0944-7113/04/11/02–03-206 $ 30.00/0  Introduction It is generally accepted that free radicals leading to ox- idative stress play an important role in pathomecha- nism of various diseases such as atherosclerosis, alco- holic liver cirrhosis and cancer etc. (Freeman and Crapo, 1982; Maxwell and Lip, 1997; Halliwell and Gutteridge, 1999). The oxidative stress is initiated by reactive oxygen species (ROS) such as superoxide anion (O 2 – ), perhydroxyl (HOO• ) and hydroxyl (HO• ) radicals. Propagation cycle of lipid peroxidation is bro- ken by either enzymatic inactivation of ROS or non-en- zymatic reactions due to the intervention of free radical scavengers and antioxidants. Recently, several experi- ments have demonstrated anti-inflammatory, anti- cancer and antioxidant properties of naturally occur- Effect of silybin on phorbol myristate actetate-induced protein kinase C translocation, NADPH oxidase activity and apoptosis in human neutrophils Zs. Varga 1 , L. Újhelyi 1 , A. Kiss 1 , J. Balla 1 , A. Czompa 2 , and S. Antus 2 1 I st Department of Medicine, Medical and Health Science Centre, 2 Department of Organic Chemistry, University of Debrecen, Debrecen, Hungary Summary Mechanism of the action of silybin (1) and its derivatives (2–4), possessing different lipid solubility in PMA-stimulated neutrophils was evaluated. Silybin (1) inhibited the calcium, phosphatidylser- ine- and diacylglycerol-dependent protein kinase C translocation and the NADPH oxidase activity in PMA-stimulated neutrophils and resulted in decreased apoptosis. Furthermore, silybin (1) inhib- ited xanthine oxidase activity and hem-mediated oxidative degradation of low-density lipoprotein, as well. Its derivatives (2–4), possessing different lipid-solubility, affected all the studied parame- ters. The lipid solubility of silybin (1) was enhanced by methylation (5′7′4′′trimethylsilybin: 2), whereas a decrease in lipid-solubility by acetylation of compound 2 (5′,7,′4′′-trimethylsilybin-ac- etate: 3) or all the hydroxyl groups of silybin (peracetyl-silybin: 4) attenuated the antioxidant ca- pacity by decreasing the inhibition in PKC translocation and NADPH oxidase activation. All the derivatives of silybin (2–4) showed no inhibition in cell free systems; e.g. did not alter the xanthine oxidase activity and the hem-mediated oxidative degradation of LDL. In conclusion, the antioxi- dant activity of (1) might be due to its ability to inhibit PKC translocation and NADPH oxidase ac- tivation in PMA-stimulated neutrophils. The increase of lipid solubility of silybin (1) supports its penetration through cell membrane and enhances its inhibitory effects. This structural modifica- tion of (1) might have pharmacological consequences. Key words: oxidative stress, silybin, antioxidants, protein kinase C, NADPH oxidase, apoptosis, neu- trophil Abbreviations: PKC – protein kinase C; NADPH – nicotin- amide adenine dinucleotide phosphate; PMA – phorbol myristate acetate; Ca – calcium; PS – phosphatidylserine; DAG – diacylglycerol; HBSS – Hanks’ balanced salt solu- tion; PMSF – phenylmethylsulphonyl fluoride; EDTA – ethylenediaminetetraacetic acid; EGTA – ethylene glycol- bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid Phytomedicine 11: 206–212, 2004 http://www.elsevier-deutschland.de/phymed