OBJECTIVETo establish a reversed-phase high performance liquid chromatorgraphy (RP-HPLC) method for detecting the dencichine in Panax notoginseng extracts and drug preparations.

METHODDencichine was extracted with the borate buffer (pH 9. 18) and the clear supernatant was used for the derivatization. Pre-column derivatization was performed using 9-fluorenylmethyl chloroformate (FMOC) to form derivatives. The mobile phase consisted of methanol and 0. 05 mol x L( -1) NaH2 PO4 (48: 52) (pH adjusted to 7.4 with NaOH solution) in a flow rate of 1.0 mL m min(-1). The ultraviolet (UV) detection wavelength was set at 262 nm.

RESULTThe linearity was demonstrated over a wide range of concentration from 1.76 mg L(-1) to 352 mg x L(-1) for dencichine. The detection limit was determined to be 60 microg x L(-1). The derivative was stable and the derivatization agent did not influence the measurement of dencichine. The average recovery rate was 95. 3% and the relative standard derivation (RSD) was 1. 7%. The method was used to determine dencichine in different P. notoginseng extracts and drug preparations.

CONCLUSIONThis method is simple, fast and sensitive, suitable for determining the dencichine in P. notoginseng extracts and drug preparations as well as for the study of the dencichine metabolism in vivo.

Amino Acids, Diamino ; analysis ; Chromatography, High Pressure Liquid ; instrumentation ; methods ; Fluorenes ; chemistry ; Panax notoginseng ; chemistry ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Reproducibility of Results

We study the techniques of synthesis of disulfide bond-bearing cyclopeptides FIK. This experimentation with the material of Fmoc-aa use Solid-Phase synthesis after condensation by HBTU/HOBt/DIEA to synthesize linear peptide, then cyclopeptide was synthesized by creation of intramolecular disulfide bond by means of 12 oxidation of bis cysteine sulfhydryl of the linear peptide. The crude production was cleaved from the resin together with all protecting group and identified and separated by MALDI-MS and RP-HPLC. The peptide yield was 18%, after purification the purity was more than 97%. It was identified on MALDI-MS and Ellman reagent detection. This method is effective, simple, rapid and obtained good yield, and it's fit for the large-scale production.
Amino Acids ; chemistry ; Combinatorial Chemistry Techniques ; methods ; Disulfides ; chemistry ; Fluorenes ; chemistry ; Molecular Structure ; Peptides, Cyclic ; chemical synthesis ; chemistry ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

No abstract available.
Antiviral Agents/*therapeutic use ; Benzimidazoles/*therapeutic use ; Female ; Fluorenes/*therapeutic use ; Hepacivirus/*classification ; Hepatitis C, Chronic/*drug therapy ; Humans ; Liver Cirrhosis/*drug therapy ; Male ; Ribavirin/*therapeutic use ; Uridine Monophosphate/*analogs & derivatives

The aromatic-ring-hydroxylating dioxygenase is a key enzyme that initiates the biodegradation of polycyclic aromatic hydrocarbons in bacteria. In the present study, a novel dioxygenase sequence was cloned from Terrabacter sp. FLO using a genome walking method. The dioxygenase was cloned into pET17 and actively expressed in E.coli BL21 (DE3) in co-expression with electron transfer chain proteins. The recombinant dioxygenase was found to transform phenanthrene, fluorene, pyrene and fluoranthene into the cis-dihydrodiol metabolites.
Actinomycetales ; enzymology ; genetics ; Bacterial Proteins ; genetics ; metabolism ; Biodegradation, Environmental ; Cloning, Molecular ; Dioxygenases ; genetics ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Fluorenes ; metabolism ; Hydroxylation ; Molecular Sequence Data ; Phenanthrenes ; metabolism ; Polycyclic Aromatic Hydrocarbons ; metabolism ; Pyrenes ; metabolism ; Recombinant Proteins ; metabolism

