Optimization Of Ultrahigh Pressure Assisted Micellar Extraction Of Dihydroquercetin From Larch And Preparation And Evaluation Of Cocrystals

Larix gmelinii is mainly distributed in the forests of Siberia,Canada,North America and the Great and Small Khing’an Mountains in northeast China,and its xylem is one of the main raw materials for dihydroquercetin extraction.Dihydroquercetin is a bioflavonoid,which is rich in phenolic hydroxyl groups,which can regulate the activity of a variety of enzymes in the human body,and then play different physiological effects,such as antioxidant,anti-tumor,antiviral,anti-cardiovascular system diseases,improve capillary microcirculation,anti-platelet aggregation and other effects,and non-toxic and harmless to infants and young children,and will not produce side effects such as developmental malformations,allergies and mutations to the fetus.However,the existing common extraction and purification methods of dihydroquercetin in larch often have the disadvantages of long extraction time,high energy consumption,cumbersome operation and large amount of solvent,which has brought great obstacles to the large-scale industrial production of dihydroquercetin.In addition,dihydroquercetin as a health product and food additive is mainly used orally.However,its poor water solubility and low oral bioavailability seriously limit the application of its oral preparation in biomedicine.In view of the above problems,this study selected Larix gmelinii as the extraction raw material,and established an efficient,green and simple extraction and purification process.In addition,two kinds of dihydroquercetin cocrystal have been developed.The antioxidant capacity and dissolution capacity in vitro were evaluated.The specific results are as follows:（1）Ethanol thermal reflux method were used to extract different parts of larch（trunk,root,root whisker,sawdust）to determine the total content of dihydroquercetin.The content of dihydroquercetin in tree trunk is the highest,with dry weight of 2.8%,wet weight of 2.38%and water content of 20.12%.With the extraction rate as the index,the surfactant used in the extraction was screened,and finally the tea saponin with the highest extraction rate（78.21%）was selected as the extraction micelle,and the tea saponin was natural,environmentally friendly,cheap and easy to obtain.Combined with single factor and response surface methodology,the ultra-high pressure assisted extraction process of tea saponins was optimized:the ratio of material to liquid was 1:11.5,the extraction pressure was 157MPa,the micelle concentration was 8%,the extraction times were 3 times,and the holding time was 5 min.Under this condition,the extraction rate of 84.35±1.20%was achieved,which was basically consistent with the predicted 84.98%.It shows that this extraction method is efficient,stable,simple and green.（2）The separation and purification of dihydroquercetin was realized by using chitosan flocculation,combined with organic reagent extraction and anti-solvent recrystallization.The optimal purification process was determined by single factor method:2%chitosan addition,ethyl acetate extraction,and the ratio of anti-solvent to main solvent was 1:150.Under the optimum conditions,the purity of dihydroquercetin can reach 95.1%.With the help of Zeta potential detection and fluorescence probe method,the mechanism of this purification method was clarified:chitosan（negative charge）interacted with the tea saponin micelle（positive charge）coated with dihydroquercetin,flocculated and precipitated,making the dihydroquercetin separated from the solution system and realized separation and purification.The product was characterized by HPLC,¹H-NMR,FTIR,DSC,TG and XRD.The results showed that the product was highly consistent with dihydroquercetin in terms of material composition,morphology,molecular structure,thermodynamic properties and crystal structure.It was comprehensively verified that the product obtained after extraction and purification was the required dihydroquercetin.It shows that this purification process can be used as a highly operational,environmentally friendly,efficient and rapid method to purify dihydroquercetin.（3）Dihydroquercetin-carbamazepine cocrystal and dihydroquercetin-isoleucine cocrystal were prepared by solvent volatilization,using the extracted and purified dihydroquercetin as the active ingredient,and using carbamazepine and isoleucine as ligands.FTIR showed that no covalent bond was formed during cocrystal formation,and its molecular structure did not change significantly compared with dihydroquercetin and ligand,but it was bound by intermolecular hydrogen bond;DSC/TG verified that the thermodynamic properties of the cocrystal changed significantly compared with dihydroquercetin and their respective ligands.The characteristic exothermic peak of dihydroquercetin-carbamazepine cocrystal changed to220℃,and the characteristic exothermic peak of dihydroquercetin-isoleucine cocrystal changed to 209℃.At the same time,it was verified that the optimal ratio of dihydroquercetin and ligand was 1:1;Through XRD characterization,in addition to retaining some of the characteristic diffraction peaks of dihydroquercetin and ligand,the two cocrystal crystals also generated new diffraction peaks,which proved that dihydroquercetin and ligand formed a new crystal structure through intermolecular hydrogen bonding.The IC₅₀of dihydroquercetin-carbamazepine cocrystal and dihydroquercetin-isoleucine cocrystal were 9.2×10^-3mg·m L^-1,8.8×10^-3mg·m L^-1respectively by DPPH free radical scavenging and in vitro dissolution test,both lower than dihydroquercetin body and their respective ligands,and their antioxidant capacity has been improved.In artificial gastric juice and artificial intestinal juice,the release rates of the two kinds of cocrystal are:dihydroquercetin-isoleucine cocrystal>dihydroquercetintechnical>dihydroquercetin-carbamazepine cocrystal.This is because isoleucine is hydrophilic,while carbamazepine is insoluble in water.They form cocrystal with dihydroquercetin through intermolecular hydrogen bonds.In the process of release,isoleucine can drive dihydroquercetin and promote its dissolution.On the contrary,carbamazepine’s hydrophobicity reduces the dissolution ability of dihydroquercetin.With the help of cocrystal technology,the antioxidant capacity of dihydroquercetin can be further enhanced,and the water solubility of dihydroquercetin can be improved,so as to improve its bioavailability.