Variations of Antioxidant Characteristics and Mineral Contents in Pulp and Peel of Different Apple (Malus domestica Borkh.) Cultivars from Pakistan

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1 Molecules 2012, 17, ; doi: /molecules Article OPEN ACCESS molecules ISSN Vritions of Antioxidnt Chrcteristics nd Minerl Contents in Pulp nd Peel of Different Apple (Mlus domestic Borkh.) Cultivrs from Pkistn Mleeh Mnzoor 1, Frooq Anwr 1,2, *, Nzmid Sri 3, * nd Muhmmd Ashrf 4, Deprtment of Chemistry nd Biochemistry, University of Agriculture, Fislbd-38040, Pkistn Deprtment of Chemistry, University of Srgodh, Srgodh-40100, Pkistn Fculty of Food Science nd Technology, Universiti Putr Mlysi, UPM Serdng, Selngor, Mlysi Deprtment of Botny, University of Agriculture, Fislbd-38040, Pkistn Deprtment of Botny nd Microbiology, King Sud University, Riydh 11451, Sudi Arbi * Authors to whom correspondence should be ddressed; E-Mils: fqnwr@yhoo.com (F.A.); nzmid@putr.upm.edu.my (N.S.); Tel.: (F.A.); (N.S.); Fx: (F.A.); (N.S.). Received: 13 October 2011; in revised form: 20 December 2011 / Accepted: 28 December 2011 / Published: 4 Jnury 2012 Abstrct: Vritions of phenolics, ntioxidnt ctivity, nd minerl contents in peel nd pulp of five pple (Mlus domestic Borkh.) cultivrs from Pkistn, nmely Red Delicious, Golden Delicious, Kshmiri Amri, Kl Kulu nd Sky Spur were pprised. The men extrct yield of ntioxidnt components obtined with 80:20 methnol-wter (v/v), ws found to be 22.1 g/100 g for peel nd 14.2 g/100 g for pulp on dry weight bsis. The mounts of totl phenolics nd totl flvonoids in peel nd pulp of different cultivrs of pple rnged from 1, ,587.9 mg gllic cid equivlent/100 g DW nd 1, ,816.4 mg ctechin equivlent/100 g DW nd 1, ,475.5 mg GAE/100 g DW nd mg CE/100 g DW, respectively. The inhibition of linoleic cid peroxidtion nd DPPH scvenging ctivity of the extrcts vried from nd % in peel, nd nd % in pulp, respectively. Reducing power of the tested fruit prt extrcts t concentrtion 12.5 mg/ml rnged from nd , respectively. With regrd to minerls nlysis, both fruit prts showed the mount of K to be the highest, followed by Mg, C, Fe, N nd Zn. The results reveled tht peel of the tested pple cultivrs in this study hd superior ntioxidnt cpcity nd minerl concentrtion thn the pulp, indicting significnt vritions between the prts

2 Molecules 2012, tested. Thus, consumption of pple fruits long with peel might be recommended to gining better nutritive benefits. Keywords: phenolic compounds; DPPH rdicl; ntioxidnt ctivity; Mlus domestic; Inter-cultivr vrition; minerl elements; fruit tissue 1. Introduction It is widely ccepted tht dietry intke of selected fruits, especilly those contining functionl bioctives such s phenolic cids, tnnins, flvonoids, nd vitmins nd essentil minerls, is linked to reduced prevlence of severl chronic diseses [1 3]. Apple (Mlus domestic Borkh.), member of the fmily Roscee, is one of the most frequently consumed fruits in mny regions cross the World. Apple fruit hs been identified s n excellent potentil source of crbohydrtes, minerls, dietry fiber nd ntioxidnt phenolics [3 5]. Recent studies revel tht consumption of pple fruit nd pple juice cn provide ntiprolifertive, nticrcinogenic, nd nti-inflmmtory helth benefits nd is strongly ssocited with lower incidence of lung cncer, virl diseses nd crdiovsculr disorders [1,3,6 8]. Apple peel, being rich source of phenolics nd dietry fiber, is lso vlued for its medicinl helth functions [5,6]. The composition nd distribution of nutrients nd high-vlue components such s phenolics minly depends upon genotypes, fruit tissue nd the mturity levels of fruits nd to smller extent on environmentl spects [9 12]. Unpeeled fruits possess higher contents of bioctive compounds s compred to peeled ones [8]. The fruit peels, lthough medicinlly importnt, re often discrded s n gro-wste nd hve not yet received ttention with regrd to their utiliztion for vlue-ddition rther thn being discrded. This might be due to the lck of commercil pplictions nd consumer s unwreness of the benefits of consuming fruit peels. Interestingly, the seed nd peel frctions hve been reported to hve higher ntioxidnt ctivity compred to the pulp frction. Gorinstein et l. [13] reported tht the minerl nd totl phenolic contents in persimmon nd pple peel were significntly higher compred to those in pulp. Similrly, Loentowicz et l. [8] reported tht pple nd per peel extrcts exhibited better potentil s nturl ntioxidnt supplements thn pulp extrct. Pkistn is blessed with wide rnge of gro-climtic conditions, which llow the production of both tropicl nd temperte fruits [14]. The most suitble soil nd climtic conditions for pple cultivtion previl in the northern hilly res of Punjb, Khyber Pukhtonkh nd Bluchistn. Apple is the fourth mjor fruit of Pkistn fter citrus, mngo nd bnn nd is widely consumed. Vrious cultivrs of pple being grown in Pkistn include Mshdy, Kshmiri Amri, Red Delicious, Sky Spur, Kl Kulu, Golden Delicious, etc. [15 17]. The totl re under pple cultivtion ws estimted to be thousnd h, while totl production ws s high s thousnd tons [18]. Severl studies hve been crried out on the phenolic profile nd ntioxidnt potentil of different cultivrs nd prts of pple fruit from vrious regions [1,4,5,9,13,19 21]. However, to the best of our knowledge, no reports on the ntioxidnt ttribute nd minerl composition of different prts of pple cultivrs commonly grown in Pkistn is vilble in the literture. Thus, this study ws undertken to

