fractions, mycorrhiza,vitamins. Key words: the lackofinformationinliterature. with dataonberryplants,including sea-buckthorn, due to However,. tocompareour results notpossible itis the impact of mycorrhiza on thechemical composition of on reports scientific earlier the confirmed partly study our well as higher antioxidant activity (40%). The results of (35%), (10%),andL-ascorbicacid(8%),as 8%, andCr-21%), (51%),totalflavonoids levels of minerals (P-27%, K-67%,Mg-4%,Na-30%,Zn- fractions ofdietary fiber. ‘Hergo’ hadmore beneficial protein (4%),crudefat(7%),fiber(30%),andall had ahighernutritionalvalue,containingmoretotal concentration of polyphenols (7%). Leaves of ‘Habego’ thepartially elevatingantioxidantactivitybyincreasing (1%), Ca(9%),Na(16%),Fe(18%),Cr(34%),and by increasinglevelsoftotalprotein(3%),Nfreeextract improved the nutritional value of leaves of sea-buckthorn Experimental StationinLipnik,Poland.Mycorrhization leaves. The studywasconducted in 2014and2015atthe composition and antioxidant activity of sea-buckthorn effect ofsymbiotic mycorrhizal fungi onthechemical leaves. The aimoftheexperimentwastodetermine there is shortage ofthe information regarding to buckthorn mainly focusonnutritiousvalueoffruitandseeds agricultural technology and climate. The studies however, substances. Their levels depend ongrowing conditions, can bearich sourceofnutrients andbiologically active The leavesofsea-buckthorn( ABSTRACT Szczecin, Poland. of PigBreeding, Animal Nutrition and Food,Judyma10,71-460 2 of Agronomy, PapieżaPawła VI 3,71-434Szczecin,Poland. 1 doi:10.4067/S0718-58392017000200155 Accepted: 23February2017. Received: 13December2016. Anna Jaroszewska buckthorn ( activity ofleavesmycorrhizedsea- Chemical compositionandantioxidant West PomeranianUniversityof Technology inSzczecin, Department West PomeranianUniversityof Technology inSzczecin, Department CHILEAN JOURNAL OF AGRICULTURAL RESEARCH 77(2)APRIL fiber chemicalcomposition, Antioxidants, * Corresponding author([email protected]). 1 , andWioletta Biel CHILEAN JOURNAL OF AGRICULTURAL RESEARCH 77(2)APRIL rhamnoides Hippophae Hippophae rhamnoides 2* L.) - JUNE 2017 ( ofsea-buckthorn andantioxidant properties composition strawberry fruits(Castellanos-Moralesetal.,2010). the phenolics in purple coneflower (Araim et al., 2009) and in of most of concentrations increased significantly to leads factor sativa has beendemonstratedinmycorrhizal roots ofalfalfa( phenols (Hazzoumietal.,2015).Highercontentofflavonoids arbuscular mycorrhizal fungi (AMF)affects thecontent of (Karagiannidis etal.,2012).Previousstudiesconfirm that affected in plants, including the pattern of secondarycompounds symbiosis, arangeofchemicalandbiologicalparameters is 2012). Duringtheestablishmentofarbuscularmycorrhizal this symbiosishasnotyetbeenreached(Karagiannidisetal., toagriculture,therealizationoffullpotential benefits these of spite In quality. soil of maintenance the to contribution multiple positiveeffects onplantgrowthandforitsimportant both medicineandhumannutrition. be anexcellentsourceofbiologicallyactivephytochemicalsin (Jaroszewska etal.,2016). Therefore, sea-buckthornleavesmay relatively high crudefat,andareagoodsourceofmacroelements et al.,2011). They containlarge amountsofcrudeprotein, (Padwad etal.,2006)andantibacterialproperties(Upadhyay leaves arearichsourceofcompoundswithanti-inflammatory its also that confirm studies some However, neglected. rather are theleaves while fruitandseeds, its concerns buckthorn usually Handique, 2013).Evaluationofthenutritionalvaluesea- protective, and immunomodulatory properties (Saikia and therapeutic activities,includingantioxidant,antitumor, hepato- of view, but also because many of them exhibit biological and compounds areofinterestnotonlyfromthechemicalpoint sterols, ,lipids,ascorbicacid,andtannins. These compounds including,amongothers,carotenoids,tocopherols, of symbiotic mycorrhizal fungi on thechemical composition impact the evaluate to experiment field a conducted we study this the qualityofberryplants,including sea-buckthorn. Therefore, in concerning thepotentialimpact ofsymbioticmycorrhizalfungion Sea-buckthorn ( improving foodquality, also thanks to their wide availability. give limitlesspossibilitiesforthediscoveryofnewdrugsand Natural products, either as extracts oras pure compounds, INTRODUCTION Hippophae rhamnoides Currently, thereislittleinformationonthechemical The arbuscularmycorrhizalsymbiosisisrecognizedforits L.) (Larose et al., 2002). The AMF root colonization - JUNE 2017 Hippophae rhamnoides L.) L.)leaves. There isalsoalackof data L.)containsaseriesof Medicago

