Food-derived hydrophilic antioxidant ergothioneine is
distributed to the brain and exerts antidepressant effect inmice
Noritaka Nakamichi1, Keigo Nakayama1, Takahiro Ishimoto1, Yusuke Masuo1, Tomohiko
Wakayama2, Hirotaka Sekiguchi3,4, Keita Sutoh3,4, Koji Usumi3, Shoichi Iseki2 & Yukio Kato1
1

Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa 920-1192, Japan
School of Medicine, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa 920-1192, Japan
3
Life Science Institute Co. Ltd., 2-6-6 Nihombashi-Horidomecho, Chuo-ku, Tokyo 103-0012, Japan
4
L•S Corporation Co. Ltd., 3-10-1 Ningyocho-Nihonbashi, Chuo-ku, Tokyo 103-0013, Japan
2

Keywords
Antidepressant effect, depression, disposition,
ergothioneine, neuronal differentiation, OCTN1
Correspondence
Yukio Kato, Faculty of Pharmacy, Institute ofMedical, Pharmaceutical and Health Sciences,
Kanazawa University, Kakuma-machi,
Kanazawa 920-1192, Japan.
Tel/Fax: (81)-76-234-4465;
E-mail: ykato@p.kanazawa-u.ac.jp
Funding Information
This study was in part supported by Grant-inAids for Scientific Research to NN [No.
25460092] and YK [No. 24390040 and
15H04664] from the Ministry of Education,
Culture, Sports, Science and Technology ofJapan, and a grant from Hoansha Foundation(Osaka, Japan).
Received: 6 November 2015; Revised: 12
February 2016; Accepted: 16 March 2016
Brain and Behavior, 2016; 6(6), e00477,
doi: 10.1002/brb3.477

Abstract
Background: Clinically used antidepressants suffer from various side effects.
Therefore, we searched for a safe antidepressant with minimal side effects
among food ingredients that are distributed to the brain. Here, we focused on
ERGO (ergothioneine), which is a hydrophilic antioxidant and contained athigh levels in edible golden oyster mushrooms. ERGO is a typical substrate ofcarnitine/organic cation transporter OCTN1/SLC22A4, which is expressed in
the brain and neuronal stem cells, although little is known about its permeation
through the BBB (blood–brain barrier) or its neurological activity. Methods:
To clarify the exposure of ERGO to brain and the possible antidepressant-like
effect after oral ingestion, ERGO or GOME (golden oyster mushroom extract)which contains 1.2% (w/w) ERGO was mixed with feed and provided to micefor 2 weeks, and then ERGO concentration and antidepressant-like effect were
evaluated by LC-MS/MS and FST (forced swimming test) or TST (tail suspension test), respectively. Results: Diet containing ERGO or GOME greatly
increased the ERGO concentrations in plasma and brain, and significantly
decreased the immobility time in both FST and TST. The required amount of
GOME (~37 mg/day) to show the antidepressant-like effect corresponds to at
most 8 g/day in humans. In mice receiving GOME-containing diet, doublecortin-positive cells showed a significant increase from the basal level, suggesting promotion of neuronal differentiation. Conclusion: Thus, orally
ingested ERGO is transported across the BBB into the brain, where it may promote neuronal differentiation and alleviate symptoms of depression at plausibly
achieved level of daily ingestion.

Introduction
Depression is a mental illness with serious social consequences, and indeed, about 20% of the population suffersfrom the mental illness at some point in their lifetime(Browne and Lucki 2013). Various antidepressant therapeutic agents, including selective serotonin reuptake inhibitors, serotonin–noradrenaline reuptake inhibitors, andtricyclic antidepressants, are currently used for the treatment of depression. However, these drugs have multipleside effects (Bet et al. 2013; Alusik et al. 2014). Therefore,
it would obviously be desirable to develop antidepressantdrugs with few or no side effects that can be safely usedin long-term treatment. From this point of view, a usefulstrategy might be to search for antidepressant activity
among edible products using animal models of depression. In fact, it has been reported that several compounds
included in edible products exert an antidepressant-like
effect. GBE (Ginkgo biloba extract) shows its antidepressant-like effect via modulation of serotonergic anddopaminergic neurotransmission, or antioxidant enzymes
in rodents (Sakakibara et al. 2006; Rojas et al. 2011).
Hypericum perforatum extract exhibits its antidepressantlike effect by biochemical mechanisms similar to the tri-ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.
Brain and Behavior, doi: 10.1002/brb3.477 (1 of 10)
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,provided the original work is properly cited.

