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Solvent effects and improvements in the deoxyribose degradation assay for hydroxyl radical-scavenging
Food Chemistry 141 (2013) 2083–2088
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Analytical Methods
Solvent effects and improvements in the deoxyribose degradation assay for hydroxyl radical-scavenging Xican Li ⇑ School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Guangzhou, China
a r t i c l e
i n f o
Article history: Received 17 October 2012 Received in revised form 30 March 2013 Accepted 8 May 2013 Available online 23 May 2013 Keywords: Antioxidant Deoxyribose degradation assay Hydroxyl radical scavenging OH Solvent effect Improvement
a b s t r a c t The deoxyribose degradation assay is widely used to evaluate the hydroxyl (OH) radical-scavenging ability of food or medicines. We compared the hydroxyl radical-scavenging activity of 25 antioxidant samples prepared in ethanol solution with samples prepared after removing the ethanol (residue). The data suggested that there was an approximately 9-fold difference between assay results for the ethanol solution and residue samples. This indicated a strong alcoholic interference. To further study the mechanism, the scavenging activities of 18 organic solvents (including ethanol) were measured by the deoxyribose assay. Most pure organic solvents (especially alcohols) could effectively scavenge hydroxyl radicals. As hydroxyl radicals have extremely high reactivities, they will quickly react with surrounding solvent molecules. This shows that any organic solvent should be completely evaporated before measurement. The proposed method is regarded as a reliable hydroxyl radical-scavenging assay, suitable for all types of antioxidants. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The deoxyribose degradation assay has been widely used to evaluate the hydroxyl (OH) radical-scavenging ability of food, nutrients, and medicines since it was established in 1987 (Halliwell, Gutteridge, & Aruoma, 1987) In the deoxyribose degradation assay, OH is obtained via Fenton reaction (Halliwell & Gutteridge, 1981; Halliwell et al., 1987). Subsequently, OH attacks deoxyribose and breaks its cyclic furan ring to generate malondialdehyde (MDA). MDA combines with 2thiobarbituric acid (TBA) to produce a chromogen with kmax at 530 nm (Cheeseman, Beavis, & Esterbauer, 1988). Therefore, the A530nm value is proportional to the produced amount of OH radicals. Higher A530nm values indicate higher levels of OH radicals. If an antioxidant sample is added, the A530nm value will decrease, suggesting that some OH radicals are scavenged by the antioxidant. This is the principle of deoxyribose degradation assay. Abbreviations: SD, standard deviation; RSD, relative standard deviation; GSH, glutathione; BHA, butylated hydroxyanisole; THF, tetrahydrofuran; DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide; ROS, reactive oxygen species; FC, final concentration; FDA, Food and Drug Administration of USA; MEAS, methanol extract of Aquilaria sinensis leaves; MEGB, methanol extract from Gynura bicolor Roxb. DC. ⇑ Address: No. 232, Waihuan East Road, Guangzhou Higher Education Mega Center, Panyu District, Guangzhou 510006, China. Tel.: +86 20 39358076; fax: +86 20 38892697. E-mail address: [email protected] URL: http://www.researchgate.net/profile/Xican_Li 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.05.084
Like most biochemical assays, the deoxyribose assay requires the preparation of sample solutions, using various organic solvents prior to measurement. However, it was observed (Wang & Li, 2011) that the organic solvents (especially alcohols) normally used to prepare these sample solutions may cause strong interferences which are often overlooked by assay users. Several research groups have clearly stated that they used organic solvents for sample solution preparation, and that the sample solution was directly used for measurement by the deoxyribose assay. For example, at least seven groups reported that they used alcohol to dissolve their samples, which was directly measured by the assay (Ganesan, Kumar, & Bhaskar, 2008; Kaurinovic, Popovic, Vlaisavljevi, & Trivic, 2011; Rop et al., 2010; Visavadiya, Soni, & Dalwadi, 2009; Wu, Yen, Wang, & Weng, 2004; You et al., 2007; Yuan, Bone, & Carrington, 2005). One research group even directly took edible wine for the assay, to estimate hydroxyl scavenging ability of the phenolic compounds in wine (Li, Wang, Li, Li, & Wang, 2009). Other groups, however, used dimethyl sulphoxide (DMSO) or ethyl acetate to dissolve the samples for direct measurement (Desmarchelier, Coussio, & Ciccia, 1998; Ekanayake, Lee, & Lee, 2004; Georgetti, Casagrande, Moura-de-Carvalho Vicentini, Verri, & Fonseca, 2006; Ozsoy, Can, Yanardag, & Akev, 2008; Puntel et al., 2008; Srinivasan, Chandrasekar, Nanjan, & Suresh, 2007). Since water solubility of antioxidant samples is generally not adequate to prepare sample stock solutions at room temperature, we believe that there are many more research groups using organic solvents for dissolving the samples and the problem may therefore
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be even more widespread. All of these investigations are therefore regarded as unreliable. Therefore, in the present study we systematically investigate the effect of various solvents in an effort to improve the deoxyribose assay performance.
tetrachloride, chloroform, cyclohexane, dichloromethane, diethyl ether, DMF (N,N-dimethylformamide), DMSO (dimethyl sulfoxide), ethanol, ethyl acetate, n-hexane, methanol, petroleum ether, THF (tetrahydrofuran) and toluene.
2. Materials and methods
2.4. Validation of the improved deoxyribose degradation method
2.1. Chemicals
The improved method was validated according to FDA guidance (US Food and Drug Administration, 2001), with regard to linearity, precision, and reproducibility. (+)-Catechin, which is a standard antioxidant, was used as the reference compound. The linearity of the method was evaluated at five concentrations. The calibration curve was prepared by plotting the mean inhibition percentages of triplicate analyses against the final concentrations. The precision was evaluated, using the relative standard deviation (RSD%) inhibition percentages of triplicate analyses.
