In vitro amoebicidal and antioxidant activity of Helianthemum ledifolium (L.) Mill. varieties

Abstract

Purpose: Acanthamoeba spp. are opportunistic protozoa causing Acanthamoeba keratitis (AK), an ocular infection frequently associated with contact lens use. Given the limitations and adverse effects of current treatments, this study aimed to discover natural, safe, and cost-effective therapeutic alternatives. Specifically, the purpose was to investigate the in vitro amoebicidal and antioxidant activities of methanol and water extracts from three varieties of Helianthemum ledifolium (L.) Mill.. Method: The study evaluated methanol and water extracts obtained from the aerial parts of three H. ledifolium varieties (H. ledifolium var. ledifolium, H. ledifolium var. microcarpum, and H. ledifolium var. lasiocarpum). Amoebicidal activity was tested in vitro against Acanthamoeba castellanii. In addition, total phenolic and flavonoid contents were calculated using Folin-Ciocalteu and AlCl3 reagents. Antioxidant activities were evaluated by measuring radical scavenging activities using the DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) assays. Findings: The amoebicidal activity against Acanthamoeba castellanii showed a dose- and time-dependent effect. Methanol extracts exhibited superior amoebicidal potency compared to water extracts, even at lower concentrations, and also had the highest total phenolic and flavonoid contents in relation to antioxidant radical scavenging activities. The most toxic effect was observed in the H. ledifolium var. lasiocarpum methanol extract, with an LD50 of 0.0034 mg/mL at 72 hours. The H. ledifolium var. microcarpum water extract exhibited the fastest effect LT50 = 0.5603 hours. Conclusion: These results suggest that H. ledifolium species represent promising candidates for the development of natural antiparasitic agents. The superior anti-Acanthamoeba activity of methanol extracts is strongly related to their significantly richer total phenolic and flavonoid contents compared to aqueous extracts, indicating that methanol is a much more effective solvent for obtaining potential bioactive compounds. H. ledifolium taxa have the potential to be an important natural resource for future antiparasitic drug research, especially in the context of increasing drug resistance and treatment failure.

Keywords

• Ameobicidal activity
• Antioxidant activity
• Helianthemum ledifolium
• Acanthamoeba castellani

Helianthemum ledifolium (L.) Mill. varyelerinin in vitro amibisidal ve antioksidan aktivitesi

Abstract

Amaç: Acanthamoeba türleri, sıklıkla kontakt lens kullanımıyla ilişkili bir göz enfeksiyonu olan Acanthamoeba keratitine (AK) neden olan fırsatçı protozoalardır. Mevcut tedavilerin sınırlamaları ve yan etkileri göz önüne alındığında, bu çalışma doğal, güvenli ve uygun maliyetli terapötik alternatifleri keşfetmeyi amaçlamaktadır. Özellikle, amaç Helianthemum ledifolium (L.) Mill'in üç varyetesinden elde edilen metanol ve su eskterelerinin in vitro amipsidal ve antioksidan aktivitelerini araştırmaktır. Yöntem: Çalışmada, üç H. ledifolium varyetesinin (H. ledifolium var. ledifolium, H. ledifolium var. microcarpum ve H. ledifolium var. lasiocarpum) toprak üstü kısımlarından elde edilen metanol ve su ekstreleri değerlendirilmiştir. Amipsidal aktivite Acanthamoeba castellanii'ye karşı in vitro test edilmiştir. Toplam Fenolik ve flavonoid içerikleri sarsasıyla Folin-Ciocalteu reaktifi ve AlCl3 reaktifi kullanılarak hesaplanmıştır. Antioksidan aktiviteleri ise DPPH (2,2-Difenil-1-pikrilhidrazil) ve ABTS (2'-Azino-bis (3-etilbenzotiyazolin-6-sülfonik asit) diamonyum tuzu) analizleri ile radikal temizleme aktiviteleri ölçülerek değerlendirildi. Bulgular: Acanthamoeba castellanii'ye karşı amip öldürücü aktivite, doza ve zamana bağlı bir etki gösterdi. Metanol ekstreleri, daha düşük konsantrasyonlarda bile, su özütlerine kıyasla daha yüksek amip öldürücü etki gösterdi. Ayrıca antioksidan radikal temizleme aktiviteleri ile ilişkili olarak en yüksek toplam fenolik ve flavonoid içeriğine sahipti. En toksik etki, 72 saatte 0,0034 mg/mL'lik bir LD50 ile H. ledifolium var. lasiocarpum metanol ekstresinde (LasM) gözlendi. H. ledifolium var. microcarpum su ekstresi (MicW) en hızlı etkiyi LT50 = 0,5603 saat ile göstermiştir. Sonuç: Bu sonuçlar H. ledifolium türlerinin doğal antiparaziter ajanların geliştirilmesi için umut verici adaylar olduğunu göstermektedir. Metanol özütlerinin yüksek anti-Acanthamoeba aktivitesi, sulu ekstrelere kıyasla önemli ölçüde daha zengin toplam fenolik ve flavonoid içerikleriyle güçlü bir şekilde ilişkilidir ve bu sonuç metanolün potansiyel biyoaktif bileşikleri elde etmek için çok daha etkili bir çözücü olduğunu göstermektedir. H. ledifolium taksonları, özellikle artan ilaç direnci ve tedavi başarısızlıkları bağlamında, gelecekteki antiparaziter ilaç araştırmaları için önemli bir doğal kaynak olabilme potansiyeline sahiptir.

