research article nutraceutical potential of tinctures from

Research ArticleNutraceutical Potential of Tinctures from Fruits Green Husksand Leaves of Juglans regia L

Urszula Gawlik-Dziki1 Agata Durak1 Aukasz Pecio2 and Iwona Kowalska2

1 Department of Biochemistry and Food Chemistry University of Life Sciences Skromna Street 8 20-704 Lublin Poland2Department of Biochemistry and Crop Quality Institute of Soil Science and Plant Cultivation State Research InstituteCzartoryskich Street 8 24-100 Pulawy Poland

Correspondence should be addressed to Urszula Gawlik-Dziki urszulagawlikuplublinpl

Received 31 August 2013 Accepted 24 October 2013 Published 28 January 2014

Academic Editors M Y Arica D Benke and S E Harris

Copyright copy 2014 Urszula Gawlik-Dziki et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The aim of this study was to assess the phenolic composition and nutraceutical potential of tinctures from fruits in two stagesof maturity (F3 younger F25 older) green husks (GH) and leaves (L) of Juglans regia L In all extracts gallic protocate-chuic 3-caffeoylquinic 3-120588-coumaroylquinic 4-caffeoylquinic 4-120588-coumaroylquinic and 120588-coumaric acids and quercetin-3-O-deoxyhexoside were detected using UPLC-MS technique Caffeic acid hexoside I and quercetin-3-O-deoxyhexoside I have beenidentified in GH tincture The highest ability to chelate Fe2+ was observed for GH tincture (EC

50= 7101 plusmn 355mgFMmL)

whereas the lowest was observed (EC50

= 13106 plusmn 655mgFMmL) for F3 tincture The highest reducing power was found forF3 and F25 (EC

50= 3247 plusmn 153 and 3607 plusmn 172mgFMmL resp) Ability of tinctures to prevent lipids against oxidation was

relatively low The highest activity (EC50= 12649 plusmn 632mgFMmL) was determined for F25 Tested tinctures showed relatively

high antiradical activitymdashEC50values ranged from 10056plusmn503 to 12904plusmn645mgFMmL for L and F25 respectivelyThe results

obtained suggest that J regia can be a source of bioactive compounds with antioxidant properties

1 Introduction

The genus Juglans (family Juglandaceae) comprises severalspecies and is widely distributed throughout theworld Greenwalnuts shells kernels and seeds bark and leaves are used inthe pharmaceutical and cosmetic industries [1 2] Walnutrsquosgreen husk is a by-product of the walnut production havingscarce use Thus using husk as a source of phytochemicalswill increase the value of the walnut production as well asoffer utilization for a by-product which is produced in a largequantity [3]

Different works demonstrated the potential antioxidantof walnut products (mainly fruits but also leaves) [1]and liqueurs produced by green fruits [2] In addition toantioxidant activity several studies have demonstrated theantimicrobial activity of phenols andor phenolic extractsof Juglans regia [4] making them a good alternative toantibiotics and chemical preservatives

Nowadays there is an increasing interest in the sub-stitution of synthetic food antioxidants by natural ones

The antioxidant compounds from waste products of foodindustry could be used for protecting the oxidative damage inliving systems by scavenging oxygen free radicals and also forincreasing the stability of foods by preventing lipid peroxida-tion [5] Special attention is focused on their extraction frominexpensive or residual sources coming from agriculturalindustries such as walnut fruit Furthermore leaves are easilyavailable in abundant amounts Walnut leaves are consideredto be a source of healthcare compounds and have beenintensively used in traditional medicine for the treatment ofvenous insufficiency hemorrhoids hypoglycemia diarrheaand fungal or microbial infections Dry walnut leaves arealso frequently used as infusions [1] The shell is used as afiltration media to separate crude oil from water [6] and thewalnut green husk is the basic material for the traditionalwalnut liqueur [2] The results obtained by Oliveira et al[3] and Carvalho et al [7] showed the potential of this lowcost natural material as source of phenolic compounds withantiradical and antimicrobial activities Phenolic compounds

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 501392 10 pageshttpdxdoiorg1011552014501392

2 The Scientific World Journal

play a number of crucial roles in the complex metabolism ofplants and also of fruit treesThey are involved in physiologi-cal processes of fruit tree growth and development and affectdifferent aspects of fruit pre- and postharvest life [8]

Foods of plant origin such as fruits and vegetables andwhole grain products have been suggested as a natural sourcefor antioxidants Antioxidants can play an important rolein disease prevention and health maintenance Plant-derivedproducts can be used either as a source of antioxidants inindustry or for medicinal purposes The antioxidant effectshown by these products proceeds fromphenolic compoundsand phytochemicals which is protected from harmful effectsof free radicals Walnut possesses a high content of 120572-tocopherol a vitamin E family compound which has antiox-idant activity mainly in the prevention of lipid oxidationprocess [1]

The aim of this study was to assess the nutraceuticalpotential of J regia fruits green husks and leaves ethanolicextracts For this purpose we have made a qualitative assess-ment of phenolic compounds Furthermore we analyzedthe total phenolic content phenolic acids and flavonoids inextracts their antioxidant activity and their effect on theactivity of some enzymes from the class of oxidoreductases

2 Materials and Methods

21 Chemicals ABTS (221015840-azino-bis(3-ethylbenzothia-zoline-6-sulphonic acid)) Folin-Ciocalteau reagent Arnovreagent gallic acid quercetin ferrozine lipoxygenasexanthine oxidase catalase and linoleic acid were purchasedfrom Sigma-Aldrich company (Poznan Poland) Acetonitrileand methanol gradient HPLC grade and formic acid LC-MSgrade for LC-UV-MS separations were purchased from JTBaker (Phillipsburg NJ) Water was purified in-house with aMilli-Q water purification system Simplicity-185 (MilliporeCo) All other chemicals were of analytical grade

22 Preparation of Samples The experimental material con-sisted of walnut fruits harvested at two stages of maturity(July 3 2012 and July 25 2012) Green husks and leaveswere harvested on September 23 2012 The research materialwas obtained from a private plantation of walnut in RadzynPodlaski PolandThe immature fruit green husks and driedleaves were shredded and were used to obtain extracts Foralcohol extraction 5 g of each plant material was extracted in50mL of EtOH (70 vv) for 2 weeks in darkness at 25∘Cand then the supernatant was recovered Obtained extractsas follows

F3 extract from walnut fruits harvested 3072012


GH extract from green husks of walnut

L extract from walnut leaves

The final extracts concentration was 01 g fresh mass (FM)mL

23 Ultraperformance Liquid Chromatography Compoundsof interest were analyzed using a Waters ACQUITY UPLCsystem (Waters Corp Milford MA USA) consisting of abinary pump system sample manager column manager andPDA detector (also from Waters Corp) Waters MassLynxsoftware v41 was used for acquisition and data processingThe samples were separated on a BEHC18 column (100mmtimes21mm id 17 120583mWaters Corp Milford MA USA) whichwas maintained at 40∘C The flow rate was adjusted to040mLmin The following solvent system mobile phase A(01 formic acid in Milli-Q water vv) and mobile phase B(01 formic acid in MeCN vv) was applied The gradientprogram was as follows 0-10min 5 B 10ndash240min 5ndash50 B 240-250min 50ndash95 B 250ndash270min 95 B 270ndash271min 95-5 B 271ndash300min 5 B Samples were keptat 8∘C in the sample manager The injection volume of thesample was 20 120583L (full loop mode) Strong needle washsolution (95 5 methanol-water vv) and weak needle washsolution (5 95 acetonitrile-water vv) were used UV-PDAdata was acquired from 220 nm to 480 nm at 5 pointsrate 36 nm resolution The separation was completed in 30minutes Peakswere assigned on the basis of theirUV spectramass to charge ratio (mz) and ESI-MSMS fragmentationpatterns

