TW201620535A - Antrodia camphorata mycelium active substance for protecting nerve cells and food composition thereof - Google Patents

Abstract

The present invention relates to an Antrodia camphorata mycelium active substance for protecting nerve cells and a food composition thereof, wherein the Antrodia camphorata mycelium active substance is capable of inhibiting cytotoxicity caused by 10 <mu>M amyloid (Amyloid <beta>(25-35)), and also inhibiting cell apoptosis arising from mitochondrial rupture caused by 1-methyl-4-phenylpyridine (MPP+) at a non-cytotoxic concentration of 50 <mu>M. Therefore, the Antrodia camphorata mycelium active substance of this invention has a potential to prevent neurodegenerative diseases.

Description

Anthraquinone mycelium active substance for protecting nerve cells and food composition thereof

The present invention relates to an anthrax mycelium active substance and a food composition thereof, and particularly to an anthrax mycelium active substance for protecting nerve cells and a food composition thereof.

Neurodegenerative Diseases

Neurodegenerative diseases are symptoms of the gradual loss of function of neuronal cells in the brain and central nervous system. The brain and spinal cord are made up of various neurons, each responsible for different functions, such as controlling movement, processing and receiving messages, and making decisions. The hippocampus in the brain area is mainly the hub responsible for learning and memory. The damage of nerve cells in the hippocampus can lead to defects in dementia and learning ability. Neurodegenerative diseases can occur as neuronal deterioration or the myelin responsible for transmitting messages loses function over time. Because the nerve cells of the brain and spinal cord are not easily regenerated, permanent damage can occur as long as they are damaged. It takes a long time for patients with neurodegenerative diseases to gradually appear. It can take several months or years for a patient to feel the impact. Symptoms are usually noticed after many brain cells have died or cell function has ceased, and a large number of nerve deaths have affected the function of the ventricles. Its obvious features are (1) the slow and progressive death of nerve cells in a specific brain region (2) the loss of neurotransmitter function (3) often occurs in middle-aged and elderly people, and the symptoms will be more serious over time. As the patient is gradually isolated from society during the onset, plus the current relevant There is a lack of medical knowledge, so the public has little understanding and attention. With the aging of the population structure, the number of patients with neurodegenerative diseases is increasing. For families, patients and the general public, the impact of economic, psychological and life forms is difficult to estimate and bear. Common neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, and Huntington's disease.

Alzheimer's disease

Alzheimer's disease is the most common neurodegenerative disease in the elderly. The main lesions are in the cerebral cortex and hippocampus. Many pathologically appear senile plaques, nerve fiber tangles. (neurofibrillary tangle) and deposition of beta-type starch (β- Amyloid ). The senile plaque is a complex diseased structure that is gradually formed by the accumulation of amyloid beta-type starch in the early stage of the disease. It is composed of about 40 to 42 amino acids. Beta-type amyloid is present in various organs of the body and is secreted in various parts of the brain. However, a single beta-type amyloid is harmless, and only polymeric beta-type amyloids are toxic, forming senescent plaques and disrupting the normal functioning of cells. Although a single beta-type amyloid is secreted in all areas of the brain, the most dense area of toxic aging spots is in the hippocampus. Stacked mature amyloid plaques often surround many dysfunctional axons and dendrites, thickening these neurites, and mature amyloid plaques can also activate microglue. Cells (microglia) help to cause inflammatory reactions and clear amyloid plaques. The accumulation of a large number of elderly and amyloid plaques in the cerebral cortex and hippocampus is the best indicator of Alzheimer's symptoms. The main physiological symptoms of Alzheimer's disease include cognitive deterioration (such as poor memory, confusion about time, people, and location), degeneration of behavioral functions (such as decreased ability to handle daily life), and mental state deterioration (for example) Depression, delusions and abnormal behaviors, etc.). The drugs currently used to treat Alzheimer's disease can only delay the symptoms of the patient and cannot be cured.

