JP6447677B2 - Composition for preventing and / or treating cognitive impairment - Google Patents

Description

  The present invention relates to a composition for prevention and / or treatment of cognitive dysfunction, and more specifically, based on the novel anti-aggregation action of amyloid β protein and antioxidant action (hydroxyl radical (· OH) scavenging ability), The present invention relates to a composition for preventing and / or treating cognitive dysfunction that can be used in the fields of medicine, food and the like.

  In Japan, which has reached a super-aging society, the most worried about medical and medical economics are Alzheimer's disease (hereinafter abbreviated as “AD”) and cerebrovascular dementia (hereinafter abbreviated as “VD”). There is an increase in patients with dementia. Since the biggest cause of dementia is “aging / aging”, an increase in the number of patients with dementia is inevitable with the aging of society.

  AD is estimated to be about 30-50% of dementia, and the proportion is gradually increasing in developed countries. In the United States, the proportion of so-called late elderly AD patients is said to increase from 40% in 2000 to 60% in 2050. Based on surveys conducted in various regions in Japan and future estimates made mainly by Director Toshio Otsuka of Tokyo's Musashino Hospital, the proportion of AD patients among all elderly people over the age of 65 was in the 1980s. It is estimated that it will increase from 1.8% to 4.4% in 2000 and to 6.5% in 2020.

  If this rate is added to the increase in the elderly population, the number of AD patients over 65 years old was approximately 1 million in 2000, but in 2020 it will be approximately 2.5 million, a 2.5-fold increase. Currently, more than one in 25 people over the age of 65 may be AD, but in 20 years it is expected to be about 1 in 15 people.

  The pathological features of the disease include senile plaque formation, abnormal tau protein phosphorylation and neurofibrillary tangles. Among these, senile plaques are formed by aggregation and accumulation of amyloid protein (hereinafter abbreviated as “Aβ protein”). It has been reported that accumulation of this protein generates active oxygen, destroys nerve cells, and causes brain atrophy. Moreover, it is known that aggregation of Aβ protein occurs several decades before the symptoms of Alzheimer's disease are observed. The current Alzheimer's disease therapeutic drugs are mainly neurostimulatory and are only coping therapy, and their effects are extremely limited.

  On the other hand, from the viewpoint of early treatment, medical associations are increasingly aware of the need for accurate diagnosis and the need for drugs with a preventive effect at the time of mild cognitive impairment, which is said to be the precursor stage of AD.

  From such a viewpoint of prevention, there has been a long-standing interest in Europe and the United States, and AD prevention effects from the restriction of calorie intake of vegetables / fruits, fish, and total intake have been accepted as such. Vitamin C and E, phytochemical antioxidants that are non-nutrients, and ω3 PUFAs such as EPA and DHA are also useful and are also sold in Japan.

EPA, DHA, rosemary, rice bran, etc. are known as natural materials that are currently promising for the prevention and improvement of cognitive function in humans (Non-Patent Documents 1 to 7). Naturally, natural antioxidants such as curcumin, quercetin, myricetin, wine polyphenol, and ferulic acid are known (Non-patent Documents 8 and above), and the patents are grape vines, peony skin extract, and cinnamon. Natural antioxidants such as extracts, onji (long-distance), licorice (licorice), catechin and green tea extract, procyanidin B _{2 and the} like are known (patent documents 1 to 9). However, it is also strongly desired to find a compound useful for the prevention / improvement of cognitive function or a substance having an Aβ aggregation inhibitory action and to examine its applicability to medicines.

JP 2006-206584 A Japanese Patent Laid-Open No. 10-226668 JP 2005-350391 A JP-A-1-246224 JP-A-1-316313 Special table 2003-519192 gazette JP-T-2006-507357 JP 2007-230938 A Japanese Patent Laid-Open No. 9-30984

J. Neurosci, 25, 3032-3040 (2005) J. Neurochem, 107, 1634-1646 (2008) J. Neurochem, 111,568-579 (2009) JAMA, 287,3223-3239 (2002) J. Neurochem, 90, 758-764 (2004) Am.J.Med., 119,751-759 (2006) J. Neurosci., 25,8807-8814 (2005) J. Bio. Chem., 280, 5922-5901 (2005) J. Neurochem., 87,172-181 (2003) J. Neurosci. Res., 75,742-750 (2004) Biochemistry, 45,6085-6094 (2006) Neurosci.Lett., 444,280-285 (2008) Tetrahedron, 34, 1389-1397 (1978) Journal of Chromatography, A (2008), 1185 (1), 117-129

  The present invention has been made under such circumstances, and screened for compounds that inhibit Aβ aggregation, mainly natural products, to find a safe substance having an AD preventive effect, and to contain cognitive dysfunction containing the same. The object is to provide a composition for prevention and / or treatment.

