View of Effect of Black Rice Bran ‘Sembada Hitam’ on T47D Breast Cancer Cells

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2023, Vol. 13, No. 3, 589 – 598 http://dx.doi.org/10.11594/jtls.13.03.17

How to cite:

Research Article

Effect of Black Rice Bran ‘Sembada Hitam’ on T47D Breast Cancer Cells

Flafiani Cios Conara, Galuh Oktavya, Yekti Asih Purwestri, Ardaning Nuriliani *

Department of Tropical Biology, Faculty of Biology, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia

Article history:

Submission November 2022 Revised May 2023

Accepted May 2023

ABSTRACT

Indonesians have a rich tradition of incorporating black rice into their diet as a functional food due to its high antioxidant content. This research investigates the effects of the ethanolic extract of "Sembada Hitam" black rice bran on T47D breast cancer cells, specifically its cytotoxic, cell growth, and apoptotic induction properties. The MTT assay method was used to evaluate the cytotoxicity of the black rice bran extract on T47D cells after 24 and 48 hours of incubation. The acridine orange/propidium iodide (AO/PI) dye double staining method was employed to assess the apoptosis- inducing properties of the extract. Additionally, a cell growth assay was conducted to evaluate the effect of the extract on cell growth, with observations recorded on days 0, 3, and 6. The results revealed that the ethanolic extract of black rice bran "Sembada Hitam," when administered at various concentrations ranging from 7.81 to 1000 µg/mL, did not exhibit cytotoxic effects on T47D cells during the 24 and 48 hours of incubation. The cell growth assay revealed that T47D cells treated with the ethanolic extract of "Sembada Hitam" black rice bran at concentrations of 250 and 500 µg/mL exhibited lower growth rates than the 1% DMSO group from the 3rd to 6th day after incubation. The ethanolic extract of "Sembada Hitam" at concentrations of 250, 500, and 1000 µg/mL resulted in cell death of 10.64 ± 2.98%, 9.99 ± 5.87%, and 5.84 ± 0.78%, respectively. In conclusion, this study found that the ethanolic extract of "Sem- bada Hitam" black rice bran did not demonstrate significant cytotoxic effects on T47D breast cancer cells and is, therefore, unlikely to be a suitable candidate for an anti- cancer agent.

Keywords: Apoptosis induction, Black rice bran, Cell growth, Cytotoxicity, Sembada hitam, T47D cells

*Corresponding author:

E-mail: [email protected]

Introduction

Breast cancer, a formidable foe, is a complex disease that disrupts the delicate balance of cell growth in both glandular and non-glandular breast tissue [1]. With its devastating impact on individ- uals and communities, it stands as a global men- ace, claiming countless lives and leaving a pro- found impact, especially among women. As one of the leading causes of cancer-related mortality worldwide, the battle against breast cancer has be- come an urgent priority in the field of oncology.

In 2012, at least there were 1.67 million deaths from breast cancer on a global scale. It is predicted that there will be 22.2 million new breast cancer cases by 2030 [2]. In Indonesia, the prevalence is predicted to rise, particularly for luminal subtype A (ER+ PR+/- HER 2-) breast cancer [3].

The development of this disease is influenced by several factors, including genetics, lifestyle, and environmental factors. Free radicals, such as reactive oxygen species (ROS) and reactive nitro- gen species (RNS), are unstable molecules that can alter ion balance or cell polarization. They can interact with nucleic acids, cell membranes, and other organelles, disrupting the balance of metabolism and may even lead to cell death [4]. Further- more, the accumulation of free radicals in cells can also cause DNA damage and increase the chance of mutations in the p53 and p21 genes [5].

A chemical or substance that can bind and de- activate radicals is required to reduce the accumulation of free radicals in cells. An antioxidant is one of the compounds that can neutralize free rad-

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icals. It reduces oxidative stress-induced cell damage to prevent gene alterations [6]. In addition, it prevents and suppresses cancer cell growth by inducing apoptosis and blocking tumorigenesis [4, 6]. Previous studies showed that antioxidants, particularly cyanidin-3-O-glucopyranoside, can trig- ger the HL-60 (human acute promyelocytic leukemia) cell line to differentiate and activate the PI3K and PKC genes, linked to the inhibition of tumorigenesis [6]. An antioxidant called myricetin is also known to inhibit the proliferation of MCF-7 breast cancer cells by blocking p21-activated ki- nase 1 through downstream signaling β-catenin and exerting apoptosis through the BRCA1- GADD45 pathway [7].

