KR20090118465A - A composition comprising a radix extract of mountain cultivated ginseng as an active ingredient for preventing and treating brain disease - Google Patents

Abstract

PURPOSE: A composition containing extract of mountain cultivated ginseng radix as an active ingredient is provided to suppress acetylcholinesterase activation and prevent or treat brain diseases. CONSTITUTION: A pharmaceutical composition for preventing and treating brain diseases contains extract of mountain cultivated ginseng radix as an active ingredient. The extract of mountain cultivated ginseng radix is isolated from leaves, flower, stem, or roots using water, low alcohol of 1-4 carbon atoms or their mixture solvent. The brain diseases are amnesia, Lewis body disease, Pick's disease, Alzheimer's disease, stroke, Parkinson's disease, or Huntington disease.

Description

A composition comprising a radix extract of Mountain Cultivated Ginseng 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 camphor ginseng extract as an active ingredient.

[Document 1] Lee Byung Yoon.精神 醫學 辭典. Seoul: Sculpture. 1990: 58-62, 390

[Document 2] Min Sung-gil. Latest mental medicine. Seoul: Sculpture. 2005: 25-36, 189-98

3 Ernest R. Hilgard, Donald George Marquis. Conditioning and learning. Appleton-Century-Crofts. New York: Teachers College. 1961: 107-9

[4] 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

[Reference 5] Ryu Young-soo, Choi Gong-han.東西 醫學 的 比較, 硏 究.東 醫 神經 精神科 學會 誌. 1996; 7 (1): 155-66

[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, Dean RL, Beer B and Lippa AS. Science . 1982; 217: 408-17

Document 9 Gold PE, Vogt J and Hall JL. Behav Neural Biol . 1986; 46: 145-55

Document 10 Wenk GL. Psychopharmacol . 1989; 99: 431-39

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

12. Anoja S, Attele Wu, Yuan CS. Biochemical Pharmacology . 1999; 58: 1685-93

Document 13 Hu SY. Economy Botany . 1976; 30: 11-28

Document 14 Lee, TH, Kim, DH. Korean J. Giseng Sci . 1999; 23: 21-37

[Research Document 15] Korea Food Research Institute, 'A Study on the Functional Characteristics of Camphor Ginseng Leaf and Development of Processed Foods Using It', Ministry of Agriculture and Forestry, Final Report, 2005.10.14

[Paper 16] 金 宇 哲.統計學 槪 論. Seoul: 英 志 文化 社. 1990: 177-9

Document 17 LeDoux JE. Behav Brain Res . 1993; 58:69

18 Ellman GL, Courtney KD, Andres V Jr, Feather-Stone RM. Biochem Pharmacol . 1961; 7: 88-95.

19 Lowry OH, et al., Journal of Biological Chemistry, 1951, 193, p265-75

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. Recent mental medicine. Seoul: Sculpture. 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 disorders, 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 Suk and others. -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).

In psychogenic forgetfulness, the loss of consciousness suddenly appears and recovers momentarily because the disability and mental disorders are not seen, the extent of memory impairment is optional, and memory impairment appears in order to obtain the benefit according to need. 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).

Neural systems related to memory are known as cholinergic nervous system, glutamatergic nervous system, GABAergic nervous system, serotonergic nervous system, adrenergic nervous system, etc. In particular, cholinergic nervous system is known to play an important role in learning and memory processes in animals and humans. In addition, there are pathways that promote learning and memory 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, but in 1973, cytotoxicity of leaf saponins was found to have neuroleptic, analgesic, hypotensive and atropine-like effects Received. 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.Afterwards, six saponin components, ginsenosides Rb1, Rb2, Rc, Rd, and Rg1, were investigated in the saponin fraction. Confirmed. Therefore, it can be seen that they are most suitable as a raw material for extracting and separating ginsenosides because of their 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, oxalic, citric, malic, succinic acid, etc. contained in ginseng leaves, and oxalic acid in camphor ginseng leaves. 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 due to environmental problems such as pesticide application (Korea Food Research Institute, 'Research of Functional Characteristics of Camphor Ginseng Leaf and Development of Processed Foods Using It') 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 the scopolamine-induced forgetful animal model of camphor ginseng extract, acetylcholine degrading enzyme. Through the (Acetylcholinesterase) test to confirm the effect of learning ability and cognitive function of camphor ginseng leaf was completed the present invention.

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

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

As defined herein, Mountain Cultivated Ginseng Radix refers to ginseng artificially grown from wild ginseng seeds or ginseng grown in the wild by the perennial vegetation of Panax ginseng CA Meyer belonging to the family Arboraceae. .

