JP2008156240A - Cerebral function-improving agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cerebral function-improving agent which can effectively improve memorizing and learning ability disorders in dementia. <P>SOLUTION: This cerebral function-improving agent is characterized by containing the specific ingredient of Ganoderma atrum. The specific ingredient is a substance exhibiting the maximum absorption peak at 280 to 330 nm and containing an aromatic ring in the molecule. The solution of the ethyl acetate-soluble content of this ingredient is colorable. This ingredient has an action for inhibiting acetylcholine esterase and prolyl endopeptidase, and is useful for improving cerebral functions. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a pharmaceutical, a quasi-drug or a food comprising a specific component of black ganoderma having a brain function improving action.

  In today's aging society, the increase in senile dementia patients has become a major social problem. Symptoms of dementia vary from person to person, but common symptoms include memory injury, disorientation, reduced judgment, thinking, and executive function.

  Dementia can be broadly classified into Alzheimer type dementia and cerebrovascular dementia according to the cause. For example, cerebral vascular dementia occurs when brain blood vessels are clogged by cerebral infarction and brain tissue is destroyed. Therefore, in cerebrovascular dementia, it is important to treat and prevent arteriosclerosis, hypertension, hyperlipidemia, diabetes, etc. that caused cerebral infarction, and treatment methods that delay the progression of dementia through this are taken. It has been.

On the other hand, the cause of Alzheimer-type dementia, which is said to occupy most dementia patients, has not been clearly elucidated, but the patient has a decreased level of acetylcholine, a neurotransmitter in the brain. Therefore, it is considered that one of the causes is a decrease in the function of cholinergic nerves (see, for example, Non-Patent Document 1). Therefore, in Alzheimer-type dementia, treatment methods aiming at preventing the functional deterioration of cholinergic nerves by increasing the concentration of acetylcholine have become mainstream. In other words, since acetylcholine is synthesized by acetylcholine transferase and decomposed by acetylcholinesterase and maintained at a constant concentration, the activity of acetylcholinesterase is inhibited to suppress the degradation of acetylcholine and to reduce the decrease of acetylcholine. It is a method to suppress. Although this method cannot completely treat symptoms, it has been found that symptoms can be improved or the progression of symptoms can be delayed.
Neurodegeneration, 4, 349-356 (1995)

  Currently, for example, acetylcholinesterase inhibitors such as tacrine and donepezil are commercially available as therapeutic agents for Alzheimer-type dementia.

  However, the above-mentioned acetylcholinesterase inhibitors such as tacrine and donepezil have problems that they cannot be taken for a long period of time and are expensive because of their liver toxicity and strong side effects.

In addition, research has been conducted to search for components that inhibit acetylcholinesterase from natural products and to treat dementia (Patent Documents 1 and 2).
JP 2006-131589 A JP 2003-286164 A

Prolyl endopeptidase is an enzyme that specifically cleaves the carboxyl side of proline in peptides containing proline, and has high activity in the hippocampal part of the brain, which is a fixed place of memory, and it is widely distributed in animal organs Is known (Non-Patent Documents 2 to 4). Similar enzymes have also been discovered in Flavobacterium bacteria (Non-Patent Document 5).
Science, 173, 827 (1971) Molecular & Cellular Biochemistry, 30, 111 (1980) Japanese Journal of Agricultural Chemistry, 58, 1147 (1984) J. et al. Biol. Chem. , 255, 4786 (1980)

This enzyme is said to have memory retention activity in substance P and neurotensin, which are considered to be neurotransmitters, THR (Thyroid Stimulating Hormone), which is thought to be related to memory, and the step-through passive avoidance learning method It is known to act on brain vasopressin and inactivate it (Non-Patent Documents 6 and 7). Vasopressin is a peptide hormone that plays a role in water reabsorption in the kidney, but it is also involved in the learning and memory processes in the brain, and it is said that memory retention is impaired when the resolution of this hormone is inactivated. ing. Moreover, it is known that the amount of vasopressin in patients with dementia is less than that of normal persons (Non-patent Document 8).
Science, 221, 1310 (1983) Nature, 308, 276 (1984) BIOINDUSTRI, 4,788 (1987)

