Basil seeds as a source of antioxidants affected by fortification with selenium

The experimental trial was carried out in 2017 and 2018 in the place of the Botanical Garden of the Department of Vegetable Production (Slovak University of Agriculture [SUA] in Nitra). Seed sowing and pre-propagation of plants were carried out in the greenhouse of the Botanical Garden SUA. The seeds were purchased from company Semo s.r.o. The pre-grown seedlings were planted in an area of 20.3 m². Investigated varieties and species of basil are summarised in Table 1.

The soil type of the experimental area is brown soil to chernozem on loess and loess loams and part along the River Nitra belongs to the area of fluvial soils, where the original soil type was fluvial and fluvial glue (Hreško et al., 2006). Fertilisation of the plants was done based on analysis of the experimental area in every evaluated year (Table 2). During the tested years, the soil from experimental area was analysed from the fertilising point of view by Department of Agro-chemistry and Plant Nutrition (Table 2).

In terms of climatic classification of the region, Nitra is situated in a warm and dry area of Slovakia. The evaluation of experimental years according to climatic normal is given in Tables 3 and 4.

The cultivation of plant material was carried out in accordance with modern agrotechnical practices of basil field cultivation. Seed sowing took place on 8 March 2017 and 15 March 2018 in greenhouse of the Botanical Garden (SUA, Nitra). Planting at the permanent place was carried out on 18 May 2017 and 10 May 2018 into well-prepared soil. Plants were planted in 3 rows of 10 plants per variety with a spacing of 0.35 × 0.40 m. The crops were cut after planting because of multiple inflorescences creation followed by plant irrigation. Based on the agrochemical analysis of the soil, fertilisation of the plants with ammonium with dolomite (27% N) at dosage 0.4 kg ∙ 100 m⁻² was carried out in two doses in both experimental years. In the phenological stage of BBCH 61 (10% of flowering flowers), an aqueous solution of sodium selenate at a concentration of 50 mg Se ∙ 1 m⁻² was applied foliarly. The plants were harvested at the stage of botanical maturity of the seeds (when two-third of the seeds on the plant changed colour from white to dark brown or black) at two different dates, as the varieties ripened gradually. The first harvest took place on 10 August 2017 in case of ‘Tulsi’ and ‘Dark Green’ and ‘Tulsi’, ‘Dark Green’ and ‘Cinamonette’ have been cut on 24 August 2017. Manual harvest of the Cinamonette and Tulsi varieties were carried out on 14 August 2018. The Dark Green variety was harvested on 17 August 2018. Subsequent drying and seed cleaning took place in well-ventilated department stores on dry web fabric.

The following reagents were used for the tests: (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox, 97%, Acros Organics™, Denmark), 2,2-diphenyl-1-picrylhydrazyl (DPPH, ≤ 100%, Sigma Aldrich), Folin–Ciocalteu reagent (Merck Germany), gallic acid (GA, Sigma Aldrich), sodium carbonate (solution 20% w/w, Merck Germany), nitric acid (HNO₃, 67%), hydrogen peroxide (H₂O₂, 30%), palladium nitrate (Pd(NO₃)₂, palladium modifier, 0.1 mol ∙ l⁻¹), ascorbic acid (AsA, solution 1%, w/w), methanol (pure pro analysis -purity grades of lab reagents, 70%, v/v, Fisher Scientific UK, Loughborough, UK).

Digestion of the plant material took place in the microwave digestion system type CEM Mars X-press (microwave digestion oven). In the digestion container, there was weighed 0.5 g of the sample. It was wetted with 1 ml double distilled water followed by the addition of 5 ml of concentrated HNO₃ and 1 ml of H₂O₂. It was digested at 150°C for a period of 20 minutes. The digestion product was refilled into volumetric flask till 25 ml. Quantitative determination of Se was done by using of ET-AAS method with Zeeman-effect background correction. Atomic absorption spectrometer (SpectrAA240FS Varian, Mulgrave Virginia, Australia) was used to measure the total selenium content. Conditions for selenium measurement were set in the equipment according to the recommendations of the manufacturer for ET-AAS technique (Rothery and Beach, 1988).

The average sample was created from the analysed basil seeds (dried at room temperature in clean laboratory conditions of the department of vegetable production) by slicing them into tiny bits and homogenised. Then 1 g of homogenised mixture and 40 ml of methanol (70%, v/v) were added into 250-ml extraction flasks. They were allowed to stand at room temperature for 20 h and then extracted with horizontal shaker for 4 h (Melicháčová et al., 2010).

