Daphnetin (7,8-Dihydroxycoumarin), a natural coumarin derivative, is a protein kinase inhibitor with inhibitory for EGFR (IC50: 7.67 μM), PKA (IC50: 9.33 μM), and PKC (IC50: 25.01 μM), also exhibit anti-oxidant and anti-inflammatory activities.

PKC:25.01 μM, EGFR:7.67 μM, PKA:9.33 μM

在水迷宫测试和强迫游泳测试中,Daphnetin（2-8 mg/kg）可增强应激小鼠的表现. Daphnetin（140 mg/kg）可使子宫重量减少39.5%.Daphnetin可使P. yoelli感染小鼠的存活时间显著延长.

Daphnetin（25-40 μM）对恶性疟原虫引起的次黄嘌呤掺入的抑制率可达50%。Daphnetin（50 μM）可使细胞周期蛋白D1的水平降低。地塞米松可影响皮质神经从而降低细胞活性,Daphnetin可剂量依赖地防止该作用。 Daphnetin能以特殊的方式剂量依赖性地抑制内源或重组TaPRK的活性。Daphnetin既对表皮细胞生长因子受体催化的外源底物酪氨酸磷酸化具有明显抑制作用,还对PKA和PKC的活性有抑制作用。Daphnetin处理24 h对MCF-7雌激素应答的人类癌细胞系生长有抑制作用（IC50：73 μM）。Daphnetin对ERK1/ERK2促有丝分裂的信号传导有抑制作用。

Assay of protein kinase C (PKC) and cAMP-dependent protein kinase (PKA) activities.: 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

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.(Only for Reference)