| In Vitro | 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. |
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| In Vivo | Tanshinone I shows antiinflammatory activity in rat carrageenan-induced
paw oedema and adjuvant-induced arthritis. In order to establish the
anti-inflammatory activity of Tanshinone I, the classical animal models
of acute and chronic inflammation [rat carrageenan (CGN)-induced paw
oedema and rat adjuvant-induced arthritis (AIA)] are employed. When
Tanshinone I is orally administered, it shows significant
anti-inflammatory activity against CGN-induced paw oedema (47%
inhibition at 160 mg/kg), while the IC50 of indomethacin is
7.1 mg/kg. In AIA, Tanshinone I gives 27% inhibition of secondary
inflammation at 18 day with an oral dose of 50 mg/kg/day, whereas
prednisolone (5 mg/kg/day) shows potent inhibition (65%) |
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