S - Type And R - Type Ginsenosides And Flavonoid Analogues Were Isolated By One - Step Immunoaffinity Chromatography

Background:The separation of chiral compounds is of great significance for pharmacological and pharmacodynamics research, and is of vital importance for clinical medication safety. On the basis of previous research achievements of our team, this paper aims to provide a novel and valuable method for the separation of chiral compounds which deserves further study.Objective:To achieve one-step separation of 20(S)-ginsenoside-Rg2 (20(S)-Rg2) and 20(R)-ginsenoside-Rg2 (20(R)-Rg2) by immunoaffinity chromatography column (IAC). To develop a one-step immunochromatographic separation method of 20(S)-ginsenoside-Rhl (20(S)-Rh1) and 20(R)-ginsenoside-Rhl (20(R)-Rhl) using monoclonal antibodies (MAbs) against ginsenoside Re. To optimize extraction conditions of IAC column based on anti-naringin MAbs and develop an on-line detection method. To separate naringin and hesperidin, neohesperidin by IAC based on anti-naringin MAbs.Methods:(1) Study on separation of 20(S)-Rg2 and 20(R)-Rg2:The immunoaffinity chromatography column (IAC) was constructed by coupling MAbs against G-Rhl to CNBr activated sepharose 4B through covalent bond. A series of mixed sample solutions with different proportions of 20(S)-Rg2 and 20(R)-Rg2 was prepared and then loaded onto the prepared IAC forthe separation of them. The samples before and after separation were analyzed by HPLC.(2) Study on separation of 20(S)-Rhl and 20(R)-Rhl:The IAC column based on anti-Re MAbs was prepared and used to separate 20(S)-Rhl and 20(R)-Rhl with the same operating procedure as above.(3) Development of an on-line detection method and investigation of extraction conditions for IAC column:The extraction conditions of IAC column based on anti-naringin MAbs were optimized, including choice of washing buffer and elution buffer, incubation time, flow rate of washing and eluting, pH and ionic strength of elution buffer.(4) Study on separation of Naringin and hesperidin, Naringin and neohesperidin: Naringin, hesperidin and neohesperidin were separated by IAC based on anti-naringin MAbs. HPLC method for simultaneously detecting the three compounds was developed and used to determine samples before and after separation.Results:(1) Separation of 20(S)-Rg2 and 20(R)-Rg2:The IAC column based on anti-Rhl MAbs was successfully prepared. The separation results was analysed by HPLC. When 20(R)-Rg2 passed through the IAC column, it was adsorbed, but the amount adsorbed was lower than that when 20(S)-Rg2 ran through the column. The relative quantities of S-type-Rg2 in the elution fraction (ELs) obtained from samples 3 (1:1) and sample 4 (4:1) significantly increased, reaching more than 2:1 and 10:1. Furthermore, the ELs obtained from sample 5 (10:1) only contained S-type-Rg2, but no R-type-Rg2.(2) Separation of 20(S)-Rhl and 20(R)-Rhl:The IAC based on anti-Re MAbs was successfully prepared used to separate 20(S) and 20(R) type ginsenoside Rhl according to the differences in adsorption between the two chiral compounds. When the mixture containing 0.2 mg/mL of 20(S)-Rhl and 0.1 mg/mL passed through the column, the ELs only contained S-type-Rh1, but no R-type-Rh1, which means that the two chiral compounds were successfully separated.(3) Development of an on-line supervision method and investigation of extraction conditions for IAC column:Taking advantage of the IAC based on anti-naringin MAbs and a Bio-Rad Duo Flow medium pressure liquid chromatography system, a simple, sensitive, and reproducible method for a one-step selective separation and purification of naringin and an on-line elution profile supervision method was developed. The extraction conditions of the IAC column were optimized:water was used as washing buffer and 0.1 M glycine-HCl buffer (pH 2.7, containing 0.5M NaCl) was used as elution buffer, the sample was incubated 30min after loading onto the column, and flow rate of washing and eluting was 2 mL/min.(4) Separation of Naringin and hesperidin, Naringin and neohesperidin:Naringin and hesperidin were separated by the IAC based on anti-naringin MAbs quickly. Naringin and neohesperidin could be separated when the loading sample contain 0.15 mg/mL of naringin, 0.3 mg/mL of neohesperidin.Conclusion:The application of IAC using MAbs against small molecules provides a totally new thought and potential method for resolving chiral compounds. The developed on-line elution profile supervision method will lay the foundation for the technology of IAC coupling with other chromatography method.