Glucuronic acid metabolites of phenolic acids target AKT-PH domain to improve glucose metabolism / 中草药·英文版

OBJECTIVE@#Phenolic acids widely exist in the human diet and exert beneficial effects such as improving glucose metabolism. It is not clear whether phenolic acids or their metabolites play a major role in vivo. In this study, caffeic acid (CA) and ferulic acid (FA), the two most ingested phenolic acids, and their glucuronic acid metabolites, caffeic-4'-O-glucuronide (CA4G) and ferulic-4'-O-glucuronide (FA4G), were investigated.@*METHODS@#Three insulin resistance models in vitro were established by using TNF-α, insulin and palmitic acid (PA) in HepG2 cells, respectively. We compared the effects of FA, FA4G, CA and CA4G on glucose metabolism in these models by measuring the glucose consumption levels. The potential targets and related pathways were predicted by network pharmacology. Fluorescence quenching measurement was used to analyze the binding between the compounds and the predicted target. To investigate the binding mode, molecular docking was performed. Then, we performed membrane recruitment assays of the AKT pleckstrin homology (PH) domain with the help of the PH-GFP plasmid. AKT enzymatic activity was determined to compare the effects between the metabolites with their parent compounds. Finally, the downstream signaling pathway of AKT was investigated by Western blot analysis.@*RESULTS@#The results showed that CA4G and FA4G were more potent than their parent compounds in increasing glucose consumption. AKT was predicted to be the key target of CA4G and FA4G by network pharmacology analysis. The fluorescence quenching test confirmed the more potent binding to AKT of the two metabolites compared to their parent compounds. The molecular docking results indicated that the carbonyl group in the glucuronic acid structure of CA4G and FA4G might bind to the PH domain of AKT at the key Arg-25 site. CA4G and FA4G inhibited the translocation of the AKT PH domain to the membrane, while increasing the activity of AKT. Western blot analysis demonstrated that the metabolites could increase the phosphorylation of AKT and downstream glycogen synthase kinase 3β in the AKT signaling pathway to increase glucose consumption.@*CONCLUSION@#In conclusion, our results suggested that the metabolites of phenolic acids, which contain glucuronic acid, are the key active substances and that they activate AKT by targeting the PH domain, thus improving glucose metabolism.

Postmortem Distribution and Postmortem Redistribution of Carbofuran-7-Phenyl Glucuronic Acid in Rabbits / 法医学杂志

OBJECTIVES@#To establish a carbofuran intragastric administration death model in rabbits, and to observe the postmortem distribution and postmortem redistribution of carbofuran-7-phenyl glucuronic acid (Glu-7PH) in rabbits.@*METHODS@#The postmortem distribution: Rabbits were given an administration of 1/2LD50, LD50, 2LD50 carbofuran. Dead rabbits were dissected immediately. Rabbits that had remained alive 2 hours were sacrificed by carbon dioxide (CO2) inhalation and dissected immediately. The myocardium, cardiac blood, liver, spleen, lung, kidney, brain and right hindlimb muscle were collected. The postmortem redistribution: After giving an administration of 4LD50 carbofuran, the myocardium, cardiac blood, liver, spleen, lung, kidney, brain, and right hindlimb muscle were collected at 0, 12, 24, 48, and 72 h postmortem in supine position at 15 ℃ room temperature. The quantity of Glu-7PH was determined by LC-MS/MS.@*RESULTS@#The postmortem distribution: Among the three dose groups, there were significant differences in the quantities of Glu-7PH in different tissues. The postmortem redistribution: There was no significant difference in the Glu-7PH quantities in cardiac blood, mycardium, spleen, kidney, brain and right hindlimb muscle, but there was a significant difference in the Glu-7PH quantities in the liver and lung.@*CONCLUSIONS@#The mycardium, cardiac blood, liver, lung, kidney, brain and hindlimb muscle of rabbits can be used as appropriate samples for Glu-7PH detection. However, it should be noted that Glu-7PH was redistributed postmortem in rabbit liver and lung.

Expression optimization and molecular modification of heparin C5 epimerase / 生物工程学报

Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.

Optimization of processing technology of Momordicae Semen cream by Box- Behnken design-response surface methodology / 中草药

Objective To optimize the optimum processing technology of Momordicae Semen cream using Box-Behnken design-response surface method (BBD-RSM). Methods The overall desirability (OD) of indexes of the content of 3-O-β-D- glucuronic acid methyl ester, β-sitosterol, and oleanolic acid and the content of fatty oil in gypsogenin was comprehensively evaluated, and BBD-RSM with three factors and three levels was used to study the effect of baking temperature and time, and pressing time on the processing technology of Momordicae Semen cream. Results According to the experiment, the optimum processing conditions were optimized: the baking time 1.68 h, the baking temperature 80 ℃, the pressing time 30 min, and the OD value was 0.777. Considering the actual situation, the optimum processing technology of Momordicae Semen was obtained by fine-tuning the baking time (A). That was, the baking time was 1.7 h, the baking temperature was 80 ℃, and the pressing time was 30 min. Also, the calculated OD values were 0.779, 0.783, 0.766, and its RSD was 1.15% through the obtained conditions in parallel with three batches of samples. Conclusion The processing technology optimized by Box-Behnken design-response surface method has the advantages of stable, good model prediction effect and a new processing technology for Momordicae Semen cream production.

