Several species of the genus Acanthamoeba cause human diseases. Treatment of infections involves various problems, emphasising the need to develop alternative antiprotozoal agents. We studied the anti-amoebic activity of Essential Oils (EOs), derived from rosemary (Rosmarinus officinalis L.) and cloves (Syzygium aromaticum L. Merr. &amp; Perry), against Acanthamoeba polyphaga strain. The amoebicidal activity of cloves and rosemary EOs was preliminary demonstrated by the morphology change (modifications in the cell shape, the presence of precipitates in the cytoplasm, autophagic vesicles, membrane blends) of the treated trophozoites. The cell-counts, carried out after staining trophozoites with a Trypan blue solution, revealed that both EOs were active in a dose-dependent manner and in relation to the exposure time. This activity was evident after few hours, with encouraging results obtained in particular with cloves EO, able to act at the lower concentrations and after 1Â&nbsp;h, probably for its high eugenol content (65.30%)

Anacarso, Immacolata

Bondi, Moreno

Condò, Carla

de Niederhäusern, Simona

Iseppi, Ramona

Messi, Patrizia

Sabia, Carla

Several species of the genus Acanthamoeba cause human diseases. Treatment of infections involves various problems, emphasising the need to develop alternative antiprotozoal agents. We studied the anti-amoebic activity of Essential Oils (EOs), derived from rosemary (Rosmarinus officinalis L.) and cloves (Syzygium aromaticum L. Merr. &amp; Perry), against Acanthamoeba polyphaga strain. The amoebicidal activity of cloves and rosemary EOs was preliminary demonstrated by the morphology change (modifications in the cell shape, the presence of precipitates in the cytoplasm, autophagic vesicles, membrane blends) of the treated trophozoites. The cell-counts, carried out after staining trophozoites with a Trypan blue solution, revealed that both EOs were active in a dose-dependent manner and in relation to the exposure time. This activity was evident after few hours, with encouraging results obtained in particular with cloves EO, able to act at the lower concentrations and after 1Â&nbsp;h, probably for its high eugenol content (65.30%)

