RESEARCH WORK

Antimicrobial potential and characterization of Melipona beecheii propolis from two agroenergetic farms

Leydi Fonte-Carballo, Yanio E. Milián-Rodríguez and Maykelis Díaz-Solares

Estación Experimental de Pastos y Forrajes Indio Hatuey, Universidad de Matanzas, Ministerio de Educación Superior Central España Republicana, CP 44280, Matanzas, Cuba
E-mail: leydis.fonte@ihatuey.cu

ABSTRACT

The study was conducted in two agroenergetic farms of Matanzas province (La Primavera and Indio Hatuey), in order to determine the in vitro antimicrobial activity of the methanolic and ethanolic extracts of propolis collected in these farms, and characterize them through infrared spectroscopy. The minimum inhibitory concentration (MIC) of such extracts was determined before studying the antimicrobial activity (by the method of agar perforations). Both biological essays were evaluated against Staphylococcus aureus, Escherichia coli, Pseudomona aeruginosa and Candida albicans. As the variance homogeneity and normality assumptions were not fulfilled, the non-parametric Kruskal Wallis test was used, to determine the existence of differences among the treatments depending on the inhibition halo. The propolis extracts from the two farms showed low MIC values for the evaluated microorganisms (between 0,07 and 0,15 mg/mL), with the exception of P. aeruginosa which was the most susceptible one (0,04 mg/mL). From the three tested dilutions: 100, 75 and 25 %, the first (belonging to the methanolic propolis extract from La Primavera farm) reached the highest inhibitory effect. The highest difference in the infrared spectra was found in the region interval from 2 000 a 500 cm^-1 of the propolis belonging to the Indio Hatuey farm. It is concluded that the propolis extractions with methanol and ethanol allow to obtain natural products with high antimicrobial activity against the four evaluated microorganisms.

Key words: infrared spectroscopy, microorganisms, honey.

INTRODUCTION

The integration of stingless bee rearing (meliponiculture) in agroforestry is considered an economic option. Meliponiculture is a common activity in Central America and Mexico, but it is carried out at small scale and in a traditional way, without any advanced technology; this procedure does not constitute a barrier, and is a convenient practice at the level of groups with few economic resources. In addition, its honey is preferred at local level because it has medicinal properties, supported on their low microbiological counts and high antimicrobial capacity, especially the Melipona beecheii honey (Landaverde et al., 2006).

The interest in introducing stingless bees in agroforestry systems and agroenergetic farms lies on the benefits they induce as pollinators. They collect the nectar and pollen from the flowers as energy and protein source. About half of the 1 000 plant species that are cultivated in the tropic for food and spice and medicine production are pollinated by bees. Among them are macadamia, chayote, coconut tree, achiote, onion, guava tree, peach palm, tamarind, avocado and citrus fruit trees (Heard, 1999).

In its pollinating action bees collect other products from the plants which are used as raw material to elaborate propolis, which is a resinous and aromatic material they elaborate from exudates of different plants. Its color varies depending on its origin and age (Wilson et al., 2015). This byproduct of the hive, besides constituting a construction material, is a «chemical weapon» that the bees use against pathogen microorganisms, because by covering the beehive walls they favor disinfection (Bankova, 2005a).

Based on the practical use that bees give to this product, it has been extensively used for many years in traditional medicine. It is estimated that 150 compounds, many of them flavonoids, have been identified in European propolis (Yang et al., 2014; Figueiredo et al., 2015).

According to Bracho (2000), several methods are used for the characterization of propolis. Among them are thin-layer chromatography (Tang et al., 2014) and infrared spectroscopy (Adib et al., 2014; González-Martín et al., 2015). The latter is a widely used technique to discriminate between pure and adulterated propolis extracts (Wu et al., 2008), and it has also been used to measure the quality and stability of the propolis-based products that are incorporated in the preservation of foodstuffs (Cattaneo et al., 2014).

