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Propolis is a resin-like material, which is collected from the buds of cone-bearing trees by the honeybees and processed to be used in their hives. Both the physicochemical composition and biological activity potential of the propolis are highly variable depending on several factors, such as the geographical location, tree source, and honeybee stra...
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... is a natural resinous substance col- lected by honeybees from the buds and barks of various plants for storage inside the hive [1]. Chestnut, beech, birch and some conifer trees in particular are well known sources of propo- lis [2]. Honeybees use propolis for various pur- poses, such as to protect the hive from micro- bial infection, to provide thermal insulation, to fill cracks or apertures within the hive and to embalm dead organisms [3,4]. Propolis has been extensively used in traditional medicine to treat various diseases since ancient times [2,5]. During the last decade, propolis from different regions of the world was extensive- ly studied to reveal its major bio-active prop- erties, such as antimicrobial [5,6] antifungal [7], antioxidant [4,8,9] anti-inflammatory [8,9] antitumoral [10,11,12,13] immunomodulatory [14], immunoregulatory [15], antidiabetic [16], antiulcerative [17,18] and antidepressant [19] activities. Due to these various critical biologi- cal properties, propolis is used in apitherapy to treat numerous diseases and also in the food industry as an additive for a range of purposes. Many researchers have reported a positive correlation between total phenolic contents (TPC) and antioxidant capacities in honey as well as in propolis [1,6,20,21,22]. Phenolic compounds are synthesized by plants as sec- ondary metabolites, such as phenolic ac- ids, flavones, flavonoids, and anthocyanins, which serve as defense mechanisms in plants to counteract reactive oxygen, a process es- sential for survival [23,24]. There are numer- ous phenolic constituents in plants, each of which has different antioxidant capacities [25]. The antioxidant and radical scavenging activ- ity of phenolic acids depends on the number and position of hydroxyl (–OH) groups, or- tho-dihydroxy groups and methoxy (–OCH 3 ) substituent in the molecules [20,22]. The chemical composition, physical structure and bioactivity properties of prop- olis largely depend on its botanical and geo- graphical characteristics. Raw propolis con- tains nearly 50% resin, made up of phenolic compounds, 30% wax, 10% essential oils, 5% pollen and 5% various organic compounds [2]. TPC and antioxidant activities of prop- olis samples have been shown to vary when collected from different parts of the hive. In our previous study, propolis samples collected from the entrance of the hives exhibited high- er antioxidant properties than those obtained from inside the hive [8]. In addition, there are studies showing that the composition and bioactivity of propolis differ due to season- al effects [26] and floral changes [1]. In recent years, several studies have investigated differ- ent propolis species from various regions of the world, such as red Brazilian propolis, [27] Korean propolis, [28] Turkish propolis [6,8] and Iranian propolis [25], which exhibit dif- ferent physical, chemical and bioactive prop- erties. Interestingly, propolis from Azerbaijan has not been studied to date. In this study, we characterized the phe- nolic composition and bioactivity properties of propolis samples collected from 15 differ- ent locations in Azerbaijan. Phenolic compo- sition was determined using HPLC, and an- tioxidant capacity was measured using TPC, FRAP assay and DPPH radical scavenging ac- tivities. Antioxidant values were compared with BHT and Trolox, used as positive con- trols. The TPC and bioactivity potential of the propolis samples varied depending on the flo- ra of the region of collection; however, there were also significant differences among sam- ples collected from the same region. 2,4,6-tripyridyl-s-triazine (TPTZ), Folin- Ciocalteu’s phenol reagent, 2, 2-diphe- nyl-1-picrylhydrazyl (DPPH), Trolox (6-hy- droxy-2,5,7,8-tetramethylchroman-2-car- boxylic acid) were purchased from Sigma Chemical Co. (St Louis, MO, USA). All chem- icals used for HPLC–DAD were of analytical grade. Standards of common phenolic com- pounds, gallic, protocatechuic acid, p-OH benzoic acid, catechin, chlorogenic acid, van- illic acid, caffeic acid, p-coumaric acid, fe- rulic acid, rutin, fisetin, quercetin, apigen- in, kaempferol, isorhamnetin and propyl- paraben were obtained from Sigma-Aldrich (Steinheim, Germany). Of the HPLC grade or- ganic reagents, acetonitrile was supplied by Sigma-Aldrich Co. (St. Louis, MO, USA) and methanol was obtained from Merck (KGaA, Darmstandt, Germany). HPLC syringe fil- ters (RC-membrane, 0.2 μm) were Sartorius Minisart RC 15, Sartorius (Germany). Propolis samples were collected as crude materials from the beehives of local beekeep- ers in 15 areas of Azerbaijan during the 2012 harvest season. Figure 1 shows the collection sites of each sample (Aktaş, Zerdap, Ismayıllı, Astara, Şemkır, Qax, Nahcıvan, Mingeçevir, Şeki, Qusar and Quba). Raw propolis samples were kept in a freez- er at –20°C until use. Samples were ground to powder; 5.0 g was placed in a falcon tube (50 mL) and 95% ethanol was added to make up a volume of 30 mL. The suspensions were stirred continuously with a shaker (Heidolph Promax 2020, Schwabach, Germany) at room temperature for 24 h and then sonicated for 3 h with a sonicator (ElmaÒ Transsonic Digital, Germany). The suspensions were fil- tered with filter paper (Whatson) and con- centrated in a rotary evaporator (IKA-Werke, Staufen-Germany) under reduced pressure at 40°C. The residue was then resuspended with a minimal volume of ethanol and kept at 4°C until use. HPLC-UV analyses were performed us- ing a Thermo Finnigan Surveyor HPLC equipped with a UV-Vis detector supplying a simultaneous double wavelength. HPLC- UV analyses were performed on a reverse phase C18 column (150 mm × 4.6 mm id, 5 mm particle; Fortis). Benzoic acid deriva- tives (such as gallic acid, protocatechuic acid, p -hydroxybenzoic acid and vanillic acid) and flavanols (such as catechin) were ana- lyzed at 280 nm; cinnamic acid derivatives such as chlorogenic acid, caffeic acid, p -cou- maric acid, ferulic acid, and flavonols such as rutin, fisetin, quercetin, apigenin, kaem- pherol, and isorhamnetin were analyzed at 315 nm. Propylparaben (IS) was analyzed at 280 nm, a normalization calibration meth- od being used. Gradient elution was used for HPLC analyses, modifying the method [29]. Mobile