Parkinson's disease

Another common neurodegenerative disease is Parkinson's disease, which occurs in people over the age of five or sixty, mainly due to a deficiency called a dopamine chemical. Dopamine is made from a nerve cell called substantia nigra in the brainstem. Degeneration of dopamine neurons in the substantia nigra of the midbrain does not produce enough neuroleptic substance dopamine to direct muscle activity; communication between neurons must rely on dopamine to smooth the movements of the body, and lack of enough dopamine to produce various activities. obstacle. Symptoms of the manifestations are balance dysfunction, muscle stiffness, slow motion, and unstable posture. Early symptoms often have a one-handed regular tremor, slower movements, and muscle stiffness gradually spread throughout the body. At the end of the day, it is almost necessary to rely on a wheelchair to act. Because Parkinson's disease is caused by the lack of dopamine, there is no obvious characteristic in the early stage of the disease. It has often been five years since the case was confirmed, and there is currently no effective drug for treatment. Loss of mitochondrial function and oxidative stress have been shown to be important pathogens in Parkinson's disease, while MPP ⁺ (1-methyl-4-phenylpyridine) is a positively charged compound. It is an inhibitor of mitochondrial complex I, which inhibits oxidative phosphorylation in mitochondria, causes ATP depletion and cell death, and is therefore toxic. In a mouse experiment, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is able to pass the Blood-brain barrier (BBB) and is nerved in the brain. Glial cells are converted to MPP ⁺ , and MPP ⁺ causes inflammation of the substantia nigra and kills dopamine-producing nerve cells in the dense part of the substantia nigra, causing the animals to develop symptoms similar to Parkinson's disease. MPP ⁺ is highly selective in triggering the production of dopaminergic neuronal death. It is through a transporter responsible for reabsorption of dopamine in the nerve endings, in which the dopamine transporter transports MPP ⁺ into the cell.

Endoplasmic Reticulum Stress

During aging, nerve cells receive excessive oxidative stress, excessive accumulation of oxides causes damage or death of nerve cells, and abnormal activation or death of glial cells is called oxidative neurotoxicity. The gradual accumulation of toxicity is the cause of neurodegenerative diseases. Tunicamycin (Taumycin, TM) can cause the production of a large number of unfolded proteins by inhibiting the N-linked glycoproteins of the protein, and causes the cells to stay in the G1 phase, resulting in Endoplasmic Reticulum Stress. In the past, it was pointed out that endoplasmic reticulum pressure is related to neurodegenerative diseases. Endoplasmic reticulum stress is the path that mainly induces neuronal apoptosis. Therefore, using TM to induce nerve cell pressure can be used as a screening mode.

Antrodia camphorata

The type of apocynum lucidum is also known as the oyster mushroom, the oyster, the scorpion scorpion, and Taiwan has a yin and yang counterpart. The body of A. camphorata is perennial and has a strong aroma of eucalyptus, which is quite different from general ganoderma lucidum. Its appearance is plate-shaped or bell-shaped. In the plate shape, the surface is orange-red (yellow) color, the whole surface has micropores, and the bottom layer of the plate has a pale yellow-white cork, whereby the cork is attached to the inner wall of the hollow material in the burdock tree. The bell-shaped type, the sub-solid layer (clock face) is also orange (yellow) color, full of micropores (4-5 bacteria/mm), with spore taste very bitter, fresh orange-red, then It becomes orange-brown or brown, and the bell body is dark green-brown. The spores were observed under a microscope, and the shape was a smooth, colorless transparent micro-bend shape.

Wild anthrax grows on the hollow inner wall of the burdock trunk, and this characteristic causes many burdocks to fall. According to the literature, Antrodia camphorata is the only wood rot fungus found in burdock trees. The disease is brown and decayed, so it is brown rot fungus. However, the pathogenicity of Antrodia camphorata is not strong, so there is very little burdock This death. Although Antrodia camphorata is a pathogen for Burdock, because Antrodia is expensive and exceeds the economic value of Burdock, it is not important that the pathogen of Burdock is not important.

