KR100502835B1 - Compositions comprising extract of ganoderma lucidum, oleamide and its structural analogue as an effective component for preventing or treating dementia - Google Patents

Abstract

The present invention is a composition for the prevention and treatment of dementia containing Ganoderma lucidum extract, oleamide and structural analogs thereof as an active ingredient.

The composition of the present invention inhibits acetylcholinesterase (AChE) activity of animal brain tissues, thereby preventing a decrease in the amount of acetylcholine in the brain and at the same time exhibiting a protective effect on brain tissues damaged by cerebral ischemia. In addition, it can be usefully used as a prophylactic and therapeutic agent for Alzheimer's dementia as well as vascular dementia.

Description

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

The present invention relates to a composition for the prevention and treatment of dementia containing Ganoderma lucidum extract, oleamide and structural analogs thereof as an active ingredient.

Dementia is a pathological phenomenon that can be distinguished from normal aging and, depending on its cause, it is classified as Alzheimer's disease, vascular dementia, other alcoholism, trauma, or aftereffects of Parkinson's disease. do.

The pathogenesis of Alzheimer's dementia is not well known, but the decrease in acetylcholine function in the central nervous system is most common. Therefore, acetylcholine precursors or drugs that inhibit the degradation of acetylcholine are used to treat the brain. Treatments that increase acetylcholine levels have been used. Therefore, acetylcholinesterase (hereinafter referred to as 'AChE') inhibitors alone or in combination with existing cholinesterase inhibitors are used as therapeutic agents, and representative drugs are tacrine and donepezil. ), Rivastigmine, galantamine, and the like.

Vascular dementia is mostly caused by damage to brain cells due to a lack of blood supply to various parts of the brain by cerebrovascular arteriosclerosis.Therapeutic agents protect against hypertension or aspirin or damaged brain cells that act on the blood vessels. A drug to make is preferable.

Existing dementia treatments mainly have an inhibitory effect on acetylcholinesterase, and therefore, there is a need for the development of drugs that have a therapeutic effect on vascular dementia.

Ganoderma lucidum is a type of mushroom that has been used as a traditional medicine in the East. It is known to have anti-inflammatory effects, anticancer effects, immunomodulatory effects, AIDS virus suppression effects, and blood pressure lowering effects. Studies on the efficacy of these ganoderma lucidum include, for example, water or ethyl acetate extract from oral administration to rats caused by carrageenan and croton oil for edema. Research papers (Stavihovha W and Slama J, The anti-inflammatory Ganoderma Lucidum Third International Symposium on Ganoderma lucidum: 9-21) and water extracts from Ganoderma lucidum inhibit the growth of cancer cells (Wang SY et al, The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages and Tlymphocytes, Int. J Cancer 70: 699-705). In addition, it is known that LZ-8, an immunomodulatory protein isolated from Ganoderma, exhibits mitogenic activity in vitro and immunomodulatory activity in vivo [Kino K et al. , Immunomodulator, LZ-8, prevents antibody production in mice, Int J Immunopharmacol 13 (8): 1109-1115].

The inventors of the present invention, while seeking safe and non-toxic natural resources capable of preventing and treating Alzheimer's dementia as well as vascular dementia, inhibit the activity of AChE from Ganoderma lucidum, which has been traditionally used in Korea, and at the same time, inhibits cerebral ischemia. Extracting the components indicated, and confirmed that the related materials are useful as a treatment for dementia completed the present invention.

An object of the present invention is to provide a composition exhibiting a prophylactic and therapeutic effect against Alzheimer's dementia as well as vascular dementia.

The present invention provides a composition for the prevention and treatment of dementia comprising Ganoderma lucidum extract, oleamide and structural analogs thereof as an active ingredient.

The composition of the present invention exhibits the prevention and treatment of Alzheimer's dementia as well as vascular dementia.

The composition of the present invention exhibits an inhibitory effect of AChE activity, thereby preventing and treating Alzheimer's dementia by restoring a decrease in the amount of acetylcholine in the brain, and at the same time, protecting the brain tissues damaged by cerebral ischemia, thereby vascular dementia. It also has a prophylactic and therapeutic effect.

Hereinafter, the composition of the present invention will be described in detail.

