Detection of the lunasin peptide in oats (Avena sativa L)

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    Received in revised form3 December 2012Accepted 3 December 2012


    and other plant sources (De Lumen, 2005; Jeong et al., 2002, 2003,2007, 2009, 2010; Nakurte et al., 2012). It is hypothesized thatlunasin might be an effector molecule that allows arrest of celldivision and initiates the second stage of seed development. It issuggested that lunasin provides a regulation of endoreduplication

    y, bioactivity andation indicate theunasin-containing

    products in the human diet (Jeong et al., 2010; Park et al., 2005).Oats belong to the Poaceae family which is the same family as

    the other grain species that contain lunasin. This study aimed todetermine if lunasin is present in oats and if so, at what levels.Previous studies have shown soy, wheat, barley, rye and triticalegenotype-dependent variations in the lunasin content. Therefore,we decided to study different oat genotypes and to monitor lunasinlevel during two years. Grain from the harvests of years 2010 and2011 grown in the eld trial of the conventional breeding program

    * Corresponding author. Tel.: 371 67362499; fax: 371 67366306.

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    Journal of Cereal Science xxx (2013) 1e6E-mail address: (R. Muceniece).associated with insulin secretion and b-cell development, proteinsynthesis and genes related to cancer diseases (Ulmius et al., 2011).

    Lunasin is a peptide that has been isolated from soybean, cereals

    2009). Findings obtained on the bioavailabilitthermostability of lunasin after oral administrreasons for recommending the inclusion of lOat (Avena sativa L) is distinct among the cereals due to itsmultifunctional characteristics and its nutritional prole (Butt et al.,2008). Recent advancements in food and nutrition have revealedthe cholesterol-lowering effects of oat dietary ber and b-glucans(Czerwinski et al., 2004; El Khoury et al., 2012; Othman et al., 2011).Oat and oat by-products are used as complementary treatments forpatients with diabetes and cardiovascular diseases. Recently,ingestion of oat bran in a meal has been shown to affect gene sets

    environmental factors, particularly temperature, on lunasin con-centration are also reported (Wang et al., 2008). The authors sug-gest that lunasin content can be improved by plant breeding andoptimization of growing conditions as well as by selecting geno-types with appropriate seed germination time (Paucar-Menachoet al., 2010). Researchers are interested in lunasin for its anti-cancer, antioxidant and anti-inammatory properties. Moreover,studies in animals have shown that lunasin can be administeredorally and can enter target tissues (De Lumen, 2005; Jeong et al.,LunasinOat genotypesLCeMS/MS

    1. Introduction0733-5210/$ e see front matter 2013 Elsevier Ltd.

    Please cite this article in press as: Nakurte,, rye, wheat, triticale). Lunasin was detected in oats using LCeMS/MS analysis. The chromatogramsand mass spectra of lunasin isolated from ve oat genotypes were compared with those of the syntheticlunasin peptide. We measured the lunasin content in harvests of two years and found that all tested oatgenotypes contained the lunasin peptide. However, we observed genotype-related uctuations in thelunasin content. Notably, the middle early oat variety Ivory contained the highest and the most stablelunasin level at 0.197 0.01 mg per g of grain in year 2010 and 0.195 0.009 mg per g of grain in 2011.We also characterized the selected oat genotypes by measuring the contents of protein, b-glucans, fat,starch and moisture in the grains. However, we did not nd correlation between lunasin and protein, andb-glucan content. Lunasin isolated from oat showed similar to the synthetic lunasin antioxidant effects.The detection of lunasin complements a list of bioactive compounds present in oats and strengthensrecommendations to use oat products.

