IN VITRO EVALUATION OF THE ANTIMICROBIAL AND THE ANTIOXIDANT ACTIVITIES OF EXTRACTS AND ESSENTIAL OILS OF Verbena bonariensis Rocha GF1, Calloni S2, Rodriguez Morcelle MI1, Apóstolo NM1, Rosso AM1, Parisi MG1 1 Departamento de Ciencias Básicas y 2Departamento de Tecnología, Universidad Nacional de Luján, Ruta 5 y Avenida Constitución, (6700) Luján, Buenos Aires, Argentina.
SUMMARY The use of traditional medicinal plants from primary health care has increased worldwide in recent years. Scientists are in the search of new phytochemicals of native and naturalized plants for nutritional and pharmaceutical purposes such as antimicrobials, antioxidants, hepatoprotective, antidiarrheal and anti-inflammatory. In the Verbenaceae family, there are six native species in Buenos Aires province, V. bonariensis, V. intermedia, V. rigida, V. gracilescens, V. montevidensis and V. litoralis. Some of them are used in popular medicine. The aim of this work is the evaluation of the antimicrobial and antioxidant activities of essential oils and the aqueous and alcoholic extracts from the aerial parts of Verbena bonariensis. Quantitative estimation of phenolic compounds were done using the FolinCiocalteu reagent. The antioxidant effect was estimated by the stable free radical 2,2diphenyl-1-picrylhidrazyl radical (DPPH) method, and the antimicrobial activity with the disc agar diffusion method. The total phenolic content found in the different extracts varied from 0,67 to 1,17 mg of gallic acid/g of dried mass and the DPPH scavenging effect was determined spectrophotometrically. It was observed that the antioxidant capacity of the hydroalcoholic extracts were between 10 to 50 µg/mL, and the potency of radical scavenging observed in ethanolic extracts from Verbena bonariensis was greater than methanolic one. Besides, antimicrobial activity towards bacteria and fungus was tested. Staphylococcus aureus was slightly inhibited by the alcoholic extracts, Salmonella enteriditis was only inhibited by the ethanolic extract and Serratia marcescens by the methanolic one. Essential oils only showed inhibitory activity against Pseudomona aeruginosa and Klebsiella pneumoniae growth. Besides, aqueous extracts did not present inhibitory effect on the bacteria tested however it inhibited the development of the mycelia fungus of Sclerotium rolfsii. a well-known phytopathogenic agent that causes disease on a wide range of agricultural and horticultural crops such as sweet potato, corn, wheat, pumpkin and peanut. No activity against Escherichia coli was observed in the different preparations. Keywords: Verbena bonariensis, antioxidant, antimicrobial activity, essential oil, extracts.
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EVALUACIÓN IN VITRO DE LAS ACTIVIDADES ANTIOXIDANTE Y ANTIMICROBIANA DE EXTRACTOS Y ACEITES ESENCIALES DE Verbena bonariensis
RESUMEN El uso de plantas medicinales tradicionales en la atención primaria de la salud ha aumentado en el mundo en los últimos años. Los científicos están en la búsqueda de nuevos fitoquímicos a partir de plantas nativas y naturalizadas con fines nutricionales y farmacéuticos como antimicrobianos, antioxidantes, hepatoprotectores, antidiarreicos y antiinflamatorios. En la familia Verbenaceae existen seis especies del género Verbena nativas de la provincia de Buenos Aires, V. bonariensis, V. intermedia, V. rigida, V. gracilescens , V. montevidensis y V. litoralis algunas de las cuales se utilizan en medicina popular. El objetivo de este trabajo es la evaluación de las actividades antimicrobiana y antioxidante de los aceites esenciales y de los extractos hidroalcohólicos de las partes aéreas de V. bonariensis. La cuantificación de los compuestos fenólicos se realizó utilizando el reactivo de Folin-Ciocalteu, la determinación de la actividad antioxidante se estimó con el método del radical libre 2,2difenil-1-picril hidracil (DPPH) y la actividad antimicrobiana con el método de difusión en agar. El contenido fenólico total en los diferentes extractos