KR20140095213A

KR20140095213A - Pharmaceutical composition for the prevention and treatment of parkinson's disease and alzheimer's disease containing ginseng flos extract

Info

Publication number: KR20140095213A
Application number: KR1020130007853A
Authority: KR
Inventors: 김형춘; 신은주; 고성권
Original assignee: 강원대학교산학협력단; 세명대학교 산학협력단
Priority date: 2013-01-24
Filing date: 2013-01-24
Publication date: 2014-08-01

Abstract

A pharmaceutical composition containing a ginseng flos extract of the present invention attentively protects dopaminergic neurons in animal models with Parkinson′s disease, boosts the level of dopamine and the expression of tyrosine hydroxylase, and consequently reduces ataxia, which is a symptom of Parkinson′s disease, with attention. The pharmaceutical composition of the present invention also accelerates the cognitive memory ability and cholinergic nervous system function in animal models with Parkinson′s disease with attention, and thus, the pharmaceutical composition can exhibit effects of prevention and treatment of Parkinson′s disease and Alzheimer′s disease by a ginseng flos extract contained in the composition of the present invention.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a composition for preventing or treating Parkinson's disease and Alzheimer's disease comprising ginseng root extract and a composition for prevention or treatment of Alzheimer's disease,

The present invention aims to provide a novel pharmaceutical composition for preventing or treating Parkinson's disease and Alzheimer's disease. More specifically, it is intended to provide a composition for preventing or treating Parkinson's disease and Alzheimer's disease containing an extract of ginseng root as an active ingredient.

Parkinson's disease (PD) is the second most prevalent brain degenerative disease following Alzheimer's disease, with approximately 1% of the population aged 50 years or older suffering from it (Science: Science , 274, 1197-1199). Parkinson's disease is a degenerative neurological disease involving progressive degeneration of dopamine-secreting neurons distributed from striatum from the substantia nigra. It is clinically characterized by 70-80% reduction in neuron terminals of the striatum and 50-60% (J. Neurol. Sci., 20, 415-455; Lancet, 337, 1321-1354). The main symptoms of Parkinson's disease are 1) bradykinesia, 2) tremor-at-rest, 3) muscle rigidity, 4) loss of postural reflexes, 5) ), Flexed posture (6), and freezing (6). Of these, two or more symptoms are present and one of them is diagnosed as Parkinson's disease when it is mild or stabilized. (Nature, 392, 605-608).

Familial Parkinson's disease is often premature (around 50 years of age) and is the hallmark of gene abnormalities such as parkin, DJ-1, PINK1, α-synuclein and LRRK2 (Nature, Idiopathic Parkinson's disease is characterized by a variety of genetic features, environmental toxins, and the age of the elderly. It is a major etiology (cf. Cell. Mol. Neurobiol., 26, 781-802). Postmortem tissue studies of Parkinson's disease have shown that dopaminergic neuronal loss and progression of lesions can be mediated by neuroinflammatory processes (Exp. Neurol., 208, 1-25), oxidative stress (J. Neurochem., 69, 1326-1329 , J. Neurosci. Res., 26, 5256-5264, Biochem. Biophys. Res. Commun., 345, 967-972, Proc. Natl. Acad. Sci. USA, 93, 2696-2701) (See Exp. Neurol., 179, 38-46). In addition, brain tissue of Parkinson's disease or animal model has been shown to induce activation of microglia (see Neurobiol. Dis., 21, 404-412), cytokines (Ann. Neurol., 44, S115-S120), cyclooxygenase Activation (Proc. Natl. Acad. Sci. USA, 100, 5473-5478) was observed, and anti-inflammatory agents were shown to have a neuroprotective effect in animal models (Ann. NY Acad. Sci. Brain Res., 909, 187-193), inflammatory response is one of the pathogenesis of neuronal death in Parkinson's disease.

The rodent model, which is mainly used for the study of the mechanism of Parkinson's disease and the pharmacological efficacy of anti-Parkinsonian compounds, includes DJ-1 gene deficient mice, PINK1 gene deficient mice, parkin gene deficient mice, α-synuclein gene-overexpressing mice. In idiopathic Parkinson's disease, 1-methyl-4-phenyl-tetrahydropyridine (MPTP), 6-hydroxydopamine, paraquat and rotenone are administered , 1384-1391).

As the economic development progresses with the remarkable development of medicine, the aging of the world is accelerating as the life expectancy increases. According to the National Statistical Office 's future population estimates, Korea is expected to enter the super aged society in 2026. Alzheimer's disease is the most common cause (60%), vascular dementia (20%), dementia with Lewy body (Parkinson's dementia, 15% ), And other causes (5%) (J. Psychopharmacol., 20, 732-755). In addition, medical advances in other areas mean that younger generations will have longer longevity, thus increasing the proportion of geriatric neurodegenerative diseases in the next generation. In the case of Alzheimer's disease, it is estimated that the number of patients worldwide will reach about 26 million in 2006, or about 106 million in 2050 (quadrupling in the world) (World Alzheimer's Association, 2006) and about 36 million by 2015 (Global Information Inc.).

