CN107773580B

CN107773580B - Crude drug composition for preventing or treating dementia or neurodegenerative disease

Info

Publication number: CN107773580B
Application number: CN201710660953.4A
Authority: CN
Inventors: 谢心范; 林煌权; 山本理
Original assignee: Xuan Yan
Current assignee: Xuan Yan
Priority date: 2016-08-25
Filing date: 2017-08-04
Publication date: 2021-09-07
Anticipated expiration: 2037-08-04
Also published as: JP7005825B2; CN107773580A; JP2019011305A

Abstract

The present invention provides a crude drug composition for preventing or treating dementia or neurodegenerative disease. The present invention relates to a composition for preventing or treating dementia or behavioral and psychological symptoms thereof or neurodegenerative diseases, which comprises notoginseng and eucommia ulmoides as active crude drug components. The present invention also relates to a composition for preventing or treating dementia or behavioral and psychological symptoms thereof or neurodegenerative diseases, which comprises notoginseng, eucommia ulmoides, and polygonatum sibiricum as effective crude drug components. The present invention also relates to a composition for preventing or treating dementia or behavioral and psychological symptoms thereof or neurodegenerative diseases, which comprises notoginseng, eucommia, polygonatum and licorice as an effective crude drug component.

Description

Crude drug composition for preventing or treating dementia or neurodegenerative disease

Technical Field

The present invention relates to a crude drug composition for preventing or treating dementia or neurodegenerative disease. More specifically, the present invention relates to a crude drug composition for preventing or treating dementia or neurodegenerative disease, which comprises notoginseng and eucommia ulmoides as effective crude drug components. The present invention also relates to a composition for preventing or treating dementia or neurodegenerative disease, which comprises notoginseng, eucommia ulmoides, and polygonatum sibiricum as active crude drug components. The present invention also relates to a composition for preventing or treating dementia or neurodegenerative disease, which comprises notoginseng, eucommia ulmoides, polygonatum sibiricum and licorice as an active crude drug component.

Background

Dementia refers to a state in which the brain function of normal activities is decreased, and cognitive functions such as memory, thinking and judgment are gradually decreased, thus impeding daily life and social life. In modern society, 1 in 4 people of people over 85 years old suffer from dementia, the greater the age is, the more easy the dementia is, the prevalence rate of under 70 years old over 65 is 1.5%, 85 years old reaches 27%, it is estimated that dementia patients over 65 years old in japan have exceeded 240 ten thousand people (Kyoho labor, Japan, "pay attention to mental health of everyone (み and な and メンタルヘルス)") of the comprehensive website http:// www.mhlw.go.jp/kokoro/know/disconnect _ recog. In recent years, dementia has become an increasingly important problem in the elderly society in the future.

According to the investigation of the police hall, as shown in the following table, 166 ten thousand persons were examined in 2016, and it is expected that the number of examinees will increase in the future. From the results of the cognitive function test in this 8 years, the published results were 2.3% for class 1 (the person with decreased memory and judgment), 26.7% for class 2 (the person with slightly decreased memory and judgment), and 71.0% for class 3 (the person with no problem at all in memory and judgment) (homepage of the association of driving schools designated throughout japan by general community law, "data of accidents and the like," change in the number of subjects in the cognitive function test "http:// www.zensiren.or.jp/kourei/data. html).

[ TABLE 1 ]

The number of persons receiving the cognitive function test (2009, 6 months-2016 years)

General corporate law people all Japan designated driving school association is written according to the data of the department of police hall traffic bureau driving license

Dementia can be roughly classified into neurodegenerative diseases, cerebrovascular diseases, and other causes (including mental diseases, brain surgery diseases, or medical diseases) from the viewpoint of the basic etiology of the diseases.

The neurodegenerative disease is a disease in which brain nerve cells gradually die, and dementia caused by the neurodegenerative disease includes, for example, dementia of the alzheimer's type, dementia of the lewy body type, cerebrovascular dementia, and dementia of the frontotemporal type.

The number of patients with dementia of the alzheimer type is the largest, and about half of the dementia is accounted for. The cause of the disease is considered to be accumulation of β -amyloid in the brain and neurotoxicity caused by the accumulation, and pathological histological features such as general atrophy, ventricular dilatation, neurofibrillary changes, senile plaques and the like of the brain are observed, and mental symptoms such as memory deterioration, disorientation, judgment deterioration and the like are gradually increased, and some cases are accompanied by depression and the like.

Second only to alzheimer-type dementia, the number of patients is dementia of the lewy body type. The lewy body is a specific protein formed in nerve cells, and it is considered that if it is concentrated in the cerebral cortex or the brain stem of the brain, nerve cells are destroyed and decreased, and thus nerve conduction cannot be smoothly performed, thereby causing dementia.

Similarly, cerebrovascular dementia is also a large number of patients, and it is caused by the fact that nutrients and oxygen such as cerebral infarction, cerebral hemorrhage, cerebral arteriosclerosis, etc. are not transferred to nerve cells, and as a result, the nerve cells in the part die, and the nerve network is destroyed.

Frontotemporal dementia is also called pick's disease, and frontal lobe and temporal lobe atrophy can be seen, which is one of the dementia of children.

Dementia is classified into core symptoms and Behavioral and Psychological Symptoms (BPSD) by symptom classification.

The core symptoms are symptoms caused by destruction of brain cells, which is a direct cause of dementia, and usually show symptoms of memory impairment, disorientation, comprehension and judgment impairment, executive dysfunction, change in emotional expression, aphasia, agnosia, and disuse.

On the other hand, Behavioral and Psychological Symptoms have been also referred to as peripheral Symptoms and also as Behavioral and Psychological Symptoms of Dementia patients (BPSD: Behavial and Psychological Symptoms of Dementia). Specific examples of the behavioral and psychological symptoms include wandering, eating behavior abnormality, refusal, anxiety and anxiety, hallucination and delusion (stolen delusion, etc.), delusion, depression, excitation, violence or \35881, abuse, incontinence, insomnia, sleep disorder or reversal of day and night, request for returning home, eating disorder and swallowing disorder, and dirty behavior.

In addition to the basic causes of the above-mentioned dementia, neurodegenerative diseases include cranial nerve diseases in which a specific nerve cell group in the central nerve gradually dies, for example, Amyotrophic Lateral Sclerosis (ALS), parkinson's disease and other parkinsonism, alzheimer's disease such as senile agnosia, Progressive Supranuclear Palsy (PSP), huntington's disease, Striatal Nigral Degeneration (SND), schey-derger syndrome (Shy-Drager syndrome), olivopontocerebellar atrophy (OPCA) and other Multiple System Atrophy (MSA), spinocerebellar ataxia (SCA3), friedred's disease and other spinocerebellar degeneration (SCD).

The brain constitutes a network of nerve cells, the smallest unit in which information transfer is performed being nerve cells (neurons). The nerve cells have neurites for forming a brain nervous system network. The neurite is generally divided into a neurite (axon) for transmitting information and a neurite (dendrite) for receiving information in terms of morphology. A long axon and a plurality of processes of the complexly branched dendrites are derived from one nerve cell, and these processes are connected with other nerve cells to form a complex neural network.

The brain gradually shrinks with age, and once damaged, the brain nerve cells are difficult to regenerate, so that the cells can not divide once after birth, and almost the same cells can continue to be used for a lifetime. Brain nerve cells grow rapidly before the age of 5 years, and young brains extend branches of nerve cells to a distance along with the growth of the nerve cells, so that a nerve network is developed. Once adult, the weight and volume of the brain decrease, and the number of brain nerve cells decreases with age. In particular, in dementia including alzheimer-type dementia or neurodegenerative disease, atrophy of neurite and reduction of synapse occur, which cause memory disorder and the like.