Determination of AOA was performed with a spectrophotometer Jenway 6301 (Bibby Scientific Ltd., UK) by the method of Hegedűs et al. (2019). DPPH inhibition and spectrophotometric measurements were performed after a constant time of 30 min. Of note, 0.1 ml of the extract was pipetted into the spectrophotometer cuvette (depending on the nature of the sample) and supplemented with 70% methanol to 2.0 ml, and 4 ml of DPPH solution of about 25 mg ∙ l⁻¹ concentration was added. Immediately after the DPPH solution was added, the absorbance of the mixture was measured at 517 nm (At₀). Thirty minutes later, the absorbance of each sample was measured at 517 nm (At₃₀). The AOA was calculated based on the following relationship:

Total polyphenol content (TPC) was estimated by using Folin–Ciocalteu assay by the method of Lachman et al. (2003) and calculated in milligram of GA equivalent (GAE) per kilogram dried weight (d.w.). GA is generally used as a standard unit for phenolic content determination because of wide spectrum of phenolic compounds. The Folin–Ciocalteu phenol reagent was added to a volumetric flask containing 100 μl of extract. The content was mixed and 5 ml of a sodium carbonate solution (20%, w/w) was added after 3 min. The volume was adjusted to 50 ml by adding distilled water. After 2 h, the samples were centrifuged for 10 min and the absorbance was measured at 765 nm of wavelength against blank (spectrophotometer Shimadzu UV/VIS-1240). The concentration of polyphenols was calculated from a standard curve plotted with known concentration of GA.

The analysis of variance (ANOVA), the multifactor analysis of variance (MANOVA) and the multiple range test were carried out using the Statgraphic Centurion XVII (StatPoint Inc., USA).

Selenium biofortification with foliar-applied sodium selenate solution in concentration of 5 mg Se ∙ 1 m⁻² significantly (p < 0.05) increased with the content of selenium in basil seeds as shown in Table 5. In 2017, the highest increase in treated variant compared with control was obtained in case of Tulsi basil (from 0.025 to 0.809 mg ∙ kg⁻¹ of Se). In 2018, the similar situation was observed for Dark Green variety with an increase from 0.078 to 0.823 mg ∙ kg⁻¹ of Se. Comparing all data from both tested years (Figure 1), the significant difference (p < 0.05) between tested variants was proved. On the other hand, the influence of variety and the year impact on selenium content was not confirmed (p > 0.05) (Table 5).

Figure 1

The content of selenium ranged from 9 to 95 μg ∙ kg⁻¹. Foliar application of inorganic selenium very significantly increased the content of selenium in basil herb according to the research study by Mezeyová et al. (2016). The most effective here seemed to be the double dose of selenium (5 mg Se ∙ m⁻²) when in ‘Red Rubin’ was incorporated in 7.859 ± 0.9 mg ∙ kg⁻¹ of selenium in comparison with 0.058 ± 0.004 mg ∙ kg⁻¹ (control variant) and in ‘Dark Green’ 4.017 ± 0.8 mg ∙ kg⁻¹ in comparison with control value 0.154 ± 0.05 mg ∙ kg⁻¹. Puccinelli et al. (2019) also tested the ability of basil plants grown in hydroponics to take up Se from the growth substrate and to study the effects of Se concentration on plant growth and Se accumulation. Se concentration increased during seedling growth, was highest in younger leaves and then declined before or on flowering.

After application of 5 mg Se ∙ m⁻² in two varieties of peas, 25-fold increase in the selenium content in seeds was reported in comparison with control in the study by Hegedűsová et al. (2015). As lot of other crops such as peas, rice, corn, wheat (Gomez-Galera et al., 2010; Ozkutlu et al., 2011; Poblaciones et al., 2014; Premarathna et al., 2012; Manojlović et al., 2019; Hawkesford and Zhao, 2007; Poblaciones and Rengel, 2018; Fernandes et al., 2014) were positive in test for incorporation of selenium in grains, there was prediction of selenium increasing possibility in basil seeds. In selenised basil seeds, the increase in selenium content in case of Tulsi was 17-fold in comparison with control variant, 12-fold in ‘Cimonette’ and 12-fold in ‘Dark Green’ compared with control.