The MS analyses were carried out on a TQD mass spec-trometer (Waters Corp) equipped with a Z-spray electro-spray interface The following instrumental parameters wereused for ESI-MS analysis of phenolic compounds (negativeionization mode) capillary voltage 28 kV cone voltage40V desolvation gas N

2800 Lh cone gas N

2100 Lh

source temp 140∘C desolvation temp 350∘C Compoundswere analyzed in full scanmode (mass range of 100ndash1600 amuwas scanned)

For ESI-MSMS selected ions were fragmented usinga collision energy of 15 V (phenolic acids derivatives) or25V (flavonoids derivatives) and collision gas (argon) at01mLmin

24 Phytochemical Analysis Total phenols were estimatedaccording to the Folin-Ciocalteau method [9] A 01mL sam-ple of the extract was mixed with 01mL of H

2O with 04mL

of Folin reagent (1 5 H2O) and after 3min with 2mL of 10

Na2CO3 After 30min the absorbance of mixed samples was

measured at a wavelength of 720 nm The amount of totalphenolics was expressed as gallic acid equivalents (GAE)

Total flavonoids were estimated according to Lamaisonmethod [10] A 1mL sample of the extract was mixed with1mL AlCl

3times 6H2O (2 vv) and after 10min the absorbance

of mixed samples was measured at a wavelength of 430 nmThe amount of flavonoids was expressed as quercetin acidequivalents (QE)

Total phenolic acids were determined according Arnovmethod [11] 1mL of distilled water was mixed with 02mL ofextract 02mLHCl (05 vv) 02mLofArnova reagent and02mL NaOH (1M) The absorbance of mixed samples wasmeasured at a wavelength of 490 nmThe amount of phenolicacids was expressed as caffeic acid equivalents (CAE)

The Scientific World Journal 3

25 Determination of Iron Chelating Activity The method ofDecker and Welch [12] was used to investigate the ferrousion chelating ability of proteins and hydrolysates Brieflythe sample (05mL) was added to 02mL 2mM FeCl

2

solution and 02mL 5mM ferrozineThemixture was shakenvigorously and incubated at room temperature for 10minThe absorbance was subsequently measured at 562 nm in thespectrophotometerThepercentage of inhibition of ferrozine-Fe2+ complex formation was given according to the formula

chelation activity = [1 minus (119860119904

119860119888

)] times 100 (1)

where 119860119904is absorbance of sample and 119860

119888is absorbance of

controlAll assays were performed in triplicate

26 Determination of Reducing Power Reducing power wasdetermined by themethod of Oyaizu [13] A 05mL of extractwasmixedwith 05mL (200mM)of sodiumphosphate buffer(pH 66) and 05mL potassium ferricyanide (1 vv) andsamples were incubated by 20min at 50∘C After that 05mLof TCA (10 vv) was added and samples were centrifuged at650 g by 10min Upper layer (1mL) of supernatant wasmixedwith 1mL of distilledwater and 02mLof ferric chloride (01vv) The absorbance was subsequently measured at 700 nmin the spectrophotometer

27 Inhibition of Linoleic Acid Peroxidation [14] Ten micro-liters of sample was added into a test tube together with037mLof 005Mphosphate buffer (pH70) containing 005Tween 20 and 4mM linoleic acid and then equilibrated at37∘C for 3min The peroxidation of linoleic acid in the abovereaction mixture was initiated by adding 20120583L of 0035hemoglobin (in water) followed by incubation at the sametemperature in a shaking bath for 10min and stopped byadding 5mL of 06 HCl (in ethanol) The hydroperoxideformed was assayed according to a ferric thiocyanate methodwith mixing in order of 002M ferrous chloride (01mL)and 30 ammonium thiocyanate (01mL) The absorbanceat 480 nm (119860

119904) was measured with a spectrophotometer

for 5min The absorbance of blank (1198600) was obtained

without adding hemoglobin to the above reaction mixturethe absorbance of control (119860

100)was obtainedwith no sample

addition to the abovemixtureThus the antioxidative activityof the sample was calculated as

inhibition = 1 minus [(119860119904minus 1198600)

(119860100minus 1198600)] times 100 (2)

28 Determination of ABTS Radical Scavenging ActivityFree radical-scavenging activity was determined by theABTS∙+ method according to [15] This reaction is basedon decolourization of the green colour of the free ABTSradical cation (ABTS∙+) The radical solution was preparedwith ABTS and potassium persulfate diluted in ethanol atfinal concentration of 245mM and left at dark for 16 h toallow radical development The solution was diluted to reach

absorbance measures around 070ndash072 at 734 nm 18mLABTS∙+ solution was mixed with 004mL of each sampleThe absorbance was measured after one minute of reaction at734 nm 70 ethanol was used as blank Percentage inhibitionof the ABTS∙+ radical was then calculated using the followingequation

Scavenging = [1 minus (119860119904

119860119888

)] times 100 (3)



control (ABTS solution)All assays were performed in triplicateAntioxidant activities (except reducing power) were

determined as EC50mdashextract concentration (mg FMmL)mdash

provided 50 of activity based on a dose-dependent modeof action In the case of reducing power EC

50is the effective

concentration at which the absorbance was 05 and wasobtained by interpolation from linear regression analysis

29 Effect on the Activity of Some Enzymes from the Class ofOxidoreductases Inhibition of lipoxygenase (LOXI) activitywas determined spectrophotometrically at a temperature of25∘C by measuring the increase of absorbance at 234 nmover a 2min period [16] The reaction mixture contained245mL of 115molL phosphate buffer 002mL of lipoxyge-nase solution (167UmL) and 005mL of inhibitor (Juglansregia extract) solution After preincubation of the mixtureat 30∘C for 10min the reaction was initiated by adding008mL 25mmolL linoleic acid One unit of LOX activitywas defined as an increase in absorbance of 0001 per minuteat 234 nm 008mL 25mmolL linoleic acid One unit of LOXactivity was defined as an increase in absorbance of 0001 perminute at 234 nm

The inhibition of xanthine oxidase (XOI) activities withxanthine as a substrate wasmeasured spectrophotometrically[17] with the following modification the assay mixtureconsisted of 05mL of test solution 13mL of 115molLphosphate buffer (pH 75) and 02mL of enzyme solution(001UmL in 115M phosphate buffer) After preincubationof the mixture at 30∘C for 10min the reaction was initiatedby adding 15mL of 015mmolL xanthine solutionThe assaymixture was incubated at 30∘C and the absorbance (295 nm)was measured every minute for 10min XO activity wasexpressed as the percentage inhibition of XO in the aboveassay mixture system and was calculated as follows

inhibition = (1 minus Δ119860mintestΔ119860minblank

) times 100 (4)

where Δ119860mintest is the linear change in absorbance perminute of test material and Δ119860minblank is the linear changein absorbance per minute of blank

