U.S. patent application number 17/003576 was filed with the patent office on 2021-02-11 for food or feed composition for improving cognitive function or memory comprising extract of desalted salicornia europaea.
The applicant listed for this patent is PHYTO CORPORATION INC.. Invention is credited to Eun-Ah CHO, Deuk-Hoi KIM, Joon Soo KIM, Mee-Hyang KWEON, Seon Yeong PARK, Hyun Joo YOON.
Application Number | 20210040133 17/003576 |
Document ID | / |
Family ID | 1000005181000 |
Filed Date | 2021-02-11 |
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United States Patent
Application |
20210040133 |
Kind Code |
A1 |
KIM; Deuk-Hoi ; et
al. |
February 11, 2021 |
FOOD OR FEED COMPOSITION FOR IMPROVING COGNITIVE FUNCTION OR MEMORY
COMPRISING EXTRACT OF DESALTED SALICORNIA EUROPAEA
Abstract
The present invention relates to a pharmaceutical composition
comprising an acanthoside B compound as an effective ingredient for
preventing or treating dementia or for improving a cognitive
function. In the present invention, a desalted glasswort extract,
and acanthoside B, which is isolated from the extract and acts as
an effective ingredient inhibitory of acetylcholine esterase, were
found to have an excellent neuroprotective activity through the
inhibition of neuroinflammation and to improve memory retention and
remarkably enhance spatial cognitive ability as measured by passive
avoidance test and Y-maze test in a scopolamine-induced amnesic
animal model. The acanthoside B or glasswort extract of the present
invention can be applied to a pharmaceutical composition for
preventing or treating dementia, a pharmaceutical composition for
improving a cognitive function, or a health functional food or feed
for improving memory retention and cognitive function.
Inventors: |
KIM; Deuk-Hoi; (Goyang-si,
KR) ; KWEON; Mee-Hyang; (Seoul, KR) ; CHO;
Eun-Ah; (Seoul, KR) ; KIM; Joon Soo;
(Chungju-si, KR) ; YOON; Hyun Joo; (Suwon-si,
KR) ; PARK; Seon Yeong; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PHYTO CORPORATION INC. |
Seoul |
|
KR |
|
|
Family ID: |
1000005181000 |
Appl. No.: |
17/003576 |
Filed: |
August 26, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16304585 |
Nov 26, 2018 |
10822367 |
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PCT/KR2018/005502 |
May 14, 2018 |
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17003576 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H 1/06 20130101; C07H
15/26 20130101; A61K 31/34 20130101; A23L 33/10 20160801; A61K
31/7034 20130101; A23K 20/121 20160501; A23L 33/105 20160801; A61P
25/28 20180101; A61K 36/21 20130101 |
International
Class: |
C07H 15/26 20060101
C07H015/26; A61P 25/28 20060101 A61P025/28; C07H 1/06 20060101
C07H001/06; A23L 33/105 20060101 A23L033/105; A61K 31/7034 20060101
A61K031/7034; A23K 20/121 20060101 A23K020/121; A23L 33/10 20060101
A23L033/10; A61K 36/21 20060101 A61K036/21; A61K 31/34 20060101
A61K031/34 |
Foreign Application Data
Date |
Code |
Application Number |
May 15, 2017 |
KR |
10-2017-0059906 |
Claims
1-10. (canceled)
11. A method for the treatment of dementia comprising the step of:
administering, to a subject, a composition comprising a desalted
Salicornia spp. extract, wherein the desalted Salicornia spp.
extract comprises acanthoside B represented by chemical formula 1
below. ##STR00007##
12. The method of claim 11, wherein the dementia is Alzheimer-type
dementia, cerebrovascular dementia, neuroinflammatory dementia,
dementia with Lewy bodies (DLB), multi-infarct dementia (MID),
frontotemporal lobar degeneration (FTLD), Pick's disease,
corticobasal degeneration (CBD), progressive supranuclear palsy
(PSP), Parkinson's disease, or Huntington's disease.
13. The method of claim 11, wherein the desalted Salicornia spp.
extract is obtained by subjecting desalted Salicornia spp. to
extraction using water or 50-100 (v/v) % of a C1 to C4 lower
alcohol.
14. The method of claim 11, wherein the desalted Salicornia spp.
extract obtained by subjecting a desalted Salicornia spp. enzymatic
hydrolysate to extraction using water or 50-100 (v/v) % of a C1 to
C4 lower alcohol.
15. A method for improvement or amelioration of cognitive ability
or memory, comprising the step of: administering, to a subject, a
composition comprising a desalted Salicornia spp. extract, wherein
the desalted Salicornia spp. extract comprises acanthoside B
represented by chemical formula 1 below. ##STR00008##
16. The method of claim 15, wherein the desalted Salicornia spp.
extract is obtained by subjecting desalted Salicornia spp. to
extraction using water or 50-100 (v/v) % of a C1 to C4 lower
alcohol.
17. The method of claim 15, wherein the desalted Salicornia spp.
extract is obtained by subjecting a desalted Salicornia spp.
enzymatic hydrolysate to extraction using water or 50-100 (v/v) %
of a C1 to C4 lower alcohol.
Description
TECHNICAL FIELD
[0001] The present invention was made with the support of the
Ministry of Agriculture, Food and Rural Affairs of the Republic of
Korea, under Project No. 116018-3, which was conducted under the
research project entitled "High value-added food technology
development project" within the project named "Development of
cognitive ability-improving functional and food substitute material
using phytomeal (halophyte-desalted material) and strategic export
commercialization thereof" by Phyto Corporation under the
management of the Korea Institute of Planning and Evaluation for
Technology in Food, Agriculture, and Forestry, from 7 Jul. 2016 to
31 Dec. 2018.
[0002] This application claims priority to and the benefit of
Korean Patent Application No. 10-2017-0059906 filed in the Korean
Intellectual Property Office on 15 May 2017, the disclosure of
which are incorporated herein by reference.
[0003] The present invention relates to a pharmaceutical
composition containing a Salicornia spp. extract for prevention or
treatment of dementia and improvement of cognitive ability and,
more specifically, to a pharmaceutical composition containing a
Salicornia spp. extract comprising acanthoside B as an active
ingredient for prevention or treatment of dementia and improvement
of cognitive ability and a method for preparing the same.
BACKGROUND ART
[0004] Dementia is defined as declines in memory and cognitive
ability to impair daily life, and Alzheimer's disease (AD) is a
clinicopathologically neurodegenerative disease involving the loss
of memory and cognitive ability and the mental and behavioral
disorders. This disease is the major cause of senile dementia, and
20-50% of AD cases occur in the elderly population over 85 years of
age. Since AD requires long-term treatment and, due to the nature
of the disease, also calls for social support for patient's family
and of caregivers, the cost of treatment is estimated to reach $64
billion globally according to the report in 2010. Already in many
developed countries, dementia is considered as a disease imposing a
serious economic burden on patients, families, and society. A
precise mechanism of this disease has not been elucidated yet, and
the disease has a complicated onset mechanism due to the nature
thereof, and thus there are many difficulties in the development of
therapy.
[0005] There are various evidences that memory decline, one of the
symptoms of dementia, is associated with the content of
acetylcholine as a neurotransmitter. After the fact that the
secretion of acetylcholine and the number of cholinergic neurons
are reduced in the brain of dementia patients has been proved, it
has become accepted that symptoms of dementia can be treated by
inhibiting the acetylcholine degrading enzyme to increase
acetylcholine on the basis of the assumption of "cholinergic
deficient hypothesis", that is, the symptoms of dementia result
from the reduction of acetylcholine in the neuron presynapses. It
has also been reported that as the concentration of acetylcholine
esterase (ACNE) increases in the cerebral blood vessels,
cholinergic neurotransmitters for neurons are deficient, thus
causing memory and cognitive impairment. Currently, four
FDA-approved medicines for treatment are tacrine (Cognex),
donepezil (Aricept), rivastigmine (Exelon), and galantamine
(Reminyl), all which are acetylcholine esterase (AChE) inhibitor
compounds. In addition to the four medicines approved by the FDA,
many medicines are currently under clinical trials. However,
Tacrine is scarcely used at present due to side effects thereof,
such as drug toxicity and hepatotoxicity, although it is a drug
showing strong acetylcholine esterase inhibitory activity, and
Rivastigmine is known to have disadvantages in that it may cause
vomiting and dizziness and is difficult to administer at high doses
effective for therapy. Therefore, the development of an AChE
inhibitor of a natural material, which will substitute for a
chemical synthetic drug and has no side effects and toxicity, is
urgently required, and studies related to the prevention of
dementia and the development of medicines using a new AChE
inhibitor compound derived from natural materials are also actively
being conducted. Recently, the development of next-generation AChE
inhibitors enabling intradermal or intravenous injection is being
conducted.
[0006] Meanwhile, neuroinflammation is known to be involved in
pathological mechanisms of neurological diseases, such as
Alzheimer's dementia, senile dementia, Parkinson's disease,
multiple sclerosis, and AIDS dementia, and inflammation responses
by hypersensitization of microglia and astrocytes are receiving
attention as major causes and effects of Parkinson's disease, which
is one of the representative neurodegenerative diseases. The
neuroglia is one type of important cells that constitute the
central nervous system. Appropriately activated neuroglia and
well-regulated inflammation responses have neuron and tissue
protective actions. However, when these cells are not properly
regulated due to excessive inflammation responses, the inflammation
mediators produced by these cells may exhibit neuronal cytotoxicity
and cause degenerative mutations of nerve tissue.
