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Citation: Hsu, C.-H.; Liao, E.-C.;
Chiang, W.-C.; Wang, K.-L.
Antioxidative Activities of
Micronized Solid-State Cultivated
Hericium erinaceus Rich in Erinacine A
against MPTP-Induced Damages.
Molecules 2023,28, 3386. https://
doi.org/10.3390/molecules28083386
Academic Editors: Hinanit Koltai
and Jih-Jung Chen
Received: 17 February 2023
Revised: 1 April 2023
Accepted: 3 April 2023
Published: 12 April 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
molecules
Article
Antioxidative Activities of Micronized Solid-State Cultivated
Hericium erinaceus Rich in Erinacine A against
MPTP-Induced Damages
Chun-Hsien Hsu 1,2,3,4 , En-Chih Liao 5,6 , Win-Chin Chiang 7and Kai-Lee Wang 8, *
1Department of Family Medicine, Taipei City Hospital, Heping Fuyou Branch, Taipei 100, Taiwan
2Department of Family Medicine, Cardinal Tien Hospital, New Taipei 231, Taiwan
3School of Medicine, College of Medicine, Fu Jen Catholic University, New Taipei 242, Taiwan
4General Education Center, University of Taipei, Taipei 100, Taiwan
5Department of Medicine, MacKay Medical College, New Taipei 252, Taiwan
6Institute of Biomedical Sciences, MacKay Medical College, New Taipei 252, Taiwan
7Jowin Biopharma Inc., New Taipei 221, Taiwan
8Department of Nursing, Ching Kuo Institute of Management and Health, Keelung 203, Taiwan
*Correspondence: d49505002@gm.ym.edu.tw or kellywang111@gmail.com; Tel.: +866-2-24372093 (ext. 286)
Abstract:
The Lion’s mane mushroom (Hericium erinaceus, HE) is a traditional medical mushroom
with high nutritional and economic value. HE possesses anticancer, antimicrobial, antioxidant, im-
munomodulating, neurotrophic, and neuroprotective activities. The present study evaluated the
protection and antioxidative activities of micronized mycelium of HE (HEM) in mice treated with
1-methyl-4-phenylpyridinium (MPTP). HEM was cultivated via solid-state fermentation and mi-
cronized using cell wall-breaking technology to increase its bioavailability when ingested. Erinacine
A, the bioactive compound in the HEM, played a pivotal role in antioxidant defense. We found that
micronized HEM could recover the dopamine level in the mice striatum in a dose-dependent manner
that had been greatly reduced during MPTP treatment. Moreover, the malondialdehyde (MDA) and
carbonyl levels were reduced in the livers and brains of the MPTP + HEM-treated groups compared
with the MPTP group. Additionally, antioxidant enzyme activities, including catalase, superoxide
dismutase (SOD), glucose-6-phosphate dehydrogenase (G6PDH), and glutathione reductase (GRd),
were elevated after the administration of HEM in MPTP-treated mice in a dose-dependent manner.
Taken together, our data indicate that HEM cultivated via solid-state fermentation and processed
with cell wall-breaking technology showed an excellent antioxidant efficacy.
Keywords:
hericium erinaceus mycelium; erinacine A; antioxidant; Parkinson’s disease; reactive
oxygen species
1. Introduction
Neurological and neurodegenerative diseases, such as Parkinson’s disease (PD),
Alzheimer’s disease (AD), and Huntington’s disease, are highly debilitating and pose signif-
icant threats to public health [
1
]. Considering the increasing older population worldwide,
neurodegenerative diseases are bound to increase over time, especially since no medica-
tion has become available to prevent or reverse the neurodegeneration induced by these
diseases. Various studies have underlined the role of oxidative stress and mitochondrial
impairment on initiating the cascade of events leading to degeneration of dopaminergic
neurons [
2
]. PD is characterized by the progressive loss of dopaminergic neurons, at least
partly due to increased reactive oxygen species (ROS) in mitochondria, lipids peroxidation,
DNA abnormalities, and proteins oxidation [
3
,
4
]. Toxicants that can increase oxidative
stress of the substantia nigra, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP),
have been used to induce PD in mice. Therefore, antioxidants capable of counteracting
oxidative stress may provide a novel potential therapy to combat PD [5].
Molecules 2023,28, 3386. https://doi.org/10.3390/molecules28083386 https://www.mdpi.com/journal/molecules
Molecules 2023,28, 3386 2 of 12
Hericium erinaceus (HE), also known as Lion’s mane mushroom or monkey’s head
mushroom, is a widespread pharmaceutical and edible fungus found in several Asian
countries. The use of HE is safe and harmless even for extended periods of time, and is
traditionally used to treat peptic ulcers and acute gastritis [
6
]. HE contains a large number
of bioactive compounds, including alkaloids, flavonoids, terpenes, polysaccharides, and
metal-chelating agents [
7
]. Recent studies have demonstrated that HE and its extracts
possess a wide range of benefits, such as anticancer, antimicrobial, antidiabetic, antioxidant,
antiaging, antihyperglycemic, antihyperlipidemic, gastroprotective, immunomodulating,
and neuroprotective activity [8–11].
Pertaining to its neuroprotective effects, which have been associated with the JNK/p38/
NF-
κ
B/CHOP/Fas/Bax signaling pathways [
12
], HE has been suggested to interrupt the
apoptosis cascade by inhibiting ROS production [
13
,
14
]. HE has also been found to re-
duce anxiety and depression through the promotion of hippocampal neurogenesis [
15
].
