KR20150050716A - Pharmaceutical and food composition of Allomyrina dichotoma for the prevention and treatment of Alzheimer's disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition including Allomyrina dichotoma as an active ingredient for preventing and treating Alzheimer′s and to a food composition for alleviating Alzheimer′s. A method for producing an Allomyrina dichotoma fragment having an anti-Alzheimer′s effect of the present invention comprises: a first step of pulverizing frozen and dried Allomyrina dichotoma; a second step of producing an Allomyrina dichotoma extraction liquid by mixing the Allomyrina dichotoma pulverized in the first step with a solvent; a third step of adding a solvent consisting of water and hexane to be mixed with the Allomyrina dichotoma extraction liquid produced from the second step, and separating only a hexane layer; and a fourth step of adding a solvent consisting of water and ethyl acetate (EtOAc) to be mixed with the Allomyrina dichotoma extraction liquid produced from the second step, and separating only ethyl acetate (EtOAc). According to a present invention, provided is pharmaceutical composition for preventing or treating Alzheimer′s and a food composition for alleviating Alzheimer′s, wherein Allomyrina dichotoma or a fragment derived from an extract thereof is included as an active ingredient, thereby ensuring an Alzheimer′s inhibiting effect by controlling β-Secretase activities.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing and treating Alzheimer's dementia and a composition for improving Alzheimer's dementia,

The present invention relates to a pharmaceutical composition for the prevention and treatment of Alzheimer's dementia comprising a long beetle, and to a composition for improving Alzheimer's dementia. More particularly, the present invention relates to a composition for preventing or treating Alzheimer's dementia such as Allomyrina dichotoma ) Or fractions derived therefrom as an active ingredient, and to a food composition for improving Alzheimer's dementia and a composition for preventing or treating Alzheimer's dementia.

Alzheimer's disease (AD), the most common cause of dementia, is characterized by general atrophy of the brain and specific histological findings such as senile plaque, neurofibrillary tangle, Granulovacuolar degeneration) is a typical degenerative neurological disorder characterized by functional degeneration of synapses and loss of nerve cells.

The most representative theories for the development of Alzheimer's disease (AD) include the mutation of the genes for the presence of precenillin (PS) and the over-production of amyloid precursor protein (APP), resulting in the formation of β-amyloid protein (Aβ) And that neurotoxicity caused by the bundle of nerve fibers inside the cell and the outer nervous system outside the cell caused by the deposition of the β-amyloid protein (Aβ) causes destruction of the nerve cell and loss of function .

Therefore, there is currently a study on inhibition of beta-secretase involved in beta amyloid protein formation.

Longevity beetle is an insect belonging to the beetle tree. So far, antioxidant effects and anti-inflammatory effects of major proteins have been reported.

Korean prior art 10-2008-0020736 entitled " Bronchoptera extracts exhibiting antioxidative activity and inhibitory activity on bone metabolism-related factors "are registered as related prior arts.

However, the prior art relates to bronchial beetle extract, and the pharmaceutical composition for the prevention and treatment of Alzheimer's dementia and the composition for improving Alzheimer's dementia, which contain the long-lived beetle fraction of the present invention as an active ingredient, are not known.

An object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of Alzheimer's dementia comprising a long-lived beetle characterized by inhibition of beta-secretase activity, which is indicated as a main cause of Alzheimer's dementia, And to provide an improved food composition.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

To achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of Alzheimer's dementia or a composition for Alzheimer's dementia improvement, which comprises as an active ingredient an Allomyrina dichotoma or a fraction derived from an extract thereof.

The fraction is characterized by comprising at least one of a hexane fraction or an ethyl acetate (EtOAc) fraction obtained by extracting the long-lived beetle with ethanol and then fractionating it.

The fraction is characterized by exhibiting the ability to inhibit the activity against beta-secretase, including any one or more of oleic acid, palmitic acid, palmitoleic acid and linoleic acid.

A method for producing a long-lived beetle fraction having an anti-Alzheimer effect of the present invention comprises: a first step of pulverizing a lyophilized long beetle; A second step of preparing a long beetle extract solution by mixing the long-lived beet pulverized in the first step with a solvent; A third step of adding a solvent consisting of water and hexane to the long beetle extract prepared in the second step and separating only the hexane layer after mixing, And a fourth step in which a solvent composed of water and ethyl acetate (EtOAc) is added to the extract and mixed with ethyl acetate (EtOAc) alone.

