Analytical Methods
Total antioxidant activity of hazelnut skin (Nocciola Piemonte PGI):Impact of different roasting conditions
Monica Locatelli *,Fabiano Travaglia,Jean Daniel Coïsson,Aldo Martelli,Caroline Stévigny 1,Marco Arlorio
Dipartimento di Scienze Chimiche,Alimentari,Farmaceutiche e Farmacologiche and DFB Center,Via Bovio,6,28100Novara,Italy
a r t i c l e i n f o Article history:
Received 31March 2009
Received in revid form 7August 2009Accepted 31August 2009
Keywords:Hazelnut skin Roasting
Polyphenols extraction Total antioxidant activity
a b s t r a c t
The thin brown perisperm (skin)that envelops hazelnut kernels is usually removed after roasting pro-cess,leading a phenolic-rich by-product.Principal aim of this work was to characteri the total antiox-idant activity of phenolic extracts obtained from roasted ‘‘Nocciola Piemonte PGI”hazelnuts skin.Different extraction solvents (methanol,acidified methanol,ethanol,acidified ethanol,and acetone/water)and different protocols (cold solvent-assisted extraction and mi-automated Soxhlet extraction)were employed.The influence of different roasting degree (180°C/10min and 180°C/20min)was also investigated.DPPH Åand ABTS Å+radical-scavenging methods,ferrous ions chelation activity and inhibition of lipid peroxidation investigated in this study demonstrated significant antioxidant properties for hazel-nut skin phenolics.The main mechanism involved appeared the antiradical activity,strictly related to the total phenolic content (r =À0.8798and À0.8285for DPPH Åand ABTS Å+assays,respectively).The acidifi-cation of extraction solvents led to a significant decrea of antiradical activity,whilst the different roast-ing conditions significantly influenced the chelation activity and the inhibition of lipid peroxidation,showing higher effectiveness for high-roasted hazelnut skin extracts.Converly,the direct measure of the antioxidant capacity of defatted hazelnut skins revealed higher ABTS Å+scavenging properties for med-ium-roasted sample.
Ó2009Elvier Ltd.All rights rerved.
1.Introduction
In food science,antioxidants are very important in that they act preventing lipid oxidation in food and decreasing the adver ef-fects of reactive species (ROS:reactive oxygen species;RNS:reac-tive nitrogen species)on normal physiological functions in humans (Huang,Ou,&Prior,2005).
Antioxidant synthetically obtained,like BHA (butylated hydroxyanisole)and BHT (butylated hydroxytoluene),are largely ud in food industry and are included in human diet.However,in recent years the u of natural antioxidants has been promoted becau of concerns regarding the safety of synthetic ones.Dietary components,including polyphenols,carotenoids and vitamins C and E,are considered effective antioxidants uful in the preven-tion of oxidative stress and related dias (Kaur &Kapoor,2001;Moure et al.,2001).
Widely distributed in the plant kingdom and abundant in our diet,polyphenols are among the most studied about class of anti-oxidants.Phenolics are the products of condary metabolism in
plants,providing esntial functions in the reproduction and the growth of the plants,acting as defens
e mechanisms against patho-gens,parasites,and predators,as well as contributing to the colour of plants (Liu,2004).In addition to their roles in plants,veral epi-demiological and clinical rearches demonstrated that phenolic antioxidants occurring in cereals,fruits and vegetables are princi-pal contributing factors for the decread incidence of veral chronical and degenerative dias (Shahidi,2000).