HE and its bioactive ingredients can promote nerve growth factor expression, thereby
improving cognitive impairments such as PD and AD [
16
]. A recent study has also con-
firmed that PD-induced neuroinflammation and oxidative stress could be inhibited by
HE [
17
]. Erinacine A (EA), a bioactive compound extracted by ethanol from HE, passes
through the blood–brain barrier and possesses neuroprotective properties by ameliorat-
ing lipopolysaccharide-induced inflammation [
18
,
19
]. EA also provided protection from
neurotoxicity by alternating the apoptosis and cell death signaling pathways [
20
]. It has
also been confirmed that EA stimulates the production of the nerve growth factor from
astroglia, thereby promoting and maintaining neural growth [
21
]. These studies clearly
demonstrate that HE possesses distinct neuroprotective activity.
In the present study, HE mycelia (HEM), which is cultivated under solid-state fer-
mentation, was micronized to increase its bioavailability. The protective and antioxidant
activities of HEM were evaluated in male C57BL/6Narl mice under MPTP treatment.
2. Results
2.1. MPTP Animal Model Set Up
The MPTP model of PD was induced, as described previously [
22
]. Mice were ran-
domly assigned into five groups, as shown in Figure 1: the control group, the MPTP group
(20 mg/kg/day for the first 5 days; Tokyo Chemical Industry, TCI, Tokyo, Japan), and
MPTP + different dosages of HEM groups (0.1 g/kg, 0.3 g/kg, and 1 g/kg, respectively).
Mice received intraperitoneal (i.p.) injection of MPTP, and the same quantity of saline was
given in the control group. Mice were orally gavaged with H2O or HEM for 30 days.
Molecules 2023, 28, x FOR PEER REVIEW 2 of 13
(MPTP), have been used to induce PD in mice. Therefore, antioxidants capable of coun-
teracting oxidative stress may provide a novel potential therapy to combat PD [5].
Hericium erinaceus (HE), also known as Lion’s mane mushroom or monkey’s head
mushroom, is a widespread pharmaceutical and edible fungus found in several Asian
countries. The use of HE is safe and harmless even for extended periods of time, and is
traditionally used to treat peptic ulcers and acute gastritis [6]. HE contains a large number
of bioactive compounds, including alkaloids, flavonoids, terpenes, polysaccharides, and
metal-chelating agents [7]. Recent studies have demonstrated that HE and its extracts pos-
sess a wide range of benefits, such as anticancer, antimicrobial, antidiabetic, antioxidant,
antiaging, antihyperglycemic, antihyperlipidemic, gastroprotective, immunomodulating,
and neuroprotective activity [8–11].
Pertaining to its neuroprotective effects, which have been associated with the
JNK/p38/NF-κB/CHOP/Fas/Bax signaling pathways [12], HE has been suggested to inter-
rupt the apoptosis cascade by inhibiting ROS production [13,14]. HE has also been found
to reduce anxiety and depression through the promotion of hippocampal neurogenesis
[15]. HE and its bioactive ingredients can promote nerve growth factor expression, thereby
improving cognitive impairments such as PD and AD [16]. A recent study has also con-
firmed that PD-induced neuroinflammation and oxidative stress could be inhibited by HE
[17]. Erinacine A (EA), a bioactive compound extracted by ethanol from HE, passes
through the blood–brain barrier and possesses neuroprotective properties by ameliorat-
ing lipopolysaccharide-induced inflammation [18,19]. EA also provided protection from
neurotoxicity by alternating the apoptosis and cell death signaling pathways [20]. It has
also been confirmed that EA stimulates the production of the nerve growth factor from
astroglia, thereby promoting and maintaining neural growth [21]. These studies clearly
demonstrate that HE possesses distinct neuroprotective activity.
In the present study, HE mycelia (HEM), which is cultivated under solid-state fer-
mentation, was micronized to increase its bioavailability. The protective and antioxidant
activities of HEM were evaluated in male C57BL/6Narl mice under MPTP treatment.
2. Results
2.1. MPTP Animal Model Set Up
The MPTP model of PD was induced, as described previously [22]. Mice were ran-
domly assigned into five groups, as shown in Figure 1: the control group, the MPTP group
(20 mg/kg/day for the first 5 days; Tokyo Chemical Industry, TCI, Tokyo, Japan), and
MPTP + different dosages of HEM groups (0.1 g/kg, 0.3 g/kg, and 1 g/kg, respectively).
Mice received intraperitoneal (i.p.) injection of MPTP, and the same quantity of saline was
given in the control group. Mice were orally gavaged with H
2
O or HEM for 30 days.
Figure 1. Treatment flow chart.
Figure 1. Treatment flow chart.
2.2. Particle Size Analysis
Our results and the corresponding electron microscope are shown in Figure 2. The
volumetric mean diameters of the particles from two different batches were 35.93 µm and
12.35 µm, respectively.
Molecules 2023,28, 3386 3 of 12
Molecules 2023, 28, x FOR PEER REVIEW 3 of 13
2.2. Particle Size Analysis
Our results and the corresponding electron microscope are shown in Figure 2. The
volumetric mean diameters of the particles from two different batches were 35.93 µm and
12.35 µm, respectively.
D
50
= 35.93 µm
D
50
= 12.35 µm
Figure 2. Particle size distribution of the HEM powder. D
50
represents the mean volumetric particle
size.
