KR20100000693A - Composition comprising the extract of platycodon grandiflorum for the improvement of age-related decrease in neurogenesis - Google Patents

Abstract

PURPOSE: A functional food for promoting differentiation of neutral stem cell containing platycodon root extract as an active ingredient is provided to suppress the reduction of differentiation of proliferation of neutral stem cells caused by aging and to promote differentiation of proliferation of neutral stem cells. CONSTITUTION: A functional food comprises platycodon root extract as an active ingredient to promote differentiation of proliferation of neutral stem cells. The nerve stem cells are derivated from dentate gyrus subgranular zone of hippocampus. The differentiation of the stem cell comprises the production of stem cells, fission, differentiation and movement. The platycodon root extract is obtained by cutting the dried platycodon root; extracting it at 20-150deg.C through enfleurage extraction, hot water extraction, ultrasonic extraction, and extraction under reflux condensation.

Description

Composition comprising the extract of Platycodon grandiflorum for the improvement of age-related decrease in neurogenesis}

The present invention relates to a functional food for promoting neural stem cell differentiation containing bellflower extract as an active ingredient.

Unlike other tissues, the neural tissue is known to be a tissue in which a series of processes such as division, differentiation, and synapse formation are all performed during development, and it has been known that little differentiation occurs after birth.

Recent studies have shown that neurons regenerate at very limited sites, which are located in the subventricular zone, the lower part of the lateral ventricles, and in the dental gyrus of the hippocampus. Is known as the subgranular zone at the base of granule cells (Kempermann et al., 2003; Larsson et al., 2004; Paizanis et al., 2007).

Cells produced in the subventricular zone migrate through the rostral migratory stream to the anterior part of the brain, resulting in differentiation into granule cells in the olfactory bulb or neuroglial cells in this region. (neuroglia) (Pencea et al., 2001; Menezes et al., 2002; Bedard and Parent, 2004; Martoncikova et al., 2006).

Cells produced in the subgranular zone are known to migrate upward in the subgranular zone to become granular cells of the dentate gyrus (Kuhn et al., 1996; Reznikov, 1991; Eckenhoff and Rakic). , 1988), the granule cells thus produced are linked to the CA3 part of the hippocampus, which is associated with the intrinsic function of the hippocampus (Li and Pleasure, 2005).

On the other hand, neurons die every day, not only in neurological diseases, 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 known as an indispensable site for 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 development and differentiation of neural stem cells in these areas can not only promote nerve regeneration but also compensate to some extent neuronal death caused by aging.

Bellflower is a perennial plant belonging to the campanula, and has been used not only for corrosion but also for medicinal plants in Korea. Natural bellflower is known to have calming, antipyretic, analgesic and anti-inflammatory effects (Lee, Kor J Phamarongr. 5, 49-56, 1974). In experiments, it has been reported that cancer cell proliferation inhibitory effect is very high (Hwang, Korean J Food Nut. 5, 13-19, 1992; Lee et al., Korean University Medical Journal 20, 39-45, 1983; Lee et al. , Korean University Medical Journal 25, 365-370, 1988). These bellflowers contain triterpenoid-based saponins, sugars, and fibers, which are quite high, and are used as herbal medicines (Lee, Chiese pharmaceutics, p129, 1981). (Kubota et al, Chemical Coommun. 22, 1314-1320, 1969; AAkiyama et al, Chem Phrm Bull. 20, 1945-1951, 1972).

The present inventors confirmed that the bellflower extract significantly increases the number of neoplastic stem cells in the dentate gyrus of the hippocampus in mice undergoing aging, thereby improving the phenomenon in which neuronal regeneration and differentiation are reduced by aging. By confirming that the present invention can be completed.

As an example for solving the above problems, the present invention provides a functional food containing bellflower extract and having the ability to promote differentiation and proliferation of neural stem cells.

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

The neural stem cells refer to neural stem cells derived from the hippocampus dental gyrus subgranular zone, and the differentiation of stem cells includes generation, division, differentiation and migration of stem cells.

Extracts as defined herein include extracts soluble in polar solvents, preferably ethanol, selected from water, methanol, butanol or mixed solvents thereof.

Bellflower as defined herein also includes dried exudates obtained from the roots of the Platycodon grandiflorum (Campanulaceae) and the roots of the same plants, ie, dendritic emulsions.

Hereinafter, the method for obtaining the extract of the present invention will be described in detail.

The bellflower extract of the present invention is chopped from the roots of dried bellflower, which is about 1 to 20 times the dry weight of water, preferably about 2 to 6 times the water, C1 to C4 lower alcohol, or a mixed solvent thereof, preferably Water or ethanol, cold sewage extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, etc. for about 1 hour to 10 days, preferably about 2 hours to 5 hours at an extraction temperature of 20 to 150 ℃, preferably 70 to 120 ℃ Extraction method obtained by the extraction method, preferably cold extraction method can be obtained by filtration, concentrated under reduced pressure or dried.

