Investigation of the acute toxicity and antimelanoma effects of total leaf extract from Annona muricata Linn

Investigation of the acute toxicity and antimelanoma effects of total leaf extract from Annona muricata Linn | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Investigation of the acute toxicity and antimelanoma effects of total leaf extract from Annona muricata Linn Huynh Thao Vy Nguyen, Hoai Nam Nguyen, Bao Han Nguyen, Vy Kha Lam, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7680956/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Melanoma, the most aggressive and lethal form of skin cancer, accounts for only 4% of all skin malignancies but is responsible for 80% of all skin cancer-related deaths. This study aimed to investigate the safety and antitumor efficacy of the total leaf extract of Annona muricata Linn (AME) in a melanoma model in Swiss albino mice. Materials and methods The safety of AME was evaluated by testing the acute toxicity of AME in healthy mice. Melanoma tumors were induced by subcutaneous implantation of the B16F10 murine melanoma cell line into either immunocompetent or immunodeficient Swiss albino mice via cyclophosphamide. B16F10-innoculated mice were treated with AME either by oral administration at a dose of 200 mg/kg, by topical application at a concentration of 20 mg/mL or by a combination of oral (100 mg/kg) and topical (10 mg/mL) treatment. Tumor progression was monitored throughout the experiment by measuring tumor size weekly. At the end of the experiment, histological analysis of excised tumor tissues was performed. Results The estimated LD 50 of AME was approximately 2180.28 ± 84.76 mg/kg. The implantation of B16F10 cells into immunodeficient Swiss albino mice resulted in the development of solid tumors the day after implantation. The tumor size increased gradually, reaching its peak at week 3. Histological analysis revealed aggressive invasion of tumor cells into skeletal muscle and neural structures. Compared with no treatment, AME treatment significantly inhibited tumor growth (p < 0.01), whereas the combination of oral and topical treatments resulted in the lowest average tumor volume with a moderate invasion profile. Conclusion AME was slightly hazardous to Swiss albino mice. This extract exhibited antimelanoma effects by inhibiting tumor growth and progression when administered orally, when applied topically, and especially when used in combination. These findings support the potential of AME as a complementary therapeutic agent for melanoma treatment. B16F10 Cyclophosphamide Melanoma Swiss albino mice Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. BACKGROUND Cancer remains a paramount global health challenge, with 19.3 million new cases and 10.0 million deaths reported worldwide in 2022 [ 1 ]. Melanoma, the most aggressive and lethal form of skin cancer, accounts for only 4% of all skin malignancies but is responsible for 80% of skin cancer-related deaths [ 2 ]. Its increasing incidence is correlated with well-established risk factors, including ultraviolet radiation exposure, genetic predispositions, and immunosuppression [ 3 ]. Despite therapeutic advances, the prognosis of metastatic melanoma remains poor; the 5-year survival rate for patients with distant metastases is less than 30% [ 4 ]. Current treatment modalities include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy [ 5 , 6 ]. Conventional chemotherapy is often limited by systemic toxicity (such as bone marrow suppression, gastrointestinal toxicity, alopecia, and neuropathies), drug resistance and suboptimal long-term outcomes [ 7 ]. These challenges underscore the critical need to explore novel therapeutic agents such as natural plant extracts to increase treatment efficacy and improve patient tolerance [ 8 , 9 ]. Soursop ( Annona muricata Linn) has long been used in traditional medicine as a remedy for various ailments. Recent studies have identified more than 120 distinct acetogenin compounds isolated from different parts of the plant, including the leaves, fruits, and seeds. Notably, approximately 60 acetogenins have been found specifically in leaves. These acetogenins are a group of structurally complex compounds that exhibit a wide range of biological activities, including anti-inflammatory, antibacterial, and particularly anticancer properties. Among them, annoreticuin and isoannoreticuin have been identified as potent compounds that exhibit cytotoxic activity against various cancer cell lines [ 10 ]. Given its promising bioactive profile, the development of soursop leaf extract for cancer prevention and supportive therapy holds considerable clinical and practical significance. This study aimed to evaluate the acute toxicity of the total leaf extract of Annona muricata and investigate its antitumor effects on Swiss albino mice bearing B16F10-induced melanoma tumors. 2. MATERIALS AND METHODS 2.1. Animals : Healthy Swiss albino mice, aged 6–7 weeks and weighing 20–30 g, were supplied by the Institute of Drug Quality Control. The animals were housed in same-sex groups of six per cage under controlled environmental conditions (temperature of 22 ± 2°C, relative humidity of 60 ± 5%, and a 12-hour light/dark cycle with lights on from 08:00 to 20:00). All the mice had ad libitum access to standard rodent chow and drinking water throughout the study. All experimental protocols were approved by the Committee on Animal Research and Ethics of the University of Medicine and Pharmacy at Ho Chi Minh City under Decision No. 3674/GCN-HĐĐĐNCTĐV, dated 20/11/2024. 2.2. Total extract from soursop leaves : Soursop leaves were harvested in May 2024, washed, air-dried, and ground into a crude powder with 5–10 mm cubes. The leaf powder was subjected to exhaustive reflux extraction via 96% ethanol. The extraction was performed at atmospheric pressure under reflux conditions at the boiling point of the solvent (three cycles of 3 hours each, with a leaf powder-to-solvent ratio of approximately 1:10 per cycle). The combined extracts were concentrated at 45°C under reduced pressure, yielding Annona muricata leaf extract (AME) with an extraction efficiency of approximately 5% and a humidity of 13%. The resulting ethanol extract was stored under refrigerated conditions for subsequent animal experiments. 2.3. Chemicals : Endoxan (cyclophosphamide 500 mg) was acquired from Baxter Oncology, Germany; 10% formalin was obtained from Xilong, China; and Tween 80 was obtained from GHTech, JHD. 2.4. Evaluation of the acute toxicity of AME The acute oral toxicity of AME was evaluated via the Behrens method [ 11 ]. Healthy mice of both sexes were fasted for 12 h prior to AME administration. The AME was suspended in distilled water containing 1% Tween 80 at different concentrations of extract ranging from 100 mg/mL to 600 mg/mL. The mice were orally administered preprepared AME at a volume of 10 mL/kg body weight, delivering extract doses between 1000 mg/kg and 6000 mg/kg. Following AME administration, the animals were continuously observed for the first 4 hours, and survival rates were recorded over the initial 72-hour period. The mice were subsequently monitored daily for 14 days to assess signs of toxicity, behavioral changes, mortality, and other clinical symptoms. The median lethal dose (LD 50 ) was estimated via the Karber and Behrens method [ 11 ]. The resulting LD 50 value was classified according to the Globally Harmonized System Globally Harmonized Classification System (GHS) for Acute Toxicity Estimate (ATE) guidelines [ 12 ]. 2.5. Evaluation of the antimelanoma effects of AME in Swiss albino mice 2.5.1. Tumor cell culture and cell preparation for implantation in mice The B16F10 murine melanoma cell line, which was originally derived from the American Type Culture Collection (ATCC, USA), was kindly provided by the Stem Cell Institute, University of Science, Vietnam National University, Ho Chi Minh City. The cells were cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 Ham (DMEM/F12) at 37°C in a humidified incubator with 5% CO 2 . On the day of implantation, B16F10 cells were detached from the culture flasks by using a mixture of trypsin-ethylene diamine tetraacetic acid and washed twice with phosphate-buffered saline (PBS). The cells were subsequently counted and resuspended in PBS to achieve the concentrations required for subcutaneous injection. Three cell concentrations were prepared as follows: 5×10 5 cells/mL for the administration of 5×10 4 cells; 5×10 6 cells/mL for the administration of 5×10 5 cells; and 5×10 7 cells/mL for the administration of 5×10 6 cells in a 0.1 mL volume of injection. 2.5.2. Establishment of a B16F10 melanoma tumor model Prior to B16F10 cell implantation, the dorsal right flank (~ 2 × 2 cm²) of each mouse was shaved to facilitate subcutaneous administration and tumor monitoring. For immunosuppression, the mice in the designated groups received intramuscular injections of cyclophosphamide at a dose of 180 mg/kg on days − 5, -3, and − 1 relative to the day of B16F10 cell inoculation. The mice were randomly assigned to two experimental groups, an immunocompetent group and an immunodeficient group, the latter of which was induced by cyclophosphamide administration. In each group, the mice were subcutaneously injected with 0.1 mL of preprepared B16F10 cell suspensions (as described in section 2.5.1 ) at three densities: 5×10 4 , 5×10 5 and 5×10 6 cells per mouse. Moreover, a normal control group (nontumor-bearing mice), in which the mice received a subcutaneous injection of 0.1 mL of PBS, was also included. 