Measurement of Antioxidant Capacity by Electron Spin Resonance

Article pubs.acs.org/ac

Measurement of Antioxidant Capacity by Electron Spin Resonance Spectroscopy Based on Copper(II) Reduction Dan Li,† Jia Jiang,† Dandan Han,‡ Xinyu Yu,‡ Kun Wang,† Shuang Zang,† Dayong Lu,‡ Aimin Yu,† and Ziwei Zhang*,† †

College of Chemistry, Jilin University, Qianjin Street 2699, Changchun 130012, People’s Republic of China Department of Materials Science and Engineering, Jilin Institute of Chemical Technology, Jilin 132022, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: A new method is proposed for measuring the antioxidant capacity by electron spin resonance spectroscopy based on the loss of electron spin resonance signal after Cu2+ is reduced to Cu+ with antioxidant. Cu+ was removed by precipitation in the presence of SCN−. The remaining Cu2+ was coordinated with diethyldithiocarbamate, extracted into n-butanol and determined by electron spin resonance spectrometry. Eight standards widely used in antioxidant capacity determination, including Trolox, ascorbic acid, ferulic acid, rutin, caffeic acid, quercetin, chlorogenic acid, and gallic acid were investigated. The standard curves for determining the eight standards were plotted, and results showed that the linear regression correlation coefficients were all high enough (r > 0.99). Trolox equivalent antioxidant capacity values for the antioxidant standards were calculated, and a good correlation (r > 0.94) between the values obtained by the present method and cupric reducing antioxidant capacity method was observed. The present method was applied to the analysis of real fruit samples and the evaluation of the antioxidant capacity of these fruits.

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Electron spin resonance (ESR) spectroscopy, also called electron paramagnetic resonance (EPR) spectroscopy, is a magnetic resonance technique for detecting chemicals with unpaired electrons including free radicals and paramagnetic transition metal ions (e.g., Cu2+).17 ESR has been applied in many fields, such as physics, chemistry, and biological science.18−20 So far, the measurment of antioxidant capacity by ESR technique mainly focus on two aspects: (1) measure the decrease of signal intensity of stable artificial free radicals (e.g., DPPH); and (2) measure the efficiency of antioxidant in scavenging short-lived radicals (e.g., hydroxyl radical) yielded from radical generation system by ESR spin-trapping technique.17 In the past decade, ESR technique was applied to the determination of antioxidant capacity of wine,21 beer,22 white tea,23 fruits and vegetables,24 wheat extracts,25 and herbal extracts.26 In this work, a new method was developed for measuring antioxidant capacity by detecting ESR signal from the remaining Cu2+ after Cu2+ was reduced to Cu+ with various antioxidant standards and real samples. Cu+ was removed by precipitation with SCN−. The remaining Cu2+ was chelated with diethyldithiocarbamate (DDC) in aqueous phase, extracted into n-butanol phase and then determined by ESR. The present method was applied to the measurement of antioxidant capacity of eight antioxidant standards and six fruit

ree radicals and reactive oxygen species generated from oxidation reactions in the human body may cause damage or death to the cell by starting chain reactions, resulting in health problems such as cancer, aging, and heart disease.1−3 Antioxidants can terminate these chain reactions by scavenging free radicals or reacting with reactive oxygen species.2,4,5 Most antioxidants are reducing agents and inhibit oxidation reactions by being oxidized themselves.2 Natural antioxidants are divided into several categories based on their common chemical structures, including phenolic acids, flavonoids, vitamins, and phenolic diterpenes.2,6 Fruits and vegetables contain a variety of antioxidants, such as vitamin C, polyphenols, and carotenoids.7 Trolox equivalent antioxidant capacity (TEAC) or vitamin C equivalent antioxidant capacity (VCEAC) is frequently used to quantify antioxidant capacity of a given substance.8,9 A number of analytical methods, especially the spectrophotometric method, have been developed to measure antioxidant capacity of antioxidants in either food/plant extracts or biological samples. The most popular methods include 1,1diphenyl-2-picrylhydrazyl (DPPH),3 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS),10 ferric reducing ability of plasma (FRAP),11 and oxygen radical absorbance capacity (ORAC).12 A new spectrophotometric method was proposed by Apak et al. in 2004 and named as cupric reducing antioxidant capacity (CUPRAC) method.13−15 In the CUPRAC assay, the Cu2+-neocuproine (Nc) complex is reduced with antioxidant to Cu+-Nc, which has an orange−yellow color and gives a maximum UV absorption at 450 nm.16 © XXXX American Chemical Society

