KR100890179B1 - Composition comprising an extract of Scrophularia buergeriana for treating or preventing the disease related to cognitive dysfunction - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of cognitive impairment-related diseases comprising the extract of Hyunsam as an active ingredient, the present extract of the present invention exhibits neuroprotective effect and short- and long-term memory improvement effect, diseases related to cognitive dysfunction It can be used in the preventive and therapeutic pharmaceutical compositions and health functional foods.

       Hyunsam, cognitive function, nerve cell, memory, pharmaceutical composition

Description

Composition comprising an extract of Scrophularia buergeriana for treating or preventing the disease related to cognitive dysfunction

      The present invention relates to a composition for the prevention and treatment of diseases related to cognitive impairment containing Hyunsam extract as an active ingredient.

Peterson RC et al., Mild cognitive impairment: clinical characteristics and outcome, Arch Neurol , 56 , pp.303-308, 1998

Collie A. & Maruff P., Neuropsychology of preclinical Alzheimers disease and mild cognitive impairment, Neuroscience & Behavioral Review , 24 , pp.365-374, 2000

[3] de la Torre JC., Alzheimer's disease; how does it start ?, J. Alzheimer's Dis ., 4 (6), pp. 497-512, 2002

4 Vajda FJ., Neuroprotection and neurodegenerative disease., J. Clin . Neurosci , 9 , pp.4-8, 2002

Monaghan, DT et al., The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system, Annual Review of Pharmacology and Toxicology , 29 , pp.365-402, 1989

Document 6 Kruk, Z.L. et al., Neurotransmitters and Drugs, Chapman & Hall, London, pp. 159-168, 1991

[7] Choi, DW, Glutamate neurotoxicity and diseases of the nervous system. Neuron , 1 , pp. 623-634, 1988

Zaczek, R. and Coyle, JT, Excitatory amino acid analogues: neurotoxicity and seizures, Neuropharmacology , 21 , pp. 15-26, 1982

Document 9 Greenamyre, JT and Young, AB, Excitatory amino acids and Alzheimer's disease, Neurobiology in Aging , 10 , pp. 593-602, 1989

Marino, MJ et al., Glutamate receptor and Parkinson's disease: opportunities and interventions, drugs Aging , 20 (5) , pp. 377-397, 2003

[11] Albers, GW et al., N-methyl-D-aspartate antagonsists: ready for clinical trial in brain ischemia, Annals of Neurology , 25 , 398-403, 1989

12.Yook Chang-soo, Asian Herbal Medicine Book, p483, 1997

[Document 13] Hwang, Sung-Yeon, Herbal Studies on Korean Ginseng Plant, Master's Thesis, Wonkwang University, 1992

Kim SR and Kim YC, Neuroprotective phenylpropanoid esters of rhamnose isolated from roots of Scrophularia buergeriana ., Phytochemistry , 54 , pp. 503-509, 2000

[Document 15] Republic of Korea Patent Publication No. 2000-0008399

[16] Kim SR et al., Four new neuroprotective iridoid glycosides from Scrophularia buergeriana roots, J Nat Prod , 65 , pp. 1696-1699, 2002

[17] Kim SR et al., E- p methoxycinnamic acid protects cultured cortical neurons from glutamate-induced neurotoxicity, Br J Pharmacol , 135 , pp. 1281-1291, 2002

18. Kim SR et al., Anti-amnestic activity of Ep- methoxycinnamic acid from Scrophularia buergeriana, Cognitive Brain Research , 17 , pp.454-461, 2003

[19] Kim, YC et al., Ginsenosides Rb1 and Rg3 protect cultured rat cortical cells from glutamate-induced neurodegeneration, Journal of Neuroscience Research , 53 , pp.426-432, 1998

      The present invention relates to a composition for the prevention and treatment of cognitive impairment-related diseases comprising the extract of Hyunsam as an active ingredient.

      Cognitive impairment refers to mental and behavioral disorders due to brain disease, brain injury, and addiction, and mental disorders due to endocrine disorders, metabolic or nutritional abnormalities, and drugs. It is a type of brain disease that occurs not only in the case of forgetfulness (anmesia), which is a temporary short-term memory disorder, but also in dementia, which causes general impairment of mental ability and judgment according to the severity of symptoms.

Mild cognitive impairment (MCI), a stage prior to the development of severe Alzhimer's disease, includes symptoms such as recent memory loss and learning, concentration, poor thinking, and poor language skills. More than 80% of MCI patients develop Alzheimer's disease in less than 10 years, much higher than only 1% of healthy population aged 65 or older (see Documents 1 and 2).

In other words, cognitive impairment is distinguished from age-associated memory impairment (AAMI) and is closely related to Alzheimer's dementia in terms of pathogenesis and course. Compared with the remarkable memory and cognitive decline. Alzheimer's dementia accounts for more than 70% of people with dementia. The cause of the dementia has not been fully understood, but recent studies have shown that cholinergic nerves appear as accumulations of neurofibrillary tangles and neuroinflammatory plaques. Decline is presumed to be the main cause (see Document 3).

