Illustration of an Annona muricata branch with fruit above a mouse running on a laboratory treadmill, with a mouse brain outline behind

Medical Research Institute · Department of Biochemistry · Alexandria University

Thesis for the degree of M.Sc. in Biochemistry

The Possible Impacts of Graviola Leaf Extract and Exercise on Depression-Like Behavior in Mice

Walaa Adel Mohamed Youssef

B.Sc. Clinical Pharmacy, Faculty of Pharmacy, Alexandria University, 2020

2026

Background

Major depressive disorder is common, and response to current antidepressants is often incomplete

11%

Lifetime prevalence of depression, upper estimate

1 in 3

Women estimated to experience a major depressive episode

Standard pharmacotherapy (SSRIs, SNRIs, tricyclic agents and MAO inhibitors) targets monoaminergic transmission.

Clinical response is frequently partial, and prolonged use is associated with adverse effects. This has prompted interest in natural products and non-pharmacological interventions.

The higher burden in women is the reason female mice were used in this study.

Lim et al., 2018 · Tam et al., 2020 · Ma et al., 2022

Pathophysiology

Mechanisms implicated in depression and measured in the hippocampus

Illustration of a hippocampal CA3 pyramidal neuron

Neurotransmission

Monoaminergic deficit

Reduced serotonin, dopamine and noradrenaline signalling.

Redox

Oxidative stress

Impaired Nrf2-dependent antioxidant response, glutathione depletion and lipid peroxidation.

Immune

Neuroinflammation

Increased pro-inflammatory cytokines, including TNF-α.

Plasticity

Neurotrophic deficit

Reduced CREB1–BDNF signalling and impaired hippocampal plasticity.

Post-transcriptional

Non-coding RNAs

miR-124 and miR-134 upregulated; LINC00473 reduced, specifically in females.

Epigenetic

SIRT1

NAD⁺-dependent deacetylase; reduced activity is associated with depressive-like phenotypes and impaired Nrf2 signalling.

Rădulescu et al., 2021 · Yao et al., 2021 · Issler et al., 2020 · Dang et al., 2022

Interventions

One reference drug, one botanical extract and one physical intervention

Fluoxetine

Selective serotonin reuptake inhibitor, used as the positive control.

20 mg/kg/day · p.o. · 4 weeks

Botanical illustration of Annona muricata

Annona muricata leaf

Anxiolytic, anti-inflammatory and antidepressant activity reported in vivo.

50 mg/kg/day · p.o. · 4 weeks

Treadmill exercise

Acts on monoamine release, neurotrophic factors, inflammation and miRNA expression.

30 min/day · forced · 4 weeks

Gavamukulya et al., 2017 · Wang et al., 2022 · Khafaga et al., 2020 · Ahmed-Farid et al., 2016

Aim of the work

To investigate the antidepressant effect of treadmill exercise and Annona muricata leaf extract, alone and in combination, compared with fluoxetine, in reserpine-treated female mice.

Behaviour: Forced swim test immobility
Neurochemistry: Hippocampal serotonin, dopamine, noradrenaline
Redox · inflammation: Nrf2, total glutathione, FRAP, MDA, TNF-α
Gene expression: CREB1, BDNF, SIRT1, LINC00473, miR-124a, miR-134
Histology: CA3 pyramidal layer, H&E

Materials and methods

Experimental timeline

56 female Swiss albino mice, 18–25 g n = 8 per group 24 ± 2 °C · 12 h light/dark IACUC approval AU012232511

Drawn to scale in days. FST: 6-min session, immobility scored over the final 4 min (Slattery & Cryan, 2012).

Materials and methods

Allocation of 56 mice to seven groups

Illustration of a female Swiss albino mouse

56female

Swiss albino mice, 18–25 g. Baseline FST did not differ between groups.

Days 1–4

Reserpine

0.1 mg/kg/day, i.p.

Day 5 FST confirmed the model. Treatments then ran daily for 4 weeks.

n = 48

Fluoxetine 20 mg/kg/day p.o. · graviola leaf extract 50 mg/kg/day p.o. · treadmill 30 min/day. These group colours are used in every result figure.

Materials and methods

Each hippocampus was divided for histology, biochemistry and gene expression

Low-magnification H&E section of a control mouse hippocampus

Control hippocampus · H&E, scale bar 200 µm. CA3 pyramidal layer was assessed in all groups.

