Use of purified mushroom beta-glucan in prevention, amelioration or treatment of alzheimer's disease, dementia or brain function degeneration

Abstract

The present invention provides the use of a purified mushroom beta-glucan in the prevention, amelioration or treatment of Alzheimer's disease, dementia or brain function degeneration, in which the purified mushroom beta-glucan is derived from mushroom mycelium or a fermentation product thereof, and the purity of the purified mushroom beta-glucan is 60% or more.

Description

Technical Field

[0001] The present disclosure relates to the use of a purified mushroom β-glucan in the preparation of a composition for preventing, improving or treating Alzheimer's disease, dementia or brain function degradation, and particularly relates to a mushroom β-glucan purified from different mushroom mycelia or their fermentation products. Background Art

[0002] Dementia is a general term used to describe a variety of symptoms, which mainly refer to the decline of memory or other thinking abilities to a degree that seriously reduces the ability to perform daily activities. Research and analysis have found that Alzheimer's Disease is the main factor causing dementia. Approximately 60% to over 80% of dementia patients are caused by Alzheimer's disease, far higher than Parkinson's Disease or vascular dementia that occurs after a stroke.

[0003] Alzheimer's disease is a neurodegenerative disease in which nerve cells in the hippocampus of the brain are damaged. From pathology, it can be found that the brain tissue atrophies, and phenomena such as amyloid β plaques, senile plaques or neurofibrillary tangles appear in the cortical region. Research has found that the concentration of amyloid β in and outside the brain cells of Alzheimer's disease patients is too high, making it difficult for brain cells to maintain health and communicate with each other. The hippocampus in the brain region is the learning and memory center of the brain, and the brain cells in this region are often the first to be damaged. Therefore, one of the earliest symptoms of Alzheimer's disease is usually memory loss. As the disease progresses, patients will have problems in recognizing time, space and people, and various cognitive function disorders will occur.

[0004] In addition to humans, elderly companion animals, including dogs and cats, also suffer from dementia. The amnesia in canines and felines is called Cognitive dysfunction syndrome (CDS). CDS in elderly pets has currently become one of the most troublesome problems in pet care. According to statistics, 1 / 3 of dogs over 11 years old suffer from CDS, and the proportion of dogs over 15 years old suffering from CDS is closer to 70%; in addition, more than 50% of cats over 15 years old also have a prevalence rate. CDS is a progressive cognitive ability degradation disease in pathology, and the clinical symptoms are mainly changes in behavior, which are similar to human Alzheimer's disease both in pathology and clinical symptoms. CDS in elderly pets not only seriously affects the health of pets, but also increases the trouble for pet owners to take care of them.

[0005] Fungal mushrooms such as Ganoderma lucidum, Schizophyllum commue, Agaricus blazei, Cordyceps sinensis, Coriolus versicolor, Taiwanofungus camphoratus, Phellinus linteus, Hericium erinaceus, Auricularia auricula, and Cordyceps militaris have been regarded by traditional Chinese medicine as medicinal materials with various excellent effects since ancient times. In recent years, many scientific studies have found that these different mushrooms have different effects such as enhancing immune ability, anti-tumor, slowing down cancer metastasis, regulating physiological functions, improving cardiovascular diseases, repairing cell tissues, anti-aging, and improving cognitive impairment. Among them, mushrooms have complex and diverse β-glucans. Many studies have found that various mushroom polysaccharides have high functional activities and have obvious medical effects on diseases such as cancer, cardiovascular diseases, and metabolic diseases that are difficult to solve in current medicine.

[0006] Currently, many studies on Alzheimer's disease or dementia are carried out with crude extracts of mushroom fruiting bodies, spores, mycelia, or their liquid fermentation products. Although it has been shown to have preventive or ameliorative effects, the crude extracts used contain various polysaccharides, glycoproteins, organic acids, and complex peptide molecules, and it is impossible to effectively identify the active ingredients that truly have efficacy. On the other hand, due to the low yield of mushroom β-glucan and the need for repeated and cumbersome purification techniques to remove impurities such as glycoproteins and small molecule peptides, only a small amount of high-purity mushroom β-glucan can be obtained. Therefore, there is currently no relevant research on the use of β-glucans from medicinal mushrooms such as Ganoderma lucidum, Schizophyllum commune, Taiwanofungus camphoratus, Hericium erinaceus, Agaricus blazei, Cordyceps sinensis, Cordyceps militaris, Phellinus linteus, Coriolus versicolor, and Auricularia auricula in the prevention, amelioration, or treatment of Alzheimer's disease or dementia.

[0007] Therefore, the need to develop high-purity mushroom β-glucan for the treatment or prevention of such diseases remains unmet. Summary of the Invention

[0008] In view of the above, the present disclosure provides the use of a purified mushroom β-glucan in the preparation of a composition for preventing, ameliorating, or treating Alzheimer's disease, dementia, or brain function degradation, wherein the purified mushroom β-glucan is derived from mushroom mycelia or its fermentation products, and wherein the purity of the purified mushroom β-glucan is 60% or more.

[0009] The present disclosure also provides a method for preventing, ameliorating or treating Alzheimer's disease, dementia or brain function degradation in an individual in need, comprising administering to the individual a composition comprising purified mushroom β-glucan, wherein the purified mushroom β-glucan is derived from mushroom mycelium or its fermentation product.

[0010] In some embodiments, the mushroom of the mushroom mycelium is selected from at least one of the group consisting of Ganoderma lucidum, Schizophyllum commune, Agaricus blazei, Cordyceps sinensis, Coriolus versicolor, Antrodia camphorata, Phellinus linteus, Hericium erinaceus, Auricularia auricula-judae and Cordyceps militaris.

[0011] In some embodiments, preventing, ameliorating or treating Alzheimer's disease, dementia or brain function degradation comprises enhancing the cell viability of nerve cells by purified mushroom β-glucan.

