KR100962010B1 - An extract of Zizyphus jujuba MILL. var spinosus and a pharmaceutical composition comprising thereof for promoting neurogenesis - Google Patents

Abstract

The present invention relates to a jujube extract and a pharmaceutical composition for promoting neuronal cell production containing the same as an active ingredient, and more specifically to a jujube extract and a pharmaceutical composition for promoting neuronal cell production by promoting neural stem cell differentiation containing the same as an active ingredient It is about.

Since the composition of the present invention promotes neuronal cell production in the gyrus of the hippocampus in animal experiments, it can be usefully used as a medicament and health food to improve neuronal cell production and promote the production of neurons.

Jujube, nerve cell generation, aging, teeth mockup

Description

Jujube extract and pharmaceutical composition for promoting neuronal production containing the same as an active ingredient {An extract of Zizyphus jujuba MILL. var spinosus and a pharmaceutical composition comprising particular for promoting neurogenesis}

The present invention relates to a jujube extract and a pharmaceutical composition for promoting neuronal cell production containing the same as an active ingredient.

Mammalian brains are the first to develop in living organisms, and neural stem cells develop complex neural networks through a series of processes including division, differentiation, survival and apoptosis, and synapse formation. It can be done.

Recently, neural stem cells have been identified in adults, and stem cells have been reported to develop and differentiate in adult brains (Johansson, C. B. et al., Cell 96, 25-34, 1999). The adult stem cells are known to occur in two extremely localized regions rather than throughout the brain. One is the subventricular zone, the lower part of the lateral ventricle, and the other is the hippocampus. subgranular zone in the dentate gyrus of hippocampus (van Praag et al., Nature 415, 1031-1034, 2002).

On the other hand, neurons die every day, not only in neurological disorders, but also in the brains of normal adults, and the number of neurons dying with age increases exponentially (Yuan and Yankner, Nature. 407, 802-809, 2000). In particular, the hippocampus is an indispensable part of external memory, and is known as the site most easily damaged by aging (Scoviller and Milner, J Neurol Neurosurg Psychiatry 20, 11-21, 1957). Therefore, by promoting the generation and differentiation of neural stem cells in these areas will be able to compensate for the neuronal cell death caused by aging by promoting neuronal production.

On the other hand, jujube is known to restore the function of the internal organs, to strengthen the whole body, to stabilize the nerves, and to control and protect the five intestines according to the <New Nongbon Carbide>, <Ilhwaboncho>, and <Baekbangbang>. Zyzyphoside, which is contained only in jujube, is a constituent of glycosides which plays a pharmacologically important role, and is contained in jujube, lysine, aspartic acid, glycine, and asparagine. ), Glutamic acid (glutamic acid), alanine (alanine), valine (valine), leucine (leucine), proline (proline) and other amino acids are also contained.

Thus, the present inventors have completed the present invention by confirming that the jujube extract significantly increases the number of new neurons in the tooth gyrus of the mouse hippocampus during the aging process.

An object of the present invention is to provide a jujube extract and a pharmaceutical composition for promoting the production of neurons and health foods containing the same as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for promoting nerve cell production containing jujube extract and the same as an active ingredient.

Jujube extract of the present invention is cut into the skin, pulp and seeds of dried jujube, about 1 to 20 times, preferably about 2 to 6 times the dry weight of water, C ₁ to C ₄ lower alcohol or a mixed solvent thereof , Cold extraction, hot water extraction, ultrasonic extraction, reflux at 20 to 150 ° C., preferably at 70 to 120 ° C. for about 1 hour to 10 days, preferably about 5 to 10 days, preferably with water or methanol The extraction liquid obtained by extraction method such as cold extraction, preferably cold needle extraction, can be obtained by filtration, concentration under reduced pressure or drying.

Specifically, when the extraction solvent is water, hot water extraction for 3 hours to 12 hours at 70 to 120 ℃ temperature, and if the extraction solvent is alcohol, cold extraction for 5 to 10 days at 20 to 70 ℃ temperature is preferable. .

Jujube is the Zizyphus tree of the Rhamnaceae family. jujuba MILL.), Zizyphus jujuba MILL.var.inermis (BGE.) REHD.) Zizyphus jujuba MILL. var. inermis) fruit flesh, skin and seed, more preferably fruit skin.

The pharmaceutical composition of the present invention preferably contains 0.01 ~ 50% jujube extract, more preferably contains 0.01 ~ 25%. However, the composition as described above is not necessarily limited thereto, and may vary depending on the condition of the subject and the type and extent of the disease.

The pharmaceutical composition of the present invention can be used to treat wound brain injury, wound spinal cord injury, Huntington's disease, Parkinson's disease or Alzheimer's disease.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. Specifically, it may be used in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods, and include extracts. Carriers, excipients and diluents that may be included in the composition 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 styrate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and 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 subject's condition and weight, extent of disease, drug form, route of administration, and 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 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

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

In another aspect, the present invention provides jujube extract and health food for promoting neuronal production containing the same as an active ingredient.

