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Free radical scavenging action of the medicinal herb Limonium wrightii from the Okinawa islands
Phytomedicine 9: 239–244, 2002 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/phytomed
Phytomedicine
Free radical scavenging action of the medicinal herb Limonium wrightii from the Okinawa islands Y. Aniya1, C. Miyagi1, A. Nakandakari1, S. Kamiya1, N. Imaizumi1, and T. Ichiba2 1
Laboratory of Physiology and Pharmacology, School of Health Sciences, Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa, Japan, 2 Okinawa Industrial Technology Center, Gushikawa, Okinawa, Japan
Summary Free radical scavenging action of Limonium wrightii O. kunthe was examined in vitro and in vivo by using electron spin resonance spectrometer and chemiluminescence analyzer. A water extract of L. wrightii showed a strong scavenging action for the 1,1-diphenyl-2-picrylhydrazyl, or superoxide anion and moderate for hydroxyl radical. The extract also depressed production of reactive oxygen species from polymorphonuclear leukocytes stimulated by phorbor-12-mysistate acetate and inhibited lipid peroxidation in rat liver microsomes. When the extract was given intraperitoneally to mice prior to carbon tetrachloride (CCl4) treatment, CCl4-induced liver toxicity, as seen by an elevation of serum aspartate aminotransferase and alanine aminotransferase activities, was significantly reduced. Gallic acid was identified as the active component of L. wrightii with a strong free radical scavenging action. Our results demonstrate the free radical scavenging action of L. wrightii and that gallic acid contributes to these actions. Key words: antioxidant, medicinal herb, carbon tetrachloride, free radical, Limonium
j Introduction Limonium Wrightii O. Kunthe. (Japanese name; Ukonisomatsu) grows wildly in a seaside district of the Okinawa Islands and is known in a folk medicine for treatment of fever or arthritis. However, biological and pharmacological actions of the herb have not been studied. As preliminary experiments, we screened the antioxidant action of various medicinal herbs by measuring the scavenging action of a stable radical, 1,1diphenyl-2-picrylhydrazyl (DPPH) and found that L. wrightii has a strong antioxidant action. Reactive oxygen species (ROS) generated endogenously or exogenously are associated with the pathogenesis of various diseases such as atherosclerosis, diabetes, cancer, arthritis and the aging process (Guyton et al., 1997; Halliwell and Gutteridge, 1999; Jaeschke and Mitchell, 1989; Kehrer, 1993; Okuda et al., 1991).
Thus, antioxidants which can scavenge ROS are expected to improve these disorders. Since carbon tetrachloride is biotransformed to a trichloromethyl radical by the cytochrome P450 system in liver microsomes followed by liver injury (McCay et al., 1984; Recknagel, 1983; Slater, 1984), this compound has been used for the evaluation of free radical scavenging action in vivo. In the present study, the radical scavenging action of L. wrightii was examined in vitro and in vivo and the antioxidant component was identified.
Abbreviations: CL – chemoluminescence; PMA – phorbol 12-myristate 13-acetate; DPPH – 1,1-diphenyl-2-picrylhydrazyl: AST – aspartate aminotransferase; ALT – alanine aminotransferase; LPO – lipid peroxidation 0944-7113/02/09/03-239 $ 15.00/0
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Reduced glutathione (GSH), glucose 6-phosphate, phorbol 12-myristate 13-acetate (PMA) and β-nicotinamide adenine dinucleotide phosphate were purchased from Sigma Chemicals (St. Louis, MO, U.S.A.). 1-Chloro-2,4dinitrobenzene (CDNB), 1,1-diphenyl-2-picrylhydrazyl (DPPH), luminol (3-aminophtalhydrazide), hypoxanthine, hydrogen peroxide (H2O2) and 2-thiobarbituric acid (TBA) were from Wako Pure Chemicals (Osaka, Japan). Glucose 6-phosphate dehydrogenase and xanthine oxidase were obtained from Oriental Yeast (Tokyo, Japan) and Boehringer Mannheim Gmbh (Mannheim, Germany). Diethylene triamine pentaacetic acid (DTPA), 5,5’dimethyl-1-pyrroline-N-oxide (DMPO) and dimethyl sulfoxide were from Dojindo Laboratories (Kumamoto, Japan). All reagents used were of analytical grade. Preparation of the Herbal Extract and Crude Antioxidant
Dried L. wrightii was supplied by a company (Nakazen Co., Ltd.) which cultivates medicinal herbs in Okinawa, Japan. Whole plant was dried at 60 °C overnight. One gram of the herb was extracted with 50 ml of water at 37 °C for 2 h and filtrated by filter paper. The filtrate thus obtained was used as the original herbal extract. Moreover, the extract of L. wrightii at 90 °C for 2 h was dried by a spray-dry method and was used as a crude antioxidant preparation. Measurement of Free Radical Scavenging Action
The antioxidant activity of the extract was examined using the DPPH radical. The reaction mixture consisted of 1 ml of 0.1 mM DPPH in ethanol, 0.95 ml of 0.05 M Tris-HCl buffer (pH 7.4), 1 ml of ethanol and 0.05 ml of the herbal extract or deionized water (control). The absorbance of the mixture was measured at 517 nm exactly 30 s after adding the extract. The radical scavenging ability of the extract from L. wrightii for superoxide anion (O2–) generated by xanthine/xanthine oxidase and hydroxyl radical (·OH) by Fenton reaction (FeSO4/H2O2) was determined by an electron spin resonance (ESR) spectrometer (JESFR30, JEOL, Tokyo, Japan), as described previously (Myagmar and Aniya, 2000). The effect of the extract on CL-production from polymorphonuclear leukocytes stimulated with PMA was also evaluated with a chemiluminescence analyzer CLD-110 (Tohoku Electronic Industrial Co. Sendai, Japan) by the same conditions as reported previously (Myagmar and Aniya, 2000) except that leukocytes which were isolated from human peripheral blood was used. The blood sample was taken from a male healthy volunteer by an authorized medical technologist.
