http://informahealthcare.com/arp ISSN: 1381-3455 (print), 1744-4160 (electronic) Arch Physiol Biochem, Early Online: 1–6 ! 2015 Informa UK Ltd. DOI: 10.3109/13813455.2015.1016974
ORIGINAL ARTICLE
Hepatoprotective activity of Peganum harmala against ethanol-induced liver damages in rats Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by Nyu Medical Center on 05/22/15 For personal use only.
Ezzeddine Bourogaa1, Raoudha Mezghani Jarraya2, Mohamed Damak2, and Abdelfattah Elfeki1 1
Laboratoire d’Ecophysiologie Animale, Faculte´ des Sciences de Sfax, University of Sfax, PB 1171, 3000 Sfax Tunisie and 2Laboratoire de Chimie des Substances Naturelles, Faculte´ des Sciences de Sfax, University of Sfax, PB 1171, 3000 Sfax Tunisie Abstract
Keywords
In this study, we investigated the protective effects of Peganum harmala seeds extract (CPH) against chronic ethanol treatment. Hepatotoxicity was induced in male Wistar rats by administrating ethanol 35% (4 g/kg/day) for 6 weeks. CPH was co-administered with ethanol, by intraperitonial (IP) injection, at a dose of 10 mg/kg bw/day. Control rats were injected by saline solution (NaCl 9%). Chronic ethanol administration intensified lipid peroxidation monitored by an increase of TBARS level in liver. Ethanol treatment caused also a drastic alteration in antioxidant defence system; hepatic superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. A co-administration of CPH during ethanol treatment inhibited lipid peroxidation and improved antioxidants activities. However, treatment with P. harmala extract protects efficiently the hepatic function of alcoholic rats by the considerable decrease of aminotransferase contents in serum of ethanol-treated rats.
Antioxidant enzymes, chloroform extract, liver, Peganum harmala, seeds
Introduction Oxidative stress and the subsequent formation of reactive oxygen species have been correlated with a number of diseases in animal models and humans. It is well documented that chronic ethanol consumption induces oxidative stress in the liver (Esterbaur, 1991). Alcohol liver disease (ALD) is the most common form of liver dysfunction in the world. ALD is also the major cause of chronic diseases and death associated with alcohol misuse (French, 1996). The liver accounts for 90% of alcohol metabolism and is the organ most adversely affected (Kaviarasan et al., 2007). Ethanol metabolism gives rise to the generation of excess amounts of reactive oxygen species which causes a profound increase in hepatic lipid synthesis and has a detrimental effect on the cellular antioxidant defence system (Navasumrit et al., 2000; Ozaras et al., 2003a). The principal damage of ethanol are hepatic induced by lipid peroxidation, decreasing activities of antioxidant enzymes (such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)) and generation of free radicals, as well as elevation of hepatic enzymes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) ¨ ner et al., 2008). ALT and AST are (Escobar et al., 1996; O Correspondence: Ezzeddine Bourogaa, Laboratoire d’Ecophysiologie Animale, Faculte´ des Sciences de Sfax, University of Sfax, PB 1171, 3000 Sfax Tunisie. Tel: (00216) 74 276 400. Fax: (00216) 74 274 437. E-mail: [email protected]
History Received 28 October 2014 Revised 6 January 2015 Accepted 4 February 2015 Published online 14 May 2015
the most sensitive biomarkers employed in the diagnosis of hepatic damage. Peganum harmala is a vivacious plant of the Zygophyllaceae family, known under the user Arabian name Harmel. It is present abundantly in the Middle East and North Africa (Chaieb & Boukhris, 1998). It is also abundant in subdesert areas of North Africa, in some regions of Europe, in Asia and in southern Russia. P. harmala is a bright-green, densely foliaged, herbaceous succulent. Each year between June and August, P. harmala produces many single white conspicuous flowers. Each flower has the potential to develop into a fruit which is a leathery, three-valve seed capsule. Each capsule measures about 3 to 8 inches in diameter and contains more than 50 dark-brown, angular seeds. P. harmala extracts are considered important for drug development, because they are reported to have numerous pharmacological activities. For a long time P. harmala has been used in traditional medicines for the relief of pain and as an antiseptic agent. P. harmala also have anti-bacterial, anti-fungal, anti-viral, anti-oxidant, anti-diabetic, anti-tumor, anti-leishmanial, insecticidal and cytotoxic activities and hepato-protective effects (Jinous & Fereshteh, 2012). The biological activity of P. harmala is essentially due to the presence of a large amount of alkaloids whose content rises suddenly during the ripping process of the fruits and condenses mainly in seeds (Hilal et al., 1978). The present work was carried out to study the effect of chloroform seeds extract on liver lipid peroxidation,
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enzymatic anti-oxidants and aminotransferase contents in ethanol-treated rats. Thus, the possible protective effect of P. harmala seeds extract against ethanol toxicity in male albino rats was detected by the determination of some biochemical parameters including activities of ALT, AST, ALP and oxidative stress parameters; such as SOD, CAT, GPx and TBARS in liver.