3 Molecules 2012, quntify nd compre the phenolic components, ntioxidnt ctivity nd minerl contents in pulp nd peel of five cultivrs widely cultivted in Pkistn. 2. Results nd Discussion 2.1. Extrct Yields, Totl Phenolic nd Totl Flvonoid Contents The peel nd pulp of fruits of the studied cultivrs of pple yielded promising mounts of extrctble mtter (dry weight bsis) with queous methnol. The men extrction yield of ntioxidnt components ws found to be 22.1 g/100 g for peel nd 14.2 g/100 g for pulp. Reltively higher extrction yield ws obtined for peel thn the pulp smples. The significnt differences in the extrction yield between peel nd pulp of sme fruits might be scribed to the vrying vilbility of extrctble components, resulting from the vried chemicl composition of the different tissues used. Li et l. [22] lso reported tht percent yield of pomegrnte peel (31.5%) extrct ws higher compred to tht of pulp (14.5%) extrct. Tble 1. Totl phenolics nd totl flvonoids contents of peel nd pulp extrcts of different cultivrs of pple (Mlus domestic Borkh.) fruit. Vriety TPC (mg gllic cid equivlent/100 g dry weight) TFC (mg ctechin equivlent/100 g dry weight) Peel Pulp Peel Pulp Golden Delicious ± 44.1 b ± ± ± 16.9 Red Delicious ± ± ± ± 19.9 Kshmiri Amri ± 43.4 b ± 24.7 b ± 27.9 b ± 15.8 Kl Kulu ± 49.4 b ± ± ± 17.7 Sky Spur ± 38.9 b ± ± 24.1 b ± 21.2 b Men ± ± 27.4 b ± ± 17.9 b Dt re men ± SD (n =3 3, P < 0.05). Different superscript letters within the sme column indicte significnt differences (P < 0.05) mong cultivrs. Different subscript letters within the Men s row indicte significnt differences (P < 0.05) between peel nd pulp. We nlyzed TPC nd TFC of the pple fruit extrcts on dry weight (DW) bsis using the spectrophotometric method. Plnt phenolics, with more thn 8,000 chemicl structures t present identified, re known to ct s n effective free rdicl scvengers nd ntioxidnts [22,23]. The most importnt nturl phenolics re flvonoids becuse of their brod spectrum biochemicl nd biologicl ctivities. Thus, the true ntioxidnt potentil is often more ccurtely reveled by expressing ntioxidnt ctivity in terms of totl phenolics nd flvonoids [23,24]. The contents of totl phenolics, clculted s gllic cid equivlent (GAE), of 80% methnolic extrcts of pple peel nd pulp re presented in Tble 1. Totl phenolic contents rnged from 1, ,587.9 mg GAE/100 g in the peel extrcts nd 1, ,475.5 mg GAE/100 g in the pulp extrcts. Of the cultivrs tested, Red Delicious nd Kl Kulu peel extrcts exhibited significntly higher contents of totl phenolics, 2,587.9 nd 2,274.8 mg/100 g, respectively, followed by Golden Delicious (2,102.4 mg/100 g) nd Kshmiri Amri (2,097.1 mg/100 g). The lowest (1,907.5 mg/100 g) content of totl phenolics ws observed for Sky Spur. In cse of pulp extrcts, highest (1,475.5 mg/100 g) mount