Various factors, such as excitotoxicity, oxidative stress, growth factor withdrawal, cytokines, or toxins, can cause nerve cell damage leading to cerebral nervous system disease. Most neuronal damage is presumed to be due to overstimulation of ionotropic or metabotropic glutamate receptors (see Document 4).

Glutamate is found at the millimolar level in the brain and acts on N-methyl-D-aspartate (NMDA) receptors and non-NMDA receptors in the brain, resulting in central excitatory neurotransmission. play an important role in excitatory neurotransmission. Exciting amino acids, such as glutamate, are involved in neuronal survival, synaptogenesis, neuronal plasticity, learning, and memory processes (see Documents 5 and 6).

However, glutamate is also known to cause neuronal cell loss in the central nervous system by two distinct mechanisms, acute and chronic (see Document 7). For example, abnormalities in the glutamate neurotransmitter system include seizures (see Document 8), Alzheimer's disease (see Document 9), Parkinson's disease (see Document 10), ischemia and spinal cord trauma. It may be involved in neurological diseases such as (see Document 11).

Hyunsam (玄 蔘) is a perennial herb of Hyunsamaceae, 80 ~ 150㎝ high, stem is straight, square, hairless, smooth and glossy. The leaves are opposite each other, and there is a leaf disease. The ovate or ovate lanceolate is somewhat rounded or slightly straight, and the tip is sharp and sharp teeth. Flower is pale yellow green and blooms from August to September. It grows on stem end bracts as a wheat plant. Corolla is in the form of a bottle, the lower end is rolled back and there is a two-stage surgery, the calyx is divided into five, and the lobe is ovoid or ovoid. Fruits are capsules, ovate, bilateral. Dried roots are known as Scrophulariae Radix and used for bleeding consonants, detoxification, fever swelling, and hunger, especially for anti-inflammatory, sore throat, rhinitis, and constipation (see Document 12). The genus of the present genus is distributed over 300 species worldwide and 5 species are known in Korea.

On the other hand, the present inventors are phenylpropanoids such as p-methoxycinnamic acid (p-methoxycinnamic acid) or p-methoxy-trans- cinnamoyl-4-α-L-rhamose It has been disclosed that phenylpropanoid esters of rhamnose, such as esters (p-methoxy-trans-cinnamoyl-4-α-L-rhamnose ester), have prophylactic or therapeutic activity for cerebral nervous system diseases (Documents 14 and 20). 15).

In addition, the inventors have disclosed that iridoid glycosides have a significant protective effect against glutamate-induced cerebral nervous system disease (see Document 15), and α, β- in phenylpropanoids. It has been disclosed that unsaturated ester moieties and para-methoxy groups play an important role in neuroprotective activity (see Document 16), and p-methoxycinnamic acid isolated from Hyunsam has anti-memory loss. It has been disclosed to have anti-amnestic activity (see Document 17).

In addition, Korean Patent Publication Nos. 10-2004-0074696 and 10-2005-0088950 disclose human fibroblast growth, expression of acid β-galactosidase, an aging marker, protein expression of p21, an aging gene, and oxidative activity. Disclosing the effect of 70% ethanol extract of brown ginseng on DNA damage and the like caused by oxidative stress, the 70% ethanol extract of brown ginseng showed excellent anti-aging activity.

However, until now, the present ginseng extract of the present invention exhibits a neuroprotective effect and a short- and long-term memory-improving effect, thereby preventing or treating a disease related to cognitive impairment has not been disclosed or taught anywhere in the document.

    The present inventors have completed the present invention by confirming that the present ginseng extract exhibits neuronal protective effect and short- and long-term memory improvement effect.

In order to accomplish the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of cognitive impairment-related diseases containing the extract of Hyunsam as an active ingredient.

The present invention provides a pharmaceutical composition for the prevention and treatment of cognitive impairment-related diseases comprising a pharmaceutically acceptable carrier, excipient or diluent in addition to the extract.

As defined herein, Hyunsam is a genus of Ssam ( Scrophularia) borealikoreana), Island figwort (Scrophularia takesimensis), large figwort (Scrophularia kakudensis ), Scrophularia koraiensis ) and Scrophularia buergeriana ), and is preferably characterized by the current ginseng ( Scrophularia buergeriana ).

The extract as defined herein is a solvent selected from water, ethanol or a mixed solvent thereof, preferably 30 to 90% water and ethanol mixed solvent, more preferably 50 to 90% water and ethanol mixed solvent, most preferably 65 To 85% is characterized in that the extract is soluble in water and ethanol mixed solvent.

Hyunsam, as defined herein, includes outposts, roots, and leaves of Hyunsam, and is preferably characterized by roots.

Cognitive impairment as defined herein includes mental and behavioral disorders due to brain disease, brain injury, addiction, etc. and endocrine disorders, metabolic or nutritional abnormalities, drugs, etc. Covering mental illness includes memory decline and memory impairment.

