EVALUATION OF SELECTED MUSHROOM EXTRACTS,
DISPIROPIPERAZINE AND DISPIROPYRROLIZIDINE DERIVATIVES
ON ACETYLCHOLINESTERASE ACTIVITY
by
Khoa Bhanthumnawin
B.Sc., University of Calgary, 2018

THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
IN
BIOCHEMISTRY

UNIVERSITY OF NORTHERN BRITISH COLUMBIA
December 2023
© Khoa Bhanthumnawin, 2023

Abstract
Acetylcholinesterase is a hydrolase enzyme known for its major role in neurotransmission
regulation by breaking down the neurotransmitter acetylcholine at the synapse after every
electrochemical signal is successfully relayed between two adjacent neurons. In a healthy human
brain, it retains itself on the extracellular surface of neurons, concordantly paints up an artistic
beautiful web of an intricate neural network upon staining. However, in an aging human brain
experiencing neurodegeneration, the network collapses into plaques that are neurotoxic to the brain.
At the center of these individual plaques are heavily aggregated amyloid-beta peptides and fibers
enveloping the neurons. This is most often one of the specific traits of Alzheimer’s and Parkinson’s
disease, two most common neurodegenerative diseases. Many studies have provided experimental
evidences of stable co-localization and fiber growth promotion existing between the amyloid-beta
peptides and acetylcholinesterase. Nevertheless, the specific chemistry and mechanism of how the
relationship between the amyloid-beta peptides and acetylcholinesterase is formed and maintained,
still remains tentative to date. Most neurodegenerative diseases remain incurable, and most of the
available medical treatments focus on alleviating the symptoms and mitigating the disease
progression, for instance through the use of acetylcholinesterase inhibitors. But with drawbacks
related to treatment effectiveness and undesirable side effects of the current available
acetylcholinesterase inhibitors, the continued search for more effective acetylcholinesterase
inhibitors has never ceased. The objectives of this in vitro study were to examine four small
molecules and twenty-one mushroom extracts for their ability to inhibit acetylcholinesterase
activity.
To perform the screen, an enzyme assay using Ellman’s reagent DTNB was used. The
assay

utilizes

thiocholine

produced

from

ii

the

hydrolysis

of

acetylthiocholine

by

acetylcholinesterase to reduce DTNB into TNB2-, a colorimetric product that can be used to
monitor the changes in the acetylcholinesterase activity level in the presence or absence of
inhibitors. Using the established acetylcholinesterase assay, none of the four small molecules
showed any inhibitory effect on acetylcholinesterase activity. On the contrary, eight of the
mushroom extracts were found to inhibit the acetylcholinesterase activity. These eight extracts
were prepared from five mushroom species that have not been extensively researched nor reported
for acetylcholinesterase inhibition. Surprisingly, for the remaining thirteen mushroom extracts that
did not show inhibition, they were found to enhance the acetylcholinesterase activity. Albeit the
unexpected irrelevance to the focus of the study, these thirteen extracts could be useful as
biological tools in the general research of regeneration, as acetylcholinesterase has been reported
to act as a mediator promoting cell proliferation and regeneration.

iii

Table of Contents
Abstract ........................................................................................................................................... ii
Table of Contents ........................................................................................................................... iv
List of Tables ............................................................................................................................... viii
List of Figures ................................................................................................................................ ix
List of Supplementary Tables ........................................................................................................ xi
List of Supplementary Figures ...................................................................................................... xii
Acknowledgement and Dedication .............................................................................................. xiii
Chapter 1 - Introduction .............................................................................................................. 1
1.1

Acetylcholinesterase Enzyme .............................................................................................. 1

1.1.1

Expression and Assembly of Acetylcholinesterase ...................................................... 1

1.1.2

The Hydrolysis of Acetylcholine by Acetylcholinesterase .......................................... 4

1.1.3

The Role of Acetylcholinesterase in Cholinergic Neurotransmission.......................... 8

1.1.4

Upstream Regulation of Acetylcholinesterase Activity ............................................. 12

1.2

Acetylcholinesterase in the Context of Neurodegeneration............................................... 16

1.2.1

Alzheimer’s Disease ................................................................................................... 18

1.2.2

Parkinson’s Disease .................................................................................................... 20

1.2.3

Huntington’s Disease.................................................................................................. 21

1.2.4

Multiple Sclerosis ....................................................................................................... 22

1.3

Acetylcholinesterase Inhibitors in the Treatment of Neurodegenerative Diseases ........... 23

1.3.1

Rivastigmine ............................................................................................................... 24

1.3.2

Donepezil .................................................................................................................... 25

1.3.3

Galanthamine .............................................................................................................. 26

1.3.4

Huperzine A................................................................................................................ 27

1.4

The Need to Search for Novel Acetylcholinesterase Inhibitors......................................... 27

1.4.1

Small Molecules: SPOPP and NP6A ......................................................................... 28

1.4.2

Small Molecules: 534_1(6) and 534_2(7) .................................................................. 29

1.4.3

MolTarPred Program .................................................................................................. 31

1.5

Mushrooms as a Potential Source of Acetylcholinesterase Inhibitors ............................... 32

1.6

Research Objectives ........................................................................................................... 36

Chapter 2 - Development of the Acetylcholinesterase Assay and Examination of the Effect
of Small Molecules on Acetylcholinesterase Activity............................................................... 37
iv

2.1

Introduction ........................................................................................................................ 37

2.1.1

Overview of the Acetylcholinesterase Assay Design ................................................. 37

2.1.2

Considerations and Selection for the Main Components of the Acetylcholinesterase
Assay .......................................................................................................................... 38

DTNB as an Ideal Reagent for Thiol Quantification ............................................................. 38
Acetylthiocholine Iodide as an Alternative Substrate of Acetylcholinesterase ..................... 40
Electrophorus electricus Acetylcholinesterase as an Economical Orthologue Prevalent in
Fundamental Research Studies .............................................................................................. 41
Sodium Phosphate Buffer as a Nurturing Cocktail for an Efficient Functionality of
Acetylcholinesterase .............................................................................................................. 43
2.2

Materials and Methods ....................................................................................................... 44

2.2.1

Preparation of the Main Components of the Acetylcholinesterase Assay – Stock
Solutions ..................................................................................................................... 44

20.2 mM of DTNB ................................................................................................................ 44
346 mM of Acetylthiocholine Iodide .................................................................................... 45
125 units/mL of Acetylcholinesterase ................................................................................... 46
0.1 M of Sodium Phosphate Buffer at pH 8.0 ....................................................................... 47
2.2.2

Preparation of the Main Components of the Acetylcholinesterase Assay – Standard
Solutions ..................................................................................................................... 48

10 mM of DTNB ................................................................................................................... 48
14 mM of Acetylthiocholine Iodide ...................................................................................... 48
9 units/mL of Acetylcholinesterase ....................................................................................... 49
4.5 units/mL of Acetylcholinesterase .................................................................................... 49
0.9 units/mL of Acetylcholinesterase .................................................................................... 49
0.09 units/mL of Acetylcholinesterase .................................................................................. 50
0.009 units/mL of Acetylcholinesterase ................................................................................ 50
2.3

Results and Discussion ...................................................................................................... 50

2.3.1

Testing the Performance of the Acetylcholinesterase Assay...................................... 51

2.3.1.1 The First Performance Test: Evaluating the Assay with Different Concentrations of
Acetylcholinesterase ............................................................................................... 51
2.3.1.1.1

Purpose of the First Performance Test ............................................................ 51

2.3.1.1.2

Experimental Procedure of the First Performance Test................................... 51

2.3.1.1.3

Raw Data Processing and Graphing Transformation of the First Performance
Test .................................................................................................................. 52
v

2.3.1.1.4

Hypothesis and Predicted Outcomes of the First Performance Test ............... 53

2.3.1.1.5

Observations and Analyses of the First Performance Test .............................. 53

2.3.1.2 The Second Performance Test: Evaluating the Assay with Different Concentrations
of Known Inhibitors of Acetylcholinesterase ......................................................... 76

2.4

2.3.1.2.1

Purpose of the Second Performance Test ........................................................ 76

2.3.1.2.2

Experimental Procedure of the Second Performance Test .............................. 77

2.3.1.2.3

Raw Data Processing and Graphing Transformation of the Second
Performance Test ............................................................................................. 79

2.3.1.2.4

Hypothesis and Predicted Outcomes of the Second Performance Test ........... 81

2.3.1.2.5

Observations and Analyses of the Second Performance Test ......................... 81

Examination of the Effect of Small Molecules on Acetylcholinesterase Activity ............ 86

2.4.1

Preparation of the Synthetic Small Molecules ........................................................... 86

2.4.1.1

Synthesis of SPOPP and NP6A ............................................................................. 86

2.4.1.2

Preparation of Flash Column Chromatography ..................................................... 89

2.4.1.3

Purification of SPOPP and NP6A .......................................................................... 92

2.4.1.4

Synthesis of 534_1(6) and 534_2(7) ...................................................................... 93

2.4.2

Evaluation of the Effect of Small Molecules on Acetylcholinesterase Activity ........ 93

2.4.2.1 Experimental Procedure .......................................................................................... 93
2.4.2.2 Hypothesis and Predicted Outcomes ...................................................................... 95
2.4.2.3 Observations and Analyses ..................................................................................... 95
Chapter 3 - Investigating the Effect of Mushroom Extracts on Acetylcholinesterase Activity
..................................................................................................................................................... 100
3.1

Introduction ...................................................................................................................... 100

3.2

Materials and Methods ..................................................................................................... 100

3.2.1

Preparation of the Mushroom Extracts ..................................................................... 100

3.2.1.1
3.3

Extraction of the Mushroom Extracts .................................................................. 100

Results and Discussion .................................................................................................... 101

3.3.1

Evaluation of the Effect of Crude Mushroom Extracts on Acetylcholinesterase
Activity ..................................................................................................................... 102

3.3.1.1 Experimental Procedure ........................................................................................ 102
3.3.1.2 Hypothesis and Predicted Outcomes .................................................................... 103
3.3.1.3 Observations and Analyses ................................................................................... 104
Chapter 4 - General Discussion ............................................................................................... 135
vi

4.1

General Remarks.............................................................................................................. 135

4.2

Establishment of the Acetylcholinesterase Assay............................................................ 135

4.3

The Effects of the Selective Small Molecules on Acetylcholinesterase Activity ............ 137

4.4

The Effects of the Selective Mushroom Extracts on Acetylcholinesterase Activity ....... 138

4.5

Concluding Remarks and Future Directions .................................................................... 146

References ................................................................................................................................... 148
Supplementary Data .................................................................................................................... 154

vii

List of Tables
Table 1. List of Example Mushroom Species Conducted in the Research of Neurodegeneration.
....................................................................................................................................................... 34
Table 2. List of Example Mushroom Species Reported for In Vitro Inhibition of
Acetylcholinesterase Activity. ...................................................................................................... 35
Table 3. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 1............................................................................................................... 56
Table 4. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 11............................................................................................................. 65
Table 5. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 12............................................................................................................. 68
Table 6. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 13............................................................................................................. 71
Table 7. Summary of the Preparation of the Mushroom Extracts. ............................................. 101
Table 8. Summary of Mushroom Extracts that Inhibited Acetylcholinesterase Activity. .......... 116
Table 9. Summary of Mushroom Extracts that Were Negative for Acetylcholinesterase Activity
Inhibition. .................................................................................................................................... 124

viii

List of Figures
Figure 1. Schematic Diagram of the Acetylcholinesterase Gene. .................................................. 2
Figure 2. Schematic Diagram of the Predominant T Form of Acetylcholinesterase in the Central
Nervous System of the Human Brain. ............................................................................................ 3
Figure 3. Schematic Diagram of the Tertiary Protein Structure of Acetylcholinesterase. ............. 6
Figure 4. Schematic Diagram of the Hydrolysis of Acetylcholine at the Catalytic Anionic Site of
Acetylcholinesterase. ...................................................................................................................... 8
Figure 5. The Chemical Structure of Rivastigmine. ..................................................................... 25
Figure 6. The Chemical Structure of Donepezil. .......................................................................... 26
Figure 7. The Chemical Structure of Galanthamine. .................................................................... 27
Figure 8. The Chemical Structure of Huperzine A. ...................................................................... 27
Figure 9. The Chemical Structures of SPOPP and NP6A. ........................................................... 29
Figure 10. The Chemical Structures of 534_1(6) and 534_2(7). .................................................. 31
Figure 11. The Reaction Mechanism of the Acetylcholinesterase Assay Using Ellman's Reagent.
....................................................................................................................................................... 38
Figure 12. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 3............................................................................................................... 58
Figure 13. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 11............................................................................................................. 66
Figure 14. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 12............................................................................................................. 69
Figure 15. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 13............................................................................................................. 72
Figure 16. The Effect of Different Acetylcholinesterase Concentrations on Acetylcholinesterase
Activity. ........................................................................................................................................ 75
Figure 17. The Effect of Different Acetylcholinesterase Concentrations on Acetylcholinesterase
Activity. ........................................................................................................................................ 76
Figure 18. The Effect of Galanthamine Hydrobromide on Acetylcholinesterase Activity. ......... 83
Figure 19. The Effect of Pyridostigmine Bromide on Acetylcholinesterase Activity. ................. 85
Figure 20. The Synthesis of SPOPP and NP6A............................................................................ 87
Figure 21. The Synthesis of SPOPP and NP6A............................................................................ 87
Figure 22. Crude Product Solids From the Synthesis of SPOPP and NP6A. ............................... 88
Figure 23. Crude Product Solids From the Synthesis of SPOPP and NP6A. ............................... 89
Figure 24. The Purification of SPOPP and NP6A. ....................................................................... 93
Figure 25. The Effect of SPOPP on Acetylcholinesterase Activity.............................................. 96
Figure 26. The Effect of NP6A on Acetylcholinesterase Activity. .............................................. 97
Figure 27. The Effect of 534_1(6) on Acetylcholinesterase Activity. .......................................... 98
Figure 28. The Effect of 534_2(7) on Acetylcholinesterase Activity. .......................................... 99
Figure 29. The Effect of Phellinus igniarius PIE3 on Acetylcholinesterase Activity at Low
Extract Concentrations. ............................................................................................................... 106
Figure 30. The Effect of Phellinus igniarius PIE2 on Acetylcholinesterase Activity at Low
Extract Concentrations. ............................................................................................................... 108
ix

Figure 31. The Effect of Ganoderma applanatum M5E3 on Acetylcholinesterase Activity at Low
Extract Concentrations. ............................................................................................................... 109
Figure 32. The Effect of Ganoderma applanatum M5E1 on Acetylcholinesterase Activity at Low
Extract Concentrations. ............................................................................................................... 111
Figure 33. The Effect of Ganoderma applanatum M5E1 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 113
Figure 34. The Effect of Hericium erinaceus 288E3 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 117
Figure 35. The Effect of Ganoderma applanatum M5E2 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 118
Figure 36. The Effect of Amanita muscaria 243E1 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 119
Figure 37. The Effect of Agaricus subrufescens 295E1 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 120
Figure 38. The Effect of Agaricus subrufescens 295E2 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 121
Figure 39. The Effect of Hericium erinaceus 288E1 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 122
Figure 40. The Effect of Inonotus obliquus 285E1 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 123
Figure 41. The Effect of Amanita muscaria 243E2 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 125
Figure 42. The Effect of Amanita muscaria 243E3 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 126
Figure 43. The Effect of Agaricus subrufescens 295E3 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 127
Figure 44. The Effect of Inonotus obliquus 285E2 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 128
Figure 45. The Effect of Inonotus obliquus 285E3 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 129
Figure 46. The Effect of Pleurotus ostreatus 68E2 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 130
Figure 47. The Effect of Phellinus pini 91E1 on Acetylcholinesterase Activity at High Extract
Concentrations. ........................................................................................................................... 131
Figure 48. The Effect of Phellinus igniarius PIE1 on Acetylcholinesterase Activity at High
Extract Concentrations. ............................................................................................................... 132
Figure 49. The Effect of Phellinus pini 91E2 on Acetylcholinesterase Activity at High Extract
Concentrations. ........................................................................................................................... 133
Figure 50. The Effect of Phellinus pini 91E3 on Acetylcholinesterase Activity at High Extract
Concentrations. ........................................................................................................................... 134

x

List of Supplementary Tables
Supplementary Table 1. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 2.......................................................... 155
Supplementary Table 2. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 3.......................................................... 158
Supplementary Table 3. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 4.......................................................... 160
Supplementary Table 4. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 5.......................................................... 163
Supplementary Table 5. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 6.......................................................... 166
Supplementary Table 6. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 7.......................................................... 169
Supplementary Table 7. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 8.......................................................... 172
Supplementary Table 8. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 9.......................................................... 175
Supplementary Table 9. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 10........................................................ 180

xi

List of Supplementary Figures
Supplementary Figure 1. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 2.......................................................... 156
Supplementary Figure 2. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test Run 4........................................................... 161
Supplementary Figure 3. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 5.......................................................... 164
Supplementary Figure 4. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 6.......................................................... 167
Supplementary Figure 5. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 7.......................................................... 170
Supplementary Figure 6. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 8.......................................................... 173
Supplementary Figure 7. Evaluating the Assay with Different Concentrations of
Acetylcholinesterase: The First Performance Test, Run 9.......................................................... 176

xii

Acknowledgement and Dedication

Set sailing towards the end of 2023, as I looked back on my journey throughout the years upon
completing this piece of writing, I would like to acknowledge and express my sincere gratitude
towards these individuals:
Dr. Chow Lee, for giving me an opportunity to get back into science when I had nowhere
else to go, and for his mentorship and constructive criticisms to help me grow as a scientist.
Dr. Kendra Furber and Dr. Kerry Reimer, for their mentorship and support with productive
feedback on my coursework and thesis.
Maggie Li, for helping me overcome many unforeseen circumstances that unexpectedly
unfolded during ongoing experiments with her creativity and advice.
Hooi Xian Lee and Victor Liu, for providing the test samples, teaching and sharing tips on
how to perform well on some of the tricky parts of certain lab techniques.
I would not have been able to come this far, had it not been for their guidance. Therefore, I am
greatly in debt to each and every one of these individuals.

Thank you for your kind guidance throughout these passing years again. It was an honor.

Sincerely,

Khoa Doan Bhanthumnawin

xiii

Chapter 1
Introduction
1.1

Acetylcholinesterase Enzyme
Acetylcholinesterase is a hydrolase enzyme known for its major role in regulating

cholinergic neurotransmission by catabolizing the neurotransmitter acetylcholine at the synapse
after every nerve impulse transmission between two adjacent neurons. The enzyme is known to
exist in four different isoforms, distinguished by the subunit types altered through various posttranslational modifications: R, S, H, and T. Each of these subunit types may also have more than
one molecular form, characterized mainly by a combination of the following three phenotypes:
homomeric or heteromeric, amphiphilic or non-amphiphilic, and soluble or membrane-bound.
In contrast to the highly conserved catalytic domain of the enzyme, the diverse subunit
types and their unique molecular forms mostly result from specific types of post-translational
modifications targeted at the flexible C-terminal domain. This domain is not necessary for the
catalytic function of the enzyme. In the central nervous system of the human brain, the
predominant form of acetylcholinesterase is a membrane-bound amphiphilic heteromer of four T
subunits attached to the proline-rich membrane anchor. The proline-rich membrane anchor is
abbreviated as PRiMA.1
1.1.1

Expression and Assembly of Acetylcholinesterase
The predominant T form is expressed from the acetylcholinesterase gene, which comprises

six exons. However, only exons 2, 3, and 4 code for the catalytic domain, while exon 6 is
alternatively spliced into one of the splice variants that codes for the unique C-terminal domains
of T subunits and contains a message directing the attachment of those subunits to the PRiMA
anchor subsequently (Figure 1).2

1

Figure 1. Schematic Diagram of the Acetylcholinesterase Gene.
The rectangles represent exons. The circle represents a regulatory intron. ATG represents the start codon
AUG. TAA and TGA represent the stop codons UAA and UGA. AATAAA represents the polyadenylation
signal.2 61

Like many other enzymes, all acetylcholinesterase mRNA transcripts are highly localized
within the nucleus and delivered to the rough endoplasmic reticulum, where translation of the
enzyme occurs. During translation, the enzyme’s protein structure undergoes several modifications,
such as glycosylation with asparagine-linked oligosaccharides and the assembly of T subunits to
the PRiMA anchor. After translation, it is a common statistic across all cell types expressing
acetylcholinesterase that 20% or fewer of the enzymes are able to exit the rough endoplasmic
reticulum as correctly folded protein structures maintaining an active catalytic activity. For the
majority that are unable to exit, they are eventually degraded within the rough endoplasmic
reticulum.
Furthermore, the asparagine-linked oligosaccharides glycosylated to the enzyme are finetuned to oligomers that are resistant to endoglycosidase H in the Golgi apparatus.3
Endoglycosidase H is a hydrolase enzyme that catabolizes oligosaccharides with a high content of
mannose monosaccharides. Because the presence of mannose monosaccharides is strongly tied to
a properly functional catalytic activity of acetylcholinesterase, it is imperative that the asparaginelinked oligosaccharides are resistant to endoglycosidase H.4
2

Upon exiting the Golgi apparatus, the fully mature acetylcholinesterase is parceled into
vesicles via a clathrin-mediated mechanism, to which these vesicles are delivered to the
extracellular surface.3 For successful presentation on the extracellular surface, the PRiMA anchor
is a crucial structural component that the enzyme cannot be without. The peptide sequence of the
anchor contains a seventeen-amino acid long proline-rich attachment domain abbreviated as
PRAD located within the N-terminal domain. It is used to bind to the tryptophan amphiphilic
tetramerization domain abbreviated as WAT located within the C-terminal domain of the peptide
sequence of acetylcholinesterase. This binding is responsible for the assembly of T subunits to the
PRiMA anchor and the presentation of the enzyme on the extracellular surface (Figure 2).5

Figure 2. Schematic Diagram of the Predominant T Form of Acetylcholinesterase in the Central
Nervous System of the Human Brain.
The predominant T form of acetylcholinesterase in the central nervous system of the human brain is a
membrane-bound amphiphilic heteromeric tetramer of T subunits attached to the proline-rich membrane
anchor PRiMA. The peptide sequence of the PRiMA anchor contains a seventeen-amino acid long prolinerich attachment domain called PRAD located within the N-terminal domain. It binds to the tryptophan
amphiphilic tetramerization domain called WAT located within the C-terminal domain of the
acetylcholinesterase peptide sequence. This binding signals the assembly of T subunits to the PRiMA
anchor and the presentation of acetylcholinesterase on the extracellular surface of the pre-synaptic neuron
at the synapse.5 62

3

Moreover, it has also been suggested to play an important role in maintaining the structural
stability and promoting the maturation of the enzyme. Incubation and reabsorption of the peptide
sequence of PRAD alone was shown to significantly increase the catalytic activity of
acetylcholinesterase on the extracellular surface by 400%.6 This indicates that PRAD may
maintain the structural stability and promote the maturation of acetylcholinesterase by increasing
the probability of the enzyme bypassing translational and post-translational checkpoints as a
correctly folded protein structure maintaining an active catalytic activity.
On the extracellular surface, recent studies experimenting with highly polarized cells in
culture have supported and suggested a common finding that all forms of acetylcholinesterase,
including the predominant T form in the central nervous system, are targeted to the apical
membrane, which translates to the growing axon tip of a neuron. In other words, the predominant
T form of acetylcholinesterase in the central nervous system has been suggested to localize on the
extracellular surface of the pre-synaptic neuron at the synapse.3
1.1.2 The Hydrolysis of Acetylcholine by Acetylcholinesterase
At the synapse, acetylcholinesterase hydrolyzes its substrate acetylcholine into acetic acid
and choline. Within its quaternary structure, there exists a deep gorge, akin to a partially cracked
soft-boiled egg. Near the outer opening of this gorge is the peripheral anionic site, abbreviated as
the P-site. The P-site is rich in aromatic residues, with tryptophan being the most prevalent.
Tryptophan’s side chain has a highly electron-rich doublet aromatic ring. In the presence of the
highly unstable cationic nitrogen of acetylcholine, they eventually form an intermolecular π-cation
bond, providing stability to the cationic nitrogen of acetylcholine. Additionally, the para-hydroxyl
group on the singlet aromatic ring of tyrosine’s side chain can form a weak intermolecular

4

hydrogen bond with the carbonyl group on the acetyl end of acetylcholine, offering further stability
to the entire substrate.
Much like the carnivorous pitcher plant, the process of luring acetylcholine into the deep
gorge begins at the P-site, starting with the doublet aromatic ring of the tryptophan-286 residue.
Once drawn into the gorge like a trapped insect, the acetylcholine molecule is guided into the lower
choline-binding pocket deep within the gorge. At this site, the molecule once again forms π-cation
bonding with the surrounding aromatic residues, including tryptophan-86 and tyrosine-337. These
residues realign and correctly position the molecule towards the catalytic triad, found at the
catalytic anionic site, abbreviated as the A-site. This is a crucial step that allows the acyl-binding
pocket, located slightly to the upper left of the choline-binding pocket, to firmly secure the acetyl
end of the molecule, preparing it for direct cleavage by the catalytic triad (Figure 3).7

5

Figure 3. Schematic Diagram of the Tertiary Protein Structure of Acetylcholinesterase.
The peripheral anionic site, abbreviated as the P-site, is responsible for drawing acetylcholine into the lower
catalytic anionic site, abbreviated as the A-site, through π-cation bonding. At the A-site, acetylcholine is
held centered, to which it will be cleaved by the catalytic triad consisting of glutamate, histidine, and serine.7
63

The catalytic triad consists of three key residues: glutamate-334, histidine-447, and serine203. They are arranged in a specific sequence where electron donation flows from glutamate to
histidine and then to serine. The hydrolysis process begins with deprotonated glutamate-334
donating a pair of electrons and abstracting a proton from histidine-447. A deprotonated histidine447 then donates a pair of electrons and abstracts a proton from serine-203, forming an anionic
oxygen on serine-203. This anionic oxygen subsequently attacks the carbonyl group, breaking the
acetyl end apart from the acetylcholine molecule.

6

As acetylcholine breaks apart, its anionic oxygen on the choline end donates a pair of
electrons and abstracts a proton from histidine-447, returning the residue to its deprotonated state
before the choline end is released from the enzyme. After separating the acetyl end, the serineacetyl complex bends backward and is stabilized by an amide backbone in the vicinity. Following
this, a water molecule infiltrates the catalytic site, and one of its protons is abstracted in exchange
for a pair of electrons from the deprotonated histidine-447. These electrons are then utilized to
attack the carbonyl group, releasing the acetyl end from the serine-acetyl complex as acetic acid.
During the release of acetic acid, the anionic oxygen on serine-203 also donates a pair of electrons
and abstracts a proton from the protonated histidine-447, restoring the residue to its deprotonated
state once again. Finally, the deprotonated histidine-447 donates a pair of electrons and abstracts
a proton from the protonated glutamate-334, completing the hydrolysis of acetylcholine by
acetylcholinesterase (Figure 4).8

7

Figure 4. Schematic Diagram of the Hydrolysis of Acetylcholine at the Catalytic Anionic Site of
Acetylcholinesterase.
The catalytic triad of the catalytic anionic site of acetylcholinesterase consists of three key residues:
glutamate-334, histidine-447, and serine-203. They are arranged in a specific sequence where electron
donation flows from glutamate to histidine and then to serine. The hydrolysis process begins with
deprotonated glutamate-334 donating a pair of electrons and abstracting a proton from histidine-447. The
small double-headed black arrow signifies the continuation of the electron flow, as explained above.

1.1.3

The Role of Acetylcholinesterase in Cholinergic Neurotransmission
As a regulator of cholinergic neurotransmission, the primary role of acetylcholinesterase is

to prevent the continued excitation of the post-synaptic neuron by catabolizing acetylcholine. To
induce an excitation or inhibition like any other neurotransmitter, acetylcholine is required to bind
to a threshold number of acetylcholine receptors located on the membrane of the dendrite or cell
body of the opposing post-synaptic neuron after it diffuses across the synapse.
Acetylcholine typically excites neurons. If it is not neutralized, it may continue binding to
these acetylcholine receptors, leading to continued excitation of the post-synaptic neuron,
potentially resulting in severe consequences. According to the Camp et al. (2005) study, a

8

homozygous knockout of exon 6 of the acetylcholinesterase gene in murine brains resulted in
homozygotes experiencing more frequent seizures than both the heterozygotes and the wild types.
These seizures exacerbated mental deficits related to memory, learning, and behavioral processes.
Since exon 6 is alternatively spliced into one of the splice variants coding for the unique C-terminal
domains of T subunits that direct the attachment of those subunits to the PRiMA anchor, the
complete loss of exon 6 would be expected to result in a complete loss of anchorage of the T
subunits to the PRiMA anchor. This would impair the presentation of acetylcholinesterase to the
extracellular

surface

of

the

pre-synaptic

neuron.

