KR20050077393A - Composition comprising the extract of rubus coreanus having neuronal cell-protecting activity for preventing and treating brain diseases - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of brain diseases, including bokbunja extract having a protective effect on neuronal cell damage induced by cerebral ischemia, the bokbunja extract of the present invention has an excellent effect of inhibiting nerve cell damage caused by cerebral ischemia Not only harmless to the human body, it can be usefully used as a pharmaceutical and health functional food for the prevention and treatment of brain diseases caused by neuronal cell death.

Description

Composition comprising the extract of Rubus coreanus having neuronal cell-protecting activity for preventing and treating brain diseases

The present invention relates to a composition for the prevention and treatment of brain diseases, including bokbunja extract having a neuronal cell damage protection activity.

According to the ratio of the elderly population in Korea, released by the National Statistical Office in October 2003, the population aged 65 and older accounted for 7.2% of the total population in 2000, entering the aging society. It is expected to enter an aging society. As the aging problem becomes a social issue in recent years, the public's interest in the characteristics of the elderly population, the elderly, such as housing, health, culture, leisure, etc. is increasing, and the demand for statistics is increasing. The key to this change is that chronic degenerative diseases are becoming more of an issue than acute infectious diseases, which have been the main cause of death over the past 50 years due to the aging population. In particular, the death from cerebrovascular disease among chronic degenerative diseases is a very important disease that ranks second in the mortality rate from a single disease.

Cerebrovascular diseases can be classified into two types. One is hemorrhagic brain disease seen in cerebral hemorrhage and the like, and the other is ischemic brain disease caused by occlusion of cerebrovascular vessels. Hemorrhagic brain disease is mainly caused by traffic accidents, and ischemic brain disease is a disease that frequently occurs in elderly people.

When transient cerebral ischemia is induced in the cerebrum, the supply of oxygen and glucose is blocked and ATP decreases and edema occurs in neurons, resulting in extensive brain damage. Neuronal death occurs after a significant time after cerebral ischemia, which is called delayed neuronal death. Delayed neuronal cell death was observed in a transient forebrain ischemic model using Mongolian gerbil. (Kirino T, Sano K., Acta. Neuropathol., 62 , pp 201-208, 1984; Kirino T., Brain Res ., 239 , pp 57-69, 1982).

There are two mechanisms of neuronal death caused by cerebral ischemia that most people accept so far. One is that excess glutamate accumulates outside the cell by cerebral ischemia. Excited neuronal cell death mechanism (Kang TC et al., J. Neurocytol., 30 , pp945-955, 2001) that causes neuronal cell death due to calcium accumulation and sudden increase of radicals in vivo due to sudden oxygen supply during ischemia-reperfusion And two mechanisms of oxidative neuronal death caused by damage to DNA and cytoplasm (Won MH. Et al., Brain Res ., 836 , pp70-78, 1999; Sun AY, Chen YM., J. Biomed. Sci ., 5 , pp 401-414, 1998; Flowers F, Zimmerman JJ., New Horiz., 6 , pp 169-180, 1998).

Based on these mechanism studies, research has been conducted to search for substances that effectively inhibit neuronal cell death in cerebral ischemia or to reveal the mechanism of the substance. However, there are few substances that effectively inhibit neuronal cell death caused by cerebral ischemia. .

Tissue plasminogen activator, a commercially available FDA and Food and Drug Administration (FDA) approved cerebrovascular ischemia, is a substance that induces rapid supply of oxygen and glucose by melting blood clots that induce cerebral ischemia . Therefore, it is not necessary to directly protect nerve cells, so it is necessary to use it quickly. Due to its thrombolytic properties, excessive use or frequent use of the wall thins down and eventually causes hemorrhagic cerebrovascular disease.

In addition, MK-801, a calcium channel blocker (calcium channel blocker) for effectively inhibiting the initial calcium influx, has been clinically tested, but the drug has been discarded due to its side effects.

On the other hand, in Korea, a large number of natural substances that have a stroke prevention effect are marketed as health foods, but most of them are not scientifically verified, and they are a cause of social food abuse.

Therefore, there is an urgent need for the development of natural materials that have a long-term use and proven safety, and have objective effects on the treatment and prevention of brain diseases.