2.3. HPLC Analysis
The chromatograms of HEM generated using HPLC are displayed in Figure 3. The
retention time of 31.867 min corresponded to erinacine A, which was identified by com-
parison with prepared standards (kindly provided by Jowin Biopharma Inc, New Taipei
City, Taiwan). The peak contents were quantified from the established calibration curve
as erinacine A is 30 µg/g dry weight of HEM.
Figure 2.
Particle size distribution of the HEM powder. D
50
represents the mean volumetric
particle size.
2.3. HPLC Analysis
The chromatograms of HEM generated using HPLC are displayed in Figure 3. The
retention time of 31.867 min corresponded to erinacine A, which was identified by compar-
ison with prepared standards (kindly provided by Jowin Biopharma Inc.,
New Taipei City
,
Taiwan). The peak contents were quantified from the established calibration curve as
erinacine A is 30 µg/g dry weight of HEM.
Molecules 2023, 28, x FOR PEER REVIEW 3 of 13
2.2. Particle Size Analysis
Our results and the corresponding electron microscope are shown in Figure 2. The
volumetric mean diameters of the particles from two different batches were 35.93 µm and
12.35 µm, respectively.
D
50
= 35.93 µm
D
50
= 12.35 µm
Figure 2. Particle size distribution of the HEM powder. D
50
represents the mean volumetric particle
size.
2.3. HPLC Analysis
The chromatograms of HEM generated using HPLC are displayed in Figure 3. The
retention time of 31.867 min corresponded to erinacine A, which was identified by com-
parison with prepared standards (kindly provided by Jowin Biopharma Inc, New Taipei
City, Taiwan). The peak contents were quantified from the established calibration curve
as erinacine A is 30 µg/g dry weight of HEM.
Figure 3.
HPLC chromatogram of HEM. The retention time of the diterpenoid erinacine A peak was
within the range of 31.000–33.000 min.
2.4. Neuroprotective Effects of HEM on MPTP-Treated Mice
To evaluate the neuroprotective effect of HEM on ameliorating MPTP-induced cyto-
toxicity and oxidative stress, the dopamine levels in the substantia nigra were determined.
MPTP was the agent that decreased the dopamine level in the brain of mice to 1535 ng/g,
as shown in Figure 4. Once coadministering mice with HEM powder at different levels,
the dopamine level was increased to 2897, 3535, and 4527 ng/g at 0.1, 0.3, and 1.0 g/kg,
respectively. The HEM powder could effectively increase the dopamine level in the brain
Molecules 2023,28, 3386 4 of 12
to 2–3 times at 0.3 and 1.0 g/kg. Our findings indicate that the HEM powder was able to re-
verse the MPTP-induced dopamine reduction in the tested mice brain in a dose-dependent
manner, as shown in Figure 4.
Molecules 2023, 28, x FOR PEER REVIEW 4 of 13
Figure 3. HPLC chromatogram of HEM. The retention time of the diterpenoid erinacine A peak was
within the range of 31.000–33.000 min.
2.4. Neuroprotective Effects of HEM on MPTP-Treated Mice
To evaluate the neuroprotective effect of HEM on ameliorating MPTP-induced cyto-
toxicity and oxidative stress, the dopamine levels in the substantia nigra were determined.
MPTP was the agent that decreased the dopamine level in the brain of mice to 1535 ng/g,
as shown in Figure 4. Once coadministering mice with HEM powder at different levels,
the dopamine level was increased to 2897, 3535, and 4527 ng/g at 0.1, 0.3, and 1.0 g/kg,
respectively. The HEM powder could effectively increase the dopamine level in the brain
to 2–3 times at 0.3 and 1.0 g/kg. Our findings indicate that the HEM powder was able to
reverse the MPTP-induced dopamine reduction in the tested mice brain in a dose-depend-
ent manner, as shown in Figure 4.
Dopamine (fold)
0
1
2
3
4
***
***
***
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
Figure 4. Dopamine levels in the substantia nigra of treated and untreated mice. A significant in-
crease in the dopamine level was found in the HEM cotreated mice compared with the MPTP group
in a dose-dependent manner. *** p < 0.001 compared with the MPTP−treated group.
We thereafter performed immunostaining for tyrosine hydroxylase (TH), the enzyme
that catalyzes the rate-limiting step in the biosynthesis of dopamine, in the right cerebrum,
and the results are shown in Figure 5. It was found that MPTP treatment could destroy
neurons and the median percentage of the positive area decreased from 12% to 4%, as
shown in Figure 5. However, HEM administration could restore MPTP-reduced TH-pos-
itive cells, and the median percentage of the positive area increased from 4% to 10% as the
administration of HEM increased from 0.1 to 1.0 g/kg. These findings demonstrated that
HEM possessed the ability to reverse MPTP-caused neurodegeneration.
Figure 4.
Dopamine levels in the substantia nigra of treated and untreated mice. A significant increase
in the dopamine level was found in the HEM cotreated mice compared with the MPTP group in a
dose-dependent manner. *** p< 0.001 compared with the MPTP-treated group.
We thereafter performed immunostaining for tyrosine hydroxylase (TH), the enzyme
that catalyzes the rate-limiting step in the biosynthesis of dopamine, in the right cerebrum,
and the results are shown in Figure 5. It was found that MPTP treatment could destroy
neurons and the median percentage of the positive area decreased from 12% to 4%, as
shown in Figure 5. However, HEM administration could restore MPTP-reduced TH-
positive cells, and the median percentage of the positive area increased from 4% to 10% as
the administration of HEM increased from 0.1 to 1.0 g/kg. These findings demonstrated
that HEM possessed the ability to reverse MPTP-caused neurodegeneration.
