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International Journal of Medicinal Mushrooms, 17(X): XXX–XXX (2015)
Lion’s
Mane,
Hericium erinaceus
and
Tiger Milk,
Lignosus
rhinocerotis
(Higher
Basidiomycetes)
Medicinal Mushrooms Stimulate
Neurite
Outgrowth
in
Dissociated
Cells of Brain, Spinal Cord,
and Retina:
An In Vitro
Study
Snehlata Samberkar,1,2,* Sivasangkary Gandhi,1,3 Murali Naidu,1,2 Kah-Hui Wong,1,2
Jegadeesh Raman,1,3 & Vikineswary Sabaratnam1,3
¹Mushroom Research Centre, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur,
Malaysia; 2Department of Anatomy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 3Institute of
Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia
* Address all correspondence to: Snehlata P. Samberkar, Department of Anatomy, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur,
Malaysia; Tel: +60379673792; Fax: +603 79674724; E-mail: snehlata@um.edu.my
ABSTRACT: Neurodegenerative disease is defined as a deterioration of the nervous system in the intellectual and
cognitive capabilities. Statistics show that more than 80–90 million individuals age 65 and above in 2050 may be
affected by neurodegenerative conditions like Alzheimer’s and Parkinson’s disease. Studies have shown that out of
2000 different types of edible and/or medicinal mushrooms, only a few countable mushrooms have been selected
until now for neurohealth activity. Hericium erinaceus is one of the well-established medicinal mushrooms for neu-
ronal health. It has been documented for its regenerative capability in peripheral nerve. Another mushroom used as
traditional medicine is Lignosus rhinocerotis, which has been used for various illnesses. It has been documented for
its neurite outgrowth potential in PC12 cells. Based on the regenerative capabilities of both the mushrooms, prior-
ity was given to select them for our study. The aim of this study was to investigate the potential of H. erinaceus and
L. rhinocerotis to stimulate neurite outgrowth in dissociated cells of brain, spinal cord, and retina from chick embryo
when compared to brain derived neurotrophic factor (BDNF). Neurite outgrowth activity was
confirmed
by the immu-
nofluorescence method in all tissue samples. Treatment with different concentrations of extracts resulted in neuronal
differentiation and neuronal elongation. H. erinaceus extract at 50 µg/mL triggered neurite outgrowth at 20.47%,
22.47%, and 21.70% in brain, spinal cord, and retinal cells. L. rhinocerotis sclerotium extract at 50 µg/mL induced
maximum neurite outgrowth of 20.77% and 24.73% in brain and spinal cord, whereas 20.77% of neurite outgrowth
was observed in retinal cells at 25µg/mL, respectively.
KEY WORDS: medicinal mushroom, Lignosus rhinocerotis, Hericium erinaceus, neurite outgrowth activity, neuro-
degenerative disease
ABBREVIATIONS: BDNF, brain=derived neurotrophic factor; DAPI, 4′,6-diamidino-2-phenylindole; DMEM,
Dulbecco’s modified Eagle’s medium; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline.
I.
INTRODUCTION
Aging is an inevitable process and the fraction of
world’s population over 65 years of age is expected
to increase to 80–90 million by the end of 2050.1
Out of many diseases that threaten aging humans,
neurodegenerative diseases such as Alzheimer ’s
disease, dementia, and Parkinson’s disease2 and
retinal diseases like diabetic retinopathy, glaucoma,
and age-related macular degeneration can be very
traumatic.3 Neurohealth is a major concern as one
ages. Retrospective studies have proved that natural
products like mushrooms are suitable candidates for
neurohealth.4 Out of 14,000 species of familiar mush-
rooms,4 roughly 20004 are acknowledged to be
fit
for
human consumption. Researchers have reported that
1045-4403/15/$35.00 © 2015 Begell House, Inc. www.begellhouse.com 1
2
Samberkar et al.
