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Rapid and efficient conversion of all-E-astaxanthin to 9Z- and 13Zisomers and assessment of their stability and antioxidant activities Cheng Yang, Lianfu Zhang, Hua Zhang, Qingrui Sun, Ronghua Liu, Jing Li, Leiyan Wu, and Rong Tsao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04962 • Publication Date (Web): 10 Jan 2017 Downloaded from http://pubs.acs.org on January 14, 2017

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Journal of Agricultural and Food Chemistry

Rapid and efficient conversion of all-E-astaxanthin to 9Z- and 13Z-isomers and assessment of their stability and antioxidant activities

Cheng Yang1, 2, Lianfu Zhang1,3,4,*, Hua Zhang2, Qingrui Sun1, 5, Ronghua Liu2, Jing Li1, Leiyan Wu6, and Rong Tsao2,*

1

School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, China

2

Guelph Research and Development Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph, Ontario N1G 5C9, Canada

3

State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, China

4

National Engineering Research Center for Functional Food, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, 214122, China

5

College of Food Science, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang 163319, China

6

College of Food Science and Engineering, Jiangxi Agricultural University, Nanchang 330045, Jiangxi, China

*

Corresponding authors:

Rong Tsao, Tel: +1 226 217 8180. Fax: +1 226 217 8183. E-mail: [email protected] Lianfu Zhang, Tel: +86-510-85917081. Fax: +86-510-85917081. E-mail:[email protected]

1

ACS Paragon Plus Environment


1

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ABSTRACT

2

An optimized isomerization method was developed by heating all-E-astaxanthin in ethyl

3

acetate (70 °C) with I-TiO2 catalyst, yielding 22.7% and 16.9% of 9Z- and 13Z-astaxanthin,

4

respectively in 2 h, with 92-95% purity after semi-preparative HPLC purification.

5

13Z-Astaxanthin had higher antioxidant activity than all-E- and 9Z-astaxanthins in oxygen

6

radical absorbing capacity assay for lipophilic compounds (ORAC-L), photochemiluminescence

7

(PCL) and cellular antioxidant activity (CAA) assays and 9Z-astaxanthin was higher in DPPH

8

radical-scavenging activity assay and lower in CAA assay. All isomers were relatively stable

9

between pH 2.0 - 11.6, except 13Z- and 9Z-astaxanthins at pH 2.0, suggesting they may be

10

converted after passing the gastric phase in vivo. Metal ions did not significantly (p < 0.05) affect

11

the stability. Results of the current study provides a means for further study into the mechanisms

12

related to in vivo transformation and bioavailability of Z-astaxanthins, and their application in the

13

development of functional foods and nutraceutical products.

14 15

KEYWORDS:

All-E-astaxanthin,

9Z-astaxanthin,

13Z-astaxanthin,

16

semi-preparative purification, antioxidant activity, stability, pH effect, metal ion

2


isomerization,

Page 3 of 36

17


INTRODUCTION

18

Astaxanthin (3, 3′-dihydroxy-4, 4′-dione-β, β′-carotene is one of the xanthophyll

19

carotenoids that widely occurs in shrimp, crab, Phaffia rhodozyma and certain algae such as

20

Haematococcus pluvialis and Chromochloris zofingiensis.1-3 It is one of the most important

21

antioxidants with many physiological functions including protection against skin cancer, diabetes

22

and macular degeneration, prevention of cardiovascular disease, improving immune system and

23

preventing neurodegenerative disorders.3 The predominant geometrical isomer of naturally

24

occurring astaxanthin is all-trans (all-E)-astaxanthin. Although all-E-astaxanthin has been the

25

subject of many studies, recent research showed that its 9Z - and 13Z- astaxanthin isomers were

26

selectively absorbed into human plasma over all-E-astaxanthin after oral administration with an

27

algae beverage rich in all-E-astaxanthin.4 The same study also showed that plasma concentration

28

of 13Z-astaxanthin was higher than 9Z-astaxanthin. More interestingly, Z-astaxanthin isomers,

29

especially 9Z-astaxanthin, have been found to exhibit higher antioxidant potential in vitro than

30

all-E-astaxanthin by DPPH (2,2-diphenyl-1-picrylhydrazyl) and lipid peroxidation assays.5

31

However, despite these findings, the physicochemical properties of Z-astaxanthin isomers and

32

their uptake mechanism inside human gastrointestinal and the metabolic systems are still

33

unknown.4-7 It is not clear how and where the Z-isomers of astaxanthin are formed in vivo, and

34

whether or not the higher antioxidant activity of Z-astaxanthin isomers observed in vitro using

35

DPPH method will also occur in vivo. Because the in vivo antioxidant system involves highly

36

complex biochemical processes, and no single in vitro chemical based assays can properly 3



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represent the antioxidant defense mechanism of a biological system, it is recommended that

38

multiple in vitro assays based on different mechanisms should be used to screen and assess the

39

antioxidant activity of natural antioxidants.8 However, only limited antioxidant assay systems

40

have been developed for lipophilic antioxidants like astaxanthin, especially for Z-isomers.9

41

The intriguing higher antioxidant potential of Z-astaxanthin isomers has instigated

42

interest in the health benefits and application of these Z-isomers in functional foods. One of the

43

most important limiting factors in research on different bioactivities and health benefits of E- and

44

Z-astaxanthin isomers is the shortage of sufficient amount of pure Z-astaxanthins. Isomerization

45

of all-E-astaxanthin is challenging due to its stereochemical stability and effects by other factors

46

including light, oxygen, iodine, microwave radiation, ultrasonic wave and thermal treatments,

47

organic solvents, presence of fatty acids and Cu (II) ion which affect the formation and yield of

48

different astaxanthin isomers.10-15 Several methods have been reported for obtaining

49

Z-astaxanthins, however, most of these methods still lack the conversion efficiency, or suffer a

50

long reaction period, or have difficulty in removing the catalyst after the reaction.5,

51

Thermal treatment and the use of different lighting are among the most common means used in

52

isomerization of the carotenoids. Higher temperature (50 ºC) was found to give greater

53

conversion rate of all-E-astaxanthin to Z-isomers in dimethyl sulfoxide (DMSO) than at 25 ºC

54

and 35 ºC.12 Solvent used in the reaction may also affect the yield and kinetics of the

55

isomerization process, and among the solvents tested, dichloromethane was found to be the best

56

for producing Z-isomers at 35 ºC.

