KR100448667B1 - Composition for preventing or treating involutional brain diseases comprising a hydrolysate of ginsenosides - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of degenerative brain disease, comprising the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient, the composition of the present invention is dementia, Parkinson's disease, stroke, Huntington's disease, etc. Has excellent prophylactic and therapeutic effects against degenerative brain diseases of:

Where

R ¹ is H or Glu-Glu,

R ² is H or OH,

R ³ is H, glucose, Ara (p) -Glu or Ara (f) -Glu.

Description

COMPOSITION FOR PREVENTING OR TREATING INVOLUTIONAL BRAIN DISEASES COMPRISING A HYDROLYSATE OF GINSENOSIDES}

In modern society, due to the rapid increase of the aged population, the development of senile dementia related to aging is a serious social problem. Despite the high incidence of this disease, there is currently no clear treatment or preventive agent, causing huge socioeconomic losses.

Alzheimer's disease is the leading disease of senile dementia, and one of the biggest reasons why the development of a cure for it so far is that the cause of the disease is not known. However, recent clinical, genetic, molecular and cytobiological studies have shown that the accumulation of beta amyloid in the brain and its neurotoxicity is a very important cause of the onset (Selkoe, Annu. Rev. Neurosci. , 17: 489-517 (1994). Therefore, in order to develop a treatment for senile dementia, it is very urgent to develop a substance having a beta amyloid inhibitory effect or a toxic inhibitory effect and a low side effect.

Current treatments for senile dementia that are approved by the US FDA include tacrine (Tacrine (Cognex)) and Donepezil (Aricept). These are all substances that increase the activity of acetylcholine, a type of neurotransmitter, which only slightly slows the progression of dementia in about 20-40% of patients.

With the disappointing effects of acetylcholine activators, drugs are being developed to activate the action of glutamate, the most common neurotransmitter in the brain. Ampacaine (Ampakine) is a drug that activates the action of glutamate, the most common neurotransmitter in the brain, and has undergone animal studies and clinical trials in Europe. Currently, development companies Cortex Pharmaceuticals and NIH Jointly in clinical trials in the United States. Successful development is expected to be more effective than acetylcholine activators.

Free radicals are known to promote the development of Alzheimer's disease due to their toxicity. Studies in the Czech Republic have also shown that the antioxidant selegiline enhances memory in Alzheimer's patients. Therefore, antioxidants such as vitamins A, C, E, selenium, and beta-carotene are thought to inhibit the development of Alzheimer's disease.

Parkinson's disease is a central nervous system degenerative disease that occurs in older people. It can cause symptoms such as limb tremor, muscle stiffness and difficulty in starting exercise, and can be accompanied by mental depression. In the brain of patients with Parkinson's disease, dopamine content in the target region, the neostriatum, is significantly reduced by the degeneration and death of dopaminergic neurons in the substantia nigra (Fahn S., Parkinsons disease in: Diseases of the nervous system , (ED) by A. Asbury, G Mckhann, pp. 1217-1238, Saunders, 1986). In other words, the cause of Parkinson's disease is known to be directly caused by the death of melanocyte dopaminergic neurons.

For the treatment of Parkinson's disease, the best way to prevent and regenerate the death of mesenchymal melanoma dopaminergic neurons is the best treatment. It is true. One reason for this is that the exact cause of neuronal death is not known. However, the accumulated research results have revealed the causes of neuronal cell death associated with Parkinson's disease. Proposed oxidative stress, abnormal energy metabolism, mutations in mitochondrial genes, and excitatory amino acid toxicity. Amino acid toxicity) and calcium concentration change are known to cause cell death. There is much evidence that oxidative stress is a major cause of dopaminergic neuronal death associated with Parkinson's disease (Fahn, S. and Cohen, G., Ann. Neurol., 32 (6): 804-812 (1992) . Foley P. and Riederer P., J. Neurol., 247 [Sppl. 2] II / 82-II / 94 (2000)). Therefore, in order to develop an effective treatment for Parkinson's disease, it is necessary to develop a substance that penetrates the brain well while preventing such a process of inducing apoptosis around oxidative stress. In addition, there is a need for the development of substances with the effect of regenerating the nervous system already undergoing degeneration.