many medicinal mushrooms promote neurotrophic
properties such as neurite outgrowth stimulation,5,6
nerve regeneration,7 neuroprotection,8 and anti-
oxidation.9 Mushrooms may have potential in the
prevention or treatment of age-related neurodegen-
erative complaints.10
The mushrooms selected for this study were
Hericium erinaceus (Bull.:Fr.)Pers. (Hericiaceae,
higher Basidiomycetes) and Lignosus rhinoc-
erotis (Cooke) Rivarden (Polyporaceae, higher
Basidiomycetes). H. erinaceus, a rare mushroom
also known as lion’s mane, monkey’s head, and
Yamabu shitake, is scattered throughout North
America, Europe, and Asia.11,12 It has been used
in Chinese and Japanese cuisine and as an herbal
remedy to treat various human diseases including
gastric ulcers for hundreds of years.12 A health syrup
called “Houtou” is prepared from dried fruit bod-
ies. H. erinaceus tablets are used to treat ulcers,
inflammation,
and tumors of the alimentary canal.13
It is also evident that H. erinaceus has facilitated
functional recovery subsequent to peripheral nerve
injury.7 Based on this study, our focus is on regen-
eration of the central nervous system (CNS) using
brain, spinal cord, and retinal explants and cells after
dissociation, using chick embryo as a model.
L. rhinocerotis is also known as tiger’s milk
mushroom or “cendawan susu rimau” in the local
language. This species is distributed only in the tropi-
cal rainforest in regions of South China, Thailand,
Indonesia, Malaysia, Philippines, and Papua New
Guinea. L. rhinocerotis has been used as a general
tonic, antipyretic, and antipruritic; it has also been
used to treat fever, cancer, food poisoning, swollen
breasts, cough, and asthma and to assist in wound
healing, among other uses.14 Its usage is limited in
spite of its medicinal properties due to unavailability.
Sclerotium of L. rhinocerotis documented enhance-
ment of neurite outgrowth activity in PC12 cells.15
Based on their potential, in this study, both mush-
rooms with different concentrations (25–100 µg/mL)
were treated with tissue samples and compared with
negative and positive controls. Neurite extensions
were confirmed by immunofluorescence staining.
II. MATERIALS
AND
METHODS
A.
Preparation
of
Mushroom
Aqueous Extracts
Mushrooms require the correct combination of
humidity, temperature, substrate (growth medium),
and inoculum (spawn) to grow. In Malaysia,
H. erinaceus is cultivated on a substrate containing
rubberwood sawdust, rice bran, and calcium carbon-
ate at a ratio of 100:5:1. After 2 months of spawn run
at 27±2 to 32±2°C at a mushroom farm (Ganofarm
Ltd., Tanjung Sepat, Selangor, Malaysia), approxi-
mately 300 g of fresh fruit body per 800 g of substrate
was harvested.16 Fresh fruiting bodies of H. erinaceus
were purchased from the mushroom farm. Fresh
fruit bodies were sliced, frozen, and freeze-dried.
The freeze-dried fruit bodies were then blended in
a Waring commercial blender and stored in airtight
containers at 4°C prior to assay.16 L. rhinocerotis,
a rare species, is found in the forests of Malaysia.
In this study, the freeze-dried powder of sclerotia
of cultivated L. rhinocerotis10,17 was purchased
from Ligno Biotek Sdn Bhd (batch no. TM02). The
freeze-dried powders of both mushrooms were then
soaked separately in distilled water (1:20, w/v) and
were agitated at 150 rpm for 24 h.15 The mixture
was then double boiled in a water bath at 100°C
for 30 min, cooled, and filtered by Whatman filter
paper No. 4. The aqueous extract was freeze-dried
and kept at –20°C prior to use.6,18
B.
