New Insights into the Exploitation of Vitis vinifera L. cv. Aglianico Leaf Extracts for Nutraceutical Purposes

an ioxidan s A icle New Insigh s in o he Exploi a ion o Vi is ini e a L. c . Aglianico Lea Ex ac s o Nu aceu ical Pu poses Fabiana Labanca 1,†, Immacola a Fa aone 1,2,†, Ma ia Rosa ia Nolè1,3, Ru h Ho nedo-O ega 3, Daniela Russo 1,2,*,†, Ma ia Ca men Ga cía-Pa illa 3, Lucia Chiummien o 1, Ma ia G azia Bonomo 1and Luigi Milella 1,2,*,† 1Dipa imen o di Scienze, Uni e si àdella Basilica a, .le A eneo Lucano 10, 85100 Po enza, I aly; [email p o ec ed] (F.L.); [email p o ec ed] (I.F.); [email p o ec ed] (M.R.N.); [email p o ec ed] (L.C.); [email p o ec ed] (M.G.B.) 2Spino BioAc iPlan s. .l., Uni e si àdella Basilica a, .le A eneo Lucano 10, 85100 Po enza, I aly 3Á ea de Nu ición y B oma ología, Facul ad de Fa macia, Uni e sidad de Se illa, C/P., Ga cıa Gonzalez 2, 41012 Se illa, Spain; [email p o ec ed] (R.H.-O.); [email p o ec ed] (M.C.G.-P.) *Co espondence: [email p o ec ed] (D.R.); [email p o ec ed] (L.M.) †Au ho s con ibu ed equally o his wo k. Recei ed: 30 June 2020; Accep ed: 30 July 2020; Published: 4 Augus 2020   Abs ac : The lea es o Vi is ini e a L. ha e been used o a long ime in adi ional medicine o he ea men o many ailmen s. G ape polyphenols, indeed, ha e been demons a ed o be able o de end agains oxida i e s ess, esponsible o a ious diso de s such as cance , diabe es and neu odegene a i e diseases. The e ec s o di e en ex ac ion echniques, Soxhle (SOX), Accele a ed Sol en (ASE 40, ASE 50) and Ul asound Assis ed Ex ac ion (UAE) we e s udied in his wo k o e alua e hei impac on he chemical p o ile and bioac i e po en ial o Vi is ini e a L. (c . Aglianico) lea ex ac s. The phy ochemical p o ile was in es iga ed by HPLC-DAD and 9 phenolic compounds we e iden i ied and quan i ied in he ex ac . Mo eo e , he an ioxidan , an icholines e ase and an i y osinase ac i i ies we e e alua ed. In de ail, he o al polyphenol con en and an ioxidan ac i i y (2,2-diphenyl-1-pic ylhyd azyl, Oxygen Radical Abso bance Capaci ies and β -Ca o ene Bleaching assays) we e e alua ed and compa ed o assess he Rela i e An ioxidan Capaci y Index (RACI). To es he inhibi o y ac i i y o ex ac s owa ds cholines e ases, ace ylcholines e ase (AChE) and bu y ylcholines e ase (BChE) inhibi ion assays we e pe o med. SOX and ASE 50 ha e shown he highes alue o RACI, 0.76 and 0.65, espec i ely. Rega ding enzyma ic inhibi o y ac i i y, ASE 50 (IC 50 =107.16 ± 8.12 µ g/mL) and SOX (IC 50 =171.34 ± 12.12 µ g/mL) ex ac s exhibi ed he highes AChE and BChE inhibi o y ac i i y, espec i ely, while UAE (IC 50 =293.2 ± 25.6 µ g/mL, ollowed by SOX (IC 50 =302.5 ± 38.3 µ g/mL) showed he highes y osinase inhibi ion alue. Ou esul s demons a ed o he i s ime ha Aglianico lea es a e impo an sou ces o phenols ha could be used o p e en oxida i e s ess and be po en ially help ul in diseases ea able wi h y osinase and cholines e ase inhibi o s, like myas henia g a is o Alzheime ’s. Keywo ds: Alzheime ’s disease; Vi is ini e a; an ioxidan p ope ies; enzyma ic inhibi o y ac i i y; ace ylcholines e ase; bu y ylcholines e ase; y osinase; polyphenol compounds 1. In oduc ion G ape ine is one o he mos widely cul i a ed plan s in he wo ld, wi h a global annual p oduc ion o nea ly 80 million ons in 2018 [ 1 ], des ined o di e en pu poses (wine, juice, able g apes, e c.). G ape ine can be conside ed an impo an sou ce o bioac i e compounds, mainly polyphenols [ 2 ]. Vi is ini e a L. lea es, commonly consumed as ood in he Medi e anean a ea, a e An ioxidan s 2020,9, 708; doi:10.3390/an iox9080708 www.mdpi.com/jou nal/an ioxidan s An ioxidan s 2020,9, 708 2 o 16 also used as a demulcen , ca ha ic, s omachic and diu e ic. Mo eo e , i has been demons a ed o be use ul in bilious dyspepsia, hemo hage, dysu ia, in ch onic b onchi is, hea diseases and gou , while in olk medicine i p e en s cons ipa ion. The ex ac ion p ocedu e is an impo an s ep in eco e ing bioac i e compounds om plan ma ices. Con en ional (mace a ion, Soxhle ex ac ion) and non-con en ional (ul asound-assis ed, mic owa e-assis ed, accele a ed sol en and subc i ical wa e ex ac ion echniques) app oaches can be applied [ 3 ]. Ex ac ion me hod e ec i eness is e iden when di e en me hods a e p ac iced on he same plan ma ices by using he same sol en and ex ac ion e iciency shows signi ican a ia ions [ 4 ]. The ex ac ion me hods should be op imized no only o i s o al phy ochemical p o ile bu also o i s biological e ec s. Se e al s udies epo ed ha he phenol composi ion o g apes is s ongly ela ed o geog aphical and clima e ac o s as well as o g ape a ie y [ 5 ]. Vi is ini e a L. c . Aglianico