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Content of phenols in wheat as affected by varietal and agricultural factors
Journal of Food Composition and Analysis 24 (2011) 785–789
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Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Original Article
Content of phenols in wheat as affected by varietal and agricultural factors Magdolna Nagy Gasztonyi a,*, Rita To¨mo¨sko¨zi Farkas a, Ma´ria Berki a, Istva´n Miha´ly Petro´czi b, Hussein Gehad Daood a a b
Department of Analytics, Central Food Research Institute, 1022 Budapest, Herman Otto´ str.15, Hungary Department of Wheat Agronomy, Cereal Research Non-Profit Ltd., 6726 Szeged, Also´ kiko¨to˝ sor. 9, Hungary
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 October 2010 Received in revised form 27 April 2011 Accepted 27 April 2011 Available online 8 May 2011
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 mg/g, the conjugated form was between 11 and 40 mg/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 mg/g, with fungicide and 189–394; 231–366; 182–324 mg/g without fungicide in varieties Be´ke´s, 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. ß 2011 Elsevier Inc. All rights reserved.
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 mg1 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).
* Corresponding author. Tel.: +36 1 2141249; fax: +36 1 2141249. E-mail address: [email protected] (M.N. Gasztonyi). 0889-1575/$ – see front matter ß 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2011.04.011
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
M.N. Gasztonyi et al. / Journal of Food Composition and Analysis 24 (2011) 785–789
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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 Be´ke´s) were obtained from Cereal Research Non-Profit Ltd. (Szeged, Hungary) in the years of 2008 and 2009. The varieties ‘‘Csillag’’, ‘‘Petur’’ and ‘‘Be´ke´s’’ 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% CaCO3–MgCO3) meadow soil rich in humus (4.5–4.8%), with good N-supplying capacity and a loamy-clay physical composition at the Fu¨lo¨psza´lla´s 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 Mu˝ szeripari Mu˝ vek 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 8C 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).
1 h at room temperature. After centrifugation at 3000 rcf for 15 min the pooled supernatants were evaporated at 40 8C 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 8C. 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 sodiumhydroxide 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 mm, 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 min1. 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.
2.2. Extraction of soluble conjugated ferulic acid 3. Results and discussion 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 Table 1 Treatments with NPK fertilizers. Treatments 1 2 3 4 5 6 7
N0P0K0 N0P1K1 N0P2K2 N2P0K0 N2P1K1 N2P2K2 N3P2K2
N
P
K
0 0 0 60 60 120 180
0 30 60 0 30 60 60
0 30 60 0 30 60 60
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 mg/ml, R2 = 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 mg/g determined in the varieties Be´ke´s, 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 mg/g for all the samples tested in the same year.
M.N. Gasztonyi et al. / Journal of Food Composition and Analysis 24 (2011) 785–789
13,465
0,120 0,110 0,100 0,090
AU
0,080 0,070 0,060 0,050 0,040 0,030 0,020 0,010 0,000 11,00
12,00
13,00
14,00
15,00
Minutes
16,00
17,00
18,00
Fig. 1. HPLC chromatogram of bound ferulic acid separated from wheat cultivar Tisza.
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 mg/g total ferulic acid determined in Be´ke´s, Csillag and Petur respectively. The total content of ferulic acid in the wheat varieties without fungicide treatment was 189–394; 231–366; 284–324 mg/g respectively in the varieties Be´ke´s, Csillag and Petur (Table 4).
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The effect of dose of NPK fertilizers on ferulic acid content of the seeds seemed to be dependent on varietal and climate factors. In Be´ke´s, 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 P1K1 and P2K2 treatments. It is interesting that the content of ferulic acid turned to the level similar to that found in the control treatment N0P0K0 when the dose of N was increased to the highest level (N3). 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 Be´ke´s, 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.
