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1 Influence of Biological Growth Regulators And Elements Of Mineral Nutrition On Antioxidant Activity And Content Of Flavonoides And Carothinoids In Kale Plants (brassica oleracea convar acephala var. Alexander LeonidovichMikhailov, Marat Ildusovich Kalimullin, Olga Arnoldovna Timofeeva Kazan Federal University, Kremlevskaya 18, Kazan, Republic of Tatarstan, ABSTRACT Leaf cabbage, or kale (Brassica oleracea convar acephala var. is a very popular leafy vegetable of the Brassicaceae family, which is actively used in the diet of the population in Northern and Central Europe, as well as in North America. It is believed that its regular intake has a beneficial effect on the human body. A number of studies have revealed a positive correlation between its consumption and human health. Kale attracts increasing attention of researchers in connection with the positive impact on human health, which is largely due to its rich phytochemical composition. Its plant tissue contain a large number of biologically active compounds, such as vitamins, mineral elements and phenolic compounds. In this connection, it was necessary to identify the factors that may enhance the synthesis of these compounds in kale plants. In our studies we used several different plant growth regulators and organomineral fertilizers. The results of the conducted experiments showed that both the first and second factors affected the antioxidant activity and the content of flavone glycosides. The data obtained can be valuable for practical application, since we conducted both field and production-scale studies. Keywords: Brassica oleracea, antioxidant activity, flavonoids, carotenoids, mineral nutrition, gibberellin, humate. Pr 3

2 1. INTRODUCTION Leaf cabbage, or kale (Brassica oleracea convar acephala var. is a very popular leafy vegetable of the Brassicaceae family, which is actively used in the diet of the population in Northern and Central Europe, as well as in North America. Kale attracts increasing attention of researchers in connection with the positive impact on human health, which is largely due to its rich phytochemical composition. It is believed that its regular intake has a beneficial effect on the human body. A number of studies have revealed a positive correlation between its consumption and human health [Lee, C. M., & Thomashow, M. F. 212]. Medical research have shown that eating Brassica vegetables reduces the risk of cardiovascular and oncological diseases [Jahangir, M., Kim, H. K., Choi, Y. H., & Verpoorte, R. 29]. Kale contains a large number of glucosinolates and polyphenols [Björkman, M., Klingen, I., Birch, A. N. E., Bones, A. M., Bruce, T. J. A., Johansen, T. J.,...Stewart, D. 211], which, as active antioxidants, largely determine its therapeutic and prophylactic properties. A large number of biologically active compounds, such as vitamins, mineral elements and phenolic compounds, have been found in the tissues of this plant. Currently, about 7 flavone glycosides have been identified in kale. They include 27 non-acylated, 3 monoacylated and 14 diacylated glycosides based on flavone quercetin, kaempferol, and isoramnetin aglycons [Schmidt, S., Zietz, M., Schreiner, M., Rohn, S., Kroh, L. W., & Krumbein, A. 21, Strand, A., Foyer, C. H., Gustafsson, P., Gardestrom, P., & Hurry, V. 23]. It is known that flavonoids have antioxidant properties, which, apparently, forms the basis of their protective effect both for plants and for humans. Therefore, it is of interest to find out factors that may enhance the synthesis of these compounds in kale plants. It is known that under the influence of stress factors plants often react with the accumulation of secondary metabolites, in particular, flavonoids [Falcone Ferreyra M.L., Rius S.P. and Casati P. 212], since these metabolites can play an important role in acclimating plants to unfavorable environmental factors. At the same time, the possibilities of enhancing the synthesis of these compounds with the help of phytohormones and other biological growth regulators have been little studied. Also, nitrogen-based fertilizers are important for both synthesis of primary and secondary compounds. It is believed that the ratio of nitrogen, carbon and sulfur determines the Pr 31

3 redistribution of precursors between primary and secondary metabolism [Bryant, J. P., Chapin, F. S., & Klein, D. R. 1983; Mugford, S. G., Lee, B. R., Koprivova, A., Matthewman, C., & Kopriva, S. 211; Scheible, W. R. D., Morcuende, R., Czechowski, T., Fritz, C., Osuna, D., Palacios-Rojas, N., Schindelasch, D., Thimm, O., Udvardi, M. K., & Stitt, M. 24]. It is assumed that, since glucosinolates contain nitrogen in their composition, their content increases under carbon deficiency. As a result, excessive nitrogen is spent on the synthesis of secondary metabolites [Bryant, J. P., Chapin, F. S., & Klein, D. R. 1983]. Nevertheless, glucosinolates also contain carbon in their composition. Therefore, the uniform application of nitrogen fertilizers throughout the growing season during the growth of kale can have a lesser effect on the balance between primary and secondary metabolism than a single application of nitrogen fertilizers [Groenbaek, M., Jensen, S., Neugart, S., Schreiner, M., Kidmose, U., & Kristensen, H. L. 214]. At the same time, the influence of other mineral elements on the synthesis of secondary metabolites of kale remains little studied. This paper is devoted to the study of the effect of natural hormones (Brassinolid and gibberellic acid), commercial plant growth and development biostimulators (ecopin - a complex of poly-beta-hydroxybutyric acid and mineral elements, Novosil - a complex of triterpenic acids derived from fir needles) and macro- and microelements, as well as organic compounds for antioxidant activity and the content of flavonoids and carotenoids in kale plants. 2. MATERIAL AND METHODS The object of the study was the kale leaves. Plants were grown in the field on sandy loamy soil. The distance between rows and plants was 45/45 cm. At the stage of 3rd-4th leave, the plants were treated with growth regulators by spraying. The investigated compounds were used in the following concentrations: Brassinolid M and gibberellic acid M, ecopin -.25 g/ha and Novosil - 5 g/ha. At the same stage of vegetation, fertilization of plants with various mineral and organomineral fertilizers was carried out. Ammonium nitrate (2 kg/ha), complex fertilizer NPK (15:15:15, 2 kg/ha) and organomineral fertilizers Humate (75% humic acids, 5% K, 2% trace elements), and Biostim (1% free amino acids, 6% N, 3% K, 5% S). The area of the plots is 1 Pr 32

