Hepatoprotective Effects of Arctium Lappa on Carbon Tetrachloride- and Acetaminophen-Induced Liver Damage
Abstract: The root of Arctium lappa Linne (A. lappa) (Compositae), a perennial herb, has been cultivated for a long time as a popular vegetable. In order to investigate the hepatoprotective effects of A. lappa, male ICR mice were injected with carbon tetrachloride (C[Cl.sub.4], 32 [micro]l/kg, i.p.) or acetaminophen (600 mg/kg, i.p.). A. lappa suppressed the SGOT and SGPT elevations induced by C[Cl.sub.4] or acetaminophen in a dose-dependent manner and alleviated the severity of liver damage based on histopathological observations. In an attempt to elucidate the possible mechanism(s) of this hepatoprotective effect, glutathione (GSH), cytochrome P-450 (P-450) and malondialdehyde (MDA) contents were studied. A. lappa reversed the decrease in GSH and P-450 induced by C[Cl.sub.4] and acetaminophen. It was also found that A. lappa decreased the malondialdehyde (MDA) content in C[Cl.sub.4] or acetaminophen-intoxicated mice. From these results, it was suggested that A. lappa could protect the liver cells from C[Cl.sub.4] or acetaminophen-induced liver damages, perhaps by its antioxidative effect on hepatocytes, hence eliminating the deleterious effects of toxic metabolites from C[Cl.sub.4] or acetaminophen.
Arctium lappa Linne (A. lappa), a perennial herb, has been cultivated as a vegetable for a long time in Taiwan and Japan (Morita et al., 1993). Lin et al. (1996) reported that it has anti-inflammatory and free radical scavenging activities. Carbon tetrachloride (C[Cl.sub.4]) induced liver damage is the best characterized animal model of xenobiotic-induced free radical-mediated hepatotoxicity (Recknagel, 1989). It is metabolized by microsomal cytochrome P-450 to the reactive trichloromethyl radical (.C[Cl.sub.3]), which may damage hepatocytes by covalently binding to polyunsaturated fatty acid (PUFA) on cellular membrane during lipid peroxidation (Comporti, 1985; Slater, 1985). Acetaminophen (paracetamol) is normally a very safe drug, but it may produce acute centrilobular hepatic necrosis, when given at high doses in humans and experimental animals (Rumore and Blaiklock, 1992; Prescott, 1983). Acetaminophen damages the liver through the formation of a highly reactive metabolite formed by P-450 (Dahlin et al., 1984; Potter et al., 1989), which is trapped and inactivated by preferential conjugation with hepatic glutathione. When acetaminophen was given in hepatotoxic dose, glutathione is depleted and the toxic metabolite binds covalently to vital proteins and enzymes, causing hepatocellular damage and necrosis (Albano et al., 1985). In this study, the hepatoprotective effects and action mechanisms of A. lappa on C[Cl.sub.4] and acetaminophen-induced liver injury were investigated.
Materials and Methods
Animals and Treatments
Male ICR mice, weighing 22-28 gm, were purchased from the Animal Center of National Taiwan University and kept on the commercial diet (Fu-so Co., Ltd., Taiwan) and tap water ad libitum.
Induction of Hepatotoxicity
C[Cl.sub.4] was dissolved in corn oil and administered i.p. to mice at a dosage of 32 [micro]l/kg. The control mice received only corn oil in a similar manner. Acetaminophen was dissolved in 0.9% normal saline with polyethylene glycol 400 as a solubilizer (1:1, v/v), then administered i.p. to mice at a concentration of 600 mg/kg, while the control group received saline only. Untreated mice served as the blank group. Animals were sacrificed at 24 hrs after i.p. injection of hepatotoxins. Blood was collected by cardiac puncture and livers were removed immediately, homogenized in 4 volumes of 1.15% KCl solution. The homogenate was centrifuged at 9,000 g for 20 min to sediment the nuclear and mitochondrial fractions. The supernatant fraction was centrifuged at 100,000 g for 60 min. The microsomal pellets obtained were suspended in 0.1 M potassium phosphate buffer (pH 7.4) and stored at -70 [degrees] C until microsomal enzyme assay was conducted.
