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. 2016 Aug 31;10(8):e0004900.
doi: 10.1371/journal.pntd.0004900. eCollection 2016 Aug.

Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis

Fernando Almeida-Souza  1   2 , Flávia de Oliveira Cardoso  1 , Bruno Vinicius da Conceição Souza  1 , Tânia Zaverucha do Valle  1 , Joicy Cortez de Sá  2 , Iara Dos Santos da Silva Oliveira  2 , Celeste da Silva Freitas de Souza  1 , Carla Junqueira Moragas Tellis  3 , Maria do Socorro Dos Santos Chagas  3 , Maria Dutra Behrens  3 , Ana Lúcia Abreu-Silva  2 , Kátia da Silva Calabrese  1
Affiliations

Morinda citrifolia Linn. Reduces Parasite Load and Modulates Cytokines and Extracellular Matrix Proteins in C57BL/6 Mice Infected with Leishmania (Leishmania) amazonensis

Fernando Almeida-Souza et al. PLoS Negl Trop Dis. .

Abstract

The absence of an effective vaccine and the debilitating chemotherapy for Leishmaniasis demonstrate the need for developing alternative treatments. Several studies conducted with Morinda citrifolia have shown various biological activities, including antileishmanial activity, however its mechanisms of action are unknown. This study aimed to analyze the in vivo activity of M. citrifolia fruit juice (Noni) against Leishmania (Leishmania) amazonensis in C57BL/6 mice. M. citrifolia fruit juice from the Brazilian Amazon has shown the same constitution of other juices produced around the world and liquid chromatography-mass spectrometry analysis identified five compounds: deacetylasperulosidic acid, asperulosidic acid, rutin, nonioside B and nonioside C. Daily intragastric treatment with Noni was carried out after 55 days of L. (L.) amazonensis infection in C57BL/6 mice. Parasitic loads, cytokine and extracellular protein matrix expressions of the lesion site were analyzed by qPCR. Histopathology of the lesion site, lymph nodes and liver were performed to evaluate the inflammatory processes. Cytokines and biochemical parameters of toxicity from sera were also evaluated. The Noni treatment at 500 mg.kg-1.day-1 for 60 days decreased the lesion size and parasitic load in the footpad infected with L. (L.) amazonensis. The site of infection also showed decreased inflammatory infiltrates and decreased cytokine expressions for IL-12, TNF-α, TGF-β and IL-10. On the other hand, Noni treatment enhanced the extracellular matrix protein expressions of collagen IV, fibronectin and laminin in the infected footpad as well collagen I and II, fibronectin and laminin in the mock-infected footpads. No toxicity was observed at the end of treatment. These data show the efficacy of Noni treatment.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Liquid chromatography–mass spectrometry analysis of Morinda citrifolia fruit juice, Noni.
(1–5) Chromatograms of compounds (m/z) identified in Noni: deacetylasperulosidic acid (389), asperulosidic acid (431), rutin (610), nonioside B (629) and nonioside C (467).
Fig 2
Fig 2. Activity of Morinda citrifolia fruit juice (Noni) treatment in C57BL/6 mice infected with Leishmania (L.) amazonensis.
(A) Kinetic of lesion of infected footpads treated with Noni (500mg.kg-1.day-1) or Glucantime (20mg.kg-1.twice a week-1). (B-C) Parasite loads in footpad and draining lymph node after 30 and 60 days of Noni treatment. Data represent mean ± SD of two independent experiments realized at least in triplicate. *p<0.05, **p<0.01, ***p<0.001 when compared with La group by two-way ANOVA and Bonferroni’s post-test. La+Noni: group infected and treated with Noni; La+Glucantime: group infected and treated with Glucantime; La: group infected and mock-treated; Normal: mock-infected and mock-treated group.
Fig 3
Fig 3. Histopathological analysis of skin and lymph nodes of C57BL/6 mice footpads infected with Leishmania (L.) amazonensis and treated with Morinda citrifolia fruit juice, Noni.
Noni group treated at 500mg.kg-1.day-1 and Glucantime group treated at 20mg.kg-1.twice a week-1; both treatments were for 60 days. At 30 days: amastigotes within macrophages (arrows in inserts) and inflammatory infiltration in dermis of all groups. At 60 days: inflammatory infiltration decreased in the Noni group, and absence of amastigotes in the Noni and Glucantime groups. La group with intense inflammatory infiltration of macrophages with parasites at the lesion site. In lymph nodes, there was a decrease of the lymphoid nodules (asterisks) hyperplasia in Noni and Glucantime groups. Images representative of two experiments realized in triplicate. Hematoxylin-eosin.
