Allium cepa Linnaeus, a worldwide culinary spice known as common onion, is a bulbous plant belonging to the Amaryllidaceae family. This species is the most widely cultivated of the Allium genus^1,2. Over the years, Allium plants have been used for many years to prevent and treat several diseases such as headaches, coughs, flu, asthma, inflammation, hemorrhage, arthritis, diabetes, and atherosclerosis.

It has also been reported that these plants, besides having pharmacological activities as antibacterial, antiviral, antifungal, anthelmintic, and antiprotozoal, also exhibited the potency of in vitro and in vivo antimotility and antiproliferative against cancer cells³. Their beneficial properties on health are due to the rich contents of bioactive phytochemicals, such as phenolic, particularly flavonoids, and some organosulfur compounds. Quercetins (quercetin-4’-monoglucoside and quercetin-3,4’-diglucoside) are the most abundant, with more than 85% of the total flavonoids found in common onions. Also, the red varieties of onions are rich in anthocyanins, especially cyanidin derivatives⁴.

Cancer is one of the significant global health problems today and responsible for most deaths after cardiovascular diseases⁵. International Agency for Research on Cancer (IARC) estimated 18.1 million new cases and 9.6 million mortalities caused by cancer in 2018 worldwide⁶. It is predicted that by 2040, the incidence of cancer will increase to 29.5 million new cases⁷. Although there are many treatment strategies to treat cancer, including chemotherapy, success in managing most issues is limited by systemic toxicity and gradual drug resistance⁸. Therefore, the research community is currently focused on searching for more efficient anticancer agents. Natural products from plants are considered potential candidates for new chemotherapeutic development because of their promising anticancer activity and low toxicity levels to normal cells⁹.

Several studies have examined the anticancer effect of Allium cepa L. on some cancer cells. Abdel-gawad et al.¹⁰ demonstrated that the methanolic extract of Allium cepa L., derived fractions, and its pure isolated compounds have high cytotoxic activity on hepatocellular carcinoma HepG2 cells. Wang et al.¹¹ reported that ethyl acetate extract of Allium cepa L. has a cytotoxic effect on human breast cancer cell lines, MDA-MB-231. Polyphenols from Allium cepa L. can inhibit in vitro the cell growth of human gastric adenocarcinoma AGS by interrupting phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, up-regulating p53, and inducing caspase-dependent apoptosis¹². However, there are no reports about the anticancer potential of Allium cepa L. on human cervical cancer HeLa cell lines. This study aimed to determine the cytotoxicity of Allium cepa L. ethanolic extract on HeLa cells.

MATERIALS AND METHODS:

Ethical Clearance:

The research procedures have been tested by the Medical and Health Research Ethics Committee, Universitas Gadjah Mada (KE/FK/0107/EC/2018).

Chemicals:

70% Ethanol, Dulbecco's modified eagle medium (DMEM) (Gibco), trypsin-ethylenediaminetetraacetic acid solution (Sigma-Aldrich), dimethyl sulfoxide (DMSO) (Sigma-Aldrich), HEPES (Sigma-Aldrich), 2% penicillin-streptomycin (Gibco), amphotericin B (Sigma-Aldrich), 10% fetal bovine serum (Rocky Mountain Biologicals, Inc.), 1× phosphate buffer saline (PBS) (Sigma-Aldrich), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich), sodium dodecyl sulfate (SDS) (Sigma-Aldrich), and HCl 0.1 N (Sigma-Aldrich).

Preparation of Allium cepa L. Extract:

The outer skins of Allium cepa L. were removed, and the bulbs were chopped small. Then they were dried at room temperature for seven days and mashed into powder using an electric blender. 148 g of the powder soaked in 70% ethanol for 24 h. The macerate was separated and concentrated using Rotavapor® R-300 at a speed of 250 rpm at 60°C, according to Jayanti et al.¹³.

Cell Culture:

HeLa cell lines were provided by the Department of Parasitology, School of Medicine, Universitas Gadjah Mada. Cells were grown in DMEM with addition of 10% fetal bovine serum, 0.5% fungizone, and 2% penicillin-streptomycin (37°C, 5% CO₂).

