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Article

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

by
Hasan Karagecili
1,*,
Mustafa Abdullah Yılmaz
2,
Adem Ertürk
3,4,
Hatice Kiziltas
5,
Leyla Güven
6,
Saleh H. Alwasel
7 and
İlhami Gulcin
4,*
1
Department of Nursing, Faculty of Health Sciences, Siirt University, Siirt 56100, Turkey
2
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Dicle University, Diyarbakır 21280, Turkey
3
Department of Pharmacy Services, Hınıs Vocational School, Ataturk University, Erzurum 25600, Turkey
4
Department of Chemistry, Faculty of Science, Ataturk University, Erzurum 25240, Turkey
5
Department of Pharmacy Services, Vocational School of Health Services, Van Yuzuncu Yil University, Van 65080, Turkey
6
Department of Pharmaceutical Botany, Faculty of Pharmacy, Ataturk University, Erzurum 25240, Turkey
7
Department of Zoology, College of Science, King Saud University, Riyadh 11362, Saudi Arabia
*
Authors to whom correspondence should be addressed.
Molecules 2023, 28(4), 1739; https://doi.org/10.3390/molecules28041739
Submission received: 5 January 2023 / Revised: 7 February 2023 / Accepted: 9 February 2023 / Published: 11 February 2023
(This article belongs to the Special Issue Extraction, Separation and Identification of Compounds from Natural Sources)
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Abstract

:
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.

1. 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, which is influenced by several variables, including the area’s floristic makeup, location, and collection time [3]. Owing to the existence of phenolic, terpenoid and alkaloids components, propolis gathered from honeybees exhibits bioactive, antibacterial, antiviral, anesthetic, antiseptic and antioxidant characteristics [4]. Overall, this complex combination is composed of approximately 50% resins, 30% wax, 10% essential oils, 5% pollen, and 5% other substances and materials, including organic compounds [5,6]. Propolis is a naturally occurring bee resin that has been used in folk remedies for many years. It was first mentioned in ancient Egyptian history on the Eberly papyrus, was used to clean the umbilical cord of newborns during the Middle Ages, and used as an antibiotic or antiseptic during World War II. It is now a natural product that is consumed worldwide and has historically been used for systemic and oral disorders as an anti-inflammatory, antibacterial, and antifungal treatment, among other ailments [7].
Propolis also has significant concentrations of minerals including copper, iron, calcium, zinc, cobalt, and potassium as well as vitamins such as B, E, C, and A [8]. Many foods use propolis, a natural substance of plant origin, as a natural preservative. For instance, it may successfully prevent oxidation and alter the quality criteria when applied to traditional Turkish sausage and dairy drinks [9]. Moreover, propolis is a non-toxic product. Approximately 70 mg per day (1.4 mg/kg body weight per day) would be a healthy quantity for humans [10]. Propolis has been increasingly utilized in the food and cosmetic industries, in addition to being used for home treatments [11].
Reactive oxygen species (ROS) are produced during oxidative stress and endogenous antioxidants in the body are unable to limit their formation [12,13]. An imbalance between endogenous antioxidant systems and ROS increases the risk of oxidative stress in the body, which can cause several diseases. ROS may serve a triggering function in the pathogenesis of inflammation, even though the inflammatory route is complex. Therefore, preventing ROS generation may be a crucial step in reducing inflammation [14]. Owing to its radical scavenging action, propolis has become a viable source of natural products in recent years [15,16]. Oxidative deterioration of lipids, proteins, and nucleic acids in living cells is enhanced by ROS. It is generally recognized that ROS, often known as free radicals, are major contributors to the onset of many serious medical conditions, including cancer, heart disease, and aging [17,18,19]. In living organisms, antioxidants delay thwart, and suppress the oxidation of lipids, carbohydrates, nucleic acids, and proteins. They contain phenols and polyphenols, which are powerful substances that reduce or eliminate the harmful effects of ROS [20]. Furthermore, antioxidants have frequently been employed as dietary supplements to protect foods from oxidative deterioration [21]. Antioxidants can halt the progression of many chronic illnesses and lipid peroxidation, shielding the human body from the damaging effects of free radicals and ROS. As a result, there is much interest in natural additives as substitutes for the most widely used antioxidants, including propyl gallate, BHA, BHT, and tert-butylhydroquinone. Liver damage and cancer development have been linked to BHA and BHT [22]. As a result, there is increasing interest in natural antioxidants and their use in culinary applications is growing [23,24].
The α-glycosidase enzyme, whose activity is critical for the breakdown of dietary polysaccharides, is one of the key targets in the treatment of diabetes [25]. α-Glycosidase inhibitors (AGIs) prevent the absorption of monomeric sugar units in the intestine; this reduces the level of postprandial plasma glucose levels. Therefore, such inhibitors can be used to treat diabetes and obesity [26,27]. The age-related neurodegenerative AD condition, which is sometimes referred to as type 3 diabetes owing to its pathophysiological resemblance to type 2 diabetes mellitus (T2DM), is triggered by both inflammatory diseases and T2DM [28].
AD is a neurological condition that progresses abruptly and causes behavioral changes, forgetfulness, cognitive impairment, and deficiencies in language. Acetylcholine (ACh) is broken down by acetylcholinesterase (AChE) into acetate (CH3COO–) and choline (Ch) [29,30]. Furthermore, the use of AChE inhibitors (AChEIs) to prevent the cholinergic breakdown of ACh is a prospective strategy for treating AD [31]. Newer, efficient 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 Zn2+ and catalyze the reversible hydration of carbon dioxide (CO2) to protons and bicarbonate (HCO3−) [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.

2. Results

2.1. 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.
The major components detected in propolis were the flavone acacetin (76.359 mg/g), caffeic acid (21.358 mg/g), p-coumaric acid (16.911 mg/g), naringenin (11.34 mg/g), chrysin (9.86 mg/g), quinic acid (7.285 mg/g), quercetin (6.223 mg/g), ferulic acid (5.11 mg/g), apigenin (4.686 mg/g), luteolin (4.394 mg/g), kaempferol (4.043 mg/g), hesperidin (2.089 mg/g), vanillic acid (1.647 mg/g) and protocatechuic acid (1.158 mg/g) (Table 2).

2.2. Reducing Abilities Results

The ferric ion (Fe3+) reductive abilities of the isolated phenolic compounds from propolis were determined according to the Oyaizu method [41]. The addition of Fe3+ to the compound causes Fe4[Fe(CN–)6]3 complex formation, which results in maximum absorption at 700 nm [42,43,44]. As summarized in Table 3, the propolis extract exhibited a potent Fe3+ reducing profile. However, the Fe3+-reducing ability of a concentration of 30 μg/mL of propolis and standards decreased in the following order: BHT (2.018, r2: 0.9466) > α-tocopherol (1.895, r2: 0.9402) > Trolox (1.545, r2: 0.9966) > BHA (1.257, r2: 0.9523) > Propolis (0.894, r2: 0.9953). All analyses were performed in triplicate.
The cupric ion (Cu2+)-reducing abilities of the phenolic composition in propolis are presented in Table 3. There was a strong relationship between the Cu2+-reducing effect and different concentrations of propolis. However, at 30 µg/mL, significant absorbance of reducing power was demonstrated by the propolis. In contrast, the Cu2+-reducing ability of propolis and the standards were found to be BHT (2.912, r2: 0.9969) > Trolox (2.323, r2: 0.9980) > BHA (1.800, r2: 0.9742) > α-Tocopherol (1.139, r2: 0.9967) > Propolis (0.778, r2: 0.9986). The CUPRAC antioxidant method is convenient, selective, inexpensive, fast, and stable [45]. According to the results provided in Table 3, the following was found: BHT (2.089, r2: 0.9581) > α-Tocopherol (1.995, r2: 0.9807) > Trolox (1.755, r2: 0.9990) > Propolis (1.114, r2: 0.9970) > BHA (0.884, r2: 0.9899) (Table 3). The higher the absorbance readings in this approach, the greater the reducing ability of the test samples.

2.3. Radical Scavenging Results

The DPPH• scavenging activity of propolis was measured, and the half maximal inhibition concentration (IC50) 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 IC50 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 IC50 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 IC50 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).

2.4. 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. IC50 values were 19.6 μg/mL (r2: 0.9327) for propolis towards CA II. For acetazolamide, the IC50 was 8.37 μg/mL (r2: 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 IC50 values of the propolis extract for enzyme inhibition. The IC50 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 IC50 of tacrine was 5.97 µg/mL (r2: 0.9706), which was utilized as a control for the AChE inhibition experiment (Figure 2B).
Propolis extracts displayed an IC50 value of 3.7 μg/mL towards α-glycosidase (r2: 0.9362, Table 5 and Figure 2C). The results reveal that propolis has inhibitory effects similar to those of α-glycosidase efficient acarbose (IC50: 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 (IC50: 90.99–876.24 μg/mL) of [51].