Preparation of Crude Drugs
The root of Arctium lappa L. was collected from local markets and authenticated by C.C. Lin, Kaoshiung Medical College.
100 gm of roots of Arctium lappa were ground to fragments and boiled with 1 liter of distilled water in a Chinese herbal decocter for 1 hr. The extract was filtered and the residue was boiled and filtered again. The filtrates were combined and lyophilized. When orally administered to mice, the crude drug powder was dissolved in normal saline at a concentration of 300 mg/10 ml/kg body weight.
Animals were lightly anesthetized with ethyl ether 24 hrs after the administration of hepatotoxins. Blood samples were allowed to coagulate at room temperature for 1 hr, serum was then separated by centrifugation at 4 [degrees] C, 5,000 rpm for 10 min. Serum concentrations of GOT and GPT were measured using a CH-100 autoanalyzer (Texas International Laboratory, USA) and Sigma GOT and GPT optimized reagents (Sigma Chemical Company, P.O. Box 14508, St. Louis, MO 63178, USA) according to the method of Bergmeyer et al. (1978). The livers were removed immediately after blood collection, and microsomes were prepared.
Lipid Peroxidation Assay in vivo
Liver lipid peroxidation was measured by the method of Buege and Aust (1978). Briefly, 1 ml of 25% microsomal supernatant was combined with 2 ml TCA-TBA-HCI
solution and mixed thoroughly. The mixture was heated for 15 min in a boiling water bath. After cooling, the flocculent precipitate was removed by centrifugation at 4 [degrees] C, 1,000 g for 10 min. The absorbance of the sample was determined at 535 nm and malondialdehyde (MDA) concentration was calculated using an extinction coefficient of 1.56 x [10.sup.5] [M.sup.-1] [cm.sup.-1].
Hepatic GSH Concentration
Total hepatic GSH (reduced glutathione) concentration was determined by the method of Tietze (1969). Each liver was excised and homogenized with phosphate buffer (pH 7.5) containing 10 mM EDTA, and centrifuged at 4 [degrees] C, 10,000 g for 10 min. The supernatant was used for total GSH assay.
Microsomal Enzyme Assay
Microsomal cytochrome P-450 and b5 contents were determined by the method of Omura and Sato (1964), using an extinction coefficient of 91 [mM.sup.-1] [cm.sup.-1] for the absorbance difference between 450 and 490 nM. NADPH-cytochrome c reductase activity was determined by the method of Phillips and Langdon (1962), which is measured by observing the increase in optical density at 550 nm, produced by reduction of cytochrome c and calculated as the change in absorbency at 550 nm/min/mg of microsomal protein. Protein concentrations of the microsomal suspensions were determined by the method described by Lowry et al., 1951.
After blood was drained, part of the hepatic tissue was collected immediately from the same lobe of the liver and fixed in 10% neutral formalin solution for at least 1 week. Subsequently, hepatic tissue was dehydrated with a series of ethanol solutions from 75% to 100% before embedding in paraffin. Cross-sections (5 [micro]m thick) were stained with hematoxylin and eosin Y (H.E.) for photomicroscopic assessment.
All experimental data are shown as means [+ or -] S.E. The statistical significance of differences between control and treated animals was evaluated by the Student’s t-test. The level of significance was chosen as p [is less than] 0.05.
There were no significant differences between the blank group and the control group on all biochemical assays in this experiment. The C[Cl.sub.4] or acetaminophen-induced SGOT and SGPT elevations were reduced in 300 mg/kg A. lappa-treated mice. These phenomena were further confirmed by histopathological examinations (Figures 1 and 2). The reverse effect of A. lappa on hepatotoxins-induced decreases of glutathione (GSH), cytochrome P-450, cytochrome b5 content and NADPH-cytochrome c reductase activity were also observed.
