Study On The Effect Of Hordei Fructus Germinatus After "Stir-frying" On Ingredients Content And Intestinal Absorption

Hordei Fructus Germinatus（HFG）is a common digestion drug,what has been used in the tradition of"stir-frying".Compared with raw HFG,the effect of resolving food stagnation in fried HFG is stronger.At present,the HFG after"stir-frying"mostly uses digestive enzymes and flavonoids as evaluation indicators.Modern research shows that the activity of digestive enzymes after"stir-frying"is inevitably reduced due to the influence of heat.Meanwhile the flavonoids and the"stir-frying"are also inextricably linked.HFG containsreducing sugars,amino acids and other"inactive ingredients"in the traditional sense.Maillard reaction may occur during the frying process,and the Maillard reaction products（MRPs）with"fragrance"is produced.Therefore,the HFG"stir-frying"process may be more related to the"inactive ingredients"in the traditional sense such as reducing sugars,amino acids,and MRPs.According to the theory of traditional Chinese medicine,the HFG after"stir-frying"can achieve the effect of"awakened spleen",that is"stir-frying and awakened spleen".Spleen governs transportation and transformation.Therefore"stir-frying and awakened spleen"can be considered as fragrant substance to restore the function of transportation and transformation of spleen,and promoting the absorption of nutrients and medicinal ingredients.This project investigated the change regulation of the content of"inactive ingredients"in the traditional sense including reducing sugar,amino acid,acrylamide,MRPs,5-HMF and"active ingredients"such as tricin,ferulic acid,catechins,quercetin,kaempferol,and evaluating the"stir-frying"process of HFG and determining the end point of the frying by HCA and PLS-DA method.On this basis,studying the effect of"stir-frying"on intestinal absorption of index components based on in vitro models,in vivo models and whole animals.And the mechanism of"stir-frying and awakened spleen"was further discussed from the perspective that MRPs promoted Intestinal absorption of ferulic acid and other"active ingredients".The main research contents are as follows:1 Effect of HFG after"stir-frying"on ingredientsHPLC was used to determine the content of Flavonoids,Quercetin,Kaempferol,Catechin,Ferulic acid,5-HMF and Acrylamide;HPLC-ELSD was used to determine the content of D-fructose,D-glucose,sucrose and D-maltose;PITC pre-column derivatization-HPLC was used to determine the content of Thr,Ala,Gly,His,Ile,Arg,γ-GABA,Tyr,Val,Pro,Met,Phe,Leu,Trp,Lys.to study the effect of Frying temperature and time on"active ingredients"（Flavonoids,Quercetin,Kaempferol,Catechin,Ferulic acid）,Maillard reactants（carbohydrate,amino acids）and Maillard reaction products（5-HMF,acrylamide,A420）.And clarifying the dynamics of each ingredients in the frying process and determine the end point of the frying by HCA and PLS-DA.The effect of the frying temperature on the active ingredients shows that the content of Flavonoids,Quercetin,Kaempferol,Catechin,Ferulic acid did not change significantly with frying temperature increasing.The effect of the frying temperature on MRPs including the sugar and amino acids showed that the contents of reducing sugar D-fructose,D-glucose,D-maltose and amino acids showed a downtrend on the whole with temperature increasing,and non-reducing sugar such as sucrose increased firstly and then decreased.The effect of the frying temperature on MRPs including5-HMF,Acrylamide and Browning shows that 5-HMF and Acrylamide are not produced when the frying temperature is below 120°C.But the three components increase slowly when the temperature is higher than 140°C,while rapidly increasing to the maximum value and reaching the steady state when reaching a certain temperature.The above results show that the"stir-frying"has little effect on the"active ingredient"in the traditional sense,but has a greater influence on the"inactive ingredients"such as Maillard reactant and products.The results of HCA further showed that 10 samples can be divided into raw malt and 90-140°C,160-180°C,200-220°C,240-260°C.PLS-DA analysis showed that the VIP value of sucrose and Maillard reaction products 5-HMF,acrylamide and A420 was larger during the frying process,which had a significant contribution rate to distinguish different temperature samples of HFG.And the contribution ratios of"active ingredients"and Maillard reactants are small.220°C was selected as the best frying temperature according to the HCA results and the pharmacological effects of ingredient with large VIP,and combined with the"color"and"fragrance"of fried HFG.The effect of the frying time on the active ingredients shows that the content of Flavonoids,Quercetin,Kaempferol,Catechin,Ferulic acid did not change significantly with frying time increasing.The effect of the frying time on MRPs including the sugar and amino acids showed that with the extension of frying time,the contents of four carbohydrates and amino acids decreased,and the contents of carbohydrates reached steady-state at 16 min and amino acids reached steady-state at8 min.The effect of the frying time on MRPs including 5-HMF,Acrylamide and Browning shows that while 5-HMF and A420 increased with the increase of the frying time,the content remained basically unchanged.Acrylamide began to be produced and gradually increased after frying 20 min.The results of HCA showed that 13 samples can be divided into raw malt,2-4 min,6 min,8-14 min,16-26 min.According to the Pharmacological effects of 5-HMF,acrylamide and melanoid,choosing 16-18 min for the best time for frying.2 Effect of HFG after"stir-frying"on