Ergothioneine Relieves Symptoms of Depression

cyclic antidepressants or serotonin reuptake inhibitors inrodents (Bukhari and Dar 2013).
ERGO (Ergothioneine) is naturally occurring antioxidant
derived from the diet and abundantly contained in golden
oyster mushroom (Pleurotus cornucopiae var. citrinopileatus),which is an edible mushroom belonging to the family Pleurotaceae. It is hydrophilic and membrane impermeable, but
is well absorbed from the gastrointestinal tract due to thepresence of a specific carrier-mediated transport system,
carnitine/organic cation transporter OCTN1/SLC22A4(Gr€
undemann et al. 2005; Kato et al. 2010). OCTN1 isexpressed ubiquitously in humans, including small intestine(Sugiura et al. 2010), neuronal bodies, and neurites(Lamhonwah et al. 2008). ERGO is taken up into neurons
by OCTN1 in a Na+-dependent manner (Nakamichi et al.
2012). ERGO serves as an antioxidant, scavenging hydrogen
peroxide, and peroxynitrite (Aruoma et al. 1999). It prevents
oxidative damage to DNA and protein (Zhu et al. 2011),
and protects neurons against b-amyloid-induced cytotoxicity (Yang et al. 2012). On the other hand, in depressed
patients, oxidative stress is increased and antioxidant levels
are decreased (Ng et al. 2008), and interestingly, preclinicalstudies have suggested that antioxidants may possess antidepressant-like effects in rodents (Eren et al. 2007; Zafir et al.
2009). In addition, ERGO exerts unique biological activities
in neural cells, including promotion of neuronal differentiation (Ishimoto et al. 2014). Some other antidepressant drugs(fluoxetine, imipramine, and reboxetine) also induce neurogenesis (Malberg et al. 2000; Santarelli et al. 2003; Ohiraet al. 2013), suggesting a possible association between neurogenesis and antidepressant effect. This could be supportedby the finding that mice developed depression-like
symptoms, as assessed with several behavioral tests, whenneurogenesis was inhibited (Snyder et al. 2011). On theother hand, neuronal atrophy was observed in the
hippocampal dentate gyrus of depressed patients (Cobb
et al. 2013) and might be involved in the pathophysiology ofdepression.
The experimental evidence mentioned above may imply
possible pharmacological relevance of the biological activity of ERGO with depression. However, there is no direct
evidence that ERGO has antidepressant activity. Therefore, in this study, we examined the disposition of ERGO,
its antidepressant-like effect, and the mechanism involved
in mice given diet containing ERGO or GOME (golden
oyster mushroom extract).

Materials and Methods
Materials
Powdered GOME (Aminothioneine), which contains
1.2% (w/w) ERGO, and GBE were supplied by L•S Cor-

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N. Nakamichi et al.

poration (Tokyo, Japan). Mouse control feed and Basal
Diet were purchased from Oriental Yeast Co. (Tokyo,
Japan) and TestDiet (St. Louis, MO), respectively, andcontained 0.2 lg/g and less than 0.01 lg ERGO/g chaw,
respectively, according to our liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination. Allother chemicals and reagents were of the highest purity
available and were purchased from commercial sources.

Animals
C57BL/6J mice were purchased from Sankyo Labo Service
Co. (Tokyo, Japan). About 5–8-week-old male mice werehoused under pathogen-free conditions at controlled temperature (21–25°C) with a 12 h light/dark cycle. The
lights remained on from 8:00 to 20:00, and food andwater were available ad libitum. The animals were cared
for in strict compliance with the guidelines outlined in
the National Institutes of Health Guide for the Care andUse of Laboratory Animals. All animal procedures used inthis work were approved by the Kanazawa University Animal Care Committee.