Stroke is the most common brain disease and is caused by the death of nerve cells following a sudden blockage of blood vessels or the loss of oxygen and energy sources due to bleeding. The main causes of neuronal death in stroke are: 1) the excitatory neurotransmitter glutamate through ionic receptors activated by NMDA, AMPA, kainic acid, etc., 2) oxidative toxicity ( Oxidative stress or free radical toxicity), 3) apoptosis (dead or precocious). After a stroke, apoptosis progresses rapidly through this series of processes, and it is necessary to develop a material that inhibits the progression and inhibits cell death. The material must penetrate the blood-brain barrier well and easily penetrate the brain. In addition, there is a need for development of substances that promote synapse formation between surviving neurons, that is, regenerate the nervous system. However, no effective stroke treatment has been developed to date.

Huntington's disease is one of the most common forms of hereditary nervous system disease that is commonly seen as a neurodegenerative disorder. Symptoms are characterized by 1) mental disorders such as personality change and depression, and 2) abnormalities in exercise. Incidence rates generally range from 4-5 per million, with ages generally in their 30s and 40s, but often begin before age 15 and even in childhood (Adams RD, Victor M. and Ropper AH, Principles of Neurology, 6th ed). Singapore: McGraw-Hill, 1997).

Huntington's disease is caused by selective loss of the basal ganglia of the brain and neurons of the cerebral cortex. Significant atrophy is found in the head of the caudate nucleus and putamen, with moderate cerebral atrophy in the frontal and temporal lobes. In 1993, the causative agent of Huntington's disease was cloned and its gene product is a protein called huntingtin. The mechanism by which neuronal cell death occurs when mutations in huntingtin occur is not known, but active research is ongoing. To date, it is known that an enzyme called caspase plays an important role and that oxidative stress plays an important role in causing cell death (Gutekunst CA, Norflus F., and Hersch SM, Curr. Opin. Neurol., 13 : 445-450 (2000)).

There is currently no clear cure for Huntington's disease. Halperidol, a dopamine antagonist, is most widely used to suppress motor disorders in patients. Other dopamine antagonists such as reserpine, clozapine, and tetrabanazine have the effect of inhibiting Huntington's disease, but have a number of side effects such as drowsiness, dyspnea, and full-scale dyskinesia (Adams et al., Supra). See literature). These drugs, including haloperidol, can alleviate symptoms such as abnormal behavior and easily changing emotions, but do not prevent the progression of the disease.

Oxidative stress caused by free radicals has been reported as a major cause of apoptosis in each tissue in the body, and has been suggested as one of the cycles of apoptosis in neurological diseases (Halliwell, B. and Gutteridge, JM, Mol. Aspects.Med ., 8 (2): 89-193 (1985); Siesjo, BK, Agardh, CD, and Bengtsson, F., Cerebrovasc. Brain Metab. Rev., 1 (3): 165-211 (1989) Schapira, AH, Curr. Opin. Neurol., 9 (4): 260-264 (1996)). Increased production of free radicals in the cell leads to the destruction of membrane lipids by lipid peroxidation, OH ^. DNA damage caused by DNA damage, double bond structure, or protein degeneration due to oxidative reaction including -SH may be the result of neuronal cell death. Evidence of the involvement of free radicals in neuronal cell death in neurological diseases includes increased production of reactive oxygen species after ischemia and inhibition of ischemic neuronal cell death by antioxidants (Flamm, ES et al ., Stroke , 9 (5): 445-447 (1978); Kogure, K. et al., Prog.Brain Res., 63 : 237-259 (1985); Chan, PH, J. Neurotrauma. , 9 Suppl 2: S417-23 (1992)), increased Fe ²⁺ , decreased glutathione, decreased glutathione peroxidase, decreased catalase, and free radical formation by dopamine oxidation in brain blackness of patients with Parkinson's disease , E. et al ., J. Neural Transm., 74 : 199-205 (1988); Fahn & Cohen, supra), increase in Fe ²⁺ in the striatum of Huntington's disease (Dexter, DT et al., Ann. Neurol , 32 Suppl: S94-100 (1992)), the production of free radicals by beta amyloid in Alzheimer's disease (Schubert, D., et al. , Proc. Natl. Acad. Sci. USA, 92 (6): 1989 -1993 (1995); Richardson, JS, Zhou, Y., and Kumar, U., Ann.NY Acad.Sci ., 777 : 362-367 (1996)), Cu / Zn Superoxide Dismutase (SOD-1) in Familial ALS Point mutations in genes (Rosen, DR et al ., Nature, 362 (6415): 59-62 (1993)). As such, various experimental results indicate that oxidative stress acts as a major cause of various degenerative neurological diseases, and it is very important to suppress neuronal cell death caused by oxidative stress in developing therapeutic agents for neurodegenerative diseases.

