Silymarin, the antioxidant component of Silybum marianum, protects against burn-induced oxidative skin injury

burns 33 (2007) 908–916

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Silymarin, the antioxidant component of Silybum marianum, protects against burn-induced oxidative skin injury Hale Z. Toklu a, Tuba Tunalı-Akbay b, Go¨zde Erkanlı c, Meral Yu¨ksel d, Feriha Ercan c, Go¨ksel S¸ener a,* a

Marmara University, School of Pharmacy, Department of Pharmacology, Tıbbiye Cad., 34668 Istanbul, Turkey School of Dentistry, Department of Biochemistry, Istanbul, Turkey c School of Medicine, Department of Histology-Embryology, Istanbul, Turkey d Vocational School of Health Related Professions, Istanbul, Turkey b

article info

abstract

Article history:

Background: Despite recent advances, severe burn is one of the most common problems

Accepted 27 October 2006

faced in the emergency room. Major thermal injury induces the activation of an inflammatory cascade resulting in local tissue damage, to contribute to the development of

Keywords:

subsequent damage of multiple organs distant from the original burn wound.

Silymarin

Objective: Silymarin, the major component of milk thistle has been shown to have anti-

Burn

oxidant properties. In the present study, we investigated the putative antioxidant effect of

Skin

local or systemic silymarin treatment on burn-induced oxidative tissue injury.

Thermal

Methods: Wistar albino rats were exposed to 90 8C bath for 10 s to induce burn. Silymarin

Oxidative

either locally (30 mg/kg) applied on 4 cm2 area or locally + systemically (50 mg/kg, p.o.) was

Injury

administered after the burn and repeated twice daily. Rats were decapitated 48 h after injury and blood was collected for tumor necrosis factor-a (TNF-a) and lactate dehydrogenase (LDH) activity. In skin tissue samples malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity, and luminol-lucigenin chemiluminescense (CL) were measured in addition to the histological evaluation. Results: Burn caused a significant increase in TNF-a and LDH levels. MDA levels were increased and GSH levels were decreased in the skin at 48 h after-burn. Both local and systemic silymarin treatments significantly reversed these parameters. The raised MPO activity and luminol-lucigenin CL were also significantly decreased. Conclusion: Results indicate that both systemic and local administration of silymarin was effective against burn-induced oxidative damage and morphological alterations in rat skin. Therefore, silymarin merits consideration as a therapeutic agent in the treatment of burns. # 2006 Elsevier Ltd and ISBI. All rights reserved.

1.

Introduction

Major burn induces the activation of an inflammatory cascade resulting in local tissue damage to contribute to the development of subsequent immunosuppression, increased susceptibility to sepsis and deleterious systemic effects in all the

organ systems distant from the original wound [1,2]. It is well known that the inflammatory response, which leads to hyperactivation of tissue neutrophils contributes to oxidative cell/tissue damage [3,4]. Macrophages are also major producers of pro-inflammatory mediators and their productive capacity for these mediators, such as prostaglandin E2,

* Corresponding author. Tel.: +90 216 414 29 62; fax: +90 216 345 29 52. E-mail addresses: [email protected], [email protected] (G. S¸ener). 0305-4179/$32.00 # 2006 Elsevier Ltd and ISBI. All rights reserved. doi:10.1016/j.burns.2006.10.407

