Ethanolic Extract of Averrhoa carambola Leaf Has an Anticancer Activity on Triple-Negative Breast Cancer Cells: An In Vitro Study

Abstract

Background/Objectives: Averrhoa carambola, or star fruit, is a shrub known for its medicinal properties, especially due to bioactive metabolites identified in its roots and fruit with anti-cancer activity. However, the biological effects of its leaves remain unexplored. This study aimed to assess the effects of ethanolic extract from A. carambola leaves on triple-negative breast cancer (TNBC), an aggressive subtype lacking specific therapy. Methods: Phytochemical analysis and HPLC profile and additional cell line evaluation employing MDA-MB-231 were carried out. Results: Phytochemical screening revealed that the ethanolic extract was rich in flavonoids, saponins, and steroids, demonstrating an antioxidant capacity of 45%. ¹H NMR analysis indicated the presence of flavonoids, terpenes, and glycoside-like compounds. Cell viability assays showed a concentration-dependent decrease in viability, with an IC₅₀ value of 20.89 μg/mL at 48 h. Clonogenic assays indicated significant inhibition of replicative immortality, with only 2.63% survival at 15 μg/mL. Migration, assessed through a wound healing assay, was reduced to 3.06% at 100 μg/mL, with only 16.23% of cells remaining attached. An additive effect was observed when combining lower concentrations of the extract with doxorubicin, indicating potential synergy. Conclusions: These results suggest that the ethanolic extract of A. carambola leaves contains metabolites with anti-cancer activity against TNBC cells, supporting further research into their bioactive compounds and therapeutic potential.

Keywords: Averrhoa carambola; MDA-MB-231 cells; breast cancer; flavonoids; polyphenols; traditional medicine; triple-negative breast cancer.

Figure 1

Chromatograms obtained at 290 nm from HPLC analysis. (A) Chromatogram of standards: gallic acid (GA, Rt 2.385 min), cinnamic acid (CA, Rt 30.795 min), anthrone (ANT, Rt 20.000 min), quercetin (Q, Rt 17.955 min), and 4-methylumbelliferone (4-ML, Rt 10.908 min). (B) Chromatogram of the ethanolic extract of A. carambola (500 ppm). (C) Chromatogram of the hydrolysate of the leaves of A. carambola (500 ppm). S: signal.

Figure 2

Viability experiments employing ethanolic extract of A. carambola on MDA-MB-231 cells. (A) No changes in viability were observed in cervical cancer cell line TC-1 exposed to A. carambola extract in increasing concentrations. (B) A concentration-dependent effect was observed on MDA-MB-231 cell line exposed to the extract. (C) The ethanolic extract of A. carambola leaves had an experimental IC₅₀ of 20.83 μg/mL in triple-negative breast cancer cell line. (D) Morphological changes and detached cells were observed from the concentration of 25 μg/mL of ethanolic extract. Magnification 10×. The p-values correspond to significant differences compared to the control, DMEM-F12 medium with 0.1% DMSO, * p < 0.05.

Figure 3

Ethanolic extract of A. carambola leaves decreases replicative immortality of MDA-MB-231 cells. (A) Photographs depict the number of colonies formed after the exposition of each treatment. It is observed that a concentration-dependent effect completely inhibits cell survival. (B) The graph shows the percentage of survival treatment. The p-values correspond to significant differences compared to the control, only DMEM medium, * p < 0.05.

Figure 4

Ethanolic extract of A. carambola leaves interferes with MDA-MB-231 cell migration. (A) Images captured at 48 h of the wound area made in MDA-MB-231 cell monolayers. Magnification 4×. (B) The graph shows the changes in the open area; a concentration-dependent inhibitory effect can be observed at 48 h that was superior to the doxorubicin effect. Comparison to 48 h control, * p < 0.05.

Figure 5

The ethanolic extract of A. carambola leaves affects the cell adhesion of MDA-MB-231 cells. (A) The micrographs show the adhesive capacity of cells recovered after exposure to A. carambola extract and reseeded for 24 h. The adhesive capacity decreases as the concentration of the extract increases. Magnification 10×. (B) The graphs show the percentage of cells adhered to the monolayer after being treated with the extract for 48, showing a concentration-dependent decrease in adhesion. (C) The graph shows the percentage of cell death after 48 h of treatment. (D) The graph shows the percentage of adhesion of detached cells after treatment that were recovered and reseeded. The p-values correspond to significant changes compared to the control, * p < 0.05.

Figure 6

The combination of a low dose of doxorubicin and intermediate doses of A. carambola extract reduces the cell viability of MDA-MB-231 cells. The graph shows the reduction in cell viability induced by the different combinations after 48 h of treatment. An additive effect was observed between the 1/5 IC₅₀ dose of doxorubicin (DOX) and the three tested concentrations of the extract. ^a 0.4 μM DOX + 15 μg/mL extract vs. 15 μg/mL of the extract, ^b 0.4 μM DOX + 25 μg/mL vs. 25 μg/mL, ^c 0.4 μM DOX + 50 μg/mL vs. 50 μg/mL, ^d 2 μM DOX + 15 μg/mL vs. 15 μg/mL, ^e 2 μM DOX + 25 μg/mL vs. 25 μg/mL, ^f 2 μM DOX + 50 μg/mL vs. 50 μg/Ml, ^a’ 0.4 μM DOX + 15 μg/mL vs. 0.4 μM DOX, ^b’ 0.4 μM DOX + 25 μg/mL vs. 0.4 μM DOX ^c’ 0.4 μM DOX + 50 μg/mL vs. 0.4 μM DOX, ^d’ 2 μM DOX + 15 μg/mL vs. 2 μM DOX, ^e’ 2 μM DOX + 25 μg/mL vs. 2 μM DOX, ^f’ 2 μM DOX + 50 μg/mL vs. 2 μM DOX, * p < 0.05.