TWI646965B - Use of Pleurotus eryngii for the treatment or prevention of dementia and its symptoms - Google Patents

Abstract

The present invention discloses a second use of Pleurotus eryngii, which refers to the use of Pleurotus eryngii for the preparation of a composition for treating and/or preventing a disease associated with an increase in phosphorylated tau-protein content and a disease associated with brain damage. Accordingly, by administering a composition containing an effective amount of Pleurotus eryngii to a body, especially an individual whose phosphorylated tau-protein content is too high or brain damage, the system can effectively improve the brain of the individual. Symptoms of impairment, memory, and learning ability decline.

Description

 Use of Pleurotus eryngii for the treatment or prevention of dementia and its symptoms  

The invention relates to the use of a fungus, in particular to the use of a Pleurotus eryngii for the treatment or prevention of dementia and its symptoms.

According to recent advances in medical technology and health care, the average life expectancy of human beings has been prolonged, and the world has entered an aging society. The incidence of many degenerative diseases has also increased. Among them, dementia has become the focus of public attention. . According to the report of the International Association of Dementia, one person suffers from dementia every 3 seconds. In 2015, the global population of dementia exceeded 40 million, and it is estimated that there will be more than 100 million people worldwide in 2050. Dementia, therefore, the cost of care for patients will become a burden on the health budget of each country. The most well-known type of dementia is Alzheimer's disease, which is mainly caused by excessive oxidative stress in the brain, resulting in amyloid β- peptide (A β ) plaques in the brain. Accumulation and neurofibrillary tangles (NFTs) promote the apoptosis of brain nerve cells, which eventually causes the cortical layer and hippocampus to shrink and lose their memory function. In addition to Alzheimer's disease can cause dementia, such as vascular disease, trauma, infectious diseases, metabolic abnormalities, etc. can cause symptoms of dementia, including memory loss, cognitive impairment, learning ability and so on.

At present, there is no clinically effective drug for the treatment of dementia. Specifically, patients with mild to moderate dementia will delay the onset of symptoms by administering a choline phosphatase inhibitor, or through exercise. The cognitive behavioral education helps patients improve their daily activities, and some people with dementia are accompanied by mental illnesses, so they will be given psychiatric drugs to alleviate their symptoms. However, these drugs cannot achieve treatment or improve. The efficacy of dementia or its related symptoms.

It can be seen from the above that dementia can cause great burden and suffering to the patients themselves or their families, and it will also greatly increase the overall medical cost, resulting in a lack of medical resources. Therefore, seeking an effective treatment and / or The effective component of preventing dementia and its symptoms is an important goal of research in the field of medicine.

The primary object of the present invention is to provide a second use of a fungus which refers to the use of Pleurotus eryngii to treat or/and ameliorate diseases associated with increased levels of phosphorylated tau-protein.

Another object of the present invention is to provide a second use of a fungus which refers to the use of Pleurotus eryngii to treat or ameliorate brain damage and related conditions.

In one embodiment of the present invention, the use of Pleurotus eryngii for the preparation of a composition for treating and/or preventing a disease associated with an increase in phosphorylated tau-protein content is disclosed. By administering a composition containing an effective amount of Pleurotus eryngii to a body, especially an individual whose phosphorylated tau-protein content is too high in the brain, the system can effectively improve the brain damage, memory and learning ability of the individual. The symptoms of depression to improve or treat the effects of diseases associated with hyperphosphorylated tau-protein levels.

Preferably, the disease is dementia, such as Alzheimer's disease.

Preferably, for a 60 kg human body, the daily effective dose of Pleurotus eryngii is at least 3.8 g, and more preferably 7.75 g or more.

In another embodiment of the present invention, the use of Pleurotus eryngii for the preparation of a composition for improving brain damage and related conditions is disclosed, wherein the relevant condition is a decrease in learning ability or a decrease in memory.

Still further, the system deposited from brain injury or brain A β plaques oxidative stress caused by too much.

For a 60 kg human body, the daily effective dose of Pleurotus eryngii is at least 3.8 g, and more preferably 7.75 g or more.

The first panel is the result of analyzing the swimming path of the space exploration experiment in each group of mice.