Trolox (±-6-hydroxyl-2,5,7,8-tetramethylchromane-2-carboxylic acid), melatonin, BHA (butylated hydroxyanisole), (+)-catechin, isoferulic acid, and sinapic acid were obtained from Sigma–Aldrich, Shanghai Trading Co. (Shanghai, China). Deoxyribose and GSH (glutathione) were purchased from Amresco Inc. (Solon, OH). Ferulic acid and caffeic acid were obtained from the National Institute for the Control of Pharmaceutical and Biological Products, China. Maclurin was from BioBioPha Co., Ltd. (Kunming, China). Proanthocyanidin, rosmarinic acid, (±)-a-tocopherol, and uracil were purchased from the Aladdin Chemistry Co. (Shanghai, China). Curcumin, thymol, 2-thiobarbituric acid (TBA), and pyridoxine hydrochloride were from Guangdong Guanghua Chemical Factory Co., Ltd. (Guangzhou, China). MEAS (methanol extract of Aquilaria sinensis leaves) and MEGB (methanol extract from Gynura bicolor Roxb. DC) were prepared in our laboratory. All other chemical reagents and organic solvents were of analytical grade. 2.2. Comparison of the hydroxyl radical-scavenging activities of 25 antioxidant compounds in ethanol solution and as residue in buffer (after removing ethanol) The assay in ethanol solution for 25 selected antioxidants (Fig. 1), was performed, based on the original deoxyribose degradation assay (Halliwell et al., 1987). In brief, the antioxidant sample was first dissolved in absolute ethanol to prepare the sample solution (at 1–4 mg/ml). An aliquot of sample solution was brought to 400 ll in phosphate buffer (0.2 M, pH 7.4). Then, 50 ll of deoxyribose (50 mM), 50 ll of Na2EDTA (1 mM), 50 ll of FeCl3 (3.2 mM) and 50 ll of H2O2 (50 mM) were added. The reaction was initiated by mixing 50 ll of ascorbic acid (1.8 mM) and the total volume of the reaction mixture was adjusted to 800 ll with buffer. After incubation at 50 °C for 20 min, the reaction was terminated by 250 ll of trichloroacetic acid (10%, w/w). The colour was then developed by addition of 150 ll of TBA (5%, in 1.25% NaOH aqueous solution) and heating in an oven at 105 °C for 15 min. The mixture was cooled and absorbance was measured at 530 nm (Unico 2100, spectrophotometer, Shanghai, China) against the buffer (as blank). The hydroxyl radical-scavenging activity was expressed as:
Inhibition % ¼
A0 A 100% A0
Here, A0 is the A530nm of the mixture without sample solution, and A is A530nm of the reaction mixture with sample solution. In the assay after removing the ethanol, an aliquot of the sample solution was added to a tube and then evaporated to dryness (water bath, 660 °C). The sample residue was subsequently determined as described above. 2.3. .Hydroxyl radical-scavenging activity of 18 pure organic solvents The hydroxyl (OH) radical-scavenging activities of 18 pure organic solvents were determined, based on the original deoxyribose method, as described in the former section. However, 18 pure organic solvents replaced the sample solutions and they were not evaporated before determination. The 18 pure organic solvents included acetone, acetonitrile, benzene, carbon disulphide, carbon
2.5. Statistical analysis Each experiment for the comparison of the 25 selected antioxidants in ethanol solution and residue was performed in triplicate and the data were recorded as Mean ± SD (standard deviation). The IC50 value for the organic solvent was defined as the volume of 50% hydroxyl radical inhibition in the 800 ll reaction system, and the unit was ll. The IC50 value for each antioxidant was defined as the final concentration of 50% hydroxyl radical inhibition, and the unit was lg/ml. Statistical comparisons were made by oneway ANOVA to detect significant difference using SPSS 13.0 (SPSS Inc., Chicago, IL) for windows. P < 0.05 was considered to be statistically significant. 3. Results and discussion In the study, we first compared the hydroxyl radical-scavenging activity of 25 antioxidant compounds in ethanol solution and as residue in buffer (after removing ethanol). (+)-Catechin, a standard antioxidant, is shown as a typical example. As shown in Fig. 2, in the assay using ethanol solution, its IC50 value was 19.7 ± 0.15 lg/ml, while an IC50 value of 281 ± 1.55 lg/ml was found using the residue. The ratio of IC50,residue:IC50,ethanolsolution values was calculated to be 14.3. Similar differences were also observed in the other 24 selected antioxidants. Table 1 illustrates that the ratio of IC50,residue:IC50,ethanolsolution values of all 25 selected antioxidants varied from 2.37 to 20.9. The average value of 8.88 suggested that the inhibition performance of the samples was enhanced by the ethanol solvent (approximately 9-fold). Further analysis indicated that, as the sample solution concentrations increased, the value of IC50,residue:IC50,ethanolsolution ratios decreased. For example, (+)-catechin at different concentrations (1, 2, and 3 mg/ml) possessed different ratio values (14.3, 12.8, and 5.40, respectively). In a word, the alcoholic interference dominated the results obtained in the assay with ethanol. To explore the mechanism of alcoholic interference, the hydroxyl-radical-scavenging ability of pure ethanol was determined, using the original deoxyribose assay. Results demonstrated that ethanol itself exhibited a dose–response curve (Supplementary material 2, Fig. S12) and its IC50 was only 1.86 ± 0.13 ll in an 800 ll reaction volume (Table 2). This means that less than 3 ll of ethanol is adequate to scavenge most hydroxyl radicals (>90%). These results are consistent with previous reports that ethanol can act as a free radical-scavenger (De La Paz & Anderson, 1992; Novogrodsky, Ravid, Rubin, & Stenzel, 1982) and can protect DNA from X-rays by scavenging hydroxyl radicals (Ellahueñe, Pérez-Alzola, & Olmedo, 2012). In summary, the alcoholic interference in the assay resulted from the hydroxyl radical-scavenging ability of ethanol itself.
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Fig. 1. The structures of 25 selected antioxidants.
Fig. 2. The dose–response curves of (+)-catechin in ethanol solution and as residue in buffer (after removing ethanol) All data are presented as the Mean ± SD for three replicates.
To systematically investigate the solvent effect, the interference of a further 17 pure organic solvents was determined, using the original deoxyribose assay. The data (Supplementary material 2) revealed that all of the solvents can effectively scavenge hydroxyl radicals. We classified the organic solvents into four main types according to their IC50 values (Table 2): (1) n-Hexane and petroleum ether were the weakest OH-scavengers (Table 2). n-Hexane and petroleum ether (a mixture of lower alkanes) are hydrocarbons that contain only r bonds (C–H and C–C r bonds), which are stable and resistant to OH damage. Additionally, the poor water solubility of these solvents also prohibited reaction with hydroxyls in the aqueous reaction system.
