Abstract
The aim of this investigation was to synthesize propolis-mediated silver nanoparticles (Pro-AgNPs) and to test their biological activities in comparison with the raw materials (Saudi propolis). The chemical–physical characterization of the product showed that Pro-AgNPs were well synthesized. For the study of biological properties, aqueous and methanolic extracts were prepared from both propolis and Pro-AgNPs. Total polyphenol contents (TPCs) and antiradical activities showed their highest values in methanolic extracts, in particular in propolis extract. But, a higher TPC was found in Pro-AgNP aqueous extract as compared to propolis aqueous extract. It seems that Pro-AgNP synthesis helped extract more phenolic compounds with water. These compounds did not enhance antiradical activity in Pro-AgNP aqueous extract but increased its antibacterial activity.
1 Introduction
Propolisor “bee glue” is a one of the most important bee products; it is a sticky material used in hive building and its defense [1,2]. It is collected by bees from a variety of resinous secretions from different plant species, then mixed with their salivary and enzymatic secretions [3]. As the origin of propolis is not a unique plant species (as it is collected from plants surrounding the hive), its composition is highly variable. Nevertheless, Thomson [4] reported an approximate composition for propolis: it is made mainly of resin and balsam (50%), followed by wax (30%). The remaining part (20%) is composed of essential oils, pollen, and impurities. In the last decades, propolis has gained increasing interest in popular medicine worldwide, which encouraged its commercial use in food, beverages, and other products like soaps and toothpastes [1]. In addition, several researches were performed with the aim to study its chemical properties and biological activities [5]. In this context, Anjum et al. [3] reported for propolis anti-fungal, anti-tumoral, anti-protozoal, anti-inflammatory, hepatoprotective, dental protective, antioxidant, anti-viral, wound healing, and anti-cancer activities.
With the improper use of antibiotics, some pathogenic bacterial strains known for their antibiotic resistance have emerged, which made the use of new classes of disinfection systems, in particular silver nanoparticles (AgNPs), an urgent need [6,7]. Currently, AgNPs are considered as the most recognized nanoparticles, thanks to their broad-spectrum antimicrobial activities [8]. AgNPs have been incorporated within a multitude of materials [7]. Although the medical uses of propolis and AgNPs, data about the use of propolis-mediated AgNPs (Pro-AgNPs) are too scarce. For this reason, Pro-AgNPs using Saudi propolis are prepared and some of its chemical–physical and biological properties are studied.
2 Materials and methods
2.1 Preparation of Pro-AgNPs
An aliquot of 25 g of Saudi propolis was added to 250 mL distilled water then incubated at 45°C for 30 min. Thereafter, the mixture was filtrated using a Whatman No. 1 filter paper. A volume of 125 mL from the obtained aqueous extract was added to 375 mL silver nitrate (AgNO3, 1 mM) solution. The mixture was maintained under continuous stirring for 48 h for the synthesis of Pro-AgNPs. Figure 1 shows AgNO3 solution, propolis extract, and Pro-AgNPs.

(a) AgNO3 solution; (b) propolis extract; and (c) Pro-AgNPs solution.
2.2 Physical–chemical properties of propolis and Pro-AgNPs
UV-Vis spectra were performed for a wavelength range of 350–600 nm using a PG Instruments spectroscopy. For XRD analysis of crystalline Pro-AgNPs, a Phillips PW 1830 instrument (40 kV; 30 mA) was used.
2.3 Total polyphenol content (TPC) and antiradical assay of propolis and Pro-AgNPs
Two types of propolis and Pro-AgNPs extracts were prepared: aqueous and methanolic extracts. For the aqueous extract, samples of 124 mg propolis and Pro-AgNPs powder were added to 5 mL water then shaken and incubated at 80°C for 3 h [9]. As regards methanolic extracts, samples of 124 mg were extracted 3 times at room temperature for 2 h at 120 rpm using 70% methanol (1:5; w-v).
The TPCs of the aqueous and methanolic extracts were determined following the method described by Bettaieb et al. [10] after evaporation under vacuum (BÜCHI Rotary Evaporators R210, Germany) at 40°C. Briefly, an aliquot of 5 µL extract was added to 0.5 mL deionized water and 0.125 mL Folin-Ciocalteu reagent then shaken. After 6 min, 1.25 mL sodium carbonate (Na2CO3, 7%) was added and the mixture was adjusted to 3 mL with deionized water then mixed thoroughly. After that, incubation at 23°C for 90 min was needed before reading the absorbance of the mixture at 760 nm. TPCs were expressed as milligrams of gallic acid equivalents per gram dry weight (mg GAE g−1 DW); the calibration curve was prepared with gallic acid from 50 to 400 mg mL−1.
The radical scavenging activities of the studied extracts were measured following the method of Nsimba et al. [11]. Each extract was mixed with 1 mL DPPH methanolic solution (0.2 mM) then shaken vigorously. After 30 min incubation in the dark, the absorbance was read at 517 nm against a blank. The radical scavenging ability was calculated using the following equation:
where A 0 represents the absorbance of the control and A 1 stands for the absorbance of the sample.
2.4 Antimicrobial activity of propolis and Pro-AgNPs
The antimicrobial activities of the aqueous and methanolic extracts of propolis and Pro-AgNPs were evaluated using three bacterial strains (Listeria monocytogenes, Salmonella typhimurium, and Escherichia coli) and three fungal strains (Alternaria solani, Helminthosporium sp., and Fusarium oxysporum).
Bacterial and fungal inoculums were prepared from fresh pure cultures in Muller Hinton broth. Each bacterial and fungal suspension was compared to 0.5 Mc Farland standard.
The antimicrobial activity of propolis and Pro-AgNPs against the selected microorganisms was evaluated using the agar well diffusion assay. The bacterial inoculum was spread on the Muller-Hinton Agar using a sterile cotton swab by lawn culture technique. After inoculation, three wells of 10 mm diameter were made in the agar plate. Then aliquots of 100 µL were added from each extract to the wells. The plates were then incubated for 72 h at 37°C. The obtained zones of inhibition around the wells were measured by a ruler.
2.5 Statistical analysis
Data were subjected to a one-way analysis of variance using SPSS 16.0 software and means were compared according to Duncan’s multiple-range test.
3 Results and discussion
3.1 Scanning electron microscopic (SEM) measurements
The SEM of silver nanoparticles is given in Figure 2. To elucidate the Ag nanoparticle morphologies, SEM was performed, which demonstrates clearly the formation of Ag nanostructures for the sample.

