Exploring the Cytotoxic Potential of Various Concentration of Extracts Tempe on Several Solvents on HSC-3 Cells: A Preliminary Study

Introduction

Cancer is a disease caused by abnormal cell growth that can spread to other parts of the body.¹ According to data from the Global Burden of Cancer (GLOBOCAN) in 2022, more than 20 million new cancer cases were recorded, with 9.7 million deaths.² A type of cancer that requires special attention is oral squamous cell carcinoma (OSCC), which primarily affects the oral cavity. This cancer not only affects the oral cavity area but can also spread to the throat, head, and neck. OSCC is the sixth most common malignancy worldwide, with more than 400,000 new cases each year, the majority of which occur in Asia, including Indonesia. It is more prevalent in males, particulary within the middle-aged to eldery population. Among the various anatomical sites in the oral cavity, the posterior lateral border of the tongue represents the most common site of occurance, accounting for approximately 50% of all OSCC cases.^3–5

The increasing number of oral cavity cancer cases has driven the development of natural agents for more effective prevention, one of which is through chemopreventive agents. Although cancer treatments such as surgery, radiotherapy, and chemotherapy are widely used, their success rates remain limited, especially for cancers that have spread. Therefore, attention to chemopreventive agents using natural substances has been growing, with the aim of preventing or slowing cancer progression, offering a safer alternative compared to conventional therapies.^6,7

One natural source with potential as a chemopreventive agent is tempe. Tempe is a traditional Indonesian food that has been consumed for generations. In addition to being a good source of nutrition, tempe contains bioactive compounds that are beneficial for health. The fermentation process of tempe by the fungus Rhizopus sp. produces enzymes that break down proteins, fats, and carbohydrates into simpler forms that are easily absorbed by the body. One of the active compounds produced is isoflavones, especially genistein, which is known for its anticancer, antioxidant, and antiproliferative properties.⁸

Several studies have shown that genistein has the ability to inhibit the growth of various types of cancer cells, such as colon, breast, pancreatic, and liver cancers. However, research on the effects of tempe on oral cavity cancer, particularly on oral squamous cell carcinoma (HSC-3), is still very limited. Given the high incidence of oral cavity cancer in Indonesia and the significant potential of tempe, this study aims to explore how tempe can kill HSC-3 cancer cells through cytotoxicity testing.

Experimental

Collection and preparation of tempe extract – The tempe used in this study was obtained from Rumah Tempe Indonesia (Bogor, West Java, Indonesia), which adheres to the Indonesian National Standard (SNI) and is made from local soybeans from Grobongan, Central Java, Indonesia. The tempe extraction was conducted at BALITRO using three different solvents: water, 70% ethanol, and 96% ethyl acetate. This study was designed as a preliminary screening of the cytotoxic activity of tempe extracts obtained using solvents of varying polarity. Water, 70% ethanol, and 96% ethyl acetate were selected to extract polar, semi-polar, and non-polar compounds, respectively. Water targets hydrophilic constituents like glycosylated isoflavones and peptides. 70% ethanol, a food-grade solvent commonly used in natural product extraction, facilitates recovery of a broad spectrum of polar to semi-polar compounds. Ethyl acetate was selected to obtain less polar, lipophilic constituents. The use of methanol or absolute ethanol (> 90%) was avoided due to toxicity concerns and limited relevance for food or nutraceutical applications. The tempe, which had been fermented for 4.5 days (108 hours), was ground using a blender.⁹ The resulting tempe powder was filtered using a 60 mesh sieve. The extraction process was performed using the maceration method, where 100 g of tempe powder was extracted with 500 mL of each solvent (water, 70% ethanol, and 96% ethyl acetate) for 3 hours in a shaker, followed by a 24-hour resting period. The extracts were then filtered using Whatman No. 42 filter paper and concentrated using a rotary evaporator. The tempe used in this study was produced according to Indonesian National Standard (SNI 3144:2015), which regulates fermentation practices, starter culture, and hygienic processing. However, chemical standardization of the extract, including quantification of key bioactive compounds such as genistein, daidzein, or total flavonoids, was not performed. This represents a limitation of the current study and will be addressed in future research through HPLC or LC-MS-based analysis.

