Article pubs.acs.org/JAFC
High-Throughput Superoxide Anion Radical Scavenging Capacity Assay Hongxun Tao,†,∥ Jinge Zhou,†,∥ Tao Wu,*,† and Zhihong Cheng*,‡ †
Key Laboratory of Standardization of Chinese Medicines of Ministry of Education, The Shanghai Key Laboratory for Compound Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China ‡ Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China S Supporting Information *
ABSTRACT: A high-throughput superoxide anion radical (O2•−) scavenging capacity assay based on the xanthine oxidase/ xanthine reaction system was developed and validated in the present study. The reaction conditions including detection wavelength, concentrations of reactant components, reaction temperature, reaction time, pH, terminator reagent, and sample dissolving solvents were optimized. The accuracy and reliability of the assay were assessed by evaluation of linearity (r2 = 0 .9513−0.9957), precision (intraday RSD 1.13−4.05% and interday RSD 2.13−5.62%), accuracy (95.64−97.42% recovery), and stability (RSD 2.62−6.19%), as well as comparison with the conventional colorimetric method. The EC50 values obtained by the current method and the conventional assay were highly correlated (r > 0.99). This high-throughput O2•− scavenging assay may be used for screening and estimating potential superoxide anion radical (O2•−) scavengers, especially food extracts and natural products with a very small amount of test material. KEYWORDS: xanthine oxidase, superoxide anion radical, high throughput, antioxidant activity
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INTRODUCTION Free radicals play an important role in physiology and pathology as mediators of many biochemical events. Excessive free radical production is associated with numerous chronic disease conditions, such as cancer,1 cardiovascular diseases,2 diabetes mellitus,3 gastric ulcer,4 and neurological disorders.5 In recent years, antioxidants have attracted a great deal of attention as potential agents for the control of diseases associated with oxidative damage. Superoxide anion radicals (O2•−), one of the most important reactive oxygen species (ROS), are produced as byproducts of metabolism, particularly in mitochondrial respiration,6 and serve as a progenitor for other toxic ROS such as hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and hydroxyl radicals (HO•). This leads to a growing interest in the discovery and development of valuable nutraceuticals and functional foods capable of scavenging O2•−. A number of O2•− scavenging assay protocols have been developed, including an electron spin resonance (ESR) method,7−14 a high-performance liquid chromatography (HPLC) method,15−17 an ultrahigh-performance liquid chromatography and triple-quadrupole mass spectrometry (UHPLC-MS) method,18 and a thin layer chromatography (TLC) bioautographic method.19 Among these, the ESR, HPLC, and UHPLC-MS methods have been shown to possess good specificity and accuracy, but require relative expensive instrumentation. TLC bioautography is an ideal method for the estimation of antioxidants present in complicated extracts and can be easily adopted in high-throughput analysis. However, this method is generally developed and used in qualitative ways. Compared to these methods, spectrophotometry is one of the most widely used techniques in biomedicinal analysis20−25 due to its © 2014 American Chemical Society
inherent ease of application and no requirement of expensive equipment. The conventional spectrophotometric methods are performed manually in cuvettes, which generally require a large sample quantity. These requirements are not compatible with the capabilities of most food chemistry laboratories charged with routine analysis of pure antioxidants. Therefore, a sensitive and high-throughput assay is needed to facilitate the rapid and automated screening of the O2•− scavengers. Superoxide anion radicals can be generated in vitro by many methods such as the hydrogen peroxide degradation system in alkaline dimethyl sulfoxide (DMSO),11,26 the phenazine methosulfate (PMS)/β-nicotinamide adenine dinucleotide (NADH) system,14,27,28 the riboflavin irradiation system,17,19,29 and the xanthine/xanthine oxidase (X/XO) system.12,18−23,28,30 Among these the X/XO system is a classical enzymatic method based on XO oxidation of xanthine giving O2•−, which subsequently reacts with a pale yellow tetrazolium salt, nitroblue tetrazolium chloride (NBT), to yield a purple formazan by monitoring its absorbance at 560 nm. In this study, a high-throughput method in a 96-well microplate format for O2•− scavenging capacity assay based on the X/XO system was developed and validated. In addition, this high-throughput assay was used to estimate the O2•− scavenging activity of some pure compounds and food extracts. Received: Revised: Accepted: Published: 9266
May 11, 2014 August 27, 2014 September 3, 2014 September 3, 2014 dx.doi.org/10.1021/jf502160d | J. Agric. Food Chem. 2014, 62, 9266−9272
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Article
solution was added to start the reaction. After gentle shaking for 10 s, the reaction mixture was incubated in a water bath at 37 °C for 20 min. Finally, 20 μL of 0.6 M HCl was added to terminate the reaction, followed by shaking for another 10 s. The absorbance at 560 nm was measured. The total volume for each reaction mixture in each well was 240 μL. A blank test without XO and a control test without antioxidants were also conducted. Allopurinol was used as a positive control for the assay. The level of O2•− scavenging capacity for each reaction was also calculated as formula 1. The EC50 values were calculated under the assay condition. Triplicate reactions were carried out for each level of every individual sample.
MATERIALS AND METHODS
Materials and Reagents. Xanthine (X0625-5G), xanthine oxidase (XO, X4875-10U), nitroblue tetrazolium chloride (NBT, N6876-5G), and allopurinol (A8003-5G) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Potassium phosphate monobasic (KH2PO4), potassium phosphate dibasic (K2HPO4·3H2O), ethylenediaminetetraacetic acid disodium salt (EDTA), sodium hydroxide (NaOH), dimethyl sulfoxide (DMSO), acetone, acetonitrile, ethanol, isopropanol, tetrahydrofuran, and hydrochloric acid (HCl) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ultrapure water was prepared by an Advantage A10 water purifier (Milli-Q Academic, Molsheim, France). All other chemicals were of analytical grade and used without further purification. Rosmarinic acid, resveratrol, and rutin (purity > 98%) were obtained from Shanghai R&D Center for Standardization of Chinese Medicines (Shanghai, China). The individual food extracts of rosemary (whole plant, 2012030), ginger (rhizoma, 2012021), and green tea (leaves, 2012020) were purchased from Sichuan Tongtai Plant Chemical Industry Co., Ltd. (Shifang, Sichuan Province, China). The lotus extract (leaves, 20120620) was purchased from Shanghai Moochun Biological Technology Co., Ltd. (Shanghai, China). A 0.2 M stock phosphate buffer solution (PBS, pH 8.0) was prepared by mixing equal volumes of 0.2 M KH2PO4 and 0.2 M K2HPO4·3H2O solutions. A 1.0 mM EDTA stock solution was prepared in ultrapure water. Stock solutions of the pure antioxidants were prepared in DMSO at concentrations of 6.0 mM, and the stock solutions of the food extracts were prepared in a mixture of DMSO/ultrapure water (1:1, v/v). A series of working solutions were made by appropriate stepwise dilutions of the above stock solutions with 0.2 M PBS buffer (pH 8.0). A fresh substrate solution containing 4 mM xanthine and 4 mM NBT was prepared in 0.05 M PBS (pH 8.0) just before use. A 0.04 U/mL XO solution (250 mL) was prepared by dilution of the 10 U XO stock solution with a mixture solution of 1 mM EDTA (125 mL) and 0.1 M PBS (pH 8.0, 125 mL). The enzyme activity of XO was determined by a reference method31 before preparation. Conventional Colorimetric Assay. The conventional colorimetric O2•− scavenging capacity assay was carried out according to a previously described laboratory protocol.32 Briefly, an aliquot of 100 μL of different concentrations of tested antioxidants was added to a 1 mL substrate solution containing 0.4 mM xanthine and 0.24 mM NBT. After the addition of 1 mL of XO solution (50 mU/mL), the reaction mixture was incubated in a water bath at 37 °C for 20 min. The reaction was then terminated by the addition of 1 mL of 0.6 M HCl, and the absorbance (A) of each reaction mixture at 560 nm was measured against a blank without XO using a microplate spectrophotometer (Power Wave, Bio-Tek, Winooski, VT, USA). The O2•− scavenging capacity for each reaction was calculated as
