Trichloroacetic acid

Pigment standards including chlorophyll a, violaxanthin, vaucheriaxanthin, and β-carotene were purchased from Sigma-Aldrich Chemical Co. (Shanghai, China; http://www.sigmaaldrich.com). Silica gel (200–300 mesh) was obtained from Qing Dao Marine Chemical Co. (Qingdao, China). HPLC-grade solvents used for HPLC analysis were purchased from Guangzhou Runhao Biotech Co. (Guangzhou, China), such as methanol, acetonitrile, acetic ether, and dichloromethane. Other analytical solvents (n-hexane, methanol, and acetone) used in extraction and isolation of violaxanthin were purchased from Guangzhou Runhao Biotech Co. (Guangzhou, China). Deionized water was prepared by a Milli-Q water purification system (Millipore Corp., Bedford, MA, USA).
Chemicals used for their antioxidant activity including 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), potassium persulfate, ferrous chloride, potassium ferricyanide, trichloroacetic acid, hydrogen peroxide, ascorbic acid, sodium dihydrogen phosphate, and disodium hydrogen phosphate were obtained from Sangon Biotech Co. (Shanghai, China).

BBB permeability was determined by visualizing and quantifying the levels of extravasated Evans blue dye (EB) in the brain. Mice were injected intraperitoneally with 0.4 ml 2% (w/v) EB (Sigma, St. Louis, MO, USA) followed by euthanization and intracardiac perfusion with PBS 1 h later. The brains were subsequently removed and photographed. For quantification, brain tissues were weighed and homogenized in 1 ml PBS, and then 1 ml 100% trichloroacetic acid (Sangon, China) was added. The mixture was vigorously shaken for 2 min to precipitate the proteins and cooled for 30 min at 4 °C. After the mixture was centrifuged (30 min at 6000 g), the absorbance of the supernatant was measured at 620 nm using a spectrophotometer. The content of EB was quantified as nanograms of dye per gram of brain tissue using a standard curve.