Quality Control of Gamma Irradiated Dwarf Mallow (Malva neglecta Wallr.)

13 Pages·2016·0.56 MB·English

molecules
Article
Quality Control of Gamma Irradiated Dwarf Mallow
(Malva neglecta Wallr.) Based on Color, Organic
Acids, Total Phenolics and Antioxidant Parameters
JoséPinela1,2,LillianBarros1,AmilcarL.Antonio1,3,AnaMariaCarvalho1,
M.BeatrizP.P.Oliveira2andIsabelC.F.R.Ferreira1,*
1 MountainResearchCentre(CIMO),ESA,PolytechnicInstituteofBragança,CampusdeSantaApolónia,
1172,5300-253Bragança,Portugal;[email protected](J.P.);[email protected](L.B.);[email protected](A.L.A.);
[email protected](A.M.C.)
2 REQUIMTE/LAQV,FacultyofPharmacy,UniversityofPorto,RuaJorgeViterboFerreira,
no.228,4050-313Porto,Portugal;[email protected]
3 CentrodeCiênciaseTecnologiasNucleares(C2TN),InstitutoSuperiorTécnico,UniversidadedeLisboa,
E.N.10,2695-066Bobadela,Portugal
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
AcademicEditor:DerekJ.McPhee
Received:7March2016;Accepted:7April2016;Published:8April2016
Abstract: Thisstudyaddressestheeffectsofgammairradiation(1,5and8kGy)oncolor,organic
acids,totalphenolics,totalflavonoids,andantioxidantactivityofdwarfmallow(MalvaneglectaWallr.).
Organicacidswereanalyzedbyultrafastliquidchromatography(UFLC)coupledtoaphotodiode
array (PDA) detector. Total phenolics and flavonoids were measured by the Folin-Ciocalteu and
aluminium chloride colorimetric methods, respectively. The antioxidant activity was evaluated
based on the DPPH‚ scavenging activity, reducing power, β-carotene bleaching inhibition and
thiobarbituricacidreactivesubstances(TBARS)formationinhibition. Analyseswereperformedin
thenon-irradiatedandirradiatedplantmaterial,aswellasindecoctionsobtainedfromthesame
samples. Thetotalamountsoforganicacidsandphenolicsrecordedindecoctedextractswerealways
higherthanthosefoundintheplantmaterialorhydromethanolicextracts,respectively. TheDPPH‚
scavengingactivityandreducingpowerwerealsohigherindecoctedextracts.Theassayedirradiation
doses affected differently the organic acids profile. The levels of total phenolics and flavonoids
werelowerinthehydromethanolicextractspreparedfromsamplesirradiatedat1kGy(dosethat
induced color changes) and in decocted extracts prepared from those irradiated at 8 kGy. The
last samples also showed a lower antioxidant activity. In turn, irradiation at 5 kGy favored the
amountsoftotalphenolicsandflavonoids. Overall,thisstudycontributestotheunderstandingof
theeffectsofirradiationinindicatorsofdwarfmallowquality,andhighlightedthedecoctionsforits
antioxidantproperties.
Keywords: Malvaneglecta;ionizingradiation;colorparameters;organicacids;antioxidantactivity;
decoction;qualitycontrol
1. Introduction
Organicacidsareprimarymetaboliteswidelyspreadthroughouttheplantkingdom. Chemically,
theyarelowweightmoleculesandareconsideredtobeanyorganiccarboxylicacidwithageneral
structureR-COOH.Althoughtheyareweakacids,thesewater-solublecompoundsmayconferacidic
propertiestofoodscontainingthemandinfluenceitsorganolepticproperties(flavor,colorandaroma)
and consequent acceptability by the consumers [1]. From a practical point of view, the organic
acidsprofile(levelsandrelativeratios)isimportantforfoodqualitycontrol. Itallowsdetermining
Molecules2016,21,467;doi:10.3390/molecules21040467 www.mdpi.com/journal/molecules
Molecules2016,21, 467 2of13
thepercentageofplantaddedtofinalproducts,thusdetectingadulterationsorpossiblemicrobial
deteriorationsoccurringduringstorage,providingapracticaladvantageforitsuseasanauthenticity
indexinplant-basedfoodsandbeverages[1–3]. Therelativeamountsandthepresenceofeachofthese
compoundsarealsousefulasameanstoevaluatetheeffectoffoodprocessing[2,4]. Thus,qualitative
andquantitativeanalysesoftheseingredientsareofgreatimportance.
Duringtheproductionprocess(fromharvestinganddryingtopackagingandstorage),rawplants
arepronetovariouscontaminationsandinfestations,whichcanleadtospoilage,qualitydeterioration
andconsequenteconomiclosses[5]. Besidesconstitutinghealthhazardstoconsumers,contaminated
productscanalsoadverselyaffecttheefficacyandstabilityoftheirbioactiveingredientsandleadto
spoilageoffinalproducts[6]. Thepresenceoforganicacidsandphenoliccompoundsisadvantageous,
as they contribute to the natural preservation process (through its antimicrobial and antioxidant
activities),butisnotenough. Thesearchfornewpreservationtreatmentswithaminimumimpacton
physical,chemicalandfunctionalparametershighlightedthegammairradiationasasafe,effective
andsustainableoptiontosanitizeplantproducts[7,8].