Therefore, it is expected that the effect of dementia is improved by recovering atrophy of neurite and reduction of synapse. The causes of dementia and neurodegenerative diseases are various, but since the direct cause of nerve dysfunction is destruction of a neural network, it would be very useful for the prevention or treatment of dementia or neurodegenerative diseases if it were possible to grow neurites and reconstruct the neural network.

Nerve growth factor, also called ngf (nerve growth factor), is one of neurotrophic factors that maintain the survival of nerve cells, promote the growth of neurites, promote the synthesis of neurotransmitters, and the like, and is a protein complex composed of 3 subunits (α, β, γ). The NGF β subunit is a polypeptide consisting of 118 amino acids (molecular weight 13kD) and exists in the form of a dimer. NGF is scattered in the peripheral nervous system in the sympathetic nervous system and the sensory nervous system, and also plays a special role in the central nervous system in basal forebrain cholinergic nerve cells projected on the cerebral cortex and hippocampus. It is well known that this nerve cell population on which NGF acts is severely exfoliated in the brain of alzheimer's disease. Therefore, it is desired to explore the development and mechanism of a method capable of increasing the number of NGF in the brain or its activity decreased due to the increase in age and alzheimer's disease, etc.

On the other hand, NGF is known to be associated with inflammation, pain and disorders (Freund V, Pons F, Joly V, Mathieu E, Martinet N, Frossard N. (2002) alignment of new growth factor expression by human air channel smooth muscle cells in inflammation conditions) Eur Respir J.20: 458-. In addition, NGF activates mast cells, releasing histamine, which may help in the treatment of histamine allergy (Aloe L, Skraper SD, Leon A, Levi-Montalcini R. (1994) New growth factor and autoimmunity diseases. autoimmunity.19: 141-. In addition, NGF, like BDNF (Brain-derived neurotrophic factor), stimulates the spread and survival of tumor cells and promotes the formation of new blood vessels in tumors (Chopin V, Lagadec C, Toillon RA, LeBourhisX. (2016) neurogenin signaling in cancer stem cells. Cell Mol Life Sci.73: 1859-1870). Thus, an increase in the amount of NGF may not necessarily lead to desirable results.

Therefore, it would be advantageous if the nerve growth activity could be increased without increasing too much the amount of NGF.

As a therapeutic drug for alzheimer disease, which is a representative example of dementia, a drug that was first developed in the world is tacrine (trade name Cognex), and approval was obtained in U.S. in 1993, but approval was not obtained in japan because of large damage to the liver. Drugs that cause little damage to the liver, inhibit decomposition in nerve cells of the neurotransmitter acetylcholine, and contribute to memory recovery, that is, donepezil (aricept (registered trademark), Eisai) which is an acetylcholine degrading enzyme inhibitor, have been developed in japan. Subsequently, therapeutic drugs such as rivastigmine, galantamine, memantine, etc. have been approved.

However, the range of choice of drugs for treating alzheimer's disease based on western medicine is still very limited, and it is known that drugs cause psychoneurosis such as excitement and mental irritability, and side effects such as digestive tract symptoms. On the other hand, in view of the limit of western medicine and the concern of side effects of conventional medicines, in recent years, the effectiveness of Chinese herbal medicines has been reported for dementia and behavioral and psychological symptoms thereof. As crude drugs or Chinese herbal medicines effective for dementia, there are used powder of suppressing liver, powder of Danggui peony root, powder of suppressing liver with dried orange peel pinellia tuber, etc.

For example, in recent years, the present invention has been applied to the mental and neurological fields such as dementia as a new possibility of yokukansan. Since the crude drugs of Danggui, Chuan Xiong and Zhuteng Gou contained in the powder for suppressing the liver disease are likely to cause gastric distention, it has been reported that the gastrointestinal burden is likely to increase if the powder for suppressing the liver disease is taken for a long time. Therefore, by adding tangerine peel and pinellia ternate into the liver-suppressing powder, and adding the liver-suppressing powder with the gastrointestinal protection function and the tangerine peel pinellia ternate, the behavior and psychological symptoms of dementia can be obviously improved, and particularly the aggressivity can be obviously improved (the eupatorium adenophorum, 14(6) in the psychiatric department, 535-542 and 2009). In addition, there are also clinical studies on yokukansan plus tangerine peel pinellia ternate (Tagetian galangal, etc., psychiatric department 23(1), 130-138, 2013).

On the other hand, as a crude drug composition effective for prevention and improvement of life-style related diseases such as hypercholesterolemia, hypertension and diabetes and improvement of liver function of a hepatitis patient, a crude drug composition containing notoginseng and eucommia ulmoides and a crude drug composition containing notoginseng, eucommia ulmoides, polygonatum sibiricum and licorice are widely known.

As a product of a crude drug composition containing notoginseng and eucommia ulmoides, a notoginseng eucommia ulmoides extract (registered trademark) is widely known and available from synechological co ltd (tokyo). This was originally developed by one of the inventors of the present invention, and was a hot water extract containing a crude drug mixture containing pseudo-ginseng and eucommia ulmoides as main components. The Notoginseng radix and Eucommiae cortex essence can preferably contain Korean Ginseng and Mel in addition to Notoginseng radix and Eucommiae cortex as main ingredients.

As a crude drug composition containing pseudo-ginseng, eucommia ulmoides, polygonatum sibiricum and licorice, sheng dan zai huang (registered trademark) is widely known and available from syngotics co. This is also a crude drug composition which was originally developed by one of the inventors of the present invention and contains pseudo-ginseng extract, eucommia bark extract, polygonatum rhizome extract and licorice root extract. In addition, hereinafter, for convenience, the crude drug composition is described as "health-preserving pien Tze Huang". Crude drug compositions containing pseudo-ginseng and eucommia ulmoides, and crude drug compositions containing pseudo-ginseng, eucommia ulmoides, and polygonatum sibiricum (and licorice) are described in japanese patent No. 4588132, U.S. patent No. 6,280,776, U.S. patent No. 6,586,017, and the like.

In addition, the radix notoginseng and eucommia ulmoides essence and the health-preserving pien Tze Huang are applied as health foods.

Prior art documents

Patent document

Patent document 1: japanese patent document No. 4588132

Patent document 2: U.S. Pat. No. 6,280,776

Patent document 3: U.S. Pat. No. 6,586,017

Non-patent document

Non-patent document 1: greene LA and Tischler AS (1976) Proc Natl Acad Sci U S A,73(7):2424-

Non-patent document 2: fukuda J, Yamaguchi K, Akimoto S, Tada Y.Neurosci Res.2:460-471,1985

Non-patent document 3: fischer W, Wictorin K, Bjoerklund A, Williams LR, Valon S, Gage FH.Nature 329:65-68,1987

Non-patent document 4: freund V, Pons F, Joly V, Mathieu E, Martinet N, Frossard N. (2002) alignment of new growth factor expression by human air estimate cell in information modulation Eur Respir J.20:458-

Non-patent document 5: aloe L, Skraper SD, Leon A, Levi-Montalcini R. (1994) New growth factor and autoimmune diseases. autoimmunity.19:141-

Non-patent document 6: chopin V, Lagadec C, Toillon RA, Le Bourhis X. (2016) Neurorphin signaling in cancer cells, cell Mol Life Sci.73:1859-

Non-patent document 7: lang Yang Hui, psychiatric department 14(6), 535-542, 2009

Non-patent document 8: tagetes et al, psychiatric department 23(1), 130-

Disclosure of Invention

Technical problem

In the prevention or treatment of dementia or neurodegenerative diseases, it is important to select a drug suitable for symptoms and physical constitutions of patients, and there is a demand for a drug that is effective for dementia, behavioral and psychological symptoms thereof, or neurodegenerative diseases. Under such circumstances, the choice of a preventive method and a therapeutic method should be expanded not only to western medicine but also to oriental medicine and the fields of crude drugs and chinese traditional medicine for patients suffering from dementia or symptoms related thereto or people around the patients, and thus there is a need for a crude drug composition which is useful for the prevention or treatment of dementia or Behavioral and Psychological Symptoms (BPSD) thereof, or neurodegenerative diseases and has few side effects.

