Trolox standard

Antioxidant activity was determined by the ABTS (2,2′-azinobis(3-ethylbenzo-thiazoline-6-sulphonic acid) assay developed by Re et al. [61 (link)], with slight modifications. In brief, a solution of the radical cation ABTS^·+ was prepared by mixing (1:1, v/v) a solution of ABTS diammonium salt (7 mM) and a solution of potassium persulfate (2.45 mM) in H₂O. The mixture was kept in the dark at room temperature for 12–18 h before use. Then, the solution was diluted with EtOH to an absorbance of 0.70 ± 0.02 at 734 nm. Stock solutions of Trolox standard (Sigma, St. Louis, MO, USA), of RA (Sigma, St. Louis, MO, USA), and of the corresponding extracts were prepared in EtOH. For the assay, 100 μL of the Trolox solution, RA solution, or extract solution were mixed with 2 mL of the ABTS⁺ solution, and the absorbance at 734 nm was measured after 6 min. Controls were prepared by adding 100 μL of EtOH to 2 mL of ABTS⁺ solution. All determinations were carried out in triplicate. The percentage of inhibition of the absorbance was calculated with the following equation: % Inhibition = [(A₀ − A₁)/A₀] × 100, where A₀ expresses the absorbance of the control and A₁ is the absorbance of the tested sample.

C-reactive protein (CRP), interleukin 6 (IL6) and tumor necrosis factor alpha (Tnf-α) ELISA kits were purchased from Elabscience Biotechnology Co., Ltd (Wuhan, China), while 1,1,3,3-tetramethoxypropane (TMP), thiobarbituric acid, potassium persulphate (K₂S₂O₈), 2,2’-azino-bis [3-ethylbenzothiazoline-6-sulphonic acid] (ABTS) reagent, trolox standard and trichloroacetic acid were purchased from Sigma Aldrich (St. Loius, MO, USA). RNA extraction kit was from RBC Bioscience Corp. (Taipei, Taiwan) and GenomeLab™ GeXP Start Kit was from Beckman Coulter Inc (Miami, FL, USA). Simvastatin, RCL2 solution and lipid profile kits were from Pfizer (New York, NY, USA), Alphelys (Toulouse, France) and Randox Laboratories Ltd (Crumlin, County Antrim, UK), respectively. Cholesterol and Cholic acid were purchased from Amresco (Solon, OH, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Standard rat pellet was from Specialty feeds (Glen Forrest, WA, USA), while palm oil was supplied by Yee Lee Edible oils Sdn. Bhd. (Perak, Malaysia). All other solvents were of analytical grade and purchased from Merck (Darmstadt, Germany). EBN supplied by Blossom View Sdn. Bhd (Terrengganu, Malaysia) was cleaned under tap water for 5 mins, dried at room temperature and ground into powder manually using mortar and pestle before incorporating it into rat pellet.

The compound 2-aminoethyl diphenylborinate (NTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and flavonoid standards (myricetin, naringenin, chrysin, and galangin) were purchased from Fluka (Steinheim, Germany); acetonitrile and formic acid (both of them MS grade), methanol (HPLC grade), sodium carbonate, hydrochloric acid, toluene, ethyl acetate, and Folin–Ciocalteu reagent from Merck (KGaA, Darmstadt, Germany); polyethyleneglycol 4000 (PEG), standards of phenolic compounds (rutin, hyperoside, astragalin, chlorogenic acid, caffeic acid, gallic acid, and ferulic acid) and Trolox standard from Sigma-Aldrich (Steinheim, Germany). All chemicals which purity is not previously emphasized were of analytical purity grade.
methanolic standard mixture of phenolic compounds was prepared with following final standard concentrations: galangin (50 ng/µL), chlorogenic acid, myricetin, chrysin and caffeic acid (100 ng/µL), gallic acid, ferulic acid and naringenin (200 ng/µL).
The cartridges for solid-phase extraction (SPE) were Strata C18–E (500 mg/3 mL) obtained from Phenomenex (Torrance, CA, USA). Ultrapure water was obtained from a TKA MicroPure (Thermo Fisher Scientific, Bremen, Germany) water purification system, 0.055 µS/cm. Syringe filters (13 mm, polyfluorotetraethylene (PTFE) membrane 0.45 µm) were purchased from Supelco (Bellefonte, PA, USA).