4 Molecules 2012, of totl phenolics (TP) ws exhibited by Red Delicious pple nd the lowest (1,185.2 mg/100 g) by Kshmiri Amri. The mount of TP in the pulp extrcts of Golden Delicious nd Sky Spur pple were 1,298.2 nd 1,201.2 mg/100 g, respectively. The mount of totl soluble flvonoids, expressed s ctechin equivlent (CE), mong peel of fruits of five pple cultivrs rnged from 1,214.3 to 1,816.4 mg CE/100 g of extrct (Tble 1). The peel extrcts of Red Delicious pple exhibited the highest (1,816.4 mg/100 g) flvonoid contents followed by Kl Kulu (1,694.6 mg/100 g). These contents were higher thn those for the peel extrcts from Golden Delicious (1,501.5 mg/100 g), Kshmir Amri (1,398.4 mg/100 g) nd Sky Spur (1,214.3 mg CE/100 g). For pulp extrcts, the mounts of totl flvonoids (TF) vried from mg CE/100 g (Sky Spur) to mg/100 g (Kl Kulu). Overll, there ws no considerble vrition in the mounts of TP nd TF mong the five pple cultivrs, however these levels differed significntly between the two prts (peel nd pulp) tested. As expected, the peel of ll the cultivrs nlyzed hd significntly higher concentrtions of TP nd TF thn those determined for pulp. The results of our present investigtion re in greement with those reported by Vieir et l. [19], who found tht peel extrct of different vrieties of pple fruit contined greter mount of phenolics, nthocynins nd flvonoids thn pulp extrct. In greement to our present dt, Abrosc et l. [1] reported tht pple peel extrcts hve higher mount of phenolics nd flvonoids thn pulp extrcts. Likewise, in nother study by Drogoudi et l. [20], pple peel extrcts exhibited 1.5 to 9.2 times greter totl ntioxidnt ctivity nd 1.2 to 3.3 times higher contents of totl phenolics thn those of flesh (pulp) extrcts. In order to ssess the dietry impct of consumption of pple fruit on the intke of ntioxidnt phenolics, their supplies on the bsis of fruit serving were clculted. The clcultion ws bsed on fresh fruit serving of 100 g which comprised of 80 g pulp + 15 g skin + 5 g ovry with seeds. On the bsis of this clcultion, TP nd TF contents were rnged from nd mg/serving, respectively. The highest contribution of TP nd TF ws provided by Red Delicious (60.2 nd 40.2 mg/serving) followed by Kl Kulu (53.9 nd 34.4 mg/serving) nd Golden Delicious (45.2 nd 30.8 mg/serving) wheres, lowest by Kshmiri Amri (38.7 nd 26.2 mg/serving) nd Sky Spur (38.1 nd 28.8 mg/serving), respectively. In generl, it is observed tht pple peel possess higher contents of phenolics nd flvonoids compred to the pulp, becuse of the nture nd distribution of phenolics nd flvonoids vries within different prts of sme fruit. The pulp of pple fruit contins ctechin, phloretin glycoside, procynidins, cffeic cid; the peel possesses ll of these compounds nd hs dditionl flvonoids such s nthocynins, quercetin glycosides nd cynidin glycoside, not present in the pulp [25]. The presence of higher mounts of totl phenolics nd flvonoids in peel, compred with pulp, in the present nlysis of pple fruits cn lso be justified by the findings of other reserchers [1,8,9,20] Reducing Power of Apple Extrct Mesurement of reducing power lso defines n importnt spect of ntioxidnt ctivity of plnt extrcts. In this ssy, the presence of reducing gent in the nlyte smple results in the reduction of the ferric/ferricynide complex to its ferrous (Fe +2 ) form. The mount of Fe 2+ is then quntittively monitored by mesuring the intensity of Perl s Prussin blue color complex t 700 nm. Higher bsorbnce vlue indictes higher reducing power nd thus higher ntioxidnt ctivity [24].

5 Molecules 2012, Tble 2 presents the reductive cpbility of peel nd pulp extrcts of different pple cultivrs. In the reducing power ssy, (extrct concentrtions used were: 2.5, 5.0, 7.5, 10.0 nd 12.5 mg/ml), lmost concentrtion-dependent increse in the bsorbnce of the smples tested ws recorded (dt not shown). Tble 2. Reducing power (bsorbnce vlues t 700 nm) of peel nd pulp extrcts (t concentrtion 12.5 mg/ml) from different cultivrs of pple (Mlus domestic Borkh.) fruit. Vriety Peel Pulp Golden Delicious 2.66 ± ± 0.04 b Red Delicious 2.89 ± ± 0.04 b Kshmiri Amri 2.60 ± ± 0.02 b Kl Kulu 2.69 ± ± 0.03 b Sky Spur 2.54 ± ± 0.03 b Dt re men ± SD (n = 3 3, P < 0.05); Different superscript letters within the sme column indicte significnt differences (P < 0.05) mong cultivrs; Different subscript letters within the sme row indicte significnt differences (P < 0.05) between peel nd pulp. The reductive cpbility of pple fruit s determined in the present nlysis ws comprble to those reported in longn fruit extrct [26]. Anlysis of vrince (ANOVA) indicted tht the reductive cpbility of pple peel extrcts ws significntly higher thn pulp extrcts. This might be due to the fct tht reductnts/phenolics compounds re present in higher concentrtions in the outer tissues (peel nd sub-epiderml tissue) of the fruits thn in the inner tissues (mesocrp nd pulp) [1,19,25]. There ws no significnt cultivr effect on the reducing power of the peel nd pulp DPPH Rdicl Scvenging Activity The chrcteristic feture of ntioxidnts to scvenge DPPH free rdicl is well ccepted nd is therefore most often selected s relible tool to evlute the free rdicl scvenging cpcity of different plnt extrcts. Interestingly, the DPPH rdicl ssy is incredibly sensitive towrds ctive ingredients even t lower concentrtions. Another beneficil spect is tht this test is time sving nd cn be used to nlyze btch of smples in shorter time. This procedure hs been often used for the ssessment of free rdicl scvenging bility of plnt-bsed ntioxidnt extrcts [27]. It is generlly predicted tht DPPH rdicl scvenging ctivity, nd with it ntioxidnt ctivity, is strongly ffected by the mount of phenolic compounds s well s the degree of hydroxyltion of the phenolic compounds [28,29]. The DPPH rdicl scvenging ctivity of peel nd pulp extrcts ws tested nd compred with BHA, TBHQ nd PG (Figure 1). The bsorbnce in this ssy ws recorded t different intervls (strting with 1 to 12 min) from the beginning of the rection. Highest vrition in scvenging ctivity, observed t 5 th min of the rection, ws used for clcultion purposes. The scvenging ctivity of positive control, BHA ws found to be higher thn those of the peel nd pulp extrcts. Other positive controls employed nmely TBHQ nd PG exhibited comprble scvenging ctivity with peel extrcts, but showed higher potentil thn the pulp extrcts.

