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Superoxide Anion Radical Scavenging Activity of Uyaku (Lindera strychnifolia), a Natural Extract Used in Traditional Medicine
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Superoxide Anion Radical Scavenging Activity of Uyaku (Lindera strychnifolia), a Natural Extract Used in Traditional Medicine Yasuko Noda, * Akitane Mori, * Kazunori Anzai, t and Lester Packer* *Department of Molecular and Cell Biology University of California Berkeley, California 94720 tNational Institute of Radiological Sciences Chiba, Japan
Uyaku (Lindera strychnifolia: Sieb. et Zucc.) is well known as a traditional Asian medicine used in the treatment of stomach and renal diseases, neuralgia, rheumatism, and aging. However, the mechanism of its effectivity is not yet known. In this study, scavenging activities of superoxide anion and hydroxyl radicals were examined by electron spin resonance (ESR) using the spin-trapping reagent 5,5'-dimethyl-l-pyrroline-N-oxide (DMPO). Watersoluble components in both Uyaku root and leaf extracts showed potent superoxide anion scavenging activity. Uyaku seed extract showed weak superoxide anion scavenging activity. In all samples of Uyaku, scavenging activity was not diminished by ascorbate oxidase treatment. The filtrate, passed through a low molecular weight cutoff microcentrifuge filter (MW < 100,000 or < 10,000), showed approximately half the activity of untreated extract samples of Uyaku roots and leaf extracts, indicating that approximately half of the scavenging activity is due to the low molecular weight Antioxidant Food Supplements in Human Health Copyright 9 1999 by Academic Press. All rights of reproduction in any form reserved.
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components. To evaluate the hydroxyl radical scavenging activity, L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H- 1benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K1), which has vitamin E and vitamin C moieties and a high potency for hydroxyl radical scavenging, was used as a standard material. Results showed little hydroxyl radical scavenging activity in root and leaf extracts, although weak scavenging activity was demonstrated in the seed extract. These results demonstrated that Uyaku roots and leaves have specific and potent superoxide anion radical scavenging activity in comparison with several other natural extracts. Thus, Uyaku may have potential for beneficial effects for health, especially for diseases in which the production of superoxide anion radical is a factor.
INTRODUCTION
Uyaku (in Japanese, Wu Yao in Chinese, Oyak in Korean) is the dried roots of L. strychnifolia (Sieb. et Zucc.) E Villar of the Lauraceae family (1-3) and a traditional Asian medicine used in China as an astringent, carminative, stomachic or tonic, for asthma, cholera, congestion, dyspepsia, dysmenorrhea, fluxes, gonorrhea, hernia, malaria, menorrhagia, stomach ache, stroke, and urinary difficulties (4). According to a traditional story, the first Emperor of Qin Dynasty, about 2300 years ago, ordered Jo Fuku (Xu Fu in Chinese) to be brought to Horai, an island on the far east sea, looking for a drug effective for longevity. After a long sailing, he and his colleagues arrived at Shingu in Wakayama prefecture, Japan, in B.c. 219 and established "Tendai-uyaku" (another name for Uyaku, used especially in this district) (5-7). Uyaku might have been brought from China from 1716-1735, according to Makino (1). Uyaku has been used as a folk drug for good health and for the treatment of diseases such as stomach and renal diseases, neuralgia, and rheumatism in some districts, including Shingu, Wakayama prefecture, Japan (8, 9), although no scientific background for these treatments has been known until now. This study examines the effects of Uyaku (root, leaf, and seed extracts for their action) on reactive oxygen species using ESR spectrometry.
MATERIALS AND METHODS Chemicals
Common chemicals were from Sigma Chemical Co. (St. Louis, MO). Xanthine oxidase (XOD) was from Boehringer Mannheim Corp. (Indi-
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anapolis, IN). The spin trap DMPO was from Labotec (Tokyo, Japan). All chemicals were the highest grade available.
