Free radical scavenging action of the medicinal herb Limonium wrightii from the Okinawa Islands.

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 phorboer-12-mysistate acetate and inhibited lipid peroxidation in rat liver microsomes. When the extract was given intraperitoneally to mice prior to carbon tetrachioride ([CCI.sub.4]) treatment, [CCI.sub.4]-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

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, 1,1 diphenyl-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 trichioromethyl 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.

Materials and Methods

Chemicals

Reduced glutathione (GSH), glucose 6-phosphate, phorbol 12-myristate 13-acetate (PMA) and [beta]-nicotinamide adenine dinucleotide phosphate were purchased from Sigma Chemicals (St. Louis, MO, U.S.A.). l-Chloro-2, 4-dinitrobenzene (CDNB), 1,1 -diphenyl-2-picrylhydrazyl (DPPH), luminol (3-aminophtalhydrazide), hypoxanthine, hydrogen peroxide ([H.sub.2][O.sub.2]) 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 [degrees]C overnight. One gram of the herb was extracted with 50 ml of water at 37 [degrees]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 [degrees]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 ([O.sup.-sub.2]) generated by xanthine/xanthine oxidase and hydroxyl radical (.OH) by Fenton reaction ([FeSO.sub.4]/[H.sub.2][O.sub.2]) was determined by an electron spin resonance (ESR) spectrometer (JESFR3O, 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 [degrees]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 [MgCl.sub.2] and 0.5 U G6PDH) and the herbal extracts or ascorbic acid in 0.1 M potassium phosphate buffer (pH 7.4) at 37 [degrees]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 [degrees]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. 1,1,3,3-Tetramethoxypropane 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 ([CCl.sub.4], [CCl.sub.4] + herbal extract and control) at random. The [CCl.sub.4] group of mice was given carbon tetrachloride ([CCl.sub.4], 2 ml/kg in 50% corn oil solution) subcutaneously (s.c.). The herbal extract plus Cd4 group was given original extract (5 ml/kg) intraperitoneally (i.p.) 1 and 15 h before [CCl.sub.4] treatment. Control mice were given corn oil (2 ml/kg, s.c.) in place of [CCl.sub.4]. Mice were killed by decapitation 72 h after [CCl.sub.4] 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 (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 Cl8, Waters, USA) to yield compound 1 (1.8 mg, a colorless solid). The structure of the compound was identified by UV, [1.sub.H] NMR and 13C NMR spectra.

Statistical Analyses

Data were expressed as the mean [+ or -] S.D. The significance of difference was calculated by Student's t-test and values of p<0.05 were used as significant.

* 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 2.1-times dilution of the original herbal extract (1 g/50 ml [H.sub.2]O). 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 original extract (x 0.5) and the original (x 1) extract, respectively. Based on the antioxidant preparation, the [IC.sub.50] (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 [IC.sub.50] 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 

Figure 3 show the effect of the intraperitoneal injection of L. wrightii extract on [CCl.sub.4]-induced liver toxicity. Serum AST and ALT activities were increased to 383% and 374% of the control, respectively, by [CCl.sub.4] treatment, and the increases were significantly decreased to 140% and 220%, respectively, when the herbal extract was given intraperitoneally twice before [CCl.sub.4] 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 [CCl.sub.4]-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, [1.sub.H] NMR and [13.sub.C] NMR spectra with those of the authentic sample (Fig. 4). Compound 1 (gallic acid): UV (MeOH) [lambda]max 215, 271 nm; APCI-MS, positive ion, M/z 171 [M+H]+; [1.sub.H] NMR (400 MHz, DMSO-d6) [delta] 6.90 (2H, s); [13.sub.C] NMR (100 MHz, DMSO-d6) [delta] 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

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 [IC.sub.50] of the crude preparation and gallic acid were 7.5 [micro]g/ml and 113 [micro]g/ml, respectively. These effects of the crude antioxidant were stronger than that of ascorbic acid ([IC.sub.50] = 3.16 [micro]g/ml). Lipid peroxidation derived from cytochrome P450 system was also inhibited by 50% at 0.2 [micro]g/ml of the crude preparation, at 50 [micro]g/ml of gallic acid and at 180 [micro]g/ml of ascorbic acid (Table 1). [IC.sub.50] values of the crude antioxidant and gallic acid for superoxide anion were 0.85 [micro]g/ml and 0.03 [micro]g/ml respectively and these are stronger than that of ascorbic acid. For hydroxyl radical, ascorbic acid showed the strongest act ion among the three antioxidants. These results are summarized in Table 1.

* 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 [CCl.sub.4]-induced liver injury was examined.

[CCl.sub.4] is known to cause liver injury via metabolic activation by the microsomal cytochrome P450-dependent monooxygenase system. [CCl.sub.4] is converted to the reactive intermediate, trichloromethyl radical ([CCl.sub.3]) and peroxyl radical ([OOCCl.sub.3]), 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 [CCl.sub.4] treatment, [CCl.sub.4]-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 [CCl.sub.4]-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 ([IC.sub.50] = 0.85 [micro]g/ml) and also markedly inhibited CL production from PMA-stimulated leukocytes (7.5 [micro]g/ml). To understand the strong scavenging action for superoxide anion we examined whether the crude antioxidant preparation can inhibit xanthine oxidase activity. The crude antioxidant inhibited xanthine oxidase activity with [IC.sub.50] 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 [H.sub.2][O.sub.2] 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 [IC.sub.50] = 113 [micro]g/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, [H.sub.2][O.sub.2] 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 [CCl.sub.4]-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., 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 [CCl.sub.4], suggesting that [CCl.sub.4]-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.

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Table 1

Comparision of radical cavenging action.

                    IC50 ([micro]g/ml)

                    Crude        Gallic acid  Vitamin C
                    preparation

DDPH                 500              2.63       2.1
Superoxid anion        0.85           0.03       6.1
Hydroxyl radical    1300         >18800         20
CL-emission            7.5          113       3160
Lipid peroxidation   200             50        180

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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Abbreviations: CL--chemoluminescence; PMA--phorbol 12-myristate 13-acetate; DPPH--1,1-dipheny-2-picrylhydrazyl: AST--aspartate aminotransferase; ALT--alanine aminotransferase; LPO--lipid peroxidation

Y. Aniya (1)

C. Miyagi (1)

A. Nakandakari (1)

S. Kamiva (1)

N. Imaizumi (1)

T. Ichiba (2)

(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

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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.

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