de Niederhäusern, Simona

Condò, Carla

Archivio istituzionale della ricerca - Università di Modena e Reggio Emilia

DOI: 10.1080/14786419.2017.1399390Journal: Natural Product ResearchManuscript ID: 1399390File Correction DetailsCorrection is made. No of Corrections: 4Online Correction Link  :    http://tandfproofs.sps.co.in/oct_tnf /index.php?token=.Iah_KmsVceZskJbgZ1wYMwx3ha4H71e24HmaSxb4Aw2hTlnJP-fbowImage Annotation DetailsNo Details FoundAttached File DetailsNo Details FoundList of  CommentsNo comments foundQuery Details1.  Please check that the givenname and surname of authors have beenset correctly.No change required2.  The disclosure statement has been inserted. Please correct if thisis inaccurate.No change required3.  The CrossRef database (www.crossref.org/) has been used tovalidate the references. Mismatches between the original manuscriptand CrossRef are tracked in red font. Please provide a revision if thechange is incorrect. Do not comment on correct changes.Resolved in textIn vitro evaluation of the amoebicidal activity of rosemary (Rosmarinusofficinalis L.) and cloves (Syzygium aromaticum L. Merr. & Perry) essentialoils against Acanthamoeba polyphaga trophozoitesRunning heads:Natural Product ResearchI. Anacarso et al.Anacarso Immacolata Sabia Carla de Niederhäusern Simona Iseppi Ramona Condò Carla Bondi Moreno Messi Patrizia  Department of Life Sciences, University of Modena and Reggio E., Modena, Italy AQ1Corresponding author: Email: patrizia.messi@unimore.it1399390_Supplemenatry_material.pdfReceived 11 Sep 2017; Accepted 15 Oct 2017© 2017 Informa UK Limited, trading as Taylor & Francis GroupAbstractSeveral species of the genus Acanthamoeba cause human diseases. Treatment of infections involves variousproblems, emphasising the need to develop alternative antiprotozoal agents. We studied the anti-amoebic activity ofEssential Oils (EOs), derived from rosemary (Rosmarinus officinalis L.) and cloves (Syzygium aromaticum L. Merr. & Perry),against Acanthamoeba polyphaga strain. The amoebicidal activity of cloves and rosemary EOs was preliminarydemonstrated by the morphology change (modifications in the cell shape, the presence of precipitates in the cytoplasm,autophagic vesicles, membrane blends) of the treated trophozoites. The cell-counts, carried out after stainingtrophozoites with a Trypan blue solution, revealed that both EOs were active in a dose-dependent manner and in relationto the exposure time. This activity was evident after few hours, with encouraging results obtained in particular with clovesEO, able to act at the lower concentrations and after 1 h, probably for its high eugenol content (65.30%).KeywordsAcanthamoeba polyphagaessential oilsclovesrosemary1.  IntroductionAcanthamoeba is a ubiquitous free-living protozoan that can exit as motile trophozoites and form cysts in response toadverse environmental conditions. Several species of Acanthamoeba are known to cause human disease, like keratitis thataffects mainly the wearers of contact lenses, skin infections and other most insidious chronic diseases like systemicdiseases or granulomatous amoebic encephalitis (GAE), especially in immunocompromised patients. Acanthamoeba can alsoserve as hosts for endosymbionts, representing a significant reservoir for environmental pathogen/opportunisticaaaaaaa, *a*serve as hosts for endosymbionts, representing a significant reservoir for environmental pathogen/opportunisticmicroorganisms (Legionella pneumophila, Aeromonas hydrophila) and food-borne pathogens (Listeria monocytogenes, Salmonellaenterica serovar Enteritidis, Yersinia enterocolitica), with important implications for human health (Snelling et al. 2006 ; Messi etal. 2011 ; Anacarso et al. 2012 ). The antiprotozoarian agents, used for the treatment of Acanthamoeba are often endowedwith unpleasant side effects, irritating and toxic for the host, and chemical disinfectants used to prevent microbial growth inman-made aquatic systems are effective only in high concentrations, representing a health risk for human exposure topotentially hazardous by-products. Therefore, it is essential to develop alternative antiprotozoal agents with high activity, lowtoxicity and high efficacy. For these reasons, there is a growing interest in using natural antimicrobial compounds and,among these, Essential Oils (EOs), generally recognised as safe (GRAS) have already shown antimicrobial activity againstbacteria of clinical origin, fungi, viruses (Cannas et al. 2016 ; Bouyahya et al. 2017 ) and, recently, special attention is alsopaid to their amoebicidal activity (Perez et al. 2012 ). There are several evidences of amoebicidal activity of essential oilsobtained from Euphorbiaceae, Asteraceae, Verbenaceae, Piperaceae plants, Peucedanum species, Allium sativum extract (Polat et al.2008 ; Rodio et al. 2008 ; Sauter et al. 2011 , 2012 ; Malatyali et al. 2012 ; Vunda et al. 2012 ; Santos et al. 2016 ; Panatieri etal. 2017 ). The aim of the present investigation was to evaluate in vitro the amoebicidal effect against Acanthamoebapolyphaga strain of essential oils derived from two widely popular spices, rosemary (Rosmarinus officinalis L.) and cloves(Syzygium aromaticum L. Merr. & Perry).2.  Results and discussion2.1.  Dist illat ion and qualitat ive–quant itat ive analysis of  essent ial o ilsFor each spice, the following total extract concentrations were obtained: 1,1 g for rosemary and 3,6 g for cloves. Theyield of the distillation process was calculated by the formula: [(g final extracted compound/g initial compound)/100]. Yieldsof 1,8% and 12% were obtained for rosemary and cloves, respectively. With regard to the chemical composition, the maincloves oil constituents were eugenol (65.30%), β-caryophyllene (15.00%), 2-methoxy-4-[2-propenyl] phenol acetate (9.75%)and α-caryophyllene (2.85%). In rosemary EO, the main compositions were 1,8-cineole (25.23%), α-pinene (18.81%), camphor(15.26%), camphene (10.82%), borneol (2.87%), β-caryophyllene (1.91%) and bornyl acetate (1.51%).2.2.  