There are several antecedents for the different uses of the N. antartica (ñire) by original people of Patagonia, including the Selknam and Tehuelche. They used the wood for firewood and the manufacture of weapons to hunt (ballers, archs) [14]. Ethnobotanical studies of the current native communities revealed the use of leaves, fruits and bark for their medicinal and dyeing properties and also as food [3,15]. Nowadays, its leaves are employed to prepare infusions, as food ingredients and for liquor and cosmetic manufacture [16]. NA infusions not only have a good taste and aroma but also contain a large amount of antioxidant compounds. The antioxidant activity can be ascribed to flavonoids and polyphenols. Recent studies demonstrated the great diversity of these compounds present in NA tea. Indeed, fifteen different phenolic compounds were identified including seven derivatives of phenolic acids, mainly represented by gallic, ellagic, quinic, caffeic, and coumaric acids, and eight flavonoid derivatives being myricetin and quercetin glycosides the most important ones [17].
In spite of the ancient and extended use of NA infusion, no thorough analysis about its potential toxicity have been performed previously. This work aimed to assess the acute and subacute toxicity of NA leaves infusion in mice and its antioxidant activity.
In general, the acute toxicity study is utilized to investigate the harmful effects of a given agent to the organism when administered as a single or short-term exposure. The studies evaluate the mortality, changes in behavior, body weight, and other spontaneous changes in the overall well-being of the mice. Following the OECD recommendations for compounds having an empiric evidence of being non-toxic, a unique limit dose of 2000 mg/kg of body weight was chosen for testing acute toxicity in this work [13]. No signs of toxicity or mortality were observed in any animal during the experimental period after the administration of the single dose. Therefore, the LD50 of extract may be considered to be higher than 2000 mg/kg. According to the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals, the substances having an LD50 value greater than 2000 mg/kg are considered as relatively safe [18].
In turn, for subacute tests repeated doses during the experiment had been employed to evaluate toxicity. A relative high dose of 800 mg/kg body weight was chosen for the tests carried out in this work. No deaths and no clinical signs of local or systemic toxic effects were registered during the subacute exposure. The behavior of animals in acute and subacute exposures was framed as normal for the species. In general, an increase or decrease in the body weight of an animal has been used as an indicator of an adverse effect of drugs and chemicals. In the present study, the body weight of treated mice did not differ significantly (P > 0.05) from those of the control groups. Moreover, the average body weight gain of mice was 720 mg ± 137 mg in 14 days, which is a normal increase of body mass when fed standard chow ad libitum [19]. These results indicate that the extract did not affect the appetite or have adverse effects on the animals' growth.
The functionality of liver and kidneys is crucial for the metabolism of ingestion and for excretion of the waste products, respectively. In this work, after the slaughter of the animals (15 days), transaminases (AST, ALT) were determined for the assessment of the hepatocellular injury, cholesterol as an indicator of metabolic dysfunction and total bilirubin for the evaluation of hepatobiliary injury [20]. The biochemical parameters of the animals acutely and subacutely treated with NA infusion, showed minor changes (statistically not-significant), when compared with the controls. Besides that, all the values were within the normal range for the Swiss albino mice [21]. The analysis of renal function showed that there were no significant differences in urea and creatinine levels between treated and control groups, indicating that NA infusion did not alter the kidney function of mice. According to the biochemical results, it is reasonable to speculate that the histology of the kidneys and liver would be normal. Indeed, photomicrographs of the liver and kidneys sections revealed a normal histoarchitecture. The potential damage at intestinal level was also evaluated by histology. The absence of clinical signs of intestinal disorders (diarrhea, weight loss) during the experiment correlated with the lack of histopathological lesions in the intestinal mucosa.
It is known that proton radical scavenging is an important attribute of antioxidants. ABTS+, a protonated radical, has characteristic absorbance maxima at 734 nm, which decreases with the scavenging of proton radicals. In the present study, NA infusion has shown 90% free radical scavenging activity at a concentration 50 µg/mL. Moreover, the free-radical scavenging activity of NA infusion was higher than that of the standard ascorbic acid, showing an IC50 of half of its value. Our results indicate that the formulation was a potent antioxidant, as determined by ABTS assay. This capacity could be ascribed to the large quantity and diversity of of polyphenols and flavonoids present in the NA infusion [17]. We also evaluated whether this antioxidant capacity observed in vitro also had a positive impact in vivo. Therefore, the ABTS assay was performed in feces and serum samples of the mice. There was a clear increase of the antioxidant capacity of feces in subacute treated mice, whereas acute treated mice showed a not-significant rise of antioxidant activity. The repetition of the doses of NA infusion during the subacute treatment could explain the greater effect observed. It is known that, the antioxidant capacity at intestinal level depends on the gut microbiota, the food intake and the scavenger activity of the enzymatic and non-enzymatic systems of the host [22]. Therefore, the increase of the antioxidant activity in feces of subacute treated mice could be ascribed to the direct influence of the polyphenols and flavonoids present in the NA infusion, to changes in gut microbiota or an increment in the host antioxidant systems. It has been reported that, polyphenols and their metabolites, such us simple phenolic acids and phenolic acids conjugated with short chain fatty acids jointly modulate the microbial community. The administration of polyphenols may increase the levels of short chain fatty acids by changing the composition of the gut microbiota, promoting the short chain fatty acids producing bacteria [23,24]. Short-chain fatty acids, such as butyrate, propionate, and acetate are produced via fermentation of indigestible carbohydrates by intestinal anaerobic commensal bacteria, including clostridia, lactobacilli and bifidobacteria species [25]. These health promoting bacteria produce numerous compounds (glutathione, butyrate, folate) with antioxidant properties and capacity to increase the expression of the host antioxidant enzymes, namely superoxide dismutase and catalase, which contributes to dismiss the oxidative stress of the gut [22,26]. Targeted studies on microbiota composition and internal antioxidant systems would clarify the real contribution of NA infusion to reduce the oxidative stress. Accordingly, the increase of antioxidant activity at intestinal level due to NA infusion could be the result of the interaction of external antioxidant compounds and host factors.