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Electron spin resonance study of free radicals formed from a procyanidin-rich pine (Pinus maritima) bark extract, pycnogenol
Free Radical Biology & Medicine, Vol. 27, Nos. 11/12, pp. 1308 –1312, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter
PII S0891-5849(99)00168-9
Original Contribution ELECTRON SPIN RESONANCE STUDY OF FREE RADICALS FORMED FROM A PROCYANIDIN-RICH PINE (PINUS MARITIMA) BARK EXTRACT, PYCNOGENOL QIONG GUO,* BAOLU ZHAO,†
and
LESTER PACKER*
*Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA and †Department of Cell Biophysics, Institute of Biophysics, Academia Sinica, Beijing, China (Received 6 April 1999; Revised 6 August 1999; Accepted 10 August 1999)
Abstract—The free radical generated from the oxidation of a French maritima pine bark extract Pycnogenol (PYC), by the horseradish peroxidase (HRP)– hydrogen peroxide (H2O2) system at pH 7.4 –10.0 was studied using electron spin resonance (ESR) spectrometer. The formation rate of the PYC radical (aH ⫽ 0.92G; g ⫽ 2.0055) was dependent on the PYC and HRP concentrations and pH; the lifetime of the radical was up to 90 min. Furthermore, it was found that the PYC radical was mainly composed of the secondary radical formed from procyanidin B3, one of major procyanidins in PYC. The primary radical signal of procyanidin B3 with hyperfine splitting constants aH ⫽ 3.67 G (1H), aH ⫽ 0.92 G (3H), and g ⫽ 2.0055 was transient and disappeared quickly, whereas its secondary radical signal appeared and increased with time. The secondary radical from dimer procyanidin B3 showed quite high stability, differing from the radical from monomer (⫹)-catechin that could not be observed possibly because of its instability. These results provide evidence to support the idea that the intramolecular hydrogen bond between the O• at the 4⬘ position in one B ring and an OH group in the other B ring of procyanidin B3 is formed during its oxidation in the presence of HRP and H2O2. © 1999 Elsevier Science Inc. Keywords—Electron spin resonance, Pycnogenol, Horseradish peroxidase, Procyanidin B3, Intramolecular hydrogen bond, Free radicals
INTRODUCTION
There has recently been growing interest in the biological properties of PYC. It has been reported that PYC protects the low-density fraction of human plasma lipoproteins against copper-induced oxidation and protects DNA from iron/ascorbate-induced damage [3]. Moreover, PYC can protect cellular systems by increasing the activities of endogenous antioxidant enzymes such as glutathione (GSH) redox enzymes (GSH reductase and GSH peroxidase), superoxide dismutase, and catalase [4] and by increasing levels of endogenous antioxidants such as GSH [4,5] and ␣-tocopherol [2]. PYC has also been demonstrated to be an efficient scavenger of reactive oxygen species such as superoxide radical anion [6 – 8], hydroxyl radical [9,10], and nitric oxide radical [9] as well as the stable radical 1,1 diphenyl-2-picrylhydrazyl (DPPH) [11,12]. In addition to acting as a free radical scavenger, PYC has been recently reported to reduce cytochrome c reversibly, possibly by donation of electrons to the iron of the heme group and competitively inhibit mitochondrial electron transport chain activity
Isolated from the bark of the French marine pine (Pinus maritima), pine bark extract, Pycnogenol (PYC) (Horphag Research, Geneva, Switzerland), is a unique mixture of bioflavonoids, which are broadly divided into monomers ((⫹)-catechin and taxifolin; about 8% of the weight) and mainly condensed flavonoids (85% of the weight) classified as procyanidins. These condensed polyphenols are mainly constituted by “bricks” of the flavan-3-ols catechin and epicatechin, linked together, from dimers (Fig. 1) up to heptamers. PYC also contains phenolic acids (such as caffeic, ferulic, and p-hydroxybenzoic acids) as minor constituents and glycosylation products, that is, glucopyranose derivatives of either flavonols or phenolic acids as lesser constituents [1,2]. Address correspondence to: Lester Packer, Department of Molecular and Cell Biology, University of California Berkeley, 251 Life Sciences Addition, Berkeley, CA 94720-3200, USA; Tel.: (510) 642-1872; Fax: (510) 642-8313; E-Mail: [email protected]. 1308
Pine bark extract free radical
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Fig. 1. Structures of oligomeric procyanidins, the major constituents of PYC.