In addition, cognitive impairment related diseases as defined herein include Alzheimer's dementia, vascular dementia, Pick's disease, Creutzfeldt-jakob's disease, dementia due to head injury, or Parkinson's disease. Preferably, it is characterized by Alzheimer's dementia.

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

The present ginseng extract of the present ginseng is about 1 to 30 times, preferably about 5 to 15 times the water, ethanol or a mixed solvent thereof, preferably water and ethanol of the outpost, leaf or root of the present ginseng, preferably the root weight As a mixed solvent, cold extraction, heating extraction, hot water extraction, ultrasonic extraction, reflux extraction for about 30 minutes to 20 hours, preferably about 1 to 5 hours at an extraction temperature of 10 to 200 ° C, preferably 50 to 100 ° C Extraction method of the present invention can be obtained by the extraction method of the present invention, preferably the heat extraction method, it can be spray-dried to obtain the Hyunsam extract powder.

The present invention provides a pharmaceutical composition for the prevention and treatment of cognitive impairment-related diseases containing the present ginseng extract obtained by the above method as an active ingredient.

     The composition for the prevention and treatment of cognitive impairment related diseases of the present invention, the total weight of the composition comprises 0.1 to 50% by weight of the extract.

     However, the composition as described above is not necessarily limited thereto, and may be modified according to the condition of the patient and the type and extent of the disease.

     Pharmaceutical compositions containing Hyunsam extract according to the present invention as an active ingredient may further comprise a surfactant and a cosolvent. The surface active agent maximizes the solubility and dissolution rate of the poorly soluble drug by forming an automatic micelle drug delivery system in which micelles, which are poorly soluble drugs, are automatically micelles when exposed to aqueous solutions and biological fluids. It is responsible for maintaining stability. In the present invention, a minimum amount of the surfactant is used to facilitate the administration.

The surfactant may use various surfactants including pharmaceutically acceptable anionic, cationic, nonionic or amphoteric surfactants. Specifically, polyoxyethylene sorbitan fatty acid ester, sorbitan fatty acid ester, polyoxyethylene fatty acid ester, myriz, polyoxyethylene polyoxypropylene block copolymer, polyoxyethylene polyoxypropylene copolymer natural or hydrogenated vegetable oil Reaction products of ethylene glycol (hereinafter referred to as cremopoa), sodium dioctylsulfosuccinate, sodium lauryl sulfonate, phospholipids, propylene glycol mono or di fatty acid esters (migliol 840), natural vegetable oil triglycerides and polyalkylenes One or more selected from the group consisting of trans esterification reaction products of polyols, mono-, di- or mono / diglycerides, sterols and derivatives thereof can be used. Preferably, the surfactant is selected from the group consisting of polyoxyethylene polyoxypropylene block copolymers, reaction products of natural or hydrogenated vegetable oils and ethylene glycol and polyoxyethylene sorbitan fatty acid esters.

The cosolvent uses one selected from the group consisting of propylene carbonate, propylene glycol, ethanol, diethylene glycol monoethyl ether, glycofurol, polyethylene glycol, dimethyl isosorbide and methyl pyrrolidone. Preferably, the cosolvent is selected from the group consisting of diethylene glycol monoethyl ether, propylene glycol and polyethylene glycol. The composition of the present invention is prepared by mixing Hyunsam extract, surfactant and cosolvent in a weight ratio of 1: 0.1 to 50: 0.1 to 50, preferably 1: 0.1 to 20: 0.1 to 20. In addition, the composition increases the solubility and absorption in the gastrointestinal tract of a substance commonly used in pharmaceuticals, such as Hyunsam extract, within the range that does not adversely affect the drug efficacy, and the dissolution and emulsification with water during oral administration increases the dissolution. Additives such as fatty acids or fatty alcohols, sugars such as white sugar, macion, malt, white sugar, gelatin, sugar and syrup, glidants such as magnesium stearate and talc, and microcrystalline cellulose Excipients such as manus, calcium dihydrogen phosphate, starch, mannitol, antioxidants, flavoring agents, preservatives, fragrances, sweeteners, pigments, pH adjusting agents and viscosity adjusting agents, which prevent the preparation from oxidizing, these may be It is preferred to add in the amount usually used for the extract,

Citric acid, oleic acid, stearyl alcohol, myristic acid, linoleic acid or lauric acid, capric acid, caprylic acid, caproic acid, etc. may be used as the fatty acid or fatty alcohol that may be used in the composition of the present invention. It is not. Antioxidants that can be used in the compositions of the present invention are butylated hydroxytoluene, sodium bisulfite, α-tocopherol, vitamins, β-carotene, tocopherol, acetate, fumaric acid, nalic acid, butylated hydroxyanisole , Propyl gallate, sodium ascorbate and the like may be used, but are not limited thereto.

Flavoring agents that can be used in the composition of the present invention may be mixed fruit flavor, apple flavor, strawberry flavor, cherry flavor, peppermint flavor, vanilla flavor, yogurt flavor, or drink flavor and the like, but are not limited thereto.

