{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,17]],"date-time":"2026-07-17T04:57:46Z","timestamp":1784264266973,"version":"3.55.0"},"reference-count":45,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,2,20]],"date-time":"2026-02-20T00:00:00Z","timestamp":1771545600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Research Fund\u2014Kenya, in cooperation with the German Academic Exchange Service"},{"name":"Apothekerstiftung Westfalen-Lippe"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>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\u201320), and one artifact (3) that was presumably formed during the acid\u2013base 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 \u03bcmol\/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 \u03bcmol\/L, SI = 43). Our findings provide further promising antiprotozoal leads for HAT and Malaria.<\/jats:p>","DOI":"10.3390\/molecules31040733","type":"journal-article","created":{"date-parts":[[2026,2,20]],"date-time":"2026-02-20T13:08:23Z","timestamp":1771592903000},"page":"733","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Identification of Steroidal Alkaloids with In Vitro Antiprotozoal Activity from Holarrhena pubescens Wall. ex G. Don"],"prefix":"10.3390","volume":"31","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9638-3250","authenticated-orcid":false,"given":"Justus Wambua","family":"Mukavi","sequence":"first","affiliation":[{"name":"University of M\u00fcnster, Institute of Pharmaceutical Biology and Phytochemistry (IPBP), PharmaCampus, Corrensstra\u00dfe 48, D-48149 M\u00fcnster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monica","family":"Cal","sequence":"additional","affiliation":[{"name":"Swiss Tropical and Public Health Institute (Swiss TPH), Kreuzstrasse 2, CH-4123 Allschwil, Switzerland"},{"name":"University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1785-7302","authenticated-orcid":false,"given":"Marcel","family":"Kaiser","sequence":"additional","affiliation":[{"name":"Swiss Tropical and Public Health Institute (Swiss TPH), Kreuzstrasse 2, CH-4123 Allschwil, Switzerland"},{"name":"University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3122-1941","authenticated-orcid":false,"given":"Pascal","family":"M\u00e4ser","sequence":"additional","affiliation":[{"name":"Swiss Tropical and Public Health Institute (Swiss TPH), Kreuzstrasse 2, CH-4123 Allschwil, Switzerland"},{"name":"University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5171-1940","authenticated-orcid":false,"given":"Njogu M.","family":"Kimani","sequence":"additional","affiliation":[{"name":"Department of Physical Sciences, University of Embu, P.O. Box 6, Embu 60100, Kenya"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5821-8307","authenticated-orcid":false,"given":"Leonidah Kerubo","family":"Omosa","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Faculty of Science & Technology, University of Nairobi, P.O. Box 30197, Nairobi 00100, Kenya"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2634-9705","authenticated-orcid":false,"given":"Thomas J.","family":"Schmidt","sequence":"additional","affiliation":[{"name":"University of M\u00fcnster, Institute of Pharmaceutical Biology and Phytochemistry (IPBP), PharmaCampus, Corrensstra\u00dfe 48, D-48149 M\u00fcnster, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,20]]},"reference":[{"key":"ref_1","unstructured":"(2026, January 05). 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