Anti-Leishmania amazonensis activity of the terpenoid fraction from Eugenia pruniformis leaves

ALBUQUERQUE, RICARDO D.D.G.; OLIVEIRA, ADRIANA P.; FERREIRA, CHRISTIAN; PASSOS, CARLOS L.A.; FIALHO, ELIANE; SOARES, DEIVID C.; AMARAL, VERONICA F.; BEZERRA, GABRIELLE B.; ESTEVES, RICARDO S.; SANTOS, MARCELO G.; ALBERT, ANDRÉ L.M.; ROCHA, LEANDRO

doi:10.1590/0001-3765202020201181

RICARDO D.D.G. ALBUQUERQUE

Universidade Federal Fluminense, Laboratório de Tecnologia de Produtos Naturais, Faculdade de Farmácia, Rua Dr. Mário Vianna, 523, Santa Rosa, 24241-000 Niterói, RJ, Brazil

https://orcid.org/0000-0002-8442-3849

ADRIANA P. OLIVEIRA

Universidade Federal do Rio de Janeiro, Laboratório Multidisciplinar de Ciências Farmacêuticas, Faculdade de Farmácia, Av. Carlos Chagas Filho, 373, Cidade Universitária, 21941-902 Rio de Janeiro, RJ, Brazil

https://orcid.org/0000-0001-6968-4269

CHRISTIAN FERREIRA

Universidade Federal do Rio de Janeiro, Laboratório de Alimentos Funcionais, Av. Carlos Chagas Filho, 373, Cidade Universitária, 21941-902 Rio de Janeiro, RJ, Brazil

https://orcid.org/0000-0003-0504-0354

CARLOS L.A. PASSOS

Universidade Federal do Rio de Janeiro, Laboratório de Alimentos Funcionais, Av. Carlos Chagas Filho, 373, Cidade Universitária, 21941-902 Rio de Janeiro, RJ, Brazil

https://orcid.org/0000-0002-9882-8222

ELIANE FIALHO

Universidade Federal do Rio de Janeiro, Laboratório de Alimentos Funcionais, Av. Carlos Chagas Filho, 373, Cidade Universitária, 21941-902 Rio de Janeiro, RJ, Brazil

https://orcid.org/0000-0001-7272-0021

DEIVID C. SOARES

Laboratório de Imunobiologia das Leishmanioses, Av. Carlos Chagas Filho 373, Cidade Universitária, 21941-902 Rio de Janeiro, RJ, Brazil

https://orcid.org/0000-0001-8078-5968

VERONICA F. AMARAL

Universidade Federal do Rio de Janeiro, Laboratório de Imunoparasitologia, Departamento de Imunobiologia, Universidade Federal Fluminense, Outeiro de São João Batista, s/n, Centro, 24020-140 Niterói, RJ, Brazil

https://orcid.org/0000-0002-1129-605X

GABRIELLE B. BEZERRA

Universidade Federal do Rio de Janeiro, Laboratório de Imunoparasitologia, Departamento de Imunobiologia, Universidade Federal Fluminense, Outeiro de São João Batista, s/n, Centro, 24020-140 Niterói, RJ, Brazil

https://orcid.org/0000-0003-2093-7186

RICARDO S. ESTEVES

Universidade Federal Fluminense, Laboratório de Tecnologia de Produtos Naturais, Faculdade de Farmácia, Rua Dr. Mário Vianna, 523, Santa Rosa, 24241-000 Niterói, RJ, Brazil

https://orcid.org/0000-0002-6611-6722

MARCELO G. SANTOS

Universidade do Estado do Rio de Janeiro, Departamento de Ciências, Faculdade de Formação de Professores, Rua Dr. Francisco Portela, 1470, Patronato, 24435-005 São Gonçalo, RJ, Brazil

https://orcid.org/0000-0002-0680-4566

ANDRÉ L.M. ALBERT

Fundação Oswaldo Cruz, Instituto Nacional de Controle de Qualidade em Saúde, Av. Brasil, 4365, Manguinhos, 21041-361 Rio de Janeiro, RJ, Brazil

https://orcid.org/0000-0002-6652-4961

LEANDRO ROCHA

Universidade Federal Fluminense, Laboratório de Tecnologia de Produtos Naturais, Faculdade de Farmácia, Rua Dr. Mário Vianna, 523, Santa Rosa, 24241-000 Niterói, RJ, Brazil

https://orcid.org/0000-0003-0484-1918

Corresponding author: Adriana Passos Oliveira E-mail: adrianapassos@pharma.ufrj.br

