Antioxidant activity and ultrastructural alterations in Leishmania amazonensis promastigotes induced by limonoid-rich fractions from andiroba oil

Atividade antioxidante e alterações ultraestruturais em Leishmania amazonensis induzidas por frações ricas em limonóides do óleo de andiroba

ABSTRACT

In the Amazon region, andiroba (Carapa guianensis) oil is among the most used products by the local population due to its medicinal properties. Previously, our group described the leishmanicidal activity of three limonoid-rich fractions from andiroba oil. This work aimed to evaluate the antioxidant activity and ultrastructural changes in Leishmania amazonensis treated with these limonoid-rich fractions from andiroba oil: 6α,11β-diacetoxygedunin; 11β-hydroxygedunin; and a fraction with both 6α,11β-diacetoxygedunin +11β-hydroxygedunin. The antioxidant activity was evaluated by the DPPH method and ultrastructural changes were evaluated by transmission electron microscopy. The fraction with both 6α,11β-diacetoxygedunin + 11β-hydroxygedunin displayed the best antioxidant activity (IC₅₀ 330.35 ± 8.60 µg mL^-1; p=0.0022) compared to the 6α,11β-diacetoxygedunin fraction. The three fractions induced structural changes to mitochondria and kinetoplasts of L. amazonensis, in addition to lipid bodies, vacuolization, and vesicles in the flagellar pocket, indicating that the limonoid-rich fractions from andiroba oil can induce structural damage to Leishmania.

KEYWORDS:
leishmaniasis; crabwood; DPPH; transmission electron microscopy; Carapa guianensis

RESUMO

Na região Amazônica, o óleo de andiroba (Carapa guianensis) é um dos produtos mais utilizados pela população local devido às suas propriedades medicinais. Anteriormente, nosso grupo descreveu a atividade leishmanicida de três frações do óleo de andiroba ricas em limonóides. Este trabalho objetivou avaliar a atividade antioxidante e alterações ultraestruturais em Leishmania amazonensis tratadas com essas frações ricas em limonóides do óleo de andiroba: 6α,11β-diacetoxigedunina; 11β-hidroxigedunina; e uma fração com ambas 6α,11β-diacetoxigedunina + 11β-hidroxigedunina. A atividade antioxidante foi avaliada pelo método DPPH, e alterações ultraestruturais foram avaliadas por microscopia eletrônica de transmissão. A fração com ambas 6α,11β-diacetoxigedunina + 11β-hidroxigedunina exibiu a melhor atividade antioxidante (IC₅₀ = 330,35 ± 8,60 µg mL^-1; p = 0.0022) comparado com a fração 6α,11β-diacetoxigedunina. As três frações induziram alterações estruturais nas mitocôndrias e cinetoplasto de L. amazonensis, além de corpúsculos lipídicos, vacuolizações e vesículas na bolsa flagelar, indicando que as frações ricas em limonóides do óleo de andiroba podem induzir dano estrutural a Leishmania.

PALAVRAS-CHAVE:
leishmaniose; andiroba; DPPH; microscopia eletrônica de transmissão; Carapa guianensis

In the Amazon region of Brazil, andiroba (Carapa sp.) (Meliaceae) stands out in popular medicine due to the properties of the oil extracted from the seeds (Costa and Marenco 2007). Andiroba oil is well known in traditional communities and used in alternative medicine in northern and northeastern Brazil to treat arthritis, cold, fever, flu, cough, and malaria, to relieve arthritis pain, to treat ear infections, wounds, bruises, insect bites in people and animals, and as an insect repellant (Henriques and Penido 2014). Carapa was described as one of the most used genera to treat and prevent leishmaniasis in Amazonian communities (Odonne et al. 2017).

Leishmaniases comprise a group of infectious-parasitic illnesses caused by several protozoa of the genus Leishmania that occur in different regions of the world, mainly in tropical and subtropical regions. The disease is endemic in the Amazon region, and cutaneous leishmaniasis is the predominant form of the disease (PAHO 2023).

