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Natural Product Letters
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Variation in the accumulation levels

of n,n-dimethyltryptamine in micro-
propagated trees and in in vitro
cultures of Mimosa tenuiflora
a b
María Del Pilar Nicasio , María Luisa Villarreal , FranÇoise Gillet
c c c
, Lamine Bensaddek & Marc-André Fliniaux
a
Centro de Investigación Biomédica del Sur Instituto Mexicano del
Seguro Social , Xochitepec, Morelos, México
b
Centro de Investigacion en Biotecnología , Universidad
Autónoma del Estado de Morelos , Cuernavaca, Morelos, México
c
Laboratoire de Phytotechnologie , Faculté de Pharmacie,
Université de Picardie Jules Verne , Amiens, France
Published online: 20 Aug 2006.

To cite this article: María Del Pilar Nicasio , María Luisa Villarreal , FranÇoise Gillet ,
Lamine Bensaddek & Marc-André Fliniaux (2005) Variation in the accumulation levels of n,n-
dimethyltryptamine in micro-propagated trees and in in vitro cultures of Mimosa tenuiflora ,
Natural Product Research: Formerly Natural Product Letters, 19:1, 61-67

To link to this article: http://dx.doi.org/10.1080/14786410410001658860

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 Natural Product Research, Vol. 19, No. 1, January 2005, pp. 61–67

VARIATION IN THE ACCUMULATION LEVELS

OF N,N-DIMETHYLTRYPTAMINE IN MICRO-
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PROPAGATED TREES AND IN IN VITRO

CULTURES OF MIMOSA TENUIFLORA
MARÍA DEL PILAR NICASIOa, MARÍA LUISA VILLARREALb,
FRANÇOISE GILLETc, LAMINE BENSADDEKc
and MARC-ANDRÉ FLINIAUXc,*
a
Centro de Investigación Biome´dica del Sur Instituto Mexicano del Seguro Social, Xochitepec,
Morelos, Me´xico; bCentro de Investigacion en Biotecnologı´a, Universidad Autónoma del Estado de
Morelos, Cuernavaca, Morelos, Me´xico; cLaboratoire de Phytotechnologie, Faculte´ de Pharmacie,
Universite´ de Picardie Jules Verne, Amiens, France

(Received 18 July 2003; In final form 3 December 2003)

The present article reports the accumulation of N,N-dimethyltryptamine and its metabolic precursors (tryp-
tophan, tryptamine) in different organs of micropropagated Mimosa tenuiflora trees (leaves, flowers and bark)
subjected to seasonal variations (January and June), as well as in in vitro cultures (plantlets and calluses) of
this plant species. The accumulation of all the tested compounds varied according to the organ, the month of
collection, and age of the plant material. In all cases, the neurotoxic compound N,N-dimethyltryptamine
(DMT) was detected with the lowest concentration 0.01% dry weight (DW) in flowers, and the highest
0.33% DW in bark. For the in vitro cultures, DMT was present in high yields in plantlets (0.1–0.2% DW),
while in calluses this compound was initially detected but its concentration decreased significantly in the sub-
sequent subcultures.

Keywords: In vitro cultures; Mimosa tenuiflora; N,N-dimethyltryptamine (DMT); Tepescohuite; Tryptamine

derivatives

INTRODUCTION

The tree Mimosa tenuiflora Willdenow-Poiret (Fabaceae) is popularly known in Mexico

as ‘‘tepescohuite’’, and its bark is used in Mexican traditional medicine to treat burns
and skin infections [1,2]. From the bark, which is applied topically to the skin, three
triterpenoidal saponins, mimonosides A, B and C were isolated and identified [3,4].
These compounds stimulate the multiplication of mouse and human breast fibroblasts,
and exert immunostimulant properties [5,6]. The observed effects of mimonosides may
explain the traditional use of the bark.

*Corresponding author. E-mail: marc-andre.fliniaux@u-picardie.fr

Natural Product Research

ISSN 1478-6419 print: ISSN 1029-2349 online ß 2005 Taylor & Francis Ltd
http://www.tandf.co.uk/journals
DOI: 10.1080/14786410410001658860
 62 M.D.P. NICASIO et al.