The second graph is the statistical result of the time required for each group of mice to reach the target quadrant.

The third panel is the result of analyzing the percentage of swimming distance in the space exploration experiment of each group of mice.

The fourth graph is the result of analyzing the number of shuttles of each group of mice in the target quadrant.

The fifth panel is the result of analyzing the swimming rate of each group of mice.

The sixth panel is the result of analyzing the T-maze test response of each group of mice.

The seventh panel is the result of analyzing the expression of phosphorylated tau-protein in the brain of each group of mice.

The eighth figure is the result of analyzing the concentration of lipid peroxidation products in the brain of each group of mice.

The ninth panel is the result of analyzing the concentration of protein carbonyl oxide in the brain of each group of mice.

The tenth figure shows the deposition of Aβ plaque in the brain of each group of mice.

The eleventh figure is the result of analyzing the percentage of Aβ plaque area in each group of mice.

To illustrate the efficacy of the present invention, the following will be further illustrated by a number of examples and with data.

Example 1: Animal feeding conditions

Seventy-five 5-week old C57BL/6J male mice were purchased from the National Laboratory Animal Center (Taiwan, R.O.C.) and weighed approximately 27 grams. The mice were pre-fed to a 16-week old laboratory animal feed (Laboratory Rodent Diet 5001) at a temperature of 25 ± 2 ° C, a relative humidity of 60 ± 10%, and a 12-hour light-dark cycle.

Example 2: Preparation of Pleurotus eryngii fruit body powder

The Pleurotus eryngii fruiting body was taken as a powder in the usual technique of the present invention, and the ergot sulfur content of the Pleurotus eryngii fruiting body powder was analyzed to be 1.3486 mg/g.

According to the US Food and Drug Administration announcement, the average daily intake (ADI) of ergot sulfur is 10.5 mg, and the average weight of mice is 27 grams based on 60 kg of adults, according to the mice. The conversion factor with human body is 12.3 (Ranter, 2005), and the daily intake of ergot sulphur in mice is known, and the ergot sulphur content of Pleurotus eryngii fruit body powder is calculated. The dose was 43 mg/mouse/day, which was used as the medium dose, while the high dose was 2.5 times the medium dose and the low dose was 0.5 times the medium dose.

Example 3: Establishing the Alzheimer Animal Model

The pre-cultured 16-week-old mice were prepared as a mouse model of Alzheimer's disease with reference to Alzheimer's disease-induced surgery (Jhoo et al., 2004).

In detail, the Aβ _1-40 was dissolved in 35% acetonitrile and 0.1% trifluoroacetic acid (pH = 2.0) solution, prepared in 0.16mM concentration of Aβ _1-40 solution. The mice were anesthetized with isoflurane (3 L/min). According to the brain anatomy of Franklin and Paxions (2005), the CA1 localization on both sides of the hippocampus was performed with a stereotaxic instrument. Taking the skull bone bregma as the origin, the left and right horizontal axis is 1.8mm (X axis ± 1.8mm), and the vertical axis is 2.3mm (Y axis - 2.3mm) for drilling. After drilling, the Aβ _1-40 solution is injected by injection. The depth of the brain is 1.5 mm, and the flow rate on each side is 2.5 μL/5 min. After completion, the skull was repaired with a bioadhesive and sutured with a suture needle and a stapler.

Example 4: Animal test

The mice pre-fed to 16 weeks old in Example 1 were divided into 6 groups, 12 in each group. The first group was normal mice, and the second to sixth groups were Alzheimer's model mice. The mice in this group were continuously fed for six weeks under the treatment conditions shown in Table 1 below. The memory learning ability test was conducted during the feeding period, and then each group of mice was sacrificed to collect blood and organs for subsequent analysis.

Example 5: Water maze test

Each group of mice in Example 4 was cultured under the treatment conditions until the 28th day, and the water maze test was performed. The dynamics of each group of mice were observed by a camera, and the data was analyzed by the image analysis software (Etho Visoin XT). The swimming pool used is about 125 cm in diameter and 45 cm high; a platform with a diameter of 12 cm and a height of 25 cm is placed in the swimming pool for the rest of the mice; the water level in the pool is higher than the platform. 2 cm, and add milk powder to form a turbid body of water to hide the platform.

