Nutritional value of vegetable Amaranthus tricolor L. seedlings grown in Moscow region

. The use of amperometric express method made it possible to measure quickly and eval-uate content of water- and alcohol-soluble antioxidants in extracts from Amaranthus tricolor L. plants. Accumulation of low molecular weight antioxidants: ascorbic acid, beta-cyanine (amaranthine) and the total content of antioxidants in various organs of Valentina amaranth seedlings were studied. The maximum amount of low molecular weight antioxidants accumulates in leaves, compared with roots and stems of seedlings grown in open and protected ground. In open ground conditions, amaranth leaves and stems have 1.5-fold and 2-fold increased level of ascorbic acid than seedlings grown in protected ground. But the total content of water-soluble antioxidants in leaves and roots of seedlings is lower compared to seedlings of protected ground. Minimum amount of antioxidants was found in alcohol extracts of stems and roots in open ground, while the total content of antioxidants in stems and roots was 1.6 fold higher in seedlings grown in protected soil. The content of amaranthine is comparable in the studied organs of amaranth seedlings of both cultivation variants. The data obtained allow to recommend use of leaves and stems of amaranth seedlings grown in open and protected ground (early spring and autumn), as a preventive antioxidant dietary product. seedlings grown in open less acid and a lower of hydrophilic and were found, which suggests a more active outflow of metabolites — antioxidants from to aboveground to recommend use of amaranth seedlings grown in open and protected ground as a dietary antioxidant for preventive


Introduction
In hot and humid regions of the world, edible species of leafy amaranths (genus Amaranthus) are considered to be popular vegetable crops: A. tricolor, A. blitum, A. dubius, A. cruentus and A. Viridis [1][2][3]. In many countries of Africa and Southeast Asia, India, southern China, amaranth leafy greens are widely used for food purposes, growing it as parsley, leaves and stems of which are used for food purposes. Young plants of vegetable species of amaranth are used in a variety of salads, appetizers, side dishes, soups, fillings for confectionery, drinks, pasta and even traditional medicine [4][5][6][7].
Such widespread use of vegetable amaranth as a food product is explained by a number of reasons, including the fact that spicy aromatic plants are practically not grown in these countries, and amaranth makes up for greens in many dishes [8,9]. The popularity of amaranth vegetables is due to their mild piquant taste and high nutritional value, leaves of which are rich in gluten-free protein, vitamins, minerals, especially calcium, iron, as well as biologically active substances [10]. In addition, in a number of countries there is a shortage of animal protein, and leaves of amaranth vegetable species contain up to 20 % of a complete protein, balanced for essential amino acids. Therefore, population of these regions replenishes the lack of dietary protein by leaves of wild vegetable amaranth, growing seedlings and preparing various diets based on them [11].
Amaranth seedlings (young plants) are a commercial product in some countries. In Indonesia, amaranth vegetables are grown on an area of 2000 hectares. In tropical countries, amaranth is sown year-round. Due to the short development cycle of seedlings (7-8 weeks), they are cut for food several times a year.
Cultivation of vegetable amaranth in non-chernozem zone faces a number of problems. For example, return cold in spring months of April-May does not allow sowing of amaranth seeds in open ground before the end of May or the beginning of June; moisture deficiency in dry years can ruin the crop, since amaranth seedlings require more watering, compared to adult plants. In addition, amaranth seedlings in open ground can suffer from weeds, whose growth rate is much higher than growth of amaranth seedlings, and later snails can harm young seedlings [12]. When growing amaranth seedlings in a protected ground, such problems do not occur. Therefore, it is important to represent change patterns in the main morphological and biochemical parameters that determine productivity and nutritional and pharmacopoeial value of seedlings grown in open and protected ground.
The aim of the work was to study morphological and biochemical parameters of amaranth seedlings when growing them in open and protected ground for food use.

Materials and methods
The object of the study are vegetable amaranth plants (Amaranthus tricolor L.) of Valentina cultivar originated in Russian Research Institute of Selection and Seed Production of Vegetable Crops (Moscow Region). Cassettes with pre-moistened peat mixture were used for sowing seeds. Sowing depth was 0.5…1 cm. After 4 weeks, the seedlings were transplanted into soil in protected and open ground [13]. Plants were grown on sod-podzolic soil with a heavy mechanical composition at night temperature of 8…14 °C and day temperature 9…25 °C in open ground, and at night temperature 14…17 °C and day temperature 22…30 °C in protected ground.
Young plants aged 6-7 weeks were cut off and morphometric indicators were studied: plant height, mass of plants, leaves, stem, root, length and width of leaf blades. In the experiment, amaranth plants of Valentina cultivar grown in protected and open ground were compared. From each experimental plot, 15 plants were collected.
Biochemical studies were carried out in the Laboratory of physiology and biochemistry of introduction and functional products of Federal Scientific Technological Center in 2018. Aqueous and alcoholic extracts of leaves, stems and roots of seedlings were used in the experiments.
Extraction of crushed leaves and other plant organs was carried out with distilled water at room temperature (water ratio 1:10), followed by centrifugation at 10,000 rpm. Amount of amaranthine in aqueous extracts was determined spectrophotometrically considering a molar extinction coefficient of 5.66⋅10 4 ⋅M -1 ⋅cm -1 and a molar weight of 726.6 [14]. Content of reduced form of ascorbic acid (AA) was determined by iodometric method based on titration of ascorbic acid in colored extracts with potassium iodate in acidic medium in presence of potassium iodide and starch [15]. The total content of antioxidants was determined by amperometric method, the result was expressed in gallic acid equivalents mEq.GK/g. The measurements were performed on a Tsvet-Yauza 01-AA device in a constant current mode [16]. The samples were crushed on a homogenizer in presence of a certain volume of extracting liquid (double-distilled water, 96% ethanol) at 20…25 °C. Then, the homogenizer was centrifuged at 10,000 g for 15 minutes at 4 °C. An aliquot of the supernatant was used to determine the total antioxidant content, if necessary diluting.
The tables 1-4 show the arithmetic mean values and standard deviations.