6 Molecules 2012, Figure 1. DPPH rdicl scvenging ctivity of 80% methnolic extrcts of peel nd pulp from different cultivrs of pple (Mlus domestic Borkh.) fruit. Error brs indicte mens ± SD (n = 3 3); BHA, butylted hydroxynisole; TBHQ, tertiry butyl hydroquinone; PG, propyl gllte. As depicted, the peel extrcts derived from ll pple cultivrs exhibited pprecibly higher scvenging ctivity, rnging from %, compred to pulp extrcts, % (Figure 1). The peel extrct from Red Delicious showed the highest (80.8%) scvenging ctivity, wheres lowest (66.6%) for Sky Spur. In cse of pulp extrcts, the highest (51.1%) nd the lowest (42.9%) scvenging cpcity ws recorded for Red Delicious nd Kshmiri Amri, respectively. In close greement to our present dt, in previous study by Abrosc et l. [1], pple peel nd pulp extrcts (t 1.0 mg/ml) showed 78% nd 55% DPPH rdicl scvenging cpcity, respectively. No significnt vrition of rdicl scvenging cpcity, ws observed mong the tested pple cultivrs, however, the difference between peel nd pulp prts of the fruits ws significnt. High rdicl scvenging ctivity of peel extrct compred with tht of pulp extrct might be linked to the presence of greter mount of phenolic compounds, flvonols nd nthocynins in the former. It hs been reported tht free rdicl scvenging ctivity of plnt bsed food (fruits nd vegetbles) extrcts is minly due to phenolic compounds, which re mostly distributed in the epiderml tissue [1,30]. It is widely ccepted tht ntioxidnt ctivity of plnt-derived extrcts is lrgely due to the bility of intrinsic phenolics to donte hydrogen toms or electrons to cpture the free rdicls [31] Antioxidnt Activity of Apple Peel nd Pulp Extrcts in Linoleic Acid Peroxidtion System The inhibition of linoleic cid peroxidtion ws lso used to pprise the ntioxidnt ctivity of pple fruit extrcts. Both pple fruit prts (peel nd pulp) exhibited pprecible inhibition of linoleic peroxidtion s shown in Figure 2. The inhibition of peroxidtion rnged from 71.7 to 84.9% for the peel extrct, wheres 43.9 to 52.8% for the pulp extrct. The peel of Kl Kul nd Kshmiri Amri exhibited the highest (84.9%) nd lowest (71.7%) inhibition of peroxidtion, wheres; in cse of pulp

7 Molecules 2012, extrct Golden Delicious nd Sky Spur offered the highest (52.8%) nd lowest (43.9%) inhibition of peroxidtion, respectively. Figure 2. Antioxidnt ctivity of 80% methnolic extrcts of peel nd pulp from different cultivrs of pple (Mlus domestic Borkh.) fruit. Error brs indicte mens ± SD (n = 3 3); BHA, butylted hydroxynisole; BHT, butylted hydroxytoluene. A significnt difference in percent inhibition of peroxidtion ws observed between peel nd pulp prts of the five cultivrs of pple fruit. However, vrition of these dt mong different cultivrs ws found to be non-significnt. Considerble inhibition of linoleic cid peroxidtion by peel nd pulp extrcts tested might be ttributed to the presence of phenolics, nthocynins, epictechin nd flvone-3-ol in these fruits [25] Minerls Minerls ply key role in vrious physiologicl functions of the body, especilly in the building nd regultion processes [32]. Apples re considered s good source of dietry minerls. The results s given in Tble 3 of the min minerl nd trce elements of the tested pple cultivrs showed tht ll the smples nlyzed contined higher mount of potssium (K) followed by clcium (C), mgnesium (Mg) nd sodium (N). The contents of K nd C in pple peel nd pulp rnged from nd mg/100 g DW nd nd mg/100 g DW, respectively. The peel nd pulp of Golden Delicious exhibited the highest contents of K (980.9 nd mg/100 g), wheres, lowest (695.3 nd mg/100 g) in Sky Spur, respectively. The concentrtion of C ws highest in peel (72.1 mg/100 g) nd pulp (48.9 mg/100 g) of Red Delicious while lowest in peel (35.6 mg/100 g) nd pulp (19.8 mg/100 g) of Sky Spur. Potssium is n essentil element for norml body growth nd mintennce. It lso plys significnt role in the osmoregultion of cells nd tissues. Potssium deficiency my cuse vriety of