    2013 Elsevier Ltd. All rights reserved.

    of DNA (De Lumen, 2005). The effects of soybean cultivar andArticle history:Received 28 June 2012We report the rst discovery of lunasin in oats (Avena sativa L). Lunasin is a novel cancer preventive, anti-inammatory and cholesterol-reducing peptide originally isolated from soy and later found in cerealsDetection of the lunasin peptide in oats

    Ilva Nakurte a, Inga Kirhnere d, Jana Namniece c, KrZaiga Vicupe e, Mara Bleidere e, Linda Legzdina d, Ra Faculty of Biology, University of Latvia, Kronvalda Blvd 4, Riga, Latviab Faculty of Chemistry, University of Latvia, Kr. Valdemara Str. 48, Riga, Latviac Faculty of Medicine, University of Latvia, Sarlotes Str. 1a, Riga LV-1001, Latviad State Priekuli Plant Breeding Institute, Zinatnes Str. 1, Priekuli LV-4126, Latviae State Stende Cereal Breeding Institute, Dizzemes, Dizstende LV 3258, Latvia

    a r t i c l e i n f o a b s t r a c t

    journal homepage: wwAll rights reserved.

    I., et al., Detection of the lunavena sativa L)

    ne Saleniece c, Liga Krigere c, Peteris Mekss b,Muceniece c,*

    SciVerse ScienceDirect

    eal Science

    elsevier .com/locate/ jcssin peptide in oats (Avena sativa L), Journal of Cereal Science (2013),

  • Cer(Latvia) was used in this study. Additionally, we evaluated anti-oxidant properties of lunasin peptide isolated from oat.

    2. Experimental

    2.1. Materials and reagents

    Acetonitrile, methanol (gradient grade), hexane (gradientgrade), formic acid (99%), triuoroacetic acid (99%), dimethylsulfoxide (DMSO), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), hydrogenperoxide (H2O2), phosphate-buffered saline (PBS) and a proteaseinhibitor cocktail were purchased from SigmaeAldrich (St. Louis,USA). The water used was puried by a Milli-Q water puricationsystem from Millipore (Billerica, Massachusetts, USA). Standardsynthetic lunasin was purchased from CASLO Laboratory ApS(Technical University of Denmark, Denmark). Working solutionswere prepared before the samples were analyzed. The standardaddition method was used to prevent matrix effects by diluting thestock solution with a sample solution containing a known lunasinconcentration. The stock solution of the standard (40 mg/mL) wasprepared by dissolving the peptide in buffer, and the resulting so-lution was stored at 4 C.

    2.2. Instrumentation

    Chromatographic analysis was performed on a modular HPLCsystem, Waters 2690 Alliance (Waters Corporation, Milford, MA,USA), equipped with a quaternary pump, an autosampler anda column thermostat coupled to an electrospray ionisation tandemmass spectrometer (Waters Micromass Quattro Micro API). HPLCseparations were performed on a reverse-phase PhenomenexSynergi Hydro-RP analytical column (4 mm, 150 2.0 mm I.D.) at30 C with a mobile phase composed of a mixture of 0.1% formicacid in water (A) and 0.1% formic acid in acetonitrile (B). Usinga ow rate of 300 mL/min, the separation of the peptide wasaccomplished with a linear gradient of 20e60% B over 8.0 min. Thefollowing gradient was used: 60% B for 3.0 min, 60e20% B over1.0 min, and 20% B for 8.0 min until the initial conditions werereached. The injection volume was 50 mL.

    The quadruple-protonated molecular ion with m/z 1258 forlunasin was detected by single-ion recording (SIR). Mass spectrawere acquired with a Micromass Quattro Micro triple-quadrupolespectrometer equipped with an ESI source. Analyses were per-formed in the positive-ion mode. The source temperature was120 C, and the desolvation temperature was 250 C. Nitrogen wasused as the nebulising gas (600 L/h), and the ES capillary was set to3.0 kV. MS analyses were performed using a cone voltage of 60 V.Data analyses were performed usingMassLynx version 4.1 software(Waters Corporation, Milford, MA, USA).

    Additionally, the HPLC puried fraction of lunasin (acetonitrile:water 1:1) was collected and concentration of lunasin determinedaccording to the synthetic lunasin standard curve. Lunasin isolatedfrom oat was used to assay its antioxidant effects.

