Pharmaceutical Biology

ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: https://www.tandfonline.com/loi/iphb20

Alkaloids from Bocconia frutescens. and Biological

Activity of their Extracts

Eugenio Sánchez-Arreola, Luis R. Hernández-Molina, José L. Sánchez-Salas &

Gudelia Martínez-Espino

To cite this article: Eugenio Sánchez-Arreola, Luis R. Hernández-Molina, José L. Sánchez-Salas

& Gudelia Martínez-Espino (2006) Alkaloids from Bocconia frutescens. and Biological Activity of
their Extracts, Pharmaceutical Biology, 44:7, 540-543, DOI: 10.1080/13880200600883106

To link to this article: https://doi.org/10.1080/13880200600883106

Published online: 07 Oct 2008.

Submit your article to this journal

Article views: 1223

View related articles

Citing articles: 1 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=iphb20
Pharmaceutical Biology
2006, Vol. 44, No. 7, pp. 540–543
Alkaloids from Bocconia frutescens and Biological Activity
of their Extracts
Eugenio S
anchez-Arreola, Luis R. Hern
andez-Molina, José L. Sanchez-Salas, and Gudelia Martı́nez-Espino
Departamento de Ciencias Quı́mico-Biol
ogicas, Escuela de Ingenierı́a y Ciencias, Universidad de las Américas,
Puebla, Cholula, Puebla, México
Abstract
In Mexican traditional medicine, Bocconia frutescens L.
(Papaveraceae) is known as ‘‘gordolobo’’ or ‘‘llorasangre.’’
Natives use this plant to treat skin ulcer, dermatitis, and
some respiratory tract infections. In this study, the aerial
parts afforded the alkaloids dihydrochelerythrine, (
)-6-
acetonyldihydrochelerythrine, (
)-6-acetonyldihydrosan-
guinarine, as well as b-amyrine acetate and 2-decanol.
The structures were determined by spectral methods
including UV, IR, NMR and mass spectrometry. The anti-
microbial activity of extracts and pure compounds was
tested with Escherichia coli, Staphylococcus aureus, Pseu-
domonas aeruginosa, and Bacillus subtilis. This study sup-
ports the use of B. frutescens in traditional medicine.
Keywords: Alkaloids, antimicrobial activity, Bocconia
frutescens, Papaveraceae, phytochemistry.
Introduction
Use of plants by human societies has a long and interesting
history. It is well-known that, since remote times, plants
satisfy diverse necessities, among which are recovery and
maintenance of health; this is very important in both rural
and urban areas in many countries. The knowledge of the
local flora by numerous ethnic groups of México repre-
sents one of the main linkages to their past culture. An
important part of this flora is traditionally used by villagers
as medicinal plants for alleviating a large number of com-
mon diseases. This is also true for many ethnic cultures of
other countries.
Various studies involving chemistry and biological
activities have been undertaken on species of the genus
Accepted: June 12, 2006
Address correspondence to: Eugenio Sanchez-Arreola, Departamento de Quı́mica y Biologı́a, Escuela de Ciencias, Universidad de
las Américas, Puebla, Santa Catarina Martir, San Andrés Cholula, Puebla, 72820 México; E-mail: eugenio.sanchez@udlap.mx
DOI: 10.1080/13880200600883106 # 2006 Informa Healthcare
Bocconia (Papaveraceae). From Bocconia latisepala were
isolated the compounds sanguinarine, protopine, cheler-
ythrine, and allocryptopine (Domı́nguez, 1965), which
were also isolated from B. arborea and B. cordata (McLean
et al., 1969). From B. cordata, Konda et al. (1985) isolated,
in addition, the compound bocconine. From the lipophilic
leaf extract of B. integrifolia was identified the compound
12-methoxydihydrochelerythrine (Oechslin et al., 1991).
Navarro et al. detected antimicrobial compounds in B.
arborea (Navarro et al., 1998). Recently isolated from
aerial parts of Bocconia arborea were the compounds
(
)-6-acetonyldihydrosanguinarine (1), (
)-6-acetonyldi-
hydrochelerythrine (2a) (Fig. 1), (
)-6-methoxydihydro-
chelerythrine, (
)-sanguidimerine, chelidimerine, and
two new compounds named bocconarborine A and B
(Julian & Delgado, 2001). From the roots of Bocconia frutes-
cens, bioactive benzo(c)phenanthridine alkaloids were iso-
lated, which inhibit specific receptors of the human
angiotensin AT1 and endothelin ETA (Caballero-George
et al., 2002, 2003).
This is the first time that the phytochemistry of the
aerial parts of Bocconia frutescens L. and the biologi-
cal activity of their extracts were studied in order to
support its use in traditional medicine. The aerial
