Leccinum molle (Bon) Bon and Leccinum vulpinum Watling

12 Pages·2016·0.61 MB·English

molecules
Article
Leccinum molle (Bon) Bon and Leccinum vulpinum
Watling: The First Study of Their Nutritional and
Antioxidant Potential
FilipaS.Reis1,2,3,4,LillianBarros1,AnabelaMartins1,M.HelenaVasconcelos3,4,5,
PatriciaMorales2andIsabelC.F.R.Ferreira1,*
1 MountainResearchCenter(CIMO),SchoolofAgriculture,PolytechnicInstituteofBragança,Apartado1172,
Bragança5301-855,Portugal;[email protected](F.S.R.);[email protected](L.B.);[email protected](A.M.)
2 Dpto.NutriciónyBromatologíaII,FacultaddeFarmacia,UniversidadComplutensedeMadrid(UCM),
PzaRamónyCajal,s/n,MadridE-28040,Spain;[email protected]
3 i3S-InstitutodeInvestigaçãoeInovaçãoemSaúdedaUniversidadedoPorto,RuaAlfredoAllen,208,
Porto4200-135,Portugal;[email protected]
4 CancerDrugResistanceGroup,InstituteofMolecularPathologyandImmunologyoftheUniversityof
Porto,IPATIMUP,RuaJúlioAmaraldeCarvalho,45,Porto4200-135,Portugal
5 LaboratoryofMicrobiology,DepartmentofBiologicalSciences,FacultyofPharmacyoftheUniversityof
Porto,RuadeJorgeViterboFerreira228,Porto4050-313,Portugal
* Correspondence:[email protected];Tel.:[phone];Fax:[phone]
AcademicEditor:DerekJ.McPhee
Received:21December2015;Accepted:18February2016;Published:20February2016
Abstract: This work presents the chemical profile of two edible species of mushrooms from the
genus Leccinum: Leccinum molle (Bon) Bon and Leccinum vulpinum Watling, both harvested on
theoutskirtsofBragança(NortheasternPortugal). Bothspecieswerepreparedandcharacterized
regardingtheircontentinnutrients(i.e.,freesugars,fattyacidsandvitamins),non-nutrients(i.e.,
phenolicandotherorganicacids)andantioxidantactivity. Tothebestofourknowledge,noprevious
studies on the chemical characterization and bioactivity of these species have been undertaken.
Accordingly,thisstudyintendstoincreasetheavailableinformationconcerningediblemushroom
species,aswellastohighlightanotherimportantfactorregardingtheconservationofthemycological
resources—theirpotentialassourcesofnutraceutical/pharmaceuticalcompounds. Overall,both
speciesrevealedsimilarnutrientprofiles, withlowfatlevels, fructose, mannitolandtrehaloseas
theforemostfreesugars,andhighpercentagesofmono-andpolyunsaturatedfattyacids. Theyalso
revealed the presence of bioactive compounds, namely phenolic (e.g., gallic acid, protocatechuic
acid and p-hydroxybenzoic acid) and organic acids (e.g., citric and fumaric acids) and presented
antioxidantproperties.
Keywords: chemical profile; nutritional value; nutraceuticals; bioactive compounds;
antioxidantpotential
1. Introduction
FewstudiesexistontheLeccinummolle(Bon)BonandLeccinumvulpinumWatlingmushroom
species. IntheliteratureitispossibletofindadescriptionofthemorphologicalfeaturesofL.molle[1],
andsomeecological[2–4]andenzymatic[5,6]studieswithL.vulpinumareavailable. Asfaraswe
know,thesearetheonlystudiesperformedtodate,soevenconsideringtheyaretwospeciesofedible
mushrooms,theydonotseemtobethemostconsumedbyaficionados. However,theirinterestfrom
thepointofviewofobtainingnutraceuticalscannotbeexcluded. Furthermore,apartfromtryingto
Molecules2016,21,246;doi:10.3390/molecules21020246 www.mdpi.com/journal/molecules
Molecules2016,21,246 2of12
valorizethesenaturalresourcesbyprovidingnewinformationabouttheirpotentialasasourceof
bioactivecompoundsandnutraceuticals,thisstudyalsoaimstohelpintheconstructionofwildedible
mushroomsdatabases.
SinceBragança(Portugal)isoneoftheregionsofEuropewithgreatermycologicalbiodiversity,
itisimportanttoundertakeacarefulidentificationandcharacterizationofalargenumberofwild
mushroom species. In addition, being also a region where mushroom picking is a tradition, the
scientificcommunityplaysanimportantroleintheformationofthegeneralpopulation. Furthermore,
publicawarenessoftheneedforacarefulandhealthydiethasincreased.Inthissense,theconsumption
ofvegetableproteinsisincreasinglyadvisedandhasbeenincreasingovertheyears.Thisisduetomany
reasonsincludingtheprevalenceofanimaldiseases,shortageofanimalproteinworldwide,thestrong
demandforhealthyfood,religion,aswellaseconomicreasons[7]. Inthisregard,mushroomshave
beenrecommendedasanalternativeproteinsourcetomeat,sincetheyarerichinproteins,essential
aminoacids,vitaminsandessentialminerals[7,8]. Additionally,asfurtherdemonstratedinthiswork,
manyresearchershaveshownthatmushroomsarealsolowincaloriesandfat[9–13]. Moreover,their
primary(e.g.,polysaccharidesandglycoproteins)andsecondary(e.g.,phenoliccompounds)bioactive
compounds,makethemasourceofnutraceuticalsandmoleculeshavingmedicinalfunctionssuchas
antioxidant,immunomodulating,andantitumouractivity[14,15].
