KR20140142871A - Composition containing gamma-mangosteen for preventing or treating degenerative brain disease - Google Patents

Abstract

The present invention relates to a composition containing gamma-mangostin as an active ingredient for preventing or treating degenerative brain diseases. The gamma-mangostin of the present invention inhibits oxidative damage in neural cells and apoptosis, has an excellent anti-oxidative effect, and inhibits excitotoxicity in neural cells, activation of Aβ peptide-induced neurotoxicity and beta-secretase so as to reduce side effects and have excellent effects for preventing nerves and suppressing the synthesis of Aβ peptide, thereby being able to be useful for preventing or treating degenerative brain diseases including dementia.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating degenerative brain diseases containing gamma-mangosteen as an active ingredient,

The present invention relates to a composition for preventing or treating degenerative brain diseases containing gamma-mangosteen as an active ingredient.

Degenerative brain disease is a generic term for all brain dysfunctions and diseases caused by degeneration of brain function. It includes dementia such as Alzheimer's disease, cognitive disorder, and stroke.

Dementia is a term derived from the Latin word meaning "lost in spirit." This means that a person who has been living normally has been impaired by various causes, and his or her cognitive function is continuously and generally lowered than before. It refers to the state to be represented. Dementia is characterized by memory loss and various other intellectual abilities, which distinguish it from the symptoms of naturally declining memory. It is known that such dementia is caused by continuous development of brain dysfunction and deterioration due to destruction of nerve cells and tissues due to a mild lesion of the brain. Concomitant disorders such as chaos after surgery can cause secondary deterioration of cognitive function, which is called 'delirium' and is a symptom different from dementia. In the past, dementia was called aggression and aging, and it was thought to be an aging phenomenon naturally occurring when an elderly person became an elderly person.

Often, they think of dementia as a disease, they all are the same, and they tend to concede that there is no cure. However, dementia is classified into more than 70 cases according to the cause. Dementia is classified into senile dementia and vascular dementia. It is known that Alzheimer's disease and vascular dementia are the most common dementia. Dementia is a degenerative brain disease that includes Lewy body dementia, anterior temporal dementia, dementia of Parkinson's disease, and is caused by various causes such as normal pressure hydrocephalus, head trauma, brain tumor, metabolic disease, .

On the other hand, the proportion of the elderly population to the total population is gradually increasing as Korea enters the aging society due to the declining fertility rate and aging population. It is known that the number of dementia patients is increasing rapidly with the increase of the elderly population. According to a study by the Korea Institute of Health and Social Research on "the status and measures of dementia of the elderly," the number of domestic dementia patients was estimated to be about 470,000 in 2010 and about 520,000 in 2012, and about 1.14 million in 2030 In addition, the prevalence of dementia was 8.76% in 2010 and is expected to increase to 9.74% in 2020.

The increase in the number of patients with dementia led to a surge in medical costs related to dementia. The total annual amount of medical expenses spent for dementia was estimated at about 47 billion won in 2002, while in 2007 it was about 302.6 billion won, which is about 6 times higher. In addition, 22.1% of demented patients among the elderly long term care insurance members were found to account for the seriousness. According to the data provided by the National Health Insurance Corporation, the actual number of health care patients is increasing every year.

In the United States, the number of patients with Alzheimer's disease in 2010 is over 500,000, and it is expected to reach about 13.5 million by 2050. In addition, the cost of medical care for Alzheimer's and dementia in the United States in 2010 is estimated at about $ 172 billion. If delaying the onset of dementia by 5 years, about $ 447 billion in medical costs are expected to be saved.

Dementia therapies currently include four types of choline esterase inhibitors: donepezil, rivastigmine, galantamine, tacrine, and NMDA receptor antagonists. Memantine has been used only limitedly, and its therapeutic effect can not be widely used. Therefore, there is a need to develop a new therapeutic agent. The dementia market in seven countries, including the United States, the United Kingdom, France, Germany, Japan, Italy and Spain, was estimated at about $ 3 billion in 2007 and is expected to reach $ 9 billion in 2017.

On the other hand, β-secretase is an enzyme involved in the production of amyloid beta (Aβ) peptide, which is known to be a major cause of dementia. In addition, beta-secretase, which is a kind of aspartic secretase, is recently attracting attention as a drug target for the development of a therapeutic agent for degenerative brain diseases including senile dementia.

