Physiological Responses of a Natural Antioxidant Flavonoid Mixture, Silymarin, in BALB/c Mice

Abstract

Silymarin is a mixture of bioactive flavonoids isolated from Milk Thistle (Silybum marianum). Crude extracts from this plant have been used for centuries as a natural remedy and silymarin is now effectively used in the treatment of inflammatory liver toxicity and disease in humans. In vitro studies show that silymarin can inhibit the production and damage caused by tumor necrosis factor α (TNFα) and is a potent antioxidant both in vitro and in vivo. Such findings suggest silymarin may impact the immune system but little information exists following in vivo exposure. Therefore, we tested the hypothesis that exposure to silymarin will modulate the inflammatory immune response. Male BABL/c mice (6/group) were treated intraperitoneally once daily for five days with 0, 10, 50 or 250 mg/kg of silymarin. Silymarin exposure did not produce any signs of overt toxicity or any changes in relative organ weights. Flow cytometric examination of splenic lymphocyte populations showed that the absolute number of CD3⁺ T-lymphocytes was reduced in the 10 and 50 mg/kg groups although significance was evident only in the 10 mg/kg group. Concomitant decreases in CD4⁺ and CD8⁺ T-cell populations were observed but only the CD4⁺ population in mice treated with 10 mg/kg of silymarin was significantly different from control. Functional examination of secondary lymphoid cells revealed that phytohemagglutinin-induced T-lymphocyte proliferation was increased in the lowest dose group only. B-lymphocyte blastogenesis induced by lipopolysaccharide was increased following exposure to 10 and 50 mg/kg of silymarin. Similarly, expression of TNFα, inducible nitric oxide synthase, IL-1β and IL-6 mRNA were increased dose-dependently. The expression of IL-2 and IL-4 were reduced in mice treated with 10 and 50 mg/kg of silymarin although only the 10 mg/kg group was significantly different from control. The results indicate that in vivo parenteral exposure to silymarin results in suppression of T-lymphocyte function at low doses and stimulation of inflammatory processes at higher doses. Further studies investigating the effects of silymarin on the immune system are warranted.

Silymarin - inflammation - natural product - T-lymphocyte - cytokine - Silybum marianum - Asteraceae