The present invention may include a food supplement acceptable food supplement to the jujube extract, and possible health foods include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and powders and granules. , Tablets, capsules or beverages. Jujube extract itself has little toxicity and side effects, so it can be used safely for prolonged periods of prevention.

The extract of the present invention may be added at 0.01 to 100% by weight of the total food weight when added to food or beverage, preferably at 90 to 99.99% by weight. The beverage may be added at a rate of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml. In addition to containing the extract as an essential ingredient in the indicated ratio, the beverage is not particularly limited in liquid component, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks.

Examples of the above-mentioned natural carbohydrates are conventional monosaccharides such as disaccharides such as glucose and fructose, such as maltose, sucrose and the like, and polysaccharides such as dextrin, cyclodextrin and the like. Sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of 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 composition 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. Others may contain pulp 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 critical but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Jujube extract of the present invention can be usefully used as a pharmaceutical composition and health food to promote the production of neurons by improving the reduction of differentiation of stem cells into neurons during aging.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

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

Example  1. Preparation of Jujube Extract

1-1. Jujube pericarp  Preparation of Extract

After purchasing dried jujube at the Chinese herbal medicine market (Daejeon, Korea), 200 g of skin was chopped and 0.8 L of 100% methanol was added and chilled for one week. The cold extract methanol extract was connected to a pressure reducing flask (aspirator) to depressurize the inside of the flask, and then filtered into a filter connected to the pressure reducing flask. The filtrate was concentrated under reduced pressure with a vacuum concentrator (BUCHI 011, Switzerland) to obtain 16.6 g of jujube bark extract, which was used as a sample of the following experimental example.

1-2. Preparation of Fruit Extract of Jujube

Except for using the pulp instead of the skin was subjected to the same reaction process as in Example 1-1 to obtain 7.2g of jujube pulp extract was used as a sample of the following experimental example.

Experimental Example  1. Measurement of Stem Cell Differentiation of Jujube Extract

In order to confirm the stem cell differentiation promoting effect of jujube extract was tested using the method of labeling the differentiation of stem cells with BrdU (Bromodeoxyuridine) in C57BL / 6J mice (Jackson Laboratory, USA).

1-1.Laboratory Animals

Experimental animals used 20 male 12-month-old C57BL / 6J mice weighing 25-30 g, and had a temperature of 23 ± 2 ° C. and a relative humidity of 55 ± 10% under constant light and dark cycles from 7 am to 7 pm. It was raised, and food and water were taken freely.

1-2. Stem Cell Differentiation Measurement

0.2 ml of 23.8 mg of the jujube extract obtained in Example 1 in 2 ml of distilled water was orally administered for at least 20 days using an esophageal needle (the dose was administered to 100 mg / kg body weight), and the control group was a solvent. Phosphorous distilled water was administered in the same amount. After administration for 20 days, 12.5 mg of BrdU (Sigma, USA) was dissolved in 1 ml of physiological saline, and then intraperitoneally administered 0.1 ml twice at 12 hour intervals to label stem cells. Since BrdU only enters the DNA of dividing cells like stem cells and not into cells that have already been formed, the presence of this substance in the cell indicates that labeled cells differentiated into neurons after BrdU was injected.

Four hours after the last BrdU administration, an intraperitoneal injection of thiopental sodium (30 g / kg, Korea) at a dose of 30 mg / kg body weight was used to anesthetize the control and experimental groups, followed by containing 1,000 IU of heparin per 1,000 ml. Physiological saline at 4 ° C. was injected into the left ventricle and washed perfusion. After the perfusion wash, the animals were immediately perfused with 4% paraformaldehyde (pH 7.4 in 0.1 M phosphate buffer) at 4 ° C.

After perfusion fixation, the brain was removed by opening the head bone space using a bone cutter, and then fixed in the same fixative for 6 hours. The post-fixed brains were submerged in a 30% sucrose solution (in 0.1 M phosphate buffer) until they settled to the bottom and then the tissues were slid into a 30 μm thickness with a sliding microtome (Reichert-Jung, Germany). Cut and stored in a 6-well plate containing a preservation solution (10% polyethylene glycol and 10% glycerol in PBS) and stored at 4 ℃ until use.

From each tissue section, tissues with well-discussed hippocampus were selected, washed three times with 0.01 M PBS for 10 minutes to wash the stock solution on the tissue, and then endogenous peroxidase present in the tissue sections. The reaction was reacted with 0.3% H ₂ O ₂ (in PBS) for 30 minutes to remove. The DNA was then denatured by reacting with a 65 ° C. 50% formamide (in 2 × standard citrate solution) solution for 2 hours and then with 37 ° C. 2N HCl for 30 minutes. To prevent nonspecific immune responses, the reacted tissue sections were reacted with 3% normal goat serum (Dako, USA) for 30 minutes.

