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2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO•) Radical-scavenging: A New and Simple Antioxidant Assay in vitro Xican Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02247 • Publication Date (Web): 10 Jul 2017 Downloaded from http://pubs.acs.org on July 10, 2017

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Journal of Agricultural and Food Chemistry

2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO•) Radical-scavenging: A New and Simple Antioxidant Assay in vitro Xican Li a,b,* a School

of Chinese Herbal Medicine, b Innovative Research & Development Laboratory of TCM, Guangzhou University of Chinese Medicine, Guangzhou, China.

E-mail address:

[email protected]

Postal addresses:

No.232, Waihuan East Road, Guangzhou Higher Education Mega Center,

Panyu District, Guangzhou, 510006, China Homepage:

http://www.researchgate.net/profile/Xican_Li

Tel:

+86 20 39358076

Fax:

+86 20 38892697

Running title: PTIO• Radical-scavenging Assay Article type: Original Articles

1

ACS Paragon Plus Environment


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ABSTRACT

2

Current in vitro antioxidant assays have several limitations, which frequently cause inconsistent

3

results.

4

2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide radical (PTIO•). After the investigation

5

of various factors, the experimental protocol was briefly recommended as follows: PTIO• and the

6

sample solution were added to phosphate buffer (pH 7.4, 50 mM), incubated at 37°C for 2 hours,

7

and then spectrophotometrically measured at 557 nm. The validation test based on 20 pure

8

compounds and 30 lyophilized-aqueous-extracts suggested that PTIO•-scavenging had a good

9

linear relationship, stability, and reproducibility. In the UPLC-ESI-Q-TOF-MS/MS analysis, PTIO•

10

was observed to give m/z 234 when encountering L-ascorbic acid. As an antioxidant assay,

11

PTIO•- scavenging possesses four advantages, i.e., oxygen-centered radical, physiological

12

aqueous solution, simple and direct measurement, and less interference from tested-sample. It

13

can also satisfactorily analyze the antioxidant structure-activity relationship. PTIO•-scavenging

14

has no stereo-specificity and is at least involved in H+-transfer.

The

study

develops

a

new

antioxidant

assay

using

15 16

Keywords: Antioxidant assay; PTIO•-scavenging; Oxygen-centered radical; Analytical method;

17

2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide; H+-transfer

18

2


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1. Introduction

20

Excessive ROS levels are known to be harmful to major biomolecules in cells. ROS-scavenging

21

thus plays a critic role in the fields of food, chemistry, medicine, nutrition, pharmacology,

22

toxicology, and traditional Chinese medicine. Nowadays, various antioxidant assays have now

23

been developed to characterize ROS scavenging levels. However, these assays frequently result in

24

inconsistent experimental results. This inconsistency may be attributed to five factors: indirect

25

measurements, non-oxygen-centered radicals, solvent effects, short wavelength determinations,

26

and limitation of experimental principle 1-3.

27

Ideally, an antioxidant assay should be based on direct scavenging of ROS. However, typical

28

ROS forms are transient and have a very short half-life, e.g., hydroxyl radicals (•OH, 10-9 s),

29

superoxide radicals (•O2-, 10-6 s), and lipid-peroxide radicals (LOO•, 10-2 s). These radicals can be

30

trapped only by specific electron spin resonance (ESR) spectroscopy at an extremely low

31

temperature. However, ESR spectroscopy cannot quantitatively evaluate the ROS-scavenging

32

level of an antioxidant 4. Thus, regular analytical technologies (e.g., spectrophotometry, HPLC,

33

and fluorimetry) are used to indirectly measure the ROS-scavenging level. For example, the

34

analysis of deoxyribose degradation can characterize •OH radical scavenging 5; the measurement

35

of pyrogallol auto-oxidation can reflect •O2- radical scavenging 6; and the determination of

36

malondialdehyde (MDA) amounts reflects LOO• radical scavenging

37

measurements undoubtedly introduce uncertainty into the evaluation of ROS scavenging.

7.