Standard Solutions SOD standard solutions were obtained as a kit (Dojindo Laboratories, Kumamoto, Japan). For measurements of hydroxyl radical scavenging activity, L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12trimethyltridecyl)-2H-l-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K1) (Senju Pharmaceutical Co. Ltd., Osaka, Japan) (8) was used as a standard. EPC-K1, which is composed of vitamin E and vitamin C by phosphate diester linkage, is water soluble and the solution is stable at room temperature. In this study, a stock solution of 10 mM EPC-K1 dissolved in 0.1 M phosphate buffer (pH 7.4) was prepared and diluted to obtain the appropriate concentrations (0-2 mM) before use. The stock solution was stored at 4~
Samples Sources Ground powder of Uyaku roots was kindly donated by Taikodo Pharmaceutical Co., Ltd. (Kobe, Japan). Dried leaves and seeds of Uyaku were collected in Wakayama, dried at room temperature for 1 month (leaves) or in a desiccator at 4~ (seeds), and ground with a mortar and pestle. To obtain the extract, the powder (5 g of Uyaku roots, 1.2 g of Uyaku leaves, or 10 g of Uyaku seeds) was added to 20 times the volume of water (1:20, by w/v), and the sample was extracted with boiling water for 10 min. After cooling to room temperature, the suspension was filtered (Whatman No. 2) and the recovered filtrate was freeze-dried. The sample was kept dry and at 4~ until use (Fig. 1). Treatments Freshly prepared sample solutions were used for assay. The extracted material was dissolved in 0.1 M phosphate buffer (pH 7.4) at concentrations from 0.1 to 0.5 mg/ml by vortex mixing (nontreatment). An aliquot of the sample was treated with ascorbate oxidase (20 units/ml of sample solution). The ascorbate oxidase stock solution dissolved in 0.1 M phosphate buffer, pH 7.4, was dialyzed at 4~ to eliminate possible contamination by sucrose. Another aliquot was passed through a centrifuge filter (UltrafreeMC Filters: 10,000 NMWL regenerated cellulose membrane; 100,000 NMWL polysulfone membrane, Millipore, MA) by centrifugation (5000 g,
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Procedure for Preparation of Uyaku Extracts Dried Uyaku roots, leaves or seeds sample (1-10 g) The sample was ground with a mortar and pestle The powder was weighed Powder was extracted with boiling water (1:20, by w/re) for 10 minutes at 100~ Extract was cooled to room temperature and filtrated using Whatman No. 2 filter paper Preparation was freeze-dried The sample was stored at 4~ in a silica-gel desiccator until use
Figure 1 Procedurefor preparation of Uyaku root, leaf, or seed extracts.
4~ to obtain materials of smaller molecular weights: less than 10,000 or 100,000.
ESR Measurements
ESR Spectrometer A new computerized, compact, highly sensitive ESR spectrometer (free radical monitor, JES FR-30, JEOL, Tokyo, japan) was used. This ESR system has the ability of normalizing all spectra for accurate calculation using manganese dioxide as an internal standard. Other details have been described in a previous report (9). Conditions for measurements in this study were as follows: magnetic field, 335.5 + 5 mT; microwave power, 4 mW; microwave frequency, 9.41 GHz; modulation width, 0.063 or 0.1 mT; sweep width, 5.0 mT; response time, 0.1 sec; amplitude, 1 • 200; and sweep time, 2 min. ESR spectra were measured at 23~ Scavenging activities were estimated using the relative peak height of ESR spectra of D M P O O O H and D M P O - O H spin adduct for superoxide anion and hydroxyl radical scavenging activities, respectively.
Measurement of Superoxide Anion Radical Scavenging Activity Fifty microliters of hypoxanthine oxidase (2.7 mg/5 ml) was first added to a test tube, followed by D M S O (30/~1), sample solution (50/zl), 4.5 M D M P O (20/xl), and X O D (0.4 units/ml) (50/zl). After vortex mixing for 10 sec at room temperature, the sample solution was transferred immediately
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into a quartz flat cell. ESR recording was started exactly 30 sec after the addition of XOD. Measurement of Hydroxyl Radical Scavenging Activity Fifty microliters of sample solution was added to a test tube followed by 0.18 M DMPO (50/,1), 2 mM H202 (50/,1), and 0.2 mM FeSO4 (50/,1). They were mixed by a vortex mixer for 10 sec. Exactly 30 sec after the addition of FeSO4 solution, ESR recording was started.