[0012] In some embodiments, preventing, ameliorating or treating Alzheimer's disease, dementia or brain function degradation comprises reducing the oxidative stress of nerve cells by purified mushroom β-glucan.

[0013] In some embodiments, reducing the oxidative stress of nerve cells comprises at least one of reducing the content of inducible nitric oxide synthase in the individual, enhancing the activity of catalase and / or superoxide dismutase, and reducing the activity of lactate dehydrogenase.

[0014] In some embodiments, preventing, ameliorating or treating Alzheimer's disease, dementia or brain function degradation comprises reducing the neurocytotoxicity caused by amyloid-β (1-40) and / or reducing amyloid accumulation by purified mushroom β-glucan.

[0015] In some embodiments, preventing, ameliorating or treating Alzheimer's disease, dementia or brain function degradation comprises preventing, ameliorating or treating cognitive dysfunction by purified mushroom β-glucan, and the cognitive dysfunction comprises at least one of memory impairment, memory decline, reduced learning ability and loss of sense of space.

[0016] In some embodiments, the purified mushroom β-glucan is obtained by the following preparation method, comprising:

[0017] Taking the mushroom mycelium and fermentation broth after liquid fermentation, and obtaining a crude extract after ultrasonic disruption treatment;

[0018] Centrifuging the crude extract and taking the upper clarified liquid;

[0019] Performing a first filtration treatment on the upper clarified liquid, and filtering the upper clarified liquid with a 0.8 to 3 μm filter membrane and a 0.22 to 0.5 μm filter membrane;

[0020] Performing a first freeze-drying treatment to prepare a mushroom polysaccharide raw material;

[0021] Redissolve the mushroom polysaccharide raw material in water to form a redissolved liquid;

[0022] Perform a second filtration treatment on the redissolved liquid, removing impurities with ultrafiltration ceramic membrane filter columns with MWCO: 2 to 10 KD and MWCO: 80 to 150 KD;

[0023] Perform a third filtration treatment, filtering with a 0.22 μm filter membrane;

[0024] Perform a second freeze-drying to produce the purified mushroom β-glucan.

[0025] In some embodiments, the composition is in the form of a powder, tablet, suppository, microcapsule, liquid, spray or plug.

[0026] In some embodiments, the composition further comprises an excipient. In some embodiments, the composition is obtained by mixing the purified and extracted β-glucan with an excipient and then drying. In some embodiments, the drying includes spray drying or freeze drying.

[0027] In some embodiments, the excipient is at least one selected from the group consisting of lactose, sucrose, glucose, fructooligosaccharide, starch, starch derivatives and dietary fiber.

[0028] In some embodiments, the composition is obtained by mixing the purified mushroom β-glucan with a solvent or buffer solution. In some embodiments, the solvent is water and the buffer solution is physiological saline.

[0029] In some embodiments, the composition is a pharmaceutical, feed, feed additive, beverage, nutritional supplement, dairy product, food, food additive, raw material for health food or finished health food.

[0030] In some embodiments, the composition further comprises an additive, and the additive is at least one selected from the group consisting of a carrier, an excipient, a preservative, a diluent, a filler, an absorption promoter, a sweetener and an adjuvant.

[0031] In some embodiments, the composition is a pharmaceutical comprising the purified mushroom β-glucan and its pharmaceutically acceptable carrier.

[0032] In some embodiments, the pharmaceutical is administered to an individual in an effective amount of 0.04 to 1 mg / kg.bw.

[0033] In some embodiments, the pharmaceutical is administered orally, rectally, transdermally, mucosally or by injection.

[0034] Based on the above invention, the present disclosure provides a purified extract of mushroom β-glucan derived from mushroom mycelium or its fermentation product, and uses it to prepare a composition for preventing, improving or treating Alzheimer's disease, dementia or brain function degradation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The above content, other objects, features and effects of the present invention will become more apparent by referring to the following description and in conjunction with the accompanying drawings.

[0036] Figure 1 In an embodiment of the present invention, the structure of purified mushroom β-glucan prepared by combining prior art and an improved purification process.

[0037] Figure 2 Showing the HPLC chromatogram and molecular weight of β-glucan purified from Ganoderma lucidum in an embodiment of the present invention.

[0038] Figure 3 Showing the HPLC chromatogram and molecular weight of β-glucan purified from Schizophyllum commune in an embodiment of the present invention.

[0039] Figure 4 Showing the memory learning test schedule of orally or injecting Alzheimer's disease rats with different doses of GL (purified Ganoderma lucidum β-glucan) and SC (purified Schizophyllum commune β-glucan) in an embodiment of the present invention.

[0040] Figure 5 Showing the effect of orally or injecting Alzheimer's disease rats with different doses of GL (purified Ganoderma lucidum β-glucan) and SC (purified Schizophyllum commune β-glucan) on the activity of acetylcholinesterase in their blood in an embodiment of the present invention.

[0041] Figure 6 Showing the effect of orally or injecting Alzheimer's disease rats with different doses of GL (purified Ganoderma lucidum β-glucan) and SC (purified Schizophyllum commune β-glucan) on the total antioxidant capacity in their blood in an embodiment of the present invention.

[0042] Figure 7 Showing the effect of orally or injecting Alzheimer's disease rats with different doses of GL (purified Ganoderma lucidum β-glucan) and SC (purified Schizophyllum commune β-glucan) on the ability to scavenge nitric oxide in their blood in an embodiment of the present invention.

[0043] Figure 8 Showing the effect of orally or injecting Alzheimer's disease rats with different doses of GL (purified Ganoderma lucidum β-glucan) and SC (purified Schizophyllum commune β-glucan) on the activity of catalase in their blood in an embodiment of the present invention.

