3 ethylbenzothiazoline 6 sulphonic acid

Orange waste used for laccase production was donated by a local restaurant and subsequently dried and milled. Cellic CTec2 enzymatic cocktail and commercial laccase (Novozymes NS-22127) were kindly donated by Novozymes^® (Bagsvaerd, Denmark). The chromatography columns Hiprep Q FF and Superdex 75 10/300 GL were acquired from GE Healthcare Life Science (Chicago, IL, USA). Aminex HPX-87P column and Precision Plus Protein TM Standards were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Malt Extract Agar, Vanillin, 3,5-dinitrosalicylic acid (DNS), sodium carbonate, and the substrates for enzymatic activities, 3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 2,6-dimethoxyphenol (DMP), xylan beechwood, carboxymethylcellulose (CMC), locust bean, debranched arabinan, β-glucan, p-nitrophenyl-α-L-arabinofuranoside, p-nitrophenyl-β-d-galactopyranoside, p-nitrophenyl-β-d-glycopyranoside, p-nitrophenyl-β-D-xylanopyranoside, and p-nitrophenyl-β-d-cellobioside, were purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents used for the assays were of analytical grade.

U87 human glioblastoma cells and cortical neurons were seeded on a 24-well plate at the concentration of 0.1 × 10⁶ cells/mL. Cells were treated with the three different GQDs at different concentrations: 250 μg/mL, 100 μg/mL, and 50 μg/mL for 24 h. After the treatment, the medium from treated and untreated cell cultures was collected, centrifuged at 1200 rpm for 5 min. and stored at −80 °C until use. Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and brain-derived neurotrophic factor (BDNF) releases were measured in culture medium using human or mouse ELISA development kits (PeproTech^® EC Ltd., UK for TNF-α, IL-6 and IL-10; MyBioSource, Inc., San Diego, CA, USA for BDNF) according to the manufacturer’s instructions. Cell supernatants and recombinant standards were serially diluted in 1 × PBS/0.05% Tween-20/0.1% bovine serum albumin (BSA) (Sigma-Aldrich^®, Dorset, UK) and added to the microplates. Factors’ binding was detected by biotin-avidin detection step, followed by chromogen 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (Sigma-Aldrich^®, Dorset, UK) incubation. Color development was monitored at 405 nm.

Most of the chemicals used for extraction and characterization were analytical grade and purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Folin and Ciocalteu’s phenol reagent, gallic acid, L-ascorbic acid, vanillin, hexahydrate aluminum chloride, quercetin, catechin, 2,2′-diphenyl-1-picrylhy-drazyl (DPPH), 2,4,6-tripyridyl-s-triazine (TPTZ) and 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) were bought from the Sigma-Aldrich (Castle Hill, NSW, Australia). The chemical reagent and reference standard for HPLC, including gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, caffeic acid, catechin, epicatechin, epicatechin gallate, quercetin and kaempferol were produced by Sigma-Aldrich (Castle Hill, NSW, Australia). Sodium carbonate anhydrous were purchased from Chem-Supply Pty Ltd. (Adelaide, SA, Australia) and 98% sulfuric acid were bought from RCI Labscan (Rongmuang, Thailand). Methanol, acetonitrile, ferric chloride (Fe[III]Cl₃•6H₂O), hydrated sodium acetate, hydrochloric acid and glacial acetic acid were purchased from Thermo Fisher Scientific Inc. (Scoresby, VIC, Australia).