Three plant genera (Anthriscus, Chaerophyllum, and Myrrhoides) of the Apiaceae family with similar leaf architecture, including annual (A. cerefolium, A. caucalis, M. nodosa), biannual (C. bulbosum), and perennial (A. sylvestris) species were investigated (Fig. 1).

Samples of Anthriscus cerefolium wild relatives (Anthriscus sylvestris, Chaerophyllum bulbosum, Anthriscus caucalis, and Myrrhoides nodosa) were gathered in 2023–2024 at the territory of Moscow region and Crimea (Table 1), while cultivated species (Anthriscus cerefolium, cv. Ogorodnic, and Chaerophyllum bulbosum of Polish production) were produced in Moscow region at the experimental fields of Federal Scientific Vegetable Center (Table 1, Fig. 2).

To evaluate the effect of habitat on the antioxidant status of A. sylvestris, the latter was also gathered in Karelia and the Arctic region of European Russia (geographical coordinates of the sampling places are presented in Table 1).

Leaves of all plants investigated were harvested at the end of June in Moscow region, Crimea, and Karelia, and in the middle of August at the Arctic.

Garden and wild chervil plants (A. cerefolium, cultivar Ogorodnik, C. bulbosum, and A. sylvestris) were grown at the experimental fields of Federal Scientific Vegetable Center in 2023–2024 in clay-loam soil with pH 6.8, 2.1% organic matter, 1.32 mg-eq 100 g⁻¹ hydrolytic acidity; 18.5 mg kg⁻¹ mineral nitrogen; 21.3 mg kg⁻¹ ammonium nitrogen; 402 mg kg⁻¹ mobile phosphorous; 198 mg kg⁻¹ exchangeable potassium; sum of absorbed bases 93.6%; cation exchange capacity 15 mg-eq 100 g⁻¹ soil. No soil fertilization was practiced during the experiment, whereas the irrigation was activated when the soil humidity dropped to 75%.

A. cerefolium and C. bulbosum were sown on 10 May, while 2-year-old plants of wild A. sylvestris were transferred from the Balashikha park (55°48′34″ N 37°57′29″ E) to the experimental plot of 3 × 3 m² with a 40 cm distance between plants.

At the 15th day of seedling development, A. cerefolium and A. sylvestris plants were sprayed with a 25 mg L⁻¹ solution of sodium selenate, as in a previous investigation on A. cerefolium Se/I biofortification, which did not cause plant toxicity [7]. Foliar supply of Se was chosen as the most effective method of Se supply because of the significantly higher assimilation of this microelement compared to soil application [32]. Harvest was practiced 30 days after the vegetation beginning. Leaves were separated from plants, dried to constant weight in an oven at 70°C, and homogenized. The obtained powder was used for the determination of AOA and TP, while the ascorbic acid and photosynthetic pigments were determined on fresh homogenized leaves.

To determine the content of photosynthetic pigments, half a gram of dry samples was homogenized in a porcelain mortar with 10 mL of 96% ethanol. The homogenized sample mixture was quantitatively transferred to a volumetric flask, bringing the volume to 25 mL, and the mixture was filtered through filter paper. In the resulting solution, analyses of chlorophyll-a, chlorophyll-b, and carotene were performed through a spectrophotometer (Unico 2804 UV, USA). The calculation of chlorophyll and carotene concentrations was achieved using appropriate equations [33]: Ch-a = 13.36A₆₆₄ − 5.19A₆₄₉; Ch-b = 27.43A₆₄₉ − 8.12A₆₆₄; C c = (1000A₄₇₀ − 2.13 Ch-a − 87.63 Ch-b)/209; where A = Absorbance at the wave length of 664, 649, and 470 nm, Ch-a = Chlorophyll a, Ch-b = Chlorophyll b and C c = Carotene.

The ascorbic acid content in leaves of A. cerefolium, A. sylvestris, and C. bulbosum plants was determined by visual titration of fresh leaf extracts in 3% trichloracetic acid with Tillman’s reagent [34]. Three grams of leaves were mixed with 5 mL of 3% trichloracetic acid and quantitatively transferred to a measuring cylinder. The volume was brought to 60 mL using 3% trichloracetic acid, and the mixture was filtered through a filter paper 15 min later. The concentration of the ascorbic acid was determined from the amount of Tillman’s reagent that went into the titration of the sample.