Alzheimer's disease is a disease characterized by histological features of amyloid plaques and neurofibrillary tangles in the hippocampus and cortex (Neuron, 6, 487-498), the pathogenesis of Alzheimer's disease The most important process is the accumulation of insoluble beta-amyloid (Aβ) peptides. Aβ (1-40) and Aβ (1-42) are the most abundant in the water-insoluble β-amyloid peptide, and Aβ (1-42) is more likely to aggregate with amyloid plaques than Aβ (See Acta Biolch. Pol., 52, 417-423). In addition, differences in genes such as amyloid precursor protein (APP), presenilin (PS) -1, PS-2 and apolipoprotein and oxidative toxicity are thought to be involved in the pathogenesis of Alzheimer's disease , 2007, 285-297), which is confirmed by establishing an animal model of Alzheimer's disease by using various genetically modified animals, such as APP-expressing mice or by administering a toxic A [beta] peptide centrally.

Oxidative damage (15-18) due to the accumulation of water-insoluble Aβ is known to be one of the major pathologies of Alzheimer's disease and is associated with increased oxidative damage indicators in brain tissue of Alzheimer's patients (Neurochem. Res. 33, 450 (Neurotox. Rex. 2, 167-168) and an endogenous antioxidant system abnormality (Neurotox. Rex. 2, 167-168) have been reported in animal models of Alzheimer's disease Res., 155, 185-196; J. Neurosci., 27, 5394-5404). In addition, the damage and dysfunction of the cholinergic system in the hippocampus and cortical area contributes to a cognitive memory loss, a characteristic symptom of Alzheimer's disease (cf. Neurology 51, S18-S29; Curr. Alzheimer Res. 1, 241-248 ).

As a conventional technique for the prevention or treatment of Alzheimer's disease, Korean Patent Laid-Open Publication No. 10-2005-0101537 discloses a method for the treatment of amyloid-β related diseases including Alzheimer's disease using 3-amino-1-propanesulfonic acid and a neuro- And Korean Patent Laid-Open Publication No. 10-2012-48105 discloses a pharmaceutical composition having the therapeutic effect of synergistic Alzheimer's disease by combining ursodeoxycholic acid and Ginkgo biloba extract . In addition, various inventions have been made on the composition for treating Alzheimer's disease or the use of medicines, but there is still a demand for development of various drugs useful for the treatment of Alzheimer's disease.

Meanwhile, ginseng roots were ginseng ( Panax ginseng CA Meyer ). Extracts of ginseng roots and active ingredients of Alzheimer's disease in vivo and in vitro models and is intended to represent a pharmacological effect in Parkinson's disease models been reported (see:... Biochim Biophys Acta, 1820, 453-460; Eur J Pharmacol, 675, 15-21; Biochim Biophys Acta, 1822, 286- 292; J Ethnopharmacol., 133, 1109-1116; Neurosci Lett., 487, 70-72; J Asian Nat Prod Res., 11, 604-612; Alzheimer Dis Assoc Disord., 22, 222-226; Zhongguo Zhong Xi Yi Jie He Za Zhi, 1992, 12, 622-623, Exp Neurol., 184,: 521-529). The extracts of ginseng roots contain many saponins of a unique dammarane structure not only in the ginseng roots but also in ginseng roots (J Agric Food Chem., 58, 868-874; Chem Pharm Bull J Korean Nat Prod Res., 1, 119-23), 55, 1034-1038; , Ginseng root may have different pharmacological mechanism, but until now, in There is no initiation or teaching applied to cerebral degenerative diseases in the central nervous system other than to have an anti-leukemia effect in vitro (see Bioorg Med Chem Lett., 20, 309-314).

Korean Patent Publication No. 10-2012-48105

Currently, dopamine replacement therapy and dopamine receptor antagonists such as L-DOPA are used for the treatment of Parkinson's disease. However, because of serious side effects such as dyskinesia during long-term administration, The development of effective drugs is urgent.

Also, in the case of Alzheimer's disease, the drug is depended on drugs such as donepezil to alleviate symptoms because a fundamental therapeutic agent has not been developed yet. Accordingly, the present inventors have made efforts to develop drugs showing efficacy against Parkinson's disease and Alzheimer's disease. As a result of intensive studies, the present inventors have found that the extract of ginseng root improves expression of tyrosine hydroxylase and dopamine levels in black and striatum in an animal model of Parkinson's disease and thus improves ataxia symptoms in Parkinson's disease. It has been found that the animal model enhances cognitive memory ability and inhibits dysfunction of the cholinergic system of hippocampal tissues, so that a pharmaceutical composition containing ginseng root extract can be used as an agent for preventing or treating Parkinson's disease and Alzheimer's disease Can be used.

In addition, since the commercialization of ginseng roots has been actively carried out so far, the ginseng roots have been discarded. Therefore, the present invention is worthy of being able to create new demand and utilize abandoned resources.

The present inventors applied an in vivo animal model in which 1-methyl-4-phenyl-tetrahydropyridine (MPTP) was repeatedly administered to mice to understand the inhibitory effect of ginseng root extract on Parkinson's disease. In the Parkinson's disease model, changes in spontaneous momentum and rota-rod test were performed to evaluate ataxia symptoms. The expression of tyrosine hydroxylase enzyme in the striatum and black parts and the dopamine concentration of the striatum were measured to determine the dopaminergic system Was evaluated.

