foods Article Nutritional and Antioxidant Potential of Fiddleheads from European Ferns Marcela Dvorakova 1, Karolína Pumprova 1,2, Žaneta Antonínová 1,2, Jan Rezek 1, Daniel Haisel 1, Libor Ekrt 3 , Tomas Vanek 1 and Lenka Langhansova 1,* 1 The Czech Academy of Sciences, Institute of Experimental Botany, Rozvojova 263, CZ-16502 Prague, Czech Republic; [email protected] (M.D.); [email protected] (K.P.); [email protected] (Ž.A.); [email protected] (J.R.); [email protected] (D.H.); [email protected] (T.V.) 2 Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 129, CZ-16521 Prague, Czech Republic 3 Faculty of Science, University of South Bohemia, Branisovska 1760, CZ-37005 Ceske Budejovice, Czech Republic; [email protected] * Correspondence: [email protected]; Tel.: +420-225-106-803 Abstract: Ferns are part of the diet and traditional medicine in East Asia, North America, and Oceania, however, their importance has been forgotten in Europe. Here, the nutritional and antioxidant potential of young fern fronds (fiddleheads) of eight families were studied. Most of the tested fern species excelled in high antioxidant capacity when compared to the reference leafy vegetables spinach and rocket. On average, the total phenol content reached 220 mg·g−1 of extract dry weight for all fiddleheads, and 15 out of 24 tested species exceeded 1 g Trolox equivalent per gram of extract dry weight in Oxygen Radical Absorbance Capacity (ORAC) assay. On the other hand, fiddleheads Citation: Dvorakova, M.; Pumprova, contained a comparable amount of carotenoids and ascorbic acid with the reference vegetables. In the K.; Antonínová, Ž.; Rezek, J.; Haisel, case of fatty acid composition, fiddleheads contained especially high amounts of essential omega-3 D.; Ekrt, L.; Vanek, T.; Langhansova, (n3) and omega-6 (n6) polyunsaturated fatty acids with a beneficial n6/n3 ratio. The n6/n3 ratio in all L. Nutritional and Antioxidant tested species was between 2 and 6.4, whereas the ratio in the reference vegetables was below 0.4. All Potential of Fiddleheads from in all, fiddleheads from European ferns are a rich source of valuable antioxidants and essential fatty European Ferns. Foods 2021, 10, 460. acids with a desirable n-6/n-3 ratio and may thus form an alternative source of these compounds, https://doi.org/10.3390/foods10020460 especially for those people not consuming fish and fish products. Academic Editors: Severino Zara and Giacomo Zara Keywords: fern species; fiddleheads; fatty acid profile; natural antioxidants; nutritional quality; alternative vegetable Received: 18 January 2021 Accepted: 13 February 2021 Published: 19 February 2021 1. Introduction Publisher’s Note: MDPI stays neutral Ferns have been recognized as edible medicinal plants for centuries, especially in with regard to jurisdictional claims in China, India, and other Asian countries. Taxonomically, they belong to pteridophytes, published maps and institutional affil- vascular plants that disperse via spores [1]. Ferns (Monilophyta) are represented by about iations. 12,000 species [2]. The habit of eating ferns goes back thousands of years when wild plant gathering was a primary food source. For example, edible ferns are mentioned in Chinese literature as early as 3000 years ago [3]. In addition, the use of fern extracts has already been known in ancient medicinal systems of India (Ayurveda) and China [3]. Although Copyright: © 2021 by the authors. today, wild plant gathering is not as widespread as thousands of years ago, there are still Licensee MDPI, Basel, Switzerland. regions where fern harvesting has remained routine or is even used commercially, such as This article is an open access article New Zealand [4], Japan, and the USA [5], Canada [6], China, Nepal, India, Malaysia, and distributed under the terms and the Philippines [3]. It may be said that it is even on the rise, as more and more people are conditions of the Creative Commons seeking healthier lifestyles and a return to traditional customs. Ferns, as plants with an Attribution (CC BY) license (https:// established impact on human health, have thus regained attention. Extracts of fern parts creativecommons.org/licenses/by/ has been studied with the aim to establish their biological activity as well as to identify 4.0/). Foods 2021, 10, 460. https://doi.org/10.3390/foods10020460 https://www.mdpi.com/journal/foods Foods 2021, 10, 460 2 of 15 the responsible chemical entities. Fern extracts exhibited potent antioxidant, antimicrobial, antibacterial, antiviral, and anti-inflammatory activities [7,8]. Simultaneously, ferns have been found to contain a high amount of phenolic compounds, glycosides, flavonoids, terpenoids, carotenoids, alkaloids, and fatty acids [7,9]. Their consumption is thus valuable not only from the medicinal point of view, but also from a nutritional perspective. When compared to other green vegetables, ferns are especially rich sources of antioxidants and of essential omega-3 and omega-6 fatty acids [10,11]. On the other hand, there is a concern about the potential toxicity of fern species, which will be discussed further. Herein, the nutritional value of 24 mainly European fern species belonging to eight families was determined in fern fiddleheads (young fronds). The total content of carotenoids, phenolic compounds, fatty acids, and ascorbate was measured along with the identification and quantification of individual carotenoids and fatty acids. The antioxidant capacity was also evaluated. For the comparison of the obtained values, two green leafy vegeta- bles, spinach, and rocket, were selected as reference vegetables as their young leaves are commonly consumed and sold in supermarkets. 