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RESULTS AND DISCUSSION Selection of the Detection Wavelength for the X/XO Reaction. The X/XO enzymatic system is based on XO oxidation of xanthine giving O2•−, which subsequently reacts with NBT to yield a purple formazan. The formation of formazan is generally detected by absorbance at 560 nm. However, several wavelengths such as 560 nm,32 510 nm,33 and 595 nm34 have also been reported for the detection of formazan production. This divergence prompted us to investigate a suitable wavelength for this reaction system. Theoretically, the individual components involved in the reaction mixture including xanthine, NBT, and XO might interfere with the formazan absorbance. As shown in Supporting Information Figure SI1A, all of the components except formazan (the reaction product) in the reaction system showed no absorption maximum between 500 and 800 nm. The absorbance of formazan from 550 to 650 nm reached a platform, among which 560 nm turned out to be stable. Therefore, 560 nm was chosen as the detection wavelength, consistent with that used in most of the X/XO literature. NBT is a classical biochemical reagent for the detection of O2•−. During the reaction process, ditetrazolium (NBT2+) is attacked by O2•−, along with one electron being transferred to turn NBT2+ into tetrazolinyl radical (NBT•+). In the following step, one more electron transfer turns NBT•+ into monoformazan (MF).35 It should be noted, therefore, that the excess of NBT or O2•− might theoretically lead to the production of only MF or diformazan (DF). The difference in molecular structures of MF and DF may result in the different maximum absorption wavelengths of these two substances. Thus, the different molar ratios of xanthine to NBT (1:1, 1:2, and 1:3) in the X/XO reaction mixture were scanned over 450−800 nm (Supporting Information Figure SI1B). The results showed that the effect of different molar ratios of xanthine to NBT was small and of marginal importance to the maximum wavelength of formazan. As the absorption behaviors of the molar ratios (1:2 and 1:3) of xanthine to NBT acted similarly, a 1:2 ratio of xanthine to NBT was chosen. Effect of Temperatures on the X/XO Reaction. Several reaction temperatures such as 25 °C20 and 37 °C32 have been used for this X/XO enzymatic reaction system. To better understand the effect of temperature on the O2•− formation, different reaction temperatures including 25, 31, 37, 43, and 49 °C on this X/XO enzymatic system were tested. As a result, the absorbance values increased when the temperature was increased from 25 to 37 °C (Supporting Information Figure SI2A), and slight decreases were observed with a temperature of >37 °C. The results indicated that the extent of the reaction reached the maximum at 37 °C. Therefore, 37 °C was selected as the reaction temperature in further experiments, which is consistent with that of O2•− physiologically generated.
O2•−scavening capacity % = [1 − (A s − A s‐b)/(Ac − Ac‐b)] × 100%
(1)
where As, As‑b, Ac, and Ac‑b represent the absorbances of samples, sample control (without addition of XO), control (without addition of tested antioxidants), and vehicle control (without addition of tested antioxidants and XO), respectively, at 560 nm after 20 min of reaction. The EC50 value is the concentration of an antioxidant capable of quenching 50% of the radicals in the reaction mixture under the assay condition. Triplicate reactions were carried out for each level of every individual sample. High-Throughput O2•− Scavenging Assay. The high-throughput O2•− scavenging assay was carried out using a 96-well microplate spectrophotometer (Power Wave, Bio-Tek). The 96-well plate was covered with a lid to prevent solvent evaporation during reaction. Six or eight different concentrations were used for each antioxidant extract and antioxidant compound in the study. The antioxidant sample solution (6.67 μL) and 193.33 μL of substrate solution (including 6.43 μL of 4 mM xanthine, 12.86 μL of 4 mM NBT, and 174.04 μL of 0.05 M PBS) were added to each well. Then 20 μL of 0.04 U/mL XO 9267
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Effect of pH on the X/XO Reaction. The effect of the PBS buffer with different pH values (6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0) on the X/XO enzymatic system was studied. As shown in Supporting Information Figure SI2B, the absorbance values increased with the increase of the pH values from 6.0 to 8.0, whereas absorbance values decreased from pH 8.0 to 9.0. A possible explanation is that the conformation of XO might be affected by the pH of the buffers.22 Therefore, a pH of 8.0 was selected in this study. Effects of Enzyme and Substrate Concentrations on the X/XO Reaction. The rate of an enzyme-catalyzed reaction depends on concentrations of enzymes and substrates.22 The effect of XO concentrations on the formazan formation was studied. At eight concentrations of XO over a 8-fold range (10−80 mU/mL), the enzyme was incubated with 4 mM substrate solution and DMSO (without antioxidants) at 37 °C for 20 min. The absorbance values gradually increased with increasing XO concentrations from 10 to 40 mU/mL, whereas there was no significant change of the absorbance values in XO concentrations up to 40 mU/mL (Supporting Information Figure SI2C). These data suggested that the optimal XO concentration in this reaction was 40 mU/mL. Similarly, several substrate concentrations (2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5 mM) with a constant molar ratio to NBT (1:2) were studied. Supporting Information Figure SI2D shows the effect of substrate concentrations on the rate of the reaction. For a given enzyme concentration (40 mU/mL), the absorbance values showed a rapid and marked increase with substrate concentrations from 2.0 to 4.0 mM and a subsequent slow increase up to 5.5 mM, which is consistent with the Michaelis−Menten kinetics theory.36 Therefore, the optimal substrate concentration in this reaction system was 4.0 mM. Effect of Reaction Time on the X/XO Reaction. Several reaction times such as 5 min,37 20 min,32 and 30 min38 have been applied for this enzymatic reaction system. For the consideration of stability, the reaction time required to reach the steady state for this enzymatic system was studied. The change in absorbance was measured for 44 min at 2 min interval, without the addition of terminators. As shown in Supporting Information Figure SI3, the absorbance values increased rapidly within 20 min, and reached a plateau from 20 to 44 min. The result indicated that the reaction mixture can be considered to reach the “steady state” at 20 min or anytime after that. Thus, a reaction time of 20 min was used. Effect of Different Sample Dissolving Solvents on the X/XO Reaction. Enzymatic reactions can be conducted not only in water but also in organic solvents.39 This suggests the feasible use of organic cosolvents for the hydrophilic and lipophilic antioxidant samples tested in the enzymatic assay. Therefore, several organic solvents including DMSO, acetone, acetonitrile, isopropanol, and tetrahydrofuran, as well as water, were selected to investigate the solvent effect on the reaction system. With a fixed ratio of solvent volume to the reaction mixture (1:30), none of these organic solvents had shown a significant effect on absorbance values compared with water (Figure 1), indicating the potential use of these solvents as sample dissolving solvents. This is especially important and useful for samples with lower solubility in water. In addition, three other different ratios of solvent volume to the reaction mixture (1:10, 1:20, and 1:40, v/v) were investigated and compared with the ratio of 1:30. As shown in Figure 1, no significant absorbance changes were observed among the 1:20, 1:30, and 1:40 ratios of solvent volume to
Figure 1. Effects of dissolving solvents and ratios of solvent volume to the reaction mixture on the xanthine/xanthine oxidase reaction. The reaction mixture contained 6.67 μL of different solvents, 193.33 μL of substrate solution (6.43 μL of 4 mM xanthine, 12.86 μL of 4 mM NBT, and 174.04 μL of 0.05 M PBS), and 20 μL of 40 mU/mL xanthine oxidase. The absorbance at 560 nm was determined for each reaction mixture after 20 min of reaction terminated by the addition of 20 μL of 0.6 M HCl. Four different volume ratios (1:10, 1:20, 1:30, and 1:40, v/v) were tested. ACE, ACN, ET, IPA, and THF represented acetone, acetonitrile, ethanol, isopropanol, and tetrahydrofuran, respectively. Data are means of triplicate measurements. Vertical bars represent the standard deviations.
