STUDY ON THE ABILITY OF EXTRACTS FROM CORDYCEPS SPP.

BIOMASS TO PREVENT LONG-TERM MEMORY IMPAIRMENT IN MICE

ABSTRACT
The memory impairment related to hippocampus, recording and recalling of
emotional memory and recognition. This area stored information and formed memories in
long-term memory and the ability to orient in space. Extracts from Cordyceps isolated
and cultured in Vietnam has been used to treat memory impairment in mice induced by
TMT (trimethyltin) at two doses of 100 mg/kg and 200 mg/kg (p.o). The behavioral
testing was carried out with the Morris water maze model, simultaneously quantify
concentration of acetylcholine (ACh) and acetylcholinesterase (AChE) in brain
hippocampus. Results showed that two extracts: Poly DL004 and n-BuOH DL0015 had
memory improvement effects at doses of 100 mg/kg in hidden platform test and probe
test but no effect on working memory test. Results of quantifying ACh and AChE also
showed there was a decrease in ACh and an increase in AChE at TMT group, and vice
versa in the other groups. However, the results did not show protective effect to prevent
memory impairment of extracts are only from the ability of anti- AChE.
Key words: acetylcholine, acetylcholinesterase, Cordyceps, memory impairment, Morris
water maze.
INTRODUCTION
Alzheimers disease (AD) is one of the most common causes of mental deterioration in
elderly people (Francis et al. 1999), ADs symptoms are memory or cognitive
impairment, emotional and behavioural disorder. Currently, causes and progression of
AD havent understand yet and there isnt really effective way to prevent the disease
although having some hypothesis about AD causes. Currently, the basic of AD treatment
method mainly relies on cholinergic hypothesis. Which said that, AD is caused by
decrease in synthesis of the neurotransmitter (ACh), and increase of AChE activity
(Orhan & Sener 2003; Francis et al. 1999). The group of drugs currently used only
reducing the progression of the disease rather than preventing degradation of nerve cells.
Hippocampus, one of the first areas of the brain is injured. When the nerve cells of the
hippocampus died, secreted ACh was not enough to transmit information (Perry etal.
1978). Drugs of anti-acetylcholinesterase group are used to prevent the decomposition of
ACh, so ACh was increased around the nerve synaptic contacts.
Cordyceps is a rare medicinal herbs in traditional Chinese, the chemical composition was
studied: all of the essential amino acids, vitamins, sugars, many complex
polysaccharides, proteins, sterols, nucleosides, macro and microelements (Holliday &
Cleaver 2008; Holliday & Wasser 2005). Some potentially bioactive constituents also
were recognized: cordycepin, cordycepic acid, nucleosid, ergosterol, -3 ergosterol,
ergosterol peroxid, 3-sitosterol, daucosterol, campeasterol,..(Paterson 2008). Nucleosides
was recorded as a substance related to effects in regulating and adjusting various
physiological processes in the nervous system. Nucleosides, especially adenosine used as
markers of Cordyceps. Adenosine release of various neurotransmitters presynaptically
and anticonvulsant activity (Li et al., 2006). Inosine, a breakdown product of adenosine,
recently has been shown to to exert immunomodulatory and neuroprotective effects.
Study in mice showed that the oral administration of inosine has antidepressant (Junko
Muto et al., 2014). In addition, adenosine also stimulates axon growth in vitro and in the
adult central nerve system and improves outcome in a rat stroke model (Irwin et al.,
2006; Lorber et al., 2009). Additional benefits of inosine after brain injury include its
anti-inflammatory effects (Hasko et al., 2004), its ability to suppress glutamate-induced
neural excitation (Shen et al., 2005); enhances the ability of undamaged neurons to
extend axon collaterals into areas that have lost their normal innervation (Chen et al.,
2002; Zai et al., 2009).
In Vietnam, the mainly researchs of Cordyceps were isolation and culture, and studies
about bioactivity or pharmacology has limited. Based on previous studies related to
acetylcholinesterase activity of 60 extracts from Cordyceps biomass (Quyen et al.,
2012), showed that three extracts polyDL004, n-BuOH DL006 and n-BuOH DL015 were
high activity. So, next researches were carried out to determine the ability of enhancing
memory impairment in mice by behavior tasks. Some results on two models Y maze and
Novel object recognition showed that the extracts had positive effect on memory and
enhanced recognition. At dose of 100 mg/kg, poly DL004 and n-BuOH DL015 have a
significant difference from TMT group (data has acepted by tp ch cng ngh sinh hc,
2014). These models evaluated extracts effect on memory at level of short-term memory.
So, the research was carried out to evaluated extracts effect on memory at level of long-