8 Molecules 2012, disorders such s musculr crmps, twitching, nd irregulr hert bet nd kidney filure [32,33]. Clcium is n importnt constituent of bones nd teeth nd it is ctively involved in the regultion of nerve nd muscle functions [34]. The contents of Mg nd N rnged from nd mg/100 g for peel while nd mg/100 g for pulp, respectively. In cse of peel, Red Delicious nd Kshmiri Amri exhibited the highest (65.9 mg/100 g) nd lowest (35.7 mg/100 g) vlues, wheres for pulp, Red Delicious nd Sky Spur showed the mximum (34.8 mg/100 g) nd minimum (15.6 mg/100 g) content of Mg, respectively. Interestingly, the peel nd pulp of Golden Delicious hd 7.3 nd 10.8 mg/100 g nd Kshmiri Amri 2.9 nd 5.3 mg/100 g of N, respectively. With regrd to the mounts of two essentil trce elements, the content of Fe in peel ( mg/100 g) nd pulp ( mg/100 g) of the tested pple cultivrs ws higher compred to Zn, mg/100 g in peel nd mg/100 g in pulp. The highest content of both of these trce elements ws found in Golden Delicious nd the lowest in Sky Spur. Being good sources of these two essentil elements tht re in short supply in the norml humn diet, the tested fruits, especilly Golden Delicious type pples, cn be explored s potentil source of Zn nd Fe [35]. The clcultion of K, C, Mg nd N on fresh pple serving portion bsis ws mde similrly s for TP nd TF contents. The mount of K, C, Mg nd N in Kl Kulu ws found to be highest (160.7, 11.8, 8.2 nd 2.8 mg/serving), followed by Golden Delicious (143.9, 13.1, 5.72 nd 1.6 mg/serving), Red Delicious (109.1, 14.1, 6.3 nd 1.1 mg/serving), Sky Spur (90.11, 5.9, 3.9 nd 1.7 mg/serving) nd Kshmiri Amri (89.9, 6.24, 3.69 nd 0.79 mg/serving), respectively. The contents of essentil trce elements, nmely Fe nd Zn rnged from nd mg/serving, with the highest vlue (0.35 nd 0.22 mg/serving) exhibited by Kl Kulu while lowest ws seen for Sky Spur (0.11 nd 0.04 mg/serving). Henriquez et l. [21] nlyzed importnt minerls in peel nd pulp of five different vrieties of pple fruit on wet bsis, nd found the following concentrtions: nd (mg/100 g of K), nd (mg/100 g of C), nd (mg/kg of Mg), nd (mg/kg of N), nd (mg/kg of Fe), nd (mg/kg of Zn), respectively. The present minerls nlysis results were in greement with those of Nour et l. [36] who reported tht potssium (K) ws the most bundnt nutrient in fruits of different pple vrieties, followed by mgnesium (Mg), clcium (C), sodium (N), nd iron (Fe). The content of K nd Mg s determined in our present investigtion ws found to lower thn those reported by Ekholm et l. [37] for pple peel (1,250 nd 70 mg/100 g DW) nd peeled Mlus sp. (960 nd 50 mg/100 g DW), respectively, while the mount of C in both the pple peel (50 mg/100 g DW) nd peeled Mlus sp. (40 mg/100 g DW) ws comprble with our present dt. Severl fctors, e.g., vriety, stte of ripeness, soil type, soil condition, nd irrigtion regime my cuse vrition in the minerl nd trce elementl contents in different types of fruits s well s within different prts of the sme fruit [38].

9 Molecules 2012, Tble 3. Minerl content in peel nd pulp tissues of different cultivrs of pple (Mlus domestic Borkh.) fruit. Vriety Prt used Minerl Contents (mg/100 g dry wt) K C Mg Golden Delicious Peel ± ± ± 1.18 Pulp ± 17.9 bc 36.7 ± 0.89 b 23.9 ± 0.49 b Red Delicious Peel ± 17.3 b 72.1 ± ± 1.29 Pulp ± 13.9 c 48.9 ± 0.99 b 34.8 ± 0.73 c Kshmir Amri Peel ± 15.5 c 44.7 ± 0.92 b 35.7 ± 0.69 c Pulp ± 11.2 d 22.9 ± 0.67 d 18.5 ± 0.37 bd Kl Kulu Peel ± 17.7 b 52.2 ± 1.12 b 55.8 ± 1.21 b Pulp ± 12.9 d 30.5 ± 0.69 bc 24.9 ± 0.49 d Sky Spur Peel ± 15.1 c 35.6 ± 0.78 bc 41.9 ± 0.87 c Pulp ± 11.7 de 19.8 ± 0.41 d 15.6 ± 0.35 d N Fe Zn Golden Delicious Peel 7.3 ± 0.21 b 2.4 ± ± 0.04 Pulp 10.8 ± ± ± 0.01 Red Delicious Peel 4.7 ± 0.18 c 1.8 ± 0.04 b 0.9 ± 0.06 Pulp 9.1 ± ± 0.05 b 0.7 ± 0.03 b Kshmir Amri Peel 2.9 ± 0.54 cd 1.6 ± 0.05 b 0.8 ± 0.05 b Pulp 5.3 ± 0.16 c 1.1 ± 0.02 c 0.5 ± 0.03 b Kl Kulu Peel 5.9 ± 0.13 c 2.2 ± ± 0.04 Pulp 8.2 ± 0.17 b 1.7 ± 0.03 b 0.8 ± 0.03 b Sky Spur Peel 7.0 ± 0.11 b 1.2 ± 0.02 bc 0.4 ± 0.02 bc Pulp 10.2 ± ± 0.13 c 0.2 ± 0.03 c Dt re men (n = 3) SD ± (n = 3, P < 0.05); Different lphbets in superscript within the sme column indicte significnt differences (P < 0.05) between peel nd pulp Correltions mong the Results Obtined from Different Antioxidnt Assys As fr s sttisticl evlution of the results of different ntioxidnt ssys is concerned, good but vrying correltion between TPC nd TFC ws observed for peel (r = ***) nd pulp (r = **) extrcts of pple fruit, s shown in Tble 4. Lin nd Tng [39] reported strong correltion between totl phenolics nd flvonoids contents in selected fruits nd vegetbles. In previous study by Vieir et l. [19], TPC nd TFC were found to be strongly ssocited with totl ntioxidnt ctivity in different prts (flesh, whole fruit nd peel) of three cultivrs of pple. These dt revel tht phenolic compounds contribute significntly towrds the ntioxidnt ctivity. Both the phenolic cids nd flvonoids, being n importnt plnt secondry metbolite, hve been commonly recognized for their potent ntioxidnt nd reductive cpcities. Antioxidnt ctivity of these compounds is minly ttributed to their redox potentil [9]. Tking into ccount the exmined prts nd cultivrs of fruits, in the present study, the extent of differences in phenolics nd flvonoids mong the pple cultivrs tested ws not so significnt compred to tht observed between the peel nd pulp. In the present investigtion, peel of different cultivrs of pple fruit ccounted for fold higher ntioxidnt ctivity compred to the pulp extrcts. Among the different cultivrs tested Red Delicious contined highest mount of TP with