Materials and methods
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Plant materials Seeds of P. harmala were collected in southern Tunisia. The plant was identified by Pr. Mohamed CHAEIB, Botanist from the Sfax Faculty of the Sciences. A voucher specimen No. LCSN100 has been deposited at the Laboratoire de Chimie des Substances Naturelles, Sfax Faculty of the Sciences in Tunisia. Extractions The dried seeds of P. harmala (300 g) were firstly extracted during 24 h by hexane. Removal of the solvent under reduced pressure gave an oil extract (yield: 6.45%). The remaining residue was damp with ammoniac during 24 h then extracted with chloroform in a Soxhlet apparatus also during 24 h. Then the solvent was evaporated to give finally dry mass (17.4 g) which was dissolved in ethanol-water (1–9, v/v), mixture to be injected into the animals. The soluble fraction (yield: 21.9%) in the ethanol-water (1–9, v/v) extract was subjected to phytochemical screening. The chemical tests performed in the last extract for sterols, triterpenoids, carotenoids, quinons, flavonoids and tropolons were negative. However, the chemical test performed for alkaloids was positive and very important.
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Blood analysis The haematological parameters, red blood cell (RBC), white blood cell (WBC) and haematocrit were determined using a blood analyser (COULTERÕ MAXMÔ/HMX haematology analyser). Preparation of liver extracts About 1 g of liver was cut into small pieces and immersed into 2 ml ice-cold lysis buffer (TBS, pH 7.4), then sonicated (10 s, twice) and centrifuged (5000 g, 30 min and 4 C). Supernatants were collected and stored at 80 C until use. Biochemical assays Assays of serum markers Activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were assayed using commercial diagnostics kits supplied by Sigma Munich (Munich, Germany) and Boehringere Mannheim (Mannheim, Germany). Estimation of LPO According to Esterbauer et al. (1991), the level of LPO was measured as thiobarbituric acid reactive substances (TBARS). For the assay, 250 ml of the supernatant was mixed with 100 ml TBS buffer, 150 ml of TCA-BHT (20–1%), vortexed and then centrifuged at 1000 rpm for 10 min. From the collected supernatant, 400 ml was mixed with 80 ml HCl (0.6 M), 320 ml of Tris-TBA (26–120 mM), vortexed and then incubated at 80 C for 10 min. The absorbance was measured at 530 nm. The amount of TBARS was calculated using an extinction coefficient of (1.56 105 M 1 cm 1) and expressed as nanomoles per milligram of protein. SOD activity
Animals and treatments The study was performed in male Wistar rats weighing 200 ± 15 g. Animals were obtained from the breeding centre of the Central Pharmacy of Tunis, Tunisia. Before any experience, all animals were acclimatized under the same laboratory conditions of photoperiod (14 h light:10 h dark cycle), and room temperature 24 ± 2 C and received a nutritionally standard diet supplied by the Socie´te´ industrielle des concentres (SICO, Sfax, Tunisia) and water was available ad libitum. The rats were randomly divided into three groups of six males, each as follows: Group 1 (control) served as untreated control and received i.p. injection of 9% NaCl. Group 2 (ethanol) received 4 g ethanol/kg b.w. (i.p.) of 35% ethanol solution for 6 weeks to induce oxidative stress. Group 3 was given the same dose of ethanol and injected by CPH 10 mg/kg b.w. (i.p.) for 6 weeks. The total rat body weights were recorded daily throughout the experimental period, at the end of which the rats were killed by decapitation. Blood was collected and livers were removed and weighed after the removal of the surrounding connective tissues.
The total (Cu-Zn and Mn) SOD activity was determined by measuring its ability to inhibit the photoreduction of nitroblue tetrazolium (NBT) (Durak et al., 1993). One unit of SOD represents the amount inhibiting the photo-reduction of NBT by 50%. The activity was expressed as units per milligram of protein. CAT activity CAT activity was measured according to Aebi (1984). The reaction mixture (1 ml) contained 100 mM phosphate buffer (pH 7), 100 mM hydrogen peroxide (H2O2) and 20 ml (about 1–1.5 mg of protein) of liver homogenate. H2O2 decomposition was followed by measuring the decrease in absorbance at 240 nm for 1 min. The enzyme activity was calculated using an extinction coefficient of 0.043 mM 1 cm 1 and expressed in international units, that is, in micromoles of H2O2 destroyed per minute per milligram of protein. GPx activity The GPx activity was assayed according to the method of Flohe and Gunzler (1984). The activity was expressed as micromoles of GSH oxidized per minute per gram of protein.
DOI: 10.3109/13813455.2015.1016974
Hepatoprotective activity of Peganum harmala against ethanol-induced liver damages in rats
Protein content Protein content in tissue extracts was determined according to Lowry’s method (Lowry et al., 1951) using bovine serum albumin as standard. Statistical analysis The results were expressed as mean ± SEM. The significance of the differences between the means of control and the means of the test studies was established by the Student’s t-test for independent samples (p50.05).
(2.98 ± 0.148). So, no significant differences were noted in the RBC and haematocrit values. Serum markers of cell damages AST, ALT and ALP were released into the blood when certain organs or tissues, particularly liver and heart, are injured. As shown in Figure 2, ethanol treatment induced an increase in the serum levels of AST, ALT and ALP in alcoholic rats (EtOH), when compared with controls. These effects were not detected in rats treated with plant extract. Estimation of TBARS levels in liver
Results Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by Nyu Medical Center on 05/22/15 For personal use only.
