UPLC-MS/MS Method for Detection of Etomidate and Its Metabolite Etomidate Acid Quantity in Blood / 法医学杂志

OBJECTIVES@#To establish a method for the simultaneous quantitative analysis of etomidate and its metabolite etomidate acid in blood, and to discuss its application value in actual cases.@*METHODS@#Acetonitrile precipitate protein method was used, and C18 column was selected. Gradient elution was performed with acetonitrile and 5 mmol/L ammonium acetate within 6 min. Electrospray ionization source in positive ion mode was used. The internal standard etomidate acid-d5 was obtained by etomidate-d5 alkaline hydrolysis reaction. Ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used for quantitative analysis. The methodological verification was conducted.@*RESULTS@#Etomidate and etomidate acid in blood showed good linear relationship in the quantitative linear range (r>0.999), with the lower limit of quantification was 2.5 ng/mL and 7.5 ng/mL, respectively. The accuracy, precision, recovery rate, and matrix effect of the method met the professional verification standards. The practical application results showed that etomidate and etomidate acid could be detected in the blood of the abusers, and their mass concentrations ranged from 17.24 to 379.93 ng/mL.@*CONCLUSIONS@#The method established in this study can simultaneously quantify etomidate and etomidate acid in blood, which is simple and convenient to operate with accuracy. It can meet the detection needs of actual cases and provide technical support for law enforcement to crack down on etomidate abuse.

Effect and mechanism of Puerariae Lobatae Radix in alleviating insulin resistance in T2DM db/db mice based on intestinal flora / 中国中药杂志

This study aimed to examine the effect and underlying mechanism of Puerariae Lobatae Radix on insulin resistance in db/db mice with type 2 diabetes mellitus(T2DM) based on the analysis of intestinal flora. Fifty db/db mice were randomly divided into a model group(M group), a metformin group(YX group), a high-dose Puerariae Lobatae Radix group(YGG group), a medium-dose Puerariae Lobatae Radix group(YGZ group), and a low-dose Puerariae Lobatae Radix group(YGD group). Another 10 db/m mice were assigned to the normal group(K group). After continuous administration for eight weeks, body weight and blood sugar of mice were measured. Enzyme linked immunosorbent assay(ELISA) was used to detect glycosylated serum protein(GSP) and fasting serum insulin(FINS), and insulin resistance index(HOMA-IR) was calculated. The histopathological changes in the pancreas were observed by HE staining. Tumor necrosis factor(TNF)-α expression in the pancreas was detected using immunohistochemistry. The structural changes in fecal intestinal flora in the K, M, and YGZ groups were detected by 16S rRNA. Western blot was used to detect the expression of farnesoid X receptor(FXR) and takeda G protein-coupled receptor 5(TGR5) in the ileum, cholesterol 7α-hydroxylase(CYP7A1) and sterol 27α-hydroxylase(CYP27A1) in the liver, and G protein-coupled receptors 41(GPR41) and 43(GPR43) in the colon. Compared with the K group, the M group showed increased body weight, blood sugar, serum GSP, fasting blood glucose(FBG), and FINS, increased HOMA-IR, inflammatory infiltration of islet cells, necrosis and degeneration of massive acinar cells, unclear boundary between islet cells and acinar cells, disturbed intestinal flora, and down-regulated FXR, TGR5, CYP7A1, CYP27A1, GPR41, and GPR43. Compared with the M group, the YX, YGG, YGZ, and YGD groups showed decreased body weight, blood sugar, serum GSP, FBG, and FINS, islet cells with intact and clumpy morphology and clear boundary, necrosis of a few acinar cells, and more visible islet cells. The intestinal flora in the YGZ group changed from phylum to genus levels, and the relative abundance of intestinal flora affecting the metabolites of intestinal flora increased. The protein expression of FXR, TGR5, CYP7A1, CYP27A1, GPR41, and GPR43 increased. The results show that Puerariae Lobatae Radix can improve the inflammatory damage of pancreatic islet cells and reduce insulin resistance in db/db mice with T2DM. The mechanism of action may be related to the increase in the abundance of Actinobacteria, Bifidobacterium, and Bacteroides in the intestinal tract and the protein expression related to metabolites of intestinal flora.