12

6, 10-13

While I2 has been used as a catalyst for better yield of 4


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Z-astaxanthin isomers, in a recent report by our laboratory, Sun et al. showed that I-TiO2 was a

58

highly effective catalyst for converting all-E-lycopene to Z-lycopene isomers, particularly the

59

(5Z- lycopene).16 UV light alone or in combination with I2 has also been frequently used to form

60

Z-carotenoids but mostly for analytical and identification purposes.17,

61

isomerization methods have been reported for astaxanthin and other carotenoids, there is still

62

room for improving the efficacy in converting and isolating pure Z-astaxanthin isomers. In

63

particular, the effectiveness of using I-TiO2 catalyst, in converting all E-astaxanthin to its

64

Z-isomers is still unknown. A systematic investigation into the optimization and effects of the

65

various factors on the formation, yield and stability of Z-astaxanthins is warranted.

18

While different

66

One of the major concerns for Z-astaxanthins, particularly the 9Z - and 13Z -isomers, to

67

be successfully used in functional foods is the stability as their stereochemistry tells they are less

68

stable than all-E-astaxanthin.19 However, just like all other carotenoids, the stability of

69

Z-astaxanthins also depends on factors such as heat, pH and metal ions. Insofar, these effects

70

have only been investigated for all-E-astaxanthin.20, 21 Also, even though dichloromethane was

71

found to be the best solvent for converting all-E-astaxanthin to its Z-isomers, it has limitation for

72

food use.12 Other food quality solvents of similar polarity such as ethyl acetate need to be

73

explored for their effect on the isomerization of all-E-astaxanthin.

74

The objectives of this study were therefore to establish a rapid and effective method to

75

produce Z-astaxanthins and to examine their physicochemical properties, i.e. the effects of pH

76

and metal ions on their stability and antioxidant activities as compared to those of the naturally 5



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occurring all-E-astaxanthin. Three different chemical based assays and a cell-based antioxidant

78

assay (CAA) were used. The Z-astaxanthins produced will be used in our future study on the

79

bioaccessibility, bioavailability and metabolism of the health promoting actions in vivo. Results

80

of this study will also provide fundamental and useful information for the production and use of

81

Z-astaxanthins as functional food and even pharmaceutical ingredients.

82 83

MATERIALS AND METHODS

84 85

Chemicals and Reagents.

86

All-E-astaxanthin standard (HPLC purity > 97%) was purchased from Sigma–Aldrich

87

(Oakville, ON, Canada). Iodine, sodium acetate, potassium iodide, ferric chloride hexahydrate,

88

sodium phosphate monobasic and sodium phosphate dibasic were purchased from Caledon

89

Laboratories Ltd. (Georgetown, ON, Canada). HPLC-grade solvents, including acetonitrile,

90

methanol, dichloromethane, ethyl acetate and formic acid were purchased from EMD Chemicals

91

(Gibbstown,

92

2-diphenyl-1-picrylhydrazyl (DPPH), trolox and 2, 2'-azobis(2-amidinopropane) dihydrochloride

93

(AAPH)] were purchased from Sigma-Aldrich (Oakville, ON, Canada). Acids were of ACS

94

(American Chemical Society) grade and supplied by Fisher Scientific (Nepean, ON, Canada).

95

Distilled and deionised water was obtained in-house from a Thermo Scientific Barnstead

96

Nanopure ultrapure water purification system (Ottawa, ON, Canada).

NJ,

USA).

Reagents

for

antioxidant

6


assays

[fluorescein,

2,

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97 98

Isomerization of all-E-astaxanthin

99

Several methods are known to induce isomerization of all-E carotenoid to its Z-isomers,

100

and two of the most commonly used methods, based on heating and lighting were chosen for

101

optimal production of Z-astaxanthins.6, 12, 16-19, 22-25

102

Thermal isomerization with or without catalyst. Two temperatures (35 ºC and 70 ºC) and

103

two catalysts (I2 and I-TiO2) were tested for their effects on isomerization. Isomerization in the

104

presence of I2 was carried out based on the method of Zechmeister with slight modification. 12, 19

105

all-E-astaxanthin (1 mL, 0.02 g/L) in dichloromethane or ethyl acetate solution containing I2 (2%,

106

w/v) was incubated in a water bath for different time intervals at 35 ºC and 70 ºC, respectively.

107

Dichloromethane was a reaction medium for the lower heating at 35 ºC and ethyl acetate for 70

108

ºC for its higher boiling point. Multiple reaction vials were incubated and reaction vials were

109

taken out from the water bath for sampling at each time interval. The reaction was terminated by

110

adding Na2S2O3 solution (1 mL, 1 mol/L) to each vial to wash out the I2 by vortexing at different

111

time intervals. This was also necessary to avoid interference by I2 (absorbs at 295 nm in

112

dichloromethane) in HPLC analysis. The organic layer was filtered through a 0.22-µm

113

membrane and analyzed directly by HPLC every 30 min - 1 h till after the reaction reached the

114

equilibrium (6-40 h depending on conditions used). All experiments were done in triplicate at

115

each time interval (Table S1).

116

Similar experiments were conducted at both temperatures in the presence of an 7



117

iodine-doped titanium dioxide (I-TiO2) (10%, w/w) which was produced according to Sun et al.16

118

The reaction mixture was centrifuged at 10,000 rpm for 10 min at different time intervals to

119

remove the catalyst. Heating of the all-E-astaxanthin (1 mL, 0.02 g/L) at 70 ºC in ethyl acetate

120

without catalyst was also conducted for comparison purposes. Formation of Z-isomers was

121

monitored by HPLC as stated above. All experiments were done in triplicate.

122

Isomerization by UV light with or without iodine. An all-E-astaxanthin dichloromethane

123

solution (0.9 mL) with I2 (2.0%, w/w) or without was added to two 1-mL quartz cuvettes and

124

kept at 10 cm distance under a UV light (UVS11, Ultra-violet Products Inc., USA) at 254 nm at

125

ambient room temperature (22 °C). The cuvettes were covered with lids and sealed with plastic

126

wrap. Temperature of each cuvette was monitored by an infrared thermometer to assure the

127

variation to be within 1 ºC. A 0.3 mL aliquot was taken at each time interval for HPLC analysis

128

of the isomers. Na2S2O3 solution was added to the sample with I2 to wash out I2 before HPLC

129

detection. All experiments were carried out in triplicate.