[0007] Degenerative diseases of the brain and nervous system are
generic terms of diseases in which specific neuronal groups of the
brain and spinal cord lose their functions and the number of
neurons is decreased, and representative examples thereof are
Alzheimer's disease (AD), Parkinson's disease, and Huntington's
disease (HD). Through molecular biology and immunohistochemical
staining studies that have been accumulated over the past 30 years,
neurodegenerative diseases are known to occur due to the apoptosis
of neurons, which are most important in the information
transmission of the brain and nervous system: problems in the
formation or functions of synapses that transmit information
between brain cells: and an abnormal increase or decrease in
electrical activity of brain nerve, and these phenomena increase
further with age. At present, the aging of the population is
rapidly progressing in the world, especially in emerging economies
and western countries. In Korea, the number of elderly people aged
65 and over was over 8 million in 2015, accounting for 15.7% of the
total population, and it is predicted to reach 40.1% by 2060.
[0008] Meanwhile, the major features of AD brain lesions are
microgliosis and neuroinflammation, and according to numerous
epidemiologic studies, the long-term taking of NSAID-based
anti-inflammatory drugs showed a tendency of lowering the
prevalence of AD. A 10-year long-term project in Canada also showed
a reduced prevalence of AD when patients with mild cognitive
impairment received NSAID treatment. It is therefore expected that
a neuroinflammation inhibitor can be used as an effective
preventive and treating agent in the events of mild and moderate
cognitive impairments. However, considering a case in which the
COX-2 inhibitor "Prexige" (lumiracoxib), as an inflammation
medicine, by Novartis has been prohibited due to side effect
thereof in Canada and Australia, the development of AD preventive
and treating agents using neuroinflammation inhibiting drugs or
brain cell protecting drugs with fewer side effects from natural
materials instead of conventional chemical synthetic drugs are
recently receiving attraction.
[0009] Salicornia spp. are annual herbs belonging to the
Chenopodiaceae family, and are obligatory halophytes or true
halophytes, which can be grown without growth reduction even in a
sand culture with 200 mM NaCl due to strong salt resistance thereof
among halophytes and absolutely require salts for growth. In
obligatory halophytes or true halophytes, solutes contributing to
osmotic control are known to be osmosis resistant organic
substances, and representative osmosis resistant substances
accumulated in cells are amino acids, onium compounds, such as
proline and glycine betaine, sugar alcohols, such as mannitol,
sorbitol, and inositol, monosaccharide trehalose, and the like.
Salicornia spp. need no "fertilizer" since they feed on seawater
and require no "agricultural pesticides" since they are not damaged
by diseases and insects due to high salinity in the body, and thus
Salicornia spp. are environmentally friendly/sustainable crops.
Salicornia spp. are naturally grown or cultivated all over the
world including Korea, USA, and Europe. Salicornia spp. can be
cultivated in triplicate for one year especially in the subtropical
and tropical regions, and therefore Salicornia spp. are optimal
model plants for "Seawater Agriculture" suitable for water shortage
and food shortage due to global warming.
[0010] Salicornia spp. are also known as "reservoirs of minerals"
since they contain large amounts of vegetable salts (NaCl, KCl,
etc.), calcium, magnesium, potassium and the like in the body.
Salicornia spp. have large amounts of nutrients, such as dietary
fibers and essential amino acids, and are rich in physiologically
active plant substances, such as chlorophyll, polyphenol and
flavonoids, and thus have long been used as a folk remedy for the
removal of toxins and fecal stasis accumulated in the body and for
the treatment of cancer, hypertension, diabetes, hepatitis, skin
diseases, arthritis and the like. As research on physiologically
active substances from native plants has been active in Korea for
the past 10 years, studies on functionalities of the halophyte
Salicornia spp., which have been naturally grown in the west coast
of Korea, have been activated by Korean researchers, and as a
result, more than 100 Korean and oversea academic papers and
numerous patents have been derived. In particular, choline of
Salicornia spp. is known to be an important precursor of
acetylcholine as a neurotransmitter, sphingomyelin as a constituent
for neurons in the brain, and lecithin as a constituent of body
constituent cells, and betaine is a substance that plays a major
role in detoxification in the liver and is known to be a crucial
contributor to reducing blood toxicity by eliminating toxicity in
the body.
[0011] Salicornia spp. contain large amounts of salts (more than
35%) as raw materials per se, and thus are utilized as a salt
substitute while keeping salty taste in Salicornia spp. related
products (powder, extract, pills, and the like) or developed as
slightly salted products through mixing with other raw materials.
In spite of the research of a variety of functionality of
Salicornia spp., Salicornia spp., extracts or powders contain a
high concentration of sodium, and thus there was a limitation in
the production of functional materials from 100% Salicornia spp.
powders or 100% Salicornia spp. extracts. The present inventors
have source techniques for functionality-enhanced desalted
Salicornia spp. materials through desalination and for
manufacturing methods therefor.
[0012] The present invention verified excellent acetylcholine
esterase inhibitory activity of the desalted Salicornia spp.
extracts; isolated acanthoside B, as an active ingredient, from the
extracts; and verified cytoprotective activity through the
inhibition of neuroinflammation and improvement effects of memory
and cognitive ability in amnesia animal models by the Salicornia
spp. extracts and acanthoside B. Therefore, the present invention
supposes a pharmaceutical composition and a functional food each
containing, a desalted Salicornia spp. extract or acanthoside B as
an active ingredient for prevention or treatment of dementia and
improvement of cognitive ability.
DETAILED DESCRIPTION OF THE INVENTION
Technical Problem
[0013] The present inventors endeavored to develop an agent for
prevention or treatment of dementia as a neuroinflammation
inhibiting medicine having fewer side effects. As a result, the
present inventors isolated acanthoside B from a Salicornia europaea
extract, and verified that said compound shows cytoprotective
activity through the inhibition of neuroinflammation and
improvement effects of memory and cognitive ability in amnesia
animal models, and thus the present inventors completed the present
invention.
[0014] Therefore, an aspect of the present invention is to provide
a pharmaceutical composition for prevention or treatment of
dementia or improvement of cognitive ability.
[0015] Another aspect of the present invention is to provide a
functional food or a feed composition for improvement of cognitive
ability and memory.
[0016] Still another aspect of the present invention is to provide
a method for isolating acanthoside B.
[0017] Still another aspect of the present invention is to provide
a method for treatment of dementia.
[0018] Still another aspect of the present invention is to provide
a method for enhancement or improvement of cognitive ability.
Technical Solution
[0019] In accordance with an aspect of the present invention, there
is provided a pharmaceutical composition for prevention or
treatment of dementia or improvement of cognitive ability, the
pharmaceutical composition containing acanthoside B represented by
chemical formula 1 below or a pharmaceutically acceptable salt
thereof.
##STR00001##
[0020] The present inventors endeavored to develop an agent for
prevention or treatment of dementia as a neuroinflammation
inhibiting medicine having fewer side effects. As a result, the
present inventors isolated acanthoside B from a Salicornia europaea
extract, and verified that said compound shows cytoprotective
activity through the inhibition of neuroinflammation and
improvement effects of memory and cognitive ability in amnesia
animal models.
[0021] The present inventors freeze-dried and then pulverized
Salicornia europaea, followed by desalination using cold water, and
performed hot-water extraction or enzymatic degradation extraction
on the desalted powder, followed by organic solvent fractionation
using chloroform and then column chromatography. After that, the
present inventors isolated a single substance by HPLC, and then
performed structural analysis thereof. As a result, the present
inventors identified the singe substance as acanthoside B and
verified cytoprotective activity thereof.
[0022] Various Salicornia spp. may be used, and for example, one or
more Salicornia spp. selected from the group consisting of
Salicornia europaea, Salicornia perennans, Salicornia procumbens,
Salicornia persica, Salicornia maritime, Salicornia bigelovii,
Salicornia depressa, Salicornia rubra, Salicornia praecox,
Salicornia senegalensis, Salicornia perrieri, Salicornia
pachystachya, Salicornia meyeriana, Salicornia uniflora and
Salicornia brachiate may be used.
[0023] A target disease of the present invention, "dementia",
includes Alzheimer-type dementia, cerebrovascular dementia,
neuroinflammatory dementia, degenerative brain diseases [e.g.,
dementia with Lewy bodies (DLB), multi-infarct dementia (MID),
frontotemporal lobar degeneration (FTLD), Pick's disease,
corticobasal degeneration (CBD), progressive supranuclear palsy
(PSP), Parkinson's disease and Huntington's disease], and the like,
but is not limited thereto.
[0024] In the present invention, the pharmaceutical composition for
improvement of cognitive ability may be used in the same meaning as
a pharmaceutical composition for enhancement of cognitive ability
or a pharmaceutical composition for amelioration of memory
disorders or enhancement of memory.
[0025] As used herein, the term "containing as an active
ingredient" refers to the inclusion of an amount that is sufficient
to attain efficacy or activity of acanthoside B below. Acanthoside
B contained in the composition of the present invention is a
compound isolated from Salicornia spp., which are natural plant
materials, and the quantitative upper limit thereof contained in
the composition of the present invention may be selected within an
appropriate range by a person skilled in the art.
[0026] Here, the acanthoside B used as an active ingredient may be
used as itself or in a form of a salt, preferably a
pharmaceutically acceptable salt.
[0027] The salt is preferably an acid addition salt formed by a
pharmaceutically acceptable free aid.
[0028] The free acid may be an inorganic acid and an organic
acid.
[0029] Examples of the organic acid include citric acid, acetic
acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic
acid, propionic acid, oxalic acid, tripleuroacetic acid, benzoic
acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic
acid, 4-toluenesulfonic acid, glutamic acid and aspartic acid, but
are not limited thereof.
[0030] Examples of the inorganic acid include hydrochloric acid,
bromic acid, sulfuric acid and phosphoric acid, but are not limited
thereto.
