IN VITRO AND IN SILICO CHARACTERIZATION OF THE 
ANTICHOLINESTERASE ACTIVITY OF SELECT 
TERPENOIDS AGAINST ANOPHELES VECTORS 

 

 

Thankhoe Abram Rants’o 

 

 

 

 

 

 

A thesis submitted to the Faculty of Health Sciences, 

University of the Witwatersrand, 

in fulfilment of the requirements for the degree 

of 

Doctor of Philosophy 

 

Johannesburg, 2023 



i 
 

DECLARATION 

 
I   Thankhoe Abram Rants’o     declare that this thesis is my own, unaided work.  It is being submitted 

for the Degree of Doctor of Philosophy at the University of the Witwatersrand, Johannesburg.  It has 

not been submitted before for any degree or examination at any other University. 

 

 

            _______________ 

 

(Signature of candidate)  

_15th day of March 2023_at__Parktown___________ 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



ii 
 

DEDICATION 

Dedicated to everyone who loved education, devoted to their studies, and hoped for a 

better future but never made it due to financial constraints. 

 I KNOW YOUR PAIN. 

 

~ Thankhoe A Rants’o 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



iii 
 

PUBLICATIONS AND PRESENTATIONS 

 

PUBLICATIONS 

Published articles 

1. Rants’o, T.A., Panayides, J-L., Koekemoer, L.L., van Zyl, R.L. Potential of essential oil-

based anticholinesterase insecticides against Anopheles vectors: A review. Molecules, 

2022; 27(20): 7026; doi: 10.3390/molecules27207026.  

Student’s contribution to the paper 

Design of the study, literature search, data management (including data collection, 

synthesis, and analysis), writing of the manuscript (Appendix F.1.1). 

 

2. Rants'o, T.A., van der Westhuizen, C. J., van Zyl, R.L. Optimization of covalent docking 

for organophosphates interaction with Anopheles acetylcholinesterase. Journal of 

Molecular Graphics and Modelling, 2022;110: 108054. doi: 10.1016/j.jmgm.2021.108054.  

Student’s contribution to the paper 

Design of the study, literature search, methodology, data management, writing of the 

manuscript and responding to the reviewers’ comments (Appendix F.1.2). 

 

3. Rants’o, T.A., van Greunen, D.G., van der Westhuizen, J.C., Riley, D.L., Panayides, J-L., 

Koekemoer, L.L., van Zyl, R.L. The in silico and in vitro analysis of donepezil derivatives 

for Anopheles acetylcholinesterase inhibition. PLoS ONE, 2022;17(11): e0277363. doi: 

10.1371/journal.pone.0277363. 

Student’s contribution to the paper 

Design of the study, literature search, methodology, data management, writing of the 

manuscript (Appendix F.1.3). 

 

4. Rants’o, T.A., Koekemoer, L.L., van Zyl, R.L. In vitro and in silico analysis of the 

Anopheles anticholinesterase activity of terpenoids. Parasitology International, 2023; 93: 

102713. doi: 10.1016/j.parint.2022.102713.   

Student’s contribution to the paper 

Design of the study, literature search, methodology, data management, writing of the 

manuscript (Appendix F.1.4). 



iv 
 

5. Rants’o, T.A., Koekemoer, L.L., van Zyl, R.L. The insecticidal activity of essential oil 

constituents against pyrethroid-resistant Anopheles funestus (Diptera: Culicidae). 

Parasitology International, 2023;95: 102749. doi: 10.1016/j.parint.2023.102749. 

Student’s contribution to the paper 

Design of the study, literature search, methodology, data management, writing of the 

manuscript (Appendix F.1.6). 

 

Manuscripts in submission 

6. Rants’o, T.A., Koekemoer, L.L., van Zyl, R.L. Bioactivity of select essential oil 

constituents against life stages of Anopheles arabiensis (Diptera: Culicidae). Submitted to 

Experimental Parasitology (Manuscript ID: EXPARA-D-22-00337) – under review. 

Student’s contribution to the paper 

Design of the study, literature search, methodology, data management, writing of the 

manuscript (Appendix F.1.5). 

 

PRESENTATIONS 

● Thankhoe A. Rants’o, Lizette L Koekemoer, Robyn L. van Zyl. Elucidation of the 

antiplasmodial mechanism of (Z)-α-santalol. 4th International Congress on Parasites of 

Wildlife. Kruger National Park, South Africa. 11-15 September 2022. [Oral]. 

● Thankhoe A. Rants’o, Lizette L Koekemoer, Jenny-Lee Panayides, Robyn L. van Zyl. 

The differential acetylcholinesterase binding of larvicidal donepezil analogues. Wits 

Research Institute for Malaria Day 2022. Faculty of Health Sciences, University of the 

Witwatersrand, 20 April 2022. [Oral]. 

● Thankhoe A. Rants’o, Jenny-Lee Panayides, Lizette L Koekemoer, Robyn L. van Zyl. 

Multi-stage activity of essential oils on Anopheles arabiensis. 6th Malaria Research 

Conference 2021. SAMRC. 3-4 August 2021 [Oral]. 

● Thankhoe A. Rants’o, Jenny-Lee Panayides, Lizette L Koekemoer, Robyn L. van Zyl. 

The potential of essential oils to overcome insecticide resistance in malaria control. 

IPUF 2021. University of Johannesburg. 5-7 July 2021. [Oral]. 

● Thankhoe A. Rants’o, Jenny-Lee Panayides, Lizette L Koekemoer, Robyn L. van Zyl. 

Structure-activity relationships for Anopheles acetylcholinesterase inhibition. Faculty 

of Health Sciences Research Day & Postgraduate Expo 2020. 15 October 2020. [Oral]. 



v 
 

● Thankhoe A. Rants’o, Jenny-Lee Panayides, Lizette L Koekemoer, Robyn L. van Zyl. 

The anticholinesterase effects of camphor on Anopheles. 53rd Annual Conference of 

the South African Society of Basic and Clinical Pharmacology 2019. Kievits Kroon 

Faircity Hotel Pretoria, South Africa. 5 - 7 October 2019. [Oral]. 

● Thankhoe A. Rants’o, Jenny-Lee Panayides, Lizette L Koekemoer, Robyn L. van Zyl. 

Identification of Anopheles-specific donepezil analogues through molecular modelling. 

School of Therapeutic Sciences Research Day 2019. Faculty of Health Sciences, 

University of the Witwatersrand. 10 September 2019. [Poster]. 

● Thankhoe A. Rants’o, Jenny-Lee Panayides, Lizette L Koekemoer, Robyn L. van Zyl. 

The isomeric effects of camphor on Anopheles. 5th Malaria Research Conference 2019. 

University of Pretoria, South Africa. 30 July - 1 August 2019. [Oral] 

 

AWARDS 

Research grants 

1. Faculty Research Committee Individual Research Grants 2019 (R 12,000) 

2. Seed Funding 2019 (R34,723) 

3. Start-up Funding 2019 (30,000) 

4. Faculty Research Committee Individual Research Grants 2020 (R 12,000) 

5. Faculty Research Committee Individual Research Grants 2021 (R 12,000) 

 

Recognition awards 

1. Award for Best Presenter (1st Prize) in PhD/Post-Doc Category at the WRIM Research 

Day 2022 (Virtual), Faculty of Health Sciences, University of the Witwatersrand, 20th-

April-2022. 

2. Outstanding Mentoring Award from the School of Therapeutic Sciences, Faculty of 

Health Sciences, University of the Witwatersrand, 17th-November-2020 

3. Postgraduate PhD Merit Award (PMA) for academic excellence from the University of 

the Witwatersrand in 2018. 

 

 

 



vi 
 

ABSTRACT 

 

Malaria is a life-threatening plasmodial disease that is transmitted by female Anopheles 

mosquitoes. Major African malaria vectors include Anopheles arabiensis, An. funestus, An. 

gambiae and An. coluzzii. Malaria vector control programs have shown effectiveness in 

reducing the Anopheles populations. The main insecticide classes used in these interventions 

include pyrethroids, organochlorines, organophosphates, carbamates, and neonicotinoids. 

