OBJECTIVE: To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation. METHODS: Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 ^-/-, arhgef10 ^+/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively. RESULTS: WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 ^-/- zebrafish had more severe symptoms than arhgef10 ^+/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis. CONCLUSION Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.
Animals ; Annexin A5 ; Apoptosis ; Blotting, Western ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line ; Cell Proliferation ; Cells, Cultured ; Flow Cytometry ; Genotype ; Humans ; In Situ Hybridization ; Larva/physiology* ; Phenotype ; RNA/isolation & purification* ; Real-Time Polymerase Chain Reaction/standards* ; Rho Guanine Nucleotide Exchange Factors/metabolism* ; Sincalide/analysis* ; Spectrophotometry/methods* ; Zebrafish/physiology*

Analysis errors can occur in the desorbing process of ginkgo diterpene lactone meglumine injection (GDMI) by a conventional analysis method, due to several factors, such as easily crystallized samples, solvent volatility, time-consuming sample pre-processing, fixed method, and offline analysis. Based on risk management, near-infrared (NIR) and mid-infrared (MIR) spectroscopy techniques were introduced to solve the above problems with the advantage of timely analysis and non-destructive nature towards samples. The objective of the present study was to identify the feasibility of using NIR or MIR spectroscopy techniques to increase the analysis accuracy of samples from the desorbing process of GDMI. Quantitative models of NIR and MIR were established based on partial least square method and the performances were calculated. Compared to NIR model, MIR model showed greater accuracy and applicability for the analysis of the GDMI desorbing solutions. The relative errors of the concentrations of Ginkgolide A (GA) and Ginkgolide B (GB) were 2.40% and 2.89%, respectively, which were less than 5.00%. The research demonstrated the potential of the MIR spectroscopy technique for the rapid and non-destructive quantitative analysis of the concentrations of GA and GB.
Chemistry, Pharmaceutical ; methods ; standards ; Drug Compounding ; standards ; Drugs, Chinese Herbal ; analysis ; chemistry ; standards ; Ginkgolides ; analysis ; chemistry ; standards ; Injections ; Lactones ; analysis ; Least-Squares Analysis ; Meglumine ; analysis ; chemistry ; standards ; Reproducibility of Results ; Risk Management ; Spectrophotometry, Infrared ; standards

Crystal structures of chemical drugs has been being investigated widely. But few attention has been paid to polymorphs-phenomena of active ingredients from Traditional Chinese Medicine(TCM). Taking anhydrous dehydroandrographolide and hydrousprim-O-glucosylcimifugin as example, differences between TCM reference substances (RSs) with different crystal structures were discussed by using microscopy, melting point determination, differential thermal analysis (DTA) and infrared (IR) methods. The results showed that different crystal structures could lead to change of melting points, thermal behaviors and IR spectrum. It's indicated that polymorphs may be considered if different physicochemical properties were obtained when applying TCM RS. Differences of chemical properties of active ingredients from TCM with different crystal structures need further investigation.
Crystallization ; Differential Thermal Analysis ; methods ; standards ; Drugs, Chinese Herbal ; chemistry ; standards ; Medicine, Chinese Traditional ; Molecular Structure ; Reference Standards ; Spectrophotometry, Infrared ; methods ; standards ; Transition Temperature

The marine biological source of mineral drugs recorded in Chinese Pharmacopoeia (2010 version) mainly including pearl, nacre, clam shell, common oyster shell, ark shell, cuttle bone, and sea-ear shell are widely used in clinical. Calcium carbonate and a small amount of protein are the main components in this type of drugs. In this paper, a systematical and comparable study were carried out by determination of calcium carbonate by EDTA titration method, the crystal of calcium carbonate by X-Ray powder diffraction and the total amino acids (TAAs) of the hydrolyzed samples by ultraviolet spectrophotometry method. As a result, the crystal structure is calcite for common oyster shell, mixture of calcite and aragonite for nacre and sea-ear shell, aragonite for the other drugs. The content of calcium carbonate ranged from 86% to 96%. Cuttle bone has the highest amount of TAAs among the seven drugs which reached 1.7% while clam shell has the lowest content of 0.16% on average. In conclusion, an effective method was developed for the quality control of marine mineral drugs by comprehensive analysis of calcium carbonate and TAAs in the seven marine mineral drugs.
Amino Acids ; analysis ; chemistry ; Animal Shells ; chemistry ; Animals ; Calcium Carbonate ; analysis ; chemistry ; Crystallization ; Edetic Acid ; chemistry ; Mollusca ; chemistry ; classification ; Pharmaceutical Preparations ; analysis ; chemistry ; standards ; Quality Control ; Reproducibility of Results ; Seawater ; Species Specificity ; Spectrophotometry, Ultraviolet ; X-Ray Diffraction