The method for extracting Ganoderma lucidum extract used as an active ingredient in the composition of the present invention is as follows.

The ganoderma lucidum is extracted with hexane, and the extraction residue is extracted again with chloroform. Then, the extraction residue is extracted with methanol again. The extract obtained is subjected to dark distillation to obtain methanol extract (hereinafter referred to as 'extract GL-M') of ganoderma lucidum. . At this time, the amount of hexane, chloroform and methanol used as the extraction solvent is sufficient to immerse the used ganoderma, respectively, and generally 2 L of hexane, chloroform and methanol per kg of ganoderma are used.

Extract GL-M, a methanol extract of Ganoderma lucidum obtained according to the above method, was subsequently fractionated by a series of silica gel column chromatography and HPTLC method to separate and purify. That is, a) silica gel column chromatography using chloroform / methanol (500: 1) as an eluent with extract GL-M, a methanol extract of ganoderma lucidum, and the obtained fractions were treated with HPTLC to extract GL-M-1 according to Rf. Obtaining 14 extracts from GL-M-14 to b) silica gel column chromatography using chloroform / methanol (100: 1) as the eluent of extract GL-M-8 in the extract obtained in step a) Treating the obtained fractions with HPTLC to obtain 7 extracts of GL-M-8-G from GL-M-8-A according to the Rf value, c) extract GL-M in the extract obtained in step b) -8-F was purified by silica gel column chromatography using chloroform / methanol (50: 1) as eluent and the resulting fractions were treated with HPTLC to obtain GL-M-8- in extract GL-M-8-Fa according to Rf. Obtaining five extracts of Fe, d) extract GL-M in the extract obtained in step c) -8-Fb was purified by silica gel column chromatography using chloroform / methanol (50: 1) as eluent and the resulting fractions were treated with HPTLC to extract GL-M-8- at extract GL-M-8-FbI according to Rf. Obtaining four extracts up to Fb-IV, e) silica gel column chromatography using chloroform / methanol (30: 1) as the eluent with extract GL-M-8-Fb-III in the extract obtained in step d) The fractions obtained by the graphing are treated with HPTLC to obtain the extract GL-M-8-Fb-III-1 according to the Rf value.

Extracts obtained according to the method described above GL-M-8, GL-M-8-F, GL-M-8-Fb, GL-M-8-Fb-III and GL-M-8-Fb-III- 1 inhibits AChE activity, respectively, as demonstrated by the examples described later, and thus may be used as an active ingredient in the composition of the present invention.

Mass spectrometry was performed on the extract GL-M-8-Fb-III-1 finally obtained according to the method of the present invention to identify the active ingredient contained in the methanol extract of Ganoderma lucidum used as an active ingredient in the composition of the present invention. This fraction was confirmed to have the same structure as 9-octadecenamide (oleamide). As a result, the ganoderma lucidum extract according to the present invention can be seen that the oleamide already known as an active ingredient inducing sleep in the brain.

The oleamide compound may also be prepared according to various chemical synthesis methods, and the method for preparing oleamide and structural analogs thereof is as follows.

The fatty acid compound (4) is reacted with thionyl chloride (SOCl ₂ ) at a temperature of 70 to 80 ° C. and when the reaction is completed, the reaction mixture is cooled to room temperature and slowly added cold ammonia water. The resulting reaction product is filtered under reduced pressure and then recrystallized to obtain oleamide and its structural analogue (1). The reaction scheme is shown in Scheme 1 below.

In Scheme 1, R ₁ is 9-octadecenoyl (oleyl), 9-hexadecenoyl (palmitoryl) or tetradecanoyl (myristyl).

After dissolving fatty acid compound (5) in dichloromethane, dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine (DMAP) were added, and 3-aminopentane (3-aminopentane) was added thereto. After time reaction, the resulting solid is filtered off and the remaining mother liquor is concentrated. The concentrated compound is subjected to column chromatography to obtain a structural analogue of oleamide (2). The reaction scheme is shown in Scheme 2 below.

In Scheme 2, R ₂ is 9-octadecenoyl (oleyl), 9,12-octadecadienoyl (linoleyl) or tetradecanoyl (myristol).

