1. Introduction
Honeybees (Apis mellifera) are known to produce propolis, a natural substance composed of collected resinous exudates, flower buds, and various plant parts. The composition of propolis is approximately 50% resins (including flavonoids, phenolics, and their esters), 5% pollen, 30% wax, 10% essential oils, and 5% other organic compounds (e.g., vitamins) ( 1 - 5 ). A more detailed breakdown reveals the presence of polyphenols (flavonoid aglycones, phenolic aldehydes, phenolic acids and esters, ketones, and alcohols), sesquiterpene quinones, coumarins, steroids, amino acids, and inorganic compounds ( 1 , 6 ). It is important to note that the content of propolis varies depending on its geographical and botanical origins ( 1 , 7 ). Propolis is a substance with a wide range of biological activities, making it a valuable component in traditional medicine and healthcare. These activities include anticancer, anti-inflammatory, antibacterial, antibiotic, antioxidative, antiviral, and anesthetic properties ( 8 - 12 ). The anti-oxidative effects of propolis contribute to the reduction of cellular damage and oxidative stress ( 12 - 14 ). Erel (2004) proposed a novel Total Antioxidant Status (TAS) method, which has been shown to be more efficient than traditional methods for evaluating antioxidant capacity. These traditional methods are often time-consuming and expensive. This method utilizes an automatic measurement system to determine Total Oxidant Status (TOS) and derive the Oxidative Stress Index (OSI) as a novel indicator of oxidative stress. The OSI is derived from the TAS and TOS values ( 16 ). The recent surge in research underscores the mounting interest in the significance of natural antioxidants for human health ( 17 ). Numerous studies have demonstrated a consistent correlation between the phenolic content of propolis and its antioxidant properties ( 7 - 19 ). Honeybee-collected propolis demonstrates compositional variations influenced by several factors, including collection time, local vegetation, and collection area ( 17 ). These geographical and ecological factors contribute significantly to the overall composition of propolis. The present study investigates the total antioxidant capacity, total oxidant capacity, and oxidative stress index of propolis samples collected from 11 different cities in Turkey.
2. Materials and Methods
2.1. Collection of Propolis Products
Propolis samples were collected from eleven geographically distinct locations across Turkey, encompassing various ecological zones (Table 1). These locations represent diverse regions within the country. These locations are as follows: Artvin-A, Balikesir-B, Duzce-C, Edirne-D, Kahramanmaras-E, Mersin-F, Mugla-G, Nigde-H, Ordu-I, Sivas-J, and Van-K. The samples were collected by beekeepers from these locations throughout 2011. It is acknowledged that propolis production can vary seasonally due to factors like plant availability. The collection of samples in different months is attributed to variations in harvest time, ecological, geographical and climatic conditions. The samples were then stored at a temperature of 4°C until analysis.
| Regions | City (Sample Code) |
|---|---|
| Northeastern Turkey | Artvin (A) |
| Marmara Region | Balikesir (B) |
| Black Sea Region | Duzce (C) |
| Black Sea Region | Ordu (I) |
| Thrace | Edirne (D) |
| Southeastern Anatolia Region | Kahramanmaras (E) |
| Mediterranean Region | Mersin (F) |
| Aegean Region | Mugla (G) |
| Central Anatolia Region | Nigde (H) |
| Central Anatolia Region | Sivas (J) |
| Eastern Anatolia Region | Van (K) |
2.2. Preparation Of the Ethanol Extract of Propolis (EEP)
The extraction of propolis samples was accomplished through the utilization of 25 mL of 70% ethanol. A sonicator was utilized for a period of 15 minutes in order to achieve a homogenate mixture (Selecta Ultrasons). Following filtration using a Whatman no. 4 paper filter, the mixture was subjected to a process of concentration under reduced pressure at 40°C in a rotary evaporator (Heildolph Heizbad HB Digit). The extracts were then stored for subsequent analysis.
