In vitro antileishmanial activity of Anacardium othonianum and isolated compounds against Leishmania amazonensis

This study analyzes the antileishmanial activity of the crude ethanol extract, fractions, and isolated compounds of A. othonianum nuts. Antileishmanial activity was evaluated against Leishmania amazonensis promastigotes in vitro. The phytochemical study was performed by high-performance liquid chromatography-high-resolution mass spectrometry-diode array detector (HPLC-HRMS-DAD) and by preparative HPLC. HPLC-HRMS-DAD analysis of the bioactive extract confirmed the presence of ten alkyl phenol derivatives that had previously been isolated from A. occidentale. Bioassay-guided isolation afforded cardanol triene, cardanol diene, cardanol monoene, cardol triene, anacardic acid triene, anacardic acid diene, and anacardic acid monoene. Cardol triene gave an IC50 of 80.66 μM. The obtained data suggest that the evaluated extract, fractions, and cardol triene had moderate activity against L. amazonensis promastigotes. This is the first description of alkyl phenols in A. othonianum.

The crude ethanol extract of A. othonianum leaves presents antifungal activity. In addition, the isolated compounds amentoflavone, gallic acid, protocatechuic acid, and ethyl 3,4,5-trimethoxybenzoate have been isolated and i also shown to display antifungal activity (Curado et al., 2016). Nevertheless, the crude extract obtained from A. othonianum nuts has not yet been investigated.
Protozoan parasites of the genus Leishmania are the etiological agent of the disease leishmaniasis, which affects the skin, mucous membranes, and internal organs. Between 700,000 and 1 million new cases of leishmaniasis are estimated to emerge annually (WHO, 2020). This disease affects poor regions in Africa, Asia, and Latin America (WHO, 2020). The treatment of leishmaniasis consists of the use of antimonials, amphotericin B, pentamidine, paromomycin, and miltefosine. However, these drugs are costly, difficult to administer, and highly toxic, not to mention the emerging resistance to them (Ghorbani & Farhoudi, 2018;Hendrickx, Caljon & Maes, 2019;Charlton, Rossi-Bergmann, Denny & Steel, 2018).
The Nuclear Magnetic Resonance data of the compounds dissolved in CDCl3 (Sigma-Aldrich) were acquired on Bruker Avance DRX 400 and 500 spectrometers (Billerica, MA, USA). The HPLC-HRMS-DAD data of the crude ethanol extract of cashew nuts were obtained on an LC-20AD HPLC (Shimadzu, Kyoto, Japan) and micrOTOF-QII ESI mass spectrometer (Bruker Daltonics, Billerica, MA, USA). The same parameters described by Bertanha et al. (2020) were applied. An ODS column (Phenomenex) was employed. The mobile phase consisted of 70:30 acetonitrile/water (+ 0.2% formic acid) for 60 min, followed by a linear gradient from 70 to 100% acetonitrile for 10 min, and 100% acetonitrile for 10 min. The flow rate was 1.0 mL/min.
The fruits of species Anacardium othonianum Riss. (Anacardiaceae) were provided by Prof. Dr. Fabiano Silva. A voucher specimen (HJ3793) was deposited in the Herbarium Jataiense Germano Guarim Neto of the Federal University of Goiás, Brazil (Herbarium HJ). The license to access genetic heritage was SisGen #AC7C911.
The CEE chromatogram was obtained between 200 and 800 nm, and in the negative mode. High-resolution mass spectra provided the exact mass for the obtained peaks and was employed to calculate the molecular formulae and errors ( Table 1). The molecular formulae already described in the literature (Correia, David & David, 2006) for compounds from the genus Anacardium were compared with the molecular formulae obtained in this study. Thus, CEE analysis by HPLC-HRMS-DAD revealed the presence of ten known alkyl phenols, namely cardol triene, cardol diene, anacardic acid triene, cardanol triene, anacardic acid diene, cardanol diene, anacardic acid monoene, cardanol monoene, anacardic acid, and cardanol. This indicates that A. othonianum is also a rich source of these compounds. Cardanol -0.44 n.i. not identified FR1 had higher yield and was purified by various chromatographic purification steps to give the main compounds ( Figure 1): cardanol triene (1), cardanol diene (2), cardanol monoene (3), cardol triene (4), anacardic acid triene (5), anacardic acid diene (6), and anacardic acid monoene (7). These compounds were identified by NMR and by comparison with previously published data (Alvarenga et al., 2016;Lomonaco et al., 2012).
Compound 4 displayed leishmanicidal activity. Its IC50 was 80.66 µM (95% confidence interval [CI], 80.44 to 80.87 µM). Compounds 1-3 and 5-7 provided IC50 > 100 µM. These data suggest that the presence of an extra hydroxyl group at the aromatic ring as well as the absence of the acid group improved the activity of compound 4 in relation to compounds 1 and 5, which also presented three double bonds at the C15 carbon chain.