In addition, after dissolving linoleic acid (6) in dichloromethane, dicyclohexylcarbodiimide and dimethylaminopyridine are added thereto, and 3-pentanol is added thereto to react for a certain time. The resulting solid is filtered off and the remaining mother liquor is concentrated and the concentrated compound is column chromatographed to give a structural analogue of oleamide (3). The reaction scheme is shown in Scheme 3 below.

In the above scheme 3 R ₃ is a octa-decamethylene 9,12- diethoxy alkanoyl (linoleyl).

Oleamide and its structural analogues prepared by the chemical synthesis method, like the extract of Ganoderma, have a prophylactic and therapeutic effect against Alzheimer's dementia as well as vascular dementia.

In addition to the active ingredients such as ganoderma lucidum extract, oleamide and structural analogues thereof, the composition of the present invention may further contain one or more active ingredients capable of treating dementia and other diseases.

The composition of the present invention, in addition to the above-described active ingredient for administration may be prepared by including one or more carriers that are acceptable in pharmaceutical or food.

Pharmaceutical or food acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, as necessary. And other conventional additives such as buffers and bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The pharmaceutical composition of the present invention can be administered parenterally or orally, and the dosage ranges depending on the weight, age, sex, health condition, diet, time of administration, administration method, excretion rate and severity of the patient. Although various, the daily dosage amount of 0.8 ~ 3.2 mg / kg is preferably administered in one to several divided doses.

The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormonal therapy, chemotherapy and biological response modifiers to prevent and treat Alzheimer's dementia, vascular dementia.

The formulation of the food composition of the present invention is prepared according to a conventional method, and may be encapsulated after drying with a carrier or formulated in the form of other tablets, granules, powders, beverages, porridges, etc. It is possible.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, this is merely provided to understand the present invention more easily, but is not limited thereto.

Example 1  : Inhibition of AChE activity by Ganoderma lucidum extract 1 (in vitro assay)

1. Preparation of Ganoderma lucidum extract

1) Preparation of Ganoderma lucidum Extract GL-H, GL-C, GL-M and GL-E

3 kg of ganoderma lucidum was placed in a crushed beaker, and about 2 times (about 6 L) of hexane was added to subtract the ganoderma lucidum, and heated at 40 to 50 ° C. for 2 hours, followed by extraction and filtration. It was distilled off under reduced pressure and dried to give the extract GL-H (17.6 g).

The extract was separated and chloroform was immersed in the remaining residue (about 6 L), heated at 40-50 ° C. for 2 hours, extracted twice, and filtered. It was distilled off under reduced pressure and dried to give the extract GL-C (55 g).

Then, the extract was separated and methanol (about 6 L) was added to the remaining residue, stirred at room temperature for two days, extracted twice, and filtered. It was distilled off under reduced pressure to give the extract GL-M (65 g).

The extract was separated and 70% ethanol (about 6 L) was added to the remaining residue, stirred for two days at room temperature, heated and extracted twice and filtered. It was distilled off under reduced pressure to give the extract GL-E (59 g).

2) Separation and Purification from Ganoderma lucidum Extract GL-M

60 g of the extract GL-M obtained in 1) was eluted in a column (10 × 100 cm) filled with silica gel using chloroform / methanol (500: 1) as a solvent. Elution fractions were taken in 100 ml each and developed with HPTLC (developing solvent chloroform / methanol 30: 1) to color with iodine, and 14 fractions were separated according to Rf. The separated fractions were distilled under reduced pressure and dried to obtain the extracts GL-M-1 to GL-M-14.

Among them, 300 mg of GL-M-8 was eluted in a column (4 × 40 cm) filled with silica gel using chloroform / methanol (100: 1) as a solvent. 50 ml of the elution fractions were collected and developed with HPTLC (developing solvent chloroform / methanol 30: 1) to develop iodine and separated into seven fractions according to Rf. Each separated fraction was distilled under reduced pressure and dried to obtain the extract GL-M-8-A to GL-M-8-G.

86 mg of the extract GL-M-8-F was then eluted in a silica gel-filled column (1.5 × 35 cm) using chloroform / methanol (50: 1) as a solvent. Elution fractions were taken in 30 ml each and developed with HPTLC (developing solvent chloroform / methanol 30: 1) and developed with iodine, and then separated into five fractions according to the Rf value. The separated fractions were distilled under reduced pressure and dried to obtain the extracts GL-M-8-F-a to GL-M-8-F-e.