reaction mixture, regardless of the nature of dissolving solvents. However, in a 1:10 ratio, all of the solvents except water and ethanol resulted in obvious decreases of the absorbance values (Figure 1). Therefore, the organic solvent effect should not be ignored in a relatively high ratio (1:10) of solvent volume to the reaction mixture (except water and ethanol). In consideration of its extensive use as a dissolving solvent in screening tests, DMSO with a 1:30 ratio of volume to the reaction mixture was used in this study. Effect of Different Terminators on the X/XO Reaction. Significant pH changes can affect enzyme activity, which might be an effective way to terminate an enzymatic reaction. HCl, a commonly used acid terminator in the X/XO reaction,40 was compared with NaOH, an alkaline terminator of other enzymes.41 As shown in Figure 2A, the absorbance values increased with increasing concentrations of NaOH solutions (0.1−0.9 M), together with the production of green substances instead of the expected purple formazan. Thus, NaOH is not suitable to terminate the reaction due to the formation of side products with absorption at 560 nm. In contrast to NaOH, a gradual decrease of the absorbance values was observed with the increase of HCl concentrations from 0.1 to 0.5 M (Figure 2B). Further increase of the HCl concentration to 1.0 M did not result in a further decrease in the absorbance values. Importantly, unlike with 0.1−0.4 M HCl solutions, the generation of the expected purple formazan cannot be observed with 0.5−1.0 M HCl, suggesting that the reaction could be totally terminated by 0.5−1.0 M HCl solutions. To further confirm the efficiency of the HCl terminator, the absorbances of the reaction mixtures at 10, 20, 40, and 60 min were determined after 10 min of reaction being terminated by 0.5−1.0 M HCl solutions. As shown in Figure 2C, no obvious increase of absorbance values was observed after the addition of 0.5−1.0 M HCl solutions at each time point, whereas that of the negative control (without addition of HCl solutions) increased rapidly from 10 to 20 min, indicating the great termination efficiency of HCl. Hence, a 0.6 M HCl solution beyond the inflection point was chosen as the optimal terminator. Surface-active agents (SAAs) are also reported to terminate the enzymatic reactions, because SAAs could neutralize the surface charges of enzyme molecules, resulting in the 9268
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Figure 2. Effects of different terminators and HCl concentrations on the xanthine/xanthine oxidase reaction (A, NaOH; B, HCl; C, 0.5−1.0 M HCl). The reaction mixture contained 6.67 μL OF DMSO, 193.33 μL of substrate solution (6.43 μL of 4 mM xanthine, 12.86 μL of 4 mM NBT, and 174.04 μL of 0.05 M PBS), and 20 μL of 40 mU/mL xanthine oxidase. The absorbance at 560 nm was determined for each reaction mixture ternminated by 20 μL of 0.1−1.0 M NaOH (A) or 20 μL of 0.2−0.7 M HCl (B) after 20 min of reaction. For the termination efficiency of HCl, 20 μL of 0 M and 0.5−1.0 M HCl solutions was added to the xanthine/xanthine oxidase reactions, followed by determination of the absorbance at 10, 20, 40, and 60 min (C). Data are means of triplicate measurements. Vertical bars represent the standard deviations.
Table 1. Linear Relationship between Antioxidant Concentrations and Percent O2•− Inhibition Obtained by the HighThroughput O2•− Scavenging Assaya sample
slope
intercept
r2
linearity range
nb
allopurinol rosmarinic acid resveratrol rutin rosemary ginger lotus green tea
2954.4 932.94 482.91 275.58 399.36 138.36 126.24 55.051
41.619 23.971 −10.518 13.265 7.1703 −4.9684 −5.9565 6.5041
0.9766 0.9808 0.9911 0.9895 0.9844 0.9957 0.9789 0.9513
2.93−14.66 μM 2.93−14.66 μM 58.65−131.97 μM 73.31−146.63 μM 18.33−146.63 μg/mL 36.66−586.51 μg/mL 73.31−586.51 μg/mL 293.26−1173.02 μg/mL
7 8 8 8 6 6 6 6
a The xanthine/xanthine oxidase reaction system included 193.33 μL of substrate solution (6.43 μL of 4 mM xanthine, 12.86 μL of 4 mM NBT, and 174.04 μL of 0.05 M PBS), 6.67 μL of antioxidant sample solution, and 20 μL of 40 mU/mL xanthine oxidase solution. The absorbance of each well was determined at 560 nm after 20 min of reaction terminated by the addition of 20 μL of 0.6 M HCl. bNumber of concentrations tested for each antioxidant.
Table 2. Precision of the High-Throughput O2•− Scavenging Assaya
sedimentation of enzyme molecules.42 As sodium dodecyl sulfonate (SDS) was previously used as a terminator for enzymatic sysytems,32 its ability as a terminator was also studied. After the X/XO reaction for 20 min, 20 μL of 69 mM SDS was added to the reaction mixture without the addition of the other terminators. As a result, the sedimentation of enzyme molecules was indeed observed. However, the purple formazan was unexpectedly adsorbed by the sediments (data not shown), which in turn interfered with the analysis. Therefore, SDS was not a suitable termination agent for this reaction. Validation of the Proposed Assay. The linear relationship between O2•− scavenging capacity and antioxidant concentrations was established using pure antioxidants and food extracts at different concentrations. Table 1 lists the linear calibration curves with their correlation coefficients r2 and linear ranges of four pure antioxidants including allopurinol, rosmarinic acid, resveratrol, and rutin, along with four selected functional food extracts including rosemary, ginger, lotus, and green tea. As a result, all calibration curves showed good linear regression (r2 > 0.9513) within test ranges. The intraday and interday precisions of the assay were determined by analyzing three runs of allopurinol (0.07, 0.10, and 0.30 mM) in hexaplicate in one day and on three consecutive days, respectively. As shown in Table 2, the RSD values of the absorbance values were 4.05 and 5.05% (0.07 mM), 1.87 and 5.62% (0.10 mM), and 1.13 and 2.13% (0.30 mM), respectively, in the intraday and interday precision tests. The accuracy of the proposed assay was assessed by quality control studies. Quality control samples were prepared in DMSO by adding known amounts of pure rosmarinic acid,
intraday
interday
allopurinol (mM)
mean ± SD
RSD%
mean ± SD
RSD%
0.07
0.25 ± 0.00 0.24 ± 0.00 0.25 ± 0.00
4.05
0.25 ± 0.00 0.24 ± 0.00 0.24 ± 0.00
5.05
0.10
0.32 ± 0.01 0.31 ± 0.01 0.32 ± 0.01
1.87
0.36 ± 0.01 0.32 ± 0.01 0.34 ± 0.01
5.62
0.30
0.45 ± 0.01 0.44 ± 0.01 0.45 ± 0.01
1.13
0.45 ± 0.01 0.44 ± 0.01 0.45 ± 0.01
2.13
The xanthine/xanthine oxidase reaction mixture contained 193.33 μL of substrate solution (6.43 μL of 4 mM xanthine, 12.86 μL of 4 mM NBT, and 174.04 μL of 0.05 M PBS), 20 μL of 40 mU/mL xanthine oxidase, and 6.67 μL of allopurinol (0.07, 0.10, and 0.30 mM). The absorbance of each well was determined at 560 nm after 20 min of reaction terminated by the addition of 20 μL of 0.6 M HCl. Values are expressed as means ± SD (n = 3). a
resveratrol, and rutin to the reaction mixture. The accuracy was determined by analyzing three runs of the three individual standards at three different concentrations (rosmarinic acid, 0.0176, 0.0264, and 0.0411 μM; resveratrol, 0.0953, 0.11, and 0.1393 μM; and rutin, 0.0953, 0.1246, and 0.1393 μM) in triplicate. The accuracy data (expressed as percent of recovery) 9269
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Table 3. Accuracy of Quality Control (QC) Samplesa QC1 (μM)
QC2 (μM)
QC3 (μM)
rosmarinic acid mean (μM) recovery (%) RSD (%)
0.0176 0.0169 96.02 ± 7.63 7.95
0.0264 0.0275 104.17 ± 3.38 3.24
0.0411 0.0356 86.62 ± 3.78 4.36
resveratrol mean (μM) recovery (%) RSD (%)
0.0953 0.0866 90.87 ± 1.92 2.11
0.1100 0.1162 105.64 ± 1.66 1.57
0.1393 0.1334 95.76 ± 1.33 1.39
rutin mean (μM) recovery (%) RSD (%)
0.0953 0.0837 87.83 ± 2.56 2.92
0.1246 0.1332 106.90 ± 2.51 2.35
0.1393 0.1316 94.47 ± 1.70 1.80
mean recovery (%)
95.64
97.42
96.40
a The xanthine/xanthine oxidase reaction mixture contained 193.33 μL substrate solution (6.43 μL of 4 mM xanthine, 12.86 μL of 4 mM NBT, and 174.04 μL of 0.05 M PBS), 20 μL of 40 mU/mL xanthine oxidase, and 6.67 μL antioxidants. Three pure antioxidants (rosmarinic acid, resveratrol, and rutin) at three different concentration levels were used for this accuracy study. The absorbance of each well was determined at 560 nm after 20 min of reaction terminated by addition of 20 μL of 0.6 M HCl. Values were expressed as means of triplicate measurements. RSD, relative standard deviation. Quality control (QC) samples including rosmarinic acid, resveratrol, and rutin at three different concentration levels (QC1, QC2, and QC3) were used for this accuracy test.
