KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY

COLLEGE OF HEALTH SCIENCES

FACULTY OF PHARMACY AND PHARMACEUTICAL SCIENCES

DEPARTMENT OF PHARMACOGNOSY

DETERMINATION OF THE HYPOTENSIVE EFFECT OF ETHANOLIC EXTRACT

OF LAUNEA TARAXACIFOLIA.

A DISSERTATION SUBMITTED TO THE DEPARTMENT OF PHARMACOGNOSY

IN PARTIAL FUFILMENT OF THE REQUIREMENTS FOR THE AWARD OF A
DEGREE IN DOCTOR OF PHARMACY, KNUST.

BY AGYEMAN AYITEY EMMANUEL

MAY 2020.

i
 KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY

COLLEGE OF HEALTH SCIENCES

FACULTY OF PHARMACY AND PHARMACEUTICAL SCIENCES

DEPARTMENT OF PHARMACOGNOSY

DETERMINATION OF THE HYPOTENSIVE EFFECT OF ETHANOLIC EXTRACT

OF LAUNEA TARAXACIFOLIA.

A DISSERTATION SUBMITTED TO THE DEPARTMENT OF PHARMACOGNOSY

IN PARTIAL FUFILMENT OF THE REQUIREMENTS FOR THE AWARD OF A
DEGREE IN DOCTOR OF PHARMACY, KNUST.

BY AGYEMAN AYITEY EMMANUEL

MAY 2020.

ii
 STUDENT’S DECLARATION
I declare that this thesis report was personally written by me under the supervision; it has not
been practically or wholly presented by anybody for the award of Doctor of Pharmacy Degree in
any department or institution. I therefore, take responsibility for any error and omission found in
it. However, all cited references have been acknowledged. This is therefore the original copy of
my work.

19-06-2020 ………………………………………
DATE AGYEMAN AYITEY EMMANUEL
(STUDENT)

SUPERVISOR’S DECLARATION
I declare that I have supervised this student in carrying out this thesis and he has my permission
to present this report for assessment.

………………………… ……………………………….
Date DR. KOMLAGA GUSTAV.
(Supervisor)

iii
DEDICATION.

I dedicate this project to the Almighty God for His guidance, protection and provision. I also
dedicate this work to my parents as a thank you for all the things they’ve done for me throughout
my academic ladder. Not forgetting my loved ones for their resolute love and support.

iv
ACKNOLEDGEMENT.

Primarily, I thank God for being able to complete this project with success. I would like to express
special thanks to my project supervisor, Dr. Gustav Komlaga for his continuous support,
encouragement and guidance during the time of research and writing of this thesis. I also thank all
lecturers, demonstrators (Yakubu) and laboratory technicians of both the Pharmacognosy and
Pharmacology departments. I’m grateful to all my friends who showed help and support in diverse
ways to make this work a success. I appreciate you all.

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ABSTRACT.

Hypertension is a major cause of many cardiovascular disorders. It underlines various

complications and is prevalent in many parts of the world. Many compounds are being researched
and used in the management of hypertension. Launea taraxacifolia is a perennial plant found
mainly in the highlands of Ethiopia and other African countries as well. This plant is recognized
for its traditional uses. In this study, the hypotensive effect of Launea taraxacifolia is experimented
both in-vitro and in-vivo. In doing this, the phytochemicals in the plant were determined and
various concentrations of its extract were made to investigate its hypotensive effect. An
anesthetized cat was subjected to varying concentrations of the extract via the femoral vein and
the heart rate, force of contraction and mean arterial pressure measured accordingly. Similar
concentrations were administered to a frog’s heart in in-vitro studies of the plant extract’s
hypotensive effect.

It was discovered that Launea taraxacifolia has a dose-dependent effect of reducing force of
contraction and mean arterial pressure in anesthetized cat. It is therefore recommended that further
studies are made to develop and validate the use of Launea taraxacifolia in management of
hypertension.

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TABLE OF CONTENT
Content
DECLARATION……………………………………………………………...............................iii
DEDICATION……………………………………………………………………………………iv
ACKNOWLEDEMENT………………………………………………………………………….v
ABSTRACT………………………………………………………………………………….......vi
TABLE OF CONTENT………………………………………………………………………….vii
CHAPTER ONE: …….………...…………………………………………………………………1
1.1 Introduction ………………...…………………………………………………………………1
1.2 Justification …………………...………………………………………………………………2
1.3 Aims and Objectives ...………………………………………………………………………..3
CHAPTER TWO: Literature Review
2.1 Launea Taraxacifolia ….………………………………………………………………………4
2.1.1 General Description …………………………………………………………………………4
2.1.2 Ethnomedicinal Uses ……….……………………………………………………………….5
2.1.3 Chemical Constituent ...…………………………………………………….….……………6
2.2 Hypertension ……………………………………………………………………………...…..7
2.2.1 Pathophysiology …….….…………………………………………………………………..8
2.3 Antihypertensives …………………………………………………………………………….9
2.3.1 Diuretics …………………………………………….….…………………………………..9
2.3.2 Adrenergic Inhibitors ………………………………………………………………………10
2.3.3 Vasodilators ………...……………………………………………………………………...11
CHAPTER THREE: MATERIALS AND METHODS
3.1 Collection and Authentication of Plant Material …………….………..…………………….13
3.2 Processing and Extraction of Plant Material….……………………………………………...13
3.3 Animals and Maintenance…………………...……………………………………………….13
3.4 Apparatus and Equipment……………………………………………………………………13
3.5 Chemicals and Drugs…………………..…………………………………………………….14
3.6 Apparatus for Determination of cat heart rate…………………………...…………………..14
3.7 Organoleptic Examination…………...………………………………………………………14