2.3. Biochemical Analyses
This study employed commercially available Rel Assay kits to determine both Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) of propolis samples. As delineated in Erel (2004) ( 15 ), the TAS assay utilizes the ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) radical cation and its color bleaching capacity. The assay's high sensitivity, with values below 3%, is a notable feature. The results of this assay are expressed in terms of mmol Trolox equivalent per liter (μmol Trolox Eq/L). The TOS determination process involves the utilization of the oxidation of ferrous ions by oxidants present within the sample, as elucidated in Erel (2005) ( 16 ). This method is reliant upon the formation of a coloured ferrous-odianisidine complex and its subsequent oxidation by sample oxidants. The oxidation of glycerol molecules also contributes to the reaction. The presence of an orange-coloured ferric ion within an acidic xylenol environment serves as an indicator for the total oxidant content in the sample. The concentration of oxidant molecules is then measured spectrophotometrically based on color intensity. Hydrogen peroxide was used for calibration, with results expressed as micromoles of hydrogen peroxide equivalent per litre (μmol H2O2 Eq/L). The oxidative stress index (OSI) is determined by calculating TAS and TOS values. The OSI is calculated using the following formula ( 16 ): OSI (optional unit) = TOS (μmol H2O2 equivalent / L) / TAC (μmol Trolox equivalent / L).
2.4. Statistical Analysis
The evaluation of all results was conducted by means of the one-way ANOVA method, with SPSS software (Chicago, IL, USA; version 16.0) being utilized for this purpose. The mean differences among the samples were then compared with Duncan's multiple tests and expressed as±SE.
3. Results
3.1. Total Antioxidative Status of Propolis Samples
As illustrated in Table 2, the antioxidant capacities of propolis samples collected from diverse geographical locations are presented. The TAS values of the propolis samples obtained from regions A to K were determined to be 4.53 ± 0.24, 3.32 ± 0.06, 3.19 ± 0.05, 3.19 ± 0.05, 3.17 ± 0.05, 3.19 ± 0.05, 3.77 ± 0.06, 3.18 ± 0.05, 3.32 ± 0.06, 112395018415001651000-228600003.18 ± 0.05 and 3.22 ± 0.06 mmol Trolox Equivalent/L. In comparison with the TAS data of propolis samples from eleven different regions, the highest TAS level was observed in region A. The TAS value of the propolis sample obtained from the G region was found to be statistically significantly lower than the TAS data of the propolis sample collected from the A region, and it was also found to be statistically significantly higher than the TAS values of the other propolis samples (P<0.01). However, no statistically significant differences were observed between the TAS values of the propolis samples from B, C, D, E, F, H, I, J and K (P>0.01).
| Parameters | N | TAS (mmol/L) | TOS (μmol/L) | OSI (TOS/TAS) |
|---|---|---|---|---|
| Locations | ||||
| Artvin(A) | 5 | 4.53±0.038 | 95.02±1.917 | 2.10±0.045 |
| Balikesir(B) | 5 | 3.32±0.041 | 45.99±0.622 | 1.39±0.026 |
| Duzce(C) | 5 | 3.19±0.042 | 37.29±0.512 | 1.17±0.009 |
| Edirne(D) | 5 | 3.19±0.044 | 21.68±0.216 | 0.68±0.008 |
| Kahramanmaras(E) | 5 | 3.17±0.032 | 42.49±0.422 | 1.34±0.018 |
| Mersin(F) | 5 | 3.19±0.024 | 45.83±0.670 | 1.44±0.017 |
| Mugla(G) | 5 | 3.77±0.049 | 71.69±0.747 | 1.90±0.039 |
| Nigde(H) | 5 | 3.18±0.038 | 12.07±0.286 | 0.38±0.005 |
| Ordu(I) | 5 | 3.32±0.044 | 59.53±0.918 | 1.79±0.039 |
| Sivas(J) | 5 | 3.18±0.050 | 29.08±0.854 | 0.91±0.026 |
| Van(K) | 5 | 3.22±0.045 | 18.44±0,586 | 0.57±0.020 |
| OVERALL | 55 | 3.39±0.055 | 43.55±3.201 | 1.24±0.073 |
3.2. Total Oxidative Status of Propolis Samples
The TOS values of the propolis samples obtained from regions A to K are presented in Table 2. These values range from 95.02±5.99 µmol H2O2 Equivalent/L in region A to 18.44±5.88 µmol H2O2 Equivalent/L in region K. In comparison with the established TOS data of propolis samples from diverse geographical locations, it was observed that the A region propolis sample exhibited the highest statistically significant TOS value (P<0.01). Conversely, the lowest TOS values (P<0.01) were detected in the E, H and K propolis samples. There was not a statistically significant difference between the TOS results of the G and I propolis samples (P>0.01). Furthermore, no statistically significant difference was observed between the TOS results of propolis samples obtained from the B, E, and F regions (P>0.01). However, a statistically significant difference was observed between the TOS results of the propolis samples collected from C and J (P>0.01). The TOS data of the G and I propolis samples were found to be statistically significantly higher than the TOS data of the B, E, and F propolis samples (P<0.01). Finally, TOS values of propolis were found to be statistically significantly higher in B, E and F regions than in C and J regions (P<0.01).
