Bioactive phytochemicals in wheat: Extraction, analysis, processing, and functional properties

Bioactive phytochemicals in wheat Extractionanalysis processing and functional properties

Devanand L Luthria Yingjian Lu KM Maria JohnUS Department of Agriculture Food Composition Methods Development Laboratory Beltsville HumanNutrition Research Center Agricultural Research Service Beltsville MD 20705 USA

A R T I C L E I N F O

Article history

Received 25 September 2014

Received in revised form 15

December 2014

Accepted 6 January 2015

Available online

A B S T R A C T

Whole wheat provides a rich source of bioactive phytochemicals namely phenolic acids

carotenoids tocopherols alkylresorcinols benzoxazinoids phytosterols and lignans This

review provides information on the distribution extractability analysis and functional prop-

erties of bioactive phytochemicals present in wheat Understanding the impact of processing

on wheat phytochemicals allows us to develop improved processes with higher retention

of bioactive compounds in processed wheat foods Details of extraction analytical meth-

odologies and processing effects on bioactive phytochemicals in wheat are presented in

tabulated form

Published by Elsevier LtdKeywords

Wheat bioactive phytochemicals

Phenolic acids

Carotenoids and tocopherols

Alkylresorcinols

Functional properties

1 Introduction

In 2013 Food and Agriculture Organization (FAO) of the UnitedNations forecasted world cereal production will reach around2500 million tonne and wheat production to be around 700million tonne (FAO 2014) Grains and its processed productsare consumed globally as important energy sources Grain-based foods provide the majority of the carbohydrates someproteins oils dietary fiber and other micronutrients in manydiets During the past few decades research publications fromuniversities governmental non-profit health industrial andtrade organizations have encouraged increased consumptionof whole grain food products due to their positive health ben-efits (Chanson-Rolle et al 2014 Shahidi amp Chandrasekara 2013)According to United States Food and Drug Administration (FDA)cereal grains that consist of the intact ground cracked or flaked

caryopsis whose principal anatomical components (the starchyendosperm germ and bran) are present in the same relativeproportions as they exist in the intact caryopsis should be con-sidered a whole grain food (FDA 2006) The germ contains theplant embryo and the endosperm contains starch and storageproteins Bran is the outermost layer which protects the innerportion of the grain from external weather insect molds andother microorganisms attack

Previous studies have indicated that consumption of wholegrain foods can significantly reduce the risk of some chronichealth conditions such as type 2 diabetes cardiovascular diseaseand cancer (Landberg Marklund Kamal-Eldin amp Aringman 2014Seo et al 2015 Tucker et al 2014) Initially the health benefi-cial effect of whole grains was primarily attributed to its highfiber content However recent research indicates that the ben-eficial effect of whole grain may arise from the combined actionof several components such as fiber vitamins phenolics

Corresponding author Research Chemist Food Composition Methods Development Lab Bldg 161 Lab 202 BARC (E) 10300 BaltimoreAvenue Agricultural Research Service US Department of Agriculture Beltsville MD 20705 USA Tel +1 301 504 7247 fax +1 301 504 8314

E-mail address DaveLuthriaarsusdagov (DL Luthria)httpdxdoiorg101016jjff2015010011756-4646Published by Elsevier Ltd

j o u rna l o f f un c t i ona l f o od s ( 2 0 1 5 ) ndash

ARTICLE IN PRESS

Please cite this article in press as Devanand L Luthria Yingjian Lu KM Maria John Bioactive phytochemicals in wheat Extraction analysis processing and functionalproperties Journal of Functional Foods (2015) doi 101016jjff201501001

Available online at wwwsciencedirectcom

ScienceDirect

journal homepage wwwelseviercom locate j ff

carotenoids alkylresorcinols and other phytochemicals (PiironenLampi Ekholm Salmenkallio-Marttila amp Liukkonen 2009)Themajor grains include wheat rice corn oats rye barley sorghumtriticale millet amaranth and teff In Asia nearly half of theannually consumed grain is rice while the major grain con-sumed in Europe and US is wheatThe bioactive phytochemicalsin wheat can be broadly subdivided into the following catego-ries phenolic acids carotenoids tocopherols alkylresorcinolsand other miscellaneous compounds (sterols steryl ferulatesbenzoxazinoids and lignans)

Phenolic acids recognized as simple phenols can be dividedinto two subgroups One is hydroxybenzoic acid derivativeswhich include vanillic syringic p-hydroxybenzoic and gallicacids The other is hydroxycinnamic acid derivatives whichinclude ferulic p-coumaric caffeic and sinapic acids (Luthriaamp Liu 2013 Verma Hucl amp Chibbar 2009) The concentrationof phenolic acids in whole wheat ranges from approximately200 to 1200 mgg dry matter basis (DMB) (Andersson DimbergAman amp Landberg 2014) Ferulic acid is the primary and mostabundant phenolic acid in wheat grain Smaller concentra-tions of p-hydroxybenzoic vanillic syringic o-coumaricp-coumaric salicylic sinapic acids are also present in wheat(Liyana-Pathirana amp Shahidi 2006 Moore et al 2005)

Whole wheat grain also provides moderate sources ofvitamin E The vitamin E includes four tocopherols andtocotrienols Tocopherols have saturated phytyl side chainswhile tocotrienols have isoprenyl side chain with three doublebonds Previous studies have confirmed the presence of α- β-δ- and γ-tocopherols in soft and hard wheat grain (Moore et al2005 Panfili Alessandra amp Irano 2003) The spelt durum andsoft wheat grains from Italy contained vitamin E in a rangefrom 565 to 743 microgg dry weight respectively with 66ndash77tocotrienols (Panfili et al 2003) In addition the concentra-tion of total tocopherols and tocotrienols in whole wheatsamples varied between 276 and 797 microgg (Lampi NurmiOllilainen amp Piironen 2008)

In whole wheat grains color has been most commonly usedas a quality indicator The color is primarily attributed to thepresence of carotenoids and their esters The concentration oftotal carotenoids in whole wheat ranged from 08 to 217 microgg(Moore et al 2005) Lutein and zeaxanthin were the mostpredominant carotenoids present in whole wheat with con-centrations of 05ndash144 and 02ndash039 microgg grain respectively(Moore et al 2005) In addition lower concentrations of ca-rotenoids including β-cryptoxanthin at 001ndash013 microgg andβ-carotene at 009ndash021 microgg have also been detected in wheat

Phenolic lipids also known as alkylresorcinols (ARs)are commonly present in wheat (Gunenc HadinezhadTamburic-Ilincic Mayer amp Hosseninian 2013) Structurally theARs are similar to tocopherols except for the presence of astraight aliphatic hydrocarbon side chain and a single pheno-lic ring The alkyl side chain may contain between 13 and 27carbon atoms 5-N-alkylresorcinols 5-alkenylresorcinols5-oxoalkylresorcinols 5-oxoalkenylresorcinols and5-hydroxyalkenylresorcinols are the five major classes of ARsreported in the whole wheat ARs in whole wheat range from489 to 1429 microgg (Ross et al 2003)

Other bioactive phytochemicals have also been extractedand identified in whole wheat In this review they are groupedtogether as a ldquomiscellaneousrdquo group The miscellaneous group

consists of benzoxazinoids lignans phytosterols and sterylferulates Benzoxazinoids are a group of potent natural com-pounds which have recently been identified in whole grainwheat and rye grains and in bakery products of these cereals(Hanhineva et al 2011 Pedersen Laursen Mortensen ampFomsgaard 2011) The total benzoxazinoids content in wheatis very low (5 microgg DMB) Phytosterols are isoprenoid com-pounds which are biosynthetically derived from squalenePhytosterols are mostly found in free or esterified forms in-cluding esters of fatty acids and phenolic acids as well asglycosides or acylated glycosides According to the study ofNurmi Nystroumlm Edelmann Lampi and Piironen (2008) thecontent of phytosterols range is 670ndash959 microgg DMB in wholegrain winter wheat and 797ndash949 microgg DMB in spring wheat Aportion of the phytosterols occurs in ferulic acid ester formie as steryl ferulates

This contribution provides an overview of bioactivephytochemicals such as phenolic acids carotenoids tocoph-erols alkylresorcinols and other miscellaneous bioactivecompounds that are commonly found in wheat grain (Table 1)The distribution of bioactive phytochemicals their extrac-tion analysis changes during processing varietal differencesand functional properties will be presented and discussed

2 Distribution of bioactive phytochemicalsin wheat

All whole wheat grain products contain bran germ and en-dosperm fractions It is well documented in the publishedliterature that the bioactive phytochemicals are not uni-formly distributed Germ and bran fractions generally containhigher concentration of bioactive phytochemicalsThis is evidentfrom number of pearling studies described in the literature(Blandino et al 2013 Liyana-Pathirana Dexter amp Shahidi 2006)Liyana-Pathirana et al (2006) studied the effect of sequentialremoval of the outermost layers (pearling) on the phenolic com-position of wheat grains Higher levels of phenolic compoundshave been reported in the bran fraction as compared to therefined grain fraction that primarily comprises endosperm Ina recent study it was reported that most of the phenolic acidsexisted in bound insoluble form (80) as compared to freesoluble form and high concentrations of phenolic acids weredetected in whole grain which contains the bran fraction (6278ndash7456 microgg dry weight) as compared to the refined grain (660ndash970 microgg dry weight) (Lu Fuerst et al 2014)

The concentration of total carotenoids in wheat ranges from07 microgg in durum wheat from Spain to as high as 136 microgg inEinkorn accessions from Italy The highest concentrations ofcarotenoids are observed in the germ fraction followed by thebran and endosperm fractions Similarly tocopherols andtocotrienols are not uniformly distributed in grain Higher con-centrations of tocopherols are found in the germ or the outerlayers and the concentration of tocopherols in the endo-sperm fraction is comparatively lower Similarly tocotrienolsare found at higher concentrations in outer layer of the grain(85 pericarp testa and aleurone) and only 15 in the en-dosperm fraction (Piironen et al 2009)

In a recent study Tanwir Fredholm Gregersen andFomsgaard (2013) investigated the levels of benzoxazinoids in

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2 j o u rna l o f f un c t i ona l f o od s ( 2 0 1 5 ) ndash

different wheat and rye fractions Higher concentrations ofbenzoxazinoids were observed in the germ fraction than the branand endosperm fractions The fine bran was the second mostenriched fraction Phytosterols are known to accumulate in the

bran and germ fractions of the wheat grain whereas sterylferulates are mostly found in the bran (Nurmi et al 2012) Theconcentration of individual phytosterols such as sitosterolcampesterol sitostanol campestanol and stigmasterol in wheat

Table 1 ndash Classification of bioactive phytochemicals present in wheat

Phytochemicalname

Example Structure

Phenolicacids

Hydroxy benzoicacid or hydroxycinnamic acidderivatives

Ferulic acid

HO

H3CO

OH

O

Tocopherol Tocopherols andtocotrienols

alpha-Tocopherol

H3C O

CH3CH3

CH3

OH

CH3

CH3 CH3 CH3

Carotenoids Carotenoids Lutein

HO

H

OH

Phenoliclipids

Alkylresorcinols 5-alkenylresorcinols

R

OH

HO

Miscellaneousgroups

Benzoxazinoids DIMBOA

N

OO OH

O

OH

Lignans Secoisolariciresinol OMe

OH

OH

MeO

OH

HO

Phytosterols Sitosterol

HO

Steryl ferulates Campesterylferulate

HO

O

H

OMe

O

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3j o u rna l o f f un c t i ona l f o od s ( 2 0 1 5 ) ndash

was highly varied in their distribution (Nurmi et al 2008) Lignansare polyphenolic substances derived from phenylalanine via di-merization of substituted cinnamic alcohols known asmonolignols to a 23-dibenzylbutane structureThey are mostlyconcentrated in the bran fraction of the whole grain

