Study On Enzymatic Synthesis,Digestion And Absorption Characteristics,Biological Activities And The Involved Mechanisms Of Puerarin Esters

Puerarin（PU）is the major active component derived from the root of Pueraria lobata（Willd.）Ohwi used as a kind of medicinal and edible plant.PU possess a number of pharmacological effects,such as protecting cardiovascular system,antioxidant,lowering blood-lipoids and anti-hyperglycemic.However,PU can hardly penetrate the epithelial cells of the small intestine,showing low digestion and absorption characteristics and oral bioavailability.These drawbacks significantly limited its activity and applications in food and pharmaceutical industries.In order to solve the above problems,a biocatalysis system was established to obtain puerarin esters（PAES）with different chain lengths using ionic liquids and acyl donor with different chain lengths.Then,their in vitro digestion and transport profiles,absorption and tissue distribution characteristics in rats,and their modulation on gut microbial composition and short chain fat acid（SCFA）were revealed.Furthermore,the protective effect and mechanism of PAES on microglia in oxygen-glucose deprivation and reperfusion（OGD/R）model were investigated.The results are as follows:（1）The feasibility of enzymatic synthesis of PAES in ionic liquids was explored.Among six ionic liquids,1-Ethyl-3-Methylimidazoliumacetate（[Emim][OAc]）was selected for advantageous reaction media.Among various sources of lipase,Novozym 435,derived from Candida.antarcita,was identified as advantageous lipase with the highest catalytic activity.The optimum reaction conditions for PAES were verified as oscillatory reaction time（200r/min）6 h,enzyme dosage 2 mg/m L,reaction temperature 50℃ and mole ratio of vinyl esters to PU 30:1 in the[Emim][OAc].Based on previous research,HPLC,HRMS and ¹³C-NMR were used to determine the structure of synthesized PAES.Moreover,the chemical stability of PU and PAES in aqueous solutions with different pH had been further explored.It was found that PAES showed the same stability as PU under acidic conditions,while PAES were unstable under alkaline conditions by hydrolysis reaction.Partial hydrolysis of PAES began when pH value reached 7.4.When the pH value was higher than 8,the recovery of puerarin hexanoate ester was less than 10%.（2）The digestive and transport profiles of PAES with different chain lengths was investigated using an in vitro digestion model and Caco-2 cells model.The results suggested that PU and PAES remained stable in gastric phases,whereas,different degrees of hydrolysis of PAES were found in the intestine digestion.PAES with less than 12 carbon chains were positively correlated with degree of hydrolysis,while those with more than 12 carbon chains（puerarin laurate ester and puerarin myristate ester）showed higher resistance to hydrolysis by the artificial human digestive juice.In the absorption and transport experiment,the absorption rate of PU in 2 h was 1.31%,and the absorption rate of puerarin hexanoate ester was 2.49times higher than that of PU.After a 2-hour transport in Caco-2 cells model,the P_app values of PAES increased with the increase of acyl donor chain lengths.The reason is that PAES with medium-long chain lengths can be more easily distributed on the surface of intestinal epithelial cell membrane due to their higher lipid solubility.（3）The relative oral absorption,including pharmacokinetics,tissue distribution after oral taking of PAES in the Sprague-Dawley（SD）rats was studied.Compared with PU,the PAES administration with different chain lengths resulted in significantly higher levels of PU and longer elimination time in the plasma and other tissues,especially puerarin myristate ester（PM）.Compartmental model and statistical moment calculation showed that the peak plasma concentrations of PM was 7.10±0.36μg/m L at 24h,which was 2.18 times that of PU.The area under curve（AUC）values of PM was 138.94±6.14μg/m L*h,which was 6.09 times higher than that of PU.Interestingly,the existence of PM could be detected in the blood and excreta.Moreover,the concentration of PU experienced the biggest growth after administration with PAES in the brain compared with pharmacological information in other tissues.The AUC of PM in cerebral cortex,hippocampus and corpus striatum were increased remarkably by 48.51,42.36 and 89.48 times than that of PU,which suggested the esters may be in favor of treating cardiovascular and cerebrovascular diseases.