10 Molecules 2012, its highest reduction potentil. The correltion between TPC nd reducing power of the peel nd pulp extrcts ws highly significnt (r = *** nd ***) s presented in Tble 4. Peel Pulp Tble 4 Comprison of results from different ntioxidnt ssys s represented by correltion coefficient (r). Vrible TPC TFC %Inhibition DPPH Reducing power TPC 1 TFC *** 1 %Inhibition ns ns 1 DPPH *** *** ns 1 Reducing power *** *** ns *** 1 TPC 1 TFC ** 1 %Inhibition ns ns 1 DPPH *** * ns 1 Reducing power *** * ns ** 1 ns: non significnt; *: level of significnce. In cse of pple peel extrct, very good correltion between TFC nd reducing power (r = ***) nd DDPH ssy (r = ***) ws observed, however, this correltion ws quite wek (r = * nd *) in the cse of the pulp extrct. A highly positive correltion between TFC nd reducing power for peel extrct might be ttributed to the fct tht in peels quercetin glycosides, flvonols nd nthocynins, due to their high concentrtion, might hve exhibited higher redox potentil nd rdicl scvenging ctivity compred to tht of the pulp. Likewise, positive correltion between TPC nd scvenging DPPH rdicl bility of pple peel nd pulp extrcts (r = *** nd ***) ws lso estblished. The correltion between these two ssys implied tht the compounds lible for free rdicl scvenging ctivity might be minly the phenolics. These phenolic compounds ct s free rdicl scvengers, chin brekers, nd electron donors. Previously, similr type of high correltion between TPC nd DPPH scvenging bility hs been reported for pple nd per fruits peel nd pulp extrcts [8]. In greement with our present dt, Abrosc et l. [1] lso found good correltion between totl phenolics contents nd DPPH free rdicl scvenging bility of different pple cultivrs. According to Wijngrd et l. [40], significnt correltion (r = 0.72) ws observed between the results of totl phenols nd DPPH rdicl scvenging cpcity of different fruits nd vegetble byproducts, including pple fruit. On the other hnd, no significnt correltion between the results of percent inhibition nd reducing power ssy for peel nd pulp ws recorded in the present experiments, which might be ttributed to the fct tht there my be some ntioxidtive compounds tht exhibited their ntioxidnt ctivity not only by donting hydrogen but lso by scvenging oxygen. Consequently, plnt extrcts with such ntioxidnt components might disply higher degree of percent inhibition s compred to their corresponding reducing power nd thus cn offer no significnt correltion. Furthermore, s expected, no significnt correltion ws observed between percent inhibition nd TFC dt of peel nd pulp extrcts which might be due to the reson tht not only the flvonoids but severl other compounds

