Effect of tamarillo (Cyphomandra betacea Sendtn.) seed ethanol extract on HSC-3 tongue cancer cells

Saqila Salfabila; Janti Sudiono

doi:10.4103/SDJ.SDJ_24_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 6 | Issue : 3 | Page : 141-145

Effect of tamarillo (Cyphomandra betacea Sendtn.) seed ethanol extract on HSC-3 tongue cancer cells

Saqila Salfabila¹, Janti Sudiono²
¹ Faculty of Dentistry, Universitas Trisakti, Jakarta, Indonesia
² Department of Oral Pathology, Faculty of Dentistry, Universitas Trisakti, Jakarta, Indonesia

Date of Submission	22-Apr-2022
Date of Decision	11-Jul-2022
Date of Acceptance	20-Aug-2022
Date of Web Publication	15-Nov-2022

Correspondence Address:
Janti Sudiono
Department of Oral Pathology, Faculty of Dentistry, Universitas Trisakti, Jakarta
Indonesia

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/SDJ.SDJ_24_22

Abstract

Background: Cancer is characterized by abnormal cell growth. Squamous cell carcinoma is the most common type oral cancer. Tamarillo fruit extract shows antiproliferative and antioxidant activity against breast cancer (MDA-MB-231) and liver cancer (HepG2) cells in rat models. Tamarillo seed extract consists of phenolics, flavonoids, triterpenoids, tannins, and alkaloids, which have antioxidant, antiproliferative, anti-inflammatory, and anticancer activity. Objectives: The aim of this study was to determine the effect of tamarillo seed ethanol extract at different concentrations on the viability of a tongue cancer cell (human oral squamous carcinoma cell line HSC-3). Methods: This experimental in vitro laboratory study comprised a treatment group comprising HSC-3 cells treated with eight concentrations of tamarillo seed extract (8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg/µL) and a positive control group treated with 3% H₂O₂. Shapiro–Wilk test showed that the data were distributed normally with P > 0.05. One-way analysis of variance (ANOVA) test showed that there were significant differences among concentrations group of the extract on HSC-3 viability cells with P ≤ 0.05. Results: At a concentration of 2 μg/µL, tamarillo seed ethanol extract showed the highest cytotoxicity against HSC-3 cells. Post hoc test revealed a significant difference (P < 0.05) in cell viability of concentration 2 μg/µL compared with that of the lower concentrations (0.25, 0.125, and 0.0625 μg/µL) and the positive control but there was no significant difference with those of higher concentration (4 and 8 μg/µL). Conclusions: A low concentration of tamarillo seed ethanol extract (2 µg/µL) had the highest cytotoxicity against HSC-3 cells significantly reducing cell viability. However, this concentration was not able to surpass the inhibition ability of the positive control (3% H₂O₂).

Keywords: HSC-3 human tongue squamous carcinoma cell line, seed extract, Tamarillo, viability

How to cite this article:
Salfabila S, Sudiono J. Effect of tamarillo (Cyphomandra betacea Sendtn.) seed ethanol extract on HSC-3 tongue cancer cells. Sci Dent J 2022;6:141-5

How to cite this URL:
Salfabila S, Sudiono J. Effect of tamarillo (Cyphomandra betacea Sendtn.) seed ethanol extract on HSC-3 tongue cancer cells. Sci Dent J [serial online] 2022 [cited 2023 Mar 20];6:141-5. Available from: https://www.scidentj.com/text.asp?2022/6/3/141/361152