burns 33 (2007) 908–916

reactive nitrogen intermediates, interleukin (IL)-6 and tumor necrosis factor (TNF)-a, is markedly enhanced following burn [4–8]. Thus, it appears that tissue injury after burn is mediated by both reactive oxygen metabolites (ROM) and activated neutrophils and macrophages [9,10]. There are many reports indicating that lipid peroxidation is increased following burn [7,8,11,12]. Lipid peroxidation is an autocatalytic mechanism leading to oxidative destruction of cellular membranes, and their destruction can lead to the production of toxic, reactive metabolites and cell death [13,14]. Furthermore, it was shown that antioxidants or free radical scavengers when given after burn, exert protective effects against burn-induced oxidative tissue damage and multiple organ failure [14–16]. Flavinoids are naturally occurring substances that possess various pharmacological actions and therapeutic applications. Some of these, due to their phenolic structures, have antioxidant effect and inhibit free radical-mediated processes [17]. The extracts of the flowers and leaves of Silybum marianum (St. Mary’s thistle, milk thistle) have been used for centuries to treat liver, spleen and gallbladder disorders. In the 1960s the biologically active molecules of the seed and fruit extracts were isolated, and the chemical structures were elucidated. The isolation led first to a mixture that was named silymarin, and it was this flavonolignan mixture, with that most of the clinical studies were carried out. The main constituents were silibinin, isosilibinin, silicristin, and silidianin [18]. One of the important issues regarding silymarin is that it may be accepted as a safe herbal product, since no health hazards or side effects are known in conjunction with the proper administration of designed therapeutic dosages. Episodes of severe sweating, abdominal cramping, nausea, vomiting, diarrhea and weakness were recently reported in Australia, but the reaction was found to be due to a substance in the milk thistle product other than silibinin [17]. Recently oxidized derivatives of silybin (the major component forming 70–80% of silymarin) and their antiradical and antioxidant activity was studied by Gazak et al. [19]. There are also several studies conducted with silymarin against oxidative stress, inflammatory responses and benzoil peroxide-induced tumor promotion in mice [20–22]. These studies have demonstrated its antioxidant, anti-inflammatory and anticarcinogenic properties [22]. Based on these findings, we investigated the putative protective role of local and systemic (oral) silymarin treatment against burn-induced oxidative damage on the skin.

2.

Materials and methods

All experimental protocols were approved by the Marmara University School of Medicine Animal Care and Use Committee.

2.1.

Animals and the induction of thermal injury

Wistar albino rats of both sexes, weighing 200–250 g, were fasted for 12 h, but allowed free access to water before the experiments. The animals were kept in individual wirebottom cages, in a room at a constant temperature (22  2 8C) with 12-h light and dark cycles, and were fed a

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standard rat chow. The rats were randomly divided into five groups of eight rats (four males and four females) each; control group, vehicle-treated burn groups, local silymarin-treated burn group, and local + oral silymarin-treated burn group. Under brief ether anesthesia, the dorsum of the rats was shaved, and exposed to 90 8C water bath for 10 s, which resulted in partial-thickness second-degree skin burn involving 30% of the total body surface area. All the burned animals were then resuscitated with physiological saline solution (10 ml/kg, subcutaneously, s.c.). In order to rule out the effects of anesthesia, the same protocol was applied in the control group, except that the dorsums were dipped in a 25 8C water bath for 10 s.

2.2.

Silymarin treatment

Silymarin either locally (30 mg/kg i.e. 6 mg silymarin in 200 ml 0.5% methyl cellulose applied on 4 cm2 area) or locally + orally (50 mg/kg suspended in saline) was administered after the injury and repeated twice daily. Vehicle-treated burn groups received either intragastric saline or 0.5% methyl cellulose on their wounds. Since the results of all parameters were not different among both vehicle groups, the data of local and oral vehicle treatments were pooled. The rats were decapitated at 48 h after burn, and trunk blood was collected to measure serum TNF-a and lactate dehydrogenase (LDH) levels. In order to evaluate the presence of oxidant injury, skin tissue samples were taken for the determination of malondialdehyde (MDA) and glutathione (GSH) levels, myelopreoxidase (MPO) activity, luminol and lucigenin chemiluminescence (CL), thromboplastic activity and protein contents. Furthermore tissue samples were examined using both light and scanning electron microscopy (SEM).

2.3.

