CROATICA CHEMICA ACTA CCACAA 80 (1) 29¿34 (2007) ISSN-0011-1643 CCA-3135 Original Scientific Paper Electrochemical and Antioxidant Properties of Anthocyanins and Anthocyanidins Andréia A. de Lima, Eliana M. Sussuchi, and Wagner F. De Giovani* Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, 14040-901, Ribeirão Preto – SP, Brazil RECEIVED JUNE 26, 2006; REVISED SEPTEMBER 29, 2006; ACCEPTED OCTOBER 27, 2006 Electrochemical properties of delphinidin, cyanidin, pelargonidin, kuromanin and callistephin were investigated by cyclic and differential pulse voltammetries at different pH values and also in methanol. On the basis of oxidation potentials, the order of antioxidant activity for antho- cyanidins is delphinidin > cyanidin > pelargonidin. Oxidation peaks for anthocyanins (kuro- manin and callistephin) are shifted to more positive potentials compared to anthocyanidins (delphinidin, cyanidin and pelargonidin). Oxidation peak currents are linearly dependent on the Keywords square root of the scan rate, which is typical of a diffusion controlled electrochemical process. anthocyanins Antioxidant activities of the compounds were evaluated using the 1,1-diphenyl-2-picrylhydra- anthocyanidins zyl (DPPH) radical-scavenging method and they are directly related to their redox potential antioxidants values. The order of the antioxidant activity is delphinidin > cyanidin > pelargonidin > kuro- electrochemical properties manin > callistephin. INTRODUCTION pharmacological properties that render them interesting potential cancer chemopreventive agents.9–11 Because Anthocyanins are one of the main classes of flavonoids anthocyanins are widely consumed, finding out addi- in red wines. They have been pointed out as the molecu- tional biological activities related to these compounds lar entities that are most likely responsible for what has would be of great interest. been described as the »French Paradox«, that is, the lo- Differences between the individual anthocyanins lie wer incidence of coronary atherosclerosis in the French in the number of hydroxyl groups, the nature and num- population compared to other Western populations, even ber of sugars attached to the molecule, the position of though the former consumes a fattier diet.1 Anthocya- this attachment, and the nature and number of aliphatic nins are representative of plant pigments widely distrib- or aromatic acids attached to sugars in the molecule. The uted in colored fruits and flowers. They also exhibit an- aglycones of anthocyanins are called anthocyanidins. tioxidant activities and therefore may contribute to pre- The antioxidant potential of anthocyanins can chan- vention of heart and inflammatory diseases.2–9 Anthocya- ge in dependence on the substituents.12,13 Some studies nins, and especially anthocyanidins, exhibit a variety of suggest that the glycosidic forms generally display a de- * Author to whom correspondence should be addressed. (E-mail: [email protected]) 30 A. A. DE LIMA et al. crease in the antioxidant capacity when compared with trochemical experiments were carried out with a PAR mo- the corresponding aglycone.14,15 This may be due to the del 273A Potentiostat/Galvanostat. Cyclic and differential steric hindrance conferred by the bulky sugars.16,17 pulse voltammetric experiments were performed in a In this work, we have studied the electrochemical one-compartment cell using a glassy carbon working elec- properties of delphinidin, cyanidin, pelargonidin, kuro- trode, a platinum wire auxiliary electrode, and a Ag/AgCl, –1 manin, and callistephin (Figure 1) in a wide range of KCl 3 mol L (0.201 vs NHE) reference electrode. Cyclic voltamograms were obtained at scan rates ranging from 25 solution conditions, by cyclic and differential pulse vol- mV s–1 to 500 mV s–1. Differential pulse voltammetric ex- tammetries. The antioxidant activities of the compounds periments were done at a pulse amplitude of 50 mV, pulse were determined by the 1,1-diphenyl-2-picrylhydrazyl width of 70 ms, and scan rate of 10 mV s–1. The glassy car- (DPPH) radical-scavenging method and correlated with bon working electrode was intensively polished with alumi- their oxidation potentials. It is assumed that both the elec- na powder prior to each measurement. → ⋅ – + trochemical oxidation (Fl–OH Fl–O +e +H) and The anthocyanins and anthocyanidins solutions, 0.1 → ⋅ ⋅ the antioxidant activity (Fl–OH Fl–O + H ) involve mmol L–1, were prepared from 1 mmol L–1 stock solutions 18 the breaking of the same O–H bond; the lower the oxi- in methanol or in buffer solutions; lithium perchlorate was dation potential, the higher the antioxidant activity of the used as the supporting electrolyte in methanol. The pH val- compound. This suggests that the electrochemical prop- ues of aqueous solutions were buffered at an