WO2006025708A2

WO2006025708A2 - Inhibitor for acetylcholinesterase containing gamma-viniferin or visitin a

Info

Publication number: WO2006025708A2
Application number: PCT/KR2005/002912
Authority: WO
Inventors: Tae-Hyung Jo; Sung-Sick Woo; Ji-Min Cha; Dong-Seon Kim; Young-Chul Lee; Seon-Gil Do; Jong-Ha Ryu; Mi-Sun Oh
Original assignee: Unigen, Inc.
Priority date: 2004-09-02
Filing date: 2005-09-02
Publication date: 2006-03-09
Also published as: WO2006025708A3; KR20060029714A

Abstract

The present invention relates to an inhibitor of acetylcholinesterase comprising vitisin A and Ϝ -viniferin that are polyphenol components in grapevine extract (Vitis vinifera) as an effective ingredient. The grapevine extract and vitisin A and Y -viniferin which are effective ingredients extracted and purified from the grapevine extract according to the present invention inhibit the dissolution of acetylcholine, neurotransmitter in human brain, by inhibiting the activity of acetylcholinesterase, and thus can be used for a pharmaceutical composition, medicine and health food product for improving brain function, preventing or treating senile dementia, blood circulatory disorder, and controlling blood pressure.

Description

INHIBITOR FOR ACETYLCHOLINESTERASE CONTAINING

GAMMA-VINIFERIN, VITISIN A OR EXTRACT OF GRAPEVINE

TECHNICAL FIELD

The present invention relates to a method for efficiently extracting and purifying

effective ingredient from grapevine (Vitis vinifera) under certain conditions, useful for

inhibition of activation of acetylcholinesterase (AChE), improvement of degenerated

memory, control of blood pressure, and blood circulation; a composition comprising

effective ingredient isolated from grapevine extract; use of the composition; and a method

for treating diseases related to inhibitory activity of acetylcholinesterase in mammal by

using the composition.

BACKGROUND ART

As the population of aged people has been increased worldwide as well as in

Korea, many degenerative senile diseases require social and economic costs. Particularly,

dementia is a serious incurable disease, and raises social and economic concern.

Dementia may be occurred from various causes, but the main cause is known as

Alzheimer's disease (65%). Dementia is characterized in that senile plaque whose main

ingredient is amyloid beta protein is deposited outside the brain cells, by which the

learning ability and memory are remarkably degenerated. Recently, great efforts have been poured into various researches including

molecular genetics to figure out the cause of dementia, but the cause is not clearly

identified yet. Thus, it is difficult to develop a medicine for dementia, but a study for

inhibiting memory degeneration that is the main symptom of dementia has been active.

In the late 1970's, it was known that dementia is closely connected to decline of the

function of cholinergic nerve system and damage of cognitive function. A study also

reported that if the concentration of acetylcholinesterase in the brain is maintained in high

level as a treatment method to Alzheimer's disease, the patient's memory is greatly

recovered. AChE is an important enzyme to hydrolyze acetylcholine, one of

neurotransmitter mediating the activity of parasympathetic nerve, into choline and acetate.

AChE is formed in the endoplasmic reticulum, and moves and functions in the cell

membrane. AChE is distributed around cholinergic nerve, particularly much at the

myoneural junction, and is found in the serum, liver and other tissues. As a result of

various efforts to develop a medicine which can improve the cognitive function by

maintaining the concentration of acetylcoline from inhibiting the activity of AchE (lyase of

acetylcholine in the brain), four medicines, tacrine, donepezil, rivastigmine and

galantamine, were developed and used for the treatment of dementia.

