An Experimental Study on the Therapeutic Efficacy of the Combined... Herbal Medicines with Atorvastatin against Hyperlipidemia in Rats

An Experimental Study on the Therapeutic Efficacy of the Combined... Herbal Medicines with Atorvastatin against Hyperlipidemia in Rats

Journal of Applied Sciences Research, 6(11): 1730-1744, 2010
© 2010, INSInet Publication
An Experimental Study on the Therapeutic Efficacy of the Combined Administration of
Herbal Medicines with Atorvastatin against Hyperlipidemia in Rats
1
M. Raouf Hamed, 1 Nahed M.A. Hassanein, 2 Azza A.Ali and
1
Toqa M.Y.EL-Nahhas
1
Department of Developmental Pharmacology, National Organization for Drug Control And
Research (NODCAR).
2
Department of Pharmacological and Toxicological, Faculty of Pharmacy, Al-Azhar University.
Abstract: Hyperlipidemia was induced experimentally in adult male Sprague-Dawley rats by feeding high
fat synthetic diet for eight weeks. Concomitantly, animals were daily treated by either atorvastatin
(1mg/kg), fresh garlic homogenate (250 mg/Kg), capsaicin (1mg/kg) or guggulipid (285mg/kg). Similar
groups of animals fed with hyperlipidemic diet received combined treatment by atorvastatin (Ator) together
with either of fresh garlic homogenate (Gar), capsaicin (Cap) or guggulipid (Gug) in the same above
mentioned dose levels. Animals were then evaluated for the serum levels of total cholesterol (TC),
triglycerides, HDL-c, LDL-c, AST and ALT after 8 weeks.The obtained results showed that administration
of either of Ator, Gar, Cap and Gug reduced serum total cholesterol and LDL-c levels, and increases that
of HDL-c as compared to non treated hyperlipidemic rats. Co-administration of Ator and each of the used
herbal drugs resulted in reduction in serum cholesterol and LDL-c levels and improving the HDL-c level
more than the corresponding single treated groups. Similar associated results were found regarding TC/
HDL-c and LDL-c / HDL-c ratios. The obtained liver pathology results confirmed that co-administration
of Gar, Cap and Gug with Ator could reduce the hyperlipidemia and the Ator-induced liver injury. Liver
histopathological examination showed that single treatment with either of Ator, Gar, Cap or Gug could
induce improvement in the degenerative fatty changes as compared to HFD non-treated rats. Combined
treatment induced a great improvement, especially in group treated with Ator and Gug where hepatocytes
of this group showed normal hepatic architecture. In conclusion, administration of either of Ator, Gar, Cap
or Gug exerted a protective effect in management of hyperlipidemia. The obtained findings point to the
possibility of optimizing the hypolipidemic efficacy of Ator as a synthetic hypolipidemic drug through its
administration in a relatively lower dose together with hypolipidemic substances of plant origin, the matter
which needs further investigations.
Key words: Hyperlipidemia – atorvastatin –garlic – capsaicin – guggulipid.
INTRODUCTION
Hyperlipidemia is a high risk factor contributing
to the development of coronary heart disease (CHD)
which is considered to be the major leading cause of
mortality and morbidity worldwide. Epidemiological
studies have shown that there is a positive correlation
between the incidence of CHD and the blood
cholesterol level[1 ].
Statins are the most powerful and efficient drugs
for treatment of hyperlipidemia [2 ] . Atorvastatin was
shown to be a highly effective member of statins for
lowering blood cholesterol, stabilizing plaque and
preventing strokes through anti-inflammatory and
other mechanisms[3 -4 ] . Atorvastatin lowers cholesterol
by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A
(HMGCoA) reductase, thus preventing the formation
of the major building block of the cholesterol
molecule. However, severe adverse events, such as
liver damage and skeletal muscle abnormalities,
including myopathy and rhabdomyolysis, sometimes
lim it the lipid-lowering therapy with these
hypolipidemic agents [5-6 ] .
Thus a harmless effective therapy for lowering
cholesterol levels would therefore be of considerable
interest. Several epidemiological studies looking for
alternative therapies have tried natural herbs and have
shown that a diet rich in plant foods may reduce the
development of certain chronic diseases such as
atherosclerosis. Among these plant foods that possess
hypocholesterolemic property in clinical, as well as
experimental studies are garlic, gum guggul and
capsicum [7 -8 ] .
Corresponding Author: M. Raouf Hamed, Department of Developmental Pharmacology, National Organization for Drug
Control And Research (NODCAR).
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J. Appl. Sci. Res., 6(11): 1730-1744, 2010
In this concern, epidemiological studies have
suggested that long-term consumption of fresh garlic
decreases plasma cholesterol and triglycerides levels
and is associated with decreasing the risk of
atherosclerosis and coronary heart disease [9 ]. In
addition, chronic oral administration of raw garlic
augment myocardial endogenous antioxidant potential
and play a role in the maintenance of optimal cardiac
function. It also protects blood vessels by holding
LDL cholesterol in a less damaging form, and by
blocking undesirable changes in blood vessel walls [10 ] .
