REGISTRO DOI: 10.69849/revistaft/ar10202502120730
Rutilene Jacondino Roll1;
Andrés Delgado-Cañedo².
Abstract: Propolis’ primary ingredients are weakly soluble in water, oil, and other medical solvents but highly soluble in ethanol. The production of propolis glycolic extracts was published 20 years ago and some authors suggested that the biological effects of these extracts could resemble those of the ethanolic extract. Despite the fact that this technique is widely used by businesses to produce non-alcoholic extracts, this communication aims to warn that glycolic solutions may have bactericidal effects per se. As a result, the glycolic solutions would be viewed as both a solubilizer and a component that will provide the extract its bactericidal properties.
Keywords: non-alcoholic extract of propolis; antibacterial effect; glycolic extracts; propolis; bee products; apitherapy.
Funding details: The authors report there are no competing interests to declare.
Propolis and other bee products have long been studied for their medicinal benefits, especially with regard to their antibacterial properties. Researchers have also looked into how the chemical makeup and organoleptic properties affect their pharmacological properties (KHALIL 2006).
The primary active components of propolis dissolve efficiently in ethanol but are very weakly in other solvents often employed in the pharmaceutical business to treat many disorders, that cannot be safely treated with ethanolic extracts (KUBILIENE et al 2015).
The first published work, using glycolic propolis extract, evaluated the effect of different diluents on the concentration of flavonoid and phenolic acids (VOLPI 2004) and the first use to evaluate the antibiotic effect occur in 2010 (VALERA et al 2010), they used commercial propolis extract and the glycolic compound was not described (VOLPI, 2004).
Using polyethylene glycol, Kubiliene et al. (2015) produced non-ethanolic propolis extracts that had greater phenolic component content but weaker antibacterial efficacy than alcoholic extracts.
Other solvents as polyethylene or propylene glycol were tested in oral health studies, food preservation or basic research, suggesting their use (MAEKAWA et al 2013; ROLLINI et al 2017, METO et al 2020, SURAN et al 2021, FREITAS et al 2022).
With the aim of producing and testing the antimicrobial potential of alcoholic and non-alcoholic propolis extracts using propolis, from the Brazilian Pampa biome, collected in the municipality of São Gabriel (Rio Grande do Sul State, Brazil), they were produced by Apis mellifera by using lateral collectors. The extract had a yellowish color and was performed according to Kubiliene and cols. (2015).
We used 5 grams of raw propolis in 15 milliliters of 80% ethanol to generate the extracts. The propolis extract was dried, weighed, and utilized to create 500 μg/mL solutions in different solvents (ethanol 80% or PEG550 20% diluted in water), always treating the microorganism with the solvent solution (water, ethanol 80% or PEG550 20% diluted in water) without propolis, as negative vehicle controls. When alcohol was utilized as a propolis solvent, 30 minutes were given for the propolis extract and the vehicle to evaporate.
For positive control, we used ampicillin (50 mg/mL) and amoxicillin (50 mg/mL) dissolved in water. These solutions were tested against Escherichia coli and Staphylococcus aureus using the inhibition halo assay, respectively. Every test was run using 5μl of the respective treatment or control solutions. Each experiment was performed in triplicate, and the treatment values were assessed against the vehicle controls utilizing the T-test. Differences with a p-value < 0.05 were deemed statistically significant. Regarding the inhibition halo assay, the ethanolic extract presents a positive effect (p < 0.0001) when compared with its control. However, the result obtained with the PEG extract was similar to its vehicle control (p = 0.59) (Table I), highlighting that the PEG+Water control produced an inhibition halo, showing an antibacterial effect per se.
Table I. Effect of propolis extract solutions with different solvents (alcoholic and non-alcoholic) on the growth of Escherichia coli and Staphylococcus aureus in diffusion tests (in disks – 6mm).
| Treatment ( propolis extract) | Size of the inhibition halo (mm) | Negative control (ethanol without propolis extract) | Size of the inhibition halo (mm) | P-value | |
| E. coli | EEP80 | 10.5 ± 0.75 | Ethanol 80% | 0 | < 0.0001 |
| NEEP (PEG550+H 2O) | 7.00 ± 2.6 | (PEG550+H 2O) | 9.00 ± 0.7 | 0.59 | |
| C+ AMP | 34.16 ± 1.61 | H20 | 0 | < 0.0001 | |
| S. aureus | EEP80 | 11.75 ±1.76 | Ethanol 80% | 0 | < 0.0001 |
| NEEP (PEG550+H 2O) | 7.00 ±1 | (PEG550+H 2O) | 7.5 ± 1 | 0.54 | |
| C+ AMC | 31.5 ± 0.9 | H20 | 0 | < 0.0001 |
In light of our findings, we performed an extensive literature search to identify articles that assessed the effect of PEG solution on bacterial viability or growth. Two papers that were published more than fifty years ago were found. In these works, the effect of polyethylene glycol (PEG) was discovered to be detrimental to the bacteria’s ability to survive in aerosol form (Cox, 1965 apud Cox, 1966). One year later, the same author (Cox, 1966) reported the cytotoxicity PEG on E. coli, Serratia marcescens, and Aerobacter aerogenes cultured in solutions containing varying concentrations of PEG (between 10 and 60%) with molecular weights ranging from 200 to 600. However, no effect was observed for PEG with molecular weights exceeding 1540. Furthermore, KINNUNEN & KOSKELA (1991) investigated the antimicrobial effects of three glycols, other than PEG, including propylene glycol, hexylene glycol, and 1,3-butylene glycol, against Candida albicans, Staphylococcus aureus, S. epidermidis and Streptococcus pyogenes A, Mitis, and E. They showed that fresh tryptic soy broth containing 10% and 30% hexylene glycol could completely eliminate all of the aforementioned bacteria; and 30% propylene glycol and 30% 1,3-butylene glycol performed similarly to 10% hexylene glycol in terms of efficacy. The results support the substitution of hexylene glycol for propylene glycol and 1,3-butylene glycol in dermatological and cosmetic products.
Based on the information provided, we decided to conduct an experiment to confirm the impact of 20% PEG solutions of various molecular weights (550, 3350, 6000, and 8000) against E. coli using the inhibition halo assay. Our results were similar to the result obtained by Cox (1966), showing antimicrobial effects only for the low molecular weight PEG (PEG550).
Reviewing the methodology of the aforementioned articles it was noted that, with the exception of Meto and colleagues study, it is unclear from these studies if negative vehicle controls, such as the glycolic solutions used to produce the extract, were employed or whether they observed no antibacterial effects.
Taken together, our results and the literature data highlight the necessity to assimilate that: the glycolic compound may present antimicrobial activity per se and it is mandatory to perform an experimental design including the vehicles together with the model microorganism to test its antimicrobial effect; each microorganism may responds differently to the many glycolic compounds and different propolis sources may present different behavior in these extracts when assayed with different methods (for example liquid vs. agar assays).
In conclusion, due to the natural antibacterial properties of the glycolic compounds, the lack of controls containing the glycolic vehicles does not allow us to state that the activity of a glycolic propolis extract is due to propolis and not to the glycolic compound used to perform the extract.
Acknowledgement: “This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001
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¹Universidade Federal do Pampa, Campus São Gabriel, Rio Grande do Sul, Brasil, Rutilene Jacondino Roll (0000-0002-8760-4389).
²Universidade Federal do Pampa, Campus São Gabriel, Rio Grande do Sul, Brasil, andrescanedo@unipampa.edu.br, Andrés Delgado-Cañedo (0000-0002-8377-6204)