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Protective effect of α- and β-amyrin, a triterpene mixture from Protium heptaphyllum (Aubl.) March. trunk wood resin, against acetaminophen-induced liver injury in mice
Journal of Ethnopharmacology 98 (2005) 103–108
Protective effect of ␣- and -amyrin, a triterpene mixture from Protium heptaphyllum (Aubl.) March. trunk wood resin, against acetaminophen-induced liver injury in mice Francisco A. Oliveira a , Mariana H. Chaves b , Fernanda R.C. Almeida b , Roberto C.P. Lima Jr. a , Regilane M. Silva a , Juliana L. Maia a , Gerly Anne A.C. Brito a , Fl´avia A. Santos a , Vietla Satyanarayana Rao a, ∗ a
Department of Physiology and Pharmacology, Federal University of Cear´a, Rua Cel Nunes de Melo 1127, Porangabussu Caixa Postal 3157, 60430-270 Fortaleza, Cear´a, Brazil b Departments of Chemistry and Pharmacology, Federal University of Piaui, Teresina, PI, Brazil Received 19 October 2004; received in revised form 6 December 2004; accepted 5 January 2005
Abstract In the search of hepatoprotective agents from natural sources, ␣- and -amyrin, a triterpene mixture isolated from the trunk wood resin of folk medicinal plant, Protium heptaphyllum was tested against acetaminophen-induced liver injury in mice. Liver injury was analysed by quantifying the serum enzyme activities and by histopathological observations. In mice, acetaminophen (500 mg/kg, p.o.) caused fulminant liver damage characterized by centrilobular necrosis with inflammatory cell infiltration, an increase in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, a decrease in hepatic glutathione (GSH) and 50% mortality. Pretreatment with ␣- and amyrin (50 and 100 mg/kg, i.p. at 48, 24, and 2 h before acetaminophen) attenuated the acetaminophen-induced acute increase in serum ALT and AST activities, replenished the depleted hepatic GSH, and considerably reduced the histopathological alterations in a manner similar to N-acetylcysteine, a sulfhydryls donor. Also, the acetaminophen-associated mortality was completely suppressed by terpenoid pretreatment. Further, ␣- and -amyrin could potentiate the pentobarbital (50 mg/kg, i.p.) sleeping time, suggesting the possible suppression of liver cytochrome-P450. These findings indicate the hepatoprotective potential of ␣- and -amyrin against toxic liver injury and suggest that the diminution in oxidative stress and toxic metabolite formation as likely mechanisms involved in its hepatoprotection. In conclusion, this study supports the traditional use of Protium heptaphyllum resin as a medicinal agent and suggests the feasibility of developing herbal drugs for treatment of liver disorders. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Protium heptaphyllum; Resin; ␣- And -amyrin; Hepatoprotection; Acetaminophen-induced liver injury
1. Introduction In the past, several studies have examined the effects of plants used traditionally by herbalists to support liver function and treat diseases of the liver. Experimental and clinical research has confirmed the efficacy of few plants like Silybum marianum (milk thistle), Picrorhiza kurroa (kutkin), ∗
Corresponding author. Tel.: +55 85 2888341; fax: +55 85 2888333. E-mail address: [email protected] (V.S. Rao).
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Curcuma longa (turmeric), Camellia sinensis (green tea), and Glycyrrhiza glabra (licorice), (Luper, 1999). In spite of significant advances in medicinal plant research and rapid strides in modern medicine, there continues to be a need for more precise, safe and effective treatments of liver disorders. In recent years, there has been a shift towards therapeutic evaluation of herbal products in liver diseases by carefully synergizing the strengths of the traditional systems of medicine with that of the modern concept of evidence-based medicinal evaluation.
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Fig. 1. Chemical structures of ␣- and -amyrin.
