KR101498724B1 - Polygalatenuifolia willdenow extract with enhanced acetylcholinesterase inhibition effect and product containing thereof for preventing or improving the memory capability - Google Patents

Abstract

The present invention relates to an extract of an extract from which an acetylcholinesterase inhibiting effect is promoted, and a composition for preventing or ameliorating memory loss comprising the same. According to the method of the present invention, it is generally possible to use an ultrafiltration method, which is not separation using a column used for separation of substances, to simply extract the saponin-based compound (ultrafiltration inner solution) and non-saponin- And the saponin-based compound in the extract is removed, thereby increasing the acetylcholinesterase inhibiting effect. Therefore, the extract is superior to the acetylcholinesterase inhibiting effect of the conventional extracts, Therefore, the extract of the present invention is not a single component but an extract of a natural substance. Since it has fewer side effects than a synthetic drug, it can be used as a health functional food composition for preventing or improving memory deficits or for preventing or treating dementia And may be usefully used as an emulsion composition.

Description

TECHNICAL FIELD The present invention relates to a method for inhibiting acetylcholinesterase inhibition and a method for preventing or reducing the memory capacity of the extract,

The present invention relates to an extract of an extract from which an acetylcholinesterase inhibiting effect is promoted, and a composition for preventing or ameliorating memory loss comprising the same.

Alzheimer's disease is the most common cause of dementia, accounting for about 50% of the disease, and it is impossible to cure. Alzheimer's disease is a progressive, degenerative disease of the brain that causes memory, thinking and behavior disorders. This disease was first reported by Alois Alzheimer in 1907.

When Alzheimer's disease progresses, various neurotransmitter changes occur in the brain. Among them, acetylcholine is the substance most closely related to cognitive function. There are various methods of enhancing the function of cholinesin such as muscarinic (M1) receptor agonist, nicotine receptor agonist, acetylcholine precursor, etc. However, up to now, it has been found that increasing the amount of acetylcholine Drugs are showing the best effect. The acetylcholinesterase inhibitor showed improvement in cognitive function in the range of about 25-40% of patients with early and middle-stage Alzheimer's disease. However, in the treatment of dementia, .

Tacrine, first approved by the US FDA in 1993, is known as a first-generation acetylcholinesterase inhibitor. In 1996, donepezil was approved as a second-generation acetylcholinesterase inhibitor. In 2000, Rivastigmine was approved in 2001, and galantamine was approved in 2001. Second generation acetylcholinesterase inhibitors have relatively few side effects and are easy to use and are widely used in clinical practice.

On the other hand, tacrine was first used by the US FDA in 1993 as an acetylcholinesterase inhibitor approved as a treatment for Alzheimer's disease. However, since the half-life of the drug is as short as 2 to 4 hours, it is troublesome to take it four times a day In addition, donepezil selectively acts on the brain as an acetylcholinesterase inhibitor, and it has been reported that adverse effects of hepatotoxicity have been reported. Therefore, Although the cold is 70 hours, it is easy to administer before bedtime, but the memory and thinking ability are not greatly improved. In addition to FDA-approved acetylcholinesterase inhibitors in 2000, rivastigmine and the FDA-approved Galantamine in 2001, there are nausea, diarrhea, loss of appetite, muscle cramps due to increased acetylcholine And sleep disorders are known to have side effects. Therefore, it is still required to develop an acetylcholinesterase inhibitor which has few side effects and can improve memory.

It is made by drying the roots of polygalatenuifolia willd, which has a spicy, bitter flavor and a warm character. It is known to have a genetic and sedative hypnotic action and is widely prescribed for forgetfulness, neurasthenia, hypertrophy and insomnia. This study has been carried out to investigate the effects of various chemicals on the central nervous system, and the neuroprotective effect, the improvement of the learning ability, the antistress effect and the antidepressant effect have been reported.