In Vitro Cytotoxicity Test:

The cytotoxicity of Allium cepa L. extract was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) method. HeLa cells were cultured in 96-well microplates (1×10⁴ cells/well) overnight. Before the treatment, the medium of all wells was removed first. Cells were then treated with four concentrations of ethanolic Allium cepa L. extract (50, 100, 200, and 400 µg/ml) for 24 h. Then the cells were administered with 100 µl of MTT solution and incubated for four h. After incubation, cell condition was examined using an inverted microscope to observe the formation of formazan crystals. Subsequently, 100 µl of 10% SDS solution was administered as the stopper and incubated at room temperature overnight. The absorbance was measured using a Benchmark Microplate Reader (Bio-Rad) at 595 nm. The percentage of cytotoxicity was calculated, and the IC₅₀ determination employed linear regression analysis using Microsoft Excel 2016 (Microsoft. Inc.)¹⁴.

RESULTS AND DISCUSSION:

The cytotoxic activity of the ethanolic extract of Allium cepa L. was evaluated by MTT assay. This technique was chosen since it is simple, reliable, and can be applied to various cell lines. This assay involved a colorimetric reaction of MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) with dehydrogenase enzyme present in living cells, reducing it into (E,Z)-5-(4,5-dimethylthiazol-2-yl)-1,3-diphenylformazan colored in a dark purple, which indicates the number of viable cells (Figure 1)^7,15,16.

Figure 1. The formation of formazan crystals in HeLa cells after being treated with Allium cepa L. extract at the concentration of (a) 50, (b) 100, (c) 200, and (d) 400 µg/ml for 24 h.

The result showed that treated HeLa cells' cytotoxicity increased gradually with increasing Allium cepa L. extract concentration. The highest inhibition of HeLa cells was found at 400 µg/ml Allium cepa L. extract concentration, where the cytotoxicity was 20.61%. Meanwhile, the lowest cell inhibition was at 50 µg/ml Allium cepa L. extract concentration, with cytotoxicity of 0.47%. Two other Allium cepa L. extract concentrations, 100 and 200 µg/ml, respectively, inhibited HeLa cells with cytotoxicity of 2.96% and 9.86%. Allium cepa L. extract showed an IC₅₀ value of 900.88 µg/ml on HeLa cells with an incubation of 24 h. The result proved that the treatment of HeLa cells with Allium cepa L. extract slowly decreased cell growth by increasing the extract concentration, and it did not reach 50% of the cells' death. The cytotoxicity graph of Allium cepa L. extract on HeLa cells is shown in Figure 2.

Figure 2. Cytotoxicity graph of Allium cepa L. extracts against HeLa cells.

In the current study, we demonstrated the anticancer activity of Allium cepa L. extracts on HeLa cells. We revealed that Allium cepa L. extract concentration-dependently increased cytotoxicity on HeLa cells with a high IC₅₀ value of 900.88 µg/ml. Zulfafamy et al.¹⁷ classified the activity index of crude extract of natural products into four class, i.e., high (≤20 µg/ml), moderate (>20-100 µg/ml), low (>100-1000 µg/ml), and inactive (>1000 µg/ml). Based on this classification, Allium cepa L. extract has a low cytotoxic effect against HeLa cells. It is similar to the previous study, which revealed that Allium cepa L. ethanolic extract did not significantly affect human hepatocellular carcinoma HepG2, human prostate cancer PC-3, and human colon adenocarcinoma HT-29 cell lines. Another study also indicated that Allium cepa L. extract was inactive against WiDr cancer cells. However, more significant results might be obtained if the active compound isolates in Allium cepa L., such as quercetins, sodium n-prophyl thiosulfates, alk(en)yl thiosulfates, and more, are used for anticancer screening. Individual quercetin glucosides showed marked effects on HepG2, PC-3, and HT-29 cells^18,19. In addition, specific compounds such as sodium n-prophyl thiosulfate showed a suppressive effect on the cell growth of human leukemia HL-60 cell lines through apoptosis induction initiated by oxidative stress²⁰.