Preservatives that can be used in the composition of the present invention may be used, but not limited to, benzoic acid, sodium benzoate, ethyl paraben, methyl paraben or propyl paraben.

Perfume which can be used in the composition of the present invention may be used, but not limited to, peppermint brain, peppermint oil, orange oil, clove oil, cinnamon oil, strawberry essence and other common fruit flavor or plant essence.

Sweeteners that can be used in the compositions of the present invention may be used, but are not limited to, white sugar, glucose, fructose, aspartame, stevioside, sorbitol, mannitol, oligosaccharide, syrup or macion syrup.

The pigments that can be used in the compositions of the present invention include green No. 3, red No. 2, red No. 3, blue No. 1, blue No. 2, yellow No. 4, yellow No. 5, water-soluble mannitol, caramel, titanium oxide or ferric oxide It may be used, but is not limited to these.

Modifiers that may be used in the compositions of the present invention may be used, but are not limited to sodium carbonate, sodium hydroxide, potassium hydroxide, triethanolamine or monoethanol amine.

Viscosity modifiers that can be used in the compositions of the present invention are hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, acacia, bentonite, alginic acid, propylene glycol alginate, polyvinylpipi Rollidone, polyvinyl alcohol, carbopul, polycarbophil and the like may be used, but are not limited thereto.

The present invention provides an oral preparation comprising a pharmaceutical composition containing the extract of the present ginseng.

The oral preparations are oral solutions containing tablets, pills, powders, hard capsules, gelatin-bended hard capsules and soft capsules, caramel or jelly type chewable tablets and aqueous solutions. Preferably, a soft capsule comprising the composition is provided. The composition according to the present invention can be formulated into an oral preparation using a conventional method after mixing and dissolving a hot ginseng extract, a surfactant and a cosolvent at 80 ℃.

The composition comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The composition comprising the extract according to the present invention is formulated in the form of an oral dosage form such as capsules, powders, granules, tablets, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Carriers, excipients and diluents which may be used in combination with the extract, and which may be included in the composition comprising the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

      Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. Administration may be administered once a day or may be divided several times. However, the dosage does not limit the scope of the invention in any aspect.

     The compositions of the present invention can be administered to a mammal, such as a human, by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous or intracerebroventricular injection.

     In addition, the present invention provides a health functional food for the prevention and improvement of cognitive impairment-related diseases comprising a hyunsam extract showing the effect of preventing and improving the cognitive impairment-related diseases.

     The composition comprising the extract of the present invention may be used in various forms, such as drugs, health foods and beverages for the prevention and improvement of cognitive impairment-related diseases, and may be used in the form of powders, granules, tablets, capsules or beverages. .

     Since the extract itself of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for long periods of time.

     The extract of the present invention may be added to a health functional food or beverage for the purpose of preventing and improving diseases related to cognitive impairment. At this time, the amount of the extract in the dietary supplement or beverage may generally be added at 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 10 g, preferably 0.3 to 1 g based on 100 ml It can be added at the ratio of.

     In addition to containing the extract as an essential ingredient in the indicated proportions, the health beverage composition of the present invention has no particular limitation on the liquid component, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates are conventional monosaccharides such as disaccharides such as glucose and fructose, such as maltose, sucrose and the like, and polysaccharides such as dextrin, cyclodextrin and the like. Sugars and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

     In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and the like. Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

    Hyunsam extract of the present invention exhibits a neuroprotective effect and short- and long-term memory improvement effect, can be used in the pharmaceutical composition and health functional food for the prevention and treatment of diseases related to cognitive impairment.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

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

Example  One. figwort  Preparation of Extract

10 L of 30% ethanol was added to 1 kg of Root of Scrophularia buergeriana , and extracted at 80 ° C. for 3 hours. The obtained extract was concentrated under reduced pressure to obtain 700 g of crude ginseng extract of about 30 brix. The obtained crude extract was spray dried to obtain 210 g of powdery ginseng 30% ethanol extract.

10% of 40% ethanol, 50% ethanol, 60% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, and 99% ethanol, respectively, using different solvents instead of 30% ethanol. Was prepared (each yield; 40% ethanol extract: 173g, 50% ethanol extract: 149g, 60% ethanol extract: 121g, 70% ethanol extract: 114g, 80% ethanol extract: 103g, 90% ethanol extract 89 g, 99% ethanol extract: 83 g).

Reference Example  One. figwort  Of extract Cinnamate  Derivative Content Determination

Cinnamate derivatives [Cinnamic acid, p-methoxycinnamic acid, hapagoside, 8-O-methoxycinna] among the extracts of Hyunsam obtained in Example 1 Mixhapa guide (8-O- (E-methoxycinnamoyl) harpagide)] was measured and the content is shown in Table 1 below.

