WO2002094302A1

WO2002094302A1 - Compositions comprising extract of ganoderma lucidum, oleamide and its structural analogue as an effective component for preventing or treating dementia

Info

Publication number: WO2002094302A1
Application number: PCT/KR2002/001012
Authority: WO
Inventors: Gil-Ja Jhon; Hwa-Jung Kim; So-Yeop Han; Hyun-Jin Cho; Ho-Sil Park; Jee-Sun Lee; Jin-Hyoung Kim; Jung-Yung Lee; Myung-Hee Lee
Original assignee: Gil-Ja Jhon; Hwa-Jung Kim; So-Yeop Han; Hyun-Jin Cho; Ho-Sil Park; Jee-Sun Lee; Jin-Hyoung Kim; Jung-Yung Lee; Myung-Hee Lee
Priority date: 2001-05-23
Filing date: 2002-05-23
Publication date: 2002-11-28
Also published as: CN1523994A; CN1788722A

Abstract

The present invention is related to a composition for preventing and treating dementia containing the extracts of ganoderma lucidum, oleamide and its structural analogues as effective components. Since the composition according to the present invention prevents reduction of the amount of acetylcholine in the brain and shows the protective effect for cerebral tissues damaged by cerebral ischemia as it inhibits the activity of acetylcholinesterase (AChE) of animal cerebral tissues, it may be used for a prevention and treatment agent not only for the Alzheimer's disease but also vascular dementia.

Description

COMPOSITIONS COMPRISING EXTRACT OF GANODERMA LUCIDUM, OLEAMIDE AND ITS STRUCTURAL ANALOGUES AS AN EFFECTIVE COMPONENT FOR PREVENTING OR TREATING DEMENTIA

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention is related to a composition for preventing and treating dementia containing the extracts of ganoderma lucidum, oleamide and its structural analogues as effective components.

Description of the Prior Art

Dementia is a pathologic phenomenon that should be distinguished from normal aging, and is divided into the Alzheimer's disease, vascular dementia, other alcoholic intoxication, and that coming from the sequel of Parkinson's disease.

The mechanism of invasion by the Alzheimer's disease has not been clarified yet. However, since the reduction of acetylcholine (ACh) functions of the central nervous system (CNS) is shown most commonly, the acetylcholine concentration of the brain is increased by administering the precursor of acetylcholine or a drug inhibiting decomposition of acetylcholine for its treatment.

Therefore, the sole inhibitor of acetylcholinesterase (hereinafter referred to as

'AChE') or the conventional drugs used in combination with the inhibitor of cholinesterase are used for its treatment. Representative drugs include tacrine, donepezil, rivastigmine, galantamine, etc.

And since the vascular dementia occurs due to the damage to brain cells as the blood supply is insufficient in many sites of the brain by hardening of brain blood vessels most of the time, it is desirable to apply a treatment agent of hypertension acting on brain blood vessels, aspirin, or a drug protecting damaged brain cells for its treatment.

Since the conventional treatment agents of dementia have had only the effect of inhibiting acetylcholinesterase mainly, it is required to develop drugs showing treatment effects on the vascular dementia. Ganoderma lucidum is a kind of mushroom. It has been used for a traditional drug in the east, and it has been known that it has had the anti- inflammation effect, anti-cancer effect, immunoregulation effect, AIDS virus inhibition effect, blood pressure lowering action, etc. The studies on the efficacy of ganoderma lucidum include a research paper in which it is confirmed that the oral application of the extract of ganoderma lucidum in water or ethyl acetate to the mice having edema occurred by carrageenan and croton oil is effective for both types of edema (Stavihovha W. and Slama J., The Anti-inflammatory Ganoderma Lucidum Third International Symposium on Ganoderma Lucidum: 9-21) and a research paper in which it is shown that the water extract of ganoderma lucidum has an effect of suppressing the growth of cancer cells (Wang S.Y. et al., The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages and T lymphocytes, Int. J. Cancer 70:699-705). In addition to the above, it is known that LZ-8, which is the immunomodulating protein separted from ganoderma lucidum, shows the mitogenic activity in vitro and the immunomodulating activity in vivo [Kino K. et al., Immunomodulator, LZ-8, prevents antibody production in mice, Int. J. Immunopharmacology 13 (8): 1109-1115].

SUMMARY OF THE INVENTION

The inventors of the present invention have looked into safe and non-toxic natural resources that can show prevention and treatment effects for not only the Alzheimer's disease but also vascular dementia. The present invention was completed as the components showing the inhibition effect of cerebral ischemia while suppressing the activity of AChE have been extracted from ganoderma lucidum which has been used traditionally in Korea, and the materials related to the above have been confirmed to be useful for the treatment agents of dementia.