The present inventors conducted research to develop a therapeutic agent for degenerative brain diseases. As a result, recently, ginsenoside Rb1 and Rg1 among the components of Panax ginseng have a strong effect of protecting nerve cells from the toxicity of beta amyloid. It has been found and has applied for a patent (Korean Patent Application No. 99-28866). Ginseng is a herbal medicine and has been used for thousands of years, so it is expected to have no significant side effects or toxicity. In addition, since it protects nerve cells from the toxicity of beta amyloid, it is highly likely that a fundamental therapeutic agent for preventing the development of senile dementia is developed.

Drugs that treat dementia, a brain disease, must cross the blood brain barrier (BBB), a closed system, making it difficult to think that the entire molecule, such as ginsenosides Rb1 and Rg1, has a molecular weight of 1,000. Therefore, the present inventors believe that small molecules that pass through the BBB to block the beta amyloid toxicity will actually be effective, and thus compounds Y and K, ginsenoside Mc, protopanacanadiol, which are hydrolysis products of ginsenosides. (Protopanaxadiol; PPD) and Protopanaxatriol (PPT) and ginsenoside Rc, whose efficacy is not yet known, have been tested for their effects on beta amyloid toxicity and production, and on neuroprotective effects. It has been found that the present invention is completed.

It is an object of the present invention to provide a pharmaceutical composition containing an active substance useful for the prevention and treatment of degenerative brain diseases.

1 is a result of the MTT test confirming the ginseng ginsenoside Rc and Mc, the toxicity of the compound Y, PPD, PPT of the hydrolysis products of ginsenosides by the beta amyloid,

2 is a MTT test result confirming the effect of preventing beta amyloid toxicity according to the concentration of PPT,

Figure 3 is a test result confirming the beta amyloid toxic effect by the antioxidant action of the compound Y, PPD, PPT hydrolyzate of ginsenosides,

4 is a test result confirming the effect of ginsenosides Rc and Mc, compounds of the hydrolysis products of ginsenosides Y, PPD, PPT on apoptosis induced by staurosporin,

5 is a Western blot result confirming that PPD and PPT increase the secretion amount of α-secretase that degrades beta amyloid,

FIG. 6 is a water maze test result confirming that administration of ginsenosides Rc and Mc and compound Y, a hydrolysis product of ginsenosides, improves spatial perception ability,

7a and 7b are Western blot results confirming the synaptic increase effect of ginsenosides Rc and Mc, compound hydrolysates of ginsenosides Y, PPD, PPT,

8A and 8B show separation peaks using LC-mass of ginsenosides Rg1 and Rb1 in brain tissue samples of rats administered ginsenosides Rg1 and Rb1, respectively.

9a and 9b show quantitative curves using LC-mass of ginsenosides Rg1 and Rb1, respectively,

10A and 10B show separation peaks using LC-mass of PPT and PPD in brain tissue samples of rats administered ginsenosides Rg1 and Rb1, respectively.

11a and 11b show the time course of concentration of ginsenosides Rg1 and Rb1 in plasma and brain tissue samples of rats administered ginsenosides Rg1 and Rb1, respectively.

12A and 12B show trends in concentrations of PPT and PPD in plasma and brain tissue samples of rats administered ginsenosides Rg1 and Rb1, respectively.

In accordance with the above object, the present invention provides a composition for the prevention or treatment of degenerative brain disease, comprising as an active ingredient a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:

Formula 1

Where

R ¹ is H or Glu-Glu-,

R ² is H or OH,

R ³ is H, glucose, Ara (p) -Glu- or Ara (f) -Glu-.

In the present invention, "arabino glucosyl (Ara (p) -Glu)" is a combination of 1 molecule of arabinose and 1 molecule of glucose in the form of pyranose, "Ara (f) -Glu" Means that one molecule of arabinose and one molecule of glucose in the form of furanose are combined.

Preferred compounds of formula 1 in the present invention are those described in Table 1 below.

compound R ¹ R ² R ³ PPD H H H PPT H OH H Compound Y H H Ara (p) -Glc- Compound K H H Glc Ginsenoside Mc H H Ara (f) -Glc- Ginsenoside Rc Glc-Glc- H Ara (f) -Glc-

Particularly preferred compounds in the present invention are protoparnaxadiol (PPD) and protoparnaxatriol (PPT).

The compounds of Formula 1 may be, for example, inorganic metal salts such as alkali metal salts (eg, sodium salts, potassium salts), alkaline earth metal salts (magnesium salts, calcium salts, etc.), ammonium salts and organic base salts according to conventional methods. (Eg, trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, etc.), and the like, and various pharmaceutically acceptable salts known in the art.