In addition, Nerve Growth Factor (NGF) has been shown to maintain the survival of nerve cells, promote the growth of neurites, promote the synthesis of neurotransmitters, etc., but since an increase in the amount of NGF does not necessarily bring about an ideal result, it is required to improve the nerve growth activity without increasing the amount of NGF.

The present inventors have made intensive studies based on the activity of nerve cells such as dementia and knowledge related to a crude drug composition, and have found a crude drug composition useful for the prevention or treatment of dementia or behavioral and psychological symptoms thereof, or neurodegenerative diseases, and have completed the present invention.

Technical scheme

The present invention relates to a composition for preventing or treating dementia or behavioral and psychological symptoms thereof, or neurodegenerative diseases, which comprises notoginseng and eucommia ulmoides as active crude drug components. The present invention also relates to a composition for preventing or treating dementia or behavioral and psychological symptoms thereof, or neurodegenerative diseases, comprising notoginseng, eucommia ulmoides, and polygonatum sibiricum as effective crude drug components. The present invention also relates to a composition for preventing or treating dementia or behavioral and psychological symptoms thereof, or neurodegenerative diseases, comprising notoginseng, eucommia, polygonatum and licorice as effective crude drug components.

Hitherto, it has not been known at all in the art that a crude drug composition containing pseudo-ginseng and eucommia ulmoides, a crude drug composition containing pseudo-ginseng, eucommia ulmoides and polygonatum sibiricum, and a crude drug composition containing pseudo-ginseng, eucommia ulmoides, polygonatum sibiricum and licorice are useful for the prevention or treatment of dementia, behavioral and psychological symptoms thereof, or neurodegenerative diseases, and nothing is suggested.

The present invention is as follows.

(1) A crude drug composition for preventing or treating dementia or behavioral and psychological symptoms thereof, or neurodegenerative diseases comprises Notoginseng radix and Eucommiae cortex as crude drug components.

(2) The crude drug composition of (1) above, further comprising Polygonatum sibiricum Red as a crude drug component.

(3) The crude drug composition according to (1) or (2) above, further comprising licorice as a crude drug component.

(4) The crude drug composition according to the item (1), wherein the content of Panax notoginseng is 10 to 90% by weight, and the content of eucommia ulmoides is 10 to 90% by weight.

(5) The crude drug composition according to the item (2), wherein the crude drug composition comprises 10 to 90 wt% of Panax notoginseng, 10 to 90 wt% of eucommia ulmoides, and 2 to 25 wt% of Polygonatum sibiricum, and the sum of the crude drug components is defined as 100 wt%.

(6) The crude drug composition according to the item (3), wherein the content of the crude drug components is limited to 100% by weight, and the content of the crude drug components is 10 to 90% by weight, the content of the eucommia ulmoides is 10 to 90% by weight, the content of the polygonatum sibiricum is 2 to 25% by weight, and the content of the licorice is 3 to 20% by weight.

(7) The crude drug composition according to the item (3), wherein the content of the crude drug components is limited to 100% by weight, and the content of the crude drug components is 10 to 90% by weight, the content of the eucommia ulmoides is 10 to 90% by weight, the content of the polygonatum sibiricum is 2 to 25% by weight, and the content of the licorice is 5 to 20% by weight.

(8) The crude drug composition according to any one of the above (1) to (7), wherein the crude drug component is an extract of each crude drug component.

(9) The crude drug composition according to any one of (1) to (8) above, wherein the dementia or behavioral and psychological symptoms thereof, or neurodegenerative disease is prevented or treated by increasing expression of neurofilaments or promoting growth of neurites.

Advantageous effects

The crude drug composition of the present invention provides a new option for the prevention or treatment of dementia or behavioral and psychological symptoms thereof, or neurodegenerative diseases.

The crude drug composition of the present invention is effective for the expression of neurofilaments and the growth of neurites, and particularly, when used together with NGF, it can promote the expression of neurofilaments and the growth of neurites even in a low use amount of NGF which does not exhibit an effect on NGF itself.

Drawings

Fig. 1 is a graph showing the effect of notoginseng eucommia ulmoides (DTS) extract and health preserving pien zaihuang (YHK) extract on the proliferation of PC12 cells; cultured cells PC12 cells were treated with DTS extract and YHK extract for 48 hours. Cell survival and proliferation assays were performed using MTT colorimetry. Taking 5 samples with n being 4 respectively as the mean value +/-SEM;

FIG. 2 is a micrograph and bar graph showing whether DTS extract and YHK extract alone induced neurite outgrowth in PC12 cells;

(A) DTS or YHK extracts (0.1, 0.5, and 5mg/mL) were applied for 48 hours in culture to PC12 cells. Cells were fixed with 4% paraformaldehyde frozen. Bars represent 50 μm. NGF was provided as a positive control. Representative images obtained from 4 independent experiments;

(B) the cultured PC12 cells were treated in the same manner as in (A). The percentage of differentiated cells and the length of the neurites were counted as described in the method section of the examples, which follows. Values are expressed as the percentage of cells in total of 100 counted cells. Mean ± standard error, n is 4;

FIG. 3 is an electrophoretic image and histogram showing the induction of neurofilament expression in PC12 cells in high concentration using YHK extract alone;

(A) DTS extract or YHK extract (0.1, 0.5, and 5mg/mL) was applied to cultured PC12 cells for 48 hours. Expression of neurofilaments (NF68 and NF160) was determined by specific antibodies. GAPDH was provided as an internal control. Representative images obtained from 4 independent experiments;

(B) the quantitative results are represented by histograms. Values are expressed as multiples of the baseline value (Basal). The control value was set to 1. Mean ± standard error, n is 4. Comparing the control values, xp < 0.05, xp < 0.01, and xp < 0.001;

FIG. 4 is a micrograph and bar graph showing that DTS extract and YHK extract promote NGF-induced neurite outgrowth;

(A) DTS extract or YHK extract (0.1, 0.5, and 5mg/mL) was used together with NGF (0.5ng/mL) for 48 hours in cultured PC12 cells. NGF (50ng/mL) was used as a positive control. The growth of neurites was observed by fixing the cells with ice-stored 4% paraformaldehyde. Bars represent 50 μm;

FIG. 5 is an electrophoresis image and histogram showing that DTS extract and YHK extract promote nerve filament expression induced by NGF;

(A) DTS extract or YHK extract (0.1, 0.5, and 5mg/mL) was treated with NGF (0.5ng/mL) for 48 hours in cultured PC12 cells. Expression of neurofilaments (NF68 and NF160) was determined by specific antibodies. GAPDH was provided as an internal control. Representative images obtained from 4 independent experiments;

FIG. 6 is a table showing examination results along with the period of taking the medicine, according to the sex and age groups;

FIG. 7 shows the contents of a predictive test for dementia of the Tabellied form;

FIG. 8 is a graph showing analysis relating to years of ingestion of test subjects taking YHK only;

FIG. 9 is a graph showing an analysis relating to the number of years of administration of a test subject taking DTS alone;

FIG. 10 is a graph showing analysis relating to the number of years taken by a subject taking both YHK and DTS;

FIG. 11 is a graph showing analysis relating to age of subjects taking YHK only;

fig. 12 is a graph showing an analysis related to the age of a test subject taking only DTS;

fig. 13 is a graph showing analysis relating to age of subjects taking both YHK and DTS.

