Antioxidant phenolic content in a cup of Macedonian “Mountain tea” (Sideritis) Petreska, J.1; Stefova, M.1; Ferreres, F.2; Moreno, D.A.2; Tomás-Barberán, F.A.2; Stefkov, G.3; Kulevanova, S.3; Medina, S.2; García-Viguera, C.; Gil-Izquierdo, A.2* 1Institute of Chemistry, Faculty of Science Sts. Cyril and Methodius University, Skopje, Republic of Macedonia 2Research Group on Quality, Safety and Bioactivity of Plant Food, CEBAS-CSIC, Murcia, Spain 3Institute of pharmacognosy, Faculty of Pharmacy Sts. Cyril and Methodius University, Skopje, Republic of Macedonia *corresponding author: angelgil@cebas.csic.es Introduction Results and discussion “Mountain tea” is a classical Mediterranean drink prepared in boiling water from plants of the genus Sideritis. This drink has been considered to have anti-ulcer, anti inflammatory and antioxidant activity (1,2) and the extent of its intake has increased due to the health beneficial properties. These properties have mainly been attributed to the flavonoids compounds present (3). The species of the Sideritis genus is also a valuable source of phenolic compounds and its traditional use in the folk medicine in treatment of gastrointestinal ailments, common colds and as a diuretic (4) is getting more interest again as related to its correlation to antioxidant activity. This work was afforded from three points of view: phytochemical evaluation with regards to its phenolic profile, identification and quantification on phenolic compounds present in extracts of Sideritis species, and relation to antioxidant activity and dietary burden of phenolics of “Mountain tea” present in the domestic prepared infusion of Sideritis. Twenty four phenolic compounds in the Sideritis extracts were identified by their UV spectra, their deprotonated molecular ions and their corresponding ion fragments, by using LC-DAD-MS/MS (Table 1). In the quantitative analysis of phenolic compounds, the methanol extracts resulted in higher phenolics contents than the extracts prepared in boiling water. Concerning to the phenolic content in the different aerial parts, leaf was the richest plant organ in phenolics followed by flower and stem with the lowest amount. Figure 1. Phenolic distribution regarding total phenolic compounds in Sideritis species expressed in %. HA, Hydroxycinnamic acid derivatives; PG, Phenylethanoid glycosides; FG, Flavonoid 7-O- glycosides; AG, Flavonoid acetylglycosides.; S.S. (w), Sideritis scardica (wild growing); S.S. (c), Sideritis scardica (cultivated), S.R. (w), Sideritis raeseri (wild growing); S.R. (c), Sideritis raeseri (cultivated). Table 1. LC-MS/MS analysis of aerial parts in Sideritis species Comparison of both extraction procedures revealed that the domestic infusion of Sideritis prepared in boiling water resulted in minor losses of hydroxycinnamic acids and phenylethanoid glycosides content. In contrast, less than 19 % of acetylglycosides from S. scardica (wild growing and cultivated), 34 % from wild growing S. raeseri and 40 % from cultivated S. raeseri originally present and quantified in the methanol extracts in Sideritis were measured in the water extracts (Figure 1). Material and methods The antioxidant activities of the extracts were evaluated through three in vitro model systems: DPPH, ABTS and FRAP assay. In these model systems the methanol extract of wild growing Sideritis raeseri was the most effective i.e. it showed the highest antioxidant capacity (Figure 3). Plant material- Aerial parts of the flowering plants were collected during July in the summer of 2008. Two wild growing species were collected: Sideritis scardica and Sideritis raeseri. Also, two more specimens were samples from plantations of the same species. The material was air dried, packed in paper bags and kept in a dark and cool place until analysis. Sample preparation- In order to analyze of the phenolic compounds in Sideritis (leaf, stem and flower), samples containing 0.2 g of powder material were processed. Two extraction procedures for sample preparation were afforded: with methanol and boiling water. The methanol extraction was performed with 20 mL of 70 % methanol at room temperature for 24 h and the water extraction was performed in boiling water for 1 h (domestic infusion) LC/DAD/ESI-MSn analysis- Chromatographic separations were carried out on 250 x 4.6 mm, 5 μm RP-18 LiChroCART column, protected with a LiChroCART guard column (4 x 4.6 mm, RP-18, 5 μm particle size). The mobile phase consisted of two solvents: water-formic acid (1 %) (A) and methanol (B). A linear gradient starting with 20 % B was installed to reach 50 % B at 25 min and 95 % B at 60 min. The flow rate was 0.7 mL min-1 and the injection volumen 80 μL. The HPLC system was equipped with an Agilent 1100 series diode array and mass detector in series (Agilent Technologies, Waldbronn, Germany). The mass detector was an Agilent G2445A Ion-Trap. MS data were acquired in the negative ionization mode and the full scan covered the mass range at m/z 100-1200. Principal Component Analysis (PCA) as multivariate statistical analysis was used and a clear separation of the samples was found according to the solvent used for extraction. Additionaly, separation of the samples was observed according to the parts of the plant (Figure 2). Figure 2. PCA based on HA; PG; FG; AG; HPLC data for the analyzed Sideritis scardica and Sideritis raeseri plant materials, SS-Sideritis scardica, SR-Sideritis raeseri, L-leaf, F-flower, S-stem, w-wild growing, c-cultivated, W-water extract, M-methanol extract. Figure 3. Free radical scavenging activity (DPPH˙ and ABTS˙+) and FRAP values of methanol and water extracts; (w) - wild growing; (c) - cultivated Conclusions In the quantitative analysis of flavonoids, the methanol extracts resulted in higher flavones contents in the Sideritis samples than the extracts prepared in boiling water. Concerning to the phenolic content in the different aerial parts, leaf was the richest plant organ in phenolic followed by flower and by stem with the lowest amount. Our results suggest that the best way to prepare a serving of “Mountain tea” is using 60% of leaf and 40% of flower of wild Sideritis raeseri will provide higher bioavailable phenolics content than a serving bag containing homogeneous distribution of aerial parts of this plant (33% leaf, 33% flower, 33% stem). 3. The methanol extract of S. raeseri (w) exhibited the highest antioxidant capacity as shown by DPPH, ABTS and FRAP assays. The antioxidant capacity was linearly correlated with phenolic content. . References (1). Villar, A., Gasco, M.A., Alcaraz, M.J. (1984). Journal of Pharmacy and Pharmacology. 36, 820-823. (2). Koleva, I., Linssen, J.P.H., Van Beek, T.A., Evstatieva, L.N., Kortenska, V., Handjieva, N. (2003). Journal of the Science of Food and Agriculture.83, 809-819. (3). Tomás-Barberán, F.A., Gil, M.I., Ferreres, F., Rivera, D., Obón, C., Tomás-Lorente, F. (1993). Biochemical Systematics And Ecology. 21, 487-497. (4). Yesilada, E., Honda, G., Sezik, E., Tabata, M., Fujita, T., Tanaka, T., Takeda, y., Takaishi, Y. (1995). Journal of Ethnopharmacology. 46, 133-152. Acknowledgement Authors are grateful to the European project (grant number FP7-204756) “Chromlab”. AGI, FF, DAM and FATB are grateful to the Spanish National project Consolider Ingenio 2010 “Fun-C-Food” (CSD2007-00063).