Effects of Ergothioneine-rich Mushroom Extract on the Oxidative

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Functional Structure/Activity Relationships

Effects of Ergothioneine-rich Mushroom Extract on the Oxidative Stability of Astaxanthin in Liposomes Jade Go Pahila, Yuki Ishikawa, and Toshiaki Ohshima J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00485 • Publication Date (Web): 21 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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Journal of Agricultural and Food Chemistry

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Effects of Ergothioneine-rich Mushroom Extract on the Oxidative Stability of

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Astaxanthin in Liposomes

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Jade Pahila, Yuki Ishikawa, and Toshiaki Ohshima*

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Department of Food Science and Technology

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Tokyo University of Marine Science and Technology

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4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan

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1*

Corresponding author:

Toshiaki Ohshima Department of Food Science and Technology Tokyo University of Marine Science and Technology 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan Phone/Fax: +81 (03) 5463 0613 E-mail: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

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Ergothioneine-rich crude extracts of Pleurotus cornucopiae were used as a source of

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antioxidative components to control the effects of lipid oxidation in astaxanthin-containing

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liposomes. This study aimed to elucidate the interactions of liposomal astaxanthin and lipids

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with ergothioneine-rich mushroom extract (ME) under radical oxidation-induced conditions,

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to provide a better understanding of the agricultural and post-harvest applications of this

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strategy. Azo compounds (2,2'-azobis(2-methylpropionamidine) dihydrochloride and 2,2'-

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azobis(2,4-dimethylvaleronitrile) were used as hydrophilic and lipophilic radical initiators,

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respectively. Results of this study demonstrate that the presence of ME significantly delayed

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the oxidative degradation of astaxanthin and controlled the progress of lipid oxidation in a

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liposomal system. The lipid hydroperoxide formation was significantly suppressed while

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polyunsaturated fatty acids were protected from degradation. In addition, Crude ME also

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demonstrated more potent DPPH radical scavenging activities and EC50 than the equimolar

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concentrations of ergothioneine alone, which suggests the presence of additional compounds

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with antioxidative properties.

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KEYWORDS: ergothioneine; mushroom extract; liposome; radical initiator; AAPH;

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AMVN; antioxidant


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INTRODUCTION

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Wild and cultured salmon meat is one of the most in-demand fishery commodities. It

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is especially valuable because of the presence of astaxanthin in the skeletal muscle tissues,

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which imparts the distinct orange to reddish coloration. Astaxanthin is a naturally occurring

39

carotenoid commonly found in aquatic organisms and is biosynthesized by certain organisms

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such as the algae Haematococcus pluvialis1, 2. Astaxanthin is known to have potent

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antioxidative properties, however, its presence in salmon meat rich in polyunsaturated fatty

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acids (PUFAs) makes it susceptible to oxidation. The oxidative process proceeds through a

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chain reaction mechanism, resulting in undesirable products compromising the nutritional

44

and organoleptic quality of this commodity.

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One strategy for preventing discoloration of salmon meat due to astaxanthin oxidation

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is the incorporation of antioxidant compounds. Ergothioneine is a hydrophilic antioxidant

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that is mainly biosynthesized by certain fungal species3. We previously demonstrated that

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crude extracts of edible mushrooms incorporated in astaxanthin-pigmented meat through in

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vitro treatment and in vivo dietary supplementation can control the progress of lipid oxidation

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and astaxanthin degradation in salmonid meat during post-harvest low temperature storage4.

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These findings strongly suggest that treatment or dietary supplementation with

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ergothioneine-rich mushroom extract (ME) suppress astaxanthin degradation. This study

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aimed to evaluate the effects of ergothioneine-rich ME on the oxidative stability of liposomal

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astaxanthin and lipid components under oxidation-induced conditions. An in vitro experiment

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was conceptualized to elucidate the interactions of astaxanthin and lipids with ergothioneine-

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rich ME in a simple liposome matrix used as a cell model. Liposome was used as a cell model

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to evaluate the effects of ergothioneine-rich ME supplementation in the stability of

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astaxanthin in a cell. Liposomes are widely used in different fields of research mostly to

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mimic cells as it structure depicts a simple structure of a cell with lipid bilayer as membrane



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and a hydrophilic core or cytoplasm. In addition, liposome was used as an in vitro model to

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be able to control the components present to be able to single out specific effects of each in

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certain conditions.

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MATERIALS AND METHODS

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Materials and chemicals. Egg yolk lecithin (95.0% purity) was purchased from

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Wako Pure Chemical Industries (Osaka, Japan). Authentic astaxanthin (97.0% purity) and L-

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ergothioneine (98.0% purity) were purchased from Abcam, Inc. (Cambridge, UK), and Focus

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Biomolecules (Plymouth Meeting, PA), respectively. Radical initiators 2,2'-azobis(2-methyl-

69

propionamidine)dihydrochloride

70

dimethylvaleronitrile) (AMVN, 98.0% purity) were purchased from Sigma-Aldrich (St.

71

Louis, MO) and Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), respectively.

(AAPH,

97.0%

purity)

and

2,2'-azobis(2,4-

72

ME preparation. A 10 g portion of lyophilized Pleurotus cornucopiae was added to

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200 mL 70% (v/v) ethanol, sonicated for 5 min, and filtered. The recovered residue was

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extracted with 100 mL 70% (v/v) ethanol, and this extraction was repeated 3 times. All

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filtrates were combined and evaporated to dryness in vacuo using a rotary evaporator. A 100

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mL aliquot of PBS pH 7.4, was added to the flask to obtain the crude ME, with total dissolved

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solids of 2.80 ± .010 °Bx. The ergothioneine content of the ME was analyzed according to

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the method of Nguyen et al. (2012)5 and amounted to 44.82 ± 0.12 mg ergothioneine per mL.

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The extract was further diluted to a final ergothioneine concentration of 0.25 mg/mL, which

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was used in the subsequent experiments.

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Liposome preparation. Liposomes were prepared according to the method of

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Moscho et al. (1996)6 with a slight modification. For the preparation of liposomes without

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astaxanthin or ME, a 0.4 mL portion of 0.1 M lecithin in chloroform was added to a 1000 mL

84

round-bottom flask containing 4 mL chloroform and 0.8 mL methanol, followed by the


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careful addition of 17 mL 10 mM PBS along the interior walls of the flask. Subsequently,

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organic solvents were evaporated using a rotary evaporator under reduced pressure at 40°C.