Figure 3. EC50 values of four selected pure antioxidant compounds (A) and four food extracts (B) determined by the high-throughput and the conventional colorimetric methods. The xanthine/xanthine oxidase (X/XO) reaction system of the conventional colorimetric method contained 1 mL of a mixture of 0.4 mM xanthine and 0.24 mM NBT, 1 mL of 50 mU/mL xanthine oxidase solution, and 0.1 mL of tested samples. The X/XO reaction mixture of the high-throughput method contained 193.33 μL of substrate solution (6.43 μL of 4 mM xanthine, 12.86 μL of 4 mM NBT, and 174.04 μL of 0.05 M PBS), and 20 μL of 40 mU/mL xanthine oxidase. The absorbance at 560 nm was measured after 20 min of reaction terminated by 1 mL of 0.6 M HCl for the conventional colorimetric method and by 20 μL of 0.6 M HCl for the high-throughput assay. Data are means of triplicate measurements. Vertical bars represent the standard deviations.
throughput assay. The EC50 value is the concentration of a selected antioxidant necessary to quench 50% O2•− radicals in the X/XO enzymatic system. A stronger antioxidant has a smaller EC50 value. The EC50 values of the four pure antioxidants are shown in Figure 3A. Their O2•− scavenging capacities were in the order allopurinol > rosmarinic acid > resveratrol > rutin determined at 20 min after the reaction was initiated. Our results were in accordance with the literature.43 Similarly, four well-known antioxidant foods including rosemary, ginger, lotus, and green tea were also estimated for their O2•− scavenging capacities. The antioxidant activities of the four food extracts can be ranked in descending order of EC50 values as follows: rosemary, ginger, lotus, and green tea (Figure 3B). The strongest antioxidant activity of rosemary might be ascribed to the presence of high levels of phenolic compounds (approximately 0.22% fresh weight, among which rosmarinic acid was 32.8 mg/100 g fresh weight).44
from the calibration standard are listed in Table 3. The recovery values ranged from 86.62 to 104.17% for rosmarinic acid, from 90.87 to 105.64% for resveratrol, and from 87.83 to 106.90% for rutin, suggesting the acceptable accuracy of the assay. The stability of the proposed assay was determined with blank (DMSO) and three different concentrations of allopurinol (0.07, 0.10, and 0.30 mM), respectively (Supporting Information Table SI1). After termination, the absorbances at 560 nm at different time periods (0, 10, and 30 min; 1, 2, 6, 12, and 24 h) were monitored. The RSD values of the absorbance values were all lotus > ginger > green tea, consistent with that of the conventional method (Figure 3B). The correlation coefficient was 0.994 between these two methods, indicating the feasibility of the present method. It is worth noting that the consumption of the enzyme (0.8 mU) was much lower than that of the conventional colorimetric method (50 mU), which makes the current procedure less expensive. In summary, a high-throughput O2•− scavenging capacity assay based on the X/XO enzymatic system was developed and validated. The method is easy to perform and time-saving, with a small amount of reagents and antioxidants consumed, and has acceptable precision, accuracy, and stability. The antioxidant data of the newly developed method are highly correlated with those of the conventional method. The high-throughput 96well plate method can be used as an alternative method for screening and investigating potential antioxidant substances.
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ASSOCIATED CONTENT
* Supporting Information S
Stability data, figures of wavelength selection, effects of temperature, pH value, xanthine oxidase concentration, substrate concentration, and reaction time on the xanthine/ xanthine oxidase reaction. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*(T.W.) Phone: +86-21-51322513. E-mail: laurawu2000@163. com Mail: 1200 Cailun Road, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. *(Z.C.) Phone: +86-21-51980157. E-mail: chengzhh@fudan. edu.cn. Mail: 826 Zhangheng Road, Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China. Author Contributions ∥
H.T. and J.Z. contributed equally to this work.
Funding
This study was supported by an Innovation Research Team grant from the Shanghai Municipal Education Commission, a Program for New Century Excellent Talents in University from the Ministry of Education of China (NCET-10-0943), a grant from the National Natural Science Foundation of China (81102775), and an Outstanding Academic Leader Plan (XBR2011057) from Shanghai Municipal Health Bureau. Notes
The authors declare no competing financial interest.