In particular, the methanol extract of the ground prevents the increase of oxidative stress by dopamine administration, reduces cytotoxicity to human SH-SY5Y neuroblastoma, and inhibits NMDA (N-methyl-D-aspartate) And BT-11, a component of the ground, has been reported to enhance the learning ability of normal adults and older adults.

On the other hand, ultrafiltration is a method of separating and concentrating a polymer material and a low molecular substance using a semi-permeable membrane. In the fields of food, pharmaceutical and medical applications, liquid phase seasoning and concentration of food materials, purification, Pyrogen removal of injected water and raw materials, sterile water production in the operating room, etc., and it is used in various fields such as water, wastewater treatment, electronic and surface treatment, fisheries, aquaculture and the like.

However, as described above, the effect of improving the learning ability of the extract is known, but research and development have not been conducted on the extract of the plant having enhanced acetylcholinesterase inhibitory effect by ultrafiltration.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention provides a method for producing an extract of an extract from which an acetylcholinesterase inhibitory effect is enhanced.

Another object of the present invention is to provide a health functional food composition for preventing or ameliorating memory impairment, which comprises an extract of green tea extract having the acetylcholinesterase inhibiting effect produced by the above method.

A further object of the present invention is to provide a pharmaceutical composition for preventing or treating dementia, which comprises an extract of a plant having the acetylcholinesterase inhibitory effect enhanced by the method.

In order to solve the above-mentioned problems, the present invention provides a method for preparing an extract of an extract having enhanced acetylcholinesterase inhibitory effect comprising the steps of:

(a) preparing a raw extract by adding one or a mixture thereof selected from the group consisting of water and lower alcohols having 1 to 4 carbon atoms to the raw paper, heating and extracting the same; And

(b) diluting the crude extract obtained in step (a) by adding purified water, and then passing the diluted water through an ultrafiltration device to obtain an ultrafiltered extract.

The present invention also provides a health functional food composition for preventing or ameliorating memory impairment, which comprises an extract of green tea extract having enhanced acetylcholinesterase inhibitory effect produced by the above method.

Further, the present invention provides a pharmaceutical composition for preventing or treating dementia, which comprises an extract of a plant having an acetylcholinesterase inhibitory effect enhanced by the method.

According to the method of the present invention, it is generally possible to use an ultrafiltration method, which is not separation using a column used for separation of substances, to simply extract the saponin-based compound (ultrafiltration inner solution) and non-saponin- And the saponin-based compound in the extract is removed, thereby increasing the acetylcholinesterase inhibiting effect. Therefore, the extract is superior to the acetylcholinesterase inhibiting effect of the conventional extracts, Therefore, the extract of the present invention is not a single component but an extract of a natural substance. Since it has fewer side effects than a synthetic drug, it can be used as a health functional food composition for preventing or improving memory deficits or for preventing or treating dementia And may be usefully used as an emulsion composition.

FIG. 1 is a graph showing the HPLC results of the crude extract-ultrafiltration internal solution of one example according to the present invention.
FIG. 2 is a graph showing the HPLC results of an extracellular ultrafiltration liquid of a raw extract of the present invention in accordance with the present invention.
FIG. 3 is a graph showing the HPLC results of the crude extract BT-11 of the comparative example according to the present invention.
FIG. 4 is a graph showing changes in the components of the ultrafiltration external fluid and the internal fluid of the raw extract of the present invention. FIG.
FIG. 5 is a graph showing an acetylcholinesterase inhibiting activity effect according to the present invention. FIG.

Hereinafter, the present invention will be described in detail.

In the present invention, the term 'ultrafiltered extract' refers to a solution permeated through ultrafiltration paper of an ultrafiltration apparatus of a raw extract, and includes ultrafiltrate outer-body dried matter.

