A Comparative Analysis of the Impacts of Cinnamomum zeylanicum and Sodium Benzoate in
Drosophila melanogaster Models for Alzheimer's Disease
Dave Achonu
[email protected]
ABSTRACT
This proposed experimental study will investigate the efficacies of sodium benzoate
(NaB) and Cinnamomum zeylanicum (CZ) as potential therapies for Alzheimer’s disease (AD)
using Drosophila melanogaster as a model organism. The fruit flies will be divided into
treatment groups and administered CZ, an equal dose of NaB, or no treatment. The cognitive
abilities of the fruit flies will be assessed using the Rapid Iterative Negative Geotaxis (RING)
Protocol and the Courtship Conditioning Assay. Statistical analyses will be performed to
compare the effects of these treatments on cognitive function in the fruit flies. The results of this
research will contribute to the growing body of literature regarding AD treatments by
highlighting the pathological mechanisms responsible for this affliction, underscoring the
pathways involved in its progression, and prompting continued drug discovery research.
Keywords: Alzheimer’s disease, Cinnamon, Sodium Benzoate, Drosophila melanogaster
INTRODUCTION
AD is a devastating terminal illness; it is the seventh most common cause of death in the
United States and the most common cause of dementia in patients over 65 years of age (National
Institute on Aging, 2021). Due to the lethal consequences of AD, researchers have developed
various treatment modalities for it, including cholinesterase inhibitors and Aducanumab
(Cummings et al., 2021). Despite the availability of these treatments, exploring innovative
interventions is imperative in the pursuit of public health. Fortunately, possible novel treatments
for this illness have emerged in recent years.
The current body of literature indicates that cinnamon may be a viable intervention for
patients suffering from cognitive disorders, such as AD. In a review by Ali Ahmadi, cinnamon
was noted to be effective in ameliorating neuroinflammatory disorders such as Parkinson’s
disease, traumatic brain injury, and AD. NaB, a known metabolite of cinnamon, has also
demonstrated neuroprotective capabilities and has even improved cognition in dementia patients
(Rangasamy, 2021; Lane, 2021). This study aims to explore both treatment modalities, compare
them, and highlight mechanisms of AD. These goals will attest the following research question:
What are the comparative effects of sodium benzoate and Cinnamomum zeylanicum on cognitive
function in Drosophila melanogaster models for Alzheimer’s disease?
LITERATURE REVIEW
Both cinnamon and its metabolite NaB may be viable treatments for AD, though it is
unclear which is the superior treatment method. Comparing the relative effects of cinnamon and
NaB in AD models will have major implications. If NaB significantly outperforms cinnamon, it
will indicate that the neuroprotective effects of cinnamon are primarily due to NaB
metabolization. Typically, greater exposure to stimuli yields greater physiological effects.
Moreover, imposing isolated NaB will yield a greater net amount of NaB when compared to
treating with cinnamon. Therefore, if the results of imposing NaB are significantly superior to
that of cinnamon, it will be apparent that the metabolite is primarily responsible for the
improvements in AD symptoms in disease models utilizing cinnamon as a treatment
intervention. This outcome furthers the discussions regarding the efficacy of NaB and
underscores multiple pathways of AD worth exploring. In a study by Chieh-Hsin Lin, NaB was
demonstrated to retard dementing. This process was attributed to a key property of NaB,
D-amino acid oxidase inhibition (Lin et al., 2014). Neuroscientist Saurav Brahmachari provides
a different perspective. In his study, NaB was demonstrated to reduce inflammation in glial cells.
He notes that the inflammation of these structures is associated with neurodegenerative diseases,
such as AD (Brahmachari et al., 2009). These two perspectives regarding the pathogenesis of AD
may be investigated further if NaB proves superior to cinnamon. However, if cinnamon exhibits
more significant effects in the AD model than NaB, it will elucidate that the inhibitory effects of
cinnamaldehyde and epicatechin—components of cinnamon—on tau tangle aggregation—a
hallmark of AD—are more potent than the effects of NaB (George, 2013). This result will
highlight these pathological mechanisms and indicate cinnamon’s potential application in a
clinical setting. Similar effects between the two groups will demonstrate that all of these avenues
substantially contribute to AD progression, and therefore, are all worth exploring when
uncovering potential treatments to this devastating affliction.
METHODOLOGY
The proposed experiment will be designed in a similar fashion to a study by Hanh Pham,
in which the health of Drosophila melanogaster expressing the Amyloid-beta 42 peptide and the
tau protein after treatment with cinnamaldehyde was assessed (2018). Fruit flies expressing these
gene products exhibit neurodegenerative symptoms similar to humans suffering from AD
(Prüßing et al., 2013).
The first portion of the experiment will be a survivor assay. This analysis will be
conducted to determine a safe and effective dose for the experiment. The survival analysis will
be based on a study by Yuling Dong (2022). The trial will consist of five treatment groups of
newly eclosed wild-type fruit flies, each receiving a different dose of NaB by mass per 100
grams of three percent yeasted banana (Control - 0g NaB/100g media, 0.01g NaB/100g media,
0.05g NaB/100g media, 0.1g NaB/100g media, 0.5g NaB/100g media). The groups will be
housed in different stock containers with their food. The trials will run until all the fruit flies are
no longer alive. After the survival analysis, mean survival rates will be calculated. The group
with the highest amount of NaB that maintains a high survival rate, comparable to the control
group, will be utilized for the study. When comparing the mean survival rate, a p-value < 0.0001
will be considered significant.
For the second portion of the study, the newly eclosed fruit flies—both gene mutated and
wild-type—will be housed in stock containers with three percent yeasted banana media, and
divided into their respective groups. Additionally, their media will contain either CZ, NaB, or no
additional treatment. These doses will be determined by the NaB survival analysis. CZ will be
equated to the determined amount by percent mass of media. This is a total of 6 experimental
groups. Drosophila will be exposed to these treatments for 21 days. After this duration of time,
the fruit flies will undergo various testing procedures. Both wild-type and mutant fruit flies will
have their cognition measured by the RING protocol and the Courtship Conditioning Assay. The
RING protocol, as outlined by Julia Warner Gargano (2005), will illustrate the locomotive and
climbing abilities of Drosophila. Six trials for each group of flies will occur. 20 fruit flies from
each group will be utilized for this assay. The mean height climbed for each group will be
calculated. Additionally, performing the Courtship Conditioning Assay, as detailed by Charles
Nichols (2012), on both sexually naive and trained male fruit flies will demonstrate memorative
ability, as trained fruit flies should remember the copulation process and perform more
efficiently. One hour after a training period with female fruit flies, male fruit flies will perform
the assay and be scored on the Courtship Index. Naive male flies will execute the same task and
be scored. These scores will be utilized to determine a mean Memory Index (MI). The data will
be analyzed utilizing a One Way Analysis of Variance (ANOVA) for the difference in mean
height climbed during the RING protocol and the difference in mean MI. A p-value of < 0.05
will be considered significant. These results will illustrate which treatment had the most robust
effect on the cognition and health of both mutant and wild-type fruit flies.
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