Therefore, an object of the present invention is to provide with a composition showing prevention and treatment effects for not only the Alzheimer's disease but also vascular dementia. The present invention provides with a composition for preventing and treating dementia containing the extracts of ganoderma lucidum, and oleamide and its structural analogues as effective components.

The composition of the present invention shows the prevention and treatment effects for the Alzheimer's disease as it shows the inhibiting effect of the AchE activity by recovering reduction of the amount of acetylcholine in the brain as well as for the vascular dementia as it shows the protective effect for brain tissues that are damaged by cerebral ischemia.

The composition of the present invention is illustrated in detail. Firstly, the method of obtaining the extracts of ganoderma lucidum that are used for effective components in the composition of the present invention is as follows:

Ganoderma lucidum is extracted with hexane, and the residuum of extraction is extracted with chloroform. Then the methanol extract of ganoderma lucidum (hereinafter referred to as 'Extract GL-M') is obtained by extracting the residuum of extraction with methanol again and distilling the extracted solution obtained under a reduced pressure. The amounts of hexane, chloroform, and methanol that are the solvents of extraction to be used are as much as ganoderma lucidum that is used is soaked in, i.e., about 2 liters of hexane, chloroform, and methanol per kg of ganoderma lucidum generally. Extract GL-M, which is the methanol extract of ganoderma lucidum obtained according to the above method, is separated and refined in fractions continuously according to a series of silica gel column chromatography and HPTLC method. In other words, the method includes the steps of a) obtaining 14 extracts, i.e., from Extracts GL-M-1 to GL-M-14, according to the Rf value by processing the fractions, obtained through the silica gel column chromatography of Extract GL-M, which is the methanol extract of ganoderma lucidum, by using chloroform/methanol (500:1) as an eluate, with HPTLC; b) obtaining 7 extracts, i.e., from Extracts GL-M- 8-A to GL-M-8-G, according to the Rf value by processing the fractions, obtained through the silica gel column chromatography of Extract GL-M-8 among the extracts obtained in the above Step a) by using chloroform/methanol (100:1) as the elute, with HPTLC; c) obtaining 5 extracts, i.e., from Extracts GL-M-8-F-a to GL-M-8-F-e, according to the Rf value by processing the fractions, obtained through the silica gel column chromatography of Extract GL-M-8-F among the extracts obtained in the above Step b) by using chloroform/methanol (50:1) as the elute, with HPTLC; d) obtaining 4 extracts, i.e., from Extracts GL-M-8-F-b-I to GL-M-8-F-b-JV, according to the Rf value by processing the fractions, obtained through the silica gel column chromatography of Extract GL-M-8-F-b among the extracts obtained in the above Step c) by using chloroform/methanol (50:1) as the elute, with HPTLC; and e) obtaining Extract GL-M-8-F-b-III-l according to the Rf value by processing the fractions, obtained through the silica gel column chromatography of Extract GL-M- 8-F-b-III among the extracts obtained in the above Step d) by using chloroform/methanol (30:1) as the elute, with HPTLC.

Extracts GL-M-8, G1-M-8-F, GL-M-8-F-b, GL-M-8-F-b-III, and GL-M-8-F- b-III-1 obtained in the above-described method may be used for active components in the composition of the present invention since they inhibit the activity of AChE as proven in preferred embodiments described below:

The qualitative analysis of Extract GL-M-8-F-b-III-l obtained finally according to the method of the present invention is performed in order to clarify active components contained in the methanol extract of ganoderma lucidum used for effective components in the composition of the present invention. And it is confirmed that the structure of the fraction is the same as that of 9-octadecenamide (oleamide). It is therefore seen that oleamide, which is known to be a material in the brain inducing sleeping, is contained in the extract of ganoderma lucidum according to the present invention as an active component. The above-described oleamide compound may be manufactured in various chemical synthetic methods, and the method of manufacturing oleamide and its structural derivatives is as follows:

The aliphatic acid compound (4) is reacted with thionyl chloride (SOCl ) at 70 to 80°C, after which the reaction mixture is cooled to the room temperature and a cool ammonium solution is added slowly. Oleamide and its Structural Derivative (1) are obtained by filtering the reaction mixture produced under a reduced pressure and re-crystallizing. The chemical equation is as in the following Equation 1 : <Equation 1>

(4) (1 )

where R- is a 9-octadecenoyl (oleyl), 9-hexadecenoyl (pahnitoleyl), or tetradecanoyl (myristyl) radical.