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Chemical characterization The extract was subjected to chemical tests. The results have allowed us to make a correlation between the nature of the products, present in the extract, and biological activities. After extraction, separation and purification by chromatographic techniques, structural studies by UV, IR, MS, 1H NMR and 13 C NMR allowed us to identify five major products: harmaline, harmine, vasicinone, vasicine and harmalacidine (Figure 1). These products have very marked biological effects, such as anti-proliferative activities (Hamsa et al., 2010; Lamchouri et al., 2010), anti-leishmanial activities (Parvaneh et al., 2011), anti-plasmodial and vasorelaxant activities (Astulla et al., 2008), Anti-nociceptive effects (Monsef et al., 2004) and insecticidal activities (Zeng et al., 2010).
The effect of seeds extract on ethanol-induced LPO in the liver is shown in Table 3. Ethanol induces a significant increase in TBARS level, which was inhibited by CPH treatment. In CPH-treated rats, TBARS levels were not significantly different from controls. Antioxidant enzymes Following the ethanol treatment, the hepatic antioxidant activities were decreased; superoxide dismutase (p50.01), catalase (p50.05) and glutathione peroxidase (p50.01) as compared with untreated control group (Table 3). The simultaneous treatment with ethanol and P. harmala seeds extract was responsible for the significant improvement in the oxidative status within rats’ livers.
Discussion
The levels of blood parameters such as RBC, WBC and haematocrit are reported in Table 2. Ethanol administration increased WBC level 3.29 ± 0.11 against 2.92 ± 0.12 in control groups (p50.05). WBC levels were significantly decreased (p50.05) in animals treated with CPH
There is increasing evidence that oxidative stress plays a vital role in the pathogenesis of alcohol liver disease (Pranoti & Gyongyi, 2009). Ethanol-induced hepatic damage is mediated by acetaldehyde and ROS (Yu et al., 2010). The removal and neutralization of these noxious metabolites are considered the crucial steps in the prevention of such pathogenesis (Ozaras et al., 2003b). Administering ethanol to rats markedly increases serum AST, ALT and ALP levels, which reflects the severity of liver injury (Lin et al., 1996). The present work revealed that ethanol administration for 6 weeks caused a significant increase in serum AST, ALT and ALP contents compared with the control group. These increases may be due to liver damage induced by EtOH intoxication. In this study, treatment of rats with the chloroform extract of P. harmala seeds resulted in significant decreases in AST, ALT and ALP activities in serum compared with the EtOH group. These results are in agreement with our previously studies (Bourogaa et al., 2013, 2014). This effect is
Table 1. Growth parameters in rat after chronic ethanol administration treated with or without P. Harmala seeds extract (CPH).
Table 2. Blood parameters in rat after chronic ethanol administration treated with or without P. Harmala seeds extract (CPH).
Growth parameters
Growth parameters
Control
Ethanol
Ethanol + CPH
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7.49 ± 1.712 2.92 ± 0.126 0.432 ± 0.003
7.62 ± 1.505 3.29 ± 0.117a 0.39 ± 0.0072
6.93 ± 1.66 2.98 ± 0.148b 0.403 ± 0.0027
Growth parameters At the end of the experiment rat body weight varied from an average of 200 ± 12 g – at the beginning of treatment – to 270 ± 20 g at sacrifice. The mean body weight gain in control rats was 35% against 22% in the ethanol-treated group (p50.01) and no significant variation between control and CPH animals was detected (38%) (Table 1). Furthermore, this treatment was correlated with the hypertrophy of the liver (Table 1). Yet, none of the observed phenomena took place when ethanol treatment was associated with CPH. Blood parameters
Control
Ethanol
Ethanol + CPH a
b
Weight gain (%) 35.83 ± 4.23 22.66 ± 3.63 38.83 ± 3.82 Relative liver weight (%) 2.38 ± 0.062 2.96 ± 0.088a 2.42 ± 0.062c Body mass index 21.85 ± 0.21 16.34 ± 0.61a 20.28 ± 0.17c
RBC (10 /mm3) WBC (103/mm3) Haematocrit
Data represent mean ± SEM of six rats. Symbols represent statistical significance. a p50.05 when compared with control group; bp50.001; cp50.05 when compared with ethanol group.
Data represent mean ± SEM of six rats. Symbols represent statistical significance. a p50.05 when compared with control group; bp50.05 when compared with ethanol group.
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Figure 1. Structure of products isolated from P. harmala seeds extract (CPH); harmaline, harmine, vasicinone, vasicine and harmalacidine.
Table 3. Effect of chronic ethanol administration on oxidative stress parameters in rat liver. Parameter
Control
Ethanol
Ethanol + CPH a
TBARS (nmol/mg protein) 0.216 ± 0.011 0.574 ± 0.012 0.343 ± 0.016b SOD (U/mg protein) 12.18 ± 0.33 6.88 ± 0.30a 11.76 ± 0.41 CAT (U/mg protein) 63.35 ± 2.86 42.6 ± 3.60b 71.3 ± 4.73c GPx (U/mg protein) 11.17 ± 1.10 6.8 ± 0.75a 10.18 ± 0.82c TBARS, thiobarbituric acid-reacting substances; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase. Data represent mean ± SEM of six rats. Symbols represent statistical significance. a p50.001; bp50.05 when compared with control group; cp50.001 when compared with ethanol group.
