Oxygen Radical Absorbance Capacity

The antioxidant activity was determined using oxygen radical absorbance capacity (ORAC) assay [22] and modified in our laboratory [23] (link). Phenolic extract dilutions were prepared with 75 mM phosphate buffer (pH 7.4). The assay was performed in black-walled 96-well plates (Corning Scientific, Corning, NY). The outside wells of the plate were not used as there was much more variation from them than from the inner wells. Each well contained 20 µL extracts or 20 µLTrolox standard (range 6.25–50 µM), and 200 µL fluoroscein (final concentration 0.96 µM), which were incubated at 37°C for 20 min. After incubation, 20 µL of 119 mM ABAP was added to each well. Fluorescence intensity was measured using Fluoroskan Ascent FL plate-reader (Thermo Labsystems, Franklin, MA) at excitation of 485 nm and emission of 520 nm for 35 cycles every 5 min. ORAC values were expressed as milligrams of TE/100 g FW. Data were reported as mean ± SD with triplicates.

Total Phenolic Content (TPC): the quantification of TPC on B. bifurcata samples was achieved using the Folin–Ciocalteu method adapted to a microscale [52 (link)], with minor modifications, and as described by Pinteus et al. [10 (link)]. Briefly, 2 µL of samples were added to 158 µL of distilled water in a 96-well microplate, followed by the addition of 10 µL of Folin–Ciocalteu reagent. The reaction mixture was pre-incubated for 2 min at room temperature, and then 30 µL of 20% Na₂CO₃ (w/v) was added and mixed. After 1 h of reaction in the dark, the absorbance was measured at 755 nm (Synergy H1 Multi-Mode Microplate Reader, BioTek^® Instruments, Winooski, VT, USA). Gallic acid was used as standard. TPC was expressed in milligrams of gallic acid equivalents per gram of dry extract (mg GAE/g of extract).
DPPH Radical Scavenging Activity: this assay was performed according to Pinteus et al. [10 (link)]. An aliquot of 2 µL of B. bifurcata samples of different concentrations was added to 198 µL of DPPH ethanol solution (0.1 mM) to obtain final concentrations of 10, 30, 100, 300 and 1000 μg/mL and the mixtures were then incubated in the dark for 30 min at room temperature. Results are expressed as mean values ± SEM (standard error of the mean). IC₅₀ values (μg/mL) were also determined for the extracts with highest activity (DPPH reduction >50%).
Oxygen Radical Absorbance Capacity (ORAC-fluorescein): this assay followed the methodology described by Dávalos et al. [53 (link)]. The reaction was carried out in 75 mM phosphate buffer (pH 7.4), and the final reaction mixture was 200 µL. B. bifurcata samples (20 µL), and fluorescein (120 µL; 70 nM, final concentration) were placed in the wells of the 96-well microplate. The mixture was pre-incubated for 15 min at 37 °C. Then, AAPH solution (60 µL; 12 mM, final concentration) was added rapidly, using a multichannel micropipette. The microplate was immediately placed in the reader and the fluorescence (λ_excitation: 458 nm; λ_emission: 520 nm), recorded every minute for 240 min, and automatically shaken prior to each reading. A blank using phosphate buffer instead of the fluorescein and eight calibration solutions, using Trolox (0–80 µM) as antioxidant standard, were also carried out in each assay. ORAC values were expressed as Trolox equivalents by using the standard curve calculated for each assay. Final results were expressed in µmol of Trolox equivalents/g of dry extract (or compound) (µmol TE/g of extract (or compound)).
Ferric Reducing Antioxidant Power (FRAP): this method was performed according to Benzie and Strain [54 (link)] and Li et al. [29 (link)], adapted to a microscale with minor modifications. FeSO₄ was used as standard. FRAP reagent was prepared with 0.3 M acetate buffer (pH = 3.6), 10 mM of TPTZ in 40 mM HCl and 20 mM ferric solution using FeCl₃ in a 96-well microplate. By freshly mixing acetate buffer, TPTZ, and ferric solutions at a ratio of 10:1:1, the final working FRAP reagent was incubated at 37 °C. Briefly, 2 µL of B. bifurcata samples were added to 198 µL of FRAP reagent and allowed to stand at 37 °C in the dark by 30 min, at which time the increase in absorbance at 593 nm was measured in the microplate reader. The difference between the absorbance of test compounds and the blank reading was calculated and expressed as µM of FeSO₄/g of extract (or compound).