Besides acetylcholinesterase, an agent for increasing synthesis of acetylchoine,

receptor agonist, etc. have been studied, but there has been no significant result. In 1998,

there has been a report showing a development possibility of medicine which can inhibit

damage of the brain cell due to toxicity action of amyloid beta protein by blocking deposit

of the protein, but no notable result has been followed yet. As stated above, although not a method to treat cause of the disease, there has been

continuous development of medicines which can improve lowered cognitive function by

supplementing and improving degenerated cholinergic nerve system known to be closely

related to memory. The medicines for dementia currently used in and out of Korea are

anti-cholinergic medicines for improving symptoms, metabolism acceleration medicines

for nonspecifically rising metabolism, brain function improvement agents which make the

blood circulation smooth, or dementia preventing and treating agents. However, their

effects are temporary and weak. Also, there is no report that brain diseases of senile

dementia patients are improved by using these medicines. Moreover, these medicines

may induce side effects such as nausea, vomiting, bronchoconstriction, etc. by stimulating

parasympathetic nerve, and cause depression, insomnia, hypertension, constipation, etc.

Therefore, there has been a need to develop a new medicine for treating dementia with less

side effects and good efficacy, and even the cause including improvement of brain

diseases.

DISCLOSURE OF THE INVENTION

There has been continuous development for dementia medicines which can

improve lowered cognitive function by supplementing and improving degenerated

cholinergic nerve system known to be closely related to memory, though not method to

treat the cause of dementia. However, the medicines for dementia currently used in and

out of Korea are anti-cholinergic medicines for improving symptoms, metabolism acceleration medicines for nonspecifically rising metabolism, brain function improvement

agents which make the blood circulation smooth, or dementia preventing and treating

agents. And, their effects are temporary and weak. Also, there is no report that brain

diseases of senile dementia patients are improved by using these medicines. Moreover,

these medicines may induce side effects such as nausea, vomiting, bronchoconstriction, etc.

by stimulating parasympathetic nerve, and cause depression, insomnia, hypertension,

constipation, etc. Therefore, there has been a need to develop a new medicine for treating

dementia with less side effects and good efficacy, even the cause including improvement

of brain diseases.

The present invention provides a medicine and health care product for the

prevention and treatment of diseases related to inhibitory activity of acetylcholinesterase

comprising extract of grapevine (Vitis vinifera), or vitisin A or γ-viniferin isolated from the

extract as effective ingredient.

The present invention also provides a use of vitisin A or γ-viniferin compound for

preventing or treating diseases related to inhibitory activity of acetylcholinesterase.

The present invention also provides a method for preventing or treating diseases

related to inhibitory activity of acetylcholinesterase comprising administering a

therapeutically effective amount of vitisin A or γ-viniferin to mammals.

The present invention also provides a method for preparing vitisin A and

γ-viniferin compounds by extracting and purifying grapevine. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a HPLC chromatography showing vitisin A or γ-viniferin in ethanol

extract of grapevine.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is characterized in the preparation of grapevine (Vitis

vinifera) extract having inhibitory activity of acetylcholinesterase, and in the method for

isolating and purifying its effective ingredient.

The inhibitory activity of acetylcholinesterase is determined by Elhnan method

which evaluates the activity of acetylcholinesterase through measuring thiocholine formed

when acetylthiocholine, synthesis substrate, is dissolved by acetylcholinesterase, and

yellow fluorescence resulting from reaction of 5,5'-dithio-bis(2-nitrobenzoic acid).

Grapevine used as material in the present invention is scientifically named Vitis

vinifera, occupies 1/3 of the fruit production throughout the world, and ranks the top in the

production amount among the fruits. Journal of Oriental Medicine,

DongEuiSuSaeBoWon, describes that the roots and cortexs of grapevine are effective for

inhibition of vomiting, in the drug name of podogeun and pododeung, respectively.

I. Active compounds

The active compound of the present invention is gamma- viniferin of the following structural formula (I) and vitisin A of the following structural formula (II).

Gamma-viniferin of the above structural formula (I) and vitisin A of the above

structural formula (II) may be synthesized by a known method in the art, or isolated from

Genus of Vitis such as Vitis vinifera, Vitis flexuosa, Vitis amurensis, Vitis coignetiae, Vitis

thunbergii, Vitis riparia, Vitis kaempferi, etc., preferably from Vitis vinifera or Vitis

flexuosa Thumb.

hi the present invention, if otherwise instructed, a pharmaceutically acceptable salt

includes one derived from pharmaceutically acceptable inorganic or organic salt, and acid.

