Study On The Differences Between "Honey-fried" And "Fried" Licorice And The Honey Frying Mechanism Based On The "Natural Deep Eutectic Solvent" Characteristic Of Honey

The development of traditional Chinese medicine compound preparation is an important direction of traditional Chinese medicine(TCM)industry.However,there are many common key problems to be solved in the development and research of classic recipes,such as the processing of decoction pieces.Due to the evolution of processing methods,the updating of processing equipment and the continuous development of TCM theory,there are great differences between the results of ancient textual research and modern clinical application."Prepared licorice" is the most representative of the main processing problems.60%of the more than 110 prescriptions in "treatise on Febrile Diseases",the core source of classic recipes,contain "prepared licorice",but its processing method is not clear."Prepared licorice" developed gradually from "fried licorice" in the Han Dynasty(A.D.25 to 220)to "honey fried licorice" in modern clinical medicine.In order to clarify the similarities and differences of different processed licorice and promote the development of high quality of classic famous compound preparations.In this study,the differences of different processed products of licorice were analyzed based on the ingredients and pharmacodynamic indexes,and the mechanism of honey fried licorice was discussed from the perspective of natural ionic liquid and deep eutectic solvents(HADES).The purposes of this study is to face up the "prepared licorice" processing way is not clear in the classical prescription that this urgent problem,clarify "fried licorice","dipped water fired licorice","honey fried licorice" material basis and pharmacological effects of similarities and differences and its causes,from two aspects of chemical benchmark and pesticide effect of benchmark scientific answer the similarities and differences among different "main licorice".To promote the development of the processing of Chinese Materia,to provide scientific basis for the correct choice of"processed licorice",to promote the development and utilization of classical prescription.In the overview section,four aspects are discussed,namely,the literature on the preparation of licorice,the traditional efficacy of "roasted licorice",the chemical composition and pharmacological effects of licorice and the characteristics of natural ionic liquid and deep eutectic solvents(NADES)of honey.It is shown that there are three traditional forms of preparation of licorice:"stir-fried","dipped water fired" and"honey fried",and the traditional effects of licorice are closely related to immunomodulatory and anti-inflammatory effects.Modern pharmacological studies have shown that licorice has significant hepatoprotective effects.Research by domestic and foreign scholars has confirmed that honey is a natural ionic liquid and deep eutectic solvent(NADES),which has the advantageous effect of improving the solubility,extraction rate,stability and in vivo absorption of ingredients.Therefore,we used the NADES properties of honey as an important entry point to study the differences in chemical composition and pharmacological activity of "fried licorice","dipped water fired licorice" and "honey fried licorice".We also investigated the differences in the chemical composition and pharmacological activity of honey fried licorice.The overall chemical composition of different processed licorice:The differences in the content of the major components were detected using UV-visible spectrophotometer.The result showed that the contents of total polysaccharides and total flavonoids in groups W and F were basically the same,and the contents in groups H and N were basically the same,and the contents in group H were higher than those in group F.The small molecule compounds of raw licorice,fried licorice,dipped water fired licorice and honey fried licorice were compared separately based on UHPLC-Q-Orbitrap metabolomics technique to find out the variability of their constituent contents.The metabolomic study showed that the chemical composition of W and F was basically the same,and the chemical composition of H and N was basically the same.46 components differing between R,F and H groups were identified.These included 21 flavonoids,8 saponins,9 small molecule sugars and their derivatives,and 8 other compounds.After that,18 compounds(including flavonoids,saponins,small molecule sugars and their derivatives)were quantified by target analysis with UHPLC-QqQ-MS.The results showed that the content of flavonoids,saponins and small molecule sugar components of licorice significantly changed after being treated with different processed.The content of dihydroflavonoid components(liquiritin apioside,liquiritin,liquiritigenin,6"-O-acetylliquiritin apioside,6"-O-acetylliquiritin)was decreased,while the content of chalcone components(isoliquiritin apioside,isoliquiritin,6"-O-acetylisoliquiritin apioside,6"-O-acetylisoliquiritin)increased,it is inferred that the chemical components in licorice may have undergone isomerization reactions as well as glycoside bond breaking reactions during the heat process,and the addition of honey would inhibit this chemical component transformation due to thermal processing,thus acting as a stabilizer of the compounds in licorice.Pharmacokinetic analysis of active ingredients:UHPLC-QqQ-MS was used to quantify the active ingredients in the blood of mice from different processed licorice groups to clarify the effects of different processing methods(fried and honey-fried)on bioavailability of of the active ingredients of licorice.The