There are only two studies on the antileishmanial activity of andiroba oil. One study tested an andiroba oil nanoemulsion against Leishmania amazonensis Lainson & Shaw, 1972, and Leishmania infantum Nicolle, 1908, however they did not describe the chemical compounds involved in the antileishmanial activity (Dhorm Pimentel de Moraes et al. 2018). In the other study, our research group did not observe antileishmanial activity of whole oil from andiroba, Carapa guianensis Aublet (IC₅₀ > 500 µg mL^{- 1}), in contrast with three of its fractions rich in diverse limonoids, which showed promising antileishmanial activity against promastigote and intracellular amastigote forms of L. amazonensis (Oliveira et al. 2018). The antileishmanial activity of limonoids was also observed with orthoacetates from Pseudocedrela kotschyi (Schweinf.) Harms. roots (Hay et al. 2007) and trigilgianin from Trichilia gilgiana Harms. stem bark (Kowa et al. 2020) against Leishmania donovani Laveran & Mesnil, 1903, in vitro. Although these studies report promising antileishmanial activity of limonoid-rich fractions, there are no reports on the effect of limonoids on ultrastructures of Leishmania parasites.

There are a few reports of andiroba oil antioxidant activity (Milhomem-Paixão et al. 2016, Araujo-Lima et al. 2018), but none associated to limonoids. Yet the antioxidant activity of limonoids is already known. For example, the antioxidant activity of limonin occurs through activation of intracellular antioxidative pathways (Yang et al. 2020).

In this study, we evaluated the antioxidant activity and the ultrastructural changes that the treatment with the abovementioned limonoid-rich fractions from C. guianensis induce in promastigote forms of L. amazonensis.

The study was authorized by the National System for Management of Genetic Heritage and Traditional Knowledge (Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado - SisGen) of the Brazilian Ministry of the Environment (authorization # A977CED for C. guianensis, and # AAE018A for L. amazonensis).

Andiroba seed oil was acquired from extractive producers in the Rosário microregion, Maranhão state, Brazil. Botanical identification was carried out at the Botany Laboratory and Herbarium of Universidade Estadual do Maranhão (voucher deposit # 4896). Details about plant acquisition, fractionation and chemical characterization are described elsewhere (Oliveira et al. 2018). We used the three fractions that previously exhibited leishmanicidal activity: fraction with 6α,11β-diacetoxygedunin (LF3); fraction with 11β-hydroxygedunin (LF5); and fraction with both 6α,11β-diacetoxygedunin and 11β-hydroxygedunin (LF4).

The antioxidant activity was evaluated by the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) (Sigma-Aldrich, St. Louis, MO, USA) free radical scavenging method as described by Wang et al. 2022. Concentrations of the fractions in methanol ranging from 1,000 to 7.8125 μg mL^-1, and 6-hydroxy-2,5,7,8- tetramethychroman-2- carboxylic (Trolox) standard curve ranging from 12.5 to 0.097 μg mL^-1, were obtained by two-folding dilutions in 96-well microplates in triplicate. Trolox is a well know antioxidant used as a standard in several commercial kits and antioxidant activity studies (Xiao et al. 2020; Rumpf et al. 2023). The inhibitory concentration of 50% (IC₅₀) of parasites was calculated by fitting a regression curve of the log concentration on the normalized response using GraphPad Prism 7.00 (GraphPad Software Inc., USA).

Ultrastructural analyses were carried out with L. amazonensis (MHOM/BR/76/MA-76) promastigote forms kept in Schneider’s Insect medium (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), penicillin (100 U mL^-1) and streptomycin (100 μg mL^-1) (Sigma-Aldrich, USA) at 26 ºC. The parasites were treated for 72 hours with the IC₅₀ of each fraction, 10.53 µg mL^-1 (6α,11β-diacetoxygedunin), 56.90 µg mL^-1 (11β-hydroxygedunin fraction), and 25.30 µg mL^-1 (6α,11β-diacetoxygedunin + 11β-hydroxygedunin). Ultrastructures of the parasites were evaluated by transmission electron microscope following Almeida-Souza et al. 2021, by treating 10⁶ parasites mL^-1 with the limonoid-rich fractions (in duplicate) and comparing the alterations in plasma membrane, nuclei, flagella and all the visualized organelles with those of untreated parasites.

