# Unveiling the cytotoxic potential of four Callistemon fruit extracts against breast and colon cancer: a combined metabolomic and in silico approach

## TL;DR

This study explores how extracts from four Callistemon species affect breast and colon cancer cells, finding that C. macropunctatus is particularly effective.

## Contribution

The study introduces a novel combination of metabolomic profiling and in silico docking to identify cytotoxic compounds in Callistemon extracts.

## Key findings

- C. macropunctatus extract showed strong cytotoxicity against breast and colon cancer cells with IC₅₀ values lower than a reference drug.
- The extract caused cell cycle arrest in both MCF-7 and Caco-2 cells, indicating broad anti-cancer potential.
- Flavonoids, lignans, and meroterpenes were identified as key metabolites with strong binding affinities to CDK6.

## Abstract

Breast cancer and colon cancer are among the most prevalent malignancies worldwide, representing significant public health challenges. This study aimed to evaluate the potentially cytotoxic effect of fruit ethanol extracts of four selected Callistemon species: Callistemon citrinus (Curtis) Skeels, Callistemon
macropunctatus (Dum.Cours.) Court, Callistemon viminalis (Sol. ex Gaertn) and Callistemon subulatus Cheel against breast (MCF-7) and colon (Caco-2) cancer cell lines in order to investigate the mechanism of action.

metabolic profiling of the four selected Callistemon species was assessed using UPLC-ESI-MS/MS analysis. The in vitro cytotoxicity effects of the tested ethanol extracts against breast (MCF-7) and colon (Caco-2) carcinoma cell lines were assessed by means of 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The most active extract cell cycle analysis was subjected to flow cytometry. In-silico docking analysis of the most abundant metabolites against cell cycle regulatory enzymes was conducted, followed by molecular docking simulations for top binders.

Among the four tested Callistemon species, the extract derived from C. macropunctatus exhibited the most potent cytotoxic activity, with IC₅₀ values of 5.45 ± 0.34 µg/mL against MCF-7 breast cancer cells and 10.24 ± 0.59 µg/mL against Caco-2 colon cancer cells. These values indicate a higher cytotoxic potency compared to the reference drug staurosporine (IC₅₀ = 7.72 ± 0.46 µg/mL for MCF-7 and 5.16 ± 0.2 µg/mL for Caco-2). As a result, C. macropunctatus was selected for further analysis related to its ability to induce apoptosis and mechanistic effects. In total, sixteen compounds were tentatively identified, with flavonoids, lignans, and meroterpenes emerging as the dominant metabolites.Specifically, the extract caused S-phase arrest in MCF-7 breast cancer cells while both G0/G1 and S-phase arrest in case of Caco-2 colon cancer cells, indicating a broad-spectrum efficacy in disrupting cell cycle progression across different cancer types. To elucidate the underlying mechanisms, in-silico docking simulations were conducted to assess the binding affinities of the identified compounds towards CDK6, a critical regulator of the cell cycle. The evaluated compounds showed promising binding affinities ranging from − 6.5 to -9.7 kcal/mol, surpassing the binding efficiency of the co-crystal ligand of cyclin-dependent kinase (CDK6). Amongst the detected phenolic compounds, avicularin, nilocitin, and quercetin 3-O-(2’’-galloyl)-β-D-galactopyranoside exhibited the highest docking scores. These compounds formed strong interactions with essential amino acid residues in the CDK6 active site, suggesting a strong potential for inhibiting CDK6 activity.

These findings warrant further exploration of C. macropunctatus extract as a promising anti-cancer agent, with a focus on elucidating its role of CDK6 inhibition and its antiproliferative effects.

The online version contains supplementary material available at 10.1186/s12906-025-05224-y.