The long-life beet extract of the second step is characterized by mixing 0.5 to 1 kg of the pulverized long beet scarab per 12 liters of the solvent, and the solvent is 70 to 80% ethanol.

The anti-Alzheimer's composition of the present invention is characterized by comprising the long-lived beetle fractions produced by the above-described method as an effective ingredient.

The method of using the anti-Alzheimer's composition of the present invention is characterized in that the anti-Alzheimer's composition is used as a food composition for improving Alzheimer's disease or a pharmaceutical composition for preventing and treating Alzheimer's disease caused by beta-secreting.

According to the present invention, it is possible to prevent Alzheimer's dementia having an Alzheimer's dementia-inhibiting effect through inhibition of beta-secretase activity by containing Allomyrina dichotoma or fractions derived from the extract thereof as an active ingredient And a therapeutic pharmaceutical composition and a food composition for improving Alzheimer's dementia.

Therefore, the present invention is expected to be advantageous in the development of anti-dementia-inducing agents, since it can pass the blood brain barrier through which the synthesis inhibitor of the polymer can not pass.

1 is a view showing the yield content of the long-lived beetle fraction of the present invention.
Fig. 2 is a graph showing inhibition of beta-secretase activity of the long-lived beetle fraction of the present invention.
FIG. 3A is a graph showing the results of performing 14 fatty acids in total of long-lived beetles by gas chromatography.
FIG. 3B is a diagram showing the component of the long-length beetle fatty acid according to the number (No.) in Table 1. FIG.
4 is a graph showing the inhibitory activity of beta-secretase activity of the main fatty acids of long-bred beetle.
5A is a graph showing the inhibition of beta-secretase activity of oleic acid, which is a representative fatty acid of the long-beetle beetle of the present invention, by Dixon plot.
FIG. 5B is a graph showing the effect of the inhibitor according to the representative fatty acid oleic acid substrate concentration of the beetle of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail, and a detailed description of known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted.

In the present invention, it is confirmed that long-lived beetles, which are generally known to be effective for liver diseases, have an effect of preventing and treating Alzheimer's dementia.

As a result, there has been a growing tendency to use grasshoppers, a larva of longevity beetle, as a health supplement, as folk remedies have been shown to be effective against liver disease. In addition, the protein isolated from longevity beetle shows strong inhibition against bacteria, and lectin extracted from longevity beetle has strong anticancer ability and anticancer effect can be expected. However, studies on physiological functions of L. longissimus larvae and larvae are widely used in folk remedies, and there is no report about prevention and treatment effects of Alzheimer 's dementia using L. longissimo.

Accordingly, the inventors of the present invention have found that the long-lived beetle of the present invention can be widely used for treating or preventing Alzheimer's dementia because the natural substance is an effective ingredient and does not cause side effects.

Alzheimer's disease is a brain disease caused by damage to nerve cells by β-amyloid protein (Aβ). In normal people, N-amyloid precursor protein (APP) is inhibited by α-Secretase - The 15th and 16th amino acids at the terminal are cleaved and cleaved by gamma-secretase to release short peptide without peptide.

On the other hand, in the case of dementia patients, amyloid fragments (Aβ _40-42 (SEQ ID NO: 2)) cleaved from APP (amyloid precursor protein) by the action of β-secretase and γ- ) Aggregate and accumulate in the brain.

In addition, β-amyloid protein (Aβ) destroys neurons in the cerebral cortex and neurons that produce acetylcholine, a neurotransmitter, to decrease the activity of acetylcholine. ROS) to induce oxidative damage, resulting in neuronal death and dementia.

Thus, beta-secretase and gamma-secretase, the major enzymes involved in the formation of beta-amyloid protein (Aβ), have emerged as a key element in the study of Alzheimer's disease. However, the inhibition of gamma-secretase showed side effects on the Notch pathway, and cytotoxicity caused by the C99 fragment resulting from the inhibition of this enzyme was observed.

Therefore, studies on inhibition of beta-secretase have been conducted at present, and inhibitors against existing beta-secretase (BACE1) are mostly based on synthetic compounds have.

The synthetic compound inhibitor has a disadvantage that most of them are oligopeptide compounds having a large molecular weight and can not pass through the blood brain barrier. Therefore, there is a limit to the development of a therapeutic agent for Alzheimer's disease in reality.

