CHAPTER I

INTRODUCTION

Background of the study

Hyperlipidemic diseases are responsible for the death of millions of people each year.

There are large numbers of epidemiological and experimental evidence pertaining to worldwide

acute hyperlipidemia disease.

Lipids performs important functions in the body, but if it is present in excess it can cause

health problems like hyperlipidemia. Hyperlipidemia is elevation of one or more fat proteins in

the blood. It is commonly referred to as high cholesterol.

Nowadays, since hyperlipidemia does not cause any symptoms it is one of the reasons of

mortality in the Philippines; Filipinos are fan of eating fatty foods without knowing the

disadvantages of it because it can significantly increase the risk of developing cardiovascular

disease, coronary artery disease, cerebrovascular disease and peripheral vascular disease.

There are some plants that can help lower cholesterol levels in the body. They are being used as

alternative medicine because in the past, people only depend on plants and used for medicinal

purposes.

Ariwat is a woody vine growing to a length of 10 meters. Stems are somewhat compressed, woody,

rough, and 1 to 2.5 centimeters in diameter. Leaves are usually 3 to 5, but sometimes 7 leaflets.

Leaflets are elliptic-oblong, 5 to 12 centimeters long, coarsely toothed, smooth and shining.

Flowers are pale green, faintly fragrant, numerous, umbellately disposed on the ultimate branches.

Fruit is smooth, brownish, rounded, fleshy, and about 1.5 centimeters in diameter.

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 In view of this, the present study will carry

out to determine the anti-hyperlipidemic activity of Ariwat (Tetrastigma harmandii Planch) leaf

ethanolic extract in induced albino Rats (Rattus).

Objectives

Generally, this study aimed to assess the Anti-hyperlipidemic activity of Ariwat

(Tetrastigma harmandii Planch) leaf ethanolic extract in induced Albino Rats (Rattus).

Specifically, the study aimed to:

1. Determine the efficiency of simvastatin on hyperlipidemic rats

a. 20mg/kg/day

2. Determine if there is a significant difference of the lipid profile of hypelipidemic rats with

Ariwat (Tetrastigma harmandii Planch) leaf ethanolic extract in different parameters:

a. Total cholesterol level (TC)

b. Triglyceride level (T)

c. Very low density lipoprotein level (VLDL)

d. Low density lipoprotein level (LDL)

e. High density lipoprotein level (HDL)

Significance of the Study

This study will provide information regarding the anti-hyperlipidemic activity of Ariwat

(Tetrastigma harmandii Planch) leaf ethanolic extract in induced albino rats. The findings of this

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study will offer additional information on the growing interest on herbal plants that possesses anti-

hyperlipidemic effect. The study can also be used as a basis for further developments.

Pharmaceutical Industry. The results of the study may serve as a basis in making

medicine especially antihyperlipidemic drugs which is of natural product origin.

The Medical Community. The information may help the medical community to

counteract the exorbitant prices of medicine. People who can’t afford the prices of commercial

drugs and those who are living in places where medicine is not available may benefit from this

study greatly.

Future Researcher. The study may serve as their reference in doing their research work.

It can serve as a basis or guide in conducting their study.

Scope and Delimitation of the Study

This study is intended only for the determination and provision of the anti-hyperlipidemic

activity of Ariwat (Tetrastigma harmandii Planch.) leaf ethanolic extract in Pig brain fed Albino

Rats.

Time and Locale of the Study

Collection of Ariwat (Tetrastigma harmandii Planch.) leaves will be done in Imurung,

Baggao, Cagayan. The experiment method will be held at the Philippine Institute of Traditional

and Alternative Health Care (PITAHC) Tuguegarao City for the plant extraction, high fat diet

preparation and anti-hyperlipidemic test. This study will be conducted on January (2017) - April

(2017).

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 CHAPTER II

REVIEW OF RELATED LITERATURE

Overview on hyperlipidemia

Hyperlipidemia is abnormally evaluated levels of any or all lipid and/or lipoproteins in

the blood. It is the most common form of dyslipidemia (which includes any abnormal lipid levels).

