KR20160081189A - Composition comprising an extract of Eisenia bicyclis for preventing and treating Alzheimers disease - Google Patents

Abstract

The present invention provides the use of rhubarb extract for the prevention, treatment or amelioration of Alzheimer's dementia. The rhubarb extract according to the present invention is characterized in that the activity of beta-site amyloid precursor protein cleaving enzyme (BACE), which is one of the enzymes involved in the production of beta-amyloid peptide which is considered to be a cause of Alzheimer's disease, And can be usefully used as an effective ingredient for preventing, treating or ameliorating Alzheimer's disease.

Description

[0001] The present invention relates to a composition for preventing and treating Alzheimer's disease,

The present invention relates to the use of rhubarb ( Eisenia bicyclis ) The present invention relates to the use of the extract for the prevention, treatment or amelioration of Alzheimer's disease.

The central nervous system, which consists of the brain and spinal cord, is the central center for the management of life phenomena. It is the central center for all the functions of the human body, from sensory and (voluntary) exercise to thinking, memory, It is an essential institution. Therefore, in all cases, such as the death of rapid nerve cells caused by stroke or trauma, or the death of slow nerve cells that cause degenerative diseases of the central nervous system such as senile dementia represented by Alzheimer's disease and Parkinson's disease, Resulting in an irreversible dysfunction of the network and eventually a permanent loss of the corresponding human function. Elderly dementia, represented by Alzheimer 's disease, is characterized by an increase in the life expectancy of the human being and a proportional increase in association with the modernization of medical welfare facilities. According to the statistics of the Korea Institute for Health and Social Affairs, the elderly population of Korea exceeded 7% in 2000 and entered the aged society. In 2003, the number of elderly people reached 8.3%, 3.97% in 2003 and 14.4% . The prevalence of dementia in elderly people over 65 years of age is estimated to be 8.2%. In the western world, approximately 10% of the population aged 65 and over, 40-50% of the population aged 80 or older are developing Alzheimer's disease, and the US already has over 5 million people with this disease, It is estimated to be in dollars. In Korea, more than 200,000 people were found to be dementia patients. In the United States, it is estimated to double to 2030 by 2030 and to reach 14 million by 2050, an increase of more than 350%.

Alzheimer's disease, which begins with cognitive dysfunction, is a degenerative disease that is destroying human nature and progressing over a long period of time. Therefore, it can not afford the socio-economic burden as a passive method of accommodating patients. Should be. However, there have not been developed therapeutic agents capable of treating the underlying cause of Alzheimer's disease so far. Common therapeutic agents include Aricept, an acetylcholinesterase inhibitor of Pfizer, Exelon of Novartis, Reminyl, and Ebixa (Memantine) from Lundbeck, a recent antagonist of the NMDA receptor that has been approved by the US FDA. However, acetylcholinesterase inhibitors improve the declining cognitive ability and do not cure the underlying cause of Alzheimer's disease. In addition, only some patients (about 40-50%) exhibit temporary symptomatic relief, and their efficacy is not long-lasting. In addition, due to the nature of the disease, a long-term use of the drug is required. However, the above-mentioned medicines have problems such as liver toxicity, vomiting, loss of appetite and various side effects. Therefore, it is urgent to develop a therapeutic agent that can prevent the progress of the disease. To this end, many multinational pharmaceutical companies are investing heavily in research and development in this field. In particular, beta or gamma secretease, which reduces the production of beta-amyloid composed of about 40 amino acids which is presumed to be a fundamental cause of Alzheimer's disease The development of inhibitors is the dominant species. In Korea, basic research on Alzheimer's disease has been done to a certain extent, but there is almost no case of development of dementia treatment itself. In the case of gamma secretase inhibitors, the outlook is unclear, as it is accompanied by significant toxicity in recent animal studies as well as in animal models. Although the period of comparative research is relatively short, it is promising as a target for development of more safe and effective dementia treatment as shown in the results of the gene-deficient transgenic animal model in the case of beta-secretease. It is also believed that targeting factors involved in the coagulation of beta-amyloid are relatively safe. In the recent development of a new drug targeting beta-amyloid, the third phase of Phenserine has been carried out in Ann Sonics of the United States. However, its efficacy was not better than that of the control group, it has the S choline has been reported that even with TB has the (cholineesterase) inhibition effect (Greig et al, J. Med Chem 44, pp.4062-4071, 2001;... MedicalNewsToday September 04, 2004 Www.medicalnewstoday.com, the Alzheimer's Association website, www.alzforum.org/drg/drc).

Another possibility is the development of a vaccine using beta-amyloid. As a result of a series of studies and clinical trials centering on Elan, it has been reported that beta-amyloid peptide can be used as a vaccine. Currently, development of vaccines using various beta-amyloid structures is underway. As a result of animal testing, beta-amyloid vaccines have been shown to reduce the number of elderly people formed in the brain and improve the cognitive performance of model animals. In addition, it may be possible to alleviate Alzheimer's disease by promoting the activity of brain cells and improving the activity of injured brain cells to improve cognitive function.

Therefore, if the activity of the acetylcholinesterase inhibitory drug is confirmed to be in the form of beta-amyloid aggregation, toxicity inhibition effect and antioxidative effect, it would be a very good therapeutic agent for the development of a new therapeutic agent for Alzheimer's disease. Such a strategy may be able to maximize the therapeutic purpose and efficacy of the disease proposed by Datamonitor et al. The development of therapeutic agents can be seen as a way of eliminating the cause and simultaneously developing symptomatic agents such as existing drugs (Nature Review Drug Discovery (2007) 6 , p.