    2.3. Lunasin content measurements

    Reversed-phase chromatography coupled to an electrosprayionisation source was used to separate and ionise lunasin. Todetermine the identity of the peptide, we performed electrosprayionization mass spectrometry (ESI-MS). The quadruple protonatedmolecular ion withm/z 1258 for lunasin was detected by single-ionrecording (SIR). Based on this method, a lunasin containing samplesolution was puried and introduced into MS by a syringe pump to

    I. Nakurte et al. / Journal of2yield a constant ow of solution (30 mL min1).

    Please cite this article in press as: Nakurte, I., et al., Detection of the luna grain was ground using a Falling Number Laboratory Mill3100 with a 0.5 mm sieve. 5 g of our were extracted with 50 mL of0.1 M PBS buffer, pH 7.4 and supplemented with fresh proteaseinhibitor cocktail (Sigma, St. Louis, MO, USA) at a concentration of1% v/v by stirring with a magnetic stir bar for 48 h at 4 C. To isolatelunasin, we used an assay similar to that of Jeong et al. (2007) withour LCeMS/MS modications (Nakurte et al., 2012). All experi-ments were performed in triplicate. A calibration curve was con-structed by plotting the average peak area against concentration,and a regression equation was computed. The assay provideda linear response over a wide range of concentrations (0e38 mg/mL). The limit of detection (0.3 ng/mL) and limit of quantication(1.0 ng/mL) were determined from the calibration curves. A mix-ture of standard solutions was injected six times and the corre-sponding peak areas were recorded. The relative standarddeviation was determined to be less than 1%. The high percentageof lunasin recovery demonstrates the accuracy of the method.

    2.4. Grain sample characterisation

    The following oat genotypes were included in the investigation:Laima, Arta, Stendes Liva (bred in Latvia) and Ivory (Germany)varieties and the breeding line S-1560 (Latvia). The crude protein, b-glucans, crude fat and starch content in dry matter and moisturecontent were determined in grain samples before grinding usinga Near Infrared Transmittance Infratec 1241 Analyzer.

    2.5. Radical scavenging assay

    The HPLC puried fraction of lunasin (acetonitrile: water 1:1)was collected and concentration of lunasin determined accordingto the synthetic lunasin standard curve. Synthetic lunasin andlunasin puried from oat at varying concentrations (0.1; 1; 10; and100 mM) was tested in DPPH radical scavenging assay according tothe published method (Tsai et al., 2009). In brief, stock solution ofDPPH was prepared in methanol, whereas acetonitrile was used assolvent control in the oat extracted lunasin assay. 25 mL of samplewas pipetted into each well of 96-well plate, and 175 mL of 0.15 mMDPPHmethanolic solution added. The mixturewas shaken 20 s andleft in the dark on ice for 30 min. The absorbance was read spec-trophotometrically at 517 nm with Bio-Tek, PowerwaveX340, Bos-ton, MA apparatus. The inhibition potency was calculated in %as follows: ((ADPPH Ablank) (Asample Asample blank))/(A DPPH Ablank) 100, where ADPPH is the absorbance of the DPPHsolution, Ablank is the absorbance of methanol,

    Asample is the absorbance of the DPPH solution in the presence ofthe sample, and A sample blank is the absorbance of methanol in thepresence of the sample.

    2.6. Cell culturing

    Human embryonic kidney HEK 293 cell line was purchased fromthe American Type Culture Collection (ATCC, Manassas, VA, catalogNo CRL-1573). The cells were cultivated in Dulbeccos ModiedEagles medium (DMEM) supplemented with 10% fetal bovineserum, 100 units/mL of penicillin and 100 mg/mL of streptomycin ina CO2 cell incubator at 37 C. The cells were plated at appropriatedensity according to each experiment in 96-well sterile clear-bottom plates or 60 mm dishes. Experiments were done whencells reached 80e90% conuence.