(2) As shown in Table 2, cyclohexane showed mild OH scavenging-activity, which may result from the ring strain inherent to the cyclic molecule (McMurry, 2003, chap 2). In addition, two typical aromatic hydrocarbon solvents, benzene and toluene, and three halohydrocarbons, carbon tetrachloride, chloroform, and dichloromethane, also possessed moderate activity. Aromatic hydrocarbon molecules contain conjugated p bonds, which are less stable than are r bonds and can react with OH radicals. The mild activity of three halohydrocarbons, however, could be attributed to the polar C–X r bonds (X = halogen). (3) A further three solvents, acetonitrile, acetone, and carbon disulphide, exhibited strong hydroxyl radical-scavenging ability (Table 2). The non-conjugated unsaturated bonds in these molecules (C„N, C@O, and C@S, respectively) are more reactive than are the conjugated p bonds in the aromatic hydrocarbon molecules. Therefore, they could easily be damaged by OH radicals. (4) Very strong OH-scavenging activity was observed in seven organic solvents, including diethyl ether, DMF, DMSO, ethanol, ethyl acetate, methanol, and THF (Table 2). Except for diethyl ether (which is still quite soluble), all of these solvents are miscible with water. All of these solvents contain at least one O atom, which forms various active chemical bonds (e.g. C–O, H–O, and S@O) or functional groups (e.g. hydroxyl, ester, amide, sulfinyl, and cyclic ether groups). These reactive chemical bonds or functional groups (McMurry, 2003, chap. 2), are responsible for the strong hydroxylradical-scavenging ability for these solvents. Importantly, DMSO, which is often used in sample solution preparation for biochemistry or animal experiments, displayed a very strong scavenging ability (IC50 = 5.76 ± 0.76 ll, Table 2). The results agree with previous studies showing that DMSO can be a hydroxyl scavenger
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Table 1 Results of deoxyribose assay of 25 selected antioxidants determined in ethanol solution and as residue in buffer (after removing ethanol). NO
Antioxidant
Resource and characterisation
IC50 value (lg/ml) Ethanol solution
(+)-Catechin BHA Caffeic acid Curcumin Ferulic acid GSH 8-Hydroxyl quinoline deriv. Isoferulic acid Maclurin Magnolol MEAS MEGB Melatonin Proanthocyanidin Protocatechuic acid Puerarin Pyridoxine Quercetin Rosmarinic acid Rutin Sinapic acid Thymol (±) a-Tocopherol Trolox Uracil
Exogenous, natural, polyphenol Exogenous, synthetic, phenol Exogenous, natural, phenolic acid Exogenous, natural, phenol Exogenous, natural, phenolic acid Endogenous, polypeptide Exogenous, synthetic, heterocyclic phenol Exogenous, natural, phenolic acid Exogenous, natural, polyphenol Exogenous, natural, phenolic phenylpropanoid Exogenous, natural,extract natural,extract Exogenous, natural,extract Endogenous, heterocyclic phenol Exogenous, natural, proanthocyanidins Exogenous, natural, phenolic acid Exogenous, natural, flavonoid glycoside Exogenous, natural, vitamin Exogenous, natural, flavonoid Exogenous, natural, polyphenol Exogenous, natural, flavonoid glycoside Exogenous, natural, phenolic acid Exogenous, natural, phenol Exogenous, natural, vitamin Exogenous, synthetic, vitamin analogue Endogenous, base pair, heterocyclic phenol Average
19.7 ± 0.15 8.69 ± 0.05a 20.4 ± 0.71a 9.46 ± 0.94a 17.3 ± 0.23a 17.1 ± 0.08a 9.93 ± 1.34a 17.0 ± 0.09a 9.27 ± 1.24a 11.6 ± 3.65a 32.8 ± 4.47a 44.9 ± 3.66a 8.96 ± 0.39a 14.5 ± 2.20a 8.32 ± 0.87a 8.97 ± 0.95b 9.82 ± 0.95b 21.9 ± 1.35a 9.84 ± 0.11a 8.89 ± 0.03a 7.09 ± 0.23a 7.66 ± 0.22 a 45.5 ± 5.71a 17.3 ± 0.13a 14.0 ± 0.13a –
b
281 ± 1.55 24.7 ± 0.15b 285 ± 2.14b 187 ± 5.82b 360 ± 1.27b 126 ± 3.27b 35.7 ± 1.45b 214 ± 1.76b 74.9 ± 2.82b 49.1 ± 4.42b 310 ± 10.21b 782 ± 20.55b 49.6 ± 1.45 b 89.1 ± 3.52b 66.9 ± 2.88b 54.0 ± 4.84b 98.1 ± 6.55b 267 ± 1.38b 62.2 ± 3.56b 37.8 ± 0.89b 17.7 ± 0.15b 103 ± 5.68 b 108 ± 4.35b 89.4 ± 3.01b 80.1 ± 2.89b –
14.3 2.84 14.0 19.7 20.9 7.39 3.60 12.6 8.06 4.25 9.46 17.4 5.53 6.14 8.03 6.02 9.98 12.2 6.32 4.25 2.50 13.3 2.37 5.17 5.73 8.88
Validation for improved method (using residue) Regression equation
R value
RSD for IC50 (%)
y = 1.49 + 0.57x y = 38.84 + 0.47x y = 5.00 + 0.55x y = 6.29 + 0.23x y = 2.32 + 0.24x y = 7.75 + 1.02x y = 4.46 + 1.28x y = 19.31 + 0.59x y = 9.19 + 0.54x y = 20.63 + 0.59x y = 12.37 + 0.12x y = 1.42 + 0.062x y = 7.32 + 0.83x y = 10.23 + 0.45x y = 9.79 + 0.60x y = 0.28 + 0.92x y = 8.06 + 0.45x y = 4.01 + 0.29x y = 7.34 + 0.68x y = 36.56 + 0.35x y = 14.34 + 1.06x y = 4.43 + 0.44x y = 0.83 + 0.48x y = 30.30 + 0.79x y = 5.30 + 0.56x –
0.99887 0.95652 0.99007 0.84743 0.98362 0.97548 0.90437 0.93745 0.91785 0.80304 0.87812 0.97781 0.97684 0.88533 0.89638 0.99956 0.87701 0.99055 0.96307 0.99679 0.83259 0.95339 0.94287 0.92516 0.92354 0.93335
0.55 0.61 0.75 3.11 0.35 2.58 6.86 0.82 3.77 9.07 3.29 2.67 2.92 3.95 4.31 8.97 6.68 0.52 5.72 2.35 0.85 2.84 4.04 3.36 3.60 3.38
IC50 value is defined as the concentration of 50% hydroxyl radical inhibition and expressed as Mean ± SD (n = 3). The linear regression was analysed by Origin 6.0 professional software. BHA, butylated hydroxyanisole. GSH, glutathione. MEAS, methanol extract of Aquilaria Sinensis leaves. MEGB, methanol extract from Gynura bicolor Roxb. DC. Mean values with different superscripts in the same row are significantly different (p < 0.05). Ratio value = IC50,residue:IC50,ethanolsolution. RSD = SD Mean 100%. Their dose–response curves were shown in Supplementary material 1.
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
a
Ratio value Residue
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X. Li / Food Chemistry 141 (2013) 2083–2088 Table 2 The IC50 values of 18 pure organic solvents by deoxyribose degradation assay. Solvent Ethanol Methanol THF DMF Diethyl ether Ethyl acetate DMSO Acetone Carbon disulphide Acetonitrile
IC50 (ll in 800 ll) a
1.86 ± 0.13 2.11 ± 0.25a 3.08 ± 0.89b 4.25 ± 0.69b 4.17 ± 1.06c 5.44 ± 1.28d 5.76 ± 0.76d 12.7 ± 0.33e 15.3 ± 0.86f 17.5 ± 1.67g
Type Very strong Very strong Very strong Very strong Very strong Very strong Very strong Strong Strong Strong
Solvent Chloroform Carbon tetrachloride Dichloromethane Toluene Benzene Cyclohexane Petroleum ether n-Hexane
IC50 (ll in 800 ll) h
26.0 ± 2.44 47.0 ± 3.56i 49.0 ± 3.52j 57.7 ± 3.54k 57.9 ± 7.66k 66.9 ± 5.99l 119 ± 5.56m 407 ± 13.3n
Type Moderate Moderate Moderate Moderate Moderate Moderate Weak Weak
The IC50 value was defined as the volume (ll) of 50% hydroxyl radical inhibition in the 800 ll reaction system, and was expressed as Mean ± SD (n = 3). The linear regression was analysed by Origin 6.0 professional software. Mean values with different superscripts (a-g) are significantly different (p < 0.05) and those with same letters are not significantly different (p < 0.05). THF, tetrahydrofuran; DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide. Their dose–response curves were shown in Supplementary material 2.
Fig. 3. The recommended procedure of the improved dexyribose degradation assay for hydroxyl radical-scavenging.