is a a ie y o ed g ape cul i a cul i a ed in Sou he n I aly as Basilica a and Campania. In Basilica a he cul i a ion o Aglianico ineya ds is mainly localized in he Vul u e a ea and he wine awa ded he Con olled Designa ion o O igin (CDO) label, in compliance wi h he legisla ion (EU 1971) [ 6 ]. P e ious s udies on Aglianico cul i a ha e la gely in es iga ed he phenolic composi ion o g ape be ies, be ies skin and wine showing he p esence o la onoid (an hocyanins, p ocyanidins, la onols, la an-3-ols, la ones, la onones) and non- la onoid compounds (phenolic acids, s ilbenes) [ 7 – 10 ], wi h an ioxidan , an imic obial, an i i al, an i-in lamma o y p ope ies [ 11 , 12 ]. Gab iele e al. [ 13 ] e alua ed he e ec o low sulphu dioxide concen a ions on he ch oma ic p ope ies, phy ochemical composi ion and an ioxidan ac i i y o Aglianico ed wines. Nowadays, one o he bigges challenges in ag icul u al p ac ices is o de elop al e na i es and alo ize he huge amoun o by-p oduc s. I is o his eason ha he Eu opean Commission has es ablished a di ec i e o was e managemen e idencing ha ecycling should be a p io i y (Di ec i e EC/2008). Al hough s ill no widely used, he ineya d p oduces a g ea quan i y o ege al byp oduc s and se e al s udies ha e demons a ed ha his biomass is also ich in polyphenolic compounds ha could be exploi ed o di e en pu poses and is a ec ed by he ex ac ion p ocedu e [ 14 – 17 ]. Thus, V. ini e a c . Aglianico lea es can be use ul as a p omising sou ce o bioac i e compounds gi ing a new pe spec i e o he use o i s by-p oduc . The phenolic composi ion and biological ac i i y o lea es was in es iga ed in di e en cul i a s o V. ini e a [ 18 , 19 ], bu , o he bes o ou knowledge, his is he i s s udy ca ied ou on lea ex ac om V. ini e a c . Aglianico. Lea seconda y me aboli es, mainly o phenolic o igin, possess impo an bene icial p ope ies o human heal h, including p o ec i e e ec agains he de elopmen and p og ession o pa hological condi ions such as cance , aging, ca dio- ascula p oblems and diabe es [ 11 , 12 , 20 ]. The oxida i e s ess is also associa ed wi h Alzheime ’s Disease (AD) and Pa kinson’s Disease (PD), wo neu odegene a i e diseases cha ac e ized by cogni i e diso de s and memo y loss. In AD, he “choline gic hypo hesis” is he mos accep ed heo y, hus he inhibi ion o ace ylcholines e ase (AchE) migh alle ia e he p og essi e de iciency o ace ylcholine (ACh) le els ha cha ac e izes he pa hology [ 21 ]. In PD, he dopamine one is dec eased and he disease is associa ed wi h y osinase-media ed neu onal damage. Al hough y osinase enzyme con ibu es o neu omelanin syn hesis in he b ain, wi h neu op o ec i e e ec s, i s o e exp ession seems o play a pi o al ole in gene a ing oxidizing compounds ha migh exace ba e dopamine oxici y [ 22 ]. AD and PD p og ession can po en ially slow down wi h he use o an ioxidan compounds [ 23 ], as well as na u al subs ances wi h an icholines e ase and an i y osinase ac i i y. Bo ai and Rizk e alua ed he neu op o ec i e and an ioxida i e po en ial o polyphenolic ex ac om V. ini e a lea es in alle ia ing aluminum chlo ide induced neu o oxici y in male a s. In ac , aluminum is a po en neu o oxic me al implica ed in he neu opa hogenesis o AD, because i induces a signi ican dec ease o ACh con en in he b ain, along wi h a signi ican inc emen o AChE ac i i y [ 24 , 25 ]. These p omising in i o esul s p o ide a conc e e base in he ield o na u al p oduc s o disco e y po en ial y osinase inhibi o s. An ioxidan s 2020,9, 708 3 o 16 The e o e, he objec i e o ou esea ch was o in es iga e he e ec o di e en ex ac ion p ocesses on he polyphenolic p o iles o V. ini e a L. c . Aglianico lea ex ac s and he an ioxidan , an icholines e ase and an i y osinase ac i i ies. 2. Ma e ials and Me hods 2.1. Chemicals Analy ical g ade me hanol and ace oni ile we e ob ained om Me ck (Da ms ad , Ge many and Molle del Vall é s, Spain); 2,2-diphenyl-1-pic yl hyd azyl (DPPH) in ee adical o m, 2,2 0 -azobis- (2-amidinop opane) dihyd ochlo ide (AAPH), T olox (6-hyd oxy-2,5,7,8- e ame hylch oman-2-ca boxylic), izma hyd ochlo ide (T is-HCl), bo ine se um albumin (BSA), Folin-Ciocal eu’s phenol eagen , β -ca o ene, linoleic acid, Tween 20, bu yla ed hyd oxy oluene (BHT, 2,6-bis (1,1-dime hyle hyl)-4-me hylphenol), 5,5 0 -di hio-bis (2-ni obenzoic acid) (DTNB), ace ylcholines e ase (AChE) om elec ic eel ( ype VI-s, lyophilized powde ), ace yl hiocholine iodide (ATCI), bu y ylcholines e ase (BChE) om equine se um (lyophilized powde ) and S-bu y yl hiocholine chlo ide (BTCC), we e pu chased om Sigma (S . Louis, MO, USA and S einheim, Ge many). Fluo escein sodium was ob ained ia Fluka (S einheim, Ge