Content of phenols in wheat as affected by varietal and agricultural factors

This study evaluates the concentration of various forms of ferulic acid in wheat and in wheat varieties grown under comparable organic and conventional conditions over two years. The effect of fungicide application in 2009 was also studied. Soluble conjugated and bound forms of ferulic acid were quantified by HPLC-PAD after extraction, the bound form was present predominantly up to 85–90% of total content. In 2008 the bound form of ferulic acid was measured in the range of 248–550 μg/g, the conjugated form was between 11 and 40 μg/g in all the wheat cultivars as a function of (NPK) treatments. Total ferulic acid content measured in 2009 varied in the range of 275–435; 267–341; 296–378 μg/g, with fungicide and 189–394; 231–366; 182–324 μg/g without fungicide in varieties Békés, Csillag and Petur respectively. In 2008 a significantly higher amount of conjugated ferulic acid was measured in all three investigated cultivars as compared to the content found in 2009 for the same cultivars. As all the samples were treated with fungicide, the main factor was the year (climate conditions). The combination of NPK, fertilizers did not affect significantly the ferulic acid concentration, on the other hand the year (climate conditions) influenced significantly the soluble conjugated ferulic acid content in all fungicide treated varieties. Keywords Wheat Cereal Food composition Food analysis Ferulic acid Phenolic polymers Tannins HPLC analysis Farming conditions Fertilization Horticulture and biodiversity Cultivar difference Genotype differences Organic agriculture 1 Introduction Dietary phenolics include phenolic acids, phenolic polymers (commonly known as tannins) and flavonoids. Phenolic acids are aromatic secondary plant metabolites, derivative from hydroxylated benzoic and cinnamic acids. The estimated range of consumption is 25 mg −1 g a day depending on diet. Epidemiological studies have associated the consumption of whole grain and whole-grain products with reduced incidence of chronic diseases such as cardiovascular disease ( Thompson, 1994; Jacobs et al., 1998 ), diabetes ( Meyer et al., 2000 ) and cancer ( Smigel, 1992; Kasum et al., 2002; Jacobs et al., 1995; Nicodemus et al., 2001 ). It is widely accepted that phenolic acids including ferulic, vanillic, and p -coumaric acids are the major antioxidants in wheat and significantly contribute to the overall antioxidant properties of wheat grain. Phenolics behave as antioxidants ( Ou and Kwok, 2004 ), due to the reactivity of the phenol moiety (hydroxyl substituent on the aromatic ring). Ferulic acid constitutes about 0.5% (w/w) of wheat bran, it is esterified to arabinose residues, which form a part of the arabinoxylan structure of the aleurone layer and pericarp of wheat bran. It was noted that genotype, growing conditions and interaction between growing condition and genotype altered the antioxidant properties of wheat samples and their levels of beneficial components, including total phenolics, phenolic acids, carotenoids and tocopherols ( Zhou et al., 2005 ). Adom et al. (2003) studied the phytochemical profiles for 11 different wheat varieties; their results showed that total phenolic content, total antioxidant activity and total flavonoid content did not vary greatly among the 11 wheat lines. However, significant differences in total ferulic acid content ( p < 0.05) and carotenoid content ( p < 0.05) among the varieties were observed. Mogren et al. (2006) showed that nitrogen fertilizer level did not affect quercetin content in onions, suggesting that nitrogen leakage from soil may be minimized without effects on flavonol content. Cultivar differences in quercetin content were significant but not consistent in all years. The objective of this work was to see whether the various farming technologies—organic and conventional—have any direct affect to the amount of total ferulic- or other phenolic-acids in various wheat varieties in two years (2008, 2009). In addition, the effect of fungicide on the ferulic acid content of wheat was also investigated in 2009. 