On the other hand, Mangosteen (Garcinia mangostana) is an evergreen arborescent tree of the mulberry tree, which is the origin of Malaysia, and is so sweet that it is fragrant, sour and sweet and is called the queen of the fruit. The pigment in the flesh of mangosteen contains tannin and can be used as a dye because its color does not change easily. Since mangosteen is seedless without fertilization, it is said that the same breed has been cultivated since ancient times and it grows only in a limited region because it is difficult to cultivate. Mangosteen is distributed in Indonesia, Malaysia, Taiwan, Philippines, India and Sri Lanka. Mangosteen is sterilized, has antibacterial and antiallergic action, and contains a large amount of beta carotene, which is known to inhibit the production of nitrosamine, a carcinogen. In addition, mangosteen is known to have effects such as prevention of osteoporosis, improvement of vision, promotion of appetite, promotion of digestion, prevention of constipation, activation of liver function, prevention of tuberculosis, and protection of heart. The skin of mangosteen contains a large amount of xanthone (or xanthone), which is reported to exhibit such excellent pharmacological effects. Xanthones are, for example, alpha mangosteen and gamma mangosteen.

The inventors of the present invention have been studying natural products and compounds separated therefrom in order to develop drugs having excellent effects for treating degenerative brain diseases such as dementia. It has been found that gamma-mangostin inhibits oxidative damage of nerve cells, Suppresses excitatory neuronal cytotoxicity, inhibits Aβ peptide-induced neurotoxicity, inhibits the activity of beta-secretase, has few side effects, protects neurons, inhibits Aβ peptide-induced neurotoxicity It is confirmed that the effect of inhibiting the formation of the compound is excellent, and the present invention has been completed.

The present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases containing gamma-mangosteen as an active ingredient.

The present invention provides a food composition for preventing or ameliorating a degenerative brain disease containing gamma-mangosteen as an active ingredient.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating degenerative brain diseases containing gamma-mangosteen as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or ameliorating degenerative brain diseases containing gamma-mangosteen as an active ingredient.

The gamma-mangosteen of the present invention inhibits oxidative damage of nerve cells, inhibits apoptosis, has excellent antioxidative effects, inhibits excitatory neuronal cytotoxicity, inhibits A [beta] peptide-induced neurotoxicity, By inhibiting the activity of cryptase, it has few side effects, is excellent in the effect of protecting neurons and inhibiting the production of A [beta] peptide, and thus can be useful for the prevention or treatment of degenerative brain diseases including dementia.

FIG. 1 is a graph showing microscopic observation of the inhibitory activity of oxidative neuronal cell damage of gamma-mangosteen according to the present invention.
FIG. 2 is a graph showing the results of measuring the reactive oxygen species inhibitory activity of gamma-mangosteen according to the present invention. FIG.
FIG. 3 is a graph showing the caspase-3 inhibitory activity of gamma-mangosteen according to the present invention measured by Western blotting.
Figure 4 shows the result of observation of the DNA fragmentation inhibitory activity of gamma-mangosteen according to the present invention by a phase contrast microscope using TUNEL method (a. Control group, b. Treated with 100 μM H ₂ O ₂ for 2 hours Experimental group, c. Experimental group treated with 100 μM H ₂ O ₂ and 10 μM gamma mangosteen for 2 hours, d. 10 μM gamma mangosteen treated with 10 μM gamma mangosteen for 2 hours, Processed).
FIG. 5 is a graph showing the results of measuring lipid peroxidation inhibitory activity and DPPH radical scavenging activity of gamma-mangosteen according to the present invention.
FIG. 6 is a graph showing the results of measuring the activity of inhibiting excitatory neuron cytotoxicity of gamma-mangosteen according to the present invention.
FIG. 7 is a graph showing the results of measuring the inhibitory activity of Aβ peptide-induced nerve cell cytotoxicity of gamma-mangosteen according to the present invention.
8 is a graph showing the results of measurement of beta-secretase inhibitory activity of gamma-mangosteen according to the present invention.
FIG. 9 is a graph showing the results of evaluation of cytotoxicity of gamma mangosteen according to the present invention. FIG.

The present invention provides a composition for preventing or treating degenerative brain diseases comprising gamma-mangosteen represented by the following formula (1) as an active ingredient.

Such compositions include pharmaceutical compositions and food compositions.