in agreement with the commonly accepted view that serum levels of transaminases return to normal with the healing of hepatic parenchyma and regeneration of hepatocytes (Thabrew et al., 1987). Alkaline phosphatise, the prototype of these enzymes, reflects the pathological alteration in biliary flow (Ploa & Hewitt, 1989). These decreases in the activities of transaminases were considered as an indicator for the improvement of the functional status of liver cells which may be due to the action of the free radical scavenging activity and antioxidant property of P. harmala seeds extract. The ethanol oxidizing system takes place in microsomes, involving an ethanol-inducible cytochrome P450 (2E1). After chronic ethanol consumption, there is a four- to ten-fold induction of P450 (2E1) expression, associated not only with increased acetaldehyde generation but also with the
production of oxygen radicals that promote lipid peroxidation (Lieber, 2004; Purohit et al., 2004). Indeed, the TBARS, product of lipid peroxidation, showed the role of remarkable free radical in liver damage which increase significantly in EtOH group. In the same time, data declared the importance of plant extract, which has shown significant decrease for TBARS levels and significant increase in anti-oxidant activities. CPH administration abolished the increase in TBARS levels in liver. This is in agreement with previous data demonstrating the protective effect of P. harmala extract and the two major alkaloids (harmine and harmaline) from the plant’s seeds against TBARS and conjugated diene formation (Berrougui et al., 2006). On the other hand, superoxide dismutase, catalase and glutathione peroxidase activities decreased significantly in ethanol-treated rats, which is in agreement with previous data demonstrating a decrease in antioxidant defence activities in rat liver exposed to ethanol (Tsvetanova et al., 2006). Treatment with plant extracts showed a protective role of P. harmala seeds. Therefore, significant increases in hepatic SOD, CAT and GPx activities were observed in rats treated with CPH. In agreement with the present finding, the protective effect of pumpkin seeds for liver damage was reported (Mohamed et al., 2009). These increases in SOD, CAT and GPx activities may be due to the anti-oxidant activity of CPH which recover the damage caused by ethanol. These results suggested that plant extract might have a direct effect on inhibiting the ROS induced membrane damage and enhancing the activity of endogenous antioxidants.
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DOI: 10.3109/13813455.2015.1016974
Hepatoprotective activity of Peganum harmala against ethanol-induced liver damages in rats
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Figure 2. Activities of AST, ALT and ALP in the liver of normal and ethanol-treated rats with or without the treatment of P. harmala seeds extract. Student’s t test: Symbols represent statistical significance ##(P50.001), #(P50.05) indicates significant difference from control rats (control). **(P50.001), *(P50.05) indicates significant difference from ethanol-treated rats (EtOH).
It could be concluded that Peganum harmala seeds extract possessing a protective role against ethanol toxicity. The protective effect of CPH may be due to its potent antioxidant activity, and/or by scavenging the free radicals and inhibition of lipid peroxidation. Finally, there is a need for further research to identify the mechanism of interaction between ethanol toxicity and P. harmala seed extract to know how this extract inhibits or prevents the ethanol toxicity.
Acknowledgements This work was financially supported by the Tunisian Ministry of Higher Education and Scientific Research and Technology.
Declaration of interest The authors declared no conflicts of interest.
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Durak I, Yurtarslanl Z, Canbolat O, Akyol O. (1993). A methodological approach to superoxide dismutase (SOD) activity assay based on inhibition of nitroblue tetrazolium (NBT) reduction. Clin Chim Acta, 214:103–4. Escobar JA, Rubio MA, Lissi EA. (1996). SOD and catalase inactivation by singlet oxygen and peroxyl radicals. Free Radical Bio Med, 20:285–90. Esterbaur H, Schaur R, Zollner H. (1991). Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Rad Biol Med, 11:81–128. Flohe L, Gunzler WA. (1984) Assays of glutathione peroxidase. Methods Enzymol, 105:114–21. French SW. (1996). Ethanol and hepatocellular injury. Clin Lab Med, 16:289–306. Hamsa TP, Kuttan G. (2010). Harmine inhibits tumour specific neo-vessel formation by regulating VEGF, MMP, TIMP and proinflammatory mediators both in vivo and in vitro. Eur J Pharmacol, 649:64–73. Hilal SH, Haggag MY, Soliman F, El-Kashoury S. (1978). Alkaloids of Peganum harmala L. Egypt Pharm Sci, 19:393–9. Jinous A, Fereshteh R. (2012). Chemistry, pharmacology and medicinal properties of Peganum harmala L. Afr J Pharm Pharmacol, 6:1573–80. Kaviarasan P, Viswanathan P, Anuradha CV. (2007). Fenugreek seed (Trigonella foenum graecum) polyphenols inihibit ethanol-induced collagen and lipid accumulation in rat liver. Cell Biol Toxicol, 23:373–83. Lamchouri F, Toufik H, Bouzzine SM, et al. (2010). Experimental and computational study of biological activities of alkaloids isolated from Peganum harmala seeds. J Mater Environ Sci, 1:343–52. Lieber CS. (2004). Alcoholic fatty liver: its pathogenesis and mechanism of progression to inflammation and fibrosis. Alcohol, 34:9–19. Lin SC, Yao CJ, Lin CC, Lin YH. (1996). Hepatoprotective activity of Taiwan folk medicine: Eclipta prostrate Linn. against various hapatotoxins induced acute hapatotoxicity. Phytotherapy Res, 10:483–90. Lowry OH, Rosebrough NJ, Randall RJ. (1951). Protein measurement with the Folin phenol reagent. J biol Chem, 193:265–78. Mohamed RA, Ramadan RS, Ahmed LA. (2009). Effect of substituting pumpkin seed protein isolate for casein on serum liver enzymes, lipid profile and antioxidant enzymes in CCl4-intoxicated rats. Adv Biol Res, 3:9–15.
