Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02776673 2014-02-20
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A method for manufacturing a composition comprising an extract of red
mold rice for treatment of Alzheimer's disease
BACKGROUND OF THE INVENTION
1) FIELD OF THE INVENTION
This invention relates to a composition and method for prevention and
treatment of Alzheimer's disease, in particular, to a composition comprising
monacolins, anti-inflammation agents and anti-oxidant compounds which are
extracted from red mold rice (RMR) and applied in various forms of pastils,
capsules, powder, beverage, etc. in prevention and treatment of Alzheimer's
disease.
2) DESCRIPTION OF THE PRIOR ART
Alzheimer's disease (AD) is associated with a progressive neurons failure.
AD is a major cause of dementia, most often dementia and AD are used
interchangably. In Alzheimer's disease (AD), the progressive loss of
cognitive,
language and emotional functions occurs. Generally, serious AD patients need
more care and require assistance in all respects, such as taking a bath,
eating,
going to toilet, etc. Therefore, there are great impacts on the families of
those
AD patients in their daily lives. Loss of memory is the most often seen
symptom of AD. At the very beginning, the symptom may not be conscious by
AD 's family around and the aforementioned symptoms do not correlate with
AD only. Some known symptoms that seriously affects a person's ability to
carry out daily activities including no recognition of a place or direction,
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without memory of recent events, bringing up a matter of the past repeatedly
or
unable to learn new things. As the disease gets worse, people often have
difficulty expressing him/herself or making decisions. Gradually there is no
recognition of family and relatives for AD patients. Some AD patients may be
in
great agitation, paranoiac, suspecious, dislike interacting with people.
Subsequently, AD patients may wander on the street or lose their way home.
New studies show that the brain damages to AD patients involved in human
vision and sense of space, so that an AD patient has the problem in
identifying a
clear direction or find the way. AD patients may have difficulty concentrating
and can not carry out daily activities by themselves. Other damage to brain
cells
of AD patients, e.g., basal forebrain and hippocampus, may lead to memory loss
and confusion. Many AD patients finally die of other causes rather than AD,
such as pneumonia. Generally, AD patients can live 6-8 years, but many AD
patients still survive for more than 20 years.
The Food and Drug Administration (FDA) of the U.S. has approved five
pharmaceutical treatments for Alzheimer's disease in clinical use, including
cholinesterase inhibitors (including tacrine and donepezil). Cholinesterase is
an
enzyme that catalyzes the hydrolysis of the neurotransmitter acetylcholine
(AChE) into choline and acetic acid, currently, those approved pharmaceutical
treatments are used to inhibit cholinesterase and repress the hydrolysis of
the
neurotransmitter acetylcholine (AChE). Both inhibitors are able to increase
AChE content in the brain, defer the lose of memory, and allow AD patients to
carry out their daily activities. Importantly, those pharmaceutical treatments
are
unable to cure Alzheimer's disease, but only relieve AD symptoms. Moreover,
the aforementioned medications are proved to cause some side effects to AD
patients, such as feel nauseated, headache, diarrhoea, insomnia, pain,
illusion or
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dizzying, etc., therefore, the aforementioned medications are not practical
Alzheimer's disease treatments.
Therefore, how to effectively prevent people from Alzheimer's disease
and cure those AD patients without noticeable side effects caused need to be
researched.
SUMMARY OF THE INVENTION
In view of the foregoing drawbacks, the invention provides a composition
and method for prevention and treatment of Alzheimer's disease for truly
achieving the effect on prevention and treatment of Alzheimer's disease
without
noticeable side effects caused.
Another objective of the invention is to provide a composition and
method for prevention and treatment of Alzheimer's disease without noticeable
side effects caused to AD patients.
The invention is a composition and method for prevention and treatment
of Alzheimer's disease, the composition comprising monacolins,
anti-inflammation agents and anti-oxidant compounds, applied in various forms
of pastils, capsules, powder, beverage, etc. in prevention and treatment of
Alzheimer's disease. The composition of the invention, which is formed by the
aforementioned three compounds, is used for the prevention and treatment of
Alzheimer's disease. Moreover, in a preferred embodiment of the invention, the
minimum effective dose of monacolins is at least greater than 100 itg, the
minimum effective dose of anti-oxidant compounds is at least greater than 40
ttg
and the minimum effective dose of anti-inflammation agents is at least greater
than 10 g; wherein the monacolines, anti-inflammation agents and anti-oxidant
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compounds have the optimum weight in the ratio of 40:2:1, enabling the
composition of the invention to achieve the objectives of prevention and
treatment of Alzheimer's disease without causing noticeable side effects to AD
patients.
Moreover, the invention is a composition and method for prevention and
treatment of Alzheimer's disease, the composition comprising monacolins,
anti-inflammation agents, wherein the minimum effective dose of monacolins is
at least greater than 200 jig and the minimum effective dose of
anti-inflammation agents is at least greater than 60 jig, wherein the
monacolines
and anti-inflammation agents have the optimum weight in the ratio of 10:1,
enabling the composition of the invention to achieve the objectives of
prevention and treatment of Alzheimer's disease without causing noticeable
side
effects to AD patients.
