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Hepatocyte Oxidative Stress Indicators of Carbon Tetrachloride Induced Hyperlipidemic Rats (Rattus norvegicus) Treated with Allium sativa Extract



Chikezie Paul Chidoka and Uwakwe Augustine Amadikwa
 
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ABSTRACT

Pathogenesis of several chronic liver diseases has been attributed to overwhelmed antioxidant protective system against Reactive Oxygen Species (ROS). The present study ascertained the capacity of short-term administration of ethanolic extract of Allium sativa to neutralize ROS and ameliorate hyperlipidemia. Hyperlipidemia was induced in rats by single intra-peritoneal injection of CCl4 (dosage = 2.0 mL kg-1), followed by treatment with ethanolic extract of A. sativa (dosage: 200 and 400 mg kg-1) at a regular interval of 16 h for 64 h. Blood samples were drawn from the rats at t = 0 and t = 76 h, i.e., 12 h after the end of 64 h treatment with CCl4 per A. sativa extract treatment, to ascertain hepatic function and Serum Lipid Profile (SLP). In addition, liver post mitochondrial supernatant (PMS) fraction was measured for oxidative stress indicators: Lipid peroxidation (LPOx), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and reduced glutathione (GSH). On the average, short-term administration of ethanolic extract of A. sativa caused reduction of SLP in the following magnitude: Total cholesterol (TC) = 19.48, triacylglycerol (TAG) = 48.59, VLDL-C = 48.57, LDL-C = 19.49 and increase in HDL-C = 32.43%. Also, improvement in oxidative stress indicators gave SOD = 10.20, GPx = 30.92, CAT = 18.18, LPOx = 35.92% and GSH = 51.09%. Although the administration of A. sativa extract to the rats did not restore full therapeutic benefits within the experimental time (t = 76 h), the capacity of the plant extract to ameliorate oxidative stress and hyperlipidemia in the animals was fairly at par with the standard hepatic drug-hepaticum.

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Chikezie Paul Chidoka and Uwakwe Augustine Amadikwa, 2014. Hepatocyte Oxidative Stress Indicators of Carbon Tetrachloride Induced Hyperlipidemic Rats (Rattus norvegicus) Treated with Allium sativa Extract. Asian Journal of Biochemistry, 9: 86-97.

DOI: 10.3923/ajb.2014.86.97

URL: https://scialert.net/abstract/?doi=ajb.2014.86.97
 
Received: November 10, 2013; Accepted: February 08, 2014; Published: April 11, 2014



INTRODUCTION

The liver is often referred to as an organ of homeostasis by virtue of the fact that the metabolic concern of the hepatocyte is to ensure constancy in the internal environment of vertebrates. The capability of the liver to achieve this physiologic feat is hinged on high vascularization of the organ, capacity to serve as storage site for macromolecules and micronutrients as well as abode for enzymes involved in carbohydrate, protein and lipid metabolism. In addition, the central roles of the liver in xenobiotic and endogenous detoxification reactions have been well reported (Sugatani et al., 2006; Shaker et al., 2010; Singh et al., 2011). The biosynthesis of most plasma lipoproteins and apolipoproteins occur in the hepatocytes (Mensenkamp et al., 2000; Jiang et al., 2006). Therefore, agents/factors that compromise hepatocellular functionality and integrity alter plasma lipid profile patterns (Wolf, 1999; Ramcharran et al., 2011). Hyperlipidemia describes the elevation in plasma lipid components; Triacylglycerol (TAG), low-density lipoprotein cholesterol (VLDL-C), low-density lipoprotein cholesterol (LDL-C) and total cholesterol (TC), but reduced levels of high-density lipoprotein cholesterol (HDL-C) (Ochani and D’Mello, 2009; Kaur and Meena, 2013). According to Shaker et al. (2010) hepatic dysfunction is associated with acute hepatitis, hepatocellular carcinoma, apoptosis, necrosis, inflammation, immune response, fibrosis, ischemia, altered gene expression and regeneration.