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Monsef HR, Ghobadi A, Iranshahi M, Abdollahi M. (2004). Antinociceptive effects of Peganum harmala L. alkaloid extract on mouse formalin test. J Pharm Pharm Sci, 7:65–9. Navasumrit P, Ward TH, Dodd NJF, O’Conner PJ. (2000). Ethanolinduced free radicals and hepatic DNA strand breaks are prevented in vivo by antioxidants: effects of acute and chronic ethanol exposure. Carcinogenesis, 21:93–9. ¨ ner AC, Mercan U, O ¨ ntu¨rk H. et al. (2008). Anti-inflammatory and O hepatoprotective activities of Trigonella foenum graecum L. Pharmacologyonline, 2:126–32. Ozaras R, Tahan V, Aydin S, et al. (2003a). N-Acetylcysteine attenuates alcohol-induced oxidative stress in animals. World J Gastroenterol, 9:791–4. Ozaras R, Tahan V, Aydin S, et al. (2003b). N-acetylcysteine attenuates alcohol-induced oxidative stress in the rat. World J Gastroenterol, 9:125–8. Parvaneh RM, Soltan AE, Hourmazd O, et al. (2011). In vitro and in vivo activities of Peganum harmala extract against Leishmania major. J Res Med Sci, 16:1032–9. Ploa GL, Hewitt WR. (1989). Detection and evaluation of chemically induced liver injury. In: Principles and Methods of Toxicology. New York: Raven Press, 399–628. Pranoti M, Gyongyi S. (2009). Signalling pathways in alcohol-induced liver inflammation. J Hepatol, 50:1258–66. Purohit V, Russo D, Coates PM. (2004). Role of fatty liver, dietary fatty acid supplements, and obesity in the progression of alcoholic liver disease: introduction and summary of the symposium. Alcohol, 34:3–8. Thabrew MI, Joice PDTM, Rajatissa WA. (1987). Comparative study of efficacy of Paetta indica and Osbeckia octandra in the treatment of liver dysfunction. Planta Medica, 53:239–41. Tsvetanova E, Kessiova M, Alexandrova A, et al. (2006). In vivo effects of CB1 receptor ligands on lipid peroxidation and antioxidant defense systems in the brain of healthy and ethanol-treated rats. Pharmacol Rep, 58:876–83. Yu HS, Tsunehiro O, Toyohi I, et al. (2010). Formation of acetaldehydederived DNA adducts due to alcohol exposure. Chemico-Biological Interactions, 188:367–75. Zeng Y, Zhang Y, Weng Q, et al. (2010). Cytotoxic and insecticidal activities of derivatives of harmine, a natural insecticidal component isolated from Peganum harmala. Molecules, 15:7775–91.
ORIGINAL ARTICLE
Hepatoprotective activity of Peganum harmala against ethanol-induced liver damages in rats Archives of Physiology and Biochemistry Downloaded from informahealthcare.com by Nyu Medical Center on 05/22/15 For personal use only.
Ezzeddine Bourogaa1, Raoudha Mezghani Jarraya2, Mohamed Damak2, and Abdelfattah Elfeki1 1
Laboratoire d’Ecophysiologie Animale, Faculte´ des Sciences de Sfax, University of Sfax, PB 1171, 3000 Sfax Tunisie and 2Laboratoire de Chimie des Substances Naturelles, Faculte´ des Sciences de Sfax, University of Sfax, PB 1171, 3000 Sfax Tunisie Abstract
Keywords
In this study, we investigated the protective effects of Peganum harmala seeds extract (CPH) against chronic ethanol treatment. Hepatotoxicity was induced in male Wistar rats by administrating ethanol 35% (4 g/kg/day) for 6 weeks. CPH was co-administered with ethanol, by intraperitonial (IP) injection, at a dose of 10 mg/kg bw/day. Control rats were injected by saline solution (NaCl 9%). Chronic ethanol administration intensified lipid peroxidation monitored by an increase of TBARS level in liver. Ethanol treatment caused also a drastic alteration in antioxidant defence system; hepatic superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. A co-administration of CPH during ethanol treatment inhibited lipid peroxidation and improved antioxidants activities. However, treatment with P. harmala extract protects efficiently the hepatic function of alcoholic rats by the considerable decrease of aminotransferase contents in serum of ethanol-treated rats.
