World Journal of Microbiology & Biotechnology (2006) 22:669–674  Springer 2006

DOI 10.1007/s11274-005-9087-z

Extracts of Acacia farnesiana and Artemisia ludoviciana inhibit growth, enterotoxin

production and adhesion of Vibrio cholerae

Santos Garcı́a, Ginebra Alarcón, Cristina Rodrı́guez and Norma Heredia*

Departamento de Microbiologı´a e Inmunologı´a, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo
León, Apdo. Postal 124-F, San Nicolás, NL, Me´xico
*Author for correspondence: Tel.: 52 (81) 8376–3044, Fax: 52 (81) 8376–3044, E-mail: norma@microbiosymas.
com

Received 3 August 2005; accepted 2 November 2005

Keywords: Acacia farnesiana, adhesion, Artemisia ludoviciana, cholera toxin, medicinal plants, Vibrio cholerae

Summary

Extracts of 32 medicinal plants commonly used in Mexico were evaluated for their effects on the growth of Vibrio
cholerae strains O1 and O139. Of these, the ethanolic extracts of Acacia farnesiana and Artemisia ludoviciana
effectively inhibited bacterial growth. The effects of these plant extracts on enterotoxin production and adhesion of
V. cholerae to Chinese hamster ovary (CHO) cells were determined. The minimal bactericidal concentration (MBC)
for growth was 4.0–7.0 mg/ml for A. farnesiana and 4.0–6.0 mg/ml in A. ludoviciana spp. mexicana. Cholera toxin
was inhibited when lower concentrations (50% or 75% of the MBC) of extracts were added to the media. Pre-
exposing bacteria or CHO cells to various concentrations of extracts affected in a different manner the adhesion
between bacteria and CHO cells.

Introduction (Garcia et al. 2002). Currently, there is no information

Cholera is an infectious diarrheal disease caused by
Vibrio cholerae, a Gram-negative, curved rod. Mobility
and mortality due to this pathogen is still to be a major
health problem worldwide. Cholera is characterized by
an acute severe and dehydrating diarrhea (Levine &
Edelman 1979). The disease is transmitted by ingesting
contaminated water (Levine et al. 1982) or food (Blake
et al. 1980; Faruque et al. 1998). To successfully infect a
human host, V. cholera cells must colonize the intestine
and produce cholera toxin (CT) (Betley et al. 1986).
Natural products derived from higher plants may
offer a new source of antibacterial agents. The use of
traditional medicine is widespread in Mexico, with large
populations relying on it. The use of plant preparations
has been well documented (Garcia-Alvarado et al.
2001), although few species have been screened for
biological activity.
The recent increased demand for minimally processed
foods with extended shelf-lives has renewed interest in
natural antimicrobials as food preservatives. Certain
metabolites, including natural products, have been
demonstrated to inhibit the production of toxins in
various bacteria (Garcia et al. 2002). Our laboratory has
shown that plant extracts can inhibit the growth and
enterotoxin production of Clostridium perfringens
about the effects of plant extracts on the production of
enterotoxin by V. cholerae.
Adhesion of bacteria to the intestinal mucosa is a
critical initial step in the pathogenesis of bacterial
infections. V. cholerae adheres to brush borders of the
villus absorptive cells in the small intestine (Nelson
et al. 1976). This is presumably followed by prolifera-
tion of the pathogen and production of the entero-
toxin, which causes diarrhea; however, symptoms may
be due in part to colonization by the pathogen itself
(Levine et al. 1988). Thus, the colonization process is
essential to the pathogenesis of cholera, as strains that
are unable to colonize the gut are also unable to cause
the disease (Srivastava et al. 1980). The CHO cell
system has been well established as a model for the
study of human intestinal infection (Alberts et al.
2000), and this cell line is known to be highly sensitive
to CT activity (Finkelstein 1973). That was the reason
we used it in this study.
Given the widespread presence of cholera, it is of
considerable interest to determine the role of natural
products on the virulence factors of V. cholerae. Here,
we have evaluated the effects of extracts of Mexican
medicinal plants on the growth and CT production of
this bacterium, as well as the adhesion of V. cholerae to
Chinese hamster ovary (CHO) cells.
670 S. Garcı´a et al.
Material and methods filter-sterilized, and maintained at 4 C for no longer
than 7 days. An aliquot was used to determine dry
Plant extracts weight.