Extracts as defined herein include those extracted from the leaves, flowers, stems or roots of camphor, preferably from leaves.

Extracts as defined herein comprise water, including purified water, lower alcohols having 1 to 4 carbon atoms or mixed solvents thereof, preferably mixed solvents of water and ethanol, even more preferably 50 to 90% ethanol It is the extract which is soluble in the solvent.

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, flowers, stems or roots of the camphor ginseng, preferably the leaves, 5 to 20 times, preferably 10 times (w / v) of water, lower alcohols having 1 to 4 carbon atoms or these A solvent selected from a mixed solvent of, preferably a mixed solvent of water and ethanol, even more preferably 50 to 90% ethanol at 10 ° C. to 50 ° C., preferably at 20 ° C. to 40 ° C. for 30 minutes to 4 hours, preferably Preferably, ultrasonic extraction, reflux extraction, hot water extraction, cooling extraction or supercritical extraction, preferably ultrasonic extraction (ultrasound extraction is a low temperature extraction method that can be extracted even at low temperature, inefficient penetration and degeneration to temperature) for 1 to 3 hours. A first step of extracting twice with an extraction method to overcome the disadvantages and to make a stable extract having high physiological activity; 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.

The present invention provides a pharmaceutical composition for the prevention and treatment of brain diseases comprising the extract obtained by the above method as an active ingredient.

The present invention also provides a health functional food for the prevention and improvement of brain diseases comprising the extract obtained by the above method 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 comprises effecting via a cholinergic nervous system, glutamatergic nervous system, GABAergic nervous system, serotonergic nervous system or adrenergic nervous system, preferably cholinergic nervous system.

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

The pharmaceutical composition containing the extract of the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

The pharmaceutical composition containing the extract of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be used. 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 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 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 extract 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. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

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 extract 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 extract may be added to food or beverages 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 for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . 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 extract of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as flavoring agents, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the extract 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 the extract of the camphor ginseng (Mountain Cultivated Ginseng Radix) as an active ingredient, more specifically, the composition containing the camphor ginseng extract as an active ingredient inhibits the acetylcholinesterase activity In addition, the scopolamine-induced forgetful rat model exhibits a memory-improving effect, and thus the composition may be usefully used as a pharmaceutical composition or health functional food for the prevention and treatment of brain diseases.

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. Preparation of camphor ginseng leaf extract

100 g of camphor ginseng leaves (Ein Manny, Yecheon Gyeongbuk Korea) was selected and washed twice with distilled water, followed by adding 10% (w / v) of camphor ginseng leaves to 70% ethanol for 2 hours to increase the extraction yield at room temperature. Ultrasonic extraction twice. This was concentrated in a vacuum distillation apparatus and dried again with a lyophilizer (EYELA FDU-1100), 35 g of a red powder obtained (yield: 35%) was used as a sample of the following experiment (hereinafter referred to as "MCGRLE").

Reference Example  1. Experimental Materials

The tacrine (9-amino-1, 2, 3, 4-tetrahydroacridine hydrochloride), (-) scopolamine hydrobromide, acetylthiocholine iodide and DTNB (5, 5'-dithiobis [2-nitrobenzoic acid]) used in this experiment were Sigma , USA), and other reagents are available on the market.

Reference Example  2. Experimental Animal

Experimental animals were used 5 weeks old male ICR mouse (SPF / VAF CrljBgi: CD-1, Orient, Gyeonggi-do, Korea). The experimental animals were acclimated for 7 days in the Daegu Haany University animal room. During the adaptation period, the temperature of the animal room was maintained at 23 ± 3 ℃, humidity of 50 ± 10%, and 12-hour cycles of light and dark. The experimental animals received unlimited feed and water during the adaptation period.

Each group was divided into 7 groups: control group, scopolamine group (Sco), THA group (positive control group), and MCGRLE group (50, 100, 200, 400 mg / kg oral administration of scopolamine). Ten animals were tested. The control group was orally administered 10% tween80 to normal rats, and the scopolamine group was intraperitoneally administered 1 mg / kg of scopolamine dissolved in distilled water 30 minutes after oral administration of 10% tween80 to normal rats. The experiment was conducted 30 minutes after scopolamine administration. The THA group was orally administered 10 mg / kg of tacrine to rats in a steady state, and intraperitoneally administered 1 mg / kg of scopolamine 30 minutes later, and the experiment was performed 30 minutes after scopolamine administration. Tacrine, sold under the trademark Cognex, is currently used as a widely recognized acetylcholine degrading enzyme inhibitor recognized by the US Food and Drug Administration. MCGRLE-administered group was intraperitoneally administered 1 mg / kg of scopolamine 30 minutes after oral administration of MCGRLE obtained in Example 1 to the steady state rats at a dose of 50, 100, 200, 400 mg / kg, The experiment 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 conducted.

