KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY

COLLEGE OF SCIENCE

FACULTY OF BIOSCIENCES

DEPARTMENT OF BIOCHEMISTRY AND BIOTECHNOLOGY

ANTIHYPERTENSIVE ACTIVITY OF COLA GIGANTEA LEAVES ON

ADRENALINE- INDUCED HYPERTENSIVE ANIMAL MODEL.

BY

STEPHEN KWABENA FREMPONG

MAY, 2019
 KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY

COLLEGE OF SCIENCE

FACULTY OF BIOSCIENCES

DEPARTMENT OF BIOCHEMISTRY AND BIOTECHNOLOGY

ANTIHYPERTENSIVE ACTIVITY OF COLA GIGANTEA LEAVES ON

ADRENALINE- INDUCED HYPERTENSIVE ANIMAL MODEL.

A THESIS SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY AND

BIOTECHNOLOGY, KWAME NKRUMAH UNIVERSITY OF SCIENCE AND

TECHNOLOGY (KNUST), IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE AWARD OF BACHELOR OF SCIENCE

(Honours) DEGREE IN BIOCHEMISTRY.

BY

STEPHEN KWABENA FREMPONG

I, Stephen Kwabena Frempong, hereby declare that with the exception of references to

other people’s work and textbooks, this dissertation is a product of my own research

under the supervision of Dr. Caleb Kesse Firempong.

Signature………………… Signature…………………..

Date………………………. Date………………………..

Mr. Stephen Kwabena Frempong Dr Caleb Kesse Firempong

(Student) (Supervisor)

i
 DEDICATION

This work is dedicated to God Almighty and my inspiring family who believe in the

pursuit of higher education for me.

ii
 ACKNOWLEDGEMENT

My profound gratitude goes to Almighty God who has been a buckler for my life. I am

grateful for the wisdom and strength granted unto me to carry out this project. I would

like to express my deepest appreciation to my supervisor, Dr. Caleb Kesse Firempong

for his constructive criticism and unflinching support towards the completion of this

work. I am also grateful for the technical support provided by Dr George Sam, Mr.

Yakubu Jibira, Mr. Edmond Dery and Mr. Prince Dagadu Okyere. I am equally grateful

to my colleagues; Victor Biney, Prince Twumasi, Daniel Odei, Angela Newton and

Kalenu Xoese for their assistance and cooperation to ensure a successful project.

iii
 TABLE OF CONTENTS
CONTENT PAGE

DECLARATION…………………………………………...................................i

DEDICATION…………………………………………………..........................ii

ACKNOWLEDGEMENT………………………………………………………iii

TABLE OF CONTENTS………………………………………………………..iv

LIST OF TABLES……………………………………………………………….x

LIST OF FIGURES……………………………………………………………...xi

ABSTRACT…………………………………………………………………….xiii

CHAPTER ONE………………………………………………………………..1

1.0 INTRODUCTION…………………………………………...........................1

1.1 BACKGROUND INFORMATION…………………………………………1

1.2 PROBLEM STATEMENT………………………………………………….3

1.3 MAIN OBJECTIVE…………………………………………………………4

1.4 SPECIFIC OBJECTIVE…………………………………………………….4

1.5 JUSTIFICATION……………………………………………………………5

CHAPTER TWO…………………………………………………………………6

2.0 LITERATURE REVIEW………………………………………………………6

2.1 HYPERTENSION……………………………………………………………...6

iv
2.1.1 Statistics on hypertension…………………………………….............................6.

2.1.2 Definition and Classification………………………………………………….7

2.1.3 Risk Factors for Hypertension………………………………………………..10

2.1.4 Diagnosis of Hypertension…………………………………………………..12

2.1.5 Biochemical Basis of Hypertension…………………………………………..13

2.1.6 Treatments for Hypertension………………………………………………….14

2.1.6.1 Lifestyle Modifications……………………………………………………...15

2.1.6.2 Conventional Drug Therapy…………………………………………………16

2.3 MEDICINAL PLANTS WITH ANTIHYPERTENSIVE EFFECTS………… .20

2.4 MARKHAMIA LUTEA………………………………………...............................23

2.4.1 Description……………………………………………………………………..23

2.4.2 Phytochemistry …………………….……………………................................24

2.4.3 Medicinal uses…………………………………………….................................25

2.4.4 Toxicity…………………………………….………………………………. .26

2.5 PHYTOCHEMICAL ANALYSIS………………………………………………27

2.5.1 Flavonoids……………………………………………………………………27

2.5.2 Terpenoids……………………………………………………………………28

2.5.3 Phenolics………………………………………………………………………29

2.5.4 Alkaloids………………………………………………………………………30

2.5.5 Glycosides…………………………………………………..............................31

v
CHAPTER THREE………………………………………………………………32

3.0 MATERIALS AND METHOD…………………………………………………..2

3.1 MATERIALS…………………………………………………………………….32

3.1.1 Study Site………………………………………………………………………32

3.1.2 Collection of Plant Materials…………………………………………………..32

3.1.3 Reagents Used…………………………………………………………………33

3.1.4 Blood Pressure Determination………………………………………………….33

3.1.5 Test Animals………………………………………………................................34

3.2 METHODS……………………………………………………………………….35

3.2.1Crude Extract Preparation of Cola gigantea…………......................... .......35