155 RESEARCHRESEARCH CHILEAN JOURNAL OF AGRICULTURAL RESEARCH 77(2)APRIL Croatia. It contains symbiotic mycorrhizal fungi ( fungi mycorrhizal symbiotic contains It Croatia. The isolate was obtained from the natural ecosystems in mycelium, which issymbiotic for plantsoftheolivefamily. Mycorrhization wasconductedwithectomicorrhizal 2 and 3 yrold shrubs, female varieties Habego and Hergo. the experimentwas20. The subjectofthestudyincluded size of single plot was 12 m = shrub (one replicates five in method randomized completely and Klevels. The experimentwasdesignedaccordingtoa of soil minerals showed high P levels and moderate Mg level ofhumusisformedfromclaysands. The analysis Ap level, the soil has a slightly acidic loamy sand. The the At (2015). WRB Group WorkingIUSS to according Soil Classification,2011), classifiedasHaplicCambisol was carriedoutbelongedtotypicalrustysoils(Polish Poland. E, 7ma.s.l.), theexperiment onwhich The soil Experimental Station in Lipnik (53°20’35” N,14°58’10” The studywasconductedin2014and2015atthe MATERIALS ANDMETHODS buckthorn. and antioxidant activity of leaves of two varieties of sea- spp., 220. Determination ofneutral detergent fiber (1991) performedwith a fiberanalyzer ANKOM al. et VanSoest to according method detergent the + crudefiber). = 100− %(moisture + crude protein + crude fat +crude ash carbohydrates werecalculated as: Nfree extract (NFE) (%) (ANKOM Technology, Macedon, New York, USA); total fiber wasdeterminedwithaanalyzer ANKOM 220 B-324 (BüchiLabortechnik AG, Flawil,Switzerland);crude 6.25) by Kjeldahl method using a Büchi Distillation Unit in amuffle furnaceat580 °C for8h;crudeprotein(N× extraction withdiethylether;crudeashbyincineration to constant weight; ether extract°C by Soxhlet 105 at by AOAC (2012).Drymatterwasevaluatedbydrying determined were (NFE) extract free N and ash crude fiber, (DW) basis.Drymatter, crudeprotein,ether extract,crude and Hergo). and halfwereusedasacontrolgroup)variety(Habego bushes from each variety were subject to mycorrhization, signs ofagingormechanicaldamage. leaves weretakenfromthe1yroldshootswithoutany from theoutsideofbush,athalftheirheight. The harvest fromshootswithoutfruiting.Leaveswerecollected development). Leavesfortheanalysiswerecollectedduring hydrogel (ensuring humidity essential for the initial fungi year ofexperiment. The mycelium contained anadditionof inthefirst therootzoneofplants applied intwoplaces and the root bacteria( onerepeat). Shrubs wereplanted in 4×3mspacing. The The fiber components were determined using All determinationswereexpressedonadryweight mycorrhiza(halfofthe were The experimentfactors Gigaspora spp., Bacillus Pochonia 2 . The total number of plots in spp.) A dose of 15mL was spp., Lecanicillium - JUNE 2017 Glomus spp.),

extract was mixedwith0.5cm 72% sulfuricacid(H (ADL) was determined by hydrolysis of ADF samplein extract and a drop of acetic acid, and then diluted to 25 cm 40 min.Blanksampleswerepreparedfrom1cm sulfuric acid(H analyses wassubjectedtomineralizationinconcentrated cellulose (CEL)asthedifference between ADF and ADL. calculated asthedifference betweenNDFand ADF, while ethanol to 25cm methanol andadropofaceticacid,thendilutedwith 1.5 cm methanol wasmixedwith1cm quercetin asareferencecompound;1cm method of Kumaran and Karunakaran (2007) using thoroughly andmadeupto10cm acid equivalent(GAE)inmgkg content insampleswascalculatedastheamountofgallic from variousconcentrations of gallic acid. Total estimate phenoliccontentusingastandardcurveobtained at 765 nm was determined. These data were used to mixture wasallowed tostandfor2h. Then theabsorbance Folin-Ciocalteu reagent (Yu et al., 2002); 0.1 cm phenolic content of extracts was determined using according toKumaranandKarunakaran(2007). The total activity determination,methanolextractswereprepared concentrated samples. diluted. Othermineralcompoundsweredeterminedin to assay Ca content, K and Mg trials were appropriately nitrate contentwasdeterminedbypotentiometry. Prior spectroscopy. The flame absorption of means by – Cu and of emulsionflamespectroscopy, Cr Zn,Fe,Pb, andMg, USA) wasusedtodetermineK,Na,andCa–bymeans Scientific, Fisher Series, Thermo Massachusetts, Waltham, An atomicabsorptionspectrometer(ASA)(iCE3000 Specol 221apparatus(866287,CarlZeissJena,Germany). ammonium molybdate,at wavelength 660nm, by using a determined bytheEgner-Riehm colorimetricmethod,with analyses wasdigestedinamixtureofnitricacid(HNO whilst thematerialformicro-componentsconcentration and perchloricacid(HClO N(CH hexadecyltrimethylammonium bromide (C Determination ofacid detergent fiber (ADF)included (822050, Merck,Kenilworth,NuevaJersey, USA). radical scavenging, asthedecreasein the absorbance and the measurement ofantioxidant capacity, orfree of astablecoloredfreeradical inaqueoussolution et al., 1999). This method isbasedonthe generation Equivalent Antioxidant Capacity(TEAC)method(Re with methanol. included sodium dodecyl sulfate (NaC (NDF) wasconductedonanash-freebasisand The material for the macro-components concentration The total flavonoids content was determined by the For total polyphenols, total flavonoids, and antioxidant The antioxidantcapacity wasassayedby Trolox 3 ) 3 20%sodiumcarbonate. The mixture was shaken 3 Br (Merck102342),whileaciddetergent lignin 3 . The absorptionat 415 nmwasreadafter 2 SO 2 4 SO ) andperchloricacid(HClO 4 ). Hemicellulose(HCEL)was 4 ). The concentrationofP was 3 Folin-Ciocalteu reagent and -1 3 sampleDW. 3 aluminumtrichloridein usingdistilledwater. The 3 plantextractin 12 H 25 3 3 16 plant plant SO H 33 4 4 3 ), ), ) ) ) 3

