Universal 32r

Soil pH measurements were carried out as described by Mandakovic et al. [45 (link)]. A portion of the BS and RSS samples was used to determine metal composition using the total reflection X-ray fluorescence (TXRF) technique. Briefly, for each sample, 1 g of soil was resuspended in 1 mL of distilled water and homogenized for 2 h at room temperature. After mixing, the samples were centrifuged at 11,440g for 10 min in a Hettich Universal 32R. The soluble fraction was recovered and measured in a Bruker S2 PICOFOX. All these analytical protocols have been previously reported [35 (link), 45 (link)]. Pearson’s correlation coefficient was calculated among all nutrients and chemical components that were present in at least 15% of the samples. For those selected variables, missing values were imputed with the missForest R package [46 (link)].

The reducing power of the P. atlantica leaf extracts was determined according to the method described by Kannan et al. (2013 ). 50-, 100-, and 200-fold methanol-diluted extracts (2.5 mL) were mixed with 2.5 mL phosphate buffer 200 mM at pH 6.6 and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50 °C for 20 min. Then, 2.5 mL of TCA (10% w/v) was added and the mixture was centrifugated for 10 min at 1000 g (Universal 32 R, Hettich, Tuttlingen, Germany). The supernatant (2.5 mL) was added to glass tubes containing 2.5 mL distilled water and 0.5 mL FeCl₃·6H₂O (0.1%, w/v). The absorbance of the resulting solution was measured at 700 nm using distilled water as control solution. The reducing power activity was calculated from a standard curve based on a set of ascorbic acid solutions (0.05–0.3 mg/mL). Results were defined as ascorbic acid equivalent antioxidant capacity (AAEAC) and expressed as milligrams of ascorbic acid equivalents per gram of dry leaf weight.

The fasted rats were euthanized by exposure to isoflurane and dissected at the end of the in vivo investigation. Blood samples were collected via cardiac punctures in all the animal groups using a sterile syringe, before being centrifuged at 3000 rpm for 10 min to separate the blood serum. The serum was preserved at −20 °C for further use to evaluate the biochemical parameters. On the other hand, the pancreas and liver were isolated from all the experimental groups and cleaned from blood, before being rinsed with a chilled saline solution. For the biochemical investigations, parts of the pancreatic and hepatic tissues were separately homogenized for 5 min in 2 mL/g tissue of cold buffer (50 mM potassium phosphate pH 7.5, 1 mM EDTA) by the means of a homogenizer (Potter-Elvehjem homogenizer). Afterward, the homogenates were clarified for 15 min at 4000 rpm and 4 °C using a centrifuge (Universal 32 R, Hettich, Tuttlingen, Germany), and the supernatants were preserved at −80 °C. For the molecular analyses, small portions of the pancreas and livers were instantly dried and preserved at −80 °C. For the histological investigations, small slices of the pancreas and liver were fixed in formalin solution (10%).