Measurement of ERGO
Mice were fed diet containing 0, 0.1, 0.3, 1, or 10% (w/w) GOME for 2 weeks, and blood was collected (Fig. 1A).
The mice were then decapitated, and brain, liver, and kidney were collected. All the tissue samples were weighed,and portions were homogenized in two volumes of distilled water. The homogenates were further diluted 5- to
4000-fold with water. The diluted homogenates, plasma,and blood were deproteinized with methanol and subjected to LC-MS/MS after centrifugation. The LC-MS/MS
system was based on a model LCMS-8040 (Shimadzu,
Kyoto, Japan). Chromatography was performed by meansof step-gradient elution (flow rate, 0.3 mL/min) as
follows: 0–0.5 min: B conc. 95%; 0.5–3.5 min: B conc.
95–30%; 3.5–5.5 min: B conc. 30%; 5.5–5.6 min: B conc.
30–95%; 5.6–8 min: B conc. 95% (A, water containing
0.1% formic acid; B, acetonitrile-containing 0.1% formicacid), using a Luna 3.0 lm HILIC column (200 A,
150 9 2.0 mm; Phenomenex, Torrance, CA) at 50°C. L(+)-Ergothioneine-d9 (Toronto Research Chemicals, Toronto, Canada) was used as the internal standard. Oral
clearance (CLoral) and tissue-to-plasma concentration
ratio (Kp) were calculated as the ratio of the daily dose of
ERGO to plasma ERGO concentration, and the ratio oftissue-to-plasma ERGO concentration measured at
2 weeks after the start of the ERGO-containing diet. To
calculate the daily dose of ERGO, the average daily foodintake of the mice was assumed to be 3.68 g (Kato et al.
2009).

ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.

N. Nakamichi et al.

The high-performance liquid chromatography (HPLC)
analysis was carried out according to the method of Katoet al. (2010) with minor modifications. All the tissue samples were weighed, and portions were homogenized in
two volumes of distilled water. Blood and homogenates
were further diluted 10–30 times with water. The diluted
samples were deproteinized with acetonitrile. The analytical column was a HILIC (4.6 9 250 mm; Nacalai Tesque,
Kyoto, Japan). The mobile phase was 0.01 mol/L ammonium acetate and acetonitrile in a ratio of 86: 14 (flowrate 1 mL/min, detection at 260 nm). Cefoperazone wasused as the internal standard.

Locomotor activity, forced swimming, open
field, and tail suspension tests
Behavioral tests were performed according to the methodsof Mouri et al. (2012) with minor modifications. Mice
were fed diet including 10% (w/w) GOME, ERGO
(120 mg/100 g diet), 10% (w/w) GBE or AA (ascorbic
acid; 120 mg/100 g diet) for 2 weeks (Fig. 1B). Pre-FST(forced swimming test; 5 min) was performed before
ingestion of each diet. After 2 weeks, to measure locomotor activity in a novel environment, each mouse wasplaced in a transparent acrylic cage. After 60 min ofhabituation, locomotion was evaluated for 5 min using avideo camera and ImageJ software (National Institutes of
Health, Bethesda, MD). Each mouse was placed in a
19 cm plastic cylinder (16 cm in diameter) containing
10 cm of water, which was maintained at 25°C, andforced to swim for 5 min. The immobility time was

Ergothioneine Relieves Symptoms of Depression

recorded on a video camera. Two days after FST, eachmouse was placed in a clear acrylic box(45 9 45 9 45 cm), which was divided into three areas(center, middle, and outer zones). The center, middle,
and outer zones were divided into 4, 12, and 20 sections,
respectively. The mouse was placed in a section of theouter zone and then allowed to freely explore its environment. The amount of time spent in each zone was evaluated for 5 min using a video camera and ImageJ software.
After the open field test, blood was collected. Brain, liver,and kidney were collected after decapitation. ERGO in
these samples was quantitated by means of HPLC.
Mice were fed diet containing 0, 0.1, 0.3, 1, or 10%
GOME for 2 weeks (Fig. 1A). The mice were suspended
from a string attached with plastic tape to the tail, andthe tail was covered with a 50 mL centrifuge tube, cut toan appropriate size, to prevent the animals climbing theirtails. The distance between the tip of the nose of eachmouse and the floor was about 40 cm. The mice weresuspended for 3 min, and the duration of immobility
from the end of min 1 to the end of min 3 was measured
using images captured on a video camera. In this procedure, the immobility time of mice treated with the tricyclic antidepressant imipramine was clearly decreased
compared to that of control mice (data not shown).

Repeated restraint stress
The restraint stress procedure was carried out according
to Kim et al. (2011) with minor modifications. Mice wererandomly divided into four groups: nonstressed mice
given control diet (n = 6), nonstressed mice given 10%
GOME diet (n = 6), stressed mice given control diet(n = 8), and stressed mice given 10% GOME diet(n = 8). Stress was imposed by forcing the animals into
an immobilizer (Natsume Seisakusho, Tokyo, Japan) for
4 h (10:00–14:00) each day, for 21 consecutive days
(Fig. 1C). The apparatus fitted tightly around the mice,
which were essentially unable to move. Body weight wasmeasured every week. After 3 weeks, the spleens were
removed and weighed.

