KR20120001977A - A composition comprising a purified extract or fraction of mountain cultivated ginseng radix leaves as an active ingredient for preventing and treating brain disease - Google Patents

Abstract

PURPOSE: A composition containing purified extract or fraction of Mountain Cultivated Ginseng Radix is provided to suppress cetylcholinesterase activity and to prevent and treat brain diseases. CONSTITUTION: A pharmaceutical composition for preventing and treating brain diseases contains purified extract or fraction of Mountain Cultivated Ginseng Radix leaves as an active ingredient. The brain diseases are amnesia, Lewis body disease, Pick's disease, Alzheimer's disease, stroke, Parkison's disease, or Huntington's disease. The fraction contains a purified material which is prepared by Kiesel gel column chromatography using chloroform and methanol. A health food for preventing and treating brain disease contains the extract or fraction as an active ingredient. The health food is powder, granule, tablet, capsule, syrup, or beverage.

Description

A composition comprising a purified extract or fraction of Mountain Cultivated Ginseng radix leaves as an active ingredient for preventing and treating brain disease}

The present invention relates to a pharmaceutical composition or health functional food for the prevention and treatment of brain diseases comprising a purified extract or fraction of camphor ginseng leaves as an active ingredient.

[Document 1] Ose-kan. Neurotransmitters and brain diseases. Seoul: Shinil Corporation. 2005:

345-64.

[Document 2] Lee Byung Yoon.精神 醫學 辭典. Seoul: Sculpture. 1990: 58-62, 390.

[Reference 3] Min Sung Gil. Latest mental medicine. Seoul: Sculpture. 2005: 25-36, 189-98.

4 Ernest R. Hilgard, et al., Conditioning and learning. Appleton-Century-Crofts. New York: Teachers College. 1961: 107-9.

[Reference 5] Lee Geun-hu, Kang Byung-jo, Kwak Dong-il, Min Sung-gil, Lee Gil-hong, Lee Mu-seok and others. Diagnostic and Statistical Manual of Mental Disorders (DSM-4). Seoul: Hana Publisher. 1995: 212-21.

[Document 6] Lee Jung-kyun. Revised Psychiatry. Seoul: Sculpture. 1992: 208-9, 421-2.

Document 7 Evans DA. et al., JAMA . 1989; 262 (18): 2552-6.

8 Bartus RT. et al., Science . 1982; 217: 408-17.

[Document 9] Park Soon-kwon, Lee Hong-jae, Kim Hyeong-taek and Hwang Eui-wan. An Experimental Study on the Dementia Treatment of Herbal Medicines. Journal of Oriental Neuropsychiatry. 1998; 9 (2): 19-35.

[Reference 10] The Korean Academy of Oriental Neuropsychiatry. Oriental Neuropsychiatry. Paju: Per house. 2006: 125.

[Document 11] Park E, Kang M, Oh JW, Jung M, Park C, Cho C, Kim C, Ji S, Lee Y, Choi H, Kim H, Ko S, Shin M, Park S, Kim HT, Hong M, Bae H. Yukmijihwang-tang derivatives enhance cognitive processing in normal young adults: a double-blinded, placebo-controlled trial. Am j chin Med . 2005; 33 (1): 107-15.

12. Tabuchi M, Yamaguchi T, Iizuka S, Imamura S, Ikarashi Y, Kase Y. Ameliorative effects of yokukansan, a traditional Japanese medicine, on learning and non-cognitive disturbances in the Tg2576 mouse model of Alzheimer's disease. J Ethnopharmacol . 2009; 122 (1) 157-62.

Document 13 Gold PE. et al., Behav Neural Biol . 1986; 46: 145-55; Wenk GL., Psychopharmacol. 1989; 99: 431-39.

[14] Ebert, U. et al., Eur J Clin Invest . 1998; 28: 944.

15. Anoja S. et al., Biochemical Pharmacology .

1999; 58: 1685-93; Hu SY. Economy Botany . 1976; 30: 11-28; Lee, TH. et al., Korean J. Giseng Sci . 1999; 23: 21-37.

[Document 16] Korea Food Research Institute, 'Research of Functional Characteristics of Camphor Ginseng Leaves and Development of Processed Foods Using It', Final Research Report p19-20, 2005.10.14.

As the amount of information to be acquired increases with the rapid development of modern society, there is a great demand for the activation of brain function in adolescents and adolescents with a large amount of learning. In addition, the symptoms of memory decline due to aging are increasing. In patients with degenerative diseases such as dementia, memory loss is a very important factor in the availability of social life (Osegwan, neurotransmitters and brain diseases. Seoul: Shinil Corp. 2005: 345-64).

Memory is the ability to regenerate past experiences based on mental processes such as learning or registration, retention, recall or recall, and recognition (Lee Byung-yun. 精神 醫學 辭典. : Piece (1990: 58-62, 390).

It is classified into the short-term memory that is morphologically active in the consciousness and the long-term memory that is not stored in the present consciousness but is stored somewhere and recalled as possible (Min Sung-gil. The latest mental medicine. 98). Looking at the hypothetical structure of memory, sensory information coming in through the eyes, ears and other sensory organs is temporarily registered in the sensory register. If the information is a human face or a visual scene, at least some of the information can be transferred directly to long-term memory. The rest of the information in the sensory register is transferred to short-term memory and remains there for a few seconds. If it is new information, it is transferred to long-term memory if not recite or think, but most of it is forgotten. However, if it is repeated repeatedly, it can be stored in long-term memory and stored semi-permanently there. But even if not recite, some of the moment's experience continues to be stored in long-term memory (Ernest R. Hilgard, et al., Conditioning and learning.Appleton-Century-Crofts.New York: Teachers College. 1961: 107-9) .

