JP2012214414A - β-SECRETASE INHIBITOR, AND FOOD AND DRINK INCLUDING β-SECRETASE INHIBITOR, AND INHIBITION RATE MEASURING METHOD TO MEASURE INHIBITION RATE THEREOF - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a β-secretase inhibitor with high safety, and food and drink including β-secretase inhibitor, and an inhibition rate measuring method to measure an inhibition rate thereof with excellent accuracy.SOLUTION: The β-secretase inhibitor includes an essential oil extracted from at least one raw material of clove, katsura, cardamon, clary sage, pencil cedar, bergamot, cabbage rose, damask rose, caraway, coriander, celery, tarragon, citrus aurantium, cumin, bay, cypress, geranium, vetiver, tagetes, patchouli, amyris, Jasminum grandiflorum, Acorus calamus, davana, curry leaf, and nigella.

Description

  The present invention relates to a β-secretase inhibitor containing a natural plant-derived essential oil as an active ingredient, a food and drink containing the β-secretase inhibitor, and an inhibition rate measuring method for measuring the inhibition rate.

  With the advent of a rapidly aging society in recent years, senile dementia has become a serious medical and social problem, and the development of effective anti-dementia drugs is strongly desired. Alzheimer-type dementia (Alzheimer's disease), a typical disease of senile dementia, is a degenerative disease characterized by brain atrophy, geriatric deposition, and neurofibrillary tangles. Is considered to be caused (Non-Patent Document 1).

  Although there is no established theory about the cause of Alzheimer's dementia, histopathological studies suggest that the elderly population is deposited, which causes neuronal loss and brain atrophy. Β-amyloid, which is the main component of the elderly group, has a cytotoxic effect, and is considered to cause neuronal cell death in Alzheimer's dementia (Non-patent Documents 2 to 5). β-amyloid is produced from the amyloid precursor protein (APP) by the action of an enzyme called β-secretase. Therefore, this compound having an inhibitory action on β-secretase is considered useful for the treatment of Alzheimer-type dementia. In recent years, research on compounds having an inhibitory effect on β-secretase has been actively carried out (Patent Document 1).

  As an inhibition rate measurement method for measuring the inhibitory effect on β-secretase, a sample solution containing a substrate peptide, β-secretase and a test compound is added to a microplate, and the fluorescence intensity is measured with a plate reader over time. A method of determining an inhibitory action on β-secretase by comparison is used (Patent Document 1). However, this method is greatly affected by the fluorescence intensity of impurities in the sample solution, and there is a problem in the accuracy of measurement results.

JP 2008-137914 A

Al zheimer, A (1907) Central Bl. Nervenheilk. Phychiatr. 30, 177-179 Yankner, B. et al. Science, 245, 417-429 (1989) Cai, X., Gold, T. & Younkin, S. Science, 259, 514-516 (1993) Rose, A. Nature Med. 2, 267-269 (1996) Scheuner, D. et al. Nature Med. 2, 864-869 (1996)

  The present invention provides a β-secretase inhibitor having high safety and an excellent inhibitory action on β-secretase activity, a food and drink containing the β-secretase inhibitor, and an inhibition rate measuring method for measuring the inhibition rate Is.

  The invention according to claim 1 includes cinnamoum cassia presl, elettaria cardamomum (L.) Maton, salvia sclarea L., juniperus virginiana L., citrus bergamia Risso et Poit. .), Centofolia rose (Rosa Centifolia L.), Damask rose (Rosa Damascena Mill), Carp carvi L., Coriandrum sativum L., Eugenia caryophyllat Thunb., Dutch honey (Apium) graveolens L. var dulce (Mill.) Pers), Estragon (Artemisia dracunculus L.), Daidai (Citrus aurantium L. spp amara), Cumin (Cuminum cyminum L.), Bay (Pimenta racemosa), Western cypress (Cupressus sempervirens) , Geranium (Pelargonium graveolens.), Vetiver (Vetiveria zizanioides), Tadget (Tagetes glanduifera Schrank (T. minuta. L.)), Patchouli (Pogostemon c) ablin, Amyris balsamifera, Sokei (Jasminum grandiflorum L.), Shobu (Acorus calamus (L.)), Davana (Artemisia pallens Wall.), Murraya koenigii (L.) Sprengel, Nigella The present invention relates to a β-secretase inhibitor comprising an essential oil extracted from at least one raw material of Sativa L.).

  The invention which concerns on Claim 2 is related with the food-drinks containing the said beta-secretase inhibitor.

