlation in transdermal permeation, simple in vitro transdermal permeation method can be adopted to study the changes in its pharmacokinetics.

Administration, Cutaneous ; Animals ; Chromatography, High Pressure Liquid ; Coumaric Acids ; administration & dosage ; pharmacokinetics ; Drugs, Chinese Herbal ; administration & dosage ; pharmacokinetics ; In Vitro Techniques ; Metabolic Clearance Rate ; Permeability ; Rats ; Rats, Nude ; Skin ; metabolism ; Skin Absorption

OBJECTIVETo study the pharmacokinetics of ferulic acid in transdermal delivery; the influence factors such as temperature, flow rate, and drug concentration on the probe recovery are also investigated to obtain accurate data.

METHODThe stability of in vitro and in vivo recovery was determined by loss but the influence factor on the recovery was determined by gain. Appling dermal microdialysis to ferulic acid analytes, the concentration of ferulic acid in the dialysate was detected by HPLC.

RESULTThere is a significant effect on the probe recovery for the in vitro recovery is much bigger at a higher temperature. When the flow rate rises, the in vitro recovery is down at index rate instead. Within the measuring rage, the drug concentration doesn't have effect on the recovery. The steady and reproduce of recovery is well within the measuring time, and the average recovery of in vitro is (24.82 +/- 1.01)% without temperature control and in vivo is (16.50 +/- 1.92)%. The C(max) of ferulic acid is 86 min and MRT is 291 min after dermal administration.

CONCLUSIONDermal microdialyis can be used in the ferulic acid study. For the ferulic acid can permeate the corneum quciky, it is practical to develop a transdermal delivery.

Administration, Cutaneous ; Animals ; Coumaric Acids ; administration & dosage ; pharmacokinetics ; Drug Stability ; Drugs, Chinese Herbal ; administration & dosage ; pharmacokinetics ; Mice ; Temperature

OBJECTIVETo explore the effects of intensive antiplatelet therapy for patients with high on-treatment platelet reactivity (HPR) after coronary stent implantation.

METHODSBetween March 2009 and February 2011, a total of 3316 consecutive acute coronary syndrome patients undergoing drug-eluting stent implanting from 3 hospitals were enrolled. Among them, 840 patients (25.3%) were identified as HPR (defined as 20 µmol/L adenosine diphosphate induced platelet aggregation of ≥ 55% at 24 hours after administration of 300 mg clopidogrel loading dose and 300 mg aspirin). The HPR patients were randomly assigned to receive standard (aspirin 300 mg/d and clopidogrel 75 mg/d, n = 280) or intensified (n = 560) antiplatelet therapy by the ratio of 1:2. Patients in the intensive group were initially treated with a double maintenance dose of clopidogrel (150 mg/d) and aspirin (300 mg/d). After 3 days, patients with unsolved HPR received additional cilostazol treatment (50 - 100 mg, bid). The reversion rate of HPR and clinical events were observed.

RESULTSIn the intensive group, HPR reversed in 304 out of 560 patients (54.3%) at 3 days post therapy and the remaining 256 patients with HPR were treated with additional cilostazol regimen for another 3 days and the total reversion rate of HPR was 81.1% (454/560). The reversion rate of HPR at 30 days in the intensified group was significantly higher than that of the standard group (69.9% vs. 55.7%, P = 0.000). At 30 days after percutaneous coronary intervention, 1 patient suffered from subacute stent thrombosis (0.2%) in intensified group and no stent thrombosis was observed in standard group (P = 1.000). There were no death, major or minor bleeding in both two groups. Minimal bleeding was also similar in the two groups (intensive: 4.28% vs. standard: 2.14%, P = 0.166).

CONCLUSIONSThe intensified antiplatelet therapy regimens could significantly increase the reversion rate of HPR in acute coronary syndrome patients undergoing coronary stenting without increasing the risk of bleeding. The clinic impact of this strategy needs to be elucidated by long term follow-up outcome studies.