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3.8 Phytochemical Analysis.……………….……………………………………………………14
3.8.1 Test for Alkaloids….………………………………………………………………………14
3.8.2 Test for Flavonoids ……..…………………………………………………………………15
3.8.3 Test for Saponins………….……………………………………………………………….15
3.8.4 Test for Terpenoids…..……………………………………………………………………15
3.8.5 Test for Tannins…..……………………………………………………………………….15
3.8.6 Test for Coumarins…..……………………………………………………………………15
3.8.7 Test sample preparation…..……………………………………………………………….15
3.9 Experimental design………..……………………………………………………………….16
3.9.1 Cat model…………….…..………………………………………………………………..16
3.9.1 Frog model…………….…..………………………………………………………………16
CHAPTER FOUR: RESULTS.
4.0 Results……………………………………………………………………………………….17
4.1 Organoleptic Examination of extract…….………………………………………………….17
4.2 Summary of Phytochemical test.............................................................................................17
4.3 Master Formula ………………….………………………………………………………….17
4.4 Effects of formulations on heart of cat……………...……………………………………….18
4.4.1Mean Arterial Pressure (in-vivo)……………………………………………...……………19
4.4.2 Force of contraction (in-vivo) ………………………………………………………….......20
4.4.3 Heart rate (in-vivo) ………………….…….……………………………………………….21
4.5 Maximum contractions from kymogram……………...………..…………………………….22
4.5.1 Heart rate (in-vitro)……………………………….…………………………...……………23
4.5.2 Force of contraction (in-vitro) ………………………………………………………….......24
4.5.3 Figure 1. (Tracings from experiment) ………………………………………………….......25
CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATIONS
5.0 Discussion….……………………………………………………………….………………..26
5.1 Conclusion……………..……………………………………………….…………………….27
5.2 Recommendations……………………………………………………………………………28
REFERENCES

viii
CHAPTER ONE.

1.1. INTRODUCTION.

Herbal medicines are plant derived products that are traditionally or globally used in treating
diseases.(Tilburt & Kaptchuk, 2008). Herbal medicines are now being integrated into health care
systems because of its increasing recognition. Across the globe, there has been an upsurge of the
sale and use of herbal medicines.

Markets, both local and international are increasing quick even up to an estimate of US$62 billion
per annum. Also, the patronage of herbal medicine has increased worldwide because it is safe,
easy and cheap to use.(Akobundu & Agyarkwa, 1998)

Personal preferences for herbal medicines exhibited amongst Asians and Africans have also
contributed to the increasing use of herbal medicines.

Herbal medicine, as the name may suggest, is not solely made from herbs. It considers all
biologically active natural products such as herbs, fungi, bee products, insects, animal parts,
minerals (shells, kaolin, bentonite).

Herbal medicines elicit toxic and efficacious effects due to the secondary metabolites they possess.
These are responsible for their activities.

The activity outcome of herbal medicines is affected by their method of preparation, route of
administration and dosage forms. Most herbal medicines come in the form of infusions,
decoctions, beverages and powders. Some commercial herbal medicinal products are commonly
seen as capsules and creams which enhances accurate dosing, compliance and the aesthetic value.
(Burkill et al, 1985)

The collection and processing of herbal products expose them to various contaminants which is a
setback in its safety. Due to less standardization of herbal medicines, they present with some
undesirable effects making their use questionable.(Michael Buenor Adinortey et al., 2018)

Generally, herbal medicines are used for both curing and managing diseases and for this reason
research into these herbs for authentication and commercial use is required.

1
Hypertension is a common disease with an estimate of 1.13 billion people in the world living with
it. Most of these people are living in developing countries. Hypertension related complications are
increasing in Sub-Saharan Africa. Traditional herbal medicine use is high among adults in the Sub-
Saharan Africa with a prevalence ranging from 38.5%-90%. [ WHO, 2011]

Even with an upsurge in the usage of herbal medicines, very few of such herbs have been
authenticated for commercial use. A lot of them still remain untapped and unauthorized for
commercial use. A thorough search on these herbal medicines tend to validate their acclaimed
benefits and encourages their use. The reason for this and many other studies on the use of herbal
medicines.