[0044] Figure 9 In one embodiment of the present invention, the effects of orally or injecting different doses of GL (purified Ganoderma lucidum β-glucan) and SC (purified Schizophyllum commune β-glucan) on the superoxide dismutase activity in the blood of Alzheimer's rats are shown.

[0045] Figure 10 In one embodiment of the present invention, the effects of orally or injecting different doses of GL (purified Ganoderma lucidum β-glucan) and SC (purified Schizophyllum commune β-glucan) on the lactate dehydrogenase activity in the blood of Alzheimer's rats are shown. Detailed implementation manners

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to implement or test the present invention, the preferred methods and materials are now described. All the published documents mentioned herein are incorporated herein by reference.

[0047] As used in this disclosure, the singular forms "a" and "the" include plural referents unless clearly and specifically limited to one referent. Unless the context clearly indicates otherwise, the term "or" can be used interchangeably with the term "and / or". The terms "first", "second", "third", etc. do not specifically imply a relative order, but are used for distinction.

[0048] As used herein, the terms "comprising", "including", "having", "containing" and any other variants thereof are meant to cover non-exclusive inclusion. For example, when describing an object that "comprises" a limitation, other components, elements, components, structures, regions, parts, devices, systems, steps or connections, etc. may also be included unless otherwise stated, and other limitations should not be excluded.

[0049] As used herein, the term "effective amount" refers to the amount of an active ingredient that achieves a clinical outcome when the composition is administered to an individual. For example, when a composition containing the purified mushroom β-glucan of the present disclosure is administered to an individual with Alzheimer's, the "clinical outcome" includes a decrease in amyloid β (1-40), an increase in total antioxidant capacity, a reduction in the severity of symptoms associated with Alzheimer's, and / or an increase in the lifespan of the individual. As will be appreciated by those skilled in the art, the effective amount may vary depending on the route of administration, the use of excipients, the possibility of co-using with other treatments, and the condition to be treated.

[0050] According to the present disclosure, terms such as "ameliorate", "treat", etc. used herein generally refer to obtaining a desired pharmacological or physiological effect. In terms of completely or partially "preventing" a disease, appearance, illness or symptom, the effect may be prophylactic, and / or in terms of partially or completely curing a disease and / or an adverse reaction attributable to the disease or illness, the effect may be therapeutic. The term "treat" as used herein encompasses any treatment of a disease, illness or abnormal behavior in a mammal (such as a human), and includes: (a) preventing a disease (such as Alzheimer's disease), an illness (such as memory impairment) or an abnormal behavior (such as loss of sense of space) from occurring in an individual who may be susceptible but has not been observed or diagnosed as having the disease; (b) inhibiting a disease, illness or symptom, i.e., causing the disease or symptom to subside; and (c) alleviating a disease, illness or symptom, i.e., causing the disease or symptom to subside.

[0051] As used herein, the terms "individual", "patient" and "subject" are used interchangeably. The term "individual" refers to a human or an animal. Examples of individuals include, but are not limited to, humans, monkeys, mice, rats, groundhogs, ferrets, rabbits, hamsters, cows, horses, pigs, deer, dogs, cats, foxes, wolves, chickens, emus, ostriches and fish.

[0052] As used herein, the terms "administer", "administer to" or "administration" refer to a method or route by which an active ingredient is at least partially localized at a desired site within an individual to produce a desired effect. The active ingredients described herein can be administered by any suitable route known in the art. For example, the pharmaceutical compositions of the present disclosure are administered orally to an individual.

[0053] As used herein, "enhance", "boost", "augment" or similar terms refer to increasing or prolonging the intended potency or duration of a drug. Thus, enhancing the efficacy of a therapeutic drug, where "enhance" refers to an increased or prolonged ability in a relevant therapeutic system, which ability is the pharmacodynamic effect or duration.

[0054] Fungal mushrooms are medicinal materials with a variety of excellent effects. In recent years, many scientific studies have found that different types of mushrooms have the effects of enhancing immune ability, anti-tumor, slowing down cancer metastasis, regulating physiological functions, improving cardiovascular diseases, repairing cell tissues, anti-aging, improving cognitive impairment, etc. Among them, β-glucan contained in mushrooms has been proven to have excellent effects of enhancing immunity and regulating physiological functions, and has been widely used in medical and health foods. Studies have found that β-glucans from different sources have different functions due to different receptors and signal transduction pathways on cell membranes. Mushroom β-glucans are complex and diverse. Many studies have found that different mushroom β-glucans not only have high functional activities, but also have obvious curative effects on many diseases that are difficult to solve in current medicine, such as cancer, cardiovascular diseases, metabolic diseases, etc.

[0055] Since the yield of mushroom β-glucan is too low and repeated and cumbersome purification techniques are required to remove impurities such as glycoproteins and small molecular peptides to obtain a small amount of purified mushroom β-glucan, there are currently no relevant studies on the use of β-glucans from medicinal mushrooms such as Ganoderma lucidum, Schizophyllum commune, Antrodia cinnamomea, Hericium erinaceus, Agaricus blazei, Cordyceps sinensis, Cordyceps militaris, Phellinus linteus, Coriolus versicolor, and Auricularia auricula in the prevention, improvement, or treatment of Alzheimer's disease or dementia. Therefore, the inventors of the present disclosure integrated existing technologies, broke through the production bottleneck, prepared purified mushroom β-glucan, and applied it to the research on the prevention, improvement, or treatment of Alzheimer's disease, dementia, or brain function degradation, and found unexpectedly effective results.

[0056] In one embodiment of the present disclosure, there is provided a use of a purified mushroom β-glucan for the preparation of a composition for preventing, improving, or treating Alzheimer's disease, dementia, or brain function degradation. The basic unit of the purified mushroom β-glucan is glucose, which is composed of a main chain connected by β1-3 glycosidic bonds and side chains connected by β1-6 glycosidic bonds. The specific structure of the mushroom β-glucan is as Figure 1 shown, and its chemical formula is (C 24 H 42 O 21 ) n .