For all the reasons,in last few years veral studies have been conduced in order to investigate the antioxidant activity of phyto-extracts obtained from vegetable sources.Particularly,agricultural and industrial residues are considered as very attractive sources of natural antioxidants (Moure et al.,2001).By-products of grape (Vitis vinifera L.)processing,such as eds and peels,are the most studied and promising antioxidants sources (Shi,Yu,Pohorly,&Kakuda,2003).The extraction and antioxidant activity of phenolic compounds from other residual materials such as apple peel (Kim et al.,2005),apple pomace (Lu &Foo,2000),sweet orange peel (Anagnostopoulou,Kefalas,Kokkalou,Assimopoulou,&Papageor-giou,2005),blanched artichoke and artichoke blanching waters (Llorach,Espín,Tomás-Barberán,&Ferreres,2002),leaves and stems of cauliflowers (Llorach,Espín,Tomás-Barberán,&Ferreres,2003),olive mill waste (Mulinacci et al.,2005),cocoa by-products (Arlorio et al.,2008;Azizah,Ruslawati Nik,&Swee Tee,1999)and nut hulls (peanut,cashew nut,hazelnut,almond,pistachios,
ossine*Corresponding author.Tel.:+390321375774;fax:+390321375621.E-mail address:monica.locatelli@pharm.unipmn.it (M.Locatelli).1
Prent address:UniversitéLibre de Bruxelles (ULB),Institute of Pharmacy,Laboratory of Pharmacognosy,Bromatology and Human Nutrition,CP 205/9,Bd du Triomphe,Brusls 1050,Belgium.
Food Chemistry 119(2010)
1647–1655
Contents lists available at ScienceDirect
Food Chemistry
j o u r n a l h o m e p a g e :/loc
ate/foodchem
Chilean hazelnut,etc.)(Goli,Barzegar,&Sahari,2005;Kamath& Rajini,2007;Moure et al.,2000;Shahidi,Alasalvar,&Liyana-Path-irana,2007;Wijeratne,Abou-Zaid,&Shahidi,2006;Yu,Ahmedna, &Goktepe,2005)have been also investigated.The prence of polyphenols in outer layers(skins,peels,and hulls)of fruits,vege-tables and eds(nuts)may offer protection against oxidative stress:it is known that hulls play the major role in the defen of the plant eds and,together with bran fractions,concentrate most phenols and tannins(Shahidi&Naczk,1995).Moreover, polyphenols play an important role in the astringent taste,causing typical long-lasting puckering,shrinking,rough,and drying nsa-tion in the oral cavity(Stark,Bareuther,&Hofmann,2005).
The antioxidant activity of nuts and their by-products has been previously studied.The studies have highlighted that nut by-products are rich sources of natural antioxidants and phenolic compounds.Among nuts,hazelnuts(Corylus avellana L.)are very interesting in that rich in phenols and,particularly,proanthocyani-dins(Gu et al.,2003).Recent studies tentatively identified veral phenolic acids in both hazelnut kernels(Alasalvar,Karamac´,Amar-owicz,&Shahidi,2006;Yurttas,Schafer,&Warthen,2000)and hazelnut by-products(skin,green leafy cover,hard shell and tree leaf)(Contini,Baccelloni,Massantini,&Anelli,2008;Shahidi et al.,2007).The works demonstrated that hazelnut skin(or peri-sperm,or testa)is a rich and low-cost source of natural phenolic antioxidants.More recently,Alasalvar et al.(2009)obtained two fractions from crude phenolic extracts of Turkish Tombul hazelnuts skin(low-molecular-weight phenolics and tannins,respectively), showing higher antioxidant/antiradical activity for tannin fraction, followed by the crude extract and low-molecular-weight phenolic compounds.However,the impact of different roasting conditions on both phenols extraction and antioxidant activity should also to be investigated,particularly considering the formation of Mail-lard products(melanoidins)during roasting.
The aim of this work was to characteri the total antioxidant activity of phenolic extracts obtained from roasted‘‘Nocciola Pie-monte PGI”hazelnuts skin,considering different approaches(free radical-s
cavenging activity,chelation of pro-oxidant ferrous ions, inhibition of lipid peroxidation).Different extraction protocols were employed(cold solvent-assisted extraction and mi-auto-mated Soxhlet extraction)and the influence of different roasting process(medium-and high-roasting degrees)was investigated. Finally,the total antioxidant capacity of hazelnut skins(defatted powders)was determined using a direct measurement protocol.