As a result of various studies to solve the above problems, the present invention has found that oleic acid, palmitic acid, palmitoleic acid, and linolenic acid, which are contained in natural products such as longevity beet, are classified into beta-secretase, which is a cause of Alzheimer's dementia ), Indicating that this species of longevity beetle ( Allomyrina dichotoma or fractions derived therefrom as an active ingredient, and a food composition for improving Alzheimer's dementia.

At this time, the long beetle ( Allomyrina dichotoma fraction is a fraction obtained by extracting and fractionating an effective parturient exhibiting beta-secretase inhibitory activity and is most suitably used as a material of the composition.

Thus, the method for producing the fractions of long-lived beetles having the anti-Alzheimer effect of the present invention will be described in detail as follows.

1. First step; Long beet pulverization

The present invention of a number of natural products having a beta-Alzheimer's dementia inhibitory effect - beetle (Allomyrina having sekeuri te rise (β-Secretase) inhibition of Alzheimer's dementia over the inhibitory effect dichotoma ).

The long beetle ( Allomyrina dichotoma ) belongs to chafer. Body length is about 30 ~ 85mm, very thick and fat, and body color is brown all over.

These long beetles ( Allomyrina dichotoma ) is recommended to use long-lived beetles containing the greatest amount of active ingredients that can prevent and cure Alzheimer's disease.

In this step, the prepared long beetle ( Allomyrina dichotoma ) is dried and then pulverized into powder form.

In other words, the longevity beetle ( Allomyrina dichotoma ) contains a large amount of water, so it needs to be dried to make it completely free of water. At this time, it is preferable to perform drying through freeze drying in order to minimize the change of the active ingredients contained in the long beetle.

The dried longevity beetle is then prepared in the form of a powder using a pulverizer for ease of subsequent extraction.

2. The second step; Production of longevity beet extract

In this step, the prepared long beetle ( Allomyrina dichotoma ) powder was extracted into a solvent and concentrated under reduced pressure to prepare a long beetle extract.

At this time, it is preferable to mix 0.5 to 1 kg of the pulverized long beetle per 12 liters of the solvent. It is preferable to use ethanol as the solvent used. Any ethanol may be used, but the active ingredient It is advisable to use 70 ~ 80% of ethanol so that the most suitable extraction can be performed.

The longevity beet pulp is completely immersed in ethanol and then extracted at 230 to 270 rpm at room temperature (20 to 25 ° C) and concentrated under reduced pressure to produce an extract containing a large amount of active ingredients of the beetle in a short period of time.

3. Steps 3 and 4; Production of long-bred beet fractions

In this step, the long-chafer ethanol extract is fractionated with water (H ₂ O), hexane (Hexane) or water (H ₂ O) and ethyl acetate (EtOAc), concentrated under reduced pressure, Ethyl acetate (EtOAc) layer is obtained.

At this time, a solution of water (H ₂ O) and hexane (Hexane) or a solution of water (H ₂ O) and ethyl acetate (EtOAc) is added to the ethanol extract of long beetle beet.

The thus-prepared long beetle ( Allomyrina dichotoma fractions have the highest yields of active ingredients showing beta -secretase activity inhibitory activity, which makes it possible to provide a pharmacological or food composition which is more economically effective for inhibiting Alzheimer's dementia.

Hereinafter, the present invention will be described in detail with reference to the following examples. Allomyrina dichotoma , or fractions derived from these extracts as an active ingredient will be described in detail as follows.

In the present invention, the above-mentioned Allomyrina dichotoma or fractions derived from these extracts are included as active ingredients in the pharmaceutical composition for the prevention and treatment of Alzheimer's dementia.

At this time, the fraction of Allomyrina dichotoma , or an extract thereof, is preferably contained in an amount of 0.01 to 50% by weight, more preferably 0.1 to 20% by weight, based on the total weight of the pharmaceutical composition. This is because when the Allomyrina dichotoma or the fractions derived from these extracts are contained in an amount of less than 0.01% by weight, the ability to inhibit beta-secretase activity deteriorates and the present invention is useful for the prevention and treatment of Alzheimer's dementia And if it is contained in an amount of more than 50% by weight, adverse effects due to the use of the organic solvent may occur.