Also, Structural Derivative (2) of oleamide is obtained by dissolving the aliphatic compound (5) in dichloromethane, adding dicyclohexylcarbodiamide (DCC) and dimethylaminopyridine (DMAP), further adding 3-aminopentane to have the reaction mixture react for a fixed amount of time, concentrating the remaining solution after filtering the solid produced, and performing column chromatography of this concentrated compound. The chemical equation is as shown in Equation 2 below: <Equation 2>

9 3-Aminopentane V

(5) (2)

where R₂ is a 9-octadecenoyl (oleyl), 9,12-octadecadienoyl (linoleyl), or tetradecanoyl (myristyl) radical. Still further, Structural Derivative (3) of oleamide is obtained by dissolving linoleic acid (6) in dichloromethane, adding dicyclohexylcarbodiarnide and dimethylaminopyridine, further adding 3-pentanol to have the reaction mixture reacted for a fixed amount of time, concentrating the remaining solution after filtering the solid produced, and performing column chromatography of this concentrated compound. The chemical equation is as shown in Equation 3 below: <Equation 3>

(6) (3)

where R₃ is a 9,12-octadecadienoyl (linoleyl) radical. As described in the above, oleamide and its structural derivatives manufactured according to the chemical synthetic method show prevention and treatment effects for not only the Alzheimer's disease but also vascular dementia as the extract of ganoderma lucidum does.

The composition of the present invention may contain additionally one or more kinds of effective components that can treat dementia and other diseases besides effective components such as the extracts of ganoderma lucidum, oleamide, and its structural derivatives.

Further, the composition of the present invention may be manufactured to include one or more kinds of carriers that are allowable pharmacologically or for the food in addition to the above-described effective components for its administration. Pharmacological carriers or those that are allowable for the food may be the saline solution, sterilized water, Ringer's solution, buffered saline solution,, dextrose solution, malto dextrine solution, glycerol, ethanol, or the mixture of one ox more of the above components. If necessary, other common additives such as an anti- oxidant, buffer solution, bacteriostatic, etc. may be added. Also, the compound may be manufactured in the form of an injection solution such as the aqueous solution, suspension, emulsion, etc., globules, capsule, granules, or tablet by adding additionally a diluent, dispersing agent, surfactant, binder, or lubricant. Still further, the composition may be preferably made as a drug according to each disease or component by using a proper method in the relevant field or a method disclosed in

Remington's Pharmaceutical Science (most updated version) published by Mack

Publishing Company in Easton in Pennsylvania of the United States.

The pharmacological composition of the present invention may be administered abrally or orally. Although the range of doses varies according to the weight, age, sex, state of health, diet, time of administration, method of administration, rate of excretion, degree of seriousness of a disease, etc., it is desirable to administer 0.8-3.2 mg/kg once or a few times a day.

The pharmacological compound of the present invention may be used solely in order to prevent and treat the Alzheimer's disease or vascular dementia or in combination with the operation, radiotherapy, hormone therapy, chemical therapy, and other methods using biological reaction controlling agents.

The form of the food composition of the present invention is made according to common methods. It may be formulated in many forms such as capsuling after being dried along with a carrier, or tablet, granules, powder, drink, porridge, etc. There are many other possible forms besides those described in the above. BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects, and advantages will ^"be better understood from the following detailed description of preferred embodiments of the invention with reference to the drawings, in which:

Figure 1 is a graph showing inhibition of the activity of AChE of the extract of ganodenna lucidum (GL-H: hexane extract of ganoderma lucidum, GL-C: chloroform extract of ganoderma lucidum, GL-M: methanol extract of ganoderma lucidum, GL-E: ethanol extract of ganoderma lucidum) according to the present invention;

Figure 2 is a diagram showing the rate of gain of the fraction separated and refined from the methanol extract of ganoderma lucidum and inhibition of the activity of AChE according to the present invention; Figure 3 is a graph showing inhibition of the activity of AChE of oleamide synthesized according to each concentration;

Figure 4 is a graph showing measurement of the selectivity of AChE/BuChE of GL-M-8-F-b-III-l which is the finally separated and refined fraction of the methanol extract of ganoderma lucidum according to the present invention; Figure 5 is a graph showing measurement of inhibition of the activity of

AChE (in vivo assay) after administering GL-M-8-F-b-III-l and synthesized oleamide according to the present invention;

Figures 6 through 8 are graphs showing the outcome of a passive avoidance experiment in which Extract GL-M-8-F-b-III-l of ganoderma lucidum according to the present invention is administered; and Figure 9 is a diagram showing the affect of Extract GL-M-8-F-b-III-l of ganoderma lucidum according to the present invention on cerebral ischemia.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Hereinafter, preferred embodiments of the present invention are presented in order to facilitate understanding of the present invention.

Preferred Embodiment 1: Effect of inhibition of the activity of AChE by the extract of ganoderma lucidum (in vitro assay)

1. Manufacture of the extracts of ganoderma lucidum

1) Manufacture of Extracts GL-H, GL-C, GL-M, and GL-E of ganoderma lucidum

The 3 kg portion of ganoderma lucidum is crushed into pieces and put into a beaker, to which about twice (about 6 liters) of hexane is added in order to have ganoderma lucidum soaked. The mixture is then heated to 40 to 50°C for 2 hours, extracted twice, and filtered. This filtrate is distilled under a reduced pressure and dried, and 17.6g of Extract GL-H is obtained.