2.Materials and methods
绯闻女孩第二季
2.1.Samples
Samples of hazelnut skins were kindly provided by Dr.Giuppe Zeppa(University of Turin,Italy).Hazelnut skins were obtained from Italian‘‘Nocciola Piemonte PGI”hazelnut kernels(C.avellana L.),namely Tonda Gentile delle Langhe cultivar,cultivated only in specific areas and according to the disciplinary of production of the protected geographical indication(PGI)‘‘Nocciola Piemonte”. Dried unshelled hazelnuts were roasted at two different condi-tions:180°C for10min(medium roasting,MR)and180°C for 20min(high roasting,HR),and hazelnut skins were recovered after spontaneous paration from the kernels after roasting.All samples were stored under vacuum and kept in the dark at À20°C until they were analyd.
2.2.Chemicals quercetin dihydrate,butylated hydroxyanisole(BHA)and diso-dium ethylenediaminetetraacetate dihydrate(Na2-EDTA))ud for the determination of total phenol content and antiradical activ-ity were purchad from Sigma–Aldrich(Milano,Italy).All chemi-cals and solvents were of reagent-grade level and purchad either from Sigma–Aldrich(Milano,Italy).
2.3.Proximate composition analysis
The moisture content of hazelnut skin samples was determined using a thermo-balance Sartorius MA30(Sartorius AG,Goettingen, Germany).Total nitrogen content and total protein content(con-version factor:6.25)were obtained according to Kjeldahl method using the Kjeltec system I(FOSS Tecator,Sweden).The ash content was determined in a muffle furnace according to AOAC(1990)pro-cedure.Lipid fraction was extracted from ground hazelnut skins (after grinding and sieving particles size<1mm)using a mi-automatic Soxhlet Büchi Extraction System B-811(Büchi Labortechnik AG,Flawil,Switzerland)for12h,employing dichlo-romethane as solvent.All the results have reported as percentage on the basis of dry weight(dw).
2.4.Extraction of phenolic fraction
The extraction of phenolic fraction from high-and medium-roasted defatted hazelnut skins was perfor
med using two different methods:(i)cold-extraction under magnetic stirring and(ii)Soxh-let extraction.For cold-extractionfive different solvents were ud:methanol,acidified methanol(hydrochloric acid0.1%,v/v), ethanol,acidified ethanol(hydrochloric acid0.1%,v/v),acetone/ water80:20,v/v;methanol was chon as solvent for Soxhlet extraction.
2.4.1.Cold-extraction under stirring
Four grams of defatted hazelnut skins powders were extracted using100mL of solvent;extraction was carried out in clod Erlen-meyerflasks and under constant magnetic stirring,in the dark at room temperature(22°C).After1h of stirring/extraction,the sus-pension wasfiltered(Buchner funnel)through Perfecte2paper filter(Superfiltro,Milan,Italy),and the solid residue was re-ex-tracted with50mL of solvent for30min.This last step was re-peated until the complete decolouration was achieved (exhaustive extraction);then,filtrates were collected.The total time required to obtain the complete extraction varied depending on the solvents employed and on the different roasting conditions (data not shown).Finally,the solvent was evaporated to dryness (vacuum,40°C)and dry extract was stored atÀ20°C until u. 2.4.2.Soxhlet extraction
Ten grams of the defatted hazelnut skin powders were extracted with Soxhlet apparatus using metha
nol for7h.The sol-vent was then evaporated to dryness(vacuum,40°C)and the dry extract was stored atÀ20°C until u.
2.5.Determination of phenolic content
The determination of total phenolic content was obtained using the classic Folin-Ciocalteu assay,as previously described in Arlorio et al.(2008).Results were expresd as catechin equivalents, through the calibration curve of(±)-catechin monohydrate.The calibration curve linearity range was50–250l g(r=0.9987).