156 CHILEAN JOURNAL OF AGRICULTURAL RESEARCH 77(2)APRIL transferred intoconicalflasksand25cm of 30 mm spectrophotometer and absorbance measured. An aliquot made bymixing50cm Japan) wasused. Kyoto, (UV-1800,Shimadzu, UV-VISspectrophotometer thesamples. as incubated underthesameconditions An cm against theappropriateblank. A typicalblankcontained 5 Absorbance ofthe aqueous phaseat 695 nmwasmeasured and incubatedat37°Cfor90minwithvigorousshaking. mM sodium phosphate, and 4mM ammonium molybdate) aliquot of ABTS aliquot cm acetic acidandthevolumewasfinallymadeupto1000 temperature overnightbeforeuse. The ABTS and allowingthemixturetostandindarkatroom Table 1.Chemicalcomposition(n=3)ofseabuckthornleaves (gkg (ABTS with aconcentrationof7mM. The ABTS radicalcation was dissolvedindeionizedwatertogiveastocksolution (ABTS) withanabsorptionmaximumof734nm. ABTS is 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid) following addition ofthe antioxidant. The radical used UV-VISspectrometer a in solution colored the of Moisture (gkg solution with1cm test tubewith5cm nitrogen freeextract. Mean values with the same letter in each line are non-significantly different at p ≤ 0.05 according to the Tukey test, DW: Dry weight, FW: fresh weight, NFE: NFE Crude fiber Crude fat Crude ash Crude protein Dry matter to giveanabsorbanceclose0.70at734nm. A 3cm was dilutedwithdeionizedwater(approximately50-fold) A samplevolumeof0.5cm hexane and were determined by Prieto et al. (1999) method. which carotenoidsdonotabsorb. respectively, and corresponding absorption coefficients at chlorophyll A andB,usingwavelengths663645nm, 440 nm, after having subtracted the concentration of (1983). These compoundsweredeterminedinwavelength and determined according to Lichtenthaler and Wellburn time thediscolorationwaseffectively complete. second absorbancereadingtakenafter6min,bywhich solution (Aslametal.,2013). extraction with mL 50 to up made was volume the finally and filtered, down, cooled was sample the Thereafter, min. was added,waterbathkeptshakingat70°Cfor40 Item Solution forextractionofwater-soluble vitamins was Tocopherols were extracted from plants samples with Carotenoids wereextracted with 80%solutionofacetone 3 3 reagentsolutionand0.5cm withdoubledistilledwater. A sampleof10gwas •+ ) was produced by reacting 9 cm 3 -1 of methanol extract was then added and a FW) •+ 3 solution was put into a cuvette in the reagent solution (0.6 M sulfuric acid, 28 reagent solution (0.6Msulfuricacid,28 3 24.5mMpotassiumpersulfate 3 acetonitrile with 10cm acetonitrilewith 680.0 ±70.3a 655.0 ±14.8b 104.8 ±10.6a 140.9 ±0.9b 953.4 ±0.9a 54.4 ±4.6a 40.9 ±1.0a 3 hexaneextract was mixedina Control 3 purehexane,anditwas Mycorrhizal effect 3 extraction solution extractionsolution 3 ABTS stock ABTS 625.9 ±0.27a 663.0 ±49.2a 101.4 ±20.2b 144.7 ±25.5a 953.1 ±19.6a 55.4 ±0.7a 39.1 ±5.6a Mycorrhiza - •+ JUNE 2017 solution 3 glacial 3

P-value -1 mm Tulsa, Oklahoma,USA). analyzed usingtheStatisticaversion12.0software(StatSoft, were 0.05. experiment ≤ the p from at results considered The independent determinations. Statistical significance was Results werepresentedasmean±standarddeviationofthree 5%). was ofchemicalcomponents error fordeterminations was comparedby Tukey’s multiplerangetests(admissible variance. The significance ofdifferences between means variables, afterassessingnormalityandhomogeneity of results usingthemycorrhizationandgenotypeasindependent and 92.41respectively. were 1.45,5.50,7.20,9.80min,and96.20,93.51,89.53, fiber content (by ~ 3.24%). In the present study variety had variety study present the In 3.24%). ~ (by content fiber respectively, relative to the control)andareductionincrude and NFE in sea-buckthorn leaves (by 2.63% and 1.21%, standard deviationof0.9. a with 95.3%, was content weight dry average The 0.05). ≤ that differs significantlyinDW contentinthedriedleaves(p Our analysis did notdistinguish a variety of sea-buckthorn leaves ofsea-buckthorncollected at theendofJuly(32%). Morgenstern etal.(2014)found thehighestDW contentin comparable with that (67% FW) of tea leaf (Li et al., 2012). peppers, andspinach)reportedbyGranadoetal.(1992),but most vegetables(lettuce,greenbeans,asparagus, in FW 96% to 82% of range the than lower was It (FW). leaves used inthe experiment was 68± 7% offreshweight (Tablebuckthorn leaves 1).Moisturecontentoffresh content ofdryweight,crudeash,andfatinsea- We foundnonsignificanteffect ofmycorrhizationonthe RESULTS ANDDISCUSSION mm, 5 µmparticle). The mobile phase was50mMKH USA) equipped with HypersilODScolumn (150 mm × 4.6 system (Series200,PerkinElmer, Shelton,Connecticut, HPLC an using (2008); al. et Kłódka by described method using ahigh-performanceliquidchromatographic(HPLC) ascorbic acid(C),andniacin(PP)inextractsweremeasured was λ = 245 nm, flow rate 1 cm 1 rate flow nm, 245 = λ was min, then this ratio was maintained for7min. Wavelength (A) andmethanol(B)ingradientfrom0%to30% A in8 0.223 0.001 0.031 0.376 0.081 0.006 0.540 DW). Two-factor ANOVA was carried out on the experimental the on out Two-factorANOVAcarried was Mycorrhization resultedinanincreaseproteincontent The contents of vitamins thiamine (B1), riboflavin (B2), riboflavin (B1), thiamine vitamins of contents The 3 . Retention times and recoveries for C, B1, PP, and B2 638.2 ±14.2a 644.2 ±36.7b 145.4 ±27.4a 953.3 ±1.1a 116.4 ±6.9a 56.7 ±1.9a 37.2 ±3.4b Habego Genotype effect 3 min 667.7 ±78.1a 673.9 ±27.3a 140.3 ±20.4b 953.2 ±0.8a 89.7 ±3.1b 53.1 ±3.2b 42.9 ±1.3a Hergo -1 , andinjection of 20 P-value 0.476 0.000 0.000 0.021 0.000 0.002 0.900 2 PO 4

