Identification And Analysis Of Bioactivity And Biological Function In Vitro Of Dihydrochalcone Compounds In Malus

Dihydrochalcone compounds(DHC)are the most abundant and various secondary metabolites in Malus.DHC not only have important biological functions in growth and development as well as stress-resistance of apple trees,but also have excellent antioxidant,anti-cancer,and hypoglycemic bioactivities.Most of the previous studies were limited to the two classic DHCs,phlorizin and phloretin.Research on other DHCs in Malus is still scarce.In this study,a series of DHCs were isolated and identified form Malus plants,then their antioxidant,antiproliferative of tumor cell lines,and hypoglycemic activities,as well as allelopathy were analyzed.The major results are as follows:1.Six DHCs were extracted,isolated and identified from Malus,and two DHC aglycones were prepared.Eight DHCs were isolated form 2 Malus plant fruits used combination of various chromatographys(high-performance liquid chromatography,liquid phase semipreparative chromatography,Sephadex,reverse chromatography,macroporous resin,and semi-preparation chromatograph).And identified 6 compounds using liquid chromatographymass spectrometry and nuclear magnetic resonance.These compounds were identified as phlorizin(P2G),trilobatin(P4G),3-hydroxyphlorizin(HP2G),sieboldin(HP4G),phloretin 2'-xyloglucoside(P2X)and 3-hydroxyphloretin 2'-xyloglucoside(HP2X).Then two DHC aglycones,phloretin(P)and 3-hydroxyphloretin(HP)were prepared.The contents of the 8 DHCs in the leaves and young fruit of 17 Malus accessions were determined,and 3 kind of germplasm resources with large-scale extraction and separation of DHCs were discovered.2.The mechanism of scavenging free radicals by dihydrochalcone compounds was clarified.The antioxidant capacity of P,P2 G,P4G,HP,HP2 G,and HP4 G was determined used ABTS and DPPH assays and then the antioxidant mechanism was analyzed.It was found that the DHCs could scavenge free radicals through hydrogen atom transfer(HAT),single electron transfer followed by proton transfer(SET-PT)and sequential proton-loss electron transfer(SPLET)mechanisms.The o-diphenolic hydroxylgroup of the DHC B-ring and the 2',6'-phenolic hydroxylgroup of the A ring are the main sites of the HAT,the phenolic hydroxylgroup at the 2'-position is an important site of the SET-PT.Through characterization of DHC dissociation found that DHC compounds can also undergo SPLET mechanism by dissociation of the phenolic hydroxylgroup at the 4'-position.A new antioxidant mechanism for DHC has also been discovered.The dissociated part of the molecule can transfer electrons to the oxidized fragment,which enhances the antioxidant capacity of the entire molecule.This is the reason why HP2 G has the strongest antioxidant capacity.3.The antiproliferative for tumor cell of DHCs were analyzed.The antiproliferativity of P2 G,P4G,P2 X,P,HP2 G,HP4G,and HP on MG-63,Hela,Hep G2,MDA-MB-231,and SKOV-3 cancer cell lines were respectively screened used Thiazolyl Blue assay.Among these compounds,P,HP2 G,HP4G,and HP have antiproliferative activities to all tumor cell lines.It was found that 2',4',6'-trihydroxyacetophenone and 3,4-o-diphenolic hydroxyl group of DHC are pharmacophores with antiproliferative activity,and the latter is more powerful than the former.Moreover,the glycosylation on A-ring of molecule could promotes the activity of the 3,4-o-diphenolic hydroxyl pharmacophore but depress the 2',4',6'-trihydroxyacetophenone.4.The inhibitory of DHC on key enzymes involved in glucose metabolism was evaluated.P2 G,P4G,P2 X,P,HP2 G,HP4G,and HP were used to inhibited ?-glucosidase and ?-amylase activity,respectively.The result shows that,most Malus dihydrochalcone compounds have strong inhibition ability for both enzymes.Among the compounds,two aglycons have best inhibitory activity.The glycosidation will reduce its activity,the phenolic hydroxyl group at the 3-position of the B-ring can partly offset the decrease of the activity caused by the glycosylation.The phenolic hydroxyl group at 4'-position is more important than the 2'-position to the inhibitory activity for the ?-glucosidase,on the contrary to the ?-amylase.5.Allelopathy of DHC through causing cell death on root tip of receptor plant.Exogenous DHC did not affect the root growth of Malus hupehensis.And its can cause defects and short of Arabidopsis root.Structure-activity relationship analysis showed that the structure of 2',6'-dihydroxyacetophenone was the key chemical shtructure to the allelopathy.The DHC with the chemical structure led to the up-regulation of genes related to Arabidopsis programmed cell death and caused cell death in the elongation zone of the root tip.