many). Glacial ace ic acid was ob ained om Pan eac (Ba celona, Spain). The s anda ds o 38 phenolic compounds we e pu chased om Fluka, [5-me hyl u u al, ace osi ingone, benzoic acid, ca eic acid, ca a ic acid, cinnamic acid, gallic acid, p-couma ic acid, (-)-epica echin, que ce in glucoside, kaemp e ol-3-O-glucoside, homo anillic acid, e hyl galla e, p-hyd oxybenzoic acid, que ce in and hyd oxy y osol], Sigma [(+)- axi olin, (-)-epica echin galla e, anillic acid, 2- u u aldehyde, ellagic acid, e ulic acid, gen isic acid, sinapic acid, sy ingic acid, (-)-ca echin, (-)-epigalloca echin, e hyl anilla e, p o oca echualdehyde, es e a ol and sy ingaldehyde], Me ck, [ anillin, gen isin], Sa c [e hyl anillin and 5-hyd oxyme hyl-2- u aldeide] and Ch omadex, [p ocyanidin-B1, p ocianydin-B2 and p ocyanidin-B3]. Double-dis illed wa e (Millipo e Co.) was used h oughou . 2.2. G ape Lea es Collec ion G ape lea es o V. ini e a L. (c . Aglianico) we e collec ed a he Ma ino wine company loca ed in Venosa, Po enza (Basilica a Region). The plan ma e ial was kep a − 20 ◦ C. Lea es we e milled manually o ob ain a ine powde be o e he ex ac ion. 2.3. Ex ac ion o An ioxidan Compounds Th ee di e en echniques we e employed o he ex ac ion o polyphenolic compounds om g ape lea es—Soxhle Ex ac ion (SOX), Accele a ed Sol en Ex ac ion (ASE) and Ul asound Assis ed Ex ac ion (UAE). 2.3.1. Soxhle Ex ac ion (SOX) SOX is a adi ional me hod which has been used o many decades [ 26 ]. The inely g ound plan ma e ial (25.40 g o d ied lea es) was placed in a cellulose himble wi h a po ous bag made o il e pape , which is inse ed on he op o he ex ac ion chambe . When he sol en (ul apu e wa e :e hanol, 50:50) is hea ed, he apo s s a o condense when hey come in o con ac wi h he condense and he sol en was collec ed in he collec ion lask. The condensed sol en will d ip in o he himble con aining he plan ma e ial. This p ocess p oceeded con inuously and i was s opped when he sol en om he siphoned ube u ned clea wi hou any dissol ed ex ac ion ma e ial (abou 6 h), meaning an exhaus ed ex ac ion. The ex ac was hen il e ed and he sol en was emo ed by using a o a y e apo a o . 2.3.2. Accele a ed Sol en Ex ac ion (ASE) The mo Scien i ic Dionex ASE 150 Accele a ed Sol en Ex ac o and 22 mL cells we e used o he ex ac ion o g ape lea es o Aglianico. The ex ac ion me hod was pe o med acco ding o a p e iously An ioxidan s 2020,9, 708 4 o 16 desc ibed p ocedu e [ 27 – 29 ], wi h sligh modi ica ions. The e ec o wo di e en empe a u es o ex ac ion, 40 ◦ C (ASE 40) and 50 ◦ C (ASE 50) was also es ed. The assays we e pe o med a 1600 psi o h ee cycles o 5 min each. Fo he ex ac ion, he d ied lea es o he Aglianico cul i a (30.29 g o ASE 40 and 26.17 g o ASE 50) we e loaded and compac ed in he cell and subjec ed o ex ac ion wi h e hanol/wa e (50:50 / ). The ex ac ed solu ion was collec ed and il e ed. The sol en was hen emo ed ia a o a y e apo a o . 2.3.3. Ul asound Assis ed Ex ac ion (UAE) The UAE me hod uses ul asound wa es o he ex ac ion o na u al compounds om g ape lea es [ 30 ]. Fo he ex ac ion by ul asonic ba h (B anson 1800 sonica o , equency o 40 Hz and ampli ude o 100%), 10.27 g o d ied lea es o Aglianico we e ex ac ed using a mix u e o 170 mL o ul apu e wa e and e hanol (50:50, / ) as sol en . Ex ac ions we e pe o med in da k bo les a 30 ◦ C o a pe iod o 6 hou s, he sol en has been changed e e y wo hou s wi h esh one o acili a e he comple e ex ac ion. Finally, ex ac s we e il e ed and he sol en was emo ed wi h a o a y e apo a o . 2.4. RP-HPLC-DAD Quali a i e and Quan i a i e Analysis o Phenolic Compounds The LC analysis o phenols was pe o med using an Agilen Se ies 1100 sys em equipped wi h a qua e na y pump (Se ies 1100 G1311A), au oma ic injec o (Se ies 122 1100 G1313A) and degasse on line (Se ies 1100 G1379A). A UV/Vis diode de ec o (Se ies 1100 G1315B) coupled o a Chems a ion HP A.10.02 (HP/Agilen ) was used o de ec ion. The column was a Me ck LiCh oCART RP-18 250-4 Supe sphe 100 RP-18, po e size 5 µ m (250 mm × 4 mm), p o ec ed by p ecolomn Me k RP-18 4.6 × 12.5 mm. Samples we e il e ed be o e injec ion h ough a Wha man il e s cellulose ace a e memb ane, po e size 0.45 µ m and he diame e size 25 mm. The ch oma og aphic condi ions ha e been p e iously desc ibed [ 31 ]. Two di e en sol en s we e used as a mobile phase—A (glacial ace ic acid/wa e , pH 2.65), B (20% A +80% ace oni ile) p og ammed in a g adien as ollows—0 min (100% A); 5 min (98% A +2% B); 10 min (96% A +4% B); 15 min (90% A +10% B); 30 min (80% A +20% B); 35 min (70% A +30% B); 40 min (100% B); 45 min (100% A); 60 min (100% A). The injec ion olume was 50 µ L. The low a e was 1.5 mL · min −1 and he empe a u e was se a 40 º C. The iden i ica ion o each compound was ob ained acco ding he e en ion ime and he UV-Visible spec a o he co esponding s anda d compound. S anda d compounds we e solubilized in me hanol a a concen a ion o 1.5 mg/mL, used as s ock solu ion and subsequen ly dilu ed o he calib a ion cu e. Quan i ica ion analysis was pe o med by ex e nal calib a ion wi h espec i e s anda ds a 280 nm o hyd oxybenzoic acids, 320 nm o hyd oxycinnamic acids and 365 nm o la onoids, in acco dance wi h he maximum abso bance o each compound. Resul s o quan i a i e analysis we e exp essed as mg o compound/kg o d ied ex ac [32]. 