For example, the pharmaceutically acceptable salt may be sodium, calcium and potassium

salt of hydroxyl group. Also, the pharmaceutically acceptable salt of amino group may be

hydrobromide, sulphate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate,

methansulphonate(mesylate) and />-toluensulphonate(tosylate). These salts can be

prepared by a known preparation method or process for salts in the art.

Preferably, an acid addition salt formed by free acid may be used. The acid

addition salt can be prepared by a conventional method, for example, one that a compound

is dissolved in an excess of acidic aqueous solution, and then precipitated in an

water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Also,

same molar amount of the compound, and acid or alcohol (for example, glycol

monomethylether) in water are heated, and then the mixture is dried by evaporation, or a

salt extracted therefrom is sucked and filtered to obtain the acid addition salt.

Here, organic acid and inorganic acid may be used as free acid. The inorganic

acid may be hydrochloric acid, phosphoric acid, sulphuric acid, nitric acid, tartaric acid,

etc.; and the organic acid maybe methansulphonic acid_{5 j}p-toluensulphonic acid, acetic acid,

trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric

acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid,

gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartatic

acid, ascorbinic acid, carbonic acid, vanillic acid, hydro-iodic acid, etc.

Also, pharmaceutically acceptable metal salt may be prepared by using a base.

For example, alkali metals or alkali earth metals may be prepared by the method that a

compound is dissolved in an excess of solution of alkali metal hydroxide or alkali earth

metal hydroxide, filtered to filter out the indissoluble compound salt, and then the

remainder is evaporated and dried. Here, preferably, sodium, potassium, or calcium salt is prepared as metal salt, and corresponding silver salt thereto may be obtained by reacting

alkali metal or alkali earth metal salt with appropriate silver salt (for example, silver

nitrate).

II. Preparation of active compound

Vitisin A and gamma- viniferin compound in the present invention can be obtained

by isolating from grapevine extract as follows.

Grapevine or its root, preferably its cortex, is properly sliced, washed, dried, and

repeatedly extracted once to five times, preferably two to three times; by a extraction

method such as hydrothermal extraction, enfleurage, reflux condensation or ultrasonic

extraction; for about 0.5 hr to 2days, preferably 1 hr to lday; at an extraction temperature

of 20 to 100 ^°C, preferably 50 to 70 ^°C; in the water, lower alcohol such as methanol or

ethanol, preferably ethanol of 50%, 70% or 95%, or mixing solvent thereof, preferably in

the mixing ratio of about 1:0.1 to 1:10, more preferably 1:0.2 to 1:3; of about volume of 2

to 100 folds of dry weight, preferably about 10 to 30 folds. Then, the extract is filtered

under reduced pressure and concentrated by decompression with a rotary vacuum

concentrator at 20 to 100^°C, preferably 50 to 70 "C, and then residue extracted thereby is

dried with vacuum freeze dryer to obtain a crude extract of grapevine.

The above crude extract of grapevine is suspended in water, and then is fractioned

by using nonpolar solvent of dichloromethane, ethyl acetate, butanol, etc. to obtain

grapevine fraction by each solvent of the present invention. Preferably, ethylacetate

soluble fraction and water soluble fraction may be obtained by using the mixing solvent of ethylacetate and water having an equivalent amount to grapevine crude extract of the

present invention.

Specifically, nonpolar soluble extract of grapevine obtained from the above

process, preferably ethylacetate soluble fraction, is isolated to 5 to 8 fractions by using the

mixing solvent of dichloromethane and methanol in the ratio 100:1 ~ 5:1 under silica gel

column chromatography, to obtain vitisin A or gamma- viniferin of the present invention

from the lower fraction 4 by using preparative HPLC.

Vitisin A and gamma-viniferin compounds of the present invention can be

synthesized by a conventional synthesis and fraction method for substituents (Herbert O.

House: Modern Synthetic Reactions, 2nd Ed., The Benjamin/Cummings Publishing Co.,

1972).

III. Composition comprising the active compound

The present invention provides a pharmaceutical composition for preventing and

treating dementia comprising grapevine extract, preferably ethanol extract, more preferably

vitisin A and gamma-viniferin compounds obtained from ethylacetate soluble fraction as

effective ingredient.