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The resultant opalescent liquid containing the liposomes was diluted to a final volume of 17

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mL. For the preparation of liposomes containing astaxanthin, 4 mL of astaxanthin solution in

89

chloroform (100 µg/mL) was used instead of chloroform. Astaxanthin was added prior the

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preparation of the liposomes so that it could incorporate into the lipid bilayer of the liposomes,

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similar as it would in an astaxanthin-pigmented salmon cells. For liposome preparation with

92

ergothioneine, 6.8 mL of ME containing 0.25 mg/mL ergothioneine was added after the

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evaporation step, and subsequently diluted to a 17-mL final volume. Ergothioneine/ME was

94

added after the preparation of liposomes as ergothioneine from the diet would be initially

95

present extracellularly before it can be transported intracellularly by specific transporters

96

(OCTN1/SLC22A4/ETT). The liposome solutions were aliquoted into 1.5 mL polypropylene

97

snap-cap microcentrifuge tubes.

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Microscopy evaluation. Liposomes containing astaxanthin were observed under the

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bright-field setting of an Olympus CKX41 Microscope (Tokyo, Japan) equipped with a

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WRAYCAM G130 Wraymer microscope camera (Osaka, Japan) to evaluate the aggregation

101

of

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yl)amino)dodecanoyl)-sn-glycero-3-phospho-choline (NBD-labeled PC, 99.0% purity,

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Avanti Polar Lipids, Inc., Alabaster, AL) was mixed with lecithin and chloroform prior to

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liposome preparation, and fluorescence in the liposomes was observed at an excitation

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wavelength of 463 nm and an emission wavelength of 536 nm to determine the structure of

106

the formed vesicles. Sizes of the liposomes were measured based on the diameter using a

107

slide micrometer.

astaxanthin

pigments.

1-Myristoyl-2-(12-((7-nitro-2-1,3-benzoxadiazol-4-

108

Oxidative stability evaluation. The different liposome preparations were subjected

109

to stability tests under oxidative conditions induced by the addition of either hydrophilic



110

AAPH or lipophilic AMVN radical initiators. The molar ratio of lecithin to the radical

111

initiators added was 2:1, which was adapted from a study7 on the influence of AAPH and

112

AMVN localization on the free radical damage of proteins in systems such as the liposome.

113

Radical initiators were separately added after the preparation of liposomes with the premise

114

that radical initiation usually starts extracellularly. The liposome preparations were then

115

incubated at 37 °C, and the progress of oxidation and stability of lipid, astaxanthin, and

116

ergothioneine were monitored quantitatively. To confirm the antioxidative effects of

117

ergothioneine in ME, parallel tests were performed using equimolar authentic ergothioneine

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solution, alongside liposomes without ME or ergothioneine.

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Total lipid hydroperoxide (HPO) quantification. Total lipid HPO was

120

quantitatively measured using the flow injection analysis (FIA) system, according to the

121

method of Sohn et al. (2005)8. Briefly, the lipid components of liposomes were extracted by

122

the Bligh and Dyer method9 (1 mL liposome, 1.1 mL methanol, and 0.8 mL chloroform) with NBD-

123

labeled PC (0.3 mL 4 µg/mL in chloroform) used as an internal standard (IS). The chloroform

124

layer was collected and subjected to analysis. A calibration curve was obtained using different

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concentrations of authentic cumene hydroperoxide (80.0 % purity, Sigma-Aldrich). All data

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are expressed as equivalent nmol cumene hydroperoxide per mL liposome.

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Fatty acid quantification. Total lipids in liposomes were extracted using the Bligh

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and Dyer method9. Fatty acid methyl esters (FAMEs) were derivatized according to the

129

Official Methods and Recommended Practices of the AOCS, using methyl tricosanoate

130

(99.0% purity, Nu-check Prep, Inc., Waterville, MN) as an IS. FAMEs were separated and

131

quantified using a Shimadzu gas chromatograph model GC-2010 (Kyoto, Japan) equipped

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with a Supelcowax™ 10 fused silica capillary column (60 m  0.32 mm i.d.  0.25 µm film

133

thickness), and a flame ionization detector. A calibration curve was obtained using different


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concentrations of FAME (99.6% purity, Nu-check Prep, Inc.). All data are expressed as mM

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FAME.

136

Determination of thiobarbituric acid reactive substances (TBARS). TBARS

137

present in liposomes were quantified as secondary products of lipid oxidation, adapting the

138

methods of Schmedes and Hølmer (1989)10 and Uchiyama and Mihara (1978)11, with slight

139

modifications. A calibration curve was obtained using varying concentrations of authentic

140

1,1,3,3'-tetraethoxypropane. All data are expressed as µM malondialdehyde (MDA)

141

equivalents.

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Total astaxanthin quantification. Total astaxanthin content was quantified using

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the method previously described4. Briefly, collected liposomes were extracted using the Bligh

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and Dyer method9 with the addition of trans-β-apo-8'-carotenal (96.0% purity, Sigma-

145

Aldrich) as an IS. The chloroform layer was collected and subjected to HPLC analysis. A

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calibration curve was obtained using different concentrations of authentic astaxanthin. All

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data are expressed as µg astaxanthin per mL liposome.

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Ergothioneine quantification. Ergothioneine content was determined following the

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methods described by Nguyen et al. (2012)5. Liposomes were subjected to Bligh and Dyer

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extraction7 with the addition of 3-methyl-1H-imidazole-2-thione (99.0% purity, Sigma-

151

Aldrich) as an IS. The water-methanolic layer was collected, evaporated to dryness in vacuo

152

using a rotary evaporator, and dissolved in 3 mL distilled water. A calibration curve was

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obtained using different concentrations of authentic ergothioneine. All data are expressed as

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µg ergothioneine per mL liposome.

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2,2-Diphenyl-1-picrylhydrazyl radical scavenging activity. The radical scavenging

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activity of ergothioneine in crude ME was determined according to the method of Giri, et al.

157

(2011)12 with a slight modification. The samples used for ergothioneine analysis were

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subjected to HPLC analysis, and bleaching of 0.1 mM methanolic 2,2-diphenyl-1-



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picrylhydrazyl (DPPH, 95.0% purity, Sigma-Aldrich) solution in the post-column reaction

160

coil was monitored at 517 nm with an SPD-M10AVP. Varying concentrations of authentic

161

ergothioneine were used to obtain a calibration curve, with all data expressed as equivalent

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mmol ergothioneine per mL sample.

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Qualitative analyses of radical scavengers in ME. Absorption thin-layer

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chromatography (TLC) was carried out on a silica gel 60 F254 plate (5 × 4 cm, Merck,

165

Darmstadt, Germany). A 10 µL portion of a two-fold diluted solution of the ME was spotted

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onto the plate. The plate was developed by methanol/water solution (4:1, v/v). The spots

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were monitored under UV light at 254 nm and subsequently visualized by spraying 1 mM

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DPPH in methanol for the evaluation of the radical scavenging activity. A 0.9 μM aqueous

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authentic ESH was used as a reference standard.