Rhei Rhizoma is the root and rhizome of the perennial herbaceous plants and related plants of Rhododendron spp., And there are about 15 species of plants. In Korea, Rheum coreanum Nakail, Pheum undulatum Linne), and dry root and rootstock are used as originals. In China, dry root and rootstocks such as Rheum palmatum Linne, Pheum tanguticum Maxim and Rheum offcinale Baill, . Studies on the components of rhubarb are continuing until recently, and studies on the sub-actions of sennoside A, prevention of gastrointestinal mucosal injury, aloe-emodin and rhein have been reported. (Chongqing Honsha, ed., Shanghai Science and Technology Publishing Co., Vol. 2, p.710, 715-716, 1996). Also, rhubarb is clinically applied to the treatment of dyspepsia, constipation, acute inflammation, infectious diseases, parasitic diseases, bleeding, thrombocytopenia, burns and skin diseases.

It is an object of the present invention to provide a medicinal composition for preventing or treating Alzheimer's dementia which is capable of inhibiting beta-secretase, that is, capable of preventing or treating Alzheimer's dementia.

The present invention provides a pharmaceutical composition for preventing and treating Alzheimer's disease with dementia comprising an extract of rhubarb as an active ingredient.

In addition, the present invention provides a food composition for preventing and alleviating Alzheimer's disease, which comprises a rhubarb extract as an active ingredient.

Also, in the present invention, 1) a step of extracting rhubarb with ethanol and fractionating the residue with a mixed solvent of hexane, water and ethanol to obtain an ethanol / water fraction; And

2) fractionating the ethanol / water fraction obtained from the step 1) with ethyl acetate to obtain an ethylacetate fraction.

Also, the present invention provides a pharmaceutical composition for preventing and treating Alzheimer's disease, comprising fucofucoole-B represented by the following formula (1) as an active ingredient.

[Chemical Formula 1]

In addition, the present invention provides a food composition for preventing and alleviating Alzheimer's disease, which comprises the aforementioned fucofructose-B as an active ingredient.

1 is a schematic diagram showing a process for producing a rhubarb extract.

FIG. 2 is a schematic diagram showing a separation and purification process of an ethyl acetate layer (ethyl acetate fraction) as the active fraction isolated in Example 1. FIG.

FIG. 3 is a graph showing the results of analyzing inhibition patterns between enzyme-substrate using the line-weaver bulk method. FIG.

Figure 4 shows the final active fraction, Fr. For B1-F1B a graph showing the results of a ¹³ C-NMR spectrum and the ¹ H-NMR spectrum.

FIG. 5 is a graph showing cytotoxicity of (A) SH-SY5Y cells and (B) BV-2 cells of fucofluoro-echol-B.

FIG. 6 is a graph showing cytoprotective effect in (A) SH-SY5Y cells and (B) BV-2 cells that induced toxicity with fucofuro-echol-B in beta-amyloid.

FIG. 7 is a graph showing the ROS inhibitory effect of fucofuro-echol-B in BV-2.

8 is a graph showing the effect of fucofuroecol-B on amyloid precursor protein action in neuroblastoma cells.

FIG. 9 is a graph showing the anti-inflammatory effect of fucofloicol-B in neuroblastoma cells induced by an inflammatory reaction with beta-amyloid.

FIG. 10 is a graph showing the effect of Fucofluoro-B on neuroblastoma cells induced by beta-amyloid to inhibit the expression of stressoxicity (HO-1) and apoptosis.

The rhubarb extract of the present invention has the activity of beta-site amyloid precursor protein cleaving enzyme (BACE) which is one of the enzymes involved in the production of beta-amyloid peptide which is considered to be the cause of Alzheimer's disease. And can be usefully used as an effective ingredient for preventing, treating or ameliorating Alzheimer's disease by inhibiting beta-amyloid formation, inhibition of beta-amyloid deposition and neurotoxicity caused by beta-amyloid.
Furthermore, fucofuroecol-B isolated from the rhubarb extract has an excellent effect for preventing, treating or improving Alzheimer's disease.

The present invention provides a pharmaceutical composition for preventing or treating Alzheimer's disease with dementia comprising an extract of rhubarb as an active ingredient.

In the production of the rhubarb extract of the present invention, dried or undried rhubarb or mixtures thereof may be used. For the efficiency of extraction, rhubarb can be crushed and used. In addition, in order to obtain an effective extract of rhubarb for the prevention or treatment of Alzheimer's dementia, branching may be performed at an elevated temperature of about 100 캜 for about 5 minutes before pulverization of the rhubarb, thereby inactivating the intracellular enzyme.

In the present invention, for the production of rhubarb extract, extraction can be carried out according to a conventional extraction method using an extraction solvent of 3 to 10 times of rhubarb.

As the extraction solvent, water, an organic solvent, or a mixed solvent thereof widely used in natural material extraction can be used. Examples of the organic solvent include ethanol, methanol, hexane, chloroform, and ethalacetate. Such an organic solvent may be used in admixture with water, but may be used without being mixed with water for easy elution of the active ingredient. In one embodiment, the rhubarb extract may be an ethanol extract.

The rhubarb extract may be further fractionated using an organic solvent having a polarity different from that of the organic solvent used in the first extraction. In this case, the rhubarb extract may be a rhubarb extract fraction obtained by extracting rhubarb with an organic solvent and fractionating the residue using an organic solvent having a different polarity.

