Free Radicals and Aging ed. bV I. Emerit & B. Chance ©1992 Birkhauser Verlag Basel/Switzerland
Free radicals, lipid peroxidation, SOD activity, neurotransmitters and choline acetyltransferase activity in the aged rat brain M. Hiramatsu, R. Edamatsua and A. Moria Yamagata Technopolis Foundation, Yamagata 990, Japan, and "Department of Neuroscience, Institute of Molecular and Cellular Medicine, Okayama University Medical School, Okayama 700, Japan Summary. The mechanism of aging is suggested to be related to oxygen free radicals. Free radicals, lipid peroxidation and SOD activity have been reported to be increased in the aged brain. A Japanese herbal medicine, Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960), which has scavenging activities against hydroxyl radicals, superoxide, 1,I-diphenyl-2-picrylhydrazyl radicals, carbon-centered radicals and alpha-tocopheroxyl radicals, decreased carbon-centered radicals and thiobarbituric acid reactive substances (TBARS) levels in the aged rat brain after a 3-week oral administration of 5% TJ-960 solution. TJ-960 elevated superoxide dismutase (SOD) activity in the cytosol fraction of the hippocampus and hypothalamus of aged rats. It decreased norepinephrine and 5-hydroxytryptamine (5-HT) levels in the hypothalamus and increased the 5-HT level in the cerebellum. TJ-960 treatment increased choline acetyltransferase activity in aged rats. As herbal medicines do not generally have harmful side effects, antioxidant TJ-960 appears to be a suitable prophylactic agent against some neuronal symptoms of aging.
Introduction Many reports have been accumulated on subjects related to free radicals and aging. The levels of antioxidants such as carotenoids, (X-tocopherol and uric acid in plasma, serum or liver have been reported to increase with aging (Cutler, 1991). It is considered that these antioxidants are synthesized to scavenge generated free radicals with aging. Thus it is important to find other excellent antioxidants against aging. A Japanese herbal medicine Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ960, Tsumura & Co., Tokyo) is a vacuum-concentrated extract of nine herbs in the following ratio: 7.0 Bupleuri radix (BupleurumJalcatum L.); 5.0 Pinelliae tuber (Pinellia ternata Breitenbach); 3.0 Scutellariae radix (Scutellaria baicalensis Georgi); 4.0 Zizyphifructus (Zizyphus Vulgaris Lamarck var. inermis Bunge); 3.0 Ginseng radix (Panax ginseng C. A. Meyer); 2.0 Glychyrrhizae radix (Glycyrrhiza glabra L. var. glandulifera Regel et Herder, Glycyrrhiza uralensis Fisher); 1.0 Ziniberis rhizoma (Zingiber officinale Roscoe); 6.0 Paeoniae radix (Paeonia albiflora Pallas var. trichocarpa Bunge); and 4.0 Cinnamomi cortex (Cinnamomum
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cassia Blume). The scavenging activity of TJ-960 on free radicals and its effect on free radicals and lipid peroxides, SOD activity, neurotransmitter levels and choline acetyl transferase activity in the aged rat brain are described here. Free radicals and lipid peroxidation
1,I-Diphenyl-2-picrylhydrazyl (DPPH) radicals are stable lipid soluble radicals. TJ-960 scavenged 30 jlM DPPH radicals in the range of 125 jlg/ml to 5 mg/ml dose-dependently (Hiramatsu et aI., 1988b). Superoxide radicals are generated by the hypoxanthine and xanthine oxidase system. These radicals of twelve signals were detected as dimethyl-l-pyrroline-l-oxide (DMPO) spin adducts using ESR spectrometry. TJ-960 scavenged superoxide dose-dependently in the range of 12.5 jlg/ml to 12.5 mg/ml (Hiramatsu et aI., 1988b). Hydroxyl radicals were generated in a (X-guanidinoglutaric acid solution and quartet signals of hydroxyl radicals were detected as DMPO spin adducts by ESR spectrometry. Carbon-centered radicals were also generated in this solution. Five mg/ml of TJ-960 significantly quenched hydroxyl and carbon-centered radicals about 45% of each in 80 mM (X-guanidinoglutaric acid solution (Hiramatsu et aI., 1988b). Carbon-centered radicals are generated by ascorbic acid and FeCl2 in the rat brain homogenate after 15 minutes incubation at 37°C, and carbon-centered radicals are detected as DMPO spin adducts using ESR spectrometry. TJ-960 solution of 22.7 mg/ml completely quenched the carbon-centered radicals and other free radicals (Hiramatsu et aI., 1988b). (X-Tocopheroxyl radicals could not be detected in microsomes and subpartic1e membranes (SPM) of rat liver, whereas lipoxygenase and arachidonic acid "generated these radicals in both membranes of rat liver after the provision of a vitamin E supplemented diet. TJ-960 scavenged (X-tocopheroxyl radicals in microsomes and SPM of rat liver and further decreased the loss of (X-tocopherol by lipoxygenase and arachidonic acid (Hiramatsu et aI., 1990). Because of its excellent scavenging activity on free radicals TJ-960 was further subjected to neurochemical study using aged male Wi star rats. The 3.5- and 24-month-old rats were divided into two groups; one group was administered a 5% TJ-960 aqueous solution orally for 3 months and the other group was given water as control. After 3 months, i.e., at their 6.5th and 27th month respectively, the animals were sacrificed by decapitation and tge cerebrum was rapidly taken out. While brain lipid peroxide and superoxide radical formation have been reported to increase with age (Sawada and Carlson, 1987), the chronic treatment of TJ-960 decreased the level of carbon-centered