[0031] The pharmaceutical composition of the present invention may
contain a pharmaceutically acceptable carrier.
[0032] The pharmaceutically acceptable carrier is normally used at
the time of formulation, and examples thereof may include lactose,
dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium
phosphate, alginate, gelatin, calcium silicate, microcrystalline
cellulose, polyvinylpyrrolidone, cellulose, water, syrup,
methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate,
talc, magnesium stearate and mineral oil, but are not limited
thereof.
[0033] The pharmaceutical composition of the present invention may
further contain a lubricant, a wetting agent, a sweetening agent, a
flavoring agent, an emulsifier, a suspending agent, a preservative
and the like, in addition to the above ingredients.
[0034] Suitable pharmaceutically acceptable carriers and agents are
described in detail in Remington's Pharmaceutical Sciences (19th
ed., 1995).
[0035] The pharmaceutical composition of the present invention may
be administered orally or parenterally.
[0036] Examples of parenteral administration may include
intravenous injection, subcutaneous injection, intramuscular
injection, intraperitoneal injection and transdermal
administration.
[0037] The appropriate dose of the pharmaceutical composition of
the present invention varies depending on factors, such as a
formulating method, a manner of administration, patient's age, body
weight, gender and morbidity, food, a time of administration, a
route of administration, an excretion rate, and response
sensitivity.
[0038] An ordinarily skilled practitioner can easily determine and
prescribe an effective dose for desired treatment or
prevention.
[0039] According to a preferable embodiment of the present
invention, the daily dose of the pharmaceutical composition of the
present invention is 0.001-10000 mg/kg.
[0040] The pharmaceutical composition of the present invention may
be formulated into a unit dosage form or may be prepared in a
multi-dose container by using a pharmaceutically acceptable carrier
and/or excipient according to a method that is easily conducted by
a person having an ordinary skill in the art to which the present
invention pertains.
[0041] Here, the dosage form may be a solution in an oily or
aqueous medium, a suspension, an emulsion, an extract, a powder,
granules, a tablet or a capsule, and may further contain a
dispersant or a stabilizer.
[0042] In accordance with another aspect of the present invention,
there is provided a functional food or a feed composition
containing acanthoside B represented by chemical formula 1 below
for improvement of cognitive ability and memory.
##STR00002##
[0043] The functional food or feed composition for improvement of
cognitive ability and memory of the present invention uses
acanthoside B, which is the same active ingredient as in the
pharmaceutical composition for prevention or treatment of dementia
or improvement of cognitive ability, and descriptions of
overlapping contents therebetween are omitted to avoid excessive
complication of the specification.
[0044] The composition of the present invention, if a food
composition, may be prepared in the form of a powder, granules, a
tablet, a capsule or a drink.
[0045] Examples thereof are various foods such as candies, drink,
gum, tea, vitamin complexes or dietary food supplements.
[0046] The food composition of the present invention may contain
not only acanthoside B as an active ingredient but also the
ingredients that are normally added at the time of food
manufacturing, for example, a protein, a carbohydrate, a fat, a
nutrient, seasoning and a flavoring agent.
[0047] Examples of the foregoing carbohydrate may include ordinary
sugars (monosaccharides, such as glucose and fructose;
disaccharides, such as maltose, sucrose, and oligosaccharides; and
polysaccharides, such as dextrin and cyclodextrin) and sugar
alcohols, such as xylitol, sorbitol, and erythritol. Examples of
the flavoring agent may include natural flavoring agents
(thaumatin, and stevia extract (e.g., rebaudioside A, glycyrrhizin,
etc.)) and synthetic flavoring agents (saccharin, aspartame,
etc.).
[0048] For example, the food composition of the present invention,
when being manufactured into a drink, may further contain, in
addition to acanthoside B of the present invention, citric acid,
liquefied fructose, sugar, glucose, acetic acid, malic acid, fruit
juice, an Eucommia ulmoides extract, a jujube extract and a
licorice extract.
[0049] Meanwhile, Salicornia spp. have long been used for an edible
purpose and as a folk drug, and thus the ingredients extracted from
Salicornia spp. or Salicornia spp. per se could be expected to have
no toxicity and side effect. For the same reasons, a Salicornia
spp. extract or a fraction ingredient thereof can also be developed
as an animal medicine and a functional feed for the same
purposes.
[0050] In accordance with still another aspect of the present
invention, there is provided a pharmaceutical composition
containing a Salicornia spp. extract for prevention or treatment of
dementia or improvement of cognitive ability.
[0051] In accordance with still another aspect of the present
invention, there is provided a functional food or a feed
composition containing a Salicornia spp. extract for improvement of
cognitive ability and memory.
[0052] The Salicornia spp. extract defined herein uses desalted
Salicornia spp. as a raw material.
[0053] According to an embodiment of the present invention,
Salicornia spp. may be desalted by mixing a dried powder of
Salicornia spp. with cold water. According to a specific embodiment
of the present invention, a desalted Salicornia spp. product (e.g.,
a desalted powder) can be obtained by washing Salicornia spp.,
followed by freeze-drying, hot-air drying, or drying in the shade,
followed by smashing, and then desalting the obtained dried powder
using cold water at 3-9.quadrature..
[0054] The Salicornia spp. extract defined herein can be extracted
by using desalted Salicornia spp. as a raw material and employing
water, a C1 to C4 lower alcohol, or a mixture solvent thereof.
Examples of the C1 to C4 lower alcohol include methanol, ethanol,
propanol, butanol, iso-propanol, and the like.
[0055] According to an embodiment of the present invention, the
Salicornia spp. extract includes an extract obtained by enzymatic
hydrolysis of desalted Salicornia spp. before extraction and then
extraction with water or an ethanol mixture solvent.
[0056] Examples of an enzyme used in the enzymatic hydrolysis
include cellulase, hemicellulase, pectinase, .beta.-glucanase and a
combination thereof, but are not limited thereto.
[0057] According to another embodiment of the present invention,
the Salicornia spp. extract can be obtained by subjecting desalted
Salicornia spp. to extraction using water or 50-100 (v/v) % of a C1
to C4 lower alcohol.
[0058] According to still another embodiment of the present
invention, the Salicornia spp. extract can be obtained by
subjecting a desalted Salicornia spp. enzymatic hydrolysate to
extraction using water or 50-100 (v/v) % of a C1 to C4 lower
alcohol. According to a particular embodiment of the present
invention, the Salicornia spp. extract may be a 50% ethanol (v/v,
%) extract of the desalted Salicornia spp. enzymatic hydrolysate
(water:ethanol=1:1, v/v).
[0059] According to still another embodiment of the present
invention, the Salicornia spp. extract may be (i) a polar solvent
extract, (ii) an enzymatic hydrolysis extract, or (iii) an alkaloid
fraction of Salicornia spp., which is to be described in a method
for isolating acanthoside B below.
[0060] In accordance with still another aspect of the present
invention, there is provided a method for isolating acanthoside B,
the method including the steps of:
[0061] (a) obtaining (i) a polar solvent extract or (ii) an
enzymatic hydrolysis extract from Salicornia spp.;
[0062] (b) adding an acidic solution to the resultant product in
step (a), followed by stirring and standing, to eliminate
precipitates;
[0063] (c) adding a basic solution to the resultant product in step
(b) and then adding a non-polar organic solvent thereto to obtain
an alkaloid fraction; and
[0064] (d) purifying the alkaloid fraction in step (c) to obtain
acanthoside B as a single substance.
[0065] Hereinafter, the method for isolating acanthoside B of the
present invention will be described in detail.
[0066] Step (a): Obtaining Salicornia Spp. Extract
[0067] First, an appropriate solvent is added to Salicornia spp.,
thereby obtaining (i) a polar solvent extract or (ii) an enzymatic
hydrolysis extract.
[0068] According to a particular embodiment of the present
invention, the Salicornia spp. represent stems, leaves or
stems/leaves thereof.
[0069] In the present invention, the extract is obtained by
treating Salicornia spp. or a desalted Salicornia spp. product with
a solvent.
[0070] For example, the desalted Salicornia spp. product (e.g., a
desalted powder) can be obtained by washing Salicornia spp.,
followed by freeze-drying, hot-air drying or drying in the shade,
followed by pulverizing, and then desalting the obtained dried
powder using cold water at 3-9.quadrature..
[0071] Examples of the polar solvent used in the isolation method
of the present invention include (i) water, (ii) an alcohol
(preferably, methanol, ethanol, propanol, butanol, n-propanol,
iso-propanol, n-butanol, 1-pentanol, 2-butoxyethanol or ethylene
glycol), (iii) acetic acid, (iv) dimethyl-formamide (DMFO), and (v)
dimethyl sulfoxide (DMSO), but are not limited thereto.
[0072] According to an embodiment of the present invention, the
polar solvent is water or a 50-100 (v/v) % C.sub.1 to C.sub.4 lower
alcohol.
[0073] Examples of the enzyme include cellulase, hemicellulase,
pectinase, .beta.-glucanase and a combination thereof, but are not
limited thereto.
[0074] Step (b): Adding Acid Solution, Followed by Stirring and
Standing, to Eliminate Precipitates
[0075] Next, an acidic solution is added to the resultant product
in step (a), followed by stirring and standing, to eliminate
precipitates;
[0076] According to an embodiment of the present invention, an
acidic solution is added to the Salicornia spp. extract obtained in
step (a), followed by stirring and then standing at 1-10.degree. C.
for 5-18 hours.
[0077] According to another embodiment of the present invention, an
acidic solution is added to the Salicornia spp. extract obtained in
step (a), followed by stirring and then standing at 2-8.degree. C.
for 10-14 hours.