Protium heptaphyllum (Aubl.) March. (Burseraceae), is a traditional medicinal plant largely encountered in the Amazon region and also in several other parts of Brazil. The resin collected from the trunk wood of Protium heptaphyllum is locally called as alm´ecega or breu branco and is widely used for skin diseases, healing of ulcers, and as an analgesic (Susunaga et al., 2001). The leaves and stem bark are also employed to treat gangrenous ulcers, inflammatory and painful conditions, diarrhoea and to promote wound healing (Lorenzi, 1972; Corrˆeia, 1984). Several species of plants that belong to Burseraceae family and in particular, the Genus Protium appears to yield large quantities of resin from their trunk wood (Corrˆeia, 1984). Studies performed on the resin and its essential oil proved its traditional use as an anti-inflammatory and analgesic agent (Siani et al., 1999; Oliveira et al., 2004a). Our previous studies have established the antipruritus and gastroprotective effects of ␣- and -amyrin (Fig. 1), a major pentacyclic triterpene obtained as an intractable isomeric mixture from the resin of Protium heptaphyllum (Oliveira et al., 2004b, c). Plant-derived triterpenoids, in general, are known to possess a wide spectrum of pharmacological activity including anti-inflammatory, anti-ulcer, anti-hyperlipidemic, anti-tumor, and hepatoprotective actions (Hu, 1988; Liu et al., 1995; James et al., 2003). Both in vitro and in vivo experiments and clinical assays demonstrate the hepatoprotective and gastroprotective properties of several triterpenoids like oleanolic acid, ursolic acid, ␣-hederin, and glycyrrhizin (Liu et al., 1994). Acetaminophen is a widely used over-the-counter drug for analgesic and antipyretic effects. Its use in overdose (suicidal or accidental) or with chronic alcohol abuse causes fulminant liver failure and contributes significantly to intensive care unit admissions and cost of hospitalization (Prescott, 2000; Gyamlani and Parikh, 2002). Acetaminophen-induced hepatic failure is the second leading cause of liver transplantation and accounts to considerable levels of morbidity and mortality (Lee, 2004). The hepatotoxicity is the result of the formation of a toxic metabolite, N-acetyl-p-benzoquinoneimine (NAPQI) by cytochrome P4502E1 that can induce a dosedependent depletion of intracellular glutathione and perturbations of calcium homeostasis (Lee et al., 1996; Chen
et al., 1999; Holownia and Braszko, 2004). Presently, Nacetylcysteine (NAC) is the treatment option since it increases glutathione levels in the hepatic cytosol and mitochondria to detoxify the highly reactive and cytotoxic NAPQI (Mitchell et al., 1973). Besides, selective inhibitors of Cytochrome P4502E1 that inhibit NAPQI formation are under investigation in managing acetaminophen-associated hepatotoxicity (Holownia and Braszko, 2004). As a part of our continuing search for naturally occurring hepatoprotective substances from folkloric plants, we have selected for the present study, ␣- and -amyrin isolated from the resin of Protium heptaphyllum, although there has been no documented evidence available on the traditional use of this resin in hepatic disorders. The option was based on the following reasons: lay people use this resin but cannot define the merits of hepatoprotectant remedies and the benefits of therapy might be actually due to improvement in liver function; ␣- and -amyrin is the major triterpenoid component of resin that exhibits anti-inflammatory, gastroprotective and antipruritus effects (Oliveira et al., 2004a, b, c); The triterpenoid mixture bears the ursane and oleanane skeletons and compounds like oleanolic acid and ursolic acid, which have chemical resemblance offer hepatoprotection against a variety of hepatotoxicants (Liu et al., 1994; Saraswat et al., 1996; Kim et al., 2004). Thus, the present study aimed to establish the hepatoprotective potential of ␣- and -amyrin, using acetaminophen-induced hepatic damage in mice as an animal model.
2. Materials and methods 2.1. Animals Adult male Wistar rats (150–200 g) obtained from the Central Animal House, Federal University of Cear´a were housed in polypropylene cages under controlled environmental conditions (22 ± 2 ◦ C, 55–60% humidity, 12:12-h light/dark cycle) and had free access to standard laboratory chow (Purina) and water. Animals were fasted overnight for experimentation, but were allowed water ad libitum.
F.A. Oliveira et al. / Journal of Ethnopharmacology 98 (2005) 103–108
Institutional Animal Care and Use Committee of the Federal University of Cear´a, Fortaleza, approved the experimental protocols in accordance with the guidelines of National Institute of Health, Bethseda, USA. 2.2. Drugs and chemicals Acetaminophen was purchased from Sigma, St. Louis, MO, USA and N-acetylcysteine (Fluimucil® ) from Zambon, S˜ao Paulo, Brazil. All reagents were commercially available and of reagent grade. The triterpenoid mixture, ␣- and -amyrin was isolated from the crude resin of Protium heptaphyllum as per methods described earlier (Oliveira et al., 2004a). For experiments, ␣- and -amyrin mixture was suspended in 3% Tween 80 in saline. Acetaminophen was dissolved in pathogen-free normal saline. 2.3. Hepatotoxicity induced by acetaminophen and treatments A total of 120 mice were used for the experiments. These were randomly divided into five groups of 20 in each. The first group received saline (10 ml/kg, i.p.) and served as normal control, while the second, third, fourth and fifth groups were respectively treated intraperitoneally with vehicle (3% Tween 80 in saline; 10 ml/kg), ␣- and -amyrin mixture (50 and 100 mg/kg), or N-acetylcysteine (750 mg/kg), 48, 24, and 2 h before the oral administration of 500 mg/kg acetaminophen. The dose selection for acetaminophen (500 mg/kg) and ␣- and -amyrin mixture (50 and 100 mg/kg) was based on our preliminary experiments. Blood samples and liver tissue specimens were collected 24 h after acetaminophen treatment. Mice that died prior to 24 h were not subjected to biochemical or histopathological analysis. 2.4. Effect of α- and β-amyrin mixture on sleeping time induced by pentobarbital sodium
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trichloroacetic acid/ethylenediaminetetraacetic acid (TCA/ EDTA) and centrifuged at 1500 × g for 15 min. The resulting supernatants were assayed calorimetrically for nonprotein sulfhydryl (NPSH) content using the Ellman’s reagent (Sedlak and Lindsay, 1968). Blood samples collected from the respective groups of mice were centrifuged for 5 min at 1000 × g to separate the serum. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using the RA-XT (automated) biochemical analyzer with respective test kits (Technicon, Bayer S.A. Diagnostic). 2.6. Histological analysis Mice under different treatments were killed at 24 h, liver tissues collected and stored in 10% phosphate buffered formalin. Liver sections (3 m) were prepared, stained with Hematoxylin and Eosin (H&E) and graded for lesions by light microscopy using the following scale (0–5). No lesions = 0; minimal lesions involving single to few necrotic cells = 1; mild lesions, 10–25% necrotic cells or mild diffuse degenerative changes = 2; moderate lesions, 25–40% necrotic or degenerative cells = 3; marked lesions, 40–50% necrotic or degenerative cells = 4; severe lesions more than 50% necrotic or degenerative cells = 5 (Silva et al., 2001). Persons unaware of treatments evaluated the liver sections from different groups. 2.7. Statistical analysis The results are presented as mean ± S.E.M. and were analyzed using a one-way analysis of variance, followed by Tukey’s test for multiple comparisons. Histological scores on liver tissues were analysed by Kruskal–Wallis nonparametric test. Differences were considered statistically significant when P < 0.05.