Statistics All data were expressed as means + SEM. Statistical analysis was performed using the Student's t test.
RESULTS Extraction of Materials Samples were extracted from ground Uyaku roots, dried Uyaku leaves, or dried Uyaku seeds using boiling water for 10 min, and the filtrates were freeze-dried overnight. The yield of the dried extracted materials was approximately 5, 12, and 5% for roots, leaves, and seeds, respectively.
Superoxide Anion Radical Scavenging Activity (SOD-like activity) Extracted materials were dissolved at concentrations of 0.1 to 0.5 mg/ml for untreated samples (control). Uyaku roots showed markedly potent scavenging activity (115.3 +_ 5.4 SOD equivalent units/mg of freezedried extract, n = 10). Uyaku leaf extracts also showed moderately potent scavenging activity (88.5 + 4.8 SOD equivalent units/mg of freeze-dried extract, n = 5), but the scavenging activity of Uyaku seed extracts was much less (16.2 + 0.2 SOD equivalent units/mg of freeze-dried extract, n = 4). These superoxide scavenging activities were not changed by the treatment with ascorbate oxidase in all Uyaku extracts. Filtered samples from Uyaku root and leaf extracts, which contain low molecular weight materials (less than 100,000 or 10,000) showed lower (roots: 56 or 48%, leaves: 57 or 51%) SOD-like activity when compared to untreated samples. The SOD-like activity of Uyaku seed extracts, was not changed by filtration (Fig. 2). Extracted samples from Uyaku roots, leaves, and seeds were dissolved at a concentration of 0.25 mg/ml, and hydroxyl radical scavenging activity
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Figure 2 Superoxide anion radical scavenging activity of Uyaku root, leaf, and seed extracts. Values are untreated (roots: n = 10; leaves: n = 5; seeds: n = 4), filtrates [MW < 100,000 (roots: n = 4; leaves: n = 4; seeds: n = 3) and MW < 10,000 (roots: n - 4; leaves: n = 4; seeds: n = 3)], and ascorbate oxidase-treated (roots: n = 3; leaves: n = 4; seeds: n = 4) samples. *p < 0.05, compared to untreated samples. Data expressed as means +_ SEM.
w a s e v a l u a t e d in c o m p a r i s o n w i t h EPC-K1. W e a k s c a v e n g i n g activities for h y d r o x y l r a d i c a l w e r e o b s e r v e d in U y a k u r o o t s , in leaves, a n d in seeds (Fig. 3).
DISCUSSION U y a k u r o o t s a n d leaves s h o w e d m a r k e d l y h i g h e r S O D - l i k e activities in c o m p a r i s o n w i t h several o t h e r n a t u r a l e x t r a c t s . A h i g h e r s c a v e n g i n g activity for free r a d i c a l s in n a t u r a l s o u r c e a n t i o x i d a n t s is s o m e t i m e s k n o w n to d e p e n d on a s c o r b a t e , w h i c h m a y be a c o m p o n e n t of this m a t e r i a l (9). C o m p a r e d w i t h o t h e r k n o w n n a t u r a l s o u r c e m a t e r i a l s u n d e r the s a m e c o n d i tions, U y a k u r o o t s , especially a s c o r b a t e o x i d a s e - t r e a t e d s a m p l e s , b e l o n g to the h i g h e s t class of S O D - l i k e activity, i.e., a l m o s t t h e s a m e activity of g r e e n
31. Uyaku 0,5
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NN untreated extracts
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0.4
B ascorbate oxidasetreated extracts
T
0
E v
0.3 r>
. ,,,.,=
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6 aUJ O l 9
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uyaku leaves
uyaku seeds
Figure
3 H y d r o x y l radical scavenging activity of U y a k u r o o t , leaf, a n d seed e x t r a c t s . Values are u n t r e a t e d (roots: n = 5; leaves: n = 5; seeds: n = 6) a n d a s c o r b a t e o x i d a s e - t r e a t e d (roots: n = 4; leaves: n = 4; seeds: n = 4) samples. D a t a e x p r e s s e d as m e a n s + SEM.