Table 2 Changes in the content of ferulic acid in four cultivars, as a function of N, P, and K treatments after fungicide treatment, in 2008. Average ferulic acid (mg/g)a
Cultivar
Form
N0P0K0
N0P1K1
N0P2K2
N2P0K0
N2P1K1
N2P2K2
N3P2K2
Be´ke´s
Conjugated Bound Total
14.46 5.52 325.26 26.71 347.83 22.51
10.20 3.70 370.63 49.92 381.43 44.03
11.33 3.34 336.51 9.87 347.22 11.71
14.34 3.81 355.55 20.4 379.72 12,24
11.55 4.36 331.72 1.70 342.37 5.18
16.28 2.17 373.87 23.60 396.73 26.07
15.32 2.76 330.97 18.25 347.47 24.97
Csillag
Conjugated Bound Total
15.72 3.19 459.27 40.22 474.99 25.09
29.63 14 247.99 24.4 276.64 13.88
16.83 0.01 282.5 77.61 299.31 71.32
14.18 4.98 383.44 33.0 391.69 19.76
18.62 5.29 368.65 61 384.62 39.96
14.78 0.79 284.40 68.05 299.18 42.35
18.31 3.25 448.54 48.02 466.85 39.31
Petur
Conjugated Bound Total
13.44 9.24 234.23 2.81 247.91 1.67
26.67 8.74 299.41 61.91 326.08 76.47
22.74 15.8 313.69 59.0 341.74 77.17
17.33 13.37 335.76 32.54 353.26 23.35
24.02 8.27 281.50 26.18 300.89 23.34
40.49 8.69 328.10 42.0 372.85 48.44
27.83 12.0 280.77 25.19 299.04 23.96
Tisza
Conjugated Bound Total
12.23 1.65 484.93 23.12 496.65 12.52
14.54 1.93 345.35 84.0 362.5 50.91
16.17 3.43 545.45 45.37 556.50 33.23
16.06 2.66 447.73 62.0 463.0 45.25
14.64 5.42 369.64 53.66 374.0 31.11
16.16 4.64 499.00 37.94 516.0 22.63
14.70 3.91 369.89 31.35 384.10 26.73
a
Average standard deviation, n = 3.
Table 3 Changes in the content of ferulic acid in three cultivars, as a function of N, P, and K treatments after fungicide treatment in 2009. Average ferulic acid (mg/g)a
Cultivar
Form
N0P0K0
N0P1K1
N0P2K2
N2P0K0
N2P1K1
N2P2K2
N3P2K2
Be´ke´s
Conjugated Bound Total
6.05 2.87 295.84 4.02 301.89 1.14
8.73 3.01 311.57 11.0 320.30 7.98
6.50 0.93 268.76 3.64 275.26 2.72
6.49 1.22 333.00 24.04 339.49 25.26
8.49 3.29 297.19 10.42 305.68 13.71
8.88 1.57 349.77 66.1 358.65 67.68
8.22 1.90 427.52 123.13 435.74 125.04
Csillag
Conjugated Bound Total
9.54 0.53 297.99 45.59 307.53 46.12
12.09 5.74 292.52 4.93 304.61 0.81
12.53 1.7 255.39 76.57 267.91 78.28
4.55 1.78 337.15 32.22 341.70 34.02
13.55 1.38 324.03 35.81 337.58 37.20
9.06 3.06 328.38 5.83 337.44 2.77
6.69 5.41 296.55 67.09 303.24 61.68
Petur
Conjugated Bound Total
6.99 2.16 361.44 40.30 368.43 42.46
10.36 6.32 357.26 9.57 367.62 15.88
14.34 1.27 363.93 62.27 378.27 68.54
7.82 1.51 325.09 64.41 332.91 63.90
7.76 2.80 307.11 4.50 314.86 1.69
6.57 0.92 302.15 29.18 308.72 28.27
6.53 2.29 289.68 20.90 296.22 18.62
a
Average standard deviation, n = 3.
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M.N. Gasztonyi et al. / Journal of Food Composition and Analysis 24 (2011) 785–789
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 ¼ 359:93 0:484 N þ 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 (Ha¨kkinen and To¨rro¨nen, 2000; Mikkonen et al., 2001).
Table 4 Changes in the content of ferulic acid in three cultivars, as a function of N, P, and K treatments without fungicide treatment in 2009. Average ferulic acid (mg/g)a
Cultivar
Form
N0P0K0
N0P1K1
N0P2K2
N2P0K0
N2P1K1
N2P2K2
N3P2K2
Be´ke´s
Conjugated Bound Total
5.65 1.89 330.37 6.76 336.02 4.87
9.01 0.35 263.86 10.17 272.87 9.82
9.70 1.4 331.36 19.78 341.06 16.96
5.73 1.45 389.22 21.07 394.46 21.29
7.76 0.93 294.82 3.7 301.28 6.46
9.47 7.01 282.05 14.88 291.53 7.87
9.25 1.19 180.62 69.34 189.86 70.54
Csillag
Conjugated Bound Total
8.99 0.27 237.38 12.28 246.37 11.99
2.99 1.14 355.31 19.0 358.30 17.86
11.69 4.25 354.60 24.88 366.29 20.62
8.04 6.51 269.01 4.43 277.05 2.07
7.23 5.52 223.75 21.94 230.98 27.45
9.96 1.28 264.52 5.51 274.48 4.22
11.58 3.76 310.95 38.26 322.53 42.02
Petur
Conjugated Bound Total
5.03 1.25 306.48 29.56 311.51 28.31
5.17 0.84 319.22 34.0 324.39 34.85
14.75 0.24 291.34 5.18 306.1 4.3
9.61 0.69 274.76 26.55 284.4 23.2
8.53 3.29 286.29 69.49 294.82 72.78
7.23 3.29 292.23 4.79 299.46 1.51
7.17 5.38 314.27 23.75 321.44 29.13
a
Average standard deviation. n = 3.
