A novel electrochemical micro - titration method for quantitative evaluation of the DPPH free radical scavenging capacity of ca ﬀ eic acid

: In this report, the stoichiometric ratio ( R ) for the interaction of diphenylpicrylhydrazyl ( DPPH ) radi - cals with the antioxidant was employed as an evaluation index for the DPPH radical scavenging activity of antiox - idants. This evaluation index was related only to the stoichiometric relationship of DPPH radicals with the antioxidant and had no relationship with the initial DPPH amount and the sample volume, which could o ﬀ er a solution to the problem of poor comparability of EC 50 values under di ﬀ erent conditions. A novel electrochemical micro - titration method was proposed for the determina - tion of the stoichiometric ratio ( R ) for the interaction of DPPH radicals with the antioxidant. This electrochemical micro - titration model was veri ﬁ ed using ca ﬀ eic acid as the DPPH radical scavenger, with the stoichiometric ratio ( R ) of DPPH radicals to ca ﬀ eic acid determined to be in the range of 2.003 – 2.046. The calculated EC 50 values were 0.513, 1.011, and 1.981 × 10 – 5 mol/L for 2.10, 4.05, and 8.02 × 10 – 7 moL of added DPPH radicals, respectively. The proposed method showed no di ﬀ erences from the conventional method, but had better precision and reliability, and used a smaller amount of sample.


Introduction
In the last decade, many analytical methods have been developed to determine antioxidant activity in vitro, measure the ability to reduce oxidant species/probes, or scavenge free radicals, such as superoxide anion, hydroxyl, peroxyl, and alkoxyl radicals [1].One such popular method for in vitro evaluation of antioxidant capacity is based on using stable free radical diphenylpicrylhydrazyl (DPPH) [2,3].The DPPH method is described as a simple, rapid, accurate, and inexpensive assay that is widely used for the determination of radical scavenging activity [4][5][6][7].Since the late 1950s, a variety of DPPH-based methods have been introduced for estimating the antioxidant activity of natural compounds or foods, including spectrophotometric [6], chemiluminescence [8], high-performance liquid chromatography with DPPH [9][10][11], gas chromatography mass spectrometry [12], fluorescence probe detection [13], and electron paramagnetic resonance methods [14].Flow injection-based methods for the determination of scavenging capacity against DPPH have also been reported [15,16].A DPPH-based optical sensor for screening antioxidant activity was introduced by Steinberg and Milardovic [17].
Electrochemical methods provide a rapid, simple, and sensitive alternative for analyzing bioactive compounds and determining antioxidant capacity and have still seen recent developments to determine the antioxidant activity [18][19][20].The relationship between the electrochemical behavior of compounds with antioxidant activity and their resulting "antioxidant power" (capacity) has attracted great interest due to "low oxidation potential" corresponding with "high antioxidant power" [21].Cyclic voltammetry (CV) on carbon electrodes seems to be a suitable method for antioxidant capacity determination, especially due to its simplicity, rapidity, and ability to be used directly in biological and crude samples, including red and white wine, tea, coffee, juices, and even blood serum [22].Differential pulse voltammetry (DPV), as a selective and sensitive technique, has also been explored in the detection of natural polyphenolic antioxidants in both complex and clinical samples [23].Malagutti et al. used square wave voltammetry with a solid carbon polyurethane electrode (rigid carbon-polyurethane composite electrode) to compare the antioxidant capacity of green tea samples [24].
Electrochemical methods based on the amperometric reduction of DPPH have also been introduced, for example, the evaluation of DPPH free radical scavenging at a glassy carbon electrode or a platinum screen-printed working electrode [25,26].Amatatongchai et al. [27] developed amperometric detection of the residual concentration of non-reacted DPPH using a carbon nanotube modifiedglassy carbon electrode, which had sensitivity more than 25 times greater than that of the bare GC electrode.Milardović et al. used amperometric detection of DPPH free radical scavenging to evaluate the antioxidant activity of some water or ethanol-soluble pure compounds from samples of tea, wine, and some other beverages [28].As mentioned earlier, electrochemical methods show good prospects in the determination of antioxidant capacity, but, to our knowledge, there have been few reports of voltammetric determination of DPPH scavenging activity.Recently, multi-walled carbon nanotubes (MWNTs) have been widely used in the fabrication of the modified electrode because of their huge specific surface area, strong mechanical performance, stable chemical property, good electrocatalytic effect, and charge transfer ability [29][30][31].
Caffeic acid, known as 3,4-dihydroxycinnamic acid, is a typical phenolic acid compound found in plants.It is the main active ingredient in many traditional herbs, such as Terminalia and Penthorum chinense Pursh [32].Pharmacological studies have proven that caffeic acid scavenges and inhibits free radical, antibiotic, antivirus, antitumor, decreasing blood lipid, hypoglycemic, and anti-aging activities, which has been widely applied in the medical industry [33].
A novel voltammetry-based electrochemical microtitration method for the quantitative assessment of the DPPH free radical scavenging capacity of caffeic acid is described herein.This method is based on the stoichiometric relationship of the interaction between caffeic acid and DPPH free radicals, and a micro-titration method was proposed to determine this stoichiometric relationship.The stoichiometric ratio (R) of the stoichiometric relationship was employed as an evaluation index for DPPH radical scavenging activity.This method offers a rapid and inexpensive solution for the evaluation of DPPH radical scavenging activity in antioxidants.
The DPPH and caffeic acid were soluble in methanol.Fresh DPPH stock solution (10 mL) at a concentration of 0.1 mg/mL was prepared on each day of analysis.The stock solutions of caffeic acid were prepared in methanol at a concentration of 1.0 mg/mL.All stock solutions were stored at 4°C.