The present invention provides a method for producing an extract of an extract of the present invention promoted by an acetylcholinesterase inhibitory effect comprising the steps of:

(a) preparing a raw extract by adding one kind or a mixture thereof selected from the group consisting of water and lower alcohols having 1 to 4 carbon atoms to the raw paper, heating and extracting the same; And

(b) diluting the crude extract obtained in step (a) by adding purified water, and then passing the diluted water through an ultrafiltration device to obtain an ultrafiltered extract.

In the step (a) according to the present invention, one kind or a mixture thereof selected from the group consisting of water and lower alcohols having 1 to 4 carbon atoms is added to the raw ground material, and the mixture is heated and extracted. .

At this time, the raw material is preferably extracted using ethanol, more preferably 70-80% ethanol mixed with water, but the present invention is not limited thereto.

On the other hand, the amount of the aqueous ethanol solution used is preferably 6-8 times the volume of the ground paper.

The method for extracting the extracts of the present invention can be carried out by an extraction method such as hot water extraction, immersion extraction, supercritical extraction, subcritical extraction, high temperature extraction, high pressure extraction, reflux cooling extraction and ultrasonic extraction or adsorption including XAD and HP- A method using a resin, and the like can be used, and it is preferable to heat, reflux or extract at room temperature, but it is not limited thereto.

The extract of the extract of the present invention is preferably 1 to 5 times, more preferably 2 to 3 times, but is not limited thereto.

On the other hand, the extraction temperature can be extracted at room temperature, but it is preferably 60 to 90 ° C.

The extract of the present invention may be extracted at room temperature without heating, but it is preferable to heat the extract to increase the yield of the extract. On the other hand, when the heating temperature is higher than 90 ° C, the components in the extract may be changed to lower the acetylcholinesterase inhibiting activity of the extract.

The extraction time of step (a) of the present invention is 1 to 24 hours, preferably 3 to 5 hours, but is not limited thereto.

The extraction time can be generally determined by a person skilled in the art.

In the step (a) of the present invention, after the extraction of the raw paper is completed, a filtration device that can be used when filtering the ground residue may be a filtering device capable of removing the paper residue according to the present invention, But it is not limited thereto.

In the step (a) of the present invention, it is preferable to carry out a concentration-reducing process, and the vacuum concentration is preferably performed using a vacuum rotary evaporator at a temperature of 30 to 40 ° C, but is not limited thereto.

Specifically, the extract of the extract of the present invention may be prepared as a soft tissue extract by concentration under reduced pressure or lyophilized, but it is preferable to concentrate the extract at a concentration of 25-30 bricks.

In the step (b) of the present invention, purified water is added to the raw extract obtained in the step (a), diluted, and then passed through an ultrafiltration device to obtain an extract of the saponin- .

At this time, the nominal molecular weight cut-off (NMWC) of the ultrafiltration membrane of the ultrafiltration apparatus of step (b) may be 3000 to 10000.

Particularly, when the molecular weight of the ultrafiltration membrane is less than 3000, there is a problem that the effective component is small. When the molecular weight exceeds 10000, the compounds having a large molecular weight are passed through the ultrafiltration device as they are, There is a problem that separation is difficult.

In the concentrate dilution of the step (b) of the present invention, the concentration is preferably 1 to 5%.

When diluting the concentrate of the extract of the present invention with a dilute concentrate of less than 1%, there is a problem that the condensed compounds are difficult to be separated. When the extract is diluted by more than 5%, a saponin- There is a problem that it can not be separated and can not be separated.

Meanwhile, in the case of drying the ultrafiltration ultrafiltration liquid of step (b) of the present invention in step (b) of the present invention, it is preferably, but not necessarily, dried under reduced pressure, vacuum dried, boiled dried, spray dried or lyophilized.

In addition, the present invention provides a raw extract for inhibiting acetylcholinesterase, wherein the ratio of TMCA and tenuifolin as an indicator component in the extract is 5 - 10: 1 (Test Example 4).

