Aydin & Kadioğlu 2022, Biologica Nyssana 13(1) 13 (1) September 2022: 33-40 DOI: 10.5281/zenodo.7117537 Total phenolic content and antioxidant activity of Thymus vulgaris, Curcuma longa, propolis and their mixtures Original Article Sinem Aydin Faculty of Science and Arts, Giresun University, Department of Biology, 28200, Giresun, Turkey sinem.aydin@giresun.edu.tr (corresponding author) Gülşah Kadioğlu Faculty of Science and Arts, Giresun University, Department of Biology, 28200, Giresun, Turkey Received: May 23, 2022 Revised: July 19, 2022 Accepted: September 16, 2022 Abstract: The current study, we investigated phenolic content and antioxidant activity of ethyl acetate and dichloromethane extracts of thyme, turmeric, propolis and their mixtures. The highest and the lowest phenolic contents were found in ethyl acetate extract of propolis (214.94±0.023 µg GAE/mL) and dichloromethane extract of thyme (21.02±0.013 µg GAE/mL). Total antioxidant capacity of ethyl acetate extracts ranges from 127.15±0.031 µg AAE/mL and 232.2±0.028 µg AAE/mL; dichloromethane extracts ranges from 61.6±0.019 µg AAE/mL and 159.95±0.035 µg AAE/mL. CUPRAC activity and DPPH radical scavenging activity of ethyl acetate extracts are higher than dichloromethane extracts. According to the obtained results, it can be said that propolis, thyme and turmeric could be an alternative to synthetic antioxidants. Key words: thyme, turmeric, propolis, antioxidant activity Apstrakt: Ukupan sadržaj fenola i antioksidativna aktivnost Thymus vulgaris, Curcuma longa, propolisa i njihovih mešavina U ovoj studiji, ispitivali smo sadržaj fenola i antioksidativnu aktivnost etil acetatnih i dihlormetanskih ekstrakata majčine dušice, kurkume, propolisa i njihovih mešavina. Najviši sadržaj fenola ustanovljen je u etil acetatnom ekstraktu propolisa (214.94±0.023 µg GAE/mL), a najniži u dihlormetanskom ekstraktu majčine dušice (21.02±0.013 µg GAE/mL). Ukupna antioksidativna aktivnost etil acetatnih ekstrakata kreće se u opsegu od 127.15±0.031 µg AAE/ mL do 232.2±0.028 µg AAE/mL, a dihlormetanskih ekstrakata od 61.6±0.019 µg AAE/mL do 159.95±0.035 µg AAE/mL. CUPRAC aktivnost i aktivnost uklanjanja DPPH radikala viša je kod etil acetatnih ekstrakata u odnosu na dihlormetanske ekstrakte. Na osnovu dobijenih rezultata, može se zaključiti da bi propolis, majčina dušica i kurkuma mogli biti alternativa sintetičkim antioksidansima. Ključne reči: majčina dušica, kurkuma, propolis, antioksidativna aktivnost Introduction Medicinal plants are utilized worldwide for the cure of many illnesses such as asthma, gastrointestinal symptoms, skin disorders, respiratory and urinary problems and cardiovascular diseases. Plants synthesize various biologically active compounds which are crucial for them to survive in the natural environment and protect them against abiotic stresses derived from temperature, water and mineral nutrient supply (Egamberdieva et al., 2017). Plants have been utilized as therapeutic resources such as herbal teas, crude extracts or pharmaceutical preparations (tinctures, pills and capsules) for many years. The World Health Organization (WHO) predicts that 65% of the world’s population still use plants as traditional medicine (Karakaş et al., 2012). Medicinal plants has been investigated for their antioxidant capacities by many researchers. Natural antioxidants are very effective to hinder the devastating effects caused by oxidative stress. Plants, vegetables and fruits have natural antioxidants such as phenolics, flavonoids, tannins and proanthocyanidins. Antioxidants present in plants may protect plants from diseases (Saeed et al., 2012). © 2022 Aydin & Kadioğlu. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 33 Thymus vulgaris L. (Thyme) growing wild in Turkey belongs to Labiatea family and possess many advantageous effects such as carminative, antiseptic, antioxidant and antimicrobial activities. Thymol and carvacrol are major components of thyme essential oil. Thymol and other phenolic components in Thymus inhibit microorganisms by increasing permeability of the cell membrane and reduction of vital intracellular substances or by disruption of bacterial enzyme systems (Tural & Turhan, 2017). Curcuma longa L. (turmeric) belongs to Zingiberaceae family generally utilized in Indian and Chinese systems of medicine. Turmeric spice are obtained from plant rhizome known as “yellow root”. It has been also utilized for the treatment of many diseases. Also, C. longa reduces risk of cancer (Schaffer et al., 2011)and has antiinflammatory, antioxidant and wound healing properties (Maheshvari et al., 2006). Propolis is a natural resinous mixture produced from substances collected from some parts of plants, buds and secretions by honey bees. Propolis is one of the “natural medicines” utilized since ancient times. More than 300 active compounds were defined in propolis. Propolis has antibacterial, antiviral and antioxidant properties. Moreover, propolis is utilized in apitheraphy, cosmetic and food industry for its antioxidant and antibacterial features (Çoşkun & İnci, 2020). In the current study, antioxidant activity and total phenolic contents of dichlorometane and ethanol extracts of thyme, turmeric, propolis and their mixtures (thyme/propolis, turmeric/propolis) at 1/1 ratio have been evaluated. We also targeted to reveal antagonistic and synergistic effects of the combination of thyme, turmeric and propolis extracts. Materials and Methods Providing of the samples Thyme, turmeric and propolis were bought from a herbal shop in Giresun, Turkey. Preparation of extracts 20 g of thyme, turmeric and propolis were extracted in a shaker for 24 h utilizing 200 mL ethyl acetate and dichloromethane, separetely. Thyme:Propolis and Curcumin:Propolis were extracted with 200 mL ethyl acetate and 200 mL dichloromethane in a shaker for 24 h, separetely. The extracts were filtered through Whatman filter paper No. 1 and residues were evaporated (40 °C) with rotary evaporator (Murugan & Parimelazhagan, 2014). Antioxidant activity Total phenolic content Total phenolic contents of the extracts were determined in accordance with the method of Slinkard & Singleton (1977) utilizing gallic acid standard. Shortly, 0.1 mL extract was diluted with 4.5 mL distilled water. Then, 0.1 mL of the Folin– Ciocalteu reagent (previously diluted 3‐fold with distilled water) was put into the mixture. After 3 minutes, 0.3 mL Na2CO3 (2%) was added. The absorbance was measured at 760 nm after incubating the mixture for 90 min. Total phenolic content of the extracts was expressed as µg gallic acid equivalents (GAE)/mL by using the calibration curve. The tests were performed in triplicate (Slimkard & Singleton, 1977). Total antioxidant capacity Phosphomolybdenum method was used to determine total antioxidant capacity of the extracts. 0.3 mL extract and 3000 µL reagent (contains 0.6 M sulfuric acid, 28 mM sodium phosphate and 28 M ammonium molybdate) was mixed and incubated at 95 °C for 90 min. Then, absorbance was read at 695 nm. Ascorbic acid was used as the standard (Prieto et al., 1999). The total antioxidant capacity was expressed as µg ascorbic acid equivalent (AAE)/mL. The tests were performed in triplicate. Cupric reducing antioxidant capacity (CUPRAC) test 0.5 mL extract (250-1000 µg/mL concentration), 1.0 mL CuCl2 solution (1x10 -2), 1.0 mL neocuproine solution (7.5x10-3 M) and 1.0 mL ammonium acetate buffer (1.0 M, pH: 7.0) were mixed in a test tube. Then, the tube was vortexed and stored in a dark place for 30 min. After this period, the absorbance was read at 450 nm. Butylated hydroxytoluene (BHT) was used as a standard antioxidant agent (Özyürek et al., 2009). DPPH radical scavenging activity DPPH radical scavenging activity of the extracts was established by DPPH. Appropriate dilution series (250-1000 µg/mL) were prepared for ethanolic extracts in DMSO. 0.75 mL of each solution was added to 1.5 mL of a 6x10-5 M methanolic solution of DPPH. The mixture was stirred vigorously and