The present study is the first to quantify the nutritional and antioxidant potential of the
aforementionedLeccinumspecies,pavingthewayfornewandfuturestudiesrelatedtothebioactivity
of these species and wild mushrooms in general. Moreover, we intend to valorize these natural
resourcesbyadvocatingspeciesconservation.
2. Results
2.1. NutrientComposition
ThenutrientcompositionofthestudiedspeciesispresentedinTable1.
Table1.NutrientscompositionofwildsamplesofLeccinummolle(Bon)BonandLeccinumvulpinum
Watling(mean˘SD).
Nutrients Leccinummolle(Bon)Bon LeccinumvulpinumWatling p-Value
Ash(g/100gdw) 5.70˘0.55 13.61˘0.86 <0.01
Proteins(g/100gdw) 13.07˘1.00 10.48˘0.50 0.0156
Fat(g/100gdw) 2.80˘0.16 2.97˘0.13 0.1090
Carbohydrates(g/100gdw) 78.43˘0.86 72.94˘0.51 0.0007
Energy(kcal/100gdw) 391.18˘0.99 360.41˘2.89 <0.001
Fructose(g/100gdw) 3.06˘0.03 4.52˘0.14 <0.0001
Mannitol(g/100gdw) 11.32˘0.30 2.68˘0.01 <0.0001
Trehalose(g/100gdw) 2.71˘0.09 8.31˘0.01 <0.0001
Totalsugars(g/100gdw) 17.09˘0.24 15.51˘0.14 0.0002
C16:0(relativepercentage) 11.23˘0.21 12.25˘0.25 0.0011
C18:0(relativepercentage) 1.79˘0.01 2.77˘00.04 <0.0001
C18:1n9(relativepercentage) 38.61˘0.33 27.06˘0.09 <0.0001
C18:2n6(relativepercentage) 43.49˘0.10 53.60˘0.37 <0.0001
SFA(relativepercentage) 16.94˘0.41 17.09˘0.31 0.4377
MUFA(relativepercentage) 39.29˘0.31 28.59˘0.04 <0.0001
PUFA(relativepercentage) 43.77˘0.10 54.32˘0.27 <0.0001
α-Tocopherol(µg/100gdw) 12.48˘0.70 14.76˘1.13 0.0137
β-Tocopherol(µg/100gdw) 12.88˘0.99 216.18˘0.90 <0.0001
γ-Tocopherol(µg/100gdw) nd 296.67˘0.19 0.0032
Totaltocopherols(µg/100gdw) 25.36˘0.29 527.61˘2.22 0.0008
dw:dryweight;nd:notdetected;mainfattyacids:C16:0(palmiticacid),C18:0(stearicacid),C18:1n9(oleic
acid)andC18:2n6(linoleicacid);20morefattyacidswereidentifiedintraceamounts;SFA:saturatedfattyacids;
MUFA:monounsaturatedfattyacids;PUFA:polyunsaturatedfattyacids.
Molecules2016,21,246 3of12
Regarding fat contents, there were no significant differences between both samples (p-value
0.1090). Moreover,bothspeciesrevealedalowfatcontent(2.80–2.97g/100gdw)comparedtoproteins
(10.48–13.07g/100gdw)andparticularlycarbohydrates(72.94–78.43g/100gdw).
Fromanutritionalstandpoint,itisalsoimportanttoidentifythesolublesugarspresentinthe
samples. Thesolublesugarsidentifiedinthestudiedspecieswerefructose(3.06–4.52g/100gdw),
mannitol(2.68–11.32g/100gdw)andtrehalose(2.71–8.31g/100gdw)(Table1).
Consideringthefattyacidscomposition,bothspeciesrevealedasimilarprofile,withpalmitic
acid (C16:0), stearic (C18:0), oleic (C18:1n9) and linoleic acid (C18:2n6) being the main fatty acids
present(Table1). Althoughpalmiticandstearicacidsaresaturatedfattyacids(SFA),thesearepresent
inmuchlowerquantitiesinbothspecies,comparedtooleic(amonounsaturatedfattyacid—MUFA)
andlinoleic(apolyunsaturatedfattyacid—PUFA)acids. Inadditiontothelowervalues,bothspecies
revealedsimilarSFAcontents,withoutsignificantdifferencesbetweenthem(16.94%and17.09%).
Apart from the abovementioned nutrients, we also assessed the presence of certain vitamins,
namelysomeisoformsofvitaminE(especiallyknownforitsantioxidantactivity). α-andβ-tocopherol
werepresentinbothspecies,butonlyL.vulpinumrevealedthepresenceoftheγ-isoform(Table1).
Infact,γ-tocopherolwastheprevailingisoforminthisspecies,whichcontributedheavilytothemuch
highervalueoftotaltocopherols(527.61µg/100gdw)comparedwithL.molle(25.36µg/100gdw).
2.2. Non-NutrientComposition
Thestudyofsomecompoundsotherthannutrients,i.e.,phenolicandotherorganicacids,was
performedinbothsamples. TheresultsaresummarizedinTable2.
