KR101374908B1 - Compound for prenventing and curing alzheimer's disease including extract of amanita verna mycelium - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating Alzheimer′s disease including extract of amanita verna mycelium, and particularly, to a pharmaceutical composition for preventing and treating Alzheimer′s disease including extract of amanita verna mycelium, in which the extract obtained by culturing amanita verna mycelium and adding alcohol thereto is able to inhibit activities of acetylcholinesterase and prolyl endopeptidase which are enzymes related to induction of Alzheimer′s disease, and thus has a possibility of being used as an agent for preventing and treating Alzheimer′s disease. The pharmaceutical composition for preventing and treating Alzheimer′s disease including extract of amanita verna mycelium, according to the present invention, is advantageous in that the extract can inhibit both activities of acetylcholinesterase and prolyl endopeptidase which are enzymes related to induction of Alzheimer′s disease, and thus provide a composition for preventing and treating Alzheimer′s disease which contains the extract. [Reference numerals] (S100) Mycelium culturing step; (S200) Alcohol extracting step; (S300) Filtering step; (S400) Step of concentrating under reduce pressure; (S500) Drying and pulverizing step

Description

Compensation for prenventing and curing alzheimer's disease including extract of amanita verna mycelium}

The present invention relates to a pharmaceutical composition for the prevention and treatment of Alzheimer's disease, comprising the extract of the white myrtle mushroom mycelium. Specifically, the extract extracted by culturing the mycelia of the white myrtle mushroom to add alcohol is the enzyme related to Alzheimer's disease. Alzheimer's including white chloroplast mushroom mycelium extract which can inhibit the activity of the terase and prolyl endopeptidase and has the inhibitory effect of acetylcholinesterase and proyl endopeptidase, which may be used as an agent for preventing and treating Alzheimer's disease. It relates to a pharmaceutical composition for preventing and treating diseases.

With the advent of an aging society, senile dementia has become a serious social problem. Geriatric dementia is mainly due to dementia caused by mutations in the nerve cells themselves (neurocytopathic dementia), and dementia caused by mutations in brain tissues other than cranial nerve cells such as blood clots in the cerebral blood vessels. Dementia). One of the neurodegenerative dementias is that symptoms of Alzheimer's disease (AD) can be defined as a decrease in cognitive function and intellectual ability to a significant degree of disability in occupational work, normal social activities or interpersonal relationships. That is, symptoms of personality change and emotional instability, including cognitive dysfunction, language, judgment, abstraction, spatial and temporal impairment, and other new skills acquisition, eventually lead to death.

Although the cause and treatment method of Alzheimer's disease are not known exactly, there is a report that the concentration of neurotransmitter acetylcholine (ACh) is particularly reduced as a result of dissecting the brain of a person who died of Alzheimer's disease.

Acetylcholinesterase (AChE) is an enzyme that specifically hydrolyzes acetylcholine (ACh), which is deeply related to memory, to choline and acetic acid. It is known that acetylcholine levels can be reduced by inhibiting the activity of acetylcholinesterase (AChE). Increasing can prevent and treat Alzheimer's disease.

Prolyl endopeptidase (PEP) is a serine protease that exhibits high substrate specificity for the proline residue of peptides and is known as an enzyme involved in the production of beta amyloid. It is known that the activity of is high. Therefore, the effect of Alzheimer's disease can be seen by inhibiting the activity of prolyl endopeptidase (PEP).

On the other hand, white bulrush mushrooms are known as poisonous mushrooms as a fungi of the fungi of the fungus. Anithin is the most lethal toxin of white oyster mushroom, a toxin with a bicyclic octapeptide structure that mainly damages the liver and kidneys, and DNA-dependent RNA polymerase that catalyzes mRNA synthesis in the nucleus of eukaryotic cells by lethal action on animals. It occurs by specifically binding to II (RNA polymerase B) and inhibiting the formation of diester phosphate bond (initiation and extension of RNA chain synthesis). It is useful for identification of eukaryotic RNA polymerase and quantification of RNA polymerase II. Similar toxins, such as α-amanitine and β-amanitine, are isolated and combined to form amatoxin. Anithin is also absorbed by the intestine and transported to hepatocytes to destroy intracellular nucleolus and interfere with gene duplication, as well as being filtered by glomeruli and resorbed in the tubules to destroy cells in the tubules.