Laboratories working towards accreditation by the International Standards Organization (ISO) 15189 standard are required to demonstrate the validity of their analytical methods. The different guidelines set by various accreditation organizations make it difficult to provide objective evidence that an in-house method is fit for the intended purpose. Besides, the required performance characteristics tests and acceptance criteria are not always detailed. The laboratory must choose the most suitable validation protocol and set the acceptance criteria. Therefore, we propose a validation protocol to evaluate the performance of an in-house method. As an example, we validated the process for the detection and quantification of lead in whole blood by electrothermal absorption spectrometry. The fundamental parameters tested were, selectivity, calibration model, precision, accuracy (and uncertainty of measurement), contamination, stability of the sample, reference interval, and analytical interference. We have developed a protocol that has been applied successfully to quantify lead in whole blood by electrothermal atomic absorption spectrometry (ETAAS). In particular, our method is selective, linear, accurate, and precise, making it suitable for use in routine diagnostics.
Accreditation ; Humans ; Laboratories/standards ; Lead/*blood/standards ; Reference Standards ; *Spectrophotometry, Atomic/standards ; *Validation Studies as Topic

A simple, fast and sensitive analytical method for the simultaneous separation and detection of 18alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B by RP-HPLC and drug quality standard was established. The structures of principal component isomer and related substances of raw material drug of ammonium glycyrrhizinate have been confirmed. Reference European Pharmacopoeia EP7.0 version, British Pharmacopoeia 2012 version, National Drug Standards of China (WS 1-XG-2002), domestic and international interrelated literature were referred to select the composition of mobile phase. The experimental parameters including salt concentration, pH, addition quantities of organic solvent, column temperature and flow rate were optimized. Finally, the assay was conducted on a Durashell-C18 column (250 mm x 4.6 mm, 5 microm) with 0.01 mol x mL(-1) ammonium perchlorate (add ammonia to adjust the pH value to 8.2) -methanol (48 : 52) as mobile phase at the flow rate of 0.8 mL x min(-1), and the detection wavelength was set at 254 nm. The column temperature was 50 degrees C and the injection volume was 10 microL. The MS, NMR, UV and RP-HPLC were used to confirm the structures of principal component isomer and related substances of raw material drug of ammonium glycyrrhizinate. Under the optimized separation conditions, the calibration curves of 18 alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B showed good linearity within the concentration of 0.50-100 microg x mL(-1) (r = 0.999 9). The detection limits for 18alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B were 0.15, 0.10, 0.10, 0.15 microg x mL(-1) respectively. The method is sensitive, reproducible and the results are accurate and reliable. It can be used for chiral resolution of 18alpha-glycyrrhizinic acid, 18Pbeta-glycyrrhizinic acid, and detection content of principal component and related substances of raw material drug of ammonium glycyrrhizinate. It is concluded that the separation of principal component isomer of raw material drug of ammonium glycyrrhizinate and the validity of the substance's structure assignments of retention time being 1.2 in the European pharmacopoeia EP7.0 version, British pharmacopoeia 2012 version remains open to question. It may be of practical value for the quality control of raw material drug, preparation, and Chinese herbal medicine of ammonium glycyrrhizinate.
Ammonium Compounds ; chemistry ; isolation & purification ; Chromatography, High Pressure Liquid ; Glycyrrhizic Acid ; chemistry ; isolation & purification ; Isomerism ; Magnetic Resonance Imaging ; Mass Spectrometry ; Molecular Structure ; Principal Component Analysis ; Quality Control ; Reference Standards ; Spectrophotometry, Ultraviolet

