Composition changes reveal relationship between color and enzymatic reaction of Magnoliae Officinalis Cortex during "sweating" process / 中国中药杂志

In this study, we employed Q Exactive to determine the main differential metabolites of Magnoliae Officinalis Cortex du-ring the "sweating" process. Further, we quantified the color parameters and determined the activities of polyphenol oxidase(PPO), peroxidase(POD), and tyrosinase of Magnoliae Officinalis Cortex during the "sweating" process. Gray correlation analysis was performed for the color, chemical composition, and enzyme activity to reveal the effect of enzymatic reaction on the color of Magnoliae Officinalis Cortex during the "sweating" process. Magnoliae Officinalis Cortex sweating in different manners showed similar metabolite changes. The primary metabolites that changed significantly included amino acids, nucleotides, and sugars, and the secondary metabolites with significant changes were phenols and phenylpropanoids. Despite the different sweating methods, eleven compounds were commonly up-regulated, including L-glutamic acid, acetylarginine, hypoxanthine, and xanthine; six compounds were commonly down-re-gulated, including L-arginine, L-aspartic acid, and phenylalanine. The brightness value(L~*), red-green value(a~*), and yellow-blue value(b~*) of Magnoliae Officinalis Cortex kept decreasing during the "sweating" process. The changes in the activities of PPO and POD during sweating were consistent with those in the color parameter values. The gray correlation analysis demonstrated that the main differential metabolites such as amino acids and phenols were closely related to the color parameters L~*, a~* and b~*; POD was correlated with amino acids and phenols; PPO had strong correlation with phenols. The results indicated that the color change of Magnoliae Officinalis Cortex during "sweating" was closely related to the reactions of enzymes dominated by PPO and POD. The study analyzed the correlations among the main differential metabolites, color parameters, and enzyme activities of Magnoliae Officinalis Cortex in the "sweating" process. It reveals the common law of material changes and ascertains the relationship between color changes and enzymatic reactions of Magnoliae Officinalis Cortex during "sweating". Therefore, this study provides a reference for studying the "sweating" mechanism of Magnoliae Officinalis Cortex and is of great significance to guarantee the quality of Magnoliae Officinalis Cortex.

Reconstitution of biosynthetic pathway for mushroom-derived cyathane diterpenes in yeast and generation of new "non-natural" analogues

Mushroom-derived cyathane-type diterpenes possess unusual chemical skeleton and diverse bioactivities. To efficiently supply bioactive cyathanes for deep studies and explore their structural diversity,

Modern research progress of Chinese materia medica diaphoretic processing method / 中草药

Diaphoretic processing is one of the essential methods aiming to speed up the drying herbs and help the preservation of medicinal materials in the initial processing of Chinese materia medica. In addition, modern research showed that diaphoretic processing also affects the chemical components pharmacological activities and pharmacodynamic action of medicinal materials. During the diaphoretic processing, the internal temperature of medicinal materials rose, which increased the enzyme activity and improved the enzyme-catalyzed reaction, and then these lead to the change of chemical components of medicinal materials. With the diaphoretic processing continues, continually elevated internal temperature of medicinal materials started a series anti-heat stress reaction and generated some secondary metabolites, which considered as main effective components of medicinal materials. In this paper, the mechanism of diaphoretic processing was revealed by analysis the biological enzyme action and anti-heat stress reaction, in order to provide a theoretical basis for the application and extension of the diaphoretic processing.

Detection ofα1 antitrypsin activity by chromogenic substrate assay with initial veloci-ty of enzymatic reaction / 军事医学

Objective To detect the activity of α1 antitrypsin(AAT) with initial velocity of enzymatic reaction in order to detect the activity of samples in the process of separating and purifying plasma protein ,chromogenic substrate assay was optimized.Methods The effect of trypsin concentration and reaction time on enzymatic reaction was acquired by the kinetic monitoring mode of the microplate reader .Initial velocity was calculated to confirm the largest concentration of trypsin which was saturated by substrate .AAT was incubated with trypsin and absorbance produced by enzymatic reaction of remaining trypsin and substrate could reflect the activity of AAT .A standard curve was established with △D fitting with the activity of AAT standard.The activity of related samples was detected and the precision and accuracy of the method were evaluated . Results Trypsin concentration was 0.0625 mg/ml.Within 20 minutes, enzymatic reaction was with initial velocity .The range of the standard curve was 200-1200 IU/ml.Correlation coefficient was more than 0.99.The activity of Cohn Ⅳ, samples of pre-processing and elution were (720.59 ±18.63), (601.84 ±19.18),and (568.09 ±24.83)IU/ml, respec-tively.The relative standard deviation was less than 10%. Sample recovery rate was 90%-110%.Conclusion The optimized chromogenic substrate assay greatly improves accuracy and precision .The method can be used for the detec-tion of AAT activity of samples in laboratories and workshops .

