Medicina Veterinária /utp shigehiro Funayama Uriel Vinicius Cotarelli de Andrade Pedro Ribas Werner

ACTION OF INHIBITOR COMPOUNDS ON GROWTH CULTURES OF STAPHYLOCOCCUS AUREUS AND PSEUDOMONAS AERUGINOSA ISOLATED FROM PROPOLIS OF APIS MELLIFERA.

Medicina Veterinária /UTP

Shigehiro Funayama

Uriel Vinicius Cotarelli de Andrade

Pedro Ribas Werner

ABSTRACT - A Brazilian propolis was investigated for its antibacterial properties. Several fractions were obtained during the partial purification: crude ethanolic extract of propolis, resinous material ethanolic solution and alkaline hydrolysis water soluble compounds solution. All fractions have no antibacterial activity against Escherichia coli, but the alkaline hydrolysis water-soluble compounds solution had a minimal inhibitory concentration of approximately 291 g/ml for Staphylococcus aureus and 1164 g/ml to Pseudomonas aeruginosa.

Key words: Brazilian propolis; Apis mellifera; Antibacterial activity; Alkaline hydrolysis.

RESUMO - Foi analisado o efeito antibacteriano de uma amostra de propolis de Apis mellifera obtida no Brasil. Foram obtidas algumas frações durante o processo de purificação parcial: extrato bruto etanólico de propolis, uma solução etanólica de um material resinoso e uma de compostos solúveis na água após hidrólise alcalina. Nenhuma das frações possui atividade antibacteriana sobre a Escherichia coli, mas a solução aquosa de compostos liberados após a hidrólise alcalina revelou uma concentração mínima inibitória de 291 g/ml para Staphylococcus aureus e de 1164 g/ml para Pseudomonas aeruginosa.

Palavras chaves: Propolis brasileiro; Apis mellifera; Atividade antibacteriana; Hidrólise alcalina.

Introduction

Propolis is a resinous substance secreted by bees, Apis mellifera, from various buds and bark of trees and shrubs, mixed with beeswax and -glucosidase which they secrete during the propolis collection (Ghisalberti 1979, König 1985, Burdock 1998, Nieva Moreno et al. 1999). Propolis is used by bees to seal openings in the hives and to protect themselves against predators as ants, hives moths, beetles and mice (Ghisalberti 1979, König 1985, Burdock 1998). Several biological activities have been reported for the ethanolic extract of propolis such as antibacterial, anti-inflammatory, antiviral, hypotensive antifungal, anaesthetic, immunostimulatory, antitumoral and cytotoxicity activities (Ghisalberti 1979, Bankova et al. 1983, Grunberger et al. 1988, Aga et al. 1993, Fernandes et al. 1997, Matsuno et al. 1997, Mirzoeva et al. 1997, Bankota et al. 1998, Burdock 1998, Park et al. 1998, Park & Ikegaki 1998, Kujumgiev et al. 1999). From these activities, the antibacterial action is the most extensively investigated and the differences between the ethanolic extracts being due to some factors such as bee species, propolis origin, extract preparation and bacteria tested (Fernandes et al., 1997; Matsuno et al., 1997; Mirzoeva et al., 1997; Nieva Moreno et. al., 1999). These pharmacological activities are probably due to a mixture of several compounds as flavonoid aglycones, cinnamic acid derivatives and terpenoids groups (Simuth et al. 1986, Takaisi & Schilcher 1994, Koo & Park 1997, Park et al. 1998, Maciejewicz 2001). So the interest shown by several researchers to obtain propolis preparations with defined chemical composition has increased because of the wide possibilities for medical applications. Besides some rare exactor extracts (Matsuno et al. 1997, Park & Ikegaki 1998), the major propolis preparations reported are ethanolic, methanolic or chloroform extracts (Ghisalberti 1979, Bankova et al. 1983, König 1985, Grunberger et al. 1988, Aga et al. 1993, Fernandes et al. 1997, Koo & Park 1997, Matsuno et al. 1997, Mirzoeva et al. 1997, Bankota et al. 1998, Burdock 1998, Park et al. 1998, Park & Ikegaki 1998, Kujumgiev et al. 1999, Nieva Moreno et al. 1999 Maciejewicz 2001). Since all of properties have shown discrete antibacterial activity, such as those against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Proteus mirabilis (Ghisalberti 1979, Bankova et al. 1983, König 1985, Grunberger et al. 1988, Aga et al. 1993, Fernandes et al. 1997, Koo & Park 1997, Matsuno et al. 1997, Mirzoeva et al. 1997, Bankota et al. 1998, Burdock 1998, Park et al. 1998, Park & Ikegaki 1998, Kujumgiev et al. 1999, Nieva Moreno et al. 1999 Maciejewicz 2001), the aim of this work was to establish extraction and purification method from displaying greater antibacterial activity.