Total polyphenols were determined in 70% ethanol extract using the Folin–Ciocalteu colorimetric method as previously described [35]. One gram of dry homogenates was extracted with 20 mL of 70% ethanol at 80°C for 1 h. The mixture was cooled down and quantitatively transferred to a volumetric flask, and the volume was adjusted to 25 mL. The mixture was filtered through filter paper, and 1 mL of the resulting solution was transferred to a 25 mL volumetric flask, to which 2.5 mL of saturated Na₂CO₃ solution and 0.25 mL of diluted (1:1) Folin–Ciocalteu reagent were added. The volume was brought to 25 mL with distilled water. One hour later, the solutions were analyzed through a spectrophotometer (Unico 2804 UV, Suite E Dayton, NJ, USA), and the concentration of polyphenols was calculated according to the absorption of the reaction mixture at 730 nm. As an external standard, 0.02% gallic acid was used. The results were expressed as mg of Gallic Acid Equivalent per g of dry weight (mg GAE g⁻¹ d.w.).

The antioxidant activity of samples was assessed using a redox titration method [35] via titration of 0.01 N KMnO₄ solution with ethanolic extracts of dry samples, produced as described in the Section 2.2. The reduction of KMnO₄ to colorless Mn⁺² in this process reflects the quantity of antioxidants dissolvable in 70% ethanol. The values were expressed in mg Gallic Acid Equivalents (mg GAE g⁻¹ d.w.).

Total Se content was analyzed using the microfluorimetric method [36]. Dried homogenized samples were digested via heating with a mixture of nitric and perchloric acids, subsequently selenate (Se + 6) was reduced to selenite (Se + 4) with a solution of 6 N HCl, and the resulting selenite was subjected to a complexation with 2,3-diaminonaphtalene to form piazoselenol. Calculation of the Se concentration was achieved by recording the piazoselenol fluorescence value in hexane at 519 nm-emission and 376 nm-excitation. Each determination was performed in triplicate. The precision of the results was verified using a reference standard of Se fortified mitsuba stem powder in each determination with a Se concentration of 1865 μg Kg⁻¹ d.w. (Federal Scientific Vegetable Center).

The appropriate determination of organic Se was achieved analogously after removal of inorganic Se species with distilled water rinsing and precipitation of water soluble proteins with trichoracetic acid solution.

To evaluate the importance of Se-biofortified A. sylvestris utilization in the Arctic, the selenium status of Nikel residents (Murmansk region) was characterized by analyzing hair samples of 15 volunteers aged 30–46 (August 2024). Hair was washed with acetone to remove fat impurities, homogenized, and subjected to the Se analysis. Selenium concentrations were also determined in dried bread samples (n = 12) and muscles of fresh water whitefish (Coregonus pidschian) of the Paz River (n = 10).

The data were processed by the analysis of variance, and mean separations were performed through Duncan’s multiple range test, with reference to a 0.05 probability level, using the SPSS software version 29 (Armonk, NY, USA). Data expressed as percentages were subjected to angular transformation before processing.

For the perspective of new functional food creation, the topic regarding the peculiarities and expediency of Se biofortification of edible wild plants arises [44]. Previous investigations indicated high prospects of Se-biofortified Allium ursinum [45], Azolla caroliniana [46], Portulaca olecracea [47], Taráxacum officinále [48], Plantago [47,49], and Rumex acetosa [47], etc. Contrary, wild A. sylvestris, A. caucalis, M. nodosa, and C. bulbosum have never been used for these purposes. In this respect, among the plants studied, A. sylvestris was chosen for Se foliar biofortification due to the chance to predict its antioxidant status in different geographical zones, as well as its high adaptability, wide habitat, and remarkable nutritional and medicinal value.

The production of functional food via plant biofortification with Se is one of the most interesting approaches for protecting human health and preventing hunger [39,46]. In this respect, wild relatives of agricultural crops are especially interesting due to their higher levels of adaptability and antioxidants [50]. Indeed, wild chervil (A. sylvestris) demonstrated unexpectedly great concentrations of total phenolics, ascorbic acid, and chlorophyll content, compared to garden chervil (A. cerefolium) (Table 4).