Nevertheless, the development of Anopheles resistance to these insecticide classes has greatly 

reduced the effectiveness of these interventions. A common resistance mechanism is through 

rapid detoxification of insecticides by overexpressed P450 monooxygenases. Although 

acetylcholinesterase (AChE) is a valid target in Anopheles vector, current anticholinesterase 

insecticides suffer from resistance and low selectivity between insect and mammal AChE 

targets. This indicates the urgent need to discover novel AChE inhibitors with higher affinity 

to Anopheles AChE compared to the mammal target, and less prone to resistance caused by the 

overexpressed monooxygenases. Identification of novel AChE inhibitors from natural sources 

and their potential to kill Anopheles during all its different life stages, presents a cost-effective 

approach. This PhD study aimed to identify such novel AChE inhibitors from essential oil 

sources and assess them for consistent activity against Anopheles species with hyperactive 

P450 monooxygenases. In this study, molecular differences between Anopheles and human 

AChEs were identified showing the opportunity to develop selective Anopheles AChE 

inhibitors. A novel approach was used to integrate the in silico and in vitro assays in assessing 

the Anopheles AChE inhibitory potential of select terpenoids and coupled these to the in vivo 

assays against different life stages of Anopheles. The terpenoids, farnesol, (-)-α-bisabolol, cis-

nerolidol, trans-nerolidol, and methyleugenol were identified as potent Anopheles AChE 

inhibitors and larvicidal agents with moderate adulticidal effects. Farnesol and (-)-α-bisabolol 

also displayed pupicidal activity, while methyleugenol inhibited the hatching of Anopheles 

eggs. Generally, farnesol and (-)-α-bisabolol were highly active across the Anopheles species, 

except in the strain with P450-based metabolic resistance. In contrast, the efficacy of cis-

nerolidol, trans-nerolidol, and methyleugenol was not affected by this resistance mechanism.  

This research suggests that cis-nerolidol, trans-nerolidol, and methyleugenol are potential 

candidates for further development as anticholinesterase bioinsecticides. 

 

Keywords: Malaria, Anopheles, life cycle, terpenoids, monooxygenases 

 



vii 
 

ACKNOWLEDGEMENTS 

 

I dearly thank my family for their continuous support. I would like to express my heartfelt 

gratitude to Wits Financial Aid and Scholarships Office that sponsored PhD studies in the very 

first year of enrolment. Without this financial assistance I could not enroll into the program, 

and for that I am grateful. I am indebted to Wits University for offering me the staff bursary 

throughout my studies. Hearty acknowledgements to my supervisors Prof Robyn L van Zyl, 

Prof Lizette L Koekemoer and Dr Jenny-Lee Panayides for the guidance they have given me 

in this research, training and funding of various studies undertaken during the course of this 

PhD. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



viii 
 

Table of Contents 
DECLARATION ......................................................................................................................... i 

DEDICATION ............................................................................................................................ ii 

PUBLICATIONS AND PRESENTATIONS .............................................................................. iii 

PUBLICATIONS ............................................................................................................... iii 

PRESENTATIONS ............................................................................................................ iv 

ABSTRACT ............................................................................................................................... vi 

ACKNOWLEDGEMENTS ...................................................................................................... vii 

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

LIST OF TABLES .................................................................................................................... xv 

LIST OF ACRONYMS AND ABBREVIATIONS ................................................................... xvii 

CHAPTER 1 ............................................................................................................................... 1 

Introduction ........................................................................................................................ 1 

1. Background ..................................................................................................................... 1 

1.1. Anopheles life cycle ................................................................................................... 1 

1.2. Malaria control ........................................................................................................ 2 

1.3. Anopheles vectors ..................................................................................................... 4 

1.4. Insecticide resistance ................................................................................................ 5 

1.5. Acetylcholinesterase and inhibitors .......................................................................... 6 

1.6. Monooxygenases ....................................................................................................... 6 

1.7. Novel AChE inhibitors ............................................................................................. 7 

1.8. Rationale of the study ............................................................................................... 8 

1.9. Aim and objectives ................................................................................................. 10 

2. Structure of the thesis ................................................................................................ 10 

References ......................................................................................................................... 14 

CHAPTER 2 ............................................................................................................................. 27 

Literature review ............................................................................................................... 27 

1. Introduction ................................................................................................................... 29 

2. Malaria Vector Control ................................................................................................. 30 

3. Conclusions and Future Perspective .............................................................................. 46 

References ......................................................................................................................... 48 

CHAPTER 3 ............................................................................................................................. 70 

Optimization of the in silico approach to assess the acetylcholinesterase inhibitory potential 
of chemical entities using molecular modelling and molecular mechanics simulations ...... 70 

1. Introduction ................................................................................................................... 72 

2. Material and methods .................................................................................................... 74 



ix 
 

3. Results and discussion ................................................................................................... 79 

4. Conclusions .................................................................................................................... 90 

References ......................................................................................................................... 92 

CHAPTER 4 ........................................................................................................................... 100 

The in vitro and in silico assessment of donepezil derivatives for the Anopheles 
acetylcholinesterase inhibition ......................................................................................... 100 

1. Introduction ............................................................................................................. 103 

2. Materials and Methods ............................................................................................ 104 

3. Results and Discussion ............................................................................................. 109 

4. In silico studies ......................................................................................................... 111 

5. Conclusions .............................................................................................................. 122 

References ....................................................................................................................... 124 

CHAPTER 5 ........................................................................................................................... 132 

The in vitro and in silico assessment of select terpenoids for the Anopheles 
acetylcholinesterase inhibition ......................................................................................... 132 

1. Introduction ............................................................................................................. 135 

2. Methods and materials ............................................................................................. 137 

3. Statistical analyses ................................................................................................... 140 

4. Results and discussion .............................................................................................. 141 

5. Conclusions .............................................................................................................. 149 

References ....................................................................................................................... 152 

CHAPTER 6 ........................................................................................................................... 163 

The activity of terpenoids against multiple stages of Anopheles life cycle ......................... 163 

CHAPTER 7 ........................................................................................................................... 192 

The insecticidal activity of essential oil constituents against insecticide resistant Anopheles 
with overexpressed P450 monooxygenases....................................................................... 192 

1. Introduction ............................................................................................................. 194 

2. Materials and methods ............................................................................................. 196 

3. Results ..................................................................................................................... 198 

4. Discussion ................................................................................................................ 206 

5. Conclusion ............................................................................................................... 208 

References ....................................................................................................................... 209 

CHAPTER 8 ........................................................................................................................... 216 

Integrative discussion ...................................................................................................... 216 

8.1 Establishment of Anopheles acetylcholinesterase as a critical target ..................... 216 

8.2 Hypothesis of essential oils as sources of novel Anopheles acetylcholinesterase ..... 217 

8.3 Optimization of the in silico approach for assessment of novel AChE inhibitors .. 218 



x 
 

8.4 In silico, in vitro and in vivo analysis of anticholinesterase activity of terpenoids .. 219 

8.5 Terpenoids bioactivity against the Anopheles life cycle ......................................... 222 

8.6 Structure-activity relationships ............................................................................ 223 

8.7 Terpenoids activity against resistant Anopheles strains ........................................ 226 

References ....................................................................................................................... 228 

CHAPTER 9 ........................................................................................................................... 236 

Conclusions and recommendations for future research ................................................... 236 

Concluding remarks ........................................................................................................ 236 

Limitations of the current study ...................................................................................... 236 

Recommendations for future research ............................................................................. 236 

CHAPTER 10 ......................................................................................................................... 238 

Appendices ...................................................................................................................... 238 

Appendix A.1: Potential of essential oil-based anticholinesterase insecticides against 
Anopheles vectors: A review (Chapter 2; published paper) ............................................. 238 

Appendix B.1: Optimization of covalent docking for organophosphates interaction with 
Anopheles acetylcholinesterase (Chapter 3; published paper) .......................................... 261 

Appendix B.2: Supplementary information for Chapter 3 (published paper)……………272 

Appendix C.1: The in silico and in vitro analysis of donepezil derivatives for Anopheles 

acetylcholinesterase inhibition (Chapter 4; published paper)……………………………...279 

Appendix C.2: Supplementary information for Chapter 4 (published paper) .................. 300 

Appendix D.1: In vitro and in silico analysis of the Anopheles anticholinesterase activity of 
terpenoids (Chapter 5; published paper)…………………………………………………….305 

Appendix E.1: The insecticidal activity of essential oil constituents against pyrethroid-
resistant Anopheles funestus (Diptera: Culicidae) (Chapter 7; published paper)………...310 

