: :

The Prevention and Cure Effects of Aspirin Eugenol Ester on

Hyperlipidemia and its Metabonomics

ISAMELDIN SULIMAN MOHAMED KARAM

2016 5

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Secrecy: No.

Chinese Academy of Agricultural Sciences

Dissertation

The Prevention and Cure Effects of Aspirin Eugenol Ester

on Hyperlipidemia and its Metabonomics

Ph.D. CandidateISAMELDIN SULIMAN MOHAMED KARAM

Supervisor Li Jian Yong
Major Basic Veterinary Medicine
Specialization Veterinary Pharmacology

May 2016

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 Abstract
Recently, hyperlipidemia and related cardiovascular disease are becoming a major health problem in
the human and even in companion animals all over the world. There are many drugs which are used as
hypolipidemic commonly known such as: statins, fibrates, ezetimibe and nicotinic acid. However, most of
them are expensive and have undesirable side-effects. So there is a need to search other alternative
medicine that can provide better safety and efficacy with less toxic and less expensive on a long term usage.
The study was conducted to investigate the prevention and cure effect of aspirin eugenol ester (AEE) on
hyperlipidemia.
1. The Preventive Effect of AEE on Hyperlipidemia
The experiment was conducted to evaluate the prevention effect of AEE on hyperlipidemia in SD male
rats. To construct hyperlipidemia disease model, the rats were fed with high fat diet for six weeks. The
drugs were administrated with the rat high fat diet at the same time to investigate the prevention effects.
Intragastrical administration of the drug in each rat based on individual weekly body weight once weekly
for six weeks, at the dosages of AEE 18, 36 and 54 mg/kg/day as low, medium and high dose respectively.
Statin at the dosage of 10 mg/kg/day and CMC-Na at the dosage of 20 mg/kg/day was used as positive and
negative control drug, respectively. Significant changes were observed on the levels of blood lipids indexes
at the end of experiment. AEE at the dosage of 54 mg/kg/day for six weeks decreased TG, TC and LDL
significantly (p<0.01), while statin decreased TC and LDL significantly (p<0.01), however, no effect on TG,
which may suggested that AEE had prevention better effect on hyperlipidemia in SD rats than statin. AEE
also showed significant change on body weight of rats during experiment which was interesting indicator
for effect on obesity.
2. The Cure Effect of AEE on Hyperlipidemia
To investigate the cure effect of AEE on hyperlipidemia, Wistar rats as experimental animals were fed
with high fat diet to induce hyperlipidemia for eight weeks. AEE were used in three different doses, and
low, medium and high dose were selected as 18, 36 and 54 mg/kg/day, respectively. To compare the effects
between AEE and its component, the mole of medium dose of AEE, aspirin, and eugenol is the same, at
0.3067 moles. In the experiment, aspirin and eugenol at the molar ratio 1:1 (0.3067 mole) were set also.
0.5% of CMC-Na at the dose of 20 mg/kg was served as the vehicle control drug and the dosage of
CMC-Na was close to equal in AEE and aspirin groups. Simvastatin 10 mg/kg was chosen as positive
control drug to compare with AEE.
The rats were divided in ten groups as blank, model groups and other eight treatment groups (n=10).
Blood samples were taken for serum, which was used with conventional pharmacological methods to

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determine classical pathology biomarkers of hyperlipidemia, including TC, TG, LDL-C and HDL-C
indexes. At the end of experiment after administrating the drug for five weeks, commercial assay kits were
used and auto analyzer machine was used to measure the biomarkers of hyperlipidemia. There were
significant differences in blood lipid indexes between each group.
Under the present study conditions, AEE at dose 54 mg/kg/day for five weeks decreased TG, TC, LDL
significantly (p<0.01) and increased HDL (p<0.05), while statin at dose 10 mg/kg and aspirin at dose 20
mg/kg decreased TG, TC, LDL significantly (p<0.01) and no increase in HDL. Eugenol at dose 18 mg/kg
decreased TC and LDL significantly (p<0.01) and had no effect on TG and HDL. Integration of aspirin and
eugenol at dose (20:18) mg/kg decreased TC and LDL significantly (p<0.01) and TG (p<0.05), however, no
effect on HDL.
Histopathological examinations for liver, stomach and intestine were performed. The results showed
obviously changes on hepatic tissues among different groups. All sections from stomach and intestine
showed no significant pathological changes.
3. Metabonomics of AEE
Hyperlipidemia with high blood lipid levels is a major risk factor for cardiovascular disease. In the
present study, hyperlipidemia disease was induced by feeding rats with HFD. A sensitive ultra-performance
liquid chromatography coupled with quadrupole time-of-flight synaptic high-definition mass spectrometry
(UPLC-Q-TOF/MS) method was used for the analysis of plasma. Principal component analysis (PCA) and
orthogonal partial least squares-discriminant analysis (OPLS-DA) were performed to investigate the
metabolic changes in rats. Potential biomarkers were detected using S-plot. 22 potential biomarkers in rat
plasma for hyperlipidemia were screened out. Furthermore, metabonomic pathway analysis was performed
with MetPA. The results revealed that the AEE treatment against hyperlipidemia may involve in regulating
the lipid metabolism, amino acid metabolism and energy metabolism.
In a conclusion, AEE at the dosage 54 mg/kg/day for six weeks had preventive effect on
hyperlipidemia in SD male rats more than simvastatin. There were significant differences in blood lipid
indexes throughout the experiment period after administrating the drug for five weeks. The AEE optimal
dose for curing hyperlipidemia in Wistar rats was considered to be 54 mg/kg/day for five weeks. The AEE
against hyperlipidemia may involve in regulating the lipid metabolism, amino acid metabolism and energy
metabolism.
Key words: Aspirin eugenol ester (AEE), hyperlipidemia, rats, high fat diet, prevention, cure,
metabonomics.

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AEE

1AEE

SD AEE 6

AEE 18 mg/kg/day

36 mg/kg/day 54 mg/kg/day10 mg/kg/day 20 mg/kg/day

54

mg/kg/day AEE AEE

AEE

2AEE

Wista 8 AEE

AEE 1836 54 mg/kg/day AEE

AEE + 1:1

0.5%20 mg/kg

10 mg/kg/day

10 5

54

mg/kg/day AEE (p<0.05)

(p<0.01)

3AEE

UPLC-Q-TOF-MS PCA

OPLS-DA S-plot

VII
 22 MetPA

AEE

SD 54 mg/kg/day AEE

Wistar

54 mg/kg/dayAEE

VIII
 CONTENT
CHAPTER I......................................................................................................................................... 1

INTRODUCTION ............................................................................................................................... 1

1.1 HYPERLIPIDEMIA ......................................................................................................................................... 1

1.1.1 Harm of Hyperlipidemia........................................................................................................................ 2
1.1.2 Cause of Hyperlipidemia ....................................................................................................................... 3
1.1.3 Type of Hyperlipidemia ......................................................................................................................... 4
1.1.4 Diagnosis of Hyperlipidemia ................................................................................................................. 5
1.1.5 Hyperlipidemia in Different Animals .................................................................................................... 7
1.1.6 Hyperlipidemia Model .......................................................................................................................... 8
1.2 LIPIDS ........................................................................................................................................................... 9
1.2.1 Lipid Metabolism ................................................................................................................................. 10
1.2.2 Cholesterol ........................................................................................................................................... 12
1.2.3 Triglycerides ........................................................................................................................................ 13
1.2.4 Lipoproteins ......................................................................................................................................... 14
1.3 TREATMENT OF HYPERLIPIDEMIA ............................................................................................................. 16
1.4 EFFICACIES OF ASPIRIN AND EUGENOL ON HYPERLIPIDEMIA ................................................................. 20
1.5 STUDY PROGRESS OF AEE ......................................................................................................................... 22
1.6 METABONOMICS ......................................................................................................................................... 24
1.7 RESEARCH OBJECTIVES ............................................................................................................................. 25

CHAPTER II ..................................................................................................................................... 27

THE PREVENTIVE EFFECT OF AEE ON HYPERLIPIDEMIA ............................................. 27

2.1 INTRODUCTION ............................................................................................................................................27

2.2 MATERIAL AND METHODS ......................................................................................................................... 28
2.2.1 Chemicals and Reagents ..................................................................................................................... 28
2.2.2 Animals ................................................................................................................................................ 28
2.2.3 Feeding ................................................................................................................................................ 28
2.2.4 Dosing .................................................................................................................................................. 29
2.2.5 Design of the Experiment .................................................................................................................... 29
2.2.6 Serum Samples .................................................................................................................................... 29
2.2.7 Statistics .............................................................................................................................................. 30
2.3 RESULTS...................................................................................................................................................... 30
2.3.1 Disease Model ..................................................................................................................................... 30
2.3.2 Body Weight ........................................................................................................................................ 30
2.3.3 Prevention Effect ................................................................................................................................. 31
2.3.4 Multiple Comparisons ......................................................................................................................... 31
2.4 DISCUSSION ................................................................................................................................................ 33

CHAPTER III.................................................................................................................................... 36

THE CURE EFFECT OF AEE ON HYPERLIPIDEMIA ............................................................ 36

3.1 INTRODUCTION ........................................................................................................................................... 36
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3.2 MATERIALS AND METHODS ....................................................................................................................... 37
3.2.1 Chemicals and Reagents ..................................................................................................................... 37
3.2.2 Animals ................................................................................................................................................ 37
3.2.3 Drug Preparation ................................................................................................................................ 37
3.2.4 Hyperlipidemia Disease Model ........................................................................................................... 38
3.2.5 Dosing .................................................................................................................................................. 38
3.2.6 Design of the Experiment .................................................................................................................... 39
3.2.7 Histopathological Preparations .......................................................................................................... 39
3.2.8 Statistics .............................................................................................................................................. 40
3.3 RESULTS...................................................................................................................................................... 40
3.3.1 Animal Disease Model ........................................................................................................................ 40
3.3.2 Body Weight ........................................................................................................................................ 41
3.3.3 Anti-hyperlipidemic Effect ................................................................................................................... 41
3.3.4 Optimal Dosage of AEE ....................................................................................................................... 42
3.3.5 Multiple Comparisons ......................................................................................................................... 44
3.3.6 Histopathology .................................................................................................................................... 45
3.4 DISCUSSION ................................................................................................................................................ 58

CHAPTER IV .................................................................................................................................... 64

MECHANISMS ON METABONOMICS OF AEE ........................................................................ 64

4.1 INTRODUCTION ........................................................................................................................................... 64

4.2 MATERIAL AND METHODS ......................................................................................................................... 65
4.2.1 Samples ............................................................................................................................................... 65
4.2.2 LCQ-T of MS Analysis of Plasma ....................................................................................................... 65
4.2.2.1 Plasma Sample Preparation ............................................................................................................ 65
4.2.2.2 Metabolic Profiling and Method Validation.................................................................................... 66
4.2.3 Data Analysis ....................................................................................................................................... 67
4.2.4 Multivariate Analysis and Identification of Potential Biomarkers .................................................... 67
4.3 RESULT ....................................................................................................................................................... 68
4.3.1 Blood Lipids ......................................................................................................................................... 68
4.3.2 Comparison of the Metabolic Profile between Control and Hyperlipidemia Groups........................ 69
4.3.3 Intervention of AEE on the Metabolic Profiling.................................................................................. 72
4.3.4 Relative Content Alteration and Pathway Analysis of Potential Biomarker ..................................... 74
4.4 DISCUSSION ................................................................................................................................................ 77

CONCLUSION .................................................................................................................................. 80

REFERENCE .................................................................................................................................... 81

ACKNOWLEDGEMENTS .............................................................................................................. 95

AUTHOR BIOGRAPHY.................................................................................................................. 96

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 Abbreviations
AEE Aspirin eugenol ester

TG Triglycerides

TCH Total cholesterol

LDL Low density lipoprotein

HDL High density lipoprotein

HFD High fat diet

CMC-Na Carboxyl methyl cellulose sodium

ASA Acetylsalicylic acid

VLDL Very low density lipoprotein cholesterol

TAG Triacylglycerol

IHD Ischemic Heart Disease

CVD Cardiovascular diseases

CHD Coronary heart disease

PLN Protein losing nephropathy

ATP III Adult Treatment Panel III

NCEP National Cholesterol Education Program

HMG Co A 3-hydroxy3methylgluataryl coenzyme -A

COX cyclooxygenase

OS Ocimum sanctum

NMR Nuclear magnetic resonance

GC-MS Gas chromatography mass spectrometry

HPLC High performance liquid chromatography

LCAT Lecithin Cholesterol Acyl Transferase

NICE National Institute for Health and Clinical Excellence

NHLBI National Heart Blood and Lung Institute

ESI Electrospray ionization

MS Mass spectrometry

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 Chapter I

Chapter I
Introduction

1.1 Hyperlipidemia

Hyperlipidemia is modifiable risk factor for atherosclerosis and related cardiovascular diseases,

including coronary heart disease, cerebral stroke, myocardial infarction and renal failurewhich are

becoming a major health problem in the world recently (Xu et al., 2014).

The term hyperlipidemia refers to increased concentrations of lipids (triglycerides, cholesterol, or both)

in the blood stream (Watson and Barrie, 1993, Ford, 1996, Johnson, 2005, Xu et al., 2014). Hyperlipidemia

can also describe as elevated total cholesterol (TC) or triglycerides (TG) or low density lipoprotein (LDL)

or low levels of high density lipoprotein cholesterol (HDL). Hyperlipidemia is a heterogeneous group of

disorders characterized by an excess of lipids in the blood stream, and these lipids include cholesterol,

cholesterol esters, phospholipids, and triglycerides. Jacobson reported that hyperlipidemia refers to elevated

levels of lipids and cholesterol in the blood and it is also identified as dyslipidemia (Jacobson, 1998), to

describe the manifestations of different disorders of lipoprotein metabolism. Dyslipidemia is the term that

is used if lipid levels are outside the normal range. Another related condition, dyslipidemia indicates

disorders of lipoprotein metabolism, including lipoprotein overproduction or deficiency. These disorders

may manifest with the elevation of serum TC, LDL, TG concentrations, and a decrease in the HDL

concentration (Xenoulis and Steiner, 2010b).

Increased blood concentrations of triglyceride are referred to as hypertriglyceridemia, which refers to

high triglyceride levels in the blood (desirable: < 200 mg /dl; borderline: 200 to 400 mg/dl; high 400 to

1000 mg/dl; very high: > 1000 mg/dl) in human, while increased blood concentrations of cholesterol are

referred to as hypercholesterolemia. Hypercholesterolemia is the term for high cholesterol levels in the

blood (desirable: < 200 mg/dl; borderline: 200 to 239 mg/dl; high: > 240 mg/dl) in human, while the mixed

hyperlipidemia is used to indicate increased cholesterol and triglyceride levels (Mahmood et al., 2009,

Lawrence et al., 2005). Hyperlipidemias are also classified according to which types of lipids are elevated,

that is hypercholesterolemia, hypertriglyceridemia or both in combined hyperlipidemia. Elevated levels of

lipoproteins may also be classified as a form of hyperlipidemia.

Hyperlipidemia can be also defined in terms of a class or classes of elevated lipoprotein in the blood,

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 Chapter I

and the term hyperlipoproteinemia was also used. The term hyperlipoproteinemia refers to increased blood

concentrations of lipoproteins, but it is often used interchangeably with the term hyperlipidemia. However,

the term hyperlipoproteinemia should ideally be used only in cases where measurement of actual

lipoproteins has been performed (Bauer, 1995, Johnson, 2005).

Hyperlipidemias are divided into primary and secondary types. Primary hyperlipidemia which occurs

as a result of high intake of diet rich in saturated fats and cholesterol, or because of some genetic defect and

heredity factor, is usually due to genetic causes (such as a mutation in a receptor protein). Secondary

hyperlipidemia occurs as a result of the predisposing factors relating to hyperlipidemia, such as some other

illnesses or metabolic disturbances, e.g., mellitus, hypothyroidism, obstructive liver disease. Blood lipids

can be raised also due to other underlying causes such as diabetes. In addition, some forms may predispose

to acute pancreatitis (Xenoulis and Steiner, 2010b).

1.1.1 Harm of Hyperlipidemia

Hyperlipidemia is a condition characterized by increased concentration of lipids (fats) in the

circulatory system (Graham et al., 1996).The main danger of hyperlipidemia is to increase the risk of

developing ischemic heart disease and cerebrovascular disease. Hyperlipidemia is a major, modifiable risk

factor for atherosclerosis and cardiovascular disease, including coronary heart disease, cerebral stroke,

myocardial infarction and renal failure (Graham et al., 1996, Xu et al., 2014). Hypercholesterolemia and

hypertriglyceridemia are involved in these disorders.

Hyperlipidemia is one of the most important factors associated with atherosclerosis. Hyperlipidemia,

atherosclerosis and related cardiovascular diseases are becoming a major health problem in the world

recently even in companion animal clinic. Hyperlipidemia has been thought to be a modifiable risk of

cardiovascular disease which is a most common cause of mortality worldwide, accounting for almost 17

million deaths annually (Smith et al., 2004). Hyperlipidemia is a major risk factor for atherosclerosis

leading to heart attack and hypercholesterolemia nevertheless can impart some degree of risk for ischemic

heart disease (IHD). Atherosclerosis and coronary heart disease are associated with elevated levels of LDL,

TC, TG and low levels of HDL, which can result in cardiovascular and cerebrovascular disease. Increased

circulating levels of LDL underlie the development of atherosclerosis. High levels of LDL cholesterol (the

so called bad cholesterol) greatly increase the risk for atherosclerosis because LDL particles contribute to

the formation of atherosclerotic plaques. Low HDL levels (good cholesterol) are an independent risk factor,

because reverse cholesterol transport by HDL works to prevent plaque formation, or even cause regression

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 Chapter I

of plaques once they have formed. HDL may also have anti-inflammatory properties that help reduce the

risk of atherosclerosis. Although elevated LDL is thought to be the best indicator of atherosclerosis risk so

LDL is consider to be pro-atherogenic factor (Jacobson, 1998). Lipid and lipoprotein abnormalities are

common in the general population, and are regarded as a modifiable risk factor for cardiovascular disease

due to their influence on atherosclerosis. So high levels of LDL increase CHD risk, HDL is consider being

anti-atherogenic factor. Thus low levels of HDL also increases CHD risk. Hyperlipidemia elevates LDL and

TG associated with increased risk. Serum levels of HDL were inversely related to risk and Am Fam

Physician mention that CVD was the leading cause of mortality accounting for 33.6 percent of all deaths in

USA in 2007 (Roger et al., 2011a, McNellis and Lewis, 2015, Last et al., 2011a). Hyperlipidemia is a

common risk factor for CVD, with 53.4 percent of adults in the United States having abnormal cholesterol

values and 32 percent having elevated LDL cholesterol levels (Roger et al., 2011b). Other complications

are coronary heart disease, ischemic cerebrovascular disease, hypertension, obesity and diabetes mellitus

(Type -II).

Cardiovascular and related illnesses are one of the most common diseases prevalent in many parts of

the world. An increased risk of coronary heart diseases is primarily associated with a high serum total

cholesterol and LDL concentration and a decrease in HDL (Ramirez and Hu, 2015).

1.1.2 Cause of Hyperlipidemia

There were many causes of hyperlipidemia. The main cause of hyperlipidemia is dietary intake.

Dietary intake is the major source of cholesterol, but it can be also synthesized endogenously by the liver

and other tissues. Postprandial hyperlipidemia is physiological and transient, and typically resolves within

712 h after a meal, depending on the fat content of the meal (Whitney, 1992, Downs et al., 1997, Bauer,

2004, Johnson, 2005). For that reason, any determination of serum lipid concentrations should always

follow a fast of at least 12 h. Hyperlipidemia can also be the result of an inherited disease in certain breeds

of dogs. Hyperlipidemia in dogs and cats can be physiological (postprandial) or pathological. Pathological

hyperlipidemia can result from increased lipoprotein synthesis or mobilization or decreased lipoprotein

clearance. It can be primary (genetic or idiopathic) or secondary to other disease processes. Veterinarians

should be familiar with how to recognize and manage this clinical disorder.

Canine hyperlipidemia is the result of an endocrine disorder, such as hypothyroidism, diabetes

mellitus, or hyperadrenocorticism, endocrine disease most commonly cause hyperlipidemia, and several

diseases have been reported to cause hyperlipidemia, (Rogers et al., 1975, Rogers, 1977, Whitney, 1992,

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 Chapter I

Bauer, 2004, Feldman et al., 2014, Johnson, 2005). Protein losing nephropathy (PLN) proteinuria

associated with hyperlipidemia, regardless of the cause, is often associated with hyperlipidemia in dogs.

The most commonly reported lipid abnormality in dogs with PLN is hypercholesterolemia, which is usually

mild or moderate (Center et al., 1987, DiBartola et al., 1989, DiBartola et al., 1990, Cook and Cowgill,

1996, Littman et al., 2000).

Hyperlipidemia can also be resulted from a single inherited gene defect in lipoprotein metabolic.