Results and discussions
Dark-colored A. tricolor seeds of Valentina cultivar germinated with an interval of two days. A slower increase in seedling height was observed in the stage of the first pair of true leaves and a sharp increase in plant height with development of subsequent leaves. Seedlings having well-developed cotyledons and first pair of true leaves were transplanted into protected and open ground.
Analysis of morphometric parameters of plant before cutting revealed that amaranth seedlings grown in sheltered soil had significantly higher morphometric indicators compared to seedlings grown in open ground.
The photosynthetic productivity of aerial parts of plants was significantly higher in seedlings in protected soil. When growing amaranth plants in protected ground at seedling stage, a more massive main shoot (stem) with a smaller mass of leaves is formed compared to open ground plants. Study of structure of seedlings' crop showed that aerial mass of seedlings grown in open ground have 52 % of leaves, 35 % of stems, while young plants in protected ground form 42 % of leaves and 44 % of stems.
It is interesting to note that root mass of seedlings of both variants was 13…14 % of the aboveground mass. This suggests that photosynthetic metabolites in plants of open ground are accumulated in large quantities in leaves, while they are distributed almost evenly between leaves and stems in plants of protected ground.
Gluten-free protein, balanced for essential amino acids, and biologically active substances with antioxidant activity that affect physiological functions of the human body, effectively participating in metabolic and protective reactions comprise nutritional and pharmacopoeial value of Valentina leaves (amaranth A. tricolor L.) [17]. Ascorbic acid is a necessary component for human life. Some vegetable crops accumulate ascorbic acid in high concentrations: bell pepperup to 200 mg%, leafy vegetable plants: vegetable chrysanthemumup to 80 mg%, watercress and corianderup to 150 mg% [18].
Amaranth seedlings accumulate reduced ascorbic acid in all organs, but in different amounts. The data presented in table 2 indicate that the maximum amount of ascorbic acid accumulates in leaves of both variants, and the minimumin stems of seedlings of protected soil and in roots of seedlings of open ground. The data obtained indicate that content of ascorbic acid in various plant organs depends on temperature conditions during cultivation. It is known that plant cell reactive oxygen species are formed at low temperature, where superoxide anion radical is the most dangerous. Ascorbic acid is able to neutralize O2-. In open ground, a decrease in night temperature to 8 °C and lower for heat-loving seedlings is a stress factor that slows down their growth and development. Therefore, under these conditions, ascorbic acid, whose level in leaves of amaranth grown in open ground, was 1.5 fold higher than that in leaves of plants of protected ground, serves as protection from action of superoxide anion radicals. Compared to leaves, 5.45-fold decreased ascorbic acid was accumulated in stems of protected seedlings and 3.7-fold less accumulated ascorbic acid was in stems of open ground seedlings. An unequal amount of ascorbic acid was found in roots of young plants grown in open and protected ground. Ascorbic acid was accumulated in large quantities in leaves and stems of seedlings grown in open ground, which might indicate its active generation under conditions of weak low-temperature stress. In addition to ascorbic acid, red-colored pigment amaranthine with antioxidant activity comparable to that of superoxide dismutase is involved in the detoxification of the superoxide anion radical [19]. Leaves and inflorescences of amaranth seedlings of both cultivation variants contained practically comparable amounts of amaranthine, while stems of young open-ground plants accumulated 1.5 times more antioxidant than stems of seedlings of protected soil.
The study of the total content of antioxidants in leaves and stems of amaranth seedlings grown in protected and open ground revealed the maximum content of antioxidants in water extracts, which was 2.5-3 fold higher than the level of antioxidants extracted in alcohol extract.
Moreover, a lower content of water-and alcohol-soluble antioxidants was found in stems of seedlings of both variants, but it was comparable with leaves.
Flavonoids have previously been shown to be contained in leaves of Valentina amaranth [19]. It should be noted that electrochemical oxidation of low molecular weight molecules with antioxidant activity of aqueous and alcoholic extracts can be described using flavonoids as an example by the following reaction: flavonoid -O-Hflavonoid -O+e+H + .
Ability of ascorbic acid, amaranthine and flavonoid molecules to oxidize on electrode at a given potential indicates the ability of these molecules to capture free radicals [21]. The high total content of water and alcohol soluble antioxidants and ascorbic acid in the leaves of amaranth seedlings indicates a high antioxidant potential of seedlings, which actively protects young open ground plants from low temperature stress factors.

Conclusions
Low night temperature (8…10 °C) in open ground has a positive effect on photosynthetic productivity, growth and development of seedlings. Moreover, their leaf mass is characterized by the maximum amount of ascorbic acid, a comparable amount of amaranthine and the total content of water-and alcohol-soluble antioxidants. At a lower night temperature, more ascorbic acid and hydrophilic antioxidants are accumulated in stems of open-ground plants compared to protected-seedlings grown at optimum temperature.
In the roots of seedlings grown in open ground, less ascorbic acid and a lower content of hydrophilic and hydrophobic antioxidants were found, which suggests a more active outflow of metabolites -antioxidants from roots to plant's aboveground organs.
The data obtained allows to recommend use of amaranth seedlings grown in open and protected ground as a dietary antioxidant product for preventive purposes.