Rural areas readily have access to more herbal medicines than orthodox medications. This fosters
the use and availability of herbal medicines for which research and authentication is required.

This research aims to determine the effects of Launea taraxacifolia also known as wild lettuce or
dandelion leaves on the heart. Launea taraxacifolia has diverse and important medicinal uses for
which it has gained popularity. It is known to be used in sickle cell anemia management – a survey
in Southern Nigeria. (Bello et al., 2018) Some also employ it directly to skin for germ eradication.

In Ghana, Gas and Ashantis are noted for using the sap to treat pain in wounds. It is known to be
used against general body weakness, dysentery, high blood pressure, malaria and to treat ulcers in
Benin. (Burkill, 1985.) It is also reported to have hypolipidemic properties for management of
water retention disorders.

L. taraxacifolia also contains many nutritional active constituents that make it perfect for both
hunger and disease alleviation in most developing countries.(M. B. Adinortey et al., 2012)

1.2 JUSTIFICATION.

• Plant preparations indicated for managing hypertension are available on the market with
most of them being liquid preparations and easy to administer.
• Increasing demand for herbal preparations which prove to be potent with less side effects.

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 • More antihypertensives being available on the market as herbal preparations is desired.

1.3 AIM AND OBJECTIVES.

1.3.1 AIM.

To determine the hypotensive effect of the ethanolic extract of Launea taraxacifolia.

1.3.2 OBJECTIVES.
• To prepare ethanolic extract of the plant material.
• To carry out phytochemical screening
• To determine the effect of the extract on the hearts of cats and frogs.

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CHAPTER 2.

2.0 LITERATURE REVIEW

2.1 LAUNEA TARAXACIFOLIA.

2.1.1 GENERAL DESCRIPTION;

Launea taraxacifolia, commonly known as Dandelion or Wild lettuce is found largely in the
highlands of Ethiopia where it originated from. It is common in the Tropical West African
regions, Mexico, West Indies, Central and West America, Asia, Atlantic Islands. It is cultivated
locally as a vegetable in Nigeria, Benin and Senegal. Despite it being a wild herb, it has been
domesticated for its culinary uses.(Akobundu & Agyarkwa, 1998)

Launea taraxacifolia is one of the plants caught between two families namely; Asteraceae and
Compositae families. Studies by Adebesi, 2004; Kuatsienu, 2012; Adetutu et al., 2016; Koukoui
et al., 2017; Owoeye et al., 2017 and Burkill, 1985 all attest that Launea taraxacifolia belongs to
the Asteraceae family. But according to the Plant list (2010), Soelberg et al., 2015; Adinortey et
al., 2012; Launea taraxacifolia was assuredly accepted to belong to the Compositae family.

Launea taraxacifolia is an annual herb that grows straight to about 3m high with a basal rosette
form of leaves. Its leaves are pinnate with dentate margins and narrowed apex. It has four main
ribs with eight thinner ribs and hairs. It produces golden yellow flowers and airborne seeds.
(Michael Buenor Adinortey et al., 2018)See Figure 1.

Launea taraxacifolia has many names by which it is identified by various ethnic groups in
different countries. In Ghana it is known as Nne noa by the Akuapems, Dadedru by the Ashantis,
Namijin dayii by the Hausas. In Benin it is called Cotafon, Watchi, Wonto, Gnari, Lanto,
Wontou, Katakpa, Yantotoe, Loto by various ethnic groups. In Nigeria, it is known as Efo yanrin
by the Yurobas and Ugu by the Ibos. The French call it “Langue de vache” which translates as
tongue of the cow. It is known as Kipo and efo nyori by the Mendes and Krios respectively.
(Kuatsienu et al., 2017)

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Figure 1.

2.1.2 ETHNOMEDICINAL USES

Launea taraxacifolia has many ethnomedicinal uses for which it is locally applied. Studies have
been conducted to confirm its use in the management of sickle cell anemia in Southern Nigeria.
The latex of the plant is also used directly to kill germs on the skin. (State & State, 2018)

Dandelion leaves are used by some people for their recreational purposes i.e. ‘to high’ or have a
hallucinogenic effect. The exposed sap is also used to treat wounds in Ghana mostly among the
Gas and Ashantis. The leaves are mixed with ash and rubbed in the sores of yaws.

Launea taraxacifolia is employed in animals to cause multiple births. (Borokini & Labunmi,
2017) The leaves of Launea taraxacifolia are used to treat ulcers in Benin. (Ojiako, 2006).