To see the effect for protecting and improving brain function or preventing and

treating dementia of grapevine extract or vitisin A and gamma-viniferin compounds

isolated therefrom, grapevine extract, vitisin A and gamma-viniferin compounds are

treated to acetylcholinesterase effective for reducing acetylcholine, which is

neurotransmitter in brain, to confirm that these compounds strongly inhibit acetylcholinesterase.

If acetylcholinesterase is inhibited, the brain function can be improved, and

dementia, blood circulatory disorder, blood pressure abnormality, myasthenia gravis,

asthenia of gastrointestinal tract and cystic smooth muscle, glaucoma, etc. can be

prevented or treated. Particularly, the present composition can be used for improving

brain function and preventing and treating dementia.

The pharmaceutical composition of the present invention comprises 0.1 to 50

weight % of the above extract or compound of the total weight of the composition.

The pharmaceutical composition comprising the present extract or compound may

further comprise appropriate carriers, excipients, and diluents, conventionally used for

preparation of pharmaceutical composition.

The pharmaceutical dosage form of the present extract or compound may be used

in the form of its pharmaceutically acceptable salt, and alone, in combination with other

pharmaceutical active compounds, and in proper set.

The pharmaceutical composition comprising the present extract or compound may

be used by formulating it into oral formulations such as power, granule, tablet, capsule,

suspension, emulsion, syrup, aerosol, etc., external preparation, suppository or sterilized

solution for injection. Carriers, excipients and diluents which can be included in the

present composition are lactose, dextrose, sucrose, solbitol, mannitol, xylitol, erythritol,

maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate,

cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water,

methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. In case of formulating, diluents or excipients such as filler, extender, bonding agent,

wetting agent, disintegrating agent, surfactant, etc. may be used. Solid preparation for

oral administration comprises tablet, pill, powder, granule, capsule, etc. and is prepared by

mixing one or more excipients, for example, starch, calcium carbonate, sucrose or lactose,

gelatin, etc., with the above extract or compound. Also, besides simple excipients,

lubricant such as magnesium stearate or talc may be used. Solution for oral

administration comprises suspension, internal solution, emulsion, syrup, etc., and besides

simple diluents such as water and liquid paraffin, various excipients, for example, wetting

agent, sweetening agent, odorant, preservative, etc. may be contained therein.

Formulation for parenteral administration includes sterilized water solution, non-aqueous

solution, suspension, emulsion, lyophilization preparation, or suppository. Propylene

glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as

ethylolate, etc. may be used as non-aqueous solution and suspension. Witepsol, macrogol,

tween 61, cacao butter, laurin butter, glycero gelatin, etc. may be used as suppository base.

IV. Dosage

The preferable dosage of extract or compound of the present invention is different

depending on condition and weight of patient; severity of disease; forms of medicine; and

route and period of administration, but may be appropriately selected by a skilled person in

the art. However, to have desirable effect, it is preferable to administer the present

extract or compound in 0.0001~100ml/kg, preferably 0.001~10ml/kg a day. The

administration can be made once or several times a day. The scope of the present invention should not be limited by the above dosage in any manner.

The extract or compound of the present invention can be administered through

various routes to mammals of rat, mouse, livestock, human, etc. The administration can

be made through all routes, for example, oral and rectal administration, or intravenous,

intramuscular, subcutaneous, intrauterine, or intracerebroventircular injection.

IV. Health care product comprising the present composition

The present invention provides health care product comprising the above extract or

compound showing the effect for protecting and improving brain function, or preventing

and treating senile dementia, blood circulatory disorder, blood pressure abnormality, and

food additive acceptable for sitology. Foods to which the present extract or compound

can be added include all sorts of foods, drinks, gums, teas, vitamin complex, and health

care products.

Preferably, the present extract or compound can be added to foods or drinks for

protecting and improving brain function, or preventing senile dementia, blood circulatory

disorder, and blood pressure abnormality. Here, the amount of the above extract or

compound in the food or drink is 0.01 to 15 weight% of total food weight, and the amount

in drink is 0.02 to 5 g, preferably 0.3 to Ig of 100 ml.

