Conclusion
This study shows that CAs are not responsible for serotonergic activity in black cohosh. New phytochemical methodology (pHZR CPC) and a sensitive dereplication method (LC-MS) led to the identification of N(omega)-methylserotonin as serotonergic active principle.
Objective
Re-isolation of the CAs was desired to permit further detailed studies. Based on the acid/base association hypothesis, a new separation scheme of the active partition was required, which separates acids from associated bases. Methodology: A new 5-HT(7) bioassay guided work-up procedure was developed that concentrates activity into one partition. The latter was subjected to a new two-step centrifugal partitioning chromatography (CPC) method, which applies pH zone refinement gradient (pHZR CPC) to dissociate the acid/base complexes. The resulting CA fraction was subjected to a second CPC step. Fractions and compounds were monitored by (1)H NMR using a structure-based spin-pattern analysis facilitating dereplication of the known acids. Bioassay
Results
A new CA was characterised. While none of the pure CAs was active, the serotonergic activity was concentrated in a single pHZR CPC fraction, which was subsequently shown to contain low levels of the potent 5-HT(7) ligand, N(omega)-methylserotonin.