(Doroshow, 1986; Repine, Pfenninger, Talmage, Berger, & Pettijohn, 1981) and could prevent DNA nicking by hydroxyl radicals (Fang & Zheng, 2002a, chap. 5). The solvent effect on the assay results arises from the hydroxylradical-scavenging ability of organic solvents themselves and can be ultimately attributed to the extreme reactivity of the hydroxyl radical. It has been demonstrated that hydroxyl radicals, once generated, will quickly react with surrounding solvent molecules (Fang & Zheng, 2002b, chap. 2; Halliwell & Gutteridge, 1984). For example, the reaction rates of OH with ethanol and DMSO were respectively 1.9 109 l mol1 s1 (Fang & Zheng, 2002b, chap. 2) and 9 109 l mol1 s1 (Nakai, Kadiiska, Jiang, Stadler, & Mason, 2006). The product of OH reaction with DMSO is CH3 (Chan et al., 1989). In a word, any organic solvent should be completely removed before measurement via the deoxyribose degradation assay, or the results will be meaningless. However, evaporation temperatures should remain below 60 °C to prevent the antioxidant samples from being destroyed. In our experiment, we found that all sample residues could be well dissolved in the aqueous reaction system. Most antioxidants are organic compounds and their water solubilities are not generally high enough to prepare sample stock solutions (room temperature), but are generally high enough to dissolve in the reaction system (50 °C). For example, the water solubility of catechin is 2.26 mg/ml (25 °C) (Srinivas, King, Howard, & Monrad, 2010), while its IC50 value was only 0.20 ± 0.0025 mg/ml (Table 1). A few typical lipid-soluble antioxidants (e.g. BHA, thymol, and tocopherol) and both extracts (MEAS and MEGB) could also be dissolved in the reaction system. This was because: (1) these antioxidants contain phenolic –OH groups that can form hydrogen bonds with the H2O molecule, increasing the water solubility; (2) the higher reaction temperature (50 °C) increased the solubility in the aqueous system; and (3) the amount of antioxidant residues was gradually reduced by OH attack in the reaction process. Based on the discussion above, the deoxyribose degradation assay can be improved as follows: first, we choose an appropriate or-
ganic solvent to prepare the sample solution. An aliquot of the sample solution was placed in a tube and this sample solution was evaporated to dryness (water bath, 660 °C). Then, the sample residue was determined as described in Section 2.2. The whole process of improved deoxyribose degradation assay is displayed in Fig. 3. It must be emphasised that, (1), if the antioxidant sample is water-soluble, we can use water (or buffer) to prepare the sample solution. Since water (or buffer) cannot react with the hydroxyl radicals, aqueous solution samples can be measured directly; (2) if the antioxidant sample is thermo-sensitive, the sample solution should be evaporated to dryness at room temperature. The improved deoxyribose assay procedure was validated, using the reference compound (+)-catechin. The regression equation of (+)-catechin was calculated as y = 1.49 + 0.57 x (y: inhibition percentage; x: final concentration) and the correlation coefficient (R) was 0.99887 (Fig. 2). The RSD% of the IC50 was 0.51% (Table 1) and met FDA requirements (US Food and Drug Administration, 2001). In addition to (+)-catechin, the other 24 selected antioxidants also exhibited good linearity (R = 0.80304– 0.99956, the average R value was 0.93335) and low RSD values (0.35–9.07%, the average value was 3.38%) (Table 1). Apparently, the improved method had good linearity and precision. In addition, the RSD% of IC50 values obtained on different days was 2.26% in the analysis of (+)-catechin (Supplementary material 3). This suggests that the improved method has good reproducibility. In summary, the improved deoxyribose assay (especially sample preparation) is reasonable and feasible. As can be seen in Fig. 1 and Table 1, the 25 selected antioxidants included all types of antioxidants, such as pure compounds and extracts; natural and synthetic; exogenous and endogenous, water-soluble and lipid-soluble, vitamins, vitamin analogues, and non-vitamins, phenols, polyphenols, phenolic acids, flavonoids, proanthocyanidins, phenylpropanoids, polypeptides, and heterocyclic phenols and base pairs. As the scavenging ability of all of these species could be successfully measured in our experiment, the improved method proved to be suitable for all types of antioxidants.
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4. Conclusion In the deoxyribose degradation assay, any organic solvent should be completely evaporated before measurement, or the result will be meaningless. Our proposed improved method is regarded as a reliable hydroxyl radical-scavenging assay, suitable for all types of antioxidants. Acknowledgements The author is thankful to his students Yuling Zhang, Junqian Xiao, Rijin Qin, Jiali Chen, Chuangli Lin, and Jianbin Liang (School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Guangzhou), for analysis work. In addition, 8-hydroxyquinoline was kindly donated by Prof. Heping Zeng (South China University of Technology, Guangzhou, China). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2013. 05.084. References Chan, J., Fujiwara, T., Brennan, P., McNeil, M., Turco, S. J., Sibille, J. C., et al. (1989). Microbial glycolipids: Possible virulence factors that scavenge oxygen radicals. Proceedings of the National Academy of Sciences, 86, 2453–2457. Cheeseman, K. H., Beavis, A., & Esterbauer, H. (1988). Hydroxyl-radical-induced iron-catalysed degradation of 2-deoxyribose, quantitative determination of malondialdehyde. Biochemical Journal, 252, 649–653. De La Paz, M. A., & Anderson, R. E. (1992). Lipid peroxidation in rod outer segments: Role of hydroxyl radical and lipid hydroperoxides. Investigative Ophthalmology and Visual Science, 33, 2091–2096. Desmarchelier, C., Coussio, J., & Ciccia, G. (1998). Antioxidant and free radical scavenging effects in extracts of the medicinal herb Achyrocline satureioides (Lam.) DC. (‘‘marcela’’). Brazilian Journal of Medical and Biological Research, 31, 1163–1170. Doroshow, J. H. (1986). Role of hydrogen peroxide and hydroxyl radical formation in the killing of Ehrlich tumor cells by anticancer quinones. Proceedings of the National Academy of Sciences, 83, 4514–4518. Ekanayake, P., Lee, Y. D., & Lee, J. (2004). Antioxidant activity offlesh and skin of Eptatretus burgeri (Hag Fish) and Enedrias nebulosus (White Spotted Eel). Food Science Technology International, 10, 171–177. Ellahueñe, M. F., Pérez-Alzola, L. P., & Olmedo, M. I. (2012). Chronic ethanol consumption in mice does not induce DNA damage in somatic or germ cells, evaluated by the bone marrow icronucleous assay and the dominant lethal mutation assay. Biological Research, 45, 27–31. Fang, Y. Z., & Zheng, R. L. (2002a). Theory and application of free radical biology (2nd ed.). Beijing: Science Press. Fang, Y. Z., & Zheng, R. L. (2002b). Theory and application of free radical biology (2nd ed.). Beijing: Science Press. Ganesan, P., Kumar, C. S., & Bhaskar, N. (2008). Antioxidant properties of methanol extract and its solvent fractions obtained from selected Indian red seaweeds. Bioresource Technology, 99, 2717–2723.