3. 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 Fe3+, CUPRAC, and Fe3+-TPTZ ion-reducing 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, Fe2+ 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 Fe3+-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) Fe3+-reducing and cupric ion (Cu2+)-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/K2S2O8 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 IC50 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 (IC50: 14.29 μg/mL) [2]. A lower IC50 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•+ (IC50: 18.32 μg/mL) and superoxide anion (O2•-, IC50: 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 IC50 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 IC50 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 IC50 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 HCO3− 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 (IC50: 1.273 μg/mL) and Giresun propolis (IC50: 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 IC50 values of 0.486 and 0.612 μg/mL [62].

4. Materials and Methods

4.1. Chemicals

Acetylcholinesterase, acetylcholine iodide, α-glycosidase, p-nitrophenyl-D-glycopyranoside, 2,2′-azino-bis 3-ethylbenzthiazoline-6-sulfonic acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), N,N-dimethyl-p-phenylenediamine (DMPD), 2,9-dimethyl-1,10-phenanthroline (Neocuproine), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), α-tocopherol, trolox, and standard phenolic compounds of LC-MS/MS were purchased from Sigma (Sigma-Aldrich GmbH, Steinheim, Germany). The other materials were procured from Sigma-Aldrich or Merck (Darmstadt, Germany), appropriately. Propolis was dissolved in ethanol for antioxidant activities, but in DMSO for enzyme inhibition tests due to the potential inhibitory effects of ethanol.

4.2. 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.

4.3. 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).

4.4. Determination of Tatal Flavonoid Content of Propolis

The total flavonoid content was determined using the aluminum chloride (AlCl3) 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% AlCl3, 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].

4.5. Test Solution for Mass Spectrometer (LC–MS/MS) and Chromatography Conditions

The analytical approach used in this investigation was conducted in accordance with recent research. The Dicle University Central Research Laboratory conducted the LC-MS/MS experiments. The analytical approach employed in this study was designed by Yılmaz [96] and adapted for propolis. To investigate the phytochemical component in propolis, the following 53 phytochemical standards were purchased from Sigma-Aldrich (Steinheim, Germany): quinic acid, fumaric acid, gallic acid, malic acid, aconitic acid, epigallocatechin, proto-catechuic acid, catechin, gentisic acid, chlorogenic acid, pro-tocatechuic aldehyde, tannic acid, epigallocatechin gallate, 4-OH benzoic acid, epicatechin, vanillic acid, caffeic acid, syringic acid, vanillin, syringic aldehyde, daidzin, epicatechin gallate, piceid, ferulic acid, p-coumaric acid, sinapic acid, coumarin, salicylic acid, cynaroside, cinnamic acid, rutin, isoquercitrin, hesperidin, o-coumaric acid, genistin, rosmarinic acid, ellagic acid, cosmosiin, quercitrin, astragalin, nicotiflorin, fisetin, cynarine, daidzein, quercetin, naringenin, luteolin, hesperetin, genistein, apigenin, kaempferol, amentoflavone and chrysin, which were used to investigate the thephytochemical component in propolis. 1,5-Dicaffeoylquinic acid and the internal standards ferulicacid-D3, rutin-D3, and quercetin-D3 were purchased from TRC (Toronto, Canada). A Shimadzu–Nexera model ultrahigh performance liquid chromatograph (UHPLC) (Shimadzu, Japan) connected to a tandem mass spectrometer was used to quantify the 53 phytochemicals. An autosampler (SIL-30AC model), column oven (CTO-10ASvp type), binary pumps (LC-30AD model), and degasser were installed on the reversed-phase UHPLC (DGU-20A3R model). The chromatographic settings were tuned to provide the optimal separation of the 53 phytochemicals while overcoming the suppression effects. The following parameters were tested and applied until the optimum conditions were achieved: different columns such as the Agilent Poroshell 120 EC-C18 model (150 mm × 2.1 mm, 2.7 µm) and RP-C18 Inertsil ODS-4 (100 mm × 2.1 mm, 2 µm), different mobile phases (B) such as acetonitrile and methanol, different mobile phase additives such as ammonium format, formic acid, ammonium acetate, and acetic acid, and different column temperatures (25, 30, 35, and 40 °C). Consequently, chromatographic separation was performed on a reversed-phase Agilent Poroshell 120 EC-C18 model analytical column. The column temperature was set to 40 °C. The elution gradient comprised eluent A (water + 5 mM ammonium formate + 0.1% formic acid) and eluent B (methanol + 5 mM ammonium formate + 0.1% formic acid). The following gradient elution profiles were used: 20–100% B (0–25 min), 100% B (25–35 min), and 20% B (35–45 min). The solvent flow rate and injection volume were settled to 0.5 mL/min and 5 µL, respectively. Mass spectrometric detection was performed using a Shimadzu LCMS-8040 model tandem mass spectrometer equipped with an electrospray ionization (ESI) source operating in both negative and positive ionization modes. LC-ESI-MS/MS data were acquired and processed using LabSolutions software (Shimadzu). Multiple reaction monitoring (MRM) mode was used for the quantification of phytochemicals. The MRM method was optimized to selectively detect and quantify phytochemical compounds based on screening of specified precursor phytochemical-to-fragment ion transitions. The collision energies (CE) were optimized to generate optimal photochemical fragmentation and maximal transmission of the desired product ions. The MS operating conditions were applied as follows: drying gas (N2) flow, 15 L/min; nebulizing gas (N2) flow, 3 L/min; DL temperature, 250 °C; heat block temperature, 400 °C; and interface temperature, 350 °C.

4.6. Ferric Ions (Fe3+) Reducing Assay

The direct reduction of Fe3+(CN—)6 and the absorbance resulting from the formation of the Perl’s Prussian Blue complex upon the addition of excess ferric ions (Fe3+) 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 (Fe3+) 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 [K3Fe(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 FeCl3 (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].

4.7. Cupric Ions (Cu2+) Reducing—CUPRAC Assay

To determine the Cu2+-reducing antioxidant capacity of propolis, the method proposed by Apak et al. [58] was used with slight modifications. Briefly, 0.25 mL CuCl2 solution (0.01 M), 0.25 mL ethanolic neocuproine solution (7.5 × 10−3 M) and 0.25 mL CH3COONH4 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].

4.8. Fe3+-TPTZ Reducing—FRAP Assay

FRAP is based on decreasing Fe3+-TPTZ in acidic media [100]. The increased absorbance of the blue color of the ferrous form of the complex (Fe2+-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 FeCl3 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.

4.9. 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.

4.10. 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].

4.11. 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].

4.12. Percentage Scavenging and IC50 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 (IC50) 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 IC50 values were calculated from these graphs [107].

4.13. 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.

4.14. 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.

4.15. 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-Tyrosine-sulfanylamide [110]. After loading the material into the affinity chromatography column, it was equilibrated with Tris-Na2SO4/HCl (pH 8.7, 22 mM/25 mM). CA II was eluted with 0.5 M sodium acetate/NaClO4 (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].

4.16. 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.

5. 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.

Author Contributions

Methodology and investigation, H.K. (Hasan Karagecili), M.A.Y., A.E., H.K. (Hatice Kiziltas), L.G.; writing—original draft preparation, review and editing, supervision, funding and acquisition, S.H.A., H.K. (Hasan Karagecili) and İ.G. All authors have read and agreed to the published version of the manuscript.

Funding

İlhami Gulcin is a member of the Turkish Academy of Sciences (TÜBA). He wants to express his heartfelt gratitude to the TÜBA for their financial assistance. In addition, Saleh Alwasel wishes to express his heartfelt gratitude to King Saud University’s Researchers Supporting Project (RSP-2023/59).