Keywords

• Amibisidal aktivite
• Antioksidan aktivite
• Helianthemum ledifolium
• Acanthamoeba castellanii

References

1. [1] Wiser, M. F. (2020). Nutrition and protozoan pathogens of humans: A primer. In Nutrition and infectious diseases: shifting the clinical paradigm (pp. 165-187). Cham: Springer International Publishing https://doi.org/10.1007/978-3-030-56913-6
2. [2] Kaya, Y., Baldemir, A., Karaman, Ü., Ildız, N., Arıcı, Y. K., Kaçmaz, G., Kolören, Z., & Konca, Y. (2019). Amebicidal effects of Trigonella foenum-graecum against Acanthamoeba cysts. Food Science & Nutrition, 7, 563-571. https://doi.org/10.1002/fsn3.849
3. [3] Petrillo, F., Tortori, A., Vallino, V., Galdiero, M., Fea, A. M., De Sanctis, U., & Reibaldi, M. (2024). Understanding Acanthamoeba keratitis: an in-depth review of a sight-threatening eye infection. Microorganisms, 12(4), 758. https://doi.org/10.3390/microorganisms12040758
4. [4] Badria, F. A., Hetta, M. H., Sarhan, R. M., & El-Din, M. E. (2014). Lethal effects of Helianthemum lippii (L.) on Acanthamoeba castellanii cysts in vitro. The Korean journal of parasitology, 52(3), 243. http://dx.doi.org/10.3347/kjp.2014.52.3.243
5. [5] Abers, M., Schroeder, S., Goelz, L., Sulser, A., St. Rose, T., Puchalski, K., & Langland, J. (2021). Antimicrobial activity of the volatile substances from essential oils. BMC complementary medicine and therapies, 21(1), 124. https://doi.org/10.1186/s12906-021-03285-3
6. [6] Hamad, A. A. (2023). In vitro Evaluation the efficacy of some new plant extracts and biocides on the viability of Acanthamoeba castellanii. Protist, 174(3), 125966. https://doi.org/10.1016/j.protis.2023.125966
7. [7] Lê, H. G., Hwang, B. S., Choi, J. S., Jeong, Y. T., Kang, J. M., Võ, T. C., ... & Na, B. K. (2024). Iris setosa Pall. ex Link Extract Reveals Amoebicidal Activity against Acanthamoeba castellanii and Acanthamoeba polyphaga with Low Toxicity to Human Corneal Cells. Microorganisms, 12(8), 1658. https://doi.org/10.3390/microorganisms12081658
8. [8] Taş, B., Kolören, Z., & Kolören, O. (2024). In vitro, amoebicidal activities of submerged plant Ceratophyllum demersum L. extract against Acanthamoeba castellanii trophozoites. Aquatic Research, 7(4), 178-188. https://doi.org/10.3153/AR24016

