Papers Effects of Phenolcarboxylic Acids on Superoxide Anion and Lipid Peroxidation Induced by Superoxide Anion Shisuo Toda1'2 Michio Kumura', and Motoyo Ohnishi' 1
Department of Pharmaceutical Science, Institute of Medical Science, Kansai Shinkyu Medical College, 990 Ogaito, Sennan,
2
Address for correspondence
Osaka 590-04, Japan
Materials and Methods
Abstract The effects of phenolcarboxylic acids, caffeic acid, p-coumaric acid, and ferulic acid on the genera-
tion of superoxide anion and the production of lipid peroxide induced by superoxide anion were studied. Only ferulic acid anion among the phenolcarboxylic
Chemicals Caffeic acid, cinnamic acid, p-coumaric acid, ferulic acid, linoleic acid, superoxide dismutase (SOD, EC 1,2,3,2), DL-a-tocopherol, and xanthine oxidase (XOD, EC 1,15,1,1) were purchased from Sigma Chemical Co. (St. Louis, Mo, U.S.A.).
Assay of inhibitory effects of phenolcarboxylic acids on the generation of superoxide anion The activities of phenolcarboxylic acids were
acids scavenged superoxide. Caffeic acid and ferulic acid
inhibited lipid peroxidation induced by superoxide anion. These effects were comparable to those of superoxide dismutase or DL-a-tocopherol.
Key words Superoxide anion, lipid peroxide production, phenolcarboxylic acids, caffeic acid, p-coumaric acid, ferulic acid, active oxygen species.
measured according to the method of Yamanaka et al. (12). The standard reaction mixture containing 0.1 mM xanthine, 0.1mM ethylenediaminetetraacetic acid, bovine serum albumine (50 pg of proteinlml), 40mM sodium carbonate (pH 10.2), 25mM nitroblue
tetrazolium, sample solution, and 7 x iO U XOD (xanthine oxidase), with a final volume of 3m1, was incubated at 25°C for 20 mm. The reaction was terminated after incubation by the addition of 0.1 ml of 6mM CuCI2 solution. The absorbance of the forma-
Introduction
zan produced was determined at 560nm; 0.1mM formazan was produced in the absence of the sample solution. Inhibitory effects of samples on the generation of superoxide anion were estimated by the equation:
Active oxygen species such as superoxide
anion, hydroxyl radical, singlet oxygen, and hydrogen peroxide, cause lipid peroxidation (1), facilitate aging (2), induce carcinogenosis by DNA injuring (3), and play some
role in inflammation (4), organ ischemia (5) and/or arteroscleosis (6).
Inhibitory ratio =
Absorbance with no — Absorbance with addition of sample addition of sample x 100 .. . Absorbance with no addition of sample
Assay of inhibitory effects of phenolcarboxylic acids on production of lipid peroxide induced by superoxide anion
Carotinoids (7, 8) or flavonoids (9, 10), a type of plant pigments, were found to scavenge superoxide anion and inhibit lipid peroxidation induced by active oxygen species such as superoxide anion or hydroxyl radical. We had found that caffeic acid and ferulic acid, which are
Superoxide anion was generated in 3 ml of standard reaction mixture containing 0.4mM xanthine, XOD (0.2 UI ml), 0.2M NaC1, 0.1mM FeCl3, 0.2mM ADP, 0.1mM ethylene-
found in plants as phenolcarboxylic acids, inhibited
diaminetetraacetic acid-Fe3 solution, and 1 mM linoleic acid
hemolysis and prevented lipid peroxidation from injuring erythrocyte membranes by hydrogen peroxide (11) in a previous investigation of the mechanism of these inhibitory effects. We have now studied the effects of phenolcarboxylic acids on the generation of superoxide anion employing a xanthine-xanthine oxidase system and on the production of lipid peroxide induced by superoxide anion.
