Inhibition of Free Radical Degradation in Medical Grade Hyaluronic Acid - Hyaluronic Acid for Biomedical and Pharmaceutical Applications - page 107

5.2 Methods and Materials for Hyaluronan
Degradation Analysis
The hyaluronan sample Lifecore P0207-1A was purchased from
Lifecore Biomedical Inc., Chaska, MN, USA (MW = 970.4 kDa).
The analytical purity grade sodium chloride (NaCl) and copper(II)
chloride (CuCl 2 × 2H 2 O) were purchased from Slavus Ltd., Bratislava,
Slovakia. l l-Ascorbic acid was the product of Merck KGaA,
Darmstadt, Germany. GSH and acetic acid were purchased from
Sigma-Aldrich, Steinheim, Germany. Deionised high-purity grade
H 2 O, with conductivity of ≤0.055 mS/cm, was produced by using the
TKA water purification system (Water Purification Systems GmbH,
Niederelbert, Germany).
The HA solution (2.5 mg/ml) was prepared in aqueous NaCl solution
(0.15 M) in the dark in two steps: first, the solvent (4.0 ml) was
added to HA (20 mg), and after 6 h of swelling, the same solvent
(3.85 ml) was added. The stock solutions of ascorbic acid (16 mM),
GSH (16 mM) and CuCl 2 (160 µM) were also dissolved in aqueous
NaCl solution (0.15 M).
The procedure for examining HA degradation by the Weissberger
biogenic oxidative system (WBOS) was as follows: a volume of 50 µl
of 160 µM CuCl 2 solution was added to the HA solution and the
mixture (7.95 ml), after stirring for 30 s was left to stand for 7 min
30 s at room temperature. Then, 50 µl of ascorbic acid solution
(16 mM) was added to the HA solution, and stirred again for 30 s.
The final reaction mixture was then immediately transferred into the
viscometer Teflon cup reservoir.
The procedures to investigate the pro- and antioxidative effects of
acetic acid and GSH were as follows:
(a) A volume of 50 ml of 160 mM CuCl 2 solution was added to the
HA solution (7.85 ml), and the mixture, after stirring for 30 s
was left to stand for 7 min 30 s at room temperature. Then, 50 ml
of 0.5% acetic acid or 50 ml of GSH (16 mM) dissolved both in