130 131

Analysis and Identification of Astaxanthin Isomers

132

Astaxanthin isomers were analyzed using an Agilent HPLC series 1100 (Agilent,

133

Waldbronn, Germany) system consisted of a degasser, a binary gradient pump, a thermostatted

134

auto-sampler, a diode array detector (DAD) and the ChemStation software (OpenLAB CDS,

135

Version: Rev. C.01.05 [35]). Separation was performed on a Kinetex XB-C18 column (100

136

mm×4.6 mm, 2.6 µm) (Phenomenex Inc., Torrance, CA, USA). The column temperature was set 8


Page 8 of 36

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at 25 °C, and the injection volume was 10 µL and flow rate was kept at 0.7 mL/ min for a total

138

run time of 26 min. The binary mobile phase consisted of A: 0.1% formic acid in water (v/v) and

139

B: 95% methanol mixed with 5% acetonitrile (v/v). The solvent gradient was as follows: 0-2.5

140

min, 60-80% B; 2.5-17.5 min, 80–100% B; 17.5-20 min, 100% B; 20-20.5 min, 100-60% B;

141

20.5-26 min, 60%B. Peaks were detected at 470 nm. The DAD scan range was 220 to 700 nm.

142

Different astaxanthin isomers were identified by matching retention times and UV/Vis spectral

143

data with those of the standard and data reported in the literature.1, 6, 14, 26 Quantification was

144

done using a calibration curve of standard all-E-astaxanthin generated between 13.5 and 135

145

mg/L. Concentrations of Z-astaxanthin isomers were expressed in all-E-astaxanthin

146

equivalents.12

147 148

Isolation and purification of Z-astaxanthin isomers

149

Heating all-E-astaxanthin in an ethyl acetate solution containing I–TiO2 at 70 ºC for 2 h

150

was chosen as a method for semi-preparative production of Z-isomers of astaxanthin. The

151

reaction mixture was first centrifuged to remove the catalyst and then dried under a nitrogen

152

stream. The dry mixture was re-dissolved in dichloromethane: methanol (1:3, v/v), filtered

153

through 0.22-µm membrane and then separated on a Kinetex C18 semi-preparative column (5

154

µm, 250 mm x 10 mm) (Phenomenex Inc., Torrance, CA, USA). The mobile phase was the same

155

as stated above. The flow rate was set at 3.0 mL/min. Under these conditions, fractions

156

containing 9Z-astaxanthin at a retention time of 30.498 min and 13Z-astaxanthin at 30.938 min 9



157

were separately collected. Fractions with a single peak were pooled and dried under a nitrogen

158

stream.

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159 160

Oxygen radical absorbing capacity for lipophilic compounds (ORAC-L) assay

161

The ORAC-L assay was conducted according to reported protocols with slight

162

modifications.27 Briefly, purified astaxanthin isomers or trolox was dissolved in DMSO and then

163

mixed in 7% randomly methylated beta-cyclodextrin (RMCD; w/v) in acetone: water 1:1 (v/v)

164

according to previously established protocol.28 To each well of the black polystyrene 96-well

165

microplate (Greiner Bio-One GmbH, Frickenhausen, Germany), 25 µL of an appropriately

166

diluted sample, blank or a series of trolox standard solutions (6.25, 12.5, 25, 50 and 100 µmol/L)

167

were added and mixed with 150 µL of fluorescein working solution (8.68 × 10-5 mmol/L

168

phosphate buffer, pH 7.4), and incubated for 30 min at 37 °C. Subsequently, 25 µL of AAPH

169

(153 mmol/L in phosphate buffer) was added to each well to initiate the reaction. Fluorescence

170

(excitation and emission wavelengths set to 485 nm and 520 nm, respectively) was read every

171

minute for 120 min in a fluorescence plate reader equipped with an automatic thermostatic

172

holder (PLX 800, Bio-Tek Instruments, Inc., Winooski, VT, USA). A calibration curve was

173

constructed daily by calculating differences of area under the fluorescein decay curve between

174

the blank and the sample. The results were expressed as µmol trolox equivalent per milligram

175

astaxanthin isomer sample (µmol TE/mg isomer) (r2 = 0.996).

176 10


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Radical scavenging activity (DPPH) assay

178

The DPPH radical-scavenging activity of the astaxanthin isomers was determined

179

spectrophotometrically on a UV/Vis plate reader (EL 340, Biotek, Inc., Winooski, VT, USA)

180

according to the procedure described by Yao et al. with slightly modifications.27 Briefly,

181

appropriately diluted samples (40 mg/L) or trolox solutions (62.5, 125, 250, 500, 750, and 1000

182

µM) were added to 200 µL of 350 µM DPPH solution (in methanol) in wells of a flat bottom

183

96-well plate (Nalge Nunc International, Denmark). To avoid the interference by astaxanthin

184

isomers (major wavelength at 470 nm) in DPPH detection at 517 nm, wells with 25 µL sample

185

solution and 200 µL methanol were set as blanks. Then the mixtures were incubated for 6 h in

186

the dark at room temperature before read at 517 nm for the absorbance of DPPH radicals. All

187

samples were tested in triplicate. The scavenging of DPPH radical was calculated by the standard

188

curve of trolox (r2 = 0.994) and expressed as µmol Trolox equivalent per milligram of

189

astaxanthin isomer (µmol TE/mg isomer).

190 191

Photochemiluminescence (PCL) assay

192

The PCL assay, which is based on the photo-induced chemiluminescence of luminol, was

193

conducted according to protocols reported by Zhang et al. using the PHOTOCHEM device and

194

system (Analytic Jena AG, Jena, Germany) with minor modifications.29 Briefly, 1.5 mL of

195

reagent 1 (sample solvent), 1.0 mL of reagent 2 (reaction buffer), 25 µL of diluted reagent 3

196

(luminol), and 5-30 µL reagent 4 (trolox) were mixed together for the calibration curve. To 11



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measure the antioxidant activity of the sample, reagent 4 was replaced by a sample solution. In

198

this system, luminol works as a photosensitiser which generates superoxide radicals and a

199

chemiluminogenic probe for free radicals. The antioxidant activities of different isomers were

200

calculated based on their inhibitory ability on luminescence generation and expressed as nmol

201

trolox equivalent per gram of astaxanthin isomers (µmol TE/g) (r2 = 0.994).