The effect of the extract on xanthine oxidase activity was measured according to the method described in the previous report (Myagmar and Aniya, 2000). Effect of Antioxidants on Lipid Peroxidation in Rat Liver Microsomes
Liver microsomes were prepared from non-treated rats (male Sprague-Dawley rats, 200–300 g) as reported previously (Aniya and Naito, 1993) and were kept at –80 °C until use. The microsomes (2–3 mg/ml) were incubated with the NADPH generating system (0.33 mM NADP, 8 mM G6P, 6 mM MgCl2 and 0.5 U G6PDH) and the herbal extracts or ascorbic acid in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C for 30 min in a total volume of 1 ml. Lipid peroxide was measured as thiobarbituric acid reactive substance. Following incubation, the peroxidation was terminated by 20% acetic acid and 0.5% 2-thiobarbituric acid was subsequently added followed by heating at 100 °C for 60 min. After cooling, 1-butanol was added followed by vigorous shaking and centrifugation. The absorbance of the butanol fraction was measured at 532 nm. 1,1,3,3Tetramethoxypropane was used as authentic standard. The protein concentration of microsomes was determined by the method of Lowry et al. (Lowry et al., 1951). Hepatoprotective Action of the Herbal Extract
Mice (ddY, 8 weeks) from Nihon SLC Co (Shizuoka) were divided into three groups (CCl4, CCl4 + herbal extract and control) at random. The CCl4 group of mice was given carbon tetrachloride (CCl4, 2 ml/kg in 50% corn oil solution) subcutaneously (s.c.). The herbal extract plus CCl4 group was given original extract (5 ml/kg) intraperitoneally (i.p.) 1 and 15 h before CCl4 treatment. Control mice were given corn oil (2 ml/kg, s.c.) in place of CCl4. Mice were killed by decapitation 72 h after CCl4 treatment after overnight starvation. To evaluate the effect of the herb alone on the parameters studied, the extract of L. wrightii (5 ml/kg) or water was given to mice intraperitoneally twice at 1 and 15 h before corn oil (2 ml/kg, s.c.) injection, then the mice were sacrificed 72 h after the last injection. In all cases blood was collected from the stump and serum was isolated by centrifugation. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in the serum were measured using an Assay Kit (Kainos, Tokyo). All experiments were conducted in conformity with the guidelines set by the Animal Care and Use Committee of the University of the Ryukyus. Isolation and Identification of Antioxidants from L. wrightii
Dried herb (50 g) was extracted two times with 25 mL of ethanol/water 1:1 by an accelerated solvent extractor
Free radical scavenging action of the medicinal herb Limonium wrightii
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Table 1. Comparision of radical cavenging action. IC50 (µg/ml)
Fig. 1. DPPH radical scavenging activity of the extract of L. wrightii. Water extract of L. wrightii was diluted, then mixed with DPPH (100 µM). Scavenging activity of the extracts for the DPPH radical was measured as described in Materials and Methods.
(ASE-200, Dionex corporation, CA, USA) to give 500 mg of a crude extract that showed the DPPH radical scavenging activity. The resulted extract was chromatographed on a Toyopearl HW-40F column (Tosoh corporation, Tokyo, Japan) with methanol/ water 1:1 followed by HPLC (Symmetry C18, Waters, USA) to yield compound 1 (1.8 mg, a colorless solid). The structure of the compound was identified by UV, 1H NMR and 13C NMR spectra. Statistical Analyses
Data were expressed as the mean ± S.D. The significance of difference was calculated by Student’s t-test and values of p < 0.05 were used as significant.
DPPH Superoxid anion Hydroxyl radical CL-emission Lipid peroxidation
Crude preparation
Gallic acid
Vitamin C
500 0.85 1300 7.5 200
2.63 2.1 0.03 6.1 > 18800 20 113 3160 50 180
j Results Radical Scavenging Action of L. wrightii
Figure 1 shows the DPPH scavenging action of L. wrightii. The DPPH radical was scavenged by the water extract of L. wrightii dose dependently, and the extract with 50% DPPH scavenging action was 2.1times dilution of the original herbal extract (1 g/50 ml H2O). Superoxide anion and hydroxyl radical scavenging actions of L. wrightii were measured by ESR spectrometer (Fig. 2). The extract at 80 times and 640 times dilution scavengd 77% and 53% of the superoxide anion, respectively and 60% and 25% of the hydroxyl radicals were scavenged by the addition of twice the
Fig. 2. Effect of L. wrightii extract on superoxide anion (A) and hydroxyl radical (B). A: hypoxanthine (1.5 µM), xanthine oxidase (0.3 unit) and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) (135 mM) were mixed with the water extract in 0.1 M phosphate buffer (pH 7.4) in the presence of diethylenetriamine penta acetic acid (DTPA) (100 µM) at room temperature, and the ESR signal for DMPO-OOH after 50 sec was monitored (g = 2.006, aN = 1.418 mT, αβH = 1.142 mT, αγH = 0.137 mT). ESR spectra were recorded at following conditions: power 4 mW, modulation width 79 µT, amplitude 200, and time constant 0.1 sec. B: FeSO4 (0.05 mM), H2O2 (2.5 mM) and DMPO (45 mM) were mixed in 0.1 M phosphate buffer (pH 7.4) in the presence of DTPA (100 µM) with the extract, and the ESR signal for DMPO-OH after 50 sec was monitored (g = 2.007, αN = 1.488 mT, αβ = 1.488 mT). Recording conditions are as follows: power 4 mW, modulation width 0.1 mT, amplitude 50 and time constant 0.1 sec. Dilution of the extract was as follows. 320 (× 320) and 80 (× 80) dilution, original extract (× 1), twice the original extract (× 0.5).
