Antioxidant, antidiabetic effects and polyphenolic contents of propolis from Siirt, Turkey

Abstract Propolis, a natural product collected by honeybees from various plant sources, has gained significant attention due to its diverse bioactive compounds and potential therapeutic properties. To further explore its contents and biological activities, this study aimed to analyze the phenolic compounds in Siirt propolis extracts obtained using different solvents, namely ethanol, water, and ethanol‐water mixtures. The primary objective of this research was to investigate the phenolic profile, as well as the antidiabetic and antioxidant activities of the propolis extracts. Chemical profiling of extracts was performed using LC–MS/MS. The antioxidant potential of the propolis extracts was evaluated through free radical scavenging methods, including DPPH and ABTS assays. As a result of these analyses, propolis extracts showed moderate radical scavenging potential with 13.86%–35.72% for DPPH and 33.62%–62.50% for ABTS at a concentration of 30 μg mL−1, respectively. This radical scavenging potential of the extracts sheds light on its ability to combat oxidative stress, which is implicated in the development of diabetes, and its potential effects on cellular health. Additionally, the study assessed the antidiabetic properties of the propolis extracts by examining their inhibition effects on α‐amylase and α‐glycosidase enzymes. Extracts with high phenolic content showed a high inhibitory effect against α‐glucosidase with an IC50 of 5.72 ± 0.83 μg mL−1. This research provided significant findings regarding the potential use of propolis in the treatment of diabetes and related metabolic disorders.

chemical content, it is important in terms of its potential to be used and developed in health, pharmaceutical production, the cosmetics industry, the food industry, and even in different fields (Devequi-Nunes et al., 2018;Doğan & Hayoğlu, 2012).
The antioxidant activities and components of propolis extracts were studied with various extraction solvents.The results of these studies showed that ethanol was more effective than water for the extraction of phenolic contents and that ethanol extracts had greater antioxidant activity than water.However, there are few studies evaluating the phenolic profiles of different ethanol/water solvents and propolis extracts (Sun et al., 2015).
The goal of this study is to compare the phenolic content profiles of Siirt Propolis (SP) extracts obtained using water and ethanol by LC-MS/MS and characterize them, and to determine their antioxidant and antidiabetic effects by various biological activity measurements.

| Preparation of propolis solvent extracts
Propolis sample was gathered from the Siirt province in the Southeastern Anatolia region of Turkey, followed by their preservation and desiccation in a light-protected environment before further processing.Extractions were carried at different solvent systems such as ethanol, water, and ethanol-water (80:20, 60:40, 40:60, 20:80, v/v).
For ethanol extraction, 10 g of liquid nitrogen-dried propolis was ground into a fine powder in a blender and mixed with 100 mL ethanol via a magnetic stirrer for 24 h.After 24 h, it was refluxed more than 1 h at 65°C.Then the ethanol extract was filtered over the Whatman No. 1 paper.The ethanol solvent was removed via a rotary evaporator (Heidolph Hei-Vap Adv ML) at 40°C to obtain a dry extract.The dried ethanolic extract was stored in a plastic bottle at −20°C until further use.
For water extraction, 10 g of liquid nitrogen-dried propolis was ground into a fine powder using a blender and then mixed with 100 mL of water and stirred with a magnetic stirrer for 24 h to extract water.After 24 h, it was refluxed 1 h at 65°C.After filtering the water extract through Whatman No. 1 paper, the resulting filtrates were frozen at −84°C in an ultra-low temperature freezer (Sanyo, Japan).They were then lyophilized in a lyophilizer under 5 mm Hg pressure at −50°C.The dried water extract was then stored in a bottle at −20°C until it was ready to be used.
For ethanol-water extractions, 10 g of liquid nitrogen-dried propolis sample was ground into a fine powder in a blender and mixed with 100 mL ethanol-water (80:20, 60:40, 40:60, 20:80, v/v) by magnetic stirrer for 24 h.After 24 h, it was refluxed at 65°C for 1 h.Then the extracts were filtered with Whatman No. 1 paper.
The ethanol solvent was first removed from the rotary evaporator after each extraction.Then, the remaining water phase was lyophilized (such as water extraction).The dried ethanol-water extracts were placed separately in a bottle and stored at -20°C until used.

