Correlation between total phenolic and flavonoid contents and biological activities of 12 ethanolic extracts of Iranian propolis

Abstract Propolis is a resinous substance produced by honey bees that is very popular as a natural remedy in traditional medicine. The current research is the first study on the biological properties of ethanolic extracts of propolis (EEP) from several different regions (12) of Iran. Total phenolic and flavonoid contents (TPC and TFC) of Iranian EEPs were variable between 26.59–221.38 mg GAE/g EEP and 4.8–100.03 mg QE/g EEP. The DPPH scavenging assay showed all the studied EEP samples, except for the sample with the lowest TPC and TFC (P6), have suitable antioxidant activity. All the EEPs inhibited both cholinesterase enzymes (acetylcholinesterase: AChE, butyrylcholinesterase: BuChE) but most of them exhibited a distinct selectivity over BuChE. Evaluation of the antibacterial activity of the EEP samples using four pathogenic bacteria (B. cereus, S. aureus, A. baumannii, and P. aeruginosa) demonstrated that the antibacterial properties of propolis are more effective on the gram‐positive bacterium. Spearman correlation analysis showed a strong positive correlation between TPC and TFC of the Iranian EEPs and their antioxidant, anticholinesterase, and antibacterial activities. Considering that there is ample evidence of anticholinesterase activity of flavonoids and a significant correlation between the anticholinesterase activity of the studied Iranian EEPs and their total flavonoid content was observed, the interaction of 17 well‐known propolis flavonoids with AChE and BuChE was explored using molecular docking. The results indicated that all the flavonoids interact with the active site gorge of both enzymes with high affinity. Summing up, the obtained results suggest that Iranian propolis possesses great potential for further studies.


| INTRODUC TI ON
Propolis (bee glue) is a natural product created by honey bees (Apis mellifera L.) by mixing their saliva, beeswax, and exudates derived from different parts of the plants (Marcucci, 1995;Silva-Carvalho et al., 2015). This resinous material forms the inner lining of the beehives and is used as a protective barrier against foreign invaders, wind, and rain. The honey bees also use propolis to repair cracks and holes in the walls of the hive and seal openings and smooth the inner walls, maintain the hive's internal temperature and cover (embalm) the corpses of large invaders that are difficult to transport out of the hive. Propolis has a very pleasant aroma and due to its antimicrobial activity, prevents the growth of bacteria and fungi inside the hives (Burdock, 1998;Martinotti & Ranzato, 2015).
The exact composition, aroma, and color of raw propolis (green, red, brown, and yellow) depend on several factors such as botanical source, collection season, and geographical area (Wang et al., 2016).
There are also a small number of enzymes in propolis including glucose 6-phosphatase, succinic dehydrogenase, acid phosphatase, adenosine triphosphatase, and beta-amylase (Bhargava et al., 2021;Lotfy, 2006). Propolis has been used by humankind for different medicinal and nonmedicinal purposes since ancient times (Kuropatnicki et al., 2013). According to the published scientific data, various biological properties have been attributed to propolis, such as anticancer, antioxidant, anticholinesterase, antihypertensive, liver protection, wound-healing, oral health, anti-inflammatory, antiulcer, immunomodulatory, antimicrobial, antiviral, antifungal, and antiparasitic (Baltas et al., 2016;Bhargava et al., 2021;Dilokthornsakul et al., 2022;Kocot et al., 2018;Pasupuleti et al., 2017;Rezvannejad et al., 2017;Saeed et al., 2021;Suran et al., 2021;Viuda-Martos et al., 2008). The data provided by in vitro studies, animal models, and human clinical trials demonstrate that propolis can reduce the manifestations of neurological and brain disorders through its protective and therapeutic effects (Zulhendri, Chandrasekaran, et al., 2021;. The current research is the first comprehensive study on the biological properties of several propolis samples collected from different regions of Iran. This study covers the evaluation of the total phenolic and flavonoid contents (TPC and TFC) and the antioxidant, anticholinesterase, and antibacterial activities of 12 ethanolic extracts of Iranian propolis. Although the antibacterial and antioxidant activities of Iranian propolis have been previously studied, its anticholinesterase activity has not been studied until now. Meanwhile, previous studies have been conducted on one or a few limited Iranian propolis samples. In this research, also for the first time, the interaction of 17 well-known propolis flavonoids with the cholinesterase enzymes (AChE and BuChE) has been investigated using molecular docking.