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Y. Aniya et al. original extract (× 0.5) and the original (× 1) extract, respectively. Based on the antioxidant preparation, the IC50 (50% inhibition concentration) for superoxide anion and hydroxyl radical was 0.85 mg/ml and 1.3 mg/ml, respectively (Table 1). The crude antioxidant preparation inhibited xanthine oxidase activity with IC50 of 2.5 mg/ml. The xanthine oxidase inhibitor allopurinol, which was used as a standard, showed 64% inhibition of the enzyme at 0.68 mg/ml. Effect of the Herbal Extract on Carbon Tetrachloride-Induced Hepatotoxicity
Fig. 3. Effect of peritoneal injection of L. wrightii on CCl4treated mice. Water (control) or the original extract from L. wrightii (UK, 5 ml/kg) was given intraperitoneally 1 and 15 h before CCl4 (2 ml/kg, 50% corn oil solution, subcutaneously) treatment. In the case of treatment with the herbal extract alone, the extract (5 ml/kg) or water (control) was given, followed by corn oil injection in place of CCl4. Mice were killed 72 h after CCl4 injection after overnight starvation. Liver and serum parameters were measured as described in Materials and Methods. Each column shows the mean ± S.D. from 6 (control), 3 (CCl4-treated), 4 (UK alone) and 5 (CCl4 + the extract treated) mice. Control values are as follows: AST; 141.0 ± 10.1 Karman unit, ALT; 21.7 ± 3.7 Karman unit. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control, # p < 0.05, ## p < 0.01 vs. CCl4-treated.
Fig. 4. Structure of the active component of L. wrightii.
Figure 3 show the effect of the intraperitoneal injection of L. wrightii extract on CCl4-induced liver toxicity. Serum AST and ALT activities were increased to 383% and 374% of the control, respectively, by CCl4 treatment, and the increases were significantly decreased to 140% and 220%, respectively, when the herbal extract was given intraperitoneally twice before CCl4 treatment. The serum enzyme activities were not changed by intraperitoneal treatment of mice with the herbal extract alone. Thus the hepatoprotective action of L. wrightii extract against CCl4-induced liver toxicity was clarified. Isolation and Identification of an Active Component of L. wrightii
The active component from L. wrightii was separated with various column chromatography steps in which the antioxidant action was evaluated by the DPPH scavenging action. The isolated compound was identified as gallic acid by direct comparison of UV, 1H NMR and 13C NMR spectra with those of the authentic sample (Fig. 4). Compound 1 (gallic acid): UV (MeOH) λmax 215, 271 nm; APCI-MS, positive ion, m/z 171 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 6.90 (2H, s); 13C NMR (100 MHz, DMSO-d6) δ 167.5 (s), 145.4 (s, x 2), 137.9 (s), 120.5 (s), 108.7 (d, x 2). Antioxidant Action of the Active Component
Fig. 5. Effect of crude antioxidant preparation and gallic acid on CL production from leukocytes stimulated by PMA. Polymorphonuclear leukocytes isolated from human blood were stimulated at 37 °C with PMA in the presence or absence of the antioxidants and thus generated luminol-CL was measured for 6 min. The inhibitory activity of antioxidants was shown as percentage of total CL count of control. d – crude antioxidant; m – gallic acid; s – ascorbic acid (V.C).
The radical scavenging action of the gallic acid was compared with the crude antioxidant preparation and ascorbic acid. Figure 5 indicates the effect of the crude antioxidant and gallic acid on the leukocytes-derived CL. The CL emission was depressed dose-dependently by an incubation of the leukocytes with the antioxidants and the values of IC50 of the crude preparation and gallic acid were 7.5 µg/ml and 113 µg/ml, respectively. These effects of the crude antioxidant were stronger than that of ascorbic acid (IC50 = 3.16 µg/ml). Lipid peroxidation derived from cytochrome P450 system was also inhibited by 50% at 0.2 µg/ml of the crude preparation, at 50 µg/ml of gallic acid and at 180 µg/ml of ascorbic acid
Free radical scavenging action of the medicinal herb Limonium wrightii (Table 1). IC50 values of the crude antioxidant and gallic acid for superoxide anion were 0.85 µg/ml and 0.03 µg/ml, respectively and these are stronger than that of ascorbic acid. For hydroxyl radical, ascorbic acid showed the strongest action among the three antioxidants. These results are summarized in Table 1.
j Discussion The antioxidant action of water extract of L. wrightii was evaluated in vitro and in vivo. The water extract from the herb markedly scavenged the superoxide anion and DPPH radicals and moderately the hydroxyl radical. DPPH can abstract labile hydrogen (Constantin et al., 1990), and the ability to scavenge the DPPH radical is related to the inhibition of lipid peroxidation (Rekka and Kourounakis, 1991). In our laboratory, it was confirmed that the DPPH scavenging activity was parallel to the inhibitory action of lipid peroxidation using an extract of mold or medicinal herbs (Aniya et al., 1999; Gyamfi et al., 1999). Since hydroxyl radicals or superoxide anions are generated under oxidative stress caused by various chemicals or disorders, it was suggested that L. wrightii may ameliorate oxidative stress-induced disorders. In the present study, the effect of the herbal extract on CCl4-induced liver injury was examined. CCl4 is known to cause liver injury via metabolic activation by the microsomal cytochrome P450-dependent monooxygenase system. CCl4 is converted to the reactive intermediate, trichloromethyl radical (·CCl3) and peroxyl radical (·OOCCl3), which in turn react with macromolecules such as lipid and protein, leading to lipid peroxidation and cell injury (McCay et al., 1984; Recknagel, 1983; Slater, 1984). When the extract from L. wrightii was given to mice intraperitoneally prior to CCl4 treatment, CCl4-induced hepatotoxicity seen in the elevation of serum AST and ALT levels was significantly depressed (Fig. 3). Thus it was clarified that the extract of L. wrightii has a strong radical scavenging action by which it ameliorates the CCl4-induced liver toxicity. Since the water extract of L. wrightii possessed a radical scavenging action, we tried to isolate active components from L. wrightii and gallic acid was identified as the main antioxidant of L. wrightii. The antioxidant action of the active component, gallic acid was compared with the crude antioxidant preparation of L. wrightii and with the water soluble antioxidant ascorbic acid. The crude preparation of L. wrightii showed very strong scavenging action for superoxide anion generated from xanthine/xanthine oxidase system (IC50 = 0.85 µg/ml) and also markedly inhibited CL production from PMA-stimulated leukocytes (7.5 µg/ml). To understand the strong scavenging action for super-