Appendix F.1: Author declarations ................................................................................. 318 

Appendix G.1: Approval of change of title....................................................................... 324 

Appendix H.1: Human Research Ethics waiver ............................................................... 325 

Appendix I.1: Biosafety Ethics certificate ........................................................................ 326 

Appendix J.1: Animal Research Ethics waiver ................................................................ 327 

 

 

 

 

 

 

 



xi 
 

LIST OF FIGURES 

 

CHAPTER 1 

Figure 1: Four Anopheles stages of growth……………………………………….…………..2 

Figure 2: The parasite developmental stages within the Anopheles host (Gitta and Kilian, 

2020)………………………………………………………………………..………………….4 

 

CHAPTER 2 

Figure 1: The nervous system ganglia of Anopheles…………………………………………33 

Figure 2: Acetylcholine release, postsynaptic receptor binding, and hydrolysis by 

acetylcholinesterase …………………………………………………………...……………..34 

Figure 3: Molecular comparison of An. gambiae wild-type (A) and resistant (B) AChE 

catalytic sites (PDB IDs: 5YDI and 6ARY, respectively) to the human AChE (PDB ID: 7E3H) 

(C), generated by Schrodinger’s Maestro 2018-2 software. The G280S mutation is shown (red 

arrow) in the resistant Anopheles AChE phenotype (B) ...............………………….………..36 

 

CHAPTER 3 

Figure 1: General OP hydrolysis by catalytic serine in the AChE catalytic site……..…..….72 

Figure 2: Chemical structures of ligand OPs………………………………………………...76  

Figure 3: Generated binding site on the AgAChE……………………………………………79 

Figure 4: Malathion (A) and malaoxon (B) docked at AgAChE with a noncovalent 

function………………………………………………………………………………...……..80 

Figure 5: Acephate (A) and methamidophos (B) docked at AgAChE with a noncovalent 

function………………………………………………………………………………...……..80 

Figure 6: Malathion (A) and malaoxon (B) re-docked at AgAChE with MM-GBSA 

method………………………………………………………………………………………..82 

Figure 7: Acephate (A) and methamidophos (B) re-docked at AgAChE with MM-GBSA 

method. ……………………………………………………………………..………………..82 

Figure 8: Malathion (A) and malaoxon (B) docked at AgAChE with new covalent 

function.……………………………………………………………………..………………..83  

Figure 9: Acephate (A) and methamidophos (B) docked at AgAChE with new covalent 

function.…………………………………………………………………..…………………..83 

Figure 10: Superimposed dimethoate in noncovalent (green) and covalent (violet) binding 

states...………………………………………………………………………………………..86 



xii 
 

Figure 11: Superimposed malathion (A) and malaoxon (B) in noncovalent (green) and 

covalent (violet) binding states……………………………………………………………….86 

Figure 12: Superimposed AgAChE active site amino acid residues non-aged (green) and aged 

by malathion or malaoxon (red) versus acephate or methamidophos (violet).  (A) Shows the 

catalytic serine reorientation (arrow) in malathion or malaoxon phosphorylation (red) and (B) 

shows the reorientation distance from the non-aged catalytic serine (2.55 Å) and increased 

distance from histidine (3.42 Å). Ligands have been undisplayed to enhance 

visibility…………………………………………………………………..…………………..87 

 

CHAPTER 4 

Figure 1: Chemical structure of donepezil showing two common sides of derivatization: A) 

indanone moiety and B) methyl linker……………………………………………………  .103 

Figure 2: Chemical structures of the nine donepezil derivatives screened. 1-benzyl-N-(2-

morpholinoethyl) piperidine-4-carboxamide 1, 1-benzyl-N-(thiazol-2-yl) piperidine-4-

carboxamide 2, 1-benzyl-N-(3,4,5-trimethoxyphenethyl) piperidine-4-carboxamide 3, 1-

benzyl-N-(pyridine-3-ylmethyl) piperidine-4-carboxamide 4, 1-benzyl-N-(furan-2-ylmethyl) 

piperidine-4-carboxamide 5, N-[2-(1H-imidazol-4-yl)ethyl]-1-benzylpiperidine-4- 

carboxamide 6, 1-benzyl-N-(1,2,3,4-tetrahydro-naphthalen-1-yl) piperidine-4-carboxamide 7, 

1-benzyl-N-(2-(piperidin-1-yl)ethyl)piperidine-4-carboxamide 8, and 1-benzyl-N-(pyridine-4-

ylmethyl) piperidine-4-carboxamide 9…………………………………..………..………...105 

Figure 3: Quality of the An. arabiensis homology model. A) Shows the quality estimate of the 

generated model based on its similarity to the template and B) shows its comparison to non-

redundant 3D structures………………………………………….……………...…………..113 

Figure 4: Validity of the model displayed by Verify3D tool (A) and scored by MolProbity (B) 

and the corresponding Ramachandran plot (C)………………………………….………….115  

Figure 5: Alignment of the catalytic sites of the generated An. arabiensis AChE (sky blue) and 

reference An. gambiae (plum)…………………………………………...…………………..116  

Figure 6: Molecular interactions of a derivative 2 with electric eel AChE (A; Score: -11.8) and 

An. arabiensis (B; Score: -7.5)………………………………………..…………………….117 

Figure 7: Superimposed amino acid residues showing the catalytic site entrance and the 

catalytic triad of electric eel (red), human (green) and Anopheles (blue) AChE. The distinct 

Cys447 at the Anopheles AChE catalytic entrance and Asp602 at the catalytic site base are shown 

by circles. ……………………………………………...……………………………………118 



xiii 
 

Figure 8: Comparison of the molecular interactions of donepezil with the AChE catalytic sites 

of electric eel (A; PDB: 1EVE; score: -15.0), human (B; PDB: 4EY7; score: -16.2) and An. 

gambiae (C; PDB: 5YDI; score: -10.2)……………………..……………………………....119 

Figure 9: Comparison of the molecular interactions of derivative 2 with the AChE catalytic 

sites of An. arabiensis model (A; score: -7.5), wild-type An. gambiae (B; score: -9.2), target 

site mutated An. gambiae (C; score: -7.8), An. coluzzii model (D; score: -9.0) and An. funestus 

model (E; score: -9.1)………………………………………………………………………..122 

 

CHAPTER 5 

Figure 1. Chemical structures of the terpenoids…………………..………………………...141 

Figure 2. Differences in farnesol poses against Anopheles AChE (A) and electric eel AChE 

(B)………………………………………………………………….………………………..146  

Figure 3. Comparison of propoxur binding pose against Anopheles (A) and electric eel 

(B)………………………………………………………………….………………………..146 

Figure 4. Crystal poses of (-)-α-bisabolol (A), cis-nerolidol (B), trans-nerolidol (C) and 

methyleugenol (D) in the Anopheles AChE binding site……………………….….………..147 

Figure 5: Superimposition of terpenoid poses on the active site of Anopheles AChE. (R)-(+)-

Limonene lodged at the active site entrance is shown in green…………….…….…………148 

 

CHAPTER 6 

Figure 1: Chemical structures of the EOCs and a positive control, propoxur, investigated for 

insecticidal activity………………………………………...………………………………..168 

Figure 2: The IC50-based comparison of the egg hatching inhibitory potential of 
EOCs………….……………………………………………………………….……………171  
Figure 3: The structural comparison of methyleugenol and propoxur, a standard insecticide. 