In the above animal model, the ginseng root extract improved the symptoms of ataxia and increased the expression of tyrosine hydroxylase enzyme and dopamine concentration in the striatum and black parts. In order to understand the inhibitory effect of ginseng root extract on Alzheimer's disease, the inventors of the present invention have found that in the case of administration of β-amyloid (Aβ) (1-42) [Aβ (1-42) vivo animal model was applied. In the above animal model, ginseng root extract inhibited cognitive memory impairment and dysfunction of the cholinergic system in hippocampal tissues. In conclusion, the present inventors have observed that ginseng roots extract significantly alleviates ataxia symptoms and significantly inhibits dopaminergic neuronal cytotoxicity in the MPTP-induced Parkinson's disease model, and that Aβ (1-42) Inhibited the decline of cognitive memory and significantly inhibited the cholinergic system dysfunction. These results suggest that the ginseng root extract is a strong candidate for the treatment of Parkinson 's disease and Alzheimer' s disease.

As a result of the study by the present inventors, ginseng roots extract inhibited dopaminergic neurotoxicity and dysfunction in black and striatum in an animal model of Parkinson's disease, thus improving ataxia symptoms in Parkinson's disease. In addition, the ginseng root extract exerted cognitive memory capacity in an animal model of Alzheimer 's disease and inhibited the functional deterioration of the cholinergic system of hippocampal tissues. This indicates that the pharmaceutical composition containing ginseng rosemary extract can be used as an agent for preventing or treating Parkinson's disease and Alzheimer's disease. In addition, since ginseng roots are commercialized only until now, ginseng roots are being discarded. Therefore, it is one of the important effects of the present invention that new demand can be created and abandoned resources can be utilized.

1 is a diagram showing an experimental schedule for evaluating the pharmacological efficacy of Ginseng Flos Extract in a Parkinson's disease model established by administration of MPTP.
Figure 2 shows the pharmacological effect of Ginseng Flos on the ataxic symptoms induced by administration of MPTP [reduction in time (A) and decrease in spontaneous momentum (B) in the rota-rod] Fig.
FIG. 3 is a graph showing the change in the concentration of dopamine (A) and its metabolites DOPAC (B), HVA (C) and dopamine turnover rate (D) in striatal tissues induced by administration of MPTP Flos. &Lt; / RTI >
4 is a graph showing the pharmacological effect of ginseng flos (ginseng flos) on reduction of expression of tyrosine hydroxylase in striatum (A) and black body (B) tissues induced by administration of MPTP.
FIG. 5 is a diagram showing an experimental schedule for evaluating the pharmacological efficacy of Ginseng Flos Extract in an Alzheimer's disease model established by administration of? -Amyloid (A?).
Fig. 6 shows the results of the evaluation of Morris water maze consisting of a reduced cognitive memory capacity (A), probe test (B) and working memory test (C) This figure shows the pharmacological effect of Korean ginseng roots extract (Ginsene Flos).
Figure 7 is a graph showing the pharmacological effect of ginseng floss extract on reduction of cognitive memory ability (evaluated by Y-maze test) due to the administration of Aβ (1-42) as a central dose.
FIG. 8 is a graph showing the pharmacological effect of ginseng flos extract on the reduction of cognitive memory ability (evaluated by the Novel object recognition test) caused by the administration of Aβ (1-42) as the central administration.
FIG. 9 is a graph showing the pharmacological effect of ginseng flos extract on reduction of cognitive memory ability (evaluated by Passive Avoidance test) due to administration of Aβ (1-42) as the central administration.
10 is a graph showing the pharmacological effect of ginseng floss extract on reduction of cognitive memory ability (evaluated by water finding test) due to the administration of Aβ (1-42) as the main ingredient.
FIG. 11 shows the pharmacokinetic profile of the ginseng flos extract for the reduction of acetylcholine level (A), acetylcholinesterase activity (B) and choline acetyltransferase activity (C) induced by the administration of Aβ (1-42) Fig.

Ginseng roots are the buds of ginseng blossoms that have been bloomed but have not yet bloomed. Ginseng roots are commonly used ginseng, such as ginseng (Panax ginseng CA Meyer), Panax quinquefolium, Panax notoginseng, Panax japonicum, Panax trifolium or Panax pseudoginseng ). &Lt; / RTI >

The ginseng roots extract can be obtained by extracting ginseng roots from dry or undried ginseng with buds and extracting with an extraction solvent. The solvent for the extraction of ginseng roots is selected from the group consisting of water, lower alcohols of C1 to C5 and mixtures thereof. For example, the ginseng roaster is washed with water to remove foreign matter and salt, dried, and then washed with water having a volume of about 5 to 50 times, preferably 10 to 30 times the weight of the ginseng roasting sample, and water such as methanol, ethanol, To a polar solvent of C5 lower alcohol or a mixed solvent thereof having a mixing ratio of about 1: 0.1 to 1:10, preferably 30 to 95% by weight of an aqueous ethanol solution at 50 to 95 DEG C for 1 to 7 days The extraction process is repeated 2 to 5 times. Alternatively, the extract may be extracted with water or a lower alcohol aqueous solution and re-extracted with butanol. The extract may then be concentrated under reduced pressure and / or lyophilized to obtain an extract of ginseng rosemary extract. In addition, the non-polar solvent-soluble extract of the present invention can be obtained by suspending the crude extract in distilled water, and then adding the non-polar solvent-soluble extract to a nonpolar solvent such as hexane, ethyl acetate or chloroform at a ratio of about 0.1 to 100 times, Followed by extraction with a nonpolar solvent and separation by adding a solvent for 1 to 10 times, preferably 2 to 5 times. Further, a normal fractionation process may be further performed.