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2,2-Diphenyl-1-picrylhydrazyl radical scavenging half maximal effective

171

concentration (EC50). The concentration of a sample that possessed a half-maximal DPPH

172

radical scavenging activity was determined following the methods of Chen and Ho (1995)13

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and Bao et al. (2010)14, with slight modifications. The aqueous sample was added to a 0.1

174

mM methanolic DPPH solution and measured at 517 nm using a Shimadzu UV-1600-PC

175

spectrophotometer. All data are expressed as DPPH radical scavenging EC50.

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Oxygen radical absorbance capacity (ORAC) assay. The assay used to determine

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the ORAC of the samples was adapted and slightly modified from the method of Ou et al.

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(2001)15, using 0.08 M fluorescein as probe in the oxidation-induced condition in the presence

179

of 0.15 mM AAPH. Fluorescence signals were monitored at 1 min intervals for 90 min using

180

a TECAN SPECTRAFluor Plus microplate reader (Männedorf, Switzerland) set at 37 °C.

181

Varying concentrations of authentic Trolox were used to obtain a calibration curve. All data

182

are expressed as µmol Trolox equivalent per g sample.


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Determination of total phenolic compounds. The total amount of phenolic

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compounds present in the ME samples was measured using the method described by Bao et

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al. (2010)14. Briefly, 0.2 mL of sample was added with 0.8 mL distilled water and 4 mL of

186

10% Folin-Ciocalteu reagent. Subsequently, 5 mL of 7.5 % aqueous sodium carbonate

187

solution was added to the mixture and was set aside for 30 min for the components to react

188

and produce a visible color. The absorbance of the mixture was measured at 765 nm using a

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Shimadzu UV-1600-PC spectrophotometer. Varying concentrations of authentic gallic acid

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were used to obtain a calibration curve, with all data expressed as µg gallic acid per mL

191

sample.

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Statistical analysis. Microsoft Excel 2016 was used to analyze the means and

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standard deviations of the collected data and to generate graphs. IBM SPSS Statistics 20 was

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used to determine significant differences among values at a 5% level of significance.

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RESULTS

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Liposome formation. Representative photos of the liposomes are shown in Figure 1.

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The bright field micrograph (Figure 1(a)) shows the aggregation of astaxanthin in liposomes

199

seen as orange-colored spherical vesicles floating in the aqueous phase, and the phase-

200

contrast micrograph (Figure 1(b)) shows the distinct circular outline of the liposomes. The

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fluorescence micrograph (Figure 1(c)) shows that NBD-labeled PC mixed with lecithin

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formed distinct circular outlines corresponding to a phospholipid bilayer emitting bright

203

fluorescence, which distinctly separated the intracellular and the extracellular aqueous phases.

204

Average sizes of the liposomes formed were 10–25 µm in diameter; thus, they can be

205

classified as giant-sized vesicles. These observations confirm the physical features of the

206

liposomes.



207

Effects of oxidation-induced conditions on liposomal lipid components. Changes

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in the total lipid HPO content of the different liposome treatments in varying incubation

209

conditions are shown in Figure 2. Liposomes without radical initiators did not show any

210

significant increase in lipid HPO (p > 0.05) after 17 days incubation at 25 °C (Figure 2(a)).

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The addition of AAPH and the increase of the incubation temperature to 37 °C resulted in a

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further increase (p < 0.05) in lipid HPO content of liposomes without ME, while liposomes

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with ME continued to have the lowest content, with no significant increase during the 24-h

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incubation period (Figure 2(b)). The addition of AMVN and the increase of the incubation

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temperature to 37 °C resulted in a higher magnitude of lipid HPO increase in all the liposome

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treatments, wherein no significant difference (p > 0.05) among treatments was observed

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(Figure 2(c)). It was confirmed that addition of both hydrophilic and lipophilic radical

218

initiators at 37 °C remarkably increased the accumulated amount of lipid HPO formed.

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Dominant constituent fatty acids of the liposomes were identified as C14:0, C16:0,

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C16:1n-7, C18:0, C18:1n-9, C18:1n-7, C18:2n-6, and C20:4n-6 (Figure 3). After 24 h

221

incubation with AAPH at 37 °C, no observable decrease (p > 0.05) was noted in most of the

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fatty acids except for PUFAs, for which concentrations in liposomes with both astaxanthin

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and ME were higher (p < 0.05) than those in all other liposomes (Figure 3(a)). Incubation

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with AMVN showed a decreasing trend for most constituent fatty acids, wherein the

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liposomes without ME had lower (p < 0.05) concentrations than the those of the liposomes

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with ME. However, no significant difference among liposomes was observed for C14:0,

227

C18:1n-7, and C20:4n-6 (Figure 3(b)).

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Effects of oxidation-induced conditions on astaxanthin content. Changes in

229

astaxanthin content of liposomes during incubation are shown in Figure 4. Decrease in

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astaxanthin content of liposomes was noted for all incubation conditions, and addition of

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radical initiators and increase of incubation temperature further accelerated the degradation


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of astaxanthin. Despite the continuous decrease in astaxanthin a difference in the degradation

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amount was observed wherein the liposomes with ME had a consistently higher (p < 0.05)

234

astaxanthin content than the liposomes without ME.

235

Liposomes incubated at 25 °C without radical initiators had the slowest rate of

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astaxanthin degradation among all incubation conditions tested (Figure 4(a). The addition of

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AAPH and the increase of the incubation temperature to 37 °C accelerated astaxanthin

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degradation during a 24-h incubation period to 66% and 32% for the liposomes with and

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without ME, respectively (Figure 4(b)). The residual amounts of astaxanthin in liposomes

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was lower when incubated with AMVN, wherein after 24 h of incubation the liposomes

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without ME contained 25% total residual astaxanthin while the liposomes with ME contained

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36% (Figure 4(c)).

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Effects of crude ME and ergothioneine on the progress of lipid oxidation.

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Changes in the lipid HPO content and TBARS of the liposomes are shown in Figure 5.

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Incubation of liposomes with AAPH at 37 °C resulted in a significant increase (p < 0.05) of

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lipid HPO in the liposomes without ME or ergothioneine, whereas the liposomes without

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AST or ME/ergothioneine had the highest HPO formed, while the liposomes containing either

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ME or ergothioneine did not exhibit any significant increase during the 24-h incubation

249

period (Figure 5(a)). Incubation with AMVN resulted in a higher magnitude of increase in

250

HPO formation in all liposomes (Figure 5(b)). Despite this increase, significant differences

251

(p < 0.05) were observed wherein the liposomes without astaxanthin or ME/ergothioneine

252

had the highest HPO content, followed by liposomes with astaxanthin and without

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ME/ergothioneine, then by liposomes with astaxanthin and ergothioneine. Liposomes with

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astaxanthin and ME had the lowest HPO content among all the liposomes that were incubated

255

with AMVN.