Guggulipid is a natural health product that has
been shown to reduce elevated blood cholesterol and
triglyceride levels, and to reduce the risk of
cardiovascular disease without any apparent risk of
liver damage [1 1 -1 2] . Gugulsterones, the main active
components of gugul resin, have the ability to boost
HDL-C levels and to ameliorate conditions associated
with the heart and vascu la r syste m . T he
cardiovascular benefits associated with gugulipid
therapy are derived from its multiple pharmacological
activities, including its hypolipidemic, antioxidant,
and antiinflammatory activities [1 3 -1 4 ].
Regarding capsaicin, the main capsaicinoid in
chili peppers, has been documented to have
pronounced hypolipidemic influence in experimental
animal and human studies . The hypocholesterolemic
action of capsaicin and its beneficial effect on overall
lipid metabolism has been evidenced under different
conditions of hyperlipemia[1 5 ] . Its physiological effects
has focused on its influence on lipid metabolism, its
action as a digestive stimulant, its beneficial influence
as a
hypocholesterolemic spice on cholesterol
gallstone disease and diabetic nephropathy and as an
antioxidant spice principle on inflammatory disease [1 6] .
The present work was designed to investigate the
hypolipidemic effects of garlic, gugulipid and capsicin
on high cholesterol diet induced hyperlipidemia in
comparison to atorvastatin and to assess the
effectiveness of each one in combination with
atorvastatin for the prevention of induction of
hyperlipidemia.
M ATERIALS AND M ETHODS
Animals: Male Sprague-Dawley rats of body weight
225±25 g were obtained from the animal house of
National Organization for Drug Control and Research
(NODCAR). They were kept under normal laboratory
conditions for at least 7 days in well ventilated
stainless steal cages where water and standard diet
were provided ad libitum.
The Vitamin M ixture Composition (g/kg) was as
Following: Vit.A, 100 IU; vit.D, 15 IU; vit.E ,0.5
mg; vit.B1, 150 mg; vit.B2, 240 mg; vit.B6 150 mg;
nicotinic acid, 1200 mg; calcium pantothenate, 1200
mg; biotin, 12 mg; vit.B12, 1.2 mg; vit.K, 1500 mg;
inositol, 3000 mg; p-aminobenzoic acid ,3000g; folic
acid, 0.33g; corn starch to 1000g [1 8 ].
The Salt M ixture Composition (g/kg) was as
Following: Sodium chloride, 105g; potassium
chloride, 289.7g; calcium phosphate, 354.2g; calcium
carbonate, 11.45g; magnesium sulphate, 90 g;
ammonium citrate, 17.9g; sodium fluoride, 0.57g;
copper sulfate, 0.2g;
alum,
0.09 g; potassium
iodide, 0.05g [1 8] .
Drugs:
Atorvastatin Ca (Ator): Atorvastatin (Ator) obtained
from EIPICO, Cairo, Egypt. It was used as oral
suspensions in 1% tween 80 in distilled water in a
dose level 1 mg/ kg, which is less than the lowest
therapeutic dose level in human [1 9 ].
Garlic (Gar): Fresh garlic (Gar) was purchased from
local markets and prepared as fresh garlic
homogenate in distilled water by a motor-driven
Teflon glass homogenizer in ice bath. The dose used
in the present study was 250 mg/kg and consumed
within a period of 30 min according to Banerjee et
al,[2 0 ] and Thomson et al,[2 1 ] .
Capsaicin (Cap): Capsaicin was obtained from
Formosa Laboratories, INC. Taoyuan, Taiwan. It was
used in a dose of 1 mg /kg according to Lee et al, [2 2 ]
and was used as suspensions in 1% tween 80 in
distilled water.
Guggulipid (Gug): Guggulipid was obtained as
c a p s u le s ( G u g g u l P le x® , G o o d ‘N N a tu r a l
Manufacturing Crop. Holbrook, New York, USA),
each capsule contains 340 mg Gum Guggl,
standardized to contain 2.5% guggulsterone. It was
used in a dose of 285 mg /kg (7.1mg/kg of
gugglesterone), which is equivalent to the therapeutic
dose [1 2 ] and was prepared as oral suspensions in 1%
tween 80 in distilled water.
All drugs concentrations were adjusted so each
100 gm body weight received 1 ml of drug
suspension and were given daily for 2 months.
