Multiskan spectrum plate reader

DPPH free radical scavenging activity of the aqueous plant extracts was carried out according to a method described by Zheleva-Dimitrova [22 (link)] with slight modifications. Briefly, 10 μL of the different concentrations of the aqueous plant extracts was reacted with 190 μL of DPPH solution (0.00625 g DPPH in 50 mL methanol) and the absorbance of the samples was determined after 30 min using a Multiskan Spectrum plate reader (Thermo Fisher Scientific, USA) at 517 nm. Free radical scavenging activity of the samples was expressed according to the equation below:
$\begin{array}{c}\text{Percent (\%) inhibition of DPPH activity}\mathrm{\hspace{0.17em}\hspace{0.17em}}\\ \hspace{1em}\hspace{1em}=\frac{A^0-A}{A^0}\times \mathrm{100},\end{array}$
where A⁰ is the absorbance of DPPH• in solution without an antioxidant and A is the absorbance of DPPH• in the presence of an antioxidant. IC₅₀ value (concentration of sample where absorbance of DPPH decreases 50% with respect to absorbance of blank) of the sample was determined. All determinations were done in triplicates.

The SRB assay is based on the measurement of cellular protein content according to our previous study [36] (link), [37] (link). Culture medium was aspirated after indicated treatment, and cells were fixed with 10% trichloroacetic acid for 10 minutes. 0.4% (w/v) SRB in 1% acetic acid was then added in each culture well and stained for 30 minutes. Unbound SRB was washed out by 1% acetic acid and SRB-bounded cells were dissolved by 10 mM Tris solution. The absorbance was measured at 515 nm by Thermo Multiskan® Spectrum plate reader.

The FRAP assay was carried out in accordance with the method described previously [23 (link)]. In a 96-well plate, 10 μL of the stock solution (1 mg/mL w/v) of the isolated compounds (1–3) was mixed with 300 μL of FRAP reagent. The FRAP reagent was prepared by mixing (10:1:1, v/v/v) of acetate buffer (300 mM, pH 3.6), tripyridyl triazine (TPTZ) (10 mM in 40 mM HCl), and FeCl_3.6H₂O (20 mM). Incubation commenced at room temperature for 30 min, and the plate was read at a wavelength of 593 nm in a Multiskan spectrum plate reader (Thermo Fisher Scientific). L-Ascorbic (Sigma-Aldrich, Cape Town, South Africa) was used as a standard, with concentrations varying between 0 and 1000 μM. Further dilutions were carried out with the samples that were highly concentrated, and such dilution factors were recorded and used for calculations of the affected samples. The results are expressed as μM ascorbic acid equivalents per milligram dry weight (μM AAE/g) of the test samples.

LMA testing was performed on protein extracted from 10 mg wholemeal flour according to the method described by Verity et al.^{59 (link)} and Newberry et al.⁶⁰ . All spectrophotometric measurements were performed using a Thermo Scientific Multiskan Spectrum plate reader. LMA testing was restricted to 24 out of 28 landraces due to grain availability.

The TEAC assay was carried out using the principle of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activity according to a method described by Ou et al. [24 (link)]. ABTS⁺ solution was prepared a day before use by mixing ABTS salt (8 mM) with potassium persulfate (3 mM) and then storing the solution in the dark until the assay could be performed. The ABTS⁺ solution was further diluted with distilled water. Twenty five microlitres (25 μL) of the diluted aqueous plant extracts was mixed with 300 μL ABTS⁺ solution in a 96-well clear microplate. The plate was read after 30 min incubation at room temperature in a Multiskan Spectrum plate reader (Thermo Fisher Scientific, USA) at 734 nm. Trolox was used as the standard and results were expressed as μmol TE/g sample. All determinations were done in triplicates.

Activities of antioxidant enzymes were determined in a clear 96-well plate using a Multiskan Spectrum plate reader (Thermo Fisher Scientific, USA). Glutathione peroxidase (GPx) activity was determined according to the method of Ellerby & Bredesen [11 (link)]. To initiate the reaction, 25 μL of H₂O₂ (15 mM) was added to a final reaction mixture containing 2.5 μL of GSH (0.1 M), 2.5 μL of GR (0.1 U/mL), 5 μL liver homogenate, 5 μL of NADPH (15 mM in 0.1 % NaHCO3), 2.5 μL of sodium azide (100 mM) and 210 μL of assay buffer (50 mM potassium phosphate, 1 mM EDTA (Ethylenediaminetetraacetic acid), pH 7.0. The rate of H₂O₂-dependent oxidation of NADPH was monitored at 340 nm at 30 s intervals for 2 min. The activity of GPx was calculated using the extinction coefficient of 6.22 mM-1 cm-1 and results expressed as nmol NADPH oxidized per min per μg protein.

The radical cation decolourization test that use the principle of 2, 2′-azinobis (3-ethylbenzothiazolie-6-sulfonate) diammonium salt (ABTS) according to the method of Re et al. (1999) (link) was used for the evaluation of TEAC of the BGN extracts. By combing 8 mM ABTS (in water) and 3mM (final concentration) of potassium persulfate and standing in the dark for 16 h, ABTS∗ was produced. This was adjusted to give an absorbance of 0.700 units at 734 nm. In a 96-well microplate, diluted extract of BGN (25 μL) was combined with 250 μL ABTS∗ solution. The plate was incubated for 30 min at room temperature after which it was read at 734 nm using a Multiskan Spectrum plate reader (Thermo Fisher Scientific, USA). The standard used for the analysis was Trolox and outcomes were communicated as micro mole Trolox Equivalent per gram sample (TE)/g.