3. Results To verify a possible influence on cytochrome-P450 enzyme system, ␣- and -amyrin was tested on pentobarbital sodium-induced sleeping time. The test was carried out in male mice (n = 10 per group) treated as follows: group 1 was treated with the vehicle of ␣- and -amyrin (10 ml/kg, p.o.) and the mice in groups 2, and 3 were given the ␣- and amyrin mixture in doses of 50 and 100 mg/kg, i.p., respectively, at 48, 24, and 2 h before the administration of pentobarbital sodium (50 mg/kg, i.p.) and the duration of sleep (min) in each animal was observed. Loss of righting reflex to recovery was recorded as the sleeping time (Darias et al., 1998).
Acetaminophen (500 mg/kg), administered orally induced a fulminant hepatic failure as evidenced by severe mortality (50%), and changes in biochemical and histological parameters (Table 1). Acetaminophen caused a significant enhancement in serum ALT and AST enzyme activities and Table 1 Effect of ␣- and -amyrin mixture on pentobarbitone (50 mg/kg, i.p.)induced sleeping time in mice Treatment
Dose (mg/kg)
Route
Sleeping time (mean ± S.E.M.) (min) 60.55 ± 4.71
Control (vehicle)
–
i.p.
2.5. Biochemical analysis
␣- and -amyrin
50 100
i.p. i.p.
Liver samples collected from mice that belong to different treatments were homogenized (20%, w/v) in 5%
Data as mean ± S.E.M.; n = 10 per group. ∗∗∗ p < 0.001 vs. Control (ANOVA followed by Tukey’s test).
86.25 ± 7.89 105.25 ± 5.29***
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Fig. 2. The representative microphotographs of H&E (×100)-stained histological sections of liver from (A) mice treated with saline showing normal architecture; (B) mice treated with acetaminophen (500 mg/kg, p.o.) and the vehicle used for ␣- and -amyrin (10 ml/kg, i.p.) showing centrilobular necrosis, congestion and an extensive area of inflammatory cell infiltration; (C) mice treated with acetaminophen (500 mg/kg, p.o.) + ␣- and -amyrin (100 mg/kg, i.p.) showing well preserved hepatocytes as well as the architecture with small areas of necrosis and inflammatory cell infiltration; and (D) mice treated with acetaminophen (500 mg/kg, p.o.) + N-acetyl cysteine (750 mg/kg, i.p.) showing partially preserved hepatocytes and architecture with small areas of necrosis and inflammatory cell infiltration.
besides, it significantly lowered the hepatic tissue level of NPSH (GSH) content as compared to saline treated controls. Histological sections showed extensive centrilobular necrosis, congestion and fatty degenerative changes with inflammatory cell infiltration in acetaminophen-treated group that received only the vehicle of ␣- and -amyrin. In contrast, pretreatment with ␣- and -amyrin at both doses (50 and 100 mg/kg, i.p.) showed a significant dose-related lowering effect on acetaminophen-induced elevation of serum ALT and AST and also replenished the depleted hepatic GSH in a significant manner similar to NAC (Table 1). Histological sections analysed for scores revealed significant differences between ␣- and -amyrin-treated and vehicle-treated
controls. Acetaminophen-induced pathological lesion scores were found to be significantly less in liver tissues from ␣- and -amyrin-treated groups (Table 1). Consistent with serological parameters, acetaminophen-induced liver congestion and centrilobular necrosis were substantially prevented by ␣- and -amyrin treatment in a manner similar to NAC (Fig. 2). Table 2 shows the pretreatment effect of ␣- and amyrin (50 and 100 mg/kg, i.p.) on pentobarbital (50 mg/kg, i.p.) induced sleeping time in mice. The sleeping time was potentiated by ␣- and -amyrin at both doses, but a statistical significance was attained only at the dose of 100 mg/kg.
Table 2 Protective effects of ␣- and -amyrin and N-acetylcysteine (NAC) against hepatotoxicity induced by acetaminophen (AAP) in mice Group
Dose (mg/Kg)
Serum ALT (IU/dl)
Control-normal Control-AAP ␣- and -amyrin + AAP ␣- and -amyrin + AAP N-acetylcysteine
– –
61.98 134.35 61.28 44.15 41.46
50 100 750
± ± ± ± ±
11.27 19.76a 12.49b 6.65b 5.34b
Serum AST (IU/dl) 43.17 107.28 32.53 2857 29.96
± ± ± ± ±
7.34 16.02a 6.98b 4.42b 6.46b
GSH (g/g liver) 655.17 432.17 612.54 642.01 611.83
± ± ± ± ±
24.10 32.37a 32.78b 28.59b 32.29b
Liver damage (histological scores) 0.0 4.83 2.17 1.50 0.50
± ± ± ± ±
0.0 0.17a 0.48b 0.22b 0.22b
Mice received intraperitoneal injections of N-acetylcysteine (NAC) or ␣-, -amyrin mixture thrice (48, 24, and 2 h) before the oral administration of acetaminophen (AAP, 500 mg/kg). Twenty-four hours latter, hepatotoxicity was determined by quantifying the serum activities of aspartate amino transferase (AST) and alaninie aminotransferase (ALT) and from histopathological analysis. Each value represents means ± S.E.M. (n = 10–20). a P < 0.05 vs. control-normal. b P < 0.05 vs. controle-AAP (one-way ANOVA and Student t-test or Kruskal–Wallis test).
