Dpph radical

Lipoid S75 (consisting of ~70% of soy phosphatidylcholine, 9% phosphatidylethanolamine and 3% lysophosphatidylcholine) was purchased from Lipoid GmbH (Ludwigshafen, Germany). Powder extract containing 5% of Z. officinalis was purchased by Farmalabor Srl (Italy). Sodium hyaluronate with low molecular weight (200–400 kDa) and a polydispersity of 1.4 Mw/Mn, was purchased from DSM Nutritional Products AG Branch Pentapharm (Switzerland). Glycerol, DPPH radical (2,2-diphenyl-1-picrylhydrazyl), mucin from porcine stomach and all other reagents of analytical grade were purchased by Sigma-Aldrich (Milan, Italy).

Fresh tender Opuntia dillenii Haw. cladodes of 2–4 month and 5–10 month specimens were collected from Donghai Island, Zhanjiang, Guangdong Province, China. Coomassie brilliant blue G250 and l-(+)-Arabinose (≥90.0%) were obtained from Fluka Chemie (Buchs, Switzerland). DPPH radical, DEAE sepharose fast flow anion exchange and Sephacryl S-400 were purchased from Sigma (Washington, WA, USA). All other chemicals and reagents used were of analytical grade.

Auricularia auricula waste residue powder was obtained by drying the powder of fruiting body after the polysaccharide was extracted. Human L02 hepatocyte cells were purchased from Fuheng Biological Technology Co.. ABTS radical and DPPH radical were purchased from Sigma‐Aldrich. Chemical reagents such as sodium hydroxide, hydrochloric acid, ethyl acetate, chloroform, ethanol, salicylic acid, ferrous sulfate, and hydrogen peroxide were purchased from Sinopharm Co..

Methanol, ethanol, ascorbic acid, chlorogenic acid, sorbitan monooleate (Span 80^®), and polysorbate 80 (Tween 80^®) were obtained from Merck (Darmstadt, Germany). Eudragit^® RS100, DPPH radical, and RU were acquired from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). Fluorescein (CAS No. 2320-96-9), trolox (CAS No. 53188-07-1), and AAPH (CAS No. 2997-92-4) were obtained from Aldrich (Milwaukee, WI). A stock solution of fluorescein (407 μmol/L) was prepared using potassium phosphate buffer (75 mmol/L; pH 7.4) and kept at 4 °C for ORAC assay. Both fluoxetine and imipramine hydrochloride were obtained from Galena^® (Porto Alegre, Brazil).

The organic solvents used in this study were Acetone, hexane, methanol, ethyl acetate, and diethyl ether were obtained from Fisher Scientific Ltd. (Ottawa, ON, Canada). The following chemicals, anhydrous sodium sulfate, sodium hydroxide, sodium chloride, dibasic and monobasic sodium phosphates, sodium nitrite, HC l, AlCl₃, potassium ferricyanide, ferrous chloride, Folin-Ciocalteu's reagent, ferric chloride, and sodium carbonate were purchased from Fisher Scientific Ltd. (Ottawa, ON, Canada) and Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). Gallic acid, catechin, Trolox, DPPH radical, and a number of phenolic acid standards were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada).

The ability of EO and MeOH extracts to reduce the color of the DPPH radical (Sigma-Aldrich, Germany) were determined according to Miguel [71 (link)]. Various concentrations (5, 10, 20, 30, 40, and 50 mg mL⁻¹) of the EOs and residues of the MeOH extract were prepared using methanol. This range of concentrations was determined based on a preliminary test using either higher concentration or lower concentration. To assess the antioxidant activity, a reaction mixture of equal volumes from the freshly prepared 0.3 mM DPPH and each concentration of the EOs or the MeOH extract, was prepared, mixed vigorously, and kept in dark for 15 min at 25 °C. Additionally, a parallel positive control, using ascorbic acid as a standard antioxidant, at concentrations of 1.0, 2.5, 5, 10, 15, and 20 mg mL⁻¹, were prepared and treated similar to the treatments. After incubation, the absorbance was measured at 517 nm using a spectrophotometer (Milton Roy Spectronic 21D UV-Visible Spectrophotometer, USA). The amount of EO or MeOH extract required to reduce the color of DPPH by 50% (IC₅₀) was calculated graphically.