Revista CENIC Ciencias Biológicas, Vol. 39, No. 1, 2008.
Antibacterial effect of Costus spiralis leaves extract on pathogenic strains of Vibrio cholerae Celso Pérez, Alina Falero, Blanca Rosa Hung, Talena Ledón and Rafael Fando.
Department of Biotechnology, National Center for Scientific Research, Ave. 25 y 158, Playa, P.O. Box 6412, Zip Code 10600 Havana, Cuba. Email: Recibido: 14 de marzo de 2007. Aceptado: 22 de junio de 2007. Palabras clave: Costus spiralis, Vibrio cholerae, antimicrobiano, patogénico, extracto vegetal.
Key words: Costus spiralis, Vibrio cholerae, antimicrobial, pathogenic, plant extract.
RESUMEN. La utilización de remedios de origen vegetal cubre una amplia variedad de enfermedades y constituye una vía alter-
nativa a la terapia con antibióticos, que por otra parte, no parece ser promisoria a largo plazo, debido la amplia diseminación de la
resistencia a los antibióticos entre los microorganismos patógenos. Los principios activos que muestran actividad antimicrobiana
pueden ser extraídos y purificados a partir de plantas para el desarrollo de nuevos medicamentos. Entre las diversas enferme-
dades que históricamente han afectado al hombre, el cólera ha sido potencialmente epidémico y una de las más sobresalientes.
La bacteria Vibrio cholerae, el agente causal, puede ser eliminado mediante antibióticos, de modo que además del tratamiento
tradicional de la enfermedad de rehidratación vía oral o intravenosa, comúnmente son aplicados antibióticos tales como la te-
traciclina, ciprofloxacina, norfloxacina o azitromicina. El efecto antimicrobiano in vitro de extractos de hojas de Costus spiralis
(Roscoe) sobre varias cepas patógenas de Vibrio cholerae fue ensayado mediante la técnica de difusión en placas de agar. Hojas
verdes de la plantas fueron colectadas, secadas en horno a 50 ºC durante 48 h, molidas y finalmente, sometidas a extracción con
etanol. Luego de secado, el material residual fue resuspendido en agua destilada a 100 mg/mL (p/v) y realizados los ensayos de
actividad antimicrobiana. Aparentemente, las cepas patógenas que representan las pandemias del siglo xx: C7258 (O1, El Tor,
Ogawa), C6706 (O1, El Tor, Inaba), O395 (O1, Clásica, Ogawa), CRC266 (O139) y 569B (O1, Clásica, Inaba) fueron matadas, a juzgar
por la presencia de halos de inhibición de crecimiento en los ensayos. Adicionalmente, se determinaron las concentraciones mí-
nimas inhibitorias de los extractos para las diferentes cepas. Los resultados anteriores fueron similares a los de la Ampicillina,
lo que sugiere que Costus spiralis pude utilizarse como fuente de principios activos contra Vibrio cholerae.
ABSTRACT. The use of remedies from plant origin covers a wide variety of maladies and constitutes an alternative way to an-
tibiotic therapy, which otherwise seems to be no longer promising due to antibiotics widespread resistance among pathogenic
microorganisms. Active principles having antimicrobial activity may be extracted and purified from plants for developing new
drugs. Among several illnesses that have historically scourged man, Cholera has been potentially epidemic and one of the most
outstanding. The bacterium Vibrio cholerae, the causal agent, can be eliminated with antibiotics so that besides the traditional
treatment of rehydration via oral or intravenous, antibiotics such as tetracycline, ciprofloxacin, norfloxacin or azithromycin are
commonly applied. The antimicrobial effect of Costus spiralis (Roscoe) leaves extracts on various pathogenic strains of Vibrio
was assayed in vitro by means of the agar plate diffusion technique. Fresh leaves from this plant were picked up, oven-
dried for 48 h at 50 ºC, powdered and finally ethanol-extracted. After drying, the remaining residue was suspended in distilled
water up to 100 mg/mL (w/v) and the antimicrobial assays carried out. The pathogenic strains representing the pandemics of
the 20th Century: C7258 (O1, El Tor, Ogawa), C6706 (O1, El Tor, Inaba), O395 (O1, Classic, Ogawa), CRC266 (O139) and 569B (O1,
Classic, Inaba) were apparently killed, as judged by halos of inhibition of growth in the assays. Furthermore, minimal inhibitory
concentrations (MICs) of the extracts for the various strains were also attempted. The above results were similar to those from
Ampicillin, suggesting that Costus spiralis may be used as a source of active principles against Vibrio cholerae.