Data Availability Statement

Data are provided in a publicly accessible repository.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Shehata, M.G.; Ahmad, F.T.; Badr, A.N.; Masry, S.H.; El-Sohaimy, S.A. Chemical analysis, antioxidant, cytotoxic and antimicrobial properties of propolis from different geographic regions. Ann. Agric. Sci. 2020, 65, 209–217. [Google Scholar] [CrossRef]
  2. Gulcin, I.; Bursal, E.; Sehitoglu, H.M.; Bilsel, M.; Goren, A.C. Polyphenol contents and antioxidant activity of lyophilized aqueous extract of propolis from Erzurum, Turkey. Food Chem. Toxicol. 2010, 48, 2227–2238. [Google Scholar] [CrossRef]
  3. Kasote, D.; Suleman, T.; Chen, W.; Sandasi, M.; Viljoen, A.; van Vuuren, S. Chemical profiling and chemometric analysis of South African propolis. Biochem. System. Ecol. 2014, 55, 156–163. [Google Scholar] [CrossRef]
  4. Bilginer, R.; Yildiz, A.A. A facile method to fabricate propolis enriched biomimetic PVA architectures by co-electrospinning. Mater. Lett. 2020, 276, 128191. [Google Scholar] [CrossRef]
  5. Freitas, A.S.; Cunha, A.; Cardoso, S.M.; Oliveira, R.; Almeida-Aguiar, C. Constancy of the bioactivities of propolis samples collected on the same apiary over four years. Food Res. Int. 2019, 119, 622–633. [Google Scholar] [CrossRef]
  6. Garzarella, E.U.; Navajas-Porras, B.; Perez-Burillo, S.; Ullah, H.; Esposito, C.; Santarcangelo, C.; Hinojosa-Nogueira, D.; Pastoriza, S.; Zaccaria, V.; Xiao, J.; et al. Evaluating the effects of a standardized polyphenol mixture extracted from poplar-type propolis on healthy and diseased human gut microbiota. Biomed. Pharmacother. 2022, 148, 112759. [Google Scholar] [CrossRef]
  7. Santos, L.A.; Rosalen, P.L.; Dias, N.A.; Grisolia, J.C.; Gomes, B.J.N.; Blosfeld-Lopes, L.; Ikegaki, M.; de Alencar, S.M.; Burger, E. Brazilian red propolis shows antifungal and immunomodulatory activities against Paracoccidioides brasiliensis. J. Ethnopharmacol. 2021, 277, 114181. [Google Scholar] [CrossRef]
  8. Abdellatif, M.M.; Elakkad, Y.E.; Elwakeel, A.A.; Allam, R.M.; Mousa, M.R. Formulation and characterization of propolis and tea tree oil nanoemulsion loaded with clindamycin hydrochloride for wound healing: In-vitro and in-vivo wound healing assessment. Saudi Pharm. J. 2021, 29, 1238–1249. [Google Scholar] [CrossRef] [PubMed]
  9. Liao, N.; Sun, L.; Wang, D.; Chen, L.; Wang, J.; Qi, X.; Zhang, H.; Tang, M.; Wu, G.; Chen, J.; et al. Antiviral properties of propolis ethanol extract against norovirus and its application in fresh juices. LWT 2021, 152, 112169. [Google Scholar] [CrossRef]
  10. Burdock, G.A. Review of the biological properties and toxicity of bee propolis (propolis). Food Chem. Toxicol. 1998, 36, 347–363. [Google Scholar] [CrossRef]
  11. Surek, M.; Fachi, M.M.; de Fátima Cobre, A.; de Oliveir, F.F.; Pontarolo, R.; Crisma, A.R.; de Souza, W.M.; Felipe, K.B. Chemical composition, cytotoxicity, and antibacterial activity of propolis from Africanized honeybees and three different Meliponini species. J. Ethnopharmacol. 2021, 269, 113662. [Google Scholar] [CrossRef] [PubMed]
  12. Gulcin, I. Antioxidants and antioxidant methods-An updated overview. Arch. Toxicol. 2020, 94, 651–715. [Google Scholar] [CrossRef] [PubMed]
  13. Durmaz, L.; Erturk, A.; Akyuz, M.; Polat Kose, L.; Uc, E.M.; Bingol, Z.; Saglamtas, R.; Alwasel, S.; Gulcin, I. Screening of carbonic anhydrase, acetylcholinesterase, butyrylcholinesterase and α-glycosidase enzymes inhibition effects and antioxidant activity of coumestrol. Molecules 2022, 27, 3091. [Google Scholar] [CrossRef]
  14. Gulcin, I. Antioxidant activity of food constituents: An overview. Arch. Toxicol. 2012, 86, 345–391. [Google Scholar] [CrossRef]
  15. Bursal, E.; Taslimi, P.; Goren, A.; Gulcin, I. Assessments of anticholinergic, antidiabetic, antioxidant activities and phenolic content of Stachys annua. Biocat. Agric. Biotechnol. 2020, 28, 101711. [Google Scholar] [CrossRef]
  16. Guzelmeric, E.; Yuksel, P.I.; Yaman, B.K.; Sipahi, H.; Celik, C.; Kırmızıbekmez, H.; Aydın, A.; Yesilada, E. Comparison of antioxidant and anti-inflammatory activity profiles of various chemically characterized Turkish propolis sub-types: Which propolis type is a promising source for pharmaceutical product development? J. Pharm. Biomed. Anal. 2021, 203, 114196. [Google Scholar] [CrossRef]
  17. Polat Kose, L.; Gulcin, I. Evaluation of the antioxidant and antiradical properties of some phyto and mammalian lignans. Molecules 2021, 26, 7099. [Google Scholar] [CrossRef] [PubMed]
  18. Taslimi, P.; Koksal, E.; Goren, A.C.; Bursal, E.; Aras, A.; Kılıc, O.; Alwasel, S.; Gulcin, I. Anti-Alzheimer, antidiabetic and antioxidant potential of Satureja cuneifolia and analysis of its phenolic contents by LC-MS/MS. Arab. J. Chem. 2020, 13, 4528–4537. [Google Scholar] [CrossRef]
  19. Kiziltas, H.; Bingol, Z.; Goren, A.C.; Pınar, S.M.; Alwasel, S.H.; Gulcin, I. LC-HRMS profiling of phytochemicals, antidiabetic, anticholinergic and antioxidant activities of evaporated ethanol extract of Astragalus brachycalyx FISCHER. J. Chem. Metrol. 2021, 15, 135–151. [Google Scholar]
  20. Bingol, Z.; Kızıltas, H.; Gören, A.C.; Polat Kose, L.; Topal, M.; Durmaz, L.; Alwasel, S.H.; Gulcin, I. Antidiabetic, anticholinergic and antioxidant activities of aerial parts of shaggy bindweed (Convulvulus betonicifolia Miller subsp.)-profiling of phenolic compounds by LC-HRMS. Heliyon 2021, 7, e06986. [Google Scholar] [CrossRef]
  21. Oztaskin, N.; Kaya, R.; Maras, A.; Sahin, E.; Gulcin, I.; Goksu, S. Synthesis and characterization of novel bromophenols: Determination of their anticholinergic, antidiabetic and antioxidant activities. Bioorg. Chem. 2019, 87, 91–102. [Google Scholar] [CrossRef]
  22. Eruygur, N.; Atas, M.; Tekin, M.; Taslimi, P.; Kocyigit, U.M.; Gulcin, I. In vitro antioxidant, antimicrobial, anticholinesterase and antidiabetic activities of Turkish endemic Achillea cucullata (Asteraceae) from ethanol extract. S. Afr. J. Bot. 2019, 120, 141–145. [Google Scholar] [CrossRef]
  23. Gulcin, I.; Buyukokuroglu, M.E.; Kufrevioglu, O.I. Metal chelating and hydrogen peroxide scavenging effects of melatonin. J. Pineal Res. 2003, 34, 278–281. [Google Scholar] [CrossRef] [PubMed]
  24. Maharramova, G.; Taslimi, P.; Sujayev, A.; Farzaliyev, F.; Durmaz, L.; Gulcin, I. Synthesis, characterization, antioxidant, antidiabetic, anticholinergic, and antiepileptic properties of novel N-substituted tetrahydropyrimidines based on phenylthiourea. J. Biochem. Mol. Toxicol. 2018, 32, e22221. [Google Scholar] [CrossRef]
  25. Takım, K.; Yigin, A.; Koyuncu, I.; Kaya, R.; Gulcin, I. Anticancer, anticholinesterase and antidiabetic activities of Tunceli garlic (Allium tuncelianum)-Determining its phytochemical content by LC-MS/MS analysis. J. Food Meas. Charact. 2021, 15, 3323–3335. [Google Scholar] [CrossRef]
  26. Taslimi, P.; Aslan, H.E.; Demir, Y.; Oztaskın, N.; Maras, A.; Gulcin, I.; Beydemir, S.; Goksu, S. Diarilmethanon, bromophenols and diarilmetan compounds: Discovery of potent aldose reductase, α-amylase and α-glycosidase inhibitors as new therapeutic approach in diabetes and functional hyperglycemia. Int. J. Biol. Macromol. 2018, 119, 857–863. [Google Scholar] [CrossRef]
  27. Gulcin, I.; Taslimi, P.; Aygun, A.; Sadeghian, N.; Bastem, E.; Kufrevioglu, O.I.; Turkan, F.; Sen, F. Antidiabetic and antiparasitic potentials: Inhibition effects of some natural antioxidant compounds on α-glycosidase, α-amylase and human glutathione S-transferase enzymes. Int. J. Biol. Macromol. 2018, 119, 741–746. [Google Scholar] [CrossRef] [PubMed]
  28. Burmaoglu, S.; Yılmaz, A.O.; Polat, M.F.; Kaya, R.; Gulcin, I.; Algul, O. Synthesis and biological evaluation of novel tris-chalcones as potent carbonic anhydrase, acetylcholinesterase, butyrylcholinesterase, and α-glycosidase inhibitors. Bioorg. Chem. 2019, 85, 191–197. [Google Scholar] [CrossRef]
  29. Cetin Cakmak, K.; Gulcin, I. Anticholinergic and antioxidant activities of USNIC acid-An activity-structure insight. Toxicol. Rep. 2019, 6, 1273–1280. [Google Scholar] [CrossRef]
