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Three new withanolides including nicphysatone A (1), an unusual withanolide lactone possessing a disconnected C-13 to C-17 linkage within the steroidal nucleus; and nicphysatone B-C (2–3) were isolated from the persistent calyx of Nicandra physaloides. Compounds 1–3 showed no cytotoxic activity in these cell lines with IC50 > 40 μM.Three new withanolides were identified from the calyces of Nicandra physaloides.Nicphysatone A is a novel withanolide possessing a disconnected C-13 to C-17 linkage.Our findings could enrich the diversity of natural withanolides.Chemical investigation on ethyl acetate extract of the calyces of Nicandra physaloides resulted in the isolation of three new withanolides named as nicphysatone A (1), nicphysatone B (2), nicphysatone C (3), together with five known withanolides, nic 17 (4), nic 7 (5), nic 2 (6), withahisolide G (7) and nicaphysalin B (8). The structures were determined by comprehensive spectroscopic experiments. The discovery enriched the diversity of natural withanolides and could serve as scaffolds for the synthesis of more potent modified withanolides.