215 radicals and thiobarbituric acid reactive substance (TBARS) formation, which is used as an index of lipid peroxidation, in the cerebrum of aged rat brain compared with the control group (Hiramatsu et at., 1988a). In summary, the above in vitro experiments showed that TJ-960 has potent scavenging activities on hydroxyl radicals, superoxide and DPPH radicals. It was further shown that TJ-960 has a quenching action on (X-tocopheroxyl radicals and carbon-centered radicals in rat liver membranes and mouse brain homogenates, respectively. The decrease in carbon-centered radicals and inhibition of TBARS formation in aged rat brain may be due to its excellent antioxidant activity against oxygen free radicals and other free radicals. SOD activity Some papers have reported and compared total SOD activity, Cu, Zn-SOD activity and Mn-SOD activity in the aged rodent brain with those of a young adult brain and have found no coincidence in the change of brain SOD activity with aging (Kellogg et aI., 1976; Mavelli et aI., 1978; Geremia et aI., 1990; Danh et aI., 1983; De Quiroga, 1990). SOD activity was measured using the ESR spectrometry method (Hiramatsu et aI., 1987) and the results showed that SOD activity in the mitochondrial fraction from the cortex, hippocampus, striatum, hypothalamus, midbrain, pons-medulla oblongata and cerebellum were markedly increased in aged rats compared with adult rats. Similarly, SOD activity in the cytosol fraction was elevated in all parts except the hypothalamus in aged rats (Hiramatsu et aI., 1992). However, the administration of TJ-960 to aged rats resulted in a decrease in SOD activity in the mitochondrial fraction of the striatum, and an increase in the hippocampus and hypothalamus. In adult rats, TJ-960 increased SOD activity in the cytosol fraction of the cortex and had no effect on the other fractions (Hiramatsu et aI., 1988a). Neurotransmitters In aged rats and mice, dopamine and norepinephrine levels are decreased (Hirschorn et aI., 1982; Estes and Simpkins, 1980, 1984; Osterburg et aI., 1981; Simpkins et aI., 1977). The hypothalamic norepinephrine level is low in aging animals (Estes and Simpkins, 1980; Osterburg et aI., 1981; Simpkins et aI., 1977) and in Alzheimer patients (Yates et aI., 1981; Gottfries et aI., 1983; Yates et aI., 1983). The dopamine-p-hydroxylase (DBH) activities in the cerebrospinal fluid of
216 aged people and patients with Alzheimer's disease are decreased (Fujita et at., 1982; Cross et at., 1981; Perry et at., 1981). In addition, cell loss in the locus coeruleus, reduced nucleolar volume in locus coeruleus neurons, and loss of norepinephrine are reported in the norepinephrinergic system of Alzheimer patients (Hardy et aI., 1985). The administration of TJ-960 for 3 weeks increased the norepinephrine level in the hypothalamus but not in any other brain regions. The dopamine level in seven parts of the aged rat brain was not affected by the treatment of TJ-960 (Hiramatsu et aI., 1986). However, the 5-hydroxytryptamine level in the hypothalamus was decreased and the level in the cerebellum was increased after chronic administration of TJ-960 (Hiramatsu et aI., 1988a). There have been Jew reports on the amino acid level in the aged rat brain. Our studies have shown that overall brain amino acid levels are lower in aged than in adult rats. Cerebral cortical y-aminobutyric acid, glutamine, and lysine levels are significantly higher in aged than in adult rats, while in the cerebellum, glutamate levels are higher and alanine levels lower. Administration of TJ-960 to rats resulted in a significant increase in cortical levels of taurine, serine and alanine in aged rats and a significant increase in cortical taurine, glutamate, glutamine, glycine and alanine in adult rats. In the cerebellum, TJ-960 produced increases in glycine and y-aminobutyric acid levels in adult rats but no significant changes were seen in aged rats. (Hiramatsu et aI., 1988c). Thus, TJ-960 increased only cerebral cortical taurine, serine and alanine in aged rats. It also increased taurine levels in adult rats. Taurine levels are not altered with age. CAT activity Previous reports have shown decreased CAT activity in the striatum and hippocampus of aged rats (Allen et aI., 1983; Haba et aI., 1988; Sims et at., 1982). CAT activity was measured according to a modification of Fonnum's method (1974) as described by Haba et ai. (1988). In comparison with adult rat brain, CAT activity in the hippocampus, pons-medulla oblongata and striatum was lower and the activity in the cerebellum was higher. The enzyme activity in the cortex of aged rats also appeared to be lower than that in adult rats but it was not significant statistically. The administration of TJ-960 increased the CAT activity in the hippocampus and striatum but it did not affect the activity in the seven brain parts of adult rats (Hiramatsu et aI., 1989). While the mechanism underlying this activity still remains to be clarified, the observed increase in CAT activity may suggest a protective effect of TJ-90 against aging in the brain.