Pigmented or black rice is a plant known for its high antioxidant content [4], and "Sembada Hitam" is a cultivar of rice that belongs to the Po- aceae family [8]. Studies have shown that black rice has six times higher antioxidant levels than white rice [9]. Cyanidin-3-glucoside, peonidin-3- glucoside, malvidin-3-glucoside, peonidin-3-glucoside, petunidin-3-glucoside, myricetin-7-O-glucoside, and other cyanidin metabolites are among the antioxidants found in black rice [10]. About 80% of the anthocyanin in black rice is cyanidin- 3-glucoside, which has anti-inflamma-tory properties known to reduce oxidative stress [11]. Other phytochemicals include steroids, phenols, tannins, phlobatannins, terpenoids, alkaloids, and flavonoids also found in black rice. Black rice is widely used as a functional food ingredient in Indonesia because of its high nutrient content and various health benefits [12]. It has been utilized commonly as a supplement to cure and prevent various diseases, including diabetes, high blood pressure, cancers, and heart disease, and has the potential for antiaging [4]. Furthermore, this rice contains bioactive substances that act as antioxidants and prevent leukemia, breast, lung, liver, cervical, and colorectal cancer [13].

A new black rice cultivar called “Sembada Hitam” has been developed in Sleman Regency, Special Region of Yogyakarta. It has a flag leaf length of 27.44 ± 3.09 cm and 1.72 ± 0.06 cm in width, with a slender grains shape and purple- black color [14]. It is known that the extract of this cultivar can boost PlGF (placental growth factor) and reduce sFlt-1 (soluble Fms-like tyrosine ki- nase) in HUVEC-induced preeclampsia. The black rice “Sembada Hitam” bran extract has an anti-angiogenic effect [15]. It lowers erythrocytic

malondialdehyde (MDA) levels in HUVEC-induced preeclampsia as an effective method for treating eclampsia [16]. The total anthocyanin content of the black rice (Oryza sativa L.) cultivar

“Sembada Hitam” was 284 mg/100 g [17]. The high levels of anthocyanin in “Sembada Hitam” is the potential to be developed as an alternative to breast cancer prevention and treatment. Despite its high antioxidant content, there is limited research on the potential use of black rice bran, specifically the "Sembada Hitam" variety, on breast cancer cells. Further research is needed to understand the effects of this cultivar on breast cancer cells.

Therefore, this study aims to investigate the cytotoxic activity, cell growth, and apoptosis induction properties of the ethanolic extract of black rice bran "Sembada Hitam" (EBRB) on T47D cells.

Material and Methods

Sample Preparation and Extraction

The black rice bran "Sembada Hitam" was sourced from Sleman Regency, Yogyakarta, and filtered using a 40-mesh sieve. A 10 g sample was then soaked in a mixture of 100 mL of 100% ethanol and 37% HCl in a ratio of 85:15. After 48 hours of maceration, the sample was filtered using Whatman paper no. 1 (Sigma). The filtrate was subsequently air-dried at room temperature to ob- tain a paste extract.

Cell Line Culture

T47D breast cancer cells were obtained from the Laboratory of Parasitology, Faculty of Medi- cine, Public Health, and Nursing (FK-KMK), Uni- versitas Gadjah Mada (UGM), Yogyakarta. The cells were cultured in complete Dulbecco’s Modi- fied Eagle Medium (DMEM) (Gibco, USA, REF 12800-058) supplemented with 10% fetal bovine serum (FBS) (Gibco, REF 10270-106), 2% peni- cillin and streptomycin (Gibco, REF 15140-122), and 0.5% amphotericin B (Gibco, REF 15290- 018). The cells were harvested using 0.5% Tryp- sin-EDTA (REF 25200-056) at 80% confluency.

MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) Assay for Cytotoxicity As- sessment

T47D cells were cultured in 96-well microplate with a density of 1 × 10⁴ cells/well in 100 µL DMEM and incubated for 24 hours at 37°C with 5% CO2. The cell cultures were treated with ethanolic extracts of black rice bran (EBRB) at 7.81,

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15.62, 31.25, 62.5, 125, 250, and 500 µg/mL and incubated at 37°C with 5% CO2 for 24 and 48 hours. Following incubation, the culture media and extract were removed and rinsed with phos- phate-buffered saline or PBS (Sigma made in the USA). Furthermore, the cells were then treated in 50 µL of MTT reagent (3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl-2H-tetrazolium bromide) and incubated for 4 hours [18]. The MTT reaction was stopped overnight by adding 10% sodium dodecyl sulfate or SDS (Merck with CAS Number 151-21- 3). The BIO-RAD Microplate Reader was used to measure the cell absorbance at the wavelength of 595 nm [3]. In this study, cells were treated with doxorubicin at concentrations of 1 and 2 µg/mL as positive controls, while 1% DMSO was used as the negative control. The percentage of cell viability was calculated using Equation 1, as follows [19]:

CV (%)=(control abs. -treated abs.)

control abs. × 100% …1

Remarks:

CV = cell viability

Control abs. = cell control absorbance Treated abs. = treated cell absorbance Cell Growth Assay

T47D cells were cultured in 6-well microplate at a 1.5 × 10⁵ cells/well density and incubated for 24 hours at 37°C with 5% CO2. The cells were treated with EBRB at 250 and 500 µg/mL concentrations, while 0.5% DMSO was used as a control.

The cells were cultured for six days, with a single passage after the third day of culture. Dead and living cells were counted using a hemocytometer after trypan blue staining on the third and sixth days of culture.

Apoptosis Test using Double Staining Method (Acridine Orange/Propidium Iodide)

The apoptosis test was conducted using the double staining method, which involved the combination of acridine orange (Merck, Germany, CAS number: 65-61-2) and propidium iodide (Millipore Sigma, USA, CAS number: 25535-16- 4) staining. T47D cells were seeded on a coverslip in 24-well microplate with a density of 1.25 x 10⁵ cells in a 1 mL/well volume and incubated for 24 hours at 37°C with 5% CO2. In addition, the cells were treated with EBRB at 250, 500, and 1000 µg/mL. Doxorubicin 0.5 µg/mL was used as the

positive control, and the cells without treatment were the negative control. The cells were incubated for 24 hours at 37°C with 5% CO2. After incubation, the coverslip was removed, mounted on a microscope slide, and stained with 10 µg/mL AO/PI dye, then incubated for 3 to 5 minutes at 37

°C. Subsequently, the samples were observed under a confocal microscope (Carl Zeiss LSM-880) at a magnification of 10×. The percentage of apoptosis was calculated as follows Equation 2:

Apoptotic cells (%)=AC count

TC Count× 100% … 2 Note:

AC count = Apoptotic cell counted TC count = Total cell counted Statistical Analysis

The data was analyzed using one-way ANOVA, followed by the Tukey HSD test when a significant difference was observed (p < 0.05).

The results are presented in the form of bar charts and graphs.

Results and Discussion

T47D cells were used as an in vitro model due to their similarity to human mammary gland epi- thelial cells in terms of their capacity to produce tumors and sensitivity to estrogen [20]. The cells are known to have high sensitivity to chemother- apy agents. Another advantage of using this model is their ability to reproduce rapidly with high ho- mogeneity, making them ideal for cytotoxicity testing [21]. Moreover, T47D cells can investigate apoptosis induction due to their capacity to ex- press caspase-3 [22].

The Ethanolic Extract Black Rice “Sembada Hitam” Bran is Non-Toxic on T47D cells

This study's findings indicate that the ethanolic extract of black rice bran "Sembada Hitam"

does not exhibit any cytotoxicity on T47D cells within 24 and 48 hours of treatment. The applica- tion of the extract did not result in any cytotoxic effects on T47D cells even at the highest concentration (500 µg/mL) after 24-hour incubation. The results of this study indicate that the ethanolic extract of black rice "Sembada Hitam" bran did not exhibit any cytotoxic effect on T47D cells after 24 and 48 hours of treatment. Moreover, no significant differences were observed when compared to the DMSO 1% control group. However, treatment

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with the EBRB extract at a concentration of 62.5 µg/mL for 24 hours resulted in the lowest cell viability of 82.80 ± 1.68%. On the other hand, the positive control group using doxorubicin at the concentrations of 1 and 2 µg/mL demonstrated inhibition of cell growth and cytotoxicity against T47D cells, with a relatively low percentage of viability below 50%, as depicted in Figure 1a. Alt- hough there was a decrease in growth, the viability of T47D cells remained above the ISO 10993-5 standard of 70%. According to the standard, an extract is considered cytotoxic if it can decrease cell viability by more than 30% [23].