These molecules share a 1,2-dimethoxybenze moiety (circled).…………….….…………..172 

Figure 4: The close structural similarities between farnesol and (-)-α-bisabolol is indicated by 

the former cyclising to the latter….………………………………………………………….175 

 

CHAPTER 7 

Figure 1: Cytochrome C oxidase standard curve (inset graph) used to determine the linearity 

of TMB substrate peroxidation (main graph) (cytochrome C concentration: 1.036 to 66.600 

µM)…………………………………………………………….……………………………199 



xiv 
 

Figure 2: Monooxygenase activity in An. funestus. (A) Comparison of the equivalent units of 

cytochrome P450/mg protein in An. funestus FUMOZ-R and An. funestus FANG; (B) 

Comparison of the P450 activity in An. funestus FUMOZ-R and An. funestus FANG…..200 

Figure 3: The susceptibility status of FANG and FUMOZ-R against three most active EOCs 

assessed at their double LC99 concentrations (a). The comparison of FANG and FUMOZ-R 

susceptibility to three less active EOCs and one EO (b). The terpenoid-induced mortalities were 

compared to deltamethrin. *Only deltamethrin in (a) showed significantly different mortality 

rates between FANG and FUMOZ-R strains (p<0.05) while all test compounds in (b) showed 

significant difference in the mortality against FANG versus FUMOZ-R (p<0.05)……….....202 

 

CHAPTER 8 

Figure 1: Chemical similarity of propoxur and methyleugenol. The common 1,2-

dimethoxybenze group is enclosed in a rectangular box…………………………………….223   

Figure 2: Propoxur (A) and methyleugenol (B) in the Anopheles AChE binding 

site…………………………………………………………………………………...……...224 

Figure 3: The close similarity of farnesol and (-)-α-bisabolol chemical structures. Farnesol is 

aliphatic while (-)-α-bisabolol is cyclic……………………………………………………..224 

Figure 4: Comparison of the molecular interactions of farnesol (A) and (-)-α-bisabolol (B) 

with the Anopheles AChE binding site……………………………………………………...225 

Figure 5: Comparison of the Anopheles AChE binding profiles of farnesol cis-nerolidol (A) 

and trans-nerolidol (B)……………………………………………………………...………226  

 

 

 

 

 

 

 

 

 

 



xv 
 

LIST OF TABLES 

CHAPTER 2 

Table 1: Anticholinesterase and insecticidal activities of essential oils……………..………..39 

Table 2: Major constituents of EOs with anticholinesterase insecticidal activity……………41 

Table 3: Anticholinesterase and insecticidal activities of essential oil constituents……..….44 

 

CHAPTER 3 

Table 1: Glide docking scores for OPs bonded to 5YDI target AgAChE protein……....……84 

Table 2: Comparison of amino acid residues involved in the binding of OPs to 5YDI target 

AgAChE protein in different docking methods…………………………………………...…..85 

 

CHAPTER 4 

Table 1: Larvicidal activities of the assessed compounds…………………………………..110 

Table 2: Comparison of the Anopheles AChE inhibitory potential of derivatives 1, 2, 5 and 

8………………………………………………………………………………………...…...111 

Table 3: Comparison of the electric eel AChE inhibitory potential of derivatives 1, 2, 5 and 

8………………………………………………………………………………………..……112 

 

CHAPTER 5 

Table 1: Comparison of the larvicidal and artemicidal activities of the assessed 

terpenoids.…………..............................................................................................................143 

Table 2: Comparison of the Anopheles and electric eel anticholinesterase activity of the 

identified terpenoids…………………………………………………………………….…..145 

Table 3: The binding scores and amino acid interactions of terpenoids and Anopheles AChE 

Table 4: The comparison of the docking scores and amino acid interactions of farnesol and 

propoxur against electric eel AChE……………………………………………………..…..149 

 

CHAPTER 6 

Table 1: Concentration-based comparison of the Anopheles egg hatching inhibitory activities 

of the select EOCs compared to propoxur…………………………………………….…….170 

Table 2: Concentration-dependent larval lethality of the assessed EOCs compared to 

propoxur…………………………………………………………………………………….173 



xvi 
 

Table 3: The EOC-induced pupal sensitivity to standardized concentrations compared to 

propoxur……………………………………………………………………...……………..174 

Table 4: Concentration-dependent adulticidal activity of EOCs against An. 

arabiensis…………………………………………………………………………...………176  

Table 5: Concentration-dependent adulticidal activity of propoxur against An. 

arabiensis…………………………………………………………………………...………176  

 

CHAPTER 7 

Table 1: WHO susceptibility analysis of An. funestus colonies, FANG and FUMOZ-R, to % 

deltamethrin…………………………………………………………………………..……..198 

Table 2: The lethality concentrations of EOCs against pyrethroid-susceptible An. 

funestus……………………………………………………………..………….………..…..201 

Table 3: Activities of select EOCs against An. funestus FUMOZ-R………………………..203 

Table 4: Comparison of terpenoid potencies between pyrethroid-susceptible and resistant An. 

funestus strains………………………………………………………………………………204 

Table 5: Evaluation of An. funestus FUMOZ-R susceptibility to deltamethrin and EOCs with 

and without a synergist…………………………………………………………………...…205 

Table 6: Co-toxicity effects of four select EOCs in combination with PBO against An. funestus 

FUMOZ-R…………………………………………………………………………………..205 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



xvii 
 

LIST OF ACRONYMS AND ABBREVIATIONS 

 

3D: 3-dimensional 

Å:  Angstrom 

ACh:  Acetylcholine  

AChE:  Acetylcholinesterase 

AgAChE:   Anopheles gambiae Acetylcholinesterase 

ANOVA: Analysis of variance 

BChE:  Butyrylcholinesterase 

CDC:  Centers for Disease Control and Prevention  

CNS:  Central Nervous System  

CYP450:  Cytochrome P450 

DDT:  Dichloro-diphenyl-trichloroethane  

DEET: N,N-diethyl-meta-toluamide 

DF: Degree of Freedom 

DMSO:  Dimethylsulfoxide  

DNA: Deoxyribonucleic acid 

DTNB: Dithiobis(2-nitrobenzoic acid)  

EO:  Essential Oil 

EOC:  Essential Oil Constituent 

et al: et alia (and others) 

FDA:  Food and Drug Administration 

GABA:  γ-Aminobutyric Acid 

GMQE:  Global Model Quality Estimate 

IC50: 50% Inhibitory Concentration 

IRS:  Indoor Residual Spraying 

ITNs:  Insecticide-Treated Mosquito Nets 

kdr:  knockdown resistance  

L:  Litre 

LC50: 50% Lethal Concentration 

LD50: 50% Lethal Dose  

LC90: 90% Lethal Concentration 

LC99: 99% Lethal Concentration 

LLINs:  Long-Lasting Insecticidal Nets 



xviii 
 

µg: microgram 

µg/ml:  microgram per millilitre 

mg: milligram 

mg/ml: milligram per millilitre 

µL: microlitre 

mL: millilitre 

µM: micromolar 

mM: millimolar 

MM-GBSA:  Molecular Mechanics - Generalised Born Surface Area  

nAChR:  Nicotinic Acetylcholine Receptor 

NPs:  Natural Products 

OP:  Organophosphate 

PBO:  Piperonyl Butoxide 

PDB:  Protein Data Bank 

QSAR:  Quantitative Structure-Activity Relationship  

QSQE:  Quaternary Structure Quality Estimation 

TMB:  3,3′,5,5′-Tetramethylbenzidine  

U/mL: Units per millilitre 

v/v: volume per volume  

w/v: weight per volume 

WHO:  World Health Organization 

 

 

 

 

 

 

 

 

 

 

 

 

 



1 
 

CHAPTER 1 

Introduction 

1. Background 

Malaria is a life-threatening disease caused mainly by Plasmodium falciparum parasites that 

are further transmitted to humans by infected female mosquitoes of the Anopheles genus 

(WHO, 2017). The recent World Health Organization (WHO) Malaria Global Report indicated 

a fourteen million increase in 2020 malaria cases (241 million) compared to 2019 (227 million), 

accompanied by a 12% increase in deaths (627 000 deaths (2020) versus 558 000 deaths 

(2019)). The WHO African Region reported exceedingly high malaria burdens in the African 

region encompassing 95% and 96% of global cases and deaths, respectively (WHO, 2021a). 

The main challenge in malaria treatment is the escalating and recurring resistance to 

antimalarial drugs and insecticides (WHO, 2016; CDC, 2020). Resistance of P. falciparum to 

previously potent antimalarial drugs including sulfadoxine-pyrimethamine and chloroquine has 

become widespread in Africa (Roux et al., 2021). Likewise, the Anopheles resistance to various 

classes of insecticides has been reported across the African continent (Pinda et al., 2020; WHO, 

2021b).  

 

1.1.  Anopheles life cycle 

As for all mosquitoes, the Anopheles mosquitoes’ life cycle is composed of four stages namely, 

egg, larva, pupa, and adult, with the first three stages being aquatic (Figure 1). At adult stage, 

certain species of the female Anopheles mosquitoes are a potential malaria vector (Service, 

1980). The adult females lay eggs in water and these hatch in a period of two to three days. 

