key: cord-1053966-dd6259rk authors: Özkök, Aslı; Keskin, Merve; Tanuğur Samancı, Aslı Elif; Yorulmaz Önder, Elif; Takma, Çiğdem title: Determination of antioxidant activity and phenolic compounds for basic standardization of Turkish propolis date: 2021-04-16 journal: Appl Biol Chem DOI: 10.1186/s13765-021-00608-3 sha: a22b368fe3bcf57085d757d3531935c9814d667a doc_id: 1053966 cord_uid: dd6259rk This study aimed to determine the standard amount of antioxidant content and compounds of the propolis for the standardization of propolis. For this purpose, the total flavonoids, total phenolic, CUPRAC antioxidant capacity content and the diversity of phenolic and flavonoid components of these propolis samples were found by HPLC determined at the 23 propolis samples which were collected different regions of Turkey. Beside that, the similarities and differences of these 23 provinces to each other according to their antioxidant capacities were investigated by multidimensional scaling analysis. The total flavonoid content in the propolis samples were determined between 21.28 and 152.56 mg CE/g. The total phenolic content in the propolis samples was found between 34.53 mg and 259.4 mg GAE/g. CUPRAC antioxidant capacity of the propolis samples and antioxidant range was found from 95.35 to 710.43 mg TE/g. Also, 4 flavonoid [Quercetin (min.1.12–max.4.14 mg/g), Galangin (min.0.72–max.40.79 mg/g), Apigenin (min.1.07–max.17.35 mg/g), Pinocembrin (min.1.32–max.39.92 mg/g] and 6 phenolic acid [Caffeic acid (min.1.20–max.7.6 mg/g), p-Coumaric acid (min.1.26–max.4.47 mg/g), trans-Ferulic acid (min.1.28–max.4.92 mg/g), Protocatechuic acid (1.78 mg/g), trans-Cinnamic acid (min.1.05–max.3.83 mg/g), Caffeic Acid Phenethyl Ester (CAPE) (min.1.41–max.30.15 mg/g)] components were detected as mg/g, in different ratios in propolis samples collected from different regions. The feature of this study, so far, is to have the maximum number of samples representing the Turkish propolis, and so is thought to help to national and international propolis standard workings. Propolis or bee glue is a substance containing a mixture of wax and resin collected by honeybees (Apis mellifera L.) from different parts (tree and flower buds, sap flows, mucilage, latex, resin etc.) of plants [1] [2] [3] [4] [5] . Honeybees, collect propolis from protective resins of flowers and trees buds with their lower jaws and carry them to the hive in the pollen sacs on their hind legs. They also add substances from their bodies during the resin collection and modeling phase. The collected propolis ensures that the hive is protected from all kinds of diseases and prevents the entrance of insects and animals by closing the small openings in the hive [6, 7] . Propolis, generally consists of 50% balsam, 30% wax, 10% essential oils and 5% pollen. Since the 1950s, scientists have started to isolate important components in propolis with the help of new analytical methods and have shown people that they have many benefits [7] . Propolis and its many compounds show a wide variety of biological and pharmacological activities [8] . It is a supplementary and supportive food that has become popular all over the world in 2000s, thanks to its antimicrobial [9] [10] [11] , antioxidant [12] , anticancer [13] , antiulcer [14] , antidiabetic [15] , anti-inflammatory [16] , antigenotoxic [17] and antiviral [18] [19] [20] [21] [22] activities. Propolis, which is Open Access *Correspondence: asozkok@gmail.com; aozkok@hacettepe.edu.tr 1 Bee and Bee Products Application and Research Center (HARUM), Hacettepe University, Ankara, Turkey Full list of author information is available at the end of the article also used in traditional and complementary medicine, is an important bee product and it is used in "Apitherapy", which is a treatment method with bee products [23] [24] [25] [26] . In particular, it has been determined by many scientists that it is effective on the corona virus in the COVID-19 pandemic in 2020 [18] [19] [20] [21] [22] . Therefore, people's interest in propolis has increased more. The content of propolis varies according to the plant source and when it is collected [27] . In addition, the variety in the content of beeswax affects the chemical composition of the raw propolis [28] . More than 300 different compounds have been detected to date in the propolis [29] [30] [31] . The majority of these components are phenolic acids and flavonoids [32] . Plant species in a geographic region determine the amount and type of compounds found in the propolis. In a study in New Zealand, dihydroflavonoids, pinobanksin and pinocembrin accounted for approximately 70% of the flavonoids in the analyzed samples. However, in a similar study conducted in