On the 28th to 30th day, the platform is fixed at the center of the fourth quadrant of the swimming pool. The mouse head enters the second quadrant of the swimming pool, and each swims for 90 seconds. If the platform is found within 90 seconds, the small platform The mouse can rest on the platform for 30 seconds, and then rest in the cage for 30 seconds, the next test, and if the platform is not found within 90 seconds, the mouse is guided to rest on the platform for 30 seconds, then The next test was performed after 30 seconds of rest in the cage. Each mouse of the group was tested 4 times a day, and the daily search time and search distance of each mouse were counted. On the 31st day, the platform was moved outside the pool, and the mouse head went out into the second quadrant of the pool, allowing it to swim for 90 seconds, and recorded the mouse in the reference recording test with the image analysis software. The quadrant dwell time, swimming distance and number of crossings and the path of the whole swimming are set. The results are shown in the first to fourth figures.

According to the statistical results of the search time, on the first day of the experiment (Day 28), the time taken by the first group to the sixth group to find the platform were: 87.17±4.57, 85.67±6.76, 84.92±7.64, 85.21 ±5.51, 84.67±6.87 and 75.15±13.73 seconds showed that the time spent in the sixth group of mice was significantly lower than that in the other groups. On the second day of the experiment, the first group of mice significantly shortened the time to find the platform (18.64±4.84 seconds), the second group spent 83.38±10.28 seconds to find the platform, and the fourth group to the sixth group found the platform. The time is 61.29±17.28, 40.67±7.73 and 40.10±8.28, respectively. Compared to the second group of mice, the fourth to sixth groups of mice were shortened by 26%, 51%, and 52%, respectively. On the third day of the experiment, the search time of the other groups of mice was shortened by 32-85% compared with the second group of mice.

In terms of search distance, the first day of the experiment, the search distance of the first group of mice was 9.90 ± 0.59 meters, significantly lower than the third group to the fifth group (11.47 ~ 11.95 meters); on the second day of the experiment, Except for the second group of mice (10.30±1.48 m), the search distances of all the groups were shortened, which were 3.61±1.28, 8.27±2.01, 5.03±1.81, 7.51±1.87 and 4.78±1.35, respectively. meter. On the third day of the experiment, the search distances of the first group to the sixth group were 3.36±0.44, 11.06±1.12, 7.94±1.68, 5.23±2.19, 3.56±1.31, and 3.46±1.78 meters, respectively, compared with the second group. The remaining groups were shortened by 70%, 28%, 53%, 68%, and 69%, respectively.

Furthermore, as can be seen from the results of the first figure, the trajectory of the second group of mouse search platforms is concentrated in the second quadrant, and there is no obvious exploration direction and target; the search trajectory of the third group of mice is concentrated in the third and the third Four quadrants; the trajectories of the fourth to sixth groups of mice are concentrated in the fourth quadrant. As can be seen from the results of the second graph, the first group of mice had a target quadrant 徘徊 time of 25.70±5.7 seconds, which was significantly higher than the second group (12.20±6.9 seconds), and the third group to the sixth group of mice. The time (21.43±4.53, 23.65±5.97, 22.25±3.62, and 29.33±3.79 seconds) was increased compared with the second group of mice.

From the results of the third graph, the percentage of the target quadrant swimming distance divided by the total distance of the first group, the third group to the sixth group was 26.34±5.88%, 22.24±3.93%, 27.19±5.60, 24.30±, respectively. 4.16 and 28.40±4.29%, which were higher than the second group (13.72±7.21%). The results showed that the time between the mice and the swimming distance was consistent, and the mice were excluded from the stagnant time due to stagnation. situation. As can be seen from the results of the fourth graph, the average number of times of the fourth quadrant of the first to sixth groups of mice was 7.25±1.16, 3.50±2.00, 6.38±1.30, 7.63±2.00, 7.25±1.06, and 8.50±2.73, respectively. Times.