Such a pharmaceutical composition of the present invention may be contained in a natural drug product having the ability to control Alzheimer's dementia for the purpose of prevention and treatment of Alzheimer's disease. In this case, according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs, the preparation is carried out in the form of a unit dose form by using a pharmaceutically acceptable carrier or excipient, .

The formulations may be in the form of tablets, capsules, powders, granules, liquids or rings, and may additionally contain dispersing or stabilizing agents. That is, it can be selected without difficulty by those skilled in the art depending on the purpose of use, and the addition amount thereof can be selected within a range that does not impair the purpose and effect of the present invention.

Examples of the pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline Cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and also pharmaceutically acceptable excipients Include, but are not limited to, lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents, preservatives and the like.

In another aspect of the present invention, the above-mentioned Allomyrina dichotoma or an extract thereof-derived fraction is contained as an active ingredient of a food composition for the purpose of improving Alzheimer's dementia. That is, when Allomyrina dichotoma or an extract derived fraction thereof is used as an active ingredient of a food composition, it can be used as it is or can be used together with components of other common food compositions and can be used appropriately according to a conventional method . The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, when the food composition is prepared, it is added in an amount of 0.01 to 10 parts by weight, preferably 0.05 to 1 part by weight, based on the raw material of the active ingredient.

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.

Such a food composition may be contained in a health food for the purpose of improving Alzheimer's dementia, and there is no particular limitation on its kind. Examples of the food to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include healthy foods in a conventional sense.

In addition to the above, the food composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may be contained as pulp for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes only and do not limit the scope of protection of the present invention.

< Example  1 > The present invention relates to a method for preventing Alzheimer's dementia, Fraction  Produce

First, a powder (WorldWeek Co., Ltd.) obtained by lyophilizing the long-bean beetle at the maximum growth stage and pulverizing the dried material in a state in which moisture was completely removed was prepared.

0.5 kg of long-lived beet powder was completely immersed in 12 L of ethanol (EtOH).

Then, the mixture was extracted at 250 rpm and 23 캜 for 3 days, and then concentrated under reduced pressure to prepare a long beetle ethanol extract.

83.80 g of the ethanol extract of longevity beetle was fractionated into a solution composed of water (H ₂ O), hexane (Hexane), water (H ₂ O) and ethyl acetate (EtOAc).

Then, a layer composed of hexane and ethyl acetate (EtOAc) was separated using a fractional funnel, and concentrated under reduced pressure to prepare each long-bred scarab fraction.

< Experimental Example  1> Fraction  Yield measurement

As in Example 1, ethanol was added to 0.5 kg of long-bean beetle to obtain 83.80 g of the extract, and the fraction was sequentially fractionated by varying the polarity of the solvent. As a result, the yield was measured as shown in FIG.

In other words, 56.01 g (66.84%) of the hexane fraction, 0.61 g (0.73%) of the ethyl acetate (EtOAc) fraction and 22.41 g (26.74%) of the water (H ₂ O) It was found that a large amount of fraction was obtained from hexane as compared with other solvents.

< Experimental Example  2 > beta-secretase (beta- Secretase, BACE1 ) Inhibition effect measurement

(1) Experimental method

BACE1 enzyme experiments were carried out to verify the anti-Alzheimer's dementia effect of the long-lived beetle fractions of Example 1 above.

Resveratrol, a well-known inhibitor of natural beta-secretase, was used as a positive control (positive control)

10 μl of a sample diluted in an assay buffer, 10 μl of 750 nM Rh-EVNLDAEFK-Quencher (substrate) (Pan Vera), and beta-secrease (1.0 U / ml) 1, and then placed in a 384-well plate, followed by reaction at 25 ° C for 60 minutes.

Then, fluorescence was measured at Ex.530 nm and Em 590 nm using a fluorescence microplate reader, and the inhibition rate was calculated by the following equation.

Inhibition rate (%) = [1 - (S - S ₀ ) / (C - C ₀ )] X 100

C: Fluorescence of control after 60 min of incubation

(Enzyme, assay buffer, substrate)

C ₀ : Fluorescence of the control at time 0

S: Fluorescence of control after 60 min of incubation

(Enzyme, sample solution, substrate)

S ₀ : Fluorescence of the tested sample (S) at time 0)

(2) Experimental results

As shown in FIG. 2, the inhibitory activity of the recombinant BACE1 activity of the long-lived beetle fraction of Example 1 was 36.36 ± 0.49 for the ethyl acetate (EtOAc) fraction, 26.75 ± 0.40 for the hexane fraction and 26.75 ± 0.40 for the 50 μg / H ₂ O) was 12.25 ± 0.36. The concentration of ethyl acetate (EtOAc) was 57.59 ± 0.92 and that of hexane (47.91 ± 3.01) was lower than that of water (H ₂ O) of 25.51 ± 0.42 It was found to have high activity.