[13]. However, the free radical formed from PYC after it is oxidized has not been studied. It has been reported that horseradish peroxidase (HRP) can react with H2O2 and generate intermediates that oxidize a large variety of compounds, including phospholipids [14] and low-density lipoprotein [15]. In particular, the HRPH2O2 system has been used to generate free radicals of some phenolic compounds [16,17] and GSH [18]. Thus the aim of the present study was to investigate the production of the free radical formed from oxidation of PYC in the HRP-H2O2 system and clarify which components of PYC will contribute to the radical signal using a direct electron spin resonance (ESR) technique. MATERIALS AND METHODS
Reagents HRP (EC 1.1 1.1.7, type VI, 250 –330 units/mg) and procyanidin B3 were obtained from Sigma (St. Louis, MO, USA). Hydrogen peroxide was from Fisher Scientific (Fair Lawn, NJ, USA). PYC was a gift from Horphag Research. Procyanidin B1, B2, C1, and C2 were gifts from Tokyo Research Laboratories, Kyowa Hakko Kogyo (Machida, Japan).
ESR detection of PYC and procyanidin B3 radicals Enzymatic oxidation of PYC or procyanidin B3 was carried out with HRP and H2O2. A typical incubation mixture for ESR measurement consisted of PYC (100 – 500 g/ml) or procyanidin B3 (400 g/ml), H2O2 (1.2 mM), and HRP (0.15 mg/ml) in 100 mM sodium carbonate buffer (pH 9.5). The reaction mixture was then immediately introduced into a quartz capillary and fitted into a flat cell, and ESR spectra were recorded with time using an IBM ER 200D-SRC ESR spectrometer (Danbury, CT, USA), except for the results shown in Fig. 2 (inset) and Fig. 4 where the Free Radical Monitor (JESFR30, JEOL, Tokyo, Japan) was used. Instrument conditions were as follows: central field, 3475 G; modulation frequency, 100 kHz; modulation amplitude, 2.0 G; microwave power, 10 mW; scan width, 100 G; gain, 5.0 ⫻ 105; temperature, 295 K. RESULTS
The PYC radical was formed on addition of HRP (0.15 mg/ml) and H2O2 (1.2 mM) to sodium carbonate buffer (pH 9.5) containing 250-g/ml PYC (Fig. 2,
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Fig. 2. Effect of PYC concentration on the time-dependent formation of the PYC radical generated by the HRP-H2O2 system over time. The reaction mixtures contained HRP (0.15 mg/ml), H2O2 (1.2 mM), and the indicated concentrations of PYC in 100-mM carbonate buffer (pH 9.5). ESR measurement conditions are those described in Materials and Methods. Inset: an ESR spectrum of the PYC radical. ESR measurement conditions: modulation frequency, 9.41 GHz; modulation amplitude, 0.63 G; microwave power, 4.0 mW; gain, 100; scan width, 25 G; temperature, 295 K.
inset). The radical signal mainly contains a six-line spectrum with a hyperfine splitting constant aH ⫽ 0.92 G and g ⫽ 2.0055 and a broadening peak. In fact, the hyperfine splitting constant of the six-line spectrum is a little higher than 0.92 G. This may be related to the fact that PYC is a mixture of different bioflavonoids, and thus the presence of other components of PYC may influence its hyperfine splitting constant. The formation of the PYC radical was dependent on the presence of both HRP and H2O2 and was detected for at least 90 min. The effect of PYC concentration on the radical formation is shown in Fig. 2. The maximal ESR signal intensity of the PYC radical (the maximal amount of the PYC radical) was proportional to PYC concentration. At 500 g/ml of PYC, the radical decayed after a rapid formation phase. On decreasing PYC concentration, the