Antioxidant enzymes, chloroform extract, liver, Peganum harmala, seeds
Introduction Oxidative stress and the subsequent formation of reactive oxygen species have been correlated with a number of diseases in animal models and humans. It is well documented that chronic ethanol consumption induces oxidative stress in the liver (Esterbaur, 1991). Alcohol liver disease (ALD) is the most common form of liver dysfunction in the world. ALD is also the major cause of chronic diseases and death associated with alcohol misuse (French, 1996). The liver accounts for 90% of alcohol metabolism and is the organ most adversely affected (Kaviarasan et al., 2007). Ethanol metabolism gives rise to the generation of excess amounts of reactive oxygen species which causes a profound increase in hepatic lipid synthesis and has a detrimental effect on the cellular antioxidant defence system (Navasumrit et al., 2000; Ozaras et al., 2003a). The principal damage of ethanol are hepatic induced by lipid peroxidation, decreasing activities of antioxidant enzymes (such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)) and generation of free radicals, as well as elevation of hepatic enzymes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) ¨ ner et al., 2008). ALT and AST are (Escobar et al., 1996; O Correspondence: Ezzeddine Bourogaa, Laboratoire d’Ecophysiologie Animale, Faculte´ des Sciences de Sfax, University of Sfax, PB 1171, 3000 Sfax Tunisie. Tel: (00216) 74 276 400. Fax: (00216) 74 274 437. E-mail: [email protected]
History Received 28 October 2014 Revised 6 January 2015 Accepted 4 February 2015 Published online 14 May 2015
the most sensitive biomarkers employed in the diagnosis of hepatic damage. Peganum harmala is a vivacious plant of the Zygophyllaceae family, known under the user Arabian name Harmel. It is present abundantly in the Middle East and North Africa (Chaieb & Boukhris, 1998). It is also abundant in subdesert areas of North Africa, in some regions of Europe, in Asia and in southern Russia. P. harmala is a bright-green, densely foliaged, herbaceous succulent. Each year between June and August, P. harmala produces many single white conspicuous flowers. Each flower has the potential to develop into a fruit which is a leathery, three-valve seed capsule. Each capsule measures about 3 to 8 inches in diameter and contains more than 50 dark-brown, angular seeds. P. harmala extracts are considered important for drug development, because they are reported to have numerous pharmacological activities. For a long time P. harmala has been used in traditional medicines for the relief of pain and as an antiseptic agent. P. harmala also have anti-bacterial, anti-fungal, anti-viral, anti-oxidant, anti-diabetic, anti-tumor, anti-leishmanial, insecticidal and cytotoxic activities and hepato-protective effects (Jinous & Fereshteh, 2012). The biological activity of P. harmala is essentially due to the presence of a large amount of alkaloids whose content rises suddenly during the ripping process of the fruits and condenses mainly in seeds (Hilal et al., 1978). The present work was carried out to study the effect of chloroform seeds extract on liver lipid peroxidation,
2
E. Bourogaa et al.
enzymatic anti-oxidants and aminotransferase contents in ethanol-treated rats. Thus, the possible protective effect of P. harmala seeds extract against ethanol toxicity in male albino rats was detected by the determination of some biochemical parameters including activities of ALT, AST, ALP and oxidative stress parameters; such as SOD, CAT, GPx and TBARS in liver.
Materials and methods
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Plant materials Seeds of P. harmala were collected in southern Tunisia. The plant was identified by Pr. Mohamed CHAEIB, Botanist from the Sfax Faculty of the Sciences. A voucher specimen No. LCSN100 has been deposited at the Laboratoire de Chimie des Substances Naturelles, Sfax Faculty of the Sciences in Tunisia. Extractions The dried seeds of P. harmala (300 g) were firstly extracted during 24 h by hexane. Removal of the solvent under reduced pressure gave an oil extract (yield: 6.45%). The remaining residue was damp with ammoniac during 24 h then extracted with chloroform in a Soxhlet apparatus also during 24 h. Then the solvent was evaporated to give finally dry mass (17.4 g) which was dissolved in ethanol-water (1–9, v/v), mixture to be injected into the animals. The soluble fraction (yield: 21.9%) in the ethanol-water (1–9, v/v) extract was subjected to phytochemical screening. The chemical tests performed in the last extract for sterols, triterpenoids, carotenoids, quinons, flavonoids and tropolons were negative. However, the chemical test performed for alkaloids was positive and very important.
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Blood analysis The haematological parameters, red blood cell (RBC), white blood cell (WBC) and haematocrit were determined using a blood analyser (COULTERÕ MAXMÔ/HMX haematology analyser). Preparation of liver extracts About 1 g of liver was cut into small pieces and immersed into 2 ml ice-cold lysis buffer (TBS, pH 7.4), then sonicated (10 s, twice) and centrifuged (5000 g, 30 min and 4 C). Supernatants were collected and stored at 80 C until use. Biochemical assays Assays of serum markers Activities of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were assayed using commercial diagnostics kits supplied by Sigma Munich (Munich, Germany) and Boehringere Mannheim (Mannheim, Germany). Estimation of LPO According to Esterbauer et al. (1991), the level of LPO was measured as thiobarbituric acid reactive substances (TBARS). For the assay, 250 ml of the supernatant was mixed with 100 ml TBS buffer, 150 ml of TCA-BHT (20–1%), vortexed and then centrifuged at 1000 rpm for 10 min. From the collected supernatant, 400 ml was mixed with 80 ml HCl (0.6 M), 320 ml of Tris-TBA (26–120 mM), vortexed and then incubated at 80 C for 10 min. The absorbance was measured at 530 nm. The amount of TBARS was calculated using an extinction coefficient of (1.56 105 M 1 cm 1) and expressed as nanomoles per milligram of protein. SOD activity
Animals and treatments The study was performed in male Wistar rats weighing 200 ± 15 g. Animals were obtained from the breeding centre of the Central Pharmacy of Tunis, Tunisia. Before any experience, all animals were acclimatized under the same laboratory conditions of photoperiod (14 h light:10 h dark cycle), and room temperature 24 ± 2 C and received a nutritionally standard diet supplied by the Socie´te´ industrielle des concentres (SICO, Sfax, Tunisia) and water was available ad libitum. The rats were randomly divided into three groups of six males, each as follows: Group 1 (control) served as untreated control and received i.p. injection of 9% NaCl. Group 2 (ethanol) received 4 g ethanol/kg b.w. (i.p.) of 35% ethanol solution for 6 weeks to induce oxidative stress. Group 3 was given the same dose of ethanol and injected by CPH 10 mg/kg b.w. (i.p.) for 6 weeks. The total rat body weights were recorded daily throughout the experimental period, at the end of which the rats were killed by decapitation. Blood was collected and livers were removed and weighed after the removal of the surrounding connective tissues.