parts of this plant are used in an infusion or pulver-
ized. The infusion is used to cure some digestive
and respiratory tract infections such as bronchitis
(Martı́nez, 1984). On the other hand, the juice of this
plant or the pulverized plant is used in the treatment
of skin ulcers and eruptions (Martı́nez, 1977). In
México, B. frutescens, traditionally known as ‘‘gordo-
lobo’’ or ‘‘llorasangre,’’ is a tree that grows in the
Tamaulipas, San Luis Potosı́, Veracruz, Oaxaca,
Chiapas, and Puebla states.
 Biological activity of Bocconia frutescens
Figure 1. Structures of Compounds 1 and 2.
Materials and Methods
General experimental procedures
Column chromatography was carried out on Macherey-
Nagel Silica gel 60 (70-230 mesh). Optical rotations were
measured on a Perkin-Elmer 341 polarimeter (Germany).
UV spectra were measured on a Varian Cary 100 spec-
trometer (Australia). IR spectra were obtained on a
Biorad FTS135 spectrometer (USA), and 200 MHz 1H
and 50.3 MHz 13C NMR spectra were recorded on a Var-
ian Gemini 2000 spectrometer (USA).
Plant material
This plant was collected in the Veracruz state, México,
during December 2001. A specimen voucher (V-5488)
was deposited at the herbarium of the Instituto de Inves-
tigaciones Biol
ogicas, Universidad Veracruzana, Xalapa
region.
Preliminary tests
Qualitative tests for leaves and stems, involving color-
ation and precipitation reactions, were performed to
detect active principles like alkaloids, leukoanthocya-
nins, saponins, triterpenes, glycosides, steroids, and
anthraquinones. These tests were performed according
to the method described by Domı́nguez (1973).
541
Extraction and isolation
Dry and pulverized leaves (200 g) of B. frutescens were
extracted with hexane to afford green oil (15 g). A por-
tion (5 g) was column chromatographed on silica gel.
Fractions eluted with hexane-CH2Cl2-ethanol (1:1:0.1)
contained (
)-6-acetonyldihydrochelerythrine (2a) as a
yellowish solid, which was recrystallized to obtain fine
and colorless crystals; the next fractions, eluted with
the same solvent, contained the mixture of (2a) and dihy-
drochelerythrine (2b) (Fig. 1), which was further purified
in a silica gel column using CHCl3-methanol.
On the other hand, dry leaves of B. frutescens (235 g)
were extracted with ethanol to afford green oil (16 g). A
portion (8 g) was also column chromatographed on silica
gel using hexane-EtOAc (0–100%) as solvent. From the
first fractions, eluted with hexane, 2-decanol was isolated.
Fractions eluted with hexane-EtOAc (19:1) contained
(
)-6-acetonyldihydrosanguinarine (1), which was puri-
fied by other column chromatography. Fractions eluted
with hexane-EtOAc (9:1) contained b-amyrine acetate,
which was purified by crystallization (Mahato & Kundu,
1994).
Biological method of evaluation
Disk diffusion method, employed to evaluate the
extracts, is one of the bioassays mostly utilized for its
simplicity and reproducibility in Müeller-Hinton
[National Committee for Clinical Laboratory Standards:
Performance standards for antimicrobial disk suscepti-
bility test (Approved standard, M2-A5). Villanova, PA,
USA, National Committee for Clinical Laboratory Stan-
dards, 1993]. The microorganisms used as reference for
this test were Staphylococcus aureus (ATCC 4012), Bacil-
lus subtilis (ATCC 465), Pseudomonas aeruginosa (ATCC
260), and Escherichia coli (ATCC 128). Positive control
was ciprofloxacin (5 mg=disk). Negative control was
sterile distilled water.
Microorganisms were inoculated in 5 ml of Trypticase
Soy Broth (TSB) and incubated during 18 h at 37C.
Then, the inoculums were adjusted in comparison with
Tube 0.5 of McFarland standard (equivalent to
1.5  106 units of colonies formed per milliliter). The
extracts to test were prepared in a solution at 5% with
the proper solvent. The paper filter disks were sterilized
and impregnated with 10 ml of samples, previously fil-
tered through a 0.22-mm filter. The negative and positive
controls were prepared as described for the samples at
the same time.
Each strain was spread with a cotton swab in three
directions. After the plates were dried, the disks impreg-
nated with each extract were placed over the agar. Each
test was made by three replications and then incubated
for 24 h at 37C. The positive cases were determined by
the presence of an inhibition zone in the disk containing
542 E. S
anchez-Arreola et al.

