Comprehensive Metabolite Profiling of Berdav Propolis Using LC-MS/MS: Determination of Antioxidant, Anticholinergic, Antiglaucoma, and Antidiabetic Effects

Propolis is a complex natural compound that honeybees obtain from plants and contributes to hive safety. It is rich in phenolic and flavonoid compounds, which contain antioxidant, antimicrobial, and anticancer properties. In this study, the chemical composition and antioxidant activities of propolis were investigated; ABTS•+, DPPH• and DMPD•+ were prepared using radical scavenging antioxidant methods. The phenolic and flavonoid contents of propolis were 53 mg of gallic acid equivalent (GAE)/g and 170.164 mg of quercetin equivalent (QE)/g, respectively. The ferric ion (Fe3+) reduction, CUPRAC and FRAP reduction capacities were also studied. The antioxidant and reducing capacities of propolis were compared with those of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol and Trolox reference standards. The half maximal inhibition concentration (IC50) values of propolis for ABTS•+, DPPH• and DMPD•+ scavenging activities were found to be 8.15, 20.55 and 86.64 μg/mL, respectively. Propolis extract demonstrated IC50 values of 3.7, 3.4 and 19.6 μg/mL against α-glycosidase, acetylcholinesterase (AChE) and carbonic anhydrase II (hCA II) enzyme, respectively. These enzymes’ inhibition was associated with diabetes, Alzheimer’s disease (AD) and glaucoma. The reducing power, antioxidant activity and enzyme inhibition capacity of propolis extract were comparable to those demonstrated by the standards. Twenty-eight phenolic compounds, including acacetin, caffeic acid, p-coumaric acid, naringenin, chrysin, quinic acid, quercetin, and ferulic acid, were determined by LC-MS/MS to be major organic compounds in propolis. The polyphenolic antioxidant-rich content of the ethanol extract of propolis appears to be a natural product that can be used in the treatment of diabetes, AD, glaucoma, epilepsy, and cancerous diseases.


Introduction
Propolis is a naturally occurring bee resin used by honeybees as a sealant for hexagonal cells as well as for protection from external threats and climatic conditions. Its biological features support a more aseptic environment, which may be obtained by embalming the bodies of invading species, whereas its mechanical properties fill cracks and promote thermal isolation, which aids in hive defense [1,2]. Geographical locations, botanical sources, and bee species all have a significant impact on the chemical makeup of propolis, and safe AChE research is required to address neurological damage since AChEIs have substantial adverse effects. Anticholinesterases or AChEIs are cholinesterase enzyme inhibitors [32]. AChEIs are often used in medicine, particularly for the treatment of AD. AChEIs and prospective lead molecules for AD have both been identified as phenolic chemicals [33,34].
Carbonic anhydrases (CAs) are metalloenzymes that contain Zn 2+ and catalyze the reversible hydration of carbon dioxide (CO 2 ) to protons and bicarbonate (HCO 3 − ) [35,36]. CAs play a variety of biochemical and metabolic roles, including ureagenesis, lipogenesis, and gluconeogenesis [37][38][39]. They also maintain a fluid balance throughout the body, particularly in the eyes, stomach, and kidneys. Glaucoma-related elevated intraocular pressure (IOP) can be relieved or treated with carbonic anhydrase inhibitors (CAIs) [40]. Various analytical techniques have been used to determine the quality of propolis. The commonly used techniques include UV spectrophotometry and LC-MS/MS.
The present study aimed to investigate the chemical ingredients and biological activities of propolis collected from the East Anatolian region of Turkey. To achieve this, the following was performed: (a) flavonoid and phenolic profiles of the samples were evaluated using LC-MS/MS; (b) the total antioxidant/phenolic capabilities of propolis equivalents were measured using DPPH, ABTS, DMPD, CUPRAC, ferric-reducing antioxidant power (FRAP), and Folin-Ciocalteu techniques; (c) the inhibitory effect of propolis on some metabolic enzymes including AChE, hCA II and α-glycosidase were investigated to determine a probable relationship with AD, diabetes mellitus, and glaucoma.