2.6.DPPHÅscavenging activity
1648M.Locatelli et al./Food Chemistry119(2010)1647–1655
et al.(2009).Samples and standard molecules were dissolved in methanol and appropriately diluted in order to obtain a calibration curve(concentration range from1to20l g mLÀ1).Antiradical activity was expresd as inhibition percentage(I%)and calculated using the following equation:
Inhibition percentageðI%Þ¼Abs controlÀAbs sample
Abs control
南京美甲Â100
Results werefinally expresd as EC50(antioxidant do re-quired to obtain a50%inhibition),calculated by probit regression analysis.
2.7.ABTSÅ+scavenging activity
The ABTS radical cation(ABTSÅ+)scavenging assay was per-formed according to the method reported by Re et al.(1999).Sam-ples and standard molecules were dissolved in ethanol and appropriately diluted in order to obtain a calibration curve(con-centrations range from10to900l g mLÀ1).Antiradical activity was expresd as inhibition percentage(I%),as previously de-scribed for DPPHÅassay.Results were expresd as EC50,calculated by linear regression analysis.
2.8.Chelation activity(ferrozine method)
The determination of ferrous ions chelation activity was per-formed according to the method reported by Liyana-Pathirana and Shahidi(2005)with some modifications.Briefly,500l L of sample or its relative solvent(control:water for Na2-EDTA,meth-anol for hazelnut skin extracts and other standard molecules), 10l L of aqueous2mM FeCl2,35l L of5mM ferrozine(3-(2-pyri-dyl)-5,6-diphenyl-1,2,4-triaz
ine-40,400-disulfonic acid sodium salt) and2.5mL of ethanol were added,adequately mixed,and left to stand for10min.Absorbance was immediately read at562nm, using a Kontron UVIKON930Spectrophotometer(Kontron Instru-ments,Milan,Italy).Samples and standard molecules were dis-solved in methanol(water in the ca of Na2-EDTA)and appropriately diluted to obtain a calibration curve.Becau of high extract concentrations employed(ranging from0.2to7mg mLÀ1), absorbance of blank solutions(without ferrozine)was measured to correct any influence due to colour of the extracts.The chelation activity,measured as inhibition percentage of ferrozine–Fe2+com-plex formation,was calculated by using the following equation:
Chelation activityðCA%Þ¼Abs controlÀðAbs sampleÀAbs blankÞ
Abs controlfriend
catti报名
Â100
Results werefinally expresd as EC50,calculated by linear regression analysis.
2.9.Inhibition of lipid peroxidation(ferric-thiocyanate method)
The determination of inhibition of lipid peroxidation was per-formed according to the ferric-thiocyanat
support是什么意思e(FTC)method reported by Zin,Hamid,Osman,and Saari(2006),with some modifications. First,100l L of linoleic acid were dissolved in4mL of EtOH,8mL of0.05M phosphate buffer(pH7.0)and3.9mL of distilled water. Three-hundred andfifty micro-litres of sample or solvent(5% methanolic ethanol,control)were added to1.4mL of the previ-ously described linoleic acid solution.This mixture was kept in a screwed-cap container in the dark and at a temperature of50°C; the accelerated oxidation of linoleic acid was measured after24, 48,72and96h of thermal treatment.The determination of oxida-tion degree(as peroxides formation)was performed according to anate and30l L of0.02M ferrous chloride in3.5%hydrochloric acid.Mixtures were shaken and exactly after3min the absorbance was measured at500nm,using a Kontron UVIKON930Spectro-photometer(Kontron Instruments,Milan,Italy).Samples and stan-dard molecules were tested at three different concentrations(10, 100,and1000l g mLÀ1).Results were expresd as inhibition of li-pid peroxidation percentage calculated by using the following equation:
Inhibition of lipid peroxidationðIP%Þ
¼100À
Abs sampleðtÞÀAbs sampleð0Þ
cmwap是什么意思
Abs controlðtÞÀAbs controlð0Þ
Â100
where(t)and(0)indicate that absorbances of the samples and the control were measured at time t(t=24,48,72and96h of thermal treatment)and at time zero(before the starting of the oxidation reaction),respectively.