activity can be extracted and purified from plants for Knowledge on medicinal properties of plants has extensively been applied all over the World to cure or Among several illnesses that have historically ameliorate suffering of mankind. The use of remedies scourged man, Cholera has been potentially epidemic from plant origin covers a wide variety of maladies and and one of the most outstanding. It is characterized by constitutes an alternative way to antibiotics, which is life-threatening secretory diarrhea with voluminous otherwise no longer promising due to the generalized an- watery stools, often accompanied by vomiting, and tibiotic resistance among pathogenic microorganisms.1 resulting in hypoglycemic shock and acidosis.4 The Theoretically, active principles having antimicrobial bacterium Vibrio cholerae, the causal agent, can be eli- minated with antibiotics so that besides the traditional treatment of rehydration via oral or intravenous, anti- Correspondence:
biotics such as tetracycline, ciprofloxacin, norfloxacin ♣To whom the correspondence should be addressed. or azithromycin are commonly applied.5 The World Revista CENIC Ciencias Biológicas, Vol. 39, No. 1, 2008.
Health Organization recommends antibiotics treatment The risk of the illness is currently higher in un- IZD diameter of inhibition zones (mm).
derdeveloped areas, with no enough sanitary condi- For determining minimal inhibitory concentrations tions, unsuitable or non-existing water purification (MICs), 20 μL of 10-fold serial dilutions from initial systems and elimination of residual waters. Although cholera has effectively been treated everywhere and even vaccination developed, plant medicines must RESULTS AND DISCUSSION
be considered, mostly in such areas where the above Inhibition of growth of Vibrio strains
strategies might not be available. In fact, there are Costus spiralis (Jacq.) Roscoe, belonging to the bota- several reports elsewhere concerning the matter.3,7 nical family Zingiberaceae, has been traditionally known The medicinal properties of Costus spiralis have by its anti-inflammatory effect on the urogenital tract, been used for treating a wide variety of illnesses, kidney, bladder and venereal diseases such as syphilis mostly dealing with the urinary tract and kidney and gonorrhea. The plant has been also applied for the disturbances.8 This report intends to demonstrate treatment of numerous illnesses including diabetes, the antimicrobial effect of Costus spiralis on Cholera. rheumatism and heart disorders. It has been also con- Here are the results of an in vitro antimicrobial assay firmed the anti-urolithiatic activity of aqueous extracts from leaves extract of this plant on pathogenic strains of Vibrio cholerae, which represent the pandemics of Concerning a possible antimicrobial activity of the plant, the antifungal principle methyl ester of para- coumaric acid was isolated from rhizomes of Costus MATERIALS AND METHODS
speciosus.12 Furthermore, a cytostatic effect of Costus Microorganisms
spiralis aqueous, alcoholic and ketonic extracts was The epidemic strains of Vibrio cholerae were used throughout this study. Table 1 shows the co- The results of inhibition of growth of the various strains of Vibrio cholerae (Table 2) clearly indicates that ethanolic extract from leaves of Costus spiralis should contain a compound or compounds, effective against The LB (Luria & Bertani) broth9 was the basal me- the bacterium. Although aqueous extracts of the plant dium for growing cholera cells. For solid media, agar (not shown) were also effective, ethanol must be better for extraction, according to the organic chemical nature of such compounds in plants. For instance, it has been Preparation of plant extracts
reported the presence of flavonol glycosides from leaves Fresh leaves from Costus spiralis were gathered and of Costus spiralis14 to which otherwise their antimicrobial cut to small pieces and oven-dried for 48 h, at 50 ºC . The activity could be attributed.3 In this case, 20 μL of the material was powdered and 40 g ethanol-extracted ethanolic extract was chosen for further assays. Indeed, (100 mL) at room temperature, for a week. After extrac- a similar effect was observed when Vibrio cells were tion and evaporation, the resulting solid material was exposed to 20 μL of the antibiotic ampicillin (1 mg/mL), picked, weighted and suspended in sterile water, up to soaked in filter paper discs, for comparison (Table 2). Moreover, the antimicrobial activity of extracts was checked by suspending O395 cells in LB liquid medium Antibacterial assays of extracts
containing the extract at 100 and 1 mg/mL . No survival The antimicrobial activity of extracts was determi- of 1.7 · 10 8 FCU/ mL were detected after 2 h of incuba- ned by the agar diffusion test, with slight modifica- tion at 37 ºC when such cultures were plated on LB, tions.10 Cultures to be assayed were pre-grown in LB suggesting a vibriocidal effect of the extract, at least in by shaking 200 r/min, at 37 ºC, up to 1.0 absorbance this case. Further assays must be carried out to the rest λ 600 nm and 106 FCU (forming colony units) plated of the strains, in order to confirm this result.
on Petri dishes containing LB. Sterile filter paper discs (6 mm), soaked with 20 μL of the extracts, Determination of minimal inhibitory concentra-
were placed on the surface of the plates. After 24 h tions (MICs).
of incubation at 37 ºC, the plates were checked and The response was similar for all strains (MIC of the diameters of clearing zones or halos of growth 1 mg/mL) except for 569B, where a MIC of 5 mg/mL was inhibition were measured in millimeters. The same observed (Table 2). This behavior might reveal a diffe- protocol was applied to paper filter discs, soaked in 20 μL ampicillin (1 mg/mL). The relative inhibition Although the exact nature of compounds having antimicrobial effect in Costus spiralis leave extracts Table 1. List of the pathogenic Vibrio cholerae strains, used in the assay.