phase (A) 2% acetic acid in water and (B), 70:30 acetonitrile/water mixtures: the following gradient was used; 0–3 min 5% B; 3–8 min 5–15% B; 8–10 min 15–20% B; 10–12 min 20–25% B; 12–20 min 25–40% B; 20–30 min 40–80% B, before returning to the initial conditions. Injection volume was 25 mL, column temperature was 30°C and flow rate was 1.2 mL/min. Limit of detection (LOD) was calculat- ed according to the EPA method as an S/N level of 3, and limit of quantification (LOQ) was calculated as an S/N level of 10 (Table 4). Calculated amounts per compound were pre- pared as follows; 0.5 mg/L for gallic acid, pro- tocatechuic acid, p -OH benzoic acid, chloro- genic acid, vanillic acid, caffeic acid, p - cou- maric acid, rutin and propylparaben, 1 mg/L for ferulic acid, fisetin, apigenin kaempher- ol, and isorhamnetin and 2 mg/L for cate- chin and Quercetin. Each mixture was inject- ed seven times to verify the LOD and LOQ of each compound and then calculated as the percentage relative standard deviation of peak area and retention time. The result was calcu- lated as mg/100 g raw propolis. Total phenolic contents (TPC) of the etha- nol extracts were determined using Folin- Ciocalteu assay [30]. All phenolic compounds, including phenolic acids, flavonoids and an- thocyanins, in the ethanolic extracts of prop- olis resulted in the formation of a blue color complex with Folin reagents whose maximum absorbance can be read at 740 nm. Gallic acid was used as the reference standard compound, and the results were expressed as mg gallic acid per g propolis. Subsequently, 680 μL dis- tilled water, 20 μL ethanol propolis extracts and 400 μL of 0.2 N Folin-Ciocalteu regents were mixed in a tube, vortexed for 2 min. Next, 400 μL of Na 2 CO 3 (7.5%) was added and the mixture incubated for 2 h at room tempera- ture. Absorbance was measured at 760 nm at the end of the incubation period. All the mea- surements were performed in triplicate The reducing power ability of ferric tripyridyl- triazine (Fe-III-TPTZ) complex (FRAP) from the ethanolic extracts of the propolis samples was measured using reported methods [31] with some modifications. The test involved the reduction of ferric tripyridyltriazine (Fe-III-TPTZ) complex to a blue-colored Fe (II) TPTZ by antioxidant agents of samples. Working FRAP reagent was prepared by mix- ing 25 mL of 300 mM acetate buffer, pH 3.6, with 2.5 mL of 10 mM TPTZ solution in 40 mM HCl and 2.5 mL of 20 mM FeCl 3 · 6H 2 O solution. Next, 3 mL freshly prepared FRAP reagent and 100 μL of the samples were mixed and incubated for 4 min at 37°C, and the ab- sorbance was read at 595 nm against reagent blank containing distilled water. Trolox was used as a positive control to construct a ref- erence curve (62.5–1000 μM). FRAP values were expressed as μM Trolox equivalent of g propolis. The higher the FRAP value, the higher the antioxidant capacity of the samples. The scavenging of 2,2-diphenyl-1-picrylhy- drazyl (DPPH) radicals was assayed using re- ported method [32]. The method is based on the radical having a purple color which de- cays after interaction with antioxidant agents, turning yellow. The change in absorbance due to colors can be spectrophotometrically mon- itored at 517 nm. Briefly, 1.5 mL of the etha- nolic extract solution was mixed with 1.5 mL of 0.1 mM DPPH (dissolved in methanol), vor- texed and ...
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... (p<0.05). Propolis from the Qax region exhibited the highest amount of TPC (79.86 mg/g GAE), while the sample from the Astara and Şeki re- gions contained the lowest (10.94 mg/g GAE; 13.41 mg/g GAE). In addition to the Qax re- gion, propolis from Zerdap, Aktaş, Ismayıllı-I and Qusar also had high phenolic contents. In terms of the differences between propo- lis samples from the same regions, significant differences were determined in TPC levels be- tween Quaba I, II and III, between Nahcivan I and II, and between Ismayıllı I and II (p<0.05). The ability of the Fe (III)-TPTZ complex re- flects the total antioxidant capacity of plants as well as of propolis and other honeybee products. Under this method, it is assumed that the higher the FRAP value, the high- er the antioxidant activity. The FRAP val- ues calculated for the propolis samples are shown in Table 1. These ranged from 170 to 438 μM Trolox/g raw propolis. The high- est FRAP values were detected in the sam- ples from the Ismayılli (I), Zerdap, Qax, and Aktaş regions, and were higher than 400 μM Trolox equivalent of g propolis. The low- est FRAP values were detected in the sam- ples from the Astara, Şeki, and Ismayılli (II) regions, at below 200 μM Trolox equivalent of g propolis. The reducing power measure- ment for the samples showed a concentra- tion-dependent pattern. A positive correlation was detected be- tween the samples’ FRAP values and TPC (r2: 0.98), which suggests that total antioxi- dant activity of the propolis samples origi- nates from the phenolic substances within the propolis (Table 2). The antioxidant agents present in the propo- lis samples enhanced the decay of the purple color of the DPPH as they interacted with the radicals and scavenged these. The change in absorbance was monitored spectrophotomet- rically at 517 nm. The propolis samples from Zerdap, Quba (II), Aktaş, Ismayıllı (I) and Qusar exhibited higher DPPH radical-scav- enging activity with lower SC 50 values, within the range of 15–22 mg/ml (Table 1). As with TPC and FRAP values, significant differences were also determined in DPPH radical scav- enging activities in propolis specimens from the same regions (p<0.05). DPPH radical scav- enging activities in the propolis samples used in the study were correlated with amounts of TPC (r2: –0,79) and FRAP values (r2: –0,81). Fifteen reference standards, including gal- lic, protocatechuic acid, p-OH benzoic acid, catechin, chlorogenic acid, vanillic acid, caf- feic acid, epicatechin, p-coumaric acid, feru- lic acid, rutin, myricetin, quercetin, apigen- in, kaempferol and isorhamnetin were used to determine and quantify amounts of phe- nolics. All of the phenolic reference stan- dards used was present in ethanolic extracts at different amount and selected samples. Only chlorogenic and catechin were absent from all samples. Caffeic acid was the most abundant phenolic acid among all propolis samples, followed by coumaric acid and feru- lic acid. Protocathechuic, p - hydroxy benzoic acid, vanillic acid and gallic acid were found in some of the samples (Table 3). Apigenin was the major flavonoid present in the samples, while quercetin, isorhamne- tin and kaempferol were present at lower concentrations among the studied flavonoids, although