In the drink composition for health care of the present invention, other ingredients

are not particularly limited as long as the above extract or compound is contained in the

composition as an essential ingredient in indicated ratio. The drink composition may also

comprise various flavors, natural carbohydrate, etc. as an additional ingredient, like conventional drinks. The examples of the above natural carbohydrate are conventional

sugars such as monosaccaride, for example, glucose, fructose, etc.; disaccharide, for

example, maltose, sucrose, etc.; and polysaccharide, for example, dextrin, cyclodextrin,

etc., and sugar alcohol such as xylitol, sorbitol, erythritol, etc. Natural flavors (taumatin,

stevia extract, for example, glycyrrhizin) and synthetic flavors (saccharin, aspartic acid

etc.) may be advantageously used as flavor besides the above. The ratio of the above

natural carbohydrate is generally of about 1 to 20 g, preferably 5 to 12g of 100 ml of the

present composition.

In addition, the extract or compound of the present invention may comprise

various nutrients, vitamins, minerals (electrolyte), flavors such as synthetic flavors and

natural flavors, etc., coloring agents and extender (cheese, chocolate etc.), pectic acid and

salt thereof, alginic acid and salt thereof, organic acid, protective colloid thickener, pH

control agent, stabilizer, aseptic, glycerin, alcohol, carbonated agent used in carbonated

drinks, etc. Further, the extract or compound of the present invention can comprise

natural fruit juice and pulp for preparing fruit juice beverage and vegetable beverage.

Such ingredients can be used alone or in combination. The ratio of the additive is not

very important, but is generally selected from the range of the weight ratio of 0 to 20 by

100 weight of the present extract or compound.

Hereinafter, the present invention will be described in more detail with reference

to the following examples and experimental examples, but the scope of the present

invention should not be construed to be limited thereto in any manner. Examples

Example 1. Determination of inhibitory activity of acetylcholinesterase

Plant sample experimental groups, and each of two positive control and two blank experimental groups were put into 96 micro well plate, and bioassay was performed. In each well for the plant sample experimental group, plant sample extract of 50mg/ml and DMSO solution were diluted with 0.1M phosphate buffer by 10 (ten) times; for the positive control group,

10 μl of 0.1M phosphate buffer including Tacrine DMSO solution (2mg/ml) was diluted by 10 (ten) times; and for the blank group, 10μ£ of 0.1M phosphate buffer containing 10% DMSO was added thereto. Then, DTNB phosphate buffer solution (5OmM) of 5/d, acetylcholinesterase 0.1M phosphate buffer solution of 2.5μ<5 (50unή7m£, the origin of electric eel), and phosphate buffer of 132.5μ6 were added, mixed, incubated at 25 ^°C for 5 minutes, and the absorbance was determined at 405nm. After determination of the absorbance, acetylthiocholine iodide distilled water solution of 50/^β (0.01Mm, Achl) was added to each experimental group, which was incubated at 25 "C for 3 minutes, and the absorbance was determined at 405nm. Inhibitory ratios to the blank and positive control experimental groups were calculated according to the following formula by with the determined absorbance.

Inhibitory ratio (%) = 100 - A/(CTRL-BLNK) X 100 A: absorbance before reaction with Achl - absorbance after reaction with Achl in the plant sample experimental group CTRL: absorbance before reaction with Achl - absorbance after reaction with

Achl (in the positive control including Tacrine DMSO solution) BLNK: absorbance before reaction with Achl - absorbance after reaction with Achl (in 0.1 % DMSO solution)

* For the CTRL and BLNK experimental groups, the mean value of absorbance was calculated for the two groups and applied to the formula. The results of measuring the inhibitory activities of acetylcholinesterase of grapevine extract, vitisin A and gamma-viniferin according to the above method are shown in the following Table 1.