3 Extraction of phytochemicals in wheat

Optimized extraction of bioactive phytochemicals is of signifi-cant importance to accurately quantify the phytochemicalcontent in foods and plant products Accurate quantificationallows establishment of proper dietary and safety guidelinesThe bioactive phytochemicals exist in multiple forms soluble(free and conjugated) and insoluble bound forms It is essen-tial to understand various forms of the bioactive phytochemicalsto accurately quantify them and understand their bioavailabilityand health promoting properties A wide array of extractionsolvents and technologies have been reported in the peer-reviewed literature (Herrero Cifuentes amp Ibantildeez 2006 Luthria2012a) The conventional extraction methods include Soxhletextraction stirring distillation manual mixing and percola-tion However in recent decades several new technologiesincluding microwave assisted extraction ultrasound-assistedextractions pressurized liquid extraction subcritical fluid ex-traction solid-phase extraction and enzyme-assisted extractionhave been used to automate andor improve the yields ofbioactive phytochemicals (Table 2)

31 Extraction of phenolic compounds

The majority of wheat phenolic acids is present mainly in thebound form linked to cell wall components such as cellu-lose lignin and proteins (Parker Ng amp Waldron 2005) Thephenolic acids in wheat have three primary states soluble freesoluble conjugated and insoluble bound (Moore et al 2005)The authors reported that most phenolic acids (~90) occurin the insoluble bound and lt9 and lt1 respectively exist insoluble conjugated and soluble free forms Alternatively a sim-plified direct method in the presence of ascorbic acid and EDTAwas used for the hydrolysis and extraction of total phenolicacids This direct method provides marginal improvement inextraction of total phenolic acids from wide array of food ma-trices including wheat (Lu Luthria et al 2014)

Ultrasonic-assisted extraction (UAE) is an inexpensive andhigh-efficient extraction method Simply its mechanismdepends on the collapse of bubbles that can accelerate therelease of extractable compounds through the disruption of bio-logical membranes and aids better solventndashsolute interactions(Vinatoru 2001) In the study of Hromadkova Kostalova andEbringerova (2008) UAE was successfully used in the extrac-tion of various phenolic compounds from wheat bran samples

Microwave-assisted extraction (MAE) uses microwave energyto facilitate partition target compound from plant cells into theextraction solvent The major advantage of MAE is the short-ened extraction time with lower quantity of solvent ascompared to conventional extraction method (Eskilsson ampBjorklund 2000)

Pressurized liquid extraction (PLE) is a relatively new tech-nology used for the extraction of phytochemicals under high

temperature and pressure With PLE application of high pres-sure allows extraction above the normal boiling point of theextraction solvent The combined use of high pressures (33ndash203 MPa) and high temperatures (40ndash200 degC) provides rapidextraction with reduced amounts of solvent (eg 20 min ex-traction with 10ndash50 mL of solvent using PLE provides similaror better extraction yields of phytochemicals as compared totraditional extraction method using 10ndash48 h and up to 200 mLof solvent) (Mendiola Herrero Cifuentes amp Ibanez 2007)Buranov and Mazza (2009) extracted and purified the ferulicacid from wheat bran by alkaline hydrolysis using PLE Theyindicated that crop residues contained pronounced levels offerulic acid after extracting with PLE

Solid-phase extraction (SPE) is widely used because it is arapid economical and sensitive technique for partial purifi-cation of extracts Different cartridges with varying numbersof sorbents can be used in SPE Irakli Samanidou Biliaderisand Papadoyannis (2012) developed and validated a high per-formance liquid chromatography (HPLC) method fordetermination of free and bound phenolic acids in cereals in-cluding wheat using solid-phase extraction on Oasis HLBcartridges with aqueous methanol as a solvent These resultsshowed that an optimized SPE method provided 95 recov-ery rate for phenolic acids from wheat (Arranz Saura-CalixtoShaha amp Kroon 2009)

32 Extraction of carotenoids and tocopherols

Supercritical fluid extraction (SFE) has been applied to the ex-traction of compounds from wheat bran and germ as it offersseveral advantages namely high selectivity and less extrac-tion time and solvent cost (Herrero et al 2006) DuranteLenucci Laddomada Mita and Caretto (2012) evaluated theeffect of encapsulation on the stability of oil extracted by SFE-CO2 from the durum wheat bran fraction The authors showedthat carotenoids were relatively stable at low temperatureHowever an increase in temperature resulted in decreased to-copherol concentrations due to hydroperoxide formation causedby lipid peroxidation They also indicated that encapsulationof SC-CO2 extracted wheat bran oil in alginate beads resultedin a protective effect on nutritionally important compoundssuch as tocopherols tocotrienols lutein zeaxanthin andβ-carotene In addition Gelmez Kincal and Yener (2009) in-vestigated the influence of SFE on the measurement of thephenolic and tocopherol content of roasted wheat germOptimal extraction conditions for total phenolics and tocoph-erols were determined to be 336 bar (pressure) 58 degC(temperature) and 10 min (extraction time)The tocopherol yieldunder these conditions corresponded to almost 100 recovery

Irakli Samanidou and Papadoyannis (2011) determined thecarotenoids tocopherols and tocotrienols in seven cereals in-cluding bread wheat and durum wheat by HPLCThe extractionmethod included sample saponification and clean-up by SPEusing OASIS HLB SPE cartridgesThe recoveries for each tocoph-erol and carotenoid compound ranged from 902 to 1101 withRSD lower than 10 In a separate research the same group op-timized a RP-HPLC method using a novel sorbent material(PerfectSil Target ODS-3 column) for the separation of tocoph-erols and tocotrienols in durum wheat and bread wheat TheSPE method was applied to extract the vitamin E components

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Table 2 ndash Methods used for the extraction of bioactive phytochemicals from wheat and its processed products

Serialno

Extraction Phytochemicals Results Changes inlevels ofphenolics a

References

1 Conventionalsolventextraction

TPC Different solvents showed different efficacies forextraction of phenolic compounds The extractproduced with 60 vv acetone had the highest contentof total phenols

+ Meneses MartinsTeixeira ampMussatto 2013

2 Ultrasonic-assistedextraction (UAE)

Phenolics-richheteroxylans

UAE represents a shortening of the process by about60 and lower consumption of the NaOH

+ HromadkovaKostalova ampEbringerova 2008

3 Microwave-assistedextraction (MAE)

TPC There was little difference of levels of phenolic acidsbetween heating with microwave irradiation and thesame conditions using a regular water bath as the heatsource

Inglett RoseStevenson Chen ampBiswas 2009

4 Pressurizedliquid extraction(PLE)

Phenolic acid The production of vanillin from crop residuescontaining greater levels of ferulic acid such as cornbran and sugar-beet pulp with PLE may be practical andcost-effective

+ Buranov amp Mazza2009

5 Enzymaticextraction

Polyphenols Enzymatic extraction seems to be more efficient thanorganic solvents for phenol bioaccessibility in wheatbread

+ Angioloni amp Collar2012

6 Solid-phaseextraction (SPE)

Phenolic acid Using an optimized solid-phase extraction (SPE)method a range of 84 to 106 recovery rate wasobtained for phenolic acids in cereals including wheat

+ Irakli SamanidouBiliaderis ampPapadoyannis 2012

7 Supercriticalfluid extraction(SFE)

Tocopherols Pressure of 4000ndash5000 psi the extracting temperature of40ndash45 degC and the carbon dioxide flow rate of 20 mLmin for 90 min were their optimal extraction conditionsto extract tocopherols from wheat germ

Ge Yan et al 2002

8 SFE Tocopherols andtocotrienols

Optimum value predicted by RSM for the concentrationof natural vitamin E was 2307 mg100g

Ge Ni Yan Chen ampCai 2002


Tocopherol yield was 033 mg tocopherolg germ at405 bar 60 degC and 10 min)

Gelmez Kincal ampYener 2009

10 SFE Tocopherolstocotrienols andcarotenoids

Encapsulation of SC-CO2 extracted wheat bran oil inalginate beads resulted in a protective effect onnutritionally important compounds such astocopherols tocotrienols lutein zeaxanthin andβ-carotene

+ Durante LenucciLaddomada Mita ampCaretto 2012


Carotenoids andtocopherols

Butanol method extraction is best for carotenoidextraction while hot saponification and the butanolprotocols have a better extraction efficiency than theother extraction method for tocopherols

+ Hidalgo BrandoliniPompei amp Piscozzi2006


Alkylresorcinols Acetone used as extraction solvent in a 140 (wv) ratiofollowed by continuous stirring (Stirrer-VWR CorningregVMS-C4) at room temperature for 24h

GunencHadinezhadTamburic-IlincicMayer ampHosseninian 2013


Alkylresorcinols Ethyl acetate used as solvent at room temperatureunder periodic hand shaking for 48 h After extractionthe solvent layer was filtered and evaporated to drynessbefore analysis

Sampietro et al2013


Alkylresorcinols Propanolwater (31) for the extraction of ARs from theprocessed food was reported

Holt MoreauDerMarderosianMcKeown ampJacques 2012

15 SFE Alkylresorcinols CO2 desired operating pressure 400 MPa extractingtemperature of 40ndash80 degC and the carbon dioxide flowrate of 15 plusmn 03 gmin was used for the extraction andthe results were compared with conventional solventextraction The extraction yield was higher for polarorganic solvents than for SC-CO2

minus Rebolleda BeltraacutenSanzGonzaacutelez-Sanjoseacute ampSolaesa 2013

Conventionalsolventextraction andSFE

Alkylresorcinols No qualitative or quantitative variations between thetwo extraction methods

Landberg DeyFrancisco Aman ampKamal-Eldin 2007

(continued on next page)

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They found that SPE provided a high extraction recovery fordurum wheat and bread wheat 870 for γ-tocotrienol and 100for δ-tocopherol (Irakli Samanidou amp Papadoyannis 2012)

Rezaee et al (2006) used dispersive liquidndashliquidmicroextraction (DLLME) for the extraction of tocopherols andtocotrienols Shammugasamy Ramakrishnan Ghazali andMuhammad (2013) determined tocopherols and tocotrienolsin cereals including wheat by reverse phase HPLC after ex-traction by combination of saponification and DLLME Theauthors concluded that DLLME method is precise and accu-rate in determining the tocopherol and tocotrienol contentsin cereals The advantages of this method were reduced ex-traction time and comparatively less solvent usage thereforecost efficient

Hidalgo Brandolini Pompei and Piscozzi (2006) used threedifferent solvents for extraction of carotenoids (water-saturated1-butanol hot saponification and tetrahydrofuran) from einkornwheat samples The authors concluded that butanol methodwas better for carotenoids extraction while hot saponifica-tion and the butanol protocols had better extraction efficiencyfor tocopherols

33 Extraction of alkylresorcinols (ARs)

Various solvents such as acetone ethyl acetate dichloromethaneand cyclohexane have been reported in literature for the ex-traction of ARs The conventional extraction method utilizesrefluxing sample with extraction solvent for period ranging from1 to 48 h The extracts are filtered evaporated dried and recon-stituted in a suitable solvent prior to analysis (Zarnowski ampSuzuki 2004) Recently a direct comparison of supercritical CO2

and conventional ethyl acetate extraction was carried outby Landberg Dey Francisco Aman and Kamal-Eldin (2007)Gunenc HadiNezhad Farah Hashem and Hosseinian (2015)The authors reported that no qualitative or quantitativevariations between the two extraction methods were de-tected Christian Anna Reinhold and Ralf (2015) reportedultrasound-assisted extraction with dichloromethane Theinteresting aspect of the Christianrsquos method was direct extrac-tion of ARs from the outer bran fraction of the grain withoutgrinding Similar extraction was carried out by Zarnowski andSuzuki (2004) by refluxing the whole grain with the selectedsolvents