Influence on catalase (CAT) activity was assayed by themethod of Claiborne [18] with some modification The assaymixture consisted of 185mL phosphate buffer (005M pH70) 10mL H

2O2(0019M) 01mL of test solution and

005mL of enzyme solution (60UmL) The decompositionof H2O2was determined directly by the extinction at 240 nm


A300

10

05

00

(au

)

2

3

56

7

8

1113

15

22

24

26

2728

29

30

31

32 34 3536

3738

39 4042 44 45 46

00 25 50 75 100 125 150

L

Elution time (min)

(a)

A300

10

05

00

(au

)

1

00 25 50 75 100 125 150

F3

Elution time (min)

(b)

A300

10

05

00

(au

)

00 25 50 75 100 125 150

F25

Elution time (min)

(c)

A300

10

05

00

(au

)

00 25 50 75 100 125 150

GH

Elution time (min)

(d)

Figure 1 UPLC chromatograms of tinctures from different parts of J regia L extract from walnut leaves F3 extract from walnut harvested3072012 F25 extract from walnut harvested 25072012 and GH extract from green husks of walnut

per unit time (3min) whichwas used as ameasure of catalaseactivity The catalase activity was expressed as lmol of H

2O2

consumed per min

210 Statistical Analysis All tests were conducted in trip-licate All experimental results were mean plusmn SD of threeparallel measurements and data were evaluated by using one-way analysis of variance The statistical differences betweenthe treatment groups were estimated through Tukeyrsquos testStatistical tests were evaluated by using the Statistica 60software (StatSoft Inc Tulsa USA) All the statistical testswere carried out at a significance level of 120572 = 005

3 Results and Discussion

31 Identification of Phenolic Compounds In all extractsprepared the following eight phenolic compounds were

detected gallic protocatechuic 3-caffeoylquinic 3-120588-coum-aroylquinic 4-caffeoylquinic 4-120588-coumaroylquinic and 120588-coumaric acids as well as quercetin-3-O-deoxyhexosideFurthermore ethanolic extract of leaves is characterized bythe greatest diversity of phenolic compounds from the testedsamples as presented in Table 1 and Figure 1

On the other hand two phenolic compounds that havebeen identified in the extract of the green husks were caffeicacid hexoside I and quercetin-3-O-deoxyhexoside I Thesecompounds in turn do not appear in the extracts of thefruits in both stages of maturity Unfortunately we wereunable to identify the dominant compounds in the extractsfrom the leaves but on the basis of literature data [1] itcan be concluded that naphthoquinones and flavonoids areconsidered as major phenolic compounds of Juglans regialeaves Juglone (5-hydroxy-14-naphthoquinone) is knownas being the characteristic compound of Juglans spp andis reported to occur in fresh walnut As dried leaves were


used juglone was not detected in any extract Further-more several hydroxycinnamic acids (3-caffeoylquinic 3-120588-coumaroylquinic and 4-120588-coumaroylquinic acids) andflavonoids (quercetin 3-galactoside quercetin 3-arabinosidequercetin 3-xyloside quercetin 3-rhamnoside and two otherpartially identified quercetin 3-pentoside and kaempferol 3-pentoside derivatives) of different walnut cultivars collectedat different times were studied by other research groups in aprevious work [19]

As with the qualitative profile extracts from Juglansregia fruits from both stages of maturity contained the samephenolic compounds However as further results presentedin Table 2 their content was significantly different

32 Phenolic Content As Table 2 presents all parts of J regiacontained significant amount ethanol-extractable phenoliccompounds (including flavonoids and phenolic acids) Tak-ing into account total phenolics it was observed that the high-est amount contained fruits in both maturity stages (11131plusmn538 and 10025 plusmn 431mgg FM for F3 and F25 resp) Inturn the highest content of flavonoids was observed in husks(16461 plusmn 823mgg FM) However the lowest amount offlavonoids (1647plusmn065mgg FM)was determined in fruits atthe youngest stage of maturity The highest concentration ofphenolic acids (32181plusmn1635 120583gg FM)was found for walnutfruits at the youngest stage of maturity while the lowest wasfound in the leaves of J regia

Consumption of certain phenolics in the food is con-sidered beneficial for human nutrition [20] Epidemiologicalevidence shows that foods rich in phenolics derived fromfruits is associated with lower risks of cancer and coronaryheart disease as well as cataracts brain and immune dysfunc-tion and stroke [21] Phenolics also contribute to astringentbut pleasant taste of the liqueur [2] Alamprese et al [22]studied the antioxidative potential of walnut liqueur andproved that antioxidant activity was directly correlated withthe total phenol content and this characteristic did not changeduring storage even for many years Halvorsen et al [23]reported that walnuts have one of the highest contents ofantioxidants among all analysed nuts and seedsThe phenoliccontent was influenced by ripeness of fruits Temperature andlength of steeping of the fruits in ethanol have little effect onthe phenolic composition of the liqueur Jakopic et al [24]in their work analyzed how much cultivar and maturationstage of the walnut influence the phenolic content in variousparts of the walnut and in traditionally prepared walnutliqueur These results indicate that early stage of maturityof walnut harvested on June 30 contained higher amountsof phenolic compounds than fruit harvested at a later stageon July 7 Similar results were obtained in the present studyChemical extracts prepared with walnuts collected on July 3were characterized by higher content of phenolic compoundsthan tinctures obtained from nuts harvested on July 25Furthermore Stampar et al [2] observed variation of certainphenolic compounds in green walnut husks during thegrowing season and they ascertained that the majority of thephenolics under investigation decreased during maturationand similar results were obtained in this study

33 Antioxidant Potential of J regia Extracts It has been rec-ognized that phenolic compounds are a class of antioxidantagents which act as free radical terminators Free radicalsare involved in many disorders such as neurodegenerativediseases cancer and AIDS Antioxidants through theirscavenging power are useful for the management of thosediseasesThemechanisms of action of flavonoids are throughscavenging or chelating processes [25ndash27] The antioxidantcapacity of walnut polyphenols has already been describedAnderson et al [28] reported the in vitro inhibition of humanplasma and low density lipoprotein (LDL) oxidation by a wal-nut extract containing ellagic acid gallic acid and flavonoidsFlavonoids can also protect cells by acting as free radicalscavengers inhibiting DNA damage and mutagenicity [29]

In the present work the antioxidant potential of ethano-lic extracts of J regia fruits green husks and leaves wasmeasured by four different assays chelating power reducingpower inhibition of lipid peroxidation and scavenging activ-ity on ABTS radicals (Figure 2)

The highest ability to chelate Fe2+ was observed fortinctures fromGH (EC

50= 7101plusmn355mgFMmL) whereas

the lowest was observed (EC50

= 13106 plusmn 655mgFMmL)for tincture from F3 All tested parts of J regia containedethanol-extractable compounds with high reducing powerThe highest activity was determined for tinctures fromF3 andF25 (EC

50= 3247 plusmn 153 and 3607 plusmn 172mgFMmL resp)

while activity of other samples were significantly lower (EC50

value averaged about 66mg FMmL) Contrary to previousresults ability of tested tinctures to prevent lipids againstoxidation was relatively low The highest EC