term memory in mice by morris water maze task. Simultaneously, ACh and AChE
concentration of hippocampus were quantified to evaluate extracts brain protective
ability.
MATERIALS AND METHODS
Animals:
All the experiments were carried out using strain of Swiss albino mice, male, 5-6 weeks
of age, strong healthy, weigh 25-30 g, purchased from Pasteur Institute Ho Chi Minh
City. Mice were fed stability in groups, each group of 6 mice in white plastic box size 28
x 30 x15 (width x length x height) (cm). After transport, mice were hold stability 3 days
before doing experiments. Mices food is rice bran produced by Vaccine Institute and
biomedical products Nha Trang, with gradients including rice flour, corn starch and
vitamines.
Extracts: All extracts were extracted from Cordyceps spp.s dried biomass, that isolated
in Vietnam and used static liquid culture at Nguyen Long company, Lam Dong provine.
Moriss Water Maze
The Morris water maze is a circular pool (1.5 m in diameter and 0.8 m in height), made
from inox, with a black decal inner surface. The pool is conceptually divided into
quadrants, designated as northeast, southeast, northwest, and southwest. The pool is filled
to a depth of 25,1 cm with water. A small platform (12 cm in diameter and 25 cm in
height) is then placed in one of the pool quadrants and submerged 1 cm below the water
surface so that it is invisible at water level. Around the water tank have four images at
four different positions, their role are oriented objects for mice.
During the test, the pools temperature is monitored closely, allowed temperature in the
range 24-28
o
C, the rooms light is not too bright, about 30-40 lux, minimize noise. Any
change in the environment can cause errors for the results of behavior test (DHooge &
De Deyn 2001). It is also important that the investigator be hidden from view of the mice
during testing.
The model includes 8 days of testing:
Hidden flatform test (4 days): five trials are carried out every day, maximum time
for each trial is 60 s, the same release points should be used for all mice. First trial of the
first day, after swiming one minute, mice was guided to the flatform and located in it for
15 s; in the case the mice found the flatform, it was permitted to remain on it for 15 s.
Next trials were carried similarly, just change release point, maximum time for mice to
swim is 60 s, the time taken to find the hidden platform was recorded using a video
camera system. After 1 minute, mice dont find the flatform, recorded time is 60 s.
During the 3 subsequent days, the mice were given five trials per day similar as first day.
Probe test (1 day): On the fifth day, the flatform is taken out of the pool, two
trials are carried out, time for each trial is 1 minute. Recording the times that mice swim
across the previous location where the platform was put.
Working memory test (3 days): 5 trials are carried out each day, similar to hidden
flatform test. However, flatform position is changed day by day. The time mice taken to
find the flatform was recorded.
Mice were devided to groups, each group was from 8 to 10 mice. Before testing 7 days,
memory impairment was induced in mice with trimethyltin (TMT) (2.4 mg/kg). Mice
have drunk galantamine or Cordycepss extracts for 3 days before injecting TMT with
dosage: Gal. (galantamine, 10 mg/kg, p.o) as a positive control, DL004.1 (poly DL004,
100 mg/kg, p.o); DL004.2( poly DL004, 200 mg/kg, p.o); DL006.1 (n-BuOH DL006,
100 mg/kg, p.o), DL006.2 (n-BuOH DL006, 200 mg/kg, p.o), DL015.1(n-BuOH DL015,
100 mg/kg, p.o), DL015.2 (n-BuOH DL015, 200 mg/kg, p.o).
Control group (Sal.) received saline solution only (i.p), and negative group received TMT
only (i.p)
Quantify ACh, AChE concentration and total protein of mice brain hippocampus:
Experimental groups are arranged similar to MWM, however, 2 days after injecting
TMT, mice brain hippocampus was received. The hippocampus was grinded with PBS
pH 7.4 (1:20), got suspension, centrifuged 12000 rpm in 15 minutes at 4
o
C, got
homogeneous solution. This solution was quantified concentration of ACh and AChE by
Amplex Red kit. Simultaneously, protein concentration of hippocampus was also
determined by Bradford method.
Statistics: values are expressed as mean SE. Data was analyzed by One way analysis of
variance test and Fisher protected least significant difference (PLSD) test
RESULTS AND DISCUSSION
Morris water maze
The change of time to find the platform in 4 days testing of hidden platform test
was shown in Figure 1. The time to find the hidden platform of Sal. (control group) is
lowest and declines through testing days. By statistical analysis, there is significant
difference between TMT and Sal. (p<0.001), this demonstrated TMT group exhibited
memory impairment.

