Chemical composition of Egyptian propolis and studying its antimicrobial activity and synergistic action with honey against some multidrug-resistant uropathogens

Abstract

Urinary tract infection (UTI) is one of the most common infections worldwide, increasing the incidence of antibiotic resistance and creating demand for alternative antimicrobial agents. Propolis, a natural antimicrobial agent, has been used in ancient folk medicine. This study evaluates the effectiveness of ethanolic extract of propolis (EEP) alone and in combination with honey against multidrug-resistant (MDR) uropathogens and also investigates the chemical composition of Egyptian propolis, which may be a potential therapeutic approach against MDR uropathogens. EEP was prepared, followed by column chromatographic fractionation using four different solvent systems. The ethyl acetate fraction was further fractionated through vacuum liquid chromatography (VLC). The antimicrobial activity of the EEP, propolis fractions, honey, and EEP-Honey mixture was studied, and the fraction with the best antimicrobial activity was analyzed by GC-MS and HPLC. The results indicated that EEP showed antimicrobial activity against the five MDR uropathogens with varying potential, while honey showed no activity against these pathogens. In comparison, the EEP-Honey mixture exhibited good antimicrobial synergy, with the MIC value decreasing by approximately 4–8 folds. In propolis fractionation, ethyl acetate was the best solvent for extracting antimicrobial substances from EEP, and fraction 5 (F5) was the most active fraction, with inhibition zone diameters of 30.33, 29.00, 21.58, 25.33, and 27.67 mm against MDR P. aeruginosa, E. coli, K. pneumoniae, S. saprophyticus, and C. albicans, respectively. GC-MS analysis of the F5 fraction revealed the presence of phenolics, flavonoids, terpenoids, acids, and alkaloids. In addition, HPLC polyphenol analysis identified 14 phenolic acids and flavonoid compounds with concentrations ranging from 117.36 to 5657.66 µg/g. Overall, the current findings highlighted the promising antimicrobial synergy of the EEP-Honey mixture against MDR urinary pathogens. The phytochemical analysis of propolis also identified potential bioactive compounds responsible for its biological and pharmaceutical properties.

Introduction

Urinary tract infections (UTIs) are among the most well-known community and hospital-associated microbial infections, affecting more than 150 million people annually worldwide^1,2. They are among the most frequent complications in critical care patients^2,3. Generally, antibiotics are prescribed as empiric therapy even as the result of urine culture, and this approach has probably contributed to the increase in antibiotic resistance worldwide⁴. Thus, the improper use of antibiotics may delay effective treatment and participate in the emergence of multidrug-resistant (MDR) microbes^5,6. The World Health Organization (WHO) and other researchers have emphasized the urgent need for novel antimicrobial approaches to combat infectious diseases^7–9.

Propolis, or bee glue, is a naturally occurring sticky material that belongs to the family of bee products^9,10. The word “propolis” originates from two ancient Greek words: pro- means “in front of” or “at the entrance,” and polis means “city” or “community,” collectively referring to a substance used to defend the hive^10,11. Propolis is collected by worker bees from the resinous secretions of a variety of plant species, such as pine, poplar, alder, conifer, beech, and birch, and then mixed with enzymatic and salivary bee secretions to form bee glue^12,13.

Propolis has been used in folk medicine since ~ 300 BC and is reported to have several biological activities, including antibacterial, fungicidal^9,14, antiviral, immunomodulator^15,16, anti-inflammatory¹⁷, antioxidant^17,18, and antitumor¹⁹. Furthermore, a previous study has demonstrated the good effect of propolis in increasing the growth rate of some types of probiotic bacteria²⁰.

Indeed, the raw propolis consists of 45 to 55% plant resin, 25 to 35% beeswax, 5 to 10% aromatic and essential oil, 5% pollen, and 5% other natural constituents²¹. Propolis also contains various sorts of secondary plant metabolites, such as phenolic acids, tannins, terpenoids, alkaloids, and flavonoids, which are responsible for several bioactivities and differ in concentrations depending on the season, plant sources, and geographical origins^9,22,23. It has many targeted sites with strong activity, attributed to its phenolic constituents, so it can potentially increase the effectiveness of other antimicrobial agents and act synergistically²⁴. The current study aimed to investigate the antimicrobial activity of the crude ethanolic extract of propolis (EEP), its synergistic effects when combined with honey, and the chemical composition of propolis responsible for its antimicrobial properties.

Materials and methods

Preparation of propolis extract, honey, and their mixture

A raw Egyptian propolis sample was obtained from the Abd El-Raheam apiary located in Marsa Matrouh, Egypt, and stored in a sterile glass container in the refrigerator, away from sunlight, until extraction. It was extracted with 70% ethanol (1:10, w/v), according to Helmy et al.⁹. The sample was incubated under agitation for seven days at 37 °C, protected from light, followed by centrifugation for 10 min. The supernatant was dried at 40 °C until ethanol evaporation occurred to obtain the pure propolis extract in powder form. This powder was weighed and dissolved in 1% DMSO (Sigma-Aldrich) to obtain EEP at a concentration of 100 mg/ml.

An Egyptian Citrus honey sample was obtained from the Egyptian Ministry of Agriculture, Giza, Egypt, and stored in a sterile glass container at room temperature. It was prepared in a 50% (v/v) concentration by adding an equal volume of honey to an equal volume of sterile distilled water in a sterile test tube. The honey solution was mixed by stirring with a vortex.