2.10.Direct measurement of total antioxidant capacity(QUENCHER approach)
The direct measurement of total antioxidant capacity of hazel-nut skin was obtained following the procedure described by Ser-pen,Gökmen,Pellegrini,and Fogliano(2008).ABTSÅ+reagent was prepared as described and further diluted in a mixture of etha-nol:water(50:50,v/v)to obtain an absorbance of0.700±0.020at 734nm.Hazelnuts skin(defatted powders)werefinely ground and sieved(particles size<250l m);then,samples were tested in the ratio of0.15mg per6mL of ABTSÅ+reagent.Absorbance mea-surements were performed at734nm after exactly6,15,30and 60min to determine the time required to reach the steady state. Results were expresd as mol of trolox equivalents per kg of sam-ple through a calibration curve(linearity range:20–160mmol; r=0.9998).
2.11.Statistical analysis
Results were expresd as mean±standard deviation(SD)of at least three independent experiments.Differences were estimated by analysis of variance(ANOVA)followed by Tukey’s‘‘Honest Sig-nificant Difference”test.Differences were considered significant at p<0.05.Pearson’s correlation analysis was ud to determine cor-relation coefficients and their statistically significance.All statisti-cal analys were performed using the free statistical software R 2.8.1version(www.R-project)(R Development Core Team,2008).
3.Results and discussion
3.1.Proximate composition of roasted hazelnut skin
First,the proximate composition of roasted hazelnut skin was determined(Table1).As expected,prolonged roasting time al-lowed the decreasing of moisture content.In order to avoid the influence of roasting conditions,lipids,proteins and ashes contents were reported on dry weight(dw)basis.Both MR and HR samples
Table1
Proximate composition of roasted hazelnut skin samples.
MR hazelnut skin HR hazelnut skin Moisture11.89±0.277.71±0.21
Lipids(dw)39.48±0.3237.41±0.76
Proteins(NÂ6.25)(dw)9.02±0.0310.42±0.20
M.Locatelli et al./Food Chemistry119(2010)1647–16551649
showed a proximate composition similar to that reported for raw hazelnut skin(Anil,2007).
护士节演讲稿
3.2.Extraction of phenolic fraction
Extraction of phenolic compounds is strongly affected by their chemical nature,the sample particles size,the extraction method employed,and the prence of interfering substances.Moreover, the solubility of phenolic substances is strictly dependent by the polarity of the solvent ud,as well as their degree of polymerisa-tion(Naczk&Shahidi,2004).In this work,the extraction of pheno-lic fraction from hazelnut skin wasfirst performed using different solvents(methanol,acidified methanol,ethanol,acidified ethanol, and acetone/water).An additional extraction by mi-automatic S
oxhlet was also performed using methanol as solvent(MeOHsox). In order to avoid the influence of lipid interfering compounds on phenols extraction and following analys,matrix was previously defatted by extraction with dichloromethane.Phenolics extraction yield,expresd as grams per100g of defatted samples,was in the order MeOH>MeOH/H+>Ac2O/H2O>EtOH/H+>EtOH>MeOHsox for MR hazelnut skin(range from43.67%to28.59%)and in the or-der Ac2O/H2O>MeOH/H+>EtOH/H+>MeOHsox>MeOH>EtOH for HR hazelnut skin(range from42.65%to27.95%).Extraction yields obtained for aqueous acetonic extracts(38.54%and42.65% for MR and HR samples,respectively)were higher than that re-ported by Contini et al.(2008),which obtained a32.6%extraction yield employing the same solvent,but using a long-time macera-tion extraction method.A recent study showed that aqueous ace-tone(80:20acetone/water,v/v)was a more effective solvent than aqueous methanol(80:20methanol/water,v/v)to extract condend tannins from hazelnut skin(Alasalvar et al.,2009). The other solvents ud in this work(MeOH,MeOH/H+,EtOH and EtOH/H+)were not previously considered for phenolic extraction from hazelnut skin.It can be obrved a different order of solvents depending on medium roasted and high-roasted samples:in fact, different thermal treatments might be induce modifications on the chemical composition and cellular structure of the original ma-trix(Saklar,Ungan,&Katnas,2003;Özdemir et al.,2001).