The ethanolic extract of the leaves showed antibacterial activities. The antimalarial activity of the
leaves was evaluated using in vitro studies. (Bello et al., 2018). Launea taraxacifolia leaves
provide anticlastogenic activity required after cancer chemotherapy using cisplatin.(Adejuwon,
2014) The leaves of the plant also showed activity against measles virus and the HEP-2 cell line
at 15mg/ml concentrations.(Adejuwon, 2014) The ethanolic extract of the leaves of Launea
taraxacifolia is stated to have neuroprotective abilities.(Michael Buenor Adinortey et al., 2018)

In toxicology studies, the ethanolic extract of Launea taraxacifolia was not lethal on the animals

5
used at all tested doses implying its safety in acute toxicity stage.(Bello et al., 2018)

2.1.3 CHEMICAL CONSTITUENTS.

For the ethnomedicinal effects and activities of the leaves of Launea taraxacifolia are the
underlying chemical compounds responsible for them. The leaves of the plant are endowed with,
tannins, saponins, terpenoids, leucoanthocyanins, steroids, phenolic acids, cardiac glycosides,
ascorbic acids, lycopene and β-carotene. (Michael Buenor Adinortey et al., 2018)

Most abundant chemical compounds found in the leaves of the plant are; palmitic acid, methyl-
II-octadecenoate, orythriol, glycerol, phytol, linelelaidic acid methyl ester.

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2.2 HYPERTENSION

The arteries carry blood at high pressures and for healthy living, there’s a threshold for which
this pressure should not exceed. Arterial pressure like weight is a continuous biological variable
with no exact distinction between normotension and hypertension. Hence, a definition of
hypertension is taken as a level of arterial blood pressure associated with doubling of long-term
cardiovascular risk. (Kaplan N.M, 2002). The continuous relation between hypertension and
cardiovascular risks makes it very prudent to keep our blood pressures in check.

Hypertension is classified by the seventh report of the Joint National Committee on Prevention,
Detection, Evaluation and Treatment of high blood pressure based on the mean of two or more
properly measured seated blood pressures on two or more occasions. (Table 1) (Chobanian et al.,
2003)

Table 1. Classification of blood pressure for adults aged ≥18 years

BP classification Systolic BP (mmHg) Diastolic BP (mmHg)

Normal <120 <80

Prehypertension 120–139 80–89
Stage 1 hypertension 140–159 90–99

Stage 2 hypertension ≥160 ≥100

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A more elaborate classification of blood pressure is provided by the European Society of
Hypertension and the European Society of Cardiology (ESH/ESC) (Table 2)

Table 2. Classification of blood pressure for adults

Category Systolic BP (mmHg) Diastolic BP (mmHg)

Optimal <120 <80

Normal 120–129 80–84

High normal 130–139 85–89

Grade 1 hypertension (mild) 140–159 90–99

Grade 2 hypertension (moderate) 160–179 100–109

Grade 3 hypertension (severe) ≥180 ≥110

Isolated systolic hypertension ≥140 <90

Hypertension is classified as either primary of secondary. It is primary when an unknown cause

leads to dysregulation of normal homeostatic control of blood pressure. 95% of hypertension
cases are primary.

Secondary hypertension is a systemic hypertension with an underlying disorder. These

underlying disorders maybe caused by renal, endocrine adrenal, neurological, postoperative
factors. (Kaplan N.M, 2002)

2.2.1 PATHOPHYSIOLOGY

Blood pressure is the product of peripheral resistance and cardiac output. Hypertension therefore
results from these parameters i.e. either an increase in cardiac output or an increase in peripheral
resistance by various mechanisms. The development of primary hypertension involves genetic
and environmental factors. These also interact with multiple physiological systems including

8
neural, renal, hormonal and vascular. For these complexities, no final common pathway has been
identified and singly targeted for hypertension treatment. Genetic factors associated with blood
pressure are the basis for variability in response to therapy as well as progression of
hypertension. Some environmental factors as smoking, obesity, physical inactivity cause
increases in blood pressure via norepinephrine release (smoking) and vasodilatory adenosine
receptor antagonism(caffeine)

Neural Mechanisms; The sympathetic nervous system controls the heart rate, cardiac output,
peripheral vasoconstriction and hence has a part to play in hypertension. (DiPiro et al., 2016)

Renal Mechanisms; Sodium contributes massively to development of hypertension due to its

related increase in stroke volume and hence cardiac output. This therefore has a link with
development of hypertension. (DiPiro et al., 2016)

Hormonal Mechanisms; Renin, produced and stored by the kidneys, is the rate determining step
in formation of angiotensin II which is a potent vasoconstrictor. High renin levels run the risk of
hypertension and this occurs via proposed mechanisms as increased sympathetic drive, defective
regulation of the RAAS and the existence of ischemic nephrons that release excess renin.

Vascular Mechanisms; The arteries decreasing in lumen size is a sure way to increase peripheral
resistance and hence induce hypertension. Remodeling of vessel or change in vascular tone
modulated by various endothelium-derived vasoactive substances, growth factors and cytokines
increases arterial stiffness and results in observed hypertension. (DiPiro et al., 2016)

2.3. ANTIHYPERTENSIVES.

These are substances or drugs that act to reduce high blood pressure by various mechanisms.
They are generally classified into;

• Diuretics.
• Adrenergic inhibitors.
• Vasodilators.

