Effect of sodium selenate foliar supplementation on Cryptotaenia japonica and Petroselinum crispum nutritional characteristics and seed quality

Оригинальные статьи / Original articles УДК 631.811.98:631.531:(635.532+635.753)

P rotection of human organism against oxidant stresses is an effective way for sustainable development of the society and improvement of human health [1]. The significance of antioxidants in human health stimulates the developm ent of functional and supple-m ental food containing high levels of antioxidants. Taking into account the protective antioxidant role of selenium against various chronic diseases, such as cardiovascular, oncological, viral caused by SARS-CoV -2 virus [2.3], special attention is paid to the production of vegetables, as the main source of antioxidants, biofortified with selenium (Se). Several investigations indicate that Se supply may enhance plant antioxidant status, increasing the accumulation of the m ost im portant antioxidants, i.e., vitamin C, polyphenols, carotenoids [4]. In this respect, Se biofortification of vegetable crops dem onstrates the greatest potential due to antioxidant properties of Se capable to im prove both plants and human immunity [5], stimulate plant secondary metabolites production, including the most powerful antioxidants such as polyphenols, carotenoids and ascorbic acid, and increase agricultural crop yield [6].

Despite such optimistic prognosis, there are still a lot of unresolved questions in the development of Se-biofor-tification technology. F irstly, narrow concentration range of Se for obtaining high beneficial agrochemical effect on plants is species and variety dependent, which makes it extremely difficult to predict the product’s quality and yield under Se supply. Secondly, Se biofortification of plants with high antioxidant activity have not yet provided sufficient data relevant to the process peculiarities. Thirdly, scant information is available on Se enriched seeds viability [7]. Taking into account the synergistic relationship between Se and organic antioxidants such as ascorbic acid, polyphenols, proteins [8,9], parsley is one of the finest object for Se biofortification, due to extrem ely high levels of natural antioxidants [10]. Indeed, parsley im portance is attributed to its high vitamins concentration (mainly vitamin C ), antioxidants [11], and som e mineral elements such as iron [12] as well as volatile oils that play an im portant role in the pharm aceutical and food industries [13]. Parsley has been used m edicinally since ancient tim es for possible m edicinal qualities including antioxidative [14], anti-carcinogenic [15], antimicrobial, laxative, anti-hyperlipidemic, anticoagulant and antihepatotoxic [16]. Because of the many antioxidants present in this plant, a diet including fresh parsley leaf can significantly increase antioxidant capacity, which plays a special role in people nutrition [10,17,18].

Nevertheless, up-to-date only few attempts have been achieved for parsley biofortification with Se, though the literature data indicate the presence of Se-M e-Se-M et (up to 21%) and Se-Me-Se-Cys (up to 4.4%), which are powerful natural anti-carcinogens [19]. No information is still available regarding Se biofortification of Japanese parsley Cryptotaenia japonica (Mitsuba), also known to be rich with natural antioxidants [20].

The aim of the present work was to assess the sodium selenate biofortification efficiency in foliar treatment of M itsuba and 5 parsley varieties, and the determination of Se-enriched seed quality.

Material and Methods

Four parsley cultivars, two of leafy type, Breeze and Moskvichka, one of curly type, Krasotka, and one of root type, Zolushka, plus the Japanese variety Mitsuba were grown at the experimental fields of F ederal Scientific Vegetable Center (Moscow region, 55°39.51' N, 37°12.23' E), in sod-podzolic clay-loam soil, pH 6.8 , 2.1% organic matter, 1.1 g·kg^-1 N, 0.045 g·kg^-1 P 2 O 5 , 0.357 g·kg^-1 K 2 O.

The mean values of temperature (°C) and relative humidity (%) as an average of 2018 and 2019 in Moscow region were the following: 16.1 and 71.8 in May; 21.0 and 73.0 in June; 23.8 and 74.9 in July; 19.0 and 76.9 in August and 14.8 and 86.0 in September.

The experimental protocol was based on the factorial combination between 5 cultivars and a selenium treatment plus an untreated control, using a split plot design with three replicates, each including 10 plants.

The seeds were sown in multicell trays filled with peat in a heated greenhouse, in the first decade of March, and the seedlings were transplanted in open field in the second decade of May. Plant care consisted of weeding, loosening row spacing and top dressing. First top dressing was carried out 2 weeks after seedlings planting. 1.5-2.0 centners of ammonium nitrate, potassium salt and 0.75-1 centners of superphosphate were applied per hectare twice: two weeks after planting and in mid-July. Foliar biofortification of plants with sodium selenate at a concentration of 25 mg Se/L (1.0 L m-²). was carried out in mid-July, the second - two weeks later. Each treatment included three repetitions. Harvesting was carried out in mid-August.

