Specord 205

The antioxidant capacity of the honey samples was determined according to Chen’s method [73 (link)] with minor modifications, using a 0.03 mM ethanolic solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH, Sigma-Aldrich, Taufkirchen, Germany). It is widely used to test the antioxidant capacity of foods, especially fruits, vegetables, juices, and even honey [74 (link),75 (link),76 (link),77 (link)]. A total of 1 mL of each prepared honey extract was taken, to which 2.5 mL of DPPH solution (Sigma-Aldrich, Taufkirchen, Germany) was added. The samples were shaken and then incubated in the dark for 30 min at room temperature. The absorbance was measured with a UV–VIS spectrophotometer (Specord 205; Analytik Jena AG, Jena, Germany) at 518 nm. An average value was calculated from the three measurements performed for each honey sample. The control was prepared under the same conditions, the difference being that, instead of the honey solution, the same volume of distilled water was used. The antioxidant activity of each sample was calculated as a percentage of the CSR (the p overof radical uptake capacity). CSR of the tested samples was calculated according to the following equation: $$\mathrm{CSR} (\%)=\frac{A_{control}-A_{sample}}{A_{control}}\times 100$$
where

A_control—the control absorbance values;

A_sample—the absorbance values tested of the samples.

Baobab flour (BF) was acquired in the North of Benin, and wheat flour (WF) type 650 was acquired at the Profil supermarket, Timisoara (Romania).
The reagents used were DPPH solution and HCl (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), Ethyl alcohol (SC Chimreactiv SRL, Bucharest, Romania), Folin-Ciocâlteu reagent (Sigma-Aldrich Chemie GmbH, Munich, Germany), and Na₂CO₃ (Geyer GmbH, Renningen, Germany). The equipment used in the study was Specord 205 (Analytik Jena AG, Jena, Germany), Chopin Mixolab (Chopin Technologies, Paris, France), the Varian 220 FAA spectrophotometer (Palo Alto, CA, USA), and Thermostat INB500 (Memmert GmbH, Schwabach, Germany).

The heat-induced haemolysis assay was conducted in accordance with the method developed by Okoli et al. [25 (link)], with some modifications exposed by Gunathilake et al. [24 (link)]. Briefly, different concentrations of essential oil (10 µL/mL, 20 µL/mL, 40 µL/mL, 80 µL/mL, 160 µL/mL) were suspended in 5 mL of PBS isotonic solution (RemedLab, Bucharest, Romania) at pH 7.4, over which 100 µL red blood cell suspension was added. After delicate shaking, the samples were incubated in a water bath at 54 °C for 20 min. The samples were centrifuged at 2500 rpm for 3 min at the end of the incubation period, and the absorbance of the supernatant was measured at 540 nm using a UV-VIS spectrophotometer (Specord 205; Analytik Jena AG, Jena, Germany). The negative control sample consisted of PBS and 100 µL erythrocyte suspension. The positive control sample consisted of 0.1 mg/mL of dexamethasone diluted in 5 mL of PBS and 100 µL erythrocyte suspension.
Formula (5) was used to determine the percentage of haemolysis inhibition: $$\% inhibition of haemolysis=100-\frac{A_1}{A_2}\ast 100$$
where:

A₁ represents the absorbance of the tested sample

A₂ represents the absorbance of the negative control.

The total phenolics content was determined according to Folin–Ciocalteu modified method [73 (link)]. An amount of 0.5 mL extract was treated with 1.25 mL Folin-Ciocalteu reagent (Sigma-Aldrich Chemie GmbH, München, Germany) diluted 1:10 with distilled water. The sample was incubated for 5 min at room temperature, then 1 mL Na₂CO₃ (Geyer GmbH, Renningen, Germany) (60 g/L aqueous salts) was added. The samples were incubated for 30 min at 50 °C in an INB500 thermostat, Memmert GmbH, Schwabach, Germany) after reading absorbance at 750 nm using a UV-VIS spectrophotometer (Specord 205; Analytik Jena AG, Jena, Germany). As a reference, ethanol (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used. The calibration curve was obtained using gallic acid (concentration range: 2.5–250 μg/mL). The results were expressed in mg GAE per g of dry matter (d.m.). All determinations were performed in triplicate.

To study the effect of mediator on decolorization of malachite green by laccase, 20 U/ml of the enzyme was added to 50.0 ml of the dye solution (100 mg/l, pH 5.5) with and without 1 mM HBT and kept at 30 °C, 150 rpm for 36 h. Decolorization of Malachite green was recorded spectrophotometrically at 619 nm (λ_max for malachite green) using a UV–vis spectrophotometer (Analytik-Jena Specord 205). The control sample containing citrate phosphate buffer (pH 5.5) in place of enzyme was run in parallel. The percentage of decolorization was calculated as follows: $$\text{Percentage of decolorization}\left(\%\right)=\left(A_{\text{c}}-A_{\text{t}}\right)/A_{\text{c}}\times 100$$ where, A_c is the absorbance of the control and, A_t is the absorbance of the test sample.