Further, the present invention provides a health functional food composition for preventing or ameliorating memory impairment, which comprises an extract of green tea extract having enhanced acetylcholinesterase inhibitory effect.

As a result of measuring the inhibitory effect of acetylcholinesterase, which is the substance most closely related to the cognitive function of the extract of the present invention, the inhibitory effect of acetylcholinesterase inhibitor BT-11 on the extracts of Korean pastes (Korean Patent Publication No. 2004-0013365) (See Experimental Example 2), it is possible to provide a health functional food composition for preventing or ameliorating memory loss or a pharmaceutical composition for preventing or treating dementia Can be usefully used as an active ingredient of < RTI ID = 0.0 >

The composition according to the present invention may be added to health functional foods such as foods, beverages and the like for the purpose of preventing or improving memory deterioration.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including dairy products, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, Beverages, alcoholic beverages and vitamin complexes, dairy products, and dairy products, all of which include health functional foods in a conventional sense.

The extract of the present invention can be added directly to the food or used together with other food or food ingredients, and can be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the extract of the present invention in the health functional food may be 0.1 to 90 parts by weight of the whole food. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention is not particularly limited to the ingredients other than the raw extract as the essential ingredient in the indicated ratios and may contain various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 of the composition of the present invention.

In addition to the above, the extract of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, Salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the extract of the present invention can contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.

These components may be used independently or in combination. The proportion of such additives is not so important, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Further, the present invention provides a pharmaceutical composition for preventing or treating dementia, which comprises an extract of an extract from which the acetylcholinesterase inhibitory effect is enhanced.

The extract of green leaf extract according to the present invention has excellent acetylcholinesterase inhibiting effect (see Experimental Example 2), and thus can be effectively used as a pharmaceutical composition for preventing or treating dementia.

Meanwhile, the pharmaceutical composition containing the extract of the extract of the present invention preferably contains 0.1 to 80% by weight of the composition based on the total weight of the composition, but is not limited thereto.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of medicaments.

The composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral preparations, suppositories and sterilized injection solutions according to a conventional method have. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may be formulated into the compositions of the present invention with at least one excipient such as starch, calcium carbonate, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a suppository base, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

The compositions of the present invention may be administered orally or parenterally, for example, orally, sublingually, nasally, rectally or subcutaneously.

The preferred dosage of the composition of the present invention may be appropriately selected by those skilled in the art depending on the age, condition and weight of the patient, the degree of disease, the type of drug, the route of administration, and the period of time. However, for the desired effect, the composition of the present invention may be administered in 1 to 3 divided doses per day within the dosage range of 10 - 1500 mg based on normal (60 kg of standard body weight), but is not necessarily limited to, Are not intended to limit the scope of the invention in any way.

According to the method of the present invention, it is generally possible to use an ultrafiltration method, which is not separation using a column used for separation of substances, to simply extract the saponin-based compound (ultrafiltration inner solution) and non-saponin- And the saponin-based compound in the extract is removed, thereby increasing the acetylcholinesterase inhibiting effect. Therefore, the extract is superior to the acetylcholinesterase inhibiting effect of the conventional extracts, Therefore, the extract of the present invention is not a single component but an extract of a natural substance. Since it has fewer side effects than a synthetic drug, it can be used as a health functional food composition for preventing or improving memory deficits or for preventing or treating dementia And may be usefully used as an emulsion composition.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

The following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited by the following Examples and Experimental Examples.

material

The dried herbs of polygala tenuifolia were used as the herb medicines used in this experiment. The roots were selected and stored in a clean warehouse. After removing the unheated part such as foreign matter, it is immersed in an appropriate amount of purified water and sufficiently absorbed. After washing cleanly (2 - 3 batches of 1 batch production standard), the water was removed using a filter net and allowed to stand at room temperature for 1 hour.

In the Examples and Experimental Examples, the 'ultrafiltration external fluid' is defined as an original extract containing a compound having a small molecular weight permeated through an ultrafiltration membrane. The 'ultrafiltration internal fluid' does not penetrate the ultrafiltration membrane, As a raw extract.