The ethanolic extract of black rice “Sembada Hitam” bran still had no cytotoxicity effect on T47D cells after 48 hours of incubation. Moreo- ver, the highest extract concentration used in the

treatment (500 µg/mL) showed a proliferative effect on T47D cells. The cell viability was found to be high, indicating up to 132.85 ± 3.875%, which is higher than the 1% DMSO control group, with a cell viability of only 100 ± 3.28%. The lowest cell viability was observed in the 48-hour treatment group with 125 µg/mL EBRB extract concentration, which was 104.82 ± 3.59%. These results are higher than the 1% DMSO control group, which had a cell viability of 100%. The extract did not decrease cell viability and instead promoted cell proliferation in T47D cells (Figure 1b).

The group treated with doxorubicin at concentrations of 1 and 2 µg/mL, which served as the positive control, demonstrated very low cell viability with values of 26.97 ± 11.35% and 7.13 ± 1.13%, respectively. The observed low cell viability indi-

a

b

Figure 1. Ethanolic extract of black rice “Sembada Hitam” bran did not show a cytotoxic effect on T47D cells within 24 hours’ incubation (a) and 48 hours’ incubation (b). EBRB: ethanolic extract of black rice bran (n = 3).

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cates the cell growth inhibitory activity of doxorubicin, significantly different from the DMSO control group, as shown in Figure 1b. These findings clearly demonstrate the cytotoxic activity of doxorubicin against T47D cells. The results are simi- lar to previous research, where the longer the doxorubicin incubation period, the more DNA and mitochondrial damage occurs, increasing the amount of cell death [24, 25]. Studies also reported that the longer T47D cells are exposed to doxorubicin, the more Fe accumulates in the mitochondria, becom- ing toxic to the cell and causing cell damage [25].

Based on the cytotoxicity test results (Figure 1), it can be concluded that the extract of black rice bran "Sembada Hitam" did not have any harmful effects on T47D cells. This may be attributed to the relatively low anthocyanin level in the "Sem- bada Hitam" black rice bran cultivar, which makes it less effective in scavenging free radicals [17].

Additionally, it is worth noting that the extraction process used ethanol as a solvent, which may have contributed to the suboptimal extraction of anthocyanins from the black rice bran. Previous research has shown that the use of methanol as a solvent for anthocyanin extraction is more effective than ethanol [26]. Therefore, selecting the right extraction method and the solvent is necessary to produce extracts with optimal levels of phytochemicals, especially anthocyanins. In this study, the black rice “Sembada Hitam” bran could not stop the growth of T47D cells probably because the extract lacks of phytochemicals in deactivating the enzyme topoisomerase II. In the development of cancer cells, the enzyme topoisomerase II plays

a crucial role in DNA strands separation to divide continuously without undergoing strand shorten- ing. Excessive proliferation can be inhibited by in- activating the enzyme topoisomerase II [27]. Dox- orubicin is a topoisomerase II inhibitor and is a widely used commercial anti-cancer drug. Fur- thermore, it can also disrupt the cell membrane and interfere with the uncoiling process in DNA, causing cell damage and triggering death through the apoptosis pathway [24, 27].

Ethanolic extract of black rice bran has no sig- nificant apoptotic effect on T47D cells.

Based on the results, the black rice "Sembada Hitam" bran extract did not exhibit cytotoxicity or significant inhibition of T47D cell proliferation.

However, further investigation is needed to determine the extract's impact on cell mortality via the apoptosis pathway. Apoptosis regulation is commonly targeted by chemotherapeutic drugs and is a promising approach for cancer treatment as it does not affect normal cells [28]. In this study, cells that died through the necrosis pathway were ignored. The positive control group of doxorubicin 0.5 µg/mL and the untreated cells exhibited the highest and lowest percentage of apoptosis, respectively. Based on the statistical analysis, the treatment groups of extracted black rice bran at 250, 500, and 1000 µg/mL concentrations showed no significant difference from the control group (Figure 2).

In this study, the death rate and morphological changes in T47D cells were assessed using the double staining method with acridine orange/pro-

Figure 2. The ethanolic extract of black rice “Sembada Hitam” bran did not significantly increase apoptosis in T47D cells compared to the cell control. EBRB: ethanolic extract of black rice bran (n = 3).

