Biotecnología

 

Antioxidant and antimicrobial activity of capulin (Prunus serotina subsp capuli) extracts

 

Actividad antioxidante y antimicrobiana de extractos de capulín (Prunus serotina subsp capuli)

 

M. Jimenez, I. Castillo, E. Azuara and C.I. Beristain*

 

Instituto de Ciencias Básicas, Universidad Veracruzana, Apdo. Postal 575, CP 91192, Xalapa, Veracruz, México. *Corresponding author. E–mail: cberistain@uv.mx Tel. +52 (228) 8 42 17 09, fax: +52 (228) 841 89 32

 

Received 24 of January 2011.
Accepted 1 of March 2011.

 

Abstract

Capulin (Prunus serotina subsp. capuli) is an annual fruit widely used in Mexico for the elaboration of several traditional products, such as medicinal tea, which is considered to present antioxidant and antimicrobial properties. The aim of this work was to evaluate the antioxidant and antimicrobial properties of aqueous, acetone, ethanol and methanol extracts. The ethanol extract presented a high anthocyanin (102±7.70 mg Cyd–3–glu/100 g extract) and polyphenol (1732±43.40 mg GAE /100 g extract) content and a high scavenging antioxidant activity (73.47±0.01%) in accordance with the reducing power (3.164±0.028), redox potential (395±2 mV) and optical density (0.921±0.08). It also presented antimicrobial activity against the majority of gram (–) bacteria: Salmonella typhimurium, Proteus mirabilis, Escherichia coli and Pseudomona aeruginosa, but only against one gram (+) bacterium: Staphylococcus aureus. In addition, said extract inhibited yeast activity, but had no effect on molds. Therefore, this ethanol extract could present potential as a food additive.

Keywords: polyphenols, anthocyanins, antibacterial, DPPH, redox potential.

 

Resumen

El capulín (Prunus serotina subsp. capuli) es una planta anual ampliamente usada en México para la elaboración de varios productos tradicionales, tales como té medicinal, el cual es considerado posee propiedades antioxidantes y antimicrobianas. El principal objetivo de este trabajo fue evaluar las propiedades antioxidantes y antimicrobianas del extracto acuoso, acetónico, etanólico y metanólico del fruto de Capulín. El extracto etanólico presentó un alto contenido de antocianinas (102±7.70 mg Cyd–3–glu/100 g extracto) y polifenoles (1732±43.40 mg GAE /100 g extracto), así como una alta actividad antioxidante (73.47±0.01%) en concordancia con el poder reductor (3.164±0.028), potencial redox (395±2 mV) y densidad óptica (0.921±0.08). Este extracto, también presentó mayor actividad antimicrobiana contra las bacterias gram (–): Salmonella typhimurium, Proteus mirabilis, Escherichia coli y Pseudomona aeruginosa, pero solo contra la bacteria gram (+): Staphylococcus aureus. Además, dicho extracto inhibió la actividad de levaduras, pero no tuvo efecto sobre mohos. Por lo tanto, el extracto etanólico podrí a presentar potencial como un aditivo en alimentos.

Palabras clave: polifenoles, antocianinas, antimicrobianos, DPPH, potencial REDOX.

 

1 Introduction

There are some components in plants that have antioxidant and antibacterial properties. This is the case of Capulin (Prunus serotina subsp capuli), which has been used for medicinal purposes since prehispanic times to treat various diseases, such as diarrhea or respiratory inflammations associated with coughing (INI, 1994). This is likely due to the fact that Capulin contains a large array of phenolic compounds such as flavonoids and tannins (Ordaz–Galindo et al. 1999), whose antioxidant and antibacterial properties are extensively documented (Einbond et al., 2004; Lachman et al., 2009). One type of flavonoid present in the skin of Capulin fruit contains anthocyanins such as cyaniding–3–glucoside and cyaniding–3–rutinoside (Ordaz–Galindo et al., 1999). Numerous studies have demonstrated that anthocyanins are effective scavengers of physiologically relevant reactive oxygen and other radicals (Han et al. 2006; Lachman et al., 2009).

