Acta Physiol Plant (2009) 31:905–914

DOI 10.1007/s11738-009-0304-5

ORIGINAL PAPER

In vitro propagation of two antidiabetic species known

as guarumbo: Cecropia obtusifolia and Cecropia peltata
Marı́a del Pilar Nicasio-Torres Æ
Juan Carlos Erazo-Gómez Æ Francisco Cruz-Sosa

Received: 22 August 2008 / Revised: 16 February 2009 / Accepted: 9 March 2009 / Published online: 26 March 2009
Ó Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2009

Abstract Two Cecropia species (Cecropia obtusifolia compounds CA and ISO, with highest concentrations in
and C. peltata), known as guarumbo, are employed in leaves from 18-month C. obtusifolia (12.28 ± 7.06 mg g-1
Mexican traditional medicine to treat diabetes mellitus; the dry leaves of CA and 8.30 ± 2.70 mg g-1 dry leaves of
leaves of both species contain phenolic bioactive com- ISO) growth in the field. Our results offer a protocol of
pounds such as chlorogenic acid (CA) and isoorientine apical-bud use for multiplication and curative-property
(ISO), which have been attributed with hypoglycemic, conservation of the two previously mentioned important
hypolipidemic, and antioxidant properties. An in vitro Mexican medicinal plants.
propagation protocol was developed from existing apical
bud meristem from C. obtusifolia seedlings; the shoot gen- Keywords Apical buds
Chlorogenic acid
Isoorientine

eration was induced on Murashige and Skoog (MS) medium Micropropagation
Multiple shoots
supplemented with varying concentrations of 6-benzyl-
aminopurine and kinetin (Kn) combined with either Abbreviations
a-naphtalene acetic acid (NAA) or indole-3-acetic acid BAP 6-Benzylaminopurine
(IAA) auxins. Best morphogenetic response was developed CA Chlorogenic acid
with Kn 26.64 lM combined with either NAA or IAA DA Diabetes mellitus
0.57 lM, respectively; likewise, C. peltata-seedling apical MSD Minimal significant difference
buds were subjected to these best selected treatments. IAA Indole-3-acetic acid
Cecropia obtusifolia and C. peltata shoots were rooted in GA3 Gibberellic acid
growth regulator-free half-strength MS medium, and ISO Isoorientine
regenerated whole plants were adapted successfully Kn Kinetin
under greenhouse conditions and field. Leaves from both MS Murashige and Skoog medium
Cecropia-micropropagated plants produced the phenolic NAA a-Naphtalene acetic acid

Communicated by E. Lojkowska.
M. del Pilar Nicasio-Torres (&)
J. C. Erazo-Gómez
Laboratorios de Biotecnologı́a, Centro de Investigación
Biomédica del Sur (CIBIS), Instituto Mexicano del Seguro
Social (IMSS), Argentina No. 1, Col. Centro, 62790 Xochitepec,
Morelos, Mexico
e-mail: pisaliva@yahoo.com.mx
M. del Pilar Nicasio-Torres
F. Cruz-Sosa
Departamento de Biotecnologı́a, Universidad Autónoma
Metropolitana-Iztapalapa (UAM-Iztapalapa), 09340 Mexico,
DF, Mexico
Introduction
Cecropia obtusifolia (Berthold) and C. peltata (Loefl) trees
(Cecropiaceae) are endangered medicinal plants distributed
widely in tropical and subtropical regions (Aguilar et al.
1994). Both species are popularly known as guarumbo in
Mexico and are utilized in folk medicine for Diabetes
mellitus (DM) treatment by means of mature leaf-extracted
infusion (Aguilar et al. 1994, 1998; Argueta et al. 1994).
Cecropia obtusifolia’s blood glucose reduction effect was