The rhubarb extract or rhubarb extract may be further purified by various chromatographies such as LH-Sephadex, silica gel column chromatography, thin layer chromatography and high performance liquid chromatography Lt; / RTI > to obtain further purified fractions. In the present invention, the 'rhubarb extract' includes all of the extract, fraction and purified product obtained in each step of extraction, fractionation or purification, and diluted solution, concentrate or dried product thereof.

In one embodiment of the present invention, the rhubarb extract may be an ethanol / water fraction obtained by extracting rhubarb with ethanol and fractionating the residue with a mixed solvent of hexane, water and ethanol.

In another embodiment of the present invention, the rhubarb extract is obtained by extracting rhubarb with ethanol and fractionating the residue with a mixed solvent of hexane, water and ethanol. Ethanol / water fraction obtained by fractionating with ethyl acetate Lt; / RTI >

The thus obtained rhubarb extract fraction may be subjected to LH-Sephadex column chromatography repeatedly, or TLC (thin layer chromatography) and HPLC (high performance liquid chromatography) may be performed to obtain further purified rhubarb extract fractions.

In the following Examples, it is confirmed through this additional purification process that the active ingredient showing the preventive or therapeutic activity for Alzheimer's dementia from the rhubarb extract is a glutarnine compound, specifically, a fluoro-ecocompound or fucofluoro-B represented by the following formula Respectively.

[Chemical Formula 1]

Accordingly, the present invention provides a medicinal composition for preventing or treating Alzheimer's dementia comprising, as an active ingredient, a rhubarb extract containing fucofurocool-B.

The present invention also

1) extracting rhubarb with ethanol and fractionating the residue with a mixed solvent of hexane, water and ethanol to obtain an ethanol / water fraction; And

2) fractionating the ethanol / water fraction obtained from the step 1) with ethyl acetate to obtain an ethylacetate fraction.

The present invention also provides a pharmaceutical composition for preventing or treating Alzheimer's dementia comprising fucofurocool-B as an active ingredient. Although Fucofluorecol-B is isolated from the rhubarb extract according to the present invention, it can be chemically synthesized, commercially available, and can be isolated from other plants.

In the following examples of the present invention, rhubarb was extracted with 100% ethanol and the resulting residue was fractionated in the order of hexane: water: ethanol and ethyl acetate to prepare a rhubarb extract fraction. Further, the ethyl acetate fraction in the rhubarb extract fraction was further fractionated and purified. The rhubarb extract, rhubarb extract and fucofructoolol-B of the present invention obtained through this process exhibit an excellent beta-secretase inhibitory effect.

Therefore, the rhubarb extract, the rhubarb extract, and the fucofucoole-B of the present invention may be included as an active ingredient for the prevention or treatment of Alzheimer's dementia.

The composition for preventing or treating Alzheimer's dementia of the present invention can be used for preventing or treating Alzheimer's dementia which is a disease caused by the activity of beta-secretase, in particular, inhibiting the activity of beta-secretase .

The composition can be used not only with the active ingredient of the present invention but also with a conventionally known preventive or therapeutic agent for Alzheimer's dementia.

The pharmaceutical composition comprising the rhubarb extract of the present invention or fucofructose-B as an active ingredient may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned effective ingredients. Examples of the adjuvants include excipients, Release agents, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants or flavors.

The content of rhubarb extract or fucofructose-B in the pharmaceutical composition can be appropriately adjusted according to the symptoms of the disease, the progress of the symptoms, the condition of the patient, the administration method, the form of the preparation, etc. For example, , 0.0001 to 10 parts by weight, or 0.001 to 1 part by weight, but is not limited thereto. The content ratio is a value based on the dried amount from which the solvent is removed.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the active ingredient described above for administration.

The form of the pharmaceutical composition may be a granule, a powder, a tablet, a coating, a capsule, a suppository, a liquid, a syrup, a juice, a suspending agent, an emulsion, a drip agent or an injectable liquid agent. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, natural sugars such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include those suitable for sterilization and in vivo such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The present invention also provides a food composition for preventing or alleviating Alzheimer's dementia comprising the above-described rhubarb extract or fucofructose-B.

The term " food composition " in the present invention means a food prepared by adding the above-described rhubarb extract or fucofructose-B to a food material such as a beverage, a tea, a spice, a gum or confectionery, or by encapsulating, This means that it takes a certain effect on health when consumed, but unlike general medicine, there is an advantage that there is no side effect that may occur when a drug is taken for a long time by using food as a raw material.

In the present invention, the food composition for preventing or ameliorating Alzheimer's dementia may be used for preventing or improving Alzheimer's dementia caused by the activity of beta-secretase such as the above-mentioned pharmaceutical composition.

The food composition according to the present invention can be formulated in the same manner as the above-mentioned pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, , Tea, a drink, an alcoholic beverage, and a vitamin complex, and includes foods in a conventional sense.

When the above-described rhubarb extract or fucofructoolole-B is used as a food additive, the rhubarb extract or fucofructose-B can be used as it is, or can be used together with other food or other food ingredients, and can be used suitably according to a conventional method have. The amount of the active ingredient to be mixed may be within a range that does not impair the inherent taste of the food, and is usually 0.01 to 50 parts by weight or 0.1 to 20 parts by weight based on 100 parts by weight of the total composition. In the case of foods in the form of granules, tablets or capsules, they may be added usually in the range of 0.1 to 100 parts by weight, or 0.5 to 80 parts by weight. However, in the case of long-term consumption intended for health or hygiene purposes or for health control purposes, 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.

The food composition may further contain various flavors or natural carbohydrates. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau Martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. In addition to the above, the food composition according to 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, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain 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 based on 100 parts by weight of the food composition of the present invention.