[Figures 1-2 ILLUSTRATION OMITTED]
C[Cl.sub.4] administration significantly decreased (P [is less than] 0.001) the glutathione concentration from 14.16 [+ or -] 0.21 (Blank group) and 12.85 [+ or -] 0.43 (Control group) to 6.4 [+ or -] 0.33 [micro]mol/gm liver. After treatment with A. lappa, the glutathione concentration significantly (P [is less than] 0.05) increased to 7.34 [+ or ] 0.31 [micro]mol/gm liver. In acetaminophen-intoxicated mice liver, the glutathione concentration decreased significantly (P [is less than] 0.001) from 19.5 [+ or -] 0.67 (Blank group) and 19.40 [+ or ] 0.92 (Control group) to 8.43 [+ or ] 0.45 [micro]mol/gm liver (P [is less than] 0.05), but returned to 11.01 [+ or ] 0.67 [micro]mol/gm liver after treatment with A. lappa.
A. lappa decreased significantly the liver malondialdehyde (MDA) content of C[Cl.sub.4] (32 [micro]l/kg) and acetaminophen (600 mg/kg) intoxicated mice (Tables 1 and 2). C[Cl.sub.4] administration significantly increased liver malondialdehyde concentration (P [is less than] 0.05) from 0.75 [+ or -] 0.08 (Blank group) and 1.11 [+ or -] 0.26 (Control group) to 1.77 [+ or -] 0.16 [micro]g/gm protein. Treatment with A. lappa decreased liver malondialdehyde concentration to 1.13 [+ or -] 0.15 [micro]g/gm protein (P [is less than] 0.001). In acetaminophen-intoxicated mice, liver malondialdehyde level increased from 0.45 [+ or -] 0.03 (Blank group) and 0.44 [+ or -] 0.03 (Control group) to 0.81 [+ or -] 0.06 [micro]g/gm protein (P [is less than] 0.01), but decreased to 0.44 [+ or -] 0.01 [micro]g/gm protein (P [is less than] 0.001) after A. lappa was administrated.
Table 1. A. Lappa (300 mg/kg, p.o.) Protected Against Acute Liver Damage Induced by Carbon Tetrachloride (C[Cl.sub.4], 32 [micro]l/kg, i.p.) Poisoning in Mice
SGOT (IU/L) 91.83 [+ or -] 10.65
SGPT(IU/L) 70.70 [+ or -] 2.80
Glutathione 14.16 [+ or -] 0.21
Malondialdehyde 0.75 [+ or -] 0.08
Cytochrome P-450 0.62 [+ or -] 0.03
Cytochrome b5 0.27 [+ or -] 0.01
NADPH-cytochrome c 21.28 [+ or -] 0.63
(nmol cyt. c reduced/mg
SGOT (IU/L) 97.25 [+ or -] 13.11
SGPT(IU/L) 58.90 [+ or -] 5.90
Glutathione 12.85 [+ or -] 0.43
Malondialdehyde 1.11 [+ or -] 0.26
Cytochrome P-450 0.52 [+ or -] 0.01
Cytochrome b5 0.29 [+ or -] 0.01
NADPH-cytochrome c 21.79 [+ or -] 0.08
(nmol cyt. c reduced/mg
SGOT (IU/L) 5405.0 [+ or -] 1134.29(***)
SGPT(IU/L) 6632.33 [+ or -] 740.34(***)
Glutathione 6.04 [+ or -] 0.33(***)
Malondialdehyde 1.77 [+ or -] 0.16(*)
Cytochrome P-450 0.27 [+ or -] 0.03(***)
Cytochrome b5 0.21 [+ or -] 0.01(**)
NADPH-cytochrome c 14.37 [+ or -] 1.34(**)
(nmol cyt. c reduced/mg
Assay C[Cl.sub.4] + A. lappa
SGOT (IU/L) 383.13 [+ or -] 60.77
SGPT(IU/L) 564.14 [+ or -] 50.80
Glutathione 7.34 [+ or -] 0.31([dagger])
Malondialdehyde 1.13 [+ or -] 0.15([dagger][dagger])
Cytochrome P-450 0.50 [+ or -] 0.02
(nmol/mg protein) ([dagger][dagger][dagger])
Cytochrome b5 0.24 [+ or -] 0.01([dagger])
NADPH-cytochrome c 20.84 [+ or -] 2.48([dagger])
(nmol cyt. c reduced/mg
Values were presented as Means [+ or -] S.E. for 15 ICR mice. Statistical significance of differences was determined by Student’s t test. (*), (**) and (***) depict P < 0.05, 0.01 and 0.001 compared to control group, ([dagger]), ([dagger][dagger]) and ([dagger][dagger][dagger]) depict P < 0.05, 0.01 and 0.001 compared to C[Cl.sub.4] group.