On the other hand, lower doses require a longer time of treatment, for example, 72 or 96 h, prior to any observed decrease in cell viability. A study reported the effect of saponin-enriched extract from Momordica charantia on human Caucasian pancreatic adenocarcinoma CFPAC-1 and human pancreatic adenocarcinoma BxPC3 cells, which did not show any significant activity in the first 24 h. This circumstance may be attributed to the differences between kinetics and cell death mechanisms, such as the ability of cells to absorb compounds during exposure time²¹. Moreover, different cell line types, plant sources, plant extract, and solvent may also contribute to differences in the plant's cytotoxic effects²². It can be exemplified by Wang et al.¹¹, which proved that ethyl acetate extract of Allium cepa L. suppresses cell growth and promotes apoptosis in fatty acid synthase (FAS) over-expressing MDA-MB-231 cells. Additionally, the varying polarity of the solvent used in the extraction process may affect each extract's phytochemical content, resulting in different cytotoxic activities²³.

While the extract's cytotoxic activity was insufficient, there is still a lot of work to be done, such as further fractionation and purification of the compounds in the extract, increasing its cytotoxic potential. However, so far, there are still no reports on Allium cepa L. extract's potential as an anticancer agent against cervical cancer HeLa cells. Thus, this study suggests that there is still a need for development in the research of Allium cepa L. species as anticancer drug candidates.

CONCLUSION:

Overall, Allium cepa L. extract exerts anticancer properties on cervical cancer HeLa cell lines through cytotoxic activity in a concentration-dependent manner with an IC₅₀ of 900.88 µg/ml, which indicates low cytotoxicity. However, the current study is the first report on the cytotoxic effect of Allium cepa L. in HeLa cell lines. On the other hand, further research is needed to fractionate and purify the extract to investigate its activity on human cancer cells, especially HeLa.

ACKNOWLEDGEMENT:

This study was supported by PMDSU Grant Funds from the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia (1341/UN3.14/LT/2018).

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. Oyewusi AJ, Oridupa OA, Saba AB, Oyewusi IK, Mshelbwala MF. Effect of the methanol extract of the red cultivar Allium cepa L. on the serum biochemistry and electrolytes of rats following sub-chronic oral administration. J Basic Clin Physiol Pharmacol. 2019; 31(2): 1-14.

2. Ansori ANM, Fadholly A, Proboningrat A, Jayanti S, Hazaya S, Susilo RJK, Sucipto TH, Soegijanto S. Efficacy of Allium cepa (amaryllidaceae) extract against dengue virus type-2 (Flaviviridae: Flavivirus) isolated from Surabaya, Indonesia. Biochem Cell Arch. 2020; 20(2): 4783-4786.

3. Asemani Y, Zamani N, Bayat M, Amirghofran Z. Allium vegetables for possible future of cancer treatment. Phytother Res. 2019; 33(12): 1–21.

4. Fredotović Ž, Šprung M, Soldo B, Ljubenkov I, Budić-Leto I, Bilušić T, Čikeš-Čulić V, Puizina J. Chemical composition and biological activity of Allium cepa L. and Allium × cornutum (Clementi ex Visiani 1842) methanolic extracts. Mol. 2017; 22(3): 448.

5. Çakır EE, Alhasan H, Çakır E, Çömelekoğlu U. Cytotoxic effect of N-acetyl cysteine in DU145 human prostate cancer cells. J Res Pharm. 2019; 23(6): 1123-1130.

6. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68(6): 394–424.

7. Suciati, Arifianti L, Elsafira A, Ilmiah LQ. In vitro anticancer property of Solanum mammosum callus culture against HeLa and Vero cell lines. J Res Pharm. 2020; 24(2): 218-224.

8. Suharty N, Wahyuni FS, Dachriyanus. Cytotoxic activity of ethanol extract of Arbuscular mycorrhizal fungi induced ginger rhizome on T47D breast cancer cell lines. Pharmacogn J. 2018; 10(6): 1133-1136.

9. Demir S, Turan I, Demir F, Ayazoglu Demir E, Aliyazicioglu Y. Cytotoxic effect of Laurocerasus officinalis extract on human cancer cell lines. Marmara Pharm J. 2017; 21(1): 121-126.