60 mg of GL-M-8-F-b was eluted in a silica gel packed column (1.2 × 30 cm) using chloroform / methanol (50: 1) as a solvent. Elution fractions were taken in 10 ml each and developed with HPTLC (developing solvent chloroform / methanol 30: 1) and developed with iodine and separated into four fractions according to Rf. Each separated fraction was distilled under reduced pressure and dried to obtain the extract GL-M-8-F-b-I to GL-M-8-F-b-IV.

40 mg of GL-M-8-F-b-III was then eluted in a silica gel packed column (1 × 25 cm) using chloroform / methanol (30: 1) as a solvent. Elution fractions were taken in 5 ml each, developed by HPTLC (developing solvent chloroform / methanol 30: 1), developed with iodine, and separated into three fractions according to Rf. The separated fractions were distilled under reduced pressure and dried to obtain the extracts GL-M-8-F-b-III-1 to GL-M-8-F-b-III-3.

2. Inhibitory Effect of AChE Activity

1) Isolation of AChE

AChE was isolated from the brains of ICR mice (20-25 g). ICR rats were taken out of the brain and weighed. 12.5 mM sodium phosphate buffer was added to the volume about twice its weight. After homogenization, the mixture was centrifuged at 4 ° C. for 10 minutes (× 1000 g). Take the supernatant, add 0.5% Triton X-100 homogenization buffer to 3 times its volume, stir at 4 ° C for 30 minutes, and centrifuge at 4 ° C for 10 minutes (× 10,000 g ). The supernatant was aliquoted into 1.5 ml tubes and stored in a deep freezer.

Care was taken not to damage the brain during extraction, and the whole process was performed quickly.

2) AChE activity inhibition measurement

50 μl of each Ganoderma lucidum extract obtained by the above 1 was mixed with 925 μl of Buffer I (0.1 M sodium phosphate buffer, pH: 8.0) and 25 μl Tween-20. Then, 33 μl of Elman sample was added and maintained at 25 ° C. for 10 minutes. Then, 20 μl of a 25 mM acetylthiocholine iodide solution as a substrate and 33 μl of AChE prepared according to the method 1) were added. Added. Then the initial reaction rate of AChE was measured for 5 minutes at 412 nm. Based on the initial reaction rate when there is no sample, the initial reaction rate was compared when there was a sample. The results are expressed as percentage inhibition.

In this way, the AChE inhibition of each Ganoderma lucidum extract obtained in Example 1 was measured. The results are shown in FIGS. 1 and 2.

As shown in Figure 1, GL-M of ganoderma lucidum extract showed the best AChE activity inhibitory effect, the yield and AChE activity inhibition of the fraction separated and purified from methanol extract (GL-M) of Ganoderma lucidum is shown in Figure 2 It was.

GL-M-8-F-b-III-1, the final separation material of ganoderma lucidum according to the present invention was the same as the structure of oleamide, and the result of measuring its inhibition of AChE activity is shown in FIG. 3.

As shown in FIG. 3, the inhibition of AChE activity increased as the concentration of oleamide increased.

3) Selectivity measurement for AChE / BuChE

Tacrine, the currently used Alzheimer's drug, inhibits both because of its lack of AChE / BuChE selectivity. However, butyrylcholinesterase (BuChE) is not associated with cholinergic transmission in the central nervous system. Accordingly, the selectivity of AChE / BuChE of GL-M-8-F-b-III-1, which is a final separation material of Ganoderma lucidum extract, was measured. BuChE was isolated from human plasma and its inhibition was measured by the same method as the inhibition of AChE activity except that butyrylthiocholine iodine was used as the substrate.

The measurement results are shown in FIG. 4.

As shown in FIG. 4, as the concentration of GL-M-8-F-b-III-1 increased, there was little inhibition of BuChE activity and showed a low inhibitory effect within 10%. In comparison, AChE showed up to 70% deactivation. From this it was confirmed that ganoderma lucidum extract according to the present invention selectively inhibits only AChE.