34 Extraction of miscellaneous bioactive phytochemicals

The miscellaneous group of phytochemicals includesbenzoxazinoids sterols lignans and steryl ferulates Extrac-tion of benzoxazinoids requires hydrolysis of the glycosides toaglycons followed by enzymatic treatment to obtain stablebenzoxazolinones The benzoxazinoids were extracted bysoaking the wheat samples in water for a period of 6 h at roomtemperature followed by boiling with water for an additional15 min The water extracts were then filtered lyophilized andpowderedThe lyophilized powder was extracted by 80 metha-nol 1 acetic acid and 19 water (vv) in a Dionex ASE 350accelerated solvent extractor The benzoxazinoids were ex-tracted using the following conditions temperature 80 degC heat5 min static time 3 min cycles 4 rinse volume 60 purge60 s (Pedersen et al 2011 Tanwir et al 2013)

Phytosterols were extracted by acid andor base hydroly-sis of the wheat sample followed by silica column purification

Table 2 ndash (continued)

Serialno

Extraction Phytochemicals Results Changes inlevels ofphenolics

References


Alkylresorcinols Dichloromethane was used under ultra-sound-assistedextraction method and the samples were sonicated for15 s at 50 amplitude During ultrasonication sampleswere cooled in an ice bath to avoid sample heating Theduration of analytical extraction was shortened frommore than 1 h to only 45 s as compared to previousmethods In addition sample weight and solvent usewere significantly reduced

+ Christian AnnaReinhold amp Ralf2015

17 Acceleratedsolventextraction (ASE)

Benzoxazinoids Dionex ASE 350 Accelerated Solvent Extractor was usedwith the extraction solvent containing 19 water 80methanol and 1 acetic acid The extraction wascarried out at 80 degC

Pedersen LaursenMortensen ampFomsgaard 2011

18 Acidbasehydrolyses

Phytosterols The phytosterols were extracted by using acid and basehydrolyses of the samples followed by silica gel columnpurification prior to the analysis

Nurmi NystroumlmEdelmann Lampi ampPiironen 2008

19 ASE Phytosterols Different extraction solvents with differenttemperature were used to optimize the extractioncondition using ASE

Dunford IrmakJonnala 2009

20 Hot acetoneunder reflux

Steryl ferulates Hot acetone under reflux followed by basendashacid for thepurification of the extract

Nurmi et al 2012

21 Alkalinehydrolysisenzyme digest

Lignans The alkaline hydrolyzed samples were column purifiedusing SPE column

Cukelj JakasaSarajlija Novotni ampCuric 2011

a(+) increase (minus) decrease or () no change in levels of plant secondary chemical

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However steryl ferulates and other phenolic acid esters wereextracted using hot acetone under reflux followed by a basendashacid wash (Nurmi et al 2012)The extraction method for lignansused an initial alkaline hydrolysis of the wheat bran followedby incubation with an enzyme (pomatia-glucuronidasesulfatase) After incubation the mixture was purified on a SPEcolumn The ethanolwater solvent mixture was used for thelignan extraction The extracts were acid hydrolyzed and fil-tered before analysis (Zhang et al 2007)

4 Analysis of phytochemicals in wheat

The analytical methods used for the determination ofphytochemicals in whole grains are continuously changing dueto rapid advances in technology The method used for analy-sis depends on the resources available and the research goals(Table 3) High performance liquid chromatography (HPLC) andgas chromatography (GC) methods have been extensively usedfor separation identification and quantification of phyto-chemical compounds in wheat samples (Gunenc et al 2013Lu Lv et al 2014 Luthria 2012b) HPLC with diode array de-tection (DAD) is the most popular and reliable technique andis commonly preferred over GC which frequently requires anadditional derivatization step Spectroscopic techniques in-cluding mass spectrometry (MS) nuclear magnetic resonance(NMR) and near-infrared (NIR) detections are commonly usedfor structural elucidation qualitative and quantitative analy-sis (Nicoletti et al 2013 Sun Sun Fowler amp Baird 2005)

41 Analysis of phenolic acids

HPLC-DAD and HPLC-MS are frequently used for the analysisof phenolic acids in wheat In previous studies Lu Lv et al(2014) determined the insoluble and soluble ferulic acid contentin ten soft wheat bran samples using HPLC The authors in-dicated that around 99 of the ferulic acid was present in aninsoluble bound form in wheat bran samples In additionNicoletti et al (2013) investigated the soluble free conju-gated and insoluble bound phenolic acids in durum wheatusing LC-MS Their results showed that the total content of thethree different forms of phenolic acids was in the order in-soluble bound gt soluble conjugated gt soluble free The authors alsopointed out that ferulic acid was the predominant phenolic acidin the soluble free and insoluble bound forms whereas sinapicacid was the predominant phenolic acid in the soluble con-jugated form Dinelli et al (2009) analyzed various phenoliccompounds including phenolic acids flavonoids coumarinsproanthocyanidins stilbenes and lignans from old and modernvarieties of durum wheat using HPLC coupled with time-of-flight (TOF)-MS They concluded that old wheat varieties mayprovide unique functional properties for their bioactive phy-tochemical content suggesting their use into a broad range ofregular and special products NMR spectroscopy is used forstructural elucidation of bioactive phytochemicals It has alsobeen used recently for metabolomic studies Sun et al (2005)identified and characterized p-hydroxybenzoic acid vanillic acidsyringic acid p-coumaric acid ferulic acid and cinnamic acidand lignin in wheat straw by using NMR and NIR In addition

minor quantities of esterified or etherified p-coumaric (006)and ferulic acid (008) were observed in the lignin prepara-tions suggesting that these two hydroxycinnamic acids arestrongly linked to lignins

42 Analysis of carotenoids and tocopherols

Tocopherols and carotenoids are also commonly analyzed byHPLC-MS and GCndashMS Hung and Hatcher (2011) developed a fastand sensitive method for separation of carotenoids from durumwheat flours by uHPLC The peaks of all carotenoid compo-nents were detected at 445 nm using a photodiode arraydetector Their results showed that two wheat varieties (Com-mander and AC Navigator) contained higher luteinconcentration (531 and 559 microgg flour respectively) than theother cultivars and wheat grown in the Taber and Swift Currentregions had greater lutein concentration (ranging from 045 to559 microgg flour) than those grown in Regina (ranging from 037to 398 microgg flour) Similarly Ndolo and Beta (2013) investi-gated the distribution of the carotenoids in endosperm germand aleurone fractions of four soft wheat cultivars using HPLCThe results indicated that the germ fraction of wheat had sig-nificant levels of lutein (2157 microgkg) and zeaxanthin (3094 microgkg) Lower amounts of lutein (557 microgkg) and zeaxanthin (notdetected) were found in the wheat endosperm In addition theaverage zeaxanthin content was 35 times higher in wheat germthan in yellow corn

Beleggia Platani Nigrp De Vita and Cattivelli (2013) ex-amined the metabolite profiles of durum wheat using GC andGCndashMSThe authors identified tocopherols phytosterols aminoacids as well as other metabolites using GCndashMS This methodcan provide a basis for developing improved wheat cultivarswith increased nutritional value and health benefits for durumwheat Lu Lv et al (2014) investigated the levels of carot-enoids and tocopherols in ten soft wheat bran samples usingHPLC Their results showed that α-tocopherol (23ndash53 microgg) andlutein (10ndash14 microgg) were the predominant tocopherols and ca-rotenoids in all wheat bran samples In another studyGiambaneli et al (2013) analyzed the tocopherol and carot-enoid contents in primitive wheat populations from Europeancountries by HPLC The authors showed that wheat geno-types affected the relative levels of α-tocopherol β-tocopherolα-tocotrienol and β-tocotrienol Lutein was the most predomi-nant catortenoid in all the samples The total carotenoidscontents in Triticum monococcum Triticum timopheevi and Triti-cum durum ranged from 0567 to 0785 mgmg Triticum dicoccumcontained the lowest relative lutein content but the highestzeaxanthin concentration (0272 mgmg total carotenoids)

Similar studies regarding the carotenoid and tocopherol de-terminations in wheat or wheat-based products were alsoreported (Lv et al 2013 Moore Luther Cheng amp Yu 2009) Ina recent study Kumar and Krishna (2013) used HPLC and NMR(13C and 1H) to determine the distribution of tocopherols andcarotenoids in the unsaponifiable matter of wheat bran andwheat germ oilsThese results showed that wheat germ oil con-tained higher concentrations of tocopherols and carotenoids(273 and 1223 mg100 g respectively) than wheat bran oil (190and 221 mg100 g respectively) The total carotenoids couldalso be determined as a measure of total xanthophylls in wheatflour and wheat grits by a spectrophotometric method (Luterotti

ARTICLE IN PRESS



Table 3 ndash Separation identification and metabolic profiling of bioactive phytochemicals in wheat and wheat-basedproducts

Serialno

Analysis Phytochemicals Results References

1 HPLC Ferulic acid Around 99 of the ferulic acid was identified as insolublebound form in wheat bran samples

Lu Lv et al 2014

2 LC-MS Phenolic acids Total content of the three different forms of phenolic acidis in the order bound gt conjugated gt free in each of durumwheat sample

Nicoletti et al2013

3 LC-MS Phenolic acidsflavonoidscoumarinsproanthocyanidinsstilbenes andlignans

Old wheat varieties may provide unique functionalproperties for their peculiar contents in bioactivephytochemicals

Dinelli et al2009

4 NMR Phenolic acidsand lignin

Phenolic acids and lignin in wheat straw by using NMR Sun Sun FowlerBaird 2005

5 NIR Phenolic acidsand lignin

Phenolic acids and lignin in wheat straw by using NIR Sun Sun FowlerBaird 2005

6 GC GCndashMS Tocopherols andphytosterols

GC and GCndashMS help to provide a basis for developingimproved wheat cultivars with increased nutritional valueand health benefits for durum wheat

Beleggia PlataniNigrp De Vitaand Cattivelli2013

7 HPLC Carotenoids andtocopherols

α-Tocopherol and lutein is the predominant tocopheroland carotenoid in all wheat bran samples

Lu Lv et al 2014


Wheat varieties affected the relative levels of all thetocopherol components and carotenoids whereas sowingdata had slight effect

Giambaneli et al2013

9 HPLC andNMR

Tocopherols andcarotenoids

Wheat germ oil contained higher concentrations oftocopherols and carotenoids (273 mg100 g 1223 mg100 g)than wheat bran oil (190 mg100 g 221 mg100 g)respectively

Kumar ampKrishna 2013

10 Colorimetricmethod

Alkylresorcinols ARs was coupled with Fast Blue RR salt in alkalinemedium yielding colored azo-derivatives and werequantified by colorimeter Good linearity was observedbetween the samples

Gajda Kulawinekamp Kozubek 2008 SampietroVattuone ampCatalaacuten 2009

11 HPLC Alkylresorcinols The ARS were analyzed by using HPLC containing KromasilC18-5 column Methanol (A) and water (B) served as mobilephase

RebolledaBeltraacuten SanzGonzaacutelez-Sanjoseacuteamp Solaesa 2013

12 GCndashMS Alkylresorcinols The extracted metabolites were silylated with 05 mLpyridineHMDSTMCS (931)

Landberg DeyFrancisco Amanamp Kamal-Eldin2007

13 GC and NMR Alkylresorcinols ARs were analyzed by GC and the characterization wasmade by using NMR

Zarnowski ampSuzuki 2004

14 HPLCMS Benzoxazinoids UPLC BEH C18 column 01formic acid in acetonitrilewater (595 vv) (phase A) and 01 formic acid inacetonitrile (phase B) ESI conditions are capillary voltageat 30 kV cone voltage at 30 eV collision energy at 3 eV and20 eV argon used as collision gas

Hanhineva et al2011

15 GC-FID Phytosterols TMS derivatized samples were analyzed by GC-FIDequipped with a 5 diphenylndash95 dimethyl polysiloxanecolumn

Nurmi et al2008

16 HPLC-MS Steryl ferulates The methanolwater (982) served as mobile phase The MSanalysis performed with APCI at the interface temperatureat 400 degC nebulizer gas pressure at 50 psi voltage of thecorona discharge needle was minus15 kV

Hakala et al2002

17 GCndashMS Lignans Pyridine and pentafluoropropionic anhydride (PFPA) wereused for the derivatization of the metabolites

