KR20050111257A - Functional food comprising magnolia kobus extracts for degenerative central nervous system disease improvement - Google Patents

Abstract

The present invention relates to a functional food for ameliorating symptoms of degenerative central nervous system disease, characterized by containing an extract of Magnolia or Epipiesmin isolated from Magnolia.

Description

FUNCTIONAL FOOD COMPRISING MAGNOLIA KOBUS EXTRACTS FOR DEGENERATIVE CENTRAL NERVOUS SYSTEM DISEASE IMPROVEMENT}

The present invention relates to a functional food for ameliorating symptoms of degenerative central nervous system disease, characterized by containing an extract of Magnolia or Epipiesmin isolated from Magnolia.

In the modern society, the elderly population is increasing rapidly as the average life span of human beings is extended due to the rapid development of biotechnology and medicine along with economic development. In the aging society, degenerative central nervous system disease, which is mainly accompanied by aging, has been the focus of attention as a challenge that must be solved in terms of prolonged quality of life.

Degenerative central nervous system disease is a disease that causes various symptoms by degenerative changes in nerve cells of the central nervous system, and mainly involves specific areas of the nervous system. Representative diseases include Parkinson's disease, Alzheimer's disease, ischemic brain disease, memory disorders, and alcoholism.

Degenerative central nervous system disease is known only to the extent that the disease is caused by genetic abnormalities and metabolic abnormalities, most of the specific causes are unknown. Degenerative central nervous system disease is characterized by a slow onset after birth, and symptoms are difficult to detect early in the symptoms, and once the onset, the disease continues for years or decades until death, and hardly treated Disease. Many patients also have a family history.

To date, research on degenerative central nervous system diseases has been mainly aimed at elucidating its mechanisms and developing mediators and selective inhibitors thereof. Recently, active cell transplantation for degenerative central nervous system diseases caused by irreversible neuronal cell death and New therapies are being tested to prevent degeneration of the central nervous system such as administration of neurotrophic factors and to promote regeneration.

In addition, oriental medicine is used to treat degenerative central nervous system diseases, such as blue fever medicine, live blood repellent medicine, hwadam medicine, flat liver wind medicine, or bohemian medicine. Although he is trying to cure the symptoms of degenerative central nervous system disease by prescribing it as (iii), modern medical interpretation and medicine development are insufficient.

Since the degenerative central nervous system disease is a disease that is difficult to diagnose and treat early, it is required to develop functional food materials in order to prevent or improve the symptoms of degenerative central nervous system disease in natural products that can be used as food.

Recently, some researchers have searched for degenerative central nervous system disease-related substances from natural products, such as Cheongung, Dansam, Doin, safflower, dew, Cheonma, Sleep, Toxic, Windproof, Seshin, Jogu etc., Gagogalpi, Seokchangpo, Jong Rhubarb, and Tangza Natural products such as trees, hooves, kwakhyang, cloves, ginger, turmeric, Hyunsam, and ginseng have been reported to be associated with the treatment of degenerative central nervous system diseases, but there is almost no connection that can lead to industrialization.

However, it is expected to develop medicinal candidates that can prevent and treat degenerative central nervous system diseases from traditional herbal prescriptions and its constituents, which have traditionally been shown to be clinically effective.

Magnolia kobus is a deciduous broad-leaved arboreous tree of the Magnolia family Magnolia, and epidermine is a substance isolated from magnolia (Latip J., Hartley TG, and Waterman PG, Phytochem., 1999, 51, 107- 110: Miyauchi T. and Ozawa S., 1998, Phytochem., 47 (4), 665-670: Miyazawa M., Kasahara H. and Kameoka H., Phytochem., 1995, 39 (5), 1027-1030: Pan ZX etc, 1987, Phytochem., 26 (5), 1377-1379).

The dried buds of magnolia are called Shin-yi, which is known to cure nasal congestion and to treat chills, fever and sweating and systemic pain due to cold caused by climate change (Lee Young-no, 1998, primary color). Korea Plant Book, etc.). In addition, Korean Patent No. 328478 discloses that Shinyi extract is used as a treatment for asthma, and Japanese Unexamined Patent Publication No. Hei 4-368334, Shinyi is known as a thromboembolism treatment agent. No information is known regarding neurological diseases.

The present inventors confirmed the neuronal outgrowth induction activity and cytotoxicity test results of epidermine isolated from magnolia during the study of functional food materials and medicinal candidates that have the effect of preventing and treating degenerative central nervous system diseases. By using epidermine isolated from magnolia extract or magnolia as a material of functional food, functional foods that are expected to improve degenerative central nervous system disease symptoms were developed to complete the present invention.

