In this study, the effect of anthocyanic extracts of strawberry (Fragaria x ananassa Duch.) variety Jacona on the in vitro growth of Staphylococcus aureus associated to bovine mastitis was evaluated. Sensitivity tests were carried out on the anthocyanic extracts by disk difussion method (10 µg/disk-100 µg/disk), using the antibiotic Dicloxacillin® as positive control (50 µg/mL, 50 µL/disk) and percentages of inhibition of bacterial growth were determined. Anthocyanic extracts managed to inhibit the bacterial growth of the strain ATCC 27543 and the isolated STA28 of S. aureus up to 54% and 40%, respectively. The results showed the antimicrobial potential of anthocyanic extracts of strawberry against S. aureus associated to bovine mastitis.
En este estudio, se evaluó el efecto de los extractos antociánicos de fresa (Fragaria x ananassa Duch.) variedad Jacona sobre el crecimiento in vitro de Staphylococcus aureus asociados a la mastitis bovina. Las pruebas de sensibilidad de los extractos antociánicos se realizaron por el método de difusión en disco (10 µg/disco a 100 µg por disco), utilizando el antibiótico Dicloxacillin® como control positivo (50 mg/ml, 50 µl/disco) y se determinaron los porcentajes de inhibición del crecimiento bacteriano. Los extractos antociánicos lograron inhibir el crecimiento de la cepa de S. aureus ATCC 27543 y del aislado STA28 hasta 54% y 40%, respectivamente. Los resultados mostraron el potencial antimicrobiano de extractos antociánicos de fresa contra S. aureus asociada a la mastitis bovina.
Bovine mastitis is considered one of the most common diseases that affects dairy cattle worldwide and causes great economic losses to producers as well as to the dairy industry, due to the decline of milk quality and yield as well as the early disposal of diseased animals (Deb
The control of S. aureus has been achieved mainly through antibiotic therapy; however, the indiscriminate use of antibiotics has promoted the selection of bacteria resistant to such compounds, which results in therapy failure; consequently, the search for new sources of antibiotics as an alternative to such conventional therapy is necessary (Adesola, 2012; Ochoa-Zarzosa
In this regard, secondary metabolites of plant origin, many of which assist in the protection of plants against herbivores, pests and pathogens, are considered an excellent source of antibiotic compounds and have been the main providers of the pharmaceutical industry since they contribute with more than 40% of the compounds that exist in the market nowadays, either as natural products or in their synthetic versions (Crozier, Jaganath & Clifford, 2007; Ochoa-Zarzosa
One of the most important sources of secondary metabolites of plant origin is fruits. The vast majority of the benefits of consuming fresh fruits has been attributed to the high concentrations of secondary metabolites that they contain and have been associated to the prevention or control of several chronic degenerative diseases (Poiroux-Gonord
The intense color that anthocyanins emit promote the dispersion of seeds and pollination due to attraction of animals and insects. Additionally, the anthocyanins participate in the protection of plants against damage caused by UV rays (Mazza & Miniati, 1993). Several studies have reported that anthocyanins possess biological activities as powerful antioxidant, anti-inflammatory and anticarcinogenic agents (Aaby, Ekeberg & Skrede, 2007; He & Giusti, 2010; Zhang, Seeram, Lee, Feng & Heber, 2008), apart from preventing cardiovascular diseases and controlling diabetes and obesity (Aaby, Mazor, Nes & Skrede, 2012; He & Giusti, 2010). Knowledge of such properties has arisen interest in studying their antifungal and antimicrobial activities on humans’ pathogens (Cisowska, Wojnizz & Hendrich, 2011; Kim
MATERIAL AND METHODS
The certified strain ATCC 27543 and the isolated STA28 of S. aureus were provided by Dr. Joel Edmundo López Meza of the Centro Multidisciplinario de Estudios en Biotecnología (CMEB-FMVZ) of the Universidad Michoacana de San Nicolás de Hidalgo. Bacteria were kept under continuous subculturing in mannitol salt agar (Bioxon®) at 37 °C. The isolate of S. aureus STA28 was characterized by observation under an optical microscope and biochemical tests such as catalase, coagulase, gelatinase and fermentation in mannitol salt agar (López-Meza
Strawberries used to obtain anthocyanic extracts were harvested from plants of F. x ananassa variety Jacona grown in the greenhouse of CIIDIR IPN Unidad Michoacan, in Jiquilpan, Michoacan, Mexico, located at an altitude of 1560 m s.n.m, delimited by the coordinates 20° 03' 02'' and 19° 52' 54'' of North latitude and meridians 102° 39' 33'' and 102° 56' 16'' West longitude.
