{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,6]],"date-time":"2026-03-06T12:05:27Z","timestamp":1772798727722,"version":"3.50.1"},"reference-count":52,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2025,2,27]],"date-time":"2025-02-27T00:00:00Z","timestamp":1740614400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Apothekerstiftung Westfalen-Lippe"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Trypanosoma brucei rhodesiense (Tbr) and Plasmodium falciparum (Pf) are protozoan parasites that cause severe diseases, namely, Human African Trypanosomiasis (HAT) and Malaria. Due to limited treatment options, there is an urgent need for new antiprotozoal drugs. Pachysandra terminalis (P. terminalis), a plant belonging to the family Buxaceae, is known as a rich source of aminosteroid alkaloids, and a previous study of our working group already showed that the alkaloid-enriched fraction of P. terminalis aerial parts showed promising activity against protozoan parasites. In the present study, the alkaloid-enriched fraction obtained from a 75% ethanol extract of aerial parts was separated to isolate a chemically diverse array of Pachysandra alkaloids for assessment of their antiprotozoal activity and later structure\u2013activity studies. This work yielded a new megastigmane alkaloid (1), 7 new aminosteroids (2, 7, 16, 17, 18, 19, 20), along with 10 known aminosteroids (3\u20135, 8, 10\u201315) and 2 artifacts (6, 9) that were formed during the isolation process. The structures were elucidated by UHPLC\/+ESI-QqTOF-MS\/MS, as well as extensive 1- and 2D-NMR measurements. The extract and its fractions, as well as the isolated compounds, were tested in vitro against Tbr and Pf, as well as cytotoxicity against mammalian cells (L6 cell line). The activity (IC50 values) of the isolated alkaloids ranged between 0.11 and 26 \u00b5M (Tbr) and 0.39 and 80 \u00b5M (Pf). 3\u03b1,4\u03b1-diapachysanaximine A (7) showed the highest activity against Tbr (IC50 = 0.11 \u00b5M) with a selectivity index (SI) of 133 and was also quite active against Pf with IC50 = 0.63 \u00b5M (SI = 23). This compound is, therefore, a promising new antiprotozoal target for further investigations.<\/jats:p>","DOI":"10.3390\/molecules30051093","type":"journal-article","created":{"date-parts":[[2025,2,28]],"date-time":"2025-02-28T02:48:37Z","timestamp":1740710917000},"page":"1093","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Antiprotozoal Aminosteroids from Pachysandra terminalis"],"prefix":"10.3390","volume":"30","author":[{"given":"Lizanne","family":"Sch\u00e4fer","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":[{"role":"author","vocabulary":"crossref"}]},{"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":[{"role":"author","vocabulary":"crossref"}]},{"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":[{"role":"author","vocabulary":"crossref"}]},{"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":[{"role":"author","vocabulary":"crossref"}]},{"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":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,2,27]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2023). World Malaria Report 2023, World Health Organization."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1038\/nrmicro.2017.161","article-title":"Drug resistance in Plasmodium","volume":"16","author":"Haldar","year":"2018","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Torreele, E., Bourdin Trunz, B., Tweats, D., Kaiser, M., Brun, R., Mazu\u00e9, G., Bray, M.A., and P\u00e9coul, B. (2010). Fexinidazole\u2014A New Oral Nitroimidazole Drug Candidate Entering Clinical Development for the Treatment of Sleeping Sickness. PLoS Negl. Trop. Dis., 4.","DOI":"10.1371\/journal.pntd.0000923"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1007\/s40262-014-0136-3","article-title":"Determination of an Optimal Dosing Regimen of Fexinidazole, a Novel Oral Drug for the Treatment of Human African Trypanosomiasis: First-in-Human Studies","volume":"53","author":"Tarral","year":"2014","journal-title":"Clin. Pharmocokinet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2128","DOI":"10.2174\/092986712800229023","article-title":"The potential of secondary metabolites from plants as drugs or leads against protozoen neglected diseases\u2014Part I","volume":"19","author":"Schmidt","year":"2012","journal-title":"Curr. Med. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2176","DOI":"10.2174\/092986712800229087","article-title":"The potential of secondary metabolites from plants as drugs or leads against protozoen neglected diseases\u2014Part II","volume":"19","author":"Schmidt","year":"2012","journal-title":"Curr. Med. Chem."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Nnadi, C.O., Nwodo, N.J., Kaiser, M., Brun, R., and Schmidt, T.J. (2017). Steroid Alkaloids from Holarrhena africana with strong activity against Trypanosoma brucei rhodesiense. Molecules, 22.","DOI":"10.1055\/s-0037-1607175"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Nnadi, C.O., Ebiloma, G.U., Black, J.A., Nwodo, N.J., Lemgruber, L., Schmidt, T.J., and de Koning, H.P. (2019). Potent Antitrypanosomal Activities of 3-Aminosteroids against African Trypanosomes: Investigation of Cellular Effects and of Cross-Resistance with Existing Drugs. Molecules, 24.","DOI":"10.3390\/molecules24020268"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"6184","DOI":"10.3390\/molecules19056184","article-title":"Antiprotozoal Activity of Buxus sempervirens and Activity-Guided Isolation of O-tigloylcyclovirobuxeine-B as the Main Constituent Active against Plasmodium falciparum","volume":"19","author":"Althaus","year":"2014","journal-title":"Molecules"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Szab\u00f3, L.U., Kaiser, M., M\u00e4ser, P., and Schmidt, T.J. (2021). Antiprotozoal Nor-Triterpene Alkaloids from Buxus sempervirens L. Antibiotics, 10.","DOI":"10.3390\/antibiotics10060696"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"M\u00fcller, F., Ritz, C.M., Welk, E., and Wesche, K. (2021). Rothmaler Exkursionsflora von Deutschland, Springer Spektrum. [22nd ed.].","DOI":"10.1007\/978-3-662-61011-4"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1381","DOI":"10.1016\/S0040-4039(00)90484-2","article-title":"Steroidal alkaloids of Pachysandra terminalis (Buxaceae)","volume":"5","author":"Knaack","year":"1964","journal-title":"Tetrahedron Lett."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1053","DOI":"10.1016\/S0040-4039(00)90429-5","article-title":"Studies on the alkaloids of Pachysandra terminalis Sieb. et Zucc.: Structure of Pachysandrine A and B","volume":"5","author":"Tomita","year":"1964","journal-title":"Tetrahedron Lett."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1817","DOI":"10.1016\/S0040-4039(01)89493-4","article-title":"Alkaloids of Pachysandra terminalis. III. Sytematic separation and characterization of alkaloids","volume":"27\u201328","author":"Kikuchi","year":"1964","journal-title":"Tetrahedron Lett."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Xiang, M., Hu, B., Qi, Z., Wang, X., Xie, T., Wang, Z., Ma, D., Zeng, Q., and Luo, X. (2022). Chemistry and bioactivities of natural steroidal alkaloids. Nat. Prod. Bioprospecting, 12.","DOI":"10.1007\/s13659-022-00345-0"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1305","DOI":"10.1021\/np300207c","article-title":"Alkaloids from Pachysandra terminalis Inhibit Breast Cancer Invasion and Have Potential for Development As Antimetastasis Therapeutic Agents","volume":"75","author":"Zhai","year":"2012","journal-title":"J. Nat. Prod."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.phytol.2021.07.014","article-title":"Pregnane alkaloids with BRD4 inhibitory and cytotoxic activities from Pachysandra terminalis","volume":"45","author":"Sun","year":"2021","journal-title":"Phytochem. Lett."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Li, X.-Y., Yu, Y., Jia, M., Jin, M.-N., Qin, N., Zhao, C., Duan, H.-Q., and Terminamines, K.-S. (2016). Antimetastatic Pregnane Alkaloids from the Whole Herb of Pachysandra terminalis. Molecules, 21.","DOI":"10.3390\/molecules21101283"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Flittner, D., Kaiser, M., M\u00e4ser, P., Lopes, N.P., and Schmidt, T.J. (2021). The Alkaloid-Enriched Fraction of Pachysandra terminalis (Buxaceae) Shows Prominent Activity against Trypanosoma brucei rhodesiense. Molecules, 26.","DOI":"10.3390\/molecules26030591"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1016\/j.pt.2011.01.004","article-title":"Malaria and trypanosomes transmission: Different parasites, same rules?","volume":"27","author":"Pollitt","year":"2011","journal-title":"Trends Parasitol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1016\/S0040-4039(00)89929-3","article-title":"Steroid alkaloids of Saracococa pruniformis lindl","volume":"6","author":"Chatterjee","year":"1965","journal-title":"Tetrahedron Lett."