Compounds of the present invention inhibit the toxicity and production of beta amyloid, have antioxidant activity to protect neurons, improve the spatial perceptual capacity through BBB in vivo and improve the synaptic density of neurons. ), Excellent prophylactic and therapeutic effects against degenerative brain diseases such as dementia, Parkinson's disease, stroke, and Huntington's disease. The compounds of the present invention increase the activity of α-secretase that cleaves beta amyloid or inhibit the activity of β-secretase, a protease that is essential for the production of beta amyloid. Thereby inhibiting the production of beta amyloid.

The compositions of the present invention can be used to prepare pharmaceutical formulations according to conventional methods. In the preparation of the formulation, it is preferred to mix the active ingredient with the carrier, to dilute it with the carrier, or to enclose it in a carrier in the form of a capsule, assay or other container. Thus, the formulation may be in the form of tablets, pills, powders, assays, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like.

Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, Propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The formulation may further comprise fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like. Compositions of the present invention may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular. Typical daily dosages of the compositions of the present invention can range from 1 to 100 mg / kg body weight, preferably from 1 to 10 mg / kg body weight, as effective ingredients, and can be administered once or in several divided doses. However, it should be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease, and therefore the dosage may be determined in any way. Also, the scope of the present invention is not limited.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are only for illustrating the present invention, the present invention is not limited to these.

Reference Example: Test Substance

Compounds Y and K, ginsenosides Mc and Rc, Protoparanaxadiol (PPD) and Protopananaxtriol (PPT) used in the examples are described in Advances in Ginseng Research, (quotation), 1998, The Korean Prepared by the Society of Ginseng.

Example 1: beta amyloid toxicity

MTT assay (Gillardon, F. et al., Brain Research , No. 6: pp. 169-172 (1996)) was performed to investigate the anti-beta amyloid toxicity effect of the isolates from ginseng.

Neuroblastoma (neuroblastoma) in a 96-well plate B103 cell line culture medium (US Salk of donor reception from Dr. David Schubert Institute) 5 X 10 3 cells 100 ㎕ [DMEM (Dullbecco's minimal Eagle's medium) + 10% FBS (fetal bovine serum)] and incubated at 8% CO ₂ , 37 ° C for one day. After changing the medium with DMEM, the test compound was treated by concentration, and after 1 hour, beta amyloid (25-35 peptide; US peptide, CA, USA) was treated at 25 μM concentration. The plate was incubated at 37 ° C. for 18 hours and then 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide was added to MTT solution [phosphate buffered saline (PBS). Solution dissolved at a concentration of 10 mg / ml] was treated and reacted at 37 ° C. for 3 to 4 hours. 100 μl of lysis buffer (10% SDS, 50% dimethylformamide, pH 4.7) was added and reacted overnight at room temperature in a box of 90% relative humidity. After measuring the absorbance of each well at 570 nm and 630 nm using a scanning multiwell spectrophotometer, the absorbance value of 630 nm was subtracted from the absorbance value of 570 nm.

When the beta amyloid-only neuronal cell group was taken as 100%, the relative survival rate of the compound treated with compound Y, ginsenoside Mc and Rc, PPD and PPT was calculated to obtain the results as shown in Table 2 and FIG. The calculated values were verified to be statistically significant.

Test compound Test substance concentration (μM) effect(%) Beta amyloid (Aβ) 25 (fixed) 100 Rc One 161.0 ± 4.9 Rc + Aβ One 106.0 ± 0.9 Rc 10 126.7 ± 3.7 Rc + Aβ 10 107.2 ± 1.9 Y One 157.7 ± 3.2 Y + Aβ One 102.8 ± 1.5 Y 10 123.5 ± 4.6 Y + Aβ 10 115.5 ± 3.5 Mc One 148.0 ± 6.3 Mc + Aβ One 106.1 ± 2.4 PPD One 137.7 ± 4.8 PPD + Aβ One 106.7 ± 6.9 PPT One 157.4 ± 3.3 PPT + Aβ One 106.6 ± 1.2 PPT 10 140.3 ± 6.4 PPT + Aβ 10 119.7 ± 8.5

In addition, the results of this experiment were almost the same when the test compound and beta amyloid were treated simultaneously with the test compound, or when the test compound was treated 1 hour after the beta amyloid treatment. It was confirmed to prevent.