The hepatocyte is well furnished with antioxidant defense systems. Notwithstanding, pathogenesis of several chronic liver diseases has been attributed to overwhelmed antioxidant protective system against ROS (Czuczejko et al., 2003; Novo et al., 2006; Chikezie, 2011). Notably, the antioxidant scavenging enzymes include superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidase (GPx), which offer primary protection to the hepatocyte and by extension, to other peripheral tissues, against oxidative injury (Halliwell, 1994; Bonnefont-Rousselot et al., 2000; Avti et al., 2006; Pasupathi et al., 2009). Some non-enzymatic antioxidant defense structures are reduced glutathione (GSH), a-tocopherol, β-carotene and ascorbate (Avti et al., 2006; Surapaneni, 2007; Singh et al., 2011; Necib et al., 2013).

Despite disparities in the distribution and metabolism plasma lipoprotein between humans and rats (Uetrecht, 2006), the use of animal model as tool for lipid and biomedical research is reliable and still popular. Also, applications of plant extracts for the treatment/management of lipidemia have been severally reported with promising prospects (Kaur and Meena, 2013; Resch and Ernst, 1995). Accordingly, among several medicinal benefits, A. sativa (garlic) have been demonstrated to be an agent of glycemic control (Banerjee and Maulik, 2002; El-Demerdash et al., 2005; Ibegbulem and Chikezie, 2013). The phytochemical and nutritive contents, coupled with previously reported medicinal usefulness of A. sativa extract (Auer et al., 1990; Resch and Ernst, 1995; Qidwai et al., 2000; Ibegbulem and Chikezie, 2013) informed the trial of A. Sativa extract in the present investigation. The present study ascertained the capacity of short-term administration of ethanolic extract of A. sativa to neutralize ROS and ameliorate hyperlipidemia in CCl4 induced hyperlipidemic rats.

MATERIALS AND METHODS

Collection of plant samples and preparation of extract: Fresh samples of A. sativa were obtained in July, 2012 from local market at Umoziri-Inyishi, Imo State, Nigeria. The plant specimen was identified and authenticated by Dr. F.N. Mbagwu at the Herbarium of the Department of Plant Science and Biotechnology, Imo State University, Owerri, Nigeria. A voucher specimen was deposited at the Herbarium for reference purposes. Ethanol/water extract (1:2 v/v) of A. sativa was prepared by methods of Ibegbulem and Chikezie (2013) with modifications according to Lam et al. (2003). Freshbulbs of A. sativa were washed under a continuous stream of distilled water for 15 min and air-dried at room temperature (25±5°C) for 5 h. The bulbs were chopped and further dried for 5 h in an oven at 60°C and subsequently ground with ceramic mortar and pestle. Twenty-five grams (25 g) of the pulverized specimen was suspended in 250 mL of ethanol/water mixture (1:2 v/v) in stoppered flask and allowed to stand in a thermostatically controlled water bath at 40°C for 24 h. The suspension was filtered with Whatman No. 24 filter paper, concentrated in a rotary evaporator at 50°C and dried in vacuum desiccator. The yield was calculated to be 3.6% (w/w). The extract was re-dissolved in 20 mL of PBS (pH = 7.4) and incubated at 37°C for 30 min with thorough shaking. The dissolved content was quickly frozen at -80°C before lyophilization. The required amount of lyophilized extract was reconstituted in 400 μL Distilled Water (DW) and administered by intra peritoneal injection to the rats at doses of 200 and 400 mg kg-1 (Giri et al., 2012) at regular time intervals of 16 for 64 h.

Experimental animals: Male rats Rattus norvegicus (8-10 weeks old) weighing 150-200 g were generous gift from Professor A.A. Uwakwe (Department of Biochemistry, University of Port Harcourt, Nigeria). The rats were maintained at 25±5°C, 30-55% of relative humidity on a 12 light 12 h dark cycle, with access to water and food ad libitum for 2 weeks acclimatization period. The handling of the animals was in accordance with the standard principles of laboratory animal care of the United States National Institutes of Health.