3.2.2 Preparation of Aqueous Extract………………………………………………..35

3.2.3 Preparation of Ethanolic Extract……………………………………………….35

3.2.4 Phytochemical Screening………………………………………………………36

3.2.4.1 Test for Flavonoids…………………………………………………………36

3.2.4.2 Test for Terpenoids…………………………………………………………36

3.2.4.3 Test for Phenols…………………………………………...............................37

3.2.4.4 Test for Alkaloids…………………………………………………………….37

3.2.4.5 Test for Glycosides…………………………………………………………37

vi
3.2.5 Preparation of dose concentrations of Powdered Extracts (Aqueous and

Ethanolic) of Markhamia lutea…………………………………………………….2.6

3.2.6 Antihypertensive Activity………………………………….................................9

3.2.7 Isolated Frog’s Heart Experiment…………………………...............................40

3.2.8 Statistical Analysis…………………………………………………………….41

CHAPTER FOUR…………………………………………………………………42

4.0RESULTS…………………………………………………………………………42

4.1 PHYTOCHEMICALANALYSIS……………………………………………….42

4.2 ANTIHYPERTENSIVE ACTIVITY OF BOTH AQUEOUS AND ETHANOLIC

EXTRACTS OF M. LUTEA ON BLOOD PRESSURE OF ADRENALINE-

INDUCED HYPERTENSIVE CAT…………………………………………………43

4.3 EFFEECTS OF BOTH AQUEOUS AND ETHANOLIC EXTRACTS OF M.

LUTEA ON ISOLATED HEART OF FROG……………………………………….46

4.4 STATISTICAL ANALYSIS…………………………………………………….48

CHAPTER FIVE…………………………………………………………………50

5.0 DISCUSSION…………………………………………………………………50

CHAPTER SIX……………………………………………………………………..61

6.0 CONCLUSION AND RECOMMENDATIONS……………..............................61

6.1 CONCLUSION…………………………………………………………………61

6.2 RECOMMENDATIONS……………………………………...............................61

REFERENCES………………………………………………………………….63

vii
 LIST OF TABLES

Table Page

Table 2.1: Classification of blood pressure for adults aged ≥ 18 years………………10

Table 2.2: Adverse effects of synthetic antihypertensive drugs……………………. 19

Table 2.3: Classification of antihypertensive herbs based on their pharmacological

activities…………………………………………………………………………….. 21

Table 2.4: List of Ghanaian plants with antihypertensive effect……………………. 22

Table 4.1: Phytochemical analysis of leaves of Cola gigantea …………………. …42

Table 4.2: Dose concentration and blood pressure responses of the cat to the aqueous

extract……………………………………………………………………………… 43

Table 4.3: Dose concentration and blood pressure responses of the cat to the ethanolic

extract………………………………………………………………………………. 44

Table 4.4: Comparison of mean blood pressure of extracts to positive……………. 48

Table 4.5: Comparison of mean blood pressure between aqueous and ethanolic
extracts…………………………………………………………………………….. 49

viii
 LIST OF FIGURES
Figure Page

Figure 2.1: Diagram of Renin Angiotensin Aldosterone system…………………….14

Figure 2.2: Overview of the mechanisms of action employed by common classes of

antihypertensive drugs……………………………………………………………….18

Figure 2.3: Image of the tree of Cola gigantea ………………………………….. …26

Figure 2.4: Image of the leaves of Cola gigantea ………………………………... ...27

Figure 2.5: General structure of Flavonoids………………………………………… 28

Figure 2.6: A structure of terpenoid………………………………………………….29

Figure 2.7: Structure of a Phenol…………………………………………………….30

Figure 2.8: Structure of alkaloids…………………………………………………….30

Figure 2.9: Structure of glycosides…………………………………………………...31

Figure 3.1: A picture of Pressure Transducer………………………………………...33

Figure 3.2: A picture of Universal Oscillograph…………………………………….. 34

Figure 3.3: A picture of anesthetized cat prepared for antihypertensive activity…….40

Figure 3.4: A picture of frog with its heart isolated………………………………….. 41

Figure 4.1: Blood pressure against increasing concentrations of aqueous extract of

Cola gigantea on anaesthetized cats……………………………………………... 44

ix
 ABSTRACT

Over the years, some medicinal plants have shown remarkable activities against certain

cancer growth which has led to the discovery of novel anticancer agents like Taxol and

Vinblastine. The objective of this study was therefore to evaluate the cytotoxic effect

of Cola gigantea using its larvicidal activities. The aqueous and ethanolic extracts of

C. gigantea were prepared using standard protocols. Phytochemical screening and

antioxidant activities of the different extracts were also carried out using standard tests

and DPPH free radical scavenging assay respectively. Mosquito larvae were employed

for the larvicidal activities. The data revealed the presence of saponins, phenols, cardiac

glycosides, tannins and terpenoids in the two different extracts, with alkaloids being

detected in only the aqueous extract. The antioxidant activities of the two extracts

(Aqueous extract-EC50 of 0.1840 mg/mL; Ethanolic extract-EC50 of 0.0840 mg/mL)

were comparable to the ascorbic acid (EC50 of 0.12440 mg/mL). The different extracts

of C. gigantea also exhibited some level of larvicidal activities with LC50 values of

33.88 mg/mL (Aqueous extract) and 1.067 mg/mL (Ethanolic extract). These findings

showed that the ethanolic extract of C. gigantea could be a potential source of cytotoxic

agents for further investigations.