Quality evaluation and discrimination of Polygonati Rhizoma from three origins based on polysaccharide composition and content / 中草药

Objective: To compare the composition and content of polysaccharides in Polygonati Rhizoma by qualitative and quantitative analysis combined with chemometrics, and provide the reference for the quality evaluation of Polygonati Rhizoma. Methods Fourier transform infrared spectroscopy (FTIR) and pre-column derivatization-HPLC (PMP-HPLC) were employed to reveal the differences of polysaccharide among the three species, and the content of total polysaccharide was determined by ultraviolet spectroscopy (UV). Results:The result indicated that the average spectra and the second derivative atlas in FTIR fingerprint of Polygonati Rhizoma was significantly different. The PCA, PLS-DA and HCA analysis provided a further refinement of the method to distinguish three species. Furthermore, the PMP-HPLC showed that the components of monosaccharide and polysaccharides of three species were different. The main common components of the 10 common peaks were identified, which were as follows: D-mannose, D-ribose, L-rhamnose, D-galacturonic acid, D-glucuronic acid, D-galactose, D-glucose, D-xylose, D-arabinose and L-fucose. The content of the total polysaccharide meeted the requirements of Chinese pharmacopoeia 2015 edition Conclusion:The study provided a new reference for quality evaluation of P. Rhizoma.

Association analysis of the UGT1A1 polymorphism and unexplained neonatal hyperbilirubinemia / 中国新生儿科杂志

Objective To study the relationship between uridine diphosphate glucuronic acid (UGT1A1) gene polymorphism and unexplained neonatal unconjugated hyperbilirubinemia in Jinhua.Method Full-term infants with unidentified non-binding hyperbilirubinemia were selected as hyperbilirubinemia group from January 2016 to December 2017 in the obstetrics or neonatal intensive care unit of Jinhua Central Hospital,healthy full-term neonates and those with physiological jaundice admitted during the same period were selected as control group.Whole blood DNA was extracted and UGT1A1 was sequenced and then annotated with human gene mutation database.The distribution and frequency of UGT1A1 genotype were analyzed.The correlation between different genotypes and unexplained unconjugated hyperbilirubinemia in neonates was also studied.Result Two hundred and forty cases were enrolled in the hyperbilirubinemia group,and 216 cases were enrolled in the control group.Four single nucleotide variation (SNV) sites associated with the disease were found on UGT1A1,which were c.211G＞A (Gly71Arg),c.686C＞A (Pro229Gln),c.1091C＞T (Pro364Leu) and c.1456T＞G (Tyr486Asp),accounting for 83.9％(141/168),1.8％(3/168),8.9％(15/168) and 5.4％(9/168) in the experimental group respectively.The genotype frequency and allele frequency analysis showed that the distribution of the two SNV sites of c.211G＞A and c.1456T＞G were statistically different between the experimental group and the control group (P＜0.05),whereas there was no statistical difference of the other two SNV sites of c.686C＞A and c.1091C＞T between the two groups.Binary Logistic regression analysis showed that c.211G＞A and c.1456T＞G were related to the occurrence of unexplained hyperbilirubinemia,The OR values (95％CI) were 5.412 (3.567～ 8.212) and 8.377 (1.052～66.670) respectively,but no correlation was found of the other two polymorphic loci.At the different genotypes of c.211G＞A locus,the levels of total bilirubin and non-binding bilirubin in infants with homozygous mutant (AA) were higher than those in infants with heterozygous mutant (GA) and wild type (GG),which was statistically significant (P＜0.05).Conclusion The most common mutation site of the UGT1A1 gene in Jinhua is c.211G＞A.The mutations of c.211G＞A and c.1456T＞G are risk factors forunconjugated hyperbilirubinemia in neonates.Of the different genotypes of c.211G＞A locus,the serum bilirubin level of homozygous mutant group was significantly higher than heterozygous mutant group and wild type group.