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A similar pattern was observed for TBARS. Incubation with AAPH resulted in a

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significant increase (p < 0.05) of TBARS in liposomes without ME or ergothioneine, while

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liposomes with either ME or ergothioneine did not show any increase (Figure 5(c)).

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Incubation with AMVN showed an increase in TBARS in all liposomes (Figure 5(d)).

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Liposomes without astaxanthin or ME/ergothioneine had the highest levels of TBARS,

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followed by liposomes with astaxanthin and without ME/ergothioneine, then by liposomes

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with astaxanthin and ergothioneine. The liposomes containing both astaxanthin and ME had

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the lowest levels of TBARS at the end of the 24-h incubation period.

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Effects of crude ME and ergothioneine on the oxidative stability of astaxanthin.

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Changes in the astaxanthin content of liposomes as an effect of the presence or absence of

266

ME or ergothioneine are shown in Figure 6. A decreasing pattern in the astaxanthin content

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was observed in all liposomes during incubation with either AAPH or AMVN. Incubation

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with AAPH showed that the liposomes without ME or ergothioneine had the lowest (p < 0.05)

269

astaxanthin content and no significant difference (p > 0.05) was observed between the

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liposomes with ME or ergothioneine (Figure 6(a)). Incubation with AMVN demonstrated a

271

faster rate of astaxanthin decrease wherein the liposomes without ME or ergothioneine had

272

the lowest (p < 0.05) astaxanthin content, followed by liposomes with ergothioneine, and

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liposomes with ME had the highest residual astaxanthin content (Figure 6(b)).

274

Stability of ergothioneine in the presence of radical initiators. Changes in the

275

ergothioneine content of liposomes with ME and authentic ergothioneine during incubation

276

with AAPH or AMVN are shown in Figure 7. Ergothioneine quantification showed that after

277

the 24-h incubation of liposomes with AAPH, a significant decrease (p < 0.05) in the

278

ergothioneine content of liposomes with authentic ergothioneine (liposome D) was observed,

279

but not in liposomes with crude ME (liposome C) (Figure 7(a)). On the other hand, no


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significant decrease in ergothioneine content was observed in both liposomes (C and D) when

281

incubated with AMVN (Figure 7(b)).

282

Changes in physical appearance of liposomes during oxidation-induced

283

conditions. Representative micrographs of liposomes before and after incubation with AAPH

284

and AMVN are shown in Figure 8. Liposomes without astaxanthin that were incubated with

285

AAPH showed no observable difference in appearance after 24 h incubation (Figure 8(a)).

286

Distinct changes in color from bright to pale orange were observed in the liposomes with

287

astaxanthin, but the sizes were still within their original size range. Liposomes incubated with

288

AMVN (Figure 8(b)) showed a notable decrease in size with an average size of 3–5 µm in

289

diameter, and no discernable orange coloration was observed in the liposomes that initially

290

contained astaxanthin. Moreover, no discernable difference was observed between the effects

291

of ME and ergothioneine on the physical appearance of the liposomes.

292

Correlation between ergothioneine content and radical scavenging activity. The

293

correlation between ergothioneine content in crude ME and corresponding DPPH radical

294

scavenging activity is shown in Figure 9. Results of ME quantitative analysis showed a high

295

correlation (R2 = 0.9984) between ergothioneine content and DPPH radical scavenging

296

activities. This correlation was relative to the values obtained from authentic ergothioneine

297

equivalents, suggesting the purity of the ergothioneine peaks separated by HPLC.

298

Representative chromatograms obtained from ergothioneine analyses are available in

299

Supporting Information.

300

Radical scavengers in the crude ME. Thin layer chromatograms of the crude ME

301

and authentic ESH tested for radical scavengers are shown in Figure 10. It can be noted that

302

the crude ME showed a clearly visible white spot on the plate sprayed with DPPH radical

303

solution having the same Rf value as that of the authentic ESH. Additional unidentified

304

compounds could also be seen to exist which can be noted as white bands on the plate, this



305

suggests that the crude ME contained a number of organic compounds having radical

306

scavenging activities in addition to ESH.

307

Comparison of the antioxidative properties of crude ME and ergothioneine.

308

Some antioxidative properties of ME and ergothioneine are compared in Table 1. The

309

comparison showed that 250 µg of ergothioneine per mL of crude ME had 2-fold higher

310

levels of total phenolics, 1.6-fold more potent DPPH EC50, and 2.6-fold higher ORAC values

311

compared to the same concentration of ergothioneine.

312

Comparison of the radical scavenging activities of crude ME and ergothioneine

313

with other hydrophilic antioxidants. Radical scavenging activities of crude ME, authentic

314

ergothioneine, ascorbic acid, and glutathione are shown in Figure 11. Ascorbic acid was used

315

in comparison as one of the most commonly used and studied antioxidants, and glutathione

316

as a commonly used and studied antioxidant with a thiol structure similar to ergothioneine.

317

The DPPH EC50 of glutathione was the highest, and there was no significant difference (p >

318

0.05) observed in the other 3 samples (Figure 11(a)); the ORAC value of ME was the highest

319

followed by authentic ergothioneine, ascorbic acid, and glutathione exhibited the lowest value

320

(Figure 11(b)).

321 322

DISCUSSION

323

This study used astaxanthin-filled liposomes as a model of the astaxanthin-pigmented

324

cells of salmonid meat to investigate the effects of ergothioneine-rich ME supplementation

325

as a strategy to protect the meat from oxidative degradation. Azo compounds such AAPH

326

and AMVN are commonly used for in vitro reactions as hydrophilic and lipophilic radical

327

initiators, respectively16, 17. The results of this study clearly demonstrate that the presence of

328

ME with astaxanthin in a liposome has potential additive antioxidative functions that


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neutralize reactive radical species to control the advancement of lipid oxidation and delay

330

astaxanthin degradation, even in the presence of both hydrophilic and lipophilic radicals.