157 CHILEAN JOURNAL OF AGRICULTURAL RESEARCH 77(2)APRIL Mean valueswiththesameletterare notsignificantlydifferent atp≤0.05according tothe Tukey test. NDF: Neutraldetergent fiber, ADF:aciddetergent fiber, ADL:aciddetergent lignin,HCEL:hemicelluloses, CEL:cellulose, DW: dryweight. g the averagecontentofNDFrangedfrom219.3to278.7 study, this In method. detergent the by obtained fractions there arenodataonthecompositionofdietaryfiber literature the In 1). (Figure fractions fiber dietary evaluated significantly more crude fiber (23%, Table 1) and all detergent lignin (ADL). The leaves of ‘Habego’ contained were nonsignificant. No such trend was observed for acid in themycorrhized leaves ofsea-buckthorn,althoughthese fraction (ADF),hemicellulose(HCEL),andcellulose(CEL) some downwardtrends in the levels ofacid detergent observed also 1). We (Figure (NDF) fiber detergent neutral significantly reducedlevelsofcrudefiber(Table 1)and Mycorrhization ‘Hergo’. than fiber crude more 23% ~ and Cummings,2009). The leavesof‘Habego’ contained inducing physiological effects onthehumanbody(Mann diverse physical and chemical properties, and iscapable of fractions. Dietaryfibercontainsmanystructureswith the source andthe mutual proportions ofthe respective buckthorn. crudefatthansea- plantain containonaverage55.6%less (2008) reportthatleavesoffodderradish,rape,chicory, and that its leaves are a goodsource oflipids. Fulkerson et al. This confirmstheresultsofotherstudies,whichshow at amorethan2.5timeslowerlevelof47gkg sea-buckthorn fruit(morefrequentlyusedasfood)stands in content protein the that noting worth is It 2012). al., et genotype and the impact of environmental conditions (Zheng crude fat levels were found in ‘Habego’ (56.7 g kg differed in crude fat content. Similar to protein, the highest (Selvamuthukumaran andFarhath,2014). Figure 1.Fiber components(n=3)ofsea-buckthornleaves. protein. ‘Habego’ leaves had 145.4gprotein kg study confirmedsea-buckthornleavesasarichsourceof ‘Habego’ contained 13.3% more ash than ‘Hergo’. This 3.72% to 4.29% DW, with a standard deviation of 0.39. from ranged DWand 4% was level average Its ash. crude by showed a considerable diversity in content measured crude fiber, andNFE. an impact on the level of crude ash, crude protein, crude fat, (120.35 gkg is morethan the levelreportedby Kashif andUllah(2013) kg The propertiesofdietaryfiberanditsvaluedependon The leaves of the studied varieties ofsea-buckthorn ofthestudiedvarieties The leaves The leaves of the studied varieties ofsea-buckthorn ofthestudiedvarieties The leaves -1 DW, andagain,itisimpossibletocomparethisdata -1 DW). The difference isprobablyduetothe - -1 JUNE 2017 DW, that -1 -1 DW). DW DW 156 to489gkg and plantain ( rape ( than 10%DW. measured inleavesofgeum,leontodon,andsalvia,atless in silene,whilethelowestHCEL concentrationswere g kg and reported HCEL content between the range of 60-220 four herbspecies( examined (2010) al. et Schädel 2014). al., et Černiauskiene cereal grains, herbs,andvegetables (Biel andJacyno,2014; to gkg much as8%. between ‘Habego’ (71.8 g) and ‘Hergo’ (65.9 g) by as influences the hardness of sea-buckthorn leaves, differed chicory, andplantaininthestudybyFulkersonetal.(2008). ADF level,justlikeNDF, wassimilartofodderradish,rape, leaves contained~18%ofthisfractionthanthose‘Hergo’. DW and varied from102 to 133.2 g kg fodder radish ( sea-buckthorn. Fulkerson et al. (2008)examined NDFin to literature due to the lack of reports relating directly to and lignin,rangedfrom167.9to205.1gkg significantly lower concentrations of P (by 0.5 g 0.5 (by P of concentrations lower significantly to control, leavesofthemycorrhized sea-buckthornhad microelements inleavesofsea-buckthorn.Compared (94%), andFe(574%). with theexceptionofK(116% greater),Mg(31%),Ca 2007) theaverage mineral content ofthe leaves was similar, the levels found in berries of sea-buckthorn (Ercisli et al., to Compared 2). (Table study our in confirmed was which conditions, maturity oftheplant, and thetime of harvesting, factors, includinggeneticcharacteristics,climate,soil (Jaroszewska etal.,2016),buttheirlevelsdependonmany contained significantlymoreCEL (~23%)than‘Hergo’. leaves ‘Habego’ study present the In plantain. and chicory Rowarth (1996)founda similar CEL level in the herbsof Salvia pratensis In this study, the average CEL content was 117.6 g kg g 117.6 was content CEL average the study, this In Hemicellulose contentwasintherangeof73.6to51.4 Acid detergent lignin (ADL),which to some extent The determinedaverage ADF, consistingofcellulose Mycorrhization significantly differentiated macro- and Sea-buckthorn leavesarearichsourceofminerals -1 Brassica napus DW. These authorsfoundhigher concentrations -1 DW. A commonsignificantsourceofHCEL are Plantago lanceolata Raphanus sativus -1 L., DW. Geum urbanum Silene flos-cuculi L.), chicory ( L. var. L., L.), which ranged from (L.) Greuter & Burdet) Cichorium intybus Leontodon hispidus -1 oleiformis DW. Fraser and -1 DW. ‘Habego’ kg Pers.), -1 DW, DW, L.) L., L., -1