FOS standards 1-kestose (GF2), 1-nystose (GF3), and 1F-fructofuranosylnystose (GF4) were purchased from Wako Pure Chemical Industries, Ltd. (Japan Company). Fructose (F), glucose (G), and sucrose (S) standards were obtained from Sigma Aldrich (St. Louis, MO, USA). A Trolox standard was obtained from Sigma Aldrich (St. Louis, MO, USA). Aguamiel was obtained from “Ejido Las Mangas” (long. −101.102500, Lat. 24.906111), Saltillo, Coahuila, Mexico from Agave salmiana in December 2017. It was sterilized by membrane filtration (0.45 μm) before its use. Orafti HSI® was obtained from Beneo Company (Belgium). Agavins were obtained from Megafarma® (Durango, Mexico). Protein marker Prestained SDS-PAGE standard #1610318 was obtained from Bio-Rad (Hercules, CA, USA). Silver BULLit™ silver stain kit was obtained from AMRESCO (Radnor, PA, USA).

Antioxidant activity was determined using the DPPH (1, 1-diphenyl-2-picryl-hydrazyl, Sigma-Aldrich Co. LLC, Saint Louis, MO, USA) radical scavenging activity method as previously described (34 (link)). The supernatant was collected after treatment of each sample extract (5 ml) with ethanol 98% (10 ml) and centrifuged at 8000 x g for 20 min. 100 μl of PB extract from each beverage was mixed with 2.9 ml of methanolic solution of DPPH (0.045 mg/ml). Absorbance was measured at 517 nm using an ultraviolet spectrophotometer (Jenway 6705 UV/Vis, Bibby Scientific Limited, ST15 OSA, UK) after the mixture was kept in the dark for 30 min. The relative antioxidant in a mixture sample to scavenge DPPH was compared with a Trolox standard (Sigma-Aldrich Co., LLC, Saint Louis, MO, USA). The results were expressed in equivalent μmol Trolox/L. The analyses were carried out in triplicate starting with a new batch of samples (three extracts). The result was calculated from nine repetitions.

The total phenolic content (TPC) was analyzed using a microvolume version of the Folin-Ciocalteu assay [21 (link)]. The calibration curve was obtained with standard gallic acid in water (Fluka, 100–800 mg/L, R² = 0.999) and hesperidin in 0.1% NaOH solution (Fluka, 200–2000 mg/L, R² = 0.998). The results were expressed both as mg of gallic acid equivalents (GAE) and hesperidin equivalents (HE) on g of dry matter (dm) of extract.
Since natural extracts are composed of different phenolic compounds with different mechanisms of antioxidant activity, the antioxidant power of citrus extracts, before and after encapsulation, was measured using two antioxidant assays: the ferric reducing antioxidant power (FRAP) and ABTS assays.
The FRAP of each extract was evaluated according to the procedure described by Bassani et al. [21 (link)]. A calibration curve was prepared with FeSO₄·7H₂O in water (Carlo Erba, 0.2–2 mmolFe(II)/L, R² = 0.999). Then the results were calculated and expressed as μmolFe(II)/g dm of extract.
The ability of the extract to reduce the ABTS radical was analyzed according to the method of Bassani et al. [21 (link)]. The calibration curve was obtained with a Trolox^® standard (Tr) in 50% ethanol (Sigma-Aldrich, 100–500 mgTr/L, R² = 0.999) and the results were calculated and expressed as μmolTr/g dm of extract.