Folin ciocalteu reagent

The total phenol content was measured using a Folin–Ciocalteu assay [38 (link)]. A 50 µL extract aliquot was added to an ELISA microplate, followed by 250 µL of Folin–Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) diluted 10× with water. After a 2 min reaction in the dark, 200 µL of Na₂CO₃ (Sigma-Aldrich, St. Louis, MO, USA) (75 mg/L) was added. The plate was incubated in the dark for 2 h, and absorbance was measured at 765 nm with an ELISA microplate reader (Thermo Scientific) using a gallic acid (Sigma-Aldrich, St. Louis, MO, USA) (in methanol 80%) calibration curve. The results are expressed in mg/mL.
We measured the total flavonoid content using the method developed by Zhishen et al. [39 (link)]. A volume of 100 µL of the extract was placed in a microcentrifuge tube, followed by 40 µL of distilled water and 30 µL of NaNO₃ (5%) (Sigma-Aldrich, St. Louis, MO, USA). The mixture was left to react for five minutes. Then, 30 µL of AlCl₃ (10%) (Sigma-Aldrich, St. Louis, MO, USA) was added, and the mixture was left to react for six minutes. Next, 200 µL of NaOH (1 N) (Sigma-Aldrich, St. Louis, MO, USA) was added, followed by 240 µL of distilled water. From this mixture, 300 µL was transferred to an ELISA microplate. Absorbance was measured at 510 nm using an ELISA microplate reader (Thermo Scientific, Waltham, Massachusetts, USA) and compared to a catechin (in 80% methanol) calibration curve. The results are expressed in mg/mL.
We measured condensed tannins using the method of Amarowicz and Pegg [40 (link)]. A volume of 50 µL of the extract was placed on an ELISA microplate, followed by 250 µL of a solution consisting of HCl (J.T. Baker^®, Radnor, PA, USA) (8% in methanol) and vanillin (Sigma-Aldrich, St. Louis, MO, USA) (1% in methanol) in a 1:1 ratio. Absorbance was measured at 492 nm using an ELISA microplate reader (Thermo Scientific, Waltham, Massachusetts, USA) and compared to a catechin calibration curve (in methanol). The results are expressed in mg/mL. All the above-mentioned tests were performed in triplicate.
We carried out the following two assays to measure the antioxidant capacity: the ferric reducing/antioxidant power (FRAP) and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Sigma-Aldrich, St. Louis, MO, USA) assays.
The FRAP assay was carried out according to the methodology described by Benzie and Strain [41 (link)] and modified by Álvarez et al. [42 (link)]. We prepared a solution of a working FRAP reagent, with acetate buffer (0.3 M, pH 3.6), TPTZ (2,4,6-tripyridyl-S-triazine) (Sigma-Aldrich, St. Louis, MO, USA) solution (10 mM in 40 mM HCl), and FeCl₃ (20 mM) (Sigma-Aldrich, St. Louis, MO, USA) in a 10:1:1 ratio. A volume of 24 µL of extract was placed on an ELISA microplate, followed by 180 µL of the working FRAP reagent. Absorbance was measured at 630 nm using an ELISA microplate reader (Thermo Scientific). We used Trolox to make a standard, and the results are expressed as Trolox equivalent (TE) (μM TE/g).
The antioxidant capacity obtained from the DPPH assay was measured according to the methodology by Brand–Williams et al. [43 (link)] and Chew et al. [44 (link)]. A solution of 1 mM DPPH in methanol was stirred. The absorbance of the solution was adjusted to 1 (<0.010) at 480 nm. Then, 20 µL of extract was placed on an ELISA microplate, followed by 280 µL of DPPH solution (Sigma-Aldrich, St. Louis, MO, USA), and incubated for 30 min in the dark covered with aluminum foil. Then, decreasing absorbance was monitored at 480 nm. A calibration curve was plotted using standard solutions of Trolox–methanol with concentrations in the range of 0 to 0.0008 µM, and the results are expressed as Trolox equivalent (TE) (μM TE/g).

2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid, ABTS) was supplied from Bio Basic (Canada). Potassium hexacyanoferrate, gallic acid, trichloroacetic acid (TCA),2,2-Diphenyl-1-picrylhydrazyl (DPPH, 90%), and Folin-ciocalteu reagent were purchased from Sigma (St. Louis, MO, USA). Butylated hydroxytoluene (BHT) was obtained from Nice Chemicals Pvt. Ltd (India). Sodium carbonate, sodium bicarbonate, and Potassium persulfate were purchased from Adwic Company, Egypt. Soy lecithin was bought from Carl Roth (Karlsruhe, Germany). Pumpkin seed oil was purchased from the oil extraction unit, National Research Centre, Egypt. Beeswax (BW) was obtained from a local market.

The total phenolic content (TPC) of the rice extracts was assessed with the Folin– Ciocalteu assay as described by [22 (link)]. The control blank was prepared using methanol while gallic acid (Sigma-Aldrich, Australia) served as the positive control.  The calibration curve was plotted, showing a linear range of concentrations between 0–500 µg/ mL (Figure S3) in the presence of Folin– Ciocalteu reagent (Sigma-Aldrich, Australia). The TPC of rice extracts was calculated as mg of gallic acid equivalent (GAE)/g dry weight (dw), using the equation obtained from the standard calibration curve as means ± standard deviation (SD) of triplicates.

The following compounds were procured from Sigma-Aldrich (St. Louis, MO, USA): Folin–Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), along with gallic acid, protocatechuic acid, 4-hydroxybenzoic acid, vanillic acid, chlorogenic acid, p-coumaric acid, 2-hydroxycinnamic acid, vanillin, ferulic acid, caffeic acid, rosmarinic acid, quercetin, apigenin, rutin, kaempferol, hesperetin, hesperidin, and 5,7-dihydroxyflavone. Additionally, potassium persulfate was also sourced from the same supplier. Sodium bicarbonate, sodium hydroxide, aluminum chloride, sodium molybdate dihydrate, and sodium nitrite were obtained from Biochem Chemopharma (Cosne-Cours-sur-Loire, France), while ethanol, copper chloride, methanol, and acetonitrile were acquired from VWR (Rosny-sous-Bois, France).

Collagen type I (COL) from scales of freshwater fish - northern pike (Esox lucius) was obtained and characterized in our laboratory^{27 (link)}. In brief, fish scales were treated with NaOH to remove non-collagenous proteins, followed by EDTA. Collagen was extracted using 0.5 M acetic acid and then separated by a salting-out process, where NaCl was added to a final concentration of 2.5 M to precipitate the collagen. The precipitate was collected by centrifugation (13,000 × g, 30 min), redissolved in acetic acid, and dialyzed to remove excess salt. Finally, the purified collagen was freeze-dried.
Chitosan (CTS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), pentasodium triphosphate (TPP), Tween 80, Span 80, Folin-Ciocalteu reagent, and gallic acid were obtained from Sigma-Aldrich (Poznan, Poland). The hydro-glycolic (propylene glycol/water 80:20) extract of pot marigold (Calendula officinalis) flowers was procured from Provital S.A. (Barcelona, Spain). All other reagents were sourced from Avantor Performance Materials Poland S.A. (Gliwice, Poland). All chemicals used were of analytical grade.