10. Abdel-gawad MM, El-hashash MA, El-sayed MM, El-wakil EA, El-sayed Abdel-lateef E. Chromatographic isolation of Allium cepa (ssp. Red onion) and its cytotoxic activity against human liver carcinoma cell lines (HepG2). Int J Pharm Pharm Sci. 2014; 6(8): 108-111.

11. Wang Y, Tian WX, Ma XF. Inhibitory effects of onion (Allium cepa L.) extract on proliferation of cancer cells and adipocytes via inhibiting fatty acid synthase. As Pac J Can Prev. 2012; 13(11): 5573-5579.

12. Lee WS, Yi SM, Yun JW, Jung JH, Kim DH, Kim HJ, Chang SH, Kim G, Ryu CH, Shin SC, Hong SC, Choi YH, Jung JM. Polyphenols isolated from Allium cepa L. induces apoptosis by induction of p53 and suppression of Bcl-2 through Inhibiting PI3K/Akt signaling pathway in AGS human cancer cells. J Cancer Prev. 2014; 19(1): 14-22.

13. Jayanti S, Nurmalita SD and Nostalia SDA. Nanoparticles from onion (Allium cepa L.) extract suppress Bcl-2 expression in MCF-7 cells in vitro. Russ J Agric Soc Econ Sci. 2019; 6(90): 45-50.

14. Proboningrat A, Fadholly A, Iskandar RPD, Achmad AB, Rantam FA, Sudjarwo SA. The potency of chitosan-based Pinus merkusii bark extract nanoparticles as anticancer on HeLa cell lines. Vet World. 2019; 12(10): 1616-1623.

15. Liu Y, Peterson DA, Kimura H, Schubert D. Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. J Neurochem. 1997; 69(2): 581-593.

16. Bahuguna A, Khan I, Bajpai VK, Kang SC. MTT assay to evaluate the cytotoxic potential of a drug. Bangladesh J Pharmacol. 2017; 12(2): 115-118.

17. Zulfafamy KE, Ardiansyah, Budijanto S. Antioxidative properties and cytotoxic activity against colon cancer cell WiDr of Rhizopus oryzae and Rhizopus oligosporus-fermented black rice bran extract. Curr Res Nutr Food Sci J. 2018; 6(1): 23-24.

18. Pan Y, Zheng YM, Ho WS. Effect of quercetin glucosides from Allium extracts on HepG2, PC-3 and HT-29 cancer cell lines. Oncol Lett. 2018; 15(4): 4657-4661.

19. Fadholly A, Ansori ANM, Shara J, Proboningrat A, Kusala MKJ, Putri N, Rantam FA, Sudjarwo SA. Cytotoxic effect of Allium cepa L. extract on human colon cancer (WiDr) cells: in vitro study. Res J Pharm Tech. 2019; 12(7): 3483-3486.

20. Chang HS, Yamato O, Yamasaki M, Ko M, Maede Y. Growth inhibitory effect of Alk(en)yl thiosulfates derived from onion and garlic in human immortalized and tumor cell lines. Cancer Lett. 2005; 223(1): 47-55.

21. Richmond RA, Vuong QV, Scarlett CJ. Cytotoxic effect of Bitter Melon (Momordica charantia L.) ethanol extract and its fractions on pancreatic cancer cells in vitro. Explor Res Hyp Med. 2017; 2(4): 139–149.

22. Nordin ML, Kadir AA, Zakaria ZA, Abdullah R, Abdullah MNH. In vitro investigation of cytotoxic and antioxidative activities of Ardisia crispa against breast cancer cell lines, MCF-7 and MDA-MB-231. BMC Complement Altern Med. 2018; 18(1): 87.

23. Tejaputri NA, Arsianti A, Qorina F, Fithrotunnisa Q, Azizah NN, Putrianingsih R. Anticancer activity of Ruellia brittoniana on HeLa cancer cells. Pharmacogn J. 2020; 12(1): 29-34.

Received on 24.11.2020 Modified on 08.02.2021

Research J. Pharm. and Tech. 2021; 14(9):4969-4972.

DOI: 10.52711/0974-360X.2021.00864