Dmso dimethyl sulfoxide

Arbutin (AR), gallic acid (GA), hydroquinone (HQ), (+)-catechin hydrate (CH), vanillic acid (VA), caffeic acid (CA), syringic acid (SA), (−)-epicatechin (EC), vanillin (VL), p-coumaric acid (PCA), trans-ferulic acid (FA), myricetin (MC), ellagic acid (EA), trans-cinnamic acid (TCA), rosmarinic acid (RA), benzoic acid (BA), quercetin (QU), rutin hydrate (RH), and kaempferol (KF) were purchased from Sigma-Aldrich (St. Louis, Missouri, United States). Acetonitrile (HPLC), methanol (HPLC), acetic acid (HPLC), ethanol, Folin-Ciocalteu's reagent, ascorbic acid, trichloroacetic acid, potassium ferricyanide, sodium carbonate, ferric chloride, DMSO (dimethyl sulfoxide), and acetic acid were obtained from Merck (Darmstadt, Germany). Diclofenac sodium and vincristine sulphate were obtained from Beximco Pharmaceuticals Ltd. and Cipla Pharmaceuticals India, respectively.

TPC of each EOs was determined according to the method of Folin-Ciocalteu (27 (link)). Concentration for gallic acid standard ranges from 20 to 1,000 μL and results were reported as GA equivalents (mg GAE/g). A total of 11 gallic acid standards were prepared. For the first standard, 1.5 mL Na₂CO₃, 0.5 mL Folin Ciocalteu's reagent (Merck 109001), 20 μL gallic acid (Sigma G7384) and 7,980 mL distilled water were added. Later, although Na₂CO₃ and Folin Ciocalteu's reagent remained constant in standards, the amount of gallic acid increased and the amount of water decreased. 20 μL of each EOs was dissolved in DMSO (dimethylsulfoxide, Merck, Germany) to yield maximum solubilization and homogenized with the same reagents. Afterwards, the samples were kept in the dark for 2 h at room temperature. The samples were then measured at 760 nm in a spectrophotometer (Shimadzu, UV 1800).

Murine neuroblastoma cell lines, N1E-115, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and culture in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (Corning, NY, USA) and 100 U/mL penicillin/streptomycin (Thermo Scientific, Waltham, MA, USA). Cells were maintained at 37 °C in humidified air containing 5% CO₂.
A5⁺ was provided by SirtLife srl, Rome, Italy. A5⁺ 10 µg is composed of ellagic acid (20%), polydatin (98%), pterostilbene (20%), honokiol (20%), mixed with recommended doses of zinc, selenium, and chromium. The polydatin and A5⁺ were dissolved in DMSO (dimethyl sulfoxide, Sigma Aldrich, Milan, Italy) at 1 mg/mL [22 (link)]. In order to establish the concentration of A5⁺ able to promote beneficial effect without exerting neurotoxic effect, N1E115 cells were treated with different concentrations (10, 50, 100 and 500 μM) for 48 h.
Neurotoxicity was induced by treating cells with different concentration (10, 30, 50, 75 and 100 μM) of 6-hydroxydopamine (6-OHDA) (Sigma Aldrich, Saint Louis, MO, USA) for 24 h.
Moreover, a combined treatment has been given: cells were pre-treated with A5⁺ for 48 h and then 6-OHDA was added in combination with A5⁺ for further 24 h.

Cell viability was determined by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Sigma Aldrich, Saint Louis, MO, USA), following manufacturer’s protocol. Briefly, 8 × 10³ cells/well were seeded into a 96 multi-well plate for 24 h and treated with 6-OHDA and A5⁺ as reported above. Then, medium was removed and cells were incubated for 3 h at 37 °C with 5 mg/mL of MTT. After 3 h, DMSO (dimethyl sulfoxide) (Sigma Aldrich, Saint Louis, MO, USA) was added and the formazan production was assessed by measuring the absorbance at 570 nm.
Following cell viability assay, we also assessed the IC50 (50% inhibitory concentration), by using GraphPad Prism 9 (La Jolla, CA, USA), by using the MTT values on Y-axis and the log of concentration on the X-axis.

The Middlebrook 7H9 and 7H10 media and the OADC (oleic acid, albumin, dextrose, and catalase) were obtained from Becton-Dickinson, (Detroit, MI, USA). The RNeasy^® Mini Kit for RNA extraction, the Omniscript^® Reverse Transcription Kit for complementary DNA acquisition, and the QuantiTectTM SYBR^® for RT-PCR were obtained from Qiagen (Germantown, MD, USA). The primers for the cytokines studied were acquired from InvitrogenTM Thermo Fisher Scientific (Waltham, MA, USA). CUR, DMSO (Dimethylsulfoxide) and ethanol was purchased from Sigma Aldrich (Zwijndrecht, The Netherlands). Primary antibody against BDNF (ab72439) was obtained from Abcam Inc. (Cambridge, MA, USA). Primary antibody against Nrf2 (T-19; sc-30915) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies against rabbit (711-035-152), goat (705-035-147) and mouse (715-035-150) were purchased from Jackson ImmunoResearch Laboratories Inc. (Jennersville, PA, USA). All other reagents were analytical grade and acquired from known commercial sources.

Peptides were synthesized at the Weizmann Institute of Science in Rehovot, Israel to perform characterization tests on their ability to influence the biological activity of chemokines. The format of the various synthesized peptides was as follows: Cyclic peptides (ACX₇CGGGSK-biotin-G) and linear peptides (X₁₂GGGSK-biotin-G) were used. The peptides were biotinylated at their C-termini; biotin served as the detector during the subsequent experiments. Each synthetic peptide was dissolved to a final concentration of 1 mg/ml (~0.6 mM) in 4% DMSO (dimethyl sulfoxide, Sigma).