Research related to white bulrush mushrooms has focused mainly on the toxin anithin, which recently combined with antibodies that recognize cancer's unique molecules to inhibit various types of cancer cells in vitro and even prevent pancreatic cancer tumors transplanted into laboratory animals. Reports of killing antitumor efficacy have been published, but research on other efficacy is very lacking.

On the other hand, in relation to a technique for inhibiting the activity of acetylcholinesterase (AChE) or prolyl endopeptidase (PEP), which is an enzyme related to Alzheimer's disease, extracts of cereals are disclosed in Korean Patent Publication No. 10-2002-0019442. A prolyl endopeptidase inhibitor comprising as an active ingredient is disclosed, and Korean Patent Laid-Open Publication No. 10-2002-0066173 discloses a dementia prevention and treatment composition effective in inhibiting the activity of acetylcholinesterase. However, there is a problem that does not inhibit both AChE and PEP activity.

In addition, Korean Patent Publication No. 10-2002-0090140 discloses a composition for the prevention and treatment of dementia containing Ganoderma lucidum extract, oleamide and structural analogues thereof as an active ingredient, but acetylcholinesterase causing Alzheimer's disease. And it has not been specifically proposed in connection with the method using the white bred mushrooms that can inhibit the activity of the proyl endopeptidase.

The present invention is derived to solve the problems as described above and to provide the necessary technology,

It is an object of the present invention to provide a pharmaceutical composition for preventing and treating Alzheimer's disease, which comprises extracts of mycelia of the Alzheimer's disease, which can inhibit both the activities of acetylcholinesterase and prolyl endopeptidase, which are related enzymes of Alzheimer's disease. .

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The pharmaceutical composition for preventing and treating Alzheimer's disease comprising the mycelial mycelium mycelium extract according to the present invention is inoculated with a mycelium of the mycelium hypnotic mushroom in a medium of pH 5-7 and cultured at a temperature of 24 ~ 35 ℃, the cultured mycelium After the ethanol of 3 to 5 times the weight to add to extract for 3 to 5 hours at 40 ~ 60 ℃ characterized in that the extract obtained by filtration and dark concentration.

At this time, the mycelium culture step is inoculated with white mycelia mushroom mycelium in YM medium of pH 5 ~ 7 characterized in that the culture for 13 to 15 days at a temperature of 24 ~ 35 ℃.

In addition, in the mycelium culture step, it is preferable to add soluble starch to YMG medium to further increase the effects of inhibiting the activities of acetylcholinesterase and prolyl endopeptidase, which are enzymes related to Alzheimer's disease.

In addition, in the extraction process by adding alcohol to the cultured mycelium, 300 to 500 parts by weight of ethanol with respect to 100 parts by weight of the cultured mycelium is characterized in that the extraction for 3 to 5 hours at 40 ~ 60 ℃.

In addition, after the reduced pressure concentration step, the concentrated may be lyophilized to powder to prepare an extract.

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The pharmaceutical composition for preventing and treating Alzheimer's disease, comprising the extract of the mycelia of the mycelia of the present invention, has the effect of inhibiting the activity of acetylcholinesterase and prolyl endopeptidase, which are enzymes related to Alzheimer's disease. There is an advantage.

In addition, the present invention has another advantage that it is possible to secure the economical efficiency of the production of the white myrtle mushroom mycelium extract due to the rapid culture speed of the white myrtle mushroom mycelium.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of Alzheimer's disease, including the extract of the white myrtle mushroom mycelium that can be used as a new use in the prevention and treatment of Alzheimer's disease by using the white mushrooms, poisonous mushrooms have.

Figure 1 shows the extraction process of the white myrtle mushroom mycelium extract according to the present invention.

Hereinafter, with reference to the accompanying drawings will be described in detail an embodiment of the present invention and experimental results that can prove the effect.

Figure 1 shows the extraction process of the white myrtle mushroom mycelium extract according to the present invention.

White Clown Mushroom  Preparation of mycelium extract

In the mycelium culture step (S100), the mycelium was isolated from wild white chlorophyll mushroom fruiting body and inoculated in YMG (Yeast extract-Malt extract-Glucose) medium at pH 4-9 and inoculated with 5% (w / v) for 14 days at 24 ° C. The cells were incubated at 120 rpm. At this time, the YMG medium was used consisting of yeast extract, malt extract, glucose.

In the alcohol extraction step (S200) was added 400 parts by weight of ethanol to 100 parts by weight of the cultured mycelium and then extracted for 4 hours at 40 ~ 60 ℃.