[0057] In one embodiment of the present disclosure, the effective purification of mushroom β-glucan can be achieved through the application and combination of various purification and extraction techniques, so that the purity of the mushroom β-glucan is 60% or higher. Moreover, the purity of the purified mushroom β-glucan can be freely adjusted according to needs. For example, the purity of the mushroom β-glucan can be between 60% and 98%, between 70% and 98%, between 80% and 98%. For example, the purity can be 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%.

[0058] In another embodiment of the present disclosure, the molecular weight of the purified mushroom β-glucan is between 3000 kDa (±15%) and 12000 kDa (±30%). For example, the molecular weight of the β-glucan from Ganoderma lucidum is between 3558 kDa and 11544 kDa (as Figure 2 shown), and the molecular weight of the β-glucan from Schizophyllum commune is between 3847 kDa and 9635 kDa (as Figure 3 shown).

[0059] In another embodiment of the present disclosure, there is also provided a method for preventing, improving, or treating Alzheimer's disease, dementia, or brain function degradation in an individual in need, including administering to the individual a composition containing a purified mushroom β-glucan with a purity higher than 60%, wherein the purified mushroom β-glucan is derived from mushroom mycelium or its fermentation product.

[0060] In one embodiment of the present disclosure, the purified mushroom β-glucan is derived from mushroom mycelia or its fermentation products. In one embodiment, the mushroom of the mushroom mycelia is at least one selected from the group consisting of Ganoderma lucidum, Schizophyllum commune, Agaricus blazei, Cordyceps sinensis, Coriolus versicolor, Antrodia cinnamomea, Phellinus linteus, Hericium erinaceus, Auricularia auricula-judae, and Cordyceps militaris.

[0061] It has been confirmed that excessive oxidative stress is an important factor accelerating aging. The accumulation of excessive oxides will produce oxidative neurotoxicity, causing damage and death of nerve cells or triggering abnormalities of glial cells, thereby leading to the occurrence of neurodegenerative diseases. Therefore, reducing the oxidative stress of nerve cells and enhancing the antioxidant capacity of nerve cells are considered to be one of the means for preventing or treating Alzheimer's disease, dementia, or other neurological diseases.

[0062] In one embodiment of the present disclosure, the prevention, improvement, or treatment of Alzheimer's disease, dementia, or brain function degradation includes increasing the cell survival rate of nerve cells through mushroom β-glucan, so as to achieve the purpose of protecting nerve cells. In some embodiments, the prevention, improvement, or treatment of Alzheimer's disease, dementia, or brain function degradation includes reducing the oxidative stress of nerve cells through the purified mushroom β-glucan. In some embodiments, reducing the oxidative stress of nerve cells includes at least one of reducing the content of inducible nitric oxide synthase in an individual's body, enhancing the activity of catalase and / or superoxide dismutase, and reducing the activity of lactate dehydrogenase. That is to say, the four results of increased catalase activity, increased superoxide dismutase activity, decreased lactate dehydrogenase activity, and decreased nitric oxide content may occur individually, or any two, any three, or all four results may occur simultaneously.

[0063] In one embodiment of the present disclosure, the prevention, improvement, or treatment of Alzheimer's disease, dementia, or brain function degradation includes reducing the neurocytotoxicity caused by amyloid-β (1-40) and / or reducing amyloid accumulation through the purified mushroom β-glucan.

[0064] In the embodiments of the present disclosure, the prevention, improvement, or treatment of Alzheimer's disease, dementia, or brain function degradation includes preventing, improving, or treating cognitive dysfunction through the purified and extracted mushroom β-glucan. In some embodiments, the cognitive dysfunction includes at least one of memory impairment, memory decline, reduced learning ability, and loss of spatial sense.

[0065] According to the present disclosure, the purified mushroom β-glucan that can prevent, improve or treat Alzheimer's disease, dementia or brain function degradation can be purified by adopting various purification and extraction techniques and combinations, so that the purity is above 60% or between 60% and 98%. In one embodiment, the purification techniques include:

[0066] Take the mushroom mycelium and fermentation broth after liquid fermentation, ultrasonically break the mushroom mycelium and mix it with its fermentation product to obtain a crude extract;

[0067] Remove the precipitate in the crude extract by centrifugation and take out the upper clarified liquid;

[0068] Subsequently, perform a first filtration treatment on the upper clarified liquid, for example, use a filtration membrane with a pore size of 0.8 to 3 μm, such as a filtration membrane with a pore size of 0.8, 0.9, 1.0, 1.5, 2, 2.5 or 3 μm, for filtration to remove impurities; then use a filtration membrane with a pore size of 0.22 to 0.5 μm, such as a filtration membrane with a pore size of 0.22, 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5 μm, for filtration to filter out impurities;

[0069] After that, perform a first freeze-drying treatment on the upper clarified liquid after the first filtration treatment to make a mushroom polysaccharide raw material;

[0070] Redissolve the mushroom polysaccharide raw material in water to form a re-dissolved liquid;

[0071] Perform a second filtration treatment on the re-dissolved liquid, which makes the re-dissolved liquid pass through ultrafiltration ceramic membrane filter columns with a molecular weight cut off (MWCO): 2 to 10 KD, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 KD and MWCO: 80 to 150 KD, such as 80, 90, 100, 110, 120, 130, 140 or 150 KD, to remove different small molecule and macromolecule impurities;

[0072] Perform a third filtration treatment on the re-dissolved liquid after the second filtration treatment, which filters out impurities with a 0.22 μm pore size mixed cellulose ester filtration membrane;

[0073] Finally, perform a second freeze-drying treatment on the re-dissolved liquid after the third filtration treatment to make a purified mushroom polysaccharide product.