Experimental Design: Animals fed HFD were
randomly divided into two main groups namely single
treatment group and combined treatment group. Each
group was further divided according to the treatment
into: Ator, Gar, Cap, Gug (for single treatment
group), and Ator+Gar, Ator+Cap and Ator+gug (for
combined treatment group). Parally two control group
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Table: Two synthetic diets nam ely standard diet (SD ) and high fat diet (H FD) were prepared according to Pedrosa et al, [1 7 ] as shown in the
following table:
Ingredients
Standard diet
(g/kg)
H igh fat diet (g/kg)
Casein
20
20
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Sucrose
10
44
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Starch
52
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Corn oil
5
20
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Vitam in m ixture*
2
2
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Salt m ixture **
6
6
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Cellulose
5
5
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Cholesterol
-1
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Sodium cholate
-2
one fed standard diet and received tween 80 and the
other one fed high fat diet without any treatment and
received only the vehicle (tween 80).
Two hours before dosing, the diet withheld from
the cages and groups treated with combined drugs,
Ator was given 2 hours before the administration of the
other combined drug. At the end of 8 weeks, blood
samples were collected from the retro-orbital venous
plexus, 24 hours after the last dosage before which
animals were fasted at least 16 hours earlier.
Biochemical Assays: Serum cholesterol was assessed
according to Allain et al[23 ] , while that of triglyceride
w a s a c c o r d in g to F o ss a ti a n d P re nc ip e [ 2 4 ] .
Determinations of each of LDL and HDL were
according to Assmann et al[2 5 ]. Lipid ratios were
determined by the methods of Lopes-Virella et al[2 6] .
Serum alanine (ALT) and aspartate transaminase (AST)
activities were determined by the method of Reitman
and Frankel,[2 7] using spectrophotometer (Shimadzu,
UV! 160).
The reagents for the estimation of lipid profile
were obtained from BioMerieux ® sa, France and for
the estimation of transaminases were obtained from
Diamond diagnostics, Egypt.
Histopathological Examination: Liver specimens of
the different groups were fixed in 10% formalin saline
for twenty four hours. Paraffin tissue blocks were
sectioned at 4 microns thickness and the tissue slide
sections were then deparaffinized, stained by
hematoxylin and eosin and examined under the light
electric microscope.
Statistical Analysis: Statistical analysis was performed
using SPSS 10.0 statistical software (SPSS Inc,
Chicago, IL). Means were used and data were analyzed
by one-way analysis of variance (ANOVA). If the test
showed a significant difference, the least significant
difference test was used as a Post hoc Tukey's test for
multiple comparisons. The differences were considered
significant if the probability was associated with
p<0.05.
RESULTS AND DISCUSSION
Effect of Atorvastatin and the Herbal M edicine and
Their Combination on Serum Cholesterol and
Triglycerides Levels: As shown in fig (1), cholesterol
level of animals fed HFD exhibited significant increase
by 257% (p<0.001) after 8 weeks as compared to the
control group fed standard diet. Administration of
either low dose of Ator or Gug induced comparable
reduction in cholesterol level by 40% (p<0.001) and 42
%( p<0.001), respectively as compared to that HFD
non-treated group. Administration of either of Gar or
Cap for 8 weeks induced reduction in serum
cholesterol level by 23% (p<0.001) and 32% (p<0.001),
respectively as compared to HDF group (fig. 1).
Combined treatment of Ator and Gar or Cap
induced pronounced reduction in cholesterol level by
47% (p<0.001) and 50% (p<0.001), respectively as
compared to HFD non-treated group (fig. 1). Similar
effect was observed after combined treatment with Ator
and Gug inducing reduction in cholesterol level by
58% (p<0.001), respectively as compared to HFD nontreated group.
Combined treatment of Ator and Gug induced
significant reduction in triglycerides level by 23%
(p<0.00) as compared to those HFD non-treated group
(fig 1).
Effect of Atorvastatin and the Herbal M edicine and
Their Combination on Serum LDL and HDL
Levels: High fat diet induced increase in LDL level by
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Fig. 1:
Effect of Garlic, Capsaicin, Guggulipid administrations on serum cholesterol levels in hyperlipidemic rats
treated or nontreated with Atorvastatin.
SD = Standard diet; HFD+ w = High fat diet (dist water); HFD+ tw = High fat diet (tween); Ator=
ato rvastatin treated group;G ar= garlic treated group; CAP= capsaicin treated group;
A+Gar=atorvastatin+garlic ;Gug= guggulipid treated group; treated group; A+Cap= atorvastatin +
capsaicin treated group; A+Gug = atorvastatin+guggulipid treated group.
a =significantly different from the standard diet group.
b=significantly different from the high fat diet group.
** = highly significant (P< 0.01).
*** = very highly significant (P<0.001)
367% (p<0.001) and decrease in HDL level by 50%
(p<0.001) as compeered to the control group (fig. 2).
