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Research Article
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Hepatocyte Oxidative Stress Indicators of Carbon Tetrachloride Induced Hyperlipidemic Rats (Rattus norvegicus) Treated with Allium sativa Extract |
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Chikezie Paul Chidoka
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Uwakwe Augustine Amadikwa
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ABSTRACT
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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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How
to cite this article:
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
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Received: November 10, 2013;
Accepted: February 08, 2014;
Published: April 11, 2014
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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 DMello, 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:
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Group C1: |
Control/Normal rats received only DW (vehicle; 2.0 mL kg-1
16 h-1, i.p.) for 64 h |
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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 |
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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 |
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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 |
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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 |
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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.
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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 |
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Table 3: |
Effects of A. sativa extract on hepatocyte SOD, GPx
and CAT activities at t = 76 h |
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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 |
|
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 DMello, 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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