Beside these saponins, a potent neurotoxic tryptophan derivative, N,N-dimethyl-

tryptamine (DMT) has also been purified from the bark of M. tenuiflora [7]. This
compound, as well as serotonin, is biosynthesized in plants from tryptophan via trypt-
amine. DMT is present in various higher plants, including other species of Mimosa
such as M. pudica and M. verrucosa, with concentrations varying from 0.06 to 0.86%
dry weight (DW) [8]. DMT has been found to be the main constituent of ‘‘ayahuasca’’,
a psychotropic hallucinogenic plant which has traditionally played a central role in
magic-religious practices and folk medicine of indigenous people from the Amazon
and Orinoco river basins [9]. DMT is active when smoked, injected and/or adminis-
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trated subcutaneously, and its regular use must be avoided due to side effects of
anxiety and stress, together with disorders of the memory and flashback [10].
In Mexico, different problems arise concerning an adequate supply of the authentic
bark of M. tenuiflora, and DMT has been reported to accumulate at a concentration of
0.03% DW in the bark of wild trees [7]. Even though it has not been scientifically docu-
mented that DMT is absorbed through the skin when the bark is applied topically in
burned patients, the above considerations limit the use of M. tenuiflora as a remedy
to treat skin burns, and the potential toxicity of this product must be taken into account
and evaluated.
There is a current need to look for alternative approaches, including use of in vitro
cultures, that could provide a safe and homogeneous supply of raw material from
M. tenuiflora. We previously reported the establishment of callus and plantlet cultures
of this tree, but the content of the hallucinogenic alkaloid DMT was only determined
in young established calluses [11,12].
In order to select plant material which contains the lowest level of neurotoxic com-
pounds, the purpose of this investigation was to evaluate the potential for accumulating
tryptamine derivatives (mainly DMT) in different plant parts and in vitro cultures of
M. tenuiflora that were subjected to seasonal variations. This information, together
with the quantified levels of the bioactive mimonosides, could eventually allow the pre-
paration of a less toxic remedy from M. tenuiflora that can be safely used in the treat-
ment of skin burns.

MATERIALS AND METHODS

Harvest of Tree Organs

The bark of wild trees was collected by the personnel of the National Union of
Tepescohuite Producers Company, in the State of Chiapas, Mexico. Flowers, leaves,
bark and seeds were collected in January and June from several six-year-old trees, pro-
duced by micropropagation and growing in the State of Morelos, Mexico [12].

In Vitro Cultures
Seeds were sterilized by several passages through ethanol for 2 min, disinfected with
0.6% sodium hypochlorite solution for 15 min and rinsed with sterile water. The seeds
were transferred to Linsmaier and Skoog medium (LS), and after two weeks, plantlets
were obtained. From each plantlet, the apical bud was collected and placed on LS
medium containing 0.1 mg/L indolacetic acid and 3 mg/L kinetin. Every four weeks,
 TRYPTAMINE DERIVATIVES IN M. TENUIFLORA 63

axillary buds from the plantlets were excised and transferred into fresh medium. Callus
cultures were developed from cotyledon explants placed on LS medium containing
2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg/L kinetin as previously
reported [11]. Every four weeks, the calluses were subcultured in fresh medium. All
in vitro cultures were maintained at 26
C under a daily photoperiod of 16 h light
with cool white fluorescent lamps at 30 mMn2s1. After each subculture, plantlet
and callus biomasses were dried, extracted, and analyzed by HPLC.