Treatment with Ator for 8 weeks reduced significantly
LDL level by 39% (p<0.001) as compared to the HFD
non-treated group (fig. 2). Treatment with either of the
traditional medicine Gar, Cap or Gug induced reduction
in HDL level by 32% (p<0.01), 35% (p<0.001) and
43% (p<0.001), respectively as compared to HFD nontreated group (fig. 2). Combined treatment of Ator with
either of Gar or Cap induced minor additive reduction
in HDL level by 44% (p<0.001) and 51.5% (p<0.001),
respectively while combined treatment of Ator and Gug
induced additive reduction by 59% as compared to
HFD (fig. 2). Concerning HDL levels, HFD induced
reduction in HDL level by 50% (p<0.005) as compared
to the standard diet control group (fig. 2). Herbal
treatment either as a single or combined treatment
could restore LDL level to its normal value (fig. 2).
Effect of Atorvastatin, Herbal M edicine and Their
Combined Treatment on Serum Tc/H DL-c and
LDL-c/ HDL-c Ratios: Regarding Tc/HDL ratio, HFD
induced significant increase in Tc/HDL ratio by 623%
(p<0.001) as compared o the control group (fig. 3).
Treatment with either of Ator or Gug induced
comparable reducing effect on Tc/HDL by 64%
(p<0.001) and 68% (p<0.001), respectively while Gar
and Cap induced reduction by 48.5% (p<0.001) and
58% (p<0.001), respectively as compared to HFD nontreated group (fig. 3).
Combined treatment of Ator with either of the
herbal medicine augmented the reduction in Tc/HDL
ratio to reach 70% (p<0.001), 71% (p<0.001) and 78%
(p<0.001) for Gar, Cap and Gug, respectively a
compared to HFD non-treated group (fig. 3).
Concerning the effect of HFD on LDL-c/HDL-c
ratio, HFD induced significant reduction in LDLc/HDL-c ratio by 867% (p<0.001) as compare to the
control group (fig. 3). Treatment with single treatment
of either of Ator, Gar, Cap, or Gug induced significant
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Fig. 2: Effect of Garlic, Capsaicin, Guggulipid admnistrations on serum LDL-Cholesterol levels in hyperlipidemic
rats treated or nontreated with Atorvastatin.
SD = Standard diet; HFD+ w = High fat diet (dist water); HFD+ tw = High fat diet (tween); Ator=
atorvastatin treated group; G ar= garlic treated group; CAP= capsaicin treated group;
A+Gar=atorvastatin+garlic; Gug= guggulipid treated group; treated group; A+Cap= atorvastatin + capsaicin
treated group; A+Gug= atorvastatin+guggulipid treated group.
a =significantly different from the standard diet group.
b=significantly different from the high fat diet group.
** = highly significant (P< 0.01).
*** = very highly significant (P<0.001).
reduction in LDL-c/HDL-c ratio by 64% (p<0.001),
52% (p<0.001), 61% (p<0.001) and 69% (p<0.001),
respectively as compare to the HFD non-treated group.
Combined treatment of Ator with either of Gar, Cap or
Gug induced more reduction in LDL-c/HDL-c ratio by
70% (p<0.01), 72% (p<0.001) and 79% (p<0.001),
respectively as compared to the HFD non-treated
group.
Effect of Atorvastatin, Herbal M edicine and Their
Combined Treatment on Serum ALT and AST
Activity: The AST activity under the influence of
feeding HFD showed marked increase by 54%
(p<0.001) as compared to the control group (fig. 4).
W hile single treatment with either of Ator or Gug
induced slight decrease in AST activity in Gug-treated
group by only 18% (p<0.05), their combined treatment
synergistically restored it to the normal value (fig. 4).
Single treatment with either of Gar or Cap as well as
their combined treatment with Ator could restore AST
activity to its normal value (fig.4).
High fat diet significantly affect ALT activity
inducing 72% (p<0.001) increase as compared to the
control group. Treatment with single or combined
herbal medication ( Gar, Cap and Gug) induced slight
decrease in ALT activity by 24% (p<0.001), 22%
(p<0.001) and 16% (p<0.01) for single treatment and
by 25.5% (p<0.001), 28% (p<0.001) and 31%
(p<0.001) for combined treatment, respectively as
compared to HFD non-treated group (fig. 4).
H istopathological Results: Histological analysis
showed that the liver samples of control rats presented
normal architecture (fig. 5). On the contrary, the
hepatocytes of hyperlipidemic rats showed an excess
deposition in the lipids vacuoles as compared to
negative control group (fig. 6). Administration of Ator
to rats fed HFD resulted in a decrease in the lipid
deposition in the hepatocytes with few inflammatory
cells infiltration and fibrosis in the portal area (fig. 7).
Hyperlipidemic rats treated with Gar showed smaller
size of lipid deposits (fig. 8), while Cap treated showed
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Fig. 3: Effect of Garlic, Capsaicin, Guggulipid admnistrations on serum T. C /HDL-c ratio in hyperlipidemic rats
treated or non-treated with Atorvastatin.