In the radical scavenging assay, the 6α,11β-diacetoxygedunin + 11β-hydroxygedunin fraction showed the best, though moderate, antioxidant activity (p = 0.0022) when compared to the 6α,11β-diacetoxygedunin fraction (Table 1). This result is similar to the IC₅₀ obtained for andiroba oils from Belém, Pará state (northeastern Amazon) (Milhomem-Paixão et al. 2016). The 11β-hydroxygedunin fraction did not exhibit antioxidant activity at the concentrations evaluated.

Table 1
Antioxidant activity of limonoid-rich fractions of andiroba (Carapa guianensis) oil. Fractions are identified by their predominant limonoid compounds. Trolox = positive control. Values are the mean ± standard deviation of three repetitions.

Fraction	DPPH IC50 (µg mL-1)
6α,11β-diacetoxygedunin	619.51 ± 11.41a
11β-hydroxygedunin	>1000b
6α,11β-diacetoxygedunin + 11β-hydroxygedunin	330.35 ± 8.60c
Trolox	6.15 ± 0.57d

• IC₅₀ = inhibitory concentration of 50% of the parasites when compared to an untreated group. Different letters in the column indicate statistical difference between groups according to a Mann-Whitney test.

Untreated parasites exhibited normal morphology (Figure 1a). The three fractions induced alteration in nuclei of the promastigotes with variations in chromatin condensation (Figure 1e,g,h). Promastigotes treated with the 6α,11β-diacetoxygedunin fraction (Figure 1b-e) showed an increase in kinetoplast and mitochondria volume, alterations in mitochondrial cristae, electron-dense material in the flagellar pocket, lipid bodies, increase of the endoplasmic reticulum and irregular membranous-shape structures near the flagellar pocket. Parasites treated with the 6α,11β-diacetoxygedunin + 11β-hydroxygedunin fraction (Figure 1f-g) displayed vacuoles and large lipid corpuscles, increase and fragmentation of the endoplasmic reticulum, and electron-dense material in the flagellar pocket. Parasites treated with the 11β-hydroxygedunin fraction exhibited electron-dense lipid bodies, large-volume vacuoles with electron-dense material close to the nucleus and to the flagellar pocket, change in mitochondrial morphology with circular membranes inside, and kinetoplast kDNA barely visible (Figure 1h).

Figure 1
Ultrastructural alterations in Leishmania amazonensis treated with limonoid-rich fractions of oil from andiroba, Carapa guianensis. Promastigotes remained untreated (A); or were treated for 72 hours with fractions containing 6α,11β-diacetoxygedunin (B-E); 6α,11β-diacetoxygedunin + 11β-hydroxygedunin (F-G); and 11β-hydroxygedunin (H). White asterisks = lipid bodies; black asterisks = vacuoles; white arrowhead = irregularly shaped vacuole with membranous structures inside near the flagellar pocket; black arrowheads = increase and fragmentation of endoplasmic reticulum; arrows = change in mitochondrial morphology with circular membranes inside; f = flagella; k = kinetoplast; m = mitochondria; n = nucleus; fp = flagellar pocket. Scale bar = 0.5 µm.

To the best of our knowledge, this is the first report of ultrastructural alterations induced by limonoid-rich andiroba oil fractions in Leishmania parasites. Leishmania infantum and L. amazonensis promastigotes treated with an andiroba oil nanoemulsion and analyzed by electron microscopy scanning showed alterations like oval cell shape and retracted flagella as early as one hour after treatment (Dhorm Pimentel de Moraes et al. 2018).

The major ultrastructural changes in our treated parasites were observed in kinetoplast/mitochondria. The kinetoplast is a structure with DNA (kDNA) in the mitochondria formed by a unique network of circular interlocked minicircles and maxicircles, and the morphological traits and segregation pattern of the kinetoplast are associated with the cell cycle stage (Minocha et al. 2011). Alterations in kDNA, such as the decrease in chromatin condensation induced by the treatments, can be related to the cell death process. Mitochondrial morphological changes, such as the swelling and cristae disorganization, are associated with a phenotype similar to apoptosis observed in mammalian cells in vitro (Basmaciyan and Casanova 2019).