2.5. To al Phenolic Con en (TPC) All ex ac s we e used o es he o al phenolic con en (TPC) by using he Folin-Ciocal eu assay [ 33 ] and 75 µ L o he dilu ed ex ac and 425 µ L o dis illed wa e was added o 500 µ L F-C eagen and 500 µ L o Na 2 CO 3 (10% w/ ). Sodium ca bona e is added o alkalise he sys em ob aining a pH alue abou 10. The solu ion was mixed and incuba ed o 1 h in he da k a oom empe a u e. A e incuba ion, he abso bance was measu ed a 723 nm using a UV-Vis spec opho ome e SPECTROs a Nano (BMG Lab ech). A s anda d calib a ion cu e was p epa ed using di e en concen a ions o gallic acid and esul s we e exp essed as µ g o Gallic Acid Equi alen s (GAE)/g o ex ac , using he eg ession equa ion be ween gallic acid s anda ds and abso bance (y =0.0871x −0.0282; R2=0.9997). Fo each sample, h ee eplica e assays we e pe o med. 2.6. An ioxidan Ac i i y The an ioxidan ac i i y was de e mined by di e en spec opho ome ic echniques which a e desc ibed below. An ioxidan s 2020,9, 708 5 o 16 2.6.1. DPPH Me hod F ee adical sca enging ac i i y o he ex ac s was e alua ed using he DPPH me hod [ 34 ] wi h some modi ica ions. This me hod e alua es he quenching abili y o ou ex ac owa d he DPPH adical, by spec opho ome ic moni o ing o he ollowing eac ion: DPPH·+R:H→DPPH −H+R, whe e R:H ep esen s an an ioxidan . Fo each ex ac , a dilu ion se ies was p epa ed ( inal concen a ion anging om 20-50 µ g/mL o lea es ex ac s). In each well, 50 µ L o he sample o sol en o he blank we e added o 150 µ L o he DPPH solu ion (120 µ M). DPPH sca enging ac i i y was moni o ed a 515 nm using a UV-Vis spec opho ome e (Syne gy HT, Bio ek ® ) a 0 min and a e 20 min, when he eac ion eached he equilib ium. The an ioxidan ac i i y was exp essed as IC 50 ( µ g/mL), he concen a ion o subs a e ha is equi ed o sca enge 50% o DPPH ee adicals [ 35 ]. To calcula e he pe cen age o inhibi ion o he adical, he abso bance alues (A) a equilib ium (T =20 min) o bo h he con ol (CT) and he sample (S) we e measu ed: % inhibi ion =[(ACT −AS)/ACT) *100]. (1) 2.6.2. Oxygen Radical An ioxidan Capaci y (ORAC) assay The ORAC assay was based on a p e iously epo ed me hod wi h sligh modi ica ions [ 36 ]. I is based on in si u gene a ion o pe oxyl ee adicals by 2,2-azobis(2-me hylp opionamidine) dihyd ochlo ide (AAPH). They in e ac wi h oxidable luo escen p obe (F · ), changing he luo escence in ensi y. In he p esence o an ioxidan s (A OH), he luo escence decay is inhibi ed, as is illus a ed in he ollowing chemical equa ion: F+A OH →FH +A O The analy ical p ocedu e was as ollows—50 µ L o sample o T olox was added o 100 µ L o Fluo escein (1.5 µ M) and 50 µ L o 2,2 0 -azobis (2-amidinop opane) dihyd ochlo ide (AAPH, 15 mM). Fo he blank, 50 µ L o phospha e bu e (75 mM, pH 7.4) was added o 100 µ L o Fluo escein and 50 µ L o AAPH, whe eas o he con ol, 50 µ L o phospha e bu e was added o 100 µ L o Fluo escein. The pla e was incuba ed o 15 min a oom empe a u e a e addi ion o AAPH. Fluo escence ( he exci a ion wa eleng h was se a 490 nm; he emission wa eleng h was 515 nm) was calcula ed e e y 5 min o 90 min a 37 ◦ C, un il i app oxima ely dec eases o 0 o o a alue less han 5% o he ini ial alue. Measu emen s we e aken in duplica e in a mul i-220 de ec o mic opla e eade (Syne gy HT, Bio ek ® , Winooski, VT, USA). T olox was used as a calib a ion s anda d (0.5–9.5 µ M). The esul s we e calcula ed as ORAC alues indica ing he di e ences be ween he blank and he sample a eas unde he luo escein decay cu e [37]. The equa ion used is he ollowing: ORAC alue =20 ×K×[(Ssample −Sblank)/(S olox −Sblank)] (2) whe e 20 is he concen a ion o T olox; K is he dilu ion ac o o sample; S is he a ea unde he cu e o he dec ease o he luo escein, he sample, he T olox o he blank. The esul s a e exp essed as µmol T olox equi alen s (TE)/mg o ex ac . 