Extraction solvent Active ingredient content by extractant (%) Cinnamic acid p-methoxycinnamic acid Hapago Said 8-O-methoxycinnamic hapaguide 99% Ethanol Extract 0.28 0.46 1.47 2.18 90% Ethanol Extract 0.14 0.21 1.09 1.36 80% Ethanol Extract 0.08 0.13 0.79 1.01 70% Ethanol Extract 0.07 0.10 0.71 0.93 60% Ethanol Extract 0.05 0.08 0.62 0.79 50% Ethanol Extract 0.07 0.09 0.50 0.66 40% Ethanol Extract 0.05 0.06 0.45 0.54 30% Ethanol Extract 0.04 0.07 0.38 0.41

As shown in Table 1, the content of cinnamate derivatives was highest when 90% or more of ethanol was used as the extraction solvent, and when 80% or less of ethanol was used as the extraction solvent, the content of cinnamate derivatives rapidly decreased. It was confirmed.

Experimental Example  One. figwort  Activity evaluation of extract

Cortical cells from fetal (17-19 days pregnant) of Sprague-Dawley rats (female, Sprague-Dawley family, 15 weeks old, 287.1-303.2 g, Seoul National University), according to Kim et al. (See Document 19). Primary cultures were prepared. Cortical cells contain 10% heat-inactivated fetal calf serum (Gibco) with penicillin (100 IU / ml, Sigma) and streptomycin (10 mg / ml, Sigma) Were incubated in DMEM (Dulbecco's modicco's modified Eagle's medium, Gibco) at 37 ° C. under a mixed gas of 95% air-5% CO ₂ . After 3 days of culture, 50 μM of 5-fluorodeoxyuridine was added to stop division of non-neuronal cells.

Each ginseng extract prepared in Example 1 was dissolved in dimethylsulfoxide (DMSO) (final concentration 0.1%). In preliminary testing, DMSO had no effect on cell survival at the concentrations used. Cortical cell cultures were washed with DMEM and incubated with Hyunsam extract for 1 hour. The cultures were treated with 50 mM glutamate and washed 30 minutes later with DMEM medium.

Neuronal viability was measured using the MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) assay method, and Hyunsam extract on glutamate toxicity by measuring LDH activity. To evaluate the neuronal protective effect of, it is shown in Table 2 below.

division Dosage Neuronal survival rate (%) MTT analysis LDH analysis Control 100 ± 4.2 100 ± 2.4 Glutamate 50mM 0.0 ± 1.3 0.0 ± 1.3 30% Ethanol Extract 50mg / ml 35.2 ± 3.2 22.5 ± 2.1 40% Ethanol Extract 50mg / ml 38.7 ± 2.1 34.9 ± 1.3 50% Ethanol Extract 50mg / ml 42.9 ± 5.2 49.2 ± 1.6 60% Ethanol Extract 50mg / ml 53.3 ± 1.9 55.7 ± 1.9 70% Ethanol Extract 50mg / ml 68.2 ± 5.5 59.2 ± 2.0 80% Ethanol Extract 50mg / ml 61.8 ± 7.9 57.0 ± 2.0 90% Ethanol Extract 50mg / ml 52.5 ± 2.7 51.5 ± 1.2 99% Ethanol Extract 50mg / ml 51.6 ± 4.2 47.2 ± 1.5

As can be seen in Table 2, it was found that the extract of Hyunsam extract obtained by using 70% and 80% of ethanol as the extraction solvent can significantly increase the survival rate of cells, compared to the extract of Hyunsam which is obtained by using other extraction solvents. Can be. This result is contrary to what is expected from the cinnamate derivative content of Example 1. That is, phenylpropanoids having an α, β-unsaturated ester moiety and a para-methoxy group (for example p-methoxycinnamic acid) play an important role in the prevention or treatment of cognitive dysfunction. Despite the report, as can be seen from the above results, it was confirmed that the extract obtained using ethanol having the ethanol content of 70% and 80% has a higher cell viability compared to the extract obtained using 90% or more of ethane.

Experimental Example  2. Manual evasion test ( Passive avoidance task )

The effect on cognitive impairment improvement (ie, memory improvement) was evaluated through the manual avoidance test described in the literature (see Document 18).

Training and testing for passive avoidance behaviors were performed in two identical light / dark square boxes (Gemini, San Francisco, CA, USA). Initially, mice (male, ICR series, 8 weeks old, 25.6-30.2 g, Seoul National University) were placed in a bright chamber and the door between compartments was opened after 10 seconds. When the mouse entered the dark compartment, the door automatically closed and an electric foot shock (0.1 mA / 10 g body weight) was applied for 2 seconds through a stainless steel rod (one training). Ninety minutes before the training attempt, the ginseng extract prepared in Example 1 was dissolved in Tween 80 solution (vehicle, 5% (w / v)) and orally administered to mice. After 60 minutes, scopolamine (1 mg / kg body weight) was injected subcutaneously to cause memory loss. After 24 hours of training attempts, the mice were placed back in the bright compartments and opened in 10 seconds in the same manner, and the time spent in the bright compartments of the animals was measured. When the mouse stays in the bright compartment for 180 seconds, the mouse avoids manual avoidance training after one training attempt, and the results of the passive avoidance experiment are shown in FIG. 1.