202 203

Cell Culture

204

The Caco2-BBe1 (clone of Caco-2) and HT-29 human intestinal cell lines were

205

purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). Caco2-BBe1

206

cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) medium (Invitrogen Life

207

Technologies Inc., Burlington, ON, Canada) with 10% fetal bovine serum (FBS; HyClone,

208

Invitrogen Life Technologies Inc., Burlington, ON, Canada) and 50 units/mL of

209

penicillin-streptomycin (Invitrogen Life Technologies Inc., Burlington, ON, Canada), and

210

incubated at 37 °C in 5% CO2 with fresh media replacements every 2-3 days. Cells between

211

passages 40 - 50 were used in cellular antioxidant activity (CAA) experiment. HT-29 cells were

212

grown in McCoy’s 5A medium (Gibco, Invitrogen Life Technologies Inc., Burlington, ON,

213

Canada) with 10% FBS and 50 units/mL penicillin-streptomycin, and incubation conditions were

214

the same as that of Caco2-BBe1. HT-29 cells between passages 10 - 20 were used in the CAA

215

experiment.

216

Determination of cellular antioxidant activity (CAA) 12


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217

The cellular antioxidant activities of different astaxanthin isomers were determined by

218

modifying methods developed by Shi et al. and Liu et al. with modifications.9, 30 Mixed cells

219

(initial seeding ratios Caco2-BBe1/HT-29: 90/10) were co-cultured at a density of 2 × 105

220

cells/well on black 96-well microplates (with transparent bottoms) in 200 µL of DMEM medium

221

with 10% FBS and 50 units/mL of penicillin-streptomycin, and incubated for 2 days at 37 °C and

222

5% CO2 to reach confluence.31, 32 Then the growth medium was removed, and the cells were

223

washed with 200 µL of DMEM medium without FBS. Triplicate wells were treated with 0.6 µM

224

of samples (final concentration, 50 µL, dissolved in DMSO) and 50 µL of 100 µM

225

2’,7’-dichlorofluorescin diacetate (DCFH-DA, dissolved in DMSO) dissolved in DMEM

226

medium without FBS at the same time, then incubated for 1 h at 37 °C and 5% CO2.33, 34 The

227

cells were washed with 200 µL of HBSS twice and treated with 100 µL 600 µM AAPH

228

(dissolved in HBSS). Then, the 96-well microplate was quickly placed in a fluorescence plate

229

reader (PLX 800, Bio-Tek Instruments, Inc., Winooski, VT, USA) at 37 °C with emission

230

wavelength at 538 nm and excitation wavelength at 485 nm and the fluorescence generation was

231

measured every 1 min for 1 h. Each plate also included triplicate control, blank, and sample

232

background wells: control wells contained cells treated with DCFH-DA and AAPH, blank wells

233

contained cells treated with DCFH-DA without AAPH, and sample background wells contained

234

cells treated with samples and DCFH-DA without oxidant. The CAA value was calculated based

235

on the following equation:

236

CAA Unit (%) = 100 - (ʃ SA - ʃ BAs)/( ʃ CA- ʃ BAc) × 100 13



237

Where, ʃ SA is the integrated area from the sample curve, ʃ BAs is the integrated area

238

from sample background curve, ʃ CA is the integrated area from the control curve, and ʃ BAc

239

background is the integrated area from the control background curve.

Page 14 of 36

240 241

Effect of pH on the stability of astaxanthin isomers

242

Stability of the above obtained three astaxanthin isomers was studied at 37 °C at selected

243

pH levels. DMSO solution of a pure isomer (6 mg/L, 20 µL) was mixed with 0.5 mL buffer

244

solution (50 mmol/L; sodium acetate buffer for pH 2.0 and 3.5; phosphate buffer for pH 7.4 and

245

pH 11.6) and incubated at 37 °C for 2 h. Buffer solutions of pH 2.0 and pH 11.6 were prepared

246

by adding acetate acid and NaOH in acetate buffer and phosphate buffer, respectively.35 After

247

incubation, 0.5 mL dichloromethane was added to the mixture, vortexed, centrifuged for 10 min

248

and then the hypophase was filtrated through a 0.22-µm membrane for HPLC analysis. Samples

249

before incubation were set as control. All experiments were done in triplicate.

250 251

Effect of metal ions on the stability of astaxanthin isomers

252

The effect of metal ions on the above obtained astaxanthin isomers was studied following

253

the same procedure as stated above for different pH conditions. Aqueous solutions (0.5 mL, 50

254

µM) of FeCl3, CuSO4, MgSO4, CaCl2, KCl and NaCl were used instead of buffers.12, 15, 36, 37

255

Samples before incubation were set as control.

256 14


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257


Statistical analysis

258

Results were expressed as mean value ± standard deviation of three independent

259

extractions or treatments. One-way analysis of variance (ANOVA) was used to compare the

260

means. Differences were considered significant at p < 0.05. All statistical analyses were

261

performed using SPSS (Version 18.0, Chicago, IL, USA).

262 263

RESULTS AND DISCUSSION

264

Isomerization of all-E-astaxanthin

265

Isomerization and oxidation often co-occur, therefore depending on the conditions of a

266

particular reaction, the yield and specificity of the resulting isomers of all E-astaxanthin can

267

vary.19, 38, 39 Results of the preliminary experiments of the present study led us to select heating

268

and UV irradiation to be the isomerization processes for further investigation on the optimal

269

conditions for obtaining Z-astaxanthins.

270

Heating all-E-astaxanthin in dichloromethane alone at 35 ºC has been reported to produce

271

similar yield of 9Z - and 13Z - isomers when the reaction reached equilibrium after nearly 50 h,

272

although initially 13Z-astaxanthin was the dominant isomer.12 The present study showed that the

273

presence of I2 in this system could significantly shorten the time from 50 to 30 h for the reaction

274

to reach the equilibrium. Addition of I2 as a catalyst did not change the isomerization pattern, and

275

had similar conversion rate for 9Z- and 13Z- isomers at 30 h (Figure 1A). This suggests that I2

276

could be a good catalyst for the thermal isomerization process of all-E-astaxanthin in 15



277

dichloromethane. Meanwhile, I-TiO2 has been shown catalyst properties in the isomerization of

278

all-E-lycopene.16 When I-TiO2 was added to the all-E-astaxanthin/dichloromethane system and

279

heated at 35 ºC, it further shortened the time to reach equilibrium of the isomers to ca. 18 h

280

without significantly affect the maximum percentages of 9Z- and 13Z- isomers, although

281

different from the above I2 system, 9Z-astaxanthin had slightly but consistently higher percentage

282

than 13Z-astaxanthin (Figure 1B). Adding I-TiO2 to the system is therefore a significant

283

improvement over that of dichloromethane alone and that with I2. Considering its catalyst effect

284

on isomerization of all-E-lycopene and all-E-astaxanthin (Figure 1B), I-TiO2 may be a good

285

catalyst for the isomerization of other all-E-carotenoids.16

Page 16 of 36

286

Yuan and Chen also found that higher temperature could markedly promote the

287

isomerization of all-E-astaxanthin in DMSO. They found that total Z-isomers (9Z + 13Z) were