An object of the present invention is to provide a functional food containing epipiesmin isolated from magnolia extract or magnolia for improving symptoms of degenerative central nervous system disease.

In order to achieve the above object, the present invention provides a functional food containing epipiesmin isolated from magnolia extract or magnolia which is expected to improve the symptoms of degenerative central nervous system disease.

Referring to the present invention in more detail as follows.

The present invention provides a functional food for improving the symptoms of degenerative central nervous system disease containing magnolia extract.

The magnolia extract according to the present invention is dried in the shade by peeling the magnolia, and the bark is pulverized and pulverized with a pulverizer and extracted using water or lower alcohol such as ethanol (Ethanol), preferably using ethanol. Extraction can be concentrated by immersion at room temperature for 3 days.

The present invention provides a functional food for ameliorating symptoms of degenerative central nervous system disease containing epidermamine isolated from magnolia. Epiudesmin isolated from the magnolia according to the present invention is expected to be developed as a functional food material and medicinal candidate material.

Epipiesmin of the present invention is a Lignan compound whose chemical formula is 3,3 ', 4,4'-tetramethoxy-7,9', 7 ', 9-diepoxylignan and the chemical structure is shown below. It is like.

Epiyudesmin is a known substance as Latip J., Hartley TG, and Waterman PG, Phytochem., 1999, 51, 107-110, Miyauchi T. and Ozawa S., 1998, Phytochem., 47 (4), 665-670), Miyazawa M., Kasahara H. and Kameoka H., Phytochem., 1995, 39 (5), 1027-1030) or pans (Pan ZX etc, 1987, Phytochem., 26) (5), 1377-1379) and the like are isolated from magnolia.

In addition, epidermine may be isolated from the magnolia extract in the following manner. The magnolia extract was fractionated using petroleum ether and ether, and then the Sepadex LH-20 column chromatography using a mixed solvent of methanol and ethanol for the ether fractions. The fractions were separated, the fractions were subjected to silica gel column chromatography using a mixed solvent of benzene and ethyl acetate, and the fractions were separated. Finally, the fractions were separated using Sephadex LH using methanol solvent. The fraction obtained by performing -20 column chromatography can be purified by a conventional purification method to isolate epiyudesmin.

The functional food of the present invention may contain components that are commonly added during food production. Commonly added ingredients in food preparation include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and salts thereof. , Organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages. In addition, the composition of the present invention may contain a natural fruit juice and a pulp for the production of fruit juice drinks and vegetable drinks. These components can be used independently or in combination.

In general, since neurological diseases are related to the death or damage of nerve cells, the treatment is to suppress the death or damage of nerve cells. More recent methods of treatment include methods of promoting cell regeneration by promoting neuronal production.

To promote the production of neurons, there are neurotrophic factors (Neuroiteic factors) involved in the survival and differentiation of nerves, or neurite outgrowth inducer (Neurite-promoting). Therefore, neurotrophic factor or neurite outgrowth-inducing substance that promotes neurite is a degenerative central nervous system disease such as Parkinson's disease, Alzheimer's disease, and ischemic brain disease. Ischemia), memory disorders, alcoholism, etc. (Gannong WF, 2003, Review of Medical Physiology, 21st ed., 62-63: Seckel BR, 1990, Muscle Nerve, 13, 785-800: Varon S. and Hagg T., 1993, Models to evaluate effects of neurotrophic factors on axonal regeneration, in Neuromethods, Vol 25, 277-289).

NGF (Nerve growth factor) is a representative material that promotes the production of neurons. When NGF pretreatment is performed on PC12 cells, PC12 cells stop dividing, differentiate into sympathetic neuron-like cells, differentiate into neurite outgrowth, and proliferate when NGF is absent Proliferation occurs, but no differentiation occurs, and cell death occurs (Greene, LA, Tischler, AS, Adv. Cell. Neurobiol., 1982, 3, 373-414). Therefore, since NGF is related to neuronal survival and differentiation, it is a neurotrophic factor and promotes neurogenesis, thereby promoting neural cell production as a neurite outgrowth inducer.

The present invention compares the pretreatment of epidermine with the pretreatment of NGF to PC12 cells, and the results of the neurite outgrowth induction experiment showed that the induction of neurite outgrowth It was confirmed to be a better material.