amount of the PYC radical formed reached the maximum later, indicating that the rate of PYC oxidation was decreased at lower concentrations. The influence of pH on the radical formation was also investigated. At pH ⬍ 9.5, ESR signal peaks of the PYC radical could not be detected, but the radical signal peak appeared at pH 9.5, and with increasing pH, the rate of the PYC oxidation and the rate of the PYC radical formation decreased (Fig. 3A). In other words, the higher pH was, the later was the appearance of the maximal amount of the PYC radical formed. This result is consistent with the fact that HRP is more active at pH 6.0, namely, HRP activity decreases with the increasing pH value (pH ⬎ 6.0). Similar results have been reported by Oubrahim et al. [19]. Because the decrease in the rate of the PYC radical formation at higher pH is probably caused by the decrease in HRP activity, it is suggested that the effect of increasing pH on the rate of the PYC radical formation should be similar to that of decreasing HRP concentration. This was confirmed by the data shown in Fig. 3B. In a mixture containing fixed concentrations of both PYC (250 g/ml) and H2O2 (1.2 mM) and varying HRP concentrations, the maximal amount of the PYC radical and the rate of its formation were decreased with the decrease in HRP concentration. This result is similar to that reported in the literature [18]. To clarify which components of PYC will contribute to the radical signal, the radical ESR signals of procyanidin B3, which is one of major procyanidins in PYC, were detected under the same conditions used to generate PYC radicals. The primary radical pyrocyanidin B3 was formed at the 4⬘-hydroxyl site of one B ring, which was confirmed by the ESR spectrum in Fig. 4, spectrum A (aH ⫽ 3.67 G [1H], aH ⫽ 0.92 G [3H]; g ⫽ 2.0055) with 2 ⫻ 4 lines (the ratio of the peak intensity is 1:3:3:1:1:3:3:1). One minute later, the secondary radical spectrum appeared; its signal intensity increased with time (Fig. 4, spectra B–D); this secondary radical was resolved (Fig. 4, spectrum E; aH ⫽
Fig. 3. (A) pH and (B) HRP concentration-dependence of the formation of the PYC radical generated by the HRP-H2O2 system over time. The reaction mixtures contained (A) 250-g/ml PYC, H2O2 (1.2 mM), and 0.15-mg/ml HRP or (B) the indicated concentrations of HRP. ESR measurement conditions are those described in Materials and Methods.
Pine bark extract free radical
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Fig. 4. Variation of ESR spectra of the procyanidin B3 radical generated by the HRP-H2O2 system. The reaction mixtures contained 100 mM carbonate buffer (pH 9.5), procyanidin B3 (400 g/ml), HRP (0.15 mg/ml), and H2O2 (1.2 mM). ESR measurement conditions are the same as those in the Fig. 2 inset except gains. Spectrum A: 1 min, gain 250; spectrum B: 2 min, gain 250; spectrum C: 3 min, gain 250; spectrum D: 5 min, gain 50; spectrum E: 6 min, gain 20; spectrum F : computer simulation of the procyanidin B3 secondary radical ESR spectrum (aH ⫽ 0.92 G [3H], aHOH ⫽ 0.61 G [1H]).
0.92 G [3H], aHOH ⫽ 0.61 G [1H], g ⫽ 2.0055) after 6 min and was relatively stable for at least 90 min. The computer simulation of this radical spectrum is shown in Fig. 4, spectrum F (aH ⫽ 0.92 G (3H), aHOH ⫽ 0.61 G (1H)). Comparing the spectrum and stability of this secondary radical with those of the PYC radical, it was found that the PYC radical signal mainly consists of the procyanidin B3 radical signal. DISCUSSION
In this investigation, the PYC radical formation was demonstrated by direct ESR technique in a system containing HRP and H2O2, where the rate of the radical
formation and decay increased with increasing PYC concentration. The increase in the rate of the radical formation may be the result of the increase in the rate of PYC oxidation, whereas rapid dimerization or polymerization of a great number of the radical molecules may result in the rapid decay of this radical. The pH dependence of the radical formation is likely to be related to the pH dependence of the enzyme HRP activity. From the PYC radical spectrum and its stability, it can be inferred that (⫹)-catechin and taxifolin, monomers accounting for 8 % of the weight of PYC, were not involved in the radical formation because the ESR spectrum of the corresponding radical formed from (⫹)-catechin was not detectable under the similar experimental conditions (data