    2.7. Cell proliferation assay

    HEK 293 cells were treated with synthetic lunasin and lunasin

    eal Science xxx (2013) 1e6puried from oat at concentrations of 0.1; 1; 10; 50 and 100 mM.

    sin peptide in oats (Avena sativa L), Journal of Cereal Science (2013),

  • After 24 h, the cell mediumwas removed and 10 mL of MTT solution(5 mg/mL in PBS) was added to each well of 96-well plate and thecells were incubated 2 h at 370 C under a 5% CO2 atmosphere. Thencells were washed with PBS and 100 mL of DMSO was added todissolve the formed formazan crystals. After 30 min of stirring, theabsorbance was measured at 560 nm using amicroplate reader (Elx808, BioTek Instruments, Inc) and results calculated with Gen5software. Experiments were performed in triplicate in three inde-pendent experiments. The results were expressed as percentage ofthe number of untreated cell control taken as 100%. Additionally,cell morphology was observed using an inverted contrasting mi-croscope (Leica, Solms, Germany).

    2.8. Measurement of glutathione peroxidase activity

    Glutathione peroxidase activity (GPx) was measured witha Cayman Chemical Company, Ann. Arbor, MI Glutathione perox-idase assay kit. No 703102. In brief, HEK 293 cells in 60 mm disheswere treatedwith synthetic lunasin and lunasin puried fromoat atconcentrations of 0.1; 1; 10 and 50 mM for 2 h. Then the cells wereharvested using a rubber policeman. The obtained cell pellet washomogenized in cold assay buffer supplied by the manufacturer.Then cells were centrifuged at 10,000 g for 15 min at 4 C andsupernatant removed for the enzyme assay according to the man-ufacturer instruction. Enzymatic reaction was initiated by adding20 mL of cumene hydroperoxide and after that, 96-well microplatewas mixed for a few s. Absorbance was measured once every minduring 5min at 340nmandGPx activity calculated as nmol/min/mL.

    software (Graph Pad Inc., USA). Data were compared by one-wayanalysis of variance (ANOVA) followed by Bonferronis multiplecomparison test with equal sample size and Students t-test. A Pvalue less than 0.05 was taken as statistically signicant.

    3. Results

    3.1. Lunasin content in oat

    Using LCeMS/MS, lunasin was fully separated in 20 min witha symmetrical peak at 8.8 min. The chromatogram of standardlunasin peptide and a representative chromatogram of the oatsample S-156 are shown in Fig. 1. The chromatograms of the othergenotypes also exhibited lunasin peaks that were clearly visible andESI-MS/MS assay conrmed identity of lunasin in oat. The ESI-MS/MS spectrum fromm/z 500 tom/z 1500 of lunasin peptide is shownin Fig. 2 wherem/z 1111.3 corresponds to sequence GDDDDDDDDDandm/z 595.1 to sequence PCEKH. Quadruple protonatedmolecularion with m/z 1258 for lunasin was detected by single-ion recording(SIR). Calculated and matched b and y ions conrmed the presenceof C-terminal amino acid sequence GDDDDDDDDD in the lunasinisolated from oat.

    The molecule of lunasin is composed of several hetero atomswhich help to bind proteins during the electrospray process. On-line calculator provided by the Institute of Systems Biology wasused to calculate a mass-charge ratio of molecular ions as well asa protein mass of the lunasin isolated from oat. For example, signalion (M 4H)4 with m/z 1256.7 multiplied by 4 gave a peptide

    I. Nakurte et al. / Journal of Cereal Science xxx (2013) 1e6 32.9. Data analysis

    Unless otherwise indicated, the data were calculated as themean standard deviation (S.D.) of three different measurements.The data were analyzed using Graph Pad Prism 3.0 statisticalFig. 1. LC-ESI-MS chromatogram of the synthetic lunasin peptide (A) and o

    Please cite this article in press as: Nakurte, I., et al., Detection of the luna of 5026.8 Da. Therefore, we suggest that the oat lunasin re-sembles here used standard synthetic 43 amino acids long lunasinpeptide with monoisotopic mass of 5025.23 Da.