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assessed in normal, hyperglycemic, and diabetic animal
models (Mellado and Lozoya 1984; Pérez et al. 1984;
Román-Ramos et al. 1991; Andrade-Cetto and Wiedenfeld
2001; Nicasio et al. 2005), although C. peltata’s blood
glucose reduction effect was only assessed in normal
mice (Nicasio et al. 2005). The hypoglycemic effect
was attributed to the following phenolic compounds:
chlorogenic acid (CA), a hydroxycinnamic acid derivative,
and isoorientine (ISO), a flavone (Andrade-Cetto and
Wiedenfeld 2001). It was also shown that C. peltata’s
hypoglycemic effect was better than that of C. obtusifolia
and that it is correlated positively with CA content (Nicasio
et al. 2005). Both compounds have been attributed with
several therapeutic properties, such as hypoglycemic and
hypolipidemic, as well as in vitro antioxidant activity; thus,
these activities might contribute to the prevention of type 2
DM (Ko et al. 1998; Andrade-Cetto and Wiedenfeld 2001;
Rodrı́guez de Sotillo and Hadley 2002; Sezik et al. 2005).
In view of that type 2 DM is one of the most common
metabolic illnesses worldwide and that the creation of new
antidiabetic medicines is necessary, the medicinal proper-
ties acknowledged for C. obtusifolia were evaluated in
humans with type 2 DM; the infusions administered pro-
duced important hypoglycemic and hypolipidemic effects
(Herrera-Arellano et al. 2004; Revilla-Monsalve et al.
2007). Cecropia obtusifolia leaves are at present employed
by some Mexican industries for the production of drugs
(Quı́micos y Vegetales Rowi, S.A. de C.V., Zapopan,
Jalisco; Ciencia y Salud: Fórmulas Herbolarias, México,
DF) and have been recently exported to Europe with the
same purpose (Gutiérrez-Domı́nguez and Betancourt-
Aguilar 2008).
The present need for an adequate C. obtusifolia and C.
peltata raw-material supply for further clinical investiga-
tion and phytopharmaceutical development requires
solving several problems, such as authentic Cecropia leaf
identification and the necessity for producing standardized
homogeneous extracts from species containing a known
concentration of both phenolic compounds. Plants used in
phytopharmaceutical preparations are obtained mainly
from natural growing areas; however, wide variations in
medicinal quality and content in phytopharmaceuti-
cal preparations are influenced principally by seasonal
collection, plant-to-plant medicinal-content variability,
medicinal-preparation adulteration with misidentified plant
species, and lack of adequate crop production and stan-
dardization methods (Rout et al. 2000; Manohar-Nalawade
and His-Sheng 2004). High raw vegetal material with
constant characteristics could be harvested from micro-
propagation-generated plants without ecosystem alteration.
Some protocols were designed to obtain high regeneration
rates of many plant species in vitro; thus, effects of explant
type, auxins, and cytokinins on shoot multiplication have
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Acta Physiol Plant (2009) 31:905–914
been reported for many medicinal plants as follows: Atropa
belladonna; Azadirachta indica; Cinchona ledgeriana, and
Camptotheca acuminata, etc., (Rout et al. 2000; Manohar-
Nalawade and His-Sheng 2004). Cecropia obtusifolia
multiplication was solely reported through vegetative
regeneration (La Pierre 2001); nonetheless, previous works
related with callus and shoot formations of these species
have not been reported. The aim of this study was to
develop a rapid and reproducible protocol for setting up an
in vitro multiplication method via C. peltata- and C. ob-
tusifolia-plantlet apical buds to preserve their ability to
produce bioactive compounds (CA and ISO).
Materials and methods
Plant material
Guarumbo-tree foliage and mature fruits were collected in
Cunduacán, Tabasco, Mexico, in February 2001 and May
2002. Three plant samples were authenticated by Abigail
Aguilar M.Sc. [Director, Instituto Mexicano del Seguro
Social México (IMSSM) Herbarium], and vouchers were
stored for reference under #14123 (for C. obtusifolia) and
#14115 (for C. peltata).
In vitro cultures
Germination
Cecropia obtusifolia seeds were surface-disinfected by
following standard procedures: 3 min embedded in 70%
ethanol (v/v) and later 10 min in 1.2% sodium hypochlorite
(v/v) with two drops of Tween-20 under constant stirring in
bath. The disinfected seeds were then thoroughly washed
five times with sterile distilled water and maintained
embedded in sterile water at room temperature for 24 h;
other seeds were embedded at 50°C for 30 min. Later, as
many as 25 disinfected seeds were transferred to a glass
container (five glass containers per treatment) with cotton
swabs wet with either sterile water or half-strength
Murashige and Skoog (1962) medium (MS) supplemented
with 15 g l-1 sucrose; gibberellic acid [(GA3) 2.89 lM]
was added to some of these latter containers. We adjusted
all media to pH 5.7 with 1 N NaOH and 1 N HCl prior to
autoclaving at 121°C for 18 min. Seeds were incubated at
26 ± 2°C during a light:dark (16:8 h) photoperiod under
50 lM m-2 s-1 warm-white fluorescent light intensity.
We recorded germinated seed numbers at 20, 30, and
40 days as percentage of germination for all treatments.
After these emerged, we transferred plantlets for growth
into 15 g l-1 sucrose and 8 g l-1 agar (final pH, 5.7)-
supplemented half-strength MS medium and incubated
Acta Physiol Plant (2009) 31:905–914
these under identical conditions. When C. obtusifolia-seed
best-germination conditions were known, we employed
identical conditions for C. peltata seeds.
Shoot generation
Apical 8–10-mm buds were aseptically excised from
20-day-old C. obtusifolia seedlings and transferred to
30 g l-1 sucrose and 8 g l-1 agar (final pH, 5.5)-supple-
mented whole MS medium (control); different treatments
were prepared on MS medium using 6-benzylaminopurine
(BAP) and kinetin (Kn) (at 8.88, 17.76, and 26.64 lM)
combined with either a-naphtalene acetic acid (NAA)- or
indole-3-acetic acid (IAA)-auxin 0.57-lM single concen-
trations, adding plant-growth regulators to the media prior
to autoclaving; explants incubated on MS basal medium
without growth regulators were used as control. All cul-
tures were maintained at 26 ± 2°C during a light:dark
(16:8 h) photoperiod under 50 lM m-2 s-1 warm-white
fluorescent light intensity, and we performed one sub-cul-
ture under identical conditions after 4 weeks. Variables
comprised explant numbers exhibiting morphogenesis,
shoot number per explant, and shoot length, as well as
number of leaves formed, all of which were recorded for
all treatments after 8 weeks. We replicated these experi-
ments three times with 20 explants per treatment. When
C. obtusifolia best-shooting conditions were known, we
employed identical conditions for C. peltata apical buds.
Shoot elongation and rooting
Formed shoots were transferred to 15 g l-1 sucrose and
8 g l-1 agar (final pH, 5.5)-supplemented growth regulator-
free, half-strength MS medium. Cultures were incubated at
26 ± 2°C during a light:dark (16:8 h) photoperiod under
50 lM s-1 m-2 warm-white fluorescent light intensity;
after 4 weeks, we sub-cultured plantlets under identical
conditions. At acclimatization time, we recorded root-
forming shoot number, root number formed per shoot, and
main-root and stem length.
Hardening and acclimatization
To acclimatize in vitro-origin 5-cm plantlets, plant roots
were rinsed thoroughly in tap water to eliminate nutritive-
medium residues, and were immediately embedded in the
following bactericidal and fungicidal chemical preventive
mixture: 0.5 g l-1 Ridomil fungicide [Novartis Metalaxil-
M (4.5%), chlorotalonil (72%), 0.5 g l-1 of bactericide
agrimycin (100 TSIZER-Streptomycin) (18.65%), and
oxytetracycline (2%)] for 3 min. Later, plantlets were
