Preparation Of Anthocyanin Monomers By Medium Pressure Rapid Separation System And Their Structure-Activity Relationships And Mechanisms On Inhibiting Fluorescent Glycation End Products

Recently,the biological activities of anthocyanins have attracted more and more attention.However,the current common methods for separating and purifying anthocyanins have some disadvantages and great limitations,namely time-consuming,low efficiency,high cost and difficulty in obtaining high amounts of high-purity anthocyanin monomers in a short period.At the same time,with the research on advanced glycation end products?AGEs?,the accumulation of AGEs has a great relationship with diabetes,Alzheimer's syndrome and aging.At present,the AGEs inhibitors used in medical research,such as aminoguanidine and aspirin,have toxic side effects.Therefore,the inhibitory effect of natural extracts on AGEs has been spotlighted.Based on the above problems,this dissertation established methods for rapid separation and purification of anthocyanins with different structures and explored the structure-activity relationships and inhibition mechanisms of anthocyanins with different structures against fluorescent AGEs by simulating different glycosylation stages in vitro models.The main findings were as follows:?1?Preparation of different anthocyanin monomers by medium pressure rapid separation systemMethods for rapid separation of seven anthocyanins from mulberry,red raspberry,strawberry,roselle,and black wolfberry fruits were established.The preparative column was flash C18?80 g,25 35 ?m,100 A?,using 2% aqueous formic acid and methanol as mobile phases,and the sample loading was 300 mg,with the gradient program for mulberry and red raspberry of: 0 2 min,20%B;22 min,20% 30%B;22 32 min,30% 40%B;and for strawberry and roselle of: 0 2 min,20%B;2 22 min,20% 30%B,22 32 min,30% 40%B,32 47 min,40% 45%B;and for black wolfberry of: 0 2 min,20%B;2 22 min,20% 30%B;22 32 min,30% 40%B;32 47 min,40% 45%B;47 57 min,45% 60%B.The separation and purification of seven structurally different anthocyanins were achieved.The purity of cyanidin 3-glucoside and cyanidin 3-rutinoside from mulberry reached 95.0% and 41.0%,respectively;while the purity of cyanidin-3-sophoroside and cyanidin-3-glucoside from red raspberry reached 60.0% and 75.0%,respectively;and the purity of pelargonidin-3-glucoside from strawberry reached 60.0%.Likewise,the purity of delphinidin-3-glucoside,delphinidin3-sambubioside and cyanidin-3-glucoside from roselle reached 26.0%,44.0% and 30.0%,respectively.Meanwhile,the purity of petunidin-3-rutinoside-?p-coumaryl?-5-glucoside from black wolfberry reached 60.0%.Apart from cyanidin-3-glucose,the other six anthocyanins with different structures were further purified to more than 90.0% using semi-preparative high-performance liquid chromatography.?2?The structure-activity relationships and inhibition mechanisms of anthocyanins with different structures against fluorescent AGEs in glucose-?-lactoglobulin simulation modelHalf inhibitory concentration(IC₅₀)values for the inhibition of fluorescent AGEs of seven structurally different anthocyanins were determined in glucose-?-lactoglobulin simulation model.The results showed that seven anthocyanins with different structures had significant inhibitory effects on fluorescent AGEs,with the subsequent inhibition order: petunidin-3-rutinoside-?p-coumaryl?-5-glucoside > cyanidin-3-sophoroside,delphinidin-3-sambubioside,cyanidin-3-rutinoside > pelargonidin-3-glucoside,delphinidin-3-glucoside > cyanidin-3-glucoside.The structure-activity relationships showed that in glucose-?-lactoglobulin model,anthocyanins with larger molecular weight had better effect on inhibiting fluorescent AGEs;when anthocyanins were substituted with diglycosides,the type of glycosyl had no significant effect on the inhibition of fluorescent AGEs;when the type of glycosyl substitution is the same,anthocyanins with only the catechol hydroxy structure in the B ring had the weakest effect on inhibiting fluorescent AGEs.Various indicators such as glucose,free lysine,free arginine,protein oxidation products,glyoxal,methylglyoxal,carboxymethyllysine and other indicators were measured in glucose-?