varió entre 0,67 a 1,17 mg de ácido gálico/g de masa seca, mientras que la capacidad antioxidante de los extractos hidroalcohólicos lo hizo entre 10 y 50 µg/mL, siendo mayor en los extractos etanólicos que en los metanólicos. Por otra parte, se evaluó la actividad antimicrobiana (hongos y bacterias) de los extractos y los aceites esenciales. Las cepas de Staphylococcus aureus fueron ligeramente inhibidas por los extractos alcohólicos, mientras que las de Salmonella enteritidis sólo fueron inhibidas por el extracto etanólico y las de Serratia marcescens por las preparaciones metanólicas. Por otra parte, los extractos acuosos no mostraron efecto inhibitorio sobre las bacterias ensayadas. Sin embargo estos extractos inhibieron el desarrollo miceliar del hongo fitopatógeno Sclerotium rolfsii, causante de enfermedades en una amplia gama de cultivos agrícolas y hortícolas como batata, maíz, trigo, calabaza y cacahuate. Los aceites esenciales sólo mostraron actividad inhibitoria del crecimiento de Pseudomona aeruginosa y Klebsiella pneumoniae. Escherichia coli no fue inhibido por las preparaciones en las condiciones estudiadas. Palabras clave: Verbena bonariensis, antioxidante, actividad antimicrobiana, aceites esenciales, extractos.
INTRODUCTION There is a growing interest in searching natural bioactive compounds for different reasons: numerous clinical and epidemiological studies have demonstrated that fruits and vegetables consumption is associated with reduced risks of developing chronic diseases such as cancer, cardiovascular disorders and diabetes, safety considerations regarding the potential harmful effects of the chronic consumption of synthetic antioxidants in foods and beverages and consumers preferences for additive-free foods as safer since preservatives can cause health hazards like asthma and cancer and are suspected to be mutagenic and neurotoxic (Dastmalchi et al., 2007; Dorman, Kos-ar, & Hiltunen, 2007) Scientists are searching new phytochemicals of native and naturalized plants for pharmaceutical and nutritional purposes such as antimicrobials, antioxidants, hepatoprotective, antidiarrheal and anti-inflammatory. In Buenos Aires province (Argentina), the genus Verbena has six native species: V. bonariensis, V. intermedia, V. rigida, V. gracilescens, V. montevidensis and V. litoralis. Except V. montevidensis all species are used in popular medicine (Barboza et al., 2009; Hernández et al., 2009). V. bonariensis is native from template and tropical regions of America and is naturalized in many countries of the world (O´Leary, 2007). It is used as digestive, hepatoprotective, anti-inflamatory, antibacterial, antifungal, febrifuge, anti-spasmodic, antiseptic, anti-diabetic, stomachic and tonic properties (Al-Amier et al., 2005; Barboza et al., 2009). The main components of this species are iridoids, phenylpropanoids, flavonoids, luteolin and terpenoids (Calvo et al., 1997; Deepak & Handa, 2000; Bilia et al., 2008). The present study was carried out to evaluate antimicrobial and antioxidant activities of the aqueous and alcoholic extracts and the essential oils from V. bonariensis aerial parts. In addition, the phenolic content of the preparations and the chemical composition of the essential oils were determined in order to establish resemblances between their composition and their potential application in phytotherapy. Materials and methods Plant material Samples of aerial parts (flowers) from Verbena bonariensis used in this study were collected from Alberti and Zárate, Buenos Aires province on February and March of 2010. In their flowering stage, plants were collected by Eng. Rodríguez Morcelle and kindly authenticated by Profesor Apóstolo, Prof. of Botany, Departamento de Ciencias Básicas, Universidad Nacional de Luján, Argentina. Samples preparation Plant extracts: Aqueous extracts were prepared as infusions by adding 100 mL of boiling water to 1 g of dried plant minced material and incubated for 10 min (Ivanova et al., 2005). For alcoholic preparations, 10 grams of dried plant samples were extracted by soxhlet for 72 h with 100 mL of ethanol and methanol at 4°C. After filtration, extracts were concentrated to dryness under reduced pressure. Finally, both extracts were reconstituted in appropriate solvents (El-Hela & Abdullah, 2012). Essential oils: Essential oils were isolated from the fresh aerial parts by hydrodistillation. After cooling, settling and drying over anhydrous sodium sulfate, oils were recovered and stored at 4°C until analysis. Quantitative and qualitative analysis