2.6.3. β-Ca o ene Bleaching Assay (BCB) The an ioxidan ac i i y was also e alua ed by β -ca o ene bleaching assay (BCB) [ 38 ]. β -Ca o ene (0.2 mg) was dissol ed in 0.2 mL o chlo o o m and hen he chlo o o m was emo ed by o a y e apo a o a oom empe a u e. The β -ca o ene solu ion was added o a lask oge he wi h linoleic acid (20 mg) and Tween 20 (200 mg) and inally oxygena ed dis illed wa e (50 mL) was added and An ioxidan s 2020,9, 708 6 o 16 mixed well. Aliquo s o he emulsion (950 µ L) we e mixed in o di e en es ubes wi h 50 µ L o sample ( he inal concen a ion o all es ed samples was 200 µ g/mL) o sol en as a blank. Bu ylhyd oxy oluene (BHT) was used as posi i e con ol. This emulsion solu ion (250 µ L) was ans e ed o a 96-well mic opla e. Then, he mic opla e was immedia ely placed a 50 ◦ C o 3 h and he abso bance was measu ed a 470 nm, using a spec opho ome e , e e y 30 min—a 0 0 , 30 0 , 60 0 , 90 0 , 120 0 , 150 0 and 180 0 un il he colo o con ol sample has changed. Resul s we e exp essed as pe cen age o An ioxidan Ac i i y (AA%) and i was calcula ed using he ollowing equa ion: (AA%)= 1−Abs sampleT00−Abs sampleT1800 Abs blankT00−Abs blankT1800!×100 (3) whe e Abs sample T 00 and Abs blank T 00 a e he abso bance be o e he incuba ion o he ex ac and he blank (wi hou ex ac ), espec i ely and Abs sample T 1800 and Abs blank T 1800 a e he abso bance a 180 min o incuba ion o ex ac and he blank, espec i ely. 2.7. Ace ylcholines e ase (AChE) and Bu y ylcholines e ase (BChE) Inhibi o y Ac i i y The inhibi ion o AChE ac i i y was de e mined based on Ellman’s me hod, as p e iously epo ed [ 38 ]. The enzyme ac i i y is measu ed by spec opho ome ic de ec ion (405 nm) o he inc ease o yellow colo p oduced om hiocholine when i eac s wi h 5,5 0 -di hio bis-2 ni obenzoa e ions (DTNB). Fo he analysis, 25 µ L o ace yl hiocholine iodide (15 mM), 125 µ L o DTNB (3 mM), 50 µ L o bu e B (50 mM T is-HCl, pH 8 con aining 0.1% bo ine se um albumin) and 25 µ L o each es sample solu ion a he di e en concen a ions we e mixed. The eac ion was s a ed by adding 25 µ L o 0.18 U/mL AChE. The abso bance was measu ed a 405 nm kine ically du ing 2 min. Nega i e con ol abso bance (Bu e A, 50 mM T is-HCl, pH 8) was also measu ed. The BChE inhibi ion assay was pe o med in a simila way [ 38 ] using 25 µ L o 15 mM S-bu y yl hiocholine chlo ide as subs a e and 0.10 U/mL o BChE as enzyme. Samples we e e alua ed a di e en concen a ions. In he same way, nega i e con ol abso bance (Bu e A) was also eco ded. The esul s we e exp essed as pe cen age o inhibi ion and calcula ed as ollows [39]: % inhibi ion =((Abssample −Absnega i e con ol)×100 −100)−1(4) Galan amine, dissol ed in Bu e A, was used as posi i e con ol o bo h assays. 2.8. Ty osinase Inhibi o y Ac i i y The inhibi ion o y osinase was pe o med by L-DOPA in i o assay [ 40 ]. A eac ion mix u e, con aining 125 mL o phospha e bu e (50 mM, pH 6.8), 25 µ L o s anda d o ex ac s a di e en concen a ions and 50 mL o y osinase (50U/mL) was incuba ed a 37 ◦ C o 15 min. Then, he L-DOPA subs a e (50 µ L) was added o he mix u e and he eac ion was moni o ed o 10 min a 475 nm. The expe imen was made in iplica e. The esul s we e exp essed as IC 50 alue, a concen a ion gi ing 50% inhibi ion o y osinase ac i i y, de e mined by in e pola ion o concen a ion- esponse cu es. Kojic acid was used as a posi i e con ol. 2.9. S a is ical Analysis Analysis o a iance was pe o med o assess he s a is ically signi ican di e ences among samples, o he polyphenolic con en o g ape lea es o Aglianico cul i a , a a con idence le el o 95% [ 41 ]. Di e ence on he mean alues was assessed by he Tukey es a a signi icance le el o p<0.05. To compa e he esul s be ween he di e en me hods, he Rela i e An ioxidan Capaci y Index (RACI) was calcula ed [42]. An ioxidan s 2020,9, 708 7 o 16 3. Resul s and Discussion 3.1. Vi is ini e a c . Aglianico Lea Ex ac ion Ex ac ion yield is a quan i a i e ep esen a ion o he e iciency o he ex ac ion p ocess o eco e na u al compounds om he plan issues [ 43 ]. Pa ame e s, such as ex ac ion echnique, sol en , empe a u e and ime, as well as he chemical na u e o he sample [ 44 ], a ec he ex ac i e yield. In his wo k, lea es om Vi is ini e a L. c . Aglianico we e ex ac ed wi h he same sol en (wa e :e hanol 50:50) bu by using di e en solid–liquid ex ac ion echniques. All ex ac s we e d ied by o a y e apo a o and yield ex ac ion was calcula ed. The esul s o ex ac i e yields we e exp essed as he pe cen age o he weigh o he c ude ex ac s. aw ma e ial. Table 1shows he di e en ex ac i e capaci ies o each echnique. Ex ac i e yields anged om 6.41 ±0.52% o 30.45 ±2.32%. Table 1. Ex ac ion pa ame e s o eco e y he bioac i e compounds om g ape lea es o Vi is ini e a L. (c . Aglianico). Me hod o Ex ac ion Sol en o Ex ac ion G Tempe a u e Time o Ex ac ion Ex ac ion Yield (%) Soxhle ex ac ion (SOX) Ul apu e