Acute Coronary Syndrome ; therapy ; Adult ; Aged ; Aspirin ; administration & dosage ; therapeutic use ; Blood Platelets ; drug effects ; Drug Therapy, Combination ; Drug-Eluting Stents ; Female ; Humans ; Male ; Middle Aged ; Percutaneous Coronary Intervention ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; administration & dosage ; therapeutic use ; Tetrazoles ; administration & dosage ; therapeutic use ; Ticlopidine ; administration & dosage ; analogs & derivatives ; therapeutic use

OBJECTIVETo evaluate the transfect results of recombinant adenovirus vector carrying tyrosinase gene (Ad-tyr) in vitro by magnetic resonance imaging (MRI) after the Ad-tyr was transfected into HepG2 cell.

METHODSThe Ad-tyr which carried the full-length cDNA of tyrosinase gene was transfected into HepG2 cell. The transfected cells were scan by MRI sequences of T1 weighted image (T1WI) , T2 weighted image (T2WI) , and short time inversion recovery (STIR) to observe the MRI signals of expressed melanin. Masson-Fontana staining was performed to search for melanin granules in transfected cells. Real-time PCR method was used to search for cDNA of tyrosinase gene.

RESULTSAd-tyr was transfected into HepG2 cells and synthesized a large amount of melanin inside. The synthesized melanin of 1 x 10(6) cells which had been transfected by Ad-tyr with the 50, 150, and 300 multiplicity of infection separately were all sufficient to be detected by MRI and showed high signals in MRI T1WI, T2WI, and STIR sequences. The signal intensities of MRI were positively correlated to the amounts of transfected Ad-tyr. The melanin granules were found in HepG2 cells in Masson-Fontana staining. The cDNA amount of tyrosinase gene in transfected HepG2 cells, which was detected by real-time PCR, was remarkably higher than that in nontransfected cells.

CONCLUSIONThe synthesized melanin of HepG2 cells, which controlled by expression of exogenous gene, can be detected by MRI, indicating that the adenovirus vector can efficiently carry the tyrosinase gene into HepG2 cells.

Adenoviridae ; genetics ; metabolism ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Hep G2 Cells ; Humans ; Magnetic Resonance Imaging ; methods ; Melanins ; analysis ; genetics ; Monophenol Monooxygenase ; biosynthesis ; genetics ; Transfection

Objective Throush identify biochemical characteristics and virulence factors of 2 strains suspected Yersinia pestis(Y.pestis)isolated from the dead Marmota himalayana(M.himalayana)to confirm the nature epidemic focus in Dege County,Sichuan Province.Methods Y.pestis was analyzed by specific staining and shape,culturing characteristics,splitting-test by bacteriophage,test of biochemical characteristics and glycolysis ability,virulence factors,virulence,nutritional requirement,plasmid,genetic test and genetic type. Results The tested strains were Gram staining bacilus.The main biochemical characteristics were Arabinose(+)、 Rhamnose(-),Maltose(+),Melibiose(-),Glycerol(+),Denitrification(+).The virulence factors with FI+.VW+, Pgm+,Pst I+;and with the common 6.0×106,45.0×106,65.0×106 plasmids,also with the virulence-relative plasmid gene.Both their absolutely lethal dose(LD100)in mice were 50 bacteria.The nutritional requirement appeared which were depended on Phenylalanine and Methionine.With the Genomovar 5 genotype characteristics of M.himalayana plague foci of Qinghai-Tibet plateau.The difference between tested strains and Yersinia pseudotubercuosis on the 3 different culture medium was obvious.The tested strains had a Y.pestis' specific 3a fragment,Pst I and FI-Ag,at 22 ℃,the strains could be split by bacteriophage completely.Conclusions According to the diagnostic criteria of plague in China,the 2 suspected strains isolated from Dege County,Sichuan Province ale confirmed as Y.pestis.both with powerful virulenceand with the characteristics of the Y.pestis of M.himahtyana in Qinghai-Tibet plateau plague natural focus.