In healthy individuals, PEP in the brain works normally, but if the regulatory mechanism is lost for some reason, vasopressin is degraded more than necessary, and memory retention appears injured. Therefore, research aimed at treating dementia by inhibiting the activity of this enzyme has been made (Patent Documents 3 to 7).
Japanese Patent No. 31483939 JP 2006-28091 A JP 2006-96709 A JP 2002-265377 A JP 2001-64197 A

Black ganoderma belongs to the genus Amanentake, a member of the family Basidiomycete, Amanitatake. Red ganoderma (Ganoderma lucidum) is common as a representative of Mannentake, and it is described in Chinese ancient pharmacology book “Shinnohon Honsokei” as “increase wisdom and forget it” (Non-patent Document 9) . In addition, it has been recognized that learning and memory functions are enhanced and maintained by long-term administration of red ganoderma (Non-patent Document 10). However, no report has been made on the effect of black ganoderma on improving brain function.
Namba Tsuneo "Primary Japanese and Chinese medicine picture book (below)" Childcare, 1980 Journal of the Japan Society for Pharmaceutical Sciences 7,388 (1990)

  In general, there are red ganoderma (ganoderma), white ganoderma, yellow ganoderma, purple ganoderma, blue ganoderma as well as black ganoderma (black turf). Although it is simply named because it is white, yellow, purple, blue and black, recently, black ganoderma has been studied separately from red ganoderma and purple ganoderma. For example, according to Non-Patent Document 2, it is reported that black ganoderma does not contain a typical triterpene component (ganoderic acid) contained in red ganoderma or purple ganoderma and has a completely different taste. From the above, there was room for further research on the medicinal effects of black ganoderma.

  Also, the brain function improving effect of a specific component contained in black ganoderma which has a different scientific name and component from red ganoderma has never been known.

  An object of the present invention is to obtain a brain function improving agent having inhibitory activity against acetylcholinesterase and prolyl endopeptidase.

  Under these circumstances, the present inventors have intensively studied and found that specific components mainly contained in black ganoderma have a very high inhibitory action on acetylcholinesterase and prolyl endopeptidase, and experimental dementia. In an animal experiment using a symptom model, it has been found that it has a high memory learning ability improving effect, and the present invention has been completed.

That is, the specific component of black ganoderma included in the brain function improving agent of the present invention is characterized by the following matters.
(1) When an infrared absorption spectrum is measured (KBr method), absorption maximums are shown at 2925, 1700, 1600, 1455, 1170, and 835 cm ⁻¹ .
(2) When the above-mentioned black ganoderma biloba components are dissolved in ethanol and the ultraviolet absorption spectrum is measured, the absorption maximum is shown at 280 to 330 nm.
(3) An aromatic ring is contained in the molecule.
(4) When ethyl acetate is added to this component, the ethyl acetate-soluble solution is colored and mainly brown to reddish brown.

Among the above, (3) can be distinguished by ¹ H, ¹³ C-NMR analysis. That is, when the solvent was measured using deuterated methanol (CD ₃ OD), spectral absorption of hydrogen atoms substituted with aromatics at 6 to 8 ppm ( ¹ H-NMR) and 100 to 150 ppm ( ¹³ C-NMR) was recognized. It is done. The above spectral analysis values may have some numerical errors depending on the analyzer, method, peak width, and the like. About (4), coloring property can be discriminate | determined by adding 1 mL of ethyl acetate to about 1-10 mg of this component.

  Moreover, the specific component of black ganoderma has the property of liberating hydroxycinnamic acid when hydrolyzed by a conventional method. For example, it can be hydrolyzed by a conventional method using an aqueous sodium hydroxide solution and can be confirmed by analysis with HPLC or the like.

The specific component of black ganoderma can be confirmed by the high performance liquid chromatograph (HPLC) shown below (see FIG. 1). The HPLC chart of the specific component of black reishi is shown. The horizontal axis represents the retention time (minutes), and the vertical axis represents the intensity. According to this analysis, a broad peak group having a retention time of about 10 to 25 minutes is a specific component of black ganoderma (the retention time and pattern may vary slightly depending on the analysis conditions).
(A) Column: Octadecylated silica gel (inner diameter 4.5 mm × 250 mm)
(B) Column temperature: 40 ° C
(C) Developing solvent: 0.2% phosphoric acid / 0.2% phosphoric acid-containing acetonitrile was used, and 0.2% phosphoric acid-containing acetonitrile was linearly changed from 20% to 100% over 20 minutes. Change solvent and hold at 100% for 25 minutes (total 45 minutes analysis).
(D) Flow rate: 1 mL / min (E) Detection: 313 nm
(F) Sample: 0.02 mL of 10 mg / mL ethanol solution introduced