Consistently,

measurements

of

acetylcholinesterase activity in brain tissues showed a complete loss of activity in the homozygotes
compared to the wild types, confirming the expected impaired presentation of acetylcholinesterase
to the extracellular surface of the pre-synaptic neuron. Without acetylcholinesterase, the loss of
control over appropriate neuron excitation may explain for the hyperexcitability, as seen with the
increased frequency of seizures observed in the homozygotes.2
In addition to its primary role, recent research studies have further linked cellular
communications between acetylcholinesterase and the muscarinic acetylcholine receptors
mAChR1 and mAChR2 as a part of biological processes involved in cholinergic neurotransmission
regulation. Both muscarinic acetylcholine receptors are abbreviated as M1 and M2 receptors. The
M1 receptor is the predominant form of the five muscarinic acetylcholine receptors in the central
nervous system and is primarily clustered on the extracellular surface of the post-synaptic neuron
at the synapse. It plays a major role in stimulating neurons to excite upon binding with
acetylcholine. The M2 receptor is moderately expressed in the central nervous system and is found
on the extracellular surface of both pre- and post-synaptic neurons at the synapse. It contributes to
the inhibition of the post-synaptic neuron and,9 notably, is involved in the autoregulation of

9

acetylcholine release into the synapse when bound by acetylcholine. Similar to the M2 receptor,
the M4 receptor is also moderately expressed in the central nervous system and plays a role in this
autoregulation. However, after an extended period of debate, it has been concluded that the M2
receptor has a more dominant role in this autoregulation.10
In murine brains, a homozygous knockout of the acetylcholinesterase gene was shown to
lead to a reduction in the localization of the M1 receptor and a complete abolition of the
localization of the M2 receptor on the extracellular surface of post- and pre-synaptic neurons,
respectively. However, the mRNA expression levels of both receptors remained largely unchanged.
This suggests that the absence of acetylcholinesterase may signal the internalization and
degradation or redistribution of both receptors within the cell.11 12 Furthermore, the absence of
acetylcholinesterase at the synapse appeared to affect the cellular responsiveness of both receptors
differently. In the case of the M1 receptor, in the same studies, treatment with one of its agonists
called oxotremorine did not substantially increase the activation signal of the MAPK/ERK
pathway in the homozygotes compared to the wild types. The MAPK/ERK pathway is one of many
that plays a crucial role in promoting brain plasticity and cognitive processes such as memory and
learning. This suggests a desensitization of the M1 receptor in the absence of acetylcholinesterase
at the synapse, and this desensitization may also extend to cholinergic signaling as a whole.11
Regarding the M2 receptor, in a separate study where the release of acetylcholine into the
synapse was monitored in slices of murine brain tissues following in vitro electrical stimulation,
treatment with one of its antagonists called atropine, while an active acetylcholinesterase was left
undisturbed, resulted in an increased efflux of acetylcholine by 66%, or 166% in net total, over the
span of 12 mins compared to the basal efflux of the control. Conversely, treatment with one of the
irreversible inhibitors of acetylcholinesterase called diisopropyl phosphorofluoridate, while an

10

active M2 receptor was left undisturbed, resulted in a decreased efflux of acetylcholine by 60%,
or 40% in net total, over the same time span compared to the basal efflux of the control. This
pattern, similar to the absence of acetylcholinesterase with the irreversible inhibition of its catalytic
activity, suggests an enhancement in the sensitivity of the M2 receptor in response to acetylcholine
binding, serving as a second line of defense to regulate the amount of acetylcholine released into
the synapse, should the primary function of acetylcholinesterase in preventing continued neuron
excitation be compromised.13
Taken together, the studies support the idea that acetylcholinesterase indirectly regulates
the M1 and M2 receptors at a certain level. The presence of an irreversibly inactive
acetylcholinesterase, or absence of an active acetylcholinesterase, was shown to not only
significantly quell the number of M1 and M2 receptors presented on the extracellular surface at
the synapse, but also impact their cellular responsiveness towards acetylcholine in opposite ways.
These changes in receptor behavior may represent cellular responses aimed at mitigating continued
neuron excitation, in order to cope with an acetylcholinesterase whose primary function is
compromised.
Hypothetically, the M1 receptor becomes less sensitive and is internalized more to prevent
acetylcholine from reaching the activation threshold required to induce excitation in the postsynaptic neuron, especially when unhydrolyzed acetylcholine is present in the synapse. On the
other hand, the M2 receptor becomes more sensitive to enhance the autoinhibition of the
acetylcholine release, preventing the excessive buildup of unhydrolyzed acetylcholine in the
synapse. As a result of preventing this buildup, more M2 receptor are internalized as a response to
balance the overall energy allocation within the cell, especially in the presence of lower levels of
unhydrolyzed acetylcholine in the synapse.

11

However, it is important to note that these cellular responses may not completely prevent
continued neuron excitation by unhydrolyzed acetylcholine. This is because acetylcholine can also
bind to other acetylcholine receptors to induce excitation, such as the nicotinic acetylcholine
receptor. Moreover, a desensitized M1 receptor does not necessarily mean it is completely
insensitive, thus unhydrolyzed acetylcholine may still stimulate neurons to excite through the
receptor. These proposed suggestions are consistent with the evidence from homozygous knockout
mice with a complete loss of exon 6 of the acetylcholinesterase gene, as they experienced more
frequent seizures compared to both the heterozygotes and the wild types.2
As for the specific mechanisms of this indirect regulation imposed on the M1 and M2
receptors in the absence of acetylcholinesterase, it is possible that extracellular signals play a role
in the internalization and desensitization of the M1 receptor, and in the internalization of the M2
receptor. This is supported by the observation that treatment with their antagonist atropine could
rescue the presentation of both receptors on the extracellular surface at the synapse. There may
also be intracellular signals involved in the hypersensitization of the M2 receptor.11 12 13 Likewise,
there may also be a different set of specific mechanisms of this indirect regulation in the presence
of an active acetylcholinesterase.
In summary, acetylcholinesterase’s role as a regulator of cholinergic neurotransmission
goes beyond preventing continued neuron excitation by catabolizing acetylcholine. It also involves
an indirect control of acetylcholine flow into the synapse and the sensitivity of cholinergic
signaling by regulating the localization and cellular responsiveness of M1 and M2 receptors
towards acetylcholine.
1.1.4

Upstream Regulation of Acetylcholinesterase Activity

12

Regulating neurotransmission is not a simple task, as it requires a delicate and steady
neutral balance avoiding the extremes. For such a crucial function, it is unlikely that
acetylcholinesterase is the sole regulator of cholinergic neurotransmission. Therefore, there may
be other upstream regulatory checkpoints that control acetylcholinesterase activity at the synapse
in a sequential order. At the post-transcriptional level, there have been reports of a microRNA
called miRNA-132 that inhibits acetylcholinesterase expression as one of its target genes. This
microRNA was found to be highly enriched in neurons.14 Either a disruption of the regulation or a
decrease in the level of miRNA-132 has been shown to upregulate acetylcholinesterase expression,
leading to mental deficits related to memory, learning, and behavioral processes.15 Similar to the
homozygous knockout of the acetylcholinesterase gene or its exon 6 alone, this suggests that
hypercatabolic activity on acetylcholine due to the overexpression of acetylcholinesterase at the
synapse may not be beneficial for brain function, further emphasizing the importance of
maintaining a neutral balance in cholinergic signaling during neurotransmission regulation.
Reports on the regulation of acetylcholinesterase activity by miRNA-132 support observations
recorded for the homozygous knockout treatment of the M1 receptor gene in the murine brain. In
both cases, excessive catabolism of acetylcholine due to the overexpression of acetylcholinesterase
and the reduction in cholinergic signaling due to the complete loss of the M1 receptor resulted in
mental deficits related to short-term memory.10
At the post-translational level, there have been reports of a protein called presenilin 1,
which appears to be an indispensable factor governing the glycosylation pattern on the protein
structure of acetylcholinesterase. This glycosylation pattern is strongly linked to the proper
catalytic activity of the enzyme on the extracellular surface of the pre-synaptic neuron at the
synapse. According to a lectin binding analysis conducted by the Cortes-Gomez et al. (2023) study,

13

the majority of acetylcholinesterase activity in brain extracts obtained from control subjects was
found to bind to Concanavalina ensiformis A and Lens culinaris lectins. These two lectins are
known to recognize and bind to α-mannose monosaccharides. While the majority of the enzyme
activity bound to the lectins, the majority of the enzyme was found to remain in fractions that did
not bind to the lectins, comprising 89% to 92% of the total. This suggests that the observed
majority of active acetylcholinesterase bound to the lectins only corresponded to a very small
portion of the total enzyme amount, in line with the common finding that only about 20% or fewer
active acetylcholinesterase can normally reach the extracellular surface. In brain extracts obtained
from Alzheimer’s disease subjects, the percentage of active acetylcholinesterase bound to the
lectins was further reduced. These findings suggest that the glycosylation pattern, enriched in
mannose monosaccharides within the protein structure of acetylcholinesterase, may play a direct
role in shaping the proper catalytic function of the catalytic anionic site of the enzyme. Changes
in this pattern may negatively impact acetylcholinesterase activity at the synapse, especially in the
context of neurodegeneration.4
The involvement of presenilin 1 in the regulation of acetylcholinesterase glycosylation was
further supported when its overexpression was shown to significantly increase the percentage of
active acetylcholinesterase bound to the lectins, with an average increase of 25% to 30% compared
to the control. This was accompanied by a larger intracellular migration across the Golgi apparatus
to the extracellular surface. Even its suppression by small interfering RNA greatly shifted the
majority of active acetylcholinesterase from being recognized by any of the lectins used, even
though there was minimal change to the total expression level of the enzyme. These findings
suggest that presenilin 1 may not only govern the glycosylation of acetylcholinesterase with highly
enriched mannose monosaccharides, but it may also play a role in directing the presentation of

14

active acetylcholinesterase to the extracellular surface, akin to the peptide sequence of PRAD.
Furthermore, disturbances affecting its functions may lead to disruptions in this specific
glycosylation pattern, resulting in a shift and reduction in the portion of active acetylcholinesterase
presented on the extracellular surface of the pre-synaptic neuron at the synapse.4
At the ligand level, apart from the acetylcholinesterase inhibitors used to alleviate the
symptoms of neurodegenerative diseases, very little is known about potential endogenous ligands
in the brain that directly bind and regulate acetylcholinesterase activity. However, to date, one
potential candidate appears to be cholesterol. According to the Paul et al. (2017) study, feeding a
hypercholesterolemic diet to mice led to an overall decrease in acetylcholinesterase activity in the
brain compared to mice fed with a healthy diet. This decrease was observed across different brain
regions, with enzyme activity reduced by at least 29%, and in some cases, as high as 57%. It was
also associated with an increase in cholesterol content in the brain and disruption of the bloodbrain barrier. At the phenotypic level, hypercholesterolemic mice also displayed mental deficits
related to short-term memory, which is consistent with the seizures experienced by murine
homozygotes when exon 6 of the acetylcholinesterase gene was deleted.2 16
Based on the general knowledge of the role of cholesterol in the brain, it is a major
component that makes up the myelin sheaths, enhancing the nerve impulse by providing an
evolutionary upgrade in the form of electrical currents racing across the nodes of Ranvier at a
propagation speed that is 500 times faster upon depolarization.17 Due to its crucial importance,
cholesterol is not permeable through the blood-brain barrier, and its level is tightly regulated in the
brain through repeated cycles of synthesis and recycling, with minimal uptake from circulation.
When there is an excess amount, it is secreted from the brain after being converted into either one
of its two main oxysterol derivatives, 24-hydroxycholesterol and 27-hydroxycholesterol. In

15

addition to aiding the excretion of excess cholesterol from the brain, these two oxysterols can also
act as potent inhibitors against the production of amyloid-beta peptides, as their levels have been
reported to be downregulated in Alzheimer’s disease.18
Returning to the aforementioned study on the effects of hypercholesterolemia in the murine
brain, although it remains unclear how high cholesterol content can disrupt the blood-brain barrier,
and how cholesterol interacts to decrease acetylcholinesterase activity in the brain, all the
evidences presented so far are in line with one another, strongly linking the pathological role of
cholesterol and the regulatory role of its oxysterol derivatives to neurodegeneration.
1.2

Acetylcholinesterase in the Context of Neurodegeneration
In a healthy human brain, acetylcholinesterase remains on the extracellular surface of pre-

synaptic neurons at the synapse, creating a beautiful and intricate neural network when stained.
However, in an aging human brain experiencing neurodegeneration, this network tends to
deteriorate into plaques, resembling chunks of cookies and their leftover crumbs of various sizes
that are neurotoxic to the brain. At the center of these individual plaques are heavily aggregated
amyloid-beta peptides and fibers that envelop the neurons. This is often one of the major
characteristic features of common neurodegenerative diseases like Alzheimer’s and Parkinson’s
diseases. In the early stages, these plaques exist as pre-plaques, which can be either small and
slightly diffused with a diameter of roughly 40 μm, or large and widely diffused with a diameter
of up to 300 μm. As the disease progresses, they eventually concentrate and become fully
developed plaques with a densely packed core.19
Various previous in vitro studies have corroborated and strongly suggested a stable colocalization of amyloid-beta peptides and acetylcholinesterase. In an experiment by Alvarez et al.
(1998), amyloid-beta peptides and acetylcholinesterase derived from cell extracts of bovine brain

16

tissues were subjected to a sedimentation velocity analysis to examine their binding affinity
towards each other and their potential for co-localization. The analysis relied on centrifuging a test
sample through a gradient of sucrose concentrations contained within a single test tube.
Theoretically, the denser a molecule is, the deeper it will be able to advance to heavier
concentrations at the bottom of the test tube. Thus, the shifts in position based on the mass of
molecules in a mixture can be used to detect co-localization when compared to controls containing
only individual molecules. By dividing the mixture into fractions and using the isotope iodine-125
to label the amyloid-beta peptides and Ellman’s reagent 5,5’-dithiobis-2-nitrobenzoic acid
abbreviated as DTNB to label the acetylcholinesterase, their relative positions along the sucrose
gradient after centrifugation were determined. The results showed that the amyloid-beta peptides
by themselves were denser and mostly concentrated in fractions derived from the bottom of the
test tube. In contrast, acetylcholinesterase by itself was mainly concentrated in fractions derived
from positions midway through the test tube. Interestingly, when amyloid-beta peptides were
mixed with acetylcholinesterase, roughly two-thirds of the labeled acetylcholinesterase shifted
together with the labeled amyloid-beta peptides to fractions derived from the bottom of the test
tube, strongly suggesting their co-localization.20
In another experiment, the amyloid-beta peptide-acetylcholinesterase complex derived
from cell extracts of bovine brain tissues was subjected to various stringent experimental
conditions of extremely high ionic strength and strong denaturation, followed by centrifugation,
to examine the overall stability of the complex. Theoretically, the heavier complex is expected to
concentrate into a pellet at the bottom of a test tube through sufficiently rough centrifugation. The
presence of acetylcholinesterase activity in the remaining supernatant at the top would determine
the relative proportion of acetylcholinesterase that manages to break free from the complex, which

17

can then be translated to the overall stability of the complex. By using DTNB to label the
acetylcholinesterase activity in the supernatant, the results showed that, except for 1% SDS and 6
M of guanidine isothiocyanate, the relative proportion of enzyme activity decreased to roughly
half or nearly completely diminished when the complex was subjected separately to stringent
experimental conditions. Overall, both experiments suggest the existence of a profoundly strong
stability inherent in the amyloid-beta peptide-acetylcholinesterase complex, bound together by
hydrophobic interactions.21
In addition to stable co-localization, other in vitro experiments have also shown that
acetylcholinesterase contributes to the growth of amyloid-beta peptides.22 Furthermore, the
interaction of acetylcholinesterase with the complex corresponds to greater neurotoxicity, as
observed by lower cell viability in the MTT assay, followed by a reduction in cell density and
truncated axon growth of neurons in cell cultures.20
1.2.1 Alzheimer’s Disease
Based on the substantial accumulation of observations and experimental data obtained from
numerous scientific studies to date, Alzheimer’s disease, the most common neurodegenerative
disease, serves as the foundation for developing several hypotheses aimed at explaining its
pathogenesis. Although these hypotheses remain incomplete, they provide valuable insights into
creating an overall depiction of Alzheimer’s disease pathogenesis, particularly from the
perspective of cholinergic neurotransmission. With hope, these insights may extend to the
pathogenesis of other neurodegenerative diseases as well.
Among these hypotheses, the amyloid-beta peptides are considered a major focal point.
According to the amyloid hypothesis, amyloid-beta peptides are generated from the amyloid
precursor protein through hydrolysis by the γ-secretase complex, of which presenilin 1 is a

18

component. This hydrolysis process depends on acetylcholinesterase. In addition to facilitating the
production of amyloid-beta peptides, acetylcholinesterase also directly binds to amyloid-beta
peptides, forming highly stable complexes that enhance their fibrilization into long fiber stretches.
Notably, acetylcholinesterase’s involvement in these three processes is independent of its catalytic
anionic site.
Overall, amyloid-beta peptides begin as short peptide sequences of approximately forty
amino acids in length. They then transform into complexes when coupled with acetylcholinesterase,
subsequently forming fibers and eventually aggregating densely enough to be termed plaques.
Consequently, the presence of amyloid-beta-acetylcholinesterase complexes is generally more
neurotoxic than amyloid-beta peptides alone. Interestingly, there is also a positive correlation
between the increase of the soluble monomeric form of acetylcholinesterase, shifting away from
its membrane-bound tetrameric form, and the increase in amyloid-beta plaque density as the
disease progresses.23
In a second hypothesis called the tau hypothesis, the focus is on the tau protein. Under
physiological conditions, it binds and stabilizes microtubules, facilitating the delivery of nutrients
and essential molecules from the cell body to the dendrite and axon within a neuron. In
Alzheimer’s disease, it detaches from microtubules and attaches to other tau proteins in a
disorderly manner, forming neurofibrillary tangles. These tangles obstruct the flow and
transportation of biomolecules, ultimately negatively impacting communication between neurons.
The abnormal behavior of the tau protein is associated with its hyperphosphorylation, and it is also
linked to an increased expression of acetylcholinesterase, neither of which occurs under
physiological conditions. Consequently, the increased expression of acetylcholinesterase results in

19

the co-localization of the enzyme with the tau protein in the cytoplasm. Uncertainly, this somehow
leads to increased acetylcholinesterase activity that depletes acetylcholine at the synapse later on.
In a third hypothesis called the cholinergic hypothesis, acetylcholinesterase becomes the
central focus. Throughout Alzheimer’s disease, acetylcholinesterase activity mildly decreases in
the early stages, but becomes overly active as the disease progresses to the late stages.
Accompanying this overactivity of acetylcholinesterase are the decreased level of miRNA-132 and
the increased apoptosis of neurons that gradually develop over time.23
1.2.2 Parkinson’s Disease
Parkinson’s disease is the second most common neurodegenerative disease. Its pathology
is mainly characterized by clumps of misfolded proteins within the neuron, of which one type is
called Lewy bodies. Similar to amyloid-beta peptides, these Lewy bodies comprise of repeating
units of the alpha-synuclein protein that can extend into long stretches of fibers. In the context of
Parkinson’s disease, the bodies are not properly degraded and overwhelm the neuron, resulting in
neurotoxicity as amyloid-beta peptides would.57 Similar to Alzheimer’s disease, the formation of
amyloid-beta plaques is also present in Parkinson’s disease, and acetylcholinesterase may also be
involved in stabilizing and enhancing their formation. Unlike Alzheimer’s disease, the neurotoxic
effects of amyloid-beta plaques may not only exacerbate dementia, but also influence mood and
physical mobility. Often, mood changes tend to be pessimistic, associated with mental states of
depression, anxiety, and apathy. Impaired physical mobility leads to disruptions in a subject’s
sense of balance, resulting in postural instability and gait difficulty. Hyperphosphorylation of the
tau protein and its association with acetylcholinesterase may contribute to visual and olfactory
dysfunctions in Parkinson’s disease as well.

20

In general, there is a slight difference from Alzheimer’s disease in that acetylcholinesterase
activity is more significantly dampened throughout Parkinson’s disease, but followed again by a
sudden increase in activity upon the apoptosis of neurons. Additionally, the significantly lower
acetylcholinesterase activity in Parkinson’s disease not only leads to more pronounced deficits
affecting cognitive processes in the central nervous system, but also affects major sensory
communications between the sense organs and the peripheral nervous system. Overall, there is a
similar mode of action for acetylcholinesterase, but a slight difference in the duration of enzyme
activity at different levels between Alzheimer’s and Parkinson’s disease. Concerning the
magnitude of the adverse effects contributed by acetylcholinesterase, it appears that Parkinson’s
disease displays more severe deficits, which tend to extend further beyond the central nervous
system compared to Alzheimer’s disease.23
1.2.3

Huntington’s Disease
Unlike Alzheimer’s and Parkinson’s diseases, Huntington’s disease does not result in the

eventual death of neurons through apoptosis. Its pathology is mainly characterized by a distinctive
mutation in the HTT gene. The HTT gene is an abbreviation for the word “Huntington”. Although
the normal function of this gene is not fully understood, it is known to be an extremely important
factor in the development of the central nervous system. In Huntington’s disease, the mutation
carried by this gene contains many repeats of the base sequence CAG, which translates to a
mutated protein consisting of an abnormally long chain of glutamine repeats. The mutated protein
is then cleaved by different proteases into many different types of fragments that cause
neurotoxicity. Of those fragments, the most studied one is called HTT exon 1. This fragment is
highly malleable, capable of transforming into different conformations that combine with other
proteins, including amyloid-beta peptides, to form neurotoxic tangles. As a result, this leads to a

21

persistent impairment of neurons.58 In addition to this type of impairment, a reduction in the
expression of acetylcholinesterase on the extracellular surface at the synapse is associated to it.
Simultaneously, a decrease in the expression of both choline acetyltransferase, an enzyme
responsible for synthesizing acetylcholine, and the vesicular acetylcholine transporter, a protein
responsible for loading acetylcholine into secretory vesicles before its release as a neurotransmitter
into the synapse, is also observed. Consequently, it has been suggested that the reduced expression
of acetylcholinesterase may represent a cellular response to balance overall energy allocation
within the cell, as the amount of acetylcholine to be synthesized, released, and subsequently
catabolized is expected to be lower under the conditions of this disease.23
1.2.4 Multiple Sclerosis
Differing from Alzheimer’s and Parkinson’s diseases, the pathology of multiple sclerosis
is mainly characterized by demyelinating lesions along the neuron. With regards to the
acetylcholinesterase enzyme, it experiences a significant and cyclic bipolar swing in activity in
multiple sclerosis, seemingly trapped in an endless cycle between the relapsing phase and the
remitting phase. In the relapsing phase, the expression of acetylcholinesterase increases by more
than 60% on average compared to normal level, followed by a rapid increase in enzyme activity
that depletes acetylcholine level. Alongside this increased enzyme activity, high levels of proinflammatory cytokines are observed. These cytokines can develop and cause permanent damage
to the myelin sheaths surrounding the axon over time.
In the remitting phase, the expression of acetylcholinesterase and its enzyme activity
decrease significantly. In some cases, a decreased activity level of both choline acetyltransferase
and the vesicular acetylcholine transporter, which coincides with the rapid increase in
acetylcholinesterase activity during the relapsing phase, and vice versa during the remitting phase,

22

is also observed. Interestingly, a decreased level of miRNA-132 is linked to the drastic increase in
acetylcholinesterase activity during the relapsing phase as well.
Taken together, in addition to its overall role in modulating cholinergic neurotransmission
in multiple sclerosis, these findings suggest a role in anti-inflammation that is inherent in
cholinergic signaling. This implies that acetylcholinesterase may have the ability to diminish the
anti-inflammatory response directed by cholinergic neurotransmission. However, the specific
mechanisms by which acetylcholinesterase contributes to the worsening severity of multiple
sclerosis during each cycle of relapsing and remitting, and how it gradually overwhelms and
desensitizes the anti-inflammatory response of cholinergic signaling over the course of the disease,
remain unclear.23
1.3

Acetylcholinesterase Inhibitors in the Treatment of Neurodegenerative Diseases
Neurodegenerative diseases remain incurable, and most available medical treatments

primarily focus on alleviating symptoms and slowing their progression for a limited time, typically
through the use of acetylcholinesterase inhibitors. Currently, the acetylcholinesterase inhibitors
used for therapeutic purposes are all reversible inhibitors. They are primarily employed to address
symptoms related to cognitive processes such as memory, learning, and judgement. Most of these
inhibitors are synthetic compounds.24 Their drug potency in treating the symptoms of
neurodegenerative diseases has an average IC50 range, which can vary from as low as 0.02 μM to
as high as 25 μM.25
Regarding treatment effectiveness, most of these inhibitors can only manage mild to
moderate stages of cognitive deterioration, and their modest effects typically last for a short period
of time, usually confined to around two years. In addition, they often come with a range of
undesirable common side effects that tend to affect the central nervous system and the digestive

23

system. These side effects can include headache, dizziness, low fever, seizures, as well as appetite
loss, abdominal pain, vomiting, and diarrhea.24 Rivastigmine, donepezil, galanthamine, and
huperzine A are among the most commonly known acetylcholinesterase inhibitors.
1.3.1

Rivastigmine
Rivastigmine is a synthetic compound that inhibits acetylcholinesterase and

butyrylcholinesterase (Figure 5). It is approved for alleviating symptoms in mild to moderate
stages of Alzheimer’s and Parkinson’s diseases, and can be administered orally or transdermally.
In some cases, transdermal administration of the drug has proven effective even against severe
stages of these diseases, making it the more popular mode of drug administration for two primary
reasons. The first reason is that it eliminates the need for patients to ingest the drug, which can be
challenging even as a simple task, particularly for those who may have difficulty remembering to
do so. The second reason is that it bypasses the gastrointestinal tract, leading to fewer side effects.
The drug primarily targets acetylcholinesterase to increase cholinergic signaling by
reducing the level of amyloid-beta peptides, thereby improving cognitive abilities necessary for
daily activities. Rivastigmine can also enhance a patient’s mood by reducing sleep disturbances,
anxiety, hallucinations, and the frequency of falls due to impaired mobility in Parkinson’s disease.
However, rivastigmine is associated with a wide range of adverse side effects, primarily related to
gastrointestinal disturbances, and also includes weight loss, skin allergies, irregular heartbeat,
vascular disorders, and psychiatric disorders, among others. Additionally, its effects typically last
only a few years, and in some cases, as short as six months to a year, in both diseases. Furthermore,
the results of rivastigmine treatment for Huntington’s disease show significant discrepancies, and
it offers insignificant improvements in the treatment of multiple sclerosis.23

24

Figure 5. The Chemical Structure of Rivastigmine.

1.3.2

Donepezil
Donepezil is a synthetic inhibitor that selectively targets acetylcholinesterase (Figure 6). It

shares many similarities with rivastigmine, but there are still some subtle differences between the
two. Originally, donepezil is approved for alleviating symptoms of mild to severe stages of
Alzheimer’s disease, although in some cases, it is used to alleviate symptoms of mild to moderate
stages of Parkinson’s disease as well. It is administered orally, with the notable advantage being
its minimal interaction and interference with the effects of other drugs when patients are
undergoing multiple drug treatments simultaneously.
In addition to reducing the level of amyloid-beta peptides by targeting acetylcholinesterase,
the drug may also inhibit the formation of neurofibrillary tangles resulting from the aggregation of
hyperphosphorylated tau protein. This capability can lead to an improvement in cognitive abilities
for both diseases. In Parkinson’s disease, it may also enhance a patient’s mood by mitigating
depression and reducing the frequency of hallucinations. However, similar to rivastigmine,
donepezil carries a significant number of side effects, including those related to gastrointestinal
disturbances, which can extend to muscle cramps and insomnia. Furthermore, its effects are
modest and not long-lasting. Additionally, the results of donepezil treatment show significant
discrepancies when it comes to both Huntington’s disease and multiple sclerosis.23

25

Figure 6. The Chemical Structure of Donepezil.

1.3.3

Galanthamine
Galanthamine is a natural alkaloid belonging to the Amaryllidaceae family that selectively

inhibits acetylcholinesterase (Figure 7). Notably, its inhibition of acetylcholinesterase is 53 times
stronger than that of butyrylcholinesterase. It is approved for alleviating symptoms in mild to
moderate stages of Alzheimer’s disease. Similar to rivastigmine, the drug enhances cognitive
abilities primarily by targeting acetylcholinesterase to increase cholinergic signaling through a
reduction in amyloid-beta peptides. One advantage over other acetylcholinesterase inhibitors is its
extensive volume of distribution, which means it circulates throughout the entire body, increasing
the likelihood of reaching the brain to exert its effects rather than being primarily confined to the
bloodstream.
In addition to Alzheimer’s disease, galanthamine may hold potential for the treatment of
Huntington’s disease. However, like most other inhibitors, it carries a significant number of side
effects, including those mentioned earlier. Furthermore, its effects are modest, not long-lasting,
and tend to diminish after one to one and a half years. Moreover, the results of galanthamine
treatment exhibit significant discrepancies in the context of Parkinson’s disease, and there is no
available recorded data on its treatment effects against multiple sclerosis.23

26

Figure 7. The Chemical Structure of Galanthamine.

1.3.4

Huperzine A
Huperzine A is a natural alkaloid derived from the Huperzia serrata species, which

selectively inhibits acetylcholinesterase (Figure 8). It has not been registered as a drug for any
neurodegenerative disease, except in China, where it has been approved for the treatment of
symptoms in mild to moderate stages of Alzheimer’s disease. Similar to rivastigmine, this
substance appears to enhance cholinergic signaling by inhibiting acetylcholinesterase and reducing
the level of amyloid-beta peptides. However, there is insufficient data to determine its side effects,
and there is no available recorded data on its treatment effects against other neurodegenerative
diseases.23

Figure 8. The Chemical Structure of Huperzine A.

1.4

The Need to Search for Novel Acetylcholinesterase Inhibitors
From

the

available

information,

it

appears

that

despite

these

well-known

acetylcholinesterase inhibitors targeting acetylcholinesterase, whether to hinder its interaction with
27

aberrant biochemical structures detrimental to cholinergic neurotransmission under disease
conditions, or to suppress its overpowering catabolism of acetylcholine upon upregulation, their
overall treatment effects are generally modest and not long-lasting.
One possible explanation for this is that their continuous inhibition of acetylcholinesterase
activity at varying strengths over an extended period of time may lead to a gradual restriction of
the activity of M1 and M2 receptors at the synapse. As a potential compensatory cellular response
to prolonged acetylcholinesterase activity suppression, three subsequent events may occur as
consequences: the desensitization of the M1 receptor’s neuronal excitatory response to
acetylcholine binding; the hypersensitization of the M2 receptor’s autoinhibitory response to
acetylcholine secretion into the synapse; and the excessive internalization of both receptors for
intracellular degradation.
Besides the modest and short-term treatment effects, these inhibitors fail to consistently
yield positive treatment results in most neurodegenerative diseases other than Alzheimer’s disease.
Additionally, their adverse side effects significantly impact on the central nervous system and the
digestive system. Consequently, these drawbacks highlight the need to search for more effective
acetylcholinesterase inhibitors capable of prolonging disease treatment while maintaining a
balanced inhibition of cholinergic neurotransmission. Such inhibitors should ideally require less
frequent administration. With this in mind, this study was undertaken to examine several small
molecules and mushroom extracts in an effort to identify potential novel acetylcholinesterase
inhibitors.
1.4.1 Small Molecules: SPOPP and NP6A
SPOPP and NP6A are dispiropiperazine derivatives synthesized from an azomethine ylide
cycloaddition reaction using acenaphthenequinone and L-proline as substrates (Figure 9). The

28

reaction mixture is simmered under reflux in methanol for 3 h at 35°C. These chemical derivatives
have been reported as excellent cell cycle checkpoint inhibitors of the G2/M phase when combined
with DNA-damaging agents or radiation treatment to combat cancer. The insufficient exploration
of this topic led to its selection as the focal point of research conducted by former team member
Victor Liu from the Lee lab.
Previously, treatment with SPOPP was demonstrated to induce cell cycle arrest at the G2/M
phase and significantly reduce cell viability through apoptosis or necrosis in various cancer cell
lines, including leukemia, glioblastoma, colon cancer, cervical cancer, ovarian cancer, and breast
cancer. The G2/M phase arrest was confirmed by examining the expression levels of cyclin B1, a
crucial regulator of the transition from the G2 phase to the M phase, and phosphohistone H3, a
biomarker signaling a disruption in this transition. Cyclin B1 level was shown to decrease, while
phosphohistone H3 level increased rapidly. Furthermore, a closer examination revealed a disarray
of mitotic spindle fiber formation misaligned at the cell’s equator and detectable DNA damage as
early as 1 h after treatment. In contrast to SPOPP, treatment with NP6A did not yield significant
results.26

Figure 9. The Chemical Structures of SPOPP and NP6A.

