The effect of the biflavonoid 2″,3″-dihydroochnaflavone on Trypanosoma cruzi Y strain

https://doi.org/10.1016/j.parint.2020.102180Get rights and content

Abstract

Trypanosoma cruzi is the causative agent of Chagas disease which affects 8 million people in Latin America. The parasite possesses high capacity to evade host immune system and the available drugs to treat Chagas disease present low efficacy combined to serious side effects to patients. Therefore, the identification of alternative therapeutics is essential. Brazilian flora exhibits an immense diversity of metabolites with great potential to be developed into new drugs. We investigated the action of 2″,3″-dihydroochnaflavone a biflavonoid extracted from Luxemburgia nobilis Eichler ex Engl. (Ochnaceae) against T. cruzi (Y strain). Our experiments showed that this compound is effective against parasite epimastigote forms, presenting IC50 value of (2.5 ± 0.1) μM after 96 h of treatment. Ultrastructure alterations were also detected in treated epimastigotes especially mitochondrial enlargement at the kinetoplast region. At the concentration of 30 μM, the compound killed (61.6 ± 3.37)% of the parasite in its amastigote form. In addition, at the same concentration, the compound killed all trypamastigotes growing within murine macrophages after 7–9 days of infection. Nonetheless, the biflavonoid concentrations were harmless to murine enriched population of lymphocytes and peritoneal macrophages. These results indicate that 2″,3″- dihydroochnaflavone presents activity against T. cruzi.

Introduction

Chagas disease is a potentially fatal infectious disease. It affects mainly poor people in tropical areas of the globe, being classified as a neglected disease according to the World Health Organization. It is endemic in 21 countries across Latin America [1]. There are around 70 million people at risk of infection. Approximately 6–7 million people are infected per year and 7.500 of them usually do not survive. The etiological agent is the flagellate protozoan Trypanosoma cruzi which is transmitted by hematophagous hemipteran insect vectors (Reduviidae family, Triatominae subfamily). Infective metacyclic trypomastigote parasite forms are released with the insect feces after a blood meal and enter the host bloodstream through the bite site. Nevertheless, other forms of transmission such as congenital, ingestion of contaminated food and blood transfusion have also been reported. Once in the mammal host bloodstream, the parasite is able to infect virtually any nucleated cell. Amastigote forms develop intracellularly where they pass through many cycles of replication and differentiate into blood trypomastigotes. These forms cause cell lysis and once released may infect other cells or be ingested by the insect vector during the blood meal. The disease is characterized by acute and chronic phases. Acute phase begins 6–10 days after infection and may last 4–8 weeks [2]. Patients at acute phase are either symptomless or present unspecific symptoms such as fever, malaise, swollen lymph nodes [3]. Around 15–30% of these patients develop a symptomatic chronic phase, which is characterized by inflammatory manifestations with cardiovascular and/or gastrointestinal tract dilatation. The resulting megacolon, megaesophagus and heart pathologies are quite debilitating [2].

Even after more than 100 years of the discovery of Chagas disease by Carlos Chagas [4] and despite medical and research advances, satisfactory treatments that lead to parasite complete elimination with minimal collateral effects are not available yet [5]. The persistent presence of the parasite in the host is directly associated to the characteristic inflammatory responses of the chronic phase [6]. Therefore, the identification of a drug that causes the elimination of the parasite is crucial for successful disease treatment [7]. In the sixties', the two drugs used in the routine treatment of Chagas disease were launched onto the market: nifurtimox [Lampit®, Bayer; (E)-N-(3-methyl-1,1-dioxo-1,4-thiazinan-4-yl)-1-(5-nitrofuran-2-yl)methanimine] and benznidazole [Rochagan®, Radanil®, Roche; N-benzil-2-(2-nitroimidazol-1-yl)acetamide]. Clinical studies showed that these two compounds presented good results at the acute phase of the disease. However, their efficacy varies according to the patient's geographic area of origin and consequently to T. cruzi strain [8]. Besides these routine drugs, only allopurinol and triazole were analyzed in clinical trials, but with no satisfactory results [9].

In the search for more efficient treatments, the usage of natural products with low toxicity to the patients, but effectively directed against T. cruzi infective forms presents great potential [[10], [11], [12], [13], [14], [15], [16]]. Plant species that belong to Ochnaceae family are abundant in Brazil being an important source of flavonoids. Among them, there is 2″,3″-dihydroochnaflavone (Fig. 1) which is extracted and isolated from leaves and branches of Luxemburgia nobilis Eichler ex Engl. (Ochnaceae) [17]. It was demonstrated that this biflavonoid presents dose-dependent toxicity to Ehrilch carcinoma lineage cells and K562 human immortalized myelogenous leukemia cell line. The mechanism of action of this compound is based in the inhibition of topoisomerases I and II α enzymes [18].

In this work, we investigated the effect of 2″,3″-dihydroochnaflavone on T. cruzi Y strain parasites as well as its cytotoxicity in host cells and consequently the putative trypanocidal action of this compound.

Section snippets

Ethics statement

All of the animal experiments were performed in strict accordance with the Brazilian animal protection law (Lei Arouca number 11.794/08) of the National Council for the Control of Animal Experimentation (CONCEA, Brazil). This protocol was approved by the Committee for Animal Use of the Institute of Veterinary of UFRRJ according to permit number 069/2014.

2″,3″-dihydroochnaflavone purification

To obtain pure 2″,3″-dihydroochnaflavone extracts, leaves of L. nobilis Eichler ex Engl. were harvested and processed as previously described

2″,3″-dihydroochnaflavone effect on the growth of T. cruzi epimastigote forms

Our results demonstrate that the treatment with the biflavonoid is efficient against T. cruzi epimastigote forms. Parasites growth inhibition caused by the compound at different concentrations was similar, around 5-fold in relation to the untreated control in a dose-independent manner (Fig. 2A). IC50 after 96 h of treatment was (2.5 ± 0.1) μM. Posteriorly, epimastigotes were incubated with 2″,3″-dihydroochnaflavone for 96 h at different concentrations, being subcultivated in fresh

Discussion

Nifurtimox and benznidazole are the current drugs utilized in the treatment of Chagas disease. These drugs heal solely patients at acute phase and do not lead to parasite load decrease as the infection advances towards chronic phase. Their efficacy also depends on the parasite strain besides causing severe side effects. Several natural products that present microbicide, antiprotozoal, antitumor and anti-HIV effects were identified in the last decades. Between 1981 and 2010, products of natural

Conclusions

In general, flavonoid effective actions against the different forms of T. cruzi depend directly on their chemical structures, evidencing structure-activity relation importance [21]. The use of 2″,3″-dihydroochnaflavone is reported in this work as promising alternative in the treatment of Chagas disease, once it caused high inhibition of epimastigotes growth, leading to considerable morphological alterations. Besides and most importantly the compound was extremely effective against amastigote

Declaration of competing interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Ackowledgments

To Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), for their financial support. To Dr. Celio Geraldo Freire de Lima for providing the LLMCK2 cells.

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