Attenuation of UVA-induced damage to human keratinocytes by silymarin



      UV radiation from sunlight is a potent environmental risk factor in skin cancer pathogenesis. UVA is the major portion of UV light reaching the earth surface (∼95%) and it is reported to lead to benign and malignant tumor formation. UVA-mediated cellular damage occurs primarily through the release of reactive oxygen species (ROS) and it is responsible for inflammation, immunosuppression, photoaging and photocarcinogenesis.


      The aim of our study was to investigate the potency of silymarin, the polyphenol fraction from the seeds of Silybum marianum, to modulate UVA-induced oxidative damage to human keratinocytes.


      Skin epidermal cell line HaCaT, extensively used for studying the influence of UV radiation, was chosen as an experimental model. Silymarin's effect on UVA-disrupted cell viability, proliferation, mitochondrial function, and intracellular ATP and GSH level was measured. Furthermore, silymarin's potency to reduce UVA-induced ROS generation, membrane lipid peroxidation, caspase-3 activation and DNA damage was monitored.


      Treatment of irradiated HaCaT (20 J/cm2) with silymarin (0.7–34 mg/l; 4 h) resulted in concentration-dependent diminution of UVA-caused oxidative stress on all studied parameters. Silymarin application extensively reduced GSH depletion and ROS production as well as lipid peroxidation in irradiated cells. Formation of UVA-induced DNA single strand breaks and caspase-3 activity was also significantly decreased by silymarin.


      The results suggest that silymarin may be beneficial in the treatment of UVA-induced skin oxidative injury and inflammation. However, further studies especially whose using human systems are needed to determine efficacy of silymarin in vivo.