transplanted to potting foam cups prepared with a sterile
commercial substrate [Sunshine fine mixture (70–80%)
907
Canadian peat moss, vermiculite, ground lime (chalk), and
a moisturizing agent]; pots were covered with transparent
plastic bags to prevent desiccation, and these were main-
tained under culture room conditions [26 ± 2°C during a
light:dark (16:8 h) photoperiod under 70 lM s-1 m-2
warm-white fluorescent light intensity] for 1 week, main-
taining 85% moisture; relative humidity was reduced
gradually. Later, pots were moved to a greenhouse facility
(27 ± 2°C, wet-walled, automatic-misted watering, and
1,110 lM s-1 m-2 light intensity); subsequently, plantlets
were fertilized weekly with a commercial fertilizer
(PetersÒ, 0.6 g l-1). When plants reached 10 and 30 cm in
height, they were transferred into larger containers
(11 9 23- and 30 9 34-cm black plastic bags, respec-
tively) filled with a 2:1 commercial substrate:garden-soil
mixture. Then, potted plants were moved to a new green-
house facility and maintained under the same 30 ± 2°C
light intensity, automatic-misted watering, and bi-weekly
fertilization. Finally, ex-vitro 6 month C. obtusifolia and
C. peltata plants were grown in ground at Xalostoc,
Morelos, Mexico (18° 460 LN y 98° 580 LW) to 1,250 msnm
with a pluvial precipitation of 912.4 mm, weather semi-warm
and sub-humid and annual temperature of 20.5°C (Rebolloza
2000). Subsequently, plants were measured, considering
height, trunk perimeter, and leaf (lobulate and lanceolate)
number at 1, 2, 3, 6, 12, and 18 months of growth.
Chemical analyses
Methanolic leaf extracts
Ex-vitro plants of C. obtusifolia and C. peltata were
evaluated for CA and ISO contents by HPLC, and their
values were compared with the wild plants. Three hetero-
geneous samples of finely ground dried leaves of adult
wild-trees (0.5 g) and ex-vitro plants at the ages of 6-, 12-,
and 18-months (0.5 g) were separately extracted by mac-
eration at room temperature three times with 10 volumes
(w/v) of methanol per 24 h, followed by filtration. Extracts
were pooled, concentrated to dryness under reduced pres-
sure, and stored in glass containers at 4°C. Samples
(500 mg) of each methanolic extract were defatted with
chloroform (10% w/v) followed by filtration; remaining
materials were redissolved in (30 ml) HPLC-grade meth-
anol and filtered through 0.45 lm PVDF filter (Whatman
Inc.) for HPLC analyses (Nicasio et al. 2005).
HPLC conditions
Extract analyses containing CA and ISO were carried out on
a Waters (Separation Module 2695) HPLC with a system
controller (Millennium System Manager Software). HPLC
analyses were performed on a 100 9 4 mm Chromolith
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RP-18e column (Merck) employing a constant temperature
of 25°C during analysis; samples (50-ll) were eluted at a
1.0 ml min-1 flow rate with (A) H2O (H3PO4 0.5%)- and
(B) CH3CN–CH3OH (1:1)-gradient mobile phases and
detected by monitoring absorbance at 254 nm using a
Waters 2996 photodiode array detector. Retention times of
chlorogenic acid (CA) (Sigma Chemical Co., St Louis, MO,
USA) and isoorientine (ISO) (Indofine Chemical Co., Inc.,
USA) were 7.3 and 15.6 min, respectively; calibration
curves constructed under 20-, 40-, 80-, and 160-lg ml-1
standard solutions (methanolic solutions utilizing com-
mercial CA and ISO compounds) exhibited an R2 of
0.996. CA and ISO analyses were performed by means of
comparatione of their absorption spectra, and amounts
were calculated by the external standard method of
quantification.
Statistical analysis
Germination, multiple budding, and CA- and ISO-content
data were subjected to analysis of variance with NCSS
version 5 statistical software (Wireframe Graphics, Kays-
ville, UT, USA) (NCSS 2001), followed by Tukey, or
Minimal significant difference (MSD) multiple-range tests,
or Student t test when means were different. Values for
p \ 0.05 were statistically significant. Values in figures are
expressed as mean ± Standard error of mean (SEM).
Results and discussion
In vitro cultures
Germination
In this work, germination percentages achieved for
C. obtusifolia seeds were low (\15%). One-way ANOVA
demonstrated non-significant differences in nutritional
medium and condition (pre-hydration and GA3)-associated
C. obtusifolia-seed germination percentages; whereas incu-
bation time showed statistical differences (F0.05,2 = 3.035)
at 30 days (12.3% non-hydrated; 9.1% pre-hydrated) of
culture (MSD0.05 = 0.954), afterward, emergence (13.1%
non-hydrated; 11.2% pre-hydrated) did not increase signif-
icantly. Previous immersion in sterile water at room
temperature or 50°C, and optimal conditions of incubation
such as temperature (16–36°C) and light did not promote this
process. Highest C. obtusifolia-seed germination percent-
ages ranged between 85 and 90% once these are obtained
from the tree; nonetheless, it is known that photoblastic
latency begins after seed collection, followed by a 20–40%
germination-percentage decrease. Additionally, these seeds
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are considered recalcitrant and possess a short longevity
period (Benavides 1994; La Pierre 2001).
Cecropia peltata seeds were sterilized as described for
C. obtusifolia seeds, pre-hydrated at room temperature, and
germinated on cotton swabs wet with sterile water.
Shoot regeneration from C. obtusifolia apical buds
Axillary shoots were produced by C. obtusifolia-plantlet
apical buds cultured on MS media supplemented with
varying concentrations of cytokinins BAP and Kn com-
bined with a single concentration of one auxin (NAA or
IAA); additionally, regarding explants (8–10 mm apical
bud) growth on MS medium-free growth regulators, only
50% developed into plantlets after 2 months. Morphoge-
netic responses are presented in Table 1: in all treatments,
multiple bud formation was observed, and response level
varied according to cytokinin type (F0.05,1 = 38.55),
assessed concentrations (F0.05,1 = 19.677), and auxin type
employed (F0.05,2 = 23.641). Only BAP presence at
26.64 lM promoted bud response in all explants indepen-
dently of the auxin used; when Kn was added to the
medium, solely less frequent bud formation (87.5%) was
recorded using IAA-combined lowest concentration
(8.88 lM). However, shoot morphology with BAP/IAA
treatments presented further differentiation, exhibiting
properly developed leaves (Fig. 1a). Furthermore, certain
apical bud-generated shoots at lowest BAP and Kn con-
centrations combined with IAA or NAA exhibited callus
formation. When callus formation occurred in apical buds,
this took place in the epicotyl’s lower part; nevertheless, in
general terms, callus formation could be considered poor.
Shoot number formed varied directly with cytokinin
concentration (F0.05,2 = 19.67); according to Tukey mul-
tiple range test (Tukey qt 0.05 = 1.802), best response
occurred at highest concentration assayed (26.64 lM).
Post-Kn treatment-obtained shoot number per explant was
higher than that for BAP combined mainly with NAA
(Table 1). Best treatments were Kn cytokinin combined
with IAA or NAA auxins alone, and NAA-combined BAP.
Cecropia obtusifolia multiplication has been reported only
via vegetative regeneration with lower production per-
centage through air layering (93%) and cuttings (58.3%)
employing auxins for rooting (La Pierre 2001); nonethe-
less, to our knowledge, these are the first results reported
on C. obtusifolia-species in vitro propagation.
In addition, it is important that this work’s results are
unlike results for many medicinal plants achieved by shoot-
tip proliferation in culture from Cinchona ledgeriana,
C. succirubra, Plantago ovata, Scopolia japonica, Scro-
phularia yoshimurae, etc. (Rout et al. 2000; Manohar-
Nalawade and His-Sheng 2004), in which BAP cytokinin
Acta Physiol Plant (2009) 31:905–914 909