[0078] According to still another embodiment of the present
invention, an acidic solution is added to the Salicornia spp.
extract obtained in step (a), followed by stirring and then
standing at 4.degree. C. for 12 hours.
[0079] According to a particular embodiment of the present
invention, hydrochloric acid is added to the Salicornia spp.
extract obtained in step (a), followed by stirring and then
standing at 4.degree. C. for 12 hours. The precipitates generated
during such a procedure are eliminated by centrifugation and
filtration under reduced pressure.
[0080] In the present invention, the acidic solution may be a weak
acid solution or a strong acidic solution.
[0081] For example, hydrochloric acid, acetic acid, sulfuric acid
or the like may be used, but the acid solution is not limited
thereto.
[0082] Step (c): Obtaining Alkaloid Fraction
[0083] Next, a basic solution is added to the resultant product in
step (b), followed by reaction, and a non-polar organic solvent is
sequentially added, thereby obtaining an alkaloid fraction.
[0084] According to an embodiment of the present invention, ammonia
water is added to the resultant product in step (b) to adjust pH to
10 or higher, and then chloroform is added to conduct distributive
fraction. Chloroform in a chloroform fraction is removed by a
vacuum evaporator, followed by freeze-drying, thereby obtaining an
alkaloid fraction of desalted Salicornia spp. (PM-AL).
[0085] In the present invention, the basic solution may be ammonia
water or caustic soda (NaOH), but is not limited thereto.
[0086] Examples of the non-polar organic solvent used in the
isolation method of the present invention include chloroform,
hexane, ethyl acetate, butanol, acetone, acetonitrile, methyl
acetate, fluoroalkane, pentane, 2,2,4-trimethylpentane, decane,
cyclohexane, cyclopentane, diisobutylene, 1-pentene,
1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether,
2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene,
diethyl ether, diethyl sulfide, dichloromethane,
1,2-dichloroethane, aniline, diethyl amine, ether, carbon
tetrachloride and THF, but are not limited thereto.
[0087] According to an embodiment of the present invention, the
non-polar solvent is chloroform.
[0088] Step (d): Isolating Acanthoside B
[0089] Last, the alkaloid fraction in step (c) is purified to
obtain acanthoside B as a single substance.
[0090] In step (d), the alkaloid fraction in step (c) can be
isolated using high-performance liquid chromatography.
[0091] The present inventors identified a molecular structure of
the compound purely isolated by the above method, and as a result,
verified that the compound is acanthoside B (chemical formula
1).
[0092] In accordance with still another aspect of the present
invention, there is provided a method for treatment of dementia,
the method including a step for administering, to a subject, a
pharmaceutical composition containing acanthoside B represented by
chemical formula 1 below or a pharmaceutically acceptable salt
thereof.
##STR00003##
[0093] In accordance with still another aspect of the present
invention, there is provided a method for improvement or
amelioration of cognitive ability, the method including a step for
administering, to a subject, a pharmaceutical composition
containing acanthoside B represented by chemical formula 1 below or
a pharmaceutically acceptable salt thereof.
##STR00004##
[0094] In accordance with still another aspect of the present
invention, there is provided a method for improvement of cognitive
ability and memory, the method including a step for administering,
to a subject, a functional food or a feed composition containing
acanthoside B represented by chemical formula 1 below.
##STR00005##
[0095] In accordance with still another aspect of the present
invention, there is provided a method for treatment of dementia,
the method including a step for administering, to a subject, a
pharmaceutical composition containing a Salicornia spp.
extract.
[0096] In accordance with still another aspect of the present
invention, there is provided a method for improvement or
amelioration of cognitive ability, the method including a step for
administering, to a subject, a pharmaceutical composition
containing a Salicornia spp. extract.
[0097] In accordance with still another aspect of the present
invention, there is provided a method for improvement of cognitive
ability and memory, the method including a step for administering,
to a subject, a functional food or a feed composition containing a
Salicornia spp. extract.
[0098] As used herein, the term "administration" refers to the
provision of a certain material for a patient by any appropriate
method, and the pharmaceutical composition of the present invention
may be administered orally or parenterally through all general
routes as long as the pharmaceutical composition can arrive at
target tissues. In addition, the composition of the present
invention may be administered using any apparatus that can deliver
active ingredients to target cells.
[0099] As used herein, the term "subject" is not particularly
limited, but means to encompass, for example, human, monkey, cow,
horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat,
rabbit or guinea pig, preferably a mammal, and more preferably a
human being.
Advantageous Effects
[0100] Features and advantages of the present invention are
summarized as follows.
[0101] (a) The present invention is directed to a pharmaceutical
composition containing acanthoside B as an active ingredient for
prevention or treatment of dementia or improvement of cognitive
ability.
[0102] (b) It was verified in the present invention that a desalted
Salicornia spp. extract and acanthoside B, which is an
acetylcholine esterase inhibiting active ingredient isolated from
the extract, had excellent neuronal protective activity through the
inhibition of neuroinflammation, and significantly improved memory
and spatial cognitive ability in the passive avoidance test and
Y-maze test in memory decline animal models induced by
scopolamine.
[0103] (c) The acanthoside B or Salicornia spp. extract of the
present invention can be applied to a pharmaceutical composition
for prevention or treatment of dementia, a pharmaceutical
composition for improvement of cognitive ability, or a health
functional food or feed for improvement of memory and cognitive
ability.
BRIEF DESCRIPTION OF THE DRAWINGS
[0104] FIGS. 1a and 1b show: HPLC chromatogram comparison among
main fractions and isolated compound S7-L3-3 (acanthoside B)
compound during a purification procedure of an acetylcholine
esterase inhibiting effective compound from a desalted Salicornia
spp. extract (PM-EE); and the UV spectrum and chemical structure of
S7-L3-3 (acanthoside B).
[0105] FIG. 2 is a schematic view of isolation and purification
procedures of an AChE inhibiting effective compound from a desalted
Salicornia spp. extract (PM-EE).
[0106] FIG. 3 shows AChE inhibitory activity comparison results
among main fractions and the isolated compound S7-L3-3 (acanthoside
B) during a purification procedure of an AChE inhibiting effective
compound from a desalted Salicornia spp. extract (PM-EE).
[0107] FIG. 4 shows ESI-MS spectra of the isolated compound
S7-L3-3: (A) positive mode, (B) negative mode.
[0108] FIGS. 5a and 5b show NMR analysis results of the isolated
compound S7-L3-3: (5a) .sup.1H-NMR spectrum, (5b) .sup.13C-NMR
spectrum.
[0109] FIGS. 6a and 6b show HMBC-NMR (6a) and .sup.1H-.sup.1H COSY
(6b) spectra of the isolated compound S7-L3-3.
[0110] FIG. 7 shows a stereoscopic structure (A) of S7-L3-through
.sup.1H and .sup.13C-NMR and 2D-NMR analysis and a chemical
structure (B) of S7-L3-3 through intramolecular carbon and hydrogen
positioning.
[0111] FIGS. 8a and 8b show neuroglial inflammation inhibitory
effects of a desalted Salicornia spp. extract (PM-EE): (8a)
LPS-induced nitrogen monoxide (NO) production inhibitory effect of
PM-EE; and (8b) neuroinflammation factor protein (iNOS and COX-2)
expression inhibitory effects of PM-EE.
[0112] FIG. 9 shows results conforming neuroinflammation gene
expression inhibitory effects of a desalted Salicornia spp. extract
(PM-EE) through RT-PCR.
[0113] FIGS. 10a to 10c show neuroglial inflammation inhibitory
effects of acanthoside B isolated from a desalted Salicornia spp.
extract (PM-EE): (10a) Cytotoxicity test of acanthoside B through
MTT assay; (10b) LPS-induced nitrogen monoxide (NO) production
inhibitory effect of acanthoside B; and (10c) neuroinflammation
factor protein (iNOS and COX-2) expression inhibitory effects of
acanthoside B.
[0114] FIG. 11 shows results conforming neuroinflammation gene
expression inhibitory effects of acanthoside B isolated from a
desalted Salicornia spp. extract (PM-EE) through RT-PCR.
[0115] FIGS. 12a and 12b show passive avoidance test results at the
time of administration of a desalted Salicornia spp. extract
(PM-EE) and acanthoside B in forgetfulness animal models using
scopolamine: (12a) PM-EE treatment effect; and (12b) acanthoside B
treatment effect.
[0116] FIGS. 13a and 13b show Y-maze test results at the time of
administration of a desalted Salicornia spp. extract (PM-EE) and
acanthoside B in forgetfulness animal models using scopolamine:
(13a) PM-EE treatment effect; and (13b) acanthoside B treatment
effect.
MODE FOR CARRYING OUT THE INVENTION
[0117] Hereinafter, the present invention will be described in
detail with reference to examples. These examples are only for
illustrating the present invention more specifically, and it will
be apparent to those skilled in the art that the scope of the
present invention is not limited by these examples.
EXAMPLES
Example 1: Preparation of Various Extracts from Desalted Salicornia
europaea Dried Powder
[0118] Before Salicornia europaea cultivated in Shinan-gun,
Jeollanam-do (Korea) have seeds in July to August, leaves and stems
thereof were harvested, washed and dried. Various extracts were
prepared from 10 g of a desalted Salicornia europaea powder
prepared through a low-temperature cold-water desalting method.
[0119] For a hot-water extract, 100 mL of distilled water was added
to 10 g of a desalted Salicornia europaea powder, followed by
15-minute ultrasonic treatment twice. Thereafter, reflux cooling
extraction was performed at 100.+-.1.degree. C. for 1 hour. Then,
the resultant extract was cooled and centrifuged (10,000 rpm, 25
minutes) to obtain a supernatant, which was then
vacuum-concentrated and freeze-dried, to thereby prepare a
hot-water extraction powder.