Detailed Description

The crude drug components in the present invention will be described.

In the present invention, the crude drug component refers to various crude drugs constituting a crude drug composition, and the various crude drugs may be in the form of various dosage forms such as granules, tablets, pills, powders, capsules, solutions (drinks), extracts, tinctures, syrups, and alcoholic preparations prepared by adjusting the medicinal parts of the base plants thereof, or the powders thereof, or the extracts thereof, or the granules obtained by drying the extracts, and the like.

In the present invention, Panax Notoginseng is also called Panax Notoginseng, Notogenseng, Radix Notoginseng, or Pseudoginseng, and is defined as the root of Panax Notoginseng Panax notogeng, or Panax pseudo-ginseng Wall. As is well known, the traditional Chinese medicine can improve dyslipidemia and inhibit hypertension and pain. In the present invention, as a plant derived from Panax notoginseng, a plant used in the art as another plant derived from Panax notoginseng can be used. In the present invention, the use site of Panax notoginseng is not particularly limited, and the root thereof is usually used.

Eucommia ulmoides, also known as Eucommia Bark, EUCOMMIAE CORTEX, generally consists of dried Bark of Eucommia ulmoides belonging to family eucommiaceae. It is known that the crude drug has effects of enhancing liver function and kidney function and lowering hypertension and hyperlipidemia concentration. In the present invention, as the plant derived from eucommia ulmoides, a plant used as another plant derived from eucommia ulmoides in the art may be used. In the present invention, the site of use of eucommia is not particularly limited, but in the present invention, it means not only the bark of the above-mentioned plant of origin such as eucommia, but also dried products of the new leaves (Eucommiae Folium), fruits (Eucommiae Berry), and/or trunk parts (Eucommiae Wood).

Sealwort, also known as Polygonatum Rhizome, Polygonati Rhizoma, or Siberian Solomonseal Rhizome, is a traditional crude drug useful for improving nutritional status and promoting health. It is known that the crude drug is effective in improving symptoms and liver functions of patients with chronic hepatitis. The site of use of Polygonatum is not particularly limited, but in the present invention, Polygonatum refers to a product obtained by drying or cooking the rhizomes of a plant of the genus Polygonatum (Polygonatum).

In the present invention, as a representative example of the plant as a base source of Polygonatum sibiricum, bupleurum falcatum (a.gray), Polygonatum sibiricum (polygon sibiricum red, polygon sibiricum dalar. ex red), Polygonatum kingianum Collett et Hemsley, and Polygonatum cyrtonema Hua (Liliaceae) are used (see seventeenth modified japanese medicine office).

In the present invention, as a base source plant of Polygonatum sibiricum, Polygonatum odoratum (Mill.) Druce, Polygonatum odoratum), Polygonatum multifluorum, Polygonatum stenophyllum Maxim, Polygonatum invocateum Maxim, Polygonatum involventium Maxim, Polygonatum macrogolum turex, Polygonatum cirrhifolium (Wall.) Royle, Polygonatum prattii Baker, Polygonatum punctium Royle, Polygonatum zanlanscinense Pamp, Polygonatum villosum Hua, Polygonatum tessellum Wang, Polygonatum kurton, Polygonatum current, Polygonatum kurton, Polygonatum kurtom, Polygonatum kurton calcium, Polygonatum kurtom, Polygonatum grandiflorum, Polygonatum calcium, Polygonatum grandiflorum major, Polygonatum, Polygonum villum, Polygonum villum, Polygonum, Melum, Polygonum, Melum, Polygonum, and so, Polygonum, and so forth.

In the present invention, licorice is also called glycyrrhiza, glycyrrhiza radix, glycyrrhiza Rhizoma, licoricce, or liquoricce, which is a crude drug widely used in Chinese herbal medicines. Is defined as the root, rhizome or product of drying stolons of the plant of origin of Glycyrrhiza (Glycyrrhiza) of Leguminosae.

In the present invention, Glycyrrhiza uralensis Fischer (Glycyrrhiza uralensis) or Glycyrrhiza glabra Linne (Leguminosae) (Glycyrrhiza glabra) recorded in the seventeenth japan pharmacopoeia is typically used as a plant of Glycyrrhiza uralensis origin.

In the present invention, as the base plant of licorice, Glycyrrhiza acanthocarpa, g.aspera, g.astragalina, g.buchorica, russian licorice g.echinata, g.eglandulosa, g.foetida, g.foetidissima, g.gottscharovii, g.iconica, g.korshinsky, american licorice g.lepidota, g.pallidiflora, g.squarulosa, g.triphylla, g.yunnanensis, new licorice g.infilfata, and the like may be used, as required.

In the present invention, as the plant of the base source of licorice, plants used in the art as other plants of the base source can also be used. In the present invention, the site of application of licorice is not particularly limited, but roots, rhizomes or stolons are generally used as described above.

In the crude drug composition of the present invention, the amount of panax notoginseng used is 10 to 90 wt%, preferably 30 to 80 wt%, more preferably 35 to 75 wt%, and still more preferably 40 to 60 wt%. The amount to be used may be less than the lowest limit or more than the highest limit of the above range, but is preferably within the above range because the synergistic effect with other biological components may not be exhibited in some cases.

In the crude drug composition of the present invention, the amount of eucommia ulmoides used is 10 to 90 wt%, preferably 15 to 80 wt%, more preferably 20 to 60 wt%, and still more preferably 30 to 50 wt%. The amount to be used may be less than the lowest limit or more than the highest limit of the above range, but is preferably within the above range because the synergistic effect with other biological components may not be exhibited in some cases.

In the crude drug composition of the present invention, polygonatum sibiricum is used in an amount of 0 to 25 wt%, preferably 2 to 25 wt%, more preferably 6 to 20 wt%, and still more preferably 10 to 20 wt%. The amount to be used may be less than the lowest limit or more than the highest limit of the above range, but is preferably within the above range because the synergistic effect with other biological components may not be exhibited in some cases. In particular, if the ratio of polygonatum is too high, it is difficult to digest it and thus it is not easily absorbed by the human body and causes discomfort to the stomach, so it is preferable to use it in the above range.

In the crude drug composition of the present invention, the amount of licorice may be 0 to 20% by weight. In the crude drug composition of the present invention, when polygonatum is used as a crude drug component, licorice is preferably used in consideration of its synergistic effect. In this case, the amount of licorice may be preferably 3 to 20% by weight, more preferably 5 to 20% by weight, and still more preferably 10 to 20% by weight. The amount of the compound to be used may be less than the lower limit of the above range or more than the upper limit, but may not exert a synergistic effect with other biological components, and therefore, it is preferably used in the above range. In addition, it is preferable that licorice be used in the above range in consideration of side effects caused by excessive ingestion thereof. Wherein, the sum of the contents of the crude drug components is limited to 100 wt% (i.e. when the crude drug components are taken within the value range of the weight percent, the sum of the weight percent of all the crude drug components is required to be 100 wt%).