Extraction Procedure
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All samples from the tree (bark, leaves and flowers) were dried at room temperature,
while plantlets and calluses were freeze-dried. The material was then powdered. Two
sets of conditions for alkaloid extraction from bark were compared. In the first process,
bark samples (2 g) were twice extracted with 50 mL chloroform–27% ammonia (49 : 1)
under reflux for 1 h. After filtration, each crude extract was evaporated to dryness
under reduced pressure. Residuals were resuspended in water–acetonitrile (85 : 15)
adjusted to pH 3 [13]. In the second process, which is used classically for the analysis
of alkaloids, the pH was varied. Bark materials (2 g) were extracted twice by maceration
with diluted HCl solution (pH 2), and after filtration, the aqueous extracts were pooled
and adjusted to pH 10.5. The alkaloidal fractions were obtained by partition with
diethylether. The organic phases, which contained alkaloids, were dried with Na2SO4,
evaporated to dryness under reduced pressure, and resuspended in methanol [7].
Samples from every tree organ (2 g) and from the in vitro cultures (200 mg) were
treated using the system chloroform–27% ammonia (49 : 1) as described before.
HPLC analyses for tryptophan, tryptamine, serotonin and DMT were performed on
a kromasil reversed-phase C18 column (5 mm, 4.6  250 mm) at room temperature.
Chromatography was carried out using a mobile gradient phase from acetonitrile
(A), and 0.1 M ammonium carbonate (B) (36 min: 10% A in 2 min, 10–20% A
in 2 min, 20–30% A in 3 min, 30–40% A in 7 min, 40–70% A in 12 min, 70–90% A
in 4 min, 90–10% A in 4 min and finally an equilibrium at 10% A for 2 min). The
flow rate of the mobile phase was 1.2 mL/min and the eluted compounds were spectro-
photometrically monitored at 280 nm. Calibration curves using authentic standards
within a concentration range of 2–40 mg/mL were used. Peak identities were confirmed
by parallel spectral analysis. Each determination was performed by triplicate, the ana-
lyses of variance for each metabolite concentrations were carried out according to a
randomized design, and mean separations were performed according to DMS and t-
student tests (0.05) [14,15].

RESULTS AND DISCUSSION

Optimization of the Analytical Procedure

The results obtained by using the two extraction procedures showed comparable yields
for both methods, since the content of the extracted compounds were not signifi-
cantly different, t0.05,2 ¼ 2.78, p > 0.05. However, the chloroform–ammonia system
was retained for further extractions of the compounds under investigation, because
of its easier use.
 64 M.D.P. NICASIO et al.

DMT, serotonin and their metabolic precursors (tryptophan, tryptamine) were

analyzed in several organs of micropropagated trees. Because the bark is the organ
routinely used in traditional medicine, the tryptophan derivatives levels obtained in
all samples were compared, for reference, to the bark content of wild trees grown
in Chiapas. The analysis performed upon several samples of this bark revealed a certain
degree of variation in the content of DMT and its metabolic precursors, even though
they are in agreement with data obtained from previous investigations, in which
serotonin (103%) and DMT (0.3  101%) were isolated from M. tenuiflora bark [7].
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DMT and Precursors Content in Micropropagated Trees

The influence of both the season and the organ of the tree upon tryptophan derivative
accumulation in trees issued from micropropagation was investigated.
The contents of tryptophan, tryptamine and serotonin in different organs of
M. tenuiflora micropropagated trees collected during two seasons of the year (January
and June) were determined (Table I). In the samples collected during January the three
compounds were detected, while in June only tryptamine was present in the leaves
and bark.
Every organ obtained from micropropagated trees showed a differential accumula-
tion profile for the tested compounds, but the leaves were the only organ that contained
all three substances, and contained the highest cumulative content of these derivatives.
For the tested organs, the amino acid tryptophan was the least abundant component
(<2.1  103%), it was only detected in the material harvested in January and its
accumulation in different organs was statistically different, F(0.05,2,4) ¼ 0.46, p < 0.05,
DMSerror ¼ 0.001. In contrast, tryptamine was accumulated in higher concentrations,
and its highest level, detected in the flowers of January (7.5  103%), was nearly
four times higher than the value registered for tryptophan in the bark and leaves of
that month. Similarly, serotonin was only accumulated in the leave samples collected
during January.
The DMT accumulation profile in different organs of micropropagated M. tenuiflora
trees is also given in Table I, where it can be seen that this metabolite was the major
tryptophan derivative present in all the collected materials, accumulating in levels
that ranged from two to three orders of magnitude higher (0.01–0.33% DW) than
the other derivatives. The total DMT content in the trees was not statistically different
for the material collected in both seasons F(0.05,2,4) ¼ 0.21, p < 0.05; however, its dis-
tribution in different organs varied statistically according to the month of collection,

