In vitro Anti-amoebic and Cytotoxic Activity of Rosa gallica and Picea orientalis Leaf Aqueous Extracts

Abstract

Acanthamoeba spp. are free-living amoebae that can cause keratitis and granulomatous encephalitis in humans. This study aimed to evaluate the anti-amoebic and cytotoxic effects of aqueous leaf extracts of Rosa gallica and Picea orientalis on Acanthamoeba spp. and two cell lines. An environmental isolate of Acanthamoeba spp. was cultured on non-nutrient agar with Escherichia coli, and viability was tested with trypan blue method and microscopic observation of morphologies after exposure to extracts. Cytotoxicity was tested on SH-SY5Y (human neuroblastoma) and HaCaT (human keratinocyte) cell lines via the MTT assay. No viable trophozoites were observed after six hours of incubation with 30 mg mL-1 P. orientalis extract, whereas 20.7% of trophozoite viability was found in R. gallica extract with same concentration after 24 hours. Both extracts demonstrated limited efficacy against the encysted stage, with cysts remaining viable after 24 hours, over 70% viability. In addition, dose-dependent cytotoxicity was observed for both extracts on SH-SY5Y and HaCaT cells. This study provides the first in vitro evidence of the anti-amoebic activity of R. gallica and P. orientalis aqueous leaf extracts. However, their limited effect on cysts and cytotoxicity highlight the need for further in vivo studies to explore therapeutic potential.

Keywords

• Acanthamoeba spp.
• Rosa gallica
• Picea orientalis
• extract
• cytotoxicity

In vitro Anti-amoebic and Cytotoxic Activity of Rosa gallica and Picea orientalis Leaf Aqueous Extracts

Abstract

Acanthamoeba spp. potansiyel patojenik serbest yaşayan amipler arasında yer almakta olup insanlarda keratit ve granulomatöz ensefalite neden olabilmektedir. Bu çalışmada, Rosa gallica ve Picea orientalis'in yaprak özütlerinin Acanthamoeba spp.’ye karşı anti-amebik ve iki faklı hücre hattında sitotoksik aktivitelerinin araştırılması amaçlanmıştır. Çevresel örnekten izole edilen Acanthamoeba spp. izolatı, Escherichia coli ile kaplanmış besleyici değeri olmayan agar kültüründe çoğaltılmıştır. Farklı konsantrasyonlardaki özütler ile inkübe edilen parazitin canlılığı, trypan mavisi yöntemiyle ve morfolojik değişiklikler gözlemlenerek takip edilmiştir. Ayrıca, özütlerin sitotoksik aktiviteleri, SH-SY5Y (insan nöroblastoma) ve HaCaT (insan keratinosit) hücre hatlarında MTT yöntemiyle test edilmiştir. Altı saatlik 30 mg mL-1 P. orientalis özütü inkübasyonu sonrasında canlı trofozoite rastlanmazken, aynı konsantrasyonda R. gallica özütü ile 24 saatlik inkübasyon sonrası trofozoitlerin ortalama %20.7’sinin canlı kaldığı gözlemlenmiştir. Trofozoit formunun aksine her iki özüt de, Acanthamoeba spp.'nin kist formuna karşı sınırlı etki göstermiştir; kistler 24 saatlik inkübasyon sonrasında %70’in üzerinde canlı kalmıştır. Ayrıca, her iki özüt de SH-SY5Y ve HaCaT hücrelerinde doz bağımlı sitotoksik etki göstermiştir. Bu in vitro çalışma sonucunda R. gallica ve P. orientalis yaprağı su özütlerinin anti-amebik aktivitesine dair ilk veriler elde edilmiştir. Özütlerin terapötik uygulama potansiyellerinin değerlendirilmesi için in-vivo çalışmalara ihtiyaç bulunmaktadır.