In the filtration step (S300), the solution extracted by adding the ethanol was filtered.

In the reduced pressure concentration step (S400), the filtered solution was concentrated under reduced pressure at 65 ~ 80 ℃.

In the dry powderization step (S500), the concentrated thing was lyophilized and powdered to complete the extract of the mycelial mushroom mycelium.

White Clown Mushroom  Of mycelium extract AChE  And PEP  Activity inhibition effect measurement

In order to confirm the inhibitory effect of acetylcholinesterase (AChE) and prolyl endopeptidase (PEP) on the mycelial extract of P. oleracea mushrooms, mycelia of pupa, fungus, leaf mushroom, shiitake, snow fungus, and mushrooms were prepared. Experiments were performed on extracts extracted by adding ethanol to the mycelium cultured in the same manner as the preparation of the white mycelia mushroom mycelium extract.

Acetylcholinesterase (AChE) activity inhibition was measured by the method of Ellman et al. 95 μl of buffer solution (50 mM phosphate buffer, pH 8.0), 15 μl of white mycelia mushroom mycelium extract and 20 μl of acetylcholinesterase (AChE) in each well of a 96-well plate Was added and reacted at 37 ° C. for 15 minutes. Then, 100 μl of substrate (Ellmans reaction solution) was added to each well and reacted at 37 ° C. for 10 minutes. Then, the absorbance was measured at 412 nm of an ELISA reader, and the change value was used. Inhibition of acetylcholinesterase (AChE) activity was measured.

In order to measure the inhibitory effect of Pyryl endopeptidase (PEP) activity, the Toda method was used.The absorbance measurement cuvette was placed in a 800 µl buffer solution (100 mM phosphate buffer, pH 7.0), 40 µl white mycelia mushroom mycelium extract and 80 Into a μl prolyl endopeptidase (PEP) was reacted for 10 minutes at 37 ℃. Subsequently, 80 μl of the substrate, Z-Gly-Pro-pNA, was added and reacted at 37 ° C. for 15 minutes, and the amount of change in absorbance was measured at 410 nm. Inhibition of endopeptidase (PEP) activity was measured.

The results of measuring the AChE and PEP activity inhibitory effects of the white myrtle mushroom mycelium extract and six mycelium extracts are shown in Table 1 below.

Strain Inhibitory effect (%) AChE inhibitory effect
(100 μg / ml, EtOH) PEP inhibitory effect
(10 μg / ml, EtOH) Amanita verna (White Clown Mushroom) 51.1 ± 0.6 22.4 ± 1.8 Cordyceps militaris (Chrysalis Cordyceps) 5.3 ± 0.6 N / D Flammulina velutipes (Enoki mushroom) 1.5 ± 0.1 N / D Grifola frondosa (Leaf mushroom) 19.8 ± 1.5 N / D Lentinus edodes (Shiitake mushrooms) 1.3 ± 0.2 2.3 ± 0.2 Paecilomyces tenuipes Snowflake Cordyceps 2.2 ± 1.6 N / D Phellinus linteus (Situation Mushroom) N / D 16.1 ± 1.2

* N / D: Not Detected

As shown in Table 1, it was confirmed that AChE and PEP activities were simultaneously inhibited in the white group of mushrooms and shiitake mushrooms, but the effect of inhibiting AChE and PEP activity of white mushrooms was 39.3 times higher than that of shiitake mushrooms (AChE ) And 9.7 times (PEP) higher. Thus, the white mycelia mushroom mycelium extract has an AChE and PEP inhibitory effect, it was confirmed that the excellent.

White Clown Mushroom  Mycelial growth rate measurement

To compare the incubation rate of various mycelium, pupa cordyceps, enoki mushroom, leaf mushroom, shiitake mushroom, snow fungus mushroom, and situation mushroom were prepared, inoculated each piece of mycelium into YMG medium and incubated for 2 weeks under the condition of pH 7, 30 ℃. It was. The size of six cultured mycelium was compared with the white mycelial mycelium by measuring the circular diameter.

The results of measuring the size of the white bulrush mushroom mycelium and six mycelium are shown in Table 2 below.

