Acidified ethanolic extract of ‘Sembada Hitam’ black rice bran protect cell cycle and cellular senescence of H2O2-induced NIH3T3 fibroblasts

Year 2026, Volume: 30 Issue: 1, 1 - 12, 11.01.2026

Arneta Yuvita , Putri Nur Aida , Ester Dewanti Yovita Wardani , Fadiah Sri Rahayu , Istiqomah Istiqomah , Matilda Jesseline Gabriela Giovanni , Syifa' Aulia Rahmah , Yekti Asih Purwestri , Ardaning Nuriliani

https://doi.org/10.12991/jrespharm.1624339

Abstract

Hydrogen peroxide (H2O2) a reactive oxygen species formed by the body, which in high concentrations leads to cellular senescence. Bran of ‘Sembada Hitam’ black rice is rich in antioxidants that may protect cells from oxidative damage. This research evaluates the potential of Sembada Hitam Bran – Acidified Ethanolic Extract (SHB-AEE) in preserving cell cycle progression and mitigating senescence in NIH3T3 fibroblasts exposed to H2O2. SHB-AEE were obtained through maceration using a mixture of 85 mL ethanol absolute and 15 mL HCl 1N as an acid modifier. The cytotoxicity of SHB-AEE was determined using the MTT assay. Based on cytotoxicity results, NIH3T3 were sybsequenly treated with specific concentrations of H2O2 and SHB-AEE for cell cycle analysis via flow cytometry and senescence-associated-galactosidase (SA-β-Gal) staining. Data were analyzed with ANOVA followed by Tukey HSD for cytotoxicity, cytoprotective, and SA-β-Gal assay or Duncan Test for cell cycle data (p ≤ 0.05). Results showed that SHB-AEE was non-cytotoxic, maintaining cell viability above 80%. At low concentrations, SHB-AEE promoted normal cell cycle progression and reduced senescence in NIH3T3 fibroblasts under oxidative stress. However, at high concentrations, it exhibited the opposite effect, indicating a strong dose-dependent response. These findings suggest that SHB-AEE has potential antioxidant and cytoprotective properties against oxidative stress-induced senescence, but further studies are required to elucidate its molecular mechanisms and optimize treatment concentration for maximum cytoprotection.

Keywords

Black rice bran , cell cycle , cellular senescence , hydrogen peroxide , oxidative stress

Ethical Statement

This study was approved by the Medical and Health Research Ethics Committee (MHREC) at Faculty of Medicine, Public Health, and Nursing Universitas Gadjah Mada – DR. Sardjito General Hospital (Approval No: KE/FK/1589/EC/2023). No human or animal subjects were directly involved in this research; the study was conducted using animal cell lines.

Supporting Institution

Faculty of Biology, Universitas Gadjah Mada

Project Number

1193/UN1/FBI.1/KSA/PT.01.03/2024

Thanks

The authors are grateful for a part of the research that was partly funded by Lecturer Students Collaboration Research Grant No: 1193/UN1/FBI.1/KSA/PT.01.03/2024, Faculty of Biology, Universitas Gadjah Mada. This grant supported the stages of data collection and analysis during the study.