In this work we analyzed plants commonly used in the Cultures

traditional medicine of Mexico to treat digestive or
gastrointestinal diseases. The plants were purchased
from retail markets in the metropolitan area of
Monterrey, NL (Table 1). All plants were identified by
Marco A. Guzmán (Department of Botany, Universi-
dad Autónoma de Nuevo León, San Nicolás, NL,
México). Dried plant material was washed, and 20-g
samples were immersed in either 100 ml of 50 mM
sodium phosphate buffer, pH 7.2 (aqueous extracts) or
96% ethanol (ethanolic extracts). The samples were
then ground with a mortar and pestle to extract sol-
uble material. Aqueous extracts were macerated at
4 C for 8 h, and ethanolic extracts were maintained at
room temperature overnight. The macerated samples
were then filtered through Whatman no.1 paper and
centrifuged at 10,000g for 20 min. Supernatants were
concentrated using a rotary evaporator (Buchi R 3000)
at 60 C until a small volume was obtained (20–30 ml).
The concentrated extracts were dried in an oven at
50 C, dissolved in 10–15 ml of phosphate buffer,
The V. cholerae toxigenic strains ATCC 25870 Inaba
O1, 7677 Ogawa O1, and 1837 Ogawa O139 were
maintained as stock cultures in Luria-Bertani agar (LB:
1% NaCl, 1% pancreatic digest of casein, 0.5% yeast
extract, and 1.5% agar) at room temperature. These
strains were originally provided by Elisa Elliot, Food
and Drug Administration, Washington, DC, USA.
Active cultures were obtained by transferring a loop of
surface growth of the stock culture into test tubes con-
taining 5 ml of LB broth (same composition but without
agar) and incubated overnight (16–18 h) at 37 C.
Antibacterial assay
Preliminary analysis of the antibacterial activity was
conducted using an agar diffusion technique, as
described previously (Garcia et al. 2002). Petri dishes
(150 mm) were filled with 25 ml of LB agar. Aliquots
containing 100 ll of the bacterial culture (1106 c.f.u.)

Table 1. Characteristics of the plants used in this study.