To the remnant from separation of this extract, about 6 liters of chloroform is added to the degree of having the remnant soaked, and the remnant is heated to 40 to

50°C for 2 hours, extracted twice, and filtered. This filtrate is distilled under a reduced pressure and dried, and 55 g of Extract GL-C is obtained. Then to the remnant of separation of the extract, about 6 liters of methanol is added, after which they are mixed at a room temperature for two days, extracted twice, and filtered. The filtrate is distilled under a reduced pressure, and 65 g of Extract GL-M is obtained. To the remnant of separation of this extract, about 6 liters of 70% ethanol is added, and the mixture is mixed at a room temperature for two days, heated, extracted twice, and filtered. The filtrate is distilled under a reduced pressure, and 59 g of Extract GL-E is obtained.

2) Separation and purification from Extract GL-M of ganoderma lucidum

The 60 g portion of Extract GL-M obtained in the above 1) is eluded with chloroform/methanol (500:1) as a solvent in a 10x100 cm column filled with silica gel. Each 100 ml of the eluded fraction is collected, developed in HPTLC using chloroform/methanol (30:1) as a developing solvent, colored with iodine, and separated into 14 fractions according to the Rf value. Extracts GL-M-1 through GL-M- 14 are obtained by distilling under a reduced pressure and drying each fraction separated.

Among them, 300 mg of GL-M-8 is eluded with chloroform/methanol (100:1) as a solvent in a 4x40 cm column filled with silica gel. Each 50 ml of the eluded fraction is collected, developed in HPTLC using chloroform/methanol (30:1) as a developing solvent, colored with iodine, and separated into 7 fractions according to the Rf value. Extracts GL-M-8-A through GL-M-8-G are obtained by distilling under a reduced pressure and drying each fraction separated.

Then, 86 mg of Extract GL-M-8-F is eluded with chloroform/methanol (50:1) as a solvent in a 1.5x35 cm column filled with silica gel. Each 30 ml of the eluded fraction is collected, developed in HPTLC using chloroform/methanol (30:1) as a developing solvent, colored with iodine, and separated into 5 fractions according to the Rf value. Extracts GL-M-8-F-a through GL-M-8-F-e are obtained by distilling under a reduced pressure and drying each fraction separated. Among thus separated and purified extracts, 60 mg of GL-M-8-F-b is eluded with chloroform/methanol (50:1) as a solvent in a 1.2x30 cm column filled with silica gel. Each 10 ml of the eluded fraction is collected, developed in HPTLC using chloroform/methanol (30:1) as a developing solvent, colored with iodine, and separated into 4 fractions according to the Rf value. Extracts GL-M-8-F-b-I through GL-M-8-F-b-IV are obtained by distilling under a reduced pressure and drying each fraction separated.

Then, 40 mg of GL-M-8-F-b-III is eluded with chloroform/methanol (30:1) as a solvent in a 1x25 cm column filled with silica gel. Each 5 ml of the eluded fraction is collected, developed in HPTLC using chloroform/methanol (30:1) as a developing solvent, colored with iodine, and separated into 3 fractions according to the Rf value. Extracts GL-M-8-F-b-III-l through GL-M-8-F-b-III-3 are obtained by distilling under a reduced pressure and drying each fraction separated.

2. Effect of inhibition of the activity of AChE

1) Separation of AChE

AChE is separated from the brains of ICR mice weighing 20-25 g. The heads of ICR mice are cut, their brains are taken out, and their weights are measured. In a volume which is about twice of their weights, 12.5 mM sodium phosphate buffer solution is added. It is homogenized and separated with a centrifuge at 4°C for 10 minutes (xl,000 g). The supernatant liquid is obtained, to which 0.5% Triton X- 100 homogenized buffer solution is added to have a volume of 3 times. The mixture is mixed at 4°C for 30 minutes, and separated with a centrifuge at 4°C for 10 minutes (xl 0,000 g). Each 1 ml of the supernatant liquid is collected in a 1.5-ml tube, and is kept in a rapid deep freezer.

Care is taken not to damage the brains when they are taken out, and the entire process of separation is performed promptly.

2) Measurement of inhibition of the activity of AChE To 50μl of each extract of ganoderma lucidum obtained in the above, 925 μl of Buffer I (0.1 M sodium phosphate buffer, pH 8.0) and 25 μl of Tween-20 are mixed. Then 33 μ .of Ellman reagent is added, and 25°C is maintained for 10 minutes. To this mixture, 20 μl of 25 mM acetylthiocholine iodide solution, which is a substrate, and 33 μl of AChE prepared for according to the method in the above 1) are added. Then the initial reaction speed of AChE is measured at 412 nm for 5 minutes. The initial reaction speed when there is a reagent is compared with that when there is no reagent. The results are shown in terms of the inhibition percentage.