288

about 27%, ca. 10-fold higher in percentage when the solution was heated at 50 ºC than 25 ºC in

289

30 h.12 However, dichloromethane is a highly restricted and regulated solvent for natural extracts

290

(maximum residue permitted: 30 ppm) or instant beverages (maximum residue permitted: 10

291

ppm), and DMSO is not a permitted carrier or extraction solvent according to Health Canada

292

(http://www.hc-sc.gc.ca/fn-an/securit/addit/list/15-extraction-eng.php), therefore they are not or

293

at least not ideal solvents for the preparation of Z-astaxanthins for functional food or beverage

294

uses. Dichloromethane is a halogenic solvent and its use is further limited by its boiling point for

295

reaction temperatures higher than 40 ºC. For this reason, ethyl acetate (boiling point 77 ºC), a

296

solvent with significantly less restrictions in food and beverage uses was chosen for further 16


Page 17 of 36


297

improving the isomerization efficacy in the present study. An ethyl acetate solution of

298

all-E-astaxanthin was heated at 70 ºC, and as shown in Figure 1C, the percentage of all three

299

isomers reached equilibrium in less than 6 h, a highly significant improvement in reaction rate

300

over the dichloromethane system. However, higher temperature caused larger difference between

301

the relative percentages of the two Z-isomers. Heating ethyl acetate to 70 ºC produced similar

302

level of 13Z-astaxanthin (21.6%) to those in the dichloromethane (35 ºC) systems, but it

303

significantly reduced the percentage of 9Z-astaxanthin (7.4%) (Figures 1A, 1B and 1C). In the

304

presence of I2, however, 9Z - and 13Z-astaxanthins maxed at 22.9% and 17.0 %, respectively and

305

reached the equilibrium at only 2 h interval (Figure 1D). This is a drastic improvement from all

306

dichloromethane systems and the plain ethyl acetate at 70 ºC. Replacing I2 with I-TiO2 in this

307

system did not lead to significant changes in reaction rate nor percentages of all isomers at

308

equilibrium (Figure 1D and 1E). However, when examined closely, we found that degradation of

309

astaxanthin was more severe in the presence of I2. Nearly 25% of total astaxanthins (sum of all

310

three isomers) was lost at 2 h when I2 was present, but only 15% was lost in the presence of

311

I-TiO2, suggesting the latter to be an ideal catalyst (Figure 1D and 1E). Overall, I-TiO2 acted as a

312

perfect catalyst for the isomerization of astaxanthin regardless of solvent type and reaction

313

temperature.

314

Photoisomerization of carotenoids occurs under direct sunlight or ultraviolet light.38, 40

315

While UV light used in this study converted all-E-astaxanthin in dichloromethane solution to 9Z-

316

and 13Z- isomers almost immediately after irradiation at room temperature (Figure 1F), 17



Page 18 of 36

317

degradation of all-E-astaxanthin also occurred rapidly and drastically. Nearly 85% of the total

318

astaxanthin was lost to degradation after 1 min (Figure 1F). Photoisomerization by UV light was

319

also conducted in the presence of I2 but all astaxanthin isomers degraded in less than 1 min (Data

320

not shown). UV degradation of total astaxanthin might be from aggregation or polymerization of

321

the astaxanthin

322

photoisomerization by UV light has been used for quick isomerization of other carotenoids, it

323

was clearly not an appropriate method for producing Z-isomers of astaxanthin.

molecules

in

addition

to possible oxidation reactions.41

Although

324 325

Semi-preparation, Isolation and Identification of Astaxanthin Isomers

326

Z-Isomers of astaxanthin were isolated from the mixture after heating all-E-astaxanthin in

327

ethyl acetate with I–TiO2 at 70 ºC for 2 h. Identification of the Z-isomers was based on

328

spectroscopic properties and Q-ratio which was defined as the absorbance ratio of the cis peak to

329

the middle maximum absorption peak. Spectroscopic features and the Q-ratio have been

330

successfully used in the identification of all-E- and Z- carotenoids, along with matching UV/Vis

331

spectra and retention time.11, 12, 18 Chromatographic retention time, UV/Vis spectra of all-E- and

332

its 9Z- and 13Z- isomers obtained in the present study are shown in Figure 2.7, 12, 14 Their Q-ratio

333

and purity are listed in Table 1, along with data reported by others.6, 14, 26

334 335 336

Antioxidant activities The antioxidant activities of the three purified astaxanthin isomers as measured by DPPH, 18


Page 19 of 36


337

ORAC-L, and PCL are presented in Figure 3. The DPPH antioxidant activity was 5.06, 6.49 and

338

8.85 µmol TE/mg for all-E-, 13Z- and 9Z-astaxanthin, respectively. The ORAC-L activity was

339

7.65, 13.22 and 11.16 µmol TE/mg for for all-E-, 13Z- and 9Z-astaxanthin, respectively.

340

13Z-Astaxanthin exhibited the highest antioxidant activity in the PCL assay (117.01 µmol TE/g)

341

followed by 9Z-astaxanthin and all-E-astaxanthin (103.41 and 92.22 µmol TE/g).

342

The results of ORAC-L and PCL showed that the antioxidant ability was in the order:

343

13Z - > 9Z - > all-E-astaxanthin, whereas in DPPH assay 9Z-astaxanthin had higher antioxidant

344

ability than 13Z - than all-E-astaxanthin. The DPPH result was the same as reported by Liu, et al.

345

who found that not only in the DPPH scavenging activity test, but in human neuroblastoma

346

SH-SY5Y cells 9Z-astaxanthin had higher antioxidant activity than 13Z-astaxanthin.5 The DPPH

347

assay is based on a mixed mechanism that involves both hydrogen atom transfer (HAT) and

348

single electron transfer (SET) reactions, and it solely relies on the synthetic free radical.