Phenolic Contents of Propolis
The total phenolic and flavonoid contents were evaluated for propolis. In this context, 53.0 mg of gallic acid equivalent (GAE)/g and 170.2 quercetin equivalent (QE)/g were calculated in propolis extract. Using 53 phenolic compounds as standards, the LC-MS/MS method was utilized to identify the major organic components in propolis preparations ( Figure 1). Phenolic compounds were elucidated by comparing their chromatographic behavior, UV spectra, and MS information with reference compounds, and 28 compounds were discovered (Table 1). Table 2 lists the mean values of each chemical based on LC-MS/MS analysis.

Radical Scavenging Results
The DPPH • scavenging activity of propolis was measured, and the half maximal inhibition concentration (IC 50 ) value was determined (Table 3). Propolis demonstrated concentration-dependent radical scavenging activity. Propolis revealed comparable and stronger anti-radical activities (20.55 µg/mL) than BHT (21.0 µg/mL), but had a better antioxidant potential in propolis samples taken from 39 different locations in Turkey. These propolis samples had antioxidant capabilities ranging from 55.98 ± 0.02% to 86.17 ± 0.16% [46]. In addition, our sample has a higher antioxidant capacity compared to various European propolis specimens (26.45 µg/mL; 27.72 µg/mL) [47]. The IC 50 values of Chinese propolis samples ranged greatly, from 71.19 ± 5.31 µg/mL to 432.08 ± 6.42 µg/mL, showing that the antioxidant activity of these propolis is not only lower but also region-dependent [48]. All analyses were performed in triplicate. Propolis IC 50 values and reference radical scavenger agents such as Trolox, α-tocopherol, BHT, and BHA were 8.157 µg/mL for propolis, 7.71 µg/mL for BHT, 7.71 µg/mL for BHA, 7.71 µg/mL for Trolox, and 8.10 µg/mL for α-tocopherol (Table 3). As indicated in Table 4, propolis was efficient in DMPD •+ scavenging at concentrations ranging from 10 to 30 µg/mL. The IC 50 of propolis was 86.64 µg/mL. The value obtained for this was 31.43 µg/mL for BHA, 14.38 µg/mL for Trolox. At all propolis concentrations, the concentration of DMPD •+ decreased significantly (p < 0.01).

Enzyme Inhibition Results
The hCA II isoform is associated with some disorders, including glaucoma, osteoporosis, and renal tubular acidosis. CA inhibitory effects of the propolis were tested and the results are presented in Table 5 and Figure 2A. IC 50 values were 19.6 µg/mL (r 2 : 0.9327) for propolis towards CA II. For acetazolamide, the IC 50 was 8.37 µg/mL (r 2 : 0.9825), which was used as a control for the carbonic anhydrase isoenzyme inhibition experiment [49].
The inhibition level of propolis extract was comparable to that of tacrine, a common reference inhibitor of the AChE. Table 5 summarizes the IC 50 values of the propolis extract for enzyme inhibition. The IC 50 value (3.4 µg/mL) of the ethanol extract for propolis against AChE showed a higher inhibition effect compared to that of tacrine ( Table 5). The IC 50 of tacrine was 5.97 µg/mL (r 2 : 0.9706), which was utilized as a control for the AChE inhibition experiment ( Figure 2B). *Acetazolamide was used as standard inhibitor for hCA, ** Tacrine was used as standard inhibitor for AChE, *** Acarbose was as used standard inhibitor for α-glycosidase, which was obtained from the literature [50]. Propolis extracts displayed an IC 50 value of 3.7 µg/mL towards α-glycosidase (r 2 : 0.9362, Table 5 and Figure 2C). The results reveal that propolis has inhibitory effects similar to those of α-glycosidase efficient acarbose (IC 50 : 22800 nM) as a typical glycosidase inhibitor [50]. In the present study, the α-glycosidase enzyme inhibition effect of propolis was higher than that of propolis samples taken from various locations in Morocco (IC 50 : 90.99-876.24 µg/mL) of [51].