Genetic defects in lipid metabolism monogenic familial hypercholesterolemia (homozygous or

heterozygous) can be due to inactive LDL receptor. Familial lipoprotein lipase deficiency can be resulted

by defect of inactive lipoprotein lipase; familial combined hyperlipidemia the reason is still unknown

(Fukai and Ushio-Fukai, 2011).

1.1.3 Type of Hyperlipidemia

There are two main types of hyperlipidemia; Primary hyperlipidemia: This may occur due to high food

intake rich of fats and cholesterol or some of genetic defect and heredity factor. Hyperlipidemia can be

primary (genetic or idiopathic), and hyperlipidemia can also be the result of an inherited disease in some

animals, or secondary to other disease processes. Secondary hyperlipidemia: This occurs due to some

diseases or metabolic disturbances, e.g., diabetes mellitus, hypothyroidism, obstructive liver disease,

secondary causes of hyperlipidemia is the most common pathologic form of hyperlipidemia in dogs

(Nelson et al., 2004). There are many diseases reported to cause hyperlipidemia. Endocrine disease is most

commonly and canine hyperlipidemia is the result of an endocrine disorder, such as hypothyroidism,

diabetes mellitus, or hyperadrenocorticism (Rogers et al., 1975, Rogers, 1977, Whitney, 1992, Bauer, 2004,

Peterson, 1988, Feldman et al., 2014, Johnson, 2005). Increases in both serum triglyceride and cholesterol

concentrations have been reported in dogs with hypothyroidism (Rogers et al., 1975, Barrie et al., 1993,

Panciera, 1994, Dixon et al., 1999, Schenck et al., 2004). In one study, hypertriglyceridemia and

hypercholesterolemia were found in 88% and 78% of dogs with hypothyroidism, respectively (Dixon et al.,

1999). Usually, lipid abnormalities resolve after treatment of hypothyroidism (Rogers et al., 1975). In dogs

with diabetes mellitus, hyperlipidemia is most commonly associated with hypertriglyceridemia but

hypercholesterolemia might also be present (Rogers et al., 1975, Wilson et al., 1986, Whitney, 1992, Barrie

et al., 1993, Peterson, 1988, Johnson, 2005). Similarly, hypertriglyceridemia usually resolves after

successful treatment of diabetes but hypercholesterolemia might persist despite therapy (Gleeson et al.,

1990, Whitney, 1992). The presence of hyperlipidemia (hypertriglyceridemia and, to a lesser degree,

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 Chapter I

hypercholesterolemia) has long been associated with naturally occurring pancreatitis in dogs (Anderson and

Low, 1965, Anderson and Strafuss, 1971, Rogers et al., 1975, Rogers, 1977, Whitney, 1992, Cook et al.,

1993, Hess et al., 1998, Hess et al., 1999, Bauer, 2004, Steiner and Williams, 2005, Johnson, 2005).

However, it remains uncertain whether hyperlipidemia develops as a result of pancreatitis or can be the

cause of pancreatitis in some cases (Whitney, 1992, Steiner and Williams, 2005).

1.1.4 Diagnosis of Hyperlipidemia

Diagnosis of hyperlipidemia depends on laboratory investigation and any determination of serum lipid

concentrations should always follow a fast of at least 12 h. Persistent fasting hyperlipidemia is always

considered abnormal and can be either secondary to other diseases or drug administration or primary.

Methods for quantification and characterization of lipids in blood routine quantitative assessment of total

cholesterol and triglyceride concentrations in serum or plasma is usually achieved by use of spectro

photometric or enzymatic methods (Nelson et al., 2004). Other methods (e.g., lipoprotein electrophoresis,

ultracentrifugation) have also been used but have limited use in the routine clinical evaluation of

hyperlipidemic animals (Whitney, 1992, Nelson et al., 2004).

As serum triglyceride concentrations increase, serum becomes turbid (cloudy) and then lactescent

(milky). In laboratory evaluation of lipid disorders, serum turbidity visual evaluation of plasma or serum

usually offers the first estimate of a samples triglyceride concentration. Samples that have normal or near

normal serum triglyceride concentrations are clear, while samples with increased serum triglyceride

concentrations are lipemic (turbid or lactescent; (Fig. 1-1) (Xenoulis and Steiner, 2010b, Watson and Barrie,

1993, Ford, 1996, Johnson, 2005). In general, turbidity appears when serum triglyceride concentrations are

2.263.39 mmol/L (200-300 mg/dL), and lactescent serum is seen when serum triglyceride concentrations

are 11.3 mmol/L (1000 mg/dL) in dogs (Bauer,1995; Johnson, 2005). However, these guidelines provide

only a rough estimate of the actual serum triglyceride concentration and measurement of the actual serum

triglyceride concentration is required. In addition, the presence or absence of hypercholesterolemia cannot

be based on serum appearance, because hypercholesterolemia does not cause increased serum turbidity

(Whitney,1992; Johnson, 2005).

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 Chapter I

Fig.1-1 Serum samples from dog with normal triglyceride concentrations are clear (left tube). As the serum
triglyceride concentration increases, serum becomes turbid (middle tube) and ultimately lactescent (right tube).
(Xenoulis and Steiner, 2010a)

Chylomicron test: the chylomicron (or refrigeration) test is performed on lipemic samples and is a

simple method to determine the specific form of lipoprotein that is responsible for hypertriglyceridemia

(Rogers,1977; Whitney, 1992; Johnson, 2005). Serum samples are refrigerated and left undisturbed for

1012 h. Due to their lower density, chylomicrons tend to move to the top of the sample and form a cream

layer (Fig. 1-2). When a cream layer is formed, then chylomicrons are in excess in the sample and account

(partially or solely) for the hypertriglyceridemia. If there is no cream layer formation, the

hypertriglyceridemia and lipemia are due to excess of other lipoproteins (usually VLDL). When a cream

layer forms, the serum below the cream layer can either be clear or turbid. In the first case,

hyperchylomicronemia is most likely solely responsible for the hypertriglyceridemia, while in the second

case other lipoproteins are also present in excess (Xenoulis and Steiner, 2010b, Whitney, 1992, Johnson,

2005).

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 Chapter I

Fig.1-2 The picture on the left shows a lipemic serum sample from a dog with hypertriglyceridemia right after
separation of the serum from the clot. The same serum sample is shown on the right after overnight refrigeration
(chylomicron test). The formation of a cream layer due to hyperchylomicronemia is obvious. The remaining
serum below the cream layer is clear (although hemolytic), which suggests that other classes of lipoproteins are
not increased in this patient (Xenoulis and Steiner, 2010a).

Interference with laboratory measurements: it is important to note that lipemia can often interfere with

the determination of several analytes, depending on the methodology and analyzer used. Analysing the

determination of which has been reported to be affected by lipemia (i.e., falsely increased or decreased)

include, but are not limited to, bilirubin, liver enzymes, amylase, lipase, electrolytes, protein, albumin, and

glucose (Nelson et al., 2004) in dogs compared to humans (Johnson, 2005).

1.1.5 Hyperlipidemia in Different Animals

Hyperlipidemia can affect different animals. Hyperlipidemia can be the result of an inherited disease

in some animals. Hyperlipidemia in dogs and cats can be physiological (postprandial) or pathological.

Increases in serum triglyceride and/or cholesterol concentrations have been observed in obese dogs

(Chikamune et al., 1995, Bailhache et al., 2003, Jeusette et al., 2005). The most profound changes were

associated with severe chronic obesity dogs (Jeusette et al., 2005). Weight loss in obese dogs leads to

significant decreases in both serum triglyceride and cholesterol concentrations (Diez et al., 2004, Jeusette et

al., 2005).

However, hyperlipidemia can also occur spontaneously after a meal of high fat foods, particularly

table scraps. After eating a meal, the nutrients in an animals body pass into the small intestine, for

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 Chapter I

digestion then to form chylomicrons, micro particles of liquid fat, are absorbed 30-60 minutes later.

Chylomicrons are in the classes of lipids, which includes both triglycerides and cholesterol, and which are

formed during the digestion of fats from food. Normally, the absorption of chylomicrons increases serum

triglycerides for 3-10 hours, but some animals will have high cholesterol and high triglyceride levels for

more than twelve hours after a meal as one of the main indications of hyperlipidemia.

In pets, hyperlipidemia most often occurs as a consequence of some other disorder, such as diabetes

mellitus (sugar diabetes), hypothyroidism (low levels of circulating thyroid hormones), Cushings disease

(excessively high cortisone levels in the body), certain liver diseases, and protein losing nephropathy (a

disease of the kidneys resulting in protein loss in the urine) (Aiba et al., 2012).

In equine poor feed quality or decrease in feed intake, particularly during a period of high energy

requirement (e g, pregnancy, systemic disease), may result in hyperlipidemia syndrome. Hyperlipidemia is

seen most commonly in ponies, miniature horses, and donkeys, and less frequently in standard size adult

horses (Duhlmeier et al., 2003, Durham, 2006).

A number of studies have shown that the feeding of high fat supplements to ruminants raises the

cholesterol concentration in the serum but not in the tissues or milk in non-ruminants (Nestel et al., 1978),

including primates and man. Hypercholesterolemia may be increased by dietary manipulations such as

feeding excessive cholesterol or fats with a high saturated fatty acid content. The serum cholesterol

concentration does not rise uniformly and hyper responsiveness has been variously attributed to excessive

absorption of cholesterol, for example, in some species of monkey, to diminished re-excretion of

cholesterol or bile acids, or to the failure of absorbed cholesterol to exert appropriate feedback inhibition on

cholesterol synthesis. As ruminants normally derive all of their cholesterol from endogenous biosynthesis,

it is reasonable to suppose that the fat induced hypercholesterolemia in ruminants is due to either an

increased synthesis of cholesterol and/or a decreased fecal excretion of cholesterol or bile acids (Nestel et

al., 1978).

1.1.6 Hyperlipidemia Model

A great number of animal models, such as pigeons, chickens, swine, cats, dogs, non-human primates,

mice, rabbits and rats, have been tested for hyperlipidemia (Moghadasian, 2002, Moghadasian et al., 2001).

Consequently, it has been tried to provoke hyperlipidemia in laboratory animals, in order to understand

better the relationship between disorders in cholesterol metabolism and atherogenesis to test possible

treatments for the reduction of circulating cholesterol level.

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 Chapter I

Commonly rat strains (i.e. Sprague Dawley, Wistar) have used as experimental animals for

hyperlipidemia studies because they typically have high levels of HDL-C and low levels of LDL-C.

Therefore, the rats had ability for hyperlipidemia model construction, hyperlipidemia can be induced in rats

by feeding the rats with high fat diet, and some studies proved that hyperlipidemia can be induced by using

standard diet with cholesterol (Cole et al., 1984). High fat diet is capable of promoting elevations in TC and

LDL in Sprague Dawley or Wistar rats likely by reducing bile acid production (Jeong et al., 2005, Horton et

al., 1995, Yokozawa et al., 2006). For inducing hypercholesterolemia in rats triglycerides rich diets

containing cholesterol, with or without cholic acid have been used (Lichtman et al., 1999); the level of

cholesterol varies substantially as well.

1.2 Lipids

Lipids are water insoluble organic compounds, which are essential for many normal functions of

living organisms: they are important components of cell membranes to store energy and play a significant

role as enzyme co-factors, hormones, and intracellular messengers (Rifai et al., 1999). A lipid profile

typically measures the levels of TC, LDL-C, HDL-C, and TG.

Four main classes of lipids can be recognized from a metabolic stand point. These are free fatty acids,

triacylglycerol, phospholipids, and cholesterol and its esters (Forrester et al., 1987). The principle functions

of lipids are to act as energy stores and to serve as important structural component of cells. To fulfill these

functions, lipids have to be transported in plasma from one tissue to another, from the intestine or the liver

to other tissues such as muscular or adipose tissue, or from the other tissues to the liver (Bishop et al.,

2000).

There are many groups of lipids and three of them are most important from a clinical perspective: fatty

acids, sterols (mainly cholesterol), and acylglycerols (mainly triglycerides) (Ginsberg, 1998, Rifai et al.,

1999). Fatty acids are relatively simple lipids and are also important components of many other lipids

(Ginsberg, 1998, Rifai et al., 1999).

The major lipids reported to be present in the plasma are fatty acids, triglycerides, cholesterol,

cholesterol esters (compounds) and phospholipids. Lipids are transported in the blood as large lipoproteins.

Other lipids soluble substances, present in much smaller amounts but of considerable physiological

importance, include steroid hormones and fat soluble vitamins (Xenoulis and Steiner, 2010b).

9
 Chapter I

1.2.1 Lipid Metabolism

Lipid metabolism can be divided into two basic pathways: the exogenous pathway, which is associated

with the metabolism of exogenous (dietary) lipids, and the endogenous pathway, which is associated with

the metabolism of endogenously produced lipids Fig. 1-3 (Ginsberg, 1998, Rifai et al., 1999, Bauer, 2004).

Exogenous pathway digestion is the first step in dietary lipid metabolism. Dietary lipids that reach the

intestine duodenum then undergo emulsification, then hydrolyzed by the pancreatic and intestinal lipases

(Bauer, 1996, Xenoulis and Steiner, 2010b). The hydrolysis of cholesterol esters in the lumen of the small

intestine is catalyzed by pancreatic cholesterol esterase, which liberates free cholesterol. Hydrolysis

products (mainly free fatty acids and mono glycerides) are then transferred to the intestinal epithelial cell,

where they diffuse through the epithelial cell membranes into the intestinal mucosal cells (Bauer, 1996,

Xenoulis and Steiner, 2010b). In the intestinal mucosal cell, free fatty acids and mono glycerides

reassemble to form triglycerides, which then combine with phospholipids, free and esterified cholesterol

and the apolipoprotein (apo) B48 to form chylomicrons (Bauer, 1995, Bauer, 1996, Bauer, 2004, Ginsberg,

1998, Rifai et al., 1999). Chylomicrons are the lipoprotein class responsible for transfer of dietary lipids.

After formation in the enterocytes, chylomicrons, which mainly contain triglycerides, are secreted into the

lacteals and enter first the lymphatic and later the blood circulation where they acquire apolipoproteins C

and apo E from circulating HDL molecules (Bauer, 1995, Bauer, 1996, Bauer, 2004, Ginsberg, 1998, Rifai

et al., 1999). Lipoprotein which is exposed on the chylomicron surface, activates the lipoprotein lipase

attached to the capillary beds in adipose and skeletal muscle tissues, which then hydrolyzes triglycerides

into free fatty acids and glycerol (Bauer, 1995, Bauer, 1996, Bauer, 2004, Ginsberg, 1998, Rifai et al.,

1999). Free fatty acids enter the muscle cells (where they are used for energy production) and/or adipocytes

(where they are re-esterified into triglycerides for storage). The cholesterol rich remaining particles

(chylomicron remnants), return their apo C-II molecule to HDL and are recognized by specific hepatic

receptors that rapidly remove them from the circulation by endocytosis (Bauer, 1995, Bauer, 1996, Bauer,

2004, Ginsberg, 1998, Rifai et al., 1999). The cholesterol found in chylomicron remnants can be used for

lipoprotein (VLDL) and/or bile acid formation, or stored as cholesteryl esters (Bauer, 1995, Bauer, 1996).

Endogenous pathway, dietary cholesterol absorption, endogenous cholesterol synthesis and biliary

cholesterol excretion regulate whole body cholesterol balance as a result of biotransformation into bile

acids or direct cholesterol excretion. Recent studies have significantly advanced our understanding of

intestinal sterol absorption at molecular level (Norata and Catapano, 2004), lipids produced endogenously

10
 Chapter I

at liver (hepatic tissue), while chylomicrons are responsible for transport of dietary lipids, VLDL, LDL and

HDL are mainly involved in the metabolism of endogenously produced lipids (Bauer, 1996). Cholesterol

(and cholesteryl esters) and triglycerides synthesized endogenously combine with phospholipids and apo

B100, and apo B48 to from VLDL (Bauer, 1996, 2004; Ginsberg, 1998; Rifai et al., 1999). After VLDL

molecules reach the vasculature, they acquire apolipoproteins C and apo E from HDL (Bauer, 1995, 2004;

Ginsberg, 1998; Rifai et al., 1999). VLDL apo C-II activates lipoprotein lipase located in the capillary beds,

which in turn leads to hydrolysis of VLDL triglycerides and the production of free fatty acids and glycerol.

The VLDL molecules remaining after hydrolysis of VLDL triglycerides (VLDL remnants) are either

removed from the circulation by the liver or undergo further transformation by lipoprotein lipase and/or

hepatic lipase to form LDL (Bauer, 1995, 1996, 2004; Ginsberg, 1998; Rifai et al., 1999; Johnson, 2005).

LDL which contains mainly cholesteryl esters and phospholipids, circulates in the blood and binds to

specific receptors that are widely distributed throughout tissues in order to deliver cholesterol, which can be

used for the synthesis of steroid hormones and cell membranes as well as for hepatic metabolism (Bauer,

1996, Ginsberg, 1998, Rifai et al., 1999).

HDLs which are synthesized primarily in the liver play an important role as donors and acceptors of

apolipoproteins C, apo E, and various lipids from other lipoproteins in the circulation (Watson and Barrie,

1993; Ginsberg, 1998; Rifai et al., 1999; Bauer, 2004), HDLs apolipoproteins have a critical role in the

reverse cholesterol transport pathway, through which cholesterol is transferred from peripheral tissues to

the small circulating discoid HDL molecules, thus converting them to nascent HDL molecules (Watson and

Barrie, 1993; Fielding and Fielding, 1995; Ginsberg, 1998; Bauer, 2004).

HDL cholesterol is then esterified by the action of (Lecithin Cholesterol Acyl Transferase) LCAT and

the resulting cholesteryl esters move to the core of the HDL molecule thus allowing more free cholesterol

to be absorbed into their surface. Continued absorption of free cholesterol and subsequent esterification by

LCAT leads to the formation of the larger, cholesteryl ester rich HDL.

11
 Chapter I

Fig. 1-3 The lipid metabolism can be divided into two basic pathways: the exogenous pathway and endogenous
pathway (Masuko Ushio-Fukai, PhD, FAHA. 2013. Anti-Atheroscrerotic Drugs. Dept of Pharmacology
University of Illinois at Chicago).

1.2.2 Cholesterol

Cholesterol is the main sterol in animal tissues, the major source of cholesterol is dietary intake, but it

can also be synthesized endogenously by the liver and other tissues. Cholesterol is absorbed from the

intestine and transported to the liver by chylomicron remnants (Jackson et al., 2010). Hepatic cholesterol

synthesis is high when animals are fed a cholesterol free diet or when there is an increased demand for

cholesterol, and it declines dramatically when cholesterol is fed (Horton et al., 1998). Hepatic cholesterol

enters the circulation as VLDL which is metabolized by lipoprotein lipase enzyme to intermediate

lipoprotein IDL and LDL which are then removed by liver or peripheral tissues. It plays a fundamental role

in central metabolic pathways, such as bile acid metabolism and steroid hormone and vitamin D synthesis

(Ginsberg, 1998, Rifai et al., 1999). In the peripheral tissues cholesterol is converted to steroid hormones or

used to form cell walls and membranes. The quantity of cholesterol transported from the liver to peripheral

tissues greatly exceeds its catabolism. So the excess amount of cholesterol is returned back to the liver by

HDL. Cholesterol is metabolized in the gluconeogenic pathway for alternative energy source (Schlumbohm

et al., 1997). Cholesterol is important in the synthesis and maintenance of cell membranes and synthesis of

12
 Chapter I

steroidal hormones (Carlson et al., 1997). Variation in blood cholesterol content has been observed during

oestrus and pregnancy in cattle, as precursor of the steroid hormones (Iriadam, 2007). (Ahmad et al., 2004)

reported that serum cholesterol concentration in cyclic cows is 199.12 9.38 mg/dL (equivalent to 5.15

mMol/L) consequently. Thus, it is not surprising that cholesterol is the most decorated molecule in

history, having contributed to as many as 13 Nobel prizes (Mahmood et al., 2009).

High cholesterol diet leading to hyperlipidemia is regarded as an important factor in the development

of Ischemic heart disease and the focus so far has been mainly on the systemic and coronary vascular

effects of cholesterol. Although only few studies questioned the effect of cholesterol diet on the heart,

several structural and functional alterations have been shown (Csont et al., 2002), suggesting that the

endogenous adaptive mechanisms against myocardial stress are impaired. Moreover, it enhances the

incorporation of cholesterol into the mixed micelle and aids transport of free cholesterol to the enterocyte.

The continuous ingestion of high amounts of fat seems to be directly related to hyperlipidemia in humans.

Hepatic synthesis of cholesterol utilizes acetyl CoA as a precursor which undergoes transformation under

the influence of the enzyme (hydroxyl methyl glutaryl-CoA) HMG- Co A reductase, to cholesterol (Jackson

et al., 2010). Because significant elevation in cholesterol levels is a risk factor for coronary heart disease,

clinicians aim to reduce cholesterol levels by prescribing HMG- Co A reductase inhibitors, also referred to

as statins, to inhibit the action of the enzyme (Jackson et al., 2010).