In this work, a rapid analysis method basing on ultraviolet spectroscopy was established for the determination of danshensu, protocatechuic aldehyde, rosmarinci acid, lithospermic acid and salvianolic acid B in the extraction process of Danhong injection. In the extraction process of Danshen and Honghua crude drugs, 44 extraction solution samples were collected and the contents of the five components were determined by HPLC analysis. The ultraviolet spectra of the samples were collected. Partial least square regression was used to establish the multivariate calibration models between the ultraviolet spectra and the contents of the five components. The results showed that the established models could predict the contents of the five components in the extraction solution accurately. The ultraviolet spectroscopy method established in this work can be used for rapid analysis of the intermediates of Danhong injection, which may be applied for the quality control in the manufacturing process.
Chemistry, Pharmaceutical ; standards ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; analysis ; isolation & purification ; Quality Control ; Salvia miltiorrhiza ; chemistry ; Spectrophotometry, Ultraviolet ; methods

BACKGROUND: The urinary iodine micromethod (UIMM) is a modification of the conventional method and its performance needs evaluation. METHODS: UIMM performance was evaluated using the method validation and 2008 Iodine Deficiency Disorders survey data obtained from four urinary iodine (UI) laboratories. Method acceptability tests and Sigma quality metrics were determined using total allowable errors (TEas) set by two external quality assurance (EQA) providers. RESULTS: UIMM obeyed various method acceptability test criteria with some discrepancies at low concentrations. Method validation data calculated against the UI Quality Program (TUIQP) TEas showed that the Sigma metrics were at 2.75, 1.80, and 3.80 for 51+/-15.50 microg/L, 108+/-32.40 microg/L, and 149+/-38.60 microg/L UI, respectively. External quality control (EQC) data showed that the performance of the laboratories was within Sigma metrics of 0.85-1.12, 1.57-4.36, and 1.46-4.98 at 46.91+/-7.05 microg/L, 135.14+/-13.53 microg/L, and 238.58+/-17.90 microg/L, respectively. No laboratory showed a calculated total error (TEcalc) Humans ; Iodine/*urine ; Laboratories/standards ; Quality Control ; Spectrophotometry/*standards ; Urinalysis/*standards

OBJECTIVETo develop a method for determining the content of mercury contained in Yuhong ointment.

METHODThe wet catalytic digestion method was adopted for the pretreatment, and the mercury content in Yuhong ointment was determined by hydride generation-atomic absorption spectrometry (HG-AAS).

RESULTThe mercury showed a good linear relation in the range from 2 to 20 microg x L(-1), with the average recovery of 104.27% and RSD of 3.37%. The RSD for real sample repeated measurement was determined to be 8.4%. The mercury content in Yuhong ointment was detected in range from 0.7 to 1.5 mg x g(-1).

CONCLUSIONThe proposed method is accurate, highly reproducible and it can be used to control mercury content of Yuhong ointment.

Catalysis ; Drugs, Chinese Herbal ; chemistry ; Mercury ; analysis ; isolation & purification ; Ointments ; Reference Standards ; Reproducibility of Results ; Spectrophotometry, Atomic ; methods

OBJECTIVETo study cultivation of Panax ginseng adventitious roots in bubble bioreactors.

METHODThe adventitious roots were obtained through tissue culture different types of bioreactors. The contents of ginsenosides Re, Rb1 and Rg1 were determined by HPLC while the contents of polysaccharides were determined by ultraviolet spectrophotometry.

RESULTThe results showed that of the three types tested, the most efficient bioreactor for cultivation of the ginseng adventitious roots was the cone-type bioreactors (with the 120 degrees ), in which, the growth curve of adventitious roots was S-shaped. The maximum biomass was obtained on the 40th day, with the fresh weight, dry weight and growth rate reaching the maximum, which were 113.15 g, 9.62 g and 63.13 times respectively, and the concomitant contents of polysaccharide and ginsenoside were 2.73% and 2.25 mg x g(-1).

CONCLUSIONThe results showed that the most efficient bioreactor for cultivation of the ginseng adventitious roots was the cone-type bioreactors (with the 120 degrees). These results provide a theoretical reference for developing an efficient production process of active metabolites of ginseng in the scale-up cultivation.

Biomass ; Bioreactors ; classification ; standards ; Chromatography, High Pressure Liquid ; Ginsenosides ; metabolism ; Panax ; genetics ; metabolism ; Plant Roots ; genetics ; metabolism ; Polysaccharides ; metabolism ; Reproducibility of Results ; Spectrophotometry, Ultraviolet ; Time Factors ; Tissue Culture Techniques ; methods