The invention also provides a method for the prevention or treatment of Alzheimer ' s dementia comprising administering to a mammal a therapeutically effective amount of a rheumatoid extract or fucofloekel-B.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

As used herein, the term "therapeutically effective amount " refers to the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, This includes an amount that induces relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. In the treatment method of the present invention, in the case of an adult, it is preferable to administer the rhubarb extract or fucofructool-B of the present invention at a dose of 1 mg / kg to 1000 mg / kg once or several times a day .

In the therapeutic method of the present invention, the composition containing the rhubarb extract or fucofructose-B as an active ingredient of the present invention can be administered orally, rectally, intravenously, intramuscularly, intraperitoneally, intramuscularly, intrasternally, transdermally, Or via the intradermal route in a conventional manner.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings, in which: FIG. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and is defined only by the scope of the claims of the invention.

[ Example ]

Example  One: rhubarb  Preparation of extract

Eiseniabycylis collected from Ulleungdo was thoroughly washed with water, dried thoroughly at 40 ℃ and then ground and used as a raw material for extraction.

Sulfur powder was extracted according to the method shown in Fig. Specifically, 3 L of 80% ethanol was added to 1 kg of powdered rhubarb, and the mixture was stirred under dark condition, and extracted twice for every 24 hours. The mixture was filtered and concentrated to obtain an ethanol extract. At this time, the extraction yield of the ethanol extract of rhubarb was 14.0%.

The concentrated ethanol extract was dissolved in 50 ml of a hexane: water: ethanol (6: 3: 3) solution, and then extracted with a liquid / liquid extraction method as shown in Fig. First, the hexane layer was separated from the ethanol extract, the hexane layer was extracted, and 50 ml of hexane was added to the remaining ethanol / water layer to perform re-separation in the same manner. After separating the hexane layer, the remaining ethanol / water layer was separated from the ethyl acetate layer by adding 100 ml of ethyl acetate. The fractionated hexane layer, ethyl acetate layer, and ethanol / water layer were concentrated under reduced pressure, fixed at a predetermined weight, and then assayed for beta-secretase inhibitory activity.

The extraction yield of the rhubarb ethanol extract was 14%, and the extraction yields of the hexane and ethyl acetate fractions were 4.8% and 3.1%, respectively.

Example  2: Beta- Secrete agent  Measurement of inhibitory activity

In order to confirm the preventive and therapeutic effects of Alzheimer's disease, a degenerative brain disease of the rhubarb extracts prepared in Example 1, the inhibitory activity of beta-secretase, which is involved in the major mechanism of Alzheimer's disease progression, Inhibitory activity assay. Extract fractions containing a large amount of beta - secretase inhibitory activity were selected among the hydrolysates.

The beta-secretase inhibitory activity was measured by modifying the method described in the beta-secretase assay kit (beta-secretase assay kit). Specifically, 70 μl of buffer (50 mM sodium acetate, pH 4.5), 10 μl of β-secretase (1.0 Unit / ml), 10 μl substrate (10 mM of MCA- [ASN670, LEU671] -AMYLOID BETA / A4 PRECURSOR PROTEIN 77, 50 mM sodium acetate buffer, pH 4.5) was mixed with 10 μl of the sample solution and reacted at 25 ° C for 20 minutes under dark condition. The fluorescence reader (Bio-Rad) was measured at excitation 360 nm and emission 405 nm. The inhibition rate of beta-secretase was calculated using the following formula 1.

<Formula 1>

Inhibition (%) = [1 - {(S-S0) / (C-C0)}] 100

C: Fluorescence value after 20 minutes from the start of the reaction in the experimental group (Enzyme + buffer + substrate) to which the sample solution was not added

C0: Fluorescence value at zero time in the experimental group (Enzyme + buffer + substrate) to which the sample solution was not added

S: Fluorescence value after 20 minutes of reaction of the sample (enzyme + sample + substrate) to which the sample solution was added

S0: Fluorescence value of the sample (enzyme + sample + substrate) to which the sample solution was added at zero time

The beta-secretase inhibitory activity of the rhubarb extract was measured by IC ₅₀ values, and the results are shown in Table 1 below.

IC ₅₀ value (mg / ml) Ethanol layer 0.70 Hexane layer 1.02 Ethyl acetate layer (EtOAcfr.) 0.24 Water layer 2.12

As shown in Table 1, the ethanol extract and beta of the extracted fraction-sikeuri protease inhibitory activity were respectively 0.70 ㎎ / ㎖ as IC ₅₀ value, appeared to 1.02 mg / ml, 0.24 mg / ml and 2.12 mg / ml , And the ethyl acetate fraction inhibitory activity is the most excellent.

Hereinafter, the ethyl acetate fraction was separated and purified by chromatography.

Example  3: LH - Sephadex  And Thin layer chromatography Beta - Secrete agent  Separation of Inhibitors

Separation and purification of the ethyl acetate layer (ethyl acetate fraction), which was the active fraction isolated in Example 1, was performed as shown in the schematic diagram of FIG. 2, and the results of the beta-secretase inhibition activity of the separated active fractions are shown in Table 3.

First, an ethyl acetate layer (ethyl acetate fraction, EtOAcfr.) Was separated from LH-Sephadex. Separation conditions were as shown in Table 2 below.