3.3. Oxidative Stress Indexes Status of Propolis Samples
The OSI values of propolis samples collected from different regions were investigated; the OSI results of the A to K propolis samples were found to be 2.10±0.22, 1.39±0.17, 1.17±0.16, 0.68±0.12, 1.34±0.17, 1.44±0.17, 1.90±0.21, 0.38±0.11, 1.79±0.20, 0.91±0.14 and 0.57±0.12 (see Table 2 for details). Upon comparison of the data, it was ascertained that the highest OSI data was observed in A and G (P<0.01), while the lowest OSI data was identified in H (P<0.01). No statistically significant differences (P>0.01) were observed in the OSI values of the propolis samples from B, E and F. However, propolis samples from B, E and F exhibited statistically significantly lower OSI levels in comparison to the propolis sample from I (P<0.01). No statistically significant difference was observed between the OSI values of C and J (P>0.01). Once more, OSI values for the B, E and F propolis samples were higher (P<0.01) than those observed for the C and J propolis samples. No statistically significant difference was observed between the OSI values of samples D and K (P>0.01). However, a statistically significant difference was observed between the OSI values of C and J and those of the D and K samples (P<0.01).
4. Discussion
Turkey's distinctive geography, encompassing three discrete floristic regions, engenders a rich tapestry of plant life that varies regionally. This study explores the potential correlation between this botanical diversity and the antioxidant capacity of propolis samples collected from various regions throughout the country. The findings of the study suggest a correlation between regional variations and the observed differences in propolis antioxidant properties, which may be influenced by the phenolic compounds present in the bees' plant sources. The TAS value of propolis obtained from the A region was observed to be higher than that of the other regions. Conversely, the OSI (TOS/TAS) and TOS values of the propolis sample collected from the H region were found to be the lowest. Consequently, it can be deduced that the antioxidant activity of the propolis sample obtained from the H region is superior to the others. Phenolic acids represent the most prevalent plant metabolites on the planet. Recent research has increasingly focused on the potential protective role of phenolic acids against damage caused by oxidative stress. The analysis of the propolis sample revealed the presence of cinnamic acid, amino acids, terpenes, phenolic acid, phenolic acid esters, flavonoids and caffeic acid. The biological activities of propolis, including its antiviral, anti-inflammatory, and antibacterial properties, are contingent upon the presence of these substances ( 20 , 21 ). Given the potential for variation in chemical constituents among diverse propolis samples, standardization is imperative for effective comparison and interpretation of biological analysis results ( 22 ). A study exploring the antioxidant properties of propolis samples collected from the Erzurum region of Turkey employed various in vitro assays to evaluate phenolic content and antioxidant capacity. These assays encompassed the determination of total antioxidant activity, ferric reducing antioxidant power (FRAP) assay, cupric ion reducing antioxidant capacity (CUPRAC) assay, scavenging activity against superoxide anion radical (O2-) and hydrogen peroxide (H2O2), and metal chelating activity. Consistent with previous reports (e.g., ( 22 )), our study observed a variation in the antioxidant activity of propolis samples, potentially linked to their phenolic content. Consistent with these observations, other researchers have documented high levels of antioxidant, antibacterial, and antifungal activity in propolis samples from Greece and Cyprus ( 23 ). The TAS values of propolis samples collected from 11 different regions in Turkey