Table 1 Cecrofolia obtusifolia apical-explant shoot proliferation after 8 weeks of culture on 0.57 lM IAA- or 0.57 lM NAA-containing MS
medium and different cytokinin sources and concentrations
Cytokinin Concentration Auxins Overall shoot Shoots with callus Shoot number Leaf number Shoot length
(lM) formation (%) formation (%) per explant per shoot (cm)

BAP 8.88 IAA 87.5 37.5 1.75 ± 0.56f 6.44 ± 1.56 1.49 ± 0.31
17.6 IAA 62.5 0 2.75 ± 0.75f 8.79 ± 1.64 2.39 ± 0.51
26.64 IAA 100 0 3.86 ± 1.02d,e,f 2.91 ± 1.00 1.14 ± 0.21
8.88 NAA 85.7 40 2.13 ± 0.44f 7.24 ± 0.75 1.50 ± 0.05
17.6 NAA 100 0 4.00 ± 1.25c,d,e 4.13 ± 1.39 0.72 ± 0.17
26.64 NAA 100 0 5.75 ± 1.19a,b,c 3.85 ± 1.06 1.18 ± 0.20
Kn 8.88 IAA 87.5 87.5 3.50 ± 1.25d,e,f 3.79 ± 1.28 2.77 ± 0.44
17.6 IAA 100 0 3.38 ± 0.47d,e,f 3.30 ± 1.14 1.91 ± 0.25
26.64 IAA 100 0 6.13 ± 0.69a,b 2.50 ± 0.58 3.50 ± 0.50
b,c,d
8.88 NAA 100 0 4.38 ± 1.38 2.52 ± 0.65 2.51 ± 0.52
17.6 NAA 100 0 6.13 ± 0.94a,b 2.97 ± 0.28 3.26 ± 0.47
26.64 NAA 100 0 6.75 ± 1.44a 3.98 ± 1.05 2.84 ± 0.34
BAP Benzylaminopurine, Kn kinetin, NAA a-naphtalene acetic acid, IAA indole-3-acetic acid, MS medium Murashige and Skoog medium
Numbers indicate mean number of responding explants as percentage of all explants and distribution. The response is among different
organogenesis degrees. Means with identical letter are not significantly different according to Tukey multiple range test (qt = 1.802)