tea (Matsucha, Kyoto, Japan) (untreated: 189.7 + 16.6, n = 4; ascorbate oxidase treated: 134.9 ___26.4, n - 2) and pine bark extracts (Pycnogenol) (untreated: 115.1 +_ 5.5, n = 6; ascorbate oxidase treated: 111.0 ___ 8.5, n = 5) (9) (Fig. 4). However, the contents of effective components in herbs or natural source materials are known to vary depending on the production process, season of harvest, or storage conditions. Therefore, such comparative evaluation should be performed more carefully in the future. SOD-like activities in Uyaku samples are not dependent on the scavenging activity of ascorbate, as these activities were not changed by treatment with ascorbate oxidase. Filtered samples from Uyaku roots and leaves, which contain low molecular weight materials (less than 10,000 or 100,000), showed approximately half of the SOD-like activity compared with untreated samples. These two filtrates had similar SOD-like activity, indicating that approxi-
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r
200-
I
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9
untreated extracts WJ ascorbate oxidasetreated extracts
E e-= r (D ._ =
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0
Figure
4 C o m p a r i s o n of superoxide anion radical scavenging activities of root, leaf, and other natural extracts. Values are untreated (roots: n = 10; leaves: n = 5; all other natural extracts: n = 6) and ascorbate oxidase-treated (roots: n = 3; leaves: n = 4; all other natural extracts: n = 5) samples. *p < 0.05, c o m p a r e d to untreated samples. Data expressed as means + SEM.
mately half of the scavenging activity is probably due to the lower molecular weight materials. Another half of the SOD-like activity observed in untreated samples would be higher molecular weight proteins (MW 100,000), which may also contribute to SOD-like activity. No significant differences in SOD-like activities were observed between the fractions containing molecules of less than 10,000 and 100,000 in all samples of Uyaku. This finding suggests that SOD-like activities in Uyaku extracts were not dependent on the enzyme SOD itself, as the molecular weights of the SODs are known to be between 10,000 and 100,000 [Cu-SOD: 32,000; Mn-SOD: 42,000-45,000 or 85,000-90,000 depending on the monomer or dimer
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(10-13)]. Because the SOD-like activity in Uyaku seed extracts was not changed by filtration, this suggests that all SOD-like activity may be dependent on lower molecular materials. Many chemical constituents of Uyaku roots are known. Examples of substances reported to be present are borneol, linderane, linderalactone, isolinderalactone, neolinderalactone, linderstrenolide, linderene, lindenenone, lindestrene, linderene acetate, isolinderoxide, linderaic acid, linderazulene, chamazulene, laurolitsine, and isogermafurene (2, 3). The precise components responsible for SOD-like activity of Uyaku roots are presently unknown. In conclusion, Uyaku roots have strong superoxide anion radical scavenging activity. SOD-like activity suggests that Uyaku extracts, both from roots and leaves, may have beneficial effects for health, especially for prevention or treatment of free radical-related diseases.