according to the method of Fukuzawa et al. (13). The incubation was initiated by addition of XOD at 37°C, and 1 mM of reaction mixture was added to 4 ml of trichloroacetic acid solution containing 0.03% butylated hydroxytoluene after incubation for 20 miii. Lipid peroxide was measured as thiobarbituric acid reactive—substance by the thiobarbituric acid method (14). The resultant values were expressed as quantities of malondialdehyde (MDA); 7.08 1.07 nrnole/ml MDAwas produced in the absence of sample solu-
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Received: February 8, 1990
Effects of Phenolcarboxylic Acids on Superoxide Anion and Lipid Peroxidation Induced by Superoxide Anion
MDA with no
Inhibitory ratio =
- MDA with
addition of sample addition of sample x 100 .. MDA with no addition of sample
Results Ferulic acid, SOD (superoxide dismutase), and DL-a-tocopherol inhibited the generation of superoxide
anion in a concentration dependent manner, whereas no inhibitory effects of caffeic acid, cinnamic acid, and pcoumaric acid were detectable as shown in Fig. 1. Inhibitoryratios of ferulic acid (66.91 3.67 %at 10 mM, 44.75 7.41% at 1 mM, and 5.33 0.80% at 10_i mM) were lower than those of SOD (98.01 0.85% at 10 mM, 97.79 at
1mM, and 88.72 2.05% at 10tmM) and higher than those ofDL-a-tocopherol (15.71 3.21% at 10mM, 2.10 1.35% at 1 mM, and 0% at 101mM).
Caffeic acid, ferulic acid, SOD, and DL-a-tocopherol inhibited the production of lipid peroxide induced by superoxide anion, but there were no detectable inhibitory effects of cinnamic acid and p-coumaric acid as shown in Fig. 2. Inhibitory ratios of caffeic acid (54.57 3.45% at 10mM and 52.47 2.16% at 1 mM) and ferulic acid (44.89 1.09% at 10mM and 35.73 8.58% at 1 mM) were comparable to those of SOD (46.93 2.04% at 10mM and 43.06 3.45% at 1mM) and DL-a-tocopherol (60.13 2.92% at 10mM and 56.14 0.73% at 1mM).
Discussion
We obtained evidence that ferulic acid scavenged superoxide anion, and that caffeic acid and
ferulic acid inhibited lipid peroxidation induced by superoxide anion. These effects were similar to those of SOD as a scavenger of superoxide anion and of aL-a-tocop herol as an inhibitor of lipid peroxidation induced by superoxide anion. In addition to SOD, flavonoids such as quercetin, myricetin, and rutin were found to be scavengers for superoxide anion (15). p-Coumaric acid, caffeic acid, and ferulic acid, all phenolcarboxylic acids, were precur-
100
sors in the biosynthesis of flavonoids. Butylated hydroxyanisole, butylated hydroxytoluene, and a-tocopherol were found to be phenolic antioxidants (16). The phenolcar-
Un:,_ _______
i02
boxylic acids, p-coumaric acid, caffeic acid, and ferulic acid, are compounds in which an OH group is added to the p-position of cinnamic acid. p-Coumaric acid, which has a proton
// 10
at the rn-position of cinnamic acid, was found not to have any effects either as a scavenger of superoxide anion or as an inhibitor of lipid peroxidation induced by superoxide anion. Caffeic acid, in which an OH group is added at the rnposition of p-coumaric acid, only had inhibitory effects on 1
10 ), n/mi)
Concentration
Fig. 1 Inhibitory effects of phenolcarboxylic acids on the generation of superoxide anion. •: caffeic acid, 0: ferulic acid, •: cinnamic acid, LII p-coumaric acid, A: SOD, A: DL-a-tocopherol. Values show mean S.E. from 4 experiments.
lipid peroxidation induced by superoxide anion. Ferulic acid, with an OCH3 group added at the rn-position of pcoumaric acid, acted as both a scavenger of superoxide anion and an inhibitor of lipid peroxidation. From these results, the scavenger effects on superoxide anion and inhibi-
tory effects on lipid peroxidation induced by superoxide anion of phenolcarboxylic acids seem to be derived from the constituents of the rn-position of cinnamic acid.
Antioxidative effects of phenolcarboxylic acid analogs on the generation of superoxide anion and the production of lipid peroxide induced by superoxide anion are under investigation.
100 r
Acknowledgements C
4
50
The authors wish to thank Dr. Junichi Kawa-
mata, Kansai Shikyu Medical College, for his advice.
____________ o_2
1o
in (oar, n/mi) Concentration
Fig. 2 Inhibitory effects of phenolcarboxylic acids on the production of lipid peroxide induced by superoxide anion. S: caffeic acid, 0: ferulic acid, •: cinnamic acid, LI: p-coumaric acid, A: SOD, A: DL-a-tocopherol. Values show
mean SE. from 4 experiments.