allowed to stand in the dark at the room temperature for 30 min. Decrease in absorbance of the solution against methanol was measured at 517 nm with a Shimadzu 1240 UV-Vis spectrophotometer (Williams et al., 1995). Rutin and Butylated hydroxytoluene (BHT) were used as standard antioxidants. The DPPH radical scavenging activity was calculated using the following equation: 34 BIOLOGICA NYSSANA ● 13 (1) September 2022: 33-40 Aydin & Kadioğlu ● Total phenolic content and antioxidant activity of Thymus vulgaris, Curcuma longa, propolis and their mixtures 35 BIOLOGICA NYSSANA ● 13 (1) September 2022: 33-40 Aydin & Kadioğlu ● Total phenolic content and antioxidant activity of Thymus vulgaris, Curcuma longa, propolis and their mixtures DPPH Radical Scavenging Activity (%)=[(A0- A1)/A0]x100 A0: Absorbance of control A1: Absorbance of extract or standard Results and discussion Total phenolic content Phenolic compounds are significant plant metabolites which have redox properties responsible for antioxidant activity (Aryal et al., 2019). Total phenolic content was determined by utilizing the Folin–Ciocalteu reagent. The results were calculated from a calibration curve (y = 0.013x, R2 = 0.9934) of gallic acid and expressed as µg Gallic Acid Equivalent (GAE)/mL (Tab. 1). The highest and the lowest phenolic contents were found in ethyl acetate of propolis (214.94±0.023 µg GAE/mL) and dichloromethane extract of thyme (21.02±0.013 µg GAE/mL). Ethyl acetate extracts exhibited higher total phenolic content than dichloromethane extracts except for ethyl acetate extract of thyme/ propolis. Total phenolic contents of ethyl acetate and dichloromethane extracts of thyme/propolis and turmeric/propolis were decreased when compared with ethyl acetate and dichloromethane extracts of thyme, turmeric and propolis except for dichloromethane extract of thyme/propolis. Total phenolic content of thyme, turmeric and propolis was also searched by many authors. For example, Bulut et al. (2020) found total phenolic content of ethanol extracts of thyme leaves as 7.01±0.13 mg GAE/g (Bulut et al., 2020). Köksal et al. (2017) determined total phenolic content of lyophilized water extract and ethanol Table 1. Total phenolic contents of the extracts (µg GAE/mL) Extract Total antioxidant capacity Ethyl acetate extract of thyme 73.25±0.030 Ethyl acetate extract of turmeric 175.74±0.050 Ethyl acetate extract of propolis 214.94±0.023 Ethyl acetate extract of thyme/propolis 129.2±0.007 Ethyl acetate extract of turmeric/propolis 173.61±0.008 Dichloromethane extract of thyme 21.02±0.013 Dichloromethane extract of turmeric 93.66±0.013 Dichloromethane extract of propolis 107.82±0.011 Dichloromethane extract of thyme/propolis 174.74±0.029 Dichloromethane extract of turmeric/propolis 82.89±0.025 extracts of thyme as 256 µg GAE/mg and 158 µg GAE/mg, respectively (Köksal et al., 2017). Erdoğan & Erbaş (2021) stated that total phenolic content of ethanol extract of turmeric was 82.47±2.70 mg GAE/g (Erdoğan & Erbaş, 2021). Yan & Asmar (2010) declared that total phenolic content of methanol extract of fresh and powder of turmeric was 348.±1.26 mg GAE/100 g and 2013.09±5.13 mg GAE/100 g, respectively (Yan & Asmah, 2010). Keskin & Kolayli (2019) reported that the total phenolic substance amount of Anatolian propolis ranged between 16.13-178.34 mg GAE/g (Keskin & Kolayli, 2019). Özdal et al. (2019) reported that the total phenolic substance amount of propolis obtained from different regions of Anatolia varies between 2,748 mg GAE/100 g and 19,969 mg GAE/100 g (Özdal et al., 2019). Collecting plants from different locations, using different extraction methods and solvents may cause discrepancy in results. Total antioxidant capacity Total antioxidant capacity method is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of green phosphate/Mo (V) complex at acid pH. Ascorbic acid was utilized to compare total antioxidant capacity of the extracts (Aliyu et al., 2012). Tab. 2 shows total antioxidant capacity of