Table 5 Investigation of the equality of samples using paired t-probe. Hypothesis
Wheat
Ferulic acid
t Value
Critical value for t-probe
Equality
Are the samples treated with or without fungicide in 2009 equal?
Be´ke´s
Conjugated Bound Total Conjugated Bound Total Conjugated Bound Total
0.8822 0.7411 0.7304 0.5628 0.5806 0.6228 0.4543 2.1053 2.1307
2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846
Yes Yes Yes Yes Yes Yes Yes Yes Yes
Conjugated Bound Total Conjugated Bound Total Conjugated Bound Total
5.7012 0.9799 1.2847 4.7831 1.3256 1.5779 4.4678 1.7567 0.7500
2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846
No Yes Yes No Yes Yes No Yes Yes
Csillag
Petur
Are the samples treated with fungicide in 2008 and 2009 equal?
Be´ke´s
Csillag
Petur
Paired t-probe
M.N. Gasztonyi et al. / Journal of Food Composition and Analysis 24 (2011) 785–789
400
Acknowledgement
Ferulic acid (µg/g)
350
Financial support from the Hungarian Scientific Research Fund (OTKA-68706) is gratefully acknowledged.
300 y = 0,211x + 359,93
250
References
200 150 100 50 0 0
20
40
60
80
100
P, K treatment (kg/ha/year) Fig. 2. The effect of P and K fertilization on Petur variety treated with fungicide in 2009.
400
Ferulic acid(µg/g)
350 300 250 200
y = -0,484x + 359,93
150 100 50 0
789
0
50
100
150
200
250
N treatment (kg/ha/year) Fig. 3. The effect of N fertilization on Petur variety treated with fungicide in 2009.
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).
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Contents lists available at ScienceDirect
Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Original Article
Content of phenols in wheat as affected by varietal and agricultural factors Magdolna Nagy Gasztonyi a,*, Rita To¨mo¨sko¨zi Farkas a, Ma´ria Berki a, Istva´n Miha´ly Petro´czi b, Hussein Gehad Daood a a b
Department of Analytics, Central Food Research Institute, 1022 Budapest, Herman Otto´ str.15, Hungary Department of Wheat Agronomy, Cereal Research Non-Profit Ltd., 6726 Szeged, Also´ kiko¨to˝ sor. 9, Hungary
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 October 2010 Received in revised form 27 April 2011 Accepted 27 April 2011 Available online 8 May 2011
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 mg/g, the conjugated form was between 11 and 40 mg/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 mg/g, with fungicide and 189–394; 231–366; 182–324 mg/g without fungicide in varieties Be´ke´s, 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. ß 2011 Elsevier Inc. All rights reserved.
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 mg1 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).
* Corresponding author. Tel.: +36 1 2141249; fax: +36 1 2141249. E-mail address: [email protected] (M.N. Gasztonyi). 0889-1575/$ – see front matter ß 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2011.04.011
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
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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 Be´ke´s) were obtained from Cereal Research Non-Profit Ltd. (Szeged, Hungary) in the years of 2008 and 2009. The varieties ‘‘Csillag’’, ‘‘Petur’’ and ‘‘Be´ke´s’’ 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% CaCO3–MgCO3) meadow soil rich in humus (4.5–4.8%), with good N-supplying capacity and a loamy-clay physical composition at the Fu¨lo¨psza´lla´s 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 Mu˝ szeripari Mu˝ vek 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 8C 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).
1 h at room temperature. After centrifugation at 3000 rcf for 15 min the pooled supernatants were evaporated at 40 8C 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 8C. 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 sodiumhydroxide 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 mm, 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 min1. 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.