Background

In worldwide, cancer is associated with substantial mortality.^[1] Oral cancer accounts for approximately 2% of all human malignancies and ranks sixth among malignancies reported worldwide.^[2] Oral cancer is common in developing countries, such as Indonesia, where it accounts for 3%–4% of all cancers.^[2],[3] Squamous cell carcinoma is the most common type of oral cancer, with a prevalence of 90% of total oral cancer cases.^[3] Squamous cell carcinomas are derived from stratified squamous cells, with the anterior two-thirds of the tongue as the most common site (40% of cases).^[4],[5] Squamous cell carcinomas on the tongue are able to invade local lymph nodes, leading to metastasis.^[6] The survival rate in such cases is poor because the tongue consists of muscles with a rich blood supply. The treatment of choice for squamous cell carcinoma on the tongue is usually surgery, followed by radio- and chemotherapy.^[7]

Today, herbal-based therapy is continued to find out the toxic effect of their phytochemical content against cancer cells. The development of herbal remedies is needed, especially for malignancies encountered in dentistry following their phytochemical potency as antimicrobial, antioxidant, and anticarcinogenic besides the price consideration of malignancy treatment needed. The herbal-based therapy is to be chosen by the population due to the easily found of this herbal plant.^[8],[9] Several herbal metabolites or their phytochemical contents have strong antioxidant potential against free radicals to reduce the redox imbalance. This redox imbalance condition may lead to different kinds of diseases such as atherosclerosis, diabetes mellitus, neurodegeneration, and cancer.^[10] Many herbal medicines with bioactive components that prevent the occurrence of the initiation phase (e.g., gene mutation that is characterized by continuous uncontrolled cell proliferation) in the carcinogenesis process have been reported.^[11]

Indonesia has a rich history of traditional natural herbal remedies.^[11]Cyhpomandrabetacea Sendtn., a member of the Solanaceae plant family, commonly known as tomato stick or tamarillo, has the potential as a herbal remedy.^[12] A previous study revealed that tamarillo (Cyhpomandrabetacea Sendtn.) has chemical compounds, including flavonoids, terpenoids, steroids, saponins, alkaloids, and tannins.^[13] Flavonoids, phenols, and tannins have very strong antioxidant activity.^[14] Flavonoid compounds also have anticancer and antiallergy properties. Flavonoid compounds can stop the production of prostaglandins, thereby relieving pain.^[15] They also exert an antibacterial effect, act as an anti-inflammatory agent, and inhibit lipid peroxidation, which reduces reactive oxygen species.^[15],[16] Thus, flavonoid compounds can slow tissue death, increase vascularity, elastin–collagen ratio, and prevent cell damage.^[15],[16]

The human body needs antioxidants to combat free radicals from sun exposure, pollution, and cigarette smoke. Antioxidants or anti-free radicals are molecules that form due to the presence of free radicals, which donate electrons and combine with free electrons from other molecules or other non-radical molecules. The formation of nonradical products resulting from the combination of two radical species can terminate this chain reaction or propagation.^[17] A study of a tongue cancer cell line HSC-3 treated with fruit peel extract of tamarillo (Cyhpomandrabetacea Sendtn.) revealed that a concentration of 5% of the extract was effective in decreasing cell viability.^[18] Another study on rats showed that extract of tamarillo fruit showed an anticancer effect, with anti-proliferative and antioxidant activity against breast cancer cells (MDA-MB-231) and liver cancer cells (HepG2).^[19] However, this extract showed no toxic effect against fibroblast or normal cells (3T3).^[18] No studies have investigated the effect of tamarillo seed extract against tongue cancer cells (HSC-3). The aim of this study was to investigate the effect of tamarillo (Cyphomandra betacea Sendtn.) seed ethanol extract on tongue cancer cell (HSC-3) viability. The hypothesis of this study was that the tamarillo seed extract would reduce the viability of HSC-3 cells.

Materials and Methods

The tamarillo used in this study was Cyphomandra betacea Sendtn., sourced from Biology LIPI Research Central. This study was conducted in the Biomedical Department and the Integrated Laboratory, from November to December 2019. The sample used was an HSC-3 cell line from the Bio Bank of the Integrated Laboratory. Tamarillo seed extract was obtained by the maceration technique by diluting 120 g of the tamarillo dried seed powder in 450 mL of 70% ethanol for 3 days, filtrated, and then evaporated in a rotary device to obtain the crude extract. The extract was diluted using the formula N1 × V1 = N2 × V2 to obtain eight concentrations: (8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg/µL) where N is the gram of extract and V is the liter volume of solution. These concentrations were used for treatment extract groups of this study based on the result of the previous study.