From the results of the 28th to 31st days, the Alzheimer's disease mice that were not administered the Pleurotus eryngii fruit body powder did not shorten the search time and distance as the number of test days increased, and could not remember the platform position. When Alzheimer's disease mice are administered with Pleurotus eryngii fruit body powder, the search time and distance are shortened as the number of test days increases, and the memory capacity can be improved.

Further, the swimming speed of each group of mice was obtained by using the test results of the 28th to 30th days. As shown in the fifth figure, the swimming rate of the second group of mice (14.95±4.09cm/s) was obvious. Lower than the first group, the third group to the sixth group of mice (18.68±2.04, 19.73±2.78, 19.49±3.60, 21.07±1.83 and 18.18±2.82cm/s). Based on previous studies (Sonn et al., 2013), it is revealed that the swimming rate is lower than that of the control group due to poor memory of Alzheimer's disease mice. Therefore, the results of the fifth figure show that the Pleurotus eryngii It can improve the swimming rate of Alzheimer's disease mice, and can improve the symptoms of memory loss caused by Alzheimer's disease.

It can be seen from the above that Alzheimer's disease-induced surgery does not establish an Alzheimer's model mouse, which makes it impossible to memorize the position of the platform, and the A. sinensis can improve the Azheimer's disease by administering the present invention. Memory and learning ability of the patient.

Example 6: T-maze test

First, a T-shaped labyrinth device is made with black acrylic, and its three arms are equal in length. The length, width and height of the device are 70, 10, and 20 cm, respectively, and are set at 50 cm from the ground.

Each group of mice that were kept for 33 days under different conditions in Example 4 was fasted for 8 hours before the test. On the first day of the experiment, food was placed at the bottom left and right side of the T-shaped labyrinth device, and each group of mice was placed in the initial channel for free movement and feeding for 5 minutes; the next day, food was placed at the bottom end of the right channel, and then left and right. The side settings are exchanged, and each mouse is trained three times; on the third day, the left channel is set to the correct path, and the right channel is blocked by the partition. When the mouse reaches the left side, the food can be safely eaten; Block the left channel. When the mouse reaches the right channel, remove the food and limit the activity range by 10 seconds, repeating 3 times; on the fourth day, set the left side to the correct path, the right side is the wrong path, record and analyze each small The number of times the mouse reached the correct path, the result is shown in the sixth picture.

From the results of the sixth graph, the correct rate of food in the first group to the sixth group was 75.56±8.82%, 44.44±13.33%, 71.11±10.54%, 75.65±13.33%, and 82.22±. 12.02% and 70.00±15.12%, of which the correct rate of the second group of mice was significantly lower than other groups.

It can be seen that since Alzheimer's disease can deteriorate the memory of the individual, even under high-intensity stimulation, it is difficult to make the individual have a deep memory, and the A. sinensis is improved by the administration of the present invention. A disorder in which individuals with poor memory can increase the accuracy of finding food. Therefore, the Pleurotus eryngii has the symptoms of improving or treating dementia or the memory loss caused by the invention.

Example 7: Analysis of the results of the mouse brain

After sacrifice of each group of mice, the brain was taken out, the right half of the brain was fixed with 10% formalin solution, and the left half of the brain was homogenized and centrifuged, and the supernatant was taken, respectively, and the commercially available tau-protein sets were respectively taken. Group (Pei Xin Biotech, Cat. No. E93893M, Taiwan), TBARS kit (Cayman, Cat. No. 10009055, USA), protein carbonyl colorimetric kit (Cayman, Cat. No. 10005020, USA) The internal phosphorylation of tau-protein, malondialdehyde and protein carbonyl oxide content, and the detailed steps of detecting the content of phosphorylated tau-protein, malondialdehyde and protein carbonyl oxide in the sample are common knowledge of those skilled in the art. Therefore, I will not repeat them here. The phosphorylated tau-protein, malondialdehyde and protein carbonyl oxide contents in the brain of each group of mice are shown in the seventh to ninth and second tables.

