Study Of Identification And Intestinal Anti-inflammatory Activity Of A.cerana Honey And A.mellifera Honey

Apis cerana cerana Fabricius(A.cerana)honey is a naturally sweet substance produced by A.cerana that collect floral nectar,plant secretions or the excretions of sucking insects on plants from diverse nectar sources,particularly flowers of herbs or traditional medicines.So,it is very popular due to its unique source.The price of A.cerana honey is commonly higher than that of Apis mellifera ligustica Spinola(A.mellifera)honey because of its low yield.However,some honey traders fraudulently substitute A.mellifera honey as A.cerana honey to earn an illegal windfall profit,which has triggered a crisis of consumer trust in Chinese apiculture.However,to our knowledge,there is no systematic method to identify the authenticity of A.cerana honey,and no theoretical data to support the biological activity of A.cerana honey.Therefore,the differences of A.cerana honey and A.mellifera honey in HS-GC-IMS and UPLC Q-Exactive Orbitrap MS fingerprints were systematically compared,and the markers were screened and confirmed using amounts of real honey samples.Then the following research will be performed by identifying its production and metabolic mechanism,clarifying the intestinal antiinflammatory activity of A.cerana honey and its characteristic markers,and constructing the correlation between intestinal anti-inflammatory activity and characteristic markers.This study laid the foundation for establishing the quality evaluation system of A.cerana honey.The main contents and result are as follows:(1)The fingerprints and discriminant analysis models of A.cerana honey and A.mellifera honey were constructed,and the markers were screened based on an untargeted strategy.Firstly,the fingerprints of volatile and nonvolatile substances in honey were constructed based on HS-GC-IMS and UPLC QExactive Orbitrap MS.Secondly,PCA and OPLS-DA models were optimized to discriminate A.cerana and A.mellifera honey.Thirdly,the VIP of the OPLS-DA model was used to initially identify the different metabolites.The p-value and fold change of univariate analysis were also applied to further screen for differentially accumulated metabolites in honey.Thus,the untargeted analyses grouped honey sampled from A.mellifera and A.cerana origins.(2)A robust and sensitive method was established and confirmed for markers in honey.Firstly,two quantitative analysis methods of HS-SPME-GC-MS and SPE-HPLC-MS/MS were established by optimizing pre-treatment and instrumental analysis conditions.The method performance results showed the developed methods were suitable for quantitative analysis of characteristic markers in honey.Finally,3-amino-2-naphthoic acid,methyl indole-3-acetate,1-nonanol,1-heptanol,and phenethyl acetate could be considered markers of A.cerana honey,as they were present in higher amounts in A.cerana honey than in A.mellifera honey,whereas kynurenic acid and phenylacetaldehyde was determined to be a marker of A.mellifera honey by determining amounts of real honey samples.And the content ranges of all markers were determined.(3)The origins of markers in A.mellifera and A.cerana honey were clarified.Firstly,it is speculated that tryptophan metabolism is a differential metabolic pathway of A.cerana and A.mellifera according to the markers in honey.Then,a method was developed for detecting the levels of tryptophan and ten metabolites in honey.Meanwhile,the contents of tryptophan and its metabolites in A.mellifera,A.cerana honey and nectar were determined,and the results confirmed there were significant differences in the contents of tryptophan and its metabolites in A.mellifera honey and A.cerana honey(p<0.05),indicating that there were differences in the tryptophan metabolism between A.mellifera and A.cerana.Kynurenine metabolism and serotonin metabolism were stronger in A.mellifera,while indole acid metabolism was dominant in A.cerana.Thus,tryptophan metabolism of honeybee may be one of the sources of markers in A.mellifera honey and A.cerana honey.(4)The antioxidant activity and intestinal anti-inflammatory activity of honey and three markers(3-amino-2-naphthoic acid,methyl indole-3-acetate and kynurenic acid)were clarified.In vitro results showed that the DPPH free radicals and ABTS free radicals of 3-amino-2-naphthoic acid,methyl indole-3-acetate and kynurenic acid had high DPPH free radicals and ABTS free radicals scavenging activities,in which methyl indole-3-acetate was the strongest.And in vitro antioxidant activity of A.cerana honey was significantly higher than A.mellifera honey.Then the LPS-challenged Caco-2 cell model was applied to investigate the anti-inflammatory activity and intestinal epithelial cell protection of honey and its markers.The results showed honey and three markers including 3-amino-2-naphthoic acid,methyl indole-3-acetate and kynurenic acid could down-regulated inflammatory expressions,and up-regulated tight junction and antioxidant expressions to exert intestinal anti-inflammatory activity.Correlation analysis showed that kynurenic acid,methyl indole-3-acetate and 3-amino-2-naphthoic acid may play an important role in antioxidant and intestinal anti-inflammatory activity in honey.