fisetin was detected only in one sample, from the Şemkır region. Rutin was detected in a sample from Quba-2 (Table 3). Propolis is a multifunctional honeybee product consisting of exudates from plants mixed with beeswax. It is used by bees for various purposes, such as temperature insula- tion and beehive repairs, embalming dead or- ganisms, etc. It has been used since as early as 3000 BC [15]. During the last two decades re- searchers have become interested in its phar- macological agents and biological activities [33,34]. The bioactivity capacity of propolis largely depends on its phenolic contents, in- cluding phenolic acids, flavonoids, anthocy- anins, and several aromatic acids and esters [35]. As stated in the introduction, the phys- ical properties and chemical composition, as well as the bioactive potential, of propolis are highly dependent on their region of ori- gin. Such factors therefore need to be investi- gated by collecting samples from different re- gions. Several studies have been performed on propolis samples from different regions of the world in order to study their biological activi- ty potentials [1,6,28,32,36]. In this study, we evaluated the TPC, an- tioxidant activity and phenolic profile of Azerbaijan propolis. TPC in the ethano- lic propolis extracts ranged from nearly 10 to 80 mg GAE/g propolis, revealing signifi- cant differences among the propolis samples from the studied regions (Table 1). This dif- ference in TPC levels may be attributed to the geographical origin of the samples; howev- er, some propolis samples collected from dif- ferent hives in the same area exhibited a wide variation in TPC. The three samples collected from Quba province, Quba I, II and III (Figure 1), had TPC values of 17.5, 61.72 and 66.02 mg GAE/g propolis, respectively. Additionally, the sample from Qusar city, which is adja- cent to Quba, had a TPC value of 69.66 mg GAE/g propolis. Similarly, the two samples from Ismayıllı city, Ismayıllı I and II, also ex- hibited variation within the same region, with TPC values of 69.73 and 23.31 mg GAE/g propolis, respectively. Our TPC results for the Azerbaijani propolis samples were similar to those for Iranian propolis [21], which may be due to similarity of geography and vegetation. [37] studied propolis samples collected from 16 different areas of the world and report- ed TPC between 31 mg GAE/g and 299 mg GAE/g propolis; Thailand propolis had the lowest TPC (31 mg GAE/g) and Hubei-China the highest (299 mg GAE/g) [1]. studied TPC in 20 propolis samples from 12 different re- gions of China and reported levels between 42 mg GAE/g and 302 mg GAE/g propolis, with Yunan region propolis having the lowest TPC (42 mg GAE/g) [21]. studied propolis sam- ples from three regions of Iran and reported TPC levels ranging from 30.80 mg GAE/g to 84.60 mg GAE/g propolis [36]. determined a TPC level in aquatic extract of propolis from the province of Erzurum in Turkey of 124 mg GAE/g propolis. In our previous study, we de- termined TPC levels in methanolic extracts of Turkish propolis within a range of 115 to 210 mg GAE/g propolis [6]. These results suggest that propolis samples collected from the same region and in the same season may exhib- it differences in terms of TPC, which may be due to some other factors, such as condition and age of the beehive, as well as the strength of the honeybee colony. Although there are various assays for measuring the antioxidant capacity of nat- ural products, the reducing power of Fe (III) test has been used as a significant indicator of total antioxidant activity [4]. Propolis ex- tracts from Azerbaijan exhibited a wide range of FRAP values, from 170 to 437.90 Trolox/g. Ismayılli-I, Zerdap, Aktaş and Quba-II sam- ples had FRAP values greater than 400 μM, while Astara, Şeki, Quba-II and Ismayılli sam- ples had the lowest FRAP values, at less than 200 μM Trolox/g. These FRAP value findings are in accordance with our previous study of propolis samples collected from Turkey, which ranged from 182 to 325 μM Trolox/g propolis. Furthermore, the FRAP values of the samples were highly correlated with their TPC values ( r 2: 0.98) . This positive correlation has also been reported in several previous propolis studies [6,21,36]. Overall, our results confirm that phenolic contents are the major determinants of the antioxidant potential of propolis on the basis of FRAP assay. All propolis samples exhibited free radical (DPPH) scavenging activity to some extent; however, considerable differences were ob- served in SC 50 values among the samples. In agreement with the FRAP results, the Zerdap, Quba-II, Ismayılli-I and Qusar samples ex- hibited higher radical scavenging capacities, and the Astara, Quba-I and Ismayılli-II sam- ples lower DPPH-scavenging activity. We thus determined positive and negative correlations between FRAP and DPPH values ( r 2 = –0,81, p<0.05), TPC and DPPH values ( r 2: –0.79, p<0.05) (Table 2). Relationships among the TPC, FRAP and DPPH activities of honey- bee products have been investigated in sever- al studies [1,6,8,24,33]. [36] demonstrated that the phenolic com- pounds present in honeybee products are re- sponsible for their antioxidant and free radi- cal-scavenging potential. More detailed anal- yses of phenolic compounds were therefore necessary to evaluate the antioxidant capac- ity of the propolis samples. In our study, sam- ples were analyzed using RP-HPLC for eight individual phenolic acids and seven individual flavonoids to quantify and evaluate the com- positions of the ethanolic propolis extracts (Table 3). All propolis samples contained caf- feic, p-coumaric and ferulic acids as the major phenolic acids and apigenin, quercetin, kae- mpherol and isoramnetin as the main flavo- noids, in parallel with other propolis studies from across the world [33,36,38,39]. The phe- nolic protocatechuic, p- hydroxybenzoic, van- illic and gallic acids were detected in a lim- ited number of the propolis samples, while chlorogenic acid was not detected in any sam- ple. Protocatechuic acid was detected in eight samples (out of 15), and Quba-III sample con- tained the highest level (918.8 mg/100 g) of propolis. In [6] study, benzoic, ferulic, caf- feic and p-coumaric acid were identified as the main phenolic acids of Turkish propolis, which is highly compatible with our findings for Azerbaijan propolis. Quercetin, apigenin, kaempherol and isoramnetin were the most abundant flavo- noids in the samples, at levels ranging from 16.20 to 697.60 mg/100g. Although rutin was detected in a limited number of samples (four out of 15), Quba-II propolis contained a significantly higher amount (683.00 mg/100 g) than other samples. ...