ionic strength erties of flavonoids may be associated with their biologi- of 0.2 mol L–1, by using Britton-Robinson buffer solu- cal activities. Hence, we propose that the simple cyclic tions.19 The pH measurements were carried out with a Mi- voltammetry technique can be used to evaluate the bio- cronal B 474 pH meter instrument. logical activity of anthocyanins and anthocyanidins. Evaluation of the Scavenging Activities R1 of Antioxidants by the DPPH Radical Method 3' OH An aliquot of methanol solution (0.1 mL) containing differ- 2' 4' B ent concentrations of standards (1, 2, 4, 6, 10, 15 and 20 HO 8 O 1' 5' mmol L–1) was added to 3.9 mL of DPPH 59.55 mmol L–1 7 2 R AC 6' 2 in methanol. Reduction of DPPH was followed by monitor- 6 3 ing the decrease of absorbance at 515 nm for 5 minutes (ti- 5 4 OR3 me necessary for the reactions to reach a plateau – steady OH state). A blank solution of DPPH was screened to estimate DPPH decompostion during the measurement time. The ex- R1 R2 R3 act initial DPPH concentration (cDPPH) in the reaction me- Pelargonidin H H H dium was calculated from the calibration curve with the Cyanidin OH H H equation determined by linear regression: Delphinidin OH OH H ⋅ Kuromanin OH H 3-O-glucoside A515 = 10034 cDPPH – 0.01824, R = 0.99975 Callistephin H H 3-O-glucoside For each antioxidant concentration, the amount of the Figure 1. Structures of delphinidin, cyanidin, pelargonidin, ku- remaining DPPH in percents was calculated as: romanin, and callistephin. DPPHremaining / % = 100 ⋅ [DPPH]t / [DPPH]t=0 EXPERIMENTAL where [DPPH]t is the concentration after 5 minutes of reac- Reagents tion. The values were plotted versus DPPH concentration to Delphinidin chloride, cyanidin chloride, pelargonidin chlo- obtain the amount of antioxidant needed to reduce the ini- ride, kuromanin chloride, and callistephin chloride were tial DPPH concentration by 50 % (EC50). purchased from Extrasynthese (Genay, France) and used without further purification.1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Aldrich Chemical Co., HPLC RESULTS AND DISCUSSION grade methanol from Mallinckrodt Inc. and lithium per- chlorate from Acros Organics. All other chemicals were of Electrochemical Properties of Anthocyanins and the highest purity and used as received. Anthocyanidins Cyclic voltamograms of the compounds studied herein Apparatus and Measurements display oxidation peaks and practically no reverse re- Routine UV-Vis spectra were obtained in 1-cm quartz cells duction peaks, indicating the occurrence of EC pro- by using a Hewlett-Packard 8453 spectrophotometer. Elec- cesses. Functional OH groups attached to the antho- Croat. Chem. Acta 80 (1) 29¿34 (2007) PROPERTIES OF ANTHOCYANINS AND ANTHOCYANIDINS 31 25 25 200 Delphinidin Pelargonidin 20 150 20 100 m /A I 15 50 15 0 A 10 0 500 1000 1500 2000 10 m (Ag/AgCl)/mVEvs. A / m I / 5 I 5 0 0 –5 –5 (a) (c) –10 –10 0 200 400 600 800 1000 1200 1400 0 200 400 600 800 1000 1200 1400 (Ag/AgCl)/mVE vs. (Ag/AgCl)/mVE vs. 25 Cyanidin 20 Kuromanin 20 15 15 10 A m 10 m /A / I I 5 5 0 0 (b/1) (d) –5 –5 0 200 400 600 800 1000 1200 1400 0 200 400 600 800 1000 1200 1400 (Ag/AgCl)/mVE vs. (Ag/AgCl)/mVE vs. 14 40 Callistephin 12 10 30 1 8 20 A m m /A I 6 2 I/ 4 10 4 3 0 2 (b/2) (e) 0 –10 200 400 600 800 1000 1200 0 200 400 600 800 1000 1200 1400 E vs. ( Ag/AgCl) /mV (E vs. Ag/AgCl) /mV Figure 2. Cyclic voltamograms of a) delphinidin (insert shows the methanol response), b/1) cyanidin, b/2) differential pulse voltamogram of cyanidin, n =10mVs–1, c) pelargonidin, d) kuromanin, and e) callistephin; 0.1 mmol L–1, 0.1 mol L–1 in methanol; 100 mV s–1, glassy carbon electrode. 20 cyanin and anthocyanidin ring structures can be elec- cinol groups. Epa data are shown in Table I. trochemically oxidized. The less positive potential Delphinidin (Figure 2a) displays four oxidation pro- peaks displayed by these flavonoids correspond to oxi- cesses; the two less positive peaks correspond to dations of the more redox-active OH groups of ring B. 3',4',5'-OH oxidations, the third process to 3-OH oxida- The potentials become successively more positive for tion, and the fourth process to 5,7-OH oxidations. The oxidations of the C-3 OH group and of the ring A resor- same processes are observed for cyanidin (Figure Croat. Chem. Acta 80 (1) 29¿34 (2007) 32 A. A. DE LIMA et al. TABLE I. Electrochemical data for anthocyanins and anthocyani- The presence of sugar moieties at position 3 of dins kuromanin and callistephin results in a shift of oxidation peaks to more positive potentials (Figures 2d, 2e). This (a) (b) Compounds (Epa vs Ag/AgCl) / mV effect is probably due to a loss of coplanarity of ring B Delphinidin 519, 608, 844, 1108 with respect to the rest of the molecule, which causes a Cyanidin 564, 646, 1084 decrease in conjugation.