SD = Standard diet; HFD+ w = High fat diet (dist water); HFD+ tw = High fat diet (tween); Ator=
atorvastatin treated group; G ar= garlic treated group; CAP= capsaicin treated group;
A+Gar=atorvastatin+garlic; Gug= guggulipid treated group; treated group; A+Cap= atorvastatin + capsaicin
treated group; A+Gug= atorvastatin+guggulipid treated group.
a =significantly different from the standard diet group.
b=significantly different from the high fat diet group.
*** = very highly significant (P<0.001).
** = highly significant (P< 0.01).
cytomegaly in most of the hepatocytes and fatty change
in few hepatocytes (fig. 9). Liver of rats fed HFD and
treated with Gug showed multiple fatty changes
associated with few fibrosis in the portal area (fig. 10).
Combined treatment of Ator with Gar, Cap and
Gug showed much more improvement in the liver
degenerative fatty changes as compared to their
corresponding single treatment. In this concern, Atror
and Gar showed moderate fatty change in some
hepatocytes (fig. 11), while Ator and Cap showed mild
fatty change (fig. 12). In addition, Ator and Gug
treatment showed an almost normal hepatic architecture
(fig. 13). Nevertheless, a fatty change was observed in
very few cells of the hepatocytes.
Discussion: Induction of hyperlipidemia by feeding
HFD for eight weeks resulted in several alterations in
the relative liver weight, serum TC, LDL-c and HDL-c
levels associated with a dramatic increase in the
atherogenic index. This effect resembles type IIa
hyperlipidemia in humans [2 8 ] and is also accompanied
by a slight decrease in TG serum level as shown in the
present study. Dietary cholesterol is known to cause a
temporary increase in the plasma cholesterol level and
a marked increase in the liver cholesterol level, biliary
excretion of bile acids and fecal excretion of sterols
and bile acids [2 9 ] .
The hypercholesterolemic effect induced by HFD
may be due to the activity of the rate-determining
enzyme in cholesterol biosynthesis, HM G-CoA
reductase, stimulating the cholesterogenesis rate [3 0 ]. On
the other hand, development of hyperlipidemia may be
also due to a decrease in catecholamine level which
leads to low â 2 - adrenergic receptor function [3 1 ] and
decrease lipolysis of fat cells [3 2 ] . Thus decrease fat
catabolism and increase the circulating lipid levels.
In the present study, HFD increased LDL levels, could
be attributed to saturated fatty acids suppress hepatic
receptor-dependent LDL uptake and increase levels of
plasma LDL[3 3 ] . Similarly, cholesterol alone also
suppresses hepatic LDL uptake and increases plasma
LDL cholesterol[3 4 -35 ] .
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Fig. 4: Effect of Garlic, Capsaicin, Guggulipid admnistrations on serum AST levels in hyperlipidemic rats treated
or non-treated with Atorvastatin.
SD = Standard diet; HFD+ w = High fat diet (dist water); HFD+ tw = High fat diet (tween); Ator=
atorvastatin treated group; G ar= garlic treated group; CAP= capsaicin treated group;
A+Gar=atorvastatin+garlic; Gug= guggulipid treated group; treated group; A+Cap= atorvastatin + capsaicin
treated group; A+Gug= atorvastatin+guggulipid treated group.
a =significantly different from the standard diet group.
b=significantly different from the high fat diet group.
* = significant ( P< 0.05) .
** = highly significant (P< 0.01).
*** = very highly significant (P<0.001).
Fig. 5: Light micrograph of liver of control rats normally fed showing normal histological architecture of the
central vein (CV) and hepatocytes (h) (H&E stain, X 64).
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Fig. 6: Light micrograph of liver of control rats fed with high fat diet and dist.H2O showing severe fatty
changes (arrow) in most of the hepatocytes (h) (H&E stain, X 64).
Fig. 7: light micrograph of liver of hyperlipidemic rats treated with atorvastatin (1 mg/kg, P.O.) daily for eight
weeks showing mild fatty changes (arrow) in some hepatocytes with few inflammatory cells infiltration
and fibrosis in portal area (Pa) (H&E stain, X 64).
Fig. 8: light micrograph of liver of hyperlipidemic rats treated with Capsaicin (1 mg/kg, P.O.) daily for eight
weeks showing mild fatty changes (arrow) with cytomegaly in most of the hepatocytes (h) (H&E stain,
X 64).
