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Tocopherols Composition of Portuguese Wild Mushrooms with Antioxidant Capacity Sandrina A. Helenoa, Lillian Barrosa, Maria Jo

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Tocopherols Composition of Portuguese Wild Mushrooms with Antioxidant Capacity Sandrina A. Helenoa, Lillian Barrosa, Maria Jo View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Biblioteca Digital do IPB 1 Tocopherols composition of Portuguese wild mushrooms with antioxidant capacity 2 3 Sandrina A. Helenoa, Lillian Barrosa, Maria João Sousab, Anabela Martinsb, 4 Isabel C.F.R. Ferreiraa,* 5 6 aMountain Research Centre (CIMO), Instituto Politécnico de Bragança, Campus de 7 Santa Apolónia, Apartado 1172, 5301-855 Bragança, Portugal. 8 bEscola Superior Agrária, Instituto Politécnico de Bragança, Campus de Santa 9 Apolónia, Apartado 1172, 5301-855 Bragança, Portugal. 10 11 12 * Author to whom correspondence should be addressed (e-mail: [email protected] 13 telephone +351-273-303219; fax +351-273-325405). 14 1 15 ABSTRACT 16 The antioxidant composition and properties of eighteen Portuguese wild mushrooms 17 (Clitocybe alexandri, Cortinarius glaucopus, Fistulina hepatica, Hydnum repandum, 18 Hygrophoropsis aurantiaca, Hypholoma capnoides, Laccaria amethystina, Laccaria 19 laccata, Lactarius aurantiacus, Lactarius salmonicolor, Lepista inversa, Lepista 20 sordida, Mycena rosea, Russula delica, Russula vesca, Suillus collinitus, Suillus 21 mediterraneensis, Tricholoma sulphureum) were evaluated, in order to contribute to the 22 overall characterization of these products. Their radical scavenging capacity, reducing 23 power and inhibition of lipid peroxidation measured in liposome solutions was fully 24 studied. Furthermore, the tocopherols composition was determined by HPLC- 25 fluorescence. The analysed mushrooms contain powerful antioxidants such as phenols 26 (0.51-7.90 mg/g) and tocopherols (0.02-8.04 µg/g). β-Tocopherol was the vitamer 27 detected in higher amounts, while δ-tocopherol was not detected in the majority of the 28 samples. All the species proved to have antioxidant activity being more significant for 29 Hygrophoropsis aurantiaca (EC50 values lower than 1.35 mg/ml) due to the 30 contribution of antioxidants such as phenols (7.90 mg/g) and tocopherols (0.02-1.94 31 µg/g). The ongoing research states the nutraceutical potential of all these unique 32 species, making the information available for a better management and conservation of 33 mushrooms and related habitats. 34 35 Keywords: Wild mushrooms; Tocopherols; HPLC; Antioxidant activity. 36 2 37 1. Introduction 38 Reactive oxygen and nitrogen species (ROS and RNS) seem to be implicated in the 39 oxidative deterioration of food products as well as in the pathogenesis of several human 40 diseases such as atherosclerosis, diabetes mellitus, chronic inflammation, 41 neurodegenerative disorders and certain types of cancer (Halliwell, 1996). There is a 42 great interest in finding natural antioxidants for use in food to retard lipid oxidative 43 rancidity or in pharmaceutical applications for chronic diseases related to the production 44 of free radicals (Prior, 2003). Macrofungi (mushrooms) are rich sources of antioxidant 45 compounds such as phenolic compounds (phenolic acids and flavonoids) and 46 tocopherols (Ferreira, Barros, & Abreu, 2009). In the last years nineteen different 47 mushroom species from the Northeast Portugal, one of the European regions with 48 higher wild mushrooms diversity, were evaluated for their antioxidant properties, in 49 order to valorise them as a source of nutraceuticals (Barros, Correia, Ferreira, Baptista, 50 & Santos-Buelga, 2008; Barros, Venturini, Baptista, Estevinho, & Ferreira, 2008; 51 Ferreira et al., 2009). Nevertheless, assuming that the proportion of mushrooms used 52 among the undiscovered and unexamined ones is only 5%, there are thousands of 53 macrofungi species potentially beneficial for mankind. Even among the already known 54 species the proportion of well investigated mushrooms is very low. Therefore, our 55 research group intends to go on studying this matrix, documenting the nutraceutical 56 potential of all unique species and making the information available for a better 57 management and conservation of this natural resource and related habitats. Particularly, 58 data on Clitocybe alexandri, Cortinarius glaucopus, Hydnum repandum, 59 Hygrophoropsis aurantiaca, Hypholoma capnoides, Laccaria amethystina, Laccaria 60 laccata, Lactarius aurantiacus, Lactarius salmonicolor, Lepista inversa, Lepista 61 sordida, Mycena rosea, Russula vesca, Suillus collinitus, Suillus mediterraneensis and 3 62 Tricholoma sulphureum have not yet been reported. Tocopherol compositions of 63 Russula delica from Turkey and Hydnum repandum from Spain were studied but there 64 are no studies on Portuguese samples. 