Moreover, the invention provides a method for prevention and treatment
of Alzheimer's disease, the method comprises the following steps:
The first step is to rinse rice and carry out sterilization under a high
pressure and high temperature environment, and then the second step is to
cultivate a specific Monascus purpureus in fresh media under a first specific
temperature, humified and a specific shaking environment during a first
specific
timeframe, subsequently, the third step is to stir the media under the first
specific temperature environment and provide a specific percentage of water
during a second specific timeframe, the following step is to properly stir the
media during a third specific timeframe under the first specific temperature
environment at fixed intervals for afterripening, subsequently, after
fermentation,
the step is to collect a Monascus -fermented product and dry the product under
a
fourth specific timeframe at a second specific temperature, the next step is
to
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grind the dried Monascus-fermented product into powder and analyze if the
Monascus-fermented product conforms to a proportion of composition required,
when the Monascus-fermented powder conforms to the proportion of
composition required in the invention for prevention and treatment of AD, the
final step is to dissolve the Monascus-fermented powder in water in a specific
proportion to make Monascus beverages or fill the Monascus-fermented powder
in a capsule or make the the Monascus-fermented powder a pastil with effect on
AD prevention and treatment. The method of the invention enables the
Monascus composition to effectively achieve the prevention and treatment of
AD without noticeable side effects caused to AD patients.
A detailed description is given in the following embodiments with
reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a prospective view of an apparatus according to the invention;
Fig. 2 is a statistical chart showing the effect of RMR on step-through
latency of multiple-trial passive avoidance task in the rats from a light
chamber
into a dark chamber;
Figs. 3-1 & 3-2 show perspective views regarding an apparatus of water
maze according to the invention;
Figs. 4-A, 4-B & 4-C show influence diagrams regarding effects of RMR
on performance of the memory and learning ability of the A040-infused rats in
the training trials of reference memory task and probe test;
Fig. 5 is a chart showing effect of RMR on performance of the memory
and learning ability of the Af340-infused rats in the training trials of
working
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memory task;
Fig. 6 is an influence diagram showing effect of RMR on activity of
acetylcholinesterase (AChE) in the hippocampus and cortex of A040-infused
rats;
Fig. 7 is a schemetic diagram showing effect of RMR on the formation of
total antioxidant status (TAS) activity in the hippocampus and cortex of
A040-infused rats;
Fig. 8 is an influence diagram showing effect of RMR on the formation of
MDA activity in the hippocampus and cortex of Ai340-infused rats;
Fig. 9 is an influence diagram showing effect of RMR on the formation of
superoxide dismutase (SOD) activity in the hippocampus and cortex of
A040-infused rats;
Figs. 10-A & 10-B are diagrams showing effects of RMR on the
formation of ROS in the hippocampus and cortex and iNOS expression of
Af340-infused rats;
Fig. 11 is a diagram showing effect of RMR on the AO 40 accumulation
in the hippocampus of A(340-infused rats; and
Figs. 12-1-12-3 are flowcharts showing the method of making the
composition of the invention for prevention and treatment of AD patients.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
To enable a further understanding of the structural features and the
technical contents of the invention, the brief description of the drawings is
followed by the detailed description of embodiments.
The invention disclosed herein is a composition and method for
prevention and treatment of Alzheimer's disease without causing noticeable
side
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effects. Prior to introduction to the composition of the invention, some
concepts
of the invention are described as follows. Agitation and accumulation of
Amyloid 0 peptide (A(3) is the pathogenic agent of Alzheimer's disease (AD),
leads to neurotransmitter deficits and repress oxidizaton and inflammatory
response in the brain resulting in the aggravation of the AD situation for AD
patients. AO is formed after sequential cleavage of the amyloid precursor
protein
(APP) through a, 0 and 7-secretase.
The formation of A0 is carried out by the proteolytic cleavage of 671 and
672 amino acid binding site perfomed by 0-secretase, as well as the cleavage
of
713 amino acid binding site of APP performed by 7-sceretase. The cleavage
performed by secretase results in not only 1-40 and 1-42 fragment but also
other
fragment. APP would be cleaved in sAPP-a and p10 fragments by a-secretase.
However, p10 would be further cleaved to p3, a part of A0, and p7 fragments.
Furthermore, the cleavage of APP performed by 0-secretase would result in the
formation of sAPP0 and p12 fragments, and futher cause p12 fragment to be
cleaved to A0 and p7 fragments though 7-secretase (Evin et al. 2003; Shoji et
al.
1992). APP is cleaved to various types of A0 though 0- and 7-secretase.
However, soluble AO without neurotoxicity would be aggregated as A.0 fibrils
with neurotoxicity which leads to neuron damage via the balance disorder of
calcium ion and oxidative stress. Ai3 fibrils also break neurite outgrowth and
cell death though promoting hyperphosphorylation of tau protein. In addition,
A0 fibrils would bind to the specific receptor on the membrane of microglia
and
astrocyte, which leads to the activation of microglia involved in the release
of
many neurotoxic factors such as proinflammatory factors: IL-6, IL-1, and
TNF-a as well as NO, ROS, and free radical. Currently, Apolipoprotein E gene
in the 19th pair of chromosome is found to associate with AD pathogenesis.
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ApoE is able to easily and rapidly bind to AA and further result in the
deposition of senile plaque involved in neurotoxicity.