The larvae stages progress through four instars where moulting occurs as each instar is 

completed to allow for the next stage of growth. Fourth instar larvae develop into a comma-

shaped pupa before maturing into an adult mosquito within a few days. A fully developed adult 

mosquito has three body sections: head, thorax and abdomen (Figure 1) (Service, 1980; 

Williams and Pinto, 2012). 



2 
 

 
Figure 1: Four Anopheles stages of growth (Adapted from Williams and Pinto, 2012). 

 

1.2.  Malaria control 

The control of malaria includes three integrated interventions: vector control, early diagnosis 

and treatment. Vector control is an essential strategy for reduction of the mosquito population 

resulting in a reduction in malaria transmission. Equally so, early diagnosis and malaria 

treatment with standard antimalarial drugs are important in preventing and reducing malaria 

transmission (WHO, 2018). Therefore, vector control interventions are used together with 

antimalarial drugs in the standard malaria control strategies (WHO, 2016). This thesis focused 

on vector control as an essential element in preventing malaria transmissions. 

 



3 
 

1.2.1. Malaria vector control 

Vector control involves two main strategies, which are the use of insecticidal agents and insect 

repellents. The repellents can be impregnated in clothing, nets or be applied as creams, lotions, 

soaps, candles, or sprays for personal (topical repellent) or area (spatial/airborne repellent) 

protection (Nguyen et al., 2018; Maia et al., 2018). The common constituent in the insect 

repellent products is N,N-diethyl-3-methylbenzamide (DEET). This synthetic repellent is 

generally safe and has been used as a repellent since the 1950s. The alternative repellents 

include natural products such as citronella oil, eucalyptus oil, and catnip oil (Nguyen et al., 

2018; Swale and Bloomquist, 2019). However, the potential effectiveness of insect repellents 

in reducing malaria transmissions has limited evidence (Chen-Hussey et al., 2013; Maia et al., 

2018).  

 

The vector control intervention with public health impact relies on the use of insecticides. With 

insecticides, there are mainly two interventions: indoor residual spraying (IRS) and insecticide-

treated mosquito nets (ITNs) or long-lasting insecticidal nets (LLINs) (WHO, 2016). 

Insecticide classes include pyrethroids, organochlorines, carbamates, organophosphates, 

neonicotinoids, bacterial larvicides, insect growth regulators, pyrroles and spinosyns (Sparks 

and Nauen, 2015; WHO, 2021c). The major insecticide classes used in malaria vector control 

are pyrethroids, organochlorines, carbamates, and organophosphates. Recently, the 

combination of a pyrethroid with a P450 monooxygenase synergist, piperonyl butoxide, has 

been added to the vector control intervention list (WHO, 2021a). The ITNs use pyrethroids and 

the IRS relies mostly on organophosphates and more recently, neonicotinoids (WHO, 2021a). 

Pyrethroids and organochlorines target voltage-gated sodium channels in which they cause 

sustained depolarization state of the neurons, which in turn leads to paralysis and death (Field 

et al., 2017). The carbamates and organophosphates inhibit the key enzyme in the cholinergic 

system, namely, acetylcholinesterase (AChE), leading to unregulated cholinergic activity with 

consistent activation of the post-synaptic membrane which eventually results in paralysis and 

death (Fukuto, 1990). Similarly, the neonicotinoids modify the insect’s cholinergic activity by 

antagonizing the binding of a neurotransmitter acetylcholine to the post-synaptic membrane 

receptors, namely nicotinic acetylcholine receptors (Simon-Delso et al., 2015). The focus of 

the current study was on the cholinergic activity of Anopheles nervous system, particularly the 

AChE activity. More detailed discussion on Anopheles AChE activity, challenges with current 



4 
 

AChE inhibitors and potential for natural product-based novel AChE inhibitors is found in 

Chapter 2. 

 

1.3.  Anopheles vectors 

1.3.1. Anopheles-human host interaction 

The female Anopheles mosquitoes transmit malaria through taking a blood meal from an 

infected human host in which they ingest Plasmodium gametocytes (sexually matured (male 

and female) erythrocytic stage of the Plasmodium parasite) (Figure 2) (CDC, 2020). The male 

(micro-gamete) and female (macro-gamete) gametocytes mate in the mosquito’s midgut to 

form zygotes. The zygotes develop into ookinetes that then penetrate the Anopheles midgut 

wall and form oocysts. These oocysts rupture and form sporozoites that are ready to infect the 

next human host. To enhance the disease transmission, sporozoites get concentrated in 

mosquito’s salivary glands in which they get released upon the bite of the human host (CDC, 

2020; Gitta and Kilian, 2020). This parasitic development process within the mosquito host is 

known as a sporogonic cycle (CDC, 2020).   

 
Figure 2: The parasite developmental stages within the Anopheles host (Adapted from Gitta 

and Kilian, 2020). 

 

1.3.2. Anopheles species in Africa 

Species from the Anopheles gambiae complex; An. gambiae, An. arabiensis, and An. coluzzii 

as well as An. funestus from the An. funestus group are the main malaria vectors in Africa 

(WHO, 2021a; Dahan-Moss et al., 2020; Sinka et al., 2012). In the recent literature, other 



5 
 

Anopheles species such as An. stephensi and An. merus are becoming more common in malaria 

transmissions (Bartilol et al., 2021; WHO, 2021a). Anopheles stephensi was localized in Asia 

before spreading to Africa where it was first reported from Djibouti in 2012 (Faulde et al., 

2014; Kweka, 2022; Mnzava et al., 2022). Anopheles merus is a member of the An. gambiae 

complex that is fast becoming an important malaria vector in East and Southern Africa 

including South Africa (Mbokazi et al., 2018; Bartilol et al., 2021). Other species identified in 

the African region include An. melas, An. nili s.l., An. moucheti, as well as An. pharoensis to 

mention but a few (Sinka et al., 2012; Antonio-Nkondjio and Simard 2013; Moyo et al., 2021; 

WHO, 2021a). 

 

1.4.  Insecticide resistance 

Significant resistance to the commonly used insecticides for vector control; organophosphates, 

organochlorides, carbamates and pyrethroids, has been reported (Engdahl et al., 2015; Brooke 

et al., 2013). This resistance can be due to various mechanisms such as target site mutation, 

metabolic enzymes detoxification, thickened cuticle, and behavioural avoidance (Weill et al., 

2003; Witzig et al., 2013; Namias et al., 2021). Target site mutations mutations have been 

detected in the insecticide targets such as AChE and sodium channels which prevents effective 

binding of the concerned insecticides to their target site, thus affecting their effectiveness. The 

overexpressed metabolic enzymes such as P450 monooxygenases, glutathione S-transferases 

and nonspecific esterases have been shown to detoxify the insecticides, and this affects the 

effectiveness of these insecticide (Witzig et al., 2013; Prasad et al., 2017; Asadi et al., 2019; 

Tchouakui et al., 2019; Namias et al., 2021). On the other hand, the cuticular mechanism 

reduces the penetration capacity of insecticides through the Anopheles epicuticle. The 

behavioural mechanism is observed whereby mosquitoes develop a sense of identification and 

avoidance of insecticide-treated areas such as pyrethroid ITNs (Ariaratnam et al., 1975; Lewis, 

1980; Wood et al., 2010; Kreppel et al., 2020; Namias et al., 2021). The latter mechanism is 

not fully understood (He et al., 2019; Namias et al., 2021).  

 

Among these resistance routes, this thesis focused on P450 monooxygenases; major metabolic 

enzymes that detoxify pyrethroids, the only class of insecticides approved for use on bed nets 

(Brooke et al., 2001; Hunt et al., 2005; WHO, 2021a). Therefore, while aiming to identify novel 

Anopheles AChE inhibitors, this study also monitored a change in their insecticidal activity 

against Anopheles species that had upregulated P450 monooxygenases. 



6 
 

 

 

1.5.  Acetylcholinesterase and inhibitors 

1.5.1. Acetylcholinesterase in Anopheles 

Two types of cholinesterase, AChE and butyrylcholinesterase (BChE), are physiologically 

present in both humans and mosquitoes. To avoid co-activity in humans, insecticide specificity 

for the mosquito’s AChE is essential (Dou et al., 2013). Localization of AChE is mainly the 

neuronal synapses while BChE is mainly found in blood plasma (Taylor et al., 2009). While 

AChE can be found in the central nervous system and neuromuscular junctions in humans, it 

is localized only in the central nervous system on insects; whereby it serves to maintain normal 

neuronal impulse transmission (Casida and Durkin, 2013).  