[Equation 1]

Control - Reagent (D/min)

Inhibition (%)= x 100

Control (D/min)

The degree of inhibition of AChE of each extract of ganoderma lucidum obtained in Preferred Embodiment 1 in such method is measured. The results are shown in Figures 1 and 2.

As shown in Figure 1, among the extracts of ganoderma lucidum, GL-M shows the best effect of inhibition of the activity of AChE. The rate of acquisition and inhibition of the activity of AChE of the fractions separated and refined from the methanol extract (GL-M) of ganoderma lucidum are shown in Figure 2.

The structure of GL-M-8-F-b-III-b, which is the final separated material of ganoderma lucidum according to the present invention is the same as that of oleamide, and the result of measurement of inhibition of the activity of AChE is shown in Figure 3. As shown in Figure 3, inhibition of the activity of AChE is increased as the concentration of oleamide is increased.

3) Measurement of the selectivity for AChE/BuChE

Tacrine, which is a treatment drug of Alzheimer's disease being used presently, has almost no selectivity for AChE/BuChE and inhibit both of them. Whereas, since butyrylcholinesterase (BuChE) is not related to the cholinergic transmission in the central nervous system, tacrine has less treatment effects and many side effects. Accordingly, the selectivity for AChE/BuChE of GL-M-8-F-b- III- 1, which is the final separated material of the extract of ganoderma lucidum according to the present invention, is measured. It is measured in the same method as that of measurement of inhibition of the activity of AChE except that BuChE is separated from human blood plasma and butyrylthiocholine iodide is used for the substrate in measuring the degree of its inhibition. The result of measurement is shown in Figure 4. As shown in Figure 4, there is shown almost no inhibition of the activity of BuChE and low inhibition effects of within 10% as the concentration of GL-M-8-F- b-III-1 is increased. Compared to this, AChE shows inhibition of the activity of up to 70%. It is confirmed from the above that the extracts of ganoderma lucidum according to the present invention inhibit only AChE selectively.

Preferred Embodiment 2: Effect 2 of inhibition of the activity of AChE according to the extracts of ganoderma lucidum (in vivo assay)

GL-M-8-F-b-III-l obtained according to Preferred Embodiment 1 and 1 mg kg and 10 mg/kg of synthesized oleamide dissolved in 10% ethanol are administered into the abdominal cavity of 5-week-old ICR mice. After one hour, AChE is separated from the cerebral tissues. The measurement of the effect of inhibition of the activity of AChE separated from the brain is performed in the same method as that of measurement of inhibition of the activity of AChE in vitro. The result is shown in Figure 5.

As shown in Figure 5, the activity of AChE is inhibited by 39% and 52% in the concentration of 1 mg/kg and 10 mg/kg, respectively, compared to the control.

And it is confirmed that the effect of inhibition of the activity of AChE of GL-M-8-

F-b-III-1 extracted from ganoderma lucidum and that of synthesized oleamide are the same.

Preferred Embodiment 3: Effect 3 of inhibition of the activity of AChE according to the extracts of ganoderma lucidum (Passive avoidance experiment)

Whether Extract GL-M-8-F-b-III-l of ganoderma lucidum according to the present invention can improve the memorizing and learning ability in vivo is investigated by performing step-through passive avoidance experiments in terms of three protocols (Passive Avoidance Experiments I, II, and III).

ICR-family female mice weighing 20-30g (about 6 weeks old) are used for the experiment after they are adapted to an animal room, in which the room temperature is maintained at 22°C and relative humidity at 50-60% and light and darkness are controlled by using an automatic control device at intervals of 12 hours, for a week or longer. The step-through passive avoidance reaction measurement instrument (O'HARA Co., Ltd., Model PAM5, France) is divided into two spaces having a hole on the partition inbetween so that the mice can pass through easily. One side (12x10x10 cm) of the box is made from a transparent acryl plate to make the inside of the box bright, and another side (16x10x10 cm) is made from a black acryl plate to make the inside of the box dark. Lattices are installed at the bottom of the dark box at intervals of 7 mm, and the electricity of 100 volts is applied through the lattices at the bottom when the mice enter the dark side.

1) Passive Avoidance Experiment 1

On the first day of experiment, the mice are put into the light side of the experimental box so that they can look through and adapt themselves as they move freely to the dark side through the hole. Then the time taken for the mice to enter from the light side to the dark side of the experimental box, i.e., latency time, is measured, and those who have the latency time of within 30 seconds are used for the experiment.