F.A. Oliveira et al. / Journal of Ethnopharmacology 98 (2005) 103–108
4. Discussion In agreement with previous studies, acetaminophen administered orally to mice at an overdose (500 mg/kg) caused a potentially fatal, centrilobular hepatic necrosis as evidenced by 50% mortality, and by histological and biochemical changes. Pretreatment with ␣- and -amyrin completely suppressed the acetaminophen-associated mortality in mice. Besides, marked elevations in serum ALT and AST activities associated with acetaminophen toxicity were reduced by ␣- and -amyrin (50 and100 mg/kg), in a dose-dependent manner. N-acetylcysteine (NAC), a known hepatoprotectant and a sulfhydryl replenisher (Prescott, 2000) also prevented the rise in serum transaminases. Improvement in the GSH level by NAC is considered the most significant mechanism of NAC against acetaminophen toxicity (Bruno et al., 1998). NAC is known to act by raising intracellular concentrations of reduced glutathione (GSH) and/or by scavenging reactive oxygen species (Aruoma et al., 1989). Similar to NAC, ␣- and -amyrin replenished the acetaminophen-induced depletion of hepatic GSH, presumably due to a diminished production of the toxic metabolite, N-acetyl-p-benzoquinoneimine (NAPQI). Histological sections of liver showed centrilobular necrosis with inflammatory cell infiltration in acetaminophenalone treated mice. Centrilobular necrosis, the pathognomonic feature of acetaminophen hepatotoxicity was strikingly reduced in ␣- and -amyrin pretreated mice. Further, the congestion and inflammatory cell infiltrationevoked by acetaminophen was considerably decreased by ␣- and -amyrin, indicating its possible anti-inflammatory action. Recently, Gardner et al. (Gardner et al., 2003) verified the potential role of inflammatory mediators using transgenic mice with a targeted disruption of the tumor necrosis factor receptor-1 (TNFR1) gene, and the results showed an exaggerated hepatotoxicity of acetaminophen in mice lacking TNFR1. We have previously reported the anti-inflammatory activity of the crude resin of Protium heptaphyllum that is rich in ␣- and -amyrin (Oliveira et al., 2004a). Interestingly, the structurally related compounds, ursolic acid and oleanolic acids were also shown to be anti-inflammatory (Baricevic et al., 2001; Banno et al., 2004). Therefore, we propose that the hepatoprotective effect evoked by ␣- and -amyrin may possibly involve, in part, an anti-inflammatory action. The triterpene, ␣- and -amyrin is devoid of any overt toxicity and no mortality was encountered in doses up to 5.0 g/kg, p.o. or 3.0 g/kg, i.p., in mice (unpublished observations). In recent years, there has been an active search for the development of cytochrome P450 inhibitors that may have therapeutic potential in the prevention of liver damage associated with acetaminophen overdose (Walubo et al., 2004). Past studies have ascribed the hepatoprotective effect of triterpene acids, oleanolic acid and ursolic acid to the inhibition of cytochrome P450 isoforms, CYP2E1, CYP1A and CYP2A (Liu et al., 1995; Kim et al., 2004) and suggest that co-administration of acetaminophen with inhibitors of
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cytochrome P450 may be of therapeutic value in the prevention of acetaminophen-associated hepatotoxicity. Considering that acetaminophen is metabolized by Cytochrome-P450 leading to the production of highly reactive toxic metabolites, which contribute to hepatotoxicity, the present study tested the effect of ␣- and -amyrin on the sleeping time induced by pentobarbital sodium in mice. It is known that nearly complete metabolism and conjugation of pentobarbital occurs in liver mainly by cytochrome-P450 (Nebert and Gon zalez, 1987). At the dose of (100 mg/kg), ␣- and -amyrin was found to potentiate the pentobarbital-induced sleeping time in a significant manner, suggesting a possible inhibition of cytochrome-P450 enzymes and the formation of NAPQI via cytochrome P450E1, which is predominantly localized to liver (Lee et al., 1996). Whether or not ␣- and -amyrin suppresses some specific isoforms of cytochrome-P450 requires further investigation. In conclusion, our study demonstrates that ␣- and -amyrin isolated from the traditional medicinal plant Protium heptaphyllum offers hepatoprotection against acetaminophen-induced hepatotoxicity. This protection appears to be, at least in part, related to its diminution of oxidative stress and to the inhibition of cytochrome-P450. Further, our findings on Protium heptaphyllum might provide leads for the design and development of novel drugs that modulate the therapeutic response of drugs like acetaminophen whose biotransformation pathways involves some cytochrome-P450 isoforms.