Cukelj JakasaSarajlijaNovotni amp Curic2011

ARTICLE IN PRESS



amp Kljak 2010) The authors indicated that the total carot-enoids levels in the wheat flours was 11ndash13 mgkg and inwheat grits 16 mgkg

43 Analysis of alkylresorcinols (ARs)

ARs are commonly analyzed by three different techniques colo-rimetric GCndashMS and HPLC-MS The colorimetric method ofdetection and quantification is simple reliable and inexpen-sive The colorimetric method based on diazonium saltFast Blue B BF4 developed by Tluscik Kozubek andMejbaum-Katzenellenbogen (1981) has been modified severaltimes Initially the modification was due to the unavailabilityof the Fast Blue BF4 reagent that was replaced by Fast Blue BZnCl2 salt that was commercially available Later chemical modi-fications were driven by optimization of sample throughputand stability of the colored complex Recently SampietroJimenez Belizaacuten Vattuone and Catalaacuten (2013) developed amicro method for the quantification of 5-n-alkylresorcinols ingrain and grain products In this method 5-N alkylresorcinolsare detected from ground and intact whole grains using FastBlue RR 1frasl2 ZnCl2 to yield colored azo derivatives that were mea-sured at 490 nm with a shorter incubation time (15 min) Thisrapid method correlated well (R2 = 09944) with the com-monly used Fast Blue B method and absorbance measurementof colored azo complex at 520 nm

Chromatographic separation using HPLC and GC with UVfluorescence (FD) Coulombic array electrochemical detection(CAED) and MS detection methods has been used for the analy-sis of alkylresorcinols Some HPLC methods have longer runtimes due to poor resolution between the unsaturated ho-mologs and saturated side chains However GC methods providecomparatively better resolution and are commonly used for theanalysis of alkylresorcinols Advances in HPLC that allow higherback pressure (gt1000 bar) and smaller particle size columns(lt3 microm) provide better resolution of ARs Ross et al (2012) de-veloped a HPLC method for the separation of alkylresorcinolsfrom 15 cereal grains and 90 cereal products and compared theperformance of three different detectors (UV FD and CAED)The limit of quantification and detection for the three methodswere significantly different CAED was the most sensitive fol-lowed by FD and UV The authors also pointed out that UVdetection generally resulted in an over-estimation ofalkylresorcinols concentration

Gas chromatography with MS and flame ionization detec-tions (FID) has been used for the analysis of alkylresorcinolsIn most procedures silyl derivatives of alkylresorcinols areprepared to improve the volatility of the compoundsLandberg Aman and Kamal Eldin (2009) used an OasisMax solidphase extraction cartridge for sample cleanup to develop a rapidGCndashMS method for the quantification of alkyl resorcinols inhuman plasma

44 Analysis of miscellaneous bioactive phytochemicals

Dinelli et al (2009) reported phytochemical profiles of wheatby HPLC-ESI-TOF-MS In this experiment a RP C18 analyticalcolumn with water and acetonitrile acidified with 05 aceticacid (vv mobile A and B) was used for the separation of thephytochemicals The ESI-MS parameters (capillary voltage

+45 kV drying gas temperature 190 degC drying gas flow 70 Lmin and nebulizing gas pressure 217 psi with the mass rangefrom mz 50 to 1000) were optimized for the profiling of 70phytochemicals including flavonoids phenolic acids andproanthocyanidins in wheat

Phytosterols and steryl ferulates were analyzed using GCand HPLC techniques For the phytosterol analysis the ex-tracts were derivatized before injecting into the GC system(Nurmi et al 2008) The individual phytosterols sitosterolcampesterol sitostanol campestanol and stigmasterol werereported HPLC (Nurmi et al 2012) and HPLC-MS (Hakala et al2002) based analysis of steryl ferulates indicated that the levelsof individual steryl ferulates (sitosteryl ferulate sitostanylferulate and campesteryl ferulate) in wheat were highly variedin their distribution Lignans were analyzed by Cukelj JakasaSarajlija Novotni and Curic (2011) using GCndashMS afterderivatizing the extracts with pentafluoropropionic anhy-dride (PFPA) to improve the volatility of the metabolites

5 Phytochemicals in grainsduring processing

Wheat is commonly processed prior to consumption Grain pro-ducers processors consumers health and nutritionalprofessionals are interested in investigating the effect of pro-cessing on bioactive phytochemicals present in wheat (WangHe amp Chen 2014) The stability of bioactive phytochemicalsis influenced by processes (milling fermentative proofingbaking enzymatic reaction extrusion cooking steamingmalting etc) and its conditions (temperature light pressuretime etc) This information is critical to the developmentof optimal processes capable of preserving bioactivephytochemicals in wheat based finished products In this reviewwe briefly summarize the effect of processing on bioactivephytochemicals namely phenolic acids carotenoids tocoph-erols alkyl resorcinols arabionxylans and other bioactivephytochemicals (Table 4)

51 Phenolic compounds during processing

Phenolic acids are the predominant phytochemicals presentin grain Ultrafine grinding used by Rosa Barron Gaiani Dufourand Micard (2013) increased the bioaccessibility of phenolic acidsfrom wheat bran The authors reported that wheat bran frac-tions had 15-fold increased antioxidant capacity when theparticle size decreased from 172 to 30 microm using ultra-fine grind-ingThe antioxidant activity was inversely related to the particlesize of the wheat bran fraction In agreement to the above studyHemery et al (2010) examined the potential of using ultra-fine grinding of wheat bran as a method to improve thebioaccessibility of p-coumaric acid sinapic acid and ferulic acidThe authors indicated that ultra-fine grinding increased theparticle surface area of wheat bran thus finally leading to therelease of more p-coumaric acid sinapic acid and ferulic acidIn another recent study Brewer Kubola Siriamornpun Heraldand Shi (2014) showed that the fine milling treatment re-sulted in higher levels of phenolic acids flavonoidsanthocyanin and carotenoids as compared to unmilledtreatment

ARTICLE IN PRESS



Table 4 ndash The effect of processing on the level of phytochemicals in wheat and its products

Serialno

Processing Phytochemicals Results Changes inlevels ofphenolics a

References

1 Ultra-finegrinding

Phenolic acids Wheat bran fractions had 15-fold increasedantioxidant capacity when the particle size decreasedfrom 172 to 30 microm using ultra-fine grinding

+ Rosa Barron GaianiDufour amp Micard 2013


Phenolic acids Ultra-fine grinding increased the particle surface areaof wheat bran thus finally leading to release of morep-coumaric acid and ferulic acid

+ Hemery et al 2010

3 Milling Phenolic acidsflavonoidanthocyanin andcarotenoid

Significant increase in extracted anthocyaninscarotenoids and ORAC value was observed andparticle size does influence the extraction ofphytochemicals in wheat bran

+ Brewer KubolaSiriamornpun Herald ampShi 2014

4 Fermentation Total phenolics The fermented wheat grains prepared with twofilamentous fungi had higher levels of phenolics ascompared to non-fermented wheat grains koji

+ Bhanja Kumari ampBanerjee 2009

5 Baking Polyphenols andflavonoids

Polyphenol content was found to be decreased in thebread samples whereas quercetin content increasedsignificantly in buckwheat with bread-making Thebaking decreased the levels of flavonoids

_ Alvarez-JubeteWijingaard Arendt ampGallagher 2010

6 Baking Phenolic acids Baking resulted in an increase in free phenolic acids inthe three products while bound phenolic acidsdecreased in bread and slightly changed in cookie andmuffin products

+minus Abdel-Aal amp Rabalski2013

7 Baking Phenolic acidstocopherols andcarotenoid

Baking reduced the concentrations of carotenoids andtocopherols however total phenolic acids contentmight not decrease during baking

minus Lu Fuerst et al 2014

8 Baking Phenolic acids The total quantified phenolic acids did not changesignificantly when preparing bread from refined andwhole wheat flour

Lu Luthria et al 2014

10 Toasting Total phenolics Longer toasting of defatted soy flakes at 150 degC resultedin increasing aglycone levels and total phenolics

+ Pananun et al 2012

11 Heating CarotenoidsTocopherols

Dry heat treatment increased the vitamin E andreduced the carotenoids in sorghum The wet heatreduced all antioxidant compounds except carotenoidswhich increased

+minus Cardoso et al 2014

12 Heating Carotenoids andtotal tocopherols

Fat-soluble compounds total tocopherols sterylferulates and carotenoids found in wheat germ startedto get the maximum reductions (0183 0034 and0004) at 130 degC

minus Kumar Swathi ampKrishna 2014

13 Baking Carotenoids Baking of β-carotene fortified bread and crackerdecreased total carotenoids from 4 to 23

minus Ranhotra GelrothLangemeier amp Rodgers1995

14 Baking Carotenoids Baking resulted in 36ndash45 losses of carotenoids inwheat-based products

minus Leehardt et al 2006

15 Fermentation Carotenoids 10 of losses in total carotenoids as a result of doughfermentation at 30 degC


16 Sprouting Polyphenols Sprouting of wheat appeared to significantly increaseits polyphenol content

+ Alvarez-JubeteWijingaard Arendt ampGallagher 2010

17 Kneading Carotenoids 66 decrease in total carotenoid contents in wheatsamples during kneading was observed


18 Mixing Tocopherols The content of tocopherol decreased during mixing forall products (214 282 and 442 for bread biscuitand pasta respectively)

minus Hidalgo amp Brandolini2010

19 Baking Alkylresorcinols To denaturation or degradation of ARs duringprocessing was reported

minus Weipert amp Al Baya 1977Winata amp Lorenz 1997

20 Wet processing Alkylresorcinols Bound with flour and starchminuslipid complexes formation Morrison Milligan ampAzudin 1984

21 Hydrothermalprocessing (HTP)and baking

Benzoxazinoids Enzymatic liberation of aglycones and their subsequentdegradation to benzoxazolinone

minus Pedersen LaursenMortensen amp Fomsgaard2011

22 Frying Phytosterols Due to the oxidation process of phytosterols theirlevels reduced in the processed food

minus Soupas Huikko Lampi ampPiironen 2007

23 Roastingwetprocessing

Lignan Decrease the lignan content due to enzymatic activity minus GerstenmeyerReimerBerghoferSchwartz amp Sontag 2013


ARTICLE IN PRESS



Fermentation processes are commonly used in the food in-dustry Bacteria and yeasts are used to convert carbohydratesinto alcohols CO2 or carboxylic acids Many metabolizingenzymes can be generated during fermentationThese enzymescan further hydrolyze the phenolic glycosides into free phe-nolic acids For instance Bhanja Kumari and Banerjee (2009)found that fermented wheat grains prepared with two fila-mentous fungi had higher levels of phenolics as compared tonon-fermented wheat grains koji

Different thermal processing methods such as bakingcooking toasting and microwaving have varying effects on thephenolic compounds in whole wheat Abdel-Aal and Rabalski(2013) studied the effect of baking on free and bound pheno-lic acids in wheat bakery products They found that ferulic acidwas the predominant phenolic acid both in the free or boundform of three whole wheat bakery products (bread cookie andmuffin) Most importantly their results showed that baking in-creased the free phenolic acids in the three bakery productswhile bound phenolic acids decreased in bread and were onlyslightly changed in cookie and muffin products Polyphenolcontent was generally found to be decreased in the breadsamples as compared to their raw grain Recently Lu Luthriaet al (2014) investigated the influence of baking on the totalphenolic acids in dough (mixed and proofed) and three breadfractions (upper crust crumbs and bottom crust) made fromrefined and whole-wheat flour of three wheat varieties Theresults showed that the total quantified phenolic acids did notchange significantly when preparing bread from refined andwhole wheat flourThe concentration of the total phenolic acidsin all whole wheat bread fractions were 55- to 98-fold greaterthan the corresponding refined wheat fractions In additionthe results indicated that the upper crust fraction had higherlevels of total phenolic acids than the dough and crumb frac-tions suggesting that total phenolic acids content did notdecrease significantly during baking (Lu Fuerst et al 2014 LuLuthria et al 2014)