331

Astaxanthin in the liposomes could control HPO formation only under limited

332

conditions at 25 °C. As incubation temperature increased, astaxanthin could not mitigate the

333

oxidative effects of higher rates of radical formation. This can be attributed to the low stability

334

of astaxanthin at higher temperatures. In a study of the stability of astaxanthin in H. pluvialis

335

powdered biomass, the low-temperature drying method resulted in a higher recovery yield of

336

astaxanthin18. In addition, it was also noted in the same study that when H. pluvialis powder

337

was stored at a higher temperature, less astaxanthin was retained18. In another study, the

338

stability of astaxanthin in a carotenoprotein of shrimp fermentation byproduct was monitored

339

under varying storage conditions, and the results showed that increased storage temperature

340

compromised astaxanthin stability due to the denaturation of protein, exposing it to air and

341

light19. The limited antioxidative functions of astaxanthin in liposomes can also be attributed

342

to the amount of astaxanthin present relative to the radicals. In the present liposome system,

343

the experimental concentration of phosphatidylcholine was approximately 3 mM, radical

344

concentration generated was estimated to be at 3 mM, and astaxanthin concentration in the

345

liposomes was 0.037 mM, which is comparable to the average astaxanthin content in the

346

muscle of pigmented salmonids4. These concentrations are within the previously reported

347

ranges for the appropriate ratio of astaxanthin to phospholipid in liposomes20,

348

Consequently, the amount of astaxanthin may not have been sufficient to counter the

349

oxidative effects of radicals present in this system.

21

.

350

The addition of ergothioneine-rich ME to liposomes showed additive effects to the

351

antioxidative properties of astaxanthin. It exhibited a delay in the progress of lipid oxidation

352

and astaxanthin degradation in both the AAPH and AMVN-initiated reactions. Ergothioneine

353

is a known antioxidant22, 23 commonly found in certain edible mushroom species3, 14, thus



354

MEs have been the focus of several natural antioxidant studies. Furthermore, ergothioneine-

355

rich MEs have been proven to have antioxidative effects when applied to certain oxidation-

356

susceptible post-harvest commodities24,

357

regarding the antioxidative properties of crude MEs against the oxidative degradation of

358

astaxanthin in salmon tissues.

25

. However, few studies have been conducted

359

The present study clearly shows that the antioxidative effects of ME addition to

360

liposomes were apparent in the suppression of lipid HPO and formation of TBARS. The

361

effects of each radical initiator on fatty acid substrates during oxidation-induced conditions

362

provided interesting outcomes. The results suggest that PUFAs (C18:2n-6 and C20:4n-6) are

363

more susceptible to oxidation caused by AAPH radicals, which is comparable to previous

364

observations in which lipid stability against peroxidation decreased with increasing

365

unsaturation26, 27. On the other hand, AMVN affected most of the fatty acids in the liposomes

366

regardless of the degree of unsaturation. These results are consistent with the mode of action

367

of AMVN as a lipophilic radical generator. Despite the decrease of certain fatty acids as an

368

effect of lipid oxidation, liposomes with ME exhibited significant lipid stability with AAPH

369

and AMVN when compared with liposomes without ME, suggesting the oxidative protective

370

effects of ME against both hydrophilic and lipophilic radicals.

371

Microscopic evaluation showed that oxidation initially targeted the astaxanthin

372

pigments present in the phospholipid membrane of the liposomes as evidenced by the distinct

373

color change after incubation. Moreover, the effect of lipophilic radicals on decreasing the

374

amount of several fatty acids was shown by the apparent decrease in the average size of the

375

liposomes after 24 h incubation with AMVN. As fatty acids are oxidized, certain regions

376

within the molecules are cleaved, compromising the integrity of the phospholipid bilayer. The

377

collective oxidation reactions could also result in a greater effect on the overall structural

378

integrity of the phospholipid bilayer of the entire vesicle. The damage in certain parts of the


Page 16 of 40

Page 17 of 40


379

bilayer, which depends on the extent of oxidation progress, could lead to the reorientation of

380

the bilayer position, which could then result in smaller vesicles. Mosca et al. (2001)28 reported

381

that azo radical-induced liposomal oxidation can lead to modifications of the z-potential of a

382

membrane bilayer, resulting in the rearrangement of the polar phosphate head in the

383

membrane. This phenomenon was observed in the microscopy analysis of the liposomes in

384

this study.

385

The parallel tests of the crude ME and ergothioneine revealed some antioxidative

386

properties present in ME. The effects of ME and authentic ergothioneine against lipid and

387

astaxanthin oxidation induced by hydrophilic radicals AAPH did not show any significant

388

difference. However, incubation with AMVN showed that crude ME exhibited more potent

389

antioxidative properties, as shown by the significant suppression of HPO and TBARS

390

formation as well as the delay in astaxanthin degradation. The kinetics of radical generation

391

from AAPH and AMVN have been established with certain formula, wherein the amount of

392

radicals generated in a simple liposomal system may also be calculated from 16,29. However,

393

the liposomes used in the present study are much more complicated since the crude ME

394

contained several unidentified compounds with radical scavenging activities. Thus, the

395

radicals generated from AAPH and AMVN may have been scavenged by ESH, as well as

396

other compounds present in ME. Furthermore, the ESH from ME remained stable after 24 h

397

of incubation at 37 °C in the presence radical initiators suggests that the amounts of azo

398

radicals generated from the imitators were not enough to degrade and significantly affect the

399

concentration of ESH under the present conditions.

400

In this study, the premise for the use of the ME as a source of antioxidants is based

401

on previous findings regarding the considerable amounts of ergothioneine present in certain

402

edible mushroom species5, 14 and its potential in post-harvest quality preservation4, 24, 25, 30.

403

Ergothioneine together with the other components in crude ME exhibited additive and



404

significantly higher antioxidative properties than just ergothioneine in terms of DPPH radical

405

scavenging activity and oxygen radical absorbance capacity. Aside from ergothioneine, a

406

wide range of antioxidant compounds including certain phenolic compounds have also been

407

identified in a variety of edible mushroom species exhibiting certain degrees of antioxidant

408

characteristics31-37. Based on the findings of this study, it can be acknowledged that other

409

components in crude ME may have contributed in the total antioxidative property.

410

In summary, this study was based on the premise that hydrophilic extracts of certain

411

edible mushroom species contain a significant amount of the potent antioxidant ergothioneine,

412

which can be applied to the post-harvest preservation of astaxanthin-pigmented salmon meat.

413

Regardless of the source of salmon, whether it is farm-raised or wild-caught, it must be

414

handled properly during transport and storage to avoid nutritional and organoleptic

415

degradation. Low-temperature storage is the most common post-harvest preservation

416

strategy; however, even in freezing conditions, lipid and astaxanthin oxidation still occur4.

417

The findings of this study successfully demonstrate the antioxidative properties of crude P.