Bromodeoxyuridine labeling and tissue
preparation

Figure 1. Schematic representation of experimental schedule. Eachtimeline shows experimental schedule for (A) Figs. 2, 5, (B) Figs. 3, 4,or (C) Fig. 6.

ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.

BrdU (Bromodeoxyuridine) labeling and tissue preparation were carried out according to the methods of Ogita
et al. (2005) with minor modifications. To detect newcells, BrdU (50 mg/10 mL/kg) dissolved in PBS (phosphate-buffered saline) was intraperitoneally injected intomice after repeated restraint stress experiments. The mice
were given two consecutive injections of BrdU at 24 hand 12 h before decapitation. Under deep anesthesia, theBrain and Behavior, doi: 10.1002/brb3.477 (3 of 10)

Ergothioneine Relieves Symptoms of Depression

mice were intracardially perfused with phosphate buffer
containing 4% paraformaldehyde. The brains were quickly
removed, and postfixed with the same fixative solution at
4°C overnight. Postfixed brains were embedded in paraffin, then coronal sections of 4-lm thickness were cut witha microtome and placed on Silan-coated slide glasses. The
paraffin-embedded brain sections were deparaffinizedwith xylene, rehydrated with ethanol at graded decreasing
concentrations of 100–50% (vol/vol), and finally washedwith PBS.

Immunostaining
Immunostaining was carried out according to the methodof Jung et al. (2013) with minor modifications. Sections
were incubated in sodium citrate at 92°C for 15 min, 0.3%
Tween 20 in PBS for 20 min, and 3% hydrogen peroxide
in PBS for 30 min. To detect BrdU incorporation, sections
were further treated with 2 mol/L HCl at 35°C for 30 min.
They were blocked with 4% Block Ace, then incubatedwith a primary antibody against BrdU (1:200; Abcam,
Cambridge, MA) or DCX (doublecortin; 1:200; Santa Cruz
Biotechnology, Santa Cruz, CA) at 4°C overnight, reactedwith a biotinylated secondary antibody against rat or goat
IgG (1:250) for 1 h at room temperature, and then reactedwith streptavidin-biotin-peroxidase complex for 30 min atroom temperature. The peroxidase reaction was visualized
by incubation of the sections in diaminobenzidine/hydrogen peroxide solution. The sections were then washedthree times with PBS for 3 min, mounted on gelatincoated slides, dehydrated in an ascending alcohol series,
and cleared in xylene. Immunopositive cells in hippocampal dentate gyrus were observed using a light microscopeand counted with Hybrid cell-counting software (KEYENCE, Osaka, Japan). To obtain the total number of
immunopositive cells per dentate gyrus, the area of dentate
gyrus was measured with ImageJ software.

Statistical analysis
Data are expressed as the mean  SEM. The statisticalsignificance of differences was determined by means ofStudent’s t-test, one-way or repeated measures ANOVA,
followed by the appropriate post hoc tests (Tukey–Kramer or Dunnett’s test), and P < 0.05 was regarded asdenoting a significant difference.

Results
Brain distribution of ERGO ingested in dietFirst, we examined the distribution of ERGO to the brain
parenchyma across the BBB (blood–brain barrier). We

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N. Nakamichi et al.

measured the concentration of ERGO in blood, plasma,
brain, liver, and kidney at 2 weeks after the mice had
been started on diet containing 0.1–10% GOME (w/w;
Fig. 1A). ERGO concentration in each tissue was dosedependently increased [Plasma F(4,15) = 133.9, P < 0.01;
Brain F(4,15) = 447.8, P < 0.01; Blood F(4,15) = 47.0,
P < 0.01; Liver F(4,15) = 87.3, P < 0.01; Kidney F(4,15) = 298.9, P < 0.01] (Fig. 2A and B). ERGO concentration in the brain was similar to that in plasma at every
dosage (Fig. 2A). ERGO concentrations in the brain andliver of the group given diet containing 10% GOME wereat most 1.5 times higher than those in animals given dietcontaining 1% GOME (w/w), indicating nonlinear behavior. A similar tendency was seen in the case of plasma,
blood, and kidney, although the differences in ERGO
concentration between the two doses were smaller(Fig. 2A and B). To directly evaluate saturation of ERGO
disposition, CLoral and Kp of ERGO were calculated. As
the dosage of GOME was increased, CLoral increased(Fig. 2C), while Kp decreased (Fig. 2D). The decrease inKp occurred at the plasma ERGO concentration of about
1–2 lg/mL (4–8 lmol/L), which is close to the Michaelis
constant (Km) for ERGO of OCTN1 (~5 lmol/L, Katoet al. 2010). These results indicate that ERGO is highly
distributed to the brain after oral ingestion.