  The black ganoderma used in the present invention belongs to the family Ganodermaaceae and Ganoderma, and the scientific name is G. atrum, G. et al. japonicum, G. et al. It is said to be sinsense. In addition, for mushrooms belonging to the genus Amanita, there are old Chinese pharmacy books, “Honcho Tsuname” and “Shinnohonsoku”, in addition to black ganoderma (Kuroshiba), red ganoderma (ganoderma), purple ganoderma ( Purple turf), blue ganoderma (blue turf), yellow ganoderma (yellow turf) and white ganoderma (white turf) are described. Black reishi can be widely distributed in China, the Japanese market, etc., or it can use native or cultivated products.

  In addition, black ganoderma has a similar appearance to purple ganoderma, so it is sometimes marketed as purple ganoderma. In this case, for example, by performing high performance liquid chromatography (HPLC) analysis under the above conditions, it is possible to detect a specific component of black ganoderma and belongs to the scope of the present invention.

  The black ganoderma used in the present invention can be used regardless of fruiting bodies, mycelium, natural products, cultivated products, cultured products and the like. Moreover, the state as it is, a crushed material, a dried material, etc. can be selected suitably as needed, and it can attach | subject to extraction operation.

  As an example of the method for extracting medicinal components of the present invention, it can be preferably obtained by solvent extraction of the fruit body of black ganoderma.

  Examples of the solvent to be extracted include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol). ), Ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, petroleum ether, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.) Etc.). Preferably, lower alcohols, ketones, acetonitrile, esters, and the like and water-containing solvents thereof are preferable, and ethanol or water-containing ethanol is particularly preferable from the viewpoint of safety. These solvents may be used alone or in combination of two or more. It is also preferable to extract under basic conditions.

  As a method for further purifying specific components of black ganoderma, solvent fraction purification can also be performed by liquid / liquid partition extraction with solvents that are not mixed with each other.

  As the solvent used for liquid / liquid partition extraction, the above-mentioned solvents can be used. For example, (water or water-containing alcohol) / hydrocarbon type, (water or water-containing alcohol) / ether type, (water or water-containing alcohol) / ester type, water / butanol type, etc. can be employed. It is not limited to the above, and any solvent system that does not mix with each other can be adopted. Preferably, extraction is performed by adding water-containing lower alcohol and hydrocarbon solvent to the solvent extract (solid), and adding the ester solvent to the remaining liquid from which the hydrocarbon solvent soluble matter has been removed, to identify black reishi. The components can be purified. Moreover, fractional extraction with an acid or a base can also be performed.

  As a method for further purifying the specific component of black ganoderma, the specific component can be purified by adsorbing the black ganoderma extract or the above solvent fraction to an adsorbent and eluting it with a solvent.

  Examples of the adsorbent include adsorbents such as styrene-divinylbenzene synthetic adsorption resin, ion exchange resin, octadecylated silica gel (ODS), silica gel, cellulose, dextran, and modified dextran. Of these, styrene-divinylbenzene synthetic adsorption resin and ODS are preferable.

  Examples of the adsorption method include, but are not particularly limited to, a method in which an adsorbent is packed in a column and the extract solution is passed through the column, and a method in which the adsorbent is directly mixed with the extract solution.

  The elution solvent for the adsorbent can be selected depending on the characteristics of each adsorbent, and includes, for example, water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid many Monohydric alcohol (1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, petroleum ether, etc.) ), Ethers (ethyl ether, tetrahydrofuran, propyl ether, etc.). A solvent such as water or a lower alcohol is preferable, and water or ethanol is particularly preferable. These solvents may be used alone or in combination of two or more. Furthermore, the pH may be adjusted by adding acid or alkali to the elution solvent.

The above extract or purified product may be used as it is, or may be used after treatment such as concentration, dilution, filtration, decolorization with activated carbon, deodorization, ethanol precipitation, etc., if necessary. Further, the extracted or purified solution may be subjected to a treatment such as concentration to dryness, spray drying, or freeze drying, and used as a dried product.