After cutting, plant fresh weight (g) of seven plants per replicate, was measured. Leaf area (cm²) was recorded for the whole plant (10 plants / replicate) using a Li-Cor 3100 area meter (Li-Cor, Lincoln, NE, USA).

Growing Conditions and Experimental Protocol

Sample Preparation

After harvesting leaves, stalks and roots were separated and weighed, roots were washed with water and dried at filter paper. Sam ples were homogenized and fresh homogenates were used for the determination of ascorbic acid, nitrates and water-soluble compounds (TDS) content. Part of samples was dried at 70°C to constant weight and was used for the determination of polyphenols, flavonoids content, total antioxidant activity and mineral composition.

Dry Matter

The dry residue was assessed gravimetrically by drying the samples in an oven at 70°C until constant weight.

Ascorbic acid

Ascorbic acid content was determined using visual titration method with sodium 2,6-dichlorophenol indophenolate (Tillmanse reagent) [21].

Preparation of ethanolic extracts

One gram of dry leaves/petioles/roots powder was extracted with 20 mL of 70% ethanol at 80°C over 1 h. The mixture was cooled and quantitatively transferred to a volumetric flask, and the volume was adjusted to 25 mL. The mixture was filtered through filter paper and used further for the determination of polyphenols, flavonoids and total antioxidant activity.

Polyphenols

Polyphenols were determined spectrophom etrically based on the F olin–C iocalteu colorim etric m ethod according to [8 ]. The concentration of polyphenols was calculated according to the absorption of the reaction mixture at 730 nm using 0.02% gallic acid as an external standard.

Antioxidant Activity (AOA)

The antioxidant activity was evaluated via titration of 0.01 N KMnO 4 solution with ethanolic extracts of dry samples [8].

Flavonoids

The total flavonoids content was determined by a spectrophotometric method based on flavonoid–aluminum chloride (AlCl₃) complexation [22]. A quantity of 0.5 mL of the ethanolic extract was diluted with 1.5 mL of 70 % ethanol, and 0.1 mL of 2% AlCl 3 , 0.5 mL of 1 M sodium acetate solution, and 1 mL of distilled water were added. The mixture was left for 30 min at room temperature and the absorption at 415 nm was measured. The total flavonoid content was determined using quercetin (Fluka, Switzerland) as an external standard

Total Dissolved Solids (TDS)

TDS was determined in water extracts using TDS-3 conductometer (HM Digital, Inc., Seoul, Korea).

Nitrates

Nitrates were assessed using ion selective electrode on ionomer Expert-001 (Econix, Russia)

Photosynthetic Pigments

Chlorophyll a, chlorophyll b and carotene accumulation levels in leaves were analyzed using 9 8 % ethanolic extracts absorption levels at 470, 649 and 664 nm according to [23].

Selenium

Fluorimetric method was used for the determination of selenium content [24]. The precision of the results was verified using a reference standard-lyophilized cabbage in each determination with Se concentration of 150 μg·kg-1.

Element Composition

Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, Hg, K, Li, Mg, Mn, N a, N i, P, Pb, Si, Sn, Sr, V , and Zn contents in dried homogenized samples were assessed using ICP-MS on quadruple mass-spectrometer N exion 300D (Perkin Elmer Inc., Shelton, CT, USA), equipped with the sevenport FAST valve and ESI SC DX4 autosampler (Elemental Scientific Inc., Omaha, NE, USA) at the Biotic Medicine Center (Moscow, Russia). Rhodium 103 Rh was used as an internal standard to eliminate instability during measurements. Quantitation was perform ed using external standard (M erck IV , m ulti-elem ent standard solution); Perkin–Elmer standard solutions for P, Si, and V, and all the standard curves were obtained at five different concentrations. F or quality control purposes, internal controls and reference materials were tested together with the samples daily. Microwave digestion of samples was carried out with sub-boiled HNO 3 diluted 1:150 with distilled deionized water (Fluka No. 02, 650 Sigma-Aldrich, Co., Saint Louis, MO, USA) in the Berghof SW-4 DAP-40 microwave system (Berghof Products + Instruments Gmb H, 72, 800 Eningen, Germany). The instrument conditions and acquisition parameters were: plasma power and argon flow, 1500 and 18 L min^-1, respectively; aux argon flow, 1.6 L min^-1; nebulizer argon flow, 0.9 8 L min^-1; sample introduction system, ESI ST PFA concentric nebulizer and ESI PF A cyclonic spray chamber (Elemental Scientific Inc., Omaha, NE, USA); sam pler and slimmer cone material, platinum; injector, ESI Quartz 2.0 m m I.D.; sam ple flow, 637 L min^-1; internal standard flow, 84 L min^-1; dwell tim e and acquisition m ode, 10–100 m s and peak hopping for all analytes; sweeps per reading, 1; reading per replicate, 10; replicate number, 3; DRC mode, 0.55 mL min^-1 ammonia (294993-Aldrich Sigma-Aldrich, C o., St. Louis, M O 63103, USA) for Ca, K, Na, Fe, Cr, V, optimized individually for RPa and RPq; STD mode, for the rest of analytes at RPa=0 and RPq=0.25 [60].