Fixed tissue

Histopathology

10% formalin, paraffin, 4.5 µm H&E sections; blinded morphometry, ImageJ; n = 5 per group

PBS homogenate

Neurochemistry and redox

5-HT, dopamine, noradrenaline by competitive ELISA; total GSH, FRAP, MDA, total protein by colorimetry

Total RNA

qRT-PCR

Nrf2, TNF-α, CREB1, BDNF, SIRT1, LINC00473 vs 18S rRNA; miR-124a, miR-134 vs U6; ΔΔCt

Results · extract characterisation

Phytochemical profile of the graviola leaf extract

HPLC · phenolic acids

mg/g

GC–MS · total ion chromatogram

retention time, min

Major constituent	RT, min	Area, %
Phytol	26.04	18.47
Hexadecanoic acid, methyl ester	26.04	14.62
Methyl linoleate	27.93	12.31
Octadecanoic acid, methyl ester	28.11	9.85
β-Caryophyllene	44.20	7.94

HPLC: Agilent 1100, C18, 280 nm · GC–MS: Agilent 7890B/5977A, HP-5MS, NIST library. Chromatogram traced from the thesis figure.

Results · model validation

Reserpine increased immobility time in the forced swim test by 69%

Illustration of a mouse in the forced swim test cylinder

+69%

Immobility relative to vehicle-treated controls after reserpine

Immobility time

s, mean ± SD

Student's t = 11.16 · p < 0.001 · n = 8 per group

Immobility scored over the last 4 min of a 6-min session. * p < 0.05 vs control.

Results · behaviour

After four weeks, G+E and TRI immobility no longer differed from control

Immobility time

s, mean ± SD

TRI · 130 s

+2% vs control; differs from untreated only (b)

G+E · 145 s

+13% vs control; differs from untreated only (b)

GRV 155 s · FLX 152 s

Graviola alone comparable to fluoxetine; single treatments remained above control (a)

n = 8. One-way ANOVA F = 28.36, p < 0.001; Tukey post hoc. a vs CTL · b vs RES · c vs FLX · d vs EX · e vs GRV · f vs G+E (p < 0.05). Dashed line: control mean.

Results · neurotransmitters

Hippocampal monoamines were partially restored in all treated groups

Serotonin

pg/mg protein

Dopamine

pg/mg protein

Noradrenaline

pg/mg protein

Every group stayed below control (a). G+E gave the highest serotonin and dopamine, fluoxetine the highest noradrenaline. TRI dopamine did not differ from untreated mice.

Mean ± SD, n = 8. Competitive ELISA. ANOVA with Tukey post hoc. a vs CTL · b vs RES · c vs FLX · d vs EX · e vs GRV · f vs G+E.

Results · redox status

Nrf2 expression increased significantly only with combined treatment

2.87×

Nrf2 fold change in G+E, significantly higher than control and every other group

Single treatments: no significant change vs untreated

Nrf2 relative expression

fold change

Mean ± SD, qRT-PCR, n = 8. ANOVA F = 36.35, p < 0.001. a vs CTL · b vs RES · c vs FLX · d vs EX · e vs GRV · f vs G+E.

Results · redox status

Glutathione, total antioxidant capacity and lipid peroxidation

Total GSH

nmol/mg protein

FRAP

µmol/mg protein

MDA

nmol/g tissue

G+E restored GSH to control level and raised FRAP to 2.1-fold of control. TRI gave the lowest MDA (−48%). Fluoxetine did not change GSH.

Mean ± SD, n = 8. ANOVA p < 0.001 for each marker; Tukey. RES vs CTL was significant for GSH only. a vs CTL · b vs RES · c vs FLX · d vs EX · e vs GRV · f vs G+E.

Results · inflammation

Reserpine doubled hippocampal TNF-α expression; G+E reduced it below control

−76%

TNF-α in G+E relative to control, significantly lower than every single treatment

TNF-α relative expression

fold change

Mean ± SD, qRT-PCR, n = 8. ANOVA F = 35.11, p < 0.001. a vs CTL · b vs RES · c vs FLX · d vs EX · e vs GRV · f vs G+E.

Results · neurotrophic signalling

BDNF expression rose to 3.85-fold of control with graviola + exercise

3.85×

G+E vs control

−86%

Untreated vs control

Fluoxetine did not restore BDNF (−65% vs control).

BDNF relative expression

fold change

Mean ± SD, qRT-PCR, n = 8. ANOVA F = 291.17, p < 0.001. a vs CTL · b vs RES · c vs FLX · d vs EX · e vs GRV · f vs G+E.

Results · gene expression

CREB1 exceeded control only with G+E; SIRT1 exceeded control with EX, G+E and TRI

CREB1

fold change

SIRT1

fold change

Fluoxetine did not significantly change either gene. TRI CREB1 (1.25) did not differ from control or untreated mice.