In addition, NGF is activated by Mitogen ativated protein kinase (hereinafter referred to as 'MAPK'), Calcium dependent protein kinase (hereinafter referred to as 'PKC') and Cyclic-AMP dependent protein kinase (hereinafter referred to as 'PKA'). It induces neurite outgrowth, so it is PD 98059 (product of SIGMA product code P-215), MGFK inhibitor, GF 109203X (product of SIGMA product code G-2911), inhibitor of PKC, which is an inhibitor of PKA. Experimental effects of epidermine as a control group of H 89 (product of SIGMA's product code B-1427) and NGF on PC12 cells at the same time resulted in the effect of epidermine inducing neurite outgrowth. It was confirmed to be an excellent material.

Based on the above results, the extract containing magnolia extract or epiudesmin isolated from magnolia can be prepared as functional foods of various formulations for the improvement of the symptoms of degenerative central nervous system diseases, and various foods, beverages, gums, teas, nutrient gangs It can be added to vitamin complexes and the like to produce a functional food.

The present invention may contain 1 to 1000 mg of epidermamine and 1 to 10 g of magnolia extract per unit formulation of the functional food formulation.

Epiyudesmin or magnolia extract isolated from the magnolia of the present invention is suitable for use as a food result of the acute toxicity test.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the examples.

<Example 1>

Preparation of Magnolia Extract

Magnolia was used as a publicly-owned species of magnolia (35 years old) collected at Gajwa Experimental Forest (Gajwa-dong, Jinju-si, Gyeongsangnam-do) in June 2001.

The magnolia was peeled and dried in the shade, and the bark was chopped and pulverized with a grinder to be powdered. The powder was extracted by immersing at room temperature for 3 days using ethanol, and the mixture was dried three times under reduced pressure, and the magnolia extract was dried. Prepared.

<Example 2>

Isolation of Epiyudesmin from Magnolia Extract

Magnolia extract dissolved in ethanol obtained before drying under reduced pressure in the manufacturing process of the magnolia extract was fractionated by successive successive extraction using petroleum ether, ether, ethyl acetate and butanol. 75 mL using a Sephadex LH-20 column chromatography (60 × 4.5 cm) using methanol-ethanol (methanol: ethanol = 3: 7, v / v) as the eluting solvent for the double ether soluble part (85.0 g). Twelve aliquots were obtained, and each aliquot was subjected to thin layer chromatography (TLC, silica gel 60 F254, developing solvent; toluene: ethyl formate: formic acid = 5: 4: 1, v / v / v). After development, the solution was divided into two fractions by searching with a UV (254 nm) lamp and a color developer (50% H ₂ SO ₄ ).

The second fraction (81.15 g) was eluted by 15 ml using silica gel column chromatography (27.5 × 7.0 cm) using benzene-ethyl acetate (benzene: ethyl acetate = 8: 1, v / v) as the elution solvent. 230 fractions were obtained and 8 fractions were obtained by UV.

The fifth fraction (3.3 g) was fractionated in 5 ml portions using Sephadex LH-20 column chromatography (50 × 4.5 cm) using methanol as the elution solvent to obtain 40 aliquots. The search result was divided into four fractions.

Compound (120 mg) isolated from the second fraction of them was identified as epiyudesmin, a lignan compound, as a result of instrumental analysis and literature search such as NMR and MS.

The MS and NMR results and epichemical structure of epidermine are as shown below.

EI-MS Spectrum m / z ; 386 (M ⁺ ).

¹ H-NMR spectral signal group (ppm); (500 MHz, acetone- d _6, δ = ppm): δ 3.12 (2H, m , H-8, H-8 ′), 3.82 (3H, s , OMe), 3.84 (3H, s , OMe), 3.86 (2H, m , H-9a, H-9a '), 4.22 (2H, m , H-9b, H-9b'), 4.73 (2H, m , H-7, H-7 '), 6.92- 7.2 (6H, m , arom).

^{Group of 13} C-NMR spectral signals (ppm); (125 MHz, acetone- d ₆ , δ = ppm): δ54. 63 (C-8, 8 '), 55.60 (C-OMe × 4), 71.67 (C-9, 9'), 85.85 (C-7, 7 '), 110.46 (C-6, 6'), 112.11 (C-5, 5 '), 118.46 (C-2, 2'), 134.81 (C-1, 1 '), 149.21 (C-4, 4'), 149.85 (C-3, 3 ').

