Citrus genus plants contain N-methylated tryptamine derivatives and their 5-hydroxylated forms

Article abstract of DOI:10.1021/jf401448q

The occurrence and distribution in Citrus genus plants of N-methylated derivatives of tryptamine and their 5-hydroxylated forms are reported. Tryptamine, N-methyltryptamine, N,N-dimethyltryptamine, N,N,N- trimethyltryptamine, 5-hydroxytryptamine (serotonin), 5-hydroxy-N- methyltryptamine, 5-hydroxy-N,N-dimethyltryptamine (bufotenine), and 5-hydroxy-N,N,N-trimethyltryptamine (bufotenidine) were quantitated by LC-ESI-MS/MS. Leaves of all citrus plants examined contained N,N,N-trimethyltryptamine, a compound that we first discovered in the bergamot plant. Interestingly, we also found out that all plants examined contained 5-hydroxy-N,N-dimethyltryptamine and 5-hydroxy-N,N,N-trimethyltryptamine, compounds never described so far in the Citrus genus. As N,N,N- trimethyltryptamine and 5-hydroxy-N,N,N-trimethyltryptamine possess nicotine-like activity by exerting their action on acetylcholine receptors, it is conceivable that both represent the arrival point of a biosynthetic pathway aimed to provide Citrus plants with chemical defense against aggressors. This hypothesis is supported by our finding that leaves and seeds, which are more frequently attacked by biotic agents, are the parts of the plant where the highest levels of those compounds were found.

Full text of DOI:10.1021/jf401448q

Article
pubs.acs.org/JAFC
Citrus Genus Plants Contain N‑Methylated Tryptamine Derivatives
and Their 5‑Hydroxylated Forms
Luigi Servillo,*,† Alfonso Giovane,† Maria Luisa Balestrieri,† Rosario Casale,† Domenico Cautela,‡
and Domenico Castaldo§,∥
†Dipartimento di Biochimica, Biofisica e Patologia Generale-II Universita
̀
degli Studi di Napoli, via L. De Crecchio, 7- 80138 Naples,
Italy
‡Stazione Sperimentale per le Industrie delle Essenze e dei derivati dagli Agrumi (SSEA), Azienda Speciale della Camera di
Commercio di Reggio Calabria, via Tommaso Campanella, 12-89125, Reggio Calabria, Italy
§Ministero dello Sviluppo Economico (MiSE), via Molise 2, Rome, Italy
ABSTRACT: The occurrence and distribution in Citrus genus plants of N-methylated derivatives of tryptamine and their 5-
hydroxylated forms are reported. Tryptamine, N-methyltryptamine, N,N-dimethyltryptamine, N,N,N-trimethyltryptamine, 5-
hydroxytryptamine (serotonin), 5-hydroxy-N-methyltryptamine, 5-hydroxy-N,N-dimethyltryptamine (bufotenine), and 5-
hydroxy-N,N,N-trimethyltryptamine (bufotenidine) were quantitated by LC-ESI-MS/MS. Leaves of all citrus plants examined
contained N,N,N-trimethyltryptamine, a compound that we first discovered in the bergamot plant. Interestingly, we also found
out that all plants examined contained 5-hydroxy-N,N-dimethyltryptamine and 5-hydroxy-N,N,N-trimethyltryptamine,
compounds never described so far in the Citrus genus. As N,N,N-trimethyltryptamine and 5-hydroxy-N,N,N-trimethyltryptamine
possess nicotine-like activity by exerting their action on acetylcholine receptors, it is conceivable that both represent the arrival
point of a biosynthetic pathway aimed to provide Citrus plants with chemical defense against aggressors. This hypothesis is
supported by our finding that leaves and seeds, which are more frequently attacked by biotic agents, are the parts of the plant
where the highest levels of those compounds were found.
