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Silymarin and its components scavenge phenylglyoxylic ketyl radicals
Fitoterapia 77 (2006) 525 – 529 www.elsevier.com/locate/fitote
Silymarin and its components scavenge phenylglyoxylic ketyl radicals František Šeršeň a,⁎, Tomaš Vencel a , Julius Annus b b
a Comenius University in Bratislava, Faculty of Natural Sciences, Institute of Chemistry, 842 15 Bratislava, Slovakia Slovak University of Technology in Bratislava, Faculty of Chemical and Food Technology, Department of Chemical Physics, Radlinského 9, 812 37 Bratislava, Slovakia
Received 27 January 2005; accepted 5 June 2006 Available online 6 July 2006
Abstract The antioxidant properties of silymarin and its flavanolignan components (silybin, silychristin and silydianin) were tested. Silymarin, silychristin and silydianin exhibit relatively good antioxidant effectiveness against phenylglyoxylic ketyl radicals and DPPH. The most effective scavengers of phenylglyoxylic ketyl radicals were silymarin and silychristin whereas silydianin was ca. 5-times less active than the first two compounds whereas silybin was ineffective. The scavenging properties of the studied compounds against DPPH radicals were in the same sequence: silymarin N silychristin N silydianin N silybin. © 2006 Elsevier B.V. All rights reserved. Keywords: Antioxidant activity; Silybin; Silychristin; Silydianin; Spin trapping
1. Introduction Silymarin is a purified extract of the seeds of Silybum marianum L., also called "milk thistle“. Silymarin consists of 70–80% of flavanolignans (silybin, isosilybin, silydianin and silychristin) and 20–30% of polyphenolic compounds [1,2]. Silymarin can help prevent or reverse liver damage caused by alcohol, recreational drugs, pesticides and some poisons. Silymarin has been used also for the treatment of poisoning by certain types of mushrooms. Some medicines used against HIV can damage the liver and silymarin might help prevent liver damage. Several anti-HIV drugs can cause stomach problems, and silymarin can help treat indigestion. No side effects of silymarin have been documented. Even very high doses do not seem to have any negative effects. However, some people get an upset stomach or have more gas when they start to use silymarin [3]. Silymarin has protective effects in the early phase of allergic asthma [4] and against stress-induced gastric ulcer [5]. Other results have shown anticarcinogenic (especially against prostate cancer) [6] and cancer chemopreventive effects [7]. Silymarin and its flavanolignan components exhibit also U antioxidant properties [8]. It was found that silybin dihemisuccinate is not a good scavenger of O2− and gives no U reaction with H2O2 but it rapidly reacts with HO radicals [9]. ⁎ Corresponding author. E-mail address: [email protected] (F. Šeršeň). 0367-326X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2006.06.005
526
F. Šeršeň et al. / Fitoterapia 77 (2006) 525–529 U
Several other authors found that silybin reacted with HO radicals [10,11]. The complex of silybin with phosphatidylcholine decreases the spin trapping of hydroxyethyl radicals in microsomes from chronic alcohol-fed rats [11,12]. Velussi et al. reported that silymarin might reduce the lipoperoxidation of cell membranes [13]. The goal of this work is to determine the effectiveness of silymarin and its flavanolignan components to scavenge phenylglyoxylic ketyl radicals. 2. Experimental 2.1. General Silymarin was a kind gift from Prof. Šimánek (Palacky University in Olomouc, Czech Republic). Silymarin contained 36.3% of silybin, 5.1% of isosilybin, 5.9% of silydianin, 15.7% of silychristin, and 1.9% of taxifolin. Silybin, silychristin and silydianin were prepared according to the procedure described by Škottová et al. [2]. D,L-2,3-diphenyltartaric acid (DPTA) was prepared as reported in Ref. [14]. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), N-tert-butyl-α-phenylnitrone (PBN), propan-2-ol (iPrOH) and methanol (MeOH) were purchased from Sigma-Aldrich Chemie GmbH. 2.2. Generation of phenylglyoxylic ketyl radicals As a source of phenylglyoxylic ketyl radicals DPTA was used, which in iPrOH undergoes spontaneous decomposition according to the reaction in Scheme 1 [14,15]. In the first phase α-carbonyl-α-hydroxybenzyl (phenylglyoxylic ketyl) radicals are formed, which then give rise to benzoyl radicals. The lifetime of these radicals is relatively short (the decay rate constant of phenylglyoxylic ketyl radicals in iPrOH is about 108 mol− 1 s− 1) [16] and they can be stabilized as spin adducts with the spin trap PBN. The lifetime of these spin adducts is longer (some tens of minutes) and therefore they can be easily recorded by a continual wave EPR spectrometer. 2.3. Scavenging of phenylglyoxilic ketyl radicals The EPR spectra of the iPrOH solution, which contained 0.025 mol dm− 3 DPTA, 0.05 mol dm− 3 PBN (control sample) and desired amounts (0.5–50 g dm− 3) of silymarin compounds were recorded. The ability of the studied compounds to scavenge phenylglyoxylic ketyl radicals was determined by searching for the lowest concentration of studied compounds, at which no EPR signal was registered. 2.4. Scavenging of DPPH radicals The effectiveness of silymarin and its flavanolignan components to scavenge DPPH radicals was carried out by EPR spectroscopy by the following procedure: into 10− 4 mol dm− 3 MeOH solution of DPPH were added desired amounts of the studied compounds and after 15 min EPR spectra were recorded. From dependencies of EPR signal intensity upon concentration SC50 values (i.e. the concentration of the studied compound which causes 50% decrease of EPR signal intensity) were calculated. 2.5. Used instruments The spectra of electron paramagnetic resonance (EPR) were recorded by the equipment ERS 230 (ZWG Akad. Wiss. Berlin, Germany), which operates in X-band (∼ 9.3 GHz) at modulation amplitude 0.1 mT and microwave power 5 mW. UV–VIS spectra were measured using diode array spectrophotometer HP 8452A. All experiments were carried out at 22 °C.
Scheme 1.
F. Šeršeň et al. / Fitoterapia 77 (2006) 525–529
527
3. Results 3.1. Generation of phenylglyoxylic ketyl radicals In Fig. 1 are presented the EPR spectra of PBN spin adducts of the spontaneous decomposition of D,L-2,3diphenyltartaric acid in iPrOH. Fig. 1A shows the EPR spectrum of iPrOH solution of 0.025 mol dm− 3 DPTA, which contains 0.05 mol dm− 3 PBN registered 5 min after their mixture. This EPR spectrum consists of three doublets, which are the result of the hyperfine interaction of unpaired electron with the nitrogen (14N) nucleus spin I = 1 (triplet) and with the hydrogen (1H) nucleus spin I = ½ (doublets). Thus, there are 6 lines of hyperfine structure with splitting constants AN = 1.4 mT and AH = 0.24 mT. The intensity of this signal increases in time and reaches maximum after ca. 100 min (Fig. 1C). Approximately after 50 min in EPR spectra appears a new signal, which is manifested at first as an unequal line intensity of doublets and as a shoulder on the first and the last doublets (in Fig. 1B denoted by asterisk). The line intensity of the first sextet after 100 min begins to decrease whereas the intensity of the new signal increases. After 120 min it is possible to observe the new sextet of lines with these splitting constants: AN = 1.4 mT and AH = 0.36 mT (Fig. 1D, E). After 140 min no lines in the EPR spectra could be observed. The rate of decomposition of DPTA was lower as it was observed in our previous work [14]. The slower decomposition is caused probably by the lower temperature (22 °C) at which the present experiments were carried out compared to the temperature in former ones (25 °C).
Fig. 1. The EPR spectra of the solution 0.025 mol dm− 3 D,L-2,3-diphenyltartaric acid and 0.05 mol dm− 3 PBN in iPrOH 5 min (A), 50 min (B), 100 min (C), 120 min (D) and 130 min (E) after mixing. Spectra of B and C were registered at half of amplification as spectra A, D and E.
F. Šeršeň et al. / Fitoterapia 77 (2006) 525–529
528 Table 1 SC50 values of scavenging of DPPH radicals Samples
Silychristin
Silydianin
Silybin
Silymarin
Vitamin C
SC50 (μg/ml) r2
51.0 0.93
60.8 0.87
156.2 0.96
50.4 0.91
1.1 0.96
The values SC50 were calculated from 3 parallel experiments. r2 = values of the standard deviation.
