B 480

Gallic acid, quercetin, 2,2-diphenyl-2-picrylhydrazyl, dichloromethane, and polyvinylpyrrolidone (PVP) were purchased from Sigma Chemical Co, USA, and were used in in vitro and in vivo experiments. All other chemicals and solvents were of analytical grade and were used without any purification.
A rotatory evaporator (Buchi, B-480, UK) was used for the efficient removal of solvents from plant extracts. A microplate reader (SpectraMax Plus, USA) was used in the determination of the total antioxidant activity of the selected medicinal plant extracts. A UV-visible, double-beam spectrophotometer (UV-1800 Shimadzu, USA) was used in the spectrophotometric bioassays. Hematological parameters were analyzed using an automated hematology analyzer (Mindray BC 5150, China). Tissue processor (Shandon, UK), microtome (Thermo Fisher, Germany), and microscope (Olympus CX 21, Japan) were used in the preparation and evaluation of hematoxylin and eosin- (H and E-) stained tissue sections.

To obtain the Spirulina biomass, the Arthrospira platensis BIO1 strain, kindly supplied by Biodesma S.L., was used. A. platensis was grown in plastic bioreactors in a greenhouse at the Universidad Politécnica de Madrid (40.446353 N-3.738341 E), as previously described [24 (link), 25 (link)]. Briefly, A. platensis was harvested when the culture reached approximately 1 g/L of dry weight. The harvest was performed from 9:00 to 10:30 hours when the protein content was higher [26 ]. Then, the cells were dried on a horizontal sheet at 50°C for approximately 4-6 h and kept in an opaque container at 4°C to prevent oxidation.
A polar Spirulina extract was prepared by solvent extraction according to the Bligh and Dyer method [27 (link)] after a prior step of Spirulina biomass sonication. Briefly, 50 mg of Spirulina biomass suspended in 1 mL of chlorophorm/methanol 1 : 2 (v/v) was submitted to 40 kHz sonication for 15 min at 25°C. The liquid phase was filtrated, and the recovered biomass was extracted with 0.5 mL of chlorophorm/methanol 1 : 2 (v/v). Next, the polar Spirulina biocomponents were separated by adding twice 0.6 mL of water with 0.58% NaCl to the chlorophorm/methanol phase. After gravimetric separation of phases for 24 h at 4°C, the aqueous phase was dried in a Buchi B480 rotary evaporator, weighed, and frozen at -70°C until use.

This analysis was carried out following the method described by [72 ]. First, 1 g of pulverized sample was added to 40 mL of 20% ethanol, homogenized and left for 4 h at 55 °C. This mixture was filtered with vacuum pump coupled with the filtrating unit. Residue was collected and re-extracted with 20 mL of 20% ethanol. Filtrates were concentrated to 40 mL in a water bath (BUCHI B-480) at 90 °C, after which the concentrate was transferred into a separating funnel. Then, 20 mL of diethyl ether was added and vigorously mixed. Afterwards, the lower fraction was collected, with the upper ether layer discarded. Butan-1-ol was added to the re-introduced lower fraction and mixed vigorously again. Then, 5 mL of 5% aqueous sodium chloride (NaCl) was added. The upper fraction (butan-1-ol) was then collected and evaporated in the oven to constant weight.
$\%\mathrm{Saponin}=\frac{\mathrm{Weight}\mathrm{of}\mathrm{fraction}}{\mathrm{Weight}\mathrm{of}\mathrm{sample}}\times 100$

The total fat content of raw olives and dehydrated and pitted olives was determined using the method developed by Folch et al. [51 (link)], with some modifications. Briefly, 200 mL of CLF:MeOH (2:1 v/v) were added to 10 g of pitted olives and mixed with an Ultraturrax T18 basic IKA (Staufen, Germany) at 11,800 and 16,200 rpm for 2 min, until a homogeneous mixture was obtained, which was maintained via magnetic stirring at 900 rpm for 60 min. The sample was then vacuum-filtered and distilled water was added to the organic phase in a 1:5 ratio. The mixture was mixed in a vortex for 1 min and centrifuged for 5 min at 10,000 rpm. The lower phases were collected and placed in a decanter for 3 h. Later, the lower phase was collected and evaporated in a rotary evaporator (Büchi B-480, Uster, Switzerland) at 40 °C under 10 mbar until a constant weight was achieved. These determinations were carried out in duplicate for each sample.