Chloroquine remains the drug of choice for the treatment of vivax malaria in Thailand. Mixed infections of falciparum and vivax malaria are also common in South-East Asia. Laboratory confirmation of malaria species is not generally available. This study aimed to find alternative regimens for treating both malaria species by using falciparum antimalarial drugs. From June 2004 to May 2005, 98 patients with Plasmodium vivax were randomly treated with either artemether-lumefantrine (n = 47) or chloroquine (n = 51). Both treatments were followed by 15 mg of primaquine over 14 days. Adverse events and clinical and parasitological outcomes were recorded and revealed similar in both groups. The cure rate was 97.4% for the artemether-lumefantrine treated group and 100% for the chloroquine treated group. We concluded that the combination of artemether-lumefantrine and primaquine was well tolerated, as effective as chloroquine and primaquine, and can be an alternative regimen for treatment of vivax malaria especially in the event that a mixed infection of falciparum and vivax malaria could not be ruled out.
Adolescent ; Aged ; Animals ; Antimalarials/adverse effects/*therapeutic use ; Artemisinins/adverse effects/*therapeutic use ; Chloroquine/adverse effects/*therapeutic use ; Drug Therapy, Combination ; Ethanolamines/adverse effects/*therapeutic use ; Female ; Fluorenes/adverse effects/*therapeutic use ; Humans ; Malaria, Vivax/*drug therapy ; Male ; Middle Aged ; Parasitemia ; Plasmodium vivax/drug effects ; Primaquine/therapeutic use ; Thailand ; Treatment Outcome

This study is designed to evaluate antioxidant and antigenotoxic activities of corn tassel extracts (CTTs). The major bioactive components of CTTs include flavonoid, saponin and polysaccharide. The antioxidant properties of the three bioactive components of CTTs were investigated by Ferric Reducing Antioxidant Property (FRAP) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) assays. The activities of the extracts were determined by assessing the inhibition of mutagenicity of the direct-acting mutagen fenaminosulf, sodium azide, and indirect-acting mutagen 2-aminofluorene using the Ames test (strains TA98 and TA100). The results showed that the extraction rates of flavonoid, saponin, and polysaccharide from the dried corn tassels were 1.67%, 2.41% and 4.76% respectively. DPPH and FRAP assay strongly demonstrated that CTTs had antioxidant properties. CTTs at doses of 625, 1250 and 2500 μg per plate reduced 2-aminofluorene mutagenicity by 12.52%, 28.76% and 36.49% in Salmonella typhimurium TA98 strain assay respectively and by 10.98%, 25.27% and 37.83%, at the same doses in Salmonella typhimurium TA100 assay system, respectively. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay showed that the different concentrations of CTTs inhibited the proliferation of MGC80-3 cells in a dose-dependent manner (P<0.01). It is concluded that these integrated approaches to antioxidant and antigenotoxicity assessment may be useful to study corn tassel as a natural herbal material.
Antimutagenic Agents ; pharmacology ; Antioxidants ; pharmacology ; Biphenyl Compounds ; antagonists & inhibitors ; metabolism ; Cell Line, Tumor ; Cell Survival ; drug effects ; Flavonoids ; pharmacology ; Fluorenes ; pharmacology ; Free Radical Scavengers ; pharmacology ; Humans ; Inflorescence ; chemistry ; Mutagens ; pharmacology ; Picrates ; antagonists & inhibitors ; metabolism ; Plant Extracts ; pharmacology ; Polysaccharides ; pharmacology ; Salmonella typhimurium ; drug effects ; genetics ; Saponins ; pharmacology ; Zea mays ; chemistry

BACKGROUNDIt has been reported that several baseline polymorphisms of direct-acting antivirals (DAAs) agents resistance-associated variants (RAVs) would affect the treatment outcomes of patients chronically infected with hepatitis C virus (CHC). The aim of this study is to investigate the prevalence of DAAs RAVs in treatment-naÏve GT1b CHC patients.

METHODSDirect sequencing and ultra-deep sequencing of the HCV NS3, NS5A, and NS5B gene were performed in baseline serum samples of treatment-naÏve patients infected with genotype 1b hepatitis C virus (HCVs).