< Example  1> Origanum extract Ultrafiltration Exogenous  Produce

Step 1: Preparation of extract

1 kg of dehydrated raw paper was added with 8 L of 75% ethanol, followed by primary extraction at 80 ° C for 4 hours. The extract was filtered, and the resulting extract was filtered through a 50-micro filter to obtain a primary extract. Subsequently, 6 times 70% ethanol was added to the raw material which had been subjected to the first extraction in the above, and then the resultant was subjected to secondary extraction at 75 ° C for 4 hours. Then, the secondary extract was passed through a 50 micro filter, &Lt; / RTI &gt; The extract obtained above was then collected and concentrated to 35 brix at 35 DEG C using a vacuum concentrator.

Step 2: Ultrafiltration fluid  And Inner  Produce

10 times as much purified water as the volume of the extract was added to the extract of step 1 to dilute the extract to a solid content of 5% or less and passed through an ultrafiltration apparatus having an NMWC pore size of 7000 of the ultrafiltration membrane to obtain an ultrafiltration outer liquid and an inner liquid &Lt; / RTI &gt;

Step 3: Sterilization and drying

The ultrafiltration external solution of the extract of the extract from the ultrafiltration ultrafiltration membrane obtained in the step 2 was heated and sterilized at 90 ° C for 30 minutes and lyophilized to prepare a powder having a moisture content of 5% or less.

< Example  2> Origin extract Ultrafiltration Exogenous  Manufacturing-2

Except that the ultrafiltration membrane having an NMWC pore size of 3000 of an ultrafiltration membrane was used instead of the ultrafiltration membrane having an NMWC pore size of 7000 in the ultrafiltration membrane in Example 1, And the ratio of TMCA: tenuifolin = 3: 1 was obtained.

< Example  3> Origanum extract Ultrafiltration Exogenous  Manufacturing-3

Except that the ultrafiltration membrane having an NMWC pore size of 5000 of the ultrafiltration membrane was used instead of the ultrafiltration membrane having the NMWC pore size of 7000 of the ultrafiltration membrane in Example 1, And the ratio of TMCA: tenuifolin = 5: 1 was obtained.

< Example  4> Origin Extract Ultrafiltration Exogenous  Manufacturing-4

Except that the ultrafiltration membrane having an NMWC pore size of 10000 in the ultrafiltration membrane was used instead of the ultrafiltration membrane having an NMWC pore size of 7000 in the ultrafiltration membrane in Example 1, And the ratio of TMCA: tenuifolin = 10: 1 was obtained.

< Comparative Example  1> Ultrafiltration membrane Pore size  Of the extract Resolution  compare

The following experiment was conducted to compare the separation ability of the extract of the leaf extract according to the pore size of the ultrafiltration membrane according to the present invention.

Except that the ultrafiltration membrane having an NMWC pore size of 15000 was used instead of the ultrafiltration membrane having an NMWC pore size of 7000 of the ultrafiltration membrane in the step 2 of Example 1 above.

result

The saponin-based compound having a high molecular weight was hardly found in the ultrafiltrate external solution of the extract of the extract of the present invention prepared in Example 1 according to the present invention. On the other hand, the ultrafiltration membrane of Comparative Example 1 was passed through an ultrafiltration apparatus having a pore size of 15000 The ultrafiltrate external solution of the extract of the leaves contained a saponin compound having a high molecular weight and was found to be similar to the saponin compound in the conventional extract BT-11 without using the ultrafiltration device.

< Experimental Example  1> HPLC  analysis

The following experiment was carried out to compare the content of the specific indicator components in the extract of the extract of Example 1 according to the present invention.

First, (1) it was identified as brown powder through visual inspection, (2) the content of TMCA (3,4,5-trimethoxycinnamic acid) was measured by using a high-performance liquid chromatograph and preparing standard solution and test solution .