  The invention according to claim 3 is an inhibition rate measuring method for measuring an inhibition rate of the β-secretase inhibitor, and first, a solution containing the β-secretase inhibitor, a solution containing a fluorescently labeled substrate peptide, and from preparing a sample solution comprising a solution containing β-secretase, and preparing a control solution comprising a dimethyl sulfoxide (DMSO) solution, a solution containing a fluorescently labeled substrate peptide and a solution containing β-secretase Then, the sample solution and the control solution are respectively subjected to high-performance liquid chromatography (HPLC) to give a fluorescently labeled degradation product of the substrate peptide, an unreacted degradation product of the substrate peptide, and an unreacted product. Separating the substrate peptide, the fluorescently labeled degradation product of the sample solution and Measuring the emission intensity of the fluorescence-labeled degradation product of the control solution, and determining the inhibition rate of the β-secretase inhibitor by comparing the intensity, and measuring the inhibition rate Law

  According to the invention according to claim 1, since an essential oil derived from a natural plant is used as an active ingredient, β that can prevent or stop senile dementia such as Alzheimer-type dementia without adversely affecting the human body. -Secretase inhibitors can be provided.

  According to the invention of claim 2, since natural plant-derived essential oil is used as an active ingredient, it is possible to prevent or stop progression of senile dementia such as Alzheimer-type dementia without adversely affecting the human body. -The food-drinks containing a secretase inhibitor can be provided.

  According to the third aspect of the present invention, the β-secretase inhibition rate can be accurately determined by providing a high performance liquid chromatography (HPLC) analysis step.

It is a figure showing the inhibition rate measurement data of the beta-secretase inhibitor of this invention.

  Hereinafter, the β-secretase inhibitor and the food and drink containing the β-secretase inhibitor according to the present invention, and the inhibition rate measuring method for measuring the inhibitory action will be described.

  The β-secretase inhibitor of the present invention contains an essential oil extracted from a natural plant as an active ingredient.

  The present invention discloses that the extracted natural oils of 25 natural plants described in claim 1 have β-secretase inhibitory action. The natural plant will be described below.

  Katsura (English name: Cassia, scientific name: Cinnamoum cassia Presl) is a dicotyledonous plant belonging to the genus Camphoraceae. The height of the tree is an evergreen small tree of about 15 m, the leaves are 12 to 15 cm long and glossy green, and many small yellowish white flowers are attached to the twigs. The fruit has an oval shape with a diameter of about 1 cm. It grows naturally in southern China and the Indochina peninsula. The skin of branches and trunks is called cinnamon bark and is used for medicinal purposes. Others are used as spices in confectionery and cooking. Branches and leaves are steam-distilled to produce cassia oil (cinnamon oil), which is used for medicinal purposes and fragrances. The main component of the essential oil is cinnamaldehyde and tannin. The β-secretase inhibitor of the present invention uses an essential oil obtained by steam distillation of leaves, stems, twigs and bark.

  Ginger crab (English name: Cardamon, scientific name: Elettaria cardamomum (L.) Maton) is a monocotyledonous plant belonging to the genus Glyceraceae. Perennial, native to southwest India. It is cultivated in South India, Sri Lanka, Malaysia, South America Guatemala, etc. The seeds are used as spices. It has a thickened woody stem in the basement, and a pseudostem with several to several tens of leaf sheaths rolled up, and leaves one after another, reaching a height of about 3 m. The leaves are needle shaped, 30-100 cm long and 7.5-15 cm wide. A flower stem of 60 to 90 cm in length is taken out from the underground stem and a white flower is attached to the conical inflorescence. Seeds have a sharp fragrance similar to camphor and are used as curry raw materials. For the β-secretase inhibitor of the present invention, an essential oil obtained by solvent extraction of fruits is used.

  Onysalvia (English name: Clarity sage, scientific name: Salvia sclarea L.) is a dicotyledonous plant belonging to the genus Salvia. It is a cold-resistant two-year-old grass that is about 1 m high, and has light purple flowers. It is distributed from southern Europe to southwest Asia and is cultivated in Europe. Essential oils are used for fragrance and medicinal purposes. The β-secretase inhibitor of the present invention uses an essential oil obtained by steam distillation of whole plants and / or flower ears.

  Empizubixin (English name: Cedarwood, scientific name: Juniperus virginiana L.) is a conifer of the Cypressaceae juniper family. The height is about 12-30 m, the crown is pyramid type, and the bark is grayish brown or reddish brown. The young leaves are needle-shaped and have a length of 3 to 6 mm, and the fruits have a blackish blue color and white powder, and have a diameter of about 5 mm and a length of 3 to 6 mm. The material is homogenous and easy to sharpen and has a fragrance. It is distributed in the eastern part of the North American Rocky Mountains, in the mountains from Hudson Bay to New Mexico, Texas, and Florida, and is exported in large quantities as pencil material. As the β-secretase inhibitor of the present invention, an essential oil obtained by steam distillation of a trunk and / or bark is used.