REFERENCES 1. Makino, T. (1989). In "Makino's New Illustrated Flora of Japan" (revised edition), p. 126. Hokuryukan, Tokyo. [in Japanese] 2. Hsu, H.-Y., Chen, Y.-P., Shen, S,-J., Hsu, C.-S., Chen, C.-C., and Chang, H.-C. (1986). In "Oriental Materia Medica: A Concise Guide," p. 420. Oriental Healing Arts Institute, Long Beach, CA. 3. Bensky, D., and Gamble, A. (1986). In "Chinese Herbal Medicine." p. 341. Eastland Press, Seattle. 4. Duke, J. A., and Ayensu, E. S. (1985). In "Medical Plants of China," Vol. 2, p. 390. Reference Publications, Algonac, Michigan. 5. Maekawa, M. (1926). "Jo Fuku." Shingu Kanko Kyokai, Shingu. [in Japanese] 6. Tsuji, S. (1926). In "Jo Fuku Densetsu," Kanko Kyokai, Shingu. [in Japanese] 7. Okuno, T. (1991). In "Roman no Hito: Jo Fuku." Gakken Okuno Tosho, Shingu. [in Japanese] 8. Mori, A., Edamatsu, R., Kohno, M., and Ohmori, S. (1989). A new hydroxyl radical scavenger: EPC-K1. Neurosciences 15, 371. 9. Noda, Y., Anzai, K., Mori, A., Kohno, M., Shimmei, M., and Packer, L. (1997). Biochem. Mol. Biol. Int. 42, 35. 10. Steinman, H. M., Naik, V. R., Abernathy, J. L., and Hill, R. L. (1974). Bovine erythrocyte superoxide dismutase: Complete amino acid sequence. J. Biol. Chem. 249, 7326. 11. Barra, D., Martini, E, Bannister, J. V., Schinia, M. E., Rotilio, G., Barrister, W. H., and Bossa, E (1980). The complete amino acid sequence of human Cu/Zn superoxide dismutase. FEBS Lett. 120, 53. 12. Keele, B. B., McCord, J. M., and Fridovich, I. (1970). Superoxide dismutase from Escherichia coli B: A new manganese-containing enzyme. J. Biol. Chem. 245, 6176. 13. Salin, M. L., Day, E. D., and Crapo, J. D. (1978). Isolation and characterization of manganese-containing superoxide dismutase from rat liver. Arc. Biochem. Biophys. 187, 223.
Superoxide Anion Radical Scavenging Activity of Uyaku (Lindera strychnifolia), a Natural Extract Used in Traditional Medicine Yasuko Noda, * Akitane Mori, * Kazunori Anzai, t and Lester Packer* *Department of Molecular and Cell Biology University of California Berkeley, California 94720 tNational Institute of Radiological Sciences Chiba, Japan
Uyaku (Lindera strychnifolia: Sieb. et Zucc.) is well known as a traditional Asian medicine used in the treatment of stomach and renal diseases, neuralgia, rheumatism, and aging. However, the mechanism of its effectivity is not yet known. In this study, scavenging activities of superoxide anion and hydroxyl radicals were examined by electron spin resonance (ESR) using the spin-trapping reagent 5,5'-dimethyl-l-pyrroline-N-oxide (DMPO). Watersoluble components in both Uyaku root and leaf extracts showed potent superoxide anion scavenging activity. Uyaku seed extract showed weak superoxide anion scavenging activity. In all samples of Uyaku, scavenging activity was not diminished by ascorbate oxidase treatment. The filtrate, passed through a low molecular weight cutoff microcentrifuge filter (MW < 100,000 or < 10,000), showed approximately half the activity of untreated extract samples of Uyaku roots and leaf extracts, indicating that approximately half of the scavenging activity is due to the low molecular weight Antioxidant Food Supplements in Human Health Copyright 9 1999 by Academic Press. All rights of reproduction in any form reserved.
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components. To evaluate the hydroxyl radical scavenging activity, L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H- 1benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K1), which has vitamin E and vitamin C moieties and a high potency for hydroxyl radical scavenging, was used as a standard material. Results showed little hydroxyl radical scavenging activity in root and leaf extracts, although weak scavenging activity was demonstrated in the seed extract. These results demonstrated that Uyaku roots and leaves have specific and potent superoxide anion radical scavenging activity in comparison with several other natural extracts. Thus, Uyaku may have potential for beneficial effects for health, especially for diseases in which the production of superoxide anion radical is a factor.
INTRODUCTION
Uyaku (in Japanese, Wu Yao in Chinese, Oyak in Korean) is the dried roots of L. strychnifolia (Sieb. et Zucc.) E Villar of the Lauraceae family (1-3) and a traditional Asian medicine used in China as an astringent, carminative, stomachic or tonic, for asthma, cholera, congestion, dyspepsia, dysmenorrhea, fluxes, gonorrhea, hernia, malaria, menorrhagia, stomach ache, stroke, and urinary difficulties (4). According to a traditional story, the first Emperor of Qin Dynasty, about 2300 years ago, ordered Jo Fuku (Xu Fu in Chinese) to be brought to Horai, an island on the far east sea, looking for a drug effective for longevity. After a long sailing, he and his colleagues arrived at Shingu in Wakayama prefecture, Japan, in B.c. 219 and established "Tendai-uyaku" (another name for Uyaku, used especially in this district) (5-7). Uyaku might have been brought from China from 1716-1735, according to Makino (1). Uyaku has been used as a folk drug for good health and for the treatment of diseases such as stomach and renal diseases, neuralgia, and rheumatism in some districts, including Shingu, Wakayama prefecture, Japan (8, 9), although no scientific background for these treatments has been known until now. This study examines the effects of Uyaku (root, leaf, and seed extracts for their action) on reactive oxygen species using ESR spectrometry.