  As a particularly preferred example, after performing the above liquid / liquid partition extraction (extraction with the above preferred solvent system), the above synthetic adsorption resin (styrene-divinylbenzene system, the above preferred solvent). The specific components of black ganoderma can be purified by elution).

  However, the specific component of black ganoderma may be used as an extract according to the purpose of use, or liquid / liquid partition extraction and purification with an adsorbent can be used alone or in combination.

  As the brain function improving agent of the present invention, a purified product such as the above extract or adsorbent may be used as it is, and a diluent can be used as long as the effect of the extract is not impaired. May be solid, liquid or semi-solid, for example: That is, excipients, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, fragrances, preservatives, solubilizers, solvents, and the like. Specifically, lactose, sucrose, sorbit, mannitol, starch, precipitated calcium carbonate, heavy magnesium oxide, talc, calcium stearate, magnesium stearate, cellulose or derivatives thereof, amylopectin, polyvinyl alcohol, gelatin, surface activity Agents, water, physiological saline, ethanol, glycerin, propylene glycol, cacao butter, laurin butter, petrolatum, paraffin, higher alcohol and the like.

  The brain function improving agent of the present invention can be used for any of pharmaceuticals, quasi drugs, and foods. Examples of the dosage form include powders, granules, tablets, sugar-coated tablets, capsules for oral use, Syrups, pills, suspensions, solutions, emulsions, etc. It can be an injection solution for parenteral use. It can also be a suppository.

  The intake of the brain function improving agent of the present invention varies depending on the administration form, purpose of use, age, weight, etc., but is 0.1 to 5,000 mg in terms of an extract or purified product containing specific components of black ganoderma. / Day, preferably 1 to 500 mg / day orally once to several times a day. Of course, as described above, since the administration method and dose vary depending on various conditions, an amount smaller than the above-mentioned administration range may be sufficient, and administration may be performed beyond the range. In addition, the method for adding the brain function improving agent in the formulation may be added in advance or during the production, and may be appropriately selected in consideration of workability.

  The brain function improving agent containing the specific component of the new black ganoderma of the present invention has a very high brain function improving action.

In order to describe the present invention in detail, examples of production of the extract used in the present invention, formulation examples of the present invention, and experimental examples are given as examples, but the present invention is not limited thereto. The part of the amount shown in the examples means part by weight, and% means% by weight.

Production Example 1 Black Lingzhi Hot Water Extract Add 2 L of purified water to 100 g of dried black reishi fruit bodies, extract at 95-100 ° C. for 2 hours, filter, concentrate the filtrate, and freeze-dry As a result, 5.4 g of a hot water extract of black ganoderma was obtained.

Production Example 2 50% Ethanol Extract of Black Reishi Shiba Add 1L of purified water and 1L of ethanol to 100g of dried Kuro Reishi fruiting bodies, extract at room temperature for 7 days, filter, concentrate the filtrate and freeze-dry As a result, 2.9 g of 50% ethanol extract of black ganoderma was obtained.

Production Example 3 Black Ganoderma Ethanol Extract Add 1 L of black ganoderma fruit body to 15 kg of ethanol, extract at room temperature for 7 days, filter and concentrate the filtrate to dryness. 20 g of extract 1 was obtained.

Production Example 4 Purified Column of Black Reishi 1
10.0 g of the hot water extract of the black reishi fruit body of Production Example 1 was dissolved in purified water and passed through a column filled with 500 mL of a styrene-divinylbenzene synthetic adsorption resin. Subsequently, after eluting with 1 L each of purified water, 10, 20, 50, 80, 90, and 100% ethanol, each was concentrated to dryness. The specific components of black ganoderma are contained in a large amount in the ethanol fraction of 80% or more, and the ethanol fractions of 80% or more were collected to obtain about 0.40 g of purified column product of black ganoderma.

Production Example 5 Purified Solvent Fraction of Black Ganoderma 2.00 g of Kuro Ganoderma Ethanol Extract of Production Example 3 was dissolved in 350 mL of ethanol and extracted with the same volume of purified water and hexane. Concentrate the aqueous layer to ½, extract by adding the same volume of ethyl acetate, extract and concentrate to dryness to obtain 0.84 g of hexane fraction, 0.56 g of ethyl acetate fraction, and 0.60 g of water fraction. It was. Among these, the ethyl acetate fraction is used as a purified solvent fraction of Kuro Reishi.