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REFERENCES
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(21) Sun, Y. I.; Oberley, L. W.; Li, Y. A simple method for clinical assay of superoxide dismutase. Clin. Chem. 1988, 34, 497−500. (22) Ukeda, H.; Maeda, S.; Ishii, T.; Sawamura, M. Spectrophotometric assay for superoxide dismutase based on tetrazolium salt 3′{1[(phenylamino)-carbonyl]-3,4- tetrazolium}-bis(4-methoxy-6nitro)benzenesulfonic acid hydrate reduction by xanthine-xanthine oxidase. Anal. Biochem. 1997, 251, 206−209. (23) Ukeda, H.; Kawana, D.; Maeda, S.; Sawamura, M. Spectrophotometric assay for superoxide dismutase based on the reduction of highly water-soluble tetrazolium salts by xanthinexanthine oxidase. Biosci., Biotechnol., Biochem. 1999, 63, 485−488. (24) Magnani, L.; Gaydou, E. M.; Hubaud, J. C. Spectrophotometric measurement of antioxidant properties of flavones and flavonols against superoxide anion. Anal. Chim. Acta 2000, 411, 209−216. (25) Ukeda, H.; Shimamura, T.; Tsubouchi, M.; Harada, Y.; Nakai, Y.; Sawamura, M. Spectrophotometric assay of superoxide anion formed in Maillard reaction based on highly water-soluble tetrazolium salt. Anal. Sci. 2002, 18, 1151−1154. (26) Hyland, K.; Voisin, E.; Banoun, H.; Auclair, C. Superoxide dismutase assay using alkaline dimethylsulfoxide as superoxide aniongenerating system. Anal. Biochem. 1983, 135, 280−287. (27) Nishikimi, M.; Appaji Rao, N.; Yagi, K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem. Biophs. Res. Commun. 1972, 46, 849− 854. (28) Valentão, P.; Fernandes, E.; Carvalho, F.; Andrade, P. B.; Seabra, R. M.; Bastos, M. L. Antioxidant activity of Centaurium erythraea infusion evidenced by its superoxide radical scavenging and xanthine oxidase inhibitory activity. J. Agric. Food Chem. 2001, 49, 3476−3479. (29) Zhao, B. L.; Li, X. J.; He, R. G.; Cheng, S. J.; Xin, W. J. Scavenging effect of extracts of green tea and natural antioxidants on active oxygen radicals. Cell Biophys. 1989, 14, 175−185. (30) Ito, A.; Krinsky, N. I.; Cunningham, M. L.; Peak, M. J. Comparison of the inactivation of Bacillus subtilis transforming DNA by the potassium superoxide and xanthine-xanthine oxidase systems for generating superoxide. Free Radical Biol. Med. 1987, 3, 111−118. (31) Bergmeyer, H. U.; Gawehn, K.; Grassl, M. Methods of Enzymatic Analysis; Academic Press: New York, 1974; Vol. 2, pp 521−522. (32) Lu, Y.; Foo, L. Y. Antioxidant and radical scavenging activities of polyphenols from apple pomace. Food Chem. 2000, 68, 81−85. (33) Aitken, R. J.; Buckingham, D.; Harkiss, D. Use of a xanthine oxidase free radical generating system to investigate the cytotoxic effects of reactive oxygen species on human spermatozoa. J. Reprod. Fertil. 1993, 97, 441−450. (34) Ries, W. L.; Key, L. L., Jr.; Rodriguiz, R. M. Nitroblue tetrazolium reduction and bone resorption by osteoclasts in vitro inhibited by a manganese-based superoxide dismutase mimic. J. Bone Miner. Res. 1992, 7, 931−939. (35) Bielski, B. H. J.; Shiue, G. G.; Bajuk, S. Reduction of nitro blue tetrazolium by CO2− and O2− radicals. J. Phys. Chem. 1980, 84, 830− 833. (36) Bassingthwaighte, J. B.; Chinn, T. M. Reexamining MichaelisMenten enzyme kinetics for xanthine oxidase. Adv. Physiol. Educ. 2013, 37, 37−48. (37) Furuno, K.; Akasako, T.; Sugihara, N. The contribution of the pyrogallol moiety to the superoxide radical scavenging activity of flavonoids. Biol. Pharm. Bull. 2002, 25, 19−23. (38) Lavelli, V.; Hippeli, S.; Peri, C.; Elstner, E. F. Evaluation of radical scavenging activity of fresh and air-dried tomatoes by three model reactions. J. Agric. Food Chem. 1999, 47, 3826−3831. (39) Klibanov, A. M. Improving enzymes by using them in organic solvents. Nature 2001, 409, 241−246. (40) Jothy, S. L.; Zuraini, Z.; Sasidharan, S. Phytochemicals screening, DPPH free radical scavenging and xanthine oxidase inhibitory activities of Cassia f istula seeds extract. J. Med. Plant Res. 2011, 5, 1941−1947. (41) Robins, S. P. An enzyme-linked immunoassay for the collagen cross-link pyridinoline. Biochem. J. 1982, 207, 617−620.
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High-Throughput Superoxide Anion Radical Scavenging Capacity Assay Hongxun Tao,†,∥ Jinge Zhou,†,∥ Tao Wu,*,† and Zhihong Cheng*,‡ †
Key Laboratory of Standardization of Chinese Medicines of Ministry of Education, The Shanghai Key Laboratory for Compound Chinese Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China ‡ Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China S Supporting Information *
ABSTRACT: A high-throughput superoxide anion radical (O2•−) scavenging capacity assay based on the xanthine oxidase/ xanthine reaction system was developed and validated in the present study. The reaction conditions including detection wavelength, concentrations of reactant components, reaction temperature, reaction time, pH, terminator reagent, and sample dissolving solvents were optimized. The accuracy and reliability of the assay were assessed by evaluation of linearity (r2 = 0 .9513−0.9957), precision (intraday RSD 1.13−4.05% and interday RSD 2.13−5.62%), accuracy (95.64−97.42% recovery), and stability (RSD 2.62−6.19%), as well as comparison with the conventional colorimetric method. The EC50 values obtained by the current method and the conventional assay were highly correlated (r > 0.99). This high-throughput O2•− scavenging assay may be used for screening and estimating potential superoxide anion radical (O2•−) scavengers, especially food extracts and natural products with a very small amount of test material. KEYWORDS: xanthine oxidase, superoxide anion radical, high throughput, antioxidant activity
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INTRODUCTION Free radicals play an important role in physiology and pathology as mediators of many biochemical events. Excessive free radical production is associated with numerous chronic disease conditions, such as cancer,1 cardiovascular diseases,2 diabetes mellitus,3 gastric ulcer,4 and neurological disorders.5 In recent years, antioxidants have attracted a great deal of attention as potential agents for the control of diseases associated with oxidative damage. Superoxide anion radicals (O2•−), one of the most important reactive oxygen species (ROS), are produced as byproducts of metabolism, particularly in mitochondrial respiration,6 and serve as a progenitor for other toxic ROS such as hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and hydroxyl radicals (HO•). This leads to a growing interest in the discovery and development of valuable nutraceuticals and functional foods capable of scavenging O2•−. A number of O2•− scavenging assay protocols have been developed, including an electron spin resonance (ESR) method,7−14 a high-performance liquid chromatography (HPLC) method,15−17 an ultrahigh-performance liquid chromatography and triple-quadrupole mass spectrometry (UHPLC-MS) method,18 and a thin layer chromatography (TLC) bioautographic method.19 Among these, the ESR, HPLC, and UHPLC-MS methods have been shown to possess good specificity and accuracy, but require relative expensive instrumentation. TLC bioautography is an ideal method for the estimation of antioxidants present in complicated extracts and can be easily adopted in high-throughput analysis. However, this method is generally developed and used in qualitative ways. Compared to these methods, spectrophotometry is one of the most widely used techniques in biomedicinal analysis20−25 due to its © 2014 American Chemical Society
inherent ease of application and no requirement of expensive equipment. The conventional spectrophotometric methods are performed manually in cuvettes, which generally require a large sample quantity. These requirements are not compatible with the capabilities of most food chemistry laboratories charged with routine analysis of pure antioxidants. Therefore, a sensitive and high-throughput assay is needed to facilitate the rapid and automated screening of the O2•− scavengers. Superoxide anion radicals can be generated in vitro by many methods such as the hydrogen peroxide degradation system in alkaline dimethyl sulfoxide (DMSO),11,26 the phenazine methosulfate (PMS)/β-nicotinamide adenine dinucleotide (NADH) system,14,27,28 the riboflavin irradiation system,17,19,29 and the xanthine/xanthine oxidase (X/XO) system.12,18−23,28,30 Among these the X/XO system is a classical enzymatic method based on XO oxidation of xanthine giving O2•−, which subsequently reacts with a pale yellow tetrazolium salt, nitroblue tetrazolium chloride (NBT), to yield a purple formazan by monitoring its absorbance at 560 nm. In this study, a high-throughput method in a 96-well microplate format for O2•− scavenging capacity assay based on the X/XO system was developed and validated. In addition, this high-throughput assay was used to estimate the O2•− scavenging activity of some pure compounds and food extracts. Received: Revised: Accepted: Published: 9266
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solution was added to start the reaction. After gentle shaking for 10 s, the reaction mixture was incubated in a water bath at 37 °C for 20 min. Finally, 20 μL of 0.6 M HCl was added to terminate the reaction, followed by shaking for another 10 s. The absorbance at 560 nm was measured. The total volume for each reaction mixture in each well was 240 μL. A blank test without XO and a control test without antioxidants were also conducted. Allopurinol was used as a positive control for the assay. The level of O2•− scavenging capacity for each reaction was also calculated as formula 1. The EC50 values were calculated under the assay condition. Triplicate reactions were carried out for each level of every individual sample.