Received: January 6, 2016 Accepted: February 29, 2016

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DOI: 10.1021/acs.analchem.6b00049 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

solution in a tube. The resulting Cu2+−DDC complex was then extracted into 750 μL of n-butanol. The resulting solution was referred to as analytical solution and measured with both electron spin resonance spectrometer and UV spectrometer. Electron Spin Resonance Measurement. The ESR spectra were collected at room temperature on a commercial Bruker A300 X-band spectrometer at a frequency of 9.84 GHz. A 40 μL aliquot of analytical solution sealed in a 1.5 mm i.d. glass capillary tube (Kimble Chase) was placed into a quartz tube (Wilmad LabGlass), and the quartz tube was put in the resonator cavity. The modulation amplitude was 2.0 G, the microwave power was 2.3 mW, the center field was 3450 G, and the sweep width was 500 G. The experiments for the measurement of the standards and the samples were carried out in two replicates and triplicate, respectively. UV Spectrometric Measurement. The UV spectra were obtained at room temperature on a Shimadzu UV-1700 PharmaSpec spectrophotometer. A 200 μL aliquot of the analytical solution was diluted four times with n-butanol, and 400 μL of the resulting solution was injected into a 1 mm quartz cuvette. Absorbance at 430 nm was recorded against a blank of n-butanol. The experiments were carried out in triplicate. DPPH Method. DPPH (0.1 mmol/L) was prepared daily by dissolving 9.86 mg DPPH in 250 mL of methanol. A 3 mL aliquot of 0.1 mmol/L DPPH solution and 100 μL of sample solution were added into a test tube and blended immediately, reacting in the dark for 60 min. The resulting solution was measured with UV spectrometer at 515 nm. The experiments were carried out in triplicate. CUPRAC Method. Nc solution (7.5 mmol/L) was prepared by dissolving 39.00 mg neocuproine in 25 mL of ethanol. Ammonium acetate (0.1 mol/L, NH4Ac) aqueous buffer was prepared and adjusted to pH 7.0 with 50% HAc. A 1 mL aliquot of 10 mmol/L CuCl2 solution, 1 mL of NH4Ac buffer solution, 1 mL of Nc solution, 100 μL of sample solution, and 1 mL of H2O were added into a test tube in sequence, and the mixture stood for 60 min. The resulting solution was measured with UV spectrometer at 450 nm. The experiments were carried out in triplicate.

juice samples. TEAC values of eight standards and six fruit juices were compared with those obtained by CUPRAC, ABTS, DPPH, and FRAP methods. The results obtained by the present method are proved to be highly correlated with those obtained by CUPRAC method.