1.2.3 Triglycerides

Triglycerides are the most common and efficient form of stored energy in mammal (Chinedum, 2012).

They can be derived from both dietary sources and endogenous (hepatic) production (Ginsberg, 1998, Rifai

et al., 1999). Triglycerides are used in animals to either generate energy or to be deposited as adipose tissue

(Silva and Dangolla, 2002). Increasing triglycerides in blood stream cause hypertriglyceridemia; which can

be factor for other disease such as Lipemia; so lipemia can be as a result of hypertriglyceridemia (Watson et

al., 1993; Bauer, 1995; Ford, 1996; Johnson, 2005). However, mild hypertriglyceridemia also does not

cause lipemia. Usually, lipemia is apparent when serum triglyceride concentrations exceed 2.26 mmol/L

(200 mg/dL) (Bauer, 1995). Many cell types and organs have the ability to synthesis triglycerides, but in

animals the liver and intestines are most active, although most of the body stores of this lipid are in adipose

tissue (Williams and Stanko, 2000). Fasting triglyceride levels are used to estimate the level of VLDL.

High levels of triglycerides are also associated with an increased risk for atherosclerosis, although the

mechanism is not entirely clear.

13
 Chapter I

Ruminal microflora can hydrolyze triglycerides to unsaturated fatty acids and glycerol (Williams and

Stanko, 2000). Fats of plant or animal origin contain the unsaturated fatty acids such as palmitoleic, oleic,

linoleic, and -linolenic acids, all of which are metabolized in the rumen (Williams and Stanko, 2000). A

high proportion of the fatty acids are then partially or completely hydrogenated and much of the glycerol is

fermented to propionic acid (Noble, 1978). Of the three major volatile fatty acids derived from ruminal

fermentation, propionic acid, in the form of propionate, is the only volatile fatty acid that contributes

directly to glucose synthesis during gluconeogenesis in the Krebs cycle (O'Boyle, 2008). Detailed reviews

regarding lipid metabolism (Jenkins, 1993), absorption and transport (Bauchart, 1993), and metabolism

(Grummer, 1993) are available. The normal concentrations vary tremendously among animals (Williams

and Stanko, 2000).

1.2.4 Lipoproteins

Because lipids are water insoluble molecules, they cannot be transported in aqueous solutions, such as

plasma. For that reason, lipids are transported in plasma as macromolecular complexes known as

lipoproteins (Mahley and Weisgraber, 1974, Whitney, 1992, Watson and Barrie, 1993, Ginsberg, 1998,

Rifai et al., 1999, Bauer, 2004, Johnson, 2005). Lipoproteins are spherical structures that consist of a

hydrophobic core containing lipids (i.e. triglycerides and/or cholesterol esters), and an amphiphilic (i.e.

both hydrophobic and hydrophilic) outer layer of phospholipids, free cholesterol, and proteins that forms a

protective envelope surrounding the lipid core (Mahley and Weisgraber, 1974, Bauer, 1996, Ginsberg, 1998,

Rifai et al., 1999, Johnson, 2005).

It is worth noting that free fatty acids are transported bound to albumin and do not require

incorporation into lipoproteins for transport (Whitney, 1992, Watson and Barrie, 1993, Ginsberg, 1998,

Rifai et al., 1999, Bauer, 2004, Johnson, 2005). Plasma lipoproteins differ in their physical and chemical

characteristics such as size, density, and composition. The proteins that are part of the lipoproteins are

known as Apo Lipoproteins (or Apo proteins) and play a significant role in lipid transport and metabolism

(Ginsberg, 1998, Rifai et al., 1999, Bauer, 2004, Johnson, 2005). Lipoproteins can contain one or a variety

of Apo lipoproteins, which regulate their metabolic in several physiological functions of lipoproteins such

as facilitation of lipid transport, maintenance of structural integrity, and activation of certain enzymes that

play key roles in lipid metabolism (Ginsberg, 1998, Rifai et al., 1999, Bauer, 2004, Johnson, 2005).

Lipoproteins are particles that contain triacylglycerol, phospholipids and cholesterol and amphipathic

proteins called Apo lipoproteins. Lipoproteins can be differentiated on the basis of their density, but also by

14
 Chapter I

the types of Apo lipoproteins they contain. The degree of lipid in a lipoprotein affects its density the lower

density of a lipoprotein contains more lipids relative to protein. The four major types of lipoproteins are

chylomicrons, very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density

lipoprotein (HDL) (Fig. 1-4). Lipoprotein species VLDL: very low density lipoproteins, LDL: low density

lipoproteins, HDL: high density lipoproteins. Plasma lipoproteins can be classified and the major property

of it is the metabolic function. Lipoproteins major metabolic function chylomicrons structural, IDL:

intermediate density lipoproteins, LDL: low density lipoproteins, Lp (a): lipoprotein abide with

phospholipids to form VLDL (Bauer, 1996, Bauer, 2004, Ginsberg, 1998, Rifai et al., 1999). Two types of

lipoproteins are triglyceride rich: the chylomicrons and VLDL. Chylomicrons are synthesized by

enterocytes from lipids absorbed in the small intestine. VLDL is synthesized in the liver. The function of

these lipoproteins is to deliver energy rich triacylglycerol (TAG) to cells in the body. TAG is stripped from

chylomicrons and VLDL through the action of lipoprotein lipase, an enzyme that is found on the surface of

endothelial cells. This enzyme digests the TAG to fatty acids and mono glycerides, which can then diffuse

into the cell to be oxidized, or in the case of an adipose cell, to be re-synthesized into TAG and stored in the

cell. LDL delivers cholesterol to cells in the body. As VLDL particles are stripped of triacylglycerol, they

become denser. These particles are remodeled at the liver and transformed into LDL. The function of LDL

is to deliver cholesterol to cells, where it is used in membranes, or for the synthesis of steroid hormones.

Cells take up cholesterol by receptor mediated endocytosis. LDL binds to a specific LDL receptor and is

internalized in an endocytic vesicle. Receptors are recycled to the cell surface, while hydrolysis in end

lysosome releases cholesterol for use in the cell. HDL is involved in reverse cholesterol transport. Excess

cholesterol is eliminated from the body via the liver, which secretes cholesterol in bile or converts it to bile

salts. The liver removes LDL and other lipoproteins from the circulation by receptor mediated endocytosis.

Additionally, excess cholesterol from cells is brought back to the liver by HDL in a process known as

reverse cholesterol transport. HDL (or really, the HDL precursor) is synthesized and secreted by the liver

and small intestine. It travels in the circulation where it gathers cholesterol to form mature HDL, which

then returns the cholesterol to the liver via various pathways. The link between cholesterol and heart

disease was recognized through the study of individuals with familial hypercholesterolemia. Individuals

with this disorder have several fold higher levels of circulating LDL due to a defect in the function of their

LDL receptors. Without functioning LDL receptors, LDL is not cleared from the circulation. As well,

because cholesterol cannot get into cells efficiently, there is no negative feedback suppression of

15
 Chapter I

cholesterol synthesis in the liver.

Fig. 1-4 The major types of lipoproteins are chylomicrons, very low density lipoprotein (VLDL), intermediate
density lipoprotein (IDL),low density lipoprotein (LDL) and high density lipoprotein (HDL) (Masuko
Ushio-Fukai, PhD, FAHA. 2013. Anti-Atheroscrerotic Drugs. Dept. of Pharmacology University of Illinois at
Chicago)

1.3 Treatment of Hyperlipidemia

Therapeutic strategies for hyperlipidemia treatment depend on reducing blood lipids, to avoid the risk

of developing ischemic heart disease, heart attack, cardiovascular and cerebrovascular disease. The main

aim of therapy in patient with hyperlipidemia is to reduce the peril of evolve atherosclerosis, coronary heart

disease, cerebral stroke, myocardial infarction and renal failure. However, to fight these problems of

hyperlipidemia, to treat hyperlipidemia in human, extensive interventions have been performed including

diet control, exercise and administration of hypolipidemic drugs (Stone, 1996). Decisions about treatment

of elevated lipids currently focus on the LDL cholesterol level as well as the cardiovascular risk status of

the individual. In 1991, the National Cholesterol Education Program (NCEP) of the National Heart Blood

and Lung Institute (NHLBI) proposed criteria for acceptable, borderline, and high levels of LDL and

TC in children and adolescents (Health and Services, 1991). These criteria have been adopted as the basis

for policy statements and treatment guidelines by many there are several secondary causes of abnormal

lipids that may occur in adolescence. To retard or prevent the formation of atherosclerosis come

hyperlipidemias on one of the present therapeutic challenges. Elevated plasma cholesterol levels have long

16
 Chapter I

been established as risk factors for CHD, and lowering cholesterol levels, particularly LDLC has been the

focus of the prevention of CHD and its squealed for almost 25 years. The U.K. national practice guidelines

U.S., U.K., and Canadian guidelines are available to help physicians manage hyperlipidemia (Grundy et al.,

2004, Genest et al., 2009). These guidelines agree that therapeutic lifestyle changes are the mainstay of

hyperlipidemia management, and that LDL cholesterol should be the primary target of therapy (Krauss et

al., 2000, Last et al., 2011a). In the past 20 years, major strides have been made in the understanding and

treatment of hypercholesterolemia and other dyslipidemias. Since its inception in 1985, the National

Cholesterol Education Program (NCEP) in USA has battled to reduce the prevalence of high blood

cholesterol through educational campaigns and science based practice guidelines (Shamir and Fisher, 2000).

However, cholesterol levels are still under treated (Mahmood et al., 2009).

One of the most important strategies in the prevention and treatment of hyperlipidemia includes

delaying fat digestion and absorption through gastrointestinal mechanisms (Adisakwattana et al., 2012),

such as the inhibition of pancreatic lipase, pancreatic cholesterol esterase activities as well as the inhibition

of cholesterol micellization, and bile acid binding (Ros, 2000). Inhibition of cholesterol esterase is expected

to limit the absorption of dietary cholesterol, resulting in delayed cholesterol absorption (Heidrich et al.,

2004). Consequently, the principal steps in the absorption of dietary cholesterol are emulsification,

hydrolysis of the ester bond by a pancreatic esterase, micellar solubilization, and absorption in the proximal

jejunum (Hui and Howles, 2005), reduction of cholesterol absorption by inhibiting cholesterol micellization

in the intestinal lumen is a new target site of intervention for the treatment of hyperlipidemia (Kirana et al.,

2005). In addition, binding bile acids by forming insoluble complexes in the intestine and increasing their

fecal excretion have been hypothesized as a possible mechanism of lowering plasma cholesterol level. This

consequently reduces the bile acid pool. As a result, greater amount of cholesterol is converted to bile acids

to maintain a steady level in the circulation (Insull, 2006). It is well known that a new attempt to reduce the

absorption of free fatty acids is by delaying triglyceride digestion with the inhibition of pancreatic lipase

(Birari and Bhutani, 2007). Pancreatic cholesterol esterase plays a pivotal role in hydrolyzing dietary

cholesterol esters (Brodt-Eppley et al., 1995).

There are many chemical drugs that lower cholesterol level in the body; commonly known as lipid

lowering drugs. Such as: statins, fibrates, ezetimibe and nicotinic acid, but most of them are unfavorable for

cost and side effects (Thomas et al., 2003). Several drugs are used to decrease LDL cholesterol such as

statins (HMG CoA reductase inhibitor), bile acid sequestrates, nicotinic acid and gemfibrozil (Panel, 2002,

17
 Chapter I

Ridker et al., 2002). The most important drugs for the treatment of dyslipidemia are by far, one group of

drugs (statins) lowers cholesterol by interfering with the cholesterol biosynthetic pathway. Most of the

drugs (statins) available today are inhibitors of 3-hydroxy3methylgluataryl coenzyme -A reductase, which

is involved in cholesterol biosynthesis in the liver. The most effective and widely used drugs for the

treatment of Hyperlipidemia are the statins. Their primary site of action is in the liver where they inhibit

HMG CoA reductase; the metabolic pathway that produces cholesterol and isoprenoids. Statins have been

shown in multiple clinical trials to reduce cardiovascular events and mortality (Adnan et al., 2011, Chilton,

2004). Statins have been shown to effectively lower LDL levels and reduce both mortality and morbidity

associated with coronary heart disease by 30% (Baigent et al., 2005). However, as noted Steinberg and

colleagues in a recent review (Steinberg, 2013); this still leaves a significant percentage of individuals for

whom statin therapy will not prevent the occurrence of adverse events. Statins have the most convincing

data for primary prevention, especially for higher risk patients. Therefore, risk stratification is essential

(Pediatrics, 1998). Statin therapy is also recommended for secondary prevention in all patients with known

cardiovascular disease or the risk equivalent. High dose statins should be initiated in patients with acute

coronary syndrome (Wang et al., 2006). National Institute for Health and Clinical Excellence (NICE)

guidelines recommend offering a fixed dose statin based on CHD risk stratification, and recommend

against checking cholesterol levels after a patient starts statin therapy (Adams, 2005). There is good

evidence for using statins in the secondary prevention of stroke and peripheral arterial disease (Health and

Services, 1991). However, based on statins medical use its importance and popularity. Elevated serum

lipids have been shown to be a major risk factor for the development of coronary heart disease and

atherosclerosis (Epstein and Ross, 1999).

Omega-3 fatty acids may be a good alternative after myocardial infarction for patients who cannot

tolerate statins. Fibrates and niacin have not been shown to reduce all-cause mortality in secondary

prevention, but may be useful adjuncts when statins alone cannot adequately control lipid levels. Other

cholesterol lowering medications used for primary or secondary prevention of cardiovascular disease have

not been shown to consistently improve patient oriented outcomes (Last et al., 2011b). On the other hand,

fibrates decrease fatty acid and triglyceride levels by stimulating the peroxisomal oxidation pathway

(Watts and Dimmitt, 1999, Ozasa et al., 1985). Apart from these drugs, ezetimibe, which selectively inhibits

intestinal cholesterol absorption (Vasudevan and Jones, 2005), Resins cholestyramine, colestipol, and

colesevelam, which sequester bile acids (Steinmetz, 2002), prevents reabsorption of cholesterol, anion

18
 Chapter I

exchange resins which bind negatively charged bile acids in the small intestine, torcetrapib, which inhibits

cholesterol ester transfer protein (Gauthier et al., 2005), avasimibe, which inhibits acyl CoA: cholesterol

acyltransferase (Kharbanda et al., 2005), implitapide, which inhibits microsomal triglyceride transfer

protein (Ueshima et al., 2005), and niacin, which modifies lipoproteins (Vasudevan and Jones, 2005), are

providing clinicians with several therapeutic options for lipid lowering Fig. 1-5. However, based on

medical use, importance, and popularity, statins and fibrates are way ahead of the others. Recent

experimental data have revealed that both statins and fibrates display a broad spectrum of activities in

addition to their lipid lowering properties. As a result, statins and fibrates are now being considered as

possible medicines in a variety of human disorders (Pahan, 2006).

Fig. 1-5 The lipid lowering drugs acting on hyperlipidemia. Such as: statins, fibrates, ezetimibe, resins and niacin
(Masuko Ushio-Fukai, PhD, FAHA. 2013. Anti-Atheroscrerotic Drugs. Dept. of Pharmacology University of
Illinois at Chicago)

The pharmacological, dietary and herbal treatment of coronary heart disease (CHD) is based on the

hypothesis that reduced cholesterol biosynthesis will lead to lower blood levels of cholesterol (Lau, 2006).

Lowering lipids and cholesterol levels by a drug or dietary interventions could reduce the risk of coronary

heart disease. Current interest in natural products has stimulated the search for new cholesterol lowering

19
 Chapter I

agents from these sources. Several synthetic hypocholesteromic agents such as statins, fibrates, resins and

nicotinic acid are capable of efficiently reducing plasma total cholesterol (TC) levels, but LDL does not

undergo any significant alteration. Also, synthetic hypolipidemic agents have one or more side effects and

are unable to increase HDL levels (Sharma et al., 2013).

Many herbal medicinal products were reported to have a potential to reduce lipid and cholesterol in

body and to enhance the safety profile by elevating HDL levels and inhibiting lipid oxidation (Zhang et al.,

2013, Thomas, 2003, Dou et al., 2008). The major portion of the global population in developing countries

still relies on botanical drugs to meet its health needs (Thomas, 2003, Sharma et al., 2013). The attention

paid by health authorities to the use of herbal medicines has increased considerably, because herbal

medicines are often only medicine available in less developed areas and are becoming a popular alternative

treatment in more developed areas (Xiao et al., 2012, Sharma et al., 2013).

There is an obvious need for more efficacious and alternative treatment options. Many Chinese herbal

medicines contain poly saccharides which can exert a wide range of pharmacological effects, including

lipid lowering drugs (Huang et al., 2010, Inoue et al., 2009, Singh et al., 1990, Kanauchi et al., 1995,

Ormrod et al., 1998). For example, berberine had regulation effect on hyperlipidemia indexes (Xiao et al.,

2012). In modern practice, there are many drugs like statins and fibrates which are in use as hypolipidemic

agent but the therapy is not cost effective, and such these drugs do not fulfill the WHO guidelines of

essential drugs (Das et al., 2012). Currently available hypolipidemic drugs have been associated with a

number of side effects. Herbal treatment of hyperlipidemia has no side effects and is relatively cheap and

locally available. So there are increasing interest in alternative/herbal medicine for the prevention and

treatment of hyperlipidemia (Mansi et al., 2009). Numerous studies have been carried out to search for

natural products with an anti-hyperlipidemic activity with minimal or no side effects.

Many of the medicinal plants widely used to reduce plasma cholesterol and to reduce the risk of

atherosclerosis related diseases (Mansi et al., 2009). Therefore it is a require of searching other materials

from natural sources that are less toxic, less expensive, which can provide better safety and efficacy on a

long term usage. Natural products from plants area rich source are used for centuries to cure

hyperlipidemia.

1.4 Efficacies of Aspirin and Eugenol on Hyperlipidemia

Aspirin has been widely used as a drug for treatment of inflammation, headache, fever, cardiovascular

20
 Chapter I

diseases for more than a century. Aspirin is well recognized as an effective anti-platelet drug for secondary

prevention in subjects at high risk of cardiovascular events. However, it is well known that the side effects

of aspirin have limited the using of this drug (Smith et al., 2000).

The biochemical mechanism or mode of action of aspirin has been described previously (Vane, 1971,

Flower et al., 1972, Vane and Botting, 1987). Also the side effect of aspirin (e.g. gastrointestinal ulcers) via

inhibition of cyclooxygenase (COX) which is a key enzyme to catalyze prostaglandin formation has been

reported (Vane, 1971). Recently, aspirin was extended to prevention and treatment of cardiovascular

diseases based on its anti-thrombotic action in platelets since inhibition of COX by blocking thromboxane

A2 production which is crucial for blood clotting (Patrono, 1989).

High dose aspirin not only lowers the inflammation mediated pathogenesis of the metabolic syndrome

(van Diepen et al., 2011), but it also diminishes hypertriglyceridemia in obese rodents (Yuan et al., 2001)

and patients with type 2 diabetes mellitus (Hundal et al., 2002). Bashir et al., 2010 reported that High dose

aspirin (120mg/kg) showed maximum decrease in serum triglyceride level (42.07%), serum total

cholesterol level (19.36%), and serum LDL level (6.18%) (Ahmad et al.). Moreover, Lin HL et al. reported

that low dose aspirin (5 mg/kg) can ameliorate high fat diet induced hyperlipidemia and hyperinsulinemia

in Sprague Dawley rats (Lin et al., 2014), also suggested that low dose aspirin may have potential in the

prevention of hyperlipidemia induced lymphocyte adhesion and inflammation. Hua Y et al. also reported

that aspirin (5 20 mg/kg) for 4 weeks can reduce total cholesterol, triglyceride, LDL and elevate HDL in

rabbits which were given with high fat diet.

Eugenol is the main component of volatile oil extracted from dry alabastrum Ocimum sanctum L of

Eugenia Caryophyllata (Clove oil). Eugenol is an allyl chain substituted guaiacol (Anandjiwala et al., 2006).

Eugenol is a member of the phenyl-propanoids class of chemical compounds. It is a clear to pale yellow

oily liquid extracted from certain essential oils especially from Ocimum sanctum L, clove oil, nutmeg,

cinnamon etc (Prakash and Gupta, 2005). It is slightly soluble in water and soluble in organic solvents such

tween 80. It has a spicy clove like aroma (Garkal et al., 2012).

Eugenol is used in perfumeries, flavorings, essential oils and in medicine as a local antiseptic and

anesthetic (Jadhav et al., 2004). It was used in the production of iso-eugenol for the manufacture of vanillin,

though most vanillin is now produced from phenol or from lignin. When mixed with zinc oxide, eugenol

forms a material which has restorative and prosthodontic applications in dentistry. Eugenol derivatives or

methoxy-phenol derivatives in wider classification are used in perfumery and flavoring (Bennett et al.).