| Propolis samples and preparation of the ethanolic extracts
Twelve Iranian propolis samples (P1-P12) used in this research are listed in Table 1, along with coordinates. Raw propolis samples were collected by experienced beekeepers from beehives located in various regions of Iran. The propolis samples were stored at −20°C until use. Preparation of the ethanolic extracts of propolis (EEP) was performed in the following steps: Raw propolis was pulverized by liquid nitrogen. Ten grams of powdered propolis with 100 mL of 80% ethanol was placed in a dark glass flask and stirred on a shaker at room temperature for 72 h and the mixture was then filtrated by centrifugation in two steps. The supernatant was concentrated using rotary evaporation at 40°C and then placed in a vacuum oven at 40°C for complete drying. The ethanolic extracts were kept in the dark containers at −20°C until further steps.

| Determination of total phenolic content
The total phenolic content (TPC) of the EEP samples was measured utilizing the colorimetric Folin-Ciocalteu method and gallic acid was used as the calibration standard (Singleton & Rossi, 1965). The results were expressed as milligrams of gallic acid equivalents (GAE) per gram of EEP (mg GAE/g EEP). For each sample, the experiment was performed in triplicate. For this purpose, 100 μL of 0.2 N Folin-Ciocalteu reagent, 590 μL of distilled water, and 10 μL of EEP in 80% ethanol (final concentration of 50 μg/mL) were placed in a cell for 1 min at room temperature in the dark. Three hundred microliters of sodium carbonate (7.5%) was then added to the cell and the mixture was incubated in the dark for 30 min at room temperature. The absorbance was read at 735 nm with a spectrophotometer (Cary 50, Australia). A solution containing 100 μL of 0.2 N Folin-Ciocalteu reagent, 600 μL of distilled water, and 300 μL of sodium carbonate (7.5%) was utilized as the blank sample.

| Determination of total flavonoid content
The aluminum chloride colorimetric method was used to measure the total flavonoid content (TFC) of EEP samples (Wang et al., 2016).
Five hundred microliters of EEP in 75% ethanol (final concentration of 50 μg/mL) was added to 500 μL of 2% aluminum chloride in a tube. The tube was then incubated in the dark for 15 min at room temperature. The absorbance was recorded at 435 nm with a spectrophotometer (Cary 50, Australia). A solution containing 500 μL of 2% aluminum chloride and 500 μL of distilled water was used as the blank. Three replications were performed for each sample.
Quercetin was applied as the calibration standard and the TFC of EEP samples was expressed in milligrams of quercetin equivalents (QE) per gram of EEP (mg QE/g EEP).

| DPPH-free radical scavenging assay
To evaluate the antioxidant activity of EEP samples, the DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging assay was used (Bondet et al., 1997). All the EEP samples were tested at six different concentrations. For each concentration, three replications were performed. Briefly, 750 μL of 0.4 mM DPPH solution was added to 250 μL of EEP dissolved in methanol. After 30 min of incubation in the dark at room temperature, the absorbance of the sample was recorded at 517 nm. A sample containing 750 μL of DPPH solution (0.4 mM) and 250 μL of methanol was considered as the control sample. Ascorbic acid was used as the positive control. For this aim, six different concentrations of ascorbic acid (0.5-12.5 μg/mL) were used and for each concentration, three replications were considered.
The following formula was used to calculate the percentage of DPPH inhibition free radical by each concentration of the EEP sample: A control , absorbance of the control sample; A sample , absorbance of the EEP sample.
The IC 50 value (the concentration of a sample that has the ability to inhibit DPPH radical by 50%) for each sample was obtained using a dose-response graph plotting the percentage of DPPH inhibition versus the concentration logarithm of the EEP sample.