1.4.2

Small Molecules: 534_1(6) and 534_2(7)

29

534_1(6) and 534_2(7) are dispiropyrrolizidine derivatives synthesized through a similar
reaction to SPOPP and NP6A, albeit under slightly different reaction conditions (Figure 10).
Simmering under reflux in either methanol or acetonitrile for as long as 1 to 48 h at a fairly hot
temperature of 65°C, in addition to the initial reaction between acenaphthenequinone and L-proline,
the by-products from the first reaction are further reacted with (2E)-2-(2-bromobenzylidine)-1indanone to produce 534_1(6) and 534_2(7). These chemical derivatives have been reported to be
bioactive molecules with a wide range of therapeutic properties, including anticancer, antioxidant,
antimicrobial, antifungal, and inhibition of acetylcholinesterase activity.
Previously, former team member Lee Hooi Xian from the Lee lab conducted research
focusing on screening 534_1(6) and 534_2(7) for their potential as novel inhibitors of KRAS, a
crucial drug target in colorectal cancer treatment. The motivation for her research stemmed from
the limited availability of effective inhibitors, with few positive treatment responses from patients
using existing drugs. Experimental results showed that treatment with either 20 μM of 534_1(6)
or 534_2(7) significantly downregulated the expression of KRAS by more than 50% after 48 h.
However, the specific mechanisms of their suppression on KRAS expression remained tentative
and required further studies.27 While dispiropyrrolizidine derivatives may exhibit an inhibitory
effect against acetylcholinesterase activity, 534_1(6) and 534_2(7) have not been reported to show
any inhibition of acetylcholinesterase activity.

30

Figure 10. The Chemical Structures of 534_1(6) and 534_2(7).

1.4.3

MolTarPred Program
Using a web-based ligand-centric computational program for small molecule target

prediction called MolTarPred, SPOPP and NP6A were predicted to bind to acetylcholinesterase.
In simple terms, the MolTarPred program comprises a large database containing numerous small
molecules, each known to bind to at least one protein target. When a new small molecule is entered
into the program, the program compares structural similarities between the new input and the small
molecules in the database to search for potential matches. When a match is found, the program
predicts the binding affinities of the new input for protein targets already known to be bound by
the matched small molecule in the database. Subsequently, based on the structural similarities
between the new input and the matched small molecule, and the known binding affinities between
the protein targets and the matched small molecule, the binding affinities between the newly input
small molecule and the protein targets are ranked by numbers, with 1 being the least reliable and
10 being the most reliable. These rankings are then considered predictions, or in mathematical
terms, probabilities.
According to the program, acetylcholinesterase was identified as the number one protein
target predicted to be bound by both SPOPP and NP6A, from a list of several potential protein
targets. The ranking for SPOPP and acetylcholinesterase was 7, while that for NP6A was 4. Later
on, former team member from the Lee lab of the University of Northern British Columbia Lee
31

Hooi Xian discovered 534_1(6) and 534_2(7) possessing molecular structures similar to SPOPP
and NP6A. In this Master’s thesis, an evaluation of these four small molecules for their potential
ability to inhibit acetylcholinesterase activity was described.
1.5

Mushrooms as a Potential Source of Acetylcholinesterase Inhibitors
In addition to small molecules, there are also several acetylcholinesterase inhibitors derived

from natural sources. Mushrooms, in particular, stand out as a unique natural resource with
numerous latent therapeutic applications yet to be fully explored. Globally, there are an estimated
three to six million mushroom species, but only 3% to 8% of them are accurately classified. Among
these, just over twenty-five are considered edible, and only a few are cultivated, mass-produced,
and consumed worldwide. Needless to say, those with significant therapeutic applications are
scarce, even more so in the context of neurodegenerative diseases.28
For those with specific therapeutic applications in neurodegenerative diseases, there are
only a handful of them. The two most commonly mentioned are Hericium erinaceus and
Volvariella volvacea. Hericium erinaceus possesses anti-aging and anti-dementia properties, and
its neuroprotective effects can support the biosynthesis of nerve growth factors. It is often used to
prevent common neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases.
Volvariella volvacea has a high antioxidant effect and can help regulate excessive neuron
excitation throughout the nervous system. It is often used to prevent cardiovascular and
neurodegenerative diseases.
In addition to these two, there are less common mushroom species like Flammulina
velutipes and Phellinus gilvus. Flammulina velutipes is often used to reduce cholesterol level in
the human body, lower pro-inflammation in the event of a food allergy, and act as a preservative
to protect meat and seafood from oxidation. It primarily exhibits anticancer effects, although its

32

inhibitory effect on acetylcholinesterase activity is also noted. Phellinus gilvus is commonly used
to treat inflammation, stomachache, and tumors. It primarily offers anticancer and antiinflammation effects, with some mention of neuroprotective effects for this species.29
While not many mushroom species are directly applied for therapeutic use, numerous
experiments are being conducted on different cell lines to explore their medicinal properties for
potential therapeutic applications in neurodegenerative diseases (Table 1). However, the scope of
these experiments is largely limited to the examination of the pathogenesis of Alzheimer’s and
Parkinson’s diseases.30 Regarding the inhibition of acetylcholinesterase activity, there are also
many mushroom species being studied, but a comprehensive understanding of the extent to which
they inhibit acetylcholinesterase activity remains mostly incomplete (Table 2).31 32 33 34 35 36 37 38 39
40 41

33

Medicinal Property

Mushroom Species

Pleurotus ostreatus
Amauroderma rugosum
Armillaria mellea
Anti-apoptosis
Morchella importuna
Pleurotus eryngii
Tremella fuciformis
Amanita caesarea
Armillaria mellea
Anti-cholinergic signaling
Flammulina velutipes
Hericium erinaceus
Amanita caesarea
Anti-neuroinflammation Antrodia camphorata
Ganoderma lucidum
Anti-neurotoxicity
Hericium erinaceus
Amanita caesarea
Auricularia auricula-judae
Cantharellus cibarius
Dictyophora indusiata
Flammulina velutipes
Antioxidant
Ganoderma atrum
Ganoderma lucidum
Inonotus obliquus
Pleurotus ostreatus
Tricholoma lobayense
Anti-amyloidogenesis

Type of Neurodegenerative Disease
Alzheimer's Disease
Neurotoxicity
Alzheimer's Disease
Neurodegenerative Diseases
Aging
Neurodegenerative Diseases
Alzheimer's Disease
Alzheimer's Disease
Cognitive Impairment
Alzheimer's Disease
Alzheimer's Disease
Parkinson's Disease
Alzheimer's Disease
Alzheimer's Disease
Alzheimer's Disease
Aging
Neurodegenerative Diseases
Neurodegenerative Diseases
Alzheimer's Disease
Aging
Alzheimer's Disease
Alzheimer's Disease
Alzheimer's Disease
Aging

Table 1. List of Example Mushroom Species Conducted in the Research of Neurodegeneration.
These mushroom species are known examples that are being experimented on different cell lines in the
research of neurodegeneration. Some have multiple medicinal properties, but most are limited against
Alzheimer’s and Parkinson’s diseases.30

34

Agaricus bisporus
Agaricus bitorquis
Agaricus essettei

Ethyl Acetate
Ethyl Acetate
Ethyl Acetate

2.27700
0.74500
0.91800

Concentration Range
(mg/mL)
N/A
N/A
N/A

Amanita strobiliformis

Hot Water

0.00727

N/A

Ganoderma adspersum

Hot Water

Ganoderma applanatum

Hot Water

Ganoderma lucidium
Ganoderma mediosinense
Ganoderma mediosinense
Ganoderma ramosissimum

Hot Water
70% Methanol
Ethyl Acetate
70% Methanol

Hericium novae-zealandiae

Water

Mushroom Species

IC50
(mg/mL)

Extract

Inonotus obliquus

80% Methanol

Phellinus igniarus

Hot Water

Phellinus pini

Hot Water

Pleurotus florida

Hydromethanol

Pleurotus ostreatus

Methanol

Pleurotus ostreatus

Hot Water

Pleurotus pulmonarius

80% Methanol

10.56% inhibition
at 0.20000 mg/mL
11.87% inhibition
at 0.20000 mg/mL
1.01000
43.86000
0.81000
58.24000

0.060 - 2.000
N/A
N/A
N/A

8.00000 - 8.70000

3.000 - 32.000

N/A
21.14% inhibition
at 0.20000 mg/mL
29.32% inhibition
at 0.20000 mg/mL
59.13000
13.00% inhibition
at 0.20000 mg/mL
8.62% inhibition
at 0.20000 mg/mL
68.60% inhibition
at 0.50000 mg/mL

N/A
N/A

N/A
N/A
N/A
N/A
0.025 - 0.200
N/A
0.063 - 1.000

Table 2. List of Example Mushroom Species Reported for In Vitro Inhibition of Acetylcholinesterase
Activity.
These mushroom species are known examples that can inhibit the in vitro acetylcholinesterase activity.
However, the extent to which they inhibit acetylcholinesterase activity remains mostly incomplete. The
highlighted species were a part of those examined for their inhibitory effect on acetylcholinesterase activity
in this study.31 32 33 34 35 36 37 38 39 40 41

Therefore, there is a need to further establish, explore, and identify mushroom species that
can be beneficial for the treatment of not only common neurodegenerative diseases, but also less
common ones. As a result, the second research objective turned to the examination of extracts from
eight mushroom species for their potential inhibitory effect on acetylcholinesterase activity. Since
35

there is a lack of comprehensive data on the full concentration range of inhibition on
acetylcholinesterase activity and the IC50 value across various studies for a few of the mushroom
species listed in Table 2, the following species were chosen for re-examination to determine
whether their potential inhibitory effect on acetylcholinesterase activity could be replicated:
Ganoderma applanatum, Inonotus obliquus, Phellinus igniarius, Phellinus pini, and Pleurotus
ostreatus. Additionally, three more species were included in the study, each of which has never
been reported to exhibit any inhibitory effect on acetylcholinesterase activity. These species were
Agaricus subrufescens, Amanita muscaria, and Hericium erinaceus.
With the exception of Agaricus subrufescens, each of these eight mushroom species has
either been reported for inhibition against acetylcholinesterase activity; has not been reported for
such inhibition but has been reported for inhibition against other targets of neurodegeneration; or
has other species from the same genus reported for inhibition against acetylcholinesterase activity
and/or inhibition against other targets of neurodegeneration. Therefore, given the significant
overlap of medicinal properties within species and across species of the same genus, it was
hypothesized that there is a high chance that each of the eight species may potentially contain at
least one extract that can exert significant inhibition on acetylcholinesterase activity.
1.6

Research Objectives
The goals of this study were to test the following hypotheses:
I.

The four small molecules SPOPP, NP6A, 534_1(6), and 534_2(7) have an
inhibitory effect against acetylcholinesterase activity.

II.

The extracts from the selected eight mushroom species collected from the
forests in Northern British Columbia have an inhibitory effect against
acetylcholinesterase activity.

36

Chapter 2
Development of the Acetylcholinesterase Assay and Examination of the Effect of Small
Molecules on Acetylcholinesterase Activity
2.1

Introduction

2.1.1

Overview of the Acetylcholinesterase Assay Design
To assess the inhibitory effect of small molecules or mushroom extracts on

acetylcholinesterase, an enzyme assay using Ellman’s reagent 5,5’-dithiobis-2-nitrobenzoic acid
(DTNB) was employed. This assay involved five key components: sodium phosphate buffer,
Electrophorus electricus (South American electric eel) acetylcholinesterase, acetylthiocholine
iodide, the test sample (either a small molecule or mushroom extract), and DTNB.
In theory to how this assay works, acetylcholinesterase initially hydrolyzes
acetylthiocholine into acetic acid and thiocholine. Thiocholine, acting as a reducing agent, then
reduces DTNB into 2-nitro-5-thiobenzoic acid, abbreviated as TNB2-, by breaking the disulfide
linkage of DTNB (Figure 11).42 The presence of TNB2- results in a yellow coloration, which its
absorbance can be measured at the wavelength of 412 nm. The intensity of the yellow color, and
therefore the absorbance, is directly proportional to the amount of TNB2-.

37

Figure 11. The Reaction Mechanism of the Acetylcholinesterase Assay Using Ellman's Reagent.
First, acetylcholinesterase hydrolyzes acetylthiocholine into acetic acid and thiocholine. Then, acting as a
reducing agent, thiocholine reduces DTNB into TNB2-. Finally, TNB2- reflects back one of the shades of
the yellow color, which can be measured at the wavelength of 412 nm.

In an ideal condition without an acetylcholinesterase inhibitor, the rate of TNB2absorbance increase is expected to follow a linear trend over time until it reaches the stationary
phase of enzyme activity, where further increases are not possible, causing the linear trend to
become logarithmic. However, in the presence of an acetylcholinesterase inhibitor, the rate of
TNB2- absorbance increase is expected to be slower compared to the absence of an inhibitor over
time.
2.1.2

Considerations and Selection for the Main Components of the Acetylcholinesterase
Assay
DTNB as an Ideal Reagent for Thiol Quantification
The use of Ellman’s reagent DTNB for thiol quantification in protein studies is well

established. The thiol side chain of cysteine, along with other thiol-containing compounds, is a
38

preferred subject of research due to its significant role in medical science as an antioxidant and
anticancer agent. While there are alternative reagents and methods for thiol quantification, such as
para-chloromercuribenzoic acid, ethylmaleimide, nitrobenzo-2-oxa-1,3-diazole, pyridine disulfide,
and the use of papain reactivation, DTNB remained the most suitable choice for this study’s
enzyme assay design.43 The specific reasons for the disadvantages of substituting DTNB with these
reagents for thiol quantification were explained in the following paragraphs.
Para-chloromercuribenzoic acid, abbreviated as pCMB, is used to react with thiols,
producing a product with its absorbance measured at the wavelength of 255 nm. However, the
product is not colorimetric, and its absorbance falls within the UV range of 100 to 400 nm, which
can also be absorbed by non-thiols. Additionally, pCMB can have non-specific reactions with
some non-thiols. Furthermore, UV detection requires experimental tools made of quartz, which
are more expensive than common materials like glass and plastic.43
As for pyridine disulfide, it reacts with thiols to generate an acidic form that quickly
rearranges into a ketone form called thiopyridone, with the absorbance of thiopyridone measured
in the range from 324 nm to 343 nm. Similar to the product of pCMB, the issues associated with
relying on UV detection are as explained. Additionally, aside from the irreversible nature of the
reaction, pyridine disulfide is poorly soluble in neutral and basic solutions,43 which are crucial
conditions for the optimal performance of the acetylcholinesterase assay. Moreover, it is capable
of permeating through biological membranes.44
Unlike pCMB and pyridine disulfide, the disadvantages of using ethylmaleimide,
nitrobenzo-2-oxa-1,3-diazole, or papain reactivation are more related to their performance
efficiency than design compatibility. Ethylmaleimide is used to react with thiols, initiating a
reaction in which its absorbance is measured as it decreases, at the wavelength of 300 nm.43 Apart

39

from the issues associated with relying on UV detection, the approach, while appropriate, may
seem counterintuitive, as it involves visualizing increasing enzyme activity corresponding to
diminishing color.
Nitrobenzo-2-oxa-1,3-diazole, abbreviated as NBD, is used to react with thiols to generate
a covalent product, with the absorbance of the product measured at the wavelength of 425 nm.
However, achieving sufficient colorimetric detection of the covalent product requires a thiol to
NBD ratio of 1:10, in contrast to a thiol to DTNB ratio of 1:1 needed to achieve the same visual
effect.43 This significant difference in the ratio leads to considerably higher costs.
Papain reactivation relies on the inactivation of a cysteine hydrolase by methyl
methanethiosulfonate, followed by its reactivation upon exposure to thiols. When subjected to its
substrate n-benzoyl-L-arginine-p-nitroanilide, the reaction generates a product with its absorbance
measured at the wavelength of 410 nm. Although the product is 100 times more sensitive than
DTNB for the same initial amount of thiols, the maximum amount of thiols that can be quantified
through papain reactivation is 100 times less than with DTNB.43 In the specific assay design of
this study, DTNB has none of the disadvantages mentioned above in a relative comparison among
all the thiol quantifiers discussed so far. Therefore, as the most suitable option by far, it was the
only appropriate thiol quantifier for this study.
Acetylthiocholine Iodide as an Alternative Substrate of Acetylcholinesterase
To facilitate the efficient reduction of DTNB, acetylthiocholine iodide was chosen as the
substrate of acetylcholinesterase, replacing the enzyme’s natural substrate acetylcholine. The
primary objective was to produce a product that contains a free thiol group for the ready reduction
of DTNB. During the hydrolysis by acetylcholinesterase, acetylthiocholine is enzymatically

40

cleaved into acetic acid and thiocholine, whereas acetylcholine is cleaved into acetic acid and
choline. Thiocholine contains a free thiol group, while choline does not (Figure 11).
Electrophorus electricus Acetylcholinesterase as an Economical Orthologue
Prevalent in Fundamental Research Studies
The selected orthologue of acetylcholinesterase was Electrophorus electricus (South
American electric eel), instead of Homo sapiens (human). In a cross-species genetic comparative
study involving Homo sapiens (human), Mus musculus (house mouse), Rattus norvegicus (brown
rat), Felis silvestris catus (cat), Oryctolagus cuniculus (European rabbit), Bos taurus (cow),
Torpedo californica (Pacific electric ray), and Drosophila melanogaster (common fruit fly), the
amino acid sequence of the primary structure of acetylcholinesterase showed a percentage identity
of 80.13% among mammals, which decreased to 56.14% when Torpedo californica was included
and further decreased to 27.68% when Drosophila melanogaster was included. However, the
amino acid sequence of the active site of the enzyme exhibited a 100.00% identity among
mammals, which decreased to 92.00% when Torpedo californica was included and further
decreased to 76.00% when Drosophila melanogaster was included. Despite the significant
differences in the overall amino acid sequence of the enzyme, the active site’s amino acid sequence
remained highly conserved across species. It was suggested that this conservation may result in a
highly similar tertiary structural fold of the enzyme among species. This, along with the
conservation of the active site, imply that the enzyme’s functionality is likely highly conserved
across species.45
Although Electrophorus electricus was not part of the comparative analysis, it was also
expected to be functionally conserved when compared to other species. According to other
scientific studies, the amino acid sequence of acetylcholinesterase showed a percentage identity of

41

50% between the Electrophorus electricus and the mammalian orthologues. As for the percentage
identity of the amino acid sequence of the enzyme’s active site, it was reported to be 83% between
these orthologues.59 60 Taken together, these two percentage identity values were reported similarly
to the ones mentioned above for the Torpedo californica orthologue, matching the given
expectation since both are electric fish species. Nevertheless, in theory, if acetylcholinesterase
activity is 100% functionally conserved across species, its binding affinity for a ligand should also
be very similar among different orthologues. This means that a small molecule inhibitor, acting as
a ligand, is expected to exhibit a very similar degree of inhibition on acetylcholinesterase from
different species.
In support of the aforementioned cross-species comparative study of the amino acid
sequence of acetylcholinesterase, another study cross-examined the effect of eight different known
inhibitors on Electrophorus electricus and Homo sapiens acetylcholinesterase. Considering the
negligible difference in the baseline activity of Homo sapiens acetylcholinesterase attributed to
biological sex and age factors from human blood donors, the study found that the variation in
acetylcholinesterase activity between species in the presence of each known inhibitor was modest
and primarily a result of the distinct chemical properties of the inhibitors. The research revealed
that Electrophorus electricus acetylcholinesterase was more sensitive to inhibitors of oxon
metabolites, whereas Homo sapiens acetylcholinesterase was more sensitive to carbamate
inhibitors.46 For instance, when tested on Electrophorus electricus acetylcholinesterase, the IC50
values for oxon metabolites chlorpyrifos-oxon (CPO), phosmet-oxon (PMO), and diazinon-oxon
(DZO) were 0.027 μM, 0.070 μM, and 1.030 μM, respectively. In contrast, the IC50 values for
carbamates pirimicarb (PI) and rivastigmine (RI) were 61 μM and 53 μM, respectively.46 When
tested on Homo sapiens acetylcholinesterase, the average IC50 values for oxon metabolites CPO,

42

PMO, and DZO were 0.300 ± 0.100 μM, 1.900 ± 1.100 μM, and 2.500 ± 0.800 μM, respectively,
while the average IC50 values for carbamates PI and RI were 20.000 ± 4.300 μM and 9.900 ± 3.100
μM,

respectively.

These

results

highlighted

that

the

interspecies

conservation

of

acetylcholinesterase activity in response to binding a ligand is rigorous, though not entirely perfect.
The findings are consistent with those of the aforementioned cross-species comparative
study of the amino acid sequence of acetylcholinesterase. They collectively reflect the overarching
reality that finding or synthesizing a species-specific acetylcholinesterase inhibitor, whether
naturally or artificially, appears to be exceedingly challenging. One plausible explanation for the
minor difference in acetylcholinesterase activity across species in the presence of each known
inhibitor could be due to minor variations in the spatial arrangement of the active site in the
acetylcholinesterase orthologues.46
In conclusion, although the choice of an acetylcholinesterase orthologue for use in the
enzyme assay design of this study may not seem essential, significant cost difference exists among
the orthologues available on the market. For example, an amount of Electrophorus electricus
acetylcholinesterase that is 100 times greater than the amount of Homo sapiens
acetylcholinesterase can be purchased for a similar price according to Sigma Aldrich. Therefore,
opting for Electrophorus electricus acetylcholinesterase appears to be the more financially
reasonable choice.
Sodium Phosphate Buffer as a Nurturing Cocktail for an Efficient Functionality of
Acetylcholinesterase
Finally, the choice of buffer in this study was 0.1 M of sodium phosphate buffer at pH 8.0.
Several critical reasons underpinned the selection of pH 8.0. First, it minimizes the protonation of
TNB2-, as the bright shade of yellow is a reflection of TNB2- in its ionized state. The pKa of TNB2-

43

is 4.53, and at pH 5.53, 6.53, and 7.53, the percentage of ionized TNB2- was reported to increase
from 50% to 91% and then greater than 99%, respectively.43 Secondly, the pH 8.0 was chosen to
keep the thiocholine, produced from the hydrolysis of acetylthiocholine by acetylcholinesterase,
in its deprotonated form. This maintains the single negative charge of its free thiol group, allowing
it to readily attack and break up the disulfide linkage of DTNB. Thirdly, the pH 8.0 falls within
the optimal range of Electrophorus electricus acetylcholinesterase activity, which ranges from pH
8.0 to 9.5.47
Regarding the concentration, 0.1 M was selected to provide the buffer with moderate ionic
strength. Excessive ionic strength on either end of the spectrum could lead to adverse consequences
for assay performance. Very high ionic strength may impede the π-cation interaction between the
positively charged nitrogen atom of acetylthiocholine and the electron-rich aromatic rings of the
side chain of multiple tryptophan residues situated within the peripheral anionic site of
acetylcholinesterase. This obstruction would hinder the efficient catabolism of the substrate by the
enzyme. Conversely, excessively low ionic strength may result in the aggregation of the side
chains of charged residues within the enzyme, potentially leading to denaturation of the enzyme.
2.2

Materials and Methods

2.2.1

Preparation of the Main Components of the Acetylcholinesterase Assay – Stock
Solutions
20.2 mM of DTNB
DTNB in powder form was accurately weighed at 0.0500 g on a small piece of weighing

paper that had been folded into a triangular shape and placed on an analytical balance. Once
weighed, the powder was carefully transferred to a 13-mL round-bottom polypropylene test tube.
This was accomplished by positioning the pointed end of the weighing paper against the tube, and

44

gently pouring the powder into it. To ensure that every speck of powder was collected, the
weighing paper was rinsed with a small amount of 100% ethanol, with all the washings directed
into the same tube.
Following this, the tube was gradually filled with 100% ethanol until it reached a total
volume of 6.25 mL. This was done using a 10-mL pipette attached to a pipette gun. Once filled,
the DTNB solution in the tube was gently mixed by pipetting the contents back and forth, using
the same 10-mL pipette attached to the pipette gun. Subsequently, the solution was divided into
seven equal portions, each comprising approximately 850 μL. All seven portions were then
separately transferred into seven 1.5-mL microcentrifuge tubes using 1000-μL pipette tips attached
to a 1000-μL pipettor.
Finally, each of the seven 1.5-mL microcentrifuge tubes was wrapped in aluminum foil to
protect the DTNB solution from exposure to light and stored in a -20°C freezer. The procedure
was conducted in a dark room with limited external light penetration due to DTNB’s sensitivity to
light, starting from the moment when the DTNB powder was transferred to the 13-mL roundbottom polypropylene test tube.
346 mM of Acetylthiocholine Iodide
Acetylthiocholine iodide in powder form was accurately weighed at 0.1000 g on a small
piece of weighing paper that had been folded into a triangular shape and placed on an analytical
balance. Once weighed, the powder was carefully transferred to a 13-mL round-bottom
polypropylene test tube. This was accomplished by positioning the pointed end of the weighing
paper against the tube, and gently pouring the powder into it. To ensure that every speck of powder
was collected, the weighing paper was rinsed with a small amount of 0.1 M of sodium phosphate
buffer at pH 8.0, with all the washings directed into the same tube.

45

Following this, the tube was gradually filled with 0.1 M of sodium phosphate buffer at pH
8.0 until it reached a total volume of 1000 μL. This was done using a 1000-μL pipette tip attached
to a 1000-μL pipettor. Once filled, the acetylthiocholine iodide solution in the tube was gently
mixed by pipetting the contents back and forth, using the same 1000-μL pipette tip attached to the
1000-μL pipettor. Subsequently, the solution was divided into five equal portions, each comprising
approximately 200 μL. All five portions were then separately transferred into five 1.5-mL
microcentrifuge tubes using 200-μL pipette tips attached to a 200-μL pipettor. Finally, all five 1.5mL microcentrifuge tubes were stored in the -20°C freezer.
125 units/mL of Acetylcholinesterase
Tris-HCl buffer at pH 7.4 with a volume of 200 μL at 1.0 M concentration was carefully
transferred to a 13-mL round-bottom polypropylene test tube. This was done using a 200-μL
pipette tip attached to the 200-μL pipettor. Following this, the tube was gradually filled with
autoclaved water until it reached a total volume of 10.00 mL. This was done using a 10-mL pipette
attached to the pipette gun. This diluted the concentration of the buffer to 20 mM.
Once filled, 2.30 mL of the buffer at 20 mM was carefully transferred to a small glass bottle
containing Electrophorus electricus acetylcholinesterase in powder form. This was done using a
5-mL pipette attached to the pipette gun. The enzyme solution in the glass bottle was gently mixed
by pipetting the contents back and forth, using a 1000-μL pipette tip attached to the 1000-μL
pipettor. Once mixed, the enzyme solution was carefully transferred back to a new 13-mL roundbottom polypropylene test tube. This was done using 1000-μL pipette tips attached to the 1000-μL
pipettor. Subsequently, the solution was divided into twenty-three equal portions, each comprising
approximately 100 μL. All twenty-three portions were then separately transferred into twenty-

46

three 1.5-mL microcentrifuge tubes using 200-μL pipette tips attached to the 200-μL pipettor.
Finally, all twenty-three 1.5-mL microcentrifuge tubes were stored in the -20°C freezer.
0.1 M of Sodium Phosphate Buffer at pH 8.0
Sodium dihydrogen phosphate in powder form was accurately weighed at 13.80 g on a
large weighing boat that had been placed on a top loading balance. Once weighed, the powder was
carefully transferred to a 250-mL Erlenmeyer flask. This was accomplished by positioning the
pointed end of the weighing boat against the flask, and gently pouring the powder into it. To ensure
that every speck of powder was collected, the weighing boat was rinsed with a small amount of
autoclaved water, with all the washings directed into the same flask. The same process was done
for 14.20 g of sodium hydrogen phosphate in powder form in another separate 250-mL Erlenmeyer
flask.
Following this, each flask was gradually filled with autoclaved water until it reached a total
volume of 80.0 mL. This was done using a 100-mL graduated cylinder to measure and transfer the
water. Once filled, both flasks were gently swirled by hand, with swift yet rhythmic movements,
to ensure thorough mixing of the contents. Subsequently, both phosphate solutions were
individually transferred back to two clean 100-mL graduated cylinders from their respective flasks.
In the graduated cylinders, both solutions were gradually filled with autoclaved water again, until
they reached a total volume of 100.0 mL. This was done using a long glass Pasteur pipette.
After that, 6.80 mL of the sodium dihydrogen phosphate solution and 93.20 mL of the
sodium hydrogen phosphate solution were transferred together to a 1000-mL glass bottle. This was
done using a 10-mL pipette and a 25-mL pipette attached to the pipette gun, respectively. This
created the sodium phosphate buffer with a concentration of 1.0 M at pH 8.0. Then, the bottle was
gradually filled with autoclaved water again, until it reached a total volume of 1000 mL. This was

47

done using a 1000-mL graduated cylinder to measure and transfer the water. This diluted the
concentration of the buffer to 0.1 M. Finally, the 1000-mL glass bottle was stored on the lab bench
at room temperature.
2.2.2

Preparation of the Main Components of the Acetylcholinesterase Assay – Standard
Solutions
10 mM of DTNB
DTNB with a volume of 500 μL at 20.2 mM concentration was carefully transferred to a

1.5-mL microcentrifuge tube. This was done using a 1000-μL pipette tip attached to the 1000-μL
pipettor. Following this, the tube was gradually filled with 100% ethanol until it reached a total
volume of 1000 μL. This was done using another 1000-μL pipette tip attached to the 1000-μL
pipettor. Once filled, the DTNB solution in the tube was gently mixed by pipetting the contents
back and forth, using the same 1000-μL pipette tip attached to the 1000-μL pipettor. This diluted
the concentration of DTNB to 10 mM. Finally, the 1.5-mL microcentrifuge tube was wrapped in
aluminum foil and stored in the -20°C freezer.
14 mM of Acetylthiocholine Iodide
Acetylthiocholine iodide with a volume of 40 μL at 346 mM concentration was carefully
transferred to a 1.5-mL microcentrifuge tube. This was done using a 200-μL pipette tip attached
to the 200-μL pipettor. Following this, the tube was gradually filled with 0.1 M of sodium
phosphate buffer at pH 8.0 until it reached a total volume of 1000 μL. This was done using a 1000μL pipette tip attached to the 1000-μL pipettor. Once filled, the acetylthiocholine iodide solution
in the tube was gently mixed by pipetting the contents back and forth, using the same 1000-μL
pipette tip attached to the 1000-μL pipettor. This diluted the concentration of acetylthiocholine
iodide to 14 mM. Finally, the 1.5-mL microcentrifuge tube was stored in the -20°C freezer.