2.3.1 DIURETICS.

These drugs induce natriuresis and reduce blood volume hence reduce cardiac output. They act
on the nephrons causing more sodium and water excretion through the urine. There are many
9
classes of diuretics;

Thiazide Diuretics – these inhibit the sodium-chloride transporter in the distal tubule causing
excretion of sodium and water in the management of hypertension. It is mostly given as first line
therapy in the management of uncomplicated hypertension. Bendroflumethiazide, indapamide,
metolazone, hydrochlorothiazide and chlorthalidone are examples under this class. They all have
their potencies in blood pressure reduction but should monitor for hypokalemia as the major side
effect. (DiPiro, 2016)

Loop Diuretics – these inhibit the sodium-potassium-chloride cotransporter in the thick

ascending limp leading to a significant loss of water reabsorption in the collecting duct and
promotes natriuresis. Most times a combination of loop and thiazide diuretics gives a synergistic
effect desirable for the management of hypertension. Bumetanide, furosemide, torsemide,
ethacrynic acid are examples of loop diuretics. They present with electrolyte imbalance and
hypokalemia as side effects and should be monitored in patients using them. (DiPiro, 2016)

Potassium-sparing Diuretics – potassium as an electrolyte is useful for various functions in the

body. There is therefore the need for its levels to be maintained for its smooth activities. Most
diuretics present with hypokalemia as untoward effect but this can be managed with the use of
potassium sparing diuretics. They oppose the actions of aldosterone at the distal tubule. This
causes more sodium and water excretion without the excretion of potassium. Amiloride,
triamterene, spironolactone, eplerenone are all under this class and they present with
hyperkalemia, gynecomastia (specific to spironolactone) as side effects. (DiPiro et al., 2016)

2.3.2 ADRENERGIC INHIBITORS.

These drugs act either centrally, peripherally, or as receptor blockers. They target receptors and
neurons of the sympathetic nervous system from brain-stem to post sympathetic adrenergic
receptors. They are also grouped as follows.

Central agonists – these drugs stimulate the α₂-adrenoreceptors (methyldopa, guanfacine,

guanabenz) and I-imidazoline receptors (rilmenidine, moxonidine) or both (clonidine). This
inhibits adenylyl cyclase activity and reduce brainstem vasomotor CNS activation leading to its
antihypertensive effect. They present with sedation, dry mouth and eyes (α₂-adrenoreceptors
agonists) liver dysfunction and hemolytic anemia (methyldopa). (Lee CC et al., 1999)

10
Peripheral adrenergic inhibitors – these drugs block adrenergic receptors preventing vessels from
constricting and hence reducing peripheral resistance. Examples under this class are reserpine,
guanethidine, guanadrel which are rarely used because of its side effects including postural
hypotension, fluid retention and erection problems. (Metha JL et al., 1987)

Selective α-adrenergic receptor blockers – as antihypertensives, these drugs became popular for
their benefits in improving insulin sensitivity, lipid levels and to relieve symptoms of benign
prostate hypertrophy. Examples under this class are prazosin, terazosin, doxazosin and they pose
the risk of heart failure, a drawback to its use. (Black HR et al., 2000)

β-adrenergic receptor blockers – these are the most popular antihypertensives after diuretics.
They act by blocking the β-adrenergic receptors which inhibits vasoconstriction hence reducing
peripheral resistance and blood pressure. They are particularly given to patients with coexisting
coronary artery disease, heart failure and arrythmias as they provide primary protection against
initial coronary events as well. Atenolol, bisoprolol, metoprolol are examples under this class
with dyspnea, bronchospasm and lethargy as their side effects. (Blumenfeld JL et al., 1999)

Combined α-& β-adrenergic receptor blockers – these sprouted from β-blockers as they were
modified structurally to provide the properties of these agents. Carvedilol has a ratio of 1:4; α: β
blocking properties, labetalol has 1:10; α: β. Their mechanism of action is mostly mediated by
the reduction in peripheral vascular resistance. Most common side effects are orthostatic
hypotension and hepatotoxicity. (Frishman WH et al., 1999)

2.3.3 VASODILATORS.

These agents have their mechanism of action being an increase in vessel lumen diameter to
decrease peripheral resistance and hence decrease blood pressure. They are grouped in the
following lines.