MATERIALS AND METHODS
Materials and Reagents. Xanthine (X0625-5G), xanthine oxidase (XO, X4875-10U), nitroblue tetrazolium chloride (NBT, N6876-5G), and allopurinol (A8003-5G) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Potassium phosphate monobasic (KH2PO4), potassium phosphate dibasic (K2HPO4·3H2O), ethylenediaminetetraacetic acid disodium salt (EDTA), sodium hydroxide (NaOH), dimethyl sulfoxide (DMSO), acetone, acetonitrile, ethanol, isopropanol, tetrahydrofuran, and hydrochloric acid (HCl) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ultrapure water was prepared by an Advantage A10 water purifier (Milli-Q Academic, Molsheim, France). All other chemicals were of analytical grade and used without further purification. Rosmarinic acid, resveratrol, and rutin (purity > 98%) were obtained from Shanghai R&D Center for Standardization of Chinese Medicines (Shanghai, China). The individual food extracts of rosemary (whole plant, 2012030), ginger (rhizoma, 2012021), and green tea (leaves, 2012020) were purchased from Sichuan Tongtai Plant Chemical Industry Co., Ltd. (Shifang, Sichuan Province, China). The lotus extract (leaves, 20120620) was purchased from Shanghai Moochun Biological Technology Co., Ltd. (Shanghai, China). A 0.2 M stock phosphate buffer solution (PBS, pH 8.0) was prepared by mixing equal volumes of 0.2 M KH2PO4 and 0.2 M K2HPO4·3H2O solutions. A 1.0 mM EDTA stock solution was prepared in ultrapure water. Stock solutions of the pure antioxidants were prepared in DMSO at concentrations of 6.0 mM, and the stock solutions of the food extracts were prepared in a mixture of DMSO/ultrapure water (1:1, v/v). A series of working solutions were made by appropriate stepwise dilutions of the above stock solutions with 0.2 M PBS buffer (pH 8.0). A fresh substrate solution containing 4 mM xanthine and 4 mM NBT was prepared in 0.05 M PBS (pH 8.0) just before use. A 0.04 U/mL XO solution (250 mL) was prepared by dilution of the 10 U XO stock solution with a mixture solution of 1 mM EDTA (125 mL) and 0.1 M PBS (pH 8.0, 125 mL). The enzyme activity of XO was determined by a reference method31 before preparation. Conventional Colorimetric Assay. The conventional colorimetric O2•− scavenging capacity assay was carried out according to a previously described laboratory protocol.32 Briefly, an aliquot of 100 μL of different concentrations of tested antioxidants was added to a 1 mL substrate solution containing 0.4 mM xanthine and 0.24 mM NBT. After the addition of 1 mL of XO solution (50 mU/mL), the reaction mixture was incubated in a water bath at 37 °C for 20 min. The reaction was then terminated by the addition of 1 mL of 0.6 M HCl, and the absorbance (A) of each reaction mixture at 560 nm was measured against a blank without XO using a microplate spectrophotometer (Power Wave, Bio-Tek, Winooski, VT, USA). The O2•− scavenging capacity for each reaction was calculated as
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RESULTS AND DISCUSSION Selection of the Detection Wavelength for the X/XO Reaction. The X/XO enzymatic system is based on XO oxidation of xanthine giving O2•−, which subsequently reacts with NBT to yield a purple formazan. The formation of formazan is generally detected by absorbance at 560 nm. However, several wavelengths such as 560 nm,32 510 nm,33 and 595 nm34 have also been reported for the detection of formazan production. This divergence prompted us to investigate a suitable wavelength for this reaction system. Theoretically, the individual components involved in the reaction mixture including xanthine, NBT, and XO might interfere with the formazan absorbance. As shown in Supporting Information Figure SI1A, all of the components except formazan (the reaction product) in the reaction system showed no absorption maximum between 500 and 800 nm. The absorbance of formazan from 550 to 650 nm reached a platform, among which 560 nm turned out to be stable. Therefore, 560 nm was chosen as the detection wavelength, consistent with that used in most of the X/XO literature. NBT is a classical biochemical reagent for the detection of O2•−. During the reaction process, ditetrazolium (NBT2+) is attacked by O2•−, along with one electron being transferred to turn NBT2+ into tetrazolinyl radical (NBT•+). In the following step, one more electron transfer turns NBT•+ into monoformazan (MF).35 It should be noted, therefore, that the excess of NBT or O2•− might theoretically lead to the production of only MF or diformazan (DF). The difference in molecular structures of MF and DF may result in the different maximum absorption wavelengths of these two substances. Thus, the different molar ratios of xanthine to NBT (1:1, 1:2, and 1:3) in the X/XO reaction mixture were scanned over 450−800 nm (Supporting Information Figure SI1B). The results showed that the effect of different molar ratios of xanthine to NBT was small and of marginal importance to the maximum wavelength of formazan. As the absorption behaviors of the molar ratios (1:2 and 1:3) of xanthine to NBT acted similarly, a 1:2 ratio of xanthine to NBT was chosen. Effect of Temperatures on the X/XO Reaction. Several reaction temperatures such as 25 °C20 and 37 °C32 have been used for this X/XO enzymatic reaction system. To better understand the effect of temperature on the O2•− formation, different reaction temperatures including 25, 31, 37, 43, and 49 °C on this X/XO enzymatic system were tested. As a result, the absorbance values increased when the temperature was increased from 25 to 37 °C (Supporting Information Figure SI2A), and slight decreases were observed with a temperature of >37 °C. The results indicated that the extent of the reaction reached the maximum at 37 °C. Therefore, 37 °C was selected as the reaction temperature in further experiments, which is consistent with that of O2•− physiologically generated.
O2•−scavening capacity % = [1 − (A s − A s‐b)/(Ac − Ac‐b)] × 100%
(1)
where As, As‑b, Ac, and Ac‑b represent the absorbances of samples, sample control (without addition of XO), control (without addition of tested antioxidants), and vehicle control (without addition of tested antioxidants and XO), respectively, at 560 nm after 20 min of reaction. The EC50 value is the concentration of an antioxidant capable of quenching 50% of the radicals in the reaction mixture under the assay condition. Triplicate reactions were carried out for each level of every individual sample. High-Throughput O2•− Scavenging Assay. The high-throughput O2•− scavenging assay was carried out using a 96-well microplate spectrophotometer (Power Wave, Bio-Tek). The 96-well plate was covered with a lid to prevent solvent evaporation during reaction. Six or eight different concentrations were used for each antioxidant extract and antioxidant compound in the study. The antioxidant sample solution (6.67 μL) and 193.33 μL of substrate solution (including 6.43 μL of 4 mM xanthine, 12.86 μL of 4 mM NBT, and 174.04 μL of 0.05 M PBS) were added to each well. Then 20 μL of 0.04 U/mL XO 9267
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Effect of pH on the X/XO Reaction. The effect of the PBS buffer with different pH values (6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0) on the X/XO enzymatic system was studied. As shown in Supporting Information Figure SI2B, the absorbance values increased with the increase of the pH values from 6.0 to 8.0, whereas absorbance values decreased from pH 8.0 to 9.0. A possible explanation is that the conformation of XO might be affected by the pH of the buffers.22 Therefore, a pH of 8.0 was selected in this study. Effects of Enzyme and Substrate Concentrations on the X/XO Reaction. The rate of an enzyme-catalyzed reaction depends on concentrations of enzymes and substrates.22 The effect of XO concentrations on the formazan formation was studied. At eight concentrations of XO over a 8-fold range (10−80 mU/mL), the enzyme was incubated with 4 mM substrate solution and DMSO (without antioxidants) at 37 °C for 20 min. The absorbance values gradually increased with increasing XO concentrations from 10 to 40 mU/mL, whereas there was no significant change of the absorbance values in XO concentrations up to 40 mU/mL (Supporting Information Figure SI2C). These data suggested that the optimal XO concentration in this reaction was 40 mU/mL. Similarly, several substrate concentrations (2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5 mM) with a constant molar ratio to NBT (1:2) were studied. Supporting Information Figure SI2D shows the effect of substrate concentrations on the rate of the reaction. For a given enzyme concentration (40 mU/mL), the absorbance values showed a rapid and marked increase with substrate concentrations from 2.0 to 4.0 mM and a subsequent slow increase up to 5.5 mM, which is consistent with the Michaelis−Menten kinetics theory.36 Therefore, the optimal substrate concentration in this reaction system was 4.0 mM. Effect of Reaction Time on the X/XO Reaction. Several reaction times such as 5 min,37 20 min,32 and 30 min38 have been applied for this enzymatic reaction system. For the consideration of stability, the reaction time required to reach the steady state for this enzymatic system was studied. The change in absorbance was measured for 44 min at 2 min interval, without the addition of terminators. As shown in Supporting Information Figure SI3, the absorbance values increased rapidly within 20 min, and reached a plateau from 20 to 44 min. The result indicated that the reaction mixture can be considered to reach the “steady state” at 20 min or anytime after that. Thus, a reaction time of 20 min was used. Effect of Different Sample Dissolving Solvents on the X/XO Reaction. Enzymatic reactions can be conducted not only in water but also in organic solvents.39 This suggests the feasible use of organic cosolvents for the hydrophilic and lipophilic antioxidant samples tested in the enzymatic assay. Therefore, several organic solvents including DMSO, acetone, acetonitrile, isopropanol, and tetrahydrofuran, as well as water, were selected to investigate the solvent effect on the reaction system. With a fixed ratio of solvent volume to the reaction mixture (1:30), none of these organic solvents had shown a significant effect on absorbance values compared with water (Figure 1), indicating the potential use of these solvents as sample dissolving solvents. This is especially important and useful for samples with lower solubility in water. In addition, three other different ratios of solvent volume to the reaction mixture (1:10, 1:20, and 1:40, v/v) were investigated and compared with the ratio of 1:30. As shown in Figure 1, no significant absorbance changes were observed among the 1:20, 1:30, and 1:40 ratios of solvent volume to
Figure 1. Effects of dissolving solvents and ratios of solvent volume to the reaction mixture on the xanthine/xanthine oxidase reaction. The reaction mixture contained 6.67 μL of different solvents, 193.33 μL of substrate solution (6.43 μL of 4 mM xanthine, 12.86 μL of 4 mM NBT, and 174.04 μL of 0.05 M PBS), and 20 μL of 40 mU/mL xanthine oxidase. The absorbance at 560 nm was determined for each reaction mixture after 20 min of reaction terminated by the addition of 20 μL of 0.6 M HCl. Four different volume ratios (1:10, 1:20, 1:30, and 1:40, v/v) were tested. ACE, ACN, ET, IPA, and THF represented acetone, acetonitrile, ethanol, isopropanol, and tetrahydrofuran, respectively. Data are means of triplicate measurements. Vertical bars represent the standard deviations.
