Antioxidant capacity, cytotoxicity and post-treatment effect of Myrtus communis L. leaf extract in a 6-hydroxydopamine-induced SH-SY5Y cell model

Abstract

Purpose: Oxidative stress drives the loss of dopaminergic neurons in Parkinson's disease, which is usually diagnosed after many of these neurons are lost. Agents acting after injury are therefore of interest. This study measured the phenolic content and antioxidant capacity of an ethanolic Myrtus communis L. leaf extract, defined its non-cytotoxic concentration range, and tested whether the extract, applied after 6-hydroxydopamine (6-OHDA) injury, improves the viability of human SH-SY5Y neuroblastoma cells. Method: Leaves of M. communis were collected in Hatay Province (Türkiye), dried, powdered and macerated in ethanol for 3 days. Total phenolic (Folin–Ciocalteu) and total flavonoid (aluminum chloride) contents, DPPH and ABTS radical scavenging, and ferric reducing antioxidant power (FRAP) were measured. Cytotoxicity was assessed in L929 fibroblasts and SH-SY5Y cells by the MTT assay after 24 and 48 h. SH-SY5Y cells were exposed to 200 µM 6-OHDA for 24 h and then treated with the extract (10–1000 µg/mL) for 24 h. Groups were compared by ANOVA and Dunnett's test. Findings: The extract (yield 16.87%) contained 256.00 ± 6.16 mg GAE/g total phenolics and 48.00 ± 11.41 mg QE/g total flavonoids, and its FRAP value was 285.66 ± 21.92 mg GAE/g extract. DPPH and ABTS IC50 values were 75.97 and 106.7 µg/mL (gallic acid: 45.25 and 64.41 µg/mL). By the ISO 10993-5 criterion (≥70% viability), L929 cells tolerated up to 31.25 µg/mL for 48 h. SH-SY5Y cells were about five times more sensitive than L929 cells (48-h IC50 31.16 vs 158.4 µg/mL). 6-OHDA reduced SH-SY5Y viability slightly at 24 h (87.5% at 200 µM) and by about half at 48 h (IC50 227.7 µM). Post-treatment increased the MTT signal only at 75 µg/mL (116.0% of 6-OHDA-only cells, p = 0.016), whereas concentrations ≥200 µg/mL lowered it to 54.0–73.1%. Conclusion: The extract showed moderate radical-scavenging activity but was tolerated only in a narrow range, and SH-SY5Y cells were more sensitive to it than fibroblasts. Evidence for a post-injury benefit was limited to a higher MTT signal at a single concentration. This exploratory finding needs confirmation with independent viability assays, appropriate controls, stronger injury and mechanistic endpoints.

Keywords

• myrtle
• phenolic compounds
• neuroprotection
• Parkinson's disease
• MTT assay

Ethical Statement

This study used commercially obtained cell lines and did not involve human participants or experimental animals; therefore, it does not require ethical approval.

Thanks

The cell culture experiments were carried out in the Pharmaceutical Toxicology Laboratories of the Pharmaceutical Sciences Center, Gülhane Institute of Health Sciences, University of Health Sciences. We thank the center for its laboratory facilities and support.

Myrtus communis L. yaprak ekstresinin antioksidan kapasitesi, sitotoksisitesi ve 6-hidroksidopamin ile indüklenen SH-SY5Y hücre modelinde hasar sonrası uygulama etkisi