52 Carotenoids and tocopherols during processing

Carotenoids are known to be sensitive to light and heat(Mercadante 2007) Ranhotra Gelroth Langemeier and Rodgers(1995) evaluated the stability of β-carotene during baking andpre-baking processing steps for wheat-based products Theauthors reported that baking caused significant reductions (74ndash85) in the all-trans β-carotene isomer content SimilarlyRanhotra et al (1995) found that carotene losses were ob-served during baking ranging from 4 to 15 for whole wheatbread products and from 18 to 23 for crackers In anotherstudy Leehardt et al (2006) reported losses in total carot-enoids of ~40 during bread baking Highest losses occurredin the crust which experienced higher temperatures than thecrumbs The authors also reported that β-cryptoxanthin levelsin bread crusts and water biscuits increased after bakingwhereas the levels of lutein zeaxanthin α- and β-carotene de-creasedThe same authors also found that the most significantdecreases (66) in total carotenoids content occur during knead-ing with a high correlation of these losses to the lipoxygenaseactivity of the wheat variety Two separate studies reported de-creases in the tocopherol and carotenoid content during theproduction of bread water biscuits and pasta from wheat flours

(Hidalgo amp Brandolini 2010) Similarly Lu Fuerst et al (2014)recently observed a decrease in the concentration of tocoph-erols and carotenoids during baking of two bread fractions(crumbs and upper crust) Recently Kumar Swathi and Krishna(2014) investigated the effect of temperature on fat-soluble com-pounds (total tocopherols and tocotrienols steryl ferulates andcarotenoids) in wheat bran and wheat germThe authors foundthat the reduction of total tocopherols and tocotrienols sterylferulates and carotenoids in wheat germ started at 130 degC

Non-thermal processing such as sprouting and kneadingtreatment may also change the levels of phytochemicals inwheat samples Alvarez-Jubete Wijingaard Arendt andGallagher (2010) reported that sprouting increased the total phe-nolic content in wheat from 531 to 110 mg GAE100 g dwbHowever Leehardt et al (2006) detected significant decreases(66) in the total carotenoid content during kneading with ahigh correlation of these losses to the lipoxygenase activity ofthe wheat varieties Furthermore the same group reported a10ndash12 loss in the tocopherol content during kneading (Leehardtet al 2006) Changes in tocopherol and tocotrienol levels werealso observed during dough making for bread biscuit and pasta(Hidalgo amp Brandolini 2010) The tocopherol content de-creased by 214 282 and 442 for bread biscuit and pastarespectively

53 ARs during processing

The processed grain products also vary in their ARs contentHigh temperature processing of grain based foods leads to de-naturation or degradation of ARs (Weipert amp Al Baya 1977Winata amp Lorenz 1997) Foods like white flour corn and rice-based grains which do not contain whole wheat or wheat branshowed low AR content (Holt Moreau DerMarderosianMcKeown amp Jacques 2012) In a recent study by Gunenc et al(2013) the authors evaluated the stability of alkylresorcinolsin breads enriched in hard and soft wheat brans The differ-ent formulas used in four bread trials were A) 100 white flour(control bread) B) 30 wheat bran C) 30 residue bran afterAR extraction and D) 30 wheat bran plus 2 crude AR extractAR content in breads varied from 11 to 829 mg100 g and washeat stable during baking After 1 h cooling all breads showedsignificantly different (P lt 005) inner temperature height andweight compared to the control bread Furthermore bread Dshowed a better height than breads B and C (P lt 005)The effectsof baking on total phenolic content (TPC) and antioxidant ac-tivity were also determined A positive correlation was observedbetween oxygen radical absorbance capacity (ORAC) and TPC(R2 = 089)

54 Miscellaneous bioactive phytochemicalsduring processing

In a study by Pedersen et al (2011) ten benzoxazinoids werequantified in durum wheat before and after five-day hydro-thermal processing (HTP) as well as in baked bread made withHTP and conventional flour The authors reported significantconcentration of benzoxazolinoids in HTP processed flour andno significant amount in conventional flour However the quan-tity of benzoxazolinoids in baked bread made from HTP flourwas reduced by around five fold but increased marginally

ARTICLE IN PRESS



in bread baked from conventional flour Leaching ofbenzoxazinoids during soaking or boiling due to the enzy-matic activity was reported by Tanwir et al (2013) Accordingto Kumar et al (2014) processing reduces the levels of sterylferulates which affect their antioxidant potentiality In addi-tion Soupas Huikko Lampi and Piironen (2007) suggested thatthe frying can affect the availability of phytosterols and reducestheir levels due to oxidation The lignans were not affected bymoderate heat processing (100 degC) whereas high tempera-ture processing (roasting at 250 degC) degraded the compoundsHowever moderate heating enhancing the extractability oflignans was reported by Gerstenmeyer Reimer BerghoferSchwartz and Sontag (2013)

6 Bioactivity of wheat

There is an increased consumption in whole grain productsbecause of their high functional property Whole wheat grainproducts are enriched in bioactive phytochemicals such as phe-nolic acids carotenoids tocopherols ARs benzoxazinoidsphytosterols and lignans Wheat phytochemicals are re-ported for their wide array of bioactivities namely high freeradical scavenging activity (Hosseinian amp Mazza 2009 LuLuthria et al 2014 Lu Lv et al 2014 Lv et al 2013) antican-cer property (Liu Winter Stevenson Morris amp Leach 2012)reduction of cholesterol (Tiwari amp Cummins 2009) decreasingcontrolling diabetes etc (Gil Ortega amp Maldonado 2011)

61 Functional properties of phenolic compounds

The wheat grains are rich in phenolic phytochemicals andare widely studied for their high antioxidant activity(Liyana-Pathirana amp Shahidi 2007 Okarter Liu Sorrells amp Liu2010) Phenolics like ferulic acid in wheat behave as physicaland chemical barriers to protect the kernel This is achievedby combating destructive radicals and deters consumption byinsects and animals because of its astringent taste There issome evidence that these compounds promote resistance tovarious diseases When compared to vegetables and fruits thefree phenolic content of grains is less as most phenolic com-pounds exist in the bound form Practically the bran is not easyto digest by the human system and hence gastrointestinal es-terase (from intestinal mucosa and microflora) facilitates therelease of ferulic acid and di-ferulic acids from branThe releaseof phenolic acids in the intestine reduces the risk of coloncancer in humans (Sang amp Zhu 2014) Nuclear factor kappa B(NF-kB) is a transcription factor regulating pro-inflammatorygenes by controlling innate immunity processes apoptosis cellproliferation and cell survival Direct correlation between theincreases in NF-kB activity was observed with several humancancers and in chronic inflammatory diseases (Hole GrimmerJensen amp Sahlstroslashm 2012) The wheat phenolic acids (free andbound) were reported to modulate NF-kB activity

62 Functional properties of carotenoids and tocopherols

Grains are one of the important sources of carotenoids and to-copherols Nutritionally carotenoids are beneficial to humans

for lowering of LDL cholesterol levels by inhibiting choles-terol biosynthesis (Tiwari amp Cummins 2009) Carotenoidsincrease iron absorption in the human body by binding ironat the aromatic ring(s) andor the conjugated double bondsystem (Garciacutea-Casal 2006) Tocopherols and tocotrienols arenaturally occurring fat soluble components that protect bio-logical membranes from oxidation and preserve immunologicalfunctions (Frank Chin Schrader Eckert amp Rimbach 2012)Theyplay a potential role in reducing various degenerative dis-eases such as cardiovascular cancer inflammatory neurologicaldisorders and cataracts

63 Bioactivity of ARs

ARs can serve as in vitro antioxidant source due to their hy-drogen donor and radical scavenging abilities (Gliwa GunencAmes Willmore amp Hosseinian 2011) Previous studies sug-gested that the membrane-located ARs are absorbed up to 80by the human body (Kamal-Eldin Pouru Eliasson amp Aman2001) Liposomes are one of the effective drug carriers due totheir ability to reduce drug toxicity and deliver the drugs tospecific cells (Torchilin 2005) The physicochemical behav-iors of ARs and their variability in structures were reported fortheir suitability as effective drug delivery systems(Zant-Przeworska Stasiuk Gubernator amp Kozubek 2010) Thestudies of Liu et al (2012) suggested that the AR fractions suchas 5-heptadecylresorcinol (IC50 = 225l gmL) 5-(16-heneicosenyl)resorcinol (trans) (IC50 = 137l gmL) 5-(14-nonadecenyl)resorcinol(trans) (IC50 = 422l gmL) and 5-(2-oxotricosanyl) resorcinol(IC50 = 1091 gmL) showed high cytotoxicity against humanprostate adenocarcinoma (PC3) cells at the IC50 concentrationof 225 137 422 and 109 microgmL (Landberg Kamal-EldinAndersson Vessby amp Aringman 2008) In a recent study it wasshown that the human plasma AR levels reach micro molarconcentrations immediately after consuming whole grain wheatand rye product (Landberg et al 2014) This indicates that theARs were absorbed by the human body in a short time with ahigh efficiency Landberg et al (2014) revealed that the enzymeinhibition potential of ARs leads to the suppression of adipocytelipolysis

64 Functional properties of miscellaneousbioactive phytochemicals

In addition to above four classes of compounds whole grainsare known to possess other bioactive phytochemicals(benzoxazinoids phytosterols lignans and etc) in minoramountsThe benzoxazinoids can serve as biomarkers of wholegrain as they are absorbed and metabolized by the body(Adhikari et al 2013) Anti-inflammatory anti-allergic and anti-carcinogenic effects of individual benzoxazinoids were reported(Poupaert Carato amp Colacillo 2005) The main function of phy-tosterols is the regulation of membrane fluidity andpermeability In humans these compounds are known for theirserum-cholesterol lowering effect (Kritchevskya amp Chen 2005)Lignans are claimed to have estrogen activity and hence servedas phytoestrogens to humans (Aehle et al 2011) A correla-tion between high intake of lignans and breast cancer wasreported by Landete (2012) Lignans have also been reportedto have anti-cancer activity against prostate cancer and

ARTICLE IN PRESS



colorectal cancer (Landete 2012) Higher levels of intake havebeen reported to reduce the risk of cardiovascular disease(Peterson et al 2010)

7 Conclusions

Whole wheat grain and its derived food products provide a greatsource of bioactive phytochemicals that provide health ben-efits to humansThe bioactive phytochemicals are not uniformlydistributed in grains bran and germ provide higher concen-trations of bioactive phytochemicals A comparison of differenttechnologies for extraction and analysis of bioactivephytochemicals from whole wheat is summarized in this reviewIt is clear that most phenolic acids are present in a bound formand improved yields are obtained after base hydrolysis usingmicrowave ultrasonication and pressurized liquid extrac-tion methods Spectrophotometric determinations are used forquick total quantification however HPLC and GC methodscoupled to mass spectrometers have been widely used for sepa-ration identification accurate quantification and metabolicprofiling There has been increased interest in understandinghow processing impacts the concentration of different bioactivephytochemicals present in whole wheat NMR techniques havebeen used in a limited way for structural elucidation and pro-filing There has been a large number of bioactivity reports onphytochemicals extracted from wheatThere is very limited re-search regarding interactions of different phytochemicals asthey exit in wheat and wheat products In addition there isalso limited research on increasing the bioavailability of boundphenolics

R E F E R E N C E S

Abdel-Aal E S M amp Rabalski I (2013) Effect of baking on freeand bound phenolic acids in whole grain bakery productsJournal of Cereal Science 57 312ndash318

Adhikari K Laursen B Gregersen P Schnoor H Witten MPoulsen L Jensen B amp Fomdsgaards I (2013) Absorptionand metabolic fate of bioactive dietary benzoxazinoids inhumans Molecular Nutrition and Food Research 57 1847ndash1858

Aehle A Muumlller U Eklund P C Willfoumlr S M Sippl W ampDraumlger B (2011) Lignans as food constituents with estrogenand antiestrogen activity Phytochemistry 72 2396ndash2405