Memory impairment is a cognitive and attention disorder that appears as a disorder of sensory centers and intellectual activities that are considered to be the main signs of cerebral disease. Memory impairment is a general term that applies to both temperamental and psychological memory impairments, including but not limited to amnesia commonly understood as forgetfulness and other conditions such as hypermemory, long-term memory deficiency, short-term memory deficit and psudoreminiscence. It is widely understood as amnesia, hypermembranes, and memory errors (Keun Hoo Lee, Kang Byung Jo, Kwak Dong Il, Min Sung Gil, Lee Gil Hong, Lee Moo Seok et al. Diagnosis and Statistical Manual of Mental Disorders (DSM-4). Seoul: Hana Publisher. 1995: 212 -21).

Forgetfulness, also called forgetfulness, can usually be limited to certain things or things. It is largely classified into organic forgetfulness and psychogenic forgetfulness (Lee Byung-yun. 精神 醫學 辭典. Seoul: Sculpture. 1990: 58-62, 390).

Temperament forgetfulness has a wide range of amnesia, forgets the entire experience of a certain period of time, the boundaries between remembered and unremembered periods and content are unclear, and memory gradually recovers, overlapping with impairments in intelligence, consciousness, and coordination. appear. If you have a brain injury, you can't remember what happened before that time, and if it recovers, then your memory will recover from a day before that time. Premature forgetfulness is the case of not remembering work and remembering the previous work correctly. Temperamental forgetfulness usually occurs due to brain chemical changes, brain trauma or progressive changes in the brain, head trauma due to accidents, epileptic seizures, acute alcoholism, cerebral arteriosclerosis, and senile diseases. Engraving.2005: 25-36, 189-98; Lee Jung-kyun, Rev. Psychiatry, Seoul: Sculpture, 1992: 208-9, 421-2).

Psychogenic forgetfulness causes sudden loss of consciousness due to sudden loss of consciousness because the disability and consciousness disorders are not seen, the extent of the memory disorder is selective, and the memory disorder appears in order to obtain the benefits required. Since such psychogenic amnesia causes some pain or anxiety, memory is restored after aging. Psychogenic forgetfulness is typically seen in hysteria (Lee Jung-kyun. Revised Psychiatry. Seoul: Sculpture. 1992: 208-9, 421-2).

Medically, amnesia is defined as a short-term memory disorder, or a temporary retrieval of the brain, especially in Lewy body disease, Pick's disease, and Alzheimer's disease (Evans DA). et al., JAMA . 1989; 262 (18): 2552-6). In addition to these diseases, forgetfulness can be caused by various chemicals or external damage.

There is an opinion that learning and memory are so closely related to the front and back that memory reflects changes in the activity of the living body, including the brain induced by training, and learning reflects the change in behavior that reflects it. However, there is an opinion that learning is a very short accumulation of memory, and it is usually expressed as learning and memory without strictly distinguishing them (Bartus RT. Et al., Science . 1982; 217: 408-17).

In Chinese medicine, memory impairment, or dementia, is a kind of forgetfulness, and it has been called as envy, hope, hope, prosperity, and so on. In addition, dementia is predicated on the basis of sickness or forgetfulness, and it is largely blasphemy (허), mentally yanghe ((), and sinjeongyu (腎精 虧虛). ), And treatment by dividing it into virtues such as non-Xinyanghe (과 陽虛), Damsimsimgyu (, 心 竅), and fish blood tank ((), Park Soon-kwon, Lee Hong-jae, Hyung-Taek Kim, Eui-Wan Hwang, An Experimental Study on the Dementia Treatment of Herbal Medicine, Journal of Oriental Neuropsychiatry, 1998; 9 (2): 19-35). Forgetfulness refers to the memory disorder and the condition between the right and the right side. Clinically, Usa-induced sarcoma and linguistic support are used. (首尾), etc. (Korean Society for Oriental Neuropsychiatry. Oriental Neuropsychiatry. Paju: Kummun Hall, 2006: 125.).

Recently, dementia due to dementia due to cardiac ratio, circulatory disorder due to soaking, and circulatory disorder of blood donation Classified as (iii), the research is actively conducted. Park et al. Reported that Yimmijihwang-tang, a typical prescription of consonant Bosin, improves memory in clinical trials. Recently, in Japan, gangansan (抑 肝) was used in the Alzheimer's model using genetic mice. The antidementia effect of yokukansan has been reported (Park E, Kang M, Oh JW, Jung M, Park C, Cho C, Kim C, Ji S, Lee Y, Choi H, Kim H, Ko S). , Shin M, Park S, Kim HT, Hong M, Bae H. Yukmijihwang-tang derivatives enhance cognitive processing in normal young adults:. a double-blinded, placebo-controlled trial Am J Chin Med . 2005; 33 (1): 107-15; Tabuchi M, Yamaguchi T, Iizuka S, Imamura S, Ikarashi Y, Kase Y. Ameliorative effects of yokukansan, a traditional Japanese medicine, on learning and non-cognitive disturbances in the Tg2576 mouse model of Alzheimer's disease. J Ethnopharmacol . 2009; 122 (1) 157-62).

Nervous systems related to memory are known as the cholinergic nervous system, the glutamatergic nervous system, the GABAergic nervous system, the serotonergic nervous system, and the adrenergic nervous system, but especially in animals and humans. It is known that the cholinergic nervous system plays an important role in learning and memory processes. In addition, pathways that enhance learning and memory include pathways that promote the release of acetylcholine or inhibit the activity of acetylcholine degrading enzymes in neurons (Gold PE. Et al., Behav) . Neural Biol . 1986; 46: 145-55; Wenk GL., Psychopharmacol . 1989; 99: 431-39). In animal experiments, the effects of anti-fogging drugs are mainly studied using scopolamine, a muscarinic receptor inhibitor, one of the subtypes of cholinergic neurons (Ebert, U. et al., Eur J). Clin Invest . 1998; 28: 944).

Jangnoesam (Mountain Cultivated Radix Ginseng) is of ginseng (Panax perennial plants belonging to Araliaceae (Araliaceae) ginseng CA Meyer) refers to ginseng that is artificially grown from wild ginseng seeds or ginseng grown in the wild. The benefits of ginseng are very effective in activating liver function, enhancing blood sugar, inhibiting cancer cell growth, lowering blood pressure and preventing arteriosclerosis in patients with hypertension, activating the body's immune function, preventing anemia, promoting metabolism in the body, and relieving stress and fatigue. Various effects are known (Anoja S. et al., Biochemical Pharmacology . 1999; 58: 1685-93; Hu SY. Economy Botany . 1976; 30: 11-28; Lee, TH. et al., Korean J. Giseng Sci . 1999; 23: 21-37).