Brazil, Uruguay and China, the dihydroflavonoid in the samples was 10% less than the samples in New Zealand [7] . Moreover, it has been found to vary the amount of flavonoids and phenolic contents in propolis samples collected from different regions of Turkey [33] [34] [35] . The most important pharmacological activity elements in propolis are flavones, flavanols and flavanones, which are common names flavonoids, and various phenolics and aromatics [7] . Propolis contains hundreds of different substances with antimicrobial properties, about 80 of which are flavonoids [1, [36] [37] [38] [39] . Phenolic compounds in propolis are found in large quantities at about 1 in 3, while flavonoids are only up to 10% (w/w) of the concentrated form of propolis [40, 41] . Among them, pinocembrin and galangin provide antibacterial activity. It also has pinocembrin, fungicidal and local anesthetic properties [36] . Cinnamyl alcohol, cinnamic acid, vanillin, benzyl alcohol, benzoic acid, caffeic acid, coumaric acid and ferulic acid are some phenolics found in propolis [7] . In recent years, studies on propolis have found that pinocembrin, pinobanksin, quercetin, chrysin and galangin flavonoids and caffeic acid and coumaric acid phenolic acids are the most common components in propolis [9, [42] [43] [44] . In this study, total flavonoids and total phenolic compounds content and total antioxidant capacity was determined at the 23 propolis samples which were collected different regions of Turkey. In addition, the diversity of phenolic and flavonoid components of these propolis samples was found by HPLC and compared with other studies. Beside that, the similarities and differences of these 23 provinces to each other according to their antioxidant capacities were investigated by multidimensional scaling analysis. On the other hand, there are marketing difficulties for propolis because of the lack of the national or international propolis standard. For this reason, national and international standard studies will progress more easily thanks to studies that reflect the general characteristics of country propolis, such as this study, and this will solve marketing problems. Propolis samples were collected from 23 different cities in Turkey in 2019. Propolis traps placed in the hives in spring season were harvested end of the summer. Traps were kept in the freezer and were removed from the freezer while preparing propolis extracts (Fig. 1) . About 30 mL of 70% ethanol solution is added to the powdered 1 g of raw propolis sample, shaken for 24 h at room temperature in shaker. The upper part is filtered through coarse filter paper and transferred to 100 mL flask. The process is repeated by adding 30 mL of 70% ethanol solution to the remaining solid part. The supernatant is added to the flask in which the first extract is collected, completed to 100 mL with 70% ethanol solution [45] . Total phenolic content was found by modifying the Meda et al. [46] , method. According to this method, the working curve was prepared using varying concentrations (0.25-0.13-0.06-0.03-0.02) of the Gallic acid (GAE) standard (0.5 mg/mL) for calibration. The dilution appropriate for the sample was done with extraction solution and 200 µL of diluted sample was put into the tubes for analysis. For the blank, 200 µL extraction solution was substituted for the sample. For the working curve, 200 µL tubes of varying concentrations of gallic acid were placed in tubes. Then, 1.5 mL of 0.2 N Folin solution was added to the tubes and left for 5 min. The tubes were then vortexed by adding 1.2 mL of NaCO 3 (7.5%) solution. It was incubated in the dark at room temperature for 90 min. Finally, the UV-spectrophotometer was read against the curve at a wavelength of 765 nm. Total flovonoid content was found by modifying the Dewanto et al. [47] , method. According to this method, the working curve was prepared using varying concentrations (0.1-0.08-0.05-0.02-0.01) of the catechin (CE) standard (1 mg/mL) for calibration. The dilution appropriate for the sample was done with extraction solution and was placed in tubes from 1 mL of diluted sample for analysis. For the blank, 1 mL of extraction solution was substituted for the sample. For the working curve, 1 mL tubes of varying concentrations of catechin were placed in each tube. The timing is started with the stopwatch and 300 µL of 5% NaNO 2 (at t = 0 time), 300 µL of 10% AlCl 3 (at t = 5 time), 2 mL of 1 M NaOH (at t = 6 time) and finally 2.4 mL distilled water was added and vortexed. Without delay, the UV-spectrophotometer was read against the curve at a wavelength of 510 nm. CUPRAC antioxidant capacity was detected according to the Apak et al. [48] , method. According to this