The total (Cu-Zn and Mn) SOD activity was determined by measuring its ability to inhibit the photoreduction of nitroblue tetrazolium (NBT) (Durak et al., 1993). One unit of SOD represents the amount inhibiting the photo-reduction of NBT by 50%. The activity was expressed as units per milligram of protein. CAT activity CAT activity was measured according to Aebi (1984). The reaction mixture (1 ml) contained 100 mM phosphate buffer (pH 7), 100 mM hydrogen peroxide (H2O2) and 20 ml (about 1–1.5 mg of protein) of liver homogenate. H2O2 decomposition was followed by measuring the decrease in absorbance at 240 nm for 1 min. The enzyme activity was calculated using an extinction coefficient of 0.043 mM 1 cm 1 and expressed in international units, that is, in micromoles of H2O2 destroyed per minute per milligram of protein. GPx activity The GPx activity was assayed according to the method of Flohe and Gunzler (1984). The activity was expressed as micromoles of GSH oxidized per minute per gram of protein.
DOI: 10.3109/13813455.2015.1016974
Hepatoprotective activity of Peganum harmala against ethanol-induced liver damages in rats
Protein content Protein content in tissue extracts was determined according to Lowry’s method (Lowry et al., 1951) using bovine serum albumin as standard. Statistical analysis The results were expressed as mean ± SEM. The significance of the differences between the means of control and the means of the test studies was established by the Student’s t-test for independent samples (p50.05).
(2.98 ± 0.148). So, no significant differences were noted in the RBC and haematocrit values. Serum markers of cell damages AST, ALT and ALP were released into the blood when certain organs or tissues, particularly liver and heart, are injured. As shown in Figure 2, ethanol treatment induced an increase in the serum levels of AST, ALT and ALP in alcoholic rats (EtOH), when compared with controls. These effects were not detected in rats treated with plant extract. Estimation of TBARS levels in liver
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Chemical characterization The extract was subjected to chemical tests. The results have allowed us to make a correlation between the nature of the products, present in the extract, and biological activities. After extraction, separation and purification by chromatographic techniques, structural studies by UV, IR, MS, 1H NMR and 13 C NMR allowed us to identify five major products: harmaline, harmine, vasicinone, vasicine and harmalacidine (Figure 1). These products have very marked biological effects, such as anti-proliferative activities (Hamsa et al., 2010; Lamchouri et al., 2010), anti-leishmanial activities (Parvaneh et al., 2011), anti-plasmodial and vasorelaxant activities (Astulla et al., 2008), Anti-nociceptive effects (Monsef et al., 2004) and insecticidal activities (Zeng et al., 2010).
The effect of seeds extract on ethanol-induced LPO in the liver is shown in Table 3. Ethanol induces a significant increase in TBARS level, which was inhibited by CPH treatment. In CPH-treated rats, TBARS levels were not significantly different from controls. Antioxidant enzymes Following the ethanol treatment, the hepatic antioxidant activities were decreased; superoxide dismutase (p50.01), catalase (p50.05) and glutathione peroxidase (p50.01) as compared with untreated control group (Table 3). The simultaneous treatment with ethanol and P. harmala seeds extract was responsible for the significant improvement in the oxidative status within rats’ livers.
Discussion
The levels of blood parameters such as RBC, WBC and haematocrit are reported in Table 2. Ethanol administration increased WBC level 3.29 ± 0.11 against 2.92 ± 0.12 in control groups (p50.05). WBC levels were significantly decreased (p50.05) in animals treated with CPH
There is increasing evidence that oxidative stress plays a vital role in the pathogenesis of alcohol liver disease (Pranoti & Gyongyi, 2009). Ethanol-induced hepatic damage is mediated by acetaldehyde and ROS (Yu et al., 2010). The removal and neutralization of these noxious metabolites are considered the crucial steps in the prevention of such pathogenesis (Ozaras et al., 2003b). Administering ethanol to rats markedly increases serum AST, ALT and ALP levels, which reflects the severity of liver injury (Lin et al., 1996). The present work revealed that ethanol administration for 6 weeks caused a significant increase in serum AST, ALT and ALP contents compared with the control group. These increases may be due to liver damage induced by EtOH intoxication. In this study, treatment of rats with the chloroform extract of P. harmala seeds resulted in significant decreases in AST, ALT and ALP activities in serum compared with the EtOH group. These results are in agreement with our previously studies (Bourogaa et al., 2013, 2014). This effect is
Table 1. Growth parameters in rat after chronic ethanol administration treated with or without P. Harmala seeds extract (CPH).
Table 2. Blood parameters in rat after chronic ethanol administration treated with or without P. Harmala seeds extract (CPH).