    In addition to identifying lunasin in all ve tested oat genotypes,we measured the lunasin content during two consecutive years(Table 1). There was a notable difference between the lunasinf lunasin extracted from the grain of the oat breeding line S-156 (B).

    sin peptide in oats (Avena sativa L), Journal of Cereal Science (2013),

  • In year 2010 we measured the highest protein content in the

    I. Nakurte et al. / Journal of Cer4breeding line S-156 followed by the varieties Arta, Stendes Liva,Ivory and Laima. In year 2011 the order of the highest crudeprotein content was a little changed as follows: S-156, Arta,Stendes Liva, Laima and Ivory. However, protein content did notcorrelate with the lunasin content of each oat genotype.

    The crude fat content in the oats was measured in the followingorder in the bothyears: S-156> Laima> Arta> Ivory> StendesLiva. However, in the harvest of year 2011 we observed approx-imately an 18% decrease in the average fat content of all tested oatgenotypes.content of the oat genotypes, as it ranged from 0.064 to 0.197 mg/gin year 2010 and from 0.034 to 0.195 mg/g in year 2011. The datademonstrate that the most lunasin-rich is the Ivory variety fol-lowed by the breeding line S-156 and the variety Arta. Lunasincontent in the Ivory variety was approximately the same in bothyears. However, in the harvest of year 2011 we observed a decreasein the lunasin production in the varieties Laima (approximately58%) and Stendes Liva (approximately 13%) whereas, in the Artavariety and breeding line S-156, we noticed a small increase.

    3.2. Content of crude proteins, fat, starch and b-glucans in the oatgenotypes

    The oat genotypes were characterised by measuring the crudeprotein, crude fat, starch, b-glucans and moisture content (Table 2).

    Fig. 2. ESI-MS analysis of the puried lunasin from oats. C-terminal fragment of thepeptide at interval from m/z 500 to m/z 1500 is shown and signal ion with m/z 1111.3corresponds to the amino acid sequence GDDDDDDDDD.The Ivory variety was the most starch-rich (49.2% in 2010 and47.7% in 2011) followed by the varieties Stendes Liva, Laima andArta and the breeding line S-156.

    Content of b-glucans ranged from 3.3% in the Stendes Liva va-riety to 4% in the Laima variety in year 2010 and from 3.1% in theStendes Liva variety to 4.63% in the S-156 breeding line. Thelunasin-rich Ivory variety contained moderate levels of b-glucans,and its lunasin content did not correlate with its b-glucans content.

    Table 1Average values of content of lunasin in oat genotypes.

    Number Genotype Lunasin mg/g grain

    2010 2011

    1 Laima 0.081 0.005 0.034 0.0022 Arta 0.084 0.003 0.117 0.0043 Stendes Liva 0.064 0.007 0.056 0.0034 Ivory 0.197 0.010 0.195 0.0095 S-156 0.091 0.005 0.117 0.004

    Data shown as mean S.D, n 3 repeats for each genotype.

    Please cite this article in press as: Nakurte, I., et al., Detection of the luna content also varied such that the highest value wasmeasured in the Stendes Liva variety and the lowest in 2010 in theLaima variety whereas in 2011 the lowest moisture content wasmeasured in the breeding line S-156. On average, in all oat geno-types, moisture content was increased approximately by 26% inyear 2011 in comparison with that in 2010.

    3.3. Antioxidant activity of lunasin

    The absorbance of DPPH without any additions was stable over30 min. As seen in Fig. 3A, radical scavenging activity of the lunasinpuried from oat did not differ from that of the standard lunasin.Both peptides showed statistically signicant inhibitory effect ata concentration of 1e100 mM.

    The cell proliferation data showed that synthetic lunasin andpuried from oat lunasin at a concentration up to 50 mM did notchange the cell number during 24 h whereas the lunasin at a con-centration of 100 mM decreased HEK 293 cell proliferation by 20%.Therefore, in the GPx assay, the peptides concentration of 50 mMwas used as the highest concentration. The synthetic lunasin andlunasin isolated from oat in a similar way increased GPx activity(Fig.3B). Statistically signicant effect was observed at concentra-tions of 10 and 50 mM.