21
 Chapter I

They are used in formulating insect attractants and UV absorbers, analgesics, biocides, and antiseptics

(Wright and Payne, 1962). The therapeutic effects of eugenol include antivirus, antibacterial, antipyretic,

analgesia, anti-inflammation, anti-platelet aggregation, anti-coagulation, anti-oxidation, anti-diarrhea,

anti-hypoxia, antiulcer, and inhibition of intestinal movement and arachidonic acid metabolism (Tragoolpua

and Jatisatienr, 2007, Chami et al., 2005, Gill and Holley, 2004, Nagababu and Lakshmaiah, 1997,

Hashimoto et al., 1988, Feng and Lipton, 1987, Raghavendra and Naidu, 2009). It is also used to treat

toothache, hepatopathy and gastrointestinal diseases. Eugenol can reduces Ca2+ influx and increases K+

efflux reduction of excitability of sensory nerves to achieve the therapeutic of toothache (da Rocha et al.,

2001). Several other pharmacological effects, such as antitumor, hepatoprotective, anti-inflammatory (oral

& topical), anti-ulcer, antimicrobial, antihyperlipidemic, and antiviral activities, have also been attributed.

They are also used in manufacturing stabilizers and anti-oxidants for plastics and rubbers. It is also used in

mouse traps (S V, 2012). However, several adverse effects were displayed following treatment with

hypolipidemic (Suanarunsawat et al., 2011, Suanarunsawat et al., 2010).

Antilipidemic actions of Ocimum sanctum essential oil drugs (Bhatnagar, 1998) is also used for its

hepatoprotective, cardioprotective and anti hyperlipidemic effects (Sharma et al., 2002, Prakash and Gupta,

2005). Some previous study showed that supplementation with OS dried leaf powder in the diet normalized

high serum lipid profile and partially protected the liver function in diabetic rats (Suanarunsawat and

Songsak, 2005). Similarly, treatment with 12 % of OS fresh leaves in the diet for four weeks significantly

decreased the serum lipid profile in normal albino rabbits (Sarkar et al., 1994). Some preliminary study also

showed that supplementation with OS dried leaf powder in the diet alleviated a high serum lipid profile in

rats fed with high cholesterol diet, and suggested that eugenol is the chemical compounds in OS leaves

possess the hypolipidemic action.

Many studies showed that both of aspirin and eugenol have some disadvantage like side effect or

resistant (Li et al., 2012). It is well known that the side effects of aspirin, such as serious gastrointestinal

damage, have limited the long time using of this classic drug (Smith et al., 2000).The disadvantages of

eugenol such as irritative and vulnerable to oxidation have limited the application of eugenol in practice (Li

et al., 2013).

1.5 Study Progress of AEE

As a new drug, AEE is pale yellow, smells and less crystal, and a candidate for treatment and therapy

22
 Chapter I

of inflammation, pain and fever and prevention of cardiovascular diseases with few side effects (Li et al.,

2010). Moreover, AEE is also a candidate drug for treatment and prevention of hyperlipidemia (Karam et

al., 2015a).

Aspirin is a classic drug and its use was extended to prevention and treatment of cardiovascular

diseases (Vane, 1971, Flower et al., 1972, Vane and Botting, 1987, Patrono, 1989, Schoemaker et al., 1998,

Wallenburg et al., 1986). Moreover, aspirin can recover hyperlipidemia indexes in rats feeding with high fat

diet, (Lin et al., 2014) also suggested that low dose aspirin may have potential in the prevention of

hyperlipidemia, reduce triglycerides in rats (Yuan et al., 2001) and type 2 diabetes mellitus (Hundal et al.,

2002).

Therapeutic effects of eugenol have been demonstrated in the effects on toothache, hepatopathy and

gastrointestinal diseases (Tragoolpua and Jatisatienr, 2007, Chami et al., 2005, Gill and Holley, 2004,

Nagababu and Lakshmaiah, 1997, Hashimoto et al., 1988, Feng and Lipton, 1987, Raghavendra and Naidu,

2009). Moreover, eugenol has been studied on treatment of hyperlipidemia (Suanarunsawat et al., 2011,

Suanarunsawat et al., 2010, Bhatnagar, 1998), and it is found that eugenol significantly decreased the serum

lipid profile in normal albino rabbits after being administrated for four weeks (Sarkar et al., 1994). Eugenol

can be used also for its hepatoprotective, cardioprotective and anti-hyperlipidemic effects (Sharma et al.,

2002, Prakash and Gupta, 2005).

Chemically carboxyl group of aspirin and hydroxyl group of eugenol are responsible for ulcer effects

and structural instability. So to overcome the disadvantages of aspirin and eugenol, AEE as a novel

medicinal compound was designed and synthesized according to the prodrug principles of structure

recombination (Liu et al., 2011b, Li et al., 2010).

In a 15 days oral dose toxicity study (Li et al., 2012), acute toxicity of AEE was less toxic 0.02 times

than aspirin and 0.27 times than eugenol. The previous research results as well as showed that AEE could

be metabolized to aspirin and eugenol in vitro and in vivo (Shen et al., 2015), and had the effects of

anti-inflammation, antipyretic, analgesia, and antioxidant. Li et al suggested that AEE was non genotoxic in

vivo or in vitro (Li et al., 2013). In addition, the previous research indicated that AEE (50 mg/kg and 160

mg/kg) could reduce the serum TC and TG in rats with standard diet (Li et al., 2012). Preventive effect of

AEE on thrombosis in rats has been demonstrated. The acute toxicity, teratogenicity, metabolism,

pharmacodynamics, stability and mutagenicity of AEE have been investigated (Ma et al., 2015). Our study

showed that AEE had a regulating effect on blood lipids indexes in Wistar rats with hyperlipidemia (Karam

23
 Chapter I

et al., 2015b).

As a promising drug candidate for prevention and treatment of hyperlipidemia, the present study was

performed to assess the efficacy of AEE on hyperlipidemia. It is important to characterize its efficacy in

rats with high fat diet. Meanwhile, this study will provide guidance for the design of further studies and

clinical trials of AEE.

1.6 Metabonomics

Metabonomics is the branch of science concerned with the quantitative understandings of the

metabolite complement of integrated living systems and its dynamic responses to the changes of both

endogenous factors (such as physiology and development) and exogenous factors (such as environmental

factors and xenobiotics) (Xu et al., 2014). Metabolomics plays an important role in systems biology

research as one of the new omics approach. It involves the detection, identification and quantitation of

small molecules involved in metabolism. With recent development of biotechnology, it is now feasible to

acquire metabonomics data with high throughputs using technical platforms such as nuclear magnetic

resonance (NMR) spectroscopy and mass spectrometry (MS). As a holistic approach, metabonomics detects,

quantifies and catalogues the time related metabolic processes of an integrated biological system, ultimately,

relates such processes to the trajectories of the pathophysiological events. Ever since its birth in 1999,

metabonomics has already been described in many scientific papers and half dozen patents, amongst which

almost most of the mare experimental articles (Tang and Wang, 2005). Now, metabonomics has been

established as an extremely powerful analytical tool and hence found successful applications in many

research areas including molecular pathology and physiology, drug efficacy and toxicity, gene

modifications and functional genomics, and environmental sciences (Tang and Wang, 2005). This holistic

approach has thus become an important part of systems biology and has now evolved to be a unique part in

global systems biology. The essence of metabonomics and some of the present applications were reviewed

to illustrate the rapid development of this extremely exciting new frontier (Tang and Wang, 2005).

Metabonomics is defined as the quantitative measurement of the dynamic multi-parametric metabolic

response of living systems to pathophysiological stimuli or genetic modification (Daviss, 2005, Nicholson,

2006).

Metabonomics is a global level tool that is employed in the prognosis or diagnosis of diseases by

investigating the endogenous levels of small molecule metabolites in clinical practices (Kaddurah-Daouk et

24
 Chapter I

al., 2008). Metabonomics is an important subject in the field of systemic biology, which includes

metabolomics, genomics, transcriptomics and proteomics and provides a wealth of information regarding

the biochemical framework in biological samples (Xu et al., 2014, Kanani et al., 2008, Koek et al., 2011).

There has been some disagreement over the exact differences between metabolomics and

metabonomics. The difference between the two terms is not related to choice of analytical platform.

Although metabonomics is more associated with nuclear magnetic resonance (NMR) spectroscopy and

metabolomics with mass spectrometry based techniques, this is simply because of usages amongst different

groups that have popularized the different terms. While there is still no absolute agreement, there is a

growing consensus that metabolomics places a greater emphasis on metabolic profiling at a cellular or

organ level and is primarily concerned with normal endogenous metabolism. Metabolomics extends

metabolic profiling to include information about perturbations of metabolism caused by environmental

factors (including diet and toxins), disease processes, and the involvement of extra genomic influences,

such as gut microflora (Robertson, 2005a).

1.7 Research Objectives

The aim of this study is to evaluate the efficacy of AEE on hyperlipidemia. Study was conduct to

determine the effective dose and evaluate the cure and prevention effects of AEE on the hyperlipidemia

biomarkers. It is also envisaged to study its mechanism on metabonomics.

There are many chemical drugs used as anti-hyperlipidemia and currently available hypolipidmic

drugs have been associated with a number of side effects (Thomas et al., 2003). So there are increasing

interest in novel drug which can provide better safety and efficacy on a long usage for prevention and

treatment of hyperlipidemia.

AEE as new drug combination between aspirin and eugenol is a promising drug candidate for

treatment and therapy of hyperlipidemia. AEE is known as novel drug for treatment of inflammation, pain,

fever and prevention of cardiovascular diseases with few side effects (Li et al., 2010). It is important to

characterize its efficacy on hyperlipidemia, compared with its component and statin as control drug. In this

study, the efficacy and prophylactic effect of AEE on hyperlipidemia were evaluated.

Statement of research hypotheses it depends on.

Rats are experimental animals for studying drugs effect on hyperlipidemia.

Establish of hyperlipidemia disease model by feeding rats with high fat diet.

25
 Chapter I

Aspirin can reduce blood lipids indexes some studies proved that.

Eugenol has been shown to lower a high serum lipid profile in hyperlipidemia.

AEE could reduce the serum TC and TG in rats with standard diet.

The study will be conducted to investigate the cure and preventive effect of AEE on hyperlipidemia

and metabonomics will be studied.

Specific objective:

-Investigate the cure efficacy of the AEE on hyperlipidemia.

-Investigate the prophylactic effect of AEE on hyperlipidemia.

-Determine the effective dose of the AEE on hyperlipidemia.

-Compare AEE with its component and statin.

-Study metabonomics of AEE.

-Study histopathological change of liver, stomach and intestine.

26
 Chapter II

Chapter II

The Preventive Effect of AEE on Hyperlipidemia

2.1 Introduction

Hyperlipidemia, which was described by the elevation of lipids in the blood stream, can affect

different kind of animals. Hyperlipidemia in small animals such as pets can be physiological or

pathological. Hyperlipidemia can be the result of an inherited disease in some animals (Xenoulis and

Steiner, 2010b). Hyperlipidemia is seen most commonly in ponies, miniature horses, and donkeys, and less

frequently in standard size adult horses. Poor feed quality or decrease in feed intake, particularly during a

period of high energy requirement (e g, pregnancy, systemic disease), may result in hyperlipidemia

syndrome in equine (Durham, 2006, Duhlmeier et al., 2003). A number of studies have shown that the

feeding of fats or excessive cholesterol supplements to ruminants raises the cholesterol concentration in

the serum (Nestel et al., 1978). A great number of animal models have been tested for hyperlipidemia

(Moghadasian, 2002, Moghadasian et al., 2001).

As a candidate for treatment and prevention of hyperlipidemia, it is important to characterize the

prevention effect of AEE on hyperlipidemia. The present experiment was performed to assess the

prevention effect of AEE on hyperlipidemia, compared with statin as model drug. Statins have been the

drugs of choice for decreasing blood plasma lipids levels, leading to substantial improvements in

cardiovascular morbidity and mortality. However, certain patients are unable to tolerate statins, such as

those with refractory familial hyperlipidemia, who are intolerant to all statin therapies (Keaney et al., 2014).

Therefore, a new approach to hypercholesterolemia treatment is needed. This study will provide guidance

for the design of further preclinical studies and clinical trials of AEE.

The study was conducted to evaluate the prevention effect of AEE on hyperlipidemia in SD male rats.

To induce hyperlipidemia in rats, the rats were feed with high fat diet for six weeks. For the prevention

experimental design, the drug was administrated with feeding rats high fat diet at the same time and

intragastrically administrated in each rat based on individual weekly body weight once weekly for six

weeks. The study was carried out for six weeks at daily doses of AEE 18, 36 and 54 mg/kg/day as low,

medium and high dose, respectively, compared with statin 10 mg/kg/day as control positive drug and with

CMC-Na 20 mg/kg/day as a vehicle control for six weeks.

Significant changes were observed on the levels of blood lipids indexes at the end of experiment. AEE

27
 Chapter II

at the dose of 54 mg/kg/day for six weeks decreased TG, TC and LDL significantly (p<0.01), which may

suggested that AEE had prevention effect on SD male rats with hyperlipidemia.

2.2 Material and Methods

2.2.1 Chemicals and Reagents

AEE as a novel drug combination between aspirin and eugenol is pale yellow and smells less

transparent crystal (purity: 99.5% with RE-HPLC) and was prepared in the Key Lab of New Animal Drug

Project of Gansu Province, Key Lab of Veterinary Pharmaceutical Development of Agricultural Ministry,

Lanzhou Institute of Husbandry and Pharmaceutical Sciences of Chinese Academy of Agricultural Science

CAAS (China). CMC-Na (carboxyl methyl cellulose sodium) as a vehicle control and simvastatin as

control positive drug was supplied by Tianjin Chemical Reagent Company (Tianjin, China).

The TG, TC, LDL and HDL kits were provided by Ningbo Medical System Biotechnology Co., Ltd

(Ningbo, China). Erba XL-640 analyzer (German) was used to measure the level of blood lipid indexes.

Drug Preparation: AEE should be crushed to be powder and then AEE and simvastatin was prepared in

0.5% of CMC-Na liquid suspension.

2.2.2 Animals

Seventy Sprague Dawley (SD) male healthy rats with clean grade (Certificate No.: SCXK (Gan)

2012-0075), aged 7 weeks and weighing 160 -180 g, were purchased from the animal breeding facilities of

Gansu traditional Chinese medicine University (Lanzhou, China). They were housed in plastic cages of

appropriate size (50cm35cm20cm, ten rats per cage) with stainless steel wire cover and chopped bedding

(wood mulch). Light and dark regime was adjusted 12 and 12 h and living temperature was about 22 2 C

with relative humidity of 55 10%. Rats feed and drinking water were supplied ad libitum.

The prevention experiment was performed in compliance with the guidelines for the care and use of

laboratory animals as described in the US National Institutes of Health and approved by Institutional

Animal Care and Use Committee of Lanzhou Institute of Husbandry and Pharmaceutical Science of CAAS

(China). Animals were allowed a 2-week quarantine and acclimation period prior to start of the study.

2.2.3 Feeding

To induce hyperlipidemia, the rats were feed with high fat diet. Blank group was fed by standard

compressed rat food for and other groups feed by high fat diet after two weeks of acclimation period. High

fat diet which was (standard rat diet 77.8%, yolk power 10%, lard 10%, cholesterol 2% and bile salts 0.2%)

consists of 41.5% lipids, 40.2% carbohydrates, and 18.3% proteins (kcal) and standard rat diet consists of

28
 Chapter II

12.3% lipids, 63.3% carbohydrates, and 24.4% proteins (kcal). The food was supplied from KeaoXieli Co.,

Ltd (Beijing, China).

2.2.4 Dosing

AEE was used in three different doses for prevention study and the high, medium, and low doses of

AEE were selected as 54, 36 and 18 mg/kg, respectively. The dosage of AEE were determined based on the

previous study about the drug effect and toxicity (Li et al., 2011a,b; Ye et al., 2011; Li et al., 2010;Li et

al.,2012). Previous study suggested that 50 mg/kg is non-toxic, safety and decreased on TC, TG in rats with

standard diet. Simvastatin as control positive drug was used in dosage 10 mg/kg. CMC-Na was used as

control negative drug in dosage 20mg/kg. AEE and simvastatin suspension liquids were prepared in 0.5%

of CMC-Na. Rats were randomized into seven groups: five test groups (n = 10 rats), model and blank

groups (n = 10 rats). Drugs were administered intragastrically in each rat based on individual weekly body

weights once weekly for six weeks.

2.2.5 Design of the Experiment

The present study was performed to assess the prevention effect of AEE on hyperlipidemia compared

with simvastatin. Animals were allowed a 2 weeks quarantine and acclimation period prior to start of the

study. Then the drugs were administrated to the rats at the same time with feeding high fat diet to model

group and treatment groups to evaluate the prevention effect of drugs specifically after repeated oral

administrations for six weeks in rats at daily dose levels of 18, 36 and 54 mg/kg as low, medium and high

dose of AEE, respectively. 10 mg/kg of simvastatin and CMC-Na 20 mg/kg were seen in Table 2-1. Serum

samples were collected from rats to determine of hyperlipidemia indexes on blood stream. After the

administration period, blood lipids levels were measured and the examinations were carried out at the end

of drug administration period. Serum samples were analyzed for hyperlipidemia indexes with a hematology

analyzer Erba XL-640 analyzer (German).

2.2.6 Serum Samples

The blood samples were collected from the rats at the end of experiment and the rats should be fasting

for 10-12 hours before collecting blood samples. The serums were got through centrifuge blood samples for

15 mins at the speed of 4000g 4C. The blood lipids indexes including TG, TC, LDL and HDL were

measured. The drugs were added for six weeks in order to make sure that there was significant difference

on blood lipids indexes among different groups.

29
 Chapter II

2.2.7 Statistics

The statistical analyses were carried out using IBM SPSS19.0 (USA). All data obtained from the

experiment were expressed as mean standard deviation (SD). Statistical differences between the

treatments groups and the model group were evaluated by using one way ANOVA with Duncan test.

p-values less than 0.05 were considered statistically significant.

Table2-1 The design of the groups in the experiment

Groups Number of rats Food Drug Dosage

Blank 10 Standard diet -- --
Model 10 HFD -- --
AEE Low 10 HFD AEE 18 mg/kg
AEE Mid 10 HFD AEE 36 mg/kg
AEE High 10 HFD AEE 54 mg/kg
Statin 10 HFD Simvastatin 10 mg/kg
CMC-Na 10 HFD CMC-Na 20 mg/kg
Note: HFD food: high fat diet food. Group statin and CMC-Na served as positive and negative control drug,
respectively.

2.3 Results

2.3.1 Disease Model

For evaluating the prevention effects of the drugs used in the experiment, the hyperlipidemia disease

model should be established in rats using high fat diet. After feeding rats with high fat diet for six weeks,

the hyperlipidemia was induced successfully in rats. According to the result of blank group and model

groups, there was significant difference between two groups among the blood lipid indexes (seen in Table

2-3). The result showed that there was significant increasing (p<0.01) in TG, TC and LDL in model group,

however, no change in HDL index between blank and model groups.

2.3.2 Body Weight

The body weights of rats were showed in Table 2-2 after acclimation period for two weeks prior to

start the experiment. The body weight of all groups during experiment was increased. The results showed

that AEE high dose could diminish the increasing rate of body weight of rats significantly rather than other

groups. There was no difference in increasing body weight rate among AEE low, medium dose, CMC-Na

and model group. Simvastatin had light change in increasing body weight rate compared with the model

group, but still not significant.

30
 Chapter II

The body weights of model and blank groups were increased during experiment. There was significant

difference (p<0.05) between two groups in weeks 3, 4 and 5. AEE low dose group had no significant

change from model group in increasing body weight rate. AEE medium dose group had significant

difference (p<0.05) from model group in week-2. AEE high dose group had significant difference from

model group from week-2 up to the end of experiment (p<0.05).

2.3.3 Prevention Effect

The prevention effect of drugs in different groups can be evaluated by comparing with model group at

the end of experiment. From Table 2-3, TC index can be decreased significantly (p<0.05) in AEE low dose

group at the end of experiment after administrating the drug for six weeks. Meanwhile, AEE low dose

group had no changes on the other blood lipids indexes. AEE medium dose group had no significant change

on all blood lipids indexes.TG, TC and LDL indexes were decreased significantly in AEE high dose group

(p<0.01) and no significant change in HDL index. Simvastatin can decrease TC and LDL significantly

(p<0.01) and no significant change on TG and HDL indexes. CMC-Na had no effect on blood lipid indexes

at the end of experiment.

2.3.4 Multiple Comparisons

Comparison between different groups in blood lipids indexes were used to evaluate the prevention

effects of different drugs. The result showed that AEE at high dose and simvastatin group had same effect

on TC and LDL in blood lipid indexes. At the end of experiment, both of AEE and statin decreased TC and

LDL significantly (p<0.01), but no significant change on HDL. However, simvastatin had no significant

change in TG index and AEE at high dose can decrease TG index significantly (p<0.01). AEE at low dose

can decrease TC index significantly (p<0.05) and no change on other indexes. AEE at medium dose had no

significant change on blood lipid index. CMC-Na had no effect on blood lipid index.