  30. Oztaskin, N.; Taslimi, P.; Maras, A.; Goksu, S.; Gulcin, I. Novel antioxidant bromophenols with acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase inhibitory actions. Bioorg. Chem. 2017, 74, 104–114. [Google Scholar] [CrossRef]
  31. Burmaoglu, S.; Kazancioglu, E.A.; Kazancioglu, M.Z.; Saglamtas, R.; Yalçın, G.; Gulcin, I.; Algul, O. Synthesis, molecular docking and some metabolic enzyme inhibition properties of biphenyl-substituted chalcone derivatives. J. Mol. Struct. 2022, 1254, 132358. [Google Scholar] [CrossRef]
  32. Kaya, Y.; Ercag, A.; Zorlu, Y.; Demir, Y.; Gulcin, I. New Pd(II) complexes of the bisthiocarbohydrazones derived from isatin and disubstituted salicylaldehydes: Synthesis, characterization, crystal structures and inhibitory properties against some metabolic enzymes. J. Biol. Inorg. Chem. 2022, 27, 271–281. [Google Scholar] [CrossRef] [PubMed]
  33. Taslimi, P.; Caglayan, C.; Gulcin, I. The impact of some natural phenolic compounds on carbonic anhydrase, acetylcholinesterase, butyrylcholinesterase, and α-glycosidase enzymes: An antidiabetic, anticholinergic, and antiepileptic study. J. Biochem. Mol. Toxicol. 2017, 31, e21995. [Google Scholar] [CrossRef] [PubMed]
  34. Kızıltas, H.; Bingol, Z.; Goren, A.C.; Alwasel, S.H.; Gulcin, I. Anticholinergic, antidiabetic and antioxidant activities of Ferula orientalis L.-Analysis of its polyphenol contents by LC-HRMS. Rec. Nat. Prod. 2021, 15, 513–528. [Google Scholar] [CrossRef]
  35. Kose, L.P.; Gulcin, I. Inhibition effects of some lignans on carbonic anhydrase, acetylcholinesterase and butyrylcholinesterase enzymes. Rec. Nat. Prod. 2017, 11, 558–561. [Google Scholar] [CrossRef]
  36. Zahedi, N.A.; Mohammadi-Khanaposhtani, M.; Rezaei, P.; Askarzadeh, M.; Alikhani, M.; Adib, M.; Mahdavi, M.; Larijani, B.; Niakan, S.; Tehrani, M.B.; et al. Dual functional cholinesterase and carbonic anhydrase inhibitors for the treatment of Alzheimer’s disease: Design, synthesis, in vitro, and in silico evaluations of coumarin-dihydropyridine derivatives. J. Mol. Struct. 2023, 1276, 134767. [Google Scholar] [CrossRef]
  37. Yiğit, B.; Taslimi, P.; Celepci, D.B.; Taskin-Tok, T.; Yiğit, M.; Aygün, M.; Ozdemir, I.; Gulcin, I. Novel PEPPSI-type N-heterocyclic carbene palladium(II) complexes: Synthesis, characterization, in silico studies and enzyme inhibitory properties against some metabolic enzymes. Inorg. Chim. Acta 2023, 544, 121239. [Google Scholar] [CrossRef]
  38. Oztaskın, N.; Goksu, S.; Demir, Y.; Maras, A.; Gulcin, I. Synthesis of novel bromophenol including diaryl Methanes-Determination of their inhibition effects on carbonic anhydrase and acetylcholinesterase. Molecules 2022, 27, 7426. [Google Scholar] [CrossRef]
  39. Erdemir, F.; Celepci, D.B.; Aktas, A.; Taslimi, P.; Gok, Y.; Karabıyık, H.; Gulcin, I. 2-Hydroxyethyl substituted NHC precursors: Synthesis, characterization, crystal structure and carbonic anhydrase, α-glycosidase, butyrylcholinesterase, and acetylcholinesterase inhibitory properties. J. Mol. Struc. 2018, 1155, 797–806. [Google Scholar] [CrossRef]
  40. Arabaci, B.; Gulcin, I.; Alwasel, S. Capsaicin: A potent inhibitor of carbonic anhydrase isoenzymes. Molecules 2014, 19, 10103–10114. [Google Scholar] [CrossRef]
  41. Oyaizu, M. Studies on products of browning reaction. Antioxidative activities of products of browning reaction prepared from glucosamine. Jpn. J. Nutr. Diet. 1986, 44, 307–315. [Google Scholar] [CrossRef] [Green Version]
  42. Gulcin, I.; Mshvildadze, V.; Gepdiremen, A.; Elias, R. Antioxidant activity of a triterpenoid glycoside isolated from the berries of Hedera colchica: 3-O-(β-D-glucopyranosyl)-hederagenin. Phytother. Res. 2006, 20, 130–134. [Google Scholar] [CrossRef] [PubMed]
  43. Bursal, E.; Aras, A.; Dogru, M.; Kilic, O. Phenolic content, antioxidant potentials of Saponaria prostrata endemi phenolic content, antioxidant potentials of Saponaria prostrata endemic plant. Int. J. Life Sci. Biotechnol. 2021, 5, 1–8. [Google Scholar]
  44. Tohma, H.; Koksal, E.; Kilic, O.; Alan, Y.; Yılmaz, M.A.; Gulcin, I.; Bursal, E.; Alwasel, S.H. RP-HPLC/MS/MS analysis of the phenolic compounds, antioxidant and antimicrobial activities of Salvia L. species. Antioxidants 2016, 5, 38. [Google Scholar] [CrossRef]
  45. Gulcin, I.; Tel, A.Z.; Kirecci, E. Antioxidant, antimicrobial, antifungal and antiradical activities of Cyclotrichium niveum (Boiss.) Manden and Scheng. Int. J. Food Prop. 2008, 11, 450–471. [Google Scholar] [CrossRef]
  46. Sarıkahya, N.N.; Goren, A.C.; Sumer Okkalı, G.; Coven, F.O.; Orman, B.; Kırcıf, D.; Yucel, B.; Kısla, D.; Demirci, B.; Altun, M.; et al. Chemical composition and biological activities of propolis samples from different geographical regions of Turkey. Phytochem. Lett. 2021, 44, 129–136. [Google Scholar] [CrossRef]
  47. Al-Ani, I.; Zimmermann, S.; Reichling, J.; Wink, M. Antimicrobial activities of European propolis collected from various geographic origins alone and in combination with antibiotics. Medicines 2018, 5, 2. [Google Scholar] [CrossRef]
  48. Jiang, X.; Tao, L.; Li, C.; You, M.; Li, G.Q.; Zhang, C.; Hu, F. Grouping, spectrum-effect relationship and antioxidant compounds of Chinese propolis from different regions using multivariate analyses and off-line anti-DPPH assay. Molecules. 2020, 25, 3243. [Google Scholar] [CrossRef]
  49. Laaroussi, H.; Ferreira-Santos, P.; Genisheva, Z.; Bakour, M.; Ousaaid, D.; Teixeira, J.A.; Lyoussi, B. Unraveling the chemical composition, antioxidant, α-amylase and α-glucosidase inhibition of Moroccan propolis. Food Biosci. 2021, 42, 101160. [Google Scholar] [CrossRef]
  50. Tao, Y.; Zhang, Y.; Cheng, Y.; Wang, Y. Rapid screening and identification of α-glucosidase inhibitors from mulberry leaves using enzyme-immobilized magnetic beads coupled with HPLC/MS and NMR. Biomed. Chromatogr. 2013, 27, 148–155. [Google Scholar] [CrossRef]
  51. Kucukoglu, K.; Gul, H.I.; Taslimi, P.; Gulcin, I.; Supuran, C.T. Investigation of inhibitory properties of some hydrazone compounds on hCA I, hCA II and AChE enzymes. Bioorg. Chem. 2019, 86, 316–321. [Google Scholar] [CrossRef]
  52. Polat Kose, L.; Gulcin, I.; Goren, A.C.; Namiesnik, J.; Martinez-Ayala, A.L.; Gorinstein, S. LC-MS/MS analysis, antioxidant and anticholinergic properties of galanga (Alpinia officinarum Hance) rhizomes. Ind. Crops Prod. 2015, 74, 712–721. [Google Scholar] [CrossRef]
  53. Gulcin, I. Antioxidant properties of resveratrol: A structure-activity insight. Innov. Food Sci. Emerg. 2010, 11, 210–218. [Google Scholar] [CrossRef]
  54. Gulcin, I. Antioxidant activity of eugenol-a structure and activity relationship study. J. Med. Food 2011, 14, 975–985. [Google Scholar] [CrossRef] [PubMed]
  55. Koksal, E.; Gulcin, I. Antioxidant activity of cauliflower (Brassica oleracea L.). Turk. J. Agric. For. 2008, 32, 65–78. [Google Scholar]
  56. Ekinci Akdemir, F.N.; Albayrak, M.; Çalik, M.; Bayir, Y.; Gulcin, I. The protective effects of p-coumaric acid on acute liver and kidney damages induced by cisplatin. Biomedicines 2017, 5, 18. [Google Scholar] [CrossRef]
  57. Ekinci Akdemir, F.N.; Gulcin, I.; GUrsul, C.; Alwasel, S.H.; Bayir, Y. Effect of p-coumaric acid against oxidative stress induced by cisplatin in brain tissue of rats. J. Anim. Plant Sci. 2017, 27, 1560–1564. [Google Scholar]
  58. Apak, R.; Calokerinos, A.; Gorinstein, S.; Segundo, M.A.; Hibbert, D.B.; Gulcin, I.; Demirci Cekic, S.; Guclu, K.; Ozyurek, M.; Esin Çelik, S.; et al. Methods to evaluate the scavenging activity of antioxidants toward reactive oxygen and nitrogen species. Pure Appl. Chem. 2022, 94, 87–144. [Google Scholar] [CrossRef]
  59. Kızıltas, H.; Bingol, Z.; Goren, A.C.; Polat Kose, L.; Durmaz, L.; Topal, F.; Alwasel, S.H.; Gulcin, I. LC-HRMS profiling, antidiabetic, anticholinergic and anti-oxidant activities of aerial parts of kınkor (Ferulago stelleta). Molecules 2021, 26, 2469. [Google Scholar] [CrossRef] [PubMed]
  60. Oleggrio, L.S.; Andrade, J.K.S.; Andrade, G.R.S.; Andrade, G.R.S.; Denadai, M.; Cavalcanti, R.L.; da Silva, M.A.A.P.; Narain, N. Chemical characterization of four Brazilian brown propolis: An insight in tracking of its geographical location of production and quality control. Food Res. Int. 2019, 123, 481–502. [Google Scholar] [CrossRef] [PubMed]