50values were

observed for F3 and L samples (22381 plusmn 1119 and 19327 plusmn966mgFMmL resp) The highest activity (EC

50= 12649 plusmn

632mgFMmL) was determined for F25 sample Testedtinctures showed relatively high antiradical activitymdashEC

50

values ranged from 10056plusmn503 to 12904plusmn645mgFMmLfor L and F25 respectively These results suggest that walnutscollected at early stage of maturity and leaves were the bestABTS radicals scavengers (Figure 2)

In several reports the antioxidant activity of J regia hasbeen described especially from walnut oil extracts [30 31]and traditional walnut liqueurs [2] Furthermore Pereira etal [1] reported the antioxidant potential of walnut leavesAntioxidant activity was accessed by the reducing powerassay and the scavenging effect on DPPH (22-diphenyl-1-picrylhydrazyl) radicals In a general way all of the studiedwalnut leaves cultivars presented high antioxidant activity(EC50values lower than 1mgmL) The main mode of action

of natural antioxidants is their ability to scavenge free radicalsbefore they can initiate free radical chain reactions in cellularmembranes or lipid-rich matrices as found in cosmeticsfoodstuffs and pharmaceutical preparations [32]

A significant role in catalysis of oxidative processesleading to the formation of hydroxyl and peroxyl radicals inthe Fenton reaction (O

2

∙minus + H2O2rarr ∙OH + HOminus + O

2)

plays presence of transition metal ions These processes canbe delayed and inactivation by chelating iron ions [26 27]Furthermore lipid peroxides and hydrogen peroxide in thepresence of the transition metals initiate the chain reaction


Table 1 Identification and occurrence of major phenolic compounds in Juglans regia fruits (F3 F25) green husks (GH) and leaves (L)tinctures Elution times correspond to the separation by UPLCMolecular mass was determined byMS as119898119911 Compounds were fragmentedin MSMS experiments and the119898119911 values for the main daughter ions are given in brackets

No Elution time (min) [M-H]minus ion MS2daughter ions UV absorbance peaks (nm) Compound identity L F3 F25 GH

1 109 169 (125) 271 Gallic acid + + + +2 199 153 (109) 259 293 Protocatechuic acid + + + +3 247 353 (191 179) 324 3-Caffeoylquinic acid + + + +4 297 181 297 Unknown + + + +5 316 341 (281 179) 325 Caffeic acid hexoside I + minus minus minus

6 340 337 (163) 310 3-p-Coumaroylquinic acid + + + +7 364 341 (281 179) 323 Caffeic acid hexoside II + minus minus +8 406 353 (179173) 325 4-Caffeoylquinic acid + + + +9 426 451 (405) 310 Unknown + minus minus minus

10 460 339 (159) 258 313 Unknown + + + +11 476 325 (265 163) 310 p-Coumaric acid hexoside + minus minus minus

12 488 177 (159 115) 259 317 Unknown minus + + +13 514 337 (173) 311 4-p-Coumaroylquinic acid + + + +14 529 281 267 Unknown minus minus minus +15 560 163 (119) 308 p-Coumaric acid + + + +16 567 193 (175) 260 364 Unknown minus + + +17 586 513 (453 409 289) 260 364 Unknown minus + + minus

18 599 197 (169 125) 273 Unknown minus + + minus

19 638 435 (285 151) 290 Unknown + minus minus minus

20 679 381 (161) 253 360 Unknown minus + + minus

21 684 435 (303 285 151) 290 Unknown + minus minus +22 724 463 (301) 255 353 Quercetin-3-O-hexoside I + minus minus +23 741 491 (331 271) 264 Unknown minus + + +24 747 463 (301) 255 352 Quercetin-3-O-hexoside II + minus minus minus

25 757 435 (303 285 151) 290 Unknown + + + +26 783 433 (301) 255 352 Quercetin-3-O-pentoside I + minus minus minus

27 802 433 (301) 255 354 Quercetin-3-O-pentoside II + minus minus minus

28 811 447 (285) 264 350 Kaempferol-3-O-hexoside + minus minus minus

29 828 433 (301) 256 352 Quercetin-3-O-pentoside III + minus minus minus

30 861 447 (301) 255 346 Quercetin-3-O-deoxyhexoside + + + +31 886 417 (285) 265 346 Kaempferol-3-O-pentoside I + minus minus minus

32 905 417 (285) 265 346 Kaempferol-3-O-pentoside II + minus minus minus


34 941 417 (285) 364 345 Kaempferol-3-O-pentoside III + minus minus minus

35 984 431 (285) 264 Kaempferol deoxyhexoside + minus minus minus

36 1037 501 (281 179) 324 Dicaffeic acid hexoside + minus minus minus

37 1051 489 (301) 252 337 Quercetin-3-O-acetyl-deoxyhexoside I + minus minus minus

38 1057 475 (301) 254 348 Quercetin-3-O-acetyl-pentoside + minus minus minus

39 1100 609 (463 301) 263 Quercetin deoxyhexoside-hexoside + minus minus minus

40 1130 489 (301) 255 361 Quercetin-3-O-acetyl-deoxyhexoside II + minus minus minus

41 1168 485 (265 163) 262 312 Unknown + minus minus minus

42 1184 473 (285) 265 312 Kaempferol acetyl-deoxyhexoside I + minus minus minus

43 1234 469 (145) 300 Unknown + minus minus minus

44 1249 473 (285) 264 311 Kaempferol acetyl-deoxyhexoside II + minus minus minus

45 1272 473 (285) 262 Kaempferol acetyl-deoxyhexoside III + minus minus minus

46 1332 285 265 363 Kaempferol + minus minus minus

Plus (+) and minus (minus) signs represent occurrence of each compound


Table 2 Comparison of total phenolics total flavonoids and phenolic acids content in different parts of J regia

Plant material Total phenolic content[mgg FM]

Total flavonoids content[mgg FM ]

Total phenolic acids content[120583gg FM ]

F3 11131 plusmn 538a 1022 plusmn 055a 32181 plusmn 1635a

F25 10025 plusmn 436b 11356 plusmn 532b 28465 plusmn 1254b

GH 5248 plusmn 123c 16461 plusmn 823c 30945 plusmn 1026c

L 672 plusmn 035d 1647 plusmn 065d 2614 plusmn 478d

F3 extract from walnut harvested 3072012 F25 extract from walnut harvested 25072012 GH extract from green husks of walnut and L extract from walnutleavesBars (means) in columns followed by the different letters differ significantly (Tukeyrsquos test 119875 lt 005)

250

200

150

100

50

0

EC50

(mg

FMm

L)