  Bergamot (English name: Bergamot, scientific name: Citrus bergamia Risso et Poit.) Is a dicotyledonous plant belonging to the genus Citrus mandarin. It is a citrus fruit that is close to lemon and is considered a hybrid of Daidai and lemon or citron. It is a tall tree resembling a lemon, with white flowers blooming, and a fruit-shaped sphere with a sharp top. Squeezing the peel gives a light green, strong fragrant bergamot oil, which is also used in cosmetics, especially for eau de cologne and soap fragrances. The scented components are linalyl acetate 34-40%, l-linanol and the like. Southern Italy and Sicily are the main production areas. For the β-secretase inhibitor of the present invention, an essential oil obtained by pressing the pericarp is used.

  Centifolia rose (English name: Cabbage Rose, scientific name: Rosa Centifolia L.) is a dicotyledonous plant belonging to the genus Rosaceae. The flower is a bulbous cabbage-shaped double valve, has a large number of petals of 60 or more, has a diameter of 5 to 6 cm, has a single or a few flowers on the stem, and is usually pink. It is of hybrid origin. The β-secretase inhibitor of the present invention uses an essential oil obtained by solvent extraction of flowers.

  Damask rose (English name: Damask Rose, scientific name: Rosa Damascena Mill) is a dicotyledonous plant belonging to the genus Rosaceae. There are 5-7 leaflets, many flowers are scattered like a cluster, there are many petals, and the flower color is red, pink, or white. The fragrance is extremely intense and rich in perfume oil components, and is used as a raw material for perfumes. It is thought that it was selected from natural hybrids. The β-secretase inhibitor of the present invention uses an essential oil obtained by steam distillation of flowers.

  Himeyuki (English name: Caraway, scientific name: Carum carvi L.) is a dicotyledonous plant belonging to the genus Caraway. The plant height is 30-60 cm and it is hairless. The lower leaf of the stem has a petiole, but the upper leaf has no petiole and multi-fissures, and the lobule deeply splits into a filament. Small flowers are white, and many bloom in a diffuse inflorescence in summer. The fruit is 3-7 cm in length, curved in a crescent shape and has 5 buds. The fruit is used as a spice, the root is as thick as a finger, yellow, fleshy, shaped and tasted like a carrot and vegetable. Originating in Europe, the fruit contains an essential oil called carvone, which is used for healthy stomach and antifungal drugs. The fruit is used as it is as a spice, or it is coarsely ground and used for flavoring dishes. For the β-secretase inhibitor of the present invention, an essential oil obtained by steam distillation of seeds is used.

  Kosuijitsu (English name: Coriander, scientific name: Coriandrum sativum L.) is a dicotyledonous plant belonging to the genus Coriander. An annual or biennial plant native to the Mediterranean coast. The stem is 30-90 cm high and sparsely branches. The leaves tear like feathers and resemble celery leaves. There is an odor peculiar to whole grass. Put a white flower at the end of the branch. The fruit is in the form of a sphere with a pair of two fruits and has a diameter of 3 to 5 mm. When the fruit is fully ripe, it has a unique aroma such as a mixture of lemon and sage. Fresh leaves are added to fish and meat dishes and used as flavorings. The β-secretase inhibitor of the present invention uses an essential oil obtained by steam distillation of leaves.

  Crested cypress (English name: Clove, scientific name: Eugenia caryophyllat Thunb.) Is a dicotyledonous plant belonging to the genus Myrtaceae. It is an evergreen tree with a tree height of 4-7m, sometimes 10m or more. Flowers gather at the end of the twig. It is currently cultivated as a spice in various tropical regions. It has a spicy and pungent flavor and has the strongest antiseptic and sterilizing power among the fragrances, so it is used for rust prevention, medicinal use, and fragrances. For the β-secretase inhibitor of the present invention, an essential oil obtained by steam distillation of this above-ground part, in particular, a dried product of flower buds, is used.

  Dutch honey bee (English name: Celery, scientific name: Apium graveolens L. var dulce (Mill.) Pers) is a dicotyledon of the genus Dutch honey genus. Generally called celery. It is a 1-2 year grass that uses petiole as an edible, and has a strong fragrance in the whole plant. The leaves are double-leafed and have a thick and long petiole, and the petiole gathers upright to become a large stock. In the summer, the stalks of 50 to 100 cm in height are put out, and many fine flowers are attached to small plural inflorescences. The flowers are white or greenish white, and the fruits are slightly spherical and small. For the β-secretase inhibitor of the present invention, an essential oil obtained by steam distillation of seeds is used.