Ginseng leaves have not been of much interest among scholars because they have not had much pharmacological meaning compared to the roots for a long time. However, in 1973, cytotoxicity of leaf saponins has neuroleptic, analgesic, hypotensive and atropine-like effects. This turned out to attract attention. When Tanaka et al. Identified and reported the presence of true aglycone in the leaf saponin fraction, they began to be energized by the saponin study. Since then, six saponin components, ginsenosides Rb1, Rb2, Rc, and Rd in the saponin fraction, , Rg1 and the like were confirmed. Therefore, it can be seen that they are most suitable as a raw material for extracting and separating ginsenosides because they have a higher content than roots or stems. In addition, as pharmacological actions on various non-saponin components separated from impurity saponin fractions have been reported, interest in volatile terpenoids, polyacetylenes, phenolic substances, lignans, alkaloids, etc. as components other than ginseng saponins has been increasing. Researcher, 'Research of Functional Characteristics of Camphor Ginseng Leaf and Development of Processed Foods Using It', Final Research Report p19-20, 2005.10.14).

Comparing the composition, content of free amino acid and free sugar of camphor and cultivated ginseng leaves, the organic acids contained in ginseng leaves are oxalic, citric, malic, succinic acid, etc. In the case of camphor ginseng leaves, the content of oxalic acid (oxalic acid) is high. The content of crude saponin was higher in cultivated ginseng than in cultivated ginseng compared to cultivated ginseng. The value is shown. On the other hand, saponin was isolated by HPLC, and the camphor ginseng leaves showed higher PD / PT ratio than cultivated ginseng. The contents of phenolic compounds of camphor ginseng leaf in each form, such as free form (FPA), soluble phenolic acid ester (SPA), and insoluble bound phenolic acid (IPA), were investigated.The total content of phenolic compounds was 734 mg%. It was 1.8 times higher than cultivated ginseng. Antioxidant activity of phenolic compounds by type revealed that camphor leaves showed higher activity than cultivated leaves.

Although ginseng root has a disadvantage that it takes at least 4-6 years to be harvested after sowing despite the excellence of the effect, the ginseng leaf has the advantage that it can be harvested every year. However, in the case of cultivated ginseng leaves, they are mostly discarded because of environmental problems such as pesticide application (Korea Food Research Institute, 'Research of functional characteristics of camphor ginseng leaves and processed food development using them', final Research Report p19-20, October 14, 2005). On the other hand, camphor ginseng grows in a clean area, so there is no problem of pesticide residues and it is possible to maximize the added value of camphor ginseng leaves, which have not been newly materialized. It is believed to be.

The inventors of the present invention confirmed the passive avoidance test and Morris water maze test using an amnesia experimental animal model derived from scopolamine of the extract or fraction of camphor ginseng leaf. The present invention was completed by confirming the effect of learning ability and cognitive function of camphor ginseng leaf through acetylcholinesterase test.

 In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of brain diseases comprising a purified extract or fraction of camphor ginseng leaves as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of brain diseases containing purified extract or fraction of camphor ginseng leaves as an active ingredient.

Mountain Cultivated Ginseng Radix, as defined herein, is a perennial vegetation of Panax ginseng ginseng CA Meyer) refers to ginseng seedlings or ginseng grown artificially in the wild and grown artificially in the wild.

A "crude extract" as defined herein comprises a solvent selected from water, including purified water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably a mixed solvent of water and ethanol, more preferably 50 to 90% ethanol. It is an extract soluble in the containing solvent.

"Polar solvent soluble extract" as defined herein includes an extract selected from water, butanol, methanol, ethanol or a mixed solvent thereof, preferably water or extracts soluble in butanol, more preferably ethanol, butanol.

"Non-polar solvent soluble extract" as defined herein includes hexane, chloroform, or extracts soluble in ethyl acetate, preferably hexane, ethyl acetate, more preferably ethyl acetate solvent.

“Fragment” as defined herein is a purified product which is separated by additionally performing the above-mentioned nonpolar solvent soluble extract, especially ethyl acetate soluble extract, using Kiesel gel column chromatography using chloroform (CHCl ₃ ) and methanol (MeOH) as eluents. , In particular developing solvent (CHCl ₃ : MeOH = 50: 50) is characterized in that it comprises a purified product (hereinafter referred to as "FKG-3").

Brain diseases as defined herein include amnesia, Lewis body disease, Pick's disease, Alzheimer's disease, stroke, Parkinson's disease or Huntington's disease, preferably forgetfulness, Lewy body disease , Pick disease, Alzheimer's dementia, and even more preferably forgetfulness.

Hereinafter, the present invention will be described in detail.

Extract of the present invention can be prepared as follows. After washing the leaves of camphor, a solvent selected from 5 to 20 times, preferably 10 times (w / v) water, lower alcohols having 1 to 4 carbon atoms or a mixed solvent thereof, preferably water And ultrasonic extraction for 30 minutes to 4 hours, preferably 1 hour to 3 hours, at 10 ° C. to 50 ° C., preferably 20 ° C. to 40 ° C., with a mixed solvent of ethanol, even more preferably 50 to 90% ethanol. Reflux extraction, hydrothermal extraction, cooling extraction or supercritical extraction, preferably ultrasonic extraction (ultrasound extraction is a low temperature extraction method that can be extracted even at low temperatures, overcoming disadvantages such as inefficient penetration and denaturation of temperature and having high physiological activity A first step of extracting three times with an extraction method to make a stable extract; The extract of the present invention can be obtained through a preparation method comprising a second step of concentrating this under reduced pressure and lyophilizing.