MATERIALS AND METHODS Chemicals
Common chemicals were from Sigma Chemical Co. (St. Louis, MO). Xanthine oxidase (XOD) was from Boehringer Mannheim Corp. (Indi-
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anapolis, IN). The spin trap DMPO was from Labotec (Tokyo, Japan). All chemicals were the highest grade available.
Standard Solutions SOD standard solutions were obtained as a kit (Dojindo Laboratories, Kumamoto, Japan). For measurements of hydroxyl radical scavenging activity, L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12trimethyltridecyl)-2H-l-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K1) (Senju Pharmaceutical Co. Ltd., Osaka, Japan) (8) was used as a standard. EPC-K1, which is composed of vitamin E and vitamin C by phosphate diester linkage, is water soluble and the solution is stable at room temperature. In this study, a stock solution of 10 mM EPC-K1 dissolved in 0.1 M phosphate buffer (pH 7.4) was prepared and diluted to obtain the appropriate concentrations (0-2 mM) before use. The stock solution was stored at 4~
Samples Sources Ground powder of Uyaku roots was kindly donated by Taikodo Pharmaceutical Co., Ltd. (Kobe, Japan). Dried leaves and seeds of Uyaku were collected in Wakayama, dried at room temperature for 1 month (leaves) or in a desiccator at 4~ (seeds), and ground with a mortar and pestle. To obtain the extract, the powder (5 g of Uyaku roots, 1.2 g of Uyaku leaves, or 10 g of Uyaku seeds) was added to 20 times the volume of water (1:20, by w/v), and the sample was extracted with boiling water for 10 min. After cooling to room temperature, the suspension was filtered (Whatman No. 2) and the recovered filtrate was freeze-dried. The sample was kept dry and at 4~ until use (Fig. 1). Treatments Freshly prepared sample solutions were used for assay. The extracted material was dissolved in 0.1 M phosphate buffer (pH 7.4) at concentrations from 0.1 to 0.5 mg/ml by vortex mixing (nontreatment). An aliquot of the sample was treated with ascorbate oxidase (20 units/ml of sample solution). The ascorbate oxidase stock solution dissolved in 0.1 M phosphate buffer, pH 7.4, was dialyzed at 4~ to eliminate possible contamination by sucrose. Another aliquot was passed through a centrifuge filter (UltrafreeMC Filters: 10,000 NMWL regenerated cellulose membrane; 100,000 NMWL polysulfone membrane, Millipore, MA) by centrifugation (5000 g,
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Procedure for Preparation of Uyaku Extracts Dried Uyaku roots, leaves or seeds sample (1-10 g) The sample was ground with a mortar and pestle The powder was weighed Powder was extracted with boiling water (1:20, by w/re) for 10 minutes at 100~ Extract was cooled to room temperature and filtrated using Whatman No. 2 filter paper Preparation was freeze-dried The sample was stored at 4~ in a silica-gel desiccator until use
Figure 1 Procedurefor preparation of Uyaku root, leaf, or seed extracts.
4~ to obtain materials of smaller molecular weights: less than 10,000 or 100,000.
