Objective:To establish and optimize a detection method of dibutyryl cyclic adenosine phosphate related substances.Methods:On the basis of the JP,the experimental conditions were optimized to obtain the optimal detection conditions.The column was CAPCEILPAK C18;mobile phase was pH 6.0 phosphate buffer solution-meth-anol with gradient elution;detection wavelength was 258 mm.The method validation was carried out.Results:In the system applicability solution,the separation degree of the main peak and adjacent peaks in each destruction solution was greater than 1.5;the solution was stable within 48 h.The content of each impurity showed a good line-ar relationship with the peak area in the range of 50％-150％of the limit concentration,R2＞0.999 4.In the spike experiment,the average recovery rate of N6-butyryl 3',5-cyclophosphatin sodium was 99.4％-106.6％.The average recoveries of 2'-0 butyryl 3,5-cycloadenosine monophosphate sodium were 88.8％-103.6％,and the average recoveries of cyclophosphaminate sodium were 96.9％-102.5％,which met the requirements.Three bat-ches of samples were tested,and the above impurities were detected.Conclusion:This method has high sensitivi-ty,good separation effect,accuracy and durability,and can be used for the detection of bucladesine sodium,provi-ding a basis for its quality control.

Objective To investigate the expression of the Toll-like receptor-4 (TLR-4) in temporomandibular joint synovitis in rats,and to discuss the correlation between the expression of TLR-4 and the synovitis.Methods Sixty male wistar rats were randomly divided into five groups,12 each.Group A was the control group in which the rats were given normal diet.In Group B,the rats' bilateral masseter muscles were cut off (masseter resection group).In Group C,An cast metal crown were bonded on the mandibular right first molar of each rat (occlusal interference group).In Group D,occlusal pad were bonded on maxillary molars of each rat (occlusal dimension increase group).In Group E,rats' bilateral masseter muscles were resected and occlusal pads were bonded on their maxillary molars (masseter resection and occlusal dimension increase group).Pathological changes of synovium were observed using hematoxylin and eosin (HE) stains and pathology scores were evaluated.The expression of TLR-4 were determined by immunohistochemical stains,and the expression of TLR-4 mRNA were determined by real-time PCR.The correlation between the expression of TLR-4,TLR-4 mRNA and the pathological score were analyzed using Spearman analysis.Results The pathological scores of Group A-E were 0.5±0.5,2.5±1.0,2.7±1.0,3.0±0.9,5.3±1.2 respectively.The expression of TLR-4 were (3.2±1.5) ％,(16.0±2.6) ％,(15.8±2.1) ％,(17.5±2.4) ％,(38.2±4.4) ％.The expression of TLR-4 mRNA were 1.07±0.09,2.12±0.33,2.07±0.29,2.17±0.34,4.53±0.46.Compared with group A,groups B-E showed significant higher pathology score (P＜0.05) and increased expression of both TLR-4 (P＜0.05) and TLR-4 mRNA (P＜0.05).An significant positive correlation was found between the expression of TLR-4 and the pathology score (r=0.785,P＜0.05),and between the expression of TLR-4 mRNA and the pathology score (r=0.720,P＜0.05).Conclusions TLR-4 may be closely associated with the development of the synovitis of TMJ of rats.

Objective To observe the correlations of conventional ultrasound and contrast-enhanced ultrasound(CEUS)features with expression level of Ki-67 of intrahepatic cholangiocarcinoma(ICC).Methods A total of 77 patients with pathologically confirmed ICC were retrospectively enrolled.According to the expression level of Ki-67 of target lesions,the patients were divided into high expression group(Ki-67≥20%,n=53)and low expression group(Ki-67＜20%,n=24).The features showed on conventional ultrasound and CEUS were compared between groups,and the variables being significantly different were analyzed with multivariate logistic regression to select ultrasonic features being correlated with Ki-67 expression level of ICC.Results The mean maximum diameter of ICC in high expression group was larger than that in low expression group([6.2±2.0]cm vs.[5.2±1.9]cm,P=0.041),and the percentage of ICC with maximum diameter>5 cm in high expression group was higher than that in low expression group(66.04%vs.33.33%,P=0.007).No significant difference of the lesions shape,border nor the internal echogenicity showed on conventional ultrasound was found between groups(all P＞0.05).The percentage of ICC with peripheral rim enhancement and enlargement after enhancement in high expression group were both higher than those in low expression group(both P＜0.05),but no significant difference of the enhancement pattern,the presence or absence of non-enhance area within the lesion nor the peak intensity of lesions was found between groups(all P＞0.05).The maximum diameter larger than 5 cm(OR=5.612,P=0.004)and peripheral rim enhancement(OR=3.880,P=0.025)were both independent factors for predicting high Ki-67 expression level of ICC.Conclusion ICC with the maximum diameter larger than 5 cm and peripheral rim enhancement on CEUS might indicate high expression level of Ki-67,which was helpful for clinical treatment decisions.

Direct acid hydrolysis of Dioscorea zingiberensis rhizomes for preparation of diosgenin is wildly used in the traditional industry， which uses a large amount of inorganic acid catalysts， with high wastewater discharge and serious environmental pollution. Therefore， exploring clean and efficient preparation methods and processes has become an inevitable choice to realize the sustainable development of industrial production of diosgenin. Herein， the author reviewed and analyzed the research progress and problems of enzymatic hydrolysis， microbial transformation and modified acid hydrolysis in the preparation of diosgenin from D. zingiberensis rhizomes during the last ten years， and their application prospects are analyzed. Enzymatic hydrolysis has mild reaction conditions， but the yield of diosgenin is low， the economic cost is high， and the purification process of active enzyme is complicated. Microorganism shows specific activity to the substrate and high efficiency for diosgenin production， and microbial transformation is clean and environmentally friendly， but microbial transformation is time-consuming and the metabolic intermediates are complicated. For the modified acid hydrolysis， two-phase acid hydrolysis can reduce the amount of acid catalyst， and sulfonic acid-functionalized ionic liquid displays good recyclable performance by replacing the traditional inorganic acid， however， the wastewater discharge should still be considered. Solid acid catalysts are non-corrosive and easy to be recycled， but the need to use ethanol as the reaction solvent has certain safety hazards， and the catalyst preparation process is cumbersome. In conclusion， exploring clean and efficient conversion methods is an important research trend for preparation of diosgenin from D. zingiberensis rhizomes. For the enzymatic hydrolysis， the key glycoside hydrolases in the bioconversion process should be explored in depth， the conversion pathway of enzymatic saponins and enzyme specificity should be fully elucidated， and efforts should be made to improve the efficiency of enzymatic hydrolysis. For the microbial transformation， we should accelerate its industrial application process based on selecting and breeding efficient transformation strains， and optimizing stable transformation systems and processes， and in-depth investigation of the mechanism of microbial transformation， fully elucidating the specific key hydrolases and its catalytic properties， and striving to improve the efficiency of microbial transformation. For the modified acid hydrolysis， novel acid catalytic system with simple structure， stable performance and good biodegradability should be explored and applied， which can effectively solve the problems of environmental pollution and production safety.