Table2.Non-nutrientcompositionofwildsamplesofLeccinummolle(Bon)BonandLeccinumvulpinum
Watling(mean˘SD).
Compounds Leccinummolle(Bon)Bon LeccinumvulpinumWatling p-Value
Oxalicacid(g/100gdw) 0.29˘0.03 0.11˘0.01 0.0002
Quinicacid(g/100gdw) nd 0.13˘0.01 <0.0001
Citricacid(g/100gdw) 2.62˘0.16 nd <0.0001
Fumaricacid(g/100gdw) 1.04˘0.02 0.25˘0.01 <0.0001
Totalorganicacids(g/100gdw) 3.95˘0.19 0.49˘0.01 <0.0001
Gallicacid(mg/100gdw) nd 0.08˘0.01 <0.0001
Protocatechuicacid(mg/100gdw) nd 0.35˘0.05 <0.0001
p-Hydroxybenzoicacid(mg/100gdw) 0.06˘0.01 0.09˘0.01 0.0002
Totalphenolicacids(mg/100gdw) 0.06˘0.01 0.52˘0.05 <0.0001
Cinnamicacid(mg/100gdw) 0.13˘0.01 0.17˘0.01 <0.0001
nd:notdetected;dw:dryweight.
Analysingtheresultsobtained,oxalic,quinic,citricandfumaricacidswerethemainorganicacids
identified. Oxalicandfumaricacidwerefoundinbothmushrooms(0.11–1.04g/100gdw). However,
theprofilesdifferduetothepresenceofquinicacidonlyinL.vulpinum(0.13g/100gdw)andcitric
acidinL.molle(2.62g/100gdw).
Regardingphenolicacids,theprofilesalsodiffersubstantiallyamongspecies(Table1;Figure1).
L.mollehasonlypresentinitschemicalconstitutionp-hydroxybenzoicacid(0.06mg/100gdw)and
therelatedcompoundcinnamicacid(0.13mg/100gdw). Ontheotherhand,theL.vulpinumprofile,
besidesp-hydroxybenzoicacid(0.09mg/100gdw)andtherelatedcompoundcinnamicacid(0.17
mg/100gdw),alsoincludesgallicacid(0.08mg/100gdw)andprotocatechuicacid(0.35mg/100g
dw).
Molecules2016,21,246 4of12
Molecules 2016, 21, 246  4 of 12 
FFiigguurree 11. .PPhheennoolilcic aaccididss pprrooffiilele ooff LL. .mmoolllele (A(A) )aanndd LL. .vvuulplpininuumm (B(B) )rreeccoorrddeedd aatt 228800 nnmm. .11: :ggaalllilci caacicdid; ;22: :
pprroottooccaatteecchhuuiicc aacciidd;; 33:: pp‐-hhyyddrrooxxyybbeennzzooiicc aacciidd aanndd 44:: cciinnnnaammiicc aacciidd. .

2.3. Antioxidant Potential 
2.3. AntioxidantPotential
The antioxidant properties of the samples were evaluated through different assays (Table 3). In 
The antioxidant properties of the samples were evaluated through different assays (Table 3).
order to estimate the reducing power of the samples, two different methodologies were performed: 
Inordertoestimatethereducingpowerofthesamples,twodifferentmethodologieswereperformed:
the  Folin‐Ciocalteu  assay  and  the  ferricyanide/Prussian  blue  assay.  To  evaluate  the  radical 
theFolin-Ciocalteuassayandtheferricyanide/Prussianblueassay. Toevaluatetheradicalscavenging
scavenging capacity, the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical was used, and to assess the 
capacity, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical was used, and to assess the lipid
lpipeirdo xpiedraotxioidnaitniohnib iintihoinb,ittihoenβ, t-hcea rβo‐tceanreo/telinneo/lleinatoeleaantde aTnBdA TRBSAaRssSa yassswayesr ewpeerref opremrfeodrm. ed. 
TTaabbllee 33.. AAnnttiiooxxiiddaanntt pprrooppeerrttiieess oofft htheem metehtahnaonloiclice xetxratrcatsctos fowf iwldilsda msapmlepsloesf Loefc cLiencucminummo lmleo(lBleo n(B)oBno)n 
Baonnd aLnedcc Lineuccminvuumlp vinuulpminWumat Wlinagtl(imnge a(mn˘eanSD ± )S.D). 
