Extracts of Acacia farnesiana and Artemisia ludoviciana inhibit growth, enterotoxin production and adhesion of Vibrio cholerae

World Journal of Microbiology & Biotechnology (2006) 22:669–674 DOI 10.1007/s11274-005-9087-z

 Springer 2006

Extracts of Acacia farnesiana and Artemisia ludoviciana inhibit growth, enterotoxin production and adhesion of Vibrio cholerae Santos Garcı´ a, Ginebra Alarco´n, Cristina Rodrı´ guez and Norma Heredia* Departamento de Microbiologı´a e Inmunologı´a, Facultad de Ciencias Biolo´gicas, Universidad Auto´noma de Nuevo Leo´n, Apdo. Postal 124-F, San Nicola´s, 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. Preexposing bacteria or CHO cells to various concentrations of extracts affected in a different manner the adhesion between bacteria and CHO cells.

Introduction 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

(Garcia et al. 2002). Currently, there is no information 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 proliferation of the pathogen and production of the enterotoxin, 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.

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S. Garcı´a et al. filter-sterilized, and maintained at 4 C for no longer than 7 days. An aliquot was used to determine dry weight.

Material and methods Plant extracts In this work we analyzed plants commonly used in the 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. Guzma´n (Department of Botany, Universidad Auto´noma de Nuevo Leo´n, San Nicola´s, NL, Me´xico). 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 soluble 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,

Cultures 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 containing 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. Mimosaceae Asteraceae Subfam. Compositae Quenopodaceae Asteraceae Subfam. Compositae Compositae Rutaceae Euphorbiaceae Euphorbiaceae Compositae Rutaceae Labiatae Euphorbiaceae Julianiaceae Cromeriaceae Zygophylilaceae Verbenaceae Malvaceae Labiatae Moraceae Labeatae Labeatae Monimiaceae Leguminoseae Mirtaceae Rizophoraceae Rosaceae Salicaceae Labiatae Solanaceae Taxodiaceae Vaccineaceae Sterculiaceae

Tropical America

Barks

North and Central America

Leaves

North America Central and Southern Mexico

Barks Aerial parts

North America Asia (China) Africa Africa Europe Asia India Africa Mexico to Peru South America Mexico Peru Europe India South America India India South America Asia and Africa Mexico and Brazil Tropical America Mediterranean Europe India Europe Tropical America Europe Subtropic regions

Branches Flowers Branches Leaves Barks Branches Leaves Leaves Barks Barks Leaves Leaves Branches Aerial parts Fruits and leaves Aerial parts Aerial parts Leaves Fruits Leaves Barks Branches Barks Branches Leaves Barks Barks Barks

Artemisia ludoviciana Nutt. ‘‘estafiate’’ Atriplex canescens (Pursh) Nutt. ‘‘cenischeo’’ Artemisia vulgaris L. ‘‘ajenjo’’ Baccharis glutinosa Pers. ‘‘jarilla’’ Citrus aurantium L. ‘‘naranja agria’’ Cnidoscolus urens (L.) Arthur ‘‘ortiga’’ E. prostrata Ait. ‘‘golondrina’’ Flourensia cernua DC. ‘‘hojasen’’ Helietta parvifolia Benth. ‘‘barreta’’ Hyptis verticillata Jacq. ‘‘hierba del negro’’ Jatropha cordata Mu¨ll.Arg. ‘‘sapo, jitotillo’’ Juliania adstringens Schlecht. ‘‘cuachalalate’’ Krameria secundiflora ex DC. ‘‘clameria, mezquitillo’’ Larrea tridentata Coville ‘‘gobernadora’’ Lippia alba N.E.Br. ‘‘salve real, te de costilla’’ Malva parviflora L. ‘‘malva’’ Mentha spicata L. ‘‘Hierba buena, menta de poleo’’ Morus alba L. ‘‘mora’’ Ocimum basilicum L. ‘‘albahaca’’ Ocinum micranthum Willd. ‘‘albahacar’’ Peumus boldus Molina ‘‘boldo’’ Prosopis juliflora (Sw.) DC. ‘‘mezquite’’ P. guajava L. ‘‘guayaba’’ Rhizophora mangle L. ‘‘mangle’’ Rosa centifolia L. ‘‘rosa de castilla’’ Salix taxifolia H.B. & K. ‘‘sauce’’ Salvia coccinea Juss. ex Murr. ‘‘mirto’’ Solanum nigrum L.’’hierba mora’’ Taxodium mucronatum Ten. ‘‘sabino’’ Vaccinium geminiflorum H.B. & K ‘‘granjeno’’. Waltheria americana L. ‘‘malva, hierba del angel’’

