On the Biosynthesis of Bioactive Tryptamines in Black Cohosh (Actaea racemosa L.)

Abstract

Botanical dietary supplements are widely used, but issues of authenticity, consistency, safety, and efficacy that complicate their poorly understood mechanism of action have prompted questions and concerns in the popular and scientific literature. Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a multicomponent botanical therapeutic used as a popular remedy for menopause and dysmenorrhea and explored as a treatment in breast and prostate cancer. However, its use and safety are controversial. A. racemosa tissues contain the bioactive serotonin analog N-methylserotonin, which is thought to contribute to the serotonergic activities of black cohosh-containing preparations. A. racemosa has several TDC-like genes hypothesized to encode tryptophan decarboxylases (TDCs) converting L-tryptophan to tryptamine, a direct serotonin precursor in plants. Expression of black cohosh TDC1, TDC2, and TDC3 in Saccharomyces cerevisiae resulted in the production of tryptamine. TDC1 and TDC3 had approximately fourfold higher activity than TDC2, which was attributable to a variable Cys/Ser active site residue identified by site-directed mutagenesis. Co-expression in yeast of the high-activity black cohosh TDCs with the next enzyme in serotonin biosynthesis, tryptamine 5-hydroxylase (T5H), from rice (Oryza sativa) resulted in the production of serotonin, whereas co-expression with low-activity TDCs did not, suggesting that TDC activity is a rate-limiting step in serotonin biosynthesis. Two T5H-like sequences were identified in A. racemose, but their co-expression with the high-activity TDCs in yeast did not result in serotonin production. TDC expression was detected in several black cohosh tissues, and phytochemical analysis using LC-MS revealed several new tryptamines, including tryptamine and serotonin, along with N-methylserotonin and, interestingly, N-N-dimethyl-5-hydroxytryptamine (bufotenine), which may contribute to hepatotoxicity. Incubation of A. racemosa leaves with tryptamine and N-methyltryptamine resulted in increased concentrations of serotonin and N-methylserotonin, respectively, suggesting that methylation of tryptamine precedes hydroxylation in the biosynthesis of N-methylserotonin. This work indicates a significantly greater variety of serotonin derivatives in A. racemosa than previously reported. Moreover, the activities of the TDCs underscore their key role in the production of serotonergic compounds in A. racemosa. Finally, it is proposed that tryptamine is first methylated and then hydroxylated to form the black cohosh signature compound N-methylserotonin.

Keywords: Actaea racemosa; Cimicifuga racemosa; Rannunculaceae; black cohosh; secondary metabolism; serotonin; tryptamines; tryptophan decarboxylase.

Figure 1

Tryptamine production in S. cerevisiae cultures expressing TDC genes from black cohosh. Panel (A) shows mass chromatograms of supernatants from galactose-induced 24-h cultures of yeast cells transformed with TDC1, TDC2, or TDC^K314R in the pYES2 vector; cultures of yeast cells transformed with the vector only were used as a negative control. Panel (B) shows mass spectra extracted from the peaks boxed in grey in panel (A). A peak eluting at ~2.47 min, indicating a compound with a mass within 20 ppm of the expected mass of 161.1079 (in ES+ mode) for tryptamine, is present in the authentic tryptamine standard as well as in TDC1- and TDC2-expressing cultures, but not in TDC^K314R (in which the L-lysine residue for PLP binding was replaced with L-arginine) or the vector-only control. Panel (C) shows the normalized concentration of tryptamine estimated from the values for total ion current (TIC) of its mass peak in Gal-induced yeast cultures transformed with TDC1, TDC2, or TDC3 (shown are means from two independent experiments; error bars indicate std dev).

Figure 2

Alignment of TDC1, TDC2, and TDC3 from black cohosh. The alignment excluded 10 amino acids at the N-terminus and 9 amino acids at the C-terminus of the TDCs corresponding to a nucleic acid sequence containing the primer-annealing sites for TDC3 amplification and that, therefore, could not be unambiguously determined. A polymorphic amino acid site, representing an indel, is present within the first 10 amino acids of the N-terminus of TDC1 and TDC2 [36] and is not included in the multiple alignment; PCR results suggested that TDC3 also contains the same indel as TDC2. Amino acid residues at positions 222, 315, and 473 of the alignment are uniquely polymorphic in TDC2 and are indicated by shaded boxes; the Cys/Ser polymorphic site at position 315 (boxed with red lines) was investigated by site-directed mutagenesis (see text and Figure 3). The L-lysine residue at position 306 required for PLP binding is indicated by a clear box.