The cultivation of Antrodia camphorata, the technique of artificial cultivation, still needs to be worked hard. Therefore, it is still obtained by means of deep mountain collection. But collecting anthocyanin is not an easy task, because the first place is to find the origin of the burdock tree. The common difficulty is the burdock tree and the lynx, which are very similar and difficult to distinguish. At present, the most direct method has been proposed by Fujita Anji. The dried oil is mainly Saforle and fifteen aldehydes, so there is the taste of scutellaria in SARS, and the sirloin is terpineol (d -terpinenol) is the main, but has the taste of camphor oil, which can distinguish the burdock and the cockroach; the second difficulty is to find the trunk with hollow holes from the large woods, which is quite difficult. If there is an ointment in the cavity, it can be collected regularly. Because the trunk of the burdock looking for a hollow hole is not easy, the unscrupulous businessman simply cut down the burdock tree, in the hope that it will grow in the future and collect and sell it. Therefore, for environmental protection and economic considerations, the development of artificial cultivation of Antrodia is the primary direction. It is a pity that artificial cultivation of Antrodia technology has not been able to break through. Antrodia camphorst grows very slowly on burdock wood chips and even stagnates. Therefore, if you can change the modern biotechnology to cultivate the mycelium of Antrodia camphorata, it will be the most economical and environmentally friendly artificial cultivation method.

In the past, it was found that the methanol extract of Antrodia camphorata fruit protects the rat's primary cortical neuron culture from neurotoxicity caused by amyloid β _(25-35) , but due to the anthraquinone fruit body The acquisition is not easy, the source of the sample is not fixed, and it is easy to change the composition of the active ingredient with the change of the eucalyptus matrix for culture, and is also susceptible to contamination by unidentified eucalyptus, thereby increasing the risk of eating. Liquid culture mycelium can fix the composition of the culture medium and can greatly reduce the risk of contamination.

Therefore, in view of: 1. Antrodia camphora is a biological species that is sent to the burdock wood. The burdock is a first-class wood species of conservation, and the hollow burdock is not easy to obtain. 2. The source of the eucalyptus matrix used to cultivate Antrodia is less fixed, and the matrix change also affects. The composition of the active ingredient; therefore, the inventors of the present invention have worked hard to develop a liquid culture mycelium, and found that the active substance of the present invention is contained in the liquid fermentation of the mycelium of the mycelium, and it is proved It has the effect of inhibiting neurotoxicity.

All scientific and technical terms used in this specification, unless otherwise defined, are defined by those of ordinary skill in the art.

The main object of the present invention is to provide an anthrax mycelium active substance for protecting nerve cells.

The mycelium mycelium active substance is prepared by the following steps: (a) fermentation: the mycelium of the anthraquinone is fermented at 28 ° C for 12 days to form a mycelium fermentation broth; (b) freeze-drying: the mycelium fermentation broth is freeze-dried to form a mycelium lyophilized powder; (c) extraction: the mycelium lyophilized powder is extracted with 10-30 times its volume of organic solvent , wherein the extraction is performed by shaking at 10-250 rpm at 15-25 ° C for 24 hours to form a supernatant; (d) concentrating and drying: the supernatant is concentrated to dryness under reduced pressure, and finally an arsenic is obtained. Mycelial active substance.

The mycelium is purchased from the Institute of Food Industry Development, deposited under the registration number: CCRC 35396 (BCRC 35396); the organic solvent of the extraction step is methanol (MeOH), ethanol (EtOH). Or ethyl ethanoate (EA); the anthrax mycelium active substance is used to antagonize the cytotoxicity caused by Amyloid β _(25-35) or MPP ⁺ .

Another object of the present invention is to provide a food composition for protecting nerve cells, wherein the food composition comprises the active substance of the mycelium of the present invention, and a pharmaceutically acceptable carrier, excipient, and diluent And adjuvants, etc.

Wherein the food composition is used to antagonize the cytotoxicity caused by Amyloid β _(25-35) or MPP ⁺ .

Figure 1 Compound Sesamin.

Embodiment 1

(1) The source of the organism

The strain A.camphorata CCRC-35396 used in the present invention was purchased from the Food Industry Development Research Institute.

(2) Fermentation of organisms

The CCRC-35396 strain was inoculated into 1 L of medium (1.0% glucose, 0.5% soy flour, 0.5% digested protein, 0.01% MgSO4, 0.01% defoamer (KM-72 antifoam), pH 4.0 ) Rotating at 28 ° C for 10 days in a 2-L Hinton flask. The cultured mycelium was transferred to a 200-L fermenter containing 120 L of the above medium in a sterile room, and fermented at 28 ° C for 12 days.