F3F25

GHL

a a aab c c

e e

d d d

bc

g

h

f

Chel Red LPO ABTS

Figure 2 Antioxidant activities of tinctures from different J regiaparts CHEL chelating power RED reducing power LPO inhi-bition of lipid peroxidation ABTS antiradical activity F3 extractfromwalnut harvested 3072012 F25 extract fromwalnut harvested25072012 GH extract from green husks of walnut and L extractfrom walnut leaves Bars (means) followed by the different lettersdiffer significantly (Tukeyrsquos test 119875 lt 005)

of lipid peroxidation that continues until it is interrupted byan antioxidant [33] Iron salts in a biological system attach tobiologicalmolecules where they cause site-specific formationof ∙OH radicals and consequent damage to lipid proteinand DNA Propagation reactions of lipid peroxidation in abiological membrane do not proceed far before they reacha protein thus lipid peroxidation in vivo causes substantialdamage to membrane proteins [34] Almeida et al [35] intheir studies have shown that aqueous extracts of the leaves ofwalnut can be a source easily accessible natural antioxidantsIn addition they showed that tested extract may be helpfulin the prevention of lipid peroxidation present in food Inaddition the antioxidant activity of the extract of J regialeaves were justified for the therapeutic use in inflammatoryconditions On the other hand studies conducted by Amaralet al [19] demonstrated that a high content of tocopherol andvitamin E in walnut prevent oxidation of lipids AlthoughFoti et al [36] showed that flavonoids are a group ofcompounds which are most active in inhibiting peroxidationof linoleic acid

34 Effect on the Activity of Some Enzymes from the Classof Oxidoreductases Research on dietary polyphenols hasintensified over the past decade mainly due to the directradical scavenging properties of many such compoundsMore recently however it has become evident that polyphe-nols may also decrease oxidative stress through indirectantioxidant action such as the inhibition of ROS-producingenzymes such as lipoxygenase (LOX) and xanthine oxidase(XO) [37]

Xanthine oxidase (EC 11322) is a member of thexanthine oxidoreductase (XOR) group found in mammalsat the highest concentration within the liver and intestineAmong several mechanisms XO may be a potential sourceof superoxide and hydrogen peroxide The predominantxanthine dehydrogenase (XDH) form can be converted intoXO under severe conditions such as ischemic injury andthereby cause increased oxidative stress [35] Both XDHand XO convert hypoxanthine to uric acid via xanthineExcessive levels of uric acid in vivo may also lead to a stateof hyperuricemia andrenal stones Various studies have alsoassociated the involvement of XO with thermal stress respi-ratory syndrome viral infection and hemorrhagic shock Itcould therefore be hypothesized that a decreased activity ofXO may be considered to be beneficial to health [37 38]

Lipoxygenase (EC 1131112 linoleate oxygen oxidore-ductase) catalyzes the oxygenation of polyunsaturated fattyacids containing a cis-cis-14-pentadiene system to hydroper-oxides The lipoxygenase pathway of the arachidonic acidmetabolism produces ROS and these reactive forms ofoxygen and other arachidonic acid metabolites might playa role in inflammation and tumor promotion Inhibitionof the arachidonic acid metabolism is also correlated withtumor promotion in animal models [27 39] The interactionof flavonoids with mammalian 15-LOX-1 merits particularattention as this enzyme is a potential target for the health-preserving effect of flavonoids [40]

As Figure 3 shows all kinds of extracts were a good sourceof LOX and XO inhibitors The highest LOX inhibition wasobserved for the extract from the leaves of walnut (EC

50=

11045mg FMmL) Other extracts showed a similar activityagainst LOX and EC

50value was from 16442mg FMmL

for F3 extract to 18642mg FMmL for F25 extract On theother hand extract from J regia fruits at second stage ofmaturity (F25) and extract fromgreen huskswere the best XOinhibitors EC

50= 10066mg FMmL and 10869mg FMmL


250

200

150

100

50

0

a

bab

cd

d d d

F3 F25 GH L

EC50

(mg

FMm

L)

LOXIXOI

Figure 3 Comparison of LOX and XO inhibitory activity oftinctures from different J regia parts F3 extract from walnutharvested 3072012 F25 extract from walnut harvested 25072012GH extract from green husks of walnut and L extract fromwalnut leaves Bars (means) followed by the different letters differsignificantly (Tukeyrsquos test 119875 lt 005)

respectively Slightly higher EC50

values were noted for thetwo other extracts

In the available literature there are no data on the effectof walnut extract on the activity of LOX and XO Thereforethe results indicating the ability of walnuts and tincturesderived froma variety of vegetative parts to inhibition of theseprooxidant enzymes should be emphasized which shows thatwalnuts and tinctures derived from a variety of vegetativeparts possess an ability to inhibit these prooxidant enzymes(Figure 3)

Dew et al [37] in their work studied the dietary role of XOinhibitors from different varieties of teas herbs fruit juicesvegetables and fruits They found that a particularly highpotential inhibitory activity of XO showed cranberry juiceand dark grape Furthermore Keszligler et al [25] demonstratedand reported cranberry juice as a protective agent againsturinary tract infection and kidney stone prevention productSaruwatari et al [41] have shown that drinking twice a dayextract prepared from 25 g of a herb (ginseng and ginger)for 5 days inhibited by 20ndash25 of the activity of XO Onthe other hand Dew et al [37] in their study compared theblack white and herbal teas The highest inhibitory activityXO had black tea closely followed by mint tea They alsoshowed that caffeine has no effect on the decrease or increasein the activity of XO

Most living organisms possess efficient enzymatic andnonenzymatic defense systems against the excess productionof ROS Antioxidant enzymes in particular superoxide dis-mutase (SOD) and catalase (CAT) are involved in cell defensemechanisms against oxidative damage Antioxidant enzymeshave an enormous theoretical advantage over exogenousantioxidants that are stoichiometrically consumed [42]

16

14

12

10

8

6

4

2

0

Cata

lase

activ

atio

n (

)

a

b

cc

F3 F25 GH L

Figure 4 Comparison of CAT-activatory abilities of tinctures fromdifferent J regia parts F3 extract from walnut harvested 3072012F25 extract from walnut harvested 25072012 GH extract fromgreen husks of walnut and L extract from walnut leaves Bars(means) followed by the different letters differ significantly (Tukeyrsquostest 119875 lt 005)

Catalase is a fundamental defense enzyme which cat-alyzes the dismutation reactions of hydrogen peroxide whichis one of the ROS

In the present study the ability of extracts to increaseCAT activity was investigated (Figure 4) It was observed thatbest CAT activators were tinctures from the leaves andwalnutgreen husks increasing CAT activity turn notify 1401 and1302 slightly weaker activity was observed for tinctures ofJ regia fruits at both stages of maturity

According to the free radical theory of ageing one mightexpect the activity of antioxidant enzymes to be altered Theactivity of these enzymes has been reported to either increaseor decrease during the ageing process Guemouri et al [43]reported that CAT activity decreased in gt65-year-old Frenchpopulation On the other hand Inal et al [44] found thatCAT activity increased with ageing The increase in CATactivities with age suggests an increase in H

2O2formation

During the ageing process steady-state concentrations oferythrocyte H

2O2might be considerably higher which could

lead to the induction of antioxidative enzymes an adaptivephenomenon For this reason the consumption of food richin CAT activators such as walnut tincture might be effectivein preventing the pathophysiological changes of ageing

4 Conclusion

Undoubtedly walnuts and ethanol extracts prepared fromdifferent vegetative parts are a rich source of phenolic com-pounds that has been proven by many researchers workingon this theme Furthermore these compounds can enhancethe effect of other antioxidants such as fat-soluble vitaminsand low molecular water soluble substances Moreover thehigh content of antioxidant components in plants decide ontheir significant role in the prevention of lifestyle diseasesThe results obtained suggest that J regia can be a sourceof bioactive compounds with antioxidant properties On thebasis of the analysis it can be concluded that the fruits of


walnut in the early stages of maturity contain significantlymore biologically active compounds than in the later stagesof fruit maturity Particularly noteworthy is the activity of thecompounds contained in the leaves of Juglans regia whichmay be easily accessible source of valuable substances