Study design:The animals were deprived of food and water for 16 h before commencement of treatments (control and test experiments) as previously described (Ibegbulem and Chikezie, 2013). Hyperlipidemia was induced in the rats by single intra-peritoneal injection of CCl4 (dosage = 2.0 mL kg-1) 16 h before commencement of study. A total of 20 rats were categorized into 5 groups of 4 (n = 4) each as follows:

Group C1: Control/Normal rats received only DW (vehicle; 2.0 mL kg-1 16 h-1, i.p.) for 64 h
Group C2: Control/Hyperlipidemic rats received 2.0 mL kg-1 CCl4+DW (vehicle; 2.0 mL kg-1 16 h-1, i.p.) for 64 h
Group T1: Hyperlipidemic rats received 2.0 mL kg-1 CCl4+A. sativa (200 mg kg-1 6 h-1, i.p.) for 64 h
Group T2: Hyperlipidemic rats received 2.0 mL kg-1 CCl4+A. sativa (400 mg kg-1 16 h-1, i.p.) for 64 h
Group T3: Hyperlipidemic rats received 2.0 mL kg-1 CCl4+Hepaticum (100 mg kg-1 16 h-1, i.p.) for 64 h

Collection of blood: Blood samples were drawn from the tail vein of each rat prior to anesthetization under light ether i.e., at experimental t = 0 h for measurement of Serum Lipid Profile (SLP) and levels of γ-glutamyl transferase (γ-GT), alanine transaminase (ALT) and aspartate transaminase (AST) activities. Finally, blood samples were obtained by carotid artery puncture for measurement of SLP and enzyme activities 12 h after the end of 64 h treatment with A. sativa extract treatment i.e., (t = 76 h).

Serum lipid profile: Total Cholesterol (TC), triacylglycerol (TAG) and high-density lipoprotein cholesterol (HDL-C) were determined using commercial kits (Randox Laboratory Ltd., UK). Low-density lipoprotein cholesterol (LDL-C) concentration was determined by difference according to the formula described by Friendwald et al. (1972): LDL-C = TC-(HDL-C)-(TAG/5), as reported by Shaker et al. (2010). Very low-density lipoprotein cholesterol (VLDL-C) concentration was estimated using the methods of Burnstein and Sammaille (1960), where the value in mg dL-1 is based on the assumption that in fasting animals, the VLDL-C to TAG ratio is relatively fixed at 1:5 (Ibegbulem and Chikezie, 2013). Atherogenic index (AI) which was a measure of atherogenesis in normal and treated rats was calculated thus: [TC-(HDL-C)]/(HDL-C) (Suanarunsawat et al., 2011).

Serum enzyme assay: AST and ALT activities were measured using the automated enzymatic methods (EliTech Diagnostic, Sees, France), whereas γ-GT activity was according to the methods as described by Fiala et al. (1972).

Preparation of liver homogenates: Organhomogenate was prepared according the procedures of Adekunle et al. (2013). Quickly, the liver was excised and placed between blotting papers to remove accompanying blood. Next, the organ was rinsed in 1.15% KCl solution to obliterate residual hemoglobin molecules. The sample was homogenized using a Teflon homogenizer in aqueous K2PO4/KHPO4 buffer (0.1 M; pH = 7.4); in 4:1 volume of buffer to organ weight. Subsequently, the homogenate was centrifuged at 10,000xg for 20 min at 4°C to obtain the post mitochondrial supernatant (PMS) fraction and collected into sample bottles. The PMS fraction was finally stored at -80°C before used for analyses. The homogenate was used to assay the following oxidative stress indicators: Lipid peroxidation (LPOx), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and GSH. Protein concentration was measured at γmax = 595 nm by methods of Bradford (1976) using bovine serum albumin as standard.

Lipid peroxidation: Measurement of LPOx was according the methods of Ohkawa et al. (1979) with minor modifications by according to Chikezie (2011). Briefly, the reaction mixture consist of PMS fraction in 50 mM Tris-HCl buffer (pH = 7.4), 500 μM ter-butyl hydroperoxide (BHP) (500 μM in ethanol) and 1.0 mM FeSO4. The reaction mixture was incubated for 90 min at 37°C, after which the reaction was stopped by introducing 0.2 mL of 8% sodium dodecyl sulfate (SDS) followed by 1.5 mL of 20% acetic acid (pH = 3.5) to the reaction mixture. The quantity of malondialdehyde (MDA) produced during the incubation period was determined by adding 1.5 mL of 0.8% thiobarbituric acid (TBA) and further heating the mixture at 95°C for 45 min at 95°C. After cooling to 24°C, the mixture was centrifuged at 3,000xg for 10 min. The TBA reactive substances (TBARS) in the supernatant were measured in supernatant at γmax = 532 nm; molar extinction coefficient (S) = 1.53x105 M-1 cm-1. The level of LPOx was expressed in terms of nM of TBARS per 90 min mg-1 protein.