Table 2 The smallest concentrations (CS) of the studied compounds in propan-2-ol solution of DPTA with PBN where no EPR signals were observed Sample
Silychristin
Silydianin
Silybin
Silymarin
Vitamin C
CS (mg/ml)
9.5 ± 1.4
48.1 ± 9.6
Inactive
9.4 ± 1.2
0.22 ± 0.035
The values were calculated from 3 parallel experiments.
3.2. DPPH test The ability of silymarin and its flavanolignan components to scavenge DPPH radicals was accompanied by a decrease of EPR signal intensity of DPPH radicals in methanol solution. From the dependencies of the signal intensity upon concentration SC50 values were calculated. The ascertained SC50 values of silymarin and its flavanolignan components as well as of vitamin C are presented in Table 1. 3.3. Test with phenylglyoxylic ketyl radicals After adding 50 mg of silymarin, silychristin and silydianin into 1 ml of the iPrOH solution with 0.025 mol dm− 3 of DPTA and 0.05 mol dm− 3 of PBN no EPR signals were observed. On the other hand, at the same concentration of silybin in the above mentioned solution the same EPR signals were observed in the control sample, whose kinetics exhibited similar character as the control sample. In the next investigation we were looking for the smallest concentration of silymarin or its flavanolignan components, where no EPR signal in the measured samples was registered. The values of such concentrations of the studied compounds are presented in Table 2. 4. Discussion The splitting constant values for spin adducts of PBN with various radicals are published in the internet database of the National Institute of Environmental Health Sciences [17]. Comparing the splitting constant values of the first EPR signal (Fig. 1A, B), which was registered in the control sample (i.e. the DPTA solution in iPrOH) with those published in the above mentioned database we suggest that this signal belongs to the PBN adduct with phenylglyoxylic ketyl radical. In a similar way, comparing the splitting constants of the second radical (AN = 1.4 mT and AH = 0.36 mT), for which the EPR spectrum is documented in Fig. 1D and E with those published in the above mentioned database we suggest that the observed adduct belongs to benzoyl radical, with splitting constants AN = 1.4 mT and AH = 0.36–0.438 mT depending on the used solvent [17]. It was found by EPR spectroscopy that silymarin and its flavanolignan components exhibit antioxidant properties. From Table 1, it is evident that the most effective scavengers of DPPH radicals are silymarin and silychristin, the less effective is silydianin and silybin is a soft scavenger. It was found that vitamin C is ca. 50-times more effective than silymarin, silychristin and silydianin and more than 142-times than silybin. Our calculated SC50 values for silymarin and vitamin C (50.4 or 1.1 μg/ml) are relatively in good accordance with those (33.8 or 1.5 μg/ml) found by Gerhäuser et al. [18]. However, our SC50 value for silybin (156.2 μg/ml) is lesser than that (841 μg/ml) found by Gažák et al. [19]. This discrepancy could be caused by different detection methods (EPR was used in our experiments and absorption spectrophotometry was used by Gažák et al.). Silymarin and silychristin are rather good scavengers of phenylglyoxylic ketyl radicals (Table 2). The same sequence of antioxidant effectiveness as for DPPH scavenging was observed also for scavenging of phenylglyoxylic