  The present inventors have studied the pharmacological action of flavonoid compounds in order to solve the above-mentioned problems. As a result, they have found several compounds having an AD preventive effect and completed the present invention.

（式中、Ｒ_１は水素原子またはグルコピラノシル基を示し、Ｒ_２は水素原子またはラ
ムノシルオキシ基を示す）

（式中、Ｒ_３は水素原子または水酸基を示す）

（式中、Ｒ_４はアラビノシル基またはラムノシル基を示す）
で表される化合物の一種または二種以上を有効成分として含有する認知機能障害の予防及び／または治療用組成物である。 That is, the present invention provides the following formulas (I) to (III):

(Wherein R ₁ represents a hydrogen atom or a glucopyranosyl group, and R ₂ represents a hydrogen atom or a ramnosyloxy group)

(Wherein R ₃ represents a hydrogen atom or a hydroxyl group)

(Wherein R ₄ represents an arabinosyl group or a rhamnosyl group)
A composition for the prevention and / or treatment of cognitive dysfunction comprising one or more compounds represented by the formula:

  In addition, the present invention is for the prevention and / or treatment of cognitive dysfunction comprising, as an active ingredient, an extract from a plant containing one or more of the compounds represented by the above formulas (I) to (III). It is a composition.

  The compounds represented by the formulas (I) to (III) of the present invention all have excellent antioxidant action and Aβ aggregation inhibitory action, and compositions for preventing and / or treating cognitive dysfunction including the same The thing is useful for the prevention and treatment of diseases caused by cognitive impairment such as AD.

  The composition for preventing and / or treating cognitive dysfunction according to the present invention contains the compound represented by the above formula (I), (II) or (III) as an active ingredient.

  Among these, the formula (I) includes the following compounds (Ia) and (Ib).

The compound of the above formula (Ia) is already known as astilbin. The astilbin (Ia) is yellow杞an evergreen tree of the family Juglandaceae; well contained in the green parts of the (Koki Engelhardtia chrysolepis), threading (Lyonia ovalifolia), dye (Chloranthus glaber), Quai No Smilax china L. (Smilax glabra ), Chidakesashi (Astilbe microphylla), triacylate Cimicifuga (Astilbe odontophylla) such that is has been confirmed also contain (Patent Document 9), is the easiest to obtain way to extract from these plants .

Among these astilbine (Ia) -containing plants, the most easily used as a raw material is “Yellow candy” ( Engelhardita chrysolepis ; HANCE). "Yellow" is an evergreen tree of the walnut family that grows naturally in the mountains of southern China, and its leaves have a subtle sweetness. Since it has been used as a priest's health tea for a long time as Tsuruyama tea, there is no problem in terms of taste and safety.

  The effects of this Tsuruyama tea are: “Fix fever and toxicity, leave toxicity by diuretic action, break down alcohol except fat, lower blood fat, make the gastrointestines strong and aid digestion, leave stains and freckles. It excluding acne, excluding mouth bitterness and bad breath, and moisturizing the skin beautifully, "and is described as being effective for health maintenance, slimming, and beauty. This Tsuruyama tea is already marketed by Maruzen Pharmaceutical Co., Ltd. in Japan as “Yellow Tea” and has anti-allergic, anti-inflammatory, antioxidant, lipid peroxide production inhibitory effects, serum lipids (cholesterol, neutral fat) Etc.) Decreases, neutral lipolysis, aldose reductase inhibition, and cancer prevention have been published in academic conferences, but there are no reports on prevention / improvement of dementia.

  This astilbine (Ia) can be obtained by a method of extracting the astilbine-containing plant with water or an organic solvent having polarity according to the method described in Non-Patent Document 13. More specifically, when an astilbine (Ia) -containing plant body, preferably a leaf part thereof, is extracted with an organic solvent having a middle polarity, a lower alcohol or water, astilbine (Ia) is extracted. This extraction treatment is performed, for example, by immersing the raw material plant in about 5 to 15 times the amount of extraction solvent at room temperature or under reflux. Specific examples of preferable extraction solvents include ethyl acetate, acetone, methanol, ethanol, isopropanol, water and the like. These organic solvents and water may be mixed and used. The obtained astilbine (Ia) -containing extract can be used as it is as a composition for the prevention and treatment of cognitive impairment, but it can be used for liquid-liquid partition extraction, chromatography, ion exchange resin treatment, membrane separation treatment, etc. Astilbin (Ia) can be obtained by purifying with the above, and can be used as a composition for the prevention and treatment of cognitive dysfunction showing stronger activity.

