1.Enzymological Characteristics of Catalytic Antibody-catalyzed enantioselective Hydrolysis of Ibuprofen Ester in Water-in-oil microemulsion
Gensheng YANG ; Yingdan QI ; Zhimin OU ; Shanjing YAO
Progress in Biochemistry and Biophysics 2009;36(2):182-189
The asymmetric hydrolyzation of racemic ibuprofen ester is one of the most important methods for chiral separation of ibuprofen. A catalytic antibody that accelerates the rate of enantioselective hydrolysis of ibuprofen methyl ester was successfully elicited against an immunogen consisting of tetrahedral sulfate hapten attached to bovine serum albumin (BSA). The rate constant enhancement factor Kcat/Kuncat was about 1.6x104. The catalytic activity of the catalytic antibody in a reverse micelle reaction system based on sodium b/s (2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane was studied. Kinetic analysis of the catalytic antibody-catalyzed reaction was found to be possible in this system. Kinetic studies showed that hydrolysis in the microemulsion system follow Michaelis-Menten kinetics. The catalytic antibody can also accelerate catalysis of S-ibuprofen methyl ester in the microemulsion system. Temperature effects, the pH profile, Km,app and Kcat were determined. The dependence of the catalytic antibody hydrolytic activity on the Wo (molar ratio of water to surfactant) showed a bell-shaped curve, presenting a maximum at about wo = 21.
2.Optimization of the Purification Technology of Total Triterpenoids of Ligustrum lucidum by Box-Behnken Re- sponse Surface Methodology
Shuang GAO ; Yuzhen WANG ; Fumin BO ; Li ZHANG ; Yue SUN ; Shanjing YANG ; Anjie FENG ; Lingjun LI
China Pharmacy 2020;31(18):2196-2201
OBJECTIVE:To i mprove the transfer rate and purity of oleanolic acid and ursolic acid in total triterpenoids from Ligustrum lucidum ,so as to optimize the purification technology. METHODS :Oleanolic acid and ursolic acid were used as representative components of total triterpenoids ,and their contents were determined by HPLC. The determination was performed on Thermo BDS Hypersil C 18 column with mobile phase consisted of methanol- 0.02% ammonium acetate solution (80∶20,V/V)at the flow rate of 1.0 mL/min. The detection wavelength was set at 210 nm,and column temperature was 30 ℃. The sample size was 20 μ L. In single factor tests,using transfer rate of oleanolic acid and ursolic acid as index ,the effects of water precipitation temperature and time ,the amount of redissolved ethanol on the purification technology was investigated ;using transfer rate and purity of two components as indexes ,the effects of the amount of activated carbon and volume fraction of crystallization ethanol were investigated. Based on it ,using the amount of redissolved ethanol and activated carbon ,volume fraction of crystallization ethanol as factors ,Box-Behnken response surface methodology was used to optimize the purification technology ,and validation tests were performed. RESULTS :The optimal purification technology was adding 4-fold(mL/g,the same below )water in L. lucidum concentrated solution ,placing for 2 hours at 0 ℃(water precipitation );adding 1-fold ethanol to dissolve (redissolution); adding 4% activated carbon (edulcoration);finally adding water to adjust the volume fraction of ethanol to 80%,placing at 4 ℃ for 12 hours(crystallization),centrifuging and drying. The results of 3 times of validation tests showed that the transfer rates of oleanolic acid and ursolic acid in total triterpenoids prepared by optimized technology were 61.11% and 65.78%,the purities of them were 53.44% and 19.79%,and RSDs were both lower than 3%. CONCLUSIONS :The optimized purification technology has high extraction efficiency and simple operation ,which can be used for industrial production of purification of total triterpenoids from L. lucidum and the development of corresponding preparations.