ABSTRACT
Alchornea cordifolia is known for its phytomedicinal properties including its antimalarial potentials. This research was aimed at isolation and characterization of bioactive components from the leaves of Alchornea cordifolia as well as investigating by in vitro, the antimalarial activities of its crude methanol extract and fractions of petroleum ether, dichloromethane, ethyl acetate and methanol, on human whole blood medium, infected with Plasmodium falciparum. Phytochemical analysis, antimalarial activity and spectral characterization were done using standard methods. Through phytochemical screening we discovered the presence of tannins, flavonoids, alkaloids, phenols, saponins, and cyanogenic glycosides. The result of the spectral analysis revealed two compounds shown to possess identical spectral properties but with a distinct addition of two olefinic protons on the NMR at C-22 and C-23 in the case of one compound. The two compounds possess a steroidal skeleton with molecular masses of 412 and 414 and molecular formulas of C29H48O and C29H50O respectively. The two compounds interpreted from the spectral results are Stigmasterol and β-sitosterol respectively. Based on the IR we also proposed a third possible structure as a stigmasterol analog. The third structure still of molecular formula C29H48O has an aldehydic functional group, CHO replacing the CH3 on C-18 but with absence of OH on C-3. On antimalarial activity, the crude methanol extract and ethyl acetate fraction showed a good dose-dependent antimalarial activity with mean IC50 values of Parasitaemia (P) = 12.2 and P = 12.94 respectively, while those of standard drugs, chloroquine and ACT, used as positive control were 13.38 and 9.2 respectively. These mean IC50 values indicate that crude methanol extract and ethyl acetate fraction gave a better antimalarial activity than chloroquine but were not as good as ACT. Using one way ANOVA for statistical analysis we confirmed our significant results. Hence the leaves of Alchornea cordifolia possess promising bioactive components that showed significant antimalarial activity against Plasmodium falciparum and may give a better activity when they work in synergy. Thus, it will be potentially effective in the fight against malaria.
TABLE
OF CONTENTS
Title Page i Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of
Contents vi
List of
Tables viii
List of
Figures ix
List of
Plates xi
List of
Charts xii
List of
Abbreviations xiii
Abstract xiv
CHAPTER
1: INTRODUCTION 1
1.1 Background of the Study 1
1.2 Statement of the Problem 2
1.3 Aim of the Study 3
1.4 Objectives of the Study 3
1.5 Justification of the Study 3
1.6 Scope of the Study 4
CHAPTER
2: LITERATURE REVIEW 5
2.1 Botanical Characteristics of Alchornea cordifolia 5
2.2 Phytomedicinal Properties 7
2.3 Phytochemical Constituents 7
2.4 Phytochemicals 8
2.5 Plasmodium
Species and Life Cycle 20
2.6 Antimalarials 21
2.6.1 Natural product antimalarials 22
2.6.2 Synthetic antimalarials 27
2.6.3 Coartem (artemether/lumefantrine) 29
2.7 Thin Layer Chromatography (TLC) 30
2.8 Column Chromatography 31
2.9 Spectroscopic Techniques 34
2.10 In
Vitro Antimalarial Activity 44
CHAPTER
3: MATERIALS AND METHODS 49
3.1 Materials 49
3.2 General Experimental Procedure 50
3.2.1 Plant material 50
3.2.2 Extraction of plant material 50
3.2.3 Isolation of constituents 51
3.2.4 Qualitative phytochemical analysis 54
3.2.5 Quantitative phytochemical determination 56
3.2.6 Structure
elucidation 60
3.2.7 In
vitro antimalarial activity 62
CHAPTER
4: RESULTS AND DISCUSSION 67
4.1 Column Chromatography 67
4.2 Thin Layer Chromatography (TLC) 69
4.3 Phytochemical Analysis 70
4.4 Spectral Analysis 73
4.5 In
Vitro Antimalarial Activity 72
CHAPTER 5: CONCLUSION AND
RECOMMENDATIONS 112
5.1 Conclusion 112
