WO2005018656A1

WO2005018656A1 - Agent for preventing and/or ameliorating brain nerve cell death

Info

Publication number: WO2005018656A1
Application number: PCT/JP2004/011037
Authority: WO
Inventors: Hiroko Isoda; Shinichi Yokota; Yukuo Abe
Original assignee: Kaneka Corporation
Priority date: 2003-08-21
Filing date: 2004-07-27
Publication date: 2005-03-03
Also published as: JPWO2005018656A1

Abstract

It is intended to provide a novel and safe agent for preventing and/or ameliorating brain nerve cell death; a drug for preventing and/or ameliorating Alzheimer’s disease characterized by containing the same; or foods and drinks containing the same. Namely, an agent for preventing and/or ameliorating brain nerve cell death containing, as the active ingredient, a wolfberry extract obtained by extraction with water or a water-based solvent mixture. This agent for preventing and/or ameliorating brain nerve cell death is useful as a drug for preventing and/or ameliorating Alzheimer’s disease.

Description

Specification

Brain nerve cell death prevention and Z or ameliorating agent

Technical field

The present invention relates to a prophylactic and / or ameliorating agent for cerebral nerve cell death and / or an agent for preventing and / or ameliorating Alzheimer's disease, comprising an extract of a wolfberry plant as an active ingredient, and an agent for preventing and / or ameliorating cerebral neuronal cell death or an Alzheimer's disease The present invention relates to a food or drink containing an agent for preventing and / or improving a disease. Background art

Alzheimer's disease is a slow but constantly progressive neurodegenerative disease with clinical symptoms centering on dementia characterized by memory loss and cognitive dysfunction.

With more than 1,000,000 patients worldwide, their prevalence tends to increase with aging, and new treatments and prophylaxis methods are particularly anticipated, especially as the population ages. It is rare. Pathological findings include senile plaques characteristic of Alzheimer's disease, and its main component is an insoluble peptide consisting of about 40 amino acid residues called amyloid 3 protein (A / 3). This amyloid 8 protein (A j3) is important as a substance that induces neuronal cell death in the brain in Alzheimer's disease. The amyloid 3 protein is formed by cleaving its precursor protein with a protease called j3 secretase, and further cleaving it with 7 secretase.

Prevention of neuronal cell death is expected to have an effective therapeutic and ameliorating effect. Presenilin (an enzyme involved in the production of β) is the same as γ-selectase, a protease required for the production of Αβ. Li, YM et al., ature, 405, 689-694, 2000) and vaccine therapy using β-amyloid as an antigen (cf. Chen, G. et al., ature, 408, 975-979, 2000). Possibilities are being considered. In neurodegenerative diseases such as Alzheimer's disease, oxidative stress has been implicated in the pathology, and oxidative stress in living tissue is an oxidative stress marker. It has been clarified that 8-hydroxy-2, 2-hydroxyguanosine (80HdG), a DNA oxide, and 8-hydroxyguanosine (80HG), an RNA oxide, are distributed in the brain of degenerative diseases (Reference: Nunomura, A. et al., J. Neurosci., 19, 1959-1964, 1999). Hemoxidase-11 has been reported to be induced by various oxidative stresses and increased in Alzheimer's disease brain (Literature: Smith, MA et al., Am. J. Pathol., 145). , 42-47, 1994), and the involvement of oxidative stress in the pathology has been suggested. In addition, abnormal cell aggregates such as A in Alzheimer's disease, intranuclear inclusions caused by polyglutamine in Huntington's disease, and α_synuclein in Parkinson's disease may also cause neuronal cell death in neurodegenerative diseases. (See Sherman, MY et al., Neuron, 29, 15-32, 2001).