All experimental results were statistically analyzed using one way analysis of variance (ANOVA), and if significant, the Student-Newman-Keuls Method (金 宇 哲. 統計學 槪 論. Seoul: 英 志 文化 社. 1990: 177- 9) was used to test the significance below 95% confidence level.

Experimental Example 1. Manual evasion test

In order to confirm the effect of MCGRLE obtained in Example 1 to suppress memory impairment using a memory decay animal model administered scopolamine (LeDoux JE., Behav Brain Res. , 1993, 58 , The experiment was conducted by applying the manual avoidance test method described in p69).

The Gemmini avoidance system (San Diego Instruments, USA) is a shuttle box (52.6 x 17.3 x 21.3 cm) divided into two equally sized rooms, with a guillotine door on the wall separating the two rooms. (guillotine door, 8.9 x 8.9 cm) is installed to automatically open and close up and down. 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. One hour before the start of the experiment, the experimental animals of Reference Example 2 were transferred to the behavior observation room and stabilized.

1-1. Training trial

Turn on the light in the right chamber of the ESC and gently lower the test animal's head to the opposite side of the guillotine door. After exploring the animals for 10 seconds, the guillotine door was opened to allow access to the dark compartment. The experimental animal instinctively moved to the dark left room, except for mice that did not enter the dark side within 40 seconds after the guillotine door was opened. Once the mouse enters the dark side, the guillotine door closes and an electric shock of 0.5 mA flows through the bottom of the grid for 3 seconds and the mouse remembers it.

The behavioral behavior of the mouse during electric shock was observed by giving 3 points for jumping, 2 points for shouting, and 0 points for no response. After the guillotine door was opened, the time until the mouse entered the dark side was measured.

As a result, as shown in Figure 1, retention time by scopolamine administration was significantly reduced compared to the control group. Meanwhile, the dose-dependent retention time was increased by a single oral administration of MCGRLE (50, 100, 200, 400 mg / kg), and it was confirmed that the MCGRLE of 400 mg / kg dose recovered to 66% of the control group. In addition, the THA administration group used as a positive control showed a high improvement effect compared to the 50% retention time of the control group.

In addition, as shown in Table 1, the administration of MCGRLE did not affect the behavioral change induced by the electric shock or the retention time shown in the learning test. It could be confirmed that not by.

Group (mg / kg) N Score ± S. E. M. Control 10 4.1 ± 0.4 Scopolamine (1) 10 4.5 ± 0.3 MCGRLE (50) 10 3.3 ± 0.2 MCGRLE (100) 10 3.1 ± 0.4 MCGRLE (200) 10 3.6 ± 0.2 MCGRLE (400) 10 4.3 ± 0.3 THA (10) 10 4.2 ± 0.3 Results are expressed as the means ± S.E.M (n = 10)

1-2. Memory test test trial )

In order to confirm the effect of MCGRLE obtained in Example 1 on long-term memory, a memory test was performed 24 hours after the learning test of Experimental Example 1-1 was completed according to the following method.

The experimental animals were placed in the circumvention test apparatus, and after 10 seconds of searching time, the time required for the guillotine door to open and the four feet of the mouse to enter the dark side was measured up to 180 seconds. The longer it takes to go to the dark, the better the learning and memory effects of passive avoidance.

As a result, as shown in Figure 2 below, the MCGRLE 50, 100, 200, 400 mg / kg single dose oral dose-dependent retention time was increased, when the 400 mg / kg dose of MCGRLE was administered 66 Recovery to% level was confirmed ( P <0.05 ).

Experimental Example 2 Morris Underwater Maze Test

MCGRLE obtained in Example 1 was tested with Morris underwater maze to confirm the effect on cognitive improvement using the scopolamine administered memory decay animal model according to the following method.