KEYWORDS: 5-hydroxy-N,N,N-trimethyltryptamine, bufotenine, psilocin, tryptamine derivatives, hydroxylated tryptamine derivatives,
citrus plants, biotic stress
a plant defense mechanism against herbivory, as its
accumulation mainly occurs in leaves. This possibility finds
support in a pharmacological study9 conducted on sections of
frog and guinea pig intestines showing that N,N,N-trimethyl-
tryptamine is a stimulant of parasympathetic ganglia and
possesses nicotine-like activity by exerting its action on
acetylcholine receptors. On the basis of those previous results
and, above all, considering that the same pharmacological
studies9 reported that 5-hydroxy-N,N,N-trimethyltryptamine,
also called bufotenidine or cinobufotenine, was an even more
potent cholinergic substance (about 10-fold) than N,N,N-
trimethyltryptamine, we sought to verify not only in bergamot
but also in many other economically important plants of the
Citrus genus plants, that is, orange, lemon, mandarin, chinotto
(Citrus myrtifolia), and citron, the presence and the distribution
in the various tissue parts of both N-methylated and 5-hydroxy-
N-methylated derivatives of tryptamine. The chemical
structures of the compounds discussed herein are shown in
Table 1.
INTRODUCTION
■
Recently, we reported the occurrence in the bergamot1 plant
(Citrus bergamia Risso et Poit) of secondary metabolites
derived from tryptamine, a substance with well-known repelling
activity on insect herbivory,2 which is biosynthesized starting
from tryptophan by the action of the enzyme tryptophan
decarboxylase.3,4 As bergamot is an agriculturally important
plant, the defense mechanisms against phytopathogenic
organisms has attracted considerable attention especially
regarding the economic impact related to the industrial
production of juice and essential oil.5−7 Our results1 showed
not only the presence of partially methylated tryptamine
derivatives, such as N-methyltryptamine and N,N-dimethyl-
tryptamine, which were distributed in all parts of the fruit (peel
and edible part) and with higher levels in leaves and seeds, but
also the occurrence of the trimethylated form of tryptamine,
that is, N,N,N-trimethyltryptamine, a metabolite never found
before in plants. The occurrence of all the N-methylated
tryptamine derivatives in the bergamot plant led us to
hypothesize the involvement of tryptophan decarboxylase in a
new metabolic pathway different from the one widely studied,
which leads to the formation of monoterpene indole alkaloids
in plants.8
In the hypothesized new pathway, the formation of N,N,N-
trimethyltryptamine from tryptamine, as a consequence of
successive methylation reactions, catalyzed by the same or more
N-methyltranferase(s), could represent the ultimate purpose of
Received: January 14, 2013
Revised: May 6, 2013
Accepted: May 7, 2013
© XXXX American Chemical Society
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Table 1. Structural Representations of Tryptophan, N-Methylated Tryptamine Derivatives, and Their 5-Hydroxylated Forms
Analyzed in This Study
Synthesis and Purification of 5-Hydroxy-N,N,N-trimethyl-
MATERIALS AND METHODS
■
tryptamine. For the conversion of 5-hydroxytryptamine into its
Reagents. Tryptophan, tryptamine, N-methyltryptamine, 5-
hydroxytryptophan, 5-hydroxytryptamine (serotonin), methyl iodide,
and 0.1% solution of formic acid in water used for LC-ESI-MS analyses
were obtained from Sigma-Aldrich (Milan, Italy). N,N-Dimethyltrypt-
amine, 4-hydroxy-N,N-dimethyltryptamine (psilocin), and 5-hydroxy-
N,N-dimethyltryptamine (bufotenine) were from LGC Standards
(Milan, Italy). SPE-C18 columns for flash chromatography (particle
size 33 μm) were obtained from Phenomenex (Anzola Emilia,Italy).
quaternary ammonium compound, that is, 5-hydroxy-N,N,N-trime-
thyltryptamine, a modified procedure proposed by Chen and
Benoiton10,11 was used, which is based on a heterogeneous phase
reaction employing methyl iodide as methylating agent in the presence
of KHCO3. Briefly, 200 mg of serotonin was dissolved in 20 mL of
methanol with 1 g of KHCO3, then 10 mL of methyl iodide (CH3I)
was added. The mixture was stirred for 12 h at room temperature. The
addition of methyl iodide (10 mL) and KHCO3 (1 g) was repeated
All other solvents and reagents used were of analytical grade.
Plant Materials. Fruits and leaves of bergamot were harvested in
October and December, 2011 in the “Pellaro” area near Reggio
Calabria (Italy). Leaves of lemon, orange, mandarin, chinotto, and
citron were harvested in December, 2011 in the SSEA Arboretum of
Reggio Calabria.