(3) Extraction of organisms

The fermented mycelium fermentation broth is freeze-dried, and the lyophilized powder after lyophilization is extracted with 10-30 volumes of 100% ethanol, and shake-extracted in a shake flask at 10-250 rpm at 15-25 ° C. After shaking for 24 hours, the extract can be repeatedly extracted several times, and the above supernatant is concentrated to dryness under reduced pressure to obtain the active substance of the mycelium of Antrodia camphorata, and then dissolved in an appropriate solvent and quantified to an appropriate concentration, and placed in 4 °C save spare.

In addition, other organic solvents, such as water, methanol, ethyl acetate, n-hexane, etc., may be used for extraction, and the mycelium fermentation broth is freeze-dried, and the freeze-dried lyophilized powder is extracted with 10-30 volumes of organic solvent. And shaking extraction with a shake flask, shaking extraction at 15-250 rpm at 15-25 ° C for 24 hours, the extraction can be repeated several times, the above supernatant is concentrated to dryness under reduced pressure to obtain the mycelium of Antrodia camphorata The active substance is reconstituted in an appropriate solvent and quantified to an appropriate concentration, and stored at 4 ° C until use.

The methanol extract of the above-mentioned Antrodia camphorata is partitioned with ethyl acetate and water, and the extract of the ethyl acetate layer is concentrated under reduced pressure to remove the solvent. After the residual ethyl acetate layer is adsorbed on silica gel and dried, The ruthenium column chromatography was used for preliminary separation, and was sequentially extracted with n-hexane-ethyl acetate (95:5→0:100) to obtain 10 divisions. The fraction obtained by hexane-ethyl acetate (80:20) was further separated by silica gel and Sephadex LH-20 column chromatography to obtain a compound, Sesamin (Fig. 1).

Example 2 Neurocytotoxic damage and protection of nerve cells

1. Antagonize Amyloid β _(25-35) Caused by primary cortical neuron toxicity

The primary brain cortex of Sprague-Dawley rats was taken out for one day, and the cortical tissue was disrupted with 0.5 mg/ml papain and filtered, and then the polylysine solution was first used. Poly-1-lysine) coated on a 24-well flat-bottom tissue culture plate with a density of 5000 cells/ml-well; after six days, each cell was properly applied to it; finally these cells were again tested (Agrocybe Silky active substance) to test. The test sample was dissolved in DMSO at concentrations of 10, 20, 40, and 50 mM using dimethyl sulfite (DMSO) as a solvent. The concentration of DMSO in the medium was maintained at no more than 1 μl/ml to ensure that it did not affect the growth of cortical neuron cells; after treatment with the test sample for two hours, 10 μM amyloid (Amyloid β _{(25-) 35)} ) Add nerve cells; two days later, remove the medium and add 300 μl/well of MTT solution (concentration: 0.5 mg/ml); place the cells in a 37 ° C, 5% CO ₂ incubator for 1 hour. The mixture of 24-well medium was removed; finally, 200 μl/well of DMSO was added to the cells, and the absorbance was measured at a absorbance of 600 nm using a spectrophotometer ELISA reader to obtain experimental results; and DMSO solvent The control group comparison can calculate the percentage of survival (%), and the percentage of survival = [absorbance of the sample (600 nm) / absorbance of DMSO (600 nm)] × 100.

As shown in Table 1, the cell viability was 53.53 ± 0.21% in the cortical neurons of the control group containing 10 μM Amyloid β _(25-35) ; and the nerve cells were treated with 20 μ/ml Antrodia methanol extract for two hours. The survival rate of the experimental group was increased to 61.65±0.50%, and the survival rate was 8% higher than that of the control group. Survival was also increased by 7% in the experimental group of ethyl acetate partition. Further purification of the extracted compound Sesamin at 10 μM concentration, the survival rate was 10% higher than the control group, and at 50 μM concentration, the survival rate was even 15% higher than the control group. *, p value<0.05, t-test.