Among the main components of propolis diverse metabolites have been identified, such as flavonoids, caffeic acid esters, diterpenes, benzophenones and volatile constituents, such as sesquiterpenes; in addition to protein substances, non-saturated fatty acids and aromatic acid esters (Barbari et al., 2011; Socha et al., 2014; Cottica et al., 2015).

It has been reported that propolis have pharmacological, bactericide, antiviral, hepatoprotective, anti-inflammatory, immunomodulatory, antioxidant and analgesic properties (Bankova, 2005b; Shi et al., 2012; Chan et al., 2013; Shpychak et al., 2015).

However, until now the microbial activity that can be shown by the propolis obtained from M. beecheii hives, as added values of the productions of agroenergetic farms, is unknown; for which the objective of this research was to determine, through in vitro studies, the antimicrobial activity of the methanolic and ethanolic extracts of propolis collected in two agroenergetic farms of Matanzas province and to know their chemical specificities through infrared spectroscopy.

To evaluate the antimicrobial activity of the propolis extracts of each farm, a simple classification design was used. Different pathogen microorganisms were used to evaluate the effectiveness of the extracts.

The propolis were collected in the hives located in two agroenergetic farms of Matanzas province (La Primavera- farm P and Indio Hatuey-farm IH), belonging to the BIOMAS-CUBA project.

The ethanolic and methanolic propolis extracts were obtained re-suspending 20 g of each propolis in 30 mL of ethanol and methanol (96 % (v/v)). The suspension was decanted after 48 h at ambient temperature. The extracts were preserved at 4 ºC until their utilization.

Preparation of the extracts and controls. The extracts were prepared using ethanol and methanol at a concentration of 4 mg/mL in Mueller-Hinton growth medium, with serial dilutions for both solvents from 1 000 to 0,95 µg/mL. For the antibacterial bioassay, ciprofloxacin, kanamycin, nystatin, clotrimazole, amoxicillin and cephalexin were used as controls, adding 5 µL of each. In addition, microorganism growth controls were used, with the following solvents: negative control 1 (CN1, referring to methanol), negative control 2 (CN2, referring to ethanol) and a sterility control of the Mueller Hinton cultivation medium for bacteria.

Preparation of the inoculants. For the preparation of the inoculants young cultures were obtained from the bacteria strains Staphylococcus aureus, Escherichia coli, Pseudomona aeruginosa and the yeast Candida albicans in the Mueller Hinton solid medium. The bacteria were incubated on plates during 24 h at 37 ºC and the yeast during 48 h at 28 ºC. For the bacteria a suspension was prepared with the strains at 0,5 in the McFarland scale and a dilution of 1:50 with Mueller Hinton medium to obtain a work suspension.

Agar perforations method. After the solidification of the medium, six perforations were made of 0,8 cm diameter, where 100 µL of the extract to be evaluated and of the negative and positive controls were placed, with 10 replications per treatment for each microorganism, and the extracts were tested using three dilutions (100, 75 and 25 %).

Incubation, microbial growth and inhibitory effect. The plates were incubated at 37 ºC for 24 h in the case of bacteria and at 28 ºC during 48 h in the case of the yeast, and afterwards the results were evaluated through the reading (in millimeters) of the diameter of the growth inhibition halo of the microorganisms. The calculation of the percentage of the relative inhibitory effect with regards to the positive control was made as follows:

Where:

IE (%): inhibitory effect of the extract with regards to the positive control.

MDIHE: mean of the diameter of the inhibition halo of the extract.

MDIHPC: mean of the diameter of the inhibition halo of the positive control.

The samples were crushed and the infrared spectra were obtained in the interval from 4 000 to 500 cm^-1, with the use of potassium bromide pills, in a Fourier Bruker-TENSOR 27 transform infrared spectrometer.

The variable inhibition halo diameter did not fulfill the variance homogeneity and normality assumptions, for which to determine the existence of difference among the treatments the non-parametric Kruskal Wallis test was carried out and InfoStat was used (Di Rienzo et al., 2014).