In addition, in order to confirm the inhibitory effect of beta amyloid toxicity according to the concentration of the test substance, the above experiment was repeated using PPT as a test substance in a concentration range of 10 nM to 10 μM. As a result, as shown in Figure 2, PPT did not show a significant effect on the inhibition of beta amyloid toxicity at low concentrations (nM range), but began to exhibit the inhibitory effect in the μM range.

Example 2: Antioxidant Effect

The mechanisms that prevent beta amyloid toxicity can be largely divided into blocking oxidative stress (or free radical damage) and blocking apoptosis (or scheduled cell death) (Becl, C. et al., Cell , 77 : pp 817-827 (1994). Yamatsuji, T. et al., Science , 272 : pp1349-1352 (1996))

To examine whether ginseng-derived compounds prevent oxidative stress, neuronal cell death was induced in the same manner as in Example 1 except for using 1 μM of H ₂ O ₂ instead of beta amyloid. As a result, as shown in Table 3 and FIG. 3, all the test compounds prevented neuronal cell death by H ₂ O ₂ , and thus it can be seen that they act as antioxidants.

Test compound Test substance concentration (μM) effect(%) H ₂ O ₂ 1 (fixed) 100 Y One 486.5 ± 2.1 Y + H ₂ O ₂ One 469.1 ± 5.2 PPD One 494.3 ± 3.5 PPD + H ₂ O ₂ One 219.6 ± 3.0 PPT One 516.1 ± 1.6 PPT + H ₂ O ₂ One 381.3 ± 0.9 PPT 10 516.5 ± 1.2 PPT + H ₂ O ₂ 10 457.8 ± 2.5

On the other hand, to investigate whether ginseng-derived compounds prevent apoptosis, induction of apoptosis of nerve cells in the same manner as in Example 1 except that 0.1 μM of staurosporin instead of beta amyloid is used, and the ginseng Relevant substances were administered to check cell viability. As a result, it was found that ginseng-derived compounds did not block apoptosis as shown in Table 4 and FIG. 4.

compound Test substance concentration (μM) effect(%) Staurosporin (S) 0.1 (fixed) 100 Rc One 191.4 ± 4.9 Rc + S One 97.0 ± 4.8 Y One 170.3 ± 2.1 Y + S One 96.7 ± 2.0 Mc One 178.6 ± 6.3 Mc + S One 101.1 ± 2.7 PPD One 176.5 ± 3.5 PPD + S One 89.2 ± 1.0 PPT One 169.7 ± 1.6 PPT + S One 107.8 ± 2.3 PPT 10 173.1 ± 1.2 PPT + S 10 104.6 ± 2.7

Thus, ginseng-derived compounds prevent toxic beta amyloid by blocking oxidative stress rather than blocking apoptosis.

Example 3: increased secretion of sAPPα (α-Secretase derived secreted form of amyloid precursor protein)

To investigate whether ginseng-derived compounds inhibit beta amyloid production, we investigated the activity of α-secretase, which cuts the middle of beta amyloid.

A neuroblastoma B103 cell line (provided by Dr. David Schubert of Salk Institute, USA) that secretes the amyloid precursor protein (APP) is 1000 K in a 100 mm dish containing DMEM + 10% FBS medium. Plated with and then incubated overnight at 37 ℃, 8% CO ₂ conditions. Change the medium to serum-free DMEM (Gibco, USA), then incubate for 18 hours further by adding PPD, PPT and PDBu (phorbol-12,13-dibutyrate: Sigma Co., USA) at concentrations of 1 μM and 10 μM, respectively. It was. Supernatants were separated from each culture and concentrated with a concentration filter (viva spin centricon, available from Vivascience, Binbrook Hill, Binbrook, UK). Proteins in the concentrate were quantified to prepare samples at a concentration of 2 μg / ml and electrophoresed on 8% SDS-PAGE gels.

The protein band on the gel was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Co., Bedford, Mass., USA), blocked with 5% skim milk and then sAPPα specific antibody 6E10 (1: 2000 dilution, Cat.No. 320-05, Senetek, Maryland heights, MO, USA) was added and reacted with PVDF membrane at room temperature for 2 hours. After the reaction, the cells were washed three times with PBS, and then reacted at room temperature for 1 hour by adding a 1: 3000 dilution of a secondary antibody, an anti-mouse IgG (Amersham Pharmacia, Piscataway, NJ, USA). Protein bands were identified using an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia, Piscataway, NJ, USA) to see the response of the antibodies. Protein bands on the membranes were quantified by image analysis.