Alternatively, an organic solvent such as n-butanol, hexane, or ethyl acetate may be added to the ginseng roots extract, preferably ginseng roots ethanol aqueous solution, obtained in the above process, in the order of a solvent having a low polarity and a solvent having a high polarity, , Ethyl acetate, and n-butanol in that order, followed by concentration under reduced pressure to obtain ginseng fermented hexane, ethyl acetate, and n-butanol fractions. The present invention provides a composition for preventing or treating Parkinson's disease and Alzheimer's disease, which comprises the ginseng root roots extract or the non-polar solvent-soluble extract obtained as described above as an active ingredient.

The composition for preventing or treating Parkinson's disease and Alzheimer's disease according to the present invention may comprise 0.1 to 99% by weight of the extract, based on the total weight of the composition.

The composition comprising the ginseng root extract of the present invention may contain carriers, excipients and diluents conventionally used in the art, and examples thereof include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, But are not limited to, lactose, mannitol, aceitol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, Stearate, and mineral oil, and the like.

In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

In addition, the composition can increase the solubility and gastrointestinal absorption of substances commonly used in pharmaceuticals, for example, ginseng root extract, and can be dispersed and emulsified with water at the time of oral administration so as not to adversely affect drug efficacy, Additives such as fatty acids or fatty alcohols that can be widely used for increasing bioavailability, saccharides such as saccharides such as white sugar, malt, black sugar, gelatin, sugar and starch syrup, lubricants such as magnesium stearate, talc, microcrystalline cellulose, Antioxidants, flavors, preservatives, fragrances, sweeteners, pigments, pH adjusting agents and viscosity control agents which prevent oxidation of the formulation, oxidants such as calcium monohydrogen phosphate, starch, mannitol, In terms of the amount usually used.

Examples of the fatty acid or fatty acid alcohol that can be used in the composition of the present invention include citric acid, oleic acid, stearyl alcohol, myristic acid, linoleic acid or lauric acid lauric acid, capric acid, caprylic acid, caproic acid, and the like, but are not limited thereto.

Antioxidants that may be used in the compositions of the present invention include but are not limited to butylated hydroxytoluene, sodium bisulfite, alpha-tocopherol, ascorbic acid, but are not limited to, β-carotin, tocopherol acetate, fumaric acid, nalic acid, butylated hydroxyanisole, propyl galate and sodium ascorbate sodium ascorbate, and the like may be used, but the present invention is not limited thereto.

Flavoring agents that can be used in the composition of the present invention include, but are not limited to, a mixed fruit, an apple, a strawberry, a cherry, a pepper, a vanilla, a yogurt or a drink.

Preservatives that can be used in the composition of the present invention include, but are not limited to, benzoic acid, sodium benzoate, ethylparaben, methylparaben or propylparaben.

Examples of the fragrance that can be used in the composition of the present invention include, but are not limited to, peppermint leaves, peppermint oil, orange oil, clove oil, cinnamon oil, strawberry essence and other common fruit or vegetable essences.

The sweetening agent that can be used in the composition of the present invention may include, but is not limited to, black sugar, glucose, fructose, aspartame, stevioside, sorbitol, mannitol, oligosaccharide,

The coloring matters which can be used in the composition of the present invention include green 3, red 2, red 3, blue 1, blue 2, yellow 4, yellow 5, water-soluble mannitol, caramel, titanium oxide, But is not limited thereto.

The pH adjusting agent which can be used in the composition of the present invention may be sodium carbonate, sodium hydroxide, potassium hydroxide, triethanolamine or monoethanolamine, but is not limited thereto.

The viscosity modifiers that can be used in the composition of the present invention include hydroxypropylcellulose (HPC), hydroxypropylmethyl cellulose (HPMC), hydroxyethyl cellulose, ethyl cellullose, methyl But are not limited to, cellulose, carboxymethyl cellulose, acacia, bentonite, alginic acid, propylene glycol alginate, polyvinyl pyrolidone, poly But are not limited to, polyvinyl alcohol, carbopol, polycarbopil, tragacanth or xanthan gum.

The present invention provides an oral preparation comprising the pharmaceutical composition containing the ginseng root extract. The oral preparation is an oral liquid containing tablets, pills, powders, hard capsules, gelatin-hard hard capsules, soft capsules, caramel or jelly type quinquer and an aqueous solution.

Meanwhile, the present invention can be manufactured by a pharmaceutical composition for parenteral administration containing ginseng root extract, and examples of the preparation for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, Dry preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. The base of suppositories may be witepsol, macrogol, tween, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention may be administered at a daily dose of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg, and may be administered once or several times a day.

The present invention provides a health functional food comprising the above extract and a pharmaceutically acceptable food-aid additive exhibiting a preventive effect of Parkinson's disease and Alzheimer's disease.

Examples of health functional foods to which ginseng roots extract can be added include various general foods, beverages, gums, tea, and vitamin complexes.