Superoxide dismutase: SOD was estimated according to the methods of Kono (1978). Briefly, the reaction mixture contained solution A (50 mM Na2CO3, 0.1 mM EDTA, pH = 10.0), solution B (96 μM nitrobluetetrazolium [NBT] in solution A) and solution C (0.6% Triton X-100 in solution A) were incubated at 37°C for 10 min. Reaction was started by introducing 100 μL of solution D (20 mM hydroxylamine hydrochloride, pH = 6.0). The rate of NBT dye reduction by anion generated due to photo-activation of hydroxylamine hydrochloride was measured at γmax = 560 nm in the absence of PMS fraction. Next, 10 μL aliquot of PMS were added to the reaction mixture and 50% inhibition in the rate of NBT reduction by SOD present in the enzyme source was measured. A unit (U) of SOD activity was defined by the 50% inhibition of NBT. SOD activity was expressed in U mg-1 protein.

Glutathione peroxidase: GPx activity was measured by the method of Paglia and valentine (1967). Briefly, the reaction mixture contained aliquot of PMS in 50 mM K2PO4/KHPO4 buffer (pH = 7.0), 1.0 mM EDTA, 1.0 mM NaN3, 0.2 mM NADPH, 1.0 U glutathione reductase and 1.0 mM GSH. The reaction mixture was allowed to equilibrate at 25°C for 5 min. The reaction was started by introducing 0.1 mL of 2.5 mM H2O2. Increase in absorbance at γmax = 340 nm was recorded for 5 min. The change in absorbance was defined as nmole of NADPH oxidized to NADP; S = 6.2x103 M-1 cm-1 at γmax = 340 nm. The levels of GPx were expressed in terms of nmole NADPH consumed min-1 mg-1 protein (U mg-1 protein).

Catalase: Measurement of PMS fraction CAT activity was according to the method of Luck (1963). The final reaction volume of 3.0 mL contained 0.05 M Tris-buffer, 5 mM EDTA (pH = 7.0) and 10 mM H2O2 (in 0.1 M K2PO4/KHPO4 buffer; pH = 7.0). A hundred microliters (100 μL) aliquot of the PMS fraction was added to the above mixture. The rate of change of absorbance min-1 at γmax = 240 nm was recorded for 5 min. CAT activity was calculated using S = 43.6 M-1 cm-1 and expressed in terms of mole H2O2 consumed min-1 mg-1 protein (U mg-1 protein).

Reduced glutathione: Level of GSH in organ homogenate was determined by the procedure according to Moron et al. (1979) with minor modification. The 100 μL aliquot of the PMS fraction was mixed with 25% of CHCl3 and centrifuged at 2000xg for 15 min to precipitate proteins. The supernatant was aspirated and diluted to 1.0 mL with 0.2 M Na2PO4/NaHPO4 buffer (pH = 8.0). Later, 2.0 mL of 0.6 mM 5, 5’-dithiobis-(2-nitrobenzoic acid) (DTNB) was added. The absorbance of the developed yellow-colour complex maintained at 25±5°C was measured at γmax = 405 nm after 10 min. A standard curve was obtained with standard μg GSH. The level of GSH was expressed as g GSH mg-1 protein.

Statistical analysis: The data collected were analyzed by the analysis of variance procedure while treatment means were separated by the Least Significance Difference (LSD) incorporated in the statistical analysis system (SAS) package of 9.1 version.