Nowadaysthereisagrowingscientificinterestinirradiation-inducedmodificationsofantioxidant
propertiesandcompoundsresponsibleforsucheffects. Theantioxidantactivityisstronglylinked
the presence of phenolic compounds [9–12], secondary metabolites frequently found attached to
sugars (glycosides), which increases their water solubility. These compounds have the ability to
donateahydrogenatomfromthearomatichydroxylgrouptoafreeradicaland/orthecapacityto
supportanunpairedelectronintheiraromaticstructures. Therefore,themethodsusedtoevaluatethe
antioxidantactivitycanbeclassifiedaccordingtothemechanismofaction,i.e.,single-electrontransfer
orhydrogenatomtransfer[13]. However,phenoliccompounds,organicacidsandotherbioactive
constituents may be affected by irradiation treatment if applied inappropriately. It is known that
ionizingradiationinteractswithwatermoleculesgeneratingfreeradicals,inareactioncommonly
knownasradiolysis[14]. Thesefreeradicalscantheninteractwithdifferentbiomolecules,leading
tobreakdownofchemicalbondsandchangesintheirstructureand,consequently,inthebioactivity
andextractabilityfromtheplantmaterial. Thereferredcompoundsmayalsobeaffectedbythedirect
impactofthegamma-rays[14,15]. Asaconsequence,thecoloroftheprocessedsamplesmaychange.
However, thisqualityattributeisdirectlyrelatedtoconsumers’appreciationofaproduct, asthey
tend to associate its color with its taste, hygienic safety, shelf-life and personal satisfaction [16,17].
Averystringentplantselectionbasedoncolorparametersalsooccursinthefood,pharmaceuticaland
cosmeceuticalindustries[18]. Therefore,thecolorevaluationisimportantinqualitycontrolparameter
ofirradiatedproducts.
Dwarf mallow (Malva neglecta Wallr.) is an annual herbaceous plant of the family Malvaceae
traditionallyeatenrawasaleafyvegetableorpreparedinherbalbeverages(mainlydecoctions)due
toclaimsofdisinfectantandanti-inflammatoryeffects[19]. Itisalsousedtotreatmultiplemedical
conditionssuchasasthma,colds,digestiveandurinaryproblems,andabdominalpains[20]. Scientific
workshavereportedantioxidant[11,20],antibacterial[21]andanti-ulcerogenic[22]propertiesforthis
plant. Nevertheless, as far as we know, the organic acids profile of this plant remains unknown,
as well as the effects induced by the gamma irradiation treatment on its physical or chemical
properties. Thus, the main purpose of this study was to investigate the dose-response effects of
gammairradiationoncolor,organicacids,totalphenolicsandflavonoids,andantioxidantproperties
(qualitycontrolindicators)ofM.neglectasamples. Theinfluenceofthepreparationmethod(decoction)
wasalsoinvestigated.
2. Results
2.1. EffectsonColorParameters
The CIE L*a*b* color values for non-irradiated and irradiated samples of M. neglecta are
presented in Figure 1. The 5 and 8 kGy doses had no influence on color parameters. However,
Molecules2016,21, 467 3of13
significant differences were found for samples irradiated with the 1 kGy dose. These samples
revealedlowerlightness(L*=43.43˘0.89)andyellowness(b*=22.29˘0.96)andhigherredness
(a*=´12.69˘0.45),whichinducedthemorepronouncedtotalcolordifference(∆E*=1.81),compared
withthenon-irradiatedcontrolsamples(L*=45.07˘0.97,b*=23.98˘0.55anda*=´13.84˘0.29).
Molecules 2016, 21, 467 3 of 12
Furthermore,whilethe∆E*upto0.5andfrom0.5to1.5indicatetracecolordifferencesandslight
0d.i5f faenredn cfreosm,r e0s.p5 etcot i1v.e5l yi,ntdhieca∆teE *trcaocrer ecsoploorn ddiifnfgerteoncthese a1nkdG sylidgohst ediinffdeirceantceessa, rneostpiceecatibvleelyd,i ftfheer eΔnEce*
cdoertreecstpabolnedbinygt htoe hthuem 1a knGeyy ed[o2s3e] .indicates a noticeable difference detectable by the human eye [23].
0.99
ΔE* 0.43 8 kGy
1.81
5 kGy
1 kGy
24.55 ±0.88a
24.14 ±0.77a 0 kGy
b*
22.29 ±0.96b
23.98 ±0.55a
-13.86 ±0.31b
-13.61 ±0.26b
a*
-12.69 ±0.45a
-13.84 ±0.29b
46.06 ±0.61a
45.47 ±0.74a
L*
43.43 ±0.89b
45.07 ±0.97a
-35 -25 -15 -5 5 15 25 35 45 55 65
CIE L*a*b* units
Figure 1. Impact of the gamma irradiation treatment on color parameters of M. neglecta powdered
Figure1. ImpactofthegammairradiationtreatmentoncolorparametersofM.neglectapowdered
ssaammpplleess.. Δ∆EE**: :tototatal lcocololor rddififfefreernecnec;e b;*b: *b:lbuleuneenses s(s–)( –↔)Ø yeylleolwlonwesnse (s+s)(; +a)*;: ag*r:eegnreneenssn e(–s)s ↔(– )rØednreesdsn (e+s)s;
a(+n)d; aLn*d: dLa*:rkdnaersksn e(0ss) (↔0) Ølighlitgnhetsnse s(1s0(01)0. 0F).oFr oeraecahc hcocloolro rpapraarmameteetre, r,ddififfefererennt tlleetttteerrss ((aa,,bb)) iinnddiiccaattee
ssttaattiissttiiccaallllyy ssiiggnniiffiiccaanntt ddiiffffeerreenncceess ((pp << 00..0055))..