"Sweating" of traditional Chinese medicinal materials during primary processing and its mechanisms of enzymatic reaction and chemical conversion / 中草药

Primary processing of traditional Chinese medicinal materials (TCMM) is an important link which closely relates to the quality of products in TCMM. The traditional primary processing method and technology systems were derived from the long-term practices and experiences, which are distinctive, colorful, diverse, and scientific. The method of "sweating" is a critical step for processing the rhizome, root, cortex, and sclerotium and helpful for distributing moisture from inside to outside of TCMM in drying process, regulating and promoting the enzymatic reaction and chemical conversion by enzyme and germs, and starting or accelerating the biotransformation and chemical conversion of primary/secondary metabolites, which could directly affect the quality of TCMM. In this paper, the authors discuss the origins and development, purpose, and significance of "sweating" processing and the mechanisms of enzymatic reaction and chemical conversion of chemical compositions during the primary processing of "sweating". These data may provide the foundation and support for processing in normalization and standardization and formulating the Standard Operating Procedure (SOP) of primary processing of TCMM.

Effects of substrates and reaction conditions on production of cyclodextrins using cyclodextrin glucanotransferase from newly isolated Bacillus agaradhaerens KSU-A11

The effects of reaction conditions on cyclodextrins (CDs) production by CGTase from newly isolated Bacillus agaradhaerens KSU-A11 is reported. Among six types of starch tested, potato starch gave highest starch conversion into CDs. In addition, CDs yield was about three fold higher when using gelatinized potato starch in comparison to raw starch. The total CDs production was increased with increasing pH, showing maximum starch conversion at pH 10. Furthermore, the proportion of gamma-CD was relatively higher under slightly acidic-neutral conditions than at alkaline pH with a maximum proportion of 35.6 percent at pH 7 compared to 7.6 percent at pH 10. Maximum starch conversion into CDs was seen at reaction temperature of 55ºC. Lower reaction temperature led to higher proportion of gamma-CD with maximum percentage at 35ºC. Cyclization reaction was significantly promoted in the presence CaCl2 (10 mM), while in the presence of ethyl alcohol there was significant decrease in CD production particularly at high concentration. beta-CD was the major product up to 1 hr reaction period with traces of alpha-CD and no detectable gamma-CD. However, as the reaction proceed, gamma-CD started to be synthesised and alpha-CD concentration increased up to 4 hrs, where the CDs ratios were 0.27:0.65:0.07 for alpha-CD:beta-CD:gamma-CD, respectively. In addition, optimum CGTase/starch ratio was obtained at 80 U/g starch, showing highest starch conversion into CDs. All the parameters involved have been shown to affect the products yield and/or specificity of B. agaradhaerens KSU-A11 CGTase.

Compatible effects of Corydalis Rhizoma and Angelicae Dahuricae Radix components on enzymatic reaction kinetics of dl-tetrahydropalmatine in rat liver microsomes / 中草药

Objective: To study the enzymatic reaction kinetics of dl- tetrahydropalmatine (TET) in total alkaloid (TA) of Corydali Rhizoma in rat liver microsomes and to investigate the compatible effects of the effective components such as coumarin (Cou) and volatile oil (VO) in Angelicae Dahuricae Radix with TA in Corydali Rhizoma on enzymatic reaction kinetics of TET. Methods: Rat liver microsomes were prepared by ultracentrifugation and the TET concentration in incubation medium was determined by HPLC. Comparative study on the enzymatic reaction kinetics of TET in each group of TA, TA-Cou, TA-VO, and TA-Cou-VO to deduce the michaelis constant (Km) and maximum reaction rate (Vmax) of TET in each group and calculate the clearance rate (CLint) of TET in each group. Results: The Km, V max, and CLint in TA group were 0.12 μmol/ (L·min·mg), 5.40 μmol/L, and 0.022 L/(min·mg), respectively; In TA-Cou group they were 0.27 μmol/(L·min·mg), 40.18 μmol/L, and 0.006 L/(min·mg), respectively; In TA-VO group they were 0.57 μmol/(L·min·mg), 22.60 μmol/L, and 0.025 L/(min·mg), respectively; In TA-Cou-VO they were 0.84 μmol/(L·min·mg), 23.25 μmol/L, and 0.036 L/(min·mg), respectively. Conclusion: The effective components of TA in Corydali Rhizoma with Cou and VO in Angelicae Dahuricae Radix could decrease CLint of TET in TA of liver.

Study on the Identification and Potency Determination of L-asparaginases from 2 Kinds of Strain / 中国药房

OBJECTIVE: To discuss the identification method of L-asparaginases prepared from E.coliASI.357 and Erwinia carotovora and the optimal enzymatic reaction conditions of potency determination.METHODS: HPLC method and isoelectric focusing electrophoresis were applied for the identification of L-asparaginase from 2 kinds of strain.The effects of category of buffer solution and pH value on enzymatic reaction of potency determination of L-asparaginase were investigated.RESULTS: HPLC chromatogram of L-asparaginases from E.coli ASI.357 was different from that from Erwinia carotovora.The retention time of the peaks were 11.0 min and 11.8 min.The isoelectric point (PI) of L-asparaginase produced from E.coliASI.357 was within 4.65～5.1 and that produced from Erwinia carotovora was within 7.1～8.20.The optimal enzymatic reaction conditions of potency determination of L-asparaginase produced from E.coliASI.35 were Tris-HCl (pH=9.0) as buffer and that produced from Erwinia carotovora was 0.2 mol?L-1 phosphate (pH=8.0) as buffer.CONCLUSION: The isoelectric point (PI) of L-asparaginases produced from 2 kinds of strain is different from each other as well as their optimal enzymatic reaction conditions of potency determination.The L-asparaginases from 2 kinds of strains should be controlled as 2 different categories.