In vitro Cytotoxicity and Genotoxicity/Antigenotoxicity Evaluation of Encapsulated Black Garlic Extracts on A549 Cells

Year 2024, , 59 - 68, 29.05.2024

Çiğdem İnci Aydemir , Hatice Elif Temiztürk , Gökçe Taner

https://doi.org/10.61678/bursamed.1452163

Abstract

Objectives: Black garlic is produced by fermenting fresh garlic under controlled temperature and humidity conditions for an extended period. Due to its sweeter taste and lack of pungent odor compared to fresh garlic, black garlic is easier to consume. Moreover, the increase in bioactive compounds such as polyphenols and flavonoids during fermentation has sparked interest in studying the health effects of black garlic. It is known that different fermentation and extraction methods can lead to variations in biological activities. Therefore, analyzing the effectiveness of black garlic processed by different methods is of critical importance. In our study, we investigated the cytotoxic, genotoxic, and antigenotoxic effects of different concentrations of encapsulated black garlic capsule extract (BGC) on lung cancer cells.

Methods: The A549 cell line was used to investigate the effects of BGC. Cells treated with BGC at different concentrations (10, 25, 50, 100, 125, 250, 500, and 1000 µg/mL) for 24 hours were subjected to MTT and NRU assays to examine the cytotoxic effects. Alkaline comet assay was performed to investigate genotoxic and antigenotoxic effects. For antigenotoxicity analysis, cells pretreated with BGC were exposed to H2O2 to explore the protective effects of BGC.

Results: According to the MTT results, cell viability remained at 90% even at concentrations higher than 125 µg/mL. However, in the NRU analysis, viability decreased to less than 70% at concentrations ranging from 50 µg/mL. Comet assay results revealed significant increases in tail length and tail intensity at different concentrations (specifically, at 250 µg/mL and above and at 50 µg/mL and 100 µg/mL, respectively). However, tail moments did not show any significant differences at any concentration. Additionally, BGC significantly reduced H2O2-induced DNA damage.

Conclusions: Our research demonstrated that BGC reduces the viability of lung cancer cells and can have genotoxic effects. Additionally, its protective effect against oxidative damage was shown at the DNA level. Based on these data, further research can be conducted on the use of BGC against cancer.

