EJBR2021v11i2art260 ISSN 2449-8955 European Journal of Biological Research Research Article European Journal of Biological Research 2021; 11(2): 260-266 DOI: http://dx.doi.org/10.5281/zenodo.4660074 Determination of total phenolic content, total flavonoid content and total antioxidant capacity in some endemic Sideritis L. (Lamiaceae) species grown in Turkey Emre Sevindik 1*, İsmail Gübeş 2, Zehra Tuğba Murathan 3, Gülendam Tümen 4 1 Faculty of Agriculture, Department of Agricultural Biotechnology, Adnan Menderes University, South Campus, Turkey 2 Anamur Forest Management Directorate Mersin, Turkey 3 Malatya Turgut Özal University, Battalgazi Vocational School, Battalgazi, Malatya, Turkey 4 Biology Department, Science and Arts Faculty, Balıkesir University, Balıkesir, Turkey * Corresponding author: E-mail: ph.d-emre@hotmail.com Received: 30 January 2021; Revised submission: 11 March 2021; Accepted: 02 April 2021 https://jbrodka.com/index.php/ejbr Copyright: © The Author(s) 2020. Licensee Joanna Bródka, Poland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) ABSTRACT: In this study, total phenolic, total flavonoid and antioxidant activities of the some endemic species Sideritis rubriflora Hub.-Mor., Sideritis libanotica Labill. subsp. violascens (P.H.Davis) P.H.Davis, Sideritis erythrantha Boıss. Et Heldr. Apus Bentham var. cedretorum P.H.Davis, Sideritis congesta P. H. Davis Et Hub.-Mor., Sideritis brevidens P.H.Davis and Sideritis vuralii H. Duman Et Başer, which were collected from Anamur district of Mersin province in Turkey, were analyzed. Total phenolic content (TPC), total flavonoid content (TFC) and total antioxidant capacity (DPPH, ABTS, FRAP) of the ground surface parts were evaluated. As a result of the study, the highest TPC value was observed in S. erythrantha subsp. cedretorum and S. rubriflora extracts as being 366.9 and 328.3 mg/g DW, respectively; the highest TFC value was observed in S. rubriflora extract as being 155.7 mg/g; the highest DPPH radical scavenging activity was observed in S. congesta and S. brevidens extracts as being 39.1% and 38.9%, respectively; the highest ABTS radical scavenging activity was observed in S. erythrantha subsp. cedretorum and S. rubriflora extracts as being 54.9% and 51.9%, respectively; the highest FRAP value was observed in S. libanotica subsp. violascens extract as being 1500.2 µ mol/g. In the light of the acquired findings, it is suggested that Sideritis species used in the study can be used as a possible natural source in the pharmaceutical and food industries. Keywords: Sideritis; Antioxidant; Total flavonoid; Total phenolic; Turkey. 1. INTRODUCTION Turkey, located in the mild regions of the world, has quite a rich diversity of habitats due to geomorphological, topographic and climatic characteristics [1]. One of the reasons for the presence of such a rich floristic diversity in Turkey is that Turkey is located at the intersection of three phytogeographic regions such as Mediterranean, Europe-Siberia and Iran Turan [2]. The Lamiaceae family encompasses important aromatic plants consisting of approximately 7173 species and 236 genera. These plants are used in the traditional and modern medicine, pesticides industries, food, cosmetic and pharmaceutical industry and they contain a high volatile oil ratio [3-6]. The genus Sideritis, a member of the Lamiaceae family, has more than Sevindik et al. Phenolic, flavonoid content and antioxidant capacity in Sideritis L. 261 European Journal of Biological Research 2021; 11(2): 260-266 150 species annual and perennial herbs and small shrubs distributed in the temperate and tropical regions of the Northern Hemisphere [7-9]. In the Flora of Turkey Sideritis L. genus is represented by 46 species and together 55 taxa, 42 taxa of which being endemic [10]. Sideritis species are calcicolous and heliophilous plants that usually grow in dry and semi-arid regions [8]. This genus contains antimicrobial and antioxidant polyphenolics such as flavonoids [11]. Sideritis species are widely used in the treatment of gastrointestinal disorders and coughs, colds and diuretic therapy, also in herbal teas and folk medicine in Turkey [12,13]. Plant chemical compounds are classified as primary and secondary metabolites according to their metabolic pathway and functions [14]. Free radicals play an important role in the pathogenesis of various diseases and therefore antioxidants have an important role in preventing diseases [15]. Phenolic substances found in plants are bioactive compounds that are important antioxidant sources [16]. Flavonoids composed a large group of polyphenolic compounds that have a benzo-p-pyron structure and are ubiquitous in plants [17]. In this study, total phenolic and total flavonoid content, and antioxidant activities of some Sideritis species spreading in Anamur/Mersin/Turkey were analyzed. 