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EXPERIMENTAL SECTION Chemicals and Materials. Eight antioxidant standards, including Trolox (a water-soluble derivative of vitamin E), one vitamin (ascorbic acid), two flavonoids (rutin and quercetin), and four phenolic acids (ferulic acid, caffeic acid, chlorogenic acid, and gallic acid) were used. Trolox was purchased from J&K Chemicals. Ascorbic acid, rutin, quercetin, and gallic acid were purchased from Aladdin Industrial Corporation. Ferulic acid and caffeic acid were obtained from National Institute for The Control of Pharmaceutical and Biological Products. Chlorogenic acid was obtained from Shanghai Yuanye BioTechnology Co., Ltd. n-Butanol, methanol, and acetic acid were purchased from Beijing Chemical Works. Sodium diethyldithiocarbamate and potassium thiocyanate were obtained from Aladdin Industrial Corporation. Copper(II) chloride and ethanol were purchased from Sinopharm Chemical Reagents Co., Ltd. Sodium acetate anhydrous was purchased from Tianjin Fengchuan Chemical Reagent Co., Ltd. DPPH was purchased from Alfa Aesar. Nc was purchased from J&K Chemicals. Preparation of Standard Solution. A stock solution of 10 mmol/L copper(II) chloride (CuCl2) was prepared by dissolving 170.48 mg CuCl2·2H2O in distilled water and diluted to 1.8 mmol/L when used. Sodium acetate (0.1 mol/L, NaAc) buffer solution was prepared in water and adjusted to pH 6.0 with 50% acetic acid (HAc). Potassium thiocyanate (50 mmol/L, KSCN) was prepared by dissolving 242.95 mg KSCN in 50 mL of water. Sodium diethyldithiocarbamate (50 mmol/ L, DDC) was prepared by dissolving 536.25 mg of DDC in 50 mL water. Stock solutions of eight standards in a concentration of 2−5 mmol/L were prepared in 100% ethanol except for ascorbic acid, which was prepared in water and quercetin, which was prepared in 100% methanol. Each stock solution, except for quercetin, which was diluted by methanol, was diluted by water to a series of concentrations in order to obtain the standard curve and time curve for each standard. Preparation of Sample Solution. Six fruits, including lemon, orange, grapefruit, mandarin orange, watermelon, and strawberry, were obtained from local supermarkets. The eaten part of each fruit was cut into pieces and pressed for juice. Milliliters of juice were filtered once with filter paper and centrifuged at 10000 rpm twice. The juices from grapefruit, mandarin orange, and watermelon were used as the sample solution without dilution. Juices from lemon, orange, and strawberry were diluted four times with water before analysis, and the resulting solution was referred to as sample solution. A total of 100 μL of sample solution were used to prepare the analytical solution. Three parallel sample solutions for each fruit were prepared and used. Preparation of Analytical Solution. A total of 200 μL of CuCl2 working solution, 200 μL of NaAc buffer solution, and 200 μL of KSCN solution were in sequence added into a test tube. Then 100 μL of antioxidant standard or sample solution were added into the tube. The resulting mixture stood for a fixed period of time (reaction time) and then was filtered with a 0.22 μm syringe filter to remove CuSCN precipitate. After filtration, 450 μL of the filtrate were mixed with 80 μL of DDC

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RESULTS AND DISCUSSION Optimization of Experimental Parameters. To optimize the experimental conditions, the effects of pH value of the buffer solution, concentration of KSCN, volume of DDC solution, and volume of n-butanol were investigated. These experiments were performed using Trolox as the antioxidant. NaAc buffer solutions with pH values ranging from 4.5 to 6.5 were prepared. The experimental results indicated that there was not an obvious difference in antioxidant capacity when pH was changed. pH 6.0, which is close to physiological pH, was selected for further experiments. Aliquots of 200 μL of KSCN solution at concentrations ranging from 0 to 100 mmol/L were used, and we found that the scavenging capacity of Trolox reached maximum once KSCN concentration was over 40 mmol/L. Thus, 50 mmol/L was selected as the concentration of KSCN in order to fully precipitate Cu+. The effect of volume ranging from 0 to 140 μL of 50 mmol/L DDC solution was investigated. The antioxidant capacity increased with volume of DDC and reached a plateau when the volume was over 40 μL. Thus, 80 μL of DDC solution were used. The volume of nbutanol was set to be 750 μL to achieve the highest extraction efficiency for Cu2+−DDC complex. B


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Analytical Chemistry Standard Curves. The ESR spectra for Cu2+−DDC in nbutanol are displayed in Figure 1. As seen in the figure, all

