Antibacterial Properties of Propolis

1Propolis is a mixture of substances used by bees to defend the hive. This protection concerns filling cavities in the walls of the hive, reducing the entrance during cold days, and also mummifying the demanded intruders, thus preventing their decay. This explains why propolis is also known as bee glue. The word, propolis, is Greek in origin and means at the entrance to the city. Discussions on the origin of propolis have been continuing since ancient times. Doubts have arisen about whether its origin comes from plants or bees. Nowadays, with the development of analytical techniques, the approximate composition of propolis and the factors influencing it are known.

2Bees collect resins from buds, exudates and other parts of plants, mix them with their own salivary enzymes and beeswax which creates propolis. The different continents, regions and plant species used to produce propolis make its composition different from each other. Even though propolis has a different chemical composition, it has similar activities such as antibacterial, antifungal, antiviral, antiparasitic, anti-inflammatory, antiproliferative and antioxidant.

3In Central Europe, including Poland, bees collect secretion from buds of poplar (Populus spp.), alder (Alnus spp.). Different poplar species are also a source of resin in other European countries, i.e., Bulgaria, Hungary, Albania and England as well as in temperate zone countries (USA, Mongolia, West Asia and North Africa). Birch (Betula spp.) is a source of propolis in Northern Europe, e.g., North-European part of Russia. Bees produce propolis also from buds of willow (Salix spp.), oak (Quercus spp.), ash (Fraxinus spp.), chestnut tree (Aesculus spp.) and bark from coniferous trees, such as spruce (Picea spp.), fir (Abies spp.) or pine (Pinus spp.). In tropical countries, bees use the secretions of such plants as Xanthorrhoea (Australia), Acacia (North Africa), Plumeria (Hawaii), Clusia (Central America) and Baccharis, Araucaria, Eucalyptus (Brazil).

4Brazil is the leading country in research on products of bees, including propolis. In Brazil, many types of propolis are distinguished because of their botanical origin. In this climate, bees collect propolis throughout the year. Bueno-Silva et al., in their study, analyzed one of the most common type of Brazilian propolis, referred to as red and whose primary plant source is Dalbergia ecastophyllum. The effect of the time of the collection of propolis, its chemical composition and antibacterial activity was examined. Seasonal variability between the concentration of vestitol, neovestitol and isoliquiritigenin was observed. The highest content of these ingredients and antibacterial activity was recorded during the rainy season (the period from January to May).

5Another type of propolis comes from stingless bees (e.g., Melipona mondury, M. scutellaris) and it is called geopropolis. It is very similar to propolis produced by bees belonging to Apis spp. in both composition and biological activity. Torres et al. in their study compared two ethanolic extracts of propolis (EEP) collected from a stingless species of bees, Melipona quadrifasciata and Tetragonisca angustula. The study showed the more significant activity of geopropolis extracts against Gram-positive bacteria (Staphylococcus aureus MSSA and MRSA, Enterococcus faecalis) than Gram-negative (Klebsiella pneumoniae, Escherichia coli). From the two geopropolis which were analyzed, the Melipona species was more effective.

6Raw propolis cannot be used directly in analysis or treatment. First, it must be extracted in order to dissolve and release the most active ingredients. The following solvents are used as the extractants: ethanol, methanol, water, hexane, acetone, dichloromethane and chloroform. Extracts contain approximately 70% concentration of propolis. In terms of antibacterial activity, the content of substances such as flavonoids and phenolic compounds is important. However, depending on the solvent used, different biological activity is found. Devequi-Nunes et al. found approximately two times higher concentrations of phenolic compounds in ethanolic extracts of brown, green and red propolis than in extracts obtained by supercritical extraction. At the same time, the levels of flavonoids were higher in ethanolic extracts of green and red propolis and lower in brown propolis, compared to supercritical extraction. Wieczyńska et al. in their studies found a stronger antimicrobial activity of ethanolic extracts from Polish propolis than hexane extracts.