Hereinafter, the present invention will be described in detail.

Gamma mangosteen, which is an active ingredient of the present invention, can be obtained by conventional extraction and fractionation methods, or commercially available reagents can be purchased and used.

In the present invention, it was obtained by the following method.

First, the shaded mangosteen hull is extracted with water, an alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, followed by filtration and concentration to obtain a crude extract. At this time, the alcohol having 1 to 4 carbon atoms is 1 to 100% (v / v) methanol, 1 to 100% (v / v) ethanol, and more preferably 1 to 100% (v / v) methanol. The obtained crude extract is suspended in distilled water and fractionated with hexane, chloroform, ethyl acetate and n-butanol in order. Among these fractions, the chloroform fraction having excellent activity for inhibiting neuronal damage is chromatographed to obtain gamma mangosteen in the form of a yellow powder.

The gamma-mangosteen of the present invention inhibits oxidative damage of nerve cells, inhibits apoptosis, has excellent antioxidative effects, inhibits excitatory neuronal cytotoxicity, inhibits A [beta] peptide-induced neurotoxicity, By inhibiting the activity of cryptase, it has few side effects, is excellent in the effect of protecting neurons and inhibiting the production of A [beta] peptide, and thus can be useful for the prevention or treatment of degenerative brain diseases including dementia.

Wherein said degenerative brain disease is selected from the group consisting of dementia, mild cognitive impairment, stroke, Creutzfeldt-Jakob disease, traumatic head injury, syphilis, acquired immunodeficiency syndrome, brain abscess, brain tumor, multiple sclerosis, hypoxia, Parkinson's disease, But are not limited to, colorimetric sclerosis, epilepsy, ischemia, paralysis, deprivation of behavior - hyperactivity disorder, schizophrenia, depression, manic depression, post traumatic stress disorder, spinal cord injury and myelitis.

The dementia includes dementia caused by senile dementia, Alzheimer's disease, vascular dementia, dementia of the frontal lobe, anterior temporal dementia, dementia caused by normal pressure hydrocephalus, head trauma, dementia caused by metabolic disease, But is not limited thereto.

The composition of the present invention, together with gamma-mangosteen, may further contain one or more known active ingredients having an effect of preventing or treating degenerative brain diseases.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral formulations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral administration 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. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For a desired effect, the gamma-mangosteen of the present invention may be administered in an amount of 1 mg / kg to 10000 mg / kg per day, or may be administered once a day or divided into several doses.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention and treatment of degenerative brain diseases.

In the present invention, the term "health functional food" refers to a food having a biological control function such as prevention and improvement of disease, bio-defense, immunity, recovery after disease and aging inhibition.

The composition of the present invention can be added to health functional foods for the purpose of preventing or improving degenerative brain diseases. When the gamma-mangosteen of the present invention is used as a food additive, the gamma-mangosteen can be added as it is or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the gamma-mangosteen of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred examples, experimental examples, and formulation examples are provided to facilitate understanding of the present invention. However, the following examples, experimental examples and preparation examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples, experimental examples and preparation examples.

[Example 1: Preparation of gamma mangosteen]

The shaded mangosteen skin (1.3 kg) was ultrasonically extracted three times with 8 L of 100% methanol (v / v%), and then filtered and concentrated under reduced pressure to obtain 401 g of methanol extract. 164 g of the methanol extract in the obtained methanol extract was suspended in distilled water and sequentially fractionated with hexane, chloroform, ethyl acetate and n-butanol, Each solvent fraction was obtained. Among these fractions, the chloroform fraction (37.9 g) showed the highest inhibitory activity against oxidative neuronal damage. Thus, the chloroform fractions were subjected to open column chromatography using gradient silica gel (593 g) using hexane and ethyl acetate as a mixed solvent to obtain a total of 49 fractions (GMC1 to GMC 49) Among them, the fraction No. 35 (3.36 g) was selected and MPLC of gradient silica gel (120 g) was performed with a mixed solvent of chloroform and acetone to obtain a total of 23 fractions. Among them, fraction 8 (GMC35-8, 698 mg) was recrystallized from methanol and distilled water to obtain a yellow powder, gamma mangosteen. The chemical structure analysis of the obtained gamma mangosteen was carried out using a mass spectrometer and a nuclear magnetic resonance spectrometer.