As shown in FIG. 1, mice whose memory has been degraded with scopolamine have shortened the appearance time toward the light, but mice treated with Hyunsam extract have a longer appearance time than 60% of the normal group. Mice treated with extracts obtained using 70% and 80% ethanol as extractant showed significantly higher memory improvement effects compared to mice administered with extracts obtained using 90% or more ethanol or 60% or less ethanol. .

After administering the 70% ethanol extract with the most excellent memory improvement effect by concentration, a manual avoidance experiment was performed in the same manner as shown above, and the results are shown in FIGS. 2 and 3. When 50 mg / kg and 100 mg / kg of 70% ethanol extract was administered, the emergence time of mice administered 50 mg / kg was 100 mg / kg compared to 51% of the normal group. It was confirmed that the memory improving activity was increased by up to 62% of the normal group (see FIG. 2). Passive avoidance test was performed after repeated administration of 70% ethanol extract at a concentration of 3, 10, 30 mg / kg once daily for 15 days. As a result, the dose-dependent increase in memory improvement activity was increased. It was confirmed (see FIG. 3).

Experimental Example  3. Morris By the sum  exam( Morris water maze test )

The effects on cognitive impairment and short and long term memory impairment were evaluated as follows.

3-1. Test Methods

Spatial memory test was performed by modifying the method of Morris (R .G. Morris, Development of a water maze procedure for studying spatial learning in the rat, J. Neurosci. Meth., 11, pp.47-60, 1984) . The water maze used a circular pool of 90 cm in diameter and 45 cm in height with a monotonous inner surface. The pool was placed in a room with posters that could serve as fixed furniture and extramaze cues, and the round paste was filled with water, and 500 ml of milk was mixed to a height of 30 cm (22 ± 1 ° C.). Divide the pool into four quadrants of equal area and install the glass platform (6 cm in diameter) 1 cm below the water surface, midway between the center and the edge of the pool in the target quadrant so that the glass platform (6 cm in diameter) is not visible. It was.

On the first day of the experiment, the mouse was inserted for 60 seconds without the locked platform to allow the mouse to become familiar with the experimental environment. Thereafter, the test was performed twice a day at 30 minute intervals for 4 days, and in all procedures, the swimming path was recorded on the map of the pool. During each test, the escape delay of the mouse was recorded and allowed to stay for 10 seconds when the mouse found a platform and rested on it. If the mouse did not find a platform within 120 seconds, the mouse was placed on the platform for 10 seconds and removed from the pool. These parameters were averaged for each test and each mouse. In Test 1 and Test 2, mice were pooled at the same point, but the point at which mice were pooled was changed on each test day. The location of the escape platform was the same, and the decrease in fms escape delay according to the date in test 1 indicates long-term memory, while the decrease in interval between tests 1 and 2 indicates short-term memory.

3-2. Dosing of the sample

Once daily, mice were orally administered Tween 80 solution (vehicle, 5% (w / v)) or 70% ethanol extract 90 minutes prior to pooling. The extract was dissolved in Tween 80 solution and administered once, for 4 days except for the first day of the experiment at doses of 10, 30 and 100 mg / kg, and at 3, 10 and 30 mg / kg for 15 days. The dose was administered once daily for 15 days, and then the experiment was conducted for 5 days in the same manner as once administration. 60 minutes after the administration of the Tween 80 solution and extract, memory deficit was induced by subcutaneous injection of scopolamine (1 mg / kg). All mice were used for the spatial memory test 30 minutes after scopolamine or 5% Tween 80 injections.

3-3. Test result

The results obtained when the 70% ethanol extract was administered once are shown in FIG. 4, and the test results are shown in FIG. 5 obtained after repeated administration for 15 days once a day.

As shown in Figures 4 and 5, the mice administered the Tween 80 solution showed a significant difference in the escape delay time between Test 1 and Test 2 on the first and second experimental days, constant on the second experimental day Escape delay time has been reached. However, mice administered scopolamine had no change in the escape delay time for 4 days in Trial 1 and Trial 2. Mice treated with 70% ethanol extract of Hyunsam after scopolamine induction significantly reduced the escape delay in Test 1 and Test 2 compared to mice administered scopolamine only. Reached.

In addition, in the animals of the group administered the present ginseng extract, including the normal model group not administered scopolamine, after the first test made on the day, the time to reach the destination in the second test was significantly reduced. In the condition-induced group administered with scopolamine only, the time taken to reach the destination in the second trial was intact, indicating that Hyunsam extract was effective in improving short-term memory. In addition, in the five-day long-term memory retention evaluation conducted after the short-term memory improvement evaluation conducted on the day, as in the previous short-term memory improvement evaluation, the time taken to reach the destination is significantly reduced in the group administered the Hyunsam extract. It can be confirmed that it plays a big role in improving long-term memory.