Cell cultures – HSC-3 cell lines were obtained from the Laboratory of the Faculty of Medicine, Yarsi University, Jakarta, Indonesia. The cells were cultured in a T75 flask and treated with 5.5 mL of Phosphate Buffered Saline (PBS). After PBS was removed, 5.5 mL of trypsin was added. The cells were incubated at 37°C in a CO₂ incubator for 5 minutes. Cell inactivation was carried out by adding Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS). The cells were then transferred to a 15 mL centrifuge tube and centrifuged for 7 minutes at 1,500 rpm. The supernatant was discarded, and the cell pellet was retained. The pellet was suspended in 1 mL of DMEM and counted using a hemocytometer. The counted cell suspension was then placed in 96-well plates and supplemented with DMEM containing 10% FBS, followed by incubation for 24 hours in a CO₂ incubator at 37°C. Subsequently, cell growth and morphology were evaluated under a microscope.

Cytotoxicity assays and IC₅₀ determination – After 24 hours of incubation, the cells were ready to be tested using cytotoxicity assays. Tempe extract at various concentrations (1000, 2000, 3000, 4000, and 5000 μg/mL) prepared beforehand was added to the 96-well plates containing HSC-3 cells. Positive control (doxorubicin), solvent control (0.0398% DMSO), and negative control (untreated media) were also prepared for comparison. The culture media in the wells were removed and replaced with the diluted tempe extract samples at different concentrations. The plates were then reincubated in a CO₂ incubator for 24 hours at 37°C. After incubation, the media mixed with the extracts were removed, and each well was treated with 110 µL of solution containing 10 µL of CCK-8 mixed with 100 µL of DMEM. The plates were incubated again for 1 hour. The cytotoxicity of the cells was measured based on the number of dead and surviving cells using the CCK-8 assay. The absorbance values were measured at a wavelength of 450 nm using a spectrophotometer. This experiment was repeated three times, and the average ± SD values were calculated. The results obtained were used to calculate the percentage of cell cytotoxicity, the IC₅₀ values, and further data analysis using the formula:¹⁰

Cell death percentage (%) = [(Absorbance A – Absorbance B) / Absorbance A] × 100

• Note:
  Absorbance A refers to the absorbance of the negative control.
  Absorbance B refers to the absorbance of the sample.

Statistical analysis – Data were presented as means ± standard deviations (SD) for the indicated number of independently performed experiments. Statistical significance (p < 0.05) was assessed by one-way analysis of variance (ANOVA) coupled with post hoc Tukey were obtained using SPSS Statistics 30.0 software (IBM, Armonk, NY, USA).

Results and Discussion

The cytotoxicity test results of SNI-standardized tempe extract from local soybeans against HSC-3 cells were obtained through absorbance measurements after 24 hours of incubation, using the colorimetric CCK-8 assay. As shown in Fig. 1, tempe extract with ethyl acetate solvent exhibited the strongest cytotoxic effect compared to ethanol and water solvents. At 1000 µg/mL, ethyl acetate extract resulted in the lowest number of viable cells. Across all tested concentrations (2000, 4000, and 5000 µg/mL), cell viability remained lower than in the doxorubicin-treated positive control, except at 3000 µg/mL. These results indicate that the active compounds in the tempe extract are better dissolved in ethyl acetate, resulting in a more optimal inhibition of cancer cells, potentially enhancing the cytotoxic effect. This study is consistent with previous research by Shabarni et al., which demonstrated that ethyl acetate exhibited the highest cytotoxic activity against T47D breast cancer cells compared to ethanol and n-hexane solvents.¹¹ Additionally, research by Ana et al. also showed that ethyl acetate had the highest cytotoxic activity against renal carcinoma cells (A498 and 786-O) compared to water, n-hexane, and n-butanol solvents.¹²

Fig. 1.

Effect of tempe extract on the viability of HSC-3 cells. HSC-3 cells were plated at 10,000 cells in 96-well plate in DMEM with 10% FBS, and then treated with various concentration (1000–5000 μg/mL) of tempe extract for 24 h. The cell cycle distribution was analyzed by flow cytometry as described in Materials and Methods. Data were presented as means ± standard deviations (SD).