2. Materials and Methods 2.1. Plant Material and Chemicals The young fern fronds (fiddleheads) were collected (24 species, 8 families) during the spring period in 2018. All tested species were of European origin, cultivated for hor- ticultural (bred cultivars) or botanical purposes (native species), or grown in the wild (supplementary Table S1). Preferentially we choose bred species (either native or horti- cultural) for our study since several species are endangered in Europe. However, neither bred nor native cultivars grown in botanical gardens were available for Preridium aquilinum. Thus, it was collected in the wild as it belongs to one of the most common edible ferns. Few fern species were collected in duplicate—horticultural and botanical garden samples such as Polystichum aculeatum, Osmunda regalis, and Polypodium vulgare; or—botanical garden and wild collected samples, such as Lastrea limbosperma, in order to detect possible variations caused by cultivation conditions (pot vs. soil, or soil vs. wild). Determination of studied taxa was provided by RNDr. Libor Ekrt, Ph.D. or by the authorities in the Botanical Garden of the Charles University in Prague, Faculty of Sciences. The collected fiddleheads were kept on dry ice during the transport and then stored under −80 ◦C until extraction for particular analyses. Reference vegetables, rocket (Eruca sativa) and spinach (Spinacia oleracea), were seeded from seeds and grown in the pots in the greenhouse for two months until young leaf collection. All chemicals and reagents for analyses (Folin-Ciocalteu reagent, fluorescein, 2,2’- azobis(2-methylpropionamidine) dihydrochloride) were purchased from Sigma-Aldrich s.r.o. (Prague, Czech Republic). Similarly, the organic solvents and standards (Trolox, gallic acid, L-ascorbic acid, nonadecanoic acid, methyl heptadecanoate-d33, Supelco 37 compo- nent FAME Mix) were purchased from Sigma-Aldrich s.r.o. (Prague, Czech Republic). 2.2. Total Phenol Content The fiddleheads were freeze-dried using a FreeZone 2.5 Labconco Freeze-Dryer (Lab- conco corp., Kansas City, Missouri, USA) equipped with a vacuum pump (Vacuubrand GMBH + CO KG, Wertheim, Germany) at −50 ◦C, 0.370 mBar. Freeze-dried and homog- enized (using a mortar and pestle) fiddleheads (0.5 g) were extracted twice in 10 mL of methanol overnight. The extraction was supported by sonication for 30 min in an ul- trasound bath at room temperature in the dark. Combined extracts were centrifuged at 1730× g, 15 min (Centrifuge Hettich Universal 32R, Tuttlingen, Germany), at room temperature and then filtered through 30 mm polyvinylidene fluoride (PVDF) 0.45 µm membrane filter (ProFill, Fisher, Pardubice, Czech Republic). Filtrates were evaporated in a vacuum on a rotary evaporator at water aspirator pressure until almost dry, and then dried completely under nitrogen flow. The average extract yield was 26%. Obtained residues Foods 2021, 10, 460 3 of 15 were redissolved in dimethyl sulfoxide (DMSO) to reach the concentration of 100 mg.mL−1 and the DMSO aliquots were kept at −80 ◦C for further analyses. Total phenol content was determined in methanolic extracts by a modified Folin- Ciocalteu colorimetric method [12]. Briefly, the DMSO aliquots were diluted in water to reach the final concentration of 80, 60, and 40 µg.mL−1 in the reaction. The samples were then incubated with Folin-Ciocalteu reagent in 96-well flat-bottom transparent microplates, and after 10 min of shaking (200 rpm) at room temperature, the reaction was terminated using 12% anhydrous sodium carbonate. The absorbance was read at 760 nm after 30 min incubation in the dark at 37 ◦C. The calculation of the phenolic content was based on a calibration curve obtained for a reference compound, gallic acid, in a concentration range from 0.39 to 25 µg·mL−1. The total phenol content was expressed in milligrams of gallic −1 acid equivalent per gram of dry extract (mg GAeqv·g DW). 2.3. Evaluation of Antioxidant Capacity The antioxidant capacity was measured using ORAC assay (Oxygen Radical Ab- sorbance Capacity) according to Silva et al. [13]. Briefly, the DMSO aliquots (prepared as described above) were diluted in 75 mM potassium phosphate buffer (pH 7.0) to reach the final concentration of 10, 5, or 2.5 µg·mL−1, respectively, in the reaction. Each sample was measured at four serial concentrations obtained by 1:1 dilutions.