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not shown). In addition, the ESR spectrum of the taxifolin radical as reported in the literature [20] is very different from that of the PYC radical and relatively unstable (data not shown). Furthermore, the radical ESR signals of procyanidin B1, B2, B3, C1, and C2, which are the major flavonoids-oligomeric procyanidins accounting for 80% of the weight of PYC, were also detected. Except for the radicals formed from procyanidin B3, all the radical spectra were completely different from that of the PYC radical and relatively unstable (data not shown). So from the secondary radical spectrum and stability of procyanidin B3, it is suggested that procyanidin B3 is the predominant species that contributes to the PYC radical signal. The mechanism of this secondary radical formation may be explained as follows: (i) intramolecular hydrogen bond between the O• (at the 4⬘ position) in one B ring (the upper ring) and an OH group in the other B ring (the lower ring) of procyanidin B3 is gradually formed; (ii) meanwhile, changes of the bond angles between carbons (at the 2 position) and B rings may occur during the formation of this hydrogen bond. On one hand, the intramolecular hydrogen bond is responsible for the delocalization of electron spin density in the -orbitals of the B ring containing unpaired electron across it onto the other B ring; this resulted in the spectral changes shown in Fig. 4. On the other hand, these signal spectra are not well resolved, probably owing to the presence of the hydrogen bond. Valoti et al. [21] have reported similar results that an ESR spectrum with better defined and sharper lines was observed in deuterated buffer because deuteration removed the hydrogen splitting of the OH group of the hydrogen bond. In addition to the intramolecular hydrogen bond causing the changes of the spectral structures, the other reason why the changes of the radical spectral structures occurred may be the changes of the bond angles between C-2 and B rings which caused the changes in the orbital overlap of the -orbitals of the B ring with the proton at C-2 of the C ring as reported in the literature [22]. In conclusion, our results demonstrate that PYC radicals can be observed in the presence of both HRP and H2O2 using ESR spectrometer. This radical is mainly derived from the procyanidin B3 radical. Although the radical of the monomer((⫹)-catechin) could not be obtained possibly because of the instability of its radical, the radical formed from the dimer (procyanidin B3) was quite stable. From this it can be confirmed that like metal ions such as Zn2⫹, Mg2⫹, and Ca2⫹ reported to stabilize o-semiquinones in aqueous media [23,24], the hydrogen bond between the O• in one B ring and an OH group in the other B ring of procyanidin B3 is an important factor that confers quite high stability to the procyanidin B3 radical by causing extensive electron delocalization. Thus it is important to study the antioxidant activity of these procyanidins and their radical species further to obtain information pertinent to the biological activities of PYC.
REFERENCES [1] Virgili, F.; Kobuchi, H.; Noda, Y.; Cossins, E.; Packer, L. Procyanidins extracted from pinus maritima bark: antioxidant activity, effects on the immune system, and modulation of nitrogen monoxide metabolism. In: Packer, L.; Hiramatsu, M.; Yoshikawa, T., eds. Antioxidant food supplements in human health. San Diego: Academic Press; 1999:323–342. [2] Virgili, F.; Kim, D.; Packer, L. Procyanidins extracted from pine bark protect alpha-tocopherol in ECV 304 endothelial cells challenged by activated RAW 264.7 macrophages: role of nitric oxide and peroxynitrite. FEBS Letts. 431: 315–318; 1998. [3] Nelson, A. B.; Lau, B. H. S.; Ide, N.; Rong, Y. Pycnogenol inhibits macrophage oxidative burst, lipoprotein oxidation and hydroxyl radical induced DNA damage. Drug Dev. Indust. Med. 24:1– 6; 1998. [4] Rong, Y.; Li, L.; Lau, B. H. Pycnogenol protects vascular endothelial cells from t-butyl hydroperoxide induced oxidant injury. Biotechnol. Ther. 5:117–126; 1994 –95. [5] Sen, C. K.; Roy, S.; Packer, L. Flow cytometric determination of cellular thiols. Methods Enzymol. 