Fig. 9: light micrograph of liver of hyperlipidemic rats treated with atorvastatin (1mg/kg, P.O.) and Capsaicin
(1mg/kg, P.O.) daily for eight weeks showing mild fatty
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Fig. 10: light micrograph of liver of hyperlipidemic rats treated with atorvastatin (1mg/kg, P.O.) and guggulipid
(285mg/kg, P.O.) daily for eight weeks showing no fatty
Fig. 11: light micrograph of liver of hyperlipidemic rats treated with atorvastatin (1mg/kg, P.O.) and guggulipid
(285mg/kg, P.O.) daily for eight weeks showing no fatty changes with cytomegaly in most of the
hepatocytes (h) (H&E stain, X 64).
Administration of Ator to rats fed HFD caused a
reduction in serum TC and LDL-c levels with a
consequence reduction in atherogenic index, while
serum HDL-c levels were elevated, a result which is in
agreem ent with B akker-Arkema et al. [ 3 6 ] and
Hunninghake et al.[3 ] . These findings could be
interpreted by the inhibitory effect of Ator on HMGCoA reductase enzyme which catalyzes the conversion
of HMG-CoA to mevalonate, a rate-limiting step in the
formation of endogenous cholesterol leading to the
decrease in the intracellular stores of cholesterol. This
in turn results in the up-regulation of LDL receptors on
the cell membrane, thus increasing the clearance of
LDL-c from plasma [3 7 -3 8] . Another possibility is that
Ator may lower LDL-c level by inhibiting hepatic
cholesterol synthesis in very low density lipoprotein
(VLDL) which is the source of LDL-c. Thus may
impair VLDL particle assembly and secretion from
liver, decrease the VLDL levels in plasma, and further
decrease the LDL level in plasma [3 6] .
Other proposed mechanism for Ator in lowering
serum cholesterol level is by inhibiting the absorption
of dietary cholesterol. Statins may reduce the
cholesterol content in the intestinal mucosal cells, with
a subsequent reduction in acyl-CoA cholesteryl
acyltransferase (ACAT) activity, which catalyzes the
intracellular esterification of cholesterol and formation
of cholesteryl esters, and thus decrease the absorption
of cholesterol[3 9 ] .
The hypolpidemic properties of garlic organosulfer
ingredients are thought to be mainly linked to their
inhibitory effect on cholesterol synthesis and to
decrease intestinal absorption of cholesterol[4 0 ]. In this
regard, organosulfur compounds decrease hepatic
cholesterogenesis by inhibition of H M G -C oA
reductase[41 ] and depress the hepatic activities of other
lipogenic, cholesterogenic enzymes such as malic
enzymes, fatty acid synthase, glucose-6-phosphate
dehydrogenase [42 ] .
In addition, the presence of high concentrations of
tellurium compounds inhibits squalene epoxidase, the
penultimate enzyme in the synthetic pathway of
cholesterol[43 ] . There is also a possibility that Gar
sulphur compounds consume NADPH that are required
for lipid synthesis, thus affecting the HMG-COA
reduction reaction by prevent the conversion of HMGCoA into mevalonate and consequently reduce the rate
of cholesterol synthesis [4 4-4 5 ] . The other possibility may
be through increase cholesterol excretion. Garlic
contain a cysteine moiety raise the level of hepatic
cholesterol 7á-hydroxylase activity, a key enzyme in
the synthesis of bile acids from cholesterol[4 6 ], thus
enhances excretion of acidic and neutral steroids [4 7 ].
Another possibility is that Gar appears to
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metabolize tissue lipids into circulation, as a result of
increase lipase activity in the adipose tissue which
suggests its catabolic role in lipid metabolism.
Administration of Gar to rats fed HFD induced
hypolipidemic effect is in agreement with those of
Adler and Holub [4 8 ] and Kojuri et al. [4 9 ].
On the other hand, the hypolipidemic effect of Cap
may be attributed to its ability to affect cholesterol
absorption and excretion rates, where Cap can bind to
the bile acids in the intestinal lumen and consequently,
decrease the dietary cholesterol absorption and increase
fecal bile acid and cholesterol excretion rate [5 0 -5 2 ] . In
addition, excessive elimination of bile acids increases
the expression of hepatic LD L-receptors and
consequently, reduces LDL cholesterol levels by
enhancing its uptake by the liver[5 3-5 4 ] . Capsaicin also
enhances energy metabolism in rats and inhibits fat
deposition in adipose tissue as well as promotes lipid
metabolism by stimulation of â-adrenergic receptor and
subsequent stimulation of adrenaline secretion from
adrenal gland[1 6 ,5 5 ] . Such increase in serum adrenaline
concentration resulted in increased synthesis of cAMP,
leading to activation of hormone-sensitive lipase (HSL)
which is the key enzyme in the regulation of lipid
stores through degradation of triacylglycerol to
diacylglycerol and frees fatty acids. In addition, it has
hydrolyzing activity against cholesterol esters [5 6 ].
These effects accompanied Cap treatment are in
co nsistent with M anjunatha and Srinivasan [ 5 7 ]
co nfirming that Cap counters the extent of
hypercholesterolemia.