Acknowledgements This study was supported by grants from the Brazilian National Agencies, CAPES (PROCAD, Proc. No. 0014/010) and CNPq (Proc. No. 472717/03-0).
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Protective effect of ␣- and -amyrin, a triterpene mixture from Protium heptaphyllum (Aubl.) March. trunk wood resin, against acetaminophen-induced liver injury in mice Francisco A. Oliveira a , Mariana H. Chaves b , Fernanda R.C. Almeida b , Roberto C.P. Lima Jr. a , Regilane M. Silva a , Juliana L. Maia a , Gerly Anne A.C. Brito a , Fl´avia A. Santos a , Vietla Satyanarayana Rao a, ∗ a
Department of Physiology and Pharmacology, Federal University of Cear´a, Rua Cel Nunes de Melo 1127, Porangabussu Caixa Postal 3157, 60430-270 Fortaleza, Cear´a, Brazil b Departments of Chemistry and Pharmacology, Federal University of Piaui, Teresina, PI, Brazil Received 19 October 2004; received in revised form 6 December 2004; accepted 5 January 2005
Abstract In the search of hepatoprotective agents from natural sources, ␣- and -amyrin, a triterpene mixture isolated from the trunk wood resin of folk medicinal plant, Protium heptaphyllum was tested against acetaminophen-induced liver injury in mice. Liver injury was analysed by quantifying the serum enzyme activities and by histopathological observations. In mice, acetaminophen (500 mg/kg, p.o.) caused fulminant liver damage characterized by centrilobular necrosis with inflammatory cell infiltration, an increase in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, a decrease in hepatic glutathione (GSH) and 50% mortality. Pretreatment with ␣- and amyrin (50 and 100 mg/kg, i.p. at 48, 24, and 2 h before acetaminophen) attenuated the acetaminophen-induced acute increase in serum ALT and AST activities, replenished the depleted hepatic GSH, and considerably reduced the histopathological alterations in a manner similar to N-acetylcysteine, a sulfhydryls donor. Also, the acetaminophen-associated mortality was completely suppressed by terpenoid pretreatment. Further, ␣- and -amyrin could potentiate the pentobarbital (50 mg/kg, i.p.) sleeping time, suggesting the possible suppression of liver cytochrome-P450. These findings indicate the hepatoprotective potential of ␣- and -amyrin against toxic liver injury and suggest that the diminution in oxidative stress and toxic metabolite formation as likely mechanisms involved in its hepatoprotection. In conclusion, this study supports the traditional use of Protium heptaphyllum resin as a medicinal agent and suggests the feasibility of developing herbal drugs for treatment of liver disorders. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Protium heptaphyllum; Resin; ␣- And -amyrin; Hepatoprotection; Acetaminophen-induced liver injury
1. Introduction In the past, several studies have examined the effects of plants used traditionally by herbalists to support liver function and treat diseases of the liver. Experimental and clinical research has confirmed the efficacy of few plants like Silybum marianum (milk thistle), Picrorhiza kurroa (kutkin), ∗
Corresponding author. Tel.: +55 85 2888341; fax: +55 85 2888333. E-mail address: [email protected] (V.S. Rao).
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Curcuma longa (turmeric), Camellia sinensis (green tea), and Glycyrrhiza glabra (licorice), (Luper, 1999). In spite of significant advances in medicinal plant research and rapid strides in modern medicine, there continues to be a need for more precise, safe and effective treatments of liver disorders. In recent years, there has been a shift towards therapeutic evaluation of herbal products in liver diseases by carefully synergizing the strengths of the traditional systems of medicine with that of the modern concept of evidence-based medicinal evaluation.
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Fig. 1. Chemical structures of ␣- and -amyrin.
Protium heptaphyllum (Aubl.) March. (Burseraceae), is a traditional medicinal plant largely encountered in the Amazon region and also in several other parts of Brazil. The resin collected from the trunk wood of Protium heptaphyllum is locally called as alm´ecega or breu branco and is widely used for skin diseases, healing of ulcers, and as an analgesic (Susunaga et al., 2001). The leaves and stem bark are also employed to treat gangrenous ulcers, inflammatory and painful conditions, diarrhoea and to promote wound healing (Lorenzi, 1972; Corrˆeia, 1984). Several species of plants that belong to Burseraceae family and in particular, the Genus Protium appears to yield large quantities of resin from their trunk wood (Corrˆeia, 1984). Studies performed on the resin and its essential oil proved its traditional use as an anti-inflammatory and analgesic agent (Siani et al., 1999; Oliveira et al., 2004a). Our previous studies have established the antipruritus and gastroprotective effects of ␣- and -amyrin (Fig. 1), a major pentacyclic triterpene obtained as an intractable isomeric mixture from the resin of Protium heptaphyllum (Oliveira et al., 2004b, c). Plant-derived triterpenoids, in general, are known to possess a wide spectrum of pharmacological activity including anti-inflammatory, anti-ulcer, anti-hyperlipidemic, anti-tumor, and hepatoprotective actions (Hu, 1988; Liu et al., 1995; James et al., 2003). Both in vitro and in vivo experiments and clinical assays demonstrate the hepatoprotective and gastroprotective properties of several triterpenoids like oleanolic acid, ursolic acid, ␣-hederin, and glycyrrhizin (Liu et al., 1994). Acetaminophen is a widely used over-the-counter drug for analgesic and antipyretic effects. Its use in overdose (suicidal or accidental) or with chronic alcohol abuse causes fulminant liver failure and contributes significantly to intensive care unit admissions and cost of hospitalization (Prescott, 2000; Gyamlani and Parikh, 2002). Acetaminophen-induced hepatic failure is the second leading cause of liver transplantation and accounts to considerable levels of morbidity and mortality (Lee, 2004). The hepatotoxicity is the result of the formation of a toxic metabolite, N-acetyl-p-benzoquinoneimine (NAPQI) by cytochrome P4502E1 that can induce a dosedependent depletion of intracellular glutathione and perturbations of calcium homeostasis (Lee et al., 1996; Chen