Considering the relatively high BACE1 inhibitory activity and high yield (66.84% of the total fatty acids) of the long-lived beetle hexane fraction, the BACE1 inhibitory effect of the long-lived beetle fractions is considered to be mostly due to the non-polar fraction. Fatty acid analysis of the same long beetle was performed.

< Experimental Example  3> Fatty acid analysis

(1) Experimental method

Gas chromatography (GC) was performed to analyze the fatty acid content of long beet powder. Before the analysis of fatty acids by GC, methylation was carried out by pretreatment before analysis.

In the pretreatment process, the longevity beet powder of freeze-dried maximum growth stage was prepared, and 2 ~ 3g of the long beetle powder (World Way Co., Ltd.) was dissolved in 5mL of tetrahydrofuran (THF) 30 mL of 1N potassium hydroxide ethanol (KOH-ethanol) was added and the mixture was refluxed at 85 DEG C for 1 hour and 30 minutes, and then filtered and washed with ethanol (EtOH).

The filtrate was subjected to neutralization titration with hydrochloric acid (HCl), and sodium sulfate (Na ₂ SO ₄ ) was added to the filtrate. The filtrate was extracted with a petroleum ether: water (4: 6) Respectively.

Sodium sulfate (Na ₂ SO ₄₎ was added to remove the water again, and then filtered and concentrated, methanol (CH ₃ OH) 40mL, and sulfuric acid (H ₂ SO ₄₎ into a 0.5mL ethyl and reflux for 3 hours at 90 ℃ Ethyl ether (30-50 mL) and distilled water (50-100 mL) were mixed in a separatory funnel, and this process was repeated once more.

Sodium sulfate (Na ₂ SO ₄₎ was added to remove water, and then allowed to stand at room temperature for 30 minutes (20 ~ 25 ℃) filtered and concentrated under reduced pressure to remove the ether (Ether), and then, gas chromatography (Gas chromatography, HP6890N GC -FID). &Lt; / RTI &gt;

Injection and detector temperatures were set at 260 ° C, and a spill ratio was measured using a Supelco ^™ SP-2560 capillary column (100 m × 0.25 mm × 0.20 μm) 50: 1, and the volume was 1 [mu] l.

Carrier gas was helium, which had no activity for the stationary phase or the sample of the column. The flow rate was set at 1 mL / min. The oven program was maintained at 140 ° C. for 5 minutes, The temperature was raised to 240 DEG C at 3 DEG C / min and then maintained for 10 minutes.

Sample concentration was used to 3,000mg / L in methylene chloride (Methylenechloride), fatty acid standard solution concentration was used 10,000mg / L of Component Supelco ^TM 37 FAME mix.

(2) Experimental results

① Fatty acid profile of longevity beetle (Profile)

The quantitative analysis using GC was carried out to reveal the representative materials of long beetle, and the retention time of 37 standard fatty acid esters was compared with the following results.

As shown in FIGS. 3A and 3B and Table 1, the fatty acid content of the long beetle was found to be 39.87%, and a total of 14 fatty acids were detected.

No. Components
(ingredient) Abbreviated form
(Abbreviation) retention time (min) ^a
(Storage time)
Amount (sample g / 100g) One Myristic acid
(Myristic acid) C14: 0 25.78 0.45 2 Myristoleic acid
(Misterol oleic acid) C14: 1n5 27.61 0.13 3 Pentadecanoic acid
(Pentadecanoic acid) C15: 0 27.80 0.13 4 Palmitic acid
(Palmitic acid) C16: 0 29.99 12.52 5 Palmitoleic acid
(Palmitoleic acid) C16: 1n7 31.45 3.71 6 Heptadecanoic acid
(Heptadecanoic acid) C17: 0
31.87 0.13 7 Stearic acid
(Stearic acid) C18: 0 33.89 0.91 8 Oleic acid
(Oleic acid) C18: 1n9c
35.37 19.13 9 Linolenic acid
(Linolenic acid) C18: 3n3 37.17 2.08 10 γ-linolenic acid
(Gamma-linolenic acid) C18: 3n6 38.62 0.12 11 α-linolenic acid
(Alpha-linolenic acid) 39.37 0.15 12 cis-11,14-Eicosenoic acid
(cis-11,14-eicosanoic acid) C20: 1n9 40.85 0.11 13 Tricosanoic Acid
(Tricosanoic acid) C23: 0 43.48 0.20 14 cis-5,8,11,14,17-Eicosapentaenoic acid
(cis-5,8,11,14,17-eicopentaenoic acid) C20: 5 45.91 0.10