Leccinum molle  Leccinum vulpinum 
Activity  Assay  p‐Value 
(Bon) LBeocnc inummolle WaLtleinccgi num
Activity Assay p-Value
Folin‐Ciocalteu assay   (Bon)Bon vulpinumWatling
17.76 ± 0.26  30.14 ± 0.70  <0.001 
Reducing  (mg GAE/g extractF) olin-Ciocalteuassay
Power  Ferricyanide/Pruss(imang bGluAeE a/ssgayex  tract) 2.13 ± 107.0.17 6˘0.26 0.543 0± .01.401˘  0.70 <0.<000.10 01
ReducingPowe(rEC50; mg/mL) 
Ferricyanide/Prussian
Radical  2.13˘0.01 0.54˘0.01 <0.001
DPPH radical‐scavbelnugeinags saacytiv(EitCy as;smayg /mL)
scavenging  50 10.68 ± 0.55  1.19 ± 0.02  <0.001 
(EC50; mg/mL) 
activity  DPPHradical-scavenging
Radicalscavengβin‐cgaraoctteivniet/ylinoleataec atisvsaityy  assay 10.68˘0.55 1.19˘0.02 <0.001
Lipid  2.23 ± 0.05  0.11 ± 0.01  <0.001 
peroxidation  (EC50; mg/mL)  (EC50;mg/mL)
TBARS assay  
inhibition  β-carotene/linoleateass1a.y48 ± 0.02  0.03 ± 0.00  <0.001 
(EC50; mg/mL)  2.23˘0.05 0.11˘0.01 <0.001
(EC ;mg/mL)
LipRidegpaerrdoixnidga tthioen Fionlhinib‐Citiioocnalteu ass5a0y, higher values mean higher reducing power; regarding the other 
TBARSassay
assays, the results are presented as EC50 values, which m1e.4a8n˘ th0a.0t 2higher val0u.0e3s ˘co0r.r0e0spond to lo<w0e.0r0 1
(EC ;mg/mL)
50
reducing power or antioxidant potential. EC50 is the extract concentration corresponding to 50% of 
RegardingtheFolin-Ciocalteuassay,highervaluesmeanhigherreducingpower;regardingtheotherassays,
antthioexriedsaunltts aacretipvrietyse onrte 0d.5a soEf Cab50svoarbluaens,cwe hfoicrh thmee aFnertrhiactyhaingihdeer/vParluuesssicaonr rbelsupeo nadsstaoylo. wTreorlroexd uEcCin5g0 vpaolwueers: 
41 oμrga/nmtiLox (irdeadnutcpiontge nptioawl.eErC),5 402is μtghe/mexLt r(aDctPcPoHnc esncatrvaetinognincogr raecstpivonitdyi)n, g18to μ5g0/%moLf (aβn‐tcioaxroidtaenntea bclteivaictyhionrg 
inh0i.b5iotifoanb)s oarnbdan 2c3e μfogr/tmheLF (eTrrBicAyRanSi dine/hPibruitsisoinan). bGluAeEa:s sgaayl.liTcr oalcoixdE eCq5u0ivvaallueenst:s4. 1µg/mL(reducingpower),
42µg/mL(DPPHscavengingactivity),18µg/mL(β-carotenebleachinginhibition)and23µg/mL(TBARS
inhibition).GAE:gallicacidequivalents.
Generally, L. vulpinum revealed the highest antioxidant potential. This species displayed the 
highest reducing power, exhibiting the highest levels of reducing compounds evaluated by the 
Generally, L. vulpinum revealed the highest antioxidant potential. This species displayed the
Folin‐Ciocalteu assay (30.14 mg GAE/g extract). Assessed through the ferricyanide/Prussian blue 
highest reducing power, exhibiting the highest levels of reducing compounds evaluated by the
assay, the highest reducing power also belonged to L. vulpinum (lowest EC50 value—0.54 mg/mL). 
Folin-Ciocalteu assay (30.14 mg GAE/g extract). Assessed through the ferricyanide/Prussian blue
This species also revealed the highest free radical scavenging activity (1.19 mg/mL) and lipid 
assay,thehighestreducingpoweralsobelongedtoL.vulpinum(lowestEC value—0.54mg/mL).
50
peroxidation inhibition, assessed through the β‐carotene bleaching inhibition (0.11 mg/mL) and by 
Thisspeciesalsorevealedthehighestfreeradicalscavengingactivity(1.19mg/mL)andlipid
the TBARS assay (0.03 mg/mL). 
peroxidationinhibition,assessedthroughtheβ-carotenebleachinginhibition(0.11mg/mL)andby

theTBARSassay(0.03mg/mL).
Molecules2016,21,246 5of12
3. Discussion
3.1. NutrientComposition
As aforementioned, the nutritional value of the studied species was evaluated in order to
recognize and value these species as healthy food. Table 1 shows that both species have low fat
content,beingricherinproteinsandmostlyincarbohydrates. Actually,thelatteraredescribedasthe
representativecompoundsofmushrooms,constitutingnearbyone-halfofmushroomdrymatter[11].
However,thisgroupincludesseveralcompounds,suchassugars(monosaccharides,theirderivatives
andoligosaccharides)andthereserveandstructuralpolysaccharides(glycans). So,giventhatitwas
intend to evaluate the nutritional value of the mushrooms, the soluble sugars presented by both
specieswasassessed. Thesolublesugarsidentifiedinthestudiedspecieswerefructose,mannitoland
trehalose(Table1). Mannitolandtrehaloseareverycommoninmushrooms[11]. Regardingfructose,
therearesomestudiesrelatingthepresenceofthissugartothetrophismofthespecies. According
to some authors [9], this sugar is mainly present in mycorrhizal species, which could justify its
identification in these Leccinum species. However, this compound has already been identified in
saprotrophicspecies[13]. Therefore,itspresenceseemsthentoberelatedtodifferentfactorsother
thanthetrophism.
Overall,bothspeciesrevealedatypicalnutritionalprofiledescribedformushrooms[10,11],so
thattheycanbereferredtoasvaluablenutritionalhealthyfoods,sincetheyarerichinproteinand
poorincaloriesandfat.