On the second day of experiment, the mice are divided into those in the vehicle (salt solution or 10% ethanol) administration group, scopolamine (1 mg/kg) administration group, simultaneous administration group of scopolamine ( 1 mg/kg) and Extract GL-M-8-F-b-III-l of ganoderma lucidum (0.5 mg/kg and 2 mg/kg), and simultaneous administration group of scopolamine and tacrine (2 mg/kg), and the drug is administered to the abdominal cavity of mice in each group. The mice are put into the light side of the box 30 minutes after the drug is administered, the latency time is measured, and an electric shock of 100 volts is given to the mice for 3 seconds when 3 seconds pass after the mice enter the dark side. They are again put into the box 15 minutes after training, and the latency time is measured up to 300 seconds to the maximum. On the third day of experiment (24 hours after the electric shock), as on the second day of experiment, the drug is administered to each mouse, and the latency time is measured after 30 minutes. The average latency time of each group obtained on the second day (15 minutes in the first training trial) and that obtained on the third day (24 hours in the retention trial) are compared with the average latency time and analysis of variance (ANOVA) test of the vehicle and scopolamine administration control group.

In the present experiment, the result of investigation of the effect of the anamnestic regeneration ability of the extracts of ganoderma lucidum through the passive avoidance reaction by using the mice, for which amnesia is induced by scopolamine, is shown in Figure 6.

As shown in Figure 6, the latency time of 6-11 minutes measured at the time of adaptation before the electric shock is given shows no difference from those of the groups in which other drugs are treated (A). The latency time measured again 15 minutes after electric shock training is significantly increased in the salt solution control group (274+17 minutes) and the vehicle control group treated with 10% ethanol which is a solvent of the extracts of ganoderma lucidum (290±9 minutes). From this result, it is confirmed that these animals in the control group have a normal anamnestic ability. In the scopolamine administration group, the passive avoidance reaction by an electric shock for 103 minutes is reduced significantly by 62% compared to that in the normal control group. From this result, it is confirmed that the models for the animals having amnesia by scopolamine are made as expected. The latency time suppressed by scopolamine by administering Extract GL-M-8-F-b- III- 1 of ganoderma lucidum is increased, which is similar to that of the administration group of tacrine which is the well-known inhibitor of acetylcholinesterase. In the group treated with 10% ethanol, which is a solvent for the extracts of ganoderma lucidum, there is no affect on 15-minute training latency of the second day, but the retention latency is reduced by about 33% 24 hours after the electric shock is given although it is not a statistically significant change compared to that of the salt solution control group. As to the latency time after 24 hours when 0.5 mg/kg of the extract of ganoderma lucidum is administered, the step-through latency suppressed by scopolamine is recovered to the same degree as that of the 10% ethanol vehicle control group (B, C).

As described in the above, Extract GL-M-8-F-b-Iϋ-l of ganoderma lucidum according to the present invention shows an effect of recovering the step-through passive avoidance reaction suppressed by scopolamine in mice, and this effect is similar to the effect of tacrine (2 mg/kg) investigated as the positive control group.

2) Passive Avoidance Experiment II

The mice are adapted as in Experiment I on the first day of this experiment. On the second ay, the mice are divided into those in the vehicle [salt solution or 0.2% methylcellulose (MC)] administration group, scopolamine (1 mg/kg) administration group, simultaneous administration group of scopolamine (1 mg/kg) and Extract GL- M-8-F-b-III-l of ganoderma lucidum (0.5 mg/kg, 2 mg/kg, and 10 mg/kg), simultaneous administration group of scopolamine and tacrine (2 mg/kg), simultaneous administration group of scopolamine and donepezil (Aricept (???) 0.5 mg/kg), sole administration group of Extract GL-M-8-F-b-III-l of ganoderma lucidum (0.5 mg/kg, 2 mg/kg, and 10 mg/kg), sole administration group of tacrine (2 mg/kg), and sole administration group of donepezil (0.5 mg/kg), and the drug is administered into the abdominal cavity of mice in each group. The mice are put into the light side of the box 30 minutes after the drug is administered, the latency time is measured, and an electric shock is given to the mice for 3 seconds when 3 seconds pass after the mice enter the dark side. They are again put into the box 15 minutes after training, and the latency time is measured up to 300 seconds to the maximum. The latency time is measured without administration of drags or electric shock 24 hours (on the third day) and 48 hours (on the fourth day) after the electric shock. The result obtained from this experiment is shown in Figure 7.

In the vehicle control group treated with 0.2% MC which is a solvent for the extracts of ganoderma lucidum, the latency time measured 15 minutes, 24 hours, and 48 hours after electric shock training shows almost no difference from that in the salt solution control group. When scopolamine and testing drags are administered simultaneously, reduction of the latency time (lowering of the anamnestic ability) induced by scopolamine by the extracts of ganoderma lucidum 15 minutes after the electric shock is recovered partially (the greatest effect of increasing at a dose of 10 mg/kg), and this effect is similar to that of tacrine, which is a well-known inhibitor of acetylcholinesterase, and donepezil. Donepezil is almost at the level of the control group 24 hours after the electric shock is given and recovers lowering of the anamnestic ability induced by scopolamine. And the extracts of ganoderma lucidum show the recovery effect of about 30-50% which is similar to that of tacrine, in all three concentration ranges investigated. The greatest effect of recovery is shown in the administration group of 2 mg kg of the extract of ganoderma. lucidum 24 hours after the electric shock. This effect is similar to that of 2 mg/kg of tacrine, which is somewhat less than the effect of donepezil.

