In Vitro Evaluation of Annona muricata Leaf Infusion as a Modulator of Antineoplastic Drug-Induced Cytotoxicity in Cancer Cell Lines

Abstract

Background/Objectives: Annona muricata (AM), commonly known as soursop or guanabana, has long been used in traditional medicine for its purported anticancer properties. However, scientific studies evaluating its potential enhancing or additive effects with conventional antineoplastic drugs (ADs) remain limited. This study aimed to assess the cytotoxic effects of an aqueous AM infusion alone and in combination with standard ADs in cancer cell lines, while also evaluating its safety in healthy cells. Additionally, we explored the potential molecular interactions of AM metabolites with therapeutic targets using silico modeling. Methods: An AM infusion (125 and 250 µg/mL) was tested on two cancer cell lines-MDA-MB-231 (human triple-negative breast cancer) and TC-1 (murine HPV16-positive cancer)-as well as healthy human leukocytes and a non-tumorigenic mouse lung cell line. Cell viability was assessed using the Alamar Blue™ assay. The combined effects of AM with multiple first-line ADs were evaluated. In silico molecular docking was performed with Molegro Virtual Docker to assess the interaction of AM metabolites (quercetin and hyperoside) with the A2B adenosine receptor. Additionally, the physicochemical properties of 13 AD were analyzed to explore correlations with cytotoxic outcomes. Results: AM infusion alone exhibited low cytotoxicity in both cancer and healthy cell types. However, when combined with ADs, it enhanced cytotoxic effects in cancer cells while sparing healthy cells at the evaluated concentrations. Docking studies revealed strong interactions between quercetin and hyperoside (major metabolites in the AM infusion) and the A2B receptor, supporting a possible mechanistic explanation for the observed effects. Conclusions: AM infusion may act as a chemical modulator, potentiating the effects of conventional ADs in cancer cells while preserving normal cell viability. These findings encourage further preclinical exploration of AM as a complementary agent in integrative oncology.

Keywords: adjuvant; antineoplastic drug; breast cancer; folk medicine; guanabana; infusion; traditional medicine.

Figure 1

Effect of the AM infusion and ADs on cell viability (%). Panel (A) shows the effect in the TC-1 cell line. Panel (B) highlights the effects in the MDA-MB-231 cell line. Results are expressed as mean ± SEM from triplicate experiments. “With” refers to treatment with Annona muricata leaf infusion at 250 µg/mL along with the AD; “Without” refers to cells treated only with the AD.

Figure 2

Relative cell viability (%) in non-tumorigenic murine lung cells (TC-1) treated with ADs alone or in combination with the AM infusion. Cells were treated with carboplatin, 5-fluorouracil, vinblastine, and epirubicin either individually (blue bars) or in combination with the AM infusion at 125 µg/mL (pink bars) or 250 µg/mL (yellow bars). Cell viability is expressed as a percentage relative to the untreated control (100%). Results are expressed as mean ± SEM from triplicate independent experiments. Statistical differences were assessed using the Mann–Whitney U test. * p < 0.05 vs. AM infusion alone at 250 µg/mL. ^a p < 0.05 vs. AM infusion alone at 125 µg/mL. “With” refers to treatment with Annona muricata leaf infusion at 250 µg/mL along with the antineoplastic drug; “Without” refers to cells treated only with the AD.

Figure 3

Spearman’s Rho correlation analysis of polar and hydrophobic molecular descriptors in TC-1 and MDA-MB-231 cell lines with and without Annona muricata infusion treatment. (A) Correlation analysis of polar and hydrophobic molecular descriptors in the TC-1 cell line with (blue) and without (orange) Annona muricata infusion treatment. (B) Correlation analysis of polar and hydrophobic molecular descriptors in the MDA-MB-231 cell line with (blue) and without (orange) Annona muricata infusion treatment. Descriptors include topological polar surface area (TPSA), consensus Log P, Silicos-IT Log P, MLOGP, WLOGP, XLOGP3, and iLOGP. Negative values indicate inverse correlations, while positive values indicate direct correlations.

Figure 4

Docking sites and interactions on the adenosine A2B receptor. (A) Binding site of quercetin. (B) Binding site of hyperoside. (C) Residue interactions of quercetin, including hydrogen bonds and π interactions. (D) Residue interactions of hyperoside, highlighting the expanded binding site and additional stabilizing interactions due to the glycosidic moiety. Docking simulations were performed using Molegro Virtual Docker 6.0, with identified cavity pockets and a custom search space to enhance accuracy. Binding interactions were categorized based on energy levels, with the MolDock Score used to rank ligand binding affinity. Strong interactions are observed with PHE 173 for both ligands (arrowhead). Mild interactions are noted with GLN 90, LEU 86, and TYR 10 for quercetin, and HIS 280 and ILE 276 for hyperoside (♦). Weak interactions are seen with ASN 186, ASN 254, and MET 182 for quercetin, and ASN 254, MET 182, MET 272, and SER 279 for hyperoside.