Study Of Functional Ingredients Of Akebia Trifoliata Pericarp And Its Anti-inflammatory Mechanism

Akebia trifoliate（Thunb.）Koidz.var.australis（Diels）Rehd,belonging to the family Lardizabalaceae and genus Akebia.Its fruit looks like a small bunch of thick bananas,cracked open longitudinally on lunar August,commonly called‘Ba Yue Gua’.It is widely distributed in North China,North South China,southeast coast and Yangtze River Basin.It has been transformed from wild fruit to base cultivation and is being widely developed as a new type of fruit resource.‘Ba Yue Gua’has been formed In Jiangxi,Hunan,Guizhou and other places,and related fruit wine,jam,fruit tea and other products have been developed.The fresh fruit of Akebia trifoliata has sweet taste and high nutritional value,but its peel accounts for more than 40%of the weight of the whole fruit.It is often discarded as waste,which not only pollutes the environment,but also wastes resources.In addition,Lardizabalaceae plant has been used as traditional Chinese medicine since ancient times,which has the functions of clearing liver,regulating qi,activating blood and relieving pain,dispersing knot and diuresis.At present,researches about Akebia mainly focus on the content of total active components and distribution in different parts,isolation and purification of single compound,in vitro antioxidant,anticancer,andα-glycosidase activity inhibition,etc.Information about the rapid qualitative and quantitative analysis of its main active components,the metabolism pattern in vivo,and the mechanism of its physiological activities such as anti-inflammatory remains deficient.Therefore,in the present study,the pericarp of Akebia was used as raw material,and the changes of its active components in the process of digestion and absorption were analyzed by both in vitro and in vivo models.The anti-inflammatory activity and possible mechanism of Akebia were explored by cell and animal inflammatory models.The development of this project will provide novel ideas and theoretical basis for the development of related products of Akebia pericarp,which is conducive to the effective and rational use of Akebia pericarp resources,reduce the generation of waste,and contribute to the long-term development of Akebia industry.The main results are as follows:1.The contents of total phenols,total flavonoids and total saponins in the three extracts were determined.The results showed that the content of 80%methanol extract was the highest,and the extraction yield was the highest.Then three different chemical antioxidant methods（DPPH,ABTS,FRAP）were used to screen the three extracts.The results showed that 80%methanol extract had the strongest antioxidant activity.Therefore,the chemical composition of 80%methanol extract was further studied qualitatively and quantitatively by UPLC-LTQ-Orbitrap-MS².Totally of 18compounds were identified including phenolics,flavonoids and saponins,the quantitative results showed that triterpenoids were the main active components.2.In vitro simulation digestion,Caco-2 monolayer cells models were used to evaluate the digestion、transport characteristics of the extract of Akebia pericarp（APE）.Results showed that（1）the total phenols,total flavonoids and total saponins in the pericarp extract of Akebia trifoliata decreased during digestion;（2）the transport efficiency of Caco-2 monolayer was different at different time points,taking 4-caffeoylquinic acid as an example,more than 60%of it can be transported in 0.5 h,and there is little change in the next 6 h.3.By analyzing the metabolites in urine and blood of rats fed with APE by gavage,the metabolic pattern of active components in APE in vivo was speculated.Results showed that ten prototypes were found in urine samples,and eight prototypes were found in blood samples,which can be considered as components absorbed by the body and excluded;a total of 42 metabolites were identified,including 39 in urine samples and 8 in blood samples.The main metabolic modes were grade I metabolism,such as dehydrogenation,dehydration,dehydroxylation,demethylation and deglucose,and grade II metabolism,such as hydroxylation,methylation,glucuronidation and sulfation.4.LPS-induced RAW264.7 cell model was used to study the anti-inflammatory activity in vitro of APE.Results displayed that APE treatment inhibited the generation of TNF-α,IL-6,IL-1β,NO and PGE₂,as well reduced their m RNA expression levels.The proteins phosphorylation in NF-κB and MAPKs also restrained by APE in concentration dependent manner,therefore ameliorated inflammation.5.DSS-induced colitis mouse model was used to explore the anti-inflammatory activity in vivo of APE.Results showed that（1）the body weight,colon length were improved after APE administration,DAI score was reduced;（2）the production and m RNA expression levels of NO、PGE₂,TNF-α,IL-6 and IL-1βwere decreased in APE-treated groups;（3）the proteins phosphorylation in NF-κB and MAPKs were inhibited by APE;（4）the composition and abundance of gut microbiota was regulated by APE;the abundance of Lachnospiraceae and Prevotellaceae which related with inflammation were reduce by APE.6.HFD+DSS induced NAFLD mouse model with intestinal injury was applied to evaluate whether the active components of APE can alleviate intestinal inflammation.Results displayed that（1）reduced weight gain,increased colon length,decreased intestinal permeability and reduced colon tissue damage were observed in APE administrated groups;（2）the expression of MUC2 and ZO-1 protein were increased by APE intervention;（3）the levels of LPS,MCP-1 and TNF-αwere decreased;（4）transcriptome analysis showed that compared with HFD+DSS group,APE intervention up-regulated 38 genes,down-regulated 49 genes;the enrichment of GO and KEGG pathway showed that the differentially expressed genes were mainly enriched in the circadian process,My D88 dependent toll like receptor signaling pathway,environmental adaptation,signal molecules and interactions,immune system,lipid metabolism and so on.（5）The results of intestinal microbial sequencing showed that APE increased the diversity and composition of intestinal microorganisms.The results showed that the active components of APE may play an anti-inflammatory role by regulating gene expression and intestinal microbial composition.7.We further analyzed the regulatory effect of APE on liver injury of NAFLD mice with intestinal injury.Results showed that（1）APE improved the liver weight and impaired glucose tolerance of mice;（2）The contents of serum TG,TC,LDL-C and TBA were decreased;（3）The contents of ALT and AST were decreased;（4）The pathological condition and lipid accumulation of liver tissue were improved;（5）The differential genes are mainly related to circadian rhythm and lipid metabolism.The results showed that the active components of APE could regulate the liver injury induced by HFD+DSS in mice.