method, the working curve was prepared using varying concentrations (0.5-0.25-0.13-0.06-0.03) of the trolox standard (1 mg/mL) for calibration. Dilution appropriate to the sample was done with extraction solution and 100 µL of diluted sample was put into the tubes for analysis. For the blank, 100 µL extraction solution was substituted for the sample. For the working curve, 100 µL of the varying concentrations of trolox were put into the tubes. Then 1 mL of CuCl 2 , 1 mL of neocuproin, 1 mL of NH 4 CH 3 COO and 1 mL of pure water were added and vortexed, respectively. Incubated for 1 h at room temperature in the dark. Finally, a 450 nm wavelength reading was made on the UV-spectrophotometer. Modified Aliyazıcıoglu et al. [33] , method was used for propolis HPLC component analysis. Powdered 1 g raw propolis sample is weighed into a 50 mL falcon tube. Add 30 mL of 70% ethanol solution, shake for 24 h on a shaker. After centrifugation, the upper phase is transferred to a 100 mL volumetric flask. The shaking process is repeated once more. The upper phase is added to the volumetric flask where the first extract is collected, and complete to 100 mL with 70% ethanol solution. The solution is filtered through a PVDF syringe filter and transferred to the vial and 20 µL is injected to the HPLC device. VWR Hitachi HLC-UV Detector (UV 280 nm) and Supelcosil LC-18 25 cm × 4.6 mm, 5 µm column is used in the HPLC. Mobile phase A: 99% Ultra pure water: 1% Acetic Acid and Mobile phase B: 100% Methanol is used and flow was 0.9 mL/min, a linear gradient was applied by increasing the B mobile phase from 10 to 90%. Multidimensional scaling (MDS) is a way to visualize the level of similarity between binary distances between a series of n objects or units. With multi-dimensional scaling analysis, objects are displayed in a k-dimensional (k > p) space based on the distance determined by p variable between n observations or units [49] . In this study, the similarities between the provinces according to the variables phenolic, flavonoid, and CUPRAC parameters were investigated using the multidimensional scaling analysis with Euclidean distance model. The similarity matrix obtained based on the variables in question was used to show the proximity and distance of the provinces to each other. The differences between provinces in relation to flavonoid was researched with One-Way analysis of variance (ANOVA). The Kruskall Wallis test was used for phenolic and CUPRAC because of normality assumptions are not valid. The differences of group means were detected by Duncan and Bonferroni multiple comparison test, parametric and nonparametric analysis, respectively. The IBM SPSS v25 program was used all statistical analysis. The total flavonoid content in the propolis samples were determined between 21.28 and 152. 56 Fig. 2 ). Many researchers have determined the total flavonoid value in propolis. Some of these researchers and their conclusions are as follows: Zarate et al. [50] in Mexico; 13-379 mg QE/g, Ozdal et al. [51] in Turkey; 522.71 mg QE/g, Narimane et al. [52] , in Algeria; 0.57-3.53 mg QE/g, Wang et al. [53] in South Korea; 21-50 mg QE/g and for Brazilia, China and Australia; 33-53 mg QE/g, Socha et al., [54] in Poland; 35.64-62.04 mg QE/g and Bonvehi and Gutierrez [55] in Spain; 72-161 mg QE/g. These results are compatible with our study. The total phenolic content in the propolis samples was found between 34.53 mg GAE/g and 259. 4 Fig. 2 ). There are many studies on total phenolic compound in propolis. Some of them are as follows: Zarate et al. [50] in Mexico; 68-500 mg GAE/g, Ozdal et al. [51] in Turkey; 314.36 mg GAE/g, Narimane et al. [52] in Algeria; 0.81-8.97 mg GAE/g, Wang et al. [53] in South Korea; 49-239 mg GAE/g and Table 1 The ANOVA means and standart errors for flavonoid, and Kruskall Wallis mean ranks for phenolic and cuprac according to provinces Different letters in the same columns show statistically differences between means (p < 0.05) CUPRAC method gives information about reductive capabilities of propolis extracts and based on reduction of Cu +2 to Cu + by antioxidants [52] . Fig. 2) . Researchers found the CUPRAC value in propolis in different countries respectively: Bayram et al. [56] in Turkey; 282.8 mg TE/g, Ozdal et al. [51] in Turkey; 1184.94 mg TE/g, Narimane et al. [52] in Algeria 8 µM TE/g, Daraban et al. [57] in Romania; 12404-35721 µM TE/100 mL. These results are similar to our study results. The results of the examination according to the similarities and differences of all provinces according to Flavonoid, Phenolic and CUPRAC antioxidant capacity contents are given in Fig. 3 . After several dimensional scaling analysis, two-dimensional (k = 2) scaling was