On the other hand, the optimum dosage of the selenium fertiliser is very important because of possible selenium toxicity in plants. It mainly depends on their ability to divert selenium away from the accumulation of selenocysteine and selenomethionine which ranges from 2 mg ∙ kg⁻¹ in non-accumulators, such as rice, and 330 mg ∙ kg⁻¹ in white clover, to several thousands of mg ∙ kg⁻¹ in the accumulator Astragalus bisulcatus. Gebreeyessus and Zewge (2019) stated that in non-accumulators selenium toxicity occurs about 10–100 mg Se ∙ kg⁻¹ d.w. Ozkutlu et al. (2011) analysed that 26 medicinal and aromatic plants O. basilicum, widely used as either a fresh or dried spice, presented the highest Se content with 1,133 ± 104 μg ∙ kg⁻¹ d.w., followed by Peganum harmala L. (951 ± 22 μg ∙ kg⁻¹ d.w.), aerial parts of Urtica dioica (content 271 ± 16 μg ∙ kg⁻¹ d.w.) and the seeds of Linum usitatissimum (193 ± 26 μg ∙ kg⁻¹ d.w.). As the basil is good accumulator of Se, it is also important to observe other important quantitative (yields of herbs or seeds) and qualitative parameters (chlorosis of herbs, interfering with chlorophylls and other antioxidants) which can also be influenced by selenium enrichment.

The values of AOA reached from 714.22% inhibition (‘Cinamonette’, 2018, selenised variant) to 1,061.03% inhibition (‘Tulsi’, 2018, selenised variant) as shown in Table 6. The impact of fortification with selenium was not significant (p > 0.05) comparing the data from both tested years (Figure 2). There were found some significant (p < 0.05) differences in varieties in relation to particular year and variant. The impact of the climatic condition in both tested years did not have significant impact on the trial results (Table 6).

Figure 2

In basil seeds, the selenium content was increased without significant impact (p > 0.05) on AOA. In our trial, in some cases selenised variant showed higher values without statistically significance, i.e. in contrast with other authors. The foliar supplementation of selenium and/or AsA, especially the mixed ones, led to significant improvement in antioxidative activities according to Oraghi Ardebili et al. (2015), tested possible impacts of foliar supplementations of Se and/or AsA on basil. Seedlings were foliarly treated with four concentrations of Se (0, 30, 60 and 120 mg ∙ L⁻¹) and/or two levels of AsA (0 and 200 mg ∙ L⁻¹).