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Journal of Dermatological Science
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Svobodova A.
        • Psotova J.
        • Walterova D.
        Natural phenolics in the prevention of UV-induced skin damage.
        Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2003; 147: 137-145
        • Van der Leun J.C.
        The ozone layer.
        Photodermatol Photoimmunol Photomed. 2004; 20: 159-162
        • Pattison D.I.
        • Davies M.J.
        Actions of ultraviolet light on cellular structures.
        EXS. 2006; 96: 131-157
        • Pinnel S.R.
        Cutaneous photodamage, oxidative stress, and topical antioxidant protection.
        J Am Acad Dermatol. 2003; 48: 1-22
        • Ischihashi M.
        • Ueda M.
        • Budiyanto A.
        • Bito T.
        • Oka M.
        • Fukunaga M.
        • et al.
        UV-induced skin damage.
        Toxicology. 2003; 189: 21-39
        • Svobodova A.
        • Walterova D.
        • Vostalova J.
        Ultraviolet light induced alteration to the skin.
        Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2006; 150: 25-38
        • Offord E.A.
        • Gautier J.C.
        • Avanti O.
        • Scaletta C.
        • Runge F.
        • Krämer K.
        • et al.
        Photoprotective potential of lycopene, β-carotene, vitamin E, vitamin C, and carnosic acid in UVA-irradiated human skin fibroblast.
        Free Radic Biol Med. 2002; 32: 1293-1303
        • Basu-Modak S.
        • Gordon M.J.
        • Dobson L.H.
        • Spencer J.P.
        • Rice-Evans C.
        • Tyrrell R.M.
        Epicatechin and its methylated metabolite attenuate UVA-induced oxidative damage to human skin fibroblasts.
        Free Radic Biol Med. 2003; 35: 910-921
        • Tobi S.E.
        • Gilbert M.
        • Paul N.
        • McMillan T.J.
        The green tea polyphenol, epigallocatechin-3-gallate, protects against the oxidative cellular and genotoxic damage of UVA radiation.
        Int J Cancer. 2002; 102: 439-444
        • Huang C.C.
        • Fang J.Y.
        • Wu W.B.
        • Chiang H.S.
        • Wei Y.J.
        • Hung C.F.
        Protective effects of (−)-epicatechin-3-gallate on UVA-induced damage in HaCaT keratinocytes.
        Arch Dermatol Res. 2005; 296: 473-481
        • Philips N.
        • Smith J.
        • Keller T.
        • Gonzales S.
        Predominant effects of Polypodium leucotomos on membrane integrity, lipid peroxidation, and expression of elastin and matrixmetalloproteinase-1 in ultraviolet radiation exposed fibroblasts, and keratinocytes.
        J Dermatol Sci. 2003; 32: 1-9
        • Erden I.M.
        • Kahramant A.
        • Kökent T.
        Beneficial effects of quercetin on oxidative stress induced by ultraviolet A.
        Clin Exp Dermatol. 2001; 26: 536-539
        • Morazzoni P.
        • Bombardelli E.
        Silybum marianum (Carduus marianus).
        Fitoterapia. 1995; 66: 3-42
        • Kren V.
        • Walterova D.
        Silybin and silymarin—new effects and applications.
        Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2005; 149: 29-41
        • Zhao J.
        • Sharma Y.
        • Agarwal R.
        Significant inhibition by the flavonoid antioxidant silymarin against 12-O-tetradecanoylphorbol 13-acetate-caused modulation of antioxidant and inflammatory enzymes, and cyclooxygenase 2 and interleukin-1alpha expression in SENCAR mouse epidermis: implications in the prevention of stage I tumor promotion.
        Mol Carcinog. 1999; 26: 321-333
        • Lahiri-Chatterjee M.
        • Katiyar S.K.
        • Mohan R.R.
        • Agarwal R.
        Flavonoid antioxidant, silymarin, affords exceptionally high protection against tumor promotion in SENCAR mouse skin tumorgenesis model.
        Cancer Res. 1999; 59: 622-632
        • Singh R.P.
        • Tyagi A.K.
        • Zhao J.
        • Agarwal R.
        Silymarin inhibits growth and causes regression of established skin tumors in SENCAR mice via modulation of mitogen-activated protein kinases and induction of apoptosis.
        Carcinogenesis. 2002; 23: 499-510
        • Zhao J.
        • Lahiri-Chatterjee M.
        • Sharma Y.
        • Agarwal R.
        Inhibitory effect of a flavonoid antioxidant silymarin on benzoyl peroxide-induced tumor promotion, oxidative stress and inflammatory responses in SENCAR mouse skin.
        Carcinogenesis. 2000; 21: 811-816
        • Katiyar S.K.
        • Korman N.J.
        • Mukhtar H.
        • Agarwal R.
        Protective effects of silymarin against photocarcinogenesis in a mouse skin model.
        J Natl Cancer Inst. 1997; 89: 556-566
        • Dhanalakshmi S.
        • Mallikarjuna G.U.
        • Singh R.P.
        • Agarwal R.
        Silibinin prevents ultraviolet radiation-caused skin damages in SKH-1 hairless mice via a decrease in thymine dimmer positive cells and an up-regulation of p53–p21/Cip1 in epidermis.
        Carcinogenesis. 2004; 64: 6349-6356
        • Katiyar S.K.
        Treatment of silymarin, a plant flavonoid, prevents ultraviolet light-induced immune suppression and oxidative stress in mouse skin.
        Int J Oncol. 2002; 21: 1213-1222
        • Mallikarjuna G.
        • Dhanalakshymi S.
        • Singh R.P.
        • Agarwal C.
        • Agarwal R.
        Silibinin protects against photocarcinogenesis via modulation of cell cycle regulators, mitogen-activated protein kinases, and Akt signalling.
        Cancer Res. 2004; 64: 6349-6356
        • Dhanalakshmi S.
        • Mallikarjuna G.U.
        • Singh R.P.
        • Agarwal R.
        Dual efficacy of silibinin in protecting or enhancing ultraviolet B radiation-caused apoptosis in HaCaT human immortalized keratinocytes.