Time Curves. The effect of the reaction time was investigated to determine the optimal period in the redox reaction for each standard. The curves of inhibition versus time obtained by ESR and UV methods are shown in Figures 2 and 3, respectively. The experimental results show that, for Trolox, ascorbic acid, ferulic acid, and rutin, the reaction is fast and completes mostly within 10 min. The time of 40 min was selected for the construction of their standard curves. However, for caffeic acid, quercetin, chlorogenic acid, and gallic acid, the redox process is slow, and it took about 40−90 min to reach equilibrium after the antioxidant was mixed with CuCl2 solution. Thus, 110, 120, 90, and 80 min were selected for the construction of the standard curves of caffeic acid, quercetin, chlorogenic acid, and gallic acid, respectively. Correlation with CUPRAC Assay. TEAC value (defined as mmol Trolox/L)29 for each standard was obtained by dividing slope of the standard curve by that of Trolox curve, and results are listed in Table 1. As shown in Table 1, the increasing order of antioxidant capacity determined by the present ESR method is ferulic acid < Trolox < ascorbic acid < gallic acid < rutin < caffeic acid < chlorogenic acid < quercetin. These results are mostly consistent with those from theoretical evaluation. In theory, antioxidant capacity of either phenolic acids or flavonoids depends largely on the number and position of their hydroxyl (−OH) groups.30 For example, free radical scavenging capability of flavonoids is highly dependent on the presence of a free 3-OH in the C ring.31,32 In this study, quercetin has a TEAC of 4.7, whereas rutin has a TEAC of only 2.9 because of the substitution of the free 3-OH with rutinose. Gallic acid has a TEAC of 2.6, which is close to its expectation value of 3.0 mmol/L Trolox corresponding to the three hydroxyl groups.30 The antioxidant abilities of hydroxycinnamic acids in increasing the resistance of low density lipoproteins (LDL) to peroxidation were investigated by Castelluccio et al., and the order of antioxidant effectiveness was chlorogenic acid ∼ caffeic acid > ferulic acid.33 The scavenging of ferulic acid was weakened by the methoxylation of the ortho-hydroxyl group when compared with the two diphenolics, chlorogenic, and caffeic acids.33 The results are consistent with ours, except for chlorogenic acid, which has a slightly higher TEAC than caffeic acid. We found that the linearity of the standard curve of chlorogenic acid becomes poor when the concentration is above 0.3 mmol/L. The highest concentration to construct the standard curve was 0.25 mmol/L, which may result in the slightly higher TEAC of chlorogenic acid. The TEAC values of these eight standards obtained by the present method were compared with the values obtained by some popular assays, including CUPRAC, 14 ABTS,34,35 DPPH,36−38 and FRAP.39,40 To obtain the correlation of

Figure 1. ESR spectra of Trolox at concentrations of 0.0, 0.8, and 1.6 mmol/L.

spectra obtained with different concentrations (0.0, 0.8, and 1.6 mmol/L) of Trolox have the same line shape. Thus, we quantified the signal simply by measuring the peak to peak amplitude of the third line among the four lines, without taking efforts to double-integrate the signals. The inhibition (%) of antioxidant is represented as the ratio of the height reduction in the presence of antioxidant to the height in the absence of antioxidant. In Figure 1, we can find a split of two peaks in the rightmost line of the ESR signal of Cu2+. We suspect that such extra splitting of two peaks comes from the two isotopes 63Cu and 65Cu existing in the CuCl2 reagent.27,28 Even with this extra signal, the quantitative analysis was not affected, especially when the third line was used for the analysis. Standard curves were constructed for each antioxidant standard by plotting the inhibition (%) versus antioxidant concentration. In order to prove the reliability of the present ESR method, the UV experiments on the same antioxidant standards were carried out. The standard curves for the eight standards obtained by ESR and UV spectrometry are displayed respectively in Figures S1 and S2. All these standard curves were simulated with linear equation. The regression equations and correlation coefficients (r) for the eight antioxidant standards are listed in Table 1. From Figures S1 and S2, as well as Table 1, it is found that the linearity for all curves is good enough and the values of r are higher than 0.994. The results obtained by ESR and UV assay are quite consistent.