Biochemical assays

Serum TNF-a levels were quantified according to the manufacturer’s instructions and guidelines using enzyme-linked immunosorbent assay (ELISA) kits specific for the previously mentioned rat cytokines (Biosource International, Nivelles, Belgium). Serum level of LDH was determined spectrophotometrically using an automated analyzer.

2.4.

MDA and GSH assays

Tissue samples were homogenized with ice-cold 150 mM KCl for the determination of MDA and GSH levels. MDA levels were assayed for products of lipid peroxidation by monitoring thiobarbituric acid reactive substance formation as described previously [23]. Lipid peroxidation was expressed in terms of MDA equivalents using an extinction coefficient of 1.56  105 M 1 cm 1 and results are expressed as nmol MDA/g tissue. GSH measurements were performed using a modification of the Ellman procedure [24]. Briefly, after centrifugation at 3000 rpm for 10 min, 0.5 ml of supernatant was added to 2 ml of 0.3 mol/l Na2HPO4
2H2O solution. A 0.2 ml solution of dithiobisnitrobenzoate (0.4 mg/ml 1% sodium citrate) was added and the absorbance at 412 nm was measured immediately after mixing. GSH levels were calculated using an extinction

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burns 33 (2007) 908–916

coefficient of 1.36  105 M mmol GSH/g tissue.

2.5.

1

cm 1. Results are expressed in

MPO activity

MPO activity in tissues was measured by a procedure similar to that described by Hillegas et al. [25]. Samples of skin tissue were homogenized in 50 mM potassium phosphate buffer (PB), with pH 6.0, and centrifuged at 41,400  g for 10 min. The pellets were then suspended in 50 mM PB containing 0.5% hexadecyltrimethylammonium bromide (HETAB). After three freeze and thaw-cycles, with sonication between cycles, the samples were centrifuged at 41,400  g for 10 min. Aliquots (0.3 ml) were added to 2.3 ml of reaction mixture containing 50 mM PB, o-dianisidine, and 20 mM H2O2 solution. One unit of enzyme activity was defined as the amount of MPO present that caused a change in absorbance, measured at 460 nm for 3 min. MPO activity was expressed as U/g tissue.

2.6.

Tissue thromboplastin activity

Thromboplastic activity of skin was evaluated according to Quick’s one stage method using normal plasma [26]. This was performed by mixing 0.1 ml tissue homogenate with 0.1 ml of 0.02 M CaCl2; the clotting reaction was started upon the addition of 0.1 ml of plasma. All reagents were brought to the reaction temperature (37 8C) before mixing. Thromboplastic activity was expressed as seconds.

histologists, who were unaware of the treatments received by the animals. Epithelial degeneration, dermal edema, hair follicular degeneration and vasocongestion were evaluated for the skin tissue. Scores for each criterion are given as 0, none; 1, mild; 2, moderate, 3, severe. Maximum score was 12. At least five microscopic areas were examined to score each specimen. For scanning electron microscopic (SEM) examination, tissue samples were fixed for 2 h in a 2.5% phosphate-buffered glutaraldehyde solution (0.1 M, pH 7.4), postfixed in a 1% phosphate-buffered osmium tetroxide solution, and passed through an increasing alcohol and amyl acetate series. After drying the tissue samples with a Bio-Rad critical point dryer and gold coating with a Bio-Rad SC 502, tissue samples were examined under a Jeol 5200 JSM (Tokyo, Japan) scanning electron microscope.

2.10.

Statistical analysis was done using a GraphPad Prism 3.0 (GraphPad Software, San Diego; CA, USA). All data are expressed as means  S.E.M. Biochemical data were compared with an analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests, while histological data were analyzed with Mann–Whitney non-parametric test. Values of p < 0.05 were considered as significant.

3. 2.7.

Protein electrophoresis

Electrophoretic examination of skin proteins was carried out by Laemmli SDS-polyacrylamide gel electrophoresis [31].

2.9.