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Conclusion TJ-960 was first developed as an anticonvulsant using an experimental animal model for epilepsy (Hiramatsu et aI., 1981; Hiramatsu et aI., 1988c; Sugaya et aI., 1988). The authors described here that TJ-960 has a free radical scavenging action in vitro and in vivo. Paeonifiorin, which is one element ofPaeoniae radix, a component ofTJ-960, does not have any scavenging action of free radicals. Traditionally, it has been believed that Chinese herbal medicine exhibits the effect only as a cocktail of complex plant extracts. It therefore remains an open question as to which specific component of TJ-960 may exert the effect seen as an antiaging effect, a free radical scavenging action and an increase in TBARS levels, SOD activity and CAT activity. Anyhow, it can be said at this point that antioxidants are excellent prophylactive agents against aging as shown in TJ-960. Allen, S., Benton, J. S., Goodhardt, M. J., Haan, E. A., Sims, N. R., Smith, C. C. T., Spillane, J. A., Bowen, D. M., and Davison, A. N. (1983) Biochemical evidence of selective nerve cell changes in the normal ageing human and rat brain. J Neurochem. 41: 256-265. Cutler, R. G. (1991) Antioxidants and aging. Am. J. Clin. Nutr. 53: 373s-379s. Cross, A. J., Crow, T. J., Perry, E. K., Perry, R. H., Blessed, G., and Tomlinson, B. E. (1981) Reduced dopamine-beta-hydroxylase activity in Alzheimer's disease. Br. Med. J. 282: 93-94. Danh, H. c., Benedetti, M. S., and Dostert, P. (1983) Differential changes in superoxide dismutase activity in brain and liver of old rats and mice. J. Neurochem. 40: 1003-1007. De Quiroga, G. B., Perez-Campo, R., and Lopezz Torres, M. (1990) Anti-oxidant defences and peroxidation in liver and brain of aged rats. Biochem. J. 272: 247-250. Estes, K. S., and Simpkins, J. W. (1980) Age-related alterations in catecholamine concentrations in discrete preoptic area and hypothalamic regions in the male rat. Brain Res. 194: 556-560. Estes, K. S., and Simpkins, J. W. (1984) Age-related alterations in dopamine and norepinephrine activity within microdissected brain regions of ovariecotomized long evans rats. Brain Res. 298: 209-218. Fonnum, F. (1974) A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 24: 407-409. Fujita, K., Maruta, K., Teradaira, R., Beppu, H., Ikegame, M., and Kawai, K. (1982) Dopamine beta-hydroxylase activity in human cerebrospinal fluid from various age groups. Clin. Chern. 28: 1403-1404. Geremia, E., Baratta, S. D., Zafarana, S., Giodarno, R., Pinizzotto, M. R., La Rossa, M. D. G., and Garozzo, A. (1990) Antioxidant enzymatic systems in neuronal and glial cell-enriched fractions of rat brain during aging. Neurochem. Res. 15: 719-723. Gottfries, C. G., Adolfsson, R., Aquilonius, S. M., Carlsson, A., Eckernas, S. A., Nordberg, A., Oreland, L., Svennerholm, L., Wiberg, A., and Winblad, B. (1983) Biochemical changes in dementia disorders of Alzheimer type (AD/SDAT). Neurobiolog. Aging 4: 261-271. Hardy, J., Adolfsson, R., Alafusoff, I., Bucht, G., Marcusson, J., Nyberg, P., Pedrahl, E., Wester, P., and Winblad, B. (1985) Transmitter deficits in Alzheimer's disease. Neurochem. Int., 7: 545-563. Haba, K., Ogawa, N., Kawata, M., and Mori, A. (1988) A method for parallel determination of choline acetyltransferase and muscarinic cholinergic receptors: Application in aged-rat brain. Neurochem. Res. 13: 951-955. Hiramatsu, M., Edamatsu, R., Kabuto, H., and Mori, A. (1988a) Effect of Sho-saiko-to-gokeishi-ka-shakuyaku-to (TJ-960) on monoamines, amino acids, lipid peroxides, and superoxide dismutase in brains of aged rats in: Recent Advances in the Pharmacology of KAMPO (Japanese herbal medicines) (E. Hosoya and Y. Yamamura, Eds). Excerpta Medica, pp. 128-135.