158 CHILEAN JOURNAL OF AGRICULTURAL RESEARCH 77(2)APRIL NO Mean valueswiththesameletterineachlinearenotsignificantlydif Pb Cu Cr Zn Fe Na, mgkg Ca Mg K P Table 2.Macro- andmicroelements (n=3)andnitratesinseabuckthornleaves. Total flavonoids,mgQEkg components, byspecific varieties cultivated in the same to absorbandcumulateinthebiomassmentioned significantly different, whichpointstothevariouscapability two examined varieties ofsea-buckthorn leaves were analyzed samplesofseabuckthornleaves,wefoundnolead. with the contamination of soil and air. Significantly, in the Plants normallycontaintraceamountsofleadwhichincrease andreducedintelligence. reproductiveproblems, disorders, neurological damage, brain anemia, severe cause can It characterized by higher nitrate (by ~ 39.4 g kg g 39.4 ~ (by nitrate higher by characterized buckthorn leaves grown in mycorrhized plots were also of Ca and Fe in fresh weight of mycorrhized lettuce. Sea- Baslam etal.(2011) reportsanincreaseintheconcentration were reportedbyJaroszewskaetal.(2016).Similarly, ofmycorrhizedsea-buckthorn inleaves higher Calevels Mean values with the same letter in each line are not significantly different at p ≤ 0.05 according to the Tukey test, GAE: Gallic acid equivalent, DW: dry DW: equivalent, acid Gallic GAE: test, Tukey the weight, QE:quercetin equivalent. to according 0.05 ≤ p at different significantly not are line each in letter same the with values Mean Carotenoids, mgkg Na (by13.9gkg Table 3.Contentofantioxidants(n=3)inseabuckthornleaves. kg g 2.7 ~ (by K 12%), i.e. Polyphenols, mgGAEkg Cr (by 0.32 g kg g kg by plants(N-NO increase in acidity increases the intensity ofnitrate uptake uptake of Nbyplant roots, and also by the soil pH. An 26%), probablyduetothemycorrhizaenablingbetter kg g 0.32 ~ (by Ca more significantly contained Cu andZninleavesofmycorrhizedlettuce. of content reduced significantly mycorrhiza the that stating (2015), al. et Kowalska of results the confirms which levels, the Cuconcentration, we observedadownwardtrendinCu on mycorrhiza of effect nonsignificant was there Although Item Item

3 The content of the tested mineral components in the Lead belongs to the group of metals hazardous to health. The tested leaves ofmycorrhized sea-buckthorn , mgkg -1 DW, i.e.33%),andZn(by~3.6gkg -1 DW -1 DW

-1 3 DW -1 - -1 ). DW, 16%),Fe(8.8gkg DW, 34%) than control. Lower Kand -1

DW -1 DW nf

152.1 ±16.2b

-1 84.2 ±16.9b 3.82 ±0.2a 0.94 ±0.0b 33.7 ±4.5a 49.5 ±3.9b 3.45 ±0.3b 2.35 ±0.1a 14.9 ±3.8a 4.75 ±0.4a DW, i.e. 18%), Mg (by ~ 0.78 ~ (by Mg DW,18%), i.e. Control 8584.7 ±2191.9b 1004.3 ±8.3a 2546.2 ±386.3a Mycorrhizal effect Control Mycorrhizal effect -1 -1 DW, 18%),and DW, i.e.11%). Microelements(mgkg 191.5 ±9.2a Macroelements(gkg 98.1 ±10.8a 3.66 ±0.3a 1.26 ±0.2a 30.1 ±1.7b 58.3 ±1.1a 3.77 ±0.2a 1.57 ±0.0b 12.2 ±4.1b 4.25 ±0.9b Mycorrhiza -1 - -1 DW, 9%), JUNE 2017 nf DW, i.e. 9174.6 ±1834.9a 2284.9 ±560.8a 946.1 ±92.9b Mycorrhiza ferent atp≤0.05accordingtothe Tukey test,nf:notfound,DW: dryweight. P-value more P (1.08 g kg had leaves Its ‘Hergo’. in found was elements analyzed the habitat conditions. A significantly higher concentration of 67%), Mg (0.7 gkg i.e. 30%),Zn(2.5mgkg antioxidant activity (by 18.2 μmol Troloxμmol kg 18.2 (by activity antioxidant higher significantly had sea-buckthorn mycorrhized the of role of AMF in increasing their concentration. The leaves et al.,2009;Baslam2011) confirmthesignificant andlettuce)(Araim inplants(purpleconeflower metabolites secondary of content the on mycorrhization of influence the et al.,2014)and380mgflavonoidskg GAE kg GAE contained significantlymorepolyphenols(by590.2mg mg quercetinequivalent [QE] kg plots. However, they had fewer total flavonoids (261.3 carotenoids (58.2mgkg berries whichcontain530to970mgcarotenoidskg were higherthanthoseinsea-buckthorn 3) (Table tested and diabetes. The averageconcentrationsofantioxidants used in the treatment of cancer, cardiovascular diseases, compounds with antibacterial and anti-stress properties, varieties ofsea-buckthorn. between content mineral in differences significant showed with previousreportsbyJaroszewskaetal.(2016),which fruits mycorrhizedtomatoes. et al.(2010),reportingincreasedantioxidantactivity in Ordookhani of research the in reflected is which 2), (Figure nitrate (~ 2.6 mg kg mg 2.6 (~ nitrate DW, i.e. 21%)than‘Habego’.‘Hergo’ leavesalsohadless 0.000 0.294 0.000 0.000 0.000 0.000 0.001 0.000 0.000 0.001