| Measurement of anticholinesterase activity
To determine the half maximal inhibitory concentration (IC 50 ) of EEP samples for acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE), the activity of the enzymes was measured in the absence and presence of six different concentrations of each EEP sample by the Ellman method (Ellman et al., 1961). Each concentration was analyzed in triplicate and a well-known inhibitor of the cholinesterase enzymes, neostigmine, was used as the positive control. All the enzyme assays were performed on a 96-well plate with a final volume of 200 μL.
The assay mixture consisted of 0.1 M potassium phosphate buffer (pH 8.0) and the enzyme (AChE or BuChE) and DTNB with the final concentrations of 0.1 unit/mL and 0.5 mM, respectively. The EEPs (in 70% ethanol) were incubated with the assay mixture for 10 min. Substrate (acetylthiocholine iodide or S-butyrylthiocholine iodide) was then added to the assay with the final concentration of 1 mM and the absorbance was read after 10 min at 405 nm by a microplate reader (Elx808 Biotek Instruments). A sample containing all assay components, except the enzyme was applied as the blank.
Finally, the percentage of enzyme activity inhibition at each concentration of the EEP sample was determined and the IC 50 value was calculated from the dose-response curve plotting the percentage of inhibition versus concentration logarithm of the EEP sample.
The results were reported as the mean ± standard deviation (SD).

| Evaluation of antibacterial activity
To evaluate the antibacterial activity of EEP samples, four pathogenic bacteria, Bacillus cereus, Staphylococcus aureus, Acinetobacter In the next step, the antibacterial activity of the EPP samples was investigated using the agar well diffusion method (Balouiri et al., 2016;Domingue et al., 1994).

| Statistical analysis
The data were calculated in the form of arithmetical mean values and standard deviations. The correlation between data was statistically analyzed using the one-way ANOVA method, Spearman correlation of SPSS software (version 13.0). The diameter of growth inhibition halo data was also statistically analyzed using the ANOVA method and SAS software (version 9.1) and means comparison has been performed by Tukey method and proc GLM.
y i denotes observations of studied parameters. x i denotes different concentrations of the EEP samples. ɛ i is the deviation vector.

| Molecular docking studies
To explore the possible binding sites of 17 well-known propolis flavonoids on the cholinesterase enzymes (AChE and BuChE) by molecular docking, AutoDock Vina 1.1.2 software (Trott & Olson, 2010) was used. For this aim, the crystal structure of human AChE (PDB entry 4M0E) (Cheung et al., 2013) and human BuChE (PDB entry 4TPK) (Brus et al., 2014) were used as the receptors. Before using the protein structures in the docking studies, the ligand and water molecules were removed and the missing residues were then added using MODELLER software (Webb & Sali, 2016). The protein structures were finally energy minimized by GROMACS (Berendsen et al., 1995). The TPC and TFC of the studied Iranian EEP samples are shown in  Table 6, there is a perfect positive correlation between TPC and TFC (R 2 = 1), which means the EEP samples with higher TPCalso possess higher TFC and vice versa. There are frequent scientific reports that the vegetation of the propolis collection area has a very significant effect on its composition (Anjum et al., 2019;Huang et al., 2014;Wieczorek et al., 2022).
Oxidative stress is mainly created as a result of the imbalance be-  Socha et al., 2015). There are also published documents demonstrating the positive correlation between the antioxidant capacity of the propolis extracts and their TPC (Ahn et al., 2007;Hamasaka et al., 2004;Kalogeropoulos et al., 2009;Moreira et al., 2008;Wang et al., 2016) or TFC (Isla et al., 2009).

| Anticholinesterase (anti-AChE and anti-BuChE) activity
Based on several published papers in recent years, propolis can be considered as a promising therapeutic natural substance to protect the brain and treat neurological injuries and disorders (Ayikobua et al., 2018;Bhargava et al., 2021;Zulhendri, Chandrasekaran, et al., 2021;.