2.5.3. Evaluation of the antimelanoma effects of AME The mice were subcutaneously injected with the optimal density of B16F10 cells to induce melanoma formation. Beginning the following day, the mice received daily treatments with AME via different administration routes: - PO200 group: Mice were orally given AME at a dose equivalent to approximately 1/10 of the previously estimated LD 50 . - TO20 group: Mice received a topical application of 30 µL/mouse of the same AME solution prepared for oral administration, which was applied to the shaved dorsal area. - Combination group (PO100 + TO10): Mice were treated with a combined regimen consisting of oral administration of AME at 1/20 of the LD 50 , along with topical application of the same solution to the shaved area. Moreover, an untreated control group in which melanoma-bearing mice received distilled water orally at a dose of 10 mL/kg body weight was included. 2.5.4. Evaluation criteria The experimental mice were weighed twice weekly during the experiment. The survival rate was recorded daily, and the latency period of tumor formation was noted. The tumor dimensions (width and length) were measured via a stereomicroscope connected to NIS-Elements software. The tumor volume (mm 3 ) was calculated according to the formula [ 13 ]: V = \(\:\frac{{\pi\:}\text{L}\text{W}²}{6}\) , where V is the tumor volume, L is the length, and W is the width of the tumor. At the end of the experiment, the mice were sacrificed, and the tumor tissue was harvested and fixed in 10% neutral buffered formalin. Tissues were embedded in paraffin wax, sectioned at a thickness of 4 µm, and stained with hematoxylin and eosin (H&E). The stained slides were examined under a light microscope for histopathological analysis at different magnifications. Tumor invasion depth was classified according to Clark levels [ 14 ]. 2.6. Statistical analysis The data were processed and analyzed via Minitab 20 software and Microsoft Excel 2016. The results are expressed as the mean ± standard error of the mean (SEM). Statistical analyses, including one-way ANOVA and the Mann‒Whitney test, were performed via Minitab 20 software. Differences were considered statistically significant when p < 0.05. 3. RESULTS 3.1. Acute toxicity of AME Within 15 to 60 min following the oral administration of AME extract, the mice exhibited a progressive decline in physical activity, accompanied by an increased respiratory rate and signs of abdominal contraction. Prior to death, affected mice exhibited convulsions and extension of the hind limbs. In the group receiving high doses of AME (> 3000 mg/kg), the majority of the mice died within two days post-administration. The mice that survived beyond the initial 2-day period gradually recovered and exhibited no abnormal clinical signs during the subsequent 14-day observation period. The mortality rate increased in a dose-dependent manner, as shown in Table 1 . The LD 50 of AME was calculated to be 2180.28 ± 84.76 mg/kg on the basis of the Karber and Behren method. According to the GHS, this LD 50 value places the extract in Category III (slightly hazadous) for oral exposure, which corresponds to substances with LD 50 values ranging from 2000 to 5000 mg/kg [ 12 ]. Table 1 Dose-dependent mortality rate of mice in the acute toxicity study of AME extract Dose (mg/kg) 1000 2000 2250 2500 3000 4000 6000 Number of mice per group 6 10 10 10 10 10 6 Number of deaths 0 3 4 6 7 8 6 Mortality rate 0 30 40 60 70 80 100 On the basis of this estimated LD 50 value, two doses were selected for oral administration in the antitumor efficacy study: 200 mg/kg and 100 mg/kg, corresponding to approximately 1/10 and 1/20 of the LD 50 , respectively. For this purpose, AME extract was dissolved in distilled water to prepare solutions at concentrations of 20 mg/mL and 10 mg/mL. A volume of 0.1 mL per 10 g of body weight was administered orally to each mouse. Additionally, the same solutions were applied topically to the shaved dorsal area of the mice to evaluate the effect of topical treatment. 3.2. Simulation of a melanoma model in Swiss albino mice In the immunocompetent group, mouse mortality was dependent on the density of B16F10 cell inoculation. At the lowest concentration (5×10 4 cells per mouse), no mortality was observed, whereas the highest concentration resulted in approximately 50% mortality. The rate of tumor formation was proportional to the number of implanted cells in immunocompetent mice. The injection of 5×10 6 cells per mouse resulted in immediate tumor formation, with visible blackening at the injection site observed as early as one day post-injection. The injection of 5×10 5 cells or 5×10 cells per mouse led to tumor development after a latency period ranging from 5–9 days (Table 2 ). Moreover, the resulting tumors in the immunocompetent group were soft and loosely textured upon palpation (Fig. 1 ). Table 2 Survival and tumor latency according to B16F10 cell density in immunodeficient vs. immunocompetent mice Group Number of cell implantation n Survival rate (%) Tumor latency (day) (Mean ± SD) Immunocompetence 5×10 4 6 100 9 ± 2 5×10 5 7 57.1 5 ± 4 5×10 6 6 50.0 1 ± 0 Immunodeficience 5×10 4 10 60.0 2 ± 1 5×10 5 10 60.0 1 ± 0 5×10 6 10 60.0 1 ± 0 Control --- 12 100.0 ---- In the immunodeficient group induced with cyclophosphamide, the survival rate was only 60%. Most tumors formed as early as the day following injection and exhibited continuous growth. Upon palpation, the tumors were firm and solid in texture (Fig. 1 ). Moreover, the blackened area at the injection site of 5×10 6 cells was noticeably larger than those observed at the two lower cell densities. The tumor size in these three groups increased rapidly from Day 1 to Week 2. Consistently, all three groups exhibited a steady increase in tumor volume from Day 1, reaching its peak at Week 3, indicating active tumor proliferation. At this time point, the group injected with 5×10 5 cells showed the most aggressive growth (~ 350 mm 3 ), followed by the group injected with 5×10 4 cells (~ 300 mm 3 ) and the group injected with 5×10 6 cells (~ 250 mm 3 ). After Week 3, the tumor volume in all the groups began to decrease. From week 5 onward, signs of surface necrosis were observed. By the end of the experiment (week 8), the group injected with 5×10 5 cells retained the largest tumor volume (Fig. 2 ). Histological examination of the tumors in the immunodeficient group confirmed the presence of malignant melanoma cells within the epidermal layer. The tumors were characterized by proliferating melanocytes with enlarged nuclei and cytoplasm containing abundant brown‒black melanin granules (Fig. 3 ). Microvascular proliferation was observed in tumor tissues across all three experimental groups. In mice injected with 5x10 5 cells, the tumor displayed a nodular architecture, with tumor cells aggressively invading skeletal muscle and neural structures, reaching a maximum invasion depth of 3 mm. In the two remaining groups of mice, tumor cells infiltrated the dermal soft tissues, including adipose tissue and skeletal muscle, with a recorded invasion depth of 1.5 mm. Notably, no evidence of vascular or perineural invasion was detected in the mice injected with 5x10 4 cells; however, such invasions were observed in the group injected with 5x10 6 cells. Therefore, for the in vivo modeling of malignant melanoma in Swiss albino individuals , a protocol utilizing immunodeficient mice pretreated with cyclophosphamide, followed by the subcutaneous injection of B16F10 melanoma cells at a density of 5x10 5 cells in 0.1 ml of PBS, was selected to investigate the antimelanoma effects of AME. 3.3. Antimelanoma effects of Annona muricata leaf extract After the 6-week experimental period, the combination of oral and topical treatments resulted in a survival rate (75.0%) comparable to that of the untreated group. In contrast, the oral administration group presented a greater survival rate, whereas the topical application group presented a lower survival rate (Table 3 ). In the untreated group, the tumor volume increased rapidly during the first three weeks (Fig. 4 ). Compared with the untreated group, the PO200 group also presented an increase in tumor volume, although to a lesser extent. Notably, high-concentration topical treatment (TO20) and combination therapy (P100 + TO10) significantly inhibited tumor growth from Week 2 to Week 6. Table 3 Survival rates of the AME-treated and untreated groups Groups n Survival rate (%) PO200 14 42.9 TO20 7 85.7 PO100 + TO10 8 75.0 Untreated 8 75.0 At the end of the experiment, the tumors in the untreated group were markedly large with dark, well-defined margins (Fig. 5 ). Specifically, the tumors in the combination treatment group were the smallest and lightest in color among all the groups. Histological analysis revealed vascular invasion in 100% of the samples from the three AME-treated and untreated groups (Fig. 6 ). The PO200 group presented the lowest average Clark level (4.25 ± 0.5) and the shallowest invasion depth (1.0 ± 0.0 mm). In contrast, both remaining AME-treated groups reached Clark level V (Table 4 ). Moreover, the topical group had the smallest average horizontal tumor diameter (3.0 mm), which was significantly different from that of the untreated group. In the PO200 group, the dermis contained tumor tissue composed of proliferating melanocytes with large nuclei and abundant cytoplasm with melanin pigment. Furthermore, the tumor cells invaded the reticular dermis. Moreover, in the TO20 group, the dermis contained tumor tissue composed of proliferating melanocytes with large nuclei and abundant cytoplasm with melanin pigment. Additionally, these tumor cells were observed to invade the subcutaneous soft tissue. Similarly, in the combination group (P100 + TO10), the dermis contained tumor tissue composed of proliferating melanocytes with large nuclei and abundant cytoplasm with melanin pigment. Moreover, these tumor cells also invade the subcutaneous soft tissue. Finally, in the untreated group, the dermis contained tumor tissue composed of proliferating melanocytes with large nuclei and abundant cytoplasm with melanin pigment. Likewise, these tumor cells were found to invade the subcutaneous soft tissue (Fig. 6 ). 