Avenanthramides and nutritional components of four mexican oat (Avena sativa L.) varieties

Avenantramidas y componentes nutricionales de cuatro variedades mexicanas de avena (Avena sativa L.)

Faviola Ortiz-Robledo¹, Ignacio Villanueva-Fierro¹*, B. Dave Oomah², Ismael Lares-Asef¹, J. Bernardo Proal-Nájera¹, J. Jesús Návar-Chaidez¹

¹ IPN CIIDIR Durango, Sigma 119 Fraccionamiento 20 Noviembre II, 34220. Durango, Durango, México. COFAA scholars.* Author for correspondence. (ifierro62@yahoo.com).

Received: July, 2012.
Approved: March, 2013.

Abstract

Oat (Avena sativa L.) is a cereal grain, its seeds are rich in nutrients, is consumed as whole grain with health benefits fot humans due to its antioxidants, such as the avenanthtamides (Avns). The contents of Avns 2c, 2f and 2p were evaluated by HPLC in groats and hulls of oat varieties Chihuahua, Cuauhtemoc, Raima and Avemex cultivated in Durango, México. The association of these Avns with protein, fat, crude fiber and ash of oat was also investigated. A 4x2 bifactorial design was used, with the number of varieties and groats ot hulls as factors. Avns contents differed significantly (p<0.05) among oat varieties, groats and hulls. Avemex groats had the highest concentrations of Avns: 3.6±0.7, 3.0±0.5 and 3.3±0.6 mg kg^-1 fot 2c, 2f and 2p Avns. Avemex groats also had the highest protein content and Avns showed positive correlation with protein content indicating that Avns could be found in oat varieties with high protein content. Karma and Avemex had highet protein content with Avemex containing three times more Avns than othet varieties.

Keywords: antioxidants, groats, hulls, oat, polyphenols.

Resumen

Palabtas clave: antioxidantes, granos descascarillados, cascarillas, avena, polifenoles.

INTRODUCTION

Chihuahua and Durango are two Mexican States accounting for 78.7 % of the total oat production in Mexico (Financiera Rural, 2010). Chihuahua and Cuauhtémoc are the most commonly grown varieties, followed by Avemex and Karma, varieties that were genetically modified by the Instituto Nacional de Investigaciones Forestales Agrícolas y Pecuarias (INIFAP), Mexico (Mariscal-Amaro et al., 2009), to confer moderate resistance to stem rust (Puccinia graminis) and crown rust (Puccinia coronata). The benefit of consuming oats for humans and livestock are due to its proteins, lipids, carbohydrates, fiber, vitamins, minerals and antioxidants (Peterson et al., 2002, Singh et al., 2013), particularly the avenanthramides (Avns); compounds formed from an anthranilic acid linked with an amide bond to a hydroxycinnamic acid (Peterson and Dimberg, 2008). Among cereals, Avns are only present in oats and are biosynthesized through the enzyme hydroxycinnamoyl CoA: hydroxyanthranilate N-hydroxycinnamoyl transferase (Wise et al., 2009). The most abundant Avns found in grain are N-(3', 4'-dihydroxy-(E)-cinnamoyl)-5-hydroxyanthranilic acid (2c), N- (4'-hydroxy-3'-methoxy- (E)-cinnamoyl)-5-hydroxyanthranilic acid (2f) and N-(4'-hydroxy-(E)-cinnamoyl)-5-hydroxyanthranilic acid (2p), 2 indicating 5-hydroxyanthranilic acid and p, f and c indicating p-coumaric, ferulic and caffeic acids (Bratr et al., 2003; Singh et al., 2013). Avns were first referred as Avns A, B and C by Collins (1989), who found over 40 different Avns-like compounds in methanolic extracts of oat groats and hulls. Other studies reported the presence of these phenolic compounds (Dimberg et al., 1993; Emmons and Peterson, 1999; Handelman et al., 1999). Besides oats, Avns are found in carnation leaves and eggs of white cabbage butrerfly (Wise et al., 2009) and oat leaves (Ishihara et al., 1999; Peterson and Dimberg, 2008; Dimberg and Peterson, 2009). Avns possess biological properties such as antioxidant activity (Peterson et al., 2002; Fagerlund et al., 2009; Ren et al., 2011) that increases with the number of radical-stabilizing groups ortho to the phenolic hydroxyl group (Fagerlund et al., 2009). Avns also exhibit anti-inflammatory (Liu et al., 2004; Guo et al., 2008; Sur et al., 2008), anti-atherogenic (Liu et al., 2004; Nie et al., 2006), anti-irritant (Sur et al., 2008) and anti-proliferative activities (Guo et al., 2010), and prevents heart disease by reducing LDL cholesterol (Ryan et al., 2007). Avns have also been associated with crown rust (Puccinia coronata) incidence and genetic resistance to the disease (Wise et al., 2008; Mariscal-Amaro et al., 2009).