The recovery of anthocyanins, polyphenols and compounds with antioxidant properties from plant materials is influenced by the solubility of the phenolic compounds in the solvent used for the extraction process. Furthermore, solvent polarity plays a key role in increasing the phenolic content, solubility and extraction of compounds with antioxidant activity in the extract (Naczk and Shahidi, 2006). However, there is no evidence that using different solvents leads to differences in the antioxidant and antimicrobial activity of Prunus serotina extracts.

Numerous antioxidant tests have been developed to evaluate antioxidant activity and to explain the function of antioxidants. The most commonly accepted assays to evaluate antioxidant activity are those of total antioxidant activity, reducing power, the DPPH assay, superoxide anion scavenging, and the hydroxyl radical scavenging assay (Frankel and Meyer, 2000; Sanchez–Moreno, 2002).

Antioxidant activity is known to be generally related with antimicrobial activity and its biological control (Escribano–Bailon et al., 2004). For example, the ethanol extract with the highest antioxidant activity was reported as having strong antibacterial activity against Staphylococcus aureus (Li et al., 2005). However, this relationship has not been documented in Capulin. As a whole, Capulin (Prunus serotina var capuli), contains anthocyanins and polyphenols compounds that confer antioxidant and antimicrobial properties, depending on the solvent used for extraction. Therefore, we hypothesized that the extracts of Capulin containing anthocyanins and polyphenols would have antioxidant and antimicrobial properties. To verify this, we studied the antioxidant and antimicrobial activity of the various extracts, their physicochemical characteristics and the correlation between antioxidant and antimicrobial activity in the different solvent extractions used.

 

2 Materials and methods

2.1 Chemicals and reagents

All of the reagents were of analytical grade. Gallic acid, Folin Ciocalteau reagent, and methanol, ethanol, DPPH (2,2–diphenyl–1–picrylhydrazyl) were purchased from Sigma–Aldrich (St. Louis, MO, USA).

2.2 Fruit material preparation

Fresh fruit samples with no apparent physical or microbial damage were collected separately, in the city of Huejotzingo, Puebla, Mexico. The fruits were authenticated by a botanist at the herbarium of the Biological Research Institute of Veracruz University, Mexico. All the fruits were selected in terms of shape (oblong), size (1–1.2 cm), color (blue–purple), and ripening index (15–17). After harvesting, they were dipped for 5 min in a solution with 0.1% of sodium hypochlorite; then, they were drained and air dried for about 1 h at 28 ºC. Thereafter, the fruit pulp was separated from the seed, placed in polyethylene bags and stored at –40ºC in a freezer (Sanyo Biomedical Freezer, MDF U5411, USA).

2.3 Preparation of the fruit extracts

The frozen fruit pulp was ground in a blender for 1 min and the sample was divided into four batches. The first batch, called "crude extract" (100 g), was extracted in an extractor (Moulinex mod. 140–1–03, Naucalpan, state of México, México). For the other batches, 100 g of complete fruit were homogenized in a blender with 500 mL of distilled water, ethanol (95%), methanol (analytical grade), or acetone being used as a solvent. The samples were left in the dark to be shaken for 24 h in an orbital shaker. Then, the extracts were filtered through a Whatman No 1 filter. Finally, the filtrates were frozen and lyophilized (Labconco, 4.5 L, USA).

2.4 Physicochemical analysis of fruit and extracts

The moisture contents were immediately measured on arrival of the fruit according to the AOAC (1995) official method 32.1.02. °Brix was determined with a digital Refractometer (digital Atago 1T model NAR–1T). The ripening index of the fruit was calculated by using the ratio of °Brix to the percentage of acidity. The yield from the dried extracts on a dry weight basis was calculated from the ratio of dried extract to the fresh fruit sample, using Eq. (1):

where W₁ was the weight of the extract after lyophilization and W₂was the dry weight of the fresh fruit sample.