418

cornucopiae extract in an astaxanthin-filled liposomal system, including delay of astaxanthin

419

degradation and suppression of lipid oxidation, and are comparable to previous findings of in

420

vitro and in vivo ME supplementation into astaxanthin-pigmented salmonid meats4. Moreover,

421

the results also provide insight into the antioxidative potency of ergothioneine and ME, which

422

was demonstrated by the specific reactions with certain types of radicals. Furthermore, the

423

findings also demonstrate the superior antioxidative potency of crude ME compared with

424

other antioxidants. The use of crude extracts is an economical approach to obtain

425

ergothioneine and other naturally-derived antioxidative compounds. Overall, these findings

426

contribute to the elucidation of the post-harvest antioxidative protective mechanism of crude

427

ME in astaxanthin-pigmented salmon meat.

428


Page 18 of 40

Page 19 of 40


429

ABBREVIATIONS

430

ME, mushroom extract; AST, astaxanthin; ESH, authentic ergothioneine; AAPH, 2,2'-

431

azobis(2-methylpropionamidine)

432

dimethylvaleronitrile); HPO, hydroperoxide; FAME, fatty acid methyl ester; TBARS,

433

thiobarbituric acid reactive substances; EC50, half maximal effective concentration; ORAC,

434

oxygen radical absorbance capacity; PUFA, polyunsaturated fatty acid.

dihydrochloride;

AMVN,

2,2'-azobis(2,4-

435 436

FUNDING

437

The study was partly supported by a scholarship awarded to J. Pahila from the Ministry of

438

Education, Culture, Sports, Science and Technology, Japan.

439 440

Supporting Information. Representative chromatograms obtained from the ergothioneine

441

analysis of authentic ergothioneine standard and crude mushroom extract.



442

REFERENCES

443

(1) Choi, Y. E.; Yun, Y. S.; Park, J. M., Evaluation of factors promoting astaxanthin

444

production by a unicellular green alga, Haematococcus pluvialis, with fractional factorial

445

design. Biotechnol. Progr. 2002, 18, 1170-1175.

446

(2) Praveenkumar, R.; Gwak, R.; Kang, M.; Shim, T. S.; Cho, S.; Lee, J.; Oh, Y. K.; Lee, K.;

447

Kim, B., Regenerative Astaxanthin Extraction from a Single Microalgal (Haematococcus

448

pluvialis) Cell Using a Gold Nano-Scalpel. ACS Appl. Mater. Inter. 2015, 7, 22702-8.

449

(3) Jones, G. W.; Doyle, S.; Fitzpatrick, D. A., The evolutionary history of the genes involved

450

in the biosynthesis of the antioxidant ergothioneine. Gene 2014, 549, 161-70.

451

(4) Pahila, J.; Kaneda, H.; Nagasaka, R.; Koyama, T.; Ohshima, T., Effects of ergothioneine-

452

rich mushroom extracts on lipid oxidation and discoloration in salmon muscle stored at low

453

temperatures. Food Chem. 2017, 233, 273-281.

454

(5) Nguyen, T. H.; Giri, A.; Ohshima, T., A rapid HPLC post-column reaction analysis for

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the quantification of ergothioneine in edible mushrooms and in animals fed a diet

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supplemented with extracts from the processing waste of cultivated mushrooms. Food Chem.

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2012, 133, 585-91.

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(6) Moscho, A.; Orwar, O.; Chiu, D. T.; Modi, B. P.; Zare, R. N., Rapid Preparation of Giant

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Unilamellar Vesicles. P. Natl. Acad. Sci. USA 1996, 93, 11443-11447.

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(7) Dean, R. T.; Hunt, J. V.; Grant, A. J.; Yamamoto, Y.; Niki, E., Free radical damage to

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proteins: The influence of the relative localization of radical generation, antioxidants, and

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target proteins. Free Radical Bio. Med. 1991, 11(2), 161-168.

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(8) Sohn, J. H.; Taki, Y.; Ushio, H.; Ohshima, T., Quantitative determination of total lipid

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hydroperoxides by a flow injection analysis system. Lipids 2005, 40, 203-209.

465

(9) Bligh, E. G.; Dyer, W. J., A rapid method of total lipid extraction and purification. Can.

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J. Biochem. Physiol. 1959, 37, 911-917.


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(10) Schmedes, A.; Hølmer, G., A new thiobarbituric acid (TBA) method for determining

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free malondialdehyde (MDA) and hydroperoxides selectively as a measure of lipid

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peroxidation. J. Am. Oil Chem. Soc. 1989, 66, 813–817.

470

(11) Uchiyama, M.; Mihara, M., Determination of malonaldehyde precursor in tissues by

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thiobarbituric acid test. Anal. Biochem. 1978, 86, 271-8.

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(12) Giri, A.; Osako, K.; Okamoto, A.; Okazaki, E.; Ohshima, T., Antioxidative properties

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of aqueous and aroma extracts of squid miso prepared with Aspergillus oryzae-inoculated

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koji. Food Res. Int. 2011, 44, 317-325.

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(13) Chen, C. W.; Ho, C. T., Antioxidant properties of polyphenols extracted from green and

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black teas. J. Food Lipids 1995, 2, 35-46.

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(14) Bao, H. N.; Osako, K.; Ohshima, T., Value-added use of mushroom ergothioneine as a

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colour stabilizer in processed fish meats. J. Agr. Food Chem. 2010, 90, 1634-1641.

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(15) Ou, B.; Hampsch-Woodill, M.; Prior, R. L., Development and Validation of an

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Improved Oxygen Radical Absorbance Capacity Assay Using Fluorescein as the Fluorescent

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Probe. J. Agr. Food Chem. 2001, 49, 4619-4626.

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(16) Niki, E., Free radical initiators as source of water- or lipid-soluble peroxyl radicals.

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Methods Enzymol. 1990, 186, 100-108.

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(17) Niki, E.; Yamamoto, Y.; Komuro, E.; Sato, K., Membrane damage due to lipid oxidation.

485

Am. J. Clin. Nutr. 1991, 53, 201S-205S.

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(18) Ahmed, F.; Li, Y.; Fanning, K.; Netzel, M.; Schenk, P. M., Effect of drying, storage

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temperature and air exposure on astaxanthin stability from Haematococcus pluvialis. Food

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Res. Int. 2015, 74, 231-236.

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(19) Armenta, R. E.; Guerrero-Legarreta, I., Stability studies on astaxanthin extracted from

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fermented shrimp byproducts. J. Agr. Food Chem. 2009, 57, 6095-100.



491

(20) Du, H. H.; Liang, R.; Han, R. M.; Zhang, J. P.; Skibsted, L. H., Astaxanthin Protecting

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Membrane Integrity against Photosensitized Oxidation through Synergism with Other

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Carotenoids. J. Agr. Food Chem. 2015, 63, 9124-30.