Figure 3

Activities of TDC1, TDC2, TDC3, and TDC variants mutated in the region of the PLP-binding active site. Panel (A) shows the activity of wild-type (wt) TDC1 and TDC2 (black bars) and their respective Cys/Ser PLP-site mutants (grey bars) measured as tryptamine production in yeast extracts [indicated are means and error bars show the standard deviation (std dev); n = 2]. Panel (B) shows the accumulation of tryptamine in yeast cultures transformed with the wild-type (wt) genes TDC1, TDC2, and TDC3, and mutant variants of TDC1 and TDC2. Data are representative of two separate experiments and were normalized to the median of all measurements; shown are the means from two biological replicates (=independent transformants), measured in duplicate. Error bars indicate std. dev.

Figure 4

Co-expression of TDCs from black cohosh with OsT5H from O. sativa, encoding tryptamine-5-hydroxylase. Yeast cells co-transformed with OsT5H in pYES-Zeo and TDCs from A. racemosa in pYES2 were Gal-induced for 24 h, and culture supernatants were analyzed for serotonin (5-HT) by LC-MS. Mass peaks in chromatograms and in extracted mass spectra (shown to the right of each chromatogram) shaded in grey correspond to 5-HT as determined by retention times and predicted and measured mass of authentic 5-HT standard (shown at the top).

Figure 5

Tryptamine compounds in young flower tissues of black cohosh. Panel (A) shows mass chromatograms of aqueous methanol extracts of flower buds and the observed masses for their peaks of bioactive tryptamines from A. racemosa plants. Chromatograms are shown for an N-methylserotonin (NMS) standard (top), and the bioactive tryptamine (TNH₂), serotonin (5-HT), N-methylserotonin (NMS), and N,N–dimethyl-5-hydroxytryptamine (N,N-dimethylserotonin or bufotenine (NdiMS). Tryptamines were identified by comparing their retention times with those of their respective authentic standards. Panel (B) shows overlaps of mass chromatograms of methanolic leaf extracts A. racemosa and standards for serotonin (5-HT), N-methylserotonin (NMS), and N,N-dimethylserotonin (NdiMS). The following masses were observed (expected masses and differences (in ppm) of expected versus observed mass are indicated in parentheses): NMS standard, 191.1170 (191.1184; 7.3 ppm); TNH2, 161.1095 (161.1079; 8.7 ppm); 5-HT, 177.1028 (177.1023; 2.8 ppm); NMS, 191.1185 (191.1184; <1 ppm); and NdiMS, 205.1347 (205.1341; 2.9 ppm).

Figure 6

Concentrations of serotonin and N-methylserotonin in detached leaves incubated with tryptamine (TNH₂) or N-methyltryptamine (M-TNH₂). Leaves were incubated with 0.1 mM of TNH₂ or M-TNH₂ for 48 h and extracted, and concentrations of serotonin and N-methylserotonin were measured by LC-MS. Panel (A) shows the concentration (as µg per g of fresh leaf tissue) of serotonin in leaves from the control (water only) and in the leaves treated with TNH₂ and M-TNH₂; panel (B) shows the concentration of N-methylserotonin in leaf tissues subjected to the same treatments. Bars show means ± std dev (n = 3); nd = not detected. The concentration of N-methylserotonin in leaf tissues incubated with M-TNH₂ was statistically significantly different from its concentration in the tissues incubated with TNH₂ or in the water-only control (p < 0.01, one-way ANOVA with Tukey’s multiple comparison test).

Figure 7

Biochemical pathways to serotonin and its methylated derivatives in plants. Solid and dashed arrows indicate confirmed and putative biochemical steps, respectively. Shading indicates biochemical routes supported by results and observations in this and previous studies. Steps with a ‘?’ symbol indicate biochemical transformations for which enzyme catalyst have not been confirmed. The shaded TDC step indicates decarboxylation catalyzed by TDC1, TDC2, and TDC3; a possible alternative route via decarboxylation of 5-hydroxy-L-tryptophan (5-OH-L-Trp) to serotonin (5-HT) has been excluded in this study for TDC1 and TDC2 and is indicated by the crossed arrow. 5-hydroxylation of tryptamine (TNH₂) to 5-HT by a cytochrome P450 monooxygenase (T5H) has been demonstrated in rice [38]. Chemical precursor feeding results of black cohosh leaves tentatively suggest that TNH₂ may be hydroxylated to 5-HT and N-methyltryptamine (M-TNH₂) to N-methylserotonin (NMS), but the enzymes at these steps are unknown. Abbreviations used: AADC, aromatic amino acid decarboxylase; diM-TNH₂, N,N-dimethyltryptamine; MT, methyltransferase; NdiMS, N,N-dimethylserotonin; SAM, S-adenosylmethionine; L-Trp, L-tryptophan.

Figure 8

TDC gene expression and concentrations of tryptamines (tryptamine, serotonin, and N-methylserotonin) in black cohosh tissues. Solid black bars show the combined concentration of all of the three tryptamines in four different A. racemosa tissues (left y-axis) and open bars show expression levels of TDC genes measured by RT-qPCR in the same tissues (right y-axis). TDC expression was normalized to the expression of EF1α and the median of expression in all tissues. Bars show the mean ± std err (n = 3–6).