Fresh leaves of Vernonia amygdalina Delile were collected in 2022 in Medan, Indonesia. African leaves used in this research were confirmed at the Indonesian Institute of Sciences, Pusat Penelitian Biologi, Cibinong, Indonesia. Annexin-V (BioLegend), dimethyl sulfoxide (Merck), ethanol (Merck), ethylacetate (Merck), n-hexane (Merck), propidium iodide (BioLegend), distilled water, and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) (Sigma) were the substances used in the research.

This study has gained ethical approval No. 1181/KEPK/2022 from the research ethics committee of Universitas Sumatera Utara.

Reflux was the method being used to make crude extract. The leaves were shade-dried, pulverized, and stored at room temperature, and then the reflux apparatus was assembled. 100 g of powdered Vernonia amygdalina Delile was extracted by 1400 mL of absolute ethanol for 3–4 hrs. The suspension was filtered and concentrated to dryness to get the crude extract using a rotary evaporator. The extractions were made in triplicate with solvent replacement and viscous extract as the result of this process.

The viscous extract was fractionated into n-hexane, ethyl acetate, and water fractions. This fractionation was done using the liquid-liquid extraction method. The viscous crude extract (100 g) was suspended in 500 ml of distilled water and fractionated using organic solvents with increasing polarity (N-hexane, ethyl acetate, and water). Each fraction from the ethanol extracts was tested as an anticancer using MCF-7/HER-2 cell lines (Alara et al. 2019).

The thin-layer chromatography method analyzes the bioactive components contained in African leaves. A plate coated with silica gel 60 GF254 (Merck, Germany) is used as the stationary phase; meanwhile, the mobile phase is used depending on the examination. After the elucidation process, the plate was removed and dried; then, it was sprayed with an appropriate spectrophotometer, heated in an oven at 110 °C for 5 minutes, and examined for color change (Wagner and Bladt 1996).

1. Flavonoid identification used mobile phase ethyl acetate-methanol-water (100:13.5:10). Then the results were stained with an AlCl ₃ 10% reagent. The yellow, green, or blue fluorescence reveals a positive reaction.
2. Alkaloid identification used mobile-phase chloroform-methanol-ammonia (85:15:1). Dragendorff’s reagent was then used as a stain. The formation of orange-brown zones reveals a positive reaction.
3. Steroids/Triterpens identification used mobile phase n hexanes: ethyl acetate (8:2). The results were stained with the Lieberman-Bouchard reagent. The formation of red-violet zones reveals a positive reaction.
4. Glycoside identification used mobile-phase ethyl acetate-methanol-water (8:1:1). The results were stained with a 50% sulfuric acid reagent. A positive reaction is revealed by forming violet-red, yellow-brown, dark blue-black, or bright blue zones.
5. Tannin identification used mobile phase chloroform-acetate acid-n butanol-water (5:2:2:1). The results were stained with a FeCl ₃ 1% reagent. The formation of gray and brown zones reveals a positive reaction.
6. Saponin identification used mobile phase chloroform-acetate acid-methanol-water (11:6:2:1). The results were stained with an anisaldehyde-sulfuric acid reagent. A positive reaction is revealed by forming blue-violet and occasionally red or yellow-brown zones.

MCF-7/HER-2 cells are the luminal breast cancer subtype purchased from the Laboratory of Parasitology, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, in Yogyakarta. MCF-7/HER-2 were inoculated in Dulbecco’s Modified Eagle Medium (DMEM) enhanced with 10% FBS (fetal bovine serum), 1% penicillin/streptomycin (Gibco), and 0.5% fungizone (Gibco) at 37 °C with 5% CO₂ (Bashari et al. 2022).

MCF-7/HER-2 cells (5000/well) seed in a 96-well plate, followed by incubation at 37 °C in a 5% CO₂ incubator for 24 hrs. The plate contained medium, and incubated cells were discarded and re-incubated by administering ethanol extract, n-hexane fraction, ethyl acetate fraction, and water fraction with different concentrations. The process continues with overnight incubation at 37 °C in a 5% CO₂ incubator. Following incubation, 0.5 mg/mL MTT was added to each well, and the plate was incubated for 4 hrs at 37 °C in 5% CO₂. The excess MTT was aspirated, and the formazan crystals formed were dissolved by administering 10% SDS (Sigma) in 0.01N HCl (Merck). The incubation of cells was done overnight at room temperature in a dimly lit place. Eventually, the absorbance was recorded at λ 595 nm wavelength using a microplate reader. The data absorbed from each well were converted to viability percentage cells (Foo et al. 2014).