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(21) Fukuzawa, K.; Inokami, Y.; Tokumura, A.; Terao, J.; Suzuki, A., Rate constants for

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quenching singlet oxygen and activities for inhibiting lipid peroxidation of carotenoids and

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alpha-tocopherol in liposomes. Lipids 1998, 33, 751-6.

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(22) Hartman, P. E., Ergothioneine as antioxidant. Methods Enzymol. 1990, 186, 310-318.

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(23) Cheah, I. K.; Halliwell, B., Ergothioneine; antioxidant potential, physiological function

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and role in disease. Biochim. Biophys. Acta 2012, 1822, 784-793.

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(24) Bao, H. N.; Ushio, H.; Ohshima, T., Antioxidative activity and antidiscoloration

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efficacy of ergothioneine in mushroom (Flammulina velutipes) extract added to beef and fish

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meats. J. Agr. Food Chem. 2008, 56, 10032-10040.

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(25) Encarnacion, A. B.; Fagutao, F.; Hirono, I.; Ushio, H.; Ohshima, T., Effects of

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ergothioneine from mushrooms (Flammulina velutipes) on melanosis and lipid oxidation of

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kuruma shrimp (Marsupenaeus japonicus). J. Agr. Food Chem. 2010, 58, 2577-85.

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(26) Triba, M. N.; Devaux, P. F.; Warschawski, D. E., Effects of lipid chain length and

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unsaturation on bicelles stability. A phosphorus NMR study. Biophys. J. 2006, 91, 1357-67.

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(27) Quinn, P. J.; Joo, F.; Vigh, L., The role of unsaturated lipids in membrane structure and

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stability. Prog. Biophys. Mol. Biol. 1989, 53, 71-103.

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(28) Mosca, M.; Ceglie, A.; Ambrosone, L., Effect of membrane composition on lipid

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oxidation in liposomes. Chem. Phys. Lipids 2011, 164, 158-65.

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(29) Niki E.; Kawakami, A.; Yamamoto, Y.; Kamiya, Y., Oxidation of lipids. VIII.

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Synergistic inhibition of phosphatidylchole lipoma in aqueous dispersion by vitamin E and

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vitamin. Bull. Chem. Soc. Jpn. 1985, 58, 1971-1975.


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(30) Encarnacion, A. B.; Fagutao, F.; Jintasataporn, O.; Worawattanamateekul, W.; Hirono,

516

I.; Ohshima, T., Application of ergothioneine-rich extract from an edible mushroom

517

Flammulina velutipes for melanosis prevention in shrimp, Penaeus monodon and

518

Litopenaeus vannamei. Food Res. Int. 2012, 45, 232-237.

519

(31) Jaworska, G.; Pogon, K.; Skrzypczak, A.; Bernas, E., Composition and antioxidant

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properties of wild mushrooms Boletus edulis and Xerocomus badius prepared for

521

consumption. J. Food Sci. Technol. 2015, 52, 7944-53.

522

(32) Elmastas, M.; Isildak, O.; Turkekul, I.; Temur, N., Determination of antioxidant activity

523

and antioxidant compounds in wild edible mushrooms. J. Food Compos. Anal. 2007, 20, 337-

524

345.

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(33) Choi, Y.; Lee, S. M.; Chun, J.; Lee, H. B.; Lee, J., Influence of heat treatment on the

526

antioxidant activities and polyphenolic compounds of Shiitake (Lentinus edodes) mushroom.

527

Food Chem. 2006, 99, 381-387.

528

(34) Zhang, Y.; Mills, G. L.; Nair, M. G., Cyclooxygenase inhibitory and antioxidant

529

compounds from the fruiting body of an edible mushroom, Agrocybe aegerita. Phytomedicine

530

2003, 10, 386-90.

531

(35) Barros, L.; Ferreira, M.-J.; Queirós, B.; Ferreira, I. C. F. R.; Baptista, P., Total phenols,

532

ascorbic acid, β-carotene and lycopene in Portuguese wild edible mushrooms and their

533

antioxidant activities. Food Chem. 2007, 103, 413-419.

534

(36) Barros, L.; Duenas, M.; Ferreira, I. C.; Baptista, P.; Santos-Buelga, C., Phenolic acids

535

determination by HPLC-DAD-ESI/MS in sixteen different Portuguese wild mushrooms

536

species. Food Chem. Toxicol. 2009, 47, 1076-9.

537

(37) Palacios, I.; Lozano, M.; Moro, C.; D’Arrigo, M.; Rostagno, M. A.; Martínez, J. A.;

538

García-Lafuente, A.; Guillamón, E.; Villares, A., Antioxidant properties of phenolic

539

compounds occurring in edible mushrooms. Food Chem. 2011, 128, 674-678.



540

FIGURE CAPTIONS

541

Figure 1. Micrograph of liposomes formed as viewed by bright-field (a), phase-contrast (b),

542

and fluorescence (c) microscopy.

543 544

Figure 2. Changes in total lipid HPO content of liposomes (3.0 mM phosphatidylcholine) at

545

25 °C without radical initiator (a), at 37 °C with 1.5 mM AAPH (b), and at 37 °C with 1.5

546

mM AMVN (c). Figure legends represent the following: - ○ - Liposome A [without

547

astaxanthin nor ME]; -●- Liposome B [with 40µM astaxanthin, without ME]; and -■-

548

Liposome C [with 40µM astaxanthin and 45 mM ESH in ME]. Data are presented as mean

549

± standard deviation (n=3). Abbreviations used: AST, astaxanthin; ME, mushroom extract.

550

Values with different superscript letters indicate significant differences among treatment

551

groups at each time point (p < 0.05).

552 553

Figure 3. Changes in the fatty acid content of liposomes (3.0 mM phosphatidylcholine) after

554

24 h incubation at 37 °C with 1.5mM AAPH (a) and 1.5mM AMVN (b). Figure legends

555

represent the following: -- Liposome A [without astaxanthin nor ME]; - - Liposome B

556

[with 40µM astaxanthin, without ME]; and -■- Liposome C [with 40µM astaxanthin and 45

557

mM ESH in ME]. Data are presented as mean ± standard deviation (n=3). Abbreviations used:

558

AST, astaxanthin; ME, mushroom extract. Values with different superscript letters indicate

559

significant differences among treatment groups at each time point (p < 0.01).

560 561

Figure 4. Changes in astaxanthin content of liposomes (3.0 mM phosphatidylcholine) at

562

25 °C without radical initiator (a), at 37 °C with 1.5 mM AAPH (b), and at 37 °C with 1.5

563

mM AMVN (c). Figure legends represent the following: -●- Liposome B [with 40µM

564

astaxanthin, without ME]; and -■- Liposome C [with 40µM astaxanthin and 45 mM ESH 24 ACS Paragon Plus Environment

Page 24 of 40

Page 25 of 40


565

in ME]. Data are presented as mean ± standard deviation (n=3). Asterisks represent

566

significant differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001) between treatments at each

567

time point.