Plant name ‘‘Spanish common name’’ Family Geographical origin Part of plant used

Acacia farnesiana (L.) Willd ‘‘huizache’’ Leguminoseae Subfam. Tropical America Barks
Mimosaceae
Artemisia ludoviciana Nutt. ‘‘estafiate’’ Asteraceae Subfam. North and Central America Leaves
Compositae
Atriplex canescens (Pursh) Nutt. ‘‘cenischeo’’ Quenopodaceae North America Barks
Artemisia vulgaris L. ‘‘ajenjo’’ Asteraceae Subfam. Central and Southern Mexico Aerial parts
Compositae
Baccharis glutinosa Pers. ‘‘jarilla’’ Compositae North America Branches
Citrus aurantium L. ‘‘naranja agria’’ Rutaceae Asia (China) Flowers
Cnidoscolus urens (L.) Arthur ‘‘ortiga’’ Euphorbiaceae Africa Branches
E. prostrata Ait. ‘‘golondrina’’ Euphorbiaceae Africa Leaves
Flourensia cernua DC. ‘‘hojasen’’ Compositae Europe Barks
Helietta parvifolia Benth. ‘‘barreta’’ Rutaceae Asia Branches
Hyptis verticillata Jacq. ‘‘hierba del negro’’ Labiatae India Leaves
Jatropha cordata Müll.Arg. ‘‘sapo, jitotillo’’ Euphorbiaceae Africa Leaves
Juliania adstringens Schlecht. ‘‘cuachalalate’’ Julianiaceae Mexico to Peru Barks
Krameria secundiflora ex DC. ‘‘clameria, mezquitillo’’ Cromeriaceae South America Barks
Larrea tridentata Coville ‘‘gobernadora’’ Zygophylilaceae Mexico Leaves
Lippia alba N.E.Br. ‘‘salve real, te de costilla’’ Verbenaceae Peru Leaves
Malva parviflora L. ‘‘malva’’ Malvaceae Europe Branches
Mentha spicata L. ‘‘Hierba buena, menta de poleo’’ Labiatae India Aerial parts
Morus alba L. ‘‘mora’’ Moraceae South America Fruits and leaves
Ocimum basilicum L. ‘‘albahaca’’ Labeatae India Aerial parts
Ocinum micranthum Willd. ‘‘albahacar’’ Labeatae India Aerial parts
Peumus boldus Molina ‘‘boldo’’ Monimiaceae South America Leaves
Prosopis juliflora (Sw.) DC. ‘‘mezquite’’ Leguminoseae Asia and Africa Fruits
P. guajava L. ‘‘guayaba’’ Mirtaceae Mexico and Brazil Leaves
Rhizophora mangle L. ‘‘mangle’’ Rizophoraceae Tropical America Barks
Rosa centifolia L. ‘‘rosa de castilla’’ Rosaceae Mediterranean Branches
Salix taxifolia H.B. & K. ‘‘sauce’’ Salicaceae Europe Barks
Salvia coccinea Juss. ex Murr. ‘‘mirto’’ Labiatae India Branches
Solanum nigrum L.’’hierba mora’’ Solanaceae Europe Leaves
Taxodium mucronatum Ten. ‘‘sabino’’ Taxodiaceae Tropical America Barks
Vaccinium geminiflorum H.B. & K ‘‘granjeno’’. Vaccineaceae Europe Barks
Waltheria americana L. ‘‘malva, hierba del angel’’ Sterculiaceae Subtropic regions Barks
Activity of plant extracts on V. cholerae
were homogeneously inoculated across the agar surface.
Five holes (12 mm in diameter) were made in the seeded
agar plate. The holes were then filled with 200 ll of each
plant extract. Sterile phosphate buffer served as control.
Dishes were then incubated for 24 h at 37 C. Inhibitory
activity was visualized as an absence of bacterial growth
in the area surrounding the holes filled with the plant
extracts.
Minimal bactericidal concentration
Of the extracts tested, only the ethanolic extracts of
Acacia farnesiana and Artemisia ludovisiana exhibited a
significant inhibitory effect on bacterial growth. The
minimal bactericidal concentration (MBC) of both
extracts was determined by the method of Rotimi et al.
(1988). Cells (1105 c.f.u.) from activated cultures of
V. cholerae were grown in culture tubes containing
3 ml of LB broth in the presence of various concen-
trations of extracts (added in increments of 0.1 mg/ml).
Cultures were then incubated at 37 C for 24 h, and
bacterial survival was determined by plate count using
LB agar. The MBC was regarded as the lowest con-
centration of the extract that did not permit any visible
bacterial colony growth on the LB agar plate after the
period of incubation. Tetracycline (2 lg/ml, Sigma-
Aldrich Quı́mica) was used as positive control (Scrascia
et al. 2003).
Effect of the extracts on CT production
Aliquots of activated cultures of V. cholera (50 ll, or
5105 c.f.u.) were inoculated in 5 ml of LB broth in the
presence of A. farnesiana and A. ludoviciana ethanolic
extracts at concentrations equal to 25%, 50%, and 75%
of the MBC. For this purpose, stocks of ethanolic
extracts were prepared, and 100 ll of the appropriate
concentration were added to the medium. After incu-
bation for 18 h at 37 C, cells were centrifuged at