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... on a reverse phase C18 column (150 mm × 4.6 mm id, 5 mm particle; Fortis). Benzoic acid deriva- tives (such as gallic acid, protocatechuic acid, p -hydroxybenzoic acid and vanillic acid) and flavanols (such as catechin) were ana- lyzed at 280 nm; cinnamic acid derivatives such as chlorogenic acid, caffeic acid, p -cou- maric acid, ferulic acid, and flavonols such as rutin, fisetin, quercetin, apigenin, kaem- pherol, and isorhamnetin were analyzed at 315 nm. Propylparaben (IS) was analyzed at 280 nm, a normalization calibration meth- od being used. Gradient elution was used for HPLC analyses, modifying the method [29]. Mobile phase (A) 2% acetic acid in water and (B), 70:30 acetonitrile/water mixtures: the following gradient was used; 0–3 min 5% B; 3–8 min 5–15% B; 8–10 min 15–20% B; 10–12 min 20–25% B; 12–20 min 25–40% B; 20–30 min 40–80% B, before returning to the initial conditions. Injection volume was 25 mL, column temperature was 30°C and flow rate was 1.2 mL/min. Limit of detection (LOD) was calculat- ed according to the EPA method as an S/N level of 3, and limit of quantification (LOQ) was calculated as an S/N level of 10 (Table 4). Calculated amounts per compound were pre- pared as follows; 0.5 mg/L for gallic acid, pro- tocatechuic acid, p -OH benzoic acid, chloro- genic acid, vanillic acid, caffeic acid, p - cou- maric acid, rutin and propylparaben, 1 mg/L for ferulic acid, fisetin, apigenin kaempher- ol, and isorhamnetin and 2 mg/L for cate- chin and Quercetin. Each mixture was inject- ed seven times to verify the LOD and LOQ of each compound and then calculated as the percentage relative standard deviation of peak area and retention time. The result was calcu- lated as mg/100 g raw propolis. Total phenolic contents (TPC) of the etha- nol extracts were determined using Folin- Ciocalteu assay [30]. All phenolic compounds, including phenolic acids, flavonoids and an- thocyanins, in the ethanolic extracts of prop- olis resulted in the formation of a blue color complex with Folin reagents whose maximum absorbance can be read at 740 nm. Gallic acid was used as the reference standard compound, and the results were expressed as mg gallic acid per g propolis. Subsequently, 680 μL dis- tilled water, 20 μL ethanol propolis extracts and 400 μL of 0.2 N Folin-Ciocalteu regents were mixed in a tube, vortexed for 2 min. Next, 400 μL of Na 2 CO 3 (7.5%) was added and the mixture incubated for 2 h at room tempera- ture. Absorbance was measured at 760 nm at the end of the incubation period. All the mea- surements were performed in triplicate The reducing power ability of ferric tripyridyl- triazine (Fe-III-TPTZ) complex (FRAP) from the ethanolic extracts of the propolis samples was measured using reported methods [31] with some modifications. The test involved the reduction of ferric tripyridyltriazine (Fe-III-TPTZ) complex to a blue-colored Fe (II) TPTZ by antioxidant agents of samples. Working FRAP reagent was prepared by mix- ing 25 mL of 300 mM acetate buffer, pH 3.6, with 2.5 mL of 10 mM TPTZ solution in 40 mM HCl and 2.5 mL of 20 mM FeCl 3 · 6H 2 O solution. Next, 3 mL freshly prepared FRAP reagent and 100 μL of the samples were mixed and incubated for 4 min at 37°C, and the ab- sorbance was read at 595 nm against reagent blank containing distilled water. Trolox was used as a positive control to construct a ref- erence curve (62.5–1000 μM). FRAP values were expressed as μM Trolox equivalent of g propolis. The higher the FRAP value, the higher the antioxidant capacity of the samples. The scavenging of 2,2-diphenyl-1-picrylhy- drazyl (DPPH) radicals was assayed using re- ported method [32]. The method is based on the radical having a purple color which de- cays after interaction with antioxidant agents, turning yellow. The change in absorbance due to colors can be spectrophotometrically mon- itored at 517 nm. Briefly, 1.5 mL of the etha- nolic extract solution was mixed with 1.5 mL of 0.1 mM DPPH (dissolved in methanol), vor- texed and incubated for 50 min in the dark at room temperature until stable absorbance values were obtained. After the incubation pe- riod, the absorbance was recorded at 517 nm against a blank and control. The control solu- tion contained DPPH solution without sample. The results were expressed as SC 50 (mg/mL), calculated from the curves by plotting ab- sorbance values. The SC 50 values represent the concentration of the extract (mg/mL) re- quired to inhibit 50% of the radicals. The results were presented as mean values and standard deviations. Data and regression analysis were performed on Microsoft Office Excel 2013 (Microsoft Corporation, Redmond, WA, USA). Data were tested using SPSS (ver- sion 9.0 for Windows 98, SPSS Inc.). Statistical analyses of the results were based on the Kruskal-Wallis test and Pearson correlation analysis, a nonparametric test. Significance was set at p < 0.05. The TPC values of the 15 propolis samples collected from different areas of Azerbaijan (Figure 1) ranged between 10.94 and 79.86 mg GAE per gram of raw propolis (Table 1). Significant differences in TPC were deter- mined among the propolis samples (p<0.05). Propolis from the Qax region exhibited the highest amount of TPC (79.86 mg/g GAE), while the sample from the Astara and Şeki re- gions contained the lowest (10.94 mg/g GAE; 13.41 mg/g GAE). In addition to the Qax re- gion, propolis from Zerdap, Aktaş, Ismayıllı-I and Qusar also had