ORAC Assay

The antioxidant capacity of the extracts was assessed by the DPPH method previously described by Mensor et al. and Payet et al. [54 (link),55 (link)]. This method is based on the reduction of alcoholic DPPH (2,2-diphenyl-1-picrylhydrazyl) solution (Sigma–Aldrich) in the presence of a hydrogen-donating antioxidant using 96-well microtiter plates. Plant extracts as described in the ORAC method were used (acetone-dissolved essential oils and absolute hydrosols). We pipetted 100 µL methanol (Kemika, Zagreb, Croatia) and 200 µL standard and/or sample into each well. We prepared serial dilutions of standard and samples by pipetting 100 µL from the first row with a multichannel pipette into the wells in the second row and so on to the last row, where 100 µL of the solution is ejected after mixing. In the first column, in 96-well plates, a blank sample was always added. For EOs, the acetone and methanolic solution were used as blank and for hydrosols, water and methanolic solution were used as blank. The reaction starts by adding 100 µL of a methanolic solution of DPPH (200 µM) to each well. The initial absorbance at 517 nm was measured immediately, using MetOH as blank value. After 60 min incubation, the absorbance was measured again and the percentage of DPPH inhibition was calculated according to the following formula by Yen and Duh [56 (link)]:
where AC(0) is the absorbance of the control at t = 0 min, and AA(t) is the absorbance of the antioxidant at t = 1 h. All measurements were performed in triplicate. The standard curve was generated by plotting the percentage of inhibition of standard with corresponding μmol of Trolox. From the standard curve, results for EOs were expressed as μmol of Trolox per g of EO and for hydrosols as μmol of Trolox per g of absolute hydrosol. Because of the data from other relevant literature we also expressed IC50 values for EOs expressed in mg/mL.
For both antioxidant methods, we also tested the activity of the most abundant compound in EOs using the same method as for total oils. We used pure standard of the hexahydropharnesyl acetone (BOC Sciences, Shirley, NY, USA ), the concentration of the solution was 1 mg per g of acetone and was then further diluted in phosphate buffer up to the concentration of 100 μg/g.

For the DPPH microplate method, the free radical scavenging activity was determined spectrophotometrically. The 2,2’-diphenyl--picrylhydrazyl radical (DPPH^•) analysis conditions were performed in a Greiner Bio-One transparent 96-well microplate (North Carolina, USA) and assayed as described by Bobo-García et al. [69 (link)], with some modifications. Samples were prepared from a stock solution (40 mg/mL) with consequent several dilutions from 28.00 to 0.88 mg/mL in methanol. The stock solution was submitted to ultrasounds (10 min) in order to improve the dissolution, and then filtered using Macherey-Nagel 0.45 µm pore size Chromafil^® PET filters (Düren, Germany). The absorbance of the mixture from a stable violet at 515 nm to yellow after the addition of different quantities of sample/standard was measured. Trolox (0.0075–0.075 mg/mL) was used as antioxidant standard. A total of 25 μL of the sample, standard or methanol (negative control), was added in each well separately to 175 μL of DPPH^• methanolic solution (60 µM) and shaken for 5 s in the Synergy H1^TM microplate reader (BioTek Instruments, Inc.). After 30 min incubation period at 23 °C kept in the dark, the absorbance was measured at 515 nm on a Synergy^TM microplate reader, and the experiment was carried out in triplicate.
The inhibition capacity expressed as IC₅₀ values indicate the concentration of the antiradical compound necessary to decrease 50% of the initial DPPH^• absorbance and was calculated using the linear regression from the concentration of sample versus percentage of inhibition [70 (link)]. To calculate the scavenging capability of DPPH radical, both DPPH^• percentage (%) of inhibition and IC₅₀ were calculated by the following equations, respectively: $\% Inhibition=\frac{A_{negative control }-A_{sample }}{A_{negative control }}\times 100$
$I{C}_{50} (\mathrm{mg}/\mathrm{mL})=\frac{50-b}{m}$
The ABTS assay was performed in a Greiner Bio-One transparent 96-well microplate (Kremsmünster, Austria), based on the inhibition by antioxidants of the absorbance of the radical cation 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS^•+), which has a characteristic wavelength absorption spectrum at 734 nm [71 (link)].
The samples used in this method were prepared the same as for the DPPH^• method mentioned above and the analysis conditions were assayed as described by Benteldjoune et al. [72 (link)], with some modifications. Briefly, the 20 µL of sample, standard or methanol (negative control), was added in each well separately and allowed to react with 180 µL of ABTS^•+ solution (prepared in methanol to an absorbance of 0.700 ± 0.02 at 734 nm) for 5 min in the dark and the absorbance was immediately recorded at 734 nm by a Synergy H1^TM microplate reader (BioTek Instruments, Inc.) and the experiment was carried out in triplicate. Trolox diluted in phosphate buffer was used as the antioxidant standard (25–175 µM). The Trolox equivalent antioxidant capacity (TEAC) was calculated using the following equation: $TEAC \left(\mathsf{\mu }\mathrm{mol}/\mathrm{g}\right)=\frac{\left(\frac{\left(IC{\%}_{Sample}-b\right)}{a}\times {10}^6\right)}{C_{Sample }\times M_{Trolox}}$
To perform the ORAC microplate method, samples were prepared from a stock solution (2.5 mg/mL) with consequent several dilutions from 2.00 to 0.06 mg/mL in a phosphate buffer (PBS) (75 mM, pH 7.4). The stock solution was submitted to ultrasounds (10 min) in order to improve the dissolution and then filtered using Macherey-Nagel 0.45 µm pore size Chromafil^® PET filters (Düren, Germany). The analysis conditions were assayed as described by Dávalos et al. [73 (link)] with some modifications. In a Thermo Fisher 96-well black microplate (Waltham, MA, USA), a volume of 20 μL of the diluted sample, standard or PBS (negative control), was added in each well separately with 120 μL of fluorescein (116.66 nM), and then the microplate was preincubated at 37 °C for 10 min. After incubation, 60 μL of 2,2′-azobis-(2-methylpropionamidine) dihydrochloride (AAPH) at 13.02 mg/mL prepared with PBS was added, and then the mixture was incubated at 37 °C for 120 min. Trolox diluted in PBS was used as antioxidant standard (10–80 µM). The fluorescent values were recorded every minute over the incubation period, at 458 nm and 520 nm using a Synergy H1^TM microplate reader (BioTek Instruments, Inc., Winooski, VT, USA, EUA) and the experiment was carried out in triplicate.
The area under the curve (AUC) over the incubation period values were calculated by subtracting the AUC of the negative control or blank from all the results. Regression equations between net AUC and antioxidant concentration were calculated. The antioxidant capacity (ORAC value) was expressed in µmoles of Trolox equivalents (TE) per grams of sample and the following equation was used [74 ]: $ORAC VALUE \left(\mathsf{\mu }\mathrm{moles}\mathrm{TE}/\mathrm{g}\right)=\left(\mathsf{\mu }\mathrm{moles}\mathrm{TE}/\mathrm{L}\right)\times DF\times \left(\mathrm{L}\mathrm{solvent}/\mathrm{g}\mathrm{sample}\right)$
where ORAC values (µmoles TE/L) correspond to the x value obtained from Trolox linear regression (y = mx + b) by replacing y from the AUC sample values, DF represents dilution factor, and L solvent/g sample is the volume of prepared mother solution/mass of sample mother solution.