et al., 1999; Holownia and Braszko, 2004). Presently, Nacetylcysteine (NAC) is the treatment option since it increases glutathione levels in the hepatic cytosol and mitochondria to detoxify the highly reactive and cytotoxic NAPQI (Mitchell et al., 1973). Besides, selective inhibitors of Cytochrome P4502E1 that inhibit NAPQI formation are under investigation in managing acetaminophen-associated hepatotoxicity (Holownia and Braszko, 2004). As a part of our continuing search for naturally occurring hepatoprotective substances from folkloric plants, we have selected for the present study, ␣- and -amyrin isolated from the resin of Protium heptaphyllum, although there has been no documented evidence available on the traditional use of this resin in hepatic disorders. The option was based on the following reasons: lay people use this resin but cannot define the merits of hepatoprotectant remedies and the benefits of therapy might be actually due to improvement in liver function; ␣- and -amyrin is the major triterpenoid component of resin that exhibits anti-inflammatory, gastroprotective and antipruritus effects (Oliveira et al., 2004a, b, c); The triterpenoid mixture bears the ursane and oleanane skeletons and compounds like oleanolic acid and ursolic acid, which have chemical resemblance offer hepatoprotection against a variety of hepatotoxicants (Liu et al., 1994; Saraswat et al., 1996; Kim et al., 2004). Thus, the present study aimed to establish the hepatoprotective potential of ␣- and -amyrin, using acetaminophen-induced hepatic damage in mice as an animal model.
2. Materials and methods 2.1. Animals Adult male Wistar rats (150–200 g) obtained from the Central Animal House, Federal University of Cear´a were housed in polypropylene cages under controlled environmental conditions (22 ± 2 ◦ C, 55–60% humidity, 12:12-h light/dark cycle) and had free access to standard laboratory chow (Purina) and water. Animals were fasted overnight for experimentation, but were allowed water ad libitum.
F.A. Oliveira et al. / Journal of Ethnopharmacology 98 (2005) 103–108
Institutional Animal Care and Use Committee of the Federal University of Cear´a, Fortaleza, approved the experimental protocols in accordance with the guidelines of National Institute of Health, Bethseda, USA. 2.2. Drugs and chemicals Acetaminophen was purchased from Sigma, St. Louis, MO, USA and N-acetylcysteine (Fluimucil® ) from Zambon, S˜ao Paulo, Brazil. All reagents were commercially available and of reagent grade. The triterpenoid mixture, ␣- and -amyrin was isolated from the crude resin of Protium heptaphyllum as per methods described earlier (Oliveira et al., 2004a). For experiments, ␣- and -amyrin mixture was suspended in 3% Tween 80 in saline. Acetaminophen was dissolved in pathogen-free normal saline. 2.3. Hepatotoxicity induced by acetaminophen and treatments A total of 120 mice were used for the experiments. These were randomly divided into five groups of 20 in each. The first group received saline (10 ml/kg, i.p.) and served as normal control, while the second, third, fourth and fifth groups were respectively treated intraperitoneally with vehicle (3% Tween 80 in saline; 10 ml/kg), ␣- and -amyrin mixture (50 and 100 mg/kg), or N-acetylcysteine (750 mg/kg), 48, 24, and 2 h before the oral administration of 500 mg/kg acetaminophen. The dose selection for acetaminophen (500 mg/kg) and ␣- and -amyrin mixture (50 and 100 mg/kg) was based on our preliminary experiments. Blood samples and liver tissue specimens were collected 24 h after acetaminophen treatment. Mice that died prior to 24 h were not subjected to biochemical or histopathological analysis. 2.4. Effect of α- and β-amyrin mixture on sleeping time induced by pentobarbital sodium
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trichloroacetic acid/ethylenediaminetetraacetic acid (TCA/ EDTA) and centrifuged at 1500 × g for 15 min. The resulting supernatants were assayed calorimetrically for nonprotein sulfhydryl (NPSH) content using the Ellman’s reagent (Sedlak and Lindsay, 1968). Blood samples collected from the respective groups of mice were centrifuged for 5 min at 1000 × g to separate the serum. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using the RA-XT (automated) biochemical analyzer with respective test kits (Technicon, Bayer S.A. Diagnostic). 2.6. Histological analysis Mice under different treatments were killed at 24 h, liver tissues collected and stored in 10% phosphate buffered formalin. Liver sections (3 m) were prepared, stained with Hematoxylin and Eosin (H&E) and graded for lesions by light microscopy using the following scale (0–5). No lesions = 0; minimal lesions involving single to few necrotic cells = 1; mild lesions, 10–25% necrotic cells or mild diffuse degenerative changes = 2; moderate lesions, 25–40% necrotic or degenerative cells = 3; marked lesions, 40–50% necrotic or degenerative cells = 4; severe lesions more than 50% necrotic or degenerative cells = 5 (Silva et al., 2001). Persons unaware of treatments evaluated the liver sections from different groups. 2.7. Statistical analysis The results are presented as mean ± S.E.M. and were analyzed using a one-way analysis of variance, followed by Tukey’s test for multiple comparisons. Histological scores on liver tissues were analysed by Kruskal–Wallis nonparametric test. Differences were considered statistically significant when P < 0.05.