31
 Chapter II

Table 2-2 Body weights of the rats during administrating the drug
Groups Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7
Blank 209.6315.18 254.3816.74 288.3817.43 333.2518.22* 346.3817.67* 371.0021.42* 385.0020.70 404.0018.96
Model 216.0011.24 249.1313.97 279.3815.81 310.2520.10 326.3820.53 349.8825.97 368.1325.69 396.2527.83
AEE Low 220.018.47 254.8816.71 283.6314.33 304.5029.19 324.1328.72 347.6329.33 366.5031.24 394.3830.62
AEE Mid 205.6317.06 *
238.3814.47 261.8813.16 291.1316.16 311.3816.56 337.3818.67 356.5020.32 387.2523.55
* * ** * **
AEE High 204.8815.40 252.2523.47 261.1312.31 285.5012.08 301.2510.82 324.1313.54 340.5015.57 366.1319.86**
Statin 220.3822.06 253.7521.41 278.5022.40 304.1325.15 321.1321.59 340.5021.69 355.2520.89 382.8823.76
CMC-Na 229.0016.19 258.7517.68 288.8816.33 302.5018.17 327.2517.89 348.2517.61 365.3820.76 391.2526.54
* **
Note: p< 0.05 significant difference from model group, p< 0.01 more significant difference from model group. The time week 0 before drug administrated and after
acclimation period 2 weeks, week7 at the end of experiment

Table2-3The blood lipids levels after drugs administration for six weeks (n=10).
Variables Blank Model CMC-Na Stain AEE 18 AEE 36 AEE 54
TG 0.530.12** 0.890.07 0.790.19 0.830.05 0.790.23 0.770.10 0.710.07**
HDL 0.720.04 0.740.08 0.690.09 0.660.13 0.730.09 0.790.10 0.650.15
** **
LDL 0.250.02 0.500.09 0.460.06 0.390.13 0.450.05 0.460.10 0.360.08**
TC 1.40.10** 2.290.26 2.130.22 1.800.16** 2.100.22* 2.150.15 1.660.15**
Note: TG: Triglyceride; HDL: High density lipoprotein; LDL: Low density lipoprotein; TC: Total cholesterol; the unit of TG, HDL, LDL and TC is mmol/L.*p< 0.05
significant difference from model group, **p< 0.01 high significant difference from model group.

32
 Chapter II

2.4 Discussion

AEE has being developed for preventing cardiovascular disease, anti-inflammatory, analgesic,

antipyretic, anti-atherosclerosis and anti-thrombosis pharmaceutical (Ma et al., 2015). Our previous studies

showed that AEE had regulation effect on blood lipids indexes which include TG, TC, LDL and HDL

(Karam et al., 2015b).

Feeding rats with high fat diet can induce hyperlipidemia in rats. For the prevention experiment design,

the rats were fed with high fat diet for six weeks and the drugs administrated at the same time. The blood

lipid indexes as TG, TC and LDL were increased in model group, which indicated that hyperlipidemia

model was successfully induced in rats at the end of experiment, compared model group with blank group

fed with standard diet. However, HDL index supposed to be decreased in model group but no change.

HDL-cholesterol in model group was similar to the blank group, this may be due to the fiber content of diet

(Matos et al., 2005, Gregorio et al., 2001), or may be due to feeding blank group long period with standard

diet rich of carbohydrate which can effect HDL level or can decrease HDL in blank group (Karam et al.,

2015b). Several studies found that the content of diet can effect HDL level (Xu et al., 2001b). Some studies

approved that HDL may not decrease after feeding high fat diet, even it can increase. As observed by Lee,

feeding mice with high fat diet for sixteen weeks showed elevated serum levels of TC, HDL-C, LDL-C,TG

and glucose, characteristics resembling hypercholesterolemia in diabetic patients (Lee et al., 2014). Karimi

reported that the TC, LDL-C, VLDL-C, HDL-C and TGs were apparently raised after rabbits were fed with

a cholesterol rich diet for four weeks (Karimi et al., 2010).

After acclimation period of two weeks, the rats were fed with high fat diet for six weeks to induce

hyperlipidemia and the drugs administrated at the same time for prevention design. The body weight of all

rats was increased during the experiment in all groups, which indicated that the rats were growing up

healthy during the experiment period. It means that the drugs was safety and had no conflict in using these

drugs in rats as experimental animals. There was significant difference among body weight between blank

and model groups in week 3, 4 and 5, which may be due to adapting the rats in model group with high fat

diet and then it growing up healthy like blank group. AEE at high dose reduced the increasing rate of body

weight of the rats significantly, which looked interesting indicator of AEE, and further studies should

evaluate the effect of AEE in obesity or decreasing the body weight. Shrestha reported that aspirin in a

mouse model effectively suppressed the increases in body weight and adipose mass without any noticeable

toxic effect (Shrestha et al., 2007). This may explain why the effect of AEE on body weight can be from
33
 Chapter II

aspirin. AEE at low dose had no significant effect on body weight compared with model group, which

mean AEE at low dose had no influence on body weight. AEE at medium dose was similar to model in

body weight increase rate except in week-2; however, there was no significant difference in other weeks.

CMC-Na also had no significant different from model in body weight increase rate at the end of experiment,

which indicated that CMC-Na had no influence on body weight and the effect of AEE at high dose on body

weight from the drug only. Simvastatin group was different from model group in body weight increase rate

but still not significant.

For evaluating the prevention effects of different drugs in different groups, the prevention effect of

AEE can be assessed by comparing with model group. The result showed that AEE at high dose can

decrease significantly TG, TC and LDL, which meant that AEE at high dose can prevent hyperlipidemia in

rats. Simvastatin can decrease significantly TC, LDL, while there was no significant change on TG index.

This indicated that AEE at high dose was more effective than simvastatin as preventive drug for

hyperlipidemia regarding TG index under experiment condition. This is why the action mechanism of

statins depends on inhibition of enzyme 3-hydroxy- 3- methyl gluataryl co enzyme A HMG-Co A reductase

inhibitors (Stein et al., 1998, Li et al., 2003). This enzyme play a crucial role on cholesterol synthesis in

liver endogenous pathway, so the main effect of statin on cholesterol biosynthesis rather than triglycerides

(Szarszoi et al., 2008, Wang et al., 2006, Lea and McTavish, 1997, Stein et al., 1998). Stein reported that

the ability of statins to reduce serum triglyceride concentration depends on the baseline of triglyceride

levels and the potency and efficacy of the statins used. However, Li et al., 2003 mentioned that statin

resulted significantly in greater reductions in TC and LDL-C indexes, respectively, and less significant

reduction was observed in TG level compared with TC and LDL-C. Moreover, there was no significant

difference in HDL cholesterol levels (Li et al., 2003). These results were matched with our results.

Both of AEE at high dose and simvastatin had no effect on HDL index, which was supposed to

increase after drugs administration but there was no change. This apparent unexpected result may be why

HDL was not decreased in model group during the experiment. Sharma reported that synthetic

hypolipidemic agents had one or more side effects and were unable to increase HDL levels (Sharma et al.,

2013) and Tang mention that there is no an effective medicine that can obviously increase HDL-C at

present (Tang et al., 2015). Bunnoy (2015) pointed out that hyperlipidemic rats treated with 5.0 mg/kg/day

of rosuvastatin for 30 days had no significant change in high HDL-C concentrations (Bunnoy et al., 2015),

however, the relationship between HDL apo CIII and the effect of statin treatment on HDL apo CIII were
34
 Chapter II

still unclear (Xiong et al., 2015).

AEE had prevention effect on hyperlipidemia in rats. However, further study is still needed for the

action mechanism of AEE. Eugenol can effect hyperlipidemia since it may act via inhibition of cholesterol

biosynthesis in liver, decrease of lipid absorption, boosted catabolism of LDL-cholesterol and TG (Mnafgui

et al., 2013, Venkadeswaran et al., 2014). Research by Shrestha (2007) showed that aspirin may restrain

raise in the plasma TG, TC and non-esterified fatty acid concentrations and thus expand its therapeutic

potential to other related metabolic diseases such as hyperlipidemia and hypercholesterolemia (Shrestha et

al., 2007), which may explain the effect of AEE on hyperlipidemia. The previous study on the metabolism

of AEE showed that AEE was decomposed into aspirin and eugenol. Salicylic acid from AEE metabolism

as a final metabolite excreted in urine was coincided with salicylic acid from aspirin metabolism. However,

the elimination time of salicylic acid was longer than aspirin, which might explain the efficacy of AEE

lasting longer than those of aspirin and eugenol. Therefore, the AEE effect on hyperlipidemia can come

mainly from eugenol and aspirin. The AEE effect on hyperlipidemia may result from synergistic action of

aspirin and eugenol together, which could display stronger drug effects than aspirin and eugenol on

hyperlipidemia (Shen et al., 2015, Karam et al., 2015a).

Therefore, AEE at dose of 54 mg/kg/day for six weeks had preventive effect on hyperlipidemia in SD

male rats under the present condition of the study. AEE showed more efficacy than simvastatin under

experiment condition. Further studies need to conduct to investigate the action mechanism of AEE.

35
 Chapter III

Chapter III
The Cure Effect of AEE on Hyperlipidemia
3.1 Introduction

Hyperlipidemia is disorder disease characterized by an excess on blood lipids which include increase

in cholesterol, triglyceride, low density lipoprotein and decrease in high density lipoprotein in blood stream

(Graham et al., 1996, Song et al., 2013). Hyperlipidemia is a risk co-factor of atherosclerosis leading to

heart attack and hypercholesterolemia, which can impart some degree of risk for ischemic heart disease

(IHD) and other cardiovascular disease including coronary heart disease, cerebral stroke, myocardial

infarction and renal failure (Xu et al., 2014).

The treatment of hyperlipidemia depends on reducing lipids in the blood stream to avoid the risk of

developing ischemic heart disease, cardiovascular and cerebrovascular disease. There are many chemical

drugs that lower blood lipids levels in the body, commonly known as lipid lowering drugs (Xu et al., 2014).

Most of lowering lipid drugs are expensive and have undesirable effect (Thomas, 2003), therefore, it is a

need of the day to search new materials from available sources that are less toxic, less expensive (Huang et

al., 2010), which can provide better safety and efficacy on a long term usage for hyperlipidemia treatment.

The experiment will be conducted to investigate the efficacy of the AEE on cure hyperlipidemia using

Wistar rats. The rats were fed with high fat diet for 8 weeks to inducing hyperlipidemia. AEE is a novel

promising drug with fewer side effects. It is important to characterize AEE efficacy on hyperlipidemia,

compared with simvastatin as control drug and with its component such as aspirin and eugenol, also

compared with integration of aspirin and eugenol together and with CMC-Na as suspension for AEE,

aspirin and simvastatin. In this study, the efficacy of AEE on hyperlipidemia was investigated.

Compared with the model group, AEE at the dosage of 54 mg/kg significantly decreased the

hyperlipidemia index including TGLDL and TC (p< 0.01), increased HDL (p< 0.05) after 5 weeks.

Meanwhile, statin had same effect on hyperlipidemia indexes TG, LDL, TC, but no significant increase in

HDL.

Our study showed that AEE was effective against the hyperlipidemia and better effect on

hyperlipidemia than its component. In the experiment, the optimal dose of AEE to cure hyperlipidemia is

54 mg/kg/day for five weeks in Wistar rats.

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 Chapter III

3.2 Materials and Methods

3.2.1 Chemicals and Reagents

AEE is a novel drug combined between aspirin and eugenol, which is a pale yellow and smells less

transparent crystal (purity: 99.5% with RE-HPLC). AEE was prepared in the Key Lab of New Animal Drug

Project of Gansu Province, Key Lab of Veterinary Drug Development of Agricultural Ministry, Lanzhou

Institute of Husbandry and Pharmaceutical Sciences of Chinese Academy of Agricultural Science (China).

CMC-Na (carboxyl methyl cellulose sodium) was used as suspension for the drugs and simvastatin was

supplied by Tianjin Chemical Reagent Company (Tianjin, China). Aspirin and Tween-80 were supplied by

Aladdin Industrial Corporation (Shanghai China). Eugenol was supplied by Sinopharm Chemical Reagent

Co., Ltd. (Shanghai China). High fat diet food (standard rat diet 77.8%, yolk powder 10%, lard 10%,

cholesterol 2% and bile salts 0.2%) consists of 41.5% lipids, 40.2% carbohydrates, and 18.3% proteins

(kcal) and standard rat diet consists of 12.3% lipids, 63.3% carbohydrates, and 24.4% proteins (kcal). The

food was supplied from Keao Xieli Co, .Ltd (Beijing, China). The TG, TC, LDL and HDL kits were

provided by Ningbo Medical System Biotechnology Co, .Ltd (Ningbo, China). Erba XL-640 analyzer

(German) was used to measure the blood lipids levels.

3.2.2 Animals

Wistar male rats used as experimental animals in this study were purchased from the animal breeding

facilities of Lanzhou Army General Hospital (Lanzhou, China) with clean grade, aged 7 weeks and

weighing 160 -180 g. They were housed in plastic cages of appropriate size (50cm35cm20 cm, ten rats

per cage) with stainless steel wire cover and chopped bedding (wood mulch). Light and dark regime was

12/12 h and living temperature was 22 2 C with relative humidity of 55 10%. Rats feed and drinking

water were supplied ad libitum. The study was performed in compliance with the guidelines for the care

and use of laboratory animals as described in the US National Institutes of Health and approved by

Institutional Animal Care and Use Committee of Lanzhou Institute of Husbandry and Pharmaceutical

Science of CAAS (China). Animals were allowed a two week quarantine and acclimation period prior to

start of the study.

3.2.3 Drug Preparation

AEE and aspirin were crushed. AEE, simvastatin and aspirin suspension liquids were prepared in 0.5%

of CMC-Na. Eugenol and Tween-80 at the mass ratio of 1:2 was mixed with the distilled water.

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 Chapter III

3.2.4 Hyperlipidemia Disease Model

After rats were arrived, they take two week as quarantine and acclimation period prior to start of the

study. Then the rats were randomized into two main groups, first one group I fed with standard diet (blank

group) and other group II fed with high fat diet for eight weeks to induce hyperlipidemia disease model in

rats. After hyperlipidemia was induced in rats, the high fat diet group (group II) were divided to nine

groups, model group and eight treatment groups, eight treatment groups (n = 10) and two groups as blank

and model (n = 10 rats) (Table 3-1).

Table 3-1 The design and dosing of the groups in the experiment.
Groups Number of rats Food Drug Dosage
A 10 Standard diet -- --
B 10 HFD -- --
C 10 HFD AEE 18 mg/kg
D 10 HFD AEE 36 mg/kg
E 10 HFD AEE 54 mg/kg
F 10 HFD Aspirin+ Eugenol (20+18) mg/kg
G 10 HFD Aspirin 20 mg/kg
H 10 HFD Eugenol 18 mg/kg
I 10 HFD Simvastatin 10 mg/kg
J 10 HFD CMC-Na (5%) 20 mg/kg
Note: HFD: high fat diet. Group A served as blank group. Group B served as model group. Group C served as
AEE low dose. Group D served as AEE medium dose. Group E served as AEE high dose. Group F served as
Integration (aspirin: eugenol 1:1). Group G served as aspirin group. Group H served as eugenol group. Group I
served as simvastatin group. Group J served as CMC-Na group.

3.2.5 Dosing

There were eight treated groups and two groups as model and blank (seen in Table 3-1). The dosage

for rats was based on individual weekly body weights for five weeks. Drugs were administered

intragastrically in each rat based on individually body weight weekly once a week. Hyperlipidemia was

induced successfully in the rats and high fat diet continued during the experiment period in model and

treatment groups.

The dosage of AEE were determined on the previous study about the drug and toxicity (Li et al.,

2011a,b; Ye et al., 2011; Li et al., 2010;Li et al.,2012). Previous studies suggested that AEE at the dosage of

50 mg/kg was non-toxic, safety and it can decrease on TC, TG in rats with standard diet. According to

38
 Chapter III

aspirin dose for human as 375 mg/person/day, the dose of AEE is 18 mg/kg as low dose, 36 mg/kg as

medium dose and 54 mg/kg as high dose.

In order to compare the effects between AEE and its component, the mole of medium dose of AEE,

aspirin, and eugenol is the same at 0.3067 moles. In the experiment, integration of aspirin and eugenol at

the molar ratio 1:1 (0.3067 mole) were also set. The 0.5% of CMC-Na at the dose of 20 mg/kg was served

as the negative control. The dosage of CMC-Na was close to equal by comparison with AEE and Aspirin

groups. Simvastatin at 10 mg/kg was chose as positive control drug to compare with AEE.

3.2.6 Design of the Experiment

After the rats were fed with high fat diet for inducing hyperlipidemia, the blood samples were

collected from the rats tail. The rats will be fasting for 10-12 hours before collecting blood samples. The

serums were got through centrifuge for 15 mins at the speed of 4000g at 4 C. The blood lipids indexes

including TG, TC, HDL and LDL were measured on week 4, 6 and week 8 to make sure the success of

hyperlipidemia animal model. After inducing hyperlipidemia successfully, blood samples were taken to

measure the changes in blood lipids on week 2 after adding the drugs and on week 5. Examinations of

serum blood lipids indexes were carried out in the same method.

3.2.7 Histopathological Preparations

The rats were anesthetized with an intraperitoneal injection of 10 % chloral hydrate (2 mL/kg) then

sacrificed, histological preparations involved passing the tissue samples throughout several steps to form

tissue paraffin blocks and sectioning, the steps according to Bancroft and Steven (Bancroft and Stevens,

1990) as the following:

Fixation: samples for histopathology were taken immediately after small pieces (about 2-3 cm)

representing the tissue collected was washed with saline and fixed in 10 % formal saline, (10 ml of

concentrated formalin add to 90 ml distilled water) the fixation period is 24 hours.

Dehydration: after histopathological samples fixed were dehydrated using the ethanol by immersing

the tissue in ascending graded concentrations of alcohol beginning from 60 % (no limited time), 90 %

ethanol for 6 hours, and to absolute ethanol for 3 hours.

Clearing: this step involved removing the alcohol from the tissues replaced by the clearing solution. In

this step xylene with chloroform was used and the tissues incubated in xylene for 20 minutes.

Impregnation and embedding: The tissue was embedded in paraffin wax to remove the clearing agent.

The tissue incubated in the bath of molten paraffin for replacing the xylene by the paraffin in three changes

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 Chapter III

of molten paraffin for 3 hours of each.

Blocking and sectioning: the tissue was then blocked with a molten wax and quickly cooled, when the

embedding time was over, tissues paraffin blocks were made and the blocks one day later were sectioned

using microtome (Leica, Germany) cut in a section of 5 m in thickness and fixed to the slides.

The section was stained with hematoxylin and eosin H&E stain, covered with cover slip, and fixed by

the Canada balsam. After drying at room temperature overnight, the section was examined microscopically

using an13395H2X microscope (Leica, Germany). The stained areas were viewed at 100X and 400X.

3.2.8 Statistics

The statistical analyses were carried out using IBM SPSS 19.0 (USA). All data obtained from the

experiment were expressed as mean standard deviation (SD). The difference between Group I and Group

II were evaluated by Students t-Test. Statistical differences between the treatments and the control were

evaluated by using one way ANOVA with Duncan test. p-values less than 0.05 were considered statistically

significant.

3.3 Results

3.3.1 Animal Disease Model

Hyperlipidemia disease model in rats was first needed to establish by feeding them with high fat diet.

In regard to blood lipid indexes levels, there was no significant difference between Group I fed with

standard diet and Group II fed with high fat diet in week 4 and 6. So the administration time of high fat diet

was prolonged. After eight weeks, there were significant increase of TG, LDL and TC indexes and decrease

of HDL index in model group (p<0.01, seen in Table 3-2). This confirmed that the hyperlipidemia animal

disease model was successfully established by feeding rats with high fat diet in the experiment for eight

weeks.

Table 3-2 Blood lipids level in Group I and II at the end of 8th week after using high fat diet.
Variables Unit TG TCH HDL LDL
Group I mmol/L 0.770.42 1.010.25 0.550.07 0.120.08
Group II mmol/L 1.510.38** 2.180.31** 0.440.06** 0.520.08**
Note: TG: Triglyceride; HDL: High density lipoprotein; LDL: Low density lipoprotein; TCH: Total cholesterol
**
p< 0.01 significant difference from blank group.

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 Chapter III

3.3.2 Body Weight

The body weight of the Wistar male rats before drug administration and after feeding rats with high fat

diet for eight weeks were recorded (seen in Table 3-3). Blank group fed with standard diet (group I) had a

lower body weight than other groups (group II) (p<0.01, seen in Table 3-3) fed with high fat diet. There

was no significant difference among other groups except blank group. After drug administration for five

weeks, the average values of body weight in drug treatment groups were less than that in model group and

there was still significant difference between blank and model group (p<0.01). The average body weight

values in CMC-Na group were similar with that model group.

Table 3-3 Body weights changes of rats before and after drug treatment on fifth week.