幼儿园六一儿童节主持词
3.3.Determination of total phenolic content
Total phenolic content of hazelnut skin is shown in Table2;re-sults are expresd as milligrams of catechin equivalent(CE)per gram of extract.The amount of phenolic compounds ranges from 637.65to380.24mg CE gÀ1and706.35to551.59mg CE gÀ1for MR and HR samples,respectively.The very low phenols content obtained for medium-roasted sample using methanol under stir-ring is presumably due to the unexpected incomplete solubility of this extract in methanol.So,this value is considered underesti-mated.Concerning both medium and high-roasted skin the most effective extraction solvent resulted ethanol(total phenolic con-tent expresd per gram of extract);however,considering results on a dw basis,the higher phenolic content was obtained using the aqueous acetonic mixture(181.51and190.88mg CE gÀ1of hazelnut skin,dw for MR and HR samples,respectively).Significant differences were obrved among the extracts.Overall,acidified solvents extracted lower amounts of phenolic compounds than not-acidified ones(p<0.05).Instead,different roasting conditions do not significantly influence the quantity of extractable polyphe-nols;the prolonged thermal treatment ems to not affect the total phenolic content of hazelnut skin(p>0.05).Results obtained in this work are similar to that obtained by other rearchers using different solvents and extraction method.Alasalvar et al.(2009)re-ported686and70
1mg CE gÀ1for crude extracts obtained from three concutive extractions at50°C(each of30min),using 80:20acetone/water(v/v)and80:20methanol/water(v/v)as sol-vent,respectively.Shahidi et al.(2007)reported a577.7mg CE gÀ1 phenolic content for hazelnut skin employing80/20(v/v)ethanol/ water mixture under reflux conditions at80°C;Contini et al. (2008)obtained499.7,588.2and546.6mg CE gÀ1for skin waste from whole roasted kernel using aqueous methanol(80/20,v/v), aqueous ethanol(80/20,v/v)and aqueous acetone(80/20,v/v), respectively,after long maceration at room temperature.Differ-ences in the total phenols content might be ascribed to different solvents and extraction methods ud,but also to different culti-vars,geographic origin and harvest ason of the samples(all the parameters are strictly related to biosynthesis of condary metab-olites such as phenolics).In this work,we analyd hazelnut skin obtained exclusively from‘‘Nocciola Piemonte PGI”hazelnut ker-nels,while authors previously cited ud by-products obtained from Turkish Tombul hazelnut(Alasalvar et al.,2009;Shahidi et al.,2007)or from a mixture of different varieties(Italian Tonda Gentile Romana,Tonda di Giffoni,Tonda Gentile delle Langhe; Turkish Tombul)(Contini et al.,2008).
3.4.Determination of antioxidant activity of hazelnut skin extracts
christmas in my heart歌词In order to better understand the antioxidant properties of hazelnut skin phenolic extracts,we perform
ed four different chem-ical in vitro assays,bad on different antioxidant mechanism. Antiradical properties were analyd using both DPPHÅand ABTSÅ+ scavenging assays,in that the methods show veral important differences in their respon to antioxidants and in their manipu-lation(Arnao,2000);then,ferrous ions chelation activity and inhi-bition of lipid oxidation(autoxidation of linoleic acid system)were determined.Trolox(uful and available as commercial standard compound in the evaluation of antioxidant properties),BHA(a syn-thetic antioxidant largely ud by food industry),some phenolic acids(gallic acid and caffeic acid)and some naturalflavonoids (epicatechin and quercetin)(qualitatively identified in hazelnut skin extracts,data not showed)were assayed for their antioxidant properties as reference compounds.
Table2
Total phenols content and antiradical activity(DPPHÅand ABTSÅ+methods)of hazelnut skin extracts.