        Carcinogenesis. 2004; 25: 784-792
        • Maines M.D.
        • Costa L.G.
        • Reed D.J.
        • Sassa S.
        • Sipes I.G.
        Current protocols in toxicology.
        John Wiley & Sons, New York1998
        • Chlopcikova S.
        • Psotova J.
        • Miketova P.
        • Sousek J.
        • Lichnovsky V.
        • Simanek V.
        Chemoprotective effect of plant phenolics against anthracycline-induced toxicity on rat cardiomyocytes. Part II. Caffeic, chlorogenic and rosmarinic acids.
        Phytoter Res. 2004; 18: 408-413
        • Lowry O.H.
        • Rosebough N.J.
        • Farr A.L.
        • Randal R.J.
        Protein measurement with the Folin phenol reagent.
        J Biol Chem. 1951; 193: 265-275
        • Sedlak J.
        • Lindsay R.H.
        Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent.
        Anal Biochem. 1968; 25: 192-205
        • Buege J.A.
        • Aust S.D.
        Microsomal lipid peroxidation.
        Methods Enzymol. 1978; 52: 302-310
        • Angelo S.D.
        • Ingrosso D.
        • Migliardi V.
        • Sorrentino A.
        • Donnarumma G.
        • Baroni A.
        • et al.
        Hydroxytyrosol, a natural antioxidant from olive oil, prevents protein damage induced by long-wave ultraviolet radiation in melanoma cells.
        Free Radic Biol Med. 2005; 38: 908-919
        • Bradford M.M.
        A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding.
        Anal Biochem. 1976; 72: 248-254
        • Tice R.R.
        • Andrews P.W.
        • Singh N.P.
        The single cell gel assay: a sensitive technique for evaluating intercellular differences in DNA damage and repair.
        Basic Life Sci. 1990; 53: 291-301
        • Ochu E.E.
        • Rothwell N.J.
        • Waters C.M.
        Caspase mediate 6-hydroxydopamine-induced apoptosis but not necrosis in PC12 cells.
        J Neurochem. 1998; 70: 2637-2640
        • Adhami V.M.
        • Aziz M.H.
        • Mukhtar H.
        • Ahmad N.
        Activation of prodeath Bcl-2 family proteins and mitochondrial apoptosis pathway by sanguinarine in immortalized human HaCaT keratinocytes.
        Clin Cancer Res. 2003; 9: 3176-3182
        • Svobodova A.
        • Walterova D.
        • Psotova J.
        Influence of silymarin and its flavonolignans on H2O2-induced oxidative stress in human keratinocytes and mice fibroblasts.
        Burns. 2006; 32: 973-979
        • Tobi S.E.
        • Paul N.
        • McMillan T.J.
        Glutathione modulates the level of free radicals produced in UVA-irradiated cells.
        J Photochem Photobiol B. 2000; 57: 102-112
        • Soo Lee Y.
        • Jin D.Q.
        • Beak S.M.
        • Lee E.S.
        • Kim J.A.
        Inhibition of ultraviolet-A-modulated signaling pathways by asiatic acid and ursolic acid in HaCaT human keratinocytes.
        Eur J Pharmacol. 2003; 476: 173-178
        • Flora K.
        • Hahn M.
        • Rosen H.
        • Bernner K.
        Milk thistle (Silybum marianum) for the therapy of liver disease.
        Am J Gastroenterol. 1998; 93: 139-143
        • Psotova J.
        • Chlopcikova S.
        • Grambal F.
        • Simanek V.
        • Ulrichova J.
        Influence of silymarin and its flavonolignans on doxorubicin-iron induced lipid peroxidation in rat heart microsomes and mitochondria in comparison with quercetin.
        Phytother Res. 2002; 16: S63-S67
        • Chlopcikova S.
        • Psotova J.
        • Miketova P.
        • Simanek V.
        Chemoprotective effect of plant phenolics against anthracycline-induced toxicity on rat cardiomyocytes. Part I. Silymarin and its flavonolignans.
        Phytother Res. 2004; 18: 107-110
        • Pietrangelo A.
        • Borella F.
        • Casalgrandi G.
        • Montosi G.
        • Ceccarelli D.
        • Gallesi D.
        • et al.
        Antioxidant activity of silybin in vivo during long-term iron overload in rats.
        Gastroenterology. 1995; 109: 1941-1949
        • Larsson P.
        • Andersson E.
        • Johansson U.
        • Ollinger K.
        • Rosdahl I.
        Ultraviolet A and B affect human melanocytes and keratinocytes differently. A study of oxidative alterations and apoptosis.
        Exp Dermatol. 2005; 14: 117-123
        • Schneide L.A.
        • Dissemond J.
        • Brenneisen P.
        • Hainzl A.
        • Briviba K.
        • Wlaschek M.
        • et al.
        Adaptive cellular protection against UVA-1-induced lipid peroxidation in human dermal fibroblasts shows donor-to-donor variability and is glutathione dependent.
        Arch Dermatol Res. 2006; 297: 324-328
        • Zhong J.L.
        • Yiakouvaki A.
        • Holley P.
        • Tyrrell M.R.
        • Pourzand C.
        Susceptibility of skin cells to UVA-induced necrotic cell death reflect the intracellular level of labile iron.
        J Invest Dermatol. 2004; 123: 771-780
        • Orrenius S.
        Mitochondrial regulation of apoptotic cell death.
        Toxicol Lett. 2004; 149: 19-23
        • Valencia A.
        • Kochevar I.E.
        • Ultraviolet
        A induces apoptosis via reactive oxygen species in a model for Smith-Lemli-Opitz syndrome.
        Free Radic Biol Med. 2006; 40: 641-650
        • Zimmermann K.C.
        • Bonzon C.
        • Green D.R.
        The machinery of programmed cell death.
        Pharmacol Ther. 2001; 92: 57-70
        • Syed D.N.
        • Malik A.
        • Hadi N.
        • Sarfaraz S.
        • Afaq F.
        • Mukhtar H.
        Photochemopreventive effect of pomegranate fruit extract on UVA-mediated activation of cellular pathways in normal human epidermal keratinocytes.
        Photochem Photobiol. 2005; 82: 398-405