349

Interpretation of the results of a DPPH assay is complicated thus cautions must be taken

350

especially when the test compounds such as carotenoids have spectra that overlap with DPPH at

351

515 nm.42

352

The ORAC-L and PCL assays are based on the HAT mechanism, and are highly sensitive

353

methods that use peroxyl radicals and superoxide ions which can be found in biological

354

systems.43 The higher antioxidant activities exhibited by the two Z-astaxanthins in these two

355

assays imply that Z-astaxanthins might have better performance than all-E astaxanthin in

356

scavenging peroxyl radicals. Similar results of Z-lycopenes were reported by Müller et al.8 One 19



Page 20 of 36

357

of the reasons for the higher antioxidant activity of the Z-astaxanthins might be their better

358

solubility in the solvent mixture used in the assays as has been found by others.8,

359

Z-Astaxanthins were also found to be at higher concentration than all-E astaxanthin in the

360

plasma.4 Higher antioxidant activity and bioavailability of Z-astaxanthins are both favorable

361

observations considering their implications in health benefits, however, further studies are

362

necessary to reveal the relationship between the two. While in vivo experiments are ideal to

363

explain this controversial result, recent advances in cell models have led us to further illustrate

364

the circumstance using a cell model. To further illustrate the antioxidant activities of three

365

astaxanthin isomers in the context of cellular activity, CAA of different astaxanthin isomers was

366

assessed using a co-culture model of Caco-2 and HT-29 cell lines. Caco2-BBe1 has a shorter

367

incubation time than Caco-2 cells, and the Caco2-BBe1/HT-29 co-culture model is considered to

368

be a more accurate system in simulating human intestinal barrier functions, but never used to

369

assess the antioxidant activity of astaxanthin isomers.31, 32

19

370

Preliminary study on cell viability showed that none of the three astaxanthin isomers was

371

cytotoxic at 0.6 μM (data not shown). In this Caco2-BBe1/HT-29 co-culture model,

372

13Z-astaxanthin possessed significantly higher activity than both all-E- and 9Z-astaxanthin (p
all-E- > 9Z-astaxanthin.

374

The reason why 9Z-astaxanthin had much lower CAA as compared to the other isomers is not

375

clear. Lower cellular uptake or faster metabolism inside the cell might be the cause.33, 34 Others

376

have found that 9Z- and 13Z-β-carotenes had lower extents of absorption than all-E-β-carotene in 20


Page 21 of 36


377

Caco-2 cells.33 Considering the higher in vivo bioavailability of Z-astaxanthins, cellular uptake

378

and bioavailability of different astaxanthin isomers may be more complex than they appear.4 The

379

lower CAA activity of 9Z-astaxanthin may have been due to the lower cellular uptake by

380

Caco2-BBe1/HT-29 co-culture system in the present study (data not shown), however, the

381

significant difference between the CAA of 9Z- and 13Z-astaxanthins still remains to be explained.

382

Furthermore, intestinal epithelial cells such as HT-29 cells produce a mucus layer that might

383

interact differently with astaxanthin isomers and hamper the uptake, bioavailability and CAA

384

results of different astaxanthin isomers.44 Cellular uptake, bioavailability and metabolism of

385

antioxidant and anti-inflammatory effects of different astaxanthin isomers are currently being

386

investigated in our laboratory, specifically aiming at the effect by the different configuration and

387

topography of the isomers.

388 389

Factors Affecting the Stability of Astaxanthin Isomers

390

All-E-astaxanthin and its cis isomers are unstable when exposed to light, oxygen or high

391

temperature, due to their highly conjugated double bond system.45 Stability of these compounds

392

or other naturally occurring carotenoids not only depends on their structural nature but also can

393

be affected by other chemical conditions such as pH and presence of metal ions.15, 35-37 Factors

394

like these are important for better understanding the stability, bioaccessibility and bioavailability

395

of carotenoids; however, existing reports are mostly about the natural form, i.e. all-E-carotenoids

396

including all-E-astaxanthin or extracts containing these natural carotenoids.20 21



Page 22 of 36

397

Stability of astaxanthin isomers was significantly affected by pH, especially at highly

398

acidic conditions (pH 2.0). 9Z-Astaxanthin was the least stable isomer among the three at all pH,

399

whereas all-E- and 13Z-isomers showed similar stability between pH 3.5-11.6 (Figure 4). Ahmat

400

et al. also reported that all-E-astaxanthin was relatively stable within the range from pH 4.0 –

401

11.0.20 However, the present study is the first report on the effect of pH on the stability of pure

402

cis astaxanthin isomers. Percent residual isomer after the incubation was around 77-91% of the

403

original concentration except for 9Z- and 13Z-astaxanthin at pH 2.0 and 9Z-astaxanthin at pH

404

11.6. Degradation occurred and the products were mostly non-carotenoids as no additional peaks

405

were detected at 470 nm. This was particularly true for all-E-astaxanthin at all pH levels,

406

however, at pH 2.0, the apparent loss of 9Z- and 13Z- isomers (65% and 43%, respectively) was

407

actually found to be due to reverse isomerization back to all-E-astaxanthin (Supplemental Figure

408

S1). This observation that isomerization only occurred to 9Z- and 13Z-astaxanthin isomers but

409

not all-E-astaxanthin at 37 ºC, pH 2.0 (acidity similar to that in human stomach) is interesting,

410

considering the two cis isomers are preferably absorbed and detected in human plasma.4 The lack

411

of isomerization of all-E-astaxanthin to 9Z- and 13Z- isomers at 37 ºC, pH 2.0 therefore suggests

412

that 9Z- and 13Z-astaxanthin in the plasma may be converted in the small intestine or after

413

absorption. Further studies on in vivo mechanism of absorption and isomerization are necessary.

414

Metal ions or anions in buffered solutions have been found to disturb the electron

415

transition and change the structure of all-E-β-carotene.39 Metal ions such as Cu2+ had been

416

reported to induce isomerization and influence the stability of all-E-astaxanthin.15 All astaxanthin 22


Page 23 of 36


417

isomers were found relatively stable in the presence of metal ions for 2 h at 37 ºC (Figure 5). 9Z -

418

and 13Z-astaxanthin isomers were more prone to degradation upon exposure to Cu2+ and K+,

419

whereas 9Z-astaxanthin was most stable in the presence of Fe3+. Different from the pH study, no

420

isomerization was observed for all isomers after incubation, and the loss of each isomer might be

421

due to oxidative degradation.20,46

422

In conclusion, an efficient method using I-TiO2 as a catalyst in ethyl acetate heated at 70

423

ºC for 2 h was developed for producing two cis-isomers, 9Z - and 13Z-astaxanthins. This method

424

allows sufficient amount of pure materials for further research on the bioaccessibility,

425

bioavailability and bioactivity of the cis isomers as these may have more important implications

426

in health benefits than naturally occurring all-E-astaxanthin due to the selective absorption of the

427

former found in vivo.4 Using purified 9Z- and 13Z-astaxanthins, the present study found that

428

13Z-astaxanthin was a stronger antioxidant than all-E- and 9Z-astaxanthins in both chemical- and

429

cell-based assay models except the DPPH assay. These observations and the fact that

430

13Z-astaxanthin is higher in plasma lead us to the conclusion that 13Z-astaxanthin may play a

431

more significant antioxidant role in vivo thus warrants further research.4 The relatively higher

432

stability of 13Z-astaxanthin at pH > 2.0 further suggests that its stability may contribute at least

433

partially to the higher bioavailability. Results of the current study provides a means for further

434

study into the mechanisms related to in vivo transformation and bioavailability of different

435

astaxanthin isomers, especially the cis-astaxanthins, and may lead to future development of

436

functional

foods

and

nutraceutical

products

based

on

23


Z-astaxanthins,

particularly


437

Page 24 of 36

13Z-astaxanthin.