Table 1. Metabolites in leaves and stems of Bocconia frutescens. Antimicrobial activity

Test Leaves Stems The results of the bioassays of antimicrobial activity for
E. coli, S. aureus, P. aeruginosa, and B. subtilis are sum-
Rapid method of þ þ
marized in Table 2, in which is shown the activity of the
Web for alkaloids
Method of Cain þ 
whole extracts against E. coli and S. aureus.
for alkaloids The diffusion test in disk was undertaken for the
Method of Cain   four purified compounds isolated from the leaves of
for leukoanthocyanins B. frutescens to detect possible antibacterial activity,
Method of Cain þ  but none of the pure compounds showed inhibitory
for saponins effects on bacteria.
Method of Cain þ 
for triterpenes
Method for   Conclusions
cardiotonic glycosides B. frutescens is distributed widely in tropical and temper-
Method for steroids þ  ate zones, pine and mesophyllic forest. In the America
Method for  
continent, its distribution goes from north of México
anthraquinones
to south of Bolivia, including the Caribbean islands.
Method for  
cyanogenic glycosides Common names in México, among others, are ‘‘cuatla-
taya,’’ ‘‘gordolobo,’’ ‘‘llora sangre,’’ ‘‘mano de le on,’’
and ‘‘palo santo.’’
the extract, which is measured with the aid of a ruler or Because of the different kinds of compounds observed
vernier. in preliminary phytochemical tests like alkaloids, sapo-
nins, terpenes, and steroids, the medicinal value of this
species was established. It is already established that anti-
Results and Discussion bacterial action is attributed to the presence of these
metabolites. Hence, detection of these kinds of metabo-
Preliminary tests for detection of secondary metabolites lites is very important to decide to test the extracts for
These tests were performed in the laboratory following antimicrobial effectiveness.
the methodology mentioned above. In Table 1 is listed With regard to the antimicrobial activity, the inhi-
the results for leaves and stems. bition grade presented by different extracts on the bac-
terial cultures clearly showed the action of leaf ethanol
extract as well as the stem hexane extract on S. aureus
Identification of secondary metabolites (Gram positive) and E. coli (Gram negative) (Table 2).
In this study, the following substances were isolated: (
)- Both leaf hexane extract and stem CH2Cl2 extract
6-acetonyldihydrosanguinarine (1), (
)-6-acetonyldihy- showed moderate activity. However, the concentration
drochelerythrine (2a), dihydrochelerythrine (2b), b-amyr- was not sufficient to cause an evident activity on P. aer-
ine acetate, and 2-decanol. The compounds isolated were uginosa and B. subtilis cultures; only the positive control
purified by column chromatography on silica gel. The showed activity against these bacteria.
structures were determined by UV, IR, 1H and 13C Because more than 90% of Staphylococcus aureus
NMR, and mass spectra. These spectra are in good strains are resistant to penicillin and other antibiotics
agreement with the spectral data reported in the litera- (Coello et al., 1994) and may cause important diseases, it
ture (Mahato & Kundu, 1994; Juli an & Delgado, 2001). is very significant that extracts of Bocconia frutescens show

Table 2. Inhibition halos of B. frutescens extracts.

