Visionlite

All enzymatic assays were carried out in 1 ml volume at 25°C using a Genesys6 UV-Vis spectrophotometer (Thermo Fisher). The insulin reduction assay was conducted as described [32 (link)]. Enzymatic activity of the recombinant protein was determined by adding varying amounts of purified PbPhLP-3, HsPhLP-3 or PbTrx-1 to a reaction mixture containing bovine insulin (44 μg ml-1) and DTT (1 mM) in a potassium phosphate buffer (100 mM potassium phosphate, 2 mM EDTA, pH 7.4). The reduction of the insulin disulfide bonds was monitored as an absorbance increase over time at 650 nm. The coupled enzymatic assays utilizing thioredoxin reductase/thioredoxin were conducted as previously described [4 (link), 48 (link)]. Briefly, oxidation of NADPH was followed as an absorption decrease at 340 nm. All assays were performed at RT in assay buffer containing 100 mM KH₂PO₄, 2 mM EDTA, pH 7.4, 200 μM NADPH (ε_{340 nm} = 6.22 mM^-1cm^-1), and 20 μM PbTrx-1 (S4 Fig). Each initial reaction was started with PbTrxR and the decrease of absorption at 340 nm was monitored during the linear phase. Initial velocities and kinetic values for each reaction were determined using the VISIONlite (Thermo Fisher) software. Enzyme kinetics were calculated using global curve fit in the Enzyme Kinetics Module of Sigma Plot 12.0.

UV spectra of the pure substances theophylline, aminophylline, ethylenediamine hydrochloride, and pharmaceutical preparations Euphyllin^® long 200 mg, and 300 mg (zr pharma&GmbH company, Wien, Austria), Theospirex^® retard 150 mg, and 300 mg (BIOFARM company, Poznan, Poland), Aminophylline 100 mg and 200 mg (advacare pharma, Cheyenne, WY, USA) were analyzed in the study. For this purpose, 1 mg of each of the tested samples was dissolved in 100 mL of 96% ethyl alcohol (POCh company, Gliwice, Poland). The samples were then mixed and poured into a quartz cuvette, which was then placed in a spectrophotometer. UV absorbance spectra were recorded in the wavelength range of 200–380 nm. UV-Vis spectrophotometer Thermo Genesys 10S produced by Thermo Scientific (Waltham, MA, USA) was used. The analysis of obtained UV spectra was performed using the programs VisionLite produced by Thermo Scientific (Waltham, MA, USA) and Origin 2016 produced by OriginLab (Northampton, MA, USA).

UV spectra of the gum arabic and tragacanth non-exposed and exposed to a tested storage conditions were analyzed in the study. For this purpose, 10 mg of each of the tested polymers were dissolved in 40 mL of water [47 ]. The samples were then thoroughly mixed and poured into a quartz cuvette with an optical path of 1 cm, which was placed in a spectrophotometer. UV absorbance spectra were recorded in the wavelength range from 200 to 380 nm [47 ,48 (link),49 (link)]. UV-Vis spectrophotometer Thermo Genesys 10S produced by Thermo Scientific (Waltham, MA, USA) was used. Analysis of recorded UV spectra was performed using the programs VisionLite produced by Thermo Scientific (Waltham, MA, USA) and Origin 2016 produced by OriginLab (Northampton, MA, USA).