Preparation of Stock and Working Solutions

The HA sample solutions (2.5 mg/ml) were prepared in the dark at room temperature in 0.15M aqueous NaCl in two steps First, 4.0 ml of the solvent was added to 20 mg of dry HA powder. Then 3.80,3.85, or 3.90 ml of the solvent was added after 6 hr. The stock solutions of L-ascorbic acid (1.6, 8, and 16 ttiM), arbutin (16 mM), and CuCl (160 pM prepared from 16 mM CuCy were also prepared in 0.15M aqueous NaCl. 

The HA degradation was indueed by the oxidative system eomprising CuCl (1.0 jM) with altering concentrations of L-ascoibic acid (10, 50, and 100 jM). The procedure was as follows the volume of 50 pi of 160 pM CuCl solution was added to the HA solution (7.90 ml) and after 30 s stirring the reaction solution was left to stand for 7.5 min at room temperature. Then 50 pi of r-ascorbic acid (1.6,8, or 16 mM) were added to the reaction vessel and the solution was gently stirred for 30 s. The reaction mixture was then immediately transferred into the viscometer Teflon cup reservoir. 

The HA degradation The procedures to test arbutin in the function of (i) preventive and (ii) chain breaking antioxidant were as follows  

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Table 3.1.4 Preparation of stock and working solutions for gas chromatography-mass spectrometry (GC-MS) selected ion monitoring (SIM) analysis. 2MBG 2-methylbutyrylglycine, BG butyrylglycine, DCA dicarboxylic acid, EMA ethylmalonic acid, GLUT glutaric acid, HG hexanoylglycine,...

Arbutin solution was prepared as specified under the paragraph Preparation of stock and working solutions . The investigated samples comprised 2 ml of the diluted ABTS + solution with addition of 50 pi of working arbutin solution (1 mM). 

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