  On the other hand, the compound of the above formula (Ib) is called eriodictyol 7-O-glucopyranoside and is a known substance contained in mountain tea (Southern) (Non-Patent Document 14). ), It has not been known that this has an effect of preventing and improving cognitive impairment.

  This eriodictyol 7-O-glucopyranoside (Ib) is obtained by extracting the eriodictyol 7-O-glucopyranoside-containing plant with water or a polar organic solvent according to the method described in Non-Patent Document 14. Is obtained. More specifically, when a plant body containing eriodictyol 7-O-glucopyranoside (Ib), preferably its aerial part, is extracted using an organic solvent having a middle polarity, a lower alcohol or water, eriodictythiol 7-O-glucopyranoside (Ib) is extracted. This extraction treatment is performed, for example, by immersing the raw material plant in about 5 to 15 times the amount of extraction solvent at room temperature or under reflux.

  Specific examples of preferable extraction solvents include ethyl acetate, acetone, methanol, ethanol, isopropanol, water and the like. These organic solvents and water may be mixed and used.

  The resulting eriodictyol 7-O-glucopyranoside (Ib) -containing extract can be used as it is as a composition for the prevention and treatment of cognitive impairment, but it can be used for liquid-liquid partition extraction, chromatography, ion exchange. By purifying by resin treatment, membrane separation treatment, etc., eriodictyol 7-O-glucopyranoside (Ib) can be obtained, and can be used as a composition for preventing and treating cognitive impairment showing stronger activity. .

  The formula (II) of the present invention includes the following compounds (IIa) and (IIb).

  The compound of the above formula (IIa) is named 6 "-O- (3,5-O-dimethylgalloyl) -cannabiscitrin [6" -O- (3,5-O-dimethylgalloyl) -cannabiscitrin] And the compound of formula (IIb) is 6 "-O- (3,5-O-dimethylgalloyl) -quercetin 3'-O-glucoside [6" -O- (3,5-O -dimethylgalloyl) -quercetin 3'-O-glucoside], both of which are novel compounds not described in any literature.

These compounds can be obtained by the method described in Example 1 from Forget-me-nots (scientific name: Myosotis scorpioides L.).

Furthermore, among the compounds of the formula (III) of the present invention, the compound in which R ₄ is a rhamnosyl group is a compound named myricetin 3-O-rhamnoside, It is a known substance contained in (Tenninka) (Non-patent Document 13).

  This myricetin 3-O-rhamnoside can be obtained by a method of extracting the myricetin 3-O-rhamnoside (III) -containing plant with water or a polar organic solvent according to the method described in Non-Patent Document 13. More specifically, when a plant body containing myricetin 3-O-rhamnoside (III), preferably its aerial part, is extracted using an organic solvent having a middle polarity, a lower alcohol or water, myricetin 3-O— Rhamnoside (III) is extracted. This extraction treatment is performed, for example, by immersing the raw material plant in about 5 to 15 times the amount of extraction solvent at room temperature or under reflux.

  Specific examples of preferable extraction solvents include ethyl acetate, acetone, methanol, ethanol, isopropanol, water and the like. These organic solvents and water may be mixed and used.

  The obtained myricetin 3-O-rhamnoside (III) -containing extract can be used as it is as a composition for the prevention and treatment of cognitive dysfunction as it is, but it can be used for liquid-liquid partition extraction, chromatography, and ion exchange resin treatment. Further, myricetin 3-O-rhamnoside (III) can be obtained by purification by membrane separation treatment or the like, and can be used as a composition for preventing and treating cognitive impairment showing stronger activity.

  The composition for preventing and / or treating cognitive dysfunction according to the present invention (hereinafter referred to as “a composition for preventing or treating dementia”) is a compound of the above formulas (I) to (III) (hereinafter referred to as “quercetin-related”). One or two or more of “sometimes referred to as“ compounds ”) are blended as active ingredients to form a pharmaceutical or health functional food.

  More specifically, in order to prepare a pharmaceutical form for the prevention and treatment of dementia, quercetin-related substances are added to appropriate carriers, excipients, diluents and the like that are usually used in the manufacture of known pharmaceutical compositions. The compound may be formulated and formulated into an oral or parenteral dosage form in accordance with conventional methods.

