Comparative study of antioxidant capacity of Rheum rhaponticum root polyphenols Piret Raudsepp1*; Kati Helmja2; Ain Raal3; Merike Vaher2; Tõnu Püssa1 1
Department of Food Science and Hygiene, Estonian University of Life Sciences, Kreutzwaldi 58 A, 51014 Tartu, Estonia; 2Department of Chemistry, Faculty of Science, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia; 3Department of Pharmacy, University of Tartu, Nooruse 1, 50411 Tartu, Estonia; *corresponding author:
[email protected] Abstract. Liquid chromatographic (LC-DAD-MS2) and capillary electrophoretic (CE) methods were used for simultaneous determination of relative contributions of a number of polyphenols into overall antioxidativity of the extract of the roots of garden rhubarb (Rheum rhaponticum L.). Antioxidativities of polyphenols were evaluated using scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. It was confirmed that hydroxystilbenes are substantially better radical scavengers than hydroxyanthraquinones. Introduction. The roots of garden rhubarb are known for extra high content of polyphenols, mainly hydroxystilbenes and -anthraquinones. Differently from the other species of Rheum, is garden rhubarb especially rich in hydroxystilbenes, derivatives of piceatannol, resveratrol, rhapontigenin and deoxyrhapontigenin (Aaviksaar et al., 2003). Matsuda and co-workers showed in the screening test for DPPH and O2 radical scavenging activity of constituents of five different rhubarbs, that stilbenes revealed activity, but anthraquinones and sennosides did not. They suggested also that galloyl moiety enhances and glucoside moiety reduces the activity (Matsuda et al., 2001). Rösch et al. have shown that the antioxidant capacities of polyphenols depend chiefly on the availability of free vicinal OH-groups (o-diphenolic arrangement) in the aglycone molecule, enabling formation during oxidation of a o-quinonic moiety, stabilized by conjugation (Rösch et al., 2003) Simultaneous comparison of antioxidativities of different polyphenols in one extract is possible to carry out by liquid chromatography or capillary electrophoresis, monitoring the disappearance of respective peaks after addition to the system of active radicals (Shui et al., 2005). Materials and methods. The rhubarb roots (50 g) were collected in two different places of Estonia. Cut and dried plant material was powdered, sieved and two parallel sub-samples by 1 g of each were macerated with a 10-fold excess (v/w) of methanol at room temperature with periodical shaking. Reversed-phase liquid chromatography at Agilent 1100 series system (Agilent Technologies, Waldbronn, Germany) with UV-Vis diode array detector (200600 nm) and ion trap equipped with an electrospray interface (ESI) with negative ionization (m/z=50-1000, target mass 400) was used to identify and monitor the disappearance of polyphenols from the extract. Column: Zorbax 300SB-C18 (2.1×150 mm – Agilent Technologies), stepwise gradient of 0.1% formic acid and acetonitrile. CE was carried out by Agilent CE System with a diode array detection (DAD). CE Chemstation (Agilent Technologies) was used for instrument control, data acquisition and data handling. Before the analyses the root extract was treated with the solutions of DPPH in the range of 1-40 mM (LC-DAD-MS2) or 1 – 5 mM (CE). The literature data (Ye et al., 2007) as well as parallel analysis of commercial standards of resveratrol, piceatannol, piceid, astringin, rhapontin, deoxyrhapontin, aloe-emodin, emodin, rhein and physcion facilitated the identification of the polyphenols.
Results and discussion. The DPPH concentrations causing 50% reduction of concentration of the single polyphenols in the extract (EC50) were calculated by decrease of heights of respective extracted ion peaks caused by increase of DPPH content. The decreasing order of antioxidativities: gallic acid glucoside > piceatannol-3’-glucoside (2) > resveratrol acetylglucoside (4) > rhapontigenin-galloylglucoside (isomer 1) (7) > deoxyrhapontigenin-galloylglucoside 1 and 2 (10 and 12) > piceatannol-4-glucoside > rhapontigenin-galloylglucoside (isomer 2) (6) > resveratroloside (1) = rhapontigenin (8) = rhapontins 1 and 2 (3 and 5) resveratrol dimer 1 (13) = deoxyrhapontigenin (14) resveratrol dimer 2 (15) = deoxyrhapontigenin-acetylglucoside (11)> deoxyrhapontin (9) > various hydroxyanthraquinones and their glucosides. As previously suggested, hydroxystilbenes are substantially better DPPH scavengers than
hydroxyanthraquinones. The exact position of a polyphenol in this sequence is determined by its structure, presence of galloyl moiety and vicinal OH-groups enhance antioxidant capacity of polyphenols.
Figure 1 UV chromatograms (306 nm) of rhubarb root extracts - dark line – initial extract, green line – extract, treated with 20 mM of DPPH. Peak numbers are explained in the text. Suitable method for the evaluating of the oxidation of the extract by CE is demonstrated. As indicated in Figure 2, CE allows successful monitoring of the appearance of new peaks as the oxidation products and also disappearance of the compounds found in the extract.
Figure 2. The electropherogram of the monitoring of oxidation: A – the extract of Rheum rhaponticum L.; B – the extract treated with 1 mM DPPH; C – the extract treated with 2.5 mM DPPH; D – the extract treated with 5 mM DPPH. The separation conditions: separation buffer composed of 25 mM sodium tetraborate (pH 9.3) and 10 % methanol, an effective length of the capillary 50 cm, voltage applied +15 kV, injections were performed hydrodynamically for 10 s. References Matsuda et al. (2001) Bioorg and Med Chem. 9: 41-50. Aaviksaar et al. (2003) Proc. Estonian Acad. Sci. Chem.,52: 99-107 Rösch et al. (2003) J. Agric. Food Chem. 51: 4233-4239. Shui et al. (2005) J. Agric. Food Chem. 53: 880-886. Ye et al. (2007) J. Am. Soc. Mass Spectrom. 18: 82-91.
XXIVth International Conference on Polyphenols (ICP 2008) - Salamanca, Spain: 7/8-11