Extraction and quantifying of anthocyanic extract of strawberry
In order to obtain the anthocyanic extract of strawberry, the methodology of Abdel-Aal & Hucl (1999) was used; it consisted of softening 1 g of fresh fruit in 5 mL of acidified ethanol (ethanol and HCl 1N; 85:15 v/v, J.T. Baker®). The pH of the mixture was adjusted to 1 with HCl 1N. The extract was shaken at 250 rpm in a rotary shaker (Heidolph®) during 16 h, at room temperature (25 ± 2 °C). The extract was centrifuged (Hettich®) at 6000 rpm for 15 min; the supernatant was recovered and diluted at 25 mL with acidified ethanol. For the extract quantifying, its absorbance was read at 535 nm in a spectrophotometer of UV/VIS light (Optizen POP®), using the pigment cyanidine 3-glycoside as standard and acidified ethanol as target. The samples were stored at -20 °C until their use. The concentration of total anthocyanins present in the extract was determined by applying the following equation: C = (A/E) × (vol/1,000) × MW × (1/weight of sample) × 106, where C = total concentration of anthocyanins (mg/kg); A = absorbance at 535 nm; E = molar absorbance of cyanidin 3-glycoside = 25 965 cm-1 M-1; and Vol = total volume of anthocyanins extract and MW= molecular weight of cyanidin 3-glycoside = 449.
In vitro inhibition tests of anthocyanic extract on S. aureus
Bioassays were carried out using the disk diffusion method for sensitivity tests on antimicrobials, with some modifications (Klančnik, Piskernik, Jeršek & Možina, 2010). Also, 100 µl of bacterial suspension, adjusted to a concentration of 1 × 108 UFC/mL, were used. This suspension was distributed over the surface of a Petri dish that contained Muller Hinton agar (Bioxon®), with the help of a sterile glass handle. Later, sterile disks of 6 mm of diameter (paper Whatman No. 1®) were saturated with the volumes corresponding to each concentration of total anthocyanins extract (10 µg, 25 µg, 50 µg, 75 µg and 100 µg = 6 µL/disk, 15 µL/disk, 30 µL/disk, 46 µL/disk and 61 µL/disk, respectively), they were dried on laminar flow bell (CHC Biolus®) during 1 h and placed on the surface of each dish.
Absolute ethanol was used as negative control (6 µL/disk, 15 µL/disk, 30 µL/disk, 46 µL/disk and 61 µL/disk) and the antibiotic Dicloxacillin® was used as positive control at a concentration of 50 µg/mL (50 µL/disk). The Petri dishes were incubated for 24 h at 37 °C. Then, the diameter of inhibition halo of treatments was measured with a digital Vernier Caliper® without considering the disk. All the treatments were performed in triplicate. The diameter of halo of inhibition was expressed as the percentage of extract inhibition which was calculated by the following formula: % of inhibition = (diameter of extract halo / diameter of positive control halo) × 100 (Corzo, 2012).
With the data obtained from the experiments, an analysis of variance (Anova) and a Tukey test (p ≤ 0.05) were carried out, by means of the program SAS® version 9.0. Data were transformed with the formula √(% inhibition )/100 (Ruiz-Sánchez, Mejía-Bautista, Cristóbal-Alejo, Valencia-Botín & Reyes-Ramírez, 2014).