The administration of cholinesterase enzymes (AChE and
BuChE) inhibitors is the main therapeutic strategy for the symptomatic treatment of mild to moderately severe forms of AD (Colovic et al., 2013;Sharma, 2019). The anticholinesterases are also used to manage other forms of neurological disorders, such as ataxia,  (Lotfi et al., 2020;Mariki et al., 2021;Sharma, 2019). The identification of new potent anticholinesterase compounds through the study of natural resources has also attracted a lot of attention (Dey et al., 2017;Dos Santos et al., 2018;Houghton et al., 2006;Mukherjee et al., 2007).
The results corresponding to the evaluation of anti-AChE and anti-BuChE activity of the EEP samples are represented in Table 3. As shown in Table 3, the anti-BuChE activity of three EEP samples (P4, P5, and P11) is slightly higher than anti-AChE activity, but the rest of the samples show a distinct selectivity over BuChE. The data published on the anticholinesterase activity of Algerian propolis methanolic extracts also demonstrated the higher ability of the extracts to inhibit BuChE than AChE (Boulechfar et al., 2019(Boulechfar et al., , 2022.
Considering that in the late stages of AD, there is a decrease in AChE level and an increase in BuChE level in the brain, selective BuChE inhibitors are of great importance in the treatment of advanced AD .
As shown in Table 6

| Antibacterial activity
The  In addition to the determination of MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) ( As mentioned earlier, the antibacterial activity of the EEP samples was also investigated using the agar well diffusion method. As shown in Table 5, seven different concentrations (7.81-500 μg/mL) of each EEP sample and one concentration (500 μg/mL) of ciprofloxacin (positive control) were studied in this test. An overview of the results indicated that as the concentration of EEP samples increases, the diameter of the growth inhibition halo also increases. The inhibition halo diameter of ciprofloxacin is 9 mm for B. cereus and S. aureus and 11 and 10 mm for A. baumannii and P. aeruginosa. The diameters of growth inhibition haloes corresponding to 500 μg/mL con-

The existence of a strong positive correlation between TPC
and TFC of the EEPs and the results obtained from agar well diffusion 1 (R 2 = .81), 2 (R 2 = .83), 3 (R 2 = .85), and 4 (R 2 = .82) ( Table 6) demonstrates that the EEP samples with higher TPC and TFC inhibit the growth of pathogenic bacteria with more power. These results are consistent with some previously published reports (da Silva et al., 2006;Górniak et al., 2019;Inui et al., 2014;Yuan et al., 2021).

| Molecular docking
As implied before, the propolis flavonoid composition, which is responsible for many of its biological and medicinal properties, is used as a criterion for the assessment of propolis quality (Huang et al., 2014;Wang et al., 2016). Scientific reports indicate the anticholinesterase activity of the flavonoid compounds (Dzoyem et al., 2017;Khan et al., 2009Khan et al., , 2018. Considering that the results obtained from the current research also represent a strong positive correlation between the anticholinesterase activity of EEP samples and their TFC, it was decided to investigate the interaction of 17 well-known propolis flavonoids (Kocot et al., 2018;Pasupuleti et al., 2017;Zhang et al., 2021) with the active site gorge of the cholinesterase enzymes (AChE and BuChE) by molecular docking studies. The structure of these flavonoid compounds is illustrated in Figure 1.
The AChE docking results demonstrated that all 17 flavonoid compounds have the ability to interact with the active site gorge of the enzyme and their best binding energy varies between −8.9 and −7 kcal/mol. The lowest docking energy is corresponding to rutin and the highest is related to fisetin (Table 7). Table 8  and Ala204) (Atanasova et al., 2015;Damuka et al., 2020;Kua et al., 2003;Wiesner et al., 2007;Zhang et al., 2002).   (Brus et al., 2014;Macdonald et al., 2012).
The amino acid residues participating in the binding of BuChE to the flavonoids are represented in Table 9. As well seen, all the TA B L E 6 The Spearman correlations between studied parameters calculated by SPSS software.  Figure 2c,d.

F I G U R E 1
The structure of 17 well-known propolis flavonoids.
According to the docking results, all the studied flavonoids bind to the gorge region of both enzymes with high affinity ( Table 7).

ACK N OWLED G M ENTS
The authors gratefully appreciate the funding support received for the project from the Research Council of the Graduate University of Advanced Technology, Kerman, Iran.

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data are available on request from the authors.