Mda kit

About 1 g of fresh plant tissue samples (or 0.01–0.05 g freeze-dried samples) were weighed, added 1 ml extract (selected the corresponding extract according to manufacturer's instructions) for ice bath homogenization, then centrifuged 10,000 g at 4°C for 20 min, took the supernatant and placed the supernatant on ice to be tested. And then the determination of antioxidant enzyme activity, MDA, OFR, and H₂O₂ content were performed with CAT Kit, SOD Kit, POD Kit, MDA Kit, OFR Kit, and H₂O₂ Kit (Solarbio, Beijing, China) according to the manufacturer's protocol.

The following hepatopancreas function indices in the serum were measured using an ADVIA2400 Automatic Biochemistry Analyzer (Siemens, Munich, Germany) and commercial kits (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) according to the manufacturer’s instructions: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), adenosine deaminase (ADA), total bilirubin (T-Bil), total protein (TP), albumin (ALB), and globulin (GLB). The activities of ALT, AST, ALP, and ADA were reported as U/L, the T-Bil content was expressed in μmol/L, and the levels of TP, ALB, and GLB were reported in g/L.
The level of MDA in the hepatopancreas tissue was measured using an MDA kit (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). The MDA content was determined based on the hepatopancreas quality, which was measured using a microplate reader (Chen et al., 2019 (link)). MDA content (reported as nmol/g tissue) was calculated by 5 × [6.45 × (A532-A600)−1.29 × A450]/0.1, where A450, A532, and A600 represented the absorbance of each sample at 450, 532, and 600 nm, respectively.

The proline content was measured at 520 nm with a UV-visible (UV-vis) spectrophotometer (Mapada, Shanghai, China) according to methods described previously by Bates et al. (80 (link)). Leaves and roots tissue (1 g) from fresh plant samples were weighed, frozen, and ground into a powder. One milliliter of an extract solution was added for ice bath homogenization, and the mixture was then centrifuged at 10,000 × g at 4°C for 20 min. The contents of the active substances MDA, H₂O₂, and O₂^·− were then determined using an MDA kit, an H₂O₂ kit, and an O₂^·− kit (Solarbio, Beijing, China), respectively, according to the manufacturer’s protocol.

H₂O₂-induced oxidative damage model was established, and the influence of the antioxidant peptide on oxidative damage was evaluated by measuring cell viability, SOD activity and MDA content. HepG2 cells suspension was absorbed and seeded on 96-well plates with the density of 1 × 104 cells/well, followed by cultivation under a humidified atmosphere (37°C, 5% CO₂) via DEME medium (with 10% FBS and 1% penicil-lin-streptomycin). After 24-h cultivation of HepG2, the cells were categorized into control and treatment groups. In addition, the control group was added with 100 μL culture medium and cultured for 4h. Besides, the treatment group (peptide-treated group) was pre-pared through supplementing 100 μL of 100 μg/mL of GSH or PGPTY (DMEM dissolved) and cultured for 4 h. Next, 10 μL H₂O₂ (800 μM) was introduced to every well, followed by incubation for 2 h. Besides, the treatment group (H₂O₂-treated group) was added with 100 μL culture medium and cultured for 4 h. Then, 10 μL H₂O₂ (800 μM) was introduced, which was also cultured for 2 h. Light density value at 595 nm was calculated, and cell viability (%) was computed as below:
Where A_{treatment group} indicates the absorbance in treatment group, A_{control group} denotes the absorbance in control group.
SOD activity and MDA content were determined using SOD kit and MDA kit (Beijing Solarbio Science and Technology Co., Ltd.).

Whole flag leaves were collected every 7 d from heading to 28 d to measure the chlorophyll, NR, POD, MDA, and TK contents.
NR: the unit was U·g⁻¹, which was determined by the NR kit of Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China); TK:TK was determined by Shanghai Ruifan Biological Co., Ltd. (Shanghai, China) Plant TK kit, the unit was U·mL⁻¹; malondialdehyde (MDA): The MDA kit was determined by Solarbio’s MDA kit, and the unit was nmol·g⁻¹; POD:POD was determined by Solarbio’s POD kit, and the unit was U·g⁻¹.

The malondialdehyde (MDA) content was quantitated by MDA kit (Solarbio, Beijing, China) using worms on the 6th day of adulthood after exposure to oxidative stress induced by 7 mM paraquat. Sample absorbance at 450, 532, and 600 nm were measured using multifunctional reader (BioTek, VT, USA). Piece bicinchoninic acid (BCA) (Thermo Fisher, Waltham, MA, USA) kit was used to quantify MDA content.