exhibited significant variations, ranging from 3.17 ± 0.05 to 4.53 ± 0.24 mmol Trolox Equivalent/L. This observation is consistent with the findings of Oses et al. ( 24 ), who reported a range of 1184.66 to 1400.86 mmol Trolox/L for the antioxidant activity of undiluted soft propolis extracts. Extensive research has underscored the multifaceted biological activities of propolis. These include its capacity to scavenge free radicals, inhibit tumor cell growth, protect cells against oxidative stress (as evidenced in germinal cells) ( 20 , 21 ), and reduce the viability of cancer cells by increasing DNA damage. In addition, Watanabe et al. investigated the tumor-inhibiting properties of propolis extracts derived from diverse geographical regions and obtained using various solvents, including ethanol, methanol, and water ( 25 ). The present study employed the commercially available Rel Assay kits to assess the Total Oxidant Status (TOS) and Oxidative Stress Index (OSI) of propolis samples collected from eleven distinct regions across Turkey. Statistical analysis was performed to evaluate potential regional variations in the antioxidant capacity (TOS), oxidant capacity (OSI), and overall oxidative stress index of the propolis samples. It is noteworthy that the Rel Assay kits utilize in vitro assays that are recognized for their high reliability and precision in measuring these parameters. The present study investigated the relationship between regional variations in Turkey and the antioxidant capacity of propolis samples. The findings of this study revealed a significant correlation, suggesting that the diverse plant communities across Turkey influence the composition and, consequently, the antioxidant properties of propolis collected by honeybees. These results underscore the pivotal role of geographical origin in shaping the biological activity of propolis.
Acknowledgment
The authors express their profound gratitude for the financial support provided by the Scientific Research Projects Unit of Nigde Omer Halisdemir University (FEB 2011/24) for this study.
Authors' Contribution
Study concept and design: Z. S, H. A.
Acquisition of data: Z. S, H. A, H. D.
Analysis and interpretation of data: H. D, B. O.
Drafting of the manuscript: Z. S, M. YN, H. YI.
Critical revision of the manuscript: M.YN, H. YI, B. O, Z, S.
Statistical analysis: H. A, H. D.
Ethics
It is hereby asserted that all ethical considerations were duly considered during the preparation of the submitted manuscript.
Conflict of Interest
The authors declare no conflict of interest.
Data Availability
The data that support the findings of this study are available on request from the corresponding author.
References
- Miguel MG, Nunes S, Dandlen SA, Cavaco AM, Antunes MD. Phenols and antioxidant activity of hydro-alcoholic extracts of propolis from Algarve, South of Portugal. Food Chem Toxicol. 2010; 48(12):3418-3423.
- Ramadan A, Soliman G, Mahmoud SS, Nofal SM, Abdel-Rahman RF. Evaluation of the safety and antioxidant activities of Crocus sativus and Propolis ethanolic extracts. Journal of Saudi Chemical Society. 2010; 6 (1):13-21.
- Potkonjak NI, Veselinović DS, Novaković MM, Gorjanović SŽ, Pezo LL, Sužnjević DŽ. Antioxidant activity of propolis extracts from Serbia: a polarographic approach. Food Chem Toxicol. 2012; 50(10):3614-3618.
- Zahid N, Alı A, Sıddıquı Y, Maqbool M. Efficacy of ethanolic extract of propolis in maintaining postharvest quality of dragon fruit during storage. Postharvest Biology and Technology. 2013; 79:69-72.
- Jug M, Karas O, Kosalec I. The Influence of Extraction Parameters on Antimicrobial Activity of Propolis Extracts. Nat Prod Commun. 2017; 12(1):47-50.
- Nakamura R, Nakamura R, Watanabe K, et al. Effects of propolis from different areas on mast cell degranulation and identification of the effective components in propolis. Int Immunopharmacol. 2010; 10(9):1107-1112.