Keywords: Antioxidant, Phytochemicals, Cytotoxicity, Larvicidal, Free radicals

x
 CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGROUND INFORMATION:

Cancer has severe health consequences, and it is a leading cause of death in the world

(Dai and Mumper, 2010). According to estimates from the International Agency for

Research on Cancer (IARC), there were 14.1 million new cancer cases and 8.2 million

cancer deaths worldwide in 2012. By 2030, the expectation of the global disease will

grow to 21.7 million new cancer cases and 13 million cancer deaths as a result of

maturation and aging of the population (WHO, 2012). In Africa, the know-how of the

pattern of cancer is very poor (Parkin et al., 2005), and the epidemiological information

on the prevalence of cancer based on the population in Sub Saharan Africa are scattered

(Wiredu and Armah, 2006). Cancer plays a major role in the health problems of both

developed and developing countries. Not only is cancer increased during growth and

aging, but also as a result of some external factors such as smoking, poor diet, infections,

sedentary lifestyles and change in reproductive patterns related to urbanization as well as

economic stability (Torre et al., 2015: WHO, 2012). The prevalence of cancer has created

the need for its control and treatment over these years.

Cancer occurs in many forms such as carcinoma, leukemia, lymphoma and melanoma

(Nagella et al., 2012). Timely detection, accurate diagnosis, and effective treatment help

i|Page
increase cancer survival rates and reduce pain (Nagella et al., 2012). Treatments of cancer

include chemotherapy, radiotherapy, surgery and other clinical trial processes. In the

course of most cancer treatment, chemotherapeutic drugs such as cyclophosphamide,

vinblastine, doxorubicin, etoposide, epirubicin, vincristine and oxaliplatin have been used

(Corrie, 2008). These drugs sometimes lack the capacity to differentiate between fast

replicating cancer cells from normal fast growing cells, like stem cells and hair cells (UK

Cancer research, 2015). These lead to conditions of anemia, nausea, vomiting and hair

loss as well (UK Cancer research, 2015).

Due to these prevailing challenges, more attention is now directed at the use of plants as

alternative treatment for cancers. Statistics have shown that 50% of cancer drugs are

obtained from plant sources (Sini et al., 2011). Some of these plants contain certain

phytochemicals which prevent cancer growth. These include curcumin from tumeric, tea

polyphenols from green tea, isothiocyanates from cruciferous vegetables, silymarin from

milk thistle, diallyl sulfide from garlic, lycopene from tomato, and gingerol from gingers

(Wang et al., 2012).

There are wide ranges of medicinal plants that contribute greatly to the physical well-

being of several people. Most of these plants and herbs have to go through a lot of ground

breaking techniques to elucidate their anti-disease potential (Priyanka et al., 2016). These

pose a challenge to many researchers in Ghana because of limited resources. Often times,

parts of the plants such as the roots, leaves, flowers, barks and stems are used by

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researchers. These plants are ubiquitous in disease treatments. For instance, the medicinal

plant Moringa oleifera is famous for the management of anemia (Devi, 2016). In the area

of cancer research, there are many people who utilize phytochemicals derived from

different parts of plants or other natural products, exclusively or concomitantly with

traditional plan for systematic therapy such as chemotherapy and/or radiation therapy (G.

Gutheil et al., 2012).

The ability of these plants to control cancer growth is as a result of some active

compounds present in their cells. These phytochemicals may contain antioxidants which

could suppress the growth of cancer cells, or sequester excess free radicals. For instance,

the chemical structure of flavonols allows them to donate hydrogen (radical scavenging)

and metal-chelating antioxidants (Martín et al., 2016). Previous studies have shown that

such plants are also toxic to certain larva of insects (Kamkaen et al., 2006). These

antioxidant and larvicidal activities of these plants provide them with the ability to inhibit

certain cancer growth. The presence of antioxidants are responsible for sequestering any

free excess reactive oxygen species (ROS) or any free radical that may pose a threat to

the cell (Li et al., 2015). Usually, it is the accumulation of these free excess ROS that

leads to oxidative stress, cell injury and subsequently to cancer (Ray et al., 2012). One of

the damages caused by ROS is manifested in carcinogenesis, where damage to DNA by

ROS is widely accepted as one of the major causes of cancer diseases (Waris and Ahsan,

2006). Mostly, the herbal medicines are readily available and cheap to obtain (Wachtel-

Galor and Benzie, 2011).

