THE CHANGES OF THE POLYPHENOL CONTENT AND ANTIOXIDANT ACTIVITY IN POTATO TUBERS ( SOLANUM TUBEROSUM L.) DUE TO NITROGEN FERTILIZATION

Cultivar is one of the most important internal factors affecting polyphenol concentration in the plants. However, influence of the grown locality, climate conditions and way of cultivation belong to important external factors. In our experiment the influence of different nitrogen doses (0 - 40 - 80 - 120 - 160 - 240 kg N.ha -1 ) applied in the form of Vermikompost on the total polyphenol content and derived total antioxidant activity in cv. Sorento were investigated. While in the 1 st - 5 th variants the determined polyphenol content in dry mater of potato tubers decreased from 399.2 to 70.40 mg.kg -1 , in the 6 th variant that was twice higher in comparison to the 5 th variants (135.6 mg.kg -1 ). The statistically significant differences in values of total polyphenol content between variants (polynomial function of 2 nd degree) were confirmed. The study also confirmed a strong statistical correlation between the content of polyphenols and the content of antioxidant activity has been confirmed (sign. F: 3.24E -10 ). The highest value of antioxidant activity was observed in the first variant. From the first to the fifth variant (7.62 - 4.84%), the value of antioxidant activity was decreasing and in the sixth variant this value increased to 6.31%.


INTRODUCTION
The polyphenols are the most abundant antioxidants in the human diet (Bystrická et al., 2010). Polyphenols are be divided into two main groups: phenolic acids and flavonoids, which create from 1/3 to 2/3 of all antioxidants (Tapiero et al., 2002). Polyphenolic substances have a wide range of physiologically beneficial effects. Polyphenols are the most widespread group of plant secondary metabolites plants that can be the most important part of defence system of plants against pests and diseases ( Mazid et al., 2001;Modrianský et al., 2003). It is known that phenolic substances are involved in maintenance of redox status of cell and its response to cold, UV radiation, the injury and the effect of pathogens (Lukaszewicz et al., 2004).
Potato tubers are, due to consumption rate, one of the major sources of antioxidant compounds in human diet. Compounds with antioxidant activity have the capability to inactivate free radicals, which negatively influence biologically important compounds (lipids, proteins and nucleic acids) (Hejtmánková et al., 2009). Although potato tubers contain a number of phenolic compounds, their content is relatively low (5 -30 mg.100 g -1 FM). There are found both in the free-form and the bound-form. Phenolic compounds are located mainly in the peel; there the content of phenolics is about ten times higher than that in the flesh. Some substances are detected only in peel. But for example, the largest content of tyrosine (monobasic phenol) is found in inside the tuber, while its small concentration in the outer layer of tube (Lister and Munro, 2000).
Flavonoids are one of the most important groups of phenolic compounds. This group consists of diverse groups of plant metabolites, including chalcones, aurons, flavanones, isoflavones, flavones, flavonols, leucoanthocyanidins, catechins and anthocyanidins. This group consist of more than 4500 compounds and the large number of derivates providing substitution of hydrogen atoms by hydroxyl, methoxyl and other groups in different positions of basic structures (Ďuračková et al., 1999;Klejdus et al., 2003). Colour of potato varieties is determined by flavonoids and anthocyanins that are found as acylated glycosides with p-coumaric acid or nonacylated glycosides. They are present mainly in the vacuolar membranes in the periderm, especially in tissues of transgenic potato tubers (Lachman and Hamouz, 2008).
Phenolic acids and their derivatives exhibit effects of primary antioxidants. The activity depends on the number of hydroxyl groups in the molecule. Generally, antioxidant derivatives of cinnamic acid and o-diphenols (for example caffeic and chlorogenic acid) are more active. Also other types of derivatives, such as amides and glycosides are active. Chlorogenic acid can constitute 90% of the total content of polyphenolic substances in potato tubers. In fresh potatoes the content of chlorogenic acid is around 100 -200 mg.kg -1 and its content gradually decreases from the edge to the centre of potato tuber. The most important componentof potato tubers is amino acid L-tyrosine

Plant material
Medium early potato cultivar Sorento was used in field trials. The potatoes were grown in loam soil. The experimental field 120 m x 250 m was divided into six parcels for different variants. In the first variant fertilizers were not applied and it was the control variant. In variants 2 -6 the granulated Vermicompost in graded doses 3.3 -6.6 -9.9 -13.2 -19.8 t.ha -1 was applied one time before the planting. These doses represented 40 -80 -120 -160 -240 kg N.ha -1 (the content of N in the Vermicompost = 1.22%). Potatoes were planted on the 27 th April 2012 and were manually harvested at the stage of full maturity on the 5 th October 2012. During the vegetation period the plants were treated three times irrigated by dose 35 L.m -2 (35 mm). Samples were taken from different variants oh four replications each 5, 20, 35 and 50-metres. All potato samples were taken with the soil.