In addition, the ginseng root extract may be added to foods or beverages for the purpose of preventing Parkinson's disease and Alzheimer's disease. At this time, the amount of the extract in the food or beverage may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 g have. The health functional beverage composition of the present invention has no particular limitation on the ingredients other than those containing the extract as an essential ingredient in the indicated ratios and may contain additional ingredients such as various flavors or natural carbohydrates such as ordinary beverages . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As other flavors other than those mentioned above, natural flavors such as tau martin, stevia extract such as rebaudioside A, glycyrrhizin and the like; And synthetic flavors such as saccharin, aspartame and the like can be advantageously used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the composition of the present invention. In addition to the above, the extract of the present invention can be applied to various kinds of nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, coloring agents and heavy stabilizers (cheese, chocolate etc.), pectic acid and its salts, alginic acid and its salts, Colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the extracts of the present invention may contain flesh for the production of natural fruit juice, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. At this time, although the proportion of the additive is not critical, it is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

The present inventors examined the preventive or therapeutic effect and the mechanism of action of Parkinson's disease in ginseng root extract. 1-methyl-4-phenyl- tetrahydropyridine in mice as an animal model for verifying the pharmacological effects by repeated administration in the (MPTP) vivo Animal models were applied. For each animal model, changes in spontaneous momentum and rota-rod test were performed to evaluate ataxia symptoms. The expression of tyrosine hydroxylase enzyme in the striatum and black parts and the dopamine concentration of the striatum were measured, System degradation and damage were evaluated. In addition, the present inventors examined the preventive or therapeutic effect and the mechanism of action of Alzheimer's disease of ginseng root extract. Examples of animal models for verifying the pharmacological effects of administration of Aβ in mice in the central vivo Animal models were applied and cognitive memory capacity was assessed by Morris water maze, Y-maze, Novel object recognition test, Passive avoidance test, Water finding test, and then the values of acetylcholine in hippocampal tissue and acetylcholine ester Pharmacological effect was measured by measuring the activity of the choline acetyltransferase and the activity of the choline acetyltransferase. One-way ANOVA or one-way ANOVA for repeated measures were used for statistical significance. Fisher's PLSD or Bonferroni's evaluation was performed as post-test. Specific examples and test examples are as follows.

Example

Example 1_ Preparation of ginseng root extract

The ginseng roaster was washed with water to remove foreign matter, dried and pulverized. To the extraction vessel, 25 g of ginseng roots and 500 ml of a 70% by weight aqueous ethanol solution were added, and the mixture was heated and extracted three times at 70 ° C for 3 hours while refluxing. The filtrate was concentrated under reduced pressure on a water bath at 40 캜 and lyophilized to obtain 5.3 g of crude extract of ginseng fermented ethanol. The following experiment was conducted with this ginseng roasted ethanol extract.

Example 2 < tb > ______________________________________ < tb > < tb > <

Repeated doses of MPTP were administered to C57BL / 6 mice at 12 weeks of age. C57BL / 6 mice were purchased from Nara Biotech (Pyeongtaek, Gyeonggi-do, Korea). Drug administration and behavioral assessment were performed as shown in Fig. In summary, MPTP (Sigma, St. Louis, Mo., USA) was dissolved in physiological saline and subcutaneously injected once for 7 days at a dose of 25 mg / kg. The ginseng roots extract (500 mg / kg) was orally administered for 5 days from the start of MPTP to the last administration day of MPTP for 12 days twice every 12 hours. Behavioral assessment was performed 1 day after the last MPTP administration, and 30 minutes after the behavioral evaluation, mice were sacrificed and stroma and black tissue were taken. As a reference drug, ropinirole was orally administered at a dose of 10 mg / kg.

Example 3_ Locomotor activity

Spontaneous momentum was measured for 30 minutes using an automatic video-tracking system (Noldus Information Technology, Wagenin, The Netherlands). The IBM computer operated four test boxes (40x40x30 cm high) at the same time. The spontaneous momentum of the mice was studied individually in each test box and they were adapted to the device for 5 minutes before starting the experiment. The distance the animals moved in centimeters for 30 minutes. Data collection and analysis took place between 9 am and 5 pm (Curr. Neuropharmacol., 9, 118-121).

Example 4 - Rota-rod test [

The Rota-Rod device (Ugo Basile Model 7650, Comerio, VA, Italy) consisted of a base platform and a non-slip swivel rod. This bar was 15 cm above the floor. The rod is 30 cm long and is divided into five equal compartments by six opaque discs (so that the objects are not confused by each other).

To adapt to the device, the mice were trained in the device for 2 minutes at a speed of 4 rpm, and evaluation was performed after 30 minutes. The evaluation started at a speed of 4 rpm and applied an acceleration paradigm to a maximum speed of 40 rpm, and the subsequent rotational speed was kept constant at 40 rpm for a maximum of 300 seconds. The period during which the animals were kept on a rotating drum was measured and the maximum cut-off time was 300 seconds (Curr. Neuropharmacol., 9, 118-121).

Example 5 Measurement of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homobanylic acid (HVA)

After weighing the striatal tissues, they were sonicated in 10% perchloric acid containing 10 ng / mg of dihydroxybenzylamine, an internal standard, and centrifuged at 20,000 x g for 10 minutes. Levels of DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homobanylic acid (HVA) in brain tissue extracts were analyzed by HPLC and detected by electrochemical detector. The fixed bed column used was a 3 μm C18 column and the mobile phase consisted of 26 mL of acetonitrile, 21 mL of tetrahydrofuran, and 960 mL of 0.15 M monochloroacetic acid (pH 3.0) containing 50 mg / L EDTA and 200 mg / L sodium octylsulphate lost. The peak area of the tissue sample and the peak area of the reference material were compared to calculate the amounts of DA, DOPAC and HVA (Neurochem. Int., 56, 229-244).

