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Hepatoprotective and antioxidant effects of methanol extractof Moringa oleifera leaves in rats Attia H. Atta1, Hamdy Soufy2, Soad M. Nasr2, Ahmed M. Soliman1, Somia A. Nassar2,Abduljalil Al Maweri1, Tamer H. Abd El-Aziz2, Hassan M. Desouky3and Aboelfetoh M. Abdalla4 1 2
Department of Pharmacology, Faculty of Veterinary medicine, Cairo University, Giza, Egypt.
Department of Parasitology &Animal Diseases, National Research Centre, 33 Bohouth St., Dokki, 12622, Giza, Egypt. 3
Department of Animal Reproduction and Artificial Insemination, National Research Centre, 33 Bohouth St., Dokki, 12622, Giza, Egypt.
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Horticultural Crop Technology Dept., Agriculture Research Division, National Research Centre, El Buhouth, St., Dokki, 12622, Cairo, Egypt.
Corresponding author: Attia H. Atta1, Department of Pharmacology, Faculty of Veterinary medicine, Cairo University, Giza P.O. box 12211, Egypt. Tel. +201006540633. E-mail:
[email protected] Abstract Methanol extract of Moringa oleiferaleaveswas evaluated for its potential hepatoprotective and antioxidant effects in CCl4-intoxicated rats. Phytochemical analysis of methanol extract of M. oleifera leaves revealed the presence of high concentration of carbohydrate, cardiac glycosides, protein and amino acids with a moderate concentration of alkaloids, reducing sugar, saponins, phenols, flavonoids, fixed oil and fats.The GC- mass analysis of the methanol extract of M. oleifera leaves revealed the presence of 25 components. The major components were trans-2, 3-Dimethoxycinnamic acid ,Flavoxate, cis,cis-Muconic acid , Z-3,17-Octadecadien-1-ol acetate and Stevioside. Acute toxicity revealed no morbidity or mortality among rats receiving oral doses up to 4500 mg/kg.BW. M. oleifera leaves methanol extract normalized the hematological and biochemical parameters which have been deteriorated in CCl4-intoxicated rats. Moreover, the level of the antioxidant parameters in liver homogenate was also returned to normal levels. These results were confirmed by the improvement of the histopathological picture of the liver of treated rats. These results confirm the hepatoprotective and antioxidant effects of methanol extract of M. oleifera leaves. Keywords: hepatoprotective, phytochemical, LD50, M. oleifera.
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Introduction Liver is a large and vital organ that functions as a center of metabolism of nutrients, drugs and xenobiotics. Additionally, it is alsoresponsible for elimination of waste products and toxic metabolites (Meyer and Kulkarini 2001; Saleem et al., 2010). Herbal treatment of many diseases including hepatopathy is increasing in many countries (Khalafalla et al., 2010). Various types of plants have been used for several centuries worldwide not only as dietary supplements but also as traditional treatments for many diseases (Wood, 1997; Iqbal and Bhanger 2006; Khalafalla et al., 2010). Among these plants, the widely cultivated Moringa oleifera (M. oleifera; Moringa or drumstick tree), was used by many tropical Asian and African countries and Latin America(Oliveira 249
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et al., 1999; Mukunzi et al., 2011). M. oleifera has long been used for the treatment of many diseases, and therefore, it was called a “miracle vegetable” (Faizi et al., 1995; Anwar et al., 2007). Several data have been published concerning its anti-atherosclerotic (Chumark et al., 2008), immune-boosting (Miyachi et al., 2004), antiviral (Murakami et al., 1998; Waiyaput et al., 2012); antioxidant (Kumar and Pari, 2003; Iqbal and Bhanger, 2006;Ndhlala et al., 2014), antimicrobial (Gupta et al., 2013), anti-inflammatory (Gupta et al., 2013), and anticancer effects (Bharali et al., 2003; Budda et al., 2011;Verma et al., 2012). The hepatoprotective effect of ethanolic extract of leaves of M. oleifera against liver damage induced by isoniazid, rifampicin, and pyrazinamide in rats (Pariand Kumar, 2002) and against acetaminophen (Fakurazi et al. 2008) was evaluated. Moreover, the hepatoprotective effect of M. oleifera seeds against diclofenac and liver fibrosis induced by the carbon tetrachloride (CCl 4) has been reported (Hamza, 2007 and 2010). The hepatoprotective effect of methanol extract M. oleifera leaves, which is the most widely used part of the plant against acute CCl4-induced hepatotoxicity has not been tested and it was the aim of this study.
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Material and Methods 2.1 Preparation of M. oleifera extract The air-dried M. oleifera leaves (Moringaceae) were moderately pulverized, and stored for further use. The powdered plant (200g) was extracted with methanol 95% for at least 24h, followed by percolation for 5 to 7 times till complete exhaustion. The methanol extracts were concentrated under reduced pressure using Rotary Evaporator at temperature not more than 50°C. The concentrated extract was kept at – 4°C until used. A voucher sample of this plant is kept in the pharmacology Department, Faculty of Veterinary, Cairo University. The extracts were freshly suspended in sterile distilled water with few drops of Tween-80. 2.2 Phytochemical analysis of M. oleifera leaves methanol extract 2.2.1 Preliminary phytochemical screening Preliminary phytochemical screening was carried for detection the following active constituents; carbohydrate, reducing sugar, phenols, flavonoids, saponins, cardiac glycosides, protein, amino acids, fixed oils and fats according to Harborne (1973), Trease and Evans (1989), Sofowora (1993).