A mixture of EEP and honey (EEP-Honey) was prepared (1:1 v/v) to get a mixture of the final concentration (100 mg EEP/ 50% honey) to study the antimicrobial synergism between propolis and honey.

Tested uropathogens

Five MDR uropathogens were used in the current study, including one yeast isolate: fluconazole-resistant Candida albicans, one strain of Gram-positive bacteria: oxacillin and mecillinam-resistant Coagulase Negative (CON) Staphylococcus saprophyticus, and 3 Gram-negative MDR bacteria included extended-spectrum beta-lactamases (ESBL) Klebsiella pneumoniae, as well as MDR E. coli and Pseudomonas aeruginosa, which resisted to ceftazidime, cefepime, aztreonam, ciprofloxacin, piperacillin, imipenem, and gentamicin.

All urinary pathogens were provided by the Clinical Microbiology Laboratory of Cleopatra Hospital, Cairo, Egypt, from urine and catheter specimens whose patients were suffering from UTIs. These pathogens were identified according to the VITEK 2 system. VITEK 2 (bioMérieux) is an automated, highly accurate microbial identification system based on phenotypic identification methods. This system accommodates colorimetric reagent cards that are automatically incubated and interpreted. Four reagent cards are used for the identification of different microorganisms: (GN) for Gram–negative bacteria; (GP) for Gram–positive cocci and non-spore-forming bacilli; (BCL) for Gram–positive spore-forming bacilli; and (YST) for yeasts and yeast-like microorganisms. Each reagent card contains 64 wells, each containing an individual test substrate for measuring different metabolic activities²⁵.

Antimicrobial activity of EEP, Propolis fractions, and honey

Agar well diffusion method

Antimicrobial activities of EEP, propolis fractions, honey, and DMSO were determined by the agar well diffusion method on Mueller-Hinton agar (Oxoid, UK) for bacteria and on 2% glucose-supplemented Mueller-Hinton agar for Candida spp²⁶. This test was carried out in the Biotechnology Lab, Faculty of Science, Al-Azhar University for Girls, Cairo, Egypt, according to the National Committee for Clinical Laboratory Standards²⁷.

Each uropathogen was freshly prepared and cultured on nutrient agar at 35–37 °C overnight. An inoculum of 1.5 × 10⁸ CFU/ml (equivalent to 0.5 McFarland) was prepared in sterile saline (0.85%) and swabbed over the surface of the prepared Mueller-Hinton agar plate. EEP and propolis fractions were prepared at a 100 mg/ml concentration. Wells were prepared in each plate using a sterile cork borer with a diameter of 8 mm and a volume of 100 µl of EEP, propolis fractions, honey (50% v/v), and DMSO (negative control) were dropped in each well. Also, cefotaxime (CTX 30), ciprofloxacin (CIP 5), and fluconazole 25 µg discs were used as positive controls, the zone of inhibition was measured, and results were interpreted according to the Clinical and Laboratory Standards Institute²⁸. The plates were placed in the refrigerator for 1–2 h to allow the extracts to diffuse into the medium⁹. Then, the plates were incubated at 35–37 °C for 18–24 h, and the inhibition zone diameters (IZDs) were measured in mm. The experiment was performed in duplicate, and the mean ± standard error (SE) of the results was calculated.

Minimum inhibitory concentration (MIC) determination

The MIC value of EEP was evaluated using the broth microdilution method in the Regional Center for Mycology and Biotechnology, Cairo, Egypt, according to Helmy et al.⁹ study. At 96 well microtiter plates (BioTek, Winooski, VT, USA), an inoculum of each uropathogen suspension (1.5 × 10⁸ CFU/ml) was incubated with broth containing different concentrations of EEP (0.12–125 mg/ml) for 24 h at 37 °C. The MIC value was estimated by visual and spectroscopic methods by absorbance measurement at 620 nm. Control tubes without uropathogen (negative controls) and without EEP (positive controls) were used. The experiment was performed in duplicate, and the MIC ± standard error (SE) of the results was calculated.

Antimicrobial synergism of the EEP-Honey mixture

Antimicrobial synergy testing of the EEP-Honey mixture against MDR uropathogens was performed using the agar well diffusion method and MIC determination, as previously described in the above sections. However, in determining the MIC, different concentrations of the EEP-Honey mixture were prepared (1:1 v/v) below the MIC value of EEP, and these mixtures were screened against MDR uropathogens as described above. The synergism was defined when the MIC value of the EEP-Honey mixture reduced the MIC value of EEP alone²⁹, as all urinary pathogens were resistant to honey.

Phytochemical analysis of propolis extract

Phytochemical analysis of propolis was performed at the Faculty of Pharmacy at Al-Azhar University for boys in Cairo, Egypt, according to Afsar et al.³⁰, with some modifications, Fig. 1. About 200 g of propolis was extracted with 70% ethanol to obtain EEP. The dried EEP was re-suspended in 300 ml of water and subjected to successive liquid-liquid extraction using petroleum ether, chloroform, ethyl acetate, and n-butanol solvent systems, and their antimicrobial activities were studied. The ethyl acetate fraction (the highest in the antimicrobial activity) was subjected to further fractionation through vacuum liquid chromatography (VLC) on silica gel column and eluted with dichloromethane (DCM), dichloromethane (DCM): ethyl acetate (80:20, 60:40, 40:60, and 20:80 v/v), ethyl acetate (EtAc): methanol (MeOH) (1:1), and methanol (MeOH) to afford seven fractions in powder form, named (F1-F7) Fig. 1. The antimicrobial activity of the seven purified fractions was studied against MDR uropathogens, and the fraction with the highest antimicrobial activity was analyzed by gas-chromatography mass-spectrometry (GC-MS) and high-performance liquid chromatography (HPLC).