Example 6_ Immunohistochemical staining

Expression of tyrosine hydroxylase in rat striatum and black tissue was evaluated by immunohistochemical staining. The sections were perfused with 0.2% Triton X-100 for 15 minutes before being subjected to 1-day overnight incubation with a tyrosine hydroxylase antibody (1: 1000, Chemicon International) after cutting brain tissue fixed with 4% p-formaldehyde Exposed and reacted with 4% normal goat serum. After 1 night, the secondary antibody was reacted for 1 hour and immunostained with 3,3-diaminobenzidine as chromogen. Each step was washed with PBS (pH 7.4) (see Neurochem. Int., 56, 229-244).

Example 7 Drug Administration and Experimental Schedule in Alzheimer's Disease Model Consisting of Central Administration of Aβ

Decreased cognitive memory by Aβ was assessed in ICR mice at 12 weeks of age. ICR mice were purchased from Nara Biotech (Pyeongtaek, Gyeonggi-do, Korea). The evaluation of drug administration and cognitive memory performance was carried out as shown in Fig. In summary, ginseng roots extract (500 mg / kg) was orally administered at intervals of 12 hours twice a day for 5 days. Toxic Aβ (1-42) (American Peptide Company, Sunnyvale, CA, USA) and the non-toxic Aβ (42-1) (American Peptide Company, Sunnyvale, CA, USA) . Cognitive memory performance was assessed 3 days after administration of Aβ. During the cognitive memory performance evaluation, the drug was administered after the behavioral and cognitive function assessments to prevent the drugs from directly affecting the behavior. After the evaluation of the cognitive memory ability, the animals were sacrificed after 30 minutes and the hippocampus tissue was taken.

Example 8_ Morris water maze test (Morris water maze test)

The Morris Underwater Maze test was performed by Behav. Brain Res. (155, 185-196) (Behav. Brain Res., 155, 185-196). The water tank used in the underwater maze test was a cylindrical water tank with a diameter of 97 cm and a height of 60 cm. The water tank was filled with 23 ± 2 ° C water diluted with milk powder during the test period. In the water tank, a transparent platform was installed 2cm below the surface of the water, and four markers were placed on the outside of the water tank. The motion trajectory of the mouse was analyzed using a video tracking system (EthoVision, Noldus, The Netherlands).

1) Reference memory evaluation ( Reference memory test )

During each trial, the mice were placed into the vats in the direction of the platform where they could not see the platform at randomly selected five starting points. In each trial, the mouse records the escape latency after being placed in the aquarium. When the mouse finds the platform, it will stay on the platform for 10 seconds and move to the home cage. If the mouse can not find the platform within 60 seconds, the escape latency is 60 seconds. Each trial was conducted four times a day for four days from three days after administration of Aβ.

2) Confirmatory evaluation ( Probe test )

A probe test was performed 7 days after administration of Aβ. After removing the platform, it was done by recording the number of times the platform had passed, allowing the mouse to swim in the tank for 60 seconds.

3) working memory assessment ( Working memory test )

Working memory testing proceeded from 8 to 10 days after Aβ administration. Similar to the reference memory evaluation, we measured the escape latency by changing the platform position daily. We used five times a day trial and escape latency for the second through fifth trials.

Example 9_Y-maze test (Y-maze test)

The Y-shaped labyrinth experiment is described in J. Alzheimers Dis. (31, 207-223) (Alzheimers Dis., 31, 207-223). The Y-maze consists of a Y-shaped arm and a black plastic box with a length of 25 cm and a height of 14 cm and a width of 5 cm. Place the mouse at the end of one arm and move the arm freely for 8 minutes. The video camera counts the number of times the mouse enters each arm. The evaluation index of the alternation behavior is compared with the control group by obtaining one point when all three arms are included. The alternation score of the mouse is converted into 100% by dividing the score obtained by the actual mouse by the possible alternation score.

Example 10_ Novel object recognition test [

The new object recognition experiment is Learn. Mem. (14, 117-125) (Learn: Mem., 14, 117-125). On the first day, the mouse is placed in a box of 40 cm × 40 cm × 30 cm and adjusted to move freely for 10 minutes. On the second day, place two objects in the box and record the reaction time for each object. After 24 hours, replace one of the two objects with a new object and record the reaction time for the new object.

Example 11_ Water finding test

The Drinking Water Finder was evaluated by Mol. Pharmacol. (71, 1598-1609) (Mol. Pharmacol., 71, 1598-1609). This test is a method of measuring attention and delayed learning ability to determine whether the position of the water tap in the training day can be remembered even after thirsty after 24 hours of water saving. On the day of training, the time to find the time and water bottle faucet for up to 5 minutes until the alcove enters the alcove with a 10 cm × 10 cm × 10 cm alcove with a faucet with a faucet tip next to the box of 50 cm × 30 cm × 15 cm . At this time, the water tap is positioned close to the wall. On the assessment day, change to a water bottle faucet filled with water and place it in the center of the alcove. Records are recorded in the same manner as training days.