The L, a, b color values of the extract were measured with a spectral photometer (Color Flex CX1115 HunterLab, USA). The instrument was standardized each time with a white ceramic plate (L = 92.8, a = —0.8; b = 0.1). After standardization the L (lightness), a (redness), b (yellowness) values were measured in the complete fruit and the extracts. Three replicate measurements were performed and the results were averaged. In addition, the color intensity (Chroma), total color difference (ΔE) with respect to the pulp and hue angle (H°) were calculated by using the following equations:

2.5 Antioxidant analysis of extracts

The total content of anthocyanin in the Capulin extracts was expressed as monomeric anthocyanins through the use of the pH differential method described by Wrolstad and Giusti (2001). The total phenolics were determined by using the Folin–Ciocalteu method described by Singleton and Rossi (1965).

The antioxidant properties were measured at specific concentrations in a solution of the extracts and all the measurements were carried out in triplicate, as follows:

• The determination of DPPH radical scavenging activity was analyzed in different concentrations of extracts with methanol used as diluent, according to the method described by Duan et al. (2007).

• The optical density was measured at 390 nm with a Spectronic Genesys 5 spectrophotometer (Milton Roy Company, Rochester, NY, USA). The extracts were diluted 1:10 v:v with distilled water to ensure that the absorbance values were in the scale.

• The redox potential was measured with a digital voltmeter (Orion 720 A, USA) in a solution of extracts with 20% of solids. The measurements were made with a platinum electrode indicator and a reference electrode of silver/silver chloride Ag/AgCl, Cl⁺_sa²_t, as described by Manzocco et al. (1998).

• The reducing power was determined according to the method of Oyaizu (1986). All determinations were carried out in triplicate and the results were averaged.

2.6 Antimicrobial activity of extracts

2.6.1 Microorganism strains

The antimicrobial activities were tested against bacteria gram (+) and gram (–), yeast and molds. The microorganisms used were supplied by the Department of Clinical Microbiology, Faculty of Pharmacobiological Chemistry, Veracruz University, Mexico; they consisted of: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Streptococcus group C, Escherichia coli ATCC 35218, Pseudomona aeroginosa ATCC 27853, Salmonella typhimurium ATCC 14028, Proteus mirabilis ATCC 12453, Candida albicans ATCC 10231, Sacharomyces cerevisae ATCC 9763, Aspergillus flavus ATCC 16872, and Aspergillus niger ATCC 11414. The bacteria and molds were grown and maintained on Nutrient agar (NA) slants and potato dextrose agar (PDA) slants, respectively. They were then stored at 4 ºC under aerobic conditions. The bacteria were cultured overnight at 35 ºC in nutrient broth, which was further diluted in saline solution (8.5 g NaCl/L water) adjusted to obtain a turbidity comparable to that of McFarland standard tube No. 5 (10⁶ CFU/mL) for further use (Vandepitte et al., 1991).

2.6.2 Agar well diffusion method

The antibacterial activities of isolates from the different extracts were evaluated by making holes in the agar, according to the Owen and Palombo (2007) method with slight modifications. Wells were made in Mueller Hinton agar with a sterile cork–borer (6 mm in diameter) and 50 uL of solution in a known concentration were pipetted into the agar wells. The inoculated plates were incubated at 37 ºC for 24 h for clinical bacterial strains and 48 h for the yeast. Antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms. A clear zone of inhibited microbial growth surrounded substances exhibiting antimicrobial properties, and zones with a diameter greater than 9 mm were considered positive.

2.6.3 Radial mycelia growth inhibition tests

The extracts were tested at the maximum concentration studied (320 mg/mL) in potato dextrose agar. Radial mycelia growth was measured after seven days of incubation. Anti–fungal activity was calculated as the percentage of radial growth obtained with the methodology proposed by Abd–El–Khair and Waffaa (2007).

2.6.4 Minimal inhibitory concentration (MIC)

The minimal inhibitory concentration (MIC) was determined according to the NCCLS (2000) by using the micro dilution method on the fruit extract. Aliquots (50 uL) of the reconstituted extract were filtered by means of Mueller–Hinton broth (Oxoid) medium. Triplicated tubes of different dilutions were inoculated with approximately 10⁶ cells CFU (according to the MacFarland scale) of the tested bacterial strain and the cultures were incubated at 37 ^◦C for 24 h. The MIC was taken as the highest dilution (least concentration) of extract showing no detectable growth.