2.2. Extraction of soluble conjugated ferulic acid 3. Results and discussion 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 Table 1 Treatments with NPK fertilizers. Treatments 1 2 3 4 5 6 7
N0P0K0 N0P1K1 N0P2K2 N2P0K0 N2P1K1 N2P2K2 N3P2K2
N
P
K
0 0 0 60 60 120 180
0 30 60 0 30 60 60
0 30 60 0 30 60 60
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 mg/ml, R2 = 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 mg/g determined in the varieties Be´ke´s, 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 mg/g for all the samples tested in the same year.
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13,465
0,120 0,110 0,100 0,090
AU
0,080 0,070 0,060 0,050 0,040 0,030 0,020 0,010 0,000 11,00
12,00
13,00
14,00
15,00
Minutes
16,00
17,00
18,00
Fig. 1. HPLC chromatogram of bound ferulic acid separated from wheat cultivar Tisza.
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 mg/g total ferulic acid determined in Be´ke´s, Csillag and Petur respectively. The total content of ferulic acid in the wheat varieties without fungicide treatment was 189–394; 231–366; 284–324 mg/g respectively in the varieties Be´ke´s, Csillag and Petur (Table 4).
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The effect of dose of NPK fertilizers on ferulic acid content of the seeds seemed to be dependent on varietal and climate factors. In Be´ke´s, 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 P1K1 and P2K2 treatments. It is interesting that the content of ferulic acid turned to the level similar to that found in the control treatment N0P0K0 when the dose of N was increased to the highest level (N3). 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 Be´ke´s, 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.
Table 2 Changes in the content of ferulic acid in four cultivars, as a function of N, P, and K treatments after fungicide treatment, in 2008. Average ferulic acid (mg/g)a
Cultivar
Form
N0P0K0
N0P1K1
N0P2K2
N2P0K0
N2P1K1
N2P2K2
N3P2K2
Be´ke´s
Conjugated Bound Total
14.46 5.52 325.26 26.71 347.83 22.51
10.20 3.70 370.63 49.92 381.43 44.03
11.33 3.34 336.51 9.87 347.22 11.71
14.34 3.81 355.55 20.4 379.72 12,24
11.55 4.36 331.72 1.70 342.37 5.18
16.28 2.17 373.87 23.60 396.73 26.07
15.32 2.76 330.97 18.25 347.47 24.97
Csillag
Conjugated Bound Total
15.72 3.19 459.27 40.22 474.99 25.09
29.63 14 247.99 24.4 276.64 13.88
16.83 0.01 282.5 77.61 299.31 71.32
14.18 4.98 383.44 33.0 391.69 19.76
18.62 5.29 368.65 61 384.62 39.96
14.78 0.79 284.40 68.05 299.18 42.35
18.31 3.25 448.54 48.02 466.85 39.31
Petur
Conjugated Bound Total
13.44 9.24 234.23 2.81 247.91 1.67
26.67 8.74 299.41 61.91 326.08 76.47
22.74 15.8 313.69 59.0 341.74 77.17
17.33 13.37 335.76 32.54 353.26 23.35
24.02 8.27 281.50 26.18 300.89 23.34
40.49 8.69 328.10 42.0 372.85 48.44
27.83 12.0 280.77 25.19 299.04 23.96
Tisza
Conjugated Bound Total
12.23 1.65 484.93 23.12 496.65 12.52
14.54 1.93 345.35 84.0 362.5 50.91
16.17 3.43 545.45 45.37 556.50 33.23
16.06 2.66 447.73 62.0 463.0 45.25
14.64 5.42 369.64 53.66 374.0 31.11
16.16 4.64 499.00 37.94 516.0 22.63
14.70 3.91 369.89 31.35 384.10 26.73
a
Average standard deviation, n = 3.