① Standard solution was prepared by the following method.

0.250 g of the standard product TMC is precisely weighed, placed in a 25 ml volumetric flask, dissolved in 70% methanol, and filled to the indicated line. Take exactly 0.5, 1, 3 and 5 ml of standard stock solutions and transfer them into 100-ml volumetric flasks as the standard solution.

② Test solution was prepared by the following method.

200 mg of the sample is precisely weighed, and exactly 70% methanol is added to make exactly 20 ml. The solution is completely dissolved and filtered through a 0.45 μm membrane filter. (Remove the first 4 ml of the filtrate before use.)

High-performance liquid chromatography conditions are as follows.

- Column: YMC J'sphere ODS H80 (C18, 5 m, 150 x 4.6 mm) or equivalent column

- Mobile phase: liquid A-50 mM dihydrogen phosphate solution (NaH ₂ PO ₄ ), liquid B-acetonitrile

- Detector and wavelength: UV absorptiometer (UV 293 nm)

- Flow rate: 1 ml / min

- Column temperature: 40 ° C

Time (min) _{A% (50 mM NaH 2 PO} 4) B% (ACN) 0 87% 13% 13 84% 16% 30 81% 19% 50 78% 22% 60 30% 70% 61 87% 13%

The quantitative test was also calculated as follows.

20 μl of standard solution and test solution is injected and tested according to the above conditions. The TMC concentration in the test solution is obtained using the calibration curve obtained by the area of TMC in the standard solution, and the TMC content (mg / 100 g) in the sample is obtained according to the following equation.

TMC content (mg / 100 g) = C 占 V / W 占 1 / 10,000

C: TMC concentration in test solution (mg / ml)

V: Total amount of test solution (ml)

W: Amount of sample (g)

1 / 10,000: Unit conversion

result

As shown in FIG. 1, the content of the saponin-based compound in the ultrafiltration external liquid was extremely low, being 10 to 20%, as shown in FIG. 1, , The content of the saponin-based compound was measured to be 70% or more in the ultrafiltration internal solution, and as shown in Fig. 3, the content of the saponin-based compound of the conventional BT-11 was found to be 40 to 50% .

It was confirmed that the fractions containing the saponin compounds and the fractions containing the non saponin compounds can be easily separated using the ultrafiltration method of the present invention, rather than using the column.

< Experimental Example  2> Ultrafiltration Outward  And Inner  Component change

The following experiments were performed to determine the changes in the components of the ultrafiltration external fluid and the internal fluid of the extract of the raw paper of the present invention. In this experiment, TMCA (3,4,5-trimethoxycinnamic acid), a non-saponin compound represented by the following formula (1), and tenuifolin, a saponin compound represented by the following formula (2) The ultrafiltration outer and inner fluid components of the extract were measured.

[Chemical Formula 1]

(2)

Step 1: Preparation of standards

A standard solution of 0.025 g of standard foamed neoprene was weighed into a 25 ml volumetric flask, dissolved in 70% methanol, and filled to the mark. The standard solution was refrigerated.

Step 2: Preparation of Test Solution

0.030 g of the sample was weighed into a 50 ml volumetric flask, completely dissolved by adding 70% methanol, and filtered with a 0.45 μm membrane filter (PTFE) to obtain a test solution.

Step 3: Quantitative test

10 μl of the standard solution obtained in step 1 and the test solution obtained in step 2 above are injected and tested according to the preceding conditions. The concentration of Tneiophorin in the standard solution is used to calculate the concentration of TMCA in the test solution using the calibration curve obtained by the area, and the content (%) of the testein in the sample is obtained according to the following equation (1). The results are shown in Table 2 below.