Lipids (water-insoluble molecules) are transported in a protein capsule. The size of that capsule,

or lipoprotein, determines its density. The lipoprotein density and type of apolipoproteins it

contains determines the fate of the particle and its influence on metabolism. Hyperlipidemias are

divided into primary and secondary subtypes. Primary hyperlipidemia is usually due to genetic

causes (such as a mutation in a receptor protein), while secondary hyperlipidemia arises due to

other underlying causes such as diabetes. 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. In addition, some forms may predispose to acute pancreatitis.

(en.wikipedia.org)

Hypolipidemic agents or antihyperlipidemic agents are a diverse group of pharmaceuticals

that are used in the treatment of high levels of fats (lipids), such as cholesterol, in the blood

(hyperlipidemia). They are called lipid-lowering drugs. (en.wikipedia.org/wiki/Lipid-lowering

agent).

Hyperlipidemia in general has no apparent symptoms and can only be discovered and

diagnosed during routine examination or evaluation for atherosclerotic cardiovascular disease.

However, deposits of cholesterol may be formed under the skin in individuals with familial form

of disorder or in persons with very high levels of cholesterol in the blood. In individuals with

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hypertriglyceridemia, several pimple-like lesions may be developed across their bodies.

Pancreatitis, a severe inflammation of the pancreas that may be life-threatening can be also be

developed due to extremely high levels of triglycerides. (www.healthscout.com).

Hyperlipidemia is a mouthful, but it’s really a fancy word for too many lipid or fats in the

blood. That can cover many conditions, but for most people, it comes down to two well-known

terms: high cholesterol and high triglycerides. Our bodies make and use a certain amount of

cholesterol every day, but sometimes that system gets out of whack, either through genetics or

diet. Higher levels of the “good” HDL cholesterol are associated with decreased risk of heart

disease and stroke. HDL helps by removing cholesterol from your arteries, which slows the

development of plaque. The “bad” LDL cholesterol, on the other hand, can lead to blockages if

there’s too much in the body. (www.heart.org)

According to Kelly (2010), the abnormal cholesterol levels are the result of an unhealthy

lifestyle including taking high-fat diet and other lifestyle factors like being overweight, heavy

alcohol used and lack of exercise. Moreover, diabetes and underactive thyroid gland have also

been reported to cause high cholesterol levels. (Tamer et al., 2011; Saeed et al., 201. Other illnesses

that may elevate cholesterol levels include polycystic ovary syndrome and kidney disease. The

higher levels of female hormones like estrogen, have been noted to increase or change cholesterol

levels. In addition, drugs like diuretics, beta-blockers and medicines used to treat depression have

also been reported to raise cholesterol levels.

A Brief History of Herbs

The use of plants as medicines dates as far back as the origin of humankind. Even

carnivorous animals are known to consume plants when ill. (A Jaguar for example, will eat leaves

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after grooming as a remedy against furballs. Everyone has seen a dog or cat eat grass, which they

may do to relieve gastric distress or to dislodge parasites.)

Since the beginning of humankind people have relied primarily on plants for nourishment.

Through trial and error they discovered that some plants are good for food, that some are

poisonous, and that some produce bodily changes such as increased perspiration, bowel movement,

urination, relief of pain, hallucination, and healing. Over the millennia these observations were

passed orally from generation to generation, with each generation adding to and refining the body

of knowledge. Every culture the world over has in this manner developed a body of herbal

knowledge as part of its tradition. (www.naturalhealthschool.com)

Herbs and plants can be processed and can be taken in different ways and forms, and they

include the whole herb, teas, syrup, essential oils, ointments, salves, rubs, capsules, and tablets that

contain a ground or powdered form of a raw herb or its dried extract. Plants and herbs extract vary

in the solvent used for extraction, temperature, and extraction time, and include alcoholic extracts

(tinctures), vinegars (acetic acid extracts), hot water extract (tisanes), long-term boiled extract,

usually roots or bark (decoctions), and cold infusion of plants (macerates). There is no

standardization, and components of an herbal extract or a product are likely to vary significantly

between batches and producers. (www.ncbi.nlm.nih.gov)

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Information of Tetrastigma harmandii Plach.

