Uv 1700 uv vis spectrophotometer

Tocopherol samples were analyzed for their capacity to scavenge the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) in accordance with Kalantzakis et al. [39 (link)], with some modifications. Briefly, a 25 μL volume of tocopherol sample (in pure form or mixture) was mixed with 975 μL of DPPH solution (100 μM in ethyl acetate) and absorbance was read at 515 nm immediately after mixing (A_515(i)) and after exactly 30 min (A_515(f)), using a Shimadzu UV-1700 UV/vis spectrophotometer (Kyoto, Japan). The capacity to scavenge the DPPH radical was expressed as shown in Equation (4): $$\mathrm{Inhibition} (\%)=\left(\frac{A_{515\left(\mathrm{i}\right)}-A_{515\left(\mathrm{f}\right)}}{A_{515\left(\mathrm{i}\right)}}\right) \times 100$$
A calibration curve was prepared using Trolox (50–1500 μM) and the results were expressed as μM Trolox equivalent antioxidant capacity (TEAC).
After determining the optimum antioxidant mixture of tocopherols, experiments were repeated with the optimum combination to validate its efficiency. Moreover, in order to examine whether the observed effect was due to addition, antagonism or synergism, the individual tocopherols were also tested for their DPPH radical scavenging activity at the concentrations specified in the optimum mixture. The scavenging activity of the mixture was compared with the theoretical sum of the activities of the individual tocopherols.

Chlorophyll and carotenoid content of the hemp seed oil samples were assessed based on the method reported by Isabel Minguez-Mosquera et al. [72 (link)] with slight modifications. Briefly, each oil sample (0.100 ± 0.001 g) was placed in a tube and the volume was made up to 3 mL using absolute diethyl ether. The solution was vortexed thoroughly and sonicated for 1 min. The absorbance of the solution was measured in the wavelength range 250–750 nm by UV-1700 UV/Vis spectrophotometer (Shimadzu, Kyoto, Japan) against a blank. Oil pigment content (µg/mL) was calculated according to the formula: $\mathrm{Chlorophyll} \mathrm{a}=9.93\times A_{663}-0.78\times A_{640}.$
$\mathrm{Chlorophyll} \mathrm{b}=17.60\times A_{640}-2.81\times A_{663}$
$\mathrm{Chlorophyll} \mathrm{a}+\mathrm{b}=7.12\times A_{663}+16.80\times A_{640}$
$\mathrm{Total} \mathrm{carotene}=\frac{\left(1000 \times A_{470} - 0.52\times Chl a-7.25\times Chl b\right)}{226}.$

The concentration of total phenolic compounds in the MeOH extracts was determined spectrometrically using the Folin–Ciocalteu method [31 (link)], using gallic acid as a standard to prepare a calibration curve. A total of 1 mL of plant extract (10 g/L) was mixed with 5 mL of Folin–Ciocalteu reagent and 4 mL (75 g/L) of sodium carbonate, and after 1 h, the absorption of the reaction mixture was measured at 765 nm against a methanol blank, using a Shimadzu UV-1700 UV/vis spectrophotometer (Tokyo, Japan). The results were expressed as milligrams of gallic acid equivalent (GAE) per gram of extract, based on the reference gallic acid calibration curve (at a linearity range of 1–10 μg/mL, with the equation y = 0.0834x + 0.0925 and R² = 0.9967) generated for this study. All determinations were performed in triplicate.