The concentration range (1000–5000 µg/mL) used in this study was determined based on preliminary tests. As shown in Fig. 2, the first assay used serial concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 µg/mL. Meanwhile, Fig. 3. That was the second assay, which used concentrations of 25, 50, 100, 200, and 400 µg/mL. The various concentrations were selected based on previous reports in breast cancer cell lines with IC₅₀ values 5.2 µg/mL. The experiment did not produce any cytotoxic effects on HSC-3 oral cancer cells in our study. Therefore, higher concentrations were then used to evaluate any potential cytotoxic activity. These findings indicate that the crude extracts may contain active constituents in low concentrations, requiring further purification and isolation to determine specific anticancer effects and to establish more precise IC₅₀ values at lower doses.

Fig. 2.

Effect of tempe extract on the viability of HSC-3 cells. HSC-3 cells were plated at 10,000 cells in 96-well plate in DMEM with 10% FBS, and then treated with various concentration (3.125–100 μg/mL) of tempe extract for 24 h. The cell cycle distribution was analyzed by flow cytometry as described in Materials and Methods. Data were presented as means ± standard deviations (SD).

Fig. 3.

Effect of tempe extract on the viability of HSC-3 cells. HSC-3 cells were plated at 10,000 cells in 96-well plate in DMEM with 10% FBS, and then treated with various concentration (25–400 μg/mL) of tempe extract for 24 h. The cell cycle distribution was analyzed by flow cytometry as described in Materials and Methods. Data were presented as means ± standard deviations (SD).

The limited cytotoxic activity observed in the aqueous extract of tempe in this study contrasts with previous reports. Divate et al., demonstrated that water extracts of tempe inhibited the proliferation of Caco-2 colon cancer cells and reduced precancerous lesions in rats.¹³ Devi et al., found that over-fermented tempe extracts exhibited significant cytotoxicity against MCF-7 breast cancer cells.⁹ These differences may be due to variations in soybean variety, fermentation conditions (e.g., duration, temperature, microbial strains), and extraction protocols (e.g., solvent type, extraction time, temperature). Such factors can influence the composition and concentration of bioactive compounds in the extracts. These factors should be carefully considered and standardized in future research to enable more consistent comparisons across different studies.

Interestingly, while several previous studies have reported significant cytotoxic effects of tempe extracts on various cancer cell lines—including colon, breast, and liver cancers—the present study did not observe comparable cytotoxicity, particularly with the aqueous extract, on HSC-3 oral cancer cells. This discrepancy highlights the context-dependent nature of natural product bioactivity. Factors such as the type of cancer cell line, extraction method, fermentation conditions, and the chemical profile of the extract may all contribute to variable outcomes. These findings underscore the importance of careful biological validation for each target system, and suggest that tempe extracts may not exert uniform effects across all cancer cell types.

For tempe extract with ethanol solvent, the viability of HSC-3 cells was relatively higher, especially at the 3000 µg/mL concentration. These results differ from previous studies that generally reported that tempe extract with ethanol solvent had anticancer activity. Ali and Rina's research showed that ethanol could dissolve bioactive compounds such as isoflavones, flavonoids, and phenolic compounds known to have anticancer activity.¹⁴ Yohanes et al.'s study also supported these findings, showing that tempe extract with ethanol solvent contained bioactive compounds with significant anticancer activity.¹⁵ For example, in a study involving breast cancer cells (MCF-7), the cytotoxic effect of tempe extract with ethanol solvent was very strong (IC₅₀ 5.20 ± 1.01 μg/mL).¹⁶ However, the different results in this study are likely due to the biological characteristics of the cancer cells used. HSC-3 cells, derived from oral squamous cell carcinoma, do not express estrogen receptors, which are a key factor in the growth of certain types of cancer, such as breast and ovarian cancer. Therefore, the absence of estrogen receptors in HSC-3 cells may lead to different responses to tempe extract compared to other cancer cells that have estrogen receptors.