Condition Contents Column strike Diameter 1.5cm, Height 50cm menstruum Methanol Flow rate 2.0 mg / ml Fraction of fraction 8.0ml

As a result of separation according to the conditions shown in Table 2, it was separated into five fractions (Fr.A. to Fr.F.), and the separated fractions were concentrated to a predetermined amount, and the β-secretase inhibitory activity Were measured. As a result, the activity of the Fr.F. fraction was highest among the five separated fractions (see Table 3).

Fr.B fractions with excellent activity were separated and purified by solid phase extraction (SPE). SPE was a column packed with C18 resin, which was separated and purified by reverse phase adsorption. Four fractions (Fr. B1 to B4) were isolated from the SPE column and the beta-secretase inhibitory activity of each fraction was measured. As a result, Fr. The beta-secretase inhibitory activity of the B1 fraction was the highest (see Table 3).

Fr. The B1 fractions were separated and purified by thin layer chromatography (TLC). TLC separation can be performed by separating the organic solvent extracts into nonpolar and polar materials. Eight fractions were isolated using hexane, ethyl acetate, and acetone in the ratio of 7: 3: 0.1. The beta-secretase inhibitory activity of each fraction was measured. As a result, Fr.beta.1-F1 among the eight fractions showed the highest beta-secretase inhibitory activity (see Table 3).

The Fr.B1-F1 fractions were separated and purified using high performance liquid chromatography (HPLC). Three fractions (Fr. B1-F1A to B1-F1C) were separated by HPLC and the beta-secretase inhibitory activity of each fraction was measured. As a result, Fr. The beta-secretase inhibitory activity of B1-F1B was the most excellent, and finally Fr. The active substance of the B1-F1B fraction was isolated.

The beta-secretase inhibitory activity of the separated final material was 18.15 μg / ml when expressed as an IC ₅₀ value, which was 13.2 times higher than that of the acetic acid fraction.

Purification step IC ₅₀ value (μg / ml) Purification fold ^* EtOAcfr. 240.01 0.08 1.00 Sephadex LH-20 (Fr. B) 160.13 + - 6.45 1.47 SPE column (Fr. B1) 91.53 + - 7.52 2.62 TLC (Fr. B1-F1) 38.24 + - 5.16 6.27 HPLC (Fr. B1-F1B) 18.15 + - 4.25 13.22 ^* Relative value of reciprocal of β? Ecretase inhibitory activity by IC ₅₀ value

FIG. 3 is a graph showing the results of analysis of the inhibition pattern between the enzyme and the substrate using the line weaver bulk method. As the graph of the separated final material treated by concentration crosses the X-ray, the beta-secretase inhibition Competitive inhibition, and a Ki value of 10.1 [mu] M. An &quot; uncompetitive inhibition &quot; between isolated enzyme-substrates allows the inhibitor to bind at or adjacent to or at the active site of the enzyme or protein and does not affect the Km value in relation to the enzyme, but decreases Vmax .

Example  4: From the rhyme  Identification of isolated active ingredients

The final active fraction, Fr. The &lt; ¹³ &gt; C-NMR spectrum and &lt; ^{1 &gt;} H-NMR spectrum of B1-F1B were carried out and the results are shown in Fig. 4, (A) shows ¹³ C-NMR spectrum and (B) shows ¹ H-NMR spectrum.

As a result, it was confirmed that the active fraction was fucofurocon-B (C ₂₄ H ₁₄ O ₁₁ ).

^{1 H-NMR (300MHz, DMSO} ) δ: 11.58 (1H, s, 14-OH), 10.05 (1H, S, 4-OH), 9.88 (1H, s, 10-OH), 9.45 (1H, s, (2H, s, 3'5'-OH), 8.22 (1H, s, 8-OH), 6.71 Hz, H-11), 6.34 (1H, s, H-3), 6.38 (1H, d, J = 1.5Hz, H- d, J = 1.5Hz, H-2, 6 &apos;).

¹³ C-NMR (300 MHz, DMSO) ?: 160.22 (C-1 '), 158.81 (C-3, 5'), 158.23 , C-15a, C-13a, C-14a, C-14a, C-14a, C- (C-4), 122.04 (C-1), 103.13 (C-6), 102.48 (C-7), 98.43 96.14 (C-13), 93.2, (C-2 ', 6') 90.68 (C-11).

Example  5: Fuko Furo Ecole Of -B Neuroblastoma cell  Protection effect

Cytotoxicity was determined in SH-SY5Y and BV-2 cells of neuroblastoma cells of fucofuroecol-B isolated from rhubarb.

Cell culture of SH-SY5Y, a neuroblastoma cell line, was performed with 10% fetal bovine serum (GIBCO, Carlsbad, CA, USA) in Dulbecco's modified Eagle medium nutrient mix F12 medium (GIBCO, Carlsbad, CA, USA) containing L-Glutamine and 15 mM HEPES , USA) and 100 U / mL penicillin-streptomycin were added and cultured at 37 ° C in 5% CO2. Cell culture of the neuroblastoma cell line BV-2 was performed by adding 5% fetal bovine serum (Hyclone, USA) and 100 U / mL penicillin-streptomycin to Dulbecco's modified Eagle medium (DMEM; Gibco, USA) Lt; / RTI &gt; The cultured cells were adjusted to 1 × 10 4 cells / well and cultured in a 96-well plate for 24 hours.

After incubation for 24 hours, 5 mg / ml of MTT solution was added to the cultured neuroblastoma cells at a concentration of 15 μl / ml (15, 25, 50, 100 and 200 μg / ml) well and cultured for 3 hours. Then, DMSO was added to 150 μl / well to solublize the cells. Absorbance was measured using a microplate reader (TECAN) instrument.