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ketyl radicals, i.e. vitamin C was ca. 43-times more effective than silymarin and silychristin, 218-times more effective than silydianin, whereas silybin was ineffective. In order to find if silymarin and its flavanolignan components inhibit the spontaneous decomposition of DPTA in iPrOH or if they interact with radical intermediates of this decomposition, an experiment by UV–VIS spectroscopy was carried out. In our previous work [15] it was found that the HU radical, which is separated from the central carbon atom of iPrOH, initiates this spontaneous decomposition of DPTA. The spontaneous DPTA decomposition is accompanied by the changes in the UV–VIS spectra, which is displayed by increasing of the absorption at 290 nm [15]. It was found that increasing of this absorption was observed also in the presence of silymarin or the flavanolignan components (not documented). From this result we assume that the spontaneous DPTA decomposition in iPrOH takes place also in the presence of silymarin or its flavanolignan components. Some ketyl radicals take place in living organisms in certain enzymatic reactions as reactive intermediates, e.g. during metabolism of xenobiotics by cytochromes P450 but also during dehydration of both endogenous and exogenous substrates by certain dehydratases [20]. Therefore, it is very important to know compounds able to perform their scavenging. The herein presented method of determining the antioxidant properties of silymarin constituents in iPrOHsolution with ε = 18 [21] can mimic surroundings occurring somewhere between the center interfacial (ε = 30) and hydrophobic parts of biological membrane [22]. Acknowledgement This work was supported by research grant of the Scientific Grant Agency of the Ministry of Education of Slovak Republic, registration number 1/0516/03. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]
Šimánek V, Walterová D, Vičar J, Urbaníková J, Křen V, Modrienský M, et al. Ceskoslov Farm 2001;50:66 [In Czech]. Škottová N, Večeřa R, Urbánek K, Váňa P, Walterová D, Cvak L. Pharmacol Res 2003;47:17. Murphy JM, Caban M, Kemper KJ. Milk Thistle (Silybum marianum). http://www.mcp.edu/herbal/milkthistle/milkthistle.pdf 2001. Breschi MC, Martinotti E, Apostoliti F, Nieri P. Eur J Pharmacol 2002;437:9. Alarcon De La Lastra C, Martin MJ, Marhuenda E. J Pharm Pharmacol 1992;44:929. Zi XL, Grasso AW, Kung HJ, Agarwal R. Cancer Res 1998;58:1920. Bhatia N, Zhao J, Douglas M, Wolf DM, Agarwal R. Cancer Lett 1999;147:77. Kvasnička F, Bíba B, Ševčík R, Voldřich M, Krátká J. J Chromatogr A 2003;990:239. Mira L, Silva M, Manso CF. Biochem Pharmacol 1994;48:753. György I, Antus S, Foldiak G. Radiat Phys Chem 1992;39:81. Basaga H, Poli G, Tekkaya C, Aras I. Cell Biochem Funct 1997;15:27. Comoglio A, Tomasi A, Malandrino S, Poli G, Albano E. Biochem Pharmacol 1995;50:1313. Velussi M, Cernigoi AM, De Monte A, Dapas F, Caffau C, Zilli M. J Hepatol 1997;26:871. Šeršeň F, Vencel T, Gáplovský A, Anus T. Ceskoslov Farm 2004;53:141 [In Slovak]. Vencel T, Gáplovská K, Gáplovský A, Toma Š, Šeršeň F. Photochem Photobiol 2004;162:53. Fujisawa T, Hammond GS. J Am Chem Soc 1972;94:4175. Li ASW, Cummings KB, RoethLing HP, Buettner GR, Chignell CF. A Spin Trapping Database Implemented on the IBM PC/AT. J Magn Reson 1988;79:140 The database is available through internet at http://epr.niehs.nih.gov/. Gerhäuser C, Klimo K, Heiss E, Neumann I, Gamal-Eldeen A, Knauft J, et al. Mutat Res 2003;523:163. Gažák R, Svobodová A, Psotová J, Sedmera P, Přikrylová V, Walterová D, et al. Bioorg Med Chem 2004;12:5677. Buckel W. FEBS Lett 1996;389:20. Janz GJ, Tomkins RPT. Nonaqueous electrolytes handbook, vol. I. New York: Academic Press; 1972. p. 59. Cevc G, Sedon JM. Physical characterization. In: Cevc G, editor. Phospholipids handbook. New York: Marcel Dekker; 1993. p. 351.