Growth parameters
Growth parameters
Control
Ethanol
Ethanol + CPH
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7.49 ± 1.712 2.92 ± 0.126 0.432 ± 0.003
7.62 ± 1.505 3.29 ± 0.117a 0.39 ± 0.0072
6.93 ± 1.66 2.98 ± 0.148b 0.403 ± 0.0027
Growth parameters At the end of the experiment rat body weight varied from an average of 200 ± 12 g – at the beginning of treatment – to 270 ± 20 g at sacrifice. The mean body weight gain in control rats was 35% against 22% in the ethanol-treated group (p50.01) and no significant variation between control and CPH animals was detected (38%) (Table 1). Furthermore, this treatment was correlated with the hypertrophy of the liver (Table 1). Yet, none of the observed phenomena took place when ethanol treatment was associated with CPH. Blood parameters
Control
Ethanol
Ethanol + CPH a
b
Weight gain (%) 35.83 ± 4.23 22.66 ± 3.63 38.83 ± 3.82 Relative liver weight (%) 2.38 ± 0.062 2.96 ± 0.088a 2.42 ± 0.062c Body mass index 21.85 ± 0.21 16.34 ± 0.61a 20.28 ± 0.17c
RBC (10 /mm3) WBC (103/mm3) Haematocrit
Data represent mean ± SEM of six rats. Symbols represent statistical significance. a p50.05 when compared with control group; bp50.001; cp50.05 when compared with ethanol group.
Data represent mean ± SEM of six rats. Symbols represent statistical significance. a p50.05 when compared with control group; bp50.05 when compared with ethanol group.
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Figure 1. Structure of products isolated from P. harmala seeds extract (CPH); harmaline, harmine, vasicinone, vasicine and harmalacidine.
Table 3. Effect of chronic ethanol administration on oxidative stress parameters in rat liver. Parameter
Control
Ethanol
Ethanol + CPH a
TBARS (nmol/mg protein) 0.216 ± 0.011 0.574 ± 0.012 0.343 ± 0.016b SOD (U/mg protein) 12.18 ± 0.33 6.88 ± 0.30a 11.76 ± 0.41 CAT (U/mg protein) 63.35 ± 2.86 42.6 ± 3.60b 71.3 ± 4.73c GPx (U/mg protein) 11.17 ± 1.10 6.8 ± 0.75a 10.18 ± 0.82c TBARS, thiobarbituric acid-reacting substances; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase. Data represent mean ± SEM of six rats. Symbols represent statistical significance. a p50.001; bp50.05 when compared with control group; cp50.001 when compared with ethanol group.
in agreement with the commonly accepted view that serum levels of transaminases return to normal with the healing of hepatic parenchyma and regeneration of hepatocytes (Thabrew et al., 1987). Alkaline phosphatise, the prototype of these enzymes, reflects the pathological alteration in biliary flow (Ploa & Hewitt, 1989). These decreases in the activities of transaminases were considered as an indicator for the improvement of the functional status of liver cells which may be due to the action of the free radical scavenging activity and antioxidant property of P. harmala seeds extract. The ethanol oxidizing system takes place in microsomes, involving an ethanol-inducible cytochrome P450 (2E1). After chronic ethanol consumption, there is a four- to ten-fold induction of P450 (2E1) expression, associated not only with increased acetaldehyde generation but also with the
production of oxygen radicals that promote lipid peroxidation (Lieber, 2004; Purohit et al., 2004). Indeed, the TBARS, product of lipid peroxidation, showed the role of remarkable free radical in liver damage which increase significantly in EtOH group. In the same time, data declared the importance of plant extract, which has shown significant decrease for TBARS levels and significant increase in anti-oxidant activities. CPH administration abolished the increase in TBARS levels in liver. This is in agreement with previous data demonstrating the protective effect of P. harmala extract and the two major alkaloids (harmine and harmaline) from the plant’s seeds against TBARS and conjugated diene formation (Berrougui et al., 2006). On the other hand, superoxide dismutase, catalase and glutathione peroxidase activities decreased significantly in ethanol-treated rats, which is in agreement with previous data demonstrating a decrease in antioxidant defence activities in rat liver exposed to ethanol (Tsvetanova et al., 2006). Treatment with plant extracts showed a protective role of P. harmala seeds. Therefore, significant increases in hepatic SOD, CAT and GPx activities were observed in rats treated with CPH. In agreement with the present finding, the protective effect of pumpkin seeds for liver damage was reported (Mohamed et al., 2009). These increases in SOD, CAT and GPx activities may be due to the anti-oxidant activity of CPH which recover the damage caused by ethanol. These results suggested that plant extract might have a direct effect on inhibiting the ROS induced membrane damage and enhancing the activity of endogenous antioxidants.
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DOI: 10.3109/13813455.2015.1016974
Hepatoprotective activity of Peganum harmala against ethanol-induced liver damages in rats
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Figure 2. Activities of AST, ALT and ALP in the liver of normal and ethanol-treated rats with or without the treatment of P. harmala seeds extract. Student’s t test: Symbols represent statistical significance ##(P50.001), #(P50.05) indicates significant difference from control rats (control). **(P50.001), *(P50.05) indicates significant difference from ethanol-treated rats (EtOH).
It could be concluded that Peganum harmala seeds extract possessing a protective role against ethanol toxicity. The protective effect of CPH may be due to its potent antioxidant activity, and/or by scavenging the free radicals and inhibition of lipid peroxidation. Finally, there is a need for further research to identify the mechanism of interaction between ethanol toxicity and P. harmala seed extract to know how this extract inhibits or prevents the ethanol toxicity.
Acknowledgements This work was financially supported by the Tunisian Ministry of Higher Education and Scientific Research and Technology.
Declaration of interest The authors declared no conflicts of interest.
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