Fig. 1 Cecropia obtusifolia plantlets on regulator-free half strength MS medium after 56 days of culture (a) Acclimatized C. obtusifolia and
Cecropia peltata plantles (b, c), C. obtusifolia (d, e) and C. peltata (f, g) plants grown in ground

levels were shown as most critical and were mainly suc-
cessfully utilized for multiplication of many medicinal
plants (Rout et al. 2000). For C. obtusifolia tree, best bud-
ding-formation treatments were obtained with Kn combined
with IAA or NAA auxins alone; this response was similar to
that achieved with certain species micropropagated by
apical shoots and testing different cytokinins: for example,
for Pichrorhiza kurroa Royle, in vitro propagation was
developed with kinetin (4.6–13.9 lM); also, addition of
IAA improves growth of generated shoots (Lal et al. 1988).
Shoot cultures from Raphanus stivus var longipinatus were
maintained with 22.2 lM of Kn (Yoeup-Paek et al. 1987)
and shoot cultures for Cephaelis ipecacuanha were estab-
lished on medium with Kn 32.22 lM and 0.4–1.0 lM of
NAA (Jha and Jha 1989); for the species Solanum triloba-
tum, highest multiple shoot-proliferation rate was produced
with Kn (9.28 lM) and BAP (8.88 lM) (Jawahar et al.
2004). Within the same context, on Kaemferia galangal
micropropagation employing axillary buds and testing dif-
ferent concentrations and combinations of auxins and