References

• [1] de Almeida AJPO, de Oliveira JCPL, da Silva Pontes LV, de Souza Júnior JF, Gonçalves TAF, Dantas SH, de Almeida Feitosa MS, Silva AO, de Medeiros IA. ROS: Basic Concepts, Sources, Cellular Signaling, and its Implications in Aging Pathways. Oxid Med Cell Longev. 2022;2022:1225578. https://doi.org/10.1155/2022/1225578
• [2] Palungwachira P, Tancharoen S, Phruksaniyom C, Klungsaeng S, Srichan R, Kikuchi K, Nararatwanchai T. Antioxidant and anti-inflammatory properties of anthocyanins extracted from Oryza sativa L. in primary dermal fibroblasts. Oxid Med Cell Longev. 2019;2019:2089817. doi: 10.1155/2019/2089817. Erratum in: Oxid Med Cell Longev. 2020 Jul 5;2020:6306104. https://doi.org/10.1155/2019/2089817.
• [3] Di Marzo N, Chisci E, Giovannoni R. The Role of Hydrogen Peroxide in Redox-Dependent Signaling: Homeostatic and Pathological Responses in Mammalian Cells. Cells. 2018;7(10):156. https://doi.org/10.3390/cells7100156
• [4] Pieńkowska N, Bartosz G, Pichla M, Grzesik-Pietrasiewicz M, Gruchala M, Sadowska-Bartosz I. Effect of antioxidants on the H2O2-induced premature senescence of human fibroblasts. Aging. 2020; 12(2): 1910-1927. https://doi.org/10.18632/aging.102730
• [5] Ortiz-Espín A, Morel E, Juarranz Á, Guerrero A, González S, Jiménez A, Sevilla F. An Extract from the Plant Deschampsia antarctica Protects Fibroblasts from Senescence Induced by Hydrogen Peroxide. Oxid Med Cell Longev. 2017;2017:2694945. https://doi.org/10.1155/2017/2694945
• [6] Venkatachalam G, Surana U, Clément MV. Replication stress-induced endogenous DNA damage drives cellular senescence induced by a sub-lethal oxidative stress. Nucleic Acids Research. 2017; 45(18): 10564-10582. https://doi.org/10.1093/nar/gkx684
• [7] Wang H, Chiu C, Wu P, Chen C. Subamolide E from Cinnamomum subavenium induces Sub-G1 cell-cycle arrest and caspase-dependent apoptosis and reduces the migration ability of human melanoma cells. J Agric Food Chem. 2011; 59(15): 8187–8192. https://doi.org/10.1021/jf2018929
• [8] Zhang S, Ma Q, Dong L, Jia X, Liu L, Huang F, Liu G, Sun Z, Chi J, Zhang M, Zhang, R. Phenolic profiles and bioactivities of different milling fractions of rice bran from black rice. Food Chem. 2022;378:132035. https://doi.org/10.1016/j.foodchem.2021.132035
• [9] Oktavya G, Purwestri YA, Saragih HTSG, Nuriliani A. Ethanolic extract of black rice‘Sembada Hitam’ bran protects the cytotoxic effect of H2O2 on NIH3T3 cells. Curr Res Nutr Food Sci. 2023; 11(1): 389-400. http://dx.doi.org/10.12944/CRNFSJ.11.1.29
• [10] Aprilianti SHU, Purwestri YA, Saragih HTSSG, Nuriliani A. Extract of black rice (Oryza sativa L. ‘Sembada Hitam’) bran protect cytotoxicity of hydrogen peroxide on vero cells in a short time incubation. J Trop Life Sci. 2024; 14(1): 1-12. http://dx.doi.org/10.11594/jtls.14.01.01
• [11] Christanto DR, Mose JC, Yuniarti T, Bestari MB, Fauziah PN, Purwestri YA, Munthe JN. Anti-angiogenic effect of black rice bran (Oryza Sativa L. ‘Sembada Hitam’) on soluble fms-like tyrosine kinase and placental growth factor in preeclampsia. Syst Rev Pharm. 2021; 12(1) :1594 – 1597. https://dx.doi.org/10.31838/srp.2021.1.225
• [12] Nguta J, Mbaria JM, Gakuya DW, Gathumbi PK, Kabasa JD, Kiama SG. Evaluation of acute toxicity of crude plant extracts from kenyan biodiversity using brine shrimp, Artemia salina L. (Artemiidae). Open Conf Procee J. 2012; 3: 30-34. http://dx.doi.org/10.2174/2210289201203010030
• [13] Xiong J, Xia L, Li L, Cui M, Gu Y, Wang P. An acetate-based NIR fluorescent probe for selectively imaging of hydrogen peroxide in living cells and in vivo. Sensors Actuators B: Chem. 2019; 288: 127-132. https://doi.org/10.1016/j.snb.2019.02.100
• [14] Sies H. Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: oxidative eustress. Redox Biol. 2017; 11: 613-619. https://doi.org/10.1016/j.redox.2016.12.035