Antidepressant-like effect of ERGO
Next, we examined the antidepressant-like effect of
ERGO. GOME or ERGO was fed to mice in the diet for
2 weeks, and FST, which is a well-established tool forassessing antidepressant-like activity in rodents, was performed (Fig. 1B). The immobility time of mice given dietcontaining 10% GOME was significantly lower than thatof mice given normal diet [F(5,70) = 2.9, P < 0.05](Fig. 3A). To examine whether this activity of GOME isdue to ERGO present in GOME, mice were fed diet containing authentic ERGO, the amount of which (120 mg
ERGO/100 g diet) was equivalent to that in diet containing 10% GOME. The immobility time in mice given dietcontaining 10% GOME was similar to that given dietcontaining the equivalent amount of authentic ERGO
(Fig. 3A). In addition, ERGO concentration in the brain
in mice given diet containing 10% GOME was also similar to that given diet containing the equivalent amount ofauthentic ERGO (Fig. 4). These data suggested that theantidepressant-like effect of GOME is predominantly due
to ERGO contained in the extract.
Ginkgo biloba extract, which is known to have anantidepressant-like effect in rodents (Sakakibara et al.
2006; Rojas et al. 2011) and was used as a positive control
in this study, also decreased the immobility time, whereas
AA, a hydrophilic antioxidant like ERGO, had no effectª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.

N. Nakamichi et al.

on the immobility time (Fig. 3A). To rule out the possibility that the decrease in the immobility time was due toan increase in motor activity, a locomotor activity test
was performed. We found no difference in motor activity
among the groups given control diet and diets containing
GOME, ERGO, GBE, or AA for 2 weeks (Fig. 3B), suggesting that differences in motor activity cannot accountfor the differences in immobility time observed in thisstudy.
To examine the antianxiety effect of ERGO in addition
to the antidepressant-like effect, we employed an open
field test. The residence time of mice in each zone showedno significant difference between the control group andthe groups given diet containing GOME, ERGO or AA(Fig. 3C). On the other hand, residence time in the centerzone after ingestion of GBE was significantly longer thanthat of the control group [F(4,40) = 7.4, P < 0.01](Fig. 3C). A similar tendency was observed in the middle
zone (Fig. 3C). Concomitantly, residence time in theouter zone was significantly shorter in the GBE ingestion
group than the control group [F(4,40) = 4.9, P < 0.01]

Ergothioneine Relieves Symptoms of Depression(Fig. 3C). These results suggest that GBE may have anantianxiety effect, but ERGO does not.
To confirm the antidepressant-like effect of ERGO andexamine its dose dependency, TST (tail suspension test)
was also performed (Fig. 1A). The immobility time ofmice given diet containing 1% or 10% GOME wasdecreased compared to that of mice given normal diet [F(4,49) = 3.8, P < 0.01] (Fig. 5), supporting the idea thatERGO possesses an antidepressant-like effect. These
results suggested that ingestion of 1% or more of GOME,
namely 12 mg/100 g diet of ERGO or more, for 2 weeks
has an antidepressant-like effect.

Promotion of neuronal differentiation by
ERGO
Some antidepressant drugs promote neurogenesis, andthis activity may be related to their antidepressant effect.
So, we next examined promotion of neurogenesis by
ERGO. It has been reported that stress decreases neurogenesis in the hippocampus (Kim et al. 2011). Thus, pro-

Figure 2. ERGO (Ergothioneine)concentrations in the body andpharmacokinetic parameters for ERGO inmice fed diet containing GOME. (A) ERGO
concentrations in plasma and brain, and (B)those in blood, liver, and kidney were
measured by LC-MS/MS in mice given
normal diet (white columns), or dietcontaining 0.1% (light gray columns),
0.3% (gray columns), 1% (dark graycolumns), or 10% (black columns) GOME
for 2 weeks. (C) CLoral of ERGO wascalculated from plasma concentration anddaily intake of ERGO. (D) Kp in blood(open squares), brain (closed circles), liver(closed triangles), and kidney (closed
diamonds) was calculated from the ERGO
concentrations in plasma and each tissue.
Each point and vertical bar represents themean  SEM. (n = 4). *P < 0.05,
significant difference from thecorresponding control value.

ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.

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Ergothioneine Relieves Symptoms of Depression

motion of neurogenesis by ERGO may be more clearlyobserved under a stress condition rather than a normal
one. In addition, since we expected to examine whether
ERGO could reverse depression-like symptoms by stress,
the repeated restraint stress procedure by Kim et al.
(2011) was employed in this study. The immobility timeof the stressed mice was significantly longer than that of
the nonstressed mice in FST and TST (Kim et al. 2011).
Therefore, 10% GOME was fed to mice for 3 weeks
under either a normal or a stress condition (Fig. 1C).
Spleen weight [F(3,8) = 7.6, P < 0.05] and body weight[First week F(3,16) = 7.8, P < 0.01; Second week F(3,16) = 8.8, P < 0.01; Third week F(3,16) = 8.9,
P < 0.01] in the stress group was significantly less than in
the control group (Fig. 6A and B), confirming the

N. Nakamichi et al.

expected effect of stress loading. However, behavioral
changes were not observed between two groups in TST(data not shown). Administration of GOME and/or stressloading did not affect the number of cells positive for
BrdU, a proliferating cell marker (Fig. 6C and E). The
number of cells positive for DCX, an immature neuronalmarker, was increased in the GOME group compared tothe control group, regardless of stress [F(3,41) = 3.7,
P < 0.05] (Fig. 6D and F), suggesting that ERGO may
promote neuronal differentiation in vivo.

Figure 4. Comparison of ERGO (ergothioneine) concentrations in the
body after oral ingestion of GOME or ERGO. (A) ERGO concentrationin brain, and (B) those in blood, liver, and kidney were measured by
HPLC in mice given normal diet (white columns), or diet containing
10% GOME (black columns), or authentic ERGO (dark gray columns)
for 2 weeks. Each vertical bar represents the mean  SEM. (n = 6).
*P < 0.05, significant difference from the corresponding control
value.

Figure 3. Behavioral test in mice given diet containing GOME, ERGO
(ergothioneine), GBE (Ginkgo biloba extract), or AA. Mice were given
normal diet (white columns), or diet containing 10% GOME (blackcolumns), authentic ERGO (dark gray columns), GBE (dotted columns),
or AA (diagonal columns) for 2 weeks. (A) Immobility time in 5 minof FST was measured in mice. Increase in immobility time wascalculated from the values after each diet treatment and before thestart of the treatment. Each value is the mean  SEM. (Control:
n = 11, GOME: n = 11, ERGO: n = 6, GBE: n = 6, AA: n = 6). (B)Locomotor activity of mice. Each mouse was placed in an individual
cage, and locomotion was assessed for 5 min after habituation. Eachvalue is shown as mean  SEM. (n = 6). (C) Time spent in the center,middle, and outside zones was measured. Each mouse was placed ina novel chamber for 5 min. Each value is the mean  SEM. (Control:
n = 15, GOME: n = 15, ERGO: n = 6, GBE: n = 3, AA: n = 6).
*P < 0.05, significant difference from the corresponding control
value.

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Figure 5. Dose-dependent reduction in immobility time in TST after
oral ingestion of GOME. Mice were given normal diet (white column,n = 15), or diet containing 0.1% (light gray column, n = 6), 0.3%
(gray column, n = 6), 1% (dark gray column, n = 12), or 10% (blackcolumn, n = 15) GOME for 2 weeks and subjected to TST. Immobility
time in 2 min was measured. Each value is the mean  SEM.
*P < 0.05, significant difference from the corresponding control
value.

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N. Nakamichi et al.

Ergothioneine Relieves Symptoms of Depression

Figure 6. Bromodeoxyuridine
incorporation or DCX expression in mice
given diet containing GOME under achronic stress condition. Mice were given
normal diet ((A), (B) white symbols, (C), (D)upper left panels) or diet containing 10%
GOME ((A), (B) diagonal symbols, (C), (D)upper right panels) under a normal
condition, or normal diet ((A), (B) graysymbols, (C), (D) lower left panels), or dietcontaining 10% GOME ((A), (B) black
symbols, (C), (D) lower right panels) undera chronic stress condition, for 21 days.
Stress was imposed by forcing the animals
into an immobilizer for 4 h/day. (A) Spleen
weight was measured and normalized bybody weight. Each value is shown as themean  SEM. (n = 3). (B) Change in bodyweight of each mouse was measured every
week and normalized with that at the startof restraint stress. Each value is shown as
the mean  SEM. (n = 5). Typical phasecontrast micrographs of (C) BrdUincorporating cells and (D) DCX-expressing
cells in each group are shown. The
numbers of (E) BrdU-incorporating cellsand (F) DCX-expressing cells were counted
by using a cell image analyzer in at least
two areas from four to eight individual
mice in each group. Vertical bar represents
the mean  SEM. (Control: n = 8, GOME:
n = 8, Control + Stress: n = 15,
GOME + Stress: n = 14). *P < 0.05,
significant difference from thecorresponding control value.