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cytokinins, highest number of shoots-per-explant was
obtained with 13.9 lM kinetin (Vincent et al. 1992); in
addition, in vitro multiplication of Coleus forskohlii Briq
utilizing nodal explants has been better achieved on MS
medium supplemented with Kn (8.88 lM) and IAA
(0.57 lM) than with BAP (Sharma et al. 1991); while for
Mimosa tenuiflora tropical tree, best response for bud for-
mation employing seedlings axillay buds was obtained with
26.64 lM of Kn mixed with 0.57 lM of IAA (Villarreal
and Rojas 1996).
With leaves formed in shoots, both cytokinins
(F0.05,1 = 25.263) and auxins (F0.05,1 = 9.788) employed
exerted a statistically significant effect on leaf number
formed. When comparing leaf production (Table 1) using
both cytokinins, shoots with more leaves occurred using
BAP (t0.05 = 12.9); best effects were recorded at 8.88 and
17.6 lM mixed with either of the two auxins. In formed
shoot length utilizing two cytokinins (BAP and Kn),
there was a significant difference according to one-way
ANOVA (F0.05,1 = 1,192.147), assayed concentrations
(F0.05,1 = 14.815), and auxins used (F0.05,1 = 25.440). In
general terms and according to Student t test
(t0.05 = 29.99), longest shoots were formed when using
Kn (t0.05 = 29.99), especially at highest concentration
(26.64 lM) mixed with either of the two auxins (Table 1).
Leaf number-per-shoot and length comprise important
parameters in considering in vitro-generated plantlet sur-
vival during acclimatization. Higher Kn concentration in
combination with IAA increased both shoot number and
length (Table 1).
Shoot regeneration from C. peltata apical buds
Morphogenetic response of C. peltata-seedling apical buds
was developed in best treatment obtained for C. obtusifolia
species, i.e., Kn (26.64 lM)-supplemented MS combined
with auxin IAA or NAA. In the first case, we obtained
2.4 ± 0.12 shoots-per-explant with 1.5 ± 0.7 cm length
and 10.5 ± 1 leaves-per-shoot, while with NAA added to
Kn, we recorded 5.8 ± 1.9 shoots-per-explant with
1.22 ± 0.3 and 3.8 ± 1.3 leaf length-per-shoot. These
results were similar to those of C. obtusifolia.
Shoot elongation and rooting
Cecropia obtusifolia and C. peltata shoots were placed
individually on growth regulator-free half-strength MS
medium for root development. After 2 months, root for-
mations on shoots were numerous and large, and were
produced at shoot bases; growth regulator addition was not
necessary (Fig. 1a). While C. obtusifolia cuttings were
exposed to NAA auxin solution, adventitious root pro-
duction was low (58.3%) (La Pierre 2001). Cecropia
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peltata plantlets exhibited a largest root average of
8.6 ± 2.6 and 3.3 ± 1.2 cm primary roots (average root
length, 6.4 ± 0.9 cm) and bore 7.5 ± 1.8 lanceolate
leaves; although C. obtusifolia plantlets possessed a largest
root average of 12.4 ± 3.4 and 3.3 ± 1.2 cm primary roots
(average root length, 7.2 ± 0.6 cm) and bore 7.0 ± 1.2
lanceolate leaves. We observed no secondary root formation,
a characteristic desirable for optimizing the acclimatization
process from seedling to greenhouse. Subsequently, generated
plantlets with leaf-number duplication were adapted to
greenhouse conditions.
Hardening and acclimatization
The success of any tissue culture protocol depends on
efficient acclimatization of in vitro-obtained plantlets
(Manohar-Nalawade and His-Sheng 2004). Additionally,
many authors have mentioned root quality-related prob-
lems when developed in agar medium, because produced
roots in vitro are not totally functional when transferred ex-
vitro (Yu et al. 2000). In this case, in vitro-obtained
Cecropia plantlets were adapted successfully to greenhouse
and field conditions.
Plant growth was noticeable, exhibiting a 100% survival
rate after 4 weeks in the greenhouse (Fig. 1c). During the in
vitro-plantlet acclimatization process, certain growth
parameters were considered in 2-, 3-, 6-, 12-, and 18-month-
old plants (Table 2). Cecropia obtusifolia plants (Fig. 1d, e)
demonstrated a 3.3-cm monthly growth rate, which later
increased to 6.4 cm when plants were removed from the
greenhouse, and of 4.4- and 8.8-cm when these were
planted in ground and totally exposed to climatic conditions
such as illumination, temperature, humidity, etc., at the site
where ex-vitro plants were planted (Xalostoc, Morelos,
Mexico: pluvial precipitation of 912.4 mm, semi-warm and
sub-humid weather, annual temperature of 20.5°C). With
C. peltata plants (Fig. 1f, g), monthly growth rate was
6.6 cm, which later increased to 9.4 and 5.3 cm, respec-
tively, even when plants were totally exposed to
environmental conditions (Table 2). Monthly growth rates
were superior to those reported (average, 1.5 cm) for wild
C. obtusifolia plants; this effect could be due to that plants
had up to large growth-regulator quantities for shoot
induction (Benavides 1994).
Although C. obtusifolia and C. peltata plantlets had
lanceolate leaves at growing-process onset (Fig. 1b, c), leaf
lobulation began after 3 months under greenhouse condi-
tions. When plants were totally exposed to environmental
conditions, as many as 95 and 73% of individual plantlets,
respectively, developed lobulated leaves (Table 2), until
acquiring completely lobulate leaves when they were
12-months-of-age old (Fig. 1d–g); this process is typical
of immature wild trees (Benavides 1994). Leaf number and
Acta Physiol Plant (2009) 31:905–914 911