Trace levels of Hg and Sn in samples were not taken into account and, accordingly, they were not included in the tables.

Statistical Analysis

In order to calculate the mean values and standard

Table 1. Biometric parameters and dry matter content of parsley leaves

Variety	Treatment	Total mass, g	Leaves mass, g	Height, cm	Dry matter, %
Breeze	Control	532.5b	444.9b (83.5%)	43.3bc	19.9a
	Se	449.7b	355.1c (79.0%)	41.3cd	21.6a
Moskvichka	Control	766.1a	626.3a (81.8%)	40.9cd	21.0a
	Se	628.5ab	476.7b (75.8%)	42.6bcd	23.9a
Krasotka	Control	679.6a	451.6b (66.5%)	35.1d	23.7a
	Se	467.1c	374.1c (80.1%)	27.2e	22.7a
Zolushka	Control	519b	369c (71.1%)	51.6b	22.5a
	Se	340d	237d (69.7%)	48.3bc	23.8a
Mitsuba	Control	452.2c	339.9c (75.2%)	78.2a	24.5a
	Se	503.3bc	365.7c (72.7%)	84.9a	23.1a

Values in columns with similar letters do not differ statistically according to Duncan test at P<0.05

errors, the R statistical version 2.5.1 (The R Project for Statistical Computing, Lyon, France) was used.

Data were processed by two-way analysis of variance, and mean separations were performed through Duncan’s multiple range test, with reference to a 0.05 probability level, using SPSS software version 21. Data expressed as percentage were subjected to angular transformation before processing.

Results and discussion

Yield and biometrical parameters

Among parsley cultivars studied no statistically significant changes in dry matter content as a result of Se treatm ent were registered, though one may indicate a tendency in yield increase of Mitsuba parsley due to Se supplem entation. On the contrary, a small decrease in leaves mass happened to be typical for other cultivars: leafy (Breeze, M oskvichka cvs), curly (Krasotka) and root parsley (Z olushka) while statistically significant total mass decrease was recorded for Krasotka and Zolushka cultivars. These data indicate lack of Se growth stimulation effect using foliar application of relatively low sodium selenate concentration (25 mg L^-1) both on Petroselinum crispum L. and Cryptotaenia japonica grown in Moscow region and parsley cultivated in the Chechen republic.

Potassium, total dissolved solids (TDS) and nitrates

Parsley is known to accumulate high levels of nitrates [25] which may be either beneficial for heart care or harmful in case of nitrates excess [26]. Data presented in Table 2 indicate the highest values of nitrates accumulation by Mitsuba and the lowest ones for curly parsley Krasotka. According to the received data (Table 3) Se decreases nitrate content in parsley leaves, the effect being as a tendency for Breeze, Moskvichka, Zolushka cvs, or statistically significant for Krasotka cv (25.0%) and Mitsuba (11.6%). These results are in good agreement with the known fact of selenate effect on nitrate reductase activity [27] but signif- ex 80

el

.-75

E- 70

Zolushka -'•

Moskvichka

Breeze

Krasotka

у = 6.8251x+24.365

R1 = 0.7924

6      6.5      7      7.5      8      8.5      9      9.5

K,g/kg

Figure 1. Relationship between TDS and K in parsley leaves fortified with Se (r-+0.890; P<0.01)

In a whole one can indicate significant inter-varietal differences in plants response to Se supplementation, the most sensitive being Zolushka and Mitsuba.

icant variations in plants sensitivity to Se supply indicate the necessity of further investigations.

Literature data indicate that the effect of Se on K accumulation depends on the dose applied and may be both positive or negative [28]. Mitsuba and parsley varieties differed significantly on the Se effect: increase in TDS was registered only for Zolushka (1.79) and Mitsuba (1.20 tim es), while K accumulation due to Se application increased only in M oskvichka, Zolushka and Mitsuba plants (1.21; 1.38 and 1.56 times accordingly). No statistically significant differences were recorded for TDS of control and Se fortified leafy parsley while lack of K changes was indicated for Breeze and Krasotka cultivars. The results also indicate that K plays the dominant role in TDS value of plants fortified with Se (Fig. 2).