Abstract

Amaç: Oksidatif stres, Parkinson hastalığında dopaminerjik nöron kaybının temel nedenlerinden biridir ve hastalık genellikle bu nöronların önemli bir kısmı kaybedildikten sonra teşhis edilir. Bu nedenle hasar sonrasında etki gösterebilen ajanlar özellikle önemlidir. Bu çalışmada Myrtus communis L. yapraklarından hazırlanan etanollü ekstrenin fenolik içeriği ve antioksidan kapasitesi ölçülmüş, sitotoksik olmayan konsantrasyon aralığı belirlenmiş ve 6-hidroksidopamin (6-OHDA) hasarından sonra uygulanan ekstrenin insan SH-SY5Y nöroblastoma hücrelerinin canlılığını artırıp artırmadığı test edilmiştir. Metod: M. communis yaprakları Hatay'dan (Türkiye) toplanmış, kurutulup toz edilmiş ve 3 gün etanolde maserasyona bırakılmıştır. Toplam fenolik (Folin–Ciocalteu) ve toplam flavonoit (alüminyum klorür) içerikleri, DPPH ve ABTS radikal süpürücü aktivite ile demir indirgeyici antioksidan güç (FRAP) ölçülmüştür. Sitotoksisite, L929 fibroblast ve SH-SY5Y hücrelerinde 24 ve 48 saat sonunda MTT testiyle değerlendirilmiştir. SH-SY5Y hücreleri 24 saat 200 µM 6-OHDA'ya maruz bırakıldıktan sonra 24 saat ekstre (10–1000 µg/mL) ile muamele edilmiştir. Gruplar tek yönlü ANOVA ve Dunnett testiyle karşılaştırılmıştır. Bulgular: Ekstre (verim %16,87) 256,00 ± 6,16 mg GAE/g toplam fenolik ve 48,00 ± 11,41 mg QE/g toplam flavonoit içermiş, FRAP değeri 285,66 ± 21,92 mg GAE/g ekstre bulunmuştur. DPPH ve ABTS IC50 değerleri 75,97 ve 106,7 µg/mL'dir (gallik asit: 45,25 ve 64,41 µg/mL). ISO 10993-5 ölçütüne (≥%70 canlılık) göre L929 hücreleri 48 saatte 31,25 µg/mL'ye kadar ekstreyi tolere etmiştir. SH-SY5Y hücreleri L929 hücrelerinden yaklaşık beş kat daha duyarlı bulunmuştur (48 saat IC50 31,16 ve 158,4 µg/mL). 6-OHDA, SH-SY5Y canlılığını 24 saatte az (200 µM'de %87,5), 48 saatte ise yaklaşık yarı yarıya azaltmıştır (IC50 227,7 µM). Hasar sonrası uygulama MTT sinyalini yalnızca 75 µg/mL'de artırmış (yalnız 6-OHDA grubuna göre %116,0; p = 0,016), ≥200 µg/mL ise %54,0–73,1'e düşürmüştür. Sonuç: Ekstre orta düzeyde radikal süpürücü aktivite göstermiş, ancak yalnızca dar bir konsantrasyon aralığında tolere edilmiş ve SH-SY5Y hücreleri ekstreye fibroblastlardan daha duyarlı bulunmuştur. Hasar sonrası yarar yönündeki kanıt, tek bir konsantrasyonda MTT sinyalindeki artışla sınırlı kalmıştır. Bu keşifsel bulgunun bağımsız canlılık testleri, uygun kontroller, daha güçlü hasar modelleri ve mekanistik parametrelerle doğrulanması gerekmektedir.

Keywords

• mersin
• fenolik bileşikler
• nöroproteksiyon
• Parkinson hastalığı
• MTT testi

Supporting Institution

Bu çalışmada ticari olarak temin edilen hücre hatları kullanılmış olup insan katılımcı veya deney hayvanı kullanılmadığından etik kurul onayı gerektirmemektedir.

Thanks

Hücre kültürü deneyleri Sağlık Bilimleri Üniversitesi, Gülhane Sağlık Bilimleri Enstitüsü, Eczacılık Bilimleri Merkezi, Farmasötik Toksikoloji Laboratuvarlarında yürütülmüştür. Laboratuvar olanakları ve destekleri için merkeze teşekkür ederiz.

References

1. [1] GBD 2021 Nervous System Disorders Collaborators. (2024). Global, regional, and national burden of disorders affecting the nervous system, 1990–2021: A systematic analysis for the Global Burden of Disease Study 2021. The Lancet Neurology, 23(4), 344–381. https://doi.org/10.1016/S1474-4422(24)00038-3
2. [2] Dias, V., Junn, E., & Mouradian, M. M. (2013). The role of oxidative stress in Parkinson's disease. Journal of Parkinson's Disease, 3(4), 461–491. https://doi.org/10.3233/JPD-130230
3. [3] Fearnley, J. M., & Lees, A. J. (1991). Ageing and Parkinson's disease: Substantia nigra regional selectivity. Brain, 114(5), 2283–2301. https://doi.org/10.1093/brain/114.5.2283
4. [4] Baytop, T. (1999). Türkiye'de bitkiler ile tedavi: Geçmişte ve bugün [Herbal therapy in Türkiye: Past and present] (2nd ed.). İstanbul: Nobel Tıp Kitabevleri.
5. [5] Dabbaghi, M. M., Fadaei, M. S., Soleimani Roudi, H., Baradaran Rahimi, V., & Askari, V. R. (2023). A review of the biological effects of Myrtus communis. Physiological Reports, 11(14), e15770. https://doi.org/10.14814/phy2.15770
6. [6] Sen, A., Kurkçuoglu, M., Yıldırım, A., Doğan, A., Bitis, L., & Baser, K. H. C. (2020). Chemical and biological profiles of essential oil from different parts of Myrtus communis L. subsp. communis from Turkey. Agriculturae Conspectus Scientificus, 85(1), 71–78.
7. [7] Aidi Wannes, W., Mhamdi, B., Sriti, J., Ben Jemia, M., Ouchikh, O., Hamdaoui, G., Kchouk, M. E., & Marzouk, B. (2010). Antioxidant activities of the essential oils and methanol extracts from myrtle (Myrtus communis var. italica L.) leaf, stem and flower. Food and Chemical Toxicology, 48(5), 1362–1370. https://doi.org/10.1016/j.fct.2010.03.002
8. [8] Amensour, M., Sendra, E., Abrini, J., Bouhdid, S., Pérez-Alvarez, J. A., & Fernández-López, J. (2009). Total phenolic content and antioxidant activity of myrtle (Myrtus communis) extracts. Natural Product Communications, 4(6), 819–824. https://doi.org/10.1177/1934578X0900400616