Table 2 Growth parameters of

Species Age Stem length Stem thick Leaf Lobular emergence
micropropagation-derived
(in months) (cm) (cm) (number) (%) and lobulated
Cecrofolia obtusifolia and
leaf number
Cecrofolia peltata plants
C. obtusifolia 2 8.7 ± 3.7 1.0 ± 0.3 5.9 ± 2.6 –
3 12.4 ± 3.9 1.0 ± 0.3 6.4 ± 1.2 (13.6) 1.0 ± 0.0
6 31.7 ± 10.0 1.6 ± 0.6 9.1 ± 1.4 (95) 2.1 ± 1.4
12 59 ± 29.7 1.9 ± 0.4 22 ± 6.3 (100)
18 112 ± 15.2 2.5 ± 0.5 28 ± 9.9 (100)
C. peltata 2 8.8 ± 4.5 1.1 ± 0.3 5.9 ± 2.1 –
3 16.2 ± 9.5 1.3 ± 0.4 6.4 ± 1.2 (32.6) 1.7 ± 0.0
6 44.3 ± 6.0 1.4 ± 0.6 9.1 ± 1.4 (72.7) 2.1 ± 1.4
12 76.2 ± 13.3 1.9 ± 0.4 17.7 ± 0.5 (100)
18 107.5 ± 22.4 2.9 ± 0.8 26.3 ± 18.2 (100)

stem diameter also doubled after 6 months and quadrupli-
cated after 12 months under greenhouse and field
conditions; stem-diameter increase was similar to that
(2 cm per year) for wild C. obtusifolia trees (Benavides
1994). Although, no abnormal morphologic variations were
observed among the population, three ex-vitro plant sam-
ples were authenticated by taxonomic determinations at the
IMSSM-Herbarium, and vouchers were stored for refer-
ence under #14860, 14861, and 14862 (for C. obtusifolia)
and #14863 and 14864 (for C. peltata).
CA and ISO contents
To our knowledge, this is the first time that the ISO
hypoglycemic compound has been quantified in wild
Cecropia tree leaves; other reports had been based solely
on CA content. Further, from practical and pharmaceutical
viewpoints, one important aim comprised micropropaga-
tion from existing meristems that yielded plants from
C. obtusifolia and C. peltata, which are genetically iden-
tical to donor plants (Rout et al. 2000); these ex-vitro plants
retained their ability to produce both bioactive compounds
(CA and ISO) (Fig. 2). Concentrations were significantly
different (F0.05,8,3 = 7.32, p = 0.0005) from those found
in wild trees and ex-vitro plants from the same species;
however, levels detected in ex-vitro plants were not sig-
nificantly different between species (F0.05,8,3 = 0.69,
p = 0.4162). In addition, the CA/ISO ratio was different
between wild and ex-vitro plants: in wild trees, CA is
accumulated in higher concentrations than those of ISO,
while in ex-vitro plants aged 6 months, ISO is accumulated
in higher concentrations than those of CA, although this
event was reverted in ex-vitro plants from 12- and 18-
months-of-age (Table 3). ISO and CA total concentration
in 6-month-old ex-vitro plants from C. obtusifolia and 6-
and 12-month ex-vitro plants from C. peltata were similar
than those in wild trees (Tukey qt = 12.377); 18-month-old
ex-vitro plants from C. obtusifolia was the species with
highest concentrations (20.58 mg g-1 of leaves), followed
by 18- and 12-month ex-vitro plants from C. peltata
(13.26 mg g-1 of leaves) and C. obtusifolia (9.85 mg g-1
of leaves), respectively (Table 3). It is important to
inform that CA (12.28 ± 7.06, 8.11 ± 3.94) and ISO
(8.30 ± 2.70, 5.15 ± 2.92) levels were tenfold higher than
those found in leaves of C. obtusifolia and C. peltata wild
plants. These differences among ex-vitro plants from dif-
ferent ages are valid, due to that younger plants are more
vulnerable to microbial attack than adult trees; thus, they
are required to produce higher concentrations of defense
compounds when grown in the field (Goralka and Lan-
genheim 1996; Nugroho and Verporte 2002; Shudha and
Ravishanka 2002). Additionally, these differences could be
related with environmental and ecology factors (tempera-
ture, humidity, altitude, soil, and soil microflora) of areas
where the ex-vitro (Xalostoc, Morelos; 1,250 msnm) and
wild (Cunduacán, Tabasco; 40 msnm) trees grow, as well
as nutrient availability and period of foliage collection
(Charlwood and Rhodes 1990; Goralka and Langenheim
1996; Nugroho and Verporte 2002; Shudha and Ravish-
anka 2002).
An increase has been reported in CA content with
increase of plant age, under mineral stress, dryness, and lack
in nitrogen for Helianthus agnus and Nicotiana tabacum
(Adams and Price 1978; Fritz et al. 2006). Further, Cucumis
sativus species produces phenolic compounds acting as
phytoalexins (antimicrobial secondary metabolites) when
their leaves are exposed to the presence of the fungus
Podosphaera xanthii; these compounds are C-glycosyl
flavonoids such as vitexin, isovitexin, orientin, and isoori-
entin (ISO), and they play a vital role in the defense
strategy of this species (McNally et al. 2003), and then for
C. obtusifolia and C. peltata.
Recorded concentrations in micropropagated plants
from C. obtusifolia (0.53 ± 0.00 mg g-1 CA leaves, and