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 concentrations 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 concentration 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, SigmaAldrich Quı´ mica) 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 incubation 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 Pharmacia 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 phosphatebuffered 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, Monterrey, Me´xico. 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, Canada), supplemented with 10% fetal bovine serum, at 37 C in an atmosphere of 5% CO2–95% air. The culture 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 resuspended 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 procedures were followed, as described below. In the first procedure, bacteria were pre-incubated with plant extracts. An aliquot of [3H]thymidine-labeled V. cholerae (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, cultures 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

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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 (Analytic 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 resuspended in 500 ll of MEM containing the same concentrations 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 resuspended 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 phytochemical compounds: polyphenolics (analysis of OH-phenolics), flavonoids (Shinoda reaction), carbohydrates (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,

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 extraction yields for these were not determined. The MBC values (V. cholerae strain-dependent) of A. farnesiana ethanolic extracts were 4.0–7.0 mg/ml and of A. ludoviciana were 4.0–6.0 mg/ml (Table 2). The ethanolic extracts 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 antibacterial 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 climates 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 (Sotohy 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 extracts against enteropathogenic organisms (Garcia et al. 2002) such as the oleuropein, a natural phenolic compound extracted from olives, inhibits the production of enterotoxin B and other exoproteins of Staphylococcus

Table 2. MBC of two medicinal plants commonly used in Mexico against three V. cholerae strains. Strain (serogroup)

MBC (mg/ml) A. farnesiana

7677 (O1) 1837 (O139) ATCC 25870 (O1) * Standard deviation (p £ 0.05).

A. ludoviciana

Ethanolic

Aqueous

Ethanolic

Aqueous

4.0±0.0* 7.0±0.0 4.0±0.1

45.0±2.9 45.0±0.0 40.0±3.0

4.0±0.0 6.0±0.7 5.0±0.1

50.0±2.9 50.0±0.9 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 production 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.

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 availability of receptors on the cell surface (Branter & Grein 1994). Phytochemical screening

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. ludoviciana affect specific bacterial properties that may alter

Several phytochemical compounds have been described in A. farnesiana and A. ludoviciana. In the case of A. farnesiana, b-sitosterol, tyramine and kaempferol have been found in leaves; anisaldehyde, benzyl alcohol, 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 salicylic 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 (serogroup)

Plant extract mg/ml (% of MBC)

CT production mg/mg protein (% of toxin reduction) A. farnesiana

7677 (O1)

ATCC 25870 (O1)

1837 (O139)

a

0 3.0c,d (75) 2.0c,d (50) 1.0c,d (25) 0 3.0c, 3.8d (75) 2.0c, 2.5d (50) 1.0c, 1.3d (25) 0 5.3c, 4.5d (75) 3.5c, 3.0d (50) 1.8c, 1.5d (25)

13.4±2.4a 0.0±0.0 (100) 0.0±0.0 (100) 0.0±0.0 (100) 12.2±3.1a 0.0±0.0 (100) 0.0±0.0 (100) 0.0±0.0 (100) 24.7±1.9a 0.0±0.0 (100) 0.0±0.0 (100) 0.0±0.0 (100)

Standard deviation (p £ 0.05). Data are expressed as a percentage of control levels. c A. farnesiana. d A. ludoviciana. b

A. ludoviciana

0.0±0.0 (100) 0.0±0.0 (100) 3.2±0.0 (76) 0.0±0.0 (100) 0.0±0.0 (100) 2.1±0.9 (83) 0.0±0.0 (100) 0.0±0.0 (100) 1.7±0.0 (93)

% of inhibition of adhesion (Statistical differences p £ 0.05) V. cholerae cells pre-incubated with plant extract

CHO cells pre-incubated with plant extract

A. farnesiana

A. farnesiana

0 81.7b (Yes) 80.3 (Yes) 41.2 (Yes) 0 89.2 (Yes) 79.1 (Yes) 71.0 (Yes) 0 89.0 (Yes) 80.3 (Yes) 27.5 (No)

A. ludoviciana

68.3 (Yes) 58.1 (Yes) 32.6 (Yes) 67.5 (Yes) 46.6 (Yes) 32.4 (Yes) 26.1 (No) 16.1 (No) 6.4 (No)

0 )20.4 )13.5 )11.8 0 )32.7 )18.1 )9.1 0 )29.9 )23.1 )20.4

A. ludoviciana

(No) (No) (No)

11.4 (No) 9.8 (No) 2.3 (No)

(Yes) (No) (No)

5.2 (No) 4.3 (No) 3.7 (No)

(Yes) (Yes) (No)

8.4 (No) 7.5 (No) 5.0 (No)

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 ethnobotanical 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.

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