In addition, many documents and literatures indicate that there is a certain
relation between cardiovascular disease and Alzheimer's disease. Researches by
Martha et al focused their experiments on elders above 65 years old and
elucidated the results that those elders who ingested saturated fats during
their
diet easily have cardiovascular disease and most of those elders suffered from
Alzheimer's disease 4 years on average after they were with diagnosed
cardiovascular disease; in contrast, there was an inverse correlation if those
elders whose diet contained abundant polyunsaturated fat and monounsaturated
fat (Freund-Levi et al. 2006). Many researches have proved that there is a
relation between AO production, which promotes Alzheimer's disease, and
intracellular lipid metabolism (Frears et al. 1999; Kuo et al. 1998; Roher and
Kuo 1999). Recent researches have found that A13 alters due to intracellular
cholesterol distribution and cholesterol esterification (Frears et al. 1999).
ApoE
increases plasma cholesterol levels and is one of the risk factors associate
with
cardiovascular disease. The experiment (Puglielli et al. 2001) showed that
there
is a positive correlation between acetylcholinesterase (AChE) activity and
cholesterol ester (CE) content in the rat cells (Puglielli et al. 2001; Zhao
et al.
2005). Therefore, it is found from researches that there is a positive
correlation
between cardiovascular disease and Alzheimer's disease.
Consequently, the composition of the invention is used to decrease the AO
accumulation in the brain and reduce saturated fats which may contribute to
cardiovascular disease, so as to remedy Alzheimer's disease.
In recent year, there are many researches associated with Monascus
species. In the future, the development of RMR ingredients and compounds
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may become part of health food with multi-functions for human beings. The
invention proves that monacolin K, -y-aminobutyric acid (GABA) and
anti-oxidant compounds in RMR are substantially enhance the feasibility for
prevention and treatment of Alzheimer's disease.
Monacolin K is lovastatin, a 3-hydroxy-3-methylglutaryl-coenzyme A
(HMG CoA) reductase inhibitor (statin) for lowering cholesterol levels, and is
clinically proved to effectively improve the symptoms of cardiovascular
disease
(CAD). In recent years, researches in epidemiology have found that statin has
great clinical effectiveness for treatment of Alzheimer's disease. In UK
General
Practitioners Research Database that recent clinical reports describe an
association between statin therapy and a reduction in the occurrence of
Alzheimer's disease by as much as 70 % (Jick et al. 2000). Similar researches
include an experiment that focused on AD patients above 60 years old in three
hospitals in the USA, their AD diagnosis is based on the standard of National
Institute of Neurological and Communicative Disorders and Stroke and the
Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA).
The experiment divided those AD patients into three groups, group A was a
control group, group B was remedied by statins, group C was remedied by other
cardiovascular disease medications (including medicine for lowering blood
pressure), the experiment results showed surprisingly that the group of AD
patients with statins remedy enabled the AD levels to decrease 70%, wherein
the lovastatin and pravastatin had the best results for AD treatment (Wolozin
et
al. 2000).
Nerve impulse transmission agents, including nicotinic, muscarinic,
serotonin, glutamate receptor and T-aminobutyric acid (GABA), norepinephrine
in the brain of AD patients are damaged. During aging process, human brain
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volumn will reduce 10%, mainly the quantity of cerebral cortex neuron, some
brain area quantity will reduce 30-50% of volumn and neurotransmitters are
also reduced, such as AChE, GABA, catecholamine, etc. In view of the
foregoing, AD patients will gradually lose their brain cognition or memory
ability. AD symptoms in early stage include memory deficit, confusion, or
difficulty making decisions. In later AD period, patients have the symptoms of
language disorder, abnormal behaviors, spasm, unable to carry out daily
activities (Mohr et al. 1994).
When 02 or H202 is deficient and OH. is excessive in mitochondria,
OH . is formed with high response. Oxidative free radicals enable Ai3 to form
insolubile AA and worsen AD symptoms (Hsieh and Tai 2003). It is found from
experiment results that oral anti-oxidants such as a combination preparation
comprising vitamin C and vitamin E is able to effectively alleviate AD
symptoms (Yallampalli et al. 1998; Yamada et al. 1999). Cerarnlde, a natural
neurilemma, is used to against neuronal damage led by Af3 and FeSO4 in
hippocampus (Abousalham et al. 2002). In 1999, Aniya et al proposed the
anti-oxidation ability of Monascus that anti-oxidant mechanism of dimerumic
acid in Monascus is known to be able to eliminate the inhibition of LPO by
=OH,
ferryl-Mb and peroxyl radicals; Monascus provides an electron for oxide
to oxidize itself into nitroxide radical, and then nitroxide radical
scavenging will
lead to an anti-oxidant effect (Taira et al. 2002). Anti-oxidant effect from
RMR
is applicable to prevention of Alzheimer's disease, so that AD patients'
condition can be improved without being aggravated due to free-radical induced
oxidation.
Ths invention is published on 30 July 2007 on Journal of Neuroscience
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Research (Lee CL, Kuo TF, Wang JJ, Pan TM: Red mold rice ameliorates
impairment of memory and learning ability in intracerebroventricular amyloid
beta-infused rat via repressing amyloid beta accumulation. J Neurosci Res.