Alvarez-Jubete L Wijingaard H Arendt E K amp Gallagher E(2010) Polyphenol composition and in vitro antioxidantactivity of amaranth quinoa buckwheat and wheat asaffected by sprouting and baking Food Chemistry 119 770ndash778

Andersson A A M Dimberg L Aman P amp Landberg R (2014)Recent findings on certain bioactive components in wholegrain wheat and rye Journal of Cereal Science 59 294ndash311

Angioloni A amp Collar C (2012) Effects of pressure treatment ofhydrated oat finger millet and sorghum flours on the qualityand nutritional properties of composite wheat breads Journalof Cereal Science 56 713ndash719

Arranz S Saura-Calixto F Shaha S amp Kroon P A (2009) Highcontents of nonextractable polyphenols in fruits suggest thatpolyphenol contents of plant foods have beenunderestimated Journal of Agricultural and Food Chemistry 577298ndash7803

Beleggia R Platani C Nigrp F De Vita P amp Cattivelli L(2013) Effect of genotype environment and

genotype-by-environment interaction on metabolite profilingin durum wheat (Triticum durum Desf) grain Journal of CerealScience 57 183ndash192

Bhanja T Kumari A amp Banerjee R (2009) Enrichment ofphenolics and free radical scavenging property of wheat kojiprepared with two filamentous fungi Bioresource Technology100 2861ndash2866

Blandino M Sovrani V Marinaccio F Reyneri A Rolle LGiacosa S Locatelli M Bordiga M Travaglia F CoissonJ D amp Arlorio M (2013) Nutritional and technological qualityof bread enriched with an intermediated pearled wheatfraction Food Chemistry 141 2549ndash2557

Brewer L R Kubola J Siriamornpun S Herald T J amp Shi Y C(2014) Wheat bran particle size influence on phytochemicalextractability and antioxidant properties Food Chemistry 152483ndash490

Buranov A amp Mazza G (2009) Extraction and purification offerulic acid from flax shives wheat and corn bran by alkalinehydrolysis and pressurized solvents Food Chemistry 115 1542ndash1548

Cardoso L D M Montini T A Pinheiro S S Pinheiro-SantAnaH M Martino H S D amp Moreira A V B (2014) Effects ofprocessing with dry heat and wet heat on the antioxidantprofile of sorghum Food Chemistry 152 210ndash217

Chanson-Rolle A Lappi J Meynier A Poutanen K Vinoy S ampBraesco V (2014) Health benefits of whole-grain Asystematic review of the evidence to propose a daily intakerecommendation The FASEB Journal 28 11ndash17

Christian H G Anna E M Reinhold C amp Ralf M S (2015)Development and validation of an HPLC method for thedetermination of alk(en)ylresorcinols using rapid ultrasound-assisted extraction of mango peels and rye grains FoodChemistry 169 261ndash269

Cukelj N Jakasa I Sarajlija H Novotni D amp Curic D (2011)Identification and quantification of lignans in wheat bran bygas chromatography-electron capture detection Talanta 84127ndash132

Dinelli G Carretero A S Silverstro R D Marotti I Fu SBenedettelli S Ghiselli L amp Gutierrez A F (2009)Determination of phenolic compounds in modern and oldvarieties of durum wheat using liquid chromatographycoupled with time-of-flight mass spectrometry Journal ofChromatography A 1216 7229ndash7240

Dunford N T Irmak S amp Jonnala R (2009) Effect of the solventtype and temperature on phytosterol contents andcompositions of wheat straw bran and germ extracts Journalof Agricultural and Food Chemistry 57 10608ndash10611

Durante M Lenucci M S Laddomada B Mita G amp Caretto S(2012) Effects of sodium alginate bead encapsulation on thestorage stability of durum wheat (Triticum durum Desf) branoil extracted by supercritical CO2 Journal of Agricultural andFood Chemistry 60 10689ndash10695

Eskilsson C S amp Bjorklund E (2000) Analytical-scalemicrowave-assisted extraction Journal of Chromatography A902 227ndash250

FAO World cereal production in 2014 to surpass the record in2013 (2014) Available from lthttpwwwfaoorgworldfoodsituationcsdbengt Accessed 171214

FDA Draft guidance whole grain label statements (2006)Available from lthttpwwwfdagovOHRMSDOCKETS98fr06d-0066-gdl0001pdfgt February 17 2006

Frank J Chin X W D Schrader C Eckert G P amp Rimbach G(2012) Do tocotrienols have potential as neuroprotectivedietary factors Ageing Research Reviews 11 163ndash180

Gajda A Kulawinek M amp Kozubek A (2008) An improvedcolorimetric method for the determination of alkylresorcinolsin cereals and whole-grain cereal products Journal of FoodComposition and Analysis 21 428ndash434

ARTICLE IN PRESS



Garciacutea-Casal M N (2006) Carotenoids increase iron absorptionfrom cereal-based food in the human Nutrition Research 26340ndash344

Ge Y Ni Y Yan H Chen Y amp Cai T (2002) Optimization of thesupercritical fluid extraction of natural vitamin E from wheatgerm using response surface methodology Journal of FoodScience 67 239ndash243

Ge Y Yan H Hui B Ni Y Wang S amp Cai T (2002) Extractionof natural vitamin E from wheat germ by supercritical carbondioxide Journal of Agricultural and Food Chemistry 50 685ndash689

Gelmez N Kincal N S amp Yener M E (2009) Optimization ofsupercritical carbon dioxide extraction of antioxidants fromroasted wheat germ based on yield total phenolic andtocopherol contents and antioxidant activities of theextracts Journal of Supercritical Fluids 48 217ndash224

Gerstenmeyer E Reimer S Berghofer E Schwartz H ampSontag G (2013) Effect of thermal heating on some lignans inflax seeds sesame seeds and rye Food Chemistry 138 1847ndash1855

Giambaneli E Ferioli F Kocaoglu B Jorjadze M Alexieva IDarbinyan N amp DrsquoAntuono L F (2013) A comparative studyof bioactive compounds in primitive wheat populations fromItaly Turkey Georgia Bulgaria and Armenia Journal of Scienceand Food Agriculture 93 3490ndash3501

Gil A Ortega M R amp Maldonado J (2011) Wholegrain cerealsand bread A duet of the Mediterranean diet for theprevention of chronic diseases Public and Health Nutrition 142316ndash2322

Gliwa J Gunenc A Ames N P Willmore W G amp Hosseinian F(2011) Antioxidant activity of alkylresorcinols from rye branand their protective effects on cell viability of PC-12 AC cellsJournal of Agricultural and Food Chemistry 59 11473ndash11482

Gunenc A Hadinezhad M Tamburic-Ilincic L Mayer P M ampHosseninian F (2013) Effects of region and cultivar onalkylresorcinols content and composition in wheat bran andtheir antioxidant activity Journal of Cereal Science 57 405ndash410

Gunenc A HadiNezhad M Farah I Hashem A amp HosseinianF (2015) Impact of supercritical CO2 and traditional solventextraction systems on the extractability of alkylresorcinolsphenolic profile and their antioxidant activity in wheat branJournal of Functional Foods 12 109ndash119

Hakala P Lampi A M Ollilainen V Werner U Murkovic MWaumlhaumllauml K Karkola S amp Piironen V (2002) Steryl phenolicacid esters in cereals and their milling fractions Journal ofAgricultural and Food Chemistry 50 5300ndash5307

Hanhineva K Rogachev I Aura A Aharoni A Poutanen K ampMykkanen H (2011) Qualitative characterization ofbenzoxazinoid derivatives in whole grain rye and wheat byLC-MS metabolite profiling Journal of Agricultural and FoodChemistry 59 921ndash927

Hemery Y M Anson N M Havenaar R Haenen G Noort Mamp Rouau X (2010) Dry-fractionation of wheat bran increasesthe bioaccessibility of phenolic acids in breads made fromprocessed bran fractions Food Research International 43 1429ndash1438

Herrero M Cifuentes A amp Ibantildeez E (2006) Sub- andsupercritical fluid extraction of functional ingredients fromdifferent natural sources Plants food-by-products algae andmicroalgae A review Food Chemistry 98 136ndash148

Hidalgo A amp Brandolini A (2010) Tocols stability during breadwater biscuit and pasta processing from wheat flours Journalof Cereal Science 52 254ndash259

Hidalgo A Brandolini A Pompei C amp Piscozzi R (2006)Carotenoids and tocols of einkorn wheat (Triticum monococcumssp monococcum L Journal of Cereal Science 44 182ndash193

Hole A S Grimmer S Jensen M R amp Sahlstroslashm S (2012)Synergistic and suppressive effects of dietary phenolic acidsand other phytochemicals from cereal extracts on

nuclear factor kappa B activity Food Chemistry 133969ndash977

Holt M D Moreau R A DerMarderosian A McKeown N ampJacques P F (2012) Accelerated solvent extraction ofalkylresorcinols in food products containing uncooked andcooked wheat Journal of Agricultural and Food Chemistry 604799ndash4802

Hosseinian F S amp Mazza G (2009) Triticale bran and strawPotential new sources of phenolic acids proanthocyanidinsand lignans Journal of Functional Foods 1 57ndash64

Hromadkova Z Kostalova Z amp Ebringerova A (2008)Comparison of conventional and ultrasound-assistedextraction of phenolics-rich heteroxylans from wheat branUltrasonics Sonochemistry 15 1062ndash1068

Hung P M amp Hatcher D W (2011) Ultra-performance liquidchromatography (UPLC) quantification of carotenoids indurum wheat Influence of genotype and environment inrelation to the colour of yellow alkaline noodles (YAN) FoodChemistry 125 1510ndash1516

Inglett G E Rose D Stevenson D G Chen D amp Biswas A(2009) Total phenolics and antioxidant activity of water andethanolic extracts from distillers dried grains with solublewith or without microwave irradiation Cereal Science 86 661ndash664

Irakli M N Samanidou V F Biliaderis C G amp PapadoyannisI N (2012) Development and validation of an HPLC-methodfor determination of free and bound phenolic acids in cerealsafter solid-phase extraction Food Chemistry 134 1624ndash1632

Irakli M N Samanidou V F amp Papadoyannis I N (2011)Development and validation of an HPLC method for thesimultaneous determination of tocopherols tocotrienols andcarotenoids in cereals after solid-phase extraction Journal ofSeparation Science 34 1375ndash1382

Irakli M N Samanidou V F amp Papadoyannis I N (2012)Optimization and validation of the reversed-phase high-performance liquid chromatography with fluorescencedetection method for the separation of tocopherol andtocotrienol isomers in cereals employing a novel sorbentmaterial Journal of Agricultural and Food Chemistry 60 2076ndash2082

Kamal-Eldin A Pouru A Eliasson C amp Aman P (2001)Alkylresorcinols as antioxidants Hydrogen donation andperoxyl radical-scavenging effects Journal of the Science of Foodand Agriculture 81 353ndash356

Kritchevskya D amp Chen C (2005) Phytosterols ndash Health benefitsand potential concerns A review Nutrition Research 25 413ndash428

Kumar G S amp Krishna A G (2013) Studies on thenutraceuticals composition of wheat derived oils wheat branand wheat germ oil Journal of Food Science and Technology 11ndash7

Kumar G S Swathi R amp Krishna A G (2014) Fat-solublenutraceuticals and their composition in heat-processedwheat germ and wheat bran International Journal of FoodScience and Nutrition 65 327ndash334

Lampi A Nurmi T Ollilainen V amp Piironen V (2008)Tocopherols and tocotrienols in wheat genotypes in theHEALTHGRAIN diversity screen Journal of Agricultural and FoodChemistry 56 9716ndash9721

Landberg R Aman P amp Kamal-Eldin A (2009) A rapid gaschromatographyndashmass spectrometry method forquantification of alkylresorcinols in human plasma AnalyticalBiochemistry 385 7ndash12