4 m 2. The content of flavonoids, carotenoids and antioxidant activity was determined 7 days after treatment. For the analysis of flavon glycosides and antioxidant activity, the samples were extracted with 9% ethanol. The content of flavonoids in terms of quercetin was calculated by the formula: X=D*25*1*1/764.6*m*2*(1-W), where D is the optical density of the test solution; specific absorption index of the quercetin and aluminum chloride complex at 43 nm; m is the mass of raw materials in grams; W is the mass loss on drying, %. Antioxidant activity was expressed as percent of inhibition of adrenaline autooxidation and was calculated by the following formula: AA, % = (D1-D2)*1/D1, where D1 is the optical density of the adrenaline hydrochloride solution added to the sodium carbonate buffer; D2 is the optical density of the extract and adrenaline hydrochloride added to the sodium carbonate buffer. The content of carotenoids was determined by the spectrophotometric method. To do this, an acetone extract was first obtained by placing the leaves (.1 g) in a porcelain mortar, added with a little calcium dioxide and washed quartz sand, and triturated with 2 ml of a 1% acetone solution. The mashed mass was added with 4 ml of acetone and triturated again for several minutes. The extract was gently poured over the stick into a funnel with a paper filter. The filter was washed several times with a solvent until the pigments were completely removed. The filtrate was then poured into a 1 ml volumetric flask. Then, the content of the flask was made up to the mark, covered with a rubber stopper, shaken carefully and used to determine the concentration of pigments. The measurements were carried out on a spectrophotometer at several wavelengths: 662 nm, 644 nm and 44.5 nm. Concentration of pigments was calculated by the equations: С chl.a+chl.b = 5.134D D 644 ; С car = 4.695D C chl.a+chl.b. Statistical processing of the data was carried out by determining the mean arithmetic values and their standard deviations. All experiments were carried out at least in three biological replication. Replication within one experiment is triple. Pr 33

5 % inhibition of autooxidation of adrenaline Influence of biological growth regulators and elements of mineral 3. RESULTS AND DISCUSSION The formation of reactive oxygen species occurs at all stages of the life of the plant. They participate in the regulation of lipid composition and permeability of membranes, stomatal movements, promote the synthesis of phytohormones and physiologically active compounds. At low concentrations, ROS induces gene expression and cell division [Makarenko O.A., Levitskii A.P. 213]. The maintenance of ROS at a low level in the cell is promoted by various antioxidants. Synthesis of these compounds is controlled by different genes, the expression of which largely depends on hormonal substances. As can be seen from Fig. 1, the growth regulators studied increased antioxidant activity in the kale leaves to varying degrees. The greatest effect was produced by gibberellic acid and Novosil. Figure 1 - The effect of biological growth regulators on the overall antioxidant activity in cabbage kale plants. The nature of the antioxidant cells is very diverse. In particular, they include flavonoids. One of the priority functions of flavonoids in leaves of many species is the protection of plants from bright light and ultraviolet (UV) radiation, potentially dangerous due to damage to DNA, proteins and membranes. The best antioxidants among flavonoids are flavonols, especially - quercetin. It is 5 times more active than vitamin C and E. Another flavonol - tempferol - is less active [Ibrahim, M. H., Jaafar, H. Z. E., Rahmat, A., & Rahman, Z. A. 211]. In our experiments, growth regulators promoted increase in the content of flavonoids, especially under the influence of Brassinolid and ecopin (Fig. 2) Control Brassinolide Gibberellic acid Novosil Ecopin Pr 34

6 % of wet weight % of dry weight Influence of biological growth regulators and elements of mineral 4 3,5 3 2,5 2 1,5 1,5 Control Brassinolide Gibberellic acid Novosil Ecopin Figure 2 - Influence of biological growth regulators on the content of flavonoids in cabbage kale plants. Lipophilic antioxidants include carotenoids, which are most effective in quenching the excessive energy of triplet chlorophylls and singlet oxygen. For humans, in addition, carotenoids are a precursor of vitamin A. According to our data, the content of carotenoids increased under the influence of Brassinolid and Novosil (Fig. 3).,5,45,4,35,3,25,2,15,1,5 Control Brassinolide Gibberellic acid Novosil Ecopin Figure 3 - The effect of biological growth regulators on the content of carotenoids in cabbage kale plants. As can be seen from Fig. 4, the content of flavonoids increased after their fertilization with organomineral fertilizer Biostim and ammonium nitrate. Pr 35