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oxide anion we examined whether the crude antioxidant preparation can inhibit xanthine oxidase activity. The crude antioxidant inhibited xanthine oxidase activity with IC50 of 2.5 mg/ml, suggesting that the crude antioxidant could directly scavenge superoxide anion. In PMA-stimulated leukocytes superoxide anion is produced via the NADPH oxidase followed by generation of H2O2 and hypochlorous acid which in turn reacts with luminol resulting in CL-emision. Thus it was suggested that the extract of L. wrightii could scavenge superoxide anion resulting in depression of CL production. Moreover, the inhibitory action of CL-emission from the leukocytes by the crude preparation was more potent than that of gallic acid (IC50 = 113 µg/ml). It is therefore assumed that although gallic acid is the main antioxidant of L. wrightii, other minor component might contribute to the radical scavenging action. The herb extract also showed hydroxyl radical scavenging action (Fig. 2). We observed that gallic acid has a Fe-chelating action, however the herbal extract shows very low iron chelating action (data not shown). Moreover hydroxyl radical scavenging action of gallic acid was low compared with that of crude preparation of the herb (Table 1). It was therefore suggested that the extract contains a component which can scavenge directly hydroxyl radical. The crude antioxidant preparation and gallic acid inhibited NADPH-derived LPO in liver microsomes as well as ascorbic acid (Table 1). It is well known that cytochrome P450 system in microsomes can generate superoxide anion, H2O2 and hydroxyl radical following by LPO. We also observed by ESR spectrometer that superoxide anion was firstly formed in rat liver microsomes in the presence of the NADPH generating system and followed by hydroxyl radical formation. This superoxide anion and also hydroxyl radical formation were inhibited by the addition of the crude antioxidant and also by gallic acid (data not shown). Thus it was suggested that superoxide anion generated from the cytochrome P450 system is mostly scavenged by the antioxidants resulting in depression of LPO. As reported elsewhere that gallic acid as a component of traditional medicine could depress CCl4-induced liver toxicity (Anand et al., 1997; Kanai and Okano, 1998), it was suggested that gallic acid in L. wrightii contribute to the in vitro and in vivo antioxidant action of the herb. L. wrightii is known in a folk medicine for the treatment of fever or arthritis. Recently anti-tumor promoting action of L. wrightii has been demonstrated (unpublished data). Gallic acid, identified as the antioxidant of L. wrightii, is well known as a natural antioxidant and has been reported to possess anti-apoptosis, antiallergic, antiinflamatory, antimutagenic and anticarcinogenic actions (Gali et al., 1991; Gali et al.,
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1992; Hayatsu et al., 1988; Perchellet et al., 1992; Sakaguchi et al., 1998; Singleton, 1981; Stich and Rosin, 1984). In addition the metabolism of gallic acid in the human body was clarified (Shahrzad and Bitsch, 1998). Thus L. wrightii may be expected to be preventive of free radical mediated disorders such as inflammation or cancer. In summary, the medicinal herb L. wrightii has a strong scavenging action of hydroxyl, superoxide anion and DPPH radicals. This herb also depressed CL-emission from PMA-stimulated leukocytes and was protective against liver injury caused by CCl4, suggesting that CCl4-derived free radicals are scavenged by L. wrightii. Gallic acid, identified as the antioxidant of L. wrightii, may contribute to the radical scavenging action of L. wrightii. Acknowledgements
The authors thank Mr. K. Nakamoto of Nakazen Company for his kindness in supplying the medicinal herb, and Ms. N. Murayama for typing the manuscript. This study was partly supported by Grant-in-Aid for University and Society Collaboration.
j References Anand, K.K., Singh, B., Saxena, A.K., Chandan, B.K., Gupta, V.N., Bhardwaj, V.: 3,4,5-Trihydroxy benzoic acid (gallic acid), the hepatoprotective principle in the fruits of Terminalia belerica-bioassay guided activity. Pharmacol. Res. 36: 315–321, 1997. Aniya, Y., Naito, A.: Oxidative stress-induced activation of microsomal glutathione S-transferase in isolated rat liver. Biochem. Pharmacol. 45: 37–42, 1993. Aniya, Y., Yokomakura, T., Yonamine, M., Shimada, K., Nagamine, T., Shimabukuro, M., Gibo, H.: Screening of antioxidant action of various molds and protection of Monascus anka against experimentally induced liver injuries of rats. Gen. Pharmacol. 32: 225–231, 1999. Constantin, M., Bromont, C., Fickat, R., Massingham, R.: Studies on the activity of bepridil as a scavenger of free radicals. Biochem. Pharmacol. 40: 1615–1622, 1990. Gali, H.U., Perchellet, E.M., Perchellet, J.P.: Inhibition of tumor promoter-induced ornithine decarboxylase activity by tannic acid and other polyphenols in mouse epidermis in vivo. Cancer Res. 51: 2820–2825, 1991. Gali, H.U., Perchellet, E.M., Klish, D.S., Johnson, J.M., Perchellet, J.P.: Antitumor-promoting activities of hydrolyzable tannins in mouse skin. Carcinogenesis 13: 715–718, 1992. Guyton, K.Z., Gorospe, M., Holbrook, N.J.: Oxidative stress and the molecular biology of antioxidant defenses. Scandalios, J.G., editor. Cold Spring Harbor Laboratory Press, New York, pp. 247-272, 1997. Gyamfi, M.A., Yonamine, M., Aniya, Y.: Free-radical scavenging action of medicinal herbs from Ghana: Thonningia sanguinea on experimentally-induced liver injuries. Gen. Pharmacol. 32: 661–667, 1999. Halliwell, B., Gutteridge, J.M.C.: Free radicals in biology and medicine. Oxford University Press, 1999.
