Antibacterial Activity of Oenothera rosea (L 'Hér) Leaf Extracts

RGF designed the study, performed the statistical analysis, wrote the protocol, and wrote the first draft of the manuscrip t. PTG managed the analyses of the study. RRM carried out the experiments and obtained his Master of Sciences degree. RQL managed the organic chemistry and the literature searches of the study. All authors read and approved the final manuscript. ABSTRACT Aims: To determine the antibacterial effect of Oenothera rosea against Escherichia coli , Salmonella enteritidis and Vibrio cholerae . Study Design: In vitro antibacterial study. Place and Duration of Study: Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento de Microbiología e Inmunología and Departamento de Química, San Nicolás de los Garza, NL. México, from June 2010 to June 2011. Methodology: The antibacterial in vitro effect of methanol and aqueous extracts of the Mexican plant O. rosea against strains of E. coli , S. enteritidis and V. cholerae was evaluated in liquid medium by the colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Results: Methanol and aqueous extracts significantly inhibited growth of all bacterium strains tested. The methanol extract caused up to 55%, 66% and 87% growth against E. coli , S. enteritidis and V. cholerae , respectively, whereas the aqueous extract induced up to 54%, 69% and 88% bacterial growth inhibition, respectively. Methanol and aqueous vehicle controls did not alter bacterial growth. Conclusion: The observed antibacterial effect of O. rosea extracts may be of benefit as an adjuvant treatment of diseases caused by the studied enterobacteria.


INTRODUCTION
Over the last decade, the interest in natural products for use in the agricultural, food and pharmaceutical industries has been renewed. Recently, scientists have focused in searching for new drugs from natural products (Cowan, 1999), since there is a continuous need to discover new antibacterial substances with diverse chemical structures and novel mechanisms of action. Another important concern is the development of antibiotics resistance in the clinics (Erturk et al., 2006) and the increase in the incidence of new and reemerging infectious diseases, particularly, in developing countries; hence, it is necessary to provide affordable health care in these countries to a greater number of people (Goud et al., 2005).
Oenothera rosea is a Mexican plant commonly known as "hit grass", which belongs to the family Onagraceae. It has been traditionally used as a treatment against cough, diarrhea and skin infections (Andrade-Cetto, 2009) and it has characteristics that makes it ideal for the study of biological activity because of previous reports indicating that the plant produces phenolic compounds, flavonoids, and coumarins, which have shown cytotoxic and antiinflammatory activity (Meckes et al., 2004), but its antibacterial potential has been only evaluated against Neisseria gonorrhoeae with minimum inhibitory concentrations ≥256 μg/mL (Cybulska et al., 2011). In addition, the species Oenothera biennis (common evening primrose) was reported to possess antibacterial activity against Streptococcus mutans (Matsumoto-Nakano et al., 2011).
The present study was undertaken to determine the in vitro antibacterial effect of O. rosea methanol and aqueous extracts on E. coli, S. enteritidis and V. cholerae growth. These microorganisms were selected because they are clinically relevant. E. coli is a major component of the normal human intestinal flora, but the enterotoxigenic, enteroinvasive, enteropathogenic and enterohemorrhagic pathotypes are frequently associated with diarrhea and other pathologies (Nataro and Koper, 1998). Although most patients may recover within 10 days, in some of them, particularly children and the elderly, the infection can be lifethreatening (Coia, 1998). Salmonella enteriditidis is another important public health problem worldwide, particularly when eggs are eaten raw or undercooked. It can cause fever, abdominal cramps, and severe diarrhea. In the elderly, infants, and immunocompromised individuals (Salmonella infections are a complication in HIV-infected people) (Fernandez-Guerrero, 1997;Center for Disease Control, 1992;Cohen et al., 1987), the infection can be fatal unless antibiotic treatment is properly provided (Chiu et al., 2002). On the other hand, V. cholerae is the causative agent of cholera and represents a great public health problem, particularly in developing countries. It causes acute diarrheal disease and about 120,000 deaths every year (Kitaoka et al., 2011); if left untreated, the cholera death rate may reach up to 50% in a few hours to days after onset of the disease (Fournier and Quilici, 2007).
The aim of the present study was to evaluate the in vitro antibacterial activity of methanol and aqueous extracts of O. rosea against E. coli, S. enteritidis and V.. cholerae, which are of clinical importance.