Table 1

Example 2. Purification of effective ingredient for inhibitinR acetylcholinesterase

in grapevine

To search an active material in grapevine, 70% alcohol aqueous solution

corresponding to 25 folds of dry weight was added to grapevine cortex (80Og), extracted

by heating, filtered, concentrated, and lyophilized to obtain dry powder (48g). Then, such obtained dry powder was fractioned by using solvent such as normal hexane,

dichloromethane, ethylacetate, normal butanol, distilled water, etc. Ethylacetate fraction

(33.6g) exhibiting the most inhibitory activity was running in the normal phase of silica gel

column, and silica gel column chromatography was conducted with increasing the ratio of

methanol by adding dichloromethane, and mixing solvent of methanol and acetic acid

thereto, to confirm the active material. After determining the specific activity to enzyme

inhibition to each effluent, purification of the active material was conducted to the fraction

exhibiting the most inhibitory activity by using 30% of acetonitrile at prep HPLC with

ODS column (9.4 X 250mm), and then active fraction was concentrated to isolate two kinds

of active materials.

Example 3. Identifying the structure of vitisin A

It was reported that one in two compounds obtained in Example 2 is vitisin A which has been reported to be isolated from Vitis coignetiae in Ito, J., Takaya, Y, Oshima, Y, and Niwa, M. [New Oligostilbenes having a benzofuran from Vitis vinifera 'Kyohou'.

Tetrahedron 55, 2529-2544, 1999] by comparing the determined value of ¹H-NMR or

C-NMR, MS, etc. with the reference value. Physicochemical characteristic and instrumental analysis data of this compound are as follows:

φ TLC : Rf value was 0.5 in 15% of methanol aqueous solution and normal phase silica gel.

If the compound was reacted with sulphuric acid solution of 10% and then heated, dark brown color came out.

(D mass spectrum m/z : 907 [MH+] W 2

(H) ¹H NMR spectrum (600 MBz, acetone-D₆) δ : 7.06(2H, d, J=8.5), 6.68(2H, d, J=8.5), 5.41(1H, d, J=3.0), 5.5O(1H, d, J=3.0), 6.11(1H, d, J=2.0), 7.22(2H, d, J=8.5), 6.86(2H, d, J=8.5), 5.91(1H, d, J=10.5), 4.26(1H, d, J=I 0.5), 6.41(1H, brs), 7.17(2H, d, J=8.5), 6.80(2H, d, J=8.5), 6.19(2H, d, J=8.5)

® ¹³C NMR spectrum (600 MHz, acetone-D₆) δ : 135.7(C-I), 129.2(C-2,6),

115.8(C-3,5), 41.0(C-7), 120.5(C-IO), 159.1(C-13), 128.3(C-2',6'), 116.5(C-3',5'), 142.6(C-9'), 156.2(C-13'), 129.2(C-I"), 115.8(C-5"), 131.4(C-7"), 123.0(C-8"), 101.3(C-12"), 130.4(C-2'",6'"), 116.4(C-3'",5'"), 107.2(C-10'",14'"),

160.6(C-ll'",13'")

Example 4. Identifying the structure of gamma- vim^'ferin

It was identified that one in the two compounds obtained in Example 2 is gamma-viniferin which was reported to be isolated from Genus of Vitis in Fulvio, M.,

Fabiano, R. [Oligostilbenes from the roots of genus Vitis. Bull. Liaison Groupe Polyphenols 16, 116-119, 1992] by comparing the determined value of ¹H-NMR or

(D TLC : Rf value was 0.5 in 20% methanol aqueous solution and normal phase silica gel.

φ mass spectrum m/z : 907 [MH+]

(H) ¹H NMR spectrum (600 MHz, acetone-D₆) δ : 4.35(1H, d, J=5.1), 5.58(1H, d,

J=5.1), 4.48(1H, d, J=5.4), 5.45(1H, d, J=5.4), 4.55(1H, d, J=4.7), 5.44(1H, d, J=4.7), 6.85(2H, d, J=8.7), 7.22(2H, d, J=8.7), 6.94(2H, d, J=8.7), 7.28(2H, d, J=8.7), 6.61(2H, d, J=8.7), 6.69(2H, d, J=8.7), 6.24(3H, brs), 6.14(2H, d, J=2.2), 6.24(1H, brs), 6.36(1H, d, J=2.1), 6.68(1H, d, J=2.1), 6.23(1H, d, J=2.1), 6.78(1H, d, J=8.4), 6.85(1H, d, J=1.9), 7.17(1H, dd, J=8.4, 1.9), 6.66(IU, d, J=16.3), 6.79(1H, d, J=16.3)