Functional food of the present invention includes the above-mentioned bellflower extract, in addition to food acceptable food supplements.

The functional food of the present invention can be used in various foods and beverages for the purpose of promoting stem cell ashing and differentiation. Foods to which the bellflower extract of the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and may be used in the form of powders, granules, tablets, capsules, or beverages. Can be.

Bellflower extract itself of the present invention is a component that can be used with confidence even when taken for a long time for the purpose of prevention because there is little toxicity and side effects.

The functional food of the present invention may be prepared by adding the bellflower extract to foods or beverages of various formulations for the purpose of promoting regeneration and differentiation of stem cells. At this time, the amount of the bellflower extract in the food or beverage can generally be added in 0.01 to 100% by weight of the total food weight, when prepared as a health beverage 0.02 to 10 g, preferably 0.3 based on 100 ml of the total composition To 1 g.

When the functional food of the present invention is prepared as a beverage, the extract contains the extract as an essential ingredient in the indicated proportions, and there is no particular limitation in the liquid component, and it contains various flavors or natural carbohydrates as additional ingredients, as in general beverages. can do.

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 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 the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the functional food of the present invention.

In addition to the above, the functional food 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, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the functional food of the present invention may contain fruit flesh for the production of natural fruit juice and fruit juice beverage and vegetable beverage.

These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the functional food of the present invention.

As described above, the bellflower extract of the present invention can be usefully used as a functional food for promoting neuronal differentiation and proliferation by aging by remarkably inhibiting the reduction of the differentiation and proliferation of neural stem cells during aging.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples, but the following Examples and Experimental Examples are merely 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 Bellflower Extract

1-1. Preparation of Extracts of Bellflower

In February 2007, dried bellflower was purchased at the Herbal Medicine Market in Seoul, and then powdered and added to 2 L of 100% ethanol for 1 week. After the cold ethanol 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 filtered filtrate was concentrated under reduced pressure with a vacuum concentrator (BUCHI 011, SWITZERLAND) to obtain 67 g of bellflower extract, which was used as a sample in the experimental example.

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

To identify the stem cell differentiation promoting effect of the bellflower extract, the differentiation of stem cells in C57BL / 6J mice was labeled with 5-bromo-2-deoxyguanosine (BrdU), doublecortin (DCX), and Ki-67 (proliferation marker). The experiment was conducted as follows.

In order to measure the stem cell differentiation promoting effect of the bellflower extract was carried out in the same manner as the animal experiment.

Experimental animals used 20 male 12-month-old C57BL / 6J mice, weighing 25 to 30 g, at 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.

25 mg of bellflower extract obtained in Example 1 was dissolved in 2 ml of distilled water, orally administered 0.2 ml for 4 weeks using an esophageal needle (dose was administered to 100 mg / kg body weight), and the control group was distilled water as a solvent. Was administered in equal amounts. After 20 days of administration, 12.5 mg of BrdU (Sigma, USA) was dissolved in 1 ml of saline solution to label stem cells, and then 0.1 ml of three doses were administered at 8 hour intervals.

The control and experimental groups were anesthetized by intraperitoneal injection of thiopental sodium at 30 mg / kg body weight 4 hours after the last BrdU administration, and then 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 directly perfused with 4% paraformaldehyde (in 0.1 M phosphate buffer; PB, pH 7.4) at 4 ° C.

After perfusion fixation, the animals were opened by using a bone cutter to remove the brain, and then fixed in the same fixative for 6 hours. After fixation, the brain was settled in a 30% sucrose solution (in 0.1 M phosphate buffer) until it settled to the bottom, and the tissue was cut into a 30 μm thickness using a sliding microtome (Reichert-Jung, Germany). 6-well plate containing a storage solution (storing solution) was stored at 4 ℃ until use.