2 Materials and methods 2.1 Grain samples and sample preparation Grain samples of various wheat varieties (Tisza, Csillag, Petur and Békés) were obtained from Cereal Research Non-Profit Ltd. (Szeged, Hungary) in the years of 2008 and 2009. The varieties “Csillag”, “Petur” and “Békés” were analysed in both years while the variety “Tisza” was involved only in 2008. The experiments were done in a long-term fertilization experiment (27 years old in 2008) set up on calcareous (22–24% CaCO 3 –MgCO 3 ) meadow soil rich in humus (4.5–4.8%), with good N-supplying capacity and a loamy-clay physical composition at the Fülöpszállás Station of the Cereal Research Non-Profit Ltd. Typical annual precipitation (30 year-average) in the area is 530 mm with 380 mm in the vegetation period. The crop year of 2008 provided optimum conditions for the wheat production with 366 mm precipitation in the vegetation period while 2009 was a drought year with 270 mm. The organic farming system was regarded on those bocks where NPK have not been applied since 27 years (Treatment 1). The conventional growing system was arranged in six combinations of N, P and K fertilizers in block system, in two years. The doses of NPK fertilizers (kg active ingredient/ha) in 7 combinations as treatments are shown in Table 1 . Furthermore, all the samples were treated with the Fungicide “Opera New”(BASF) in 2008. In the following year all the samples were grown with and without application of the fungicide. Wheat grain of each cultivar was milled using a laboratory mill (Labour Műszeripari Művek 00124, Esztergom) containing a 1 mm mesh sieve. The whole ground sample (10 g) was transferred to an Erlenmeyer flask, defatted five times with hexane at a 2:1 ratio (v/w), filtered through a Whatman No. 1 filter paper, the final defatted samples were dried at room temperature and stored at −20 °C until analysis. The measurements were started within two weeks with the stored samples. Phenolic acid standards were (≥99.0%; HPLC) purchased from Sigma–Aldrich. All other chemicals and solvents were of analytical or HPLC-grade purity were received from Reanal (Hungary) and Forr-Lab (Hungary). 2.2 Extraction of soluble conjugated ferulic acid Wheat grain of each cultivar was milled using a laboratory mill containing a 1 mm mesh sieve. The fine flour (10 g) was transferred to an Erlenmeyer flask, defatted five times with hexane at a 2:1 ratio (v/w), filtered through a Whatman No. 1 filter paper, and the final defatted samples were dried at room temperature. Bound and soluble conjugated ferulic acids were extracted according to the method of Adom and Liu (2002) , with slight modification on the HPLC gradient elution. The defatted flour was extracted twice with 80% chilled ethanol at a 4:1 ratio (v/w) for 1 h at room temperature. After centrifugation at 3000 rcf for 15 min the pooled supernatants were evaporated at 40 °C and reconstituted with water to a final volume of 5–10 ml. The extract (1 ml) was digested with 3 M NaOH for 1 h with shaking, under nitrogen gas, and the solution was neutralized with an appropriate amount of HCL. The mixture was extracted five times with ethyl acetate, and the ethyl acetate fraction was evaporated to dryness at 35 °C. Ferulic acid was recovered for analysis in 3–6 ml of HPLC water. 2.3 Extraction of bound ferulic acid Following a two-time extraction with 80% chilled ethanol and centrifugation, the residues were digested with 3 M sodium-hydroxide at room temperature for 1 h with shaking under nitrogen gas. The process of neutralization and extraction with ethyl acetate were carried out in a separation funnel. After evaporation of ethyl acetate, ferulic acid was re-dissolved in a 100× diluted solution. 