3. Results To verify a possible influence on cytochrome-P450 enzyme system, ␣- and -amyrin was tested on pentobarbital sodium-induced sleeping time. The test was carried out in male mice (n = 10 per group) treated as follows: group 1 was treated with the vehicle of ␣- and -amyrin (10 ml/kg, p.o.) and the mice in groups 2, and 3 were given the ␣- and amyrin mixture in doses of 50 and 100 mg/kg, i.p., respectively, at 48, 24, and 2 h before the administration of pentobarbital sodium (50 mg/kg, i.p.) and the duration of sleep (min) in each animal was observed. Loss of righting reflex to recovery was recorded as the sleeping time (Darias et al., 1998).
Acetaminophen (500 mg/kg), administered orally induced a fulminant hepatic failure as evidenced by severe mortality (50%), and changes in biochemical and histological parameters (Table 1). Acetaminophen caused a significant enhancement in serum ALT and AST enzyme activities and Table 1 Effect of ␣- and -amyrin mixture on pentobarbitone (50 mg/kg, i.p.)induced sleeping time in mice Treatment
Dose (mg/kg)
Route
Sleeping time (mean ± S.E.M.) (min) 60.55 ± 4.71
Control (vehicle)
–
i.p.
2.5. Biochemical analysis
␣- and -amyrin
50 100
i.p. i.p.
Liver samples collected from mice that belong to different treatments were homogenized (20%, w/v) in 5%
Data as mean ± S.E.M.; n = 10 per group. ∗∗∗ p < 0.001 vs. Control (ANOVA followed by Tukey’s test).
86.25 ± 7.89 105.25 ± 5.29***
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Fig. 2. The representative microphotographs of H&E (×100)-stained histological sections of liver from (A) mice treated with saline showing normal architecture; (B) mice treated with acetaminophen (500 mg/kg, p.o.) and the vehicle used for ␣- and -amyrin (10 ml/kg, i.p.) showing centrilobular necrosis, congestion and an extensive area of inflammatory cell infiltration; (C) mice treated with acetaminophen (500 mg/kg, p.o.) + ␣- and -amyrin (100 mg/kg, i.p.) showing well preserved hepatocytes as well as the architecture with small areas of necrosis and inflammatory cell infiltration; and (D) mice treated with acetaminophen (500 mg/kg, p.o.) + N-acetyl cysteine (750 mg/kg, i.p.) showing partially preserved hepatocytes and architecture with small areas of necrosis and inflammatory cell infiltration.
besides, it significantly lowered the hepatic tissue level of NPSH (GSH) content as compared to saline treated controls. Histological sections showed extensive centrilobular necrosis, congestion and fatty degenerative changes with inflammatory cell infiltration in acetaminophen-treated group that received only the vehicle of ␣- and -amyrin. In contrast, pretreatment with ␣- and -amyrin at both doses (50 and 100 mg/kg, i.p.) showed a significant dose-related lowering effect on acetaminophen-induced elevation of serum ALT and AST and also replenished the depleted hepatic GSH in a significant manner similar to NAC (Table 1). Histological sections analysed for scores revealed significant differences between ␣- and -amyrin-treated and vehicle-treated
controls. Acetaminophen-induced pathological lesion scores were found to be significantly less in liver tissues from ␣- and -amyrin-treated groups (Table 1). Consistent with serological parameters, acetaminophen-induced liver congestion and centrilobular necrosis were substantially prevented by ␣- and -amyrin treatment in a manner similar to NAC (Fig. 2). Table 2 shows the pretreatment effect of ␣- and amyrin (50 and 100 mg/kg, i.p.) on pentobarbital (50 mg/kg, i.p.) induced sleeping time in mice. The sleeping time was potentiated by ␣- and -amyrin at both doses, but a statistical significance was attained only at the dose of 100 mg/kg.
Table 2 Protective effects of ␣- and -amyrin and N-acetylcysteine (NAC) against hepatotoxicity induced by acetaminophen (AAP) in mice Group
Dose (mg/Kg)
Serum ALT (IU/dl)
Control-normal Control-AAP ␣- and -amyrin + AAP ␣- and -amyrin + AAP N-acetylcysteine
– –
61.98 134.35 61.28 44.15 41.46
50 100 750
± ± ± ± ±
11.27 19.76a 12.49b 6.65b 5.34b
Serum AST (IU/dl) 43.17 107.28 32.53 2857 29.96
± ± ± ± ±
7.34 16.02a 6.98b 4.42b 6.46b
GSH (g/g liver) 655.17 432.17 612.54 642.01 611.83
± ± ± ± ±
24.10 32.37a 32.78b 28.59b 32.29b
Liver damage (histological scores) 0.0 4.83 2.17 1.50 0.50
± ± ± ± ±
0.0 0.17a 0.48b 0.22b 0.22b
Mice received intraperitoneal injections of N-acetylcysteine (NAC) or ␣-, -amyrin mixture thrice (48, 24, and 2 h) before the oral administration of acetaminophen (AAP, 500 mg/kg). Twenty-four hours latter, hepatotoxicity was determined by quantifying the serum activities of aspartate amino transferase (AST) and alaninie aminotransferase (ALT) and from histopathological analysis. Each value represents means ± S.E.M. (n = 10–20). a P < 0.05 vs. control-normal. b P < 0.05 vs. controle-AAP (one-way ANOVA and Student t-test or Kruskal–Wallis test).