Kingdom: Plantae

(unranked): Angiosperms

(unranked): Eudicots

(unranked): Rosids

Order: Vitales

Family: Vitaceae

Genus: Tetrastigma
Species:
Harmandii Planch

Tetrastigma is a genus of plants in the grape family, Vitaceae. The plants are vines that

climb with tendrils and have palmately compound leaves. The species are found in subtropical

and tropical regions of Asia, Malesia, and Australia, where they grow in primary

rainforest, gallery forest and monsoon forest and moister woodland. Species of this genus are

notable as being the sole hosts of parasitic plants in the family Rafflesiaceae, one of

which, Rafflesia arnoldii, produces the largest flower in the world.

Ayo is a climbing shrub producing stems up to 10 meters long. The stems are 10 - 25mm

in diameter and climb into the surrounding vegetation for support, attaching themselves by

means of tendrils.

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 The plant is found in thickets at low and medium altitudes in Cagayan, Apayao, Abra,

Lepanto, La Union, Nueva Viscaya, Bataan, Bulacan, Rizal, Laguna, and Quezon Provinces in

Luzon; and in Mindoro and Panay.

The plant is harvested from the wild and used locally as a source of food, medicines and fibre .

Fruits are sour but edible. Similar in flavour to the muscadine grape (Vitis rotundifolia). Made

into jellies, and A decoction of the plant is used internally as a powerful diuretic.

The decoction is applied to the skin as a treatment against scabis.

CHAPTER III

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 METHODS AND MATERIALS

Materials

The following materials will be used in the study: capillary micro tubes, filter paper, syringes,

water bath, vacuo, vial, rotary evaporator, butter, coconut oil, rat chow pellets, simvastatin 20mg

and Ariwat leaves.

The following reagents were utilized: distilled water and 80% ethanol, normal saline

solution.

Experimental Design and Treatments

The experimental rats will randomly distributed employing completely randomized design with

five treatments. Each group will be replicate with 4 mice.

The following treatments that will be used to conduct the study are:

T1- Normal Control

T2- Hyperlipidemic Control

T3-Positive Control – Hyperlipidemic rats. Treated with simvastatin 1.8mg/kg body weight

T4- Hyperlipidemic rats treated with Ariwat (Tetrastigma harmandii Planch) leaf ethanolic extract

400mg/kg body weight

T5- Hyperlipidemic rats treated with Ariwat (Tetrastigma harmandii Planch) leaf ethanolic extract

500mg/kg body weight

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Plant material

Ariwat (Tetrastigma harmandii Planch.) will be collected at Imurung Baggao, Cagayan.

The plant will be cleaned with distilled water and will air dried for 7 days under normal

environmental condition.

Preparation of the Extract

Ariwat (Tetrastigma harmandii Planch) leaves will be air dried for seven (7) days in

preparation for the extraction. Air dried plant will be soaked in 80% ethanol for 48 hours. Using a

filtered paper, the residue will be separated and discarded from the filtrate. The filtrate will be

concentrated under vacuo using a water bath. Flame test will be used to determine the presence of

alcohol in the extract. The volume of the concentrated extract will be recorded as well as the

concentration of the extract as to the grams of the dried plant material per ml of the extract will be

obtained. The vial will properly labeled and refrigerated until used. 50% of the pure extract will

also prepared by adding 10 ml of distilled water to the 10 ml of the extract. (Guevarra)

Experimental Animals

Adult Male albino Rats about 2 months old weighing about 180-200g, will be used in the

study. It will be obtained from the Food and Drug Administration Philippines (FDA Phils.), and

will be kept at the Philippine Institute of Traditional and Alternative Health Care (PITAHC)

Tuguegarao City.

Animal Test Preparation:

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 Rats will be housed in clean steel cages, four rats in each cage, in a controlled environment

(24-260C) with a 12-hour light and 12-hour darkness cycle and fed with normal rat chow and water

ad libitum. The rats are allowed to acclimatize to these conditions for one week. After the

adaptation period, rats will be weighed, recorded, numbered and randomly assigned to five groups

of four rats each. For 31 days, rats will be fed and induced orally by gavage with high fat diet

except for the control group. The animals will not be administered with any test substance during

these days. This will be done to ensure that the rats become hyperlipidemic before the initiation of

the treatment. Subsequently, after the initiation of hyperlipidemia the animals will be treated for

14 days. The Ariwat (Tetrastigma harmandii Planch) leaf ethanolic extract and simvastatin will

be administered once daily as a single oral suspension. All the experimental procedure will be

carried out between 8 am to 5 pm. The test substance will be administered between 9:00 am to

10:00 am. The animals will be maintained as per the Institutional Animal Ethical Committee

guidelines.