The composition of the crude drug composition may contain other crude drug components as described below, as long as each crude drug component is within the above composition range. For example, the crude drug composition of the present invention may further contain at least one selected from the group consisting of korean ginseng and honey as an arbitrary crude drug component. Wherein, the sum of the contents of the crude drug components is limited to 100 wt% (i.e. when the crude drug components are taken within the value range of the weight percent, the sum of the weight percent of all the crude drug components is required to be 100 wt%).

In the crude drug composition of the present invention, Korean ginseng, which is an arbitrary crude drug component, may be used in an amount of 0 to 20% by weight, and honey, which is an arbitrary crude drug component, may be used in an amount of 0 to 30% by weight. These components are preferably used in the above range in view of easy ingestion of the crude drug composition.

The crude drug components and optional crude drug components vary depending on the year, season, moisture content, etc. of collection of each raw material, and therefore the amount of the crude drug components used may be appropriately changed or may be outside the above range.

The crude drug composition of the present invention, by containing these optional components, has advantages that it is easy to ingest and easily absorbed by the human body, and has an effect even when taken for a short period of time, and the effect can be sustained even after the administration is terminated. In this regard, when polygonatum is used, it is desirable for many japanese people to include glycyrrhiza, but depending on individual differences, there are some cases where it can be absorbed by the human body even without glycyrrhiza.

In the present invention, any crude drug component may be a generally used crude drug. Any crude drug component may be used if it is, for example, one recorded in the japanese pharmacopoeia, but may be other than this. In addition, commercially available Panax notoginseng extract and anti-inflammatory agent as described above can be used as the crude drug components of the present invention.

In the present invention, when referring to each crude drug component or crude drug composition such as pseudo-ginseng, eucommia ulmoides, polygonatum sibiricum, licorice, the medicinal part of the plant of origin, that is, the medicinal part corresponding to the plant of origin, may mean any one of the root, stolon, bark, new leaf, fruit, trunk part, rhizome and/or extract thereof. In addition, the dosage form can be any one of powder, granule, tablet, pill, powder, capsule, solution (beverage), extract, tincture, syrup, alcohol, etc.

In the present invention, the term "extract of crude drug components" or "extract" refers to an extract of crude drug components to be extracted, which is an extract of the crude drug components, with water at room temperature to 60 ℃ or hot water at 60 ℃ to 100 ℃, or with an ethanol solution at room temperature to 100 ℃ or with a mixed solution of water and ethanol (100: 0 to 0: 100). The extraction time may be, for example, 0.5 to 2 hours, but a shorter or longer time may be suitably employed depending on the purpose. The kind, temperature and time of the extraction solvent are examples, and may be appropriately changed according to the purpose.

Further, when the crude drug component in the crude drug composition is composed of a medicinal part of the original plant, i.e., the root, the stolon, the bark, the new leaf, the fruit, the trunk part, the rhizome, etc., which is not subjected to extraction treatment, and/or is in a form of a preparation containing the same, it can be used after extraction as described above before use.

Crude drugs are a general term for drugs used without purifying active ingredients from products having naturally occurring drug effects, and the active ingredients are not limited to one kind, and many ingredients are associated with each other in a complicated manner in many cases. Therefore, it is now becoming a problem in the future that the crude drug component or its extract is composed of what effective chemical components, or how to directly specify the crude drug composition or its extract by structure or characteristics.

The crude drug composition of the present invention containing pseudo-ginseng and eucommia ulmoides (not containing polygonatum kingianum) may contain ginseng, honey, etc. as optional components in addition to the main crude drug components pseudo-ginseng and eucommia ulmoides. As a crude drug composition containing notoginseng and eucommia ulmoides (not containing polygonatum rhizome), as described above, notoginseng eucommia ulmoides essence (registered trademark, synechological co., ltd., tokyo) is widely known and commercially available. In the present specification, for convenience, a crude drug composition containing notoginseng and eucommia ulmoides but not containing polygonatum sibiricum is sometimes referred to as DTS.

The crude drug composition of the present invention containing panax notoginseng, eucommia ulmoides, and polygonatum sibiricum is preferably prepared by including glycyrrhiza uralensis. As described above, a health-preserving pien zaihuang (registered trademark, synusiologic co ltd (tokyo)) is widely known and commercially available as a crude drug composition containing pseudo-ginseng, eucommia ulmoides, polygonatum sibiricum and licorice. It contains hot water extract of crude drug mixture containing Notoginseng radix, Eucommiae cortex, rhizoma Polygonati and Glycyrrhrizae radix as main ingredients. For convenience, the crude drug composition containing notoginseng, eucommia ulmoides, polygonatum sibiricum and licorice is sometimes referred to as YHK in the specification of the present application.

Next, a method for producing the crude drug composition of the present invention will be described.

The crude drug composition of the present invention can be produced by a method generally used in the art using the above crude drug components.

For example, when the crude drug component in the crude drug composition is composed of roots, stolons, barks, leaves, fruits, trunk parts, rhizomes, etc. which have not been subjected to extraction treatment, and/or when the crude drug component is a formulation containing them, the crude drug composition of the present invention can be produced by weighing and mixing them in a fixed amount.

When crude drug components not subjected to extraction treatment are used, they may be used in the form of, for example, powder.

The crude drug composition can be divided into 1 times of prescription amount for use. In this case, the crude drug composition of the present invention to be prescribed may be extracted and administered as described above before use. The obtained liquid extract can be made into granule by drying, and can be made into granule, tablet, pill, powder, capsule, solution (beverage), extract, tincture, syrup, alcohol, etc., as described below.

In another aspect of the crude drug composition of the present invention, each crude drug component may be an extract of the crude drug component, for example. The crude drug components are prepared into fine pieces and quantitatively weighed, and the weighed crude drug components or a mixture obtained by mixing the raw drug components and the raw drug components are extracted with water, hot water, ethanol, or a mixed solution of water and ethanol as described above, and subjected to various steps of filtration, purification, and concentration as necessary, thereby producing a crude drug composition of a liquid extract.

The 1-time prescription amount may be packaged in a sealed bag such as an aluminum bag. In this case, the crude drug composition of the present invention may be administered by heating or diluting before use.

In the present invention, the method for extracting the crude drug component is not limited to the above method, and any extraction method that is conventionally used according to the general knowledge of a person of ordinary skill in the art can be used.

The obtained crude drug component extract can be made into granule by drying, and made into granule, tablet, pill, powder, capsule, solution (beverage), extract, tincture, syrup, alcohol, etc.

The obtained two extracts may be mixed with Korean ginseng extract and honey as optional components (mixing step). The crude drug composition of the present invention can be obtained in powder or powder form by hot air drying the mixture (drying step).

Further, by adding an excipient such as an emulsifier to the obtained mixture, molding and hot air drying, a dosage form such as a tablet can be obtained. As the emulsifier, honey, vegetable oil, xylitol, etc. can be used. The weight ratio of the emulsifier is preferably 0 to 20 wt%.

The content of the crude drug composition of the present invention in the mixed powder of the above crude drug component extracts and the tablet obtained by hot air drying the emulsifier as necessary is 0.1 to 0.5g, and particularly preferably 0.25g per tablet.