11 Molecules 2012, such s vitmins, nthocynins, crotenoids etc. lso contribute to ntioxidnt ctivity in terms of mesurement of percent inhibition of peroxidtion [41]. Similrly, no significnt correltion between TPC nd percent inhibition for peel nd pulp extrct ws observed in the present nlysis which could be explined on the bsis tht not only the phenolics but some other compounds such s tocopherols nd crotenoids etc. lso donte ntioxidnt ctivity in terms of mesurement of percent inhibition of peroxidtion [41]. Likewise our present trends, totl phenolics nd percent inhibition of linoleic peroxidtion of different stored pple fruits showed wek correltion [42]. The present vrition in correltion coefficient mong different ntioxidnt ssys indictes tht single ssy is not sufficient to fully pprise the ntioxidnt ttributes of specific plnt mtrix [43,44]. Besides, synergistic effects mong different endogenous compounds re nother fctor which my lso contribute to enhncing the totl ntioxidnt cpcity of plnt extrcts [45]. 3. Experimentl 3.1. Smples Fresh fruits of five different pple cultivrs, nmely Golden Delicious, Red Delicious, Kshmiri Amri, Kl Kulu nd Sky Spur, were collected from the vicinity of Swt city, Khyber Pukhtonkh, Pkistn nd University of Azd Jmmu & Kshmir, Rwlkot cmpus, Rwlkot, Azd Kshmir, Pkistn. Three different smples of ech cultivr were rndomly hrvested. To ensure tht the cultivrs were the sme, the fruits were further identified nd uthenticted by the Deprtment of Horticulture, University of Agriculture, Fislbd, Pkistn Chemicls nd Regents Linoleic cid, 2,2-diphenyl-1-picrylhydrzyl (DPPH) rdicl, Folin-Cioclteu regent, butylted hydroxynisole (BHA) butylted hydroxytoluene (BHT), tertiry butyl hydroquinone (TBHQ), propyl gllte (PG) nd gllic cid were purchsed from Sigm Chemicl Co. (St. Louis, MO, USA). All other chemicls nd regents (nlyticl grde) used were from Merck (Drmstdt, Germny) or Sigm Aldrich (Buchs, Switzerlnd), unless stted otherwise Smple Preprtion After wshing with tp wter, the fruits were peeled off with the id of steel knife. The seed were removed from the pulp mnully nd the pulp nd peel recovered were sliced into pprox. 1 1 cm slices Dry Mtter Determintion In view of the vrying levels of moisture mong pple fruit vrieties, we mde ll clcultions on dry mtter bsis. For dry mtter determintion, AOAC procedure (method ) ws followed [46]. Briefly, known weight (5.0 g) of the smple ws subjected to n electric-oven (Memmert, Germny) drying using 105 C temperture, until constnt weight chieved.

12 Molecules 2012, Antioxidnt Activity of Fruits Extrction Homogenized fruit smple (ech 20 g) ws extrcted with queous methnol (200 ml, methnol-wter, 80:20, v/v) t room temperture for 8 hours using n orbitl shker (Gllenkmp, UK). The residue ws seprted from the extrct by filtering through Whtmn filter pper No. 1 nd re-extrcted twice with fresh solvent. The three recovered extrcts were pooled nd then excess of the solvent ws distilled off in vcuum rotry evportor (EYELA, Tokyo, Jpn) t 45 C. The semi-solid crude concentrted extrcts obtined were stored t 4 C, until used for further experimenttion Determintion of Extrct Yield The yield of extrct (extrctble components) expressed on dry weight bsis of peel nd pulp ws clculted from the following eqution: Yield (g/100 g) = (W 1 100)/W 2 where W 1 is the weight of the extrct residue obtined fter solvent removl nd W 2 is the weight of peel or pulp tken Determintion of Totl Phenolics Content (TPC) A clorimetric technique, bsed on Folin-Cioclteu regent, ws employed to determine the mount of totl phenolics [47]. The procedure involved the mixing of crude extrct (50 mg) with Folin-Cioclteu regent (0.5 ml) nd deionized wter (7.5 ml). After llowing the mixture to stnd for ten minutes t mbient (room) temperture, 20% queous sodium crbonte (w/v, 1.5 ml) ws dded. The mixture ws incubted t 40 C in wter bth for 20 min, followed by cooling using n ice bth. The bsorbnce of the finl mixture ws recorded t 755 nm (Spectrophotometer U-2001, Hitchi Instruments Inc., Tokyo, Jpn). For clcultion of TP mount, stndrd gllic cid clibrtion curve prepred by running solutions in the concentrtion rnge of ppm (R 2 = ) ws used. The results were expressed s gllic cid equivlents (GAE) mg/100 g of dry mtter Determintion of Totl Flvonoid Contents (TFC) Amounts of TF were estimted clorimetriclly. A previously described method [48] ws used wherein fruit extrct (1 ml contining 0.1 mg/ml dry mtter) ws mixed with wter (4 ml) in 10 ml volumetric flsk. At the strt, queous 5% NNO 2 (3 ml) ws dded to the volumetric flsk then t 5 min, 10% AlCl 3 (0.3 ml) nd t 6 min, 1.0 M NOH (2 ml) were dded sequentilly. Finlly, the volume ws mde up to 10 ml by dding more distilled wter nd the rection mixture in the flsk ws mixed thoroughly for homogeniztion. The bsorbnce ws noted t 510 nm using spectrophotometer. TFC were clculted using stndrd clibrtion curve, prepred by nlyzing ctechin stndrd solutions within the concentrtion rnge of mg/l (R 2 = ). The mounts were reported s ctechin equivlents (mg CE/100 g of dry mtter).