wa e and e hanol, 50:50 25.40 g 100 ◦C 6 h 30.45 ±2.32 b Ul asound Assis ed Ex ac ion (UAE) Ul apu e wa e and e hanol, 50:50 10.27 g 30 ◦C 6 h 13.81 ±1.13 c Accele a ed Sol en Ex ac ion (ASE 40) Ul apu e wa e and e hanol, 50:50 30.29 g 40 ◦Cs a ic ime 5 min × 3 cycles 6.44 ±0.48 a Accele a ed Sol en Ex ac ion (ASE 50) Ul apu e wa e and e hanol, 50:50 26.17 g 50 ◦Cs a ic ime 5 min × 3 cycles 6.41 ±0.52 a Ex ac ions we e epea ed in iplica e and exp essed as mean ± s anda d de ia ion. Signi ican di e ences (p<0.05) a e highligh ed wi h di e en supe sc ip le e s (a, b, and c). The choice o his mix o sol en s allows us o pe o m an eco-sus ainable ex ac ion, ob aining good ex ac i e yields. Ou esul s, indeed, a e compa able wi h hese o Ma loub [ 45 ] who achie ed yields anging om 12.10 ± 1.81% o 44.99 ± 1.19%, by using di e en mix o ace one and me hanol. These oxic sol en s a e mo e expensi e and equi e highe disposal cos s, a he expense o he en i onmen . ASE condi ions ( empe a u e, cycles and sol en ) ha e been selec ed basing on he esul s ob ained by p e ious s udies [ 20 ] and op imized in o de o ob ain he comple e ex ac ion o me aboli es. Howe e , ASE sys em epo ed he lowes ex ac i e yield a bo h used empe a u e (40 and 50 ◦ C), whe eas he highes ex ac ion yield was obse ed in SOX ex ac . The ac ha he sample is epea edly in con ac wi h esh sol en and he high empe a u e eached by SOX me hod, could be he eason o he highe con en o seconda y me aboli es ex ac ed om lea ma e ial. In ac , empe a u e is an impo an pa ame e con ibu ing o he ex ac ion yield. Usually, ele a ed empe a u es lead o he imp o ed ex ac ion e iciencies [46] bu hey can p esen he incon enience o he deg ada ion o he mo labile compounds. 3.2. Iden i ica ion and Quan i ica ion o Phenolic Compounds The iden i ica ion and quan i ica ion o phenolic compounds in he lea es o Aglianico was ca ied ou using he HPLC-DAD me hod. As epo ed in Figu e 1, a o al o 9 phenolic compounds [gallic acid, (+)-ca echin, benzoic, ca a ic and ca eic acids, u in, que ce in, que ce in-3-O-glucoside and kaemp e ol-3-O-glucoside] we e iden i ied and quan i ied in ou samples (Table 2). Mo eo e , o he 3 la onols could be p esumably iden i ied on he basis o he li e a u e [que ce in-3-O-galac oside, que ce in-3-O-glucu onide and que ce in-3-O-glycoside]. SOX ex ac o lea issue allowed he iden i ica ion o hese phenolic compounds. Howe e , gallic and ca a ic acid, que ce in-3-O-glucoside and que ce in we e no p esen in ASE 40, ASE 50 and UAE. An ioxidan s 2020,9, 708 8 o 16 An ioxidan s 2020, 9, x 8 o 16 Figu e 1. HPLC ch oma og am o Soxhle (SOX) lea ex ac o Vi is ini e a c Aglianico (320 nm). Peaks: 1, gallic acid; 2, ca a ic acid; 3, (+)-ca echin; 4, ca eic acid; 5, benzoic acid; 6, u in; 7, que ce in- 3-O-galac oside; 8, que ce in-3-O-glucoside; 9, que ce in -3-O-glucu onide; 10, que ce in-3-O- glycoside; 11, kaemp e ol-3-O-glucoside; 12, que ce in. Table 2. Quan i ica ion o compounds in Vi is ini e a (c . Aglianico) lea ex ac s. Peak Analy e RT (min) λmax SOX ASE 40 ASE 50 UAE 1 Gallic acid 3.9 280 159.91 ± 1.54 a Nd Nd Nd 2 Ca a ic acid 6.7 320 5706.97 ± 77.20 a 3651.71 ± 52.91 b 4075.15 ± 23.40 c 6047.84 ± 41.30 d 3 (+)-Ca echin 14.7 280 1176.00 ± 17.32 a 300.52 ± 0.41 b 331.72 ± 0.52 c 682.81 ± 2.72 d 4 Ca eic acid 16.3 320 557.31 ± 2.68 a Nd Nd Nd 5 Benzoic acid 21.8 280 131.23 ± 4.89 a 452.83 ± 2.76 b 315.78 ± 1.85 c 408.27 ± 0.00 d 6 Ru in 28.1 365 205.69 ± 15.34 a 210.64 ± 7.20 a 192.23 ± 18.89 a 319.75 ± 1.49 b 7 Que ce in-3-O-galac oside 29.5 365 2938.56 ± 6.79 a 1976.24 ± 20.78 b 2003.21 ± 16.80 b 2449.91 ± 3.43 c 8 Que ce in-3-O-glucoside 29.9 365 2352.24 ± 41.34 a Nd Nd Nd 9 Que ce in -3-O-glucu onide 30.3 365 2893.19 ± 21.59 a 2009.87 ± 5.99 b 2023.01 ± 3.45 b 2465.27 ± 3.43 c 10 Que ce in-3-O-glycoside 30.8 365 3362.17 ± 4.94 a 2219.28 ± 1.60 b 2450.58 ± 23.26 c 3150.82 ± 10.29 d 11 Kaemp e ol-3-O-glucoside 33.6 365 666.64 ± 6.10 a 562.06 ± 24.41 b 535.27 ± 24.99 b 572.65 ± 10.39 b 12 Que ce in 38.1 365 490.71 ± 0.63 a Nd Nd Nd TOTAL 20640.62 ± 200.36 11383.15 ± 116.06 11926.95 ± 96.36 16097.32 ± 73.05 Resul s a e exp essed as mean ± s anda d de ia ion as mg o single s anda d/Kg o ex ac ; RT = e en ion ime; λmax = wa eleng h o maximum abso bance; samples a e ex ac s ob ained by Soxhle ex ac i e echnique (SOX), Accele a ed Sol en Ex ac ion a 40 °C (ASE 40), Accele a ed Sol en Ex ac ion a 50 °C (ASE 50) and Ul asound Assis ed Ex ac ion (UAE); nd = no de ec ed; di e en supe sc ip le e s (a, b, c and d) deno e s a is ically signi ican di e ences in he same column (p < 0.05). Mo eo e , o he 3 la onols could be p esumably iden i ied on he basis o