In vitro activity: Daphnetin strongly inhibits not only EGF receptor-catalyzed tyrosine phosphorylation of exogenous substrate but also PKA and PKC activities. Daphnetin inhibits MCF-7 estrogen-responsive human carcinoma cell line at 24-hour exposure with IC50 of 73 μM. Daphnetin reduces the level of cyclin D1 even at 50 μM. Daphnetin protects the cortical neurons against dexamethasone-induced reduction of cell viability in a dose-dependent manner. Daphnetin inhibits the activities of endogenous or recombinant TaPRK in a specific and dose-dependent manner. Daphnetin causes a 50% inhibition (IC50) of 3H-hypoxanthine incorporation by Plasmodium falciparum at concentrations between 25 μM and 40 μM. Daphnetin inhibits the mitogenic signalling of ERK1/ERK2.

Kinase Assay: PKC and PKA activities are detected. Briefly, 5 mL of PKC or PKA is mixed with 5 mL of lipid preparation containing 100 mM phorbol 12-myristate 13-acetate, 2.8 mg/mL phosphatidyl serine, and Triton X-100 mixed micelles for PKC assay or 5 mL of 40 mM cAMP in 50 mM Tris-HCl, pH 7.5, for PKA assay, and 5 mL of daphnetin. The reaction is started by adding 10 mL of PKC substrate solution containing 250 mM acetylated myelin basic protein, 100 mM ATP, 5 mM CaCl2, 10 mM MgCl2, 20 mM Tris-HCl, pH 7.5, or 10 mL of PKA substrate solution containing 200 mM Kemptide, 400 mM ATP, 40 mM MgCl2, 1 mg/mL BSA, 50 mM Tris-HCl, pH 7.5, and 20-25 mCi/mL[g-32P] ATP. After incubation at 25 °C for 5 minutes, 20 mL of each mixture is spotted on a piece of phosphocellulose disc which is immediately put into 1% H3PO4. After free [g-32P] ATP on the discs is removed, the peptide-incorporated 32P on the discs is counted in a scintillation counte.

Cell Assay: The cytostatic effect of Daphnetin tested on the MCF-7 tumor cells is estimated using the microculture MTT assay. The assay is based on the reduction of soluble tetrazolium salt by mitochondria of viable cells. The reduced product, an insoluble purple-colored formazan, is dissolved in dimethyl sulfoxide and measured spectrophotometrically (570 nm). The amount of formazan formed is proportional to the number of viable cells. Cells (3 × 103) are seeded in each of the 96 microplate wells in a 200 μL medium containing the corresponding concentration of daphnetin. Daphnetin is tested at five concentrations (12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM). After 24-hour, 48-hour, and 72-hour exposure, the percentage of proliferative inhibition of treated cells is estimated against the solvent-treated control cells (PI% = [(T/C) – 1] × 100). PI = proliferation inhibition; T = treated, C = control. IC50 is calculated from the least square concentration-response regressions.

Biochem Biophys Res Commun. 1999 Jul 14;260(3):682-5; Eur J Pharmacol. 2011 Oct 1;668(1-2):35-41.