To explore novel lead compounds for botanical pesticides from natural sources, a new pregnane glycoside (periplocoside) P2 as well as its isomer, a known pregnane glycoside P1, were isolated from the root barks of Periploca sepium using a bioactivity-guided method, and their structures were confirmed by ID NMR, 2D NMR, IR, ESI-MSn and HRMS. Their insecticidal activities were evaluated against 3rd instar larvae of M separata, and the results indicated that P2 exhibited excellent insecticidal activity with LC₅₀ values of 2.9 and 2.2 mg/mL after 24 and 48 h, respectively, much lower than those of the positive control celangulin-V (24.7 and 21.0 mg/mL after 24 and 48 h, respectively). This work demonstrated that pregnane glycosides from Periploca sepium could be promising lead compounds for developing botanical pesticides urgently needed in agriculture.

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