218 Hiramatsu, M., Edamatsu, R., Kohno, M., and Mori, A. (1988b) Scavenging of free radicals by Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960) in: Recent Advances in the Pharmacology of KAMPO (Japanese herbal medicines) (E. Hosoya and Y. Yamamura, Eds). Excerpta Medica, pp. 120-127. Hiramatsu, M., Haba, K., Edamatsu, R., Hamada, H., and Mori, A. (1989) Increased choline acetyltransferase activity by Chinese herbal medicine Sho-saiko-to-go-keishi-ka-shakuyakuto in aged rat brain. Neurochem. Res. 14: 249-251. Hiramatsu, M., Kabuto, H., and Mori, A. (1986) Effects of shosaiko-to-go-keishi-kashakuyaku-to (TJ-960) on brain catecholamine level of aged rats. IRCS Med. Sci. 14: 189-190. Hiramatsu, M., Kabuto, H., and Mori, A. (1988c) Effects of Sho-saiko-to-go-keishi-kashakuyaku-to (TJ-960) on convulsions and brain 5-hydroxytryptamine in EI mice, in: Recent Advances in the Pharmacology of KAMPO (Japanese herbal medicines) E. Hosoya and Y. Yamamura, Eds. Excerpta Medica, pp. 69-73. Hiramatsu, M., and Kohno, M. (1987) Determination of superoxide activity by electron spin resonance spectrometry using the spin trap method. JEOL News 23A: 7. Hiramatsu, M., Kohno, M., Edamatsu, R., and Mori, A. (1992) Increased superoxide dismutase activity in aged human cerebrospinal fluid and rat brain by electron spin resonance spectrometry using the spin trap method. J. Neurochem. 58: 1160-1164. Hiramatsu, M., Velasco, R. D., and Packer, L. (1990) Vitamin E radical reaction with antioxidants in rat liver membranes. Free Rad. BioI. Med. 9: 459-464. Hischorrn, I. D., Marman, M. H., and Sharpless, N. S. (1982) Dopamine receptor sensitivity following nigrostriatallesion in the aged rat. Brain Res. 234: 357-368. Kellogg, E. W., and Fridovich, I. (1976) Superoxide dismutase in the rat and mouse as a function of age and longevity. J. Gerontol. 31: 405-408. Mavelli, I., Mondovi, B., Federico, R., and Rotilio, G. (1978) Superoxide dismutase activity in developing rat brain. J. Neurochem. 31: 363-364. Osterburg, H. H., Donahue, H. G., Severson, J. A., and Finch, C. E. (1981) Catecholamine levels and turnover during aging in brain regions of male C57BLj6J mice. Brain Res. 224: 337-352. Perry, E. K., Blessed, G., Tomlinson, B. E., Perry, R. H., Crow, T. J., Cross, A. J., Dockray, G. J., Dimaline, R., and Arregui, A. (1981) Neurochemical activities in human temporal lobe related to aging and Alzheimer-type changes. Neurobiol. Aging 2: 251-256. Sawada, M., and Carlson, J. C. (1987) Changes in superoxide radical and lipid peroxide formation in the brain, heart and liver during the lifetime of the rat. Mech. Aging Dev. 41: 125-137. Simpkins, J. W., Mueller, G. P., Huang, H. H., and Meites, J. (1977) Evidence for depressed catecholamine and enhanced sereotonin metabolism in aging male rats: possible relation to gonadotropin secretion. Endocrinology 100: 1672-1678. Sims, N. R., Marek, K. L., Bowen, D. M., and Davison, A. N. (l982) Production of [14C]acetylcholine and [l4C]carbon dioxide from [U-1 4C]glucose in tissue prisms from aging rat brain. J. Neurochem. 38: 488-492. Sugaya, E., Ishige, A., Sekiguchi, K., Iizuka, S., Sugimoto, A., Yuzurihara, M., and Hosoya, E. (1988) Inhibitory effect,of mixture of herbal drugs (TJ-960, SK) on pentylenetetrazol-induced convulsions in EI mice. Epilepsy Res. 2: 337-339. Yates, C. M., Ritchie, I. M., Simpson, J., Maloney, A. F. J., and Gordon, A. (1981) Noradrenaline in Alzheimer-type dementia and Down syndrome. Lancet ii: 39-40. Yates, C. M., Simpson, J., Gordon, A., Maloney, A. F. J., Allison, Y., Ritchie, I. M., and Urquhart, A. (1983) Catecholamines and cholinergic enzymes in pre-senile and senile Alzheimer-type dementia and Down's syndrome. Brain Res. 280: 119-126.