Georgetti, S. R., Casagrande, R., Moura-de-Carvalho Vicentini, F. T., Verri, W. A., & Fonseca, M. J. (2006). Evaluation of the antioxidant activity of soybean extract by different in vitro methods and investigation of this activity after its incorporation in topical formulations. European Journal of Pharmaceutics and Biopharmaceutics, 64, 99–106. Halliwell, B., & Gutteridge, J. M. C. (1981). Formation of a thiobarbituric-acidreactive substance from deoxyribose in the presence of iron salts. FEBS Letters, 128, 2. Halliwell, B., & Gutteridge, J. M. C. (1984). Oxygen toxicity, oxygen radicals, transition metals and disease. Biochemical Journal, 219, 1–14. Halliwell, B., Gutteridge, J. M. C., & Aruoma, O. I. (1987). The deoxyribose method: A simple ‘‘test-tube’’ assay for determination of rate constants for reactions of hydroxyl radicals. Analytical Biochemistry, 165, 215–219. Kaurinovic, B., Popovic, M., Vlaisavljevi, S., & Trivic, S. (2011). Antioxidant capacity of Ocimum basilicum L. and Origanum vulgare L. extracts. Molecules, 16, 7401–7414. Li, H., Wang, X., Li, Y., Li, P., & Wang, H. (2009). Polyphenolic compounds and antioxidant properties of selected China wines. Food Chemistry, 112, 454–460. McMurry, J. (2003). Fundamentals of organic chemistry. Beijing: China Machine Press. Nakai, K., Kadiiska, M. B., Jiang, J. J., Stadler, K., & Mason, R. P. (2006). Free radical production requires both inducible nitric oxide synthase and xanthine oxidase in LPS-treated skin. Proceedings of the National Academy of Sciences, 103, 4616–4621. Novogrodsky, A., Ravid, A., Rubin, A. L., & Stenzel, K. H. (1982). Hydroxyl radical scavengers inhibit lymphocyte mitogenesis. Proceedings of the National Academy of Sciences, 79, 1171–1174. Ozsoy, N., Can, A., Yanardag, R., & Akev, N. (2008). Antioxidant activity of Smilax excelsa L. leaf extracts. Food Chemistry, 110, 571–583. Puntel, G. O., Gubert, P., Peres, G. L., Bresolin, L., Rocha, J. B., Pereira, M. E., et al. (2008). Antioxidant properties of oxime 3-(phenylhydrazono) butan-2-one. Archives of Toxicology, 82, 755–762. Repine, J. E., Pfenninger, O. W., Talmage, D. W., Berger, E. M., & Pettijohn, D. E. (1981). Dimethyl sulfoxide prevents DNA nicking mediated by ionizing radiation or iron/hydrogen peroxide-generated hydroxyl radical. Proceedings of the National Academy of Sciences, 78, 1001–1003. Rop, O., Mlcek, J., Jurikova, T., Valsikova, M., Socho, R. J., & Daniel, V. R. (2010). Phenolic content, antioxidant capacity, radical oxygen species scavenging and lipid peroxidation inhibiting activities of extracts of five black chokeberry (Aronia melanocarpa (Michx.) Elliot) cultivars. Journal of Medicinal Plants Research, 22, 2431–2437. Srinivas, K., King, J. W., Howard, L. R., & Monrad, J. K. (2010). Solubility of gallic acid, catechin, and protocatechuic acid in subcritical water from (298.75 to 415.85) K. Journal of Chemical & Engineering Data, 55, 3101–3108. Srinivasan, R., Chandrasekar, M. J. N., Nanjan, M. J., & Suresh, B. (2007). Antioxidant activity of Caesalpinia digyna root. Journal of Ethnopharmacology, 113, 284–291. US Food and Drug Administration, US Department of Health and Human Services, Center for Drug Evaluation and Research. (2001). FDA guidance for industry bioanalytical method validation. Visavadiya, N. P., Soni, B., & Dalwadi, N. (2009). Evaluation of antioxidant and antiatherogenic properties of Glycyrrhiza glabra root using in vitro models. International Journal Food Science and Nutrition, 60, 135–149. Wang, X. Z., & Li, X. C. (2011). Evaluation of antioxidant activity of isoferulic acid in vitro. Natural Product Communications, 6, 1285–1288. Wu, M. J., Yen, J. H., Wang, L., & Weng, C. Y. (2004). Antioxidant activity of porcelainberry (Ampelopsis brevipedunculata (Maxim.) Trautv.). American Journal of Chinese Medicine, 32, 681–693. You, Y., Duan, X., Wei, X., Su, X., Zhao, M., Sun, J., et al. (2007). Identification of major phenolic compounds of Chinese water chestnut and their antioxidant activity. Molecules, 12, 842–852. Yuan, Y. V., Bone, D. E., & Carrington, M. F. (2005). Antioxidant activity of dulse (Palmaria palmata) extract evaluated in vitro. Food Chemistry, 91, 485–494.
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Analytical Methods
Solvent effects and improvements in the deoxyribose degradation assay for hydroxyl radical-scavenging Xican Li ⇑ School of Chinese Herbal Medicine, Guangzhou University of Chinese Medicine, Guangzhou, China
a r t i c l e
i n f o
Article history: Received 17 October 2012 Received in revised form 30 March 2013 Accepted 8 May 2013 Available online 23 May 2013 Keywords: Antioxidant Deoxyribose degradation assay Hydroxyl radical scavenging OH Solvent effect Improvement
a b s t r a c t The deoxyribose degradation assay is widely used to evaluate the hydroxyl (OH) radical-scavenging ability of food or medicines. We compared the hydroxyl radical-scavenging activity of 25 antioxidant samples prepared in ethanol solution with samples prepared after removing the ethanol (residue). The data suggested that there was an approximately 9-fold difference between assay results for the ethanol solution and residue samples. This indicated a strong alcoholic interference. To further study the mechanism, the scavenging activities of 18 organic solvents (including ethanol) were measured by the deoxyribose assay. Most pure organic solvents (especially alcohols) could effectively scavenge hydroxyl radicals. As hydroxyl radicals have extremely high reactivities, they will quickly react with surrounding solvent molecules. This shows that any organic solvent should be completely evaporated before measurement. The proposed method is regarded as a reliable hydroxyl radical-scavenging assay, suitable for all types of antioxidants. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The deoxyribose degradation assay has been widely used to evaluate the hydroxyl (OH) radical-scavenging ability of food, nutrients, and medicines since it was established in 1987 (Halliwell, Gutteridge, & Aruoma, 1987) In the deoxyribose degradation assay, OH is obtained via Fenton reaction (Halliwell & Gutteridge, 1981; Halliwell et al., 1987). Subsequently, OH attacks deoxyribose and breaks its cyclic furan ring to generate malondialdehyde (MDA). MDA combines with 2thiobarbituric acid (TBA) to produce a chromogen with kmax at 530 nm (Cheeseman, Beavis, & Esterbauer, 1988). Therefore, the A530nm value is proportional to the produced amount of OH radicals. Higher A530nm values indicate higher levels of OH radicals. If an antioxidant sample is added, the A530nm value will decrease, suggesting that some OH radicals are scavenged by the antioxidant. This is the principle of deoxyribose degradation assay. Abbreviations: SD, standard deviation; RSD, relative standard deviation; GSH, glutathione; BHA, butylated hydroxyanisole; THF, tetrahydrofuran; DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide; ROS, reactive oxygen species; FC, final concentration; FDA, Food and Drug Administration of USA; MEAS, methanol extract of Aquilaria sinensis leaves; MEGB, methanol extract from Gynura bicolor Roxb. DC. ⇑ Address: No. 232, Waihuan East Road, Guangzhou Higher Education Mega Center, Panyu District, Guangzhou 510006, China. Tel.: +86 20 39358076; fax: +86 20 38892697. E-mail address: [email protected] URL: http://www.researchgate.net/profile/Xican_Li 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.05.084
Like most biochemical assays, the deoxyribose assay requires the preparation of sample solutions, using various organic solvents prior to measurement. However, it was observed (Wang & Li, 2011) that the organic solvents (especially alcohols) normally used to prepare these sample solutions may cause strong interferences which are often overlooked by assay users. Several research groups have clearly stated that they used organic solvents for sample solution preparation, and that the sample solution was directly used for measurement by the deoxyribose assay. For example, at least seven groups reported that they used alcohol to dissolve their samples, which was directly measured by the assay (Ganesan, Kumar, & Bhaskar, 2008; Kaurinovic, Popovic, Vlaisavljevi, & Trivic, 2011; Rop et al., 2010; Visavadiya, Soni, & Dalwadi, 2009; Wu, Yen, Wang, & Weng, 2004; You et al., 2007; Yuan, Bone, & Carrington, 2005). One research group even directly took edible wine for the assay, to estimate hydroxyl scavenging ability of the phenolic compounds in wine (Li, Wang, Li, Li, & Wang, 2009). Other groups, however, used dimethyl sulphoxide (DMSO) or ethyl acetate to dissolve the samples for direct measurement (Desmarchelier, Coussio, & Ciccia, 1998; Ekanayake, Lee, & Lee, 2004; Georgetti, Casagrande, Moura-de-Carvalho Vicentini, Verri, & Fonseca, 2006; Ozsoy, Can, Yanardag, & Akev, 2008; Puntel et al., 2008; Srinivasan, Chandrasekar, Nanjan, & Suresh, 2007). Since water solubility of antioxidant samples is generally not adequate to prepare sample stock solutions at room temperature, we believe that there are many more research groups using organic solvents for dissolving the samples and the problem may therefore
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be even more widespread. All of these investigations are therefore regarded as unreliable. Therefore, in the present study we systematically investigate the effect of various solvents in an effort to improve the deoxyribose assay performance.
