WO2000004912A1

WO2000004912A1 - Therapeutic agent for dementia, comprising ginseng extract

Info

Publication number: WO2000004912A1
Application number: PCT/KR1999/000381
Authority: WO
Inventors: Inhee Mook; Kyoon Huh; In-Soo Joo; Min-Whan Jung
Original assignee: Digital Biotech Co., Ltd.
Priority date: 1998-07-20
Filing date: 1999-07-19
Publication date: 2000-02-03
Also published as: AU4934499A; KR20000006625A

Abstract

Disclosed is a therapeutic agent for dementia and a method for treatment of dementia. It comprises as medicinally effective component the ginsenoside Rb1 or Rg1, both being ginseng extracts. The therapeutic agent has an efficacy against neuronal cell death ascribed to beta-amyloid, a fundamental factor causing dementia such as Alzheimer's disease, by virtue of the advantages of the ginsenoside Rb1 and Rg1 acting specially on the hippocampus to activate the formation of synapses as well as the signal transduction between synapses, leading to an improvement in learning and memory.

Description

THERAPEUTIC AGENT FOR DEMENTIA, COMPRISING GINSENG

EXTRACT

Field of the Invention

The present invention relates to a therapeutic agent

for dementia, comprising a ginseng extract as a medicinally

effective component and a method for the treatment of

dementia which comprises administering a ginseng extract.

Description of the Prior Art

The fundamental cause of dementia resides in neuronal

cell death. In view of this point, conventional medicines

which have been used to treat dementia cannot be said to be

genuine therapeutic agents because they all function only to

enhance the activity of acetylcholine, a neurotransmitter,

but are unable to prevent neuronal cell death itself. It is

true that, in an early stage of dementia, the neuronal cells in which acetylcholine is implicated as a transmitter are

damaged. However, dementia is also caused by the damage of

the neuronal cells in the electric transmission mechanism of

which other neurotransmitters, such as glutamate, are

involved. Therefore, increasing the activity of

acetylcholine shows a temporary effect only for the patients

who are in the early stage of dementia.

Since glutamate serves as a neurotransmitter with the

most abundance in the brain, it is expected that a glutamate

activator, if it is developed, could have more potent

activity against dementia than could acetylcholine

activators. However, the glutamate activator functions only

to enhance the interaction between the neurons which still

remain intact after the occurrence of neuronal cell death,

rather than prevent the neuronal cells from being damaged.

Accordingly, the glutamate activator cannot be a fundamental

therapeutic agent.

Extensive clinical and genetic research have recently

been directed to dementia, indicating that, when beta- amyloid is accumulated in the brain, it damages neuronal

cells, leading to causing dementia such as Alzheimer's

disease .

In order to cure dementia, therefore, there is a strong

demand for the development of a therapeutic agent which can

restrain beta-amyloid from being formed as well as eliminate

the toxicity of beta-amyloid.

Disclosure of the Invention

It is, therefore, an object of the present invention to

overcome the above problems encountered in prior arts and to

provide a dementia-curing agent which is able to prevent the

neuronal cell death attributable to the accumulation of

beta-amyloid in the brain and regenerate the neuronal cells

which are undergoing a cell death program.

In accordance with the present invention, the above

object could be accomplished by a provision of a therapeutic

agent for dementia, comprising ginsenoside Rbl or Rgl as a

medicinally effective component. The intensive and thorough research on the prophylaxis

and treatment of dementia, repeated by the present

inventors, resulted in the finding that ginseng extracts,

especially ginsenosides Rbl and Rgl, are strongly preventive

of neuronal cell death attributable to beta-amyloid and

curative of neuronal cell damage.

From ancient time, ginseng has been used as a crude

drug in the Orient. Now, ginseng is found to produce no

harmful effects. With this advantage in mind, the present

invention utilizes ginseng extracts, especially ginsenosides

Rbl and Rgl, in treating the neuronal cell damage ascribed

to the accumulation of beta-amyloid.

Brief Description of the attached Drawings

The above and other objects, features and other

advantages of the present invention will be more clearly

understood from the following detailed description taken in

conjunction with the accompanying drawings, in which: Fig. 1 is a graph showing the results of the space

perception ability test using the Morris water maze

technique for the control and the test groups administered

with ginsenosides Rgl and Rbl;

Fig. 2 shows Western blots of synaptophysin 38 kDa in

molecular weight, obtained from the control and the groups

administered with ginsenosides Rbl and Rgl; and

Fig. 3 is a histogram showing the average synaptophysin

levels, along with the standard deviations, in the

hippocampus and the cortex for the control, the Rbl-

administered group and the Rgl-administered group.

Modes for Carrying Out the Invention

Below, a detailed description will be given of the

curative effect of ginsenosides Rbl and Rgl on dementia,

which was demonstrated by the experiments the present

inventors had performed.