2.2.2 GC-mass analysis The analysis of M. oleifera methanol extract was carried out using a GC (Agilent Technologies 7890A) interfaced with a mass-selective detector (MSD, Agilent 7000 Triple Quad) equipped with an apolar Agilent HP-5ms (5%-phenyl methyl poly siloxane), capillary column (30 m × 0.25 mm i. d. and 0.25 μm film thickness). The carrier gas was helium with the linear velocity of 1 ml/min. The 250
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injector and detector temperatures were 200º C and 250º C, respectively. Injection mode, split; split ratio 1: 10, volume injected 1μl of the sample. The MS operating parameters were as follows: ionization potential 70 eV, interface temperature 250º C, and acquisition mass range 50–600.The identification of components was based on a comparison of their mass spectra and retention time with those of the authentic compounds and by computer matching with NIST and WILEY library as well as by comparison of the fragmentation pattern of the mass spectral data with those reported in the literature
2.3 Determination of LD50 LD50 of the studied extract was determined as described by Kerber, (1941). In this experiment, nine groups of five Swiss mice were used. Mice in each group received an oral dose of methanol extract of M. oleifera leaves of 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, and 4500 mg/kg .b.wt. Mice were observed for morbidity and /or mortality over 72 hours.
2.4 hepatoprotective effect: 2.4.1 Experimental design This experiment was carried out at the Experimental rat Unit of Lab Animal House, Department of Pharmacology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. Thirty, six weeks old, Sprague Dawley male and female rats (190–200 g) were used. Rats were randomly divided into six equal groups. Rats of group I was kept as environmental control. Rats of group II. were Injected CCl 4 30% in corn oil at a dose of 3 ml kg-1BW via the intraperitoneal (I/P) route twice weekly for 4 weeks. Rats of group III –V were administered Moringa oleifera methanol extract orally in a dose of 100, 200 and 400 mg/kg BW in corn oil 24 h after CCl4 30% I/P injection. Rats of group VI were Administered silymarin (as a standard drug) orally in a dose of 100 mg/kg BW in corn oil 24 h after CCl4 30% I/P injection. The experiment persisted for 4 weeks. Rats were kept in ordinary cages at room temperature of 25 ± 3°C with a 12 h dark/light cycle. They were allowed standard laboratory feed and water ad libitum and reared according to guidelines approved by the Institutional Animal Care and Use Committee, Faculty of Veterinary Medicine, Cairo University.
2.4.2 Blood Samples At the end of the experiment, blood samples were collected by puncture of retro orbital plexus of veins from each rat 24 hours after the CCl4 administration. The blood collected with anti-coagulant EDTA (Ethylene diamine tetra acetic acid tripotassium) was used for hematology. Another blood sample was collected in a plain tube, and was used for serum separation. Sera were stored in -20ºC until used in biochemical analysis.
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2.4.3 Hematological investigations Complete blood count was performed on blood samples with anticoagulant using hematological analyzer (Exigo Eos Vet, Sweden). The erythrogram consisted of red blood cell count (RBCs), hematocrit (HCT), hemoglobin (Hb) concentration and red cell indices, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and red blood cell distribution width absolute (RDWa), leukogram (includes white blood cell count (WBCs), lymphocytes (%), monocytes (%), granulocytes (%)platelet count (PLT) and mean platelet volume (MPV).
2.4.4 Serum Biochemical assessments Serum total proteins and albumin were determined according to the methods of Doumas et al. (1971) and Henary et al.(1974), respectively. The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) were determined according to the procedure of Reitman and Frankel (1957). Serum urea and creatinine were determined according to the methods of Patton and Crouch (1977) and Houot (1985), respectively. Serum total cholesterol and triglycerides were estimated according to Allain et al. (1974) and Fossati and Prencipe (1982), respectively. Test kits supplied by bioMérieuxFrance, were used.
2.4.5 Tissue sampling and preparation of liver homogenate At the end of the experiment (4 weeks) all rats were euthanized by over dose of chloroform. Specimen from each liver was rapidly removed and washed with 0.9% ice-cold NaCl and stored at 80ºC until homogenate preparation to determine the oxidant/antioxidant status. For Preparation of liver homogenate, one gram of liver tissue was homogenized in ice-cold 1.15% solution of potassium chloride in 50 mmol L-1 potassium phosphate buffer solution (pH 7.4) to yield a liver homogenate 10% (W/V). Homogenization was performed using Sonicator, 4710 Ultrasonics Homogenizer (ColeParmer Instrument Co., USA).The homogenate was centrifuged at 4,000×g for 5 min at 4°C. The supernatant was collected and stored at -80ºC until used.
2.4.6 Assessment of total protein concentrations and oxidant/antioxidant biomarkers Total protein concentration was determined in liver homogenate according to the method of Lowry et al. (1951), using bovine serum as a standard. The concentration of glutathione reduced (GSH) using Ellman’s reagent according to the method described by Ellman (1959) and its concentration was expressed as mM g-1 tissue homogenate., lipid peroxidation by-products (LPx) expressed as malondialdehyde (nM g-1) according to Ohkawa et al. (1979). The activity of glutathione peroxidase (GPx) expressed as U g-1 wet liver tissue was estimated according to Paglia and Valentine (1967). 252
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and catalase activity (CAT) expressed as U g-1 wet liver tissue (Aebi, 1984). All parameters were analyzed using Spectrophotometer (T80 UV/VIS PG instrument Ltd, UK) using Test kits from Biodiagnostic, Dokki, Egypt.
2.4.7 Pathological studies Liver specimens from each rat were collected immediately after sacrifice and fixed in 10% formol saline, dehydrated, cleared, and embedded in paraffin blocks. Paraffin sections of 5-μm thickness were prepared, stained by haematoxylin and eosin (H&E), and examined microscopically for histopathological alterations (Bancroft et al. 1996).
2.5Statistical analysis Data were expressed as mean ±SE. Differences between treated groups were tested for significance using a one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test using SPSS version 16 computer program. Differences were considered significant at P