| Instruments and chromatographic conditions
A liquid chromatograph-mass spectrometer (LC-MS/MS) was used to analyze 18 distinct phenolic compounds.A Shimadzu Nexera X2 UHPLC (Shimadzu, Japan) liquid chromatograph equipped with an InertSustain Swift C18 (2.1 mm*100 mm, 3 μm) column was used, and the system was coupled to an 8050 triple quadruple detector (Shimadzu, Japan) controlled by LabSolution 5.60 SP2 software.Ultra-pure water was prepared on a Milli-Q IQ 7000 system (Millipore Company, USA).Propolis samples (SP1-SP6) were extracted by the Reflux Mechanism.

| Determination of LC-MS/MS analysis conditions
Binary gradient LC-40D XS-Autosampler Model SIL-40C XS-Oven Model CTO-40S-Shimadzu 8050 model liquid chromatography device LC-MS/MS analysis of the samples was performed by triple quadrupole MS/MS detector.The elution gradient was established with mobile phase A (water and 0.1% formic acid) and mobile phase B (methanol and 0.1% formic acid).The gradient elution program started with 20% B for 0 min, increasing linearly from 50% for 8 min, then to 95% B at 12 min.Finally, the system returned to its original conditions over 0.1 min and equilibrated for 3 min (total run time 15 min).Multiple reaction monitoring processing (MRM) mode was used to quantify the analysis.Two applications were made for each compound analysis in the experiment.The first was performed for quantitative results and the second was analyzed for confirmation.
The optimized MS parameters for phenolic compounds are given in Table 1.Optimum Electrospray Ionization (ESI) parameters; 350°C interface temperature, 250°C DL temperature, 400°C nebulizer gas (nitrogen) temperature, 3 L/min, gas flow 15 L/min, drying gas flow was determined.

| Sample preparation
For phenolic component analysis, Touzani et al. (2021) was followed.0.01 g of propolis sample (with an accuracy of 0.001 g) was weighed into a 50 mL centrifuge tube, 15 mL of methanol was added to it.It was kept in ultrasonic for 20 min.It was then centrifuged at 4000 rpm for 10 min.After centrifugation, make up to 50 mL of methanol.It was passed through filter paper and passed through a 0.45 μm filter tip, taken into a vial to give LC-MS/MS, and the measurement was made.The experiments were conducted in triplicate (n = 3), and the outcomes were presented as the mean value accompanied by the standard deviation.

| Method validation parameters
The validation parameters were determined to be linearity, precision (reproducibility and, recovery), the limits of detection (LOD), and quantification (LOQ) experiments.The signal-to-noise ratio was determined by measuring the signal-to-noise ratio by injecting until the S/N ratio was 3 for LOD and 10 for LOQ.The linearity for the phenolic compounds at the concentration levels of 0.1, 0.2, 0.5, 0.75, TA B L E 1 MS parameters for the tested compounds.

| DPPH radical removal potential
The DPPH free radical scavenging potential was assessed using the Blois method (Blois, 1958;Kavaz Yüksel et al., 2021).A freshly prepared solution of DPPH (10 −3 M) was utilized as the free radical source.The samples were pipetted into test tubes at concentrations of 10, 20, and 30 μg/μL in a total volume of 1 mL, followed by the addition of 96% ethanol to complete the final volume to 1.5 mL.
Subsequently, 0.5 mL of the freshly prepared DPPH solution was added and mixed using a vortex.After incubating for 30 min, the percentage of radical scavenging activity was expressed by measuring the absorbance at 517 nm.

| ABTS radical removal potential
The ABTS radical scavenging potential was evaluated following the method introduced by Re et al. (Re et al., 1999).ABTS radicals were generated by combining persulfate (2.45 nM) solution with a prepared ABTS (7 mM) solution.Controls were set up using 1000 μL of ABTS radical solution and 500 μL of 96% ethanol.Next, different concentrations (ranging from 10 to 40 μg mL −1 ) of the samples were mixed with 1 mL of ABTS radical solution, and the total volume was adjusted to 1.5 mL with 96% ethanol.After 30 min of incubation in the dark, the absorbance was measured at 734 nm with respect to the blank.The ABTS radical scavenging potential was measured and expressed as a percentage in the results.

| α-Amylase inhibitory activity
A slightly modified α-amylase inhibition test method, as established by Yu et al. (2012), was employed in this study.Firstly, 10 μL of α-amylase solution (1 unit/mL in distilled water) was combined with 10 μL of samples with varying concentrations and incubated at 37.0°C for 15 min.The reaction was started by adding 500 μL of substrate, which was made from a 1% starch solution in 20 mmol/L sodium phosphate buffer (pH 6.9).The sample was then incubated for 10 min at 37°C.300 μL of DNS reagent consisting of 12% Na-K tartrate in 0.4 mol/L NaOH and 1% 3,5 dinitrosalicylic acid was added to stop the reaction.Subsequently, the test tubes were subjected to a boiling water bath for 10 min, followed by cooling at room temperature, and 5 mL of distilled water was added.Finally, the results of the enzymatic reaction were measured at 540 nm.