Production Example 6 Purified HPLC Product of Black Ganoderma 0.50 g of Kuro Ganoderma Ethanol Extract of Production Example 3 was purified by preparative HPLC to obtain 0.21 g of purified Kuro Ganoderma HPLC. The HPLC fraction range was the range described in the paragraph “0019”.
<HPLC fractionation conditions>
(A) Filler: ODS (inner diameter 20 mm x length 250 mm)
(B) Developing solvent Using acetonitrile containing 0.2% phosphoric acid / 0.2% phosphoric acid, acetonitrile containing 0.2% phosphoric acid is linearly solvent from 20% to 100% over 20 minutes. And hold at 100% for 25 minutes.
(C) Flow rate: 10 mL / min (D) Detection: 350 nm

Production Example 7 Purified Column 2 of Black Reishi
2.0 g of the ethanol extract of Black Reishi in Production Example 3 was dissolved in 350 mL of ethanol and extracted by adding the same volume of purified water and hexane. The aqueous layer portion was concentrated to 1/2, extracted with the same volume of ethyl acetate, and then concentrated to dryness to obtain 0.56 g of an ethyl acetate fraction. This was dissolved in 50% ethanol and passed through a column packed with 100 mL of a styrene-divinylbenzene synthetic adsorption resin. Next, after eluting 1 L each of purified water, 10, 20, 50, 80, 90, and 100% ethanol, 80% or more ethanol fractions were combined to obtain 0.24 g of Kuro Reishi column purified product 2. .

<Spectral analysis of Production Examples 6 and 7>
Infrared absorption spectrum analysis Infrared absorption spectra were measured by the KBr method for the purified product of Kuro Reishi in Preparation Example 6 and the purified product of Kuro Reishi column in Preparation Example 7. As a result, the absorption maximum was shown at 3294, 2926, 1698, 1604, 1456, 1169, and 823 cm ⁻¹ .
Ultraviolet Absorption Spectrum Analysis 5 mg of the purified product of black reishi from Production Example 6 and the purified product of black reishi from Production Example 7 was dissolved in 100 mL of ethanol, and the ultraviolet absorption spectrum was measured. As a result, the maximum of absorption was shown at 295.3 nm.
NMR spectrum analysis As a result of ¹ H, ¹³ C-NMR analysis by a conventional method using the HPLC purified product of Kuro Reishi in Production Example 6 and the purified product of Kuro Reishi in Production Example 7, 6.6 to 7.6 ppm Spectral absorption was observed at ( ¹ H-NMR), 100 to 150 ppm ( ¹³ C-NMR), and the presence of an aromatic ring was confirmed. Note that deuterated methanol (CD ₃ OD) was used as the solvent.
Color of Solution 5 mL of ethyl acetate was added to 10 mg of the HPLC purified product of Black Reishi in Preparation Example 6 and the purified product of Black Reishi in Preparation Example 7. After the ethyl acetate soluble component was filtered, the color of the solution was visually confirmed. As a result, the color of the solution was reddish brown.

Comparative Production Example 1 Red Ganoderma Hot Water Extract To 100 g of dried red ganoderma fruit body, 2 L of purified water was added, extracted at 95-100 ° C. for 2 hours, filtered, and the filtrate was concentrated and frozen. After drying, 6.0 g of hot water extract of red ganoderma was obtained.

Comparative Production Example 2 Purified Solvent Fraction of Red Ganoderma The same procedure was carried out except that the black ganoder of Production Example 5 was replaced with red ganoderma turf to obtain 0.64 g of ethyl acetate fraction (purified solvent fraction).

  The brain function improving agent of the present invention can be formulated as the following formulation examples.

Formulation Example 1 Powder Formulation Formulation 1. 1. Purified HPLC product of Kuro Reishi (Production Example 6) 20 parts Dried corn starch 30
3. Microcrystalline cellulose 50
[Production method] Components 1 to 3 are mixed to obtain a powder.

Formulation Example 2 Tablet formulation Formulation amount 1. Purified column 2 of Kuro Reishi (Production Example 7) 5 parts Dried corn starch 25
3. Carboxymethylcellulose calcium 20
4). Microcrystalline cellulose 40
5. Polyvinylpyrrolidone 7
6). Talc 3
[Manufacturing method] Components 1 to 5 are mixed, then 10% water is added as a binder, dried after extrusion granulation. Ingredient 6 is added to the molded granules, mixed and compressed into tablets. One tablet is 0.52 g.