Cell viability was calculated by the following equation and is shown in Fig.

<Formula 1>

Cell viability (%) = Ab570 (sample untreated group) -Ab (sample group) / Ab (sample untreated group) X100

FIG. 5 is a graph showing cytotoxicity in (A) SH-SY5Y cells and (B) BV-2 cells of Fucofluoroecol-B, showing that no cytotoxicity was observed up to a concentration of 200 μg / ml of fucofluoro-B Can be confirmed.

In addition, the protective effect of fucofuroxol-B on neuroblastoma cells in neuroblastoma cells that caused toxicity with beta-amyloid was measured.

The beta-amyloid peptide was completely dissolved in 100% DMSO at a concentration of 250 uM and then diluted 1: 10 with PBS to a concentration of 25 μM. After inducing coagulation reaction at room temperature for 4 days, they were stored frozen in 1.5 ml tubes. SH-SY5Y and BV-2 cells, neuroblastoma cells cultured confluently at about 80% in a 100-mm dish, were treated with 500 μl trypsin-EDTA solution (1X) and dropped from the dish. After 5 ml of the culture medium was suspended, the cells were counted using a hemocytometer. Cell suspension was added to 96 well plate at 1 × 10 ⁴ cells / well, and incubated at 37 ° C in 5% CO ₂ And cultured in a cell incubator for 4 hours or more. After confirming that the cells were attached to the plate, 100 μl / well of the medium without serum was changed and incubated for 1 hour. Fucofluoro-Echol-B was diluted in 50% DMSO concentration, and then added with 5 μl / well and incubated for 1 hour. The aggregated beta-amyloid was treated with 1 μM of the cells and cultured for 18-20 hours. 5 mg / ml of MTT solution was treated with 15 μl / well of cells and cultured for 3 hours. Then, DMSO was added to 150 μl / well to solublize the cells. Absorbance was measured at 570 nm using a microplate reader (TECAN) instrument.

Cytotoxicity was calculated according to the above formula 1 and is shown in FIG.

FIG. 6 is a graph showing cytoprotective effect in (A) SH-SY5Y cells and (B) BV-2 cells that induced toxicity with fucofuro-echol-B in beta-amyloid. It can be confirmed that the treatment effect is dependent on the treatment with fucofuro-echol-B.

Example  6: DCFH - DA Using Intracellular ROS  Inhibitory effect

The intracellular formation of ROS was measured by Engelmann et al. (Engelmann, J., Volk, J., Leyhausen, G., & Geurtsen, W. (2005) ROS formation and glutathione using oxidative sensitive dye DCFH- levels in human oral fibroblasts exposed to TEGDMA and camphorquinone. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 75B, 272-276).

The neuroblastoma cells, BV-2 cells, were cultured on a black microtiter 96-well plate, labeled with 20 μM DCFH-DA dissolved in HBSS (Hanks balanced salt solution) and kept in the dark for 20 minutes. The non-fluorescent DCFH-DA dye, which is freely permeabilized into the cell, is hydrolyzed by the cell neste-8-lase into 2 ', 7'-dichlorodehydrofluorescein (DCFH) and captured inside the cell. The cells were then treated with different concentrations of the samples and incubated for 1 hour. Cells were washed three times with PBS and then 500 μM H2O2 (dissolved in HBSS) was added. The formation of fluorescent DCF by oxidation of DCFH in the presence of various ROS was read out every 30 minutes at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The dose-dependent and time-dependent effects of the treatment are plotted in Fig. 7 as compared to the fluorescence intensity of the control of untreated samples after plotting.

In the present invention, Fig. 7 is a graph showing the effect of ROS inhibition of fucofructoolecol-B in BV-2. As shown in FIG. 7, it can be confirmed that the effect of free radical scavenging in the nerve is concentration-dependent in fucofuroecol-B. Compared with the comparative group ascorbic acid, the higher radical scavenging effect was observed at the sample treatment concentration of 50 μg / ml.

The results of Examples 5 and 6 indicate that fucofuroxole-B, a beta-secretory inhibitor isolated from rhubarb, effectively protects neuroblastoma cells that induced toxicity with beta-amyloid.

Example  7: In neuroblastoma cells  Amyloid precursor protein degradation process Fucoproxecol Impact of -B

Beta-amyloid forms oligomers and aggregates with each other to cause closure of neuroblastoma cells, resulting in irreversible degenerative destruction. In particular, beta - amyloid in the brain and spinal fluid of patients with dementia is detected more frequently than in normal individuals. The SH-SY5Y APP695swe cell line used in the experiment is similar to the pathology of demented patients, and the amount of beta-amyloid secretion is increased more than two times as compared with that of normal cells, so that secretion inhibitory activity is easy to be detected. A sandwich ELISA was performed to determine the amount of beta-amyloid secreted from the cell line transduced in the form of SH-SY5Y APP695swe. 1 × 10 ⁶ cells were cultured in a 60 mm dish for 24 hours, and the culture was recovered in the presence of a protease inhibitor and used as a sample. A 100-μL sample was added to each plate coated with a beta-amyloid-specific monoclonal antibody or a beta-amyloid-specific polyclonal antibody, reacted at 4 ° C for 16 hours, washed seven times, and HRPconjugated Aβ (11-28) specific monoclonal antibody was reacted at 4 ° C for 1 hour. After washing again 9 times, tetramethylbenzidine (TMB) substrate solution was added and reacted at room temperature for 30 min. After adding 100 정지 of stop solution, the absorbance was measured at 450 nm using a Microplate Reader (Model 680, Bio-Rad, CA).