A better understanding of the present invention may be

obtained in light of the following examples which are set forth to illustrate, but are not to be construed to limit

the present invention. In the following examples, an in

vi tro experiment was described in Example I while the

ginsenosides were directly administered to test animals in

Example II { in vivo experiment) .

EXAMPLE I

There was established a system in which neuronal cells

were growing, but showed a cell death program when being

added with beta-amyloid. An examination was made of whether

the cell death program of the neuronal cells was prevented

or ceased or not when they were subjected to the

pretreatment , cotreatment or posttreatment with a crude

ginseng extract, commercially available from Sigma Co.,

U.S.A., with the standard of the time when beta-amyloid was

added. As a result, an effect of restraining neuronal cell

death, even though the effect was slight, was obtained.

Ginsenosides Rbl and Rgl, commercially available from

Wako Co, were also investigated for their activity against the neuronal cell death ascribed to the accumulation of

beta-amyloid. For all cases of the pretreatment , the

cotreatment and the posttreatment , a potent inhibitory

activity against cell death were observed.

In more detail, by adding various concentrations of

beta-amyloid, believed to be a cause of Alzheimer disease,

to media in which neuronal cells were being cultured, there

was established the concentration of beta-amyloid at which

the neuronal cells started to die. The neuronal cells were

pretreated, cotreated or posttreated with the ginseng

extracts with respect to the addition of beta-amyloid and

then, cultured for 18 hours, followed by measuring the

inhibitory effect of the ginseng extracts on neuronal cell

death attributable to beta-amyloid. With regard to the

measurement, an MTT (3- (4, 5-dimethyl-2-thiazoly) -2, 5-

diphenyl-2H-tetrasolium bromide) assay was taken.

MTT assay In a culture medium in which neuronal cells were

growing, 15μl of an MTT solution were added, after which

they were cultured at 37°C for 3-4 hours. The MTT solution

was prepared by dissolving 2-5 mg of the MTT reagent per ml

of PBS. After being added with lOOμl of a solubilization

buffer containing 10 % of sodium dodecyl sulfate (SDS) and

50 % of dimethylformaldehyde with pH 4.7, the culture medium

was let to stand at room temperature for 24 hours. Then,

the medium was measured for absorbance at 570 nm and 630 nm.

Since the absorbance at 630 nm was a reference level, the

values resulting from subtraction from the absorbance at 570

nm to that at 630 nm were recorded.

The results of the MTT assay are given in Tables 1 and

2, below.

While the absorbance data obtained from the neuronal

cell group which was treated with beta-amyloid only, were

regarded zero %, the absorbance data from the neuronal cell

group which was treated with the ginseng extract components

in the absence of beta-amyloid were set as 100 %. When neuronal cells were treated with beta-amyloid and ginseng

extract both, how many neuronal cells were survived could be

represented as relative percentages.

TABLE 1

Efficacy Test of Rbl

In Table 1, the control was treated with neither beta-

amyloid nor the ginseng extract Rbl. For the neuronal cell group treated with beta-amyloid only, 50μM of beta-amyloid

were used.

As shown in Table 1, the addition of beta-amyloid had

the neuronal cell group begin to undergo a cell death

process. However, when a variety of Rbl concentrations were

added to the neuronal cell group which had already undergone

a cell death process, the cells were restored to life again,

with an increase in the absorbance. Calculating an average,

the increase rate obtained was 1.45% upon addition of Rbl at

an amount of lOnM, 4.76% upon addition of Rbl at an amount

of 50nM, and 66.04% upon addition of Rbl at an amount of

lμM. Consequently, the most potent efficacy against cell

death attributed to beta-amyloid was expressed when Rbl was

used at an amount of lμM in this experiment.

TABLE 2

Efficacy Test of Rgl

In Table 2, the control was treated with neither beta-

amyloid nor the ginseng extract Rgl. For the neuronal cell

group treated with beta-amyloid only, 50nM of beta-amyloid

were used.

As shown in Table 2, the presence of beta-amyloid

induced the neuronal cell group to undergo apoptosis.

However, when a variety of Rbl concentrations were added to

the neuronal cell group which had already undergone a cell

death program, the cells were regenerated with an increase

in the absorbance. Calculating an average, the increase rate of the absorbance was 4.23% upon addition of Rgl at an

amount of 10 nM, 13.03% upon addition of Rgl at an amount of

50nM, and 32.5% upon addition of Rgl at an amount of lμM.

Consequently, the most potent efficacy against cell death

attributed to beta-amyloid was expressed when Rbl was used

at an amount of lμM in this experiment.

EXAMPLE II

In this example, after ginsenosides Rbl and Rgl were

injected to live mice, a test was made of space perception

ability, one of the most representative criteria of

dementia, and a cerebrotomy operation was conducted to

investigate the synapse density in the brain.