[Experimental Example 1: Measurement of oxidative neuronal damage inhibitory activity of gamma-mangosteen]

1. Cell preparation and culture

Cultures of cerebral cortical neurons obtained from fetuses of SD (Sprague-Dawley) rats on the 17th day of pregnancy were cultured as follows.

Specifically, the brain of the rat was removed to remove the brain membrane, and the cortical portion was separated and cut into small pieces. Then, the cells were separated into single cells using three Pasteur pipettes each having a small hole size. Poly -L- lysine to 6 × 10 ⁵ cells / well or 6 × 10 (poly-L- lysine) and laminin (well plate) 24- well plates coated with a preloaded (laminin) or 35 mm culture dish (dish) ⁶ cells / culture Dissociated densities of the rat neurons were transplanted, respectively. The neurons were placed in MEM (minimum essential medium, containing Earle's salts) containing 5% FBS (fetal bovine serum), 5% HS (horse serum), 2 mM glutamine and 25 mM glucose, ₂ < / RTI > in a 37 < 0 > C incubator maintained at ambient conditions. After 7 days of incubation, the cells were treated with 10 [mu] M cytosine arabinoside to inhibit growth of cells other than neurons, and then used for experiments after 10 days or more of incubation.

2. Oxidative Cell Damage Induced by Neuronal Cells

 In order to induce oxidative cell damage of the neuron cultured in the above 1, the following experiment was conducted.

Specifically, the cerebral cortical neurons of the primary cultured rat were suspended in HEPES-normal saline (HCSS; 120 mM NaCl, 5.4 mM KCl, 1.6 mM MgCl ₂ , 2.3 mM CaCl ₂ , 14.9 mM glucose, 16.8 mM HEPES, NaOH) and treated with HCSS containing 100 μM H ₂ O ₂ or 0.5 mM / 10 mU / ml xanthine / XO (xanthine oxidase) for 5 min or 10 min, respectively. Then, the culture medium was exchanged with MEM medium containing 25 mM glucose and cultured for 18-20 hours in an incubator at 37 ° C while maintaining the environmental conditions at 95% air and 5% CO ₂ concentration, Lt; / RTI >

3. Measurement of oxidative neuronal damage inhibitory activity of gamma-mangosteen

In order to measure the inhibitory activity of the gamma-mangosteen of the present invention on damage to neurons, MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide , a yellow tetrazole method) was performed to measure cell viability of primary cultured rat cerebral cortical neurons.

 Specifically, 250 占 퐇 of MTT reagent (2 mg / ml concentration) was added to each well and cultured in a 37 占 폚 incubator for 3 hours. Then, the supernatant of the culture was removed, and formazan ) Crystals were dissolved in 500 [mu] l of DMSO. Absorbance was measured at 550 nm using a microplate reader (SpectraMax M2 microplate reader; Molecular Devices, USA). The morphological changes of the neurons were observed using a phase contrast microscope, The degree of damage was confirmed. The results are shown in Fig.

As shown in FIG. 1, the gamma-mangosteen of the present invention significantly increased the cell survival rate of rat cerebral cortical neurons induced by oxidative cell damage by H ₂ O ₂ and X / XO at a concentration of 10 μM In particular, it was confirmed that the cell viability of rat cerebral cortical neurons induced by oxidative cell damage by H ₂ O ₂ was significantly increased to 107.9%.

Therefore, the gamma-mangosteen of the present invention significantly inhibits oxidative cell damage induced by oxidative stress in primary cultured rat cerebral cortical neurons in a concentration-dependent manner, and thus inhibits oxidative damage and neuroprotective effect of neurons .

4. Measurement of reactive oxygen species inhibitory activity of gamma-mangosteen

The reactive oxygen species (ROS) for the gamma-mangosteen of the present invention In order to measure the inhibitory activity, the reactive oxygen species inhibitory activity induced by H ₂ O ₂ , X / XO in nerve cells cultured in the above 2 was measured using DCF-DA as follows.

Specifically, the cultured cells were washed with HCSS, cultured in MEM medium containing 10 μM DCF-DA for 30 minutes, and then cultured in 200 μM H ₂ O ₂ , 0.5 mM / 10 mU / ml X / XO for 2 hours And the appropriate concentration of gamma-mangosteen was treated at the same time. After a certain period of time, the fluorescence was measured at 490 nm and 520 nm using a microplate reader (SpectraMax M2 ^e microplate reader) and the IC ₅₀ value was measured . The reactive oxygen species inhibitory activity of gamma-mangosteen is shown in FIG. 2, and the IC ₅₀ values thereof are shown in Table 1 below.