Free radicals, lipid peroxidation, SOD activity, neurotransmitters and choline acetyltransferase activity in the aged rat brain M. Hiramatsu, R. Edamatsua and A. Moria Yamagata Technopolis Foundation, Yamagata 990, Japan, and "Department of Neuroscience, Institute of Molecular and Cellular Medicine, Okayama University Medical School, Okayama 700, Japan Summary. The mechanism of aging is suggested to be related to oxygen free radicals. Free radicals, lipid peroxidation and SOD activity have been reported to be increased in the aged brain. A Japanese herbal medicine, Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960), which has scavenging activities against hydroxyl radicals, superoxide, 1,I-diphenyl-2-picrylhydrazyl radicals, carbon-centered radicals and alpha-tocopheroxyl radicals, decreased carbon-centered radicals and thiobarbituric acid reactive substances (TBARS) levels in the aged rat brain after a 3-week oral administration of 5% TJ-960 solution. TJ-960 elevated superoxide dismutase (SOD) activity in the cytosol fraction of the hippocampus and hypothalamus of aged rats. It decreased norepinephrine and 5-hydroxytryptamine (5-HT) levels in the hypothalamus and increased the 5-HT level in the cerebellum. TJ-960 treatment increased choline acetyltransferase activity in aged rats. As herbal medicines do not generally have harmful side effects, antioxidant TJ-960 appears to be a suitable prophylactic agent against some neuronal symptoms of aging.
Introduction Many reports have been accumulated on subjects related to free radicals and aging. The levels of antioxidants such as carotenoids, (X-tocopherol and uric acid in plasma, serum or liver have been reported to increase with aging (Cutler, 1991). It is considered that these antioxidants are synthesized to scavenge generated free radicals with aging. Thus it is important to find other excellent antioxidants against aging. A Japanese herbal medicine Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ960, Tsumura & Co., Tokyo) is a vacuum-concentrated extract of nine herbs in the following ratio: 7.0 Bupleuri radix (BupleurumJalcatum L.); 5.0 Pinelliae tuber (Pinellia ternata Breitenbach); 3.0 Scutellariae radix (Scutellaria baicalensis Georgi); 4.0 Zizyphifructus (Zizyphus Vulgaris Lamarck var. inermis Bunge); 3.0 Ginseng radix (Panax ginseng C. A. Meyer); 2.0 Glychyrrhizae radix (Glycyrrhiza glabra L. var. glandulifera Regel et Herder, Glycyrrhiza uralensis Fisher); 1.0 Ziniberis rhizoma (Zingiber officinale Roscoe); 6.0 Paeoniae radix (Paeonia albiflora Pallas var. trichocarpa Bunge); and 4.0 Cinnamomi cortex (Cinnamomum
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cassia Blume). The scavenging activity of TJ-960 on free radicals and its effect on free radicals and lipid peroxides, SOD activity, neurotransmitter levels and choline acetyl transferase activity in the aged rat brain are described here. Free radicals and lipid peroxidation
1,I-Diphenyl-2-picrylhydrazyl (DPPH) radicals are stable lipid soluble radicals. TJ-960 scavenged 30 jlM DPPH radicals in the range of 125 jlg/ml to 5 mg/ml dose-dependently (Hiramatsu et aI., 1988b). Superoxide radicals are generated by the hypoxanthine and xanthine oxidase system. These radicals of twelve signals were detected as dimethyl-l-pyrroline-l-oxide (DMPO) spin adducts using ESR spectrometry. TJ-960 scavenged superoxide dose-dependently in the range of 12.5 jlg/ml to 12.5 mg/ml (Hiramatsu et aI., 1988b). Hydroxyl radicals were generated in a (X-guanidinoglutaric acid solution and quartet signals of hydroxyl radicals were detected as DMPO spin adducts by ESR spectrometry. Carbon-centered radicals were also generated in this solution. Five mg/ml of TJ-960 significantly quenched hydroxyl and carbon-centered radicals about 45% of each in 80 mM (X-guanidinoglutaric acid solution (Hiramatsu et aI., 1988b). Carbon-centered radicals are generated by ascorbic acid and FeCl2 in the rat brain homogenate after 15 minutes incubation at 37°C, and carbon-centered radicals are detected as DMPO spin adducts using ESR spectrometry. TJ-960 solution of 22.7 mg/ml completely quenched the carbon-centered radicals and other free radicals (Hiramatsu et aI., 1988b). (X-Tocopheroxyl radicals could not be detected in microsomes and subpartic1e membranes (SPM) of rat liver, whereas lipoxygenase and arachidonic acid "generated these radicals in both membranes of rat liver after the provision of a vitamin E supplemented diet. TJ-960 scavenged (X-tocopheroxyl radicals in microsomes and SPM of rat liver and further decreased the loss of (X-tocopherol by lipoxygenase and arachidonic acid (Hiramatsu et aI., 1990). Because of its excellent scavenging activity on free radicals TJ-960 was further subjected to neurochemical study using aged male Wi star rats. The 3.5- and 24-month-old rats were divided into two groups; one group was administered a 5% TJ-960 aqueous solution orally for 3 months and the other group was given water as control. After 3 months, i.e., at their 6.5th and 27th month respectively, the animals were sacrificed by decapitation and tge cerebrum was rapidly taken out. While brain lipid peroxide and superoxide radical formation have been reported to increase with age (Sawada and Carlson, 1987), the chronic treatment of TJ-960 decreased the level of carbon-centered