Results

Luminol and lucigenin CL assay

To assess the role of reactive oxygen species in burn-induced tissue damage, luminol and lucigenin CL were measured as indicators of radical formation. Measurements were made at room temperature using Junior LB 9509 luminometer (EG&G Berthold, Germany). Specimens were put into vials containing HEPES buffer (0.5 M phosphate buffer containing 20 mM HEPES, pH 7.2). Reactive oxygen species were quantitated after the addition of enhancers such as lucigenin or luminol for a final concentration of 0.2 mM. Luminol is used to detect a group of reactive species, i.e. OH, H2O2 and HOCl, and lucigenin is used for O2 detection [27–29]. Counts were obtained at 1 min intervals and the results were given as the area under curve (AUC) for a counting period of 5 min. Counts were corrected for wet tissue weight (rlu/mg tissue) [30].

2.8.

Statistical analysis

Light and scanning electron microscopic preparation

For light microscopic evaluation skin tissue samples were fixed in 10% formaldehyde and processed routinely for embedding in paraffin. Following dehydration in ascending series of ethyl alcohol, tissue samples were cleared in toluene. Paraffin sections of 5–6 mm thick were stained with hematoxylin and eosin (H&E) to indicate histological degeneration. Microscopic scoring was done by experienced

In the vehicle-treated burn groups, serum TNF-a levels were significantly increased when compared to control group ( p < 0.001). Although the TNF-a levels in the silymarin-treated groups were still significantly higher than that of the control group, both local and local + oral silymarin treatments significantly reduced the raised TNF-a levels ( p < 0.001) (Fig. 1A). Serum LDH activity, as a marker of generalized tissue damage, showed a significant increase in burn group ( p < 0.01), while silymarin administration reversed this effect significantly ( p < 0.05) (Fig. 1B).

3.1.

MDA and GSH levels

The levels of MDA, measured as an index of tissue lipid peroxidation, were significantly higher in the burn group as compared to control group ( p < 0.001; Fig. 2A) while the endogenous antioxidant, GSH levels in the skin tissues of vehicle-treated rats significantly decreased following burn ( p < 0.001; Fig. 2B). However, treatment with silymarin significantly ( p < 0.01–0.001) reversed the MDA and GSH levels.

3.2.

MPO activity

Severe skin scald injury (30% of total body surface area) caused significant increase ( p < 0.001) in MPO activity, as an indicator of tissue neutrophil infiltration, in 48 h-burn group (Fig. 3A) while this increase in skin MPO activity was significantly abolished by both treatment procedures ( p < 0.001).

burns 33 (2007) 908–916

Fig. 1 – Serum (A) TNF-a and (B) LDH levels in the control ‘C’ and vehicle- or silymarin-treated (oral: 50 mg/kg; local: 6 mg/rat) burn groups decapitated at 48 h after burn trauma (n = 8 in each group). *p < 0.05, **p < 0.01, ***p < 0.001: compared with the control ‘C’ group. +p < 0. 05, +++ p < 0.001: compared with the vehicle-treated burn group.

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Fig. 3 – (A) Myeloperoxidase (MPO) and (B) thromboplastin activity in the skin tissues of control ‘C’ and vehicle- or silymarin-treated (oral: 50 mg/kg; local: 6 mg/rat) burn groups decapitated at 48 h after burn trauma (n = 8 in each group). *p < 0.05, **p < 0.01, ***p < 0.001: compared with the control ‘C’ group. +p < 0. 05, ++p < 0.01, +++p < 0.001: compared with the vehicle-treated burn group.

3.3.

Thromboplastin activity

Numerically decreased thromboplastic activity in tissue samples contributes to high thromboplastin levels. Burn groups revealed a significant increase in thromboplastic activity. In the local silymarin treated group skin thromboplastic decreased significantly when compared with the burn group ( p < 0.001) while treatment with local + oral silymarin treatment also showed a slight, but significant decrease in thromboplastic activity when compared to the burn group ( p < 0.05) (Fig. 3B).