568 569

Figure 5. Changes in total lipid HPO content of liposomes (3.0 mM phosphatidylcholine)

570

incubated at 37 °C with 1.5 mM AAPH (a), 1.5 mM AMVN (b), TBARS in the liposomes

571

(3.0 mM phosphatidylcholine) incubated at 37 °C with 1.5 mM AAPH (c), and 1.5 mM

572

AMVN (d). Figure legends represent the following: -○- Liposome A [without astaxanthin

573

nor ME/ESH]; -●- Liposome B [with 40µM astaxanthin, without ME/ESH]; -■- Liposome

574

C [with 40µM astaxanthin and 45 mM ESH in ME]; and -▲- D [with 40µM astaxanthin and

575

45 mM authentic ESH]. Data are presented as mean ± standard deviation (n=3). Superscript

576

letters indicate significant differences among treatment groups at each time point (p < 0.05).

577 578

Figure 6. Changes in astaxanthin contents of liposomes (3.0 mM phosphatidylcholine)

579

incubated at 37 °C with 1.5 mM AAPH (a), and 1.5 mM AMVN (b). Figure legends represent

580

the following: -●- Liposome B [with 40µM astaxanthin, without ME/ESH]; -■- Liposome

581

C [with 40µM astaxanthin and 45 mM ESH in ME]; and -▲- D [with 40µM astaxanthin and

582

45 mM authentic ESH]. Data are presented as mean ± standard deviation (n=3).

583

Abbreviations used: AST, astaxanthin; ME, mushroom extract; ESH, authentic ergothioneine.

584

Values with different superscript letters indicate significant differences among treatment

585

groups at each time point (p < 0.05).

586 587

Figure

588

phosphatidylcholine) incubated at 37 °C with 1.5 mM AAPH (a), and 1.5 mM AMVN (b).

589

Figure legends represent the following: -■- Liposome C [with 40µM astaxanthin and 45

7.

Changes

in

total

ergothioneine

contents


of

liposomes

(3.0

mM


590

mM ESH in ME]; and -■- D [with 40µM astaxanthin and 45 mM authentic ESH]. Data are

591

presented as mean ± standard deviation (n=3). Abbreviations used: AST, astaxanthin; ME,

592

mushroom extract; ESH, authentic ergothioneine. Values with asterisks represent significant

593

differences among treatment groups (p < 0.05).

594 595

Figure 8. Changes in physical appearance of liposomes observed in micrographs taken before

596

(a) and after incubation (b) at 37 °C. Abbreviations used: AST, astaxanthin; ME, mushroom

597

extract; ESH, authentic ergothioneine.

598 599

Figure 9. Relationship between the ergothioneine content in the crude ME and the DPPH

600

radical scavenging activities relative to the ESH standard equivalents. Data are presented as

601

mean ± standard deviation (n=3).

602 603

Figure 10. Thin layer chromatographic separation of authentic ergothioneine (ESH) and

604

mushroom extract (ME). Visualized by spraying by DPPH radical solution (a) and by UV

605

light irradiation at 254 nm (b).

606 607

Figure 11. DPPH radical EC50 (a) and ORAC (b) of the different antioxidant samples. Data

608

are presented as mean ± standard deviation (n=3). Abbreviations used: ME, mushroom

609

extract; ESH, authentic ergothioneine. Values with different superscript letters indicate

610

significant differences among samples (p < 0.05).

611 612


Page 26 of 40

Page 27 of 40


TABLES Table 1. Total phenolics, DPPH EC50, and oxygen radical absorbance capacity comparison of authentic ergothioneine and crude mushroom extract. Data are presented as mean ± standard deviation (n=3). Antioxidative indices

Authentic ergothioneine

Crude mushroom extract

Total phenolics (eqv µg gallic acid)

43.2 ± 1.2a

89.3 ± 0.9a

DPPH radical scavenging EC50b (µg)

4.6 ± 0.1

2.8 ± 0.1

7375.3 ± 99.7

19042.3 ± 264.1

ORACc (µmol Trolox eqv/g) a

1mM ergothioneine, bHalf maximal effective concentration. cORAC, oxygen radical absorbance

capacity.



Figure 1


Page 28 of 40

Page 29 of 40


Figure 2

Lipid hydroperoxide content (µM eqv cumene-OOH)

(a) 50

A [w/o AST/ME]

B [w/ 40µM AST, w/o ME]

40

C [w/ 40µM AST & 45mM ESH in ME]

30 20 10 0

b b a

0

aa a

aa a

aa a

3

6

bb a 9 12

15

18

Days


(b) 50 40

A B C

30

c b

20 b a 10 b a b a aa a 0 0 6

b b a

a

12

18

24

Hours


(c) 500 400

A B C

300 200 a a a aa 0 a 0 6

b b a

b ab a

b a a

100

12

18

24

Hours 29 ACS Paragon Plus Environment


Figure 3


Page 30 of 40

Page 31 of 40




Figure 4

(a)

30

Astaxanthin (µg/mL)

25

**

*

20

15 10

B [w/ 40µM AST, w/o ME]

5

C [w/ 40µM AST & 45mM ESH in ME]

0

0

(b) 30 Astaxanthin (µg/mL)

***

***

3

6

9 12 Days

15

18

B

25

C

20

* ***

15

**

***

10 5 0

0

6

12

18

24

Hours

(c) 30

B C

Astaxanthin (µg/mL)

25

20 15

*

*

10

***

5

0 0

6

12

18

24

Hours 32 ACS Paragon Plus Environment

Page 32 of 40

Page 33 of 40


Figure 5



Figure 6


Page 34 of 40

Page 35 of 40


Figure 7

(b)

(a)

0.8

C [w/ 40µM AST & 45mM ESH in ME] D [w/ 40µM AST & 45mM ESH]

Ergothionine (mMl)

Ergothionine (mMl)

0.8 0.6

0.4

*

0.2 0

C D

0.6 0.4 0.2 0

0

24

0

Hours

24

Hours



Figure 8


Page 36 of 40

Page 37 of 40


Figure 9



Figure 10


Page 38 of 40

Page 39 of 40


Figure 11

(b) Oxygen radical absorbance capacity (µmol trolox equivalent/g)

DPPH radical scavenging EC 50 (mM)

(a) 0.5 b

0.4 0.3 0.2 0.1

a

a

a

0.0

20000

c

16000 12000 8000

b

4000 a 0


a


For TOC Graphics


Page 40 of 40

Effects of Ergothioneine-rich Mushroom Extract on the Oxidative

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