In the present experiment, in order to study whether the extracts of ganoderma lucidum have an affect on the normal anamnestic ability, only testing drugs are administered without administering scopolamine and the passive avoidance experiment is performed in the same method as in the above.

As shown on the left part of Figure 7, all of the extracts of ganoderma lucidum, and tacrine and donepezil which are positive control drugs do not have any significant affect on the normal anamnestic ability. Rather, they are shown to have a tendency of further improving the normal anamnestic ability 24 hours after the electric shock is given.

3) Passive Avoidance Experiment III

In the present experiment, in order to investigate the continuous administration effect of the extracts of ganoderma lucidum, the mice are adapted as in Experiments I and II on the first day of experiment. On the second day of experiment, the mice are divided into those in the vehicle [salt solution or 0.2% methylcellulose (MC)] administration group, scopolamine (1 mg/kg) administration group, simultaneous administration group of scopolamine (1 mg/kg) and Extract GL- M-8-F-b-III-l of ganoderma lucidum (0.5 mg/kg, 2 mg/kg, and 10 mg/kg), simultaneous administration group of scopolamine and tacrine (2 mg/kg), and simultaneous administration group of scopolamine and donepezil (0.5 mg/kg), and the drug is administered into the abdominal cavity of mice in each group. The same electric shock training as in the above is performed 30 minutes after the drug is administered, the mice are put into the experimental box again after 15 minutes to measured the latency time, and the second electric shock training is performed. The extracts of ganoderma lucidum (0.5 mg/kg, 2 mg/kg, and 10 mg/kg), tacrine (2 mg/kg), and donepezil (0.5 mg/kg) are administered continuously for five days without the electric shock from the third day of the experiment. The result is shown in Figure 8.

As shown in Figure 8, the anamnestic ability which is lowered significantly by scopolamine is recovered at a level of that of the control group after the extracts of ganoderma lucidum according to the present invention are administered continuously.

Preferred Embodiment 4: Effect of protection of damaged cerebral tissues induced by cerebral ischemia by the extracts of ganoderma lucidum

In order to observe the effect of protection of cerebral infarction induced by cerebral ischemia by the extracts of ganoderma lucidum, the models of animals having cerebral ischemia are made through the middle coronary artery occlusion (MCAO) of mice (Longa et al, 1986).

White female mice (Sprague-Dawley) weighing 250-300 g are used. They are allowed to take water and food freely before and after the induction of cerebral ischemia, and are used for the experiment after they go through the term of adaptation of about a week.

They are induced to be anesthetized by introducing 3.0-3.5% enflurane (manufactured by Choongwae Pharmaceutical Company) under mixed gases of N₂O and O₂ in an anaesthesia chamber, fixed to an operation table, and kept to be anesthetized during the operation by introducing 1.5-2.0% enflurane on a mask again. Their body temperature is maintained at 37+0.5 °C by using a heating pad during the operation in order to prevent lowering of their body temperature. About 1 cm of their necks is cut along the central line of the neck, and the right common carotid artery is separated from the vagus nerve carefully and exposed. The external carotid artery and internal carotid artery are separated from peripheral nerves of common carotid artery, and strings are suspended loosely on separated common carotid artery, external carotid artery, and internal carotid artery in advance. And ischemia is induced by binding the strings suspended on the common carotid artery and external carotid artery to band them, making a hole on the original portion of internal carotid artery with an injection needle, closing the middle cerebral artery by pushing a 17-mm probe into the internal carotid artery through the cut portion, and fixing the probe with the string suspended on the internal carotid artery. At this time, the prove is used for a stopper by making the diameter of its end 0.3 mm by heating one end of 4-0 nylon surgical thread near a small iron. It is cut to have a length of about 17 mm and coated with silicone (Xantopren, Bayer Dental) to which a hardening agent (Optosil, Bayer Dental) is added. The operated portions of mice are stitched together, and they are put in another cage until they regain consciousness from anesthesia. The neurologic defect is confirmed when a fixed amount of time of about 30 minutes passes after inducement of cerebral ischemia. As to the neurologic defect, whether the left hemiparesis occurs when the tail of a white mouse is lifted up to the air completely and whether circling to the left occurs spontaneously when the tail is lifted up are observed (Bederson et al., 1986). In the present experiment, the middle cerebral artery is closed 30 minutes after Extract GL-M-8-F- b-III-1 of ganoderma lucidum (0.5 mg/kg, 2 mg/kg, and 10 mg/kg) is pre-treated into the abdominal cavity in order to induce cerebral ischemia for 9 hours, after which the head is cut and the brain is taken out promptly. Then the degree of cerebral infarction is measured by adding 2% TTC (2,3,5-triphenyltetrazolium chloride) solution made from 0.9% physiological salt solution to the third slice (5-7 mm) among fresh brain coronal slices divided continuously in the thickness of 2 mm from the spot 1 mm from the frontal pole by using the brain matrix (Neurotec, Korea) and dyeing at 37°C for 30 minutes. The result of measurement is shown in Figure 9.