Samples Total phenols(mg CE gÀ1of extract)DPPHÅ(EC50;l g mLÀ1)ABTSÅ+(EC50;l g mLÀ1) MR HR MR HR MR HR
MeOH380±24a701±107a,c10.11(8.49–12.09)a 3.67(3.19–4.21)a655±6a350±1a MeOH/H+513±50b564±66b,d 5.43(4.55–6.49)b 6.33(5.53–7.31)b484±26b439±27b EtOH638±35c,
d706±104a 4.70(3.75–5.90)c 3.91(3.38–4.52)a318±20c364±6c EtOH/H+575±21c,b552±64b,d 5.31(4.29–6.61)c,a 4.71(4.00–5.54)c374±19d379±14c Ac2O/H2O631±19d631±88a,b,c 5.02(3.99–6.31)c,b 5.00(4.13–6.06)c336±29c,d351±10a,c MeOHsox562±29b594±8c,d 5.46(4.30–6.94)c 4.01(3.33–4.83)a317±13c361±10a,c 1650M.Locatelli et al./Food Chemistry119(2010)1647–1655
3.4.1.DPPHÅscavenging activity
DPPHÅis one of the most ud synthetic radicals to evaluate antiradical properties of bioactive compounds and food extracts. It is more stable than common natural radicals(hydroxyl and superoxide radicals)and it is unaffected by certain side reactions, such as metal–ion chelation and enzyme inhibition.In this work, DPPHÅscavenging properties were evaluated testing at least six dif-ferent concentrations for each extract and repeating experiments at least in triplicate.Results were reported as concentration re-quired to obtained a50%radical inhibition(EC50,expresd as l g of extract per millilitre of solvent;Table2);higher antiradical activity corresponds to lower EC50values.Becau of the restricted linearity range between antioxidant concentration and radical inhibition(I%),EC50values were calculated on the basis of probit regression,according to the method reported by Locatelli et al. (2009).As previously obrved for total phenolic content,metha-nolic extract obtained from MR hazelnut skin showed the lowest DPPH antiradical activity(EC50:10.11l g
mLÀ1),in accordance with their incomplete solubility in the reaction solvent(methanol); so,antiradical activity of this sample has to considered underesti-mated.Ethanolic and methanolic extracts were characterid by higher scavenging properties for MR and HR samples,respectively. DPPH antiradical activity ranged from3.67to10.11l g mLÀ1;MR and HR samples activity was not significantly different(p>0.05), whilst acidified solvents extracts were less actives than corre-sponding not-acidified ones(p<0.05,methanolic extract from MR hazelnut skin was not considered becau of their incomplete solubility).Antiradical activity of hazelnut skin extracts was com-pared with that of antioxidant standard compounds(Table3).Ex-cept for MR methanolic extract,all hazelnut skin extracts were at least1.5-fold more active than BHA;HR methanolic extract was comparable to trolox(no significant difference was obrved). Compared to the other standard molecules,hazelnut skin extracts appeared less effective to scavenge DPPH radical.Becau of exper-imental differences among the DPPH methods reported in litera-ture,it is difficult to compare our results with that obtained by other authors.