Preparation
of Explants
Culture
and Trypsinization
Fertilized chicken eggs were collected from Charoen
Pokphand Jaya Farm (M) Sdn Bhd (Negeri Sembilan,
Malaysia) and then were incubated at 39°C in a
humidified
incubator. The brain (day 4), spinal cord
(day 6), and retina (day 9) were dissected on their
respective days.19 Based on a modified method of
Gibco Life Technologies (Selangor, Malaysia), the
tissue samples (brain, spinal cord, and retina) were
finely chopped, washed with phosphate-buffered
saline (PBS) twice, and centrifuged at 3000 rpm
for 3 min after adding trypsin. The supernatant
International Journal of Medicinal Mushrooms
Medicinal Mushrooms Induce Neurite Outgrowth in Brain, Spinal Cord, and Retinal Cells
3
2
2
was discarded and the cell pellet was resuspended
twice with 2–5 mL of prewarmed (37°C) complete
media. The samples were centrifuged at 3000 rpm
for 3–5 min. The supernatant was discarded and
the cell pellet was added to fresh complete media
dilution in blocking buffer) for 1 h. The cells were
washed and then incubated with secondary antibody,
anti-rabbit IgG-fluorescein isothiocyanate (FITC)
antibody produced in sheep (Sigma, St. Louis, MO,
USA) (1:80 dilution in blocking buffer) for 1 h in
and incubated at 37±2°C in a 5% CO
incubator for 24 h.
C. Neurite
Outgrowth
Assay
humidified the dark at room temperature. The cells were then
washed thrice. The coverslips were then mounted
with 4′,6-diamidino-2-phenylindole (DAPI), which
stained the nucleus. Images were observed and cap-
tured with a
fluorescent
microscope (Nikon Eclipse
Two-day-old cultured cells were seeded into 12-well
plates at a cell density of 5×104 cells per well. The
mushroom aqueous extracts in Dulbecco’s modified
Eagle’s medium (DMEM) at concentrations of 25,
50, 75, and 100 µg/mL (w/v), and brain-derived
neurotrophic factor (BDNF) at 10 ng/mL (w/v) were
tested for neurite outgrowth stimulation activity.
Cells in complete DMEM without treatment served
as the negative control. Plates were incubated at
80i microscope using FITC and DAPI filters).
F. Statistical
Analysis
All experiments were carried out in three repli-
cates. Results were expressed as the means ± SD.
All data were subjected to analysis of variance
using GraphPad Prism Statistical Software version
7 (GraphPad Software Inc., La Jolla, CA, USA). The
37±2°C in a 5% CO incubator for 2 days.15 differences among samples were evaluated using
Duncan’s multiple range test, where p<0.05 was
D.
Scoring
of
Neurites
Neurite extensions were scored under an inverted
microscope (Nikon Eclipse TS100) with the aid of
a handheld counter. A cell was scored positive for
bearing neurites if it had at least one thin extension
longer than the diameter of its cell body.20 In a well,
10 fields with an average of randomly chosen 250–
300 cells per well were examined and photographed
using a Nikon DS-Fi1 camera and were processed
with Nikon’s NIS-Elements D imaging software.6
E.
Neurofilament Staining
A
neurofilament
was used as an indicator for neurite
outgrowth and immunofluorescence staining was
used for confirming neuronal extension, which is
an increase in axonal length. Based on the standard
method,21 primary neuronal cells were seeded in
12-well plates and exposed to treatment for 2 days.
The cells were fixed with 4% paraformaldehyde
for 20 min at room temperature. After two washes
with PBS, the cells were incubated with primary
antibody,
antineurofilament
200 antibody produced
in rabbits (Sigma, St. Louis, MO, USA) (1:80
considered significant.
III. RESULTS
AND
DISCUSSION
Neurons in the mature CNS are unable to regenerate
injured axons and the neurons that remain unin-
jured are unable to form novel connections that
might compensate for ones that have been lost.22
Subsequently, due to a break in the communication
between healthy neurons, a cascade of events takes
place that leads to neuronal degeneration and cell
death. The factors responsible for failure of regen-
eration are several and include poor regenerative
ability of CNS neurons, inhibitory properties of
astrocytes,23 and inhibitory molecules produced
by oligodendrocytes and myelin.24 Overpowering
these issues will facilitate the nerve regeneration for
restoration of function following damage, through
accident, injury, or neurodegenerative disease.
Natural products have been traditionally
accepted as remedies due to the popular belief that
they present minor side effects.25 In traditional
Chinese medicine, mushrooms have always been
prepared for medicinal use by hot water extraction.
The number of mushrooms, however, studied for
Volume 17, Number X, 2015
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Samberkar et al.