determined the best because of giving the lowest The Kruskal's stress value and higher the coefficient of determination (R 2 ), as 0.004 and 0.99, respectively. Therefore, the results were given, and comments were made on twodimension scaling. The stimulus coordinates of provinces and configurations of provinces showed that Muğla, Isparta, Düzce, Rize, Adana and Maraş were found similar, Tekirdağ, Karaman, Kocaeli, Bolu, Ankara, Balıkesir, Giresun and Kastamonu were found similar; Zonguldak, İstanbul, Kırklareli, Bursa, Sakarya, Amasya and Ordu were found similar and Konya and İzmir were found similar among each other. Optimal two-dimensional configuration of provinces based on stimulus coordinates was illustrated in Fig. 3 . Propolis has many biological and pharmacological activities thanks to its large number of phenolic and flavonoid components [33] . For the HPLC method validation of the study, the repeatability, reproducibility, recovery, linearity, limit of detection limit (LOD) and limit of quantitation (LOQ) validation parameters were examined and presented in Table 2 . In this study, 4 flavonoid [quercetin (min.1.12max.4.14 mg/g), galangin (min.0.72-max. 40 .79 mg/g), (Table 3 , Fig. 4 ). Cunha et al. [58] found caffeic acid, p-coumaric acid and ferulic acid in all of the extracts prepared in their study using different solvents to determine the phenolic content of Brazilian propolis. Also, Choi et al., [59] stated that in many propolis samples obtained from Korea contains caffeic acid (min.1.0-8.7 mg/g), p-coumaric acid (min.1.2-7.1 mg/g), ferulic acid (min.0.5-1.9 mg/g), apigenin (min.0.6-2.4 mg/g), pinocembrin (min.1.5-87.8 mg/g) and galangin (min.4.9-max.26.3 mg/g). These results are in line with our results. On the other hand, Lagouri et al., [60] 19 .06 mg/g) at the Italian propolis samples. Also, these results close to our study results. As a result, in this study, we found caffeic acid, caffeic acid phenethyl ester (CAPE), galangin and pinocembrin as major components for the Turkish propolis. Because these components have been detected in almost all provinces and these components can be used for quality determination and standardization of Turkish propolis. As a similar, Sorucu and Oruc [62] determined pinocembrin, CAPE, caffeic acid highest amounts at the propolis samples from the northwest of Turkey. As a result, the content of raw propolis varies according to the botanical origin of the region where it is obtained. Turkey, where they grow different plant species, is a country with rich botanical resources. Because, there are three phytogeographical regions in Turkey (Euro & Siberian, Mediterranean, Irano & Turanian) and the plant diversity varies from region to region [63] . So, there are about 12,000 plant species in Turkey and 3000 of them are endemic. About 500 plant species are nectar plants and are preferred by honeybees [64] . Since propolis is in very different phytogeographic regions in the 23 cities studied, the botanic origin varies. For this reason, propolis contents also differ greatly and it is very important to making content analysis for propolis standardization. In this study, total phenolics, total flavonoids and antioxidant capacity amounts were determined and compared statistically at the propolis samples, which were collected from different regions of Turkey. By illuminating the phenolic and flavonoid components contained in propolis samples, components [caffeic acid, caffeic acid phenethyl ester (CAPE), galangin and pinocembrin] that could be markers for Turkish propolis were determined. Thus, for the basic standardization of Turkish propolis, the range in which the total phenolic substance and flavonoid substance amounts should be and the components it should contain were determined. These compounds can be used in the marketing quality control of Turkish propolis. High performance liquid chromatografic analysis of flavonoids from propolis Propolis: a wonder bees product and its pharmacological potentials Biological properties and therapeutic applications of propolis The chemical analysis of propolis, which are produced in western of Turkey Determination of biochemical profile of bilecik propolis Propolis: a review Value-added products from beekeeping Other products of the hive. In the hive and the honeybee. Editorial Graham In vitro antimicrobial activity of propolis samples from different geographical origins against certain oral pathogens Antibacterial activity of bee propolis samples from different geographical regions of Turkey against two foodborne pathogens, Salmonella enteritidis and Listeria monocytogenes In vitro antimicrobial assessment of Cuban propolis extracts Chilean propolis: antioxidant activity and