5.2 Recommendations 112
References 114
LIST OF
TABLES
2.5 Duration
of Phases of Plasmodium Species 21
2.6.1 Examples
of Antimalarial Herbal Isolates
22
2.10.7 Summary of Assay Methods 48
4.1.1 Column
Chromatography (First Phase) 67
4.1.2 Column
Chromatography (Second Phase) 67
4.2.1 TLC
of Pure Components from Column Chromatography
(First Phase) 69
4.2.2 TLC
of Pure Components from Column Chromatography
(Second Phase)
69
4.3.1 Phytochemical
Analysis 70
4.3.2 Cyanogenic
Glycosides 71
4.4.1 DEPT,
13C NMR and 1H NMR Chemical shifts of Compound 1
(stigmasterol)
recorded in CDCl3 97
4.4.2 13C NMR and 1H
NMR Chemical Shift Values of Compound 2
(β-sitosterol)
recorded in CDCl3 87
4.5.1 In vitro Antimalarial Activity of BC 95
4.5.2 In vitro Antimalarial Activity of CME 96
4.5.3 In vitro Antimalarial Activity of PEF 96
4.5.4 In vitro Antimalarial Activity of DMF 97
4.5.5 In vitro Antimalarial Activity of EAF 97
4.5.6 In vitro Antimalarial Activity of MF 98
4.5.7 In vitro Antimalarial Activity of CQ 98
4.5.8 In vitro Antimalarial
Activity of ACT 99
4.11 Statistical
Analysis using One Way Anova including Turkey HSD 108
LIST OF FIGURES
2.4.1 Phenolic Compounds Isolated from Alchornea Species 10
2.4.2 Flavonoids Isolated from Alchornea Species 14
2.4.3 Alkaloids Isolated from Alchornea Species 16
2.4.4 Steroids Isolated from Alchornea Species 19
2.5 Plasmodium Species and Life Cycle 20
2.6.2.1 Chloroquine 27
2.6.2.2 Quinine derivatives 27
2.6.2.3 Lumefantrine 28
2.6.2.4 Artemisinin and its derivatives 28
2.9.2 IR absorption range and fingerprint region 37
2.9.3.1 Proton Chemical shifts and
Chemical Environments 40
2.9.3.2: Carbon-13 Chemical Shifts and Chemical
Environments 41
2.9.4.1: DEPT-Distortionless Enhancement by
Polarisation Transfer 41
2.9.4.2: DEPT-Distortionless Enhancement by
Polarisation Transfer 42
2.9.5: 2D COSY 43
4.4.1 FT-IR Spectrum of ND-2 from EA21 73
4.4.2 1H-NMR Spectrum of ND-2 from EA21 75
4.4.3 COSY Spectrum of ND-2 from EA21 76
4.4.4 13CNMR Spectrum of ND-2 from EA21 77
4.4.5 13CNMR Spectrum of ND-2 from EA21 78
4.4.6 DEPT Spectrum of ND-2 from EA21 79
4.4.7 DEPT Spectrum of ND-2 from EA21 80
4.4.8 1H-NMR Spectrum of ND-2 from EAF3 84
4.4.9 COSY Spectrum of ND-1 from EAF3 85
4.4.10: DEPT Spectrum of ND-1 from
EAF3 86
4.4.11: MS Spectrum of ND-1 from EAF3 88
4.4.12: Stigmasterol
(Compound 1) 90
4.4.13: β-Sitosterol
(Compound 2) 90
4.4.14: The
Third Proposed Structure 91
4.5.1 In
vitro Antimalarial Plot of BC 100
4.5.2 In
vitro Antimalarial Plot of CME 101
4.5.3 In
vitro Antimalarial Plot of PEF 102
4.5.4 In
vitro Antimalarial Plot of DMF 103
4.5.5 In
vitro antimalarial Plot of EAF 104
4.5.6 In
vitro Antimalarial Plot of MF 105
4.5.7 In
vitro Antimalarial Plot of CQ 106
4.5.8 In
vitro Antimalarial Plot of ACT 107
LIST
OF PLATES
2.1 Leaves of Alchornea cordifolia 6
LIST OF CHARTS
3.1 Fractionation
of Crude Methanol Extract from the Leaves of
Alchornea
cordifolia 66
LIST
OF ABBREVIATIONS
A.cordifolia
– Alchornea cordifolia
BC – Blood Control wells
CME – Crude
Methanol extract
DM – Dichloromethane
DMF –
Dichloromethane fraction
EA – Ethyl acetate
EAF – Ethyl
acetate fraction
M – Methanol
ME – Methanol extract
P- Parasitaemia
PE – Petroleum ether
PEF – Petroleum ether fraction
PW- Precautionary
wells
SW – Supplementary wells
CHAPTER
1
INTRODUCTION
The last decade
has witnessed an upsurge in the use of herbs as a source of medication (WHO,
2004). Some herbs like Artemisia annua
have also become the basis for some pharmaceutical medications. The
“phytochemical world” has of recent attracted many researchers due to the
healthy phytochemicals derived from plants (WHO, 2004; 2006). Prominent among
herbs are those for the treatment of malaria because malaria poses a great
threat to more than half of the world’s population (WHO, 2015).