Discussion
The present findings indicate that the food-derived
antioxidant ERGO is highly distributed to the brain
across the BBB after oral ingestion and exert the antidepressant-like effect despite its hydrophilic property. ERGO
has been detected in the brain of mice under usual dietary conditions (Kato et al. 2010), but there has been noª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.

direct evidence that ERGO can permeate the BBB and be
distributed to the brain. In this study, we clarified therelationship between oral intake and brain parenchymal
content of ERGO (Fig. 2). The observed increase in CLoral(Fig. 2C) and decrease in Kp in various organs including
the brain (Fig. 2D) with increasing level of ERGO in thediet suggest saturation in the disposition kinetics of
ERGO in the body. In particular, since the decrease in Kp

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Ergothioneine Relieves Symptoms of Depression

was observed at the plasma concentration close to theKm (5 lmol/L) of ERGO for OCTN1 (Fig. 2D), it seems
likely that the distribution of ERGO to each organ ismainly governed by OCTN1. However, OCTN1 is minimally expressed in the BBB (Okura et al. 2008). Therefore, ERGO may be delivered to the brain parenchyma bytransporters other than OCTN1. OCTN1 is, on the other
hand, functionally expressed in brain neurons (Nakamichi
et al. 2012), and therefore, the saturation of brain distribution of ERGO (Fig. 2D) may reflect saturation of
uptake into neurons after passage through the BBB.
The oral ingestion of diet containing 10% GOME andthe same amount of authentic ERGO as it had similareffects on immobility time of mice in FST (Fig. 3A), andthe concentration of ERGO in the brain was also similar
in mice given the two diets (Fig. 4A), suggesting thatthe antidepressant-like effect of GOME may be predominantly due to ERGO. ERGO shows low membrane permeability due to its high water-solubility, but is a goodsubstrate of OCTN1. We have previously reported that
disposition of food-derived ERGO under normal dietaryconditions is mainly regulated by OCTN1, since ERGO
is present in various organs of wild-type mice, butabsent in those of octn1 gene knockout mice (Kato et al.
2010). In this study, we found that even when a higher
amount of ERGO was loaded into mice by ingestion of
GOME, the ERGO disposition was still governed by
OCTN1, showing nonlinear kinetics in plasma concentration and tissue distribution (Fig. 2). This result is
consistent with the idea that disposition of ERGO iscontrolled by the transporter. Such transporter-mediateddistribution of ERGO to the body is clearly distinct fromthe case of flavonoids, major components of GBE exerting various biological activities, which do not have specific uptake transporters in the body. On the other hand,ingestion of AA, which is a water-soluble antioxidant
like ERGO, did not decrease the immobility time ofmice in FST (Fig. 3A). AA minimally permeates the BBB
and is distributed to the brain only in the form of oxidized dehydroascorbic acid (Gess et al. 2011). Moreover,AA did not affect neuronal differentiation, whereas
ERGO promoted it in cultured neural stem cells (Ishimoto et al. 2014). These may be the reason why oral
ingestion of AA failed to show any antidepressant-like
effect.
Ginkgo biloba extract also decreased the immobility
time of mice as effectively as ERGO in FST (Fig. 3A). It
has already been reported that GBE exhibits its antidepressant-like effect via modulation of serotonergic anddopaminergic neurotransmission, or antioxidant enzymes
in rodents (Sakakibara et al. 2006; Rojas et al. 2011).
Spontaneous locomotor activity showed no difference
among the groups given GOME, ERGO, and GBE in thisBrain and Behavior, doi: 10.1002/brb3.477 (8 of 10)