Discussion
Phenolic chemicals present in all plants are vital components of the human diet. They have received a great deal of attention because of their biological activities, which include antioxidant characteristics [52][53][54]. Propolis contains an appreciably high number of phenolic compounds, which have positive effects on human health. Phenolic compounds also hinder oxidation and improve the chemical stability of food products [55]. Flavonoids, such as flavones, flavonols, flavanones, and dihydroflavonols, as well as other polyphenols, are primarily responsible for the biological action of propolis [16,56,57]. Caffeic acid phenylethyl ester, gallic acid, cinnamic acid, galangin, caffeic acid, naringenin, luteolin, kaempferol, quercetin, pinocembrin, rutin, p-coumaric acid, and ferulic acid are among the components of propolis. They ultimately enhance the effective digestion, antioxidant capacity, and metabolic, physiological, and immune capabilities of body tissues [58,59]. More than 300 substances, including phenolic acids and their esters, flavonoids, terpenes, triterpenes, alcohols, aromatic aldehydes, fatty acids, stilbenes and steroids, lignans, amino acids, and sugars, among others, have been observed in various propolis species [60]. Considering the phenolic content determinations of propolis performed as gallic acid equivalent, the propolis samples obtained from different locations in Turkey displayed effective phenolic (88.7-261.1 mg GAE/g propolis) and total flavonoid (37.5-150.4 mg QE/g propolis) contents of the ethanolic propolis extracts [61]. In another study, aqueous extract of propolis expressed 124.3 µg GAE and 8.15 µg QE per g of aqueous extract of propolis [2]. In addition, Aygul and co-workers determined that Ankara propolis (8.50 mg GAE/g propolis) and Giresun propolis (7.88 mg GAE/g propolis) exhibited notable total phenolic contents [62].
Therefore, it is important to choose the most appropriate method to determine the antioxidant capacity of herbal extracts or biological samples. In the present study, many bioanalytical methods, such as reducing effects and radical removal methods, were used to determine the antioxidant capacity of the extract [63,64]. The diversity, high number of ingredients, and rich phenolic content may explain the antioxidant potential of propolis. The reduction potentials of phenolic compounds isolated from propolis were determined using three different reduction systems, including Fe 3+ , CUPRAC, and Fe 3+ -TPTZ ionreducing abilities [65,66]. The radical scavenging properties of the propolis were examined using DPPH, ABTS, and DMPD radical scavenging assays. Plants, natural compounds, and propolis samples can exhibit reducing properties, thereby neutralizing oxidants and ROS.
The total reducing capacity of the pure antioxidant substances and plant extracts was determined using the FRAP test. Ferric salt, which is the basis of the FRAP test, was utilized as an oxidant in the electron transfer process [67]. Owing to its colored combination with TPTZ, which has a maximum absorbance at 593 nm, Fe 2+ may be detected spectrophotometrically [68]. Depending on the reducing power of the antioxidant samples, the yellow color of the test solution changed to different colors of green or blue in this assay. The reducing capacity of a compound may be a good predictor of its potential antioxidant activity. The Fe 3+ -TPTZ-reducing test was used to assess the reducing capabilities of propolis and standards [69]. In our previous study, we determined that the aqueous extract of propolis exhibited concentration-dependent (10-30 µg/mL) Fe 3+ -reducing and cupric ion (Cu 2+ )-reducing abilities with statistically significant differences (p < 0.01) [2].
The DPPH method is based on the DPPH • removal of antioxidant components in the plant extracts. The scavenging effect of ABTS •+ radicals is based on a similar mechanism [70,71]. The DPPH • test, which is based on reducing DPPH • to the non-radical form DPPH-H, is commonly used to assess the antioxidant capacity [72,73]. Propolis is thought to have natural antioxidant potential if it possesses DPPH • scavenging capability. In an ABTS/K 2 S 2 O 8 system, radicals of ABTS were produced [74]. This test is a decolorization approach in which the radical is created directly in a stable state prior to treatment with suspected antioxidants. The improved approach for producing ABTS •+ reported here involves the direct creation of a blue-green ABTS •+ chromophore via the reaction between ABTS and potassium persulfate [75]. A prior study revealed that aqueous extract of propolis displayed effective DPPH • radical scavenging with an IC 50 value of 31.81 µg/mL [2].