Example 12_ Passive avoidance test (passive avoidance test)

The passive avoidance experiment was performed by the Behav. Brain Res. (See, Behav. Brain Res., 155, 185-196) by the Gemini Avoidance System (San Diego Instrument, San Diego, Calif. The system consists of two spaces with a shock generator on the floor and a guillotine door between them. In the Acquisition trial, the mice are placed in a start chamber and adapted for 20 seconds. After 20 seconds, the space filled with the mice is lit and the guilin door is opened in the middle. As soon as the mouse enters the opposite room without lighting, the guillotine door is closed and an electric shock (0.3 mA, 3 s, 1 time) is given do. Twenty-four hours later, in the retention trial, place the mice back in the starter chamber, turn on the lights, and record the step-through latency (up to 300 seconds).

Example 13 Measurement of activity of acetylcholine (ACh), acetylcholinesterase (AChE) and choline acetyltransferase (ChAT)

ACh levels and AChE activity in the hippocampus and cortical tissues were measured using the AmplexRed Acetylcholine / Acetylcholinesterase Assay Kit (Molecular Probes, Inc., Eugene, OR) according to the manufacturer's protocol. The activity of ChAT was determined as follows (J. Neurosci. Res. 87, 3658-3670). 25 μl of a solution of 0.5 M sodium phosphate buffer (pH 7.2), acetyl-CoA (6.2 mM), choline chloride (1 M), neostigmine sulfate (0.76 mM), NaCl (3 M) and EDTA (1.1 mM) , The volume was adjusted to 400 μl, the solution was pre-incubated at 37 ° C for 5 minutes, and then 100 μl of the sample was added and incubated at 37 ° C for 20 minutes. After the sample was boiled for 2 minutes to terminate the reaction, 1 ml of distilled water was added. The denatured protein was removed by centrifugation, and 1 ml of supernatant was added to a test tube containing 30 μl of 4,4'-dithiopyridine (10 mM). Absorbance was measured at 324 nm after 15 minutes. ChAT activity was expressed as coenzyme nmol / hrg protein. Protein concentrations were measured using a BCA protein assay reagent (Pierce, Rockford, Ill., USA) according to the manufacturer's protocol.

Experimental Example 1 Pharmacological effect of ginseng root extract in Parkinson's disease model induced by MPTP

One) MPTP Ataxia due to

MPTP administration in mice significantly reduced the time to maintain balance on the rotating rod in the rotor - rod evaluation ( P <0.01) and significantly decreased spontaneous momentum ( P <0.05). The administration of ginseng roots extract in mice significantly improved these ataxia symptoms by MPTP [Rota-rod test: P <0.01, ginseng roots extract (500 mg / kg) + MPTP. Saline + MPTP; locomotor activity: P <0.05, ginseng root extract (500 mg / kg) + MPTP vs. Saline + MPTP; ]. The effect of ginseng roots extract was similar to or superior to that of the reference drug ropinirole (10 mg / kg, po). These results indicate that ginseng roots extract has significant pharmacological effects on ataxia induced by MPTP (Fig. 2).

2) MPTP Of dopamine levels and dopamine metabolite levels

MPTP administration in mice significantly reduced the levels of dopamine and its metabolites (dopamine, DOPAC, HVA, P <0.01) and dopamine turnover significantly ( P <0.01). The administration of ginseng roots extract in mice significantly inhibited the dopamine and dopamine turnover rate ( P <0.01, ginseng roots extract (500 mg / kg) + MPTP vs. dopamine turnover) by MPTP. Saline + MPTP]. On the other hand, the reference drug ropinirole (10 mg / kg, po) showed no significant pharmacological effect. The above results indicate that the ginseng root extract has significant pharmacological effects on dopamine deficiency and dopamine turnover increase in striatum induced by MPTP (FIG. 3).

3) MPTP because of tyrosine hydroxylase Reduction of expression

Immunohistochemical staining showed that administration of MPTP in mice significantly reduced tyrosine hydroxylase expression in striatum and melanoma ( P <0.01). The administration of ginseng roots extract significantly inhibited the decrease of tyrosine hydroxylase expression by MPTP. [Striatal and Brucellosis: P <0.01, Extract of ginseng root (500 mg / kg) + MPTP. Saline + MPTP]. The effect of ginseng roots extract was similar to or superior to that of the reference drug ropinirole (10 mg / kg, po). These results indicate that the ginseng root extract has significant pharmacological effects on the reduction of expression of tyrosine hydroxylase on striatum induced by MPTP and on black body (FIG. 4).

Experimental Example 2 Pharmacological effects of ginseng root extract on the reduction of cognitive memory capacity and cholinergic dysfunction induced by administration of Aβ (1-42)

1) Morris Underwater Maze Experiment Morris water maze )

The administration of Aβ (1-42) in mice significantly delayed the escape latency in the reference memory test of Morris water maze ( P <0.01), indicating a decrease in spatial reference memory . Administration of ginseng roots extract in mice significantly reduced the prolongation of escape latency by Aβ (1-42) (500 mg / kg, P <0.01) (Fig. 6A). The same result was obtained in the probe test again confirming the spatial reference memory (Fig. 6B).

The administration of Aβ (1-42) in mice significantly extended escape latency in the working memory test of Morris water maze ( P <0.01), indicating a decrease in spatial working memory. Administration of ginseng roots extract in mice significantly reduced the prolongation of escape latency by Aβ (1-42) (500 mg / kg, P <0.05) (Fig. 6C). The effect of ginseng roots extract was similar to that of reference extract (40 mg / kg, po). These results indicate that ginseng roots extract has significant pharmacological effects on spatial reference memory and spatial working memory reduction induced by Aβ (1-42) (Fig. 6).