Example 2  : Inhibitory Effect of Ganoderma lucidum Extract on AChE Activity 2 ( in vivo  assay)

Intraperitoneally administer 1 mg / kg and 10 mg / kg of oleamide synthesized with GL-M-8-Fb-III-1 obtained according to Example 1 in 10% ethanol to 5-week-old ICR rats. After time, AChE was isolated from brain tissue. Inhibitory effect of AChE isolated from the brain was performed in the same manner as in vitro AChE inhibitory assay.

The results are shown in FIG.

As shown in FIG. 5, AChE activity was inhibited by 39% at 1 mg / kg concentration and 52% at 10 mg / kg concentration, compared to the control, and GL-M-8-Fb- extracted from Ganoderma lucidum. It was confirmed that AChE activity inhibitory effect of oleamide synthesized with III-1 was the same.

Example 3  : Inhibitory Effect of AChE Activity 3 by Ganoderma lucidum Extract (Manual Evasion Experiment)

Whether the GL-M-8-Fb-III-1 extract of Ganoderma lucidum according to the present invention can improve memory and learning ability in vivo can be tested in three protocols: , II, III) to investigate.

After ICR male rats with a body weight of 20-30g (approximately 6 weeks) are kept at room temperature of 22 ℃ and relative humidity of 50-60%, they are acclimated for 12 hours in an animal room that controls the contrast using an automatic control device for 12 hours or more. It was used for the experiment. O'HARA Co. Ltd., Model PAM5, France, a step-through passive evasion measurement device, is divided into two spaces, with a hole in the partition between them, allowing the mouse to pass easily. One part (12 × 10 × 10 cm) of the box is made of a transparent acrylic plate and the inside is bright, while the other part (16 × 10 × 10 cm) is made of a black acrylic plate and the box is dark. On the bottom of the dark box there is a grid of 7 mm intervals so that when the mouse enters the dark area, 100 volts of electricity will be given through the grid at the bottom.

1) Manual Evasion Experiment I

On the first day of the experiment, the rats were placed in the bright part of the test box so that the rats could freely navigate and adapt to the dark areas through the holes. At this time, the time (latency time, delay time) from the light part to the dark part of the test box was measured, and only mice having a delay time of 30 seconds or less were used in the experiment.

On the second day of the experiment, mice were treated with vehicle (saline or 10% ethanol), scopolamine (1 mg / kg) group, scopolamine (1 mg / kg) and ganoderma lucidum GL-M-8-Fb-III-1. Drugs were intraperitoneally administered to rats by dividing into the co-administration group of the extract (0.5 mg / kg, 2 mg / kg) and the co-administration of scopolamine and tacrine (2 mg / kg). 30 minutes after drug administration, the rats were placed in the bright part of the test box and the delay time was measured. After 3 seconds after the rats entered the dark part, the rats were given electrical stimulation (100 volts) for 3 seconds. After 15 minutes of training, the test box was put back into the test box and the delay time was measured and measured up to 300 seconds.

On the third day of the experiment (after 24 hours of electrical stimulation), as in the second day of the experiment, the delay time was measured again 30 minutes after the drug was administered to each rat. The mean delay time (15 minutes; 1st training trial) and the mean delay time (24 hours; retention trial) for each group obtained on day 2 were compared with the mean delay time and variance analysis (ANOVA; vehicle and scopolamine-treated controls). Analysis of Variance) test.

In this experiment, the effects of the ganoderma lucidum extract on the memory regeneration ability using the mice induced memory loss by scopolamine are shown in FIG. 6.

As shown in Figure 6, the delay time (6-11 minutes) measured at the time of adaptation before the electrical stimulation was given, no difference was observed between different drug treatment groups (A). Delay times measured after 15 minutes of electrostimulation training were significantly increased in the saline control group (274 ± 17 minutes) and in the vehicle control group treated with 10% ethanol (290 ± 9 minutes) as a solvent for Ganoderma lucidum extract. Normal memory capacity of control animals was confirmed. Compared to the normal control group, the scopolamine-treated group significantly reduced the passive avoidance response (103 minutes) to electrical stimulation (62%). You can see that it was created. The delay time inhibited by scopolamine was increased by the administration of GL-M-8-F-b-III-1 extract of Ganoderma lucidum, and the effect was similar to that of the well known acetylcholinesterase inhibitor tacrine. The 10% ethanol treated solvent of Ganoderma lucidum extract had no effect on the 15-minute training latency on the second day, but after 24 hours of electrical stimulation, there was no statistically significant change compared to the saline control group. Retention latency is reduced by about 33%. The delay time after 24 hours at 0.5 mg / kg administration of Ganoderma lucidum extract recovered to the same extent that the step-through delay inhibited by scopolamine was equivalent to that of the 10% ethanol vehicle control (B, C).