2.5. Antioxidant Activity of HEM on the Brain
To further evaluate the antioxidant activities of HEM on the brains of MPTP-treated
mice, oxidative stress biomarkers, including protein carbonyl (PC) content and malondi-
aldehyde (MDA) levels in the homogenized brain, were evaluated. As shown in Figure 6,
there was no difference in the PC levels in the brains of the MPTP-induced group com-
pared with those in the control group. Similar results were also observed with respect
to the MDA levels, as shown in Figure 6, which is the oxidative product of polyunsatu-
rated fatty acids peroxidation. However, both PC and MDA levels decreased significantly
in the
MPTP + HEM
(1 g/kg) group compared to the MPTP group (p< 0.01), as shown
in Figure 6.
Molecules 2023,28, 3386 5 of 12
Molecules 2023, 28, x FOR PEER REVIEW 5 of 13
Control
MPTP
MPTP+
HEM (0.1 g/kg)
MPTP+
HEM (0.3 g/kg)
MPTP+
HEM (1 g/kg)
(A)
(B)
Figure 5. (A) Immunohistochemical staining of tyrosine hydroxylase (TH). The MPTP group exhib-
ited dopamine deficiency syndrome. Co-treatment of HEM ameliorated MPTP-reduced TH expres-
sion in a dose-dependent manner. (B) The positive area of striatum is expressed in a Box and
Whisker Plot. The results reflect the mean values of cells. The leer “a” represents a statistical dif-
ference (p < 0.01) compared to the control group, while the leer “b” represents a statistical differ-
ence (p < 0.01) compared to the MPTP treatment group. The statistical analysis was performed using
Holm-Sidak tests.
2.5. Antioxidant Activity of HEM on the Brain
To further evaluate the antioxidant activities of HEM on the brains of MPTP-treated
mice, oxidative stress biomarkers, including protein carbonyl (PC) content and malondial-
dehyde (MDA) levels in the homogenized brain, were evaluated. As shown in Figure 6,
there was no difference in the PC levels in the brains of the MPTP-induced group com-
pared with those in the control group. Similar results were also observed with respect to
the MDA levels, as shown in Figure 6, which is the oxidative product of polyunsaturated
fay acids peroxidation. However, both PC and MDA levels decreased significantly in the
MPTP + HEM (1 g/kg) group compared to the MPTP group (p < 0.01), as shown in Figure
6.
Figure 5.
(
A
) Immunohistochemical staining of tyrosine hydroxylase (TH). The MPTP group exhibited
dopamine deficiency syndrome. Co-treatment of HEM ameliorated MPTP-reduced TH expression in
a dose-dependent manner. (
B
) The positive area of striatum is expressed in a Box and Whisker Plot.
The results reflect the mean values of cells. The letter “a” represents a statistical difference (p< 0.01)
compared to the control group, while the letter “b” represents a statistical difference (p< 0.01) compared
to the MPTP treatment group. The statistical analysis was performed using Holm-Sidak tests.
Molecules 2023, 28, x FOR PEER REVIEW 6 of 13
Carbonyl content
(nmol/mg)
0
50
100
150
200
250
300
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
***
MDA (μM/g)
0
50
100
150
200
250
300
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
**
(a) (b)
Figure 6. Effects of MPTP treatment in the presence or absence of HEM in the brains of mice on the
(a) protein carbonyl content and (b) MDA levels. A significant decrease in the PC and MDA levels
was found in the brains of mice obtained from treatment with a high concentration of HEM (1 g/kg)
compared with the MPTP group. Values are expressed as mean ± SD. ** p < 0.01;*** p < 0.001 com-
pared with the MPTP−treated group.
2.6. Antioxidant Activity of HEM on Livers
Our results showed that there was no difference in the PC and MDA levels of the
MPTP-induced group compared with the control group. However, there was a significant
reduction in both PC and MDA levels in the MPTP + HEM (0.3 and 1 g/kg) group com-
pared to the MPTP group (p < 0.01 and p < 0.001), as shown in Figure 7.
Carbonyl content
(nmol/mg)
0
300
600
900
1200
1500
∗∗∗
∗∗∗
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
MDA (μM/g)
0
600
900
1200
1500
1800
∗∗∗
∗∗
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
(a) (b)
Figure 7. Effects of MPTP treatment in the presence or absence of HEM in the livers of mice on the
(a) protein carbonyl content and (b) MDA levels. A significant decrease in the PC and MDA levels
was found in the livers of mice treated with MPTP + HEM (0.3 and 1 g/kg) compared with the MPTP
group. Values are expressed as mean ± SD. ** p < 0.01; *** p < 0.001 compared with the MPTP−treated
group.
2.7. Effect of HEM Treatment on Oxidative Stress Parameters of RBCs
Oxidative stress biomarkers, such as SOD, catalase, G6PDH, and GRd, were evalu-
ated in the red blood cells (RBCs) of male mice exposed to MPTP in the presence or ab-
sence of HEM at different concentration. As shown in Figure 8, the antioxidant biomarkers
of RBC were reduced after MPTP treatment, although most differences were only slightly
significant. However, HEM administration at different concentrations for 30 days could
Figure 6.
Effects of MPTP treatment in the presence or absence of HEM in the brains of mice on
the (
a
) protein carbonyl content and (
b
) MDA levels. A significant decrease in the PC and MDA
levels was found in the brains of mice obtained from treatment with a high concentration of HEM
(
1 g/kg
) compared with the MPTP group. Values are expressed as mean
±
SD. ** p< 0.01;*** p< 0.001
compared with the MPTP-treated group.