Table 2. A. Lappa (300 mg/kg, p.o.) Protected Against Acute Liver Damage Induced by Acetaminophen (acetaminophen, 600 mg/kg, i.p.) Poisoning in Mice
SGOT (IU/L) 85.17 [+ or -] 14.67
SGPT (IU/L) 75.13 [+ or -] 15.10
Glutathione 19.5 [+ or -] 0.67
Malondialdehyde 0.45 [+ or -] 0.03
Cytochrome P-450 0.68 [+ or -] 0.01
Cytochrome b5 0.26 [+ or -] 0.01
reductase 19.97 [+ or -] 0.86
(nmol cyt. c
SGOT (IU/L) 101.25 [+ or -] 11.16
SGPT (IU/L) 76.60 [+ or -] 5.90
Glutathione 19.40 [+ or -] 0.92
Malondialdehyde 0.44 [+ or -] 0.03
Cytochrome P-450 0.62 [+ or -] 0.04
Cytochrome b5 0.31 [+ or -] 0.06
reductase 18.92 [+ or -] 0.67
(nmol cyt. c
SGOT (IU/L) 730.33 [+ or -] 155.25(*)
SGPT (IU/L) 2665.80 [+ or -] 794.76(*)
Glutathione 8.43 [+ or -] 0.45(***)
Malondialdehyde 0.81 [+ or -] 0.06(***)
Cytochrome P-450 0.29 [+ or -] 0.01(***)
Cytochrome b5 0.17 [+ or -] 0.01(***)
reductase 14.80 [+ or -] 2.20
(nmol cyt. c
Assay Acetaminophen + A. lappa
SGOT (IU/L) 178.30 [+ or -] 21.34([dagger])
SGPT (IU/L) 111.29 [+ or -] 12.72([dagger])
Glutathione 11.01 [+ or -] 0.67([dagger])
Malondialdehyde 0.44 [+ or -] 0.01
([micro]g/gm protein) ([dagger][dagger][dagger])
Cytochrome P-450 0.44 [+ or -] 0.01
(nmol/mg protein) ([dagger][dagger][dagger])
Cytochrome b5 0.25 [+ or -] 0.01
(nmol/mg protein) ([dagger][dagger][dagger])
reductase 21.36 [+ or -] 0.95([dagger])
(nmol cyt. c
Values were presented as Means [+ or -] S.E. for 15 ICR mice. Statistical significance of differences was determined by Student’s t test. (*) and (***) depict P < 0.05, 0.001 compared to control group, ([dagger]) and ([dagger][dagger][dagger]) depict P < 0.05, 0.001 compared to acetaminophen group.
Cytochromion P-450, Cytochrome b5 and NADPH-cytocrome c Reductase Concentrations
Cytochrome P-450, cytochrome b5 and NADPH-cytocrome c reductase concentrations decreased significantly after C[Cl.sub.4] administration, while acetaminophen administration decreased significantly cytochrome P-450 and cytochrome b5, but with little change in NADPH-cytocrome c reductase concentration. A. lappa returned all of these liver metabolic enzyme concentrations to normal values.
The hepatoprotective effect of A. lappa was further confirmed by histopathological examination. Livers of C[Cl.sub.4]-treated mice showed many small uniform sizes, round to ovoid shape, a yellow white necrotic area on the liver capsule and in the parenchyma grossly, marked bridging necrosis, mild to moderate ballooning degeneration, pyknotic cell, and cell debris of hepatocytes, and inflammatory cell infiltration in the central area microscopically. After the treatment with A. lappa, fewer necrotic areas with mild inflammatory cell infiltration and some microvesicular change were observed in the central zone; binuclear cells increased. Acetaminophen toxicity showed prominent and diffuse, yellow-white and bacillus-like necrotic areas on the liver capsule and parenchyma grossly, severe zonal necrosis and congestion in the perivenous area microscopically. Treatment with A. lappa reversed the acetaminophen-induced hepatic lesions, presence of arrangement of liver plates and many binuclear cells are found. No necrotic lesions were seen, indicating that the hepatocytes were in regeneration after the administration of A. lappa.