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pidium iodide dye. Using the AO/PI method, live cells emitting green fluorescence can be identified under a confocal microscope, while cells undergoing apoptosis exhibit reddish-orange and brown- ish-yellow staining (as shown in Figure 3). The control group primarily consists of viable cells, as evidenced by the green fluorescent stain. Mean- while, numerous T47D cells observed in the doxorubicin group at 0.5 µg/mL concentration were stained red, indicating cell death through the apoptotic pathway. The density of T47D cells is relatively loose with a rounded cell shape, as shown in Figure 3b. It was also found that cells that colored an orange-yellow indicated death from early apoptosis. The cell densities were in the treatment groups of black rice “Sembada Hitam” bran at the concentrations of 250, 500, and 1000 µg/mL (Fig- ures 3c, 3d, and 3e.) higher than the control. Cell death with apoptosis pathway was seen in all treatment groups of the ethanolic extract of black rice

“Sembada Hitam” bran.

The black rice bran “Sembada Hitam” extract treatment showed a low and insignificant induction compared to the control group of cells without treatment, based on the apoptosis double assay results with AO/PI staining. It is consistent with the cytotoxic test results after a 24-hour treatment period, which showed a slight reduction in cell via-

bility. Furthermore, the induction of apoptosis may be caused by the anthocyanin content in the extract of black rice, especially cyanidin-3-glucoside and peonidin-3-glucoside compounds. Cya- nidin-3-glucoside and peonidin-3-glucoside induce apoptosis by stopping the G2/M phase of the cell cycle, condensing chromatin, increasing antioxidant activity, and triggering DNA fragmenta- tion [29]. It promotes the activity of caspase-3, a key element in the induction of apoptosis in cells [30].

Apart from its anthocyanin activity, the bran extract of "Sembada Hitam" black rice may also induce apoptosis in T47D cells through the action of tocotrienol compounds. Tocotrienols exert their pro-apoptotic effect by arresting the cell cycle at the G0/G1 phase and activating caspase-3 and caspase-9. Furthermore, γ-tocotrienol enhances the Bax/Bcl-2 ratio, which facilitates the process of mitochondrial outer membrane permeabilization [31]. The permeabilization promotes the re- lease of cytochrome c and activates the caspase cascade resulting in apoptosis. T47D cell apoptosis may also be induced through the poly ADP-ribose polymerase (PARP) cleavage pathway by tocotrienols. A previous study reported that tocotrienols enhanced the occurrence of apoptosis by PARP cleavage in MDA-MB-231 and MCF-7 hu- Figure 3. T47D cells morphology following treatment with ethanolic extract of black rice “Sembada Hitam” ex-

tract bran. Cell control group without treatment (A), doxorubicin at the concentration of 0.5 µg/mL (B), ethanolic extract of black rice bran at the concentration of 250, 500, and 1000 µg/mL (C, D, E). Acridine orange/propidium iodide double staining method with 10 × 10 magnification. Orange arrows indicate live cells, and light-blue arrows indicate apoptotic cells.

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man breast cancer cells [32].

Sembada Hitam" bran extract contains compounds that induce apoptosis, the double staining assay indicates that T47D cells undergo only a small amount of apoptosis. This is probably due to the extract containing the compound oryzanol, which acts as an antagonistic apoptosis inhibitor.

In H2O2 -induced SH-SY5Y neuroblastoma cells, oryzanol has been shown to reduce the expression of the Bax protein, lower oxidative stress, and prevent neurotoxicity [33]. The downregulation of the proteins Bax, caspase-9 and caspase-3 may have contributed to the prevention of the apoptotic process in T47D cells, inhibiting the activation of the mitochondrial apoptotic pathway. These findings contradict other studies, where black rice extract promotes caspase-9 and caspase-3 activity in MDA-MB-453 breast cancer cells and induces apoptosis [34]. This study contradicts other findings, demonstrating that the black rice fraction

“Woja Laka” promotes cell cycle arrest in the G0- G1 phase of HepG2 cells and triggers apoptosis [35]. The poor induction in T47D cells indicated that the black rice “Sembada Hitam” bran extract could not be used as an anti-cancer agent.

In this research, the apoptosis double staining assay showed that doxorubicin 0.5 µg/mL exhibited relatively strong apoptosis induction activity compared to the EBRB treatment group. Doxoru- bicin triggers apoptosis in T47D cells by increasing reactive oxygen species (ROS), raising caspase-3 levels, and initiating cell degradation [36]. Additionally, it induces apoptosis in MBA- MB-231 and MCF-10F breast cancer cells by up- regulating the Bax protein and downregulating the expression of the apoptosis-inhibitory gene Bcl-2 [37].

The extract of black rice “Sembada Hitam”

bran induced apoptosis of T47D cells with a relatively low percentage compared to doxorubicin.