  At this time, the quercetin-related compound is desirably contained in an amount of 0.01 to 99.9% by mass (hereinafter simply referred to as “%”), and more preferably 0.1 to 90%. However, the composition as described above is not necessarily limited to this, and can be changed according to the condition of the patient, the type of disease, and the degree of progression.

  Examples of the oral administration agent include oral solid preparations such as powders, granules, tablets, pills and capsules, and oral liquids such as suspensions, liquids for internal use, oils, syrups, emulsions and aerosols. . Examples of parenteral administration agents include external preparations, suppositories, sterile injection solutions, sterile aqueous solutions, non-aqueous solvents, suspensions, oils, and lyophilized preparations.

  Examples of carriers, excipients, and diluents that can be used in the above-mentioned pharmaceutical form for preventing or treating dementia include lactose, dextrose, sucrose, sorbitol, mannitol, zyitol, erythritol, maltitol, starch, acacia Rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, calcium carbonate, liquid paraffin and mineral oils Can be mentioned.

  Further, in formulating, normally used fillers, extenders, binders, wetting agents, disintegrants, surfactants, sweeteners, fragrances, preservatives and the like may be used.

  In particular, in a preparation for parenteral administration, vegetable oils such as propylene glycol, polyethylene glycol and olive oil, and injectable esters such as ethyl oleate can be used as non-aqueous solvents and suspensions. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

  The composition for preventing or treating dementia in a pharmaceutical form prepared as described above can be administered by various routes to humans, mammals such as domestic animals, mice and mice. All modes of administration are predictable, but can be administered, for example, by oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine dura mater, or intracerebroventricular injection.

  The desired dosage of the quercetin-related compound in the above administration varies depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the administration route, and the period, and can be appropriately selected by those skilled in the art. However, for desirable effects, the quercetin-related compound should be administered at a dose of 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. This administration can be performed once a day, or can be divided into several times. Accordingly, the above doses do not limit the scope of the present invention in any way.

  Further, in order to make the composition for preventing or treating dementia of the present invention into the form of health function food, a quercetin-related compound may be added to a known food material to make a food. Examples of the food form include various foods, beverages, gums, teas, vitamin composites, health supplements, and the like, and can be used in the form of powder, granules, tablets, capsules, health drinks, and the like.

  In the composition for preventing or treating dementia in the form of health functional food, those in the form of powder, granules, tablets, and capsules can be prepared according to the manufacturing method of the pharmaceutical form, but in the form of health drink Is produced by blending a quercetin-related compound as an essential component and adding various flavoring agents or natural carbohydrates used in ordinary beverages as additional components to the liquid component as necessary.

  Examples of the above-mentioned natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), polysaccharides (eg, dextrin, cyclodextrin, etc.), and xylitol, sorbitol, erythritol. Such as sugar alcohol. As flavoring agents other than those mentioned above, natural flavoring agents (such as thaumatin, stevia extract (eg rebaudioside), glycyrrhizin, etc.) and synthetic flavoring agents (such as saccharin, aspartame) can be advantageously used.

  The ratio of the natural carbohydrate is generally about 1 to 20 g, desirably about 5 to 12 g, per 100 ml of the composition of the present invention.

  In addition to the above, the composition for preventing or treating dementia in the form of a health drink of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors, and flavoring agents such as natural flavoring agents, coloring agents, And fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonated beverages It is possible to contain a carbonating agent to be added.

  In addition, the composition in the form of a health drink of the present invention can contain natural fruit juice, fruit juice drink, and pulp for the production of vegetable drink. Such components can be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

  Since the quercetin-related compound itself of the present invention has almost no toxicity and side effects, it can be used with confidence even when it is taken for a long time for the purpose of prevention and improvement.

  The quercetin-related compound of the present invention is added to a food material or beverage material for the purpose of preventing and improving cognitive dysfunction, and can be used as a health functional food. At that time, the amount of the compound added to the food or beverage is In general, in the case of health food compositions, it is about 0.01 to 15% of the total food weight, and in the case of health drinks, 0.02 to 10 g, preferably 0 to 100 ml. .3 to 1 g can be added.

  As another embodiment of the present invention, there is provided a composition for preventing or treating dementia comprising an extract from a plant containing a quercetin-related compound, that is, an extract containing a quercetin-related compound at a high concentration as an active ingredient. Can be mentioned. Such an extract contains at least 5% of a quercetin-related compound.