299:247–258; 1998. [6] Blazso, G.; Gabor, M.; Sibbel, R.; Rohdewald, P. Antiinflammatory and superoxide radical scavenging activities of procyanidins containing extract from the bark of pinus pinaster sol. and its fractions. Pharm. Pharmacol. Lett. 3:217– 220; 1994. [7] Noda, Y.; Anzai, K.; Mori, K.; Kohno, M.; Shinmei, M.; Packer, L. Hydroxyl and superoxide anion radical scavenging activities of natural source of antioxidants using the computerized JES-FR 30 ESR spectrometer system. Biochem. Mol. Biol. Int. 42:35– 44; 1997. [8] Elstner, E. F.; Kleber, E. Radical scavenger properties of leucocyanidin. In: Das, N. P., ed. Flavonoids in biology and medicine. III. Current issues in flavonoid research. Singapore: National University of Singapore Press; 1990: 227–235. [9] Virgili, F.; Kobuchi, H.; Packer, L. Procyanidins extracted from Pinus maritima (Pycnogenol威): scavengers of free radical species and modulators of nitrogen monoxide metabolism in activated murine RAW 264.7 macrophages. Free Radic. Biol. Med. 24:1120 –1129; 1998. [10] Macrides, T. A.; Shihata, A.; Kalafatis, N.; Wright, P. F. A. A comparison of the hydroxyl radical scavenging properties of the shark bile steroid 5--scymnol and plant pycnogenols. Biochem. Mol. Biol. Int. 42:1249 –1260; 1997. [11] Uchida, S.; Edamatsu, R.; Hiramatsu, M.; Mori, A.; Nonaka, G.; Nishioka, I.; Niwa, M.; Ozaki, M. Condensed tannins scavenge active oxygen radicals. Med. Sci. Res. 15:831– 832; 1987. [12] Packer, L.; Rimbach, G.; Virgili, F. Antioxidant activity and biologic properties of a procyanidin-rich extract from pine (Pinus maritima) bark, Pycnogenol. Free Radic. Biol. Med. 27:704 –724; 1999. [13] Moini, H.; Arroyo, A.; Vaya, J.; Packer, L. Bioflavonoid effects on the mitochondrial respiratory electron transport chain and cytochrome c redox state. Redox Rep. 4:35– 41; 1999. [14] Kim, E. H.; Sevanian, A. Hematin- and peroxide-catalyzed peroxidation of phospholipid liposomes. Arch. Biochem. Biophys. 288:324 –330; 1991. [15] Wieland, E.; Parthasarathy, S.; Steinbero, D. Peroxidae-dependent metal-independent oxidation of low density lipoprotein in vitro: a model for in vivo oxidation? Proc. Natl. Acad. Sci. USA 90:5929 –5933; 1993. [16] Schwartner, C.; Michel, C.; Stettmaier, K.; Wagner, H.; Bors, W. Marchantins and related polyphenols from liverwort: physico-chemical studies of their radical-scavenging properties. Free Radic. Biol. Med. 20:237–244; 1996. [17] Bors, W.; Heller, W.; Michel, C. The chemistry of flavonoids. In: Rice-Evans, C. A.; Packer, L., eds. Flavonoids in health and disease. New York: Marcel Dekker, Inc.; 1998:111–136. [18] Harman, L. S.; Carver, D. K.; Schreiber, J.; Mason, R. P. One- and two-electron oxidation of reduced glutathione by peroxidases. J. Biol. Chem. 261:1642– 1648; 1986. [19] Oubrahim, H.; Richard, J. M.; Cantin-Esnault, D. Peroxidase-mediated oxidation, a possible pathway for activation of the fungal nephrotoxin orellanine and related compounds: ESR and spin-trapping studies. Free Radic. Res. 28:497– 505; 1998. [20] Bors, W.; Michel, C.; Stettmaier, K. The interaction of flavonoids and ascorbate as studied by EPR spectroscopy. J. Magnetic Resonance Anal. 3:1– 6; 1997. [21] Valoti, M.; Sipe, H. J., Jr.; Sgaragli, G.; Mason, R. P. Free radical intermediates during peroxidase oxidation of 2-t-butyl-4-methoxyphenol, 2,6-di-t-butyl-4-methylphenol, and related phenol compounds. Arch. Biochem. Biophys. 269:423– 432; 1989. [22] Kuhnle, J. A.; Windle, J. J.; Waiss, A. C. Electron paramagnetic resonance spectra of flavonoid anion-radicals. J. Chem. Soc. (B). 6:613– 616; 1969. [23] Yamasaki, H.; Grace, S. C. EPR detection of phytophenoxyl radicals stabilized by zinc ions: evidence for the redox coupling of plant phenolics with ascorbate in the H2O2-peroxidase system. FEBS Letts. 422:377–380; 1998. [24] Kalyanaraman, B. Characterization of o-semiquinone radicals in biological systems. Methods Enzymol. 186:333–343; 1990.