7The aim of this review is to show the main active substances of propolis and antibacterial activity of this bee product.

8The chemical composition of propolis is closely related to the resins and balsams of plant sources used to produce it. Along with the progress of research, more than 300 chemical components of propolis have been identified. The main groups of chemical compounds found to be present in propolis except resins are waxes, polyphenols (phenolic acids, flavonoids) and terpenoids (Figure 1).

9Polyphenols and terpenoids are also considered to be the most active. The flavonoid group includes chrysin, pinocembrin, apigenin, galangin, kaempferol, quercetin, tectochrysin, pinostrobin and others (Figure 2). Another critical group of compounds of propolis are aromatic acids, among which the most often occur in ferulic, cinnamic, caffeic, benzoic, salicylic and p-cumaric acids (Figure 3). In Polish propolis, the content of flavonoid compounds ranged from 6.2 to 18.8%. Among the flavonoids, the highest amounts were pinocembrin (mean 4.7%), pinobenchin (mean 3.1%), galangin (mean 2.2%) and chrysin (mean 2.1%). In addition, propolis also includes other phenolic compounds (e.g., artepillin C), and terpenes (terpineol, camphor, geraniol, nerol, farnesol) which are responsible for its characteristic fragrance (Figure 4). In propolis, micro and macroelements (Mn, Fe, Si, Mg, Zn, Se, Ca, K, Na, Cu) and vitamins B1, B2, B6, C and E can be found. This diversity of the chemical composition gives propolis an additional advantage as an antibacterial agent. The combination of many active ingredients and their presence in various proportions prevents the bacterial resistance from occurring.

10The antibacterial activity of propolis should be considered on two levels. First, it is connected with the direct action on the microorganism, and the other with stimulation of the immune system resulting in activation of natural defenses of the organism. The analysis of the mechanisms of propolis allow it to infer its effect on the permeability of the cellular membrane of microorganism, disruption of membrane potential and adenosine triphosphate (ATP) production as well as decreasing bacterial mobility (Figure 5). Generally, it is observed that the antimicrobial activity of propolis is higher in relation to Gram-positive than Gram-negative bacteria. This is explained by the species-specific structure of the outer membrane of the Gram-negative bacteria and the production of hydrolytic enzymes which break down the active ingredients of propolis. Artepillin C (3,5-diprenyl-p-coumaric acid) is one of the numerous phenolic compounds (prenyl derivative of p-coumaric acid) found in propolis. Research carried out in Brazil by Veiga et al. shows a higher concentration of artepillin C in ethanolic extracts of propolis compared with hexane extracts. These extracts also showed high antibacterial activity on MRSA S. aureus. In studies against anaerobic bacterium Porphyromonas gingivalis, it was found that artepillin C has bacteriostatic activity with membrane blebbing. Artepillin C shows additionally anti-inflammatory effects mediated with modulation of NF-kappaB and inhibition of prostaglandin E(2) and nitric oxide.

11Other prenyl derivatives found in propolis, i.e., 3-prenyl-cinnamic acid allyl ester and 2-dimethyl-8-prenylchromene, also have similar antimicrobial activity. The antimicrobial effect on skin infection-related microbes, such as S. aureus also presents kaempferide. The ethanolic extract of propolis containing high concentrations of kaempferide, artepillin-C, drupanin and p-coumaric acid showed antioxidant activity and antibacterial against S. aureus, S. saprophyticus, Listeria monocytogenes and E. faecalis.