FIG. 1: Effect of varying concentrations of H. erinaceus and Lignosus rhinocerotis aqueous extracts on neurite out-
growth stimulation on chick embryo’s brain cells.
neurohealth activity are few and H. erinaceus10 is
one of them. Its chemical composition was docu-
mented by Kawagishi et
al.
5
Further study on aqueous
extracts proved that polysaccharides could induce
neuronal differentiation and promote neuronal sur-
vival.26 Ongoing research in our laboratory shows
that H. erinaceus,6 L. rhinocerotis,15,27 and Pleurotus
giganteus (Berk.) Karunarathna & K.D. Hyde
(morning glory mushroom, cow’s stomach mush-
room)28 exhibit neurite outgrowth stimulatory effects
in NG108-15 and PC12 cell lines. Inspired by this,
researchers focused their goal on peripheral nerve
regeneration following crush injury to the rat pero-
neal nerve by using aqueous extract of H. erinaceus.
This study proved that daily administration of aque-
ous extract of H. erinaceus has
beneficial
effects on
recovery of the injured rat peroneal nerve in early
stages of regeneration.7
Another medicinal mushroom is L. rhinocerotis.
Retrospective studies have shown that an aqueous
extract of L. rhinocerotis sclerotium induced neu-
rite outgrowths of 24.4% and 42.1% at 20 µg/mL
(w/v) of aqueous extract alone and a combination of
20 µg/mL (w/v) aqueous extract and 30 ng/mL (w/v)
of NGF, respectively, in rat pheochromocytoma cells
(PC12 cells).15 Similarly, the present study showed
neurite outgrowth of 20.77% and 24.73% at 50 µg/mL
in brain and spinal cord cells and 20.77% at 25 µg/mL
in retinal cells. Consistent with our previous study,
the sclerotia of L. rhinocerotis reported neurite
outgrowth in N2a.29 It was recently shown that scle-
rotial extract performs better than mycelial extract.29
Advanced study shows maximum neurite extension
for L. rhinocerotis and curcumin at 21.1% at 20
µg/mL and 29.47% at 10 µg/mL on PC-12 cells.30
Combining 20 µg/mL of L. rhinocerotis with 1 µg/
mL curcumin gave 27.2% neurite extension in PC12
cells.30 Taken as a whole, these medicinal mushrooms
have shown neurological properties such as neuronal
survival and neurite outgrowth activities including
improvement in recovery and function in both in vivo
and in vitro mammalian nervous systems.31
The aqueous extracts of H. erinaceus and L. rhi-
nocerotis showed a gradual dose-dependent twofold
increase in neurite outgrowth stimulation at 25 and
50 µg/mL concentration compared to the negative
control. Figure 1 shows the neurite outgrowth stimu-
lation on brain cells after 48 h incubation decreased
the extension in a dose-dependent manner observed
at 75 and 100 µg/mL concentrations. The maximal
stimulated outgrowth on brain cells treated with
aqueous extracts of H. erinaceus and L. rhinocerotis
was 20.47% and 20.77%, respectively, at 50 µg/mL,
comparable to that of the BDNF-treated cells
(positive control), whereas H. erinaceus showed
significant (p<0.05) neurite outgrowth of 18.73%
International Journal of Medicinal Mushrooms
Medicinal Mushrooms Induce Neurite Outgrowth in Brain, Spinal Cord, and Retinal Cells
5
FIG. 2: Effects of varying concentrations of H. erinaceus and Lingnosus rhinocerotis aqueous extract on in vitro neu-
rite outgrowth stimulation on chick embryo’s spinal cells.
at a concentration of 25 µg/mL. H. erinaceus and
L. rhinocerotis aqueous extracts showed signifi-
cant (p<0.05) neurite outgrowth of 22.47% and
24.73%, respectively, at 50 µg/mL on the spinal
cord (Fig. 2). The aqueous extract of L. rhinocero-
tis showed significant (p<0.05) neurite outgrowth
(20.77%) at 25 µg/mL on retinal cells; in contrast,
the aqueous extract of H. erinaceus (21.70%)
exerted its maximum neurite growth at 50 µg/mL
(Fig. 3). Comparing both of the aqueous extracts,
H. erinaceus exerted a significantly potent neurite
outgrowth on retinal cells at a lower concentra-
tion compared to L. rhinocerotis aqueous extract.
