1700 pharmaspec uv vis spectrophotometer

The measurement of the DPPH (2,2-diphenylpicrylhydrazyl hydrate) radical scavenging activity was carried out according to Yen and Chen [81 (link)] with modifications concerning the time of reaction [82 ]. Dry hydroethanolic extract (0.25 mg) was dissolved in 1 mL of methanol (2×, 4×, 8×, 16× diluted solutions). Essential oils were also diluted in methanol (concentrations 0.20, 0.40, 0.80, 1.60, and 3.20 mg × mL⁻¹) [83 (link)]. Then, 3 mL of methanol and 1 mL of DPPH methanolic solution (0.12 mg × mL⁻¹) were added to 1 mL of the different concentrations of extracts and essential oils. Absorbance was measured after 10 min at 517 nm using a 1700 PharmaSpec UV/Vis spectrophotometer (Shimadzu, Warsaw, Poland). The antioxidant activity of extracts and essential oils was calculated as I = [(AB − AA)/AB] × 100, where I is DPPH inhibition (%); AB is the absorbance of a blank sample (t = 0 min); AA is the absorption of extract solution (t = 10 min). Trolox was used to estimate a standard curve. Results are expressed in μmol Trolox equivalents per g of extract and essential oil.

For the estimation of the peroxidase activity of α-Syn in the absence and presence of heme, 2 μM heme bound or unbound α-Syn (WT or H50Q, α-Syn/heme 25:1) was treated at 25 °C with 200 µM H₂O₂ and 10 mM guaiacol. The product formation kinetics was followed using a Shimadzu 1700 Pharmaspec UV-VIS spectrophotometer. The rate of decomposition of hydrogen peroxide (H₂O₂) by peroxidase using guaiacol as a hydrogen donor, was determined by measuring the rate of colour development spectrophotometrically at 470 nm using the extinction coefficient of 2.66 × 10⁴ M⁻¹ cm^{−164 (link)}.

ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid radical caption) was measured according to the method described by Re et al. [84 (link)] and Arts et al. [85 (link)]. A stock solution was prepared by stirring 7 mM ABTS and 2.45 mM (final concentration) potassium persulfate in water and incubating at room temperature in the dark, for 16 h before use. The concentrated ABTS was diluted with phosphate buffered saline (PBS) to a final absorbance of 0.72 (±02) at 734 nm. Then 1 mL ABTS solution was added to 100 μL of extracts (2×, 4×, 8×, 16× diluted solutions) and 100 μL of essential oil (0.20, 0.40, 0.80, 1.60, and 3.20 mg/ml concentrations). Absorbance of ABTS was measured on a 1700 PharmaSpec UV/Vis spectrophotometer (Shimadzu) after 6 min incubation in dark, at 734 nm. The ability of the test sample to scavenge ABTS was compared to the Trolox standard. The solutions of Trolox were prepared in PBS, such that the final concentrations of the standards were 0.0, 1.25, 2.5, 5.0, 7.5, 10.0 mg × 100 mL⁻¹. The percentage inhibition of ABTS of the test samples were calculated according to the following formula:
where AB is the absorbance of a blank sample and AA is the absorbance of the test sample. Results are expressed in mg Trolox equivalents per 100 mL of extract.