In vitro activity:  Tanshinone I, an active principle isolated from Salvia miltiorrhiza (Danshen), is structurally similar to tanshinone IIA and may possess similar cytotoxic effects on tumor cells. Tanshinone I inhibits PGE2 formation from LPS-induced RAW macrophages (IC50 = 38 μM). However, this compound does not affect COX-2 activity or COX-2 expression. Tanshinone I is an inhibitor of type IIA human recombinant phospholipase A2 (PLA2) with IC50 of 11 μM. In preliminary data, tanshinone I possesses the strongest inhibitory effect on tumor necrosis factor-α (TNF-α)-induced adhesion molecules.

Kinase Assay: As sources of PLA2, human recombinant sPLA₂ (type IIA) is purified from CHO cells transfected with the PLA₂ gene and rabbit recombinant platelet cPLA2 is obtained through its expression in baculovirus. The standard reaction mixture (200 μL) contained 100 mM Tris-HCl buffer (pH 9.0) with 6 mM CaCl₂ and 20 nmol 1-acyl-[1-¹⁴C]-arachidonyl-sn-glycerophosphoethanolamine (2000 cpm/nmol) in the presence or absence of Tanshinone I. The reaction is started by adding 50 ng purified sPLA₂ or cPLA₂. After 20 min at 37°C, the free fatty acid generated is analysed. Under these standard conditions, the reaction mixture in the absence of Tanshinone I released approximately 10% of free fatty acid from the phospholipid substrate added.

Cell Assay: RAW 264.7 cells are cultured with DMEM supplemented with 10% FBS and 1% antibiotics under 5% CO₂ at 37°C. Briefly, cells are plated in 96-well plates (2×10⁵ cells/well). LPS (1 ug/mL) and Tanshinone I are simultaneously added and incubated for 24 h, unless otherwise specified. The PGE₂ concentration in the medium is measured using an EIA kit for PGE2. In order to determine the effects of Tanshinone I on PGE2 production after induction of COX-2, cells are incubated with LPS (1 ug/mL) for 24 h and thoroughly washed. Then, Tanshinone I is added without LPS and the cells are incubated for another 24 h. From the medium, PGE2 concentrations are measured. The cytotoxicity of Tanshinone I to RAW cells is checked using the MTT assay. Tanshinone I does not show any cytotoxicity up to 100 uM.