Synergistic Anticancer Effects of Flavonoid and Acetogenin Fractions from Annona muricata Leaves in Leukemic and Peripheral Blood Cells

Suganya Ilango¹*, Priyanka Jayachandran², Ramalingam Nirmaladevi³

¹Department of Biochemistry, Faculty of Science and Humanities, SRM Institute of Science and Technology, Kattankulathur Campus, Chengalpattu-603203, Tamil Nadu, India.

²Department of Biochemistry and Biotechnology, Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore 641108, Tamil Nadu, India.

³Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore-641043, Tamil Nadu, India.

*Corresponding Author E-mail: suganyai@srmist.edu.in

ABSTRACT:

Annona muricata (soursop) leaves are rich in bioactive compounds with known anticancer properties. Flavonoids and acetogenins, key constituents of these leaves, have shown potential antileukemic effects individually, but their combined efficacy and selectivity remain underexplored. The primary aim of the research is to examine the synergistic anticancer effects of flavonoid and acetogenin enriched fractions from A. muricata leaves on leukemic (Molt-3) cells and assess their cytotoxicity on normal peripheral blood lymphocytes (PBL). Ethanolic extracts of A. muricata leaves were subjected to bioassay-guided fractionation and chromatographic separation to isolate flavonoid and acetogenin enriched fractions. GC-MS was used to identify phytoconstituents. Cytotoxicity was assessed using MTT assay, and synergy was analysed using Combenefit software based on Loewe, Bliss, and Highest Single Agent models. Apoptosis and cell cycle changes were evaluated using flow cytometry. GC-MS analysis revealed the detection of flavonoids such as myricetin and luteolin glucosides, and acetogenins like muricatacin and α-muurolene. The combination of flavonoid and acetogenin fractions exhibited enhanced cytotoxicity in Molt-3 cells (IC₅₀: 40.21 μg/ml) compared to individual treatments. Synergy was confirmed across all models, particularly at 60 μg/ml of flavonoids and 80–100 μg/ml of acetogenins. Flow cytometry showed increased G0/G1 arrest and apoptosis in Molt-3 cells, while PBLs remained largely unaffected, indicating selectivity. Flavonoid and acetogenin fractions from A. muricata exert synergistic, selective cytotoxic effects on leukemic cells while sparing normal cells, suggesting their potential as a natural, targeted therapeutic approach for leukemia.

KEYWORDS: Synergistic effect, Annona muricata, antileukemic activity, bioactive components, leukemia cells.

INTRODUCTION:

Leukemia, a hematological malignancy originating from blood-forming tissues, continues to represent a substantial global health concern despite ongoing advancements in therapeutic interventions. Conventional chemotherapy, though effective, is often associated with severe side effects and resistance in patients, underlining the need for alternative therapeutic approaches that are both effective and selective in targeting cancer cells. In recent years, natural plant products have acquired substantial recognition as potential anticancer properties attributed to their diverse bioactive compounds and lower toxicity profiles ^1,2,3.

Annona muricata, commonly referred to as soursop or graviola, is a tropical plant widely recognized for its traditional use in ethnomedicine across various cultures due to its diverse pharmacological properties. Several studies have highlighted the anticancer potential of A. muricata, particularly attributing its effects to two major classes of phytochemicals—acetogenins and flavonoids ^4,5. Acetogenins are a group of polyketide-derived fatty acid derivatives that have been reported to inhibit mitochondrial complex I and promote apoptosis within cancerous cells ^6,7. Flavonoids are a class of polyphenolic compounds recognized for their antioxidant, anti-inflammatory, and anticancer properties, and are known to influence key signaling pathways that regulate cell proliferation and                  apoptosis ^8,9.

Although both flavonoids and acetogenins have individually demonstrated antileukemic properties, their combined effect has not been thoroughly investigated. It is hypothesized that a combination of these bioactive fractions may exert a synergistic cytotoxic effect on leukemic cells while minimizing toxicity to normal cells. The present study aims to evaluate the synergistic anticancer effects of flavonoid and acetogenin enriched fractions from A. muricata leaves in leukemic Molt-3 cells and assess their safety in peripheral blood lymphocytes (PBL), providing insights into their potential as natural therapeutic agents.