Silymarin and its components scavenge phenylglyoxylic ketyl radicals František Šeršeň a,⁎, Tomaš Vencel a , Julius Annus b b
a Comenius University in Bratislava, Faculty of Natural Sciences, Institute of Chemistry, 842 15 Bratislava, Slovakia Slovak University of Technology in Bratislava, Faculty of Chemical and Food Technology, Department of Chemical Physics, Radlinského 9, 812 37 Bratislava, Slovakia
Received 27 January 2005; accepted 5 June 2006 Available online 6 July 2006
Abstract The antioxidant properties of silymarin and its flavanolignan components (silybin, silychristin and silydianin) were tested. Silymarin, silychristin and silydianin exhibit relatively good antioxidant effectiveness against phenylglyoxylic ketyl radicals and DPPH. The most effective scavengers of phenylglyoxylic ketyl radicals were silymarin and silychristin whereas silydianin was ca. 5-times less active than the first two compounds whereas silybin was ineffective. The scavenging properties of the studied compounds against DPPH radicals were in the same sequence: silymarin N silychristin N silydianin N silybin. © 2006 Elsevier B.V. All rights reserved. Keywords: Antioxidant activity; Silybin; Silychristin; Silydianin; Spin trapping
1. Introduction Silymarin is a purified extract of the seeds of Silybum marianum L., also called "milk thistle“. Silymarin consists of 70–80% of flavanolignans (silybin, isosilybin, silydianin and silychristin) and 20–30% of polyphenolic compounds [1,2]. Silymarin can help prevent or reverse liver damage caused by alcohol, recreational drugs, pesticides and some poisons. Silymarin has been used also for the treatment of poisoning by certain types of mushrooms. Some medicines used against HIV can damage the liver and silymarin might help prevent liver damage. Several anti-HIV drugs can cause stomach problems, and silymarin can help treat indigestion. No side effects of silymarin have been documented. Even very high doses do not seem to have any negative effects. However, some people get an upset stomach or have more gas when they start to use silymarin [3]. Silymarin has protective effects in the early phase of allergic asthma [4] and against stress-induced gastric ulcer [5]. Other results have shown anticarcinogenic (especially against prostate cancer) [6] and cancer chemopreventive effects [7]. Silymarin and its flavanolignan components exhibit also U antioxidant properties [8]. It was found that silybin dihemisuccinate is not a good scavenger of O2− and gives no U reaction with H2O2 but it rapidly reacts with HO radicals [9]. ⁎ Corresponding author. E-mail address: [email protected] (F. Šeršeň). 0367-326X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2006.06.005
526
F. Šeršeň et al. / Fitoterapia 77 (2006) 525–529 U
Several other authors found that silybin reacted with HO radicals [10,11]. The complex of silybin with phosphatidylcholine decreases the spin trapping of hydroxyethyl radicals in microsomes from chronic alcohol-fed rats [11,12]. Velussi et al. reported that silymarin might reduce the lipoperoxidation of cell membranes [13]. The goal of this work is to determine the effectiveness of silymarin and its flavanolignan components to scavenge phenylglyoxylic ketyl radicals. 2. Experimental 2.1. General Silymarin was a kind gift from Prof. Šimánek (Palacky University in Olomouc, Czech Republic). Silymarin contained 36.3% of silybin, 5.1% of isosilybin, 5.9% of silydianin, 15.7% of silychristin, and 1.9% of taxifolin. Silybin, silychristin and silydianin were prepared according to the procedure described by Škottová et al. [2]. D,L-2,3-diphenyltartaric acid (DPTA) was prepared as reported in Ref. [14]. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), N-tert-butyl-α-phenylnitrone (PBN), propan-2-ol (iPrOH) and methanol (MeOH) were purchased from Sigma-Aldrich Chemie GmbH. 2.2. Generation of phenylglyoxylic ketyl radicals As a source of phenylglyoxylic ketyl radicals DPTA was used, which in iPrOH undergoes spontaneous decomposition according to the reaction in Scheme 1 [14,15]. In the first phase α-carbonyl-α-hydroxybenzyl (phenylglyoxylic ketyl) radicals are formed, which then give rise to benzoyl radicals. The lifetime of these radicals is relatively short (the decay rate constant of phenylglyoxylic ketyl radicals in iPrOH is about 108 mol− 1 s− 1) [16] and they can be stabilized as spin adducts with the spin trap PBN. The lifetime of these spin adducts is longer (some tens of minutes) and therefore they can be easily recorded by a continual wave EPR spectrometer. 2.3. Scavenging of phenylglyoxilic ketyl radicals The EPR spectra of the iPrOH solution, which contained 0.025 mol dm− 3 DPTA, 0.05 mol dm− 3 PBN (control sample) and desired amounts (0.5–50 g dm− 3) of silymarin compounds were recorded. The ability of the studied compounds to scavenge phenylglyoxylic ketyl radicals was determined by searching for the lowest concentration of studied compounds, at which no EPR signal was registered. 2.4. Scavenging of DPPH radicals The effectiveness of silymarin and its flavanolignan components to scavenge DPPH radicals was carried out by EPR spectroscopy by the following procedure: into 10− 4 mol dm− 3 MeOH solution of DPPH were added desired amounts of the studied compounds and after 15 min EPR spectra were recorded. From dependencies of EPR signal intensity upon concentration SC50 values (i.e. the concentration of the studied compound which causes 50% decrease of EPR signal intensity) were calculated. 2.5. Used instruments The spectra of electron paramagnetic resonance (EPR) were recorded by the equipment ERS 230 (ZWG Akad. Wiss. Berlin, Germany), which operates in X-band (∼ 9.3 GHz) at modulation amplitude 0.1 mT and microwave power 5 mW. UV–VIS spectra were measured using diode array spectrophotometer HP 8452A. All experiments were carried out at 22 °C.