[0120] For methanol and ethanol extraction, 100 mL of methanol and
ethanol were added to 10 g of a desalted Salicornia europaea
powder, respectively, followed by 15-minute ultrasonic treatment
twice. Thereafter, reflux cooling extraction was performed at
around melting points of the respective solvents for 3 hours. Then,
the resultant extracts were cooled, filtered under reduced
pressure, and centrifuged (10,000 rpm, 25 minutes), to thereby
obtain supernatants, respectively. The obtained supernatants were
concentrated through drying under reduced pressure to remove
alcohols, respectively, and then suspended in distilled water. The
suspensions were freeze-dried to prepare methanol and ethanol
extraction powders, respectively. In addition, for hydrolyzed
alcohol extracts, water and methanol or water and ethanol were
mixed at a (v/v) ratio of 1:1 or 3:7 to prepare 50% or 70% methanol
or 50% and 70% ethanol, respectively, and then each 100 mL was
added to 10 g of a desalted Salicornia europaea powder,
respectively. Thereafter, 15-minute ultrasonic treatment was
conducted twice, and then reflux cooling extraction was performed
at about melting points of the solvents for 3 hours. Thereafter,
the resultant extracts were cooled, filtered under reduced
pressure, and centrifuged to obtain supernatants. The obtained
supernatants were concentrated through drying under reduced
pressure to remove alcohols, followed by freeze-drying, to thereby
prepare 50% or 70% methanol and ethanol extraction powders.
Example 2: Preparation of Various Extracts from Desalted Salicornia
europaea Enzymatic Hydrolysate
[0121] Salicornia europaea leaves and stems harvested in July to
August were washed and dried. 60 mL of distilled water was added to
10 g of a desalted Salicornia europaea powder prepared through a
low-temperature cold-water desalting method, followed by 15-minute
ultrasonic treatment twice. Thereafter, cellulase, hemicellulase,
pectinase and .beta.-glucanase (Sigma Co, USA) each were added at
0.1% (v/v), followed by enzymatic hydrolysis at 50.degree. C. for
18 hours, thereby obtaining a desalted Salicornia europaea
enzymatic hydrolysate.
[0122] A hot-water extract of an enzymatic hydrolysate was prepared
by adding 40 mL of distilled water to the desalted Salicornia
europaea enzymatic hydrolysate and then carrying out reflux cooling
extraction through the same method as in example 1.
[0123] Methanol and ethanol extracts of a desalted Salicornia
europaea enzymatic hydrolysate were prepared as follows. 100 mL of
methanol and ethanol were added to a dried powder obtained by
freeze-drying the desalted Salicornia europaea enzymatic
hydrolysate, followed by 15-minute ultrasonic treatment twice.
Thereafter, reflux cooling extraction was performed at around
melting points of the respective solvents for 3 hours. Then, the
resultant extracts were cooled, filtered under reduced pressure,
and centrifuged (10,000 rpm, 25 minutes), to thereby obtain
supernatants. The obtained supernatants were concentrated through
drying under reduced pressure to remove alcohols, and then
suspended in distilled water. Thereafter, the suspensions were
freeze-dried to prepare methanol and ethanol extraction powders of
the desalted Salicornia neuropaea enzymatic hydrolysate.
[0124] 50% methanol and 50% ethanol extracts of the desalted
Salicornia europaea enzymatic hydrolysate were prepared by, after
the enzymatic hydrolysis, adding an equivalent amount (60 mL,
v/v=1:1) of methanol or ethanol and then carrying out extraction
through the same method as in example 1. 70% methanol and 70%
ethanol extracts of the desalted Salicornia europaea enzymatic
hydrolysate were prepared by, after the enzymatic hydrolysis,
adding 140 mL of methanol or ethanol and then carrying out
extraction through the same as in example 1.
Example 3: Measurement of Acetylcholine Esterase Inhibitory
Activity
[0125] It has been reported that as the concentration of
acetylcholine esterase (AChE) increases in the cerebral blood
vessels, cholinergic neurotransmitters for neurons are deficient in
neurons, causing memory and cognitive impairment. Therefore, the
measurement of AChE inhibitory activity can be used as a tool for
the development of a drug or health functional raw material for
prevention and treatment of dementia or enhancement of cognitive
ability. In the present invention, the AChE inhibitory activity of
a desalted Salicornia europaea extract, a desalted Salicornia
europaea enzymatic hydrolysis extract, or purified fractions
obtained from a desalted Salicornia europaea extract were measured,
and the measurement was conducted by partially correcting Ellman's
coupled enzyme assay. That is, 170 .mu.L of 100 mM phosphate buffer
(pH 8), 170 .mu.L of 2 mM dithiobisnitrobenzoic acid (DTNB), and 20
.mu.L of an extraction sample were added to on a 96-well
microplate. Then, 20 .mu.L of AChE 0.25 U/mL in the buffer was
dispensed, followed by pre-incubation at 37.degree. C. for 10
minutes, and then a 3.75 mM substrate solution of acetylcholine
Iodide was added. The enzymatic reaction solution was incubated at
37.degree. C. for 10 minutes, and then the absorbance was measured
by a UV-VIS microreader at 410 nm. The AChE inhibitory activity was
calculated as below by comparison between the absorbance (Ac) of a
control group not containing a substrate and a sample and the
absorbance (As) of a test group.
*AChE inhibitory activity (%)=[1-(As/Ac)].times.100 [0126] Ac:
absorbance of a control group, [0127] As: absorbance of a sample
group.
Test Example 1. Yields of Desalted Salicornia europaea Powder
Extracts and AChE Inhibitory Activity Thereof
[0128] The measurement results of AChE inhibitory activity of
various desalted Salicornia europaea extracts prepared in example 1
at the same concentration (100 .mu.g/mL) and the yields of the
respective extracts are shown in Table 1.
TABLE-US-00001 TABLE 1 50% 50% 70% 70% Hot Meth- Eth- meth- eth-
meth- eth- -- water anol anol anol anol anol anol Yeild (%) 15.6
8.7 7.8 11.6 10.9 9.8 9.2 AChE 52.6 58.9 61.2 62.5 65.2 60.5 62.8
Inhibtory activity (%) * sample concentration: 100 .mu.g/mL
As shown in Table 1 above, it could be verified that all of the
hot-water extract, methanol and ethanol extracts, or hydrolyzed
methanol and ethanol extracts of desalted Salicornia europaea
showed high AChE inhibitory activity of 52.6% or higher at the
concentration of 100 .mu.g/mL. Especially, it could be seen that
the 50% ethanol extract showed the highest AChE inhibitory activity
(65.2%) and the hot-water extract showed the highest yield
(15.6%).
Test Example 2. Yields of Desalted Salicornia europaea Enzymatic
Hydrolysate and AChE Inhibitory Activity Thereof
[0129] The measurement results of AChE inhibitory activity of
various extracts of desalted Salicornia europaea enzymatic
hydrolysate, prepared in example 2, at the same concentration (100
.mu.g/mL) and the yields of the respective extracts are shown in
Table 2.
TABLE-US-00002 TABLE 2 50% 50% 70% 70% Hot Meth- Eth- meth- eth-
meth- eth- -- water anol anol anol anol anol anol Yeild (%) 32.6
19.1 17.4 28.7 29.3 21.5 21.1 AChE 54.0 60.9 65.2 62.7 66.4 61.8
63.6 Inhibtory activity (%) * sample concentration: 100
.mu.g/mL
[0130] As shown in Table 2 above, it could be verified that the
hot-water extract, methanol and ethanol extracts, or hydrolyzed
methanol and ethanol extracts of desalted Salicornia europaea
enzymatic hydrolysate showed increased AChE inhibitory activity at
the concentration of 100 .mu.g/mL compared with the extracts before
enzymatic hydrolysis. Furthermore, it could be particularly seen
that the yields were remarkably increased. The reason seems that
polymer dietary fibers in Salicornia europaea were hydrolyzed
through actions of enzymes, such as cellulase, hemicellulose,
pectinase, and .beta.-glucanase, and therefore soluble components
in water and alcohols were increased. Especially, the highest AChE
inhibitory activity (67.2%) and a significant yield (29.3%) were
shown in the 50% ethanol extract. Hence, 50% ethanol extraction on
the desalted Salicornia europaea enzymatic hydrolysate was
conducted for a mass-extraction condition for isolation of an
active ingredient showing AChE inhibitory activity in the desalted
Salicornia europaea extract.
Example 4: Isolation and Purification of Substance Having AChE
Inhibitory Activity from Desalted Salicornia europaea
[0131] 4-1. Preparation of Desalted Salicornia europaea Extract
(PM-EE) and Alkaloid Fraction Thereof
[0132] On the basis of the results of test examples 1 and 2, an
active ingredient showing AChE inhibitory activity was isolated
from the desalted Salicornia europaea extract. Leaves and stems of
Salicornia europaea harvested in July to August were washed and
freeze-dried. Thereafter, 3 L of distilled water was added to 500 g
of a desalted Salicornia europaea powder obtained through
cold-water desalination, followed by sufficient mixing. Then, a
composite enzyme (Optivin Mash, Connell Bros Company, Australia)
containing cellulose, hemicellulose, pectinase, and
.beta.-glucanase was added at 0.3% (v/v). In addition, enzymatic
hydrolysis was conducted at 50.degree. C. for 18 hours. An
equivalent amount of ethanol was added to the produced enzymatic
hydrolysate, and the mixture was subjected to reflux cooling
extraction at 85.+-.1.degree. C. for 3 hours, followed by cooling.