tetrachloride, chloroform, cyclohexane, dichloromethane, diethyl ether, DMF (N,N-dimethylformamide), DMSO (dimethyl sulfoxide), ethanol, ethyl acetate, n-hexane, methanol, petroleum ether, THF (tetrahydrofuran) and toluene.
2. Materials and methods
2.4. Validation of the improved deoxyribose degradation method
2.1. Chemicals
The improved method was validated according to FDA guidance (US Food and Drug Administration, 2001), with regard to linearity, precision, and reproducibility. (+)-Catechin, which is a standard antioxidant, was used as the reference compound. The linearity of the method was evaluated at five concentrations. The calibration curve was prepared by plotting the mean inhibition percentages of triplicate analyses against the final concentrations. The precision was evaluated, using the relative standard deviation (RSD%) inhibition percentages of triplicate analyses.
Trolox (±-6-hydroxyl-2,5,7,8-tetramethylchromane-2-carboxylic acid), melatonin, BHA (butylated hydroxyanisole), (+)-catechin, isoferulic acid, and sinapic acid were obtained from Sigma–Aldrich, Shanghai Trading Co. (Shanghai, China). Deoxyribose and GSH (glutathione) were purchased from Amresco Inc. (Solon, OH). Ferulic acid and caffeic acid were obtained from the National Institute for the Control of Pharmaceutical and Biological Products, China. Maclurin was from BioBioPha Co., Ltd. (Kunming, China). Proanthocyanidin, rosmarinic acid, (±)-a-tocopherol, and uracil were purchased from the Aladdin Chemistry Co. (Shanghai, China). Curcumin, thymol, 2-thiobarbituric acid (TBA), and pyridoxine hydrochloride were from Guangdong Guanghua Chemical Factory Co., Ltd. (Guangzhou, China). MEAS (methanol extract of Aquilaria sinensis leaves) and MEGB (methanol extract from Gynura bicolor Roxb. DC) were prepared in our laboratory. All other chemical reagents and organic solvents were of analytical grade. 2.2. Comparison of the hydroxyl radical-scavenging activities of 25 antioxidant compounds in ethanol solution and as residue in buffer (after removing ethanol) The assay in ethanol solution for 25 selected antioxidants (Fig. 1), was performed, based on the original deoxyribose degradation assay (Halliwell et al., 1987). In brief, the antioxidant sample was first dissolved in absolute ethanol to prepare the sample solution (at 1–4 mg/ml). An aliquot of sample solution was brought to 400 ll in phosphate buffer (0.2 M, pH 7.4). Then, 50 ll of deoxyribose (50 mM), 50 ll of Na2EDTA (1 mM), 50 ll of FeCl3 (3.2 mM) and 50 ll of H2O2 (50 mM) were added. The reaction was initiated by mixing 50 ll of ascorbic acid (1.8 mM) and the total volume of the reaction mixture was adjusted to 800 ll with buffer. After incubation at 50 °C for 20 min, the reaction was terminated by 250 ll of trichloroacetic acid (10%, w/w). The colour was then developed by addition of 150 ll of TBA (5%, in 1.25% NaOH aqueous solution) and heating in an oven at 105 °C for 15 min. The mixture was cooled and absorbance was measured at 530 nm (Unico 2100, spectrophotometer, Shanghai, China) against the buffer (as blank). The hydroxyl radical-scavenging activity was expressed as:
Inhibition % ¼
A0 A 100% A0
Here, A0 is the A530nm of the mixture without sample solution, and A is A530nm of the reaction mixture with sample solution. In the assay after removing the ethanol, an aliquot of the sample solution was added to a tube and then evaporated to dryness (water bath, 660 °C). The sample residue was subsequently determined as described above. 2.3. .Hydroxyl radical-scavenging activity of 18 pure organic solvents The hydroxyl (OH) radical-scavenging activities of 18 pure organic solvents were determined, based on the original deoxyribose method, as described in the former section. However, 18 pure organic solvents replaced the sample solutions and they were not evaporated before determination. The 18 pure organic solvents included acetone, acetonitrile, benzene, carbon disulphide, carbon
2.5. Statistical analysis Each experiment for the comparison of the 25 selected antioxidants in ethanol solution and residue was performed in triplicate and the data were recorded as Mean ± SD (standard deviation). The IC50 value for the organic solvent was defined as the volume of 50% hydroxyl radical inhibition in the 800 ll reaction system, and the unit was ll. The IC50 value for each antioxidant was defined as the final concentration of 50% hydroxyl radical inhibition, and the unit was lg/ml. Statistical comparisons were made by oneway ANOVA to detect significant difference using SPSS 13.0 (SPSS Inc., Chicago, IL) for windows. P < 0.05 was considered to be statistically significant. 3. Results and discussion In the study, we first compared the hydroxyl radical-scavenging activity of 25 antioxidant compounds in ethanol solution and as residue in buffer (after removing ethanol). (+)-Catechin, a standard antioxidant, is shown as a typical example. As shown in Fig. 2, in the assay using ethanol solution, its IC50 value was 19.7 ± 0.15 lg/ml, while an IC50 value of 281 ± 1.55 lg/ml was found using the residue. The ratio of IC50,residue:IC50,ethanolsolution values was calculated to be 14.3. Similar differences were also observed in the other 24 selected antioxidants. Table 1 illustrates that the ratio of IC50,residue:IC50,ethanolsolution values of all 25 selected antioxidants varied from 2.37 to 20.9. The average value of 8.88 suggested that the inhibition performance of the samples was enhanced by the ethanol solvent (approximately 9-fold). Further analysis indicated that, as the sample solution concentrations increased, the value of IC50,residue:IC50,ethanolsolution ratios decreased. For example, (+)-catechin at different concentrations (1, 2, and 3 mg/ml) possessed different ratio values (14.3, 12.8, and 5.40, respectively). In a word, the alcoholic interference dominated the results obtained in the assay with ethanol. To explore the mechanism of alcoholic interference, the hydroxyl-radical-scavenging ability of pure ethanol was determined, using the original deoxyribose assay. Results demonstrated that ethanol itself exhibited a dose–response curve (Supplementary material 2, Fig. S12) and its IC50 was only 1.86 ± 0.13 ll in an 800 ll reaction volume (Table 2). This means that less than 3 ll of ethanol is adequate to scavenge most hydroxyl radicals (>90%). These results are consistent with previous reports that ethanol can act as a free radical-scavenger (De La Paz & Anderson, 1992; Novogrodsky, Ravid, Rubin, & Stenzel, 1982) and can protect DNA from X-rays by scavenging hydroxyl radicals (Ellahueñe, Pérez-Alzola, & Olmedo, 2012). In summary, the alcoholic interference in the assay resulted from the hydroxyl radical-scavenging ability of ethanol itself.