10,000g at 4 C for 15 min. The supernatant was
removed, freeze-dried, and resuspended in a small
volume of distilled water. Levels of CT were determined
by ELISA, as previously reported (Tamplin et al. 1988).
Water was used as a negative control.
Radiolabeling of bacterial cells
The radiolabeling of bacterial cells was performed by a
modification of the assay described by Heredia et al.
(1998). An aliquot of the activated cultures containing
5105 c.f.u. was inoculated into tubes with 3 ml of fresh
LB broth. Methyl-1-2-[3H]thymidine (Amersham Phar-
macia Biotech, UK) was added to the tubes at a final
concentration of 10 lCi/ml. The samples were then
mixed and incubated at 37 C for 3 h. Bacterial cells
were centrifuged and washed twice with phosphate-
buffered saline (PBS: 0.01 M, pH 7.2) and resuspended
in 3 ml of fresh non-radioactive LB.
671
Tissue culture
CHO-K1 cells (ATCC CCL61) were provided by Javier
Vargas, Instituto Mexicano del Seguro Social, Monter-
rey, México. Monolayers of CHO cells were prepared in
Falcon tissue culture flasks (Becton Dickinson Labware,
Oxnard, CA, USA). Cells were routinely grown in
Minimal Essential Medium (MEM, Gibco BRL, Can-
ada), supplemented with 10% fetal bovine serum, at
37 C in an atmosphere of 5% CO2–95% air. The cul-
ture medium was changed daily.
For adherence assays, post-confluent cells were used
after 2 or 3 days in culture. Cells were dispersed by the
addition of 4 ml of trypsin solution (25% w/v, Gibco,
BRL, Canada). After centrifugation, cells were resus-
pended in MEM, counted, and diluted appropriately for
the various assays.
Adhesion assay
An experiment was conducted to determine if extract
concentrations lower than the MBC (75%, 50%, and
25%) affected the viability of V. cholerae or morphology
of CHO cells. An aliquot of V. cholerae (5105 c.f.u.) or
5104 CHO cells were inoculated in 2.5 ml of LB or in
500 ll of MEM, respectively, containing the extracts.
After 3 h of incubation at 37 C, (a) viable bacterial cells
were determined by plate count, (b) CHO cells were
washed, new MEM media was added, and the number
and morphology of the cells were determined after
incubation at 37 C by 24 h. In both cases no effect was
detected in the number of viable bacteria or in the
morphology and number of CHO cells when compared
with the control (without extracts).
The adherence of V. cholerae to CHO cells in the
presence of A. farnesiana and A. ludoviciana extracts was
examined by a modification of the assay described by
Henriksson and Conway (1996). Two different proce-
dures were followed, as described below. In the first
procedure, bacteria were pre-incubated with plant
extracts. An aliquot of [3H]thymidine-labeled V. chole-
rae (5105 c.f.u.) was inoculated in 2.5 ml of LB in the
presence of A. farnesiana or A. ludoviciana extracts at
25%, 50%, or 75% of the MBC. These concentrations
varied slightly among the different strains of V. cholerae
tested, and ranged from 0.225 to 0.300 mg/ml for 25%
of the MBC, 0.150–0.200 mg/ml for 50% of the MBC,
and 0.075–0.100 mg/ml for 75% of the MBC. Sterile
distilled water was added to the tubes to bring the final
volumes to 3 ml. After 3 h of incubation at 37 C, cul-
tures were centrifuged at 10,000g for 10 min at room
temperature and washed twice with PBS. Cells were then
resuspended in 3 ml of MEM. Aliquots from this culture
(500 ll) were added to an Eppendorf tube containing
500 ll of a suspension of 5104 CHO cells. The mixture
was incubated for 1 h at 37 C in an atmosphere of 5%
CO2–95% air. The suspension was then centrifuged at
1000g for 10 min, the pellet was washed twice with
PBS, and the final pellet was resuspended in 500 ll of
672
PBS. The number of 3H-labeled V. cholerae that adhered
to CHO cells in a 50-ll aliquot of the final suspension
was determined by liquid scintillation counting (Ana-
lytic Delta, model 300). Bacterial cells pre-incubated
with phosphate buffer were used as a control.
In the second procedure, CHO cells were pre- incubated
with plant extracts. In this case, 5104 CHO cells (in a
volume of 500 ll) were washed twice in PBS and resus-
pended in 500 ll of MEM containing the same concen-
trations of extracts used in the first procedure (where
bacteria were pre-incubated). After 1 h of incubation at
