2.1 Reagents

The pure flavonoid, rutin, was obtained from commercial source (Sigma-Aldrich) and used as received. The chemical structure of rutin is shown in Fig. 1.

Two types of Chinese green tea, Sencha (S) and Gun Powder (GP), in the form of dry leaves were obtained from Natur-Vit (Poland). Their extraction was performed in 90% ethanol using an OMC SER 148 apparatus from Envag, consisting of three extraction columns and three containers with thimbles, in which samples of powdered and dried tea were placed. The process was carried out in boiling ethanol (temperature, 78.5 °C) for 8 h.

The chemicals used for the preparation of the extract solutions were as follows:

• • acetonitrile (CH₃CN) pure p.a. from POCh Gliwice, Poland;
• • tetrabutylammonium perchlorate ([C₄H₉]₄NClO₄) from Fluka, which was used as a supporting electrolyte.

2.2 Measurement methods

Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements were performed using an Autolab analytical unit (Eco Chemie, Holland). A three-electrode system was used for the measurements with the platinum test and auxiliary electrodes. The potential of the tested electrode was measured versus a ferricinium/ferrocene reference electrode (Fc⁺/Fc), the standard potential of which is defined as zero, independent of the solvent used. Prior to taking the measurements, all solutions were degassed with argon. During the measurements, an argon atmosphere was maintained over the solution. The effect of the polarisation rate on the electro-oxidation of rutin and green tea extracts in an anhydrous medium was assessed.

UV-VIS spectra were recorded in the wavelength range from 190 to 800 nm using a UV-VIS spectrophotometer (Shimadzu UV-24001 PC).

3.1 Cyclic and differential pulse voltammetry of rutin and green tea

The electrode reactions characterising the electrochemical oxidation of flavonoids on a platinum electrode were examined by means of cyclic and DPV. The cyclic voltammetric profile is an important tool for assessing the presence of homogenous chemical reactions following the electrochemical oxidation. DPV has a higher resolution, which enables better separation of the peaks characterising the subsequent steps of an electro-oxidation reaction. The half-wave potential of a peak in a cyclic voltammogram corresponds to the potential of a peak occurring in a differential pulse curve and is characteristic of each of the subsequent steps of the electrode reaction being investigated.

Select cyclic and differential pulse voltammograms recorded in the solution of the rutin, green tea extracts and the supporting electrolyte are presented in Fig. 2.

Within the potential range in which the flavonoid oxidation peaks appear, the supporting electrolyte (tetrabutylammonium perchlorate in acetonitrile – 0.1 mol L^-1) shows no characteristic peaks except for the charging of the electrical double layer (Fig. 2A, B, curve 4). However, a small wave appears in the supporting electrolyte within the potential range from 0.5 to 1.0 V in the voltammograms. This wave can be attributed to the presence of (C₄H₉)₄NClO₄ and perchlorate ion oxidation. Fortunately, this wave current is relatively low in comparison with the peak currents attributed to the flavonoid oxidation. Zieja, Gadowska-Trzos, and Stojek [20] have reported that this wave can also be caused by the oxidation of impurities such as water and other organic substances.

The voltammograms recorded by CV show that the oxidation of rutin on a platinum electrode proceeds in at least four electrode steps before the oxidation potential of the electrolyte (1.8 V). Four oxidation peaks (peaks I, II, III and IV) are observed at the potentials 0.36 V (E_1/2I – half-wave potential), 0.69 V (E_1/2II), 1.06 V (E_1/2III), and 1.50 V (E_1/2IV) (Fig. 2A, curve 1). The electrode reactions investigated are irreversible reactions. The cyclic voltammograms of rutin and the extract of GP green tea are similar. GP is oxidised irreversibly, as is rutin, in at least in four electrode steps: 0.36 V (E_1/2I), 0.73 V (E_1/2II), 1.08 V (E_1/2III), and 1.56 V (E_1/2IV) (Fig. 2A, curve 2). In contrast, the extract of S green tea is oxidised in at least two electrode steps at potentials 1.11 V (E_1/2III) and 1.62 V (E_1/2IV) (Fig. 2A, curve 3). With the use of DPV, rutin and GP solutions are oxidised in at least in four electrode steps. Their DPV voltammograms show four peaks at the following potentials: for rutin, 0.33 V (E_pI), 0.69 V (E_pII), 1.04 V (E_pIII), and 1.50 V (E_pIV) (Fig. 1B, curve 1), and for GP extract, 0.32 V (E_pI), 0.70 V (E_pII), 0.98 V (E_pIII), and 1.56 V (E_pIV) (Fig. 2B, curve 2). The solution of sample S is oxidised in at least one electrode step (Fig. 1B) at a potential of 1.56 V (E_pIV) (Fig. 2B, curve 3). It can be concluded that the extract of GP contains primarily rutin and other compounds, while the S extract contains no rutin but contains other phenol compounds.

3.2 UV-VIS analysis

The rutin and green tea solutions were examined by UV-VIS spectrophotometry, and the results are shown in Fig. 3. Three characteristic absorption bands of rutin were present at 202 nm and 252 nm (which correspond to the transfer of π-π* electrons in the benzene ring) and at 349 nm (which corresponds to the transfer of π-π* electrons in B and C rings). The intensity and the positions of these two bands depend on the number and positions of hydroxyl groups in the rutin molecule. The weaker band at 296 nm corresponds to keto-hydroxyl tautomerism between the four and seven positions in the rutin molecule. (Fig. 3A, curve 1) [17]. These are characteristic bands of flavonoids. The spectra of GP and S extracts were not identical. There were absorption bands at 217, 274, 407, 598 and 667 nm. Spectrum of GP extract includes additional band at 350 nm, which is characteristic for rutin. It can be concluded that the extract of GP contains primarily rutin and other compounds, while the S extract does not contains rutin but contains other phenol compounds. In comparison to the spectrum of rutin, spectra of green tea extracts showed additional absorption bands at 407, 589 and 659 nm characteristic for chlorophyll (Fig. 3B, curve 2 and 3) [21–23]. Based on the analysis of the UV-VIS spectra, it can be concluded that, in addition to rutin and other phenol compounds, the samples of green tea contain chlorophyll, which imparts colour to both GP and S green tea.