Keywords

Black Garlic, Antigenotoxicity, Cytotoxicity, Comet Assay

References

• Shang A, Cao SY, Xu XY, Gan RY, Tang GY, Corke H, et al. Bioactive Compounds and Biological Functions of Garlic (Allium sativum L.). Foods 2019;8(7):246.
• Kilic-Buyukkurt O, Kelebek H, Bordiga M, Keskin M, Selli S. Changes in the aroma and key odorants from white garlic to black garlic using approaches of molecular sensory science: A review. Heliyon 2023;9(8).
• Stępień AE, Trojniak J, Tabarkiewicz J. Anti-Cancer and Anti-Inflammatory Properties of Black Garlic. Int J Mol Sci 2024;25(3):1801.
• Kim JS, Kang OJ, Gweon OC. Comparison of phenolic acids and flavonoids in black garlic at different thermal processing steps. J Funct Foods 2013;5(1).
• Sato E, Kohno M, Hamano H, Niwano Y. Increased anti-oxidative potency of garlic by spontaneous short-term fermentation. Plant Foods for Human Nutrition 2006;61(4).
• Toledano Medina MA, Pérez-Aparicio J, Moreno-Rojas R, Merinas-Amo T. Evolution of some physicochemical and antioxidant properties of black garlic whole bulbs and peeled cloves. Food Chem 2016;199.
• Shin JH, Lee CW, Oh SJ, Yun J, Kang MR, Han SB, et al. Hepatoprotective effect of aged black garlic extract in rodents. Toxicol Res 2014;30(1).
• Tsai JC, Chen YA, Wu JT, Cheng KC, Lai PS, Liu KF, et al. Extracts from fermented black garlic exhibit a hepatoprotective effect on acute hepatic injury. Molecules 2019;24(6).
• Jung YM, Lee SH, Lee DS, You MJ, Chung IK, Cheon WH, et al. Fermented garlic protects diabetic, obese mice when fed a high-fat diet by antioxidant effects. Nutrition Research 2011;31(5).
• Purev U, Chung MJ, Oh DH. Individual differences on immunostimulatory activity of raw and black garlic extract in human primary immune cells. Immunopharmacol Immunotoxicol 2012;34(4):651–60.
• Tabari M, Ebrahimpour S. Effect of aged garlic extract on immune responses to experimental fibrosarcoma tumor in BALB/c mice. Indian J Cancer 2014;51(4).
• Medina MÁT, Merinas-Amo T, Fernández-Bedmar Z, Font R, Del Río-Celestino M, Pérez-Aparicio J, et al. Physicochemical characterization and biological activities of black and white garlic: In vivo and in vitro assays. Foods 2019;8(6).
• Zhang Y, Li HY, Zhang ZH, Bian HL, Lin G. Garlic-derived compound S-allylmercaptocysteine inhibits cell growth and induces apoptosis via the JNK and p38 pathways in human colorectal carcinoma cells. Oncol Lett 2014;8(6).
• Jikihara H, Qi G, Nozoe K, Hirokawa M, Sato H, Sugihara Y, et al. Aged garlic extract inhibits 1,2-dimethylhydrazine-induced colon tumor development by suppressing cell proliferation. Oncol Rep 2015;33(3).
• Saha SK, Lee S Bin, Won J, Choi HY, Kim K, Yang GM, et al. Correlation between oxidative stress, nutrition, and cancer initiation. Vol. 18, International Journal of Molecular Sciences 2017.
• Forni C, Rossi M, Borromeo I, Feriotto G, Platamone G, Tabolacci C, et al. Flavonoids: A myth or a reality for cancer therapy? Molecules 2021;26(12).
• Jang HJ, Lee HJ, Yoon DK, Ji DS, Kim JH, Lee CH. Antioxidant and antimicrobial activities of fresh garlic and aged garlic by-products extracted with different solvents. Food Sci Biotechnol 2018;27(1).
• Jeong YY, Ryu JH, Shin JH, Kang MJ, Kang JR, Han J, et al. Comparison of anti-oxidant and anti-inflammatory effects between fresh and aged black garlic extracts. Molecules 2016;21(4).
• Zhang Z, Lei M, Liu R, Gao Y, Xu M, Zhang M. Evaluation of Alliin, Saccharide Contents and Antioxidant Activities of Black Garlic during Thermal Processing. J Food Biochem 2015;39(1).
• Xiong F, Dai CH, Hou FR, Zhu PP, He RH, Ma H Le. Study on the ageing method and antioxidant activity of black garlic residues. Czech Journal of Food Sciences 2018;36(1).
• Lee KC, Teng CC, Shen CH, Huang WS, Lu CC, Kuo HC, et al. Protective effect of black garlic extracts on tert-Butyl hydroperoxide-induced injury in hepatocytes via a c-Jun N-terminal kinase-dependent mechanism. Exp Ther Med 2018;15(3).
• Cinar A, Altuntas S, Demircan H, Dundar AN, Taner G, Oral RA. Encapsulated black garlic: Comparison with black garlic extract in terms of physicochemical properties, biological activities, production efficiency and storage stability. Food Biosci 2022;50.
• Dong M, Yang G, Liu H, Liu X, Lin S, Sun D, et al. Aged black garlic extract inhibits HT29 colon cancer cell growth via the PI3K/Akt signaling pathway. Biomed Rep 2014;2(2):250–4.
• Bae SE, Cho SY, Won YD, Lee SH, Park HJ. Changes in S-allyl cysteine contents and physicochemical properties of black garlic during heat treatment. LWT 2014;55(1).
• Choi IS, Cha HS, Lee YS. Physicochemical and antioxidant properties of black garlic. Molecules 2014;19(10):16811–23.
• Bontempo P, Stiuso P, Lama S, Napolitano A, Piacente S, Altucci L, et al. Metabolite Profile and In Vitro Beneficial Effects of Black Garlic (Allium sativum L.) Polar Extract. Nutrients 2021;13(8).
• Farhat Z, Scheving T, Aga DS, Hershberger PA, Freudenheim JL, Hageman Blair R, et al. Antioxidant and Antiproliferative Activities of Several Garlic Forms. Nutrients 2023;15(19).
• Park C, Park S, Chung YH, Kim GY, Choi YW, Kim BW, et al. Induction of apoptosis by a hexane extract of aged black garlic in the human leukemic U937 cells. Nutr Res Pract 2014;8(2).
• Permatasari E, Farida, Widiyanto S. Cytotoxic effects and apoptosis of solo black garlic (Allium sativum L.) extract on T47D breast cancer cell Line. In: AIP Conference Proceedings. American Institute of Physics Inc 2020.
• Wang X, Jiao F, Wang QW, Wang J, Yang K, Hu RR, et al. Aged black garlic extract induces inhibition of gastric cancer cell growth in vitro and in vivo. Mol Med Rep 2012;5(1):66–72.
• Furdak P, Pieńkowska N, Kapusta I, Bartosz G, Sadowska-Bartosz I. Comparison of Antioxidant and Antiproliferative Effects of Various Forms of Garlic and Ramsons. Molecules 2023;28(18).
• Putnam KP, Bombick DW, Doolittle DJ. Evaluation of eight in vitro assays for assessing the cytotoxicity of cigarette smoke condensate. Toxicology in Vitro 2002;16(5).
• Fotakis G, Timbrell JA. In vitro cytotoxicity assays: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicol Lett 2006;160(2).
• Sun YE, Wang W. Changes in nutritional and bio-functional compounds and antioxidant capacity during black garlic processing. J Food Sci Technol 2018;55(2).