In addition, the polar or non-polar solvent soluble extract of the present invention, after washing the camphor ginseng leaves, 5 to 20 times, preferably 10 times the volume (v / w%) of ethanol, and then 1 at room temperature After immersion extraction method, ultrasonic extraction method, reflux extraction method, hot water extraction method, cooling extraction method or supercritical extraction method for 3 hours to 30 hours, preferably 5 to 25 hours, preferably immersion extraction method is repeated three times, and then filtered with a filter paper. Concentrated under reduced pressure, lyophilized to dilute the resulting product with water, and then subjected to a conventional fractionation process using n-hexane, ethyl acetate, and butanol, and a non-polar solvent-soluble extractable fraction available for non-polar solvents such as n-hexane and ethyl acetate. ; And polar solvent soluble extract fractions soluble in polar solvents such as butanol, water and the like.

The present inventors inhibited acetylcholinesterase activity of purified extracts or fractions of camphor ginseng leaves obtained by the above method, and confirmed the memory-improving effect in scopolamine-induced forgetful rat model. It was confirmed that the present invention is useful for providing useful pharmaceutical compositions and health functional foods for the prevention and treatment of brain diseases.

The present invention provides a pharmaceutical composition for the prevention and treatment of brain diseases, including the purified extract or fractions obtained by the preparation method as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of brain diseases comprising a purified extract or fraction obtained as the active ingredient as an active ingredient.

Compositions of the present invention inhibit acetylcholinesterase activity and include memory-improving effects in scopolamine-induced forgetful rat models.

The composition of the present invention is passed through the cholinergic nervous system, the glutamatergic nervous system, the GABAergic nervous system, the serotonergic nervous system or the adrenergic nervous system, preferably via the cholinergic nervous system. To exhibit effects.

The pharmaceutical composition for preventing and treating brain diseases containing the purified extract or fraction of the present invention comprises 0.1 to 50% by weight of the extract based on the total weight of the composition.

Pharmaceutical compositions containing tablet extracts or fractions of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical compositions containing tablet extracts or fractions of the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively. It can be formulated and used in the form. Carriers, excipients and diluents which may be contained in the composition containing the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, 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 such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, 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.

The amount of tablet extract or fraction of the present invention may vary depending on the age, sex, and weight of the patient, but 0.1 to 100 mg / kg may be administered once to several times daily. The dosage may also be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Accordingly, the dosage is not limited in any way to the scope of the present invention.

The pharmaceutical composition may be administered to various mammals such as mice, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a health functional food for the prevention and improvement of brain diseases comprising the purified extract or fraction as an active ingredient. Foods to which it may be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gums, teas, vitamin complexes, and health functional foods.

In addition, the purified extract or fraction may be added to food or beverage for the purpose of preventing and improving brain diseases. At this time, the amount of the extract in the food or beverage may be added to 0.01 to 50% by weight of the total food weight, the health functional beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.01 to 1 g based on 100 ml.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except the tablet extract or fractions as essential ingredients in the indicated ratios, and contains various flavors or natural carbohydrates as additional ingredients, as in general beverages. can do. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclodextrin and the like and xylitol, sorbitol, erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tautin, stevia extract) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 3-15 g per 100 ml of the composition of the present invention.

In addition to the above, the purified extracts or fractions of the present invention are various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the purified extract or fraction of the present invention may contain a pulp for the production of natural fruit juices and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 50 parts by weight per 100 parts by weight of the extract of the present invention.

The present invention relates to a composition comprising a purified extract or fraction of the leaves of Mountain Cultivated Ginseng Radix as an active ingredient, more specifically, a composition comprising the purified extract or fraction of the leaves of camphor ginseng as an active ingredient is acetyl It inhibits choline degrading enzyme (acetylcholinesterase) activity and shows memory-improving effect in scopolamine-induced forgetful rat model, so the composition is useful as a pharmaceutical composition or health functional food for the prevention and treatment of brain diseases. It is available.

1 is a diagram showing the separation product of camphor ginseng leaf,
2 is a diagram showing a separation product of ethyl acetate (EtOAc) from camphor leaves,
Figure 3 is a diagram showing the results of passive avoidance test of ethyl acetate (EtOAc) fraction,
Figure 4 is a diagram showing the results of passive avoidance test of n-butanol (BuOH) fraction,
Figure 5 is a diagram showing the results of passive avoidance test of the FKG-3 fraction,
Figure 6 is a diagram showing the learning effect of the Morris underwater maze device of the FKG-3 fraction,
7 is a diagram showing the memory effect of the Morris underwater maze device of the FKG-3 fraction,
8 is a diagram showing the results of the Y-maze test of the FKG-3 fraction,
9 is a diagram showing the results of immunohistochemistry of the FKG fraction,
10 is a diagram showing the results of measuring the acetylcholine (AChE) activity of the FKG fraction.

Hereinafter, the present invention will be described in detail. However, the following Examples, Reference Examples and Experimental Examples are merely illustrative of the present invention, but the content of the present invention is not limited thereto.

Example  1. Extraction and Fraction of Camphor Leaves

1-1. Camphor leaf Crude extract  detach

Wash 100g of camphor ginseng leaves more than 4 years old, purchased from Eo-In-Man (Gyeongbuk, Korea) twice with distilled water, and add 70% ethanol to 10 times (w / v) volume of camphor ginseng leaves for 3 hours. Ultrasonic extraction times. This was concentrated in a vacuum distillation apparatus and lyophilized again, and 7.2 g of the red powder obtained was used for the experiment (yield: 7.2%).

1-2. Fraction of Soluble Extract with Polar Solvent and Nonpolar Solvent

6L ethanol was added to 717 g of fresh camphor ginseng leaf, and the immersion extraction was repeated three times at room temperature for 24 hours, followed by filtering with Whatman No. 1 filter paper, and concentrating using a vacuum concentrator at 40 ° C. or lower to obtain 31.68 g of a powder sample. Got it. 3.9 g of the concentrated product was diluted in water, followed by liquid-liquid partition using the same amount of organic solvent (n-Hexane, EtOAc, n-BuOH, etc.) to n-hexane (Hexane) fraction (hereinafter “PgL-nHx”). 0.17g, Etal Acetate (EtOAc) fraction (hereinafter referred to as “PgL-EtOAc”) 8.8 g, n-butanol fraction (hereinafter referred to as “PgL-nBU”) 1.4 g, H ₂ O fraction (hereinafter referred to as “PgL-EtOAc”) 1.4 g were each separated by polarity (see FIG. 1).