PII S0891-5849(99)00168-9
Original Contribution ELECTRON SPIN RESONANCE STUDY OF FREE RADICALS FORMED FROM A PROCYANIDIN-RICH PINE (PINUS MARITIMA) BARK EXTRACT, PYCNOGENOL QIONG GUO,* BAOLU ZHAO,†
and
LESTER PACKER*
*Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA and †Department of Cell Biophysics, Institute of Biophysics, Academia Sinica, Beijing, China (Received 6 April 1999; Revised 6 August 1999; Accepted 10 August 1999)
Abstract—The free radical generated from the oxidation of a French maritima pine bark extract Pycnogenol (PYC), by the horseradish peroxidase (HRP)– hydrogen peroxide (H2O2) system at pH 7.4 –10.0 was studied using electron spin resonance (ESR) spectrometer. The formation rate of the PYC radical (aH ⫽ 0.92G; g ⫽ 2.0055) was dependent on the PYC and HRP concentrations and pH; the lifetime of the radical was up to 90 min. Furthermore, it was found that the PYC radical was mainly composed of the secondary radical formed from procyanidin B3, one of major procyanidins in PYC. The primary radical signal of procyanidin B3 with hyperfine splitting constants aH ⫽ 3.67 G (1H), aH ⫽ 0.92 G (3H), and g ⫽ 2.0055 was transient and disappeared quickly, whereas its secondary radical signal appeared and increased with time. The secondary radical from dimer procyanidin B3 showed quite high stability, differing from the radical from monomer (⫹)-catechin that could not be observed possibly because of its instability. These results provide evidence to support the idea that the intramolecular hydrogen bond between the O• at the 4⬘ position in one B ring and an OH group in the other B ring of procyanidin B3 is formed during its oxidation in the presence of HRP and H2O2. © 1999 Elsevier Science Inc. Keywords—Electron spin resonance, Pycnogenol, Horseradish peroxidase, Procyanidin B3, Intramolecular hydrogen bond, Free radicals
INTRODUCTION
There has recently been growing interest in the biological properties of PYC. It has been reported that PYC protects the low-density fraction of human plasma lipoproteins against copper-induced oxidation and protects DNA from iron/ascorbate-induced damage [3]. Moreover, PYC can protect cellular systems by increasing the activities of endogenous antioxidant enzymes such as glutathione (GSH) redox enzymes (GSH reductase and GSH peroxidase), superoxide dismutase, and catalase [4] and by increasing levels of endogenous antioxidants such as GSH [4,5] and ␣-tocopherol [2]. PYC has also been demonstrated to be an efficient scavenger of reactive oxygen species such as superoxide radical anion [6 – 8], hydroxyl radical [9,10], and nitric oxide radical [9] as well as the stable radical 1,1 diphenyl-2-picrylhydrazyl (DPPH) [11,12]. In addition to acting as a free radical scavenger, PYC has been recently reported to reduce cytochrome c reversibly, possibly by donation of electrons to the iron of the heme group and competitively inhibit mitochondrial electron transport chain activity
Isolated from the bark of the French marine pine (Pinus maritima), pine bark extract, Pycnogenol (PYC) (Horphag Research, Geneva, Switzerland), is a unique mixture of bioflavonoids, which are broadly divided into monomers ((⫹)-catechin and taxifolin; about 8% of the weight) and mainly condensed flavonoids (85% of the weight) classified as procyanidins. These condensed polyphenols are mainly constituted by “bricks” of the flavan-3-ols catechin and epicatechin, linked together, from dimers (Fig. 1) up to heptamers. PYC also contains phenolic acids (such as caffeic, ferulic, and p-hydroxybenzoic acids) as minor constituents and glycosylation products, that is, glucopyranose derivatives of either flavonols or phenolic acids as lesser constituents [1,2]. Address correspondence to: Lester Packer, Department of Molecular and Cell Biology, University of California Berkeley, 251 Life Sciences Addition, Berkeley, CA 94720-3200, USA; Tel.: (510) 642-1872; Fax: (510) 642-8313; E-Mail: [email protected]. 1308
Pine bark extract free radical
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Fig. 1. Structures of oligomeric procyanidins, the major constituents of PYC.
[13]. However, the free radical formed from PYC after it is oxidized has not been studied. It has been reported that horseradish peroxidase (HRP) can react with H2O2 and generate intermediates that oxidize a large variety of compounds, including phospholipids [14] and low-density lipoprotein [15]. In particular, the HRPH2O2 system has been used to generate free radicals of some phenolic compounds [16,17] and GSH [18]. Thus the aim of the present study was to investigate the production of the free radical formed from oxidation of PYC in the HRP-H2O2 system and clarify which components of PYC will contribute to the radical signal using a direct electron spin resonance (ESR) technique. MATERIALS AND METHODS
Reagents HRP (EC 1.1 1.1.7, type VI, 250 –330 units/mg) and procyanidin B3 were obtained from Sigma (St. Louis, MO, USA). Hydrogen peroxide was from Fisher Scientific (Fair Lawn, NJ, USA). PYC was a gift from Horphag Research. Procyanidin B1, B2, C1, and C2 were gifts from Tokyo Research Laboratories, Kyowa Hakko Kogyo (Machida, Japan).