  Furthermore, as another embodiment of the present invention, a composition for preventing / treating dementia can be mentioned which comprises a dried plant pulverized product having a high quercetin-related compound content as an active ingredient. Such a dry pulverized plant body contains at least 20% of a quercetin-related compound by dry matter weight.

  EXAMPLES Hereinafter, although an Example and a test example are given and this invention is demonstrated further in detail, this invention is not restrict | limited at all by these Examples.

Example 1
6 "-O- (3,5-O-dimethylgalloyl) -cannabicitrin (IIa)
6 "-O- (3,5-O-dimethylgalloyl) -quercetin 3'-O-glucoside (IIb):
Forget-me-nots (forget-me-nots; (scientific name) Myosotis scorpioides L.) flowers 100 g were placed in a flask, 50 mL of aqueous alcohol 1000 mL was added thereto, heated under reflux for 2 hours, and filtered. The same operation was again performed on the solid matter after filtration to obtain a filtrate. The combined filtrates were concentrated under reduced pressure at a temperature of 50 ° C. or lower and then dried under reduced pressure at 40 ° C. to obtain 32 g of forget-me-not flower extracts.

  To 32 g of the obtained forget-me-not flower extract, 100 mL of water is added and suspended, and the mixture is applied on a porous resin (DIAION HP20, 500 mL), followed by 1.5 L of water, 1.5 L of methanol, and 1.5 L of acetone in this order. Elute. Methanol was distilled off from the fraction eluted with 1.5 L of methanol to obtain 8.9 g of methanol eluted fraction.

  This methanol elution fraction was dissolved in a mixed solution of chloroform / methanol / water = 10: 5: 1 (volume ratio) and flowed from the top of a glass column packed with silica gel (trade name: silica gel 60, manufactured by Merck). And adsorbed onto silica gel. Next, a mixed solution of chloroform / methanol / water = 10: 5: 1 (volume ratio) was flowed as a mobile phase, and the eluate was collected and desolvated to obtain a flavonoid glycoside concentrate (1.4 g). It was. The obtained flavonoid glycoside concentrate was fractionated using liquid chromatography under the following conditions.

Stationary phase: JAIGEL GS-310 (manufactured by Nippon Analytical Industry)
Column diameter: 20mm
Column length: 500mm (two connected)
Moving bed: methanol Moving bed flow rate: 5 ml / min Detection: UV280nm and RI

  By this liquid chromatography, a fraction flowing out from a retention time of 75 minutes to 90 minutes was purified by recycling to obtain Compound 1 (43 mg), Compound 2 (48 mg), and Compound 3 (183 mg).

The compounds 1 to 3 obtained by extraction, separation by silica gel and purification by liquid chromatography were examined for their structures from ¹³ CNMR data and two-dimensional NMR analysis shown in Table 1. -O- (3,5-O-dimethylgalloyl) -quercetin 3'-O-glucoside (general formula IIb) and compound 2 are 6 "-O- (3,5-O-dimethylgalloyl)- Cannabiscitrin (general formula IIa) and compound 3 were identified as isoorientin, respectively.

Example 2
Astilbine (Ia);
Take 200 g of yellow leaves ( Engelhardtia chrysolepis ) leaves in a flask, add 2 L of 90% aqueous ethanol to this and extract for 2 hours. Concentrate the resulting extract under reduced pressure at a temperature of 50 ° C. or lower, then 40 ° C. or lower. Was dried under reduced pressure to obtain 34 g of jaundice extract.

  34 g of the obtained jaundice extract was suspended in 200 mL of water, extracted three times with 200 mL of chloroform, and then extracted three times with 200 mL of ethyl acetate. The obtained ethyl acetate extract was further separated by high performance liquid chromatography (column: TSKgel ODS-120T, 21.5 mm × 30 cm; elution solvent: acetonitrile / 0.05% trifluoroacetic acid = 20: 80). 0.29 g, 0.22 g of neoastilbine, 0.21 g of isoastilbine, and 0.18 g of neoisoastilbine were obtained.

Example 3
Eriodictyol 7-O-glucopyranoside (Ib):
42.7 g of the above-ground part of sancha (sanza: (scientific name) Elsholtzia bodinieri) was placed in a flask, 500 mL of 50% aqueous alcohol was added thereto, heated under reflux for 2 hours, and filtered. The same operation was again performed on the solid matter after filtration to obtain a filtrate. The combined filtrates were concentrated under reduced pressure at a temperature of 50 ° C. or lower, and then lyophilized to obtain 12.6 g of a Yamacha ground extract.