4. DISCUSSIONS Annona muricata Linn is a tropical plant species recognized for its edible fruit, which exhibits significant medicinal properties alongside reported toxicological concerns. In our study, oral administration of Annona muricata leaf extract (AME) resulted in an estimated LD 50 of approximately 2180.28 ± 84.76 mg/kg, which is considered “slightly hazardous” according to the GHS classification. In vivo studies by De Sousa OV et al. (2010) demonstrated that oral administration of the ethanol extract of Annona muricata leaves resulted in an LD 50 of 1670 mg/kg (95% CI: 1240–2260 mg/kg) [ 15 ]. Similarly, Acesio NO et al. (2017) reported 100% mortality in all experimental groups within 24 hours following oral administration of a 96% ethanol extract of Annona muricata leaves at doses ranging from 1000 to 2000 mg/kg [ 16 ]. In contrast, an acute toxicity study by Siti Norliyana Zubaidi et al. (2023) indicated that the 80% ethanol extract of Annona muricata leaves could be safely administered at a dose of 2000 mg/kg [ 17 ]. Furthermore, Agu et al. (2017) reported a moderately hazardous LD 50 value of 1918.33 mg/kg for leaf and fruit extracts from methanol [ 18 ], which may be attributed to the use of methanol as the extraction solvent. The methanol extract demonstrated a distinc extraction efficiency compared to the ethanol extract and was associated with the co-extraction of a broader range of potentially toxic compounds, which may contribute to the increased toxicity observed. The leaf extract of Annona muricata contains several bioactive constituents, primarily acetogenins, alkaloids, and flavonoids. Among these, acetogenins are the predominant compounds and are considered chiefly responsible for the acute toxicity of the leaf, particularly its neurotoxic effects [ 19 ]. Notably, several studies have demonstrated that acetogenins isolated from Annona muricata leaves exhibit cytotoxic activity against tumor cells [ 20 – 26 ], suggesting their potential therapeutic relevance. However, the presence of these compounds may also contribute to the observed toxic effects of ethanol-based extracts. Therefore, the future development of ethanol extracts from Annona muricata leaves as therapeutic agents requires rigorous standardization of both the raw material and the extraction process to ensure consistent safety and efficacy. In our study, determining acute toxicity is a critical step in establishing the safety profile of AME and was used to define the therapeutic doses (100 and 200 mg/kg, equivalent to 1/20 and 1/10 of the LD 50 , respectively) for evaluating its antimelanoma effects. Moreover, further investigations should include subchronic toxicity assessments to evaluate potential long-term cumulative effects, as well as detailed chemical characterization to elucidate the relationships between specific acetogenin contents and both toxicological and pharmacological outcomes. Melanoma is recognized as the most dangerous type of skin cancer and is characterized by high mortality rates and a propensity for developing metastatic lesions that evade immune surveillance [ 27 ]. The B16F10 cell line, which is used extensively in melanoma research, represents the 10th subclone of the parental B16 cell line. Originally derived from C57BL/6 mice, this pigment-producing melanoma cell line has strong metastatic potential, with the ability to spread to organs such as the spleen, liver, and lungs [ 28 , 29 ]. Although implantation of the B16F10 melanoma cell line in the C57BL/6 inbred mouse strain is widely employed in experimental oncology models because of its immunocompatibility and reproducibility [ 30 , 31 ], maintenance and breeding of the C57BL/6 inbred mouse strain can present logistical and financial challenges in certain research settings. As a practical alternative, Swiss albino mice offer several advantages, including ease of handling, lower cost, and broader availability. When combined with an appropriate immunosuppression protocol, Swiss albino mice are capable of supporting B16F10 cell inoculation and can serve as a viable model for melanoma research, providing a more accessible and cost-effective platform for preclinical evaluation. Our results revealed significant differences in tumor development between immunocompetent and immunodeficient Swiss albino mice. In the group that did not receive immune inhibitors, the tumors were soft and loosely textured upon palpation. In contrast, mice pretreated with cyclophosphamide developed solid tumors with firm consistency, indicating more aggressive growth characteristics. These findings are consistent with those of previous studies worldwide [ 32 , 33 ] and suggest that immunosuppression plays a critical role in facilitating solid tumor formation and progression in this melanoma model. Melanoma tumors in the immunodeficient group were observed as early as one day post-injection and exhibited continuous growth throughout the study period. Interestingly, the growth dynamics varied depending on the number of cells injected. The group that received 5×10 6 cells presented the earliest tumor formation, whereas the group that received 5×10 5 cells presented the most aggressive growth, reaching approximately 350 mm 3 by week 3. This was followed by the 5×10 4 cell group (approximately 300 mm 3 ) and the 5×10 6 cell group (approximately 250 mm 3 ). The relatively slower tumor growth observed in the group injected with 5×10 6 cells may be attributed to a 'cell overcrowding' effect. In a nutrient- and oxygen-limited microenvironment, excessive cell density can lead to competition for resources, thereby restricting tumor expansion [ 34 , 35 ]. Rapidly growing tumors consume large amounts of oxygen and nutrients; if the vascular system does not develop in time to meet this demand, regions within the tumor may become necrotic and shrink. To compensate, tumors can stimulate angiogenesis to restore the oxygen supply, enabling cancer cells to survive and regrow. In this study, necrosis was observed in both groups injected with 5×10 5 and 5×10 6 cells, likely due to rapid tumor growth and subsequent ischemia. In contrast, no necrosis was detected in the group injected with 5×10 4 cells, possibly due to the smaller tumor size and reduced metabolic demand [ 34 , 36 ]. The present study provides preliminary yet valuable evidence of the antitumor efficacy of Annona muricata leaf extract (AME) against melanoma in a murine model. Both oral and topical routes of administration exhibited distinct therapeutic effects. Oral administration of AME in the PO200 group was associated with the lowest average Clark level (4.25 ± 0.5) and the shallowest invasion depth (1.0 ± 0.0 mm), suggesting that systemic delivery may play a pivotal role in suppressing vertical tumor invasion and limiting deep tissue penetration. In contrast, high-concentration topical treatment in the TO20 group significantly restricted horizontal tumor expansion, resulting in the smallest average tumor diameter (3.0 mm), highlighting its efficacy in controlling the local tumor burden. Compared with the other groups, the combination group (P100 + TO10) presented the greatest overall therapeutic effect, with consistent tumor volume suppression from Week 2 to Week 6 and visibly smaller, lighter tumors. These findings highlight the complementary benefits of dual-route administration: oral delivery enables the systemic distribution of bioactive compounds to target disseminated tumor cells, whereas topical application ensures high local concentrations at the tumor site to inhibit surface-level proliferation. This synergistic approach aligns with current evidence supporting multimodal delivery strategies in cancer therapy, which often outperform monotherapies by addressing both systemic and localized disease components [ 37 , 38 ]. Despite these promising antitumor effects observed with AME, several limitations remain. Histological analyses revealed persistent vascular invasion and subcutaneous infiltration across all treatment groups, indicating that tumor progression was not completely suppressed. Additionally, the study employed only a single dose for each administration route, which precludes a comprehensive evaluation of dose‒response relationships and optimal therapeutic windows. The absence of biochemical and molecular data further limits mechanistic interpretation, making distinguishing between the systemic and localized effects of AME difficult. These limitations warrant careful consideration and underscore important directions for future research. First, dose-escalation experiments are essential for determining both the minimum effective dose and the maximum tolerated dose of AME. Second, biochemical and immunohistochemical analyses should be conducted to elucidate the molecular mechanisms underlying its antitumor effects. Third, long-term safety assessments, including subchronic toxicity and recurrence monitoring, are necessary to assess the sustainability and potential risks of prolonged use. Moreover, future investigations should explore combination strategies involving AME and established therapies, such as immune checkpoint inhibitors or BRAF/MEK inhibitors, to evaluate potential synergistic effects and broaden its clinical applicability in melanoma treatment. Integrative therapeutic approaches may facilitate the positioning of Annona muricata leaf extract as an adjuvant agent in melanoma management, thereby bridging traditional herbal medicine with contemporary oncological practice. 