9. [9] Belahcene, S., Kebsa, W., Akingbade, T. V., Umar, H. I., Omoboyowa, D. A., Alshihri, A. A., Abo Mansour, A., Alhasaniah, A. H., Oraig, M. A., Bakkour, Y., & Leghouchi, E. (2024). Chemical composition, antioxidant and anti-inflammatory activities of Myrtus communis L. leaf extract: Forecasting ADMET profiling and anti-inflammatory targets using molecular docking tools. Molecules, 29(4), 849. https://doi.org/10.3390/molecules29040849
10. [10] Parlar, Z., Diker, N. Y., & Çankaya, İ. İ. (2023). Total content analysis, in vitro antioxidant, and enzyme inhibition tests on Myrtus communis L. Journal of Research in Pharmacy, 27(Special Issue 7), 108. https://doi.org/10.29228/jrp.665
11. [11] Parlar, Z., Özkurt, M. C., Tel, B. C., Bedir, E., & Tatlı Çankaya, İ. İ. (2025). Phytochemical profiling and in vitro biological activities on Myrtus communis L. Phytochemistry Letters, 69, 103629. https://doi.org/10.1016/j.phytol.2025.103629
12. [12] Harassi, Y., Tilaoui, M., Idir, A., Frédéric, J., Baudino, S., Ajouaoi, S., Mouse, H. A., & Zyad, A. (2019). Phytochemical analysis, cytotoxic and antioxidant activities of Myrtus communis essential oil from Morocco. Journal of Complementary and Integrative Medicine, 16(3), 20180100. https://doi.org/10.1515/jcim-2018-0100
13. [13] Simola, N., Morelli, M., & Carta, A. R. (2007). The 6-hydroxydopamine model of Parkinson's disease. Neurotoxicity Research, 11(3–4), 151–167. https://doi.org/10.1007/BF03033565
14. [14] Xicoy, H., Wieringa, B., & Martens, G. J. M. (2017). The SH-SY5Y cell line in Parkinson's disease research: A systematic review. Molecular Neurodegeneration, 12, 10. https://doi.org/10.1186/s13024-017-0149-0
15. [15] da Silva Bastos, K. V. L., Martínez, G. P., Batista Alves, V. S., de Souza, A. B., Tomé, A. C., & de Moura Souza, F. (2026). Neuroprotective properties of plant extracts: An evaluation of in vitro evidence and future directions for clinical translation. Current Organocatalysis, 13(4), e22133372423645. https://doi.org/10.2174/0122133372423645251213035231
16. [16] Dessì, D., Fais, G., Follesa, P., & Sarais, G. (2025). Neuroprotective effects of myrtle berry by-product extracts on 6-OHDA-induced cytotoxicity in PC12 cells. Antioxidants, 14(1), 88. https://doi.org/10.3390/antiox14010088
17. [17] Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. American Journal of Enology and Viticulture, 16(3), 144–158. https://doi.org/10.5344/ajev.1965.16.3.144
18. [18] Chang, C. C., Yang, M. H., Wen, H. M., & Chern, J. C. (2002). Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Journal of Food and Drug Analysis, 10(3), 178–182. https://doi.org/10.38212/2224-6614.2748
19. [19] Brand-Williams, W., Cuvelier, M. E., & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. LWT – Food Science and Technology, 28(1), 25–30. https://doi.org/10.1016/S0023-6438(95)80008-5
20. [20] Baldemir Kılıç, A., Karaman, Ü., Yusufbeyoğlu, S., Yeşilyurt, E. B., Turan Buğatekin, A., Güral, Y., & Yar, T. M. (2026). In vitro amoebicidal and antioxidant activity of Helianthemum ledifolium (L.) Mill. varieties. Biological Diversity and Conservation, 19(1), 74–86. https://doi.org/10.46309/biodicon.2026.1818866
21. [21] Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26(9–10), 1231–1237. https://doi.org/10.1016/S0891-5849(98)00315-3
22. [22] Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": The FRAP assay. Analytical Biochemistry, 239(1), 70–76. https://doi.org/10.1006/abio.1996.0292
23. [23] Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods, 65(1–2), 55–63. https://doi.org/10.1016/0022-1759(83)90303-4
24. [24] International Organization for Standardization. (2009). Biological evaluation of medical devices — Part 5: Tests for in vitro cytotoxicity (ISO Standard No. 10993-5:2009). Geneva: ISO.
25. [25] Karakaş, D., Arı, F., & Ulukaya, E. (2017). The MTT viability assay yields strikingly false-positive viabilities although the cells are killed by some plant extracts. Turkish Journal of Biology, 41(6), 919–925. https://doi.org/10.3906/biy-1703-104