 

Inhibition of AChE has been the target site for two of the four major classes of insecticides: 

organophosphates and carbamates. Exposure to these insecticides leads to insect death from 

neuronal hyperexcitation (English and Webster, 2012). However, mutations in the AChE genes 

have greatly decreased the effectiveness of these insecticides (Casida et al. 2004). Unlike 

humans where a single ace gene codes for AChE, mosquitoes have two ace genes (ace-1 and 

ace-2) coding for AChE1 and AChE2 genotypes, respectively (Weill et al., 2002; Hoffmann et 

al., 1992). The DNA sequence showed that there is about 53% similarity between ace-1 and 

ace-2 genes in Anopheles mosquitoes (Weill et al., 2002; Elamathi et al., 2016). The AChE1 is 

responsible for the enzymatic activity in the cholinergic system while AChE2 is involved in 

the reproductive system development of female mosquitoes (Lu et al., 2012). The mutation 

responsible for insecticide resistance has been reported in AChE1 (ace-1R) whereby a glycine 

was converted to serine at position 119 (G119S) in An. gambiae, and at lower intensities on 

An. coluzzii and An. arabiensis (Essandoh et al., 2013; Weill et al., 2003; Keita et al., 2020).  

 

1.6.  Monooxygenases 

Multiple P450 monooxygenases have been associated with insecticide resistance (Cassone et 

al., 2014; Witzig et al., 2013). These vary between species and even geographical variations 

within individual species. The amplified P450 monooxygenases mainly reduce the activity of 

pyrethroids though other insecticides including organochlorines, carbamates and 

organophosphates are affected at comparatively lower intensities (Nardini et al., 2013; Matowo 

et al., 2022). The main P450 genes implicated in pyrethroid resistance include CYP6P4, 



7 
 

CYP6M2, CYP6P3 (Müller et al., 2008; Wagah et al., 2021; Matowo et al., 2022), CYP6P9, 

CYP12F2, CYP6AG1, CYP6Z1 and CYP6Z3 (Nardini et al., 2013; Chiu et al. 2008, Ibrahim et 

al., 2016; Norden et al., 2022) just to name a few. The overexpressed P450 monooxygenases 

have been detected in An. funestus and An. arabiensis, An. gambiae and An. coluzzii (Christian 

et al. 2011; Koekemoer et al., 2011, Munhenga and Koekemoer, 2011; Venter et al., 2017; 

Ingham et al., 2017; Wagah et al., 2021; Nolden et al., 2022). 

 

1.7.  Novel AChE inhibitors 

Due to the increasing resistance to insecticides, there is an urgent need for the discovery of 

novel insecticides with capacity to overcome resistance (Pinda et al., 2020; WHO, 2021b). 

AChE serves as a critical target for the neuroactive agents (discussed extensively in Chapter 2) 

(Ĉolović et al., 2013). A molecular mechanism of current AChE inhibitors shows that they 

form an irreversible complex with the AChE catalytic serine residues that is responsible for 

acetylcholine hydrolysis (Hörnberg et al., 2007; Rants’o et al., 2022). However, this catalytic 

serine residue is also present in mammals and is often targeted by the AChE inhibitors used in 

the treatment of Alzheimer's disease (Dou et al., 2013). Using the in silico studies, this thesis 

showed key differences between the AChE binding sites of mammals and Anopheles 

mosquitoes, indicating the possibility of identifying Anopheles selective anticholinesterases. 

 

The AChE inhibitors that have been originally designed for the treatment of Alzheimer's 

disease but showed no activity against the human target have been suggested as a promising 

group of novel compounds that may act against the Anopheles AChE (Pang et al., 2009; Hartsel 

et al., 2012; Dou et al., 2013; Souto et al., 2021; Montgomery et al., 2022). Structural analogues 

of the most potent AChE inhibitor, donepezil, a piperidine derivative used for Alzheimer’s 

disease, that lacked significant activity against mammal AChE (van Greunen et al., 2019) have 

been explored for potential activity against Anopheles AChE in Chapter 4 of this thesis. 

 

Among the plant-derived natural products (NPs), essential oils (EOs) have been widely studied 

for their insecticidal activity (Gnankiné and Bassolé, 2017; Luo et al., 2022; Wangrawa et al., 

2022). Though these NPs have been implicated in AChE inhibition of other mosquito species 

(Seo et al., 2015; Liu et al., 2021; Santos et al., 2021), AChE inhibition as the mechanism of 

insecticidal activity in Anopheles species by EOs and essential oil constituents (EOCs) has not 

been well studied and characterized.   



8 
 

 

In this study, the anticholinesterase activity of select EOCs was studied in vitro and their 

insecticidal activities evaluated using in vivo bioassays. With molecular modelling being a 

substantial tool for studying the mechanism of action of compounds at a molecular level 

(Kanitkar et al., 2021; Rants’o et al., 2022), various techniques of molecular modelling have 

been used to elucidate the Anopheles AChE inhibition by terpenoids in this thesis for the first 

time. Moreover, the effectiveness of Anopheles anticholinesterases against Anopheles colonies 

with overexpressed monooxygenases is reported in this study.  

 

In general, AChE is a critical enzyme in the Anopheles central nervous system. Biochemical 

and molecular studies have suggested critical differences between mammal and Anopheles that 

create opportunity for identification of Anopheles selective AChE inhibitors. In order to 

identify selective Anopheles anticholinesterases, molecular modelling technology can be used 

to study the binding differences of potential AChE inhibitors between the Anopheles and 

mammal targets. On the other hand, the upregulated P450 monooxygenases is the most 

concerning Anopheles resistance mechanism that compromises vector control interventions. 

Hence, the goal of this project was to identify novel Anopheles AChE selective inhibitors with 

sustained insecticidal activity against Anopheles species with overexpressed P450 

monooxygenases. 

 

1.8.  Rationale of the study 

The resistance against insecticides by all main African malaria vectors has increased in both 

intensity and geographical distribution (WHO, 2021b). The target site for almost all approved 

insecticides including pyrethroids, organochlorines, organophosphates, carbamates, and 

neonicotinoids is the insect nervous system. Due to small sizes, the mosquitoes have a short 

pathway to the nervous system making it easy for the penetration and subsequent systemic 

distribution of an insecticide from contact exposure (Davies et al., 2007). The pyrethroids and 

organochlorines target the voltage-gated sodium channels in the peripheral and central nervous 

system (Silver et al., 2014). The organophosphates, carbamates, and neonicotinoids modify the 

cholinergic activity of the central nervous system through alteration of the AChE activity 

(organophosphates and carbamates) or agonism at nicotinic acetylcholine receptors 

(neonicotinoids). Acetylcholine is a key excitatory neurotransmitter in the insect nervous 

system making AChE the main nervous system enzyme (English and Webster, 2012; Simon-

Delso et al., 2015).  AChE is therefore a critical target for the discovery of novel insecticides 



9 
 

hence current studies are aimed at identifying novel AChE inhibitors with high selectivity 

towards Anopheles over the corresponding mammalian targets (Engdahl et al., 2016; Carlier et 

al., 2017).  

 

A potent human AChE inhibitor, donepezil, has been shown to be active against insect AChE, 

but is about 40 times more selective to human than the corresponding Anopheles target 

(Williamson et al., 2013; Engdahl et al., 2015). The current study assessed the donepezil 

derivatives prepared by van Greunen et al. (2019) for potential selective Anopheles AChE 

inhibition and rationalised their binding profiles through molecular docking. On the side of 

NPs, previous studies have identified essential oils as potential sources of cost-effective and 

eco-friendly bioinsecticides (Pavela, 2015; Isman, 2020). Various terpenoid classes including 

sesquiterpenes, monoterpenes, and diterpenes form the main chemical constituent repository 

of essential oils (Sharifi-Rad et al., 2017). Jankowska et al. (2017) stated that some essential 

oils display the insect neurotoxic effects characterized by rapid paralysis and death. This is 

consistent with neurotoxic effects caused by AChE inhibition (Colović et al., 2013).  Indeed, 

some essential oils and EOCs have shown the anticholinesterase potential (Miyazawa and 

Yamafuji, 2005; Alout et al., 2012). This study assessed several EOCs including sesquiterpene 

alcohols, monoterpenes and phenylpropanoids for Anopheles AChE. All in vitro AChE 

screening assays were carried out on both Anopheles and electric eel AChE targets for 

selectivity analysis. 