When compared to positive controls, the data clearly revealed that propolis has effective ABTS •+ scavenging activity. Our sample showed a higher ABTS •+ scavenging effect than that of lyophilized aqueous extract of propolis from Turkey's Erzurum province (IC 50 : 14.29 µg/mL) [2]. A lower IC 50 value similar to the DPPH free radical scavenging activity suggests higher ABTS •+ scavenging activity. According to previous reports, the principal disadvantage of the DMPD •+ approach is that its sensitivity and repeatability are significantly reduced when hydrophobic antioxidants such as α-Tocopherol or BHT are utilized [76]. Considering the literature, it seems that Moroccan propolis, which was collected from different locations, exhibited effective superoxide anion and nitric oxide radicals scavenging activity and metal chelating properties [77]. Our previous study demonstrated that aqueous extract of propolis had effective DMPD •+ (IC 50 : 18.32 µg/mL) and superoxide anion (O 2 •-, IC 50 : 9.89 µg/mL) radicals scavenging activities [2]. α-Glycosidase suppression causes delays in sugar absorption during digestion. Clinical trials using acarbose and miglitol as α-glycosidase inhibitors have revealed lower postprandial hyperglycemia and greater insulin sensitivity [78]. These inhibitors block the α-glycosidase enzyme in the small intestine, which is responsible for the digestion of complex carbohydrates. This enzymatic process lowers postpartum blood glucose levels by reducing carbohydrate breakdown and glucose absorption [79]. When the literature was searched, it was observed that Moroccan propolis collected from different regions inhibited the α-glycosidase enzyme with IC 50 values between 0.01-0.07 mg/mL. Moreover, it was observed that the same propolis samples inhibited α-amylase as another digestive enzyme, with IC 50 values between 0.09 and 0.52 mg/mL [77].
AD is the most prevalent neurodegenerative ailment, and the leading cause of dementia among the elderly. The reduction in AChE levels in the brain is the most significant biochemical alteration in AD [80]. AChEIs are used for the treatment of AD; however, these drugs have several negative side effects. As a result, research and use of novel potent antioxidants and AChE agents are greatly needed [81]. It was also found that the predominant AChE inhibitory effects were related to aromatic chemicals, and to a lesser extent, aliphatic molecules [82]. Medicinal herbs are always rich in cholinesterase inhibitors [83]. The cholinesterase inhibitory properties of propolis extract were evaluated in the current study using AChE. The ethanol extract was shown to effectively inhibit AChE. In a previous study, the inhibition effects of propolis, which was obtained from different locations, on some crucial enzymes, such as urease, xanthine oxidase and AChE, were investigated. They found that the propolis sample inhibited AChE enzyme with IC 50 values ranging from 0.221 to 1340 mg/mL [61]. Previous studies have shown that there was a relationship between propolis phenolic contents and AChE inhibition, which importantly suggests that the enzyme was probably inhibited by phenolic substances [61]. It is known that propolis is a complex resinous material. Therefore, it is very important that the specific phenolics in this complex mixture exhibit high AChE inhibitions even at low concentrations.
CA II has been linked to epilepsy, glaucoma, edema, and assumable altitude sickness [84]. The activation and inhibition of CA isoforms have important therapeutic goals in the treatment of a variety of disorders including glaucoma, edema, cancer, obesity, hypertension, epilepsy, and osteoporosis [85]. CA II suppression reduces HCO 3 − generation and, as a result, aqueous humor secretion, resulting in lower ocular pressure [86]. Glaucoma is a multifactorial optical disease characterized by optical nerve degeneration, which is mostly associated with high IOP, which can result in blindness. Because hCA inhibitors such as acetazolamide, brinzolamide, and dorzolamide are effective in lowering IOP after topical treatment, novel therapeutic considerations are required [87]. In another study on propolis, it was determined that Ankara propolis (IC 50 : 1.273 µg/mL) and Giresun propolis (IC 50 : 1.374 µg/mL) had quite high cytosolic hCA I isoform. On the other hand, both propolis samples inhibited predominant and cytosolic hCA II isoenzyme with IC 50 values of 0.486 and 0.612 µg/mL [62].