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1002\/hlca.200490042","article-title":"New Cholinesterase-Inhibiting Steroidal Alkaloids from Sarcococca saligna","volume":"87","author":"Feroz","year":"2004","journal-title":"Helv. Chim. Acta"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1248\/yakushi1947.87.3_215","article-title":"Pachysandra Alkaloids. I. Systematic isolation and characterization of alkaloids","volume":"87","author":"Tomita","year":"1967","journal-title":"Yakugaku Zasshi"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1993","DOI":"10.1016\/S0040-4039(01)83899-5","article-title":"Alkaloids of Pachysandra terminalis. IV. Structure of epipachysamine B, epipachysamine C and terminaline","volume":"24","author":"Kikuchi","year":"1965","journal-title":"Tetrahedron Lett."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Zhang, Y., Li, Y., Xu, Y., Fan, H., Huang, W., Zhang, H., Wang, W., and Song, X. (2020). A new C21 steroidal compound from the whole herb of Pachysandra terminalis Sieb. et Zucc. Biochem. Syst. Ecol., 91.","DOI":"10.1016\/j.bse.2020.104056"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1641","DOI":"10.1016\/0040-4039(64)83107-5","article-title":"Studies on the alkaloids of Pachysandra terminalis Sieb. et Zucc. (2).: Structure of pachysamine -A and -B","volume":"25","author":"Tomita","year":"1964","journal-title":"Tetrahedron Lett."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1248\/cpb.15.207","article-title":"Pachysandra alkaloids. III. Structures of pachysandrine-B, -C and -D","volume":"15","author":"Kikuchi","year":"1967","journal-title":"Chem. Pharm. Bull."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"295","DOI":"10.1016\/j.steroids.2004.11.007","article-title":"Cholinesterase inhibitory pregnane-type steroidal alkaloids from Sarcococca hookeriana","volume":"4","author":"Choudhary","year":"2005","journal-title":"Steroids"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1248\/cpb.15.307","article-title":"Pachysandra Alkaloids. V. Structure of Epipachysamine-A, -B, -C, -D, -E and -F","volume":"15","author":"Kikuchi","year":"1967","journal-title":"Chem. Pharm. Bull."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"416","DOI":"10.1248\/cpb.23.416","article-title":"Pachysandra Alkaloids. XIII. Structure and stereochemistry of spiropachysine, a novel spiro-lactam alkaloid","volume":"23","author":"Kikuchi","year":"1975","journal-title":"Chem. Pharm. Bull."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1016\/j.steroids.2011.11.001","article-title":"Rapid characterization and identification of steroidal alkaloids in Sarcococca coriacea using liquid chromatography coupled with electrospray ionization quadropole time-of-flight mass spectrometry","volume":"77","author":"Musharraf","year":"2012","journal-title":"Steroids"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1007\/s10600-015-1267-y","article-title":"A new megastigmane alkaloid from Pachysandra terminalis with antitumor metastasis effect","volume":"51","author":"Jin","year":"2015","journal-title":"Chem. Nat. Compd."},{"key":"ref_33","first-page":"626","article-title":"Three new steroidal alkaloids from Pachysandra axillaris","volume":"32","author":"Qui","year":"1990","journal-title":"Zhiwu Xuebao"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/j.phytol.2015.10.009","article-title":"New immunomodulatory steroidal alkaloids from Sarcococa saligna","volume":"14","author":"Iqbal","year":"2015","journal-title":"Phytochem. Lett."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1022","DOI":"10.1055\/s-0029-1240896","article-title":"Bioactive Steroidal Alkaloids from Sarcococca hookeriana","volume":"76","author":"Devkota","year":"2010","journal-title":"Planta Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"818","DOI":"10.1016\/j.steroids.2010.05.005","article-title":"Pregnane alkaloids from Pachysandra axillaris","volume":"75","author":"Sun","year":"2010","journal-title":"Steroids"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Hesse, M., Meier, H., and Zeeh, B. (2005). Spektroskopische Methoden in der organischen Chemie, Thieme.","DOI":"10.1055\/b-002-46985"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1833","DOI":"10.1016\/S0040-4039(97)00163-9","article-title":"Isolation of the first natural 6-hydroximino-4-en-3-one-steroids from the sponges Cinachyrella spp","volume":"38","author":"Peixinho","year":"1997","journal-title":"Tetrahedron Lett."