intestinal absorption of active ingredients2.1 Everted gut sac modelUsing everted gut sac model,and determining the contents of ferulic acid,catechin,kaempferol and quercetin in raw HFG（ferulic acid,catechin,kaempferol,quercetin+3 kinds of reducing sugars+15 kinds of amino acids）and fried HFG（ferulic acid,catechin,kaempferol,quercetin+MRPs）by HPLC to investigate the effect of"stir-frying"on four components.The results showed that the four components were absorbed in different intestinal segments of rats.Compared with the raw HFG,the Ka and Papp of quercetin and kaempferol in the fried HFG increased significantly in the duodenum,jejunum and ileum（p<0.05）.Meanwhile,the Ka and Papp of catechins in fried HFG increased significantly in the duodenum and jejunum（p<0.05）.Compared with raw HFG,ferulic acid of the fried HFG showed a significant increase in Ka and Papp in the ileum（p<0.05）.The results indicate that"stir-frying"can promote the absorption of active components of HFG to a certain extent in the small intestine.2.2 Situ single-pass intestinal perfusion modelUsing situ single-pass intestinal perfusion model,and determining the contents of ferulic acid,catechin,kaempferol and quercetin in raw HFG and fried HFG by HPLC to investigate the effect of"stir-frying"on four components.The results showed that compared with the raw HFG,the Ka and Papp of catechin,kaempferol and quercetin were significantly increased in duodenum,jejunum and ileum（p<0.05）,and the Ka and Papp of ferulic acid in the duodenum and jejunum were significantly increased（p<0.05）.The results futher indicate that"stir-frying"can promote the intestinal absorption of active components.Comprehensive comparison of Ka and Papp,the intestinal absorption of four ingredients is better in the duodenum.2.3 Whole animal modelUsing whole animal model,and determining the contents of ferulic acid,catechin,kaempferol and quercetin in the plasma of HFG by HPLC-MS/MS,drawing the concentration-time curve and compareing Ka,AUC_0-t,C_max and other pharmacokinetic parameters to investigate the effect of"stir-frying"the pharmacokinetics of the four components in the HFG.The results showed that compared with the raw HFG,the Ka,AUC0-t and Cmax of catechin,ferulic acid,quercetin and kaempferol in fried HFG increased significantly（p<0.05）.The result again shows that"stir-frying"can promote the absorption of active ingredients in HFG3 Study on intestinal absorption mechanism of active constituents in HFG3.1 Effect of concentration and verapamil,EDTA and mannitol on intestinal absorption of active ingredientsInvestigating the absorption mechanism of ferulic acid,catechin,kaempferol and quercetin in the small intestine by everted gut sac model.The results was that the absorption of kaempferol and quercetin did significant change with increasing concentration.It indicates that the intestinal absorption mechanism of kaempferol and quercetin is passive transport.Compared with the medium concentration,there was no significant change in the Ka and Papp of low concentrations of catechin and ferulic acid.However,with the further increase of concentration,the Ka and Papp of catechin and ferulic acid decreased significantly（p<0.05）,suggesting that there was saturation in the absorption of catechin and ferulic acid and there may be vector involvement in the transport process.There was no significant change in Ka and Papp of the ferulic acid,catechin,kaempferol and quercetin after adding verapamil and mannitol,showing that four ingredients were not substrates for P-gp and SGLT protein.After adding EDTA,the Ka and Papp of catechin,quercetin and kaempferol increased significantly（p<0.05）,indicating that its absorption of three components may be related to TJP.3.2 Effect of HFG after"stir-frying"on the expression of TJPBased on TJP mediated intestinal absorption of catechin,kaempferol and quercetin,studying the effect of HFG after"stir-frying"on the expression of Claudin-1 and ZO-1 in duodenum by western blot and q-PCR.The results of western blot showed that compared with the control group,the expression of ZO-1 and Claudin-1 in the raw HFG did not change significantly.Compared with the raw HFG,the expression of ZO-1 and Claudin-1 in fried HFG and MRPs was also significantly decreased（p<0.05）.The results of q-PCR showed that compared with the control group,the expression of ZO-1 and Claudin-1 mRNA did not change significantly in the raw HFG.Compared with the raw HFG,the expression of ZO-1 and Claudin-1mRNA was significantly decreased in the fried HFG and MRPs group（p<0.05）.The above results indicate that the HFG after"stir-frying"can"down-regulate"the expression of TJP,improve the efficiency of bypass absorption,and promote intestinal absorption.And this function may be related to MRPs.The research results of this project show that the HFG after"stir-frying"has a greater impact on"inactive ingredients"and less on"active ingredients".Therefore,maillard reaction products including 5-HMF,acrylamide,A420 and other traditional"inactive ingredients"is selected as the evaluation index of the"stir-frying"to determine the end point of the frying.It provides a new perspective for the research of Chinese medicine frying process.Meanwhile,MRPs formed after HFG"stir-frying"has a greater impact on the intestinal absorption of"active ingredient".It may be one of the mechanisms of"awakened spleen"in HFG.From the perspective of Maillard,this project studies the"stir-frying"of HFG and clarifies the mechanism of"stir-frying and awakened spleen".It provides new ideas for the research of traditional Chinese medicine processing and traditional Chinese medicine theory.