in vivo exposure of liquiritin apioside,liquiritin,liquiritigenin,and glycyrrhetinic acid were significantly increased after honey frying of licorice,and the pharmacokinetic curves of liquiritin and liquiritigenin in the honey-fried licorice group showed a double peak phenomenon.Furthermore,on the basis of raw licorice decoction,honey or NADES was added to verify the promotion effect of honey on the in vivo absorption of the active ingredients of licorice.The bioavailability of liquiritin apioside,liquiritin,liquiritigenin,and glycyrrhetinic acid was detected to be increased in both H and N groups,suggesting that the NADES properties of honey could facilitate the absorption of the active compounds of licorice.The pharmacological effects of raw licorice,fried licorice,honey fried licorice and NADES fried licorice were mainly evaluated in the following three aspects:comparison of the spleen tonifying effect and intestinal flora regulating effect;comparison of the anti-acute inflammatory and anti-acute liver injury effects.In vivo immune assays showed that honey fried licorice significantly increased the phagocytic index,spleen index and thymus index in mice compared with raw licorice and fried licorice,and enhanced the immunomodulatory effects of licorice by increasing the phagocytic capacity of macrophages and improving spleen and thymus functions.The results of intestinal flora assay showed that honey fried licorice significantly altered the species diversity and abundance of intestinal flora in mice compared to raw and fried licorice.The anti-inflammatory assay also showed that honey fried licorice could significantly inhibit the ear swelling induced by croton oil in mice.The results of liver injury experiment showed that compared with raw licorice,both fried licorice and honey fried licorice could reduce the abnormal increase of ALT and AST levels in serum and liver caused by carbon tetrachloride,and reduce the degree of liver swelling;especially the effect of honey fried licorice was more obvious and stronger than fried licorice.The data of metabolomics screened out the content measurement of different components constituted the "chemical data" collection,and the pharmacological index data constituted the "pharmacological effect" data set,and the binary correlation analysis and linear-regression analysis were performed by SPSS-22.0 to find out the chemical components that were highly correlated with pharmacological index,and these components were the potential material basis for the different effects of licorice.The compounds that positively correlated with immunomodulatory effects was glycyrrhetinic acid.The compounds that negatively correlated with immunomodulatory effect were acetyl compounds(22β-acetoxylglycyrrhizic acid,6"-O-acetylliquiritin apioside,6"-O-acetylliquiritin)uralsaponin B,and liquiritigenin.Glycyrrhizic acid may be the main active component exerting anti-inflammatory and hepatoprotective effects,while isoliquiritin may also be positively correlated with hepatoprotective activity.Uralsaponin B,liquiritin,and acetyl compounds(22β-acetoxylglycyrrhizic acid,6"-Oacetylliquiritin apioside and 6"-O-acetylliquiritin)were negatively associated with hepatoprotective activity.The physiological status of mice after 7 days of administration in pharmacological experiments was constructed to quantify the in vivo blood concentrations of the active index components in different groups of mice.To observe the accumulation concentration of the indicator component in vivo after longterm administration.Quantification of the accumulation concentrations of liquiritin,liquiritigenin,isoliquiritin,glycyrrhizic acid and glycyrrhetinic acid in vivo after multiple doses showed that the exposure of liquiritin,liquiritigenin,isoliquiritin and glycyrrhetinic acid in vivo increased in the H/N group after 7 days of continuous dosing,but the increase of liquiritin was not statistically significant.The accumulation concentration of glycyrrhizic acid was significantly decreased in the H/N group.It is suggested that liquiritigenin,isoliquiritin and glycyrrhetinic acid may be the key components for the enhancement of pharmacological activity of honey-fried licorice.All the above experimental results showed no significant difference between honey fried licorice and NADES fried licorice,suggesting that the potency enhancement of honey fried licorice is mainly related to the NADES structure of honey.The NADES fraction of honey has the ability to improve the extraction rate of medicinal components,enhance the stability of medicinal components during heating and concoction,promote the in vivo absorption of medicinal components and improve their biological activity.Further experimental results based on FT-IR technique to analyze the interaction between the NADES structure of honey and the pharmacodynamic components of licorice revealed the existence of multiple interaction forces between NADES and the active ingredients to improve the extraction rate,stability,and bioavailability of the active ingredients in licorice.This study shows that there is no difference between the historically existing "dipped water fired licorice" and "fried licorice",fried licorice and honey fried licorice have different pharmacological activities and different pharmacological substance bases,so clinically they should be used separately.In terms of regulating immune function,antiinflammatory and liver protection,honey fried licorice is preferred.The mechanism of honey roasted licorice potentiation may be related to the NADES property of honey which has the ability to improve the solubility,extraction rate,stability and in vivo absorption of ingredients.