These indirect

38

On the other hand, some direct radical-scavenging assays are not based on oxygen-centered

39

radicals. For example, 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH•) and 2,2'-azino-bis

40

(3-ethylbenzthiazoline-6-sulfonic acid) radical ion (ABTS+•), which are widely used for in vitro

41

antioxidant assays, are nitrogen-centered radicals. Hence, the DPPH• assay and the ABTS•+ assay

42

are preferred for "reactive nitrogen species (RNS) scavenging" models, not "ROS scavenging"

43

models. In a radical adduct formation (RAF)-based antioxidant process 8, the difference between

44

RNS scavenging and ROS scavenging will be enlarged. An O atom in a phenolic antioxidant can

45

link a N atom in RNS to form a stable O-N σ-bond and hardly link another O to yield a O-O σ-bond.

46

A superoxide bond (O-O) is well known to be very unstable. Thereby, it is scientifically unsound

47

to use an RNS scavenging model to estimate the ROS-scavenging level of a phenolic antioxidant.

48

A phenolic antioxidant may also undergo a hydrogen transfer pathway to scavenge ROS. The 3



49

so-called hydrogen transfer actually includes several subtypes, i.e., proton loss single electron

50

transfer (SPLET) 9, hydrogen atom transfer (HAT) 10-11, proton-coupled electron transfer (PCET)

51

12,

52

proton (H+). Thus, the pH value of the experimental solution can cause strong interference 6, 14,15.

53

Moreover, because the protonation extents of the solvents are different from one another, there

54

is a great solvent effect, i.e., an antioxidant may present different ROS-scavenging activities in

55

some antioxidant assays among different solvents

56

have considerably different DPPH•- scavenging rate constants in ethanol (k=95.1 M-1 s-1) and in

57

ethyl acetate (k=1.6 M-1 s-1) 17.

sequential electron proton transfer (SEPT)

13,

and so on. Most of these transfers involve the

5,13-16.

For example, resveratrol was shown to

58

Finally, the background absorbance of tested samples can also disturb the assay. For

59

example, an H2O2-scavenging assay based on a redox reaction is performed under short

60

wavelength (λ= 230 nm) 18. A galvinoxyl radical-scavenging assay, however, is carried out using

61

432 nm 1. The background absorbance from the tested samples may easily interfere with such

62

short wavelength absorbances.

63

Therefore, it is vital to search for a stable, hydrophilic, oxygen-centered radical to evaluate

64

ROS-scavenging levels. The 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical

65

(PTIO•), a species currently used to detect NO levels 19-20, is hypothesized to be such a candidate

66

in the present study. As shown in Fig. 1A, the unpaired electron is located in the O atom, and thus,

67

it is an oxygen-centered radical; the amine oxide zwitterion moiety makes it a hydrophilic species.

68

A benzene ring linked to a C=N double bond is thought to produce a large π-π conjugative system

69

with long wavelength absorbance. Thus, as a navy blue powder, PTIO• is stable under usual

70

temperature. It's scavenging may be easily and directly detected by a spectrophotometer.

71

The present study therefore attempts to develop PTIO• scavenging as a new in vitro

72

antioxidant assay. To the best of our knowledge, this is the first report regarding the application

73

of PTIO• in the field.

74 75

2. Materials and methods

76

2.1 Materials

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PTIO• (CAS 18390-00-6, >98.0%), catechol and glutathione (GSH) were purchased from TCI

78

Chemical Co. (Shanghai, China). Trolox (±-6-hydroxyl-2,5,7,8-tetramethlychromane-2-carboxylic 4


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acid) was obtained from Sigma-Aldrich Shanghai Trading Co. (Shanghai, China). D-ascorbic acid

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and melatonin were from J&K Scientific (Beijing, China). Proanthocyanidin, chlorogenic acid,

81

sodium nitroprusside (SNP), and resveratrol were purchased from Aladdin Chemistry Co.

82

(Shanghai, China). Caffeic acid and ferulic acid were purchased from the National Institute for the

83

Control of Pharmaceutical and Biological Products (Beijing, China). Ellagic acid and gallic acid

84

were purchased from Guangdong Guanghua Chemical Plants Co., Ltd. (Shantou, China). Quercetin,

85

sinapine and daidzein were obtained from Sichuan Weikeqi Biological Technology Co., Ltd.