Table 3 Changes in the content of ferulic acid in three cultivars, as a function of N, P, and K treatments after fungicide treatment in 2009. Average ferulic acid (mg/g)a
Cultivar
Form
N0P0K0
N0P1K1
N0P2K2
N2P0K0
N2P1K1
N2P2K2
N3P2K2
Be´ke´s
Conjugated Bound Total
6.05 2.87 295.84 4.02 301.89 1.14
8.73 3.01 311.57 11.0 320.30 7.98
6.50 0.93 268.76 3.64 275.26 2.72
6.49 1.22 333.00 24.04 339.49 25.26
8.49 3.29 297.19 10.42 305.68 13.71
8.88 1.57 349.77 66.1 358.65 67.68
8.22 1.90 427.52 123.13 435.74 125.04
Csillag
Conjugated Bound Total
9.54 0.53 297.99 45.59 307.53 46.12
12.09 5.74 292.52 4.93 304.61 0.81
12.53 1.7 255.39 76.57 267.91 78.28
4.55 1.78 337.15 32.22 341.70 34.02
13.55 1.38 324.03 35.81 337.58 37.20
9.06 3.06 328.38 5.83 337.44 2.77
6.69 5.41 296.55 67.09 303.24 61.68
Petur
Conjugated Bound Total
6.99 2.16 361.44 40.30 368.43 42.46
10.36 6.32 357.26 9.57 367.62 15.88
14.34 1.27 363.93 62.27 378.27 68.54
7.82 1.51 325.09 64.41 332.91 63.90
7.76 2.80 307.11 4.50 314.86 1.69
6.57 0.92 302.15 29.18 308.72 28.27
6.53 2.29 289.68 20.90 296.22 18.62
a
Average standard deviation, n = 3.
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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 ¼ 359:93 0:484 N þ 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 (Ha¨kkinen and To¨rro¨nen, 2000; Mikkonen et al., 2001).
Table 4 Changes in the content of ferulic acid in three cultivars, as a function of N, P, and K treatments without fungicide treatment in 2009. Average ferulic acid (mg/g)a
Cultivar
Form
N0P0K0
N0P1K1
N0P2K2
N2P0K0
N2P1K1
N2P2K2
N3P2K2
Be´ke´s
Conjugated Bound Total
5.65 1.89 330.37 6.76 336.02 4.87
9.01 0.35 263.86 10.17 272.87 9.82
9.70 1.4 331.36 19.78 341.06 16.96
5.73 1.45 389.22 21.07 394.46 21.29
7.76 0.93 294.82 3.7 301.28 6.46
9.47 7.01 282.05 14.88 291.53 7.87
9.25 1.19 180.62 69.34 189.86 70.54
Csillag
Conjugated Bound Total
8.99 0.27 237.38 12.28 246.37 11.99
2.99 1.14 355.31 19.0 358.30 17.86
11.69 4.25 354.60 24.88 366.29 20.62
8.04 6.51 269.01 4.43 277.05 2.07
7.23 5.52 223.75 21.94 230.98 27.45
9.96 1.28 264.52 5.51 274.48 4.22
11.58 3.76 310.95 38.26 322.53 42.02
Petur
Conjugated Bound Total
5.03 1.25 306.48 29.56 311.51 28.31
5.17 0.84 319.22 34.0 324.39 34.85
14.75 0.24 291.34 5.18 306.1 4.3
9.61 0.69 274.76 26.55 284.4 23.2
8.53 3.29 286.29 69.49 294.82 72.78
7.23 3.29 292.23 4.79 299.46 1.51
7.17 5.38 314.27 23.75 321.44 29.13
a
Average standard deviation. n = 3.
Table 5 Investigation of the equality of samples using paired t-probe. Hypothesis
Wheat
Ferulic acid
t Value
Critical value for t-probe
Equality
Are the samples treated with or without fungicide in 2009 equal?
Be´ke´s
Conjugated Bound Total Conjugated Bound Total Conjugated Bound Total
0.8822 0.7411 0.7304 0.5628 0.5806 0.6228 0.4543 2.1053 2.1307
2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846
Yes Yes Yes Yes Yes Yes Yes Yes Yes
Conjugated Bound Total Conjugated Bound Total Conjugated Bound Total
5.7012 0.9799 1.2847 4.7831 1.3256 1.5779 4.4678 1.7567 0.7500
2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846 2.446911846
No Yes Yes No Yes Yes No Yes Yes
Csillag
Petur
Are the samples treated with fungicide in 2008 and 2009 equal?
Be´ke´s
Csillag
Petur
Paired t-probe
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400
Acknowledgement
Ferulic acid (µg/g)
350
Financial support from the Hungarian Scientific Research Fund (OTKA-68706) is gratefully acknowledged.
300 y = 0,211x + 359,93
250
References
200 150 100 50 0 0
20
40
60
80
100
P, K treatment (kg/ha/year) Fig. 2. The effect of P and K fertilization on Petur variety treated with fungicide in 2009.
400
Ferulic acid(µg/g)
350 300 250 200
y = -0,484x + 359,93
150 100 50 0
789
0
50
100
150
200
250
N treatment (kg/ha/year) Fig. 3. The effect of N fertilization on Petur variety treated with fungicide in 2009.
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).
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