Primary fraction in in vitro and in The second fraction was searched after identifying the substance with the highest activity through in vivo experiments. 4.38 g of a portion of the active PgL-EtOAc (EtOAc) fractions were adsorbed onto a Kiesel 60 (C-18, 0.063-0.2 mesh, 1.8 id × 41 cm) gel, followed by chloroform (CHCl ₃ ) and methanol (MeOH). Gradient eluted with CHCl ₃ : MeOH = 100: 0 (hereinafter referred to as "FKG-1") 0.48 g, CHCl ₃ : MeOH = 75: 25 (hereinafter referred to as "FKG-2") 2.42 g, CHCl ₃ MeOH = 50: 50 (hereinafter referred to as "FKG-3") 0.92 g, CHCl ₃ MeOH = 25: 75 (hereinafter referred to as "FKG-4") 0.16 g, CHCl ₃ : MeOH = 0: 100 (hereinafter referred to as "FKG-5") 0.08 g and 70% Acetone (hereinafter referred to as "FKG-6") were obtained. Each extract was concentrated and used as a sample of this experiment while being stored in the refrigerator after lyophilization (see Fig. 2).

Reference Example  1. Experimental animals and test equipment

1-1. Experimental animal

The experimental animals were supplied with 5 weeks of male ICR mouse (SPF / VAF CrljBgi: CD-1) from Orient Co., Ltd. (Gyeonggi-do, Korea). The experimental animals were used for 7 days in the animal room of Daegu Haany University and divided into normal group, control group and camphor cedar leaf fraction group. Experimental animals were housed in a polycarbonate mice cage with five animals in a constant temperature room at a temperature of 23 ± 3 ° C, a humidity of 50 ± 10%, and a 12-hour cycle of light and dark cycles.

1-2. Test equipment

The test apparatus used in this experiment is as follows.

Reference Example  2. Preparation before manual evasion test

2-1. Conditional avoiding reaction device

The evacuation unit is divided into two rooms of equal size, with a shuttle box (52.6 × 17.3 × 21.3 cm), and the guillotine door (8.9 × 8.9 cm) is installed on the wall separating the two rooms. You can open and close the In the room on the right, a very bright light bulb is installed, which creates a bright environment that the animals do not like. The light on the left side of the room did not enter at all, making the animals feel relatively comfortable.

2-2. Classification of experimental animals

Experimental animals were experimented with 10 animals in each group. Dosage was adjusted according to the yield of each material.

2-2-1. Control group

Normal rats were orally administered 10% Tween 80, followed by learning and memory tests.

2-2-2. Sco County

Normal rats were given oral administration of 10% Tween 80 one hour before the study test, and after 30 minutes, scopolamine (scopolamine) dissolved in distilled water was intraperitoneally administered at a dose of 1 mg / kg and scopolamine (scopolamine). Learning test was conducted 30 minutes after administration. After the study test, water and feed were sufficiently supplied for 24 hours without any treatment, and the memory test was performed.

2-2-3. Positive control group

Tackine (tacrine, THA, 10 mg / kg) was orally administered 1 hour prior to the study test in steady-state rats, and 30 minutes later, scopolamine was intraperitoneally administered at a dose of 1 mg / kg, A study test was conducted 30 minutes after scopolamine administration. After the study test, water and feed were sufficiently supplied for 24 hours without any treatment, and the memory test was performed.

2-2-4. Fraction of Korean mountain ginseng leaves (FKG) group

FKG was administered orally by dose at 1 hour before the study test was conducted in steady-state rats. After 30 minutes, scopolamine was administered at a dose of 1 mg / kg, and 30 minutes after scopolamine administration. Later, a study test was conducted. After the study test, water and feed were sufficiently supplied for 24 hours without any treatment, and the memory test was performed.

All experimental results were statistically analyzed using one way analysis of variance (ANOVA). When significance was recognized, the significance test was performed below 95% confidence level using Student-Newman-Keuls Method.

Experimental Example  1. Manual Evasion Test Passive avoidance test )

In order to measure the learning test and the memory test of the samples obtained in Example 1, the experiment was conducted by applying the method disclosed in the literature as follows (Ebert U, Kirch W. Scopolamine model of dementia: electroencephalogram findings and cognitive performance. Eur J Clin Invest . 1998; 28 (11): 944-9).

One hour before the start of the experiment, the rats were transferred to the behavior observation room, and the drugs were stabilized after administration.

1-1. Ethyl acetate fraction ( PgL - EtOAc Learning test training trial ) And memory test ( test trial )

Turn on the light in the right room of the shuttle box and gently lower the mouse's head to the opposite side of the gillotin door. The rats were searched for 10 seconds and then the guillotin doors were opened for entry into the dark compartment. The rat instinctively moved to the dark left chamber, where rats that did not enter the dark side within 40 seconds of opening the gillotin door were excluded from the experiment. Once the rat enters the dark side, the guillotin door closes and a 0.5 mA electric shock flows through the grid floor for three seconds, which the mouse remembers. The time from the opening of the guillotin door until the rat entered the dark side was measured.

Memory test was performed to determine the effect of camphor ginseng leaf fraction on long-term memory 24 hours after the study. The rats were placed in a round-trip box, and after 10 seconds of searching time, the guillotin door was opened, and the time required for all four feet of the mouse to enter the dark was measured up to 180 seconds. The longer it takes to go to the dark, the better the learning and memory of passive avoidance.

Whether or not a single extract of 10, 50, 100 and 200 mg / kg of ethyl acetate fraction (PgL-EtOAc) was used to inhibit memory impairment in memory-depleted animal models administered Scopolamine. As a result of using a manual evasion measuring device,

In the memory test, the scopolamine group had a statistically significant decrease in latency from 32.13 ± 2.54 seconds to 149.12 ± 11.41 seconds in the control group ( P <0.05). It is judged that the electric shock during the learning test cannot be remembered, and the memory loss model by scopolamine seems to be well made.