Fig. 1.

Propolis fractionation and purification of the most active antimicrobial substance. VLC: vacuum liquid chromatography, DCM: dichloromethane, EtAc: ethyl acetate, MeOH: methanol, GC-MS: gas chromatography-mass spectrometry, HPLC: high-performance liquid chromatography, +++ ve: strong positive activity, F1- F7: fraction 1- fraction 7.

Characterization of the most active propolis fraction

The most active fraction (F5) with the highest antimicrobial activity was analyzed by GC-MS and HPLC polyphenol analysis to predict the empirical chemical structure, molecular formula, and nomenclature of the most active compounds.

GC-MS analysis of propolis active fraction (F5)

The GC-MS system (Agilent Technologies) was equipped with a gas chromatograph (7890B) and mass spectrometer detector (5977 A) at Central Laboratories Network, National Research Centre; Cairo, Egypt. The active propolis fraction (F5) derivatizations were carried out using trimethylsilyl (TMS) derivatization and based on the optimized protocol described by Villas-Bôas et al.³¹. In summary, dried samples were re-suspended in 20 µL of pyridine and 100 µL of N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) and incubated in a dry block heater at 70 °C for 60 min. The GC was equipped with an HP-5MS column (30 m x 0.25 mm internal diameter and 0.25 µm film thickness). Analyses were carried out using helium as the carrier gas at a flow rate of 1.0 ml/min at a splitless injection volume of 2 µl and the following temperature program: 50 °C for 10 min; rising at 8 °C/min to 300 °C and held for 10 min. The injector and detector were held at 280 °C and 220 °C, respectively. Mass spectra were obtained by electron ionization (EI) at 70 eV using a spectral range of 50–550 m/z and a solvent delay of 10 min. The mass temperature was 230 °C and Quad 150 °C. Identification of different constituents was determined by comparing the spectrum fragmentation pattern with those stored in Wiley and NIST Mass Spectral Library data.

HPLC analysis of propolis active fraction (F5)

HPLC polyphenol analysis of the most active fraction was carried out using an Agilent 1260 series. The separation was carried out using a Kromasil C18 column (4.6 mm x 250 mm i.d., 5 μm). The mobile phase consisted of water (A) and 0.05% trifluoroacetic acid in acetonitrile (B) at a flow rate of 1 ml/min. The mobile phase was programmed consecutively in a linear gradient as follows: 0 min (82% A); 0–5 min (80% A); 5–8 min (60% A); 8–12 min (60% A); 12–15 min (85% A) and 15–16 min (82% A). The injection volume was 10 µl for each of the sample solutions (15 mg/ml). The column temperature was maintained at 35 °C. Polyphenol compounds were assayed by external standard calibration at 280 nm. HPLC analysis was carried out in the Central Laboratories Network, National Research Centre, Cairo, Egypt³².

Data analysis

Data were presented as mean, and standard error (SE) was determined. Data were normally distributed, and statistical analysis was performed using ANOVA one-way LSD tests. The software package CoStat for Windows version 6.45 was used for data analysis. A P value of < 0.05 was considered statistically significant for all analyses.

Results

Antimicrobial activity of EEP and its synergistic action with honey

In this study, the antimicrobial activity of EEP and its synergistic action with honey was determined by recording their IZDs and MIC values on MDR uropathogens. DMSO (negative control) was considered an inert solvent and didn’t report any inhibitory action on all MDR uropathogens. Also, cefotaxime (CTX 30), ciprofloxacin (CIP 5), and fluconazole 25-µg discs were used as positive controls and didn’t report inhibitory action on all MDR uropathogens since IZD less than 12 mm —for most antibiotics— was considered as resistant according to the Clinical and Laboratory Standards Institute breakpoints. Honey has not recorded any antimicrobial action, while EEP showed an inhibitory effect with variable action on all MDR uropathogens. Indeed, EEP showed higher activity against C. albicans and S. saprophyticus with IZDs of 21.67 and 21.33 mm and MIC values of 3.51 and 3.90 mg/ml, respectively, followed by P. aeruginosa, K. pneumonia, and E. coli with IZDs of 15.00, 13.83, and 12.58 mm, respectively, and MIC value ranged between 14.66 and 31.25 mg/ml; as shown in Table 1.

Table 1.

Antimicrobial activity of positive and negative controls, EEP, honey, and EEP-Honey mixture against MDR uropathogens.

pathogen	Mean IZD (mm) ± SE							MIC (mg/ml)
	CTX 30	CIP 5	Flu 25	DMSO	EEP (100 mg/ml)	Honey (50%)	EEP + Honey	EEP	EEP + Honey
E. coli	0.00 ± 0.00	8.00 ± 0.58	NA	0.00 ± 0.00	12.85c, B ± 0.67	0.00 ± 0.00	16.58c, A ± 0.17	31.25a, A ± 1.52	7.81c, B ± 0.64
K. pneumoniae	0.00 ± 0.00	9.33 ± 0.67	NA	0.00 ± 0.00	13.83bc, B ± 0.44	0.00 ± 0.00	20.67b, A ± 0.58	15.63b, A ± 0.87	3.9c, B ± 0.51
P. aeruginosa	0.00 ± 0.00	0.00 ± 0.00	NA	0.00 ± 0.00	15.00b, B ± 0.58	0.00 ± 0.00	21.33b, A ± 0.33	14.66b, A ± 0.45	1.95bc, B ± 0.19
S. saprophyticus	7.00 ± 0.00	0.00 ± 0.00	NA	0.00 ± 0.00	21.33a, B ± 0.58	0.00 ± 0.00	25.67a, A ± 0.33	3.90c, A ± 0.37	0.98b, B ± 0.21
C. albicans	NA	NA	0.00 ± 0.00	0.00 ± 0.00	21.67a, B ± 0.33	0.00 ± 0.00	26.00a, A ± 0.67	3.51c, A ± 0.72	0.95b, B ± 0.48

SE = Standard error, CTX 30: Cefotaxime, CIP 5: Ciprofloxacin, Flu 25: Fluconazole, NA: Not applicable.