5. CONCLUSION Annona muricata leaf extract demonstrated a favorable safety profile (LD 50 : 2180.28 ± 84.76 mg/kg). In a B16F10 melanoma mouse model in immunodeficient Swiss albino mice, oral administration or topical application, especially combination treatment, significantly reduced the tumor burden and inhibited tumor progression. These findings support its potential as a promising adjuvant therapy for melanoma. Declarations Ethics approval and consent to participate All experimental protocols were approved by the Committee on Animal Research and Ethics of the University of Medicine and Pharmacy at Ho Chi Minh City under Decision No. 3674/GCN-HĐĐĐNCTĐV, dated 20/11/2024. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. Competing interests All authors declare no competing interest. Funding This study was not funded by any organisations. Authors' contributions Conceived and designed experiments: NT-H, HP-D and HTV-N. Performed experiments: HN-N, BH-N, VK-L and HTV-N. Analyzed data: NT-H and HTV-N. Manuscript drafting and revision: NT-H, HP-D and HTV-N. All authors read and agreed to the final manuscript. Acknowledgements We acknowledge University of Medicine and Pharmacy at Ho Chi Minh city, Pham Ngoc Thach University of Medicine (Ho Chi Minh City, Vietnam) for supporting this study. Clinical trial number Not applicable. References Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians. 2024;74(3):229-63. Arnold M, Singh D, Laversanne M, Vignat J, Vaccarella S, Meheus F, et al. Global Burden of Cutaneous Melanoma in 2020 and Projections to 2040. JAMA dermatology. 2022;158(5):495-503. Institute NC. SEER Cancer Stat Facts: Melanoma of the Skin2024. Available from: https://seer.cancer.gov/statfacts/html/melan.html. 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Molecules (Basel, Switzerland). 2014;19(8):11679-721. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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07:36:48","extension":"xml","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":112760,"visible":true,"origin":"","legend":"","description":"","filename":"e78775f4012249a1bb087761792c344e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7680956/v1/1bcca793dc60336b6d9ccab5.xml"},{"id":92057756,"identity":"d38b05b0-946e-4bba-bf8f-d88a1e7f44a5","added_by":"auto","created_at":"2025-09-24 07:20:48","extension":"html","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127084,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7680956/v1/5cbaec9868d6fcd02f12afb3.html"},{"id":92057732,"identity":"7729758e-b365-4cec-982a-ffad18feec77","added_by":"auto","created_at":"2025-09-24 07:20:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":781834,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopic images of tumors from immunocompetent (A) and immunodeficient (B) mice under a stereomicroscope after surgery at the end of the experiment.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7680956/v1/bdd7cbf8fd3ecfcf6cf626e2.png"},{"id":92057731,"identity":"af1f27c2-da44-4c48-9416-6de52f2dd706","added_by":"auto","created_at":"2025-09-24 07:20:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":152765,"visible":true,"origin":"","legend":"\u003cp\u003eTumor volume (mm\u003csup\u003e3\u003c/sup\u003e) by B16F10 cell density in the immunodeficient group\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNote: * = p \u0026lt; 0.05 vs 5x10\u003c/em\u003e\u003csup\u003e\u003cem\u003e5\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e cells/mouse\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7680956/v1/07d681a7793f950ed811a072.png"},{"id":92057733,"identity":"9e4104d8-64a5-4406-8ef2-97fdcfb636ea","added_by":"auto","created_at":"2025-09-24 07:20:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1638430,"visible":true,"origin":"","legend":"\u003cp\u003eHistological images of tumor sections under 40X objective magnification\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e(A) 5x10\u003c/em\u003e\u003csup\u003e\u003cem\u003e4\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e cells/mouse, (B) 5x10\u003c/em\u003e\u003csup\u003e\u003cem\u003e5\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e cells/mouse, (C) 5x10\u003c/em\u003e\u003csup\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e cells/mouse\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7680956/v1/237dea37337c96c999fcaca8.png"},{"id":92057734,"identity":"bd2d1f01-748a-4a8c-bb42-cfca2cfa6f6b","added_by":"auto","created_at":"2025-09-24 07:20:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":148202,"visible":true,"origin":"","legend":"\u003cp\u003eTumor volume (mm\u003csup\u003e3\u003c/sup\u003e) in the AME-treated and untreated \u003cem\u003egroups\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNote: * = p \u0026lt; 0.05 vs untreated\u003c/em\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7680956/v1/75f77810c02b398634c85123.png"},{"id":92059220,"identity":"7c906de9-54e2-40d8-83ad-707198c00f95","added_by":"auto","created_at":"2025-09-24 07:44:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":984809,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopic tumor features of AME-treated mice after oral administration (A), topical application (B) or combination (C) and the untreated group (D)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7680956/v1/87deffe50f8a753abe979f99.png"},{"id":92058460,"identity":"31578607-2b65-4205-aa3f-2cb2089c8078","added_by":"auto","created_at":"2025-09-24 07:28:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1121939,"visible":true,"origin":"","legend":"\u003cp\u003eHistological images of tumor sections under 40X objective magnification\u003c/p\u003e\n\u003cp\u003e(A) PO200; (B) TO20; (C) PO100+TO10; (D) Untreated\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7680956/v1/fdc7599dc5799b7f7a2569d4.png"},{"id":92070863,"identity":"996a1603-d429-4f44-9cfc-6ec5db0b325e","added_by":"auto","created_at":"2025-09-24 09:39:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8389237,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7680956/v1/60b388e2-20ba-45f4-a440-dac74f78f334.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigation of the acute toxicity and antimelanoma effects of total leaf extract from Annona muricata Linn","fulltext":[{"header":"1. BACKGROUND","content":"\u003cp\u003eCancer remains a paramount global health challenge, with 19.3\u0026nbsp;million new cases and 10.0\u0026nbsp;million deaths reported worldwide in 2022 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Melanoma, the most aggressive and lethal form of skin cancer, accounts for only 4% of all skin malignancies but is responsible for 80% of skin cancer-related deaths [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Its increasing incidence is correlated with well-established risk factors, including ultraviolet radiation exposure, genetic predispositions, and immunosuppression [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite therapeutic advances, the prognosis of metastatic melanoma remains poor; the 5-year survival rate for patients with distant metastases is less than 30% [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Current treatment modalities include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Conventional chemotherapy is often limited by systemic toxicity (such as bone marrow suppression, gastrointestinal toxicity, alopecia, and neuropathies), drug resistance and suboptimal long-term outcomes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These challenges underscore the critical need to explore novel therapeutic agents such as natural plant extracts to increase treatment efficacy and improve patient tolerance [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSoursop (\u003cem\u003eAnnona muricata\u003c/em\u003e Linn) has long been used in traditional medicine as a remedy for various ailments. Recent studies have identified more than 120 distinct acetogenin compounds isolated from different parts of the plant, including the leaves, fruits, and seeds. Notably, approximately 60 acetogenins have been found specifically in leaves. These acetogenins are a group of structurally complex compounds that exhibit a wide range of biological activities, including anti-inflammatory, antibacterial, and particularly anticancer properties. Among them, annoreticuin and isoannoreticuin have been identified as potent compounds that exhibit cytotoxic activity against various cancer cell lines [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Given its promising bioactive profile, the development of soursop leaf extract for cancer prevention and supportive therapy holds considerable clinical and practical significance. This study aimed to evaluate the acute toxicity of the total leaf extract of \u003cem\u003eAnnona muricata\u003c/em\u003e and investigate its antitumor effects on Swiss \u003cem\u003ealbino\u003c/em\u003e mice bearing B16F10-induced melanoma tumors.