Protective Effects of Aqueous Propolis Extract from the Artvin-Hatila Region of Türkiye Against Zinc Oxide-Induced Genotoxicity: Antimutagenic, Antioxidant, and Phytochemical Evaluation

Abstract

This study investigated the protective effects of an aqueous propolis extract obtained from the Artvin-Hatila region of Türkiye against genotoxic stress induced by zinc oxide (ZnO) exposure in Allium cepa root meristem cells. Cytogenetic parameters such as mitotic index, chromosomal abnormalities, and micronucleus formation were evaluated. Antioxidant capacity was measured using DPPH, FRAP, and CUPRAC assays. The phenolic compound profile was determined via HPLC, and total phenolic and flavonoid contents were spectrophotometrically quantified. The results demonstrated that the 100 mg/L concentration of propolis significantly reduced ZnO-induced cytogenetic damage. At both lower and higher concentrations, this effect was diminished, and genotoxicity increased in some parameters. The findings suggest that the phenolic compounds in propolis may suppress ZnO-related genotoxic stress and, when applied within an appropriate dose range, contribute to maintaining cellular structural integrity in biological systems. These results indicate that propolis may represent a promising natural resource for the development of alternative protective strategies against environmental toxic agents.

Keywords

• Propolis
• Antimutagenicity
• Antioxidant activity
• Phenolic composition
• Allium cepa
• HPLC analysis

Türkiye'nin Artvin-Hatila Bölgesinden Elde Edilen Propolisin Sulu Ekstraktının Çinko Oksit Kaynaklı Genotoksisiteye Karşı Koruyucu Etkileri: Antimutajenik, Antioksidan ve Fitokimyasal Değerlendirme

Abstract

Bu çalışmada, Türkiye’nin Artvin-Hatila bölgesinden elde edilen propolisin sulu ekstraktının, çinko oksit (ZnO) maruziyeti ile oluşturulan genotoksik strese karşı Allium cepa kök hücrelerindeki koruyucu etkileri araştırılmıştır. Mitotik indeks, kromozomal anormallikler ve mikronükleus oluşumu gibi sitogenetik parametreler değerlendirilmiş; ayrıca propolisin antioksidan kapasitesi DPPH, FRAP ve CUPRAC analizleriyle belirlenmiştir. HPLC ile fenolik bileşik profili analiz edilmiş, toplam fenolik ve flavonoid içeriği spektrofotometrik olarak ölçülmüştür. Sonuçlar, 100 mg/L konsantrasyonundaki propolis ekstraktının ZnO kaynaklı sitogenetik hasarı anlamlı şekilde azalttığını göstermektedir. Düşük ve yüksek konsantrasyonlarda bu etki zayıflamış, hatta bazı parametrelerde genotoksik etki artmıştır. Elde edilen bulgular, propolisin içerdiği fenolik bileşiklerin ZnO kaynaklı genotoksik stresi baskılayabileceğini ve uygun doz aralığında uygulandığında, biyolojik sistemlerde hücresel düzeyde yapısal bütünlüğü korumaya katkı sağlayabilecek doğal bir biyokoruyucu ajan niteliği taşıyabileceğini göstermektedir. Bu sonuçlar, propolisin çevresel toksik ajanlara karşı alternatif koruyucu stratejilerin geliştirilmesinde değerlendirilebilecek potansiyel bir doğal kaynak olabileceğine işaret etmektedir.

Keywords

• Propolis
• Antimutajenik Etki
• Antioksidan Aktivite
• Fenolik Bileşik Profili
• Allium cepa
• HPLC Analizi