Different small letters indicate significant differences within the same column (p-value < 0.05).

Different capital letters indicate significant differences (p-value < 0.05) between columns in the same test.

A p-value < 0.05 was set as representing statistical significance for all analyses.

When propolis was mixed with honey, the mixture (EEP-Honey) showed a significant increase in its IZD in the range of 16.58–26.00 mm and a decrease in its MIC values compared to EEP alone against MDR uropathogens, as a good synergistic action was recorded. The results of the synergistic effect in Table 1 indicated that MDR Gram-negative uropathogens showed a decrease in their MIC value from 14.66 to 31.25 mg/ml in EEP to 1.95–7.81 mg/ml in the EEP-Honey mixture. Moreover, MDR S. saprophyticus and fluconazole-resistant C. albicans showed a decrease in the MIC values from 3.90 to 3.51 mg/ml in EEP to 0.98 and 0.95 mg/ml in the EEP-Honey mixture, respectively, when treated with (EEP-Honey) mixture.

The p-values ​​indicated that the EEP-Honey treatment option had a significantly greater antimicrobial effect than EEP alone across all tested pathogens. Thus, the statistical analysis suggests that honey enhances the antimicrobial properties of EEP, making EEP + Honey mixture a more effective treatment option.

Antimicrobial activity of the extracted substances from EEP using different solvent systems

The data in Table 2; Fig. 2 indicated that, among all four solvent systems (petroleum ether, chloroform, ethyl acetate, and n-butanol), ethyl acetate was the best solvent for extracting the antimicrobial substance from EEP against MDR uropathogens (p-value < 0.05), except for K. pneumonia, followed by n-butanol, as it gave higher activity on this pathogen.

Table 2.

Effect of different solvent systems on the antimicrobial activity of EEP against MDR uropathogens.

pathogen	Mean diameter of inhibition zone(mm) ± SE
	Petroleum ether	Chloroform	Ethyl acetate	N-butanol
E. coli	9.00c ± 0.67	10.67b ± 0.67	12.00a ± 0.00	11.00ab ± 0.00
K. pneumoniae	0.00c ± 0.00	10.33b ± 0.58	10.88ab ± 0.33	11.67a ± 0.67
P. aeruginosa	10.58b ± 0.17	11.00b ± 0.17	13.67a ± 0.67	13.00a ± 0.10
S. saprophyticus	11.33c ± 0.33	15.00b ± 0.00	20.33a ± 0.88	19.33a ± 0.58
C. albicans	15.33d ± 0.67	18.33c ± 0.33	28.00a ± 0.58	24.67b ± 0.33

Different small letters indicate significant differences (p-value < 0.05) between columns in the same test, representing the effect of the different solvent systems on the antimicrobial activity of EEP against each uropathogen. A p-value < 0.05 was set as representing statistical significance for all analyses.

Fig. 2.

Effect of different solvent systems on antimicrobial activity of EEP against MDR uropathogens.

Antimicrobial activity of the fractions produced from VLC of Ethyl acetate EEP fraction

Using a VLC silica gel column, the ethyl acetate EEP fraction was separated into seven further fractions named F1, F2, F3, F4, F5, F6, and F7. Results in Table 3 and Plate 1 declared that the antimicrobial activity of the seven fractions was ordered as follows: F5 > F6 > F4 > F3 > F7 > F2 > F1. Fraction 5 (F5) that was eluted with dichloromethane (DCM): ethyl acetate (20:80) was the most active and gave significant antimicrobial activity among other fractions, with IZD of 30.33, 29.00, 27.67, 25.33, and 21.58 mm against the MDR P. aeruginosa, E. coli, C. albicans, S. saprophyticus, and K. pneumoniae, respectively. The p-values ​​indicated Fraction 5 (F5) had a significantly greater antimicrobial effect than other fractions across all tested pathogens.

Table 3.

Antimicrobial activity of the fractions produced from VLC of ethyl acetate EEP fraction against MDR uropathogens.

Propolis Fractions	pathogens
	E. coli	K. pneumoniae	P. aeruginosa	S. saprophyticus	C. albicans
	Mean diameter of inhibition zone(mm) ± SE
F1	10.33f ± 0.33	0.00e ± 0.00	0.00e ± 0.00	10.58d ± 0.17	11.00e ± 0.58
F2	12.00e ± 0.58	12.00d ± 0.00	13.33d ± 0.33	20.33b ± 0.67	20.00c ± 0.00
F3	14.67cd ± 0.33	13.33cd ± 0.33	15.00c ± 0.00	24.00a ± 0.00	23.67b ± 0.67
F4	15.33c ± 0.67	14.00c ± 0.33	15.33c ± 0.33	22.00b ± 0.58	23.00b ± 0.00
F5	29.00a ± 0.00	21.58a ± 1.17	30.33a ± 0.33	25.33a ± 0.67	27.67a ± 0.33
F6	27.33b ± 0.33	18.00b ± 0.67	24.00b ± 0.00	20.67b ± 0.33	21.33c ± 0.58
F7	14.00d ± 0.00	13.00cd ± 0.10	15.33c ± 0.33	13.58c ± 0.17	14.67d ± 0.33

Different small letters indicate significant differences within the same column (p-value < 0.05). A p-value < 0.05 was set as representing statistical significance for all analyses.