The hypolipidemic effect of Gug administration to
rats fed HFD is consistent with the findings of Singh
et al.[1 3 ] , Urizar and Moore[5 8 ] and Ulbricht et al.[5 9 ] .
Several possible mechanisms have been proposed for
hypolipidemic activity of Gug. One of these
mechanisms is that guggul may inhibit lipogenic
enzymes and HMG-CoA reductase in the liver through
down modulation of their gene expression leading to
inhibition of endogenous cholesterol synthesis and
reduction of hepatic TG and VLDL secretion [6 0 -6 2 ].
Guggulsterone may greatly reduce LDL cholesterol
levels by enhancing uptake of excess serum LDLcholesterol particles by the liver. This is accomplished
by increasing hepatic binding sites for LDL through
receptor-mediated endocytosis, located on the surface
of the liver cell membranes, thus increasing LDL
clearance [5 8 ,6 3 -6 6 ] . Guggulsterones have been also
reported to antagonize the farsenoid X receptor (FXR)
and down regulate FXR target genes which mediates
the conversion of cholesterol to bile acids [6 7 -6 8 ].
Antagonizing this receptor leads to increasing the
release of cholesterol 7á-hydroxylase, the rate-limiting
enzyme in the bile acid synthesis in the liver which in
turn increases the cholesterol catabolism [5 8 ] .
Moreover, guggulesterone remove bile acids from
the liver by its stimulatory effect on bile salt export
pump (BSEP), the rate limiting bile acid transporter
and synergistically up-regulates BSEP expression and
consequently increases secretion of bile acids. Such
effect is clearly in contrast to the expected inhibitory
effect of guggulsterone on BSEP as an FXR
antagonist[6 9] . In addition, guggulsterone antagonized the
bile acid receptor, another nuclear hormone receptor,
which in turn reduces the absorption of cholesterol in
the gut via its effect on the intestinal bile acid
transporter[6 8 ] .Thus the action of guggulesterone, which
is involved in the cholesterol metabolism and bile acid
regulation, may be attributed to the potent effect of
Gug on hyperlipidemia. Another possibility is that Gug
directly activate the thyroid gland and consequently
increase thyroid hormone levels, increase metabolic rate
and decrease body mass and body fat[14 ] .
The reduction in HDL cholesterol level in animals
fed HFD, in the present study, may be due to the
decrease in lecithin-cholesterol acyltransferase (LCAT)
activity, the enzyme involved in the transesterification
of cholesterol, the maturation of HDL and the flux of
cholesterol from cell membranes into HDL [7 0 -7 1 ].
Atrvasatatin treatment could restore HDL-c levels
may be attributed to the reduction of cholesteryl ester
transfer protein (CETP) which plays an important role
in HDL metabolism [36 ] . In plasma, CETP facilitates the
transfer of TG and cholesteryl esters (CE) between
apoB-containing lipoproteins and HDL, resulting in the
net transfer of CE mass from HDL to apoB-containing
lipoproteins[72 ] . The increased level of HDL would
facilitate and increase the clearance of free cholesterol
which is produced by peripheral tissues from plasma,
and thus help to further decrease the cholesterol level
in plasma.
In the present study, Cap treatment could prevent
the decreased plasma HDL-c level induced by HFD,
such effect of Cap may be interpreted on the basis that
Cap increase lecithin cholesterol acyl transferase
(LCAT) activity[7 2] , thus counter act the effect of HFD.
The mechanism by which Cap may elevate HDL-c
concentration is through increase LCAT activity, which
transforms the discoidal nacscent-HDL particles to
spherical HDL by conversion of free-cholesterol esters.
The spherical HDL is capable of acting as a scavenger
of tissue cholesterol thus, maintaining cholesterol
homeostasis[7 2 ] . In addition, Cap probably lowers
lipogenesis by lowering the fatty acid synthesizing
enzyme, fatty acid synthase and tended to increase
hepatic triglyceride lipase activities, which relates to fat
uptake into the liver and converts IDL into LDL [5 1 -52 ] .
In addition Cap enhancs lipoprotein lipase (LPL)
activity which catalyze lipolysis of triglyceride rich
lipoproteins (VLDL) the major source of HDL [7 2 ].
1739
J. Appl. Sci. Res., 6(11): 1730-1744, 2010
Increase LPL activity may account for the increased fat
uptake in muscles and resulted in low serum
triglyceride [7 ,7 3 ].
Combined treatment of Ator and Gar induced more
pronounced hypolipidemic effect than single treatment
may be attributed to their synergistic effect via similar
mechanisms of action rather than their different
mechanisms. Inhibition of the HMG-CoA reductase, the
main mechanism of action of Ator, may be augmented
by organosulpher compounds.