In the past several years, the chemical constituents, pharmacological researches and clinical applications of Arctium lappa Linne had been reported by many authors. Herein, we would like to review shortly these excellent research papers.
In 1993, Wang et al. reported that there are six compounds which could be isolated from the seeds of Arctium lappa Linne. One of them is a new lignan named neoarctin B. The other five compounds were identified as daucosterol, arctigenin, arctiin, matairesinol and lappaol F (Wang et al., 1993).
The fruit of Arctium lappa Linne is an often-used herbal drug in traditional Chinese medicine for the treatment of common cold caused by wind and heat. This drug contains many constituents, principally arctiin, with arctigenin in smaller amount (Sun et al., 1992).
In 1992, Nose et al. indicated that Arctiin and tracheloside are two major lignans of Arctium lappa. Both lignans were stable in rat gastric juice and arctiin was rapidly transformed to arctigenin in rat large intestinal flora, followed by conversion to the major metabolite, 2-(3″,4″-dlhydroxybenzyl)-3-(3′,4′-dimethoxy-benzyl)-butyrolactone. On the other hand, tracheloside also decreased dependently accordingly with time and was converted to trachelogenin and its major metabolite, 2-(3″,4″-dihydroxy-benzyl)-3-(3′,4’dimethoxy-benzyl)-2-hydroxybutyrolactone (Nose, 1992).
In 1993, Kato et al. reported that another constituent xyloglucan could be isolated from the 24% KOH extract of edible Arctium lappa L., which was built up predominantly of repeating-oligosaccharide units of hepta-(Glc:Xyl = 4:3), nona-(Glc:Xyl:Gal:Fuc= 4:3:1:1) and deca-(Glc:Xyl:Gal:Fuc = 4:3:2:1) saccharides in an approximate molar ratio of 14:12:5 (Kato et al., 1993).
The differentiation inducing activities of lignoids from the fruits of Arctium lappa Linne against mouse myelold leukemia cells (M1) has been reported by Umehara et al., 1996. The active components were confirmed as three new dilignans. The most active lignan was found as arctigenin, and its aliphatic esters were found more effective in inducing the differentiation of M1 cells than its aromatic esters, Especially, n-decanoate, which was the most active aliphatic esters derivative of arctigenin, induced more than half of the M1 cells into phagocytic cells at a concentration of 2 microM (Umehara et al., 1996).
In 1996, Lin et al. suggested that the hot aqueous extracts of Arctium lappa Linne (root) possess free radical scavenging activity. The inhibitory effects of A. Lappa on carrageenan-induced rat paw edema and C[Cl.sub.4]-induced hepatotoxicity could be due to its free radical scavenging effect (Lin et al., 1996).
The effects of Arctium lappa, with suspected application to prevent and treat kidney stone formation, have been studied using female Wistar rats by Grases et al., 1994. It was suggested that beneficial effects caused by Arctium lappa infusions on urolithiasis can be attributed to some disinfectant action, and tentatively to the presence of saponins (Grases, 1994).
Arctium lappa was also reported to have the preventive effect of growth retardation. The inclusion of 8% mineral oil in a fat-free diet causes severe growth retardation in rats. This growth retardation, found to be primarily due to the reduction in nutrient intake, could be prevented by the concurrent inclusion of 10% water-insoluble dietary fiber [gobo fiber] prepared from Arctium lappa Linne. It was suggested that the prevention of growth retardation by Arctium lappa was due to its ability to inhibit mineral oil absorption from the intestinal lumen (Morita et al., 1993).
Iwakami et al. has reported that the hot aqueous extracts of Arctium lappa Linne contained lignans and sesquiterpenes, that showed significant inhibitory activities on the binding of platelet activating factor (PAF) to rabbit platelets (Iwakami et al., 1992).
Anonymous authors had reported in the Bulletin of the World Health Organization that Arctium lappa had inhibitory activity against HIV (human immunodeficiency virus) (Anonymous, 1989).