Method of Preparation of High Fat Diet:

Edible coconut oil and butter will procured from the market and a mixture of the two is

prepared in a ratio of 2: 3 respectively v/v as per method of Shyamala et al. This high fat diet at a

dose of 10 ml/kg body weight is fed to the animals per orally daily in addition to the high fat pellets

that were also given to rats 10g daily which is a mixture of butter and pellets at a ratio of 10:90.

These diets will be given to rats for the experimental period of 31 days to produce hyperlipidemia.

ssssss

Method of Preparation of Simvastatin Suspension:

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 The stock solution is prepared by dissolving 20 mg of simvastatin in 70 ml of normal saline

and used as a standard drug in a dose of 1.8 mg/kg body weight for the respective group. The daily

dose of simvastatin for mice was calculated by extrapolation from the human dose (20 mg/day) as

described by Ghosh.

Blood Sample Collection

Blood sample collection from rats will be done following the hot compress procedure due

to the cold weather. First, rats is covered with cloth to protect them from too much heat. Then,

they were placed above the hot compress bag (2-3 minutes) to bring back their normal temperature

and to dilate their blood vessels. After which, .5ml of blood samples were extracted through the

tail vein of twelve hours fasted mice by wounding their tail. This blood sample collection was

conducted after the 31th day of hyperlipidemia induction for the initial findings and after the 14 th

day of treatment for the final results.

Biochemical Estimations

The blood samples will be collected in capillary micro test tubes. The serum obtained from

blood will be used for the estimation of the serum lipid profile like total cholesterol, triglyceride,

HDL‐cholesterol, LDL-cholesterol, and VLDL-cholesterol.

Statistical analysis
Quantitative data were expressed as the geometric mean from a log transformed lipid values. The

statistical analysis was carried out using SPSS Statistics 17.0 software (SPSS Inc., Chicago, IL,

USA). Statistical differences between control group and other groups were compared using the

Tukey kramer test for P-value calculation. All P-values lower than 0.05 were considered

significant.

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 Chapter 4
Results and Discussion
Antihyperlipidemic activity of Ariwat
The lipid profile of rats fed pig brain extract was evaluated for the activity of ariwat
extract. The results of the antihyperlipidemic medication is shown in table 1. Compared activity
for triglycerides, LDL and VLDL showed mean levels were not significantly different between
treatments (figure 2, 4 and 5; appendix table 3, 5 and 6 with a p value of 0.49, 0.204 and 0.906
). And, compared with the negative control, the untreated rats, shows significantly low in total
cholesterol and HDL levels (figure 1 and 3; appendix table 2 and 3 with a p value of 0.035 and
0.001). Administration with 5g/kg of the ariwat extract have the strongest activity of the
experimental group though the activity was not adequate to reduce the levels compared to the
standards of the hyperlipidemic group pig brain fed rats.
Table 1. Total cholesterol Triglycerides, HDL, LDL and VLDL mmol/L to the control group (1)
Negative Control, (2) Hyperlipidemic Control (3) Simvastatin (4) 5g/Kg Of Ariwat (5) 10g/Kg
Of Ariwat extract
=====================================================================
Treatments Total Triglycerides, HDL LDL VLDL
cholesterol

1 1.611 a 0.6093a 0.5619a 1.0251a 0.2771a

2 3.9536 b 1.5275a 3.3410b 0.8020a 0.3775a
3 2.3736b 0.6119a 2.8580b 0.8241a 0.2803a
4 2.3081b 0.7592a 1.7348b 0.7903a 0.3435a
5 2.9735b 0.8633a 2.2027b 0.8832a 0.4167a
Distinct letters at the same column indicate statistically significant difference p ≤ 0.05
Use as food and medicine, the plant extract showed minor effect to lipid metabolism by
unknown mechanism. The biosynthesis of cholesterol requires liver enzyme cytochrome p450
in the process and drugs used to inhibit the function of these enzymes blocks the accumulation
of cholesterol in the blood (Cholesterol: Synthesis, Metabolism, Regulation, 2017).