37 C in an atmosphere of 5% CO2–95% air, cells were
centrifuged at 1000g at room temperature and washed
twice with PBS. Cells were then resuspended in 500 ll of
MEM and then added to an Eppendorf tube containing
500 ll of radioactive V. cholerae (5105 c.f.u.). The
sample was incubated for an additional 1 h at 37 C in an
atmosphere of 5% CO2–95% air. The suspension was
then centrifuged at 1000g for 10 min, the pellet was
washed twice with PBS, and the final pellet was resus-
pended in 500 ll of PBS. The amount of radioactive
V. cholerae that adhered to CHO cells in a 50-ll aliquot of
the final suspension was determined by liquid scintillation
counting. CHO cells pre-incubated with phosphate buffer
were used as a control.
All experiments (MBC, enterotoxin determination,
and adhesion assays) were performed in duplicate a total
of three times. The ANOVA test (p £ 0.05) was used to
determine statistical significance.
Partial characterization of the extracts of A. farnesiana
and A. ludoviciana
Each extract was subjected to screening for phyto-
chemical compounds: polyphenolics (analysis of
OH-phenolics), flavonoids (Shinoda reaction), carbo-
hydrates (anthrone test), and alkaloids (Dragendorff
test) (Silva et al. 1998).
Results and discussion
Antimicrobial testing and MBC determination
In this study, 32 plants were analysed for their ability to
inhibit bacterial growth of V. cholerae (Table 1). Of these,
Table 2. MBC of two medicinal plants commonly used in Mexico against three V. cholerae strains.
Strain (serogroup) MBC (mg/ml)
A. farnesiana
Ethanolic
7677 (O1) 4.0±0.0*
1837 (O139) 7.0±0.0
ATCC 25870 (O1) 4.0±0.1
* Standard deviation (p £ 0.05).
S. Garcı´a et al.
the ethanolic extracts of A. farnesiana and A. ludoviciana
most efficiently inhibited bacterial growth, while the other
extracts exhibited no detectable antimicrobial activity.
When 20 g of dried plant was macerated with 100 ml of
ethanol, 420 and 731 mg of dry extract was obtained for
A. farnesiana and A. ludoviciana respectively. Since the
activity of the aqueous extracts was very low, the extrac-
tion yields for these were not determined. The MBC val-
ues (V. cholerae strain-dependent) of A. farnesiana
ethanolic extracts were 4.0–7.0 mg/ml and of A. ludovi-
ciana were 4.0–6.0 mg/ml (Table 2). The ethanolic ex-
tracts of both plants were 10-fold more effective
compared to the aqueous samples (Table 2). This suggests
that ethanol is a better solvent for isolating the antibac-
terial compounds. All the tested strains were sensitive to
tetracycline (MBC was 2 lg/ml for all the strains) that
was used as an assay control.
A. ludoviciana (also known as estafiate) was originally
brought to Mexico by the Spanish but is now considered
a weed. Bark of estafiate is commonly sold in medicinal
plant stores in Mexico. A solution of the boiled chips has
been reported to have many medicinal properties against
stomach aches, diarrhea, and vomiting (Martinez 1936).
A. farnesiana (also known as huizache, cassie, sweet
Acacia, popinac, or sponge tree) thrives abundantly in
the Sonora desert and in tropical and subtropical cli-
mates throughout Mexico. Folkloric Mexican medicine
uses the flowers, leaves, and roots to make soothing teas
and washes, to treat bladder problems or as a topical
antiseptic for oral inflammations. The astringent fruit is
also used to treat dysentery (Garcia-Alvarado et al. 2001).
The extract of this plant is active against C. perfringens
but not against Salmonella spp. or Escherichia coli (Sot-
ohy et al. 1995). No information is available about its
effect on V. cholerae.
CT production
To treat or prevent a disease, it is important not only to
kill the pathogen but also to inhibit the production or
activity of its virulence factors. Previous studies have
evaluated the antimicrobial activity of various plant ex-
tracts against enteropathogenic organisms (Garcia et al.
2002) such as the oleuropein, a natural phenolic com-
pound extracted from olives, inhibits the production of
enterotoxin B and other exoproteins of Staphylococcus
A. ludoviciana
Aqueous Ethanolic Aqueous
45.0±2.9 4.0±0.0 50.0±2.9
45.0±0.0 6.0±0.7 50.0±0.9
40.0±3.0 5.0±0.1 50.0±2.0
Activity of plant extracts on V. cholerae 673