Hayatsu, H., Arimoto, S., Negishi, T.: Dietary inhibitors of mutagenesis and carcinogenesis. Mutat. Res. 202: 429–446, 1988. Jaeschke, H., Mitchell, J.R.: Mitochondria and xanthine oxidase both generate reactive oxygen species in isolated perfused rat liver after hypoxic injury. Biochem. Biophys. Res. Commun. 160: 140-147, 1989. Kanai, S., Okano, H.: Mechanism of the protective effects of sumac gall extract and gallic acid on the progression of CCl4-induced acute liver injury in rats. Am. J. Chin. Med. 26: 333–341, 1998. Kehrer, J.P.: Free radicals as mediators of tissue injury and disease. Crit. Rev. Toxicol. 23: 21–48, 1993. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951. McCay, P.B., Lai, E.K., Poyer, J.L., DuBose, C.M., Janzen, E.G.: Oxygen- and carbon-centered free radical formation during carbon tetrachloride metabolism. Observation of lipid radicals in vivo and in vitro. J. Biol. Chem. 259: 2135–2143, 1984. Myagmar, B.-E., Aniya, Y.: Free radical scavenging action of medicinal herbs from Mongolia. Phytomedicine 7: 221–229, 2000. Okuda, M., Ikai, I., Chance, B., Kumar, C.: Oxygen radical production during ischemia-reperfusion in the isolated perfused rat liver as monitored by luminol enhanced chemiluminescence. Biochem. Biophys. Res. Commun. 174: 217–221, 1991. Perchellet, J.P., Gali, H.U., Perchellet, E.M., Klish, D.S., Armbrust, A.D.: Antitumor-promoting activities of tannic acid, ellagic acid, and several gallic acid derivatives in mouse skin. Basic Life Sci. 59: 783–801, 1992. Recknagel, R.O.: Carbon tetrachloride hepatotoxicity: status quo and future prospects. Trends Pharmacol. Sci. 4: 129–131, 1983. Rekka, E., Kourounakis, P.N.: Effect of hydroxyethyl rutosides and related compounds on lipid peroxidation and free radical scavenging activity. Some structural aspects. J. Pharm. Pharmacol. 43: 486–491, 1991. Sakaguchi, N., Inoue, M., Ogihara, Y.: Reactive oxygen species and intracellular Ca2+, common signals for apoptosis induced by gallic acid. Biochem. Pharmacol. 55: 1973–1981, 1998. Shahrzad, S., Bitsch, I.: Determination of gallic acid and its metabolites in human plasma and urine by high-performance liquid chromatography. J. Chromatogr. B 705: 87–95, 1998. Singleton, V.L.: Naturally occurring food toxicants: phenolic substances of plant origin common in foods. Adv. Food Res. 27: 149–242, 1981. Slater, T.F.: Free-radical mechanisms in tissue injury. Biochem. J. 222: 1–15, 1984. Stich, H.F., Rosin, M.P.: Naturally occurring phenolics as antimutagenic and anticarcinogenic agents. Adv. Exp. Med. Biol. 177: 1–29, 1984.
j Address Yoko Aniya, Ph.D. Laboratory of Physiology and Pharmacology, School of Health Sciences, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan. Tel: ++81-98-895-1251; Fax: ++81-98-895-1443; e-mail: [email protected]
Phytomedicine
Free radical scavenging action of the medicinal herb Limonium wrightii from the Okinawa islands Y. Aniya1, C. Miyagi1, A. Nakandakari1, S. Kamiya1, N. Imaizumi1, and T. Ichiba2 1
Laboratory of Physiology and Pharmacology, School of Health Sciences, Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa, Japan, 2 Okinawa Industrial Technology Center, Gushikawa, Okinawa, Japan
Summary Free radical scavenging action of Limonium wrightii O. kunthe was examined in vitro and in vivo by using electron spin resonance spectrometer and chemiluminescence analyzer. A water extract of L. wrightii showed a strong scavenging action for the 1,1-diphenyl-2-picrylhydrazyl, or superoxide anion and moderate for hydroxyl radical. The extract also depressed production of reactive oxygen species from polymorphonuclear leukocytes stimulated by phorbor-12-mysistate acetate and inhibited lipid peroxidation in rat liver microsomes. When the extract was given intraperitoneally to mice prior to carbon tetrachloride (CCl4) treatment, CCl4-induced liver toxicity, as seen by an elevation of serum aspartate aminotransferase and alanine aminotransferase activities, was significantly reduced. Gallic acid was identified as the active component of L. wrightii with a strong free radical scavenging action. Our results demonstrate the free radical scavenging action of L. wrightii and that gallic acid contributes to these actions. Key words: antioxidant, medicinal herb, carbon tetrachloride, free radical, Limonium
j Introduction Limonium Wrightii O. Kunthe. (Japanese name; Ukonisomatsu) grows wildly in a seaside district of the Okinawa Islands and is known in a folk medicine for treatment of fever or arthritis. However, biological and pharmacological actions of the herb have not been studied. As preliminary experiments, we screened the antioxidant action of various medicinal herbs by measuring the scavenging action of a stable radical, 1,1diphenyl-2-picrylhydrazyl (DPPH) and found that L. wrightii has a strong antioxidant action. Reactive oxygen species (ROS) generated endogenously or exogenously are associated with the pathogenesis of various diseases such as atherosclerosis, diabetes, cancer, arthritis and the aging process (Guyton et al., 1997; Halliwell and Gutteridge, 1999; Jaeschke and Mitchell, 1989; Kehrer, 1993; Okuda et al., 1991).
Thus, antioxidants which can scavenge ROS are expected to improve these disorders. Since carbon tetrachloride is biotransformed to a trichloromethyl radical by the cytochrome P450 system in liver microsomes followed by liver injury (McCay et al., 1984; Recknagel, 1983; Slater, 1984), this compound has been used for the evaluation of free radical scavenging action in vivo. In the present study, the radical scavenging action of L. wrightii was examined in vitro and in vivo and the antioxidant component was identified.