RESULTSOne hundred and sixty CHC patients were studied. Complete sequence information was obtained for 145 patients (NS3), 148 patients (NS5A), and 137 patients (NS5B). Treatment-failure associated variants of DAAs were detected: 56.6% (82/145) of the patients presented S122G for simeprevir (NS3 protease inhibitor); 10.1% (14/148) of the patients presented Y93H for daclatasvir and ledipasvir (NS5A protein inhibitors); 94.2% (129/137) of the patients presented C316N for sofosbuvir (NS5B polymerase inhibitor). Nearly, all of the DAAs RAVs detected by ultra-deep sequencing could be detected by direct sequencing.

CONCLUSIONSThe majority of genotype 1b CHC patients in China present a virus population carrying HCV DAAs RAVs. Pretreatment sequencing of HCV genome might need to be performed when patients infected with GT1b HCV receiving DAAs-containing regimens in China. Population sequencing would be quite quantified for the work.

Adult ; Aged ; Antiviral Agents ; therapeutic use ; Benzimidazoles ; therapeutic use ; China ; Drug Resistance, Viral ; genetics ; Female ; Fluorenes ; therapeutic use ; Genotype ; Hepacivirus ; drug effects ; pathogenicity ; Hepatitis C ; drug therapy ; High-Throughput Nucleotide Sequencing ; Humans ; Imidazoles ; therapeutic use ; Male ; Middle Aged ; Polymorphism, Genetic ; genetics ; Simeprevir ; therapeutic use

OBJECTIVETo investigate the changes of oval cell proliferation rate in the rat 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) model.

METHODSLivers were collected from 2-AAF/PH rats at different time points after hepatectomy. Paraffin sections were investigated by double immunofluorescent staining with confocal microscopy for oval cell marker epithelial cell adhesion molecule and proliferative index proliferating cell nuclear antigen, or epithelial cell adhesion molecule and alpha-smooth muscle actin. Deposition of matrix in liver tissue was detected by sirius red staining.

RESULTSResponse of ductular oval cells could be observed in portal area at 2 days after PH, and the number of oval cells reached its peak at 9 days and then gradually declined. Oval cell proliferation rate decreased from (91.3 +/- 1.6)% at 2 days after PH to (53.6 +/- 4.4)% at 12 days (P < 0.01). In addition, oval cells infiltrating into liver parenchyma were closely associated with activated hepatic stellate cells and extracellular matrix.

CONCLUSIONSOval cell proliferation rate starts decreasing before its number reaches a peak in 2-AAF/PH model. Hepatic stellate cells probably tightly regulate oval cell number through secreting several factors and producing extracellular matrix.

2-Acetylaminofluorene ; pharmacology ; Animals ; Cell Division ; Cell Proliferation ; Hepatectomy ; Liver ; cytology ; Male ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology

Objective: To investigate the dynamic expression pattern of carcinoembryonic Wnt3a and its early monitoring value using a hepatocellular carcinoma model. Methods: Forty-eight Sprague Dawley (SD) rats were fed with pellet feed containing 2-acetylaminofluorene (2-AAF, 0.05%) to induce hepatocarcinogenesis, and control rats were fed a pellet diet. Liver tissue and blood samples were collected every two weeks. Liver tissues were pathologically examined using HE staining and grouped. The gene and Wnt3a mRNA expression were analyzed by genome-wide microarray. The expression and distribution of Wnt3a in liver tissue were analyzed by immunohistochemistry. Wnt3a concentration in liver tissue and serum was quantified by enzyme-linked immunosorbent assay. Statistical methods such as χ² test, Mann-Whitney test and analysis of variance were used to analyze the differences between groups. Results: According to the pathological examination results, the rat livers were divided into four groups: control, hepatocyte degeneration, precancerous lesions and hepatocellular carcinoma. Genome-wide expression profiling analysis and comparison with the control group revealed that 268 and 312 genes were up-regulated and 57 and 201 genes were down-regulated in the precancerous and cancerous group when signal logarithm ratio (SLR) was >8 log₂^cy5/cy3, and these significantly altered genes mainly involved in cell proliferation, signal transduction, tumor metastasis, and apoptosis. The expression of Wnt3a at mRNA level was significantly increased in all stages of cancer induction, including degeneration group (1.15±0.24, q=8.227), precancerous group (1.85±0.18, q=12.361) and cancerous group (2.59±0.55, q=18.082). Compared with the control group (0.25±0.11, F=121.103, P<0.001), the degeneration group, the precancerous group and the liver cancer group were up-regulated by 4.6, 7.4 and 10.4-folds, respectively. Immunohistochemistry showed that compared with the control group, the positive rate of Wnt3a in the degeneration group was 66.7% (12/18, χ²=10.701, P=0.001), and both the precancerous and liver cancer groups were positive (9/9, χ²=17.115, P<0.001). Wnt3a expression was gradually increased in liver and blood samples during the process of carcinogenesis, and the difference between two groups was statistically significant (F=176.711, P<0.001). Wnt3a overexpression was secreted into blood stream via cancerous liver tissue, and there was a linear correlation between Wnt3a levels in blood and liver samples (r=0.732, P<0.001). Conclusions: Wnt3a overexpression is closely related with hepatocellular carcinogenesis, and thus may become a new monitoring marker.
Rats ; Animals ; Carcinoma, Hepatocellular/pathology* ; Liver Neoplasms/pathology* ; Rats, Sprague-Dawley ; Carcinogenesis/metabolism* ; 2-Acetylaminofluorene ; RNA, Messenger/metabolism* ; Precancerous Conditions

Three different chemical carcinogens, 2-acetylaminofluorene (AAF), diethylnitrosamine(DENA), and 3'-methyl-4dimethylaminoazobenzene (3'-Me DAB) were used to induce hepatomas in rats. Plasma membrane surface proteins of normal rat liver cells and rat hepatomas were extracted with 3M KCI. From the analysis of the proteins of normal rat liver and rat hepatoma induced by 3'-Me DAB by discontinuous polyacrylamide gel electrophoresis(Disc-PAGE), under nonreducing and nondenaturing conditions polyacrylamide gel electrophoresis in the presence of SDS and 2-mercaptoethanol (SDS-PAGE), Sephadex G-200 gel permeation chromatography, DEAE-A50 ion-exchange chromatography and two-dimensional gel electrophoresis, at least three tumor specific antigens were identified. One had a molecular weigh of 66,000 (pl=6.79) while the other two had the same molecular weight 73,000 but differed in their isoelectric points (7.58 and 7.81). For immunological analysis of tumor specific antigens, the absorbed antiserum was prepared. Plasma membrane surface proteins of rat hepatoma induced by 3'-Me DAB were used to obtain New Zealand White male rabbit antiserum. Rabbit antiserum was then reacted with the proteins isolated from the plasma membrane surface of normal rat liver and the absorbed antiserum reacting specifically with the tumor specific antigens derived by 3'-Me DAB was obtained. Using the absorbed antiserum, the immunoreactivities of plasma membrane surface proteins isolated from rat hepatomas induced by 3'-Me DAB, AAF, and DENA were compared by Ouchterlony double immunodiffusion analysis and immunoelectrophoresis. To characterize the proteins reacting to the absorbed antiserum, immunoglobulin G was separated from the absorbed antiserum and coupled to cyanogen bromide-activated Sepharose CL-4B. The isolated proteins from the plasma membrane surface proteins of 3'-Me DAB-induced hepatoma using this immunoaffinity chromatography had molecular weights of 66,000 and 73,000. The localization of these proteins on surface plasma membranes of rat hepatomas induced by 3'-Me DAB was confirmed by an immunofluorescence technique. The experimental results revealed the existence of cross-reacting common antigens on the plasma membrane surface of rat hepatomas induced by different hepatocarcinogens.
2-Acetylaminofluorene ; Animal ; Antigens, Neoplasm/*isolation and purification ; Antigens, Surface/isolation and purification ; Diethylnitrosamine ; Liver Neoplasms, Experimental/chemically induced/*immunology ; Methyldimethylaminoazobenzene ; Rats ; Rats, Inbred Strains ; Support, Non-U.S. Gov't