Keywords

• Acanthamoeba spp.
• Rose gallica
• Picea orientalis
• özüt
• sitotoksisite

References

1. Chan LL, Mak JW, Low YT, Koh TT, Ithoi I, Mohamed SM. Isolation and characterization of Acanthamoeba spp. from air-conditioners in Kuala Lumpur, Malaysia. Acta Trop. 2011;117:23-30.
2. Bonkowski M. Protozoa and plant growth: the microbial loop in soil revisited. New Phytologist. 2004;162(3):617–631.
3. Raghavan A, Rammohan R. Acanthamoeba keratitis - A review. Indian J Ophthalmol. 2024;72:473-82.
4. Sharma G, Kalra SK, Tejan N, Ghoshal U. Nanoparticles based therapeutic efficacy against Acanthamoeba: Updates and future prospect. Exp Parasitol. 2020;218:108008.
5. Carnt N, Robaei D, Minassian DC, Dart JKG. Acanthamoeba keratitis in 194 patients: risk factors for bad outcomes and severe inflammatory complications. Br J Ophthalmol. 2018;102:1431-5.
6. Bullé DJ, Benittez LB, Rott MB. Occurrence of Acanthamoeba in hospitals: a literature review. RECI. 2020;10(2):174-80.
7. Yera H, Ok V, Lee Koy Kuet F, Dahane N, Ariey F, Hasseine L, et al. PCR and culture for diagnosis of Acanthamoeba keratitis. Br J Ophthalmol. 2021;105:1302-6.
8. Goh JWY, Harrison R, Hau S, Alexander CL, Tole DM, Avadhanam VS. Comparison of in vivo confocal microscopy, PCR and culture of corneal scrapes in the diagnosis of Acanthamoeba Keratitis. Cornea. 2018;37:480-5.

