In vitro serotonergic activity of black cohosh and identification of N(omega)-methylserotonin as a potential active constituent

Abstract

Cimicifuga racemosa (L.) Nutt. (syn. Actaea racemosa L., black cohosh) is used to relieve menopausal hot flashes, although clinical studies have provided conflicting data, and the active constituent(s) and mechanism(s) of action remain unknown. Because serotonergic receptors and transporters are involved with thermoregulation, black cohosh and its phytoconstituents were evaluated for serotonergic activity using 5-HT7 receptor binding, cAMP induction, and serotonin selective re-uptake inhibitor (SSRI) assays. Crude extracts displayed 5-HT7 receptor binding activity and induced cAMP production. Fractionation of the methanol extract led to isolation of phenolic acids and identification of N(omega)-methylserotonin by LC-MS/MS. Cimicifuga triterpenoids and phenolic acids bound weakly to the 5-HT7 receptor with no cAMP or SSRI activity. In contrast, N(omega)-methylserotonin showed 5-HT7 receptor binding (IC50 = 23 pM), induced cAMP (EC50 = 22 nM), and blocked serotonin re-uptake (IC50 = 490 nM). These data suggest N(omega)-methylserotonin may be responsible for the serotonergic activity of black cohosh.

Figure 1

Key phytoconstituents of black cohosh.

Figure 2. Bioassay guided fractionation scheme

The isolation scheme following the fractionation and subfractionation of the crude MeOH extract. The clinical extract (75% EtOH) had an IC₅₀ of 55 μg/mL. The structures of the most relevant triterpenes, phenolic acids, and N_ω-methylserotonin are shown in Figure 1.

Figure 3. Black cohosh induces intracellular cAMP

Dose-dependent induction of cyclic AMP (pmol/mL) for the clinical black cohosh extract (■) and XAD-MeOH extract, EC₅₀ 70 μg/mL (□) mediated through the 5-HT₇ receptor.

Figure 4. Positive ion electrospray LC-MS analysis and tandem mass spectra of fraction 7 and N_ω-methylserotonin

Total ion chromatograms of A) N_ω-methylserotonin standard. B) Fraction 7. A comparison of the product ion tandem mass spectra of N_ω-methylserotonin and a compound from fraction 7 eluting at 22 min. The major fragment ion corresponds to the loss of methylamine from the side chain. C) N_ω-methylserotonin standard. D) Black cohosh fraction 7.

Figure 5. N_ω-methylserotonin can bind to the 5-HT₇ receptor

Percent inhibition of 5-HT₇ receptor binding with increasing doses of N_ω-methylserotonin. IC₅₀ 23 pM.

Figure 6. N_ω-methylserotonin induces intracellular cAMP

(A) Cyclic AMP intracellular levels of human 5-HT₇ transfected HEK293 cells after treatment with N_ω-methylserotonin (100 nM). (B) Intracellular cyclic AMP (pmol/mL) as a response of increasing dosage from N_ω-methylserotonin mediated through the 5-HT₇ receptor. *p < 0.05.

Figure 7. N_ω-methylserotonin exhibits SSRI activity

Dose-dependent percent inhibition of selective serotonin reuptake with N_ω-methylserotonin. The IC₅₀ value was determined to be 490 nM.