© ¹³C NMR spectrum (600 MHz, acetone-D₆) δ : 134.0(C-I), 127.8(C-2,6), 116.1(C-3,5), 93.8(C-7), 107.0(C-10,14), 159.7(C-Il, 13), 125.5(C-2'), 110.5(C-5'), 126.5(C-6'), 123.9(C-7'), 130.2(C-8'), 159.4(C-13'), 132.5(C-I"), 115.9(C-3",5"), 93.9(C-7'"), 52.0(C-8"), 96.5(C-12"), 127.8(C-2'",6'"), 116.3(C-3'",5'"), 106.9(C-10'",14'"), 158.9(C-11'",13'")

Example 5. Content analysis of γ-viniferin and vitisin by genus and part of

grapevine

To 1Og of each branch cortex and twig extract of Jingzaojing, Niunai, Cambell early, Sekirei, Rizamart, Menicure finger, Red nehelescol, Ruby seedless, Black olympia, Mario, Honey juice, Muscat bailey A, the contents of gamma- viniferin and vitisin A were analyzed by part of grapevine.

The instrument was L-7000 series of Hitachi Co.; Zorbax XDB-C 18,

(4.6X150mm) was used as stationary phase; and in mobile phase, B solution was 100%

ACN, C solution was ACN: Water=l:9 , and D solution was ACN: Water=9:l. The

condition was shown in the following Table 2.

Table 2.

The temperature of the stationary phase was 40 ^°C, the wavelength of detector was UV 205nm, and the sample of lOμβ was injected by 2000ppm. The analysis result was shown in the following Table 3.

Table 3.

Active ingredients of gamma-viniferin; vitisin; or lower alcohol extract, ethylacetate fraction or butanol fraction of grapevine containing them, etc. in the present invention may be differently administered depending on age of patient, degree of dementia, and weight of patient, but may be administered by 0.1 to 500mg, once or several times a day. This amount may be increased or decreased depending on severity of diseases, sex, age, etc.

The formulation examples are exemplified below: Formulation Example 1. (Preparation of tablet)

ethylacetate fraction of grapevine 500. Omg

lactose 500.0mg

talc 5. Omg

magnesium stearate l.Omg

The above-mentioned ingredients were formulated according to a conventional

preparation method for tablet.

Formulation Example 2. (Preparation of tablet)

butanol fraction of grapevine lOO.Omg

lactose lOO.Omg

talc l.Omg

magnesium stearate proper quantity The above-mentioned ingredients were formulated according to a conventional

preparation method for tablet.

Formulation Example 3. (Preparation of tablet)

vitisinA 2.0mg

lactose lO.Omg

talc proper quantity

magnesium stearate proper quantity

The above-mentioned ingredients were formulated according to a conventional preparation method for tablet.

Formulation Example 4. (Preparation of capsule) γ-viniferin 500.0mg

lactose 500.0mg

talc 5.0mg

magnesium stearate 5.0mg

The above-mentioned ingredients were formulated according to a conventional preparation method for capsule.

Formulation Example 5. (Preparation of capsule)

vitisinA 2.0mg

lactose 30.0mg

talc l.Omg

magnesium stearate l.Omg

Formulation Example 6. (Preparation of solution)

grapevine ethanol extract 500mg

isomerized sugar 20.Og

antioxidant proper quantity

methyl paraoxybenzoate proper quantity

total amount after adding purified water of proper quantity 100.0ml

The above-mentioned ingredients were formulated according to a conventional preparation method for solution, and then filled into brown bottle to give solution.

Formulation Example 7. (Preparation of solution)

vitisinA 50.0mg γ-viniferin 50.0mg

isomerized sugar 50.0mg

antioxidant proper quantity

methyl paraoxybenzoate proper quantity

total amount after adding purified water of proper quantity 100.0ml

Formulation Example 8. (Preparation of powder)

polyphenol fraction of grapevine 50. Omg

lactose lOO.Omg

talc 5.0mg

The above-mentioned ingredients were mixed, put into vinyl pack, and sealed off according to a conventional preparation method for powder to give powder.