3.4.2.ABTSÅ+scavenging activity
The basis of the method is to monitor the decay of the radical-cation ABTSÅ+produced by the oxidation of2,20-azinobis(3-ethyl-benzothiazoline-6-sulphonate)(ABTS)caud by the addition of anti
oxidants.Generally,results are expresd relative to trolox (trolox equivalents);however,on analogy to DPPHÅassay,in this work results were expresd as EC50values.For each extract and standard molecule at least nine concentrations were tested and at least three different experiments were performed.EC50values, expresd as l g of extract per millilitre of solvent(Table2),were calculated by linear regression analysis;linearity range between antioxidant concentration and antiradical activity(ABTSÅ+inhibi-tion percentage,I%)was verified for I%values upper than90%. Hazelnut skin extracts were solved in ethanol;both methanolic and acidified methanolic extracts from medium-roasted sample were partially insoluble in the solvent(solid insoluble residue was higher for methanolic extract than for acidified methanolic one),so values obtained for the extracts should be considered underestimated.ABTSÅ+scavenging activity(EC50values)ranged from655.15to317.24l g mLÀ1.Methanolic extracts obtained by Soxhlet apparatus(317.24l g mLÀ1)and cold-extraction under stirring(350.05l g mLÀ1)exhibited the highest ABTS antiradical properties for MR and HR hazelnut skin,respectively.As previously reported for DPPHÅmethod,MR and HR samples activity was not significantly different(p>0.05),whilst acidification of solvents led to a significant decrea of antiradical activity(p<0.01,par-tially insoluble extracts were not considered in this analysis).Com-pared to standard compounds,MR hazelnut skin extracts showed ABTSÅ+scavenging properties similar to epicatechin and trolox (p>0.05,partially insoluble extracts not included).The study of Alasal
var et al.(2009)showed for acetonic and methanolic aque-ous extracts of hazelnut skin6.33and6.36mmol of trolox equiva-lents per gram of crude extract,respectively.Shahidi et al.(2007) obtained for hazelnut skin ethanolic extract an ABTS radical anion (ABTSÅÀ)scavenging activity equal to132.0mg of trolox equivalent per gram of extract(TEAC);at the same concentration the aqueous ethanolic(80%ethanol)extract of almond brown skin showed a 52.9TEAC(Siriwardhana&Shahidi,2002).Kamath and Rajini (2007)reported that ethanolic extract of cashew nut skin was equally potent as BHA in ABTSÅ+scavenging assay(EC50of cashew nut skin extract:1.30l g mLÀ1).
3.4.3.Chelation activity
Metal-mediated formation of free radicals may cau various modifications to DNA bas,enhanced lipid peroxidation,and changes in calcium and sulphydryl homeostasis.Becau of high reactivity,iron is one of most important lipid oxidation pro-oxi-dants,particularly in its ferrous state.So,the effective Fe2+chela-tors may afford protection against oxidative damage by inhibiting production of ROS and lipid peroxidation(Liyana-Pathir-ana&Shahidi,2007).In this work,the ferrous chelation activity of hazelnut skin extracts was evaluated using the ferrozine method. At least six different concentrations for each extract were tested and experiments were repeated at least in triplicate.EC50values, expres
d as mg of extract per millilitre of solvent,were calculated by linear regression analysis;linearity range between antioxidant concentration and chelation activity(expresd as percentage, CA%)was verified for CA%values up to75–90%,depending on dif-ferent samples analyd.
Table4shows the results obtained measuring the ferrous che-lation activity of roasted hazelnut skin extracts.It is important to highlight that it was not possible to evaluate the activity of the samples extracted using acidified solvents(their absorbance values resulted higher than control and so not considered).
If compared to the antiradical activity,iron chelation capacity of hazelnut skin extracts were relatively weaker,ranging from
Table3
Antioxidant activity of standard compounds.
Samples Antiradical activity(EC50;l g mLÀ1)Chelation activity(EC50;mg mLÀ1)Inhibition lipid peroxidation(IP%;t=96h)
DPPHÅABTSÅ+100l g mLÀ11000l g mLÀ1
Trolox 3.32(3.05–3.62)a300±9a–17.2±3.2a100.0±0.7a Gallic acid 1.03(0.96–1.1)b84±5b–46.6±0.5a,b82.6±0.4b Caffeic acid 2.93(2.73–3.14)c214±10c–29.4±4.7a,b78.7±1.4b BHA8.18(7.44–8.99)d213±13c–97.6±0.8c97.4±0.3a Quercetin 1.99(1.77–2.23)e206±12c 5.07±0.4256.4±2.5b99.1±1.7a Epicatechin 3.11(2.88–3.34)a309±21a–30.8±1.9a,b68.1±4.8c
M.Locatelli et al./Food Chemistry119(2010)1647–16551651