 

In this study, a novel in silico approach of the integrated homology modelling, molecular 

modelling, and molecular dynamic simulations was used to characterize the AChE binding 

profiles of donepezil derivatives and terpenoids. Molecular structures of human, electric eel 

and Anopheles AChE binding sites have been extensively studied to identify key differences 

that may explain the differences observed in the in vitro activities between these targets and 

permit the rational design of selective Anopheles AChE inhibitors for future studies. 

For relevance to field application, the identified AChE inhibitors from in vitro and in silico 

studies were assessed for in vivo activity against all four stages of Anopheles life cycle 

including egg, larva, pupa and adult. The laboratory strains of four main African malaria 

vectors An. gambiae (COGS), An. arabiensis (KWAG), An. funestus (FANG, FUMOZ and 

FUMOZ-R) and An. coluzzii (G3) were used in this study. The cytotoxicity potential of the 

identified Anopheles AChE inhibitors was assessed through artemicidal assay using Artemia 

franciscana (Hübsch et al., 2014). 



10 
 

 

1.9. Aim and objectives 

1.9.1.  Aim 

The overall aim of this thesis was to identify novel Anopheles AChE selective inhibitors with 

unique molecular binding profiles coupled to the insecticidal activity that is sustained in 

Anopheles species with upregulated P450 monooxygenases.  

1.9.2. Specific objectives 

To achieve the aim of this project, the following objectives were set:  

A. To determine and characterize the AChE inhibition of select terpenoids by evaluating 

them against AChEs from electric eel and Anopheles using in vitro enzyme assays and 

in silico modelling technology.  

B. To assess the aquatic toxicological profile of the identified AChE inhibitors against 

Artemia franciscana.  

C. To evaluate the identified Anopheles anticholinesterases for insecticidal activity against 

Anopheles vector life stages including egg, larva, pupa and adult. 

D. To assess the change in insecticidal activity of novel Anopheles AChE inhibitors when 

screened against the Anopheles species with upregulated P450 monooxygenases. 

E. To assess the combination effect of novel Anopheles AChE inhibitors with synergists 

on Anopheles species with overexpressed P450 monooxygenases 

 

2. Structure of the thesis 

The structure of this thesis follows the integrated format outlined in the University of the 

Witwatersrand’s Faculty of Health Sciences guide for PhD thesis formats. Other chapters of 

this thesis are presented in the form of published work, manuscript submitted for publication 

and final manuscript ready for submission to the identified journal. For this reason, the general 

formatting and referencing styles of such chapters are aligned with the requirements of their 

specific journals. 

 
Chapter 1: The introduction chapter discusses the background information of the study, the 

study rationale, significance of the study, outline the study aim and objectives, and conceptual 

framework. 

 



11 
 

Chapter 2: The literature review chapter. This chapter is presented as a published paper 

entitled “Potential of essential oil-based anticholinesterase insecticides against Anopheles 

vectors: A review”. The paper discusses acetylcholinesterase as a critical target in the 

Anopheles nervous system and essential oils as potential sources of novel inhibitors of this 

target. Therefore, this paper establishes the base for this thesis. It has been published in the 

special edition (Essential Oils: Characterization, Biological Activity and Application) in 

Molecules (Molecules 2022;27(20): 7026; https://doi.org/10.3390/molecules27207026).  

 

Chapter 3: This chapter outlines the study methodology. The chapter consists of a published 

paper. As indicated in objective A, this study aimed to evaluate the acetylcholinesterase 

inhibition potential through both the in vitro and in silico methods.  The paper presents the 

optimization of in silico methods including molecular modelling and molecular mechanics 

simulations. These optimized methods were further used in the following chapters of this thesis 

to assess the binding modes of the identified novel Anopheles AChE inhibitors. This paper was 

published in the Journal of Molecular Graphics and Modelling (J Mol Graph Model. 2022;110: 

108054; https://doi.org/10.1016/j.jmgm.2021.108054). 

 

Chapter 4: This chapter is presented as a published paper. As done in Chapter 3, molecular 

modelling is performed using a 3-dimensional (3D) structure of the target protein. If available, 

these proteins can be obtained in Protein Data Bank (PDB). In the case where the required 

protein is unavailable, the method named homology modelling is used to generate new 3D 

protein structures. These new models should be validated and meet the set quality standards 

before use in the molecular modelling work. Among the Anopheles species used in this thesis 

(An. gambiae, An. arabiensis, An. coluzzii and An. funestus), only An. gambiae had its AChE 

crystal structure available in the PDB repository at the time of writing this thesis. Therefore, 

Chapter 4 focuses mainly on the application of homology modelling to generate AChE crystal 

structures for An. arabiensis, An. coluzzii and An. funestus.  

 

Moreover, the in vitro assessment of Anopheles AChE inhibition, in vivo larvicidal activity 

against the assessed Anopheles species along with aquatic toxicity assessment through 

artemicidal assay were conducted in this published paper. This paper used a set of compounds 

derived from a known potent human AChE inhibitor, donepezil. These compounds were used 

to optimize the methodology before assessing the EOCs. Moreover, this was also based on the 

hypothesis that donepezil derivatives that lack activity against mammal AChE target may 

https://www.mdpi.com/journal/molecules/special_issues/essential_oils_characterization_biological_activity_application
https://doi.org/10.3390/molecules27207026
https://doi.org/10.1016/j.jmgm.2021.108054


12 
 

selectively possess activity against insect target. This paper was published in PLoS ONE (PLoS 

ONE, 2022;9;17(11): e0277363; https://doi.org/10.1371/journal.pone.0277363). Both the 

objectives A and B have been fulfilled in Chapter 4. 

 

Chapter 5: This chapter consists of a published paper. The paper outlines the in vitro and in 

silico assessment of select terpenoids for Anopheles AChE inhibition. This chapter utilized the 

same methods used in Chapter 3 but with a different set of compounds being the terpenoids. 

This chapter therefore addresses both objectives A and B by identifying selective Anopheles 

AChE inhibitors and assessing their artemicidal toxicity potentials. The paper has been 

published in Parasitology International (Parasitol Int. 2023;93: 102713; 

https://doi.org/10.1016/j.parint.2022.102713). 

 

Chapter 6: The chapter presents a manuscript titled “Bioactivity of select essential oil 

constituents against life stages of Anopheles arabiensis (Diptera: Culicidae)”. The manuscript 

has been submitted to Experimental Parasitology and is currently undergoing the review 

process (Manuscript ID: EXPARA-D-22-00337). This chapter addresses the objective C by 

identifying the Anopheles anticholinesterase EOCs with egg hatching inhibitory activity, 

larvicidal, pupicidal and adulticidal effects. 

 

Chapter 7: This chapter presents evidence on the potential of essential oil constituents to 

reverse insecticide resistance. It is presented in the form of a published paper entitled “The 

insecticidal activity of essential oil constituents against pyrethroid-resistant Anopheles funestus 

(Diptera: Culicidae)”. The paper has been published in Parasitology International (Parasitol 

Int. 2023;95: 102749; https://doi.org/10.1016/j.parint.2023.102749). Chapter 7 addresses 

objective D by identifying the Anopheles anticholinesterase EOCs that were not affected by 

the upregulated Anopheles CYP450 monooxygenases. It also addresses objective E by 

identification of the Anopheles anticholinesterase EOCs whose considerable activities against 

resistant Anopheles were only obtained through combination with standard CYP450 synergist, 

piperonyl butoxide. 

 

Chapter 8: In this chapter, the broader study findings are discussed.  

 

Chapter 9: In this chapter the study implications are outlined, and the conclusions are drawn. 

Furthermore, the study limitations as well as future recommendations are outined. 

https://doi.org/10.1371/journal.pone.0277363
https://doi.org/10.1016/j.parint.2022.102713
https://doi.org/10.1016/j.parint.2023.102749


13 
 

 

Chapter 10: Appendices. This section presents the published papers for Chapters 2 and 3, 

supplementary information for Chapters 3 and 4, author declarations, title approval letter and 

the ethical clearance certificates. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



14 
 

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malaria vectors [Internet]. Rijeka (HR): InTech. 