Landberg R Dey E S Francisco J D C Aman P amp Kamal-Eldin A (2007) Comparison of supercritical carbon dioxideand ethyl acetate extraction of alkylresorcinols fromwheat and rye Journal of Food Composition and Analysis 20534ndash538

ARTICLE IN PRESS



Landberg R Kamal-Eldin A Andersson A Vessby B amp AringmanP (2008) Alkylresorcinols as biomarkers of whole-grain wheatand rye intake Plasma concentration and intake estimatedfrom dietary records American Journal of Clinical Nutrition 87832ndash838

Landberg R Marklund M Kamal-Eldin A amp Aringman P (2014) Anupdate on alkylresorcinols ndash Occurrence bioavailabilitybioactivity and utility as biomarkers Journal of FunctionalFoods 7 77ndash89

Landete J M (2012) Plant and mammalian lignans A review ofsource intake metabolism intestinal bacteria and healthFood Research International 46 410ndash424

Leehardt F Lyan B Rock E Boussard A Potus J ChanliaudE amp Remesy C (2006) Wheat lipoxygenase activity inducesgreater loss of carotenoids than vitamin E duringbreadmaking Journal of Agricultural and Food Chemistry 541710ndash1715

Liu L Winter K M Stevenson L Morris C amp Leach D N(2012) Wheat bran lipophilic compounds with in vitroanticancer effects Food Chemistry 130 156ndash164

Liyana-Pathirana C M Dexter J amp Shahidi F (2006)Antioxidant properties of wheat as affected bypearling Journal of Agricultural and Food Chemistry 546177ndash6184

Liyana-Pathirana C M amp Shahidi F (2006) Importance ofinsoluble-bound phenolics to antioxidant properties ofwheat Journal of Agricultural and Food Chemistry 541256ndash1264

Liyana-Pathirana C M amp Shahidi F (2007) Antioxidant and freeradical scavenging activities of whole wheat and millingfractions Food Chemistry 101 1151ndash1157

Lu Y Fuerst P Lv J Yu L Fletcher A Morris C F Kiszonas AM Yu L amp Luthria D (2015) Phytochemicals Profile andAntiproliferative Activity of Dough and Bread Fractions Madefrom Refined and Whole Wheat Flours Cereal Chemistry(accepted)

Lu Y Luthria D Fuerst E P Kiszonas A M Yu L amp MorrisC F (2014) Effect of processing on phenolic composition ofdough and bread fractions made from refined and whole-wheat flour of three wheat varieties Journal of Agricultural andFood Chemistry 62 10431ndash10436

Lu Y Lv J Yu L Bayard A Costa J Yu L amp Luthria D (2014)Phytochemical composition and antiproliferative activities ofbran fraction of ten Maryland-grown soft winter wheatcultivars Comparison of different radical scavenging assaysJournal of Food Composition and Analysis 36 51ndash58

Luterotti S amp Kljak K (2010) Spectrophotometric estimation oftotal carotenoids in cereal grain products Acta ChimicaSlovenica 57 781ndash787

Luthria D L (2012a) Optimization of extraction of phenolicacids from a vegetable waste product using a pressurizedliquid extractor Journal of Functional Foods 4842ndash850

Luthria D L (2012b) A simplified UV spectral scan method forthe estimation of phenolic acids and antioxidant capacity ineggplant pulp extracts Journal of Functional Foods 4 238ndash242

Luthria D L amp Liu K (2013) Localization of phenolic acids andantioxidant activity in sorghum kernels Journal of FunctionalFoods 5 1751ndash1760

Lv J Lu Y Niu Y Whent M Ramadan M F Costa J LuthriaD amp Yu L (2013) Effect of genotype environment and theirinteraction on phytochemical compositions and antioxidantproperties of soft winter wheat flour Food Chemistry 138454ndash462

Mendiola J A Herrero M Cifuentes A amp Ibanez E (2007)Use of compressed fluids for sample preparation Foodapplications Journal of Chromatography A 1152234ndash246

Meneses N Martins S Teixeira J A amp Mussatto S I (2013)Influence of extraction solvents on the recovery ofantioxidant phenolic compounds from brewerrsquos spentgrains Separation and Purification Technology 108152ndash158

Mercadante A Z (2007) Carotenoids in foods Sources andstability during processing and storage In C Socaciu (Ed)Food colorants Chemical and functional properties (pp 213ndash240)FL USA CRC Press (Taylor and Francis Group)

Moore J Hao Z Zhou K Luther M Costa J amp Yu L (2005)Carotenoid tocopherol phenolic acid and antioxidantproperties of Maryland-grown soft wheat Journal ofAgricultural and Food Chemistry 53 6649ndash6657

Moore J Luther M Cheng Z amp Yu L (2009) Effects of bakingconditions dough fermentation and bran particle size onantioxidant properties of whole-wheat pizza crusts Journal ofAgricultural and Food Chemistry 57 832ndash839

Morrison W R Milligan T P amp Azudin M N (1984) Arelationship between the amylose and lipid contents ofstarches from diploid cereals Journal of Cereal Science 2257ndash271

Ndolo V U amp Beta T (2013) Distribution of carotenoids inendosperm germ and aleurone fractions of cereal grainkernels Food Chemistry 139 663ndash671

Nicoletti I Martini D De Rossi A Taddei F DrsquoEgidio M G ampCorradini D (2013) Identification and quantification ofsoluble free soluble conjugated and insoluble boundphenolic acids in durum wheat (Triticum turgidum L vardurum) and derived products by RP-HPLC on a semimicroseparation scale Journal of Agricultural and Food Chemistry 6111800ndash11807

Nurmi T Lampi A M Nystroumlm L Hemery Y Rouau X ampPiironen V (2012) Distribution and composition ofphytosterols and steryl ferulates in wheat grain and branfractions Journal of Cereal Science 56 379ndash388

Nurmi T Nystroumlm L Edelmann M Lampi A M amp Piironen V(2008) Phytosterols in wheat genotypes in the HEALTHGRAINdiversity screen Journal of Agricultural and Food Chemistry 569710ndash9715

Okarter N Liu C S Sorrells M E amp Liu R H (2010)Phytochemical content and antioxidant activity of six diversevarieties of whole wheat Food Chemistry 119 249ndash257

Pananun T Montalbo-Lomboy M Noomhorm A Grewell D ampLamsal B (2012) High-power ultrasonication-assistedextraction of soybean isoflavones and effect of toasting LWT-Food Science and Technology 47 199ndash207

Panfili G Alessandra F amp Irano M (2003) Normal phasehigh-performance liquid chromatography method forthe determination of tocopherols and tocotrienols incereals Journal of Agricultural and Food Chemistry 513940ndash3944

Parker M L Ng A amp Waldron K W (2005) The phenolic acidand polysaccharide composition of cell walls bran layers ofmature wheat (Triticum aestivum L cv Avalon) grains Journal ofAgricultural and Food Chemistry 85 2539ndash2547

Pedersen H Laursen B Mortensen A amp Fomsgaard I (2011)Bread from common cereal cultivars contains an importantarray of neglected bioactive benzoxazinoids Food Chemistry127 1814ndash1820

Peterson J Dwyer J Adlercreutz H Scalbert A Jaques P ampMcCollough M L (2010) Dietary lignans Physiology andpotential for cardiovascular disease risk reduction NutritionReviews 68 571ndash603

Piironen V Lampi A M Ekholm P Salmenkallio-Marttila M ampLiukkonen K H (2009) Micronutrients and phytochemicalsin wheat grain In K Khan amp P R Shewry (Eds) Wheatchemistry and technology (4th ed pp 179ndash222) St Paul MNUSA AACC International

ARTICLE IN PRESS



Poupaert J Carato P amp Colacillo E (2005) 2(3H)-benzoxazoloneand bioisosters as lsquolsquoprivileged scaffoldrsquorsquo in the design ofpharmacological probes Current Medicinal Chemistry 12 877ndash885

Ranhotra G S Gelroth J A Langemeier J amp Rodgers D E(1995) Stability and contribution of beta carotene added towhole wheat bread and crackers Cereal Chemistry 72139ndash141

Rebolleda S Beltraacuten S Sanz M T Gonzaacutelez-Sanjoseacute M L ampSolaesa Aacute G (2013) Extraction of alkylresorcinols fromwheat bran with supercritical CO2 Journal of Food Engineering119 814ndash821

Rezaee M Assadi Y Hosseini M R M Aghaee E Ahmadi F ampBerijani S (2006) Determination of organic compounds inwater using dispersive liquid-liquid microextraction Journal ofChromatography A 1116 1ndash9

Rosa N N Barron C Gaiani C Dufour C amp Micard V (2013)Ultra-fine grinding increases the antioxidant capacity ofwheat bran Journal of Cereal Science 57 84ndash90

Ross A B Bourgeois A Macharia H N Kochhar S Jebb S ABrownlee I A amp Seal C J (2012) Plasma alkylresorcinols as abiomarker of whole-grain food consumption in a largepopulation Results from the WHOLE heart InterventionStudy American Journal of Clinic Nutrition 95 204ndash211

Ross A B Shepherd M J Schuumlpphaus M Sinclair V Alfaro BKamal-Eldin A amp Aman P (2003) Alkylresorcinols in cerealsand cereal products Journal of Agricultural and Food Chemistry51 4111ndash4118

Sampietro D A Jimenez C M Belizaacuten M M Vattuone M A ampCatalaacuten C A (2013) Development and validation of amicromethod for fast quantification of 5-n-alkylresorcinols ingrains and whole grain products Food Chemistry 141 3546ndash3551

Sampietro D A Vattuone M A amp Catalaacuten C A N (2009) Anew colorimetric method for determination ofalkylresorcinols in ground and whole-cereal grains using thediazonium salt Fast Blue RR Food Chemistry 115 1170ndash1174

Sang S amp Zhu Y (2014) Bioactive phytochemicals in wheat branfor colon cancer prevention In R R Watson V Preedy amp SZibadi (Eds) Wheat and rice in disease prevention and healthBenefits risks and mechanisms of whole grains in health promotion(pp 121ndash129) Waltham Massachusetts USA Academic Press

Seo C Yi B Oh S Kwon S Kim S Song N Cho J Park KAhn J Hong J Kim M Lee J amp Park K (2015) Aqueousextracts of hulled barley containing coumaric acid and ferulicacid inhibit adipogenesis in vitro and obesity in vivo Journalof Functional Foods 12 208ndash218

Shahidi F amp Chandrasekara A (2013) Millet grain phenolics andtheir role in disease risk reduction and health promotion Areview Journal of Functional Foods 5 570ndash581

Shammugasamy B Ramakrishnan Y Ghazali H M ampMuhammad K (2013) Combination of saponification anddispersive liquidndashliquid microextraction for thedetermination of tocopherols and tocotrienols in cereals byreversed-phase high-performance liquid chromatographyJournal of Chromatography A 1300 31ndash37

Soupas L Huikko L Lampi A amp Piironen V (2007) Pan-fryingmay induce phytosterol oxidation Food Chemistry 101 286ndash297

Sun X Sun R Fowler P amp Baird M (2005) Extraction andcharacterization of original lignin and hemicelluloses fromwheat straw Journal of Agricultural and Food Chemistry 53 860ndash870

Tanwir F Fredholm M Gregersen P amp Fomsgaard I (2013)Comparison of the levels of bioactive benzoxazinoids indifferent wheat and rye fractions and the transformation ofthese compounds in homemade foods Food Chemistry 141444ndash450

Tiwari U amp Cummins E (2009) Nutritional importance andeffect of processing on tocols in cereals Trends in Food Scienceamp Technology 20 511ndash520

Tluscik F Kozubek A amp Mejbaum-Katzenellenbogen W (1981)Alkylresorcinols in rye (Secale cereale L) grains VI Colorimetricmicromethod for the determination of alkylresorcinols withthe use of diazonium salt Fast Blue B Acta Societas BotanicaPolonica 50 645ndash651

Torchilin V P (2005) Recent advances with liposomes aspharmaceutical carriers Nature Reviews Drug Discovery 4 145ndash160