438 439

Supporting Information

440 441

Table S1. Experimental Conditions for all-E-Astaxanthin Isomerization

442 443

Figure S1. Stability at pH 2.0 at 37 ºC for 2 h. a) all-E astaxanthin; b) 13Z-astaxanthin; c)

444

9Z-astaxanthin. Reverse isomerization was observed with 13Z-astaxanthin and 9Z-astaxanthin.

445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465

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C934-40. 7. Subramanian, B.; Tchoukanova, N.; Djaoued, Y.; Pelletier, C.; Ferron, M.; Robichaud, J., Investigations on the geometrical isomers of astaxanthin: Raman spectroscopy of conjugated polyene chain with electronic and mechanical confinement. J. Raman Spectrosc. 2014, 45, 299-304. 8. Müller, L.; Goupy, P.; Fröhlich, K.; Dangles, O.; Caris-Veyrat, C.; Böhm, V., Comparative study on antioxidant activity of lycopene (Z)-isomers in different assays. J. Agric. Food Chem. 2011, 59, 4504-4511. 9. Liu, X.; Luo, Q.; Rakariyatham, K.; Cao, Y.; Goulette, T.; Liu, X.; Xiao, H., Antioxidation and anti-ageing activities of different stereoisomeric astaxanthin in vitro and in vivo. J. Funct. Foods 2016, 25, 50-61. 10. Bjerkeng, B.; Følling, M.; Lagocki, S.; Storebakken, T.; Olli, J. J.; Alsted, N., Bioavailability of all-E-astaxanthin and Z-isomers of astaxanthin in rainbow trout (Oncorhynchus mykiss). Aquaculture 1997, 157, 63-82. 11. Zhao, L.; Zhao, G.; Chen, F.; Wang, Z.; Wu, J.; Hu, X., Different effects of microwave and ultrasound on the stability of (all-E)-astaxanthin. J. Agric. Food Chem. 2006, 54, 8346-8351. 12. Yuan, J.-P.; Chen, F., Isomerization of trans-astaxanthin to cis-isomers in organic solvents. J. Agric. Food Chem. 1999, 47, 3656-3660. 13. Yuan, J.-P.; Chen, F., Kinetics for the reversible isomerization reaction of trans-astaxanthin. Food Chem. 2001, 73, 131-137. 14. de Bruijn, W. J.; Weesepoel, Y.; Vincken, J.-P.; Gruppen, H., Fatty acids attached to all-trans-astaxanthin alter its cis–trans equilibrium, and consequently its stability, upon light-accelerated autoxidation. Food Chem. 2016, 194, 1108-1115. 15. Zhao, L.; Chen, F.; Zhao, G.; Wang, Z.; Liao, X.; Hu, X., Isomerization of trans-astaxanthin induced by copper (II) ion in ethanol. J. Agric. Food Chem. 2005, 53, 9620-9623. 16. Sun, Q.; Yang, C.; Li, J.; Aboshora, W.; Raza, H.; Zhang, L., Highly efficient trans– cis isomerization of lycopene catalyzed by iodine-doped TiO2 nanoparticles. RSC Advances 2016, 6, 1885-1893. 17. Qiu, D.; Chen, Z.-R.; Li, H.-R., Effect of heating on solid β-carotene. Food Chem. 2009, 112, 344-349. 18. Li, H.; Deng, Z.; Liu, R.; Loewen, S.; Tsao, R., Ultra-performance liquid chromatographic separation of geometric isomers of carotenoids and antioxidant activities of 20 tomato cultivars and breeding lines. Food Chem. 2012, 132, 508-17. 19. Zechmeister, L., Cis-trans isomeric carotenoids, vitamins A and arylpolyenes. Elsevier: 2014. 20. Ahmat, M.; Amihan, T.; Aineiwaer, A., Extraction and stability research of shrimp astaxanthin. J. Food Saf. Qual. 2013, 4, 905-910. 25



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21. Zhang, Y.; Wu, L.; Luo, Z.; Lin, Q.; Duan, S., Extraction of Astaxanthin from Shrimp and its Stability. Mod. Food Sci. Technol. 2008, 12, 032. 22. Aman, R.; Schieber, A.; Carle, R., Effects of heating and illumination on trans-cis isomerization and degradation of β-carotene and lutein in isolated spinach chloroplasts. J. Agric. Food Chem. 2005, 53, 9512-9518. 23. Lambelet, P.; Richelle, M.; Bortlik, K.; Franceschi, F.; Giori, A. M., Improving the stability of lycopene Z-isomers in isomerised tomato extracts. Food Chem. 2009, 112, 156-161. 24. Honda, M.; Takahashi, N.; Kuwa, T.; Takehara, M.; Inoue, Y.; Kumagai, T., Spectral characterisation of Z-isomers of lycopene formed during heat treatment and solvent effects on the E/Z isomerisation process. Food Chem. 2015, 171, 323-329. 25. Molnár, P.; Szabolcs, J., (Z/E)-photoisomerization of C 40-carotenoids by iodine. J. Chem. Soc., Perkin Trans. 2. 1993, 261-266. 26. Grynbaum, M. D.; Hentschel, P.; Putzbach, K.; Rehbein, J.; Krucker, M.; Nicholson, G.; Albert, K., Unambiguous detection of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superba Dana). J. Sep. Sci. 2005, 28, 1685-1693. 27. Tang, Y.; Li, X.; Zhang, B.; Chen, P. X.; Liu, R.; Tsao, R., Characterisation of phenolics, betanins and antioxidant activities in seeds of three Chenopodium quinoa Willd. genotypes. Food Chem. 2015, 166, 380-388. 28. Huang, D.; Ou, B.; Hampsch-Woodill, M.; Flanagan, J. A.; Deemer, E. K., Development and validation of oxygen radical absorbance capacity assay for lipophilic antioxidants using randomly methylated β-cyclodextrin as the solubility enhancer. J. Agric. Food Chem. 2002, 50, 1815-1821. 29. Zhang, B.; Deng, Z.; Tang, Y.; Chen, P.; Liu, R.; Ramdath, D. D.; Liu, Q.; Hernandez, M.; Tsao, R., Fatty acid, carotenoid and tocopherol compositions of 20 Canadian lentil cultivars and synergistic contribution to antioxidant activities. Food Chem. 2014, 161, 296-304. 30. Shi, Y.; Kovacs-Nolan, J.; Jiang, B.; Tsao, R.; Mine, Y., Antioxidant activity of enzymatic hydrolysates from eggshell membrane proteins and its protective capacity in human intestinal epithelial Caco-2 cells. J. Funct. Foods 2014, 10, 35-45. 31. Pan, F.; Han, L.; Zhang, Y.; Yu, Y.; Liu, J., Optimization of Caco-2 and HT29 co-culture in vitro cell models for permeability studies. Int. J. Food Sci. Nutr. 2015, 66, 680-685. 32. Gupta, V.; Doshi, N.; Mitragotri, S., Permeation of insulin, calcitonin and exenatide across Caco-2 monolayers: measurement using a rapid, 3-day system. PLoS One 2013, 8, e57136. 33. During, A.; Hussain, M. M.; Morel, D. W.; Harrison, E. H., Carotenoid uptake and secretion by CaCo-2 cells β-carotene isomer selectivity and carotenoid interactions. J. Lipid Res. 2002, 43, 1086-1095. 34. During, A.; Dawson, H. D.; Harrison, E. H., Carotenoid transport is decreased and expression of the lipid transporters SR-BI, NPC1L1, and ABCA1 is downregulated in Caco-2 26