As shown in Figure 9, it is seen that the damaged area of the cerebral cortex and striate corpus induced by ischemia for 9 hours (shown in white) is reduced by the administration of Extract GL-M-8-F-b-III-l of ganoderma lucidum according to the present invention.

Preferred Embodiment 5: Effect of inhibition of the activity of AChE by oleamide and its structural analogues

The effect of inhibition of the activity of AChE of oleamide and its structural analogues, i.e., palmitoleylamide [CH₃(CH₂)₅CH=CH(CH₂)₇-CONH₂], myristylamide [CH₃(CH₂)₁ -CONH₂], n-pentyl oleoaniide

[CH₃(CH₂)₇CH=CH(CH₂)₇-CONHCH(CH₂CH₃)₂], n-pentyl linoleoamide

[CH3(CH₂) CH=CHCH₂CH=CH(CH₂)₇-CONHCH(CH₂CH₃)₂], n-pentyl myristaniide [CH₃(CH₂)₁₂-CONHCH(CH₂CH₃)₂], and pentyl linolate [CH₃(CH₂)₄CH=CHCH₂CH=CH(CH₂)₇-COOCH(CH₂CH₃)₂], is confirmed in the same method as those in Preferred Embodiments 1 and 2. Donepezil and tacrine are used for the control group. The result of measurement is shown in Table 1 as follows:

[Table 1]

As shown in Table 1, the effect of inhibition of the activity of AChE of oleamide and its structural analogues is increased as the concentration is increased. The composition according to the present invention shows the prevention and treatment effect for not only the Alzheimer's disease but also vascular dementia since it prevents reduction of the amount of acetylcholine in the brain and shows the protective effect for cerebral tissues damaged by cerebral ischemia as it inhibits the activity of AChE of animal cerebral tissues.

While the invention has been described in terms of a few preferred embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims.

Claims

What is claimed is:

1. A composition for the prevention and treatment of dementia containing the extracts of ganoderma lucidum as effective components.

2. The composition of claim 1, wherein said composition is a pharmacological composition characterized by the prevention and treatment of the Alzheimer's disease and vascular dementia simultaneously.

3. The composition of claim 1, wherein said composition is a food composition characterized by the prevention and treatment of the Alzheimer's disease and vascular dementia simultaneously.

4. The composition of claims 1 to 3 for the prevention and treatment of dementia characterized by that said extracts of ganoderma lucidum include GL-M,

GL-M-8, GL-M-8-F, GL-M-8-F-b, GL-M-8-F-b-III, and GL-M-8-F-III-1.

5. The composition of claim 4 for the prevention and treatment of dementia characterized by that one of said extracts of ganoderma lucidum is GL-M- 8-F-b-III-l.

6. A composition for the prevention and treatment of dementia containing a chemical compound having the following chemical formula 1 as an effective component: <Chemical Formula 1>

where Ri is a 9-octadecenoyl (oleyl), 9-hexadecenoyl (palmitoleyl), or tetradecanoyl (myristyl) radical.

7. A composition for the prevention and treatment of dementia containing a chemical compound having the following chemical formula 2 as an effective component: <Chemical Formula 2>

where R₂ is a 9-octadecenoyl (oleyl), 9,12-octadecadienoyl (linoleyl), or tetradecanoyl (myristyl) radical.

8. A composition for the prevention and treatment of dementia containing a chemical compound having the following chemical formula 3 as an effective component: <Chemical Formula 3>

9. The composition of claims 6 to 8, wherein said composition is a pharmacological composition characterized by the prevention and treatment of the Alzheimer's disease and vascular dementia simultaneously.

10. The composition of claims 6 to 8, wherein said composition is a food composition characterized by the prevention and treatment of the Alzheimer's disease and vascular dementia simultaneously.

11. A method of extraction of the extracts of ganoderma lucidum comprising the steps of: extraction of ganoderma lucidum with hexane; extraction of the remnant of extraction with chloroform; extraction of the remnant of extraction with methanol to obtain a methanol extract of ganoderma lucidum (GL-M); and separation and refinement of said methanol extract of ganoderma lucidum according to the silica gel column chromatography and HPTLC.