Tucker A J Vandermey J S Robinson L E Graham T EBakovic M amp Duncan A M (2014) Effects of breads ofvarying carbohydrate quality on postprandial glycaemicincretin and lipidaemic response after first and second mealsin adults with diet-controlled type-2 diabetes Journal ofFunctional Foods 6 116ndash125

Verma B Hucl P amp Chibbar R N (2009) Phenolic acidcomposition and antioxidant capacity of acid and alkalihydrolysed wheat bran fractions Food Chemistry 116 947ndash954

Vinatoru M (2001) An overview of the ultrasonically assistedextraction of bioactive principles from herbs UltrasonicsSonochemistry 8 303ndash313

Wang T He F amp Chen G (2014) Improving bioaccessibility andbioavailability of phenolic compounds in cereal grainsthrough processing technologies A concise review Journal ofFunctional Foods 7 101ndash111

Weipert D amp Al Baya A W (1977) 5-Alky-Resorcin in getreideund getreideprodukten (5-alkylresorcinols in cereals andcereal products Getreide Mehl und Brot 31 225ndash229

Winata A amp Lorenz K (1997) Effects of fermentation andbaking of whole wheat and whole rye sourdough breads oncereal alkylresorcinols Cereal Chemistry 74 284ndash287

Zant-Przeworska E Stasiuk M Gubernator J amp Kozubek A(2010) Resorcinolic lipids improve the properties ofsphingomyelinndashcholesterol liposomes Chemistry and Physics ofLipids 163 648ndash654

Zarnowski R amp Suzuki Y (2004) Expedient Soxhlet extraction ofresorcinolic lipids from wheat grains Journal of FoodComposition and Analysis 17 649ndash663

Zhang Z Li D Wang L Ozkan N Chen X Mao Z amp Yang H(2007) Optimization of ethanolndashwater extraction of lignansfrom flaxseed Separation and Purification 5717ndash24

ARTICLE IN PRESS



carotenoids alkylresorcinols and other phytochemicals (PiironenLampi Ekholm Salmenkallio-Marttila amp Liukkonen 2009)Themajor grains include wheat rice corn oats rye barley sorghumtriticale millet amaranth and teff In Asia nearly half of theannually consumed grain is rice while the major grain con-sumed in Europe and US is wheatThe bioactive phytochemicalsin wheat can be broadly subdivided into the following catego-ries phenolic acids carotenoids tocopherols alkylresorcinolsand other miscellaneous compounds (sterols steryl ferulatesbenzoxazinoids and lignans)

Phenolic acids recognized as simple phenols can be dividedinto two subgroups One is hydroxybenzoic acid derivativeswhich include vanillic syringic p-hydroxybenzoic and gallicacids The other is hydroxycinnamic acid derivatives whichinclude ferulic p-coumaric caffeic and sinapic acids (Luthriaamp Liu 2013 Verma Hucl amp Chibbar 2009) The concentrationof phenolic acids in whole wheat ranges from approximately200 to 1200 mgg dry matter basis (DMB) (Andersson DimbergAman amp Landberg 2014) Ferulic acid is the primary and mostabundant phenolic acid in wheat grain Smaller concentra-tions of p-hydroxybenzoic vanillic syringic o-coumaricp-coumaric salicylic sinapic acids are also present in wheat(Liyana-Pathirana amp Shahidi 2006 Moore et al 2005)

Whole wheat grain also provides moderate sources ofvitamin E The vitamin E includes four tocopherols andtocotrienols Tocopherols have saturated phytyl side chainswhile tocotrienols have isoprenyl side chain with three doublebonds Previous studies have confirmed the presence of α- β-δ- and γ-tocopherols in soft and hard wheat grain (Moore et al2005 Panfili Alessandra amp Irano 2003) The spelt durum andsoft wheat grains from Italy contained vitamin E in a rangefrom 565 to 743 microgg dry weight respectively with 66ndash77tocotrienols (Panfili et al 2003) In addition the concentra-tion of total tocopherols and tocotrienols in whole wheatsamples varied between 276 and 797 microgg (Lampi NurmiOllilainen amp Piironen 2008)

In whole wheat grains color has been most commonly usedas a quality indicator The color is primarily attributed to thepresence of carotenoids and their esters The concentration oftotal carotenoids in whole wheat ranged from 08 to 217 microgg(Moore et al 2005) Lutein and zeaxanthin were the mostpredominant carotenoids present in whole wheat with con-centrations of 05ndash144 and 02ndash039 microgg grain respectively(Moore et al 2005) In addition lower concentrations of ca-rotenoids including β-cryptoxanthin at 001ndash013 microgg andβ-carotene at 009ndash021 microgg have also been detected in wheat

Phenolic lipids also known as alkylresorcinols (ARs)are commonly present in wheat (Gunenc HadinezhadTamburic-Ilincic Mayer amp Hosseninian 2013) Structurally theARs are similar to tocopherols except for the presence of astraight aliphatic hydrocarbon side chain and a single pheno-lic ring The alkyl side chain may contain between 13 and 27carbon atoms 5-N-alkylresorcinols 5-alkenylresorcinols5-oxoalkylresorcinols 5-oxoalkenylresorcinols and5-hydroxyalkenylresorcinols are the five major classes of ARsreported in the whole wheat ARs in whole wheat range from489 to 1429 microgg (Ross et al 2003)

Other bioactive phytochemicals have also been extractedand identified in whole wheat In this review they are groupedtogether as a ldquomiscellaneousrdquo group The miscellaneous group

consists of benzoxazinoids lignans phytosterols and sterylferulates Benzoxazinoids are a group of potent natural com-pounds which have recently been identified in whole grainwheat and rye grains and in bakery products of these cereals(Hanhineva et al 2011 Pedersen Laursen Mortensen ampFomsgaard 2011) The total benzoxazinoids content in wheatis very low (5 microgg DMB) Phytosterols are isoprenoid com-pounds which are biosynthetically derived from squalenePhytosterols are mostly found in free or esterified forms in-cluding esters of fatty acids and phenolic acids as well asglycosides or acylated glycosides According to the study ofNurmi Nystroumlm Edelmann Lampi and Piironen (2008) thecontent of phytosterols range is 670ndash959 microgg DMB in wholegrain winter wheat and 797ndash949 microgg DMB in spring wheat Aportion of the phytosterols occurs in ferulic acid ester formie as steryl ferulates

This contribution provides an overview of bioactivephytochemicals such as phenolic acids carotenoids tocoph-erols alkylresorcinols and other miscellaneous bioactivecompounds that are commonly found in wheat grain (Table 1)The distribution of bioactive phytochemicals their extrac-tion analysis changes during processing varietal differencesand functional properties will be presented and discussed

2 Distribution of bioactive phytochemicalsin wheat

All whole wheat grain products contain bran germ and en-dosperm fractions It is well documented in the publishedliterature that the bioactive phytochemicals are not uni-formly distributed Germ and bran fractions generally containhigher concentration of bioactive phytochemicalsThis is evidentfrom number of pearling studies described in the literature(Blandino et al 2013 Liyana-Pathirana Dexter amp Shahidi 2006)Liyana-Pathirana et al (2006) studied the effect of sequentialremoval of the outermost layers (pearling) on the phenolic com-position of wheat grains Higher levels of phenolic compoundshave been reported in the bran fraction as compared to therefined grain fraction that primarily comprises endosperm Ina recent study it was reported that most of the phenolic acidsexisted in bound insoluble form (80) as compared to freesoluble form and high concentrations of phenolic acids weredetected in whole grain which contains the bran fraction (6278ndash7456 microgg dry weight) as compared to the refined grain (660ndash970 microgg dry weight) (Lu Fuerst et al 2014)

The concentration of total carotenoids in wheat ranges from07 microgg in durum wheat from Spain to as high as 136 microgg inEinkorn accessions from Italy The highest concentrations ofcarotenoids are observed in the germ fraction followed by thebran and endosperm fractions Similarly tocopherols andtocotrienols are not uniformly distributed in grain Higher con-centrations of tocopherols are found in the germ or the outerlayers and the concentration of tocopherols in the endo-sperm fraction is comparatively lower Similarly tocotrienolsare found at higher concentrations in outer layer of the grain(85 pericarp testa and aleurone) and only 15 in the en-dosperm fraction (Piironen et al 2009)

In a recent study Tanwir Fredholm Gregersen andFomsgaard (2013) investigated the levels of benzoxazinoids in

ARTICLE IN PRESS






Phytochemicalname

Example Structure

Phenolicacids


Ferulic acid

HO

H3CO

OH

O


alpha-Tocopherol

H3C O

CH3CH3

CH3

OH

CH3

CH3 CH3 CH3


HO

H

OH

Phenoliclipids


R

OH

HO

Miscellaneousgroups


N

OO OH

O

OH


OH

OH

MeO

OH

HO


HO


HO

O

H

OMe

O

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Serialno


References






















Ge Yan et al 2002



















Sampietro et al2013










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Serialno


References














Nurmi et al 2012





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Serialno



Lu Lv et al 2014


Nicoletti et al2013



Dinelli et al2009










Lu Lv et al 2014




9 HPLC andNMR














Hanhineva et al2011


Nurmi et al2008


Hakala et al2002



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Serialno


References






+ Hemery et al 2010















































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7 Conclusions


R E F E R E N C E S


























ARTICLE IN PRESS




































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Phytochemicalname

Example Structure

Phenolicacids


Ferulic acid

HO

H3CO

OH

O


alpha-Tocopherol

H3C O

CH3CH3

CH3

OH

CH3

CH3 CH3 CH3


HO

H

OH

Phenoliclipids


R

OH

HO

Miscellaneousgroups


N

OO OH

O

OH


OH

OH

MeO

OH

HO


HO


HO

O

H

OMe

O

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Serialno


References






















Ge Yan et al 2002



















Sampietro et al2013










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Serialno


References














Nurmi et al 2012





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Serialno



Lu Lv et al 2014


Nicoletti et al2013



Dinelli et al2009










Lu Lv et al 2014




9 HPLC andNMR














Hanhineva et al2011


Nurmi et al2008


Hakala et al2002



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Serialno


References






+ Hemery et al 2010















































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7 Conclusions


R E F E R E N C E S


























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Ge Yan et al 2002



















Sampietro et al2013










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Serialno


References














Nurmi et al 2012





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Serialno



Lu Lv et al 2014


Nicoletti et al2013



Dinelli et al2009










Lu Lv et al 2014




9 HPLC andNMR














Hanhineva et al2011


Nurmi et al2008


Hakala et al2002



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Serialno


References






+ Hemery et al 2010















































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7 Conclusions


R E F E R E N C E S


























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Ge Yan et al 2002



















Sampietro et al2013










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Nurmi et al 2012





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Nicoletti et al2013



Dinelli et al2009










Lu Lv et al 2014




9 HPLC andNMR














Hanhineva et al2011


Nurmi et al2008


Hakala et al2002



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+ Hemery et al 2010















































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Nurmi et al 2012





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Serialno



Lu Lv et al 2014


Nicoletti et al2013



Dinelli et al2009










Lu Lv et al 2014




9 HPLC andNMR














Hanhineva et al2011


Nurmi et al2008


Hakala et al2002



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+ Hemery et al 2010















































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Lu Lv et al 2014


Nicoletti et al2013



Dinelli et al2009










Lu Lv et al 2014




9 HPLC andNMR














Hanhineva et al2011


Nurmi et al2008


Hakala et al2002



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+ Hemery et al 2010















































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Lu Lv et al 2014


Nicoletti et al2013



Dinelli et al2009










Lu Lv et al 2014




9 HPLC andNMR














Hanhineva et al2011


Nurmi et al2008


Hakala et al2002



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+ Hemery et al 2010















































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+ Hemery et al 2010















































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Bioactive phytochemicals in wheat: Extraction, analysis, processing, and functional properties

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Transcript of Bioactive phytochemicals in wheat: Extraction, analysis, processing, and functional properties