[Equation 1]

(Mg / g) = C 占 V / W 占 1 / 1,000

   C: Tenuifolin concentration in test solution (ppm)

   V: Total amount of test solution (ml)

   W: Amount of sample (g)

1 / 1,000: Unit conversion

TMCA Tenuifolin Example 1
(Extract of ultrafiltrate from extracts of earth) 1.45 0.21 Comparative Example 1
(Extract of yeast extract ultrafiltration inner solution) 0.32 1.48 Comparative Example 2
(BT-11) 0.8 1.24

As a result of measuring the changes of the surface components of the ultrafiltration external fluid and the internal aqueous solution of the extract of the present invention, the content of the non-saponin-based compound (1.45%) in the ultrafiltration external fluid of Example 1 according to the present invention It was confirmed that the TMCA content in BT-11 (0.8%) was about twice as high as that in the conventional extract of BT-11. In the case of the ultrafiltration inner solution of Comparative Example 1, the saponin- Comparative Example 2). &Lt; tb &gt;&lt; TABLE &gt;

< Experimental Example  3> Inhibition of acetylcholinesterase ( Ellman's method )

The following experiment was carried out to confirm the acetylcholinesterase inhibitory effect of the ultrafiltrate inner solution and the outer solution of the crude extract of Example 1 according to the present invention.

reagent

1) Buffer solution I: 100 mM phoshate, pH 8.0

2) Buffer solution II: 100 mM phosphate pH 7.0

3) a buffer screen Elman reagent: DTNB 10mM, NaHCO ₃ 17.85mM

4) Acetylthioclorine iodine 75 mM

Experimental Method

Each of Example 1 and Comparative Example 1, Comparative Example 2, and the control group according to the present invention was dissolved in DMSO and then diluted to 200 μg / ml in buffer I (diluted to a final concentration of 5% for DMSO). 1.5 ml of diluted BT-11, 1.5 ml of buffer solution I, 20 μl of acetylcholine solution and 100 μl of buffered Ellman's reagent were mixed and cultured at 25 ° C. for 10 minutes. 20 μl of the enzyme sample was added, followed by inversion and the absorbance was measured at intervals of 30 seconds. The absorbance was measured for 5 minutes to confirm the linear reaction.

The untreated group (Blank) used saline instead of the enzyme sample. In addition, it was also confirmed that the absorbance was measured without adding acetylthiocolchine to measure a slight nonspecific reaction in the enzyme activity measurement of each of Example 1, Comparative Example 1, Comparative Example 2, and the control group according to the present invention.

AChE activity was shown as 100% in the case of no addition of each of Example 1 and Comparative Example 1, Comparative Example 2, and the control group according to the present invention, and the results of Example 1 and Comparative Example 1, Comparative Example 2, The percent inhibition of AChE activity when each of the control groups was added was expressed as percent inhibition. Each experiment was repeated 5 times and the significance of the results was verified by Student t-test.

Enzyme reaction rate measurement

The dose-response curve of BT-11 was obtained in order to measure the 50% enzyme inhibitory concentration (IC ₅₀ ) of BT-11 in Example 1 and Comparative Example 1, Comparative Example 2 and the control group according to the present invention, respectively (Ellman's method) The rate of enzyme reaction was calculated from the graph.

The values were measured five times for each concentration. The enzyme reaction rates of Example 1 and Comparative Example 1, Comparative Example 2 and Control group according to the present invention were measured in the same manner as in Example 1 and Comparative Example 1 and Comparative Example 2 according to the present invention while changing the substrate concentration to 23 to 230 μM. And AChE inhibition on the substrate was measured according to the presence or absence of each of the control groups (40 μM), and Km and Vmax values were obtained from the line-weaver plot.

The results are shown in Table 3 and FIG.