As shown in Table 1 and FIGS. 3A and 3B, oleic acid (47.97%) was the highest in the total fatty acid content, and oleic acid (19.13 g) was present per 100 g of the long beetle powder .

The content of palmitic acid, palmitoleic acid and linoleic acid were 31.39%, 9.30% and 5.22%, respectively, and the content of four main fatty acids was 93.88% of total fatty acids.

② Inhibitory effect of BACE1 (β-Secretase)

The results of measuring the inhibitory effect of BACE1 (β-Secretase) on oleic acid, palmitic acid, palmitoleic acid and linoleic acid which are typical fatty acids of the long-beetle, as shown in FIG.

As shown in FIG. 4, palmitic acid (14.3 ± 1.3%, 36.1 ± 4.9%), palmitoleic acid (17.2 ± 1.4%, 37.5 ± 2.8%) and linoleic acid (13.9 ± 1.3% 1.1%) showed a beta-secretase activity inhibitory effect.

In particular, oleic acid showed excellent inhibition of BACE1 inhibition by showing 30.8 ± 0.7% and 76.7 ± 1.6% at 10 and 100 μM levels, respectively.

In addition, an enzyme inhibition pattern of oleic acid showing a high BACEl inhibitory effect was measured using a Dixon plot.

As a result, as shown in FIGS. 5A and 5B, FIG. 5A is a graph showing the beta-secreotide inhibition performance of IC ₅₀ 6.1 X 10 ^-5 M, FIG. 5B shows K _i 3.43 X 10 ^-5 M, K _i Given that the value is the x-intercept, oleic acid has been shown to inhibit BACE1 in a non-competitive manner.

< Experimental Example  4> Study on enzyme specificity and inhibition pattern of long-chafer fatty acid

(1) Experimental method

In order to confirm the enzyme specificity for the beta-secretase by using the long-lived beet fractions of Example 1 and the representative fatty acids (oleic acid, palmitic acid, palmitoleic acid, and linoleic acid) of the beetle of Experimental Example 3, chymotrypsin Chymotrypsin) and trypsin (serine proteases).

Chymotrypsin and trypsin were assayed for inhibitory activity using 0.05 M Tris-HCI buffer (pH 8.2, 0.02 M calcium chloride (CaCl ₂ )) and substrates were 1.25 mM z-Arg-pNA 1.25 mM zL -Tyr-pNA was used.

OD values were measured at 410 nM using a 96-well plate.

Α-Secretase inhibition activity was measured by fluorescence ELISA using recombinant TACE (0.1 ppm in 25 mM Tris buffer), samples, and substrate at 25 ° C for 1 hour, The fluorescence intensity was measured at Em 450 nM and the inhibition rate was calculated in the same manner as the inhibition of the activity of the beta-secretase of Experimental Example 2.

(2) Experimental results

The enzyme specificities of beta-secretase of representative long-chain fatty acids (oleic acid, palmitic acid, palmitoleic acid, linoleic acid) of long-lived beetle fractions of Example 1 and Experimental Example 3 were examined, appear.