According to Sarfraz et al. (2011) total AOA of O. sanctum seed and leaf extract displayed 84.59% and 79.39% inhibition activities, respectively. Ocimum sanctum L. leaves reached 73.70% ± 5.87% by DPPH method in the study by Farrukh et al. (2006). In relation to seeds, 11 varieties of bean were studied by Armendáriz-Fernández et al. (2019) and they were classified into three main groups: (1) high levels, (2) medium levels and (3) low levels of antioxidant capacity. Within the high level of antioxidant capacity are the following bean varieties: Sangre de Toro, Blanco Michigan, Pinto Americano and Flor de Mayo. The Sangre de Toro variety has the highest antioxidant capacity (82.1%), followed by ‘Blanco Michigan’ (81.8%), ‘Pinto Americano’ (80.6%) and ‘Flor de Mayo’ (79.1%). In comparison to our results, there are differences in AA inhibiton. The problem in the antioxidant capacity determination is that the research laboratories use different methods of determination. Result of measurement depends on the initial DPPH concentration and the chosen reaction time, which has been chosen by the authors. The results are difficult to compare or not comparable at all. Due to the mutual comparison of various agricultural products was calculation of the original Lachman method modified according to Hegedűs et al. (2019), which takes into account the dilution of the extract due to varying antioxidant activities of individual horticultural crops. For comparability of our results with the results of other authors, we also expressed the AOA results as TEAC. Total AOA varied from 26.26 mmol Trolox ∙ kg⁻¹ d.w. (‘Dark Green’) to 28.67 mmol Trolox ∙ kg⁻¹ d.w. (‘Tulsi’) in controlled variant and from 23.50 mmol Trolox ∙ kg⁻¹ d.w. (‘Cinamonette’) to 28.97 mmol Trolox ∙ kg⁻¹ d.w. (‘Tulsi’) in selenised variant when following the average of both tested years (Table 7). Influence of selenisation on TEAC was not confirmed as shown in Figure 3 (p > 0.05). Similarly, in the study by Mezeyová et al. (2016), in selenised variant significant influence of variety was found whereas Purple Ruffles variety reached significantly lower TEAC (p < 0.05) in comparison with ‘Dark Green’ and ‘Tulsi’. Total AOA varied from 10.8 to 35.7 μmol TE ∙ g⁻¹ d.w. in Dezful I and Babol accessions, respectively, in the study by Javanmardi et al. (2003) as they determined total AOA in 23 Iranian basil accessions as micromoles of Trolox equivalents per gram of dry weight. To compare AOA with the seeds of basil was difficult so far as there were not scientific studies oriented to this kind of topic. According to Cherian (2019) the basil seeds are often underutilised, despite having a high concentration of powerful compounds and active ingredients that can impact human health. Some of the key active ingredients in basil seeds are dietary fibre, vitamin K, iron, protein, phytochemicals, polyphenolic flavonoids like orientin and vicenin and other powerful antioxidants. Their gelatin-like consistence in fluid started to be interesting and comparable with chia seeds. In the study by Marineli et al. (2014), the commercial chia seeds from Chile were chemically and nutritionally characterised and were investigated for their antioxidant potential by different in vitro methods. Average TEAC was estimated 523.78 μmol Trolox ∙ g⁻¹. All the methods evaluated in their study provided quite similar results to the ones reported by Capitani et al. (2012) for Argentina chia meals (557.2 μmol Trolox ∙ g⁻¹) and fibrous fractions (446.4 μmol Trolox ∙ g⁻¹) obtained by pressing extraction and by Vázquez-Ovando et al. (2009) for a Mexican chia fibrous fraction (488.8 μmol Trolox ∙ g⁻¹). Comparing the selenisation influence on AOA of basil seeds with other authors is very difficult, because of the absence of scientific studies. The importance of agrological technologies was confirmed by Falco et al. (2018), where the AOA of chia seed was negatively affected by irrigation and ranged from 1.317 ± 0.027 to 2.174 ± 0.010 mmol Trolox ∙ g⁻¹.

Figure 3

The content of polyphenols in average ranges from 1414.61 μg GA ∙ g⁻¹ d.w. (‘Tulsi’, selenised variant) to 1681.75 μg GA ∙ g⁻¹ d.w. (‘Dark Green’, control) as shown in Table 8. Statistical significance of variety was confirmed (p < 0.05) evaluating every year, but in average of 2 years this difference was not significant. Influence of the year on TPC was also not found (p > 0.05).

Total phenolic content ranged from 22.9 to 65.5 mg GA ∙ g⁻¹ d.w. in 23 Iranian basil accessions according to Javanmardi et al. (2003) as they determined total phenolic contents by using a spectrophotometric technique based on the Folin–Ciocalteu reagent. In frozen sample of Cinnamon, basil leaves were found 4.4 ± 0.1 (mg CAE ∙ g⁻¹ fresh weight) according to Abramovič et al. (2018). Regarding the total phenolic content in seeds, Marineli et al. (2014) found out in chia seeds samples from Chile 0.94 mg GA ∙ g⁻¹, which was similar to previous reports Reyes-Caudillo et al. (2008), where chia seed from two different regions in Mexico showed values between 0.88 and 0.92 mg GA ∙ g⁻¹. In climate conditions of Slovakia, it has not been possible to grow the seeds of Salvia hispanica so chia has to be imported. On the other hand, the basil seeds are easily grown and fully matured. According to Pérez-Jiménez et al. (2010), dried sweet basil has 322 mg ∙ 100 g of total polyphenols and it is sorted at 26th place within the 100 richest food sources. In Figure 4, average values in case of control are lower compared with selenised variant but this difference was not statistically significant (p > 0.05). The effort to compare the influence of selenisation on TPC with other authors was not successful as this question was not solved in the seeds of basil. Impact of selenium fortification on Se accumulation and total polyphenols was tested in grains of pea in Hegedűsová et al. (2017). The significantly positive influence of Se application on the total polyphenols content (TPC) has been confirmed in two pea varieties after application of dosage in 100 g Se/ha (52% and 33%). TPC ranged in interval from 1471 to 2236 mg GA ∙ kg⁻¹ d.w. for Ambassador variety and from 1417 to 1880 mg GA ∙ kg⁻¹ d.w. for ‘Premium’ in dependence on observed variant.

Figure 4