Table 1. Regression Equations and TEAC Values (mmol Trolox/L) of Antioxidant Standards Determined by ESR and UV Assay r

regression equation std Trolox ascorbic acid ferulic acid rutin caffeic acid quercetin chlorogenic acid gallic acid

ESR y y y y y y y y

= = = = = = = =

0.502c + 0.013 0.561c + 0.008 0.429c − 0.023 1.44c + 0.024 1.51c + 0.037 2.44c − 0.008 1.86c − 0.002 1.28c + 0.015

UV y y y y y y y y

= = = = = = = =

0.500c + 0.001 0.568c − 0.009 0.406c − 0.026 1.45c + 0.018 1.46c + 0.015 2.23c + 0.010 1.69c + 0.010 1.28c − 0.004 C

TEAC

ESR

UV

ESR

UV

0.997 0.999 0.994 0.996 0.994 0.997 0.997 0.995

0.998 0.999 0.997 0.996 0.999 0.998 0.997 0.998

1.00 1.12 0.85 2.87 3.01 4.86 3.71 2.55

1.00 1.14 0.81 2.90 2.92 4.46 3.38 2.56


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Figure 2. Change of inhibition with time for the antioxidants obtained by ESR spectroscopy: (A) Trolox, (B) ascorbic acid, (C) ferulic acid, (D) rutin, (E) caffeic acid, (F) quercetin, (G) chlorogenic acid, and (H) gallic acid.

Figure 3. Change of inhibition with time for the antioxidants obtained by UV spectrometry: (A) Trolox, (B) ascorbic acid, (C) ferulic acid, (D) rutin, (E) caffeic acid, (F) quercetin, (G) chlorogenic acid, and (H) gallic acid.

by ABTS (r = 0.59), DPPH (r = 0.66), and FRAP (r = 0.73) are relatively low. Analysis of Real Fruit Juice Samples. The present method was applied to the analysis of six real fruit juice samples, including lemon, orange, grapefruit, mandarin orange, watermelon, and strawberry juices. The reaction time is 60 min. The values of TEAC for these juice samples are calculated as mmol TEAC per liter of juice, and listed in Table 3. From these

TEAC values between any two methods, the calibration curves were constructed by plotting TEAC values obtained by first method versus those obtained by second method. The slope of the calibration curve is taken as the correlation coefficient (r). As seen in Table 2 and Figure 4, TEAC values obtained by the present method has a high correlation with those obtained by CUPRAC method (r = 0.94). However, the correlations of TEAC values obtained by the ESR method with those obtained D


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Table 2. Comparison of TEAC Values (mmol Trolox/L) of Antioxidant Standards Obtained by ESR with Other Methods std

ESR

CUPRACN14

ABTS34,35

DPPH36−38

FRAP39,40

Trolox ascorbic acid ferulic acid rutin caffeic acid quercetin chlorogenic acid gallic acid