86

(Chengdu, China). 3,5-Dicaffeoylquinic acid, baicalein, scutellarein, and baicalin were purchased

87

from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). (+)Catechin and L-ascorbic acid

88

were from Guangzhou Chemical Reagent Factory (Guangzhou, China). All other chemical reagents

89

were of analytical grade. Thirty lyophilized aqueous extracts from medicinal or edible plants were

90

prepared in our laboratory (Suppl. 1).

91 92

2.2 UV spectra of PTIO•

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PTIO• radicals were dissolved in distilled water at concentrations of 1.1 and 2.2 mmol/L. The

94

aqueous solutions were scanned from 300-1000 nm by a UV/Vis spectrophotometer (Jinhua 754

95

PC, Shanghai, China). The above experimental protocol was repeated using methanol and DMSO

96

instead of distilled water.

97 98 99 100

2.3 Solvent effect In the experiment, we used (+)catechin, chlorogenic acid, and proanthocyanidin as reference compounds. The initial experimental protocol was based on the DPPH assay 21.

101

In the (+)catechin comparative experiment between different solvents, a (+)catechin

102

aqueous solution was added to a PTIO• aqueous solution, and then, the solution was thoroughly

103

mixed. After it was incubated at room temperature for 30 min, the absorbance of the mixture was

104

determined at 557 nm using a UV/Vis spectrophotometer (Jinhua 754 PC, Shanghai, China). The

105

percentage of PTIO• inhibition was calculated using the following formula:

106

107

Inhibition % =

A0 − A × 100 % , A0

where A0 is the absorbance without sample, and A is the absorbance with sample. 5



108

For comparison, a (+)catechin methanolic solution was added to a PTIO• methanolic solution,

109

and then, the absorbance was determined at 586 nm. Additionally, a (+)catechin DMSO solution

110

was mixed with a PTIO• DMSO solution and the absorbance was then measured at 584 nm. The

111

percentages of PTIO• inhibition of (+)catechin were similarly calculated using the above formula.

112 113

The above comparative experiments were repeated using chlorogenic acid and proanthocyanidin.

114 115

2.4 Stability and reaction time test

116

To study the stability of the PTIO• radical, a 1.0 mmol/L PTIO• aqueous solution was

117

kinetically monitored at 557 nm for 20 hours at room temperature using a UV/Vis

118

spectrophotometer (Jinhua 754 PC, Shanghai, China). To determine the best reaction time, the

119

reaction mixtures of PTIO• with (+)catechin (0.25 and 0.5 mg/mL), chlorogenic acid (0.25 and 0.5

120

mg/mL), and proanthocyanidin (0.25 and 0.5 mg/mL) were kinetically monitored at 557 nm for

121

20 hours at room temperature.

122 123

2.5 Temperature and irradiation effect

124

Similarly, (+)catechin, chlorogenic acid, and proanthocyanidin were used as reference

125

compounds in the experiments. Each reference compound (in an aqueous solution) was added to

126

a PTIO• aqueous solution and mixed thoroughly. After incubation in a 37°C water-bath for 2

127

hours, the absorbance of the mixture was determined at 557 nm using a UV/Vis

128

spectrophotometer (Jinhua 754 PC, Shanghai, China). The percentage of PTIO• inhibition was

129

calculated using the above formula. The above experiment was repeated at 15°C, 25°C, 45°C, and

130

55 °C.

131

To explore the irradiation effect between irradiation and non-irradiation, each of the three

132

reference compounds was comparatively determined at 37°C in aqueous solution for 2 hours.

133

The UV irradiation was carried out using a ZXF-LCA UV-light catalytic reactor (Zhengxin

134

Instrument Factory, Binhai, China.)