For a space perception ability test, 60 B6 mice were

used. A solution of a ginseng extract containing

ginsenoside Rbl or Rgl in physiological saline was

intraperitoneally injected into the mice at a dose of lmg

per kg of the body weight of the mice at 24 hour intervals

for 4 days. After completion of the four-day- administration, the behavior of the mice was observed from

the fifth day.

In order to test the mice for space perception ability,

the Morris water-maze technique was adopted. Testing was

conducted twice a day for 5 days while the water was

maintained at 21-24 °C. Upon each test, the testees were

positioned at any starting point and allowed to discover a

platform concealed in the maze. Where the testees did not

reach the platform within a maximum of 60 seconds, they were

regarded as to have failed the test. If so, the mice were

let to be positioned at the platform for 10 seconds. In the

course that the mice sought for the platform, the latency,

the swimming speed and the swimming trace were recorded at

every trial.

With reference to Fig. 1, there is a graph showing the

space perception ability change with the trial number for

the control and the test groups administered with

ginsenosides Rgl and Rbl. As apparent in this graph, the

group administered with ginsenoside Rbl or Rgl is superior in space perception ability to the control. From the second

day of the space perception ability test, the administration

of the ginsenosides showed an effect of enabling the mice to

reach the platform within shorter periods of time compared

with those of the control. The difference in the latency

between the ginsenoside-administered groups and the control

got wider as the learning was repeated.

Next, the brains were enucleated from the mice which

were administered with the ginsenosides Rgl and Rbl and with

physiological saline, in order to examine the density of the

synapses therein. To this end, synaptophysin, a

representative of synaptic vesicles, which are densely

distributed near the cytoplasmic membrane of the presynaptic

axon, was quantified and its changes in quantity were

compared between the administered groups and the control.

In relation to this change, if the region in which the

density of synapses is increased is coincident with the

region which is responsible for learning and memory of the

brain, the difference in space perception ability between pre- and post-administration of the ginsenosides might be

believed to be closely connected with the mechanism of

increasing the density of synapses in the brain.

As a standard to detect the density of synapses,

synaptophysin, a synapse-unique protein, was measured for

the level in the brain of the mice which had experienced the

space perception ability test, through a Western blotting

technique. The target tissue which was suitable for this

goal was the hippocampus because it takes charge of the

memory of the brain, while the cortex was used for

comparison.

With reference to Fig. 2, there are Western blots of

synaptophysin 38kDa in molecular weight, obtained from the

control and the groups administered with ginsenosides Rbl

and Rgl. As indicated from the Western blots of Fig. 2,

both of the groups administered with the ginsenosides Rbl

and Rgl showed that the level of synaptophysin was increased

specifically in the hippocampus, but not in the cortex. For the control, no observation was made of the level increase

of synaptophysin in the hippocampus.

Amounts of synaptophysin in the brain were measured

with the aid of a densitometer, followed by calculating

average values and their standard deviation from the

measurements and then, comparing them between the

administered groups and the control.

Referring to Fig. 3, there are histograms showing the

average synaptophysin levels, along with the standard

deviations, in the hippocampus and the cortex for the

control, the Rbl-administered group and the Rgl-administered

group. As seen in Fig. 3a, the level of synaptophysin in

the hippocampus was increased about 2.1 times for the Rbl-

administered group and about 1.8 times for the Rgl-

administered group greater than for the control. In the

case of the cortex, which is seen in Fig. 3b, almost no

differences in the level of synaptophysin were found among

the control, the Rbl-administered group and the Rgl-

administered group. Taken together, the data obtained in the above examples

indicate that the ginsenosides Rbl and Rgl of ginseng

extracts act specifically on the hippocampus to activate the

formation of synapses as well as the signal transduction

between synapses, leading to an improvement in the learning

and memory. Based on these experimental data, therefore,

the present invention verifies the efficacy of the

ginsenosides Rbl and Rgl as therapeutic agents for dementia.

Further, the present invention provides a basis on which

clinical experiments can be carried out to produce dementia-

curing agents.

As described hereinbefore, the therapeutic agent for

dementia, according to the present invention, based on

ginsenoside Rbl or Rgl, has advantages of being preventive

of neuronal cell death ascribed to beta-amyloid, a

fundamental factor causing dementia such as Alzheimer

disease, and acting specifically on the hippocampus.

Claims

CLAIMES

1. A therapeutic agent for dementia, comprising ginsenoside

Rbl or Rgl as a medicinally effective component.

2. A pharmaceutical composition comprising an effective

amount of ginsenoside Rbl or Rgl together with a

pharmaceutically acceptable carrier or diluent.

3. A method for the treatment of dementia which comprises

administering ginsenoside Rbl or Rgl.