215 radicals and thiobarbituric acid reactive substance (TBARS) formation, which is used as an index of lipid peroxidation, in the cerebrum of aged rat brain compared with the control group (Hiramatsu et at., 1988a). In summary, the above in vitro experiments showed that TJ-960 has potent scavenging activities on hydroxyl radicals, superoxide and DPPH radicals. It was further shown that TJ-960 has a quenching action on (X-tocopheroxyl radicals and carbon-centered radicals in rat liver membranes and mouse brain homogenates, respectively. The decrease in carbon-centered radicals and inhibition of TBARS formation in aged rat brain may be due to its excellent antioxidant activity against oxygen free radicals and other free radicals. SOD activity Some papers have reported and compared total SOD activity, Cu, Zn-SOD activity and Mn-SOD activity in the aged rodent brain with those of a young adult brain and have found no coincidence in the change of brain SOD activity with aging (Kellogg et aI., 1976; Mavelli et aI., 1978; Geremia et aI., 1990; Danh et aI., 1983; De Quiroga, 1990). SOD activity was measured using the ESR spectrometry method (Hiramatsu et aI., 1987) and the results showed that SOD activity in the mitochondrial fraction from the cortex, hippocampus, striatum, hypothalamus, midbrain, pons-medulla oblongata and cerebellum were markedly increased in aged rats compared with adult rats. Similarly, SOD activity in the cytosol fraction was elevated in all parts except the hypothalamus in aged rats (Hiramatsu et aI., 1992). However, the administration of TJ-960 to aged rats resulted in a decrease in SOD activity in the mitochondrial fraction of the striatum, and an increase in the hippocampus and hypothalamus. In adult rats, TJ-960 increased SOD activity in the cytosol fraction of the cortex and had no effect on the other fractions (Hiramatsu et aI., 1988a). Neurotransmitters In aged rats and mice, dopamine and norepinephrine levels are decreased (Hirschorn et aI., 1982; Estes and Simpkins, 1980, 1984; Osterburg et aI., 1981; Simpkins et aI., 1977). The hypothalamic norepinephrine level is low in aging animals (Estes and Simpkins, 1980; Osterburg et aI., 1981; Simpkins et aI., 1977) and in Alzheimer patients (Yates et aI., 1981; Gottfries et aI., 1983; Yates et aI., 1983). The dopamine-p-hydroxylase (DBH) activities in the cerebrospinal fluid of
216 aged people and patients with Alzheimer's disease are decreased (Fujita et at., 1982; Cross et at., 1981; Perry et at., 1981). In addition, cell loss in the locus coeruleus, reduced nucleolar volume in locus coeruleus neurons, and loss of norepinephrine are reported in the norepinephrinergic system of Alzheimer patients (Hardy et aI., 1985). The administration of TJ-960 for 3 weeks increased the norepinephrine level in the hypothalamus but not in any other brain regions. The dopamine level in seven parts of the aged rat brain was not affected by the treatment of TJ-960 (Hiramatsu et aI., 1986). However, the 5-hydroxytryptamine level in the hypothalamus was decreased and the level in the cerebellum was increased after chronic administration of TJ-960 (Hiramatsu et aI., 1988a). There have been Jew reports on the amino acid level in the aged rat brain. Our studies have shown that overall brain amino acid levels are lower in aged than in adult rats. Cerebral cortical y-aminobutyric acid, glutamine, and lysine levels are significantly higher in aged than in adult rats, while in the cerebellum, glutamate levels are higher and alanine levels lower. Administration of TJ-960 to rats resulted in a significant increase in cortical levels of taurine, serine and alanine in aged rats and a significant increase in cortical taurine, glutamate, glutamine, glycine and alanine in adult rats. In the cerebellum, TJ-960 produced increases in glycine and y-aminobutyric acid levels in adult rats but no significant changes were seen in aged rats. (Hiramatsu et aI., 1988c). Thus, TJ-960 increased only cerebral cortical taurine, serine and alanine in aged rats. It also increased taurine levels in adult rats. Taurine levels are not altered with age. CAT activity Previous reports have shown decreased CAT activity in the striatum and hippocampus of aged rats (Allen et aI., 1983; Haba et aI., 1988; Sims et at., 1982). CAT activity was measured according to a modification of Fonnum's method (1974) as described by Haba et ai. (1988). In comparison with adult rat brain, CAT activity in the hippocampus, pons-medulla oblongata and striatum was lower and the activity in the cerebellum was higher. The enzyme activity in the cortex of aged rats also appeared to be lower than that in adult rats but it was not significant statistically. The administration of TJ-960 increased the CAT activity in the hippocampus and striatum but it did not affect the activity in the seven brain parts of adult rats (Hiramatsu et aI., 1989). While the mechanism underlying this activity still remains to be clarified, the observed increase in CAT activity may suggest a protective effect of TJ-90 against aging in the brain.