In our study, leaves of the mycorrhized sea-buckthorn mycorrhized the of leaves study, our In Sea-buckthorn is a natural source of bioactive P-value 0.023 0.000 0.171 -1 DW) -1 -1 DW) DW, i.e. 7%),compared to plants from control 173.1 ±30.4a 2054.1 ±243.5b 79.1 ±11.0b 3.82 ±0.1a 1.00 ±0.1b 30.6 ±1.0b 56.0 ±3.6a 3.69 ±0.1a 1.92 ±0.4b 10.2 ±1.8b 3.96 ±0.5b 929.9 ±74.9b 7067 ±570.3b Habego -1 nf DW, i.e. 27%), K (6.8 g kg -1 Habego -1 DW, i.e.1%). The resultscorrespond DW, i.e.4%),Na(24.1g kg Genotype effect -1 DW, i.e.6%).Previousstudieson -1 Genotype effect , i.e.8%),andCr(0.21mgkg 10691.7 ±417.9a 103.2 ±5.0a 170.5 ±14.9b 1020.5 ±14.2a 2777.1 ±362.9a 3.66 ±0.3a 1.21 ±0.3a 33.1 ±5.2a 51.7 ±6.5b 3.57 ±0.4b 1.99 ±0.5a 17.0 ±1.4a 5.04 ±0.1a -1 -1 DW (Chuetal.,2003). Hergo nf DW, i.e.16%)and Hergo -1 DW, i.e.3%) -1 -1 DW (Pop DW, i.e. P-value P-value -1 0.000 0.293 0.000 0.000 0.000 0.000 0.026 0.000 0.000 0.000 0.000 0.000 0.003 DW, DW, -1

159 CHILEAN JOURNAL OF AGRICULTURAL RESEARCH 77(2)APRIL of 2139.6mgkg et al.,2011). The examinedleavescontainedanaverage 188.3 μmol Trolox μmol 188.3 ~ (by activities antioxidant higher by distinguished DW, i.e.10%). The leavesofthisvarietywerealso acid atlevelsrangingfrom360to2500mg100g arereportedtocontainL-ascorbicbuckthorn berries ascorbic acid(Table 4).Dependingontheecotype,sea- variety significantly differentiated only the level of studied sea-buckthornleaves,mycorrhizationand the In vegetables. and fruits in abundant E, and C, A, by antioxidants, which include, among others, vitamins content ofantioxidantsinberryplants. importance ofgenotypeasacrucialfactoraffecting the well asotheranalysis(Alfaroetal.,2013)highlightthe the same conditions ofclimate and soil. The results as varietal characteristics,astheplants weregrownunder varieties ofsea-buckthornwereprobablyduetothe differences in antioxidant levels between the two rich inbeneficialphenoliccompounds. The significant et al.(2015)showedthatseabuckthornleavesarevery mg GAE kg GAE mg properties, with significantly more polyphenols (3624.7 Figure 2. Antioxidant activities(n=3)measured by ABTS assayinsea-buckthornleaves. had a164.1mgkg mycorrhized lettuce. The leaves of ‘Hergo’ sea-buckthorn significant increaseinL-ascorbicacidfreshweight in (7%) in control. Baslam et al. (2011) demonstrated a of theremainingvitamins(tocopherols,thiamine, did notfindsignificantdifferences intheconcentrations of‘Habego’. acidthanleaves L-ascorbic weAlthough QE kg Table 4.Contentofvitamins(n=3)inseabuckthornleaves. Mean valueswith the sameletterineachlinearenotsignificantly dif Niacin Riboflavin Thiamine L-ascorbic acid Tocopherols Item The harmful effects offree radicals are counteracted The studied ‘Hergo’ had clearly superior antioxidant -1 DW, i.e.35%),andcarotenoids(90.6mgkg -1 DW, i.e. 51%), total flavonoids (723 mg -1 DW, comparedto155.3mgkg -1 -1 DW: Dryweight, ABTS: 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid). Mean valueswiththesameletterarenotsignificantlydifferent atp≤0.05accordingtothe Tukey test. DW, 40%)(Figure2).Morgenstern DW (8%)higherconcentrationof 2217.3 ±188a 40.98 ±4.9a 4.86 ±0.4a 5.86 ±0.3a 1.40 ±0.1a Control mgkg Mycorrhizal effect -1 DW 2062.0 ±16.0b 38.19 ±2.7a 4.57 ±0.2a 5.68 ±0.2a 1.36 ±0.2a Mycorrhiza - JUNE 2017 -1 -1 (Bal DW DW ferent atp≤0.05according tothe Tukey test. -1

P-value of non-mycorrhizedsea-buckthornand‘Hergo’ leaves. was atendencytowardstheirhigherlevelsintheleaves riboflavin, andniacin)insea-buckthornleaves,there research inthisareaseemsnecessary. the lack of information in the literature. Therefore, further with dataonberryplants,includingsea-buckthorn,due to leaves). However, it is not possible to compare our results mycorrhiza onthechemicalcompositionofplants(fruits, confirmed theearlierscientificreportsonimpact of their antioxidantactivity. The results ofour study partly on thechemicalcompositionofseabuckthornleavesand assess theimpactofarbuscularmycorrhizalfungi(AMF) activity. acid. The leavesof‘Hergo’ alsoshowedhigherantioxidant L-ascorbic and carotenoids, flavonoids, total polyphenols, buckthorn hadmoreminerals(P, K,Mg,Na,Zn,andCr), dietary fiber. Ontheotherhand,leavesof‘Hergo’ sea- protein, crudefat,fiber, andalltestedfractionsof by ahighernutritionalvalue,containingmoretotal characteristics. The Habego variety was characterized different varietieswasclearlydependentonthevarietal buckthorn alsoshowedanincreasedantioxidantactivity. their consumption. Leaves collected from mycorrhized sea- these compounds with diet without theneedto increase mycorrhized sea-buckthorn may improve the intake of and polyphenols,whichmeansthatthecultivationof content of total protein, Nfree extract, Ca, Na, Fe, Cr, The leavesofmycorrhized sea-buckthornhadahigher CONCLUSION 0.187 0.143 0.609 0.007 0.094 Summing up the results of the experiment is difficult to difficult is experiment the of results the up Summing The concentrationoftestcompoundsintheleaves 2057.6 ±17.4b 4.66 ±0.3a 5.67 ±0.2a 1.40 ±0.1a 38.1 ±2.4a Habego Genotype effect mg kg -1 DW 2221.7 ±184a 4.77 ±0.4a 5.87 ±0.3a 1.38 ±0.2a 41.0 ±5.1a Hergo P-value 0.583 0.113 0.849 0.000 0.080