Lycium plants are plants of the Solanaceae family, and include species such as Lycium chinensis, Lycium barbarum, Lycium turcomanicum, Lycium potaninn, and Lycium dasystemum. The leaves are called lycopodium leaves, the fruits are called lycopodium, and the root bark is called skeletal bark. It is said to enhance liver function, hypertension, atherosclerosis, prevent presbyopia, etc., and in addition to carotene 'vitamines,] 3 sitosterol Contains linoleic acid, zeaxanthin and betaine-fezaliene. As a pharmacological effect, it has been reported that betaine has an effect on lipid metabolism and anti-fatty liver, and an anti-fatty liver effect of a water extract on the liver is known. On the other hand, regarding neuronal cell death and neuronal cell death in Alzheimer's disease, there have been reports on compositions aimed at improving learning ability and memory ability using organic solvent extracts (Republic of Korea No. 2002-0038381). However, there is no report that the water-soluble fraction of the wolfberry plant material has an activity of suppressing neuronal cell death or progression of Alzheimer's disease. DISCLOSURE OF THE INVENTION An object of the present invention is to provide an agent for preventing and / or improving brain nerve cell death or an agent for preventing and / or improving Alzheimer's disease from a safe food material or an extract thereof. The present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, the extract obtained by contacting the wolfberry plant material with water or a mixed solvent mainly composed of water yielded an extract of cerebral nerve cells. The present inventors have found that the compound has a prophylactic and / or ameliorating effect or a prophylactic and / or ameliorating effect on Alzheimer's disease, and has completed the present invention. That is, the first aspect of the present invention relates to a preventive and / or ameliorating agent for cerebral nerve cell death, comprising a Lycium plant material or an extract therefrom as an active ingredient. (Lycium) The present invention relates to a preventive and / or ameliorating agent for Alzheimer's disease comprising a plant material or an extract therefrom as an active ingredient.

Hereinafter, embodiments of the present invention will be described in detail.

The method for producing Lycium plants used in the present invention is not particularly limited. The wolfberry plant used in the present invention may be not only a naturally grown plant but also an artificially cultivated plant. In addition, there are no particular restrictions on the timing of collection, the number of years of growth, the cultivation method, and the cultivation period of the wolfberry plant material used in the present invention.

As the wolfberry plant used in the present invention, only one kind may be selected from those produced by various production methods, or a plurality of species may be used in combination. Preferably, a Lycium chinensis plant is used.

The extract is obtained by extracting various parts of the plant body (whole plant, flower, leaf, etc.) as they are or after pulverizing them and extracting them with an extraction solvent. Further, they can be used in combination with each solvent extract.

As the extraction solvent, water or a mixed solvent mainly composed of water can be used. The water-based mixed solvent referred to in the present invention is mainly composed of water (water content is about 21% or more, preferably about 31% or more, more preferably about 41% or more, and most preferably about 5% or more. 1% or more), and a polar organic solvent such as an alcohol (for example, a lower alcohol such as ethanol or methanol, or a polyhydric alcohol such as glycerin) alone or in any combination of two or more liquids. . The amount of the water-based mixed solvent used for the extraction is such that the water content is about 51% or more from the viewpoint of improving the extraction efficiency of the active ingredient contained in the wolfberry plant material. It is preferable to use an amount capable of sufficiently dissolving. When extracted mainly with water, the components to be extracted are different because the polarity as the solvent is different compared to when mainly extracted with an organic solvent.

When water is used as the extraction solvent, it is preferable to use hot water from the viewpoint of improving the extraction efficiency of the active ingredient contained in the wolfberry plant material. The amount of hot water used for the extraction is preferably an amount capable of sufficiently dissolving the active ingredient contained in the wolfberry plant material.

The production method is not particularly limited, except for the type of the extraction solvent. Usually, the extraction may be carried out using an extraction solvent at normal temperature and normal pressure, and after the extraction, the solution may be concentrated to dryness or oil or fat. It may be in the form of paste, paste, gel, or powder. The extraction temperature can be, for example, from about 0 to 100 ° C, about 30 to 100 ° C, or about 60 to 100 ° C. In some cases, extraction under conditions of about 30 to 120 ° C, and extraction under supercritical conditions using autoclave, carbon dioxide, etc. are also possible. The extraction time can be, for example, about 30 minutes to about 10 days, about 1 hour to 5 days, or about 2 hours to 1 day. If necessary, it can be further purified by an optional operation using activated carbon ram-ion exchange resin or the like. The amount of the extraction solvent may be, for example, about 1 to 100 parts by weight, about 2 to 100 parts by weight, or about 5 to 50 parts by weight, based on 1 part by weight of the wolfberry plant material. .