reaction mixture, regardless of the nature of dissolving solvents. However, in a 1:10 ratio, all of the solvents except water and ethanol resulted in obvious decreases of the absorbance values (Figure 1). Therefore, the organic solvent effect should not be ignored in a relatively high ratio (1:10) of solvent volume to the reaction mixture (except water and ethanol). In consideration of its extensive use as a dissolving solvent in screening tests, DMSO with a 1:30 ratio of volume to the reaction mixture was used in this study. Effect of Different Terminators on the X/XO Reaction. Significant pH changes can affect enzyme activity, which might be an effective way to terminate an enzymatic reaction. HCl, a commonly used acid terminator in the X/XO reaction,40 was compared with NaOH, an alkaline terminator of other enzymes.41 As shown in Figure 2A, the absorbance values increased with increasing concentrations of NaOH solutions (0.1−0.9 M), together with the production of green substances instead of the expected purple formazan. Thus, NaOH is not suitable to terminate the reaction due to the formation of side products with absorption at 560 nm. In contrast to NaOH, a gradual decrease of the absorbance values was observed with the increase of HCl concentrations from 0.1 to 0.5 M (Figure 2B). Further increase of the HCl concentration to 1.0 M did not result in a further decrease in the absorbance values. Importantly, unlike with 0.1−0.4 M HCl solutions, the generation of the expected purple formazan cannot be observed with 0.5−1.0 M HCl, suggesting that the reaction could be totally terminated by 0.5−1.0 M HCl solutions. To further confirm the efficiency of the HCl terminator, the absorbances of the reaction mixtures at 10, 20, 40, and 60 min were determined after 10 min of reaction being terminated by 0.5−1.0 M HCl solutions. As shown in Figure 2C, no obvious increase of absorbance values was observed after the addition of 0.5−1.0 M HCl solutions at each time point, whereas that of the negative control (without addition of HCl solutions) increased rapidly from 10 to 20 min, indicating the great termination efficiency of HCl. Hence, a 0.6 M HCl solution beyond the inflection point was chosen as the optimal terminator. Surface-active agents (SAAs) are also reported to terminate the enzymatic reactions, because SAAs could neutralize the surface charges of enzyme molecules, resulting in the 9268
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Figure 2. Effects of different terminators and HCl concentrations on the xanthine/xanthine oxidase reaction (A, NaOH; B, HCl; C, 0.5−1.0 M HCl). The reaction mixture contained 6.67 μL OF DMSO, 193.33 μL of substrate solution (6.43 μL of 4 mM xanthine, 12.86 μL of 4 mM NBT, and 174.04 μL of 0.05 M PBS), and 20 μL of 40 mU/mL xanthine oxidase. The absorbance at 560 nm was determined for each reaction mixture ternminated by 20 μL of 0.1−1.0 M NaOH (A) or 20 μL of 0.2−0.7 M HCl (B) after 20 min of reaction. For the termination efficiency of HCl, 20 μL of 0 M and 0.5−1.0 M HCl solutions was added to the xanthine/xanthine oxidase reactions, followed by determination of the absorbance at 10, 20, 40, and 60 min (C). Data are means of triplicate measurements. Vertical bars represent the standard deviations.
Table 1. Linear Relationship between Antioxidant Concentrations and Percent O2•− Inhibition Obtained by the HighThroughput O2•− Scavenging Assaya sample
slope
intercept
r2
linearity range
nb
allopurinol rosmarinic acid resveratrol rutin rosemary ginger lotus green tea
2954.4 932.94 482.91 275.58 399.36 138.36 126.24 55.051
41.619 23.971 −10.518 13.265 7.1703 −4.9684 −5.9565 6.5041
0.9766 0.9808 0.9911 0.9895 0.9844 0.9957 0.9789 0.9513
2.93−14.66 μM 2.93−14.66 μM 58.65−131.97 μM 73.31−146.63 μM 18.33−146.63 μg/mL 36.66−586.51 μg/mL 73.31−586.51 μg/mL 293.26−1173.02 μg/mL
7 8 8 8 6 6 6 6
a The xanthine/xanthine oxidase reaction system included 193.33 μL of substrate solution (6.43 μL of 4 mM xanthine, 12.86 μL of 4 mM NBT, and 174.04 μL of 0.05 M PBS), 6.67 μL of antioxidant sample solution, and 20 μL of 40 mU/mL xanthine oxidase solution. The absorbance of each well was determined at 560 nm after 20 min of reaction terminated by the addition of 20 μL of 0.6 M HCl. bNumber of concentrations tested for each antioxidant.