Analysis
Soil. In soil samples agrochemical parameters (content of organic carbon and content of humus, values of active and exchange soil reaction) were determined. These parameters were determined according to the method of Fiala et al., (1999).
The contents of available macronutrients nutrients and micronutrients were determined according to the method of Mehlich. The Mehlich III soil extractant and SOP was used for extracting Ca, Mg, P and K from soil samples. The flame atomic absorption spectrometer AAS Varian AASpectr DUO 240FS/240Z/UltrAA equipped with a D2 lamp background correction system, using an air -acetylene flame (Varian, Ltd., Mulgrave, Australia) was used for the determination of macroelements Ca, Mg, and K as output analytical method and P was determined spectrophotometrically using spectrophotometer UV-VIS 1800 (Shimadzu, Kyoto, Japan) at λ = 666 nm with ascorbic acid after 2 h standing and colouring.
Plant material. The content of N in potato tubers was determined by Kjeldahl method, the content of P spectrophotometrically, and K, Ca, Mg by AAS method, as described previously. Samples of fresh matter were prepared by homogenisation of all potato tubers from individual containers and consequently lyophilised. The potato samples were incinerated in the Nabertherm muffle furnace MARS X Press Microwave Oven (CEM, Matthews, USA) at 200 °C and dissolved ash was diluted to a certain volume with redistilled water. The mixture was heated in a digestion block according to the following sequence: 20 -175 °C/15 min, 175 °C/15 min, 175 -80°C/20 min. Minerals concentrations were determined on a dry weight basis as mg.kg -1 .
The total content of polyphenols (TP) in extracts of lyophilized samples (without peels) with 80% ethanol was determined by the modified method of Lachman et al., (2006) using Folin-Ciocalteau reagent at λ = 765 nm. Content of total polyphenols was expressed as the content of gallic acid and was calculated on dry mater.
Antioxidant activity (AOA) was determined using 2,2-diphenyl-1-picrylhydrazyl which in ethanol solution is in colourless stable radical form. Its reduction is manifested by the change of colour of solution and is measured spectrophotometrically. Gallic acid was used as the standard and on its equivalent particular values of AOA samples were calculated (Brand-Williams et al.,

1995). Statistics
To examine the impact of nitrogen application on the production of polyphenols and antioxidant activity the regression and correlation analysis (Microsoft Excel) was used.