RESULTS

At the end of the experimental time, t = 76 h, serum γ-GT, ALT and AST activities of group C1 (C1γ-GT, C1ALT and C1AST) did not show significant difference (p>0.05) compared to corresponding enzyme activities at t = 0 h. Table 1 showed that the ratio of C1ALT activity to C1AST activity at t = 0 h and t = 76 h was 1:2 approx. In addition, relative marginal variation in C1γ-GT activity within the experimental time was 0.73%; p>0.05. Although γ-GT, ALT and AST activities of group C2 were significantly (p<0.05) elevated compared to group C1, group C2 exhibited marginal variations in the three serum enzyme activities at t = 76 h compared to the values at t = 0 h; increase in C2γ-GT activity = 7.90%, decrease in C2ALT activity = 10.06% and increase in C2ALT activity = 1.02%. However, group C2 serum γ-GT, ALT and AST activities were relatively elevated at t = 76 h compared to groups C1, T1, T2 and T3. Specifically, at t = 76 h, C2γ-GT, C2ALT and C2AST activities represented 2.31, 1.48 and 1.27 folds increase in corresponding enzyme activity compared to group C1; p<0.05.

At the beginning of the experiment, i.e., at t = 0 h, serum γ-GT, ALT and AST activities of rats in groups C2, T1, T2 and T3 were comparatively not significantly (p>0.05) different. The three serum enzyme activities were within the range: γ-GT = 38.08±1.05, 41.43±0.99 U L-1; ALT = 71.89±1.57, 75.68±0.95 U L-1 and AST = 123.68±1.99, 130.80±0.94 U L-1 (Table 1). Furthermore, within the experimental time, serum γ-GT, ALT and AST activities of groups C2, T1, T2 and T3 were significantly different (p<0.05) compared to group C1. Specifically, C2γ-GTactivity represented 2.4 folds increase compared to C1γ-GT activity at t = 76 h; p<0.05.Again, at t = 0 h, serum γ-GT, ALT and AST activities of groups T1, T2 and T3 were significantly different (p<0.05) compared to group C2, whereas at t = 76 h, the three serum enzymes activities were not significantly different (p>0.05).

Table 1: Serum γ-GT, ALT and AST activities of normal and hyperlipidemic rats treated with A. sativa extract
Image for - Hepatocyte Oxidative Stress Indicators of Carbon Tetrachloride Induced Hyperlipidemic Rats (Rattus norvegicus) Treated with Allium sativa Extract
Mean (X)±SD of three (n = 3) determinations, Mean in the columns with the same letter are not significantly different at p>0.05 according to LSD

Although at t = 76 h, T1γ-GT activity was significantly (p<0.05) elevated compared to C1γ-GT activity, serum T1γ-GT represented 32.33% decrease in enzyme activity relative to T1γ-GT activity at t = 0 h. Likewise, decreases in serum enzyme activities at t = 76 h relative to t = 0 h were: T1ALT activity = 22.56% and T1AST activity =11.40%. The reduction in serum enzyme activities in group T2 was in the order: T2γ-GTactivity = 37.24%>T2ALT activity = 32.31%>T2AST activity = 12.19%. T3γ-GT activity at t = 76 h represented 2 folds decrease compared to T3γ-GT activity at t = 76 h. T3ALT and T3AST activities at t = 76 h decreased by 1.60 and 1.20 folds, respectively compared to the corresponding enzyme activity at t = 0 h.

Although, T1ALT activity and was not significantly different (p>0.05) activity from T2ALT activity; these values represented corresponding 18.29 and 26.57% reduction in enzyme activities relative to C1ALT activity; p<0.05. Conversely, T3ALT activity = 47.09±0.99 U L-1<C1ALT activity = 46.14±1.64 U L-1; p>0.05 (Table 1). Likewise, T3ALT activity was not significantly different (p>0.05) from T2ALT activity. Peak value of serum AST activity was registered in group C2; C2AST activity = 124.94±2.64 U L-1 (Table 1). Serum AST activity was in the order: T1AST activity = 115.89±1.95 U L-1>T2AST activity = 109.96±1.62 U L-1>T3AST activity = 102.08±1.91 U L-1 (Table 1). These values corresponded to 7.34, 11.99 and 18.30% reduction in T1AST, T2AST and T3AST activities, respectively compared to C2AST activity.