RESULTS AND DISCUSSION
Several phenolic compounds of fruits and vegetables such as quercetin, kaempferol, gallic acid, cinnamic acid and coumarins, among others, have been studied due to their antioxidant and antimicrobial qualities against several pathogen microorganisms (Hafidh
Anthocyanic extracts of F. x ananassa var. Jacona showed significant antibacterial activity (p ≤ 0.05) on the growth of S. aureus ATCC 27543, since all the assayed amounts inhibited its growth. Inhibition percentages observed oscillated between 2.4% and 53.6%, which increased in proportion to the applied amount (table 1). Similarly, the assays with the isolated STA28 revealed a significant antimicrobial activity (p ≤ 0.05) in all the amounts of anthocyanic extract assayed. Inhibition percentages observed were from 2.56% to 40.05% (table 1).
|Table 1||Percentage of inhibition of anthocyanic extracts of strawberry var. Jacona on S. aureus|
|Treatments (µg)||Inhibition (%)*|
|10||2.4 ± 0.05 F||2.56 ± 0.17 E|
|25||13.9 ± 0.05 E||20.45 ± 0.2 D|
|50||32.5 ± 0.05 D||23.1 ± 0.36 C|
|75||39.6 ± 0.15 C||25.66 ± 0.36 C|
|100||53.6 ± 0.15 B||40.05 ± 0.6 B|
|**Positive Control||100 ± 0.2 A||100 ± 0.8 A|
*Mean and standard deviation of inhibition percentages are presented. Different letters within a column indicate significant differences based on Tukey test (P < 0.05, n = 3).
**Dicloxacillin® at 50 µg/mL (50 µL/disk).
Source: Author's own elaboration. Download
Although the results of the present work revealed the antimicrobial effect of strawberry anthocyanic extract against bacteria associated to bovine mastitis for the first time, the use of plant extracts to control this disease has already been documented. Mubarack, Doss, Dhanabalan & Venkataswamy (2011) evaluated the antibacterial activity of ethanolic extracts of four medicinal plants of India (C. ciliaris, C. grandis, Brachiaria sp. and A. indicum) against pathogenic isolates that cause bovine mastitis (Streptococcus agalactiae, Kleibsella pneumoniae, Escherichia coli and S. aureus). The authors reported that, with concentrations of 100 mg/mL and 200 mg/mL, inhibition from 34% to 82% were generated, in contrast to positive control (Ciprofloxacin, 200 mg/mL). Dhanabalan
It is important to note that the low concentration of the anthocyanin extract evaluated in the present study (less than or equal to 100 μg/disc) allowed an inhibition greater than 40% in both strains of S. aureus., while in other reports, greater amounts of anthocyanin extracts were used on humans’ pathogenic strains, Gram (+) as well as Gram (-). For example, anthocyanin extracts of Sysygium cumini fruits (5 mg/disk and 10 mg/disk) against Enterococcus faecalis, Bacillus cereus, B. subtilis, Meticillin Resistant Staphylococcus Aureus (MRSA), besides Salmonella typhimorium, E. coli and S. paratyphi B; assays targeted to S. aureus showed inhibition in both concentrations, with 10 mm and 12.5 mm of inhibition diameter (25% and 31%, respectively), contrasting with 40 mm of inhibition of positive control (Ampicillin 10µg/disk) (Priya, Devi, Eganathan & Kingsley, 2013). Similarly, the results matched the ones obtained by Nohynek
The above results show the antibacterial potential of strawberry anthocyanic extract against S. aureus, the main pathogenic bacteria associated to bovine mastitis; this could increase the possible pharmaceutical uses of these compounds in veterinarian medicine. However, deeper research should be done to know the effect of the application of greater amounts of anthocyanins on the growth of S. aureus, to determine the antibacterial activity of anthocyanic extracts of different varieties of strawberry on more S. aureus strains of clinical interest, and to identify the compounds responsible for inhibiting their growth.
Authors want to thank the support given by the Consejo Nacional de Ciencia y Tecnología (Conacyt) (CB-2009-131769) and the Instituto Politécnico Nacional (IPN-SIP-20171398).
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