Amoebicidal act ivity of  essent ial o ilsThe amoebicidal activity against A. polyphaga ApUP of cloves and rosemary EOs, studied by microtitre plates method, ispreliminary demonstrated by the morphology change of the treated trophozoites, compared to the control. Figure 1  (a, b, c,d) shows the most indicative changes in trophozoites morphology, after the last time of contact (144 h). The mainmorphological alterations were the modifications in the cell shape, the presence of precipitates in the cytoplasm, autophagicvesicles, membrane blends, observable at the highest and lowest concentration of 4 mg/mL and 0,1 mg/mL for rosemary EOand cloves EO, respectively. The cell count, carried out after staining trophozoites with a Trypan blue solution, revealed thatboth EOs were active in a dose-dependent manner and in relation to the exposure time. In particular, the rosemary EO(Figure 2 ) showed a good amoebicidal activity, but only at the higher concentrations (4 and 2 mg/mL); in both cases the totalkilling of cells was obtained at the end of the experiment (144 h), with a percentage reduction of 100%. At the concentrationsof 1 and 0.1 mg/mL, the EO displayed a lower amoebicidal capacity, and gradual over time: after the first hour the adding of1 mg/mL solution determined a 27% reduction, whereas a good killing percentage (86%) was observed after 144 h. At0.1 mg/mL, a decrease in cell count of Acanthamoeba of about 50% was obtained, but only at the end of the experiment. Thecloves EO showed a better amoebicidal activity than the rosemary EO (Figure 3 ). A total killing of A. polyphaga was obtainedafter 1 h using 1, 2, 4 mg/mL concentrations. With the concentration of 0.1 mg/mL a 43.37% reduction of amoeba was onlyobserved but, even in this case, the total elimination of vital cells was obtained after 24 hours’ incubation.Figure  1. Effects o f EOs o f rosem ary (a ) and cloves (c)  on A. polyphaga trophozo ites vs. contro l (b and d, respective ly). Trophozo ites a fte rexposure  to  the  EOs appears as spheric and dam aged fo rm s o r as few disaggregated fo rm s and am orphous m ass fo r rosem ary and cloves,respective ly.Note: Inverted microscope (200× magnification).Figure  2. Am oebicida l act ivity o f rosem ary EO a t d iffe rent concentra t ions (0.1, 1, 2, 4 m g/m L) presented as percentage  o f reduction o f A.polyphaga trophozo ites.Figure  3. Am oebicida l act ivity o f cloves EO a t d iffe rent concentra t ions (0.1, 1, 2, 4 m g/m L) presented as percentage  o f reduction o f A. polyphagatrophozo ites.3.  ConclusionsRecently, the antiprotozoal activity of many essential oils has been reported. Laboratory tests have been carried out todetermine the effect of various essential oils against A. polyphaga. In the present investigation, we report the amoebicidalactivity of two widely used natural substances after few hours of contact, with encouraging results obtained in particular forthe cloves EO. In fact, a total killing of A. polyphaga trophozoites was obtained after 1 h using concentrations of 1, 2, 4 mg/mL.Both EOs were also able to prevent cysts formation. This capability is very important because trophozoites can turn into cystforms during the therapy, causing difficulties and treatment failures. Other factors, as the emerging resistance topharmacotherapy (Turner et al. 2000 ; Johnston et al. 2009 ) can also lead to delayed suitable therapy. Even in this case, asalready emerged for the problem of antibiotic resistance in pathogenic bacteria (Yap et al. 2014 ), the combination ofessential oils with antiprotozoal agents could be also useful in the fight against drug-resistant Acanthamoeba. Actually, inmany fields there is a «return to the natural», and the results here reported fall in this philosophy. The essential oils fromrosemary (R. officinalis L.) and clove (Syzygium aromaticum L. Merr. & Perry), in particular, are obtained from natural popularspices that could represent a promising alternative to treat infectious diseases or to employ as food preservative (Budri et al.2015 ; Vital et al. 2016 ; Jardak et al. 2017 ; Ugalde et al. 2017 ). Further studies will be necessary to determine the toxicity, themolecular mechanisms involved in the amoebicidal activity, the pharmacokinetic and pharmacodynamics parameters andthe molecular targets of these two spices.Supplemental dataSupplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1399390.AQ2AQ3Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1399390.Disclosure statementNo potential conflict of interest was reported by the authors.Ref erencesAnacarso I , de Niederhäusern S , Messi P , Guerrieri E , Iseppi R , Sabia C , Bondi M . 2012. Acanthamoeba polyphaga, a potentialenvironmental vector for the transmission of food-borne and opportunistic pathogens. J Basic Microbiol. 52:261–268.Bouyahya A , Et-Touys A , Bakri Y , Talbaui A , Fellah H , Abrini J , Dakka N . 2017. 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In vitro evaluation of the amoebicidal activity of rosemary (Rosmarinus officinalis L.) and cloves (Syzygium aromaticum L. Merr. & Perry) essential oils against Acanthamoeba polyphaga trophozoites

In vitro evaluation of the amoebicidal activity of rosemary (Rosmarinus officinalis L.) and cloves (Syzygium aromaticum L. Merr. & Perry) essential oils against Acanthamoeba polyphaga trophozoites

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