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Cola gigantea is a member of the Sterculiaceae family, and it is usually known as giant

cola. Watapuo is the local Asante-Twi name, in Ghana. It is a large tree in dry semi-

deciduous forest in West Africa and the West Indies (Agyare et al., 2012). The nuts (kola)

are often used in the treatment of whooping cough, asthma, malaria, and fever. Other

traditional uses include increasing the capacity for physical exertion and for enduring

fatigue without food, stimulating a weak heart, and treating nervous debility, weakness,

lack of emotion, depression, anxiety, and sea sickness (Odugbemi, 2006). The leaf

ethanolic extract of Cola gigantea has been shown to have some activity against Candida

albicans and phytochemical screening of the leaf extract indicated the presence of

alkaloids, saponins, tannins, and cardenolides (Sonibare et al., 2009). These vital

phytochemicals may suppress the growth of tumour (Agyare et al., 2012).

1.2 PROBLEM STATEMENT

There is limited information on the antitumour properties of Cola gigantea. Additionally,

investigations concerning indigenous herbs with the potential to treat tumours are at its

infant stage in Ghana. There is also poor documentation and screening systems on such

medicinal plants which restrict their use in managing abnormal tissue growth in Ghana.

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1.3 MAIN OBJECTIVE.

The purpose of the present study was aimed at investigating extracts of Cola gigantea for

their antioxidant and cytotoxic activities.

1.4 SPECIFIC OBJECTIVES.

1. To determine the phytochemicals present in the aqueous and ethanolic leaf

extracts of Cola gigantea.

2. To evaluate the antioxidant properties of the two different extracts.

3. To determine the cytotoxicity of the two extracts using their larvicidal activities.

1.5 JUSTIFICATION

Cola gigantea has been proven to cure a variety of diseases by the indigenes in the rural

areas (Sonibare et al., 2009). The phytochemicals responsible for the efficacy of this

medicinal plant is not fully known. Cytotoxic studies on Cola gigantea could provide

scientific bases for the use of the plant in treating abnormal tissue growth. Any positive

data on the plant could also serve as a foundation for further antitumour studies. The

screening model could be used to identify other potential medicinal plants in Ghana.

v|Page
 CHAPTER THREE

3.0 MATERIALS AND METHOD

3.1 MATERIALS

3.1.1 Study Site

The evaluation of the cytotoxicity of Cola gigantea was carried out at the Project

Laboratory of the Department of Biochemistry, KNUST-Kumasi, Ghana. The

phytochemical screening and the antioxidant activity test was also carried out at the

Project Laboratory and the general Biochemistry laboratory of same department,

respectively.

3.1.2 Collection of Plant Material

Fresh leaves of Cola gigantea were obtained from KNUST campus, precisely adjacent

the KNUST main administration building. The fresh Cola gigantea leaves were

authenticated by Mr. Osafo Asare, a herbalist at the Department of Herbal Medicine,

KNUST-Kumasi, Ghana.

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3.1.3 Reagents

The reagents used in the study were Ferric chloride, Ferric acid, Hydrochloric acid,

Sodium hydroxide, Wagner’s reagent, Chloroform, Sulphuric acid, Glacial acetic acid,

Ascorbic acid, Methanol, Ethanol, 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) and Dimethyl

Sulfoxide (DMSO. They were obtained from the Department of Biochemistry and

Biotechnology, KNUST-Kumasi, Ghana.

3.1.4 Test Organism

Mosquito larvae were used for the cytotoxicity evaluation. The mosquito larvae were

trapped close to the garden at Biological Science Department.

3.2 METHODS

3.2.1 Crude Extract Preparation of Cola gigantea Leaves

The leaves of the Cola gigantea were washed thoroughly for about 3 times with tap water

and finally with distilled water to remove any unwanted particles. The leaves were sliced

into bits and shade-dried at room temperature for about 2 weeks. The dried materials were

then milled into powdered form using corn milling machine. The powdered dry leaves

were stored in airtight bags.

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3.2.2 Preparation of Aqueous Extract

A portion of the powdered dry leaves (100 g) were soaked in 1L of disilled water for 30

minutes. It was later boiled for 2 hours and allowed to simmer for another 30 minutes.

The boiled extract was cooled and filtered through a clean linen cloth. The filtrate was

oven dried and stored in airtight bags and kept in a cool dry place.

3.2.3 Preparation of Ethanol Extract

Another portion of the powdered leaves (100 g) were boiled by refluxing in about 1 L of

80% ethanol for three times. The mixture was filtered through two-fold linen. The filtrate

was partially dried at a temperature of 60˚C in a rotary evaporator. The concentrates

obtained were oven-dried, stored in airtight containers and kept in a cool dry place.

3.2.4 Phytochemical Screening

Qualitative phytochemical screening was carried out on both the aqueous and ethanol

extracts of the plant using standard procedures as described by Trease and Evans (1989)

and Sofowara (1993).

3.2.4.1 Test for Flavonoids - Alkaline Test

The extracts (1 mL concentration each) were pipetted into test tubes and three drops of

20% Sodium hydroxide were added to each extract. Hydrochloric acid (1 mL) was added

viii | P a g e
to each extract. The formation of an intense yellow colour which changes to colourless

upon standing shows the presence of flavonoids.