217
Conclusion TJ-960 was first developed as an anticonvulsant using an experimental animal model for epilepsy (Hiramatsu et aI., 1981; Hiramatsu et aI., 1988c; Sugaya et aI., 1988). The authors described here that TJ-960 has a free radical scavenging action in vitro and in vivo. Paeonifiorin, which is one element ofPaeoniae radix, a component ofTJ-960, does not have any scavenging action of free radicals. Traditionally, it has been believed that Chinese herbal medicine exhibits the effect only as a cocktail of complex plant extracts. It therefore remains an open question as to which specific component of TJ-960 may exert the effect seen as an antiaging effect, a free radical scavenging action and an increase in TBARS levels, SOD activity and CAT activity. Anyhow, it can be said at this point that antioxidants are excellent prophylactive agents against aging as shown in TJ-960. Allen, S., Benton, J. S., Goodhardt, M. J., Haan, E. A., Sims, N. R., Smith, C. C. T., Spillane, J. A., Bowen, D. M., and Davison, A. N. (1983) Biochemical evidence of selective nerve cell changes in the normal ageing human and rat brain. J Neurochem. 41: 256-265. Cutler, R. G. (1991) Antioxidants and aging. Am. J. Clin. Nutr. 53: 373s-379s. Cross, A. J., Crow, T. J., Perry, E. K., Perry, R. H., Blessed, G., and Tomlinson, B. E. (1981) Reduced dopamine-beta-hydroxylase activity in Alzheimer's disease. Br. Med. J. 282: 93-94. Danh, H. c., Benedetti, M. S., and Dostert, P. (1983) Differential changes in superoxide dismutase activity in brain and liver of old rats and mice. J. Neurochem. 40: 1003-1007. De Quiroga, G. B., Perez-Campo, R., and Lopezz Torres, M. (1990) Anti-oxidant defences and peroxidation in liver and brain of aged rats. Biochem. J. 272: 247-250. Estes, K. S., and Simpkins, J. W. (1980) Age-related alterations in catecholamine concentrations in discrete preoptic area and hypothalamic regions in the male rat. Brain Res. 194: 556-560. Estes, K. S., and Simpkins, J. W. (1984) Age-related alterations in dopamine and norepinephrine activity within microdissected brain regions of ovariecotomized long evans rats. Brain Res. 298: 209-218. Fonnum, F. (1974) A rapid radiochemical method for the determination of choline acetyltransferase. J. Neurochem. 24: 407-409. Fujita, K., Maruta, K., Teradaira, R., Beppu, H., Ikegame, M., and Kawai, K. (1982) Dopamine beta-hydroxylase activity in human cerebrospinal fluid from various age groups. Clin. Chern. 28: 1403-1404. Geremia, E., Baratta, S. D., Zafarana, S., Giodarno, R., Pinizzotto, M. R., La Rossa, M. D. G., and Garozzo, A. (1990) Antioxidant enzymatic systems in neuronal and glial cell-enriched fractions of rat brain during aging. Neurochem. Res. 15: 719-723. Gottfries, C. G., Adolfsson, R., Aquilonius, S. M., Carlsson, A., Eckernas, S. A., Nordberg, A., Oreland, L., Svennerholm, L., Wiberg, A., and Winblad, B. (1983) Biochemical changes in dementia disorders of Alzheimer type (AD/SDAT). Neurobiolog. Aging 4: 261-271. Hardy, J., Adolfsson, R., Alafusoff, I., Bucht, G., Marcusson, J., Nyberg, P., Pedrahl, E., Wester, P., and Winblad, B. (1985) Transmitter deficits in Alzheimer's disease. Neurochem. Int., 7: 545-563. Haba, K., Ogawa, N., Kawata, M., and Mori, A. (1988) A method for parallel determination of choline acetyltransferase and muscarinic cholinergic receptors: Application in aged-rat brain. Neurochem. Res. 13: 951-955. Hiramatsu, M., Edamatsu, R., Kabuto, H., and Mori, A. (1988a) Effect of Sho-saiko-to-gokeishi-ka-shakuyaku-to (TJ-960) on monoamines, amino acids, lipid peroxides, and superoxide dismutase in brains of aged rats in: Recent Advances in the Pharmacology of KAMPO (Japanese herbal medicines) (E. Hosoya and Y. Yamamura, Eds). Excerpta Medica, pp. 128-135.