Thus, GL-M-8-Fb-III-1 extract of Ganoderma lucidum according to the present invention showed the effect of restoring the step-through passive avoidance reaction inhibited by scopolamine in mice, which was investigated as a positive control. Similar to the effect of tacrine (2 mg / kg).

2) Manual Evasion Experiment II

On the first day of the experiment, the mice were adapted in the same manner as in Experiment I, and on the second day of the experiment, the rats were treated with vehicle [saline or 0.2% methylcellulose (MC)], scopolamine (1 mg / kg) group, scopolamine (1 mg / kg). Kg) and co-administration of GL-M-8-Fb-III-1 extract (0.5 mg / kg, 2 mg / kg, 10 mg / kg) of Ganoderma lucidum, of scopolamine and tacrine (2 mg / kg) Co-administration group, co-administration of scopolamine and donepezil (Aricept; 0.5 mg / kg), GL-M-8-Fb-III-1 extract of Ganoderma lucidum (0.5 mg / kg, 2 mg / kg, 10 mg / kg) The drug was administered to the rats (all intraperitoneally) by dividing into a single administration group, tacrine (2 mg / kg) alone administration group and donepezil (0.5 mg / kg) alone administration group. 30 minutes after drug administration, the rats were placed in the bright part of the test box and the delay time was measured. After 3 seconds after the rats entered the dark part, electric stimulation was given for 3 seconds. After 15 minutes of training, the test box was put back into the test box and the delay time was measured and measured up to 300 seconds. Delay times were measured 24 hours after the electrical stimulation (day 3) and after 48 hours (day 4) without drug administration or electrical stimulation. The results investigated in this experiment are shown in FIG. 7.

The delay time measured after 15 minutes, 24 hours, and 48 hours of electric stimulation training in the vehicle control group treated with 0.2% MC of the solvent of Ganoderma lucidum extract showed little difference from the saline control group. When co-administration of scopolamine and test drugs, the reduction of scopolamine-induced delay (lowering memory) was partially recovered (maximum at dose of 10 mg / kg) by Ganoderma lucidum extract after 15 minutes of electrical stimulation. Increasing effect), this effect was similar to the effects of well-known acetylcholinesterase inhibitors tacrine and donepezil. After 24 hours of electrical stimulation, donepezil recovered the scopolamine-induced memory loss at almost the control level, and Ganoderma lucidum extract had a recovery effect of about 30-50%, similar to tacrine at all three concentrations investigated. Indicated. After 24 hours of electrostimulation, the ganoderma lucidum extract showed the greatest recovery effect in the 2mg / kg administration group, which was similar to that of tacrine 2mg / kg and slightly less than that of donepezil.

In this experiment, passive evacuation experiments were performed in the same manner as above only after the test drug was administered without scopolamine to investigate whether Ganoderma lucidum extract affects normal memory.

As shown in the left part of Figure 7, ganoderma lucidum extract and positive control drugs tacrine and donepezil did not have a significant effect on the normal memory, but after 24 hours of electrical stimulation showed a tendency to further improve the normal memory.

3) Manual Evasion Experiment III

In this experiment, the first day of the experiment was conducted to investigate the effect of continuous administration of Ganoderma lucidum extract. On the second day of the experiment, mice were treated with vehicle [saline or 0.2% methylcellulose (MC)] group, scopolamine (1 mg / kg) group, scopolamine (1 mg / kg) and ganoderma lucidum. Co-administration group of GL-M-8-Fb-III-1 extract (0.5 mg / kg, 2 mg / kg, 10 mg / kg), co-administration of scopolamine and tacrine (2 mg / kg) and scopol The amine and donepezil (0.5 mg / kg) were divided into the co-administration group, and the drug was administered to the rats in each group (all intraperitoneally). Thirty minutes after the drug administration, the same electrical stimulation training was conducted, and after 15 minutes, the test box was put back into the test box to measure the delay time and a second electrical stimulation training was performed. Ganoderma lucidum extract (0.5 mg / kg, 2 mg / kg, 10 mg / kg), tacrine (2 mg / kg) and donepezil (0.5 mg / kg) were continuously administered for 5 days from the third day of the experiment without electric stimulation.

