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Campomanesia xanthocarpa (Mart.) O. Berg essential oil induces antileishmanial activity and remodeling of the cytoplasm organelles Fabiana Borges Padilha Ferreira , Áquila Carolina Fernandes Herculano Ramos-Milaré , José Eduardo Gonçalves , Danielle Lazarin-Bidóia , Celso Vataru Nakamura , Rosangela Rumi Sugauara , Carla Maria Mariano Fernandez , Zilda Cristiani Gazim , Izabel Galhardo Demarchi , Thaís Gomes Verzignassi Silveira & Maria Valdrinez Campana Lonardoni To cite this article: Fabiana Borges Padilha Ferreira , Áquila Carolina Fernandes Herculano Ramos-Milaré , José Eduardo Gonçalves , Danielle Lazarin-Bidóia , Celso Vataru Nakamura , Rosangela Rumi Sugauara , Carla Maria Mariano Fernandez , Zilda Cristiani Gazim , Izabel Galhardo Demarchi , Thaís Gomes Verzignassi Silveira & Maria Valdrinez Campana Lonardoni (2020): Campomanesia�xanthocarpa (Mart.) O. Berg essential oil induces antileishmanial activity and remodeling of the cytoplasm organelles, Natural Product Research, DOI: 10.1080/14786419.2020.1827401 To link to this article: https://doi.org/10.1080/14786419.2020.1827401

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NATURAL PRODUCT RESEARCH https://doi.org/10.1080/14786419.2020.1827401

SHORT COMMUNICATION

Campomanesia xanthocarpa (Mart.) O. Berg essential oil induces antileishmanial activity and remodeling of the cytoplasm organelles
Fabiana Borges Padilha Ferreiraa, Aquila Carolina Fernandes Herculano
iad, Ramos-Milare
a, Jos
e Eduardo Gonc¸alvesb,c, Danielle Lazarin-Bido d e Celso Vataru Nakamura , Rosangela Rumi Sugauara , Carla Maria Mariano Fernandeze, Zilda Cristiani Gazime, Izabel Galhardo Demarchif, Tha
ıs Gomes Verzignassi Silveiraa,g and Maria Valdrinez Campana Lonardonia,g a Posgraduate Program in Health Sciences, Universidade Estadual de Maring
a, Maring
a, Paran
a State, Brazil; bPosgraduate in Clean Technologies, UniCesumar, Maring
a, Paran
a State, Brazil; cCesumar Instituto de Ci^encias, Tecnologia e Inovac¸~ao – ICETI, Maring
a, Paran
a State, Brazil; dLaboratory of Innovation in Development of Medicines and Cosmetics, Department of Health Sciences, Universidade Estadual de Maring
a, Maring
a, Paran
a State, Brazil; ePosgraduate in Biotechnology Applied to Agriculture, Universidade Paranaense, Umuarama, Paran
a State, Brazil; fFederal University of Santa Catarina, Florian
opolis, Santa Catarina, Brazil; gDepartment of Clinical Analysis and Biomedicine, Universidade Estadual de Maring
a, Maring
a, Paran
a State, Brazil

ABSTRACT

ARTICLE HISTORY

Leishmaniasis is a neglected disease that affects millions of people worldwide. This study aimed to analyze antileishmanial activity of Campomanesia xanthocarpa leaf essential oil (EO) on promastigote and amastigote forms of Leishmania amazonensis, cytotoxicity in murine macrophages and sheep erythrocytes. The essential oil (EO) was analyzed by gas chromatography/mass spectrophotometry. The main and most abundant compounds were sesquiterpene hydrocarbons (71.22%) such as trans-caryophyllene (7.87%), bicyclogermacrene (11.28%), and d-cadinene (8.34%). The IC50 for promastigote and amastigote forms of L. amazonensis was 70 mg mL 1 and 6 mg mL 1, respectively. C. xanthocarpa EO was not cytotoxic for murine macrophages (CC50 1860 mg mL 1) and sheep erythrocytes (1.5%), presenting high selectivity index for protozoan (310). C. xanthocarpa EO induced effects on the morphology and ultrastructure of this parasite. The high activity for intracellular amastigote forms, low toxicity to murine macrophages, and erythrocytes, suggest that C. xanthocarpa EO is promising for the treatment of leishmaniasis.