Gamma mangosteen (IC _50, uM) H ₂ O ₂ 9.1 [mu] M X / XO 26.0 [mu] M

As shown in Table 1 above, the IC ₅₀ values of the gamma mangosteen of the present invention were measured to be lower than X / XO in H ₂ O ₂ , indicating that the gamma mangosteen had a stronger ROS produced by H ₂ O ₂ Respectively.

As shown in FIG. 2, gamma-mangosteen of the present invention inhibited the ROS of the rat cerebral cortical neurons induced by H ₂ O ₂ and X / XO, respectively, in a concentration-dependent manner.

Therefore, the gamma-mangosteen of the present invention significantly inhibits ROS produced by oxidative stress in primary cultured rat cerebral cortical neurons in a concentration-dependent manner, Respectively.

[Experimental Example 2: Measurement of inhibitory activity of gamma mangosteen on apoptosis]

1. Measurement of caspase-3 inhibitory activity of gamma mangosteen

In order to examine the apoptosis inhibitory activity of gamma-mangosteen of the present invention, western blotting was performed according to a conventionally known method. The primary cultured rat cerebral cortical neurons prepared in 2 of Experimental Example 2 were treated with H ₂ O ₂ for 2 hours to measure active caspase-3 produced from procaspase-3.

Specifically, the cerebral cortical neurons cultured in Experimental Example 2 were treated with a reaction solution containing 100 mM H ₂ O ₂ or 100 mM H ₂ O ₂ and an appropriate concentration of gamma-mangosteen for a predetermined period of time. The cells were washed with PBS (pH 7.3), and then lysed in a lysis buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 4.5 mM sodium pyrophosphate, Glycerophosphate, 1 mM NaF, 1 mM Na ₃ VO ₄ , 1% Triton X-100, 0.5% Nonidet P-40 and protease inhibitor cocktail (Boehringer, Indianapolis, USA )) At a temperature of 4 째 C for 30 minutes and centrifuged at 14,000 rpm for 30 minutes. The supernatant was then taken and the protein concentration was assessed with BioRad Dc protein assay kit (BioRad, Hercules, USA). The same amount (30 μg) of the protein was separated by electrophoresis using a 12% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Whatman, Boston, USA) Non-fat dry milk for 1.5 hours at room temperature to block nonspecific reactions. After incubation at 4 ° C for 24 hours with a primary antibody (rabbit antiboby), the cells were washed with TBS (tris-buffered saline with Tween) and incubated with anti-rabbit secondary antibody It is reacted with an IgG-HRP) were analyzed using the ECL detection system (detection system) (Thermo Scientific, using ^{Rockford, USA) Chemi-Doc TM} MP imaging system (imaging system) (BioRad, Hercules , CA). The control group was set to contain only DMSO solvent instead of gamma-mangosteen, and alpha-mangosteen was set to the same concentration gradient instead of gamma-mangosteen as a positive control group. The results are shown in Fig.

As shown in FIG. 3, gamma-mangosteen of the present invention significantly inhibited the production of active form of caspase-3 (cleaved caspase 3) as compared with the control group. On the other hand, the positive control group, alpha mangosteen significantly inhibited caspase-3, but was found to have lower inhibitory activity than gamma mangosteen.

Therefore, the gamma-mangosteen of the present invention significantly inhibits the activity of caspase-3 involved in apoptosis in a concentration-dependent manner and has a more excellent inhibitory effect than the other xanthone, alpha mangosteen, Inhibitory effect was excellent.

2. Measurement of DNA fragmentation inhibitory activity of gamma-mangosteen

In order to examine the apoptosis inhibitory activity of gamma-mangosteen of the present invention, the primary cultured rat cerebral cortical neurons prepared in Experimental Example 2 were treated with H ₂ O ₂ for 2 hours, And DNA fragmentation was confirmed by staining with the TUNEL method.