Plate 1.

Plate’s photos reveal the antimicrobial activity of the fractions produced from VLC of ethyl acetate EEP fraction on MDR (A): E. coli, (B): K. pneumonia, (C): P. aeruginosa, (D): CON S. saprophyticus, and (E): C. albicans. Numbers from 1 to 7 indicated fraction numbers.

Characterization and identification of the most active Propolis fraction

Fraction 5 (F5) was the most active fraction and gave significant antimicrobial activity among other fractions and was consequently subjected to (GC-MS) and (HPLC) polyphenol analysis.

GC-MS analysis

The GC-MS analysis of the most active fraction allowed the detection of ninety compounds in different concentrations (99.1%), including phenolics, flavonoids, terpenoids, alkaloids, and organic acids, as shown in Table 4; Fig. 3. The major compounds present in the most active fraction of the propolis (F5) were identified as caffeic acid dimethyl ether (14.63%, compound 33, RT 33.925), monopalmitoylglycerol (9.79%, compound 54, RT 38.76), and 1,6-dihydroxy-8-methoxy-3-methylanthraquinone (9.12%, compound 69, RT 41.244), which contributed to the biological activity of propolis.

Table 4.

Results of GC/MS analysis of the most active fraction of propolis.

Compound No.	Retention Time (RT)	compound	Formula	Area Sum %
1	18.338	Butoxyethanol	C6H14O2	0.16
2	22.092	Silanol	SiH4O	0.24
3	22.309	Cyclohexanediol	C6H12O2	0.27
4	22.515	Trans-cyclohexanediol	C6H12O2	0.24
5	23.025	Benzeneacetic acid	C8H8O2	0.28
6	23.391	Acetin	C5H10O4	1.37
7	24.713	Disiloxane, 1,3-bis(1,1 dimethylethyl)	C12H30OSi2	0.38
8	24.793	Resorcinol	C6H6O2	0.22
9	25.05	Butoxyacetate	C10H20O3	0.15
10	25.256	2-Aminoisobutyric acid	H2N-C(CH3)2-COOH	0.18
11	25.909	Oxyvaleric acid	CH3(CH2)3COOH	0.2
12	26.155	Kojic acid	C6H6O4	0.2
13	27.013	Cinnamic acid	C9H8O2	0.72
14	27.076	Pyrogallol	C6H6O3	0.59
15	27.396	Tyrosol	C8H10O2	0.32
16	28.192	-4-Hydroxybenzoic acid	C7H6O3	0.56
17	28.472	Dodecenoic acid	C12H22O2	0.28
18	29.204	Dimethoxybenzene	C8H10O2	0.18
19	29.433	10-Undecynoic acid	C11H18O2	0.15
20	30.017	Phloretic acid	C9H10O3	0.22
21	30.074	4-Methox benzoic acid	C8H8O3	0.41
22	30.417	Azelaic acid	C9H16O4	0.33
23	30.824	Protocatechoic acid	C7H6O4	0.7
24	30.95	p-methoxy Cinnamic acid	C10H10O3	0.19
25	31.07	Falcarinol	C17H24O	0.2
26	31.762	2,4-Dihydroxyacetophenone	C8H8O3	0.47
27	32.077	Hydroxydihydrosafrole	C10H12O3	0.25
28	32.386	Caffeic acid	C9H8O4	0.65
29	32.569	Gallic acid	C7H6O5	1.16
30	33.015	Ninhydrin	C9H6O4	0.16
31	33.324	Dimethyl caffeic acid	C11H12O4	1.4
32	33.404	Palmitic Acid	C16H32O2	1.29
33	33.925	Caffeic acid dimethyl ether	C11H12O5	14.63
34	34.16	beta.-Cholestane-alpha.,7.alpha.,12.alpha.,24.alpha.,25-pentol	C27H44O5	0.37
35	34.429	Benzothiophene-3-carbonitrile, 4,5,6,7-tetrahydro-2-(4-tert-butylbenzylidenamino	C9H10O2S	0.18
36	34.492	1,3-Bis(pentamethyldisilanyloxy)propane	C17H20O6S2	0.31
37	34.989	Beta- Eudesmol	C15H26O	0.17
38	35.316	13-Octadecenoic acid	C18H34O2	0.23
39	35.35	alpha.-D-Glucopyranoside	C7H14O6	0.29
40	35.573	Stearic acid	C17H35CO2H	0.37
41	36.408	Heneicosanoic acid	C21H42O2	1.06
42	36.494	5-dimethyl-6,8-dioxooctahydro-1 H-1,4-methanoinden-1-yl)propanoate	C13H22O2	0.42
43	36.586	− (3a,5-dimethyl-6,8-dioxooctahydro-1 H-1,4-methanoinden-1-yl)propanoate	C16H30O2	0.75
44	36.689	Vanillic acid	C8H8O4	0.29
45	36.918	1-ethyl-2-benzimidazolyl (2-methoxyphenyl)	C11H14O3	0.2
46	37.021	9-Octadecenamide	C18H35NO	1.14
