Rutin

Styrene (99%, extra pure, stabilized), antioxidants (trolox (97%), rutin (97+%), ellagic acid (≥97%)), and polyvinylpyrrolidone (PVP, K-30, Mw 50,000) were purchased from Acros Organics (Geel, Belgium). The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (95%) was bought from Alfa Aesar (Kandel, Germany). Potassium chloride (KCl), potassium peroxodisulphate (KPS), ethanol, and methanol were acquired from VWR (Debrecen, Hungary). Nitrogen (4.5) gas was obtained from Messer (Budapest, Hungary). All chemicals were used without further purification. Ultrapure water with a resistivity higher than 18.2 MΩ∙cm was obtained from a three-stage VWR Puranity TU+ (Debrecen, Hungary) device. The measurements were carried out at 25 °C and at pH 10 unless otherwise indicated.

The test was based on the protocol of Duarte-Almeida et al. [25 (link)], with some modifications. An ethanolic solution of 2,2-diphenyl-1-picryl hydrazyl (DPPH) (Sigma, St. Louis, MO, USA) was prepared in order to produce an absorbance between 0.8 and 0.99 at 517 nm. One hundred microliters of each extract diluted in ethanol (Synth, Diadema, Brazil) at initial concentrations of 0.004, 0.01, 0.04, 0.1, 0.4 and 1.0 mg/mL were pipetted in a cuvette and after the addition of 900 μL of DPPH the cuvette was placed in a spectrophotometer (PG Instruments LTD, Leicestershire, United Kingdom) and read during 2 min at 517 nm. ethanol (vehicle) was used as control and rutin (Acros Organics, New Jersey, USA) as positive control. The percentage of DPPH scavenger (S) was calculated by the formula: S_DPPH = [(A_c - A_s)/A_c] × 100, where A_c = absorbance for the control and A_s = absorbance for the sample. The concentration of each extract that quenches 50% of DPPH (EC₅₀) was calculated by linear regression (% of scavenge vs final concentration) using the mean of 4 assays.

Reagents used in the cell culture, including Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA, were obtained from Gibco, Thermo Fisher Scientific, Inc. (Waltham, MA, USA). CML was purchased from Cayman Chemical (Ann Arbor, MI, USA). Sodium dodecyl sulfate (SDS), 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and potassium phosphate-potassium hydroxide (KH₂PO₄-KOH) were purchased from USB Corporation (Cleveland, OH, USA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), potassium ferricyanide(III) (K₃Fe(CN)₆), ferric chloride (FeCl₃), ascorbic acid, phenazine methosulfate (PMS), β-nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH), nitrotetrazolium blue chloride (NBT), butylated hydroxyanisole (BHA), 2-deoxy-D-ribose, thiobarbituric acid (TBA), trichloroacetic acid (TCA), mannitol, ferrous chloride (FeCl₂), ethylenediaminetetraacetic acid (EDTA), 2′, 7′-dichlorofluorescin-diacetate (H₂DCFDA), 2-propanol and 20-hydroxyecdysone were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Rutin was obtained from ACROS Organics (Morris Plains, NJ, USA). HPLC-grade acetonitrile and formic acid were obtained from J.T. Baker, Thermo Fisher Scientific, Inc. (Waltham, MA, USA). All other chemicals used were of analytical grade or higher grade.

Alloxan monohydrate (Sigma-Aldrich Co., St. Louis, MO, USA), nicotinamide (Sigma-Aldrich Co., St. Louis, MO, USA), acarbose (LGM Pharma, USA), metformin (Bristol laboratories Ltd.), gallic acid (Merck KGaA, USA), rutin (Acros organics, USA), ascorbic acid (Carl Roth, Germany), quercetin (Alfa Aesar, UK), p-nitropheynyl glucopyranoside (pNPG) (BBI Biotech, China), Rat Insulin ELISA kit, Rat Glucagon-ELISA kit, and Rat GLP-1 ELISA kit (Wuxi Donglin Sci & Tech Development Co. Ltd., China) were used in the study. All other solvents and chemicals used in the study were of analytical grade.

All chemicals and commercial standards were purchased from Sigma (Sigma, St. Louis, MO, USA). Acetone, methanol, hexane, acetic and hydrochloric acid were from BioLab (BioLab, Jerusalem, Israel). Ethanol was from Gadot (Gadot, Netanya, Israel). Rutin and trolox were purchased from Acros Organics (Acros Organics, New Jersey, USA). Lanthanum chloride was from EMD (Millipore Darmstadt, Germany).

Cuprizone [bis(cyclohexanone) oxaldihydrazone] was purchased from Sigma-Aldrich Co. (USA). Rutin was obtained from Acros Organics (Belgium). Dimethyl fumarate was purchased from Hikma (Egypt). Spectrophotometric kits for glutathione peroxidase (GPx) and malondialdehyde (MDA) were purchased from Biodiagnostic (Giza, Egypt). Mouse TNF-α and IL-1β ELISA kits were purchased from MyBioSource (San Diego, USA) and Sunred (Shanghai; China), respectively. All other chemicals were of the highest analytical grade and purchased from Sigma-Aldrich Co. (USA) or Merck (Germany).