he li e a u e [que ce in-3-O-galac oside, que ce in-3-O-glucu onide and que ce in-3-O-glycoside]. SOX ex ac o lea issue allowed he iden i ica ion o hese phenolic compounds. Howe e , gallic and ca a ic acid, que ce in-3-O-glucoside and que ce in we e no p esen in ASE 40, ASE 50 and UAE. Fo quan i ica ion analysis, pu e compounds we e used as s anda ds. All he analyzed compounds, de ec ion wa eleng hs, maximum abso p ions, e en ion imes and concen a ions a e lis ed in Table 2. Thei o al amoun (exp essed as mean ± s anda d de ia ion /Kg o ex ac ) anged om 20,640.62 ± 200.36 mg /Kg in SOX ex ac o 11383.15 ± 116.06 mg /Kg in ASE 40 ex ac .The iden i ied compounds we e in acco dance wi h o he wo ks al eady published on di e en lea ex ac s o V. ini e a L. [18,47]. The hyd oxycinnamic and hyd oxybenzoic acids a e no mally ound in g ape peels, howe e lea es can be also conside ed a new sou ce o phenolic acid [48,49]. Among hyd oxybenzoic acids, gallic acid was ound in SOX ex ac (159.91 ± 1.54 mg/Kg). Mo eo e , benzoic acid is p esen in all samples wi h a mean alue o 327.03 mg/Kg; ASE 40 was he ex ac wi h he highes con en o benzoic acid (452.83 ± 2.76 mg/Kg). In compa ison, he hyd oxycinnamic acid con en was highe han ha o hyd oxybenzoic acids. The mos abundan hyd oxycinnamic acid Figu e 1. HPLC ch oma og am o Soxhle (SOX) lea ex ac o Vi is ini e a c . Aglianico (320 nm). Peaks: 1, gallic acid; 2, ca a ic acid; 3, (+)-ca echin; 4, ca eic acid; 5, benzoic acid; 6, u in; 7, que ce in-3-O- galac oside; 8, que ce in-3-O-glucoside; 9, que ce in -3-O-glucu onide; 10, que ce in-3-O-glycoside; 11, kaemp e ol-3-O-glucoside; 12, que ce in. Table 2. Quan i ica ion o compounds in Vi is ini e a (c . Aglianico) lea ex ac s. Peak Analy e RT (min) λmax SOX ASE 40 ASE 50 UAE 1 Gallic acid 3.9 280 159.91 ±1.54 aNd Nd Nd 2 Ca a ic acid 6.7 320 5706.97 ±77.20 a3651.71 ±52.91 b 4075.15 ± 23.40 c 6047.84 ± 41.30 d 3 (+)-Ca echin 14.7 280 1176.00 ±17.32 a300.52 ±0.41 b331.72 ±0.52 c682.81 ±2.72 d 4 Ca eic acid 16.3 320 557.31 ±2.68 aNd Nd Nd 5 Benzoic acid 21.8 280 131.23 ±4.89 a452.83 ±2.76 b315.78 ±1.85 c408.27 ±0.00 d 6 Ru in 28.1 365 205.69 ±15.34 a210.64 ±7.20 a192.23 ±18.89 a319.75 ±1.49 b 7 Que ce in-3-O-galac oside 29.5 365 2938.56 ±6.79 a1976.24 ±20.78 b 2003.21 ± 16.80 b2449.91 ±3.43 c 8 Que ce in-3-O-glucoside 29.9 365 2352.24 ±41.34 aNd Nd Nd 9Que ce in -3-O-glucu onide 30.3 365 2893.19 ±21.59 a2009.87 ±5.99 b2023.01 ±3.45 b2465.27 ±3.43 c 10 Que ce in-3-O-glycoside 30.8 365 3362.17 ±4.94 a2219.28 ±1.60 b 2450.58 ± 23.26 c 3150.82 ± 10.29 d 11 Kaemp e ol-3-O-glucoside 33.6 365 666.64 ±6.10 a562.06 ±24.41 b535.27 ±24.99 b572.65 ±10.39 b 12 Que ce in 38.1 365 490.71 ±0.63 aNd Nd Nd TOTAL 20640.62 ±200.36 11383.15 ±116.06 11926.95 ±96.36 16097.32 ±73.05 Resul s a e exp essed as mean ± s anda d de ia ion as mg o single s anda d/Kg o ex ac ; RT = e en ion ime; λ max =wa eleng h o maximum abso bance; samples a e ex ac s ob ained by Soxhle ex ac i e echnique (SOX), Accele a ed Sol en Ex ac ion a 40 ◦ C (ASE 40), Accele a ed Sol en Ex ac ion a 50 ◦ C (ASE 50) and Ul asound Assis ed Ex ac ion (UAE); nd =no de ec ed; di e en supe sc ip le e s (a, b, c and d) deno e s a is ically signi ican di e ences in he same column (p<0.05). Fo quan i ica ion analysis, pu e compounds we e used as s anda ds. All he analyzed compounds, de ec ion wa eleng hs, maximum abso p ions, e en ion imes and concen a ions a e lis ed in Table 2. Thei o al amoun (exp essed as mean ± s anda d de ia ion /Kg o ex ac ) anged om 20,640.62 ± 200.36 mg /Kg in SOX ex ac o 11383.15 ± 116.06 mg /Kg in ASE 40 ex ac .The iden i ied compounds we e in acco dance wi h o he wo ks al eady published on di e en lea ex ac s o V. ini e a L. [ 18 , 47 ]. The hyd oxycinnamic and hyd oxybenzoic acids a e no mally ound in g ape peels, howe e lea es can be also conside ed a new sou ce o phenolic acid [ 48 , 49 ]. Among hyd oxybenzoic acids, gallic acid was ound in SOX ex ac (159.91 ± 1.54 mg/Kg). Mo eo e , benzoic acid is p esen in all samples wi h a mean alue o 327.03 mg/Kg; ASE 40 was he ex ac wi h he highes con en o benzoic acid (452.83 ± 2.76 mg/Kg). In compa ison, he hyd oxycinnamic acid con en was highe han ha o hyd oxybenzoic acids. The mos abundan hyd oxycinnamic acid was ca a ic acid; as shown in Table 2, he highes con en was shown in UAE ex ac (6047.84 ± 41.30 mg/Kg). Guidoni e al. [ 50 ] iden i ied a a oyl es e s o ans-ca eic acid and ans-couma ic acid as he p incipal hyd oxycinnamic acid de i a i es in g ape ine lea es. The An ioxidan s 2020,9, 708 9 o 16 con en o la an-3-ols in he lea ex ac s o Aglianico g ape ine was highe han o he s udies [ 51 ]. In pa