bioactivity-guided isolation and structural identification of toxic cucurbitacin steroidal glucosides from stemodia kingii

A histologically validated murine model for the ovine intoxication by Stemodia kingii was used as a bioassay to guide the isolation of several groups of toxins from Stemodia kingii. Two of the toxins from one group were purified sufficiently to allow structural analysis and a determination of their median lethal doses (LD₅₀) for oral administration to mice. A combination of acid hydrolysis, elemental analysis, HPLC-MS, 1D-NMR (¹H, ¹³C) and 2D-NMR (¹H-¹H COSY, ¹³C-¹H HSQC and HMBC, and gNOESY) was used to define stemodiosides B3 and B4 as cucurbitacin steroidal glucosides. Thus stemodioside B3 is (24Z)-3α-(β-glucopyranosyloxy)-2β,20,27-trihydroxy-19-(10 to 9β)-abeo-10α-lanost-5,24-diene-11-one and stemodioside B4 is (23E)-3α-(β-glucopyranosyloxy)-2β,20,22,27-tetrahydroxy-19-(10 to 9β)-abeo-10α-lanost-5,23-diene-11-one. The approximate oral LD₅₀s for stemodiosides B3 and B4 in mice were estimated to be 99 and 42 mg/kg body weight, respectively.