2.4 Determination of ferulic acid content In this work we focused on only ferulic acid as being the main phenolic acid in wheat samples. Ferulic acid in sample extracts was quantified by a RP-HPLC procedure (Waters 2695), using a Nucleodur Sphinx RP column (3 μm, 150 mm × 4.6 mm). Gradient elution was applied with solutions A (methanol), B (water/formic acid 99/1) and C (acetonitrile) as follows: linear gradient from 100% B/0% A/0% C to 20% A/60% B/20% C, 0–10 min; linear gradient from 20% A/60% B/20% C to 30% A/30% B/40% C, 10–20 min; isocratic elution 30% A/30% B/40% C, 20–25 min; gradient elution from 30% A/30% B/40% C to 0% A/100% B/0% C, 25–28 min; isocratic elution 0% A/100% B/0% C, 28–30 min. The flow rate was 0.7 ml min −1 . The ferulic acid was detected at 320 nm with PAD detector. 2.5 Statistical analysis Significance of the results and statistical differences were analysed using Microsoft Office Excel. Correlations between Nitrogen, Phosphor, Potassium treatments and the studied ferulic acid content were evaluated using regression analysis. To investigate the effect of the years and application of fungicide paired t -test was applied. The least significant difference test was applied to determine differences among means p < 0.05. 3 Results and discussion All the extractions and HPLC analysis were repeated three times for all the wheat samples harvested in 2008 and 2009. We also determined the main parameters of validation according the ferulic acid calibration curve: LOD: 3.2 ng/ml; LOQ: 10.7 ng/ml. Linearity range: 0.1–12 μg/ml, R 2 = 0.9529. The results of recovery tests were: bound ferulic acid 88 ± 9; conjugated ferulic acid 96 ± 8. Precision (RSD): 7.7. Fig. 1 shows the chromatogram of bound ferulic acid in Tisza. Ferulic acid (4-hydroxy-3-methoxycinnamic) was found to be the major phenolic acid in wheat, and it was present mostly in the bound form (up to 85–90%). Data in Table 2 show the change in the ferulic acid content in wheat grains from plant as a function of treatment with fungicide in addition to NPK fertilizations. The amounts of bound form were in the ranges of 325–374, 248–459, 234–335, 345–545 μg/g determined in the varieties Békés, Csillag, Petur and Tisza respectively. As concerns the conjugated ferulic acid, it was found to be in a range between 10.2 and 40.5 μg/g for all the samples tested in the same year. In 2009, the experiments were performed to include application of NPK fertilisers with and without fungicide treatment. The results are shown in Tables 3 and 4 . The values of ferulic acid content of wheat cultivars as a function of application of various fertilizer doses, after fungicide treatment in 2009 varied from 275 to 435; 267 to 341; 296 to 378 μg/g total ferulic acid determined in Békés, Csillag and Petur respectively. The total content of ferulic acid in the wheat varieties without fungicide treatment was 189–394; 231–366; 284–324 μg/g respectively in the varieties Békés, Csillag and Petur ( Table 4 ). The effect of dose of NPK fertilizers on ferulic acid content of the seeds seemed to be dependent on varietal and climate factors. In Békés, Petur and Tisza varieties no significant change was found in the content of conjugated and bound ferulic acid in the grains as a function of increasing dose of NPK fertilizers, while in the variety Csillag the increase of dose of NPK fertilisers caused the level of bound ferulic acid to decrease particularly at P 1 K 1 and P 2 K 2 treatments. It is interesting that the content of ferulic acid turned to the level similar to that found in the control treatment N 0 P 0 K 0 when the dose of N was increased to the highest level (N 3 ). However such a tendency was not observed for the same variety cultivated in 2009. This indicates that the impact of NPK treatment and dose is influenced by the complex interaction between genetic and environmental factors that require further research work and more specific investigation to be clarified. The statistical analysis ( Table 5 ) showed that the content of conjugated ferulic acid of wheat cultivated in 2008 is significantly higher than that of wheat grain obtained in 2009 for Békés, Csillag and Petur varieties. As all the samples were treated with fungicide the seasonal variation (climate conditions) is the most likely affecting factor. Several studies have been conducted to gain information on the effect of the production method on the carotenoid and polyphenol concentrations in different fruits and vegetables, their results showed quite different tendencies. For example some studies demonstrated lower amounts of phenolic compounds in organically grown tomatoes and broccoli ( Robbins et al., 2005 ). These inconsistencies suggest that factors other than the production method alone might affect the phytochemical concentrations, for example cultivar, microclimate, stage of ripeness and soil conditions. The doses of Phosphor and Potassium ( Table 1 ) were the same in the fertilization experiments, so we considered those elements as one variable. During the correlation analysis we investigated the effect of these two different variables N, PK and the collective correlation of these variables on the content of ferulic acid. As the results showed, except in some samples there is no relationship characterised by linear function between the ferulic acid content and the amount of fertilizers. In those cases where the regression analysis found linear coherence, the effect originated from the fertilizers is infinitesimal comparing to total ferulic acid content. As Fig. 2 shows, for example in the case the effect of P and K on the fungicide treated Petur in 2009, the axial cutting is higher with order of magnitude than the gradient of the equation: Y = 359.93 − 0.484 × N + 0.211 × PK where Y is the calculated ferulic acid content; N the amount of Nitrogen fertilizer; PK the amount of Phosphor an Potassium fertilization. This means that the maximal doses of N (N = 180 kg/ha/year) resulted in about 20% decrease of the content of ferulic acid, and the upper doses of PK (PK = 60 kg/ha/year) caused slight increase in ferulic acid content in Petur, in 2009. Figs. 2 and 3 show the linear functions between fertilizers and ferulic acid concentration in the case of fungicide treated Petur in 2009. In the present study the organically produced wheat exhibited phytochemical concentrations similar to those of the conventionally grown ones. In other studies the authors reported higher phytochemical concentrations in the organically grown fruits and vegetables than in the conventionally produced ones ( Young et al., 2005; Weibel et al., 2000 ). An explanation might be that plants change their metabolism toward carbon-containing compounds (starch, cellulose and non-nitrogen-containing secondary metabolites such as phenolic acids and terpenoids) when nitrogen availability is limited for growth, due to a different fertilising strategy than in the conventional production method ( Werner, 1996 ). Otherwise, when nitrogen is readily available, plants will primarily form compounds with high nitrogen content, for example, proteins for growth and nitrogen-containing secondary metabolites such as alkaloids ( Toor et al., 2006 ). Additionally, the accumulation of nitrogen differs between plant organs. In leaves and roots as well as stems the nitrogen accumulation is higher than in fruits and seeds ( Stracke et al., 2009 ). Therefore, different nitrogen fertilising strategies might have a greater influence on phytochemical concentrations in leaves, roots and stems than in fruits and seeds, it cannot be excluded that differences may occur in other organs. No differences in the phytochemical concentrations between the organic and conventional farming systems were reported for yellow plums and strawberries as well as black currants ( Häkkinen and Törrönen, 2000; Mikkonen et al., 2001 ). 