Table 2. Precision of the High-Throughput O2•− Scavenging Assaya
sedimentation of enzyme molecules.42 As sodium dodecyl sulfonate (SDS) was previously used as a terminator for enzymatic sysytems,32 its ability as a terminator was also studied. After the X/XO reaction for 20 min, 20 μL of 69 mM SDS was added to the reaction mixture without the addition of the other terminators. As a result, the sedimentation of enzyme molecules was indeed observed. However, the purple formazan was unexpectedly adsorbed by the sediments (data not shown), which in turn interfered with the analysis. Therefore, SDS was not a suitable termination agent for this reaction. Validation of the Proposed Assay. The linear relationship between O2•− scavenging capacity and antioxidant concentrations was established using pure antioxidants and food extracts at different concentrations. Table 1 lists the linear calibration curves with their correlation coefficients r2 and linear ranges of four pure antioxidants including allopurinol, rosmarinic acid, resveratrol, and rutin, along with four selected functional food extracts including rosemary, ginger, lotus, and green tea. As a result, all calibration curves showed good linear regression (r2 > 0.9513) within test ranges. The intraday and interday precisions of the assay were determined by analyzing three runs of allopurinol (0.07, 0.10, and 0.30 mM) in hexaplicate in one day and on three consecutive days, respectively. As shown in Table 2, the RSD values of the absorbance values were 4.05 and 5.05% (0.07 mM), 1.87 and 5.62% (0.10 mM), and 1.13 and 2.13% (0.30 mM), respectively, in the intraday and interday precision tests. The accuracy of the proposed assay was assessed by quality control studies. Quality control samples were prepared in DMSO by adding known amounts of pure rosmarinic acid,
intraday
interday
allopurinol (mM)
mean ± SD
RSD%
mean ± SD
RSD%
0.07
0.25 ± 0.00 0.24 ± 0.00 0.25 ± 0.00
4.05
0.25 ± 0.00 0.24 ± 0.00 0.24 ± 0.00
5.05
0.10
0.32 ± 0.01 0.31 ± 0.01 0.32 ± 0.01
1.87
0.36 ± 0.01 0.32 ± 0.01 0.34 ± 0.01
5.62
0.30
0.45 ± 0.01 0.44 ± 0.01 0.45 ± 0.01
1.13
0.45 ± 0.01 0.44 ± 0.01 0.45 ± 0.01
2.13
The xanthine/xanthine oxidase reaction mixture contained 193.33 μL of substrate solution (6.43 μL of 4 mM xanthine, 12.86 μL of 4 mM NBT, and 174.04 μL of 0.05 M PBS), 20 μL of 40 mU/mL xanthine oxidase, and 6.67 μL of allopurinol (0.07, 0.10, and 0.30 mM). The absorbance of each well was determined at 560 nm after 20 min of reaction terminated by the addition of 20 μL of 0.6 M HCl. Values are expressed as means ± SD (n = 3). a
resveratrol, and rutin to the reaction mixture. The accuracy was determined by analyzing three runs of the three individual standards at three different concentrations (rosmarinic acid, 0.0176, 0.0264, and 0.0411 μM; resveratrol, 0.0953, 0.11, and 0.1393 μM; and rutin, 0.0953, 0.1246, and 0.1393 μM) in triplicate. The accuracy data (expressed as percent of recovery) 9269
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Table 3. Accuracy of Quality Control (QC) Samplesa QC1 (μM)
QC2 (μM)
QC3 (μM)
rosmarinic acid mean (μM) recovery (%) RSD (%)
0.0176 0.0169 96.02 ± 7.63 7.95
0.0264 0.0275 104.17 ± 3.38 3.24
0.0411 0.0356 86.62 ± 3.78 4.36
resveratrol mean (μM) recovery (%) RSD (%)
0.0953 0.0866 90.87 ± 1.92 2.11
0.1100 0.1162 105.64 ± 1.66 1.57
0.1393 0.1334 95.76 ± 1.33 1.39
rutin mean (μM) recovery (%) RSD (%)
0.0953 0.0837 87.83 ± 2.56 2.92
0.1246 0.1332 106.90 ± 2.51 2.35
0.1393 0.1316 94.47 ± 1.70 1.80
mean recovery (%)
95.64
97.42
96.40
a The xanthine/xanthine oxidase reaction mixture contained 193.33 μL substrate solution (6.43 μL of 4 mM xanthine, 12.86 μL of 4 mM NBT, and 174.04 μL of 0.05 M PBS), 20 μL of 40 mU/mL xanthine oxidase, and 6.67 μL antioxidants. Three pure antioxidants (rosmarinic acid, resveratrol, and rutin) at three different concentration levels were used for this accuracy study. The absorbance of each well was determined at 560 nm after 20 min of reaction terminated by addition of 20 μL of 0.6 M HCl. Values were expressed as means of triplicate measurements. RSD, relative standard deviation. Quality control (QC) samples including rosmarinic acid, resveratrol, and rutin at three different concentration levels (QC1, QC2, and QC3) were used for this accuracy test.
Figure 3. EC50 values of four selected pure antioxidant compounds (A) and four food extracts (B) determined by the high-throughput and the conventional colorimetric methods. The xanthine/xanthine oxidase (X/XO) reaction system of the conventional colorimetric method contained 1 mL of a mixture of 0.4 mM xanthine and 0.24 mM NBT, 1 mL of 50 mU/mL xanthine oxidase solution, and 0.1 mL of tested samples. The X/XO reaction mixture of the high-throughput method contained 193.33 μL of substrate solution (6.43 μL of 4 mM xanthine, 12.86 μL of 4 mM NBT, and 174.04 μL of 0.05 M PBS), and 20 μL of 40 mU/mL xanthine oxidase. The absorbance at 560 nm was measured after 20 min of reaction terminated by 1 mL of 0.6 M HCl for the conventional colorimetric method and by 20 μL of 0.6 M HCl for the high-throughput assay. Data are means of triplicate measurements. Vertical bars represent the standard deviations.
throughput assay. The EC50 value is the concentration of a selected antioxidant necessary to quench 50% O2•− radicals in the X/XO enzymatic system. A stronger antioxidant has a smaller EC50 value. The EC50 values of the four pure antioxidants are shown in Figure 3A. Their O2•− scavenging capacities were in the order allopurinol > rosmarinic acid > resveratrol > rutin determined at 20 min after the reaction was initiated. Our results were in accordance with the literature.43 Similarly, four well-known antioxidant foods including rosemary, ginger, lotus, and green tea were also estimated for their O2•− scavenging capacities. The antioxidant activities of the four food extracts can be ranked in descending order of EC50 values as follows: rosemary, ginger, lotus, and green tea (Figure 3B). The strongest antioxidant activity of rosemary might be ascribed to the presence of high levels of phenolic compounds (approximately 0.22% fresh weight, among which rosmarinic acid was 32.8 mg/100 g fresh weight).44
from the calibration standard are listed in Table 3. The recovery values ranged from 86.62 to 104.17% for rosmarinic acid, from 90.87 to 105.64% for resveratrol, and from 87.83 to 106.90% for rutin, suggesting the acceptable accuracy of the assay. The stability of the proposed assay was determined with blank (DMSO) and three different concentrations of allopurinol (0.07, 0.10, and 0.30 mM), respectively (Supporting Information Table SI1). After termination, the absorbances at 560 nm at different time periods (0, 10, and 30 min; 1, 2, 6, 12, and 24 h) were monitored. The RSD values of the absorbance values were all lotus > ginger > green tea, consistent with that of the conventional method (Figure 3B). The correlation coefficient was 0.994 between these two methods, indicating the feasibility of the present method. It is worth noting that the consumption of the enzyme (0.8 mU) was much lower than that of the conventional colorimetric method (50 mU), which makes the current procedure less expensive. In summary, a high-throughput O2•− scavenging capacity assay based on the X/XO enzymatic system was developed and validated. The method is easy to perform and time-saving, with a small amount of reagents and antioxidants consumed, and has acceptable precision, accuracy, and stability. The antioxidant data of the newly developed method are highly correlated with those of the conventional method. The high-throughput 96well plate method can be used as an alternative method for screening and investigating potential antioxidant substances.
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ASSOCIATED CONTENT
* Supporting Information S
Stability data, figures of wavelength selection, effects of temperature, pH value, xanthine oxidase concentration, substrate concentration, and reaction time on the xanthine/ xanthine oxidase reaction. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*(T.W.) Phone: +86-21-51322513. E-mail: laurawu2000@163. com Mail: 1200 Cailun Road, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. *(Z.C.) Phone: +86-21-51980157. E-mail: chengzhh@fudan. edu.cn. Mail: 826 Zhangheng Road, Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, China. Author Contributions ∥
H.T. and J.Z. contributed equally to this work.
Funding
This study was supported by an Innovation Research Team grant from the Shanghai Municipal Education Commission, a Program for New Century Excellent Talents in University from the Ministry of Education of China (NCET-10-0943), a grant from the National Natural Science Foundation of China (81102775), and an Outstanding Academic Leader Plan (XBR2011057) from Shanghai Municipal Health Bureau. Notes
The authors declare no competing financial interest.
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