2007,
85, 3171-3182). In the experiment of the invention, male Wistar rats (weighing
250 g, 8 weeks old) were obtained from the Laboratory Animal Center of
National Taiwan University College of Medicine. They were divided into 7 rats
per group and kept in a temperature-controlled room (23 1 0) under a 12-hr
light: 12-hr dark cycle (light on at 08:00 and off at 20:00) and were given
free
access to food and water. When the weight is approximately 300 g, those rats
were divided into groups with brain infusion. The daily RMR dietary and Af140
infustion are as shown in Table 1. The rats were divided to groups including
1)
vehicle infused rats (vehicle group), surgery for i.c.v. with vehicle
solution; 2)
A(340 infused (A(3 group), which were infused i.c.v. with A1340 solution; 3)
A040 infused with administration of lovastatin (LS group), 4) A(340 infused
with administration of a onefold dosage of RMR (RL group) and 5) A040
infused with administration of a fivefold dosage of RMR (RH group). The
dosage of RMR is calculated in accordance with Boyd's formula of body
surface area as recommended by the Food and Drug Administration (Boyd 1935;
Lee et al. 2006). 2 g of RMR was used as the onefold dosage for an adult with
a
weight of 65 kg and a height of 170 cm. RH group of rats were daily
administered high dosage of RMR, LS and RL groups of rats were administered
the same dosage of lovastatin (1.43 mg/kg/day, per rat) for comparing the
effects between applying simply monacolin K and applying RMR composition,
including comprising monacolins, anti-inflammation agents and anti-oxidant
compounds, for the prevention and treatment of AD.
Rats were injected with A340 infusion for 28 consective days in the brain
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to develop Alzheimer's disease. The daily dietary dose of RMR products or the
same lovastatin contents was used for those rats to find out the RMR outcome
against AO-induced neurotoxicity and evaluate effect of RMR on the memory
and learning ability of the rats. The results of the experiment of the
invention
show that infusion of A(340 for 28 consective days in the brain increased the
AChE activity, reactive oxygen species (ROS) content and lipid peroxide (LPO)
levels, and at the same time reduce Total antioxidant capacity (T-AOC) and
superoxide dismutase (SOD) activity. The dietary dose of RMR significantly
repressed the Af340 infusion damage to the brain and had better effect
compared
with lovastatin group. Moreover, A040 infusion was not able to considerably
accumulate in hippocampus through repressing oxidizaton and inflammatory
response. Red mold rice (RMR) is at the first time proved to have the effect
on
decreasing the risk factor for memory deficit and dementia in AD patients and
the effects is significant compared with lovastatin group.
The animals grouping and experiment schedule of the invention are as
follows:
Groups AO RMR (monacolin K) Lovastatin
Infusion (mg/kg rat per day) (mg/kg rat per day)
_
Vehicle - - -
AO + - -
_
LS + - 1.43
_
RL + 151 (1.44) -
RH + 755 (7.20) -
_
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Table 1
With reference to Fig. 1, an apparatus 10 consisted of a light chamber 11
and a dark chamber 12; the chambers were both the same size. A shuttle door 13
of 10 (W) X 10 (D) cm was set for separation of the two chambers. When the
shuttle door 13 was opened, the light chamber 11 and the dark chamber 12 are
open to each other. The light chamber 11 is with illumination, whereas the
dark
chamber 12 is not. A plurality of electric wires 14 arranged with a parallel
interval of 1 cm were set through the floor of the dark chamber 12 and
delivered
an electric shock when the door was closed. In each trial, a rat was placed
into
the light chamber 11 first and the shuttle door 13 was opened. After the rat
entered the dark chamber 12, the shuttle door 13 was immediately closed and
the retention time was recorded, and an inescapable electric shock (100 V, 0.3
mA, 2 sec) was delivered through the electric wires 14, the rat was removed
from the dark chamber after 5 sec. of foot shock. If the rat was indifferent
to go
into the dark chamber 12 after 300 sec., the rat was compelled to go into the
dark chamber 12 and received electric shock through the electric wires 14
arranged on the floor of the dark chamber 12 after the shuttle door 13 was
closed. Subsequently, the rat was again placed into the light chamber 11 with
the shuttle door 13 opened, and the step-through latency of the rat in the
light
chamber 11 was measured in the retention test performed 24 hours and 48 hours
after the training trial. If the time the rat stayed in the light chamber 11
was
measured above 300 sec., memory and learning ability of the rat was considered
normal without problem.
The step-through latency of a rat from the light chamber 11 to the dark
chamber 12 was used as a marker to evaluate the memory and learning ability in
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the passive avoidance task. As shown in Fig. 2, all the rats would immediately
go into the dark chamber 12 in the first trial because of skoto-taxis, but the
electric shock in the dark chamber 12 should intimidate and prevent rats with
normal memory ability from going into the dark chamber 12 the next time.
Therefore, the step-through latency among each group of rats in the light
chamber 11 would show the most significant difference in the second trial. The
results of the second trial clearly indicate that Aig-infused rats still spent
shorter
times staying in the light chamber than vehicle-infused rats. However, RL and
RH groups are able to ensure staying in the light chamber for a longer time
compared with the AO group (p<0.05). The effect on lovastatin group is lower
than that on RL group with the same monacolin K contents, but the performance
on the memory and learning ability improvement of lovastatin group is
significantly higher than that of AO group.
