Genesys 150 uv vis

The measurement of antiradical activity of samples before and after digestion was carried out via DPPH stable radical (2,2-diphenyl-1- picrylhydrazyl) spectroscopy [3 (link)].
Absorbance was measured at a wavelength of 517 nm, and the UV-VIS spectrophotometer (Genesys 150 UV-VIS, Thermo Scientific, Waltham, MA, USA) was calibrated to pure methanol. Measurements were performed every 5 min until a plateau was reached. This allowed the changes in absorbance to be monitored over time and indicated when the plateau was reached. The following formula was used to calculate the radical scavenging potential of the extracts: $$\%RSA=\left[\frac{(A_0-A_{1)}}{A_0}\right]\times 100$$

A₀—the absorbance of the sample except for the tested extracts;

A₁—the absorbance of the sample with tested extracts.

Phlorotannin content was estimated using the Folin–Ciocalteu method [30 ]. In brief, 0.5 mL of extract, standard, or extraction solvent (blank), along with 0.25 mL of Folin–Ciocalteu reagent (diluted 1:2 with deionized water), was sequentially transferred to 3.75 mL of deionized water. The solution was homogenized, and 0.5 mL of 10% w/v Na₂CO₃ was added. The solution was mixed and left to stand in the dark for 1 h at room temperature. The absorbances were measured at 750 nm (Genesys 150 UV-Vis, Thermo Fisher Scientific, Waltham, MA, USA) and compared to a phloroglucinol calibration curve (15–150 mg/L). All analyses were carried out in duplicate. The results were expressed as mg of phloroglucinol equivalents (mg PE) per g of dry seaweed and g of dry extract.

The capacity of light transmittance of chia films was measured according to Dick et al. [15 (link)] with minor modifications. Chia film samples (4 cm × 0.8 cm) were placed in a spectrophotometer cell and transmittance was measured in triplicate by spectrum scanning (wavelengths from 200 to 900 nm) with a spectrophotometer UV-Vis GENESYS 150 (Thermo Fisher Scientific, Waltham, MA, USA). Air was used as reference and the transmittance values (expressed as % of transmittance) were measured in triplicate.

Proteins contained in HSE were quantified by two different methods including Bicinchoninic Acid Protein Assay (BCA proteins assay kit, Sigma-Aldrich) and Bradford Protein Assay. The BCA protein assay was performed as described in literature [18 ]. Bradford assay for protein determination was performed with slight modifications to the original assay [19 (link)]. Carbohydrates contained in HSE were quantified by the Phenol-Sulfuric Acid method as previously performed by Albalasmeh and coworkers, with slight differences [20 (link)]. Total Phenolic Content (TPC) in HSE (including hydrolysable tannins and polyphenols) was quantified as previously proposed by Aruwa and his collaborators [21 (link)]. The antioxidant activity of HSE, quantified by DPPH assay, aimed to investigate the capability of our extracts to inhibit free radicals. DPPH solution and L-ascorbic acid solution (5 mg/mL) were used, respectively, as negative and positive controls. The percentage radical scavenging activity was normalized as a function of HSE concentrations. The percentage DPPH inhibition was calculated using the following equation [22 ]:

All quantifications were performed with a spectrophotometer (UV-Vis Genesys 150^®-ThermoScientific).

The Total Phenolic Content (TPC) of the aromatic plant extracts was determined in triplicate using Folin-Ciocalteu reagent as described by Singleton and Rossi [39 ] with modifications. Briefly, 200 µL of EOs and EEs dissolved in methanol (1 mg/mL) were mixed with 2.5 mL of Folin-Ciocalteu (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:10 in distilled water. The samples were incubated at room temperature for 5 min and vortexed. Next, two mL of 7.5% w/v Na₂CO₃ (Sigma-Aldrich, St. Louis, MO, USA) was added and the tubes were incubated for 90 min in darkness. The absorbance of samples was measured at 765 nm using a spectrophotometer UV-Vis GENESYS 150 (Thermo Fisher Scientific, Waltham, MA, USA) against a blank of distilled water. A standard curve with concentrations of gallic acid in methanol ranging from 0.05 to 2 mg/mL was performed. The amount of TPC was determined as milligrams of gallic acid equivalent per gram of extract (mg GAE/g extract) using the standard curve.

We also investigated the direct effect of nutraceutical, compared to placebo, in a cellular model of hepatic steatosis. Polyphenols from nutraceutical and placebo capsule were extracts. At one capsule of nutraceutical and placebo was added with 50 mL of water:ethanol mixture (1:3). Then, the extracts were first purified by centrifuge at 2500 rpm/5 min, followed by two centrifuges of 20×g/8 min. Subsequently the extracts were characterized for the antioxidant activity. The antioxidant activity of nutraceutical and placebo extract was quantified by DPPH assay, to investigate the capability of two extracts to inhibit free radicals. DPPH solution and l-ascorbic acid solution (5 mg/mL) were used as negative and positive controls, respectively (x) [52 ]. All quantifications were performed with a spectrophotometer (UV–Vis Genesys 150®-ThermoScientific).