This finding contradicts previous studies, where the black rice bran “Cempo Ireng” fraction had higher apoptosis induction activity than doxorubicin [38]. The difference is probably due to the extraction methods, types of solvents, and black rice cultivars used. The difference in plant cultivar af- fects the phytochemical content of the extract [39];

hence black rice bran “Sembada hitam” may have lower anthocyanin contents than the “Cempo Ireng” variety. It is confirmed by the results from a previous study, where the variety of black rice known as “Cempo Ireng” has an anthocyanin con-

tent of 428.38 mg/100 g. Meanwhile, “Sembada Hitam” has a lower amount at 284.05 mg/100 g [17]. The induction of apoptosis in T47D cells is probably not optimal due to the comparatively low anthocyanin content. Therefore, further research is required to determine the types and quantities of phytochemical compounds in the black rice bran

“Sembada Hitam” extract.

Black rice bran extract slows T47D cell prolifer- ation

Even though EBRB did not exhibit cytotoxic potential against T47D cells after 24 and 48 hours of incubation, a cell growth assay was carried out to determine the prolonged effect of EBRB on T47D cells. T47D cell growth in the control group (1% DMSO) decreased from day 0 of treatment to day 3. However, the cell growth started to increase from the fourth to the sixth day of culture. The reduction in the first three days was most likely due to T47D cells entering the lag phase after planting and suffering stress from the treatment of 1%

DMSO and “Sembada Hitam” black rice bran extract at doses of 250 and 500 µg/mL. The increase from day 3 to 6 was most probably related to the cells’ ability to adapt. From the 3^rd to 6^th day after incubation, the extract of black rice “Sembada Hitam” bran group with a concentration of 250 µg/mL had lower cell growth than 500 µg/mL and 1% DMSO. Furthermore, the treatment with 500 µg/mL “Sembada Hitam” black rice bran extract showed lower cell growth than 1% DMSO. A low- ering in cell growth rate compared to the 1%

DMSO control group suggests that the extract may suppress the growth of T47D breast cancer cells, as shown in Figure 4.

Based on the graph of Figure 4, the growth trend showed a growth arrest from the 1^st to the 3^rd day. Based on these patterns, T47D cells may con- tinue to grow slowly when incubated for a prolonged time. Lower cell growth rate in the EBRB group after day 3 was most likely induced by anthocyanin activity in the ethanolic extract of black rice ”Sembada Hitam” bran. Previous research has shown that anthocyanins can suppress the development of some cancer cells and prevent transfor- mation [40]. Anthocyanin and anthocyanidin have been shown to potentially target receptor tyrosine kinases (RTKs) such as EGFR, PDGFR, and VEGF/VEGFR, and inhibit cancer cell proliferation by interfering with the Ras-MAPK and PI3K/Akt signaling pathways [6]. Additionally,

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cyanidin-3-glucoside isolated from strawberry plants showed antioxidant activities and prevented the proliferation of human colorectal adenocarci- noma. This is achieved by inhibiting the growth of 3D spheroid in HT-29 and HCT-116 colon cancer cells [41].

Despite having a relatively low level of apoptosis induction and no cytotoxic activity on T47D cells, the ethanolic extract of black rice bran

“Sembada Hitam” is still beneficial for consump- tion. Further research on the variety as an anti-cancer agent is required, particularly phytochemical screening and selecting the best extraction method. Meanwhile, the selection of the extraction solvent used, the effect of time, and the extract storage temperature should be investigated to ob- tain extracts of good quality and quantity.

Conclusion

The study evaluated the effects of ethanolic extract of black rice bran "Sembada Hitam" on T47D breast cancer cells in terms of cytotoxicity, cell growth, and apoptotic induction. Results indicate that the extract did not exhibit cytotoxic effects on T47D cells during the 24 and 48 hours of incubation. At concentrations of 250, 500, and 1000 µg/mL, the extract induced cell death of 10.64 ± 2.98%, 9.99 ± 5.87%, and 5.84 ± 0.78%, respectively. However, the findings suggest that the extract may not be a potent anti-cancer agent.

Acknowledgment

Funding for this research was provided by the Faculty of Biology, Gadjah Mada University through the Lecturer and Student Collaboration

Grant in 2021 and 2022 (Research agreement no.

1020/UN1/FBI/KSA/PT.01.03/2021 and no.

1185/UN1/FBI.1/KSA/PT.01.03/2022).

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