icula , he con en o (+)-ca echin in SOX was 1176.00 ± 17.32 mg/Kg. Conce ning he la onols and in acco dance wi h li e a u e [ 51 ], he amoun o que ce in de i a i es in lea es was highe han in he kaemp e ol de i a i es. Webe (1993) de ec ed di e en O-glycosides o que ce in and kaemp e ol in g ape ine lea es [ 52 ]. Consequen ly, we could suppose, wi h good app oxima ion, ha he peaks 7, 9 and 10 co esponded o some O-glycoside de i a i es, p obably que ce ine-3-O-galac oside, -3-O-glucu onide and -3-O-glycoside, espec i ely, basing on li e a u e e idence [ 52 ]. In he case o kaemp e ol glycosides, i co esponded o he -3-O-glucoside. Que ce in-3-O-glucoside is p esen only in SOX sample in huge quan i y (2352.24 ± 41.34 mg/Kg). In ela ion o he aglycones, que ce in was ound a low le els and i was de ec ed only in SOX sample wi h 490.71 ±0.63 mg/Kg. 3.3. To al Polyphenol Con en and An ioxidan Ac i i y The e alua ion o To al Polyphenol Con en (TPC) was ca ied ou using Folin-Ciocal eu me hod and he esul s we e exp essed as mg Gallic Acid Equi alen s (GAE)/g o d ied ex ac [ 33 ]. The o al phenolic con en o ex ac s anged om 143.37 ± 2.33 o 312.78 ± 13.56 mg GAE/g in ASE 40 and ASE 50 ex ac s, espec i ely. These esul s a e highe han hese epo ed by Fe hi, San aniello, Ze ize , C uciani, Fadda, Sanna, Do e, Maioli and D’hallewin [ 20 ], con i ming ha ou a emp o op imize ex ac ion condi ion has been success ul. High TPC alue was also obse ed in SOX ex ac (236.43 ± 2.62 mg GAE/g), as shown Figu e 2A. Di e en phenolic con en s o g ape lea es a e epo ed in he li e a u e. Uysal e al. [ 53 ] showed ha he o al phenolic con en o lea ex ac anged om 60.14 ± 4.33 and 64.66 ± 0.35 mg GAE/g by a Soxhle appa a us using me hanol and wa e espec i ely. Once again, ou choice o sol en s is no only cheape and mo e eco- iendly bu also p o ides be e esul s o he eco e y o polyphenols. The ee adical-sca enging ac i i y o ex ac s is consis en wi h TPC, he highes adical sca enging ac i i y was indeed obse ed o ASE 50 ex ac wi h IC 50 alue o 25.40 ± 0.54 µ g/mL (Figu e 2B), ollowed by SOX ex ac . Bo h ex ac s epo ed high con en o polyphenols ha can jus i y he good adical sca enging ac i i y. Howe e , ou esul s a e be e han hose p e iously epo ed o g ape lea ASE hyd oe hanolic ex ac (0.09 ±0.32 mg/mL) [20]. Conce ning he ORAC (exp essed as µmol o T olox equi alen (TE)/g o ex ac ), he alues anged om 3702.71 ± 366.90 o 5227.09 ± 261.32 µ mol TE/g (Figu e 2C) and SOX ex ac epo ed he highes ORAC alue. I is impo an o highligh ha he lea es o Aglianico p esen ed highe ORAC alues in compa ison wi h g ape lea ex ac s epo ed in o he s udies ( anging om 1.52 o 2.55 µmol T olox equi alen s/mg o ex ac ) [51]. Lipid pe oxida ion inhibi ion, ca ied ou by β -ca o ene bleaching (BCB) assay, showed ha all ex ac s exhibi ed mode a e β -ca o ene bleaching inhibi ion ac i i y, lowe han 50% a a inal sample concen a ion o 0.2 mg/mL. In ac , esul s anged om 4.50 ± 0.40 o 45.80 ± 3.20%AA in he UAE and SOX ex ac s, espec i ely (Figu e 2D). Se e al s udies showed no co ela ion be ween TPC and BCB [ 54 ]. In his con ex , i TPC gi es an indica ion o he le els o bo h lipophilic and hyd ophilic compounds, BCB, in con as , only gi es an indica ion o he le els o lipophilic compounds [ 55 ]. ASE 40 and ASE 50 ex ac s ha e simila alue o lipid pe oxida ion inhibi ion in spi e o e y la ge di e ences in TPC. This implies he p esence o app oxima ely simila amoun s o lipophilic an ioxidan s in bo h samples. A new concep , Rela i e An ioxidan Capaci y Index (RACI), was applied, in eg a ing an ioxidan capaci y da a de e mined by se e al me hods [ 42 ]. To compa e he an ioxidan capaci y o ex ac s de i ed om di e en chemical me hods, esul s o TPC, DPPH, ORAC and BCB we e used o calcula e he RACI. Da a o ela i e an ioxidan ac i i y we e ep esen ed in Figu e 3. Acco ding o ob ained esul s, he lea ex ac ob ained by Soxhle echnique showed he highes RACI alue o 0.76, ollowed by ASE 50 (0.65). An ioxidan s 2020,9, 708 16 o 16 59. Ga cia-Jimenez, A.; Te uel-Puche, J.A.; Ga cia-Ruiz, P.A.; Sau a-Sanma in, A.; Be na, J.; Rod í guez-L ó pez, J.N.; Ga cia-Cano as, F. Ac ion o y osinase on ca eic acid and i s n-nonyl es e . Ca alysis and suicide inac i a ion. In . J. Biol. Mac omol. 2018,107, 2650–2659. [C ossRe ] [PubMed] 60. Honisch, C.; Os o, A.; de Ma os, A.D.; Vincenzi, S.; Ruzza, P. Isola ion o a y osinase inhibi o om un ipe g apes juice: A spec opho ome ic s udy. Food Chem. 2020,305, 125506. [C ossRe ] Da a A ailabili y: All da ase used o his s udy is a ailable on eques . © 2020 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h p://c ea i ecommons.o g/licenses/by/4.0/).