The amount of the crude drug composition of the present invention to be ingested is not particularly limited from the viewpoint of effectiveness in a human body, but is 0.1 to 2g/Kg, preferably 0.1 to 0.25g/Kg, about 1 day from the viewpoint of safety.

However, from the viewpoint of effectiveness and safety, the content and intake amount of these crude drug compositions can be set to an amount that does not cause side effects or is controlled to a minimum and can sufficiently exert the drug effect, according to the technical common knowledge of those skilled in the art.

The crude drug composition of the present invention can be administered in various dosage forms depending on the administration form. For formulation, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. are generally used. Suitable for oral administration, the dosage forms include powder, granule, tablet, pill, powder, capsule, solution (drink), extract, tincture, syrup, alcohol, etc., and the crude drug composition of the present invention can be obtained in the form of these dosage forms by using a diluent and an excipient. The solid preparation can be prepared by using at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc., and a lubricant such as magnesium stearate, talc, etc., other than the simple excipient. The liquid preparation may be in the form of a suspension, an emulsifier, a syrup, etc., or may be in the form of water, a wetting agent, a sweetener, an aromatic agent, a preservative, etc., as a simple diluent.

The crude drug composition of the present invention prepared as a preparation can be divided into individual packets and commercialized as appropriate according to the purpose. For example, the packaging can be carried out by packaging with a sealing bag such as an aluminum bag, aluminum foil, and moisture-proof fiber paper, or with a packaging paper.

The method for producing the crude drug composition of the present invention, in which the crude drug component is in the form of other dosage forms such as powder, granule, tablet, pill, powder, capsule, solution (drink), extract, tincture, syrup, alcohol, etc., can be easily understood by those skilled in the art from the general knowledge of crude drugs and pharmaceutical industry and the above description.

The pharmacological effects of the crude drug composition of the present invention on dementia, behavioral and psychological symptoms thereof, or neurodegenerative diseases will be described below.

It is known that PC12 cells are cell lines derived from pheochromocytoma derived from rat adrenal medulla (Greene LA and Tischler AS (1976) Proc Natl Acad Sci USA,73(7): 2424-. Thus, PC12 cells were used for research since a long time as an in vitro cell model for elucidating the growth of neurites, the differentiation mechanism in nerve cells, and the mechanism of NGF action, and were commercially available (Das KP, Freudenrich TM, Mundy WR. (2004) Assessment of PC12 cell differentiation and neural growth: a comprison of morphological and neural measurements (evaluation of cell differentiation and neurite growth: comparison of morphological and neurochemical assays) Neurooxicol Terate. 26: 397-.

NGF is scattered in the peripheral nervous system in the sympathetic nervous system and the sensory nervous system, and in addition, plays a special role in the central nervous system for basal forebrain cholinergic nerve cells projected on the cerebral cortex and hippocampus. It is well known that this nerve cell population on which NGF acts is severely exfoliated in the brain of alzheimer's disease. NGF also shows a stretching effect of nerve fibers on sympathetic ganglion neurons in elderly people and rats (Fukuda J, Yamaguchi K, Akimoto S, Tada Y. NGF-dependent and-independent growth of neurites from sympathogenic neurons of the aged in serum-free medium) Neurosci Res.2:460-471, 1985). Furthermore, it has been suggested that the decrease in the activity of NGF in the brain is associated with the decrease in learning ability due to aging (Fischer W, Wictorin K, Bjoerkund A, Williams LR, Varon S, gap FH. Electron: atomic of cholinergic neuron atrophy and spatial memory in induced by nerve growth factor in aged rats by neural growth factor) Nature329:65-68,1987).

Neurofilaments are special intermediate fibers in nerve cells, sparsely located in nerve cell bodies and dendrites and densely located in axons, interconnected by numerous cross-links. It has been found that the intrinsic protein in nerve fibers is composed of 3 homologous isomers having different molecular weights, namely NF-H having a molecular weight of 200kD, NF-M or NF160 having a molecular weight of 160kD, and NF-L or NF68 having a molecular weight of 68 kD.

The high expression of the Neurofilament (NF) protein, which is controlled between axonal growths, suggests that neurofilaments are important for axonal development. According to some studies, it was suggested that the elongation of the neurite filaments within the axon could be promoted by more effective elongation towards the long axis process (Walker KL, Yoo HK, Undamatala J, SzaroBG. (2001) Loss of neurofilaments altering the axon growth dynamics.) J Neurosci.21: 9655-.

It was found that the crude drug composition of the present invention induced neurofilament expression in PC12 cells, and therefore had a significant effect on the initiation of neurite outgrowth in PC12 cells. In addition, when the crude drug composition of the present invention is used together with NGF, the expression of neurofilaments in PC12 cells induced by NGF is further promoted. Furthermore, when the crude drug composition of the present invention is used together with NGF, the growth of neurite outgrowth induced by NGF is significantly promoted.

It is judged that the prevention or treatment of dementia or behavioral and psychological symptoms thereof, or neurodegenerative diseases is performed by increasing the expression of neurofilaments induced by Nerve Growth Factor (NGF), and/or promoting the growth of neurites in nerve cells. Therefore, the crude drug composition of the present invention has a significant effect on differentiation and growth of nerve cells including expression of nerve filaments and growth of nerve processes of nerve cells, and thus has a good effect on prevention or treatment of dementia or behavioral and psychological symptoms thereof, or neurodegenerative diseases.

The effect of the present invention relating to the prevention or treatment of dementia or behavioral and psychological symptoms thereof, or neurodegenerative diseases can also be verified by answering a questionnaire to a test subject who has taken DTS or YHK for a certain period of time.

The Dayou dementia prediction test (extracted from the homepage http:// www.mainichi.co.jp/nichou/expression. html of the dementia prevention treasury of the public welfare treasury legal for people) was used in the questionnaire for a simple examination of dementia. The contents of which are shown in fig. 6. The test is designed to be simple for the elderly themselves or the spouse or the same resident to predict the very early stage of dementia, the early stage of dementia or a state that is likely to progress to dementia.

From the results of the questionnaire, it was found that the prevention and treatment of dementia was effective by administering YHK, DTS or both YHK and DTS of the present invention.

The present invention will be described in more detail with reference to the following examples, which are merely illustrative and are not intended to limit the scope of the present invention defined by the claims below.

Examples

Example 1

[ purpose ]

In order to explore the functions of the pseudo-ginseng eucommia ulmoides essence and the health-preserving pien Tze Huang on nerve cells, water extracts of the pseudo-ginseng eucommia ulmoides essence and the health-preserving pien Tze Huang are prepared, and the effects of the pseudo-ginseng eucommia ulmoides essence and the health-preserving pien Tze Huang are evaluated according to neuropathology. The neurobiological functions of notoginseng and eucommia ulmoides essence and health-preserving pien Tze Huang are evaluated from two aspects of (i) differentiation of nerve cells and (ii) expression of synapsin.

[ Material and method ]

(1) Preparation of crude drug composition (extract)

The used notoginseng extract was purchased from synechological co ltd (tokyo) and composed of 33g (55 wt%) of notoginseng and 22g (45 wt%) of eucommia ulmoides. Hereinafter, the crude drug composition is abbreviated as DTS.

The health-preserving pien Tze Huang is composed of 6g (40 wt%) of pseudo-ginseng, 4.5g (30 wt%) of eucommia bark, 2.25g (15 wt%) of rhizoma polygonati and 2.25g (15 wt%) of liquorice. Hereinafter, the crude drug composition is abbreviated as YHK.

