2.5.2. Inhibition of erythrocyte hemolysis mediated by peroxyl free
radicals
The antioxidant activity of the mushroom extracts was measured as the inhibition of erythrocyte hemolysis. Blood was
obtained from a male ram (churra galega transmontana) of body weight 67 kg.
Erythrocytes separated from the plasma and
the buffy coat were washed three times with phosphate buffer saline
(PBS, 10 mM, 10 ml) at pH 7.4 (prepared by mixing 10 mM of
NaH2PO4 and Na2HPO4, and 125 mM of NaCl in 1 l of distilled
water) and centrifuged at 1500g for 5 min. During the last washing, the erythrocytes were obtained by centrifugation
at 1500g for 10 min. A suspension of erythrocytes in PBS (20%, 0.1 ml)
was added to 2,20-azobis(2-amidinopropane)dihydrochloride
(AAPH, 200 mM, 0.2 ml) solution (in PBS) and mushroom methanolic extracts of different concentrations (0.1 ml).
The reaction mixture was shaken gently (30 rpm) while being incubated at
37 C for 3 h. The reaction mixture was diluted with PBS (8 ml)
and centrifuged at 3000g for 10 min; the absorbance of its supernatant was then read at k = 540 nm
by a spectrophotometer, after filtration with a syringe filter (cellulose membrane 30 mm