A desmutagenic factor was isolated from burdock (Arctium lappa Linne). This factor reduced the mutagenicity of mutagens that are active without metabolic activation, such as 4-NO2-1,2-DAB and 2-NO2-1,4-DAB, as well as mutagens such as ethidium bromide, 2-aminoanthracene, Trp-P-1 and Trp-P-2 requiring S9 for metabolic activation. This desmutagenic factor is resistant to heat and proteolytic enzymes. The partially purified principles had a molecular weight higher than 300,000 and showed characteristics of a polyanionic substance. An irreversible diminution of the mutagen was confirmed by treatment of 2-NO2-1,4-DAB or Trp-P-2 with the burdock factor (Morita et al., 1984).
In the present study, the hepatoprotective effects of Arctium lappa on carbon tetrachloride and acetaminophen-induced liver damage were investigated in mice.
It has been proposed that the C[Cl.sub.4]-induced liver injury is brought about by * C[Cl.sub.3] radical, which is formed by cytochrome P-450 enzymes. This * C[Cl.sub.3] radical could induce the peroxidation of the unsaturated fatty acids that constitute the cell membrane, which leads to membrane injury (Clawson, 1989; Corongiu et al., 1986; Jeffrey et al., 1993). * C[Cl.sub.3] free radical was also reported to induce fatty degeneration of the liver and centrilobular necrosis (Slater et al., 1985). Lipid accumulation due to the * C[Cl.sub.3]-damaged liver prevented transport of triglyceride-rich very low density lipoprotein (VLDL) into blood (Pencil et al., 1984).
The antipyretic and analgesic drug acetaminophen (paracetamol) is safe in the therapeutic range, but an overdose often causes severe hepatotoxicity in experimental animals and humans (Mitchell, 1988). The acute changes in the liver produced by a single dose of acetaminophen include hemorrhagic and gross centrilobular necrosis (Zieve et al., 1985a; 1985b). Vacuolization around the necrotic area and inflammatory cellular infiltration were rarely found. Nuclear pyknosis, karyolysis and eosinophilia of the cytoplasm of the necrotic centrilobular hepatocytes, and vascular congestion have also been observed.
It was established that a fraction of the administered acetaminophen is metabolized by the microsomal monooxygenase system to a toxic reactive metabolite, which has been identified as N-acetyl-p-benzoquinone imine (NAPQI) (Nelson, 1990). NAPQI can react with sulfhydryl substances such as glutathione (GSH) (Potter and Hinson, 1986) or protein-thiols (Hoffmann et al., 1985). When acetaminophen was given at the hepatotoxic dose, glutathione will be depleted, then the toxic metabolite NAPQI turned out to bind covalently with other macromolecules in hepatocytes; these conjugates will lead to severe cellular dysfunction, enzyme inactivation, etc. (Albano et al., 1985; Manson and Fischer, 1986; Fischer et al., 1985), eventually resulting in cell necrosis (Miller and Jollow, 1987). It was suggested that A. lappa may protect the liver cells from C[Cl.sub.4]- or acetaminophen-induced liver damages, perhaps by its antioxidant effect (due to increase in GSH) and as a free radical scavenger (due to decrease in MDA) hepatocytes, hence eliminating the toxic metabolites from C[Cl.sub.4] or acetaminophen and inducing liver regeneration.
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Song-chow Lin(1)(*), Tsao-chuen Chung(2),(3), Chun-ching Lin(4), Tzuu-Huei Ueng(5), Yun-ho Lin(6), Shuw-yuan Lin(7) and Li-ya Wang(1)
(1) Department of Pharmacology, Taipei Medical College, 250, Wu-hsing Street, Taipei, Taiwan (2) School of Medical Technology, Taipei Medical College, Taipei, Taiwan (3) Graduate Institute of Medical Technology, College of Medicine, National Taiwan University, Taiwan (4) School of Pharmacy, Kaohsiung Medical College, Kaohsiung, Taiwan (5) Institutes of Toxicology, College of Medicine, National Taiwan University, Taiwan (6) Department of Pathology, Taipei Medical College, Taipei, Taiwan (7) Department of Food and Nutrition, Hung-Kuang Institute of Technology, Taichung Hsien, Taiwan (*) Corresponding author (Accepted for publication October 1, 1999)
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