MATERIALS AND METHODS

Annona muricata leaf powder was subjected to maceration in 95% ethanol for a duration of five days. The ethanol was subsequently removed using a rotary evaporator, and the resulting residue was redissolved in acetone to reduce chlorophyll content. The solution was then filtered through a Buchner funnel using silica gel 60 layered on filter paper. Fractionation of the crude extract was performed by sequential leaching with different solvent systems: water, water-ethanol (7:3 v/v), and water-ethanol (1:1 v/v), yielding fractions F1, F2, and F3, respectively. A combination of ethanol, ethanol-ethyl acetate (1:1 v/v), and ethyl acetate was used to obtain fraction F4. Phytochemical analysis of F4 confirmed the presence of flavonoids. The sample was concentrated at 50ºC and designated as the Flavonoid Enriched Fraction (FEF) for use in subsequent experimental investigations. Further fractionation of the FEF was performed using a 50 cm open column chromatography setup. The F4 fraction was subjected to elution with a series of solvent systems—hexane, hexane-chloroform (8:2 v/v), hexane-chloroform (1:1 v/v), and ethyl acetate—on a silica gel column. The resulting F4C eluate was analysed for the presence of annonaceous acetogenins using Kedde’s reagent. Fractions testing positive were concentrated at 50ºC and labelled as the Acetogenin Enriched Fraction (AEF) for experimental purposes.

GC-MS analysis was carried out on the FEF, and AEF obtained from Annona muricata leaves to identify their phytochemical constituents. Cytotoxicity was evaluated using the MTT assay, which measures cell viability based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in cell lines. To assess potential combinatorial effects, the synergistic interaction between the FEF and AEF was analysed using Combenefit software, which quantifies synergy, additivity, or antagonism between compound pairs. Cell cycle analysis was carried out using flow cytometry (BD FACSVerse) to determine the influence of the ethanolic crude extract, FEF, AEF and combination of FEF &AEF on the cell cycle progression in Molt-3 cells and peripheral blood lymphocytes (PBL), following standard flow cytometric protocols. To elucidate the mechanism of cell death, apoptosis induction by the crude extract, FEF, AEF and combination of FEF &AEF was examined using Annexin V/FITC-PI staining. The proportion of apoptotic cells was measured using the Annexin V/FITC Apoptosis Detection Kit (BD Biosciences) through flow cytometry. Data, expressed as mean ± SD, were statistically evaluated using one-way ANOVA followed by Tukey’s post hoc test in GraphPad Prism version 8.4.1, with a significance threshold set at P < 0.05.

RESULTS AND DISCUSSION:

GCMS analysis:

Bioactive components present in the FEF were identified from the GC–MS profile (Table 1) by comparing their mass spectra with the NIST library database ¹⁰.

Table 1. Bioactive constituents identified in FEF of Annona muricata leaves

Apex RT	Start RT	End RT	Area	%Area	Height	%Height	Compound
7.81	7.48	7.99	1667.946	2.73	242.389	3.43	Isoferulic acid
7.92	7.72	7.99	7711.012	12.64	966.360	13.67	Pentanedioic acid
8.42	8.24	8.66	5496.529	9.01	655.096	10.27	Myricetin
8.63	8.42	8.81	9509.375	15.59	1026.307	13.92	Apigenin-6-C-glucoside
8.82	8.62	8.91	21415.233	35.10	2216.587	31.36	Luteolin 3 ́7-di-O-glucoside
9.94	9.64	9.99	3751.875	6.15	483.352	6.84	Glycitein

Table 2. Bioactive constituents identified in AEF of Annona muricata leaves

Apex RT	Start RT	End RT	Area	%Area	Height	%Height	Compound
9.40	9.37	9.48	3968.079	14.82	961.089	20.82	α-Muurolene
11.79	11.71	11.83	3120.326	11.65	738.019	15.99	Cis-solamin
11.95	11.86	12.09	8600.178	32.12	1685.298	36.51	Muricatacin
12.73	12.82	13.15	11087.702	41.41	1231.612	26.68	Germacrene B

Table 2 contains comprehensive tabulations of the AEF's GC-MS analysis, was identified by comparing the mass spectra components with the NIST library.

MTT analysis:

MTT assay results demonstrated that the ethanolic leaf extract of Annona muricata and its derived fractions were effective in inducing cell death even at lower concentrations. Upon 24-hour exposure of Molt-3 leukemic cells to increasing concentrations of the ethanolic extract and its fractions, a significant increase in cell mortality was observed compared to the control group. Among the various treatments, the combination of the FEF and AEF exhibited the highest cytotoxic activity, followed by AEF, FEF, and the crude extract at a concentration of 500 μg/mL. The IC₅₀ values were determined to be 93.28 μg/mL for the ethanolic crude extract, 79.67 μg/mL for FEF, 64.37 μg/mL for AEF, and 40.21 μg/mL for the combined FEF and AEF treatment, respectively.