aureus (Tranter et al. 1993). Garlic oil and onion oil have
been shown to diminish toxin production by
C. botulinum type A in meat slurry (De Wit et al. 1979).
More recently, our laboratory reported that extracts of
Euphorbia postrata, Haematoxylon brasiletto, and
Psidium guajava completely inhibited enterotoxin pro-
duction by C. perfringens (Garcia et al. 2002) and that
H. brasiletto extracts inhibited growth and enterotoxin
production of V. cholerae (Garcia et al. 2005)
Our results showed that both A. farnesiana and
A. ludoviciana were able to inhibit CT production. No
CT formation was detected at levels of plant extracts
lower than the MBC equivalent to 25%, 50%, and 75%
for A. farnesiana and 50% and 75% for A. ludoviciana
(Table 3). At 25% of the MBC (1.0–1.5 mg/ml), extracts
of A. ludoviciana reduced CT production by 76–93%.
Plant extracts would be expected to act by interfering
either directly or indirectly with a physiological process
to reduce CT production. The ANOVA test showed
differences (p £ 0.05) in all the treatments when these
were compared with controls.
Adhesion assay
To examine the effect of A. farnesiana and A. ludoviciana
extracts on bacterial adherence we used the CHO cells
monolayer system. This system has been well established
as a model for human intestinal infection (Alberts et al.
2000), and this cell line is known to be highly sensitive to
CT activity (Finkelstein 1973).
Adhesion was reduced in a concentration-dependent
manner when V. cholerae bacteria were pre-incubated
with plant extracts (Table 3). This could suggest that
ethanol-soluble extracts of A. farnesiana and A. ludovi-
ciana affect specific bacterial properties that may alter
bacterial adhesion, perhaps blocking bacterial receptors.
In most cases, the statistical analysis (p £ 0.05) showed
differences between treatments and controls.
Dramatically different results were observed when
CHO cells were pre-incubated with plant extracts;
adhesion was increased in almost all strains with the
A. farnesiana extract (Table 3). This suggests that the
extract alters the cell surface in such a way that
adhesion is favored. However, no statistical differences
(p £ 0.05) were observed in the case of A. ludoviciana
extract.
Previous investigations of natural anti-plaque agents
from ‘‘Nigerian chewing sticks’’ identified compounds
that significantly altered the in vitro adherence of
selected oral streptococci to glass and synthetic
hydroxylapatite substrates (Wolinski & Sote 1983,
1984). The results indicated that these plant compounds
might bind to bacterial adhesins and disrupt the avail-
ability of receptors on the cell surface (Branter & Grein
1994).
Phytochemical screening
Several phytochemical compounds have been de-
scribed in A. farnesiana and A. ludoviciana. In the case
of A. farnesiana, b-sitosterol, tyramine and kaempferol
have been found in leaves; anisaldehyde, benzyl alco-
hol, benzaldehyde, p-cresol, methyl salycilate and
eugenol were reported in flowers (Garcia-Alvarado et
al. 2001). Also, alpha-terpineol, anisaldehyde, benzoic
acid, beta-ionone, coumanine, cuminaldehyde, ellagic
acid, eugenol, gallic acid, isorhamnetin-3-rutinoside,
kaempherol, methyl eugenol, methyl gallate and sali-
cylic acid have been described in this plant (Duke
1992). In the case of a related plant of A. ludoviciana,