Molecules 2023,28, 3386 6 of 12
2.6. Antioxidant Activity of HEM on Livers
Our results showed that there was no difference in the PC and MDA levels of the
MPTP-induced group compared with the control group. However, there was a significant
reduction in both PC and MDA levels in the MPTP + HEM (0.3 and 1 g/kg) group compared
to the MPTP group (p< 0.01 and p< 0.001), as shown in Figure 7.
Molecules 2023, 28, x FOR PEER REVIEW 6 of 13
Carbonyl content
(nmol/mg)
0
50
100
150
200
250
300
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
***
MDA (μM/g)
0
50
100
150
200
250
300
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
**
(a) (b)
Figure 6. Effects of MPTP treatment in the presence or absence of HEM in the brains of mice on the
(a) protein carbonyl content and (b) MDA levels. A significant decrease in the PC and MDA levels
was found in the brains of mice obtained from treatment with a high concentration of HEM (1 g/kg)
compared with the MPTP group. Values are expressed as mean ± SD. ** p < 0.01;*** p < 0.001 com-
pared with the MPTP−treated group.
2.6. Antioxidant Activity of HEM on Livers
Our results showed that there was no difference in the PC and MDA levels of the
MPTP-induced group compared with the control group. However, there was a significant
reduction in both PC and MDA levels in the MPTP + HEM (0.3 and 1 g/kg) group com-
pared to the MPTP group (p < 0.01 and p < 0.001), as shown in Figure 7.
Carbonyl content
(nmol/mg)
0
300
600
900
1200
1500
∗∗∗
∗∗∗
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
MDA (μM/g)
0
600
900
1200
1500
1800
∗∗∗
∗∗
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
(a) (b)
Figure 7. Effects of MPTP treatment in the presence or absence of HEM in the livers of mice on the
(a) protein carbonyl content and (b) MDA levels. A significant decrease in the PC and MDA levels
was found in the livers of mice treated with MPTP + HEM (0.3 and 1 g/kg) compared with the MPTP
group. Values are expressed as mean ± SD. ** p < 0.01; *** p < 0.001 compared with the MPTP−treated
group.
2.7. Effect of HEM Treatment on Oxidative Stress Parameters of RBCs
Oxidative stress biomarkers, such as SOD, catalase, G6PDH, and GRd, were evalu-
ated in the red blood cells (RBCs) of male mice exposed to MPTP in the presence or ab-
sence of HEM at different concentration. As shown in Figure 8, the antioxidant biomarkers
of RBC were reduced after MPTP treatment, although most differences were only slightly
significant. However, HEM administration at different concentrations for 30 days could
Figure 7.
Effects of MPTP treatment in the presence or absence of HEM in the livers of mice on the
(a) protein
carbonyl content and (
b
) MDA levels. A significant decrease in the PC and MDA levels was
found in the livers of mice treated with MPTP + HEM (0.3 and 1 g/kg) compared with the MPTP group.
Values are expressed as mean ±SD. ** p< 0.01; *** p< 0.001 compared with the MPTP-treated group.
2.7. Effect of HEM Treatment on Oxidative Stress Parameters of RBCs
Oxidative stress biomarkers, such as SOD, catalase, G6PDH, and GRd, were evaluated
in the red blood cells (RBCs) of male mice exposed to MPTP in the presence or absence of
HEM at different concentration. As shown in Figure 8, the antioxidant biomarkers of RBC
were reduced after MPTP treatment, although most differences were only slightly signifi-
cant. However, HEM administration at different concentrations for 30 days could reverse
the reduced MPTP-causing enzyme activities in a dose-dependent manner (Figure 8).
Molecules 2023, 28, x FOR PEER REVIEW 7 of 13
reverse the reduced MPTP-causing enzyme activities in a dose-dependent manner (Figure
8).
SOD activity (U/ml)
0
60
80
100
120
∗∗∗
∗
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
Catalase activity
(nmol/min/ml)
0
50
100
150
200
250
∗
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
(a) (b)
G6PDH activity
(nmol/min/ml)
0
600
800
1000
1200
∗∗∗
∗∗
∗∗∗
MPTP - + + + +
HEM - - 0.1 0 .3 1
(g/kg)
GRd activity (nmol/min/ml)
0
50
100
150
200
250
∗∗
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
(c) (d)
Figure 8. Effect of HEM treatment on the oxidative stress parameters in RBCs. Different parameters
were determined, including (a) SOD, (b) catalase, (c) G6PDH, and (d) GRd. Values are expressed as
mean ± SD. * p < 0.05; ** p < 0.01 and *** p < 0.001 as compared with the MPTP−treated group.
3. Discussion
Herein, we established a micronized HEM powder using a spiral jet mill to break the
cell walls and accelerate the release of active ingredients from HEM. Erinacine A (EA), the
main natural antioxidant compound of HE, was 30 µg/g dry weight in HEM. HEM could
increase the dopamine level in the brain, suggesting that HEM could recover the function
of the substantia nigra. TH expression in the striatum was also recovered to almost full
levels using HEM powder in MPTP-treated mice. This effect is, at least in part, due to
reduced oxidative stress in the body (Figure 9).
Figure 8.
Effect of HEM treatment on the oxidative stress parameters in RBCs. Different parameters
were determined, including (
a
) SOD, (
b
) catalase, (
c
) G6PDH, and (
d
) GRd. Values are expressed as
mean ±SD. * p< 0.05; ** p< 0.01 and *** p< 0.001 as compared with the MPTP-treated group.