Furthermore, compared to C2γ-GT activity, T1γ-GT activity was lower (p<0.05) than C2r-GT which was 59.20% reduction in enzyme activity. However, T1γ-GT activity was raised compared to C1γ-GT activity; t = 76 h, T1γ-GT activity = 27.06±1.96 U L-1 >C1γ-GT activity =17.78±0.75 U L-1; p<0.05 (Table 1). T2γ-GT activity was lower than T1γ-GT activity by 6.10%; p>0.05. Nevertheless, T2γ-GT activity was significantly (p<0.05) lower than C2γ-GT activity. T3γ-GT activity was not significantly (p>0.05) different from C1γ-GT activity; specifically, T3γ-GT activity = 20.98±0.92 U L-1 >C1γ-GT activity =17.78±0.75 U L-1; t = 76 h (Table 1). C2ALT activity was highest, representing 1.48 folds increase in enzyme activity compared to C1ALT activity (p<0.05).

SLP indicated C1TC = 33.75±1.02 mg dL-1 (Fig. 1), of which serum concentrations of VLDL-C, LDL-C and HDL-C accounted for 11.02, 53.21 and 35.76% of TC concentration, respectively; AI = 0.54 (Table 2). C2SLP showed that serum lipids concentrations were profoundly altered. For instance, serum TAG, TC, VLDL-C and LDL-C concentrations were significantly (p<0.05) elevated in group C2 by factors of 2.72, 1.76, 2.72 and 2.42, respectively, compared to group C1. The reduced levels of serum HDL-C in group C2 caused corresponding increase in AI (Table 2). Generally, T1SLP was not significantly different (p>0.05) from T2SLP. However, these values represented significant (p<0.05) alteration in T1SLP compared to C1SLP.

Image for - Hepatocyte Oxidative Stress Indicators of Carbon Tetrachloride Induced Hyperlipidemic Rats (Rattus norvegicus) Treated with Allium sativa Extract
Fig. 1: SLP at t = 76 h of normal and hyperlipidemic rats treated with A. sativa extract

Table 2: Atherogenic index at t = 76 h of normal and hyperlipidemic rats treated with A. sativa extract
Image for - Hepatocyte Oxidative Stress Indicators of Carbon Tetrachloride Induced Hyperlipidemic Rats (Rattus norvegicus) Treated with Allium sativa Extract

Table 3: Effects of A. sativa extract on hepatocyte SOD, GPx and CAT activities at t = 76 h
Image for - Hepatocyte Oxidative Stress Indicators of Carbon Tetrachloride Induced Hyperlipidemic Rats (Rattus norvegicus) Treated with Allium sativa Extract
Mean (X)±SD of three (n = 3) determinations, Mean in the columns with the same letter are not significantly different at p>0.05 according to LSD

The use of group C2 as reference point indicated decreased T1TAG and T1VLDL-C (p<0.05), whereas T1TC and T1LDL-C (p>0.05) were not significantly different. Furthermore, T1HDL-C was not significantly (p>0.05) elevated. Accordingly, T2SLP was significantly different (p<0.05) from C2SLP. Conversely, T3SLP showed no significant difference (p>0.05) from T2SLP, except in LDL-C concentration. An overview of Table 2 showed that the AI was in the order: C2 >T1> T2>T3>C1.

Hepatocyte C2SOD gave the highest level of enzyme activity, representing 3.77 folds increase in activity compared to C1SOD activity (p<0.05). Furthermore, hepatocyte T1SOD, T2SOD and T3SOD exhibited elevated activities, which was significantly different (p<0.05) from C1SOD activity. However, hepatocyte T1SOD and T2SOD activities were reduced compared to C2SOD activity (p>0.05). Specifically, T3SOD activity gave 0.770±0.7 U mg-1 protein (Table 3), corresponding to 21.48% reduction in SOD activity compared to C2SOD activity. C2GPx, T1GPx, T2GPx and T3GPx activities were reduced relative to C1GPx activity. GPx showed progressive increase in enzyme activity in the order: T3GPx = 7.09±0.08 U mg-1 protein> T2GPx = 6.44±0.09U mg-1 protein> T1GPx = 6.39±0.14U mg-1 protein>C2GPx= 4.90±0.10 U mg-1 protein (Table 3). A cursory look at Table 3 showed that hepatocyte CAT activity of the various experimental groups followed the same pattern as hepatocyte GPx activity. T1CAT, T2CAT and T3CAT activities were reduced compared to C1CAT activity (p>0.05). However, levels of activity of T1CAT, T2CAT and T3CAT were not significantly different (p>0.05).