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4. Discussion In agreement with previous studies, acetaminophen administered orally to mice at an overdose (500 mg/kg) caused a potentially fatal, centrilobular hepatic necrosis as evidenced by 50% mortality, and by histological and biochemical changes. Pretreatment with ␣- and -amyrin completely suppressed the acetaminophen-associated mortality in mice. Besides, marked elevations in serum ALT and AST activities associated with acetaminophen toxicity were reduced by ␣- and -amyrin (50 and100 mg/kg), in a dose-dependent manner. N-acetylcysteine (NAC), a known hepatoprotectant and a sulfhydryl replenisher (Prescott, 2000) also prevented the rise in serum transaminases. Improvement in the GSH level by NAC is considered the most significant mechanism of NAC against acetaminophen toxicity (Bruno et al., 1998). NAC is known to act by raising intracellular concentrations of reduced glutathione (GSH) and/or by scavenging reactive oxygen species (Aruoma et al., 1989). Similar to NAC, ␣- and -amyrin replenished the acetaminophen-induced depletion of hepatic GSH, presumably due to a diminished production of the toxic metabolite, N-acetyl-p-benzoquinoneimine (NAPQI). Histological sections of liver showed centrilobular necrosis with inflammatory cell infiltration in acetaminophenalone treated mice. Centrilobular necrosis, the pathognomonic feature of acetaminophen hepatotoxicity was strikingly reduced in ␣- and -amyrin pretreated mice. Further, the congestion and inflammatory cell infiltrationevoked by acetaminophen was considerably decreased by ␣- and -amyrin, indicating its possible anti-inflammatory action. Recently, Gardner et al. (Gardner et al., 2003) verified the potential role of inflammatory mediators using transgenic mice with a targeted disruption of the tumor necrosis factor receptor-1 (TNFR1) gene, and the results showed an exaggerated hepatotoxicity of acetaminophen in mice lacking TNFR1. We have previously reported the anti-inflammatory activity of the crude resin of Protium heptaphyllum that is rich in ␣- and -amyrin (Oliveira et al., 2004a). Interestingly, the structurally related compounds, ursolic acid and oleanolic acids were also shown to be anti-inflammatory (Baricevic et al., 2001; Banno et al., 2004). Therefore, we propose that the hepatoprotective effect evoked by ␣- and -amyrin may possibly involve, in part, an anti-inflammatory action. The triterpene, ␣- and -amyrin is devoid of any overt toxicity and no mortality was encountered in doses up to 5.0 g/kg, p.o. or 3.0 g/kg, i.p., in mice (unpublished observations). In recent years, there has been an active search for the development of cytochrome P450 inhibitors that may have therapeutic potential in the prevention of liver damage associated with acetaminophen overdose (Walubo et al., 2004). Past studies have ascribed the hepatoprotective effect of triterpene acids, oleanolic acid and ursolic acid to the inhibition of cytochrome P450 isoforms, CYP2E1, CYP1A and CYP2A (Liu et al., 1995; Kim et al., 2004) and suggest that co-administration of acetaminophen with inhibitors of
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cytochrome P450 may be of therapeutic value in the prevention of acetaminophen-associated hepatotoxicity. Considering that acetaminophen is metabolized by Cytochrome-P450 leading to the production of highly reactive toxic metabolites, which contribute to hepatotoxicity, the present study tested the effect of ␣- and -amyrin on the sleeping time induced by pentobarbital sodium in mice. It is known that nearly complete metabolism and conjugation of pentobarbital occurs in liver mainly by cytochrome-P450 (Nebert and Gon zalez, 1987). At the dose of (100 mg/kg), ␣- and -amyrin was found to potentiate the pentobarbital-induced sleeping time in a significant manner, suggesting a possible inhibition of cytochrome-P450 enzymes and the formation of NAPQI via cytochrome P450E1, which is predominantly localized to liver (Lee et al., 1996). Whether or not ␣- and -amyrin suppresses some specific isoforms of cytochrome-P450 requires further investigation. In conclusion, our study demonstrates that ␣- and -amyrin isolated from the traditional medicinal plant Protium heptaphyllum offers hepatoprotection against acetaminophen-induced hepatotoxicity. This protection appears to be, at least in part, related to its diminution of oxidative stress and to the inhibition of cytochrome-P450. Further, our findings on Protium heptaphyllum might provide leads for the design and development of novel drugs that modulate the therapeutic response of drugs like acetaminophen whose biotransformation pathways involves some cytochrome-P450 isoforms.
Acknowledgements This study was supported by grants from the Brazilian National Agencies, CAPES (PROCAD, Proc. No. 0014/010) and CNPq (Proc. No. 472717/03-0).
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