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e2.1. Animals\u003c/strong\u003e: Healthy Swiss \u003cem\u003ealbino\u003c/em\u003e mice, aged 6\u0026ndash;7 weeks and weighing 20\u0026ndash;30 g, were supplied by the Institute of Drug Quality Control. The animals were housed in same-sex groups of six per cage under controlled environmental conditions (temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, relative humidity of 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5%, and a 12-hour light/dark cycle with lights on from 08:00 to 20:00). All the mice had \u003cem\u003ead libitum\u003c/em\u003e access to standard rodent chow and drinking water throughout the study.\u003c/p\u003e\n\u003cp\u003eAll experimental protocols were approved by the Committee on Animal Research and Ethics of the University of Medicine and Pharmacy at Ho Chi Minh City under Decision No. 3674/GCN-HĐĐĐNCTĐV, dated 20/11/2024.\u003c/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003cstrong\u003e2.2. Total extract from soursop leaves\u003c/strong\u003e: Soursop leaves were harvested in May 2024, washed, air-dried, and ground into a crude powder with 5\u0026ndash;10 mm cubes. The leaf powder was subjected to exhaustive reflux extraction via 96% ethanol. The extraction was performed at atmospheric pressure under reflux conditions at the boiling point of the solvent (three cycles of 3 hours each, with a leaf powder-to-solvent ratio of approximately 1:10 per cycle). The combined extracts were concentrated at 45\u0026deg;C under reduced pressure, yielding\u0026nbsp;\u003cem\u003eAnnona muricata\u003c/em\u003e leaf extract (AME) with an extraction efficiency of approximately 5% and a humidity of 13%. The resulting ethanol extract was stored under refrigerated conditions for subsequent animal experiments.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e\u003cstrong\u003e2.3. Chemicals\u003c/strong\u003e: Endoxan (cyclophosphamide 500 mg) was acquired from Baxter Oncology, Germany; 10% formalin was obtained from Xilong, China; and Tween 80 was obtained from GHTech, JHD.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e\u003cstrong\u003e2.4. Evaluation of the acute toxicity of AME\u003c/strong\u003e\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eThe acute oral toxicity of AME was evaluated via the Behrens method [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. Healthy mice of both sexes were fasted for 12 h prior to AME administration. The AME was suspended in distilled water containing 1% Tween 80 at different concentrations of extract ranging from 100 mg/mL to 600 mg/mL. The mice were orally administered preprepared AME at a volume of 10 mL/kg body weight, delivering extract doses between 1000 mg/kg and 6000 mg/kg. Following AME administration, the animals were continuously observed for the first 4 hours, and survival rates were recorded over the initial 72-hour period. The mice were subsequently monitored daily for 14 days to assess signs of toxicity, behavioral changes, mortality, and other clinical symptoms. The median lethal dose (LD\u003csub\u003e50\u003c/sub\u003e) was estimated via the Karber and Behrens method [\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. The resulting LD\u003csub\u003e50\u003c/sub\u003e value was classified according to the Globally Harmonized System Globally Harmonized Classification System (GHS) for Acute Toxicity Estimate (ATE) guidelines [\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Evaluation of the antimelanoma effects of AME in Swiss \u003cem\u003ealbino\u003c/em\u003e mice\u003c/h2\u003e\n \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e\n \u003ch2\u003e2.5.1. Tumor cell culture and cell preparation for implantation in mice\u003c/h2\u003e\n \u003cp\u003eThe B16F10 murine melanoma cell line, which was originally derived from the American Type Culture Collection (ATCC, USA), was kindly provided by the Stem Cell Institute, University of Science, Vietnam National University, Ho Chi Minh City. The cells were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium/nutrient mixture F-12 Ham (DMEM/F12) at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003eOn the day of implantation, B16F10 cells were detached from the culture flasks by using a mixture of trypsin-ethylene diamine tetraacetic acid and washed twice with phosphate-buffered saline (PBS). The cells were subsequently counted and resuspended in PBS to achieve the concentrations required for subcutaneous injection. Three cell concentrations were prepared as follows: 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL for the administration of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells; 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL for the administration of 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells; and 5\u0026times;10\u003csup\u003e7\u003c/sup\u003e cells/mL for the administration of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells in a 0.1 mL volume of injection.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\n \u003ch2\u003e2.5.2. Establishment of a B16F10 melanoma tumor model\u003c/h2\u003e\n \u003cp\u003ePrior to B16F10 cell implantation, the dorsal right flank (~\u0026thinsp;2 \u0026times; 2 cm\u0026sup2;) of each mouse was shaved to facilitate subcutaneous administration and tumor monitoring. For immunosuppression, the mice in the designated groups received intramuscular injections of cyclophosphamide at a dose of 180 mg/kg on days \u0026minus;\u0026thinsp;5, -3, and \u0026minus;\u0026thinsp;1 relative to the day of B16F10 cell inoculation. The mice were randomly assigned to two experimental groups, an immunocompetent group and an immunodeficient group, the latter of which was induced by cyclophosphamide administration. In each group, the mice were subcutaneously injected with 0.1 mL of preprepared B16F10 cell suspensions (as described in section \u003cspan class=\"InternalRef\"\u003e2.5.1\u003c/span\u003e) at three densities: 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e, 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e and 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells per mouse. Moreover, a normal control group (nontumor-bearing mice), in which the mice received a subcutaneous injection of 0.1 mL of PBS, was also included.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\n \u003ch2\u003e2.5.3. Evaluation of the antimelanoma effects of AME\u003c/h2\u003e\n \u003cp\u003eThe mice were subcutaneously injected with the optimal density of B16F10 cells to induce melanoma formation. Beginning the following day, the mice received daily treatments with AME via different administration routes:\u003c/p\u003e- PO200 group: Mice were orally given AME at a dose equivalent to approximately 1/10 of the previously estimated LD\u003csub\u003e50\u003c/sub\u003e.\u003cp\u003e- TO20 group: Mice received a topical application of 30 \u0026micro;L/mouse of the same AME solution prepared for oral administration, which was applied to the shaved dorsal area.\u003c/p\u003e\n \u003cp\u003e- Combination group (PO100\u0026thinsp;+\u0026thinsp;TO10): Mice were treated with a combined regimen consisting of oral administration of AME at 1/20 of the LD\u003csub\u003e50\u003c/sub\u003e, along with topical application of the same solution to the shaved area.\u003c/p\u003eMoreover, an untreated control group in which melanoma-bearing mice received distilled water orally at a dose of 10 mL/kg body weight was included.\n \u003c/div\u003e\n \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\n \u003ch2\u003e2.5.4. Evaluation criteria\u003c/h2\u003e\n \u003cp\u003eThe experimental mice were weighed twice weekly during the experiment. The survival rate was recorded daily, and the latency period of tumor formation was noted. The tumor dimensions (width and length) were measured via a stereomicroscope connected to NIS-Elements software. The tumor volume (mm\u003csup\u003e3\u003c/sup\u003e) was calculated according to the formula [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]: V = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{{\\pi\\:}\\text{L}\\text{W}\u0026sup2;}{6}\\)\u003c/span\u003e\u003c/span\u003e, where V is the tumor volume, L is the length, and W is the width of the tumor.