The bokbunja ( Rubus coreanus MIQ) of the present invention is an immature fruit of the bokbunja strawberry belonging to the Rosaceae, the fruit is large enough around July and is not ripe yet when the blue color is removed and used to remove the stem and leaves. Bokbunja contains carbon (Carvone), sugars and small amounts of vitamin C. It is used for cold, fertilization, and tonic medicine. It is effective in relieving weakness, tone, energetic improvement, tonic protection, gangjeong, diabetes, and jogging. Efficacy in weakening, tinnitus, etc., is known to be effective in women's skin beauty, vision disorders (Bo Bo-seop and Shin Min-kyo, Hyangjedae, Yeonglimsa, pp652-653, 1998). Bokbun is especially used for diabetes and sexual decline (Moon, GS, Constituents and Uses of Medicinal Herbs, Ilweolseogak, Seoul, pp310-311, 1991), tannin, diterpene, and catechin. Separately reported (Kim SW et al., Arch. Pharm. Res. , 24 , pp412-415, 2001; Tanaka T. et al., Chem. Pharm. Bull., 41 , pp1214-1220, 1993) .

Accordingly, the present inventors completed the present invention by confirming that the bokbunja leaf extract of the present invention has an activity capable of significantly inhibiting cerebral ischemic neuronal cell death while searching for a natural substance having a treatment and prophylactic effect on brain disease.

An object of the present invention is to provide a composition for the prevention and treatment of brain diseases containing bokbunja extract and an active ingredient which is harmless to the human body and can prevent neuronal cell damage caused by cerebral ischemia.

In order to achieve the above object, the present invention provides a crude extract or a non-polar solvent soluble extract and a composition for the prevention and treatment of brain diseases caused by neuronal cell death containing the same as an active ingredient.

The bokbunja may use immature fruit, mature fruit, leaves or seeds, preferably bokbunja leaves may be used.

The crude extract includes an extract available in water, a polar solvent of C ₁ to C ₄ lower alcohol or a mixed solvent thereof.

In addition, the nonpolar solvent soluble extract includes a non-polar solvent such as lower acetate, such as ethyl acetate, chloroform, hexane, acetone, dichloromethane, carbon tetrachloride, preferably an extract soluble in ethyl acetate.

Brain diseases caused by neuronal death may include stroke, stroke, dementia, Parkinson's disease, Huntington's disease, Pick disease and Creutzfeld-Jakob disease.

Hereinafter, the present invention will be described in detail.

The bokbunja crude extract and the non-polar solvent soluble extract of the present invention are each dried bokbunja ripe fruit, immature fruit, about 1 to 20 times the weight of leaves and seeds, preferably about 5 to 10 times the volume of water, lower alcohol or their Mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably 1: 0.2 to 1: 3, at a extraction temperature of 10 to 100 ° C, preferably 10 to 60 ° C, for about 0.5 hour to 1 day, Preferably, the extraction method such as ultrasonic extraction, reflux extraction, etc. for about 0.5 to 12 hours is repeated 1 to 5 times, preferably 2 to 5 times, so as to be a soluble extract according to water, lower alcohol or a mixed solvent thereof. Crude extracts can be obtained.

After the crude extract is suspended in a polar solvent such as water or methanol, it is dissolved in a polar solvent by adding a nonpolar solvent such as hexane, ethyl acetate or chloroform in a volume of about 1 to 100 times, preferably about 1 to 50 times, of the suspension. The nonpolar solvent soluble layer may be fractionated once to 10 times, preferably 2 times to 5 times, to obtain a nonpolar solvent soluble part and a polar solvent soluble part. The nonpolar solvent soluble layer may be subjected to flash column silica gel chromatography, liquid, and the like. Chromatography (LC) and thin layer column chromatography (TLC) can be separated into 1 to 15 fractions, preferably using flash column silica gel chromatography, which in turn is characterized by gas chromatography (Gas Chromatogra phy) and Mass spectrometry can be used to analyze markers that exhibit activity.

Additionally, the non-polar solvent soluble extract of the present invention may be prepared by suspending the water soluble extract, the lower alcohol soluble extract, and the soluble extract by a mixed solvent thereof in water, and then about 1 to 10 times of the suspension, preferably about Non-polar solvents such as lower acetate, chloroform, acetone, dichloromethane, carbon tetrachloride, etc., such as 1 to 5 times the volume of ethyl acetate, can be obtained by fractionation of 1 to 5 times, preferably 2 to 4 times. In addition, conventional fractionation processes can also be carried out (Harborne JB; Phytochemical methods: A guide to modern techniques of plant analysis, 3rd Ed. , Pp 6-7, 1998).