160 CHILEAN JOURNAL OF AGRICULTURAL RESEARCH 77(2)APRIL Chu, Q.C., Qu, W.Q., Peng, Y.Y., Cao, Q.H., and Ye, J.N. 2003. E., Jariene, H., Danilcenko, J., Kulaitiene, J., Černiauskiene, Hazzoumi, Z., Moustakime, Hazzoumi, Z.,Moustakime, Y., K.A. Elharchli,E.,andJoutei, Granado, F., Olmedilla,B.,Blanco,I.,andRojas-Hidalgo,E.1992. and I., Barchia, J.S., Neal, A., W.J.,Horadagoda, Fulkerson, Fraser, T.J., and Rowarth, J.S.1996.Legumes, herbsorgrassfor Ercisli, S.,Orhan,E.,Ozdemir, andSengul,M.2007. O., The H., Vierheilig,Wendelin,S., V.,J., Castellanos-Morales, Villegas, Biel, W., andJacyno,E.2014.Chemical composition and nutritive 2011.Arbuscular N. Goicoechea, and I., Garmendia, M., Baslam, buckthorn Sea 2011. S. Satya, and S.N., V.,Naik, Meda, L.M., Bal, Aslam, J.,Khan,S.H.,andS.A.2013.Quantificationof 2009. C. Charest, and J.T., Amason, A., Saleem, G., Araim, AOAC. 2012.Official 18 methodsofanalysis. and I., Seguel, A., Quiroz, R., Palma, A., Mutis, S., Alfaro, REFERENCES Determination of flavonoids in flavonoids of Determination Agrobotanici ClujNapoca for foodenrichment in dietary fiber. Notulae Botanicae Horti and Jukneviciene,E.2014.Pumpkinfruitflourasasource Chemistry 90:1774-1782. at different nitrogenlevels.Journal Agricultural andFood and Biological Technologies in Agriculture 2(10):1-11. yield ofessentialoilinbasil( stress ongrowth,phenoliccompounds, glandularhairs,and 2015. Effect ofarbuscularmycorrhizal fungi (AMF)andwater Journal of Agricultural FoodChemistry 40:2135-2140. compositioninraw and cookedSpanishvegetables. and graincrops.LivestockScience114:75-83. the warmtemperateclimateof Australia for dairy cows:Herbs Nandra, K.S.2008.Nutritivevalueofforagespeciesgrownin Association 58:49-52. Grassland Zealand New the of Proceedings performance? lamb grown in Turkey. ScientiaHorticulturae115:27-33. activity ofseabuckthorn( genotypic effects onthechemicalcompositionand antioxidant electrochemical detection.Chromatographia58:67-71. its phytopharmaceuticalsbycapillaryelectrophoresiswith the quality of strawberry fruits ( by the arbuscular mycorrhizal fungus Eder, R., and Cárdenas-Navarro, R.E.R. 2010. Root colonisation lipid profileinrats. value ofprotein in hulled dwarf oat lines and the effect onserum Food Chemistry59:5504-5515. quality ofgreenhouse-grownlettuce. Journal Agricultural and mycorrhizal fungi (AMF) improved growth and nutritional and cosmoceuticals.FoodResearchInternational44:1718-1727. berries: A potentialsourceofvaluablenutrientsfornutraceuticals Saudi ChemicalSociety17:9-16. through highperformanceliquidchromatography. Journalof L.) cultivar’s fruitsgrowinginDubai,United Arab Emirates, water soluble vitamins in six date palm ( Agricultural andFoodChemistry57:2255-2258. coneflower, fungus increasesgrowthandsecondarymetabolismofpurple Root colonization by an arbuscular mycorrhizal (AM) Maryland, USA. Gaithersburg, (AOAC), Chemists Analytical Official of 13(1):67-78. molinae on polyphenol content and antioxidant activity in murtilla ( year harvest and genotype of Influence 2013. E. Scheuermann, Turcz) fruit.JournalofSoilScience and PlantNutrition Echinacea purpurea

Italian JournalofFoodScience26:203-209.

42(1):19-23. Hippophae rhamnoides Ocimum gratissimum Hippophae rhamnoides Fragaria (L.) Moench.Journal of Glomus intraradices Phoenix dactylifera × th ananassa ed. Association ed. Association L.) Chemical L.)Chemical - L.)berries JUNE 2017 Duch.) L.and alters Ugni