Similar results were previously reported by Kirkin et al. [24]. Those authors found that the 7 kGy
SimilarresultswerepreviouslyreportedbyKirkinetal.[24]. Thoseauthorsfoundthatthe7kGy
dose had a greater effect on color parameters of Rosmarinus officinalis L. than higher doses of 12 and 17
dosehadagreatereffectoncolorparametersofRosmarinusofficinalisL.thanhigherdosesof12and
kGy. In these samples, the L* value was decreased and the a* value was increased, in accordance to our
17kGy. Inthesesamples,theL*valuewasdecreasedandthea*valuewasincreased,inaccordance
results. The discoloration of Piper nigrum L. by decreasing the a* and b* values and increasing the L*
toourresults. ThediscolorationofPipernigrumL.bydecreasingthea*andb*valuesandincreasing
value was also reported, as well as the suitability of the applied doses to maintain the color parameters
theL*valuewasalsoreported,aswellasthesuitabilityoftheapplieddosestomaintainthecolor
of Thymus vulgaris L. and Cuminum cyminum L. A tendency for lightness decrease was also reported by
parametersofThymusvulgarisL.andCuminumcyminumL.Atendencyforlightnessdecreasewas
Pereira et al. [25] in Ginkgo biloba L. samples irradiated at 10 kGy. Nevertheless, browning is unwanted
also reported by Pereira et al. [25] in Ginkgo biloba L. samples irradiated at 10 kGy. Nevertheless,
in dried products [26]. On the other hand, Pinela et al. [9] reported that gamma irradiation treatment
browningisunwantedindriedproducts[26]. Ontheotherhand,Pinelaetal.[9]reportedthatgamma
up to 10 kGy had no effect on the color parameters of shade- and freeze-dried samples of Tuberaria
irradiationtreatmentupto10kGyhadnoeffectonthecolorparametersofshade-andfreeze-dried
lignosa (Sweet) Samp. Therefore, we can conclude that the irradiation-induced modifications on color
samplesofTuberarialignosa(Sweet)Samp. Therefore,wecanconcludethattheirradiation-induced
parameters not only depend on the applied dose, but also on the plant material under investigation.
modificationsoncolorparametersnotonlydependontheapplieddose,butalsoontheplantmaterial
underinvestigation.
2.2. Effects on Organic Acids
2.2. ETfafebcltes o1n sOhorwgasn itchAe coirdgsanic acids content found in M. neglecta dry material submitted to gamma
irradiation, as well as in the decocted extracts (the form traditionally used in folk medicine) prepared
Table1showstheorganicacidscontentfoundinM.neglectadrymaterialsubmittedtogamma
from that material. Oxalic, quinic, malic, citric, succinic and fumaric acids were quantified, being
irradiation,aswellasinthedecoctedextracts(theformtraditionallyusedinfolkmedicine)prepared
oxalic acid the most abundant organic acid (Figure 2). The total amounts recorded in the decocted
fromthatmaterial. Oxalic,quinic,malic,citric,succinicandfumaricacidswerequantified,beingoxalic
extracts were always higher than those found in the plant dry material, as well as the individual
acidthemostabundantorganicacid(Figure2). Thetotalamountsrecordedinthedecoctedextracts
levels of each organic acid (except for citric acid) (Table 1). This difference may be related to a better
were always higher than those found in the plant dry material, as well as the individual levels of
extraction efficiency achieved at elevated temperature, but that can lead to citric acid degradation
eachorganicacid(exceptforcitricacid)(Table1). Thisdifferencemayberelatedtoabetterextraction
[27]. These results are in agreement with those previously reported by Guimarães et al. [10] for
Matricaria recutita L. dry material and decocted extracts.
Molecules 2016, 21, 467 4 of 12
Table 1. Organic acids content in the dry material and decocted extracts of M. neglecta samples
submitted to gamma irradiation.
Dose Oxalic Acid Quinic Acid Malic Acid Citric Acid Succinic Acid Fumaric Acid Total
Dry material (mg/g dw)
0 kGy 67.50 ± 0.16 b 6.63 ± 0.21 b 3.16 ± 0.28 b 21.56 ± 0.41 b 7.50 ± 0.13 a 15.07 ± 0.03 c 121.43 ± 1.17 b
1 kGy 67.29 ± 0.05 b 7.50 ± 0.42 a 3.09 ± 0.16 b 19.84 ± 0.14 c 7.30 ± 0.41 a 18.04 ± 0.05 a 123.07 ± 0.02 a
5 kGy 65.30 ± 0.17 c 6.34 ± 0.28 b 3.42 ± 0.33 ab 22.28 ± 0.31 a 7.29 ± 0.12 a 13.59 ± 0.07 d 118.22 ± 1.15 c
8 kGy 71.75 ± 0.10 a 6.45 ± 0.08 b 3.64 ± 0.05 a 19.02 ± 0.08 d 6.23 ± 0.13 b 15.85 ± 0.05 b 122.94 ± 0.30 ab
Decocted extracts (mg/g dw)
Molecules2016,21, 467 4of13
0 kGy 106.71 ± 0.50 a 3.59 ± 0.25 d 9.78 ± 0.67 b 7.92 ± 0.09 d 47.43 ± 0.67 b 20.61 ± 0.14 c 196.03 ± 0.80 b
1 kGy 79.80 ± 0.05 c 6.96 ± 0.20 c 10.52 ± 0.08 a 9.80 ± 0.15 b 47.36 ± 0.38 b 21.54 ± 0.19 b 175.99 ± 0.89 c
5 kGy 88.72 ± 0.03 b 8.90 ± 0.09 a 9.72 ± 0.19 b 12.76 ± 0.07 a 54.17 ± 0.06 a 20.81 ± 0.08 c 195.09 ± 0.01 b
efficiencyachievedatelevatedtemperature,butthatcanleadtocitricaciddegradation[27]. These
8 kGy 106.33 ± 0.10 a 8.37 ± 0.39 b 7.89 ± 0.29 c 8.58 ± 0.35 c 41.42 ± 0.50 c 25.17 ± 0.09 a 197.76 ± 0.95 a
resultsareinagreementwiththosepreviouslyreportedbyGuimarãesetal.[10]forMatricariarecutitaL.
The results are presented as mean ± standard deviation. In each column and for each sample (dry
drymaterialanddecoctedextracts.
material and decoction), different letters (a–d) indicate statistically significant differences (p < 0.05).