N. Nakamichi et al.

study (Fig. 3B), indicating that the decrease in immobility
time after ingestion of these materials is not due to anincrease in motor activity. On the other hand, in the
open field test to evaluate anxiety, GBE increased the residence time in the center zone and decreased that in theouter zone compared to the control group, as reported
previously (Ma et al. 2012), whereas GOME and ERGO
had no such effect (Fig. 3C). Mice usually prefer to stayon the wall side (outer zone) in a novel environment(Crawley 1999), and administration of drugs withantianxiety and/or excitatory effects, such as cocaine,decreases the residence time in the outer zone (Prut andBelzung 2003; Carey et al. 2008). Thus, since ERGO
shows the different pattern of neurological effect from
GBE, and further studies are required to clarify whether
ERGO exerts the antidepressant-like effect via a different
mechanism from GBE.
The concentration of ERGO in the brain wasincreased dose-dependently after oral ingestion of GOME
[13.1 lg/mL (57 lmol/L) in the group given 1% GOME
diet and 21.5 lg/mL (94 lmol/L) in the group given
10% GOME diet (Fig. 2A)], suggesting that 57 lmol/L
ERGO is a sufficient concentration to exert an antidepressant-like effect. On the other hand, the concentrationof ERGO in blood of mice given control diet was 37 lg/
mL (161 lmol/L), which is similar to the blood concentration in healthy volunteers, 40 lg/mL (174 lmol/L)(Kato et al. 2010). In addition, GOME used in this study
contains approximately 1.2% ERGO and mice (20 gbody weight) took 3.68 g of diet per day (Kato et al.
2009), so that daily intake of ERGO was about 22 mg/
kg body weight in mice given diet containing 1%
GOME. The body surface area per weight of human isabout 15.4 times smaller than that of mice (Davies andMorris 1993), so ERGO may exert an antidepressanteffect in human at 1/15.4 of the concentration in mice.
For a 70 kg human, the required intake of ERGO per
day would be 22 9 70/15.4 = 100 mg (about 8 g
GOME), and this seems to be an amount that could be
easily ingested. Thus, it is plausible that an antidepressant effect in humans could be achieved by taking moderate amounts of GOME.
This study is the first to indicate that ERGO-containing
diet GOME promotes neuronal differentiation in the hippocampus dentate gyrus in vivo (Fig. 6), and this result iscompatible with the previous report that exposure of cultured neural stem cells to ERGO markedly increased thenumber of cells immunoreactive for the neuronal markerbIII-tubulin, suggesting that ERGO promotes neuronaldifferentiation in vitro (Ishimoto et al. 2014). In thisstudy, whereas an ERGO-containing diet increased DCXpositive neuronal cells (Fig. 6E and F), the diet did notaffect the number of BrdU-positive proliferating cellsª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.

N. Nakamichi et al.

(Fig. 6C and D). ERGO suppresses proliferation of neuralstem cells by reducing cellular oxidative stress at leastin vitro since exposure of cultured neural stem cells to
ERGO or other antioxidants (edaravone and AA) led to asignificant decrease in the area of neurospheres, which areformed from clusters of proliferating cells, with concomitant elimination of intracellular reactive oxygen species(Ishimoto et al. 2014). Such two different effects of
ERGO, suppression of proliferation and promotion ofneuronal differentiation, on neural stem cells (Ishimoto
et al. 2014) could be one of the reasons for the discrepancy of the number between BrdU- and DCX-positive
cells (Fig. 6). Several antidepressant drugs such as fluoxetine, imipramine, and reboxetine, promote neurogenesis(Malberg et al. 2000; Santarelli et al. 2003; Ohira et al.
2013). Thus, ERGO may exert its antidepressant-like
effect at least partially via promotion of neuronal differentiation in vivo. However, further studies would be
required to confirm this.
In conclusion, orally ingested ERGO is transported
across the BBB into the brain and exerts the antidepressant-like effect in mice. Since ERGO is abundantly contained in edible golden oyster mushrooms, and its
disposition is limited by the uptake transporter OCTN1,
it is likely to be safe for long-term use with few or noside effects in human. Furthermore, ERGO could exert an
antidepressant effect at plausibly achieved level of daily
ingestion according to the estimate from the present
results. Thus, we suggest that ERGO is a promising candidate for prevention and/or treatment of depression. In
the future, however, we need to show that oral ingestionof ERGO or its derivatives prevents onset of depression
in stressed people and alleviates their symptoms in
depressed patients.

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Acknowledgment
The authors thank L. Ishida in Kanazawa University fortechnical assistance.

Conflict of Interest
There is no conflict of interest statement.
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ª 2016 The Authors. Brain and Behavior published by Wiley Periodicals, Inc.