Medicinal Chemistry Research https://doi.org/10.1007/s00044-023-03064-4 MEDICINAL CHEMISTR Y RESEARCH ORIGINAL RESEARCH Antiproliferative activity of secondary metabolites isolated from Humboldtia unijuga Bedd. Reshma Vijayakumari Raveendran Nair 1,2 ● Ajikumaran Nair Sadasivan Nair 1 ● Anil John Johnson 1 ● Ananthakrishnan Jayakumaran Nair 3 ● Sabulal Baby 1 Received: 25 February 2023 / Accepted: 14 April 2023 © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023 Abstract Humboldtia unijuga Bedd. (Fabaceae), a tree species endemic to southern region of Western Ghats in India, is least explored for its chemical constituents and biological activities. Phytochemical analysis of H. unijuga roots and stems resulted in the isolation of twenty-nine secondary metabolites with four new molecules, viz., methyl 3-(tetradecanoyloxy)olean-12-en-28- oate (3), lup-20(29)-en-3-yl 3-hydroxyheptadecanoate (26), methyl 3-(dodecanoyloxy)olean-12-en-28-oate (27), 28- oxoolean-12-en-3-yl myristate (28). Antiproliferative (cytotoxicity) activity of the twenty-nine isolated compounds was evaluated by MTT assay in five cancer cell lines, viz., C6, Hepa 1-6, RIN-5F, DLA and PC-12. These in vitro screening assays revealed promising anticancer activity of oleanolic acid-3-acetate (5) and (2S)-1-O-palmitoyl-2-O-(9Z-octadecenoyl)- 3-O-(6’-sulfo-α-D-quinovopyranosyl) glycerol (16). The mechanism of anticancer activity of these two molecules was elucidated by phase contrast and fluorescent microscopy, caspase 3 activity assay and flow cytometric cell cycle/apoptotic analyses. These compounds demonstrated anticancer activity on C6 and DLA cells by inducing cell specific apoptosis. Briefly, this study led to the isolation of twenty nine secondary metabolites, with four new ones, from H. unijuga, and two of the isolated molecules demonstrated promising anticancer activity. Graphical abstract * Sabulal Baby sabulal@gmail.com 1 Phytochemistry and Phytopharmacology Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Thiruvananthapuram, Kerala, India 2 University of Kerala, Thiruvananthapuram, Kerala, India 3 Department of Biotechnology, University of Kerala, Thiruvananthapuram, Kerala, India Supplementary information The online version contains supplementary material available at https://doi.org/10.1007/s00044- 023-03064-4. 1234567890();,: 1234567890();,: