The manufacture of ethanol-free propolis solutions offers a broader application. A few trials with Lithuanian propolis have been conducted. The aims of the study are to manufacture propolis water and water-free solutions and evaluate the quality and antimicrobial activity of these solutions. The studied solutions containing 2.5%, 5%, and 10% propolis are prepared. As solvents, purified water, 70% v/v ethanol, 96.3% v/v ethanol, propylene glycol, and their systems were used. Determination of total levels of phenolic compounds (FAE mg/g) is based on colour oxidation-reduction reaction using Folin-Ciocalteu reagent under alkaline conditions and performed at 765 nm wavelength using UV spectrophotometer. The highest content of phenolic compounds was determined in solutions containing 10% propolis extracts, and the lowest amounts in 2.5% propolis extracts. The water extracted the lowest amount of phenolic compounds from crude propolis, ethanol extracted the highest amount, and propylene glycol ranked the middle position. It is determined that technological parameters (stirring, temperature) contribute to content of phenolic compounds. During microbiological study, MICs were determined. The studies showed that water extracted propolis solutions and solvents mixture did not inhibit the growth of the studied microorganisms, and propolis solutions in propylene glycol were found to have antimicrobial activity.
Propolis as an active substance is attractive due to its antimicrobial and antimycotic properties and as a natural substance whose effect was proven by biological experiments [
Current applications of propolis include over-the-counter preparations for cold syndrome (upper respiratory tract infections, common cold, and flu-like infection) as well as dermatological preparations useful in wound healing, treatment of burns, acne, herpes simplex and genitalis, and neurodermatitis [
The manufacture of ethanol-free propolis solutions offers a broader application in medicine and everyday use. There are many data on chemical composition of propolis solutions in ethanol and antimicrobial activity. However, a few trials with Lithuanian propylene glycol extracted propolis, its water solutions, and appropriate solvent mixtures have been conducted.
The aims of the study are to manufacture propolis water and water-free solutions and evaluate quality as well as antimicrobial activity of these solutions.
The studied solutions containing 2.5%, 5%, and 10% propolis 200 mL are prepared. As solvents, purified water, 70% v/v ethanol, 96.3% v/v ethanol, propylene glycol (1,2-propanediol), and their systems composing of 6.25 mL 96.3% w/w ethanol, 2.5 g propylene glycol, and purified water up to 25 mL of total volume are used. Crushed crude propolis is soaked in an appropriate amount of solvent and left for maceration for seven days [
Determination of total levels of phenolic compounds is based on colour-oxidation-reduction reaction using Folin-Ciocalteu reagent under alkaline conditions and performed at 765 nm wavelength using Unicam Helios
Colour reaction: into 100 mL measurement flask 15 mL of purified water, 4 mL of Folin-Ciocalteu reagent depending on concentration and studied solution, and then 6 mL of 20% sodium bicarbonate are added. Diluted with purified water up to 100 mL measurement. The manufactured solution is stored for 2 h at room temperature for reaction to take place. Absorbtion is measured by reference solution using purified water [
The study is performed following the Ph. Eur. 01/2002, 2.6.12. Microbiological study is conducted under aseptic conditions. During microbiological study, MIC (minimum inhibitory concentration)—the highest dilution of preparation (the lowest concentration of preparation), which inhibits a certain growth of standard microorganism culture was determined. When the main solutions were prepared, dilutions were performed with 10 mL of Mueller-Hinton agar (Mueller-Hinton Agar, Becton, Dickinson and Company) to obtain working solutions in Mueller-Hinton agar, in which MIC effect of the studied preparations on the growth of standard microorganisms was determined. Then, every Petri dish containing dilutions and covered with Mueller-Hinton agar was inoculated with standard bacteria:
According to the data of the literature, different concentrations (2.5%, 5%, and 10% propolis) of water and water-free extracts of propolis were made [
The data presented in Table
Total amount of phenolic compounds expressed as FAE mg/g in propolis extracts. Data presented as mean ± SD,
Solvents | Total amount of phenolic compounds (mg/g) | |||
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Propolis concentration | ||||
2.5% | 5% | 10% | ||
Propolis extracts | Water |
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Ethanol |
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Propylene glycol |
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Three-solvent system |
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In order to improve the extraction of active substances, extracts with three-solvent system and propylene glycol propolis extracts were stirred with magnetic stirring. Stirring has been performed for 3 h; samples for the analysis of phenolic compounds were taken after 1 h, after 2 h, and after 3 h of stirring, respectively.
The results of the study showed that stirring had an effect on the release of phenolic compounds from extracted raw material—after 1 h of stirring the highest amount of phenolic compounds was released from 2.5% propolis extract, and the lowest from 10%, and after 3 h the lowest amount of phenolic compounds was released from 2.5% propolis extract, but the highest amount from 10% propolis extract. However, a comparison of the data after two hours of stirring did not determine significant higher amounts of phenolic compounds. Differences between the total amount of phenolic compounds in the all studied propolis propylene glycol extracts and 2.5% propolis in three-solvent system stirred for 2 and 3 h were not statistically significant (
Effect of stirring ontotal amount of phenolic compounds.
To evaluate the influence of temperature, 5% propolis solutions in propylene glycol were used. Stirring was performed at different temperatures (40, 50, and 60°C), time of stirring—2 h. For the analysis, 5% propolis solutions in propylene glycol were used.
The results showed that the lowest amounts of phenolic compounds were released when solutions were stirred for 2 h at 40°C temperature—
Influence of temperature on total amount of phenolic compounds in 5% propolis propylene glycol extracts.
The results of the study showed that water extracted propolis and propolis solution in three-solvent system (water-ethanol-propylene glycol) were not effective against the studied strains of microorganisms. Ethanol 2.5% propolis extracts were more effective against the studied microorganisms compared with other investigated extracts. The result showed that the growth of
Microbiological activity of propolis phenolic compounds in propylene glycol solutions.
These solutions were also active against the other studied microorganisms (Figure
Crude propolis concentration has an influence on the amounts of phenolic compounds in propolis extracts when water, ethanol, or propylene glycol are used in the process of extraction. The highest content of phenolic compounds was determined in solutions containing 10% propolis extracts, and the lowest amounts—2.5% propolis extracts. The results revealed that water used in the process of extraction extracts the lowest amount of phenolic compounds from crude propolis, ethanol—the highest amount, and propylene glycol ranks the middle position.
It is determined that technological parameters (stirring, temperature) contribute to the content of phenolic compounds when propylene glycol as solvent was used. The highest amounts of phenolic compounds was determined when temperature was 60°C, and stirring time—2 h.
The studies showed that higher content of active substances is obtained when ethanol was used. However, the data also demonstrated that both solvents used were suitable for crude propolis extraction because propylene glycol extracted propolis solutions had antimicrobial activity. Since propylene glycol is nonvolatile and contains no water, it may be widely used as solvent in manufacturing of propolis solutions.
There is no conflict of interests to declare.