[0074] In some embodiments, the above filtration treatment can be repeated several times before proceeding to the subsequent steps, so that the purity of the purified mushroom polysaccharide reaches above 60% or between 60% and 98%, such as 60, 65, 70, 75, 80, 85, 90, 95 or 98%, for use in different state requirements.

[0075] In one embodiment of the present disclosure, mushroom polysaccharide powder of a specific purity, such as greater than 90%, is formulated into purified mushroom polysaccharide solutions of different concentrations according to different requirements, filtered through a 0.22 μm sterile injection filter membrane, bottled, and then sterilized by high-pressure steam, such as sterilized at 121 °C for 20 minutes, thereby preparing bottled injections or oral liquids.

[0076] In one embodiment of the present disclosure, the composition of the present disclosure can be in any suitable form, including but not limited to powders, tablets, suppositories, microcapsules, liquids, sprays or inserts.

[0077] In one embodiment of the present disclosure, the composition further includes excipients. In one embodiment, the composition is obtained by mixing purified mushroom β-glucan with excipients and then drying, and the resulting composition is in the form of a powder, for example. In another embodiment, the drying is, for example, spray drying or freeze drying. In yet another embodiment, the excipients include lactose, sucrose, glucose, fructooligosaccharides, starch, starch derivatives or dietary fiber, but are not limited thereto.

[0078] In one embodiment of the present disclosure, the composition is obtained by mixing purified mushroom β-glucan with a solvent or buffer solution, and the resulting composition is in the form of a liquid, for example. In yet another embodiment, the solvent can be water, and the buffer solution can be physiological saline or other pharmaceutically acceptable buffer solutions, not limited thereto.

[0079] In one embodiment of the present disclosure, the composition of the present disclosure includes pharmaceuticals, feeds, feed additives, beverages, nutritional supplements, dairy products, foods, food additives or raw materials and finished products of health foods, but are not limited thereto. In one embodiment, the composition of the present disclosure is a pharmaceutical, which includes purified mushroom β-glucan and its pharmaceutically acceptable carrier. In one embodiment, the pharmaceutical of the present disclosure is for human or animal use.

[0080] In some embodiments, the pharmaceutical is administered to an individual in an effective amount for the prevention, improvement or treatment of Alzheimer's disease, dementia or brain function degradation, wherein the effective amount is between 0.04 mg and 1 mg per kilogram of human body weight (0.04 to 1 mg / kg.bw), such as 0.04, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 or 1 mg / kg.bw, and other animals can be converted according to pharmacokinetics. In other embodiments, the pharmaceutical is administered orally, rectally, transdermally, mucosally or by injection, but is not limited thereto.

[0081] In one embodiment of the present disclosure, the composition of the present disclosure may comprise at least one additive selected from the group consisting of carriers, excipients, preservatives, diluents, fillers, absorption promoters, sweeteners, and adjuvants, but not limited thereto.

[0082] Needless to say, it is believed that those skilled in the art can make the most use of the present disclosure based on the foregoing description. Therefore, the following examples are for illustrative purposes only and do not limit the scope of the present disclosure in any way.

[0083] Examples

[0084] Preparation Example: Preparation of High-Purity Mushroom β-Glucan from Mushroom Mycelium

[0085] The purification technique of the purified mushroom β-glucan of the present invention is further improved based on the purification methods of the applicant's previous patents (Patent Nos. I481718 and I686480), the entire content of which is incorporated herein by reference. Specifically, one liter of culture medium is prepared with sterile water as the solvent in a fermentation vessel, and trehalose, mannose, and glucose are used as the carbon sources of the culture medium, and the weight ratio of the trehalose, mannose, and glucose is 1:1:1, and 0.5 wt% yeast extract is used as the nitrogen source of the culture medium; then, the prepared production medium together with the fermentation vessel is placed in an autoclave for high-temperature and high-pressure sterilization, and the sterilization conditions are 121 °C for 15 minutes. After sterilization, it is cooled at room temperature, and then Ganoderma lucidum mycelium is added, and cultured at a rotation speed of 30 to 100 revolutions per minute for about 15 days, so that the mushroom mycelium can synthesize a large amount of mushroom β-glucan in the culture medium.

[0086] Take the mushroom mycelium and fermentation broth after liquid fermentation, break the mushroom mycelium by ultrasonic wave and mix it with its fermentation product to obtain a crude extract; remove the precipitate in the crude extract by centrifugation, take out the upper clarified liquid, and then use a 0.8 μm filter membrane (AAWP04700, MF- ) and a 0.45 μm mixed cellulose ester filter membrane (HAWP04700, MF- ) for filtration to remove impurities; then remove the moisture by freeze-drying to make a mushroom polysaccharide raw material.

[0087] Dissolve the dried mushroom polysaccharide raw material in water, and filter it through ultrafiltration ceramic membrane filter columns with MWCO: 2 to 10 KD and MWCO: 80 to 150 KD to remove different small molecule and macromolecule impurities. Then filter it through a 0.22 μm mixed cellulose ester filter membrane (GSWP04700, MF- ) and then freeze-dry it to make a purified mushroom β-glucan with a purity of 60 to 98%.

[0088] Purified mushroom β-glucan solutions of various purities are prepared into various required concentrations, and then filtered through a hydrophilic sterilized polysulfide syringe filter with a pore size of 0.22 μm and a diameter of 33 mm (SLGPR33RB, ), bottled after filtration, and then autoclaved at 121 °C for 20 minutes to prepare bottled injections or oral liquids.

[0089] Other mushroom mycelia such as Ganoderma lucidum and Schizophyllum commune are purified according to the aforementioned steps and used for the analysis of subsequent examples.

[0090] The mushroom β-glucan obtained by this purification method can reach a purity of more than 60% under the condition of re-dissolving in water, and further separation and purification can make the purity as high as 98% (this is limited by the current β-glucan reagent grade reference sample (Beta Glucan, United States Pharmacopia (USP) Refference Standard, Sigma-Aldrich, 1048288), and the purity analysis can only detect up to 98%).