9. Papa V, Rama P, Radford C, Minassian DC, Dart JKG. Acanthamoeba keratitis therapy: time to cure and visual outcome analysis for different antiamoebic therapies in 227 cases. Br J Ophthalmol. 2020;104:575-81.
10. Rayamajhee B, Williams NLR, Siboni N, Rodgers K, Willcox M, Henriquez FL, et al. Identification and quantification of Acanthamoeba spp. within seawater at four coastal lagoons on the east coast of Australia. Sci Total Environ. 2023;901:165862.
11. Buchele MLC, Nunes BF, Filippin-Monteiro FB, Caumo KS. Diagnosis and treatment of Acanthamoeba Keratitis: A scoping review demonstrating unfavorable outcomes. Cont Lens Anterior Eye. 2023;46:101844.
12. Mileva M, Ilieva Y, Jovtchev G, Gateva S, Zaharieva MM, Georgieva A, et al. Rose flowers-a delicate perfume or a natural healer? Biomolecules. 2021;11.
13. Babich O, Zhikhreva A, Kulikova Y. Phytochemical composition of Rosa Gallica and prospects of its use. IJCBS 2024;25(19):1078-1084.
14. Abdelbaky AS, Mohamed A, Alharthi SS. Antioxidant and antimicrobial evaluation and chemical investigation of Rosa gallica var. aegyptiaca leaf extracts. Molecules. 2021;26.
15. Vargas-Segura AI, Silva-Belmares SY, Segura-Ceniceros EP, Ascacio-Valdés JA, Méndez-González L, Ilyina A. Screening and characterization of medicinal plants extracts with bactericidal activity against Streptococcus mutans. Nat Prod Res. 2020;34(18):2672-2676.
16. Alicandri E, Paolacci AR, Osadolor S, Sorgona A, Badiani M, Ciaffi M. On the evolution and functional diversity of terpene synthases in the Pinus species: A review. J Mol Evol. 2020;88:253-83.
17. Salem MZ, Elansary HO, Elkelish AA, Zeidler A, Ali HM, El-Hefny M, et al. In vitro bioactivity and antimicrobial activity of Picea abies and Larix decidua wood and bark extracts. Bio Resources. 2016;11(4):9421-9437.
18. Tanase C, Boz I, Oroian S, Coșarcă S, Toma F, Mare A, et al. Antibacterial activity of spruce bark (Picea abies l.) extract against E. coli. Acta Biol Maris. 2018;1(1):5-9.
19. Niyyati M, Dodangeh S, Lorenzo-Morales J. A Review of the current research trends in the application of medicinal plants as a source for novel therapeutic agents against Acanthamoeba infections. Iran J Pharm Res. 2016;15:893-900.
20. Erdoğan Ö, Paşa S, Demirbolat GM, Birtekocak F, Abbak M, Çevik Ö. Synthesis, characterization, and anticarcinogenic potent of green-synthesized zinc oxide nanoparticles via Citrus aurantium aqueous peel extract. Inorg Nano-Met Chem. 2025;55(1),67-75.
21. Strober W. Trypan Blue Exclusion Test of Cell Viability. Curr Protoc Immunol. 2015;111:A3 B 1-A3 B.
22. Wang F. Culture of animal cells: a manual of basic technique. In Vitro Cell Dev Biol Anim. 2006;42(5):169-169.
23. Supino R. MTT Assays. In vitro toxicity testing protocols; O’Hare S and Atterwill CK, Eds.; Humana Press: Totowa, NJ, 1995;137-149.
24. Campolo A, Pifer R, Walters R, Thomas M, Miller E, Harris V, et al. Acanthamoeba spp. aggregate and encyst on contact lens material increasing resistance to disinfection. Front Microbiol. 2022;13:1089092.
25. Gabriel AA, Panaligan DC. Heat and chlorine resistance of a soil Acanthamoeba sp. cysts in water. J Appl Microbiol. 2020;129:453-64.
26. Coulon C, Collignon A, McDonnell G, Thomas V. Resistance of Acanthamoeba cysts to disinfection treatments used in health care settings. J Clin Microbiol. 2010;48:2689-97.
27. Ithoi I, Ahmad AF, Mak JW, Nissapatorn V, Lau YL, Mahmud R. Morphological characteristics of developmental stages of Acanthamoeba and Naegleria species before and after staining by various techniques. Southeast Asian J Trop Med Public Health. 2011;42:1327-38.
28. Masek A, Latos M, Chrzescijanska E, Zaborski M. Antioxidant properties of rose extract (Rosa villosa L.) measured using electrochemical and UV/Vis spectrophotometric methods. Int J Electrochem Sci. 2017;12(11): 10994–11005.
29. Lee MH, Nam TG, Lee I, Shin EJ, Han AR, Lee P, et al. Skin anti-inflammatory activity of rose petal extract (Rosa gallica) through reduction of MAPK signalling pathway. Food Sci Nutr. 2018;6:2560-7.
30. Moein M, Zarshenas MM, Delnavaz S. Chemical composition analysis of rose water samples from Iran. Pharm Biol. 2014;52:1358-61.
31. Wang H. Beneficial medicinal effects and material applications of rose. Heliyon. 2024;10:e23530.
32. Cendrowski A, Krasniewska K, Przybyl JL, Zielinska A, Kalisz S. Antibacterial and antioxidant activity of extracts from rose fruits (Rosa rugosa). Molecules. 2020;25.