| α-Glycosidase enzyme inhibition measurement
Analysis was performed using a modified developed method for αglucosidase activity determination (Xu et al., 2014).4-nitrophenyl β-D-glucopyranoside (pNPG) substrate (5 mM) was dissolved in 25 mL of phosphate buffer (0.1 M).The reaction was prepared using specific ratios of substrate (pNPG), Phosphate Buffer (5.0 mM, pH 6.9), enzyme solution in Phosphate buffer (0.15 U/mL, pH 7.4).Then, the absorbance value at 540 nm was read every minute, and at the end of 5 min, the difference between the value in the first minute and the value read in the fifth minute was taken.This experiment was repeated for 5 different inhibitor concentrations.The IC 50 value of all substances was calculated.

| Statistical studies
This study employed statistical analysis using the R programming language to assess the relationship between variables.Pearson correlation analysis was utilized to evaluate the association between two variables.The results were exhibited as mean ± standard error of the mean (95% confidence intervals).Differences between data sets were considered statistically significant when the p-value was less than .05.

| Phenolic contents of propolis
The assessment of total phenolic and flavonoid contents was con-  4).
Among the extracts obtained from all solvent systems, the most common phenolics were caffeic acid, pinocembrin, chrysin, CAPE, and galangin (Figure 1).Although the solubility of propolis was very low, the highest solubility and the highest content of phenolic substances were observed in absolute ethanol.In the study was obtained highest yield in ethanol extract (2.12 g/100 mL at 25°C).

| Antioxidant and antidiabetic effect of propolis extracts
The results of the antioxidant and antidiabetic evaluations of propolis extracts are shown in Table 5.The antioxidant effect was assessed using DPPH and ABTS analyses, while the antidiabetic effect was determined based on α-amylase and α-glucosidase inhibition activity.

F I G U R E 1
Major phenolic compounds according to LC-MS/MS results (μg mL −1 ).inhibitory effect on α-amylase, the lowest effect on α-glucosidase was observed in SP2.It was determined that samples SP4 and SP5 did not have any effect on the α-glucosidase enzyme.

| DISCUSS ION
Propolis is a natural product resulting from the mixing of bee secretions with botanical exudates and Phenolic components in its content are vital components of the human diet.It is defined as an extremely complex resinous material containing biologically active molecules with antioxidant, antibacterial, antiparasitic, antiviral, hepatoprotective, antifungal, antidiabetic, and immunomodulatory activities (de et al., 2017;Kocot et al., 2018;Santos et al., 2020).Phenolic contents (flavonoids), common especially in its composition, are plant metabolites commonly distributed in the plant kingdom.
Recent focus of attention on phenolic acids stems from their potential protective role against oxidative damage diseases (DM, coronary heart disease, stroke, and cancers) through the ingestion of fruits and vegetables.In addition, it was reported that the application of propolis extracts has been tried in therapies metabolic diseases, inflammation, and against cancer, since propolis is rich in flavonoids such as kaempferol, genistein, apigenin, chrysin, and cinnamic acid derivatives.Studies on animal and cellular models indicate that propolis can regulate the accumulation of AGEs, oxidative stress, and inflammation in adipose tissue, all of which contribute to insulin defects (Kitamura, 2019).Studies have reported that the primary components of propolis are phenolic compounds, which consist of caffeic, ferulic, p-coumaric, and cinnamic acids.The pharmacological benefits of propolis are associated with its phenolic compounds and flavonoid skeleton structure (Gülçin et al., 2010;Kumazawa et al., 2004).Phenolic contents with and without flavonoid structure are widely included in the content of propolis produced by bees in different regions of the world (Jordan, Brazil, Turkey, etc.), and solutions obtained from propolis are used extensively in traditional treatment methods and in folk medicine.For centuries, propolis has been used medicinally to treat infections and promote wound healing (Alencar et al., 2007;Naik et al., 2021).
The current COVID-19 pandemic also shows that food supplements that strengthen the immune system, such as propolis, are very important.The contents of propolis produced in different regions may also contain different components.Phenolic contents share the same general structure that carries the aromatic hydroxyl functional group and form a class of approximately 8000 molecules in nature (Karaman et al., 2010).
There are uncertainties about the content of commercial products prepared from available propolis extracts.Solubility of propolis in water due to its organic structure and in ethanol due to its resinous structure is very low.In recent years, solutions (tincture) "claimed to have 20 percent propolis content" are also widely sold as food supplements.
For this reason, it is significant to obtain the contents of propolis in different regions with new techniques and to evaluate their biological activities.In our study, phenolic compounds were determined by LC-MS/MS analysis of Siirt propolis extracts obtained by using different solvents such as (ethanol, water, and ethanol-water mixtures).While the solubility is highest in ethanol (21%), it gradually decreases in the transition to the water phase.The extract obtained a yield of 7% in the water phase.
In the study, phenol and flavonoid contents of the extracts obtained using different solvents were determined.In 100% ethyl alcohol solvent, phenolic compounds were detected CAPE 602.43 μg mL Values are expressed as percent radical scavenging particles at 30 μg mL −1 . b Values are expressed as percent inhibition at 30 μg mL −1 .c ND effect could not be determined.
e Glycosidase and amylase enzyme inhibitor.
TA B L E 5 Radical scavenging potentials of propolis extracts and inhibition effects on diabetes-related enzymes α-amylase and α-glucosidase.