The phytochemical properties of the extract were examined using qualitative and also quantitative technique tests for flavonoid. The total analysis for flavonoids in this study was done using colorimetric method analysis. Colorimetric analysis method used to measure total flavonoid is based on the occurrence of complex reactions between flavonoid and aluminum chloride (AlCl₃). The solution of AlCl₃ will form the stable complex with C4 group and hydroxil group of C3 or C5 of flavon and flavonol.^[20] The 10 mg of extract was solved into 10-mL ethanol 96% and as much as 0.5 mL extract sample was piped into reaction tube then 2.2 mL aquabidest and 0.15 mL of 5% sodium nitrite were added into the tube, incubated for 5 min, added with 0.15 mL of 10% AlCl_3, mixed, and incubated again at room temperature for 6 min. Then added 2.0 mL NaOH 1 M and incubated at room temperature for 15 min. The absorbance of the solution was measured using a spectrophotometer with a wavelength of 510 nm. A subculture of HSC-3 cells in nitrogen liquid was warmed in a water bath at 37°C and then diluted in the culture media up to 10 times, centrifuged to obtain the cell pellet, and re-suspended with culture media. A T-flask was used for the cell culture, with incubation from 24 h up to 7 days. The cells were then collected for further analysis. The total sample used for each well was 15,000 cells that were cultured for group samples consisting of nine treatment extract groups, a positive control, and a negative control. Let it in an incubator (37°C) for 24 h and then put Cell Counting Kit-8 (CCK-8) into the well. After 1 h, the viability test was measured using a microplate reader with ƛ = 450 nm. The measurement was done three times.

The cytotoxicity test in this study used the Cell Counting Kit-8 (CCK-8) test (Sigma Aldrich, Germany). In contrast with the MTT assay that determined the cytotoxic cells, the CCK-8 test counts the viability cells based on the color changes of the HSC-3 cells. This CCK-8 test used in this study determines viability cells using 96 wells, which consisted of 15,000 cells with 200 µL in each well. CCK-8 reagent was added to each well and the 96-well plate was placed in an incubator (37°C) for 24 h. After 1 h, the viability of the HSC-3 cells was measured using a microplate reader with ƛ = 450 nm. The scheme of the research protocol is shown in [Figure 1]. The cell viability measurement was performed three times. Cell viability was determined based on color changes of the HSC-3 cells treated with the CCK-8 reagent. Cell viability was determined using the following formula as follows:^[21]

Figure 1: The study protocol and different concentrations of seed extract tested

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Statistical analysis

This study used Shapiro–Wilk test to know the homogeneity of data within groups. The data of this study were normally distributed; therefore, parametric statistical analysis was done to find out the differences between groups.

Results

The results of the viability of HSC-3 cells are shown in [Figure 2]. Shapiro–Wilk test showed that the data were distributed normally with P > 0.05. One-way analysis of variance (ANOVA) and least significant difference post hoc test showed that there were significant differences among concentrations group of the extract on HSC-3 viability cells with P < 0.05. The phytochemical contents of the tamarillo seed extract are shown in [Table 1]. The colorimetric method analysis showed that the mean value of flavonoid content within tamarillo seed extract was 275.30 mg/g extract.