13 Molecules 2012, DPPH. Scvenging Assy 2,2 -Diphenyl-1-picrylhydrzyl (DPPH) free rdicl scvenging cpcity of the extrcts ws ssessed following previously described procedure [49]. In brief, freshly prepred DPPH methnolic solution (5.0 ml, g/l) nd the extrct (1.0 ml, contining 25 μg/ml of dry mtter in methnol) were mixed in test tube. Absorbnce of the rection mixture ws recorded t different time intervls (strting with 0 time to 10 min) t wvelength of 515 nm ginst control. A control, contining ll regents, except the fruit extrct ws processed under similr conditions. Synthetic ntioxidnts, nmely tert-butylhydroquinone (TBHQ), butylted hydroxynisole (BHA) nd propyl gllte (PG) t concentrtions of 25 ppm were used s positive controls. The percent remining mounts of DPPH rdicl (DPPH. ) were clculted from the clibrtion curve. Absorbnce mesured t the 5th min ws used for comprison of the rdicl scvenging ctivity of the extrcts Determintion of Antioxidnt Activity in Linoleic Acid System The ntioxidnt ctivity of the tested pple fruit extrcts ws lso determined by mesuring the inhibition of linoleic cid peroxidtion [50]. For this purposes, the extrct (5 mg of ech fruit) were dded seprtely to n emulsion which contined solution of linoleic cid (0.13 ml), 99.8% ethnol (10 ml) nd 10 ml of 0.2 M sodium phosphte buffer (ph 7). The mixture ws mde up to 25 ml with distilled wter nd incubted t 40 C up to 360 h. The mgnitude of linoleic cid oxidtion ws mesured by peroxide dt using the thiocynte method s described by Yen et l. [51]. Briefly, in flsk, ethnol (10 ml, 75% v/v), queous solution of mmonium thiocynte (0.2 ml, 30% w/v), smple solution (0.2 ml) nd ferrous chloride (FeCl 2 ) solution (0.2 ml, 20 mm in 3.5% HCl v/v) were dded sequentilly. After 3 min of vortexing, the bsorbnce of the rection mixture ws recorded t 500 nm with spectrophotometer. A control, contining ll regents, except the extrcts ws lso processed under similr conditions. Synthetic ntioxidnts, butylted hydroxytoluene (BHT) nd butylted hydroxynisole (BHA) were used s positive controls. Percent inhibition of linoleic cid oxidtion ws clculted using following eqution: 100 [(Abs. increse of smple t 360 h/abs. increse of control t 360 h) 100] Determintion of Reducing Power The reducing power of the extrcts ws determined ccording to previously described procedure [52] with slight modifictions. The extrcts ( mg/ml) were mixed with sodium phosphte buffer (5.0 ml, 0.2 M, ph 6.6) nd potssium ferricynide (5.0 ml, 1.0%) in test tube. The rection mixture ws incubted t 50 C for 20 min in wter bth nd then 10% trichlorocetic cid (5 ml) ws dded followed by centrifugtion of the mixture t 980 g for 10 min t 5 C (CHM-17; Kokusn Denki, Tokyo, Jpn). After centrifugtion, the upper lyer of the solution (5.0 ml) ws collected nd diluted further by dding distilled wter (5.0 ml) nd ferric chloride (1 ml, 0.1%). The bsorbnce of the finl solution ws red t 700 nm using spectrophotometer.

14 Molecules 2012, Minerl Composition Preprtion of Smples for Minerl Anlysis Briefly, dried smples of pple peel nd pulp (1.0 g in ech cse) were trnsferred into digestion flsks contining concentrted HNO 3 (5 ml). Similrly, in nother digestion flsk, blnk smple ws prepred by dding HNO 3 (5 ml), but without the smple [53]. The flsks were heted for 2 h on n electric hot plte (HP 220, UTEC Products Inc., Albny, NY, USA) t 80 to 90 C, fter tht, temperture ws rised to 150 C, letting to boiling the solution. Menwhile, pproprite volume of concentrted HNO 3 nd 30% hydrogen peroxide (H 2 O 2 ) were dded to the flsks, occsionlly nd boiling continued until cler solutions were obtined indicting the completion of the digestion process nd oxidtion of the orgnic mtter. The contents of the flsks were llowed to cool t room temperture then smll mount of distilled wter ws dded nd the solution filtered through Whtmn No. 42 (<0.45-μm Millipore) filter pper. Finlly, the volume ws mde up to 25-mL in volumetric flsk by dding distilled wter Preprtion of Stndrds nd Anlysis of Smples For quntittive purposes, working stndrd solutions of the elements nmely sodium (N), clcium (C), potssium (K), mgnesium (Mg), iron (Fe), nd zinc (Zn) were prepred from the stock stndrd solutions contining 1,000 mg/l of n element in 2 N nitric cid. Clibrtion nd mesurement of C, Mg, Fe nd Zn ws done on n tomic bsorption spectrophotometer (A Anlyst 300, Perkins Elmer, Wlthm, MA, USA) wheres, N nd K were nlyzed on flme photometer (Sherwood, UK). Stndrd clibrtion curves were constructed for ech element individully using liner correltion by lest squre method. A blnk reding ws lso recorded to mke necessry corrections during clcultion of elementl concentrtions Sttisticl Anlysis Three different smples of ech fruit cultivr were rndomly ssyed. Ech smple ws nlyzed individully in triplicte nd dt re reported s men (n = 3 3 1) ± SD (n = 9). The dt generted ws subjected to nlysis of vrince (ANOVA) using Minitb 2000 Version 13.2 sttisticl softwre (Minitb Inc., Stte College, PA, USA). The correltion nlysis ws done using the Person liner correltion method t the significnce level of Conclusions The phenolic components, ntioxidnt ctivity nd minerl composition of peel nd pulp tissues of different cultivrs of pple fruit grown in Pkistn were compred. Nutritionlly, mong pple cultivrs nlyzed, the highest per serving bsis contribution of totl phenolics nd totl flvonoids ws provided by Red Delicious, nd the lowest by Sky Spur. On the other hnd the Kl Kulu cultivr offered the mximum mount of minerls on per serving bsis thn others. The peel of the pple cultivrs tested exhibited higher contents of phenolics nd better ntioxidnt ctivity coupled with higher mounts of vluble minerls compred to those in the pulp suggesting tht fruit peel removl

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