E. coli S. aureus P. aeruginosa B. subtilis

Sample ATCC 128 ATCC 4012 ATCC 260 ATCC 465

1. Leaf, EtOH 17 mm 21 mm 0 0
2. Leaf, hexane 5 mm 6 mm 0 0
3. Leaf, CH2Cl2 0 0 0 0
4. Leaf, MeOH 0 0 0 0
5. Stem, hexane 11 mm 14 mm 0 0
6. Stem, CH2Cl2 9 mm 10 mm 0 0
(þ) Ciprofloxacin 5 mg=d 32 mm 32 mm 32 mm 26 mm
() Sterile distilled water 0 0 0 0
 Biological activity of Bocconia frutescens
activity against this microorganism. S. aureus may mainly
cause infections in the respiratory tract and in skin
wounds. On the other hand, E. coli may cause infections
in the digestive tract. Based on the fact that the extracts
of Bocconia frutescens show activity against these micro-
organisms, its traditional medicine use, like those described
in the ‘‘Introduction’’ section, is totally supported.
The chemical study on leaf ethanol and hexane extracts
afforded (
)-6-acetonyldihydrochelerythrine (2a), (
)-6-
acetonyldihydrosanguinarine (1), b-amyrine acetate, and
2-decanol, but no compound showed activity with the
bacteria used. This fact can be due to the following factors:
(a) the activity is due to a synergic action; (b) the active
substance is a product of the metabolic transformation
of the isolated products; (c) the isolated metabolites really
have no activity against the bacteria used. More studies
should be performed to elucidate these suggestions.
This plant is used in many countries of the Americans
as a medicinal; however, in other countries of the world,
this species is considered as a pest. For example, in
Hawaii, where this plant is known as plume poppy, it is
being fought due to its invasive ability (ISCs & CGAPS,
2003; Chimera, 2004; Klasner et al., 2004). This plant
was first cultivated as ornamental, but now grows wild.
No medicinal use is given in Hawaii; however, in light
of these investigations, it would be very important and
useful to take advantage of this plant as a medicinal,
instead of eradicating it from Hawaii’s forests.
Acknowledgment
We wish to thank to M.S. Santiago Mario Vazquez
Torres for plant collection and its classification.
References
Caballero-George C, Vanderheyden PML, Apers S, Van den
Heuvel H, Solis PN, Gupta MP, Claeys M, Pieters L,
Vauquelin G, Vlietinck AJ (2002): Inhibitory activity
on binding of specific ligands to the human angiotensin
II AT1 and endothelin 1 ETA receptors: Bioactive benzo
[c]phenanthridine alkaloids from the root of Bocconia
frutescens. Planta Med 68: 770–775.
Caballero-George C, Vanderheyden PML, Solis PN, Gupta
MP, Pieters L, Vauquelin G, Vlietinck AJ (2003): In
vitro effect of sanguinarine alkaloid on binding of
[3H]candesartan to the human angiotensin AT1 recep-
tor. Eur J Pharmacol 458: 257–262.
543
Chimera C (2004): Bird-facilitated dispersal of the invasive
tree Bocconia (Bocconia frutescens) in a Hawaiian dry
forest. Hawaii Conservation Conference Abstracts p. 5.
Coordinating Group on Alien Pest Species (CGAPS).
Honolulu, HI.
Coello R, Jiménez J, Garcı́a M, Arroyo P, Minguez D,
Fern andez C, Cruzet F, Gaspar C (1994): Prospective
study of infection, colonization and carriage of methi-
cillin-resistant Staphylococcus aureus in an outbreak
affecting 990 patients. Eur J Clin Microbiol Infect Dis
13: 74–81.
Domı́nguez XA (1973): Métodos de Investigaci on Fitoquı́-
mica. México, Noriega Editores, Editorial LIMUSA.
Domı́nguez XA, Garcı́a J, Monroy A (1965): A chemical
study of Bocconia latisepala Wats. Can J Chem 43:
679–682.
ISCs and CGAPS (Invasive Species Committees of Hawaii
and Coordinating Group on Alien Pest Species)
(2003): Report for the Calendar 2003 year.
Juli
an A, Delgado G (2001): (
)-Bocconarborines A and B,
novel 1,3-bis-benzo[c]phenanthridinyl acetone alkaloids
from Bocconia arborea. Rev Soc Quı́m Méx 45: 189–194.
Klasner F, Hu D, Dicus G (2004): National Park service
invasive species inventories and ‘‘vital signs’’ monitoring.
Hawaii Conservation Conference, Abstracts, p. 15.
Coordinating Group on Alien Pest Species (CGAPS).
Honolulu, HI.
Konda Y, Harigaya Y, Onda M (1985): Studies on the con-
stituents of Bocconia cordata. Structure elucidation of
bocconine by means of nuclear magnetic resonance
spectroscopic studies. J Heterocycl Chem 23: 877–879.
Mahato SB, Kundu AP (1994): 13C NMR spectra of penta-
cyclic triterpenoids—A compilation and some salient
features. Phytochemistry 37: 1517–1575.
Martı́nez OE (1977): Flora de Veracruz, fascı́culo 77. Vera-
cruz, Instituto de Ecologı́a, A. C. Xalapa, Riverside,
CA, University of California, pp. 25–27.
Martı́nez M (1984): In: Las Plantas Medicinales De México,
3rd ed. Bota: Mexico city, MX, México.
McLean DB, Gracey DE, Saunders JK, Rodrigo R, Manske
RHF (1969): Some benzophenanthridine alkaloids
from Bocconia arborea. Can J Chem 29: 1951–1956.
Navarro V, Rojas G, Delgado G, Lozoya X (1998): Anti-
microbial compounds detected in Bocconia arborea
extracts by a direct bioautographic method. Arch Med
Res 29: 191–194.
Oechslin SM, Oechslinmerkel K, Wright AD (1991): A
NMR-study of 4-benzophenanthridine alkaloids. J Nat
Prod 54: 519–524.