[0091] Example 1: Analysis of Purified Mushroom β-Glucan

[0092] The basic unit of the purified mushroom β-glucan prepared according to the aforementioned preparation example is glucose, which is composed of a main chain connected by β1-3 glycosidic bonds and a side chain of β1-6 glycosidic bonds. The specific structure of the purified mushroom β-glucan is as Figure 1 shown.

[0093] Using a High Performance Liquid Chromatography (HPLC) high performance liquid chromatograph (HPLC Pump, JASCO; Autosampler, AS-4050) combined with a refractive index detector (Refractive Index Detector, RIdetector, RI-4030), the Shodex SUGAR KS-804, KS-805, and KS-806 columns are connected in series to analyze the molecular weights of different mushroom β-glucans. The results show that the molecular weight of the purified Ganoderma lucidum β-glucan is between 3558 kDa and 11544 kDa (as Figure 2 shown), and the molecular weight of the purified Schizophyllum commune β-glucan is between 3847 kDa and 9635 kDa (as Figure 3 shown).

[0094] Protein quantitative analysis was performed on the β-glucan produced by the present invention (Bio-Rad Protein Assay Kit II (5000002EDU)). The results showed that in the samples with a concentration of 1500 ppm, the average protein concentration of the purified Ganoderma lucidum β-glucan product was 2.031 μg / mL after three consecutive detections, and the average protein concentration of the purified Schizophyllum commune β-glucan product was 3.665 μg / mL. The results are shown in Table 1.

[0095] Table 1. Protein concentration analysis of purified Ganoderma lucidum β-glucan (GL) and purified Schizophyllum commune β-glucan (SC)

[0096]

[0097] Example 2: Effects of purified mushroom β-glucan on human neuroblastoma cell line and mouse neuroblastoma cell line

[0098] To evaluate the neuroprotective ability of purified mushroom β-glucan, taking purified Ganoderma lucidum β-glucan as an example, a 48-hour cell viability assay was performed on the human neuroblastoma cell line (IMR 32) and the mouse neuroblastoma cell line (Neuro 2a). The results showed that purified Ganoderma lucidum β-glucan did not reduce cell viability. On the contrary, 24 hours after the addition dose reached 25 μg / mL, the cell viability was significantly increased (Table 2), indicating that β-glucan has the function of protecting nerve cells.

[0099] Table 2. Cell viability of IMR 32 and Neuro 2a cells treated with different concentrations of GL (purified Ganoderma lucidum β-glucan) samples for 24 and 48 hours.

[0100]

[0101] Example 3: Effects of purified Ganoderma lucidum β-glucan on learning and memory, spatial exploration and working memory in mice with Alzheimer's disease

[0102] Alzheimer's disease can cause damage to the hippocampus of the brain, affecting responses to different behaviors such as learning ability, memory ability, and spatial judgment. In this study, 56 Crl:SD rats were divided into 9 groups. Microinjectors were installed in the hippocampal region of all rats' brains, and Aβ was injected daily. 1-40Amyloid proteins can induce Alzheimer's disease. After installation, the following groups were established: The AM group was the drug control group (Positive control), and the common Alzheimer's disease drug Aricept was administered daily; The GLI1 group was injected with 0.47 mg / kg.bw of purified Ganoderma lucidum β-glucan daily; The SCI1 group was injected with 0.47 mg / kg.bw of purified Schizophyllum commune β-glucan daily; The GL1 group was administered 0.47 mg / kg.bw of purified Ganoderma lucidum β-glucan daily; The GL5 group was administered 2.35 mg / kg.bw of purified Ganoderma lucidum β-glucan daily; The SC1 group was administered 0.47 mg / kg.bw of purified high-purity Schizophyllum commune β-glucan product daily; The SC5 group was administered 2.35 mg / kg.bw of purified high-purity Schizophyllum commune β-glucan product daily; The A group was the blank control group (No-treatment control), and no drugs were administered after inducing Alzheimer's disease; The C group was the control group (Control), and neither Alzheimer's disease was induced nor any drugs were administered. The Morris water maze (MWM) test was started 22 days after the operation, and the differences in learning and memory, spatial exploration, and working memory were evaluated respectively. The detailed test schedule is as Figure 4 shown.

[0103] In the MWM test, the pool where the rats swam was divided into four quadrants, and the learning, memory, and search abilities of the rats for the target were observed. The shorter the swimming time and distance required to find the target, the better the related abilities.

[0104] Regarding the evaluation of learning and memory, it can be found from the data in Tables 3 to 5 that the experimental groups injected or administered with purified Ganoderma lucidum and Schizophyllum commune β-glucans performed better than the drug-administered group (AM group) on the first to third days of the learning and memory test, and even far better than the control group (C group) that did not induce Alzheimer's disease and was not administered drugs.

[0105] Table 3. The residence time and total moving distance of rats orally or injected with different doses of GL and SC in different quadrants on the first day of the learning and memory test.

[0106]

[0107] 1. Each value is expressed as mean ± SD (n = 6).

[0108] 2. a,b,c The value represents a significant difference between the stationary platform quadrant and other quadrants (p < 0.05).

[0109] Table 4. The residence time and total moving distance of rats orally or injected with different doses of purified GL and SC in different quadrants on the second day of the learning and memory test.

[0110]

[0111] 1. Each value is expressed as mean ± SD (n = 6).

[0112] 2. a,b,c The value represents a significant difference between the stationary platform quadrant and other quadrants (p < 0.05).

[0113] Table 5. Residence time and total moving distance of rats orally or injectedly administered different doses of GL and SC in different quadrants on the third day of the learning and memory test.

[0114]

[0115] 1. Each value is expressed as mean ± SD (n = 6).