Revista CENIC Ciencias Biológicas, Vol. 39, No. 1, 2008.
Table 2. Antimicrobial effect of Costus spiralis leaves
1. Kardar S.S. Antibiotic resistance: new approaches to a his- torical problem. kardar.html (Revised: January 20th 2007.) 2. Hostettmann K. Strategy for the biological and chemical evaluation of plant extracts. Pure Appl. Chem., 70, 2122-
2123, 1998. phuket97/hostettmann.html (Revised: February 5th 2007.) 3. Murphy Cowan M. Plant products as antimicrobial agents. Clinical Microbiol. Rev., 12, 564-582, 1999. (Revised: 4. Finkelstein R.A. Cholera, Vibrio cholerae O1 and O139, and other pathogenic vibrios. Medical Microbiology. 1 The antimicrobial effect was determined by the agar diffusion Fourth Edition, Samuel Baron (ed.), New York, 2002. http:// method. Paper filter discs, soaked with 20 μL of the extract or (Revised: Fre- ampicillin were placed on LB plates, previously seeded with Vibrio cells. After 24 h of incubation at 37 ºC, the plates were 5. Deen J. and Clemens J. Antimicrobial treatment for chol- checked and diameters of halos of growth inhibition were era. The Lancet, 366, 9491, 1054-1055, 2005.
6. World Health Organization Fact Sheet. “Cholera.” Fact 2 Relative inhibition zones (RIZ) are expressed as follows: sheet No. 107, March 2000. tre/factsheets/fs107/en/. (Revised: March 2nd 2007.) 7. García S., Alarcón G., Gómez M. and Heredia N. Haema- IZD diameter of inhibition zones (mm).
toxylon brasiletto extracts inhibit growth, enterotoxin 3 MIC minimal inhibitory concentrations.
production, and adhesion of Vibrio cholerae. Food Bio-
technology, 19, 15-26, 2005.
8. Alves da Fonseca Z. Plantamed – Plantas e ervas medici- is unknown, these results might be useful for further nais e fitoterápicos. Costus spiralis (Jacq.) Roscoe. http:// assays concerning purification of active principles. (Revised: Since few folk applications concerning intestinal disorders have been suggested for this plant,8,15 Cos- 9. Sambrook J., Fritsch E.F. and Maniatis T. Molecular tus spiralis may be considered as an alternative way Cloning – A laboratory manual. Second Edition, Cold to antibiotic therapy for cholera. The above results Spring Harbor Laboratory Press, Cold Spring Harbor- must be supported by further in vivo studies in expe- rimental animal models involving pathogenic strains 10. Nostro A., Germanò M.P., D’Angelo V., Marino A. and Can- natelli M.A. Extraction methods and bioautography for evaluation of medicinal plant antimicrobial activity. Lett.
In principle, this phytotherapy could be assumed as Appl. Microbiol., 30, 379-384, 2000. http://www.blackwell-
highly effective, mostly in those cases where the malady becomes epidemic and sanitary conditions were not promptly available. Additional research must be done, 11. Araújo T., Diogo C., Da Silva A.P., Riggio M.T., Lapa A.J. in order to elucidate the actual chemical composition and Souccar C. Evaluation of the antiurolithiatic activity of the compounds involved in the possible vibriocidal of the extract of Costus spiralis Roscoe in rats. J. Ethnop-
harmacol., 66, 193-198, 1999.
12. Bandara B.M.R., Hewage C.M., Karunaratne V. and Adika- CONCLUSIONS
ram N.K.B. Methylester of para-coumaric acid, antifungal principle of rhizome of Costus speciosus. Planta Med., 54,
The ethanolic extracts from leaves of Costus spiralis must contain active principles showing antimicrobial 13. López A.M., Rojas N.M. and Jiménez C.A. Plant extracts activity against pathogenic strains of Vibrio cholerae. with cytostatic properties growing in Cuba. Rev. Cub. Med
Costus spiralis may be considered as an alternative Trop., 31, 97-104, 1979.
way to antibiotic therapy for cholera, mostly in those 14. Antunes A.S., Da Silva B.P., Parente J.P. Flavonol gly- cases where the malady becomes epidemic and sanitary cosides from leaves of Costus spiralis. Fitoterapia, 71,
conditions were not promptly available.
15. Kamesgwara Rao C. Database of medicinal plants. May ACKNOWLEGMENTS
2000. Appendix 6. Specimen profiles from the database of plants with antimicrobial activity (Costus speciosus and The technical assistance of María Emilia Hervé is Piper nigrum).



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