Of
the prominent antimalarial herbs is Alchornea
cordifolia (A. cordifolia). It is mostly used for medicinal purposes and
widely distributed in the east, south, west and central Africa (www.prota4u.org).
Among its phytochemical benefits is its traditional use as an antimalarial. A.
cordifolia is thus the focus of this research because of its traditional
effectiveness.
1.1 BACKGROUND OF THE STUDY
The term malaria originates from
Medieval Italian: “mala aria” meaning
“bad air” (Reiter, 1999). The disease is endemic in the tropical and sub-tropical
zones around the equator (Caraballo, 2014) due to high temperatures and high
humidity, heavy rainfall as well as stagnant water which is a breeding ground
for mosquitoes. These regions are more of sub-Saharan Africa, Asia and Latin
America (WHO Malaria Fact Sheet, 2014). The worldwide malaria report states
that in 2018, there were about 228 million cases of malaria, with 85% of these
cases in sub-Saharan Africa and India. Of all the malaria cases worldwide, six
countries were accountable for more than half. To our dismay, our country
Nigeria ranks topmost on this statistics, being accountable for about 25%. This
is alarming! Mortality rate is still on the high side globally, reaching up to
405,000. (WHO, 2019). This rapid spread of malaria and high mortality rate call
for prompt actions which might include the use of more antimalarial
phytomedicines.
1.2 STATEMENT OF THE PROBLEM
Malaria is a deadly disease caused by parasites (Plasmodium falciparum, Plasmodium vivax,
Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi), transmitted to
humans when bitten by infected female Anopheles
mosquitoes. Plasmodium falciparum and
Plasmodium vivax pose the greatest
threat. Plasmodium Knowlesi was
recognized by World Health Organization, WHO in 2008 and rarely causes diseases
in humans (WHO, 2008; WHO, 2017).
The most prominent transmission
medium is through bites from infected female Anopheles mosquitoes that had a blood meal from an individual
infected with the parasitaemia. Other media of transmission include blood
transfusion, organ transplant, unsterilized needles, and from a pregnant mother
to her fetus. Symptoms of malaria which usually begin
within fifteen days include fever, vomiting, headaches and tiredness (Caraballo,
2014). Severe symptoms result in jaundice, coma, seizures, cerebral malaria or
even death (Bartoloni and Zammarchi, 2012). In pregnant women, malaria results
in stillbirths, decrease in birth weight, abortion or infant mortality.
Disastrous consequences indeed!
Methods
used to prevent malaria include medications, vector control and prevention of
bites. Treatment of malaria with antimalarial medications is highly endorsed. From
a public health perspective, one of the goals of treatment of malaria is to
prevent offshoot and recurrence of resistance to antimalarial medicines (WHO,
2018).
1.3 AIM OF THE STUDY
To investigate the potentiality of A.
cordifolia as an antimalarial agent.
1.4 OBJECTIVES OF THE STUDY
Isolation and characterization of the bioactive
components from the leaves of A. cordifolia as well as investigating by in vitro its antimalarial
potentials.
1.5 JUSTIFICATION OF THE STUDY
Malaria has posed a great threat to a greater number
of the world’s population. Thus, there is a prompt need for more antimalarials
to curb drug resistance and reduce mortality rate. If A. cordifolia is investigated and adopted as an
antimalarial, it may result in great benefits to malaria endemic zones and
humanity in general with the following possible benefits:
i.
Possibility of being
proposed and tested as an alternative to existing antimalarials;
ii.
Ability to serve as a
good source of phytochemicals for further production of conventional
antimalarials;
iii.
The phytochemical marker
from A. cordifolia can provide the basis for parallel drug development
of synthetic antimalarials;
iv.
The fact that it is a
widely distributed herb (www.prota4u.org), implies it will be easily accessible
by patients in remote areas; hence it may serve as a complement to existing
antimalarials.
1.6 SCOPE OF THE STUDY
Based on its traditional use as a cure for malaria,
this research work aims at isolating, purifying and investigating the
antimalarial potentials of the bioactive components from the leaves of A.
cordifolia.
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