Abbreviations: CL – chemoluminescence; PMA – phorbol 12-myristate 13-acetate; DPPH – 1,1-diphenyl-2-picrylhydrazyl: AST – aspartate aminotransferase; ALT – alanine aminotransferase; LPO – lipid peroxidation 0944-7113/02/09/03-239 $ 15.00/0
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Reduced glutathione (GSH), glucose 6-phosphate, phorbol 12-myristate 13-acetate (PMA) and β-nicotinamide adenine dinucleotide phosphate were purchased from Sigma Chemicals (St. Louis, MO, U.S.A.). 1-Chloro-2,4dinitrobenzene (CDNB), 1,1-diphenyl-2-picrylhydrazyl (DPPH), luminol (3-aminophtalhydrazide), hypoxanthine, hydrogen peroxide (H2O2) and 2-thiobarbituric acid (TBA) were from Wako Pure Chemicals (Osaka, Japan). Glucose 6-phosphate dehydrogenase and xanthine oxidase were obtained from Oriental Yeast (Tokyo, Japan) and Boehringer Mannheim Gmbh (Mannheim, Germany). Diethylene triamine pentaacetic acid (DTPA), 5,5’dimethyl-1-pyrroline-N-oxide (DMPO) and dimethyl sulfoxide were from Dojindo Laboratories (Kumamoto, Japan). All reagents used were of analytical grade. Preparation of the Herbal Extract and Crude Antioxidant
Dried L. wrightii was supplied by a company (Nakazen Co., Ltd.) which cultivates medicinal herbs in Okinawa, Japan. Whole plant was dried at 60 °C overnight. One gram of the herb was extracted with 50 ml of water at 37 °C for 2 h and filtrated by filter paper. The filtrate thus obtained was used as the original herbal extract. Moreover, the extract of L. wrightii at 90 °C for 2 h was dried by a spray-dry method and was used as a crude antioxidant preparation. Measurement of Free Radical Scavenging Action
The antioxidant activity of the extract was examined using the DPPH radical. The reaction mixture consisted of 1 ml of 0.1 mM DPPH in ethanol, 0.95 ml of 0.05 M Tris-HCl buffer (pH 7.4), 1 ml of ethanol and 0.05 ml of the herbal extract or deionized water (control). The absorbance of the mixture was measured at 517 nm exactly 30 s after adding the extract. The radical scavenging ability of the extract from L. wrightii for superoxide anion (O2–) generated by xanthine/xanthine oxidase and hydroxyl radical (·OH) by Fenton reaction (FeSO4/H2O2) was determined by an electron spin resonance (ESR) spectrometer (JESFR30, JEOL, Tokyo, Japan), as described previously (Myagmar and Aniya, 2000). The effect of the extract on CL-production from polymorphonuclear leukocytes stimulated with PMA was also evaluated with a chemiluminescence analyzer CLD-110 (Tohoku Electronic Industrial Co. Sendai, Japan) by the same conditions as reported previously (Myagmar and Aniya, 2000) except that leukocytes which were isolated from human peripheral blood was used. The blood sample was taken from a male healthy volunteer by an authorized medical technologist.
The effect of the extract on xanthine oxidase activity was measured according to the method described in the previous report (Myagmar and Aniya, 2000). Effect of Antioxidants on Lipid Peroxidation in Rat Liver Microsomes
Liver microsomes were prepared from non-treated rats (male Sprague-Dawley rats, 200–300 g) as reported previously (Aniya and Naito, 1993) and were kept at –80 °C until use. The microsomes (2–3 mg/ml) were incubated with the NADPH generating system (0.33 mM NADP, 8 mM G6P, 6 mM MgCl2 and 0.5 U G6PDH) and the herbal extracts or ascorbic acid in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C for 30 min in a total volume of 1 ml. Lipid peroxide was measured as thiobarbituric acid reactive substance. Following incubation, the peroxidation was terminated by 20% acetic acid and 0.5% 2-thiobarbituric acid was subsequently added followed by heating at 100 °C for 60 min. After cooling, 1-butanol was added followed by vigorous shaking and centrifugation. The absorbance of the butanol fraction was measured at 532 nm. 1,1,3,3Tetramethoxypropane was used as authentic standard. The protein concentration of microsomes was determined by the method of Lowry et al. (Lowry et al., 1951). Hepatoprotective Action of the Herbal Extract
Mice (ddY, 8 weeks) from Nihon SLC Co (Shizuoka) were divided into three groups (CCl4, CCl4 + herbal extract and control) at random. The CCl4 group of mice was given carbon tetrachloride (CCl4, 2 ml/kg in 50% corn oil solution) subcutaneously (s.c.). The herbal extract plus CCl4 group was given original extract (5 ml/kg) intraperitoneally (i.p.) 1 and 15 h before CCl4 treatment. Control mice were given corn oil (2 ml/kg, s.c.) in place of CCl4. Mice were killed by decapitation 72 h after CCl4 treatment after overnight starvation. To evaluate the effect of the herb alone on the parameters studied, the extract of L. wrightii (5 ml/kg) or water was given to mice intraperitoneally twice at 1 and 15 h before corn oil (2 ml/kg, s.c.) injection, then the mice were sacrificed 72 h after the last injection. In all cases blood was collected from the stump and serum was isolated by centrifugation. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in the serum were measured using an Assay Kit (Kainos, Tokyo). All experiments were conducted in conformity with the guidelines set by the Animal Care and Use Committee of the University of the Ryukyus. Isolation and Identification of Antioxidants from L. wrightii
Dried herb (50 g) was extracted two times with 25 mL of ethanol/water 1:1 by an accelerated solvent extractor
Free radical scavenging action of the medicinal herb Limonium wrightii
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Table 1. Comparision of radical cavenging action. IC50 (µg/ml)
Fig. 1. DPPH radical scavenging activity of the extract of L. wrightii. Water extract of L. wrightii was diluted, then mixed with DPPH (100 µM). Scavenging activity of the extracts for the DPPH radical was measured as described in Materials and Methods.
(ASE-200, Dionex corporation, CA, USA) to give 500 mg of a crude extract that showed the DPPH radical scavenging activity. The resulted extract was chromatographed on a Toyopearl HW-40F column (Tosoh corporation, Tokyo, Japan) with methanol/ water 1:1 followed by HPLC (Symmetry C18, Waters, USA) to yield compound 1 (1.8 mg, a colorless solid). The structure of the compound was identified by UV, 1H NMR and 13C NMR spectra. Statistical Analyses
Data were expressed as the mean ± S.D. The significance of difference was calculated by Student’s t-test and values of p < 0.05 were used as significant.