References

1. [1] Sun Z., Xiong T., Li S., Zhang Y., Wang Y., Liu X. Comparative study of the toxicity of ZnO nanoparticles suspension, Zn²⁺, and insoluble particles on Allium cepa, Ecotoxicology and Environmental Safety, 186 109754, 2019.
2. [2] Kumari M., Khan S. S., Pakrashi S., Mukherjee A., Chandrasekaran N. Cytogenetic and genotoxic effects of zinc oxide nanoparticles on root cells of Allium cepa, Journal of Hazardous Materials, 190 613-621, 2011.
3. [3] Shaymurat T., Gu J., Xu C., Yang Z., Zhao Q., Liu Y., Liu Y. Phytotoxic and genotoxic effects of ZnO nanoparticles on garlic (Allium sativum L.): A morphological study, Nanotoxicology, 6 241-248, 2012.
4. [4] Bozkuş T., Karakaş D., Yıldız A. Evaluation of genotoxic and cytotoxic effects of propolis against oxidative stress, Environmental Toxicology and Pharmacology, 87 103695, 2021.
5. [5] Castro M. L., Valente I. M., Monteiro C., Louro Martins M. A., Pintado M., Rodrigues J. A. Assessment of bioactive properties of propolis extracts: A comparative approach, Talanta, 130 329-336, 2014.
6. [6] Medana C., Carbone F., Aigotti R., Appendino G., Baiocchi C. Selective analysis of phenolic compounds in propolis by HPLC-MS/MS, Phytochemical Analysis, 19 32-39, 2008.
7. [7] Volpi G., Bergonzini G. Analysis of flavonoids from propolis by on-line HPLC detection, Journal of Pharmaceutical and Biomedical Analysis, 42 354-361, 2006.
8. [8] Daleprane J. B., Abdalla D. S. P. Emerging roles of propolis: Antioxidant, cardioprotective, and antiangiogenic actions, Evidence-Based Complementary and Alternative Medicine, 175135, 2013.

