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

## TL;DR

This study identifies new steroidal alkaloids from a tropical tree that show strong antiprotozoal activity against diseases like malaria and African trypanosomiasis.

## Contribution

A new steroidal alkaloid with potent antiplasmodial activity and high selectivity was isolated and characterized.

## Key findings

- Compounds 2 and 16 showed the highest antitrypanosomal activity with IC50 of 0.2 μmol/L.
- New compound 5 exhibited the strongest antiplasmodial activity with IC50 of 0.7 μmol/L and SI of 43.
- PLS regression modeling helped identify compounds responsible for antiplasmodial activity.

## 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 Holarrhena pubescens (Apocynaceae). H. pubescens 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 Trypanosoma brucei rhodesiense (Tbr) and Plasmodium falciparum (Pf). 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 (5), along with 18 known compounds (1, 2, 4, 6–20), and one artifact (3) 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 2 and 16 (IC50 = 0.2 μmol/L) demonstrated the highest antitrypanosomal activity, with compound 2 showing the highest selectivity (SI = 127). The new compound 5 exhibited the strongest antiplasmodial activity and selectivity against Pf (IC50 = 0.7 μmol/L, SI = 43). Our findings provide further promising antiprotozoal leads for HAT and Malaria.