33. Milala J, Piekarska-Radzik L, Sojka M, Klewicki R, Matysiak B, Klewicka E. Rosa spp. extracts as a factor that limits the growth of Staphylococcus spp. bacteria, a food contaminant. Molecules. 2021;26.
34. Ren G, Xue P, Sun X, Zhao G. Determination of the volatile and polyphenol constituents and the antimicrobial, antioxidant, and tyrosinase inhibitory activities of the bioactive compounds from the by-product of Rosa rugosa Thunb. var. plena Regal tea. BMC Complement Altern Med. 2018;18:307.
35. Öz M. Chemical composition and antimicrobial properties of essential oils obtained from the resin of Picea orientalis. J Essent Oil-Bear Plants. 2022;25(2),326–37.
36. Hofmann T, Albert L, Németh L, Vršanská M, Schlosserová N, Voběrková S, et al. Antioxidant and antibacterial properties of Norway Spruce (Picea abies H. Karst.) and Eastern Hemlock (Tsuga canadensis (L.) Carrière) Cone Extracts. Forests. 2021;12,1189.
37. Salem MZM, Elansary HO, Elkelish AA, Zeidler A, Ali HM, Mervat E H, et al. In vitro bioactivity and antimicrobial activity of Picea abies and Larix decidua wood and bark extracts. Bio Res. 2016;11(4):9421-37.
38. Chu DM, Miles H, Toney D, Ngyuen C, Marciano-Cabral F. Amebicidal activity of plant extracts from Southeast Asia on Acanthamoeba spp. Parasitol Res. 1998;84:746-52.
39. Tas B, Koloren Z, Koloren O. In vitro, amoebicidal activities of submerged plant Ceratophyllum demersum L. extract against Acanthamoeba castellanii trophozoites. Aquatic Res. 2024;7(4),178-88.
40. Degerli S, Berk S, Malatyali E, Tepe B. Screening of the in vitro amoebicidal activities of Pastinaca armenea (Fisch. & C.A.Mey.) and Inula oculus-christi (L.) on Acanthamoeba castellanii cysts and trophozoites. Parasitol Res. 2012;110:565-70.
41. Polat ZA, Vural A, Ozan F, Tepe B, Ozcelik S, Cetin A. In vitro evaluation of the amoebicidal activity of garlic (Allium sativum) extract on Acanthamoeba castellanii and its cytotoxic potential on corneal cells. J Ocul Pharmacol Ther. 2008;24:8-14.
42. Ayna A, Tunc A, Ozbolat SN, Bengu AS, Aykutoğlu G, Canlı D, et al. Anticancer, and antioxidant activities of royal jelly on HT-29 colon cancer cells and melissopalynological analysis. Turk J Bot. 2021;45(8):809-819.
43. Ayna A, Ozbolat SN, Darendelioglu E. Quercetin, chrysin, caffeic acid and ferulic acid ameliorate cyclophosphamide-induced toxicities in SH-SY5Y cells. Mol Biol Rep. 2020;47:8535-43.
44. Ozbolat SN, Ayna A. Chrysin suppresses HT-29 cell death induced by diclofenac through apoptosis and oxidative damage. Nutr Cancer. 2021;73:1419-28.
45. Choudhari AS, Mandave PC, Deshpande M, Ranjekar P, Prakash O. Corrigendum: Phytochemicals in cancer treatment: from preclinical studies to clinical practice. Front Pharmacol. 2020;11:175.
46. Puladze M, Mulkijanyan K, Gogitidze N, Sulakvelidze M, Novikova Z, Mushkiashvili N, et al. Phytochemical and pharmacological study of Rosa gallica L. Georgian cultivar essential oil production waste. GSC Biol Pharm Sci. 2022;19:213-22.
47. Kashami DA, Rasooli I, Rezaee MB, Owlia P. Antioxidative properties and toxicity of white rose extract. Iran. J Med Aromat Plants. 2011;5:415-425.
48. Meimandi K, Yaghoobi MM. Effects of aqueous and ethanolic extract of Rosa damascena Mill L. against human gastric cancer cells. J Mol Cell Res. 2015;28(2):299-309.
49. Gerasimova T, Gateva S, Jovtchev G, Angelova T, Topashka-Ancheva M, Dobreva A, et al. Rosa damascena Mill. essential oil: analysis of in vitro and in vivo genotoxic and cytotoxic potentials by employing three cytogenetic endpoints. Molecules. 2024;30.
50. Coșarcă SL, Moacă EA, Tanase C, Muntean DL, Pavel IZ, et al. Spruce and beech bark aqueous extracts: Source of polyphenols, tannins and antioxidants correlated to in vitro antitumor potential on two different cell lines. Wood Sci Technol. 2019;53:313-330.
51. Namshir J, Shatar A, Khandaa O, Tserennadmid R, Shiretorova VG, Nguyen MC. Antimicrobial, antioxidant and cytotoxic activity on human breast cancer cells of essential oil from Pinus sylvestris. var mongolica needle. Mong J Chem. 2020;21(47):19-26.
52. Diouf PN, Stevanovic T, Cloutier A. Study on chemical composition, antioxidant and anti-inflammatory activities of hot water extract from Picea mariana bark and its proanthocyanidin-rich fractions. Food Chem. 2009;113(4):897-902.