9. [9] Kaynak, B., Aydoğdu, G., & Kolören, Z. (2024). Investigation of Sambucus ebulus Plant Extract with Regard to its In-vitro DNA Protective Action, Cytotoxic Effect and Amoebicidal Amoebicidal on Acanthamoeba castellanii Trophozoites. Karadeniz Fen Bilimleri Dergisi, 14(4), 2172-2189. https://doi.org/10.31466/kfbd.1537169
10. [10] Kaynak, B., Aydoğdu, G., & Kolören, Z. (2025). Onopordum acanthium Özütünün Acanthamoeba castellanii Trofozoitleri Üzerinde Amoebisidal Etkisinin, DNA Hasarına Karşı Koruyucu ve Sitotoksik Aktivitelerinin Araştırılması. Karadeniz Fen Bilimleri Dergisi, 15(1), 434-447. https://doi.org/10.31466/kfbd.1586799
11. [11] Plescia, F., Venturella, F., D’Anneo, A., Catania, V., Gargano, M. L., Polito, G., ... & Raffa, D. (2022). Phytochemical-rich extracts of Helianthemum lippii possess antimicrobial, anticancer, and anti-biofilm activities. Plant Biosystems-An International Journal Dealing with all Aspects of Plant Biology, 156(6), 1314-1324. https://doi.org/10.1080/11263504.2022.2048274
12. [12] Rubio-Moraga, Á., Argandoña, J., Mota, B., Pérez, J., Verde, A., Fajardo, J., ... & Gómez-Gómez, L. (2013). Screening for polyphenols, antioxidant and antimicrobial activitiesof extracts from eleven Helianthemum taxa (Cistaceae) used in folk medicine in south-eastern Spain. Journal of Ethnopharmacology, 148(1), 287-296. https://doi.org/10.1016/j.jep.2013.04.028
13. [13] Kilic, A. B., Karaman, U., Yusufbeyoglu, S., Yesilyurt, E. B., & Yar, T. M. (2023). Investigation of in vitro anti-trichomoniasis effect of Helianthemum ledifolium L.(Mill.) varieties against Trichomonas vaginalis. https://doi.org/10.5455/annalsmedres.2023.11.296
14. [14] Yeşilyurt, E. B., Erik, S., Baysal, E. Ö., & Akaydın, G. (2015). Taxonomic studies on endemic species from Turkey: Helianthemum nummularium L. Miller subsp. lycaonicum Coode&Cullen Cistaceae. Biological Diversity and Conservation, 8(3), 181-187. ISSN 1308-8084 Online; ISSN 1308-5301 Print [15] Davis, P. H. (1970). Flora of Turkey and the East Aegean Islands. Vol.2. (pp 515-516). Edinburgh : Edinburgh University Press
15. [16] Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). [14] Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. In Methods in enzymology (Vol. 299, pp. 152-178). Academic press. https://doi.org/10.1016/S0076-6879(99)99017-1
16. [17] Sun, L., Zhang, J., Lu, X., Zhang, L., & Zhang, Y. (2011). Evaluation to the antioxidant activity of total flavonoids extract from persimmon (Diospyros kaki L.) leaves. Food and chemical toxicology, 49(10), 2689-2696. https://doi.org/10.1016/j.fct.2011.07.042
17. [18] Brand-Williams, W., Cuvelier, M. E., & Berset, C. L. W. T. (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
18. [19] 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
19. [20] BALDEMİR, A., KARAMAN, Ü., YUSUFBEYOĞLU, S., EKEN, A., ILDIZ, N., İLGÜN, S., ... & ÇANKAYA, S. (2018). Laurocerausus officinalis Roemer (Taflan) Meyve Ekstrelerinden Nanoçiçek Sentezi ile Akantamoebisidal Aktivitenin Arttırılmasında Yeni Bir Strateji. Mikrobiyoloji Bülteni, 52(1), 56-71. https://doi.org/10.5578/MB.66400
20. [21] Baldemir, A., Gökşen, N., Ildız, N., Karatoprak, G. Ş., & Koşar, M. (2017). Phytochemical Profile and Biological Activities of Helianthemum canum l. baumg. from Turkey. Chemistry & Biodiversity, 14(7), e1700052. https://doi.org/10.1002/cbdv.201700052
21. [22] Benabdelaziz, I., Marcourt, L., Benkhaled, M., Wolfender, J. L., & Haba, H. (2017). Antioxidant and antibacterial activities and polyphenolic constituents of Helianthemum sessiliflorum Pers. Natural product research, 31(6), 686-690. https://doi.org/10.1080/14786419.2016.1209669
22. [23] Laib, I., & Djahra, A. B. (2022). Phytochemical investigation of Helianthemum lippii l. aerial Dum. Cours part and evaluation for its antioxidant activities. International Journal of Secondary Metabolite, 9(2), 229-237. https://doi.org/10.21448/ijsm.999518
23. [24] Djahra, A. B., Khaoula, Z., Ahmed, G., Mounia, B., Salah, B., Katrenada, O., & Khaoula, J. (2018). Evaluation of total phenolic contents and antioxidant potentials of ten medicinal plants from Algerian Sahara. International Journal of Biological and Agricultural Research, 1(2), 28-36. https://dspace.univ-eloued.dz/handle/123456789/7070
24. [25] Terfassi, S., Dauvergne, X., Cérantola, S., Lemoine, C., Bensouici, C., Fadila, B., ... & Benayache, S. (2022). First report on phytochemical investigation, antioxidant and antidiabetic activities of Helianthemum getulum. Natural Product Research, 36(11), 2806-2813. https://doi.org/10.1080/14786419.2021.1928664