Experimental Example  4. Comparative component analysis

Industrial usefulness, such as the manufacturing process and manufacturing cost of the present ginseng 70% ethanol extract and extract powder X of the present invention showed the most excellent activity in the experimental example was compared with the extract known in the prior art as follows.

(1) Preparation of Standard Solution

Each standard was precisely taken in 16.0 mg, 15.5 mg, 10.1 mg, and 3.1 mg, dissolved in 90% methanol, and labeled in 1000 ml to use as a standard solution.

(2) Preparation of Sample Liquid

The chloroform-methanol (CHCl ₃ -MeOH, 1: 1, hereinafter, CM) extract powder extract of the present ginseng described in Patent Publication No. 2000-0008399 compared to the powdery ginseng 70% ethanol extract of Example 1 It was dissolved in 90% methanol and used as a sample liquid.

(3) analysis conditions

Four types of HS (Harpagoside), HG (Harpagide), MCA (p-methoxycinnamic acid) and CA (cinnamic acid) were quantitatively analyzed using high pressure liquid chromatography-ultraviolet detector. The column used in the analysis was a reverse phase C18 column (18.5 micrometer filler, inner diameter 4.6 millimeters, length 150 millimeters), which was mixed with 1% acetic acid and acetonitrile at 77:33 as a mobile phase, and the flow rate was 1.0 ml per minute. The detection wavelength of the detector was 280 nm, and the column temperature was maintained at 30 ° C. The analysis results are shown in Table 3 and FIG. 6.

sample Yield (%) Content of each ingredient extracted from 1g of sample (mg / g) HS HG MCA CA system Brown Ginseng Extract 70% Ethanol 24 1.76 2.55 1.19 0.17 5.67 Hyunsam chloroform-methanol extraction drying extract (CM) 1.7 0.25 0.45 0.28 0.04 1.02 HS: Harpagoside, HG: Harpagide, MCA: p-methoxycinnamic acid, CA: Cinnamic acid

As a result, as shown in Table 3 and Figure 6, the present ginseng 70% ethanol extract of the present invention shows an excellent yield of 14 times or more in the yield, compared to the comparison CM, which is compared to the comparison ginseng chloroform-methanol extract dry extract ( When using CM) industrially, not only 14 times more raw ginseng herbal medicines are used but also the production cost of chloroform and methanol used as extractant is higher than that of ethanol in the present invention. .

In addition, in the contents of HS (Harpagoside) and HG (Harpagide), 70% ethanol extract of the present ginseng of the present invention is not only about 7 times and about 5.6 times higher than the comparative CM extract, respectively, MCA (p-methoxycinnamic acid) and CA (Cinnamic acid) also showed an average of 4.2 times higher content.

Experimental Example  5. Anti-dementia efficacy comparison test

The excellent dementia efficacy of the present ginseng 70% ethanol extract of the present invention showed the most excellent activity in the experimental example was compared with the extract known in the prior art as follows.

(1) Experimental method

Rats (male, Sprague-Dawley family, 10 weeks old, 305.8 ~ 333.4 g, Central Experimental Animal Co., Ltd.) were obtained and purified for 7 days, and used as test animals. Twelve animals were separated from each group, and the test was divided into a normal group, a condition group, and a drug administration group.

Normal saline and pathophysiological group were administered saline solution, and drug administration group was administered orally with the present substance 70% ethanol extract and CM of the present invention, 1 hour and 30 minutes later, normal saline to the normal group, pathologic group and drug administration group Escolopamine was administered subcutaneously at a 2 mg dose per kilogram. After 30 minutes, the evacuation shuttle box (Med. Associates Inc, USA) with a 3-mm-thick lattice type electrical stimulator at 0.5 cm intervals was divided into a bright room and a dark room. When I entered the room, I trained for 2 seconds to give 5mA of electrical stimulation through the lattice type electrical stimulation device. After 24 hours of training, the test was carried out in the same manner as above with the electrical stimulation removed, and the time taken for the test animal to stay in the bright room was measured. Time was measured in seconds, and compared with the normal group and the condition group to evaluate the degree of memory improvement of the drug administration group, the results are shown in Table 4 and FIG.

Latency (sec) Normal 173.6 ± 2.9 Negative 40.0 ± 4.0 Brown Ginseng 70% Ethanol Extract 100mg / kg 117.9 ± 5.4 Brown Ginseng Chloroform-Methanol Extract (CM) 100mg / kg 71.6 ± 6.2 Brown Ginseng Chloroform-Methanol Extract (CM) 17mg / kg 51.6 ± 3.8 Donepezil 5mg / kg 84.4 ± 5.4 * CM 17mg / kg dosage is the same as 70% ethanol extract 100mg / kg

As a result of the experiment, as shown in Table 4 and Figure 7, the 70% ethanol extract of the present ginseng of the present invention compared to the CM extract (17mg / kg) of the comparative effect of about 6.7 times more excellent cognitive dysfunction (ie, memory improvement) effect It confirmed that it represents.