Bergamot Peel Extracts. The preparations started with water
washing of the bergamot fruits followed by the manual scraping of the
exocarp in order to remove the essential oils from the utricles on the
fruit surface. Then, after washing again with water, the peel and seed
were separated manually. The peel (flavedo and albedo) was
homogenized in a mixer with water in the 1:1 (w/w) ratio. The
homogenate was allowed to stand for 2 h under constant agitation and
then centrifuged at 18000g for 30 min at 4 °C. The supernatant was
finally frozen at −20 °C until used for the successive determinations.
Bergamot Endocarp Extracts. The endocarp (the edible part of the
fruit, constituted by juice and pulp) deprived of seeds was
homogenized in a mixer and then centrifuged at 18000g for 30 min
at 4 °C and the supernatant frozen at −20 °C until used for the
successive determinations.
twice more. Finally, the mixture was centrifuged and the supernatant
collected and evaporated to dryness at 40 °C in a rotary evaporator.
The residue, containing the 5-hydroxy-N,N,N-trimethyltryptamine,
was dissolved in 10 mL of Milli Q grade water and purified by flash
chromatography on a SepPac C18 cartridge (Phenomenex, Anzola
Emilia, Italy). The sample-loaded column was washed with 100 mL of
Milli Q water and then the retained 5-hydroxy-N,N,N-trimethyltrypt-
amine was eluted with a 50 mL solution of H2O/acetonitrile (80:20).
The eluate was evaporated to dryness under a stream of air and dried
overnight under vacuum in the presence of P2O5. The yield was 55%.
Standard and Sample Preparations. The standard stock
solutions of tryptophan (1), 5-hydroxytryptophan (2), tryptamine
(3), N-methyltryptamine (4), N,N-dimethyltryptamine (5), N,N,N-
trimethyltryptamine (6), 5-hydroxytryptamine (7), 5-hydroxy-N-
methyltryptamine (8), 5-hydroxy-N,N-dimethyltryptamine (9), 5-
hydroxy-N,N,N-trimethyltryptamine (10), and 4-hydroxy-N,N-dime-
thyltryptamine (11) were prepared at a concentration of 2000 ng/mL
and stored at 4 °C. Prior to injection, stock solutions were
appropriately diluted with water containing 0.1% formic acid before
being used as working solutions.
Bergamot Seed Extracts. The seeds recovered from each lot were
initially washed with water, drained, and dried on filter paper.
Successively, 2−4 g of the seeds was homogenized in a mixer with 20
mL of Milli-Q water. The homogenate was kept for 3 h under constant
agitation, then centrifuged at 18000g for 30 min at 4 °C and the
supernatant frozen at −20 °C until used for the successive
determinations.
Bergamot and Other Citrus Plant Leaf Extracts. The citrus leaves
were washed with distilled water and dried with filter paper. Then 25 g
of product, finely chopped, was homogenized in a blender with 100
mL of 0.1% formic acid in Milli Q grade water and then kept under
stirring for 3 h. The homogenate was finally centrifuged at 18000g for
30 min, and the supernatant was stored in 20 mL vials at −20 °C.
Instrumental Conditions for HPLC-ESI-MS/MS Analyses. The
optimization of the instrumental parameters for the analyses of the
tryptamine and 5-hydroxytryptamine derivatives was performed by
continuous infusion of 5 μM standard solution in 0.1% formic acid.
The mass cutoff and the fragmentation amplitude were optimized in
order to obtain the most efficient MS/MS transitions from the
positively charged precursor ion [M+H+] to the fragment ions. The
transitions utilized for quantification were 205.1→188 for tryptophan,
161.1→144 for tryptamine, 175.1→144 for N-methyltryptamine,
189.1→144 for N,N-dimethyltryptamine, 203.2→144 for N,N,N-
trimethyltryptamine, 221.2→204 for 5-hydroxytryptophan, 177.1→
160 for 5-hydroxytryptamine, 191.1→160 for 5-hydroxy-N-methyl-
tryptamine, 205.1→160 for 5-hydroxy-N,N-dimethyltryptamine,
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205.1→160 for 4-hydroxy-N,N-dimethyltryptamine, and 219.1→160
for 5-hydroxy-N,N,N-trimethyltryptamine.
The substances were analyzed by HPLC-ESI/MS/MS, as described
by Servillo et al.1,12 for analysis of betaines and methylated tryptamine
derivatives. Briefly, the chromatography, isocratically conducted with
0.1% formic acid in water, was performed with a Supelco Discovery-C8
column, 100 × 3.0 mm, particle size 5 μm, at a flow rate of 100 μL/
min. Volumes of 10−20 μL of standard solutions or samples were
injected.