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] J A Pereira I Oliveira A Sousa I C F R Ferreira ABento and L Estevinho ldquoBioactive properties and chemicalcomposition of six walnut (Juglans regia L) cultivarsrdquo Food andChemical Toxicology vol 46 no 6 pp 2103ndash2111 2008

[2] F Stampar A Solar M Hudina R Veberic and M ColaricldquoTraditional walnut liqueurmdashcocktail of phenolicsrdquo FoodChemistry vol 95 no 4 pp 627ndash631 2006

[3] I Oliveira A Sousa I C F R Ferreira A Bento L Estevinhoand J A Pereira ldquoTotal phenols antioxidant potential andantimicrobial activity of walnut (Juglans regia L) green husksrdquoFood and Chemical Toxicology vol 46 no 7 pp 2326ndash23312008

[4] C Proestos N Chorianopoulos G-J E Nychas and MKomaitis ldquoRP-HPLC analysis of the phenolic compoundsof plant extracts Investigation of their antioxidant capacityand antimicrobial activityrdquo Journal of Agricultural and FoodChemistry vol 53 no 4 pp 1190ndash1195 2005

[5] D P Makris G Boskou and N K Andrikopoulos ldquoPolyphe-nolic content and in vitro antioxidant characteristics of wineindustry and other agri-food solid waste extractsrdquo Journal ofFood Composition and Analysis vol 20 no 2 pp 125ndash132 2007

[6] A Srinivasan and T Viraraghavan ldquoRemoval of oil by walnutshell mediardquo Bioresource Technology vol 99 no 17 pp 8217ndash8220 2008

[7] M Carvalho P J Ferreira V S Mendes et al ldquoHuman cancercell antiproliferative and antioxidant activities of Juglans regiaLrdquo Food and Chemical Toxicology vol 48 no 1 pp 441ndash4472010

[8] V Usenik G Osterc M Mikulic-Petkovsek et al ldquoThe involve-ment of phenolic compounds in the metabolism of fruit treesrdquoin Razprave IV Razreda SAZU vol 45 pp 187ndash204 SAZULjubljana Slovenia 2004

[9] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstics acid reagentsrdquoAmer-ican Journal of Enology andViticulture vol 16 no 3 pp 144ndash1581965

[10] T Bahorun B Gressier F Trotin et al ldquoOxygen speciesscavenging activity of phenolic extracts from hawthorn freshplant organs and pharmaceutical preparationsrdquo Arzneimittel-ForschungDrug Research vol 46 no 11 pp 1086ndash1089 1996

[11] M Szaufer-Hajdrych ldquoPhenolic acids in leaves of species of theAquilegia genusrdquoHerba Polonica vol 50 no 2 pp 10ndash14 2004

[12] E A Decker and B Welch ldquoRole of ferritin as a lipid oxidationcatalyst in muscle foodrdquo Journal of Agricultural and FoodChemistry vol 38 no 3 pp 674ndash677 1990

[13] M Oyaizu ldquoStudies on products of browning reaction- antiox-idative activities of products of browning reaction prepared

from glucosaminerdquo Japan Journal of Nutrition vol 44 no 6pp 307ndash315 1986

[14] J-M Kuo D-B Yeh and B S Pan ldquoRapid photometricassay evaluating antioxidative activity in edible plant materialrdquoJournal of Agricultural and Food Chemistry vol 47 no 8 pp3206ndash3209 1999

[15] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[16] B Axelrod T M Cheesbrough and S Laakso ldquo[53] Lipoxyge-nase from soybeans EC 1131112 Linoleateoxygen oxidoreduc-taserdquoMethods in Enzymology vol 71 pp 441ndash451 1981

[17] A P Sweeney S G Wyllie R A Shalliker and J L MarkhamldquoXanthine oxidase inhibitory activity of selected Australiannative plantsrdquo Journal of Ethnopharmacology vol 75 no 2-3pp 273ndash277 2001

[18] A Claiborne ldquoCatalase activityrdquo in CRC Handbook of Methodsfor Oxygen Radical Research R A Greenwald Ed pp 283ndash284CRC Press Boca Raton Fla USA 1985

[19] J S Amaral M R Alves R M Seabra and B P P OliveiraldquoVitamin E composition of walnuts (Juglans regia L) a 3-yearcomparative study of different cultivarsrdquo Journal of Agriculturaland Food Chemistry vol 53 no 13 pp 5467ndash5472 2005

[20] J B Golding W Barry McGlasson S Grant Wyllie and DN Leach ldquoFate of apple peel phenolics during cool storagerdquoJournal of Agricultural and Food Chemistry vol 49 no 5 pp2283ndash2289 2001

[21] V Lattanzio ldquoBioactive polyphenols their role in quality andstorability of fruit and vegetablesrdquo Journal of Applied Botanyvol 77 no 5-6 pp 128ndash146 2003

[22] C Alamprese C Pompei and F Scaramuzzi ldquoCharacterizationand antioxidant activity of nocino liqueurrdquo Food Chemistry vol90 no 4 pp 495ndash502 2005

[23] B LHalvorsen KHolteMCWMyhrstad et al ldquoA systematicscreening of total antioxidants in dietary plantsrdquo Journal ofNutrition vol 132 no 3 pp 461ndash471 2002

[24] J Jakopic M Colaric R Veberic M Hudina A Solar and FStampar ldquoHow much do cultivar and preparation time influ-ence on phenolics content in walnut liqueurrdquo Food Chemistryvol 104 no 1 pp 100ndash105 2007

[25] T Keszligler B Jansen and A Hesse ldquoEffect of blackcurrant- cranberry- and plum juice consumption on risk factorsassociated with kidney stone formationrdquo European Journal ofClinical Nutrition vol 56 no 10 pp 1020ndash1023 2002

[26] U Gawlik-Dziki M Jezyna M Swieca D Dziki B Baraniakand J Czyz ldquoEffect of bioaccessibility of phenolic compoundson in vitro anticancer activity of broccoli sproutsrdquo FoodResearch International vol 49 no 1 pp 469ndash476 2012

[27] U Gawlik-Dziki M Swieca M Sułkowski D Dziki B Bara-niak and J Czyz ldquoAntioxidant and anticancer activities ofChenopodium quinoa leaves extractsmdashin vitro studyrdquo Food andChemical Toxicology vol 57 pp 154ndash160 2013

[28] K J Anderson S S Teuber A Gobeille P Cremin A LWaterhouse and FM Steinberg ldquoWalnut polyphenolics inhibitin vitro human plasma and LDLoxidationrdquo Journal of Nutritionvol 131 no 11 pp 2837ndash2842 2001

[29] L Salter T Clifford N Morley D Gould S Campbell andA Curnow ldquoThe use of comet assay data with a simplereaction mechanism to evaluate the relative effectiveness of freeradical scavenging by quercetin epigallocatechin gallate andN-acetylcysteine in UV-irradiatedMRC5 lung fibroblastsrdquo Journal


of Photochemistry and Photobiology B vol 75 no 1-2 pp 57ndash612004

[30] J C Espın C Soler-Rivas and H J Wichers ldquoCharacterizationof the total free radical scavenger capacity of vegetable oilsand oil fractions using 22-diphenyl-1-picrylhydrazyl radicalrdquoJournal of Agricultural and Food Chemistry vol 48 no 3 pp648ndash656 2000