9
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
tion. Inhibitory effects of samples on lipid peroxidation induced by superoxide anion were estimated by the following equation:
Planta Med. 57(1991)
10 Planta Med. 57(1991)
1
° Sorata, H., Takahama, U., Kimura, M. (1984) Biochim. Biophys. Acta. 799, 798—803.
Toda, S., Ohnishi, M., Kimura, M., Nakashima, K. (1988) J.
Chau, W-S. H. (1987) in: Autooxidation of Unsaturated Lipid (Chau,
W-S. H., ed), pp. 1—16, Academic Press, London. 2 Jozwiak, Z., Jasnowska, B. (1985) Mech. Aging Dev. 32, 77—83. O'Brien, P. J. (1988) Free Radical Biol. Med. 4, 169—183. Oyanagi, Y. (1976) Biochem. Pharmacol. 25, 1465—1472. Tapple, A. L., (1973) Federation Proc. 32, 1870—1874. 6 Yagi, K., Ohkawa, H., Ohnishi, N., Yamashita, M., Nakashima, T. (1981) J. App!. Biocheni. 3, 58—65. Foote, C. S., Denng, H. W. (1968) J. Am. Chem. Soc. 90,6233—6235. 8 Burton, G. W., !ngo!d, K. U. (1984) Science 224, 569— 573. Ueno, I., Kohno, M., Haraikawa, K., Hirano, I. (1984) J. Pharm. Dyn. 7, 798—803.
12
13
14
16
Ethnopharmac. 23, 105—108. Yamanaka, N., Nishida, K., Ota, K. (1979) Physio!. Chem. Physics. 11,253—256. Fukuzawa, K., Takase, S., Tsukatani, H. (1985) Arch. Biochem. Biophys. 240, 117—120. Fukuzawa, K., Chida, H., Tokumura, A., Tsukatani, H. (1981) Arch. Biochem. Biophys. 206, 173—180. Roback, J., Gryg!ewski, H. J. (1988) Biochem. Pharmaco!. 37, 837— 841. Pryor, W. A., Strickland, T., Church, F. (1988) J. Am. Chem. Soc. 110, 2224— 2230.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
References
Shisco Toda et al.
Department of Pharmaceutical Science, Institute of Medical Science, Kansai Shinkyu Medical College, 990 Ogaito, Sennan,
2
Address for correspondence
Osaka 590-04, Japan
Materials and Methods
Abstract The effects of phenolcarboxylic acids, caffeic acid, p-coumaric acid, and ferulic acid on the genera-
tion of superoxide anion and the production of lipid peroxide induced by superoxide anion were studied. Only ferulic acid anion among the phenolcarboxylic
Chemicals Caffeic acid, cinnamic acid, p-coumaric acid, ferulic acid, linoleic acid, superoxide dismutase (SOD, EC 1,2,3,2), DL-a-tocopherol, and xanthine oxidase (XOD, EC 1,15,1,1) were purchased from Sigma Chemical Co. (St. Louis, Mo, U.S.A.).
Assay of inhibitory effects of phenolcarboxylic acids on the generation of superoxide anion The activities of phenolcarboxylic acids were
acids scavenged superoxide. Caffeic acid and ferulic acid
inhibited lipid peroxidation induced by superoxide anion. These effects were comparable to those of superoxide dismutase or DL-a-tocopherol.
Key words Superoxide anion, lipid peroxide production, phenolcarboxylic acids, caffeic acid, p-coumaric acid, ferulic acid, active oxygen species.
measured according to the method of Yamanaka et al. (12). The standard reaction mixture containing 0.1 mM xanthine, 0.1mM ethylenediaminetetraacetic acid, bovine serum albumine (50 pg of proteinlml), 40mM sodium carbonate (pH 10.2), 25mM nitroblue
tetrazolium, sample solution, and 7 x iO U XOD (xanthine oxidase), with a final volume of 3m1, was incubated at 25°C for 20 mm. The reaction was terminated after incubation by the addition of 0.1 ml of 6mM CuCI2 solution. The absorbance of the forma-
Introduction
zan produced was determined at 560nm; 0.1mM formazan was produced in the absence of the sample solution. Inhibitory effects of samples on the generation of superoxide anion were estimated by the equation:
Active oxygen species such as superoxide
anion, hydroxyl radical, singlet oxygen, and hydrogen peroxide, cause lipid peroxidation (1), facilitate aging (2), induce carcinogenosis by DNA injuring (3), and play some
role in inflammation (4), organ ischemia (5) and/or arteroscleosis (6).