• [15] Han X, Chen H, Gong H, Tang X, Huang N, Xu W, Tai H, Zhang G, Zhao T, Gong C, Wang S, Yang Y, Xiao H. Autolysosomal degradation of cytosolic chromatin fragments antagonizes oxidative stres–induced senescence. J Biol Chem. 2020; 295(14): 4451-4463. https://doi.org/10.1074/jbc.ra119.010734
• [16] Boonyanuphong P, Tobgay U. Protective effect of two Thai pigmented rice cultivars against H2O2-induced oxidative damage in HT-29 cell culture. Food Res. 2022; 6(1): 27–33. https://doi.org/10.26656/fr.2017.6(1).206
• [17] Kristamtini, Indrasari SD, Widyayanti S, Andriyanto R. Molecular, morphological, and biochemical identification of sembada merah and sembada hitam rice (Oryza sativa L). J Phys: Conf Series. 2021; 1918(5): 052017. https://doi.org/10.1088/1742-6596/1918/5/052017 [18] Kumar N, Murali RD. Black rice: a novel ingredient in food processing. J Nutr Food Sci. 2020; 10(2): 771. https://doi.org/10.35248/2155-9600.20.10.771
• [19] Khammanit R, Lomaratb P, Anantachokec N, Satoa VH, Ungsurungsied M, Mangmool S. Inhibition of oxidative stress through the induction of antioxidant enzymes of pigmented rice bran in HEK-293 cells. Nat Prod Commun. 2017; 12(7): 1107-1110. http://dx.doi.org/10.1177/1934578X1701200727
• [20] 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. 2018; 6(1): 23-34. http://dx.doi.org/10.12944/CRNFSJ.6.1.03
• [21] Arifin AS, Yuliana ND, Rafi M. Aktivitas antioksidan pada beras berpigmen dan dampaknya terhadap kesehatan. Jurnal Pangan, 2019; 1-12. https://jurnalpangan.com/index.php/pangan/article/view/416/349
• [22] Hetharia GE, Briliannita A, Astuti M, Marsono Y. Antioxidant extraction based on black rice (Oryza Sativa L. Indica) to prevent free radical. Mater Sci Eng. 2020; 823(1):012002. http://dx.doi.org/10.1088/1757-899X/823/1/012002
• [23] Giordano ME, Caricato R, Lionetto MG. Concentration dependence of the antioxidant and prooxidant activity of trolox in Hela cells: involvement in the induction of apoptotic volume decrease. Antioxidants (Basel). 2020;9(11):1058. https://doi.org/10.3390/antiox9111058
• [24] Eghbaliferiz S, Iranshahi M. Prooxidant activity of polyphenols, flavonoids, anthocyanins, and carotenoids: updated review of mechanisms and catalyzing metals. Phytother Res. 2016; 30: 1379-1391. https://doi.org/10.1002/ptr.5643
• [25] Maadi H, Soheilifar MH, Wang Z, Analysis of cell cycle by flow cytometry, In Cell-Cycle Synchronization: Methods and Protocols., Springer, New York, USA 2022.
• [26] Bae IY, An JS, Oh IK, Lee HG. Optimized preparation of anthocyanin-rich extract from black rice and its effects on in vitro digestibility. Food Sci Biotechnol. 2017; 26: 1415-1422. https://doi.org/10.1007/s10068-017-0188-x
• [27] CCRC. MTT cytotoxicity assay. https://ccrc.farmasi.ugm.ac.id/wp-content/uploads/sites/1439/03.010.02-uji-sitotoksik-MTT-1.pdf (accessed 10 January 2024)
• [28] Rukmana RM, Soesilo NP, Rumiyati, Pratiwi R. Chemopreventive activities of ‘Woja Laka’ black rice bran fractions on liver carcinoma HepG2 cells. Biomed Pharmacol J. 2017; 10(4): 1677-1684. https://dx.doi.org/10.13005/bpj/1279
• [29] CCRC. Flow cytometry protocol. http://ccrc.farmasi.ugm.ac.id/wp-content/uploads/sites/1439/03.014.02-flowcytometry.pdf (accessed 10 January 2024)
• [30] Nuriliani A, Nakahata Y, Ahmed R, Khaidizar FD, Matsui T, Bessho Y. Over‐expression of Nicotinamide phosphoribosyltransferase in mouse cells confers protective effect against oxidative and ER stressinduced premature senescence. Genes Cells. 2020; 25(8): 593-602. https://doi.org/10.1111/gtc.12794
• [31] Cayman Chemical. https://www.caymanchem.com/product/602010/senescence-associated-%CE%B2-galactosidase-staining-kit (accessed 10 January 2024)