9. [9] Bruschi M. L., Lara E. H. G., Martins C. H. G., Vinholis A. H. C., Lucarini R., Panini G. C. Preparation and antimicrobial activity of gelatin microparticles containing propolis against oral pathogens, Drug Development and Industrial Pharmacy, 29 375-381, 2003.
10. [10] Bozkuş T. N., Değer O., Yaşar A. Chemical characterization of water and ethanolic extracts of Turkish propolis by HPLC-DAD and GC-MS, Journal of Liquid Chromatography & Related Technologies, 44 77-86, 2021.
11. [11] Kaur P., Mehta S. K., Goyal D. Allium cepa assay-based comparative assessment of genotoxicity of nanomaterials. In D. Das (Ed.), Nanotoxicity: Toxicity Evaluation, Risk Assessment and Management, 185-205, 2019.
12. [12] Özkar A., Akyıl D., Konuk M., Çakır Ö. Potential genotoxic and cytotoxic effects of deltamethrin in Allium cepa root apical meristem cells, Environmental Toxicology and Pharmacology, 39(2) 638-644, 2015.
13. [13] Bayram N. E., Karadayı M., Güllüce M., Bayram S., Sorkun K., Öz G. C., Aydoğan M. N., Koç T. Y., Alaylar B., Salih B. Genotoxic and antigenotoxic evaluation of propolis by using in vitro bacterial assay systems. Mellifera, 15(1) 29-36, 2015.
14. [14] Mocanu A., Isopencu G., Busuioc C., Popa O. M., Dietrich P., Socaciu-Siebert L. Bacterial cellulose films with ZnO nanoparticles and propolis extracts: Synergistic antimicrobial effect, Scientific Reports, 9 17687, 2019.
15. [15] Ajeng D. A. P., Plashintania D. R., Putri R. M., Wibowo I., Ramli Y., Herdianto S., Indarto A. Synthesis of zinc oxide nanoparticles using methanol propolis extract (Pro-ZnO NPs) as antidiabetic and antioxidant, Plos One, 18(7) e0289125, 2023.
16. [16] Roberto M. M., Jamal C. M. Antigenotoxicity and antimutagenicity of ethanolic extracts of Brazilian green propolis and its main botanical source determined by the Allium cepa test system, Genetics and Molecular Research, 15(2), 2016.
17. [17] Singleton V. L., Orthofer R., Lamuela-Raventos R. M. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin–Ciocalteu reagent, Methods in Enzymology, 299 152-178, 1999.
18. [18] Zhishen J., Mengcheng T., Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals, Food Chemistry, 64(4) 555-559, 1999.
19. [19] Brand-Williams W., Cuvelier M. E., Berset C. Use of a free radical method to evaluate antioxidant activity, LWT – Food Science and Technology, 28(1) 25-30, 1995.
20. [20] Cüce M., Bekircan T., Laghari A. H., Sökmen M., Sökmen A., Uçar E. Ö., Kılıç A. O. Antioxidant phenolic constituents, antimicrobial and cytotoxic properties of Stachys annua L. from both natural resources and micropropagated plantlets, Indian Journal of Traditional Knowledge, 16(3) 407-416, 2017.
21. [21] Cüce M., Bekircan T., Laghari A., Sokmen M., Sokmen A., Ucar E., Kılıç A. Phenolic Profiles, Antimicrobial and Cytotoxic Properties of Both Micropropagated and Naturally Growing Plantlets of Calamintha sylvatica subsp. sylvatica Bromf. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 47(4) 1145-1152, 2019.
22. [22] Benzie, I. F. F., Strain, J. J. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay, Analytical Biochemistry, 239(1), 70–76, 1996.
23. [23] Pulido, R., Bravo, L., Saura-Calixto, F. Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay, Journal of Agricultural and Food Chemistry, 48(8) 3396–3402, 2000.
24. [24] Kalogeropoulos, N., Konteles, S. J., Troullidou, E., Mourtzinos, I., Karathanos, V. T. Chemical composition, antioxidant activity and antimicrobial properties of propolis extracts from Greece and Cyprus, Food Chemistry, 116(2) 452-461, 2009.
25. [25] Kumazawa, S., Hamasaka, T., Nakayama, T. Antioxidant activity of propolis of various geographic origins, Food Chemistry, 84(3) 329-339, 2004.
26. [26] Bankova, V., de Castro, S. L., Marcucci, M. C. Propolis: Recent advances in chemistry and plant origin, Apidologie, 31(1) 3-15, 2002.
27. [27] Firuzi, O., Lacanna, A., Petrucci, R., Marrosu, G., Saso, L. Evaluation of the antioxidant activity of flavonoids by “ferric reducing antioxidant power” assay and cyclic voltammetry, Biochimica et Biophysica Acta, 1721(1–3) 174-184, 2005.
28. [28] Garrido, L., Delgado, R., Bosch, F., Munoz, A. Antioxidant properties of caffeic acid derivatives: Structure–activity relationship, Food Chemistry, 135(4) 2408-2414, 2012.
29. [29] Apak, R., Güçlü, K., Demirata, B., Ozyurek, M., Celik, S. E., Bektasoglu, B., Berker, K. I., Ozyurt, D. Comparative evaluation of various total antioxidant capacity assays applied to phenolic compounds with the CUPRAC assay, Molecules, 12(7) 1496-1547, 2004.