In the extraction of the present invention, the collected wolfberry material is washed to remove dirt and organisms attached to the surface, and then crushed by cutting, pole mill, ultrasonic treatment, homogenizer, etc., and then extracted. it can. Prior to crushing, air drying or freeze drying may be performed. The crushed wolfberry plant material is preferably extracted several times with an extraction solvent such as hot water while stirring. This extract can be used as it is as an agent for preventing and / or improving brain nerve cell death or an agent for preventing and / or improving Alzheimer's disease. The invention also relates to a method for preventing and / or ameliorating said condition in a mammal.

The extract of the present invention can be adjusted to an appropriate concentration by diluting or concentrating as necessary. Further, by spray-drying the extract, a preventive and / or ameliorating agent for powdery neuronal cell death or prevention and / or prevention of Alzheimer's disease can be obtained. It can also be an improver. As described above, the concentration and shape of the extract of the present invention are not particularly limited, and can be appropriately determined according to the use.

The extract of the present invention can also be used as an orally or non-orally ingestible composition containing components other than an extract of a wolfberry plant material. The composition can be used in foods and drinks, or pharmaceuticals, cosmetics, and baths to which other nutrients or active ingredients are added as long as the effect of wolfberry is not reduced.

The extract of the present invention is highly safe for living organisms because the extract of edible genus moss plant material is used as an active ingredient. Therefore, the extract of the present invention can be contained in a wide range of products such as pharmaceuticals, functional foods, foods and drinks, quasi-drugs, cosmetics, and bath preparations.

According to the present invention, the preventive and / or ameliorating agent for cerebral nerve cell death is preferably an agent for preventing and / or ameliorating a neurodegenerative disease, more preferably an agent for preventing and / or ameliorating Alzheimer's disease. Neurodegenerative diseases can include, but are not limited to, Alzheimer's disease, Parkinson's disease, Pick's disease, Korsakoff's disease, and frontal or subcortical dementia of blood vessels or other progeny.

The agent for preventing and / or ameliorating brain nerve cell death or the agent for preventing and / or ameliorating Alzheimer's disease according to the present invention comprises an extract as an active ingredient, and may be an extract or an extract itself, or a known agent. It may be a composition to which carriers, auxiliaries, food and drink materials, other pharmaceutically acceptable formulation materials, etc. are added. The concentration of the extract can be appropriately selected within a range in which a desired effect is exhibited, and the dose to be administered to the human body is, for example, about 0.01 to 50 mg / kg body weight per day. The drug can be administered in one to several doses, depending on the purpose and route of administration of the drug, depending on the purpose, route of administration, tablets, capsules, injections, drops, powders, suppositories, Granules, ointments, It can be made into turbidity, emulsion, syrup, cream, etc. Also, in this composition, binders, excipients, lubricants, disintegrants, stabilizers, emulsifiers generally used in pharmaceutical preparations And additives such as buffering agents, etc. Preferred examples of the binder include guar gum, acacia powder and the like. Preferred examples of excipients include starch, trehalose, dextrin and the like. Preferred examples of the lubricant include stearic acid, talc, sucrose fatty acid ester, polyethylene glycol and the like. Preferable examples of the disintegrant include starch, carboxymethylcellulose, corn starch and the like. Preferred examples of the stabilizer include fats and oils, propylene glycol, cyclodextrin and the like. Preferable examples of the emulsifier include an anionic surfactant, a nonionic surfactant, and polybutyl alcohol. Preferred examples of the buffer include buffers such as phosphate, carbonate, and citrate.

Furthermore, the extract of the present invention can be contained in foods and drinks, cosmetics and bath preparations. The content thereof is, for example, usually about 0.0001 to 100% by weight as food or drink, and about 0.1 to 100% by weight in view of the fact that it can be ingested efficiently. About 100% by weight, usually about 0.0001% by weight or more as a bath agent, and about 0.001% to 100% by weight is preferable because it can be efficiently mixed.