UPLC-Q-TOF-MS metabolomic study on improvement of acute myocardial ischemia in rats by Dalbergia cochinchinensis heartwood / 中国中药杂志

This paper aimed to study the effect of Dalbergia cochinchinensis heartwood on plasma endogenous metabolites in rats with ligation of the left anterior descending coronary artery, and to analyze the mechanism of D. cochinchinensis heartwood in improving acute myocardial ischemic injury. The stability and consistency of the components in the D. cochinchinensis heartwood were verified by the establishment of fingerprint, and 30 male SD rats were randomly divided into a sham group, a model group, and a D. cochinchinensis heartwood(6 g·kg~(-1)) group, with 10 rats in each group. The sham group only opened the chest without ligation, while the other groups established the model of ligation. Ten days after administration, the hearts were taken for hematoxylin-eosin(HE) staining, and the content of heart injury indexes in the plasma creatine kinase isoenzyme(CK-MB) and lactate dehydrogenase(LDH), energy metabolism-related index glucose(Glu) content, and vascular endothelial function index nitric oxide(NO) was determined. The endogenous metabolites were detected by ultra-high-performance liquid chromatography-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS). The results showed that the D. cochinchinensis heartwood reduced the content of CK-MB and LDH in the plasma of rats to relieve myocardial injury, reduced the content of Glu in the plasma, improved myocardial energy metabolism, increased the content of NO, cured the vascular endothelial injury, and promoted vasodilation. D. cochinchinensis heartwood improved the increase of intercellular space, myocardial inflammatory cell infiltration, and myofilament rupture caused by ligation of the left anterior descending coronary artery. The metabolomic study showed that the content of 26 metabolites in the plasma of rats in the model group increased significantly, while the content of 27 metabolites decreased significantly. Twenty metabolites were significantly adjusted after the administration of D. cochinchinensis heartwood. D. cochinchinensis heartwood can significantly adjust the metabolic abnormality in rats with ligation of the left anterior descending coronary artery, and its mechanism may be related to the regulation of cardiac energy metabolism, NO production, and inflammation. The results provide a corresponding basis for further explaining the effect of D. cochinchinensis on the acute myocardial injury.

Comparative study of rat serum pharmacochemistry between Puerariae Lobatae Radix and Puerariae Thomsonii Radix based on UPLC-Q-TOF-MS / 中国中药杂志

UPLC-Q-TOF-MS and serum pharmacochemistry were employed to study the migrating components in rat sera after intragastric administration of the water extracts of Puerariae Lobatae Radix(PLR) and Puerariae Thomsonii Radix(PTR). After the respective intragastric administration of PLR and PTR extracts, blood samples were collected from the orbital vein. The serum samples were treated by protein precipitation method with methanol and acetonitrile at a ratio of 1∶1 and then passed through Agilent ZORBAX RRHD SB-C_(18) column(3 mm×100 mm, 1.8 μm) and Agilent SB-C_(18) pre-column(3 mm×5 mm, 1.8 μm) with 0.1% formic acid aqueous solution(A)-acetonitrile(B) as the mobile phase. The elution was performed at the flow rate of 0.25 mL·min~(-1), the column temperature of 40 ℃, and the injection volume of 2 μL. By comparison of the total ion chromatogram and secondary fragment ion information of PLR and PTR water extracts, PLR-and PTR-containing sera, and blank serum, we found 42 migrating components(including 17 prototype components and 25 metabolites) in the sera of rats treated with PLR and 35 migrating components(including 15 prototype components and 20 metabolites) in the sera of rats treated with PTR. Thirty-three common components were shared by the two treatments, including 13 prototype components and 20 metabolites. The differences of migrating components in the PLR-and PTR-treated rat sera provide a scientific basis for further study of the active components and quality markers of PLR and PTR.

Molecular Mechanism of Ligustri Lucidi Fructus-Astragali Radix for Anti-cancer Based on Network Pharmacology / 中国实验方剂学杂志

Objective: To screen out active ingredients, and predict the anti-cancer targets of Ligustri Lucidi Fructus-Astragali Radix based on the "herbs-active ingredient-action targets" network. Method: The traditional Chinese medicine (TCM) system pharmacology platform (TCMSP) was employed to screen out the active ingredients and putative targets of anti-cancer of glossy privet fruit and astragalus. DisGeNET database and Online Mendelian Inheritance in Man (OMIM database) were employed to predict the targets for treating cancer, and then "herbs-active ingredients-key targets" network was constructed by using Cytoscape software. The omicshare platform was employed to match the putative targets of ingredients and the targets for treating cancer. Finally, the protein interaction network of key targets was constructed by using String database, and the analysis of biological functions and pathways of them was carried out by using DAVID database. Result: Totally 33 drug active ingredients were screened out, involving a total of 203 targets, and 14 of them were related to cancer. These 14 key targets played an therapeutic role in treating cancer by regulating target proteins, such as ERBB2, AR, SRC, EGFR, ESR1, as well as proteoglycans in cancer, cancer pathways, microRNAs in cancer and other pathways. Conclusion: The therapeutic mechanism of Ligustri Lucidi Fructus-Astragali Radix reflects the multi-component, multi-target, and multi-pathway characteristics of TCMs, providing the scientific basis for further study and the material basis of Ligustri Lucidi Fructus-Astragali Radix against cancer.