ESR Measurements
ESR Spectrometer A new computerized, compact, highly sensitive ESR spectrometer (free radical monitor, JES FR-30, JEOL, Tokyo, japan) was used. This ESR system has the ability of normalizing all spectra for accurate calculation using manganese dioxide as an internal standard. Other details have been described in a previous report (9). Conditions for measurements in this study were as follows: magnetic field, 335.5 + 5 mT; microwave power, 4 mW; microwave frequency, 9.41 GHz; modulation width, 0.063 or 0.1 mT; sweep width, 5.0 mT; response time, 0.1 sec; amplitude, 1 • 200; and sweep time, 2 min. ESR spectra were measured at 23~ Scavenging activities were estimated using the relative peak height of ESR spectra of D M P O O O H and D M P O - O H spin adduct for superoxide anion and hydroxyl radical scavenging activities, respectively.
Measurement of Superoxide Anion Radical Scavenging Activity Fifty microliters of hypoxanthine oxidase (2.7 mg/5 ml) was first added to a test tube, followed by D M S O (30/~1), sample solution (50/zl), 4.5 M D M P O (20/xl), and X O D (0.4 units/ml) (50/zl). After vortex mixing for 10 sec at room temperature, the sample solution was transferred immediately
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into a quartz flat cell. ESR recording was started exactly 30 sec after the addition of XOD. Measurement of Hydroxyl Radical Scavenging Activity Fifty microliters of sample solution was added to a test tube followed by 0.18 M DMPO (50/,1), 2 mM H202 (50/,1), and 0.2 mM FeSO4 (50/,1). They were mixed by a vortex mixer for 10 sec. Exactly 30 sec after the addition of FeSO4 solution, ESR recording was started.
Statistics All data were expressed as means + SEM. Statistical analysis was performed using the Student's t test.
RESULTS Extraction of Materials Samples were extracted from ground Uyaku roots, dried Uyaku leaves, or dried Uyaku seeds using boiling water for 10 min, and the filtrates were freeze-dried overnight. The yield of the dried extracted materials was approximately 5, 12, and 5% for roots, leaves, and seeds, respectively.
Superoxide Anion Radical Scavenging Activity (SOD-like activity) Extracted materials were dissolved at concentrations of 0.1 to 0.5 mg/ml for untreated samples (control). Uyaku roots showed markedly potent scavenging activity (115.3 +_ 5.4 SOD equivalent units/mg of freezedried extract, n = 10). Uyaku leaf extracts also showed moderately potent scavenging activity (88.5 + 4.8 SOD equivalent units/mg of freeze-dried extract, n = 5), but the scavenging activity of Uyaku seed extracts was much less (16.2 + 0.2 SOD equivalent units/mg of freeze-dried extract, n = 4). These superoxide scavenging activities were not changed by the treatment with ascorbate oxidase in all Uyaku extracts. Filtered samples from Uyaku root and leaf extracts, which contain low molecular weight materials (less than 100,000 or 10,000) showed lower (roots: 56 or 48%, leaves: 57 or 51%) SOD-like activity when compared to untreated samples. The SOD-like activity of Uyaku seed extracts, was not changed by filtration (Fig. 2). Extracted samples from Uyaku roots, leaves, and seeds were dissolved at a concentration of 0.25 mg/ml, and hydroxyl radical scavenging activity
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Figure 2 Superoxide anion radical scavenging activity of Uyaku root, leaf, and seed extracts. Values are untreated (roots: n = 10; leaves: n = 5; seeds: n = 4), filtrates [MW < 100,000 (roots: n = 4; leaves: n = 4; seeds: n = 3) and MW < 10,000 (roots: n - 4; leaves: n = 4; seeds: n = 3)], and ascorbate oxidase-treated (roots: n = 3; leaves: n = 4; seeds: n = 4) samples. *p < 0.05, compared to untreated samples. Data expressed as means +_ SEM.
w a s e v a l u a t e d in c o m p a r i s o n w i t h EPC-K1. W e a k s c a v e n g i n g activities for h y d r o x y l r a d i c a l w e r e o b s e r v e d in U y a k u r o o t s , in leaves, a n d in seeds (Fig. 3).
