Folin ciocalteu reagent

The influents used in this work were produced from a hot water treatment of hardwood chips at 80 °C for 1 h before a thermomechanical pulping process, and they were received from a pulping company in Northern Ontario, Canada. Fly ash was collected from the bark boiler of the same pulping company, in which wastewater sludges, sawdust and barks from softwood and hardwood species were burned for heat generation, and fly ash was produced as a by-product. Activated sludge was obtained from the secondary wastewater treatment system of the same pulping company. Potassium dihydrogen phosphate (KH₂PO₄), ammonium chloride (NH₄Cl), sodium hydroxide (NaOH, >99.0%) and sodium chloride (NaCl) were obtained from Sigma-Aldrich Inc., USA and sodium hydroxide was diluted to 1 mol/L prior to use. The protein standard bovine serum albumin and polysaccharides standard glucose were purchased from Sigma-Aldrich company, USA. The protein assay kit including Lowry reagent and folin-ciocalteu reagent were purchased from Thermo Fisher Scientific Inc., USA. The chemical oxygen demand (COD) kits (K-7365) were purchased from CHEMetrix Inc., USA. The biological oxygen demand (BOD) nutrient buffer solution was purchased from HACH Company, USA.

Fatty acid methyl ester, tocopherols (α, β, γ, and δ), tocotrienols (α, β, γ, and δ), phytosterol, squalene, 4-hydroxybenzoic acid, caffeic acid, chlorogenic acid, cinnamic acid, gallic acid, vanillic acid, p-coumaric acid, sinapic acid, ferulic acid, ellagic acid, salicylic acid, vanillin, epicatechin, quercetin and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were obtained from Sigma-Aldrich Chemical Co., Ltd. (Shanghai, China). Folin-Ciocalteu reagent, chromatographic grade methanol, acetonitrile, formic acid and n-hexane were purchased from Thermo Scientific (Beijing, China). All reagents and chemicals were kept at analytical grade and were purchased from the Sinopharm Chemical Reagent Beijing Co., Ltd., (Beijing, China).
The E. mollis seeds used in this study were bought from a local market in Yi cheng, Shanxi province, China. The collected seeds were manually de-hulled and air-dried until the moisture content was below 5%. Then, the kernels were ground with a disintegrator and passed through a 40-mesh sieve to obtain a homogeneous and fine powder, which was stored in polyethylene containers at 4 °C until analysis.

Following the procedure outlined by Singleton et al., 1999 [12 ], the total phenolic contents were determined. In fact, the extracts were dissolved in DMSO at a concentration of 100 mg/mL and then diluted to a final concentration of 1 mg/mL in distilled water. The Folin–Ciocalteu reagent (0.2 N, TECNO PHARMCHEM HARYANA) was added to each test tube corresponding to each dilution that contained 0.5 mL of the solutions obtained after dilution, 2.5 mL of the reagent had been diluted 10 times, and the test tubes were then allowed to sit at room temperature for 5 minutes. Following the incubation time, 2 mL of a sodium carbonate solution (75 mg/mL) was added, and the mixture was then agitated. The test tubes were once again incubated for two hours at room temperature in the dark. The etalon for the calibration curve was made up using gallic at different concentrations of 0, 20, 40, 60, 80, and 100 mg/L. The optical density was obtained by a spectrophotometer (Thermo Scientific Evolution 300) at 760 nm against the blank (0.5 mL of Folin–Ciocalteu reagent and 0.5 mL of sodium carbonate). The test was carried out twice, and the outcome was represented as mg of gallic acid (Sigma-Aldrich) equivalents per 100 mg of extract (mg GAE/100 mg extract) [13 (link)].

Ciprofloxacin and gentamicin (Microxpress, a division of Tulip Diagnostics Pvt. Ltd.) antibiotic discs were utilized as standard drugs for antibacterial assay. Nutrient broth (HiMedia Laboratories Pvt. Ltd., Mumbai), Mueller Hinton Agar (MHA) (HiMedia Laboratories Pvt. Ltd., Mumbai), 3 (4, 5 dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide (MTT) (Beyotime Biotechnology, China), DPPH (Thermo Fisher scientific, India Pvt. Ltd; Mumbai), Barium chloride (HiMedia Laboratories Pvt. Ltd., Mumbai), Folin-Ciocalteu reagent, quercetin, ascorbic acid dimethyl sulfoxide (Thermo Fisher Scientific, India Pvt. Ltd., Mumbai).

The successful synthesis and cleavage of the peptide was confirmed on a test sample using liquid chromatography/mass spectrometry (LCMS). Liquid chromatography separation was performed with a gradient between mobile phase A of water with 0.1% formic acid (Thermo Fisher, 99.0+%, Optima LC/MS Grade) and mobile phase B of acetonitrile (Thermo Fisher, HPLC grade) with 0.1% formic acid, using a 30 mm by 2.1 mm C18 column with 3 μm sized particles (Waters) using a Q-TOF Premier LCMS system (Waters). The dried peptide was then dissolved in 1.5 ml of 50:50 water:acetonitrile and purified using a 25 g C8 column (Interchim) on a puriFlash XS520Plus system (Interchim), with a mobile phase A of water with 0.1% TFA and a mobile phase B of acetonitrile with 0.1% TFA, and collected in fractions. The fractions were checked by LCMS to identify those containing pure peptide, and these fractions were pooled, dried using a centrifugal Genevac EZ-2 Personal Evaporator, and stored at −20 °C. Peptide concentrations were determined by the Lowry protein assay using Folin & Ciocalteu Reagent (Thermo Fisher).

Gallic acid, ascorbic acid, quercetin, and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) were purchased from Hi‐Media Laboratories, India. Folin–Ciocalteu reagent, sodium carbonate, methanol, ethanol, sodium nitrite, sodium bisulfite, anthrone, sulfuric acid, D‐glucose, and hydrochloric acid were purchased from Thermo Fisher Scientific, India. Similarly, aluminum chloride was purchased from SD Fine‐Chem Limited, India, and acetaldehyde was purchased from Nike Chemicals, India.

HPLC grade water and solvents methanol, acetone, hexane, acetonitrile, ethanol, and glacial acetic acid; and Folin–Ciocalteu reagent, sodium carbonate anhydrous, and ammonium acetate were purchased from Thermo Fisher Scientific. Analytical standards of gallic acid, (+)‐catechin, (−)‐catechin, and (−)‐epicatechin were obtained from MilliporeSigma. (+)‐epicatechin was purchased from Nacalai USA, Inc. The Reference Material (RM) 8403 cocoa flavanol extract was obtained from the National Institute of Standards and Technology (NIST).