high phenolic contents. In terms of the differences between propo- lis samples from the same regions, significant differences were determined in TPC levels be- tween Quaba I, II and III, between Nahcivan I and II, and between Ismayıllı I and II (p<0.05). The ability of the Fe (III)-TPTZ complex re- flects the total antioxidant capacity of plants as well as of propolis and other honeybee products. Under this method, it is assumed that the higher the FRAP value, the high- er the antioxidant activity. The FRAP val- ues calculated for the propolis samples are shown in Table 1. These ranged from 170 to 438 μM Trolox/g raw propolis. The high- est FRAP values were detected in the sam- ples from the Ismayılli (I), Zerdap, Qax, and Aktaş regions, and were higher than 400 μM Trolox equivalent of g propolis. The low- est FRAP values were detected in the sam- ples from the Astara, Şeki, and Ismayılli (II) regions, at below 200 μM Trolox equivalent of g propolis. The reducing power measure- ment for the samples showed a concentra- tion-dependent pattern. A positive correlation was detected be- tween the samples’ FRAP values and TPC (r2: 0.98), which suggests that total antioxi- dant activity of the propolis samples origi- nates from the phenolic substances within the propolis (Table 2). The antioxidant agents present in the propo- lis samples enhanced the decay of the purple color of the DPPH as they interacted with the radicals and scavenged these. The change in absorbance was monitored spectrophotomet- rically at 517 nm. The propolis samples from Zerdap, Quba (II), Aktaş, Ismayıllı (I) and Qusar exhibited higher DPPH radical-scav- enging activity with lower SC 50 values, within the range of 15–22 mg/ml (Table 1). As with TPC and FRAP values, significant differences were also determined in DPPH radical scav- enging activities in propolis specimens from the same regions (p<0.05). DPPH radical scav- enging activities in the propolis samples used in the study were correlated with amounts of TPC (r2: –0,79) and FRAP values (r2: –0,81). Fifteen reference standards, including gal- lic, protocatechuic acid, p-OH benzoic acid, catechin, chlorogenic acid, vanillic acid, caf- feic acid, epicatechin, p-coumaric acid, feru- lic acid, rutin, myricetin, quercetin, apigen- in, kaempferol and isorhamnetin were used to determine and quantify amounts of phe- nolics. All of the phenolic reference stan- dards used was present in ethanolic extracts at different amount and selected samples. Only chlorogenic and catechin were absent from all samples. Caffeic acid was the most abundant phenolic acid among all propolis samples, followed by coumaric acid and feru- lic acid. Protocathechuic, p - hydroxy benzoic acid, vanillic acid and gallic acid were found in some of the samples (Table 3). Apigenin was the major flavonoid present in the samples, while quercetin, isorhamne- tin and kaempferol were present at lower concentrations among the studied flavonoids, although fisetin was detected only in one sample, from the Şemkır region. Rutin was detected in a sample from Quba-2 (Table 3). Propolis is a multifunctional honeybee product consisting of exudates from plants mixed with beeswax. It is used by bees for various purposes, such as temperature insula- tion and beehive repairs, embalming dead or- ganisms, etc. It has been used since as early as 3000 BC [15]. During the last two decades re- searchers have become interested in its phar- macological agents and biological activities [33,34]. The bioactivity capacity of propolis largely depends on its phenolic contents, in- cluding phenolic acids, flavonoids, anthocy- anins, and several aromatic acids and esters [35]. As stated in the introduction, the phys- ical properties and chemical composition, as well as the bioactive potential, of propolis are highly dependent on their region of ori- gin. Such factors therefore need to be investi- gated by collecting samples from different re- gions. Several studies have been performed on propolis samples from different regions of the world in order to study their biological activi- ty potentials [1,6,28,32,36]. In this study, we evaluated the TPC, an- tioxidant activity and phenolic profile of Azerbaijan propolis. TPC in the ethano- lic propolis extracts ranged from nearly 10 to 80 mg GAE/g propolis, revealing signifi- cant differences among the propolis samples from the studied regions (Table 1). This dif- ference in TPC levels may be attributed to the geographical origin of the samples; howev- er, some ...
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The aim of this study was to evaluate the potential use of crop wastes (stems and leaves) from broccoli, beans and tomato to extract antioxidant polyphenols. Two different methods of extraction were performed: ultrasound (40 kHz) and conventional with 70 °C. In both methods, alkaline and acidic solvents were used. The polyphenol composition was ass...
This study aimed to investigate HPLC-DAD based chemical compositions, total phenolic compounds, antioxidant, enzyme inhibition and anti-cancer activities of three edible mushrooms: Clavariadelphus truncatus Donk, Craterellus tubaeformis (Fr.) Quél. and Hygrophoruspudorinus (Fr.) Fr. p-Hydroxy benzoic acid was specified as the main compound in C. tr...
Citations
... mg/kg), apigenin (177-1807.2 mg/kg), kaempferol (124-2159 mg/ kg) and isorhamnetin (75-593 mg/kg) [52]. Sixty-seven phenolic compounds were identified in Portuguese propolis [53]. ...
