Study On The Formation Mechanism Of "Black As Lacquer,Sweet As Candy" In The Processed Radix Rehmanniae From The Reactions Of "Cross-Linking Color-Maillard" And "Sugar Hydrolysis"

Radix Rehmanniae（RR）is derived from the fresh or dried roots of Rehmannia glutinosa Libosch.Fresh Radix Rehmanniae（FRR）,raw Radix Rehmanniae（RRR）,processed Radix Rehmanniae（PRR）are three different forms of clinical application of RR.RRR possesses a“cold”character and can“reduce the heat in blood,promote the production of body fluids”.However,PRR possesses a“warm”character as a tonic and can“nourishing yin,replenish blood,reinforcing essence and marrow”.Even though processing standard of PRR is“black as lacquer,sweet as candy”,the exact mechanisms of its scientific connotation are poorly understood.Previous research mainly focused on the natural constitutes changes of amino acids,sugars,and iridoid glycosides,the formation of new reaction products in the processing process has not been systematically carried out.In view of the above problems,relevant research work has been carried out systematically.RR was processed by referring to the processing methods of RRR and PRR in the“National Standard for processing of traditional Chinese Medicine”.The extraction and enrichment methods of RR were optimized according to the material basis of“black as lacquer”in the processed products of RR.The reaction products of cross-linking color and Maillard were enriched by porous resin column chromatography and dialysis,respectively.To system study the effects of processing on iridoid glycosides in RR,iridoid glycosides,nucleosides and phenylethanoid glycosides in the processed products of RR were analysed by UPLC-UV.In the processing of FRR–RRR–PRR,the content of iridoid glycosides decreased significantly,especially catalpol,digitalis D and geniposide.Focusing on the content changes of amino acids and sugars during the processing of RR,amino acids and sugars in the processed products of RR were determined by precolumn derivatization of UPLC and HPLC-ELSD.FRR were processed to PRR,basic amino acids（Arg,Lys,His）,acidic amino acids（Asp,Glu）and some neutral amino acids（Trp,Gln）decreased sharply,by more than 78%.Stachyose（tetrasaccharide）decreased from 562 mg/g to 42 mg/g,while the content of fructose,glucose（monosaccharide）and mannotriose（trisaccharide）increased significantly,which revealed that the hydrolysis of oligosaccharide to monosaccharide during high temperature steaming is the formation mechanism of“sweet as candy”in PRR.Focusing on the formation mechanism of“black as lacquer”of cross-linking color reaction,cross-linking color reaction of different types of iridoid glycosides with amino acids were systematically developed.Simulated the processing conditions of RRR（p H=6,50℃,β-glucosidase）,the cross-linking color reaction of cyclopentene（geniposide）,cyclopentane（leonuride）and epoxy ether（catalpol）iridoid glycosides with amino acids were conducted.The absorption curve of the sample solution in the wavelength range of 200–800 nm was determined by UV-Vis spectrophotometry.The results showed that genipin was generated from cyclopentene iridoid glycosides（geniposide）under the action ofβ-glucosidase,which was cross-linking with amino acids and showed a characteristic absorption peak at 600 nm,neither epoxy ether nor cyclopentane type iridoid glycosides reacted.Focusing on the formation mechanism of“black as lacquer”of Maillard reaction,caramelization reaction of oligosaccharides and monosaccharides and Maillard reaction with amino acids（Maillard reaction）were systemtically studied.Simulated the processing conditions of PRR（p H=4.8,100℃）,the absorption curve of the reaction products of caramelization and Maillard in the wavelength range of 200–800 nm were determined by UV-Vis spectrophotometry.It was found that oligosaccharides,monosaccharides and amino acids had no obvious absorption in the wavelength range of 200–800 nm,the reaction products of caramelization and Maillard have strong absorption peaks appeared in the range of200–800 nm(λ_max=280 nm),and part of the Maillard reaction products（Fru–Arg,Sta–Arg）appeared characteristic abosrption peak at 450 nm.In this experiment,three rapid quantitative methods for the determination of iridoid glycosides,amino acids and sugars of RR were established,and was applied to law of composition change in the processing of RR.The cross-linking color reaction,caramelization reaction and Maillard reaction rules were systematically studied.It is revealed that the scientific connotation of“black as lacquer,sweet as candy”of PRR,which provided the basis for the processing technology and quality control of RR.