  61. Baltas, N.; Yildiz, O.; Kolayli, S. Inhibition properties of propolis extracts to some clinically important enzymes. J. Enzyme Inhib. Med. Chem. 2016, 31 (Suppl. S1), 52–55. [Google Scholar] [CrossRef]
  62. Aygul, I.; Yaylaci Karahalil, F.; Supuran, C.T. Investigation of the inhibitory properties of some phenolic standards and bee products against human carbonic anhydrase I and II. J. Enzyme Inhib. Med. Chem. 2016, 31 (Suppl. S4), 119–124. [Google Scholar] [CrossRef] [PubMed]
  63. Gulcin, I. The antioxidant and radical scavenging activities of black pepper (Piper nigrum) seeds. Int. J. Food Sci. Nutr. 2005, 56, 491–499. [Google Scholar] [CrossRef] [PubMed]
  64. Artunc, T.; Menzek, A.; Taslimi, P.; Gulcin, I.; Kazaz, C.; Sahin, E. Synthesis and antioxidant activities of phenol derivatives from 1,6-bis(dimethoxyphenyl)hexane-1,6-dione. Bioorg. Chem. 2020, 100, 103884. [Google Scholar] [CrossRef]
  65. Balaydın, H.T.; Gulcin, I.; Menzek, A.; Goksu, S.; Sahin, E. Synthesis and antioxidant properties of diphenylmethane derivative bromophenols including a natural product. J. Enzyme Inhib. Med. Chem. 2010, 25, 685–695. [Google Scholar] [CrossRef] [PubMed]
  66. Gulcin, I.; Oktay, M.; Koksal, E.; Serbetci, H.; Beydemir, S.; Kufrevioglu, Ö.I. Antioxidant and radical scavenging activities of uric acid. Asian J. Chem. 2008, 20, 2079–2090. [Google Scholar]
  67. Gulcin, I.; Topal, F.; Cakmakcı, R.; Goren, A.C.; Bilsel, M.; Erdogan, U. Pomological features, nutritional quality, polyphenol content analysis and antioxidant properties of domesticated and three wild ecotype forms of raspberries (Rubus idaeus L.). J. Food Sci. 2011, 76, C585–C593. [Google Scholar] [CrossRef] [PubMed]
  68. Gulcin, I.; Goren, A.C.; Taslimi, P.; Alwasel, S.H.; Kilic, O.; Bursal, E. Anticholinergic, antidiabetic and antioxidant activities of Anatolian pennyroyal (Mentha pulegium)-Analysis of its polyphenol contents by LC-MS/MS. Biocat. Agric. Biotechnol. 2020, 23, 101441. [Google Scholar] [CrossRef]
  69. Kiziltas, H.; GOren, A.C.; Alwasel, S.; Gulcin, I. Sahlep (Dactylorhiza osmanica): Phytochemical analyses by LC-HRMS, molecular docking, antioxidant activity and enzyme inhibition profiles. Molecules 2022, 27, 6907. [Google Scholar] [CrossRef]
  70. Turkan, F.; Atalar, M.N.; Aras, A.; Gulcin, I.; Bursal, E. ICP-MS and HPLC analyses, enzyme inhibition and antioxidant potential of Achillea schischkinii Sosn. Bioorg. Chem. 2020, 94, 103333. [Google Scholar] [CrossRef]
  71. Talaz, O.; Gulcin, I.; Goksu, S.; Saracoglu, N. Antioxidant activity of 5,10-dihydroindeno[1,2-b]indoles containing substituents on dihydroindeno part. Bioorg. Med. Chem. 2009, 17, 6583–6589. [Google Scholar] [CrossRef] [PubMed]
  72. Gulcin, I.; Kufrevioglu, O.I.; Oktay, M.; Buyukokuroglu, M.E. Antioxidant, antimicrobial, antiulcer and analgesic activities of nettle (Urtica dioica L.). J. Ethnopharmacol. 2004, 90, 205–215. [Google Scholar] [CrossRef]
  73. Han, H.; Yılmaz, H.; Gulcin, I. Antioxidant activity of flaxseed (Linum usitatissimum L.) shell and analysis of its polyphenol contents by LC-MS/MS. Rec. Nat. Prod. 2018, 12, 397–402. [Google Scholar] [CrossRef]
  74. Bursal, E.; Aras, A.; Kilic, O.; Taslimi, P.; Goren, A.C.; Gulcin, I. Phytochemical content, antioxidant activity and enzyme inhibition effect of Salvia eriophora Boiss. & Kotschy against acetylcholinesterase, α-amylase, butyrylcholinesterase and α-glycosidase enzymes. J. Food Biochem. 2019, 43, e12776. [Google Scholar]
  75. Hamad, H.O.; Alma, M.H.; Gulcin, I.; Yılmaz, M.A.; Karaogul, E. Evaluation of phenolic contents and bioactivity of root and nutgall extracts from Iraqian Quercus infectoria Olivier. Rec. Nat. Prod. 2017, 11, 205–210. [Google Scholar]
  76. Bayrak, C.; Uc, E.M.; Rezaei, M.; Gulcin, I.; Menzek, A. Synthesis and antioxidant activities of benzylic bromophenols inclusive of natural products. Turk. J. Chem. 2022, 46, 1405–1416. [Google Scholar] [CrossRef]
  77. Popova, M.; Lyoussi, B.; Aazza, S.; Antunes, D.; Bankova, V.; Miguel, G. Antioxidant and α-glucosidase inhibitory properties and chemical profiles of Moroccan propolis. Nat. Prod. Commun. 2015, 10, 1961–1964. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  78. Aktas, A.; Yakalı, G.; Demir, Y.; Gulcin, I.; Aygun, M.; Gok, Y. The palladium-based complexes bearing 1,3-dibenzylbenzimidazolium with morpholine, triphenylphosphine, and pyridine derivate ligands: Synthesis, characterization, structure and enzyme inhibitions. Heliyon 2022, 8, e10625. [Google Scholar] [CrossRef]
  79. Bora, R.E.; Bilgicli, H.G.; Uc, E.M.; Alagoz, M.A.; Zengin, M.; Gulcin, I. Synthesis, characterization, evaluation of metabolic enzyme inhibitors and in silico studies of thymol based 2-amino thiol and sulfonic acid compounds. Chem. Biol. Interact. 2022, 366, 110134. [Google Scholar] [CrossRef]
  80. Aktas Anıl, D.; Polat, M.F.; Saglamtas, R.; Tarikogulları, A.H.; Alagöz, M.A.; Gulcin, I.; Algul, O.; Burmaoglu, S. Exploring enzyme inhibition profiles of novel halogenated chalcone derivatives on some metabolic enzymes: Synthesis, characterization and molecular modeling studies. Comp. Biol. Chem. 2022, 100, 107748. [Google Scholar] [CrossRef]
  81. Gulcin, I.; Scozzafava, A.; Supuran, C.T.; Akıncıoglu, H.; Koksal, Z.; Turkan, F.; Alwasel, S. The effect of caffeic acid phenethyl ester (CAPE) on metabolic enzymes including acetylcholinesterase, butyrylcholinesterase, glutathione S-transferase, lactoperoxidase, and carbonic anhydrase isoenzymes I., II, IX, and XII. J. Enzyme Inhib. Med. Chem. 2016, 31, 1095–1101. [Google Scholar] [CrossRef] [PubMed]
  82. Topal, M.; Gulcin, I. Evaluation of the in vitro antioxidant, antidiabetic and anticholinergic properties of rosmarinic acid from rosemary (Rosmarinus officinalis L.). Biocat. Agric. Biotechnol. 2022, 43, 102417. [Google Scholar] [CrossRef]
  83. Gulcin, I.; Petrova, O.V.; Taslimi, P.; Malysheva, S.F.; Schmidt, E.Y.; Sobenina, L.N.; Gusarova, N.K.; Trofimov, B.A.; Tuzun, B.; Farzaliyev, V.M.; et al. Synthesis, characterization, molecular docking, acetylcholinesterase and α-glycosidase inhibition profiles of nitrogen-based novel heterocyclic compounds. ChemistrySelect 2022, 7, e20220037. [Google Scholar] [CrossRef]
  84. Gumus, M.; Babacan, Ş.N.; Demir, Y.; Sert, Y.; Koca, I.; Gulcin, I. Discovery of sulfadrug-pyrrole conjugates as carbonic anhydrase and acetylcholinesterase inhibitors. Arch. Pharm. 2022, 355, e2100242. [Google Scholar] [CrossRef]
  85. Yigit, M.; Celepci, D.B.; Taslimi, P.; Yigit, B.; Cetinkaya, B.; Ozdemir, I.; Aygun, M.; Gulcin, I. Selenourea and thiourea derivatives of chiral and achiral enetetramines: Synthesis, characterization and enzyme inhibitory properties. Bioorg. Chem. 2022, 120, 105566. [Google Scholar] [CrossRef]
  86. Topal, F.; Aksu, K.; Gulcin, I.; Tumer, F.; Goksu, S. Inhibition profiles of some symmetric sulfamides derived from phenethylamines on human carbonic anhydrase I., and II isoenzymes. Chem. Biodiver. 2021, 18, e2100422. [Google Scholar] [CrossRef]
  87. Gulcin, I.; Buyukokuroglu, M.E.; Oktay, M.; Kufrevioglu, O.I. Antioxidant and analgesic activities of turpentine of Pinus nigra Arn. Subsp. pallsiana (Lamb.) Holmboe. J. Ethnopharmacol. 2003, 86, 51–58. [Google Scholar] [CrossRef]
  88. Gulcin, I.; Kaya, R.; Gören, A.C.; Akıncıoğlu, H.; Topal, M.; Bingöl, Z.; Cetin Cakmak, K.; Ozturk Sarikaya, S.B.; Durmaz, L.; Alwasel, S. Anticholinergic, antidiabetic and antioxidant activities of cinnamon (Cinnamomum verum) bark extracts: Polyphenol contents analysis by LC-MS/MS. Int. J. Food Prop. 2019, 22, 1511–1526. [Google Scholar] [CrossRef]
  89. Koksal, E.; Bursal, E.; Gulcin, I.; Korkmaz, M.; Caglayan, C.; Goren, A.C.; Alwasel, S.H. Antioxidant activity and polyphenol content of Turkish thyme (Thymus vulgaris) monitored by LC-MS/MS. Int. J. Food Prop. 2017, 20, 514–525. [Google Scholar] [CrossRef]
  90. Singleton, V.L.; Rossi, J.A. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 1965, 16, 144–158. [Google Scholar]
  91. Gulcin, I.; Beydemir, Ş.; Sat, I.G.; Kufrevioglu, O.I. Evaluation of antioxidant activity of cornelian cherry (Cornus mas L.). Acta Aliment. Hung. 2005, 34, 193–202. [Google Scholar] [CrossRef]