ESR detection of PYC and procyanidin B3 radicals Enzymatic oxidation of PYC or procyanidin B3 was carried out with HRP and H2O2. A typical incubation mixture for ESR measurement consisted of PYC (100 – 500 g/ml) or procyanidin B3 (400 g/ml), H2O2 (1.2 mM), and HRP (0.15 mg/ml) in 100 mM sodium carbonate buffer (pH 9.5). The reaction mixture was then immediately introduced into a quartz capillary and fitted into a flat cell, and ESR spectra were recorded with time using an IBM ER 200D-SRC ESR spectrometer (Danbury, CT, USA), except for the results shown in Fig. 2 (inset) and Fig. 4 where the Free Radical Monitor (JESFR30, JEOL, Tokyo, Japan) was used. Instrument conditions were as follows: central field, 3475 G; modulation frequency, 100 kHz; modulation amplitude, 2.0 G; microwave power, 10 mW; scan width, 100 G; gain, 5.0 ⫻ 105; temperature, 295 K. RESULTS
The PYC radical was formed on addition of HRP (0.15 mg/ml) and H2O2 (1.2 mM) to sodium carbonate buffer (pH 9.5) containing 250-g/ml PYC (Fig. 2,
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Fig. 2. Effect of PYC concentration on the time-dependent formation of the PYC radical generated by the HRP-H2O2 system over time. The reaction mixtures contained HRP (0.15 mg/ml), H2O2 (1.2 mM), and the indicated concentrations of PYC in 100-mM carbonate buffer (pH 9.5). ESR measurement conditions are those described in Materials and Methods. Inset: an ESR spectrum of the PYC radical. ESR measurement conditions: modulation frequency, 9.41 GHz; modulation amplitude, 0.63 G; microwave power, 4.0 mW; gain, 100; scan width, 25 G; temperature, 295 K.
inset). The radical signal mainly contains a six-line spectrum with a hyperfine splitting constant aH ⫽ 0.92 G and g ⫽ 2.0055 and a broadening peak. In fact, the hyperfine splitting constant of the six-line spectrum is a little higher than 0.92 G. This may be related to the fact that PYC is a mixture of different bioflavonoids, and thus the presence of other components of PYC may influence its hyperfine splitting constant. The formation of the PYC radical was dependent on the presence of both HRP and H2O2 and was detected for at least 90 min. The effect of PYC concentration on the radical formation is shown in Fig. 2. The maximal ESR signal intensity of the PYC radical (the maximal amount of the PYC radical) was proportional to PYC concentration. At 500 g/ml of PYC, the radical decayed after a rapid formation phase. On decreasing PYC concentration, the
amount of the PYC radical formed reached the maximum later, indicating that the rate of PYC oxidation was decreased at lower concentrations. The influence of pH on the radical formation was also investigated. At pH ⬍ 9.5, ESR signal peaks of the PYC radical could not be detected, but the radical signal peak appeared at pH 9.5, and with increasing pH, the rate of the PYC oxidation and the rate of the PYC radical formation decreased (Fig. 3A). In other words, the higher pH was, the later was the appearance of the maximal amount of the PYC radical formed. This result is consistent with the fact that HRP is more active at pH 6.0, namely, HRP activity decreases with the increasing pH value (pH ⬎ 6.0). Similar results have been reported by Oubrahim et al. [19]. Because the decrease in the rate of the PYC radical formation at higher pH is probably caused by the decrease in HRP activity, it is suggested that the effect of increasing pH on the rate of the PYC radical formation should be similar to that of decreasing HRP concentration. This was confirmed by the data shown in Fig. 3B. In a mixture containing fixed concentrations of both PYC (250 g/ml) and H2O2 (1.2 mM) and varying HRP concentrations, the maximal amount of the PYC radical and the rate of its formation were decreased with the decrease in HRP concentration. This result is similar to that reported in the literature [18]. To clarify which components of PYC will contribute to the radical signal, the radical ESR signals of procyanidin B3, which is one of major procyanidins in PYC, were detected under the same conditions used to generate PYC radicals. The primary radical pyrocyanidin B3 was formed at the 4⬘-hydroxyl site of one B ring, which was confirmed by the ESR spectrum in Fig. 4, spectrum A (aH ⫽ 3.67 G [1H], aH ⫽ 0.92 G [3H]; g ⫽ 2.0055) with 2 ⫻ 4 lines (the ratio of the peak intensity is 1:3:3:1:1:3:3:1). One minute later, the secondary radical spectrum appeared; its signal intensity increased with time (Fig. 4, spectra B–D); this secondary radical was resolved (Fig. 4, spectrum E; aH ⫽
Fig. 3. (A) pH and (B) HRP concentration-dependence of the formation of the PYC radical generated by the HRP-H2O2 system over time. The reaction mixtures contained (A) 250-g/ml PYC, H2O2 (1.2 mM), and 0.15-mg/ml HRP or (B) the indicated concentrations of HRP. ESR measurement conditions are those described in Materials and Methods.