Table 4 showed that hepatocyte level of C2LPOx doubled that of C1LPOx. However, levels of T1LPOx, T2LPOx and T3LPOx were not significantly different (p>0.05), but with values significantly lower than those of C2LPOx; p<0.05 and C1LPOx; p>0.05. Level of C2GSH was relatively lowest, whereas C1GSH registered the highest concentration.

Table 4: Effects of A. sativa extract on hepatocyte LPOx and GSH levels
Image for - Hepatocyte Oxidative Stress Indicators of Carbon Tetrachloride Induced Hyperlipidemic Rats (Rattus norvegicus) Treated with Allium sativa Extract
Mean (X)±SD of three (n = 3) determinations, Mean in the rows with the same letter are not significantly different at p>0.05 according to LSD

Table 4 showed progressive increase in levels of hepatocyte GSH in the order: T3GSH = 12.78±0.55 μg GSH mg-1 protein> T2GSH = 10.32±0.85 μg GSH mg-1 protein>T1GSH = 9.05±0.35 μg GSH mg-1 protein, p>0.05.

DISCUSSION

Short-term administration of CCl4 to the experimental rats induced hepatocellular damage typified by raised levels of diagnostic liver functional enzymes in serum; γ-GT, ALT and AST (Table 1). The measurement of serum γ-GT, ALT and AST activities as a basis for ascertaining and confirmation of hepatocellular damage and dysfunction have been widely reported (Sugatani et al., 2006; Abdel-Moneim and Ghafeer, 2007; Shaker et al., 2010; Singh et al., 2011; Al-Dosari, 2011). The serum γ-GT, ALT and AST activities in groups T1, T2 and T3 relative to the group C2 was obvious indication of improvement of functional status of rats in groups T1, T2 and T3. The result of the present study confirmed the implication of ROS as promoters of hepatic damage which was indicated by disturbances in antioxidant defense systems and alterations of biopsy oxidative stress indicators. This mechanism by which CCl4 compromised hepatic functionality and integrity was previously suggested by Shaker et al. (2010). They reported that the biotransformation of CCl4 caused the production of highly unstable free radicals (CCl3 or CCl3O2), engendering endoplasmic reticulum lipid peroxidation and cellular damage. Mayes (1983) in another report stated that the short-term hepatotoxic effect of CCl4 was because of the capability of CCl4 to inhibit secretory mechanism and conjugation of lipids with apolipoproteins within the hepatocytes and thereby causing fatty liver. In this regard preceding studies have revealed distortions in plasma lipoproteins and lipid profile in animals with induced hepatocellular damage or impairments (Ooi et al., 2005; Jiang et al., 2006; Ramcharran et al., 2011). The reports presented here showed perturbation in SLP patterns in the experimental rats which was in concordance with previous observations. The alterations in SLP were reflections of compromised structural and functional integrity of the hepatocytes. Ooi et al. (2005) posited that the low serum level of HDL-C was a reflection of pathologic conditions and could indicate the severity of hepatic dysfunction. The hyperlipidemic ameliorative property of A. sativa extract is exemplified by its serum TC, TAG, VLDL-C and LDL-C lowering effect in a dose dependent manner (Fig. 1) in the experimental rat groups (T1 and T2). In similar manner, Lau et al. (1983) had demonstrated by the use of both animal and human studies, that components of garlic lowered plasma TC and TAG levels with changes in blood lipoproteins and coagulation parameters. They further posited that their available data suggested that garlic may be of value in either the prevention or treatment of atherosclerotic diseases. In another study, El-Demerdash et al. (2005) reported the presence of cysteine derivatives, notably, S-alkyl cysteine sulfoxides in A. sativa. They noted that during extraction these compounds are converted by allinase into thiosulfinates and polysulfides compounds which possess hypocholesterolaemic as well as antidiabetic, antibiotic and fibrinolytic properties.