    4. Discussion

    Obtained results conrm our hypothesis that oats containlunasin. Recently, detailed studies on soy-derived lunasin haverevealed an asparagine residue at the C-terminal of the peptidewith monoisotopic mass of 5139.25 Da (Seber et al., 2012). Our goalwas not to specify the sequence of the oat lunasin. However, weanalyzed MS spectra of the causing debate C-terminal amino acidsequence of the lunasin isolated from oat and did not nd an extraamino acid residue in comparison with MS spectra of the synthetic43 amino acids long lunasin. Moreover, we calculated the mass ofthe oat peptide to be close to that reported earlier for 43 amino acidlong lunasin (De Mejia et al., 2009; Dia et al., 2009). However, weaccept the possibility that our calculations are less precise than themethods used by Seber et al. and may not detect a 0.1 Da differencein peptidemass. Up to now there is no data that length of peptide C-

    Table 2Content of crude protein, fat, starch, b-glucans and moisture in oat genotypes (%).

    Genotype Proteins Fat Starch b-Glucans Moisture

    2010/2011 2010/2011 2010/2011 2010/2011 2010/2011

    Laima 9.9/11.1 7.8/5.8 46.4/45.6 4.0/3.6 7.5/11.8Arta 14.7/12.1 6.6/5.3 43.8/46.5 3.5/3.4 8.8/12.1Stendes Liva 13.7/11.2 5.0/4.9 47.8/47.3 3.3/3.1 12.4/12.8Ivory 10.5/10.8 5.1/5 49.2/47.7 3.6/3.1 8.6/12.1S-156 16.1/16 12.2/9 35.7/34.9 3.63/4.63 9.8/10.7

    eal Science xxx (2013) 1e6terminal inuences the bioactivity of lunasin.Herein, we provide the rst report of the presence of lunasin

    peptide in oat and show genotype-related variations during dif-ferent years. Lunasin is a peptide that was initially isolated from theprotein fraction of soybeans and up to now soy is found to be themost lunasin-rich plant. Nowadays the presence of the lunasinpeptide has been reported in many soybean varieties with con-centrations ranging from 4.4 to 70.5 mg/g of protein or 0.5e8.1 mg/g of seed (De Lumen, 2005; De Mejia et al., 2004; Hernandez-Ledesma et al., 2009a (review)). The investigation of large num-ber of soy varieties has given information how widely the contentof lunasin may change. The results on the effect of soybean cultivarand environmental factors on lunasin concentration are also

    sin peptide in oats (Avena sativa L), Journal of Cereal Science (2013),

  • xtraars)nitr

    Cerreported. The highest lunasin concentration, 11.7 0.3 mg/g our,was found in Loda soybean cultivar grown at 23 C; the lowestconcentration, 5.4 0.4 mg/g our, was found in Imari soybeancultivar grown at 28 C. It was found that lunasin production in soycultivars depends on environmental temperature during germi-nation time (Paucar-Menacho et al., 2010; Wang et al., 2008).

    In Europe, soybeans are not as popular in the human dietcompared to cereals, such as barley, wheat, rye and oats. Wedetected lunasin in different oat genotypes and conrmed this bymonitoring lunasin production during two years. The following vedistinct oat genotypes were included in the investigation: thehulled oat varieties Laima, Arta, Stendes Liva and Ivory, and thenaked breeding line S-1560. Themiddle-latematuring, high yieldingand lodging resistant variety Laima is the most widely grown oatin Latvia. Arta is an early-ripening variety with high grain quality(low hull content) and protein and fat content. Stendes Liva isa late maturing, tall-height feed oat variety suitable for grain andforage. The middle early oat variety Ivory showed balancedagronomic features and good quality under the conditions in Latvia.The naked oat breeding line S-156 has the potential for high yields,excellent threshability and grain quality. In our study, the middleearly oat variety Ivory showed the most stable production oflunasin during two harvests whereas the middle-late maturingvariety Laima in year 2011 produced approximately 52% lesslunasin compared with that in 2010. The lunasin content in theearly-ripening variety Arta and the late maturing variety Stendes