Scheme 1.
F. Šeršeň et al. / Fitoterapia 77 (2006) 525–529
527
3. Results 3.1. Generation of phenylglyoxylic ketyl radicals In Fig. 1 are presented the EPR spectra of PBN spin adducts of the spontaneous decomposition of D,L-2,3diphenyltartaric acid in iPrOH. Fig. 1A shows the EPR spectrum of iPrOH solution of 0.025 mol dm− 3 DPTA, which contains 0.05 mol dm− 3 PBN registered 5 min after their mixture. This EPR spectrum consists of three doublets, which are the result of the hyperfine interaction of unpaired electron with the nitrogen (14N) nucleus spin I = 1 (triplet) and with the hydrogen (1H) nucleus spin I = ½ (doublets). Thus, there are 6 lines of hyperfine structure with splitting constants AN = 1.4 mT and AH = 0.24 mT. The intensity of this signal increases in time and reaches maximum after ca. 100 min (Fig. 1C). Approximately after 50 min in EPR spectra appears a new signal, which is manifested at first as an unequal line intensity of doublets and as a shoulder on the first and the last doublets (in Fig. 1B denoted by asterisk). The line intensity of the first sextet after 100 min begins to decrease whereas the intensity of the new signal increases. After 120 min it is possible to observe the new sextet of lines with these splitting constants: AN = 1.4 mT and AH = 0.36 mT (Fig. 1D, E). After 140 min no lines in the EPR spectra could be observed. The rate of decomposition of DPTA was lower as it was observed in our previous work [14]. The slower decomposition is caused probably by the lower temperature (22 °C) at which the present experiments were carried out compared to the temperature in former ones (25 °C).
Fig. 1. The EPR spectra of the solution 0.025 mol dm− 3 D,L-2,3-diphenyltartaric acid and 0.05 mol dm− 3 PBN in iPrOH 5 min (A), 50 min (B), 100 min (C), 120 min (D) and 130 min (E) after mixing. Spectra of B and C were registered at half of amplification as spectra A, D and E.
F. Šeršeň et al. / Fitoterapia 77 (2006) 525–529
528 Table 1 SC50 values of scavenging of DPPH radicals Samples
Silychristin
Silydianin
Silybin
Silymarin
Vitamin C
SC50 (μg/ml) r2
51.0 0.93
60.8 0.87
156.2 0.96
50.4 0.91
1.1 0.96
The values SC50 were calculated from 3 parallel experiments. r2 = values of the standard deviation.
Table 2 The smallest concentrations (CS) of the studied compounds in propan-2-ol solution of DPTA with PBN where no EPR signals were observed Sample
Silychristin
Silydianin
Silybin
Silymarin
Vitamin C
CS (mg/ml)
9.5 ± 1.4
48.1 ± 9.6
Inactive
9.4 ± 1.2
0.22 ± 0.035
The values were calculated from 3 parallel experiments.