135 136 137

2.6 pH value effect Considering the complexity of the pH effect, 20 common antioxidants (C1-C20, Fig. 2 & 6


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Table 1) were selected for the investigation, including (+)catechin, chlorogenic acid,

139

proanthocyanidin, caffeic acid, ferulic acid, GSH, (+)ascorbic acid, (-)ascorbic acid, ellagic acid,

140

gallic acid, Trolox, resveratrol, and quercetin. The experiment was conducted without UV

141

irradiation, and the distilled water previously used was replaced by a 50 mM phosphate buffer at

142

pH 5.0, 6.0, 7.4, 8.0, and 9.0. Then, the percentages of PTIO• inhibition were calculated using the

143

above formula.

144 145

2.7 Validation of the PTIO•-scavenging assay

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In the validation experiment, 20 pure compounds (C1-C20, Fig. 2 & Table 1) and 30 plant

147

extracts (lyophilized powders, E1-E30, Table 1) were investigated using the following protocol:

148

PTIO• radicals were dissolved in phosphate buffer (pH 7.4, 50 mM) at approximately 0.05 mg/mL

149

and then increased by sample solution. The total volume of the reaction mixture was adjusted by

150

the buffer. After being thoroughly mixed, the reaction solution was incubated at 37°C in a

151

water-bath without UV irradiation for 2 hours, and then, the absorbance was measured at 557 nm.

152

The tested sample could be prepared using distilled water, buffer, or organic solvents. According

153

to the A 557nm value, the PTIO• inhibition percentages were calculated, and dose response curves

154

were created.

155

On the basis of the dose response curves, the linearity of the method was evaluated at five

156

concentrations. A calibration curve was prepared by plotting the mean inhibition percentages of

157

triplicate analyses against the final concentrations. The precision was evaluated using the relative

158

standard deviation (RSD %) inhibition percentages of the triplicate analyses. In addition,

159

reproducibility was also assessed using FDA guidance

160

guidance were used to validate the PTIO•-scavenging assay in the study.

22.

The three parameters based on FDA

161 162

2.8 UPLC-ESI-Q-TOF-MS/MS analysis for the reaction product of PTIO• with SNP and ascorbic acid

163

Four milligrams of SNP (or L-ascorbic acid) was added to 1 mL of a PTIO• aqueous solution (4

164

mg/mL). After being ultrasonically incubated for 20 min, the mixture was left standing for 24 hr.

165

The product mixture was diluted 40 times and then filtered using a 0.22-μm filter and further

166

analyzed using a UPLC-ESI-Q-TOF-MS/MS system equipped with a C18 column (2.1 mm i.d. × 100

167

mm, 1.6 μm, Phenomenex, USA). The mobile phase was 100% methanol with a flow rate of 0.2 7



168

mL/min. The injection volume was 3 μL and was applied for separation. Q-TOF-MS/MS was

169

performed on a Triple TOF 5600plus Mass spectrometer (AB SCIEX, Framingham, USA) equipped

170

with an ESI source. The scan range was 100-2000 Da. The following parameter settings were

171

used: ion spray voltage, +4500 V; ion source heater, 550°C; curtain gas (CUR, N2), 30 psi;

172

nebulizing gas (GS1, Air), 50 psi; and Tis gas (GS2, Air), 50 psi. The declustering potential (DP)

173

was +100 V; the collision energy (CE) was +40 V with a collision energy spread (CES) of 20 V.

174 175

For comparison, 1 µL of a PTIO• aqueous solution (50 μg/mL) was also injected for the analysis using the above conditions.

176 177

2.9 Statistical analysis

178

Each experiment was performed in triplicate, and the data were recorded as the mean±SD

179

(standard deviation). The IC50 value for each antioxidant was defined as the final concentration

180

needed for 50% PTIO• inhibition. Statistical comparisons were made by one-way ANOVA to

181

detect the significant difference using SPSS 13.0 (SPSS Inc., Chicago, IL) for Windows. P < 0.05

182

was considered to be statistically significant.

183 184

3 Results

185

3.1 UV spectra

186 187

The UV spectra of PTIO• (Fig. 3) suggested that PTIO• showed a long wavelength absorbance at 557 nm, 586 nm, and 584 nm in water, methanol, and DMSO, respectively.