3.4.

Luminol and lucigenin chemilumiscence

Luminol and lucigenin CL level showed significant increases in the skin tissues of the burn group compared to control. On the other hand, silymarin treatment to the burn group decreased these values and reversed back to the control levels (Fig. 4).

3.5. Fig. 2 – (A) Malondialdehyde (MDA) and (B) glutathione (GSH) levels in the skin tissues of control ‘C’ and vehicleor silymarin-treated (oral: 50 mg/kg; local: 6 mg/rat) burn groups decapitated at 48 h after burn trauma (n = 8 in each group). **p < 0.01, ***p < 0.001: compared with the control ‘C’ group. ++p < 0.01, +++p < 0.001: compared with the vehicletreated burn group.

Protein electrophoresis

The skin protein bands obtained by SDS polyacrylamide gel electrophoresis were influenced by positions. They were found to differ between control (C) and burn groups. Band potency decreased roughly at 28–30 Da protein at 48 h after burn. Local + oral administration of silymarin increased this response more than local administration (Fig. 5).

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Fig. 4 – (A) Luminol and (B) lucigenin chemilumiscence (CL) levels in the skin tissues of control ‘C’ and vehicle- or silymarin-treated (oral: 50 mg/kg; local: 6 mg/rat) burn groups decapitated at 48 h after burn trauma (n = 8 in each group). ***p < 0.001: compared with the control ‘C’ group. +++ p < 0.001: compared with the vehicle-treated burn group.

3.6.

Histopathological observations

The effects of systemic and local antioxidant treatments on severe burn have been extensively studied [32–36]. The results showed that prevention of local tissue damage is an important issue since skin burn may cause damage to multiple organs distant from the original burn wound and may lead to sepsis and multiple organ failure [7,8,15,37]. Many studies showed that burn is associated with enhanced generation of reactive oxygen metabolites (ROM), which cause lipid peroxidation of the membranes. Although it is difficult to quantitate ROM because of their reactive nature and short lives, CL is a simple but a reproducible technique. The two CL probes, luminol and lucigenin, differ in selectivity. Luminol detects H2O2, OH , hypochloride, peroxynitrite, and lipid peroxyl radicals, whereas lucigenin is particularly sensitive to superoxide radical [27–29]. Our luminol- and lucigenin-enhanced CL data support the notion that skin injury induced by burn involves toxic oxygen metabolites. It is well known that oxygen free radicals can injure lipids, protein, and DNA and thus may contribute to the loss of enzymatic activity or structural integrity. As a free radical generating system, lipid peroxidation is suggested to be closely related with burn-induced tissue damage, and MDA is a good indicator of the degree of lipid peroxidation [7,8]. In this study, increased CL levels support this hypothesis since there was also significant increase in the MDA levels, indicating the increased lipid peroxidation in the skin tissue. Moreover, in the present study silymarin prevented burn-induced free radical generation that causes dramatic increases in lipid peroxidation. In accordance with the increases in oxidant production and lipid peroxidation, GSH levels were decreased. Since GSH is essential for the protection of thiol and other nucleophilic

Burn-induced cellular damage was observed on the skin tissues of burn group (Fig. 6C and D) demonstrating structural degeneration of epidermis and dermis with necrosis, vasocongestion and edema, accompanied by inflammatory cell infiltration. These changes were reversed by either local (Fig. 6E and F) or local + oral silymarin treatment (Fig. 6G and H). Burn groups revealed extreme damage of epidermal hair follicles (Fig. 6D), and this seemed to be normal in silymarin treated groups (Fig. 6F and H). Both local and local + oral administrations of silymarin attenuated burn-induced dermal degeneration, where the increased total damage scores in burn groups (11.50  0.27 and 10.75  0.54) were also reduced significantly ( p < 0.001) in local and local + oral silymarin treated groups (7.25  1.91 and 7.25  1.39, respectively).