The HPLC-ESI-MS/MS analyses were performed with an HPLC
Agilent 1100 series equipped with an on line degasser and automatic
injector coupled online with an Agilent LC-MSD SL quadrupole ion
trap. The MS acquisition was performed by using ESI in positive ion
mode, with nitrogen as the nebulizing and drying gas under the
following conditions: nebulizer pressure, 30 psi; drying temperature,
350 °C; and drying gas, 7 L/min. The ion charge control (ICC) was
applied with the target set at 30000 and maximum accumulation time
at 20 ms. The measurements were performed from the peak area of
the extracted ion chromatogram (EIC). The quantitation was achieved
by comparison with the calibration curves obtained with standard
solutions. The retention time (min) and peak areas of the monitored
fragment ions were determined by the Agilent software Chemstation,
version 4.2.
RESULTS AND DISCUSSION
■
Identification of Bufotenine in Citrus Plants. In our
previous work,1 aimed to verify the occurrence of tryptophan
derived metabolites in the bergamot plant, we observed in the
MS full scan chromatogram of leaf extracts the presence of two
well resolved peaks both corresponding to m/z 205.1. The
more retained peak, emerging at 14.2 min, was tryptophan,
whereas the less retained one, emerging at 12.1 min, was not
identified. We have herein focused our attention on this latter
peak, performing the chromatographic analysis of bergamot leaf
extracts by isolating at m/z 205.1 and subjecting ions to MS2
fragmentation (Figure 1A and B). The fragmentation pattern of
the peak at retention time 12.1 min is dominated by the
fragment at m/z 160, which might indicate the presence of
hydroxyvinylindole in the molecular structure of the unknown
compound. This indication was confirmed by successive MS3
analyses showing that the fragmentation of the MS2 ions,
isolated at m/z 160, gives a pattern dominated by a fragment at
m/z 132 (Figure 1C). This fragment has been proposed to be
produced from the hydroxyvinylindole ion by a neutral loss of
CO from the benzene ring. The consequent benzene ring
contraction generates two fused five-membered rings with the
Figure 1. (A) LC-ESI-MS chromatogram conducted in zoom scan
mode on a bergamot leaf extract in the m/z range 201−209 amu,
extracted at m/z 205.1 (red line) and m/z 203.2 (black line). Peak 2
corresponds to tryptophan and peak 3 to N,N,N-trimethyltryptamine
as reported in ref 1. (B) MS2 fragmentation pattern of peak 1 in A. (C)
MS3 fragmentation pattern of the ion at m/z 160 in B. In B the
proposed structure for the MS2 ion at m/z 160 and in C that for the
MS3 ion at m/z 132 as reported in ref 13 are also indicated .
stances are considered psychoactive. Psilocin is particularly
present in some hallucinogenic species of mushrooms,
bufotenine has been found in animals, mushrooms, and higher
plants.14,15 In order to determine which of the two substances
corresponds to the peak at r.t. 12.1 min, standard solutions of
both were analyzed by LC-ESI-MS/MS utilizing the same
chromatographic conditions, which we previously used for the
determinations of betaines in Citrus plants.12,16 In these
conditions, bufotenine and psilocin were separated; the first
emerged at r.t. 10.5 min and the second at r.t. 12.1 min (Figure
2A). It is also interesting to note that the two compounds, in
the same instrumental fragmentation conditions, behave
differently. In fact, for psilocin, the two MS2 fragments at m/
z 160 and m/z 58 showed almost identical intensities (Figure
2C), whereas, for bufotenine, the fragment at m/z 160 showed
an intensity about twice that of the fragment at m/z 58 (Figure
2B).
On the basis of these results, the extract of tissue parts of the
various citrus plants were analyzed as described above. In all
cases, we observed the peak at m/z 205.1, emerging at r.t. 10.5
min, which showed the same fragmentation pattern of
bufotenine. As an example, the chromatogram of a bergamot
leaf extract is reported in Figure 3A and B, where the
bufotenine identification, besides the presence of the peak at r.t.