Preparation of Propolis
A sample of propolis (50 g) was collected in May 2022 from one of the beehives of Yuksel Gulcin, a farmer located in Berdav, a village in Tutak in the district of Agri, and stored before processing. The extraction process was completed as previously mentioned [2,88]. For preparation of propolis, a 25 g sample was milled into a fine powder and combined with ethanol. The prepared extract was filtered using Whatman No.1 paper and the filtrate was collected before removing the ethanol using a rotary evaporator (RE 100 Bibby, Stone Staffordshire, England) at 50 • C to obtain a dry extract. The yield of propolis was calculated as 75% and 18.75 g extract was placed in a dark plastic bottle and kept at −20 • C until use.

Determination of Total Soluble Phenolic Contents of Propolis
Total phenol content was used to determine the amount of phenolic compound present in propolis as gallic acid equivalents. [89]. The procedure was based on that described by Singleton and Rossi [90], with slight modifications [91]. Propolis extract (0.5 mL) was added to 1.0 mL of Folin-Ciocalteu reagent [92] as described in detail in [93]. Afterward, carbonate (0.5 mL, 1%) was added and the mixture was stirred vigorously. Absorbance was measured at 760 nm against a water-containing blank sample after 2 h of incubation in the dark at room temperature. The quantity of phenol in one gram of propolis extract was calculated as mg of gallic acid equivalents (GAE).

Determination of Tatal Flavonoid Content of Propolis
The total flavonoid content was determined using the aluminum chloride (AlCl 3 ) technique [94]. Briefly, 0.5 mL of the propolis extraction solution was combined with 1.5 mL of 95% methanol, 1.5 mL of 10% AlCl 3 , 0.5 mL of 1.0 M potassium acetate solution, and 2.3 mL of distilled deionized water. The absorbance was measured at 415 nm after incubation in the dark (25 • C, 40 min). Water was used as the blank sample. The total amount of flavonoids was calculated as mg quercetin equivalents (QE)/g of propolis extract [95].

Ferric Ions (Fe 3+ ) Reducing Assay
The direct reduction of Fe 3+ (CN -) 6 and the absorbance resulting from the formation of the Perl's Prussian Blue complex upon the addition of excess ferric ions (Fe 3+ ) were used to the test ferric-reducing antioxidant capacity. Thus, the FRAP method was employed to assess the lowering capacity of propolis [97]. The reduction of (Fe 3+ ) ferricyanide in stoichiometric excess relative to the antioxidants is the basis for this approach. In 0.75 mL of distilled water, different doses of propolis (10-30 µg/mL) were combined with 1.25 mL of 0.2 M, pH 6.6 sodium phosphate buffer and 1.25 mL of potassium ferricyanide [K 3 Fe(CN) 6 ] (1%). For 30 min, the mixture was incubated at 50 • C. After 30 min, the reaction mixture was acidified with 1 mL 10% trichloroacetic acid and incubated in the dark for 30 min. Finally, 0.25 mL of FeCl 3 (0.1%) was added to the solution, and the absorbance at 700 nm was measured. Increased absorbance of the reaction mixture implies a greater reduction capacity [98].