Moreover, although the fat levels are low, these include primarily unsaturated fatty acids.
As presented in Table 1, although the saturated palmitic and stearic acids represented part of the
mainfattyacidsfoundinthetargetspecies,oleic(MUFA)andlinoleic(PUFA)acidswereidentifiedin
muchhigherquantities. Thesefindingsareimportantbothforthechemicalcharacterizationofthe
speciesaswellastopromotetheirhealthbenefits. Itiswellknownthatmanybeneficialeffectshave
beenattributedtotheconsumptionofunsaturatedfattyacids,particularlyinrelationtopreventionof
cardiovasculardiseases[16,17].
Asdescribedintheprevioussection(Results), vitaminEwasalsofoundinthepresentstudy.
Giventheimportanceofthisvitamininpreventinglipidperoxidation[18],itsconsumptionthrough
ourdailydietisoftheutmostimportance. Regardingtheobtainedresults,α-andβ-isoformswere
presentinbothspecies,whileγ-tocopherolwasonlypresentinL.vulpinum(Table1). Thisdifference
canbeexplainedbythespecificityofproductionofdifferentmetabolitescharacteristicofeachspecies.
As described in literature [19], some compounds may be produced by all varieties of a particular
speciesorgenusduringtheirnormalmetabolism,whileothersmaybespecificofanorganism.
Accordingtothefindingsofthiswork,andpresentedinTable1,bothL.molleandL.vulpinumseem
tobevaluablehealthyfoods,beingasourceofmono-andoligosaccharides,mono-andpolyunsaturated
fattyacidsandvitaminE.
3.2. Non-NutrientComposition
Thestudyofnutrientcompositionisveryimportanttovalorisethe studiedLeccinumspecies,
sinceitprovidesinformationabouttheimportance/benefitsoftheirconsumption. However,itisalso
importanttostudyothermoleculesthatmaybebiologicallyactive. Forthisreason,ananalysisof
phenolicandotherorganicacidswasundertakeninbothsamples(Table2).
As mentioned above, both profiles were quite different between species. Therefore, some
particular compounds may be mostly produced by a specific species, or their production may be
relatedtothesurroundingenvironment(e.g., possiblesubjectiontostressconditions). Infact, the
activationofsecondarymetabolismandincreasedproductionofphenoliccompoundsinresponseto
variousstressconditionsiswelldocumented[20].
Aspreviousreferred,althoughorganicacidsareconsiderednon-nutrients,theyareconsidered
importantmoleculesgiventheirbiologicalactivities. Forexample,citricacidisknownworldwidefor
Molecules2016,21,246 6of12
itsantioxidantactivityandasanaturalconservative. ThismakesL.molleasanalternativesourceof
suchinterestingmolecules. Quinicacidisalsoknownforitsantioxidantpotential[21],whichmaybe
anindicatorofsuchactivitybyL.vulpinumspecies. Theotherorganicacids,oxalicandfumaricacids
arealsorecognisedbioactivecompoundswithpharmacologicalproperties[22,23].
AspresentedinTable2andFigure1, thechemicalprofileofL.vulpinumisricherinphenolic
acids. Thepresenceofthesecompoundsisimportantbecausetheyarepointedoutasbiologically
activemolecules. Theyareoftenassociatedwithhyperglycaemiaandhypertensionprevention[24],
beneficialcardiovasculareffects[25],ortheantioxidant,antimicrobialandantitumorpotentialofthe
species[26,27]. Althoughthereisaplethoraofphenoliccompoundsoccurringinnature,inourstudy
(asinmanystudiesaboutmushrooms)onlytheprecursorcinnamicacidandtwogroupsofphenolics
wereidentified: somehydroxybenzoicacids(gallic,protocatechuicandp-hydroxybenzoicacids)and
hydroxycinnamicacids(p-coumaricacid). Itshouldbenotedthatalthoughflavonoidsareoccasionally
reportedascompoundspresentinmushrooms,thesematricesdonothavethebiosyntheticpathway
toproducethem. Thus,itisbelievedthattheflavonoidsexceptionallypresentinsomemushroom
species,couldbeduetothesymbioticrelationshipsestablishedwithplants[28]. Wealsoshouldbear
inmindthatthechemicalcompositionofmushroomsdependsontheenvironmentinwhichthey
grow. Thisisappliedevenmoretosecondarymetabolites,suchasphenoliccompounds,whichare
usuallyproducedinresponsetostressconditions[29]. Nonetheless,thesecompoundsarepresent
insmallamountsinoursample,andthebioactivitypresentinthisstudycouldbecorrelatedwith
thesecompounds,butalsocouldberelatedtoothercompoundsnotidentifiedinthisstudy. Thus,the
obtainedresultsindicatethatLeccinumspeciesareagoodsourceofnutrientsandnutraceuticals,and
theirconsumptionisalsoawaytoobtainphenolicandotherorganicacids.
3.3. AntioxidantPotential
The antioxidant properties of the samples were evaluated through different assays (Table 3).
As mentioned, L. vulpinum revealed the highest antioxidant potential in all the performed assays.