Conc. (UM)
(density) Trypsin
(Trypsin) Chymotrypsin
(Chymotrypsin) a-Secretase
(Alpha-
Secrecy) Hexane
(Hexane) 50 2.61 ± 0.65 3.57 + - 0.10 5.13 ± 1.22 100 1.73 + - 0.40 7.29 ± 1.54 10.73 ± 0.40 EtOAc
(Ethyl acetate) 50 1.89 0.90 4.06 ± 0.59 8.01 + - 1.68 100 1.90 ± 1.40 8.18 ± 2.40 13.47 ± 3.03 H ₂ O (water) 50 5.99 ± 0.87 2.03 ± 0.10 8.28 ± 0.99 100 7.38 ± 0.97 14.64 ± 2.11 14.21 + - 2.54 Oleic acid
(Oleic acid) 50 5.50 ± 1.97 5.00 ± 0.19 8.31 ± 1.73 100 3.71 ± 1.97 9.45 + 0.39 8.62 ± 1.31 Palmitic acid
(Palmitic acid) 50 5.36 + 0.11 7.17 ± 0.49 3.83 ± 0.22 100 11.18 ± 1.32 3.25 + - 0.99 9.34 ± 3.51 Palminoleic acid
(Palmitoleic acid) 50 8.28 ± 1.41 2.46 0.41 4.27 ± 0.62 100 9.57 ± 0.81 2.95 ± 0.22 5.01 ± 0.65 linoleic acid
(Linoleic acid) 50 8.62 ± 1.21 3.41 0.30 5.34 ± 1.72 100 8.54 0.44 1.62 0.35 11.62 + - 1.95

As shown in Table 2, no significant inhibitory effect on the chymotrypsin, trypsin and alpha-secretase of the long-lived beetle fractions and major fatty acids of the present invention is shown, It was confirmed that scarab was an inhibitor with selective specificity for beta-secretase.

The present invention relates to a pharmaceutical composition for preventing and treating Alzheimer's dementia which inhibits Alzheimer's dementia through inhibition of β-Secretase activity by containing Allomyrina dichotoma or fractions derived from the extract thereof as an active ingredient. It is possible to provide a composition and a food composition for improving Alzheimer's dementia.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be possible. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

Claims (11)

Which comprises as an active ingredient an allomyrina dichotoma or a fraction derived from an extract thereof,
A pharmaceutical composition for preventing and treating Alzheimer's dementia.
The method according to claim 1,
Wherein the fraction comprises at least one of a hexane fraction or an ethyl acetate (EtOAc) fraction obtained by extracting the long-lived beetle with ethanol and fractionating the same.
A pharmaceutical composition for preventing and treating Alzheimer's dementia.
3. The method of claim 2,
Wherein said fraction exhibits an ability to inhibit the activity against beta-secretase, including any one or more of oleic acid, palmitic acid, palmitoleic acid, and linoleic acid.
A pharmaceutical composition for preventing and treating Alzheimer's dementia.
Which comprises as an active ingredient an allomyrina dichotoma or a fraction derived from an extract thereof,
A food composition for improving Alzheimer's dementia.
5. The method of claim 4,
Wherein the fraction comprises at least one of a hexane fraction or an ethyl acetate (EtOAc) fraction obtained by extracting the long-lived beetle with ethanol and fractionating the same.
A food composition for improving Alzheimer's dementia.
6. The method of claim 5,
Wherein said fraction exhibits an ability to inhibit the activity against beta-secretase, including any one or more of oleic acid, palmitic acid, palmitoleic acid, and linoleic acid.
A food composition for improving Alzheimer's dementia.
A first step of pulverizing the lyophilized long beetle;
A second step of preparing a long beetle extract solution by mixing the long-lived beet pulverized in the first step with a solvent;
A third step of adding a solvent consisting of water and hexane to the long beetle extract solution prepared in the second step, and then separating only the hexane layer (hexane);
Adding a solvent consisting of water and ethyl acetate (EtOAc) to the extract of the long beetle produced in the second step, and then separating only EtOAc with ethyl acetate.
A method for producing long-lived beetle fractions having an anti-Alzheimer's effect.
8. The method of claim 7,
Wherein the long-life beetle extract of the second stage is prepared by mixing 0.5 to 1 kg of the pulverized long beetle beetle per 12 liters of the solvent.
A method for producing long-lived beetle fractions having an anti-Alzheimer's effect.
8. The method of claim 7,
Wherein the solvent in the second step is 70-80% ethanol.
A method for producing long-lived beetle fractions having an anti-Alzheimer's effect.
9. An anti-Alzheimer's composition comprising as an active ingredient a long-lived beetle fraction produced by the method of any one of claims 7 to 9.
The anti-Alzheimer's composition of claim 10 is used as a food composition for prevention and improvement of Alzheimer's disease caused by beta-secretase or a pharmaceutical composition for prevention and treatment of Alzheimer's disease,
A method of using an anti-Alzheimer's composition.

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