6
mAU
2500
2250
2000
1750 1
1500
1250
1000
750
500
250
2 3 4 5
0
2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0
Time(min)
FFiigguurree 22.. CChhrroommaattooggrraapphhiicc pprrooffiillee ooff oorrggaanniicc aacciiddss iinn tthhee ddeeccooccttiioonn pprreeppaarreedd ffrroomm MM.. nneegglleeccttaa ssaammpplleess
iirrrraaddiiaatteedd aatt 55 kkGGyy,, oobbttaaiinneedd uussiinngg UUFFLLCC--PPDDAA.. PPeeaakk iiddeennttiifificcaattiioonn:: 11:: ooxxaalliicc aacciidd,, 22:: qquuiinniicc aacciidd,, 33::
mmaalliicc aacciidd,, 44:: cciittrriicc aacciidd,, 55:: ssuucccciinniicc aacciidd,, aanndd 66:: ffuummaarriicc aacciidd..
In the plant material, the relative amounts of oxalic, citric and fumaric acids were the most affected
Table 1. Organic acids content in the dry material and decocted extracts of M. neglecta samples
by the irradiation treatment (Table 1); however, this variation was more marked in the decocted
submittedtogammairradiation.
extracts, mainly in the oxalic and succinic acids, probably due to the combined effects induced by
gaDmosmea irrOaxdaliiactAiocind andQ purineipcaArcaidtion mMaeltihcAocdid. TherCeiftorirceA, ctihde levSueclcsi noifc oArcigdaniFcu amcairdicsA acnidd relatiTvoeta rlatios
in the decocted extracts were somewhat dDifrfyermeantetr ifarlo(mmg /tghdowse) found in the dry material, e.g., citric acid
w0aks Gthye se6c7.o50n˘d 0m.16obst ab6u.6n3d˘a0n.2t1 obrgan3i.1c6 a˘ci0d.2 8inb the2 1p.5la6n˘t0 m.41abteria7l. 5a0n˘d0 p.13reasent1e5d.0 7a˘ r0e.d03ucctio1n2 1o.4f3 ~˘112.%17 bin
1kGy 67.29˘0.05b 7.50˘0.42a 3.09˘0.16b 19.84˘0.14c 7.30˘0.41a 18.04˘0.05a 123.07˘0.02a
the decocted extracts, while the relative ratios of succinic acid increased ~19% in those preparations.
5kGy 65.30˘0.17c 6.34˘0.28b 3.42˘0.33ab 22.28˘0.31a 7.29˘0.12a 13.59˘0.07d 118.22˘1.15c
N8ekvGerythel7e1s.7s5, ˘al0t.h10oaugh6 o.4x5a˘li0c.0, 8mbalic,3 .c64it˘ric0. 0a5nad fu1m9.0a2r˘ic0 .a0c8idds h6a.2v3e˘ b0e.1e3nb alre15a.d85y˘ d0e.0s5cbribe1d22 .i9n4 ˘flo0.w30eabrs
and flowering shoots of Malva sylvestris DLe. c[o2c8te]d, qexutriancitsc (amngd/g sduwc)cinic acids were not detected or present
in0 tkrGayce a1m06o.7u1n˘t0s. 5i0na thi3s. 5s9p˘e0c.i2e5sd, so a9. 7q8u˘a0l.i6t7abtive 7a.n92a˘ly0s.0is9 dof th4e7. 4o3r˘g0a.n67icb aci2d0.s6 1p˘ro0.f1i4lec can19 6b.0e3 u˘s0e.8d0 bto
de1tkeGcty pos7s9.i8b0l˘e 0a.d05ucltera6.t9i6o˘ns0 .i2n0 cthe s10a.m52p˘l0e.s08. a 9.80˘0.15b 47.36˘0.38b 21.54˘0.19b 175.99˘0.89c
5kGy 88.72˘0.03b 8.90˘0.09a 9.72˘0.19b 12.76˘0.07a 54.17˘0.06a 20.81˘0.08c 195.09˘0.01b
8kGIyn th1e0 6d.3r3y˘ m0.1a0taerial8,. 3t7h˘e 01.3 k9Gb y d7o.s89e ˘d0id.2 9ncot in8d.5u8c˘e0 a.3n5yc adv41e.4r2se˘ e0f.5f0ecct on25 t.1h7e˘ o0r.0g9aanic 1a9c7i.d76s˘ p0r.o95fiale
and increased the total levels. Lower amounts of total organic acids were however detected in samples
Theresultsarepresentedasmean˘standarddeviation.Ineachcolumnandforeachsample(drymaterialand
irradiadteecdo catito n5) ,kdGiffyer,e dntuleet tteors a(a –dde)cinrdeiacsaete ostfa toisxtiacalilcly asingdni fifcuamntadrififce raenccidess(.p T<h0e.0 5h).ighest levels of citric and

Intheplantmaterial,therelativeamountsofoxalic,citricandfumaricacidswerethemostaffected
bytheirradiationtreatment(Table1);however,thisvariationwasmoremarkedinthedecoctedextracts,
mainly in the oxalic and succinic acids, probably due to the combined effects induced by gamma
irradiationandpreparationmethod. Therefore,thelevelsoforganicacidsandrelativeratiosinthe
decoctedextractsweresomewhatdifferentfromthosefoundinthedrymaterial,e.g.,citricacidwas
thesecondmostabundantorganicacidintheplantmaterialandpresentedareductionof~12%in
thedecoctedextracts,whiletherelativeratiosofsuccinicacidincreased~19%inthosepreparations.
Molecules2016,21, 467 5of13
Nevertheless,althoughoxalic,malic,citricandfumaricacidshavebeenalreadydescribedinflowers
andfloweringshootsofMalvasylvestrisL.[28],quinicandsuccinicacidswerenotdetectedorpresent
intraceamountsinthisspecies,soaqualitativeanalysisoftheorganicacidsprofilecanbeusedto
detectpossibleadulterationsinthesamples.