Although oat is recognized as a nutrient-rich cereal with human health benefits, mainly by the presence of antioxidants, there is limited information about Avns content in oats and its anatomical fractions (Liu et al., 2011; Hole et al., 2012). Thus, the objective of this study was to determine the content of Avns 2p, 2f and 2c in four oat varieties grown in the State of Durango, México, as well as their correlation with the chemical composition of the grain in order to provide nutritious and nutraceutical profiles. This study aims to show that oat varieties resistant to stem and crown rusts may have high Avns contents and therefore are more nutritious than susceptible varieties.

MATERIALS AND METHODS

Oats varieties Chihuahua, Cuauhtémoc, Avemex and Karma were used in this study. The first two were grown during 2010 in Nuevo Ideal, Durango (24° 53' 51.3" N; 105° 02' 32" W; altitude 1990 m). The soil, sandy clay loam and pH 8.28, was not fertilized and no pathogens were observed. Avemex and Karma were genetically modified by INIFAP, in order to confer moderate resistance to stem (Puccinia graminis) and crown rusts (Puccinia coronatd) (Mariscal-Amaro et al., 2009). These varieties were grown during 2010 in Valle del Guadiana, Durango (23° 59' 12.4" N; 104° 37' 38.7" W; altitude 1878 m). The loam textured soil (pH 8.0) was fertilized with 120 and 60 kg ha^-1 of nitrate and phosphate, respectively. Since the soil had no observable pathogens it was not treated with any pesticides.

Chemicals. Synthetic Avns N-(3', 4'-dihydroxy-(E)-cinnamoyl)-5-hydroxyanthranilic acid (2c), N-(4'-hydroxy-3'-methoxy-(E)-cinnarrioyl)-5-hydroxyanthranilic acid (2f) and N-(4'-hydroxy-(E)-cinnamoyl)-5-hydroxyanthranilic acid (2p) were provided by Dr. Mitchell L. Wise (Cereal Crops Research, ARS, USDA, Madison, WI, United States). Methanol and orthophosphoric acid were obtained from Fermont (Monterrey, México) and acetonitrile from Aldrich (D.F., México).

Avns extraction and analysis. Extraction and analysis of Avns were performed as described by Bryngelsson et al. (2002). Groats and hulls were separated by hand and ground in a blender until the powder passed through a 0.5 mm sieve. 5 g of each sample (in triplicate) were used to extract Avns with methanol (36 mL) by stirring, for 30 min, twice at room temperature the same day that the grinding was performed. The mixture was centrifuged for 10 min, the supernatants pooled, filtered through Whatman filter paper No. 41, and the solvent evaporated under vacuum at 40 °C. The residue was resuspended to 2 mL in methanol and stored at -20 °C until further analysis. Avns were analyzed by HPLC in an Agilent HPLC 1100 equipped with a degasser, quaternary pump and diodearray detector using a reversed phase column (Waters C-18 Symmetry 3.9x150 mm). The mobile phase consisted of two solvents: solvent A (0.01 M phosphoric acid in acetonitrile:water 5:95, v/v) and acetonitrile as solvent B. Samples were run with a linear gradient for 60 min from 0-40 % B at 1 mL min^-1 flow rate. The analytes were detected at 340 nm, with a bandwidth of 8 nm, and 500 nm as reference with a bandwidth of 50 nm. Synthetic standards of each Avns were used to identify and quantify oats Avns.

Validation of analytical system. Accuracy and relative errors were parameters considered in this study. Precision was measured in terms of repeatability of values (standard deviation) obtained under the same (day, analyst, sample, and machine) operating conditions; linearity (the slope of the calibration curves) was measured to determine if results were directly proportional to the concentration of analyte (coefficient of determination); noise was taken as two standard deviations of the baseline, and limit of quantification was 10 times the noise.

Experimental design. A 4x2 completely fue randomized bifactorial design was used: factor 4 the number of varieties and factor 2 groats or hulls of each variety. Three extractions of each sampled variety were injected twice in the HPLC.

