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 Cer
Journal of Cereal Science xxx (2013) 1e6E-mail address: Ruta.Muceniece@lu.lv (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.http://dx.doi.org/10.1016/j.jcs.2012.12.008
Please cite this article in press as: Nakurte,http://dx.doi.org/10.1016/j.jcs.2012.12.008(barley, 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
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I., et al., Detection of the lunavena sativa L)
ne Saleniece c, Liga Krigere c, Peteris Mekss b,Muceniece c,*
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.
2.1. 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.
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 lunahttp://dx.doi.org/10.1016/j.jcs.2012.12.008The 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.