AUTHOR CONTRIBUTIONS

RDDGA (PhD. student) contributed to collecting plant samples, running the laboratory work, analysis of the data and drafted the paper. MGS contributed to plant identification and herbarium confection. RSE contributed to the phytochemical analysis. VFA and GBB contributed to anti-promastigote assay in strain MHOM / BR / 77LTB0016. DCS contributed to anti-amastigote studies. CF, CLAP, and EF designed the leishmanicidal studies in strain WHOM / BR / 75 / Josefa analysis of the data and drafted the paper. ALMM contributed to the chromatographic analysis. APO and LMR designed the study, supervised the laboratory work and contributed to the critical reading of the manuscript. All the authors have read the final manuscript and approved the submission.

Universidade Federal Fluminense, Laboratório de Tecnologia de Produtos Naturais, Faculdade de Farmácia, Rua Dr. Mário Vianna, 523, Santa Rosa, 24241-000 Niterói, RJ, Brazil

Universidade Federal do Rio de Janeiro, Laboratório Multidisciplinar de Ciências Farmacêuticas, Faculdade de Farmácia, Av. Carlos Chagas Filho, 373, Cidade Universitária, 21941-902 Rio de Janeiro, RJ, Brazil

Universidade Federal do Rio de Janeiro, Laboratório de Alimentos Funcionais, Av. Carlos Chagas Filho, 373, Cidade Universitária, 21941-902 Rio de Janeiro, RJ, Brazil

Laboratório de Imunobiologia das Leishmanioses, Av. Carlos Chagas Filho 373, Cidade Universitária, 21941-902 Rio de Janeiro, RJ, Brazil

Universidade Federal do Rio de Janeiro, Laboratório de Imunoparasitologia, Departamento de Imunobiologia, Universidade Federal Fluminense, Outeiro de São João Batista, s/n, Centro, 24020-140 Niterói, RJ, Brazil

Universidade do Estado do Rio de Janeiro, Departamento de Ciências, Faculdade de Formação de Professores, Rua Dr. Francisco Portela, 1470, Patronato, 24435-005 São Gonçalo, RJ, Brazil

Fundação Oswaldo Cruz, Instituto Nacional de Controle de Qualidade em Saúde, Av. Brasil, 4365, Manguinhos, 21041-361 Rio de Janeiro, RJ, Brazil

Figures (5)
Tables (1)

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Figure 1
Inhibition of L. amazonensis proliferation by terpenoid fraction of E. pruniformis leaves (TF-EpL) and cytotoxicity against murine peritoneal macrophages in vitro. (a) Promastigotes (10⁷ / mL) were grown in the presence or absence of TF-EpL, or diluent (DMSO), at the indicated concentrations, during 24 h. Parasite viability was checked by the dehydrogenases activity using the MTT assay. The results were expressed as the percentage of viable promastigotes in relation to the untreated control (100 %) and are shown as the mean ± SEM of 3 independent experiments. (b) Peritoneal macrophages (10⁵) from BALB/c mice were infected with promastigotes at a ratio of 10 parasites to 1 macrophage for 24 h and were either left untreated or were treated with the indicated concentrations of TF-EpL for 24 h, washed, fed with Schneider’s complete medium, and cultured at 26 °C. The macrophage parasite load was evaluated after 48 h using Alamar Blue. (c) Cells were cultured with 1, 10, 25, 50, 100 and 200 µg/mL TF-EpL for 24 h at 37 °C in 5 % CO₂. Viability was measured by the XTT method. The results represent the means ± SEM from 3 experiments performed in triplicate. p < 0.05 (*), p < 0.001 (**) and p < 0.0001 (***) compared to the control.

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Figure 2
Evaluation of cell cycle, mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) production by Leishmania amazonensis promastigotes. Promastigotes were treated or not with TF-EpL and cell cycle was evaluated after 48h after PI staining by flow cytometry analysis. Untreated parasites control (a), 1 % DMSO (b), 43.60 μg / mL TF-EpL (c) and 0.1 μg/mL Amphotericin B (d). Representative plots of at least three independent experiments with similar results. (e) Promastigotes treated or not with 43.60 µg/mL TF-EpL was evaluated using a JC-1 assay. The results are expressed as red / green fluorescence ratios and represent the averages ± SEM from 3 independent experiments. (f) Promastigotes treated or not with 43.60 µg/mL TF-EpL or 1 % DMSO were then stained with 50 μM DCFDA and ROS was measured by flow cytometer. The results represent the means ± SEM from 3 experiments performed in triplicate. p < 0.05 (*), p < 0.001 (**), and p < 0.0001 (***) compared to the untreated control.