48

9 units/mL of Acetylcholinesterase
Acetylcholinesterase with a volume of 72 μL at 125 units/mL concentration was carefully
transferred to a 1.5-mL microcentrifuge tube. This was done using a 200-μL pipette tip attached
to the 200-μL pipettor. Following this, the tube was gradually filled with 20 mM of Tris-HCl buffer
at pH 7.4 until it reached a total volume of 1000 μL. This was done using a 1000-μL pipette tip
attached to the 1000-μL pipettor. Once filled, the enzyme solution in the tube was gently mixed
by pipetting the contents back and forth, using the same 1000-μL pipette tip attached to the 1000μL pipettor. This diluted the concentration of acetylcholinesterase to 9 units/mL. Finally, the 1.5mL microcentrifuge tube was stored in the -20°C freezer.
4.5 units/mL of Acetylcholinesterase
Acetylcholinesterase with a volume of 50 μL at 9 units/mL concentration was carefully
transferred to a 1.5-mL microcentrifuge tube. This was done using a 200-μL pipette tip attached
to the 200-μL pipettor. Following this, the tube was gradually filled with 20 mM of Tris-HCl buffer
at pH 7.4 until it reached a total volume of 100 μL. This was done using another 200-μL pipette
tip attached to the 200-μL pipettor. Once filled, the enzyme solution in the tube was gently mixed
by pipetting the contents back and forth, using the same 200-μL pipette tip attached to the 200-μL
pipettor. This diluted the concentration of acetylcholinesterase to 4.5 units/mL. Finally, the 1.5mL microcentrifuge tube was stored in the -20°C freezer.
0.9 units/mL of Acetylcholinesterase
Acetylcholinesterase with a volume of 20 μL at 9 units/mL concentration was carefully
transferred to a 1.5-mL microcentrifuge tube. This was done using a 200-μL pipette tip attached
to the 200-μL pipettor. Following this, the tube was gradually filled with 20 mM of Tris-HCl buffer
at pH 7.4 until it reached a total volume of 200 μL. This was done using another 200-μL pipette

49

tip attached to the 200-μL pipettor. Once filled, the enzyme solution in the tube was gently mixed
by pipetting the contents back and forth, using the same 200-μL pipette tip attached to the 200-μL
pipettor. This diluted the concentration of acetylcholinesterase to 0.9 units/mL. Finally, the 1.5mL microcentrifuge tube was stored in the -20°C freezer.
0.09 units/mL of Acetylcholinesterase
Acetylcholinesterase with a volume of 10 μL at 0.9 units/mL concentration was carefully
transferred to a 1.5-mL microcentrifuge tube. This was done using a 200-μL pipette tip attached
to the 200-μL pipettor. Following this, the tube was gradually filled with 20 mM of Tris-HCl buffer
at pH 7.4 until it reached a total volume of 100 μL. This was done using another 200-μL pipette
tip attached to the 200-μL pipettor. Once filled, the enzyme solution in the tube was gently mixed
by pipetting the contents back and forth, using the same 200-μL pipette tip attached to the 200-μL
pipettor. This diluted the concentration of acetylcholinesterase to 0.09 units/mL. Finally, the 1.5mL microcentrifuge tube was stored in the -20°C freezer.
0.009 units/mL of Acetylcholinesterase
Acetylcholinesterase with a volume of 10 μL at 0.09 units/mL concentration was carefully
transferred to a 1.5-mL microcentrifuge tube. This was done using a 200-μL pipette tip attached
to the 200-μL pipettor. Following this, the tube was gradually filled with 20 mM of Tris-HCl buffer
at pH 7.4 until it reached a total volume of 100 μL. This was done using another 200-μL pipette
tip attached to the 200-μL pipettor. Once filled, the enzyme solution in the tube was gently mixed
by pipetting the contents back and forth, using the same 200-μL pipette tip attached to the 200-μL
pipettor. This diluted the concentration of acetylcholinesterase to 0.009 units/mL. Finally, the 1.5mL microcentrifuge tube was stored in the -20°C freezer.
2.3

Results and Discussion

50

2.3.1

Testing the Performance of the Acetylcholinesterase Assay

2.3.1.1

The First Performance Test: Evaluating the Assay with Different
Concentrations of Acetylcholinesterase

2.3.1.1.1

Purpose of the First Performance Test

Before the acetylcholinesterase assay could be used to experiment with the test samples, it
underwent two different performance tests to assess its ability to detect potential
acetylcholinesterase inhibitors. The first performance test focused on the assay’s capability to
generate appropriate absorbance readings with increasing concentrations of acetylcholinesterase.
This test served two purposes. First, it aimed to confirm that the assay would function correctly
without the addition of any test sample. Hypothetically, assuming all other factors, including time,
remain constant, the change in TNB2- absorbance should increase as the concentration of
acetylcholinesterase increases. This is because a greater amount of enzyme leads to the production
of more thiocholine, resulting in more DTNB reduction and TNB2- generation, ultimately causing
a larger change in absorbance over the same time span.
The second purpose was to identify one specific concentration of acetylcholinesterase that
would produce a suitably steep slope representing a constant linear change in TNB2- absorbance
over time. This choice would simplify the detection of potential acetylcholinesterase inhibitors.
By assessing the steepness of the linear slope, it becomes easier to distinguish the absorbance
signal in the presence of a potential enzyme inhibitor. With a lower amount of thiocholine
generated due to inhibited enzyme activity, the change in absorbance is smaller, resulting in a
slower rate of increase. As a result, the slope of the constant linear change in TNB2- absorbance is
shallower when compared to the absence of the inhibitor.
2.3.1.1.2

Experimental Procedure of the First Performance Test

51

Experiments were carried out using 96-well microtiter plates. In this test, only four out of
five components of the assay were included: sodium phosphate buffer, Electrophorus electricus
acetylcholinesterase, acetylthiocholine iodide, and DTNB. On the plate, each row was divided into
one control well and five different treatment wells.
In the control well, the following components were added: 180 μL of 0.1 M of sodium
phosphate buffer at pH 8.0, 0 μL of acetylcholinesterase, 10 μL of 14 mM of acetylthiocholine
iodide, and 10 μL of 10 mM of DTNB.
In each treatment well, the following components were added: 160 μL of 0.1 M of sodium
phosphate buffer at pH 8.0, 20 μL of a specific concentration of acetylcholinesterase, 10 μL of 14
mM of acetylthiocholine iodide, and 10 μL of 10 mM of DTNB. The concentration of
acetylcholinesterase was divided into five increasing concentrations ordered from least to most
concentrated: 0.009, 0.09, 0.9, 4.5, and 9 units/mL of acetylcholinesterase.
There were three rows in total, each representing one technical replicate. The three
technical replicates were combined to form one experimental replicate corresponding to a single
run. For each experimental replicate, the change in the absorbance of TNB2- was detected at the
wavelength of 412 nm and recorded every 2 mins for a total of 30 mins using the Synergy 2 plate
reader machine. A total of three experimental replicates were performed.
2.3.1.1.3

Raw Data Processing and Graphing Transformation of the First
Performance Test

For each of the five different concentrations of acetylcholinesterase used in the treatment,
the raw absorbance values were recorded every 2 mins for a total of 30 mins and then directly
plotted onto a graph of absorbance against time. In cases where values were marked with an

52

“overflow” signal, they were automatically assigned a value of 4.000 to indicate that they had
exceeded the upper limit of the plate reader machine’s detection range.
The objective was to demonstrate that the rate of TNB2- absorbance increase corresponding
to the level of acetylcholinesterase activity exhibited significant changes over the 30-min time
span when treated with various concentrations of acetylcholinesterase. Each raw absorbance value
at every time point was derived from the average of nine different raw absorbance values recorded
across nine different technical replicates, corresponding to three different experimental replicates.
Each absorbance value plotted on the graph was accompanied by a standard deviation value
calculated from the provided raw data.
2.3.1.1.4

Hypothesis and Predicted Outcomes of the First Performance Test

According to the hypothesis, as the concentration of acetylcholinesterase increases, the rate
of TNB2- absorbance increase should accelerate, leading to a more significant change in
absorbance. It may even reach the stationary phase of enzyme activity at a specific enzyme
concentration, gradually transitioning from an initially linear trend to a logarithmic trend as
saturation is achieved.
2.3.1.1.5

Observations and Analyses of the First Performance Test

In run 1, as the concentration of acetylcholinesterase increased, the rate of TNB2absorbance showed an initial slow linear increase, which gradually transitioned into a logarithmic
trend, starting from 0.9 units/mL of acetylcholinesterase or higher. This pattern was consistently
observed across three technical replicates.
However, the graphical representation of the rate of TNB2- absorbance increase in all three
technical replicates were suboptimal. The lines were neither smooth nor steady as expected, given
that they should have followed proper linear or logarithmic trends. Instead, they appeared jagged,

53

indicating fluctuations in the absorbance readings. These fluctuations sometimes were minor,
while at other times, they were significant. Notably, in a few instances, lower enzyme
concentrations yielded higher absorbance readings than higher concentrations at the same time
point.
After the run, it was suspected that the issue may have been related to inadequate mixing
of the wells. The only mixing action during the run involved swirling the microtiter plate for a few
secs after all components had been added to the wells. Additionally, many absorbance readings
from time points in the second half of the 30-min time span for wells treated with 0.9 units/mL of
acetylcholinesterase or higher exceeded the upper limit of the plate reader machine’s detection
range. Due to this, and given the similarity of their graphical data, the treatments with
acetylcholinesterase concentrations of 4.5 units/mL and 9.0 units/mL were discontinued in
subsequent runs. The data of the treatment with the 0.9 units/mL concentration was used to make
relative comparisons instead (Table 3).

54

Replicate 1
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.116
0.131
0.137
0.143
0.149
0.155
0.159
0.162
0.166
0.171
0.175
0.179
0.182
0.187
0.191
0.196

0.127
0.159
0.190
0.222
0.255
0.261
0.302
0.331
0.365
0.451
0.488
0.532
0.563
0.583
0.602
0.620

0.189
0.275
0.273
0.444
0.484
0.514
0.549
0.590
0.629
0.666
0.705
0.742
0.802
0.875
1.141
1.185

0.620
1.094
1.490
1.474
1.563
1.852
2.246
2.646
3.287
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

1.077
1.944
2.345
2.696
2.757
3.052
3.026
2.939
3.186
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

0.785
2.365
2.605
2.868
2.937
2.956
3.021
3.106
3.167
3.213
3.242
3.271
3.301
3.342
3.342
3.386

Replicate 2
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.116
0.128
0.133
0.141
0.215
0.354
0.431
0.455
0.465
0.483
0.545
0.551
0.551
0.560
0.566
0.573

0.254
0.319
0.374
0.393
0.408
0.424
0.445
0.457
0.468
0.474
0.482
0.489
0.497
0.500
0.513
0.518

0.182
0.257
0.368
0.392
0.433
0.485
0.544
0.577
0.640
0.695
0.816
0.888
0.968
1.045
1.113
1.182

0.261
0.823
1.196
1.569
1.884
2.242
2.559
2.785
2.958
3.098
3.200
3.340
3.404
3.477
3.554
3.648

1.360
2.131
2.459
2.517
2.488
2.797
2.792
2.830
2.908
3.017
3.326
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

0.608
0.781
0.756
0.731
0.751
0.816
1.040
1.309
1.748
2.093
2.337
2.221
2.500
3.681
OVRFLW
OVRFLW

55

Replicate 3
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.107
0.118
0.124
0.129
0.134
0.139
0.143
0.148
0.153
0.159
0.165
0.170
0.177
0.182
0.186
0.191

0.207
0.314
0.239
0.213
0.299
0.379
0.399
0.422
0.442
0.492
0.491
0.493
0.500
0.506
0.511
0.516

0.159
0.196
0.293
0.357
0.400
0.436
0.469
0.498
0.527
0.561
0.597
0.634
0.664
0.692
0.742
0.808

0.199
0.923
1.154
1.681
2.188
2.550
2.951
3.342
3.541
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

0.436
0.881
1.343
1.896
2.238
2.329
2.411
2.455
2.512
2.558
2.588
2.633
2.669
2.709
2.757
2.802

0.762
3.461
3.732
3.486
OVRFLW
3.945
3.840
3.448
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

Table 3. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 1.
The test aimed to assess the assay’s ability to generate appropriate absorbance readings in response to
increasing concentrations of acetylcholinesterase. It analyzed the rate of TNB2- absorbance increase over a
30-min time span as acetylcholinesterase concentration increased. The term “OVRFLW”, or “Overflow”,
denoted absorbance readings that exceeded the upper limit of the Synergy 2 plate reader machine’s
detection range and could not be measured. Black highlighted sections in the tables represented unusual
data where absorbance readings resulting from treatments with lower enzyme concentrations were higher
than those from treatments with higher enzyme concentrations. The lower panels of graphs depicted the
changes in TNB2- absorbance in response to increasing concentrations of acetylcholinesterase over the
course of 30 mins. The experiment was repeated three times (n = 3).

In run 2, experimental conditions from run 1 were replicated, with one notable change: the
order of the components added to the treatment wells was adjusted. In run 1, the order was as
follows: 160 μL of 0.1 M of sodium phosphate buffer at pH 8.0, 20 μL of a specific concentration
of acetylcholinesterase, 10 μL of 10 mM of DTNB, and 10 μL of 14 mM of acetylthiocholine
56

iodide. In run 2, the order was adjusted to: 20 μL of a specific concentration of acetylcholinesterase,
10 μL of 10 mM of DTNB, 160 μL of 0.1 M of sodium phosphate buffer at pH 8.0, and 10 μL of
14 mM of acetylthiocholine iodide. Unfortunately, this adjustment significantly impaired the
assay’s performance.
The graphical representation of the rate of TNB2- absorbance increase almost entirely
flattened out across most wells in the three technical replicates throughout the 30-min time span.
Additionally, none of the wells displayed the characteristic bright shade of yellow associated with
TNB2- production. It was suspected that adding components with smaller volumes first may have
caused them to dry up, especially the critical acetylcholinesterase, leading to aggregation and loss
of functionality. This damage could not be reversed by adding the sodium phosphate buffer later
in an attempt to rehydrate the enzyme. Reordering the components to the original sequence from
run 1 in subsequent runs was necessary, at least until any further adjustments were made
(Supplementary Table 1 and Supplementary Figure 1).
In run 3, experimental conditions from run 1 were replicated, but with an increased mixing
time. After adding all components to the wells, the swirling time of the microtiter plate was
increased to 30 secs before the plate was loaded into the plate reader machine. The results closely
resembled those of run 1 for all three technical replicates, albeit with some improvement in assay
performance quality.
The graphical representation of the rate of TNB2- absorbance increase was smoother but
still not completely consistent and steady. As such, minor fluctuations in the absorbance readings
persisted. Surprisingly, the formation of crystals in some wells was noticed for the first time
(Figure 12). The formation of these crystals corresponded to wells where absorbance readings
exhibited minor fluctuations.

57

Figure 12. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 3.
The test aimed to assess the assay’s ability to generate appropriate absorbance readings in response to
increasing concentrations of acetylcholinesterase. It analyzed the rate of TNB2- absorbance increase over a
30-min time span as acetylcholinesterase concentration increased. A photo of the microtiter plate (the
second 4x3 set on the right within the red box) used for the run was also attached to illustrate the unexpected
results. The experiment was repeated three times (n = 3).

After the run, it was strongly suspected that these unusual crystals were the root cause of
the inconsistency in absorbance readings and the fluctuations in the graphical representation of the
rate of TNB2- absorbance increase. However, at the time, the exact cause of the crystal formation
was uncertain. To address this, additional literature research was conducted, leading to the
identification of five main possible factors contributing to the formation of these crystals: low pH,
58

temperature, contamination, mixing, and concentration. In the subsequent runs, these factors were
either eliminated or carefully controlled to investigate whether the problem with crystallization
could be resolved (Supplementary Table 2).
In run 4, experimental conditions from run 1 were replicated, with a few notable exceptions.
To ensure the pH of the sodium phosphate buffer was correct, a piece of pH test strip was soaked
with a few drops of the buffer. The pH was recorded to be approximately 8.0. To maintain the
correct temperature of all components, they were placed in a cool area on the lab bench to slowly
thaw to room temperature, avoiding reliance on the warmth of human hands. To prevent
contamination, the control wells with 0 units/mL of acetylcholinesterase and the treatment wells
with three different acetylcholinesterase concentrations of 0.009, 0.09, and 0.9 units/mL from all
three technical replicates were arranged and separated horizontally by one column of wells based
on the order of enzyme concentration.
Regarding mixing, instead of swirling the microtiter plate for 30 secs after adding all
components to the wells, before the plate was loaded into the plate reader machine, the solution in
each individual well was gently pipetted up and down with a small volume of approximately 10 to
20 μL each time a component was added, until all components were included. The results closely
resembled what was observed in run 3 for all three technical replicates. The most noteworthy
observation was the absence of crystal formation in any wells before the plate was loaded into the
plate reader machine. However, after completing the run, crystals had formed in two of twelve
wells. Additionally, the wells with crystals displayed significantly higher absorbance readings and
fluctuations compared to those without crystals, even among wells treated with the same enzyme
concentration.

59

This further confirmed with high certainty that these peculiar crystals were the root cause
of the inconsistency in absorbance readings and the fluctuations in the graphical representation of
the rate of TNB2- absorbance increase. Simultaneously, this shed light on the nature of the crystal
formation. One significant finding was that the time taken for the crystals to form appeared to vary
considerably. They could form immediately after all components were added to the wells before
mixing, during the mixing process, or even while the microtiter plate was being processed in the
plate reader machine.
Overall, while the crystallization problem persisted, the three main factors suspected to
contribute to the crystal formation were eliminated in this run. Since the issue remained unresolved
despite careful control and monitoring of these factors, the two remaining factors, mixing and
concentration, were examined in the subsequent runs to determine if any progress towards
resolution of the problem could be made (Supplementary Table 3 and Supplementary Figure 2).
In run 5, experimental conditions from run 4 were replicated, with one exception.
Concerning the mixing action, instead of gently pipetting the solution up and down with a small
volume of approximately 10 to 20 μL each time a component was added to each individual well,
until all components were included, the solution in each individual well was gently pipetted up
and down with a larger volume of 100 μL, but only after all components were added to the wells.
Following this, the microtiter plate was swirled again for 30 secs before being loaded into the plate
reader machine.
The results closely mirrored those observed in run 1 for all three technical replicates.
However, a notable difference was the graphical representation of the rate of TNB2- absorbance
increase, which was smooth and consistent, following either a linear or logarithmic trend. There
were no fluctuations in the graphical lines over the entire 30-min time span, and the absorbance

60

readings exhibited high consistency across the technical replicates. Notably, there was no
crystallization at all during this run.
Initially, the mixing factor was suspected as the cause of the crystal formation
(Supplementary Table 4 and Supplementary Figure 3). However, run 6 did not reproduce the
results observed in run 5. Despite repeating everything exactly as in run 5, the crystallization
problem resurfaced, and the results resembled those observed in runs 3 and 4. This ruled out the
mixing factor as the possible cause of the crystal formation. Consequently, concentration became
the sole remaining factor under scrutiny. Building on the observations from run 4 of the first
performance test, another significant detail emerged in run 6 was the appearance of crystallization
immediately after the addition of acetylthiocholine iodide to the wells. As such, the concentration
of acetylthiocholine iodide became the primary suspect. It was examined in the subsequent runs to
determine whether any resolution to the problem could be achieved. If the concentration of
acetylthiocholine iodide was indeed the cause of crystallization, then the crystallization process
should cease when the concentration of acetylthiocholine iodide was diluted (Supplementary Table
5 and Supplementary Figure 4).
In run 7, experimental conditions from run 5 were replicated, except for the adjustment in
the added amount and concentration of acetylthiocholine iodide, which was changed from 10 μL
of 14 mM of acetylthiocholine iodide to 10 μL of 1.4 mM of acetylthiocholine iodide. This
adjustment resulted in a 10-fold dilution of the acetylthiocholine iodide concentration. The results
were fairly reproducible to what was observed in run 5, with the only significant difference being
that the displayed absorbance readings were much lower due to the substrate concentration being
diluted (Supplementary Table 6 and Supplementary Figure 5).

61

Similarly, in run 8, experimental conditions from run 5 were replicated, except the
adjustment in the added amount and concentration of acetylthiocholine iodide, which was changed
from 10 μL of 14 mM of acetylthiocholine iodide to 10 μL of 7 mM of acetylthiocholine iodide.
This adjustment resulted in a 2-fold dilution of the acetylthiocholine iodide concentration. Once
again, the results were sufficiently reproducible to what was observed in run 5, with the only
significant difference being that the displayed absorbance readings were slightly lower due to the
substrate concentration being diluted. However, the absorbance readings were much higher than
those in run 7.
Overall, at this point, the results from both runs 7 and 8 seemed to suggest that the
concentration of acetylthiocholine iodide was the primary source of the problem leading to the
crystal formation. However, it was later discovered that the concentration of the sodium phosphate
buffer used had been incorrect throughout the first eight runs of the first performance test. Instead
of 0.1 M, it had mistakenly been prepared as 1.0 M. Recalling back to the time during the
preparation of both the stock and standard solutions of acetylthiocholine iodide, heavy
precipitation quickly formed over time shortly after mixing the sodium phosphate buffer with the
acetylthiocholine iodide powder. Although the precipitates had slightly different shapes, their
appearance closely resembled the crystals formed during the runs.
As a result, the suspicion shifted to the sodium phosphate buffer in the following runs, and
it became the focus of the examination. Assuming that the real source of the problem lied with the
buffer’s concentration, it was expected that the crystallization process would not recur even if 14
mM of acetylthiocholine iodide were to be reused, as long as the buffer’s concentration used to
dissolve it was indeed 0.1 M (Supplementary Table 7 and Supplementary Figure 6).

62

In run 9, experimental conditions from run 5 were replicated, with the only change being
the adjustment of the sodium phosphate buffer concentration used to prepare 14 mM of
acetylthiocholine iodide, which was corrected to 0.1 M. The results were almost entirely
reproducible to what was observed in run 5. The only minor difference of note was that the
treatment with 0.009 units/mL of acetylcholinesterase resulted in slightly lower absorbance
readings across the 30-min time span compared to the treatment with 0 units/mL of
acetylcholinesterase, even though the total change in absorbance over 30 mins was much greater
for the treatment with 0.009 units/mL of acetylcholinesterase than for the treatment with 0
units/mL of acetylcholinesterase.
To confirm the previously incorrect concentration of sodium phosphate buffer as the real
source of the crystallization problem, four more runs were performed to assess the reproducibility
of run 9. The results were completely reproducible to what was observed in run 5, and even better
than what was observed in run 9 for runs 11 through 13 (Tables 4-6 and Figures 13-15,
respectively).

63

Replicate 1
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.138
0.139
0.139
0.139
0.140
0.140
0.140
0.140
0.140
0.141
0.141
0.141
0.141
0.141
0.143
0.143

0.148
0.149
0.152
0.153
0.156
0.158
0.160
0.162
0.164
0.166
0.167
0.169
0.171
0.173
0.175
0.177

0.222
0.254
0.284
0.316
0.347
0.379
0.408
0.438
0.468
0.496
0.524
0.555
0.585
0.612
0.641
0.667

1.169
1.773
2.357
2.872
3.338
3.704
3.992
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

Replicate 2
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.128
0.133
0.134
0.134
0.135
0.136
0.136
0.136
0.137
0.137
0.137
0.138
0.138
0.138
0.138
0.139

0.139
0.141
0.143
0.145
0.147
0.150
0.152
0.155
0.157
0.159
0.162
0.164
0.166
0.168
0.170
0.172

0.208
0.241
0.274
0.308
0.340
0.371
0.403
0.434
0.465
0.495
0.526
0.556
0.586
0.615
0.645
0.677

1.192
1.817
2.423
2.981
3.403
3.848
4.012
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

64

Replicate 3
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.138
0.138
0.138
0.139
0.138
0.139
0.140
0.141
0.140
0.141
0.141
0.143
0.142
0.141
0.142
0.143

0.141
0.142
0.145
0.146
0.149
0.152
0.154
0.156
0.158
0.160
0.162
0.164
0.166
0.169
0.171
0.172

0.221
0.253
0.288
0.322
0.357
0.391
0.425
0.458
0.491
0.524
0.555
0.585
0.617
0.648
0.679
0.708

1.132
1.778
2.396
2.942
3.425
3.779
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

Table 4. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 11.
The test aimed to assess the assay’s ability to generate appropriate absorbance readings in response to
increasing concentrations of acetylcholinesterase. It analyzed the rate of TNB2- absorbance increase over a
30-min time span as acetylcholinesterase concentration increased. The term “OVRFLW”, or “Overflow”,
denoted absorbance readings that exceeded the upper limit of the Synergy 2 plate reader machine’s
detection range and could not be measured. The lower panels of graphs depicted the changes in TNB2absorbance in response to increasing concentrations of acetylcholinesterase over the course of 30 mins. The
experiment was repeated three times (n = 3).

65

Figure 13. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 11.
A photo of the microtiter plate (inside the red box) used for the run was also attached to illustrate the
complete reproducibility of the results obtained from run 5 when treated with the correct amount and
concentration of acetylthiocholine iodide, along with the correct concentration of sodium phosphate buffer
in which the acetylthiocholine iodide was dissolved. This was evident from the absence of the crystal
formation in all wells, and from the consistent absorbance readings across the 30-min time span for all three
technical replicates.

66

Replicate 1
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.156
0.157
0.157
0.157
0.157
0.157
0.157
0.158
0.157
0.157
0.158
0.158
0.158
0.158
0.158
0.159

0.152
0.154
0.153
0.156
0.157
0.160
0.163
0.163
0.164
0.167
0.168
0.171
0.172
0.174
0.176
0.178

0.261
0.298
0.333
0.367
0.405
0.438
0.468
0.506
0.548
0.587
0.625
0.661
0.697
0.730
0.766
0.799

1.520
2.146
2.723
3.235
3.651
3.885
4.019
OVRFLW
4.029
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

Replicate 2
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.149
0.150
0.150
0.151
0.151
0.151
0.151
0.152
0.152
0.152
0.152
0.152
0.153
0.152
0.153
0.153

0.161
0.164
0.165
0.166
0.168
0.168
0.170
0.172
0.173
0.175
0.177
0.178
0.180
0.180
0.181
0.184

0.270
0.307
0.344
0.380
0.414
0.448
0.483
0.520
0.556
0.590
0.629
0.650
0.694
0.729
0.764
0.805

1.310
1.889
2.412
2.877
3.289
3.555
3.815
3.918
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

67

Replicate 3
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.157
0.157
0.158
0.159
0.159
0.158
0.159
0.158
0.158
0.159
0.158
0.158
0.159
0.154
0.157
0.153

0.159
0.161
0.164
0.165
0.168
0.170
0.173
0.175
0.178
0.180
0.181
0.184
0.188
0.186
0.187
0.193

0.237
0.266
0.294
0.322
0.349
0.377
0.406
0.428
0.457
0.475
0.504
0.536
0.569
0.608
0.635
0.665

1.129
1.688
2.201
2.663
3.066
3.462
3.748
4.038
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

Table 5. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 12.
The test aimed to assess the assay’s ability to generate appropriate absorbance readings in response to
increasing concentrations of acetylcholinesterase. It analyzed the rate of TNB2- absorbance increase over a
30-min time span as acetylcholinesterase concentration increased. The term “OVRFLW”, or “Overflow”,
denoted absorbance readings that exceeded the upper limit of the Synergy 2 plate reader machine’s
detection range and could not be measured. Black highlighted sections in the tables represented unusual
data where absorbance readings resulting from treatments with lower enzyme concentrations were higher
than those from treatments with higher enzyme concentrations. The lower panels of graphs depicted the
changes in TNB2- absorbance in response to increasing concentrations of acetylcholinesterase over the
course of 30 mins. The experiment was repeated three times (n = 3).

68

Figure 14. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 12.
A photo of the microtiter plate (inside the red box) used for the run was also attached to illustrate the
complete reproducibility of the results obtained from run 5 when treated with the correct amount and
concentration of acetylthiocholine iodide, along with the correct concentration of sodium phosphate buffer
in which the acetylthiocholine iodide was dissolved. This was evident from the absence of the crystal
formation in all wells, and from the consistent absorbance readings across the 30-min time span for all three
technical replicates.

69

Replicate 1
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.157
0.157
0.157
0.157
0.157
0.156
0.154
0.157
0.152
0.150
0.156
0.159
0.159
0.160
0.161
0.162

0.160
0.163
0.165
0.167
0.169
0.169
0.169
0.173
0.183
0.188
0.194
0.198
0.201
0.205
0.208
0.212

0.222
0.247
0.268
0.292
0.313
0.341
0.366
0.392
0.415
0.440
0.462
0.486
0.507
0.529
0.552
0.575

1.293
1.893
2.477
3.025
3.529
3.957
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

Replicate 2
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.147
0.147
0.148
0.148
0.149
0.149
0.149
0.149
0.149
0.149
0.149
0.150
0.149
0.150
0.150
0.150

0.152
0.154
0.156
0.158
0.159
0.162
0.164
0.165
0.166
0.168
0.170
0.169
0.171
0.175
0.179
0.184

0.207
0.229
0.252
0.273
0.296
0.319
0.341
0.363
0.385
0.407
0.428
0.450
0.469
0.489
0.512
0.534

0.981
1.466
1.889
2.316
2.745
3.056
3.360
3.590
3.846
3.890
4.042
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

70

Replicate 3
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.156
0.156
0.157
0.158
0.158
0.159
0.159
0.160
0.161
0.162
0.163
0.163
0.164
0.164
0.165
0.166

0.159
0.163
0.166
0.170
0.173
0.177
0.180
0.184
0.187
0.191
0.194
0.197
0.201
0.203
0.207
0.210

0.253
0.290
0.326
0.362
0.398
0.435
0.472
0.504
0.540
0.580
0.614
0.647
0.682
0.717
0.749
0.782

1.040
1.600
2.131
2.613
3.089
3.451
3.796
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

Table 6. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 13.
The test aimed to assess the assay’s ability to generate appropriate absorbance readings in response to
increasing concentrations of acetylcholinesterase. It analyzed the rate of TNB2- absorbance increase over a
30-min time span as acetylcholinesterase concentration increased. The term “OVRFLW”, or “Overflow”,
denoted absorbance readings that exceeded the upper limit of the Synergy 2 plate reader machine’s
detection range and could not be measured. The lower panels of graphs depicted the changes in TNB2absorbance in response to increasing concentrations of acetylcholinesterase over the course of 30 mins. The
experiment was repeated three times (n = 3).