Figure 1. Mean plot of the total cholesterol (mmol) levels of rats

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Figure 2. Mean plot of the triglycerides (mmol) levels of rats

Figure 3. Mean plot of the HDL (mmol) levels of rats

Figure 4. Mean plot of the LDL (mmol) levels of rats

Figure 5. Mean plot of the VLDL (mmol) levels of rats

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 CHAPTER 5
SUMMARY, CONCLUSION AND RECOMMENDATION
Summary
The study was conducted to determine the activity of ariwat extract (Tetrastigma
harmandii Planch) in pig brain fed induced hyperlipidemic rats. Using 2 experimental group
medicated with the extract for 5 and 10 ml/kg and 3 control group (1-negative control, 2
hyperlipidemic pig fed rats and 3-hyperlipidemic pig brain rats medicated with simvastatin were
used in the study. The force gavage feeding of the brain extract to rats by gavage was done in 7
days. Medication with the plant extract was done in 3 days. Lipid profile for total cholesterol,
triglycerides, Hdl , LDl, and VLDl were evaluated from blood extracted from tail vein thereafter
treatment. Results showed a mild anti-hyperlipidemia (total cholesterol and HDL) activity of the
extract with both the extract not significantly different against the hyperlipidemic control rats ( p
value > 0.05.
Conclusion
The plant extract possess mild activity in the control of total cholesterol and HDL levels in
pig fed rat induced hyperlipidemic model. The study also concludes that feeding of pig brain did
not affect the lipid in triglycerides, LDL and VLDL levels of rats.
Recommendation
The ariwat plant is recommended as a mild total cholesterol inhibitor based the graphical
presentation of effect .

Reference

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Tetrastigma harmandii - Useful Tropical Plants
http://tropical.theferns.info/viewtropical.php?id=Tetrastigma+harmandii
Cholesterol: Synthesis, Metabolism, Regulation, 2017 .
https://themedicalbiochemistrypage.org/cholesterol.php

Appendix Table 1. Total cholesterol Triglycerides, HDL, LDL and VLDL mmol/L to the control
group (1) Negative Control, (2) Hyperlipidemic Control (3) Simvastatin (4) 5g/Kg Of Ariwat (5)
10g/Kg Of Ariwat extract
1 Total Triglycerides, HDL LDL √ x+0.5 VLDL
cholesterol

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 1 1.07 0.51 0.72 0.12 0.79 0.23
2 1.82 0.42 1.42 0.21 0.85 0.19
3 1.86 0.99 0.25 1.16 1.29 0.45
4 1.86 0.65 0.39 1.17 1.29 0.30
2
1 4.68 1.45 3.72 0.19 0.83 0.46
2 4.59 2.41 3.85 0.10 0.77 0.64
3 2.67 O.34 2.41 0.11 0.78 0.15
4 4.26 1.02 3.61 0.19 0.83 0.46
3
1 3.91 0.63 3.42 0.20 0.84 0.29
2 2.65 1.23 1.86 0.10 0.77 0.56
3 4.79 0.43 4.56 0.22 0.85 0.20
4 2.61 0.42 2.30 0.16 0.81 0.19
4
1 1.32 0.20 1.23 0.00 0.71 0.09
2 2.65 0.46 2.40 0.00 0.71 0.21
3 4.69 2.96 2.60 0.69 1.09 1.34
4 1.73 1.22 1.18 0.00 0.71 0.55
5
1 2.20 1.38 1.41 0.16 0.81 0.63
2 1.95 0.49 1.53 0.24 0.86 0.22
3 4.59 1.91 3.41 0.21 0.85 0.87
4 3.97 0.43 3.20 0.58 1.04 0.25

Appendix Table 2. ANOVA of the total cholesterol (mmol) to the different treatments

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Appendix Table 3. ANOVA of the triglycerides (mmol) to the different treatments

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Appendix Table 4. ANOVA of the HDL(mmol) to the different treatments

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Appendix Table 5. ANOVA of the LDL(mmol) to the different treatments

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Appendix Table6. ANOVA of the VLDL(mmol) to the different treatments

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