The amount of beta-amyloid secreted in the culture solution after treatment with fucoprooctol-B was measured by the sandwich ELISA method, and the results were compared and shown in FIG. 8 (B). In the present invention, FIG. 8 is a graph showing the effect of fucofurocool-B on amyloid precursor protein in neuroblastoma cells, wherein 8 (B) shows inhibition of beta-amyloid formation. The positive control group was treated with ascorbic acid. The amount of beta-amyloid secreted by 50 μM of fucoprooctol-B was about 49.5 and 30.9%, respectively, in the negative control group.

In addition, western blot analysis was performed for protein expression in SH-SY5Y cells. 1x10 &lt; ⁶ &gt; cells were cultured in a 60 mm dish and treated with fucoprochorol-B dissolved in DMSO for 12 hours. After 24 hours, the culture was recovered under the above conditions. Cell lysis buffer (RIFA buffer, Sigma Aldrich, USA) supplemented with protease inhibitor was added to the cells washed with PBS and pulverized. 50 μg of the protein was separated by 12% Tris-glycine SDS-PAGE, and the protein pattern of sAPPβ (soluble APPβ) was measured by immunoblotting, and the result was shown in FIG. 8 (a). FIG. 9 (a) is a graph showing sAPP-beta formation inhibition, and it can be confirmed that the amount of sAPPβ protein expression is decreased in a concentration-dependent manner. sAPP beta is an amyloid precursor protein present in the neuroblastoma cell membrane, which is produced by degradation of beta-secretase by APP. It is an indicator of the function of beta-secretase.

Example  8: In neuroblastoma cells  Anti-inflammatory effect

The neuroblastoma cells, BV-2 cells, were cultured in 96-well plates and cultured after treatment with various concentrations of fucoprococcal-B and beta-amyloid. Nitrite accumulation in the supernatant was mixed with the same volume of Griess reagent (0.1% N- (1-naphthyl) -ethylenediamine, 1% sulfanilamide in 5% phosphoric acid) and incubated at room temperature for 10 min. The absorbance at 550 nm was measured in an automated microplate reader and sodium nitrite of known concentration was used as a standard. In addition, measurement of the concentration of prostaglandin is -2 for 24 hours to the culture of nerve cells subspecies BV-2 cells in 48-well plate and treated by 24 hours after serum starvation one, the concentration Foucault furo ekkol -B 37 ℃, 5% CO ₂ Lt; 0 &gt; C for 30 minutes. Subsequently, cultured medium supernatants were treated with beta-amyloid for 18 hours and subjected to PGE2 ELISA assay.

RNA was extracted from BV-2 cells, a neuroblastoma cell, and the cells were harvested and total RNA was extracted using RNeasyMiNi Kit (Quiagen, Valencia, Calif.) According to the manufacturer's instructions. CDNA was synthesized by RT-PCR using a Superscript pre-amplification system (GIBCO BRL, Grand Island, NY). PCR was carried out using primers specific for iNOS, COX-2, IL-1 ?, TNF-? and GAPDH: 40 sec at 94 ° C, 45 sec at 55 ° C and 50 sec at 72 ° C followed by 30 cycles Lt; RTI ID = 0.0 &gt; 72 C &lt; / RTI &gt; for 10 minutes. The sequence of the primers was as follows.

Primer Sequence Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) F: 5'-AATGACCCCTTCATTGAC-3 '
R: 5'-TCCACGACGTACTCAGCGC-3 ' iNOS 5'-AAGCACATGCAGAATGAGTACCG-3 '
Gt; COX-2 5'-GGAGAGACTATCAAGATAGTGATC-3 '
5'-ATGGTCAGTAGACTTTTACGACTA-3 ' TNF-a 5'-CCCCTCAGCAAACCACCAAGT-3 '
5'-CTTGGGCAGATTGACCTCAGC-3 ' IL-1? 5'-AATCTCACAGCAGCACATCAA-3 '
5'-AGCCCATACTTTAGGAAGACA-3 '

The PCR products were electrophoresed using 2% agarose gel in TAE (40 mM Tris acetate containing 1 mM EDTA) and visualized by ethidium bromide staining. The level of mRNA was expressed as a percentage of the gene PCR product for the corresponding [beta] -actin PCR product and normalized to the control sample.

The neuroblastoma cells, BV-2 cells, were homogenized using protein extraction solution (PRO-PREP, Intron Biotechnology, Korea), incubated for 1 hour in ice and dissolved, and centrifuged at 15000 rpm for 15 minutes. Protein concentration was measured by Bradford method (Bio-Rad Laboratories Inc. Hercules, Calif., USA) and the same amount of protein (50 μg) was separated on SDS / 12% -polyacrylamide gel, ECL nitrocellulose membrane (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). The blots were incubated with 5% (w / v) skim milk in TBST [Tris-Buffered Saline Tween-20: 150 mM NaCl solution containing 10 mM Tris (pH 8.0) and 0.05% Tween- Lt; / RTI &gt; After briefly washing in TBST, the membranes were incubated with specific antibodies for 5 hours at room temperature. Rabbit polyclonal antibody (1: 1000) (Cayman Chemical, Ann Arbor, Mich., USA) for iNOS and COX-2 and rabbit polyclonal antibody (1: 1000) for TNF- (Santa Cruz Biotechnology Inc. Santa Cruz, Calif, USA). The blots were then reacted with the corresponding conjugated anti-rabbit or anti-mouse antibody (1: 10,000 dilution, Cell signaling). Immunoreactive proteins were detected using an ECL Western blotting detection system. The relative density of the protein bands was scanned by density measurement using MyImage (SLB) and quantified by Labworks 4.0 software (UVP Inc.).