  Estragon (English name: Estragon, scientific name: Artemisia dracunculus L.) is a dicotyledonous plant belonging to the genus Artemisia. Also called tarragon. It is a perennial widely distributed in the region from Central Asia to Siberia. The stem is upright, 40-120 cm in height, and the rhizome crawls sideways. The foliage is linear and has a pattern of 3-7 cm in length, 2-8 mm in width, and no pattern. The inflorescence is a large cone with many flowers. It is often used in French cuisine using escargots, and is also cultivated mainly in Europe because it is used in spices such as salads, omelets, gratin and sauces. The β-secretase inhibitor of the present invention uses an essential oil obtained by steam distillation of the whole plant.

  Daidai (English name: Neroli, scientific name: Citrus aurantium L. spp amara) is a dicotyledonous plant belonging to the genus Citrus mandarin. It is an evergreen small tree with a height of 5m. The leaves are oval, oval, 6-8 cm long, sharp at the tip, round at the base, blunt saw leaves at the edges, leathery, scattered with oil spots, and wide wings on the petiole. The flowers are white and about 4 cm in diameter, the fruits are spherical and about 8 cm in diameter, ripen in winter, and the outer skin is orange, smooth and shiny. The β-secretase inhibitor of the present invention uses an essential oil obtained by steam distillation of flowers.

  Cumin (English name: Cumin, scientific name: Cuminum cyminum L.) is a dicotyledonous plant belonging to the genus Cuminaceae. 20-30cm in height, the leaf attached to the lower part of the stem is oval with a long handle, the petiole of the upper leaf is 1-2cm in length, the leaf blade is completely split, and the end is 1-5cm in length Become a thread. Add light red or white florets in late spring. The fruit is an elongated oval shape, 4-7 mm in length, and the ingredients include cuminal, which is the source of the pungent taste that burns. As a spice, it is mixed with cheese, curry powder ingredients, and chili powder, and it is also used in soups, stews, and rice dishes. It is also used for flavoring bread and cakes. For the β-secretase inhibitor of the present invention, an essential oil obtained by steam distillation of seeds is used.

  Bay (English: Bay, scientific name: Pimenta racemosa) is a dicotyledonous plant belonging to the genus Pimenta. It is a small Takagi tree native to the West Indies, Guyana and Venezuela, and is about 6 to 15 meters high. The leaves are cortical, fallen oval to oval, about 15 cm long and face each other. A lot of white flowers are attached to the conical inflorescence from the leaf buds at the end of the branch, and the fruit ripens black. The leaves contain 1-1.4% essential oil and are fragrant. This is called bay oil and is used as a fragrance for hair styling, soap, toilet deodorant and the like. The β-secretase inhibitor of the present invention uses an essential oil obtained by steam distillation of leaves.

  Western cypress (English name: Italian Cypress, scientific name: Cupressus sempervirens) is a conifer of the genus Cypress. The leaves are scaly leaves like cypress, and cypress oil (Oil Of Sypress) is collected from the branches and used for perfumes and cough medicines. As the β-secretase inhibitor of the present invention, an essential oil obtained by steam distillation of branches and leaves is used.

  Geranium (English name: Rose Geranium, scientific name: Pelargonium graveolens.) Is a dicotyledonous plant belonging to the genus Asteraceae. A perennial plant native to South Africa, cultivated due to the fragrance of roses. As the β-secretase inhibitor of the present invention, an essential oil obtained by steam distillation of branches and leaves is used.

  Vetiver (English name: Vetiver, scientific name: Vetiveria zizanioides) is a monocotyledonous plant belonging to the genus Vetiveraceae. It is a perennial plant that is cultivated mainly in the tropics and subtropics of Asia, Africa, South America, etc. in order to extract aromatic oil from the roots and to prevent soil erosion and windbreak. The place of origin is from India to Southeast Asia. The plant height is 2m and grows like a large stock. The leaves are elongated, about 30-100 cm long and about 10 mm wide. A 10-30 cm long spike is placed on the stem length. The roots are distilled to obtain Vetiver oil. It is brown and fragrant and is used as a perfume for perfumes, cosmetics, soaps, and the like. The β-secretase inhibitor of the present invention uses an essential oil obtained by steam distillation of roots.

  Tadget (English name: Tagete, scientific name: Tagetes glanduifera Schrank (T. minuta. L.)) is a dicotyledonous plant belonging to the genus Manjugiku. It is an annual grass with a strong fragrance. Naturalized in a warm region in the Southern Hemisphere. It is one of the basic plants of the essential oil Tagetes Oil. As the β-secretase inhibitor of the present invention, an essential oil obtained by steam distillation of flowers and whole plants is used.