10.5 min, is confirmed by the higher intensity of the MS2
fragment at m/z 160 than that of the fragment at m/z 58
(Figure 3C). It is noteworthy that an intense fragment at m/z
188 is also observed in the MS2 fragmentation pattern of the
peak at r.t 10.5 min, for leaf extracts. This is due to the presence
13
positive charge on the nitrogen atom of the pyrrole ring
(Figure 1C).
The same type of determination conducted on the leaf
extracts of all Citrus genus plants examined showed the
presence of the peak at m/z 205.1 with r.t. 12.1 min having the
same MS2 and MS3 fragmentation patterns reported above.
The generation from the ion at m/z 205.1 of the MS2
fragment at m/z 160 and the MS3 fragment at m/z 132 from
the MS2 fragment at m/z 160 gives a strong indication of the
occurrence of hydroxyvinylindole in the structure of the
unknown compound. Moreover, the presence in its MS2
fragmentation pattern of another fragment at m/z 58, which
also occurs in the MS2 spectrum of N,N-dimethyltryptamine,1
led us to hypothesize that the unknown compound is a
hydroxylated N,N-dimethyltryptamine. However, two com-
pounds have been found in the natural world, both having m/z
205.1, which differ in the OH position in the indole structure,
that is, psilocin (4-hydroxy-N,N-dimethyltryptamine) and
bufotenine (5-hydroxy-N,N-dimethyltryptamine). Both sub-
C
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Figure 2. (A) Total ion current (TIC) chromatogram (black line) and
MS2 extracted ion chromatogram (EIC) at m/z 160 (red line) of a
standard mixture of bufotenine and psilocin. The chromatographic
conditions are reported in the text. (B) MS2 fragmentation pattern of
the bufotenine peak. (C) MS2 fragmentation pattern of the psilocin
peak.
of tryptophan in the extract. Actually, tryptophan (m/z 205.1)
elutes at r.t. of 12.7 min, but it is much more abundant than
bufotenine; therefore, the ascending part of the tryptophan
peak partially coelutes with the descending part of the
bufotenine peak (Figure 3A). However, in order to get further
experimental evidence for bufotenine identification, we
performed chromatographic analysis also with a longer C8
column (15 cm) in the same isocratic elution conditions. In this
way, complete separation among bufotenine, psilocin, and
tryptophan (Figure 3C and D) and the disappearance of the
fragment at m/z 188 in the fragmentation pattern of the peak
corresponding to bufotenine in the bergamot leaf extract
analysis (Figure 3D, inset) were obtained.
Identification of 5-Hydroxytryptamine and Its N-
Methyl Derivatives in Citrus Plants. The presence of
bufotenine in all the examined citrus plants prompted us to
look for the occurrence in the same sources of 5-hydroxytrypt-
amine (serotonin) and possibly other compounds deriving
from its N-methylation, that is, 5-hydroxy-N-methyltryptamine
and 5-hydroxy-N,N,N-trimethyltryptamine. Moreover, 5-hy-
droxytryptophan, as a possible precursor of 5-hydroxytrypt-
amine, was also searched for. In the MS2 analyses, the
monitored transitions were 177.1→160 for 5-hydroxytrypt-
amine, 191.1→160 for 5-hydroxy-N-methyltryptamine, and
221.2→204 for 5-hydroxytryptophan.
Figure 3. (A) MS2 EIC chromatograms at m/z 188 (black line) and at
m/z 160 (blue line) of bergamot leaf extract. The chromatography was
performed with a C8 column (10 cm) eluted as reported in the text.
(B) MS2 EIC chromatogram of the peak at r.t. 10.4 min. In the inset,
the MS2 fragmentation pattern of the peak at r.t. 10.4 min is reported.
The intense fragment at m/z 188, typical of the MS2 tryptophan
fragmentation, is due to the partial overlap of the tryptophan peak at
r.t. 12.4 min, as is seen in A. (C) Standard mixture of bufotenine,
psilocin, and tryptophan chromatographed on a longer C8 column (15
cm). (D) MS2 EIC chromatograms at m/z 188 (black line) and at m/z
160 (blue line) of the same bergamot leaf extract as that in A but
chromatographed on a longer C8 column (15 cm). In the inset, the
fragmentation pattern of the peak corresponding to bufotenine is
reported, in which the fragment at m/z 188 is no longer present.