Cytotoxic effect on PBL, results indicated that as the concentration of the fractions increased, the extent of cell death decreased. The IC₅₀ values were 5.28 μg/mL for the ethanolic crude extract, 10.21 μg/mL for the FEF, 18.92 μg/mL for the AEF, and 37.67 μg/mL for the combination of FEF and AEF. These findings confirm the non-cytotoxic nature of the treatment groups in normal peripheral blood lymphocytes (PBLs) while selectively targeting cancerous cells. This study demonstrates that the combination of flavonoid and acetogenin enriched fractions from Annona muricata leaves exerts a synergistic cytotoxic effect on Molt-3 leukemic cells, with minimal toxicity to normal PBLs. This selective cytotoxicity aligns with recent findings that highlight the promising anticancer potential of A. muricata phytoconstituents, especially in selectively targeting malignant cells while sparing normal            ones ^{3,11,12,13,14}. The observed synergistic effect between flavonoid and acetogenin enriched fractions of Annona muricata in leukemic cells underscores the therapeutic potential of combinatorial phytotherapy. The enhanced cytotoxicity in Molt-3 cells, accompanied by minimal impact on peripheral blood lymphocytes (PBL), is indicative of a selective mechanism that targets malignant cells—a hallmark of effective and safe anticancer agents ^{2, 15}.

Synergistic effect of FEF and AEF:

Synergy is the term used to characterize combined effects that are stronger than the sum of their separate effects. The surface responses of FEF and AEF on Molt-3 cells are shown in Figure 1. The level of synergism is indicated by the dark blue coloured regions. The interaction between FEF and AEF was very certainly additive/synergistic over the entire dose-response matrix, as shown by the Loewe synergy and antagonism surface map (Figure 1a), with the largest synergistic effect centred at 60μg/ml of FEF and 80 or 100μg/ml of AEF. The plot, on the other hand, presented some antagonistic pairings. In general, there was a high level of agreement between the Loewe additive approach and the other two methods utilised (Highest Single Agent and Bliss) (Figure 1b and 1c), indicating that the overall results for each combination were appropriate.

The observed synergistic effect can be attributed to the distinct but complementary mechanisms of action of the two bioactive groups. Acetogenins, such as muricatacin and cis-uvariamicin IV, have been reported to disrupt mitochondrial function by inhibiting complex I, resulting in ATP depletion and activation of intrinsic apoptotic pathways. In silico docking studies support this mechanism by demonstrating strong binding affinities of these acetogenins to apoptosis-regulating proteins like caspase-3 ². Furthermore, these compounds have been shown to induce G0/G1 cell cycle arrest in cancer cells, contributing to their antiproliferative effects ¹. Acetogenins are well-documented inhibitors of mitochondrial complex I, leading to decreased ATP synthesis and induction of the intrinsic apoptotic pathway^16,17,18. These compounds, including muricatacin and cis-solamin identified in our study, have previously demonstrated strong pro-apoptotic effects in various cancer cell lines ^19,20,21. Docking studies have corroborated their high binding affinity to pro-apoptotic proteins like caspase-3 and Bax, offering molecular insights into their activity ^22,23,24,25. The use of Combenefit software to validate synergy across Loewe, Bliss, and HSA models supports the robustness of our findings. Similar combinatorial studies using plant-based fractions have shown that synergy not only increases efficacy but also allows dose reductions, which is critical for minimizing toxicity ^{26,27,28,29,30,31.}

Figure 1. Synergistic effect of FEF and AEF. a) Loewe model, b) Highest Single Agent model, c) Bliss model

Cell cycle analysis:

Cell cycle profiling was performed to assess the phase at which arrest occurred. In the control group, cells were evenly distributed across all cell cycle phases, reflecting normal progression. However, treatment with the crude extract, FEF, and AEF led to a notable accumulation of cells in the G0-G1 phase, indicating cell cycle arrest at this checkpoint. This effect was most pronounced in cells treated with the combined FEF and AEF fractions, suggesting a disruption in cell cycle progression at the G0-G1 checkpoint (Figure 2). For normal peripheral blood lymphocytes, cell cycle distribution remained largely balanced. However, some treated groups exhibited mild cell cycle arrest, with a significantly lower proportion of affected cells compared to the Molt-3 treated cell lines (Figure 3). These observations further underscore the selective and non-toxic nature of the       Annona muricata fractions on normal cells.

Figure 2. Distribution of Molt-3 cells in various phases of cell cycle

Figure 3. Distribution of PBL in various phases of cell cycle

Annexin V-FITC and PI staining:

The mode of cell death was assessed through flow cytometric analysis employing Annexin V-FITC and PI staining. Results demonstrated a higher proportion of viable cells in the control group relative to the treated groups. Administration of the combined FEF and AEF fractions led to a pronounced induction of apoptotic cell death, with a marked increase in cells at both early and late apoptotic stages (Figure 4). Analysis of the ethanolic crude extract, along with the individual FEF and AEF fractions of Annona muricata, and their combined application, revealed a significant elevation in the percentage of cells undergoing apoptosis across both early and late phases.