The other alterations are related to the flagellar pocket, an invagination of the cell membrane around the proximal end of the flagellum. In Leishmania parasites, endo/exocytosis occurs exclusively through the flagellar pocket (Halliday et al. 2021). The electron dense material observed in the flagellar pocket, and the irregular shaped vacuole with membranous structures inside or near the flagellar pocket, indicate an improvement in exocytosis by the promastigotes. The increase in vacuolization, with some vacuoles presenting material inside, as well as the increase in lipid bodies, is a signal of exacerbated production/recycling of proteins by the cells to survive. The increase in exocytic activity, that is also associated with drug resistance (Perea et al. 2016), demonstrates the need of the parasite cell to remove intracellular material as an attempt to survive drug action (Almeida-Souza et al. 2020).

The set of ultrastructural alterations, some related to accidental cell death, such as the increase in the exocytic activity, the increase in lipid bodies and vacuolization, and some related to the regulation of cell death, like the mitochondria/kinetoplast alterations, indicate anti-Leishmania effects of the limonoid-rich fractions that may act by different mechanisms. Further studies should isolate the limonoid compounds in the tested fractions and elucidate the mechanisms involved in the tendency to leishmanicidal and antioxidant activity observed in our study.

ACKNOWLEDGMENTS

This research was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES (Finance Code 001), and by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (26/211.680/2021). FA-S is a postdoctoral research fellow (CAPES 88887.363006/2019-00). KSC (315225/2021-1) and ALA-S (313348/2021-9) are research productivity fellows by Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq.