Specifically, the measurement of apoptosis by the TUNEL method was performed using the Deadend ^TM Colorimetric TUNEL assay kit and the method proposed by the manufacturer was slightly modified. Separated cerebral cortical cells were transplanted into a 96-well plate at a density of 1 × 10 ⁵ cells / well and cultured at 37 ° C. for 14-15 days in an incubator maintained at 95% air and 5% CO ₂ . The cultured cells were treated with a solution containing 100 mM H ₂ O ₂ or 100 mM H ₂ O ₂ and 10 mM gamma-mangosteen for 2 hours, and then treated with 4% paraformaldehyde (methanol free) For 25 minutes. Then, the cells were washed twice with phosphate-buffered saline (PBS) for 5 minutes, treated with 0.2% Triton X-100 for 5 minutes, and then washed twice with PBS. The cells were treated with equilibration buffer for 10 minutes, washed with PBS, and then treated with TdT reaction mix at room temperature for 60 minutes. After washing with PBS, the cells were treated with 2X SSC (saline-sodium citrate) for 15 minutes, washed with PBS, and then treated with 0.3% H ₂ O ₂ for 5 minutes. After washing with PBS, the cells were treated with horseradish peroxidase-labeled streptavidin for 30 minutes at room temperature, washed again with PBS, treated with diaminobenzidine for 10 minutes, ≪ / RTI > three times and then confirmed by a Nikon eclipse TS100 phase contrast microscope. The results are shown in Fig.

As shown in FIG. 4, it was confirmed that the number of cells stained in the experimental group treated with H ₂ O ₂ , which is an oxidative cell damage inducer, and 10 μM of gamma-mangostin of the present invention, was almost the same as that of the control group.

Accordingly, it was confirmed that the gamma-mangosteen of the present invention significantly inhibited DNA fragmentation involved in apoptosis, and thus, it was confirmed that the effect of inhibiting damage to nerve cells was excellent.

[Experimental Example 3: Antioxidant activity measurement of gamma mangosteen]

In order to measure the antioxidative activity of gamma-mangosteen of the present invention, the lipid peroxidation inhibitory activity and DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of gamma mangosteen prepared in Example 1 were measured, Brain homogenate was obtained from Weinung SD rats.

Measurement of lipid peroxidation inhibitory activity of gamma mangosteen was performed as follows.

First, a certain amount of brain homogenate; 10 μM Fe ²⁺ ; 100 [mu] M ascorbic acid; And gamma-mangosteen at an appropriate concentration were reacted at 37 DEG C for 1 hour, and then trichloroacetic acid and TBA were added sequentially and mixed. After heating at 100 ° C for 15 minutes and centrifugation, the supernatant was separated and the absorbance was measured at 532 nm using a microplate reader (SpectraMax M2 ^e microplate reader). The percent inhibition of lipid peroxidation in the experimental group and the control group was calculated using the following equation (1).

The control group was set up by treating only the solvent in which the sample was dissolved. All experiments were expressed as the average value ± SEM calculated from the results obtained by repeating at least 3 times in 2 groups at once.

The DPPH radical scavenging activity of gamma mangosteen was measured as follows.

First, the reaction solution containing 150 μM of DPPH dissolved in methanol and the appropriate concentration of gamma-mangosteen was reacted at 37 ° C. for 30 minutes, and the absorbance at 520 nm was measured using a Microplate reader (SpectraMax M2 ^e microplate) Respectively. The DPPH radical scavenging ability of the experimental group and the control group was calculated using the above equation (1). The results are shown in Fig.

The results are shown in Fig.

As shown in FIG. 5, in the gamma-mangosteen of the present invention, a high lipid peroxidation inhibitory activity of 80% was measured from a concentration of 10 μM or more, wherein the IC ₅₀ value was 5.8 μM. Meanwhile, the gamma-mangosteen of the present invention had a DPPH radical scavenging activity of 17.2% at 10 μM and 51% at 30 μM, and the IC ₅₀ value was 29.3 μM.

Therefore, the gamma-mangosteen of the present invention has a strong lipid peroxidation inhibitory activity and a small but significant DPPH radical scavenging ability, and thus it is confirmed that the antioxidant activity is excellent and the nerve cell damage inhibition and neuroprotective effect are excellent.