71

Figure 15. Evaluating the Assay with Different Concentrations of Acetylcholinesterase: The First
Performance Test, Run 13.
A photo of the microtiter plate (inside the red box) used for the run was also attached to illustrate the
complete reproducibility of the results obtained from run 5 when treated with the correct amount and
concentration of acetylthiocholine iodide, along with the correct concentration of sodium phosphate buffer
in which the acetylthiocholine iodide was dissolved. This was evident from the absence of the crystal
formation in all wells, and from the consistent absorbance readings across the 30-min time span for all three
technical replicates.

In conclusion, this confirmed with absolute certainty that the previously incorrect
concentration of sodium phosphate buffer at 1.0 M was the sole error causing the formation of
crystals within some of the microtiter plate wells. These crystals could form at various stages,
either immediately after all components were added to the wells before mixing, during mixing, or
while the microtiter plate was being processed in the plate reader machine. The major negative
72

impact of these crystals on the assay’s performance was their disruption of the direct pathway of
light passing through the wells, leading to the inconsistency in absorbance readings, and substantial,
unsteady fluctuations in the graphical representation of the rate of TNB2- absorbance increase
(Supplementary Table 8 and Supplementary Figure 7).
As the issue of crystallization was being resolved, some additional adjustments were made
to the assay’s procedure during the last four runs of the first performance test. In run 10, six
technical replicates were performed over five consecutive periods spanning roughly 8 h. The first
period consisted two technical replicates, followed by the remaining four technical replicates, each
within one of the remaining four periods. It was observed that the rate of TNB2- absorbance
increase for treatments with lower acetylcholinesterase concentrations slowed down over time, to
the point where it became so sluggish that it almost resembled a flat line on the graph. This pattern
was consistent for the same treatment with one enzyme concentration over the span of 8 h,
regardless of the acetylcholinesterase concentration used.
It was suspected that the enzyme lost its stability over time, including its functionality,
even when stored on ice throughout the experiment. To prevent this from recurring, a new batch
of fresh standard solutions of acetylcholinesterase was prepared every time a run was performed,
to maintain the structural and functional stability of the enzyme. Additionally, the step of swirling
the microtiter plate for 30 secs before loading it into the plate reader machine was removed. It was
considered unnecessary as it did not significantly contribute to the efficiency of well mixing. It
was evident that within the confined space of each well, swirling the microtiter plate by hand could
not generate enough force to create a swirling motion adequate for efficient mixing. Therefore,
gently pipetting the solution up and down in every well with a large volume of 100 μL after adding

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all components to the wells was the sole practical option to achieve efficient mixing. These two
new adjustments were subsequently applied in runs 11 through 13 (Supplementary Table 9).
Of all thirteen runs conducted in the first performance test, runs 11 through 13 stood out as
the only ones where the assay’s performance reached the highest level of optimization.
Consequently, their data were selected for processing to serve as the final results of the first
performance test. Notably, the two acetylcholinesterase concentrations of 4.5 units/mL and 9.0
units/mL, which had been excluded after the first run due to a significant number of absorbance
readings going beyond the upper limit of the plate reader machine’s detection range, were
reintroduced in runs 10 through 13 for the purpose of data completion.
Based on the mean data from the last three runs of the first performance test, corresponding
to three experimental replicates, a graph of absorbance plotted against time for five different
concentrations of acetylcholinesterase was created. As the concentration of acetylcholinesterase
increased, the rate of TNB2- absorbance increase became faster, resulting in a more substantial
change in absorbance over the same 30-min time frame. Additionally, the rate of TNB2- absorbance
increase became increasingly rapid, to the point where saturation could instantly be reached at the
zeroth min with extremely high concentrations of acetylcholinesterase. This shift in the graphical
trend indicated that the rate of TNB2- absorbance increase transitioned from initially being linear
at low enzyme concentrations to logarithmic at higher concentrations (Figure 16).

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Figure 16. The Effect of Different Acetylcholinesterase Concentrations on Acetylcholinesterase
Activity.
The variation in the in vitro acetylcholinesterase activity was determined by the change in absorbance over
a 30-min time span, with the acetylcholinesterase concentration primarily increasing by factors of ten. Each
data point on the graph represented the mean absorbance reading recorded for the treatment with one
specific acetylcholinesterase concentration at one specific timepoint, with a 2-min interval between
measurements. Each mean absorbance reading was averaged from runs 11 through 13 of the first
performance test, representing an average obtained from three experimental replicates, corresponding to
nine technical replicates (n = 9). The absorbance value of 4.0 represented the upper limit of the absorbance
detection range of the Synergy 2 plate reader machine, beyond which no numerical value could be recorded.
Owing to extremely small standard deviations, the error bars for most data points were not visible.

The results not only supported the hypothesis, but also demonstrated that the assay
functioned correctly on its own without the addition of any test sample. Focusing on the results,
the acetylcholinesterase concentration of 0.09 units/mL was the only one to produce a suitably
steep slope representing a consistent linear change in TNB2- absorbance over time. This meant that
should there be a potential inhibitor of the enzyme, the decline in the steepness of the linear slope
would be easily noticeable. A decrease in thiocholine content would result in a lower TNB2amount and a slower rate of TNB2- absorbance increase over time, causing the previously straight
incline to become flatter and more horizontally leveled. With its favorable range of absorbance
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and moderate incline, the concentration of 0.09 units/mL was chosen for use in runs where test
samples were added (Figure 17).

Figure 17. The Effect of Different Acetylcholinesterase Concentrations on Acetylcholinesterase
Activity.
The data from Figure 6 were re-plotted to more effectively illustrate the effect of lower acetylcholinesterase
concentrations on acetylcholinesterase activity.

2.3.1.2

The Second Performance Test: Evaluating the Assay with Different
Concentrations of Known Inhibitors of Acetylcholinesterase

2.3.1.2.1

Purpose of the Second Performance Test

The second performance test aimed to assess the assay’s ability to generate decreasing
absorbance readings in response to increasing concentrations of a known acetylcholinesterase
inhibitor. This test served two purposes. First, it sought to ensure that the assay operated effectively
when a test sample was introduced. Hypothetically, assuming all other factors, including time,
remain constant, the change in TNB2- absorbance should decrease as the concentration of a
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potential acetylcholinesterase inhibitor increases. This is because a higher concentration of the
enzyme inhibitor would result in a reduced production of thiocholine, leading to a lower amount
of DTNB reduction and TNB2- generation, ultimately yielding a smaller change in absorbance over
the same time span.
The second purpose was to utilize the inhibition strength on acetylcholinesterase activity
of the known inhibitor as a reference point for comparison. This reference would help assess the
inhibition strength on the enzyme’s activity by any potential inhibitors discovered in this study.
2.3.1.2.2

Experimental Procedure of the Second Performance Test

Experiments were carried out using 96-well microtiter plates. In this test, all five
components of the assay were included: sodium phosphate buffer, Electrophorus electricus
acetylcholinesterase, acetylthiocholine iodide, DTNB, and a small molecule as the test sample. On
the plate, each row was divided into four different control wells and six different treatment wells.
For the control wells, there were four different types: first negative control, second negative
control, first positive control, and second positive control.
In the first negative control well, the following components were added: 180 μL of 0.1 M
of sodium phosphate buffer at pH 8.0, 10 μL of 10 mM of DTNB, and 10 μL of 14 mM of
acetylthiocholine iodide.
In the second negative control well, the following components were added: 170 μL of 0.1
M of sodium phosphate buffer at pH 8.0, 20 μL of 0.09 units/mL of acetylcholinesterase, and 10
μL of 10 mM of DTNB. The purpose of the two negative controls was to ensure that the assay
would not exhibit any activity in case one of the two crucial components, the enzyme or its
substrate, was missing.

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In the first positive control well, the following components were added: 160 μL of 0.1 M
of sodium phosphate buffer at pH 8.0, 20 μL of 0.09 units/mL of acetylcholinesterase, 10 μL of 10
mM of DTNB, and 10 μL of 14 mM of acetylthiocholine iodide. Repeating what was done in the
first performance test, including the first positive control was essential to ensure that the assay
would function correctly without the addition of any test sample.
In the second positive control well, the following components were added: 140 μL of 0.1
M of sodium phosphate buffer at pH 8.0, 20 μL of 0.09 units/mL of acetylcholinesterase, 10 μL of
10 mM of DTNB, 20 μL of a specific concentration of a solvent used to dissolve the known
inhibitor of acetylcholinesterase, and 10 μL of 14 mM of acetylthiocholine iodide. In contrast to
the first positive control, this was intended to ensure that any inhibitory effect on
acetylcholinesterase activity would not likely be attributed to the solvent used to dissolve the
known enzyme inhibitor.
In each treatment well, the following components were added: 140 μL of 0.1 M of sodium
phosphate buffer at pH 8.0, 20 μL of 0.09 units/mL of acetylcholinesterase, 10 μL of 10 mM of
DTNB, 20 μL of a specific concentration of a known inhibitor of acetylcholinesterase, and 10 μL
of 14 mM of acetylthiocholine iodide. The concentration of the known enzyme inhibitor was
divided into six increasing concentrations ordered from least to most concentrated: 0.02, 0.2, 2, 4,
20, and 40 μM. Once all concentrations of the known enzyme inhibitor were added to the treatment
wells, the wells underwent a 15-min incubation period before acetylthiocholine iodide was
introduced to initiate the assay.
There were three rows in total, each representing one technical replicate. The three
technical replicates were combined to form one experimental replicate corresponding to a single
run. For each experimental replicate, the change in the absorbance of TNB2- was detected at the

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wavelength of 412 nm and recorded every 2 mins for a total of 30 mins using the Synergy 2 plate
reader machine. A total of three experimental replicates were performed.
In addition to the treatment with the known acetylcholinesterase inhibitor, the experimental
procedure of the second performance test also included a treatment with a known non-inhibitor of
the enzyme. Testing a pair of test samples with opposite effects on acetylcholinesterase would
ensure that only compounds structurally compatible with the enzyme’s active site and chemically
compatible with the surrounding atoms within the active site could exhibit an inhibitory effect on
the enzyme’s activity. Aside from the known non-inhibitor of acetylcholinesterase, a second
known inhibitor of the enzyme was also included.
Aspirin, which has no reported history of inhibiting acetylcholinesterase, was selected as
the known non-inhibitor. The known inhibitors of acetylcholinesterase chosen were galanthamine
hydrobromide and pyridostigmine bromide. Both are well-known and well-studied reversible
inhibitors that selectively target different sites within the enzyme to inhibit its activity.
Galanthamine hydrobromide selectively binds to aromatic residues within the peripheral anionic
site of the enzyme, while pyridostigmine bromide selectively binds to the hydroxyl side chain of
the serine residue in the catalytic triad located within the catalytic anionic site of the enzyme.24
The effect of aspirin on acetylcholinesterase activity was tested individually with either
galanthamine hydrobromide or pyridostigmine bromide, forming two separate experimental sets.
2.3.1.2.3

Raw Data Processing and Graphing Transformation of the Second
Performance Test

In each technical replicate, for each of the negative control, positive control, and treatment
with one concentration of a small molecule, the total change in TNB2- absorbance over the 30-min
time span was calculated by subtracting the raw absorbance value recorded at the zeroth min from

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the value recorded at the thirtieth min. As there were two negative controls, the final total change
in TNB2- absorbance was determined by averaging the total changes from the two controls. The
same process was repeated for the two positive controls.
Next, the true average total change in TNB2- absorbance observed for the positive control
was calculated by subtracting the average total change for the negative control from the average
total change for the positive control. This step removed background absorbance contributed by
any solution in the positive control well apart from DTNB. This revealed the actual change in
absorbance due to DTNB reduction to TNB2-.
The true average total change in TNB2- absorbance observed for the positive control, along
with the total changes for the treatments with the six different concentrations of the small molecule,
were normalized to a range from 0% to 100% enzyme activity. The 100% mark was represented
by the true average total change in TNB2- absorbance observed for the positive control. This
normalization allowed a relative comparison of how acetylcholinesterase activity would decrease
as the concentration of the small molecule increased by factors of ten, if the small molecule acted
as an actual inhibitor of acetylcholinesterase.
In cases where an inhibitory effect occurred and sufficient data were available, a sigmoidal
dosage-response graph was created. This graph plotted percentage values of enzyme activity
against the increasing concentrations of the small molecule. Each percentage value at a specific
concentration was an average of nine different percentage values calculated from raw absorbance
data recorded in nine technical replicates, corresponding to three different experimental replicates.
A standard deviation value for each percentage value was also provided based on the raw data.
Following this, the IC50 value was calculated based on the shape of the sigmoidal curve,
considering the length and depth of both the upper and lower portions surrounding the inflection

80

point. The normalization and the IC50 value calculation were computed using the PRISM graphing
software. The x-axis of the graph was presented in a logarithmic scale to match the S-shaped
sigmoidal trend of a dosage-response graph.
Once the graph for the small molecule was completed, it was merged into a final overall
graph with the graph of another small molecule that did not inhibit acetylcholinesterase activity.
This comparison allowed for a clear distinction in the impact on the acetylcholinesterase activity
when exposed to an inhibitor versus a non-inhibitor.
2.3.1.2.4

Hypothesis and Predicted Outcomes of the Second Performance Test

According to the hypothesis, as the concentration of a known acetylcholinesterase inhibitor
increases, the linear rate of TNB2- absorbance increase should slow down, leading to a smaller
change in absorbance. This gradual decrease would cause the initially steep incline, which starts
at a certain degree, to flatten into a straight horizontal line.
On a dose-response graph where the percentage of enzyme activity is plotted against the
concentration of small molecule, an increase in the concentration of a known acetylcholinesterase
inhibitor by factors of ten should result in a decrease in the percentage of enzyme activity. This
decrease should follow a sigmoidal trend, producing a reverse S-shaped curve.
2.3.1.2.5

Observations and Analyses of the Second Performance Test

In the second performance test, a graph of the percentage of enzyme activity plotted against
concentration was created based on the mean data from three experimental replicates. In the first
experimental set, the effects of aspirin and galanthamine hydrobromide on acetylcholinesterase
activity were compared.
As the concentration of aspirin increased, mainly by a factor of ten within the range of 0.02
to 40 μM, the percentage of enzyme activity remained relatively stable and close to the levels

81

observed in the positive controls. This suggested that aspirin had no significant effect on
acetylcholinesterase activity. In contrast, as the concentration of galanthamine hydrobromide
increased in a similar manner, the percentage of enzyme activity significantly decreased, following
a sigmoidal trend and ultimately reaching complete inhibition at 100%. The standard deviations of
the mean data for the aspirin treatment were substantial, indicating random fluctuations in the
percentage of enzyme activity, while the standard deviations of the mean data for the galanthamine
hydrobromide treatment were small, suggesting minimal variability.
In summary, the data showed that aspirin had no impact on acetylcholinesterase activity,
while galanthamine hydrobromide clearly inhibited the enzyme’s activity. The concentration of
galanthamine hydrobromide required to inhibit acetylcholinesterase activity by 50%, or the IC50
value, was calculated to be 0.313 μM. Excluding eight outliers from a total of eighty-one search
results, which represent scientific studies conducted from the year 2000 to 2021 and were sourced
from the ChEMBL database of the European Molecular Biology Laboratory at that time, the
reported IC50 values for the inhibition of galanthamine hydrobromide on the activity of
Electrophorus electricus acetylcholinesterase in the nanomolar unit showed a range from 0.200
μM to 1.500 μM. Among these results, thirty-three reported IC50 values were below 0.700 μM.
The most frequently occurring value among those thirty-three search results was in the six hundred
nanomolar range.48 The IC50 value obtained in this study for galathamine hydrobromide, required
to inhibit acetylcholinesterase activity, fell within this reported range. It was notably close to the
most common value found in the research studies. As a result, this value was considered consistent
and reliable (Figure 18).

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Figure 18. The Effect of Galanthamine Hydrobromide on Acetylcholinesterase Activity.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
different small molecules at increasing concentrations primarily by factors of ten, over a 30-min time span.
Aspirin is a known non-inhibitor of acetylcholinesterase. Galanthamine hydrobromide is a known inhibitor
of acetylcholinesterase. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific small molecule, and at one specific
small molecule concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from three experimental replicates, corresponding to nine technical replicates (n = 9). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity. The IC50 value represented the concentration of galanthamine
hydrobromide required to inhibit acetylcholinesterase activity by 50%.

In the second experimental set, the effect of aspirin was compared to the effect of
pyridostigmine bromide on acetylcholinesterase activity. As the concentration of aspirin increased,
mainly by a factor of ten within the range of 0.02 to 40 μM, the percentage of enzyme activity
remained relatively stable and close to the levels observed in the positive controls again. While the
trendline appeared to show a slight decrease after the third data point, it did not seem to be a
substantial decrease, as the last three data points remained fairly consistent, with a slight increase
observed from the fourth data point to the sixth data point. Similar to the testing of aspirin in the
first experimental set, the presence of aspirin did not significantly alter acetylcholinesterase
83

activity in comparison to the levels observed in the positive controls in the second experimental
set. The results, characterized by large standard deviations, confirmed that aspirin had no
substantial effect on the activity of acetylcholinesterase.
In contrast to aspirin, as the concentration of pyridostigmine bromide increased in a similar
manner, the percentage of enzyme activity significantly decreased, following a sigmoidal trend.
However, the trend did not reach a complete inhibition of enzyme activity at 100%, as seen with
the testing of galanthamine hydrobromide in the first experimental set. Examining data variability
between experimental replicates, it was evident that the standard deviations of the mean data for
the pyridostigmine bromide treatment were small, indicating that pyridostigmine bromide also had
an inhibitory effect on acetylcholinesterase activity. However, its inhibitory effect on the enzyme
activity was approximately eighteen times weaker than the effect of galanthamine hydrobromide,
as reflected in the IC50 value of 5.530 μM. The IC50 value of pyridostigmine bromide demonstrated
that a significantly higher concentration was required to achieve the same degree of inhibition on
acetylcholinesterase activity compared to what was observed with galanthamine hydrobromide.
Contrary to galanthamine hydrobromide, there were no reliable, up-to-date extensive research
studies nor reports on the inhibitory effect of pyridostigmine bromide specifically on
Electrophorus electricus acetylcholinesterase activity, as no relevant information was found in the
ChEMBL database of the European Molecular Biology Laboratory, nor on the National Center for
Biotechnology Information website at the time. Despite the absence of literature values for
comparison, the experimental IC50 value of 5.530 μM was considered reliable due to the precise
data with low variability between experimental replicates. In addition to a weaker inhibitory effect,
the effective concentration range was also smaller for pyridostigmine bromide, as indicated by the

84

narrower and steeper slope around the inflection point of the sigmoidal trend compared to the slope
observed for galanthamine hydrobromide (Figure 19).

Figure 19. The Effect of Pyridostigmine Bromide on Acetylcholinesterase Activity.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
different small molecules at increasing concentrations primarily by folds of ten, over a 30-min time span.
Aspirin is a known non-inhibitor of acetylcholinesterase. Pyridostigmine bromide is a known inhibitor of
acetylcholinesterase. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific small molecule, and at one specific
small molecule concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from three experimental replicates, corresponding to nine technical replicates (n = 9). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity. The IC50 value represented the concentration of pyridostigmine
bromide required to inhibit acetylcholinesterase activity by 50%.

In conclusion, the final results supported the hypothesis and demonstrated the reliability of
the developed assay in this study. The evidence was the characteristic reverse S-shaped curve on
the dosage-response graph, representing the sigmoidal decrease in enzyme activity with increasing
inhibitor concentrations, primarily by a factor of ten. With two known acetylcholinesterase
inhibitors examined, the results also provided insights into the potential range of inhibition strength
85

for an acetylcholinesterase inhibitor. This information would be useful for comparing the
inhibition strength on acetylcholinesterase activity for the four small molecules SPOPP, NP6A,
534_1(6), and 534_2(7) against that of the two known acetylcholinesterase inhibitors, serving as
positive controls, to assess the effects of the small molecules on acetylcholinesterase activity as
described below.
2.4

Examination of the Effect of Small Molecules on Acetylcholinesterase Activity
The rationale for examining the effects of SPOPP, NP6A, 534_1(6) and 534_2(7) on

acetylcholinesterase activity was already provided on pages 30 to 33 in Chapter 1.
2.4.1

Preparation of the Synthetic Small Molecules

2.4.1.1

Synthesis of SPOPP and NP6A
In two separate small weighing boats, 1.8200 g of acenaphthenequinone and 1.1500 g of

L-proline in powder form were weighed on an analytical balance. Mass measurements were
typically accurate to three decimal places, occasionally varying slightly at the fourth decimal place.
After weighing, 200 mL of 100% high-performance liquid chromatography-grade methanol was
measured and transferred to a 1000-mL round-bottom flask using a 500-mL graduated cylinder.
The pre-weighed acenaphthenequinone and L-proline were also transferred to the same 1000-mL
round-bottom flask. To ensure no powder was left behind in the weighing boats, the boats were
rinsed with a small amount of 100% normal-grade methanol. The flask was then gently swirled by
hand for 10 mins to thoroughly mix the acenaphthenequinone, which was only partially soluble,
with the highly soluble L-proline in 100% normal-grade methanol.
Before preparing the reaction mixture, a water reflux apparatus was set up and maintained
at a temperature of 35°C. After the 10-min swirl, a 25×7-mm magnetic stir bar was added to the
flask. The flask was then placed under the water reflux apparatus. Through reflux, the reaction

86

mixture was continuously stirred at 280 rpm while being simmered at 35°C for precisely 3 h to
synthesize SPOPP and NP6A (Figures 20-21). Concurrently, a rotary evaporator was set up at
30°C.

Figure 20. The Synthesis of SPOPP and NP6A.
Acenaphthenequinone (4) and L-proline (5) was dissolved in methanol, continuously stirred and warmed
at 35°C for exactly three hours while being placed under reflux to synthesize SPOPP (8) and NP6A (9).26
64

Figure 21. The Synthesis of SPOPP and NP6A.
A photo showing the completion of the reaction described in Figure 20.

Following the 3-h simmer, the flask was carefully removed quickly and attached to the
rotary evaporator. During rotary evaporation, 100% methanol was evaporated from the reaction
mixture at 30°C until no solvent remained and product solids began to reappear. After the solids
reappeared, the flask was left under the same rotary evaporation conditions for an additional 3 h
87

to ensure thorough removal of any remaining residual methanol integrated within the solids. This
step was crucial to prevent the solids from turning into sticky chunks of sludge.
Once the solids were completely dried, the magnetic stir bar was removed by tilting the
flask to allow it to slide out naturally. The removed stir bar was rinsed in a small beaker with a
small amount of 100% normal-grade methanol to redissolve any remaining adhering solids. A selfmade fishing rod, equipped with a magnetic stir bar at its end, was used to pull the stir bar out
through magnetism. After that, the solution from rinsing the stir bar was transferred back to the
same round-bottom flask containing the bulk of the product solids.
The flask was then reattached to the rotary evaporator and left under the same rotary
evaporation conditions for another 2 h to further dry the residual methanol used for rinsing (Figure
22). Finally, the product solids were scraped into a vial and stored at 4°C. The product solids were
considered crude, containing various substances including SPOPP and NP6A, resulting in a dark
green color (Figure 23).

Figure 22. Crude Product Solids From the Synthesis of SPOPP and NP6A.

A photo showing the crude product solids obtained after the reaction mixture shown in Figure 21
was completely evaporated through rotary evaporation.

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Figure 23. Crude Product Solids From the Synthesis of SPOPP and NP6A.

A photo showing the dark green crude product solids after they were scraped into a vial from the
1000-mL round-bottom flask shown in Figure 22.
2.4.1.2

Preparation of Flash Column Chromatography
In two separate small weighing boats, 0.5000 g of the crude product, which included

SPOPP and NP6A, and 4.00 g of silica in powder form, were weighed on an analytical balance
and a top loading balance, respectively. Mass measurements were typically accurate to three
decimal places, occasionally varying slightly at the fourth decimal place. After weighing, 500 mL
of 100% high-performance liquid chromatography-grade methanol was measured and transferred
to a 1000-mL round-bottom flask using the 500-mL graduated cylinder. The pre-weighed crude
product and silica were then added to the same 1000-mL round-bottom flask. To ensure no material
was left behind in the weighing boats, the boats were rinsed with a small amount of 100% normalgrade methanol to collect every speck of both materials into the flask. The flask was then gently
swirled by hand for 10 mins to thoroughly mix the crude product and silica.
During this time, the rotary evaporator was set up at 30°C. After the 10-min swirl, the flask
was attached to the rotary evaporator. Using rotary evaporation, 100% methanol was evaporated
from the solution at 30°C until no solvent remained and solids began to reappear. After the solids
reappeared, the flask was left under the same rotary evaporation conditions for an additional 5
mins to ensure the thorough removal of any remaining residual methanol integrated within the
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solids. Given the extremely dry nature of the solids, 5 mins of drying was considered sufficient.
Once the solids were completely dried, they were scraped into a vial and stored at 4°C. These
solids consisted of the crude product adhering to silica particles, and were intended for the
subsequent purification process using flash column chromatography. The whole sample’s color
had transformed from dark green to bright orange.
In a 1000-mL Erlenmeyer flask, 180.00 g of silica was weighed on a top loading balance.
Mass measurements were typically accurate to three decimal places, occasionally varying slightly
at the fourth decimal place. After weighing, 3 L of high-performance liquid chromatography-grade
hexane, mixed with normal-grade ethyl acetate in a 9:1 ratio, was divided into smaller portions
and distributed into a 600-mL beaker and two 1000-mL Erlenmeyer flasks. One of the Erlenmeyer
flasks contained the pre-weighed 180.00 g of silica. The 1000-mL graduated cylinder was used to
measure and transfer the solvent.
Next, the bottom of the glass column, the entire V-shaped zone, was entirely filled with
sand. After filling the V-shaped zone, hexane-ethyl acetate was carefully pipetted down the side
of the column in small drips, gradually raising the liquid level to a point where even significant
disturbance, such as splashing large volumes of liquid, would not distort the sand layer’s flat
surface. Subsequently, a substantial volume of hexane-ethyl acetate was poured into the column,
filling it to half of its total height. The silica solution from one of the two 1000-mL Erlenmeyer
flasks was then introduced into the column. After transferring, the flask was rinsed with a small
amount of hexane-ethyl acetate to ensure that any remaining residual silica was washed into the
column.
With the aid of an air pump, the hexane-ethyl acetate solution in the column was rapidly
drained. The combination of the solution’s large volume and the air pressure released by the pump

90

compacted the silica layer tightly on top of the sand layer. Simultaneously, as the hexane-ethyl
acetate solution was being drained, any residual silica particles adhering to the column’s side were
washed down by gently pipetting tiny streams of hexane-ethyl acetate alongside the column’s wall.
Since the silica particles were denser than the hexane-ethyl acetate solution, the silica layer sank
faster and became compacted before the solution could follow. Once the level of the hexane-ethyl
acetate solution descended to the top surface of the tightly packed silica layer, the drainage was
immediately halted. The volume of hexane-ethyl acetate used to pack the silica layer was then
recollected for reuse in the upcoming purification process.
Prepared and ready for sample loading, the previously acquired bright orange solids, which
included the crude product adhering to silica particles, were gently sprinkled onto the top surface
of the tightly packed silica layer. To ensure thorough distribution of the sample, small drips of
hexane-ethyl acetate were carefully pipetted down along the column’s wall, washing any residual
solids onto the silica layer. This process continued until the solvent sufficiently covered the sample
layer, without overflowing. The objective was to uniformly moisten and flatten the sample layer
while preventing sample dilution.
Following this, an additional layer of sand was sprinkled on top of the sample layer. This
second sand layer was intentionally made thicker than the first one located in the V-shaped zone
at the base of the glass column. Its purpose was to create a thick barrier that would shield the
evenly distributed sample layer from disturbances when the hexane-ethyl acetate was introduced
to initiate the purification process. Once the sand layer was in place, more small drips of hexaneethyl acetate were carefully pipetted down along the column’s wall again, until the liquid reached
a specific height where even intense disturbances, like splashing large volumes of liquid, would
not distort the flat surface of the second sand layer. Finally, to complete this setup, a substantial

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volume of hexane-ethyl acetate was transferred into the column until it was filled nearly to its total
height.
2.4.1.3

Purification of SPOPP and NP6A
With the assistance of the air pump, the purification process was initiated and continuously

maintained using hexane-ethyl acetate at a flow rate of 2 psi/min. Whenever the level of hexaneethyl acetate dropped low near the second sand layer, the air pump was temporarily halted and
removed to allow for the addition of more hexane-ethyl acetate. This was done without interrupting
the drainage flow during the process.
As the sample layer separated into distinct bands while migrating through the silica layer,
SPOPP in the orange band and NP6A in the yellow band were collected separately into two 100mL round-bottom flasks (Figure 24). These flasks were then attached to the rotary evaporator.
Through rotary evaporation at 30°C, the hexane-ethyl acetate eluent was evaporated until no
solvent remained and purified solids began to reappear. After the solids reappeared, the flasks were
left under the same rotary evaporation conditions for an additional 5 mins to ensure the complete
removal of any remaining residual hexane-ethyl acetate. Subsequently, the flasks were removed
and stored at 4°C. Upon evaluating the quality of the purification, the purified solids were
redissolved in 100% acetonitrile before being subjected to high performance liquid
chromatography.

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Figure 24. The Purification of SPOPP and NP6A.

