Investigation of the spectroelectrochemical behavior of quercetin isolated from Zanthoxylum bungeanum

Flavonoids are common bioactive components in plants. Quercetin is the most abundant flavonoid in the human diet, accounting for more than half of the total daily consumption of flavonoids. In this study, adsorption and electrocatalytic activities of quercetin isolated from Zanthoxylum bungeanum on an electrode was studied via homemade electrodes. An in situUV-Visible thinlayer spectroelectrochemical method was used to study the electrochemical behavior of quercetin in detail and to explore its electrochemical reaction mechanism. This experiment proves that UV-Vis thin-layer spectroelectrochemistry is a feasible way for studying the electrochemical reaction mechanism of flavonoids in plants.


Introduction
Zanthoxylum bungeanum is a kind of plant resource with high edible and medicinal value [1,2]. The chemical components of Z. bungeanum mainly include volatile oils, alkaloids, amides, coumarins, and flavones. Flavonoids are a kind of common bioactive component in plants [3,4]. In recent years, quercetin, hyperoside, anisidine, arbutin, and rutin have been isolated from the pericarp of Z. bungeanum [5,6].
Quercetin is the most abundant flavonoid in the human diet, accounting for more than half of the total daily consumption of flavonoids. Ionizing radiation can produce a large number of free radicals and cause a series of free radical reactions, including lipid peroxidation [7][8][9]. Excessive free radicals are harmful and can lead to changes in the cellular structure and the destruction of cellular functions, which may cause cancer, increased ageing, cardiovascular disease, and other health concerns. Quercetin can combine with Fe(III) and Mn(II) ions, and its role as an antioxidant may be achieved by affecting the internal balance of metal ions, thus changing their oxidation state in cells [10][11][12][13]. NO is a messenger molecule found in recent years, which is a part of many physiological activities. Excessive NO production in the brain can lead to neurodegenerative diseases. Quercetin can inhibit the production of NO in a dosedependent manner and thus shows a preventive effect on neurodegenerative diseases. Furthermore, the incubation of 10 mg of quercetin with the HepG2 cell line can effectively inhibit the binding activity of nuclear factor kappa B and protect against injuries induced by H 2 O 2 [14][15][16].
Electrochemical methods have been widely used for studying the antioxidation activity of natural antioxidants; in addition, these methods have been used for the analysis and detection of natural antioxidants [17][18][19]. The main research contents of electrochemical methods can be summarized as the electrochemical detection of flavonoids and the kinetic study of electrode processes. However, the evaluation of the antioxidant activity of natural antioxidants and the interaction between natural antioxidants and natural oxidants have also been reported [20,21]. Cyclic voltammetry, as a simple and fast electrochemical method, has been used by many researchers to detect antioxidants [22][23][24]. Differential pulse voltammetry, oscillopolarography, and flow injection amperometric methods have also been used in the analysis of natural antioxidants [25][26][27]. Choosing a suitable method is the key for improving detection sensitivity. By using electrochemical theories and methods, the reversibility of a reaction can be obtained from the electrochemical data, and the apparent kinetic parameters, such as the number of electron transfers, rate constants, exchange current densities, and transfer coefficients, can be obtained; furthermore, the intermediate particles and products can be detected [28,29].
Spectroelectrochemistry is an interdisciplinary field developed in chronology. A simple electrochemical measurement technique can only obtain indirect information about the interfacial structure and reaction history of the electrode solution [30,31]. The main disadvantage of electrochemical techniques is that they only have pure electrochemical measurements and lack the characteristics of the electrode reaction molecules; thus, there is no useful information about reaction products or intermediates [32]. Spectroelectrochemistry is a method that combines spectroscopy and electrochemical methods to simultaneously measure an electrolytic cell. Generally, the spectroelectrochemical spectrum uses electrochemistry as the excitation signal, and the response of the system to the electrical excitation signal is monitored by the spectroscopic technology [33,34]. The two are closely combined to exert their respective advantages. In this way, a variety of information can be obtained at the same time, which provides a very powerful research method for studying the electrode process mechanism and electrode surface characteristics, along with identifying the intermediates of reaction processes, transient states, and product properties; moreover, certain electrochemical parameters can be measured [35,36].
It is often difficult to study the redox reaction of drugs in vivo. According to the research characteristics of medicinal chemistry, people often choose electrochemical techniques to study the electron transfer properties of drugs for simulating or assisting the exploration of the redox process of drugs in vivo [37,38]. Chemical reactions of drug molecules often occur in multiple steps, and the reaction mechanism is complex; therefore, it is difficult to accurately determine them by general methods [39][40][41]. UV-Vis spectroscopy has unique advantages in identifying reactive substances, especially the transient states and intermediates of the reaction. Therefore, UV-Vis spectroscopy can immediately provide considerable information about reactants, intermediates, and products, thereby making it a powerful method to study electrochemical reactions and mechanisms [42,43]. Many drug molecules have characteristic absorption in the ultraviolet-visible region, which makes it possible to study the properties of drug molecules by spectroelectrochemistry. The thin-layer spectroelectrochemical measurement requires a short electrolysis time, which makes it easy to control the directional electron transfer properties of drug molecules, and the drug distribution in the thin-layer solution is uniform [44,45]. Thin-layer spectroelectrochemistry can also be used to study the adsorption properties of drugs on electrode surfaces.
The purpose of this study is to enhance adsorption and electrocatalytic activities of quercetin on the electrode by using homemade electrodes and to use in situ UV-Visible thin-layer spectroelectrochemical methods to study the electrochemical behavior of quercetin in detail and explore its electrochemical reaction mechanism.