Inthedrymaterial,the1kGydosedidnotinduceanyadverseeffectontheorganicacidsprofile
andincreasedthetotallevels. Loweramountsoftotalorganicacidswerehoweverdetectedinsamples
irradiated at 5 kGy, due to a decrease of oxalic and fumaric acids. The highest levels of citric and
succinicacidsandareductionoffumaricaciddetectedinbothsamples(drymaterialanddecocted
extract)werealsoassociatedwiththe5kGydose. Finally,thesamplesirradiatedat8kGygavethe
highestlevelsoftotalorganicacids. Itcanbeconcludedthat,ingeneral,theeffectsinducedbythe
differentdoseswerepositive,asthedecreaseintotalorganicacidswasmainlyassociatedwiththe
oxalicacidcontent. Despitetheimportanceofthisacidinthepharmaceuticalindustry[29],ithasbeen
associatedwithsomehealthproblems,e.g.,oxalicacidcancombinewithcalciuminthekidneysto
formkidneystonesinsusceptiblepeople[30].
Theidentifiedorganicacidshavebeenusedasfoodadditivesorpharmaceuticalandcosmetic
excipients [31,32]. Citric acid is a widely used food additive in many kinds of beverages due to
itsmildandrefreshingsourness[1,33],succinicacidisalsousedinthefoodindustryaswellasin
pharmaceuticalsandantibiotics[31],fumaricacidisusedduetoitseffectivenessagainstpsoriasis
andinflammationandduetoitsneuro-andchemoprotectiveeffects[34],andmalicandcitricacids
are reported to have bactericidal effects [35]. Additionally, the consumption of these compounds
in moderate amounts can promote appetite, help digestion and be beneficial to human health [4].
Nevertheless,excessivedosesofcertainorganicacidsshouldbeavoided[32].
2.3. EffectsonTotalPhenolics,TotalFlavonoidsandAntioxidantActivity
Theeffectsofgammairradiationonthetotalphenolicandflavonoidcontentsandantioxidant
activityoftheM.neglectahydromethanolicanddecoctedextractscanbeaccessedfromtheanalysisof
Table2. TheantioxidantactivityresultsareexpressedinEC values;thus,thelowertheEC value,
50 50
thehighertheantioxidantactivity. Ingeneral,thedecoctedextractsrevealedhigheramountsoftotal
phenolicsandflavonoidsthanthehydromethanolicextracts,aswellasanincreasedDPPH‚scavenging
activityandreducingpower. Thesamplesirradiatedat1kGyandextractedwiththehydromethanolic
mixturerevealedlowerlevelsofbothtotalphenolicandflavonoidsanddecreasedDPPH‚scavenging
activity,reducingpower,andTBARSformationinhibitioncapacity. Thisresultisinagreementwith
thehigher∆E*foundinthesesamples. Contrariwise,thehydromethanolicextractspreparedfromthe
samplesirradiatedat5kGyrevealedhigherlevelsoftotalphenolicsandflavonoidsandanincreased
DPPH‚ scavengingactivityandlipidperoxidationinhibitioncapacity(accessedbytheβ-carotene
bleachinginhibitionandTBARSformationinhibitioninbraincellhomogenates)incomparisonwith
those prepared from the non-irradiated control samples. In fact, the lipid peroxidation inhibition
capacityoftheseextractswasfavoredbytheirradiationtreatment(exceptfortheTBARSformation
inhibitionoftheextractspreparedfromthesamplesirradiatedat1kGy). Thisresultcanbeexplained
bytheaffinityofthesetwoinvitroassaysforlipophilicantioxidantsandbythefactthattheirradiation
treatmentaffectsthedifferentantioxidantmoleculesdifferently[14,15].
Molecules2016,21, 467 6of13
Table2.AntioxidantactivityofthehydromethanolicanddecoctedextractspreparedfromtheM.neglectadrymaterialsubmittedtogammairradiation.
β-CaroteneBleaching TBARSFormation
Dose DPPH‚ScavengingActivity ReducingPower(EC50 Inhibition Inhibition TotalPhenolics TotalFlavonoids
(EC50values,mg/mL) values,mg/mL) (EC50values,mg/mL) (EC50values,mg/mL) (mgGAE/gExtract) (mgCE/gExtract)
Hydromethanolicextracts
0kGy 1.15˘0.02c 0.52˘0.01c 0.46˘0.01a 0.56˘0.05b 69.54˘0.21b 22.85˘0.52b
1kGy 1.57˘0.02b 0.69˘0.01a 0.41˘0.03b 0.58˘0.03a 55.04˘0.36d 19.56˘0.08c
5kGy 1.06˘0.07d 0.57˘0.02b 0.41˘0.01b 0.11˘0.03d 78.55˘0.67a 27.30˘0.20a
8kGy 1.75˘0.04a 0.56˘0.01b 0.40˘0.01b 0.22˘0.01c 64.78˘1.51c 22.70˘0.51b
Decoctedextracts
0kGy 0.37˘0.01c 0.268˘0.002b 0.16˘0.01c 0.403˘0.004b 91.05˘1.14b 25.14˘0.53b
1kGy 0.40˘0.01b 0.264˘0.003c 0.17˘0.02c 0.35˘0.01c 96.92˘3.73a 28.03˘0.07a
5kGy 0.36˘0.01c 0.253˘0.003d 0.41˘0.01b 0.32˘0.01d 96.76˘0.60a 27.63˘0.35a
8kGy 0.46˘0.02a 0.326˘0.001a 0.46˘0.01a 0.69˘0.02a 78.99˘0.30c 21.98˘0.47c
Theresultsarepresentedasmean˘standarddeviation.Ineachcolumnandforeachsample(hydromethanolicanddecoctedextracts),differentletters(a–d)indicatestatistically
significantdifferences(p<0.05). EC50:Extractconcentrationcorrespondingto50%ofantioxidantactivityor0.5ofabsorbanceinthereducingpowerassay. TroloxEC50values:
42µg/mL(DPPH‚scavengingactivity),41µg/mL(reducingpower),18µg/mL(β-carotenebleachinginhibition)and23µg/mL(TBARSinhibition).GAE:gallicacidequivalents;
CE:catechinequivalents.