were performed by GC-MS. Analytical methods Total phenolic compounds were assayed spectrophotometrically according to the Folin– Ciocalteu method (FC) and expressed as gallic acid equivalents. Verbena´s extracts were mixed with 500 µL of FC reagent, previously diluted 10–fold with distilled water, and allowed to stand at room temperature for 5 minutes, then 400 µL of Na2CO3 (60 g/L) solution were added to the mixture. After 15 minutes at 50°C, the absorbance was measured at 760 nm. Results were expressed as % gallic acid equivalents in dry weight plant (g of gallic acid/100 g dry weight plant) (Singleton & Rossi, 1965). Antioxidant activity was determined according to the stable DPPH radical technique. In the DPPH assay, the hydrogen atom or electron donation abilities of the corresponding extracts were measured from the bleaching of the purple-coloured methanol solution of 2,2-diphenyl-1-picrylhidrazyl radical (DPPH). Different extracts were added to 0.2 mL of DPPH 0.5 mM in 0.004% methanol solution of DPPH, and final volume was adjusted to 1 mL. Mixtures were vigorously shaken and left for 30 min in the dark. Absorbance was measured at 517 nm using MEOH as blank and DPPH diluted in MEOH (1:4) was used as control. Neutralization of DPPH radical (S%) was calculated as follow: S % = (Ablank – Asample / Ablank) x 100 where Ablank is the absorbance of the control reaction (containing all reagents except the test compound), and Asample is the absorbance of the test compound. Tests were carried out in triplicate. The SC50 value represented the concentration of the extract that caused 50 % of neutralization (Steff et al., 2009). In vitro assays for antibacterial activity were tested with the disc-agar diffusion method. American Type Culture Collection of Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 35218, Pseudomona aeruginosa, Serratia marcescens, Salmonella enteritidis and Klebsiella peumoniae (from the Food Microbiology Laboratory, Departamento de Tecnología de la Universidad Nacional de Luján) were used in this study. All the microorganisms mentioned above were incubated at 37±0.1°C (30±0.1°C for Serratia marcescens) for 24 h by inoculation into Trypteinesoy broth. Volatile oils and extracts were impregnated on 6 mm discs of filter paper (Whatmann N° 3) which were placed on the agar surface cultivated with the selected microorganisms. Petri dishes were incubated at 37°C for 24 h. Inhibitory activity of extracts and essential oil were also tested. Antifungal activity was performed by type confrontational dual bioassays. Potato dextrose agar (PDA) medium at pH 5.8 was used for Macrophomina phaseolina and Sclerotium rolfsii and V8 medium (34% of commercial V8 juice, Campbell 8 vegetables with 3% agar) to Phytophthora capsici. Fungal strains were provided by the Plant Pathology Laboratory, Universidad Nacional de Luján. Discs of filter paper Whatmann N° 3 (6 mm) were impregnated on volatile oils and extracts, placed at a distance of 8 cm from the inoculum and incubated at room temperature. Results measuring the effect of the Verbena preparations on the mycelial growth of the fungus were observed from 3 to 10 culture days and compared with the control without the preparations.
Statistical analysis. Results were expressed as means standard deviation of at least three independent experiments using the InfoStat software (2008). All data were analyzed by ANOVA followed by Tukey test. Differences were considered significant when p
