Inhibition of Free Radical Degradation in Medical Grade Hyaluronic Acid - Hyaluronic Acid for Biomedical and Pharmaceutical Applications - page 108

saline or 0.5% acetic acid were added to the solution followed
by a repeated 30 s stirring. Finally, 50 ml of ascorbic acid solution
(16 mM) was added to the reaction mixture, stirred for 30 s and
immediately transferred into the viscometer Teflon cup reservoir.

(b) In the second experimental setting, a procedure similar to that
described in (a) was applied; however, after standing for 7 min
30 s at room temperature, 50 ml of ascorbic acid solution (16 mM)
was added to the reaction mixture and stirred for 30 s. After 1 h,
finally 50 ml of 0.5% acetic acid or GSH (16 mM) was added to
the reaction mixture, stirred for 30 s and immediately transferred
into the viscometer Teflon cup reservoir.

The resulting reaction mixture (8.0 ml) was transferred into the
Teflon cup reservoir of a Brookfield LVDV-II-PRO digital rotational
viscometer (Brookfield Engineering Labs. Inc., Middleboro, MA,
USA). Recording of the viscometer output parameters started 2 min
after the onset of the experiment. The changes of dynamic viscosity
of the system were measured at 25.0 ± 0.1°C at 3-min intervals for
up to 5 h. The viscometer Teflon spindle rotated at 180 rpm, i.e., at
the shear rate of 237.6 s -1 [26].

5.3 Results and Discussion

According to WBOS, hydrogen peroxide (H 2 O 2 ) was generated as a
result of Cu(II) catalysed ascorbic acid oxidation ( Scheme 5.1 ) [27].

During the reaction, Cu(II) ions are reduced to Cu(I) ions by ascorbate
(AscH - ) to produce · OH radicals as shown in Equation 5.1 . DHA
is dehydroascorbate.

Cu(I) + H 2 O 2 → Cu(II) + · OH + OH- (5.1)

Figure 5.3 displays the results of a potential pro-oxidative effect of
acetic acid itself in both experimental settings (a, b), thus promoting
the oxidative degradation of HA macromolecules induced by WBOS