Molecules2016,21, 467 7of13
Regardingthedecoctedextracts,the8kGydosedecreasedtheantioxidantpropertiesandlevels
oftotalphenolicsandflavonoids. However,theotherassayeddosesreinforcedthereducingpower,
theTBARSformationinhibitioncapacity, andthelevelsoftotalphenolicsandflavonoids. Similar
trendshavebeenobservedinotherstudies. AhigherDPPH‚ scavengingactivityandreducingpower
ofdecoctedandinfusedextractsofT.vulgaris,comparedtothehydroalcoholicones,wasfoundby
Martinsetal.[12]. Decoctionwasalsothepreferablepreparationmethodforobtainingincreasedlevels
ofphenolicacidsandflavonoids. Thesuitabilityofdecoctionsandinfusionsforextractingphenolic
compoundsfromcommercialsamplesofAchilleamillefoliumL.wasdemonstratedbyDiasetal.[36],
whopreparedaqueousextractsthatalsohadinterestingantioxidantproperties. Furthermore,despite
theplantextractsrichinphenoliccompoundsarecommonlyreferredtoashavingincreasedantioxidant
properties[9–11],thepresenceoforganicacids,asphenolics,mayalsocontributetotheseeffects[37].
ThetotalphenolicandflavonoidcontentsofdifferentpartsofM.neglectawerealreadyreportedby
Dalaretal.[11]forsamplesfromtheEasternAnatoliaRegionofTurkey. Lowertotalphenoliccontents
of17.4˘0.3mgGAE/gextractand6.6˘0.3mgGAE/gextractwerereportedinleafandwholeplant
extracts,respectively,obtainedusingacidifiedmethanol(80%methanoland1%HCl(v/v)inwater)as
extractionsolvent. Regardingtotalflavonoids,7.21˘0.28mgRE(rutinequivalents)/gextractand
2.95˘0.16mgRE/gextractwerefoundintheleafandwholeplantextracts,respectively. Thesame
authorsalsoevaluatedtheantioxidantactivitythroughtheferricreducingantioxidantpower(FRAP)
andoxygenradicalabsorbancecapacity(ORAC)assays. Thehighestbioactivitywasassignedtothe
leafextractsandthentotheflowerextracts. Thesedifferencesinthephenoliccontentmaybejustified
byvariationsinedafoclimaticconditionsofthelocationswherethesampleswerecollected,which
may affect the plant composition during the growing season [38]. Additionally, as verified in our
study,theextractionmethodalsocausessignificantvariationsintheevaluatedresponses. Theused
ofdifferentvariablesinobtaininghydromethanolicanddecoctedextracts,namelydifferentsolvents,
extraction times, temperatures, and sample to solvent ratios, may have favored the extraction of
differentmolecules.
Although nothing has been reported on the impact of gamma irradiation on the antioxidant
activity or phenolic composition of M. neglecta, different effects have been reported in other plant
materials. AstudyconductedbyPinelaetal.[9]concludedthatirradiationdosesupto10kGydid
notsignificantlyaffecttheantioxidantactivityandphenoliccompositionofdecoctionsandinfusions
preparedfromT.lignosasamples. Ontheotherhand, Pereiraetal. reportedincreasedantioxidant
propertiesininfusionsandmethanolicextractsofborututu(CochlospermumangolensisWelw.)[39]and
G. biloba samples [25] irradiated at 10 kGy, respectively. The authors also verified that the 10 kGy
doseimprovestheextractabilityofphenoliccompoundsfromtheG.bilobasamples[40]. Anincreased
totalphenoliccontentandenhancedantioxidantactivityofgammairradiatedalmondskinsextracted
with40%ethanolwasreportedbyHarrisonandWere[41]. Thoseauthorsattributedtheseresultsto
thereleaseofphenoliccompoundsfromglycosidiccomponentsanddegradationoflargerphenolic
compounds into smaller ones by the gamma irradiation treatment. In fact, the direct impact of
gamma-rays and the indirect action of radiolytic products may change the structure of different
antioxidantmoleculesand/orbreaksomechemicalbonds,thusleadingtoitsdecompositionoraltered
extractabilityfromtheplantmaterial[15,25,41]. Thatiswhythebioactivityofirradiatedsamplescan
eitherdecreaseorimprove.
3. MaterialsandMethods
3.1. Dosimeters,StandardsandReagents
Amber Perspex routine dosimeters, Batch V, were purchased from Harwell Company
(Oxfordshire,UK).Organicacids(oxalic,quinic,malic,citric,succinicandfumaricacids)andTrolox
(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid)werepurchasedfromSigma(St. Louis,MO,
USA).2,2-Diphenyl-1-picrylhydrazyl(DPPH‚)wasobtainedfromAlfaAesar(WardHill,MA,USA).
Molecules2016,21, 467 8of13
Allotherchemicalsandsolventswereofanalyticalgradeandpurchasedfromcommonsources. Water
wastreatedinaModelA10Milli-Qwaterpurificationsystem(Millipore,Billerica,MA,USA).
3.2. SampleCollectionandIrradiationExperiments
Plantmaterial(leafyfloweringstems)fromdwarfmallow(MalvaneglectaWallr. Fam. Malvaceae)
was sustainably harvested from the wild in June at Miranda do Douro, Northeastern Portugal,
consideringlocalmedicinalusesaswellashealers’andselectedconsumers’criteria,whicharerelated
toparticulargatheringsitesandrequirementsforsafeherbaldosagesforms,namelydecoctions[42].
