Cytotoxic and Apoptotic Properties of the Flavonoid-rich Ethyl Acetate Fraction of the Crude Methanol Leaf Extract of Syzygium samarangense (Blume) Merr. (Myrtaceae) against MCF-7 Breast Cancer Cells

Abstract

Introduction: Breast cancer is the most common cancer among women in the Philippines and about 3 in every 100 Filipina will be diagnosed with breast cancer in their lifetime. There is a need to discover safe, yet inexpensive herbal extracts with potential cytotoxic properties as potential treatment modalities to treat breast cancer.

Objectives: This study seeks to explore the cytotoxic and apoptotic properties of the ethyl acetate fraction of the defatted crude methanol leaf extract of Syzygium samarangense in MCF-7 breast cancer cell lines.

Methods: Screening for flavonoids of the extracts was performed using TLC, total flavonoids, total phenols, FTIR and LC-MS spectroscopy. The hydrogen peroxide and ferric reducing anti-oxidant power were used as substrates to assess in vitro anti-oxidative properties of the extracts. The MTT dye viability assay was used to assess the cytotoxic properties of the extracts against MCF-7 cells. Apoptotic properties of the extracts in MCF-7 cells were determined by caspase-3 activation assay, DNA fragmentation patterns and fluorescence microscopy after annexin-V and propidium iodide staining.

Results: The abundance of flavonoids in the ethyl acetate fraction of the crude methanol leaf extract was established by TLC, FTIR, LC-MS/MS, total flavonoid and total phenol analyses. The in vitro anti-oxidative properties of this extract was comparable to ascorbic acid. The median inhibitory concentration (IC₅₀) of this extract in MCF-7 breast cancer cell lines was 7.2 mcg/mL while doxorubicin registered an IC₅₀ of 1.2 mcg/mL. At this concentration, the extract was not cytotoxic to normally-dividing breast epithelial cells. Cytotoxicity of the extract was mediated via apoptosis as demonstrated by DNA fragmentation, caspase-3 activation and fluorescence microscopic analyses.

Conclusion: The study shows that the flavonoid-rich ethyl acetate fraction of the crude methanol leaf extract of S. samarangense possesses potent apoptotic and cytotoxic properties against MCF-7 breast cancer cell lines at low concentrations.

Keywords: MCF-7 breastcancer cell lines; Syzygium samarangensi; apoptotic; cytotoxic; makopa.

Figure 1

Thin-layer chromatograms of the Ethyl Acetate Fraction of the Crude Methanol Leaf Extract of S. samarangense at (A) UV 254 nm and with (B) K₂Fe (CN)₆ - FeCl₃ TS with Quercetin as Marker Substance on Right Lanes.

Figure 2

FTIR Spectrum of the Ethyl Acetate Fraction of the (A) Crude Methanol Leaf Extract of S. samarangense and the (B) Marker Substance Quercetin.

Figure 3

LC-EI-MS/MS Spectrum of the Ethyl Acetate Fraction of the Crude Methanol Leaf Extract of S. samarangense.

Figure 4

Comparative Concentration - H₂0₂ Scavenging Inhibition Curves Between the Crude Methanol Leaf Extract of S. samarangense and its Ethyl Acetate Fraction.

Figure 5

Comparative Inhibition - Concentration Curves of MCF-7 Breast Cancer Cell Lines Exposed to the Crude Methanol Leaf Extract of S. samarengense and its Ethyl Acetate Fractions.

Figure 6

Comparative Inhibition - Concentration Curves of Normal Human Epithelial Breast Myoblast Cell Lines Exposed to the Crude Methanol Leaf Extract of S. samarengense and its Ethyl Acetate Fractions.

Figure 7

Electrophoresis Plate Showing DNA Fragmentation in MCF-7 Breast Cancer Cells Exposed to (2) 2% DMSO; (3) Crude Methanol Leaf Extract of S. samarengense and its (4-5) Ethyl Acetate Fraction and (6) Doxorubicin at IC₅₀ Concentrations.

Figure 8

Caspase-3 Activation in MCF-7 Breast Cancer Cell Lines Exposed to the Crude Methanol Leaf Extract of S. samarengense at IC₅₀ Concentrations.

Figure 9

The Extrinsic and Intrinsic Pathways of Apoptosis. (Eskandari and Eaves, 2022)

Figure 10

Fluorescence Micrograph of Apoptotic MCF-7 Breast Cancer Cells (Green) Exposed to (A) 2% DMSO, (B) Crude Methanol Leaf Extract of S. samarengense and its Ethyl Acetate Fraction (B & C) and Doxorubicin (D) at IC₅₀ Concentrations.(*p < 0.001 vs control)

Figure 11

Interaction of a Flavonoid in the Active Site of Estrogen A Receptor.