2.7 Statistical analyses

Three analytical replicates (n = 3) were carried out for each analysis of the extracts. Measurements were averaged, and the results are given as mean and standard deviation (SD). The analyses were performed with the Statistical package by Minitab, Inc., v. 12 (State College, PA, USA).

 

3 Results and discussion

3.1 Physicochemical analysis of Capulin fruit

The ethanol extract had the highest extraction yield percentage (66.97%) in comparison with the other extracts. No significant differences were found among yields from the aqueous (46%), acetone (47.80%) and methanol (48%) extracts (Table 1). The moisture content of the fruit was 75%, the total acidity evaluation (expressed as malic acid) was 1.1% and the pH found in pulp from the fruit was 3.96, this was a value significantly higher than those found in the different extracts, thus assuring the stability of the anthocyanins and phenolic compounds present in same (Markakis, 1982).

The presence of anthocyanins and polyphenols provide specific colorations which can be estimated through the magnitude of color parameters. In this work, the "a*", "b*" and "L*" color parameters were highest in the aqueous extract (11.63, 11.85, and 9.61% respectively), indicating a higher presence of anthocyanins and polyphenols. No significant differences in total color change with respect to the pulp were found among the acetone (19.53±1.67), ethanol (17.22±1.67) and methanol (18.16±1.34) extracts but significant differences were found with the aqueous extract (8.89±0.50), indicating a low color change with respect to the pulp color.

3.2 Antioxidant analysis of extracts

After 30 min of incubation the absorbance of the reaction with the antioxidant present and the free radical DPPH did not change, which suggested that 30 min were enough to determine the antioxidant activity in vitro of the extracts. Said extracts inhibited a DPPH radical in a dose scavenging manner, which reached maximum values at a concentration of 10 mg/mL (Fig. 1). No significant differences were found between the ethanol (73.47±0.00%) and methanol (72.74 ± 1.03 %) extracts and they had the highest scavenging activity, followed by the aqueous (51.38%) and acetone (44.03 ± 0.42%) extracts, respectively (Table 2). The ethanol and methanol extracts had a high polyphenol content and no significant differences were found between them; this is in accordance with some studies which mention that the free radical–scavenging activity is greatly influenced by the phenolic composition, and that the structural conformation of phenolic compounds distinguishes different type of antioxidant mechanisms (Bors et al., 1997; Larrauri et al., 1996).

The acetone extract had the highest anthocyanin concentration (141 ± 3 Cyd–3–glu /100 g extract) and polyphenol content (1937 mg GAE /100 g extract), followed by ethanol and methanol, which did not show significant differences, whereas the aqueous extract had the lowest anthocyanin concentration (3 ± 1 mg Cyd–3–glu /100 g extract) and polyphenol content (747 mg GAE /100 g extract). The acetone extract had the highest anthocyanin and polyphenol concentrations; these possessed high reactivity, which permitted them to act as hydrogen or electron donors. It has been reported that various anthocyanins and flavonoids (mainly, cyanidin–3 glucoside and cyainidin 3–rutinoside) were found in samples of Capulin extracted with acethone (Ordaz–Galindo et al., 1999), which are probably responsible for the antioxidant activities detected. In this case, however, the antioxidant activity was low (45 ca) meaning that the polyphenolic content of the extract did not seem to be the only factor determining the antioxidant activity (Larrauri, Ruperez and Calixto–Saura, 1996).

On the other hand, the aqueous extracts presented the highest value of redox potential (429±2 mV), whereas the methanol extract had the lowest redox potential (236±2 mV). No significant differences in redox potential were found among the ethanolic, acetone and aqueous extracts. The reducing power of the extracts at 1 ml/ml varied within the range of 1.99 to 3.16, in the following order: ethanol (3.16) > methanol (2.99) > acetone (2.94) > aqueous (1.99) extracts. The ethanolic extract also had a high anthocyanin (102±7.70 Cyd–3–glu /100 g extract) and polyphenol (1732±43.40 mg GAE /100 g extract) content and a capacity for scavenging, which was about 73% in comparison with the others. This extract also had high reducing power (3.164), optical density (0.921±0.08), and redox potential (395±2) factors which provide constitutes more precise information about the antioxidant ability of the molecules present in these extracts. The same can be said in regard to the efficient hydrogen and electron donating properties of their compounds, capable of neutralizing free radicals and forming stable products (Liyana–Pathirana et al., 2006).