Inhibition rate (%) Example 1
(Extract of ultrafiltrate from extracts of earth) 48.101 Comparative Example 1
(Extract of yeast extract ultrafiltration inner solution) 12.041 Comparative Example 2
(BT-11) 29.774 Control group
CTL (DHED) 44.538

As shown in Table 3, the acetylcholinesterase inhibitory effect was about 48% or more in the case of the ultrafiltration ultrafiltrate extract of Example 1 according to the present invention, and that of the conventional extract BT-11 (Comparative Example 2) (About 29.8%), and it was found to be about 1.6 times higher than that of acetylcholinesterase (about 29.8%), and about 1.1 times higher than that of DHEA (Dehydroevo diamine) .

On the other hand, in the case of the ultrafiltration internal solution of Comparative Example 1, it was confirmed that the acetylcholinesterase inhibiting effect was as low as 12%.

Therefore, the extract of the present invention can be effectively used for the treatment of dementia because the acetylcholinesterase inhibiting effect of the extract of the present invention is more excellent than that of the original extract, and the extract of the present invention is not a single component , Which is less effective than synthetic drugs and therefore can be used as an acetylcholinesterase inhibitor.

< Experimental Example  4> Acetylcholinesterase inhibitory effect according to the ratio of indicator components

The following experiment was carried out to confirm acetylcholinesterase inhibitory effect according to the ratio of the indicator components in the extracts of the present invention.

The acetylcholinase inhibitory effect was measured using the same method except that the extract of the present invention was used instead of the original extract in Experimental Example 3, except that the extract of the present invention had a different ratio of the indicator components obtained by modifying the porosity of the ultrafiltration membrane in Example 1 Respectively. The results are shown in Table 4 below.

division Surface composition ratio Inhibition rate (%) TMCA Tenuifolin Example 1 7 One 48.1% Example 2 3 One 33.7% Example 3 5 One 44.6% Example 4 10 One 47.2%

As shown in Table 4, the acetylcholinesterase inhibitory effect of the extract of Example 1 according to the present invention was found to be about 48% or more, while that of the extract of Example 4 was higher than that of TMCA The content was higher than that of Example 1, but the acetylcholinesterase inhibitory effect was confirmed to be similar to that of Example 1. On the other hand, as shown in the results of Example 2, it was confirmed that the acetylcholinesterase inhibiting effect was lowered when the difference in the content of TMCA and the amount of cinnamon was small.

Therefore, in accordance with the method of the present invention, it is generally possible to separate the saponin-based compound (ultrafiltration internal solution) and the non-saponin-based compound The ultrafiltration outer fluid) can be separated. By removing the saponin-based compounds in the extract, the acetylcholinesterase inhibiting effect is raised. Therefore, it is superior to the acetylcholinesterase inhibiting effect than the conventional extracts, And the extract of natural origin is not a single component and the extract of natural origin is less harmful than the synthetic drug. Therefore, the composition of health functional food for prevention or improvement of memory loss, prevention or treatment of dementia And can be usefully used as a pharmaceutical composition.

Claims (7)

(a) preparing a raw extract by adding one kind or a mixture thereof selected from the group consisting of water and lower alcohols having 1 to 4 carbon atoms to the raw paper, heating and extracting the same; And
(b) Purified water is added to the crude extract obtained in step (a) and diluted, and then the solution is passed through an ultrafiltration apparatus having a nominal molecular weight cutoff (NMWC) of 3000 to 10,000 in the ultrafiltration membrane to obtain an ultrafiltrate The method according to any one of claims 1 to 3, wherein the step of extracting acetylcholinesterase inhibitor comprises the steps of:
The method according to claim 1,
Wherein the extraction temperature is 60-90 ° C. in heating the step (a).
delete The method according to claim 1,
Wherein the concentration of the diluted concentrate of step (b) is 1 to 5% by weight.
delete A health functional food composition for preventing or ameliorating memory impairment, which comprises an extract of an acetylcholinesterase inhibitor extract prepared by the method of claim 1.
A pharmaceutical composition for preventing or treating dementia, which comprises an extract of a plant extract for inhibiting acetylcholinesterase produced by the method of claim 1.

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