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It was stated that samples with high total phenolic and flavonoid content showed high activity (Stavropoulou et al., 2021).There are numerous other studies in the literature that provide supportive evidence for this claim (Al-Khayri et al., 2022;Kavaz et al., 2022;Kavaz Yüksel et al., 2021;Tohma et al., 2015).According to the results obtained from our study, higher radical scavenging activity was found at the concentration of 30 μg mL −1 compared to other literature studies mentioned above.In addition, samples with high phenolic content, especially SP1 and SP2, exhibited higher radical scavenging activity as stated in the literature (Stavropoulou et al., 2021).
Treating diabetes mellitus (DM) involves therapeutic approaches that focus on inhibiting enzymes responsible for breaking down polysaccharide molecules into simpler sugars or monosaccharides.This inhibition, particularly of α-glycosidase and α-amylase enzymes, slows down the digestion and absorption of simple carbohydrates in the intestine.Consequently, it helps regulate postprandial blood glucose levels, contributing to the treatment of DM.Certain clinically recommended medications, such as acarbose and voglibose, act as α-glycosidase inhibitors, facilitating the control and treatment of DM (Karagecili et al., 2023).In a study, antioxidant and antidiabetic properties of Berdav propolis were determined.Propolis extract showed an inhibitory effect against α-glycosidase with IC 50 values of 3.7 μg mL −1 (Karagecili et al., 2023).In another study, they examined the inhibitory effects of natural phenolic compounds on α-glycosidase and α-amylase enzymes in vitro.These compounds showed significant inhibition against α-amylase and α-glycosidase with IC 50 values ranging from 137.36 to 737.23 nM and 29.01 to 157.96 nM, respectively.It has also been reported that caffeic acid phenethyl ester (CAPE) is a bioactive molecule of propolis and has a high inhibitory potential on these two enzymes (Karagecili et al., 2023).When the aforementioned and other literature studies were evaluated, it was stated that the inhibition of α-glycosidase and α-amylase increased due to the increase in phenolic content.
When our results are evaluated, it can be stated that samples with high phenolic content exhibit higher inhibitory potential.In this context, other literature studies and these results support each other (Chaudhry et al., 2017;Taslimi & Gulçin, 2017).Caffeic acid, p-coumaric acid, kaempferol, geistein, apigenin, pinocembrin, CAPE, chrysin, and galangin are above 50 μg mL −1 among the 18 different phenolic compounds.In addition, SP1 and SP2 crude extracts are the samples in which the phenolic components are obtained at the highest rate, and especially the SP1 extract is much richer in flavonoids such as caempferol, genistein, and apigenin.It can be said that SP1 has the highest antioxidant levels in DPPH (35.72 ± 4.03%) and ABTS (62.50 ± 5.96%) analyses due to increased phenolic structures in the matrix and it also has an antidiabetic effect by showing the highest inhibition on the αamylase enzyme (49.45 ± 5.18%) and α-glucosidase enzyme (IC 50 of 5.72 ± 0.83 μg mL −1 ).These effects gradually decrease as the water rate in the solvent increases, but as the water rate in the solvent increases, the amount of caffeic acid (1565.89μg mL −1 ) increases at least twice compared to the SP1 extract (Table 4).
According to the results of Pearson correlation analysis (Figure 2), there is a significant positive correlation between the DPPH radical scavenging potential of all other components, especially CAPE (p < .001,R = .743)and p-Coumaric Acid (p = .001,R = .691)in propolis.
Caffeic acid and p-Coumaric acid also showed a significant effect on ABTS radical scavenging activity.When these results are evaluated, the high radical scavenging activity in SP1, SP5, and SP6 samples can be attributed to these compounds.Genistein (p = .001,R = .658),apigenin (p = .002,R = .633),pinocembrine (p = .001,R = .673),chrysin (p = .001,R = .662),kaempferol (p = .003,R = .623),and galangin (p = .002,R = .642)compounds caused high α-amylase inhibition effect with positive correlation results.The high α-amylase inhibition potential in SP1, SP2, and SP6 samples can be attributed to these compounds.In addition, a positive correlation was found between the increase in DPPH radical scavenging potential of the samples and the α-amylase inhibition effect (p = .006,R = .575).All compounds except caffeic acid and p-Coumaric acid in the extracted content show a positive correlation with each other proportionally (p < .001,R > .800).
A study conducted in 2011 examined the anti-radical activities of CAPE.They stated that CAPE has a good radical scavenging capacity (IC 50 : 9.8 and 3.3 mg mL −1 , respectively) against DPPH and ABTS radicals compared to standard antioxidant compounds such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol, and trolox (Göçer & Gülçin, 2011).It has also been reported that caffeic acid has the highest peroxyl radical scavenging compared to the standard antioxidant trolox.In particular, it has been shown in in vitro analyses that caffeic acid has the radical scavenging potential of ABTS (92.9% at 25 mg mL −1 ) and DPPH (92.1% at 20 mg mL −1 ) (Khan et al., 2016).
As a result, when the above-mentioned literature results and the effects of the components within the scope of this study on bioactivity were evaluated together according to Pearson correlation (Figure 2), it was seen that they were compatible with the literature studies.The results indicate that the antioxidant and antidiabetic effects of the propolis samples may vary depending on factors such as the propolis source, collection region, and extraction method.It is essential to conduct further investigations to understand the variations in the antioxidant properties of different propolis samples thoroughly.