Experimental
All reagents were of analytical grade. Paraffin wax and graphite powder were spectrally pure, and other reagents were analytically pure. Quercetin was isolated from Z. bungeanum (inset of Figure 1). When designing a thin-layer electrochemical cell, to avoid modifying the sample cell of the spectrophotometer, a common cuvette was directly used as the cell body. The research electrode should be easily fixed and removed for grinding and cleaning before the experimental test.
To obtain the changes in the concentrations of reactants and products in the thin-layer liquid phase during the electrode reaction, the parallel incidence method was adopted, that is, the incident light was projected across the electrode surface in parallel with the electrode surface. For the light path of the Shimadzu UV-Vis 2550 spectrophotometer used in the experiment, the electrode surface should be inserted vertically into the cuvette. A certain slit was formed by a gasket of a certain thickness at the upper and lower ends of the electrode plate, and another baffle was used in the cuvette. This slit formed a thin layer of electrolyte, and the thickness was the thickness of the gasket. The thickness of the thin pool could be adjusted by changing the thickness of the gasket.
A certain amount of quercetin stock solution was taken and diluted with a certain amount of buffer to obtain the required concentration of quercetin solution. A cuvette was filled with a certain amount of quercetin solution. The research electrode was inserted, and the thin cell was filled with solution to drive out air bubbles. The reference electrode and the platinum mesh auxiliary electrode were inserted, and a syringe was used to aspirate excess solution. The cuvette was inserted into the sample cell of the spectrophotometer, and leads of the three electrodes were led out of the test system. The quercetin was scanned by cyclic voltammetry and potentiostatic electrolysis, and the scanning spectrum and absorbance-time curve of the thin-layer cell solution were recorded.
Ethical approval: The conducted research is not related to either human or animal use. Figure 1 shows the large range of CV curves of quercetin in B-R buffer solution (0.1 M) at different pH values. It can be seen from the figure that with the increase in the pH value, redox peak potentials of quercetin are negatively shifted, and the peak current tends to be smaller. When the pH value reaches 12, almost no redox peak appears. When pH = 1.8, the system shows the best CV curve, with a good peak shape and large peak current. This behavior change may be caused by the hydrolysis of quercetin. Therefore, pH of 1.8 was selected for further study. Figure 2 shows the repeated scanning of the UV-Vis spectrum of the solution in the thin-layer cell during the constant potential oxidation process that was within the potential range of the first oxidation peak. At the beginning of the scanning, there are two maximum absorption peaks at 369 and 255 nm, which were the characteristic absorption peaks of quercetin. The absorption band of the first long wavelength is mainly due to the transition between the highest occupied molecular orbital and the lowest unoccupied molecular orbital (HOMO-LUMO) [46].