  92. Folin, O.; Ciocalteu, V. On tyrosine and tryptophane determinations in proteins. J. Biol. Chem. 1927, 73, 627–650. [Google Scholar] [CrossRef]
  93. Cakmakci, S.; Topdas, E.F.; Kalin, P.; Han, H.; Sekerci, P.; Polat Kose, L.; Gulcin, I. Antioxidant capacity and functionality of oleaster (Elaeagnus angustifolia L.) flour and crust in a new kind of fruity ice cream. Int. J. Food Sci. Technol. 2015, 50, 472–481. [Google Scholar] [CrossRef]
  94. Gulcin, I.; Tel, A.Z.; Goren, A.C.; Taslimi, P.; Alwasel, S. Sage (Salvia pilifera): Determination its polyphenol contents, anticholinergic, antidiabetic and antioxidant activities. J. Food Meas. Charact. 2019, 13, 2062–2074. [Google Scholar] [CrossRef]
  95. Kalin, P.; Gulcin, I.; Goren, A.C. Antioxidant activity and polyphenol content of cranberries (Vaccinium macrocarpon). Rec. Nat. Prod. 2015, 9, 496–502. [Google Scholar]
  96. Yilmaz, M.A. Simultaneous quantitative screening of 53 phytochemicals in 33 species of medicinal and aromatic plants: A detailed, robust and comprehensive LC–MS/MS method validation. Ind. Crops Prod. 2020, 149, 112347. [Google Scholar] [CrossRef]
  97. Elmastas, M.; Turkekul, I.; Ozturk, L.; Gulcin, I.; İsıldak, O.; Aboul-Enein, H.Y. The antioxidant activity of two wild edible mushrooms (Morchella vulgaris and Morchella esculanta). Comb. Chem. High Throughput Screen. 2006, 9, 443–448. [Google Scholar] [CrossRef]
  98. Cetinkaya, Y.; Gocer, H.; Menzek, A.; Gulcin, I. Synthesis and antioxidant properties of (3,4-dihydroxyphenyl)(2,3,4-trihydroxyphenyl)methanone and its derivatives. Arch. Pharm. 2012, 345, 323–334. [Google Scholar] [CrossRef]
  99. Taslimi, P.; Gulcin, I. Antioxidant and anticholinergic properties of olivetol. J. Food Biochem. 2018, 42, e12516. [Google Scholar] [CrossRef]
  100. Rezai, M.; Bayrak, C.; Taslimi, P.; Gulcin, I.; Menzek, A. The first synthesis, antioxidant and anticholinergic activities of 1-(4,5-dihydroxybenzyl)pyrrolidin-2-one derivative bromophenols including natural products. Turk. J. Chem. 2018, 42, 808–825. [Google Scholar]
  101. Gulcin, I.; Elias, R.; Gepdiremen, A.; Boyer, L.; Koksal, E. A comparative study on the antioxidant activity of fringe tree (Chionanthus virginicus L.) extracts. Afr. J. Biotechnol. 2007, 6, 410–418. [Google Scholar]
  102. Blois, M.S. Antioxidant determinations by the use of a stable free radical. Nature 1958, 26, 1199–1200. [Google Scholar] [CrossRef]
  103. Huyut, Z.; Beydemir, S.; Gulcin, I. Antioxidant and antiradical properties of some flavonoids and phenolic compounds. Biochem. Res. Int. 2017, 2017, 7616791. [Google Scholar] [CrossRef]
  104. Tohma, H.; Gulcin, I.; Bursal, E.; Goren, A.C.; Alwasel, S.H.; Koksal, E. Antioxidant activity and phenolic compounds of ginger (Zingiber officinale Rosc.) determined by HPLC-MS/MS. J. Food Meas. Charact. 2017, 11, 556–566. [Google Scholar] [CrossRef]
  105. Fogliano, V.; Verde, V.; Randazzo, G.; Ritieni, A. Method for measuring antioxidant activity and its application to monitoring the antioxidant capacity of wines. J. Agric. Food Chem. 1999, 47, 1035–1040. [Google Scholar] [CrossRef] [PubMed]
  106. Gulcin, I.; Beydemir, S.; Topal, F.; Gagua, N.; Bakuridze, A.; Bayram, R.; Gepdiremen, A. Apoptotic, antioxidant and antiradical effects of majdine and isomajdine from Vinca herbacea Waldst. and kit. J. Enzyme Inhib. Med. Chem. 2012, 27, 587–594. [Google Scholar] [CrossRef]
  107. Gulcin, I.; Alwasel, S.H. Metal ions, metal chelators and metal chelating assay as antioxidant method. Processes 2022, 10, 132. [Google Scholar] [CrossRef]
  108. Ellman, G.L.; Courtney, K.D.; Andres, V.R.M.F. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–90. [Google Scholar] [CrossRef]
  109. Verpoorte, J.A.; Mehta, S.; Edsall, J.T. Esterase activities of human carbonic anhydrases B and C. J. Biol. Chem. 1967, 242, 4221–4229. [Google Scholar] [CrossRef]
  110. Mutlu, M.; Bingol, Z.; Uç, E.M.; Koksal, E.; Goren, A.C.; Alwasel, S.H.; Gulcin, I. Comprehensive metabolite profiling of cinnamon (Cinnamomum zeylanicum) leaf oil using LC-HR/MS, GC/MS, and GC-FID: Determination of antiglaucoma, antioxidant, anticholinergic, and antidiabetic profiles. Life 2023, 13, 136. [Google Scholar] [CrossRef] [PubMed]
  111. Gocer, H.; Akincioglu, A.; Goksu, S.; Gulcin, I. Carbonic anhydrase inhibitory properties of phenolic sulfonamides derived from dopamine related compounds. Arab. J. Chem. 2017, 10, 398–402. [Google Scholar] [CrossRef]
Molecules 28 01739 g001 550
Figure 1. (A). Chromatogram of all standard phenolic compounds analyzed using the LC-MS/MS method 1: Quinic acid, 2: Fumaric acid, 4: Gallic acid 6: Protocatechuic acid, 9: Chlorogenic acid, 10: Protocatechuic aldehyde, 14: 4-OH Benzoic acid, 16: Vanillic acid, 17: Caffeic acid, 19: Vanillin, 24: p-Coumaric acid, 26: Ferulic acid, 31: Miquelianin, 33: Rutin, 34: isoquercitrin, 35: Hesperidin, 40: Cosmosiin, 41: Quercitrin, 42: Astragalin, 43: Nicotiflorin, 47: Quercetin, 48: Naringenin, 49: Hesperetin, 50: Luteolin, 52: Kaempferol, 53: Apigenin, 55: Chrysin, and 56: Acacetin. (B). Chromatogram of propolis ethanol extract compounds.
Figure 1. (A). Chromatogram of all standard phenolic compounds analyzed using the LC-MS/MS method 1: Quinic acid, 2: Fumaric acid, 4: Gallic acid 6: Protocatechuic acid, 9: Chlorogenic acid, 10: Protocatechuic aldehyde, 14: 4-OH Benzoic acid, 16: Vanillic acid, 17: Caffeic acid, 19: Vanillin, 24: p-Coumaric acid, 26: Ferulic acid, 31: Miquelianin, 33: Rutin, 34: isoquercitrin, 35: Hesperidin, 40: Cosmosiin, 41: Quercitrin, 42: Astragalin, 43: Nicotiflorin, 47: Quercetin, 48: Naringenin, 49: Hesperetin, 50: Luteolin, 52: Kaempferol, 53: Apigenin, 55: Chrysin, and 56: Acacetin. (B). Chromatogram of propolis ethanol extract compounds.
Molecules 28 01739 g001
Molecules 28 01739 g002 550
Figure 2. The half-maximal inhibitory concentration (IC50) graphs of propolis against human carbonic anhydrase II (hCA II) (A), acetylcholinesterase (AChE) (B) and α-glycosidase (C) enzymes.
Figure 2. The half-maximal inhibitory concentration (IC50) graphs of propolis against human carbonic anhydrase II (hCA II) (A), acetylcholinesterase (AChE) (B) and α-glycosidase (C) enzymes.
Molecules 28 01739 g002
Table
Table 1. Analytical method validation parameters that belong to the LC-MS/MS method.
Table 1. Analytical method validation parameters that belong to the LC-MS/MS method.
No.AnalytesRT aM.I. (m/z) bF.I. (m/z) cIon. ModeEquationR2 dRSD% eLinearity LOD/LOQ (µg/L) fRecovery (%)U gGr. No. i
InterdayIntradayInterdayIntraday
1Quinic acid3.0190.893.0Negativey = −0.0129989 + 2.97989x0.9960.690.510.1–525.7/33.31.00111.00830.03721
2Fumaric aid3.9115.240.9Negativey = −0.0817862 + 1.03467x0.9951.051.021–50135.7/167.90.99631.00160.00911
3Aconitic acid4.0172.8129.0Negativey = −0.7014530 + 32.9994x0.9712.070.930.1–516.4/31.40.99681.00680.02471
4Gallic acid4.4168.879.0Negativey = 0.0547697 + 20.8152x0.9991.600.810.1–513.2/17.01.00100.99470.01121
5Epigallocatechin6.7304.8219.0Negativey = −0.00494986 + 0.0483704x0.9981.220.731–50237.5/265.90.99691.00400.01843
6Protocatechuic acid6.8152.8108.0Negativey = 0.211373 + 12.8622x0.9571.430.760.1–521.9/38.60.99721.00550.03501
7Catechin7.4288.8203.1Negativey = −0.00370053 + 0.431369x0.9992.141.080.2–1055.0/78.01.00241.00450.02213
8Gentisic acid8.3152.8109.0Negativey = −0.0238983 + 12.1494x0.9971.811.220.1–518.5/28.20.99631.00770.01671
9Chlorogenic acid8.4353.085.0Negativey = 0.289983 + 36.3926x0.9952.151.520.1–513.1/17.61.00001.00230.02131
10Protocatechuic aldehyde8.5137.292.0Negativey = 0.257085 + 25.4657x0.9962.080.570.1–515.4/22.21.00020.99880.03961
11Tannic acid9.2182.878.0Negativey = 0.0126307 + 26.9263x0.9992.401.160.05–2.515.3/22.70.99700.99500.01901
12Epigallocatechin gallate9.4457.0305.1Negativey = −0.0380744 + 1.61233x0.9991.300.630.2–1061.0/86.00.99811.00790.01473
131,5-Dicaffeoylquinic acid9.8515.0191.0Negativey = −0.0164044 + 16.6535x0.9992.421.480.1–55.8/9.40.99830.99970.03061
144-OH Benzoic acid10.5137.265.0Negativey = −0.0240747 + 5.06492x0.9991.240.970.2–1068.4/88.11.00321.00680.02371