Preparation of O. rosea Leaf Extracts
The plant material used in this study was obtained from a local market in downtown Monterrey, Nuevo Leon, Mexico and was identified as O. rosea by M.Sci. María del Consuelo González de la Rosa, Chief of the Herbarium of the Biological Sciences College at Autonomous University of Nuevo Leon. The aerial parts of O. rosea were dried in an oven at 40ºC, powdered using a Moulinex blender (Goldsmith 38, Colonia Polanco, Mexico DF) and stored. To prepare the methanol extract and the vehicle control, 100 ml of 100% methanol alone (methanol vehicle control) and methanol containing 10 grams of leaves powder were allowed to stand for 24 hours at room temperature. The resulting extract and vehicle control were centrifuged at 2800 rpm for 15 minutes and supernatants were placed in 1 mL Eppendorf tubes, previously weighted, after which they were dried under vacuum using a speed-vac concentrator (Savant Instruments Inc., Hicksville, NY). To prepare the aqueous extract and the vehicle control, 100 ml boiled water alone (aqueous vehicle control) or boiled water containing 10 grams of leaves powder were allowed to stand for 10 minutes, lyophilized (Labconco corporation, KC), and stored at -20ºC until use. The methanol and the aqueous extracts and the vehicle controls were suspended in sterile culture medium and then filter-sterilized through 0.22 -pore size diameter filters (Millipore, Bedford, MA).

Antibacterial Activity of O. rosea Extracts
Aqueous and methanol O. rosea leaf extracts were prepared to evaluate their in vitro antibacterial activity. We selected E. coli, S. enteritidis and V. cholerae species because they are clinically relevant, particularly in immunocompromised individuals. The percentage of microbial growth inhibition by O. rosea leaf extracts in liquid medium by a colorimetric technique (Gomez-Flores et al., 1995) was determined; for this, 100 l of E. coli and S. enteritidis cultures were placed in 10 mL brain heart infusion culture medium (Becton Dickinson, Cockeysville, MD) or 10 mL of Luria-Bertani (LB) culture medium (Difco Laboratories, Detroit, MI) for V. cholerae cultures, and were incubated at 37ºC for 24 hour. Aliquots of 800 µL from these culture suspensions were taken, mixed with 200 µL of sterile glycerol and frozen at -70ºC, until use.
In order to evaluate the in vitro antibacterial activity of O. rosea extracts, bacteria frozen cultures were thawed at 4ºC and then they were activated by inoculating 10 µL of the bacteria suspensions in 1 mL of BHI medium for E. coli and S. enteritidis and LB medium for V. cholera, and incubated at 37ºC for 24 h. Next, bacterial concentration was determined in a Neubauer hematocytometer (Fisher Scientific Co., Pittsburgh, PA) and adjusted to 1x10 3 cells/mL. To determine the antibacterial activity of the extracts, cell viability was measured by the MTT reduction assay (Gomez-Flores et al., 2007, 1995. MTT was prepared at a concentration of 5 mg/mL and sterilized by filtering through a 0.22-µm filter (Millipore, Carrigtwahill, CO, Ireland). Fifty microliters of the microbial suspensions were plated in their specific culture media, in flat-bottomed 96-well plates (Corning Incorporated, Corning, NY), in the presence or absence of serial dilutions (1:2) of the O. rosea leaf methanol and aqueous extracts (50 L), tetracycline control (3 g/mL; Lot # R32874, Research Organics, Cleveland, OH) and vehicle controls (methanol and culture medium). The vehicle controls were similarly processed as with plant methanol and aqueous extractions, but without plant material. Plates were then incubated for 6 hour at 37ºC, after which the tetrazolium salt MTT was added to all wells at a final concentration of 0.5 mg/mL and plates were incubated for 4 additional hours. At the end of the incubation period, 50 L of extraction buffer were added to all wells and plates were incubated overnight at 37ºC. Optical densities resulting from dissolved formazan crystals were then read in a microplate reader (Beckman Coulter, Inc., Fullerton, CA) at 570 nm.