Groups Before drug treatment After drug treatment

A 276.16.4aa 330.15.6 bb
B 295.47.1 358.87.2
C 298.45.6 347.36.8 b
D 303.67.3 340.8 5.9 bb
E 299.56.9 340.88.1 bb
F 301.15.9 339.3 6.4 bb
G 305.66.9 343.45.1 bb
H 292.65.3 321.15.9 bb
I 306.85.9 342.47.1 bb
J 302.76.7 352.3 6.6
Note: A: blank group, B: model group, C: AEE low dose, D: AEE medium dose, E: AEE high dose, F:
integration group (aspirin: eugenol, molar ratio 1:1, 0.11 mmol), G: aspirin group, H: eugenol group, I:
simvastatin group, J: CMC-Na group. ap< 0.05, aap< 0.01 significant difference from model group before drugs
administration. bp< 0.05,bbp< 0.01 significant difference from model group after drugs administration. The time
before drug treatment was the end of 8th week after HFD was used and fifth week after drug treatment was the
end of 13th week after HFD and drugs was used.

3.3.3 Anti-hyperlipidemic Effect

To evaluate the drugs effect on hyperlipidemia indexes, blood samples were taken for blood lipids

examination after the drugs were given for two weeks. However, there was no significant difference among

groups. So the time was extended for five weeks. From Table 3-4, the difference effects of drugs on

hyperlipidemia indexes among different groups appeared after the drugs were given for five weeks.

Meanwhile, there was no statistical difference between model group and CMC-Na group as negative

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control, which indicated that CMC-Na had no effect on hyperlipidemia indexes as a vehicle control.

After administrating the drugs to the rats for five weeks, the blood lipid analysis (seen in Table 3-4)

showed different values. The blood lipid indexes TG, TC and LDL were significantly decreased in varying

degrees in comparison with model group. These changes mean that there were significant differences

between the treated groups and the model group among the hyperlipidemia indexes at the end of 13th

weeks.

There was a significant increase in the serum TG levels of model group as compared with blank group.

On the other hand, treated groups with AEE high dose, integration of aspirin and eugenol (1:1) group,

aspirin group and simvastatin group significantly decreased the levels of TG (p<0.01) as compared with

model group. AEE at medium dose can decrease TG index significantly (p<0.05) compared with model

group. Compared with those groups together, AEE at high dose group was more effective than other groups

except simvastatin group as control positive drug of hyperlipidemia. Both of AEE at high dose and

simvastatin had similar effect on TG. However, AEE at low dose group, eugenol group, CMC-Na group had

no significant decrease on TG index. With the increase of AEE doses, the mean values of TG were

decreased, which showed that TG values were dose dependence in AEE.

There was a significant increase in the serum indexes of LDL and TC levels in model group as

compared with blank group. On the other hand, treated groups at AEE low, medium, high dose, integration

of aspirin and eugenol (1:1) group, aspirin group, eugenol group, simvastatin group significantly

decreased levels of LDL and TC (p<0.01) as compared with model group. CMC-Na group had no

significant decrease on LDL and TC indexes.

There was a no significant decrease in the serum HDL index levels in model group compared with

blank group at week 13. There was no significant difference in HDL index in related groups as AEE low,

medium dose, integration aspirin: eugenol (1:1) group, aspirin group, eugenol group, simvastatin group,

CMC-Na group as compared with model group. However, AEE at high dose group was the only one group

of significant increase on HDL index as compared with model group (p<0.05).

3.3.4 Optimal Dosage of AEE

For optimal dose of AEE in our experiment regarding the blood lipid indexes which included TG, TC.

LDL and HDL, the results demonstrated that AEE had significant effect on ameliorative hyperlipidemia

indexes (Fig.3-1). Through the evaluation of AEE groups with different doses, AEE at high dose could

decrease more significantly the levels of TG, TC, LDL (p<0.01), and at the same time increase HDL level

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 Chapter III

(p<0.05). The effects of AEE at high, medium and low dose on LDL and TC were similar. However, AEE

at high dose had more significant effect on TG (p<0.01) and can reduce TG. AEE at low dose had no

significant effect on TG, AEE at medium dose had significant effect on TG (p<0.05), but AEE at high dose

had more significant effect on TG index (p<0.01). AEE at high dose can increase significantly HDL as

good cholesterol (p<0.05) and can prevent hyperlipidemia, while AEE at medium and low dose had no

significant effect on HDL index.

Fig. 3-1 The influence of AEE different dosage on hyperlipemia indexes after administration for five weeks
(n=10). TG: Triglyceride; HDL: High density lipoprotein; LDL: Low density lipoprotein; TCH: Total cholesterol.
The blank group and AEE groups were used to compare with the model group. ap< 0.05,aap< 0.01 significant
difference of HDL; bp< 0.05 , bbp< 0.01significant difference of LDL; cp< 0.05,ccp< 0.01 significant difference of
TG. dp< 0.05,ddp< 0.01 significant difference of TCH. There was no significant difference among three AEE
groups in influence on hyperlipidemia indexes.

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 Chapter III

3.3.5 Multiple Comparisons

Compared different drugs in different groups among the blood lipid indexes, the changes in TC, TG,

LDL and HDL were interesting (Fig.3-2) and this showed the different effects of drugs on blood lipids

levels.

When compared with aspirin group, there was significant decrease of TG, LDL and TC indexes in

model group. However, simvastatin had significant (p<0.05) effect on LDL more than aspirin and aspirin

group had no significant different from other groups. There was significant decrease of LDL and TC in

eugenol group from model group (p<0.01). AEE at high dose and integration (aspirin: eugenol 1:1) had

significant effect on HDL and more than eugenol. Integration (aspirin: eugenol 1:1) can decrease

significantly LDL and TC compared with model group (p<0.01), while TG index decrease significantly

(p<0.05). However, AEE at high dose had significant effect on LDL and HDL and more than integration

(aspirin: eugenol 1:1) (p<0.05).

According to the result of statistical analyses, the effect of AEE at high dose was similar to simvastatin

as control positive drug for hyperlipidemia. AEE at high dose decreased TG, LDL and TC significantly

(p<0.01) and increased HDL significantly (p<0.05). On the other hand, simvastatin had no significant

increase of HDL index and decreased significantly TG, LDL, TC when compared with model group

(p<0.01), however simvastatin significant effect on TG more than AEE low dose (p<0.01) and medium

dose (p<0.05). There was significant different effect of simvastatin on LDL more than aspirin (p<0.05),

while significant different effect of simvastatin on LDL more than integration (aspirin: eugenol 1:1)

(p<0.01). There was significant different of simvastatin on TC from CMC-Na group (p<0.05). Compared

with aspirin and eugenol group, there was no significant difference between AEE and other groups, except

the HDL index in AEE high dose group (Fig.3-2).

Compared with model group, AEE high dose and simvastatin significantly reduced TG, LDL and TC

(p<0.01). Meanwhile, AEE high dose increased significantly HDL (p<0.05), while simvastatin had no

significant increase of HDL. The results showed that simvastatin had stronger effect than low and medium

dose of AEE on TG and LDL value. From the results (Fig.3-2), there was no statistical difference between

simvastatin and AEE high dose groups on blood lipid indexes TG, LDL and TC. This suggested that the

anti-hyperlipidemic effects of high dose AEE and simvastatin were similar among these indexes TG, LDL

and TC. However, AEE high dose significantly increased HDL index (p<0.05), while simvastatin had no

significant effect on HDL index and AEE more effective than simvastatin among HDL index.

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 Chapter III

Fig. 3-2 Effects of different drugs on hyperlipidemia indexes after drugs administration for five weeks (n=10).
TG: Triglyceride; HDL: High density lipoprotein; LDL: Low density lipoprotein; TCH: Total cholesterol.
Integration: aspirin and eugenol at the molar ratio 1:1. ap< 0.05 significant difference from aspirin group. aap<
0.01 significant difference from aspirin group. bp< 0.05 significant difference from eugenol group. bb
p< 0.01
c cc
significant difference from eugenol group. p< 0.05 significant difference from integration group. p< 0.01
significant difference from integration group. dp< 0.05 significant difference from simvastatin group. dd
p< 0.01
significant difference from simvastatin group.

3.3.6 Histopathology

The slides were stained with hematoxylin and eosin H&E stain was performed to access the changes

on tested organs such as the liver, stomach and intestine. Microscopic liver, stomach and intestine tissues

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 Chapter III

are shown in (Fig. 3-3). Liver histological examination of the blank group showed normal cell architecture

(Fig. 3-3), while significant morphological changes were observed in hepatic tissue in the model group (Fig.

3-4). Liver sections in the model group showed less cells and lipid vacuolization, which are observed in Fig.

3-4. Liver section was also showed that there was infiltration of lipid. Any other histopathological changes

were not found in stomach and intestine. There was increase in liver centro lobular hypertrophy. There was

a trend that the rats receiving high fat diet had a higher ratio of fat cells in their livers compared with blank

group. Finally, an increased ratio of lipid hepatocytes was observed in exposed livers.

In treated groups, liver section from group C (AEE at low dose) showed improvement in structural

organization of hepatic lobules as compared with model group. The hepatocytes exhibited reduction in

vacuoles accumulation. The fat vacuoles in hepatocyte were less than that seen in model group (Fig. 3-5).

In liver section from group D (AEE at medium dose), the hepatic lobules were less distinct than those in the

section from group AEE at low dose. Many cells contained varied size vacuoles and other cells showed

granular cytoplasm. The vacuolation was moderate in section from group AEE at low dose but less than

those in model group (Fig. 3-6). In liver section from group E (AEE high dose), the vacuolar hepatocyte

were more than that seen in group C and D of AEE low and medium dose but the vacuolation were less

than that seen in model group. Many hepatocytes were granular with moderate cell degeneration and

necrosis. The low dose had better effect than the medium and high dose. The high dose had worse effect on

the liver than the low and medium dose (Fig. 3-7). Liver section from group F (integration of aspirin and

eugenol 1:1) showed slight vacuolar degeneration. The hepatic lobes looked better than those in model

group (Fig. 3-8). In liver section from group G (aspirin), the hepatic lobes were less distinct and vacuolar

degeneration was more moderate than those in group F (integration of aspirin and eugenol 1:1) (Fig. 3-9).

In liver section from group H (eugenol), the changes were similar to the changes in section G (aspirin) (Fig.

3-10). In liver section from group I (statin), liver sections of this group exhibited a normal pattern of liver

parenchyma and some hepatocytes showed slight vacuolar degeneration with remarkable reduction in fatty

droplets (Fig. 3-11). In liver section from group J (CMC-Na), no significant pathological alteration was

seen in Fig. 3-12. All sections from stomach and intestine showed no significant pathological changes.

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 Chapter III

Table 3-4 The blood lipids levels at the end of 13th week (after drugs administration for five weeks, n=10).
Variables Blank Model CMC-Na Simvastatin AEE 18 AEE 36 AEE 54 Integration Aspirin Eugenol
TG 0.770.2** 1.650.22 1.630.34 1.120.15** 1.430.11 1.360.16* 1.230.15** 1.350.17* 1.270.15** 1.390.22
*
HDL 0.460.04 0.520.05 0.580.02 0.560.05 0.550.04 0.560.03 0.620.05 0.610.02 0.610.05 0.560.01
** ** ** ** ** ** **
LDL 0.110.03 0.540.04 0.510.05 0.230.04 0.310.04 0.30.03 0.250.04 0.310.02 0.300.04 0.280.05**
TCH 1.010.18** 2.490.14 2.110.38 1.610.23** 1.820.16** 1.730.21** 1.550.27** 1.770.11** 1.800.10** 1.680.13**
Note: TG: Triglyceride; HDL: High density lipoprotein; LDL: Low density lipoprotein; TCH: Total cholesterol; Integration: aspirin: eugenol (molar ratio 1:1, 0.11 mmol).
The unit of TG, HDL, LDL and TC is mmol/L.*P< 0.05 significant difference from model group. **P< 0.01 significant difference from model group.

47
 Chapter III

Fig.3-3 Group A (blank) the liver section from rats, this group showed normal structural organization of hepatic lobules and normal hepatocytes, stomach and intestine normal
cell architecture (overhead row 100X, down row 400X),liver in the left, intestine in the middle and stomach in the right.

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 Chapter III

Fig.3-4 Group B (model) the liver section from rats, this group showed general impairment of the normal structural organization of hepatic lobules in many areas. The
hepatocytes showed varied size of regular cytoplasmic vacuoles (fatty change appeared). Hepatocytes showed degeneration, swollen and vacuolation, stomach and intestine
showed no significant pathological changes (overhead row 100X, down row 400X), liver in the left, intestine in the middle and stomach in the right.

49
 Chapter III

Fig. 3-5 Group C (AEE low dose) Sections from rats liver, this group showed improvement in structural organization of hepatic lobules as compare to group B. The
hepatocytes exhibited reduction in vacuoles accumulation. The fat vacuoles in hepatocyte were less than that seen in group B, stomach and intestine showed no significant
pathological changes (overhead row 100X, down row 400X), liver in the left, intestine in the middle and stomach in the right.

50
 Chapter III

Fig.3-6 Group D (AEE medium dose) liver section from rats the hepatic lobules were less distinct than those in the section from group C. Many cells contained varied size
vacuoles and other cells showed granular cytoplasm, stomach and intestine showed no significant pathological changes (overhead row 100X, down row 400X), liver in the
left, intestine in the middle and stomach in the right.

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 Chapter III

Fig.3-7 Group E (AEE high dose) liver section from rats the vacuolation were less than that seen in group B. Many hepatocytes were granular with moderate cell
degeneration and necrosis, stomach and intestine showed no significant pathological changes (overhead row 100X, down row 400X), liver in the left, intestine in the middle
and stomach in the right.

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 Chapter III

Fig.3-8 Group F (integration of aspirin and eugenol 1:1) liver section from rats showed slight vacuolar degeneration. The hepatic lobes looked better than those in group B,
stomach and intestine showed no significant pathological changes (overhead row 100X, down row 400X), liver in the left, intestine in the middle and stomach in the right.

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 Chapter III

Fig.3-9 Group G (aspirin) liver section from rats the hepatic lobes were less distinct and vacuolar degeneration was more moderate than those in group F, stomach and
intestine showed no significant pathological changes (overhead row 100X, down row 400X), liver in the left, intestine in the middle and stomach in the right.

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 Chapter III

Fig.3-10 Group H (eugenol) liver section from rats the changes were similar to the changes in section G, stomach and intestine showed no significant pathological changes
(overhead row 100X, down row 400X), liver in the left, intestine in the middle and stomach in the right.

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 Chapter III

Fig.3-11 Group I (statin) Liver sections from rats of this group exhibited a normal pattern of liver parenchyma; some hepatocytes showed slight vacuolar degeneration with
remarkable reduction in fatty droplets i.e. (reversed towards control sections were considerably high), stomach and intestine showed no significant pathological changes
(overhead row 100X, down row 400X), liver in the left, intestine in the middle and stomach in the right.

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 Chapter III

Fig.3-12 Group J (CMC-Na) No significant pathological alteration was seen on those organs from rats, stomach and intestine showed any significant pathological changes
(overhead row 100X, down row 400X), liver in the left, intestine in the middle and stomach in the right.

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3.4 Discussion

Hyperlipidemia is a group of metabolic disorders characterized by the elevated levels of lipids in the

blood stream. Hyperlipidemia is a major modifiable risk factor for atherosclerosis and related

cardiovascular disease (Bauer, 1995). These lipids include cholesterol, cholesterol esters, phospholipids,

and triglycerides. Increased levels of LDL are related to the development of atherosclerosis (Smith et al.,

2004, Jacobson, 1998). HDL plays an important role in removing cholesterol from tissues and protecting

against cardiovascular disease. Hyperlipidemia can be the result of an inherited disease in certain breeds of

dogs (Xenoulis and Steiner, 2010b). In small animals such as pets, hyperlipidemia most often occurs as a

consequence of some disorder and can be physiological (postprandial) or pathological. Hyperlipidemia

even can occur spontaneously after a meal of high fat foods, particularly table scraps (Whitney, 1992, Bauer,

2004). Hyperlipidemia is seen most commonly in ponies, miniature horses, and donkeys, and less

frequently in standard size adult horses (Durham, 2006, Duhlmeier et al., 2003). In non-ruminants,

including primates and man, hyperlipidemia may be increased by dietary manipulations such as feeding

excessive cholesterol or fats with high saturated fatty acid content (Nestel et al., 1978). A great number of

animal models, such as pigeons, chickens, swine, cats, dogs, monkeys, mice, rabbits and rats, have been

tested for hyperlipidemia (Moghadasian et al., 2001). Commonly used rats strains (i.e. Sprague Dawley,

Wistar) typically have high levels of HDL-C and low levels of LDL-C. HFD is capable of promoting

elevations in TC and LDL in Sprague Dawley or Wistar rats, likely by reducing bile acid production (Jeong

et al., 2005, Yokozawa et al., 2006, Horton et al., 1995).

There are many chemical drugs that could ameliorate hyperlipidemia such as statins, fibrates,

ezetimibe and nicotinic acid, but most of them are expensive and have undesirable effects (Thomas et al.,

2003). So there are increasing interests in alternative drug for the prevention and treatment of

hyperlipidemia. Currently, available hypolipidemic drugs have been associated with a number of side

effects. Therefore, it is important to develop a novel drug that is less toxic, less expensive in order to

provide better safety and efficacy on a long term usage.

In this study, the anti-hyperlipidaemic effects of AEE were investigated in rats with induced

hyperlipidemia by high fat diet. Rats as experimental animal model can be used for testing drugs for

hyperlipidemia (Jeong et al., 2005, Yokozawa et al., 2006, Horton et al., 1995). To induce hyperlipidemia,

rats were fed with high fat diet. Many studies supported that feeding animals with dietary manipulations

such as feeding excessive cholesterol or fats with high saturated fatty acid content can increase
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 Chapter III

hyperlipidemia in most animals (Nestel et al., 1978). The blood lipid indexes were observed during the

experiment especially first eight weeks. When compared the blood lipids indexes between blank and model

group at week 8, there was a significant elevation in the levels of serum LDL, TG and TC indexes, and

decrease of HDL index. Changed levels of these parameters in serum are presumptive markers of

hyperlipidemia, which mean that hyperlipidemia was induced successfully in the rats.

LDL is more related to hyperlipidemia and atherosclerosis, which is an important index among blood

lipids indexes. On other hand, HDL in this experiment decreased at week 8 in model group. However, HDL

value in model group showed no significant difference from blank group at week 13. This apparently

unexpected result could be explained by the light increase of HDL in model group and decrease in blank

group. Some studies found that the food content can affect HDL level (Xu et al., 2001a), light decrease of

HDL in blank group may be due to the feeding long period with standard diet rich of carbohydrate. Some

studies found that the increased plasma apo E of apolipoproteins in aged hyperlipidemia rats can increase

HDL after a long period (Xu et al., 2001a, Van Lenten and Roheim, 1982, Brown and Goldstein, 1976, Hui

et al., 1981, Sparks and Marsh, 1981).

The body weight of the rats was increased during experiment in all groups, which indicated that the

rats grew up healthy during the experiment. These mean the drugs are safety and no conflict in using on

rats. The purchased rats were aged seven weeks and weight 160-180g. After acclimation period for two

weeks, the rats were fed with high fat diet for eight weeks to induce hyperlipidemia. Table 3-3 showed that

there was significant difference between model and blank groups on body weight after feeding rats with

high fat diet for eight weeks. These results indicated that the high fat diet had a remarkable influence on

body weight, which may suggest the close relationship between hyperlipidemia and obesity. Some studies

mentioned that increased serum TG and/or TC concentrations have been observed in obese dogs

(Chikamune et al., 1995, Bailhache et al., 2003, Jeusette et al., 2005). Jeusette reported that the most

profound changes were associated with severe chronic obesity dogs (Jeusette et al., 2005). Meanwhile,

some studies found that weight loss in obese dogs lead to significant decreases in both serum TG and TC

concentrations (Diez et al., 2004, Jeusette et al., 2005). After drugs administration for five weeks, the

average values of body weight in treatment groups were less than model group significantly, which meant

there was relation between hyperlipidemia and body weight. When hyperlipidemia was induced in rats, the

body weight was increased significantly; however, when hyperlipidemia was cured in treatment groups, the

body weight was decreased significantly compared with model group. CMC-Na group were similar with
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model group in hyperlipidemia indexes and body weight, which mean CMC-Na as negative control drug

had no effect on hyperlipidemia or body weight.