Cupric Ions (Cu 2+ ) Reducing-CUPRAC Assay
To determine the Cu 2+ -reducing antioxidant capacity of propolis, the method proposed by Apak et al. [58] was used with slight modifications. Briefly, 0.25 mL CuCl 2 solution (0.01 M), 0.25 mL ethanolic neocuproine solution (7.5 × 10 −3 M) and 0.25 mL CH 3 COONH 4 buffer solution (1.0 M) were added to a test tube, which was then mixed with various concentrations of propolis (10-30 µg/mL). The whole volume was then reduced to 2 mL by adding distilled water and vigorously mixing. The tubes were sealed and stored at room temperature. After 30 min, absorbance was measured at 450 nm against a reagent blank. The increased absorbance of the reaction mixture suggests an increased reduction capacity [99].

Fe 3+ -TPTZ Reducing-FRAP Assay
FRAP is based on decreasing Fe 3+ -TPTZ in acidic media [100]. The increased absorbance of the blue color of the ferrous form of the complex (Fe 2+ -TPTZ) was measured spectrophotometrically at 593 nm [101]. Briefly, 2.25 mL of the newly created TPTZ solution (10 mM) in HCl (40 mM) was poured into 2.5 mL of acetate buffer (pH 3.6, 0.3 M) and FeCl 3 solution in water (2.25 mL, 20 mM). Then, propolis (10-50 µg/mL) was dissolved in a buffer solution (5 mL) and the mixture was incubated in the dark at 37 • C for 30 min. Finally, the absorbance of each sample was measured.

DPPH • Scavenging Activity
The DPPH • scavenging activity of propolis was assessed using the DPPH • scavenging method [102]. The DPPH solution was prepared the day before measurement. The solution flask was coated with aluminum foil, stirred for 16 h and kept in the dark at 4 • C. Briefly, a 0.1 mM DPPH solution was prepared in ethanol, and 0.5 mL of this solution was added to 2 mL of propolis sample solution in ethanol at different concentrations (10-30 µg/mL). The propolis samples were vortexed and incubated at 30 • C in the dark for 30 min. Absorbance was measured at 517 nm against blank samples. A decrease in absorbance indicated DPPH free radical scavenging activity. The decrease in absorbance indicated that DPPH actively scavenges free radicals.

ABTS •+ Scavenging Activity
The ABTS •+ radical scavenging activity of propolis was determined using a previous method [103]. ABTS •+ has a blue-green color and a distinctive absorbance at 734 nm. The reaction of ABTS (2 mM) in water and potassium persulfate (2.45 mM) at room temperature, which was vortexed for 30 min in a flask coated with aluminum foil, led to the production of the ABTS •+ cation radical. At 734 nm, an absorbance of 0.800 ± 0.05 was obtained by diluting the ABTS •+ solution with phosphate buffer (0.1 M, pH 7.4). Then, 0.25 mL of the ABTS •+ solution was added to 1.75 mL of the sample solution in ethanol at various propolis concentrations (10-30 µg/mL). The propolis sample was vortexed and left in the dark for 30 min. After 30 min, the absorbance at 734 nm for each concentration was measured and compared with that of the blank. The decreased absorbance of the sample suggests ABTS •+ cation radical scavenging activity [104].

DMPD •+ Scavenging Activity
The radical scavenging capacity of propolis against DMPD was measured using the method described by Fogliano et al. [105]. This assessment is based on the ability of the extract to suppress the production of DMPD •+ cation radicals. Briefly, 105 mg was added to 5 mL DMPD •+ solution. Then, 1 mL of this solution was added to 100 mL acetate buffer (pH 5.3, 0.1 M), and agitated for 5 min in the dark. Ferric chloride (0.2 mL, 0.05 M) was added to this solution to form DMPD •+ . Standard antioxidants or propolis at varying doses (10-30 µg/mL) were added and the total volume was adjusted with distilled water (0.5 mL). The DMPD •+ solution (1 mL) was immediately added to the reaction mixture, which was thoroughly mixed and incubated for 50 min in the dark. The absorbance was measured at 505 nm [106].