With reference to Fig. 3, the invention provides a water maze 20,
comprising a circular tank 21 (diameter 140 cm, height 45 cm), which was used
as an apparatus of the water maze 20 in which a movable escape platform P1
(diameter 12 cm, height 25 cm.) was located inside the tank 21. Prior to the
experiment, the circular tank 21 was filled with water to a height of 27 cm.
The
circular tank 21 was divided into four quadrants ( I, II, III and IV), there
were
five starting positions set in the tank 21 and a position with equal distance
from
center and edge in the middle of each quadrant was marked for the location of
platform Pl. During the experiment, a camera was set at the ceiling above the
center of the water tank 21 for recording swimming routes of those rats.
Fig. 4 shows the influence effects of RMR on performance of the
memory and learning ability of the A040-infused rats. The time that rats
started
from a starting point of the apparatus of water maze 20 to search the escape
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platform P1 is regarded as the index for evaluation of the reference memory
task. The experiment results show that the AO-infused group always has longer
escape latency of finding the escape platform P1 from the second trial to the
ninth trial compared with the vehicle group. The dietary administration of RMR
(RL and RH groups), however, signigicantly decreases escape latency from the
second trial to the ninth trial compared with the AO-infused group (p<0.05).
Lovastatin administration also results in shorter escape latency compared with
the AO group, but the effect would be weaker than in the RL and RH groups.
Changes in path length produced by training trials in each group of rats
showed
a pattern similar to that of escape latency. There also were no significant
differences in swiming speed among those groups of animal during the course
of the training trials (data not shown) (p>0.05).
Probe test was immediately carried out after the training trial of the last
reference memory task on day 24. The escape platform P1 was removed from
the apparatus of water maze 20 (water tank 21). The time that rats wandered in
the original quadrant with the escape platform P1 placed therein is regarded
as
the index of the probe test for evaluation of the memory and learning ability.
The results showed in Fig. 4-A that the AO group spent less time searching the
target quadrant than the vehicle group (p<0.05). Administration with RMR at
onefold dosage in RL group or fivefold dosage in RH group results in
significant increase on search time in the target quadrant by 38.2% (p<0.05)
and
48.0% (p<0.01), respectively, compared with the AO group, suggesting that the
increase in search time in the RL and RH groups is attributable to the effects
of
RMR. It proves that red mold rice is able to improve the memory and learning
ability for AD. Swiming pathway is helpful in understanding the truth of the
memory and learning ability of rats in spatial probe trial. Fig. 4-B clearly
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indicated that AO-infused rats searched the target quadrant with directionless
escape and around the whole circular tank 21. In contrast, rats with better
memory and learning ability, such as RL, RH and vehicle groups, swam directly
to the target quadrant and lingered for a long time. However, the lovastatin
group always resulted in an ordinary ameliorative effect between RL group and
AO group on impairment of memory and learning ability (Figs. 4-B and 4-C).
The working memory task is a method for evaluation of short-term the memory
and learning ability. With reference to Fig. 5, AO-infused rats are unable to
shorten the escape latency time in searching the escape platform P1 compared
with vehicle group (p<0.01). However, both RL and RH groups are able to
perform memory and learning as fast as the vehicle group in the working
memory task, and RL and RH groups significantly decrease escape latency time
by 57.3% and 58.9% compared with the AO group (p<0.01). LS group
significantly decreases escape latency time by 26.7% compared with the AO
group (p<0.05), but the effect was weaker than with the RL and RH groups
(p<0.05).
AO has been proved to be the risk factor for memory deficit and
dementia in AD patients (Hashimoto et al. 2005; Stephan and Phillips 2005) and
intracerebroventricular (i.c.v.) infusion of AO40 into lateral ventricle of
rats has
been demonstrated as a successful method for establishing an AD animal model
(Stephan and Phillips 2005). Learning ability and memory deficit were tested
in
the behavior of AO-infused AD rats (Kar et al. 1998; Schubert et al. 1995;
Townsend and Pratico 2005). The free radials and ROS induced by AO deposit
damaged the neuron and synaptic junction and finally led to memory deficit and
lack of and learning ability (Townsend and Pratico 2005). Therefore,
antioxidants and anti-inflammation agents have usually been tried to reduce
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these AD risk factors in brain and ameliorate the impairment of memory and
learning ability (Chauhan et al. 2004; Cordle et al. 2005).
Moms water maze task is used to evaluate the memory and learning
ability, the reference memory task is a method of evaluating long-term memory
ability and the working memory task is a method of evaluating short-term
memory ability. According to the results of the aforementioned memory and
learning tasks, the AD rats with onefold dosage and fivefold dosage of RMR
shortened the escape latency time in the reference memory task and the working
memory task (p<0.05). In addition, RMR dietary rats prolonged their searching
to time in the target wuadrant in probe trial. The experiment results
clearly showed
that rats with RMR dietary are able to improve the memory and learning ability
to shorten the time in finding the escape platform in water maze. In contrast,
AD
rats without RMR dietary only searched the whole water maze with
directionless escape and spent more time in the reference memory task and
working memory task.