    Fig. 3. DPPH radical scavenging activity (A) of synthetic lunasin (cycles) and lunasin econtrol. (B). Effect of synthetic lunasin (white bars) and lunasin isolated from oat (gray bcontrol with buffer, #P 0.05 vs. control with correspondingly to sample diluted aceto

    I. Nakurte et al. / Journal ofLiva was less variable. In studies on soybeans, light and dark con-ditions were found not to inuence the amount of the lunasinproduced (Park et al., 2005), but cultivar and growth conditions,such as soil moisture and temperature, did affect lunasin content inseeds (Wang et al., 2008). As all oat genotypes were grown bya similar conventional agricultural way, we suggest the cultivarreaction to environmental temperature during germination time.Despite the uctuations in the content of the lunasin in oat, thisamount is comparable to that in barley, wheat and rye. The lunasincontents of 9 barley cultivars are reported to be from 0.0127 to0.099 mg/g of seed (Jeong et al., 2010) and 5.9e8.7 mg/g of protein(Hernandez-Ledesma et al., 2009a). In developing wheat seeds,lunasin was detectable at a level of about 0.0284e0.290 mg oflunasin/g of seed (Jeong et al., 2007). Jeong et al. (2009) rstreported the existence of lunasin in rye cultivars. Lunasin waspresent in 15 out of 21 cultivars of rye analyzed, and the lunasincontent was 0.05e0.150 mg/g of seed on average. Recently we havefound lunasin in triticale where lunasin was present at a level ofabout 0.429e6.458 mg/g of seed (Nakurte et al., 2012). Thechanging lunasin content measured within the different species of

    Please cite this article in press as: Nakurte, I., et al., Detection of the luna and cereals indicates that the levels of this important bioactivepeptide can be genetically manipulated. Recently, lunasin has beenreported to be present in different non cereal plants e.g. Amaranth,a traditional Mexican plant (9.5e12.1mg of lunasin per g of protein)(Silva-Sanchez et al., 2008), in seeds of the Solanaceae family plants(approximately 22e36 mg of lunasin per g of protein) (Hernandez-Ledesma et al., 2009a; Jeong et al., 2007a), winter cherry (17 mg oflunasin/g protein) and Jimson weed (10.3 mg of lunasin/g protein)(Hernandez-Ledesma et al., 2009a).

    Lunasin was found at an average concentration of 32.8 mg/gkidney and 3.5 mg/g liver of lunasin enriched barley fed rats (ratsreceived 300 mg of lunasin during 4 weeks) (Jeong et al., 2010).Thus, it was concluded that lunasin present in barley is bioavailableand bioactive. However, large-scale animal studies and humanclinical trials to determine the efcacy of lunasin in vivo have beenhampered by the cost of synthetic lunasin. Therefore, very prom-ising is the recently published method of isolation of 99% purelunasin from soywith a yield of 442mg/kg defatted soy our (Seberet al., 2012) and, moreover, recombinant production of lunasin(Kyle et al., 2012).

    Protein content of the hull-less oat kernel (groat) ranges from 12to 24% (Butt et al., 2008). Here, we measured on average similarprotein content. However, we did not observe correlation betweenlunasin and protein content. We measured content of protein, fatand starch mainly to analyze the nutritional prole of oat geno-types in parallel with contents of lunasin and b-glucans which are

    cted from oat (squares). *P 0.05 vs. absorbance of DPPH without peptides taken ason activity of GPx in HEK 293 cells after 2 h treatment with the peptides. *P 0.05 vs.ile (solvent control).

    eal Science xxx (2013) 1e6 5bioactive substances with proved pharmacological effects. Co-existence of lunasin and b-glucans in oat could be preferable foroat genotype selection for breeding. Wemeasured the content of b-glucans in oats to be an average of 3.1e4.63%. These values arecomparable with those from other studies, demonstrating that di-etary ber and b-glucan content do vary between different oatgenotypes. However, a typical b-glucan concentration of approx-imately 4% dry weight per g of grains is maintained (Manthey et al.,1999). Signicant scientic data support the regulatory inuence ofdietary ber and oat b-glucans on blood cholesterol levels (Charltonet al., 2011; Othman et al., 2011; Manthey et al., 1999). Moredetailed studies have shown that the ability of oat and barleyproducts to attenuate postprandial glycemic and insulinemicresponse is related to content of (1 / 3)(1 / 4)-b-D-glucan (b-glucan) (Wood, 2007).