Asexpected,thebioactivepropertiesoffoodsaredirectlyrelatedtothebioactivemoleculespresentin
theirchemicalconstitution. Therefore,theantioxidantpropertiesofthestudiedfruitingbodiesmust
berelatedtosomeofthecompoundsstudiedinthisworktopresentsuchantioxidantactivity. L.molle
showedhigherlevelsoforganicacids(3.95g/100gdw),whichmaybethebasisofitsantioxidant
activity. Ontheotherhand,L.vulpinumexhibitedgreatercontentsofphenolicacidsandmuchhigher
values of tocopherols (527.61 µg/100 g dw). Thus, vitamin E and phenolic acids may be largely
responsibleforthehigherantioxidantactivityofthespeciesL.vulpinum. However,theseresultsdo
notinvalidatethatothermolecules(e.g.,vitaminC,carotenoids,lycopene)mayalsoberesponsiblefor
theseproperties.
4. ExperimentalSection
4.1. StandardsandReagents
Acetonitrile 99.9%, n-hexane 95% and ethyl acetate 99.8% were of HPLC grade and obtained
from Fisher Scientific (Lisbon, Portugal). The fatty acids methyl ester (FAME) reference standard
mixture 37 (standard 47885-U) was purchased from Sigma (St. Louis, MO, USA) as were also
other individual fatty acid isomers, standards of sugars, tocopherols, and organic acids, Trolox
(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) and phenolic standards. Racemic tocol
(50mg/mL),waspurchasedfromMatreya(PleasantGap,PA,USA).2,2-Diphenyl-1-picrylhydrazyl
(DPPH)wasobtainedfromAlfaAesar(WardHill,MA,USA).Allotherreagentswereacquiredfrom
specializedsellers. WaterwastreatedinaMilli-Qwaterpurificationsystem(TGIPureWaterSystems,
Greenville,SC,USA).
Molecules2016,21,246 7of12
4.2. MushroomSpeciesandSamplePreparation
Leccinummolle(Bon)BonandLeccinumvulpinumWatlingwildsampleswerecollectedinBragança
inthenortheasternregionofPortugalintheautumnof2012. Authenticationswereundertakenat
thePolytechnicInstituteofBragançaandvoucherspecimensweredepositedattheherbariumofthe
SchoolofAgricultureofthePolytechnicInstituteofBragança,Portugal. Afterlyophilisation(FreeZone
4.5model7750031,Labconco,KansasCity,MO,USA),allthesamplesweremilledandmixedtoobtain
afinedriedandhomogeneouspowder(«20mesh),andthenstoredinadesiccator,untilthetimeof
theanalysis.
4.3. NutrientComposition
4.3.1. NutritionalValue
Thenutritionalvalueofthestudiedsampleswascalculatedbasedonprotein,fat,carbohydrate
andashcontentsobtainedfollowingstandardprocedures[30]. Thecrudeproteincontent(Nˆ4.38)
of the samples was estimated as nitrogen content by the macro-Kjeldahl method; the crude fat
by extracting a known weight of milled samples with petroleum ether, by means of a Soxhlet
apparatus;andtheashcontentassessedafterincinerationat600˘15˝C.Totalcarbohydrateswere
calculatedbydifference. EnergywascalculatedaccordingtoRegulation(EC)No. 1169/2011ofthe
EuropeanParliamentandoftheCouncil,of25October2011,ontheProvisionofFoodInformationto
Consumers[31],followingtheequation: Energy(kcal/100gdw)=4ˆ(gprotein+gcarbohydrate)+
9ˆ(gfat).
4.3.2. SolubleSugars
Solublesugarswereassessedthroughchromatographictechniques—highperformanceliquid
chromatograph(HPLC)—followingaprocedurealreadydescribed[32].TheHPLCapparatusconsisted
ofapump(Smartlinesystem1000,Knauer,Berlin,Germany),adegassersystem(Smartlinemanager
5000) and an auto-sampler (AS-2057 Jasco, Easton, MD, USA), coupled to a refraction index (RI)
detector(KnauerSmartline2300). ThechromatographicseparationwasachievedwithaEurospher
100-5NH column(5µm,4.6ˆ250mm,Knauer)operatingat30˝C(7971RGraceoven). Themobile
2
phasewasacetonitrile/deionizedwater,70:30(v/v)ataflowrateof1mL/min. Theidentificationofthe
sugarswasundertakenbycomparingtheirpeakrelativeretentiontimeswithcommercialstandards.
Data were analysed through the Clarity 2.4 Software (DataApex, Podohradska, Czech Republic).
Finally,thequantificationwasbasedontheRIsignalresponseofeachstandard,relyingontheinternal
standard(IS,raffinose)methodandusingcalibrationcurvesobtainedfromthecommercialstandards
ofeachcompound. Theresultswereexpressedingper100gofdryweight.