\u003c/p\u003e\u003cp\u003eAt the end of the experiment, the mice were sacrificed, and the tumor tissue was harvested and fixed in 10% neutral buffered formalin. Tissues were embedded in paraffin wax, sectioned at a thickness of 4 \u0026micro;m, and stained with hematoxylin and eosin (H\u0026amp;E). The stained slides were examined under a light microscope for histopathological analysis at different magnifications. Tumor invasion depth was classified according to Clark levels [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e\u003cp\u003eThe data were processed and analyzed via Minitab 20 software and Microsoft Excel 2016. The results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analyses, including one-way ANOVA and the Mann‒Whitney test, were performed via Minitab 20 software. Differences were considered statistically significant when p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Acute toxicity of AME\u003c/h2\u003e\u003cp\u003eWithin 15 to 60 min following the oral administration of AME extract, the mice exhibited a progressive decline in physical activity, accompanied by an increased respiratory rate and signs of abdominal contraction. Prior to death, affected mice exhibited convulsions and extension of the hind limbs. In the group receiving high doses of AME (\u0026gt;\u0026thinsp;3000 mg/kg), the majority of the mice died within two days post-administration. The mice that survived beyond the initial 2-day period gradually recovered and exhibited no abnormal clinical signs during the subsequent 14-day observation period. The mortality rate increased in a dose-dependent manner, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The LD\u003csub\u003e50\u003c/sub\u003e of AME was calculated to be 2180.28\u0026thinsp;\u0026plusmn;\u0026thinsp;84.76 mg/kg on the basis of the Karber and Behren method. According to the GHS, this LD\u003csub\u003e50\u003c/sub\u003e value places the extract in Category III (slightly hazadous) for oral exposure, which corresponds to substances with LD\u003csub\u003e50\u003c/sub\u003e values ranging from 2000 to 5000 mg/kg [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDose-dependent mortality rate of mice in the acute toxicity study of AME extract\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDose (mg/kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2250\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2500\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4000\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e6000\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of mice per group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber of deaths\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMortality rate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eOn the basis of this estimated LD\u003csub\u003e50\u003c/sub\u003e value, two doses were selected for oral administration in the antitumor efficacy study: 200 mg/kg and 100 mg/kg, corresponding to approximately 1/10 and 1/20 of the LD\u003csub\u003e50\u003c/sub\u003e, respectively. For this purpose, AME extract was dissolved in distilled water to prepare solutions at concentrations of 20 mg/mL and 10 mg/mL. A volume of 0.1 mL per 10 g of body weight was administered orally to each mouse. Additionally, the same solutions were applied topically to the shaved dorsal area of the mice to evaluate the effect of topical treatment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Simulation of a melanoma model in Swiss \u003cem\u003ealbino\u003c/em\u003e mice\u003c/h2\u003e\u003cp\u003eIn the immunocompetent group, mouse mortality was dependent on the density of B16F10 cell inoculation. At the lowest concentration (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per mouse), no mortality was observed, whereas the highest concentration resulted in approximately 50% mortality. The rate of tumor formation was proportional to the number of implanted cells in immunocompetent mice. The injection of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells per mouse resulted in immediate tumor formation, with visible blackening at the injection site observed as early as one day post-injection. The injection of 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells or 5\u0026times;10 cells per mouse led to tumor development after a latency period ranging from 5\u0026ndash;9 days (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Moreover, the resulting tumors in the immunocompetent group were soft and loosely textured upon palpation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSurvival and tumor latency according to B16F10 cell density in immunodeficient \u003cem\u003evs.\u003c/em\u003e immunocompetent mice\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroup\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNumber of \u003c/p\u003e\u003cp\u003ecell implantation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSurvival rate (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTumor latency (day)\u003c/p\u003e\u003cp\u003e(Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eImmunocompetence\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9 \u0026plusmn; 2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e57.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5 \u0026plusmn; 4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026times;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1 \u0026plusmn; 0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eImmunodeficience\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026times;10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2 \u0026plusmn; 1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026times;10\u003csup\u003e5\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1 \u0026plusmn; 0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u0026times;10\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1 \u0026plusmn; 0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e---\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e100.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e----\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn the immunodeficient group induced with cyclophosphamide, the survival rate was only 60%. Most tumors formed as early as the day following injection and exhibited continuous growth. Upon palpation, the tumors were firm and solid in texture (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Moreover, the blackened area at the injection site of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells was noticeably larger than those observed at the two lower cell densities. The tumor size in these three groups increased rapidly from Day 1 to Week 2. Consistently, all three groups exhibited a steady increase in tumor volume from Day 1, reaching its peak at Week 3, indicating active tumor proliferation. At this time point, the group injected with 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells showed the most aggressive growth (~\u0026thinsp;350 mm\u003csup\u003e3\u003c/sup\u003e), followed by the group injected with 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells (~\u0026thinsp;300 mm\u003csup\u003e3\u003c/sup\u003e) and the group injected with 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells (~\u0026thinsp;250 mm\u003csup\u003e3\u003c/sup\u003e). After Week 3, the tumor volume in all the groups began to decrease. From week 5 onward, signs of surface necrosis were observed. By the end of the experiment (week 8), the group injected with 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells retained the largest tumor volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHistological examination of the tumors in the immunodeficient group confirmed the presence of malignant melanoma cells within the epidermal layer. The tumors were characterized by proliferating melanocytes with enlarged nuclei and cytoplasm containing abundant brown‒black melanin granules (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Microvascular proliferation was observed in tumor tissues across all three experimental groups. In mice injected with 5x10\u003csup\u003e5\u003c/sup\u003e cells, the tumor displayed a nodular architecture, with tumor cells aggressively invading skeletal muscle and neural structures, reaching a maximum invasion depth of 3 mm. In the two remaining groups of mice, tumor cells infiltrated the dermal soft tissues, including adipose tissue and skeletal muscle, with a recorded invasion depth of 1.5 mm. Notably, no evidence of vascular or perineural invasion was detected in the mice injected with 5x10\u003csup\u003e4\u003c/sup\u003e cells; however, such invasions were observed in the group injected with 5x10\u003csup\u003e6\u003c/sup\u003e cells.\u003c/p\u003e\u003cp\u003eTherefore, for the \u003cem\u003ein vivo\u003c/em\u003e modeling of malignant melanoma in Swiss \u003cem\u003ealbino individuals\u003c/em\u003e, a protocol utilizing immunodeficient mice pretreated with cyclophosphamide, followed by the subcutaneous injection of B16F10 melanoma cells at a density of 5x10\u003csup\u003e5\u003c/sup\u003e cells in 0.1 ml of PBS, was selected to investigate the antimelanoma effects of AME.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Antimelanoma effects of \u003cem\u003eAnnona muricata\u003c/em\u003e leaf extract\u003c/h2\u003e\u003cp\u003eAfter the 6-week experimental period, the combination of oral and topical treatments resulted in a survival rate (75.0%) comparable to that of the untreated group. In contrast, the oral administration group presented a greater survival rate, whereas the topical application group presented a lower survival rate (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the untreated group, the tumor volume increased rapidly during the first three weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Compared with the untreated group, the PO200 group also presented an increase in tumor volume, although to a lesser extent. Notably, high-concentration topical treatment (TO20) and combination therapy (P100\u0026thinsp;+\u0026thinsp;TO10) significantly inhibited tumor growth from Week 2 to Week 6.