The results are shown in FIG.

As shown in Figure 8, after the continuous administration of Ganoderma lucidum extract according to the present invention was remarkably reduced memory capacity by scopolamine to the control level.

Example 4  : Protective effect of brain injury tissue caused by cerebral ischemia by Ganoderma lucidum extract

In order to observe the protective effect of Ganoderma lucidum extract on brain tissue infarction induced by cerebral ischemia, a rat coronary artery occlusion (MCAO) was used to create an animal model of cerebral ischemia (Longa et al., 1986). ).

Male and female rats (Sprague-Dawley) weighing 250-300g were used, and water and food were freely consumed before and after the induction of cerebral ischemia.

Introduced 3.0 ~ 3.5% of enflurane (NFOL) in the anesthesia chamber under mixed gas of N ₂ O and O ₂ , fix it to the operating table after anesthesia induction, and introduce 1.5 ~ 2.0% of enfloran as mask Anesthesia was continued during surgery. The body temperature was maintained at 37 ± 0.5 ℃ using a heating pad during surgery in order to prevent the temperature decrease. The neck was incised about 1 cm along the midline of the neck and the right common carotid artery was carefully separated from the vagus nerve and exposed. The external carotid artery and the internal carotid artery were separated from the surrounding nerves from the carotid carotid artery and the thread was hung loosely in advance on the detached carotid artery, the external carotid artery and the internal carotid artery. Ligation of the common carotid and external carotid arteries is used to ligate the carotid and external carotid arteries and to puncture the carotid artery at the base of the carotid artery. In the carotid artery, the thread was tied to the internal carotid artery and the probe was fixed to induce ischemia. At this time, probe is used as a stopper by heating one end of 4 ~ 0 nylon surgical thread near the pharynx to make the end diameter 0.3mm and cut it to about 17mm length. Next, a coating with silicone (Xantopren, Bayer Dental) to which a curing agent (Optosil, Bayer Dental) was added was used. The surgical site was sutured and placed in another cage until awake from anesthesia. Neurological deficits were identified after a period of time (approximately 30 minutes) after cerebral ischemia. Neurological deficits observed whether left hemiparesis occurred when the tail of the white rat was fully lifted in the air and circling to the left when the tail was lifted up or spontaneously (Bederson et al., 1986). In this experiment, 30 minutes after pre-treatment of GL-M-8-Fb-III-1 extract of Ganoderma lucidum (0.5mg / kg, 2mg / kg, 10mg / kg) After the brain was closed for 9 hours to induce cerebral ischemia, the brain was quickly removed. Using the brain matrix (Neurotec. Korea), the third piece (5 ~ 7mm) of consecutively divided fresh brain coronal slices of 2mm thickness from 1mm from the frontal pole was used. 2% TTC (2,3,5-triphenyltetrazolium chloride) solution prepared with 0.9% saline was added and stained at 37 ° C. for 30 minutes to determine the degree of brain damage.

The measurement results are shown in FIG. 9.

As shown in FIG. 9, the extent of injury in the cerebral cortex and striatum induced by 9 hours ischemia (white area) is reduced by administration of GL-M-8-Fb-III-1 extract of Ganoderma lucidum according to the present invention. It can be seen.

Example 5  : Effect of Inhibiting AChE Activity by Oleamide and Its Structural Analogues

Oleamide and its structural analogs palmitoleamide [CH ₃ (CH ₂ ) ₅ CH═CH (CH ₂ ) ₇ -CONH ₂ ], myristylamide [CH ₃ (CH ₂ ) ₁₂ -CONH ₂ ], normal- Pentyl oreoamide [CH ₃ (CH ₂ ) ₇ CH = CH (CH ₂ ) ₇ -CONHCH (CH ₂ CH ₃ ) ₂ ], normal-pentyl-linoleamide [CH ₃ (CH ₂ ) ₄ CH = CHCH ₂ CH = CH (CH ₂ ) ₇ -CONHCH (CH ₂ CH ₃ ) ₂ ], normal-pentyl-myrimidamide [CH ₃ (CH ₂ ) ₁₂ -CONHCH (CH ₂ CH ₃ ) ₂ ], pentolinolate [CH ₃ (CH ₂ ) ₄ CH = CHCH ₂ CH = CH (CH ₂ ) ₇ -COOCH (CH ₂ CH ₃ ) ₂ ] The effect of inhibiting AChE activity was confirmed in the same manner as in Example 2.