Direct Vasodilators - these drugs act directly on smooth muscles to produce vasodilation.
Minoxidil and hydralazine are examples. Their vasodilatory effects cause an increase in heart
rate, stroke volume, cardiac output as a result of baroreceptor-mediated reflex which increases
sympathetic discharge hence their limited use in hypertension management. (Leenen FH et al.,
1987)

11
Calcium Channel Blockers – they are classified into;

• Dihydropyridines – Amlodipine, nifedipine, felodipine

• Non-dihydropyridines – Phenylalkylamines and Benzothiazepines (verapamil and
diltiazem respectively)

They produce vasodilatory effects by interacting with L-type voltage gated plasma membrane
channels. They inhibit aldosterone production resulting in natriuresis and maintain effective
blood flow to the kidneys improving the glomerular filtration rate. They exhibit peripheral
edema, bradycardia, constipation, reflex tachycardia, gingival hyperplasia as side effects. They
are admirable for their ability to avoid interference with NSAIDs as seen in other classes. (Celis
H et al., 2001)

Angiotensin Converting Enzyme Inhibitors – they are classified by the ligand of zinc ion of
angiotensin converting enzyme; sulfhydryl, phosphoryl, carboxyl. Examples of ACEIs are
lisinopril, enalapril, captopril, monopril. Their mechanism of action is by reducing the levels of
Angiotensin II, thereby removing its vasoconstricting effect. Some of its effects are mediated by
inhibition of breakdown of bradykinin which is the role of angiotensin converting enzyme.
Angiotensin converting enzyme inhibitors also prevent the expected reflex increase in
sympathetic activity after vasodilation. They are popular for the cardioprotective and reno-
protective effects. It is therefore recommended for most patients at high risks of coronary heart
diseases. A dry cough is the most frequent side effect of ACEI therapy. Bronchospasm may also
occur. For these, angiotensin receptor blockers may be preferred. (Yusuf S et al., 2000)

Angiotensin Receptor Blockers – these drugs act by blocking angiotensin II from the AT1
receptor hence antagonizing its effects resulting in a fall in peripheral vascular resistance. It
differs from ACEIs by bradykinin levels being reduced. Examples of drugs under this class are
losartan, valsartan, irbesartan. They present with angioedema as their common side effect.
(Velazquez EJ et al., 2000)

12
CHAPTER 3.

3.0 MATERIALS AND METHODS.

3.1 COLLECTION AND AUTHENTICATION OF PLANT MATERIAL.

The fresh leaves of Launea taraxacifolia were collected from the Physic garden of the Faculty of
Pharmacy and Pharmaceutical Sciences in Kwame Nkrumah University of Science and
Technology. This was done in the month of October, 2019. The leaves harvested were
authenticated by Dr. Sam.

3.2 PROCESSING AND EXTRACTION OF PLANT MATERIAL

The leaves were sun-dried and subsequently milled into a coarse powder.

About 144 g of powdered plant material was extracted using 70% ethanol over 48 hours with
intermittent stirring. The extract was filtered and the filtrate was freeze-dried into semisolid
extract.

3.3 ANIMALS AND MAINTENANCE.

Three (3) cats each weighing between 2-3 kg and three (3) frogs of average weight 50 g were
used. These animals were acquired from the KNUST, FPPS – Department of Pharmacology’s
Animal Farm where they were maintained and fed appropriately.

3.4 APPARATUS AND EQUIPMENT.

• Weighing balance
• Syringes
• Kymograph
• Beakers
• Conical flask
• Measuring cylinders
• Test tubes
• Equipment for hypotension testing

13
3.5 CHEMICALS AND DRUGS.

• Acetylcholine (20ug/ml)
• Norepinephrine (20ug/ml)
• Normal saline
• Ringer’s physiological solution
• Fehling’s solutions
• 1% Lead acetate solution
• Dragendorff’s reagent
• 10% Tannic acid solution

3.6 APPARATUS FOR DETERMINATION OF CAT HEART RATE, FORCE OF

CONTRACTION AND TONE.

The apparatus consists mainly of a table with a metallic surface, a device for recording blood
pressure, a clamp holding tubes connecting to selected vessels of the cat and a writing point
attached to a graph sheet for recording contractions. The cat is placed on the metallic surface and
fastened to it appropriately using soft plastic tubes.

METHODS

3.7 ORGANOLEPTIC EXAMINATION.

The color, odor and taste of the extract of Launea taraxacifolia was examined and recorded.

3.8 PHYTOCHEMICAL ANALYSIS.

These tests were carried out on the freeze-dried extracts of Launea taraxacifolia

3.8.1 TEST FOR ALKALOIDS

About 3 ml of extract was evaporated to dryness on a water bath. The residue was dissolved in 1%
H2SO4 and then filtered. The filtrate was made distinctly alkaline with dilute ammonia solution.
The solution was extracted with chloroform and the chloroformic fraction evaporated to dryness.
The residue was dissolved in 1% H2SO4 and tested for alkaloids using Dragendorff’s reagent and
observed for orange precipitate.
14
3.8.2. TEST FOR FLAVONOIDS

Few drops of dilute NaOH was added to the plant extract in solution and an intense yellow color
appeared. Few drops of dilute H2SO4 was then added and observed for colorless appearance.

3.8.3. TEST FOR SAPONINS

About 2ml extract was shaken vigorously with 3ml of water in a test tube and observed for stable
froth formation.

3.8.4. TEST FOR TERPENOIDS

About 3ml of plant extract was made using chloroform. Concentrated H2SO4 was then added to
form a layer, and observe for reddish brown color interface.

3.8.5. TEST FOR TANNINS

The plant material was extracted using ethanol.