TaxonomicidentificationoftheplantmaterialwasconfirmedbyDr. AnaMariaCarvalhofromthe
PolytechnicInstituteofBragança,Portugal. AvoucherspecimenwasdepositedintheHerbariumof
theSchoolofAgricultureofBragança. Sampleswerethenlyophilized(FreeZone4.5,Labconco,Kansas
City,MO,USA),reducedtoafinedriedpowder(20mesh)andmixedtoobtainahomogeneoussample.
Theobtainedpowderedsamplewasdividedintofourportionsandsubmittedto1,5and8kGy
ofgamma-rays(predicteddoses). Anon-irradiatedcontrol(0kGy)followedalltheexperiments. The
irradiationwasperformedinacobalt-60experimentalchamber(Precisa22,GravinerManufacturing
Company Ltd., London, UK) located at the University of Lisbon Centre for Nuclear Sciences and
Technologies(C2TN,Lisbon,Portugal),withfoursourcesandatotalactivityof177TBq(4.78kCi;
January2014).Duringtheirradiationprocess,AmberPerspexroutinedosimeterswereusedtomeasure
thedistributionoftheabsorbedenergyandtodeterminethemaximum(D )andtheminimum(D )
max min
doseabsorbedbythesamples,followingtheprocedurepreviouslydescribedbyFernandesetal.[43].
The measured average doses were 1.10 ˘ 0.16 kGy, 4.82 ˘ 0.10 kGy and 8.07˘0.46kGy for the
samplesirradiatedatthepredicteddosesof1,5and8kGy,respectively. Theestimateddoseratefor
theirradiationposition,obtainedwithaFrickedosimeter[43],was1.9kGy/handthedoseuniformity
ratio(D /D )was1.2. Forsimplicity,thepredicteddoseswereconsideredinthetext.
max min
3.3. ColorMeasurement
Thepowderedsampleswereplacedonaadapterforgranularmaterials(modelCR-A50,Konica
MinoltaSensing,Inc.,Sakai,Osaka,Japan)toreduceexternalinterferencesanddatawerecollectedin
threedifferentpointsoneachsetofsampleswithacolorimeter(modelCR-400,Konica)previously
calibratedusingthestandardwhiteplate. UsingilluminantCandthediaphragmopeningof8mm,the
CIEL*a*b*colorspacevalueswereregisteredthroughthecomputerizedsystemusingtheSpectraMagic
Nx(versionCM-S100W)colordatasoftware. Averagevalueswereconsideredtodeterminethecolor
coordinates,whereL*representslightness,a*representschromaticityonagreen(–)tored(+)axis,
andb*representschromaticityonablue(–)toyellow(+)axis. Thetotalcolordifference(∆E*)was
calculatedaccordingtotheCIEDE2000equation[44].
3.4. PreparationofDecoctionsandHydromethanolicExtracts
Decoctionswerepreparedaccordingtofolkrecipes/formulations[42]. Briefly,eachpowdered
sample (1 g) was added to distilled water (200 mL) and boiled for 5 min. The mixture was left to
stand at room temperature for 5 min more and then was filtered through Whatman No. 4 paper.
Theobtaineddecoctionswerefrozenandlyophilized.
Hydromethanolic extractions were performed by stirring each powdered sample (1 g) with
methanol/water (30 mL, 80:20, v/v) at 25 ˝C and 150 rpm for 1 h. After filtering the supernatant
through Whatman No. 4 paper, the residue was extracted with an additional portion (30 mL) of
the hydromethanolic mixture. The combined extracts were concentrated under reduced pressure
(R-210rotaryevaporator,Büchi,Flawil,Switzerland)andthenfrozenandlyophilized.
The lyophilized decocted and hydromethanolic extracts were redissolved in water and
methanol/water(80:20,v/v),respectively,toobtain4mg/mLstocksolutionswhichweresuccessively
diluted to different concentrations for evaluation of the antioxidant activity and total phenolics
andflavonoids.
Molecules2016,21, 467 9of13
3.5. AnalysisofOrganicAcids
Organic acids were analyzed by ultra fast liquid chromatography (UFLC) coupled with
photodiode array (PDA) detection according to the procedures previously described by
Pereiraetal.[28]. Briefly,thepowderedsamples(~1g)wereextractedbystirringwithmetaphosphoric
acid(25mL,25˝Cat150rpm)for45minandthensubsequentlyfilteringthroughWhatmanNo.4
paper. Fordecoctions,thelyophilizedextracts(~10mg)weredissolvedinmetaphosphoricacid(1mL).
Allsampleswerefilteredthrough0.2µmnylonfiltersbeforeanalysis. Theorganicacidsfoundwere
quantifiedbycomparisonoftheareaoftheirpeaksrecordedat215nmwithcalibrationcurvesobtained
fromcommercialstandardsofeachcompound. Theresultswereexpressedinmgpergofdryweight
orlyophilizeddecoction(dw).
3.6. EvaluationoftheTotalPhenolicandFlavonoidContent
The total phenolic content was determined in the hydromethanolic and decocted extracts
concentratedat1.25mg/mLbytheFolin-Ciocalteumethod[45,46]withslightmodifications[47]. This
assayisbasedontheformationofablue-coloredcomplexbetweenthemolybdenumandtungsten
present in the Folin-Ciocalteu reagent upon reaction with reducing agents, which is monitored at
765nm. Thestandardcurvewascalculatedusinggallicacidandtheresultswereexpressedasmgof
gallicacidequivalents(GAE)pergofextract.
The total flavonoid content was determined in the hydromethanolic and decocted extracts
concentrated at 2.5 mg/mL using the aluminium chloride colorimetric method [48] with slight
modifications as described by the authors [49]. This assay is based on the formation of a
flavonoid-aluminiumcomplex,whichismonitoredat510nm. Thestandardcurvewascalculated
usingcatechinandtheresultswereexpressedasmgofcatechinequivalents(CE)pergofextract.