The potent anti hyperlipidemic effect of Ator and
Cap combined treatment, in the present study,
countered the extent of hypercholesterolemia induced
by HFD may be attributed to their action via different
mechanisms which enable these two compounds to act
concurrently to reduce serum lipids and poteniate their
effect.
Interestingly, Ator and Gug combined treatment
induced the most powerful hypolipidemic effect which
meets the European and United States guidelines
requirement for the recommended target of serum total
and LDL-c levels in management of hyperlipidemia [7 4 ].
Such hypolipidmic effect of this combined treatment
may be attributed to their effect via similar and/or
d iffe re n t m e c h an ism s o f a c tio n which m a y
synergistically potentiate their hypolipidemic efficacy.
Each of Ator and Gug act on HMG-CoA reductase, the
rate limiting enzyme of cholesterol biosynthesis,
through either competition with this enzyme or down
regulation of its gene expression[3 7 -3 8 ,6 0 -61 ] . In addition,
they also prevent cholesterol absorption from the
gut[3 9 ,6 8 ,75] .
In
the
present
work,
the
produced
hypercholesterolemia induced in rats by consuming
HFD was accompanied by increase in serum ALT and
AST activities and is in agreement with the findings of
Bolkent et al.[76 ] and Abd El-Twab [7 7] . Injury of liver
tissues and cell membrane damage caused by
hyperlipidemia resulted in a leak of the liver enzymes
(normally located in the cytosol) into the blood stream
and increased their levels in serum. In addition, this
elevation may be attributed to the role of free radical
reactions and lipid peroxidation in hyperlipidemia in
the pathomechanism of fatty liver [7 8 ]. Elevated serum
ALT and AST activities usually indicate hepatocyte
damage and the most common presentation is fatty
liver [7 9 ].
W hile Ator treatment failed to ameliorate the effect
of HFD on serum AST and ALT, single as well as
combined treatment with Gar, Cap or Gug caused
amelioration in the activity of these enzymes
suggesting that they may play an important role in
improving liver function.
The amelioration in serum ALT and AST activities
by Gar treatment is in agreement with Abdel –
Rahman et al.,[8 0 ] . It was known that Gar extract
contains a wide range of antioxidants that can act in
synergistic or additive fashion and protect cells against
oxidative damage [81 ] . Garlic organosulphur compounds
exerted its antioxidant action by scavenging reactive
o xygen sp ecies (R O S) required fo r lip id
peroxidation [ 4 5 , 8 2 ] a nd e nha nc ing the c e llular
antioxidants, like reduced glutathione, superoxide
dismutase, catalase and glutathione peroxidase of
vascular endothelial cells along with reduction of basal
lipid peroxidation [2 0 ,8 3 -8 4 ] . The improvement of liver
function by Gug and Cap was similar to the results
obtained by Kumar et al. [8 5 ] and Manjunatha and
Srinivasan[8 6 ] , an effect which is attributed to the
antioxidant properties of Gug and Cap inhibiting the
generation of oxygen free radicals and protect organs
against oxidative modifications in lipid and protein
components restoring the functional and structural
integrity of cells and prevents the oxidative
damage [1 1 ,8 7 ].
In the present study, the histological observations
were parallel to the obtained biochemical findings.
Inductions of hyperlipidemia in rats showed sever fatty
changes in liver hepatocytes. This effect may be due to
the accumulation and deposition of abundant fat
droplets in hepatocytes which occupied the entire cell
cytoplasm [8 ,7 6 ,8 5] .
The histological examination of liver sections of
rats fed HFD treated with either of Ator or Gar or Gug
showed mild fatty changes in few hepatocytes
associated with few inflammatory cells infiltration in
the portal area. These findings agree with that obtained
by Martín-Castillo et al.[8 8 ] , Abdel – Rahman et al. [8 0]
and Kumar et al.[8 5 ] . The histological examination of
liver sections of rats fed HFD treated with Cap showed
mild fatty changes in few hepatocytes. This effect may
be due to the Cap capability to inhibit fat deposition
through enhancing the energy metabolism [5 1 ,8 9 ].
Combined treatment showed marked improvements
in the overall hepatic architecture more than that
obtained by single treatment especially with Gug which
showed overall hepatic architecture within normal
limits. A result which is consistent with the
biochemical parameters. In conclusion, administration
of either of Ator, Gar, Cap or Gug exerted a protective
effect in management of hyperlipidemia. Furthermore,
combined treatment of Ator with each of the utilized
herbs resulted in much more protection against
hyperlipidemia. Finally, the obtained findings point to
the possibility of optimizing the hypolipidemic efficacy
of Ator as a synthetic hypolipidemic drug through its
administration in a relatively lower dose together with
hypolipidemic substances of plant origin. Consequently
the side effect of synthetic drugs can be diminished,
the matter which needs further clinical investigations.
1740
J. Appl. Sci. Res., 6(11): 1730-1744, 2010
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