extracts. While total antioxidant capacity of ethyl acetate extracts ranges from 127.15±0.031 µg AAE/mL and 232.2±0.028 µg AAE/mL; dichloromethane extracts ranges from 61.6±0.019 028 µg AAE/mL and 159.95±0.035 µg AAE/ mL. Ethyl acetate extracts exhibited higher total antioxidant capacity than dichloromethane extracts except for ethyl acetate extract of thyme/propolis. Total phenolic contents of ethyl acetate and dichloromethane extracts of thyme/propolis and turmeric/ propolis were decreased when compared with ethyl acetate and dichloromethane extracts of thyme, turmeric and propolis except for dichloromethane extract of thyme/propolis. This situation might be arised by the interactions among the active substances in propolis and thyme or turmeric. The presence of phenolic compounds could be attributable to the observed high total antioxidant capacity. Many surveys were done by other researchers about total antioxidant capacity of propolis, thyme and turmeric. Yılmaz et al. (2017) investigated total antioxidant capacity of Propolis collected from Sakyatan (KS) and Kızılören BIOLOGICA NYSSANA ● 13 (1) September 2022: 33-40 36 (KK) regions of Konya and they found total antioxidant capacities of KS propolis and KK propolis as 2.21±0.11 mmol TEs/g extract and 2.40±0.15 mmol TEs/g extract, respectively (Yılmaz et al., 2017). Özcan & Özkan (2018) investigated total antioxidant activity of different extracts of thyme and they found that total antioxidant activity of thyme ranges from 91.14±0.87 -123.34±0.95 mg AAE/g. Bulus et al. (2017) determined that total antioxidant capacity of butanol extract of turmeric was 370 AAE/g. Our results and literature results are different. This differences can be explained with collecting sample from different locations, using different solvents and extraction techniques. Table 2. Total antioxidant capacity of the extracts (µg AAE/mL) Extract Total antioxidant capacity Ethyl acetate extract of thyme 127.15±0.031 Ethyl acetate extract of turmeric 177.16±0.021 Ethyl acetate extract of propolis 232.2±0.028 Ethyl acetate extract of thyme/propolis 147.52±0.031 Ethyl acetate extract of turmeric/propolis 174.6±0.046 Dichloromethane extract of thyme 61.6±0.019 Dichloromethane extract of turmeric 113.85±0.019 Dichloromethane extract of propolis 101±0.010 Dichloromethane extract of thyme/propolis 159.95±0.035 Dichloromethane extract of turmeric/propolis 85.04±0.038 Extract concentration (µg/mL) Cuprac activity Ethyl acetate extract of thyme 2501.4479±0.031 5002.2108±0.0006 7502.3390±0.020 10002.3562±0.088 Ethyl acetate extract of turmeric 2501.7463±0.039 5001.9936±0.003 7502.0067±0.022 10002.0151±0.017 Ethyl acetate extract of propolis 2501.9856±0.073 5001.9761±0.024 7502.0374±0.019 10002.0573±0.109 Ethyl acetate extract of thyme/propolis 2501.801±0.043 5001.8249±0.030 7501.9752±0.075 10002.0055±0.049 Ethyl acetate extract of turmeric/propolis 2501.8199±0.018 5002.1059±0.0462 7502.0493±0.019 10001.9689±0.041 Dichloromethane extract of thymus 2500.5513±0.032 5000.6634±0.049 7501.1269±0.018 10001.3479±0.037 Table 3. Cuprac activity of the extracts Extract concentration (µg/mL) Cuprac activity Dichloromethane extract of turmeric 2501.3326±0.042 5001.8600±0.029 7501.8966±0.017 10001.9426±0.055 Dichloromethane extract of propolis 2501.6693±0.059 5001.8658±0.005 7502.0466±0.021 10002.1393±0.013 Dichloromethane extract of thyme/propolis 2501.8020±0.015 5001.9255±0.011 7501.9895±0.011 10002.0625±0.078 Dichloromethane extract of turmeric/propolis 2500.9922±0.016 5001.4591±0.051 7501.7229±0.044 10001.7569±0.007 BHT 2500.6635±0.023 5000.7016±0.021 7500.8283±0.024 10000.9716±0.014 Aydin & Kadioğlu ● Total phenolic content and antioxidant activity of Thymus vulgaris, Curcuma longa, propolis and their mixtures Aydin & Kadioğlu ● Total phenolic content and antioxidant activity of Thymus vulgaris, Curcuma longa, propolis and their mixtures BIOLOGICA NYSSANA ● 13 (1) September 2022: 33-40 37 CUPRAC test Tab. 3 presents CUPRAC activity of the extracts. Ethyl