188

According to the maximum absorbance values and the PTIO• concentrations, the molar

189

absorption coefficients (ε) of PTIO• were calculated as 1022 L mol-1 cm -1, 677 L mol-1 cm -1, and

190

477 L mol-1 cm-1 in water, methanol, and DMSO, respectively.

191 192

3.2 Solvent effects

193

The dose response curves are plotted in Suppl. 2. It was observed that each reference

194

compound exhibited a dose-dependent effect; however, the IC50 values varied with the types of

195

solvents. In general, the lowest IC50 values were observed in the aqueous solution, while

196

intermediate IC50 values were found in the methanol solution, and the highest IC50 values were

197

found in the DMSO solution (Fig. 4). This result strongly indicated a solvent effect. 8


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198 199

3.3 Stability and reaction time test The A 557 nm decay of the PTIO• aqueous solution (1 mg/mL) within 20 hours is shown in Fig. 5.

200 201

The fact that the A

202

that the A

203

that the PTIO• aqueous solution can be stored at room temperature for at least 20 hours, and the

204

reaction of PTIO• with an antioxidant can reach a balance within 2 hours.

557 nm

557 nm

value of the PTIO• aqueous solution remained stable for 20 hours and

value of the PTIO• reactant reached a stable minimum within 2 hours indicates

205 206

3.4 Temperature and irradiation effect

207

In the temperature effect experiment, each of the three reference compounds had a

208

dose-dependent effect (Suppl. 3). Nevertheless, the IC50 values of each reference compound

209

decayed with the reaction temperature. This finding suggests that a higher temperature

210

accelerated the PTIO• scavenging reaction. According to the temperature-IC50 curves, we

211

conducted a first order exponential decay fit, in which an inflection point was found at

212

approximately 37℃(Fig. 6A). In the irradiation effect experiment, however, none of the reference compounds showed a

213 214

significant (p baicalein (without 4'-OH

300

group and with a glucoside). All these results agree with previous studies about the antioxidant

301

structure-activity relationship 23-26, suggesting that PTIO•-scavenging assay can also be used for

302

the antioxidant structure-activity relationship analysis.

303

As mentioned above, PTIO• radicals are currently used to measure the levels of NO 19.

19-20.

In

304

the reaction with NO, PTIO• is reported to donate O to NO

305

UPLC-ESI-Q-TOF-MS/MS analysis of the product mixture of PTIO• and SNP, a loss of the O atom

306

was observed (m/z 234-218). However, in its reaction with L-ascorbic acid, only a loss of H/H+

307

(m/z 235-234) was found without O atom transfer. These findings suggest that PTIO• scavenging

308

by the phenolic antioxidant is involved in a hydrogen transfer, especially H+ transfer. The

309

possibility of H+-transfer is further supported by the abovementioned pH effect and solvent effect.

310

As illustrated in Table 1, 20 reference compounds (C1-C20) similarly presented the weakest

311

PTIO• scavenging at pH 5.0 or 6.0, regardless of whether they had an acidic –COOH group or an

312

alkaline N atom. This finding means that under a lower pH value, a higher proton (H+) 12


In fact, in our

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313

concentration in the solution can considerably inhibit H+-transfer-based PTIO• scavenging. In the

314

context of the solvent effect, three reference compounds (C1-C3) decreased their PTIO•

315

scavenging levels in the order of water>methanol>DMSO (Fig. 4). This sequence is parallel with

316

their extents of protonation (water>methanol>DMSO) but not with their polarity indexes: water

317

(10.2) >DMSO (7.2) >methanol (5.1). In short, our new observations about the pH effect, the

318

solvent effect, and the UPLC-ESI-Q-TOF-MS/MS analysis further support the previous hypothesis

319

involving H+-transfer

320

happen early or late. Thus, there are three possibilities in PTIO• scavenging, i.e., PCET, SPLET, or

321

SEPT subtypes. However, further work is required to precisely identify which subtypes are

322

involved.

19.

Electron-transfer (redox reaction), accompanied by H+-transfer, should

323

As shown in Table 1 and Suppl. 4, two stereoisomers, D-ascorbic acid and L-ascorbic acid,

324

did not present a significant (p

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