4.3.3. FattyAcids
Beforetheanalysisoffattyacids,atrans-esterificationprocedurewasperformed,followinga
procedurepreviouslyreported[32]. Thedetectionwasalsomadebychromatographictechniques,
in this specific case, by gas chromatography (GC). The GC equipment was composed by a gas
chromatograph (DANI 1000, Contone, Switzerland), equipped with a split/splitless injector and
aflameionizationdetector(FID).SeparationwasachievedusingaMacherey–Nagel(Düren,Germany)
column(50%cyanopropyl-methyl-50%phenylmethylpolysiloxane,30mˆ0.32mmi.d. ˆ0.25µm
df). Theoventemperatureprogramwasasfollows: theinitialtemperatureofthecolumnwas50˝C,
held for 2 min, then a 30 ˝C/min ramp to 125 ˝C, 5 ˝C/min ramp to 160 ºC, 20 ºC/ min ramp to
180˝C,3˝C/minrampto200˝C,20˝C/minrampto220˝Candheldfor15min. Thecarriergas
(hydrogen)flow-ratewas4.0mL/min(0.61bar),measuredat50˝C.Splitinjection(1:40)wascarried
outat250˝C.Theidentificationwascarriedoutbycomparingtherelativeretentiontimesofthefatty
acidmethylesters(FAME)ofthesampleswithcommercialstandards. Thequantificationwasmade
Molecules2016,21,246 8of12
usingtheClarity4.0.1.7Software(DataApex),beingtheresultsexpressedasrelativepercentagesof
eachfattyacid.
4.3.4. Tocopherols
VitaminEisoformswerealsoseparatedbyHPLC(abovementionedanddescribed),accordingto
aprocedurepreviouslydescribedbyotherauthors[33]. Theanalysiswasperformedbycouplingwith
afluorescencedetector(FP-2020;Jasco,Easton,MD,USA),whichwasprogrammedforexcitationat
290nmandemissionat330nm. ThechromatographicseparationwasachievedwithaPolyamideII
(5µm,250ˆ4.6mm)normal-phasecolumnfromYMCWaters(YMCAmerica,Inc.,Allentown,PA,
USA)operatingat30˝C.Themobilephaseusedwasamixtureofn-hexaneandethylacetate(70:30,
v/v) at a flow rate of 1 mL/min. The peaks detected were compared with commercial standards,
beingthequantificationbasedonthefluorescencesignal,usingtheinternalstandard(tocol)method.
Theresultswereexpressedinµgper100gofdryweight.
4.4. Non-NutrientComposition
4.4.1. OrganicAcids
OrganicacidsweredetectedbyUltraFastLiquidChromatography(UFLC).Detectionwascarried
outcouplingthechromatograph(Shimadzu20Aseries,ShimadzuCorporation,Kyoto,Japan)toa
photodiodearraydetector(PDA),using215nmand245nmaspreferredwavelengths. Separation
was achieved on a SphereClone (Phenomenex, Torrance, CA, USA) reverse phase C18 column
(5µm,250mmˆ4.6mmi.d.) thermostatted at 35 ˝C. The elution was performed with sulphuric
acid(3.6mM)usingaflowrateof0.8mL/min. Theorganicacidsfoundwerequantifiedcomparing
theareaoftheirpeaksrecordedat215nmwithcalibrationcurvesobtainedfromcommercialstandards
ofeachcompound[34]. Theresultswereexpressedingper100gofdryweight.
4.4.2. PhenolicAcids
Phenolic acids analysis was performed using the Shimadzu 20A series ultra-fast liquid
chromatograph(UFLC,ShimadzuCorporation, equipmentdescribedabove)[35]. Separationwas
achievedusingaWatersSpherisorbS3ODS-2C (3µm,4.6mmˆ150mm)columnthermostatted
18
at 35 ˝C. The solvents used were: (A) 0.1% formic acid in water; (B) acetonitrile. The elution
gradient established was 10% A to 15% B over 5 min, 15%–25% A in B over 5 min, 25%–35%
A in B over 10 min, isocratic 50% B for 10 min, and re-equilibration of the column, using a
flow rate of 0.5 mL/min. Double online detection was carried out in the PDA using 280 nm as
preferred wavelength and in a mass spectrometer (MS) connected to HPLC system via the DAD
cell outlet. The quantification of the phenolic compounds identified was made by comparison of
the area of their peaks recorded at 280 nm with calibration curves obtained from the commercial
phenolic standards: Gallic acid (y=224,587x´129,097; R2 = 0.9997; LOD 0.19 µg/mL; LOQ 0.63
µg/mL); protocatechuic acid (y=116,749x´38733; R2 = 1; LOD 0.29 µg/mL; LOQ 0.97 µg/mL);
p-hydroxybenzoic acid (y=164,204x+12,917; R2 = 0.9999; LOD 0.11 µg/mL; LOQ 0.36 µg/mL);
cinnamicacid(y=863,668x´884,517;R2=0.9998;LOD0.42µg/mL;LOQ1.41µg/mL).Theresults
wereexpressedinmgper100gofdryweight.
4.5. AntioxidantProperties
4.5.1. ExtractionProcedure
The lyophilized samples (1 g) were extracted by stirring with 40 mL of methanol at room
temperature for 1 h and subsequently filtered through Whatman No. 4 paper. The residue was
then extracted with an additional portion of methanol (20 mL) for 1 h. The combined methanolic
Molecules2016,21,246 9of12
extractswereevaporatedunderreducedpressureat40˝C(R-210rotaryevaporator,Büchi,Flawil,
Switzerland)andre-dissolvedinmethanol(20mg/mL)andstoredat4˝Cforfurtheruse.
4.5.2. AntioxidantActivityEvaluation
From the stock solution, successive dilutions were made and submitted to the invitro assays
alreadydescribed[35]. Thesampleconcentrations(mg/mL)providing50%ofantioxidantactivityor
0.5ofabsorbance(EC )werecalculatedfromthegraphsofantioxidantactivitypercentages(DPPH,
50
β-carotene/linoleateandTBARSassays)orabsorbanceat690nm(ferricyanide/Prussianblueassay)
againstsampleconcentrations. Troloxwasusedasapositivecontrol.