From the result in this study, there was significant change on blood lipid indexes after administrating

the drug for five weeks. The examination of hyperlipidemia indexes after two weeks of drug administration

showed no significant changes in most groups. So the time was extended to five weeks. The changes in TC,

TG, LDL and HDL indexes confirmed that AEE had influence on hyperlipidemia. Previous study on AEE

showed that it had effect on TG and TC in Wistar rats with standard diet (Li et al., 2012). Compared with

simvastatin and CMC-Na as positive and negative control drugs respectively, high dose of AEE had

positive effects on anti-hyperlipidemia indexes. High dose of AEE significantly decreased TG, LDL, TC

and increased HDL index, but simvastatin had no significant in increasing HDL. This result confirmed that

AEE more benefit on curing hyperlipidemia than simvastatin under the experiment condition. Based on the

results (Table 3-4) regarding TG index the decrease in the treated groups AEE high dose, integration of

Aspirin: Eugenol (1:1) group, Aspirin group and simvastatin group was high significantly reduced the

levels of TG. Compared those groups together among TG index, it was found that AEE high dose group

was more effective than other groups except simvastatin group. Statin is control positive drug of

hyperlipidemia and both of AEE high dose and simvastatin had similar effect on TG. Since eugenol had no

significant difference on TG, it may be speculated that aspirin may play a key role on TG reduction. So the

effect of AEE on TG may be due to aspirin rather than eugenol. The result showed same effects of different

drugs on TC and LDL and indicated that the effects of simvastatin, AEE and its component on LDL, TC

were similar, when compared with model group. HDL in all treated groups had no significant difference

from model group. However, AEE high dose group was the only one group in significant increase on HDL

index. AEE at high dose was more effective on hyperlipidemia rather than other drugs including

simvastatin.

To choose the optimal dose, AEE was used in three different doses (18, 36 and 54 mg/kg) as low,

medium and high dose, respectively. Regarding blood lipid indexes, the results demonstrated that different

doses of AEE had different effects on hyperlipidemia indexes. Through the evaluation of AEE groups with

different doses in this study, AEE high dose could more significantly decrease the levels of TG, TC, LDL,

and increase HDL level at the same time. The effects of AEE at different doses on LDL and TC were

similar (Fig.3-1). However, AEE at high dose had more significant effect on TG index, can reduce

triglycerides more significantly. AEE at low dose had no significant effect on TG. AEE at medium dose had
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significant effect on TG but still less than AEE at high dose, which had more significance on TG index.

Regarding TG index, AEE high dose was more effective than low and medium dose. AEE high dose also

could increase HDL significantly. HDL as good cholesterol can prevent hyperlipidemia and plays an

important role in removing cholesterol from tissues and protecting against cardiovascular disease. AEE

medium and low dose had no significant effect on HDL index. So AEE high dose more effective on HDL

index than AEE low and medium dose. The optimal dose of AEE to reduce hyperlipidemia in rats is 54

mg/kg/day for five weeks under our experimental condition, of course, further studies is needed for action

mechanism of AEE on anti-hyperlipidemia.

The changes in TC, TG, LDL and HDL are interesting since they appeared to be most important for

hyperlipidemia. This is very beneficial to the therapy of hyperlipidemia. The result in Figure 3-2 showed

that simvastatin was more effective than aspirin, integration, and AEE low and medium dose on LDL index.

Simvastatin had significant influence on LDL because the action mechanism of simvastatin depended on

HMG-Co A reductase inhibiter, which can reduce cholesterol synthesis in the liver in endogenous pathway.

Furthermore, simvastatin was more effective than AEE low and medium dose on TG indexes, which looked

logic result for simvastatin as positive control drug for hyperlipidemia.

The result showed that the hyperlipidemia disease model was constructed successfully in rats by

feeding with high fat diet. Causing histopathological changes in liver was partly supported by H&E

staining. These results were consistent with those of previous studies (Feng et al., 2011), and indicated that

a high fat diet accumulated fat in hepatic tissue cells (Wu et al., 2014). High fat diet can finally cause fatty

liver. In addition, Table 3-4 showed significant differences in the blood lipid index of the model group

compared with the blank group. There were histological changes in hepatic tissue which confirmed the

previous result of blood lipid indexes. That means high fat diet successfully induced hyperlipidemia in rats

to make disease model. Certainly, these changes may be able to be explained in part by the elevated cholic

acids. Cholic acid is synthesized in the liver and secreted in the gallbladder or intestine. In the treatment

groups, the liver recovered gradually. Statin group showed liver recovery with remarkable reduction in fatty

droplets. The action mechanism of statins depends on inhibition of enzyme 3-hydroxy- 3- methyl gluataryl

co enzyme A HMG-CoA reductase inhibitors (Stein et al., 1998, Li et al., 2003), which is play a crucial role

on cholesterol synthesis in liver endogenous pathway. AEE in different doses showed less recovery than

statin. With the increase of AEE doses, the fatty change in liver recover was inversely, which may indicate

that the action mechanism of AEE is different than statin. Statin is working on cholesterol synthesis in liver
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while AEE may is different mechanism on blood stream, because the blood lipid analysis showed that AEE

had good result better than statin.

All Sections from stomach and intestine showed no significant pathological changes. These indicated

there was no harm on those organs, which may mean the drugs were safety for using on experimental

animals for long period.

The action mechanism of AEE on curing hyperlipidemia was still unclear. AEE was decomposed by

enzyme into aspirin and eugenol, which may act synergistically. The effect of AEE on hyperlipidemia may

be due to either aspirin or eugenol or both of them. Many studies supported that aspirin and eugenol had

influence on hyperlipidemia. Previous study showed that eugenol had effect on hyperlipidemia since it was

probably mediated through inhibition of hepatic cholesterol biosynthesis, reduction of lipid absorption,

enhanced catabolism of LDL-cholesterol, and catabolism of TG (Mnafgui et al., 2013, Venkadeswaran et

al., 2014). Shrestha reported that aspirin can prevent increases of TG, TC and non-esterified fatty acid

concentrations in the plasma. Thus, therapeutic potential of aspirin was expanded to other related metabolic

diseases such as hyperlipidemia and hypercholesterolemia. Moreover, aspirin was also tested in a mouse

model for its efficacy to prevent diet induced obesity and effectively suppressed the increases in body

weight and adipose mass without any noticeable toxic effect (Shrestha et al., 2007). Lin HL et al. reported

that low dose aspirin (5 mg/kg) can ameliorate high fat diet induced hyperlipidemia and hyperinsulinemia

in Sprague Dawley rats (Lin et al., 2014). Ahmad reported that high dose aspirin (120 mg/kg) showed

maximum decrease in serum TG level, serum TC level, and serum LDL level (Ahmad et al.). In our study,

the results (Table 3-4) showed that eugenol could significantly reduce the LDL and TC indexes. However,

there was no statistical difference between eugenol and model groups in the TG and HDL indexes.

Noteworthy, aspirin could reduce TG, LDL and TCH index (Table 3-4) significantly, which showed that

aspirin possessed positive drug effects on hyperlipidemia.

The study on the metabolism of AEE showed that AEE was decomposed into aspirin and eugenol.

Salicylic acid (SA) from AEE metabolism as a final metabolite excreted in urine was coincided with SA

from aspirin metabolism. However, the elimination time of SA was longer than aspirin, which might

explain the efficacy of AEE lasting longer than those of aspirin and eugenol. Therefore, the AEE effect on

hyperlipidemia can come mainly from eugenol and aspirin, and the effect of AEE on hyperlipidemia may

result from synergetic action of aspirin and eugenol together, which could display stronger drug effects than

aspirin and eugenol.

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In summary, there were significant differences in blood lipid indexes throughout the experiment period

under the present study conditions. For Wistar rats, the optimal dose for curing hyperlipidemia was

considered to be 54 mg/kg/day administrating for five weeks. Further studies should be conducted to

investigate its effect and the action mechanism of AEE on anti-hyperlipidemia.

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Chapter IV
Mechanisms on Metabonomics of AEE
4.1 Introduction

Metabonomics has been widely used in discovering biomarkers and in monitoring therapeutic efficacy

and pathogenesis in early detection and clinical diagnosis for many diseases, including tumors (Zhou et al.,

2012, Patterson et al., 2011), heart disease (Chen et al., 2008, Rhee and Gerszten, 2012) and diabetes

mellitus (Newgard et al., 2009, Leslie and Beyan, 2011, Robertson, 2005b). Moreover, small changes in the

activities of individual enzymes, genes and proteins or other biomarkers can be amplified in metabonomics

analysis. Many studies have successfully carried out with quadruple time of flight mass spectrometry

(LC-Q-TOF-MS) based on metabonomics approaches to study hyperlipidemia by analyzing the serum or

urine of rats or humans (Zhang et al., 2009, Ma et al., 2011, Ma et al., 2013).

Metabonomics aims at the detection and quantitation of metabolites within a biological system. As the

most direct representation of biological changes, metabonomics is an important component in system

biology research. Recent development on high resolution, high accuracy mass spectrometers enable the

simultaneous study of hundreds or even thousands of metabolites in one experiment. Liquid

chromatography mass spectrometry (LC-MS) is a commonly used as an instrument for metabonomics

studies due to its high sensitivity and broad coverage of metabolome (Zhou, 2011, Gika et al., 2007,

Morrison et al., 2007). Metabonomics plays an important role in systems biology research as one of the

new omics approach. It involves the detection, identification and quantitation of small molecules

involved in metabolism. With recent development of biotechnology, it is now feasible to acquire

metabonomics data with high throughputs using technical platforms such as nuclear magnetic resonance

(NMR) spectroscopy and mass spectrometry (MS). Liquid chromatography mass spectrometry (LC-MS),

which couples MS detection with chromatography separation, has gained popularity for metabonomics

analyses due to its sensitivity, accuracy and coverage of metabolome. By analyzing spectral features from

LC-MS data, metabolites can be detected and their abundances in samples can be quantitated, relatively or

absolutely (Zhou, 2011, Soga et al., 2003, Bundy et al., 2009).

Metabolism is the set of chemical reactions which happens in a biological organism. These chemical

reactions are usually divided into two categories. Catabolism breaks down large molecules to harvest

energy and provides building blocks to synthesize organic compounds. Anabolism uses the energy and

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building blocks from catabolism to synthesize protein, lipids, nucleotides and etc. The substrates,

intermediates and products involved in metabolism are metabolites. Common types of metabolites include

amino acids, carbohydrates, nucleic acids, and lipids. By organizing the chemical reactions within

metabolism into metabolic pathways, one metabolite is sequentially transformed into another metabolite or

its polymers through a chain of reactions, with the catalysis of enzymes (Zhou, 2011, Griffin, 2003,

Beckonert et al., 2007).

Metabolism plays an important role in supporting the normal function of a biological system by

allowing the growth and reproduction of the organism, maintaining its structure and adapting to ever

changing environment. At present, metabonomics investigations have been applied in various research

areas including environmental and biological stress studies, functional genomics, biomarker discovery, and

integrative systems biology (van der Greef et al., 2004, Khoo and Al-Rubeai, 2007, Goodacre et al., 2004).

Those studies facilitate understandings of biochemical fluxes and discoveries of metabolites which are

indicative of unusual biological or environmental perturbations (Chiang et al., 2006, Gibney et al., 2005).

This study was conducted to investigate the metabonomics of AEE on hyperlipidemia during the

experiment using SD male rats.

4.2 Material and Methods

4.2.1 Samples

The investigation of AEE in three different doses on hyperlipidemia in SD male rats as experimental

animals was carried out with the same methods in experiment in chapter . The plasma samples were

collected from rats in the experiment for metabonomics investigation. At the end of experiment, each rat

was isolated in separate metabolic stainless steel cages for three days and fasted overnight (10-12 h allowed

free access to water). Blood samples were collected at the end of experiment. Then the rats were

anesthetized with an intraperitoneal injection of 10 % chloral hydrate (2 mL/kg), the plasma were got

through centrifuge the blood samples for 15 mins at the speed of 4000g at 4 C.

4.2.2 LCQ-T of MS Analysis of Plasma

4.2.2.1 Plasma Sample Preparation

Plasma samples were thawed at room temperature prior to analysis. 600 L cold acetonitrile (-20 C)

was added into 200 L of sample in 2 ml centrifuge tube. After vortex-mixing for 30 s, the mixture was

centrifuged at 12000 rpm for 10 mins to precipitate the proteins. The supernatant was filtered through a

0.22 m nylon filter. An aliquot of 4 L was injected for UPLC/MS analysis.

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4.2.2.2 Metabolic Profiling and Method Validation

Chromatographic separation was performed on an Agilent ZORBAX SB-C18 threaded column

(3.0100 mm, 1.8 m, Agilent Corp., USA) using HPLC system consisted of a degasser, thermostattwo

binary pumps and autos-ampler (1290, Agilent). The column was maintained at 35 C and eluted at a

flowing rate of 0.3 mL/min, using a mobile phase of solvent A - water with 0.1% formic acid (by volume)

and solvent B acetonitrile with 0.1% formic acid (by volume). The gradient program was optimized as

follows: 0 min: 95% A; 0-4 min 95% to 60% A; 4-8 min 60% to 40% A; 8-18 min 40% to 5% A; 18-23min

5% A. The post time was set to 5 mins for equilibration with 95% A and 5% B. The eluent from the column

was directed to the mass spectrometer without split.

Agilent 6530 Q-TOF (Agilent Corp., USA) was used to carry out the mass spectrometry with a dual

electrospray ionization source (ESI) operating in positive and negative ion mode. The sample cone voltage

was set at 135 V in both model. The capillary voltages were set at 4.0 KV in positive model and 3.5 KV in

negative model, respectively. Using dry gas nitrogen, the desolvation gas rate was set to 10 L/min at 350 C.

The scan time was set at 1 spectra/second. Data was collected in centroid mode from m/z 50 to m/z 1000.

During the analysis two reference masses: 121.0509 m/z (C5H4N4) and 922.0098 m/z (C18H18O6N3P3F24)

were continuously measured to allow constant mass correction, and obtain the accurate mass.

To ensure the stability and reproducibly of the metabolic system, a quality control sample was pooled

from 30 l of supernatant drawn from each sample. During sequence analysis, the QC sample was operated

every 4 plasma samples to evaluate stability. Five consecutive runs of QC samples in positive and negative

model were shown in Fig. 4-1. The results showed that the chromatograms were tightly over lapped and the

retention time was stable without obvious drift, which indicated the metabolic analysis system was highly

stable and reproducible.

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Fig. 4-1 Five consecutive runs of QC samples in positive and negative model

4.2.3 Data Analysis

The results of the blood lipids were expressed as mean standard deviation (SD). The significance of

differences between groups had been evaluated by one-way ANOVA with least significant difference (LSD)

test using the Statistical Package for Social Science program (SPSS 16.0, Chicago, IL, USA). The

significance threshold was set at p< 0.05 for this test.

4.2.4 Multivariate Analysis and Identification of Potential Biomarkers

The raw MS spectra were firstly processed by Agilent chromatographic work software, which allowed

deconvolution, alignment and filter to give a list of mass and retention time pairs with corresponding peak

area for all the detected peaks from each file in the data set. The resulting data as comma-separated value

(CSV) format was exported to SIMCA-P 13.0 (Umetrics, Sweden) for subsequent multivariate analyses.

The two data sets (1304 MS features, ESI+; 197 MS features, ESI-) were separately imported into SIMCA

to perform unsupervised principal component analysis (PCA) and orthogonal partial least squares

discriminant analysis (OPLS-DA). Data were Pareto-scaled before multivariate statistical analysis. The

OPLS-DA models were validated by ANOVA of the cross-validated residuals (CV-ANOVA) method.

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Potential biomarkers were identified according to variable of importance in the project (VIP) values and

S-plots. A potential metabolic biomarker was selected when its VIP value was more than 1.00. Potential

biomarkers were also performed LSD post-hoc test (SPSS, Chicago, IL, USA). A probability of (p< 0.05)

was considered to be a statistically significant difference.

Identification of the marker metabolites was achieved through a mass-based search followed by

manual verification. First, TOF-MS accurate mass value of the molecular ion of interest was searched

against the Human Metabolome Database (HMDB) and Bio fluid Metabolites Database (Metlin). Then, the

putative identifications were verified by comparing the MS2 fragmentations. Parts of the metabolites were

further identified by reference standards. Biochemical reactions involved were found through KEGG and

HMDB.

4.3 Result

4.3.1 Blood Lipids

There were significant increase of TG, LDL and TC indexes and decrease of HDL index of model

group compared with blank group (p<0.01, seen in Table 4-1). This confirmed that the hyperlipidemia

animal disease model was successfully established by feeding rats with high fat diet in the experiment.

The blood lipid indexes TG, TC and LDL were significantly decreased in varying degrees in

comparison with model group. These changes mean that there were significant differences between the

AEE groups and the model group among the hyperlipidemia indexes at the end of experiment. AEE high

dose more significantly decrease TG, TC and LDL (p<0.01), while AEE medium dose significantly

decrease TG, TC and LDL (p<0.05), however, AEE low dose had no effect on TG, TC and LDL but it

increase HDL more significantly (p<0.01).

Table 4-1 The blood lipids levels after drugs administration at the end of experiment
Variables Blank Model AEE 18 AEE 36 AEE 54
TG 0.44 0.15** 0.67 0.13 0.59 0.16 0.53 0.15* 0.49 0.16**
HDL 0.52 0.05** 0.35 0.11 0.48 0.1** 0.33 0.07 0.32 0.09
** *
LDL 0.25 0.04 0.46 0.07 0.48 0.07 0.35 0.11 0.31 0.09**
TC 1.24 0.13** 1.63 0.16 1.53 0.17 1.41 0.18* 1.22 0.2**
Note: TG: Triglyceride; HDL: High density lipoprotein; LDL: Low density lipoprotein; TCH: Total cholesterol; The unit of
TG, HDL, LDL and TC is mmol/L.* p< 0.05 significant difference from model group **p< 0.01 significant difference from
model group.

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4.3.2 Comparison of the Metabolic Profile between Control and Hyperlipidemia Groups

The representative total ion chromatograms (TICs) of the plasma samples in control and model group

analyzed by Q-TOF in positive and negative model were shown in Fig. 4-2. TICs showed good separations

and strong signals for analysis. Clear discriminations in profile and abundance were observed between

control and model group. Thus, the differences in metabolite profiles of two groups were directly reflected

through the TIC identified by UPLC-Q-TOF.

Fig. 4-2 TICs (total ion chromatograms) showed good separations and strong signals for analysis.

In order to understand the global metabolic differences between control and model group and to get an

overview of the data, PCA approaches were used to distinguish the differences and screen the variations

contributing to the classification. PCA is an unsupervised technique, meaning that it shows the main

structure in the data without considering a special direction or type of information. The multivariate data

was firstly displayed by PCA. The PCA score plots (Fig. 4-3) showed that the model group exhibited a

tendency to be away from control group. Meanwhile, a clear separation was observed between control and

model group. Plasma metabolic profiles in the model group deviated from the control, which indicated

significant biochemical changes were induced by HFD. The parameters for the classification model were

R2X = 0.752 and Q2= 0.661 in positive model and R2X = 0.615 and Q2= 0.489 in negative model,

respectively. These results indicated that the models were good to fit and predict. Meanwhile, all samples

fell inside the 95% confidence interval, which was represented by an ellipse. The PCA results did not find

any outliers which may disturb continued analysis. Therefore, the hyperlipidemia disease model induced by

HFD was successful and could be used in this study to explore the regulation effects of AEE on blood

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lipids.

Fig. 4-3 The PCA score plots showed that the model group exhibited a tendency to be away from control group.

In order to bring out the potential biomarkers of hyperlipidemia, the OPLS-DA method was employed

between the model and control groups. In this work, a well-fitting two-component OPLS-DA model (ESI+,

R2Y = 0.968, Q2Y = 0.918; ESI-, R2Y = 0.995, Q2Y = 0.981; p-value of CV-ANOVA of the models less

than 0.001) was constructed to identify and reveal the differential metabolites (Fig. 4-4). The good

parameters and the clear separation indicated that the OPLS-DA model was successful and there was

significantly difference in plasma metabolite profiles from the two groups. S-plot as a tool was employed to

visualize covariance and correlation between the metabolites and the model class. Those ions far from the

origin made contribution to the clustering significantly. As shown in Fig. 5, S-plot based on plasma

metabolic profiles indicated that ions away from center contributed to the clustering. These ions contributed

significantly to differentiate the clustering of model group from control, which could be considered as

potential biomarkers responsible for hyperlipidemia induced by HFD. In addition, the VIP values of these

ions were all above 1 and p-values were less than 0.05.

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Fig. 4-4 The clear separation indicated that the OPLS-DA (orthogonal partial least squares discriminant analysis)
model was successful.

Fig. 4-5 S-plot based on plasma metabolic profiles indicated that ions away from center contributed to the
clustering.

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4.3.3 Intervention of AEE on the Metabolic Profiling

OPLS-DA was conducted to determine whether AEE influenced the metabolic pattern. The score plots

of the OPLS-DA model were shown in Fig. 4-6. The parameters for the classification (ESI+: R2Y = 0.96,

Q2= 0.86; ESI-: R2Y = 0.95, Q2= 0.90) were good to fit and predict, respectively. Both of the p-values of

CV-ANOVA in two models were less than 0.001. A clear separation was observed among the control,

model and AEE groups. According to score plots of all samples, samples of model group clustered away

from those of control group, indicating that hyperlipidemia was successfully induced by HFD. In ESI+

model, the metabolic profile of rats in AEE treated groups overlapped each other. Compared with the model

group, AEE groups were all located at the opposite direction along latent variable 2. Moreover, AEE groups

showed a trend to be close to the control. In ESI- model, a clear separation was observed among AEE

groups. Compared with the model group, AEE groups were located at the opposite direction along latent

variable 2. AEE medium and high groups were on the same side of latent variable 1 and showed a trend to

be close to the control group. Therefore, the results indicated the deviations induced by HFD were

significantly improved after treatment of AEE.