Fig 4
Fig 4. Cytokine gene expression in C57BL/6 mouse footpads infected with Leishmania (L.) amazonensis and treated with Morinda citrifolia fruit juice.
Relative quantification of IFN-γ (A), iNOS (B), IL-12 (C), TNF-α (D), IL-10 (E), TGF-β (F) and IL-4 (G) with RPLP0 as endogenous control. Noni group treated at 500mg.kg-1.day-1 and Glucantime group treated at 20mg.kg-1.twice a week-1, both during 60 days. Data represent mean ± SD of two experiments realized in triplicate. *p<0.05, **p<0.01, ***p<0.001 when compared with control group or between group brackets by two-way ANOVA and Bonferroni’s post-test. RQ: relative quantification; RPLP0: ribosomal protein large P0; La+Noni: group infected and treated with Noni; La+Glucantime: group infected and treated with Glucantime; La: group infected and mock-treated; Noni: group mock-infected and treated with Noni; Normal: mock-infected and mock-treated group.
Fig 5
Fig 5. Quantification of serum cytokines from C57BL/6 mice infected with Leishmania (L.) amazonensis and treated with Morinda citrifolia fruit juice.
Cytokines levels of IFN-γ (A), IL-12 (B), TNF-α (C), IL-4 (D), IL-10 (E) and TGF-β (F). Noni group treated at 500mg.kg-1.day-1 and Glucantime group treated at 20mg.kg-1.twice a week-1, both for 60 days. Data represent mean ± SD of two experiments realized in duplicate. *p<0.05, ***p<0.001 when compared with control group or between group brackets by two-way ANOVA and Bonferroni’s post-test. La+Noni: group infected and treated with Noni; La+Glucantime: group infected and treated with Glucantime; La: group infected and mock-treated; Noni: group mock-infected and treated with Noni; Normal: mock-infected and mock-treated group.
Fig 6
Fig 6. Extracellular matrix protein analysis of C57BL/6 mice footpads infected with Leishmania amazonensis and treated for 60 days with Morinda citrifolia fruit juice, Noni.
Noni group treated at 500mg.kg-1.day-1 and Glucantime group treated at 20mg.kg-1.twice a week-1. Histopathology (A) and extracellular matrix protein gene expressions (B-F) of skin. Images are representative of two independent experiments realized in triplicate. HE: hematoxylin-eosin. Data represent mean ± SD of two independent experiments realized in triplicate. *p<0.05, **p<0.01, ***p<0.001 when compared with control group or between group brackets by one-way ANOVA and Bonferroni’s post-test. RQ: relative quantification; RPLP0: ribosomal protein large P0; La+Noni: group infected and treated with Noni; La+Glucantime: group infected and treated with Glucantime; La: group infected and mock-treated; Noni: group mock-infected and treated with Noni; Normal: mock-infected and mock-treated group.
Fig 7
Fig 7. Quantification of alanine aminotransferase (ALT) and histopathology of liver from C57BL/6 mice infected with Leishmania amazonensis and treated for 60 days with Morinda citrifolia fruit juice, Noni.
Noni group treated at 500mg.kg-1.day-1 and Glucantime group treated at 20mg.kg-1.twice a week-1. Diffuse inflammatory infiltration (arrows) and periportal infiltration (arrow-heads) in the liver of infected mice. The inflammatory intensity of infiltration decreases with Glucantime and Noni treatments. Hematoxylin-eosin. Data represent mean ± SD of two independent experiments realized in duplicate. *p<0.05, **p<0.01 when compared with control group or between group brackets by one-way ANOVA and Bonferroni’s post-test. La+Noni: group infected and treated with Noni; La+Glucantime: group infected and treated with Glucantime; La: group infected and mock-treated; Noni: group mock-infected and treated with Noni; Normal: mock-infected and mock-treated group.
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Grants and funding

This work was supported by Fundação de Amparo à Pesquisa e Desenvolvimento Científico do Maranhão (http://www.fapema.br/) grant number APP-00844/09, and Ponex-241709/2014 to ALAS; Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (http://www.faperj.br/) grant number E-26/111.252/2014 to KSC and Conselho Nacional de Desenvolvimento Científico e Tecnológico (http://cnpq.br/) grant numbers 407831/2012.6 and 309542/2013-8 to ALAS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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