For the MTT assay, the cells were incubated in 96-well plates at two cells/well density. The cells with the same density rate were made into three 96-well microplates, followed by incubation at 37 °C in a 5% CO₂ incubator overnight. The plate contained medium, and incubated cells were discarded and then re-incubated with ethanol extract and ethylacetate fraction with different concentrations in a 5% CO₂ incubator at 37 °C for 24, 48, and 72 hrs after the given extract and fraction. The cells were allowed to attach and proliferate during the desired incubation time. Following incubation, 0.5 mg/mL MTT was administered in each well, and the plate was incubated for 4 hrs at 37 °C. After four hours, the cells were given 10% SDS (Sigma) in 0.01N HCl (Merck) to dissolve the formazan crystals. The cells were incubated for 24 hrs at room temperature in a dimly lit place. Eventually, the absorbance was recorded at λ 595 nm wavelength using a microplate reader. The data absorbed from each well were converted to viability percentage cells (Wei et al. 2017).

The MCF-7/HER-2 cells (500.000 cells/mL) were inoculated in a 6-well plate following incubation for 24 hrs at 37 °C in 5% CO₂. The conical tube was used to put the media in and rinsed with PBS (Phosphate Buffer of Standard), then the cells were given ethyl acetate and doxorubicin with different concentrations following incubation for 24 hrs at 37 °C in 5% CO₂. After administering 0.025% trypsin to the cells, they were collected into labeled conical tubes. PBS was used to rinse and was collected in the labeled conical tube. The media was centrifuged at 2.500 rpm for 5 minutes, and the sediment was compiled, while the supernatant was discarded (Hasibuan and Fachrunisa 2019).

As can be seen in Table 1, the phytochemical screening result of the crude ethanol extract of Vernonia amygdalina using thin-layer chromatography showed the presence of several of the bioactive components tested, including flavonoids, tannins, saponins, glycosides, and steroids and triterpenes (Harahap et al. 2021b). N-hexane and ethyl acetate are the solvents used in this research for liquid-liquid extraction. N-hexanes fraction, ethylacetate fraction, and water fraction were fractionated from ethanol extract. The N-hexane fraction contained bioactive components such as flavonoids, tannins, glycosides, and steroids and triterpenes. Ethylacetate fractions contained flavonoids, tannins, glycosides, saponins, steroids, and triterpenes. The water fraction contained bioactive compounds such as flavonoids, alkaloids, saponins, and glycosides. These bioactive compounds have a pivotal role in the pharmacological activities of plants (Alebiosu and Yusuf 2015; Hasibuan et al. 2021).

The MTT technique (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) was used in determining the inhibition value of a 50% death cell, which is the parameter in this study (Juwitaningsih et al. 2022). The mitochondrial activity of cells is the ground principle in this research. The result found that the ethyl acetate fraction IC₅₀ value is 66 μg/mL, which is classified as a strong cytotoxic effect compared to other extracts and fractions (Table 2). According to the IC₅₀ value classification, the activity of anticancer is divided into several groups: IC₅₀ value ≤ 10 μg/mL is classified as highly active; IC₅₀: 20–100 μg/mL is classified as active; IC₅₀: 100–500 μg/mL is classified as moderate cytotoxic effect (Fithrotunnisa et al. 2020). Based on the screening result, it was found that the ethyl acetate fraction contained secondary metabolites, which were classified as anticancer substances, for instance, flavonoids. Flavonoid compounds have not only great potential as a breast anticancer by inhibiting the activity of DNA topoisomerase I/II, resulting in the induction of apoptosis in cells, but also have great potential in decreasing and inhibiting the activity of endonuclease and the expression of Bcl-2 and Bcl-XL genes because of the presence of flavonoid bioactive compounds (Hasibuan and Fachrunisa 2019; Hasibuan et al. 2021).

As a basis for further characterization of Vernonia amygdalina Delile of cullar response in MCF-7/HER-2, the ethanolic extract and ethylacetate fraction were examined for inhibiting cell proliferation by using the MTT assay (Hasibuan et al. 2020a). The result indicated that treating MCF-7/HER-2 cells with different ethylacetate fraction concentrations and ethanol extracts induced cell proliferation rates.

The bar chart (Fig. 1) shows that the viability cells of MCF-7/HER-2 treated with an ethylacetate fraction concentration of 66 g/mL showed the best result in inhibiting cell proliferation. The percentage of cell viability increased from 64.46% within 24 hrs of incubation to 61.67% within 48 hrs, a decrease in the percentage of cell viability to 53.89% within 72 hrs of incubation, which means the cells experience saturation. As it also shows no increase in cell growth until 72 hrs of incubation, this is probably due to cell death. This death occurs through a cell cycle arrest mechanism, so that the ability of MCF-7/HER-2 cells to proliferate decreases. It proves that the ethylacetate fraction of African leaves has the potential to inhibit cell proliferation (Wong et al. 2013; Burhan et al. 2021).

Figure 1. 

Proliferation profiles of ethanol extract with different concentrations.