Material and Methods

Propolis Propolis of Apis mellifera was obtained from bees hives raised al Tijucas do Sul, State of Paraná, Brazil.

Microorganisms: Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa ATCC 25853

Extraction and partial purification

An amount of 250 g Apis mellifera propolis was used. The samples of propolis were stored at 0.5 -1^o C to harden and then ground to powder means of an electric blender. A total of 600 ml of 75% ethanol was added and the apparatus was turned on five times during a period of 3 minutes each. These operations were repeated after 3 hours and the suspension allowed to stand for 16 hours in maceration and then filtered through filter paper. The yield was approximately 450 ml of crude ethanolic extract of propolis (CEEP), which was concentrated by aeration during 6 hours. After evaporation of the ethanol two fractions obtained: an aqueous solution and a yellowish water insoluble resinous material. About 110 g of the latter was washed with distilled water until the rinse water did not seem cloudy. The washed resinous material was dissolved in 150 ml of 99.8% ethanol and tagged as resinous material ethanolic solution (RMES). The pH of this solution was adjusted to 7.5 by addition of 15 ml NaOH 2.5 N and stored at 20^o C for at least 20 hours. After this period, the volume of this hydrolyzed mixture was reduced to half by aeration and both liquid and resinous fractions were collected. The water-soluble compounds released by alkaline hydrolysis were collected from the resinous material by washing it carefully 3 times with 50 ml of distilled water or until the rinse water did not seem cloudy. All fractions of rinsed water were added to the former one. After storage at 0.5^o–2^o C for 48 hours, the aqueous solution became cloudy. Keeping it under the same temperature, the preparation was spumdown at 10,000 x g for 10 minutes, the supernatant was collected, tagged as alkaline hydrolysis water-soluble compounds alkaline hydrolysis water-soluble compounds (AHWSCS) and the pellet was discarded.

Preparation of filter paper disks containing propolis extracts

The filter paper disks (8 x 1 mm) were incubated at 50^o C for 48 hours and their weights determined. The extracts of propolis disks were prepared applying 10 l of extract for 5 times on each filter paper disk and incubated at 30^o C during 20 minutes between subsequent applications and kept at the same temperature during 24 hours for drying, after which their weights were again determined.

Determination of antibacterial specific activities of propolis extracts

Estimation of bacteria susceptibility to propolis extracts was performed by inoculating 0.1 ml of a Brain Heart Infusion culture for microorganisms in Mueller-Hinton agar plates. The culture was spread using Drigalski's handle in circular movements until the agar plates had absorbed the broth culture. The filter paper disks containing propolis extracts were placed on the inoculated Mueller-Hinton agar plates and incubated at 37^o C for 24 hours.