References

• 1 Hahn G, Lehmann H D, Kurten M, Uebel H, Vogel G. On the pharmacology and toxicology of silymarin, and antihepatotoxic active principle from Silybum marianum .  Arzneimittel-Forschung/Drug Research. 1968;  18 698-704
• 2 Lahiri-Chatterjee M, Katiyar S K, Mohan R R, Agarwal R. A flavonoid antioxidant, silymarin, affords exceptionally high protection against tumor promotion in the SNECAR mouse skin tumorogenisis model.  Cancer Research. 1999;  59 622-32
• 3 Manna S K, Mukhopadhyay A, Van N T, Aggarwal B B. Silymarin suppresses TNF-induced activation of NK-κB, c-Jun N-terminal kinase, and apoptosis.  The Journal of Immunology. 1999;  163 6800-9
• 4 Ferenci P, Dragosics B, Dittrich H, Frank H, Benda L, Lochs H, Meryn S, Base W, Schneider B. Randomized controlled trial of silymarin treatment in patients with cirrhosis of the liver.  Journal of Hepatology. 1989;  9 105-13
• 5 Greimel A, Koch H. Silymarin - An inhibitor of horseradish peroxidase.  Experimentia. 1977;  33 1417-8
• 6 Lang I, Deak G, Muzes G, Pronai L, Feher J. Effect of natural bioflavonoid antioxidant silymarin on superoxide dismutase (SOD) activity and expression in vitro .  Biotechnology therapeutics. 1993;  4 263-70
• 7 Lang I, Deak G Y, Nekam K, Muzes G Y, Gonzalez-Cabello R, Gergely P, Feher J. Hepatoprotective and immunomodulatory effects of antioxidant therapy.  Acta Medica Hungaria. 1988;  45 287-95
• 8 Agoston M, Cabello R G, Blazovics A, Feher J, Vereckei A. The effect of amiodarone and/or antioxidant treatment on splenocyte blast transformation.  Clinica Chimica Acta. 2001;  303 87-94
• 9 Meroni P L, Barcellini W, Borghi M O, Vismara A, Ferraro G, Ciani D, Zanussi C. Silybin inhibition of human T-lymphocyte activation.  International Journal of Tissue Reactions. 1988;  10 177-81
• 10 Tager M, Dietzmann J, Thiel U, Hinrich Neumann K, Ansorge S. Restoration of the cellular thiol status of peritoneal macrophages from CAPD patients by the flavonoids silibinin and silymarin.  Free Radical Research. 2001;  34 137-51
• 11 Kang S N, Lee M H, Kim K M, Cho D, Kim T S. Induction of human promyelocytic leukemia HL-60 cell differentiation into monocytes by silibinin: involvement of protein kinase C.  Biochemical Pharmacology. 2001;  61 1487-95
• 12 Zi X, Mukhtar H, Agarwal R. Novel cancer chemopreventive effects of a flavonoid antioxidant silymarin: inhibition of mRNA expression of an endogenous tumor promoter TNFα.  Biochemical and Biophysical Research Communications. 1997;  239 334-9
• 13 Cho J Y, Kim P S, Park J, Yoo E S, Baik K U, Kim Y, Park M H. Inhibition of tumor necrosis factor-a production in lipopolysaccharide-stimulated RAW264.7 cells from Amorpha fruticosa .  Journal of Ethnopharmacology. 2000;  70 127-33
• 14 Saliou C, Rihn B, Cillard J, Okamoto T, Packer L. Selective inhibition of NF-κB activation by the flavonoid hepatoprotector silymarin in HepG2: Evidence for different activating pathways.  FEBS Letters. 1998;  440 8-12
• 15 Letteron P, Labbe G, Degott C, Berson A, Fromenty B, Delaforge M, Larrey D, Pessayre D. Mechanism for the protective effects of silymarin against carbon tetrachloride-induced lipid peroxidation and hepatotoxicity in mice. Evidence that silymarin acts both as an inhibitor of metabolic activation and as a chain-breaking antioxidant.  Biochemical Pharmacology. 1990;  39 2027-34
• 16 He Q, Osuchowski M F, Johnson V J, Sharma R P. Physiological responses of a natural antioxidant flavonoid, silymarin, in BALB/c mice: I. Induction of transforming growth factor β₁ and c-myc in liver with marginal effects on other genes.  Planta Medica. 2002;  68 676-9
• 17 Johnson V J, Sharma R P. Gender-dependent immunosuppression following subacute exposure to fumonisin B₁ .  International Immunopharmacology. 2001;  1 2023-34
• 18 Valenzuela A, Aspillaga M, Vial S, Guerra R. Selectivity of silymarin on the increase of the glutathione content in different tissues of the rat.  Planta Medica. 1989;  55 420-2
• 19 Lang I, Deak G, Muzes G, Pronai L, Feher J. Effect of the natural bioflavonoid antioxidant silymarin on superoxide dismutase (SOD) activity and expression in vitro.  Biotechnology Therapeutics. 1993;  4 263-70
• 20 Schmidt K N, Amstad P, Cerutti P, Baeuerle P A. The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-kappa B.  Chemistry and Biology. 1995;  2 13-22
• 21 Li J J, Oberley L W, Fan M, Colburn N H. Inhibition of AP-1 and NF-κB by manganese-containing superoxide dismutase in human breast cancer cells.  FASEB Journal. 1998;  12 1713-23
• 22 Han Y J, Kwon Y G, Chung H T, Lee S K, Simmons R L, Billiar T R, Kim Y M. Antioxidant enzymes suppress nitric oxide production through the inhibition of NF-kappaB activation: Role of H₂O₂ and nitric oxide in inducible nitric oxide synthase expression in macrophages.  Nitric Oxide. 2001;  5 504-13
• 23 Kita H, Lian Z X, Van de Water J, He X S, Matsumura S, Kaplan M, Luketic V, Coppel R L, Ansari A A, Gershwin M E. Identification of HLA-A2-restricted CD8(+) cytotoxic T cell responses in primary biliary cirrhosis: T cell activation is augmented by immune complexes cross-presented by dendritic cells.  Journal of Experimental Medicine. 2002;  195 113-23