TABLE I Seasonal variation in the accumulation of tryptophan, tryptamine, serotonin

and DMT (103%) in different organs of micropropagated M. tenuiflora trees

Compound January June

Bark Flowers Leaves Bark Flowers Leaves

Tryptophan 2.1 0.7 2.15 – – –

Tryptamine 2.2 7.5 3.7 7.1 – 7.4
Serotonin – – 9.0 – – –
DMT 350 30 10.0 110 – 90
Concentrations are expressed in percentages in relation to DW. The values are the mean of three
samples.
 TRYPTAMINE DERIVATIVES IN M. TENUIFLORA 65

since in January the highest accumulation was for the bark, while in June, leaves and
bark contained comparable amounts F(0.05,2,4) ¼ 1.04, p < 0.05, DMSerror ¼ 0.124.
From these results, it appears that the season deeply influences the tryptophan
derivatives content of the micropropagated trees. All the compounds were accumulated
in January where DMT reached its highest value in the bark. In June, DMT was at a
low concentration, and tryptamine was the only other compound present exhibiting
high levels. It is important to underline that in the bark, the tryptamine concentration
was significantly lower in January than in June, and a possible explanation could be
that in the first month, most of this compound was metabolized to DMT, following
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its biosynthetic pathway [16].

Tryptophan Derivative Content of In Vitro Plantlets and Calluses

In the plantlets obtained by micropropagation, every metabolite was detected (Table II)
all throughout 10 subculture periods, in concentrations statistically equivalent
F(0.05,2,4) ¼ 0.321, p < 0.05. The ranges of concentrations were 1–2  101% for trypto-
phan, 0.7–1  101% for tryptamine, 0.8–2  101% for serotonin and 1–2  101%
for DMT. Even though a slightly higher concentration (F(0.05,2,4) ¼ 8.03, p < 0.05,
DMSerror ¼ 0.054) was recorded for DMT, analogous levels of tryptophan, tryptamine
and serotonin were also accumulated in plantlets.
In the case of calluses, the main accumulated metabolite was DMT, followed by tryp-
tophan, tryptamine and serotonin (Table II). In the cell line CAF of M. tenuiflora,
DMT and serotonin were only detected in the first (50  103% and 3  103%), and
second (40  103% and 9  103%) subcultures, and disappeared thereafter. In a
different investigation using the cell line CXM, the complete absence of tryptophan
derivatives was only registered until subculture numbers 43–46 were reached. It can
be hypothesized that DMT accumulation in calluses was shut down by the presence
of the auxin 2,4-D which had been added to the culture medium. This situation has
been reported for other alkaloids in Catharanthus roseus and Nicotiana tabacum cell
cultures [17,18], and could be corroborated by eliminating the auxin from the culture

TABLE II Accumulation of tryptophan and its derivatives in M. tenuiflora

plantlets and calluses during different times in culture

Subculture Concentration ( 103%)

No
Tryptophan Tryptamine Serotonin DMT

Plantlets 100–200 70–100 80–200 100–200

Calluses
1 18 10 3 50
2 20 80 9 40
3 25 14 – –
4 25 14 – –
5 25 8 – –
6 20 3 – –
7 30 3 – –
8 30 2 – –
9 30 – – –
10 35 – – –
Concentrations are expressed in percentages in relation to. The values are the mean of three
samples.
 66 M.D.P. NICASIO et al.