On the other hand, single doses of ethyl acetate fraction (PgL-EtOAc) at 10, 50, 100 and 200 mg / kg resulted in scopolamine with 67.13 ± 5.62 sec, 63.63 ± 8.17 sec, 114.50 ± 14.92 sec and 145.50 ± 11.66 sec, respectively. The statistically significant latency time was increased for 32.13 ± 2.54 seconds in the (scopolamine) group, especially at a dose of 200 mg / kg, recovering to 97.3% of the control ( P <0.05). As well as showing a 65.1% level of retention time of the THA control used as a positive control showed a much higher improvement effect (Table 1; see FIG. 3b).

1-2. Butanol  Fraction ( PgL - nBU Learning test training trial ) And memory test ( test trial )

Turn on the light in the right room of the shuttle box and gently lower the mouse's head to the opposite side of the gillotin door. The rats were searched for 10 seconds and then the guillotin doors were opened for entry into the dark compartment. The rat instinctively moved to the dark left chamber, where rats that did not enter the dark side within 40 seconds of opening the gillotin door were excluded from the experiment. Once the rat enters the dark side, the guillotin door closes and a 0.5 mA electric shock flows through the grid floor for three seconds, which the mouse remembers. The time from the opening of the guillotin door until the rat entered the dark side was measured.

The memory test was performed to determine the effect of the camphor cedar leaf fraction on organ memory after 24 hours after the learning test. The rats were placed in a round-trip box, and after 10 seconds of searching time, the guillotin door was opened, and the time required for all four feet of the mouse to enter the dark was measured up to 180 seconds. The longer it takes to go to the dark, the better the learning and memory of passive avoidance.

Extracts were obtained by oral single doses of butanol fraction (PgL-nBU) at 2.5, 5, 10 and 20 mg / kg, using a memory decay animal model administered Scopolamine (1 mg / kg, ip). Whether there is an effect of suppressing memory impairment was checked using a manual avoidance measuring device.

As a result of confirming the memory impairment by administration of scopolamine (1 mg / kg, i.p.),

In the scopolamine group, the retention time in the memory test was 54.25 ± 16.95 seconds, which was statistically significantly reduced for 161.00 ± 5.01 seconds in the control group ( P <0.05), but the butanol fraction (PgL-nBU) Of the tacrine in the positive control group had a retention time of 110.75 ± 15.62 seconds in the tacrine group of the positive control group after the scopolamine administration. However, butanol fraction (PgL-nBU) did not show any significant increase compared to 54.25 ± 16.95 sec. Through these results, it was determined that there was an active ingredient in the ethyl acetate fraction (PgL-EtOAc), and the separation was continued (Table 2; FIG. 4B).

1-3. FKG  Fraction ( FKG -3) study test ( training trial ) And memory test ( test trial )

Turn on the light in the right room of the shuttle box and gently lower the mouse's head to the opposite side of the gillotin door. The rats were searched for 10 seconds and then the guillotin doors were opened for entry into the dark compartment. The rat instinctively moved to the dark left chamber, where rats that did not enter the dark side within 40 seconds of opening the gillotin door were excluded from the experiment. Once the rat enters the dark side, the guillotin door closes and a 0.5 mA electric shock flows through the grid floor for three seconds, which the mouse remembers. The time from the opening of the guillotin door until the rat entered the dark side was measured.

The memory test was performed to determine the effect of the camphor cedar leaf fraction on organ memory after 24 hours after the learning test. The rats were placed in a round-trip box, and after 10 seconds of searching time, the guillotin door was opened, and the time required for all four feet of the mouse to enter the dark was measured up to 180 seconds. The longer it takes to go to the dark, the better the learning and memory of passive avoidance.

As a result of measuring the inhibitory activity of acetylcholine (AChE) in the ethyl acetate fraction (PgL-EtOAc), FKG-3 was the best activity, and FKG-3 was selected for manual avoidance test. Single doses of FKG-3 at 0.5, 1, 2 and 4 mg / kg resulted in statistical results for 17.88 ± 2.29 seconds in the scopolamine group at 120.88 ± 21.07 and 136.63 ± 15.52 seconds at 2 and 4 mg / kg, respectively. Significantly increased latency time ( P <0.05) (Table 3; see FIG. 5B).

Experimental Example  2. Morris Underwater maze  Learning and memory measurement by device

An experiment was conducted to measure the learning and memory of FKG-3 obtained in Example 1-2.

2-1. Morris Underwater maze  Learning measurement

Experiments on cognitive improvement through Morris water maze are already known as a method of screening dementia improving drugs. The maze specification is 90cm in diameter and 45cm in height, and the diameter of the white platform is 6cm. The periphery of the underwater maze kept the space cues always constant, such as computer systems and video thermostats connected to video cameras.

As an experimental method, the maze was filled with water so that the height of the water was 30 cm, and the mouse was installed below 1 cm of the water height so that the mouse could not see the platform. Maze was divided into four quadrants using four markers and divided into northeast (NE), northwest (NW), southeast (SE), and southwest (SW), and a platform was installed in one quadrant of maze. The Morris water maze test runs for six days, on the first day each rat is free to swim in the maze for one minute to adapt to the water, with no platform installed, and the second From day 5 to day 5, each rat was allowed to swim in maze for 1 minute at 10 minute intervals, four times a day. One test method for 4 days on the 5th day from the second day shows that a mouse that has been raised for 10 seconds within 1 minute on a platform already installed in maze does not find a platform within 1 minute after completing the experiment or does not climb on the platform for 10 seconds. Rats were artificially placed on the platform for 10 seconds after the end of the experiment, and the experiment was terminated. At this time, the position of the platform was fixed at the same position. On the 6th day, the platform was removed from the maze and the rats stayed at the platform where the platform was located.