On the other hand, tempe contains isoflavones, which are phytoestrogens with a chemical structure resembling human estrogen. These isoflavones are known to interact with estrogen receptors in hormone-based cancer cells, resulting in a more significant anticancer effect.¹⁷ Previous studies, such as the one conducted by Fahrul et al., showed that isoflavones in tempe could induce apoptosis and inhibit the proliferation of breast cancer cells through their interaction with estrogen receptors.¹⁸ Additionally, Iqra et al.'s research supported this finding, stating that the phytoestrogens could reduce the risk of hormone-based cancer development, such as breast cancer.¹⁹ However, in this study, the anticancer effects of isoflavones on oral cancer cells were not significant, which could be influenced by the lack of estrogen receptors in the HSC-3 cell line. Without the interaction between isoflavones and estrogen receptors, the mechanism of isoflavones in inhibiting cancer cell proliferation becomes less effective. These results suggest that oral cancer and other types of cancers, such as breast or ovarian cancer, have very different characteristics, both in terms of receptors and growth mechanisms. Therefore, cancer prevention and treatment efforts cannot be generalized and must be tailored to the specific characteristics of each type of cancer. The use of HSC-3 cells, which are estrogen receptor (ER)-negative, limits the direct extrapolation of our findings to hormone-dependent cancers. While flavonoids such as genistein and daidzein in tempe have demonstrated estrogenic activity in other models, the cytotoxic effects observed in this study are likely mediated through ER-independent pathways. Further research using ER-positive cancer cell lines is needed to determine whether tempe extracts exert differential effects in hormone-sensitive tumors.

The stronger cytotoxic effect of tempe extract with ethyl acetate solvent is likely due to ethyl acetate's ability to better dissolve semipolar compounds such as flavonoids, and phenolics. These compounds are known to have significant anticancer activity.²⁰ Flavonoids, particularly isoflavones like genistein found in tempe, play a crucial role in inhibiting cancer cell growth, directly contributing to higher cytotoxicity. This study aligns with previous research by Kristin et al., which showed that ethyl acetate solvent produced higher total flavonoids compared to ethanol solvent.²¹

Flavonoids can suppress cancer cell proliferation and induce apoptosis through various molecular mechanisms. First, flavonoids inhibit critical signaling pathways for cancer cell growth, such as EGFR/MAPK, PI3K/Akt, and NF-κB, thereby slowing cell proliferation. Additionally, flavonoids modulate the ratio between pro-apoptotic proteins (such as Bax and Bak) and anti-apoptotic proteins (such as Bcl-2 and Bcl-xL), triggering the release of cytochrome c from mitochondria, thereby activating the apoptosis cascade. Caspase activation, especially caspase-3, caspase-7, and caspase-9, also plays an essential role in cell death. Flavonoids like genistein can upregulate p53, triggering apoptosis through the intrinsic pathway. Overall, flavonoids work by inhibiting proliferation and activating cell death pathways, making them potential anticancer agents.²²

The morphological observations using the colorimetric CCK-8 assay on HSC-3 cells post-treatment, as shown in Fig. 4, support the results of the cytotoxicity test. With ethanol as the solvent, the cells maintained their normal morphology at concentrations of 1000, 2000, and 3000 µg/mL. However, at concentrations of 4000 µg/mL and 5000 µg/mL, shrinkage and a reduction in the number of viable cells were observed, although some cells remained. In contrast, with ethyl acetate, more significant morphological changes occurred, especially at concentrations of 2000, 3000, 4000, and 5000 µg/mL, where most of the cells showed shrinkage and blebbing, characteristic of apoptosis. At 5000 µg/mL, nearly all cells were dead, indicating a very strong cytotoxic effect. Water also caused a reduction in the number of viable cells at high concentrations, although it was not as effective as ethyl acetate. Doxorubicin used as a positive control, effectively induced the death of nearly all cells, while the solvent control and untreated groups showed cells that remained intact without significant morphological changes.

Fig. 4.

Morphological changes mediated by tempe extract in HSC-3 cells. Cells were treated with or without various concentrations of tempe extract for 24 h. Cells were photographed by inverted microscopy (magnification 100×).

Although morphological observation showed more pronounced structural damage in cells treated with ethyl acetate extract at higher concentrations, the quantitative IC₅₀ value derived from the MTT assay was lower for the ethanol extract (60.57 µg/mL) than for ethyl acetate (122.7 µg/mL). This discrepancy may reflect differences in the mechanisms of action, onset of effects, or extract composition. Morphological changes are often visually apparent but not always proportional to loss of viability, which is more reliably assessed through metabolic activity-based assays such as MTT.