medium or by introducing selected precursors for the biosynthesis of DMT, e.g. trypto-
phan or tryptamine.
We can conclude that M. tenuiflora accumulated high yields of DMT, as it is the case
for some other Mimosa species. Indeed, this compound has been detected in bark,
leaves and flowers of M. tenuiflora micropropagated trees. From all the tested trypt-
amine derivatives, DMT is the one that accumulated at the highest level. The bark of
the January collect was the organ that contained the highest amount of the toxic
alkaloid, and this content (3  101%) was higher than the values we were able to
detect for wild trees (2  101%) t0.05,2 ¼ 2.78, p > 0.05. As the bark is the organ tradi-
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tionally used for medical purposes, and variations were registered in various samples,
it is necessary to make a systematic analytical control of the drug samples prior to
its use. Bark should be preferentially harvested during winter.
Our results have shown that there are natural regulatory controls for the synthesis of
tryptamine derivatives in M. tenuiflora. Following these results, it would be interesting
to look for a correlation of the investigated material with the accumulation of the
saponins involved in cell proliferation promotion (mimonosides A, B and C). Further
investigations will be focused on the establishment of cell and root suspension cultures
free of toxic compounds and otherwise capable of accumulating the mimonosides A, B
and C.
To our knowledge, this is the first report on the analysis of tryptophan derivatives
from different organs as well as from in vitro cultures of M. tenuiflora trees.

Acknowledgements
This work was taken in part from the M.Sc. thesis of Pilar Nicasio, and was carried
out in collaboration with the Mexican Institute of Social Security (IMMS) and the
University of Picardie Jules Verne.
The authors gratefully acknowledge the Mexican Institute of Social Security (IMMS)
for a scolarship given to Pilar Nicasio, and Dr. Eduardo Aranda for supervising the
statistical analysis. The investigation also benefited from a mobility program supported
by ECOS-ANUIES.

References
[1] R. Grether (1988). Boletı´n de la Sociedad Botánica (México), 48, 151–152.
[2] X. Lozoya (1988). Investigación Cientı´fica y Tecnológica (México), 135, 9–11.
[3] Y. Jiang, G. Massiot, C. Lavaud, J.M. Teulon, C. Guéchot, M. Haag-Berruier and R. Anton (1991).
Journal of Natural Products, 54, 1247–1253.
[4] Y. Jiang, G. Massiot, C. Lavaud, J.M. Teulon, C. Guechot, M. Haag-Berruier and R. Anton (1991).
Phytochemistry, 30, 2357–2360.
[5] Y. Jiang, B. Weniger, M. Haag-Berruier and R. Anton (1992). Phytotherapy Research, 6, 310–313.
[6] R. Anton, Y. Jiang, B. Weniger, J.P. Beck and L. Rivier (1993). Journal of Ethnopharmacology,
38, 153–157.
[7] M. Meckes-Lozoya, X. Lozoya, R. Marles, C. Soucy-Breau, Q.A. Varason and J.T. Arnason (1990).
Archivos de Investigación Médica (México), 2, 175–177.
[8] J. Ott (1999). Journal of Psychoactive Drugs, 3, 171–177.
[9] M. Yritia, J. Riba, A. Ramirez, A. Castillo, Y. Alfaro, R. de la Torre and M. Barbanoj (2002). Journal of
Chromatography, 779, 271–281.
[10] J. Rick and M.D. Strassman (1995). Journal of Nervous Mental Disease, 3, 127–138.
[11] M.L. Villarreal, G. Rojas, M. Meckes and P. Nicasio (1993). Biotechnology Letters, 7, 721–726.
[12] M.L.Villarreal and G. Rojas (1996). Plant Cell Reports, 16, 80–82.
 TRYPTAMINE DERIVATIVES IN M. TENUIFLORA 67

[13] M.A. Fliniaux, F. Manceau and A. Jacquin-Dubreuil (1993). Journal of Chromatography, 644, 193–197.
[14] C. Dowdy and A. Wearden (1983). Statistics for Research. Wiley-Interscience, New York.
[15] V. Sharp (1979). Statistics for Social Sciences. Little Brown and Company, Boston.
[16] E.L. Fellows and E.A. Bell (1971). Phytochemistry, 10, 2083–2091.
[17] O.J.M. Goddijn, R.J. De Kam, R.A. Zanetti, R.A. Schilperoot and J.H.C. Hoge (1992). Plant Molecular
Biology, 18, 1113–1120.
[18] O.J.M. Goddijn, F.P. Lohman, R.J. De Kam, R.A. Schilperoot and J.H.C. Hoge (1994). Molecular
Genetics, 242, 217–225.
Downloaded by [Memorial University of Newfoundland] at 07:17 09 October 2014