Scopolamine was used as an amnesia-causing substance, and the dose was administered intraperitoneally at 1 mg / kg 30 minutes before the first acquisition daily, and the 10% tween and FKG-3 fractions of the control group and the FKG-3 group, respectively. Oral administration at a 2 mg / kg dose. On the last 5 days after dosing for 4 days, the time is given up to 60 seconds and no medication is administered. All experimental data were recorded and measured using the Ethovision program (Noldus, Netherlands).

In the study of Morris aquatic maze, the time required to reach the platform within 60 seconds for 4 days was 46.69 ± 2.88 seconds for the control group and 55.75 ± 1.36 seconds for the scopolamine treatment group. , Scopolamine + FKG-3 fraction 2 mg / kg group was 54.19 ± 2.20 seconds, scopolamine + tacrine group 50.53 ± 3.16 seconds, there was no significant difference between groups In the last 4 days, the time required to reach the platform was 14.84 ± 2.10 seconds in the control group, 56.56 ± 1.40 seconds in the scopolamine-treated group, and scopolamine + FKG-3 2 In the mg / kg group, 19.59 ± 1.42 seconds, the scopolamine + tacrine group was 33.81 ± 4.15 seconds, showing a significant difference ( P <0.05 ) between the groups. As a result of the post hoc test according to the measurement days, the learning ability of the scopolamine-administered group was lower than that of the control group from the second day.

In comparison, the FKG-3 2 mg / kg group showed a significant improvement in learning performance. The time to reach the platform from day 3 was statistically significant ( P <0.05 ) compared to the scopolamine group. Decrease (Table 4; see FIG. 6).

2-2. Morris Underwater maze  Memory measurement

After the platform was removed to perform the memory test on the fifth day of the last day of the study with Morris underwater maze, free swimming was performed for 60 seconds, and the time spent in the quadrant of the total time was measured by the Ethovision program. The memory measurement results of each group were 24.09 ± 2.20 seconds in the control group, 14.68 ± 2.04 seconds in the scopolamine group, 25.25 ± 2.36 seconds in the scopolamine + FKG-3 group, and scopolamine ) + Tacrine group showed a significant difference ( P <0.05) between the groups of 23.38 ± 0.47 seconds. As a result of the post hoc test on the degree of staying on the platform, the scopolamine injury group showed a significant decrease ( P <0.05) compared to the control group and the FKG-3 administration group (scopolamine ± FKG). -3) (2 mg / kg) showed a significant increase ( P <0.05) compared to the scopolamine injury group (Table 5; see Figure 7).

Experimental Example  3.Y-maze test mase test )

Y-maze experiment of FKG-3 obtained in Example 1-2 was carried out.

Drug administration was performed in the same manner as in the passive avoidance test. The instrument used for the Y-maze test consisted of three arms, each arm was 42 cm long, 3 cm wide, 12 cm high, and the angle of contact between the three arms was 120 °. All experimental devices consisted of black polyvinyl plastic. Each branch was set to A, B, and C, and the mouse was carefully placed on one branch and allowed to move freely for 8 minutes. Only when the tail is completely entered, it is also recorded when the branch went back. One point (actual alternation) was given to three different branches. Alteration behavior is defined as entering all three in turn and is calculated by the following equation.

As a result of the experiment, administration of scopolamine (1 mg / kg, ip) significantly reduced the spontaneous alternation compared to the control group ( P <0.05). On the other hand, spontaneous alternation was significantly increased by FKG-3 group compared with scopolamine group ( P <0.05). Increased spontaneous alternation means that learning and memory have recovered. On the other hand, there was no change in the total entry indicating the total number of entrances to each zone, indicating that spontaneous alternation was not caused by the change in the activity of the rat, as confirmed by manual avoidance test (FIG. 8). Reference).

Experimental Example  4. Immunohistochemistry  ( Immunihistochemistry )

The immunohistochemistry of FKG-3 obtained in Example 1-2 was performed.

Rats were anesthetized with pentobabital sodium (60 mg / kg, i.p.) and fixed by perfusion through 4% paraformaldehyde (PFA) solution made with 0.1 M phosphate buffer (pH 7.4). After perfusion fixation, the brain was removed and placed overnight in PFA solution and refrigerated for 48 hours in 30% sucrose solution made with 0.05 M phosphate-buffered saline (PBS, pH 7.4). It was. A brain slice of 30 μm thickness was prepared using a parietal slicer equipped with a cryostat. Brain sections were treated with PBS containing pERK or pCREB, BDNF antibody (1: 1000, Santa cruz, USA) for 24 hours, 0.3% Triton X-100, 0.5 mg / ml bovine serum albumin, and 1.5% normal horse plasma (4 ° C). Incubated overnight). Then incubate in a solution prepared by diluting the secondary antibody solution (Vector, Burlingame, USA) at 1: 200 for 90 minutes, and in the avidin-biotin-peroxidase complex (1: 100 Vector, Burlingame, USA) solution at room temperature for 1 hour. During incubation. Finally, brain slices were washed three times in PBS solution for 5 minutes and then sealed and fixed.

Expression of pERK, pCREB and BDNF in rat hippocampus to identify pERK, pCREB and BDNF pathways related to learning and memory in addition to the cholinergic nervous system, a major nervous system Was observed by immunostaining, FKG-3 2 mg / kg administration did not affect the expression of pCREB and BDNF, but CA1 and CA3, dentate of hippocampus (hippocampus) by administration of FKG-3 4 mg / kg pERK expression increased in the gyrus region. In particular, activation of the ERK pathway seems to be an important key to explain the memory-improving effect of FKG because it is very important in the function of nerves, such as synaptic plasticity as well as learning and memory (see Figure 9).

Experimental Example  5. FKG  Determination of Acetylcholinease Activity from Fractions AChE activity assay )

In order to measure the acetylcholine degrading enzyme activity of FKG-3 obtained in Example 1-2, experiments were conducted using acetylcholine iodide as a base as described by Ellman et al. (Oh MH, . Houghton PJ, Whang WK, Cho JH Screening of Korean herbal medicines used to improve cognitive function for anti-cholinesterase activity Phytomedicine 2004; 11 (6):.. 544-8).