Statistical analysis. The experimental design was used to determine the influence of variety and fraction on Avns content. Analysis of variance was performed with the GLM procedure, means comparison with Duncan's test to determine variety, fraction, and variety-fraction effects and Pearson correlation performed according to SAS Institute Inc. (Cary, North Carolina, 1990).

RESULTS AND DISCUSSION

The results of accuracy showed relative errors of 2.79, 5.67 and 0.85 %, for Avs 2c, 2p and 2f, at 1 mg L^-1 concentration; a relative error of 10 %o is considered accurate. Precision (expressed as standard deviation) was 0.42, 0.088 and 0.214 mg kg^-1, for Avns 2c, 2p and 2f. For linearity, the coefficients of determination (R²) were 0.999, 0.998 and 0.997 for Avns 2c, 2p and 2f. Limits of quantification were 0.152, 0.147 and 0.143 mg kg^-1, for Avns 2c, 2p and 2f. Chihuahua hulls had the highest ash content and the groats the highest fat content. Avemex groats had the highest protein (20.7 %) content, and Cuauhtémoc hulls the richest in crude fiber content. Protein content of Karma and Avemex were significantly (p<0.05) higher than those of Chihuahua and Cuauhtémoc for groats and hulls (Table 1). Our results are consistent with those reported (Shewry et al., 2008) on ash and fiber rich oat hulls and (Zwer, 2010; Welch, 2011) for protein and lipid rich groats.

Avns content was significantly different (p<0.05) among varieties and between fractions (groats and hulls). Avemex had the highest concentration of the three measured Avns, in both oats hull and groats. The lowest concentration for Avn 2c and 2p occurred in Chihuahua hulls and Karma hulls. Chihuahua, Cuauhtémoc and Karma hulls showed the lowest values for Avn 2f and Karma hulls for Avn 2p. The hulls-groats ratio (%) revealed that Avemex, Karma and Chihuahua synthesized greater amount of Avn 2c in groats than hulls. Cuauhtémoc variety had greater amount of Avn 2c in hull than groats in hull than in groats. Avn 2f content was similar in Chihuahua hull and groats, whereas Chihuahua and Cuauhtémoc had higher Avn 2p contents in hulls than other varieties (Table 2).

Avns concentration depended on variety and fraction confirming previous reports (Emmons and Peterson, 1999; Emmons and Peterson, 2001; Dimberg et al., 2005). However, Avns concentrations (in groats and hulls) in this study were lower than those reported by Emmons and Peterson (2001), Bratr et al. (2003) and Dokuyucu et al. (2003), but similar to those observed by Peterson et al. (2005). These differences can be explained by the genotype and the strong influence of growing environment that influences the Avns synthesis.

Protein in oats showed positive correlation with fat, Avns 2f and 2c, and it is inversely associated with ash and fiber (Table 3). Dimberg et al. (2005) found correlation among protein and Avns in a study of oat grain grown in conventional and organic systems. Correlation among protein and Avns 2f and 2c suggests that oat grain rich in protein will also be rich in Avns. In the present study fat was negatively correlated with ash and fiber, whereas Avns showed high positive correlations among them (Table 3). High correlation among the three Avns indicates that their production in oat grain is related, that is, if any one of the Avns is found in grain it is very likely that the other two Avns are also found.

In oat hulls, Avns showed positive correlation among them, but correlation with protein was no significant, this may be due to low amount of protein in the hulls. Protein was inversely associated with ash content, while fat showed positive correlation with ash content and inverse correlation with fiber. In groats, protein content showed a negative correlation with fat, while protein content showed positive correlation with Avns 2p and 2c (Table 3). Correlation among protein content and Avns suggests again that oat grains with high protein content produce higher concentrations of Avns than oat grains with low protein content.

CONCLUSIONS

The oats of Avemex and Karma varieties had the highest protein contents, but Avemex contained three times higher Avns concentrations than the rest. Karma also considered as a stem rust resistant variety had Avns content similar to non-resistant varieties Cuauhtémoc and Chihuahua. The use of Avemex variety is suggested for the preparation of food like snacks, meals, or cereals, as well as alcoholic extracts, but it is necessary to study the range of safe use.

ACKNOWLEDGEMENT

We would like to thank a M.Sc. Jesús López Hernandez (INIFAP) and M.Sc. Jésus Herrera (CIIDIR-IPN, Durango) for providing oats samples and Dr Mitchell L Wise for providing synthetic avenanthramides. Support was provided by COCYTED No. DGO 2008-C04-94500 and SIP from Instituto Politécnico Nacional No. 20091283, 20101637 and 20110855.

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