DISCUSSION U y a k u r o o t s a n d leaves s h o w e d m a r k e d l y h i g h e r S O D - l i k e activities in c o m p a r i s o n w i t h several o t h e r n a t u r a l e x t r a c t s . A h i g h e r s c a v e n g i n g activity for free r a d i c a l s in n a t u r a l s o u r c e a n t i o x i d a n t s is s o m e t i m e s k n o w n to d e p e n d on a s c o r b a t e , w h i c h m a y be a c o m p o n e n t of this m a t e r i a l (9). C o m p a r e d w i t h o t h e r k n o w n n a t u r a l s o u r c e m a t e r i a l s u n d e r the s a m e c o n d i tions, U y a k u r o o t s , especially a s c o r b a t e o x i d a s e - t r e a t e d s a m p l e s , b e l o n g to the h i g h e s t class of S O D - l i k e activity, i.e., a l m o s t t h e s a m e activity of g r e e n
31. Uyaku 0,5
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T
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0.0 uyaku roots
uyaku leaves
uyaku seeds
Figure
3 H y d r o x y l radical scavenging activity of U y a k u r o o t , leaf, a n d seed e x t r a c t s . Values are u n t r e a t e d (roots: n = 5; leaves: n = 5; seeds: n = 6) a n d a s c o r b a t e o x i d a s e - t r e a t e d (roots: n = 4; leaves: n = 4; seeds: n = 4) samples. D a t a e x p r e s s e d as m e a n s + SEM.
tea (Matsucha, Kyoto, Japan) (untreated: 189.7 + 16.6, n = 4; ascorbate oxidase treated: 134.9 ___26.4, n - 2) and pine bark extracts (Pycnogenol) (untreated: 115.1 +_ 5.5, n = 6; ascorbate oxidase treated: 111.0 ___ 8.5, n = 5) (9) (Fig. 4). However, the contents of effective components in herbs or natural source materials are known to vary depending on the production process, season of harvest, or storage conditions. Therefore, such comparative evaluation should be performed more carefully in the future. SOD-like activities in Uyaku samples are not dependent on the scavenging activity of ascorbate, as these activities were not changed by treatment with ascorbate oxidase. Filtered samples from Uyaku roots and leaves, which contain low molecular weight materials (less than 10,000 or 100,000), showed approximately half of the SOD-like activity compared with untreated samples. These two filtrates had similar SOD-like activity, indicating that approxi-
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untreated extracts WJ ascorbate oxidasetreated extracts
E e-= r (D ._ =
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Figure
4 C o m p a r i s o n of superoxide anion radical scavenging activities of root, leaf, and other natural extracts. Values are untreated (roots: n = 10; leaves: n = 5; all other natural extracts: n = 6) and ascorbate oxidase-treated (roots: n = 3; leaves: n = 4; all other natural extracts: n = 5) samples. *p < 0.05, c o m p a r e d to untreated samples. Data expressed as means + SEM.
mately half of the scavenging activity is probably due to the lower molecular weight materials. Another half of the SOD-like activity observed in untreated samples would be higher molecular weight proteins (MW 100,000), which may also contribute to SOD-like activity. No significant differences in SOD-like activities were observed between the fractions containing molecules of less than 10,000 and 100,000 in all samples of Uyaku. This finding suggests that SOD-like activities in Uyaku extracts were not dependent on the enzyme SOD itself, as the molecular weights of the SODs are known to be between 10,000 and 100,000 [Cu-SOD: 32,000; Mn-SOD: 42,000-45,000 or 85,000-90,000 depending on the monomer or dimer
31. Uyaku
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(10-13)]. Because the SOD-like activity in Uyaku seed extracts was not changed by filtration, this suggests that all SOD-like activity may be dependent on lower molecular materials. Many chemical constituents of Uyaku roots are known. Examples of substances reported to be present are borneol, linderane, linderalactone, isolinderalactone, neolinderalactone, linderstrenolide, linderene, lindenenone, lindestrene, linderene acetate, isolinderoxide, linderaic acid, linderazulene, chamazulene, laurolitsine, and isogermafurene (2, 3). The precise components responsible for SOD-like activity of Uyaku roots are presently unknown. In conclusion, Uyaku roots have strong superoxide anion radical scavenging activity. SOD-like activity suggests that Uyaku extracts, both from roots and leaves, may have beneficial effects for health, especially for prevention or treatment of free radical-related diseases.
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