In the present invention, FIG. 9 is a graph showing the anti-inflammatory effect of fucofurocool-B on neuroblastoma cells induced by beta-amyloid, wherein (1) represents the amount of nitric oxide produced, (2) represents the amount of prostaglandin- (TNF-α) and interleukin-beta (IL-1β), and (4) the amount of expression of nitric oxide synthase (iNOS) and cyclooxygenase 2 &lt; / RTI &gt; (COX-2).

The results showed that fucofuroxole-B decreased the production of inflammation response factors such as sodium nitrite and prostaglandin-2 in the neuroblastoma cells induced by β-amyloid, and iNOS, COX-2, IL-1 [beta] and TNF- [alpha] in a dose-dependent manner.

Example  9: Neuroblastoma cell  Killing effect

In order to measure the inhibitory effect of SH-SY5Y cells on beta-amyloid, the expression of HO-1, an intracellular detoxifying enzyme, Bax, a cell death-suppressing gene, and Bcl-2, a cell death gene, -PCR method. PCR was performed using primers specific for HO-1, Bax, Bcl-2 and GAPDH: 40 sec at 94 ° C, 45 sec at 55 ° C and 50 sec at 72 ° C, followed by 30 cycles and 72 ° C Lt; / RTI &gt; for 10 minutes. The sequence of the primers was as follows.

Primer Sequence Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) F: 5'-AATGACCCCTTCATTGAC-3 '
R: 5'-TCCACGACGTACTCAGCGC-3 ' Heme oxygenase-1 F: 5'-CACGCCTACACCCGCTACCT-3 '
R: 5'-TCTGTCACCCTGTGCTTGAC-3 ' Bax F: 5'-GGGGACGAACTGGACAGTAA-3 '
R: 5'-CAGTTGAAGTTGCCGTCAGA-3 Bcl-2 F: 5'-CGACTTCGCCGAGATGTCCAGCCAG-3 '
R: 5'-ACTTGTGGCCCAGATAGGCACCCAG-3 '

The PCR products were electrophoresed using 2% agarose gel in TAE (40 mM Tris acetate containing 1 mM EDTA) and visualized by ethidium bromide staining. The level of mRNA was expressed as a percentage of the gene PCR product for the corresponding [beta] -actin PCR product and normalized to the control sample.

In the present invention, FIG. 10 is a graph showing the effect of Fucofluoro-B on neuroblastoma cells induced by beta-amyloid toxicity, and suppression of intestinal phenotype (HO-1) and apoptosis.

As a result, the expression of HO-1, an intracellular detoxifying enzyme, was increased in a concentration-dependent manner. When the bax / bcl-2 ratio of the neuroblastoma cells was inhibited by the bax / bcl-2 ratio, .

Claims (15)

Containing the rhubarb extract as an active ingredient
A pharmaceutical composition for preventing or treating Alzheimer's disease.
The method according to claim 1,
The rhubarb extract is an ethanol extract of rhubarb
A pharmaceutical composition for preventing or treating Alzheimer's disease.
The method according to claim 1,
The rhubarb extract was obtained by extracting rhubarb with ethanol and fractionating the residue with a mixed solvent of hexane, water and ethanol. The ethanol / water fraction obtained was fractionated with ethyl acetate
A pharmaceutical composition for preventing or treating Alzheimer's disease.
The method according to claim 1,
Rhubarb extracts contain a fluorotannine compound
A pharmaceutical composition for preventing or treating Alzheimer's disease.
The method according to claim 1,
The rhubarb extract contains fucofoolecohol-B represented by the following formula
A pharmaceutical composition for preventing or treating Alzheimer's disease.
[Chemical Formula 1]

Containing the rhubarb extract as an active ingredient
A food composition for preventing or ameliorating Alzheimer's disease.
The method according to claim 6,
The rhubarb extract contains fucofoolecohol-B represented by the following formula
A food composition for preventing or ameliorating Alzheimer's disease.
[Chemical Formula 1]

1) extracting rhubarb with ethanol and fractionating the residue with a mixed solvent of hexane, water and ethanol to obtain an ethanol / water fraction; And
2) fractionating the ethanol / water fraction obtained from the step 1) with ethyl acetate to obtain an ethylacetate fraction.
A pharmaceutical composition for preventing or treating Alzheimer's disease, comprising fucofructose-B represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

10. The method of claim 9,
The composition for preventing or treating Alzheimer's disease according to claim 1, wherein the fucofucoole-B is 0.0001 to 10 parts by weight based on 100 parts by weight of the pharmaceutical composition.
10. The method of claim 9,
A pharmaceutical composition for preventing or treating Alzheimer's disease, which further comprises a pharmaceutically acceptable carrier.
10. The method of claim 9,
A pharmaceutical composition for preventing or treating Alzheimer's disease, which is in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, solutions, aerosols, excipients, injections, transdermal drugs or suppositories.
1. A food composition for preventing or alleviating Alzheimer's disease, comprising fucofucoole-B represented by the following formula (1) as an active ingredient.
[Chemical Formula 1]

14. The method of claim 13,
The composition for preventing or alleviating Alzheimer's disease according to claim 1, wherein the fucofluorocyclo-B is contained in an amount of 0.01 to 50 parts by weight based on 100 parts by weight of the food composition.
14. The method of claim 13,
A food composition for preventing or improving Alzheimer's disease, which further comprises a food-acceptable food supplementary additive.

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