Percentage Scavenging and IC 50 Determination
The antioxidant (DPPH • , ABTS •+ , and DMPD •+ ) scavenging potentials were calculated by comparing them to the typical antioxidant substances (BHA, BHT, Trolox, and α-tocopherol). The dose-dependent antioxidant potential of propolis was investigated using various concentrations (10-30 µg/mL) of the sample and reference standards. The propolis concentrations that caused 50% enzyme inhibition (IC 50 ) values were calculated from the activity (%) versus propolis plots. First, enzyme inhibition was studied at several propolis concentrations. The obtained values were plotted as % activity against propolis concentrations. The IC 50 values were calculated from these graphs [107].

AChE Enzyme Inhibition Assay
The AChE inhibitory action of propolis was assessed as previously described by Ellman et al. [108]. AChE activity was measured spectrophotometrically at 412 nm using acetylthiocholine iodide as a substrate for the enzymatic reaction, and AChE activity was measured using 5,5 -dithio-bis (2-nitro-benzoic) acid.

α-Glycosidase Enzyme Inhibition Assay
The inhibitory effect of these substances on α-glycosidase enzyme activity were tested using a p-nitrophenyl-D-glycopyranoside (p-NPG) substrate [50]. First, 40 µL of the sample solution was mixed with 200 µL phosphate buffer (0.15 EU/mL, pH 7.4). Furthermore, after preincubation, 50 µL p-NPG in phosphate buffer (5 mM, pH 7.4) was added and incubated again at 30 • C. Absorbance was measured spectrophotometrically at 405 nm according to a previous study.

hCA II Isoenzyme Inhibition Assay
The method established by Verpoorte et al. [109] previously described the inhibition of both hCA isoenzymes. The dominant cytosolic CA II isoenzyme was isolated from human erythrocytes using affinity column chromatography with Sepharose-4B-Tyrosinesulfanylamide [110]. After loading the material into the affinity chromatography column, it was equilibrated with Tris-Na 2 SO 4 /HCl (pH 8.7, 22 mM/25 mM). CA II was eluted with 0.5 M sodium acetate/NaClO 4 (pH 5.6, 25 • C). The differences in absorbance were measured over 3 min at 348 nm using p-nitrophenylacetate (PNA) as a substrate, which was transformed into the p-nitrophenolate ion by both isoenzymes. One enzyme unit of CA esterase activity was defined as the hydrolysis of 1 mol PNA in 1 min to p-nitrophenolate and acetate. The Bradford assay was used to quantify the amount of protein throughout the purification process. As a reference protein, bovine serum albumin was employed. SDS-PAGE was used to control the purity of the CA II isoform [111].

Statistical Analysis
All measurements were performed in triplicate for each sample. Data are presented as means (n = 3) and evaluated using one-way ANOVA followed by Tukey's post hoc test; p < 0.001 was considered statistically significant.

Conclusions
Propolis, which is vital for bees themselves, their offspring and their hives, has a rich natural content obtained from different parts of the surrounding plants. This product, which it believed to be rich, nutritious and contributes to the formation of honey, has been used by mankind for thousands of years. According to the LC-MS/MS analysis, the major components detected in propolis are acacetin, caffeic acid, p-coumaric acid, naringenin, chrysin, quinic acid, quercetin, ferulic acid, apigenin, luteolin, kaempferol, hesperetin, vanillic acid, and protocatechuic acid. Furthermore, the propolis ethanol extract had increased antioxidant activity, reducing power, and phenolic contents, and inhibited AChE, α-glycosidase, and hCA II. Propolis can be used as a natural product in the treatment of serious and common T2DM, AD and glaucoma diseases, neurodegenerative, hormonal, and metabolic diseases, as well as in the food and pharmaceutical industries, owing to its phenolic and flavonoid contents, which have antioxidant, reducing and radical scavenging capacities.

Conflicts of Interest:
The authors declare no conflict of interest.