Donepezil and tacrine were used as controls.

The measurement results are shown in Table 1.

% Inhibition 10 ^-2 (M) 10 ^-3 (M) 10 ^-4 (M) 10 ^-5 (M) 10 ^-6 (M) 10 ^-7 (M) 10 ^-8 (M) R ₁ Orail 98 60 8 - - - Palmito Rail 99 58 44 - - - - Myristeel - 75 10 - - - - R ₂ Orail - 96 42 0.2 - - - Linoleale - 70 3 - - - - Myristeel - 97 44 5 - - - 96 32 0.5 - - - - Control Donepezil - - - - 96 74 8 Taclean - - - 99 83 43

As shown in Table 1, oleamide and its structural analogues increased the effect of inhibiting AChE activity with increasing concentration.

The composition of the present invention inhibits AChE activity of animal brain tissues, thereby preventing a decrease in the amount of acetylcholine in the brain, and at the same time exhibits a protective effect on brain tissues damaged by cerebral ischemia, thus preventing Alzheimer's disease as well as vascular dementia. It has a prophylactic and therapeutic effect.

1 is the inhibition of AChE activity of Ganoderma lucidum extract (GL-H: Ganoderma lucidum hexane extract, GL-C: Ganoderma lucidum chloroform extract, GL-M: Ganoderma lucidum extract, GL-E: Ganoderma lucidum extract) Is a diagram showing.

2 is a diagram showing the yield and AChE activity inhibition of the fraction separated and purified from the methanol extract of Ganoderma lucidum according to the present invention.

3 is a diagram showing the inhibition of AChE activity of the synthesized oleamide by concentration.

Figure 4 is a measure of the selectivity of AChE / BuChE of GL-M-8-F-b-III-1, the final separated purified fraction of methanol extract of Ganoderma lucidum according to the present invention.

5 is a diagram measuring the inhibition of AChE activity after administration of oleamide synthesized with GL-M-8-F-b-III-1 according to the present invention (in vivo assay).

6 to 8 are diagrams showing the results of the passive avoidance experiment administered with the extract GL-M-8-F-b-III-1 of Ganoderma lucidum according to the present invention.

9 is a view showing the effect of the extract GL-M-8-F-b-III-1 of Ganoderma lucidum according to the present invention on cerebral ischemia.

Claims (11)

delete delete delete One selected from GL-M, GL-M-8, GL-M-8-F, GL-M-8-Fb, GL-M-8-Fb-III, GL-M-8-Fb-III-1 Composition for the prevention and treatment of dementia containing Ganoderma lucidum extract containing the above effective fraction 5. The composition for preventing and treating dementia of claim 4, wherein the ganoderma lucidum extract is GL-M-8-F-b-III-1. A composition for preventing and treating dementia containing the compound of formula 1 as an active ingredient In Formula 1, R ₁ is 9-octadecenoyl (oleyl), 9-hexadecenoyl (palmitoreyl) or tetradecanoyl (myristol). A composition for preventing and treating dementia containing a compound of Formula 2 as an active ingredient In Formula 2, R ₂ is 9-octadecenoyl (oleyl), 9,12-octadecadienoyl (linoleyl) or tetradecanoyl (myristyl). A composition for preventing and treating dementia containing a compound of formula 3 as an active ingredient A pharmaceutical composition according to any one of claims 4 to 8, characterized in that it simultaneously prevents and treats Alzheimer's dementia and vascular dementia. delete Extracting the ganoderma lucidum with hexane, then extracting the extracted residue with chloroform and then extracting the extracted residue with methanol, further separating and purifying the methanol extract (GL-M) obtained by silica gel column chromatography and HPTLC. Extraction method of ganoderma lucidum extract for prevention and treatment of dementia