    In addition to exhibiting anti-cancer activity, lunasin is alsoa valuable peptide for the cardiovascular system. Lunasin has beenreported to lower serum LDL cholesterol levels by selectively dis-rupting a necessary step in the production of a key enzyme, HMG-CoA reductase, and by upregulating the expression of the

    sin peptide in oats (Avena sativa L), Journal of Cereal Science (2013),

  • CerLDL-receptor gene (Hernandez-Ledesma et al., 2009a). Synthetic 43amino acids long lunasin possesses antioxidant properties as itshows linoleic acid oxidation inhibitory, free radical scavenging(ABTS radical) activities and reactive oxygen species (ROS) leveldecreasing effect in lipopolysaccharides-activated macrophages(Hernandez-Ledesma et al., 2009b).

    Several authors have proved bioavailability and bioactivity oflunasin or lunasin-like peptides isolated from non-cereal plants.Indeed, lunasin-like peptides puried from Amaranth inhibitedchemical carcinogen-induced transformation of NIH-3T3 cells tocancerous foci (Maldonado-Cervantes et al., 2010) whereas lunasinpuried from Solanum nigrum has been found to protect againstoxidative DNA damage (Jeong et al., 2010a). Herein, we comparedantioxidant activity of the lunasin puried from oat with that of thesynthetic lunasin. We found antioxidant activity of the peptides inDPPH radical scavenging assay and GPx activity test in HEK 293cells. Effects of the lunasin puried from oats did not differ fromthat of the synthetic lunasin.

    We measured the highest lunasin content in the Ivory varietybut the Laima variety and the breeding line S-156 were the mostb-glucans-rich. Thus, we cannot recommend the most healthy oatgenotype based on lunasin and b-glucans content.

    Oats are considered as high level lipid containing cereals (up to18% oil content) (Banas et al., 2007) and as valuable protein cereals(Butt et al., 2008). In our study, the most fat, protein and b-glucanrich was naked oat breeding line S-156, however, with moderatecontent of the lunasin. Probably, because the amount of testedsamples was too small we cannot make general conclusions aboutthe relationships between content of lunasin and other compounds.

    5. Conslusions

    We provide the rst reported presence of lunasin peptide in oatgenotypes and suggest that this discovery will strengthen generalassumption that oat is a healthy food. Additionally, we havemonitored changes in lunasin content during different years andhave demonstrated genotype-dependent variations of the lunasincontent. In the future, we suggest studying a broader range of ge-notypes and investigating the inuence of the farming system, cropmanagement and climate conditions on lunasin content in oat ge-notypes as well as clarifying if consumption of lunasin-containingoat foods plays an important role in cancer and cardiovasculardisease prevention. In antioxidant assays obtained results indicatethat oat lunasin is bioactive.


    This research was supported by the European Social Fund co-nanced project Nr. 2009/0218/1DP/


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    sin peptide in oats (Avena sativa L), Journal of Cereal Science (2013),

    Detection of the lunasin peptide in oats (Avena sativa L)1. Introduction2. Experimental2.1. Materials and reagents2.2. Instrumentation2.3. Lunasin content measurements2.4. Grain sample characterisation2.5. Radical scavenging assay2.6. Cell culturing2.7. Cell proliferation assay2.8. Measurement of glutathione peroxidase activity2.9. Data analysis

    3. Results3.1. Lunasin content in oat3.2. Content of crude proteins, fat, starch and -glucans in the oat genotypes3.3. Antioxidant activity of lunasin

    4. Discussion5. ConslusionsAcknowledgmentsReferences


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