SPOPP in the orange band and NP6A in the yellow band were isolated separately through flash
column chromatography.
2.4.1.4

Synthesis of 534_1(6) and 534_2(7)
Both 534_1(6) and 534_2(7) were readily available and provided by Dr. Hooi Xian Lee, a

former team member from the Lee research group.49
2.4.2
2.4.2.1

Evaluation of the Effect of Small Molecules on Acetylcholinesterase Activity
Experimental Procedure
The experimental setup closely mirrored the one planned and established for the second

performance test. Experiments were carried out using 96-well microtiter plates. In this test, all five
components of the assay were included: sodium phosphate buffer, Electrophorus electricus
acetylcholinesterase, acetylthiocholine iodide, DTNB, and a small molecule as the test sample. On
the plate, each row was divided into four different control wells and six different treatment wells.
For the control wells, there were four different types: first negative control, second negative
control, first positive control, and second positive control.

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In the first negative control well, the following components were added: 180 μL of 0.1 M
of sodium phosphate buffer at pH 8.0, 10 μL of 10 mM of DTNB, and 10 μL of 14 mM of
acetylthiocholine iodide.
In the second negative control well, the following components were added: 170 μL of 0.1
M of sodium phosphate buffer at pH 8.0, 10 μL of 10 mM of DTNB, and 20 μL of 0.09 units/mL
of acetylcholinesterase.
In the first positive control well, the following components were added: 160 μL of 0.1 M
of sodium phosphate buffer at pH 8.0, 10 μL of 10 mM of DTNB, 20 μL of 0.09 units/mL of
acetylcholinesterase, and 10 μL of 14 mM of acetylthiocholine iodide.
In the second positive control well, the following components were added: 140 μL of 0.1
M of sodium phosphate buffer at pH 8.0, 10 μL of 10 mM of DTNB, 20 μL of 0.09 units/mL of
acetylcholinesterase, 20 μL of a specific concentration of a solvent used to dissolve the small
molecule, and 10 μL of 14 mM of acetylthiocholine iodide.
In each treatment well, the following components were added: 140 μL of 0.1 M of sodium
phosphate buffer at pH 8.0, 10 μL of 10 mM of DTNB, 20 μL of 0.09 units/mL of
acetylcholinesterase, 20 μL of a specific concentration of a small molecule, and 10 μL of 14 mM
of acetylthiocholine iodide. The concentration of the small molecule was divided into six
increasing concentrations ordered from least to most concentrated: 0.02, 0.2, 2, 4, 20, and 40 μM.
Once all concentrations of the small molecule were added to the treatment wells, the wells
underwent a 15-min incubation period before acetylthiocholine iodide was introduced to initiate
the assay.
There were three rows in total, each representing one technical replicate. The three
technical replicates were combined to form one experimental replicate corresponding to a single

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run. For each experimental replicate, the change in the absorbance of TNB2- was detected at the
wavelength of 412 nm and recorded every 2 mins for a total of 30 mins using the Synergy 2 plate
reader machine. A total of three experimental replicates were performed for the known noninhibitor aspirin, or each small molecule. A total of six experimental replicates were performed for
each pair of aspirin and one of the small molecules, forming one experimental set. The only minor
difference was a slight alteration in the order of component addition. To maintain the enzyme’s
freshness, acetylcholinesterase was introduced immediately after DTNB, before adding the sample
and acetylthiocholine iodide.
2.4.2.2

Hypothesis and Predicted Outcomes
According to the hypothesis, if a small molecule acts as an acetylcholinesterase inhibitor,

as its concentration increases, the linear rate of TNB2- absorbance increase should slow down,
leading to a smaller change in the absorbance. This gradual decrease would cause the initially steep
incline, which starts at a certain degree, to flatten into a straight horizontal line.
On a dose-response graph where the percentage of enzyme activity is plotted against the
concentration of small molecule, an increase in the concentration of the small molecule by factors
of ten should result in a decrease in the percentage of enzyme activity. This decrease should follow
a sigmoidal trend, producing a reverse S-shaped curve.
2.4.2.3

Observations and Analyses
Based on the mean data from three experimental replicates, a graph illustrating the

percentage of enzyme activity versus the concentration of two different types of small molecules
was generated. In all four experimental sets, where the effect of aspirin was compared to that of
four small molecules: SPOPP, NP6A, 534_1(6), and 534_2(7), it was observed that as the
concentration of aspirin increased, mainly by a factor of ten within the range of 0.02 to 40 μM, the

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percentage of enzyme activity remained quite similar to the positive controls. In the presence of
aspirin, the acetylcholinesterase activity did not significantly deviate from that observed in the
positive controls. However, there were substantial variations in the data as indicated by the large
standard deviations observed for the aspirin treatment. Disappointingly, both the trend and data
variability were similarly observed for the other four small molecules as well (Figures 25-28).

Figure 25. The Effect of SPOPP on Acetylcholinesterase Activity.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
different small molecules at increasing concentrations primarily by factors of ten, over a 30-min time span.
Aspirin is a known non-inhibitor of acetylcholinesterase. SPOPP is a small molecule that was used to test
for an inhibitory effect on acetylcholinesterase activity. Each data point on the graph represented the mean
percentage value of acetylcholinesterase activity recorded for the treatment with one specific small
molecule, and at one specific small molecule concentration. Each mean percentage value of
acetylcholinesterase activity was averaged from three experimental replicates, corresponding to nine
technical replicates (n = 9). Each set of upper and lower bars surrounding each data point on the graph
represented the standard deviation of a mean percentage value of acetylcholinesterase activity.

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Figure 26. The Effect of NP6A on Acetylcholinesterase Activity.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
different small molecules at increasing concentrations primarily by factors of ten, over a 30-min time span.
Aspirin is a known non-inhibitor of acetylcholinesterase. NP6A is a small molecule that was used to test
for an inhibitory effect on acetylcholinesterase activity. Each data point on the graph represented the mean
percentage value of acetylcholinesterase activity recorded for the treatment with one specific small
molecule, and at one specific small molecule concentration. Each mean percentage value of
acetylcholinesterase activity was averaged from three experimental replicates, corresponding to nine
technical replicates (n = 9). Each set of upper and lower bars surrounding each data point on the graph
represented the standard deviation of a mean percentage value of acetylcholinesterase activity.

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Figure 27. The Effect of 534_1(6) on Acetylcholinesterase Activity.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
different small molecules at increasing concentrations primarily by factors of ten, over a 30-min time span.
Aspirin is a known non-inhibitor of acetylcholinesterase. 534_1(6) is a small molecule that was used to test
for an inhibitory effect on acetylcholinesterase activity. Each data point on the graph represented the mean
percentage value of acetylcholinesterase activity recorded for the treatment with one specific small
molecule, and at one specific small molecule concentration. Each mean percentage value of
acetylcholinesterase activity was averaged from three experimental replicates, corresponding to nine
technical replicates (n = 9). Each set of upper and lower bars surrounding each data point on the graph
represented the standard deviation of a mean percentage value of acetylcholinesterase activity.

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Figure 28. The Effect of 534_2(7) on Acetylcholinesterase Activity.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
different small molecules at increasing concentrations primarily by factors of ten, over a 30-min time span.
Aspirin is a known non-inhibitor of acetylcholinesterase. 534_2(7) is a small molecule that was used to test
for an inhibitory effect on acetylcholinesterase activity. Each data point on the graph represented the mean
percentage value of acetylcholinesterase activity recorded for the treatment with one specific small
molecule, and at one specific small molecule concentration. Each mean percentage value of
acetylcholinesterase activity was averaged from three experimental replicates, corresponding to nine
technical replicates (n = 9). Each set of upper and lower bars surrounding each data point on the graph
represented the standard deviation of a mean percentage value of acetylcholinesterase activity.

Although the treatments with SPOPP and NP6A showed a slight reduction in enzyme
activity as the concentration of these small molecules increased from 20 μM onwards, there was
substantial overlap between the standard deviations of the mean data at concentrations of 20 μM
and 40 μM, making it difficult to conclude a true inhibition. Since none of the treatments with the
four small molecules exhibited sufficient inhibition of enzyme activity, the determination of their
IC50 values was not possible (Figures 25-28). In summary, the final results suggested that none of
the four small molecules was able to inhibit acetylcholinesterase activity, as there were no
noticeable signs of inhibition.

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Chapter 3
Investigating the Effect of Mushroom Extracts on Acetylcholinesterase Activity
3.1

Introduction
The rationale for examining the effects of the following mushroom extracts on

acetylcholinesterase activity was already provided on pages 34 to 38 in Chapter 1: Agaricus
subrufescens 295E1-E3, Amanita muscaria 243E1-E3, Ganoderma applanatum M5E1-E3,
Hericium erinaceus 288E1 and E3, Inonotus obliquus 285E1-E3, Phellinus igniarius PIE1-E3,
Phellinus pini 91E1-E3, Pleurotus ostreatus 68E1 and E2.
3.2

Materials and Methods

3.2.1 Preparation of the Mushroom Extracts
3.2.1.1

Extraction of the Mushroom Extracts
All mushroom extracts, available in powder form, were provided by members of the Lee

research group. The crude extracts were prepared using the following procedure.
Before the extraction commenced, all mushroom specimens were manually ground into
fine powder. Subsequently, for one specimen, approximately 1 g of powder was extracted with 10
mL of 80% ethanol at 65°C for 3 h while being continuously stirred in a small beaker. The resulting
solution was filtered through gravity filtration and labeled as E1.
The unfiltered residues remaining from the first extraction were subjected to a second
extraction with 10 mL of 50% methanol at 65°C for 3 h, also with continuous stirring. Afterwards,
this solution was filtered through gravity filtration again and labeled as E2.
Following the same procedure, the unfiltered residues remaining from the second
extraction were subjected to a third extraction with 10 mL of autoclaved water at 65°C for 3 h

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while being continuously stirred. Afterwards, this solution was filtered through gravity filtration
again and labeled as E3.
Subsequently, all three types of solutions were subjected to rotary evaporation to remove
the solvents. The remaining substances were further dried to form crude extracts through
lyophilization with a Labconco freeze dryer. Finally, the crude extracts of all mushroom specimens
were individually resuspended in different solvents, as outlined in the table below (Table 7).50

Mushroom Species
Agaricus subrufescens
Amanita muscaria
Ganoderma applanatum
Hericium erinaceus
Inonotus obliquus
Phellinus igniarius
Phellinus pini
Pleurotus ostreatus

Extract
295E1
295E2
295E3
243E1
243E2
243E3
M5E1
M5E2
M5E3
288E1
288E3
285E1
285E2
285E3
PIE1
PIE2
PIE3
91E1
91E2
91E3
68E1
68E2

Solvent Used to Extract
80% Ethanol
50% Methanol
Water
80% Ethanol
50% Methanol
Water
80% Ethanol
50% Methanol
Water
80% Ethanol
Water
80% Ethanol
50% Methanol
Water
80% Ethanol
50% Methanol
Water
80% Ethanol
50% Methanol
Water
80% Ethanol
50% Methanol

Table 7. Summary of the Preparation of the Mushroom Extracts.
The crude extracts of all mushroom specimens were resuspended in different solvents as indicated.

3.3

Results and Discussion

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3.3.1

Evaluation of the Effect of Crude Mushroom Extracts on Acetylcholinesterase
Activity

3.3.1.1

Experimental Procedure
The experiment setup was similar to what was previously planned and setup for the

experimentation with the small molecules, as described in Chapter 2, with a few minor adjustments.
Experiments were carried out using 96-well microtiter plates. In this test, all five components of
the assay were included: sodium phosphate buffer, Electrophorus electricus acetylcholinesterase,
acetylthiocholine iodide, DTNB, and a mushroom extract as the test sample. On the plate, each
row was divided into four different control wells and five different treatment wells.
For the control wells, there were four different types: first negative control, second negative
control, first positive control, and second positive control.
In the first negative control well, the following components were added: 180 μL of 0.1 M
of sodium phosphate buffer at pH 8.0, 10 μL of 10 mM of DTNB, and 10 μL of 14 mM of
acetylthiocholine iodide.
In the second negative control well, the following components were added: 170 μL of 0.1
M of sodium phosphate buffer at pH 8.0, 10 μL of 10 mM of DTNB, and 20 μL of 0.09 units/mL
of acetylcholinesterase.
In the first positive control well, the following components were added: 160 μL of 0.1 M
of sodium phosphate buffer at pH 8.0, 10 μL of 10 mM of DTNB, 20 μL of 0.09 units/mL of
acetylcholinesterase, and 10 μL of 14 mM of acetylthiocholine iodide.
In the second positive control well, the following components were added: 140 μL of 0.1
M of sodium phosphate buffer at pH 8.0, 10 μL of 10 mM of DTNB, 20 μL of 0.09 units/mL of

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acetylcholinesterase, 20 μL of a specific concentration of a solvent used to resuspend the powder
of the mushroom extract, and 10 μL of 14 mM of acetylthiocholine iodide.
In each treatment well, the following components were added: 140 μL of 0.1 M of sodium
phosphate buffer at pH 8.0, 10 μL of 10 mM of DTNB, 20 μL of 0.09 units/mL of
acetylcholinesterase, 20 μL of a specific concentration of a mushroom extract, and 10 μL of 14
mM of acetylthiocholine iodide. The concentration of the crude mushroom extract was divided
into five increasing concentrations ordered from least to most concentrated: 0.1875, 0.375, 0.75,
1.5, and 3.0 mg/mL. Once all concentrations of the mushroom extract were added to the treatment
wells, the wells underwent a 15-min incubation period before acetylthiocholine iodide was
introduced to initiate the assay.
There were three rows in total, each representing one technical replicate. The three
technical replicates were combined to form one experimental replicate corresponding to a single
run. For each experimental replicate, the change in the absorbance of TNB2- was detected at the
412-nm wavelength and recorded every 2 mins for a total of 30 mins using the Synergy 2 plate
reader machine. A total of two experimental replicates were performed for each of two mushroom
extracts, one known to inhibit acetylcholinesterase activity and another extract from the group
under investigation. A total of four experimental replicates were performed, pairing one extract
known to inhibit acetylcholinesterase activity and another extract from the group under
investigation, forming one experimental set.
3.3.1.2

Hypothesis and Predicted Outcomes
According to the hypothesis, if a mushroom extract contains any substance that acts as an

acetylcholinesterase inhibitor, as the extract concentration increases, the linear rate of TNB2absorbance increase should slow down, leading to a smaller change in the absorbance. This gradual

103

decrease would cause the initially steep incline, which starts at a certain degree, to flatten into a
straight horizontal line.
On a dose-response graph where the percentage of enzyme activity is plotted against the
concentration of mushroom extract, an increase in the concentration of the mushroom extract by
factors of ten should result in a decrease in the percentage of enzyme activity. This decrease should
follow a sigmoidal trend, producing a reverse S-shaped curve.
3.3.1.3

Observations and Analyses
With the current limited knowledge of the in vitro bioactivity of mushroom extracts on

acetylcholinesterase activity, the range of extract concentration used in this experimentation
initially ranged from 0.0937 mg/mL to 1.5 mg/mL. This choice was based on a common
observation that various mushroom species, which have previously been studied, contain natural
substances primarily extracted using either methanol or water. These substances typically exhibit
an inhibition of acetylcholinesterase activity in the range of approximately 10% to 30% when their
concentration is around 0.2 mg/mL. Utilizing this critical information as a reference point, a
reasonable and appropriate extrapolation was made in both directions to establish a concentration
range from 0.0937 mg/mL to 1.5 mg/mL.
After determining this range, it was applied to conduct the experimentation on the
following crude mushroom extracts: Phellinus igniarius PIE3, Phellinus igniarius PIE2,
Ganoderma applanatum M5E3, and Ganoderma applanatum M5E1. Furthermore, considering
that only Pleurotus ostreatus has been extensively studied as possessing an inhibitory effect on
acetylcholinesterase activity, the Pleurotus ostreatus 68E1 extract was used as the control in the
initial phase of the experimentation.31

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Based on the mean data from two experimental replicates, a graph showing the percentage
of enzyme activity plotted against concentration for two different mushroom extracts was created.
In each experimental set, the potential inhibitory effect of the Pleurotus ostreatus 68E1 extract
was used as the positive control to establish a relative standard for determining the inhibition
strength on acetylcholinesterase activity for the four mushroom extracts mentioned earlier, should
any of them inhibit the enzyme activity.
Throughout the four experimental sets, despite a successive increase in the concentration
of the Pleurotus ostreatus 68E1 extract by a factor of two, ranging from 0.0937 mg/mL to 1.5
mg/mL, the percentage of enzyme activity was reduced by at most approximately 15% (Figures
29-32). Considering the low data variability, as indicated by the relatively small standard deviation
bars around each mean data point, it was evident that the extent of acetylcholinesterase activity
inhibition upon treatment with the Pleurotus ostreatus 68E1 extract was consistent with the
common 13% inhibition value reported in the current literature when acetylcholinesterase was
treated with the methanol extract of Pleurotus ostreatus at a concentration exceeding 0.2 mg/mL
(Figures 29-32).31
Unlike the control Pleurotus ostreatus 68E1, there appeared to be no inhibition of
acetylcholinesterase activity upon treatment with the Phellinus igniarius PIE3, Phellinus igniarius
PIE2, nor Ganoderma applanatum M5E3 extract (Figures 29-31). Surprisingly, there was a sudden
and consistent increase in the percentage of enzyme activity when acetylcholinesterase was treated
individually with any of the three extracts. This abnormality was accompanied by high data
variability, indicated by slightly broader standard deviation bars around each mean data point,
raising questions about the legitimacy of the observed trend (Figures 29-31). Even if it were the
case, it remained unclear what could be causing this seemingly stimulatory effect on

105

acetylcholinesterase activity, as no further conclusive evidences were obtained from subsequent
experimentation. Nonetheless, within the scope of this study, these three mushroom extracts of
Phellinus igniarius PIE3, Phellinus igniarius PIE2, and Ganoderma applanatum M5E3 were ruled
out as potential candidates for acetylcholinesterase inhibition.

Figure 29. The Effect of Phellinus igniarius PIE3 on Acetylcholinesterase Activity at Low Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.0937 mg/mL to 1.5 mg/mL, over a 30-min
time span. Pleurotus ostreatus 68E1 is a mushroom extract known to inhibit acetylcholinesterase. Phellinus
igniarius PIE3 is a mushroom extract that was used to test for an inhibitory effect on acetylcholinesterase
activity. Each data point on the graph represented the mean percentage value of acetylcholinesterase activity
recorded for the treatment with one specific mushroom extract, and at one specific extract concentration.
Each mean percentage value of acetylcholinesterase activity was averaged from two experimental replicates,
corresponding to six technical replicates (n = 6). Each set of upper and lower bars surrounding each data
point on the graph represented the standard deviation of a mean percentage value of acetylcholinesterase
activity.

On a side note, regarding the two missing mean data points at the higher extract
concentration end of the Phellinus igniarius PIE2 data, they were excluded due to the solution’s
opaqueness in the treatment wells of the last two highest extract concentrations. This opaqueness
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resulted in an extremely high background level, far exceeding the upper limit of the plate reader
machine’s absorbance detection range. Theoretically, at this level of intensity, even the
background absorbance, excluding the true absorbance from TNB2- as a product of the reduction
of DTNB, could not be accurately measured as a numerical value. This is because the designated
wavelength, which is supposed to pass through the solution after being emitted from the plate
reader machine, may barely penetrate the solution, leading the machine to mistakenly determine
that the solution has an infinitely high absorbance. Therefore, with careful consideration, these two
missing mean data points representing the outcome of the treatment of acetylcholinesterase with
either 0.75 mg/mL or 1.5 mg/mL of the Phellinus igniarius PIE2 extract were disregarded (Figure
30).

107

Figure 30. The Effect of Phellinus igniarius PIE2 on Acetylcholinesterase Activity at Low Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.0937 mg/mL to 1.5 mg/mL, over a 30-min
time span. Pleurotus ostreatus 68E1 is a mushroom extract known to inhibit acetylcholinesterase. Phellinus
igniarius PIE2 is a mushroom extract that was used to test for an inhibitory effect on acetylcholinesterase
activity. Each data point on the graph represented the mean percentage value of acetylcholinesterase activity
recorded for the treatment with one specific mushroom extract, and at one specific extract concentration.
Each mean percentage value of acetylcholinesterase activity was averaged from two experimental replicates,
corresponding to six technical replicates (n = 6). Each set of upper and lower bars surrounding each data
point on the graph represented the standard deviation of a mean percentage value of acetylcholinesterase
activity.

108

Figure 31. The Effect of Ganoderma applanatum M5E3 on Acetylcholinesterase Activity at Low
Extract Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.0937 mg/mL to 1.5 mg/mL, over a 30-min
time span. Pleurotus ostreatus 68E1 is a mushroom extract known to inhibit acetylcholinesterase.
Ganoderma applanatum M5E3 is a mushroom extract that was used to test for an inhibitory effect on
acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity.

Among the four mushroom extracts experimented in the initial phase, only the Ganoderma
applanatum M5E1 extract exhibited an inhibition of acetylcholinesterase activity. When
comparing the inhibition strength displayed by the Pleurotus ostreatus 68E1 extract, there was a
similarity in strength when the enzyme was treated individually with either of the extracts at
concentrations ranging from 0.0937 mg/mL to 0.375 mg/mL. However, this similarity gradually
diverged as the extract concentration increased beyond 0.375 mg/mL.
During the transition from 0.375 mg/mL to 1.5 mg/mL, the Pleurotus ostreatus 68E1
extract actually exhibited a slightly greater inhibition strength than the Ganoderma applanatum

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M5E1 extract. This trend continued until the extract concentration approached very close to 1.5
mg/mL, at which point there was a sharp decline in the percentage of acetylcholinesterase activity
observed for the treatment with the Ganoderma applanatum M5E1 extract. This decline allowed
the inhibition strength exerted by the Ganoderma applanatum M5E1 extract to surpass that of the
Pleurotus ostreatus 68E1 extract.
Visualizing the data at the intersection point between the Pleurotus ostreatus 68E1 and the
Ganoderma applanatum M5E1 data, assuming there was an imaginary line perpendicular to the
trendline of the Pleurotus ostreatus 68E1 data passing through the intersection point, it could be
observed that the sharp decline in the trendline of the Ganoderma applanatum M5E1 data near the
1.5 mg/mL-concentration mark deviated almost 45° degrees as it surpassed away from the
trendline of the Pleurotus ostreatus 68E1 data. At 1.5 mg/mL, exceeding the approximate 15%
inhibition strength exerted by the Pleurotus ostreatus 68E1 extract, the Ganoderma applanatum
M5E1 extract exhibited an approximate inhibition strength of 21% (Figure 32).
Based on this observation, it was postulated that the Ganoderma applanatum M5E1 data
may hold promise as the most potent candidate for acetylcholinesterase inhibition in the initial
phase of the experimentation. Consequently, it was decided that a higher concentration range from
0.375 mg/mL to 5.0 mg/mL of Ganoderma applanatum M5E1 should be further investigated.

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Figure 32. The Effect of Ganoderma applanatum M5E1 on Acetylcholinesterase Activity at Low
Extract Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.0937 mg/mL to 1.5 mg/mL, over a 30-min
time span. Pleurotus ostreatus 68E1 is a mushroom extract known to inhibit acetylcholinesterase.
Ganoderma applanatum M5E1 is a mushroom extract that was used to test for an inhibitory effect on
acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity.

Utilizing the established null results of the Phellinus igniarius PIE3, Phellinus igniarius
PIE2, and Ganoderma applanatum M5E3 data to an advantage, one of these three extracts was
selected to be the negative control and paired with the Ganoderma applantum M5E1 data in the
second phase of the experimentation. That chosen extract was the Ganoderma applanatum M5E3
extract. In terms of the experimental results, there was no particularly significant detail that favored
the selection of the Ganoderma applanatum M5E3 extract as the negative control over the other
two. The choice was based on one of the fundamental principles of scientific experimentation.

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When applying the scientific method to any experiment, apart from varying levels of one
or more independent variable of interest, all other variables should be tightly controlled to maintain
their constant state. This is done to ensure that the influence exerted by the independent variable(s)
on the subject of interest is directly responsible for the observed outcome measured by the
dependent variable with an intrinsic connection to the subject of interest. In this experimentation,
there were two independent variables: the concentration of a mushroom extract and the type of
mushroom extract. Regarding the type of extract, as there were different mushroom species, and
each species had different extracts extracted using different solvents or solvent combinations, it
was preferrable to have a relative comparison between two types of extract derived from the same
species on a graph whenever possible. This approach helped maintain consistency and aesthetics
in presenting the experimental results.
By extending the extract concentration range to a higher level, the inhibitory effect on
acetylcholinesterase activity exerted by the Ganoderma applanatum M5E1 extract became more
apparent (Figure 33). At 1.5 mg/mL, the approximate inhibition strength was 31%. With a standard
deviation of approximately 10%, the mean data point closely matched the approximate inhibition
strength of 21% observed in the Ganoderma applanatum M5E1 data during its participation in the
initial phase of the experimentation. At 3.0 mg/mL, the approximate inhibition strength further
increased to 62%, which was twice the strength observed for the previous mean data point.
Although the standard deviation of the mean data point at 3.0 mg/mL had slightly more variation
than the one at 1.5 mg/mL, there was no overlap between the two mean data points, indicating that
the robust increase in inhibition strength was authentic. Unfortunately, similar to the previous case
of the two missing mean data points for the last two highest extract concentrations of the Phellinus
igniarius PIE2 data, the solution at 5.0 mg/mL was too opaque, resulting in an extremely high

112

background absorbance beyond the detection capability of the plate reader machine. As a result,
the mean data point at 5.0 mg/mL of the Ganoderma applanatum M5E1 data was also disregarded.
Nonetheless, the approximate inhibition strength exerted by the extract at 5.0 mg/mL could still
be estimated to be around 80% through trendline extrapolation. Despite these challenges, the entire
Ganoderma applanatum M5E1 dataset was sufficient to determine an IC50 value of 2.320 mg/mL
(Figure 33).

Figure 33. The Effect of Ganoderma applanatum M5E1 on Acetylcholinesterase Activity at High
Extract Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E3 is a mushroom extract used as the negative control. Ganoderma
applanatum M5E1 is a mushroom extract that was used to test for an inhibitory effect on
acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity. The IC50 value represented the concentration of Ganoderma
applanatum M5E1 required to inhibit acetylcholinesterase activity by 50%.

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In contrast to the Ganoderma applanatum M5E1 data, the Ganoderma applanatum M5E3
data exhibited a significant spike in the percentage of enzyme activity when acetylcholinesterase
was treated with the extract. Furthermore, when compared to the Ganoderma applanatum M5E3
data from its previous experimental set during the initial phase of the experimentation, the
percentage of enzyme activity for all mean data points, except the one at 5.0 mg/mL, increased by
approximately 30%, and even as high as 50% for the one at 3.0 mg/mL. This spike in activity was
also in line with the previous results seen in the Phellinus igniarius PIE3 data. As previously
mentioned, whether this apparent stimulatory effect on acetylcholinesterase activity induced by
the Ganoderma applanatum M5E3 extract was real or not would require further experimentation
to obtain more conclusive evidences. However, at the moment, based solely on the inconsistency
in the data between the four experimental replicates and the fairly large overlapping standard
deviation among most of the mean data points, it appeared that there may not be any significant
biochemical effect on acetylcholinesterase activity exerted by the Ganoderma applanatum M5E3
extract.
Overall, with the data gathered from the second phase of the experimentation, it was
confirmed that the Ganoderma applanatum M5E1 extract did indeed inhibit acetylcholinesterase
activity, and this inhibitory effect was significant. Additionally, as there was a potential issue with
solutions containing a mushroom extract at the concentration of 5.0 mg/mL being too opaque for
their absorbance to be measured by the plate reader machine, the extract concentration range was
subsequently adjusted to range from 0.1875 mg/mL to 3.0 mg/mL. This range was then used to
experiment with the remaining mushroom extracts to assess their potential inhibitory effects on
acetylcholinesterase activity in the final phase of the experimentation (Figure 33).

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In many ways, the third phase of the experimentation resembled the initial phase. The only
change made was in the choice of the control. Instead of using Pleurotus ostreatus 68E1, the
Ganoderma applanatum M5E1 extract was employed as the positive control. This decision was
based on the fact that the Ganoderma applanatum M5E1 extract exhibited a stronger inhibitory
effect on acetylcholinesterase activity, making it a more suitable relative standard for evaluating
the inhibition strength of the remaining mushroom extracts, should any of them exhibit a
significant inhibitory effect.
Out of the remaining extracts, an additional seven were found to inhibit
acetylcholinesterase. These extracts, along with the Ganoderma applanatum M5E1 extract, were
ranked in the following order from the weakest to the strongest inhibition strength, as indicated by
their IC50 values, from highest to lowest: Hericium erinaceus 288E3 with an IC50 value of 3.866
mg/mL, Ganoderma applanatum M5E2 with an IC50 value of 3.038 mg/mL, Ganoderma
applanatum M5E1 with an IC50 value of 2.320 mg/mL, Amanita muscaria 243E1 with an IC50
value of 1.380 mg/mL, Agaricus subrufescens 295E1 with an IC50 value of 1.230 mg/mL, Agaricus
subrufescens 295E2 with an IC50 value of 1.110 mg/mL, Hericium erinaceus 288E1 with an IC50
value of 1.060 mg/mL, and Inonotus obliquus 285E1 with an IC50 value of 0.980 mg/mL (Table 8
and Figures 34-40).

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Mushroom Species
Agaricus subrufescens
Amanita muscaria
Ganoderma applanatum
Hericium erinaceus
Inonotus obliquus
Pleurotus ostreatus

Extract
295E1
295E2
243E1
M5E1
M5E2
288E1
288E3
285E1
68E1

IC50 [mg/mL]
1.230
1.110
1.380
2.320
3.038
1.060
3.866
0.980
N/A

Maximum Inhibition
83.00% at 3.000 mg/mL
87.00% at 3.000 mg/mL
67.00% at 3.000 mg/mL
62.00% at 3.000 mg/mL
48.00% at 3.000 mg/mL
78.00% at 3.000 mg/mL
39.00% at 3.000 mg/mL
91.00% at 3.000 mg/mL
16.00% at 1.500 mg/mL

Table 8. Summary of Mushroom Extracts that Inhibited Acetylcholinesterase Activity.
A list of mushroom extracts found to inhibit the in vitro acetylcholinesterase activity in this study. The IC50
value represented the concentration of a mushroom extract required to inhibit acetylcholinesterase activity
by 50%. The maximum inhibition represented a mushroom extract’s highest level of inhibition on
acetylcholinesterase activity, based on observations recorded from the conducted experiments.

