In vitro Anti-Leishmanial Activities of Methanol Extract of Brucea antidysenterica J.F. Mill Seeds and Its Solvent Fractions

Abstract

Introduction

Leishmaniasis is one of the neglected tropical diseases, threatening lives of about 350 million people globally. Brucea antidysenterica seeds are used for the treatment of cutaneous leishmaniasis in the traditional medicine in Ethiopia.

Objective

This study aimed to evaluate Brucea antidysenterica seeds’ anti-leishmanial activity in vitro.

Methods

The crude (80% methanol) extract of Brucea antidysenterica seeds and its fractions were evaluated for their anti-leishmanial activities against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania aethiopica, and for their cytotoxic effects against mammalian cells. The quantitative estimations of total phenolic compounds (TPCs), flavonoids (TFCs) and alkaloids (TACs) were determined, spectrophotometrically. Median inhibitory concentration (IC₅₀) and median cytotoxic concentration (CC₅₀) of the extract and its solvent fractions were calculated using GraphPad Prism 9.1.0 computer software. Data was presented as mean ± standard error of the mean (SEM).

Results

The crude extract and its hexane, ethyl acetate and butanol fractions showed anti-leishmanial activities, with IC₅₀ values of 4.14–60.12 µg/mL against promastigotes, and 6.16–40.12 µg/mL against amastigotes of both Leishmania species. They showed moderate cytotoxicity against Vero cell lines and peritoneal mice macrophages, with CC₅₀ values of 100–500 µg/mL, but >1600 µg/mL against red blood cells. Selectivity indices ranged from 7.97 to 30.97. The crude extract, and its ethyl acetate and hexane fractions possessed 54.78–127.72 mg of gallic acid equivalent TPC, 18.30–79.21 mg of quercetin equivalent TFC, and 27.62–97.22 mg of atropine equivalent TAC per gram of extracts.

Conclusion

The seeds of the plant possessed anti-leishmanial activities against L. aethiopica and L. donovani that might provide a scientific justification for its use in the treatment of leishmaniasis by traditional healers. Future works are recommended to isolate, purify and identify the possible secondary metabolites attributed to the anti-leishmanial activity.

Keywords:

• anti-leishmanial activity
• Brucea antidysenterica seeds
• Leishmania aethiopica
• Leishmania donovani
• promastigote
• amastigote

Abbreviations

AMB, Amphotericin B; AE/g, Atropine equivalent per gram of extract/fraction; ATCC, American Type Culture Collection; CC50, Cytotoxic Concentrations that kill 50% of the cells; CL, Cutaneous leishmaniasis; DMSO, Dimethylsulfoxide; GAE/g, Gallic acid equivalent per gram of extract/fraction; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HINBCS, Heat Inactivated New Born Calf Serum; GAE/g, Gallic acid equivalent per gram of extract/fraction; IC50, Effective Concentration that inhibits 50% of the cells; LRDL, Leishmaniasis Research and Diagnostic Laboratory; M-199, Medium −199 with Earle’s salts; MEM, Minimum Essential Media; QE/g, Quercetin equivalent per gram of extract/fraction; RPMI, Roswell Park Memorial Institute; SI, Selectivity Index; TAC, Total alkaloidal content; TFC, Total flavonoid content; TPC, Total phenolic content; VL, Visceral leishmaniasis; WHO, World Health Organization.

Data Sharing Statement

Data supporting the results reported in the manuscript will be available upon request.

Acknowledgments

We are grateful to Dr. Daniel Gizaw and Dr. Dereje Shegu from National Animal Health Diagnosis and Investigation Center, Sebeta, Ethiopia for providing Vero cell lines. We also thank Mr. Dawit Araya and Mr. Mulugeta Gichile, the staff of Leishmania Research and Diagnostic Laboratory (LRDL) of Addis Ababa University, for all their support and assistance throughout the study period. This paper is based on a thesis by Mr. Tasisa Ketema. It has been published on the institutional website: http://etd.aau.edu.et/handle/123456789/.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no competing interests in this work.

Additional information

Funding

Center for Innovative Drug Development and Therapeutic Trials for Africa (CDT-Africa) and Addis Ababa University through its thematic research grant supported this research. The funders have no involvement in the study design or submission of the paper for publication.