Table 3. Effect of A. farnesiana and A. ludoviciana ethanolic extracts on CT production of V. cholerae strains and relative levels of adhesion of
V. cholerae to CHO cells.

V. cholerae strain Plant extract mg/ml CT production % of inhibition of adhesion (Statistical differences p £ 0.05)
(serogroup) (% of MBC) mg/mg protein
(% of toxin reduction) V. cholerae cells pre-incubated CHO cells pre-incubated with
with plant extract plant extract

A. farnesiana A. ludoviciana A. farnesiana A. ludoviciana A. farnesiana A. ludoviciana

0 13.4±2.4a 0 0
7677 (O1) 3.0c,d (75) 0.0±0.0 (100) 0.0±0.0 (100) 81.7b (Yes) 68.3 (Yes) )20.4 (No) 11.4 (No)
2.0c,d (50) 0.0±0.0 (100) 0.0±0.0 (100) 80.3 (Yes) 58.1 (Yes) )13.5 (No) 9.8 (No)
1.0c,d (25) 0.0±0.0 (100) 3.2±0.0 (76) 41.2 (Yes) 32.6 (Yes) )11.8 (No) 2.3 (No)
0 12.2±3.1a 0 0
ATCC 25870 (O1) 3.0c, 3.8d (75) 0.0±0.0 (100) 0.0±0.0 (100) 89.2 (Yes) 67.5 (Yes) )32.7 (Yes) 5.2 (No)
2.0c, 2.5d (50) 0.0±0.0 (100) 0.0±0.0 (100) 79.1 (Yes) 46.6 (Yes) )18.1 (No) 4.3 (No)
1.0c, 1.3d (25) 0.0±0.0 (100) 2.1±0.9 (83) 71.0 (Yes) 32.4 (Yes) )9.1 (No) 3.7 (No)
0 24.7±1.9a 0 0
1837 (O139) 5.3c, 4.5d (75) 0.0±0.0 (100) 0.0±0.0 (100) 89.0 (Yes) 26.1 (No) )29.9 (Yes) 8.4 (No)
3.5c, 3.0d (50) 0.0±0.0 (100) 0.0±0.0 (100) 80.3 (Yes) 16.1 (No) )23.1 (Yes) 7.5 (No)
1.8c, 1.5d (25) 0.0±0.0 (100) 1.7±0.0 (93) 27.5 (No) 6.4 (No) )20.4 (No) 5.0 (No)
a
Standard deviation (p £ 0.05).
b
Data are expressed as a percentage of control levels.
c
A. farnesiana.
d
A. ludoviciana.
674
A. vulgaris L. compounds such as cineol, thujone,
tannins, bitter juice and santonine have been described
(Garcia-Alvarado et al. 2001).
Preliminary chemical analyses performed on A.
farnesiana and A. ludoviciana showed the presence of
flavonoids, and carbohydrates. Saponins and alkaloids
were not detected in both plants. At this moment work
is in progress on the complete characterization of the
anti-V. cholerae active compounds.
Conclusion
This study supports the selection of plants by ethnobo-
tanical criteria to enhance the probability of finding
species with activity against V. cholerae. Our results
point to A. farnesiana and A. ludoviciana as potential
sources of compounds active against V. cholerae. These
extracts could potentially be used as preservatives in
foods or as therapeutic agents for the control of cholera.
Future studies will be aimed at addressing these
important issues.
Acknowledgements
This work was supported by the Consejo Nacional de
Ciencia y Tecnologı́a de México (CONACYT) and by
Universidad Autónoma de Nuevo León. Ginebra Ala-
rcón was a CONACYT Fellow.
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