Production Example 3 Tablet Confection Formulation Ethanol extract of black reishi (Production Example 3) 1.5 parts Dried corn starch 50.0
3. Erythritol 40.0
4). Citric acid 5.0
5. Sucrose fatty acid ester 3.4
6). Fragrance 0.1
[Production Method] Components 1 to 4 are mixed, and 10% water is added as a binder to increase the size of the fluidized bed. Ingredients 5 and 6 are added to the formed granules, mixed and compressed into tablets. One tablet is 1.0 g.

Formulation Example 4 Beverage Formulation Purified solvent fraction of Kuro Reishi (Production Example 5) 0.05 part2. Stevia 0.05
3. Malic acid 5.0
4). Glycerin fatty acid ester 0.5
5. Fragrance 0.1
6). Water 94.3
[Manufacturing method] Components 1 to 5 are stirred and dissolved in a part of the water of component 6. The remaining water of component 6 is then added and mixed.

Next, experimental examples will be given to explain the effects of the present invention in detail.

Experimental Example 1 Acetylcholinesterase inhibition test The activity inhibitory action of acetylcholinesterase (hereinafter referred to as AChE) was tested. The measurement of AChE inhibitory activity uses rat brain homogenate as an enzyme, acetylthiocholine as a substrate, and DTNB (5,5-dithio-bis (2-nitrobenzonic acid) as thiocholine released from the substrate by the action of AChE. ) Was reacted by measuring the absorbance of TNB (5-thio-2-nitrobenzoic acid) at 412 nm.

  0.05 mL of AChE dissolved in 0.54 mL of 50 mM sodium phosphate buffer (pH 7.0, hereinafter referred to as phosphate buffer), 0.01 mL each sample dissolved in DMSO (dimethyl sulfoxide), 2.5 mM acetylthioiodide 0.2 mL of choline phosphate buffer solution and 0.2 mL of 1.5 mM DTNB phosphate buffer solution were added and reacted at 37 ° C., and the slope of absorbance at 412 nm per minute (SX) was calculated.

  The reaction was performed in the same manner as described above using 0.01 mL of phosphate buffer instead of AChE, and the absorbance gradient (SXB) was calculated.

  The reaction was performed in the same manner as described above using 0.01 mL of DMSO instead of the sample solution, and the absorbance gradient (SC) was calculated.

  The reaction was carried out in the same manner as above using 0.01 mL of DMSO instead of the sample solution and 0.01 mL of phosphate buffer instead of AChE, and the absorbance gradient (SB) was calculated.

Evaluation of the AChE inhibition rate of each sample was calculated from the obtained slopes by the following formula.

AChE inhibition rate (%) = [1- (SX-SXB) / (SC-SB)] x 100

  As a result, as shown in Table 1, high specific acetylcholinesterase inhibitory activity was recognized as compared with other hot water and ethanol extracts as specific components of black ganoderma.

Experimental Example 2 Prolyl Endopeptidase Inhibition Test The activity inhibitory action of prolyl endopeptidase (hereinafter referred to as PEP) was tested. The measurement of PEP inhibitory activity was carried out using the enzyme Flavobacterium (Flavobacterium).
PEP derived from meningoseepticum) and Z-Gly-Pro-pNA as a substrate were used, and the absorbance of p-NA (paranitroaniline) released from the substrate was measured at 410 nm by the esterase action of PEP. The final concentration of each sample used in this measurement method was 0.2 mg / mL.

  To 0.1 mL of 0.1 M sodium phosphate buffer (pH 7.0), 0.125 mL of a sample dissolved in a 40% dioxane solution and 0.125 mL of 2 mM Z-Gly-Pro-pNA are added, and the mixture is added at 30 ° C. for 5 minutes. Preincubated. After 5 minutes, 0.1 mL of PEP (0.175 U / mL) dissolved in 0.05 M sodium phosphate buffer was added, and the reaction was carried out for 10 minutes. Thereafter, the reaction was stopped with 2.0 mL of Triton X-100 solution, and the absorbance at 410 nm was measured.