47	37.158	Urocanic Acid	C6H6N2O2	2.42
48	37.244	Silane, diethyl(3-methylbutoxy)octadecyloxy	C5H11Cl3Si	1.31
49	37.33	t-Butyldimethyl(10-octylundec-10-enyloxy) silane	C25H52OSi	3.63
50	37.45	15-Isopropenyl-oxacyclopentadecan-2-one	C20H38O2Si	0.24
51	37.696	Hexakis(fluorodimethylsilyl)benzebe	C18H36F6Si6	0.5
52	37.942	1,4-benzenediacetonitrile, .alpha.,.alpha.‘-bis[[4-(diethylamino)-2-methoxyphenyl]methylene	C34H38N4O2	3.22
53	38.068	Linoleic acid (LA)	C18H32O2	1.65
54	38.76	Monopalmitoylglycerol	C19H38O4	9.79
55	39.184	3,7,11,15-tetramethylhexadecan-1,3-diol	C20H42O2	1.51
56	39.361	Erythro-Pentonic acid, 2-deoxy-3,4,5-tris-OH	C17H42O5	0.65
57	39.487	1-Monoferuloylglycerol	C13H16O6	0.26
58	39.75	5,8,11-Eicosatriynoic acid	C20H28O2	2.43
59	40.048	Tofisopam	C22H26N2O4	0.7
60	40.271	Arachidonic acid	C20H24O2	0.48
61	40.397	Silane, diethyloctyloxytetradecyloxy	C26H56O2Si	1.02
62	40.494	2-Monostearin	C21H42O4	0.23
63	40.614	1-Monooleoylglycerol	C21H40O4	1.07
64	40.706	Genistein	C15H10O5	0.55
65	40.774	Catechine	C15H14O6	2.33
66	40.826	Glycerol monostearate	C21H42O4	2.63
67	40.917	D-Glucopyranuronic acid	C6H10O7	0.99
68	41.106	[(2-{3,4-Bisoxy]phenyl}- oxy]-3,4-dihydro-2 H-chromen-7-yl-oxysilane	C30H54O6Si5	1.74
69	41.244	1,6-Dihydroxy-8-methoxy-3-methylanthraquinone	C16H12O5	9.12
70	41.318	(4-(1-(3,5-Dimethyl-4phenyl)-1,3-dimethylbutyl)-2,6-dimethylphenoxysilane	C28H46O2Si2	0.6
71	41.358	[(2-{3,4-Bisoxy]phenyl}-3,5-bis-3,4-dihydro-2 H-chromen-7-yl)oxy](trimethyl)silane	C30H54O6Si5	1.21
72	41.427	17.alpha.-Methyltestosterone	C20H30O2	1.14
73	41.621	4-Androsten-4-ol-3,17-dionel	C19H26O3	0.32
74	41.661	Monoketal adduct	C29H32O5	0.39
75	42.296	Saponarin	C27H30O15	0.24
76	43.172	3,5-Dihydroxybenzyl alcohol	C7H8O3	0.15
77	43.698	delta.(1)-Tetrahydrocannabinolic acid	C22H30O4	0.54
78	43.784	17-(1,5-Dimethylhexyl)-10,13-dimethyl-3-styrylhexadecahydrocyclopenta[a] phenanthren-2-one	C35H52O	0.43
79	44.036	-unknown		1.91
80	44.665	1,2,8-Trihydroxy-3-methoxy-6-methylanthraquinone	C16H12O6	0.66
81	44.974	Monoolein	C21H40O4	0.48
82	45.592	Homogentisic acid	C8H8O4	0.4
83	45.712	2,4-Imidazolidinedione, 5- oxy]phenyl]-3-methyl-5-phenyl	C16H16N2O4	0.54
84	45.884	Naringenin	C15H12O5	1.48
85	46.205	2-Oxabicyclo[3.3.0]octa-3,7-dien-6-one, 3-acetyl-4-methyl-1,5,7,8-tetrakis	C22H42O7	2.99
86	46.519	6-[Nonadecenyl]salicylic acid	C26H42O3	0.89
87	47.074	Octasiloxane, hexadecamethyl	C16H50O7	0.27
88	47.944	2-Monomyristin	C17H34O4	0.65
89	49.552	Heptasiloxane, tetradecamethyl ester	C14H42O7Si7	0.16
90	49.855	1-Monolinoleoylglycerol	C27H54O4	0.65
Total				99.1

Fig. 3.

GC/MS analysis of the most active fraction of propolis.

HPLC analysis

HPLC polyphenol analysis of the most active fraction (F5) in propolis revealed the presence of 14 phenolic acids and flavonoid compounds in the range of 117.36–5657.66 µg/g. These compounds included 4 flavonoids (catechin, naringenin, taxifolin, and kaempferol) and 10 phenolic acids (gallic acid, chlorogenic acid, catechin, methyl gallate, caffeic acid, syringic acid, pyrocatechol, ellagic acid, coumaric acid, vanillin, and cinnamic acid). The biological activity of the propolis fraction contributed to the most abundant compounds, which included naringenin (5657.66 µg/g), gallic acid (5217.66 µg/g), taxifolin (5192.48 µg/g), pyrocatechol (2182.90 µg/g), and kaempferol (1029.27 µg/g), as shown in Table 5; Fig. 4.

Table 5.

Results of HPLC analysis of fraction 5.