3.2.4.2 Test for Alkaloids - Wagner’s Test

The extracts (1 mL concentration each) were pipetted into test tubes and five drops of

Wagner’s reagent were added to each extract, and mixed thoroughly. The formation of

reddish-brown precipitate shows the presence of alkaloids.

3.2.4.3 Test for Saponins - Foam Test

Distilled water (3 mL each) was added to 2 mL each of each extract concentration in test

tubes. The mixture were shaken vigorously and observed for the presence of persistent

foam which indicates the presence of saponins.

3.2.4.4 Test for Tannins - Braymer’s Test

Three drops of 10 % Ferric chloride was added to 1 mL of each extract concentration in

test tubes. The formation of a blue or greenish colour shows the presence of tannins.

3.2.4.5 Test for Phenols - Ferric Chloride Test

Drops of 5% ferric chloride reagent (3) were added to 1 mL of each extract concentration.

The presence of a deep blue or black colouration shows the presence of phenols.

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3.2.4.6 Test for Terpenoids - Salkowski’s Test

Chloroform (1 mL concentration each) was added to 2 mL each of the extract

concentration. Three drops of concentrated sulphuric acid were also added. The formation

of a reddish brown precipitate shows the presence of terpenoids.

3.2.4.7 Test for Cardiac Glycosides - Keller- Kelliani’s Test

A volume of 5 mL each of the extract concentration were pipetted into test tubes and 1

mL of glacial acetic acid added. Concentrated sulphuric acid were carefully added to the

sides of the test tubes. Formation of a brown ring indicates the presence of cardiac

glycosides.

3.2.5 Test for Antioxidant Activity

Stock solutions of the aqueous and hydroethanolic extracts were prepared by dissolving

10 mg of each of the dried samples in 1 mL of distilled water and 70% hydroethanol

respectively. Additionally, stock solutions of 10 mM of the standard compound (Ascorbic

acid) and 0.5 mM of DPPH were prepared by dissolving 0.176 mg of Ascorbic acid and

3 mg of DPPH in 1 mL of water and 15 mL absolute methanol respectively. The solutions

were then vortexed until complete dissolution was obtained. The DPPH solution was

quickly placed in the dark as it photo-bleaches in the light. The extracts were serially

diluted with water (for aqueous extract) and 70% hydroethanol (for hydroethanolic

extract) to obtain a concentration range of 0.156–10 mg/ml in 1.5 mL eppendorf tubes.

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Each concentration of the test sample (100 𝜇L) was transferred into a 96 well plate. This

was followed by the addition of 100 𝜇L of 0.5 mM (DPPH). For positive control or

standard, ascorbic acid was used at a concentration of 0.156–10 mg/mL in distilled water.

The solvents (80% hydroethanol) and distilled water were used as blanks. Triplicate

experiments were performed. The plates were covered with aluminum foil, shaken gently

and kept in the dark for 20 minutes after which the absorbance was read on a Synergy H1

plate reader at 517 nm. The percentage scavenging activity was determined using the

equation below:

% Scavenging = [Absorbance of control (OD0) - Absorbance of sample (OD1)] × 100

The mean antioxidant activity for the triplicate experiment was plotted for the standard

and extracts. Their effective concentration at 50% (EC50) values, which is the amount of

antioxidant necessary to decrease the initial DPPH concentration by 50%, were

determined by nonlinear regression analysis.

Absorbance of control= Blank (Solvent) - Colour control

Absorbance of sample= Mean absorbance – Colour control

Colour control= DPPH + Methanol

Mean absorbance= Average absorbance of triplicate

Blank = Methanol

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A graph of the mean antioxidant activity will be plotted against the concentrations and

the EC50 values of each of the extract calculated.

3.2.6 Growing of Larvae

Empty containers were collected and filled with water. The containers were placed on an

open lawn. After some weeks, the eggs laid in the stagnant water by the mosquitoes

hatched into larvae. The larvae were fed with algae and allowed to grow until they were

visible to the naked eye. Once visible in the water, they were harvested and kept until

when needed.

3.2.7 Test for Larvicidal Activity

The cytotoxic activity test was performed on both the aqueous and ethanolic extracts of

Cola gigantea. Stock solutions (10 mg/mL each) of both the aqueous and ethanolic

extracts were prepared. The ethanolic extract powder (1 g) was first dissolved in 5 mL

DMSO and later topped up to 100 mL with distilled water. The aqueous extract powder

(1 g) was dissolved in 100 mL of distilled water only. The stock solutions were serially

diluted (two-fold) for both extracts to obtain concentrations of 5.0, 2.5, 1.25, 0.625 and

0.3125 mg/mL. Five larvae were added to each concentration of both extracts, and

mortality observed at different times (24, 48 and 72 hours). A control for the aqueous was

setup using only distilled water. Control for the ethanol extracts consists of 5ml DMSO

and 95 mL distilled water. Larva mortality was determined by the inability of the larvae

xii | P a g e
to wiggle or move when probed with a stirrer. The mean percentage mortalities were

calculated for each concentration. The mean percentage mortality result was plotted

against the concentration using Microsoft Excel software. The concentration that killed

50% of the larvae (LC50) was calculated from the regression equations obtained from the

graph.