Few drops (about 8 drops) of 1% lead acetate solution added and observed for brown precipitate.

3.8.6. TEST FOR COUMARINS

The plant material was extracted using ethanol. A filter paper was dipped into the extract and
afterwards, it was dipped into water. The filter paper was then placed into a UV spectrophotometer
and observed for intense light blue fluorescence.

3.2.2 TEST SAMPLE PREPARATION

Approximately 10 g of the freeze-dried material was weighed into a flask. About 1mL of tween
80 and small about of water was added. The mixture was triturated with mortar and pestle and
the paste topped up with water to 100 mL and labelled appropriately.

15
3.9 EXPERIMENTAL DESIGN.

3.9.1 CAT MODEL.

Three cats were used for this in vivo experiment. Pentobarbital was used to anesthetize the cat
via injection. The animal was properly set unto the dissecting table after its consciousness was
lost. The cat’s limbs were properly clipped to the table to prevent unnecessary movements of the
cat. The left femoral vein was exposed and tied just as the right carotid artery was also exposed
and cannulated for blood pressure measurement. The basal blood pressure was recorded on a
filter paper of micro-dynamometer after administration of normal saline to prevent blood
clotting. Drugs and the extract were administered through the cannula inserted in the femoral
vein.

The first set of drugs were prepared extracts and the graded dose response for each extract noted.
Following this was the administration of adrenaline and acetylcholine. The dose which caused
the maximum effect was recorded as the experimental dose. Washing was properly done after
every administration of extract and drugs till blood returned back to ‘normal’.

3.9.2 FROG MODEL.

An average-sized frog was used for this experiment. The frog’s head suffered a blow to kill it.
Skin and muscle incisions were made to expose its spine. Pithing was done to deactivate the
nerves to prevent interferences when working on the frog. Dissection was carefully done after
which the heart of the pithed frog was carefully freed of connective tissue and exposed for drug
administration. The kymograph was set for the experiment. The heart was moistened by
irrigation with frog Ringer’s solution continuously. A clip which held the base of the ventricles
was tied to a lever system with a writing point moving on the kymograph for recording
contractions. Control drugs (Adrenaline, acetylcholine) and extracts of varying concentrations
were administered directly to the heart and the various contractions recorded appropriately.

16
CHAPTER 4

4.0 RESULTS.

4.1 ORGANOLEPTIC EXAMINATION OF EXTRACTS.

Table 4.1: Showing the physical (organoleptic) characteristics of the freeze-dried Extract.

Launea taraxacifolia extract.

Taste Characteristic
Color Dark-Green.
Odor Characteristic.

Table 4.2: Summary of phytochemical tests on the freeze-dried extract of Launea

taraxacifolia.

Phytoconstituents. Results.

Tannins +

Saponins +

Glycosides +

Alkaloids _

Flavonoids +

Steroids +

Terpenoids +

Key: + Present - Absent

Table 4.3: Showing Master Formula of Formulated Test Samples.

INGREDIENTS QUANTITY

Launea taraxacifolia (freeze-dried) 10g

Tween 80 1mL
Water to 100mL

17
Table 4.4: Showing the effects of various concentrations of test sample and standard drugs
on the heart of an anesthetized cat.

HR FC Tone MAP
(mmHg)
N/S 168 3.9 1.2 109
3.9 1.3 103
4.1 1.3
10mg/kg LTE 170 2 1 87
2.3 1.1 93
2.4 1.1
N/S 164 4.1 1.5 104
4.2 1.3 102
4.3 1.4
30mg/kg LTE 162 3.5 1.3 82
3.7 1.2 81
3.5 1.3
N/S 146 4.4 1.5 103
4.6 1.6 104
4.7 1.5
50mg/kg LTE 150 3 1 72
3.2 1 64
3.1 1.1
N/S 140 4.1 1.3 105
4.3 1.2 107
4.4 1.1
Adrenaline 168 6 0.5 172
6.2 0.8 124
6.3 0.9
Acetylcholine 162 2.7 1.3 35
2.8 1.4 29

18
 2.9 1.3

Key: N/S – Normal saline LTE – Launea taraxacifolia extract. HR – Heart rate.

FC – Force of contraction. MAP – Mean arterial pressure.

Graph 1: Showing the average mean arterial pressure for the various formulations (in vivo).

19
Graph 2: Showing the average force of contraction for the various formulations (in vivo).

20
Graph 3: Showing the average heart rate for the various formulations. (in vivo)

21
Table 4.5: Showing the maximum contractions obtained from the kymogram by the drugs
used (Frog model).

HR FC
N/S 20bpm 1.5cm
18bpm 1.4cm
21bpm 1.3cm

10mg/kg LTE 20bpm 1.5cm

19bpm 1.4cm
19bpm 1.4cm

N/S 22bpm 1.4cm

20bpm 1.4cm
19bpm 1.5cm

30mg/kg LTE 21bpm 1.4cm

20bpm 1.3cm
21bpm 1.4cm

N/S 19bpm 1.5cm

19bpm 1.3cm
20bpm 1.3cm

50mg/kg LTE 20bpm 1.3cm

21bpm 1.4cm
21bpm 1.3cm

N/S 20bpm 1.2cm

21bpm 1.3cm
19bpm 1.3cm

Adrenaline 20bpm 1.3cm

21bpm 1.2cm
20bpm 1.2cm

Acetylcholine 21bpm 1.3cm

20bpm 1.2cm

22
 21bpm 1.2cm
Key: N/S – Normal saline LTE – Launea taraxacifolia extract. HR – Heart rate.