Content Comparison and Correlation Analysis of Gypsogenin 3-O-β-D-glucuronic Acid Methyl Ester in Mo-mordica cochinchinensis from 14 Production Places of 8 Provinces / 中国药房

OBJECTIVE:To provide reference for variety breeding,high yield and high quality cultivation of Momordica cochi-nchinensis. METHODS:Based on Chinese Pharmacopoeia (2015 edition,Vol Ⅰ),HPLC for determining the content of gyp-sogenin 3-O-β-D-glucuronic acid methyl ester in M. cochinchinensis was optimized,the contents of gypsogenin 3-O-β-D-glucuronic acid methyl ester in M. cochinchinensis from 14 production places of 8 provinces were compared,and cluster analysis was conduct-ed. Correlation of longitude,latitude and altitude with content of gypsogenin 3-O-β-D-glucuronic acid methyl ester in M. cochinchi-nensis was analyzed by SPSS17.0 software. RESULTS:Gypsogenin 3-O-β-D-glucuronic acid methyl ester in M. cochinchinensis showed good linear relationship in 0.05-0.5 mg/mL,regression equation was Y=4361.95X+67.3808(R2=0.9997);the limits of quantification and detection were 15.62 ng and 4.67 ng,respectively. Average recovery was 99.76%(RSD=1.36%,n=9). The content of gypsogenin 3-O-β-D-glucuronic acid methyl ester in M. cochinchinensis from 14 production places of 8 provinces had ex-tremely significant differences(P<0.01). Clustering analysis results indicated that M. cochinchinensis from 14 production places of 8 provinces were divided into Ⅰ,Ⅱ,Ⅲ,Ⅳ groups. Contents of gypsogenin 3-O-β-D-glucuronic acid methyl ester in M. cochi-nchinensis of Ⅲ,Ⅳ groups were relatively high,including Guangxi Pingnan,Guizhou Jiangkou,Guizhou Dejiang,Fujian Jian-yang and Hunan Huitong,in which,Guangxi Pingnan,Guizhou Jiangkou,Guizhou Dejiang and Hunan Huitong had high altitude and low latitude,Fujian Jianyang had high altitude. The content of gypsogenin 3-O-β-D-glucuronic acid methyl ester in M. cochi-nchinensis was positively correlated with altitude,while negatively correlated with longitude and latitude. CONCLUSIONS:HPLC is simple,accurate and reproducible for determining the content of gypsogenin 3-O-β-D-glucuronic acid methyl ester in M. cochi-nchinensis,and the determined contents of gypsogenin 3-O-β-D-glucuronic acid methyl ester in M. cochinchinensis from 14 produc-tion places of 8 provinces have reached the standard of Chinese Pharmacopoeia (2015 edition,Vol Ⅰ). Cultivation in Guangxi Pingnan,Guizhou Jiangkou,Guizhou Dejiang,Hunan Huitong and other areas with high altitude and low latitude helps to improve the content accumulation of gypsogenin 3-O-β-D-glucuronic acid methyl ester in M. cochinchinensis.

Role of UGT1A1 gene polymorphism in the pathogenesis of Gilbert syndrome / 临床肝胆病杂志

As a bilirubin metabolic disorder, Gilbert syndrome belongs to the category of congenital non-hemolytic jaundice. Deficiency or decrease in the activity of bilirubin-uridine diphosphate glucuronyltransferase (UGT) is an important reason for the pathogenesis of Gilbert syndrome. UGT1A1, an isoenzyme of UGT, is a key enzyme to direct bilirubin in the liver. Mutations in UGT1A1 gene lead to the structural abnormality of UGT, and thus result in the decrease or loss of the ability of UGT to bind bilirubin. This article summarizes the research advances in the role of UGTA1 and its polymorphism in the pathogenesis and diagnosis of Gilbert syndrome.

Determination of naringenin and its glucuronic acid conjugate in rat plasma by LC-MS/MS / 中国药学杂志

OBJECTIVE: To establish an LC-MS/MS method for determination of naringenin and its glucuronic acid conjugate in rat plasma. METHODS: A Shimpack ODS C18 column (2.3 mm × 75 mm, 3 μm) was used with a mobile phase consisting of 0.1% acetic acid solution and methanol at the flow rate of 0.2 mL·min-1. The detection mode was MRM, and the precursor/product ion pair was m/z 271. 1→151.0 for naringenin and m/z 301.2→T64.1 for the IS. RESULTS: The calibration curve was linear in the range of 0.01-4 μg·mL-1 (r=0.9997) for naringenin. The average recoveries at high, medium, and low concentrations were between 85%-115%. The method was used to determine the plasma concentration of naringenin after gastric gavages of different dosages to Wister rats. CONCLUSION: The method is rapid, sensitive and reproducible. Naringenin, reaching peak concentration quickly, has low concentration in plasma and most became its glucuronic acid conjugate.

Investigate the Compatible Stabilities of Sodium Glucuronic Acid Injection and Ganlixin Injection / 医学研究杂志

Objective Investigate the compatible stabilities of Sodium Glucuronic Acid Injection and Ganlixin Injection.Methods The Sodium Glucuronic Acid Injection mixed with Ganlixin Injection,to study the content of Sodium Glucuronic Acid Injection and Ganlixin Injection in different time by uv spectrophotometry,Investigate the appearance of the mixed solution,detect PH values and Insoluble Particles.Results At experimental temperature(15℃),the mixed solution,PH values,Insoluble Particles and the relative percentage have no obvious change in 4 hours.Conclusions The Sodium Glucuronic Acid Injection mixed with Ganlixin Injection is stabilization,so Sodium Glucuronic Acid Injection can combined with Ganlixin Injection.