  11.3 g of the above-extracted mountain tea ground extract was suspended by adding 100 mL of water, and placed on a porous resin (DIAION HP20, 500 mL), 1.5 L of water, 1.5 L of 50% aqueous methanol, 1 methanol. Elution was carried out in the order of 0.5 L and 1.5 L of acetone. Methanol was distilled off from the fraction eluted with 1.5 L of 50% aqueous methanol to obtain 5.2 g of 50% aqueous methanol eluted fraction.

3.4 g of this 50% aqueous methanol elution fraction was dissolved in 40% aqueous methanol and flowed from the top of a glass column packed with octadecyl silica gel (trade name: Chromatorex ODS, manufactured by Fuji Silysia Chemical Co., Ltd.). Adsorbed. Subsequently, 40% aqueous methanol was allowed to flow as a mobile phase, and the eluate was collected and desolvated to obtain 1.3 g of flavonoid glycoside concentrate A and 0.7 g of flavonoid glycoside concentrate B.
The obtained flavonoid glycoside concentrate A was fractionated using liquid chromatography under the following conditions.

Stationary phase: JAIGEL GS-310 (manufactured by Nippon Analytical Industry)
Column diameter: 20mm
Column length: 500mm (two connected)
Moving bed: methanol Moving bed flow rate: 5 ml / min Detection: UV280nm and RI

  By this liquid chromatography, a fraction flowing out from a retention time of 75 minutes to 90 minutes was purified by recycling to obtain Compound 1 (703 mg). Flavonoid glycoside concentrate B was recrystallized from aqueous methanol to obtain Compound 2 (312 mg).

As a result of examining the structure of compounds 1-2 obtained by extraction, silica gel separation, and further purification by liquid chromatography, the structure was examined from ¹³ CNMR data and two-dimensional NMR analysis shown in Table 2, compound 1 was found to be (2RS ) Eriodictyol 7-O-glucopyranoside and Compound 2 were identified as luteolin 7-O-glucopyranoside, respectively.

< References >
１.Roemmelt S., Zimmermann N., Rademacher W., Treutter D., Phytochemistry,
64, 709 (2003).
２.Mizuno M., Kato M., Iinuma M., Tanaka T., Kimura A., Ohashi H., Sakai
H., Phtochemistry, 26, 2418-2480(1987)

<References>
1. Roemmelt S., Zimmermann N., Rademacher W., Treutter D., Phytochemistry,
64, 709 (2003).
2. Mizuno M., Kato M., Iinuma M., Tanaka T., Kimura A., Ohashi H., Sakai
H., Phtochemistry, 26, 2418-2480 (1987)

Example 4
Myricetin 3-O-rhamnoside (III):
300 g leaves of Tenninka ((Scientific name) Rhodomyrtus tomentosa ) were placed in a flask, 3000 mL of 80% aqueous alcohol was added thereto , heated under reflux for 2 hours, and filtered. The same operation was again performed on the solid matter after filtration to obtain a filtrate. The combined filtrates were concentrated under reduced pressure at a temperature of 50 ° C. or lower and then dried under reduced pressure at 40 ° C. to obtain 75 g of Tenjin flower leaf extract.

  Water (100 mL) was added to and suspended in 72 g of the resulting Tenjin flower leaf extract, applied to a porous resin (DIAION HP20, 1000 mL), and eluted in the order of 3 L of water, 3 L of methanol, and 3 L of acetone. Methanol was distilled off from the fraction eluted with 3 L of methanol to obtain 26.5 g of methanol-eluted fraction.

  This methanol elution fraction was dissolved in a mixed solution of chloroform / methanol / water = 10: 5: 1 (volume ratio) and flowed from the top of a glass column packed with silica gel (trade name: silica gel 60, manufactured by Merck). And adsorbed onto silica gel. Next, a mixed solution of chloroform / methanol / water = 10: 5: 1 (volume ratio) was passed as a mobile phase, the eluate was collected, and the concentrate obtained by desolvation was dissolved in 40% aqueous methanol. It flowed in from the upper part of the glass column packed with octadecyl silica gel (trade name: Chromatorex ODS, manufactured by Fuji Silysia Chemical Co., Ltd.) and was adsorbed on the octadecyl silica gel. Subsequently, 40% aqueous methanol was poured as a mobile phase, and the eluate was collected and desolvated to obtain 1.2 g of a flavonoid glycoside concentrate.

  The obtained flavonoid glycoside concentrate was fractionated using liquid chromatography under the following conditions.