[31] L Li R Tsao R Yang C LiuH Zhu and J C Young ldquoPolyphe-nolic profiles and antioxidant activities of heartnut (Juglansailanthifolia var cordiformis) and Persian walnut (Juglans regiaL)rdquo Journal of Agricultural and Food Chemistry vol 54 no 21pp 8033ndash8040 2006

[32] M Kosar H J D Dorman and R Hiltunen ldquoEffect of an acidtreatment on the phytochemical and antioxidant characteristicsof extracts from selected Lamiaceae speciesrdquo Food Chemistryvol 91 no 3 pp 525ndash533 2005

[33] I A Siddiqui A JaleelW Tamimi andHM F Al Kadri ldquoRoleof oxidative stress in the pathogenesis of preeclampsiardquoArchivesof Gynecology and Obstetrics vol 282 no 5 pp 469ndash474 2010

[34] J M C Gutteridge ldquoLipid peroxidation and antioxidants asbiomarkers of tissue damagerdquo Clinical Chemistry vol 41 no 12pp 1819ndash1828 1995

[35] I F Almeida E Fernandes J L F C Lima P C Costa and MFernanda Bahia ldquoWalnut (Juglans regia) leaf extracts are strongscavengers of pro-oxidant reactive speciesrdquo FoodChemistry vol106 no 3 pp 1014ndash1020 2008

[36] M Foti M Piattelli M T Baratta and G Ruberto ldquoFlavonidscoumarins and cinnamic acids as antioxidants in a micellarsystem Structure-activity relationshiprdquo Journal of Agriculturaland Food Chemistry vol 44 no 2 pp 497ndash501 1996

[37] T P Dew A J Day and M R A Morgan ldquoXanthine oxidaseactivity in vitro effects of food extracts and componentsrdquoJournal of Agricultural and Food Chemistry vol 53 no 16 pp6510ndash6515 2005

[38] C J Wallwork D A Parks and G W Schmid-SchonbeinldquoXanthine oxidase activity in the dexamethasone-inducedhypertensive ratrdquoMicrovascular Research vol 66 no 1 pp 30ndash37 2003

[39] T Juntachote and E Berghofer ldquoAntioxidative properties andstability of ethanolic extracts of Holy basil and Galangalrdquo FoodChemistry vol 92 no 2 pp 193ndash202 2005

[40] C D Sadik H Sies and T Schewe ldquoInhibition of 15-lipoxygenases by flavonoids structure-activity relations andmode of actionrdquo Biochemical Pharmacology vol 65 no 5 pp773ndash781 2003

[41] J Saruwatari K Nakagawa J Shindo S Nachi H Echizen andT Ishizaki ldquoThe in-vivo effects of sho-saiko-to a traditionalChinese herbal medicine on two cytochrome P450 enzymes(1A2 and 3A) and xanthine oxidase in manrdquo Journal of Phar-macy and Pharmacology vol 55 no 11 pp 1553ndash1559 2003

[42] S K Nelson S K Bose G K Grunwald P Myhill and J MMcCord ldquoThe induction of human superoxide dismutase andcatalase in vivo a fundamentally new approach to antioxidanttherapyrdquo Free Radical Biology and Medicine vol 40 no 2 pp341ndash347 2006

[43] L Guemouri Y Artur B Herbeth C Jeandel G Cuny andG Siest ldquoBiological variability of superoxide dismutase glu-tathione peroxidase and catalase in bloodrdquo Clinical Chemistryvol 37 no 11 pp 1932ndash1937 1991

[44] M E Inal G Kanbak and E Sunal ldquoAntioxidant enzymeactivities and malondialdehyde levels related to agingrdquo ClinicaChimica Acta vol 305 no 1-2 pp 75ndash80 2001

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


play a number of crucial roles in the complex metabolism ofplants and also of fruit treesThey are involved in physiologi-cal processes of fruit tree growth and development and affectdifferent aspects of fruit pre- and postharvest life [8]

Foods of plant origin such as fruits and vegetables andwhole grain products have been suggested as a natural sourcefor antioxidants Antioxidants can play an important rolein disease prevention and health maintenance Plant-derivedproducts can be used either as a source of antioxidants inindustry or for medicinal purposes The antioxidant effectshown by these products proceeds fromphenolic compoundsand phytochemicals which is protected from harmful effectsof free radicals Walnut possesses a high content of 120572-tocopherol a vitamin E family compound which has antiox-idant activity mainly in the prevention of lipid oxidationprocess [1]

The aim of this study was to assess the nutraceuticalpotential of J regia fruits green husks and leaves ethanolicextracts For this purpose we have made a qualitative assess-ment of phenolic compounds Furthermore we analyzedthe total phenolic content phenolic acids and flavonoids inextracts their antioxidant activity and their effect on theactivity of some enzymes from the class of oxidoreductases

2 Materials and Methods

21 Chemicals ABTS (221015840-azino-bis(3-ethylbenzothia-zoline-6-sulphonic acid)) Folin-Ciocalteau reagent Arnovreagent gallic acid quercetin ferrozine lipoxygenasexanthine oxidase catalase and linoleic acid were purchasedfrom Sigma-Aldrich company (Poznan Poland) Acetonitrileand methanol gradient HPLC grade and formic acid LC-MSgrade for LC-UV-MS separations were purchased from JTBaker (Phillipsburg NJ) Water was purified in-house with aMilli-Q water purification system Simplicity-185 (MilliporeCo) All other chemicals were of analytical grade

22 Preparation of Samples The experimental material con-sisted of walnut fruits harvested at two stages of maturity(July 3 2012 and July 25 2012) Green husks and leaveswere harvested on September 23 2012 The research materialwas obtained from a private plantation of walnut in RadzynPodlaski PolandThe immature fruit green husks and driedleaves were shredded and were used to obtain extracts Foralcohol extraction 5 g of each plant material was extracted in50mL of EtOH (70 vv) for 2 weeks in darkness at 25∘Cand then the supernatant was recovered Obtained extractsas follows



GH extract from green husks of walnut

L extract from walnut leaves

The final extracts concentration was 01 g fresh mass (FM)mL

23 Ultraperformance Liquid Chromatography Compoundsof interest were analyzed using a Waters ACQUITY UPLCsystem (Waters Corp Milford MA USA) consisting of abinary pump system sample manager column manager andPDA detector (also from Waters Corp) Waters MassLynxsoftware v41 was used for acquisition and data processingThe samples were separated on a BEHC18 column (100mmtimes21mm id 17 120583mWaters Corp Milford MA USA) whichwas maintained at 40∘C The flow rate was adjusted to040mLmin The following solvent system mobile phase A(01 formic acid in Milli-Q water vv) and mobile phase B(01 formic acid in MeCN vv) was applied The gradientprogram was as follows 0-10min 5 B 10ndash240min 5ndash50 B 240-250min 50ndash95 B 250ndash270min 95 B 270ndash271min 95-5 B 271ndash300min 5 B Samples were keptat 8∘C in the sample manager The injection volume of thesample was 20 120583L (full loop mode) Strong needle washsolution (95 5 methanol-water vv) and weak needle washsolution (5 95 acetonitrile-water vv) were used UV-PDAdata was acquired from 220 nm to 480 nm at 5 pointsrate 36 nm resolution The separation was completed in 30minutes Peakswere assigned on the basis of theirUV spectramass to charge ratio (mz) and ESI-MSMS fragmentationpatterns