‚ Reducingpower
ThereducingpowerofthesampleswasmeasuredthroughtheFolin-Ciocalteuassayandthe
Ferricyanide/Prussianblueassays.
TheFolin-Ciocalteuassayallowstoquantifythetotalphenolicsandotherreducingspeciespresent
inthesamples,relyingontheirelectrontransfercapability. Theabsorbancewasmeasuredat765nm
(Analytikijena200-2004spectrophotometer,Jena,Germany)andgallicacidwasusedtoobtainthe
standardcurve. Theresultswereexpressedasmgofgallicacidequivalents(GAE)pergofextract.
Regarding the Ferricyanide/Prussian blue assay, the methodology of which is based on the
capacitytoconvertFe3+ intoFe2+,theabsorbancewasmeasuredat690nminaELX800Microplate
Reader(Bio-TekInstruments,Inc;Winooski,VT,USA).
‚ Radicalscavengingactivity
Intheperformedassay,thepurplechromogen2,2-diphenyl-1-picrylhydrazyl(DPPH)radicalis
reducedbyantioxidantcompoundstothecorrespondingpaleyellowhydrazine. Thismethodology
wasaccomplishedusingthemicroplatereadermentionedabove,andtheabsorptionmeasuredat515
nm. Theradicalscavengingactivity(RSA)wascalculatedasapercentageofDPPHdiscolorationusing
theequation: %RSA=[(ADPPH´AS)/ADPPH]ˆ100,whereASistheabsorbanceofthesolution
containingthesample,andADPPH,theabsorbanceoftheDPPHsolution.
‚ Lipidperoxidationinhibition
Thelipidperoxidationinhibitionwasevaluatedthroughtheinhibitionofβ-carotenebleaching
(orβ-carotene/linoleateassay)andtheTBARSassay.
Intheβ-carotene/linoleateassay,thepresenceofantioxidantsinthesamplesandtheircapacityto
neutralizethelinoleatefreeradicals,avoidsβ-carotenebleaching,whichcanbeevaluatedmeasuring
the absorbance at 470 nm. Therefore, β-carotene bleaching inhibition was calculated using the
followingformula:
pAbsorbanceafter2hofassay{initialabsorbanceqˆ100.
Thethiobarbituricacidreactivesubstances(TBARS)assayisbasedonthedecreasingofTBARS
caused by the lipid peroxidation inhibition in porcine (Sus scrofa) brain homogenates due to
the presence of antioxidants. This decrease can be measured spectrophotometrically at 532 nm.
Theinhibitionratio(%)wascalculatedusingthefollowingformula:
Inhibitionratiop%q “ rpA´Bq{Asˆ100%,
whereAandBweretheabsorbanceofthecontrolandsamplesolution,respectively.
4.6. StatisticalAnalysis
Foreachspecies,threerandomlychosensampleswereusedandallassayswerecarriedoutin
triplicate. Resultswereexpressedasmeanvaluesandstandarddeviation(SD).Theresultsofeach
Molecules2016,21,246 10of12
parameterwerecomparedbymeansofaStudent’sttesttodeterminethesignificantdifferenceamong
samples,withα=0.05. ThisanalysiswascarriedoutusingJMPStatisticalDiscoveryV.10program
(IBMSPSSsoftware,Armonk,NY,USA).
5. Conclusions
This work is the first to report the nutritional, chemical and bioactive properties of Leccinum
molle(Bon)BonandLeccinumvulpinumWatling. Giventhechemicalcompositionofbothspecies,we
concludethattheyareagoodfoodoptionforinclusionintoday’sdiet, beingasourceofessential
nutrients,aswellasnutraceuticalsandotherbiologicallyactivecompounds. Theyrevealedagood
nutritionalvalue(lowinfat)andprovedtoberichinMUFAandPUFA,solublesugars(otherthan
sucroseorglucose)andvitaminE.Additionally,theyalsoexhibitedantioxidantproperties,which
corroboratesthestatementofthesemushroomsashealthfoods.
Acknowledgments: IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT,
the Portuguese Foundation for Science and Technology. The authors are grateful to the Foundation for
Science and Technology (FCT, Portugal) for the grant of F.S. Reis (SFRH/BD/111753/2015) and L. Barros
(SFRH/BPD/107855/2015), and COMPETE/QREN/EU for the financial support to CIMO (strategic project
PEst-OE/AGR/UI0690/2014).
AuthorContributions:F.S.Reis,I.C.F.R.Ferreira,P.MoralesandM.H.Vasconcelosconceivedthestudy.F.S.Reis,
P.MoralesandI.C.F.R.Ferreiradesignedtheexperiments. F.S.ReisandA.Martinsperformedthetaxonomic
identificationofthespecies, andtheF.S.Reiscarriedouttheassays. F.S.ReisandL.Barrosperformeddata
organizationandanalysisoftheresults.F.S.Reis,L.BarrosandI.C.F.R.Ferreirawrotethemanuscript.A.Martins,
P.MoralesandM.H.Vasconcelosrevisedthemanuscript.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
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