acetate extracts had better CUPRAC activity than dichloromethane extracts at 1000 µg/ml concentration. CUPRAC activity of ethyl acetate and dichloromethane extracts of thyme/propolis and turmeric/propolis were decreased when compared with ethyl acetate and dichloromethane extracts of thyme, turmeric and propolis except for dichloromethane extract of thyme/propolis at concentration of 1,000 µg/mL. This situation might be a consequence of the interactions among active substances in propolis and thyme or turmeric. The results indicate a concentration dependent CUPRAC activity. All extracts had higher activity than BHT. DPPH radical scavenging activity Tab. 4 demonstrates DPPH radical scavenging potentials of extracts at different concentrations (250-1000 µg/mL) measured as a degree of discoloration displayed the extracts’ scavenging potential. Dichloromethane extract of thyme showed no activity, while all the extracts exhibited lower activity than BHT and Rutin. Table 4. DPPH radical scavenging activity of the extracts and standards (% inhibition) Extract Concentration (µg/mL) DPPH radical scavenging activity Ethyl acetate extract of thyme 250 not activity 500 7.54±0.002 750 39.04±0.0009 1000 67.56±0.004 Ethyl acetate extract of turmeric 250 10.3±0.002 500 38.53±0.0013 750 55±0.004 1000 75.25±0.006 Ethyl acetate extract of propolis 250 66.4±0.005 500 67.99±0.004 750 73.36±0.001 1000 79.53±0.001 Ethyl acetate extract of thyme/propolis 250 45.63±0.008 500 73.19±0.005 750 77.57±0.005 1000 81.64±0.003 Ethyl acetate extract of turmeric/propolis 250 38.02±0.036 500 61.68±0.009 750 65.23±0.008 1000 74.16±0.004 Dichloromethane extract of thymus 250 not activity 500 not activity 750 not activity 1000 not activity Dichloromethane extract of turmeric 250 50.94±0.001 500 55.22±0.002 750 59.94±0.001 1000 70.39±0.014 BIOLOGICA NYSSANA ● 13 (1) September 2022: 33-40 Aydin & Kadioğlu ● Total phenolic content and antioxidant activity of Thymus vulgaris, Curcuma longa, propolis and their mixtures DPPH radical scavenging activity of ethyl acetate and dichloromethane extracts of thyme/ propolis and turmeric/propolis were increased when compared with ethyl acetate and dichloromethane extracts of thyme, turmeric and propolis at 1,000 µg/mL concentration. The best activity was detected in ethyl acetate extract of thyme/propolis (81.64%) and the worst activity was detected in ethyl acetate extract of thyme (67.56%) concentration of 1,000 µg/mL. Ethyl acetate extracts generally showed better activity than dichloromethane extracts. DPPH radical scavenging activity was searched by many authors. Can et al. (2015) concluded that DPPH scavenging activity of propolis from Azerbaijan ranges from 15±1.00-198±3.40 (Can et al., 2015). Köksal et al. (2017) found DPPH scavenging activity (IC50 value) of lyophilized water extract and ethanol extract of thyme as 13.4 and 12.1, respectively (Köksal et al., 2017). Priyanka et al. (2017) investigated DPPH scavenging activity (% inhibition) of turmeric cultivars and they found that activity ranges from 49.63±2.97 to 59.58±2.95 (Priyanka et al., 2017). These differences might be a consequence of used solvent and different location of material collection. Conclusions The results suggest that the thyme, turmeric and propolis utilized in the current study possess antioxidant properties. Thyme, turmeric and propolis also can be used as ingredients for development of a new antioxidant agents. Further work should be focused on the isolation and elucidation of secondary metabolites in thyme, turmeric and propolis responsible for the antioxidant activity. Antioxidant activity of mixtures are lower than thyme, turmeric and propolis because of interactions of active substances in mixtures. Since the antioxidant activity of plant mixtures with propolis is lower than the antioxidant activities of these plants and propolis alone, plants and propolis should be consumed individually, not as a mixture. 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