The MS analyses were carried out on a TQD mass spec-trometer (Waters Corp) equipped with a Z-spray electro-spray interface The following instrumental parameters wereused for ESI-MS analysis of phenolic compounds (negativeionization mode) capillary voltage 28 kV cone voltage40V desolvation gas N

2800 Lh cone gas N

2100 Lh

source temp 140∘C desolvation temp 350∘C Compoundswere analyzed in full scanmode (mass range of 100ndash1600 amuwas scanned)

For ESI-MSMS selected ions were fragmented usinga collision energy of 15 V (phenolic acids derivatives) or25V (flavonoids derivatives) and collision gas (argon) at01mLmin

24 Phytochemical Analysis Total phenols were estimatedaccording to the Folin-Ciocalteau method [9] A 01mL sam-ple of the extract was mixed with 01mL of H

2O with 04mL

of Folin reagent (1 5 H2O) and after 3min with 2mL of 10

Na2CO3 After 30min the absorbance of mixed samples was

measured at a wavelength of 720 nm The amount of totalphenolics was expressed as gallic acid equivalents (GAE)

Total flavonoids were estimated according to Lamaisonmethod [10] A 1mL sample of the extract was mixed with1mL AlCl

3times 6H2O (2 vv) and after 10min the absorbance

of mixed samples was measured at a wavelength of 430 nmThe amount of flavonoids was expressed as quercetin acidequivalents (QE)

Total phenolic acids were determined according Arnovmethod [11] 1mL of distilled water was mixed with 02mL ofextract 02mLHCl (05 vv) 02mLofArnova reagent and02mL NaOH (1M) The absorbance of mixed samples wasmeasured at a wavelength of 490 nmThe amount of phenolicacids was expressed as caffeic acid equivalents (CAE)



2



119860119888

)] times 100 (1)












(119860100minus 1198600)] times 100 (2)




119860119888

)] times 100 (3)






50is the effective





) times 100 (4)






A300

10

05

00

(au

)

2

3

56

7

8

1113

15

22

24

26

2728

29

30

31

32 34 3536

3738

39 4042 44 45 46

00 25 50 75 100 125 150

L

Elution time (min)

(a)

A300

10

05

00

(au

)

1

00 25 50 75 100 125 150

F3

Elution time (min)

(b)

A300

10

05

00

(au

)

00 25 50 75 100 125 150

F25

Elution time (min)

(c)

A300

10

05

00

(au

)

00 25 50 75 100 125 150

GH

Elution time (min)

(d)



2O2

consumed per min




















50values were


50= 12649 plusmn


50





2


2)












































250

200

150

100

50

0

EC50

(mg

FMm

L)

F3F25

GHL

a a aab c c

e e

d d d

bc

g

h

f

Chel Red LPO ABTS







50=






250

200

150

100

50

0

a

bab

cd

d d d

F3 F25 GH L

EC50

(mg

FMm

L)

LOXIXOI







16

14

12

10

8

6

4

2

0

Cata

lase

activ

atio

n (

)

a

b

cc

F3 F25 GH L





2O2formation




4 Conclusion






References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



2



119860119888

)] times 100 (1)












(119860100minus 1198600)] times 100 (2)




119860119888

)] times 100 (3)






50is the effective





) times 100 (4)






A300

10

05

00

(au

)

2

3

56

7

8

1113

15

22

24

26

2728

29

30

31

32 34 3536

3738

39 4042 44 45 46

00 25 50 75 100 125 150

L

Elution time (min)

(a)

A300

10

05

00

(au

)

1

00 25 50 75 100 125 150

F3

Elution time (min)

(b)

A300

10

05

00

(au

)

00 25 50 75 100 125 150

F25

Elution time (min)

(c)

A300

10

05

00

(au

)

00 25 50 75 100 125 150

GH

Elution time (min)

(d)



2O2

consumed per min




















50values were


50= 12649 plusmn


50





2


2)












































250

200

150

100

50

0

EC50

(mg

FMm

L)

F3F25

GHL

a a aab c c

e e

d d d

bc

g

h

f

Chel Red LPO ABTS







50=






250

200

150

100

50

0

a

bab

cd

d d d

F3 F25 GH L

EC50

(mg

FMm

L)

LOXIXOI







16

14

12

10

8

6

4

2

0

Cata

lase

activ

atio

n (

)

a

b

cc

F3 F25 GH L





2O2formation




4 Conclusion






References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


A300

10

05

00

(au

)

2

3

56

7

8

1113

15

22

24

26

2728

29

30

31

32 34 3536

3738

39 4042 44 45 46

00 25 50 75 100 125 150

L

Elution time (min)

(a)

A300

10

05

00

(au

)

1

00 25 50 75 100 125 150

F3

Elution time (min)

(b)

A300

10

05

00

(au

)

00 25 50 75 100 125 150

F25

Elution time (min)

(c)

A300

10

05

00

(au

)

00 25 50 75 100 125 150

GH

Elution time (min)

(d)



2O2

consumed per min




















50values were


50= 12649 plusmn


50





2


2)












































250

200

150

100

50

0

EC50

(mg

FMm

L)

F3F25

GHL

a a aab c c

e e

d d d

bc

g

h

f

Chel Red LPO ABTS







50=






250

200

150

100

50

0

a

bab

cd

d d d

F3 F25 GH L

EC50

(mg

FMm

L)

LOXIXOI







16

14

12

10

8

6

4

2

0

Cata

lase

activ

atio

n (

)

a

b

cc

F3 F25 GH L





2O2formation




4 Conclusion






References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology















50values were


50= 12649 plusmn


50





2


2)












































250

200

150

100

50

0

EC50

(mg

FMm

L)

F3F25

GHL

a a aab c c

e e

d d d

bc

g

h

f

Chel Red LPO ABTS







50=






250

200

150

100

50

0

a

bab

cd

d d d

F3 F25 GH L

EC50

(mg

FMm

L)

LOXIXOI







16

14

12

10

8

6

4

2

0

Cata

lase

activ

atio

n (

)

a

b

cc

F3 F25 GH L





2O2formation




4 Conclusion






References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











































250

200

150

100

50

0

EC50

(mg

FMm

L)

F3F25

GHL

a a aab c c

e e

d d d

bc

g

h

f

Chel Red LPO ABTS







50=






250

200

150

100

50

0

a

bab

cd

d d d

F3 F25 GH L

EC50

(mg

FMm

L)

LOXIXOI







16

14

12

10

8

6

4

2

0

Cata

lase

activ

atio

n (

)

a

b

cc

F3 F25 GH L





2O2formation




4 Conclusion






References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


250

200

150

100

50

0

a

bab

cd

d d d

F3 F25 GH L

EC50

(mg

FMm

L)

LOXIXOI







16

14

12

10

8

6

4

2

0

Cata

lase

activ

atio

n (

)

a

b

cc

F3 F25 GH L





2O2formation




4 Conclusion






References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





References























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article nutraceutical potential of tinctures from

Documents