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Fig. 1. The structures of 25 selected antioxidants.
Fig. 2. The dose–response curves of (+)-catechin in ethanol solution and as residue in buffer (after removing ethanol) All data are presented as the Mean ± SD for three replicates.
To systematically investigate the solvent effect, the interference of a further 17 pure organic solvents was determined, using the original deoxyribose assay. The data (Supplementary material 2) revealed that all of the solvents can effectively scavenge hydroxyl radicals. We classified the organic solvents into four main types according to their IC50 values (Table 2): (1) n-Hexane and petroleum ether were the weakest OH-scavengers (Table 2). n-Hexane and petroleum ether (a mixture of lower alkanes) are hydrocarbons that contain only r bonds (C–H and C–C r bonds), which are stable and resistant to OH damage. Additionally, the poor water solubility of these solvents also prohibited reaction with hydroxyls in the aqueous reaction system.
(2) As shown in Table 2, cyclohexane showed mild OH scavenging-activity, which may result from the ring strain inherent to the cyclic molecule (McMurry, 2003, chap 2). In addition, two typical aromatic hydrocarbon solvents, benzene and toluene, and three halohydrocarbons, carbon tetrachloride, chloroform, and dichloromethane, also possessed moderate activity. Aromatic hydrocarbon molecules contain conjugated p bonds, which are less stable than are r bonds and can react with OH radicals. The mild activity of three halohydrocarbons, however, could be attributed to the polar C–X r bonds (X = halogen). (3) A further three solvents, acetonitrile, acetone, and carbon disulphide, exhibited strong hydroxyl radical-scavenging ability (Table 2). The non-conjugated unsaturated bonds in these molecules (C„N, C@O, and C@S, respectively) are more reactive than are the conjugated p bonds in the aromatic hydrocarbon molecules. Therefore, they could easily be damaged by OH radicals. (4) Very strong OH-scavenging activity was observed in seven organic solvents, including diethyl ether, DMF, DMSO, ethanol, ethyl acetate, methanol, and THF (Table 2). Except for diethyl ether (which is still quite soluble), all of these solvents are miscible with water. All of these solvents contain at least one O atom, which forms various active chemical bonds (e.g. C–O, H–O, and S@O) or functional groups (e.g. hydroxyl, ester, amide, sulfinyl, and cyclic ether groups). These reactive chemical bonds or functional groups (McMurry, 2003, chap. 2), are responsible for the strong hydroxylradical-scavenging ability for these solvents. Importantly, DMSO, which is often used in sample solution preparation for biochemistry or animal experiments, displayed a very strong scavenging ability (IC50 = 5.76 ± 0.76 ll, Table 2). The results agree with previous studies showing that DMSO can be a hydroxyl scavenger
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Table 1 Results of deoxyribose assay of 25 selected antioxidants determined in ethanol solution and as residue in buffer (after removing ethanol). NO
Antioxidant
Resource and characterisation
IC50 value (lg/ml) Ethanol solution
(+)-Catechin BHA Caffeic acid Curcumin Ferulic acid GSH 8-Hydroxyl quinoline deriv. Isoferulic acid Maclurin Magnolol MEAS MEGB Melatonin Proanthocyanidin Protocatechuic acid Puerarin Pyridoxine Quercetin Rosmarinic acid Rutin Sinapic acid Thymol (±) a-Tocopherol Trolox Uracil
Exogenous, natural, polyphenol Exogenous, synthetic, phenol Exogenous, natural, phenolic acid Exogenous, natural, phenol Exogenous, natural, phenolic acid Endogenous, polypeptide Exogenous, synthetic, heterocyclic phenol Exogenous, natural, phenolic acid Exogenous, natural, polyphenol Exogenous, natural, phenolic phenylpropanoid Exogenous, natural,extract natural,extract Exogenous, natural,extract Endogenous, heterocyclic phenol Exogenous, natural, proanthocyanidins Exogenous, natural, phenolic acid Exogenous, natural, flavonoid glycoside Exogenous, natural, vitamin Exogenous, natural, flavonoid Exogenous, natural, polyphenol Exogenous, natural, flavonoid glycoside Exogenous, natural, phenolic acid Exogenous, natural, phenol Exogenous, natural, vitamin Exogenous, synthetic, vitamin analogue Endogenous, base pair, heterocyclic phenol Average
19.7 ± 0.15 8.69 ± 0.05a 20.4 ± 0.71a 9.46 ± 0.94a 17.3 ± 0.23a 17.1 ± 0.08a 9.93 ± 1.34a 17.0 ± 0.09a 9.27 ± 1.24a 11.6 ± 3.65a 32.8 ± 4.47a 44.9 ± 3.66a 8.96 ± 0.39a 14.5 ± 2.20a 8.32 ± 0.87a 8.97 ± 0.95b 9.82 ± 0.95b 21.9 ± 1.35a 9.84 ± 0.11a 8.89 ± 0.03a 7.09 ± 0.23a 7.66 ± 0.22 a 45.5 ± 5.71a 17.3 ± 0.13a 14.0 ± 0.13a –
b
281 ± 1.55 24.7 ± 0.15b 285 ± 2.14b 187 ± 5.82b 360 ± 1.27b 126 ± 3.27b 35.7 ± 1.45b 214 ± 1.76b 74.9 ± 2.82b 49.1 ± 4.42b 310 ± 10.21b 782 ± 20.55b 49.6 ± 1.45 b 89.1 ± 3.52b 66.9 ± 2.88b 54.0 ± 4.84b 98.1 ± 6.55b 267 ± 1.38b 62.2 ± 3.56b 37.8 ± 0.89b 17.7 ± 0.15b 103 ± 5.68 b 108 ± 4.35b 89.4 ± 3.01b 80.1 ± 2.89b –
14.3 2.84 14.0 19.7 20.9 7.39 3.60 12.6 8.06 4.25 9.46 17.4 5.53 6.14 8.03 6.02 9.98 12.2 6.32 4.25 2.50 13.3 2.37 5.17 5.73 8.88
Validation for improved method (using residue) Regression equation
R value
RSD for IC50 (%)
y = 1.49 + 0.57x y = 38.84 + 0.47x y = 5.00 + 0.55x y = 6.29 + 0.23x y = 2.32 + 0.24x y = 7.75 + 1.02x y = 4.46 + 1.28x y = 19.31 + 0.59x y = 9.19 + 0.54x y = 20.63 + 0.59x y = 12.37 + 0.12x y = 1.42 + 0.062x y = 7.32 + 0.83x y = 10.23 + 0.45x y = 9.79 + 0.60x y = 0.28 + 0.92x y = 8.06 + 0.45x y = 4.01 + 0.29x y = 7.34 + 0.68x y = 36.56 + 0.35x y = 14.34 + 1.06x y = 4.43 + 0.44x y = 0.83 + 0.48x y = 30.30 + 0.79x y = 5.30 + 0.56x –
0.99887 0.95652 0.99007 0.84743 0.98362 0.97548 0.90437 0.93745 0.91785 0.80304 0.87812 0.97781 0.97684 0.88533 0.89638 0.99956 0.87701 0.99055 0.96307 0.99679 0.83259 0.95339 0.94287 0.92516 0.92354 0.93335
0.55 0.61 0.75 3.11 0.35 2.58 6.86 0.82 3.77 9.07 3.29 2.67 2.92 3.95 4.31 8.97 6.68 0.52 5.72 2.35 0.85 2.84 4.04 3.36 3.60 3.38
IC50 value is defined as the concentration of 50% hydroxyl radical inhibition and expressed as Mean ± SD (n = 3). The linear regression was analysed by Origin 6.0 professional software. BHA, butylated hydroxyanisole. GSH, glutathione. MEAS, methanol extract of Aquilaria Sinensis leaves. MEGB, methanol extract from Gynura bicolor Roxb. DC. Mean values with different superscripts in the same row are significantly different (p < 0.05). Ratio value = IC50,residue:IC50,ethanolsolution. RSD = SD Mean 100%. Their dose–response curves were shown in Supplementary material 1.