15Epicatechin11.6289.0203.0Negativey = −0.0172078 + 0.0833424x0.9961.470.621–50139.6/161.61.00131.00120.02213
16Vanillic acid11.8166.8108.0Negativey = −0.0480183 + 0.779564x0.9991.920.761–50141.9/164.91.00220.99980.01451
17Caffeic acid12.1179.0134.0Negativey = 0.120319 + 95.4610x0.9991.111.250.05–2.57.7/9.51.00151.00420.01521
18Syringic acid12.6196.8166.9Negativey = −0.0458599 + 0.663948x0.9981.181.091–5082.3/104.51.00061.00720.01291
19Vanillin13.9153.1125.0Positivey = 0.00185898 + 20.7382x0.9961.100.850.1–524.5/30.41.00090.99670.01221
20Syringic aldehyde14.6181.0151.1Negativey = −0.0128684 + 7.90153x0.9992.510.770.4–2019.7/28.01.00010.99640.02151
21Daidzin15.2417.1199.0Positivey = 9.45747 + 152.338x0.9962.251.320.05–2.57.0/9.50.99551.00170.02022
22Epicatechin gallate15.5441.0289.0Negativey = −0.0142216 + 1.06768x0.9971.631.280.1–519.5/28.50.99840.99460.02293
23Piceid17.2391.0135.0/106.9Positivey = 0.00772525 + 25.4181x0.9991.941.160.05–2.513.8/17.81.00420.99790.01991
24p-Coumaric acid17.8163.093.0Negativey = 0.0249034 + 18.5180x0.9991.921.430.1–525.9/34.91.00491.00010.01941
25Ferulic acid-D3-IS h18.8196.2152.1NegativeN.A.N.A.N.A.N.A.N.A.N.A.N.A.N.A.0.01701
26Ferulic acid18.8192.8149.0Negativey = −0.0735254 + 1.34476x0.9991.440.531–5011.8/15.60.99510.99760.01811
27Sinapic acid18.9222.8193.0Negativey = −0.0929932 + 0.836324x0.9991.450.520.2–1065.2/82.31.00311.00370.03171
28Coumarin20.9146.9103.1Positivey = 0.0633397 + 136.508x0.9992.111.540.05–2.5214.2/247.30.99500.99580.03831
29Salicylic acid21.8137.265.0Negativey = 0.239287 + 153.659x0.9991.481.180.05–2.56.0/8.30.99500.99980.01581
30Cynaroside23.7447.0284.0Negativey = 0.280246 + 6.13360x0.9971.561.120.05–2.512.1/16.01.00721.00020.03662
31Miquelianin24.1477.0150.9Negativey = −0.00991585 + 5.50334x0.9991.310.950.1–510.6/14.70.99340.99650.02202
33Rutin25.6608.9301.0Negativey = −0.0771907 + 2.89868x0.9991.381.090.1–515.7/22.70.99771.00330.02472
34Isoquercitrin25.6463.0271.0Negativey = −0.111120 + 4.10546x0.9982.130.780.1–58.7/13.51.00570.99630.02202
35Hesperidin25.8611.2449.0Positivey = 0.139055 + 13.2785x0.9991.841.350.1–519.0/26.00.99671.00430.03352
36o-Coumaric acid26.1162.893.0Negativey = 0.00837193 + 11.2147x0.9992.111.460.1–531.8/40.41.00440.99860.01471
37Genistin26.3431.0239.0Negativey = 1.65808 + 7.57459x0.9912.011.280.1–514.9/21.71.00621.00470.00832
38Rosmarinic acid26.6359.0197.0Negativey = −0.0117238 + 8.04377x0.9991.240.860.1–516.2/21.21.00561.00020.01301
39Ellagic acid27.6301.0284.0Negativey = 0.00877034 + 0.663741x0.9991.571.230.4–2056.9/71.01.00051.00480.03641
40Cosmosiin28.2431.0269.0Negativey = −0.708662 + 8.62498x0.9981.651.300.1–56.3/9.20.99400.99730.00832
41Quercitrin29.8447.0301.0Negativey = −0.00153274 + 3.20368x0.9992.241.160.1–54.8/6.40.99600.99780.02682
42Astragalin30.4447.0255.0Negativey = 0.00825333 + 3.51189x0.9992.081.720.1–56.6/8.20.99680.99570.01142
43Nicotiflorin30.6592.9255.0/284.0Negativey = 0.00499333 + 2.62351x0.9991.481.230.05–2.511.9/16.70.99541.00440.01082
44Fisetin30.6285.0163.0Negativey = 0.0365705 + 8.09472x0.9991.751.190.1–510.1/12.70.99801.00420.02313
45Daidzein34.0253.0223.0Negativey = −0.0329252 + 6.23004x0.9992.181.730.1–59.8/11.60.99260.99630.03703
47Quercetin35.7301.0272.9Negativey = +0.00597342 + 3.39417x0.9991.891.380.1–515.5/19.00.99670.99710.01753
48Naringenin35.9270.9119.0Negativey = −0.00393403 + 14.6424x0.9992.341.690.1–52.6/3.91.00621.00200.03923
49Hesperetin36.7301.0136.0/286.0Negativey = +0.0442350 + 6.07160x0.9992.472.130.1–57.1/9.10.99980.99630.03213
50Luteolin36.7284.8151.0/175.0Negativey = −0.0541723 + 30.7422x0.9991.671.280.05–2.52.6/4.10.99521.00290.03133
51Genistein36.9269.0135.0Negativey = −0.00507501 + 12.1933x0.9991.481.190.05–2.53.7/5.31.00691.00120.03373
52Kaempferol37.9285.0239.0Negativey = −0.00459557 + 3.13754x0.9991.491.260.05–2.510.2/15.40.99920.99900.02123
53Apigenin38.2268.8151.0/149.0Negativey = 0.119018 + 34.8730x0.9981.170.960.05–2.51.3/2.00.99851.00030.01783
54Amentoflavone39.7537.0417.0Negativey = 0.727280 + 33.3658x0.9921.351.120.05–2.52.8/5.10.99911.00440.03403
55Chrysin40.5252.8145.0/119.0Negativey = −0.0777300 + 18.8873x0.9991.461.210.05–2.51.5/2.80.99221.00500.03233
56Acacetin40.7283.0239.0Negativey = −0.559818 + 163.062x0.9971.671.280.02–11.5/2.50.99491.00110.03633
a R.T.: retention time, b MI (m/z): molecular ions of the standard analytes (m/z ratio), c FI (m/z): fragment ions d r2: coefficient of determination, e RSD: relative standard deviation, f LOD/LOQ (µg/L): limit of detection/quantification, g U (%): relative uncertainty percentage at the 95% confidence level (k = 2), h IS: internal standard, i Gr. No: represents the grouping of internal standards; these numbers indicate which IS stands for which phenolic compounds.
Table
Table 2. LC–MS/MS parameters of selected compounds and amount of antioxidants in propolis in mg/g concentration.
Table 2. LC–MS/MS parameters of selected compounds and amount of antioxidants in propolis in mg/g concentration.
CompoundsAnalytePhenolics (mg Analyte/g Propolis)
1Quinic acid7.285
2Fumaric aid0.134
3Aconitic acidN.D.
4Gallic acid0.150
5EpigallocatechinN.D.
6Protocatechuic acid1,158
7CatechinN.D.
8Gentisic acidN.D.
9Chlorogenic acid0.018
10Protocatechuic aldehyde0.338
11Tannic acidN.D.
12Epigallocatechin gallateN.D.
131,5-Dicaffeoylquinic acidN.D.
144-OH Benzoic acid0.306
15EpicatechinN.D.
16Vanillic acid1.647
17Caffeic acid21.358
18Syringic acidN.D.
19Vanillin0.116
20Syringic aldehydeN.D.
21DaidzinN.D.
22Epicatechin gallateN.D.
23PiceidN.D.
24p-Coumaric acid16.911
25Ferulic acid-D3-ISN.D.
26Ferulic acid5.110
27Sinapic acidN.D.
28CoumarinN.D.
29Salicylic acidN.D.
30CyranosideN.D.
31Miquelianin0.020
32Rutin-D3-ISN.D.
33Rutin0.035
34Isoquercitrin0.045
35Hesperidin0.045
36O-Coumaric acidN.D.
37GenistinN.D.
38Rosmarinic acidN.D.
39Ellagic acidN.D.
40Cosmosiin0.026
41Quercitrin0.087
42Astragalin0.030
43Nicotiflorin0.024
44FisetinN.D.
45DaidzeinN.D.
46Quercetin-D3-ISN.D.
47Quercetin6.223
48Naringenin11.340
49Hesperetin2.089
50Luteolin4.394
51GenisteinN.D.
52Kaempferol4.043
53Apigenin4.686
54AmentoflavoneN.D.
55Chrysin9.860
56Acacetin76.359
N.D.: not detected, IS: internal standard.
Table
Table 3. The reducing abilities of propolis and standards a concentration of 30 μg/mL.
Table 3. The reducing abilities of propolis and standards a concentration of 30 μg/mL.
AntioxidantsFe3+-ReducingCu2+-ReducingFRAP-Reducing
λ700r2λ700r2λ700r2
BHA1.257 ± 0.0880.95231.800 ± 0.1560.97420.884 ± 0.1160.9899
BHT2.018 ± 0.0290.94662.912 ± 0.0120.99692.089 ± 0.0270.9581
α-Tocopherol1.895 ± 0.0080.94021.139 ± 0.0960.99671.995 ± 0.0160.9807
Trolox1.545 ± 0.0190.99662.323 ± 0.0490.99801.755 ± 0.0930.9990
Propolis0.894 ± 0.0200.99530.778 ± 0.0540.99861.114 ± 0.0450.9970
Table
Table 4. The reducing abilities of propolis and standards at concentration of 30 μg/mL.
Table 4. The reducing abilities of propolis and standards at concentration of 30 μg/mL.
AntioxidantsDPPH• ScavengingABTS•+ ScavengingDMPD•+ Scavenging
IC50r2IC50r2IC50r2
BHA9.000.93997.710.933031.430.9993
BHT21.000.96687.710.9330--
α-Tocopherol5.920.97707.710.933014.380.9349
Trolox9.630.99478.100.9550--
Propolis20.550.99898.1570.998586.640.9855
Table
Table 5. Half-maximal inhibition concentration (IC50, μg/mL) of propolis against human carbonic anhydrase isoenzyme II (hCA II) acetylcholinesterase (AChE), and α-glycosidase (α-Gly) enzymes.
Table 5. Half-maximal inhibition concentration (IC50, μg/mL) of propolis against human carbonic anhydrase isoenzyme II (hCA II) acetylcholinesterase (AChE), and α-glycosidase (α-Gly) enzymes.
AntioxidantshCA IIAChEα-Glycosidase
IC50r2IC50r2IC50r2
Propolis19.60.93273.40.98693.70.9362
Acetazolamide *8.370.9825----
Tacrine **--5.970.9706--
Acarbose ***----22,800-
*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].
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Karagecili, H.; Yılmaz, M.A.; Ertürk, A.; Kiziltas, H.; Güven, L.; Alwasel, S.H.; Gulcin, İ. Comprehensive Metabolite Profiling of Berdav Propolis Using LC-MS/MS: Determination of Antioxidant, Anticholinergic, Antiglaucoma, and Antidiabetic Effects. Molecules 2023, 28, 1739. https://doi.org/10.3390/molecules28041739