In addition to the above operation (A), the PEP inhibition rate was evaluated by adding (B) Triton X-100 solution before the enzymatic reaction for control of A, and (C) 40% dioxane solution instead of the sample solution. (D) The Triton X-100 solution was added before the enzyme reaction for the control of C, and the calculated value of the absorbance at each stage was substituted into the following equation.

PEP inhibitory activity (%) = [(C−D) − (A−B)] / (C−D) × 100

  As a result, as shown in Table 2, the specific component of black ganoderma was found to have a higher PEP inhibitory activity than other hot water and ethanol extracts.

Experimental Example 3 Morris Water Maze Test The memory / learning improvement effect of the present invention was examined by the Morris water maze test.

  A platform having a diameter of 5 cm and a height of 10 cm was installed in a water tank having a diameter of 120 cm and a height of 20 cm, and the surface of the water was made 1 cm higher than the platform, and was made cloudy with skim milk. The water temperature was adjusted to 18 ± 1 ° C., and all positions were fixed during the test.

  Four points on the edge of the aquarium were arbitrarily designated as north, south, east, and west. The mouse was swam from each point, and the time to reach the platform was measured up to 90 seconds. Trials were performed four times, one from each direction, and the trial for the day was completed. When the mouse reached its own strength, the mouse was left as it was for 15 seconds, and one trial was completed. If the mouse could not reach the platform after 90 seconds, the experimenter guided the mouse to the platform and left it on it for 15 seconds to complete one trial. The interval between the four trials was 2 minutes, and the average value of the arrival times of 4 trials per day was used as the data for one day.

  After performing the training trial for 2 days, the trial was performed without administering the drug on the 3rd day, and the arrival time and average body weight of each group were made uniform. On the 4th and 5th days, scopolamine (muscarinic acetylcholine receptor antagonist) 1 mg / kg was intraperitoneally administered 30 minutes before the trial, and the test trial was conducted under conditions that induced memory / learning impairment. The effect of improving the memory / learning disorders of the samples was evaluated. The sample suspended in distilled water was orally administered 60 minutes before the trial, and the same amount of distilled water was administered to the control group.

  Table 3 shows the arrival time for each sample administration on the fifth day of the test. As a result, the specific component of black ganoderma was found to significantly shorten the swimming time compared to other hot water and ethanol extracts.

  The brain function improving agent of the present invention showed high acetylcholinesterase inhibitory action and prolyl endopeptidase inhibitory action. Therefore, it is expected to be used as a pharmaceutical, quasi-drug or food having a safe and excellent brain function improving action.

The HPLC chart of the specific component of black reishi is shown. The horizontal axis represents the retention time (minutes), and the vertical axis represents the intensity.

Claims (5)

A brain function improving agent containing a specific component extracted from black ganoderma and having the physical properties of (1) to (4).
(1) When the infrared absorption spectrum is measured (KBr method), the absorption maximum is shown at 2925, 1700, 1600, 1455, 1170, 835 cm ⁻¹ ,
(2) When this component is dissolved in ethanol and the ultraviolet absorption spectrum is measured, the absorption maximum is shown at 280 to 330 nm.
(3) contains an aromatic ring in the molecule,
(4) When ethyl acetate is added to this component, the ethyl acetate-soluble solution is colored and exhibits brown to reddish brown color. The brain function improving agent according to claim 1, comprising a solvent extract of black ganoderma. The solvent extract of Kuro Reishi is a solvent fraction purification by liquid / liquid partition extraction. As a method of liquid / liquid partition,
(1) Extract the solvent extract (solid) by adding a hydrous lower alcohol and a hydrocarbon solvent,
(2) An ester solvent is added to the residual liquid from which the hydrocarbon solvent-soluble component has been removed, followed by extraction.
(3) contained in the ester layer,
The brain function improving agent according to any one of claims 1 to 2, comprising a component extracted by the above operation. Adsorbing a solvent extract of Kuroshikishiba or its solvent fraction purified product selected from styrene-divinylbenzene synthetic adsorption resin, ion exchange resin, octadotesilated silica gel (ODS), silica gel, cellulose, dextran, and modified dextran The brain function improving agent according to any one of claims 1 to 3, comprising a component purified by an agent. A pharmaceutical, quasi-drug, or food comprising the brain function improving agent according to any one of claims 1 to 4.

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