Identification of Steroidal Alkaloids with In Vitro Antiprotozoal Activity from Holarrhena pubescens Wall. ex G. Don

Abstract

Human African Trypanosomiasis (HAT) and Malaria are serious infectious diseases endemic in tropical regions, caused by protozoan parasites, and necessitating an urgent development of new antiprotozoal drugs. As part of our ongoing search for new antiprotozoal steroidal alkaloids from plants, we investigated the methanolic stem bark extract of <i>Holarrhena pubescens</i> (Apocynaceae). <i>H. pubescens</i> is a tropical tree that some Kenyan coastal communities have long used to treat various ailments, including fever and stomach pain. The crude extract, alkaloid fraction, and 16 subfractions acquired through centrifugal partition chromatography (CPC) displayed promising in vitro antiprotozoal activity against <i>Trypanosoma brucei rhodesiense</i> (<i>Tbr</i>) and <i>Plasmodium falciparum</i> (<i>Pf</i>). Partial least squares (PLS) regression modeling of UHPLC/+ESI QqTOF-MS data and the antiprotozoal activity data of the crude extract and its fractions was performed to predict compounds that may be responsible for the observed antiplasmodial activity. Chromatographic separation of the alkaloid fraction afforded one new steroidal alkaloid (<b>5</b>), along with 18 known compounds (<b>1</b>, <b>2</b>, <b>4</b>, <b>6</b>-<b>20</b>), and one artifact (<b>3</b>) that was presumably formed during the acid-base extraction process. The structural characterization of the isolated compounds was accomplished using UHPLC/+ESI-QqTOF-MS/MS and NMR spectroscopy. The isolated compounds were tested for their in vitro antiprotozoal properties against the two aforementioned pathogens, as well as for their cytotoxicity against mammalian cells (L6 cell line). Compounds <b>2</b> and <b>16</b> (IC₅₀ = 0.2 μmol/L) demonstrated the highest antitrypanosomal activity, with compound <b>2</b> showing the highest selectivity (SI = 127). The new compound <b>5</b> exhibited the strongest antiplasmodial activity and selectivity against <i>Pf</i> (IC₅₀ = 0.7 μmol/L, SI = 43). Our findings provide further promising antiprotozoal leads for HAT and Malaria.