[0116] 2. a,b,c The value represents a significant difference between the stationary platform quadrant and other quadrants (p < 0.05).

[0117] In the 90 - second spatial probe test in the water maze, it was found that when rats were placed in the second and third quadrants, the experimental groups fed or injected with purified mushroom β - glucan had significant differences in the speed of spatial exploration compared with the C group and the AM group, especially the high - dose feeding group (GL5 / SC5) was particularly significant. The detailed data are shown in Table 6.

[0118] Table 6. 90 - second spatial probe test: Residence time of rats orally or injectedly administered different doses of purified GL and SC in different quadrants

[0119]

[0120]

[0121] a,b,c The value represents a significant difference between the stationary platform quadrant and other quadrants (p < 0.05).

[0122] In the continuous three - day working memory assessment in the water maze, it can be found from the data in Tables 7 to 9 that the experimental groups fed or injected with purified mushroom β - glucan had significant advantages in the performance of working memory. It can be seen from the first day that the high - dose feeding group showed significantly better performance in terms of time and speed. Although the control group and the drug - using group also improved working memory, overall they were still inferior to the group fed with purified mushroom β - glucan.

[0123] Table 7. Residence time and total moving distance of rats orally or injectedly administered different doses of purified GL and SC in different quadrants on the first day of the working memory test.

[0124]

[0125] 1. Each value is expressed as mean ± SD (n = 6).

[0126] 2. a,b The value represents a significant difference between the stationary platform quadrant and the other quadrants (p < 0.05).

[0127] Table 8. Residence time and total moving distance of rats orally or injectively administered with different doses of GL and SC in different quadrants on the second day of the working memory test.

[0128]

[0129] 1. Each value is expressed as mean ± SD (n = 6).

[0130] 2. a,b The value represents a significant difference between the stationary platform quadrant and the other quadrants (p < 0.05).

[0131] Table 9. Residence time and total moving distance of rats orally or injectively administered with different doses of GL and SC in different quadrants on the third day of the working memory test.

[0132]

[0133] 1. Each value is expressed as mean ± SD (n = 6).

[0134] 2. a,b The value represents a significant difference between the stationary platform quadrant and the other quadrants (p < 0.05).

[0135] Example 4: Effects of Purified Mushroom β-Glucan on Acetylcholinesterase Activity and Oxidative Stress in Rats

[0136] In rats induced with Alzheimer's disease, due to damaged brain cells and impaired nerve conduction, the acetylcholinesterase activity in their bodies will increase significantly. Therefore, the acetylcholinesterase activity in the experimental rats was analyzed 28 days after the surgery. As Figure 5 shown, it can be found that the acetylcholinesterase activity in rats injected or orally administered with purified Ganoderma lucidum or Schizophyllum commune β-glucan is not only lower than that in the induced group, but also shows a tendency to approach the control group as the dose increases. This result indicates that purified mushroom β-glucan has the function of protecting nerve cells, and there is no significant difference between it and the AM group. Each value in the figure is expressed as mean ± SD (n = 6); # indicates a significant difference compared with the β-amyloid injection group (Group A) (p < 0.05); * indicates a significant difference compared with the normal group (Group C) (p < 0.05).

[0137] In addition, brain nerve cells are damaged due to increased oxidative stress and the long-term accumulation of the toxicity of oxidative stress. Therefore, 28 days after the operation, blood was drawn from the rats participating in the experiment to analyze their total antioxidant capacity and nitric oxide scavenging capacity in vivo, as well as the changes in the activities of catalase, superoxide dismutase, and lactate dehydrogenase in their bodies. As Figures 6 to 10 shown, it can be found that when the total antioxidant capacity of rats injected or orally administered with purified Ganoderma lucidum or Schizophyllum commune β-glucan was evaluated using Trolox equivalent antioxidant capacity equivalents as the standard for measuring antioxidant capacity, the total antioxidant capacity in their bodies was significantly increased, the content of inducible nitric oxide synthase (iNOS), which can increase the nitric oxide content in the body, decreased (which means that the nitric oxide scavenging capacity was improved), the activities of catalase and superoxide dismutase with antioxidant functions increased, and the activity of lactate dehydrogenase, which represents the degree of cell damage in the body, decreased. This result shows that purified mushroom β-glucan has excellent effects on reducing oxidative stress, and all the analysis results have significant differences compared with the AM group. Each value in the figure is expressed as the mean ± SD (n = 6); # indicates a significant difference compared with the β-amyloid injection group (Group A) (p < 0.05); * indicates a significant difference compared with the normal group (Group C) (p < 0.05).

[0138] Example 5: Safety evaluation of purified mushroom β-glucan

[0139] To evaluate the safety of purified mushroom β-glucan, the physiological indexes of the rats participating in this study were detected.

[0140] Starting from after the operation, purified mushroom β-glucan was fed and injected to the rats for a total of four weeks, and the weights of all the rats participating in the experiment were recorded every week. The results showed that purified mushroom β-glucan had no significant effect on the body weight of the rats (Table 10).

[0141] Table 10. Weekly average body weight changes of rats orally administered or injected with different doses of GL and SC

[0142]

[0143] Each value is expressed as the mean ± SD (n = 6)

[0144] On the 28th day after the operation, the total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and creatine phosphokinase in the blood of all the rats participating in the experiment were analyzed. The results showed that there were no significant differences in the relevant indexes in the bodies of all the rats participating in the experiment (Table 11).

[0145] Table 11. Changes in the concentrations of cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and creatine phosphokinase (CPK) in the blood of rats orally or injectively administered with different doses of GL and SC

[0146] Each value is expressed as mean ± SD (n = 6)

[0147] On the 28th day after surgery, the aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total protein, albumin, and globulin in the blood of all rats participating in the experiment were analyzed. The results showed that there were no significant differences in the relevant indicators in all rats participating in the experiment (Table 12).