A complete chromatographic separation of all examined
metabolites was obtained using the 10 cm Supelco Discovery-
C8 column in a time less than 16 min and in isocratic condition
with 0.1% formic acid in water as eluent. In particular, 5-
hydroxytryptophan elutes first at r.t. 6.2 min, followed by 5-
hydroxytryptamine at r.t. 8.1 min, and 5-hydroxy-N-methyl-
tryptamine at r.t. 10 min (Figure 4A). The MS2 fragmentation
patterns obtained from standard solutions of the metabolites
are reported in Figure 4C−E. The chromatographic analysis
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Figure 4. (A) MS2 EIC of a standard mixture of 5-hydroxytryptophan
(2), 5-hydroxytryptamine (7), and 5-hydroxy-N-methyltryptamine (8).
The MS2 transitions followed were 221.2→204 for (2) (red line),
177.1→160 for (7) (blue line), and 191.1→160 for (8) (black line).
(B) MS2 EIC of a bergamot leaf extract, where no signal from 2 is
observed. (C, D, and E) MS2 fragmentation patterns of the peaks
depicted in A. The MS2 fragmentation patterns of the peaks
corresponding to 7 and 8 from the bergamot leaf extract being
identical to those of the standard compounds were not reported. The
chromatographic conditions are described in the text.
Figure 5. (A) Representative chromatogram of a bergamot leaf extract
following the MS2 transitions 219.1→160. (B) Chromatography of the
synthesized 5-hydroxy-N,N,N-trimethyltryptamine (10) standard
solution performed in the same experimental conditions. (C) MS2
fragmentation pattern of the peak reported in A. Inset of C: MS3
fragmentation pattern of the peak in A by isolating at m/z 160. The
MS2 and MS3 fragmentation patterns of the peak in B (standard 5-
hydroxy-N,N,N-trimethyltryptamine) being identical to those of the
peak in A were omitted.
performed in the same conditions of a bergamot leaf extract is
reported in Figure 4B. The occurrence in the extract of 5-
hydroxytryptamine and 5-hydroxy-N-methyltryptamine was
confirmed by the identity of the retention times and MS2
fragmentation patterns with the respective standards. 5-
Hydroxytryptamine and 5-hydroxy-N-methyltryptamine were
found in the leaf extracts of all citrus plants examined. However,
5-hydroxytryptophan was not detected in leaf extracts (Figure
4B).
available, it was synthesized according to the Chen and
Benoiton method.10,11 The synthesized 5-hydroxy-N,N,N-
trimethyltriptamine was purified and subjected to mass
spectrometric analysis in order to find the optimal instrumental
conditions for detection and quantitation. The MS2 fragmenta-
tion pattern of 5-hydroxy-N,N,N-trimethyltriptamine standard
solution is reported in Figure 5A. The MS3 fragmentation
pattern was obtained isolating the MS2 fragment at m/z 160.
This pattern was dominated by the product ion at m/z 132
(Figure 5C). Then, the chromatographic behavior and MS2
fragmentation pattern of the synthesized 5-hydroxy-N,N,N-
trimethyltriptamine were compared with those of the unknown
compound from the leaf extract (Figure 5). Both compounds
showed the same mass at m/z 219.1 of the parent ions, the
same MS2 fragmentation patterns, and the same intensity ratio
between the two main fragments at m/z 160 and 60. Moreover,
they eluted at the same retention time (Figure 5A and B).