The bar chart (Fig. 2) shows that the viability cells of MCF-7/HER-2 treated with an ethyl acetate fraction concentration of 66 g/mL showed the best result in inhibiting cell proliferation. The percentage of cell viability decreased from 56.0% within 24 hrs of incubation to 50.19% within 48 hrs. There was a decrease in the percentage of cell viability to 58.67% within 72 hrs of incubation, which means the cells experienced saturation at 48 hrs. The ethanol extract from African leaves has the potential to inhibit cell proliferation through dose-dependent concentration (Hasibuan and Fachrunisa 2019; Hasibuan et al. 2021).

Figure 2. 

Proliferation profiles of ethylacetate with different concentrations.

Based on the bar chart (Fig. 3), doxorubicin has a tremendous potential to inhibit cell proliferation through time-dependent concentrations. Doxorubicin is a chemotherapeutic agent for various types of cancer, such as breast cancer. The mechanism of action of doxorubicin is by inhibiting the topoisomerase II enzyme; this enzyme works by binding to cancer cell DNA and blocking enzymes necessary for cancer cell growth, such as the topoisomerase II enzyme (Hasibuan and Fachrunisa 2019).

Figure 3. 

Proliferation profiles of doxorubicin with different concentrations.

MCF-7/HER-2 was incubated with 33.0 μg/mL ethyl acetate to examine the effects on cell cycle inhibition by flow cytometry. The result indicated for the G₀-G₁ phase of cell control was 38.3% (Table 3 and Fig. 4). After 24 hrs of treatment with 33.0 μg/mL ethylacetate at 38.6%, it was revealed that ethyl acetate induced the G₀-G₁ phase. In phases S and G₂-M, cells were treated with 33.0 μg/mL ethylacetate with 20.9% and 17.3. It can be concluded that the ethylacetate fraction has tremendous potential to inhibit the cell cycle of MCF-7/HER-2 compared to the cell control in phase G₂-M with 15.7%. Inducing apoptosis and arresting the cell cycle in the G₀-G₁, S, and G₂-M phases are the best ways to inhibit the growth of cancer cells, as many anticancer agents do. Ethylacetate in the S phase and DNA repair in the G₂-M phase of cycle cells trigger the death cell mechanism (apoptosis), which is crucial during cell growth regulation (Hasibuan and Fachrunisa 2019; Simanullang et al. 2022). To be more specific, the most important target to be utilized as an anticancer therapy is the phase during the G₂-M checkpoint in the cell cycle, resulting not only in the prevention of cell DNA damage by letting it enter the mitosis but also giving the space and time for repairing the DNA damage, which can enhance the cytotoxic chemotherapy effect (Kwan et al. 2016; Hasibuan et al. 2020b). Flavonoid compounds have great potential as breast anticancers by inhibiting DNA topoisomerase I/II activity, resulting in cell apoptosis (Mahmod and Talib 2021). The ethyl acetate fraction can enhance the accumulation of G₂-M cell phases in the cell cycle, indicating damaged DNA repair. Not only are flavonoid compounds known to have anticancer potential, but saponin compounds contained in African leaves also have the potential to be anticancer. Saponins act as an anticancer by inducing cell cycle arrest in the G1 phase (Burhan et al. 2021; Hudan and Patricia 2022).

Figure 4. 

Cell cycle analysis of apoptosis of MCF-7/HER-2 cells: A. Control cell; B. Treatment with 33.0 µg/mL ethylacetate fraction; C. Treatment with 0.9 µg/mL doxorubicin.

The flow cytometry method was used to determine apoptosis on purpose to calculate the number of living cells, cell necrosis, and apoptosis within a short period. MCF-7/HER-2 was given Annexin V to bind phosphatidylserine found in the cell plasma membrane during fluorescence apoptosis (Hasibuan and Fachrunisa 2019). As seen in Table 4 and Fig. 5, MCF-7/HER-2 cells were administered with ethylacetate fraction 33.0 μg/mL, resulting in early apoptosis of 21.8%, late apoptosis of 11.6%, and final necrosis of 26.1% compared to cell control, which was not treated with ethylacetate in the early apoptosis with 7.1%, which is lower than ethyl acetate. It indicates that the ethyl acetate fraction can be used to promote apoptosis. The ethylacetate fraction of African leaves has the potential to induce apoptosis. Based on the phytochemical screening displayed, the ethyl acetate fraction contained tannins, bioactive compounds that function as anticancer, initiating apoptosis pathways, and decreasing protein kinase activation (Okoduwa et al. 2020; Burhan et al. 2021).

Figure 5. 

Flow cytometry analysis of apoptosis of MCF-7/HER-2 cells: A. Control cell; B. Treatment with 33.0 µg/mL ethylacetate fraction; C. Treatment with 0.9 µg/mL doxorubicin.