Walnut (Juglans regia) leaf extracts are strong scavengers of pro-oxidant reactive species
Introduction
The prevention of the oxidative reactions in foods, pharmaceuticals and cosmetics and the management of oxidative stress-related diseases are some of the potential applications of antioxidants. The most widely used antioxidants, to prevent the oxidation of lipids in foods, are butylated hydroxyanisole (BHA), propyl gallate and 2-tert-butylhydroquinone (TBHQ) (Moure et al., 2001). However, there has been growing concern over the safety of some of the commercial antioxidants because several studies documented the mutagenesis and carcinogenesis potential associated with some synthetic antioxidants (Ito et al., 1986, Witschi, 1986), although other authors reported anti-carcinogenic effects (Williams, Iatropoulos, & Whysner, 1999). Therefore, the extraction and characterisation of natural antioxidants are of considerable interest. Natural antioxidants, such as tocopherols, ascorbic acid and plant polyphenols have gained increasing importance.
Walnut (Juglans regia L.) is a deciduous tree native in southeastern Europe of the Juglandaceae family. The walnut fruits are a highly nutritious food, which are rich in oil composed of unsaturated fatty acids. Antioxidant effects of isolated polyphenols obtained from walnuts have been previously reported (Fukuda, Ito, & Yoshida, 2003). Scavenging of hydroxyl radicals and superoxide radicals is documented for water and methanol extracts of the kernel of J. regia (Ohsugi et al., 1999). Walnut liqueur, obtained with green walnuts, also presents antioxidant activity which was correlated with its polyphenolic composition (Alamprese, Pompei, & Scaramuzzi, 2005). Walnut leaf has been widely used in folk medicine for the treatment of skin inflammations, hyperhidrosis and ulcers and for its antidiarriec, anti-helmintic, antiseptic and astringent properties (Bruneton, 1999, Proença da Cunha et al., 2003).
No studies concerning the scavenging effect of reactive oxygen species (ROS) or reactive nitrogen species (RNS) have been reported for J. regia leaves.
The objective of the present study is thus to assess the in vitro scavenging activity of pro-oxidant reactive species exerted by the J. regia leaf ethanol:water (4:6) extract, namely on the ROS [hydroxyl radical (HO), superoxide radical , peroxyl radical (ROO) and hydrogen peroxide (H2O2)] and the RNS [nitric oxide (NO) and peroxynitrite anion (ONOO−)].
Section snippets
Plant material
J. regia leaves were collected during Summer, July 2003, in Mirandela, Northern Portugal, in a highland with high thermal amplitude. The leaves were dried at room temperature. Voucher specimens were preserved in our laboratory for further reference.
Chemicals
Dihydrorhodamine 123 (DHR), 4,5-diaminofluorescein (DAF-2), 3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene (NOC-5), nitroblue tetrazolium chloride (NBT), lucigenin, diethylenetriaminepentaacetic acid (DTPA), β-nicotinamide adenine nucleotide
Total phenols
The total phenolic content of the J. regia ethanol:water (4:6) extract was 270 ± 3 mg GAE per g of lyophilised extract (mean ± SE). The extract yield was 0.804 g, which corresponds to a recovery of 20.1%. Several polyphenols have been identified on J. regia leaves, namely cinnamic acids (3-caffeoylquinic acid, 3-p-coumaroylquinic and 4-p-coumaroilquinic acids) and flavonoids (quercetin 3-galactoside, quercetin 3-arabinoside, quercetin 3-xyloside, quercetin 3-rhamnoside and a kaempferol derivative).
Conclusions
The studied J. regia (ethanol:water) extract was shown to be very effective against the evaluated pro-oxidant species. The scavenging effects of the studied reactive species have been described for some of the polyphenols that have been identified in J. regia leaves. These data imply that at least part of the observed antioxidant activity may be a result of the extract’s phenolic compounds.
The results of the present study showed that the J. regia leaf can be used as an easily accessible source
Acknowledgement
The authors greatly acknowledge José Madureira for the collection of Juglans regia leaves.
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