Molecules 2023,28, 3386 7 of 12
3. Discussion
Herein, we established a micronized HEM powder using a spiral jet mill to break the
cell walls and accelerate the release of active ingredients from HEM. Erinacine A (EA), the
main natural antioxidant compound of HE, was 30
µ
g/g dry weight in HEM. HEM could
increase the dopamine level in the brain, suggesting that HEM could recover the function
of the substantia nigra. TH expression in the striatum was also recovered to almost full
levels using HEM powder in MPTP-treated mice. This effect is, at least in part, due to
reduced oxidative stress in the body (Figure 9).
Molecules 2023, 28, x FOR PEER REVIEW 7 of 13
reverse the reduced MPTP-causing enzyme activities in a dose-dependent manner (Figure
8).
SOD activity (U/ml)
0
60
80
100
120
∗∗∗
∗
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
Catalase activity
(nmol/min/ml)
0
50
100
150
200
250
∗
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
(a) (b)
G6PDH activity
(nmol/min/ml)
0
600
800
1000
1200
∗∗∗
∗∗
∗∗∗
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
GRd activity (nmol/min/ml)
0
50
100
150
200
250
∗∗
MPTP - + + + +
HEM - - 0.1 0.3 1
(g/kg)
(c) (d)
Figure 8. Effect of HEM treatment on the oxidative stress parameters in RBCs. Different parameters
were determined, including (a) SOD, (b) catalase, (c) G6PDH, and (d) GRd. Values are expressed as
mean ± SD. * p < 0.05; ** p < 0.01 and *** p < 0.001 as compared with the MPTP−treated group.
3. Discussion
Herein, we established a micronized HEM powder using a spiral jet mill to break the
cell walls and accelerate the release of active ingredients from HEM. Erinacine A (EA), the
main natural antioxidant compound of HE, was 30 µg/g dry weight in HEM. HEM could
increase the dopamine level in the brain, suggesting that HEM could recover the function
of the substantia nigra. TH expression in the striatum was also recovered to almost full
levels using HEM powder in MPTP-treated mice. This effect is, at least in part, due to
reduced oxidative stress in the body (Figure 9).
Figure 9.
A schematic diagram of HEM preventing MPTP toxicity. Deep red represents the results of
MPTP treatment; blue represents the results of HEM treatment. MDA, malondialdehyde; PC, protein
carbonyl; TH, tyrosine hydroxylase. Deep red arrows represent the results of MPTP treatment; blue
arrows represent the results of HEM treatment.
Lipophilic MPTP can easily penetrate the blood–brain barrier (BBB) and is then con-
verted into 1-methyl-4-phenylpyridinium (MPP+) by enzyme monoamine oxidase B, which
activates cell death signaling pathways and induces dopaminergic neurotoxicity [
23
]. The
present study demonstrated that MPTP administration (20 mg/kg/day for 5 days) reduced
dopamine release and TH expression in the striatum, indicating successful induction of
dopaminergic neurotoxicity in mice. Furthermore, video evidence (Supplementary Materi-
als Video S1) confirmed the success of the behavioral model. These results are consistent
with previous experiments [
24
] and confirm the validity and efficiency of our animal model.
However, it should be noted that protein oxidation levels did not significantly change after
administering 20 mg/kg of MPTP. Possible reasons for this include: (1) oxidative stress is
an early indicator of cell damage, and the cellular damage caused by MPTP may gradually
diminish over time. In our study, the observation period was longer (25 days) compared
to the usual period (20 days). Additionally, (2) the dosage of MPTP we administered was
lower, and (3) younger mice tend to have better recovery characteristics [
25
,
26
]. In our
current study, the oral administration of HEM could restore MPTP-induced dopaminergic
neuron degeneration and reduced dopamine levels. It is common knowledge that PD is
caused by the loss of nerve cells in the patient’s brain, leading to the reduction of dopamine
levels, which plays a vital role in regulating body movement. Therefore, the present
findings may suggest that HEM could be beneficial to increasing the dopamine levels in
patients with PD. Shimbo et al. have found that erinacine A, a bioactive compound in HEM,
stimulates the secretion of the nerve growth factor, an essential protein for supporting
neuron’s growth and maintenance, in the rat locus coeruleus. Moreover, erinacine A has
also been shown to stimulate dopamine metabolites production [
27
]. These experimental
results are also consistent with our experiments.
MPTP neurotoxicity is rapid (as early as 2 h) and stabilizes within 7 days. In addition,
90% of striatal dopamine depletion and 70% loss of dopaminergic neurons were induced af-
ter four injections of MPTP at a daily dose of 20 mg/kg, thereby causing motor deficits [
28
].
MPTP neurotoxicity is associated with the inhibition of ATP production and stimulation
Molecules 2023,28, 3386 8 of 12
of multiple ROS production, which then declines and damages protein function through
oxidation and nitration [
24
]. In SY5Y neuroblastoma cells, MPTP (50
µ
M) exposure stimu-
lates intracellular ROS production, reaching its peak at 6–12 h, and then declining to near
baseline after 48 h of exposure [
29
]. ROS are known to play a key role in the aging process
and have also been implicated in aging-related neurodegenerative diseases such as PD [
29
].
Therefore, reinforcement of the antioxidant defense system or scavenger administration is
critical because it may combat these diseases [
30
]. Furthermore, reducing free radicals via
antioxidants has been shown to combat toxin-induced degenerative diseases [
20
]. Herein,
we found that HEM significantly reduced free radical production both in the brain and liver.