Fig. 1. The change of time that mice used to find the hidden platform among experiment
groups
Comparing TMT and Gal., there is significant difference in time to find hidden
platform (p<0.001), showed that galantamine has effected to against memory impairment,
also viewed models confidence. When comparing the time to find hidden platform
among testing days, there is significant difference between: first day and second day
(p<0.001), second day and third day (p<0.01), third day and fourth day (p<0.01). That
showed time factor (testing day) have effected to the change (declining) of the time to
find the platform. Through training process with unchanging platform position during all
trials, mice progress in shortening the time to find the platform.
There is significant difference between DL0004.1 and TMT (p<0.05), DL0015.1
and TMT (p<0.01). Thus, mice of two groups DL0004 and DL0015.1 have a memory
improvement in this test. So, extracts of Poly DL0004 and n-BuOH DL0015 proved
protective effects on mice to prevent memory impairment at dose of 100 mg/kg.
Probe test
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Result of the change in the number of times mice swim cross the platform
position was shown in figure 2.

Fig. 2. The change in the number of times mice swim cross the platform position on fifth
day of probe test. Statistical significant difference compare with TMT group: * (p<0.05),
** (p<0.01), *** (p<0.001).

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Fig. 3. The difference in time to find the platform on sixth, seventh and eighth day in
working memory test
The number of times mice swim cross the platform position of Sal. group is
highest. Other group have the lower number of the times, especially TMT group.
Comparing with TMT group, Gal. group has significant difference in the number of times
mice swim cross the platform position (p<0.001). In addition, there are also DL0004.1
group (p<0.05) and DL0015.1 (p<0.05). That showed dose of respective extracts has
effect to improve memory in this test. This result is logical when comparing with hidden
platform test.
Working memory test
In this test, there is a change of platform position among quadrants of the pool.
The results showed that there is no significant difference when comparing other groups
with TMT (unless Sal. and Gal. group) (Figure 3). This means dosage of extracts and
model of preventing disease are not enough to maintain effect to protect brain.

In previous experiments, when testing in models: Y-maze and Novel object recognition,
the result showed that dose of 100 mg/kg of Poly DL0004 and n-BuOH DL0015 extract
has also effect on mice brain. But in working memmory test, they havent expressed. We
suggest that dose of extracts will be changed (50 mg/kg or 150 mg/kg, p.o) to find
suitable dosage. Or increasing the number of days for treatment more than 3 days, that
can be 7 or 14 days; or when time mice were tested, extract oral therapy will be still
continuous.
ACh and AChE concentration in hippocampus
After data was statistical analysed, the results was shown in Figure 4. The
significant difference in ACh concentration between TMT and Sal. (p<0.01) showed that
ACh concentration of TMT group declined. This demonstrated TMT is an agent causing
memory impairment. Between Gal. and TMT group (p<0,001), Gal. group has a better
ACh concentration, so galantamine has positive effect on mice.
In addition, comparing TMT with other groups, results are also different from
DL0004.1 (p<0.001), DL0004.2 (p<0.01), DL0006.2 (p<0.01). Specially, ACh
concentration of DL0015.1 group has no significant difference comparing with TMT
group. Although dose of extract of DL004.2 and DL006.2 group havent effect on
behavior tasks, ACh concentration still be remained. This result showed cholinergic
hypothesis has also limited role on enhancing memory in mice.
AChE concentration in hippocampus
AChE concentration of TMT group is highest, and there is significant difference
from other groups (unless DL006.1). (Figure 5). This showed AChE activity increased
when induced with TMT.
Results from quantitating ACh and AChE concentration showed no correlate with
behavior tasks. Thereby, the mechanism of preventing memory impairment in mice is
very complex, beyond the influence of the neurotransmitter (ACh), there are other
mechanisms.
The difference in the concentration of ACh and AChE between different dosages
of an extract had no statistical significance. Thus, there is no correlation between dose
and effect of extracts to protect brain against memory impairment.


Fig. 4. The difference in acetylcholine concentration (nM/mg protein) of hippocampus
among testing groups. Statistical significant difference compare with TMT group: *
(p<0,05), ** (p<0,01), *** (p<0,001).

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Fig. 5. The difference in acetylcholinesterase concentration (mU/mg protein) of
hippocampus among testing groups. Statistical significant difference compare with TMT
group: * (p<0,05), ** (p<0,01), *** (p<0,001).


Some researches showed that cause of AD is related to oxidative stress, the
injured brain area has many free radicals (Orhan et al, 2008; Windelborn and Lipton,
2008). Therefore, the drugs used to treat AD must have high antioxidant activity. In 2007,
Yu et al compared ability to against oxidative damage between two strains of cultured C.
militaris and natural C. sinensis on lipids, proteins and lipoproteins, the results show that
both of them contain high antioxidant activity. Also in this year, Gu et al concluded both
natural and cultured Cordyceps have high potential in the production of antioxidant
compounds. Along with the research about antioxidant activity of extracts from
Cordyceps isolated and cultured in Vietnam (Thu Huynh et al, 2012), extracts from
buthanol and polysacharide extract also have high antioxidant activity. This paralells to
above results, showed extracts positive effects on the brain in mice can through not only
anti-acetylcholinesterase but also antioxidant activity.

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