Elisa kit

The content of H₂O₂ was determined according to the method given by Gong et al. (2008) (link). H₂O₂ content was measured using the ELISA kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the instructions. The H₂O₂ content was calculated based on the standard curve.
The malondialdehyde (MDA) was determined according to the method of Zhang et al. (2007) (link). The concentration of MDA was determined using the colorimetric method of thiobarbituric acid (TBA) with Micro MDA Assay Kit (BC0025; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer’s instructions.

Culture supernatants and sediments from Mtb-infected RAW 264.7 cells were harvested at 0 h, 4 h, 8 h, 12 h, 24 h post-infection and stored at − 80 °C for cytokine measurement. The concentrations of TNF-α and IL-6 in culture supernatant were detected using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (Solarbio, Beijing, China) [15 (link)]. The mRNA level of TNF-α and IL-6 in culture sediments were determined using qPCR. In simple terms, the total RNA was extracted with Trizol method according to the instructions of the manufacturers [16 (link)]. After treatment with DNaseI (TaKaRa, Dalian, China), the cDNAs were reverse-transcribed from 5 µg of total RNA with the PrimeScript™ II 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). Real time PCR (qPCR) was carried out in triplicates for each sample using TB Green^® Premix Ex Taq™ II (TaKaRa, Dalian, China) in the CFX96 touch Real-Time PCR System (Bio-Rad) [17 (link)]. Primers for transcriptional level analysis are listed in Additional file 3: Table S2. GAPDH were used as the internal control of the respective qPCR experiments.

After supernatant of brain tissue and cells were obtained, total protein concentrations were determined by a BCA Kit as described above. Total proteins were extracted and L-18, TNF-α and IL-1β were, respectively, analyzed using an ELISA Kit (Solarbio, Beijing, China), according to the manufacturer’s instructions. Samples were tested at least in duplicate.

The total glutathione (GSH and GSSG) was extracted using an ELISA kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer’s instructions. Either leaf or root (0.100 g) was homogenized and centrifuged at 7,100 × g (total glutathione) or 1,100 × g (GSSG) at 4°C for 10 min. The supernatant was collected to determine total glutathione and GSSG.
The contents of AsA and DHA were analyzed according to the method as described by Arakawa et al. (1981) (link). The AsA and DHA contents were analyzed according to the following steps. The first extract reagent (5 mL) was added to the frozen samples (0.1 g), and it was homogenized in an ice bath. Then, the homogenate was centrifuged at 8,000 × g at 4°C for 20 min for AsA or 16,000 × g at 4°C for 20 min for DHA, respectively. The analysis was performed using the AsA ELISA kit and DHA ELISA kit, respectively.

The superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) activities are measured with an ELISA kit (Solarbio Science & Technology, Beijing, China) according to the manufacturer’s instructions.

HaCaT cells were inoculated in a 96-wells plate for 24 h, modeled by a UV lamp (8.64 mJ/cm²), and treated with MITC, MITC/NPs, HACE/NPs, and HACE/MITC NPs (20 μg/mL) for 24 h. The GSH, MDA, SOD, and ROS released from cell culture supernatants were investigated using an ELISA kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) as per the manufacturer’s instructions. The color reaction was developed using a chromogenic agent, and the optical density was measured using a microplate reader.