WO2001056585A1

WO2001056585A1 - Cognitive enhancer comprising ginseng extract

Info

Publication number: WO2001056585A1
Application number: PCT/KR2000/000323
Authority: WO
Inventors: Min Whan Jung; Inhee Mook
Original assignee: Digital Biotech Co., Ltd
Priority date: 2000-01-31
Filing date: 2000-04-07
Publication date: 2001-08-09
Also published as: KR20010077131A; KR100375018B1; AU2000241467A1

Abstract

Disclosed is a cognitive enhancer. It comprises as a principle the ginsenoside Rb1 or Rg1. These cognitive enhancing principles exert action specifically on the hippocampus to facilitate the formation of synapses as well as synaptic transmission between synapses, and lead to an improvement in the learning and memory for which the hippocampus is responsible.

Description

COGNITIVE ENHANCER COMPRISING GINSENG EXTRACT

Field of the Invention

The present invention relates, in general, to a cognitive

enhancer comprising ginseng extract as a main component and,

more particularly, to ginsenosides Rbl and Rgl as cognition

enhancing principles.

Description of the Prior Art

Based on the research results which have been known thus

far, memory can be divided into two types: explicit memory and

implicit memory. Usually, the commonly used memory is referred

to as explicit memory, which is exemplified by the recalling

of human faces or events that one has experienced.

Since the Scoville and Milner' s report published in 1957, extensive research on the neural mechanisms of memory has been

conducted, revealing that the hippocampus and its neighboring

regions are essentially responsible for explicit memory.

Spatial memory is a representative example of explicit

memory. In other animals but humans, it has been suggested that

the function of the hippocampus might be confined to spatial

memory and other forms of explicit memory might have evolved

from spatial memory. Accordingly, spatial memory is the most

widely adopted to analyze the function of the hippocampus in

animals, such as rats or mice. In this regard, the Morris water

maze task is the most favorite among many learning tasks.

In connection with the efficacy of ginseng on memory

enhancement, avoidance tasks have been most widely used. Mainly

responsible for the avoidance task is the amygdala rather than

the hippocampus. Thus, the avoidance task is not suitable as

a paradigm for the explicit memory in which the hippocampus

plays an important role.

Extensive research has been conducted, reporting that ginseng extract can enhance learning and memory ability. For

the most part, however, previous research is concerned with

ginseng effects on the activity of acetylcholine, focused on

the extracts rather than ginsenosides Rbl and Rgl, tested ginseng

effects on brain damaged animals, or did not analyze spatial

learning ability which is directly relevant to the hippocampus.

Particularly, Ying asserted, in his article entitled "Study

on the neurotropic mechanism of ginsenoside Rbl and Rgl —

influence on mouse brain development", that ginsenosides Rbl

and Rgl function to increase the number of the synapse in the

hippocampal CA3 region. Because this research focuses on the

development of the nerve systems of weaning mice, it is relevant

to brain development rather than learning and memory.

In an article of Sakanaka, entitled "Neurotrophic actions

of ginsenosides Rbl, peptide growth factors and cytokines",

it was reported that the animal group administered with Rbl

exhibits improvement in spatial learning in the Morris water

maze compared to control. However, this research result was obtained from an ischemic animal model, but not from normal

animals. They measured the extent Rbl blocks impairment of

learning ability which is induced by ischemic neuronal death

in hippocampal CA1 area. This research is more relevant to

neuroprotective effects of Rbl against ischemic insult, thus

quite different from the research of enhancing spatial learning

ability of normal animals.

In consequence, no research results are disclosed as to

the enhancement of spatial learning and memory directly related

to the hippocampus of normal animals.

Disclosure of the Invention

Therefore, it is an object of the present invention to

provide cognitive enhancers which facilitate synaptogenesis

in the hippocampus and enhance explicit memory capability.

Based on the present invention, the above object could

be accomplished by a provision of a cognitive enhancer comprising

ginsenoside Rbl or Rgl as a main component, which facilitates the formation of hippocampal synapses

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 spatial learning

ability test using the Morris water maze technique for the control

and the test groups administered with ginsenosides Rbl and Rgl;

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;

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;

Fig.4 shows the results of the synaptic plasticity measured from the control, the Rbl-administered group and the

Rgl-administered group; and

Fig. 5 shows the results measured for the intensity of

response to given stimuli in the control, the Rbl-administered

group and the Rgl-administered group.

Modes for Carrying Out the Invention

A detailed description will be given of the cognitive

enhancer comprising ginseng extract as a main component, below.

After ginsenosides Rbl and Rgl were administered to live

mice, a cerebrotomy operation gave information about synapse

density in the brain: the synapse density was increased in the

hippocampus, but not in the neocortex. In other words, the

synapse density of the hippocampus is selectively increased

upon the administration of ginsenoside Rbl and Rgl.

In addition, after ginsenosides Rbl and Rgl were administered

to live mice, they were tested for spatial learning in the Morris watermaze. Inthistest, anoticeable improvement in the learning

ability was detected from the administered mice, compared to

control .

After ginsenosides Rbl and Rgl were administered to live

mice, the hippocampus was excised from them, sliced and cultured.

As an intensive neurophysiological research, it was found that

the intensity of the response to a given stimulus was greater

in an Rbl administered group than other groups. This result,

along with the finding that no changes occurred for the

physiological indices of each synapse, indicates that Rbl has

no influence on the function of each synapse, but increases

the number of synapses to improve the learning ability.

To sum up, the research of the present inventors demonstrates

that the ginseng extracts Rbl and Rgl facilitate the formation

of synapses in the hippocampus nerve system and enhance the

learning and memory ability for which the hippocampus is

responsible for.