30. [30] Apak, R., Özyürek, M., Güçlü, K., Çapanoğlu, E. Antioxidant activity/capacity measurement, 1. Classification, physicochemical principles, mechanisms, and electron transfer (ET)–based assays, Journal of Agricultural and Food Chemistry, 64(5) 997-1027, 2016.
31. [31] Aliyazicioglu, R., Sahin, H., Erturk, O., Ulusoy, E., Kolayli, S. Properties of phenolic content and biological activity of propolis samples from different geographical origins, International Journal of Food Properties, 16(2) 277–287, 2013.
32. [32] Buratti, S., Benedetti, S., Cosio, M. S. Evaluation of the antioxidant power of honey, propolis and royal jelly by amperometric flow injection analysis, Talanta, 71(3) 1387-1392, 2007.
33. [33] Nagai, T., Sakai, M., Inoue, R., Inoue, H., Suzuki, N. Antioxidative activities of some commercially honeys, royal jelly, and propolis, Food Chemistry, 80(1) 29-33, 2003.
34. [34] Russo, A., Troncoso, N., Sanchez, F., Garbarino, J. A., Vanella, A. Propolis protects human spermatozoa from DNA damage caused by benzo[a]pyrene and exogenous reactive oxygen species, Life Sciences, 70(8) 989-1000, 2002.
35. [35] Rojas, E., Lopez, M. C., Valverde, M. Single cell gel electrophoresis assay: methodology and applications, Journal of Chromatography B: Biomedical Sciences and Applications, 722(1–2) 225-254, 1993.
36. [36] Sudhakar, R., et al. Cytogenetic evaluation of the genotoxic potential of fenitrothion in mouse bone marrow cells, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 490(2) 113-119, 2001.
37. [37] Leme, D. M., Marin-Morales, M. A. Allium cepa test in environmental monitoring: A review on its application, Mutation Research/Reviews in Mutation Research, 682(1) 71-81, 2009.
38. [38] Mercykutty, V. C., Stephen, J. Induction of chromosome aberrations in Allium cepa by pollution of Cochin backwater, Cytologia, 45(4) 769-777, 1980.
39. [39] Celik, T. A., Aslanturk, O. S. Evaluation of cytotoxicity and genotoxicity of Inula viscosa leaf extracts with Allium test, Journal of Biomedicine and Biotechnology, 2010, 189252.
40. [40] Oyeyemi, I. T., Bakare, A. A. Genotoxicity of treated pharmaceutical effluent and potential protective effects of vitamins C and E using Allium cepa assay, Journal of Toxicology and Environmental Health Sciences, 5(3) 43-51, 2013.
41. [41] Chukwujekwu, J. C., Van Staden, J., Genotoxic effects of propolis from different geographical origins, South African Journal of Botany, 91 33-38, 2014.
42. [42] Roberto, D., et al., Cytogenotoxic effects of Brazilian green propolis on human cells, Toxicology in Vitro, 34 212-219, 2016.
43. [43] Levan, A. The effect of colchicine on root mitoses in Allium, Hereditas, 24(4), 471–486, 1938.
44. [44] Liu, D., et al. Evaluation of genotoxic effects of linuron in root meristem cells of Allium cepa, Mutation Research, 334(2) 253-258, 1995.
45. [45] Patil, B. C., Bhat, G. I. A comparative study of cytological effects induced by ethyl methane sulphonate and maleic hydrazide in Trigonella foenum-graecum L., Cytologia, 57(3), 327–334, 1992.
46. [46] Fiskesjö, G., Levan, A. Evaluation of the genotoxicity of surface water samples with the Allium test, Science of the Total Environment, 109–110, 343–354, 1993.
47. [47] Darlington, C. D., McLeish, J. Action of colchicine and related compounds on the mitotic spindle, Nature, 168(4273), 403–406, 1951.
48. [48] Yi, H., Meng, Q. DNA bridge formation and chromosome breakage, Mutation Research, 554(1–2), 71–78, 2003.
49. [49] Bianchi, F., et al. Genotoxicity biomarkers in human populations exposed to environmental and dietary agents, Mutation Research, 752(1), 1–9, 2013.
50. [50] Çavuşoğlu, K. The effect of propolis extract on Allium cepa root meristem cells, Environmental Toxicology and Pharmacology, 78, 103382, 2020.
51. [51] Çavuşoğlu, K. Determination of antigenotoxic potential of propolis on mercury-induced genotoxicity in Allium cepa test system, Environmental Science and Pollution Research, 27 23029-23038, 2020.
52. [52] Fenech, M. The in vitro micronucleus technique, Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 455(1–2) 81-95, 2000.
53. [53] Gebel, T., Kunz, B. M., Dunkelberg, H. Comparison of mutagenicity of dichloroacetic acid and trichloroacetic acid in mammalian cell systems, Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 395(1) 63-72, 1997.
54. [54] Dantas, F. J. S., et al. Protective effects of propolis extract on DNA damage and oxidative stress in Allium cepa cells, Biomedicine & Pharmacotherapy, 132 110835, 2020.