Cox 2 inhibitor screening kit

Acetonitrile of HPLC grade was purchased from TEDIA (Fairfield, OH), and analytical reagent grade trifluoroacetic acid was purchased from Nanjing Chemical Reagent Company (Nanjing, China). Ultrapure water was prepared in the lab using a Millipore water purification system (Milford, MA). Paeoniflorin (PubChem ID 442534, purity >98%) and amygdalin (PubChem ID 656516, purity >98%) were purchased from MedChem Express, and paeonol (PubChem ID 24895579, purity >99%) and cinnamaldehyde (PubChem ID 24900954, purity >95%) were purchased from Sigma Aldrich (Shanghai, China).
Antibodies against COX2 and β-actin were purchased from Santa Cruz. HRP-conjugated secondary antibodies were purchased from Sigma Aldrich. The ECL reagent kit was purchased from Thermo Fisher. A COX2 inhibitor screening kit was purchased from Beyotime Technologies (#S0168, Nantong, China).

For the enzyme activity detection of cPLA2, small intestine and hippocampal tissues were perfused with PBS containing 0.16 mg/ml heparin to remove red blood cells and clots. Then, the tissues (50 mg) were homogenized in 1 ml pre-cold buffer (50 mM HEPES, pH 7.4, containing 1 mM EDTA). After being centrifuged, the supernatant was analyzed according to the instructions of the commercial kit (Cayman Chemical, Ann Arbor, MI, United States). The samples were diluted at 1:5 and 1:10, respectively, for hippocampal tissues and small intestines. The absorbance was recorded at 414 nm using a plate reader. To detect the enzyme activity of COX-2, the COX-2 inhibitor screening kit (Beyotime Biotechnology, China) was used. The hippocampal tissues and small intestines were homogenized using lysis buffer at a ratio of 1:5 (mg/μl) and quantified by the BCA protein assay kit (Thermo, United States). The enzyme activity of COX-2 of the samples was analyzed following the instructions of the commercial kit and corrected as Relative Fluorescence Unit (RFU) per gram protein.

Anti-inflammatory activity was assessed by measuring COX-2 enzymatic inhibition on the basis of a described method by using the COX-2 Inhibitor Screening Kit (Beyotime, No. S0168) [5]. Celecoxib, a COX-2 inhibitor, was used as a positive control. The results of anti-COX-2 activities are presented as IC₅₀ values (μg/ml or μM), a measurement of the inhibition of enzyme activity by each sample by 50%.

PNF was acquired from Hebei Jiahenglengbei Co., Ltd., (Yunnan; batch numbers C20071810 and C20071809, respectively). Distilled water was acquired from Guangzhou Watsons Food & Beverage Co., Ltd. D101 macroporous resin, standard saponins (ginsenosides Rb1, Rb2, Rb3, Rc, Rd, Re, Rg1, and notoginsenoside R1), and vanillin were acquired from Shanghai Yuanye Biotechnology Co., Ltd. (batch numbers B21053, B21054, B21055, B21099, B210056, B21050, B21051, and B21052, respectively). Anhydrous ethanol and glacial acetic acid were acquired from Fuchen Chemical Reagent Co., Ltd. (Tianjin, China), and chromatographic grade methanol and acetonitrile were acquired from Aladdin Reagent Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium, heat-inactivated fetal bovine serum, Dulbecco’s phosphate-buffered saline (PBS), penicillin–streptomycin, and trypsin 2.5% were acquired from Life Technologies, Inc. (Grand Island, NY, USA). β-actin, PGE-2, IL-1β, and TNF-α antibodies were acquired from Cell Signaling Technology (Beverly, MA, USA). PGE-2, IL-1β, and TNF-α enzyme-linked immunosorbent assay (ELISA) kits were purchased from BioChell (Shanghai, China). The COX-2 inhibitor screening kit was acquired from Beyotime Biotechnology (Shanghai, China).