Acetylcholine (AChE) was prepared by using a Teflon homogenizer (Eyela, Japan) to isolate rat cerebral cortex 10-fold phosphate buffer solution (12.5 mM phosphate buffer solution containing 400 mM NaCl, pH 7.0). Pulverized cerebral cortex with a tissue grinder (Potter-Elvehjem Tissue Grinders, DaiHan, Korea) and centrifuged at 1,000 × g for 10 minutes at a temperature of 4 ℃ to obtain a supernatant, and the supernatant was acetylcholine (AChE). ) As a sample.

FKG was diluted to a concentration of 200 μg / ml with phosphate buffer solution (100 mM, sodium phosphate buffer, pH 8.0) at each concentration, and 1.5 ml of the target substance diluted, 2.6 ml of buffer solution, and 75 mM acetylcholine iodide. 20 μl of acetylthiocholine iodide solution and buffered Ellman's reagent: 10 mM DTNB (5.5'-dithiobis-bis (2-nitrobenzoic acid)) and 15 mM sodium bicarbonate (NaHCO) 100 μl of the solution consisting of ₃ ) were mixed and reacted at a temperature of 25 ° C. for 30 minutes. Then, 400 μl of enzyme source was added to the solution, and absorbance was measured at 410 nm after 5 minutes (Optizen 2120UV, Mecasys Co. Ltd., Korea). At this time, as a control, a reaction solution in which an acetylcholine (AChE) sample was not added but a salt (saline) was added instead, and reacted without addition of acetylcholine iodide was measured by measuring absorbance. , Non-specific reaction between the FKG and the enzyme activity measurement reagent was confirmed that the acetylcholine (AChE) activity of the reaction solution without FKG is 100% acetyl Inhibition of choline (AChE) activity was confirmed.

FKG ethyl acetate fraction (PgL-EtOAc) and its gradient eluents, FKG-1, FKG-2, FKG-3 and FKG-4, inhibited the activity of acetylcholine (AChE) to improve memory and cognitive ability Acetylcholine (AChE) activity test was performed to determine whether the positive control tacrine inhibited acetylcholine (AChE) activity in a dose-dependent manner, and FKG-2 (CHCl ₃ : MeOH = 75: 25). And FKG-3 (CHCl ₃ : MeOH = 50: 50) it was found that the active effect is excellent in the fraction eluent (see Fig. 10).

It describes a formulation example of a composition comprising the camphor ginseng extract of the present invention, the present invention is not intended to limit it, it is intended to explain in detail only.

Formulation example  One. Liquid  Produce

PgL-EtOAc 459 mg

10 mg potassium sorbate

0.04 ml of polysorbate 80

Copulation

Flavoring Agent

Purified water

The above components are mixed to prepare a liquid according to a conventional liquid preparation method.

Formulation example  2. Suspension  Produce

PgL-nBU 459 mg

Hydroxyethyl Cellulose 30 mg

Agar 30 mg

1.6 g of D-sorbitol 70%

Flavoring Agent

Purified water

The above components are mixed to prepare a suspending agent according to a conventional suspending agent production method.

Formulation example  3. Preparation of Tablets

PgL-nHx 459 mg

Starch 100 mg

Magnesium stearate proper amount

Coating base amount

The above components are mixed and tablets are prepared according to a conventional tablet production method.

Formulation example  4. Preparation of Capsules

FKG-3 459 mg

Starch 100 mg

Talc 1 mg

Magnesium stearate proper amount

The above ingredients are mixed to produce a capsule according to a conventional method for producing a capsule.

Formulation example  5. Preparation of Injectables

PgL-nBU 459 mg

Mannitol 180 mg

_{Na 2 HPO 4 · 12H 2 O} 26 mg

Sterile distilled water for injection

Usually, it is prepared in the above-described content of ingredients per one vial (20 ml) according to the preparation method of the injection.

Formulation example  6. Manufacture of health food

FKG-3 1000 mg

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

Nicotinamide 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5 mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

15 mg potassium monophosphate

Dicalcium 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 a composition that is relatively suitable for the health functional food, the composition is mixed in a preferred embodiment, but the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health functional food manufacturing method. Then, the granules may be prepared and used for preparing the nutraceutical composition according to a conventional method.

Formulation example  7. Manufacture of health drinks

PgL-EtOAc 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

900 ml of purified water whole

After mixing the above components according to the 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 Used to prepare the healthy beverage composition of the invention.

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.

Claims (8)

Jangnoesam (Mountain Cultivated Ginseng Radix ) A pharmaceutical composition for the prevention and treatment of brain diseases, comprising a purified extract or fraction of the leaf as an active ingredient.
The pharmaceutical composition of claim 1, wherein the brain disease is amnesia, Lewis body disease, Pick's disease, Alzheimer's disease, stroke, Parkinson's disease or Huntington's disease. According to claim 1, wherein the purified extract is a pharmaceutical composition, characterized in that the polar solvent soluble extract or non-polar solvent soluble extract of camphor ginseng leaves. The pharmaceutical composition of claim 3, wherein the polar solvent soluble extract comprises an extract available from a solvent selected from water, butanol, methanol, ethanol or a mixed solvent thereof. The pharmaceutical composition of claim 3, wherein the nonpolar solvent soluble extract comprises an extract soluble in hexane, chloroform, or ethyl acetate. According to claim 1, wherein the fraction is purified by separating the non-polar solvent soluble extract, in particular ethyl acetate soluble extract by additionally performing Kiesel gel column chromatography method using chloroform (CHCl ₃ ) and methanol (MeOH) as the eluent. Water, especially developing solvent (CHCl ₃ : MeOH = 50: 50) pharmaceutical composition characterized in that it comprises a purified product (FKG-3) that can be separated. Health functional food for the prevention and improvement of brain disease, comprising purified extract or fraction of leaves of Mountain Cultivated Ginseng Radix . The dietary supplement of claim 7 which is a powder, granule, tablet, capsule, syrup or beverage.

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