These observations sufficiently prove that the unknown
compound is 5-hydroxy-N,N,N-trimethyltriptamine. Anyway,
to obtain further evidence of the identity of the compound, 5-
hydroxy-N,N,N-trimethyltriptamine standard solutions were
added to bergamot leaf extracts and subjected to chromatog-
raphy by utilizing C8 columns of 10 and 15 cm and two
different isocratic elution conditions, that is, 100% water with
0.1% formic acid and a mixture of 75% water with 0.1% formic
acid containing 25% methanol. In all cases, only one
symmetrical peak was seen in the EIC chromatograms obtained
by isolating the ion at m/z 219.1, thus confirming the identity
Identification 5-Hydroxy-N,N,N-trimethyltriptamine
(Bufotenidine) in Citrus Plants. The absence of 5-
hydroxytryptophan in all of the citrus plant leaf extracts
examined led us to hypothesize that 5-hydroxytryptamine and
its N-methyl derivatives do not stem from decarboxylation of 5-
hydroxytryptophan but rather that they come from tryptamine
and/or its N-methyl derivatives by successive hydroxylation. As
we recently discovered the occurrence in bergamot of
noticeable levels of N,N,N-trimethyltryptamine,1 we examined
the possibility that the hydroxylation product of N,N,N-
trimethyltryptamine was also present in this plant source. To
this end, a bergamot leaf extract was analyzed by LC-ESI-MS/
MS by isolating at m/z 219.1. As shown in Figure 5A, an
intense total ion current signal is seen in the chromatogram
region between 11 and 15 min, and the extracted ion
chromatogram (Figure 5C) shows the presence of two
fragments: the first at m/z 160 and the second, less intense,
at m/z 60, indicative of the probable presence of the
trimethylammonium group in the structure of the substance.1,12
Moreover, the unknown compound also shows a retention time
higher than that of 5-hydroxy-N,N-dimethyltryptamine, provid-
ing further support to the hypothesis that it was the
hydroxylated form of N,N,N-trimethyltryptamine. This is
analogous to our finding, previously reported for N-methylated
tryptamines, that the retention time increases when methyl-
ation on amino nitrogen increases.1 To confirm that hypothesis,
as 5-hydroxy-N,N,N-trimethyltriptamine was not commercially
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of the unknown compound from the leaf as 5-hydroxy-N,N,N-
trimethyltriptamine.
bergamot leaves.1 The data in Table 3 show the common
presence of these indole metabolites in all the Citrus species
examined. However, their levels significantly vary among the
species. In particular, leaves of bergamot, lemon, and citron
contain levels of N,N,N-trimethyltryptamine and 5-hydroxy-
N,N,N-trimethyltryptamine consistently higher than those of
orange, mandarin, and chinotto.
Distribution of Tryptamine Derivatives in Various
Parts of the Bergamot Plant and in Leaves of Other
Citrus Plants. The quantitative distribution of the hydroxy-
lated tryptamine derivatives in fruits and leaves of the bergamot
plant is reported in Table 2.
Tryptamine Metabolism in Citrus Plants. In plant
metabolism, tryptamine represents an intermediate of the
metabolic flow that leads from tryptophan to compounds
important for plant physiology and defense. Tryptamine derives
from tryptophan decarboxylation by the activity of the
tryptophan decarboxylase enzyme. Once formed, it is converted
into a variety of metabolites such as the phytohormone indole-
3-acetic acid17 and the monoterpene indole alkaloids such as
the well-known anticancer agents vinblastine and vincristine.8
However, the role of tryptamine as a precursor of simpler
secondary metabolites involved in plant defense against biotic
agents has not been well defined. Although there are no reports
in the literature on the presence in Citrus genus plants of the
enzyme tryptophan decarboxylase, the presence at noticeable
levels of tryptamine and its N-methylated derivatives makes it
reasonable to hypothesize the presence in this plant genus of
tryptophan decarboxylase. Moreover, the occurrence of 5-
hydroxytryptamine and its N-methylated derivatives in all the
citrus plants we examined and the absence of 5-hydroxy-
tryptophan also support the likelihood of tryptamine 5-
hydroxylase activity. In fact, the absence of 5-hydroxytrypto-
phan suggests that 5-hydroxytryptamine in Citrus plants is
generated by the hydroxylation of tryptamine by tryptamine 5-
hydroxylase, as it also occurs in the majority of plants.20
Instead, in mammals and some plants such as Hypericum
perforatum18,19 (St. John’s wort), 5-hydroxytryptamine is
formed by the decarboxylation of 5-hydroxytryptophan.
Therefore, the presence of the N-methylated derivatives of
serotonin that we have found in the Citrus plants (Table 2 and
3) suggests that they may arise either from the methylation of
serotonin or from hydroxylation of the N-methylated
derivatives of tryptamine, which are also present at noticeable
levels in this plant genus. However, the presence of both
pathways is a possibility. Figure 6 summarizes the hypotheses
for the formation of all of these tryptamine derivatives. Of
course, further studies are required to support such hypo-
thetical pathways.