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 CHAPTER FOUR

4.0 RESULTS

4.1 PHYTOCHEMICAL ANALYSIS

From the phytochemical analysis, saponins, phenols, cardiac glycosides, tannins and

terpenoids were present in both the aqueous and ethanolic extracts, but flavonoids were

clearly absent in these extracts (Table 4.1). Alkaloids were also present in only the

aqueous extract.

Table 4.1: Phytochemical constituents of different Cola gigantea extracts.

Phytochemical Aqueous Hydroethanol (80% ethanol)

Saponins + +

Phenols + +

Cardiac Glycosides + +

Flavonoids - -

Alkaloids + -

Tannins + +

Terpenoids + +

[+] = present [-] = absent

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4.2 ANTIOXIDANT ACTIVITY

The aqueous extract of Cola gigantea had an EC50 value of 0.184 mg/mL (Figure 4.1)

which was the highest among the extracts. The ethanolic extract of Cola gigantea also

had EC50 value of 0.084 mg/mL (Figure 4.2) which was the lowest. Finally, the ascorbic

acid produced an EC50 value of 0.124407 mg/mL (Figure 4.3).

Aqueous Extract EC50 = 0.184 ± 0.00724 mg/mL

100
% Antioxidant Activity

80

60

40

20

0
0 0.15625 0.3125 0.625 2.5
Concentration (mg/ml)

Figure 4.1: Antioxidant activity of aqueous extract of Cola gigantea

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 Ethanolic Extract EC50 = 0.084 ± 0.00322 mg/mL
120
% Antioxidant Activity

100
80
60
40
20
0
0 0.15625 0.3125 0.625 2.5 5
Concentration (mg/mL)

Figure 4.2: Antioxidant activity of ethanolic extract of Cola gigantea.

Ascorbic Acid EC50= 0.124407 ± 0.0222 mg/mL

80

60

40

20

0
0 0.0781 0.1563 0.3125 0.625 1.25 2.5 5
Concentration (mg/ml)

Figure 4.3: Antioxidant activity of ascorbic acid (standard drug)

From Table 4.2, the EC50 values of the ethanolic extract and the standard (ascorbic acid)

showed no significant statistical difference. However, there were significant statistical

xvi | P a g e
difference of EC50 values between the ascorbic acid and the aqueous extract as well as

the ethanolic extract and the aqueous extract at 0.05 α-value.

Table 4.2: Multiple Comparisons of antioxidant activities of the different Cola

gigantea extracts.

(I) Antioxidant (J) Antioxidant Mean Difference (I-J) Sig.

Ascorbic Aqueous -.0748731* .001

Ethanol .0250796 .051

Aqueous Ascorbic .0748731* .001

Ethanol .0999527* .000

Ethanol Ascorbic -.0250796 .051

Aqueous -.0999527* .000

*The mean difference is significant at the 0.05 level.

4.3 LARVICIDAL ACTIVITY

The LC50 of the aqueous extract was 33.88 mg/mL, where as that of the ethanol extract

was 1.067 mg/mL. From figure 4.4, the percentage mortality of larvae was high with

increase of time in the ethanolic extract as compared to the aqueous extract. Also, figures

4.5 and 4.6 shows the larvicidal activities of ethanolic and aqueous extracts respectively,

with high percentage mortality from the ethanolic extract of C. gigantea.

xvii | P a g e
 100
90 48, 90 72, 90
Percentage Mortality (%)

80
70
60 48, 60 72, 60
mortality
50
(ethanolic)
40
mortality
30
(aqueous)
20
10
0 24, 0 24, 0
0 20 40 60 80
Time (hours)

Figure 4.4: Larvicidal activity of different Cola gigantea extracts on mosquito

larvae over a given time.

100
90
Percentage mortality (%)

80
70
60
50
40
30
20
10
0
0.3125 0.625 1.25 2.5 5
Concentration (mg/ml)

Figure 4.5: Larvicidal activity of different ethanolic extract concentrations of Cola

gigantea.

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 Percentage mortality (%) 70
60
50
40
30
20
10
0
0.3125 0.625 1.25 2.5 5
Concentration (mg/ml)

Figure 4.6: Larvicidal activity of different aqueous extract concentrations of Cola

gigantea.

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CHAPTER FIVE

5.0 DISCUSSION

The phytochemicals present in the extracts of Cola gigantea confer certain variety of

pharmacological importance to the plant. Thus, saponins, phenols, cardiac glycosides,

tannins and terpenoids were present in the two extracts, with alkaloids being detected in

only the aqueous extract. However, flavonoids were clearly absent in both the aqueous

and ethanolic extracts of the plant material.

Tannins have the potential to reduce mutagenic activities. Their anti-carcinogenic and

mutagenic potentials may be associated with their antioxidant activity, which protect

cellular oxidative damage (Chung et al., 1998). Saponins possess cytotoxic activities.