12Other flavonoids found in propolis are pinocembrin and apigenin. The study conducted by Veloz et al. on Chilean propolis found that antibacterial activity of both of these compounds against Streptococcus mutans is higher than activity of polyphenols mixture or even chlorhexidine (MICs = 1.6 µg/mL), with minimum inhibitory concentrations (MICs) 1.4 µg/mL and 1.3 µg/mL, respectively. Several studies have demonstrated the antibacterial activity of isolated pinocembrin against S. mutans, S. sobrinus, S. aureus, E. faecalis, L. monocytogenes, Pseudomonas aeruginosa and K. pneumoniae. Isolated apigenin acts against Gram-negative bacteria: P. aeruginosa, K. pneumoniae, Salmonella enterica serotype Typhimurium, Proteus mirabilis and Enterobacter aerogenes. It was also observed the synergistic antibacterial effect of apigenin with β-lactam antibiotics against methicillin-resistant S. aureus (MRSA), and apigenin with ceftazidime against ceftazidime-resistant Enterobacter cloacae.

13Cinnamic acid and its derivatives are a group of aromatic, carboxylic acids commonly found in the plant kingdom. They are present both in green parts of plants and in flowers. Propolis, as a material composed to a large extent of plants secretions, is a rich source of cinnamic acid and esters. Many studies documented the antimicrobial activity of cinnamic acid against Aeromonas spp., Vibrio spp., E. coli, L. monocytogenes, Mycobacterium tuberculosis, Bacillus spp., Staphylococcus spp. Streptococcus pyogenes, Micrococcus flavus, P. aeruginosa, S. enterica serotype Typhimurium, Enterobacter cloacae and Yersinia ruckeri. Cinnamic acid and its derivatives inhibit bacteria by damaging the cell membrane, inhibiting ATPases, cell division and biofilm formation. Moreover, they had anti-quorum sensing activity.

14Interestingly, there are many other ingredients of propolis, such as terpenoid lupeol, flavonoids: quercetin, chrysin, kaempferol, fisetin or decanoic acids, i.e., 10-hydroxyl-2-decenoic acid. Some studies are analyzing the antibacterial and anti-inflammatory activity of quercetin, chrysin and kaempferol.

15In the research conducted in Korea by Park et al., scientists noticed the effectiveness of lipases in decreasing fatty acids levels in propolis extract. Beeswax and resins are the main components of propolis and both are hydrophobic. The use of fat-degrading enzymes helps to improve the extraction and isolation of active propolis compounds which could make it be used much more widely. The reaction involving lipozyme TL IM increases antimicrobial activity against Staphylococcus epidermidis and Propionibacterium acne.

16Kubiliene et al. compared composition and biological activities of propolis extracts prepared with an alternative, nonalcoholic mixture of solvents such as polyethylene glycol 400 (PEG), water and olive oil. They found no significant differences in the total content of phenolic compounds compared with ethanolic extracts. However, the determination of different classes of compounds was not specific. PEG-water solutions presented naringenin, galangin and kaempferol. The addition of PEG connected with exposition for higher temperature enabled extraction of ferulic, caffeic, p-coumaric acids, quercetin and artepillin C. Nonalcoholic extract of propolis was prepared by mixing water, oil and PEG and presented similar or higher antimicrobial activity against S. aureus, Bacillus cereus, P. aeruginosa and K. pneumoniae then EEP.

17Recently, the evaluation of the antibacterial activity of propolis extracts is based on the determination of total phenolics content (TP) and flavonoids (FP). In their study, Bridi et al. found that TP and FP tests do not always adequately reflect antimicrobial activity in vitro. The results of the TP in the samples with the highest and the lowest content were directly proportional to the content of flavonoids and antioxidant properties. However, they were not unambiguous in the case of antibacterial activity. It is suggested that other tests, e.g., ORAC (Oxygen Radical Absorbance Capacity) and antimicrobial tests, should be considered in setting international quality standards for propolis.

18Assuming that propolis and products of other bees have antibacterial activity, their combination should intensify this effect. Al-Waili et al. examined such a dependence on the combination of propolis and honey collected in Saudi Arabia and Egypt. The results of this study confirm that the combination of propolis and honey enhances their antimicrobial effect (for S. aureus and E. coli). This effect was significant for Saudi propolis (almost two times lowering the MIC value). Egyptian raw materials showed much less activity.