2) Y-shaped labyrinth experiment (Y- maze test )

Administration of Aβ (1-42) in mice significantly reduced the alternation behavior in the Y-shaped labyrinth ( P <0.01), suggesting a decrease in spatial working memory . Administration of ginseng roots extract in mice significantly inhibited the reduction of space replacement behavior by Aβ (1-42) (500 mg / kg, P <0.01). The effect of ginseng roots extract was similar to that of reference extract (40 mg / kg, po). These results indicate that the ginseng root extract has significant pharmacological effects on the reduction of spatial working memory induced by A? (1-42) (Fig. 7).

3) New object recognition experiment Novel object recognition test )

The administration of Aβ (1-42) in mice significantly reduced the search behaviors for new objects in new object recognition experiments ( P <0.01), which means a decrease in visual recognition memory. Administration of ginseng roots extract in mice significantly inhibited the reduction of Aβ (1-42) search behavior on new objects (500 mg / kg, P <0.01). The effect of ginseng roots extract was similar to that of reference extract (40 mg / kg, po). These results indicate that the ginseng root extract has significant pharmacological effects on the retinal or memory retention induced by A [beta] (1-42) (Fig. 8).

4) Passive avoidance test method ( Passive avoidance test )

The administration of Aβ (1-42) in mice significantly reduced the step-through latency to the dark room in the passive avoidance experiment ( P <0.01), which implies a decrease in associative learning. Administration of ginseng roots extract in mice significantly inhibited the reduction of step-through lanticity by Aβ (1-42) (500 mg / kg, P <0.01). The effect of ginseng roots extract showed better pharmacological effect than that of reference extract (40 mg / kg, po). These results indicate that the ginseng root extract has significant pharmacological effects on the reduction of the associative learning ability induced by A [beta] (1-42) (Fig. 9).

5) Drinking water finding evaluation ( Water finding test )

The administration of Aβ (1-42) in mice significantly increased the finding latency of finding a water tap in the drinking water finding assessment ( P <0.01), indicating a decrease in latent learning. Administration of ginseng roots extract in mice significantly inhibited the prolongation of finding latency by Aβ (1-42) (500 mg / kg, P <0.01). The effect of ginseng roots extract was similar to that of reference extract (40 mg / kg, po). These results indicate that the ginseng root extract has significant pharmacological effects on the reduction of delayed learning ability induced by A? (1-42) (Fig. 10).

6) In the sea horse Acetylcholine concentration and Acetylcholinesterase and Choline Acetyl trans The measurement results of the activity of perilla

The administration of Aβ (1-42) in mice significantly reduced the acetylcholine concentration of hippocampus tissue ( P <0.01) and the activity of acetylcholine synthetase, choline acetyltransferase ( P <0.05) The activity of acetylcholinesterase, acetylcholinesterase, was significantly increased ( P <0.01), indicating that the cholinergic function of the hippocampus was impaired. Administration of ginseng roots extract in mice significantly inhibited this cholinergic change by Aβ (1-42). [acetylcholine: P <0.01, ginseng root extract (500 mg / kg) + Aβ (1-42) Saline + A? (1-42); acetylcholinesterase and choline acetyltransferase: P <0.05, ginseng root extract (500 mg / kg) + Aβ (1-42) Saline + A? (1-42)]. The effect of ginseng roots extract was similar to that of reference extract (40 mg / kg, po). The above results indicate that the extract of P. ginseng has significant pharmacological effects on the cholinergic dysfunction of the hippocampus by Aβ (1-42) (FIG. 11).

The present invention provides a pharmaceutical composition for preventing or treating Parkinson's disease and Alzheimer's disease containing ginseng root extract as an active ingredient. The present invention has therapeutic effects on Parkinson's disease and Alzheimer's disease, and can be manufactured into oral administration and parenteral pharmaceutical preparation through appropriate formulation. Particularly, since the ginseng roots have been discarded so far, it is one of the important industrial applicability of the present invention that it is possible to utilize abandoned resources.

Claims

A pharmaceutical composition for preventing or treating Parkinson's disease or Alzheimer's disease comprising ginseng root extract as an active ingredient.

[Claim 2] The ginseng root extract of claim 1, wherein the ginseng root extract is extracted and concentrated with a solvent selected from the group consisting of water, C1 to C5 lower alcohols or a mixture thereof, to prevent or treat Parkinson's disease or Alzheimer's disease A pharmaceutical composition.

The ginseng root extract according to claim 1, wherein the ginseng root extract is obtained by extracting and concentrating the ginseng root with a solvent selected from the group consisting of water, C1 to C5 lower alcohols or a mixture thereof, and concentrating the crude extract to obtain a final extract of butanol &Lt; / RTI > or a pharmaceutically acceptable salt thereof, for the prophylaxis or treatment of Parkinson ' s disease or Alzheimer ' s disease.

The ginseng root extract according to claim 1, wherein the ginseng root extract is extracted and concentrated with a solvent selected from the group consisting of water, C1 to C5 lower alcohols or a mixture thereof, to obtain a crude extract, which is suspended in water, And butanol. The pharmaceutical composition for preventing or treating Parkinson ' s disease or Alzheimer ' s disease.

The pharmaceutical composition for preventing or treating Parkinson's disease or Alzheimer's disease according to claim 2, wherein the C1 to C5 lower alcohol is ethanol.