- Do Nascimento TG, dos Santos Arruda RE, da Cruz Almeida ET, dos Santos Oliveira JM, Basílio-Júnior ID, Celerino de Moraes Porto IC, Watson DG. Comprehensive multivariate correlations between climatic effect, metabolite-profile, antioxidant capacity and antibacterial activity of Brazilian red propolis metabolites during seasonal study. Scientific Reports. 2019; 9(1)
- Fischer G, Conceição FR, Leite FP, et al. Immunomodulation produced by a green propolis extract on humoral and cellular responses of mice immunized with SuHV-1. Vaccine. 2007; 25(7):1250-1256.
- Sangiovanni E, Dell'Agli M. Special Issue: Anti-Inflammatory Activity of Plant Polyphenols. Biomedicines. 2020; 8(3):64.
- Talas ZS, Gulhan MF. Effects of various propolis concentrations on biochemical and hematological parameters of rainbow trout (Oncorhynchus mykiss). Ecotoxicol Environ Saf. 2009; 72(7):1994-1998.
- Nori MP, Favaro-Trındade CS, Alencar SM, Thomazınıı M, Balıeıro JCC, Castıllo CJC. Microencapsulation of propolis extract by complex coacervation. LWT. Food Science and Technology. 2011; 44:429-435.
- Kandiel MMM, El-Asely A, Radwan HA, Abbass AA. Modulation of genotoxicity and endocrine disruptive effects of malathion by dietary honeybee pollen and propolis in Nile tilapia (Oreochromis niloticus). Journal of Advanced Research. 2013; 5(6):671-684.
- Braakhuis A. Evidence on the Health Benefits of Supplemental Propolis. Nutrients. 2019; 11(11): 2705.
- Mushtaq W, Baba H, Akata İ, Sevindik M. Antioxidant Potential and Element Contents of Wild Edible Mushroom Suillus granulatus. Kahramanmaraş Sütçü İmam Üniversitesi Tarım ve Doğa Dergisi. 2020; 23(3): 592-595.
- Erel O. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin Biochem. 2004; 37(4):277-285.
- Erel O. A new automated colorimetric method for measuring total oxidant status. Clin Biochem. 2005; 38(12):1103-1111.
- Dogan H, Akyol E, Akgul H, Talas ZS. Biologic Activities of Honeybee Products Obtained From Different Phytogeographical Regions of Turkey. Turkish Journal of Agriculture - Food Science and Technology. 2014; 2 (6): 273-276.
- Pasdaran A, Nahar L, Asnaasharı S, Sarker SD, Delazar A. Gc-ms analysis, free-radical-scavenging and insecticidal activities of essential oil of scrophularia oxysepala boiss. Pharmaceutical Sciences. 2013; 19(1):1.
- Pasdaran A, Delazar A, Ayatollahı SA, Nahar L, Sarker SD. Phytochemical and Bioactivity Evaluation of Scrophularia amplexicaulis Benth. Records of Natural Products. 2016; 10 (4): 519-525.
- Khan MA, Rahman AA, Islam S, et al. A comparative study on the antioxidant activity of methanolic extracts from different parts of Morus alba L. (Moraceae). BMC Res Notes. 2013; 6:24.
- Khatoon M, Islam E, Islam R, et al. Estimation of total phenol and in vitro antioxidant activity of Albizia procera leaves. BMC Res Notes. 2013; 6:121.
- Sforcin JM, Bankova V. Propolis: is there a potential for the development of new drugs? J Ethnopharmacol. 2011; 133(2):253-260.
- Kalogeropoulos N, Konteles SJ, Troullıdou E, Mourtzınos I, Karathanos VT. Chemical composition, antioxidant activity and antimicrobial properties of propolis extracts from Greece and Cyprus. Food. Chem2009; 116(2):452-461.
- Osés SM, Pascual-Maté A, Fernández-Muiño MA, López-Díaz TM, Sancho MT. Bioactive properties of honey with propolis [published correction appears in Food Chem. 2016;201:361. Food Chem. 2016; 196:1215-1223.
- Watanabe MA, Amarante MK, Conti BJ, Sforcin JM. Cytotoxic constituents of propolis inducing anticancer effects: a review. J Pharm Pharmacol. 2011; 63(11):1378-1386.
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