[0148] Table 12. Changes in aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (Alk-p), total protein, albumin, and globulin in the blood of rats orally or injectively administered with different doses of GL and SC

[0149]

[0150] Each value is expressed as mean ± SD (n = 6)

[0151] On the 28th day after surgery, the renal function indicators blood urea nitrogen, creatinine, and uric acid in the blood of all rats participating in the experiment were analyzed. The results showed that there were no significant differences in the relevant indicators in all rats participating in the experiment (Table 13).

[0152] Table 13. Changes in the renal function indicators blood urea nitrogen (BUN), creatinine, and uric acid in the blood of rats orally or injectively administered with different doses of GL and SC

[0153]

[0154]

[0155] Each value is expressed as mean ± SD (n = 6)

[0156] On the 28th day after surgery, the electrolytes sodium, potassium, chloride, calcium, and phosphorus in the blood of all rats participating in the experiment were analyzed. The results showed that there were no significant differences in the relevant indicators in all rats participating in the experiment (Table 14).

[0157] Table 14. Changes in the electrolytes sodium, potassium, chloride, calcium, and phosphorus in the blood of rats orally or injectively administered with different doses of GL and SC

[0158]

[0159] Each value is expressed as mean ± SD (n = 6)

[0160] From the above results, it can be seen that the purified mushroom β-glucan extracted from Ganoderma lucidum or Schizophyllum commune has no significant effect on the physiological functions and blood indices of rats, confirming its excellent safety for animals and making it available for further development as food or medicine.

Claims

1. Use of purified mushroom β-glucan in preparing a composition for preventing, improving or treating Alzheimer's disease, dementia or brain function degeneration, wherein: The purified mushroom β-glucan is derived from mushroom mycelium or a fermentation product thereof, wherein the purity of the purified mushroom β-glucan is 60% or more.

2. The use according to claim 1, wherein The mushroom of the mushroom mycelium is at least one selected from the group consisting of Ganoderma lucidum, Schizophyllum, Agaricus blazei, Cordyceps sinensis, Coriolus versicolor, Antrodia cinnamomea, Phellinus linteus, Hericium erinaceus, Auriculariale and Cordyceps militaris.

3. The use according to claim 1, wherein The prevention, improvement or treatment of Alzheimer's disease, dementia or brain function degeneration includes improving the cell survival rate of nerve cells by using the purified mushroom β-glucan.

4. The use according to claim 1, wherein The prevention, improvement or treatment of Alzheimer's disease, dementia or brain function degeneration comprises reducing the oxidative stress of nerve cells by the purified mushroom β-glucan.

5. The use according to claim 4, wherein The reducing the oxidative stress of nerve cells comprises at least one of reducing the content of inducible nitric oxide synthase in the individual, increasing the activity of catalase and / or superoxide dismutase, and reducing the activity of lactate dehydrogenase.

6. The use according to claim 1, wherein The prevention, improvement or treatment of Alzheimer's disease, dementia or brain function degeneration includes reducing the neuronal cell toxicity caused by amyloid protein (Amyloid β (1-40)) and / or reducing the accumulation of amyloid protein by the purified mushroom β-glucan.

7. The use according to claim 1, wherein The prevention, improvement or treatment of Alzheimer's disease, dementia or brain function degeneration includes preventing, improving or treating cognitive dysfunction by the purified mushroom β-glucan, and the cognitive dysfunction includes at least one of memory impairment, memory decline, reduced learning ability and loss of spatial sense.

8. The use according to claim 1, wherein The purified mushroom β-glucan is obtained by the following preparation method, comprising: The liquid-fermented mushroom mycelium and fermentation liquid are subjected to ultrasonic crushing to obtain a crude extract; Centrifuging the crude extract and taking the upper clear liquid; The upper clarified liquid is subjected to a first filtration treatment, wherein the upper clarified liquid is filtered through a 0.8 to 3 μm filter membrane and a 0.22 to 0.5 μm filter membrane; Performing a first freeze-drying process to prepare mushroom polysaccharide raw material; Redissolving the mushroom polysaccharide raw material in water to form a reconstituted liquid; The reconstituted liquid is subjected to a second filtration treatment, which uses an ultrafiltration ceramic membrane filter column with a molecular weight cutoff (MWCO): 2 to 10KD and MWCO: 80 to 150KD to remove impurities; A third filtration process is performed, which is filtering with a 0.22 μm filter membrane; A second freeze-drying step is performed to produce the purified mushroom β-glucan.

9. The use according to claim 1, wherein The composition is in the form of powder, tablet, suppository, microcapsule, liquid, spray or plug.

10. The use according to claim 1, wherein The composition further comprises an excipient.

11. The use according to claim 10, wherein The composition is obtained by mixing the purified mushroom beta-glucan and an excipient, and then drying the mixture.

12. The use according to claim 10, wherein The excipient is at least one selected from the group consisting of lactose, sucrose, glucose, fructooligosaccharides, starch, starch derivatives and dietary fiber.

13. The use according to claim 9, wherein The composition is obtained by mixing the purified mushroom β-glucan with a solvent or a buffer solution.

14. The use according to claim 1, wherein The composition is a medicine, feed, feed additive, beverage, nutritional supplement, dairy product, food, food additive, health food raw material or health food finished product.

15. The use according to claim 1, wherein The composition further comprises an additive, wherein the additive is selected from at least one of the group consisting of a carrier, a preservative, a diluent, a filler, an absorption enhancer, a sweetener, and an adjuvant.

16. The use according to claim 14, wherein The composition is a medicine, which comprises the purified mushroom β-glucan and a pharmaceutically acceptable carrier thereof.

17. The use according to claim 16, wherein The pharmaceutical product is administered to a subject in an effective amount of 0.04 to 1 mg / kg.bw.

18. The use according to claim 16, wherein The medicine is administered orally, rectally, through the skin, through the mucosa or by injection.

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