Abstract: Because of the serious spectral overlap in several regions of the ¹Hspectrum, the 1Hchemical shifts of an ocotillol-type saponin, which was first isolated from the leaves of panax Quinquetolium L., are completely assigned by recently developed two dimensional homonuclear Hartmann-Hahn spectroscopy (2D-HOHAHa) and rotating frame nuclear Overhauser effect spectroscopy (ROESY).The 2D-HOHAHAexperiment is able to obtain the phase sensitive two dimensional spectrum with all peaks in the near absorption mode, and the cross peaks multiplets are nearly in-phase.So the high detection resolution and sensitivity of 2D-HOHAHAexperiment make it possible to observe the cross peaks overlapped such as H13-H17 and H23a-H23e, and that covered by diagonal peaks with high strength in COSYspectrum, such as H2a-H2e.On the other hand, during the spin locking period of 2D-HOHAHAexperiment, relayed magnetization transfer will occurs.For shorter mixing period (shorter than 20 ms) little RELAY signals can be observed, but for longer mixing times single-RELAY and multiple-RELAY effects become significant.Through the RELAY cross peaks appeared in 2D-HOHAHA spectrum of OTS, the accurate ¹Hchemical shifts of some proton signals in severe spectral overlap regions are assigned, such as, the ¹Hchemical shifts of H22a, H22e, Hla, H1e and H9 proton are 1.592, 1.857, 1.026, 1.640 and 1.586 ppm respectively.The spin-locking NOEexperiment is used to provide through-space connectivity between the various coupling networks of protons.The linking position of glucosyl with aglycone in OTSand the chemical shift assignments of methyl protons which are easily distinguished but difficultly assigned are determined by ROESYspectrum.

Key words: 2D-HOHAHA, ROESY, Spectral assignments, Ocotillol-type saponin