DPPH Superoxid anion Hydroxyl radical CL-emission Lipid peroxidation
Crude preparation
Gallic acid
Vitamin C
500 0.85 1300 7.5 200
2.63 2.1 0.03 6.1 > 18800 20 113 3160 50 180
j Results Radical Scavenging Action of L. wrightii
Figure 1 shows the DPPH scavenging action of L. wrightii. The DPPH radical was scavenged by the water extract of L. wrightii dose dependently, and the extract with 50% DPPH scavenging action was 2.1times dilution of the original herbal extract (1 g/50 ml H2O). Superoxide anion and hydroxyl radical scavenging actions of L. wrightii were measured by ESR spectrometer (Fig. 2). The extract at 80 times and 640 times dilution scavengd 77% and 53% of the superoxide anion, respectively and 60% and 25% of the hydroxyl radicals were scavenged by the addition of twice the
Fig. 2. Effect of L. wrightii extract on superoxide anion (A) and hydroxyl radical (B). A: hypoxanthine (1.5 µM), xanthine oxidase (0.3 unit) and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) (135 mM) were mixed with the water extract in 0.1 M phosphate buffer (pH 7.4) in the presence of diethylenetriamine penta acetic acid (DTPA) (100 µM) at room temperature, and the ESR signal for DMPO-OOH after 50 sec was monitored (g = 2.006, aN = 1.418 mT, αβH = 1.142 mT, αγH = 0.137 mT). ESR spectra were recorded at following conditions: power 4 mW, modulation width 79 µT, amplitude 200, and time constant 0.1 sec. B: FeSO4 (0.05 mM), H2O2 (2.5 mM) and DMPO (45 mM) were mixed in 0.1 M phosphate buffer (pH 7.4) in the presence of DTPA (100 µM) with the extract, and the ESR signal for DMPO-OH after 50 sec was monitored (g = 2.007, αN = 1.488 mT, αβ = 1.488 mT). Recording conditions are as follows: power 4 mW, modulation width 0.1 mT, amplitude 50 and time constant 0.1 sec. Dilution of the extract was as follows. 320 (× 320) and 80 (× 80) dilution, original extract (× 1), twice the original extract (× 0.5).
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Y. Aniya et al. original extract (× 0.5) and the original (× 1) extract, respectively. Based on the antioxidant preparation, the IC50 (50% inhibition concentration) for superoxide anion and hydroxyl radical was 0.85 mg/ml and 1.3 mg/ml, respectively (Table 1). The crude antioxidant preparation inhibited xanthine oxidase activity with IC50 of 2.5 mg/ml. The xanthine oxidase inhibitor allopurinol, which was used as a standard, showed 64% inhibition of the enzyme at 0.68 mg/ml. Effect of the Herbal Extract on Carbon Tetrachloride-Induced Hepatotoxicity
Fig. 3. Effect of peritoneal injection of L. wrightii on CCl4treated mice. Water (control) or the original extract from L. wrightii (UK, 5 ml/kg) was given intraperitoneally 1 and 15 h before CCl4 (2 ml/kg, 50% corn oil solution, subcutaneously) treatment. In the case of treatment with the herbal extract alone, the extract (5 ml/kg) or water (control) was given, followed by corn oil injection in place of CCl4. Mice were killed 72 h after CCl4 injection after overnight starvation. Liver and serum parameters were measured as described in Materials and Methods. Each column shows the mean ± S.D. from 6 (control), 3 (CCl4-treated), 4 (UK alone) and 5 (CCl4 + the extract treated) mice. Control values are as follows: AST; 141.0 ± 10.1 Karman unit, ALT; 21.7 ± 3.7 Karman unit. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control, # p < 0.05, ## p < 0.01 vs. CCl4-treated.
Fig. 4. Structure of the active component of L. wrightii.
Figure 3 show the effect of the intraperitoneal injection of L. wrightii extract on CCl4-induced liver toxicity. Serum AST and ALT activities were increased to 383% and 374% of the control, respectively, by CCl4 treatment, and the increases were significantly decreased to 140% and 220%, respectively, when the herbal extract was given intraperitoneally twice before CCl4 treatment. The serum enzyme activities were not changed by intraperitoneal treatment of mice with the herbal extract alone. Thus the hepatoprotective action of L. wrightii extract against CCl4-induced liver toxicity was clarified. Isolation and Identification of an Active Component of L. wrightii
The active component from L. wrightii was separated with various column chromatography steps in which the antioxidant action was evaluated by the DPPH scavenging action. The isolated compound was identified as gallic acid by direct comparison of UV, 1H NMR and 13C NMR spectra with those of the authentic sample (Fig. 4). Compound 1 (gallic acid): UV (MeOH) λmax 215, 271 nm; APCI-MS, positive ion, m/z 171 [M+H]+; 1H NMR (400 MHz, DMSO-d6) δ 6.90 (2H, s); 13C NMR (100 MHz, DMSO-d6) δ 167.5 (s), 145.4 (s, x 2), 137.9 (s), 120.5 (s), 108.7 (d, x 2). Antioxidant Action of the Active Component
Fig. 5. Effect of crude antioxidant preparation and gallic acid on CL production from leukocytes stimulated by PMA. Polymorphonuclear leukocytes isolated from human blood were stimulated at 37 °C with PMA in the presence or absence of the antioxidants and thus generated luminol-CL was measured for 6 min. The inhibitory activity of antioxidants was shown as percentage of total CL count of control. d – crude antioxidant; m – gallic acid; s – ascorbic acid (V.C).
The radical scavenging action of the gallic acid was compared with the crude antioxidant preparation and ascorbic acid. Figure 5 indicates the effect of the crude antioxidant and gallic acid on the leukocytes-derived CL. The CL emission was depressed dose-dependently by an incubation of the leukocytes with the antioxidants and the values of IC50 of the crude preparation and gallic acid were 7.5 µg/ml and 113 µg/ml, respectively. These effects of the crude antioxidant were stronger than that of ascorbic acid (IC50 = 3.16 µg/ml). Lipid peroxidation derived from cytochrome P450 system was also inhibited by 50% at 0.2 µg/ml of the crude preparation, at 50 µg/ml of gallic acid and at 180 µg/ml of ascorbic acid
Free radical scavenging action of the medicinal herb Limonium wrightii (Table 1). IC50 values of the crude antioxidant and gallic acid for superoxide anion were 0.85 µg/ml and 0.03 µg/ml, respectively and these are stronger than that of ascorbic acid. For hydroxyl radical, ascorbic acid showed the strongest action among the three antioxidants. These results are summarized in Table 1.