Inhibitory ratio =
Absorbance with no — Absorbance with addition of sample addition of sample x 100 .. . Absorbance with no addition of sample
Assay of inhibitory effects of phenolcarboxylic acids on production of lipid peroxide induced by superoxide anion
Carotinoids (7, 8) or flavonoids (9, 10), a type of plant pigments, were found to scavenge superoxide anion and inhibit lipid peroxidation induced by active oxygen species such as superoxide anion or hydroxyl radical. We had found that caffeic acid and ferulic acid, which are
Superoxide anion was generated in 3 ml of standard reaction mixture containing 0.4mM xanthine, XOD (0.2 UI ml), 0.2M NaC1, 0.1mM FeCl3, 0.2mM ADP, 0.1mM ethylene-
found in plants as phenolcarboxylic acids, inhibited
diaminetetraacetic acid-Fe3 solution, and 1 mM linoleic acid
hemolysis and prevented lipid peroxidation from injuring erythrocyte membranes by hydrogen peroxide (11) in a previous investigation of the mechanism of these inhibitory effects. We have now studied the effects of phenolcarboxylic acids on the generation of superoxide anion employing a xanthine-xanthine oxidase system and on the production of lipid peroxide induced by superoxide anion.
according to the method of Fukuzawa et al. (13). The incubation was initiated by addition of XOD at 37°C, and 1 mM of reaction mixture was added to 4 ml of trichloroacetic acid solution containing 0.03% butylated hydroxytoluene after incubation for 20 miii. Lipid peroxide was measured as thiobarbituric acid reactive—substance by the thiobarbituric acid method (14). The resultant values were expressed as quantities of malondialdehyde (MDA); 7.08 1.07 nrnole/ml MDAwas produced in the absence of sample solu-
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
Received: February 8, 1990
Effects of Phenolcarboxylic Acids on Superoxide Anion and Lipid Peroxidation Induced by Superoxide Anion
MDA with no
Inhibitory ratio =
- MDA with
addition of sample addition of sample x 100 .. MDA with no addition of sample
Results Ferulic acid, SOD (superoxide dismutase), and DL-a-tocopherol inhibited the generation of superoxide
anion in a concentration dependent manner, whereas no inhibitory effects of caffeic acid, cinnamic acid, and pcoumaric acid were detectable as shown in Fig. 1. Inhibitoryratios of ferulic acid (66.91 3.67 %at 10 mM, 44.75 7.41% at 1 mM, and 5.33 0.80% at 10_i mM) were lower than those of SOD (98.01 0.85% at 10 mM, 97.79 at
1mM, and 88.72 2.05% at 10tmM) and higher than those ofDL-a-tocopherol (15.71 3.21% at 10mM, 2.10 1.35% at 1 mM, and 0% at 101mM).
Caffeic acid, ferulic acid, SOD, and DL-a-tocopherol inhibited the production of lipid peroxide induced by superoxide anion, but there were no detectable inhibitory effects of cinnamic acid and p-coumaric acid as shown in Fig. 2. Inhibitory ratios of caffeic acid (54.57 3.45% at 10mM and 52.47 2.16% at 1 mM) and ferulic acid (44.89 1.09% at 10mM and 35.73 8.58% at 1 mM) were comparable to those of SOD (46.93 2.04% at 10mM and 43.06 3.45% at 1mM) and DL-a-tocopherol (60.13 2.92% at 10mM and 56.14 0.73% at 1mM).