The comparison of foliar Se biofortification effect on wild A. sylvestris and cultivated A. cerefolium showed that, despite the higher initial antioxidant characteristics of A. sylvestris, compared to A. cerefolium, the mentioned plants displayed similar levels of resistance to Se treatment and increase of chlorophyll a, b, carotene, ascorbic acid, and Se biofortification (Table 4, Fig. 5). A beneficial effect of Se on the biosynthesis of plant antioxidants was recorded in many agricultural crops, suggesting the high prospect of Se biofortification [8,51]. Notably, in our research a significantly higher increase in the total antioxidant activity and polyphenol content was recorded in wild chervil under Se supplementation, compared to A. cerefolium, with values of 72.0 and 36.4 mg GAE g⁻¹ d.w., respectively (Fig. 5). The data presented in Table 4 indicate that the consumption of 10 g of Se-fortified wild chervil powder may provide up to 60% of the Recommended Dietary Allowance for Se equal to 55 μg per day, not exceeding the Tolerable Upper Intake Level (UL) for adults of 400 μg [52]. Furthermore, the amount of organic Se in Se fortified plants was as high as 81.2% for Se biofortified A. sylvestris and 77.9% for A. cerefolium. These results are in accordance with the known ability of plants to use the external Se to synthesize various organic Se compounds, such as Se containing amino acids (selenomethionine, selenocysteine), the corresponding peptides, polysaccharides, etc., using the chemical similarity of Se and S [44]. As an essential microelement for human health, Se in the form of amino acids is incorporated into different enzymes, providing the antioxidant activity of glutathione peroxidases and thioredoxin reductase, participating in thyroid hormone metabolism in triiodothyronine deiodinases, ensuring Se transport in the form of selenoprotein P, regulating the inflammation processes in the form of selenoprotein S [10].

From a practical point of view, the new Se-biofortified product shows the highest values of the AOA and TP content in plants from the Moscow region, similar to those recorded in the Arctic A. sylvestris. Future investigations may provide additional data, as the effect of altitude on the efficiency of Se biofortification in A. sylvestris in Arctic conditions has never been investigated previously. Supposedly, Se supply will further enhance plant tolerance to severe northern climate due to the antioxidant properties of Se, allowing to protect plants against all forms of oxidant stresses [8,51]. Moreover, rather high levels of Se may become significant for the northern zones of selenium deficient soil and low human Se status [53].

Indeed, epidemiological investigations in Nikel settlement indicated rather low Se levels in human hair: 377 ± 13 μg kg⁻¹ d.w. on average (358–394 μg kg⁻¹ d.w.; n = 15), along with low Se content in bread (58 ± 3 μg kg⁻¹ d.w. on average; 54–63; n = 9) produced from grain of the neighboring Se-deficient regions and in whitefish muscles (Coregonus pidschian) of the Paz river (359 ± 22 μg kg⁻¹ f.w.; n = 12). These results confirm the low Se status of the mentioned region and are in agreement with the previous investigations carried out in Syktyvkar, Komi republic (human serum Se concentration range of 54–91 μg L⁻¹) [53] and the evaluation of Se content in whitefish from Arkhangelsk region (340 μg kg⁻¹ f.w.; Pechora river; Narian-Mar; 67°38′16″ N.; 53°00′24″ E) [54].

A. sylvestris is widespread in the territory of the Pechenga district of Murmansk region, where the Nikel settlement is situated. According to our observation, this plant grows especially at unpolluted territories, with the exception of Nikel settlement, where imported soil has been used after the closure of Pechenganikel Cu/Ni smelting plant, which entails the possibility of A. sylvestris Se biofortified production in the Murmansk region, for the human Se status optimization. Unfortunately, no attempts have been made to assess the efficiency of plant Se biofortification in the Arctic zone, where severe climate may hamper plant growth and, therefore, the results may significantly differ from those obtained in the Moscow region; however, the latter hypothesis needs experimental confirmation.

The production of Se biofortified A. sylvestris plants may become important for the residents of other Se-deficient areas in Russia and other countries, due to the significant levels of antioxidants, sufficient Se levels predominantly in organic form, and low-cost technology of this new functional food. At present, A. sylvestris is used in salads, pastry, and soups [4,16], while the present results provide some new aspects of Se-enriched plant utilization as a spice and biologically active food additive.