j Discussion The antioxidant action of water extract of L. wrightii was evaluated in vitro and in vivo. The water extract from the herb markedly scavenged the superoxide anion and DPPH radicals and moderately the hydroxyl radical. DPPH can abstract labile hydrogen (Constantin et al., 1990), and the ability to scavenge the DPPH radical is related to the inhibition of lipid peroxidation (Rekka and Kourounakis, 1991). In our laboratory, it was confirmed that the DPPH scavenging activity was parallel to the inhibitory action of lipid peroxidation using an extract of mold or medicinal herbs (Aniya et al., 1999; Gyamfi et al., 1999). Since hydroxyl radicals or superoxide anions are generated under oxidative stress caused by various chemicals or disorders, it was suggested that L. wrightii may ameliorate oxidative stress-induced disorders. In the present study, the effect of the herbal extract on CCl4-induced liver injury was examined. CCl4 is known to cause liver injury via metabolic activation by the microsomal cytochrome P450-dependent monooxygenase system. CCl4 is converted to the reactive intermediate, trichloromethyl radical (·CCl3) and peroxyl radical (·OOCCl3), which in turn react with macromolecules such as lipid and protein, leading to lipid peroxidation and cell injury (McCay et al., 1984; Recknagel, 1983; Slater, 1984). When the extract from L. wrightii was given to mice intraperitoneally prior to CCl4 treatment, CCl4-induced hepatotoxicity seen in the elevation of serum AST and ALT levels was significantly depressed (Fig. 3). Thus it was clarified that the extract of L. wrightii has a strong radical scavenging action by which it ameliorates the CCl4-induced liver toxicity. Since the water extract of L. wrightii possessed a radical scavenging action, we tried to isolate active components from L. wrightii and gallic acid was identified as the main antioxidant of L. wrightii. The antioxidant action of the active component, gallic acid was compared with the crude antioxidant preparation of L. wrightii and with the water soluble antioxidant ascorbic acid. The crude preparation of L. wrightii showed very strong scavenging action for superoxide anion generated from xanthine/xanthine oxidase system (IC50 = 0.85 µg/ml) and also markedly inhibited CL production from PMA-stimulated leukocytes (7.5 µg/ml). To understand the strong scavenging action for super-
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oxide anion we examined whether the crude antioxidant preparation can inhibit xanthine oxidase activity. The crude antioxidant inhibited xanthine oxidase activity with IC50 of 2.5 mg/ml, suggesting that the crude antioxidant could directly scavenge superoxide anion. In PMA-stimulated leukocytes superoxide anion is produced via the NADPH oxidase followed by generation of H2O2 and hypochlorous acid which in turn reacts with luminol resulting in CL-emision. Thus it was suggested that the extract of L. wrightii could scavenge superoxide anion resulting in depression of CL production. Moreover, the inhibitory action of CL-emission from the leukocytes by the crude preparation was more potent than that of gallic acid (IC50 = 113 µg/ml). It is therefore assumed that although gallic acid is the main antioxidant of L. wrightii, other minor component might contribute to the radical scavenging action. The herb extract also showed hydroxyl radical scavenging action (Fig. 2). We observed that gallic acid has a Fe-chelating action, however the herbal extract shows very low iron chelating action (data not shown). Moreover hydroxyl radical scavenging action of gallic acid was low compared with that of crude preparation of the herb (Table 1). It was therefore suggested that the extract contains a component which can scavenge directly hydroxyl radical. The crude antioxidant preparation and gallic acid inhibited NADPH-derived LPO in liver microsomes as well as ascorbic acid (Table 1). It is well known that cytochrome P450 system in microsomes can generate superoxide anion, H2O2 and hydroxyl radical following by LPO. We also observed by ESR spectrometer that superoxide anion was firstly formed in rat liver microsomes in the presence of the NADPH generating system and followed by hydroxyl radical formation. This superoxide anion and also hydroxyl radical formation were inhibited by the addition of the crude antioxidant and also by gallic acid (data not shown). Thus it was suggested that superoxide anion generated from the cytochrome P450 system is mostly scavenged by the antioxidants resulting in depression of LPO. As reported elsewhere that gallic acid as a component of traditional medicine could depress CCl4-induced liver toxicity (Anand et al., 1997; Kanai and Okano, 1998), it was suggested that gallic acid in L. wrightii contribute to the in vitro and in vivo antioxidant action of the herb. L. wrightii is known in a folk medicine for the treatment of fever or arthritis. Recently anti-tumor promoting action of L. wrightii has been demonstrated (unpublished data). Gallic acid, identified as the antioxidant of L. wrightii, is well known as a natural antioxidant and has been reported to possess anti-apoptosis, antiallergic, antiinflamatory, antimutagenic and anticarcinogenic actions (Gali et al., 1991; Gali et al.,
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1992; Hayatsu et al., 1988; Perchellet et al., 1992; Sakaguchi et al., 1998; Singleton, 1981; Stich and Rosin, 1984). In addition the metabolism of gallic acid in the human body was clarified (Shahrzad and Bitsch, 1998). Thus L. wrightii may be expected to be preventive of free radical mediated disorders such as inflammation or cancer. In summary, the medicinal herb L. wrightii has a strong scavenging action of hydroxyl, superoxide anion and DPPH radicals. This herb also depressed CL-emission from PMA-stimulated leukocytes and was protective against liver injury caused by CCl4, suggesting that CCl4-derived free radicals are scavenged by L. wrightii. Gallic acid, identified as the antioxidant of L. wrightii, may contribute to the radical scavenging action of L. wrightii. Acknowledgements
The authors thank Mr. K. Nakamoto of Nakazen Company for his kindness in supplying the medicinal herb, and Ms. N. Murayama for typing the manuscript. This study was partly supported by Grant-in-Aid for University and Society Collaboration.
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j Address Yoko Aniya, Ph.D. Laboratory of Physiology and Pharmacology, School of Health Sciences, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan. Tel: ++81-98-895-1251; Fax: ++81-98-895-1443; e-mail: [email protected]