Karagiannidis, N., Thomidis, T., andFilotheou,E.P. 2012.Effects P.2016. Bośko, and S., W.,Stankowski, Biel, A., Jaroszewska, soil for base reference World2015. WRB. Group WorkingIUSS Kashif, M., and Ullah, S. 2013. Chemical composition and Kumaran, A., and Karunakaran, R.J. 2007. In vitro antioxidant vitro In 2007. R.J. Karunakaran, and A., Kumaran, of Effect 2015. M. Gąstoł, and Konieczny,A., I., Kowalska, Estimating 2008. M. Bońkowski, and Telesiński,A., D., Kłódka, Morgenstern, A., Ekholm, A., Scheewe,P., andRumpunen,K. for implications Possible 2009. J.H. Cummings, and J.I., Mann, Lichtenthaler, H.K.,and Wellburn, A.R. 1983.Determinations Li, X.,Xie,C.,He, Y., Qiu,Z.,andZhang, Y. 2012.Characterizing Y.,Piché, S., P.,Gagné, Moutoglis, R., Chênevert, G., Larose, Polish SoilClassification(PSC). 2011. SoilScience Annual Banerjee, D., Karan, S., Chanda, M., Jain, L., Y.,Padwad, Ganju, Ordookhani, K., Khavazi, K.,Moezzi, A., and Rejali, F. Morgenstern, A., Ekholm, A., Scheewe,P., andRumpunen, Agricultural Science and uptake in selected medicinal plants. Journal of of leaves. JournalofElementology21(4):1029-1041. on basic chemical compounds andminerals of sea buckthorn Evaluation oftheinfluencesymbioticmycorrhizalfungi 106. 203p.FAO, Rome,Italy. resources 2014,update 2015. World Soil Resources Reports indica minerals analysisof activities of methanol extracts of five of extracts methanol of activities substrates. JournalofElementology20(3):631-642. oflettucegrown onvariouson theyieldandnutritionalstatus mycorrhiza andthephosphoruscontentinanutrientsolution in English). Chemia Toksykolgiczna 41(4):957-963 (inPolish,withabstract vitamins indifferent kindsofteainfusions.Bromatologiai selected of and fluorine of content the between dependence the Journal DairyandFoodSciences leaf development of sea buckthorn( 2014. Changes in content of major phenolic compounds during Metabolism andCardiovascularDiseases19:226-229. health of the different definitionsofdietaryfibre.Nutrition 11:591-592. in different solvents. Biochemical Society Transactions of totalcarotenoidsandchlorophyllableafextracts 12(7):9847-9861. using wavelettransformandmultivariate analysis.Sensors spectroscopy diffusereflectance with tea of content moisture the fungus. JournalofPlantPhysiology159(12):1329-1339. symbiosis and the root colonizing arbuscular mycorrhizal sativa of roots in levels 2002. H. Vierheilig,and India. FoodScienceand Technology 40(2):344-352. 62(3):1-193. (in Polish withEnglishsummary). macrophages. InternationalImmunopharmacology 6:46-52. lipopolysaccharide induced inflammatory response in murine P.K., etal.2006.Effect ofleafextractsea-buckthornon Agricultural Research contentsintomato. andpotassium of Journal African activity,antioxidant on AMF and PGPR of Influence 2010. 14-16 October. Helsinki,Finland. Finland. Naantali, Institute, Resources Natural quality. Petruneva, E.(eds.)Producingseabuckthornofhigh and S., Kauppinen, In 74-77. p. leaves. buckthorn K. 2015.Majorphenoliccompoundsinprocessedsea Agricultural andFoodScience23(3):207-219. Glomus lamellosum , aremodulatedbythedevelopmentalstageof Punica granatu

4(3):137-144.

5(10):1108-1116. Hippophae rhamnoides ongrowth,essentialoilproduction and Ocimum sanctum

8(1):67-73. Hippophae rhamnoides Phyllanthus leaves. World , speciesfrom Azadirachta Medicago L.)

161 CHILEAN JOURNAL OF AGRICULTURAL RESEARCH 77(2)APRIL Schädel, Ch., Richter, A., Blochl, A., and Hoch, G. 2010. G. Hoch, and A., Blochl, Richter,A., Ch., Schädel, Saikia, M., andHandique, P.J. 2013. Antioxidant and antibacterial Re, R.,Pellegrini,N.,Proteggente, A., Pannala, A., Yang,and M., Prieto, P., Pineda,M.,and Aguilar, M.1999. Weesepoel,R.M., Vincke,J.P.,Pintea, A., Y.,Pop, C., and Socaci, Plantarum 139:241-255. under extremecarbonsource–sinkimbalances.Physiologia Hemicellulose concentration and composition in plant cell walls Life SciencesBiotechnologyandPharmaResearch2(1):80-91. salicifolia ofseabuckthorn( activity ofleafandbarkextracts and Medicine26(9-10):1231-1237. ABTS radicalcation decolorization assay. FreeRadical Biology Rice-Evans, C. 1999. Antioxidant activity applying an improved Analytical Biochemistry269:337-341. applicationtothedeterminationofvitaminE.Specific through theformationofaphosphomolybdenumcomplex: Spectrophotometric quantitation ofantioxidant capacity varieties. FoodChemistry147:1 from sixRomanianseabuckthorn( Gruppen, H. 2014. Carotenoid composition of berries and leaves D. Don) of north East India. International Journal of Journal International India. East north of Don) D. - 9. Hippophae rhamnoides - JUNE 2017 Hippophae L.) Upadhyay, N.K., Yogendra Kumar, M.S., and Gupta, A. 2011. A. Gupta, and M.S., YogendraKumar,N.K., Upadhyay, Zheng, J., Yang, B., Trépanier, M., and Kallio, H. 2012. Effects of Effects 2012. H. J., Yang,Kallio, Zheng, Trépanier,and B., M., Yu, L.,Haley, S.,Perret,J.,Harris, M., Wilson, J., and Qian, M. Methods 1991. B.A. Lewis, and J.B., P.J.,Robertson, VanSoest, K andFarhath, M., Selvamuthukumaran, buckthorn ( Antioxidant, cytoprotectiveandantibacterialeffects ofSea Food ResearchInternational21:759-765. shelf stabilityofantioxidantrichseabuckthornfruityoghurt. Journal of Agricultural andFoodChemistry buckthorn ( acidin sea andascorbic alcohols,fruitacids, sugar of sugars, genotype, latitude, and weather conditions on the composition Journal of Agricultural andFoodChemistry50:1619-1624. 2002. Freeradicalscavengingpropertiesofwheatextracts. Sciences 74(10):3583-3597. polysaccharides in relation to animal nutrition. Journal ofDairy for dietaryfiber, neutraldetergent fiberandnonstarch Chemical Toxicology48(12):3443-3448. Hippophaë rhamnoides Hippophae rhamnoides ssp. L.)leaves.Foodand mongolica . 2014.Evaluationof

60(12):3180-3189. ) berryjuice.

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