19There is limited research on the action of propolis on the anaerobic bacteria. However, this research indicates a high activity of this bee product against Clostridium, Bacteroides, Porphyromonas, Prevotella, Fusobacterium, Actinomyces and Propionibacterium species. In Polish studies, anaerobic bacteria of the Fusobacterium genus were the most susceptible to low concentrations (0.01–0.06 mg/mL) of ethanolic extract from propolis (EEP). However, bacteria of the genus Actinomyces, Bacteroides, Clostridium, Peptococcus, Peptostreptococcus and Propionibacterium were sensitive to EEP in high concentrations (1–3 mg/mL).

20The minimal inhibitory concentration (MIC) is the lowest concentration of an antimicrobial agent in which no growth was observed of a microorganism in an agar or broth dilution susceptibility test. This study included scientific publications specifying the MIC for extracts of propolis using the broth dilution method. The MIC is expressed in micrograms per milliliter [µg/mL].

21The analysis of the literature completed for this review included a total of approximately 600 propolis MIC values for different groups of microorganisms. In order to visualize the collected results in the form of arithmetic mean values, microorganisms of less than 3 MIC values were excluded from the analysis. These were Actinomyces naeslundii (1 record), B. cereus (1), Bifidobacterium bifidum (1), B. infantis (2), B. longum (1), Clostridium butyricum (2), C. paraputrificum (1), C. perfringens (1), L. monocytogenes (2), and P. gingivalis (1).

22The ethanol extract of propolis (EEP) was most commonly used in the analyzes, i.e., in 324 records. Methanol (MeEP) was used as the extractant at a similar frequency (209 records). The non-alcoholic extracts are much less frequently analyzed. In such cases, the solvents were water (WEP) in 19 records, dimethyl sulfoxide (DMSO) in 12 records, dichloromethane (DCM) in 10 records, and hexane and supercritical fluid (SCEP) in six records.

23The antibacterial activity of propolis is most often tested on E. coli, S. aureus, Salmonella spp. and P. aeruginosa. The ten most frequently analyzed bacteria are shown in Figure 6.

24The analysis of the average MIC values for propolis extracts confirmed their higher efficacy against Gram-positive than Gram-negative bacteria. The MIC values for EEP were 117–1840 µg/mL for the first group and 34–5000 µg/mL for the second group of bacteria. All data are shown in Table 1 for Gram-positive bacteria and Table 2 for Gram-negative bacteria.

25It is best to compare the effect of propolis (EEP) on the basis of geographical origin for S. aureus as a representative of Gram-positive bacteria and E. coli representing Gram-negative strains. This is the largest number of available tests on the listed species.

26In S. aureus, the highest activity was observed for EEP from Turkey, Taiwan and Oman with MIC values of 8, 10 and 81 µg/mL, respectively. The lowest activity was observed for samples of propolis from Chile, Australia and Germany. The MIC values for EEP were 1445, 1200 and 750 µg/mL, respectively. Against E. coli, the most active were ethanol extracts of propolis obtained from Turkey, Oman and Slovakia, with MICs of 116, 302 and 510 µg/mL, respectively. At the end, with the lowest activity, there were propolis samples from Germany, Korea and Ireland with MIC values between 1200–5000 µg/mL. Interestingly, Brazil, despite having the largest amount of research on propolis, was in the middle for both Gram-positive and Gram-negative bacteria. The results for all analyzed bacterial species are presented in Table 3 and Table 4.

27The most active in both cases were samples of propolis collected in Turkey and Oman. Both countries can be included in the Middle East countries, famous for the trade in fragrances. The same plants are probably a source for bees for the production of propolis. It would also be interesting to analyze the impact of the degree of urbanization of the country on the quality and biological activity of propolis. However, research on this topic is unavailable.

28Propolis is a significant antimicrobial bee product. It acts both against Gram-positive and Gram-negative, as well as aerobic and anaerobic bacteria. The activity of propolis depends on chemical composition and is different in individual countries.