3.3 Antimicrobial properties of the extracts

The results showed that the extracts had different levels of sensitivity to the same bacteria. The data indicated that the ethanol and acetone extracts exhibited higher antimicrobial activity (with halo inhibition up to 11 mm) against the microorganisms (bacteria and yeast) tested; whereas the methanol extract was the least effective against gram (–) bacteria (Table 3). The results obtained from the disc diffusion method indicate that all the extracts studied produced slight inhibition zone (with halo < 2 mm) on gram (+) bacteria. The ethanol extract showed high inhibition on gram (–) bacteria especially against Proteus mirabilis and the yeast Sacchraromyces cerevisiae, but it did not show any effect on Enterococcus faecalis; On the other hand, the methanol extracts inhibited the gram (+) bacteria Enterococcus faecali s and Staphylococcus aureus but were only slightly effective against Salmonella typhimurium.

All the extracts show differences in the effect of their antioxidant compounds on gram (+) and gram (–) bacteria, these being due to the structural differences between these groups (Russel, 1991). Gram–negative bacteria possess an outer membrane surrounding the cell wall, which restricts the diffusion of hydrophobic compounds through its lipopolysaccharide covering. The results show that the ethanol extract possesses major antibacterial activity towards gram (–) and gram (+) bacteria, and that this is associated with its redox properties as hydrogen donators and single oxygen quenchers (Rice–Evans et al. 1997). In addition this extract contains more phenolic compounds which have been responsible for several properties, including antimicrobial ones (Chanwitheesuk et al., 2007; Panizii et al., 2002).

The values obtained suggested a broad antimicrobial activity of the extracts in a concentration–dependent manner against the tested bacteria and yeast (Table 4). The ethanol extract showed the highest inhibitory effect on cell viability. The MIC of the ethanol extract on gram (–) bacteria varied from 20 to 160 mg/mL for Salmonella typhimurium and Pseudomona aeruginosa, respectively. All the extracts had a slight antibacterial activity against at least one bacterium, but none of them showed antifungal activity against Aspergillus flavus and Aspergillus niger.

On the other hand, all the extracts studied showed the lowest MIC against Sacharomyces cerevisiae, this being the most sensitive microorganism, acetone and ethanol showed an MIC of 20 mg/mL, whereas, the aqueous extract had no effect on this microorganism. Pseudomona aeruginosa and Enterococcus feacalis showed the most resistance to the extracts, with an MIC of about 160 mg/mL. The aqueous extract was the least efficient growth inhibitor against all types of bacteria tested (MIC 320 mg/mL), with the exception of Sacharomyces cereviasiae. The degree of antibacterial activity of the extracts according to the number of microorganisms inhibited and to the MIC can be expressed in the following order: ethanol > acetone > aqueous > methanol.

As expected, the MIC of the extracts was highest in the ethanol extract, which showed the greatest antioxidant activity, thus revealing that this ethanol extract is more effective than the others. All the extracts, with the exception of the methanol extract had but a little effect against the gram (–) pathogen Salmonella typhimurium,which is an extensively studied microorganism due to the infections it produces. Thus, adding the ethanol extract to foods may lower the risk of bacterial infections, particularly in the intestinal tract, mainly due to the protective action provided by its antioxidant compounds.

 

Conclusion

The redox potential, reducing power and optical density confirmed the antioxidant activity of the acetone, methanol and ethanol extracts. Of the four tested, the ethanol extract presented the most antioxidant activity observed with the antioxidant test, and the most antimicrobial activity against Proteus mirabilis, Salmonella typhimurium, Escherichia coli and Pseudomona aeruginosa which are gram (–) and against the gram (+) Staphylococcus aureus. All of this suggests that these extracts represent an interesting food additive for incorporation into functional foods, due to the presence of both antioxidant and antimicrobial (against gram (–) bacteria and yeast) effects.

 

Acknowledgements

This work was supported by the financial aid we received by PROMEP.

 

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