As confirmed in Experimental Example 4 and Experimental Example 5, the present ginseng 70% ethanol extract of the present invention is significantly improved in yield compared to the comparative CM (Korean Patent Publication No. 2000-0008399), and also in the component content In the present ginseng ethanol extract of the present invention was confirmed that the average content of four times or more. In particular, a decrease in the content of HS (Harpagoside) and HG (Harpagide) of the comparative CM is expected to be the cause of the decrease in efficacy in Experimental Example 4.

In addition, compared to the current ginseng chloroform-methanol extract or extract powder X is a trace amount of chloroform used as a solvent, it remains, there is a risk of side effects when taking a long time, and to remove the chloroform from the extract or extract powder X There is a problem that the production cost.

That is, the present invention has a very high yield compared to the chloroform-methanol (CHCl ₃ -MeOH, 1: 1, CM) extract of the present ginseng described in the Republic of Korea Patent Publication No. 2000-0008399 and the extract dry extract thereof, Cost reduction is expected, and the content of ingredients is excellent, and the effect of improving cognitive dysfunction is also excellent. Also, in the improvement of cognitive dysfunction and brain neuroprotection effect, the water and ethanol mixed solvent It was confirmed that the extract was very excellent.

Hereinafter, the preparation examples of the composition containing the compound of the present invention will be described, but the present invention is not intended to limit the present invention, but is only intended to be described in detail.

Formulation example  1. Preparation of tablets 1

100 mg of Ginseng 80% ethanol extract of Example 1

750mg of microcrystalline cellulose

Silicon dioxide 2mg

20 mg sodium starch glycolate

Magnesium Stearate 3mg

Hydroxypropyl Methyl Cellulose

Ethyl Cellulose

Polyvinyl alcohol

Talc proper amount

Xanthan Gum

Quinoline yellow ws aluminum lake

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

Formulation example  2. Preparation of Tablets 2

100 mg of brown ginseng 70% ethanol extract of Example 1

Anhydrous calcium hydrogen phosphate 750 mg

Silicon dioxide 2mg

20 mg sodium starch glycolate

Magnesium Stearate 3mg

Titanium oxide appropriate

Diethylphthalate Proper

Alluraed ac aluminum lake

Lecithin

Indigo carmine aluminum lake appropriate amount

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

Formulation example  3. Powder  Produce

20 mg of brown ginseng 70% ethanol extract of Example 1

Lactose 100 mg

Talc 10 mg

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

Formulation example  4. Manufacture of capsule

10 mg of ginseng 60% ethanol extract of Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

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

Formulation example  5. Preparation of Injectables

10 mg of Ginseng 50% ethanol extract of Example 1

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  6. Liquid  Produce

20 mg of brown ginseng 90% ethanol extract of Example 1

10 g of isomerized sugar

5 g of mannitol

Microcrystalline cellulose

Calcium monohydrogen phosphate

Purified water

According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added to adjust the total amount to 100 ml, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation example  7. Manufacture of Healthy Foods

1000 mg of Hyunsam 40% ethanol extract of Example 1

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation example  8. Manufacture of health drinks

1000 mg of Hyunsam 70% ethanol extract of Example 1

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components in accordance with a conventional healthy beverage production method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is a composition that is relatively suitable for the preferred beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

1 is a diagram showing the results of passive avoidance test after administration of Hyunsam extract,

Figure 2 is a diagram showing the results of passive avoidance test after administration of 70% ethanol extract of Hyunsam,

Figure 3 is a diagram showing the results of passive avoidance test after repeated administration of 70% ethanol extract once a day for 15 days,

Figure 4 is a diagram showing the results of several maze test after administration of 70% ethanol extract of Hyunsam,

Figure 5 is a diagram showing the results of several labyrinth test after repeated administration of 70% ethanol extract once a day for 15 days,

6 is a diagram showing a relative composition ratio of p-methoxycinnamic acid (MCA) of Hyunsam ginseng chloroform-methanol extract (CM) compared to 70% ethanol extract (70% EtOH Ex.)

7 is a view showing a comparison of the passive avoidance experiment results of Hyunsam 70% ethanol extract (70% EtOH Ex.) And Hyunsam chloroform-methanol extract (CM).

Claims (13)

A pharmaceutical composition for the prevention and treatment of Alzheimer's dementia or vascular dementia, containing as an active ingredient an extract available in 65-85% water and ethanol mixed solvent of Hyunsam. A pharmaceutical composition according to claim 1 comprising a pharmaceutically acceptable carrier, excipient or diluent in addition to said extract. delete delete delete delete The pharmaceutical composition of claim 1 in the form of capsules, powders, granules, tablets, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories and sterile injectable solutions. Health functional food for the prevention and improvement of Alzheimer's dementia or vascular dementia containing as an active ingredient an extract available in 65-85% water and ethanol mixed solvent of Hyunsam. delete delete delete delete The dietary supplement of claim 8 which is a powder, granule, tablet, capsule or beverage.

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