（4）New potential targets and pathwats of PAES on ischemic stroke were identified using network pharmacology.A total of 75 and 1432 related targets of PAES and ischemic stroke,respectively,were identified in the databases and and Swiss Target Prediction Data Bank.18common targets of PAES and ischemic stroke were obteined by Venn diagram.GO and KEEG analysis obtained 16 biological processes（BP）,4 cell composition（CC）and 3 molecular functions（MF）.Acoording to the 152 KEGG pathways,the main signaling pathways between PAES and ischemic stroke were ERK,P13K,RAS,TNF,IL-17,and NF-κB signaling pathway.（5）The protective effect and mechanism of PAES in the BV-2 cells were systematically investigated,performed by oxygen and glucose deprivation（OGD）and oxygen and glucose deprivation-reoxygenation（OGD/R）.These models mimic the different ischemic stroke insults.In the oxygen-glucose deprivation（OGD）model,the viability of BV-2 cells was significantly increased upon PAES pretreatment.PAES pretreatment suppressed the production of inflammatory cytokines and significantly promoted the polarization of BV-2cells to the M2 phenotype.In the OGD/R model,PAES suppressed the expression of cleaved-caspase 3 and decreased the Bax/Bcl-2 ratio,resulting in the inhibition of mitochondrial disorders and apoptosis induced by OGD/R.PAES,by activating the Nrf2/Akt pathway,up-regulated the expression of SOD1 and inhibited the oxidative stress response.Furthermore,PAES treatment significantly decreased the levels of TNF-αand IL-1βin the supernatant and downregulated the expression of MMP2 and MMP9,which alleviated the OGD/R-induced inflammatory response via P38-JNK signaling pathway and NF-κB translocation.（6）The modulation of PU and PAES on gut microbial composition and SCFA were compared.CK group,PU group and PAES group（PP,PH and PM groups）shared 294 OTU.Compare with PU group,the PAES group had a significantly higher gut microbiota abundance based on the values of Chao and ACE.The values of Shannon and Simpson indicated that the diversity of the gut microbiota decreased with the increased chain lengths of PAES.At the phylum level,the relative abundance of Firmicutes,Acidobacteria and Actinobacteria decreased in PAES group,while Bacteroides and Verrucomicrobia phylum increased.At the order level,compared with PU group,the relative abundance of Clostridiales,Bacteroidales and Verrucomicrobiales increased in the PAES group（p<0.05）,while the relative abundance of Bifidobacteriales,Coriobacteriales and Enterobacteriales decreased（p<0.05）.At the genus level,supplementation by puerarin hexanoate ester was found to enrich Lactobacillus（45.88%）,Clostridium Xl Va（7.71%,p<0.05）,Sporobacter（7.84%,p<0.01）,Ruminococcus（5.27%,p<0.05）,Allobaculum（4.44%,p<0.01）,Blautia（2.17%）.Compared with PU group,the concentrations of acetic acid,propionic acid and butyric acid were significantly increased in the PAES group.Moreover,supplementation by puerarin propanoate ester group showed the capacity to deliver specific propionic acid to the colon,while PAES with medium-long chain lengths had more opportunity to regulate the composition of gut microbiota for enhancing the short chain fat acid production.In summary,aiming at the problems presented in the application of PU,this study explored the biocatalysis system of acylation reaction catalyzed by lipase in ionic liquid.Then,a series of PAES with different chain lengths were synthesized.Compared with PU,PAES showed significantly enhanced lipid solubility and higher resistance to hydrolysis by the artificial human digestive juice.The PAES administration resulted in significantly higher levels of PU and longer elimination time in the plasma and other tissues,regulated the composition of gut microbiota,enhanced the short chain fat acids production and resisted OGD and OGD/R induced BV-2 cells apoptosis.