RESULTS AND DISCUSSION
Potatoes do not have special land and climatic requirements. Typical lands for potatoes are light or medium soils with a permeable underclass. Sandy land is also suitable but it has to contain 8 -10% clay particles and more than 2% of humus. The content of humus in the soil (Hum. = 1.56%) was determined on the basis of the content of organic carbon (C ox = 0.91%). Suitability of land for potatoes is associated with the soil reaction. High and stable yields of potatoes are achieved in the weakly acidic soils (pH 6.7). There determined values of active soil reaction (pH/H 2 O) in the examined soil ranged in interval from 6.83 to 8.17 and exchange soil reaction (pH/KCl) from 5.35 to 7.14. Investigated soil is characterized by medium supply of phosphorus (P = 66.90 mg.kg -1 ), good supply of potassium (K = 210.8 mg.kg -1 ) and a high supply of magnesium (Mg = 260.4 mg.kg -1 ). The results of soil analysis (average values from all sampling sites) are presented in Table 1.
The content of macroelements in dry matter (DM) of potato tubers is presented in Table 2. Minerals make up about 1.1% of the total weight of tubers (Vreugdenhil et al., 2007). The average consumption of potatoes (300 g) provides at about 21% of iron, 9.6% of phosphorus, 3.4% of calcium and 80% of potassium of recommended daily dose. It means that potassium with the content around 1.7 -2.02% in dry weight is the most important element of potato tuber, followed by phosphorus which is located in different forms and different contents of the inorganic compounds (12 -39%) and in starch (20 - The average values of TP and AOA at different doses of nitrogen applied to the soil are presented in Table 3. It appears that graded nitrogen doses application can decrease the formation of polyphenolic compounds and relating antioxidant activity to a certain N concentration in the soil. The total content of polyphenol determined in dry mass of the potato tubers decreased from 399.3 mg.kg -1 (0 kg N.ha -1 ) to 70.40 mg.kg -1 (160 kg N.ha -1 ). But after application of 240 kg N.ha -1 the total content of polyphenol steeply increased 1.93 times than in the previous variant (Table 3). The same tendency was observed in obtained values of the total antioxidant activity because from the second to the fifth variant the values of AOA decreased by 2.5% -36.7%, while in the 6 th variant (240 kg N.ha -1 ) the AOA value increased by 30.28% compared with the control variant.
Polynomial function of the second degree was used to examine the correlation between TP content and different nitrogen applied to the soil: (y = 0.0112x 2 -3.9443x + 426.38; significance F = 3.852E -11 ) (Figure 1). This regression model explains the variability of TP to 66%.
Total content of polyphenol (TP) in potatoes is mainly influenced by the cultivar ( Although not all polyphenolic compounds are characterized as antioxidants, the strong statistical significance (significance F = 3.24E -10 ) between TP and AOA was confirmed. The regression coefficient is statistically significant and the line explains the variability for 58% of the AOA (Figure 2). Increasing of AOA around 0.007% depends on increasing of TP in potato tubers.   Obtained results in this study are in agreement with studies related to organic and conventional way of cultivation of other crops. Phenolic and hydrophilic antioxidant profiles of organically and conventionally produced tomato juices demonstrated statistically higher levels (P < 0.05) for organic tomato juices (Vallverdú -Queralt et al., 2012). This increase corresponded not only with increasing contents of soil organic matter accumulating in organic plots but also with reduced manure application rates once soils in the organic systems had reached equilibrium levels of organic matter. Principal component analysis documented that phenolic compounds and hydrophilic antioxidant capacity were responsible for the differentiation between organically and conventionally produced tomato juices. Comparison of mineral nutrient, chicken manure and grass-clover mulch used for tomato grooving showed that the mean total phenolic and ascorbic acid content of tomatoes treated with chicken manure and grass-clover mulch was 17.6% and 29% higher, respectively, than the tomatoes grown with in mineral nutrient solutions (Toor et al., 2006). The antioxidant activity in the ammonium treated plants was 14% lower compared with other variants. However, a two-years study examining of the concentration of various forms of ferulic acid in wheat grown under comparable organic and conventional conditions has shown that the combination of NPK, fertilizers do not significantly the ferulic acid concentration, on the other hand the year (climate conditions) significantly influenced the soluble conjugated ferulic acid content in all fungicide treated varieties (Gasztonyi et al., 2011). Likewise a negative effect of nitrogen amount on the concentration of annthocyanins and flavanols in the grape peels (increasing the amount of nitrogen from 120 kg.ha -1 N to 180 kg.ha -1 N using urea) An overview of recent research of Stefanelli et al., (2010) it is notable that the lowest N application rate resulted in the highest flavonoid content. Low or even zero N applications resulted in the highest content of phenolic compounds in grapes, Chinese cabbage, broccoli heads, apple, basil, pak choi, lettuce, tomato, olives, and strawberries. Also study of the influence of nitrogen fertilization on the content of phenolic compounds in walnut kernels revealed that N fertilization had a significant negative effect on the phenolic compounds content (Verardo et al., 2013). Polyphenol concentration declined as N availability increased in olives, diminishing the resulting oil quality. Tomato fruit antioxidant capacity and vitamin C content were the highest with N as chicken manure or grass/clover mulch compared with nitrate (NO 3 -)/ammonium (NH 4 + ) fertilizers . Organic fertilizers such as codahumus 20, mixed compost, sheep manure with mixed compost and codahumus 20 may be better fertilizer combinations that enhance tomato quality and antioxidant activity (Riahi and Hdider, 2013). In comparison of using mineral NPK fertilizer, organic fertilizer (chicken manure) and bioorganic-fertilizer composed of a mixture 50% Azotobacter chrococcum and 50% Bacillus megaterium it was indicated in broccoli cultivars that there is a good margin for enhancing antioxidant compounds in broccoli for economic production using organic fertilization (Naguib et al., 2012). Phenylalanine ammonia-lyase (PAL) is a key enzyme under low N supply or deficiency, which increased activity under N stress increases phenolic compounds and in addition, furthermore releases nitrogen from phenylalanine, thereby providing N for redistribution.

CONCLUSION
Analysis of the potatoes grown under the same climatic conditions and using different doses of nitrogen confirmed their effect on the formation of polyphenolic compounds. Higher doses of N significantly reduced the formation of polyphenols. Their content was from the second to the fifth variant by 17.06%, 53.04, 75.62 resp. 82.37% lower than the content of TP in the first (control) variant. After application of Vermicompost in the amount of 240 kg N.ha -1 the TP content was even by 1.9 higher than after application of Vermicompost in the amount (160 kg N.ha -1 ). The strong statistical relationship between TP and AOA (y = 0.0071x + 4.8665; significance F = 3.21E -10 ) was confirmed. Also the values of AOA decreased from the second to the fifth variant. In N-rate 160 kg.ha -1 the determined AOA was about 36.39% lower than that in Nrate 0 kg.ha -1 . In N-rate 240 kg.ha -1 , the AOA has increased from 4.844% (160 kg N.ha -1 ) to 6.311% (Δ = +30.28%).
In potato tubers also the content of macroelements was determined, which in different variants ranged in intervals 28.25 -58.21 mg Na.kg -1 DM; 440.46 -510.41 mg P.kg -1 DM; 3967.38 -5725.59 mg K.kg -1 DM; 41.52 -54.40 mg Ca.kg -1 DM; 144.81 -177.68 mg Mg.kg -1 DM and 28.25 -58.21 mg N.kg -1 DM. No statistically significant differences between Na, P, K, Ca, Mg and N contents in potato tubers and the amount of applied N into the soil were confirmed.
While the above study indicated that increased generally resulted in lower polyphenolic content, there should be