In the following examples, the influence of ginsenosides Rbl and Rgl on spatial learning ability of mice and the formation

of synapses in the brain will be better understood with reference

to the accompanying drawings.

For a spatial learning 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 1 mg per kg of the body weight of

the mice at 24 hour intervals for 4 days. Into a control group,

a physiological saline was administered in the same manner.

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 spatial learning 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 testee was positioned at an

arbitrary starting point and allowed to discover a platform

concealed in the maze . Where the testee 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. While the mice sought for the

platform, the latency, the swimming speed and the swimming trace

were recorded in each trial.

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

learning 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 spatial learning ability

to the control. From the second day of spatial learning 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 isolated from the mice which were

administered with the ginsenosides Rgl, Rbl or physiological

saline, in order to examine the synaptic density. To this end, synaptophysin, which is a synaptic marker protein, was quantified

using Western blotting technique and its levels were compared

between the administered groups and the control. Synaptophysin

levels were measured in the same group of animals that experienced

Morris water maze test so that changes in synaptic density and

learning ability can be compared in the same animals. The target

tissue formeasurement of synaptophysin is the hippocampus because

it is essential for spatial learning and memory The neocortex

was used for comparison.

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

synaptophysin 38 kDa 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 (Fig. 2a), but not in the cortex (Fig. 2b).

In the control, the level of synaptophysin in the hippocampus

was not changed. 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 for 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 (Fig. 3a) and in the cortex (Fig. 3b) 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 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.

Next, an examination was made of the change in the

physiological index of the brain. After being administered with ginsenosides Rgl and Rbl,

mice were rendered to undergo a cerebrotomy operation to isoalte

the brain. The brain was sliced into a thickness of 400 μm,

and the slices were cultured for a short period of time (3 to

6 hours) while their physiological indices were measured.

Schaffer collateral/commissural fibers of the hippocampus

were stimulated with a metal electrode. A glass electrode was

used to measure the initial slope of field potential responses

in the CA1 stratum radiatum region . In order to measure long-term

potentiation, which is a form of long-term synaptic plasticity

and a leading candidate for the substrate of memory, stimulation

was conducted in three steps . First, low-frequency stimulation

was delivered until stable baseline responses were obtained

for at least for 10 min. Then, theta burst stimulation (TBS),

a type of high-frequency stimulation, was delivered for 2 seconds,

and the stimulation pattern was changed back to the original

low frequency stimulation. The degree of long-term potentiation was assessed by measuring comparing field potential responses

to low frequency stimulation before and after TBS. For the

measurement of the paired-pulse facilitation, a formof short-term

synaptic plasticity, two stimuli were delivered at an interval

of 50 ms. A measurement was made of the extent to which the

response to the second stimulus was increased compared to that

of the response to the first stimulus. The input-output

relationship was determined by measuring field potential

responses while stimulation intensity was allowed to vary several

steps.

Turning to Fig.4, results of synapticplasticity experiments

are shown for the control group administered with no ginseng

extracts, the ginsenoside Rbl-administered group, and the

ginsenoside Rgl-administered group.

Fig.4a shows results of long-termpotentiation experiments .

AfterTBS, as shown in Fig.4a, the initial slopes, which correspond

the magnitudes of the response, were all increased.

Electrophysiological records are drawn on the upper portion of Fig. 4a. The responses before and after TBS are shown

overlapped. As apparent from these patterns, the response after

TBS was increased. The graph at the lower right side in Fig.

4a shows the mean values of the response magnitudes after TBS

(n=3) and the standard error means. As shown, the responses

were increased by as much as about 50 %, compared to the baseline

responses. No statistically noticeable differences were found

among the experimental groups.

In Fig. 4b are given the measurements of the paired-pulse

facilitation at an interval of 50 ms . The upper illustrations

are typical examples of paired-pulse facilitation. As shown

in Fig. 4b, when the synapses are stimulated twice at an interval

of 50 ms, the response to the second stimulus is increased in

comparison with the first. Also, the mean values over three

experiments and the standard error means are shown for the

experimental groups. In all cases, the response to the second

stimulus was increased by as much as about 80 % compared to the first. Among the experimental groups, no statistically

noticeable differences were found.

With reference to Fig. 5, input-output curves that relate

input stimulus intensity and output response size are shown

for the control which was administered with no ginseng extracts,

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

as mean values along with their standard error means. While

no significant differences are found between the control and

the Rgl-administered group, the Rbl-administered group responded

to the stimulation at considerably higher magnitudes than did

the control .

Taken together, the results obtained in the above experiments

demonstrate that ginsenosides Rbl and Rgl exert action

specifically on the hippocampus to facilitate the formation

of synapses as well as the synaptic transmission 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 cognitive enhancers. Further, the present invention provides a basis

on which clinical experiments can be carried out to produce

cognitive enhancers.

As described hereinbefore, a cognitive enhancer based on

ginsenoside Rbl or Rgl is provided. These cognitive enhancing

principles activate the synapse formation in the hippocampal

nervous system of the brain, and lead to enhanced learning and

memory for which the hippocampus is responsible for.

The present invention has been described in an illustrative

manner, and it is to be understood that the terminology used

is intended to be in the nature of description rather than of

limitation. Many modifications and variations of the present

invention are possible in light of the above teachings . Therefore,

it is to be understood that within the scope of the appended

claims, the invention may be practiced otherwise than as

specifically described.

Claims

CLAIM

1. A cognitive enhancer, which comprises ginsenoside Rbl

or Rgl as a main principle and plays a role in facilitating

the formation of hippocampal synapses in the brain.

2. A cognitive enhancer, which comprises ginsenoside Rbl

or Rgl as a main principle and plays a role in facilitating

explicit learning and memory capability.