Evaluation of in vitro anti-inflammatory effects of the remaining water subextract of Cistus laurifolius leaves

Cistus laurifolius L. (Cistaceae) is known as 'laden' in Anatolia and the leaves of the plant are used against rheumatismal diseases traditionally [1]. The ethanol extract of the leaves were submitted to solvent-solvent extractions and n-hexane, chloroform, ethyl acetate, n-butanol, remaining water subextracts were obtained. The extract and subextracts were investigated for their inhibitory effects on Nuclear Factor kappa B (NF-?B) transcrption factor on lipopolysaccharide induced Raw 264.7 macrophages. Only remaining water subextract exerted 20% inhibition. The subextract was fractionated by chromatographic methods but the fractions were found to be not effective. Thus, remaining water subextract was directed for further in vitro anti-inflammatory investigations. The effects on nitric oxide (NO), prostaglandine E2 (PGE2), tumor necrosis factor (TNF?) and interleukins (IL-1?, IL-1?, IL-2, IL-6) were studied by Griess and ELISA. The effects on iNOS and COX-2 protein levels and the phosphorylation levels of mitogen activated protein kinases (ERK1/2, JNK, p38) and I kappa B alpha (I?B?) were examined by Western Blot method. The subextract was applied to Raw 264.7 macrophage cells at 25, 50 and 100 µg/mL concentrations and it provided up to 57% decrease of NO production and up to 55% decrease of PGE2 production of cells. The iNOS and COX-2 protein levels were decreased accordingly. The subextract exerted inhibitory effect on TNF? (37%). This subextract inhibited the phosphorylation of I?B? at 50 and 100 µg/mL concentrations while JNK phosphorylation was concentration-dependently inhibited.