[Experimental Example 4: Measurement of nerve cell toxicity inhibitory activity of gamma mangosteen]

1. Measurement of excitatory nerve cell toxicity inhibitory activity

In order to measure the excitatory neuron cytotoxicity inhibitory activity of gamma-mangosteen according to the present invention, the primary cultured rat cerebral cortical neurons prepared in Experimental Example 2 were suspended in 300 μM N-methyl-D-aspartate or 100 treated with glutamate for 15 minutes and then cultured for 20 to 24 hours. Then, the MTT method was performed in the same manner as described in Experimental Example 3, and the cell viability and the method described in 4 of Experimental Example 1 Similarly, ROS was measured using DCF-DA.

Specifically, the cultured neurons were washed with HCSS, treated with Mg ²⁺ -free HCSS containing 100 μM glutamic acid or 300 μM NMDA for 15 minutes, and the culture medium was exchanged with MEM containing 25 mM glucose And incubated for 20-24 hours at 37 ° C in an incubator maintained at 95% air and 5% CO ₂ environment to induce excitatory neuronal cytotoxicity. The induced nerve cell toxicity was measured according to the above measurement method, and the results are shown in FIG.

As shown in FIG. 6, the gamma-mangosteen of the present invention significantly inhibited NMDA or glutamate-induced excitatory neuronal cytotoxicity in primary cultured rat cerebral cortical neurons in a concentration range of 0.3 to 3 μM, %. In addition, it was confirmed that the gamma-mangosteen of the present invention significantly decreased the concentration of ROS produced by NMDA or glutamate, respectively.

Therefore, the gamma-mangosteen of the present invention significantly inhibited NMDA and glutamate-induced excitatory neuronal cytotoxicity in a concentration-dependent manner, thus confirming that the effect of inhibiting neuronal damage is excellent.

2. Measurement of Aβ peptide-induced neuronal cytotoxicity inhibitory activity

The primary cultured rat cerebral cortical neurons prepared in Experimental Example 2 were suspended in 40 [mu] M A [beta] _(25-35) (Amyloid [beta] ₎ to measure the inhibitory activity of A [beta] peptide- protein fragment (25-35)) for 24 hours, and the cell survival rate was measured by MTT method in the same manner as described in Experimental Example 3.

Specifically, Aβ _(25-35) was dissolved at a concentration of 1 mM using a slight modification of the above Experimental Method 1, followed by aggregation in an incubator at 37 ° C. for one week, and then used. Then, the washing and the cultured neurons with HCSS, and then 40 μM Aβ and added to the MEM culture medium containing 25 mM glucose dissolved the _(25-35) keep the environmental conditions of 95% air, 5% CO ₂ 37 ℃ Incubated for 24 hours in a temperature incubator to induce neuronal cytotoxicity. The induced A [beta] peptide-induced neuronal cytotoxicity was measured according to the above measurement method, and the results are shown in Fig.

As shown in Fig. 7, gamma-mangosteen of the present invention was confirmed to have a cell survival ratio in the range of 30 to 40% as compared with the control group. Aβ _(25-35) and gamma mangosteen at 0.3 to 10 μM concentration were treated together, but the toxicity of neurons was inhibited significantly.

Therefore, it was confirmed that the gamma-mangosteen of the present invention significantly inhibited A [beta] peptide-induced neuronal cell toxicity, and thus it was superior in the effect of inhibiting neuronal damage.

[Experimental Example 5: Measurement of beta-secretase inhibitory activity of gamma mangosteen]

In order to measure the beta-secretase inhibitory activity of the gamma-mangosteen of the present invention, beta-secretase fluorescence resonance energy transfer (BACE1 FRET) was performed according to the conventional method. The beta-secretase is a protein involved in the production of A [beta] peptide from amyloid precursor protein (APP).

Specifically, the inhibitory activity of the beta-secretase was measured by using a BACE1 FRET with a slight modification of the method proposed by the manufacturer. Specifically, 10 μM gamma-mangostin at an appropriate concentration was added to a 96-well plate (the control was replaced with an Esey buffer solution) and 20 μM of the substrate was added to the 96-well plate. The plate was carefully mixed with 10 μM of beta-secretase, ^The fluorescence was measured at 545 nm excitation and 585 nm emission using a microplate reader. After reacting at room temperature for 2 hours, fluorescence was measured under the same conditions. The inhibition rate (%) of the activity of beta-secretase was calculated using the above formula (1). The results are shown in Fig.

As shown in FIG. 8, the gamma-mangosteen of the present invention significantly inhibited beta-secretase in a concentration-dependent manner, wherein the IC ₅₀ value was 0.96 μM.