The aim of this study was to analyze the total phenolic content, phenolic profile, and antioxidant activity of honey, bee pollen, and propolis samples from Turkey's Black Sea Region. The total phenolic content of these bee products was found using Folin–Ciocalteu's method, and their antioxidant capacity was found using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and cupric ion reducing antioxidant capacity (CUPRAC) assays. Phenolic compounds of honey samples were characterized by high-performance liquid chromatography coupled to a photodiode array detector and mass spectrometer (HPLC–DAD–ESI-MS), while phenolic compounds of bee pollen and propolis samples were quantified ultra-fast liquid chromatography (UFLC). The total phenolic values for honey, bee pollen and propolis were 6.32–18.21 mg GAE/100 g, 547.64–769.4 mg GAE/100 gr, and 6096.1–11,564 mg GAE/100 gr, respectively. DPPH and CUPRAC values of honey, pollen and propolis were 11.05–21.38% and 0.38–1.48 µmol Trolox/g; 24.67–38.63% and 0.03–0.05 mmol Trolox/g; 11.81–34.12% and 0.47–0.89 mmol Trolox/g, respectively. About 30 different phenolic compounds were identified as quantitative. Our findings have shown that all bee products examined in the study are found to contain gallic acid, naringenin, and caffeic acid. In terms of total phenolic content and antioxidant activity, the bee product extracts were ranked as follows: propolis > bee pollen > honey. The high concentration of phenolic compounds in propolis explains its remarkable antioxidant effect.
... In the current study, 12 reference standards were used to determine and quantify the amount of phenolics ( Figure S2). The presence of GA, coumaric acid, rutin, vanillic acid, sinapic acid, and kaempferol documented herein (Table 4) have previously been commonly identified in propolis from Europe, Asia, and North and South America (Can et al., 2015;Coneac et al., 2009;Paulino et al., 2010). These compounds have also been found to be associated with the antioxidant potential in propolis. ...
... The highest amount of phenolic contents were found in propolis from the Qax region, which was rich in coumaric acid, kaempferol, and vanillic acid. It also exhibited a higher antioxidant capacity compared to propolis from other regions of Azerbaijan (Can et al., 2015). The identification of rutin (2.096 mg/g) and kaempferol (0.222 mg/g) from propolis collected from the west side of Romania showed a good correlation with the antioxidant activity (Coneac et al., 2009). ...
... Similar to the present study, 42.9-303 mg GAE/g of TPC has been reported from Chinese propolis (Ahn et al., 2007). TPC values varied between 31 and 72 mg GAE/g in Azerbaijan propolis in another study (Can et al., 2015). A previous study by Keskin et al. (2020) reported that TPC ranged from 28.8 to 80.4 mg GAE/ g in propolis samples from different regions of Turkey. ...
... P1 and P6 samples exhibited the highest FRAP, and also had the highest TPC and TFC values. The antioxidant capacity of bee products, such as honey, pollen, and propolis was reported to be related to their phenolic composition (Can et al., 2015;Mohammadzadeh et al., 2007). The results of the current study support this idea. ...
... A previous study investigated the antioxidant capacity of Azerbaijan propolis and reported FRAP values of 170.3-377.9 lM Trolox/g (Can et al., 2015). FRAP values were higher in the present study. ...
This study investigated various properties of raw propolis samples from Marmara region of Turkey. Total phenolic content (TPF), total flavonoid (TFC) values, condensed tannin content (CTC) and phenolic profiles of ethanolic extracts (70%) were analyzed. Antioxidant activities were tested using the Ferric (III) reduction/antioxidant capacity (FRAP) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging methods. Phenolic profiles of the samples were analyzed using high performance liquid chromatography (RP-HPLC-UV) method. TPC, TFC and CTC values in the samples ranged from 52.9 to 203.5 mg GAE/g, 13.6 to 43.9 mg QUE/g, and 2.0 and 2.9 mg CTE/g, respectively. Caffeic acid phenyl ester (CAPE), chrysin, pinocembrin, rutin, and t-cinnamic acid were detected as the common phenolic components in all samples. In conclusion, Marmara region propolis is a rich source of bioactive compounds, especially flavonoids with antioxidant properties.
... The extract (P3) from Sibi was seen to have greatest antioxidant activity with smallest IC 50 value of (27:07 ± 0:73 mg mL −1 ), and the sample (P8) collected from Jaffar Abad with highest IC 50 value (94:43 ± 2:07 mg mL −1 ) showed lowest antioxidant potential as presented in (Table 3). According to Zehra, Yildiz, Şahin, Asadov, and Kolayli [23], the antioxidant potential of propolis extracts have a positive correlation with their phenolic, flavonoids, and other bioactive contents. Recentlym Shahbaz et al. [1] reported the DPPH free radical scavenging activity of propolis up to 70% which is in accord to the findings of current study. ...
Propolis is a well-known resinous natural substance collected by honeybees (Apis mellifera L.) from plants exudations. Variations in chemical composition of propolis are due to different sources from which it is collected and change in climate and geographical location. In this study, different propolis samples were collected from different regions of Balochistan and examined for its chemical composition, total phenolics and total flavonoid contents, and antioxidant potential by using DPPH radical scavenging assay and antimicrobial activity. Bioactive components analysis revealed the presence of steroids, carbohydrates, flavonoids, coumarins, cardiac glycosides, quinones, anthraquinones, terpenoids, tannins, and phlobatannins at different levels. The total phenolics contents were ranged from 2.9343 ± 1.247 to 6.0216 ± 2.873 mg GAE g-1, and flavonoid contents were found to be 0.1546 ± 0.087 to 0.6586 ± 0.329 mg QE g-1, respectively. The antioxidant ability of each extract was analyzed by their concentration having 50% inhibition ( I C 50 ). The propolis sample P3 possessed lower I C 50 27.07 ± 0.73 mg mL−1 with higher % inhibition of DPPH radical, and P8 showed lower % inhibition by having I C 50 84.43 ± 2.07 mg mL−1. The antibacterial activity of all samples was analyzed against a wide group of bacteria including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumonia and propolis extract (P4) was highly active against Klebsiella pneumoniae with the maximum diameter of zone of inhibition 20.33 ± 1.52 mm, and propolis extract (P3) showed maximum zone of inhibition against Escherichia coli 19.06 ± 1.90 , while propolis extract (P2) was found less active with minimum diameter of zone of inhibition 7.46 ± 1.50 mm . The antifungal activity of extract was considered as active against the fungal species. Propolis extract (P3) showed 82% of zone of inhibition against Aspergillus Niger, and propolis extract (P1) was highly active against Aspergillus parasiticus with 80% of zone of inhibition. By comparing the vibration frequencies in wave numbers of the sample spectrograph acquired from an FTIR spectrophotometer, the functional groups present in the extracts were identified. The presence of seven elements (Fe, Zn, Mn, Ni, Pb, Cd, and Cr) was analyzed through atomic absorption spectrophotometer. The obtained concentrations were within the permissible ranges established by the World Health Organization. The GC-MS analysis revealed the presence of 80 different compounds belonged to different classes. The obtained results confirmed the imperative potential of propolis which can be used in various biological applications.