To evaluate the free radical scavenging activity of NNF, DPPH- [38 (link)] and ABTS-radical-scavenging assay [39 (link)] was performed in which ascorbic acid was used as a positive control. The following equation was used to calculate the percent inhibition: $\mathrm{Radical}-\mathrm{scavenging}\mathrm{activity}\left(\%\mathrm{inhibition}\right)=\left\{\frac{\left({\mathrm{Abs}}_{\mathrm{c}}-{\mathrm{Abs}}_{\mathrm{s}}\right)}{{\mathrm{Abs}}_{\mathrm{c}}}\right\}\times 100$
where Abs_c and Abs_s denotes the absorbance of the control and the experimental sample, respectively. All samples were analyzed in triplicate.
A non-enzymatic phenazine methosulfate-nicotinamide adenine dinucleotide (PMS/NADH) system was used to generate the superoxide radical (O₂^•−), which reduces NBT to a purple color formazan. The method described by Kumar and Chattopadhyay [12 (link)] was adopted for the superoxide scavenging activity of NNF and percent inhibition was calculated by using Equation (1).
The Fe³⁺-ascorbate-EDTA-H₂O₂ system (Fenton reaction) was carried out to generate the hydroxyl radical (OH^•). The hydroxyl radical (OH^•) scavenging activity of NNF was measured as described in previous method [40 (link)] and equation 1 was used to determine the percent inhibition.
For the measurement of reducing power, the ferric reducing antioxidant power (FRAP) assay [41 (link)] and the cupric-reducing antioxidant capacity (CUPRAC) [42 (link)] was performed and the results were expressed as the ascorbic acid-equivalent antioxidant value (μM).
The oxygen radical absorbance capacity (ORAC) assay [43 (link)] was carried out using Trolox, a water-soluble analogue of vitamin E, as a positive control. The experiment was conducted at 37 °C and at pH 7.4 with a blank sample in parallel, and the antioxidant potentiality was calculated as a trolox-equivalent antioxidant value (µM).

An oxygen radical absorbance capacity (ORAC) assay was conducted per the method in the literature^{65 (link)}. Briefly, 100 μL of fluorescein (10 μM) and a 50 μL sample (0.1 mg/mL) of Trolox (15–250 μM) were transferred into a 96-well plate. Subsequently, 50 μL of 2, 2’-azobis(2-methylpropionamidine) dihydrochloride (500 mM) was added and the fluorescence of the mixture was recorded for 2 h at 485-nm excitation and 528-nm emission in a Synergy LX microplate reader (BioTek, Winooski, VT, USA). The ORAC was reported in terms of micromoles of Trolox equivalents (TE) per gram of dry RPH sample (µmol TE/g RPH)^{65 (link)}. $$\text{ROO}·+\text{AH}\to \text{ROOH}+\text{A}·$$ $$\text{ROO}·+\text{FLH}\to \text{ROOH}+\text{FL}·$$

To evaluate antioxidant activities of hydrolytes in vitro, DPPH free radical scavenging activity (DPPH RSA) assay, hydroxyl free radical (OH) scavenging activity (OH RSA) assay and oxygen radical absorbance capacity (ORAC) assay were conducted according to previous studies, with minor modifications [42 (link),43 (link),44 (link)]. The DPPH and OH radical scavenging rate were calculated.
The ORAC was defined as Trolox equivalents (mmol TE/g sample or mmol TE/mmol sample) according to the area under the curve (AUC) and calculated using the following formula:
where AUC_sample, AUC_control and AUC_Trolox were the integral areas under the fluorescence decay curve of the sample, PBS and Trolox, and M_Tolox and M_sample were the concentrations of the Trolox and sample, respectively.