Therefore, the gamma mangosteen of the present invention significantly inhibited the production of A [beta] peptide by significantly inhibiting beta-secretase in a concentration-dependent manner, and thus it was confirmed that the effect of treating dementia by inhibiting the dementia inducing factor is excellent.

[Experimental Example 6: Cytotoxicity measurement of primary cultured cortical cells of gamma-mangosteen]

In order to evaluate the cytotoxicity of the gamma mangosteen of the present invention, the gamma mangosteen prepared in Example 1 was added to the primary cultured rat cerebral cortical neurons prepared in Experimental Example 2 at a concentration of 0.3 to 30 μM ), And cell viability was measured by MTT method in the same manner as described in Experimental Example 1, 3. The results are shown in FIG.

As shown in FIG. 9, the gamma-mangosteen of the present invention did not significantly affect the cell viability of the neuron at the concentration of 0.3-10 μM, but the cell viability was decreased from 30 μM concentration to 33.4% Respectively.

Therefore, it was confirmed that the gamma-mangosteen of the present invention had no significant cytotoxicity at a concentration of less than 30 μM, but significant cytotoxicity at a concentration of 30 μM or more.

Hereinafter, the pharmaceutical compositions for preventing or treating degenerative brain diseases containing gamma-mangosteen according to the present invention and the pharmaceutical compositions for preventing or ameliorating degenerative brain diseases will be described. However, the present invention is not limited to the specific examples I want to explain.

[Preparation Example 1: Preparation of pharmaceutical preparation]

1. Manufacturing of powder

Gamma Mangosteen 20 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

2. Preparation of tablets

Gamma Mangosteen 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

3. Preparation of capsules

Gamma Mangosteen 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

4. Preparation of injections

Gamma Mangosteen 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

5. Manufacture of liquids

Gamma Mangosteen 20 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

[Formulation example 2. Preparation of food preparation]

1. Manufacture of health food

Gamma Mangosteen 100 mg

Vitamin mixture quantity

70 g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 g of vitamin B12

Vitamin C 10 mg

Biotin 10 g

Nicotinic acid amide 1.7 mg

Folate 50 g

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary 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 mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

2. Manufacture of health drinks

Gamma Mangosteen 100 mg

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3g

Water quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 liter container, It is used in the production of the health beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (8)

A pharmaceutical composition for preventing or treating degenerative brain diseases, comprising gamma-mangosteen represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

The pharmaceutical composition according to claim 1, wherein the gamma mangosteen is obtained from mangosteen hull.
The method according to claim 1, wherein the gamma mangosteen is obtained by extracting mangosteen hull with water, an alcohol having 1 to 4 carbon atoms or a mixed solvent thereof to obtain a crude extract, and then extracting the crude extract with hexane, chloroform, Wherein the composition is obtained by fractionation of the chloroform fraction obtained by successive fractionation with ethyl acetate, n-butanol, in order to prevent or treat degenerative brain diseases.
4. The method according to claim 3, wherein the alcohol having 1 to 4 carbon atoms is 1 to 100% (v / v) methanol or 1 to 100% (v / v) A pharmaceutical composition.
5. The method of any one of claims 1 to 4 wherein the degenerative brain disease is selected from the group consisting of dementia, mild cognitive impairment, stroke, Creutzfeldt-Jakob disease, traumatic head injury, syphilis, acquired immune deficiency syndrome, brain abscess, Hypersomnia, Parkinson's disease, schizophrenia, Huntington's disease, Pick's disease, amyotrophic lateral sclerosis, epilepsy, ischemia, paralysis, paresis, hyperactivity disorder, schizophrenia, depression, mania, posttraumatic stress disorder, , Wherein the compound is at least one kind of degenerative brain disease selected from the group consisting of:
6. The method according to claim 5, wherein the dementia is selected from the group consisting of senile dementia, Alzheimer's disease, vascular dementia, dementia of the frontal lobe, dementia due to normal pressure hydrocephalus, dementia caused by head trauma, dementia caused by metabolic diseases, Wherein the dementia is one or more dementia selected from the group consisting of dementia, dementia, and dementia.
1. A food composition for preventing or improving degenerative brain diseases, comprising gamma-mangosteen represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

8. The food composition according to claim 7, wherein the gamma mangosteen is obtained from mangosteen peel.

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