Cytotoxic Activity of Ethyl Acetate Extract from Voacanga foetida (Bl.) Rolfe Leaves Against T47D Breast Cancer Cells

The cytotoxicity of ethyl acetate extract from Tampa badak ( Voacanga foetida (Bl.) Rolfe) leaves against Artemia salina leach larvae was determined using the brine shrimp lethality test (BSLT) method and was evaluated against T47D breast cancer cells using MTT assay method. The result of BSLT showed a consistent result with MTT assay, which is the result obtained that ethyl acetate extract is very toxic against A. salina Leach larvae with LC 50 value of 8.61 µg/mL and very cytotoxic against T47D breast cancer cells with IC 50 values of 0.87; 0.66; and 0.95 µg/mL at the 24, 48 and 72 hours of incubation times, respectively. The MTT assay data were analyzed using a two-way ANOVA statistical method to see the effect of the dependent variable (concentration and time) on the independent variable (% viability). Based on the statistical test result, there is a difference in % viability between concentrations of 0.1; 1; and 10 µg/mL (p <0.05), but the length of incubation does not affect % viability (p >0.05).


INTRODUCTION
Breast cancer attacks the epithelial tissue of the breast (carcinoma) and generally originates from the glands, gland ducts, and supporting tissues of the breast 1 . The risk factors for breast cancer include a family history of breast cancer, obesity, consumption of fast food that contains a lot of saturated fat, alcohol consumption, menopause at an older age (> 50 years), early menarche, namely the first menstruation at a relatively young age (≤12 years), long-term use of hormonal contraceptives, radiation exposure, having had a benign breast tumor or breast cancer, never giving birth or giving birth for the first time at the age of more than 35 years, and not breastfeeding are also risk factors for breast cancer 2, 3 . Treatment of cancer patients can be done with surgery, radiotherapy, and chemotherapy. Chemotherapy is a cancer treatment using cytotoxic chemicals 4 . The working principle of chemotherapy is to kill cancer cells, control their growth, and stop their growth from spreading or reduce the symptoms caused by cancer 5 . Treatment with chemotherapy has not given satisfactory results because it does not work precisely. It can also cause normal cell damage and cause some side effects, such as hair loss, nausea, vomiting, diarrhea, susceptibility to infection, thrombocytopenia, neuropathy, and myalgia. Treatment with radiation causes side effects such as nausea and vomiting 6 . Meanwhile, surgical treatment cannot entirely remove body tissue damaged by cancer 7 . Because of these conditions, it is necessary to look for alternative drugs, one of which is developing anticancer agents derived from natural ingredients or

Abstract
The cytotoxicity of ethyl acetate extract from Tampa badak (Voacanga foetida (Bl.) Rolfe) leaves against Artemia salina leach larvae was determined using the brine shrimp lethality test (BSLT) method and was evaluated against T47D breast cancer cells using MTT assay method. The result of BSLT showed a consistent result with MTT assay, which is the result obtained that ethyl acetate extract is very toxic against A. salina Leach larvae with LC50 value of 8.61 µg/mL and very cytotoxic against T47D breast cancer cells with IC50 values of 0.87; 0.66; and 0.95 µg/mL at the 24, 48 and 72 hours of incubation times, respectively. The MTT assay data were analyzed using a twoway ANOVA statistical method to see the effect of the dependent variable (concentration and time) on the independent variable (% viability). Based on the statistical test result, there is a difference in % viability between concentrations of 0.1; 1; and 10 µg/mL (p <0.05), but the length of incubation does not affect % viability (p >0.05). 9 chemopreventives 8 . Anticancer drugs from natural ingredients can treat the source of the disease by repairing damaged cells, tissues, and organs by increasing the immune system 9 . Tampa badak (Voacanga foetida (Bl.) Rolfe) is an Indonesian medicinal plant from the Apocynaceae family. Based on our previous work, the leaves methanolic extracts, their fractions (n-hexane, ethyl acetate, and butanol fractions), and some isolated compounds from this plant were reported to have highly cytotoxic activity against L1210 blood cancer cells 10 . One isolated compound from this plant's butanol fraction of leaves ethanolic extract was also tested against various cancer cell lines (K562, A549, HeLa), and the isolate exhibited a highly cytotoxic activity 11 . In addition, the ethanolic extract also demonstrated in vivo anticarcinogenic activity on the dose of 200 mg/kgBW, and based on the acute toxicity test, this extract was also categorized as not toxic (LD50 >15000 mg/kgBW) 12 . Therefore, it becomes fascinating to explore more widely the potential of V. foetida as a natural source of bioactive agents to treat various types of cancers. Our previous work also reported that the leaves ethyl acetate extract of V. foetida exhibited highly cytotoxic activity against HTB-38 colon cancer cells 13 . However, the potency of the cytotoxic activity of this extract against other cancer cell lines has never been explored. Therefore, this work aimed to investigate the cytotoxic potency of ethyl acetate extract of V. foetida leaves by BSLT method and then continued with an in vitro evaluation against T47D breast cancer cells.