In addition, the antioxidant activity was significantly increased with the oral administration
of HEM powder.
The brain is easily affected by the aging processes caused by oxidative stress. Our
experimental study showed that MDA production was reduced in the HEM (0.1 and
0.3 g/kg
) groups compared with the control and MPTP-treated groups, although this effect
was not significant at low doses of HEM. The administration of high doses of HEM (1 g/kg)
could significantly (p< 0.01) reduce MDA levels. Similar results were also found regarding
the increase in PC contents, suggesting that HEM could effectively counteract oxidative
stress in brain tissues. Furthermore, hepatic MDA and PC levels were also reduced in
MPTP-treated mice. Antioxidative stress parameters, including SOD, catalase, G6PDH,
and GRd activities, were elevated in RBCs of the MPTP + HEM treatment group compared
with the MPTP-treated group.
HE possesses neuroprotective effects and its bioavailability can be determined using
erinacine A and erinacine S, its two major compounds. Erinacine A can be detected in
plasma at 1 min after the oral administration of HE as it penetrates the BBB via passive
diffusion. Consequently, it was detected in the brain 4 h post-administration and reached
its maximum level after 8 h. Moreover, the binding of erinacine A was found to be the
highest (28.94%
±
9.29%) in the brain. The absolute bioavailabilities of erinacine A and
erinacine S were 24.39% and 15.13%, respectively [31,32].
Conclusively, HEM powder can be very beneficial in combating diseases that follow
dopaminergic pathways in the brain, including nigrostriatal, mesolimbic, mesocortical, and
tuberinfundibular systems that play vital roles in regulating many important physiological
functions. This study also found that HEM could reduce ROS levels in the brain, liver,
and blood. ROS are mainly produced by the mitochondria during both physiological and
pathological conditions, and by endothelial and inflammatory cells. Despite the fact that
these organelles have intrinsic ROS scavenging capacities, these may not be enough to
address the cellular need of clearing ROS generated by the mitochondria [
33
]. Hence, HEM
powder enrichment may provide answers to this question, and thus protect individuals’
wellness and health from ROS-induced cellular damages.
4. Conclusions
In conclusion, the study found that HEM powder has the potential to fight diseases
that affect the dopaminergic pathways and lower ROS levels in the brain, liver, and blood,
thus safeguarding individuals from cellular damage caused by ROS. HEM enrichment may
address the cellular need for clearing ROS generated by the mitochondria, thus protecting
individuals’ health and wellness from ROS-induced cellular damage.
5. Materials and Methods
5.1. Preparation of HE Mycelium
HEM powder was purchased from Fungus Biotech, Co., Ltd., Yilan, Taiwan, which
used the HE strain (BCRC 36470, Bioresource Collection and Research Center, Hsinchu,
Taiwan) and was produced under solid-state fermentation. The HEM powder was dried
at 60
◦
C in a tray dryer and grinded into 100-mesh powder at Fungus Biotech. It was
then further ground into smaller particles through a spiral jet mill (spiral jet Mill, OM2
Micronizer, Sturtevant, Int., Hanover, MA, USA) to undergo the cell wall-breaking effect
Molecules 2023,28, 3386 9 of 12
with a particle size distribution of D75 < 50
µ
m micronized powder at Formosan Nano
Biology Co., Ltd., Taichung, Taiwan. The cell wall-breaking technology greatly contributes
to the increased rate of releasing active ingredients from the fine HEM powder.
5.2. Particle Size Analysis
HEM particle size distributions were evaluated using a Beckman Coulter LS230 particle
size analyzer, which can measure particles ranging from 40 nm to 2 mm in size [
34
]. HEM
particles were measured in an ethanol dispersed solution.
5.3. High Performance Liquid Chromatography (HPLC)
We weighed 1 g of the Hericium erinaceus mycelium powder and extracted it with 5 mL
of 50% methanol using ultrasonic technology. The resulting mixture was then centrifuged
at 3000
×
gfor 5 min, and this procedure was repeated once. The supernatant was filtered
using ADVANTEC NO.1 membranes and diluted with 50% methanol to a final volume of
10 mL. Prior to HPLC analysis, the solution was filtered through a 0.22
µ
m PVDF syringe
filter and degassed.
HPLC analysis of erinacine A was performed on a Thermo Scientific Dionex Ultimate
3000 HPLC system (Thermo Scientific, Bremen, Germany) equipped with a quaternary
rapid separation pump (LPG-3400SD), TCC-3000 temperature-controlled column (40
◦
C),
and DAD-3000 diode array detector, as previously described, with minor modifications [
35
].
Chromatographic separations were achieved on an InertSustain C-18 (250
×
4.6 mm,
5µm
)
with a linear A–B gradient (0–20 min 66% B to 70% B, 25–35 min 70% B to 100% B) at a
constant flow rate of 1 mL/min and a total run time of 35 min. Solvent A consisted of
0.2% H
3
PO
4
in Milli-Q water and solvent B of 100% methanol. The absorption spectra of
eluted compounds were detected at 340 nm using Dionix Chromeleon software (Version
6.80, Service Release SR14).
5.4. Animals Groups and Experimental Procedure
Adult (8–12 weeks old) male C57BL/6Narl mice, weighing 20–30 g, were purchased
from the National Laboratory Animal Center (Taipei, Taiwan). The animals were housed at
a temperature of 22
±
1
◦
C, with 14 h of automatic illumination daily (06:00–20:00) in the
Animal Center of the National Yang-Ming University, Taiwan. Animal care conformed to the
Guidelines of the Animal Use and Care Committee of National Yang-Ming University, Taiwan.