Among the tissue sections, tissues with well-formed hippocampus were selected, and washed three times with 0.01 M PBS for 10 minutes to wash the stock solution on the tissue, and then endogenous peroxidase ( In order to remove peroxidase) it was reacted with 0.3% H 2 O 2 (in PBS) for 30 minutes. Then, the mixture was reacted with 65 ° C. 50% formamide (in 2X standard citate solution) solution for 2 hours and then reacted with 37 ° C. 2N HCl for 30 minutes to denature DNA. In order to prevent nonspecific immune responses, the reacted tissue sections were reacted with 3% normal goat serum for 30 minutes. Rabbit anti-BrdU (Chmicon Internationl, USA), goat anti-DCX (SantaCruz, USA), which is a primary antibody diluted 1: 1000 in order to label stem cells of tissue sections reacted with the 3% normal goat serum Or rabbit anti-Ki-67 (Abcam, UK) was reacted for 48 hours at 4 ℃. After completion of the reaction, the tissue sections were reacted with anti-rabbit IgG or anti-goat IgG (Vector), a secondary antibody diluted 1: 200, respectively, and then reacted with ABC solution (Vector), a third antibody diluted 1: 200. ), And 3,3'-diaminobezidine (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 on a gelatin-coated slide glass 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 axioM1 microscope (Carl Zeiss, Germany) attached to a digital camera (Axiocam, Cal Zeiss, Germany) with a total of 200 times the entire tooth gyrus and shown in FIG. (See Figure 1)

As shown in FIG. 1, A, C and E are photographs of the entire area of the teeth of the solvent-administered group and B, D and F are the entire areas of the tooth and the entire area of the bellflower extract-administered group, and image stained cells (Optimas 6.5, CyberMetrics , USA) program was used to count the number of neurons is shown in FIG. (See Figure 2)

Student t-test was performed to verify the significance of each group, and the data were expressed as mean and standard mean error. The dentate gyrus was photographed using an AxioM1 microscope (Carl Zeiss, Germany), and the results are shown in FIG. 1.

As a result, in FIG. 1, the control group (A) administered with the solvent was able to confirm staining of BrdU in a very small number of cells, whereas in the experimental group administered with 100 mg / kg of bellflower extract (B) A number of cells stained with BrdU could be confirmed. Dyeing of DCX, a marker for differentiation of endogenous neural stem cells into neurons, was observed in the control group (C) with solvent, while a few cells showing DCX staining were observed, whereas 100 mg / kg of bellflower extract was administered. In the experimental group (D), a large number of cells stained with DCX were observed. In addition, in the case of Ki-67, which is known as a proliferation marker of neurons, the control group (E) was able to confirm immunoreactivity only in a small number of cells, but compared to the (F) control group in the bellflower group. As a result, more Ki-67 responsive cells were identified.

In addition, as a result of confirming the cells stained with BrdU according to the administration of the bellflower extract in Figure 2, the administration of 100 mg / kg bellflower extract increased about 7.5 cells per tooth gyrus section for the control group (180%) The cells stained with DCX increased about 3.7 per tooth gyrus section (155%) when the bellflower extract was administered, and the cells stained with Ki-67 were sectioned when the bellflower extract was administered. It was confirmed that about 5.6 increased (800%). Therefore, it was confirmed that the administration of bellflower extract has an excellent effect on the differentiation and proliferation of neural stem cells.

Hereinafter, the preparation examples of the composition including 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  One. Powder  Produce

Bellflower Extract 100 mg

Lactose 100 mg

Talc 10 mg

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

Formulation example  2. Preparation of Tablets

Bellflower Extract 100 mg

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.

Formulation example  3. Preparation of Capsule

Bellflower Extract 100 mg

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.

Formulation example  4. Liquid  Produce

Bellflower Extract 100 mg

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 and dissolved in purified water, lemon flavor is added, the above ingredients are mixed, and then, purified water is added to adjust the total amount to 100 ml, and then sterilized by filling into a brown bottle. Manufacture.

Formulation example  5. Preparation of dietary supplements

Bellflower Extract 2500 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B 10.13 mg

Vitamin B 20.15 mg

Vitamin B 60.5 mg

120.2 μg of vitamin B

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 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.

1 is oral administration of a solvent (water) (A, C and E) or 100 mg / kg (B, D and F) for 4 weeks to confirm the neuronal cell regeneration promoting effect of the bellflower extract, immediately before sacrifice BrdU was administered three times at 8-hour intervals, and after 8 hours, gingival tissue was collected and stained with BrdU antibodies (A and B), DCX antibodies (C and D), and Ki-67 antibodies (E and F). One image photo,

Figure 2 is oral administration of bellflower extract solvent or bellflower extract at 100 mg / kg, then three times the BrdU every 8 hours, showing the number of cells stained BrdU, DCX and Ki-67 after 8 hours It is a graph.

Claims (4)

Functional food containing bellflower extract and has the ability to promote differentiation and proliferation of neural stem cells. The method according to claim 1, The neural stem cell is a functional food, characterized in that the nerve stem cells derived from the hippocampus (dentate gyrus) subgranular zone (subgranular zone). The method according to claim 1, Functional food, characterized in that the differentiation of stem cells, including the generation, division, differentiation and migration of stem cells. The method according to claim 1, The extract is a functional food, characterized in that the extract available in a solvent selected from water, methanol, ethanol, butanol or a mixed solvent thereof.

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