218 Hiramatsu, M., Edamatsu, R., Kohno, M., and Mori, A. (1988b) Scavenging of free radicals by Sho-saiko-to-go-keishi-ka-shakuyaku-to (TJ-960) in: Recent Advances in the Pharmacology of KAMPO (Japanese herbal medicines) (E. Hosoya and Y. Yamamura, Eds). Excerpta Medica, pp. 120-127. Hiramatsu, M., Haba, K., Edamatsu, R., Hamada, H., and Mori, A. (1989) Increased choline acetyltransferase activity by Chinese herbal medicine Sho-saiko-to-go-keishi-ka-shakuyakuto in aged rat brain. Neurochem. Res. 14: 249-251. Hiramatsu, M., Kabuto, H., and Mori, A. (1986) Effects of shosaiko-to-go-keishi-kashakuyaku-to (TJ-960) on brain catecholamine level of aged rats. IRCS Med. Sci. 14: 189-190. Hiramatsu, M., Kabuto, H., and Mori, A. (1988c) Effects of Sho-saiko-to-go-keishi-kashakuyaku-to (TJ-960) on convulsions and brain 5-hydroxytryptamine in EI mice, in: Recent Advances in the Pharmacology of KAMPO (Japanese herbal medicines) E. Hosoya and Y. Yamamura, Eds. Excerpta Medica, pp. 69-73. Hiramatsu, M., and Kohno, M. (1987) Determination of superoxide activity by electron spin resonance spectrometry using the spin trap method. JEOL News 23A: 7. Hiramatsu, M., Kohno, M., Edamatsu, R., and Mori, A. (1992) Increased superoxide dismutase activity in aged human cerebrospinal fluid and rat brain by electron spin resonance spectrometry using the spin trap method. J. Neurochem. 58: 1160-1164. Hiramatsu, M., Velasco, R. D., and Packer, L. (1990) Vitamin E radical reaction with antioxidants in rat liver membranes. Free Rad. BioI. Med. 9: 459-464. Hischorrn, I. D., Marman, M. H., and Sharpless, N. S. (1982) Dopamine receptor sensitivity following nigrostriatallesion in the aged rat. Brain Res. 234: 357-368. Kellogg, E. W., and Fridovich, I. (1976) Superoxide dismutase in the rat and mouse as a function of age and longevity. J. Gerontol. 31: 405-408. Mavelli, I., Mondovi, B., Federico, R., and Rotilio, G. (1978) Superoxide dismutase activity in developing rat brain. J. Neurochem. 31: 363-364. Osterburg, H. H., Donahue, H. G., Severson, J. A., and Finch, C. E. (1981) Catecholamine levels and turnover during aging in brain regions of male C57BLj6J mice. Brain Res. 224: 337-352. Perry, E. K., Blessed, G., Tomlinson, B. E., Perry, R. H., Crow, T. J., Cross, A. J., Dockray, G. J., Dimaline, R., and Arregui, A. (1981) Neurochemical activities in human temporal lobe related to aging and Alzheimer-type changes. Neurobiol. Aging 2: 251-256. Sawada, M., and Carlson, J. C. (1987) Changes in superoxide radical and lipid peroxide formation in the brain, heart and liver during the lifetime of the rat. Mech. Aging Dev. 41: 125-137. Simpkins, J. W., Mueller, G. P., Huang, H. H., and Meites, J. (1977) Evidence for depressed catecholamine and enhanced sereotonin metabolism in aging male rats: possible relation to gonadotropin secretion. Endocrinology 100: 1672-1678. Sims, N. R., Marek, K. L., Bowen, D. M., and Davison, A. N. (l982) Production of [14C]acetylcholine and [l4C]carbon dioxide from [U-1 4C]glucose in tissue prisms from aging rat brain. J. Neurochem. 38: 488-492. Sugaya, E., Ishige, A., Sekiguchi, K., Iizuka, S., Sugimoto, A., Yuzurihara, M., and Hosoya, E. (1988) Inhibitory effect,of mixture of herbal drugs (TJ-960, SK) on pentylenetetrazol-induced convulsions in EI mice. Epilepsy Res. 2: 337-339. Yates, C. M., Ritchie, I. M., Simpson, J., Maloney, A. F. J., and Gordon, A. (1981) Noradrenaline in Alzheimer-type dementia and Down syndrome. Lancet ii: 39-40. Yates, C. M., Simpson, J., Gordon, A., Maloney, A. F. J., Allison, Y., Ritchie, I. M., and Urquhart, A. (1983) Catecholamines and cholinergic enzymes in pre-senile and senile Alzheimer-type dementia and Down's syndrome. Brain Res. 280: 119-126.
