α 4 pyridyl 1 oxide n tert butylnitrone

For composite preparation, medium molecular weight chitosan (190–310 kDa), aqueous solutions of acetic acid (99.8%) and hydroxyapatite were purchased from Sigma-Aldrich (USA). Synthetic riboflavin (Sigma-Aldrich) was used as a control for the RF naturally obtained. For the antioxidant activity of the compound, ABTS (Pol-Aura, Poland) was used. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide thiazolyl blue tetrazolium bromide (MTT) and dimethylsulfoxide for the characterization of yeast riboflavin, were purchased from Sigma-Aldrich (USA) and Stanlab (Poland), respectively. For EPR measurements, potassium hydroxide and hydrogen peroxide (Chempur, Poland) as well as α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (Sigma-Aldrich, USA) were used.

Fetal bovine serum (FBS), trypsin-EDTA, and penicillin-streptomycin (P/S) were purchased from Gibco-BRL (Grand Island, NY, USA). 5,5-Dimethyl-1-pyrroline N-oxide, dimethyl sulfoxide (DMSO), acridine orange, DPPH, 2,2-azobis(2-amidinopropane) hydrochloride, α-(4-pyridyl-1-oxide)-N-tert-butylnitrone, H₂O₂, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1,3-bis (diphenylphosphino) propane (DPPP), and 2,7-dichlorofluorescein diacetate (DCFH₂-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Fenton’s reagent was prepared using hydrogen peroxide (H₂O₂) (Sigma-Aldrich Chemie GmbH, Germany) and ferrous sulfate (FeSO₄.7H₂O) (BDH Laboratory Supplies, United Kingdom). A fixed concentration of FeSO₄ (500 μM) and a variable concentration of H₂O₂ (100 μM/1 mM) was used_. Spin trapping reagent, POBN [α-(4-Pyridyl 1-oxide)-N-tert-butylnitrone] was purchased (Sigma-Aldrich Chemie GmbH, Germany).