Although the ranking of the inhibition strength followed this order, the ranking of the
approximate maximum inhibition strength at 3.0 mg/mL, the highest extract concentration tested
in the third phase of the experimentation, did not necessarily align with the same order. It was
ranked from the weakest to the strongest approximate maximum inhibition strength, as indicated
by the percentage of acetylcholinesterase activity inhibited: Hericium erinaceus 288E3 at 39%,
Ganoderma applanatum M5E2 at 48%, Ganoderma applanatum M5E1 at 62%, Amanita muscaria
243E1 at 67%, Hericium erinaceus 288E1 at 78%, Agaricus subrufescens 295E1 at 83%, Agaricus
subrufescens 295E2 at 87%, and Inonotus obliquus 285E1 at 91% (Table 8 and Figures 34-40).
Aside from the inhibition strength, the determination of the inhibition range for these
mushroom extracts also took into consideration three key characteristics of the sigmoidal dosageresponse curve: the breadth of the curve during the initial transition before it starts to climb or dive,
the steepness of the linear segment around the inflection point, and the total length of the linear
segment before the curve levels off.

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Starting with Hericium erinaceus 288E3, its inhibition range was estimated to be from 0.75
mg/mL to 3.0 mg/mL. Despite having a fairly broad initial dive, and a moderately steep linear
segment at the inflection point, its inhibition range was mostly limited by a very short total length
of the linear segment, which quickly leveled off after 1.5 mg/mL to 3.0 mg/mL (Figure 34).

Figure 34. The Effect of Hericium erinaceus 288E3 on Acetylcholinesterase Activity at High Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Hericium erinaceus 288E3 is a mushroom extract that was used to test for an inhibitory effect
on acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity. The IC50 value represented the concentration of Hericium erinaceus
288E3 required to inhibit acetylcholinesterase activity by 50%.

Moving to Ganoderma applanatum M5E2, its inhibition range was estimated to be from
2.75 mg/mL to 3.5 mg/mL. In contrast to Hericium erinaceus 288E3, its inhibition range was very

117

restricted due to an acute initial dive, resulting in an extremely steep and extended linear plunge
at the inflection point (Figure 35).

Figure 35. The Effect of Ganoderma applanatum M5E2 on Acetylcholinesterase Activity at High
Extract Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Ganoderma applanatum M5E2 is a mushroom extract that was used to test for an inhibitory
effect on acetylcholinesterase activity. Each data point on the graph represented the mean percentage value
of acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity. The IC50 value represented the concentration of Ganoderma
applanatum M5E2 required to inhibit acetylcholinesterase activity by 50%.

Amanita muscaria 243E1 had an estimated inhibition range from 0.375 mg/mL to 4.0
mg/mL. Similar to Hericium erinaceus 288E3, but with a broader initial dive, and a slightly steeper
and longer total length of the linear segment at the inflection point, this allowed it to have an
inhibition range generally wider than Hericium erinaceus 288E3 (Figure 36).

118

Figure 36. The Effect of Amanita muscaria 243E1 on Acetylcholinesterase Activity at High Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Amanita muscaria 243E1 is a mushroom extract that was used to test for an inhibitory effect
on acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity. The IC50 value represented the concentration of Amanita muscaria
243E1 required to inhibit acetylcholinesterase activity by 50%.

Remarkably, the sigmoidal dosage-response curves of Agaricus subrufescens 295E1 and
Agaricus subrufescens 295E2 closely resembled that of Amanita muscaria 243E1. They all
displayed nearly identical broad initial dives and similarly matched total lengths of the linear
segment at the inflection point which also leveled off just after 3.0 mg/mL. The only difference
was the steepness of the linear segment at the inflection point, with Agaricus subrufescens 295E2
being the steepest, and Agaricus subrufescens 295E1 and Amanita muscaria 243E1 being
somewhat less steep but similar. As a result, both Agaricus subrufescens 295E1 and Agaricus

119

subrufescens 295E2 had an estimated inhibition range from 0.375 mg/mL to 4.0 mg/mL (Figures
37-38).

Figure 37. The Effect of Agaricus subrufescens 295E1 on Acetylcholinesterase Activity at High
Extract Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Agaricus subrufescens 295E1 is a mushroom extract that was used to test for an inhibitory
effect on acetylcholinesterase activity. Each data point on the graph represented the mean percentage value
of acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity. The IC50 value represented the concentration of Agaricus
subrufescens 295E1 required to inhibit acetylcholinesterase activity by 50%.

120

Figure 38. The Effect of Agaricus subrufescens 295E2 on Acetylcholinesterase Activity at High
Extract Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Agaricus subrufescens 295E2 is a mushroom extract that was used to test for an inhibitory
effect on acetylcholinesterase activity. Each data point on the graph represented the mean percentage value
of acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity. The IC50 value represented the concentration of Agaricus
subrufescens 295E2 required to inhibit acetylcholinesterase activity by 50%.

Moving to Hericium erinaceus 288E1, its sigmoidal dosage-response curve resembled that
of Hericium erinaceus 288E3 in terms of the steepness of the linear segment at the inflection point.
However, the initial dive was much broader, even more extensive than that of Amanita muscaria
243E1, Agaricus subrufescens 295E1, and Agaricus subrufescens 295E2. Additionally, the total
length of the linear segment of the curve extended to the degree where the point the curve began
leveling off was beyond clear sight. This combination of characteristics led to an estimated
inhibition range for Hericium erinaceus 288E1 from 0.0937 mg/mL to 5.0 mg/mL (Figure 39).

121

Figure 39. The Effect of Hericium erinaceus 288E1 on Acetylcholinesterase Activity at High Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Hericium erinaceus 288E1 is a mushroom extract that was used to test for an inhibitory effect
on acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity. The IC50 value represented the concentration of Hericium erinaceus
288E1 required to inhibit acetylcholinesterase activity by 50%.

Lastly, the sigmoidal dosage-response curve of Inonotus obliquus 285E1 closely resembled
that of Hericium erinaceus 288E1 in every aspect, except for the steepness of the linear segment
at the inflection point, where it was the steepest among all eight extracts exhibiting
acetylcholinesterase inhibition. Consequently, the inhibition range of Inonotus obliquus 285E1
was estimated to be from 0.0937 mg/mL to 6.0 mg/mL (Figure 40).

122

Figure 40. The Effect of Inonotus obliquus 285E1 on Acetylcholinesterase Activity at High Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Inonotus obliquus 285E1 is a mushroom extract that was used to test for an inhibitory effect
on acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity. The IC50 value represented the concentration of Inonotus obliquus
285E1 required to inhibit acetylcholinesterase activity by 50%.

Returning to Ganoderma applanatum M5E1, its initial dive was the broadest and most
gradual of all mushroom extract datasets showing acetylcholinesterase inhibition. Combined with
a steady decline in the percentage of acetylcholinesterase activity along its sigmoidal dosageresponse curve, the inhibition range for Ganoderma applanatum M5E1 could potentially be
extrapolated from 0.375 mg/mL up to 10.0 mg/mL (Figure 33).
In summary, the estimated acetylcholinesterase inhibition ranges for all eight mushroom
extracts exhibiting acetylcholinesterase inhibition were ranked in the following order from the
smallest to the largest range: Ganoderma applanatum M5E2 with a projected range from 2.75
123

mg/mL to 3.5 mg/mL, Hericium erinaceus 288E3 with a projected range from 0.75 mg/mL to 3.0
mg/mL, Amanita muscaria 243E1 and Agaricus subrufescens 295E1 and Agaricus subrufescens
295E2 with a projected range from 0.375 mg/mL to 4.0 mg/mL, Hericium erinaceus 288E1 with
a projected range from 0.0937 mg/mL to 5.0 mg/mL, Inonotus obliquus 285E1 with a projected
range from 0.0937 mg/mL to 6.0 mg/mL, and Ganoderma applanatum M5E1 with a projected
range from 0.375 mg/mL to 10.0 mg/mL (Figures 33-40).
Regarding the mushroom extracts that did not inhibit acetylcholinesterase activity, their
presence resulted in a higher percentage of acetylcholinesterase activity compared to the control
wells lacking these extracts (Table 9 and Figures 41-50).

Mushroom Species
Agaricus subrufescens
Amanita muscaria
Ganoderma applanatum
Inonotus obliquus
Phellinus igniarius
Phellinus pini
Pleurotus ostreatus

Extract
295E3
243E2
243E3
M5E3
285E2
285E3
PIE1
PIE2
PIE3
91E1
91E2
91E3
68E2

Solvent Used to Extract
Water
50% Methanol
Water
Water
50% Methanol
Water
80% Ethanol
50% Methanol
Water
80% Ethanol
50% Methanol
Water
50% Methanol

Table 9. Summary of Mushroom Extracts that Were Negative for Acetylcholinesterase Activity
Inhibition.
A list of mushroom extracts that did not inhibit the in vitro acetylcholinesterase activity in this study.

For some of these extracts, the increase in acetylcholinesterase activity compared to the
standard controls was moderate, with a small standard deviation that was consistent across the
extract concentration range, increasing successively by factors of two from 0.1875 mg/mL to 3.0
mg/mL. These extracts included Amanita muscaria 243E2, Amanita muscaria 243E3, Agaricus
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subrufescens 295E3, Inonotus obliquus 285E2, Inonotus obliquus 285E3, and Pleurotus ostreatus
68E2 (Figures 41-46).

Figure 41. The Effect of Amanita muscaria 243E2 on Acetylcholinesterase Activity at High Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Amanita muscaria 243E2 is a mushroom extract that was used to test for an inhibitory effect
on acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity.

125

Figure 42. The Effect of Amanita muscaria 243E3 on Acetylcholinesterase Activity at High Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Amanita muscaria 243E3 is a mushroom extract that was used to test for an inhibitory effect
on acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity.

126

Figure 43. The Effect of Agaricus subrufescens 295E3 on Acetylcholinesterase Activity at High
Extract Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Agaricus subrufescens 295E3 is a mushroom extract that was used to test for an inhibitory
effect on acetylcholinesterase activity. Each data point on the graph represented the mean percentage value
of acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity.

127

Figure 44. The Effect of Inonotus obliquus 285E2 on Acetylcholinesterase Activity at High Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Inonotus obliquus 285E2 is a mushroom extract that was used to test for an inhibitory effect
on acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity.

128

Figure 45. The Effect of Inonotus obliquus 285E3 on Acetylcholinesterase Activity at High Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Inonotus obliquus 285E3 is a mushroom extract that was used to test for an inhibitory effect
on acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity.

129

Figure 46. The Effect of Pleurotus ostreatus 68E2 on Acetylcholinesterase Activity at High Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Pleurotus ostreatus 68E2 is a mushroom extract that was used to test for an inhibitory effect
on acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity.

However, for some other extracts, the increase in acetylcholinesterase activity was
abnormally high, accompanied by a large standard deviation that fluctuated tremendously across
the same extract concentration range (Figures 47-50).

130

Figure 47. The Effect of Phellinus pini 91E1 on Acetylcholinesterase Activity at High Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Phellinus pini 91E1 is a mushroom extract that was used to test for an inhibitory effect on
acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity.

131

Figure 48. The Effect of Phellinus igniarius PIE1 on Acetylcholinesterase Activity at High Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Phellinus igniarius PIE1 is a mushroom extract that was used to test for an inhibitory effect
on acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity.

Notably, Phellinus pini 91E2 and Phellinus pini 91E3 displayed instability that even led to
a much lower percentage of acetylcholinesterase activity at higher extract concentrations compared
to the standard controls. This peculiar trend, where the acetylcholinesterase activity was much
higher at lower extract concentrations, and then rapidly decreased to a significantly lower
percentage of enzyme activity at higher extract concentrations, raised questions about its
credibility as a true acetylcholinesterase inhibition event (Figures 49 and 50). As to what in these
extracts may induce this strange anomaly remained largely undetermined.

132

Figure 49. The Effect of Phellinus pini 91E2 on Acetylcholinesterase Activity at High Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Phellinus pini 91E2 is a mushroom extract that was used to test for an inhibitory effect on
acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity.

133

Figure 50. The Effect of Phellinus pini 91E3 on Acetylcholinesterase Activity at High Extract
Concentrations.
The shift in percentage of in vitro acetylcholinesterase activity when the enzyme was separately exposed to
increasing concentrations of mushroom extracts, ranging from 0.1875 mg/mL to 3.0 mg/mL, over a 30-min
time span. Ganoderma applanatum M5E1 is a mushroom extract used as a controlled reference for
comparison. Phellinus pini 91E3 is a mushroom extract that was used to test for an inhibitory effect on
acetylcholinesterase activity. Each data point on the graph represented the mean percentage value of
acetylcholinesterase activity recorded for the treatment with one specific mushroom extract, and at one
specific extract concentration. Each mean percentage value of acetylcholinesterase activity was averaged
from two experimental replicates, corresponding to six technical replicates (n = 6). Each set of upper and
lower bars surrounding each data point on the graph represented the standard deviation of a mean percentage
value of acetylcholinesterase activity.

Due to the abnormal trend observed with Phellinus pini 91E2 and Phellinus pini 91E3, it
was challenging to definitively determine whether these extracts had a genuine
acetylcholinesterase inhibition effect. Therefore, further future experimentation through
alternative biochemical analysis methods would be necessary to provide more conclusive answers
to this mystery.

134

Chapter 4
General Discussion
4.1

General Remarks
This study successfully achieved its original research objectives within a reasonable

timeframe yielding some new findings. The primary research objectives of this study were to
investigate four small molecules and mushroom extracts from eight different mushroom species
to screen for their potential inhibitory effects on acetylcholinesterase activity in vitro. Throughout
the experimentation process, none of the small molecules exhibited inhibition, but eight mushroom
extracts were identified as inhibitors of acetylcholinesterase activity. Notably, the strength of
inhibition on the enzyme activity was substantial, with approximate maximum inhibition strengths
ranging from 39% to 91%. Furthermore, these significant inhibitory effects were achieved with
relatively small quantities, as indicated by IC50 values ranging from 4.0 mg/mL to slightly less
than 1.0 mg/mL. The eight mushroom extracts that demonstrated inhibition were as follows:
Agaricus subrufescens 295E1, Agaricus subrufescens 295E2, Amanita muscaria 243E1,
Ganoderma applanatum M5E1, Ganoderma applanatum M5E2, Hericium erinaceus 288E1,
Hericium erinaceus 288E3, and Inonotus obliquus 285E1.
4.2

Establishment of the Acetylcholinesterase Assay
In order to successfully perform the screening and achieve the presented results, the

acetylcholinesterase assay was used. The assay was designed based on five main components:
sodium phosphate buffer, Electrophorus electricus acetylcholinesterase, acetylthiocholine iodide,
a small molecule or a mushroom extract as the test sample, and DTNB. Together, they allow the
assay to specifically quantify the level of enzyme activity of acetylcholinesterase through the
absorbance of TNB2-, the reduced counterpart of DTNB after its reduction by one of the byproducts

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produced from the hydrolysis of acetylthiocholine iodide by acetylcholinesterase. In the order of
the reaction sequence, acetylcholinesterase first hydrolyzes acetylthiocholine into acetic acid and
thiocholine. Next, thiocholine acts as a reducing agent and reduces DTNB into TNB2- by severing
the disulfide linkage of DTNB. As a compound reflecting one shade of yellow, a colorimetric
detection of TNB2- can be translated into absorbance at the wavelength of 412 nm. Thus, the more
TNB2- there is, the greater the absorbance will be, reflecting a higher level of acetylcholinesterase
activity, and vice versa.
First, the sodium phosphate buffer was made to 0.1 M at pH 8.0. The pH was adjusted to
8.0 to minimize the protonation of TNB2-, as the ionized form is the only form capable of reflecting
the yellow color. It was also adjusted to maintain the thiocholine in its deprotonated form, allowing
it to readily attack the disulfide linkage of DTNB by the free thiol group. Furthermore, the pH falls
within the optimal range for Electrophorus electricus acetylcholinesterase activity. The choice of
a 0.1 M concentration was made to maintain a balanced ionic strength that does not interfere with
the π-cation interaction between the positively charged nitrogen atom of acetylthiocholine and the
electron-rich aromatic rings of the side chain of tryptophan residues within the peripheral anionic
site of acetylcholinesterase in the case of excessively high ionic strength, nor cause aggregation of
the side chains of charged residues of the enzyme in the case of excessively low ionic strength.
Secondly, Electrophorus electricus acetylcholinesterase was selected due to its costeffectiveness compared to the Homo sapiens orthologue. There was not much concern with the
enzyme performance based on the chosen orthologue, as the amino acid sequence of its active site
is highly conserved across species, giving rise to activity redundancy. Thirdly, the
acetylthiocholine iodide was chosen for its special free thiol group that readily reduces DTNB
once hydrolyzed by acetylcholinesterase. Lastly, DTNB was selected as a reagent for thiol

136

quantification because it does not react non-specifically with non-thiols, and the TNB2- absorbance
outside of the UV range eliminates the need for expensive experimental tools for UV detection.
Once the assay design was established and all necessary materials were prepared, the assay
was initially tested to ensure proper functionality without any test sample added. Then, the assay
was tested with five different concentrations of acetylcholinesterase to determine one appropriate
concentration that could produce a sufficiently steep linear increase in TNB2- absorbance over time,
making it easier to detect the inhibition of acetylcholinesterase activity in the presence of a
potential novel inhibitor. Following this, the assay was tested with known inhibitors of
acetylcholinesterase to verify that inhibition would occur in the presence of a true inhibitor.
Simultaneously, the results from the experiments with the known inhibitors were used as a point
of reference for comparing the acetylcholinesterase inhibition strength with other potential novel
inhibitors that may be discovered in this study. Only after the results from these four testing
procedures were satisfactory that it was officially qualified for testing with the small molecules
and mushroom extracts.
4.3

The Effects of the Selective Small Molecules on Acetylcholinesterase Activity
Previously, SPOPP, 534_1(6), and 534_2(7) were known for their in vitro anticancer

properties. Using the web-based ligand-centric computational program for small molecule target
prediction called MolTarPred, acetylcholinesterase was identified as the top target for binding by
SPOPP and NP6A. Since dispiropyrrolizidine derivatives have molecular structures closely related
to SPOPP and NP6A, it was predicted that 534_1(6) and 534_2(7) may also bind to
acetylcholinesterase. However, this study firmly established that none of these small molecules
possesses an inhibitory effect against acetylcholinesterase activity.

137

Although the results were negative, they provided a confident conclusion that these small
molecules do not exhibit inhibitory effects on acetylcholinesterase activity. These findings also
raised questions about the reliability of small molecule target prediction programs, such as
MolTarPred. These programs should generally only be used as supportive tools to assist in
experimental design, but should not be solely relied upon to draw decisive conclusions in research
studies without supporting experimental evidences, as demonstrated in this study.
4.4

The Effects of the Selective Mushroom Extracts on Acetylcholinesterase Activity
Contrary to the small molecules, this study extensively established, and was the first to

report eight novel mushroom extracts with significant inhibition of acetylcholinesterase activity.
These extracts exhibited a maximum inhibition strength ranging from 39% to 91% at relatively
low concentrations, with IC50 values ranging from 4.0 mg/mL to slightly less than 1.0 mg/mL. In
comparison to the IC50 value range of the example mushroom species reported for in vitro
inhibition of acetylcholinesterase activity in Table 2, the IC50 values of mushroom extracts shown
to inhibit acetylcholinesterase activity in this study were relatively low. This indicated that their
inhibition strength on acetylcholinesterase activity was relatively moderate to strong. However,
until further isolation and purification of substances from these extracts were done, it should be
carefully noted that these IC50 values represented an extract as a whole, rather than an individual
small molecule. Therefore, an effective comparison between the efficacy of acetylcholinesterase
inhibition of an extract versus that of a purified molecule would deem to be difficult to achieve.
Supposedly there are a few existing novel small molecules responsible for the inhibitory
effect seen in these extracts, whether or not the potency of these small molecules falls within the
average IC50 range from as low as 0.02 μM to as high as 25 μM observed for acetylcholinesterase
inhibitors,25 or even falling below the lower end of the average range, and whether or not they are

138

any more effective than the current inhibitors in any way, remained tentative for further
investigation in future in vitro and in vivo studies.
As any biological extract is a complex mixture of numerous known and unknown small
molecules, achieving consistent inhibition of acetylcholinesterase activity across a wide
concentration range in six technical replicates suggests that there may be one or multiple inhibitors
present in each extract, possibly in significant quantities. Alternatively, there could be one or more
highly

abundant

components

that

suppress

one

or

multiple

enhancers

promoting

acetylcholinesterase activity. If these findings are consistently replicated in future studies, further
analyses of the content within each of these eight mushroom extracts could be conducted to
identify the specific small molecule or group of small molecules responsible for the observed
inhibition of acetylcholinesterase activity in this study.
One potential approach for future analyses of the extracts could be through the application
of chromatography, one of the well-established biochemical laboratory techniques that the Lee lab
at the University of Northern British Columbia is proficient at. With various types of
chromatography to be utilized, one way to approach the analyses could involve a combination of
size-exclusion chromatography and high-performance liquid chromatography. The process would
begin with size-exclusion chromatography to fractionate the content of each extract based on size.
Subsequently, each fraction collected could be further subdivided into smaller sub-fractions using
high-performance liquid chromatography, employing a gradient mobile phase to optimize
separation based on solubility. Rather than relying on a single solvent, a gradient comprising a
mixture of two or more solvents could be used to continuously alter the solubility condition over
time in a dynamic fashion. This approach would provide a limitless range of solubility conditions
confined within the gradient for the small molecules within each fraction to dissolve into. Finally,

139

each collected sub-fraction containing a single small molecule could be separately tested in vitro
to assess its impact on acetylcholinesterase activity. If the molecule demonstrates significant
inhibitory activity, further analyses, including kinetics and structural analysis could be further
conducted to elucidate its mode of inhibition on the enzyme activity and determine its chemical
identity.
In addition to the points mentioned earlier, the new findings provided further evidences
and reaffirmed the therapeutic potential of several mushroom species with medicinal properties to
combat against neurodegenerative diseases. These properties have been well-documented in
numerous past and recent research studies, particularly in the cases of Hericium erinaceus and
Inonotus obliquus. Previous research has mainly focused on the 80% methanol,51 and 70% ethanol
extracts of the two species, which contain substances demonstrating strong antioxidative
properties.52 The antioxidant properties exhibited by these extracts were mostly found targeting
and upregulating the nuclear factor erythroid 2-related factor 2 signaling pathway. This pathway
assists cells in resisting the harmful effects of reactive oxygen species during the cells’ response
to oxidative stress. This role of antioxidative properties is crucial in the context of
neurodegeneration, as oxidative stress-induced apoptosis, leading to the loss of neurons, is another
common pathological event in various neurodegenerative diseases.51 Nonetheless, given that
acetylcholinesterase specializes in the regulation of cholinergic neurotransmission within neurons,
as opposed to the more general cellular response to oxidative stress observed across all cell types,
the new findings presented in this study possibly marked the first emphasis on the therapeutic role
of Hericium erinaceus and Inonotus obliquus, directed towards an enzyme with a highly localized
function exclusive to a distinct cell type, playing a more direct and substantial role in the
development of neurodegenerative diseases.

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As for Agaricus subrufescens, Amanita muscaria, and Ganoderma applanatum, it appears
that there have been no prior reports nor extensive research conducted on their potential roles in
the treatment of neurodegenerative diseases. Any specific biological targets that these mushrooms
may inhibit in this context have also not been reported. Therefore, the findings presented in this
study were the first to highlight and comprehensively establish the novel medicinal properties of
these three mushrooms, particularly their targeting of acetylcholinesterase as a potential
therapeutic approach for neurodegenerative diseases.
Beside being the first to report the acetylcholinesterase inhibitory property possessed by a
few mushroom species, these findings also contained important observations worth mentioning as
well. In consistent with the 80% methanol and 70% ethanol extracts of Hericium erinaceus and
Inonotus obliquus reported to demonstrate strong oxidative properties in previous research, almost
all mushroom extracts reported to inhibit acetylcholinesterase activity in this study were also
extracted using methanol and ethanol. The only difference was the percentage of methanol and
ethanol used to extract, which were 50% methanol for the E2 extracts and 80% ethanol for the E1
extracts. With respect to the E1 and E2 mushroom extracts used in this study, the substances
contained within these extracts were previously predicted to be small molecules, in contrast to the
E3 extracts that were predicted to contain macromolecules such as polysaccharides.50 In
comparison to known acetylcholinesterase inhibitors such as rivastigmine, galanthamine, and
donepezil, the small molecules contained within the E1 extracts may likely have a similar solubility,
as all are soluble in ethanol to various degrees. Taken altogether, these observations suggested that
mushroom extracts derived from methanol and ethanol extractions may contain more substances
that possess an inhibitory property on acetylcholinesterase activity. Nevertheless, until these
substances are isolated, purified, and further analyzed according to the approach suggested above,

141

their identity and the degree to which they inhibit acetylcholinesterase activity in comparison to
the current acetylcholinesterase inhibitors remains tentative.
Regarding the mushroom extracts that enhanced acetylcholinesterase activity, this elevated
enzyme activity level was of particular interest because it may indicate neurotoxicity in the context
of neurodegeneration. For instance, Melo et al. (2003) conducted an in vitro study demonstrating
how amyloid plaques contribute to an increased acetylcholinesterase activity in Alzheimer’s
disease using a retinal cell-based model. Their study showed that cells incubated with amyloid
beta-peptides alone for 24 h exhibited an enhanced enzyme activity level beyond that observed in
the control group without amyloid beta-peptides. This enhanced level of enzyme activity was
consistently observed to be between 120% to 130%, similar to the moderate increase in the
percentage of acetylcholinesterase activity seen in response to the individual treatments with the
following nine mushroom extracts in this study: Amanita muscaria 243E2, Amanita muscaria
243E3, Agaricus subrufescens 295E3, Ganoderma applanatum M5E3, Inonotus obliquus 285E2,
Inonotus obliquus 285E3, Phellinus igniarius PIE2, Phellinus igniarius PIE3, and Pleurotus
ostreatus 68E2.53
Most notably, cases involving extracts from natural products other than mushrooms
enhancing acetylcholinesterase activity have also been documented.54 Unlike the eight mushroom
extracts that displayed a significant inhibitory effect on the acetylcholinesterase activity, these
particular extracts were hypothesized to potentially contain enhancers of acetylcholinesterase.
These enhancers could act as toxins exacerbating the detrimental effects associated with
neurodegeneration.
Likewise, a similar reasoning could be inferred for the following remaining four mushroom
extracts: Phellinus igniarius PIE1, Phellinus pini 91E1, Phellinus pini 91E2, and Phellinus pini

142

91E3. The only more surprising exception was that these extracts not only induced an unusually
high percentage of acetylcholinesterase activity, but also exhibited a moderate to drastic decline
in enzyme activity as the extract concentration increased progressively from 0.1875 mg/mL to 3.0
mg/mL. As peculiar as the trend was, it was not unique and have been observed in several other
related studies. For instance, Akbari et al. (2022) reported an in vivo study on how vitamin B12
administration prevents ethanol-induced learning and memory impairment. A comparative
analysis showed the hippocampal acetylcholinesterase activity of mice fed with water containing
20% ethanol throughout their adolescence intensified to over 200% of the normal enzyme activity
level observed in the untreated group. On top of the ethanol treatment, further administration of
vitamin B12, starting with a dosage from 0.5 mg/kg to 1.5 mg/kg in the murine hippocampus
already displaying intensified hippocampal acetylcholinesterase activity level induced by 20%
ethanol, and subsequently, administration of vitamin B12 alone at a dosage of 1.5 mg/kg to the
murine hippocampus with normal hippocampal acetylcholinesterase activity level, gradually
mitigated the level of hippocampal acetylcholinesterase activity, to the extension where the
enzyme activity level was reduced to 60% of the normal enzyme activity level observed in the
control with no treatment of 20% ethanol and vitamin B12.55
Based on the observed pattern in the in vivo study, it was hypothesized that the peculiar
pattern of acetylcholinesterase activity in response to the increasing dosages of either the Phellinus
igniarius PIE1, Phellinus pini 91E1, Phellinus pini 91E2, or Phellinus pini 91E3 extract may also
result from a similar circumstance. It was hypothesized that within each of these four extracts,
there may be one or more acetylcholinesterase enhancers in extremely great abundance to the point
of saturation, making their enhancing effects on acetylcholinesterase activity nearly completely
dosage-independent. Conversely, within each of these same extracts again, there may be one or

143

more acetylcholinesterase inhibitors in scarcity compared to the enhancers, making their inhibitory
effects on acetylcholinesterase activity lean more towards being dosage-dependent instead. Then
at some point forward, there may come a point where the concentration of the inhibitors may
become sufficient to reverse the enhancing effects of the enhancers on acetylcholinesterase activity,
leading to a drastic decline after reaching elevated levels beyond the norm. This hypothesis was
reminiscent of the peculiar response of acetylcholinesterase activity to the increasing dosages of
these four extracts.
Considering both hypotheses above, the enhanced acetylcholinesterase activity observed
in response to the separate treatments with Amanita muscaria 243E2, Amanita muscaria 243E3,
Agaricus subrufescens 295E3, Ganoderma applanatum M5E3, Inonotus obliquus 285E2, Inonotus
obliquus 285E3, Phellinus igniarius PIE1, Phellinus igniarius PIE2, Phellinus igniarius PIE3,
Phellinus pini 91E1, Phellinus pini 91E2, Phellinus pini 91E3, and Pleurotus ostreatus 68E2 all
pointed towards the existence of one or more acetylcholinesterase enhancers in extreme abundance
within each of these thirteen extracts. If this holds true, then there should also exist an extremely
narrow concentration range between zero and the point of saturation for the enhancers, where their
enhancing effects on acetylcholinesterase activity briefly become dosage-dependent. If this is the
case, the extremely narrow concentration range should exist somewhere below 0.1875 mg/mL.
Further testing was conducted under the same experiment setup, extending into an ultralow concentration range from 9.37 × 10-6 mg/mL to 9.37 × 10-2 mg/mL, specifically for the
Agaricus subrufescens 295E3 and Inonotus obliquus 285E3 extracts. The results revealed a
concentration range between 0.01 mg/mL and 0.1 mg/mL where the enhancing effects of these
two extracts on acetylcholinesterase activity became dosage-dependent. Thus, to some extent, the
hypotheses were supported, although the existence of one or more acetylcholinesterase enhancers

144

in extreme abundance may not hold true for the remaining eleven extracts. Consequently, these
test results should be considered preliminary, and further studies would be required to conduct.
Moreover, a bioactivity-guided approach should be employed to isolate compounds with inhibitory
or stimulatory activity on acetylcholinesterase.
Considering that the mushroom extracts enhancing acetylcholinesterase activity to be
detrimental to the process of neurodegeneration, utilizing them in research for the therapeutics of
neurodegenerative diseases may be risky and ineffective. However, it is worth noting that an
enhanced acetylcholinesterase activity level may not necessarily be an undesirable biological
effect in cell types other than neurons. In addition to its major role in neurotransmission regulation,
it has been reported that the acetylcholinesterase enzyme can also act as a mediator promoting cell
proliferation and regeneration. There are substantial evidences of its involvement in a cell’s life
cycle in various studies using invertebrate and vertebrate models, such as sea slugs, octopuses, and
salamanders.
For example, in a study by Fossati et al. (2013) on limb regeneration in octopuses, the
acetylcholinesterase activity was shown to increase and become most active when a small hook
sprouted from an amputated tentacle between day 17 to day 28. Afterwards, the enzyme activity
level gradually dissipated as the full functional structure was completely regrown. Most notably,
acetylcholinesterase was mobilized from the nervous system to participate in the limb regeneration
process.56 This suggests that the process of regeneration is nerve-dependent, and implies that
acetylcholinesterase can serve as a dual mediator with both inhibitory and enhancing effects on
the enzyme that can be beneficial for the development of diverse life functionalities and disease
treatments under the right conditions and circumstances. Therefore, even though deviating from
the original research objectives, the discovery of these acetylcholinesterase activity-enhancing

145

mushroom extracts in this study could still prove to be useful in future studies related to cellular
regeneration.
4.5

Concluding Remarks and Future Directions
In conclusion, this study found that none of the four small molecules inhibited

acetylcholinesterase activity, but eight mushroom extracts did. While the negative results with the
small molecules were not particularly exciting, they still provided valuable additional knowledge
contributing to the understanding of the medicinal properties of dispiropiperazine and
dispiropyrrolizidine derivatives. Moreover, these results paved way for future studies to shift focus
onto exploring other potential medicinal properties of these molecules. Additionally, this study
highlighted the need for caution when using small molecule target prediction programs like
MolTarPred.
Moving forward, this study has made a significant contribution by identifying eight novel
mushroom extracts that showed inhibition of acetylcholinesterase activity. These extracts, from
Hericium erinaceus and Inonotus obliquus, were found to exhibit an inhibitory effect against
acetylcholinesterase activity for the first time. Simultaneously, the study was the first to report
extracts from Agaricus subrufescens, Amanita muscaria, and Ganoderma applanatum as having
potential beneficial medicinal properties against neurodegeneration. Collectively, these extracts
may provide new sources of compounds for potential drug development in the treatment of
neurodegenerative diseases.
Even for the extracts that were found to enhance acetylcholinesterase activity, they may
prove useful as biological tools for understanding tissue development, or as sources of compounds
for drug development to treat other types of diseases. Nonetheless, the discovery of the inhibitory
properties against acetylcholinesterase activity in the mentioned eight novel mushroom extracts in

146

this study served as a reminder of the latent medicinal potentials of mushrooms as medicines for
the treatment of a wide range of diseases. Given the vast number of mushroom species, many of
which contain undiscovered compounds, future studies should be encouraged to conduct further
research in this area.