3.7. EvaluationoftheAntioxidantActivity
The hydromethanolic and decocted extracts at different concentrations were submitted to
four distinct invitro assays to evaluate its antioxidant capacity [9]. Briefly, the DPPH‚ scavenging
activityandthereducingpowerassayswereperformedusinganELX800MicroplateReader(Bio-Tek
Instruments,Inc.;Winooski,VT,USA).ThereductionofDPPH‚wasdeterminedafterincubationof
thedifferentextractswithamethanolicsolutioncontainingDPPH‚ (6ˆ10´5 M)for60mininthe
darkbymeasuringtheabsorbanceat515nm. Theradicalscavengingactivity(RSA)wascalculatedas
apercentageofDPPH‚discolorationusingtheEquation(1):
RSAp%q “ rpA ´A q{A sˆ100 (1)
DPPH S DPPH
where A is the absorbance of the DPPH‚ solution and A is the absorbance of the solution
DPPH S
containing the sample extract. The reducing power was evaluated by the ferricyanide/Prussian
blue assay, whereby the capacity of the extracts to convert potassium ferricyanide (K [Fe(CN) ])
3 6
into potassium ferrocyanide (K [Fe(CN) ]), which then reacts with ferric chloride (FeCl ) to form
4 6 3
a ferric-ferrous complex that is measuring spectrophotometrically, was monitored at 690 nm. The
β-carotene bleaching inhibition (CBI) was evaluated by measuring the capacity of the extracts to
neutralize linoleate free radicals. The different extracts were mixed with an emulsion containing
β-carotene,linoleicacidandTween80emulsifierandtheabsorbancewasimmediatelymeasuredat
470nminaModel200spectrophotometer(AnalytikJena,Jena,Germany). After2hofincubationat
50˝C,theabsorbancewasreadagain. TheCBIwascalculatedusingtheEquation(2):
CBIp%q“ pA {A qˆ100 (2)
120 0
whereA istheabsorbanceoftheemulsionafter2hofincubationandA istheinitialabsorbance.The
120 0
thiobarbituricacidreactivesubstances(TBARS)formationinhibitionwasevaluatedinporcinebrain
Molecules2016,21, 467 10of13
homogenates. Thecolorintensityofthemalondialdehyde-thiobarbituricacid(MDA-TBA)complex
formedduringheatingofthereactionmixtureat80˝Cwasmeasuredat532nm. Theinhibitionratio
wascalculatedusingtheEquation(3):
Inhibitionratiop%q “ rpA´Bq{Asˆ100 (3)
whereAandBcorrespondtotheabsorbanceofthecontrolandthesamplesolution,respectively. The
resultswereexpressedinEC values(mg/mL),i.e.,sampleconcentrationproviding50%ofantioxidant
50
activityor0.5ofabsorbanceinthereducingpowerassay. Troloxwasusedaspositivecontrol.
3.8. StatisticalAnalysis
Inallcases,analyseswerecarriedoutusingthreesamplesseparatelyprocessed,andalltheassays
werecarriedoutintriplicate. Theresultswereexpressedasmean˘standarddeviationandanalyzed
using one-way analysis of variance (ANOVA) followed by Tukey’s HSD test with α = 0.05. This
treatmentwascarriedoutusingIBMSPSSStatisticsforWindows,Version22.0(IBMCorp.,Armonk,
NY,USA).
4. Conclusions
This study demonstrated the adequacy of gamma irradiation at 5 and 8 kGy to preserve
the color parameters of M. neglecta dry material. Moreover, it was confirmed that the observed
irradiation-induced modifications of color parameters not only depend on the applied dose, but
alsoontheplantmaterialunderstudy. Oxalic,quinic,malic,citric,succinicandfumaricacidswere
identifiedinthisplantforthefirsttime. Thetotallevelsrecordedindecoctedextractswerealways
higher than those found in the plant dry material, as well as the individual levels of each organic
acid(exceptforcitricacid). Irradiationat5kGyincreasedtheamountsofcitricandsuccinicacids
and decreased the fumaric acid levels in both matrices. In general, decoctions were preferred for
theirhigherlevelsoftotalphenolicsandflavonoids,DPPH‚scavengingactivityandreducingpower.
Inthesepreparations,theantioxidantpropertiesandlevelsoftotalphenolicsandflavonoidswere
decreasedwiththe8kGydose. Inturn,thehydromethanolicextractsobtainedfromsamplesirradiated
at1kGyshoweddecreasedlevelsoftotalphenolic,totalflavonoids,andlowerantioxidantproperties.
Thus,decoctionswerehighlightedbyinterestingantioxidantpropertiesandlevelsoftotalphenolics
and organic acids. Nevertheless, further studies are of interest to evaluate the decontamination
effectivenessofthistechnologyandtoinvestigatetheeffectinotherqualityparameters.
Acknowledgments: TheauthorsaregratefultoPRODERresearchprojectno. 53514,AROMAP,forfinancial
supportoftheworkandtotheFoundationforScienceandTechnology(FCT,Portugal)forfinancialsupportto
CIMO(PEst-OE/AGR/UI0690/2014),REQUIMTE(UID/QUI/50006/2013),C2TN(RECI/AAG-TEC/0400/2012),
J.Pinela(SFRH/BD/92994/2013)andL.Barros(SFRH/BPD/107855/2015).
Author Contributions: J. Pinela, L. Barros, A.L. Antonio and I.C.F.R. Ferreira designed the experiments;
A.M.Carvalhoperformedthetaxonomicidentificationofthespecies;A.L.Antonioperformedtheirradiation
treatment; J. Pinela and L. Barros performed the analytical assays and analyzed the data; J. Pinela and
I.C.F.R.Ferreirawrotethemanuscript;A.L.Antonio,A.M.CarvalhoandM.B.P.P.Oliveirarevisedthemanuscript.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
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