As a result, in the test group treated with PPT, the secretion amount of sAPPα was further increased compared to the control group not treated with PPT (FIG. 5).

Example 4: β-secretase Inhibitory Effect

To determine whether ginseng-derived compounds inhibit the activity of beta secretase, a protease that is essential for the production of beta amyloid, the following experiment was performed. As a comparative group, peptide H4848 (purchased from Bachem), which is already known as an inhibitor of beta secretase, was used, and PPD and PPT were used as experimental materials.

Neuroblastoma B103 cell line secreting APP was plated at 1500 K in a 100 mm dish containing DMEM + 10% FBS medium and incubated overnight, followed by MEST buffer (20 mM MES, 0.15 M NaCl, 0.5% Triton X-100, Cell extracts were made by bursting the cells with pH 6.0 (Bamchem).

In an amount as shown in Table 5, 5 μM of M2465 (Bachem) and MEST buffer as 200 m sodium acetate (pH 4.5) buffer, distilled water, β-secretase inhibitor, and β-secretase substrate were mixed in this order. The mixed solution was reacted at 37 ° C. for 10 minutes and cell extracts were added in the amounts shown in Table 5.

Added amount (μl) Buffer Distilled water Enzyme inhibitor temperament MEST Buffer Cell extract Blank test group 5 37.5 . 2.5 5 . Control 5 37.5 . 2.5 3.8 1.2 Comparative group H4848 (100 nM) 5 32.5 5 2.5 3.8 1.2 Test group PPT (100 nM) 5 32.5 5 2.5 3.8 1.2 PPT (1 μM) 5 32.5 5 2.5 3.8 1.2 PPT (10 μM) 5 32.5 5 2.5 3.8 1.2 PPT (100 μM) 5 32.5 5 2.5 3.8 1.2

The obtained mixed solution was reacted at 37 ° C. for 2 hours, and then read by fluorometer at 508 nm emission wavelength after 360 nm excitation. Inhibitory effect of the enzyme was shown as the degree of inhibition compared to the control.

As a result, as shown in Table 6 below, the phenomenon of blocking the activity of beta-secretase was observed in the experimental group similar to the comparison group, the higher the concentration of PPT was confirmed that the greater the inhibitory effect of the enzyme.

PPT Treatment Concentration 100 nM 1 μM 10 μM 100 μM Enzyme inhibitory effect (% of control) 0 0.6 3.31 18.1

Example 5: In vivo ( in vivo Behavioral experiment

Water maze to measure spatial perception ability to check in vivo activity of ginsenosides Rc and Mc, compounds Y and K, PPD, and PPT that showed beta amyloid toxicity in vitro Tests were performed (Mook-Jung et al., J. Neuro. Res. , 63 : pp509-515 (2001)).

As a result, as shown in Figure 6, the group administered ginsenoside Rc or Mc, or Compound Y was much faster to go to the platform than the control group administered only saline. Similar results were also observed in the group administered with compound K, PPD or PPT.

These results indicate that cognitive function, in particular, spatial perception ability is improved by administration of ginsenosides Rc and Mc, compounds Y and K, PPD, and PPT derived from ginseng.

Example 6: Increase Synaptic Density

Intraperitoneally in C57BL / 6 mice (9 weeks old, 25g), ginsenoside Rc or Mc, or compound Y or K in an amount of 1 mg / kg, or PPD or PPT in an amount of 0.5 mg / kg daily for 4 days It was administered once. After 4 days, the brain of the rat was removed, and the hippocampus and cortex of the brain were separated, and the amount of synaptophysin protein used as a marker of each synaptic vesicle was determined using the specific antibody (Cat. # 902 314, Boehringer Mannheim, Germany) was used to quantify by Western blot LAS100 (densitometer, Fujifilm, Japan) and image analysis method.

As a result, as shown in FIGS. 7A and 7B, there was no change in the group treated with saline, but the amount of synaptopycin was significantly increased in the hippocampus in the rats treated with PPD and PPT, and no change was observed in the cortex. Did. These results indicate that PPD, PPT, etc. administered intraperitoneally enter the brain and increase the synaptic density of brain neurons. Similar results were also obtained when ginsenoside Rc or Mc, or compound Y or K was administered.

Example 7 BBB transmittance

When ginseng-derived compounds were administered in vivo, they were carried out as follows to identify substances exhibiting activity by penetrating BBB.