2. MATERIALS AND METHODS 2.1. Plant materials In this study, Sideritis rubriflora, S. libanotica subsp. violascens, S. erythrantha var. cedretorum, S. congesta, S. brevidens and S. vuralii species were collected from Anamur district of Mersin/Turkey province in 2017 (Figure 1) and moved to the herbarium. Specimens were identified and prepared for experimental study. Figure 1. Location of the Mersin/Anamur (https://www.google.com/maps). Sevindik et al. Phenolic, flavonoid content and antioxidant capacity in Sideritis L. 262 European Journal of Biological Research 2021; 11(2): 260-266 2.2. Extraction Dried aerial parts were ground with an electrical blender. The powdered aerial parts (30 g) were placed in Soxhlet apparatus, and extraction was performed in 300 mL of methanol (polarity index: 5.1) for 6 h. The solution was filtered and concentrated at 40°C by vacuum (SCILOGEX RE100-Pro, USA). Extracts were frozen (–18°C) until used for determining the TPC, TFC and antioxidant activities. 2.3. Total Phenolic Content (TPC) TPC of Sideritis sp. extracts was determined using Folin-Ciocalteu procedure described by Spanos and Wrolstad [18] with slight modifications. The absorbance was measured by UV-Vis spectrophotometer (UNICO S1205, USA) at 765 nm. The results are expressed as milligrams of gallic acid equivalents (GAE) per g of dry weight (DW). 2.4. Total Flavonoid Content (TFC) TFC of extracts was detected using spectrophotometric method described by Quettier et al. [19]. The absorbance was measured at 415 nm. The results are expressed as milligrams of quercetin equivalents per g of dry weight (DW). 2.5. Antioxidant activity According to 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3 ethylbenzothiazoline-6-sulfonic acid (ABTS), and Ferric Reducing Antioxidant Power (FRAP) methods antioxidant activity of extracts was determined. 2.6. DPPH method The diluted extract (50µ L) was mixed with DPPH solution (950 µ L, 0.1 N). The mixture was placed in a shaker at room temperature in the dark for 30 min. The sample was then measured at 515 nm by UV-Vis spectrophotometer. The inhibition of the DPPH radical by the sample was calculated using the formula: (Absorbance control-absorbance sample/Absorbance control)×100 [20]. 2.7. ABTS method This assay was carried out as described earlier [21]. ABTS solution and 2.45 mM potassium persulfate solution were stirred (1:1 v/v). The mixture was left for 12-16 h at room temperature in the dark. After an absorbance value of 0.70 at 734 nm was reached, the mixture was diluted with methanol. The diluted extract (0.15 mL) was then mixed with 2.85 mL of diluted ABTS solution followed by incubation for 2 h at room temperature in the dark. Absorbance was measured at 734 nm by spectrophotometer. Percentage of ABTS was calculated using the formula: (Absorbance control–absorbance sample/Absorbance control)×100. 2.8. FRAP method FRAP assay was performed according to the procedure described by Benzie and Strain [22] with slight modifications. To prepare the FRAP reagent, 25 mL of 300 mM sodium acetate buffer (pH 3.6), 2.5 mL of 2,4,6-tripiridil-s-triazin (TPTZ), 10 mM of 40 mM HCl, and 2.5 mL of iron(III) chloride hexahydrate (FeCl3 6H2O) (20 mM) were mixed. The initial absorbance value of 900 µ L of reagent was measured at 593 nm. The diluted extract (20 µ L) and 2.98 mL of FRAP reagent were mixed followed by incubation for 10 min at room temperature. Absorbance was measured at 593 nm using spectrophotometer. The ferric ion reducing ability of Sevindik et al. Phenolic, flavonoid content and antioxidant capacity in Sideritis L. 263 European Journal of Biological Research 2021; 11(2): 260-266 extracts was determined using the calibration curve and reported as µ mol of FeSO4 equivalents per gram of sample. 