Received 7 May 2020 Accepted 5 September 2020 KEYWORDS

Leishmania amazonensis; cutaneous leishmaniasis; Myrtaceae; gabiroba; essential oil

CONTACT Fabiana Borges Padilha Ferreira [email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1827401. ß 2020 Informa UK Limited, trading as Taylor & Francis Group

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F. B. P. FERREIRA ET AL.

1. Introduction Leishmaniasis is caused by intracellular protozoa from the genus Leishmania. The main clinical manifestations include cutaneous, mucocutaneous, and visceral lesions. L. amazonensis causes cutaneous leishmaniasis (CL) and diffuse anergic cutaneous leishmaniasis (DCL), a rare but very severe clinical form that affects about 1% of all CL cases each year in Brazil (Machado et al. 2019). The current treatment for leishmaniasis is based mainly on pentavalent antimony and amphotericin B, which are drugs with serious side effects and high toxicity, high cost and drug resistance (Garcia et al. 2018). These limitations stimulate the search for new therapeutic options more efficient, safer, and with low-cost. Campomanesia xanthocarpa (Mart.) O. Berg it’s a fruitful species, native of Brazil from the Myrtaceae family, also popularly known as gabiroba, that has of yet been poorly explored. It is found in the southern and southeastern regions of Brazil and is also found in Argentina, Paraguay and Uruguay (Lorenzi, 1992). Several therapeutic activities have been attributed to C. xanthocarpa, such as anti-inflammatory (Da Silva et al. 2016), antidiarrheal, antimicrobial, hypercholesterolemia and diuretic (Garlet, 2019), hypotensive (Morais et al. 2020). As far as we know, there are no reports in the literature on the antileishmanial activity of the essential oil (EO) from C. xanthocarpa leaves. In this context, this study aimed to chemically characterize the EO of the leaves of C. xanthocarpa and investigate its effect on the promastigote and amastigote forms of L. amazonensis, in addition to the ultrastructural changes induced, and cytotoxicity to macrophages and erythrocytes.

2. Results and discussion The chemical characterization of C. xanthocarpa EO by GC-MS showed a total of 45 compounds (Table S1, supplementary material). The predominant class of compounds identified was sesquiterpene hydrocarbons (71.22%). The main compounds were transcaryophyllene (7.87%), bicyclogermacrene (11.28%), and d-cadinene (8.34%) (Figure S1,