TPC, DPPH, ABTS and ORAC assays were selected for the characterisation of SBP extracts. Detailed description of these methods is provided elsewhere [27 (link)]. Briefly, for TPC assay extract solutions were mixed with Folin–Ciocalteau reagent and 7% Na₂CO₃ in a 96-well microplate. The absorbance was measured at 765 nm after 30 min in a FLUOstar Omega Reader (BMG Labtech, Offenburg, Germany). TPC was expressed in mg of GAE/g dry extract weight (DWE) and DWP.
For ABTS^•+ decolourisation 6 µL of sample were added to 294 µL of ABTS^•+ working solution, while for DPPH• scavenging 8 μL of sample were mixed with 292 µL of DPPH• methanolic solution. The absorbance was measured in 96-well microplates using a FLUOstar Omega Reader (BMG Labtech, Ortenberg, Germany) during 30 min at 734 nm and 60 min at 515 nm for ABTS^•+ and DPPH•, respectively. Trolox was used as a standard, antioxidant capacity of the extracts was determined from the calibration curves and the results were expressed as µM TE/g DWE and DWP. Each analysis was carried out in six replicates.
For ORAC assay 25 µL of sample and 150 µL (14 μM) fluorescein solutions were placed into the wells of a black 96-well microplate. Then the mixture was preincubated in a FLUOstar Omega Reader for 15 min at 37 °C and 25 µL of AAPH (240 mM) were pipetted into each well. The fluorescence was recorded every cycle (in total, 120 cycles) using 485 excitation and 530 emission fluorescence filters. Antioxidant curves (fluorescence versus time) were first normalized and from the normalized curves the net area under the fluorescein decay curve (AUC) was measured. The results were expressed in µM TE/g DWE and DWP.

The ORAC assay was carried out using a plate reader (Synergy™ HTX, Agilent, Santa Clara, CA, USA) with an adapted methodology [85 (link),86 (link),87 (link),88 (link),89 (link)]. Samples were extracted into acetone:water:acetic acid at a volume ratio of 70:29.5:0.5, then diluted in phosphate buffer. Diluted samples were put into a 96-well plate with fluorescein solution. The reaction was initiated by the addition of 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) solution. Trolox was used as standard, so the results are expressed as µmol of Trolox equivalent (TE) per g of sample. The experiment was performed in triplicate.

The antioxidative capability of TSE and its primary flavonoids was determined using the ORAC assay according to the previously described method [44 ,45 ]. Both Trolox and AAPH were dissolved in potassium phosphate buffer (PBS) immediately before the ORAC assay. The reaction mixture was prepared with 20 μL of 75 mmol L⁻¹ PBS, 20 μL TSE or eight flavonoids solution and 20 μL fluorescein sodium in the presence of 140 μL AAPH. The ORAC procedure was carried out on a Synergy H1 fluorescence microplate reader (BioTek, Winooski, VT, USA) with an excitation/emission wavelength of 485/527 nm. Briefly, fluorescein sodium was served as the substrate. AAPH was used to generated peroxyl radicals. The results were calculated based on the area of under the fluorescence decay curve with the Trolox as a standard. All results were expressed as ORAC value which is defined as a protection area under curve of one Trolox unit.

An ORAC assay was carried out as described by Prior et al. [24 (link)]. The excitation wavelength was 485 nm, while the emission wavelength was 520 nm. The results are given as µM of Trolox per 100 g of BPF (µM T/100 g).