... Phenolic composition of the bee products is very important and is mostly determined by chromatographic analysis techniques. HPLC-UV and HPLC-PDA are the most used practical techniques, preparation of the bee products for analysis and validation of phenolic components are important analytical studies [11][12][13]. The aim this study was to obtain a quick, reliable, and simplified methodology for the detection and quantification of phenolic acids and flavonoids in some bee products. ...
In this work, a reverse phase high-performance liquid chromatographic method (RP-HPLC) with photo diode array (PDA) at 4 different wavelengths (250, 280, 320 and 360) for the determination of some polyphenols in some bee products (honey, pollen, and propolis) is developed. The analyses were carried out on C18 column (250 mm x 4.6 mm, 5 µm; GL Sciences), and the gradient program was used with a mobile phase A reservoir with 70% acetonitrile and the B reservoir with 2% acetic acid. The HPLC method was founded that limit of detection was in the range 0.022-0.0908 µg/mL; the limit of quantification was in the range 0.074-0.3027 µg/mL, all calibration curves were linear R 2 >0.995 within the range, the recovery range was 91.43-111.37% for 10 ppm and 98.44-101.68% for 40 ppm and relative error levels 0.0330-0.0290 respectively. The developed method was applied to some bee products available on the Turkey market. The study aimed at phenolic profiles of the bee products extracts were revealed by using 25 phenolic standards. The proposed method was optimized, quickly, and simple validated by evaluating the linear range, the limits of detection and quantification, the accuracy, the precious, the repeatability, and recoveries suitable for the phenolic analysis. It is concluded that evaluated and the quantitative determination of the bee products can be made quickly and reliably with the optimized method. HIGHLIGHTS • RP-HPLC-PDA method was developed for the simultaneous determination of 25 phenolic compounds. • The reliability of the developed method was confirmed by the validation test. • An HPLC method has been developed to quantify phenolic acid and flavonoids in some bee products. • The method showed good results in a run time of only 60 min. Kara, Y; et al. 2
... Taking into account the predominance of important flavanoids in local propolis [6] samples during the biochemical study of bee propolis produced in Azerbaijan, it can be concluded that propolis is a very effective agent against respiratory viral diseases. Predominance of flavonoids such as quercetin and campherol, which have specific antiviral activity in locally produced propolis, actualizes the use of the product as an antibacterial and antiviral agent. ...
... The purpose of using local products is to ensure that propolis is rich in antimicrobial and antiviral chemicals [6]. ...
Currently, propolis is used as an antibacterial, anti-inflammatory, antiviral, antioxidant, analgesic, anti-tumor, anti-mucor, cytotoxic, antiseptic, antimutagenic, antihepatotoxic agent. The important antimicrobial and antiviral properties of mountain mint (Ziziphora Clinopodioides), one of the beneficial herbal plants in our flora, have been scientifically studied and important results obtained. The main goal of the study is to use bee wax, natural bee honey and mountain mint, which combine antimicrobial and antiviral properties to prevent respiratory infections. This necessiates the use of locally produced bee wax as an antibacterial and antiviral agent. The obtained materials have been developed and applied in the form of oral and nasal sprays. The active parts of propolis have been included in the solvent, and the residual and insoluble parts have been separated in the spray prepared for respiratory diseases with the addition of 10% extract of propolis in 70° ethyl alcohol, alcoholic extract of mountain mint and honey. To prepare a 10% propolis-based spray, the following ingredients have been used: 90 g of 10% extract of propolis in 70% alcohol; 5 grams of natural honey; aqueous extract of mountain mint-5 g. The obtained product was packed in special spray cans and used by volunteers for several months. No infectious diseases of the respiratory tract have been detected during the period of use.
... However, very little is known about the chemical composition, biological effects, and antimicrobial properties of the propolis collected by the Arabian honey bee from the Asir region of Saudi Arabia. Although it is obvious that the propolis is a plant-originated bee product, its chemical composition, physical properties, and biological activities vary according to available local flora, harvesting season, and type of bee species [19,20]. Due to the adeptness in the local environment, foraging behavior on local flora, and morphological characteristics of Arabian honey bee, we hypothesize that the propolis produced by this race has distinctive composition and biological activity. ...
... In a similar way, the stock solution for each extract of propolis was composed by deliquescing 1 gram of propolis extract in 100 mL of 99 percent methanol. Then, this stock solution of propolis extract was used to make their respective serial dilutions of 5,10,20,40,60,80,100,150,200, and 250 micrograms per milliliter. Standard ascorbic acid (SAA) was used as a reference, and it was dissolved in 99 percent methanol to provide a stock solution containing identical amounts of Met, BuT, Hex, and Eac. ...