Monacolin K, an inhibitor of HMG-CoA reductase, was regarded as an
important metabolite with hypolipidemic ability in RMR, statin are reported as
a
novel remedy for AD via the repression of AO formation and AO-induced
inflammatory response (Chauhan et al. 2004; Li et al. 2006). Many studies
related to AD have used statins to lower AO formation in an AD transgenic
mouse model (Yamada et al. 1999). Lovastatin has never been used to
ameliorate the impairment of memory and learning ability in the AO-infused AD
rat model. In this study, lovastatin was used to substitute for monacolin K of
RMR in order to investigate whether ameliorating the impairment of memory
ability by RMR administration resulted only from monacolin K. The results on
the memory task clarified that lovastatin results in a weaker effect on
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ameliorating the impairment of memory than in the RL group even though the
two groups included equal levels of monacolin K or lovastatin. Therefore,
monacolin K is not the only functional ingredient to ameliorate AO-induced
memory impairment.
Currently, the treatment and remedy of AD mostly focus on inhibition of
AChE activity to increase AChE concentration and improve brain cognition and
memory ability (Nabeshima and Nitta 1994). Relevant researches by
establishing AD animal model also proved that the neurotransmitter AChE in
the brain would be significantly decreased in the AO-infused rats. Some
researches also proved that AO-infused rats have substantially less AChE
contents than normal rats (Arendt et al. 1984; Darvesh et al. 2004). The
decrease in AChE concentration and an increase in AChE activity caused
serious neuron loss (Stephan and Phillips 2005). In addition, the increase in
AChE activity was proved to stimulate AO aggregation in vitro and to form
stable complexes with AO fibrils (Stephan and Phillips 2005). Therefore,
inhibition of AChE activity is regarded as neuro-protection mechanism to
indirectly lower AO-induced memory deficit. Fig. 6 is an influence diagram
showing effect of RMR on activity of acetylcholinesterase in the hippocampus
and cortex of A040-infused rats. AO i.c.v. infusion leads to an increase in
AChE
activity in cortex by 50.5% and by 179.1% in hippocampus compared with the
vehicle group. Administration with onefold or fivefold dosages of RMR
significantly inhibits the AO-increased AChE activity, but lovastatin is
ineffective at inhibiting AChE activity in cortex and hippocampus. The results
suggest that the ingredient in RMR with an inhibitory effect on AO-raised AChE
activity should be not monacolin K but the other functional metabolites. Not
many researches elucidated that lovastatin is able to inhibit AChE activity,
even
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some researches pointed out that lovastatin does not have significantly
inhibition effect on AChE activity. This research result matches the invention
tendency. In addition to monacolin K, metabolite with effectiveness of RMR
may include other AChE inhibitors. However, the metabolite, GABA of RMR,
is also a neurotransmitter and is useful to improve cognition and memory
ability
of AD patients.
AO has been demonstrated to cause oxidative stress damage in AD brain.
Therefore, repressing Af3-induced oxidative stress was regarded as an
important
goal in drug development for AD. In Fig. 7, AO infusion decreases the total
antioxidants status (TAS) levels of cortex and hippocampus by 20.9% and
20.4%, respectively, compared with the vehicle group. Lovastatin
administration is able to increase TAS levels by 13.9% in cortex compared with
the AO group, but it is ineffective in hippocampus. However, significant
increases by 24.6% and 46.2% in TAS levels in cortex and hippocampus were
seen in the RL group. However, the experiment results showed that fivefold
dosage RMR is able to increase TAS levels in cortex and hippocampus in other
groups compared with the A13 group.
The effects of RMR administration on MDA levels of cortex and
hippocampus are shown in Fig. 8. MDA levels are significantly increased by
95.3% and 112% in cortex and hippocampus, respectively, through A(3 infusion;
but the MDA increase would be remarkably reversed with dose-response by
increasing the dosage of RMR.
RL and RH groups also showed similar neuroprotective effects on
superoxide dismutase (SOD) activity of cortex and hippocampus as shown in
Fig. 9. Although SOD activity of cortex and hippocampus were reduced by AO
infusion by 19.8% and 25.2%, respectively, RL and RH groups exhibited a
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significant increase in AO-reduced SOD activity by 27.2% and 52.7% in cortex
and hippocampus with onefold dosage RMR and by 27.2% and 60.9% in cortex
and hippocampus with fivefold dosage RMR.
In view of the foregoing, the rats in the experiment have serious oxidative
stress damage in cortex and hippocampus. The oxidative stress damage can be
improved by daily RMR dietary with dose-response and better effect than
lovastatin group. Anti-oxidant compounds that are extracted from
Monascus-fermented products include dimerumic acid, tannin, phenol,
monounsaturated fatty acid and sterols (Aniya et al. 1999; Wang et al. 2006).
Therefore, the invention indicates that anti-oxidant compounds are able to
be selected from either of the following compounds: dimenimic acid, tannin,
phenol, monounsaturated fatty acid, sterols and superoxide dismutase (SOD).
The experiment showed that the results of reactive oxygen species (ROS)
levels in cortex and hippocampus as shown in Fig. 10-A. AO infusion
significantly stimulates the increase of ROS levels in cortex and hippocampus
by 39.8% and 28.7% (p<0.05). However, daily administration with RMR in RL
and RH groups remarkably reduced AO-induced ROS levels in cortex and
hippocampus by 16.0% (p<0.05) and 21.2% (p<0.05), respectively, in RL group
and by 35.4% (p<0.01) and 21.3% (p<0.05), respectively, in RH group. The
decrease in ROS levels by 29.3% in cortex and by 15.7% (p<0.05) in
hippocampus were also found in the lovastatin group. The iNOS expression in
hippocampus was shown in the immunohistochemical stain in Fig. 10-B. AO
infusion resulted in a significant increase of iNOS expression, but the
expression would be inhibited in the RL and RH groups. Importantly, iNOS
expression of hippocampus in RL and RH groups is lower than that in the
lovastatin group. The experiment results also showed that the anti-
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effect of lovastatin group is lower than that of RL and RH groups with dietary
onefold and fivefold dosage RMR.