The total polyphenol fruit and leaf extracts were preliminary evaluated for the detection of flavonoids (Shinoda test), steroids and terpenoids (Liebermann–Buchard test), alkaloids (Dragendorff test), and sapponins (foam test) [43 (link),44 ]. Soluble solids were determined in a refractometer (ATAGO) as the percentage °Brix.
The total polyphenol content (TPC) was determined by the Folin–Ciocalteu spectrophotometric method, as described by Rojas-Garbanzo et al. [45 (link)]. Fruit and leaf extracts were incubated with Folin–Ciocalteu reagent and sodium carbonate solution (75 g L⁻¹) at 50 °C for 15 min, and the absorbance was measured at λ = 620 nm (FLUOstar OPTIMA, BMG LABTECH, Ortenberg, Germany). TPC was calculated against an external calibration curve of gallic acid (10–80 mg GAE L⁻¹, r² = 0.9909), and it is expressed as µg of gallic acid (>99% purity, Sigma-Aldrich, St Louis, MO, USA) equivalents (GAE) per gram of extract. The results are shown as the mean ± SD (n = 3).
The total flavonoid content (TFC) was analyzed by the aluminum chloride method described by Fernandes et al. [46 (link)]. Fruit and leaf extracts (10 mg extract mL⁻¹ in methanol) with 2% aluminum chloride solution (in methanol) were incubated for 10 min at room temperature. The absorbance was measured at λ = 450 nm (FLUOstar OPTIMA, BMG LABTECH, Ortenberg, Germany), and TFC was calculated against an external calibration curve of quercetin (0–160 mg QE L⁻¹, r² = 0.9981) and is expressed as mg of quercetin (>99% purity, Sigma-Aldrich, St Louis, MO, USA) equivalents (QE) per gram of extract. The results are shown as the mean ± SD (n = 3).
To determine the chlorophyll and carotenoid content, the UV-VIS spectrum was recorded at λ = 200–750 nm (Shimadzu UV1800), with dimethyl-sulfoxide (DMSO) as a blank probe. The pigment content based on the absorbance was calculated as previously described [47 ], as follows: $$\begin{gathered} Chlorophyll A \left(\mathsf{\mu }\mathrm{g}/\mathrm{mL}\right)=12.47 A_{665.1}-3.62 A_{649.1} \\ Chlorophyll B \left(\mathsf{\mu }\mathrm{g}/\mathrm{mL}\right)=25.06 A_{649}-16.5 A_{665.1} \\ Total carotenoids \left(\mathsf{\mu }\mathrm{g}/\mathrm{mL}\right)=\frac{1000 A_{480}-1.29 C_A-53.78 C_B}{220} \end{gathered}$$
The protein content was analyzed using the Quick Start^TM Bradford Protein Assay Kit (BIO-RAD), following the manufacturer’s protocol. The total protein concentration was calculated by comparing it against an external bovine serum albumin (BSA) standard curve (0–2 mg mL⁻¹, r² = 0.973).
The antioxidant activity was determined by the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay, as described by Wang et al. [48 (link)]. Leaf samples (10 mg extract mL⁻¹ in 70% ethanol) diluted in methanol were incubated with DPPH (0.5 mM in methanol) at 37 ºC for 30 min in the dark. Absorbance was measured at λ = 544 nm (FLUOstar OPTIMA, BMG LABTECH), and the radical scavenging activity is shown as the DPPH percentage inhibition = [Ac − As − Ab)]/(Ac × 100), where Ac is the absorbance of the negative control (DDPH without sample), As is the absorbance of the sample, and Ab is the absorbance of the sample without DPPH. The IC₅₀ (the concentration of the sample required to inhibit the DPPH response by 50% with respect to the untreated control) was calculated from the linear equation of each curve (Graph-Pad Prism, v. 9.1.1; GraphPad Software, San Diego, CA, USA). The ORAC (Oxygen Radical Absorbance Capacity) was determined in the fruit and leaf total polyphenol extracts by the QUIMED Laboratory at Universidad Nacional de Costa Rica, based on the method described by Zamora et al. [49 (link)].