3.2. DPPH test The ability of silymarin and its flavanolignan components to scavenge DPPH radicals was accompanied by a decrease of EPR signal intensity of DPPH radicals in methanol solution. From the dependencies of the signal intensity upon concentration SC50 values were calculated. The ascertained SC50 values of silymarin and its flavanolignan components as well as of vitamin C are presented in Table 1. 3.3. Test with phenylglyoxylic ketyl radicals After adding 50 mg of silymarin, silychristin and silydianin into 1 ml of the iPrOH solution with 0.025 mol dm− 3 of DPTA and 0.05 mol dm− 3 of PBN no EPR signals were observed. On the other hand, at the same concentration of silybin in the above mentioned solution the same EPR signals were observed in the control sample, whose kinetics exhibited similar character as the control sample. In the next investigation we were looking for the smallest concentration of silymarin or its flavanolignan components, where no EPR signal in the measured samples was registered. The values of such concentrations of the studied compounds are presented in Table 2. 4. Discussion The splitting constant values for spin adducts of PBN with various radicals are published in the internet database of the National Institute of Environmental Health Sciences [17]. Comparing the splitting constant values of the first EPR signal (Fig. 1A, B), which was registered in the control sample (i.e. the DPTA solution in iPrOH) with those published in the above mentioned database we suggest that this signal belongs to the PBN adduct with phenylglyoxylic ketyl radical. In a similar way, comparing the splitting constants of the second radical (AN = 1.4 mT and AH = 0.36 mT), for which the EPR spectrum is documented in Fig. 1D and E with those published in the above mentioned database we suggest that the observed adduct belongs to benzoyl radical, with splitting constants AN = 1.4 mT and AH = 0.36–0.438 mT depending on the used solvent [17]. It was found by EPR spectroscopy that silymarin and its flavanolignan components exhibit antioxidant properties. From Table 1, it is evident that the most effective scavengers of DPPH radicals are silymarin and silychristin, the less effective is silydianin and silybin is a soft scavenger. It was found that vitamin C is ca. 50-times more effective than silymarin, silychristin and silydianin and more than 142-times than silybin. Our calculated SC50 values for silymarin and vitamin C (50.4 or 1.1 μg/ml) are relatively in good accordance with those (33.8 or 1.5 μg/ml) found by Gerhäuser et al. [18]. However, our SC50 value for silybin (156.2 μg/ml) is lesser than that (841 μg/ml) found by Gažák et al. [19]. This discrepancy could be caused by different detection methods (EPR was used in our experiments and absorption spectrophotometry was used by Gažák et al.). Silymarin and silychristin are rather good scavengers of phenylglyoxylic ketyl radicals (Table 2). The same sequence of antioxidant effectiveness as for DPPH scavenging was observed also for scavenging of phenylglyoxylic
F. Šeršeň et al. / Fitoterapia 77 (2006) 525–529
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ketyl radicals, i.e. vitamin C was ca. 43-times more effective than silymarin and silychristin, 218-times more effective than silydianin, whereas silybin was ineffective. In order to find if silymarin and its flavanolignan components inhibit the spontaneous decomposition of DPTA in iPrOH or if they interact with radical intermediates of this decomposition, an experiment by UV–VIS spectroscopy was carried out. In our previous work [15] it was found that the HU radical, which is separated from the central carbon atom of iPrOH, initiates this spontaneous decomposition of DPTA. The spontaneous DPTA decomposition is accompanied by the changes in the UV–VIS spectra, which is displayed by increasing of the absorption at 290 nm [15]. It was found that increasing of this absorption was observed also in the presence of silymarin or the flavanolignan components (not documented). From this result we assume that the spontaneous DPTA decomposition in iPrOH takes place also in the presence of silymarin or its flavanolignan components. Some ketyl radicals take place in living organisms in certain enzymatic reactions as reactive intermediates, e.g. during metabolism of xenobiotics by cytochromes P450 but also during dehydration of both endogenous and exogenous substrates by certain dehydratases [20]. Therefore, it is very important to know compounds able to perform their scavenging. The herein presented method of determining the antioxidant properties of silymarin constituents in iPrOHsolution with ε = 18 [21] can mimic surroundings occurring somewhere between the center interfacial (ε = 30) and hydrophobic parts of biological membrane [22]. Acknowledgement This work was supported by research grant of the Scientific Grant Agency of the Ministry of Education of Slovak Republic, registration number 1/0516/03. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]
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