Results and discussion
Due to the HOMO-LUMO conversion, the electron charge density of the B-ring shifts to the carbonyl bond of the C-ring [47]. Other transformations are the redistribution of charge throughout the molecule. For these compounds, the UV-Vis spectrum cannot distinguish the detailed structure of the molecule. Figure 3 shows the change in the absorbance of quercetin on the quercetin electrode in the thin-layer cell under open-circuit conditions. As time passes, Abs 369nm starts to decrease from the initial maximum value to a limit value, indicating that quercetin has a certain adsorption on the electrode surface. If the concentration of quercetin is increased, the time needed to reach adsorption equilibrium will increase accordingly [48]. When the   surface is replaced by a polyethylene plate, Abs 369nm did not change over time, indicating that quercetin adsorption is carried out on the electrode surface rather than on the polyethylene plate. Figure 4 shows the in situ kinetic tests of quercetin and its oxidation products in the thin-layer cell at 369 and 293 nm, respectively. In the course of repeated EMF scanning, the absorbance of the thin-layer solution shows periodic changes at both wavelengths. During forward scanning, Abs 369nm decreases with time, while Abs 293nm increases, which corresponds to the decrease in the quercetin concentration and the increase in the product concentration. In the back sweep of potential, due to the opposite electrode process, the product is reduced, so the corresponding absorbance change is also opposite to that of the forward sweep. However, the amplitude of the change is smaller than that during forward scanning, especially in the case of a slow scan (Figure 4a). With the increase in the number of scanning cycles, the overall Abs 369nm shows a decreasing trend, while the overall Abs 293nm shows an increasing trend (Figure 4a and b). At the same time, Figure 4a and b clearly shows that not only quercetin is preadsorbed on the electrode surface before the power is turned on participating in the electron transfer reaction but also quercetin and its products in solution participate in the reaction through the preadsorption and subsequent desorption steps [49,50].
It is generally believed that for a quasi-reversible reaction, the reversibility of the system will decrease with the increasing scanning speed. Comparing Figure  4a and b, it can be seen that the studied reaction has high reversibility at high scanning speeds, which is contrary to the performance of typical quasi-reversible reactions [51,52]. When the scanning speed is fast enough (Figure 4c), Abs 369nm and Abs 293nm hardly change with the number of cycles, showing the properties of a nearly ideal reversible system. Figure 5 shows the cyclic voltammetry curve obtained from a wide range of potential scans. A significant feature is that in the subsequent cycle, the oxidation peaks (except A2) and reduction peaks decrease significantly. A2 becomes the main oxidation peak. In addition, at least from the second cycle, the reactants of A2 did not come from the products of A1. It can also be seen that A3 is a composite peak. In the second cycle, there are two small peaks attributed to A3 in the potential range. In addition, the reduction peaks on the curve are all very wide composite peaks. Figure 6a shows the absorbance time curve of the quercetin solution during cyclic potential scanning at a rate of 5 mV/s and in the range of 0-0.9 V. It can be seen that Abs 369nm decreases to a very small value in the first  forward scanning process, which makes the overall change in the subsequent scanning process small. Accordingly, Abs 293nm also reaches a high value in the first scan, which is similar to when the sweep speed is high. This result indicates that most quercetin in the thin-layer solution takes part in the reaction during the first forward scan, but the products in the solution are not significantly reduced back to their original forms during the reverse scan. It can be concluded that quercetin has been continuously adsorbed on the electrode surface to participate in the aforementioned oxidation reaction over the whole potential range of a 0.9 V sweep potential [53]. During the subsequent cycle, Abs 293nm shows periodic absorbance changes that are synchronized with the cyclic potential scanning, especially when the scanning speed is high (Figure 6b and c). However, it is remarkable that Abs 293nm decreases in the forward scan and increases in the reverse scan, which is the opposite of the periodicity of oxidation products and reduction products during cyclic scanning. The main reason for this phenomenon can be attributed to the influence of the added electrode potential on the adsorption of the product. In the back sweep of the potential, the product is driven by the interfacial electric field to desorb from the electrode surface into the solution and then adsorb to the electrode surface again during the forward sweep of the potential. During forward scanning, the electrode surface is covered by the continuous adsorption of the final product [54]. Figure 6a also shows that Abs 369nm shows a less obvious periodic change, which is opposite to that of Abs 293nm , indicating the competitive adsorption between the residual quercetin and final oxidation products on the electrode surface. In Figure 6a, we see a more complex periodic absorption wave, but it disappears when the scanning speed increases (Figure 6b and c). This complex wave appears simultaneously with the cyclic voltammetry peak, which is clearly related to the formation of oxidation products. When these products are formed on the electrode surface, they diffuse to the thin-layer solution due to supersaturation on the electrode surface. Then, with the positive change in potential, these products are adsorbed on the electrode surface, which results in the absorption wave. Because the adsorption and desorption process of oxidation products is relatively slow, this step is skipped when the scanning speed is fast.

Conclusion
In the homemade 0.2 mm-thick thin-layer electrochemical cell, the absorbance at two wavelengths expresses a good response to the electrochemical reaction process, thereby quickly and accurately showing the electrochemical reaction process. The oxidation behavior of quercetin is a chain reaction. The results show that quercetin adsorbed on the electrode surface and in the solution participates in the electrode reaction at the same time, and the electrode has strong adsorption behavior toward quercetin and its various oxidation products at high potentials. This experiment proves that UV-Vis thin-layer spectroelectrochemistry is a feasible way to study the electrochemical reaction mechanism of flavonoids in plants.

Conflict of interest:
The authors declare no conflict of interest.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.