(Step 1) Preparation of Sample

(1) Preparation and Quantification of Ginsenoside Sample

A 1 mg / ml stock solution of Ginsenoside Rg1 and Rb1 (ICN, USA) was serially diluted with acetonitrile to prepare solutions with final concentrations of 0.1, 0.2, 0.4, 0.6 and 1.0 μg / ml, respectively. 300 µl of this solution was placed in a plastic tube, evaporated to dryness in a speed-vac, and the same amount of acetonitrile was added to prepare 0.3 ml of standard solution. 20 μl of standard solution is injected into an LC mass system [Finnigan LCQ DECA (LC / MSn System)] equipped with an HPLC column (Zorbax Eclipse XDB-C18, 2.1 mm x 15 cm) and a mass selective detector. It was. The separation and detection conditions for each substance are as follows:

Quantification of Rg1

Mobile Phase-Acetonitrile: Deionized Water (25%: 75%)

Flow rate: 0.5 ml / min

Retention time of the peak: 2.1 minutes

Detected ionic species: RSI of [M + H] (m / z) 801, [M + H] (m / z) 823 and fragment ions (m / z) 643, followed by ESI SIM ms [822.40-825.40]. Detect and quantify signal.

Quantification of Rb1

Mobile phase: 20% to 90% gradient of acetonitrile fraction to the total amount of acetonitrile and deionized water mixture (gradient rate: up to 5 minutes (20% → 90%), then 2 minutes (90% → 20%))

Flow rate: 0.5 ml / min

Retention time of the peak: 5.4 minutes

Detected ionic species: ESI SIM ms [1130.50-1133.50 after confirming Rb1 with [M + H] (m / z) 1109, [M + H] (m / z) 1131 and fragment ions (m / z) 325/365 ] Signal is detected and quantified.

(2) Preparation and Quantification of Blood Samples

A 1 mg / ml stock solution of ginsenosides Rg1 and Rb1 was serially diluted with acetonitrile to prepare a solution having a final concentration of 0.1, 0.2, 0.4, 0.6, 1.0 μg / ml. 300 μl of this solution was placed in a plastic tube, evaporated to dryness in a speed-bag, and then 100 μl of plasma was added and mixed by vortex. 200 µl of acetonitrile was added to the mixed solution, and the mixture was mixed by vortex for 5 minutes and centrifuged at 3,000 rpm for 20 minutes. 270 μl of the supernatant was evaporated to dryness in a speed-bag, reconstituted by addition of 270 μl of acetonitrile, and then 20 μl was injected into an LC mass system as described in (1).

(Step 2) Investigation of Drug Dynamics in Blood and Brain

Male Sprague-Dawley rats (250-300 g body weight) were lightly anesthetized with ether and then catheter was inserted into the femoral sinus, into a vein with polyethylene tube (PE-50). After restoring the experimental animals, 1 mg / kg of Rg1 and Rb1 saline solution were intravenously administered to the femoral vein (IV bolus administration), respectively. About 2, 5, 10, 15, 20, 30, 45, and 60 minutes after administration, about 300 μl of blood was collected from the femoral artery and centrifuged to obtain 100 μl of plasma. From the obtained plasma, a blood sample was prepared in the same manner as in step (2). 20 μl of sample was injected into the LC mass system as in step 1 and the ginsenosides Rg1 and Rb1 and their metabolites PPD and PPT were quantified to determine the concentration in plasma per ml of plasma (for ginsenosides) or peak size (metabolism). Sieve).

Immediately after taking the blood sample, the brain tissue obtained from the rat head was lightly washed with ice-cold saline, and then weighed in a weighed 15-ml Falcon tube and weighed again. Acetonitrile equal to twice the weight of brain tissue was added to the tube and homogenized with a tissue homogenizer (Ultra-Turrax T25, JANKE & KUNKEL IKA-Labortechnik). The mixed mixture was centrifuged at 3,000 rpm for 20 minutes, approximately 300 μl of the supernatant was evaporated to dryness in a speed-bag, and reconstituted with 300 μl of acetonitrile. 20 μl of this mixture was injected into the LC mass system as in Step 1 to quantify the concentration of ginsenosides and their metabolites in the brain and expressed as the weight or peak size of each substance per gram of brain tissue.

Separation and detection conditions of ginsenosides Rg1 and Rb1 are the same as in Example 1 (1), and the separation conditions and detection conditions for the metabolites PPD and PPT are as follows:

Quantification of PPT

Mobile Phase-Acetonitrile: Deionized Water (25%: 75%)

Flow rate: 0.5 ml / min

Retention time of the peak: 2.1 minutes

Detected ionic species: PPT confirmed with [M + H] (m / z) 477 and [M + Na] (m / z) 499, followed by detection and quantification of signals in ESI SIM mode [497.50-500.50].