Strain Mycelial growth (mm) Amanita verna (White Clown Mushroom) 54.2 ± 2.5 Cordyceps militaris (Chrysalis Cordyceps) 45.3 ± 1.5 Flammulina velutipes (Enoki mushroom) 50.1 ± 2.2 Grifola frondosa (Leaf mushroom) 35.5 ± 1.2 Lentinus edodes (Shiitake mushrooms) 48.4 ± 2.3 Paecilomyces tenuipes Snowflake Cordyceps 51.2 ± 1.5 Phellinus linteus (Situation Mushroom) 45.3 ± 2.7

As shown in Table 2, the size of the mycelium of the white bulrush mushroom was measured the largest in comparison with the six other myceliums, and thus, the culture rate was the fastest during the same period. Therefore, it could be confirmed that economic feasibility according to the increase in the production of the white myrtle mushroom mycelium extract due to the rapid culture rate.

White Clown Mushroom  Optimum Condition Experiment for Mycelial Culture

For the same period, YMG medium was adjusted to pH 4-8 at 30 ° C to find the optimal pH for mycelial growth of white chloroplast mushroom. In order to find the optimum temperature, the incubation temperature at pH 7 was 17, 24 and 30, respectively. It was incubated for 2 weeks by adjusting to 35 and 40 ℃.

The results of measuring the size of the white cultivated mushroom mycelium cultured for 2 weeks are shown in Table 3 below.

Initial pH (30 ℃) Mycelial growth (mm) Incubation temperature (℃, pH 7)) Mycelial growth (mm) 4 41.3 ± 1.2 17 46.2 ± 2.2 5 52.5 ± 2.3 24 51.8 ± 1.4 6 54.4 ± 2.5 30 53.9 ± 2.2 7 53.3 ± 2.6 35 50.2 ± 1.7 8 50.7 ± 1.5 40 44.6 ± 1.2

As shown in Table 3, the size of the white myrtle mycelium was the largest in the culture of white myrtle mushroom mycelium at pH 5-7, and the largest size of mycelial mycelium at the temperature of 24 ~ 35 ℃. From the results, it was determined that the optimum pH and temperature for the mycelial culture of white chloroplast mushroom were pH 5-7 and 24 ~ 30 ° C.

According to additives White Clown Mushroom  Mycelium extract AChE  And PEP  Activity inhibition effect measurement

White bulrush mushroom mycelia extract (control) inoculated in YMG medium and soluble starch in YMG medium were compared with Tacrine and Z-Pro-prolinal, which have the effect of inhibiting AChE and PEP activity. The inhibitory effects of AChE and PEP activity of mushroom mycelium extracts, toxins of white Algae mushroom, Tacrine and Z-Pro-prolinal were measured.

As described in Example 1, white chlorophyll mushroom mycelium extract inoculated in YMG medium and cultured by adding soluble starch (soluble starch) were extracted using ethanol.

Sample
Inhibitory effect (%) AChE inhibitory effect
(100 μg / ml, EtOH) PEP inhibitory effect
(10 μg / ml, EtOH) Control group (YMG) 51.1 ± 0.6 22.4 ± 1.8 soluble starch group 82.2 ± 1.6 62.9 ± 1.3 Tacrine 97.5 ± 2.1 N / A Z-Pro-prolinal N / A 90.4 ± 2.3 Amanitin N / D N / D

* N / A: Not Applicable

As shown in Table 4, the samples showing lower levels than Tacrine and Z-Pro-prolinal, but inhibiting the activity of AChE and PEP at the same time, were added to the mycelium mycelial extract (YMG) and soluble starch inoculated in YMG medium. It was possible to extract only mycelial mycelium mycelium culture. Among them, mycelial mycelium extract with soluble starch was 1.6 times (AChE) and 2.8 times higher (PEP) than the control group (YMG).

On the other hand, amaranthine, which is a representative toxin of chloroplasts, did not show inhibitory activity in both AChE and PEP enzymes. It is considered to be a bioactive substance.

In conclusion, it was confirmed that the addition of soluble starch increased the effect of inhibiting the activity of Alzheimer's disease inducing enzymes (AChE and PEP).

Claims (8)

Inoculate the mycelium of white bulrush mushroom to the medium of pH 5-7 and incubate for 13-15 days at the temperature of 24-30 ° C, add 3-5 times the weight of ethanol to the cultured mycelium, and then add 3% at 40-60 ° C. A pharmaceutical composition for preventing and treating Alzheimer's disease comprising a white myrtle mushroom mycelium extract obtained by darkening the extract obtained by extracting for 5 hours and then filtering it.
delete The method according to claim 1,
The medium is YMG medium (YMG) as a medium for Alzheimer's disease prevention and treatment comprising a white myrtle mushroom mycelium extract, characterized in that soluble starch is added thereto.
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