The use of doxorubicin as a positive control in this study is based on its ability to inhibit DNA and RNA synthesis, with a mechanism of action involving DNA intercalation, inhibition of topoisomerase II, and free radical production. Doxorubicin has been widely used as an anticancer drug, inducing cancer cell death through various pathways, including apoptosis, autophagy, cellular senescence, and necrosis.²³ Additionally, DMSO at a concentration of 0.0398% was chosen as the solvent control due to its amphiphilic nature, which does not affect cell viability at low concentrations, making it an ideal component in cytotoxicity testing.²⁴

The cytotoxic activity of tempe extract against HSC-3 cells in this study was evaluated using the CCK-8 colorimetric method, which utilizes a water-soluble tetrazolium salt to detect intracellular dehydrogenase activity. The CCK-8 method has higher sensitivity compared to other assays. In CCK-8 measurements, WST-8 dye [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt] is reduced by intracellular dehydrogenases to form an orange water-soluble product.²⁵ The primary parameter used for cytotoxicity testing is the IC₅₀ value, which is the concentration required to inhibit 50% of the cancer cells tested.

Based on the cytotoxicity assay presented in Fig. 5, tempe extract in high concentration (1000–5000 µg/mL) in ethanol, ethyl acetate, and water solvents showed significant cytotoxic activity, with IC₅₀ values of 72.59 µg/mL, 103.4 µg/mL, and 197 µg/mL, respectively. According to the US National Cancer Institute's cytotoxicity classification, a compound is considered highly active if its IC₅₀ ≤ 20 µg/mL, active (IC₅₀ 21–200 µg/mL), weakly active (IC₅₀ 201–500 µg/mL), and inactive (IC₅₀ ≥ 501 µg/mL). These results indicate that tempe extract with ethanol solvent is the most effective in inhibiting HSC-3 cancer cell growth, as it has the lowest IC₅₀ value (60.57 µg/mL). On the other hand, although ethyl acetate solvent caused higher cell death at higher concentrations, its IC₅₀ of 122.7 µg/mL indicates that higher concentrations are needed to achieve 50% inhibition of cancer cells. This suggests variations in the mechanism of action or composition of active compounds extracted by each solvent. Ethanol, which is polar, tends to be more efficient in extracting active compounds at lower concentrations. Conversely, ethyl acetate, which is semipolar, extracts compounds such as flavonoids, terpenoids, and phenolics, which are known to have cytotoxic activity but require higher concentrations to achieve the same effectiveness.²⁶

Fig. 5.

Effect of tempe extract in high concentration on the cytotoxicity of HSC-3 oral cancer cells. Cytotoxicity was assessed using the CCK-8 assay, and IC50 values, representing the concentration required to inhibit 50% of cell viability, were calculated using regression analysis (y = ax + b) with GraphPad Prism software (California, USA).

This study was designed as an initial investigation to assess the cytotoxic potential of tempe extracts, and did not include molecular analyses to elucidate the underlying mechanisms. Although flavonoids and phenolic compounds found in tempe have been reported to induce apoptosis in cancer cells through pathways involving p53, Bax, Bcl-2, and caspases, the present study did not confirm these mechanisms experimentally. This remains a limitation of our study and will be addressed in future research using appropriate molecular assays such as Western blotting or RT-PCR. A key limitation of this preliminary study is the absence of chemical analysis and standardization of the extracts. Although the tempe used was produced under standardized conditions (SNI 3144:2015), the active components within the ethanol, ethyl acetate, and water extracts were not identified or quantified. Future studies will include HPLC or LC-MS analysis to determine the concentrations of key compounds such as genistein, daidzein, and other phenolic constituents, in order to establish standardized and reproducible extract profiles.

In conclusion, SNI-standardized tempe extract from local soybeans has cytotoxic potential against oral cancer cell line HSC-3, with varying effectiveness depending on the solvent used. Among the three solvents tested, ethyl acetate showed the highest cancer cell inhibition at higher concentrations, supported by morphological changes such as shrinkage and blebbing, indicating apoptosis in HSC-3 cells. However, ethyl acetate has a higher IC₅₀ value compared to ethanol, suggesting that the bioactive compounds extracted by ethanol are more effective at lower concentrations in inhibiting HSC-3 cell growth. Additionally, the biological characteristics of HSC-3 cells, which do not express estrogen receptors, may influence the cell's response to isoflavones in tempe extract.

Acknowledgments

This research was conducted with the assistance of the Laboratory at the Faculty of Medicine, Yarsi University, Jakarta.

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