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSurvival rates of the AME-treated and untreated groups\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGroups\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003en\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSurvival rate (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePO200\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e42.9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTO20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e85.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePO100\u0026thinsp;+\u0026thinsp;TO10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e75.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUntreated\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e75.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAt the end of the experiment, the tumors in the untreated group were markedly large with dark, well-defined margins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Specifically, the tumors in the combination treatment group were the smallest and lightest in color among all the groups. Histological analysis revealed vascular invasion in 100% of the samples from the three AME-treated and untreated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The PO200 group presented the lowest average Clark level (4.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5) and the shallowest invasion depth (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 mm). In contrast, both remaining AME-treated groups reached Clark level V (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Moreover, the topical group had the smallest average horizontal tumor diameter (3.0 mm), which was significantly different from that of the untreated group. In the PO200 group, the dermis contained tumor tissue composed of proliferating melanocytes with large nuclei and abundant cytoplasm with melanin pigment. Furthermore, the tumor cells invaded the reticular dermis. Moreover, in the TO20 group, the dermis contained tumor tissue composed of proliferating melanocytes with large nuclei and abundant cytoplasm with melanin pigment. Additionally, these tumor cells were observed to invade the subcutaneous soft tissue. Similarly, in the combination group (P100\u0026thinsp;+\u0026thinsp;TO10), the dermis contained tumor tissue composed of proliferating melanocytes with large nuclei and abundant cytoplasm with melanin pigment. Moreover, these tumor cells also invade the subcutaneous soft tissue. Finally, in the untreated group, the dermis contained tumor tissue composed of proliferating melanocytes with large nuclei and abundant cytoplasm with melanin pigment. Likewise, these tumor cells were found to invade the subcutaneous soft tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n"},{"header":"4. DISCUSSIONS","content":"\u003cp\u003e\u003cem\u003eAnnona muricata\u003c/em\u003e Linn is a tropical plant species recognized for its edible fruit, which exhibits significant medicinal properties alongside reported toxicological concerns. In our study, oral administration of \u003cem\u003eAnnona muricata\u003c/em\u003e leaf extract (AME) resulted in an estimated LD\u003csub\u003e50\u003c/sub\u003e of approximately 2180.28\u0026thinsp;\u0026plusmn;\u0026thinsp;84.76 mg/kg, which is considered \u0026ldquo;slightly hazardous\u0026rdquo; according to the GHS classification. \u003cem\u003eIn vivo\u003c/em\u003e studies by De Sousa OV et al. (2010) demonstrated that oral administration of the ethanol extract of \u003cem\u003eAnnona muricata\u003c/em\u003e leaves resulted in an LD\u003csub\u003e50\u003c/sub\u003e of 1670 mg/kg (95% CI: 1240\u0026ndash;2260 mg/kg) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Similarly, Acesio NO et al. (2017) reported 100% mortality in all experimental groups within 24 hours following oral administration of a 96% ethanol extract of \u003cem\u003eAnnona muricata\u003c/em\u003e leaves at doses ranging from 1000 to 2000 mg/kg [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In contrast, an acute toxicity study by Siti Norliyana Zubaidi et al. (2023) indicated that the 80% ethanol extract of \u003cem\u003eAnnona muricata\u003c/em\u003e leaves could be safely administered at a dose of 2000 mg/kg [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, Agu et al. (2017) reported a moderately hazardous LD\u003csub\u003e50\u003c/sub\u003e value of 1918.33 mg/kg for leaf and fruit extracts from methanol [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], which may be attributed to the use of methanol as the extraction solvent. The methanol extract demonstrated a distinc extraction efficiency compared to the ethanol extract and was associated with the co-extraction of a broader range of potentially toxic compounds, which may contribute to the increased toxicity observed.\u003c/p\u003e\u003cp\u003eThe leaf extract of \u003cem\u003eAnnona muricata\u003c/em\u003e contains several bioactive constituents, primarily acetogenins, alkaloids, and flavonoids. Among these, acetogenins are the predominant compounds and are considered chiefly responsible for the acute toxicity of the leaf, particularly its neurotoxic effects [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Notably, several studies have demonstrated that acetogenins isolated from \u003cem\u003eAnnona muricata\u003c/em\u003e leaves exhibit cytotoxic activity against tumor cells [\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], suggesting their potential therapeutic relevance. However, the presence of these compounds may also contribute to the observed toxic effects of ethanol-based extracts. Therefore, the future development of ethanol extracts from \u003cem\u003eAnnona muricata\u003c/em\u003e leaves as therapeutic agents requires rigorous standardization of both the raw material and the extraction process to ensure consistent safety and efficacy. In our study, determining acute toxicity is a critical step in establishing the safety profile of AME and was used to define the therapeutic doses (100 and 200 mg/kg, equivalent to 1/20 and 1/10 of the LD\u003csub\u003e50\u003c/sub\u003e, respectively) for evaluating its antimelanoma effects. Moreover, further investigations should include subchronic toxicity assessments to evaluate potential long-term cumulative effects, as well as detailed chemical characterization to elucidate the relationships between specific acetogenin contents and both toxicological and pharmacological outcomes.\u003c/p\u003e\u003cp\u003eMelanoma is recognized as the most dangerous type of skin cancer and is characterized by high mortality rates and a propensity for developing metastatic lesions that evade immune surveillance [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The B16F10 cell line, which is used extensively in melanoma research, represents the 10th subclone of the parental B16 cell line. Originally derived from C57BL/6 mice, this pigment-producing melanoma cell line has strong metastatic potential, with the ability to spread to organs such as the spleen, liver, and lungs [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Although implantation of the B16F10 melanoma cell line in the C57BL/6 inbred mouse strain is widely employed in experimental oncology models because of its immunocompatibility and reproducibility [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], maintenance and breeding of the C57BL/6 inbred mouse strain can present logistical and financial challenges in certain research settings. As a practical alternative, Swiss \u003cem\u003ealbino\u003c/em\u003e mice offer several advantages, including ease of handling, lower cost, and broader availability. When combined with an appropriate immunosuppression protocol, Swiss \u003cem\u003ealbino\u003c/em\u003e mice are capable of supporting B16F10 cell inoculation and can serve as a viable model for melanoma research, providing a more accessible and cost-effective platform for preclinical evaluation.\u003c/p\u003e\u003cp\u003eOur results revealed significant differences in tumor development between immunocompetent and immunodeficient Swiss \u003cem\u003ealbino\u003c/em\u003e mice. In the group that did not receive immune inhibitors, the tumors were soft and loosely textured upon palpation. In contrast, mice pretreated with cyclophosphamide developed solid tumors with firm consistency, indicating more aggressive growth characteristics. These findings are consistent with those of previous studies worldwide [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and suggest that immunosuppression plays a critical role in facilitating solid tumor formation and progression in this melanoma model. Melanoma tumors in the immunodeficient group were observed as early as one day post-injection and exhibited continuous growth throughout the study period. Interestingly, the growth dynamics varied depending on the number of cells injected. The group that received 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells presented the earliest tumor formation, whereas the group that received 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells presented the most aggressive growth, reaching approximately 350 mm\u003csup\u003e3\u003c/sup\u003e by week 3. This was followed by the 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cell group (approximately 300 mm\u003csup\u003e3\u003c/sup\u003e) and the 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cell group (approximately 250 mm\u003csup\u003e3\u003c/sup\u003e). The relatively slower tumor growth observed in the group injected with 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells may be attributed to a 'cell overcrowding' effect. In a nutrient- and oxygen-limited microenvironment, excessive cell density can lead to competition for resources, thereby restricting tumor expansion [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Rapidly growing tumors consume large amounts of oxygen and nutrients; if the vascular system does not develop in time to meet this demand, regions within the tumor may become necrotic and shrink. To compensate, tumors can stimulate angiogenesis to restore the oxygen supply, enabling cancer cells to survive and regrow. In this study, necrosis was observed in both groups injected with 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e and 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells, likely due to rapid tumor growth and subsequent ischemia. In contrast, no necrosis was detected in the group injected with 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells, possibly due to the smaller tumor size and reduced metabolic demand [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe present study provides preliminary yet valuable evidence of the antitumor efficacy of \u003cem\u003eAnnona muricata\u003c/em\u003e leaf extract (AME) against melanoma in a murine model. Both oral and topical routes of administration exhibited distinct therapeutic effects. Oral administration of AME in the PO200 group was associated with the lowest average Clark level (4.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5) and the shallowest invasion depth (1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 mm), suggesting that systemic delivery may play a pivotal role in suppressing vertical tumor invasion and limiting deep tissue penetration. In contrast, high-concentration topical treatment in the TO20 group significantly restricted horizontal tumor expansion, resulting in the smallest average tumor diameter (3.0 mm), highlighting its efficacy in controlling the local tumor burden. Compared with the other groups, the combination group (P100\u0026thinsp;+\u0026thinsp;TO10) presented the greatest overall therapeutic effect, with consistent tumor volume suppression from Week 2 to Week 6 and visibly smaller, lighter tumors. These findings highlight the complementary benefits of dual-route administration: oral delivery enables the systemic distribution of bioactive compounds to target disseminated tumor cells, whereas topical application ensures high local concentrations at the tumor site to inhibit surface-level proliferation. This synergistic approach aligns with current evidence supporting multimodal delivery strategies in cancer therapy, which often outperform monotherapies by addressing both systemic and localized disease components [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite these promising antitumor effects observed with AME, several limitations remain. Histological analyses revealed persistent vascular invasion and subcutaneous infiltration across all treatment groups, indicating that tumor progression was not completely suppressed. Additionally, the study employed only a single dose for each administration route, which precludes a comprehensive evaluation of dose‒response relationships and optimal therapeutic windows. The absence of biochemical and molecular data further limits mechanistic interpretation, making distinguishing between the systemic and localized effects of AME difficult. These limitations warrant careful consideration and underscore important directions for future research. First, dose-escalation experiments are essential for determining both the minimum effective dose and the maximum tolerated dose of AME. Second, biochemical and immunohistochemical analyses should be conducted to elucidate the molecular mechanisms underlying its antitumor effects. Third, long-term safety assessments, including subchronic toxicity and recurrence monitoring, are necessary to assess the sustainability and potential risks of prolonged use. Moreover, future investigations should explore combination strategies involving AME and established therapies, such as immune checkpoint inhibitors or BRAF/MEK inhibitors, to evaluate potential synergistic effects and broaden its clinical applicability in melanoma treatment. Integrative therapeutic approaches may facilitate the positioning of \u003cem\u003eAnnona muricata\u003c/em\u003e leaf extract as an adjuvant agent in melanoma management, thereby bridging traditional herbal medicine with contemporary oncological practice.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003e\u003cem\u003eAnnona muricata\u003c/em\u003e leaf extract demonstrated a favorable safety profile (LD\u003csub\u003e50\u003c/sub\u003e: 2180.28\u0026thinsp;\u0026plusmn;\u0026thinsp;84.76 mg/kg). In a B16F10 melanoma mouse model in immunodeficient Swiss \u003cem\u003ealbino\u003c/em\u003e mice, oral administration or topical application, especially combination treatment, significantly reduced the tumor burden and inhibited tumor progression. These findings support its potential as a promising adjuvant therapy for melanoma.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols were approved by the Committee on Animal Research and Ethics of the University of Medicine and Pharmacy at Ho Chi Minh City under Decision No. 3674/GCN-HĐĐĐNCTĐV, dated 20/11/2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was not funded by any organisations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceived and designed experiments: NT-H, HP-D and HTV-N. Performed experiments: HN-N, BH-N, VK-L and HTV-N. Analyzed data: NT-H and HTV-N. Manuscript drafting and revision: NT-H, HP-D and HTV-N. All authors read and agreed to the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge University of Medicine and Pharmacy at Ho Chi Minh city, Pham Ngoc Thach University of Medicine (Ho Chi Minh City, Vietnam) for supporting this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. 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Molecules (Basel, Switzerland). 2014;19(8):11679-721.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"B16F10, Cyclophosphamide, Melanoma, Swiss albino mice","lastPublishedDoi":"10.21203/rs.3.rs-7680956/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7680956/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eMelanoma, the most aggressive and lethal form of skin cancer, accounts for only 4% of all skin malignancies but is responsible for 80% of all skin cancer-related deaths. This study aimed to investigate the safety and antitumor efficacy of the total leaf extract of \u003cem\u003eAnnona muricata\u003c/em\u003e Linn (AME) in a melanoma model in Swiss \u003cem\u003ealbino\u003c/em\u003e mice.\u003c/p\u003e\u003ch2\u003eMaterials and methods\u003c/h2\u003e\u003cp\u003eThe safety of AME was evaluated by testing the acute toxicity of AME in healthy mice. Melanoma tumors were induced by subcutaneous implantation of the B16F10 murine melanoma cell line into either immunocompetent or immunodeficient Swiss \u003cem\u003ealbino\u003c/em\u003e mice \u003cem\u003evia\u003c/em\u003e cyclophosphamide. B16F10-innoculated mice were treated with AME either by oral administration at a dose of 200 mg/kg, by topical application at a concentration of 20 mg/mL or by a combination of oral (100 mg/kg) and topical (10 mg/mL) treatment. Tumor progression was monitored throughout the experiment by measuring tumor size weekly. At the end of the experiment, histological analysis of excised tumor tissues was performed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe estimated LD\u003csub\u003e50\u003c/sub\u003e of AME was approximately 2180.28\u0026thinsp;\u0026plusmn;\u0026thinsp;84.76 mg/kg. The implantation of B16F10 cells into immunodeficient Swiss \u003cem\u003ealbino\u003c/em\u003e mice resulted in the development of solid tumors the day after implantation. The tumor size increased gradually, reaching its peak at week 3. Histological analysis revealed aggressive invasion of tumor cells into skeletal muscle and neural structures. Compared with no treatment, AME treatment significantly inhibited tumor growth (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas the combination of oral and topical treatments resulted in the lowest average tumor volume with a moderate invasion profile.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eAME was slightly hazardous to Swiss \u003cem\u003ealbino\u003c/em\u003e mice. This extract exhibited antimelanoma effects by inhibiting tumor growth and progression when administered orally, when applied topically, and especially when used in combination. These findings support the potential of AME as a complementary therapeutic agent for melanoma treatment.\u003c/p\u003e","manuscriptTitle":"Investigation of the acute toxicity and antimelanoma effects of total leaf extract from Annona muricata Linn","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-24 07:20:43","doi":"10.21203/rs.3.rs-7680956/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"557598ec-10d0-4210-99db-d804381b333c","owner":[],"postedDate":"September 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-24T09:38:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-24 07:20:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7680956","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7680956","identity":"rs-7680956","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}