As revealed in the previous literature, Alzheimer's disease increases the content of phosphorylated tau-proteins and free radicals in the brain, and the phosphorylated tau-protein neurotubules are tightly joined and separated, resulting in the loss of nerve disintegration. Function, while free radical molecules attack the white matter side chain, causing damage to nerve cells; due to Alzheimer's disease, brain damage, the content of lipid oxidation products in the brain: malondialdehyde will increase, and when malondialdehyde The higher the content, the more serious the brain damage. Therefore, the levels of phosphorylated tau-protein, malondialdehyde and protein carbonyl oxide in the brain of the second group of mice were significantly higher than those in the first group of mice.

By administering the Pleurotus eryngii to the individual suffering from Alzheimer's disease, the content of phosphorylated tau-protein, malondialdehyde and protein carbonyl oxide in the brain is significantly lower than that of the apricot. The fruit body powder of the mushroom is low, indicating that the Pleurotus eryngii is reduced in the brain by reducing the oxidative stress in the brain, thereby reducing the concentration of phosphorylated tau-protein in the brain, and also removing free radicals in the brain and making the protein carbonyl in the brain. The concentration of oxides is reduced, reducing the damage of nerve cells. Therefore, the Pleurotus eryngii has the efficacy of treating or/and preventing dementia and causing nerve damage thereof.

Example 8: Results of immunohistochemical staining

The brains of each of the mice of the group were subjected to immunohistochemical staining analysis, wherein the steps of tissue sectioning and staining are the general knowledge of the technical field to which the present invention pertains, and thus will not be redundant. The stained tissue sections were taken under a 40-fold microscope field, and the results were as shown in the tenth figure, and the mouse brain A was analyzed by the image software (Image J, pro plus 6.3, MediaCybernetics Inc, USA). Percentage of beta plaque deposition, where the formula: A β plaque area percentage = (A β plaque area / brain area) x 100%, the results are shown in Figure 11 and Table 3.

From the results of the present example, the second group of mice A β plaques in the brain area than the first set of lines, compared to the second group of mice, the third to sixth group A β plaques in mice brains The area system is significantly reduced. Furthermore, it can be seen from the previous research that melatonin reduces the amount of Aβ plaque deposits in Alzheimer's mice by reducing the pressure on the brain, and thus it is known that the apricot abalone disclosed in the present invention By reducing the oxidative stress in the brain, the mushroom system can reduce the expression of the pre-existing starch and β-secretase activity in the brain to reduce the deposition of Aβ plaque in the brain. Accordingly, the Pleurotus eryngii system disclosed in the present invention can effectively prevent or/and treat dementia or its related symptoms.

It can be clearly seen from the above examples and examples that the actual system of the Pleurotus eryngii mushroom of the present invention can reduce the damage of nerve cells, enhance the learning ability and memory of the individual, and therefore, the effective amount of the present invention is administered. Uncovering Pleurotus eryngii to a body, especially those suffering from dementia or Alzheimer's disease, can effectively improve or treat dementia or its related symptoms, such as learning disabilities, cognitive decline, and memory loss.

Furthermore, the results of the above examples show that mice must ingest 21.5 mg of Pleurotus eryngii (low dose) daily to prevent or treat dementia and its related symptoms, and to eat more than 43 mg. Pleurotus eryngii has a better effect. According to the conversion coefficient between human and mouse is 12.3, and the content of ergot sulfur in the oyster mushroom (1.3486mg/g), it is known that for adults of 60 kilograms, at least 3.875g of apricot must be ingested daily. Pleurotus ostreatus can achieve the effect of preventing or treating dementia and its related symptoms, and the effect is best when the food intake of Pleurotus eryngii reaches 7.75 g or more. Considering the daily intake of ergot sulfur in the human body, it is recommended that the daily intake of 60 kg of adults should not exceed 8 grams of Pleurotus eryngii.

Claims (6)

A use of Pleurotus eryngii for the preparation of a phosphorylated tau-protein inhibitor. The use according to the first aspect of the invention, wherein the phosphorylated tau-protein inhibitor is for the treatment of Alzheimer's disease. The use according to claim 1, wherein the phosphorylated tau-protein inhibitor is for treating dementia. A use of Pleurotus eryngii for the preparation of A beta plaque inhibitors. The use according to claim 4, wherein the A beta plaque inhibitor is for the treatment of Alzheimer's disease. The use according to claim 4, wherein the A beta plaque inhibitor is for treating dementia.