65 Vitamin E is a term frequently used to designate a family of chemically related 66 compounds, namely tocopherols and tocotrienols, which share a common structure 67 having a chromanol head and isoprene side chain. Due to its role as a scavenger of free 68 radicals, vitamin E is also believed to protect our bodies against degenerative 69 malfunctions, mainly cancer and cardiovascular diseases (Burton & Traber, 1990; 70 Kamal-Eldin & Appelqvist, 1996). In the past α-tocopherol was considered the most 71 active form of vitamin E in humans and it was reported to exhibit the highest biological 72 activity. However, many recent publications have been focused on the health effects of 73 the other vitamin E isoforms (Traber, 1999; Schwenke, 2002). 74 Some reports have been published on mushrooms tocopherols contents (Mau, Chao, & 75 Wu, 2001; Mau, Lin, & Chen, 2002; Mau, Lin, & Song, 2002; Yang, Lin, & Mau, 2002; 76 Mau, Chang, Huang, & Chen, 2004; Elmastas, Isildak, Turkekul, & Temur, 2007; Lee, 77 Kim, Jang, Jung, & Yun, 2007; Tsai, Tsai, & Mau, 2007; Barros, Correia et al., 2008; 78 Barros, Venturini et al., 2008; Lee, Jian, Lian, & Mau, 2008; Jayakumar, Thomas, & 79 Geraldine, 2009). They all described the same methodology including saponification in 80 the extraction process and analysis by HPLC coupled to UV detector. Our research 81 group (Barros, Correia et al., 2008) was the only describing an extraction process 82 without saponification, by adding an antioxidant to avoid tocopherols oxidation, while 83 protecting the samples from light and heat, followed by HPLC-fluorescence analysis. 84 Using this process, α-, β-, δ- and γ-tocopherols were identified and quantified in wild 85 mushrooms, while tocotrienols were not detected in any of the cited studies. 86 4 87 2. Materials and methods 88 89 2.1. Mushroom species 90 Samples of Clitocybe alexandri (Gillet) Konrad, Cortinarius glaucopus (Schaeff), 91 Fistulina hepatica (Schaeff.: Fr.), Hydnum repandum (L.: Fr.), Hygrophoropsis 92 aurantiaca (Wulf.: Fr.) Mre., Hypholoma capnoides (Fr.) Quel., Laccaria amethystina 93 (Bolt. ex Fr.) R.Maire, Laccaria laccata (scop.: Fr.) Berk. & Broome, Lactarius 94 aurantiacus (Fr.), Lactarius salmonicolor (Heim y Leclair), Lepista inversa (Scop.: Fr.) 95 Pat., Lepista sordida (Fr.) Singer, Mycena rosea (Schumach.) Gramberg, Russula 96 delica (Fr.), Russula vesca (Fr.), Suillus collinitus (Fr.) Kuntz, Suillus mediterraneensis 97 (Jacquetant & Blum) Redeuilh, Tricholoma sulphureum (Bull.: Fr.) Kumm. were 98 collected under Quercus pyrenaica Willd. and mixed stands of Quercus sp. and Pinus 99 sylvestris Ait., in Bragança (Northeast Portugal), in autumn 2008. Mycena rosea and 100 Tricholoma sulphureum are not edible species and Cortinarius glaucopus is a species of 101 unknown edibility. Taxonomic identification of sporocarps was made according to 102 several authors (Marchand, 1971-1986; Moser, 1983; Breitenbach & Kränzlin, 1984- 103 2000); Alessio, 1985; Bon, 1988; Noordeloos (1988-2001); Candusso & Lanzoni, 1990; 104 Courtecuisse, 1999; Courtecuisse & Duhem, 2005) and online keys 105 (http://www.mycokey.com/), and representative voucher specimens were deposited at 106 the herbarium of Escola Superior Agrária of Instituto Politécnico de Bragança. All the 107 samples were lyophilised (Ly-8-FM-ULE, Snijders) and reduced to a fine dried powder 108 (20 mesh). 109 110 2.2. Standards and reagents 111 The eluents n-hexane 95% and ethyl acetate 99.98% were of HPLC grade from Lab- 112 Scan (Lisbon, Portugal). Methanol was of analytical grade purity and supplied by 5 113 Pronalab (Lisbon, Portugal). Tocopherol standards (α, β, γ and δ), and the standards 114 used in the antioxidant activity assays: BHA (butylhydroxyanisole), TBHQ (tert- 115 butylhydroquinone), L-ascorbic acid, α-Tocopherol and gallic acid were purchased 116 from Sigma (St. Louis, MO, USA). Racemic tocol, 50 mg/ml, was purchased from 117 Matreya (PA, USA). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) was obtained from 118 Alfa Aesar (Ward Hill, MA, USA). All other chemicals were obtained from Sigma 119 Chemical Co. (St. Louis, MO, USA). Water was treated in a Milli-Q water purification 120 system (TGI Pure Water Systems, USA). 121 122 2.3. Determination of phenols and tocopherols 123 For phenols content determination, the sample (1 ml) was mixed with Folin-Ciocalteu 124 phenol reagent (1 ml). After 3 min, saturated sodium carbonate solution (1 ml) was 125 added to the mixture and adjusted to 10 ml with distilled water. The reaction was kept in 126 the dark for 90 min (following Barros, Venturini et al., 2008, after which the absorbance 127 was read at 725 nm (Analytikijena 200-2004 spectrophotometer). Gallic acid was used 128 to calculate the standard curve of absorbance vs. concentration (10−5-4×10−4 mol⋅l−1; Y 129 = 2.8557X−0.0021; R2 = 0.9999) and the results were expressed as mg of gallic acid 130 equivalents (GAEs) per g of extract. 131 Tocopherols content was determined following
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