Food and water were available ad libitum. The animal use protocol was approved by the
Institutional Animal Care and Used Committee (Approval Number: 1050306 & 1060509).
The MPTP model of PD was induced, as described previously [
22
]. Mice were ran-
domly assigned into five groups, as shown in Figure 1: the control group, the MPTP group
(20 mg/kg/day for the first 5 days; Tokyo Chemical Industry, TCI), and MPTP + different
dosages of HEM groups (0.1 g/kg, 0.3 g/kg, and 1 g/kg, respectively). Mice received an
intraperitoneal (i.p.) injection of MPTP, and the same quantity of saline was given in the
control group. Mice were orally gavaged with H2O or HEM for 30 days.
5.5. Dopamine Measurement
Mice were sacrificed, and the striatum was quickly dissected on ice and homogenized
in a stock solution containing 0.1 M HClO
4
, 0.1 mM EDTA, and 0.1 mM Na
2
S
2
O
5
and
centrifuged at 13,000 rpm for 10 min at 4
◦
C. The supernatant was filtered with 0.45-
µ
m
membranes before HPLC analysis. The dopamine level in this isolated substantia nigra
homogenate was measured with electrochemical detection, as described previously [22].
5.6. Tyrosine Hydroxylase Measurement
Tyrosine hydroxylase (TH), a key precursor for dopamine production, was measured
using immunohistochemistry (IHC) [
36
,
37
]. The right cerebrum was immersed in cold
paraformaldehyde in 0.1 M phosphate buffer (pH: 7.4) and sectioned into 10
µ
m thick
slides. All sections were stained for TH determination. The optical density of areas with
Molecules 2023,28, 3386 10 of 12
TH expression was determined by measuring at least three randomly selected microscopic
fields on each slide. The average integral optical density was defined as the percentage of
positive area ×optical density/total area [38].
5.7. Protein Carbonyl Content Measurement
The liver and brain of the tested mice were collected, and the protein carbonyl con-
tent was then evaluated [
24
]. Approximately 150–200 mg of brain or liver tissues were
homogenized separately in 50 mM of MES buffer (1–2 mL, pH 6.7, containing 1 mM EDTA)
and centrifuged. Each supernatant was collected and stored at
−
80
◦
C. The sample was
then determined using a protein carbonyl colorimetric assay kit (No. 10005020, Cayman,
MI, USA).
5.8. Lipid Peroxidation Level Determination
The lipid peroxidation level in the brain and liver was determined as described in
a previously published method [
39
] and expressed as the MDA value. MDA was mea-
sured with a thiobarbituric acid-reactive substance (TBARS) assay kit (Item No. 10009005,
Cayman, MI, USA). Briefly, the brain and liver were isolated and homogenized in cooled
RIPA buffer. Consequently, all samples were centrifuged at 1600
×
gfor 10 min at 4
◦
C. The
supernatant was then stored at −80 ◦C, and the MDA values were determined.
5.9. Antioxidant Status Activity
Antioxidant enzyme activities of RBCs were evaluated using a previously published
method [
40
]. Whole blood with heparin was collected and centrifuged. RBCs were washed
with normal saline twice and lysed using 50 mM phosphate buffer (pH: 6.6). The super-
natant was collected and determined within one month. Superoxide dismutase (SOD,
Item No. 706002), catalase (CAT, Item No. 707002), glutathione peroxidase (GPx, Item
No. 703102), glutathione reductase (GRd, Item No. 703202), and glucose-6-phosphate
dehydrogenase (G6PDH, Item No. 700300) activities were determined using commercial
kits (Cayman, MI, USA).
5.10. Data Analysis and Statistical Assessment
Data collected were expressed as mean
±
SD. Analysis of variance was used to access
the statistical significance for repeated data measurements, and the differences among
individual mean values in different groups were analyzed using the Holm-Sidak post-hoc
test followed by one way Analysis of Variance (ANOVA). Differences were considered to
be significant at p< 0.05.
Supplementary Materials:
The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/molecules28083386/s1, Video S1.
Author Contributions:
Study concepts, quality control of data and algorithms: C.-H.H. and W.-C.C.
Study design, data acquisition, data analysis and interpretation, statistical analysis: E.-C.L. and
W.-C.C. Manuscript preparation & editing: C.-H.H. and K.-L.W. Manuscript review: C.-H.H. and
K.-L.W. All authors have read and agreed to the published version of the manuscript.
Funding:
This study was funded by the Shin Kong Wu Ho-Su Memorial Hospital (2020SKHCDR002,
2020SKHADR021, and 2020SKHADR022), the Department of Health, Taipei City Government (No. 11101-
62-022 and 11201-62-033), Taipei City Hospital (TPCH-112-33), and the National Science and Technology
Council (MOST110-2320-B-254-001, MOST111-2320-B-254-001, MOST111-2314-B-532-001), Taiwan.
Institutional Review Board Statement:
The animal study protocol was approved by the Institutional
Animal Care and Used Committee (IACUC) of National Yang-Ming University (protocol code
1050306 & 1060509).
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Molecules 2023,28, 3386 11 of 12
Acknowledgments:
Cartoons in Figures 1and 9, and graphical abstract were created with BioRender.
com (accessed on 16 February 2023).
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds will not be available.
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