Stationary phase: JAIGEL GS-310 (manufactured by Nippon Analytical Industry)
Column diameter: 20mm
Column length: 500mm (two connected)
Moving bed: methanol Moving bed flow rate: 5 ml / min Detection: UV280nm and RI

  By this liquid chromatography, the fraction flowing out at a retention time of 75 to 90 minutes was purified by recycling to obtain Compound 1 (385 mg) and Compound 2 (171 mg).

As a result of examining the structure of compounds 1 and 2 obtained by extraction, separation with silica gel, and purification by liquid chromatography, the structure was examined from ¹³ CNMR data and two-dimensional NMR analysis shown in Table 3, compound 1 was myricetin 3 -O-rhamnoside and Compound 2 were identified as myricetin 3-O-arabinofuranoside, respectively.

< References >
1) Tetrahedron, 34, 1389-1397 (1978)
2) Phytochemistry, 29, 1707-1708 (1990)

<References>
1) Tetrahedron, 34, 1389-1397 (1978)
2) Phytochemistry, 29, 1707-1708 (1990)

Test example 1
Antioxidant evaluation:
About the compound shown in following Table 4, the antioxidant action (hydroxyl radical (* OH) scavenging ability) was investigated with the following method. The results are shown in Table 5.

<Test compound>

<Test method>
(Reagents and equipment used)
As reagents, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO; Labotech Co., Ltd.), Trolox (SIGMA) and acetone (Kokusan Chemical Co., Ltd.) were obtained and used. Other reagents were purchased from Wako Pure Chemical. Also, as ESR equipment to be used, JEOL TE-100 (JEOL Ltd.) was used, and ESR flat cell (LC-12: effective volume 130 μL) was used. Measured. The specific measurement conditions were as follows, referring to the conditions of “Pharmaceutical Journal”, Vol. 118, pp. 609 to 615 (1998), magnetic field 335.5 ± 5 mT, output 4 mW, frequency 9.41 GHz, sweep time 2 minutes, The modulation width is 79 μT, the amplitude is 200, and the time constant is 0.3 s.

(Preparation of measurement sample)
The measurement sample was adjusted to about 0.5 mM with acetone (final solution concentration: 0.125 mM), and measurement was started. A substance that cannot erase hydroxyl radicals generated at that concentration was judged to have no scavenging ability. Moreover, the substance which erase | eliminates all the hydroxyl radicals (strong active substance) was further diluted with acetone and measured. Curcumin and quercetin were used as positive controls, and these were prepared to about 0.5 mM and 0.3 mM (final solution concentrations of about 0.125 and 0.075 mM), respectively, and used for measurement.

(Preparation of standard solution)
Trolox (J. Agric. Food Chem. 2003, 51, 3273-3279) used as a standard substance for antioxidant ability in the ORAC method was used as a standard substance. A standard solution was prepared by diluting this product with acetone to a concentration of about 0.05 to 0.4 mM (final solution concentration: about 0.0125 to 0.1 mM).

(Generation of hydroxyl radical and ESR measurement)
In reference to the description of “Pharmaceutical Journal”, Vol. 118, pp. 609-615 (1998), add 0.064M DMPO solution (final solution concentration 0.016M), 1.36 mM hydrogen peroxide (final solution concentration 0) to the test tube. .34 mM) and 50 μL each of the standard solution or the sample solution were mixed with a mixer for 1 to 2 seconds. 50 μL of a 0.136 mM iron sulfate solution (final solution concentration: 0.034 mM) was added to the solution, and after mixing for about 5 seconds, the iron sulfate solution was added to a flat cell for ESR measurement, and ESR measurement was performed 60 seconds later. .

(Creation of Trolox calibration curve and calculation of Trolox-like activity value)
Using a solution of each Trolox concentration, an SOD-like activity value calculation method for JSR's ESR application (JEOL Ltd. HP ESR Application Note ER-040004 (http://www.jeol.co.jp/technical/ai /esr/esr-an/er-040004/index.htm) to create a calibration curve (1) between Trolox concentration (X) and elimination ability (I ₀ / I-1) according to the following formula Further, the Trolox-like activity value (A: .OH elimination ability) was determined from the formula (2).

I ₀ /I-1=40.436X−0.3424 (1)
I: Relative intensity obtained from ESR spectrum
[R (DMPO-OH peak height) / Mn (Mn ²⁺ peak height)]
I ₀ : Relative intensity when Trolox concentration is 0 mM
X: Trolox concentration (mM)

A = [(I ₀ / I−1) −intercept] / slope / C × 1000 (2)
A: Trolox-like activity value
C: Sample solution concentration (μM)
The intercept and inclination are determined from the created Trolox calibration curve (1).