Preparation for the modified electrode
First, 6 cm graphite rod was taken and sealed in the polyethylene pipe using an epoxy resin binder.One of the ends was used as an electrode connection, and the other end was polished to a mirror-like surface with the different sizes of the metallographic sandpaper.Then, the mirror side was carefully polished for about 15 min using functionalized MWNTs on parchment paper, in order to embed MWNTs uniformly on the surface of the electrode and got the inlaid MWNTs/GE.Finally, the prepared electrode was electrochemically cleaned in a based solution by cycling potentials between −0.4 and +2.0 V at 0.1 V/s until a steady cyclic voltammogram was obtained.peak was observed for caffeic acid at 0.35 V (vs SCE).The oxidation peak current of caffeic acid decreased remarkably with the addition of DPPH, as depicted by curves b-e in Figure 2. A good linear relationship was observed between the oxidation peak current (i pa ) and the caffeic acid concentration (c), with a linear regression equation of i pa (mA) = 53.444c(μmol/L) + 2.7513 (r = 0.9991), meaning that the interaction of caffeic acid with DPPH could be more clearly evaluated via DPV by adding DPPH gradually into caffeic acid solution.Furthermore, a good linear relationship between the oxidation peak current of caffeic acid decreasing (Δi pa ) and the cumulative amount of added DPPH (n DPPH ) was obtained when varying its concentration from 1.0 × 10 -5 to 6.0 × 10 -5 moL/L, and the linear regression equation could be described as follows: Δi pa (μA) = 10.708nDPPH -0.242 (r = 0.9980).These results suggested that caffeic acid and DPPH interacted according to a certain stoichiometric relationship.

Mathematical model of electrochemical micro-titration
The concentration of caffeic acid showed a good linear relationship with the oxidation peak current (i pa ), as mentioned earlier, which can be depicted as follows: where c CA and i pa represent the concentration and oxidation peak current of caffeic acid, respectively.c CA decreases with the addition of DPPH, resulting in a decrease in i pa , as described by equation (2): where i pa 0 , c CA 0 , and n CA 0 represent the initial oxidation peak current, concentration, and amount of caffeic acid, respectively; V 0 is the initial volume of the solution mixture for analysis; n CA is the amount of caffeic acid; V i is the volume for a single addition of DPPH; and ∑ = V i N 1 i is the volume change during the titration process.In our experiment, V 0 and V i were 10.0 mL and 10.0 μL, respectively, and the number of additions was about 10; thus, the volume change in the titration process was less than 1.0%.equation (2) can be reformulated as follows: where Δn CA is the amount of caffeic acid consumed by DPPH in the titration process.According to the aforementioned experimental result, the interaction between caffeic acid and DPPH was based on a certain stoichiometric relationship, meaning that DPPH might react with caffeic acid according to a stoichiometric ratio of R, R = 1.983,Δn CA = Rn DPPH .Therefore, equation (3) can be further rewritten as: where R is the stoichiometric ratio, n DPPH is the amount of DPPH consumed in the titration process, K is the slope of the linear regression equation (equation ( 2)) for the oxidation peak current (i pa ), and the caffeic acid concentration (c) can be calculated using equation (1).As shown in equation ( 2), Δn CA (in equation ( 2)) could be transformed when n DPPH was added, which meant that the stoichiometric ratio (R) for DPPH and caffeic acid could be easily obtained using the proposed electrochemical micro-titration method.The evaluation of DPPH radical scavenging activity of antioxidants is normally based on the median elimination concentration (EC 50 ), as defined in equation ( 5): where c A50 and n A50 are the median elimination concentration and amount of antioxidants that cause the initial DPPH concentration to decrease by 50%, respectively.Substituting the stoichiometric ratio of R = n DPPH /Δn CA into equation (5) gives the following equation: where n DPPH 0 is the initial amount of DPPH.equation (6) shows an alternative way to calculate EC 50 using the stoichiometric ratio of DPPH and antioxidant.Moreover, the stoichiometric ratio (R) could also be used as a key parameter to characterize the antioxidant capacity of antioxidant compounds.For a certain antioxidant compound, a higher R value denotes the ability to scavenge more DPPH radicals and possession of a higher antioxidant capacity.