Then, centrifugation was conducted at 4.degree. C. and 10,000 rpm
for 25 hours. The obtained centrifuged supernatant was dried under
reduced pressure at 45.degree. C. to completely remove ethanol,
followed by freeze-drying, thereby obtaining a desalted Salicornia
europaea extract (PM-EE).
[0133] The obtained extract PM-EE (100 g) was dissolved in 2 L of
distilled water, and then 6 N hydrochloric acid was added to adjust
pH to 2.0. Then, the resultant solution was stirred for 30 minutes
and allowed to stand at 4.degree. C. for 12 hours. The precipitates
generated during such a procedure were eliminated by centrifugation
and filtration under reduced pressure. In addition, 6 N ammonia
water was added to adjust pH to 10 or higher, and the resultant
solution was transferred into a funnel, followed by distributive
fraction using an equivalent amount of chloroform. An equivalent
amount of chloroform was again added to a water layer, followed by
second distributive fraction in the funnel. Thereafter, chloroform
in the primary and secondary chloroform fractions was removed using
a vacuum evaporator. Last, the fractions were suspended in
distilled water, followed by freeze-drying, thereby obtaining an
alkaloid fraction of desalted Salicornia europaea (PM-AL).
[0134] 4-2: Column Chromatography Purification
[0135] The PM-AL fraction (8 g) obtained in example 4-1 was loaded
a column (3.3.times.40 cm) charged with polar silica gel (60 G,
Merck, Germany). The fraction was released at a flow rate of 0.3
mL/min using a mobile solvent with different mixing proportions of
chloroform and methanol. As a result, 100 mL of eight fractions
(PM-1, PM-2, PM-3, PM-4, PM-5, PM-6, PM-7, and PM-8) were obtained.
Of these, a fraction (PM-S7, 187 mg) having excellent AChE
inhibitory activity was dried under reduced pressure, and then
dissolved in 3 mL of methanol. Then, the solution was introduced
into the third column (2.5.times.33 cm) charged with gel filtration
Sephadex LH-20 for a low molecular weight, and then while 100%
methanol (flow rate: 0.2 mL/min) as a mobile solvent is allowed to
flow therethrough, a total of seven fractions (PM-7-L1, -L2, -L3,
-L4, -L5, -L6, -L7). Finally, a PM-S7-L3 fraction, which has the
most excellent AChE inhibitory activity among the seven fractions,
was concentrated under reduced pressure, and freeze-dried to obtain
90 mg.
[0136] 4-3. Pure Isolation by High Performance Liquid
Chromatography (HPLC)
[0137] The PM-S7-L3 fraction (90 mg) obtained in example 4-2 was
dissolved in 2 mL of methanol for HPLC, and filtered through a
0.22-.mu.m filter, and then a single substance having strong AChE
inhibitory activity (S3-L3-3) was isolated using analytical and
preparative high-performance liquid chromatography. As analytical
HPLC, a model (1260 Infinity, Agilent, USA) equipped with Zorbax
Eclips C18 (5 .mu.m, 4.5.times.250 mm, Agilent) was used. As
preparative high-performance liquid chromatography, a model
(Multiple Preparative HPLC (LC-forte/R, YMC, Japan)) equipped with
a prep column (Triart C18, 20 mm.times.150 mm, 5 .mu.m, YMC, Japan)
was used. The mobile phase solvent conditions were as follows: the
mobile phase solvent was allowed to flow through the column at a
flow rate of 1 mL/min under gradient conditions using acetonitrile
and tertiary distilled water containing 0.04% of trifluoroacetic
acid (TFA). Agilent, 1200 DAD detector or YMC-YUV-3400 UV detector
was used. As a result of pure fraction of compounds using
absorbance at two wavelength regions (254 and 210 nm), compound
S7-L3-1 (38.5 mg) could be obtained at a retention time of 26.5
minutes. In FIG. 1, analytical HPLC chromatogram profiles of the
desalted Salicornia europaea extract (PM-EE), the column
purification fractions PM-S7 and PM-S7-L3, and the compound S7-L3-3
isolated finally by analytical HPLC were compared, and it could be
verified that as the purification proceeded, the intensity of the
peak at 26.5 min, which corresponds to a retention time of the
active ingredient S7-L3-3, was increased and the entire
chromatogram was simplified. FIG. 1 shows the UV spectrum and
chemical structure of acanthoside B, which were established by
structural analysis of purely purified S7-L3-3. In addition, FIG. 2
shows a schematic diagram of the entire isolation and purification
procedures of an AChE inhibiting active ingredient.
[0138] 4-4. Confirmation of AChE Inhibitory Activity and IC.sub.50
Values of Desalted Salicornia europaea Extraction and Purification
Fractions
[0139] AChE inhibitory activity of the desalted Salicornia europaea
extract (PM-EE), the alkaloid fraction of desalted Salicornia
europaea (PM-AL), and the column chromatography purification
fractions (PM-S7 and PM-S7-L3), and the finally isolated compound
S7-L3-3, which were obtained during the purification procedure of
the AChE inhibiting compound S7-L3-3 in examples 4-1 to 4-3 above,
were compared and measured. The average values obtained from the
test repeated three times or more at concentrations of 100, 50, and
10 .mu.g/mL of each sample are shown in FIG. 3. The AChE inhibitory
activity, 65.2%, of the desalted Salicornia europaea extract
(PM-EE) at a concentration of 100 .mu.g/mL was significantly
excellent compared with a fermented Aronia melanocarpa extract
first disclosed in Korean Patent Publication No. 10-2016-0088622.
It could also be verified that as the purification proceeded, the
AChE inhibitory activity of each fractions were gradually
increased. It could be especially verified that at the
concentration of 10 .mu.g/mL, the AChE inhibitory activity of the
finally purified compound S7-L3-3 was remarkably increased (93.2%)
compared with that of PM-EE (25.8%). In addition, the IC.sub.50
values of the extracts in respective steps and the purification
fractions, at which the AChE inhibitory activity was reduced by
50%, were compared with those of galantamine as a positive control
group, which is an AChE inhibiting synthetic medicine prescribed as
a dementia drug, and berberine, which is an AChE inhibiting
ingredient isolated from a natural material (Table 3). The
structural analysis of example 5 below identified that the finally
purified compound S7-L3-3 was acanthoside B. The measurement and
comparison with eleutheroside E as a phenylpropanoid
glycoside-based substance having a similar structure to acanthoside
B is also shown in Table 3.
TABLE-US-00003 TABLE 3 Sample name IC.sub.50 (.mu.g/mL) PM-EE 78.9
.+-. 3.90 PM-AL 20.7 .+-. 1.23 PM-S7 10.5 .+-. 0.8 PM-S7-L3 4.4
.+-. 0.5 S7-L3-3 (acanthoside B) 2.8 .+-. 0.21 Tacrine 0.036 .+-.
0.01 Galantamine 3.6 .+-. 0.41 Berberine 10.7 .+-. 0.59
Elutheroside E 8.2 .+-. 0.37
[0140] As a result of measurement of AChE inhibitory activity
IC.sub.50 values of the respective samples, the desalted Salicornia
europaea extract (PM-EE) showed a IC.sub.50 value of 78.9.+-.3.9,
as shown in Table 3 above, and it could be verified that as the
purification proceeded, such a value was gradually decreased. The
finally purified S7-L3-3 (acanthoside B) showed an IC.sub.50 value
of 2.8.+-.0.21, indicating that the AChE inhibitory activity
increased by about 28 times due to the purification. The AChE
inhibitory activity of S7-L3-3 (acanthoside B) was lower than that
of tacrine (IC.sub.50 value: 0.038.+-.0.01), which is a drug which
was initially prescribed as a FDA approved anti-dementia drug but
for which clinical prescription is prohibited due to
hepatotoxicity. However, the AChE inhibitory activity of S7-L3-3
(acanthoside B) was equal to or higher than that of the synthetic
medicine galantamine (IC.sub.50 value: 3.6.+-.0.41) as an AChE
inhibitor and was about 3.8 times better than that of the natural
material-derived AChE inhibitory compound berberine (IC.sub.50
value: 5.6.+-.0.19). Meanwhile, eleutheroside E, which has a
phenylpropanoid glycoside-based similar structure, like acanthoside
B, and has one more glucose molecule than acanthoside B, was
measured to have an IC.sub.50 value of 8.2.+-.0.37, indicating that
the AChE inhibitory activity of S7-L3-3 (acanthoside B), which is a
purified sample in the Salicornia europaea extract, was about three
times stronger than that of eleutheroside E. Therefore, it could be
seen that such a difference in AChE inhibitory activity between
eleutheroside E and acanthoside B indicates that the degree of
substitution of glucose in the phenylpropanoid molecule functions
as an important factor in the AChE inhibitory activity. The AChE
inhibitory activity of the Salicornia europaea extract (PM-EE,
IC.sub.50: 78.9.+-.3.90) was also higher that the antecedently
reported AChE inhibitory activity values of an Aster yomena extract
[Kor. J. Herbology 2009; 24(4):121-126] and a fermented Aronia
melanocarpa extract [Korean Patent Publication No.
10-2016-0088622], and therefore it was suggested that both of
acanthoside B and the Salicornia europaea extract (PM-EE) are AChE
inhibiting natural materials having no side effects and toxicity
and can be developed for uses of a medicine and a functional food
for prevention and treatment of dementia and improvement of
cognitive ability.
