Fitoterapia 75 (2004) 696 – 701

www.elsevier.com/locate/fitote

Analysis of essential oils from wild and

micropropagated plants of damiana (Turnera diffusa)
Lilia Alcaraz-Meléndez*, Javier Delgado-Rodrı́guez,
Sergio Real-Cosı́o
Programa de Agricultura de Zonas Áridas, Centro de Investigaciones Biológicas del Noroeste (CIBNOR),
P.O. Box 128, La Paz, Baja California Sur 23000, México
Received 24 June 2004; accepted 2 September 2004
Available online 22 October 2004

Abstract

Damiana is a medicinal plant with many traditional uses and a reputation as an aphrodisiac.
Essential oils produced by this plant are used in traditional medicine, and for the preparation of
liquors and tea. The composition of essential oils from wild damiana, plants grown with
micropropagated methods involving cell suspension, and explants in solid medium, is presented.
Relevant differences are observed in oils coming from wild and micropropagated plants, where
micropropagated plants being more uniform with respect to quality and quantity. The most
abundant constituents of the oils were caryophyllene oxide, caryophyllene, y-cadinene, elemene
and 1,8-cineol.
D 2004 Elsevier B.V. All rights reserved.

Keywords: Damiana; Turnera diffusa; Essential oils

1. Introduction

Damiana (Turnera diffusa, Turneraceae) is a wild shrub found throughout Mexico,

Central America and the West Indies, as well as in some parts of South America. In
Mexico, it grows in arid and semi-arid zones in the Los Cabos Region, the southernmost

* Corresponding author. Tel.: +52 612 123 8439; fax: +52 612 125 3625.
E-mail address: lalcaraz04@cibnor.mx (L. Alcaraz-Meléndez).

0367-326X/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2004.09.001
 L. Alcaraz-Meléndez et al. / Fitoterapia 75 (2004) 696–701 697

part of the state of Baja California Sur [1]. Damiana has great socio-economic importance
because it is used as tea, as flavoring for liquors and as medicine. It has a long history of
use in traditional herbal medicine throughout the world, mainly as an aphrodisiac. It has
been reported as an astringent, diuretic, expectorant, laxative, nervine, purgative,
stimulant, tonic, and in venereal disease treatments. It is used in treating common cold,
diabetes, dysentery, dysmenorrea, dyspepsia, enuresis, headache, hormone imbalance,
infection, liver ailments, depression, nerve disorders, pain, poor appetite and stomach ache
[2]. This plant is collected by native people on wild arid lands. In 2002, a technique for
propagation from tissue culture [3–5] was implemented.
Content of essential oils in wild damiana has been investigated by different
authors [2,6,7]. This report concerns studies of essential oils of dry and fresh plant
material, comparing (1) wild plants, (2) plants micropropagated on solid medium and
(3) a stable variant of the plant with distinct morphology that originated in liquid
culture [5].

2. Experimental

2.1. Biological material

Samples were gathered from wild plants under cultivation and propagated plants by
tissue cultures techniques [3,5] and then transplanted to an experimental field at the Centro
de Investigaciones Biológicas del Noroeste (CIBNOR). Plants of homogeneous size were
selected for these studies, and they were also selected for appearance of healthiness,
including high density of brilliant green leaves, new shoots of reddish brown color and
yellow flowers. The plants ranged in height from 1 to 1.5 m with abundant branches and
free of obvious diseases.
To perform extraction and analysis of essential oils, samples were divided into two
portions: The first extract was made immediately after collecting samples and the
second extract from samples dried for 3 days in ventilated plastic containers that
allowed air circulation. For wild plants, seven samples of fresh and dry material were
analyzed. For plants derived from propagation of cells in suspension culture and for
plants propagated in solid medium culture, three samples of fresh and dry material
were analyzed.

2.2. Essential oils extraction

Each sample for analysis was 30 g, including leaves, stems and flowers, fresh or dry
weight, and placed in a steam distiller [8]. After distillation, essential oils were separated
with a 500-ml funnel. To each 450 ml distilled sample, 35–40 ml of anhydrous
diethylether (Baker, HPLC grade) was added. The solution was agitated carefully and was
maintained in repose for 15–20 min. This step was repeated twice. The water phase was
discarded and the ether phase was dried over anhydrous sodium sulfate and evaporated to
dryness. To each recovered sample, 2 ml hexane (HPLC grade, Sigma-Aldrich) was added.
The sample was concentrated another time, 0.5 ml of hexane was added and the sample
698 L. Alcaraz-Meléndez et al. / Fitoterapia 75 (2004) 696–701

transferred to a 1-ml amber vial. The vial was sealed with aluminum lids with Teflon
(Supelco) and stored at 5 8C until analyzed by gas chromatography.

