3 5 dinitrosalicilic acid

Folin-Ciocalteu reagent, HPLC grade methanol, tannic acid ACS reagent, Quillaja saponin, 3,5-dinitrosalicilic acid (98 %), aluminum trichloride, sulfuric acid (18 M), glucose (99.5 %), stigmasterol (95 %), hydroquinone (99 %), ursolic acid (98.5 %), digitoxin (92 %) and bromocresol green (95 %) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Quercetin dihydrate (98 %) and dragendorff’s reagent were obtained from Aldrich (Milwaukee, WI, USA). Ferric Chloride (FeCl₃•6H₂O) (97.0-102.0 %) was obtained from Spectrum Chemical and Lab Products (Gardena, California, USA) and potassium ferrocyanide (K₄Fe(CN)₆•3H₂O), HCl (12 M), sodium hydrogen phosphate, ethyl acetate (EtOac), acetic anhydride and dichloromethane were obtained from Thermo Fisher Scientific (New Jersey, USA). Nicotine (99 %) was obtained from VWR (New Jersey, USA). All chemicals were used without further purification.

Sumner reagent was prepared by adding 500 mL of deionized water to a 2 L Erlenmeyer flask and heating on a hot plate at a low temperature (about 40 °C). Then, 300 g of potassium sodium tartarate (S-2377 Sigma Aldrich, St. Louis, MO, USA) was added and mixed. Then, 200 mL of NaOH 2N (MERCK 6498) was added. Next, 10 g of 3,5-dinitrosalicilic acid (D-0550 Sigma Aldrich, St. Louis, MO, USA) was added and dissolved completely. The solution was cooled and completed to 1 L with dH₂O. Then, the solution was kept in a dark bottle at room temperature for later use. To quantitatively test the reducing sugars in the wash solution, 0.5 mL of reaction products in film wash (5 mL) of different enzyme concentrations were added to 0.5 mL of Sumner reagent. The mixture was incubated in a boiling water bath for 5 min. Then, color change was examined using UV-vis absorbance at 510 nm. Absorption studies were carried out at room temperature on a UV-vis spectrophotometer (Hewlett-Packard, model 8453, Agilent Technologies, CA, USA) from 200 to 600 nm using a quartz cell (10 mm path length). For calibration, known amounts of glucose were subjected to the same process. Then, the amount of glucose in the wash solution was calculated and given as the percentage of the original CMC weight added to the film.