Example 5: Structural Analysis of Compound (S7-L3-3) Isolated from
Desalted Salicornia europaea Extract (PM-EE) Showing AChE
Inhibitory Activity
[0141] 5-1. Determination of Molecular Weight and UV .lamda.max of
S7-L3-3
[0142] For determination of the molecular weight of S7-L3-3, the
compound isolated in example 4-3, 1 mg of compound A was subjected
to positive and negative scanning using an electrospray ionization
(ESI) mass spectrometer (LC-ESI mass spectrometer, AGILENT 1100,
USA Micromass Quattro II), and high-resolution MS was measured
(FIGS. 4a and 4b). The maximum UV absorption range of the isolated
compound S7-L3-3 was measured in the range of 190-400 nm using a UV
spectrophotometer (Genesys 10S UV-VIS spectrophotometer, Thermo
Scientific, USA) by dissolving the sample at a concentration of 1
mg/mL in methanol.
[0143] 5-2. Nuclear Magnetic Resonance (NMR) Analysis
[0144] NMR spectroscopy was performed in a manner in which compound
S7-L3-3 (5 mg) was completely dried, dissolved in CDCl.sub.3 (0.5
ml), placed in a 5-mm NMR tube, and analyzed using a Jeol model
(JNM-ECA 600, Jeol, Japan), and .sup.1H-NMR (FIG. 5a) was measured
at 600 MHz, and .sup.13C-NMR (FIG. 5b) was measured at 150 MHz.
Through HMBC-NMR (FIG. 6a) and .sup.1H-.sup.1H COSY-NMR (FIG. 6b)
measurement, the positions and stereoscopic structure of hydrogen
and carbon in the compound S7-L3-3 were determined (FIG. 7).
[0145] As a result of the measurement above, the compound S7-L3-3
was identified to be acanthoside
((2S,3R,4S,5S,6R)-2-[4-[(3S,3aR,6S,6aR)-3-(4-hydroxy-3,5-dimethoxyphenyl)-
-1,3,3a,4,6,6a-hexahydrofuro[3,4-c]furan-6-yl]-2,6-dimethoxyphenoxy]-6-(hy-
droxymethyl)oxane-3,4,5-triol) having a molecular weight of 580,
which has not been reported in Salicornia europaea until now, and
the physical and chemical properties thereof are as follows.
[0146] (1) Molecular formula: C.sub.28H.sub.36O.sub.13
[0147] (2) Molecular weight: 580, ESI-MS: m/z 579.0 [M-H].sup.+,
m/z 602.9 [M.sup.+Na].sup.+ (FIG. 4)
[0148] (3) UV .lamda.max: 210 nm, 238 sh, 272 nm
[0149] (4) Appearance: white powder
[0150] (5) Solubility: soluble in methanol, ethanol, ethyl acetate,
ethyl acetate, chloroform, or pyridine
[0151] (6) .sup.1H and .sup.13C-NMR (CDCl.sub.3, 600 MHz): d 6.47
(2H, s, H-2 and H-6), 4.62 (1H, d, J 4.6 Hz, H-7), 3.00 (1H, m,
H-8), 3.81 (1H, m, H-9a), 4.19 (1H, m, H-9b), 6.51 (2H, 1H, H-2'
and H-6'), 4.65 (1H, H-7'), 4.50 (1H. H-1''), 3.49 (1H, H-2''),
3.37 (1H, H-3''), 3.41 (1H, H-4''), 3.16 (1H. H-5''), 3.71 (1H,
H-6''a), 3.64 (1H, H-6''b), 3.77 (6H, s, 2-00H3), 3.78 (6H, s,
2-OCH3) (FIG. 5A); .sup.13C-NMR (CDCl.sub.3, 150 MHz): d131.2
(C-1), 102.3 (0-2), 147.3 (C-3), 134.4 (C-4), 147.3 (C-5), 102.7
(C-6), 86.0 (C-7), 53.9 (C-8), 71.6 (C-9), 56.1 (2 X--OCH3), 56.2
(2 Y--OCH3), 138.2 (C-1'), 102.9 (C-2'), 152.6 (C-3'), 134.4
(C-4'), 152.6 (C-5'), 102.9 (C-6'), 85.6 (C-7'), 54.2 (C-8'), 71.7
(C-9'), 105.4 (C-1''), 73.9 (C-2''), 75.9 (C-3''), 69.5 (C-4''),
76.2 (C-5''), 61.5 (C-6'') (FIG. 5B)
##STR00006##
Example 6: Neuroglia Protective Effect of Desalted Salicornia
europaea Extract (PM-EE)
Test Example 1. Confirmation of Neuroinflammation Factor Protein
Expression Inhibitory Effect of PM-EE
[0152] In order to investigate the effects of LPS (200 ng/ml),
which is a neuroinflammation inducing substance, and a desalted
Salicornia europaea extract (PM-EE, 0-100 .mu.g/mL), which is a
test sample, on neuroglia in BV2 microglia, which are
LPS-stimulated neuroglia, the cytotoxicity test was conducted
through MMT assay. As a result, it was verified that cell viability
was not significantly changed in all the test material
concentrations of LPS and PM-EE alone or together compared with
control groups. Therefore, in order to analyze the
neuroinflammation inhibitory ability of PM-EE at concentrations
(20, 50, 100 .mu.g/mL) without cytotoxicity, as for whether the
nitric oxide (NO) produced in LPS (neuroinflammation inducing
factor, 200 ng/ml)-stimulated mouse BV-2 microglia was inhibited by
PM-EE treatment, the content of intracellular LPS-induced nitric
oxide (NO) was measured through NO assay using Griess reagent. As
can be seen in the results of FIG. 8a, the content of LPS-induced
amplified intracellular nitric oxide (NO) was increased compared
with a control group by about nine times. However, as a result of
treatment with PM-EE at different concentrations (20, 50, and 100
.mu.g/mL), the amount of nitric oxide (NO) was remarkably reduced
dose-dependently (FIG. 8a). In addition, the expressions of
inducible nitric oxide synthetase (iNOS) protein, which is an
LPS-induced nitric oxide (NO) synthesis inducing enzyme, and
cyclooxygenase type 2 (COX-2) protein known as a neuroinflammation
factor, were examined by western blotting. As a result, it could be
verified that PM-EE inhibited the expressions of iNOS and COX-2
dose-dependently in a protein stage (FIG. 8b).
Test Example 2. Confirmation of Neuroinflammation Gene Expression
Inhibitory Effect of PM-EE
[0153] BV2 microglia as neuroglia were treated with LPS and the
desalted Salicornia europaea extract (PM-EE) at different
concentrations (0-100 .mu.g/mL). After 1 hour, cells were
stimulated by LPS, and after 3 hours, an RNA sample was isolated by
an RNA extraction buffer. Thereafter, RT-PCR assay was conducted
through RNA purification procedure. Table 4 below shows primers
used in RT-PCR of the present test (SEQ ID NO: 1 to SEQ ID NO: 12).
RNA isolated from BV2 microglia was subjected to denaturation at
95.degree. C. for 30 minutes, 45 times of chain reactions in
conditions of 95.degree. C. for 5 seconds and 60.degree. C. for 20
seconds, and annealing to 95.degree. C. at 0.2.degree. C./15 sec,
using the primers in Table 4, and then the reaction was stopped.
Last, separation by bp size was conducted through agarose gel
electrophoresis. Then, bands were checked under UV, and
fluorescence was imaged by a camera. As shown in the results in
FIG. 9, the expressions of neuroinflammation-related genes
(IL-1.beta., iNOS, COX-2, and TNF-.alpha.) were not or slightly
observed in a control group treated without LPS. However, it could
be verified that the mRNA expression levels of these genes were
significantly increased in test groups treated with LPS. It could
also be verified that the expression pattern was reduced
dose-dependently at the time of the treatment with the desalted
Salicornia europaea extract (PM-EE). It was especially verified
that LPS-induced amplified neuroinflammation-related genes
((IL-1.beta., iNOS, COX-2, and TNF-.alpha.) at a high concentration
(100 .mu.g/mL) was restored to almost the same level as that of a
control group before LPS induction. These results suggest that the
desalted Salicornia europaea extract (PM-EE) can strongly inhibit
neuroinflammation from mRNA gene stages as well as the
neuroinflammation factor protein expression, in BV2 microglia as
neuroglia. These results verified that PM-EE can improve brain
cognitive ability by suppressing neuroinflammation to prevent brain
impairment.