The significant decrease in ROS levels in cortex and hippocampus can be
seen with dose response in the RMR administration groups. Although
lovastatin has been used to lower AO-induced inflammation response in cell
model, the neuroprotective effect of lovastatin has never been used to
ameliorate the impairment of memory ability in AO-induced AD rat model
according to our information. In this study, lovastatin administration also
showed a significant decrease in ROS levels and iNOS expression compared
with the AO group, but the effect was less than in the RL and RH groups. This
is not the first report of the anti-inflammatory ability of RMR, the
anti-inflammatory metabolites of RMR have been reported including various
forms of monacolins; six azaphilones: monascin, ankaflavin, rubropunctatin,
monascorburin, rubropunctamine and monascorburamine; two
furanoisophthalides: xanthomonasin A and xanthomonasin B; and two amino
acids: (+)-monascumic acid and (-)-monascumic acid (Schubert et al. 1995). It
is clear that monacolin K is not the only functional metabolite repressing the
AO-induced inflammation response. Another experiment (Akihisa et al 2005)
showed that red mold rice repressed inflammation response induced by
12-0-tetradecanoylphorbol-13-acetate (TPA), the research also proved that
major anti-inflammation agents are chemical compounds of azaphilones and
furanoisophthalides. The results from this study and related researches
regarding RMR against inflammation suggest that RMR repressing
AO-induced inflammation response mainly results from the coordination
mechanism collectively worked by monacolin K and other anti-inflammation
agents.
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Accordingly, the invention provides inflammation agents, which is able to
be selected from the following compounds of 7-aminobutyric acid (GABA),
monascin, ankaflavin, rubropunctatin, monascorburin, rubropunctamine,
monascorburamine, xanthomonasin A, xanthomonasin B, (+)-monascumic acid
and (-)-monascumic acid.
During the experiment, A(340 was continuously infused for 28 days into
the brain in hippocampus of the rats. The results in Fig. 11 showed that A040
accumulation in hippocampus of the Ar3 group was higher than that in the
vehicle group. It is known from the foregoing results and researches that
A(340
infusion will cause oxidative stress and inflammation response in the brain,
and
progressively cause Af340 accumulation. More A(340 accumulation will cause
more serious oxidative stress and inflammation response, so as to continuously
aggravate brain damage in a vicious circle. Lovastatin has the effect of
inhibiting Ai340-induced inflammation response, but is weaker against
oxidative
stress. It is found from the experiment results that A(340 content of LS group
in
hippocampus was slightly lower than that of AO group, however, there was
significant A(340 accumulation in the brain. The RL group and RH group with
dietary dose of RMR had less Af340 accumulation in hippocampus than the Af3
group. The main reason why red mold rice is able to reduce A1340 accumulation
in hippocampus lies in the ability of inhibiting oxidative stress and
inflammation response. A(340-infused rats were not influenced by A1340
accumulation due to oxidative inflammation agents, so that A1340 was unable to
cause damage to the brain so as to effectively improve the memory and learning
ability.
Monacolins, anti-inflammation agents and anti-oxidant compounds
included in the composition and method for prevention and treatment of
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Alzheimer's disease of the invention are extracted from red mold rice (RMR) to
improve existing symptoms or slow Alzheimer's disease. In the first
embodiment of the invention, the minimum effective dose of monacolins in 1 g
of red mold rice is at least greater than 100 pg, the minimum effective dose
of
anti-oxidant compounds in 1 g of red mold rice is at least greater than 40
jig,
and the minimum effective dose of anti-inflammation agents in 1 g of red mold
rice is at least greater than 10 jig, wherein monacolins, anti-inflammation
agents
and anti-oxidant compounds have the optimum weight in ratio of 40:2:1 as
shown in the first embodiment to achieve the best effectiveness. In addition,
monacolins, anti-inflammation agents and anti-oxidant compounds have the
optimum weights in ratio from 10:4:1 to 90:2:1 and the composition of the
invention is able to be applied to in various forms of pastils, capsules,
powder,
beverage, etc.
Moreover, in the second embodiment of the invention, the composition
comprising the minimum effective dose of monacolins in 1 g of red mold rice is
at least greater than 200 pig and the minimum effective dose of
anti-inflammation agents in 1 g of red mold rice is at least greater than 60
pg;
wherein monacolins and and anti-inflammation agents have the optimum weight
in ratio of 10 : 1 as shown in the second embodiment to achieve the best
effectiveness; in addition, monacolins and anti-inflammation agents have the
optimum weight ratio range from 10:3 to 45:1 and the composition in the
second embodiment of the invention is proved to achieve the same effect as in
the first embodiment of the invention. In the second embodiment of the
invention, the composition comprising both monacolins and anti-inflammation
agents is able to be used for prevention and treatment of Alzheimer's disease
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without causing noticeable side effects and applied in various forms of
pastils,
capsules, powder, beverage, etc. in prevention and treatment of Alzheimer's
disease.