-lactoglobulin model during the glycosylation reaction using phenol sulfuric acid,ophthalaldehyde,fluorescence microscopy,high performance liquid chromatography and dodecyl sulfate sodium salt-polyacrylamide gel electrophoresis approaches.The results showed that after 7 days of incubation at 37 °C,the highest levels of glucose,free lysine and free arginine were found in the anthocyanin-added group with values of 6.9,3.4 and 1.6 times of the blank group.The highest inhibition of anthocyanins on the formation of glyoxal,methylglyoxal and carboxymethyllysine were 90.3%,77.2% and 66.2%,respectively.The above results indicated that anthocyanins had massive inhibitory effects on the early glycation reaction of carbonylamine,the middle and late products.The results of molecular docking showed that anthocyanins with larger molecular weight interacted more glycation sites with protein,more effectively shielded the carbonyl compounds and inhibited the glycosylation reaction.?3?The structure-activity relationships and underlying mechanisms of anthocyanins on fluorescent AGEs in glyoxal-?-lactoglobulin and methylglyoxal-?-lactoglobulin modelsIC₅₀ values of seven anthocyanins for inhibiting fluorescent AGEs were measured in glyoxal-?-lactoglobulin and methylglyoxal-?-lactoglobulin models.The inhibitory effect of seven structurally different anthocyanins on fluorescent AGEs in glyoxal-?-lactoglobulin model was as follow: petunidin-3-rutinoside-?p-coumaryl?-5-glucoside > cyanidin-3-sophoroside,delphinidin-3-sambubioside,cyanidin-3-rutinoside > delphinidin-3-glucoside > pelargonidin-3-glucoside > cyanidin-3-glucoside.The structure-activity relationships showed that in glyoxal-?-lactoglobulin model,anthocyanins with larger molecular weight had better effect against fluorescent AGEs;when anthocyanins were substituted with diglycosides,the type of glycosyl had no significant effect on the inhibition of fluorescent AGEs;when the type of glycosyl substitution was the same,anthocyanins with pyrogallol structure had the best effect on inhibiting fluorescent AGEs.The inhibitory effect of seven structurally different anthocyanins on fluorescent AGEs in methylglyoxal-?-lactoglobulin model was as follow: petunidin-3-rutinoside-?p-coumaryl?-5-glucoside > delphinidin-3-sambubioside > cyanidin-3-rutinoside > cyanidin-3-sophoroside > delphinidin-3-glucoside > cyanidin-3-glucoside > pelargonidin-3-glucoside.The structureactivity relationships showed that in methylglyoxal-?-lactoglobulin model,anthocyanins with larger molecular weight had better effect on inhibiting fluorescent AGEs;when anthocyanins with the same core and substituted with diglycosides,anthocyanins with rutinoside substitution were more effective on inhibiting fluorescent AGEs than anthocyanins with sophoroside substitution;when the type of glycosyl substitution is the same,anthocyanins with pyrogallol structure had the best effect on inhibiting fluorescent AGEs,followed by anthocyanins with catechol structure.The key indicators for different stages of glycosylation in the two simulated models,including free lysine,free arginine,glyoxal,methylglyoxal,protein oxidation products,thiol,protein carbonyl,protein molecular weight and non-fluorescent AGEs were determined.The results showed that anthocyanins could shield the glycosylation sites of ?-lactoglobulin and reduce the consumption of free lysine and free arginine.The removal rate of glyoxal and methylglyoxal could be as high as 67.4% and 49.2%,separately,preventing the formation of protein cross-linked products and inhibiting protein carbonyl and carboxymethyllysine up to 52.9% and 92.8%,respectively.UPLC-Q-TOF-MS analysis showed that glyoxal and methylglyoxal could be trapped by anthocyanins,forming mono-adducts or di-adducts,thereby inhibiting the glycosylation...