Pine bark extract free radical
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Fig. 4. Variation of ESR spectra of the procyanidin B3 radical generated by the HRP-H2O2 system. The reaction mixtures contained 100 mM carbonate buffer (pH 9.5), procyanidin B3 (400 g/ml), HRP (0.15 mg/ml), and H2O2 (1.2 mM). ESR measurement conditions are the same as those in the Fig. 2 inset except gains. Spectrum A: 1 min, gain 250; spectrum B: 2 min, gain 250; spectrum C: 3 min, gain 250; spectrum D: 5 min, gain 50; spectrum E: 6 min, gain 20; spectrum F : computer simulation of the procyanidin B3 secondary radical ESR spectrum (aH ⫽ 0.92 G [3H], aHOH ⫽ 0.61 G [1H]).
0.92 G [3H], aHOH ⫽ 0.61 G [1H], g ⫽ 2.0055) after 6 min and was relatively stable for at least 90 min. The computer simulation of this radical spectrum is shown in Fig. 4, spectrum F (aH ⫽ 0.92 G (3H), aHOH ⫽ 0.61 G (1H)). Comparing the spectrum and stability of this secondary radical with those of the PYC radical, it was found that the PYC radical signal mainly consists of the procyanidin B3 radical signal. DISCUSSION
In this investigation, the PYC radical formation was demonstrated by direct ESR technique in a system containing HRP and H2O2, where the rate of the radical
formation and decay increased with increasing PYC concentration. The increase in the rate of the radical formation may be the result of the increase in the rate of PYC oxidation, whereas rapid dimerization or polymerization of a great number of the radical molecules may result in the rapid decay of this radical. The pH dependence of the radical formation is likely to be related to the pH dependence of the enzyme HRP activity. From the PYC radical spectrum and its stability, it can be inferred that (⫹)-catechin and taxifolin, monomers accounting for 8 % of the weight of PYC, were not involved in the radical formation because the ESR spectrum of the corresponding radical formed from (⫹)-catechin was not detectable under the similar experimental conditions (data
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not shown). In addition, the ESR spectrum of the taxifolin radical as reported in the literature [20] is very different from that of the PYC radical and relatively unstable (data not shown). Furthermore, the radical ESR signals of procyanidin B1, B2, B3, C1, and C2, which are the major flavonoids-oligomeric procyanidins accounting for 80% of the weight of PYC, were also detected. Except for the radicals formed from procyanidin B3, all the radical spectra were completely different from that of the PYC radical and relatively unstable (data not shown). So from the secondary radical spectrum and stability of procyanidin B3, it is suggested that procyanidin B3 is the predominant species that contributes to the PYC radical signal. The mechanism of this secondary radical formation may be explained as follows: (i) intramolecular hydrogen bond between the O• (at the 4⬘ position) in one B ring (the upper ring) and an OH group in the other B ring (the lower ring) of procyanidin B3 is gradually formed; (ii) meanwhile, changes of the bond angles between carbons (at the 2 position) and B rings may occur during the formation of this hydrogen bond. On one hand, the intramolecular hydrogen bond is responsible for the delocalization of electron spin density in the -orbitals of the B ring containing unpaired electron across it onto the other B ring; this resulted in the spectral changes shown in Fig. 4. On the other hand, these signal spectra are not well resolved, probably owing to the presence of the hydrogen bond. Valoti et al. [21] have reported similar results that an ESR spectrum with better defined and sharper lines was observed in deuterated buffer because deuteration removed the hydrogen splitting of the OH group of the hydrogen bond. In addition to the intramolecular hydrogen bond causing the changes of the spectral structures, the other reason why the changes of the radical spectral structures occurred may be the changes of the bond angles between C-2 and B rings which caused the changes in the orbital overlap of the -orbitals of the B ring with the proton at C-2 of the C ring as reported in the literature [22]. In conclusion, our results demonstrate that PYC radicals can be observed in the presence of both HRP and H2O2 using ESR spectrometer. This radical is mainly derived from the procyanidin B3 radical. Although the radical of the monomer((⫹)-catechin) could not be obtained possibly because of the instability of its radical, the radical formed from the dimer (procyanidin B3) was quite stable. From this it can be confirmed that like metal ions such as Zn2⫹, Mg2⫹, and Ca2⫹ reported to stabilize o-semiquinones in aqueous media [23,24], the hydrogen bond between the O• in one B ring and an OH group in the other B ring of procyanidin B3 is an important factor that confers quite high stability to the procyanidin B3 radical by causing extensive electron delocalization. Thus it is important to study the antioxidant activity of these procyanidins and their radical species further to obtain information pertinent to the biological activities of PYC.
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