¹ H- ¹ H COSY correlations: H-7 / H-7'↔H-8 / H-8 ', H-9a / H-9a'↔H-9b / H-9b' / H-8 / H- 8'.

HMBC correlations: H-7 → C-2 / C-6 / C-8 / C-9, H-9 → C-7 / C-8.

Mass (MS) spectra of the isolated compounds were determined by JEOL JMS-600W, nuclear magnetic resonance such as ¹ H-, ¹³ C-NMR, ¹ H- ¹ H Correlation Spectroscopy (COSY), and ¹ H Detected Multiple Bond Connectivity (HMBC). (NMR) spectra were measured using the Varian UI 500 of the Seoul Institute of Basic Science and Technology.

<Example 3>

Neurite outgrowth induction experiment

(1) Culture of PC12 Cells and Pretreatment of Samples

PC12 cells were used as a medium containing serum (5% FBS + 10% HS) RPMI 1640, the culture was incubated in accordance with the conventional culture method in a 37 ℃ incubator. Epiudesmin was added to the serum-containing medium (60 mm culture dish), incubated for 7 days, medium was changed if necessary, and neurite bearing & Neurite outgrowth was measured at each time period.

(2) Measurement of neurite bearing and neurite outgrowth

After fixation by adding 4% paraformaldehyde to PC12 cells, using a phase-contrast microscope (x100-125 magnification), the cellular process was longer than 1 cell diameter (in 1x1-mm fields). ) Was counted and measured. The neuritic index was calculated by dividing the number of neurite forming cells by the total number of cells in this field (minimum 200 per count). Comparison of neurite outgrowth by pretreatment of NGF and epidermine was examined by measuring cell number and length of nerite after single or combination pretreatment. The number of neurite forming cells was counted by the substance and measured as a ratio with the total cell number.

After 96 hours of epiyudesmin (20 μg / ml) pretreatment, PC12 cells showed significant neuronal outgrowth induction. Neurite forming cell numbers increased concentration-dependently with the treatment of epidermine (20 μg / ml).

After 96 hours of epiyudesmin (20 μg / ml) pretreatment, PC12 cells showed significant neuronal outgrowth induction compared to the control (FIG. 1) (FIG. 2).

When co-administration of epidermine and NGF showed a significant neurite outgrowth induction effect compared to the NGF alone group (FIG. 3) (FIG. 4).

In addition, when the neurite length was combined with epidermamine (20 µg / ml) and NGF (2 ng / ml) (250 µm, contrast to scale bar 50 µm), NGF alone administration group (150 µm, contrast to scale bar) As much as 5/3 times longer than that of 50 μm), it can be seen that it showed a significant neurite outgrowth induction action.

<Example 4>

Influence of MAPK inhibitors, PKC inhibitors and PKA inhibitors

(1) Effect of MAPK, PKC and PKA Inhibitors

Neuronal outgrowth induction of NGF is activated by MAPK, PKC, and PKA. Therefore, after pretreatment with PD 98059, an inhibitor of MAPK, GF 109203X, an inhibitor of PKC, and H 89, an inhibitor of PKA, it was determined whether neurogenesis of PC12 cells by epidermine. Neurite outgrowth induction was carried out according to Example 3- (2) above.

The pretreatment of PD 98059 (3 mM), a MAPK inhibitor, inhibited the neurite outgrowth induction of epidermine (20 µg / ml), and NGF (2 ng / ml) and epidermide (20). Μg / ml) When PD 98059 was administered to the combination treatment group, the number of neurite forming cells was significantly reduced compared to the NGF-only group (FIG. 5).

Pretreatment of PKC inhibitor GF 109203X (100 nM) and PKA inhibitor H 89 (100 nM) inhibited the neurite outgrowth induction of epidermine (20 μg / ml), and NGF (2 ng / ml) The number of neurite forming cells increased in the co-administration of and epiyudesmin (20 μg / ml) was decreased by GF 109203X and H 89 pretreatment (FIGS. 6 and 7).

Taken together, it can be seen that epidermine is a neurite outgrowth inducer.

Therefore, epidermine or magnolia extract isolated from magnolia can be used as a material of functional food for improving symptoms of degenerative central nervous system disease. In addition, functional foods containing the material can be prepared.

Example 5

Cytotoxicity test

A test on the cytotoxicity of PC12 cells of epiyudesmin isolated from the magnolia of the present invention was performed.

In order to confirm whether the epidermis of the present invention is toxic to humans, it was determined by MTT / LDH activity assay.