2. Antagonize MPP ⁺ Toxic toxicity of NG108-15 cell line

The cell line of NG108-15 was plated in a 96-well flat-bottom tissue culture plate at a density of 2×10 ⁴ cells/ml-well; after 24 hours, each cell was appropriately applied thereto; finally, these cells were further tested samples. (Astragalus mycelium active substance) to test. The test sample was dissolved in DMSO at a concentration of 20 and 40 mM using dimethyl sulfite (DMSO) as a solvent. The concentration of DMSO in the medium was maintained at no more than 1 μl/ml to ensure that it did not affect the growth of NG108-15 cells; after treatment with the test sample for 2 days, the concentrations were 20, 40, 60, 80 μM, respectively. NP108-15 cells were treated with MPP ⁺ (1-methyl-4-phenylpyridine); four days later, the medium was removed and 100 μl/well of MTT solution (concentration 0.5 mg/ml) was added; the cells were placed at 37 ° C. After culturing for 1 hour in a 5% CO ₂ incubator, the mixture of 96-well medium was removed; finally, 30 μl/well of DMSO was added to the cells, and the ELISA reader was used as a spectrophotometer at an absorption wavelength of 570 nm. The absorbance was measured to obtain the experimental results; and the percentage of survival (%) was calculated in comparison with the DMSO solvent control group, and the percentage of survival = [absorbance of the sample (570 nm) / absorbance of DMSO (570 nm)] × 100.

In the cell viability data (Table 2), in the experimental group of Antrodia camphorata-ethyl acetate, the cytotoxicity caused by low concentration of 20 μM MPP ⁺ increased the survival rate from 66.19% to 87.61%; There was no significant difference in the toxicity of high concentration MPP ⁺ ; in the experimental group of Antrodia camphora - methanol, the cytotoxicity induced by different concentrations of MPP ⁺ had obvious protective effect, and the survival rate was increased at 20 μM MPP ⁺ concentration. 23%, 40 μM MPP ⁺ concentration can increase survival by 20%. *, p value<0.05, **, p value<0.01, t-test.

The anthraquinone active substance of the present invention is fermented by a natural medicinal fungus, so that in addition to being more safe, it has the potential to treat and improve neurodegenerative diseases.

The detailed description of the present invention is intended to be illustrative of the preferred embodiments of the present invention, and is not intended to limit the scope of the present invention. In the scope of patents.

In summary, this case is not only innovative in terms of technical thinking, but also has the potential to treat or prevent neurodegenerative diseases. It should have fully complied with the statutory invention patent requirements of novelty and progressiveness, and the application will be submitted according to law, and you are requested to approve it. This invention patent application, in order to invent invention, to the sense of virtue.

Claims (8)

An active substance of Antrodia camphorata which protects nerve cells, which is prepared by the following steps: (a) Fermentation: The mycelium of Antrodia camphorata is fermented at 28 ° C for 12 days to form a hyphae. (b) freeze-drying: the mycelium fermentation broth is freeze-dried to form a mycelium lyophilized powder; (c) extraction: the mycelium lyophilized powder is 10-30 times its volume Extraction by organic solvent, wherein the extraction is shock-extracted at 10-250 rpm at 15-25 ° C for 24 hours to form a supernatant; (d) concentrated and dried: the supernatant is concentrated to dryness under reduced pressure, and finally A medicinal mycelium active substance is obtained. For example, the Mycelium mycelium active substance for protecting nerve cells according to the first aspect of the patent application, wherein the mycelium is deposited in the Food Industry Development Research Institute of the corporation, the accession number: CCRC 35396. The mycelium mycelium active substance for protecting nerve cells according to the first aspect of the patent application, wherein the organic solvent is methanol, ethanol or ethyl acetate. The mycelium mycelium active substance for protecting nerve cells according to the first aspect of the patent application, wherein the anthrax mycelium active substance is used for antagonizing the cytotoxicity caused by amyloid β _(25-35) . The mycelium mycelium active substance for protecting nerve cells according to the first aspect of the patent application, wherein the anthrax mycelium active substance is used for antagonizing cells caused by 1-methyl-4-phenylpyridine (MPP ⁺ ) toxicity. A food composition for protecting nerve cells, comprising the mycelium mycelium active material according to claim 1 of the patent application, and a pharmaceutically acceptable carrier, an excipient, a diluent, an adjuvant, and the like. A food composition for protecting nerve cells according to claim 6, wherein the food composition is for antagonizing cytotoxicity caused by amyloid β _(25-35) . A food composition for protecting nerve cells according to claim 6, wherein the food composition is for antagonizing cytotoxicity caused by 1-methyl-4-phenylpyridine (MPP ⁺ ).