  Patchouli (English name: Patchouli, scientific name: Pogostemon cablin) is a dicotyledonous plant belonging to the genus Lamiaceae. It is a perennial plant cultivated mainly in Southeast Asia and India as a raw material plant for obtaining essential oils. The stem is 30-80 cm high and branches off from the bottom. The leaves are wide oval, wavy and irregular heavy sawtooth, 8-10 cm long, with fine hairs on the back. The flower spikes are 5 to 15 cm long and have a light purple, two-lip flower at the tip of the branch. Many distill leaves that have been dried for 4.5 days to obtain essential oils with a strong fragrance. In India, it is used as a medicinal bath with fragrances such as clothes and pillows, or in baths. The β-secretase inhibitor of the present invention uses an essential oil obtained by steam distillation of leaves.

  Amiris (English name: Amyris, scientific name: Amyris balsamifera) is a dicotyledonous plant belonging to the genus Amiris. For the β-secretase inhibitor of the present invention, an essential oil obtained by steam distillation of a trunk is used.

  Sokei (English name: Jasmin, scientific name: Jasminum grandiflorum L.) is a dicotyledonous plant belonging to the genus Sokei. It is an evergreen shrub with a height of 1 to 2 m and is all hairless. Around July to September, 2-10 white flowers are laid out in a star shape and have an aroma. The β-secretase inhibitor of the present invention uses an essential oil obtained by solvent extraction of flowers.

  Shobu (English name: Calamas, scientific name: Acorus calamus (L.)) is a monocotyledonous plant of the genus Araceae. It is a summer green perennial with thick rhizomes that crawls across the surface of the earth and leaves leaves in two rows. Dried rhizomes are used for pain relief and aromatic stomach medicine and contain about 3% essential oil. The β-secretase inhibitor of the present invention uses an essential oil obtained by steam distillation of rhizomes.

  Davana (English name: Davana, scientific name: Artemisia pallens Wall.) Is a dicotyledonous plant belonging to the genus Artemisia. An aromatic annual plant, distributed in southern India. It is cultivated for collecting essential oils. As the β-secretase inhibitor of the present invention, an essential oil obtained by steam distillation of the above-ground part of grass is used.

  Nanyo Sanroku (English name: Curry leaf, Scientific name: Murraya koenigii (L.) Sprengel) is a dicotyledonous plant belonging to the genus Geckiaceae. An evergreen shrub with fragrance, distributed from Ryukyu, South China to India. Leaves are widely used as a spice for aromatizing curry and are also used as a tonic. The β-secretase inhibitor of the present invention uses an essential oil obtained by steam distillation of the whole plant.

  Nigera (English name: Black Cummin, scientific name: Nigella sativa L.) is a dicotyledonous plant belonging to the genus Aspergillus. Also called odor black seed grass. It is distributed from the Mediterranean coast to central Europe and Southwest Asia, and is cultivated in the Mediterranean region, West Asia and India. Seeds are placed in bread alone or mixed with sesame. Oil has a unique fragrance. For the β-secretase inhibitor of the present invention, an essential oil obtained by steam distillation of seeds is used.

  These essential oils may be essential oils other than those described above, and the method for collecting essential oils is not limited to the above method. These essential oils may be used singly or in combination of two or more, and have strong β-secretase inhibitory activity as detailed in the following examples. The secretase inhibitor of the present invention contained as a component can be used for various applications.

  For example, when the β-secretase inhibitor of the present invention is used as a pharmaceutical, it is used as a preventive agent for senile dementia in mammals (particularly humans), particularly as a preventive agent for Alzheimer's dementia.

  The β-secretase inhibitor of the present invention is a tablet, powder, fine granule, granule, coated tablet, capsule, syrup, troche, inhalant, suppository, injection, ointment by a conventional method. , Ophthalmic ointments, eye drops, nasal drops, ear drops, poultices, lotions and the like.

  Excipients, binders, lubricants, coloring agents, flavoring agents, and if necessary stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, Antioxidants and the like can be used, and in general, ingredients and blending amounts used as raw materials for pharmaceutical preparations are appropriately selected and formulated by a conventional method.

  When administering the pharmaceutical formulation of this invention, the form is not specifically limited, What is necessary is just the method used normally, and oral administration or parenteral administration may be sufficient. The pharmaceutical dosage according to the present invention can be appropriately selected from pharmacologically effective doses according to the degree of symptoms, age, sex, body weight, dosage form, specific type of disease, and the like. As an example of the dosage, in the case of oral administration, the amount of active ingredient is usually about 0.001 to 1000 mg / kg for an adult, and if this is administered once to several times a day, Good.