147

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153

Supplementary Data
Replicate 1
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.216
0.239
0.305
0.354
0.366
0.376
0.398
0.407
0.408
0.404
0.407
0.413
0.421
0.425
0.428
0.431

0.212
0.179
0.169
0.166
0.165
0.170
0.173
0.174
0.176
0.179
0.182
0.184
0.187
0.189
0.192
0.194

0.204
0.206
0.194
0.186
0.181
0.181
0.179
0.179
0.180
0.182
0.184
0.185
0.187
0.189
0.191
0.193

0.161
0.141
0.137
0.143
0.146
0.149
0.152
0.157
0.160
0.163
0.166
0.169
0.172
0.175
0.178
0.181

Replicate 2
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26

0.0

0.009

0.09

0.9

0.502
0.626
0.704
0.744
0.778
0.802
0.814
0.822
0.827
0.834
0.834
0.839
0.843
0.848

0.256
0.258
0.259
0.260
0.261
0.261
0.260
0.260
0.258
0.256
0.255
0.253
0.252
0.250

0.231
0.235
0.190
0.181
0.203
0.260
0.353
0.531
0.631
0.654
0.666
0.670
0.665
0.670

0.225
0.234
0.225
0.212
0.206
0.205
0.206
0.208
0.211
0.214
0.217
0.219
0.222
0.224

154

28
30

0.849
0.852

0.249
0.248

0.673
0.671

0.227
0.229

Replicate 3
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.200
0.186
0.189
0.191
0.192
0.192
0.193
0.194
0.194
0.194
0.196
0.198
0.201
0.204
0.209
0.215

0.227
0.227
0.222
0.218
0.214
0.211
0.208
0.206
0.204
0.203
0.203
0.203
0.204
0.205
0.206
0.208

0.221
0.218
0.190
0.148
0.145
0.161
0.175
0.205
0.250
0.507
0.704
0.604
0.549
0.494
0.490
0.489

0.136
0.146
0.154
0.160
0.164
0.167
0.171
0.175
0.179
0.183
0.186
0.189
0.192
0.195
0.198
0.201

Supplementary Table 1. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 2.
The test aimed to assess the assay’s ability to generate appropriate absorbance readings in response to
increasing concentrations of acetylcholinesterase. It analyzed the rate of TNB2- absorbance increase over a
30-min time span as acetylcholinesterase increased. The lower panels of graphs depicted the changes in
TNB2- absorbance in response to increasing concentrations of acetylcholinesterase over the course of 30
mins. The experiment was repeated three times (n = 3).

155

Supplementary Figure 1. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 2.
A photo of the microtiter plate used for the run was also attached to illustrate the unexpected results.

156

Replicate 1
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.172
0.181
0.395
0.489
0.503
0.514
0.520
0.542
0.571
0.583
0.591
0.596
0.602
0.606
0.607
0.605

0.174
0.178
0.182
0.187
0.191
0.196
0.201
0.206
0.211
0.216
0.220
0.225
0.229
0.233
0.235
0.238

0.177
0.220
0.245
0.246
0.291
0.386
0.455
0.479
0.493
0.499
0.525
0.536
0.533
0.554
0.645
0.652

0.490
0.984
1.375
1.566
1.725
1.881
2.044
2.157
2.266
2.346
2.404
2.454
2.502
2.531
2.572
2.492

Replicate 2
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.259
0.317
0.290
0.388
0.424
0.448
0.469
0.491
0.548
0.554
0.557
0.564
0.567
0.574
0.576
0.579

0.146
0.157
0.172
0.176
0.175
0.177
0.179
0.182
0.183
0.188
0.201
0.238
0.298
0.392
0.444
0.629

0.193
0.223
0.262
0.282
0.281
0.281
0.289
0.300
0.309
0.329
0.354
0.384
0.418
0.516
0.637
0.759

0.472
1.038
1.423
1.679
1.809
1.895
2.023
2.063
2.127
2.258
2.421
2.605
2.796
2.924
3.226
OVRFLW

157

Replicate 3
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.158
0.161
0.165
0.169
0.172
0.175
0.178
0.18
0.183
0.186
0.189
0.193
0.197
0.200
0.205
0.209

0.182
0.182
0.184
0.187
0.190
0.192
0.195
0.197
0.200
0.202
0.205
0.208
0.212
0.216
0.220
0.224

0.155
0.172
0.190
0.207
0.225
0.242
0.260
0.277
0.294
0.311
0.328
0.345
0.361
0.377
0.394
0.411

0.394
0.932
1.338
1.604
1.788
1.926
2.048
2.148
2.222
2.298
2.362
2.428
2.479
2.509
2.525
2.551

Supplementary Table 2. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 3.
The test aimed to assess the assay’s ability to generate appropriate absorbance readings in response to
increasing concentrations of acetylcholinesterase. It analyzed the rate of TNB2- absorbance increase over a
30-min time span as acetylcholinesterase concentration increased. The term “OVRFLW”, or “Overflow”,
denoted absorbance readings that exceeded the upper limit of the Synergy 2 plate reader machine’s
detection range and could not be measured. Black highlighted sections in the tables represented unusual
data where absorbance readings resulting from treatments with lower enzyme concentrations were higher
than those from treatments with higher enzyme concentrations. The lower panels of graphs depicted the
changes in TNB2- absorbance in response to increasing concentrations of acetylcholinesterase over the
course of 30 mins. The experiment was repeated three times (n = 3).

158

Replicate 1
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.578
0.645
0.677
0.679
0.679
0.691
0.711
0.719
0.730
0.730
0.735
0.737
0.740
0.738
0.738
0.734

0.319
0.320
0.319
0.319
0.318
0.318
0.317
0.317
0.317
0.317
0.317
0.317
0.317
0.317
0.318
0.319

0.296
0.327
0.357
0.385
0.412
0.437
0.463
0.488
0.512
0.535
0.558
0.580
0.602
0.625
0.649
0.673

1.010
1.431
1.745
1.937
2.055
2.155
2.216
2.248
2.274
2.295
2.276
2.279
2.297
2.314
2.353
2.363

Replicate 2
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.235
0.227
0.244
0.267
0.287
0.312
0.341
0.369
0.395
0.415
0.435
0.447
0.464
0.474
0.492
0.502

0.302
0.312
0.323
0.329
0.333
0.336
0.337
0.337
0.337
0.337
0.336
0.337
0.336
0.336
0.336
0.337

0.265
0.291
0.313
0.333
0.354
0.375
0.396
0.416
0.437
0.457
0.476
0.496
0.516
0.535
0.555
0.576

0.571
0.810
0.950
1.029
1.093
1.151
1.203
1.256
1.305
1.351
1.392
1.436
1.478
1.520
1.557
1.598

159

Replicate 3
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.238
0.245
0.249
0.255
0.261
0.267
0.272
0.276
0.279
0.282
0.285
0.287
0.290
0.292
0.294
0.296

0.316
0.318
0.318
0.318
0.318
0.318
0.318
0.318
0.318
0.319
0.319
0.320
0.322
0.322
0.323
0.325

0.315
0.344
0.366
0.384
0.401
0.418
0.434
0.452
0.469
0.486
0.504
0.521
0.540
0.558
0.577
0.596

0.298
0.359
0.397
0.432
0.470
0.509
0.548
0.587
0.626
0.666
0.707
0.749
0.796
0.845
0.897
0.952

Supplementary Table 3. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 4.
The test aimed to assess the assay’s ability to generate appropriate absorbance readings in response to
increasing concentrations of acetylcholinesterase. It analyzed the rate of TNB2- absorbance increase over a
30-min time span as acetylcholinesterase concentration increased. Black highlighted sections in the tables
represented unusual data where absorbance readings resulting from treatments with lower enzyme
concentrations were higher than those from treatments with higher enzyme concentrations. The lower
panels of graphs depicted the changes in TNB2- absorbance in response to increasing concentrations of
acetylcholinesterase over the course of 30 mins. The experiment was repeated three times (n = 3).

160

Supplementary Figure 2. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test Run 4.
A photo of the microtiter plate (the third 4x3 set at the bottom within the red box) used for the run was also
attached to illustrate the crystal formation that likely contributed to the higher absorbance readings and the
greater fluctuations along the absorbance readings of the two wells from replicates 1 and 2, which were
both treated with the same enzyme concentration of 0 units/mL.

161

Replicate 1
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.256
0.252
0.240
0.239
0.236
0.241
0.243
0.243
0.245
0.249
0.250
0.253
0.255
0.257
0.259
0.261

0.313
0.287
0.274
0.271
0.274
0.277
0.279
0.282
0.285
0.288
0.290
0.293
0.295
0.297
0.300
0.302

0.315
0.334
0.349
0.368
0.391
0.416
0.441
0.466
0.492
0.517
0.544
0.570
0.597
0.624
0.652
0.680

0.562
1.111
1.472
1.615
1.728
1.831
1.932
2.017
2.094
2.157
2.225
2.285
2.337
2.389
2.437
2.486

Replicate 2
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.221
0.218
0.212
0.211
0.215
0.219
0.223
0.227
0.232
0.234
0.237
0.241
0.245
0.249
0.253
0.256

0.363
0.344
0.328
0.316
0.308
0.301
0.297
0.296
0.294
0.293
0.293
0.294
0.295
0.296
0.298
0.301

0.388
0.388
0.385
0.396
0.410
0.432
0.457
0.481
0.510
0.537
0.566
0.595
0.626
0.658
0.689
0.723

0.660
1.264
1.818
2.201
2.426
2.563
2.663
2.732
2.812
2.846
2.913
2.947
3.021
3.071
3.105
3.175

162

Replicate 3
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.246
0.255
0.242
0.236
0.236
0.235
0.239
0.241
0.243
0.245
0.249
0.253
0.256
0.259
0.262
0.266

0.283
0.282
0.279
0.281
0.282
0.285
0.287
0.290
0.293
0.295
0.297
0.299
0.302
0.304
0.306
0.307

0.298
0.319
0.342
0.363
0.383
0.404
0.424
0.446
0.469
0.492
0.517
0.542
0.567
0.594
0.621
0.649

0.588
1.161
1.660
1.988
2.095
2.188
2.247
2.313
2.365
2.415
2.460
2.496
2.535
2.567
2.581
2.631

Supplementary Table 4. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 5.
The test aimed to assess the assay’s ability to generate appropriate absorbance readings in response to
increasing concentrations of acetylcholinesterase. It analyzed the rate of TNB2- absorbance increase over a
30-min time span as acetylcholinesterase concentration increased. The lower panels of graphs depicted the
changes in TNB2- absorbance in response to increasing concentrations of acetylcholinesterase over the
course of 30 mins. The experiment was repeated three times (n = 3).

163

Supplementary Figure 3. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 5.
A photo of the microtiter plate (ignoring the first column located on the furthest left of the plate, the 4x3
set starting from the second column within the red box) used for the run was also attached to illustrate the
surprising disappearance of the crystal formation after the mixing factor had been considered for.

164

Replicate 1
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.184
0.184
0.185
0.187
0.188
0.190
0.193
0.194
0.197
0.200
0.201
0.202
0.205
0.209
0.213
0.210

0.245
0.248
0.249
0.250
0.253
0.254
0.255
0.257
0.259
0.260
0.262
0.263
0.265
0.267
0.268
0.270

0.240
0.246
0.252
0.257
0.261
0.266
0.272
0.276
0.281
0.286
0.290
0.295
0.299
0.305
0.308
0.313

0.875
1.469
1.703
1.901
2.116
2.244
2.321
2.420
2.557
2.632
2.727
2.798
2.857
2.903
2.945
2.979

Replicate 2
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.226
0.215
0.208
0.220
0.217
0.224
0.223
0.225
0.231
0.230
0.238
0.238
0.238
0.243
0.246
0.249

0.328
0.354
0.374
0.387
0.388
0.400
0.411
0.424
0.431
0.438
0.441
0.444
0.447
0.450
0.453
0.455

0.262
0.266
0.270
0.275
0.280
0.286
0.291
0.297
0.302
0.307
0.312
0.317
0.323
0.328
0.333
0.338

0.907
1.443
1.556
1.725
1.793
1.863
1.954
2.043
2.083
2.166
2.182
2.258
2.284
2.327
2.366
2.403

165

Replicate 3
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.239
0.242
0.241
0.244
0.246
0.248
0.250
0.252
0.254
0.256
0.258
0.261
0.262
0.265
0.267
0.269

0.241
0.247
0.250
0.273
0.375
0.543
0.710
0.781
0.799
0.819
0.840
0.857
0.867
0.878
0.882
0.888

0.247
0.252
0.263
0.270
0.277
0.282
0.288
0.301
0.377
0.558
0.575
0.593
0.582
0.581
0.587
0.598

0.890
1.587
1.976
2.513
3.007
3.723
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

Supplementary Table 5. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 6.
The test aimed to assess the assay’s ability to generate appropriate absorbance readings in response to
increasing concentrations of acetylcholinesterase. It analyzed the rate of TNB2- absorbance increase over a
30-min time span as acetylcholinesterase concentration increased. The term “OVRFLW”, or “Overflow”,
denoted absorbance readings that exceeded the upper limit of the Synergy 2 plate reader machine’s
detection range and could not be measured. Black highlighted sections in the tables represented unusual
data where absorbance readings resulting from treatments with lower enzyme concentrations were higher
than those from treatments with higher enzyme concentrations. The lower panels of graphs depicted the
changes in TNB2- absorbance in response to increasing concentrations of acetylcholinesterase over the
course of 30 mins. The experiment was repeated three times (n = 3).

166

Supplementary Figure 4. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 6.
A photo of the microtiter plate used for the run was also attached to illustrate the irreproducibility of the
results obtained from run 5, shown by the recurrence of the crystal formation in many wells.

167

Replicate 1
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.078
0.080
0.081
0.082
0.083
0.083
0.084
0.085
0.086
0.087
0.088
0.088
0.090
0.090
0.091
0.092

0.082
0.083
0.084
0.085
0.087
0.088
0.089
0.090
0.092
0.094
0.095
0.097
0.098
0.099
0.101
0.102

0.101
0.110
0.117
0.125
0.133
0.140
0.147
0.154
0.161
0.168
0.175
0.182
0.188
0.195
0.201
0.207

0.329
0.406
0.460
0.489
0.513
0.526
0.533
0.538
0.539
0.540
0.539
0.537
0.536
0.534
0.533
0.530

Replicate 2
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.082
0.085
0.086
0.085
0.086
0.088
0.088
0.088
0.090
0.090
0.091
0.094
0.094
0.096
0.096
0.097

0.084
0.086
0.087
0.088
0.088
0.090
0.091
0.092
0.094
0.095
0.096
0.097
0.098
0.100
0.101
0.101

0.101
0.108
0.115
0.123
0.131
0.137
0.144
0.151
0.157
0.165
0.171
0.177
0.183
0.189
0.195
0.200

0.332
0.422
0.479
0.512
0.534
0.551
0.556
0.558
0.563
0.561
0.558
0.558
0.553
0.552
0.548
0.546

168

Replicate 3
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.094
0.095
0.095
0.096
0.097
0.098
0.099
0.099
0.100
0.101
0.102
0.103
0.104
0.104
0.106
0.106

0.084
0.085
0.086
0.087
0.088
0.090
0.091
0.092
0.093
0.094
0.096
0.097
0.098
0.099
0.100
0.101

0.111
0.123
0.136
0.147
0.160
0.172
0.182
0.193
0.203
0.214
0.222
0.232
0.240
0.249
0.258
0.265

0.350
0.431
0.481
0.514
0.534
0.547
0.551
0.551
0.553
0.555
0.554
0.552
0.550
0.548
0.546
0.544

Supplementary Table 6. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 7.
The test aimed to assess the assay’s ability to generate appropriate absorbance readings in response to
increasing concentrations of acetylcholinesterase. It analyzed the rate of TNB2- absorbance increase over a
30-min time span as acetylcholinesterase concentration increased. Black highlighted sections in the tables
represented unusual data where absorbance readings resulting from treatments with lower enzyme
concentrations were higher than those from treatments with higher enzyme concentrations. The lower
panels of graphs depicted the changes in TNB2- absorbance in response to increasing concentrations of
acetylcholinesterase over the course of 30 mins. The experiment was repeated three times (n = 3).

169

Supplementary Figure 5. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 7.
A photo of the microtiter plate used for the run was also attached to illustrate the fair reproducibility of the
results obtained from run 5 when treated with the same amount but at a diluted concentration of
acetylthiocholine iodide by a factor of 10, as indicated by the disappearance of the crystal formation in all
wells.

170

Replicate 1
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.131
0.132
0.133
0.134
0.137
0.138
0.139
0.141
0.142
0.144
0.146
0.147
0.149
0.151
0.153
0.155

0.159
0.160
0.164
0.166
0.168
0.170
0.172
0.174
0.177
0.179
0.181
0.183
0.185
0.187
0.189
0.191

0.192
0.218
0.245
0.271
0.298
0.325
0.352
0.378
0.404
0.429
0.454
0.480
0.505
0.531
0.557
0.583

0.618
0.823
0.918
1.024
1.108
1.140
1.182
1.253
1.296
1.333
1.373
1.399
1.418
1.446
1.462
1.483

Replicate 2
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.133
0.133
0.134
0.135
0.136
0.139
0.141
0.142
0.145
0.147
0.148
0.151
0.153
0.155
0.158
0.160

0.153
0.157
0.160
0.163
0.167
0.170
0.173
0.176
0.179
0.183
0.186
0.189
0.193
0.196
0.199
0.202

0.208
0.238
0.267
0.293
0.322
0.349
0.377
0.403
0.428
0.456
0.480
0.506
0.531
0.557
0.582
0.607

0.727
1.168
1.465
1.573
1.637
1.667
1.759
1.810
1.818
1.850
1.897
1.896
1.913
1.927
1.923
1.938

171

Replicate 3
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.139
0.140
0.142
0.144
0.147
0.148
0.151
0.153
0.155
0.157
0.160
0.161
0.163
0.165
0.167
0.169

0.143
0.146
0.149
0.152
0.153
0.158
0.160
0.163
0.166
0.170
0.173
0.176
0.180
0.183
0.186
0.189

0.189
0.213
0.236
0.261
0.284
0.306
0.329
0.351
0.374
0.396
0.418
0.440
0.462
0.483
0.505
0.527

0.627
0.798
0.896
1.008
1.075
1.158
1.196
1.252
1.305
1.338
1.381
1.383
1.413
1.443
1.466
1.498

Supplementary Table 7. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 8.
The test aimed to assess the assay’s ability to generate appropriate absorbance readings in response to
increasing concentrations of acetylcholinesterase. It analyzed the rate of TNB2- absorbance increase over a
30-min time span as acetylcholinesterase concentration increased. The lower panels of graphs depicted the
changes in TNB2- absorbance in response to increasing concentrations of acetylcholinesterase over the
course of 30 mins. The experiment was repeated three times (n = 3).

172

Supplementary Figure 6. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 8.
A photo of the microtiter plate used for the run was also attached to illustrate the fair reproducibility of the
results obtained from run 5 when treated with the same amount but at a diluted concentration of
acetylthiocholine iodide by a factor of 2. Even though the crystal formation recurred in a few wells, the
overall absorbance data was stable and replicable to what was observed in run 5.

173

Replicate 1
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.138
0.139
0.139
0.139
0.137
0.138
0.143
0.143
0.142
0.143
0.143
0.143
0.144
0.144
0.145
0.145

0.120
0.120
0.121
0.123
0.124
0.125
0.126
0.127
0.128
0.128
0.129
0.130
0.131
0.132
0.133
0.134

0.181
0.206
0.233
0.260
0.285
0.310
0.334
0.362
0.387
0.413
0.441
0.471
0.493
0.502
0.522
0.539

1.372
2.032
2.569
3.025
3.307
3.555
3.509
3.629
3.620
3.701
3.746
3.769
3.770
3.831
3.735
3.751

Replicate 2
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.141
0.141
0.141
0.141
0.141
0.141
0.139
0.138
0.141
0.143
0.145
0.145
0.146
0.146
0.147
0.147

0.134
0.132
0.132
0.130
0.131
0.130
0.129
0.129
0.132
0.136
0.138
0.140
0.142
0.143
0.145
0.146

0.198
0.230
0.264
0.297
0.328
0.357
0.388
0.418
0.445
0.477
0.515
0.558
0.597
0.633
0.668
0.706

1.447
2.212
2.858
3.221
3.387
3.457
3.494
3.527
3.548
3.544
3.590
3.654
3.609
3.630
3.655
3.719

174

Replicate 3
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

0.137
0.140
0.138
0.138
0.136
0.136
0.137
0.138
0.138
0.139
0.140
0.140
0.141
0.141
0.142
0.143

0.111
0.112
0.114
0.116
0.117
0.118
0.119
0.120
0.122
0.123
0.124
0.125
0.126
0.127
0.128
0.129

0.180
0.210
0.241
0.271
0.301
0.329
0.357
0.385
0.415
0.443
0.471
0.499
0.526
0.554
0.582
0.610

1.365
2.122
2.801
3.368
3.762
3.970
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

Supplementary Table 8. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 9.
The test aimed to assess the assay’s ability to generate appropriate absorbance readings in response to
increasing concentrations of acetylcholinesterase. It analyzed the rate of TNB2- absorbance increase over a
30-min time span as acetylcholinesterase concentration increased. The term “OVRFLW”, or “Overflow”,
denoted absorbance readings that exceeded the upper limit of the Synergy 2 plate reader machine’s
detection range and could not be measured. Black highlighted sections in the tables represented unusual
data where absorbance readings resulting from treatments with lower enzyme concentrations were higher
than those from treatments with higher enzyme concentrations. The lower panels of graphs depicted the
changes in TNB2- absorbance in response to increasing concentrations of acetylcholinesterase over the
course of 30 mins. The experiment was repeated three times (n = 3).

175

Supplementary Figure 7. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 9.
A photo of the microtiter plate used for the run was also attached to illustrate the almost complete
reproducibility of the results obtained from run 5 when treated with the correct amount and the correct
concentration of acetylthiocholine iodide, along with the correct concentration of sodium phosphate buffer
in which the acetylthiocholine iodide was dissolved in. This was shown by the disappearance of the crystal
formation in all wells.

176

Replicate 1
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.150
0.152
0.153
0.156
0.158
0.159
0.160
0.160
0.161
0.161
0.161
0.161
0.161
0.161
0.162
0.162

0.175
0.175
0.175
0.175
0.175
0.175
0.175
0.175
0.175
0.175
0.175
0.175
0.176
0.176
0.176
0.176

0.655
0.667
0.677
0.687
0.699
0.710
0.721
0.733
0.744
0.756
0.766
0.776
0.787
0.799
0.809
0.821

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

Replicate 2
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.173
0.172
0.170
0.168
0.165
0.172
0.170
0.169
0.169
0.169
0.169
0.169
0.169
0.170
0.170
0.170

0.165
0.159
0.167
0.170
0.174
0.177
0.180
0.183
0.186
0.188
0.192
0.194
0.197
0.200
0.202
0.205

0.236
0.292
0.348
0.402
0.455
0.508
0.560
0.611
0.662
0.712
0.761
0.811
0.857
0.906
0.952
0.999

0.958
1.671
2.319
2.920
3.426
3.806
4.025
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

3.107
OVRFLW
OVRFLW
4.048
3.973
OVRFLW
3.965
4.002
3.974
3.978
3.990
3.921
4.007
3.928
3.962
3.907

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

177

Replicate 3
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.177
0.176
0.176
0.173
0.167
0.169
0.169
0.173
0.173
0.173
0.173
0.173
0.173
0.174
0.173
0.173

0.182
0.178
0.177
0.181
0.184
0.187
0.188
0.190
0.191
0.192
0.194
0.195
0.196
0.197
0.198
0.199

0.218
0.239
0.265
0.290
0.316
0.340
0.365
0.388
0.412
0.436
0.459
0.481
0.503
0.525
0.546
0.568

0.844
1.550
2.215
2.788
3.273
3.663
3.927
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

3.565
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

Replicate 4
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24

0.0

0.009

0.09

0.9

4.5

9.0

0.190
0.183
0.188
0.189
0.190
0.191
0.191
0.191
0.191
0.191
0.192
0.192
0.192

0.194
0.196
0.197
0.199
0.200
0.200
0.201
0.201
0.202
0.200
0.199
0.202
0.206

0.226
0.248
0.270
0.292
0.311
0.331
0.349
0.367
0.383
0.397
0.425
0.452
0.474

0.757
1.341
1.874
2.361
2.798
3.150
3.416
3.624
3.781
3.870
3.922
3.953
3.858

3.647
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
3.971
3.912
3.899
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

178

26
28
30

0.192
0.192
0.193

0.208
0.211
0.212

0.495
0.515
0.533

3.858
3.683
3.801

OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW

Replicate 5
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30

0.0

0.009

0.09

0.9

4.5

9.0

0.182
0.178
0.178
0.172
0.179
0.181
0.182
0.181
0.182
0.181
0.181
0.180
0.180
0.180
0.180
0.180

0.188
0.184
0.181
0.177
0.183
0.185
0.187
0.189
0.190
0.190
0.192
0.193
0.194
0.196
0.196
0.197

0.206
0.219
0.228
0.239
0.247
0.254
0.262
0.268
0.280
0.304
0.316
0.328
0.342
0.355
0.367
0.377

0.854
1.443
1.977
2.469
2.930
3.322
3.671
3.808
4.035
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

2.923
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
4.039
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
3.974
OVRFLW
3.959
OVRFLW
OVRFLW
3.988
3.986
3.942
3.958
3.921
3.885
3.867
3.859

Replicate 6
[unit/mL]
Minutes
0
2
4
6
8
10
12
14
16
18

0.0

0.009

0.09

0.9

4.5

9.0

0.189
0.188
0.188
0.188
0.186
0.187
0.187
0.185
0.184
0.182

0.183
0.172
0.170
0.153
0.167
0.173
0.178
0.182
0.185
0.186

0.217
0.223
0.231
0.235
0.241
0.246
0.249
0.254
0.256
0.263

0.781
1.264
1.715
2.109
2.466
2.711
2.954
3.147
3.268
3.287

3.821
OVRFLW
OVRFLW
OVRFLW
OVRFLW
3.989
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

179

20
22
24
26
28
30

0.186
0.183
0.181
0.188
0.190
0.186

0.187
0.188
0.188
0.189
0.189
0.190

0.270
0.276
0.278
0.278
0.289
0.304

3.549
3.834
3.865
3.971
OVRFLW
4.040

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW
OVRFLW

Supplementary Table 9. Evaluating the Assay with Different Concentrations of Acetylcholinesterase:
The First Performance Test, Run 10.
The test aimed to assess the assay’s ability to generate appropriate absorbance readings in response to
increasing concentrations of acetylcholinesterase. It analyzed the rate of TNB2- absorbance increase over a
30-min time span as acetylcholinesterase concentration increased. The term “OVRFLW”, or “Overflow”,
denoted absorbance readings that exceeded the upper limit of the Synergy 2 plate reader machine’s
detection range and could not be measured. Black highlighted sections in the tables represented unusual
data where absorbance readings resulting from treatments with lower enzyme concentrations were higher
than those from treatments with higher enzyme concentrations. The lower panels of graphs depicted the
changes in TNB2- absorbance in response to increasing concentrations of acetylcholinesterase over the
course of 30 mins. The experiment was repeated three times (n = 3).

180