<Test results>
The hydroxyl radical scavenging ability of each test sample was measured according to the above test method together with quercetin and curcumin as positive controls. The hydroxyl radical scavenging ability of each sample in Table 5 was calculated as a relative value with the trolox-like activity value of quercetin as 100%.

  From Table 5, all compounds showed an antioxidant effect (hydroxyl radical (.OH) scavenging ability) equal to or higher than that of curcumin, though not as much as quercetin.

Test example 2
Aggregation inhibitory action of Aβ protein:
The compounds shown in Table 6 below were examined for their Aβ protein aggregation inhibitory action by the following method. The results are shown in Table 7.

<Test compound>

<Test method>
The amount of amyloid β (Aβ) protein aggregation was measured using thioflavin T. Specifically, since thioflavin T is known to emit fluorescence by binding to a β sheet of aggregated Aβ protein, this fluorescence intensity is detected with a plate reader, and this is used as an index of the amount of Aβ aggregation. . That is, the Aβ protein aggregation inhibitory effect of the test substance was calculated as an attenuation rate of fluorescence intensity.

The specific protocol is as follows.
1. A test substance diluted in DMSO was diluted with phosphate buffered saline (hereinafter referred to as PBS) so as to be 20 μM, and 25 μL was dispensed into a 96-well plate.
2. Aβ (1-42) was dissolved in PBS to a concentration of 40 μM, and dispensed into the above plate by 25 μM.
3. 10 μM of the test substance and 20 μM of Aβ (1-42) were incubated at 300 rpm and 37 ° C. for 48 hours.
4. Thioflavin T was dissolved in PBS to a concentration of 10 μM, and 50 μL was dispensed onto the plate to a final concentration of 5 μM, followed by stirring at 300 rpm and 37 ° C. for 30 minutes.
5. Thereafter, the fluorescence intensity of each well was measured at an excitation wavelength of 442 nm and an absorption wavelength of 485 nm.
6. From the fluorescence intensity emitted from wells containing only Aβ (1-42), each test substance and A
The value obtained by subtracting the fluorescence intensity emitted from the well mixed with β (1-42) was regarded as the Aβ (1-42) aggregation inhibitory effect of the test substance.
7. The Aβ (1-42) aggregation inhibitory action possessed by 10 μM quercetin was defined as 100%, and the Aβ (1-42) aggregation inhibitory action possessed by each test substance was calculated as a ratio to this and is shown in Table 7.

<Test results>

  From Table 7, it was shown that each test substance has an excellent amyloid β protein aggregation inhibitory effect equivalent to quercetin and curcumin.

  All of the compounds represented by the formulas (I) to (III) of the present invention have excellent antioxidant action and Aβ aggregation inhibitory action. Therefore, the composition for the prevention and / or treatment of cognitive dysfunction including this is useful for the prevention and treatment of diseases caused by cognitive dysfunction such as AD as a pharmaceutical or a health functional food.

Claims (10)

The following formula (III)

( Wherein R ₄ represents an arabinosyl group )
The amyloid protein aggregation inhibitor which contains the compound represented by these as an active ingredient. The amyloid protein aggregation inhibitor according to claim 1, which is used for preventing senile plaque formation in the brain. Total weight with respect to the amyloid protein aggregation inhibitor according to claim 1 or 2 wherein comprises from 0.1 to 90% by weight of compounds represented by the formula (III) of the agent. The amyloid protein aggregation inhibitor according to any one of claims 1 to 3, which is in the form of a pharmaceutical or a health functional food. The amyloid protein aggregation inhibitor according to any one of claims 1 to 4, which is a powder, granule, tablet, capsule or beverage dosage form. The following formula (III)

( Wherein R ₄ represents an arabinosyl group )
A amyloid protein aggregation inhibitor as an active ingredient an extract from a plant containing a compound represented by the plant is, amyloid protein aggregation inhibitor is rhodomyrtus tomentosa. The amyloid protein aggregation inhibitor according to claim 6, which is used for preventing senile plaque formation in the brain. Total weight with respect to the amyloid protein aggregation inhibitor according to claim 6, wherein comprising said extract 0.1 to 90 wt% of the agent. The amyloid protein aggregation inhibitor according to any one of claims 6 to 8 , which is in the form of a pharmaceutical or a health functional food. The amyloid protein aggregation inhibitor according to any one of claims 6 to 9 , which is a powder, granule, tablet, capsule or beverage dosage form.