7 % of dry weight Influence of biological growth regulators and elements of mineral 4 3,5 3 2,5 2 1,5 1,5 Control Ammonium nitrate NPK Humate Biostim Figure 4 - Effect of elements of mineral nutrition on the content of flavonoids in cabbage kale plants. It is assumed that a decrease in the nitrogen content enhances the synthesis of secondary compounds with a high carbon content, such as flavone glycosides. The activity of phenylalanine ammonia lyase, an enzyme that catalyzes one of the three main stages of the synthesis of flavonoids, is inhibited by high nitrogen concentrations [Ibrahim, M. H., Jaafar, H. Z. E., Rahmat, A., & Rahman, Z. A. 211]. Studies conducted on kale cabbage have shown that a decrease in nitrogen sorptospecifically affects the content of glucosinolates and flavonoids. A decrease in the nitrogen dose reduced the content of individual glucosinolates, quercetin and common flavone glycosides in the Reflex F1 hybrid, in contrast to traditional varieties that are less sensitive to nitrogen. The introduction of nitrogen into nitrogen-depleted growth media of Arabidopsis thaliana sprouts enhanced the expression of compounds stimulating the synthesis of chalcone synthase and flavone synthase - enzymes involved in the synthesis of flavonoids [Olsen, K. M., Slimestad, R., Lea, U. S., Brede, C., Lovdal, T., Ruoff, P. Lillo, C. 29]. Carotenoids, in addition to performing the functions of auxiliary pigments during photosynthesis, also have a number of properties important for plants. They play a crucial role in protecting plants from active forms of oxygen, in particular, singlet oxygen and triplet chlorophyll. In addition, after getting into the human body, they form vitamin A. In our experiments, organomineral fertilizers Humate and Biostim increased the content of carotenoids (Fig. 5). Pr 36

8 % inhibition of autooxidation of adrenaline % of wet weight Influence of biological growth regulators and elements of mineral,35,3,25,2,15,1,5 Control Ammonium nitrate NPK Humate Biostim Figure 5 - Effect of elements of mineral nutrition on the content of carotenoids in cabbage kale plants Figure 6 - Effect of elements of mineral nutrition on the overall antioxidant activity in plants of cabbage kale. Since both flavonoids and carotenoids protect cells from reactive oxygen species, we studied antioxidant activity in kale plants. As can be seen from Fig. 6, despite the increase in the content of flavonoids after fertilizing plants with ammonium nitrate, the total antioxidant activity increased only under the influence of organomineral fertilizers Humate and Biostim (Fig. 6). 4. SUMMARY Control Ammonium nitrate NPK Humate Biostim The results obtained by us in field studies show that the antioxidant activity in the cells of kale plant changes, and the content of carotenoids and flavone glycosides increases under the influence of plant growth regulators and organomineral fertilizers. Pr 37

9 5. CONCLUSION Influence of biological growth regulators and elements of mineral The conducted studies testify to the possibility of regulation of the general antioxidant system of plants with the help of biological growth regulators. The most effective were di- and triterpenoids. An increase in the antioxidant activity of kale plants under the influence of biological growth regulators was mainly due to an increase in the content of secondary metabolites, in particular, flavonoids. In addition, triterpenoids increase the content of carotenoids (provitamin A). Also, fertilizers with a low nitrogen content and a relatively high content of organic compounds can be considered more promising for growing kale, as they increase the antioxidant properties of carotenoids and flavonoids. 6. ACKNOWLEDGEMENTS The work is performed according to the Russian Government Program of Competitive Growth of Kazan Federal University. 7. REFERENCES Björkman, M., Klingen, I., Birch, A. N. E., Bones, A. M., Bruce, T. J. A., Johansen, T. J.,...Stewart, D. Phytochemicals of Brassicaceae in plant protection and human health Influences of climate, environment and agronomic practice // Phytochemistry V.72(7). - C Bryant, J. P., Chapin, F. S., & Klein, D. R. Carbon nutrient balance of boreal plants in relation to vertebrate herbivory // Oikos V. 4(3). C Falcone Ferreyra M.L., Rius S.P. and Casati P. Flavonoids: biosynthesis, biological functions and biotechnological applications // Front.PlantSci V. 3. C doi:1.3389/fpls Jahangir, M., Kim, H. K., Choi, Y. H., & Verpoorte, R. Health-affecting compounds in Brassicaceae // CRC Critical Reviews in Food Science and Nutrition V C Groenbaek, M., Jensen, S., Neugart, S., Schreiner, M., Kidmose, U., & Kristensen, H. L. Influence of cultivar and fertilizer approach on curly kale (Brassica oleracea L. var. sabellica). 1. Genetic diversity reflected in agronomic characteristics and phytochemical concentration // Journal of Agricultural and Food Chemistry V. 62(47). C Pr 38

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