Peak No.	Retention time (RT)		Concentration (µg/g)	Identified compounds
	Standard	Test
1.	3.263	3.334	5217.66	Gallic acid
2.	4.046	3.970	594.05	Chlorogenic acid
3.	4.475	4.460	1183.75	Catechin
4.	5.668	5.694	194.71	Methyl gallate
5.	5.974	5.993	121.32	Coffeic acid
6.	6.513	6.504	153.70	Syringic acid
7.	7.193	7.328	2182.90	Pyrocatechol
8.	8.147	8.301	336.62	Ellagic acid
9.	9.139	9.149	117.36	Coumaric acid
10.	9.894	9.848	154.83	Vanillin
11.	10.218	10.223	5657.66	Naringenin
12.	13.431	13.019	5192.48	Taxifolin
13.	14.332	14.590	322.58	Cinnamic acid
14.	14.715	14.907	1029.27	Kaempferol

Fig. 4.

HPLC chromatogram of (A) reference standards polyphenol compounds and (B) fraction 5 in propolis with identified marker compounds. 1: gallic acid, 2: chlorogenic acid, 3: catechin, 4: methyl gallate, 5: coffeic acid, 6: syringic acid, 7: pyrocatechol, 8: ellagic acid, 9: coumaric acid, 10: vanillin, 11: naringenin, 12: taxifolin, 13: cinnamic acid, and 14: kaempferol.

Discussion

Propolis (bee glue) is as old as honey and has been used by humans as a traditional medicine since antiquity. There are many records recommending its utilization in medication by the ancient Egyptians, Persians, and Romans³³. The results of the in vitro antimicrobial assay indicated that EEP inhibited the growth of Gram-positive CON S. saprophyticus bacteria and Candida albicans yeast better than Gram-negative P. aeruginosa, K. pneumonia, and E. coli bacteria. This is explained by the presence of the outer membrane in the Gram-negative bacteria^14,34. The low sensitivity of E. coli toward propolis has also been reported by many researchers, as this bacterium showed either very low sensitivity or a total lack of sensitivity to propolis^35,36. Moreover, a previous study by Taher³⁷ supported our results and reported that MDR K. pneumoniae was sensitive to Iraq EEP with an average inhibition zone of 12.6 mm at a concentration of 100 mg/ml, while another study by Al-Ani et al.²² found P. aeruginosa displayed high resistance to European propolis, which is in contrast to our results. In the current study, the MIC value of EEP on C. albicans and S. saprophyticus was 3.51 and 3.90 mg/ml, respectively, while against P. aeruginosa, K. pneumonia, and E. coli were in the range of 14.66–31.25 mg/ml, which were higher than a previous study of ours on Turkish propolis that reported MIC values in the range of 0.185-3.50 mg/ml on K. pneumonia, P. aeruginosa, S. aureus, and C. albicans⁹. On the other hand, previous studies have concluded that natural honey can inhibit the growth of some pathogenic bacteria with varying inhibition degrees^9,38,39, while honey in our study showed no effect on the isolated pathogens, which may be due to the extreme resistance of urinary pathogens to antibiotics.

The addition of propolis to honey resulted in an increase in IZDs of EEP from 12.85 to 21.67 mm to 16.58–26.00 mm in the EEP-Honey mixture as well as a decrease in the MIC values of EEP from 3.51 to 31.25 mg/ml to 0.95–7.81 mg/ml in the EEP-Honey mixture, where significant synergy has been recorded. Basically, the antimicrobial synergism of propolis and honey may be related to the synergistic effect of their various flavonoids and phenolic compounds, which have been supported by previous studies^24,29,40. In line with our findings, another study by Hamouda et al.⁴¹ reported the good antimicrobial effect of Egyptian fennel honey and EEP against 19 strains of methicillin-resistant Staphylococcus aureus (MRSA), where honey and EEP showed a synergistic effect when added together (MIC = 7.84 mg/ml).

Propolis fractionation was performed using a three-step sequential extraction: first with 70% ethanol, followed by the use of four solvent systems (petroleum ether, chloroform, ethyl acetate, and n-butanol), where ethyl acetate was the best solvent for the extraction of the antimicrobial substance from EEP. These results were supported by the study of Bouaroura et al.⁴² using Algerian propolis, as the ethyl acetate extract displayed increased contents of antimicrobial substances. Further, findings in the Chen et al.⁴³ study reported the ethyl acetate fraction showed the highest total phenolic and flavonoid contents, which have potential antioxidant and antimicrobial activity, compared to the n-butanol fraction, chloroform fraction, and petroleum ether fractions. The standard protocols for chemical fractionation and bioactivity-guided chemical analysis were used to identify the bioactive ethyl acetate fraction, as it contained the highest polyphenol contents⁴⁴. The third step was carried out using a VLC silica gel column, as the ethyl acetate EEP fraction was separated into seven further fractions named F1, F2, F3, F4, F5, F6, and F7, on which the pure F5 fraction gave the best antimicrobial activity against MDR uropathogens and was subsequently analyzed by GC-MS and HPLC.

The GC-MS analysis of the F5 fraction revealed the presence of 90 chemical compounds at different concentrations, including phenolic acids, flavonoids, terpenoids, alkaloids, steroids, organic acids, fatty acids, hydrocarbon esters, ketones, and sugars. These compounds act synergistically and are responsible for the therapeutical and pharmacological properties of propolis^12,45. Polyphenol compounds (phenolic acids and flavonoids) are considered the main antimicrobial and bioactive antioxidant components in propolis^9,46. In our study, caffeic acid esters are strong bactericidal agents and inhibit bacterial growth by disrupting membrane permeability or through oxidative stress mechanisms^47,48. Also, 1,6-Dihydroxy-8-methoxy-3-methylanthraquinone and monopalmitoylglycerol were reported in propolis in Kurek-Górecka et al.⁴⁸ and Shi et al.⁴⁹. studies with anti-inflammatory properties. Further, 4-hydroxycinnamic acid is a derivative of cinnamic acid and disrupts the outer membrane of Gram-negative bacteria, causing cytoplasmic leakage⁵⁰.