1.00 1.12 0.85 2.87 3.01 4.86 3.71 2.55

1.00 0.96 1.20 2.56 2.89 4.38 2.47 2.62

1.00 0.99 1.90 2.42 1.26 4.72 1.24 3.01

1.00 0.91 0.60 2.77 1.11 2.55 4.7 2.66

1.00 1.04 1.01 1.99 2.32 3.50 1.05 2.27

The TEAC values for the standards and juice samples obtained by the present method have respectively low (r = 0.66) and high (r = 0.97) correlation with those obtained by DPPH method. The results should be due to the difference of the matrices for standard and sample. Some similar experimental results were found in some publications.1,14,43 Method Evaluation. The advantages of the present ESR method are as follows: (1) The agents used, including CuCl2, KSCN, and DDC, are low cost, stable, and insensitive to heat or light; (2) When UV spectroscopy was applied too high absorbance (such as >1.0) is not suitable. However, when the analyte concentration in the final analytical solution is too high, no dilution is needed before measurement for ESR signal collection; (3) In ESR spectroscopy, the resonance signal from Cu2+ was measured, and color from any sample itself (e.g., color of juices) will not influence the quantification; (4) Complex of Cu2+−DDC was finally extracted into the n-butanol phase so that interference of sample matrix was eliminated; (5) The redox reaction is performed at pH 6.0, which is close to physiology pH in human body; (6) Samples were filtered after the redox reaction completes and Cu2+−DDC complex was extracted into the organic phase for the final analysis. Thus, it is not necessary for the sample to be measured immediately after the reaction. We measured the sample within several days and experimental results were repeatable. However, compared with CUPRAC method, when the present method was applied, more operation steps were required.

Figure 4. Correlation of TEAC values obtained by ESR method with those obtained by CUPRAC method (r = 0.94).

Table 3. TEAC Values (mmol Trolox/L juice) of Six Fruit Juices Obtained by ESR, DPPH, and CUPRAC fruit lemon orange grapefruit mandarin orange watermelon strawberry

ESR 1.92 0.79 1.45 1.21 0.31 3.99

± ± ± ± ± ±

0.25 0.33 0.01 0.06 0.06 0.49

DPPH 2.21 1.82 1.49 1.80 0.38 6.36

± ± ± ± ± ±

0.02 0.03 0.03 0.02 0.00 0.31

CUPRAC 1.38 2.01 1.27 2.29 0.39 7.06

± ± ± ± ± ±

0.03 0.04 0.02 0.02 0.01 0.16

TEAC values, we could find that the increasing order of antioxidant capacity determined by the present ESR method is watermelon < grapefruit < mandarin orange < orange < lemon < strawberry. We also measured each fruit juice sample by DPPH and CUPRAC assays, and the TEAC values are listed in Table 3. As seen in Table 3, the TEAC values obtained by the present method has a high correlation with those obtained by either DPPH (r = 0.97) or CUPRAC method (r = 0.91). The DPPH method involves an electron transfer of the free radical, whereas CUPRAC method and the present method involve measurement of the reducing potential of juices.41 The present method is based on the reaction: Cu2+ + e + SCN− → CuSCN, and CUPRAC method is based on the reaction: Cu(Nc)22+ + e → Cu(Nc)2+. The same sample matrix may have significantly different effect on the redox potentials of the two reactions. The difference in the redox potentials may result in the difference in the TEAC values. The matrices of juices are much more complicated than those of the eight standards that we studied. The complicated juice sample matrices may have very different effect on the potentials of the two reactions. Various antioxidants may interact with each other, as might affect the total antioxidant capacity.42 So, for some juice samples there is no good agreement of the TEAC values between CUPRAC and ESR methods. Although different methods gave different TEAC values for specific sample (e.g., orange), the correlations between methods were generally good.

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CONCLUSIONS

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ASSOCIATED CONTENT

A new ESR method was proposed for measuring antioxidant capacity taking advantage of the reduction of Cu2+ into Cu+. In order to evaluate the method, the present method was applied to the measurement of both antioxidant standards and real fruit juice samples. Standard curves for the eight antioxidant standards show good linearity, and the TEAC values obtained by the present method has a high correlation with those obtained by CUPRAC method. The present method could be applied to the determination of thiols in medicine and biological samples by changing the experimental conditions.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b00049. Standard curves for the antioxidants obtained by ESR spectroscopy and by UV spectrometry (PDF). E


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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 13610709861. Fax: +86 431 85112355. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project is supported by the Research Startup Foundation of Jilin University (No. 450060521178). REFERENCES

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Measurement of Antioxidant Capacity by Electron Spin Resonance

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