Table 2. Distribution of the 5-Hydroxylated Forms of
Methylated Tryptamine Derivatives in Various Parts of the
Bergamot Plant
a
parts of the bergamot fruit
compd
albedo
edible part
seed
leaf
2
absent/trace
trace
absent/trace
trace
absent/trace
0.5−6.0
absent/trace
0.1−1.0
7
8
trace
trace-0.05
1.0−2.0
trace-0.1
5.0−25.0
5.0−30.0
2.0−6.0
1.0−3.0
1.0−3.5
6.0−12
9
0.03−0.05
0.4−0.8
10
a
(2) 5-hydroxytryptophan, (7) 5-hydroxytryptamine, (8) 5-hydroxy-
N-methyltryptamine, (9) 5-hydroxy-N,N-dimethyltryptamine, and
(10) 5-hydroxy-N,N,N-trimethyltryptamine. Ranges are expressed as
mg/kg. Trace = values less than 0.02 mg/kg.
It appears from Table 2 that 5-hydroxytryptophan is absent
in all tissue parts of the fruit and leaves. Conversely, 5-
hydroxytryptamine was relatively abundant in bergamot seeds
and leaves but only present at trace level in the edible part of
the fruit (fruit juice). As for 5-hydroxy-N-methyltryptamine, the
highest levels were found in seeds (up to 25 ppm), while it is
only at trace levels in the albedo and the edible part of the fruit,
and intermediate amounts were found in leaves. Also, 5-
hydroxy-N,N-dimethyltryptamine shows a distribution similar
to that of 5-hydroxy-N-methyltryptamine, with the highest
levels in seeds (up to 30 ppm) and the lowest in the fruit juice
(up to 2 ppm) and intermediate levels in leaves. The highest
amounts of 5-hydroxy-N,N,N-trimethyltryptamine were found
in leaves (up to 12 ppm) and the lowest in albedo and fruit
juice, while intermediate levels were present in seeds (up to 6
ppm).
Table 3 reports the ranges of tryptophan, tryptamine, and its
N-methylated derivatives and those of 5-hydroxytryptamine
and its N-methylated derivatives in leaves of the various citrus
plants. For the sake of clarity, the last column of Table 3 lists
the amounts of these compounds previously reported in
a
Table 3. Distribution of Tryptophan and Tryptamine Derivatives in Leaves of Citrus Plants
compd
lemon
orange
mandarin
chinotto
citron
bergamot
1
2
8.0−24.0
absent/trace
0.7−3.0
trace
15.0−44.0
absent/trace
trace −0.3
trace
27.0−77.0
absent/trace
0.3−1.5
27.0−80.0
absent/trace
0.2−1.0
trace
31.0−92.0
absent/trace
0.4−2.0
trace
21.0−60.0
absent/trace
0.5−2.0
3
4
trace
0.2−0.5
5
0.1−0.2
2.0−4.5
0.1−0.5
0.2−0.8
0.1−0.2
3.0−7.0
trace −0.1
0.6−1.5
trace −0.1
0.1−0.3
0.4−2.0
trace −0.1
trace −0.1
trace −0.1
trace −0.1
trace −0.1
trace
0.2−0.5
0.4−1.0
0.1−0.6
0.5−3.0
1.5−5.5
trace −0.1
0.10−0.20
1.2−2.8
0.1−1.0
0.2−0.9
1.2−4.6
3.5−7.5
0.2−0.4
6.0−12.0
0.1−1.0
1.0−3.0
1.0 −3.5
6.0 −12.0
6
7
8
9
10
trace −0.1
a
(1) tryptophan, (2) 5-hydroxytryptophan, (3) tryptamine, (4) N-methyltryptamine, (5) N,N-dimethyltryptamine, (6) N,N,N-trimethyltryptamine,
(7) 5-hydroxytryptamine, (8) 5-hydroxy-N-dimethyltryptamine, (9) 5-hydroxy-N,N-dimethyltryptamine, and (10) 5-hydroxy-N,N,N-trimethyltrypt-
amine. Ranges are expressed as mg/kg. Trace = values less than 0.02 mg/kg.
F
dx.doi.org/10.1021/jf401448q | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
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AUTHOR INFORMATION
Corresponding Author
*Phone: +390815665865. Fax: +390815665863. E-mail: luigi.
servillo@unina2.it.
■
Present Address
∥D.C.: Dipartimento di Ingegneria Industriale e ProdAL scarl,
̀
Universita degli Studi di Salerno, via Ponte Don Melillo, I-
84084 Fisciano (SA), Italy.
Notes
The authors declare no competing financial interest.
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dx.doi.org/10.1021/jf401448q | J. Agric. Food Chem. XXXX, XXX, XXX−XXX