When the extract of the present invention is contained in cosmetics and used as a preventive and / or ameliorating agent for cerebral nerve cell death or as a preventive and / or ameliorating agent for Alzheimer's disease, it can be efficiently incorporated at about 0.0001% by weight or more. , About 0.01 to 20 weight. /. It is preferred that

Since the extract of the present invention has a preventive and / or ameliorating effect on cerebral nerve cell death and / or a preventive and / or ameliorating effect on Alheimer's disease, by including it in food, the extract can prevent and / or prevent cerebral nerve cell death. Alternatively, it can be a functional food for the purpose of improvement or prevention and / or improvement of Alzheimer's disease. The kind of the food to be targeted is not particularly limited as long as the extract does not inhibit the preventive and / or ameliorating effect of the cerebral nerve cell death or the preventive and / or ameliorating effect of Alzheimer's disease. For example, juices, soft drinks, beverages such as tea, processed foods such as bread and rice cakes, sweets such as candy, instant foods such as cup ramen, fats and oils such as putters, salad oils, dressings, mayonnaise, sauces, soy sauce, etc. It can be included in a wide range of foods and drinks, such as seasonings such as mirin, sprinkles, and miso. Of course, instead of the extract, wolfberry can be crushed as it is, and it can be added directly to food etc., and even if the crushed material is ingested directly, It is presumed that the active ingredient of the wolfberry plant material is extracted by ingested water or digestive fluid in the body.

Since the extract of the wolfberry plant material of the present invention has a neuronal cell death inhibitory action by an amyloid protein which is an Alzheimer's disease-causing substance, it is an agent for preventing and / or ameliorating brain neuronal cell death or preventing and / or ameliorating Alzheimer's disease. Useful as an agent.

The present invention can improve the quality of life by preventing or delaying the progress of symptoms mainly by preventing and / or improving neurodegenerative death due to oxidative stress of brain nerve cells, and improving the quality of life simply. It is not intended to improve learning ability or memory.

Amyloid protein, a causative substance of Alzheimer's disease, deposits in nerve cells in the brain and generates radicals and the like, causing cell damage and cell death. In addition, there are many reports that a common mechanism of neurodegenerative diseases is caused by accumulation of abnormal proteins, for example, amyloid protein in Alzheimer's disease and polyglutamine in Huntington's disease.

The anti-neural cell death evaluation performed in the present invention revealed that the extract of the wolfberry plant material had a cytoprotective effect on amyloid protein. Anti-neuronal cell death evaluation involves, for example, adding a sample extract to human neuroblastoma cell line SH-SY5Y cells, adding amyloid J3 protein, a causative agent of Alzheimer's disease, culturing the cells, and then measuring the number of viable cells. Is possible. The number of viable cells can be easily measured, for example, by a protein quantification method using 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2Htetrazolium bromide (MTT). be able to. The anti-neural cell death evaluation or the anti-Alzheimer effect can be evaluated by measuring the number of viable cells after completion of the culture using the above-described evaluation system. That is, the number of viable cells after the above operation is larger when the plant extract sample has a cytoprotective effect, as compared with the control without the plant extract sample. The relative viability, which indicates the ratio of the number of viable cells in the plant extract sample-treated group to the plant extract-untreated group, exceeds about 1.0 This indicates that the plant extract sample has a preventive and / or ameliorating effect on brain nerve cell death or a preventive and / or ameliorating effect on Alzheimer's disease. Brief Description of Drawings

FIG. 1 shows the results of evaluating anti-neural cell death using human neuroblastoma cell line SH-SY5Y cells.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically with reference to Production Examples, Examples, and Formulation Examples. However, the scope of the present invention is not construed as being limited by these Production Examples, Examples, and Formulation Examples.

(Production Example 1)

1 g of dry weight of Lycium cMnensis plant material was cut and crushed, and 10 ml of water was added, followed by autoclaving at 105 ° C for 15 minutes. After centrifugation at 3000 rpm at 4 ° C for 5 minutes, the supernatant was filtered with a 0.22 μιη filter, and the filtrate was used as a hot water extract sample.

(Production Example 2)

Lycium chinensis (Lycium chinensis) Plant material 1 g of dry weight was cut and crushed. They were placed in ethanol and left in a dark place out of direct sunlight for 4 weeks. After centrifugation at 3000 rpm at 4 ° C for 5 minutes, the supernatant was filtered through a 0.22 μm filter to obtain a 70% ethanol extract sample.