TABLE-US-00004 TABLE 4 Gene Sequence (5'.fwdarw.3') INOS Forward
TGAAGAAAACCCCTTGTGCT (SEQ ID NO: 1) iNOS Reverse
TTCTGTGCTGTCCCAGTGAG (SEQ ID NO: 2) COX2 Forward
CAAGACAGATCATAAGCGAGGA (SEQ ID NO: 3) COX2 Reverse
GGCGCAGTTTATGTTGTCTGT (SEQ ID NO: 4) TNF-.alpha. Forward
CCACCACGCTCTTCTGTCTAC (SEQ ID NO: 5) TNF-.alpha. Reverse
AGGGTCTGGGCCATAGAACT (SEQ ID NO: 6) IL-1 .beta. Forward
TGTGAAATGCCACCTTTTGA (SEQ ID NO: 7) IL-1 .beta. Reverse
GGTCAAAGGTTTGGAAGCAG (SEQ ID NO: 8) IL-6 Forward
TGATGCACTTGCAGAAAACA (SEQ ID NO: 9) IL-6 Reverse
ACCAGAGGAAATTTTCAATAGGC (SEQ ID NO: 10) GAPDH Forward
AAGGGCTCATGACCACAGTC (SEQ ID NO: 11) GAPDH Reverse
TTCAGCTCTGGGATGACCTT (SEQ ID NO: 12)
Example 7: Neuroglia Protective Effect of Active Ingredient
Acanthoside B Isolated from PM-EE
Test Example 1: Confirmation of Neuroinflammation Factor Protein
Expression Inhibitory Effect of Acanthioside B
[0154] A test was conducted to investigate effects of the
neuroinflammation inducing substance LPS (200 mg/mL) and the active
ingredient acanthoside B of PM-EE on brain glia cells in
LPS-stimulated brain glia cells BV2 microglia. The cells were
treated with each material at different concentrations (1, 5, 10
.mu.g/mL), and a cytotoxicity test was conducted through MTT
analysis. As a result, it was verified that the cell viability was
not significantly changed in all the test material concentration
groups treated with LPS and acanthoside B alone or together
compared with a control group (FIG. 10a). Therefore, in order to
analyze the neuroinflammation inhibitory ability of acanthoside B
at concentrations (1, 5, 100 .mu.g/mL) without cytotoxicity, as for
whether the nitric oxide (NO) produced in LPS (200
ng/ml)-stimulated mouse BV-2 microglia was inhibited by acanthoside
B, the content of intracellular LPS-induced nitric oxide (NO) was
measured through NO assay using Griess reagent. It could be
verified from the results of FIG. 10b that LPS-induced amplified
intracellular nitric oxide (NO) was remarkably decreased
dependently on the treatment concentration of acanthoside B. In
addition, it was verified through western blotting that the
expressions of iNOS, which is an LPS-induced nitric oxide (NO)
synthesis inducing enzyme, and COX-2, which is an neuroinflammation
factor, were remarkably inhibited (FIG. 10c). Therefore, it was
verified that acanthoside B, which is a neuroinflammation
inhibiting active ingredient in PM-EE, inhibited the expressions of
iNOS and COX-2 proteins even at a remarkably low concentration
compared with PM-EE.
Test Example 2. Confirmation of Neuroinflammation Factor Gene
Expression Inhibitory Effect of Acanthioside B
[0155] A test was conducted to investigate an effect of acanthoside
B on neuroinflammation factor gene expression. The BV2 microglia as
neuroglia were treated with LPS and acanthoside B at different
concentrations (1, 5, 10 .mu.g/mL), and after 1 hour, cells were
stimulated by LPS. After 3 hours, RNA sample was separated by RNA
extraction buffer to synthesize cDNA, and RT-PCR was conducted by
the same method as in example 6 using the primers in Table 4. As
shown in the results in FIG. 11, the expressions of
neuroinflammation-related genes (IL-1.beta., iNOS, COX-2, and
TNF-.alpha.) were not or slightly observed in a control group
treated without LPS. However, it could be verified that the mRNA
expression levels of these genes were significantly increased in
test groups treated with LPS. It could also be verified that the
expression pattern was reduced dose-dependently at the time of the
treatment with acanthoside B. It was especially verified that
LPS-induced amplified neuroinflammation-related genes (IL-1.beta.,
iNOS, COX-2, and TNF-.alpha.) at a high concentration (10 .mu.g/mL)
was restored to almost the same level as that of a control group
before LPS induction. Therefore, it could be verified that
acanthoside B as an AChE inhibiting active ingredient isolated from
PM-EE acts on neuroinflammation from mRNA gene stages as well as
the neuroinflammation factor protein expression, thereby inhibiting
neuroinflammation to prevent brain impairment, thus improving brain
cognitive ability.
Example 8: Confirmation of Improvement of Memory and Cognitive
Ability of Desalted Salicornia europaea Extract (PM-EE) and Active
Ingredient (Acanthoside B) Thereof in Cognitive Ability Impairment
Animal Models
Test Example 1. Analysis of Cognitive Ability and Memory
Improvement Efficacy of PM-EE and Acanthoside B in In-Vivo
Cognitive Ability Impairment Models
[0156] A test was conducted to investigate the improvement efficacy
of cognitive ability and memory of the desalted Salicornia europaea
extract (PM-EE) obtained in example 4-1 above and acanthoside B as
an AChE inhibitory and neuroinflammation inhibiting substance
isolated from PM-EE in examples 4-2 and 4-3. The learning ability
effect was measured through a passive avoidance test in
forgetfulness animal models using scopolamine. Mice were placed in
the bright section with shining light and allowed to go over for 20
seconds. Subsequently, the guillotine door was opened and the mouse
was allowed to enter into the dark section. Here, mice that did not
enter the dark section within 60 seconds after the guillotine door
was opened were excluded from the test. The time from when the
guillotine door was opened to when the mouse entered the dark
section was measured. Once the mouse entered the dark section, the
guillotine door was closed, and an electric shock of 0.25 mA flows
through a grid bottom for 3 seconds, and the mouse remembered this
electrical action. The present test was conducted 24 hours after
the learning test. For the test, 10 SD rates (240-260 g) per group
were treated with PM-EE and acanthoside B at different
concentrations, and at 30 minutes after the final administration of
a test sample, scopolamine (Sigma-Aldrich, Co. USA) dissolved in
distilled water was intraperitoneally administered at a dose of 1
mg/kg. At 30 minutes after the administration of scopolamine, the
mouse was allowed to go over for 10 seconds, and then the time
taken for four feet of the mouse to enter the dark section after
the opening of the guillotine door (transfer latency time, TLT:
retention time) was measured up to 150 seconds. Here, the longer
the time taken, the better the cognitive ability and memory in
passive avoidance. In addition, separately from PM-EE, which is the
desalted Salicornia europaea extract sample, and acanthoside B,
mg/kg of the dementia drug galantamine (Sigma-Aldrich, Co. USA) as
a positive control group was administered. As a result of recording
the transfer latency time (TLT) through a computer, the retention
time in the groups administered with only scopolamine was
significantly reduced in all the experiments, confirming that
memory and cognitive ability decline models were fabricated. It
could also be verified that in cognitive ability and memory
impairment mouse models, the impaired cognitive ability was
improved by the treatment with PM-EE (FIG. 12a) and acanthoside B
(FIG. 12b), confirming that TLT was significantly increased. All of
the above effects are considered to bed dose-dependent, and
especially the administration of PM-EE and acanthoside B at high
concentrations showed more superior cognitive ability and memory
improvement effects compared with the dementia drug,
galantamine.
Test Example 2. Analysis of Cognitive Ability and Memory
Improvement Efficacy in Y-Maze Test
[0157] A test was conducted to investigate the improvement efficacy
of cognitive ability and memory of the desalted Salicornia europaea
extract (PM-EE) obtained in example 4-1 above and acanthoside B as
an AChE inhibitory and neuroinflammation inhibiting substance
isolated from PM-EE in examples 4-2 and 4-3. The Y-maze test was
conducted in forgetfulness animal models using scopolamine. In the
present test, PM-EE and acanthoside B were dissolved in 10% Tween
80, and then the mixture was orally administered at different
concentrations (PM-EE and acanthoside B). In addition, 10 mg/kg of
the dementia drug galantamine (Sigma-Aldrich, Co. USA) as a
positive control group was administered. For the Y-maze test, a
test animal was carefully placed in a test apparatus composed of
three arms (A, B, C) of a black polyvinyl plastic, each of the arms
being 50 cm in length, 10 cm in width, and 20 cm in height, and the
folding angle of the three arms being 120 degrees, and the test
animal was allowed to freely move for 8 minutes, and then the
number of arm entries was recorded. If the test animal sequentially
entered the three different arms, 1 point (actual alternation) was
given. The alternation behavior was calculated by the following
formula.
Alternation behavior (actual alternation)/(maximum
alternation).times.100 (maximum alternation: the total number of
arm entries-2)
[0158] As test results, the behavior of a normal animal, that is, a
control group, scored 50 points, but reduced to 45.5 points by the
administration of scopolamine, showing declines in cognitive
ability and memory. It could also be verified that the alternation
behavior, that is, spatial cognitive ability was again restored
dose-dependently due to the administration of PM-EE and acanthoside
B. Therefore, it was verified that PM-EE (FIG. 13a) and acanthoside
B (FIG. 13b) showed superior cognitive ability and memory
improvement effects to galantamine as a dementia medicine.
Example 9: Single Doing Toxicity Test
[0159] A single dosing toxicity test on a desalted Salicornia
europaea extract (PM-EE) was conducted using mice. As a result of
the single dosing toxicity test, no death cases were observed when
PM-EE was administered at 2 g/kg, which is an available dose
defined by ICH, for 2 weeks. In addition, no significant
abnormality was observed in weight gain, feed intake, and the like.
Therefore, it could be seen that the desalted Salicornia europaea
extract (PM-EE) containing acanthoside of the present invention can
be developed as a safe drug for prevention and treatment of
dementia, a health functional food raw material and a feed.
Sequence CWU 1
1
12120DNAArtificial SequenceSynthetic Construct 1tgaagaaaac
cccttgtgct 20220DNAArtificial SequenceSynthetic Construct
2ttctgtgctg tcccagtgag 20322DNAArtificial SequenceSynthetic
Construct 3caagacagat cataagcgag ga 22421DNAArtificial
SequenceSynthetic Construct 4ggcgcagttt atgttgtctg t
21521DNAArtificial SequenceSynthetic Construct 5ccaccacgct
cttctgtcta c 21620DNAArtificial SequenceSynthetic Construct
6agggtctggg ccatagaact 20720DNAArtificial SequenceSynthetic
Construct 7tgtgaaatgc caccttttga 20820DNAArtificial
SequenceSynthetic Construct 8ggtcaaaggt ttggaagcag
20920DNAArtificial SequenceSynthetic Construct 9tgatgcactt
gcagaaaaca 201023DNAArtificial SequenceSynthetic Construct
10accagaggaa attttcaata ggc 231120DNAArtificial SequenceSynthetic
Construct 11aagggctcat gaccacagtc 201220DNAArtificial
SequenceSynthetic Construct 12ttcagctctg ggatgacctt 20
* * * * *