Statistical Analysis
The results were expressed as mean  SEM of triplicate determinations from three independent experiments. Level of significance was assessed by Dunnet's t test.
Medicinal plants have become part of alternative medicine worldwide because of their potential health benefits. These plants can be ingested or directly applied to treat infections (Rojas et al., 2006) and this may be useful to overcome the increased resistance of microorganisms to conventional antibiotics from bacteria and fungi (Chinedum, 2005). Compounds synthesized by plants have a broad therapeutic potential due to their chemical structures, for which the evaluation of their biological activity is important to develop new products with pharmacological potential and to validate treatments traditionally used by the Mexican population and other people from developing countries (Rodriguez-Fragoso et al., 2008).
Plant antibiotics are not currently used in a health program because of their low activity, unless their MICs are in the range of 0.1 to 1 mg/mL (Tegos et al., 2002;Drusano, 2004); thus, the results of the present study may be an indication of an important antibiotic activity of O. rosea extracts. Novel antimicrobial activity of plant extracts is an alternative to be considered as a result of the increasing resistance of microorganisms, mostly bacteria, to antibiotics (Russell, 2000;Moreillon, 2000). Because of this, isolation and evaluation of potential natural antibiotic agents, particularly, O. rosea leaves, may lead to the discovery of antibiotics for which bacteria and other organisms are susceptible (Diallo et al., 1999). Compounds synthesized by plants have a broad therapeutic potential due to their chemical structures, for which the evaluation of their biological activity is important to develop new products with pharmacological potential and to validate treatments traditionally used by the Mexican population and other people from developing countries (Rodriguez-Fragoso et al., 2008).
Plant antibiotics are not currently used in a health program because of their low activity, unless their MICs are in the range of 0.1 to 1 mg/mL (Tegos et al., 2002;Drusano, 2004); thus, the results of the present study may be an indication of an important antibiotic activity of O. rosea extracts. Novel antimicrobial activity of plant extracts is an alternative to be considered as a result of the increasing resistance of microorganisms, mostly bacteria, to antibiotics (Russell, 2000;Moreillon, 2000). Because of this, isolation and evaluation of potential natural antibiotic agents, particularly, O. rosea leaves, may lead to the discovery of antibiotics for which bacteria and other organisms are susceptible (Diallo et al., 1999). Compounds synthesized by plants have a broad therapeutic potential due to their chemical structures, for which the evaluation of their biological activity is important to develop new products with pharmacological potential and to validate treatments traditionally used by the Mexican population and other people from developing countries (Rodriguez-Fragoso et al., 2008).
Plant antibiotics are not currently used in a health program because of their low activity, unless their MICs are in the range of 0.1 to 1 mg/mL (Tegos et al., 2002;Drusano, 2004); thus, the results of the present study may be an indication of an important antibiotic activity of O. rosea extracts. Novel antimicrobial activity of plant extracts is an alternative to be considered as a result of the increasing resistance of microorganisms, mostly bacteria, to antibiotics (Russell, 2000;Moreillon, 2000). Because of this, isolation and evaluation of potential natural antibiotic agents, particularly, O. rosea leaves, may lead to the discovery of antibiotics for which bacteria and other organisms are susceptible (Diallo et al., 1999 The higher antibacterial activity of the aqueous extracts, compared with the methanol ones, may be related to alterations of the active compound (s) present in the fresh plant after the methanol extraction and because of the water soluble nature of the anionic substances which are naturally occurring in most plant materials (Darout et al., 2000;El Astal et al., 2005). This may be of relevance since aqueous infusions are commonly administered or ingested by many cultures to treat diverse maladies.

CONCLUSION
To our knowledge, this is the first report showing that O. rosea leaf extracts inhibit E. coli, S. enteritidis and Vibrio cholera growth. The respective observed MICs were 0.5 mg/mL, 0.125 mg/mL and 0.5 mg/mL for the methanol extract, and 0.25 mg/mL, 0.125 mg/mL and 0.031 mg/mL for the aqueous extract, with the order of potency of S. enteritidis > E. coli = Vibrio cholera for the methanol extract and Vibrio cholera > S. enteritidis > E. coli for the aqueous extract. There are still a number of plant compounds that remain to be evaluated at the molecular, cellular and physiological levels for their potential to treat human diseases. Further studies are underway to evaluate the O. rosea leaf extracts and active compounds in an in vivo model of infection, and to characterize the antibacterial active compound (s).