  In addition, the β-secretase inhibitor of the present invention has strong β-secretase inhibitory activity, so that it is used in addition to products such as bathing agents, soaps, fragrances, aromatherapy essential oils, perfumes, hair styling agents, etc. be able to.

  The β-secretase inhibitor of the present invention is usually contained in an amount of about 0.001 to 100% by weight, preferably about 0.1 to 5% by weight, based on the entire product.

  When the above-mentioned product containing the β-secretase inhibitor of the present invention is used, the relaxation effect and refreshing effect due to the scent of essential oil are exhibited, and the healing effect of the mind and body is exhibited in a modern society with many stresses. By using the above products in an environment where elderly people, sick people, etc. live, the effect of preventing / stopping senile dementia is also exhibited. The effects of the essential oil used, such as antiseptic and antibacterial effects, are also exhibited.

  The β-secretase inhibitor of the present invention can be used as a mixture with other natural plant essential oils when used as an essential oil for aromatherapy. The essential oil for aromatherapy can also be used in a form that can be spread and diffused in a predetermined place and inhaled. The oil can be spread and diffused using a diffuser or the like. The distribution / spreading place can be distributed, for example, in a house room, a hotel room, a conference room, a hospital room, an event hall where people gather, a break plaza, various relaxation facilities, and the like. In particular, it is preferably used in hospitals, facilities, etc. where senile dementia patients live.

  The β-secretase inhibitor of the present invention is a food additive such as soft drinks, dairy products (processed milk, yogurt), confectionery (jelly, chocolate, biscuit, gum, tablet confectionery) or various foods and beverages such as supplements. It can also be blended.

  When used as a food additive, the amount added is not particularly limited, and may be appropriately determined according to the type of food. As an example, what is necessary is just to add so that content may become the range of about 0.0005 to 50 weight% as dry weight of an above-described extract.

In addition to the β-secretase inhibitor of the present invention, the food or drink contains excipients, flavoring agents, colorants, preservatives, thickeners, stabilizers, gelling agents, antioxidants, and the like. Also good.
Among these, excipients include, but are not limited to, powders such as fine particle silicon dioxide, sucrose fatty acid ester, crystalline cellulose / sodium carboxymethylcellulose, calcium hydrogen phosphate, wheat starch, rice starch, corn starch , Starches such as potato starch, dextrin, cyclodextrin, crystalline cellulose, lactose, glucose, sugar, reduced maltose, starch syrup, fructooligosaccharide, galactooligosaccharide, soybean oligosaccharide, isomaltoligosaccharide, xylooligosaccharide, maltooligosaccharide, milk fruit Examples thereof include sugars such as oligosaccharides, and sugar alcohols such as sorbitol, erythritol, xylitol, lactitol, and mannitol. These excipients can be used alone or in combination of two or more.

  Examples of the flavoring agent include, but are not limited to, for example, bontang extract, which is a fruit juice extract, lychee extract, apple juice, orange juice, citron extract, peach flavor, ume flavor, sweetener acesulfame K, erystol, oligosaccharide , Mannose, xylitol, isomerized sugar, green tea, oolong tea, banaba tea, Tochu tea, iron kannon tea, pearl barley tea, amacha tea, macomo tea, kelp tea, and yogurt flavor.

  The inhibition rate measurement method for measuring the inhibition rate of the β-secretase inhibitor of the present invention will be described in detail.

  The inhibition rate of the β-secretase inhibitor of the present invention refers to the rate at which degradation of the substrate peptide by β-secretase is inhibited. As the β-secretase inhibitor, the above-mentioned essential oil derived from a natural plant is used.

As the substrate peptide, a peptide in which fluorescence is labeled at the N-terminus and a quenching group is labeled at the residue on the opposite side can be used. -Methylcoumarin-4-yl) quenching peptide in which acetyl group (hereinafter referred to as MOCAs group) is linked to lysine residue and dinitrophenyl group (hereinafter referred to as Dnp group): MOCAc-ser-Glu-Val-Asn the -Leu-Asp-Ala-Glu- Phe-Arg-Lys (Dnp) -Arg-Arg-NH 2 can be used. This substrate peptide is a substrate that emits fluorescence when the MOCAs group and Dnp group inhibit the respective fluorescence and are cleaved. The substrate peptide is used after dissolved in a solvent such as a dimethyl sulfoxide (DMSO) solution.

  β-secretase can be obtained by any method. β-secretase is an enzyme that degrades substrate peptides. β-secretase is used after diluted with a buffer solution such as sodium acetate buffer.