REFERENCES

• Almeida-Souza, F.; Silva, V.D.D.; Silva, G.X.; Taniwaki, N.N.; Hardoim, D.J.; Buarque, C.D.; et al. 2020. 1,4-disubstituted-1,2,3-triazole compounds induce ultrastructural alterations in Leishmania amazonensis promastigotes: An in vitro antileishmanial and in silico pharmacokinetic study. International Journal of Molecular Sciences 21: 6839. doi: 10.3390/ijms21186839
  » https://doi.org/10.3390/ijms21186839
• Almeida-Souza, F.; Silva, V.D.D.; Taniwaki, N.N.; Hardoim, D.J.; Mendonça Filho, A.RD.; Moreira, W.F.F.; et al. 2021. Nitric oxide induction in peritoneal macrophages by a 1,2,3-triazole derivative improves its efficacy upon Leishmania amazonensis in vitro infection. Journal of Medicinal Chemistry 64: 12691-12704.
• Araujo-Lima, C.F.; Fernandes, A.S.; Gomes, E.M.; Oliveira, L.L.; Macedo, A.F.; Antoniassi, R.; et al. 2018. Antioxidant activity and genotoxic assessment of crabwood (andiroba, Carapa guianensis Aublet) seed oils. Oxidative Medicine and Cellular Longevity 2018: 3246719.
• Basmaciyan, L.; Casanova, M. 2019. Cell death in Leishmania Parasite 26: 71. doi: 10.1051/parasite/2019071.
  » https://doi.org/10.1051/parasite/2019071
• Costa, G.d.; Marenco, R.A. 2007. Fotossíntese, condutância estomática e potencial hídrico foliar em árvores jovens de andiroba (Carapa guianensis). Acta Amazonica 37: 229-234.
• Dhorm Pimentel de Moraes, A.R.; Tavares, G.D.; Soares Rocha, F.J.; de Paula. E.; Giorgio, S. 2018. Effects of nanoemulsions prepared with essential oils of copaiba- and andiroba against Leishmania infantum and Leishmania amazonensis infections. Experimental Parasitology 187: 12-21.
• Halliday, C.; de Castro-Neto, A.; Alcantara, C.L.; Cunha-E-Silva, N.L.; Vaughan, S.; Sunter, J.D. 2021. Trypanosomatid flagellar pocket from structure to function. Trends in Parasitology 37: 317-329.
• Hay, A.E.; Ioset, J.R.; Ahua, K.M.; Diallo, D.; Brun, R.; Hostettmann, K. 2007. Limonoid orthoacetates and antiprotozoal compounds from the roots of Pseudocedrela kotschyi Journal of Natural Products 70: 9-13.
• Henriques, M.; Penido, C. 2014. The therapeutic properties of Carapa guianensis Current Pharmaceutical Design 20: 850-856.
• Kowa, T.K.; Tchokouaha, L.R.Y.; Cieckiewicz, E.; Philips, T.J.; Dotse, E.; Wabo, H.K.; et al. 2020. Antileishmanial and cytotoxic activities of a new limonoid and a new phenyl alkene from the stem bark of Trichilia gilgiana (Meliaceae). Natural Product Research 34: 3182-3188.
• Milhomem-Paixão, S.S.; Fascineli, M.L.; Roll, M.M.; Longo, J.P.; Azevedo, R.B.; Pieczarka, J.C.; et al. 2016. The lipidome, genotoxicity, hematotoxicity and antioxidant properties of andiroba oil from the Brazilian Amazon. Genetics and Molecular Biology 39: 248-256.
• Minocha, N.; Kumar, D.; Rajanala, K.; Saha, S. 2011. Kinetoplast morphology and segregation pattern as a marker for cell cycle progression in Leishmania donovani The Journal of Eukaryotic Microbiology 58: 249-253.
• Odonne, G.; Houel, E.; Bourdy, G.; Stien, D. 2017. Treating leishmaniasis in Amazonia: A review of ethnomedicinal concepts and pharmaco-chemical analysis of traditional treatments to inspire modern phytotherapies. Journal of Ethnopharmacology 199: 211-230.
• Oliveira, I.S.S; Moragas Tellis, C.J.; Chagas, M.; Behrens, M.D.; Calabrese, K.d.S.; Abreu-Silva, A.L.; et al. 2018. Carapa guianensis Aublet (Andiroba) seed oil: Chemical composition and antileishmanial activity of limonoid-rich fractions. Biomed Research International 2018: 5032816.
• PAHO. 2023. Leishmaniasis: Epidemiological Report for the Americas. No.11 (December 2022) - PAHO/WHO | Pan American Health Organization. PAHO. ( (https://www.paho.org/en/documents/leishmaniasis-epidemiological-report-americas-no11-december-2022 ). Accessed on 13 Feb 2023.
  » https://www.paho.org/en/documents/leishmaniasis-epidemiological-report-americas-no11-december-2022
• Perea, A.; Manzano, J.I.; Castanys, S.; Gamarro, F. 2016. The LABCG2 Transporter from the protozoan parasite Leishmania is involved in antimony resistance. Antimicrobial Agents and Chemotherapy 60: 3489-3496.
• Rumpf, J.; Burger, R.; Schulze, M. 2023. Statistical evaluation of DPPH, ABTS, FRAP, and Folin-Ciocalteu assays to assess the antioxidant capacity of lignins. International Journal of Biological Macromolecules 233: 123470.
• Wang, Z.; Feng, Y.; Yang, N.; Jiang, T.; Xu, H.; Lei, H. 2022. Fermentation of kiwifruit juice from two cultivars by probiotic bacteria: Bioactive phenolics, antioxidant activities and flavor volatiles. Food Chemistry 37: 131455.
• Xiao, F.; Xu, T.; Lu, B.; Liu, R. 2020. Guidelines for antioxidant assays for food components. Food Frontiers 1: 60-69.
• Yang, R.; Song, C.; Chen, J.; Zhou, L.; Jiang, X.; Cao, X.; et al. 2020 Limonin ameliorates acetaminophen-induced hepatotoxicity by activating Nrf2 antioxidative pathway and inhibiting NF-κB inflammatory response via upregulating Sirt1. Phytomedicine: International Journal of Phytotherapy and Phytopharmacology 69: 153211.

• CITE AS:

  Almeida-Souza, F.; Oliveira, I.S.S.; Magalhães, I.F.B.; Taniwaki, N.N.; Calabrese, K.S.; Abreu-Silva, A.L. 2024. Antioxidant activity and ultrastructural alterations in Leishmania amazonensis promastigotes induced by limonoid-rich fractions from andiroba oil. Acta Amazonica 54: e54bc23113

Data availability

The data that support the findings of this study are available, upon reasonable request, from the corresponding author, Kátia da Silva Calabrese.

Edited by

ASSOCIATE EDITOR:

Jorge M. David

Publication Dates

• Publication in this collection
  08 Jan 2024
• Date of issue
  Jan-Mar 2024

History

• Received
  18 Apr 2023
• Accepted
  26 Oct 2023