The pattern of AI of the various experimental groups (Table 2) showed the propensity of hyperlipidemia, occasioned by hepatic dysfunction, to promote atherogenic conditions. Studies have confirmed that hyperlipidemia elicits oxidative stress in organs such as the heart, kidney and liver (Suanarunsawat et al., 2011; Shaker et al., 2010) which plays a major role in the etiology of atherosclerosis, hypertension, diabetes and several degenerative diseases (Vijayakumar et al., 2004; Du et al., 2010). In addition, mechanism generated by ROS cause the oxidation of LDL-C, engendering cytotoxic events in endothelial cells and selective accumulation of modified LDL-C (Torres et al., 1999). This pathologic event is one of the various major contributing and causative factors of atherosclerosis. The present study has shown the capacity of A. sativa extract to reverse oxidative stress and hyperlipidemia in the experimental rats (T1 and T2) which was comparable to those treated with the standardhypolipidemic drug-hepaticum (T3). However, the short-term treatments did not provide for the animals, the requisit and anticipated full ther apeutic benefits. Likewise, previous authors have reported the therapeutic usefullness of A. sativa for the treatment and management of cardiovascular diseases (Mahmoodi et al., 2006), hypertension (Benavides et al., 2007), Alzheimer's disease (Peng et al., 2002), inflammation, thrombosis (Fukao et al., 2007) malignancy (Hsing et al., 2002) fatty liver (Sahebkar, 2011) and as antimicrobial (Gull et al., 2012).

Liver biopsy showed perturbations of enzymatic (SOD, GPx and CAT) and non-enzymatic (LPOx and GSH) oxidative stress indicators of experimental rats (Tables 3 and 4). In agreement with the present findings, Durendic-Brenesel et al. (2013) reported increased SOD activity in the liver homogenates of the hyperlipidemic rats (Table 3). The reduced levels of C2GPx and C2CAT activities were the effect of raised and overwhelming levels of ROS (El-Demerdash et al., 2005; Avti et al., 2006); ROS has inhibitory effect on ROS scavenging enzymes such as CAT and GPx activities (Hassan and Fridovich, 1978; Avti et al., 2006). Consequently, raised levels of cytotoxic ROS engendered membrane lipid peroxidation with the productions of associated by-products such as malondialdehyde (MDA) and 4-hydroxyalkenals (4HNE) (Shaker et al., 2010; Al-Dosari, 2011; Durendic-Brenesel et al., 2013). Depleting C2GSH concentration confirmed increased oxidative stress (Surapaneni, 2007; Abdel-Moneim and Ghafeer, 2007) through ROS oxidation of sulfhydryl groups involved in cellular enzymatic cofactor and non-enzymatic reduction pathways. The present investigations showed that administration of A. sativa extract caused relief in oxidative stress to the experimental rats as indicated by decreased SOD but increasedGPx and CAT activities; decreased LPOx but increased GSH content in groups T1 and T2 compared to group C2 (Tables 3). Equally, oxidative stress indicators showed that short-term administration of A. sativa extract did not restore full therapeutic benefits to the experimental rats. However, the capacities of the two experimental doses (200 and 400 mg kg-1) of A. sativa extractto alleviate oxidative stress were comparable to the standard hepatic drug-hepaticum. Previous studies have shown that Buckwheat (Fagopyrum esculentum) (Durendic-Brenesel et al., 2013), Ocimum sanctum L. (Suanarunsawat et al., 2011) and Roselle (Hibiscus sabdariffa Linn.) (Ochani and D’Mello, 2009) share similar antioxidant phytochemicals with A. sativa extract (Ibegbulem and Chikezie, 2013). Accordingly, the presence of phytochemicals such phenolics, tannins and flavonoids in A. sativa extract, coupled with high content of antioxidant element-selenium (Banerjee and Maulik, 2002) contributed to the antioxidant property of A. sativa extract.

Although the administration of A. sativa extract to the rats did not restore full therapeutic benefits within the experimental time (t = 76 h), the capacity of the plant extract to ameliorate oxidative stress and hyperlipidemia in the animals was fairly at par with the standard hepatic drug-hepaticum.

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