The minimum inhibitory concentration (MIC) is defined as the lowest concentration of extract that is capable of inhibiting cell growth.

The methanolic and ethanolic propolis extracts of M. beecheii showed low MIC values for the different microorganisms (table 1). In the case of P. aeruginosa, the most susceptible bacteria within the Gram-negative group, these values reached up to 0,04 mg/mL in the ethanolic extract of the Indio Hatuey farm.

The results of the antimicrobial activity of plant extracts depend on the specific characteristics of the active principles present in them and on their solubility in the solvents used for the extraction. This explains the finding of active extracts only against fungi or only against bacteria, Gram-positive or Gram-negative; or against both groups of bacteria, when the extract was capable of extracting a mixture of active components present in the plant (Rojas and Rodríguez, 2008).

In general, the methanolic propolis extract from the Primavera farm in the 100 % dilution was the best treatment against the four evaluated microorganisms (tables 2 and 3), showing the highest values in the inhibition halos. In addition, S. aureus was the most susceptible Gram-positive bacteria to this extract, showing the highest inhibition halos. These results coincide with the statement by Krolicka et al. (2008) and Chouna et al. (2009), regarding the fact that Gram-positive bacteria are more susceptible than Gram-negative ones, because the cell wall of the former is less complex and lacks an effective filtration for large molecules, due to the pore size in their cell wrap, for which they are less selective.

The infrared spectra of each propolis were obtained, compared among themselves and the existing differences in their chemical composition were proven (fig. 1). No spectra were obtained with very intense or well defined bands, as it was expected in purified or previously treated compounds, because the sample is a complex matrix formed by a large number of compounds of different chemical composition; however, marked differences were noted among the samples of each farm, which proved that there was variation in their chemical composition. In addition, some bands appeared which provide information of the chemical species that constitute the propolis from both farms.

The highest differences in the spectra were found in the interval of the region from 2 500 to 500 cm^-1, mainly the bands that appear at 2 510,26; 1 034,77; 874,68 and 668,31 cm^-1, in the propolis from the Indio Hatuey farm (table 4), corresponding to valence vibrations between the simple and double bonds, strongly coupled, such as ½_C-O o ½_C-F; besides doubling vibrations outside the plane and low energy valence vibrations, such as ³ _CH of olefins and aromatics, ³ _OH present in associated alcohols and acids or ³ _NH present in associated amines and amides, which indicates that this propolis shows higher content of this type of compound than that of the Primavera farm.

In 1 450 cm^-1 a medium intensity band assigned to the valence vibration of the C H» C (½_{C H» C}) bond of aromatic rings was observed, besides a band in 1 034 cm^-1 assigned to ½_C-O; all these groups are present in some of the compounds responsible for the biological activity of the propolis, such as flavonoids and anthraquinones (Moþ et al., 2011; Figueiredo et al., 2015). Besides, another band was observed at 1 709 cm^-1, characteristic of the C = O (½_{C = O}) bond stretching band corresponding to groups of ketones or aldehydes, possible substituents of terpenes, compounds to which antimicrobial properties are also ascribed (Silva Frozza et al., 2013); which would explain the results referring to the diameter of the inhibition halos shown by the methanolic and ethanolic extracts from the two farms (tables 2 and 3), and especially the result of the former extract belonging to La Primavera farm.

The propolis extractions with methanol and ethanol allowed to obtain natural products with high antimicrobial activity against the four evaluated microorganisms.

The propolis methanol extract of La Primavera farm showed a higher inhibitory effect, and Staphylococcus aureus was the most susceptible bacteria to this treatment.

The infrared spectra of the propolis allowed to identify functional groups present in some of the chemical compounds, such as flavonoids, anthraquinones and terpenes, responsible for the antimicrobial activity of this byproduct of the M. beecheii hive.