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Latscha, H.P., Kazmaier, U., and Klein, H. (2023). Organische Chemie\u2014Chemie-Basiswissen II, Springer Spektrum.","DOI":"10.1007\/978-3-662-67010-1"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"4292","DOI":"10.1021\/jo100276m","article-title":"Reaction of Dichlormethane with Pyridine Derivatives under Ambient Conditions","volume":"75","author":"Rudine","year":"2010","journal-title":"J. Org. Chem."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Nnadi, C.O., Althaus, J.B., Nwodo, N.J., and Schmidt, T.J. (2018). 3D-QSAR Study on the Antitrypanosomal and Cytotoxic Activities of Steroid Alkaloids by Comparative Molecular Field Analysis. Molecules, 23.","DOI":"10.20944\/preprints201804.0282.v1"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Szab\u00f3, L., and Schmidt, T.J. (2020). Target-Guided Isolation of O-tigloylcyclovirobuxeine-B from Buxus sempervirens L. by Centrifugal Partition Chromatography. Molecules, 25.","DOI":"10.3390\/molecules25204804"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"906","DOI":"10.1046\/j.1365-3156.2001.00775.x","article-title":"The phenomenon of treatment failures in human African trypanosomiasis","volume":"6","author":"Brun","year":"2001","journal-title":"Trop. Med. Int. Health"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1016\/0001-706X(93)90083-N","article-title":"A comparison of three methods of estimating EC50 in studies of drug resistance of malaria parasites","volume":"55","author":"Huber","year":"1993","journal-title":"Acta Trop."},{"key":"ref_45","first-page":"50","article-title":"Chloroquine sensitivity of isolates of Plasmodium falciparum adapted to in vitro culture","volume":"33","author":"Ponnudurai","year":"1981","journal-title":"Trop. Geogr. Med."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"710","DOI":"10.1128\/AAC.16.6.710","article-title":"Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique","volume":"16","author":"Desjardins","year":"1979","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_47","unstructured":"Lefkovits, I., and Pernis, B. (1990). Plasmodium falciparum Malaria Parasite Cultures and Their Use in Immunology, Academic Press. Immunological Methods."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"269","DOI":"10.1038\/374269a0","article-title":"Malarial haemozoin\/beta-haematin supports haem polymerization in the absence of protein","volume":"374","author":"Dorn","year":"1995","journal-title":"Nature"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"673","DOI":"10.1126\/science.781840","article-title":"Human malaria parasites in continuous culture","volume":"193","author":"Trager","year":"1976","journal-title":"Science"},{"key":"ref_50","unstructured":"(2025, February 11). American Type Culture Collection. Available online: https:\/\/www.atcc.org\/products\/crl-1458?matchtype=&network=x&device=c&adposition=&keyword=&gad_source=1&gclid=Cj0KCQiAqL28BhCrARIsACYJvkc4HaNakUs4XdDO-iagFhY7g-WUWkR3dcXPQQJmUXVK2-SkbQY31t4aAogjEALw_wcB."},{"key":"ref_51","first-page":"1317","article-title":"Antiprotozoal activity of alkaloid fractions and isolation of a new megastigmane alkaloid from leaves of Pachysandra terminalis. In Proceedings of the 71st International Congress of the Society for Medicinal Plant and Natural Product Research (GA), Dublin, Ireland, 2\u20135 July 2023","volume":"89","author":"Kaiser","year":"2023","journal-title":"Planta Med."},{"key":"ref_52","unstructured":"Sch\u00e4fer, L., Kaiser, M., M\u00e4ser, P., and Schmidt, T.J. (2024, January 13\u201317). Isolation, structural characterization and antiprotozoal activity of steroidal alkaloids from Pachysandra terminalis. Proceedings of the International Congress on Natural Products Research (ICNPR), Krakow, Poland. Available online: https:\/\/www.icnpr2024.org\/AbstractBook."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/30\/5\/1093\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T16:43:57Z","timestamp":1760028237000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/30\/5\/1093"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,27]]},"references-count":52,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2025,3]]}},"alternative-id":["molecules30051093"],"URL":"https:\/\/doi.org\/10.3390\/molecules30051093","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,2,27]]}}}