| CON CLUS IONS
Propolis, which has a natural and rich content of pollen collected by bees from the habitats around them, is of critical importance for the bees themselves, their offspring, and their hives.Due to the rich -Nunes et al. (2018) conducted an evaluation of propolis extracts obtained through ethanolic extraction and supercritical extraction from three different propolis species (brown red and green) collected in various regions of Brazil (specifically, the state of Bahia).The study involved the determination of phenolic compounds, flavonoids, in vitro antioxidant activity (DPPH), as well as luteolin, ferulic acid, and catechin content in the propolis extracts.The results indicated that the ethanol extracts yielded the most favorable outcomes, displaying significant selectivity for extracting antioxidant compounds (Devequi-Nunes et al., 2018).On the other hand, Woźniak et al. (2023) investigated the chemical composition and biological properties of propolis extracts obtained from different regions in Poland.The study revealed moderate DPPH free radical scavenging activity ranging from 29.22% to 35.14% at the concentration 0.1 mg mL −1

F
I G U R E 2 A correlation heatmap based on assessing the relationship between variables.The number in brackets in each cell represents the p-values and the other represents the R-value.R-values are a measure of correlation strength and range from −1 to +1.
a All results were calculated based on 10 g of propolis.−1.The samples exhibited radical scavenging activity in the range of 13.86%-35.72%forDPPHand33.62%-62.50%forABTSataconcentration of 30 μg mL −1 , respectively.At 30 μg mL −1 , samples SP1 and SP5 showed the highest DPPH radical scavenging potential at about 35%, while ABTS radical scavenging potential was observed in samples SP1 and SP1.1 at about 62%.In all samples, the lowest radical scavenging potential was observed in sample SP3 for DPPH and SP2 for ABTS.Inhibitory effects on α-amylase and α-glucosidase enzymes of all propolis samples were shown (Table5).The highest inhibitory effect on the enzymes was determined for SP1.At 30 μg mL −1 , sample SP1 had an inhibitory effect on α-amylase enzyme with 49.45 ± 5.18%, while it had an inhibitory effect on α-glucosidase with an IC 50 of 5.72 ± 0.83 μg mL −1 .While examples SP3 showed the lowestTA B L E 4Note: Phenolic compounds amount were expressed as μg mL −1 .Abbreviation: ND, not detected.