4 Conclusion It was concluded that organic farming did not affect ferulic acid concentration. NPK treatments are not useful in improving the content of ferulic acid in wheat when applied at 30–180 kg active ingredient/ha concentrations in seven combination. In fungicide treated wheat it is important to consider the seasonal variation (change in climate), which can significantly influence ferulic acid content in different varieties of wheat. These results were in agreement with conclusions of Stracke et al. (2009) that climate factors have a greater impact on the phytochemical concentrations in whole wheat than the production method (organic/conventional). Acknowledgement Financial support from the Hungarian Scientific Research Fund ( OTKA-68706 ) is gratefully acknowledged. References Adom et al., 2003 K.K. Adom M.E. Sorrells R.H. Liu Phytochemicals profiles and antioxidant activity of wheat varieties Journal of Agricultural and Food Chemistry 51 2003 7825 7834 Adom and Liu, 2002 K.K. Adom R.H. Liu Antioxidant activity of grains Journal of Agricultural and Food Chemistry 50 2002 6182 6187 Häkkinen and Törrönen, 2000 S.H. Häkkinen A.R. Törrönen Content of flavonols and selected phenolic acids in strawberries and Vaccinium species: influence of cultivar, cultivation site and technique Food Research International 33 2000 517 524 Jacobs et al., 1998 D.R. Jacobs Jr. K.A. Meyer L.H. Kushi A.R. Folsom Whole grain intake may reduce risk of coronary heart disease death in postmenopausal women: the Iowa Women's Health Study American Journal of Clinical Nutrition 68 1998 248 257 Jacobs et al., 1995 D.R. Jacobs Jr. J. Slavin L. Marquart Whole grain intake and cancer: a review of literature Nutrition and Cancer 22 1995 221 229 Kasum et al., 2002 C.M. Kasum D.R. Jacobs Jr. K. Nicodemus A.R. Folsom Dietary risk factors for upper aerodigestive tract cancers International Journal of Cancer 99 2002 267 272 Meyer et al., 2000 K.A. Meyer L.H. Kushi D.R. Jacobs Jr. J. Slavin T.A. Sellers A.R. Folsom Carbohydrates, dietary fiber, incident type 2 diabetes mellitus in older women American Journal of Clinical Nutrition 71 2000 921 930 Mikkonen et al., 2001 T.P. Mikkonen K.R. Maatta A.T. Hukkanen Flavonol content varies among black currant cultivars Journal of Agricultural and Food Chemistry 49 2001 3274 3277 Mogren et al., 2006 L.M. Mogren M.E. Olsson U.E. Gertsson Quercetin content in field-cured onions ( Allium cepa L.): effects of cultivar, lifting time, and Nitrogen fertlizer level Journal of Agricultural and Food Chemistry 54 2006 6185 6191 Nicodemus et al., 2001 K.K. Nicodemus D.R. Jacobs Jr. A.R. Folsom Whole and refined grain intake and risk of incident postmenopausal breast cancer Cancer Causes Control 12 2001 917 925 Ou and Kwok, 2004 S. Ou K.C. Kwok Ferulic acid: pharmaceutical functions, preparation and applications in foods Journal of Agricultural and Food Chemistry 84 2004 1261 1269 Robbins et al., 2005 R.J. Robbins A.S. Keck G. Banuelos J.W. Finley Cultivation conditions and selenium fertilization alter the phenolic profile, glucosinolate, and sulphoraphane content of broccoli Journal of Medicinal Food 8 2005 204 214 Smigel, 1992 K. Smigel Fewer colon polyps found in men with high-fiber, low fat diets Journal of the National Cancer Institute 84 1992 80 81 Stracke et al., 2009 B.A. Stracke J. Eitel B. Watzl P. Mader Influence of the production method on phytochemical concentrations in whole wheat ( Triticum aestivum L.): a comparative study Journal of Agricultural and Food Chemistry 57 2009 10116 10121 Thompson, 1994 L.U. Thompson Antioxidants and hormone-mediated health benefits of whole grains Critical Reviews in Food Science and Nutrition 34 1994 473 497 Toor et al., 2006 R.K. Toor G.P. Savage A. Heeb Influence of different types of fertilizers on the major antioxidant components of tomatoes Journal of Food Composition and Analysis 19 2006 20 27 Weibel et al., 2000 F.P. Weibel R. Bickel S. Leuthold T. Alföldi Are organically grown apples tastier and healthier? A comparative field study using conventional and alternative methods to measure fruit quality Acta Horticulture 7 2000 417 427 Werner, 1996 M.R. Werner Soil quality characteristics during conversion to organic orchard management Ecology 5 1996 151 167 Young et al., 2005 J.E. Young X. Zhao E.E. Carey R. Welti S.S. Yang W. Wang Phytochemical phenolics in organically grown vegetables Molecular Nutrition and Food Research 49 2005 1136 1142 Zhou et al., 2005 K. Zhou J.-J. Yin L. Yu Phenolic acid, tocopherols and carotenoid composition, and antioxidant functions of hard red winter wheat bran Journal of Agricultural and Food Chemistry 53 2005 3916 3922