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cells treated with ezetimibe. J. Nutr. 2005, 135, 2305-2312. 35. Bustos-Garza, C.; Yáñez-Fernández, J.; Barragán-Huerta, B. E., Thermal and pH stability of spray-dried encapsulated astaxanthin oleoresin from Haematococcus pluvialis using several encapsulation wall materials. Food Res. Int. 2013, 54, 641-649. 36. Chen, C.-S.; Wu, S.-H.; Wu, Y.-Y.; Fang, J.-M.; Wu, T.-H., Properties of astaxanthin/Ca2+ complex formation in the deceleration of cis/trans isomerization. Org. Lett. 2007, 9, 2985-2988. 37. Kreißig, F.; Schäfer, C.; Ulrich, J., Prevention of Solvent‐Mediated Isomer Transfer of Carotenoids. Chem. Eng. Technol. 2014, 37, 1358-1362. 38. Dugave, C.; Demange, L., Cis-trans isomerization of organic molecules and biomolecules: implications and applications. Chem. Rev. 2003, 103, 2475-2532. 39. Gao, Y.; Kispert, L. D., Reaction of carotenoids and ferric chloride: Equilibria, isomerization, and products. J. Phys. Chem. B. 2003, 107, 5333-5338. 40. Liu, H.; Kao, T.; Chen, B., Determination of carotenoids in the chinese medical herb Jiao-Gu-Lan (Gynostemma Pentaphyllum MAKINO) by liquid chromatography. Chromatographia 2004, 60, 411-417. 41. Adamkiewicz, P.; Sujak, A.; Gruszecki, W. I., Spectroscopic study on formation of aggregated structures by carotenoids: role of water. J. Mol. Struct. 2013, 1046, 44-51. 42. Nomura, T.; Kikuchi, M.; Kubodera, A.; Kawakami, Y., Proton ‐ donative antioxidant activity of fucoxanthin with 1, 1‐diphenyl‐2‐picrylhydrazyl (DPPH). IUBMB Life 1997, 42, 361-370. 43. Prior, R. L.; Wu, X.; Schaich, K., Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J. Agric. Food Chem. 2005, 53, 4290-4302. 44. Mahler, G. J.; Shuler, M. L.; Glahn, R. P., Characterization of Caco-2 and HT29-MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability. J. Nutr. Biochem. 2009, 20, 494-502. 45. Yang, S.; Zhou, Q.; Yang, L.; Xue, Y.; Xu, J.; Xue, C., Effect of thermal processing on astaxanthin and astaxanthin esters in pacific white shrimp Litopenaeus vannamei. J. Oleo Sci. 2015, 64, 243-253. 46. Gao, G.; Deng, Y.; Kispert, L. D., Semiconductor photocatalysis: photodegradation and trans-cis photoisomerization of carotenoids. J. Phys. Chem. B. 1998, 102, 3897-3901.

576 577

Funding

578

This work was funded by grants from the Natural Sciences Foundation of China

579

(31171724) and the Fundamental Research Funds for the Central Universities (JUSRP51501) 27



Page 28 of 36

580

and the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP

581

20130093110008). Cheng Yang is a visiting Ph. D. student funded by China Scholarship Council

582

through the Agriculture & Agri-Food Canada-Ministry of Education of China (AAFC-MOE

583

Ph.D. Research Program. This study was also partially supported by the Abase fund of AAFC

584

(Project # J-000283.001.01).

585 586

Note

587

The authors declare no competing financial interest.

28


Page 29 of 36


588

Figure Captions

589

Figure

590

dichloromethane with I2 at 35 ºC; (B) heating all-E-astaxanthin in dichloromethane with I–TiO2

591

catalyst at 35 ºC; (C) heating all-E-astaxanthin in ethyl acetate without I2 or I–TiO2 catalyst at 70

592

ºC; (D) heating all-E-astaxanthin in ethyl acetate with I2 at 70 ºC; (E) heating all-E-astaxanthin in

593

ethyl acetate with I–TiO2 catalyst at 70 ºC; (F) UV illumination (254 nm).

1.

Isomerization

of

all-E-astaxanthin

by:

(A)

heating

all-E-astaxanthin

in

594 595

Figure 2. A: Semi-preparative HPLC isolation and purification of three astaxanthin isomers

596

(inserts: purified isomer); B: UV-Vis spectrum of three astaxanthin isomers. (a) all-E-astaxanthin;

597

(b) 9Z-astaxanthin; (c) 13Z-astaxanthin.

598 599

Figure 3. Antioxidant activities as measured by DPPH (A), ORAC-L (B), PCL (C) and CAA (D)

600

assays. Values with different letters in same figure are significantly different from each other

601

(values are mean ± SD, n = 3, p < 0.05).

602 603

Figure 4. pH Stability of astaxanthin isomers at 37 ºC for 2 h. Values with different letters in the

604

same concentration are significantly different from each other (values are mean ± SD, n = 3, p

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