4.

Discussion

As evidenced by alterations in MDA and GSH levels, and MPO activity, the results of the present study demonstrate that the burn-induced skin damage is a consequence of oxidative injury, and ameliorated by silymarin treatment. Moreover, morphological changes in the injured skin tissue and thromboplastin activity due to burn trauma were also improved by silymarin treatment. Silymarin also reduced serum levels of LDH and the proinflammatory cytokine TNF-a. These findings suggest that silymarin, appears to have a protective role in the burn-induced oxidative injury of the skin.

Fig. 5 – Electrophoretic pattern of skin samples. Columns: (A) control, (B) burn, (C) burn + local silymarin treatment, and (D) burn + local + oral silymarin treatment.

burns 33 (2007) 908–916

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Fig. 6 – The light microscopic (A, C, E, and G) and scanning electron microscopic (B, D, F, and H) evaluation of the skin tissue. Normal epidermis, hair follicles and dermis in the control group (A and B). Severe degeneration in epidermis (arrow), hair follicles (*) and inflammatory cell infiltration (arrow head) in saline-treated burn groups (C and D). Mild degeneration in epidermis (arrow), hair follicles (*) and inflammatory cell infiltration in burn groups treated with either local (E and F) or local + oral (G and H) silymarin. Original magnifications—A, C, E and G: 100T; insets: 400T; B, D, F, and H: 500T.

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groups in proteins from the toxic oxygen radicals, the concentration of intracellular GSH, therefore, is the key determinant of the extent of burn-induced skin injury [7,8,38]. It was demonstrated that cellular GSH concentration can be influenced by exogenous administration of antioxidants [39,40]. In our study, depletion of GSH stores following burn was restored by silymarin treatment. Considering the mechanism of antioxidant activity, silymarin, which is a wellknown free radical scavenger and a stimulator of several antioxidative enzymes, may have an important role in determining GSH homeostasis within the cell and also determine the total amount of GSH within the cell [41,42]. Thus, modulation of GSH metabolism by silymarin might present a useful adjuvant therapy in the burn trauma. Silymarin is a mixture of bioactive flavonolignans isolated from S. marianum (L.) Gaertn., employed routinely in the treatment of alcoholic liver disease and is used as hepatoprotective agent in humans [17,34]. In a recent study by Ramakrishnan et al. [42], investigating the mechanism underlying the protective effects of silymarin in hepatic carcinogenesis, it was demonstrated that a number of endogenous antioxidants, including GSH, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PD) can be induced by silymarin, and that this chemically mediated upregulation of cellular defenses is accompanied by a markedly increased resistance to hepatic cell injury elicited by reactive oxygen species [42]. Silymarin also reduces the increase of hepatic stellate cells and TGFbeta1 production in the CCl(4)-treated rats suggesting that silymarin prevents hepatic fibrosis through suppression of inflammation and hypoxia in the hepatic fibrogenesis [43]. Moreover, oral silymarin administration has been shown to reduce lipid peroxidation of brain tissues in acetaminopheninduced toxicity by enhancing antioxidant enzymes [41]. On the other hand silymarin improving antioxidant status in blood and liver, positively affects plasma lipoprotein profile in an experimental model of dietary induced hypertriglyceridemia [44]. Both clinical and experimental studies showed that any noxious event is perceived by tissue macrophages and monocytes, which in turn secrete cytokines such as interleukin-1 and TNF-a [45]. As evidenced in the present study, burn resulted in increased serum TNF-a, indicating the role of this cytokine in the burn-induced injury, while silymarin depressed the TNF-a response. Thus, it seems likely that the alleviation of burn-induced oxidative tissue damage by silymarin involves suppression of a variety of pro-inflammatory mediators produced by leukocytes and macrophages. It was demonstrated that some phytochemicals, including silymarin, are known to suppress cancer cell proliferation, inhibit growth factor signaling pathways, induce apoptosis, and inhibit NF-kappaB [46]. Chang et al. who studied the mechanism of the inhibition of NF-kB, demonstrated that silymarin inhibited TNF-alpha-induced calcium-dependent NF-kappaB activation irrespective of its antioxidant effect [47]. However, since proinflammatory cytokines are triggered by ROS, antioxidants may contribute the inhibition of cytokine release by the inhibition of radicals. As shown in our study, silymarin by its antioxidant effects reduced the