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supplementary material). The major compounds identified by Limberger et al. (2001) in the same plant were the sesquiterpenes, spathulenol and globulol. The IC50 of C. xanthocarpa EO on the promastigote and amastigote forms of L. amazonensis were 70 mg mL 1 and 6 mg mL 1, respectively. The IC50 of AmB (amphotericin B) was 0.6 mg mL 1 for promastigote forms (Table S2, supplementary material). The EO was able to reduce the infection index in 71.4%, 67.7%, 41.1% and 19.1% at concentrations of 200, 100, 10 and 1 lg mL 1, respectively. These values indicate that the amastigotes forms were more sensitive to the leishmanicidal effect of the EO. These results are very interesting because the amastigotes forms are the main target of chemotherapy for leishmaniasis and are responsible for the clinical manifestations in the vertebrate host (Cunningham 2002; McConville and Handman 2007). The biological activities of EO are usually attributed to their major compounds. The sesquiterpenes are considered potent promoters of cutaneous permeation because they can interact with the compounds of the lipid membrane altering its fluidity, and those with leishmanicidal activity could be promising candidates to integrate nanocarriers in the treatment of cutaneous leishmaniasis (Moreira et al. 2019). When going through the cell membrane, these compounds can cause impermeability loss to intracellular electrolytes, which can result in cell death (Medeiros et al. 2011). The C. xanthocarpa EO was not cytotoxic to macrophages at a concentration under less than 1 mg mL 1 in 24 h. The cytotoxic concentration for 50% of the macrophages (CC50) was 1860 mg mL 1 for C. xanthocarpa EO. The selectivity index of C. xanthocarpa EO was 26.6 for promastigotes and 310 for amastigotes, showing that EO was more selective for amastigote and promastigote forms than for macrophages. The CC50 of AmB was 11.0 mg mL 1 at 24 h, indicating higher toxicity in relation to C. xanthocarpa EO (Table S2, supplementary material). C. xanthocarpa EO did not present hemolytic activity at lower concentrations than 1 mg mL 1 (Table S2, supplementary material). The hemolytic activity was 1.5% at the higher tested concentration when compared to the positive control (p < 0.001). The low toxicity found in C. xanthocarpa EO is in accordance with the data obtained by Markman et al. (2004), who evaluated the extract of C. xanthocarpa leaves applied on mice at doses of 5 g/kg without presenting toxicity. In vivo assays with Wistar rats showed that C. xanthocarpa extract did not induce mutagenicity (Sousa et al. 2019) or acute toxicity (Da Silva et al. 2016), demonstrating the low toxic risk of this species. The analyses by scanning electron microscopy allowed to verify changes in the structure and size of parasites such as cellular retraction and rounded shape (Figure S2C and S2D, supplementary material) were observed when compared to the untreated control (Figure S2A and S2B, supplementary material). The untreated group presented typically elongated shape, well-characterized flagella, and regular-looking organelles. Alterations were also seen in intracellular amastigote forms with damage and rupture of the cell membrane at a concentration of 200 lg mL 1 and reduction of the number of amastigotes per cell at a 10 lg mL 1 (Figure S2E, S2F and S2G, supplementary material). Structural damages were identified, such as mitochondrial edema and increase in the kinetoplast volume (Figure S3B and S3E, supplementary material), atypical intense cytoplasmic vacuolization, the appearance of autophagy characteristics (Figure S3B and S3C, supplementary material) and accumulation of lipid body (Figure S3B, S3C,

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F. B. P. FERREIRA ET AL.

S3D and S3F, supplementary material). Nuclear fragmentation was also observed with alterations in the chromatin condensation pattern with the presence of membranous profiles (Figure S3C and S3D, supplementary material), deformation of flagellar pocket, and formation of intracellular vesicles (Figure S3F, supplementary material). On the other hand, untreated cells presented normal characteristic nuclear morphology with preserved cytoplasm organelles (Figure S3A, supplementary material). The accumulation of lipid drops in the cytoplasm was found in promastigotes forms, suggesting remodeling of the cytoplasm organelles (Medeiros et al. 2011). Another important observed finding was the kinetoplast swelling. Kinetoplast is an organelle unique to kinetoplastids, consisting of mitochondrial DNA, which is
n 2010). Mitochondria considered a target to drug action (Adade and Souto-Padro also showed alterations, which could be related to possible alterations in the lipid composition (Garcia et al. 2017). Mitochondria are source of cellular energy, and damage to the mitochondrial membrane can induce intrinsic cell death (Machado et al. 2012). Promastigotes treated with IC50 of C. xanthocarpa EO presented slight reduction of marking intensity by Rhodamine 123, weakly inducing depolarization of the mitochondrial membrane that can be evident from the negative value of IV (Figure S4A, S4B, S4C and S4D, supplementary material), therefore, it is not possible to state that the EO interferes with mitochondrial bioenergetics in the parasites’ cells, despite C. xanthocarpa EO being an apoptosis inducer.

3. Conclusion C. xanthocarpa EO presented antileishmanial activity against both L. amazonensis promastigote and amastigote forms, showing high selectivity index for the parasite. C. xanthocarpa EO caused damage suggestive of an autophagic phenotype. However, characteristic changes similar to apoptosis were observed. These promising results point out C. xanthocarpa EO as a natural agent for the treatment of leishmaniasis, revealing its potential for future in vivo studies.

Disclosure statement No potential conflict of interest was reported by the authors.

Funding This study was financed in part by Fundac¸~ao Arauc
aria the Coordenac¸
ao de Aperfeic¸oamento de Pessoal de N
ıvel Superior – Brazil (CAPES) Finance Code 001.

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