Figure 4. Distribution of Molt-3 cells in apoptosis phase

Figure 5. Distribution of PBL in live phase

In the PBL-treated groups, the incidence of apoptotic cells was minimal. Nevertheless, certain treated groups displayed features indicative of both early and late apoptotic events (Figure 5). Despite this, the overall percentage of apoptotic cells was consistently lower than that observed in the treated Molt-3 cell lines. These findings reinforce the non-toxic profile of the fractions in PBL. Notably, in the group exposed to the combined fractions, 93.40% of the cells remained viable, demonstrating a significant protective effect on normal peripheral blood lymphocytes. When comparing the outcomes between Molt-3 cells and PBL, it can be concluded that the combined fractions demonstrate a cytotoxic effect on leukemic cells while showing no toxic impact on PBL. This suggests that acetogenins may have worked synergistically with flavonoids. Additionally, the results imply that cancer cells are selectively targeted, triggering apoptosis specifically in them while preserving normal PBL, thereby confirming the specificity of the mechanism.

Flavonoids, widely studied for their antioxidant and anticancer properties, can modulate cellular redox status and affect signaling pathways involved in apoptosis. These compounds regulate key molecular targets such as p53, Bcl-2, and caspases, which play crucial roles in cancer cell death³. Their antioxidant action also helps reduce oxidative stress, which may sensitize cancer cells to the cytotoxic action of acetogenins, explaining the enhanced efficacy when used in combination. The selectivity of the combined treatment for leukemic cells over PBLs is particularly significant. Previous studies have confirmed the low toxicity of A. muricata extracts in normal cells, suggesting that the plant’s constituents exhibit tumor-specific actions ². This selective cytotoxicity is critical in minimizing adverse effects and enhancing therapeutic safety. Moreover, the combined fractions demonstrated the lowest IC₅₀ value among all treatments, indicating a potent synergistic interaction^32,33,34,35. Such interactions allow for effective dose reductions, lowering the risk of side effects while maintaining efficacy. This supports the principle of combination therapy, which is increasingly being adopted in cancer treatment protocols for improved outcomes ¹.  Flavonoids such as myricetin, luteolin derivatives, and apigenin glucosides have established roles in modulating oxidative stress, inhibiting proliferation, and promoting apoptosis through p53 activation and inhibition of PI3K/Akt and NF-κB pathways^36,37,38,39. These mechanisms likely complement the mitochondrial inhibition by acetogenins, explaining the pronounced synergy observed in our combinatorial treatments^40,41. Notably, our results demonstrate significant G0/G1 arrest in Molt-3 cells following combined treatment. This cell cycle arrest has been previously reported for both flavonoids and acetogenins and is associated with upregulation of CDK inhibitors like p21 and p27 and downregulation of cyclins ^42,43,44. The minimal cytotoxicity observed in PBLs corroborates findings from other studies indicating the tumor-specific cytotoxicity of A. muricata extracts ^45,46,47,48. This selectivity can be attributed to the unique metabolic and signaling pathways in cancer cells, including elevated ROS levels and altered mitochondrial membrane potentials, which render them more susceptible to phytochemical-induced apoptosis ^49,50,51,52.

Moreover, our findings are in alignment with previous in vitro studies where Annona muricata extracts selectively targeted breast, colon, and prostate cancer cells ^53,54,55,56. In vivo models have further supported these outcomes, with reduced tumor volumes and preserved normal tissue histology upon treatment with A. muricata derivatives ^57,58,59,60.

The flavonoid-acetogenin combination also fits within the broader framework of multi-targeted therapy—an approach increasingly advocated for complex diseases like cancer. Natural product combinations have been shown to modulate multiple oncogenic pathways simultaneously, reducing the likelihood of           resistance ^61,62,63,64. Our study also opens avenues for nanocarrier-based delivery of these phytoconstituents. Encapsulation of flavonoids and acetogenins in liposomes or nanoparticles has been shown to enhance bioavailability, improve cellular uptake, and increase therapeutic indices in preclinical models ^{65,66,67,68,69,70,71}. In summary, the synergistic interaction between flavonoid and acetogenin fractions from Annona muricata offers a compelling basis for further development as a dual-action, selective anticancer therapy. The current findings advocate for in vivo validation and mechanistic studies using gene expression and protein quantification tools to elucidate the precise apoptotic and cell cycle arrest pathways involved.

CONCLUSION:

In conclusion, the flavonoid and acetogenin fractions from A. muricata show strong synergistic effects against leukemic cells with minimal harm to normal cells, emphasizing their potential as natural therapeutic agents. Further studies, including molecular mechanism exploration and in vivo validation, are warranted to translate these findings into clinical application.

CONFLICT OF INTEREST:

All authors have none to declare.

ABBREVIATIONS:

FEF- Flavonoid enriched fraction, AEF- Acetogenin enriched fraction, PBL- Peripheral Blood Lymphocytes, A. muricata – Annona muricata.

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Received on 08.05.2025      Revised on 17.09.2025

Accepted on 09.12.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2329-2335.

DOI: 10.52711/0974-360X.2026.00334

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