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Figure 3
Assessment of Annexin-V expression on Leishmania induced by terpenoid fraction of E. pruniformis leaves (TF-EpL) treatment. Promastigotes were treated or not with TF-EpL and the expression of Annexin-V / Propidium Iodide (PI) measured. Untreated parasites control (a), parasites treated with 1 % DMSO (b) and 43.60 μg/mL TF-EpL (c) are shown as a representative result out of 3 independent experiments with similar results. The percentage of PI positive (d), Annexin-V positive (e) and Annexin-V-PI positive promastigotes (f), treated as above, shown as mean ± SEM of 3 independent experiments. p < 0.001 (**) compared to the control.

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Figure 4
Effect of terpenoid fraction of E. pruniformis leaves (TF-EpL) on the synthesis of total lipids on Leishmania. Promastigotes were incubated in the presence or absence of 43.60 μg/mL TF-EpL for 24 h. Cells were labeled with Nile Red (1 μg/mL) and the percentage of neutral lipids (a) and phospholipids (b) were estimated in flow cytometry. Histograms are representative of 3 independent experiments with similar results. p < 0.001 (**), compared to control.

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Figure 5
Triterpenoids from E. pruniformis leaves. 1 – β-Sitosterol; 2 – Ursolic acid; 3 – Corosolic acid; 4 – Asiatic acid.

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Table I
¹H. and ¹³C-NMR Data of Compounds 1, 2, 3 and 4, in ppm. Assignments of ¹³C-NMR data were accomplished with the aid of DEPT, HSQC and HMBC experiments.

	β-sitosterol		Ursolic Acid		Corosolic Acid		Asiatic Acid
Position	δ H	δ C	δ H	δ C	δ H	δ C	δ H	δ C
1		37.2		40.0		46.9		46.6
2		31.6	1.84	28.8	3.63 (ddd)	68.2	3.69 (ddd)	68.2
3	3.51 (m)	71.8	3.48 (dd)	78.8	2.91 (d)	83.1	3.36 (dd)	76.8
4		42.3		39.7		39.1		42.7
5	5.33 (d)	140.7		56.5		55.3		46.8
6		121.7	Hα 1.59 (t)	19.4		18.1		17.7
7		31.9	Hα 1.60 (t)	34.2		32.8		32.2
8		31.9		40.6		39.4		39.4
9		50.1	1.65 (t)	48.7		47.6		47.4
10		36.5		37.9		37.8		37.6
11		21.1	1.99 (m)	24.3		23.0		23.2
12		39.8	5.51 (t)	126.3	5.24 (t)	125.3	5.26 (t)	125.2
13		42.3		139.9		138.3		138.4
14		56.8		43.2		41.9		41.9
15		26.1	Hβ 2.35 (td)	29.3		27.8		27.7
16	0.66 (s)	28.2	2.15 (td)	25.6		23.9		23.9
17		56.0		48.7		47.6		48.4
18		36.1	2.66 (d)	54.2	2.21 (d)	53.0	2.21 (d)	52.8
19	0.99 (s)	19.0	1.49 (t)	40.1		39.0		39.0
20		34.0	1.07	40.1		39.0		39.0
21	0.90 (d)	26.1	Hα 1.40 (t)	31.7		30.4		30.3
22		45.8	1.96 (m)	37.9		36.7		36.7
23		23.1	1.26 (s)	29.5	1.02 (s)	27.9	3.50 (d)	64.9
24	0.84 (t)	12.0	1.04 (s)	17.2	0.81 (s)	16.1	0.70 (s)	12.5
25		29.2		16.3	1.01 (s)	15.8	1.03 (s)	16.2
26	0.81 (d)	19.8	1.08 (s)	18.1	0.85 (s)	16.4	0.85 (s)	16.4
27	0.79 (d)	19.4	1.25 (s)	24.6		22.7	1.14 (s)	22.7
28		18.8		180.2		180.2		180.2
29		11.9	1.03 (d)	18.2	0.89 (d)	16.2	0.89 (d)	16.2
30	-	-	0.98 (d)	22.1	0.97 (s)	20.1	0.97 (s)	20.1
U.S.	1.12-2.32 (m)

[1] Corresponding author: Adriana Passos Oliveira E-mail: adrianapassos@pharma.ufrj.br

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Anti-Leishmania amazonensis activity of the terpenoid fraction from Eugenia pruniformis leaves

Abstract