They have unique biological ability to lyse erythrocytes or to foam (Francis et al., 2002).

They exert wide ranges of pharmacological importance which include anti-inflammatory,

hypocholesterolemic, immunomodulatory, hypoglycaemic and antifungal properties

(Podolak et al., 2010).

Phenols have the potential to inhibit bacterial, fungal, protozoan and parasitic growth.

This ability depends on their interaction with proteins or membrane disturbing properties

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(Stasiuk and Kozubek, 2010). Cardiac glycosides are organic molecules containing one

structure of glycoside which act on the contractile muscle of the heart (Ibraheem and

Maimako, 2014). They are classified as extremely toxic with a narrow therapeutic range.

Alkaloids have some pharmaceutical applications which include anesthetics, antibiotics,

analgesic, and disinfectant (Ibraheem and Maimako, 2014). Several alkaloids obtained

naturally from plants have shown antineoplastic effects on various forms of cancer.

Berberine (isoquinoline alkaloid) prevents the growth of many cancer cell lines. It arrests

cell cycle at M phases and also through apoptosis (Sun et al., 2009).

The alkaloids were present in aqueous extract but absent in hydroethanol extract. This

implies that the leaves generally contain alkaloids. Additionally, the absence of the

alkaloids could be as a result of the choice of solvent used. Generally, the solubilities of

different alkaloids and their salts is a true reflection of their varied complex nature and

chemical structure (El-sakka, 2010). Studies have shown that free alkaloids bases are

usually fairly soluble in ethanol but insoluble in water (Ashutosh, 2003). Alkaloidal salts

on the other hand are less soluble in ethanol, but readily soluble in water (Ibraheem and

Maimako, 2014). Therefore, it could be that the alkaloids present in the leaves were

alkaloidal salts other than free alkaloids. These salts have polar properties just as water

so their solubility in aqueous solvent was greatly enhanced.

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Similar study on the same plant observed both flavonoids and alkaloids in Cola gigantea

(Agyare et al., 2012). This could be due to the differences in the entire extraction

protocols.

Most of the terpenes involved in anticancer activities are made of other phytochemical.

Example is camptothecin, which is a modified monoterpene indole alkaloid, used for

colon cancer treatment (Demain and Vaishnav, 2011). The absence of flavonoids in both

the aqueous and ethanolic extraction could be due to the type of extraction method used.

According to Biesaga (2011), the extraction mode as well as the chemical structure of the

flavonoid affects their degradation. Most of the modes of extraction come with harsh

conditions which easily degrades the flavonoids. According to Okuda et al. (1993), high

temperatures cause flavonoid levels to fall.

The choice of solvent for extraction could also play a role in the absence of the flavonoids.

Solubility of flavonoids is dependent to the extent on whether they appear to be bound to

one or more sugar residues, which render them highly polar (Bohm, 1998). In their free

form, they tend to be less polar (Bohm, 1998). Therefore, it is likely that the flavonoids

in the two extracts were below detection limits.

Antioxidant scavenging activity is based on the potential to donate a lone pair of electrons

to neutralize free radicals (Molyneux, 2004). Usually, the donation is done by a sample

with the ability to donate these lone pair of electrons (a free radical scavenger). The

molecule of 2, 2-diphenyl-1-picryl-hydrazyl (DPPH) is characterized as a stable free

radical by virtue of the delocalization of the spare electron over the molecule as a whole

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(Molyneux, 2004). The DPPH picks up electrons from the free radical scavenger. This

results in a decrease in absorbance and a corresponding change in the original violet

colour of the DPPH to yellow (Molyneux, 2004). The colour change is usually dependent

on the extent of electrons gained. The EC50 value, which is the effective concentration at

which 50% of the free radicals are scavenged, shows how strong or weak an antioxidant

is. According to Molyneux (2004), EC50 values below the standard compound show good

scavenging activity of the plant.

The EC50 value of the standard ascorbic acid was 0.124407 mg/mL. Even though the EC50

value (0.184 mg/mL) of the aqueous was slightly greater than the standard, it still has the

potential to mop up free radicals as the ascorbic acid. The good antioxidant activity of the

aqueous could be as a result of the numerous phytochemicals present in the aqueous

extract. These phytochemicals provides large amount of lone pair electrons (Martín et al.,

2016). Thus, they give good effective concentration for scavenging much of the free

radicals.

However, the EC50 value (0.084 mg/mL) of the ethanolic extract was below that of the

standard. This shows its ability to mop up free radicals readily. Like the aqueous extract,

the ethanolic extract has phytochemicals which contribute numerous electrons for

scavenging free radicals. However, some phytochemicals have very high antioxidant

activity and so they reflect in the total antioxidant activity of the ethanolic extract.

Therefore, the reason for the high antioxidant activity and low EC50 value of the ethanolic

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extract could be that the solvent of extraction was able to isolate almost all the antioxidant

phytochemicals present.

