*2.7. Biochemical Determinations: Salivary Amylase Activity and IL-1*β *Concentration*

The salivary amylase activity (EC 3.2.1.1) was assessed spectrophotometrically using an alkaline solution of 3,5-dinitrosalicylic acid (DNS). The absorbance of samples was measured at 540 nm, accompanying the increased concentration of reducing sugars released during the hydrolysis of starch catalyzed by salivary amylase. Salivary amylase activity was determined in duplicate samples and expressed in μmol/mg protein [23].

Salivary interleukin 1β (IL-1β) concentration was determined by ELISA using a commercially available kit from EIAab Science Inc. Wuhan (Wuhan, China) in accordance with the manufacturer's instructions provided in the package.

#### *2.8. Biochemical Determinations: Redox Assay*

All analyses were performed in duplicate samples. Absorbance and fluorescence were measured with Infinite M200 PRO Multimode Tecan microplate reader. The results were standardized to 1 mg of total protein. The content of protein was evaluated by the bicinchoninic method (BCA) using a ready-made reagent kit (Thermo Scientific Pierce BCA Protein Assay Kit, Rockford, IL, USA) and a bovine serum albumin standard (BSA).