Each sample of DTS and YHK was pulverized into a powder, 50mg was weighed into 1.5mL of a microtube of Ebende, 1mL of water was added thereto, ultrasonic irradiation was performed for 30 minutes, and centrifugation was performed at 13200rpm for 10 minutes. The supernatant was collected and filtered through a PTFE syringe filter (0.22 μm). Stock solution of 50mg/mL, and storing at-20 deg.C.

(2) Cell culture

PC12 cells derived from rat adrenal medulla were purchased from the American Type Culture Collection (ATCC). Cells were cultured in Darbeke's Modified Eagle's Medium (DMEM) supplemented with 6% Fetal Bovine Serum (FBS) and Horse Serum (HS), 100 units/mL of penicillin, and 100 μ g/mL of streptomycin, and placed in a humidified CO2 (7.5%) incubator (incubator) at 37 ℃. Fresh medium was applied daily. The medium was replaced with fresh DMEM 3 hours before the extraction treatment, together with 1% FBS and HS, 100 units/MI penicillin and 100. mu.g/mL streptomycin. In the extraction process, the extract was diluted with DMEM containing 1% FBS and HS, 100 units/mL penicillin and 100. mu.g/mL streptomycin.

(3) Cell survival assay

Cell survival was assessed by the MTT (3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2H-tetramethylazole) assay. The cells were allowed to stand in a 96-well plate for 24 hours, and treated with various concentrations of the extract of the crude drug composition for 48 hours. Then, the cells were incubated with MTT at 37 ℃ for 3 hours. Then, the medium was discarded, and 100. mu.L of DMSO was added to each well. The plate was shaken in the dark for 15 minutes. Then, the absorbance at 570nm was measured by a microplate reader (Thermo Scientific, Flymont, Calif.).

(4) Treatment of crude drug composition in extract

The effect of extracts on neurite outgrowth was investigated with cultured PC12 cells. As a positive control showing neuronal processes, 50ng/mL of Nerve Growth Factor (NGF) was provided (purchased from Alomone Labs, Ltd., Cat #: N-100). PC12 cells (5 × 104 cells/well) were seeded into 6-well plates. After 24 hours, the cells were replaced with DMEM medium containing 1% FBS, 1% HS and 1% P/S. In the extraction treatment, a range of concentrations of the extract (0.1, 0.5, and 5mg/mL) was treated 2 times every 24 hours. Subsequently, the cells were collected for further experiments.

(5) Determination of neurite outgrowth

After processing, images were captured by manual setup using an optical microscope (Diagnostic Instruments) with a phase difference light collector and a 10-fold objective lens and a digital camera (Diagnostic Instruments, stirling elevation, michigan). To analyze the number and length of neurites, approximately 100 cells were counted from at least 10 fields of view randomly selected for each medium. Then, the number and length of the neurites of the cells were analyzed using Photoshop (registered trademark) software. When one or more neurites are longer than the diameter of a cell body, the cells are classified into different groups according to the length of the neurites of the cell body. Namely, the particles are classified into less than 30 μm, 30 to 60 μm, 60 to 90 μm and more than 90 μm. In this test, NGF (50ng/mL) was used as a positive control.

(6) Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE)

After the extraction treatment, the protein for neural differentiation was extracted as a low-salt lysis buffer (10mM HEPES, pH7.4, 150mM NaCl, 1mM EDTA, 1mM EGTA, 0.5% Triton X-100, 5mM benzamidine HCl, 10. mu.M aprotinin, 10. mu.M leupeptin). After centrifugation at 13200rpm (16100 Xg) at 4 ℃ for 20 minutes, the supernatant was collected and the total amount of protein was determined by protein assay. All samples were normalized to the same grade of protein, treated with 2 xSDS-PAGE sample buffer (0.125M Tris-Cl, pH6.8, 4% SDS, 20% glycerol, 2% 2-mercaptoethanol and 0.02% bromophenol blue), and boiled for 15 minutes before SDS-PAGE.

(7) Western blot analysis

After electrophoresis, the protein was transferred to a nitrocellulose membrane using a Mini wet transfer tank (Mini Trans-Blot (registered trademark) CeLL). The transfer conditions are as follows. 1 Xtransfer buffer (25mM Tris, 192mM glycine, 15% ethanol, 0.1% SDS), 16 hours, 40V and 0.1A. Confirmation was made by staining the load with ponceau S in an amount equal to the sample transfer. Nitrocellulose membranes were blocked with 5% skim milk for 2 hours at room temperature in Tris buffered saline/0.1% Tween20(TBS-T), followed by incubation with primary antibody diluted into 2.5% skim milk in TBS-T at 4 ℃. Then, the nitrocellulose membrane was rinsed with TBS-T, and incubated with peroxidase (HRP) -conjugated anti-mouse secondary antibody or peroxidase (HRP) -conjugated anti-rabbit secondary antibody diluted in 2.5% skim milk for 2 hours at room temperature in TBS-T. After washing with TBS-T, the immunocomplexes were visualized by Enhanced Chemiluminescence (ECL). The intensity of the bands in the control and various samples run on the same gel under strictly normalized ECL conditions was compared using an image analyzer based on a calibration curve constructed by parallel gels with one serial dilution in the sample.

The antibodies used in the experiment are shown below.

[ TABLE 2 ]

(8) Other tests

Protein concentration was determined according to the Bradford method using a kit obtained from Bio-Rad corporation (Heracles, Calif.). Statistical tests were performed using a univariate variance analysis. Differences derived from baseline or control values are classified as follows.

Tp < 0.05, Tp < 0.01, and Tp < 0.001.

[ results and examination ]

(1)Effects of DTS extract and YHK extract on PC12 cell proliferation

PC12 cells are a well-established model of cells for neural differentiation that can respond to various stimuli such as NGF. First, a cell survival test was performed to determine the safe concentration range (0-5 mg/mL) of all extracts in each extract that did not cause cell proliferation and cell death. As a result, the cell viability was not significantly affected by the DTS extract and YHK extract at concentrations of 0 to 5mg/mL, and three different concentrations of 0.1, 0.5, and 5mg/mL (low, medium, and high) were selected for the following experiments (fig. 1).

(2)Biological evaluation of neuro-differentiated DTS and YHK extracts

PC12 cells showed similar characteristics to sympathetic neurons such as neurofilament expression and neurite outgrowth. Here, the number and length of neurite outgrowth of PC12 cells and the expression of neurofilaments (NF68 and NF160) were examined, and morphological and biological changes after treatment of DTS extract and YHK extract were examined.

2-1)Induction of neurite outgrowth of PC12 cells with DTS extract and YHK extract alone

The neural differentiation of PC12 cells was morphologically investigated by measuring the number and length of neurites. In this case, NGF was used at a concentration of 50ng/mL as a positive control. Significant neurite outgrowth of PC12 cells was observed after 48 hours of treatment with 50ng/mL NGF. No significant growth of the neurites of the cultures was observed in the treatments with DTS extract or YHK extract at 0.1, 0.5 and 5mg/mL (concentrations selected in previous experiments) (FIGS. 2A and B). However, as shown in the following experimental results, YHK extract induced neurofilament expression in PC12 cells at high concentration, and in addition, DTS extract and YHK extract promoted neurite outgrowth induced by NGF and NGF-induced expression by neurofilament.

2-2)Induction of neural filament expression in PC12 cells with YHK extract alone at high concentration

Neural differentiation can be biochemically measured by analyzing the expression of neurofilaments, the main structural component of differentiated nerve cells (neurons). During neurite outgrowth, heterodimers are formed when the neurofilament subunits NF68 (about 68kDa) and NF160 (about 160kDa) of mammals construct the domain of a neurite.

