Characteristics And Functional Properties Of Polyphenols In Aronia Berries

Aronia berries(Aronia melanocarpa and Aronia mitschurinii)are rich in cyanidin-type anthocyanin polyphenols,but their bioactives are underutilized as of functional food.Currently,polyphenol bioactivity and health benefit studies or industry application of aronia berry that focus exclusively on extractable polyphenols(EPPs)by using aqueous-organic solvents extraction,ignoring an important fraction of polyphenol,the non-extractable polyphenols(NEPPs).The objective of this study is to understand the composition and content of EPPs and NEPPs in aronia berry,as well as the bioavailability,physiology relevant bioactivities and mechanisms.Specified as follows:(1)The extractable polyphenols(EPPs)in aronia berries were extracted by acetone/water/acetic acid(70?29.5?0.5,v/v/v),then fractioned to isolate anthocyanins,neutral phenols and proanthocyanidins.Anthocyanins and neutral phenols were isolated by sequential elution from C18 solid phase extraction(SPE)cartridges,whereas proanthocyanidins were isolated by a Sephadex LH-20 column.The non-extractable polyphenols(NEPPs)were released by 20% acidified methanol water from freeze-dried aronia berry powder.The polyphenol profiling in EPPs and NEPPs were determined by UHPLC.The dominant compounds in aronia EPPs is cyanidin-type anthocyanin polyphenols,with 3-galactoside 32176± 815 nmol/g,3-arabinoside 9515 ± 174 nmol/g.The proanthoycanins fraction in aronia berries is mainly polymers(degree of polymerization DP ? 12),with content of 12302 ± 2889 nmol/g.After acid hydrolysis,NEPPs were released.The highest content among NEPPs is cyanidin,with content of 69104 ± 6553 nmol/g,followed by 2391 ± 212 nmol/g of cyanidin-3-galactoside,1992 ± 145 nmol/g of quercetin.In all,freeze-dried aronia berries contain a total of 30947.85± 993 ?g/g DW NEPPs,account for a proportion of 58.86 % in total polyphenols,whereas EPPs is 41.14%,with 21631 ± 694 ?g/g DW.(2)With the hypothesis of non-extractable aronia polyphenols would differentially affect microbiota and colonic anthocyanin content relative to aronia extractable polyphenols,we examined the differential effects of freeze-dried whole aronia berry(AB,contains both EPPs and NEPPs),aronia extract(AE,contains EPPs),and polyphenol-depleted dried aronia berry(D,contains NEPPs)on the gut microbiota and fecal composition of healthy C57BL/6J mice.AB and D increased fecal anthocyanins more than AE.Diets differentially affected SCFA and microbiota.AB fecal SCFA was associated with increased metabolism of succinate and pyruvate to butyrate.D increased acetic acid production,was associated with increased abundance of predicted genes for fermentation of carbohydrates to acetyl-co A.AB and D also increased predicted abundance of microbial catechol metabolism pathway I relative to C,which was attributed to enrichment of Lachnospiraceae.Therefore,the berry matrix impacts how aronia polyphenols interact with the gut microbiota in healthy mice.(3)We further tested how aronia berry,aronia EPPs,NEPPs and phenolic constituents improve intestinal barrier function.Caco-2 cells were cultured on transwell plates and allowed differentiate to form a model intestinal barrier.Barrier function of differentiated Caco-2 cells was challenged by the addition of an inflammatory cocktail(IC: TNF-?,IL-1?,and IFN-? to the basolateral media and lipopolysaccharide to the apical media).Transepithelial electric resistance(TEER)value was determined by chopstick electrode,permeability was determined by application of Lucifer yellow and expression of tight junction was assessed by q RT-PCR and western blot analysis.1.5 mg/m L whole aronia berries(contains both EPPs and NEPPs),0.15 mg/m L aronia ethanolic extract(EPPs)and 0.15 mg/m L aronia ethanolic extract residue(NEPPs)improved IC induced epithelial barrier dysfunction by inhibition TEER loss and prevention of permeability of Lucifer yellow(LY)through increased tight junction ZO-1,OLCN and CLDN1 gene and protein expression levels.NEPPs and EPPs differentially improved the gastrointestinal barrier function,NEPPs prevented significant higher flux of fluorescence molecules across cell monolayers relative to EPPs,upregulated the gene and protein expression of ZO-1 by 20.1% and 32.8%,respectively.Except quercetin-3-glucuronide(Q3glu),aonia phenolic constituents,cyanidin-3-galactoside(C3gal),quercetin and catabolites3,4-dihydroxyphenylacetic acid(DOPAC)also improved barrier function by upregulation gene and protein expression of tight junctions ZO-1,OLCN and CLDN1.Quercetin was able to exert highest barrier protection by increased TEER% of 69.4% and reduced LY flux by 52.3%relative to IC,the increased expression of tight junctions reach up to 182.6%.(4)In vitro and in vivo evidences related the aronia berry assumption with inflammatory inhibition,thus we tested the effect of aronia polyphenols and its catabolites on T cell meditated autoimmune responses modulation.Based on the na(?)ve CD4+ T cell isolated from murine splenocytes by using magnetic-activated cell sorting(MACS)technology,an in vitro model of regulatory T cell(Treg)generation from na(?)ve CD4+ cell was established and optimized.The Treg cells and subpopulations were analyzed by flow cytometer after stained the cell surface antigens CD3,CD4,CD25 and intracellular marker of Foxp3 and IL-10.2 ?M quercetin significant increased the CD4+CD25 Treg differentiated from na(?)ve CD4+ T cells by 22.3%relative to untreated control.Moreover,compared to control,CD4+CD25 Tregs subpopulations:IL-10+ Treg and Foxp3+IL-10+ Treg were also increased 21-fold and 34-fold,respectively.Catabolite 3,4-dihydroxyphenylacetic acid(DOPAC)have a same trend of Treg and subpopulations promotion.These findings supported that aronia polyphenol and its catabolite inhibited the excessive immune responses by promoting the differentiation of Treg cells and anti-inflammatory cytokine IL-10.(5)Angiotensin-converting enzyme 2(ACE2)is a host receptor for severe acute respiratory syndrome coronavirus 2(SARS-Co V-2).Inhibiting the interaction between the envelope spike glycoproteins(S-proteins)of SARS-Co V-2 and ACE2 is a potential antiviral therapeutic approach.Based the preliminary computational molecular docking study identified quercetin,luteolin,and eriodictyol as potential inhibitors of the interaction between S protein and ACE2,we tested the inhibition activity of aronia flavonoids and other polyphenols with Bring 3',4'-hydroxylation inhibit recombinant human(rh)ACE2 activity.rh ACE2 activity was assessed with the fluorogenic substrate Mca-APK(Dnp).Aronia polyphenols reduced rh ACE2 activity by 15 ? 66% at 10 ?M.Rutin,quercetin-3-O-glucoside,tamarixetin,and 3,4-dihydroxyphenylacetic acid inhibited rh ACE2 activity by 42 ? 48%.Quercetin was the most potent rh ACE2 inhibitor among the polyphenols tested,with an IC50 of 4.48 ?M.Thus,structure-functional relationship of polyphenols with B-ring 3',4'-hydroxylation anti-viral activity was established.Quercetin,its metabolites,and polyphenols with B-ring 3',4'-hydroxylation inhibited rh ACE2 activity at physiologically relevant concentrations in vitro.