  A solution containing a substrate peptide is added to a solution obtained by mixing a solution containing the β-secretase inhibitor, a solution containing β-secretase, and a buffer solution such as a sodium acetate buffer to prepare a sample solution. The sample solution preferably contains a surfactant. After incubating the sample solution for at least 3 hours, a buffer solution such as phosphate buffer is added to stop the reaction.

  As a sample solution control, a solution containing a dimethyl sulfoxide (DMSO) solution instead of a β-secretase inhibitor, that is, a dimethyl sulfoxide (DMSO) solution, a solution containing β-secretase, and a buffer solution such as a sodium acetate buffer are mixed. A solution containing the substrate peptide is added to the prepared solution to prepare a control solution. After incubating the control solution for at least 3 hours, a buffer solution such as phosphate buffer is added to stop the reaction.

  Through the above-described steps, the sample solution and the control solution include a decomposition product of a substrate peptide labeled with a fluorescent group (hereinafter referred to as a fluorescent label decomposition product), a substrate peptide labeled with a quenching group, that is, not fluorescently labeled. There will be degradation products and unreacted substrate peptides. These materials are separated by high performance liquid chromatography (HPLC). The luminescence intensity of the separated fluorescent label decomposition product in the sample solution and the fluorescent label decomposition product in the control solution is measured.

The β-secretase inhibition rate of β-secretase agent is expressed by the following formula:
Secretase inhibition rate (%) = {1- (S / C)} × 100
In the formula, S represents the luminescence intensity of the fluorescent label degradation product in the sample solution, and C represents the luminescence intensity of the fluorescence label degradation product in the control solution.

  Hereinafter, the effects of the present invention will be made clearer by describing examples of the present invention. However, the present invention is not limited to the following examples.

(Synthesis example)
(Reagent Preparation-1)
To 4.1 g of sodium acetate (molecular weight: 82.03), 400 ml of acetic acid was added and dissolved to adjust the pH to 4.5. The solution was made up to 500 ml with pure water to obtain a 0.1 M acetate buffer (1).
Substrate peptide MOCAc-ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Arg-Lys (Dnp) -Arg-Arg-NH2 ((Trifluoroacetate Form), molecular weight: 2001.08, Peptide Institute 3212- v) was dissolved in 550 μL of DMSO to 1 mM to obtain a substrate peptide solution (2).

(Preparation of reagent-2)
The 0.1M acetate buffer (1) was diluted 5-fold with water, and 0.1% Triton X-100 was added to obtain a 20 mM acetate buffer (3).
β-secretase (β-Secretase, extecellular domain human, recombinant Sigma S-4195) was diluted 50-fold with 20 mM acetate buffer (3) and adjusted to 100 Units / ml to obtain β-secretase solution (4) .
Substrate peptide solution (2) was diluted 10-fold with 20 mM acetate buffer (3) to obtain substrate diluted solution (5).

(Preparation of test sample)
78 μL of 20 mM acetate buffer (3), 10 μL of β-secretase solution (4), and 2 μL of an essential oil sample extracted from a natural plant were mixed to prepare a solution (6). A mixture of 78 μL of 20 mM acetate buffer (3), 10 μL of β-secretase solution (4), and 2 μL of DMSO was mixed to obtain a solution (7). Solution (6) and solution (7) were added to a 96-well plate (black) and incubated at 37 ° C. for 10 minutes to obtain a sample solution and a control solution, respectively. 10 μL of substrate peptide dilution solution (5) and 20 mM acetate buffer were added thereto, and the sample concentration was adjusted to 1.25% (250 μg / ml) and 0.25% (50 μg / ml), and at 37 ° C. for 3 hours. Incubated. 900 μL of a phosphate buffer (pH 7.0) having an ionic strength I = 0.15 was added to stop the reaction.

(Measurement of β-secretase inhibition rate)
This reaction product was separated by HPLC, and the luminescence intensity of the fluorescence labeled decomposition product of each of the sample solution and the control solution was measured.
(HPLC conditions)
A liquid: 0.1% acetonitrile formic acid, B liquid: 0.1% formic acid water 0 min: A liquid 10%, B liquid 90%
Gradient 25 minutes: Solution A 60%, Solution B 40%
25.01 min: A solution 90%, B solution 10%
30 minutes: A solution 10%, B solution 90%
Detector: Fluorescence detector EX328nm, EM393nm
Flow Rate 1ml / min
Column: L-column ODS 250 mm, 4.6 mm

Secretase inhibition rate (%) = {1- (S / C)} × 100
In the formula, C represents the luminescence intensity of the fluorescent label degradation product in the control solution, and S represents the luminescence intensity of the fluorescence label degradation product in the sample solution. The results are shown in FIG.