Experiments with Morris water maze are known as a method of screening cognitive-improving drugs. The labyrinth has a diameter of 90 cm and a height of 45 cm and a white platform of 6 cm in diameter. The periphery of the underwater maze kept constant space cues, such as a video system and a computer system connected to the water temperature control. As an experimental method, the labyrinth was filled with water so that the height of the water was 30 cm, and the mouse was installed below 1 cm of the height so that the mouse could not see the platform. In the labyrinth, four markers were used to divide the labyrinth into four quadrants, divided into northeast (NE), northwest (NW), southeast (SE), and southwest (SW), and a platform was installed in one quadrant of the maze. The Morris Underwater Maze test is conducted for six days, on the first day each mouse is free to swim in the maze for one minute to adjust to the water, with no platform installed and one day from the second day to the fifth day. Each mouse was allowed to swim in the labyrinth at 20 minutes intervals for 2 minutes, 4 times a day. One experiment for four days on the fifth day from the second day is that a mouse that is already in the labyrinth for 10 seconds on a platform that has already been installed in a maze does not find a platform within 1 minute of the experiment or does not climb on the platform for 10 seconds. After the end of the experiment artificially placed on the platform for 10 seconds to end the experiment, where the position of the platform was fixed in the same place. On day 6, the platform was removed from the maze and the time the mouse stayed at the platform was measured. Scopolamine administration was administered intraperitoneally at 1 mg / kg 30 minutes before the first acquisition daily, and all experimental data were recorded and measured using the Ethovision program (Noldus, Netherlands).

2-1. Learning test at Morris Underwater Maze

The time to reach the platform within 60 seconds was measured for 5 days to measure the learning test in the Morris underwater maze test.

As a result, as shown in Table 2 and Figure 3, the control group on the first day 50.06 ± 3.76 seconds, 60.00 ± 0.00 seconds in the scopolamine group, 57.93 ± 7.28 seconds in the MCGRLE group, 54.5 ± 2.59 seconds in the THA group There was no significant difference between the groups, but as learning progressed, the time to reach the platform in the last 5 days was 17.04 ± 3.08 seconds in the control group, 42.59 ± 7.52 seconds in the scopolamine group and 21.45 ± 2.39 seconds in the MCGRLE group. , THA group showed a significant difference ( P <0.05 ) between the groups at 26.75 ± 2.32 seconds. As a result of the post hoc test according to the measurement date, the scopolamine-administered group showed significantly lower learning ability than the control group from the second day. On the other hand, the MCGRLE group (scopolamine 1 mg / kg + MCGRLE 400 mg / kg) showed a significant enhancement in learning performance. That is, the time required to reach the platform from the 3rd day was statistically compared to that of the scopolamine group. P <0.05 ) was significantly reduced.

Group (mg / kg) N 1st day 2nd day 3rd day 4th day Control 10 50.1 ± 3.8 25.0 ± 4.7 22.4 ± 3.2 17.0 ± 3.1 Scopolamine (1) 10 60.0 ± 0.0 51.2 ± 3.0 ^* 52.1 ± 3.5 ^* 42.6 ± 7.5 ^* MCGRLE (400) 10 57.9 ± 7.3 45.1 ± 4.4 33.3 ± 3.1 ^# 21.4 ± 2.4 ^# THA (10) 10 54.5 ± 2.6 43.2 ± 4.5 30.7 ± 3.2 ^# 26.7 ± 2.3 ^# Each value represents the means ± SEM (n = 10). * P <0.05 as compared with the control group, # P <0.05 as compared with the scopolamine-treated group.

2-2. Memory test in Morris underwater maze test

In order to measure the memory test in the Morris underwater maze test, the platform was removed on the 6th day of the last day, and free swimming was performed for 60 seconds, and the time spent in the quadrant of the total time was measured by the Ethovision program.

As a result, as shown in Table 3 and Figure 4, the memory measurement results of each group was 35.06 ± 1.84 seconds in the control group, 25.34 ± 1.83 seconds in the scopolamine group, 32.96 ± 1.91 seconds in the MCGRLE group, 33.11 in the THA group There was a significant difference ( P <0.05 ) among the groups at ± 1.78 seconds. As a result of the post hoc test on the degree of staying on the platform, the scopolamine-treated group showed a significant decrease ( P <0.05 ) compared with the control group, and the scopolamine-treated group in the MCGRLE-treated group (scopolamine + MCGRLE 400 mg / kg) It can be seen that significantly increased compared to ( P <0.05 ).

Group (mg / kg) N Duration of 5th day Retention time (sec) Control 10 35.1 ± 1.8 Scopolamine (1) 10 25.3 ± 1.8 ^* MCGRLE (50) 10 33.1 ± 1.8 MCGRLE (100) 10 30.7 ± 1.9 MCGRLE (200) 10 29.4 ± 1.4 MCGRLE (400) 10 31.1 ± 1.3 ^# THA (10) 10 32.9 ± 1.9 ^# Each value represents the means ± SEM (n = 10) * P <0.05 as compared with the control group, # P <0.05 as compared with the scopolamine-induced group.