2.3. Gas chromatography analysis

Samples (1 Al) were analyzed by gas chromatography (Hewlett-Packard model

G1800B) with an electron ionization detector FID (110 11 AFS) (Supelco). The
capillary column was made of Supelcowax-10 fused silica, 60 m long with a 0.25 Am film
thickness. The procedure used a 25 cm/s helium gas flow at 75 8C for 4 min, then
programmed to reach 200 8C at an increment of 4 8C min 1, and held at the final
temperature for 5 min. The program bHPCHEMQ was used for the chromatographic

Table 1
Essential oils extracted from fresh and dry material of six wild damiana plants
Rt (min) Library/ID CAS no. Wild plants (% area)
Fresh material Dry material
(average/standard (average/standard
deviation) deviation)
11.97 a-Pinene 000080-56-8 0.19F0.1 0.45F0.3
14.69 h-Pinene 000127-91-3 0.38F0.30a 1.97F1.31
17.81 Limonene 005989-27-5 0.09b 0.16F0.08a
18.1 1,8-Cineole 000470-82-6 1.846F1.26a 2.79F0.43
26.78 a-Cubebene 017699-14-8 0.16F0.12 2.27F0.40c
27.16 (+)-3-Carene 013466-78-9 0.55F0.27 0.54F0.08d
28.04 Copaene 003856-25-5 0.70F0.33 0.69F0.43a
29.11 Linalool D 000078-70-6 0.00 0.000
29.43 h-Cubebene 013744-15-5 0.19b 0.000
30.55 Pinocarvone 016812-40-1 0.62b 1.34F0.83c
30.71 Aristolene 006831-16-9 2.17F0.57a 0.000
30.9 (+)-Norinone 038651-65-9 0.64F0.36d 1.63F1.33c
31.07 h-Elemene 000515-13-9 3.29F0.51 2.66b
31.26 Caryophyllene 000087-44-5 1.22F0.42 2.37F0.90d
32.29 Alloaromadendrene 025246-27-9 0.62F0.38 1.31F0.61e
32.39 Myrtenal 000564-94-3 0.77F0.39e 1.82F1.14c
33.54 y-Cadinene 000483-76-1 5.54F1.02 7.73F2.43
34.08 g-Amorphene 030021-74-0 1.08b 0.99F0.27d
34.74 Verbenone 001196-01-6 0.00 0.28b
35.49 h-Germacrene 015423-57-1 0.43F0.24 0.88b
36.22 g-Cadinene 039029-41-9 0.63F0.30d 0.000
36.78 Myrtenol 000515-00-4 1.11F0.66e 1.22F0.59c
42.56 Caryophyllene oxide 001139-30-6 2.94F1.63 5.30F2.48a
44.74 Globulol 000489-41-8 0.00 0.000
46.24 ( )Spathulenol 077171-55-2 0.65F0.27 1.24F0.29
a
Obtained in five of six plants.
b
Obtained in one of six plants.
c
Obtained in three of six plants.
d
Obtained in two of six plants.
e
Obtained in four of six plants.
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analysis. The compounds were identified with the database NBS75K.L comparing the
mass spectrum obtained with this database.

3. Results and discussion

Essential oils of damiana (T. diffusa) extracted by steam distillation from wild plants,
plants micropropagated in solid medium and plants propagated in liquid medium are listed
in Tables 1–3. The main constituents of the essential oils in all samples are caryophyllene
oxide, caryophyllene, y-cadinene , h-elemene and 1,8-cineole. One report [9] indicates that
y-cadinene is a testosterone inducer and 1,8-cineole is testosterone hydroxylase inducer.
Fresh and dry samples of all forms of damiana contained both compounds, but wild plants
contain more y-cadinene than cultivated plants. This may support the common belief that
damiana is useful as an aphrodisiac. Results show high variability between plants from

Table 2
Essential oils extracted from fresh and dry material of three damiana plants micropropagated in solid medium
Rt (min) Library/ID CAS no. Solid media (% area)
Fresh material Dry material
(average/standard (average/standard
deviation) deviation)
11.97 a-Pinene 000080-56-8 0.27F0.04a 0.29F.06
14.69 h-Pinene 000127-91-3 0.28F0.27 0.45F0.04
17.81 Limonene 005989-27-5 0.00 0.00
18.1 1,8-Cineole 000470-82-6 0.32F0.17 0.82F0.43
26.78 a-Cubebene 17699-14-8 0.16F0.15 0.23F0.20
27.16 (+)-3-Carene 13466-78-9 0.57F0.06a 0.55F0.09a
28.04 Copaene 003856-25-5 0.67F0.27 0.54F0.15
29.11 Linalool D 000078-70-6 0.1b 0.14F0.03
29.43 h-Cubebene 013744-15-5 0.15F0.06 0.15b
30.55 Pinocarvone 016812-40-1 0.75b 0.87b
30.71 Aristolene 006831-16-9 0.00 0.00
30.9 (+)-Norinone 038651-65-9 0.51F0.06 0.78F0.16
31.07 h-Elemene 000515-13-9 2.26F0.42a 2.28F0.16a
31.26 Caryophyllene 000087-44-5 4.77F0.37 5.57F1.78a
32.29 Alloaromadendrene 025246-27-9 1.52F0.32a 0.27b
32.39 Myrtenal 000564-94-3 0.84b 1.25F0.32
33.54 y-Cadinene 000483-76-1 2.70F1.68 2.31F2.09
34.08 g-Amorphene 030021-74-0 0.00 0.74F0.03a
34.74 Verbenone 001196-01-6 0.89F0.23 1.18F0.17a
35.49 h-Germacrene 015423-57-1 0.71F0.08 0.39F0.23
36.22 g-Cadinene 039029-41-9 1.13F0.27 1.59F0.17a
36.78 Myrtenol 000515-00-4 0.00 0.00
42.56 Caryophyllene oxide 001139-30-6 11.86F1.79 11.35F1.51a
44.74 Globulol 000489-41-8 0.00 0.00
46.24 ( )Spathulenol 077171-55-2 0.87F0.1a 0.96F0.04
a
Obtained in two of three plants.
b
Obtained in one of three plants.
700 L. Alcaraz-Meléndez et al. / Fitoterapia 75 (2004) 696–701