From Figures 4.1 and 4.2, the antioxidant activities of both the aqueous and ethanolic

extracts were concentration dependent, respectively. It is therefore assumed that the

increase in concentration was proportional to the increase in the number of electron lone

pairs of the phytochemicals. Often times, the EC50 values could be affected by some

molecules in the extracts that quench the antioxidant activities of the extracts. So the

calculated EC50 values might not reflect their true EC50 value. The difference in the

statistical mean of EC50 value of the aqueous extract and that of the ascorbic acid (Table

4.2) could mean that the antioxidant activity of the aqueous is not as effective as that of

the ascorbic acid. However, the ethanolic extract could have the same antioxidant strength

as the ascorbic acid due to the insignificant statistical difference between its EC 50 value

and that of the ascorbic acid. Finally, the antioxidant strengths of both the aqueous extract

and ethanolic extract are different. These observations could be accounted for by the

variations in the amount of phytochemicals present in the extracts of Cola gigantea.

The larvicidal activity test is done to predict whether the plant extracts contain some

bioactive compounds that are toxic to the larvae. Mosquito larvae at the onset of

maturation undergo various cell divisions and differentiations in order to metamorphose

into another stage of their life cycle (Farnesi et al., 2012). This biological activity mimics

the rapid division of cancer cells. In the present study, the mosquito larvae were used as

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a model to draw preliminary conclusions as to whether the plant extracts had some

potential cytotoxic compounds present in them, for further investigations.

From the results, both the aqueous and ethanol extract showed some levels of toxicity. At

the start of the test, an assumption was made that any first larvae death could be recorded

in the highest concentration of both extracts, and so 5 mg/mL was the ideal concentration

which was used for the comparison analysis in Figure 4.4. Within 24 hours of the test, no

death was recorded in all the concentrations for both extracts. The first point of interaction

by the active compounds could be through the membrane of the larvae. Mosquitoes in all

the stages of post-embryonic life, have their bodies covered with an integument composed

of an innermost epidermal monolayer and an outermost complex extracellular matrix

known as cuticle (Farnesi et al., 2012). As a result of this, it will take quite a long time

for the bioactive compounds to cross the cuticle of the larvae. This could be the reason

for the negative mortality recorded during the first 24 hours period. After 48 hours of the

test, percentage mortalities of both aqueous and ethanolic extracts increased rapidly to

60% and 90% respectively. At this stage, it could be that most of the bioactive compounds

have successfully crossed the membrane barrier of the larvae to their internal organs to

induce death. Also, the larva undergoes moulting as it grows (Norbert, 2010). Thus, as

the previously hard exoskeleton is shed off, a soft new one emerges. The bioactive

compounds could penetrate more through the new exoskeleton, to induce death. So the

percentage of death could directly be proportional to the percentage of larvae that moulted

during that period. The comparable high percentage mortality in the ethanol extract could

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be due to the presence of some high amounts of certain bioactive compounds which

induces death more readily in the larvae. After 72 hours, no change in percentage

mortality was observed. This could be due to the fact that the number of larvae left had

developed resistance to the bioactive compounds or probably the working threshold of

the extracts was reached.

The larvicidal activities of the ethanolic extract (LC50= 1.067 mg/mL) of Cola gigantea

was far better than the aqueous extract (LC50= 33.88 mg/mL), Figures 4.5 and 4.6. The

choice of solvent could have played a significant role in the larvicidal activity (Ghosh et

al., 2012). The aqueous extract at 1.25 mg/mL concentration recorded no mortality, which

implies that the larvae in that extract concentration could survive (Figure 4.6). The

findings also showed that the different extracts were concentration dependent, thus as

concentration increases, the percentage mortality also increases to that extent. The high

mortality could be attributed to the shortage of oxygen to the larvae caused by the

turbidity of the extract as a result of high extract concentration (Vinayagam et al., 2008).

The high extract concentration also concentrates much of the bioactive compounds which

could lead to the high mortality rate. Previous work done by Jose and Adesina (2015)

using Cola gigantea extract also showed a high larvicidal activity.

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 CHAPTER SIX

6.0 CONCLUSION AND RECOMMENDATIONS

6.1 CONCLUSION

Cola gigantea showed the presence of saponins, phenols, cardiac glycosides, tannins and

terpenoids in both aqueous and ethanolic extract. Alkaloids were only found to be present

in aqueous exract. Ethanolic extract of C. gigantea also showed high antioxidant activity

(EC50=0.084 mg/mL), whereas that of the aqueous extract showed less antioxidant

activity (EC50=0.184 mg/mL), but comparable to the standard ascorbic acid (EC50=

0.1244 mg/mL). The larvicidal activity of the ethanolic extract (LC50= 1.067 mg/mL) was

far better than the aqueous extract (LC50= 33.88 mg/mL). Taken together, it can be said

that the Cola gigantea has shown some prospects of cytotoxic activities.

6.2 RECOMMENDATIONS

 Further studies can be undertaken on the plant to obtain the exact phytochemicals that

contribute to the good antioxidant activity.

 Investigations can also be done on other parts of the plant for more improved

antioxidant and cytotoxic activities.

 The ethanolic extract of Cola gigantea can further be tested on cancer cell lines to see

their direct effect.

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