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912 Acta Physiol Plant (2009) 31:905–914

Fig. 2 Chromatograms of a chlorogenic acid (CA) and isoorientine (ISO) pure compounds, and contents in b Cecropia obtusifolia wild, c
Cecropia obtusifolia ex-vitro, d Cecropia peltata wild, and e Cecropia peltata ex-vitro tree leaves

0.82 ± 0.00 mg g-1 ISO leaves) were similar to those
previously detected (0.66–3.6 mg g-1 CA leaves and 0.27–
1.30 mg g-1 of ISO leaves) in plants obtained from stem
explants (data not shown).
Measured CA concentrations (1.48 ± 0.09 mg g-1 of
C. obtusifolia leaf and 1.86 ± 0.34 mg g-1 for C. peltata
leaf) in leaves of wild trees were similar than those pre-
viously reported for leaves from the same wild species, in
which C. obtusifolia leaves had CA leaf levels of 2.0, 2.99,
0.22, and 0.7 mg g-1, as well as 0.18 mg g-1 of ISO leaf,
and C. peltata leaves had 1.5 mg g-1 of leaf (Herrera-
Arellano et al. 2004; Nicasio et al. 2005; Revilla-Monsalve
et al. 2007).
In addition ex-vitro plants retained their pharmacology
properties, methanolic extracts from leaves of wild and 6-
month ex-vitro plants administrated oral via in normal Balb
C mice (0.5 g kg-1 body weight) showed reduced blood-
glucose level after 2 and 4 h of their administration at 27.6
and 24% for wild plants, and 13.2 and 5.0% for ex-vitro
plants from C. obtusifolia; 25.0 and 37.9% for wild plants,
and 7.1 and 19.7% for ex-vitro plants from C. peltata,
respectively.

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Acta Physiol Plant (2009) 31:905–914 913

Table 3 Chlorogenic acid (CA)

Species Origin Concentration (mg g-1 of dried leaves)
and isoorientine (ISO) contents
in Cecropia obtusifolia- and CA ISO Total
Cecropia peltata-tree leaves
C. obtusifolia Wild 1.48 ± 0.12 0.83 ± 0.06 2.31b
Micro-propagated
6 months of age 0.53 ± 0.00 0.82 ± 0.00 1.35b
12 months of age 6.12 ± 0.06 3.73 ± 0.59 9.85a,b
18 months of age 12.28 ± 7.06 8.30 ± 2.70 20.58a
C. peltata Wild 1.86 ± 0.34 0.86 ± 0.21 2.72b
Micro-propagated
Means with identical letters
are not significantly different 6 months of age 0.71 ± 0.06 1.62 ± 0.33 2.33b
according to Tukey multiple 12 months of age 3.39 ± 2.30 2.21 ± 0.89 5.60b
range test (qt = 0.05, 24) 18 months of age 8.11 ± 3.94 5.15 ± 2.92 13.26a,b
(CA and ISO = 12.377)

Conclusions Fritz C, Palacios N, Fiel R, Stitt M (2006) Regulation of secondary

In vitro culture protocol represents an alternative and
interesting perspective for C. obtusifolia- and C. peltata-
trees multiplication for an adequate supply of raw mate-
rials; besides, serve as the basis for micropropagation of
other Cecropia plants. In these conditions, it may be
possible to obtain up to 200 ex-vitro plants per seedling
apical-bud in 6 months and we created an experimental
field with 200 trees. Micropropagated Cecropia-plant
leaves retained their ability to produce bioactive com-
pounds and hypoglycemic activity was corroborated in
normal mice.
Acknowledgments This work was supported by Basic grant 181150
from the Consejo Nacional de Ciencia y Tecnologı́a, México
(CONACyT, México) for PhD studies in the UAM-Iztapalapa Bio-
technology Program and from FOFOI-IMSS grant 2004/077.
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