Table 3
Essential oils extracted from fresh and dry material of three damiana plants micropropagated in cell suspension
media
Rt (min) Library/ID CAS no. Cell suspension (% area)
Fresh material Dry material
(average/standard (average/standard
deviation) deviation)
11.97 a-Pinene 000080-56-8 0.2a 0.00
14.69 h-Pinene 000127-91-3 0.00 0.00
17.81 Limonene 005989-27-5 0.00 0.00
18.1 1,8-Cineole 000470-82-6 2.36F0.73 2.16F1.49
26.78 a-Cubebene 17699-14-8 0.49F0.11 0.37F0.25
27.16 (+)-3-Carene 13466-78-9 0.29F0.23 0.54F0.09
28.04 Copaene 003856-25-5 0.38a 0.37a
29.11 Linalool D 000078-70-6 0.17a 0.09F0.01b
29.43 h-Cubebene 013744-15-5 0.17F0.05 0.22F0.01b
30.55 Pinocarvone 016812-40-1 0.1F0.03b 0.12a
30.71 Aristolene 006831-16-9 0.00 0.00
30.9 (+)-Norinone 038651-65-9 0.00 0.09a
31.07 h-Elemene 000515-13-9 2.15F0.25b 1.68a
31.26 Caryophyllene 000087-44-5 4.19F0.36 3.94F0.2
32.29 Alloaromadendrene 025246-27-9 1.75F0.03b 1.83a
32.39 Myrtenal 000564-94-3 0.00 0.00
33.54 y-Cadinene 000483-76-1 1.77F1.37b 1.31F1.35
34.08 g-Amorphene 030021-74-0 0.00 0.38F0.2
34.74 Verbenone 001196-01-6 0.00 0.00
35.49 h-Germacrene 015423-57-1 0.62F0.08 0.55F0.01
36.22 g-Cadinene 039029-41-9 0.99F.01b 1.12F0.02
36.78 Myrtenol 000515-00-4 0.00 0.00
42.56 Caryophyllene oxide 001139-30-6 12.57F2.06 13.73F0.35b
44.74 Globulol 000489-41-8 0.29F.03 0.39a
46.24 ( )Spathulenol 077171-55-2 0.88F0.05 1.03F0.11
a
Obtained in one of three plants.
b
Obtained in two of three plants.

different sources. Micropropagated plants had more homogeneity of essential oils than
wild plants, whose contents of essential oils had large standard deviations. Damiana is a
wild plant in a process of domestication. High variability of essential oils between
individual specimens support previous conclusions that wild populations of medicinal
plants typically have a high degree of genetic variability, and additionally, represent a
substantial part of the available genetic diversity in the world [10]. The active chemical
constituents of medicinal plants are affected by numerous factors, including genetic
differences that are not morphologically obvious and the ontogeny of individual plants or
populations that affect secondary metabolite production [11]. From our analyses of the
non-aggregated, individual assay data, we determined which ecotype or variety is best
endowed with biologically active compounds. This variation could form the basis of an
industry dedicated to cultivating specialized ecotypes for different medicinal applications
[12]. Our results support a program of continued selection of damiana with identified
important compounds, and to propagate them by tissue culture. We continue to analyze
 L. Alcaraz-Meléndez et al. / Fitoterapia 75 (2004) 696–701 701

different ecotypes of wild damiana from Baja California Sur to select plants with important
compounds for commercial uses.

Acknowledgements

This research was supported by a grant from the Consejo Nacional de Ciencia y
Tecnologia (CONACYT) and Sistema de Investigación del Mar de Cortéz (SIMAC No.
6005) of Mexico. At CIBNOR, Bertha Olivia Arredondo provided help in GC analysis,
Teodoro Reynoso assisted in preparing this paper and the staff editor clarified and
improved the text.

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