If NGF in the culture of 50ng/mL on PC12 cells was applied for 48 hours, expression of both NF68 and NF160 was reliably induced. DTS extracts on cultured PC12 cells were used alone, and at all concentrations, expression of F68 and NF160 was not controlled. On the other hand, YHK extract alone, at high concentration (5mg/mL), up-regulated protein expression of NF68 and NF160 (about 4-fold) (fig. 3A and B).

2-3)DTS extract and YHK extract promoted neurite outgrowth induced by NGF

In this example, as an appropriate concentration of NGF that is almost completely ineffective in inducing neurite outgrowth and nerve filament expression, a concentration of NGF of 0.5ng/mL was selected.

In neurite outgrowth assays, the percentage of cells differentiated by the synergistic treatment of DTS or YHK extract (0.1, 0.5, and 5mg/mL) and NGF (0.5ng/mL) was dependent on the amount used. The synergistic treatment with DTS and NGF showed an increase in PC12 cells with longer neurites, for example, 30-60 μm, 60-90 μm and over 90 μm neurites. The same observation was made for YHK and NGF synergy (fig. 4A and B).

2-4)DTS extract and YHK extract promoted NGF-induced neurofilament expression

To further investigate the effect of DTS extract and YHK extract on promoting neural differentiation, DTS extract or YHK extract (0.1, 0.5, and 5mg/mL) and NGF (0.5ng/mL) were co-treated with PC12 cells. DTS was treated synergistically with NGF at all concentrations, resulting in a slight increase in NF68 expression, and NF68 was expressed 2-3 fold in synergistic treatments with YHK and NGF at 0.1 and 0.5 mg/mL. A significant increase (about 8-fold) in NF68 was observed in 5mg/mL YHK in combination with NGF. The expression of NF160 was increased more significantly in all groups than NF 68. In the synergistic treatment of DTS and NGF, 0.1mg/mL of DTS showed the highest effect (about 6-fold) on NF160 expression. Of the synergistic treatments of YHK and NGF, 5mg/mL of YHK showed the highest effect (approximately 9-fold) on NF160 expression. Refer to fig. 5A and B.

[ conclusion ]

When the DTS extract and YHK extract were used alone, if NGF was used together, it promoted the induction of neurite outgrowth and the increase in neurofilament expression even in a low use amount of NGF that did not exhibit the effect of NGF itself.

Example 2

The subjects who took DTS or YHK of example 1 for a certain period of time were asked to answer a questionnaire to verify the effects of the present invention on the prevention and treatment of dementia.

As a questionnaire for simple examination of dementia, the dative dementia prediction test of fig. 6 was employed as described above.

The test subjects to be examined were those who took YHK or both YHK and DTS for 3 years or more (the longest duration of taking was 15 years). However, liver function abnormality (80% in patients who had hepatitis C and 20% in other patients with liver function abnormality) occurred in all the subjects, and patients who had other diseases were unknown.

The method comprises the following steps: and (4) dispatching a questionnaire to the test object, and sending back the test object after filling.

During the period: optionally filled in before the end of 2017 at 2 months, and a reply is given in 3 months.

Fig. 7 shows the total number of test subjects of 62 subjects, and the number of test subjects per individual age group and age group.

The approximate score is determined as 0 to 8 points for normality, 9 to 13 points for caution, and 14 to 20 points for the possibility of early symptoms of dementia.

The results are summarized in bar graphs as shown in FIGS. 8 to 13.

1. Analysis relating to years of administration

(1) Among the test subjects in the whole group administered with YHK alone, DTS alone and YHK + DTS, no test subject showing early symptoms of dementia appeared.

(2) In the group with YHK alone, 50% of the patients who took YHK alone were cautious for less than 3 years, and all were normal for 3 years or more and 6 years or more, as shown in FIG. 8.

(3) In the group that took only DTS, 50% of the patients who took 3 to 6 years needed attention, and all the patients who took 6 years or more were normal, as shown in fig. 9.

(4) Both YHK and DTS were taken normally even less than 3 years, as shown in fig. 10. Although 18% of patients who had taken the medicine for 3 to 6 years (2 of 11 cases) need to be paid attention, the age distribution in this study is an age group close to the upper limit of 76 to 80 years, and therefore, it can be considered that the patient is affected by an increase in age.

2. Age-related analysis of test subjects

(1) The subjects who took only YHK, as shown in fig. 11, were all normal as long as the age group was known.

(2) In the test subjects who took only DTS, as shown in fig. 12, patients who needed attention (33% of the total) appeared in the group aged 61 to 65.

(3) In the test subjects who took both YHK and DTS, as shown in fig. 13, 6% of the total were noticed in the group aged 76 to 80 years, and all others were normal.

According to the above results, no patients with early dementia and requiring attention were observed in the test subjects who took YHK3 or more alone. It is judged that if DTS is taken alone, the result of improving liver and kidney functions can be obtained if DTS is taken for a long period of 6 years or more, and brain cells are easily activated. In the case of YHK and DTS combined, all patients who had not developed early dementia and who needed attention even after taking them for less than 3 years were normal. From this, it was determined that YHK or a combination of YHK and DTS is effective for the prevention of dementia.

Data published in police where elderly people over 70 years old are checked for updated driver's license as described in the background section: (http://www.zensiren.or.jp/kourei/data/data.html) Of these, class 1 (one with a decreased memory and judgment) was 2.3%, class 2 (one with a slightly decreased memory and judgment) was 26.7%, and class 3 (one with a slightly decreased judgment)Memory and judgment without any problem) was 71.0%. As compared with this data, in this example, the total of 26 subjects over 70 years old who took YHK, DTS, or both YHK and DTS were 7.7% of 2 subjects (corresponding to the above-mentioned group 2) and 92.3% of normal (corresponding to the above-mentioned group 3) subjects, and it was found that the dementia prevention and treatment effect of the present invention was good.

Claims

1. An application of a crude drug composition containing Notoginseng radix and Eucommiae cortex as effective crude drug components in preparing medicine for preventing or treating dementia is provided, wherein the Notoginseng radix accounts for 10-90 wt%, and the Eucommiae cortex accounts for 10-90 wt%.

2. The use according to claim 1, wherein the prevention or treatment of dementia is by increasing the expression of neurofilaments or promoting the growth of neurites.

3. An application of a crude drug composition taking pseudo-ginseng, eucommia ulmoides, rhizoma polygonati and liquorice as effective crude drug components in preparing a medicine for preventing or treating dementia is disclosed, wherein the pseudo-ginseng accounts for 40-60 wt%, the eucommia ulmoides accounts for 30-50 wt%, the rhizoma polygonati accounts for 2-25 wt%, the liquorice accounts for 3-20 wt%, and the sum of the crude drug components is limited to 100 wt%.

4. The use according to claim 3, wherein the prevention or treatment of dementia is by increasing the expression of neurofilaments or promoting the growth of neurites.

5. The use according to claim 3 or 4, wherein the content of the above-mentioned Panax notoginseng is 40 to 60 wt%, the content of the above-mentioned eucommia ulmoides is 30 to 50 wt%, the content of the above-mentioned Polygonatum sibiricum is 2 to 25 wt%, and the content of the above-mentioned Glycyrrhiza glabra is 5 to 20 wt%, with the total content of the above-mentioned crude drug components being 100 wt%.

6. The use according to any one of claims 1 to 4, wherein the effective crude drug component is an extract of each crude drug component.