Anti-inflammation agents in red mold rice (RMR) is 7-aminobutyric acid
(GABA), red mold rice is regarded as natural food and is fermented and
extracted through specific methods without causing side effects to human
bodies. Therefore, RMR applications for the prevention and treatment of
Alzheimer's disease only generate significant effectiveness without causing
side
effects on patients like general pharmaceuticals.
With reference to Figs. 12-1 ¨ 12-3, the invention provides a method for
the prevention and treatment of Alzheimer's disease, the method comprises the
following steps:
First of all, the step is to rinse rice and carry out sterilization under a
high
pressure and high temperature environment (step 100); the rice is logn-grain
rice (Oryza sativa L., Japonica) purchased from a local supermarket in Taiwan
to be used for RMR production under solid-state cultivation and the
aforementioned high pressure and high temperature environment means under
the environment at 1210 by the pressure of 1 kg/cm2. The second step is to
cultivate a specific Monascus purpureus in fresh media under a first specific
temperature, humified and a specific shaking environment during a first
specific
timeframe (step 110), wherein the Monascus purpureus in fresh media means at
least 5 g of the rice is soaked in 100 mL of sterile distilled water, the
first
specific timeframe is after 48 hours and the first specific temperature is
maintained at 300, the specific shaking environment is maintained at a
rotational speed between 100 and 150 revolutions per minute (rpm); in other
words, the Monascus purpureus is cultivated in the media and maintained in an
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environment at a temperature of 300, a rotational speed 125 rpm and after 48
hours the cultivation is completed for collection. 500 g of solid-state
cultivation
is then collected, the Monascus is immersed in water for 6-8 hours, filtered
by
cotton cloth and placed on a cloth in a plate for sterilization (121 C, 20-25
min).
Another sterilization (121 C, 20 min) is then carried out after 100 mL of
water
sprinkling. After cooling, another Monascus cultivation is carried out by
placing
Monascus in solid-state media (5%). Subsequently, the step is to sufficiently
stir the media under the first specific temperature environment and provide a
specific percentage of water during a second specific timeframe (step 120),
wherein the second specific timeframe means within 72 hours, the specific
percentage of water means 20% of sterile distilled water is supplemented; in
other words, the Monascus is cultivated in a thermostated container at 30 C by
way of stirring the media every 24 hours within 72 hours and supplementing
with 20% of sterile distilled water. The next step is to properly stir the
media
during a third specific timeframe under the first specific temperature
environment at intervals for afterripening (step 130), wherein the
afterripening
is the formation stage of metabolites and the third specific timeframe is
within
96 hours. The fixed intervals are equal to every 10 hours. That is, the
Monascus
media is properly stirred every 10 hours within a total of 96 hours.
Subsequently,
after fermentation, the step is to collect a Monascus-fermented product and
dry
the product under a fourth specific timeframe at a second specific temperature
(step 140), wherein the fourth specific timeframe means within 24 hours and
the
second specific temperature is maintained at the temperature range of 55-60 C.
This step is to collect Monascus and carry out the procedure of drying the
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Monascus-fermented product for 24 hours at 60 C. The next step is to grind the
dried Monascus-fermented product into powder and analyze if the
Monascus-fermented product conforms to a proportion of composition required
(step 150), wherein the proportion of composition required means the
composition comprising monacolins, anti-inflammation agents and anti-oxidant
compounds at the effective weight ratios ranged from 10:4:1 to 90:2:1. If the
Monascus-fermented powder does not conform to the proportion of composition
required, the process is ended by failure. When the Monascus-fermented
powder conforms to the proportion of composition required in the invention for
prevention and treatment of AD, there are step A and step B to follow as shown
in Figs. 12-2 and 12-3. Following step A, the final step is to dissolve the
Monascus-fermented powder in water in a specific proportion to make
Monascus beverages with effect on AD prevention and treatment (step 160) and
the process is completed. The specific proportion is between 1.0% and 4.0%.
Following step B, the final step is to fill the Monascus-fermented powder
in a capsule or make the the Monascus-fermented powder a pastil (step 170).
The method of the invention is then completed.
The experiment for the invention is carried out through conventional
media for Monascus production under solid-state cultivation, the koji dish
size
is 20 (L) x 30 (W) X 5 cm (H) and a cloth is placed on the bottom of the media
for easily stirring and maintaining the humidity, there is another cloth on
the top
for separation of external contamination and maintaining the moisture of
Monascus during fermentation. The cultivation is carried out in an opening
space. Consequently, the composition of the invention through the method
proposed herein is able to be applied to in various forms of pastils,
capsules,
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powder, beverage, etc.
New characteristics and advantages of the invention covered by this
document have been set forth in the foregoing description. It is of course to
be
understood, however, that this disclosure is, in many respects, only
illustrative.
Changes may be made in details without exceeding the scope of the invention
by those who are skilled in the art under the doctrine of equivalents, such as
monacolins, anti-inflammation agents and anti-oxidant compounds, or other
composition against Alzheimer's disease (not limit to red mold rice extracts).
The scope of the invention is, of course, defined in the language in which the
appended claims are expressed.
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