Quantification of PPD

Mobile phase: 20% to 90% gradient of acetonitrile fraction to the total amount of acetonitrile and deionized water mixture (gradient rate: up to 5 minutes (20% → 90%), then 2 minutes (90% → 20%))

Flow rate: 0.5 ml / min

Retention time of the peak: 7.1 minutes

Detected ionic species: PPD confirmed by [M + H] (m / z) 447 and [M + Na] (m / z) 469, followed by detection and quantification of signals in ESI SIM mode [467.50-470.50].

(Step 3) Results and Interpretation

(1) Quantification of Rg1, Rb1, PPD and PPT

The brain transition rate of the substance (K _brain ) can be expressed as the ratio of the mean concentration in the _brain (C _{mean, brain} ) and the mean concentration in the plasma (C _{mean, plasma} ) of the brain as shown in Equation 1 below.

However, the average concentration of a substance can be found by dividing the area from 0 to t time by t by the concentration-time curve. Therefore, the brain transition rate of the material (K _brain ) can be written as Equation 2.

According to the LC-mass assay developed in this example, ginsenoside Rg1 and Rb1 peaks in brain tissue samples obtained by administering 1 mg / kg doses of Rg1 and Rb1 to SD rats were well separated from endogenous substances (FIG. 8a and 8b). In addition, the minimum Rg1 and Rb1 concentrations quantifiable from blood samples obtained from the SD rats by this method were 50 ng / ml and 100 ng / ml, respectively (FIGS. 9A and 9B). Therefore, this method can be easily applied to the analysis of ginsenoside concentration in biological samples.

In addition, peaks of PPT and PPD were sufficiently detected by LC-mass method in brain tissue samples obtained 15 minutes after administration of 1 mg / kg doses of Rg1 and Rb1 to SD rats (FIGS. 10A and 10B). ).

(2) Trend of Ginsenosides Rg1 and Rb1, PPD and PPT in Blood and Brain

The concentrations of ginsenosides Rg1 and Rb1 in plasma and brain samples obtained by administering ginsenosides Rg1 and Rb1 at a dose of 1 mg / kg are shown in FIGS. 11A and 11B, respectively. As shown in Figures 11a and 11b, the drug concentration in the brain was less than the quantitative sensitivity, the drug concentration in the brain was very low (5.8% or less) compared to the plasma concentration up to 60 minutes. In addition, the concentration of a series of metabolites of ginsenosides Rg1 and Rb1 was detected in plasma and brain tissue samples. Except for PPT (the final metabolite of Rb1), the concentration ratio of plasma to brain tissue was less than 15%. Therefore, it was confirmed that the metabolites of Rb1, Ginsenoside Rb1 and Rg1 and their metabolites except these PPT did not migrate well to the brain. The trends in the concentrations of PPT and PPD in plasma and brain samples are shown in FIGS. 12A and 12B, respectively, and in the case of PPT, plasma and brain concentrations were almost similar. The brain migration rate of PPT calculated by Equations 1 and 2 was 130 ± 46.4%, which was very good.

Ginseng ginsenosides Rc and Mc, the hydrolysates of ginsenosides Y, K, PPD, and PPT prevent the production and toxicity of beta amyloid, improve spatial perception, and increase the synaptic density of brain neurons. In addition, since it exhibits strong antioxidant activity, it can be used as an excellent preventive and therapeutic agent for degenerative brain diseases such as dementia, Parkinson's disease, stroke, and Huntington's disease.

Claims (7)

A composition for preventing or treating degenerative brain disease, comprising the compound of formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: Formula 1 Where R ¹ is H or Glu-Glu, R ² is H or OH, R ³ is H, glucose, Ara (p) -Glu or Ara (f) -Glu. The method of claim 1, The compound of formula 1 is ginsenoside Rc, ginsenoside Mc, compound Y, compound K, protopanaxanadiol (PPD) or protopanaxanatriol (PPT). The method of claim 1, The degenerative brain disease is Alzheimer's dementia, Parkinson's disease, stroke or Huntington's disease. The method of claim 1, A composition, characterized in that the composition exhibits an antioxidant effect. The method of claim 1, The composition is characterized in that the composition exhibits an α-secretase activating effect. The method of claim 1, The composition is characterized in that the β-secretase (β-secretase) activity inhibitory effect. The method of claim 1, The composition is characterized in that the synaptic density increase effect.