The dosage of AEE made remarkably influence on blood lipids results. Metabolic patterns in three

AEE groups were evaluated by OPLS-DA approach. The score plots of AEE samples were shown in Fig.

4-7 (ESI+R2Y = 0.95Q2Y= 0.74ESI-R2Y = 0.95Q2Y= 0.89; p-value of CV-ANOVA less than 0.001).

A clear separation was observed between AEE groups. Samples in AEE high group were clustered away

from low and medium groups both in ESI+ and ESI- models, indicating that high dosage of AEE made

different effects on hyperlipidemia rats. These results were in accordance with the results of biochemical

parameters and pathological changes observation. The blood lipids indexes including TG, TCH, HDL and

LDL in high dose AEE group were the best among AEE groups. With the dose increasing, the pathologic

changes of liver tissue were more severe than in low and medium dose AEE group.

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Fig. 4-6 The score plots of the OPLS-DA model In ESI+ model, the metabolic profile of AEE treated groups
overlapped each other. AEE groups were all located at the opposite direction along latent variable 2. In ESI-
model, a clear separation was observed among AEE groups. AEE medium and high groups were on the same
side of latent variable 1 and showed a trend to be close to the control group.

Fig. 4-7 The score plots of AEE samples clear separation was observed between AEE groups.

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4.3.4 Relative Content Alteration and Pathway Analysis of Potential Biomarker

The relative content of all the potential biomarkers were calculated and the results were listed in Table

4-2. Compared with the control group, HFD made significantly influence on all potential biomarkers. The

relative contents of potential biomarker were changed significantly by AEE administration.

In order to identify and visualize the affected metabolic pathways in hyperlipidemia rats, metabonomic

pathway analysis was performed with MetPA. Based on the KEGG ID of potential biomarker, the Rattus

norvegicus (rat) as pathway library was selected; the algorithms for pathway enrichment analysis and

topological analysis were set as default. The results revealed that the disturbed pathways in response to

hyperlipidemia and AEE treatment were ether lipid metabolism, citrate cycle (TCA cycle),

glycerophospholipid metabolism, cysteine and methionine metabolism, tryptophan metabolism, arginine

and proline metabolism (Fig.4-8 and Table 4-3).

Fig. 4-8 The disturbed pathways in response to hyperlipidemia and AEE treatment groups metabolism. a: Ether
lipid metabolism, b: Citrate cycle (TCA cycle), c: Glycerophospholipid metabolism, d: Cysteine and methionine
metabolism, e: Arginine and proline metabolism, f: Tryptophan metabolism.

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Table 4-2 Potential biomarkers related to hyperlipidemia detected by UPLC-Q/TOF MS and relative content of biomarker.
No. Compound Formula RT VIP SM Relative intensities (mean SD) Pathway
Con Mod AEE L AEE M AEE H
A A A
1 LysoPC(17:0) C25H52NO7P 13.77 1.43 + 12.190.32 18.130.35 40.20 2.50.20 4.10.17 A Glycerophospholipid metabolism
A A A
2 CE(5:0) C32H54O2 3.79 1.43 + 5.00.22 17.820.34 1.50.23 1.30.18 2.90.09 Fat digestion and absorption
A A A A
3 Carnosine C9H14N4O3 3.26 1.43 + 7.20.31 15.460.27 8.20.12 5.60.26 7.40.31 Histidine metabolism
A A A A
4 LysoPC(15:0) C23H48NO7P 11.23 1.43 + 8.740.38 17.120.27 4.00.31 2.550.24 4.10.21 Glycerophospholipid metabolism
A A A A
5 Methylisocitric acid C7H10O7 8.91 1.43 + 6.300.22 15.570.10 8.20.17 6.20.31 4.30.16 Propanoate metabolism
A A A
6 L-Cystathionine C7H14N2O4S 8.91 1.35 + 20.210.18 19.620.10 5.60.19 2.50.25 4.10.11 Glycine, serine and threonine metabolism
a A A A
7 Anserine C10H16N4O3 8.91 1.32 + 17.060.20 16.430.13 3.00.19 6.70.17 4.910.18 Histidine metabolism
A A A A
8 LysoPC(P-16:0) C24H50NO6P 13.21 1.30 + 6.760.39 15.370.37 4.10.41 4.00.26 3.250.07 Glycerophospholipid metabolism
9 LysoPC(20:1(11Z)) C28H56NO7P 15.78 1.29 + 9.520.39 A 18.130.32 2.00.19 A 2.50.13 A 4.10.15 A Glycerophospholipid metabolism
A A A A
10 LysoPC(16:0) C24H50NO7P 12.42 1.25 + 5.220.28 24.490.20 0.40.09 0.250.11 0.410.12 Glycerophospholipid metabolism
A A A A
11 LysoPC(P-18:1(9Z)) C26H52NO6P 15.21 1.23 + 6.630.33 15.700.32 5.200.19 2.850.27 3.360.18 Glycerophospholipid metabolism
A A A A
12 LysoPC(18:0) C26H54NO7P 15.21 1.23 + 5.110.33 24.230.28 8.560.19 1.840.28 4.560.83 Glycerophospholipid metabolism
A A A A
13 LysoPC(P-18:0) C26H54NO6P 13.79 1.21 + 4.210.44 17.130.34 0.310.05 0.250.08 0.410.14 Glycerophospholipid metabolism
A A A A
14 LysoPC(O-18:0) C26H56NO6P 16.21 1.20 + 3.720.52 15.520.39 1.20.03 0.870.42 1.450.51 Ether lipid metabolism
A A A A
15 Docosahexaenoic acid C22H32O2 18.17 1.28 + 1.614.97 14.840.44 0.130.19 0.160.05 0.310.74 Biosynthesis of unsaturated fatty acids
A A a A A
16 Glycerophosphocholine C8H20NO6P 1.53 1.68 - 3.980.46 16.694.77 19.51.02 3.420.13 3.480.57 Glycerophospholipid metabolism
A A A A
17 L-Tryptophan C11H12N2O2 2.84 1.75 - 17.795.49 4.980.83 0.400.19 9.150.28 4.721.41 Glycine, serine and threonine metabolism
A A A A
18 L-Malic acid C4H6O5 1.72 1.75 - 5.110.69 17.330.47 191.42 1.420.13 5.720.69 Citrate cycle
19 Succinic acid C4H6O4 3.71 1.91 - 4.020.46 A 18.850.66 211.15 A 6.570.66 A 4.480.57 A Citrate cycle
A A A A
20 L-Lysine C6H14N2O2 1.35 1.62 - 4.50.45 14.80.10 16.10.52 4.220.752 1.20.60 Lysine biosynthesis
A A A A
21 Deoxycholic acid C24H40O4 11.49 1.26 - 11.290.92 1.080.892 6.120.67 7.960.53 0.460.09 Bile acid biosynthesis
A A A A
22 L-Arginine C6H14N4O2 4.99 1.33 - 3.640.73 14.40.40 11.80.85 1.360.61 1.40.62 Arginine biosynthesis
a A
SMScan model; significant difference from model group (p<0.05); significant difference from model group (p<0.01).

75
 Chapter IV

Table 4-3 Pathway analysis result with MetPA

No. Pathway name Total Expected Hits Raw p -log (P) Impact
1 Ether lipid metabolism 13 0.148 2 0.0089 4.73 0.357
2 Citrate cycle (TCA cycle) 20 0.228 2 0.0206 3.88 0.071
3 Aminoacyl-tRNA biosynthesis 67 0.765 3 0.0374 3.29 0
4 Glycerophospholipid metabolism 30 0.342 2 0.0441 3.12 0.066
5 Biotin metabolism 5 0.057 1 0.0559 2.89 0
6 Nitrogen metabolism 9 0.103 1 0.0984 2.32 0
7 Histidine metabolism 15 0.171 1 0.1589 1.84 0
8 Glyoxylate and dicarboxylate metabolism 16 0.183 1 0.1686 1.78 0
9 beta-Alanine metabolism 19 0.217 1 0.1971 1.62 0
10 Propanoate metabolism 20 0.228 1 0.2064 1.58 0
11 Butanoate metabolism 20 0.228 1 0.2064 1.58 0
12 Pyruvate metabolism 22 0.251 1 0.2247 1.49 0
13 Alanine, aspartate and glutamate metabolism 24 0.274 1 0.2425 1.42 0
14 Cysteine and methionine metabolism 28 0.320 1 0.2771 1.28 0.166
15 Glycine, serine and threonine metabolism 32 0.365 1 0.3103 1.17 0
16 Steroid biosynthesis 35 0.399 1 0.3342 1.10 0
17 Tryptophan metabolism 41 0.468 1 0.3797 0.97 0.157
18 Biosynthesis of unsaturated fatty acids 42 0.479 1 0.3870 0.95 0
19 Arginine and proline metabolism 44 0.502 1 0.4013 0.91 0.082
The total number of compounds in the pathways; the hits are the actually matched number from the user upload data; the raw p is the original p value calculated from the
enrichment analysis; the impact is the pathway impact value calculated from pathway topology analysis.

76
 Chapter IV

4.4 Discussion

Metabonomics study of metabolite composition and abundance in a biological organism, can be dated

back to ancient China, where doctors used ants to detect high glucose level in human urine, and hence

diagnose diabetes (van der Greef and Smilde, 2005). The concept that individuals might have a metabolic

profile that reflected in the makeup of their biological fluids was introduced by Roger Williams in the late

1940s who used paper chromatography to identify characteristic metabolic patterns in urine and saliva

(Williams, 1956), which were associated with a variety of subjects samples groups, including alcoholics,

schizophrenics, and residents of mental hospitals, and produced what he considered to be very suggestive

evidence that there were characteristic metabolic patterns associated with each of these groups (Gates and

Sweeley, 1978). However, these methods are largely qualitative and can detect only a few metabolites in

one experiment. In recent years, the development of new methodologies for studying pharmacological

efficacies, mechanisms and metabonomics has become a hotspot for pharmacology and pharmacodynamics

research (Kaddurah-Daouk et al., 2008).

The result of blood lipids examination and metabonomics confirmed that the hyperlipidemia disease

model was constructed successfully. Based on the UPLC-Q-TOF/MS analysis method, metabolite profiling

of serum was evaluated to provide comprehensive insights into hyperlipidemia. Meanwhile, MetPA

platform was introduced to identify the most relevant pathway and elucidate the underlying mechanism of

hyperlipidemia from the metabolic pathways analysis in a global view (Zhou et al., 2015). The

hyperlipidemia disease was induced by HFD. The biochemical indexes examination demonstrated that

administration of HFD could remarkably alter the blood lipids level. In this study, 22 potential biomarkers

were selected, which were related to hyperlipidemia. Metabolomic results from pathway analysis indicated

that endogenous metabolites in SD rats were remarkably infected by HFD administration and AEE

treatment.

Increased cholesterol intake from HFD was associated with hyperlipidemia. The rat fed with high fat

diet was widely used as hyperlipidemia disease model. The evaluation of disease model was carried out by

blood lipid levels examination including TG, TCH, HDL and LDL. The increased levels of TCH, TG and

LDL and the decreased level of HDL were observed in model group in this study, which indicated the blood

lipid profile was significantly changed by HFD and the success of the disease model. AEE treatment

significantly changed the blood lipid levels in rat including the reduction in TCHTG, LDL and increase in

HDL. The disorder of lipid metabolism played a pivotal pathogenetic role in the initiation and progression

77
 Chapter IV

of hyperlipidemia, which has been proved by numerous studies. The metabonomic results demonstrated

that HFD can markedly alter the endogenous metabolites in SD rats. The major pathways related to these

metabolites were ether lipid metabolism, TCA cycle, glycerophospholipid metabolism, cysteine and

methionine metabolism, tryptophan metabolism, arginine and proline metabolism (Reuter and Heybey,

1984, Safwat et al., 2009). The metabonomic pathways were related with lipid metabolism, amino acid

metabolism and energy metabolism (Qi et al., 2015).

Lipid Metabolism

Lysophosphatidyl cholines (LysoPCs) consist of a class of phospholipids and are formed by hydrolysis

of phosphatidyl cholines (PC). LysoPCs play key roles in lipid metabolism and the progress of many

diseases including inflammation, atherosclerosis, diabetes, cancer and blood lipid disorders. High

concentrations of LysoPCs can cause inflammation and the autoimmune response. The total LysoPCs in

serum have been reported to increase gradually in animals on a high cholesterol diet (Kim et al., 2013). In

present study, LysoPCs were significantly increased in comparison with the control rat, which was in good

agreement with previous reports. The levels of LysoPCs in hyperlipidemia rat may indicate that the changes

of LysoPCs could be relevant to the pathogenesis of hyperlipidemia. AEE treatment showed favorable

inhibition on LysoPCs, indicated that the depression of AEE on LysoPCs might contribute to lowering

blood lipids level (Ou et al., 2012).

Bile acids are physiological detergents that facilitate the excretion, absorption, and transport of fats

and sterols in the intestine and liver. Hence, bile acid plays a key role in lipid metabolism. Deoxycholic

acid is derived from the bacterial degradation of the primary bile acid, which is a cholesterol derivative

with detergent like properties. Deoxycholic acid could inhibit rate limiting enzyme HMG-CoA and the

absorption of cholesterol in gut. In model group, amount of deoxycholic acid was lower than control group.

The results suggested that HFD may increase the blood lipid levels by the inhibition of deoxycholic acid.

The amount of deoxycholic acid was increased by AEE treatment, which may make contribution to down

regulation blood lipid levels (Porsch-Ozcurumez et al., 2001).

Energy Metabolism

TCA cycle is the main pathway of glucose degradation and the primary energy supplier for universal

organisms. Succinic acid and malic acid are metabolites in TCA cycle and the most important energy

source in mammals. In this study, succinic acid and malic acid were significantly increased in the model

group compared with the control group. These result suggested that HFD may disturb the TCA cycle

78
 Chapter IV

markedly and ultimately alter energy metabolism (Li et al., 2015). Increased amount of succinic acid and

malic acid may be as a consequence of increased fatty catabolism. AEE at the dose of 36 and 54 mg/kg

could significantly reduce the amount of succinic acid and malic acid, which indicated that the energy

metabolism may be normalized by AEE treatment (Calderon-Santiago et al., 2013).

Amino acid metabolism

The liver is the hub of amino acids metabolism, and any hepatic injury might induce amino acids

metabolic disturbances. The previous study had proved that fatty liver could be induced by HFD

administration in rat, which could cause damage to liver structure and function (Long et al., 2015). The

study found that amino acid metabolism was affected in the HFD group, leading to changes in the levels of

many amino acids, including L-lysine, L-arginine and L- tryptophan. In the study, the levels of L-lysine and

L-arginine were increased and L- tryptophan was decreased in model group. These results may be related to

disorder function of liver caused by HFD and the perturbation of energy metabolism. The trend of amino

acid was changed by AEE treatment. In addition, the alteration of amino acid was frequently reported to be

related to modulation of the activity or population of gut microbiota (Kurowska and Carroll, 1994). Further

investigation about AEE effects on gut microbiota should be performed in further studies.

In this study, an UPLC-Q/TOF MS-based metabonomic was carried out to characterize the global

plasma metabolic profile associated with hyperlipidemia in rat fed with HFD. 22 metabolites involving into

the alteration of energy metabolism, lipid metabolism and amino acid metabolism were identified as

potential biomarkers related to hyperlipidemia. Among them, lipid pathways played vital roles (Liu et al.,

2011a). With the metabonomic method, the therapeutic effects of AEE were delineated in this study.

Treatment of AEE could effectively change blood lipid levels and inhibit the metabolic alternations induced

by hyperlipidemia. The findings suggested that hyperlipidemia induced by HFD may lead to perturbation of

LysoPCs, bile acids, TCA cycle and amino acids, which might be the pharmacological basis of AEE against

hyperlipidemia.

This result may explain the good effect of AEE on hyperlipidemia. Further studies should be

conducted to investigate the action mechanism of AEE.

79
 Conclusion

Conclusion
1. Hyperlipidemia was constructed successfully in rats by feeding with high fat diet. Significant

increases on blood lipid indexes TG, TC and LDL were confirmed by meatbonomics study and

significant fatty change among the hyperlipidemia groups on hepatic tissue by histopathology.

2. The high fat diet had a remarkable influence on body weight. AEE can reduce the increasing rate

of body weight in the rats significantly.

3. The preventive effects of AEE on hyperlipidemia in SD male rats were confirmed in the

experiment. AEE at dose 54 mg/kg/day for six weeks decreased TG, TC and LDL significantly

(p<0.01).

4. AEE had better effect on curing hyperlipidemia than its component. The optimal dose of AEE to

cure hyperlipidemia was 54 mg/kg/day.

5. In cure and preventing hyperlipidemia, AEE had better effects than statin under the present

condition of the study.

6. Since statin showed remarkable reduction in fatty droplets on hepatic tissue than AEE, the

mechanism of action between AEE and statin may be quite different.

7. The drugs were safe for use on experimental animals for long period in all sections from stomach

and intestine.

8. AEE treatment against hyperlipidemia may involve in regulating the lipid metabolism, amino acid

metabolism and energy metabolism.

80
 Reference

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 Acknowledgements

Acknowledgements
First and foremost, I would like to express my ever grateful to Almighty God. Thanks to God for

giving me the strength needed to conduct the present study to continue and be a positive person to

successfully achieve my goal.

I am grateful to China Scholarship Council (CSC) and Graduate School of Chinese Academy of

Agricultural Science (GSCAAS) and Lanzhou Institute of Husbandry and Pharmaceutical Sciences CAAS

for full scholarship support for Ph. D study.

I would like to express my deep gratitude to my supervisor, Professor Li Jian Yong. His wide

knowledge, logical way of thinking and personal guidance has provided a good basis for the present thesis,

great thanks to his team work specially Ya Jun Yang, Xi Wang Liu and Dr. Qin Zhe for their valuable

suggestions and helpful criticism until the end of this investigation, for providing me a good environment

for experimental work.

My thanks are extended to the staff of Lanzhou institute of husbandry and pharmaceutical science of

CAAS and I am deeply grateful to my colleague Ma Ning for considerable help, valuable advices and

continuous encouragement.

The words of gratitude to International Education Office of GSCAAS for good treatment, facilitate

and cooperation that I have received during my study in China.

I would like to acknowledge the financial support of National Natural Science Foundation of China

(31402254), Science and Technology Foundation for Youth of Gansu Province (1506RJYA148).

I would also like to acknowledge the support of Sudan government specially the ministry of livestock

& fisheries and range lands (Animal Resources Research Corporation ARRC), for their help to facilitate my

nomination, agree and their supports to join the study in China and complete my Ph.D. study here.

I am grateful to Prof. Ahmed Abd Elrhaeem Gameel and Dr. Hala Ali in Department of Pathology

Faculty of Veterinary Medicine Sudan University of Science and Technology.

I am deeply grateful to my wife and my kids for continuous support, encouragement and

understanding.

I express my heartiest thanks to my father, mother and all who helped directly and indirectly for

building-up my career.

95
 Author Biography

Author Biography
Personal data

Name: Isameldin Suliman Mohamed Karam

Sex: Male
Address: Lanzhou Institute of Husbandry and Pharmaceutical Science of CAAS

E-mail: isamkarm@yahoo.com

Tel.: +8618294414277.

Date of Birth: 11/07/1974.

Place of Birth: Sudan.

Nationality: Sudanese.

Marital Status: Married.

Education: Qualification
2013-2016 Appeared Ph. D studies in Veterinary pharmacology in Lanzhou Institute of

Husbandry and Pharmaceutical Science of Chinese Academy of Agricultural

Science, China.

2002-2005 I was granted master MSc. degree in Vet. Microbiology entitled Studies on

Contagious Bovine Pleuropneumonia in Khartoum state-Sudan. Faculty of

Veterinary Medicine, University of Khartoum Sudan

1993-1998 Awarded bachelor degree BSc. Faculty of veterinary medicine, University of

Khartoum Sudan

Work: Employments history

2014- Researcher at Central Laboratory of Veterinary Research, Soba (Animal Resources

Research Corporation ARRC Sudan).

2005-2013 Working in the Ministry of Livestock & fisheries and range lands, Khartoum, Sudan.

1998-2005 During this period I worked as a supervisor for private animal farms, Khartoum,

Sudan.

Publication:

I published paper entitled: Regulation effect of Aspirin Eugenol Ester on blood lipids in Wistar rats

with hyperlipidaemia. BMC veterinary research.2015

96
 Author Biography

Training Courses

Attended a training course of 2 weeks entitled embryo transfer, Umbaba Veterinary Institute during

2007, Cairo Egypt

Workshop on the Control of Contagious Bovine Pleuropneumonia CBPP Khartoum, Sudan PACE

Program in the Sudan July, 2003

Countries visited:

Egypt, Saudi Arabia, Ukraine, Turkey, Qatar, China.

Society membership

A member of the Sudanese Veterinary Association

A member of the Sudanese Veterinary Council

97