Mean ± SD, qRT-PCR, n = 8. CREB1 F = 25.64; SIRT1 F = 25.31; both p < 0.001. a vs CTL · b vs RES · c vs FLX · d vs EX · e vs GRV · f vs G+E.

Results · non-coding RNAs

Both miRNAs were lowest in G+E; LINC00473 was restored by the combinations

miR-124a

fold change

miR-134

fold change

LINC00473

fold change

LINC00473 fell by 82% and fluoxetine did not restore it. G+E returned it to control level (1.15-fold); TRI exceeded control (1.70-fold).

Mean ± SD, qRT-PCR, n = 8. ANOVA p < 0.001 for each transcript; Tukey. a vs CTL · b vs RES · c vs FLX · d vs EX · e vs GRV · f vs G+E.

Summary of results

Change from control in every outcome: G+E reverses most of the reserpine pattern

Values: % change vs control, from the thesis tables. Colour: above control / below control, saturating at ±100%. Dot: significant vs untreated (RES row: vs control), Tukey p < 0.05.

Results · correlation analysis

SIRT1 and miR-134 are the most connected markers among treated mice

Strongest pairs

BDNF, CREB1, Nrf2 and SIRT1 rise together and fall as TNF-α and the two miRNAs rise.

Pearson r, n = 40 treated mice; all 26 pairs shown are p < 0.05. Line width ∝ |r|. Positive · negative.

Results · histopathology

CA3 pyramidal layer: control, reserpine and fluoxetine

CTL

Evenly arranged neurons with vesicular nuclei, prominent nucleoli and Nissl granules.

RES

Shrunken hyperchromatic, pyknotic and karyorrhectic nuclei; foci of neuronal loss.

FLX

Most pyramidal cells shrunken and hyperchromatic; pyknotic and karyorrhectic nuclei persist.

H&E, scale bar 50 µm. n = 5 per group, blinded examination.

Results · histopathology

CA3 pyramidal layer: exercise, graviola and the combinations

EX

Partial improvement

Shrunken and apoptotic neurons, few normal cells

GRV

Partial improvement

Hyperchromatic and apoptotic neurons, few normal cells

G+E

Closest to control

Densely packed pyramidal neurons with basophilic cytoplasm, vesicular nuclei and Nissl granules; occasional ghost cells

TRI

Marked improvement

Many vesicular nuclei; few pyknotic and ghost cells

H&E, scale bar 50 µm. n = 5 per group, blinded examination.

Discussion

Proposed mechanism: the SIRT1–miR-134–CREB1–BDNF axis

Arrows: activation · bars: inhibition. Directions (↑ ↓) are the G+E changes vs untreated. Links are inferred from expression changes, correlations and the literature; they were not tested individually.

Discussion

Limitations and future work

Limitations

The reserpine model reflects acute monoamine depletion rather than chronic MDD.
Only female mice and a single dose of each agent were studied.
Gene-level findings are based on transcript abundance, without protein confirmation.
The divergent effects of the triple combination on dopamine, FRAP and Nrf2 are not explained.

Future work

Evaluation in a chronic stress model and in male animals.
Dose–response studies that define the margin against acetogenin-related neurotoxicity.
Pharmacokinetic study of the graviola–fluoxetine interaction.
Standardisation of the extract and protein-level validation of key targets.

Conclusions

GRV

Graviola leaf extract alone reduced immobility to a degree comparable with fluoxetine.

G+E

Combined with exercise, it produced the largest changes in Nrf2, BDNF, CREB1, TNF-α and both miRNAs, and the best-preserved CA3 morphology.

TRI

Adding fluoxetine normalised immobility time but attenuated the effects on dopamine, FRAP and Nrf2.

Acknowledgements

Thank you

Supervisors

Prof. Dr. Wafaa Mahmoud Abdel-Rehim
Assist. Prof. Dr. Rasha Abd El-Mottleb EL-Tahan
Assist. Prof. Dr. Shimaa Abdel Raheem Mahmoud
Dr. Rana Hassan Mahmoud Hassan Khafaga

Examining committee

Prof. Dr. Wafaa Mahmoud EL-Sayed Abdel-Rehim
Prof. Dr. Nagwa Mohamed Assem
Prof. Dr. Hussein Khamis Hussein
Assist. Prof. Dr. Shimaa Abdel Raheem Mahmoud

Histopathology: Dr. Noura Matar, Department of Cell Biology and Histochemistry