The present invention provides a pharmaceutical composition for the prevention and treatment of brain diseases caused by neuronal cell death, including bokbunja crude extract or a non-polar solvent soluble extract obtained by the above manufacturing process.

The bokbunja leaf extract obtained as described above of the present invention can be confirmed that by significantly reducing the volume of edema caused by nerve cell damage in vivo in the brain ischemia animal model, inhibits neuronal cell death caused by cerebral ischemia.

The bokbunja has been used for a long time as a herbal medicine and edible extracts of the present invention extracted from them also have no problems such as toxicity and side effects.

The pharmaceutical composition for preventing and treating brain diseases of the present invention comprises 0.1 to 50% by weight of the extract based on the total weight of the composition.

Pharmaceutical compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, or may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

Pharmaceutical compositions comprising extracts according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The extract of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a health functional food comprising the extract and a food acceptable food supplement additive exhibiting a prophylactic effect of brain disease. Examples of the food to which the bokbunja extract can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of preventing brain diseases. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100ml. have.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for having the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the extract of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extract of the present invention may contain fruit flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Below, The invention is illustrated in detail by the following examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example 1. Preparation of Bokbunja Crude Extracts

Bokbun, grown in Korea (Gochang), was purchased and used at the Gochang Gunbok Molecule Research Laboratory. 300 g of each dried bokbunja ripe fruit, immature fruit, leaves and seeds were cut and finely rinsed, and 2 liters of 85% methanol aqueous solution was sonicated at 15 ° C. twice for 1 hour, and then combined with the filter paper (Whatman, USA). ), Then concentrated under reduced pressure with a vacuum concentrator (EYELA, N-1000, Japan) and dried, dried bokbunja ripe fruit (yield 35g), immature fruit (yield 45g), leaves (yield 24g) and Crude extracts of each of the seeds (yield 15 g) were obtained.

Example 2. Preparation of Bokbunja Leaf Polar Solvent and Nonpolar Solvent Soluble Extract

24 g of the bokbunja leaf crude extract obtained in Example 1 was suspended in 500 ml of water, and ethyl acetate was added to the same amount. Soluble parts were collected. The same volume of ethyl acetate solvent as the lower layer (water layer) was repeated three times in the same manner as described above to obtain a substance that can be dissolved in the ethyl acetate layer as much as possible until the color of the solution becomes light. The ethyl acetate soluble extract (8.8 g) and bokbunja leaf water soluble extract (21.2 g) were obtained.

Example 3. Analysis of Indicators

8.8 g of the bokbunja leaf ethyl acetate soluble extract obtained in Example 2 was subjected to flash column silica gel chromatography (flash column silica gel chromatography, Biotage, Autflash 70) using an eluent (hexane: ethyl acetate = 100: 1 → 1: 1). ), 15 fractions were obtained. Of the 15 fractions, three small fractions showing nitric oxide generation inhibitory activity were subjected to gas chromatography (Gas Chromatography, Japan JEOL, LTD, LTD, JMS_AX505WA) and mass spectrometry (Japan JEOL, LTD, JMS_AX505WA). Indicators present in the fractions were analyzed.

As a result, compounds exhibiting activity in these fractions were identified as Aglycone and derivatives thereof of Kaempferol, and are shown in Table 1 below.

ppm Equol Enterodiol Enterlaclactone Catechin Genistein Kaempferil Apienin Quercitrin Bokbunja Immature Fruit 0.14 nd nd 2343.36 6.56 4.82 2.59 nd Bokbunja Ripe Fruit nd nd nd 4.54 4.02 0.80 0.65 59.40 Bokbunja Leaf nd nd nd 4.86 nd 121.18 nd nd Bokbunja Seed 0.13 0.04 0.08 nd 3.61 1.08 0.70 nd     nd: not detected

Experimental Example 1. Measurement of inhibitory effect on cell damage by induction of cerebral ischemia

To investigate the effect of Bokbunja extract on the local cerebral ischemia model, we used an intraluminal suture method (Quartremain, D. et al, Cerebrovascular Disease , 16 (4) , pp346-355, 2003). The effect of inhibiting neuronal damage by induction of cerebral ischemia was measured.