Sample collection and extraction
The plant sample, as shown in Figure 1, was collected from Biological Education and Research Forest Universitas Andalas, Padang, West Sumatra, Indonesia 14 . The identification document number 315/K-ID/ANDA/IX/2020 identified it as Voacanga foetida (Bl.) Rolfe. The leaves part of V. foetida was washed, dried, and sorted. As much as 2.15 kg of chopped simplicia was macerated using n-hexane for five days at room temperature and in triplicate. The n-hexane macerates were separated by filtration, and the residues were macerated using ethyl acetate for five days at room temperature, triplicate, and then filtered to afford ethyl acetate macerates. The ethyl acetate macerates were concentrated by a vacuum rotary evaporator to afford 70 g of ethyl acetate concentrated extract. 10

Brine Shrimp Lethality test (BSLT)
The BSLT is a preliminary screening to determine whether a plant has bioactive compounds that have the potential as anticancer. The advantages of the BSLT method are that it is fast, easy, and does not require expensive costs. The toxicity test against A. salina followed the standard protocol from previous literature 15, 16 . The extract was tested in various concentrations (1000, 100, and 10 μg/mL), and the LC50 was calculated.

Cytotoxic test using MTT assay
The cytotoxic test was used to measure cancer cell viability after adding a sample solution. The advantages of this method are that the work is relatively fast, the results are accurate, the interpretation of the results is relatively easy, and the equipment used is simple. In this work, the cytotoxic test was performed using an MTT assay, a colorimetric assay. The principle of this assay is the reduction of the yellow tetrazolium salt to form purple formazan crystals that are insoluble in water by the succinate reductase enzyme present in the mitochondria of living cells. The addition of a stopper reagent will dissolve the formazan crystals, which are then measured for absorbance using an ELISA reader at a wavelength of 570 nm. The intensity of the purple color formed is proportional to the number of living cells. The greater the absorbance, the greater the number of living cells. The cytotoxicity assay using MTT assay followed the standard protocol from the previous literature 17 . In this work, in vitro cytotoxic activity of the ethyl acetate extract at various concentrations (10, 1, and 0.1 μg/mL) and various incubation times were evaluated against the T47D breast cancer cell line, and the IC50 values were calculated. The test on the normal cell has not been conducted because the Vero cell was not available in the laboratory where we worked.

Brine Shrimp Lethality test (BSLT)
The toxic effect of an extract based on the BSLT test was determined by determining the LC50 value. LC50 is the concentration of the tested extract that can cause the death of 50% of A. salina. The death of the larvae was caused by the tested compound, which acted as stomach poisoning. The test compound that entered the larval body interfered with the larval digestive system and inhibited taste receptors in the larval mouth area. This causes the larvae to fail to get a taste stimulus, so they cannot recognize their food, and as a result, the larvae starve to death 18 . The resulting toxic effect indicates the disruption of the cell formation process, which is assumed to be cancer cells 19 .
In this test, a negative control was also used to see whether the response to the test animals' death was caused by the extract and not caused by the solvent used 18 . The standard criterion for measuring the mortality of A. salina larvae is if the larvae do not show movement during observation 20 . Based on the BSLT result presented in Table I, the ethyl acetate extract of V.
foetida leaves exhibited an LC50 value of less than 10 µg/mL. Based on this result, the ethyl acetate extract can be categorized as very toxic against A. salina.

Cytotoxic test using MTT assay
The MTT assay showed a consistent result with BSLT results, where the ethyl acetate extract of V. foetida leaves also exhibited an IC50 value of less than 10 µg/mL against T47D breast cancer cells, as presented in Table II  Based on Figure 2, generally, there is a decrease in the percentage of cell viability when the incubation time is extended from 24 to 48 hours. After 48 hours, the viability percentage tends to increase again, and it cause the IC50 value to rise to 0.95 μg/mL at the incubation time of 72 hours. This is a common phenomenon in cytotoxic assay against cancer cells, and previous researchers have also reported similar things, and the different plant extracts might show a different phenomenon 23 . However, the difference in incubation times from 24 to 72 hours in this work was observed not to cause a difference in the category of their cytotoxic activities. In other word, it can be observed that the variation in incubation time did not affect the percentage of cell viability. All extracts in various incubation times still exhibited the same cytotoxic category. To support this hypothesis, the data were analyzed using the two-way ANOVA statistical method to see the effect of the independent variables (concentration and time) on the dependent variable (percentage of cell viability). Furthermore, the Tukey test was also performed to see the difference between the concentration and time group variables on the percentage of cell viability. The result of ANOVA analysis gave a significant difference if p <0.05. Based on the two-way ANOVA analysis, it was found that the variation in incubation times (24,48, and 72 hours) did not show a significant effect on the percentage of cell viability that was indicated by a p-value of 0.606 (p >0.05). While the variation in tested concentrations (0.1, 1.0, and 10 µg/mL) showed a significant effect on the percentage of cell viability that was indicated by the p-value of 0.000 (p <0.05). Furthermore, the Tukey test was conducted to see the difference in concentration variables. Tukey's test is a further test that assesses significant differences in variables in a group. Based on this test, it is known that each concentration is significantly different, where the concentration of 0.1 μg/mL is significantly different from the concentration of 1.0 and 10 µg/mL and vice versa.
The cytotoxic effect of the ethyl acetate extract of V. foetida leaves is thought to be due to secondary metabolites such as alkaloids, steroids, and terpenoids 24 . Some alkaloids are used as cancer drugs and can induce apoptosis by binding to DNA by inhibiting the topoisomerase I enzyme in the DNA replication process so that it will cause permanent DNA double strands damage that triggers apoptosis 25 . Some steroids also have anticancer activity by occupying estrogen hormone