3. RESULTS AND DISCUSSION Natural products derived from plants provide many opportunities for new medicines [23,24]. Phenolic compounds, also known as secondary metabolites, are among the most important and functional components produced by plants. These components are involved in activities such as color formation, taste formation, aroma formation, and plant defense systems in plants [25]. The amount of phenolic compounds found in plants varies depending on the variety, soil structure, habitat, climatic and seasonal characteristics [26]. The results of TPC and TFC of Sideritis extracts are presented in Table 1. Table 1. Total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activities of endemic Sideritis species. Sideritis species TPC (mg/g) TFC (mg/g) DPPH (%) ABTS (%) FRAP (µmol/g) S. rubriflora 328.3±14.8a 155.7±38.9a 18.3±4.3c 51.9±5.2a 1160.3±29.2d S. libanotica subsp. violascens 172.3±13.5c 74.8±1.9c 33.3±1.6b 43.6±2.5b 1500.2±38.6a S. erythrantha var .cedretorum 366.9±19.4a 93.5±8.4b 21.8±3.8c 54.9±1a 970.8±5.8e S. congesta 52.5±2.7e 31.7±4.7d 39.1±8.2a 12.5±1.7e 1390.3±20.1b S. brevidens 205.5±12.8b 105.9±4.4ab 38.9±9.9a 42.8±1.1b 1170.2±18.4d S. vuralli 35.5±2.9f 14.2±0.9f 32.8±5.5b 18.9±2.9d 1230.8±5.6c All values a represented as means ±SD (n = 3). Different letters (a-f) within the columns indicate statistically significant differences by Duncan’s multiple range test at p<0.05. Statistically significant differences among samples were observed (p<0.05). The highest TPC value was detected in S. erythrantha subsp. cedretorum and S. rubriflora extracts as being 366.9 and 328.3 mg/g DW, while the lowest TPC value was determined in S. rubiflora as 328.3 mg/g DW. It was determined that TFC values of samples vary between 14.2 mg/g DW (S. vuralli) and 155.7 mg/g (S. rubriflora). Gökbulut et al. [27] reported that the amount of total phenolic matter in the methanol extracts of the ground surface parts of Sideritis argyrea, S. congesta and S. erythrantha var. cedretorum species obtained from local markets varied between 121.7 and 190.8 mg GA/g DW, and that the highest TPC value was observed in S. erythrantha var. cedretorum. Radojevic et al. [28] reported that TPC amount in methanolic extract of S. montana L. species was found as 97.85 mg/g, and TFC amount was found as 159.54 mg/g. Also, Tadić et al. [29] reported that TPC amount in ethanolic extracts of S. scardica Griseb was found as 188.5 mg/g. Alipieva et al. [30] found out that in the tea of S. scardica x S. syriaca hybrid plants, TPC content was 32.2 mg/g, and TFC content was 9.6 mg/g. Nakiboğlu et al. [31] reported that TPC value was 0.089 µ g (GAE/µ g extract) in the methanolic extract of S. spylea species. Tunalıer et al. [32] reported that TPC values varied between 191.6 and 402.5 mg/g among 27 Sideritis species. Sağdıç et al. [33] reported TPC values as 39.35 and 93.79 mg/g in two endemic Sideritis species. In general, the results we obtained in our study are similar to the results reported in the literature. In the study, by using 3 different methods, antioxidant activity values of plant extracts were determined. Antioxidant capacity results of samples are given in Table 1. According to this, DPPH radical scavenging activity of the samples ranged from 18.3 to 39.8%. The highest DPPH radical scavenging activity was observed in S. congesta and S. brevidens extracts as being 39.1% and 38.9%, and the lowest activity was observed in S. brevidens extracts as being 38.9. ABTS radical scavenging Sevindik et al. Phenolic, flavonoid content and antioxidant capacity in Sideritis L. 264 European Journal of Biological Research 2021; 11(2): 260-266 activity results of plant extracts were in parallel with TPC and TFC results. The highest ABTS radical sweep activity was observed in S. erythrantha subsp. cedretorum and S. rubriflora extracts as being 54.9% and 51.9%, respectively; the highest FRAP value was observed in S. libanotica subsp. violascens extract as being 1500.2 µmol/g. Gökbulut et al. [27] reported that the highest antioxidant activity in the methanol extracts of Sideritis argyrea, S. congesta and S. erythrantha var. cedretorum species was observed in S. erythrantha var. cedretorum. Sağdıç et al. [33] reported that DPPH radical sweep activities of S. ozturkii and S. caesarea plant extracts were 41.68% and 72.47%, respectively. Koleva et al. [34] reported that the radical sweep activity in the extracts of Sideritis scardica, S. syriaca and S. montana which were obtained by using different solvents was above 90%. The differences between the results we obtained in our study and those reported in the literature may be due to differences in species, methods, growing conditions or solvent. 5. CONCLUSIONS As a result, the maximum TPC value in this study; in Sideritis erythrantha subsp. cedretorum and S. rubriflora species, the highest TFC value was in S. rubriflora species detected. 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