(Production Example 3)

1 kg of dry weight of Lycium chinensis (Lycium chinensis) plant material was cut and crushed, 10 L of water was added, and autoclaving was performed at 105 ° C for 15 minutes. The mixture was centrifuged at 3000 rpm at 4 ° C for 5 minutes, and the supernatant was filtered through a 0.22 μm filter to obtain an extract. The obtained liquid was concentrated to dryness to obtain about 87 g of a solid.

(Example 1) Evaluation of anti-neural cell death 15% FBS, DMEM / Ham's F-12 (1: 1 mixture) 1% non-essential MEM amino acid, 1% penicillin (SOOO ^ ug / ml)-Streptomycin (5000 IU / ml) Using a medium, a human neuroblastoma cell line SH-SY5Y cell (ATCCCRL-2266) was seeded at a concentration of 1.6 × 10 ⁵ cells / ml on a 96-well plate coated with collagen (ΙΟΟ / ml). 5% C0 ₂ under after 37 ° C De晚culture, washed with 100 mu 1 of 0PTI MEM medium (GIBCO-BRL, Inc.), then obtained in Production Example 1 to 0PTI- MEM medium 100 ul of per each Ueru Each of the obtained hot water extract of Mushrooms diluted with a culture medium to a desired concentration was used. Add at / _o (v / v). After amyloid β protein (10_ ⁴ Micromax) was added 10 1 / well are further causative agent of Alzheimer's disease, and cultured for 3 days. Next, add 20 μl of the ΜΤΤ solution (6 mg of 3- (4,5-dimethyl-2-thiazolyl bromide) -2,5-diphenyl-2H tetrazolium bromide (manufactured by Wako Pure Chemical Industries) to lm 1 of PBS. Was added to each well, and the culture was continued for another 4 hours. Thereafter, the MTT formazan produced by adding a 10% SDS solution was dissolved, and the culture was continued for another 12 hours. MTT formazan produced by cells was quantified by measuring absorbance at 570ηπι. This procedure utilizes the fact that the number of surviving cells is proportional to the protein quantification value, and the number of surviving cells was quantified quantitatively. The ratio of the number of viable cells in the plant extract sample-treated group to the plant extract-untreated group is expressed as a relative survival rate. The results are shown in Figure 1.

(Comparative Example 1)

The procedure was performed in the same manner as in Example 1 except that the 70% ethanol extract of Kumikoko obtained in Preparation Example 2 was used instead of the hot water extract of Mushroom plant material obtained in Preparation Example 1. . The results are shown in Figure 1.

The vertical axis in FIG. 1 shows the number of surviving cells (quantitative protein value) in the sample-added group versus the number of surviving cells (quantitative protein value) in the sample-free group, and indicates the relative survival rate. That is, a value greater than 1.0 indicates that the survival rate is higher than that of the group without the sample. The horizontal axis represents the concentration of sampnore used.

As a result, the low concentration cytoprotection i.e. anti Alzheimer effect of 10- ⁸ ~10- ⁴ g / ml in hot water extract sample was observed, observed cytoprotection in 70% ethanol extract sample I couldn't. (Formulation Example 1) Production of wolfberry plant material extract-containing tablets

Wolfberry extract (Production Example 2) 45 parts by weight

Lactose 35 parts by weight

Crystalline cellulose 15 parts by weight

5 parts by weight of sucrose fatty acid ester

The above composition was mixed and tableted to produce a dietary tablet containing a wolfberry plant material extract.

(Formulation Example 2) Production of soft strength peptide containing wolfberry plant material extract

Wolfberry extract (Production Example 2) 40 parts by weight

Olive oil 55 parts by weight

Glycerin fatty acid ester 5 parts by weight

Mix and crush with the above composition, fill into gelatin capsules, contain wolfberry plant material extract

A food and drink power capsule was manufactured.

(Formulation Example 3) Production of soft drink containing wolfberry plant material extract

Wolfberry plant material extract (Production Example 2) 1 part by weight

Liquid bran 30 parts by weight

0.1 parts by weight of citric acid

0.5 parts by weight of fragrance

Water 150 parts by weight

By mixing with the above composition, a soft drink containing a wolfberry plant material extract was produced.