Figure 2: Percentage of viable HSC-3 cells treated with the extract of tamarillo (Cyphomandra betacea Sendtn.) seed and the positive control

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Table 1: Phytochemical content of tamarillo seed extract

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The results of post hoc test revealed significant differences in the viability of cells between the positive control and three of the treatment extract groups (0.25, 0.125, and 0.0625 μg/µL). There was no significant difference in the viability of cells of the positive control group versus that of other treatment extract groups (8, 4, 2, 1, and 0.5 μg/µL). Among the concentrations of extract tested, the concentration of 2 μg/µL showed the highest potential to decrease the viability of HSC-3 cells, higher than that of the other extract concentrations but not as high as that of the positive control, as shown in [Figure 2]. There was no significant difference in cytotoxicity effect between the concentrations of 2 and 8 μg/µL which means the two concentrations have similar toxic effects on HSC-3 cells. The cytotoxicity effect of the positive control was higher than that of all the extract treatment groups, with significant differences among the groups. The extract with a concentration of 2 μg/µL was more toxic than that of extracts with higher concentrations (4–8 μg/µL)

Discussion

Ethanol extract of tamarillo seed reduced the viability of cancer cells, especially at a concentration of 2 μg/µL. This concentration was as effective (potent) as the highest concentration (8 μg/µL) of ethanol extract tested. The cytotoxicity of ethanol extract of tamarillo seed against cancer cells as shown in this study is attributed to its phytochemical content (i.e., phenolics, flavonoids, triterpenoids, tannins, and alkaloids), particularly polyphenol flavonoid compounds, which contribute to its anticancer properties. In this study, the flavonoid content was as high as 275.30 mg/g extract

The results of this study are in accordance with those of previous studies that one of which revealed ethanol extract of tamarillo fruit showed potent anticancer, antioxidant, and antiproliferative activity against liver cancer cells (HepG2) and breast cancer cells (MDA-MB-231) due to its phytochemical (flavonoid) content.^[18] A study found that tamarillo (Cyphomandra betacea Sendtn.) is a low-fat and calorie fruit with high nutritional value that provides a number of micronutrients, vitamins, and minerals and contains the chemical compounds flavonoids, terpenoids, steroids, saponins, alkaloids, and tannins.^[13] Polyphenolic compound flavonoids play a role in the cytotoxicity of cancer cells by influencing the cell cycle in the initiation phase of carcinogenesis, especially in the interaction phase between DNA and the first exposure to the carcinogen. According to Heravi et al.’s (2013) study, flavonoids also influence other stages of cell cycles, such as proliferation and gene mutation, by inhibiting the G1/S and G2/M phases of the cell cycle to give a period of repair of DNA damage.^[22] Flavonoids can decrease the gene mutation of cancer cells, which depends on the P53 protein, which has been mutant.^[23] Flavonoids as an anticancer agent can inhibit tyrosine kinase protein activity in a similar manner to growth signal factor for the nuclear.^[24] All of this potency was proven in this study, in which ethanol extract of tamarillo (Cyphomandra betacea Sendtn.) seed with its high flavonoid content as much as 275.30 mg/g extract showed cytotoxicity effect against HSC-3 cancer cells started from low concentration which was at 0.0625 μg/µL extract.

The crude extract used in this study may account for the cytotoxicity potency of cancer cells. The higher concentrations may contain more un-dissolved phytochemical compounds, which may have contributed to the cytotoxicity potency of the extract against the HSC-3 cells. This would be a limitation of this study.

Conclusions

Ethanol extract of tamarillo (Cyphomandra betacea Sendtn.) seed showed cytotoxicity effect against HSC-3 cancer cells starting from the concentration of 0.0625 μg/µL with the optimum concentration of 2 μg/ µL. However, at this concentration, the potency of the extract was still lower than that of the positive control (3% H₂O₂).

The cytotoxicity of ethanol extract of tamarillo (Cyphomandra betacea Sendtn.) seed against cancer cells was due to the phytochemical content of the extract, especially its high flavonoid content as much as 275.30 mg/g extract. A study using high-performance liquid chromatography of Tamarillo seed extract is needed to confirm its potential cytotoxicity against cancer cells.

Financial support and sponsorship

Not applicable.

Conflicts of interest

There are no conflicts of interest.

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