Tissue sections reacted with 3% normal goat serum were reacted with rabbit anti-BrdU (Chmicon Internationl, USA), a primary antibody diluted 1: 1000, to label stem cells for 48 hours at 4 ° C. After completion of the reaction, the tissue sections were reacted with anti-rabbit IgG (Vector, USA), a secondary antibody diluted at 1: 200, for 2 hours, and then, with ABC solution (Vector), a tertiary antibody diluted at 1: 200, for 2 hours. The reaction was performed time, and 3,3'-diaminobenzidine (DAB) was developed as a substrate. Each step was washed three times for 10 minutes using 0.01 M PBS. After tertiary reaction, the tissue sections were plated in gelatin-coated slide glass (Superior, Germany) and dried at room temperature for 12 hours, and the dried tissue sections were enclosed through a conventional dehydration and transparency process.

Each tissue was photographed using the AxioM1 microscope (AxioM1 microscope, Carl Zeiss, Germany) attached with a digital camera (Axiocam, Cal Zeiss, Germany) 100 times the entire tooth gyrus and 400 times the subgranular cell area. 1 is shown. The darkly stained cells were shown in FIG. 2 by counting the relative number of neurons using an image analyzer (Optimas 6.5, CyberMetrics, USA) program. Student t-test was performed to verify the significance of each group, and the data were expressed as mean and standard mean error.

As a result, in FIG. 1, the control group (A and C) to which the solvent was administered was able to confirm staining of BrdU in a few cells, whereas in the experimental group to which the jujube extract 100 mg / kg was administered (B and D), In the gyrus was able to identify a number of cells stained with BrdU.

In addition, as a result of confirming the stem cells stained with BrdU according to the administration of jujube extract in Figure 2, 347.2% of BrdU stained cells were observed in the control group when 100 mg / kg jujube extract was administered, It was confirmed that the administration of jujube extract drastically changed the number of stem cells.

Therefore, the jujube extract of the present invention promotes neural stem cell differentiation and consequently promotes the production of neurons significantly.

Hereinafter, the preparation examples of the composition comprising the extract of the present invention, but the present invention is not intended to limit it, but is intended to explain in detail only.

Formulation example  1. Preparation of Pharmaceutical Composition

1-1. Powder  Produce

100 mg of jujube extract of Example 1

Lactose 100 mg

Talc 10 mg

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

1-2. Manufacture of tablets

100 mg of jujube extract of Example 1

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

1-3. Preparation of Capsules

100 mg of jujube extract of Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

1-4. Liquid  Produce

100 mg of jujube extract of Example 1

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve it, and then, lemon flavor is added, the above components are mixed, and then purified water is added to adjust the total amount to 100 ml. Manufacture.

Formulation example  2. Manufacturing of health food

2-1. Preparation of Granules

2500 mg of jujube extract of Example 1

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 10.13 mg

Vitamin B ₂ 20.15 mg

Vitamin B ₆ 60.5 mg

120.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

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 vitamin and mineral mixture is a composition suitable for a relatively healthy food in a preferred embodiment, the composition 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.

1 is an oral administration of jujube extract followed by staining the gyrus tissue with BrdU is a diagram observed,

A and C: tooth gyrus whole area

B and D: subgranular area

Figure 2 is an oral administration of jujube extract, and then administered BrdU is a diagram showing the number of cells stained in BrdU as a ratio to the control group.

Claims (13)

A pharmaceutical composition for promoting neuronal cell production comprising jujube extract prepared by extracting jujube with a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, and a mixture thereof as an active ingredient. The method according to claim 1, The extraction is characterized in that cold extraction, hot water extraction, ultrasonic extraction or reflux cooling extraction for 1 hour to 10 days at a temperature of 20 to 150 ℃ with a solvent of 1 to 20 times the dry weight of jujube. The method according to claim 1, The extraction is characterized in that the cold extraction for 5 to 10 days at a temperature of 70 to 120 ℃ with 2 to 6 times the weight of the jujube dry weight. delete The method according to claim 1, The neuron production is characterized in that by the neural stem cell differentiation promotion. The method according to claim 1, A composition, which is used to treat wound brain injury, wound spinal cord injury, Huntington's disease, Parkinson's disease or Alzheimer's disease. The method according to claim 1, The active ingredient is a composition, characterized in that 0.01 to 50% by weight based on the total weight of the pharmaceutical composition. The method according to claim 1, The active ingredient is a composition, characterized in that 0.01 to 25% by weight based on the total weight of the pharmaceutical composition. A health food for promoting neuronal cell production comprising a jujube extract prepared by extracting jujube with a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, and a mixture thereof. The method according to claim 9, The neuron production is a health food, characterized in that by promoting neural stem cell differentiation. The method according to claim 9, Healthy foods that are powders, granules, tablets, capsules or beverages. The method according to claim 9, The active ingredient is a health food, characterized in that contained 0.01 to 100% by weight relative to the total weight of the health food. The method according to claim 9, The active ingredient is a health food, characterized in that 90 to 99.99% by weight based on the total weight of the health food.

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