Electrochemical micro-titration of caffeic acid scavenging DPPH radicals
Electrochemical micro-titration was performed according to the method in Section 2. Figure 3 shows the titration curve, which depicts the cumulative added amounts of DPPH (n DPPH ) and the change in oxidation peak current value of caffeic acid (Δi pa ), calculated using equation (2).
Δi pa increased rapidly with the addition of DPPH and remained almost unchanged after DPPH addition reached a certain amount, indicating that caffeic acid was consumed almost completely.The inflexion in the titration curve can be characterized as the endpoint.
The stoichiometric ratio (R) of the reaction of caffeic acid with DPPH radicals was calculated using equation (4) based on the titration curve in Figure 3.And the result of stoichiometric ratio (R) is shown in Table 1.Moreover, the EC 50 of CA was calculated using the stoichiometric ratio (R) according to equation (6).The EC 50 result was compared with conventional photometric analysis, as shown in Table 2.The R value was consistent for different n DPPH values, and the EC 50 increased with the increase in n DPPH .The EC 50 resulting from this method was in agreement with that of the conventional method, and had a lower RSD, indicating that this method was reliable.

The spectroscopy study of CA scavenging DPPH radical
The DPPH radical scavenging activity of CA was also determined by conventional photometric analysis [34], the result is shown in Figure 4, a good linear relationship between CA concentration and scavenging rate was obtained, with a linear regression equation of E (%) = 14,518c + 0.1980 (r = 0.9910) before reaching the maximum, which ensure experimental results possesses better accuracy.The EC 50 was calculated based on the linear regression equation, as shown in Table 2.
Traditional evaluation methods for estimating the DPPH radical scavenging activity of antioxidants are mainly based on the median elimination concentration (EC 50 ).However, the inadequacies of this method are that EC 50 values for the same antioxidants under different conditions are quite different due to clear changes in EC 50 with changing initial DPPH amounts and sample volumes used for determination.Therefore, an electrochemical micro-titration method was used to obtain the stoichiometric ratio (R) of CA for DPPH radical scavenging.And the stoichiometric ratio (R) for the interaction of DPPH radicals with the antioxidant was employed as an evaluation index for the DPPH radical scavenging activity of  antioxidants.And the result indicated that this evaluation index was related only to the stoichiometric relationship of DPPH radicals with the antioxidant and had no relationship with the initial DPPH amount and the sample volume, which could overcome the problem of poor comparability of EC 50 values under different conditions.Furthermore, the EC 50 % values corresponding to different initial DPPH can be accurately deduced according to the R value, and the results are consistent with the conventional method.Therefore, the stoichiometric ratio (R) is superior to the conventional methods in terms of comparability, applicability, and reliability in evaluating antioxidant activity, and the amount of reagent is significantly reduced, which reduces the analysis cost and it has a good application prospect.

Conclusion
In this report, a novel sensitive electrochemical microtitration method for determination and estimation of the DPPH free radical scavenging activity of active materials was proposed based on the interaction between caffeic acid and DPPH.An electrochemical micro-titration model was established based on titration curves between oxidation peak current change of the antioxidant (Δi pa ) and the added amount of DPPH radicals in the titration process.The stoichiometric ratio (R) for the reaction of antioxidant with the amount of DPPH radicals added was determined using the obtained titration equation, while the DPPH median elimination concentration (EC 50 ) of antioxidants was calculated from the stoichiometric ratio (R).The above-mentioned electrochemical micro-titration model, verified using caffeic acid, indicated that the proposed method was simple, quick, sensitive, and low cost, providing a novel approach to the evaluation of antioxidant properties.

2. 3 . 2
Figure 1 shows the cyclic voltammograms (CV) of the interaction of caffeic acid with DPPH free radicals in HAc-NaAc buffer solution (pH = 4.0) at the graphite electrode.A pair of quasi-reversible redox peaks with peak

Table 2 :
Results of half of the scavenging rate (EC 50 ) (n = 6)