FC – Force of contraction.

Graph 4: Showing the average heart rate for the various formulations. (in vitro)

23
Graph 5: Showing the average force of contraction for the various formulations. (in vitro)

24
Figure 1. Tracings obtained from the experiment.

25
CHAPTER 5.

5.0 DISCUSSION.

Drugs for managing hypertension vary in their mechanisms of action. They all seek to reduce either
cardiac output or peripheral resistance in order to reduce blood pressure. (Gordan et al., 2015)

For this reason, compounds possessing the ability to reduce cardiac output or peripheral resistance
are studied for their effective use in managing hypertension. From literature, Launea taraxacifolia
has suspected effects on blood pressure and this paper studies its effect on the heart using in-vivo
and in-vitro experiments. (Bello et al., 2018)

In the in-vivo study, the extract of Launea taraxacifolia was assessed on its ability to affect the
force of contraction and heart rate of an anesthetized cat. It was found that, 10mg/kg of the extract
reduced the heart’s force of contraction by 10.81% from basal contraction given by normal saline.
30mg/kg of the extract reduced the force of contraction by 14.28% from the basal contractility and
50mg/kg of extract reduced the force of contraction by 32.60% from basal contraction.

This activity may be due to the extract stimulating receptors on the heart as beta 2 adrenergic
receptors responsible for vasodilation and muscarinic (M₂) receptor responsible for the reduction
in contractility experienced.(Gordan et al., 2015)

The control drugs, adrenaline and acetylcholine increased contractility by 51.22% and decreased
contractility by 56.45% of basal contraction respectively.

Adrenaline acts on the alpha- and beta-adrenergic receptors of the heart causing increased heart
rate, increased contractility and vasodilation peculiar to alpha 1 stimulation. This therefore
accounts for its positive ionotropic and chronotropic effects experienced. Acetylcholine acts on
muscarinic (M₂) receptors of the heart to reduce heart rate and force of contraction thereby causing
a reduced blood pressure as seen in this experiment.(Stiles & Lefkowitz, 1984)

To add up, the mean arterial pressure of the cat was recorded. 10mg/kg of extract caused a 15.53%
decrease in basal mean arterial pressure. 30mg/kg of extract caused a 21.15% decrease in basal
mean arterial pressure and 50mg/kg of extract caused a 30.39% decrease in basal mean arterial
pressure. With force of contraction having a bearing on blood pressure, it is therefore
correspondent for the various concentrations of extract to cause a decrease in mean arterial pressure

26
respectively. Adrenaline increased mean arterial pressure by 38.95% whilst acetylcholine
decreased mean arterial pressure by 66.67% from basal mean arterial pressure.

The heart rate of the cat after injection of various concentrations of the extract remained almost in
the same range as that of the normal saline. This may be due to a diminished effect of the extract
on the heart rate of cat.

In the in vitro study, frog’s heart was used. The frog’s heart has several receptors as adrenergic
(alpha and beta) and cholinergic. These receptors regulate heart contractility and rate, the basis for
the use of frog heart for this study. 10mg/kg, 30mg/kg, 50mg/kg concentrations of the extract failed
to show distinct changes in both heart rate and force of contraction of the frog’s heart as was
expected. The control drugs too had this similar effect. This may be due to unforeseen undesirable
circumstances affecting the experiment. The tracings therefore seemed to have similar height and
spacing.

With these, it can be concluded that the extract of Launea taraxacifolia could cause dose-
dependent decrease in contractility and mean arterial pressure of the anesthetized cat mainly due
to its affinity and activity on the M₂ and beta 2 receptors.

The phytochemical screening revealed the presence of certain secondary metabolites in the extract
of Launea taraxacifolia which are responsible for its hypotensive effect as well as other effects of
the plant.(Michael Buenor Adinortey et al., 2018)

5.1 CONCLUSION.

Launea taraxacifolia demonstrated hypotensive activity in vivo and can be further studied and
developed in management of hypertension. However, more studies should be done on its in-vitro
activity since its hypotensive activity was not ascertained in this study.

27
5.2 RECOMMENDATION.

I recommend the following;

• The in-vitro experiment should be repeated for consistency in results to ascertain the
hypotensive activity of Launea taraxacifolia.
• More experiments should be conducted on this extract to determine the various
mechanisms of its hypotensive activity.

28
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