ROS generation as assessed by chemiluminescence, and also inhibited TNF-a. Although we did not evaluate the NF-kB, it can be speculated that inhibition of proinflammatory cytokine, TNF-a may cause the inhibition of NF-kB. Furthermore, silymarin was also shown to modulate immune response, by augmenting synthesis of anti-inflammatory cytokines, such as IL-10, IL-12 [48,49]. Besides their direct damaging effects on tissues, ROM trigger the accumulation of leukocytes, which further enhance tissue injury when activated [50]. MPO activity is used as indirect evidence of neutrophil infiltration in oxidant-induced tissue injury. In our study, burn caused significant increase in MPO activity, while silymarin reversed this effect. Increasing evidence suggests that mesengial cells and neutrophils release chemotactic substances, which further promote neutrophil migration to tissues, activating neutrophils and increasing injury [51]. Thus, observations of the present study demonstrate that burn induces tissue injury either directly by promoting oxidative damage and by binding to tissue proteins, or indirectly by stimulating neutrophil infiltration. Cruz et al. [52] who studied the anti-inflammatory effects of silymarin, has shown that silymarin by reducing colonic MPO activity and improving colonic oxidative status protected intestinal tissues in the colitic animals. This suggests that the wellknown antioxidant properties of silymarin participate in its anti-inflammatory activity. Also, the present study, for the first time, investigates the effect of silymarin on skin thromboplastic activity. An increase in thromboplastic activity in skin samples following burn was reversed by both local and local + oral treatment. Thromboplastin, known as tissue factor or Factor III, is an important coagulation factor that initiates extrinsic blood coagulation with FVII. It is not actively found in the blood but it is the cell component of the membranes [53]. It was been shown that some tissues and fluids of the body have thromboplastic activities [54–56]. Thromboplastin is also a thermolabile protein and can easily be affected from the thermal changes. Increased thromboplastic activity in tissue samples contributes to low thromboplastin levels. In the present study thromboplastic activity is increased in burn group. This result supports the thermolabile feature of thromboplastin. On the other hand thromboplastic activity was decreased in rats treated with both local and local + oral silymarin administration. Locally administered silymarin decreased the activity more effectively than locally + orally administered silymarin. The effect of local + oral administration of silymarin on skin thromboplastic activity needs further investigation. In this study we observed that some protein bands disappeared after burn as evidenced with skin protein electrophoresis, while local + oral silymarin treatment changed this appearance. In vitro experiments with isolated cell nuclei and nucleoli, demonstrated that the enzymatic activity of DNA-dependent RNA-polymerase I is stimulated by silymarin. Thus, protein biosynthesis is indirectly intensified. Stimulation of RNA and protein biosynthesis was actually found to result in an increase in the rate of DNA biosynthesis [18]. In conclusion, findings of the present study demonstrated for the first time that both systemic and local administration

burns 33 (2007) 908–916

of silymarin, a phenolic compound, with its potent free radical scavenging and antioxidant properties, reduce the burninduced oxidative skin damage in the rat. Since silymarin may be accepted as a safe herbal product, and no health hazards or side effects are known in conjunction with the proper administration of designed therapeutic dosages [17], it may have a therapeutic value in the treatment of burn.

[15]

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Acknowledgement The authors are grateful to Ozgur Goknel, the Medical Director, in MIKROGEN Pharmaceutical, for supplying silymarin.

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