Minimal inhibitory concentration of AHWSCS

Serial AHWSCS concentrations (145.5 - 2328 g/ml) were performed in Petri dishes containing 20 ml of Mueller-Hinton agar to test bacterial growth. Simultaneously, plates containing medium without AHWSCS were used as control. The agar was allowed to solidify and 0.1 ml of a culture containing approximately 10³ cells/ml was inoculated on each plate and incubated at 37^o C for 24 hours. The minimal inhibitory concentration (MIC) is defined as the lowest concentration that totally inhibits bacterial growth after incubation at 37^o C for 24 hours.

Results

Table 1 and Figure 1 summarize the extraction, partial purification and specific activities determination of propolis extracts fractions on Staphylococcus aureus culture.

Table 1 - Isolation, partial purification and antibacterial specific activities of propolis fractions

Fractions	Total volume (ml)	Dry weight (mg/50 l)	Diameter of inhibition zone on S .aureus culture (mm)	Specific activity (Inhibition zone/dry weight)	Purification
CEEP	450	10.4	11	1.0	----
RMES	230	5.3	13	2.4	2.4
AHWSCS	240	2.5	18	7.2	7.2

Figure 1. Antibacterial specific activities of propolis extracts.

Disk 1 contained 50l of CEEP, disk 2 50l of RMES and disk 3 50l of AHWSCS.

Table 2 summarizes the minimal inhibitory concentrations determinations of AHWSCS. The MIC determined for S. aureus was 291 g/ml and to P. aeruginosa was 1164 g/ml.

Table 2- Determination of minimal inhibitory concentrations of AHWSCS

Mueller & Hinton;Agar plate containing AHWSCS (g/ml)	^aStaphylococcus. aureus culture (10^3cells/ml) (ml)	^bPseudomonas aeruginosa culture (10^3cells/ml) (ml)	^cCFU Number	^dCFU Number
0	0,10	0,10	174	180
145,5	0,10	0,10	154	176
291	0,10	0,10	0	158
582	0,10	0,10	0	95
873	0,10	0,10	0	57
1162	0,10	0,10	0	0
1746	0,10	0,10	0	0

(a) Mueller & Hinton agar plate inoculated with S. aureus; (b) Mueller & Hinton agar plate inoculated with P. aeruginosa;(c) S. aureus colony formation unit; (d)P. aeruginosa colony formation unit.

Discussion

Although the chemical analysis of AHWSCS from this propolis preparation was not performed, certainly their antibacterial activity is due to a complex mixture of propolis compounds. This statement is based on differences found in Brazilian propolis chemical composition reported by Bankova et al (1983). The growth results on Mueller-Hinton agar plate (see Figure 1) and data of Table 1 show the specific activities of each purification step of propolis compounds and their effects on Staphylococcus aureus. An evidently interesting point is that extraction of propolis antibacterial compounds was increased 7 times with alkaline hydrolysis when compared to former ethanolic extraction. The AHWSCS has no antibacterial activity against Escherichia coli (data not shown), but had approximately MIC of 291 g/ml for Staphylococcus aureus (Gram-positive bacteria) and 1164 g/ml to Pseudomonas aeruginosa (Gram-negative bacteria). These results indicate that these antibacterial activities were not due to the presence of one particular compound, but to the actions of an active compounds mixture. So that AHWSCS contains some active compounds, which possess different mechanisms of antibacterial action. Since AHWSCS is an aqueous solution and it has antibacterial activity against Gram-positive and Gram-negative bacteria, these results contest the finding reported by Nieva Moreno (1999), who stated that ethanolic extract of propolis is the most active. Finally the present investigation suggests possible use of AHWSCS as a bactericidal agent in human and veterinary medicine, and the need of additional investigation on this matter.

Conclusions

The alkaline treatment of RMES hydrolyzes esters releasing active water-soluble compounds that provide a significant increase of antibacterial activity of AHWSCS. Since AHWSCS has antibacterial activity against Staphylococcus aureus (Gram-positive bacteria) and Pseudomonas aeruginosa (Gram-negative bacteria), there was a strong evidence that these antibacterial activities were due to an active compounds mixture.

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