The HPLC analysis of the most active propolis fraction (F5) resulted in the presence of 14 polyphenol compounds, including naringenin, gallic acid, taxifolin, pyrocatechol, kaempferol, catechin, chlorogenic acid, methyl gallate, coffeic acid, syringic acid, ellagic acid, coumaric acid, vanillin, and cinnamic acid, which contributed to the antimicrobial activities of propolis. Previous studies on African, Asian, and European propolis concluded that propolis contains predominantly polyphenols such as naringenin, galangin, pinocembrin, apigenin, pinobanksin, quercetin, cinnamic acid and its esters, aromatic acids and their esters, kaempferol, chrysin, p-coumaric acid, cinnamyl caffeate, cinnamylidene acetic acid, and caffeic acid^46,51,52. The composition of propolis varies depending on the plant’s origin, geographical location, and collection seasons^9,22.

The high concentration of naringenin and taxifolin flavonoids in our study may be the main reason for the significant inhibitory effect of F5 fraction against MDR uropathogens, especially P. aeruginosa (IZD = 30.33 mm), where Vandeputte et al.⁵³ and Shariati et al.⁵⁴ studies reported the inhibitory effect of naringenin and taxifolin on the expression of various quorum sensing (QS) controlled genes in P. aeruginosa and thus reduce the virulence factors of pathogenic bacteria. Furthermore, naringenin possesses anti-staphylococcal activity^55,56 and antibacterial properties against E. coli⁵⁶. Likewise, some phenolic acids, such as cinnamic acid and its derivatives, have anti-QS activity and inhibit bacteria by damaging the cell membrane, inhibiting ATPases, cell division, and biofilm formation⁵⁷. Also, other phenolic acids like ferulic, caffeic, and chlorogenic acids are effective in preventing bacterial adhesion^58,59; while gallic acid inhibits efflux pump mechanisms in MDR Staphylococcus spp. and E. coli strains. The mixture of phenolic catechin and vanillic acids possesses antioxidant and antimicrobial synergy and is effective in preventing cell adhesion and biofilm formation in uropathogenic E. coli compared to single compounds or nitrofurantoin antibiotics⁶⁰. Accordingly, the combination of many active components in propolis and their presence in various proportions prevents the occurrence of bacterial resistance^48,61,62. Finally, the design of the study and the important findings were presented as a schematic drawing in Fig. 5.

Fig. 5.

A Schematic drawing illustrating the design of the study and the important findings.

Conclusion

Over recent years, the increasing empiric treatment with antibiotics in UTIs and the emergence of MDR urinary pathogens have stimulated the use of traditional medicine and attempts to improve it for therapeutic use, such as propolis, honey, and their mixtures. In the current study, propolis inhibited all MDR uropathogens, while honey did not. Interestingly, the combination of propolis and honey showed a great synergistic effect with a significant increase in the antimicrobial activity of propolis, which was expressed by reducing the MIC value of EEP for all uropathogens by approximately 4–8 folds. Propolis fractionation was carried out, as ethyl acetate was the best solvent to extract the antimicrobial substances from EEP. The ethyl acetate EEP fraction was further fractionated using a VLC silica gel column to get seven fractions, of which the F5 fraction was the most active with the best antimicrobial activity against MDR uropathogens and was analyzed by GC-MS and HPLC. HPLC analysis of the polyphenols in the F5 fraction of propolis revealed the presence of 14 phenolic acids and flavonoid compounds, where the most abundant compounds were naringenin, gallic acid, taxifolin, and pyrocatechol. GC-MS analysis indicated the presence of many constituents in different concentrations, including phenolic acids, flavonoids, terpenoids, and alkaloids, which are responsible for the biological and pharmaceutical properties of propolis. Thus, the combination of many active ingredients in propolis and their presence in various proportions prevents bacterial resistance.

Abbreviations

CON

Coagulase negative

EEP

Ethanolic extract of propolis

ESBL

Extended spectrum β-lactam antibiotic

F1

F7:Fraction 1:fraction 7

GC-MS

Gas chromatography-mass spectrometry

HPLC

High-performance liquid chromatography

IZD

Inhibition zone diameter

MDR

Multidrug-resistant

MIC

Minimum inhibitory concentration

MRSA

Methicillin-resistant Staphylococcus aureus

RT

Retention time

SE

Standard error

UTI

Urinary tract infection

VLC

Vacuum liquid chromatography

Author contributions

NMS, MMA, and AAE developed and supervised the work. AKH, NMS, MMA, and AAE designed the study. MMA performed the MIC test. AAE performed the phytochemical analysis of propolis. AKH collected the uropathogens, performed the antimicrobial and synergy experimental work, analyzed and interpreted the data, and wrote the first draft of the manuscript. NMS reviewed and editing the initial and final drafts of the manuscript. All authors read and approved the final manuscript.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Data availability

All information created or analyzed during the present study are included in the manuscript.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

Not applicable. This manuscript does not refer to or imply the use of any animal or human data or tissues.