Discussion
We obtained evidence that ferulic acid scavenged superoxide anion, and that caffeic acid and
ferulic acid inhibited lipid peroxidation induced by superoxide anion. These effects were similar to those of SOD as a scavenger of superoxide anion and of aL-a-tocop herol as an inhibitor of lipid peroxidation induced by superoxide anion. In addition to SOD, flavonoids such as quercetin, myricetin, and rutin were found to be scavengers for superoxide anion (15). p-Coumaric acid, caffeic acid, and ferulic acid, all phenolcarboxylic acids, were precur-
100
sors in the biosynthesis of flavonoids. Butylated hydroxyanisole, butylated hydroxytoluene, and a-tocopherol were found to be phenolic antioxidants (16). The phenolcar-
Un:,_ _______
i02
boxylic acids, p-coumaric acid, caffeic acid, and ferulic acid, are compounds in which an OH group is added to the p-position of cinnamic acid. p-Coumaric acid, which has a proton
// 10
at the rn-position of cinnamic acid, was found not to have any effects either as a scavenger of superoxide anion or as an inhibitor of lipid peroxidation induced by superoxide anion. Caffeic acid, in which an OH group is added at the rnposition of p-coumaric acid, only had inhibitory effects on 1
10 ), n/mi)
Concentration
Fig. 1 Inhibitory effects of phenolcarboxylic acids on the generation of superoxide anion. •: caffeic acid, 0: ferulic acid, •: cinnamic acid, LII p-coumaric acid, A: SOD, A: DL-a-tocopherol. Values show mean S.E. from 4 experiments.
lipid peroxidation induced by superoxide anion. Ferulic acid, with an OCH3 group added at the rn-position of pcoumaric acid, acted as both a scavenger of superoxide anion and an inhibitor of lipid peroxidation. From these results, the scavenger effects on superoxide anion and inhibi-
tory effects on lipid peroxidation induced by superoxide anion of phenolcarboxylic acids seem to be derived from the constituents of the rn-position of cinnamic acid.
Antioxidative effects of phenolcarboxylic acid analogs on the generation of superoxide anion and the production of lipid peroxide induced by superoxide anion are under investigation.
100 r
Acknowledgements C
4
50
The authors wish to thank Dr. Junichi Kawa-
mata, Kansai Shikyu Medical College, for his advice.
____________ o_2
1o
in (oar, n/mi) Concentration
Fig. 2 Inhibitory effects of phenolcarboxylic acids on the production of lipid peroxide induced by superoxide anion. S: caffeic acid, 0: ferulic acid, •: cinnamic acid, LI: p-coumaric acid, A: SOD, A: DL-a-tocopherol. Values show
mean SE. from 4 experiments.
9
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
tion. Inhibitory effects of samples on lipid peroxidation induced by superoxide anion were estimated by the following equation:
Planta Med. 57(1991)
10 Planta Med. 57(1991)
1
° Sorata, H., Takahama, U., Kimura, M. (1984) Biochim. Biophys. Acta. 799, 798—803.
Toda, S., Ohnishi, M., Kimura, M., Nakashima, K. (1988) J.
Chau, W-S. H. (1987) in: Autooxidation of Unsaturated Lipid (Chau,
W-S. H., ed), pp. 1—16, Academic Press, London. 2 Jozwiak, Z., Jasnowska, B. (1985) Mech. Aging Dev. 32, 77—83. O'Brien, P. J. (1988) Free Radical Biol. Med. 4, 169—183. Oyanagi, Y. (1976) Biochem. Pharmacol. 25, 1465—1472. Tapple, A. L., (1973) Federation Proc. 32, 1870—1874. 6 Yagi, K., Ohkawa, H., Ohnishi, N., Yamashita, M., Nakashima, T. (1981) J. App!. Biocheni. 3, 58—65. Foote, C. S., Denng, H. W. (1968) J. Am. Chem. Soc. 90,6233—6235. 8 Burton, G. W., !ngo!d, K. U. (1984) Science 224, 569— 573. Ueno, I., Kohno, M., Haraikawa, K., Hirano, I. (1984) J. Pharm. Dyn. 7, 798—803.
12
13
14
16
Ethnopharmac. 23, 105—108. Yamanaka, N., Nishida, K., Ota, K. (1979) Physio!. Chem. Physics. 11,253—256. Fukuzawa, K., Takase, S., Tsukatani, H. (1985) Arch. Biochem. Biophys. 240, 117—120. Fukuzawa, K., Chida, H., Tokumura, A., Tsukatani, H. (1981) Arch. Biochem. Biophys. 206, 173—180. Roback, J., Gryg!ewski, H. J. (1988) Biochem. Pharmaco!. 37, 837— 841. Pryor, W. A., Strickland, T., Church, F. (1988) J. Am. Chem. Soc. 110, 2224— 2230.
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
References
Shisco Toda et al.