Formulation Example 9 (Preparation of injection)

viniferin l.Omg

antioxidant proper quantity

tween 80 proper quantity

total amount after adding distilled water for injection of proper quantity 2.0ml

The above-mentioned ingredients were filled into ampule of 2.0ml volume, and sterilized according to a conventional preparation method for injection to give injection.

Formulation Example 10 (Preparation of injection)

ethylacetate fraction of grapevine 10.Omg

antioxidant proper quantity tween 80 proper quantity

INDUSTRIAL APPLICABILITY

The gamma-viniferin and vitisin A compounds according to the present invention

are effective inhibitors of acetylcholine lyase capable of inhibiting activity of

acetylcholinesterase, and can be used for effectively preventing and treating all kinds of

symptoms caused by senile dementia including Alzheimer's disease and blood circulation

disorder or decrease of acetylcholine content in the form of pharmaceutical formulation

comprising the above compounds as an effective ingredient.

Claims

WHAT IS CLAIMED IS

1. A pharmaceutical composition for preventing or treating diseases related

to inhibitory activity of acetylcholinesterase comprising gamma- viniferin compound of the

following structural formula (I) or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition for preventing or treating diseases related to

inhibitory activity of acetylcholinesterase comprising vitisin A compound of the following

structural formula (II) or a pharmaceutically acceptable salt thereof.

3. The composition according to claim 1 or 2, wherein the disease related to

inhibitory activity of acetylcholinesterase is selected from the group consisting of

abnormality of brain function, dementia, blood circulatory disorder, blood pressure abnormality, myasthenia gravis, asthenia of gastrointestinal tract and cystic smooth muscle,

and glaucoma.

4. The composition according to claim 1 or 2, characterized in that the

gamma-viniferin compound or vitisin A compound is isolated from any one of Vitis

vinifera, Vitis flexuosa, Vitis amurensis, Vitis coignetiae, Vitis thunbergii, Vitis riparia or

Vitis kaempferi.

5. The composition according to claim 3, wherein the disease related to

inhibitory activity of acetylcholinesterase is dementia.

6. Use of gamma-viniferin compound of the following structural formula (I)

for preventing or treating diseases related to inhibitory activity of acetylcholinesterase.

7. Use of vitisin A compound of the following structural formula (II) for

8. Use according to claim 6 or 7, wherein the disease related to inhibitory

activity of acetylcholinesterase is selected from the group consisting of abnormality of

brain function, dementia, blood circulatory disturbance, blood pressure abnormality,

myasthenia gravis, asthenia of gastrointestinal tract and cystic smooth muscle, and

glaucoma.

9. Use according to claim 6 or 7, characterized in that the gamma-viniferin

compound or vitisin A compound is isolated from any one of Vitis vinifera, Vitis flexuosa,

Vitis amurensis, Vitis coignetiae, Vitis thunbergii, Vitis riparia or Vitis kaempferi.

10. Use according to claim 8, wherein the disease related to inhibitory activity

of acetylcholinesterase is dementia.

11. A method for preventing or treating diseases related to inhibitory activity

of acetylcholinesterase comprising administering a therapeutically effective amount of

gamma-viniferin compound of the following structural formula (I) to mammal.

12. A method for preventing or treating diseases related to inhibitory activity

vitisin A compound of the following structural formula (II) to mammal.

13. The method according to claim 11 or 12, wherein the disease related to

abnormality of brain function, dementia, blood circulatory disorder, blood pressure

abnormality, myasthenia gravis, asthenia of gastrointestinal tract and cystic smooth muscle,

and glaucoma.

14. A method for preparing gamma- viniferin compound of the following structural formula (I) and vitisin A compound of the following structural formula QI)

comprising,

heating after adding any one of water, lower alcohol or mixing solvent thereof to

grapevine to obtain extract;

obtaining the crude extract by filtering, concentrating, and lyophilizing the above

extract; and

fractioning and then purifying the above crude extract with nonpolar solvent.