Retinal cell stimulation was significant (p<0.05)
at 50 µg/mL by H. erinaceus. Neurite outgrowth
is confirmed by neurofilament staining as shown
in Fig. 4. Figures 4A, D, and G show BDNF at
the concentration of 10 ng/mL used as a positive
control in the brain, spinal cord, and retina. Figures
4B, E, and H show the negative control in all three
FIG. 3: Effects of varying concentrations of H. erinaceus and Lignosus rhinocerotis aqueous extract on in vitro neu-
rite outgrowth stimulation on chick embryo’s retina cells.
Volume 17, Number X, 2015
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Samberkar et al.
FIG. 4: Fluorescent microscopy image of in vitro neurite outgrowth in chicken embryo’s brain, spinal cord. and retinal
cells by Lignosus rhinocerotis aqueous extracts. Brain and spinal cord cells were treated with hot aqueous extract
L. rhinocerotis at a concentration of 50 and 25 µg/mL for retinal cells after 48 h of incubation at 37±2°C in a 5% CO
2
humidified incubator. (A) Brain positive control: BDNF(10 ng/mL). (B) Negative control of brain cells without extract.
(C) Brain cells treated with hot aqueous extract of L. rhinocerotis at 50 µg/mL. (D) Spinal cord positive control: BDNF
(10 ng/mL). (E) Negative control of spinal cord cells without extract. (F) Spinal cord cells treated with hot extract of L.
rhinocerotis at 50 µg/mL. (G) Retina positive control: BDNF (10 ng/mL). (H) Negative control of retinal cells without
extract. (I) Retinal cells treated with hot aqueous extract of L. rhinocerotis at 25 µg/mL. (C), (F), and (I) Cells show
an exuberant long neurite outgrowth (arrow) as compared to (A) (D), and (G).
samples, which have either no neurite, or neurite
with insufficient length to be scored as positive.
Figures 4C, F, and I show neurite outgrowth in the
brain, spinal cord, and retinal cells confirmed by
neurofilament
staining. Neurite extension is marked
by an arrow. Recent research suggested that these
neurofilaments are closely related to many neuro-
degenerative diseases, such as amyotrophic lateral
sclerosis, Parkinson’s diseae, and Alzheimer’s dis-
ease. Using in vitro assays, cultures, and transgenic
mice, these studies provided new insights into neuro-
filament function. The function of each subunit,
the relationship of neurofilaments with other cyto-
skeletal elements and their clinical significance are
topics of increasing attention.
IV.
CONCLUSIONS
H. erinaceus and L. rhinocerotis aqueous extracts
were examined for neurite outgrowth activity in
International Journal of Medicinal Mushrooms
Medicinal Mushrooms Induce Neurite Outgrowth in Brain, Spinal Cord, and Retinal Cells
7
the brain, spinal cord, and retinal cells of chicken
embryo. Four different concentrations (25, 50, 75,
and 100 µg/mL) were tested. Among the concen-
trations, H. erinaceus aqueous extract, 50 µg/mL,
stimulated neurite outgrowth in brain and spi-
nal cord cells. L. rhinocerotis sclerotium extract
showed similar neurite outgrowth activity in reti-
nal cells at 25 µg/mL after 48 h of incubation.
Immunofluorescence staining confirmed neurite
outgrowth.
ACKNOWLEDGMENTS
The authors thank the University of Malaya for
Bantuan Kecil Penyelidikan (BKP) grant BK003-
013, RG 193/11AFR, and UMRG RP 005B-13
AFR, and Mrs. Cheng Poh Guat of Vita Agrotech,
a mushroom farm in Tanjung Sepat, Selangor,
Malaysia for the continuous supply of fresh
H. erinaceus mushroom. The technical assistance
by the staff of the Department of Anatomy is greatly
appreciated.
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International Journal of Medicinal Mushrooms