From 5,000 crude plant and fungi extracts screening to leishmanicidal compounds identification: The challenge goes on

Leishmaniasis are diseases caused by protozoa parasites of the Leishmania genus. About 0.7 million to 1.3 million new cases occur worldwide annually of cutaneous leishmaniasis [1]. Furthermore, the number of cases is increasing globally at an alarming rate. The need for alternative treatments for leishmaniasis has been stimulating bioprospecting programs. Thus, we screened more than 5,000 thousands plant and fungi ethanol extracts (EX) at 20 µg/ml using the amastigote-like forms (ALF) of Leishmania (leishmania) amazonensis (IFLA/BR/67/PH8 strain) [2]. The active extracts were assayed for cytotoxic activity against macrophages (Mθ). Those without cytotoxicity were tested on Mθ infected with the amastigote form of L. amazonensis. Twenty-seven extracts passed the screen and were selected for chemical fractionation aiming at the identification of the leishmanicidal compounds. We hereby describe the investigation of two of them: Baccharis platypoda (B) and Cyrtocymura scorpioides (C), from the Asteraceae family. Both EX from the leaves were subjected to HSCCC. From B, 241 fractions were collected and grouped on 21 fractions according to TLC. These fractions were evaluated against the intracellular parasites. One of the fractions killed 100% of the amastigotes forms at 50 µg/mL and was fractionated (RP-HPLC) to afford a white amorphous powder. This compound killed 72% of the amastigotes at 50 µg/mL (155µM). Analysis of MS and NMR data allowed us to identify a new clerodane diterpene: (1R,3R,4R,8aR)-3-(hydroxymethyl)-4-[(E)-5-hydroxy-3-methyl-pent-3-enyl]-4,8,8a-trimethyl-1,2,3,4a,5,6-hexahydronaphthalen-1-ol. From C, 260 fractions afforded 24 groups after TLC analysis. Group 2 (20 µg/mL) killed 89% of the ALF. RP-HPLC separation afforded chlorogenic acid (CAc) which killed 70% of the ALF (56µM, 20 µg/mL). Amphotericin B was used as control. LC-MS/MS of the other six active fractions indicated the presence of rutin, isoquercitrin, quercitrin.

Financial support: CNPq/FAPEMIG.

Keywords: Leishmania amazonensis, Asteraceae, diterpene, chlorogenic acid, flavonoids

References:

[1] WHO, 2014 http://www.who.int/mediacentre/factsheets/fs375/en/accessed: May 2014

[2] Teixeira MCA, et AL. (2002). A simple and reproducible method to obtain large numbers of axenic amastigotes of different Leishmania species. Parasitology Research 88: 963 – 968.