Ariaratnam, V.; Georghiou, G. P., (1975). Carbamate resistance in Anopheles albimanus: 

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http://www.who.int/entity/malaria/publications/world-malaria-report%202016/report/en/index.html
http://www.who.int/malaria/publications/country-profiles/profile_zaf_en.pdf
https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2021
https://www.who.int/news-room/fact-sheets/detail/malaria
https://apps.who.int/iris/handle/10665/345573
https://www.researchgate.net/publication/262450744_Training_Manual_on_Malaria_Entomology
https://www.researchgate.net/publication/262450744_Training_Manual_on_Malaria_Entomology


26 
 

Witzig C, Parry M, Morgan J, Irving H, Steven A, Cuamba N, Kerah-Hinzoumbé C, Ranson 

H, and Wondji CS. (2013). Genetic mapping identifies a major locus spanning P450 clusters 

associated with pyrethroid resistance in kdr-free Anopheles arabiensis from Chad. Heredity 

110: 389–397. 

Wood OR, Hanrahan S, Coetzee M, Koekemoer LL. (2010). Brooke BD. Cuticle thickening 

associated with pyrethroid resistance in the major malaria vector Anopheles funestus. Parasites 

& Vectors 3: 1-7. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



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CHAPTER 2 

Literature review 

This chapter presents a detailed literature review that was carried out in line with the aim of 

this study to assess the potential of identifying novel AChE inhibitors from the natural sources, 

mainly the essential oils. The chapter is presented in the form of a completed review 

manuscript.  

This manuscript paved a way for the assessment of essential oil constituents in this study, for 

the Anopheles acetylcholinesterase inhibitory activity.  

 

Chapter type: Published ISI paper (Appendix A.1) 

Title of manuscript: Potential of essential oil-based anticholinesterase insecticides against 

Anopheles vectors: A review 

Authors: Thankhoe A. Rants’o1,2, Lizette L. Koekemoer2,3,4, Jenny-Lee Panayides5, Robyn 

L. van Zyl1,2 
1   Pharmacology Division, Department of Pharmacy and Pharmacology, Faculty of Health 

Sciences,  

University of the Witwatersrand, 7 York Road, Parktown, Johannesburg 2193, South 

Africa; thankhoe.rantso@wits.ac.za; robyn.vanzyl@wits.ac.za  
2 WITS Research Institute for Malaria, Faculty of Health Sciences, University of the 

Witwatersrand,  

7 York Road, Parktown, Johannesburg 2193, South Africa; thankhoe.rantso@wits.ac.za; 

robyn.vanzyl@wits.ac.za; lizette.koekemoer@wits.ac.za  
3 School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, 7 York 

Road, Parktown, Johannesburg 2193, South Africa; lizette.koekemoer@wits.ac.za  
4 Centre for Emerging Zoonotic and Parasitic Diseases, National Institute for Communicable 

Diseases of the National Health Laboratory Service, 1 Modderfontein Road, Sandringham, 

Johannesburg 2192, South Africa; lizette.koekemoer@wits.ac.za  
5 Pharmaceutical Technologies, CSIR Future Production: Chemicals, Meiring Naude Road, 

Pretoria, 0184, South Africa; JPanayides@csir.co.za  
 

Journal/DOI: Molecules 2022;27(20): 7026; https://doi.org/10.3390/molecules27207026  

Impact factor: 4.93 

mailto:thankhoe.rantso@wits.ac.za
mailto:robyn.vanzyl@wits.ac.za
mailto:thankhoe.rantso@wits.ac.za
mailto:robyn.vanzyl@wits.ac.za
mailto:lizette.koekemoer@wits.ac.za
mailto:lizette.koekemoer@wits.ac.za
mailto:lizette.koekemoer@wits.ac.za
mailto:JPanayides@csir.co.za
https://doi.org/10.3390/molecules27207026


28 
 

Author contributions: 

Contribution Thankhoe A. 
Rants’o 

Lizette L. 
Koekemoer 

Jenny-Lee 
Panayides 

Robyn L.  
van Zyl 

Conceptualization     
Literature search     
Data analysis     
Writing – original draft 
preparation     

Writing - review and editing     
Supervision     
Funding acquisition     

 

Abstract: The insect nervous system is critical for its functional integrity. The cholinergic 

system, of which acetylcholinesterase (AChE) is a key enzyme, is essential to the Anopheles 

(consisting of major malaria vector species) nervous system. Furthermore, the nervous system 

is also the primary target site for insecticides used in malaria vector control programs. 

Insecticides, incorporated in insecticide-treated nets and used for indoor residual spraying, are 

a core intervention employed in malaria vector control. However, Anopheles resistance against 

these insecticides has grown rapidly. Due to this major setback, novel agents with potential 

activity against resistant Anopheles and/or capacity to overcome resistance against current 

WHO-approved insecticides are urgently needed. The essential oils have the potential to be 

natural sources of novel insecticides with potential to inhibit the Anopheles AChE target. In 

the current review, the scientific evidence highlights the ability of essential oils and specific 

essential oil constituents to serve as anticholinesterase insecticides. For this reason, the 

published data from scientific databases on the essential oils and essential oil constituents on 

anticholinesterase, ovicidal, larvicidal, pupicidal and adulticidal activities were analyzed. The 

identification of major constituents in active essential oils and their possible influence on the 

biological activity have also been critically evaluated. Furthermore, the toxicity to mammals 

as well as potential activity against the mammalian AChE target has also been reviewed. The 

importance of identifying novel potent insecticides from essential oils has been discussed, in 

relation to human safety and cost-effectiveness. Finally, the critical insights from this review 

can be used to inform future researchers towards potent and safe anticholinesterase insecticides 

for the management of Anopheles malaria vectors. 

Keywords: malaria; insecticides; terpenoids; acetylcholinesterase 

 

 



29 
 

1. Introduction 

Malaria is a devastating disease caused by a protozoan parasite, namely Plasmodium 

falciparum which is the major causative agent in the pathogenesis of this infectious disease [1–

3]. Anopheles vectors are infected with malaria after they ingest blood from an infected human 

host. The female Anopheles vectors effectively bite the human hosts between dusk and dawn 

[3] and it is during this time that she ingests gametocytes. The Plasmodium gametocytes 

develop into an oocyst in the mosquito midgut, which then matures into sporozoites. The 

sporozoites are released into the hemolymph and migrate to the salivary glands [1,4]. This 

parasite developmental process within the vector takes approximately 11–16 days before the 

female mosquito is able to transmit the parasite to the next human host during a blood feeding. 

This means that a long lifespan of the Anopheles vector is required for the successful 

completion of the parasite development and reinfection of the human host. Vertebrate blood is 

needed every 2–3 days by the female mosquito for nutrition, as well as egg development. The 

eggs are oviposited into water and fertile eggs hatch into larvae a few days later. Larvae will 

develop into pupae and finally adults will emerge after a few days [3]. There are more than 400 

Anopheles species of which about 30 are major malaria vectors. The African Anopheles vectors 

have both long lifespans and a higher preference for human feeding and, collectively, these 

account for the high malaria cases and mortality that is recorded in Africa [3,5]. Other factors, 

such as climate conditions and political and economic stability, also affect the intensity of 

transmission and enhance the problem [3]. 

The malaria vectors have long been controlled by using insecticides. Insecticide classes 

include organophosphates, carbamates, pyrethroids, organochlorines and neonicotinoids [6]. 

Larvicides including insect growth inhibitors as well as bacterial larvicides, such as Bacillus 

thuringiensis subspecies israelensis, Bacillus sphaericus and spinosyns from 

Saccharopolyspora species, have also gained popularity in mosquito control activities [6–8]. 

The implementation of large-scale larviciding is however challenging in Sub-Saharan Africa 

and these may be used as a complementary intervention [6,9–11]. The Anopheles vectors have 

developed substantial resistance against almost all current insecticides [12–15]. To compound 

the issue, the commercial development of insecticides through various and often complicated 

synthetic mechanisms is expensive and time-consuming [16,17]. We propose that the 

identification of potential insecticides from natural product resources, such as essential oils 

(EOs), is a relatively cost-effective and faster alternative. Target identification and the 

corresponding mechanism of action are critical components of the drug discovery process [18]. 



30 
 

Acetylcholinesterase (AChE) is a validated target in the insect nervous system and inhibitors 

of this critical enzyme have been useful in the control of m