Propolis (bee glue) is a complex, phyto-based resinous material obtained from beehives. Its chemical and biological properties vary with respect to bee species, type of plants, geographical location, and climate of a particular area. This study was planned with the aim of determining the chemical composition and to investigate various properties (against oxidants and microbes) of different extracts of Saudi propolis collected from Arabian honey bee (Apis mellifera jemenitica) colonies headed by young queens. Chemical analysis of propolis extracts with different solvents, i.e., ethyl acetate (Eac), methanol (Met), butanol (BuT), and hexane (Hex) was done through colorimetry for the total phenolic content (TPC) and total flavonoid content (TFC) evaluation. For separation and extensive characterization of the Met extract, chromatography and 1H NMR were deployed. Six different microorganisms were selected to analyze the Saudi-propolis-based extract’s antimicrobial nature by measuring zones of inhibition (ZOI) and minimum inhibitory concentration (MIC). Molecular docking was done by utilizing AutodDock, and sketching of ligands was performed through Marvin Chem Sketch (MCS), and the resultant data after 2D and 3D clean were stored in .mol format. The highest TFC (96.65 mg quercetin equivalents (QE)/g of propolis and TPC (325 mg gallic acid equivalents (GAE)/g of propolis) were noted for Met. Six familiar compounds were isolated, and recognition was done with NMR. Met extract showed the greatest 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) free radical scavenging activity and Ferric Reducing Antioxidant Power (FRAP). Met showed max microbial activity against Staphylococcus aureus (ZOI = 18.67 mm, MIC = 0.625 mg/mL), whereas the minimum was observed in Hex against E. coli (ZOI = 6.33 mm, MIC = 2.50 mg/mL). Furthermore, the molecular docking process established the biological activity of separated compounds against HCK (Hematopoietic cell kinase) and Gyrase B of S. aureus. Moreover, the stability of protein–ligand complexes was further established through molecular dynamic simulation studies, which showed that the receptor–ligand complexes were quite stable. Results of this research will pave the way for further consolidated analysis of propolis obtained from Arabian honey bees (A. m. jemenitica).
... It may be concluded that this difference is due to different extraction conditions. In another study, 95% ethanol was used for the extraction of propolis obtained from Azerbaijan, the authors reporting DPPH cleaning activity in the range of 15-198 mg/mL (Can et al. 2015). Another propolis study from different locations in Indonesia reported that the IC 50 values of the DPPH test for Trigona sp. ...
The biological activity of propolis is mostly due to its polyphenols. Raw propolis is consumed after being extracted in various solvents, ethanol being the most commonly used. However, the ideal percentage and ratio of ethanol for use in the preparation of propolis extracts are uncertain. The aim of this study is to compare the phenolic compositions and antioxidant activities of propolis extracts extracted with different ethanol concentrations and extraction conditions. Two different extraction methods were used, normal and ultrasonic extraction. The propolis extracts were evaluated in terms of total phenolic content, total flavonoid content, ferric reducing/antioxidant power,1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, and phenolic compositions using RP-HPLC–PDA. The propolis extracts with the highest activity were determined beforehand by preparing ethanol–water mixtures at different percentages, ranging from 0 to 99.9%. The most suitable propolis-to-solvent ratio was then determined. The optimum ethanolic percentage varied between 60 and 70%, the ultrasonic extraction process increased the efficiency, and the ideal solvent-propolis ratio (w/v) ranged between 1:5 and 1:10.
... The TPC value of the pollen extract in this study was similar to those in the range of 9.15 to 462.02 mg gAE/g pollen [38,[46][47][48][49][50]. Also, the TPC value of the propolis extract was similar to those reported in various propolis samples from different regions with TPCs ranging from 31.00 to 302.00 mg gAE/g propolis [42,[51][52][53][54]. numerous studies on TPC analyzes in bee products describe various ranges for total phenolics of pollen and propolis from different geographical origins, depending on the standard (gallic acid, pinocembrin, chlorogenic acid) and solvent (methanol, ethanol) used [55][56][57][58]. ...
Bee pollen and propolis are considered as health-promoting foods with many therapeutic(antibacterial, antifungal and antioxidant) activities. This study analyzed the phenolic profile andthe antioxidant properties of Turkish bee pollen and propolis ethanolic extracts and assayedtheir antiproliferative effect on myeloma cells and in vitro antibacterial activity againstStaphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The antibacterial activityassays included agar well diffusion and microdilution methods. The phenolic profile and severalaromatic compounds of the extracts were determined by high-performance liquid chromatographywith diode-array detection (HPLC-DAD). The antiproliferative activity on myeloma cells wasdetermined by MTT test. The propolis extract had higher total phenolic content (TPC), free-radicalscavenging activity (DPPH) and half-maximal inhibitory concentration (IC 50) than the pollenethanolic extract. Benzoic and cinnamic acid were the most abundant aromatic substances inthe pollen and propolis extracts, respectively. The IC 50 values of pollen and propolis extracts onmyeloma cells were 1.49% and 2.88%, respectively. The propolis extract was active against S.aureus and E. coli, but not P. aeruginosa. The pollen extract presented no detectable inhibitionzone against the three bacterial strains. The minimum inhibitory concentration (MIC) of bothextracts for S. aureus and E. coli was 0.63% (w/v ). The minimum bactericidal concentration (MBC)of the propolis extract was 1.25% for S. aureus and E. coli. MIC could not be determined for thepollen extract in the tested bacteria. The pollen and propolis extracts did not exert antimicrobialactivity against P. aeruginosa up to 2.5% concentration.
... During the research, the quantity of vanillic acid was significantly higher than caffeic acid. However, the studies of other researchers showed that the quantity of caffeic acid in propolis extract was higher than vanillic acid (14). Such differences may be due to different propolis collection sites and different types of plants used for making propolis (15). ...
The aim of this study was to produce propolis extracts, assess their quality and effect on skin cells and determine the penetration of active ingredients from designed semi-solid topical formulations. The use of higher-concentration ethanol and a larger amount of raw material allows extracting a larger quantity of active ingredients from raw propolis. Ultrasound extraction is an effective method for the production of aqueous extracts of propolis. The results show that depending on concentration, propolis extracts reduce the viability of keratinocytes. The phenolic compounds under observation penetrated the epidermis and dermis from designed formulations. The base of semi-solid formulation influences the efficacy of propolis preparations. The overall quantity of phenolic compounds that penetrated the skin was around 2 % from the ointment and 1.5 % from the cream.