Experimental Example 3. Acetylcholine degrading enzyme activity (AChE activity assay)

3-1. In vitro  acetylcholinesterase activity measurement

In vitro acetylcholine degrading enzyme (AChE) activity measurement of MCGRLE obtained in Example 1 is described in Ellman et al. (Ellman GL. Et al.,Biochem Pharmacol., 1961,7, p88-95) was tested by measuring the absorbance using acetylcholine iodide as a substrate as described below.

The preparation of AChE was performed by separating the cerebral cortex of mice using a Teflon homogenizer (Eyela, Tokyo, Japan) and adding 10-fold phosphate buffer solution (12.5 mM phosphate buffer solution including 400 mM NaCl, pH 7.0). The cerebral cortex was pulverized with a homogenizer, and the homogenate was centrifuged at 1,000 × g for 10 minutes at 4 ° C. to obtain a supernatant. The supernatant was used as the AChE sample of the following experiment.

MCGRLE was diluted to a concentration of 200 ㎍ / ml with phosphate buffer solution (100 mM, sodium phosphate buffer, pH 8.0) at each concentration, and 1.5 ㎖ of diluted target substance, 2.6 ml of buffer solution, and 20 ㎖ of 75 mM acetylthiocholine iodide solution. And 100 μl of a buffered Ellman's reagent (a solution consisting of 10 mM DTNB (5.5'-dithiobis-bis (2-nitrobenzoic acid)) and 15 mM NaHCO ₃ ) are reacted at 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 containing no AChE sample but saline was used instead, and the absorbance was measured by reacting without adding acetylthiocholine iodide, thereby measuring MCGRLE and enzyme activity. It was confirmed that non-specific reaction between the reagents did not occur, and when the AChE activity of the reaction solution without adding MCGRLE was 100%, the inhibition of AChE activity of the reaction solution to which each searched substance was added was confirmed.

As shown in FIG. 5, MCGRLE inhibited acetylcholine degrading enzyme activity in a dose-dependent manner, and the IC ₅₀ value was 654 μg / ml.

3-2. Ex-vivo  acetylcholinesterase activity measurement

In vitro acetylcholine degrading enzyme (Ex-vivo In order to measure acetylcholinesterase activity, the brain of the mouse was extracted 1 hour after oral administration in the MCGRLE 400 mg / kg administration group of Reference Example 2, and the hippocampus, cerebral cortex, and striatum were separated, respectively. Acetycholine degrading enzyme activity was measured in the same manner as in Experimental Example 3-1.

As a result, as shown in Figure 6 in the ex-vivo experiment MCGRLE was found to inhibit the acetylcholine degrading enzyme effects of 43%, 24%, 24% in the hippocampus, cerebral cortex and striatum, respectively. Protein quantitation was determined using the Lowry method (Lowry OH, et al., Journal of Biological Chemistry , 1951, 193 , p265-75).

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 1 Preparation of Liquid

MCGRLE 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 Preparation of Suspending Agent

MCGRLE 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 Tablet

MCGRLE 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 Capsule

MCGRLE 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 Injection

MCGRLE 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

MCGRLE 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

MCGRLE 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.

1 is a diagram measuring the latency time of a training trial in a passive avoidance experiment,

Figure 2 is a measure of the retention time (* P <0.05 as compared with the control group, # P <0.05 as compared with the scopolamine-treated group) in the test trial in a passive avoidance test ,

3 is a view measuring the retention time of the learning test in the Morris water maze test,

Figure 4 is a measure of the retention time of the memory test in Morris water maze test (* P <0.05 versus control group, # P <0.05 versus scopolamine-treated group in graph B),

5 is in vitro of MCGRLE ( in In vitro ) (Each value represent means ± SEM (n = 3)), which shows the inhibitory effect on acetylcholine degrading enzyme (IC ₅₀ ),

Figure 6 ex vivo of MCGRLE ( ex In vivo ) (Each value represent means ± SEM (n = 3)) showing the inhibitory effect (%) on acetylcholine degrading enzyme.

Claims (6)

A pharmaceutical composition for the prevention and treatment of brain diseases, comprising the extract of Mountain Cultivated Ginseng Radix . The pharmaceutical composition according to claim 1, wherein the extract is extracted from leaves, flowers, stems, or roots of camphor ginseng. The pharmaceutical composition according to claim 1, wherein the extract is an extract available in a solvent selected from water including purified water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. 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. Jangnoesam (Mountain Cultivated Ginseng Radix ) A dietary supplement for the prevention and improvement of brain diseases, including extract as an active ingredient. 6. The dietary supplement of claim 5 which is a powder, granule, tablet, capsule, syrup or beverage.

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