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^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
In order to evaluate the inhibitory activity of Tanshinone I against animal models of acute and chronic inflammation, rat carrageenan (CGN)-induced paw oedema and adjuvant-induced arthritis (AIA) models are employed. Briefly, 1% CGN dissolved in pyrogen-free saline (0.05 mL) is injected into right hind paw of rats for the paw oedema test. After 5 h, swelling of the treated paw is measured using a plethysmometer. Tanshinone I dissolved in 0.5% CMC is administered orally 1 h prior to CGN injection. For the AIA test, an arthritic inflammation is provoked by injection of Mycobacterium Butyricum (0.6 mL/rat) dissolved in mineral oil to the right hind paw of rats. Tanshinone I is orally administered every day. The swelling of the treated and untreated paws is measured using a plethysmometer.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Kim SY, et al. Effects of Tanshinone I isolated from Salvia miltiorrhiza bunge on arachidonic acid metabolism and in vivo inflammatory responses. Phytother Res. 2002 Nov;16(7):616-20.  [Content Brief]