After induction of the local cerebral ischemia model, 0 minutes and 2 hours, that is, after reperfusion, 100 mg / kg of bokbunja leaf extract obtained in Example 1 and 20% Tween were simultaneously administered to the experimental group. After inducing cerebral ischemia in the brain of Wiarar rats, the cervical dislocation is used to extract the brain and the suture junction (bregma) according to the brain dissection guide, Bri. J. Neurosurg , 10 (2) , pp 155-160, 1996. ) Was cut to a thickness of 2 mm from the +5 mm area, and then discarded on both sides, and the obtained brain sections were stained with TTC to measure the volume of the brain injury site.

As a result, when the bokbunja leaf extract is administered at a concentration of 100 mg / kg it was confirmed that by suppressing the cell damage site by more than 40% by inhibiting neuronal cell death caused by cerebral ischemia (see FIGS. 1 and 2).

Experimental Example 2. Toxicity Test

2-1. Oral administration

ICR-based mice and Sprague Dawley were divided into four groups of 10 animals each, followed by oral administration of the bokbunja extract of the present invention at doses of 500, 725, 1000 and 5000 mg / kg, respectively, and observed for toxicity for 2 weeks. As a result, none of the four groups died, and no symptoms were apparent from the control group.

2-2. Intraperitoneal administration

Toxicity for 24 hours after intraperitoneal administration of bokbunja extract of the present invention at doses of 25, 250, 500 and 725 mg / kg, respectively, divided into four groups of 10 mice each weighing 25 grams 5g and Sprague Dawley As a result, none of the four groups died, and no symptoms were apparent from the control group.

As a result, the bokbunja extract of the present invention was confirmed that there is little acute toxicity, it is safe.

Bokbunja extract of the present invention can be administered in the following formulations, the following formulation examples are merely to illustrate the invention, whereby the content of the present invention is not limited.

Formulation Example 1 Preparation of Powder

100 mg of extract of Example 1

Corn Starch 100mg

Lactose 100mg

Talc 10mg

The above ingredients are mixed and filled in airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

100 mg of extract of Example 2

Corn Starch 100mg

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

100 mg of extract of Example 1

Lactose 50mg

1 mg magnesium stearate

The above ingredients are mixed and compressed into tablets according to a conventional method for preparing capsules to fill gelatin capsules.

Formulation Example 4 Preparation of Liquid

100 mg of extract of Example 2

10 g of isomerized sugar

10g per book

Lemon flavor

Purified water

According to the conventional method for preparing a liquid solution, each component is added to the purified water, dissolved, and lemon flavor is added, and then purified water is added to adjust the total amount to 100 ml, and then filled into a brown bottle to prepare a liquid solution.

Formulation Example 5 Preparation of Healthy Food

1000 mg of extract of Example 1

Vitamin Mixture

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 6 Preparation of Healthy Drink

1000 mg of extract of Example 2

Revitalization extract 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is a composition suitable for a preferred beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, use purpose.

As described above, the bokbunja extract of the present invention not only has an excellent effect of inhibiting neuronal cell death generated in cerebral ischemia, but also harmless to the human body, medicines and health functions for preventing and treating brain diseases caused by neuronal cell death. It can be effectively used in food.

1 is a diagram showing the neuroprotective effect of bokbunja leaf extract (100mg / ㎏) in in vivo cerebral ischemia model (MCAo),

Figure 2 is a diagram showing the effect of bokbunja leaf extract on edema volume inhibition of brain tissue in cerebral ischemia induced rats.

Claims (8)

Rubus coreanus MIQ crude extract or nonpolar solvent soluble extract and pharmaceutical composition for the prevention and treatment of brain diseases containing the same as an active ingredient. The pharmaceutical composition of claim 1, wherein the crude extract is extracted with water, a polar solvent of C ₁ to C ₄ lower alcohol, or a mixed solvent thereof. The pharmaceutical composition according to claim 1, wherein the nonpolar solvent soluble extract is extracted with lower acetate, such as ethyl acetate, chloroform, hexane, acetone, dichloromethane or carbon tetrachloride. 4. The pharmaceutical composition according to claim 3, wherein the nonpolar solvent soluble extract is extracted with ethyl acetate. The pharmaceutical composition according to claim 1, wherein the bokbunja use leaves. The pharmaceutical composition of claim 1, wherein the brain disease is stroke, stroke, dementia, Parkinson's disease, Huntington's disease, Pick disease, or Creutzfeld-Jakob disease. A health functional food comprising a bokbunja crude extract or a nonpolar solvent soluble extract and a food supplement acceptable food additive which exhibit a prophylactic effect of a brain disease. The health functional food according to claim 7, which is a health beverage.