SEM of silver nanostructures.
The SEM micrograph indicates that there are many micropores among the nanocrystals silver.
3.2 Physical and chemical characterization of Pro-AgNPs
UV-Vis spectrum showed a marked increase in the absorbance of the aqueous Pro-AgNPs extract with a peak around 437 nm (Figure 3). Our results are in agreement with those of Kothai and Jayanthi [12] who found a similar peak around 413 characteristics of AgNPs.

UV-Vis spectrum of AgNPs aqueous extract.
XRD analysis showed four distinct peaks at 38°, 44°, 64°, and 78° (Figure 4). Similar results were also obtained by Kothai and Jayanthi [12] and Ghadiri et al. [13], were identified as AgNPs with obvious face-centered cubic crystal nature.

XRD pattern of Pro-AgNPs.
Based on this chemical–physical characterization, it is clear that of Pro-AgNPs were well formed in our study.
3.3 TPC and antiradical activities of propolis and Pro-AgNPs
All extracts showed high levels for these two parameters, values ranged from 87 to 284 mg GAE g−1 DW for TPC and from 10 to 56% for antiradical activities. But the highest values were found in methanolic extracts of propolis followed by methanolic extracts of Pro-AgNPs (Table 1). The higher TPC recorded in Pro-AgNPs (167 mg GAE g−1 DW) as compared to that of propolis (87 mg GAE g−1 DW) could be explained by the fact that the formation of AgNPs may make more phenolic compounds water extractible.
TPC and antioxidant activities (DPPH test) in aqueous and methanolic extracts of propolis and Pro-AgNPs.
Parameters | Aqueous extracts | Methanolic extracts | ||
---|---|---|---|---|
Propolis | Pro-AgNPs | Propolis | Pro-AgNPs | |
TPC (mg GAE g−1 DW) | 87 ± 3d | 167 ± 4c | 284 ± 13a | 242 ± 4b |
Anti-radical activity (%) | 16.1 ± 1.1c | 10.2 ± 0.5d | 55.9 ± 2.9a | 24.2 ± 2.2b |
Means followed by different letters in superscript are significantly different according to Duncan’s multiple range test.
Means are of 4 replicates ±SE.
3.4 Antimicrobial activities of propolis and Pro-AgNPs
The inhibition zones of the studied extracts are presented in Table 2, and representative samples are illustrated in Figure 5. Our results showed no antifungal activity in the four extracts. As regards antibacterial activities, they depended on both material (propolis or Pro-AgNPs) and extract type (aqueous or methanolic). The widest inhibition zone was recorded in S. typhimurium treated with propolis followed by E. coli treated with Pro-AgNPs. The absence of antibacterial activity in propolis aqueous extract can be attributed to AgNPs themselves and probably to phenolic compounds that were made water extractible through the synthesis of AgNPs.
Diameters of the inhibition zones following the incubation of bacteria and fungi in the presence of 10-millimeter wells within the agar plates containing aqueous and methanolic extracts of propolis and Pro-AgNPs
Test organisms | Aqueous extracts | Methanolic extracts | ||
---|---|---|---|---|
Propolis | Pro-AgNPs | Propolis | Pro-AgNPs | |
Bacteria | ||||
L. monocytogenes | — | 13.50a | 13.17a | 12.00b |
S. typhmurium | — | 15.17b | 20.67a | 16.00b |
E. coli | — | 18.33b | 18.17b | 19.50a |
Fungi | ||||
A. solani | — | — | — | — |
Helminthosporium sp. | — | — | — | — |
F. oxysporum | — | — | — | — |
Values are means of 3 replicates. Means followed by different letters in superscript are significantly different according to Duncan’s multiple range test.

Correlation between TPCs and antioxidant activities in propolis and Pro-AgNPs aqueous and methanolic extracts.
4 Conclusion
In this study, Pro-AgNPs were synthesized and the chemical–physical characterization of the obtained product showed that the nanoparticles were well formed. The comparison between aqueous and methanolic extracts of propolis and Pro-AgNPs exhibited differential biological properties depending on both material and solvent. It seems that Pro-AgNPs synthesis helped extract more phenolic compounds with water. These compounds did not enhance antiradical activity in Pro-AgNPs aqueous extract but increased its antibacterial activity.
-
Funding information: The authors gratefully acknowledge Qassim University, represented by the Deanship of Scientific Research, on the financial support for this research under the number 10209-cos-2020-1-3-1, during the academic year 1442 AH/2020 AD.
-
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
-
Conflict of interest: The authors state no conflict of interest.
References
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© 2021 Maged S. Al-Fakeh et al., published by De Gruyter
This work is licensed under the Creative Commons Attribution 4.0 International License.
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