  From the results of FIG. 1, the average β-secretase inhibition rate of three times of 1.25% (250 μg / ml) of each test sample is 17.51% to 69.96%, and the essential oil of each sample is β-secretase. It was shown to inhibit the activity.

  Next, the inhibition rate measurement method of the β-secretase inhibitor of the present invention is compared with the conventional inhibition rate measurement method (plate method).

(Sample preparation)
10 g of Ryutan (Tochimoto Tenkaido Lot 0903C010901) was powdered, and 100 ml of 50% EtOH was added thereto, followed by extraction under reflux conditions for 2 hours. Filtration was performed while hot to obtain a filtrate. This operation was repeated once more, and the obtained filtrate was combined with the first filtrate, and the solvent was distilled off under reduced pressure to obtain Sample 3.
About 1 g of the obtained sample 3, the solvent was partitioned with 100 ml of water and hexane to obtain respective layers. The obtained aqueous layer was extracted once again with 100 ml of hexane, and the obtained aqueous layer was concentrated under reduced pressure to distill off hexane. Thereafter, the same operation was sequentially performed with ethyl acetate, chloroform, and 1-butanol. The water fraction was Sample 1, and the butanol fraction was Sample 2.

The obtained samples 1, 2 and 3 were used as essential oil samples, and the β-secretase inhibition rate was measured by the inhibition rate measurement method using HPLC of the present invention in the same manner as in the above Examples.
For comparison, the β-secretase inhibition rate was measured by a conventional plate method.
The results are shown in Table 1.
Although the inhibition rate by the conventional plate method is -0.1% to 50.5% and the variation is as large as 50.6%, the inhibition rate by the inhibition rate measurement method using HPLC of the present invention is -21.1. The variation was as small as 25.8% between% and 4.7%.
The β-secretase inhibition rate could be measured with high accuracy by the inhibition rate measurement method using HPLC of the present invention.

  INDUSTRIAL APPLICABILITY The present invention can be suitably used as a pharmaceutical product for preventing Alzheimer-type dementia, and includes products such as bathing agents, soaps, fragrances, aromatherapy essential oils, perfumes, hair styling products, soft drinks, milk It can be suitably used as an additive for various foods and beverages such as products, confectionery or supplements.

Claims (3)

  Katsura (Cinnamoum cassia Presl), Shouzoku (Elettaria cardamomum (L.) Maton), Onysarubia (Salvia sclarea L.), Empizubikshin (Juniperus virginiana L.), Bergamot (Citrus bergamia Risso et Poit.), Rosa Centifolia L., Damask rose (Rosa Damascena Mill), Carp carvi L., Koitsurum (Coriandrum sativum L.), Eugenia caryophyllat Thunb., Apium graveolens L. var dulce (Mill. ) Pers), Estragon (Artemisia dracunculus L.), Daidai (Citrus aurantium L. spp amara), Cumin (Cuminum cyminum L.), Bay (Pimenta racemosa), Western cedar (Cupressus sempervirens), Geranium (Pelargonium graveolens.), Vetiver zizanioides, Tadgets (Tagetes glanduifera Schrank (T. minuta. L.)), Patchouli (Pogostemon cablin), Amyris (Amyris) among balsamifera), Sokei (Jasminum grandiflorum L.), Shabu (Acorus calamus (L.)), Davana (Artemisia pallens Wall.), Nanyo Sanroku (Murraya koenigii (L.) Sprengel), Nigera (Nigella sativa L.) A β-secretase inhibitor comprising an essential oil extracted from at least one raw material.   A food or drink comprising the β-secretase inhibitor. An inhibition rate measuring method for measuring an inhibition rate of the β-secretase inhibitor,
First, preparing a sample solution comprising a solution containing the β-secretase inhibitor, a solution containing a fluorescently labeled substrate peptide, and a solution containing β-secretase;
And a step of preparing a control solution comprising a dimethyl sulfoxide (DMSO) solution, a solution containing a fluorescently labeled substrate peptide, and a solution containing β-secretase,
Next, the sample solution and the control solution are each subjected to high-performance liquid chromatography (HPLC) to give a fluorescently labeled degradation product of the substrate peptide, an unlabeled degradation product of the substrate peptide, and an unreacted substrate. Separated into peptides, measured the luminescence intensity of the fluorescence-labeled degradation product of the sample solution and the fluorescence-labeled degradation product of the control solution, by comparing each of the emission intensity of the β-secretase inhibitor Determining the inhibition rate; and
An inhibition rate measuring method comprising:

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