{"paper_id":"1a46f90c-1e40-47d5-a121-673c0ff272ae","body_text":"bioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \nClove Aqueous Extract Triggers a Multi-Organellar Stress Crisis through Lysosomal \nDestabilisation and Mitochondrial Hyperpolarisation to Suppress Patient-Derived \nOvarian Cancer Cells     \nYazid Ghanem1, Hadeel Odwan2, Musha Yang3,4, Victoria Malone3,4, Fawza Alenazi5, Fears     \nAbu Saadeh4,6, Steven G. Gray4,7, Derek Doherty8, Cara Martin3,4, Sharon O`Toole3,4,9, John    \nJ. O`Leary3,4 and Bashir M. Mohamed3,4,9\n     \n1 \n School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland                   \n2 \n Food Science & Environmental Health, Technology university, Dublin 7, Ireland                     \n3 \nDepartment of Histopathology, Trinity College Dublin, Dublin 8, Ireland                           \n4 \nTrinity St James's Cancer Institute, Dublin 8, Ireland                                                           \n5 \nDepartment of Biochemistry, King Saud University, Riyadh, Saudi Arabia                                          \n6 \nDivision of Gynaecological Oncology, St. James’s Hospital; Dublin 8, Ireland                                       \n7 \nThoracic Oncology Research Group, St. James's Hospital, Dublin 8, Ireland                        \n8 \nDepartment of Immunology, Trinity College Dublin, Dublin 8, Ireland                          \n9 \nDepartment of Obstetrics and Gynaecology, Trinity College Dublin, Dublin 8, Ireland     \n     \n     \n     \n     \n     \n     \n*Corresponding author email: bashmohamed@gmail.com     \n     \n          \n        \n      \n  \n  \n  \n  \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \n  \n  \n  \nAbstract:     \nOvarian cancer (OC) remains a lethal malignancy with limited therapeutic options, \nunderscoring the need for the identification of novel agents. Natural products like clove \n(Syzygium aromaticum) have shown promising anti -cancer activity, but their mechanism in \nOC is poorly understood. This study investigates the anti -tumour effects and underlying \nmechanisms of a clove aqueous extract (CAE) on a panel of patient -derived OC cells. We \nfound that CAE significantly inhibited cellular proliferation and induced cell death in a time- \nand dose -dependent manner. Mechanistically, CAE induced profound cellular stress, \nactivating the transcription factor ATF-2. This was accompanied by a significantly increased \nlysosomal stress response, as evidenced by increased lysosomal  mass/acidity, and a \npathogenic hyperpolarisation of the mitochondrial membrane potential ( ΔΨm). The \nbioenergetic crisis induced as a consequence resulted in a sharp reduction in cellular oxygen \nconsumption rate (OCR). Notably, the sensitivity to CAE -induced lysosomal and \nmitochondrial dysfunction varied across cell lines, revealing distinct phenotypic responses. \nOur results demonstrate that clove extract exerts its anti -tumour effects by orchestrating a \nmulti-organellar stress response, positioning lysosom al disruption as a central event in its \nmechanism of action. This study provides a strong rationale for the further development of \nclove-based interventions for OC.     \n     \n     \n     \n     \n     \n     \n     \n     \n    \n    \n    \n    \n    \n    \n     \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \nKeywords:  Clove extract, Ovarian cancer, Patient Derived Model, Patient Derived Organoid \noxygen consumption rate, mitochondrial membrane potential     \n          \n        \nIntroduction     \nOvarian cancer (OC) remains the most lethal gynaecologic malignancy worldwide, and \ndespite advances in treatment, survival outcomes have improved only modestly. In the United \nStates, recent surveillance estimates project approximately 20,890 new cases and 12,730 \ndeaths in 2025 (1). The 5 -year relative survival rate remains at roughly 50 –55%, reflecting \nonly incremental gains over several decades (1 –3). A major contributor to the persistently \npoor prognosis is the fact that most patients are diagnosed at an advanced stage; nearly 70% \npresent with FIGO stage III or IV disease. This is largely due to the nonspecific nature of early \nsymptoms and the continued absence of an effective screening strategy for the general \npopulation (4–7).     \nThe current standard first -line treatment for OC consists of maximal cytoreductive \n(debulking) surgery followed by platinum-based chemotherapy, most commonly carboplatin \nor cisplatin in combination with paclitaxel (8 –11). Although initial response rates ca n \napproach 80%, more than half of patients eventually experience disease recurrence, primarily \ndue to the development of intrinsic or acquired resistance to chemotherapy (8–11). Moreover, \nplatinum and taxane regimens are also associated with cumulative tox icities. Nephrotoxicity \nand peripheral neurotoxicity can become dose -limiting and may restrict further treatment \noptions (12–15). Collectively, these challenges highlight the urgent need for novel therapeutic \nstrategies that act through alternative mechani sms and exhibit improved safety profiles. \nNatural products have historically played a central role in cancer drug discovery, with an \nestimated 60% of currently used chemotherapeutic agents derived from natural compounds \nor their structural analogues (16–18). Paclitaxel, a cornerstone of OC therapy, is itself derived \nfrom the Pacific yew tree, underscoring the enduring relevance of plant -based molecules in \noncology (19,20). Beyond their direct cytotoxic effects, natural products are increasingly \nbeing investigated for their ability to overcome chemoresistance and mitigate treatmentrelated \ntoxicities (16,21).     \nClove (Syzygium aromaticum L.), a spice widely used in traditional Chinese medicine, \nAyurvedic medicine, and Kampo practices, contains a diverse array of bioactive constituents, \nincluding eugenol, β-caryophyllene, flavonoids, and various triterpenoids (22 –24). Clove \nextracts have demonstrated antioxidant, anti -inflammatory, antimicrobial, and anticancer \nactivities in various experimental models (25 –30). Of particular relevance to OC, the clove \nderived flavonoid kumatakenin has been shown to induce apoptosis  and inhibit tumour \nassociated macrophage activation in preclinical studies (31,32). More broadly, clove extracts \nexert cytotoxic and pro -apoptotic effects across multiple cancer types, largely through \nmodulation of cell -cycle regulation, DNA damage respon ses, and mitochondrial stress \npathways (25–32). Given the metabolic vulnerabilities and organelle -specific dysfunctions \ncharacteristic of OC cells, we hypothesised that clove aqueous extract (CAE) may exert \nantitumor effects by coordinating stress response s within both lysosomes and mitochondria. \nTo test this hypothesis, we employed a panel of patient-derived OC models representing major \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \nepithelial subtypes, including high -grade serous ovarian cancer (HGSOC) and \ncarcinosarcoma, thereby capturing the molecular and clinical heterogeneity of the disease  \n(33–36).     \nThis experimental approach allowed us to determine whether CAE exhibits broad antitumor \nactivity across OC subtypes or displays selective efficacy, to delineate the temporal sequence \nof lysosomal and mitochondrial disruption, and to identify the downstream pathways leading \nto cell death. Together, these findings provide mechanistic insight into the anticancer activity \nof clove-derived compounds and support the potential of organelle -targeting strategies as a \nnovel avenue for improving OC treatment.     \n                    \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \nMaterials and methods     \nEthical Approval     \nThis study was approved by St. James’s Hospital and Adelaide and Meath Hospital, Dublin, \nincorporating the National Children’s Hospital Research Ethics Committee (Reference: \n2012/11/04). All procedures were conducted in accordance with the Declaration of H elsinki \nand relevant institutional and national guidelines. Written informed consent was obtained \nfrom all participants or their legal guardians prior to sample collection.     \nPatient-Derived OC Cells Isolation and Expansion      \nPatient-Derived OC cells with high-grade serous (HGSOC) and carcinosarcoma  subtypes \nwere isolated from either ascites fluid obtained from three patients (designated OCAS12, \nOCAS14 and OCAS17) and/or from ovarian tumour tissue (OCAST16) and used throughout \nthis study. As previously described (37), cells were maintained in RPMI 16 40 medium \n(GIBCO, Invitrogen, Ireland) supplemented with 10% (v/v) fetal calf serum, 20 mM HEPES, \n10 μM nicotinamide, 10 μM SB202190, 1.25 mM N-acetyl-L-cysteine, 10 ng/mL FGF-10, 1 \nng/mL FGF-2, 1× B27 supplement, Primocin (1:100, v/v), 10 μM Y-27632, 2 mM Lglutamine, \nand 100 U/mL penicillin -streptomycin. Cultures were maintained at 37°C in a humidified \nincubator with 5% CO . Four days after initial plating, non -adherent cells were removed by \nwashing with phosphate -buffered saline (PBS), and fresh culture medium was added. Cells \nwere subsequently expanded for experiments under standard conditions.     \nPatient-Derived OC Cell Phenotypic Characterisation     \nPatient-Derived OC cells were characterised for cancer -associated and epithelial – \nmesenchymal transition (EMT) markers, including mucin -1 β-catenin, ErbB -3, EGFR, \nclaudin7, folate receptor -α, and mesothelin. Cells were seeded at 1 × 10 cells per well in \n96well plates and incubated overnight at 37°C with 5% CO . The following day, cells were \nwashed with PBS and fixed in 3% paraformaldehyde (PFA; Sigma -Aldrich, Ireland) for 20 \nminutes at room temperature (RT). After fixation, cells were blocked with 5% bovine serum \nalbumin (BSA; Sigma-Aldrich, Ireland) for 1 hour at RT.     \nCells were then incubated overnight at 4°C with mouse monoclonal primary antibodies \n(1:200; Santa Cruz Biotechnology, Germany). The next day, cells were washed with PBS and \nincubated with a goat anti-mouse FITC-conjugated secondary antibody (1:300; ThermoFisher \nScientific, Dublin, Ireland) for 1 hour at RT. Nuclear staining was performed using Hoechst \n33342 (1:1000; ThermoFisher Scientific, Dublin, Ireland) for 20 minutes at RT. Following \nfinal PBS washes, cells were imaged using an EVOS inverted fluorescence microscope  \n(Figure 1).     \nConstruction and treatment of scaffold-free PDOs      \nAll Patient -Derived OC Cells  (OCAS12,OCAS14 OCAS17 and OCAST16 cells) were \nseeded in a and fed with DMEM/RPMI (Invitrogen, Ireland) supplemented with 10% (v/v) \nfoetal calf serum, 20 mM HEPES, 10 µM SB202190, 1.25 mM N -Acetyl-L-cysteine, 10 \nng/mL FGF-10, 1 ng/mL FGF-2, 1X B27 Additive, 1:100 (v/v) Primocin, and 10 µM Y27632, \n2 mM Lglutamine and 100 U/mL penicillin-streptomycin. The cells were maintained at 37ºC \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \nin humidified air with 5% CO 2 until spherical organoids formed within approximately eight \ndays.     \n     \nPreparation of Clove Crude Aqueous Extract (CAE)     \nDried clove buds (Syzygium aromaticum L.) were purchased from a local commercial supplier \n(Dublin). The buds were finely ground using a Krups coffee grinder. For each extraction, 5 g \nof ground clove powder was suspended in 50 mL of cell culture medium (RPMI). The \nmixtures were incubated on a Stu art Scientific digital roller mixer for 24 hours at room \ntemperature. Following incubation, samples were centrifuged at 5,000 × g for 30 minutes at \n4°C using a Hettich Rotina 35 R refrigerated centrifuge. The resulti ng supernatants of CAE \nwere collected and filtered under sterile conditions through a 0.22 µm PES membrane filter \n(Millex-GP, Merck) using a 50 mL Terumo syringe. Filtered CAE were transferred into sterile \n50 mL Sarstedt screw-cap tubes and stored at 4°C until use.     \nCytotoxic effects of CAE      \nAssessment of Cell Viability using CCK-8 Assay     \nTo measure cell viability, we used the Cell Counting Kit-8 (CCK-8, Selleckchem). The CCK8 \nsolution was diluted tenfold in complete RPMI medium and then added to the patient-derived \nOC cells in 96 -well plates. The plates were incubated for 90 minutes accord ing to the \nmanufacturer's instructions. Finally, the absorbance was read at 450 nm using a Varioskan \nLUX microplate reader (ThermoFisher Scientific).     \nATF-2 and P-ATF-2 Expression Analysis by Cytell Imaging System      \nAll Patient-Derived OC Cells were seeded in 96 -well plates overnight at a density of 5000 \ncells per well and subsequently exposed to several concentrations of CAE (20ug, 40, and \n80ug/ml), then incubated for 72h.  Exposed cells were then washed in PBS, fixe d with 3% \nPFA and stained for either ATF-2 and/or p -ATF-2 (Santa Cruz) and nuclei (Hoechst). Cells \nwere scanned and analysed using the Cytell™ imaging system (38,39).     \nReal-Time Metabolic Analysis     \nThe oxygen consumption rate (OCR) of four patient-derived OC models (OCAS12, OCAS14, \nOCAS17, and OCAST16) was assessed in real-time using the Resipher system® (40). Briefly, \ncells were seeded in 96-well plates at 5,000 cells per well and incubated overnight. Then the \nbaseline level of OCR was measured for an additional 24h. Subsequently, the cultured \nmedium was replaced with fresh medium containing CAE at concentrations of 20, 40, or 80 \nµg/mL. OCR measurements were recorded continuously for 72 hours post -treatment. An \noverview of this experimental flow is shown in Figure 1.     \nLysosomal Mass/Acidity and mitochondrial membrane potential measurements      \nIn this study, patient-Derived OC cells (OCAS12, OCAS14, OCAS17 and OCAST16) were \nseeded in 96 -well plates overnight at a density of 5000 cells per well and subsequently \nexposed to several concentrations of CAE (20, 40, or 80 µg/mL), then incubated for 72h.  \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \nFollowing exposure to CAE cells were fixed with 3% PFA, washed in PBS and then imaged \nusing an inverted fluorescent microscope, and changes in mitochondrial membrane potential \n(MMP) and lysosomal mass/pH were scanned and analysed using the Cytell ™ imaging \nsystem (38,39).     \n     \nStatistical Analysis     \nAll the raw data from the investigated biological parameters were analysed using GraphPad \nPrism 8. All treatments were compared to the untreated cells for statistical significance. \nStatistical significance was determined using one-way ANOV A coupled with a nonparametric \nTukey’s post hoc test multiple comparison test for “*” for p<0.05, “**” for p<0.01, and “***” \nfor p<0.001. The data is presented as the mean ± standard error of the mean (n=3), and \nstatistical significance was defined by p<0.05.     \n               \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \nResults:     \nAssessment of cancer-associated and EMT markers in Patient-Derived OC cells     \nPatient-Derived OC cells were characterised for a number of cancer-associated and epithelial– \nmesenchymal transition (EMT) markers by immunocytochemistry, and the results are shown \nin Figure 2.     \n     \nPatient-Derived OC Models Display Distinct Sensitivity Patterns to Clove Extract     \nThe response of OCAS12 cells to CAE showed a clear time -dependent pattern. At 24 hours, \nthe CCK-8 assay revealed a robust dose-response relationship (Figure 3a): all concentrations \n(20, 40, and 80 µg/mL) significantly reduced cell viability compared with untreated controls \n(all p <0.001). The 40 -µg dose produced a significantly greater effect than 20 µg (p <0.01) \nbut increasing the dose from 40 µg to 80 µg offered no additional benefit, indicating an early \nplateau. By 72 hours (Figure 3b), this profile shif ted markedly; the lowest concentration (20 \nµg) produced a much stronger reduction in viability, nearly triple that seen at 24 hours and \nthe previous dose-dependency disappeared entirely. At this later time point, 20 µg performed \nequivalently to 40 µg and 80 µg (all p = ns), suggesting that prolonged exposure saturates the \ncytotoxic effect. OCAS14 cells demonstrated the opposite path. After 24 hours, all doses of \nCAEs significantly reduced viability relative to the control (p <0.001), but the concentrations \nwere indistinguishable from one another, suggesting immediate maximal efficacy (Figure 3c). \nAt 72 hours, however, OCAS14 cells developed a strong dose -response pattern (Figure 3d). \nBoth 40 µg and 80 µg achieved significantly greater cytotoxicity than 20 µg  (p <0.001), \nthough the effect again plateaued between the top two doses (p = ns).     \nOCAS17 cells exhibited an intermediate profile. At 24 hours, all doses significantly reduced \nviability, but only the comparison between 20 µg and 80 µg reached significance (p <0.05), \nindicating an early partial plateau (Figure 3e). By 72 hours, a clear do se-response emerged; \nboth 40 µg and 80 µg were significantly more effective than 20 µg (p <0.001), although the \ndifference between 40 µg and 80 µg remained non -significant (Figure 3f). OCAST16 cells \nshowed a more consistent behaviour across time. At both 24 and 72 hours (Figure 3 g&h), the \nCAE produced a clear dose-response relationship, with each concentration significantly more \ncytotoxic than the last (all p <0.001). The only exception occurred between 40 µg and 80 µg \nat 72 hours, where the effect reached  a plateau (p = ns), suggesting that 40 µg represents an \noptimal prolonged -exposure dose. Together, the four patient -derived models exhibited \nstrikingly contradictory response profiles. OCAS12 lost dose -dependency over time, while \nOCAS14 gained it only aft er prolonged exposure. OCAST16 displayed consistent \ndoseresponsiveness at both time points, whereas OCAS17 showed a gradual strengthening of \ndose dependence.      \n     \nCAE Differentially Regulates ATF2 Expression and Activates ATF2 Stress Signalling     \nCAE exerted distinct, model -specific effects on total ATF2 expression while consistently \nactivating ATF2-mediated stress signalling across all four OC models. In OCAS12 cells, total  \nATF2 levels were strongly and dose-dependently suppressed at all concentrations (all p <   \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \n0.001), with clear stepwise reductions between 20 µg and 40 µg and between 20 µg and 80 \nµg; however, suppression plateaued between the two highest doses (Figure 4a). OCAS14 cells \nexhibited a similar profile, showing pronounced ATF2 suppression across all concentrations \nwith preserved dose-dependent differences at the lower end of the range (Figure 4b). OCAS17 \ncells demonstrated a weaker but statistically significant reduction in ATF2 expression at all \ndoses (Figure 4c), with significance increasing in a concentration-dependent manner (20 µg, \np < 0.05; 40 µg, p < 0.01; 80 µg, p < 0.001). In contrast, OCAST16 cells uniquely displayed \na paradoxical increase in total ATF2 expression at all concentrations (Figure 4d), suggesting \nthe engagement of a distinct compe nsatory or adaptive regulatory mechanism that \ndifferentiates this model from the suppressive ATF2 profiles observed in OCAS12, OCAS14, \nand OCAS17.     \nDespite these divergent effects on total ATF2 abundance, CAE consistently increased \nphosphorylated ATF2 (P-ATF2) levels across all models, although the magnitude and dose \nsensitivity varied. OCAS12 cells showed a robust, concentration -dependent increase in  \nPATF2 (Figure 5a) at all doses (all p < 0.001), with the response plateauing at the highest \nconcentration. OCAS14 exhibited the most pronounced activation (Figure 5b), maintaining \nstatistically significant discrimination even between higher doses (40 µg vs 80 µg, p < 0.01). \nOCAS17 cells required higher concentrations to elicit significant P -ATF2 induction; 20 µg \nhad no effect, whereas both 40 µg and 80 µg produced significant increases (Figure 5c). In \nOCAST16 cells, P -ATF2 levels increased steadily across concentrations, with significant \ndifferences observed between the lowest and highest doses (Figure 5 d). Collectively, these \nfindings demonstrate that CAE broadly engages ATF2 -mediated stress signalling across \npatient-derived OC models, independent of its effects on total ATF2 protein levels, and \nhighlight cell-type-specific thresholds that govern the balance between ATF2 suppression and \nactivation.     \n     \nCAE Disrupts Mitochondrial Metabolism in 2D and 3D Models      \nIn two-dimensional (2D) cultures, CAE impaired mitochondrial oxidative phosphorylation \nacross all models, as measured by OCR (Figure 6 a -h). OCAS12 cells exhibited \ndosedependent OCR suppression (Figure 6 b), with a 17.24% reduction at the lowest \nconcentration (p < 0.05). OCAS14 showed a more modest response (Figure 6 d), with \nstatistical significance achieved only at 20 µg (p < 0.05). In contrast, OCAS17 was highly \nsensitive, showing a 27.70% reduction in OCR at 20 µg and near -complete suppression of \nmitochondrial respiration at higher concentrations (Figure 6 f). OCAST16 was comparatively \nresistant, displaying significant effects only when compared with untreated controls (Figure \n6 h). Longterm metabolic effects were further evaluated in three-dimensional patient-derived \norganoids (PDOs) over 8 days. CAE induced a model -dependent hypermetabolic response \ncharacterised by early metabolic stimulation followed by energetic collapse, consistent with \nprogressive mitochondrial dysfunction (Figure 7). Ascites-derived high-grade serous ovarian \ncancer (HGSOC) organoids (OCAS12 and OCAS14) responded earliest (Figures 8 &9), at \nDays 3 and 4, respectively (all p < 0.001), whereas tumour tissue –derived OCAS16 (Figure \n10) and carcinosarcoma-derived OCAS17 (Figure 11) organoids showed significant responses \nby Day   5 (p < 0.001 or p < 0.01). Although the timing of response initiation converged, the \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \nmetabolic curves diverged substantially. OCAS12 exhibited a transient metabolic peak \nfollowed by a decline, whereas OCAS14, OCAS16, and OCAS17 displayed sustained, \nexponential escalation indicative of uncontrolled compensatory metabolism. Notably, \nOCAS16 a nd OCAS17 developed the most severe and persistent hypermetabolic states, \nconsistent with heightened mitochondrial stress and reduced adaptive capacity.      \n     \nCAE-Induced Lysosomal Perturbation      \nIt is well established that certain cytotoxic agents disrupt cellular function through several \nmechanisms. These include an increase in mitochondrial membrane potential (MMP), damage \nto the cell membrane, and impaired organelle function, specifically throu gh the alteration of \nlysosomal pH or an increase in lysosome production (38, 39). To determine whether \nCAEinduced stress extended to the lysosomal compartment, lysosomal mass and acidity were \nmeasured across the four patient -derived OC cell lines. In OCAS1 2 cells, CAE induced a \nsignificant and progressive increase in lysosomal signal across all concentrations compared \nwith nontreated controls (20 µg and 40 µg, p < 0.05; 80 µg, p < 0.01), with no significant \ndifferences between doses, indicating a robust res ponse even at the lowest concentration \n(Figure 12a). OCAS14 cells exhibited a clear dose -dependent lysosomal response. While 20 \nµg had no effect, both 40 µg and 80 µg produced highly significant increases (p < 0.01 and p \n< 0.001, respectively). Significant stepwise differences were observed between 20 µg and 40 \nµg, 20 µg and 80 µg, and 40 µg and 80 µg, demonstrating strong concentration -dependent \nlysosomal perturbation (Figure 12b). Similarly to OCAS12, OCAS17 cells were highly \nsensitive, showing significant increases in lysosomal mass/acidity at all concentrations (20 µg \nand 40 µg, p < 0.01; 80 µg, p < 0.001), with effects plateauing at the lowest dose (Figure 12c). \nIn contrast, OCAST16 cells displayed a distinct threshold response. The 20 µg dose had no \neffect, whereas both 40 µg and 80 µg induced sharp and significant increases (p < 0.05 and p \n< 0.01, respectively), confirming that a critical concentration is required to disrupt lysosomal \nfunction in this model (Figure 12d).      \n     \nCAE Induces Mitochondrial Hyperpolarisation      \nMitochondrial membrane potential ( ΔΨm) was assessed to further evaluate mitochondrial \nstress. Rather than inducing depolarisation, CAE caused significant mitochondrial \nhyperpolarisation, a recognised early event in stress -induced cell death pathways. OCAS12 \ncells showed pronounced hyperpolarisation at all concentrations (20 µg and 40 µg, p < 0.01; \n80 µg, p < 0.001), with the 80 µg dose significantly exceeding the 40 µg response (p < 0.05), \nindicating dose -dependent intensification (Figure 13a). OCAS14 cells  exhibited \ndosedependent hyperpolarisation, with significant effects observed at 40 µg (p < 0.05) and 80 \nµg (p < 0.01), and a stronger response at 80 µg compared with 20 µg (p < 0.05) (Figure 13b). \nOCAS17 cells demonstrated a higher threshold for mitochond rial involvement, with \nsignificant hyperpolarisation detected only at the highest concentration (80 µg, p < 0.05), \nwhile lower doses had no effect (Figure 13c). Consistent with its behaviour in other assays, \nOCAST16 cells exhibited a strong threshold -dependent response, with significant \nhyperpolarisation induced at 40 µg (p < 0.05) and 80 µg (p < 0.01). Notably, the magnitude \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \nof this response was the greatest among all models, indicating particularly severe \nmitochondrial stress (Figure 13d).   Discussion   This study demonstrates that CAE exerts \npotent, multilayered cytotoxicity in OC through a coordinated lysosomal and mitochondrial \nstress program, revealing a previously unrecognised organelle -centric mechanism of action \nfor a natural product. By leveragin g patient -derived OC models, we captured clinically \nrelevant inter -patient heterogeneity, revealing that CAE induces cell dea th via distinct \ntemporal and dose-dependent pathways in different OC tumour subtypes, insights that would \nhave been masked in conventional immortalised cell lines.     \nWhile natural products have long contributed to anticancer drug discovery (17 –33), \ncomparatively few studies have explored their capacity to exploit organelle -specific \nvulnerabilities in OC. By employing PDO models, we captured clinically relevant \nheterogeneity that would likely be overlooked in conventional immortalised cell lines, \nhighlighting the translational importance of modelling patient diversity (41–45).     \nAt the molecular level, CAE differentially modulated ATF2, a stress-responsive transcription \nfactor implicated in DNA repair, invasion, and chemoresistance (46 –48). In OCAS12, \nOCAS14, and OCAS17, total ATF2 expression was reduced, whereas OCAST16 showed a \nparadoxical increase, likely reflecting a compensatory stress response rather than sustained \npathway activation. Notably, all models displayed elevated phosphorylated ATF2 (P-ATF2), \nalbeit with varying magnitude and dose sensitivity (49 –51), revealing that  CAE engages a \nconserved ATF2-mediated stress signalling axis independent of total protein levels. These \nfindings highlight the dynamic, context -dependent regulation of stress signalling in OC and \ndemonstrate that consistent cytotoxic outcomes can emerge f rom divergent molecular \nresponses.     \nCAE also profoundly disrupted mitochondrial function, reducing oxidative phosphorylation \nand inducing mitochondrial hyperpolarisation (ΔΨm), a recognised early apoptotic event (52– \n62). This metabolic collapse, reflected by dose-dependent reductions in oxygen consumption \nrate (OCR), highlights the bioenergetic vulnerability of OC cells. Concurrently, CAE \ntriggered lysosomal perturbations, evidenced by increased mass and acidity in a model - and \ndosedependent manner (63 -65). The parallel disruption of lysosom al and mitochondrial \ncompartments illustrates organelle crosstalk as a critical determinant of cell fate, consistent \nwith emerging literature on the role of lysosome -mitochondria interactions in apoptosis and \nchemoresistance (66-69).     \nThe heterogeneity in response across PDO models underscores the importance of \npatientstratified therapeutic strategies. OCAS12 exhibited early and robust sensitivity, \nsaturating at lower doses, whereas OCAS14 developed a delayed dose -response, and \nOCAST16 required higher concentrations to engage organelle stress. OCAS17, derived from \ncarcinosarcoma, demonstrated relative resistance, consistent with its intrinsic chemoresistant \nphenotype (37,70). These observations support the potential for functional precis ion \nmedicine, where dose and schedule are customised to each tumour’s vulnerabilities.     \nImportantly, CAE’s dual organelle targeting distinguishes it mechanistically from \nconventional chemotherapeutics, which predominantly disrupt DNA replication or \nmicrotubule dynamics (66-69). By simultaneously compromising mitochondrial metabolism \nand lysosomal function pathways central to chemoresistance, CAE or its purified constituents \ncould serve as noncrossresistant agents, either alone or as sensitisers to platinum - and \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \ntaxanebased therapy (71– 74). Moreover, the heterogeneity observed should not be viewed as \na limitation but as clinically actionable information, enabling the identification of tumours \nmost likely to benefit from CAEbased strategies through predictive biom arkers, such as \nmetabolic signatures, lysosomal regulators, or ATF2 activation states.     \nIn summary, CAE exerts potent anticancer activity in OC via a coordinated lysosomal – \nmitochondrial stress program, producing apoptosis through context -dependent yet \nmechanistically convergent pathways. The PDO platform proved essential, capturing \ninterpatient variability, informing personalised dosing, and uncovering mechanistic \ncomplexity whereby consistent cytotoxic outcomes arise from diverse molecular responses. \nThese findings establish a framework for the development of organelle -targeted natural \nproducts in OC and highlight the translational potential of clove -derived compounds in \nprecision oncology.        \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. 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The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \n   \nFigure 2:  Patient-Derived OC Cells and Phenotypical Characterisation.  OC cells \nestablished from the ascites samples were phenotypically profiled by cancer markers and \nEMT markers, including human epidermal growth factor receptor 2 (HER2/Neu), folate \nreceptor alpha (FRα), EGFR, βcatenin, -Claudin7 and Mucin-1.      \n     \n  \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \n     \n    \nFigure 3.  Cytotoxic Responses to \nCAE in Patient -Derived OC cells.  \nPanels (a-h) show cell viability assessed \nby CCK-8 assay at 24- and 72-hours for \nfour distinct patient -derived OC Cells \n(OCAS12, OCAS14, OCAS17, \nOCAST16). Cells were treated with no \ntreatment control (NT), 20, 40, and 80 \nµg/mL. Data are presented as mean ± \nSEM of n=3 independent experiments. \nStatistical significance is indicated as \n*p < 0.05, **p < 0.01, ***p < 0.001 \nversus control; brackets indicate \nsignificance between doses.      \n     \n     \n     \n     \n     \n     \n     \n     \n     \n     \n     \n \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \n     \n    \n \nFigure 4. Modulation of ATF2 Expression by CAE in OC Models.  Analysis of ATF2 \nprotein or mRNA levels in four patient -derived OC cells (OCAS12, OCAS14, OCAS17, \nOCAST16) following 24-hour treatment with CAE (0, 20, 40, 80 µg/mL). Data are presented \nas mean ±   SEM of n=3 independent experiments. Statistical significance was determined \nby one-way ANOV A with a post-hoc Tukey test. *p < 0.05, **p < 0.01, ***p < 0.001, ns = \nnot significant.     \n \n     \n     \n     \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \n \n          \nFigure 5. CAE activates the stress-responsive P-ATF-2: Quantification of P-ATF-2 level \nin (A) OCAS12, (B) OCAS14, (C) OCAS17, and (D) OCAST16 cells following treatment \nwith CAE. Data are normalised to the non -treated (NT) control and presented as mean ± \nSEM of n=3 independent experiments. Statistical significance was determined by one -way \nANOV A with a post-hoc Tukey test. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.      \n \n     \n  \n  \n  \n  \n  \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \n \nFigure 6. Clove aqueous extract acutely impairs mitochondrial oxidative \nphosphorylation in a cell line -dependent manner. Measurement of OCR, an indicator of \nmitochondrial oxida -   tive phosphorylation, in (a&b) OCAS12, (c&d) OCAS14, (e&f) \nOCAS17, and (g&h)  OCAST16 cells following 72 -hour treatment with CAE. Data are \nnormalised to the nontreated  (NT) control and presented as mean ± SEM of n=3 independent \nexperiments. Statistical significance was determined by one -way ANOV A with a post-hoc \nTukey test. *p < 0.05, **p <   0.01, ***p < 0.001, ns = not significant.     \n     \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n   \n  \n  \n  \n  \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \n \nFigure 7. Clove aqueous extract induces a biphasic hypermetabolic response preceding \nenergetic collapse in all PDOs (OCAS12, OCAS14, OCAS17 and OCAST16). Metabolic \nresponse curves of all OCPDOs treated with CAE over an 8-day interval, reflecting long-term \ntreatment effects in a model that closely mimics the patient tumour microenvironment.     \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \n  \n      \n \n     \nFigure 8. Clove aqueous extract induces a biphasic hypermetabolic response culminating \nin metabolic suppression in OCAS12 PDOs. Time-course analysis of metabolic activity in \nOCAS12 PDOs treated with CAE over 8 days. Data are normalised to the non -treated (NT) \ncontrol for each time point and presented as mean ± SEM of n=4 independent experiments. \nStatistical significance was determined by two-way ANOV A with a post-hoc Tukey test. *p <    \n0.05, **p < 0.01, ***p < 0.001, ns = not significant.     \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \n \nFigure 9. Clove aqueous extract induces a biphasic hypermetabolic response culminating \nin metabolic suppression in OCAS14 PDOs. Time-course analysis of metabolic activity in \nOCAS14 PDOs treated with CAE over 8 days. Data are normalised to the non -treated (NT) \ncontrol for each time point and presented as mean ± SEM of n=4 independent experiments. \nStatistical significance was determined by two-way ANOV A with a post-hoc Tukey test. *p <  \n0.05, **p < 0.01, ***p < 0.001, ns = not significant.     \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \n \nFigure 10. Clove aqueous extract induces a biphasic hypermetabolic response \nculminating in metabolic suppression in OCAS17 PDOs. Time-course analysis of \nmetabolic activity in OCAS17 PDOs treated with CAE over 8 days. Data are normalised to \nthe non -treated (NT) control for each time point and presented as mean ± SEM of n=4 \nindependent experiments. Statistical significance was determi ned by two-way ANOV A with \na post-hoc Tukey test. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.     \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \n \nFigure 11. Clove aqueous extract induces a biphasic hypermetabolic response \nculminating in metabolic suppression in OCAST16 PDOs.  Time-course analysis of \nmetabolic activity in OCAST16 PDOs treated with CAE over 8 days. Data are normalised to \nthe non -treated (NT) control for each time point and presented as mean ± SEM of n=4 \nindependent experiments. Statistical significance was deter mined by two-way ANOV A with \na post-hoc Tukey test. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.     \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \n \n       \n     \nFigure 12. Clove aqueous extract induces lysosomal distress in a cell line -dependent \nmanner. Quantification of lysosomal mass/pH in (A) OCAS12, (B) OCAS14, (C) OCAS17, \nand (D) OCAST16 cells following 24 -hour treatment with clove aqueous extract. Data are \nnormalised to the non-treated (NT) control and presented as mean ± SEM of n=3 independent \nexperiments. Statistical significance was determined by one -way ANOV A with a post-hoc \nTukey test. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.     \n     \n     \n     \n     \n         \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint \n\nbioRxiv preprint doi: https://doi.org/10.64898/2026.01.28.702206; this version posted January 30, 2026. The copyright holder has placed this \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nremix, or adapt this material for any purpose without crediting the original authors.  \n     \n \nFigure 13. Clove aqueous extract triggers mitochondrial membrane potential   \nhyperpolarisation. Quantification of mitochondrial membrane potential in (A) OCAS12, (B) \nOCAS14, (C) OCAS17, and (D) OCAST16 cells following treatment with clove aqueous \nextract. An increase in the metric indicates mitochondrial hyperpolarisation. Data are \nnormalised to the non-treated (NT) control and presented as mean ± SEM of n=3 independent \nexperiments. Statistical significance was determined by one -way ANOV A with a post-hoc \nTukey test. *p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant.     \n     \n     \n     \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \nremix, or adapt this material for any purpose without crediting the original authors. \npreprint (which was not certified by peer review) in the Public Domain. It is no longer restricted by copyright. Anyone can legally share, reuse, \nThe copyright holder has placed thisthis version posted February 2, 2026. ; https://doi.org/10.64898/2026.01.28.702206doi: bioRxiv preprint","source_license":"Public-Domain","license_restricted":false}