As a result, epiudesamine did not show a cytotoxic effect in the range of 40 ~ 80㎍ / ㎖ (search by LDH activity measurement), but showed a cytotoxic action above 80㎍ / ㎖.

Through these safety experiments, it is determined that the epidermine of the present invention does not adversely affect the human body.

The results of all experiments were expressed as mean and standard error, and the significance between means of each group was verified by ANOVA followed by Turkey's test. The statistical significance level between the control group and the experimental group was p <0.05.

<Production Example 1>

Manufacture of tablets

45.0% by weight of the magnolia extract prepared in Example 1, 5.0% by weight of glucoseamine, 7.5% by weight of mucodose, 5.0% by weight of riboflavin, 20.0% by weight of lactose, 2.5% by weight of isoflavone, 15.0% by weight of carboxymethylcellulose Purified tablets were prepared.

<Production Example 2>

Manufacture of tea bags

Teabag tea was prepared by sealing 0.7 g of the magnolia extract prepared in Example 1 in a tea bag film (3-4 × 5-6 cm).

<Production Example 3>

Manufacture of Yokan

3.5% by weight of the magnolia extract prepared in Example 1 was continuously stirred and heated at 90-100 ° C. with a mixture of yolk raw material consisting of 3.5% by weight of lima bean rock, 25% by weight of sugar, 10% by weight of maltooligosaccharide, and 2% by weight of agar. After mixing, the mixture was cooked and then cooled at room temperature to prepare yokan.

Production Example 4

Preparation of Functional Drinks

10 mg of epiderminin prepared in Example 2, 600 mg of citric acid, 100 g of oligosaccharide, 2 g of apple concentrate, 1 g of taurine, and purified water were added and mixed to a total of 900 mg, followed by a method for preparing a general functional beverage for about 1 hour. After stirring and heating at 85 ℃ the resulting solution was filtered and obtained in a sterilized 2L container, sealed sterilization and refrigerated and then prepared in the form of the functional food of the present invention.

As described above, the functional food containing the extract of the present invention and epidermine isolated from the magnolia can be used to improve the symptoms of degenerative central nervous system disease.

In particular, the present invention shows that the epidermis isolated from magnolia exhibits neurite outgrowth inducing action, so the functional food containing the extract of magnolia and the epidermis isolated from magnolia promotes the regeneration of central nerve cells. It is expected to improve the symptoms of degenerative central nervous system diseases such as Parkinson's disease, Alzheimer's disease, ischemic brain disease, memory disorders and alcoholism.

1 is a graph showing the induction of neurite outgrowth in PC12 cells not pretreated as a control,

FIG. 2 is a graph showing the induction of neurite generation factor in PC12 cells by pretreatment of Epiudesmin (20 μg / ml) for 96 hours. FIG.

Figure 3 is a graph showing the induction of new light production factor in PC12 cells by pretreatment of NGF (Nerve growth factor: 2ng / ㎖) for 96 hours,

FIG. 4 is a graph showing the induction of neurite generation factor in PC12 cells by pretreatment of epiyudesmin (20 µg / ml) and NGF (2ng / ml) for 96 hours.

FIG. 5 is a graph showing the effect of PD 98059, a miogen activated protein kinase (MAPK) inhibitor, on the induction of neuronal factor production of epidermamine and NGF,

FIG. 6 is a graph showing the effect of GF 109203X, a calcium dependent protein kinase (PKC) inhibitor, on the induction of neurogenesis factor of epiudesamine and NGF,

7 is a graph showing the effect of H 89, a Cyclic-AMP dependent protein kinase (PKA) inhibitor, on the neutrophil inducer action of epidermamine and NGF.

Claims (6)

Functional food for improving the symptoms of degenerative central nervous system disease containing magnolia extract. The functional food for ameliorating symptoms of degenerative central nervous system disease according to claim 1, wherein the magnolia extract contains Epiudesmin. The functional food for improving symptoms of degenerative central nervous system disease according to claim 1 or 2, wherein the food is selected from a tablet, granule, capsule or liquid formulation. According to claim 1 or 2, wherein the functional food is a functional food for improving the symptoms of degenerative central nervous system disease, characterized in that the solid food. According to claim 1 or 2, wherein the functional food is a functional food for improving the symptoms of degenerative central nervous system disease, characterized in that the beverage or tea. A food additive for improving the symptoms of degenerative central nervous system disease, containing Epipiesmin isolated from Magnolia as an active ingredient.