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
a
Ratio value Residue
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X. Li / Food Chemistry 141 (2013) 2083–2088 Table 2 The IC50 values of 18 pure organic solvents by deoxyribose degradation assay. Solvent Ethanol Methanol THF DMF Diethyl ether Ethyl acetate DMSO Acetone Carbon disulphide Acetonitrile
IC50 (ll in 800 ll) a
1.86 ± 0.13 2.11 ± 0.25a 3.08 ± 0.89b 4.25 ± 0.69b 4.17 ± 1.06c 5.44 ± 1.28d 5.76 ± 0.76d 12.7 ± 0.33e 15.3 ± 0.86f 17.5 ± 1.67g
Type Very strong Very strong Very strong Very strong Very strong Very strong Very strong Strong Strong Strong
Solvent Chloroform Carbon tetrachloride Dichloromethane Toluene Benzene Cyclohexane Petroleum ether n-Hexane
IC50 (ll in 800 ll) h
26.0 ± 2.44 47.0 ± 3.56i 49.0 ± 3.52j 57.7 ± 3.54k 57.9 ± 7.66k 66.9 ± 5.99l 119 ± 5.56m 407 ± 13.3n
Type Moderate Moderate Moderate Moderate Moderate Moderate Weak Weak
The IC50 value was defined as the volume (ll) of 50% hydroxyl radical inhibition in the 800 ll reaction system, and was expressed as Mean ± SD (n = 3). The linear regression was analysed by Origin 6.0 professional software. Mean values with different superscripts (a-g) are significantly different (p < 0.05) and those with same letters are not significantly different (p < 0.05). THF, tetrahydrofuran; DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide. Their dose–response curves were shown in Supplementary material 2.
Fig. 3. The recommended procedure of the improved dexyribose degradation assay for hydroxyl radical-scavenging.
(Doroshow, 1986; Repine, Pfenninger, Talmage, Berger, & Pettijohn, 1981) and could prevent DNA nicking by hydroxyl radicals (Fang & Zheng, 2002a, chap. 5). The solvent effect on the assay results arises from the hydroxylradical-scavenging ability of organic solvents themselves and can be ultimately attributed to the extreme reactivity of the hydroxyl radical. It has been demonstrated that hydroxyl radicals, once generated, will quickly react with surrounding solvent molecules (Fang & Zheng, 2002b, chap. 2; Halliwell & Gutteridge, 1984). For example, the reaction rates of OH with ethanol and DMSO were respectively 1.9 109 l mol1 s1 (Fang & Zheng, 2002b, chap. 2) and 9 109 l mol1 s1 (Nakai, Kadiiska, Jiang, Stadler, & Mason, 2006). The product of OH reaction with DMSO is CH3 (Chan et al., 1989). In a word, any organic solvent should be completely removed before measurement via the deoxyribose degradation assay, or the results will be meaningless. However, evaporation temperatures should remain below 60 °C to prevent the antioxidant samples from being destroyed. In our experiment, we found that all sample residues could be well dissolved in the aqueous reaction system. Most antioxidants are organic compounds and their water solubilities are not generally high enough to prepare sample stock solutions (room temperature), but are generally high enough to dissolve in the reaction system (50 °C). For example, the water solubility of catechin is 2.26 mg/ml (25 °C) (Srinivas, King, Howard, & Monrad, 2010), while its IC50 value was only 0.20 ± 0.0025 mg/ml (Table 1). A few typical lipid-soluble antioxidants (e.g. BHA, thymol, and tocopherol) and both extracts (MEAS and MEGB) could also be dissolved in the reaction system. This was because: (1) these antioxidants contain phenolic –OH groups that can form hydrogen bonds with the H2O molecule, increasing the water solubility; (2) the higher reaction temperature (50 °C) increased the solubility in the aqueous system; and (3) the amount of antioxidant residues was gradually reduced by OH attack in the reaction process. Based on the discussion above, the deoxyribose degradation assay can be improved as follows: first, we choose an appropriate or-
ganic solvent to prepare the sample solution. An aliquot of the sample solution was placed in a tube and this sample solution was evaporated to dryness (water bath, 660 °C). Then, the sample residue was determined as described in Section 2.2. The whole process of improved deoxyribose degradation assay is displayed in Fig. 3. It must be emphasised that, (1), if the antioxidant sample is water-soluble, we can use water (or buffer) to prepare the sample solution. Since water (or buffer) cannot react with the hydroxyl radicals, aqueous solution samples can be measured directly; (2) if the antioxidant sample is thermo-sensitive, the sample solution should be evaporated to dryness at room temperature. The improved deoxyribose assay procedure was validated, using the reference compound (+)-catechin. The regression equation of (+)-catechin was calculated as y = 1.49 + 0.57 x (y: inhibition percentage; x: final concentration) and the correlation coefficient (R) was 0.99887 (Fig. 2). The RSD% of the IC50 was 0.51% (Table 1) and met FDA requirements (US Food and Drug Administration, 2001). In addition to (+)-catechin, the other 24 selected antioxidants also exhibited good linearity (R = 0.80304– 0.99956, the average R value was 0.93335) and low RSD values (0.35–9.07%, the average value was 3.38%) (Table 1). Apparently, the improved method had good linearity and precision. In addition, the RSD% of IC50 values obtained on different days was 2.26% in the analysis of (+)-catechin (Supplementary material 3). This suggests that the improved method has good reproducibility. In summary, the improved deoxyribose assay (especially sample preparation) is reasonable and feasible. As can be seen in Fig. 1 and Table 1, the 25 selected antioxidants included all types of antioxidants, such as pure compounds and extracts; natural and synthetic; exogenous and endogenous, water-soluble and lipid-soluble, vitamins, vitamin analogues, and non-vitamins, phenols, polyphenols, phenolic acids, flavonoids, proanthocyanidins, phenylpropanoids, polypeptides, and heterocyclic phenols and base pairs. As the scavenging ability of all of these species could be successfully measured in our experiment, the improved method proved to be suitable for all types of antioxidants.
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