antiproliferative action in human tumor cell lines The immunomodulatory and anticancer properties of propolis Evaluation of antiulcer activity of the main phenolic acids found in Brazilian Green Propolis Potential antidiabetic and hypolipidemic effects of propolis extract in streptozotocininduced diabetic rats Identification and quantification of phytochemical composition and anti-inflammatory and radical scavenging properties of methanolic extracts of Chinese propolis Genotoxic and antigenotoxic evaluation of propolis by using in vitro bacterial assay systems Back to the basics: propolis and COVID-19 Propolis and its potential against SARS-CoV-2 infection mechanisms and COVID-19 disease The effect of propolis supplementation on clinical symptoms in patients with coronavirus (COVID-19): a structured summary of a study protocol for a randomised controlled trial Molecular interaction analysis of Sulawesi propolis compounds with SARS-CoV-2 main protease as preliminary study for COVID-19 drug discovery Molecular interaction analysis of Sulawesi propolis compounds with SARS-CoV-2 main protease as preliminary study for COVID-19 drug discovery Apitherapy. The complete guide to complementary therapies in cancer care: essential information for patients, survivors and health professionals Effect of Turkish propolis extracts on proteome of prostate cancer cell line Cytotoxic effect of Turkish propolis on liver, colon, breast, cervix and prostate cancer cell lines Effects of propolis in an experimental rat model of allergic rhinitis Propolis research and the chemistry of plant products Bees and beekeeping. Science, practice and world resources Studies on the constituents of yellow Cuban Propolis: GC-MS determination of triterpenoids and flavonoids Beneficial effects of propolis on human health and neurological diseases Recent advances in the chemical composition of propolis Antibacterial diterpenic acids from Brazilian propolis Properties of phenolic composition and biological activity of propolis from Turkey Standardization of Propolis, is it possible? Profiling of Turkish propolis subtypes: comparative evaluation of their phytochemical compositions, antioxidant and antimicrobial activities A study on flavonoids of propolis The composition and plant origins of propolis: a report of work at Oxford Propolis: chemical composition, biological properties and therapeutic activity Comparison of the flavonoid aglycone contents of Apis mellifera propolis from various regions of Brazil The composition, active components and bacteriostatic activity of propolis in dietetics Characterisation of flavonoids in three hive products: bee pollen, propolis, and honey Phenolic characterization of Northeast Portuguese propolis: usual and unusual compounds Chrysin: a histone deacetylase 8 inhibitor with anticancer activity and suitable candidate for the standardization of Chinese propolis Chemical profiling and chemometric analysis of South African propolis Standard methods for Apis mellifera propolis research Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC method Multidimensional scaling Flavonoids, phenolic content, and antioxidant activity of propolis various areas of Guanajuato Evaluation of Turkish propolis for its chemical composition, antioxidant capacity, anti-proliferative effect on several human breast cancer cell lines and proliferative effect on fibroblasts and mouse mesenchymal stem cell line Correlation between antioxidant activity and phenolic acids profile and content of Algerian propolis: influence of solvent Relationship between total phenolic contents and biological properties of propolis from 20 different regions in South Korea Phenolic composition and antioxidant activity of propolis from various regions of Poland Antioxidant activity and total phenolics of propolis from the Basque Country (Northeastern Spain) Cevahir Öz G (2020) Screening for antioxidant capacity, pollen types and phytochemical profile by GC/MS and UHPLC from propolis The evaluation of antioxidant capacity of propolis originating from Western Romania Factors that influence the yield and composition of Brazilian propolis extracts Antioxidant properties and phenolic composition of propolis from diverse geographic regions in Korea Antioxidant properties and phenolic composition of Greek propolis extracts Effectiveness of different analytical methods for the characterization of propolis: a case of study in Northern Italy Determination of biologically active phenolic compounds in propolis by LC-MS/MS according to seasons and altitudes The floristic regions of Turkey and a geographical approach for anatolian diagonal Turkey's nectarious plants, pollen, honey Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.