Table 2 The analysis method of biomarker and enzymes activity related parameters

Reactive oxygen species (ROS)

2,7-dichlorofluorescin-diacetate (DCFH-DA) method, reactive oxygen species can oxidize non-fluorescent DCFH to produce fluorescent DCF, and the absorbance was assessed at the characteristic absorption peak.

S0033M

Malondialdehyde (MDA)

Thibabituric acid method, MDA can be condensed with thibabituric acid to form a red product and the absorbance was assessed at the characteristic absorption peak.

A003–1-2

Protein carbonyl (PC)

The 2,4-dinitrophenylhydrazine (DNPH) method, the carbonyl group reacts with DNPH to form a red product, and the absorbance was assessed at the characteristic absorption peak.

A087–1-2

Anti-superoxide anion (ASA)

Spectrophotometric method, superoxide anion free radical, electron transfer substance and chromogenic agent react together to show purplish red, and the absorbance was assessed at the characteristic absorption peak.

A052–1-1

Anti-hydroxy radical (AHR)

Fenton reaction method, the Fenton reaction produces hydroxyl radicals, which can react with griess reagent to produce red products after combining with electron acceptor, and the absorbance was assessed at the characteristic absorption peak.

A018–1-1

Superoxide dismutase (SOD)

Hydroxylamine method, the reaction system of xanthine and xanthine oxidase produces superoxide anion free radicals, which oxidize hydroxylamine to form nitrite, and show purplish red products under the action of chromogenic agents, and the absorbance was assessed at the characteristic absorption peak.

A001–2

Catalase (CAT)

Ammonium molybdate method, the decomposition of H₂O₂ by catalase can be halted by the addition of ammonium molybbate, and the remaining hydrogen peroxide acts with ammonium molybbate to produce a yellow complex, and the absorbance was assessed at the characteristic absorption peak.

A007–1-1

Glutathione peroxidase (GPx)

Spectrophotometric method, GPx can promote the reaction of H₂O₂ with reduced glutathione (GSH) to form H₂O and oxidized glutathione (GSSG), GSH reacts with dinitrobenzoic acid to form 5-thio-dinitrobenzoic acid anion, which shows a stable yellow color, and the absorbance was assessed at the characteristic absorption peak.

A005–1-2

Glutathione-S-transferase (GST)

Spectrophotometric method, GST has the ability to catalyse the binding of reduced GSH to 1-chloro-2,4-dinitrobenzene (CDNB substrate). Within a certain reaction time, the activity of GST is linearly related to the change of substrate concentration before and after the reaction, and the absorbance was assessed at the characteristic absorption peak.

A004–1-1

Glutathione reductase (GR)

Ultraviolet spectrophotometry method, when GSSG is catalyzed by glutathione reductase GR and supplied with hydrogen by NADPH, GSSG is reduced to reduced GSH, GSH increases and NADPH decreases, and the absorbance was assessed at the characteristic absorption peak.

A062–1-1

Glutathione (GSH)

Spectrophotometric method, Reduced GSH can react with dithio-dinitrobenzoic acid (DTNB) to form a yellow compound, and the absorbance was assessed at the characteristic absorption peak.

A006–2-1

Protein concentrations

Coomassie brilliant blue method, Protein molecules have -NH₃⁺ groups, and when the brown-red Coomassie brilliant blue chromogenic agent is added to the protein standard solution or sample, the anion on the Coomassie brilliant blue dye binds to the protein-NH₃⁺, turning the solution blue, and the absorbance was assessed at the characteristic absorption peak.

A045–2-2