(Formulation example 4) Preparation of a cracker containing wolfberry plant material extract

Wolfberry extract (Production Example 2) 1 part by weight

Soft flour 1 20 parts by weight

Salt

Baking pad

30 parts by weight of putter

40 parts by weight of water A wolfberry plant material extract-containing cracker having the above composition was prepared by a conventional method.

(Formulation Example 5) Preparation of dressing containing wolfberry plant material extract

Wolfberry extract (Production Example 2) 10 parts by weight

Olive oil 80 parts by weight

60% by weight vinegar

Salt 3 parts by weight

1 part by weight of pepper

5 parts by weight of lemon water

A dressing containing the wolfberry plant material extract was prepared by the usual method with the above composition.

(Formulation Example 6) Preparation of wolfberry plant material extract-containing chewing gum

Wolfberry extract (Production Example 2) 0.5 parts by weight

Gum base 20 parts by weight

Sugar 70 parts by weight

0.5 parts by weight of fragrance

Water

A chewing gum containing wolfberry plant material extract was prepared with the above composition by a conventional method.

(Formulation Example 7) Preparation of tea containing wolfberry plant material extract

Extract of wolfberry extract material (Production Example 2) Dissolve an appropriate amount in hot water Industrial applicability

The wolfberry plant material extract described herein is useful for preventing and / or ameliorating cerebral nerve cell death or preventing and / or improving Alzheimer's disease. Since the extract of wolfberry plant material of the present invention is highly safe and can be prepared by a simple method, the extract is expected to be extremely applicable to pharmaceuticals, foods and drinks, and to have a very wide range of application. .

Claims

The scope of the claims

1. A preventive and / or ameliorating agent for cerebral neuronal cell death, comprising a Lycium plant material or an extract therefrom as an active ingredient.

2. The preventive and / or ameliorating agent for brain nerve cell death according to claim 1, wherein the wolfberry plant is Lycium chinensis.

3. The extract of cerebral nerve cell death according to claim 1 or 2, wherein the extract from Lycium plant material is an extract obtained by extracting wolfberry with water or a mixed solvent mainly composed of water. Preventive and / or ameliorating agent.

4. A preventive and / or ameliorating agent for Alheimer's disease, comprising a Lycium plant material or an extract therefrom as an active ingredient.

5. The preventive and / or ameliorating agent for Alzheimer's disease according to claim 3, wherein the wolfberry plant is Lycium chinensis.

6. The prevention and Z or amelioration of Alzheimer's disease according to claim 4 or 5, wherein the Lycium plant extract is an extract obtained by extracting wolfberry with water or a water-based mixed solvent. Agent.

7. The preventive and / or ameliorating agent for cerebral nerve cell death according to any one of claims 1 to 3 or the agent for preventing and / or ameliorating Alzheimer's disease according to any one of claims 4 to 6, and is acceptable for eating and drinking. Food or drink composition comprising a carrier.

8. A preventive and / or ameliorating agent for cerebral nerve cell death according to any one of claims 1 to 3, or an agent for preventing and / or ameliorating Alzheimer's disease according to any of claims 4 to 6, and is also pharmaceutically acceptable. A pharmaceutical composition comprising a carrier.

9. A preventive and / or ameliorating agent for cerebral nerve cell death according to any one of claims 1 to 3 or an agent for preventing and / or ameliorating Alzheimer's disease Z or ameliorating agent according to any of claims 4 to 6, and is acceptable for cosmetics. A cosmetic composition comprising a carrier.

10. The preventive and / or ameliorating agent for Z or cerebral nerve cell death according to any one of claims 1 to 3, which comprises contacting a wolfberry plant material with water or a water-based mixed solvent. The method for preventing and / or modifying Alzheimer's disease according to the above.

11. The method for producing a composition according to any one of claims 7 to 9, comprising contacting the wolfberry plant material with water or a water-based mixed solvent.

12. A method for preventing and / or ameliorating cerebral nerve cell death, comprising administering to a mammalian subject an effective amount of any one of claims 1 to 3.