AMA Style

Karagecili H, Yılmaz MA, Ertürk A, Kiziltas H, Güven L, Alwasel SH, Gulcin İ. Comprehensive Metabolite Profiling of Berdav Propolis Using LC-MS/MS: Determination of Antioxidant, Anticholinergic, Antiglaucoma, and Antidiabetic Effects. Molecules. 2023; 28(4):1739. https://doi.org/10.3390/molecules28041739

Chicago/Turabian Style

Karagecili, Hasan, Mustafa Abdullah Yılmaz, Adem Ertürk, Hatice Kiziltas, Leyla Güven, Saleh H. Alwasel, and İlhami Gulcin. 2023. "Comprehensive Metabolite Profiling of Berdav Propolis Using LC-MS/MS: Determination of Antioxidant, Anticholinergic, Antiglaucoma, and Antidiabetic Effects" Molecules 28, no. 4: 1739. https://doi.org/10.3390/molecules28041739

APA Style

Karagecili, H., Yılmaz, M. A., Ertürk, A., Kiziltas, H., Güven, L., Alwasel, S. H., & Gulcin, İ. (2023). Comprehensive Metabolite Profiling of Berdav Propolis Using LC-MS/MS: Determination of Antioxidant, Anticholinergic, Antiglaucoma, and Antidiabetic Effects. Molecules, 28(4), 1739. https://doi.org/10.3390/molecules28041739

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