TWI780840B - An extract of sarcodia and uses thereof - Google Patents

Abstract

The present invention provides a use of a composition for preparing a drug for providing neuroprotection, promoting bone formation and treating atopic dermatitis, wherein the composition comprises an extract of sarcodia.

Description

A kind of sea fungus extract and its application

The present invention relates to a novel use of sea fungus extract, in particular to the use of the sea fungus extract for preparing medicines for providing neuroprotection, promoting bone formation and treating allergic dermatitis.

Sea fungus is mainly distributed in the Indian Ocean and the Western Pacific. Sea fungus is a large marine heterotroph (heterotroph) algae. Because of its high content of micronutrients, it is also called longevity vegetable in Japan, and sea fungus is called the first sea vegetable in Europe and America. At present, sea fungus is mostly used for direct consumption or food additives. Although sea fungus has a long history of consumption, there is little research on its efficacy.

Bone is a hard organ that makes up the endoskeleton of vertebrates, which includes mineralized skeletal tissue, bone marrow, periosteum, nerves, blood vessels, cartilage and other soft tissues, as well as a small number of skeletal cells. Bone defects or defects may result from congenital defects or acquired disease, aging or trauma. Although small bone defects can heal on their own, larger bone defects or bone defects on smaller bones will be difficult to heal completely and will require bone grafting (including autologous bone grafting, allogeneic bone grafting and xenografting) , artificial bone (including bone cement and bioceramics), tissue engineered bone, or bone handling techniques. Therefore, in the face of bone diseases of various causes (such as osteoporosis, bone defect or fracture disease, etc.), it is necessary to administer different drugs or perform surgery.

In addition, as the global population ages, the number of patients with neurodegenerative diseases continues to increase, leading to impairment of cognition, memory, and motor ability in more and more elderly people. According to reports, the World Health Organization predicts that neurodegenerative diseases affecting motor function will become the second leading cause of epidemic death in the next two decades. Therefore, the development of therapeutic methods for such diseases has become one of the key research projects of modern biomedicine. Since the pathogenic mechanisms of most neurodegenerative diseases are not yet clear, there is currently a lack of effective treatments for these diseases, and available treatments are limited to symptom management and arrest of disease progression.

Atopic dermatitis is a chronic disease highly related to immunity and inflammation. Atopic dermatitis is an inflammatory skin disease accompanied by itching, and it is a chronic disease and it usually begins in infancy. Atopic dermatitis has constant itching as the main symptom, and has a nature of repeated recovery and deterioration without a specific cause. Although many studies have been conducted on atopic dermatitis recently, the etiology of atopic dermatitis has been unclear until now.

The natural compounds purified from seaweed have been proven to have many biological activities, such as: anticoagulant, antiviral, antioxidative, antiallergic, anticancer, anti-inflammatory and anti-obesity activities. Therefore, sea fungus in seaweed also has great potential for research and development, and there is still much room for research and development in its medical applications.

The present invention is to dry the sea fungus, then extract it with alcohol to obtain an alcohol extract; then soak the alcohol extract in water to suspend it; then use ethyl acetate to distribute and extract, and finally A sea fungus ethyl acetate extract EE was obtained from the ethyl acetate layer.

In neuroprotection experiments, the sea fungus ethyl acetate extract EE can increase the survival rate of SH-SY5Y nerve cells damaged by 6-hydroxydopamine (6-OHDA). at the same time, The sea fungus ethyl acetate extract EE can improve the swimming ability of zebrafish with Parkinson's disease-like behavior. The results show that the sea fungus ethyl acetate extract EE has neuroprotective effect and can improve the symptoms of neurodegenerative diseases.

In the experiment of osteogenesis, the sea fungus ethyl acetate extract EE promoted the increase of the number of joints in zebrafish. At the same time, the sea fungus ethyl acetate extract can improve the recovery rate of the skull defect in rats. This result shows that the sea fungus ethyl acetate extract EE can promote bone formation and be used for the treatment of bone defects.

In the experiment of atopic dermatitis, the sea fungus ethyl acetate extract EE can improve the symptoms of atopic dermatitis in mice, such as erythema, edema, peeling and dryness. At the same time, the sea fungus ethyl acetate extract EE can reduce the expression level of IgE increased due to atopic dermatitis. In addition, because atopic dermatitis can cause enlargement of the spleen and lymph nodes, after administration of the sea fungus ethyl acetate extract EE, the enlargement of the spleen and lymph nodes can be improved. The results show that the sea fungus ethyl acetate extract EE can treat atopic dermatitis.

The term "a" or "an" herein is used to describe elements and components of the present invention. This term is only for convenience of description and to give the basic concept of the present invention. This statement should be read to include one or at least one, and the singular also includes the plural unless it is clearly stated otherwise. When used in conjunction with the word "comprising" in the claims, the word "a" may mean one or more than one.

The term "or" used herein in claims means "and/or" unless it is expressly intended to mean only the other option, or unless the other options are mutually exclusive.

The invention provides a preparation method of sea fungus extract, which comprises the following steps: (a) extracting sea fungus with an alcohol solvent to obtain a sea fungus alcohol extract, which wherein the alcoholic solvent is methanol or ethanol; and (b) partitioning and extracting the sea fungus alcoholic extract with an organic solution or supercritical extraction to form an organic solvent layer and an aqueous layer, and then collect the organic solvent layer to obtain the sea fungus extract, wherein the organic solution is an organic solvent or a mixture of water and an organic solvent, and the organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptyl alkane, octane, nonane, methyl ether, diethyl ether, methylene chloride, chloroform, or carbon tetrachloride.

In a specific embodiment, the sea fungus is Sarcodia ceylanica .

In a specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the sea fungus alcohol extract is a sea fungus ethanol extract.

In another specific embodiment, the organic solvent is ethyl acetate. In a preferred embodiment, the sea fungus extract is a sea fungus ethyl acetate extract.

In a specific embodiment, the method includes a step (a1), before the step (a), wherein the step (a1) includes drying the sea fungus. In a preferred embodiment, the sea fungus is dried at a temperature of 20-70°C. In a more preferred embodiment, the sea fungus is dried at a temperature of 40-60°C. In another specific embodiment, the sea fungus is dried at 50°C.

In a specific embodiment, the drying time of the sea fungus is 12-48 hours. In a preferred embodiment, the drying time of the sea fungus is 16-32 hours. In a preferred embodiment, the drying time of the sea fungus is 20-24 hours.

According to the present invention, after the sea fungus is dried, it is filtered through a sieve with a certain mesh size and then extracted with ethanol. in a specific embodiment Among them, the method includes a step (a2), before the step (a), wherein the step (a2) includes filtering the sea fungus with a 30-70 mesh sieve. In a preferred embodiment, the method includes a step (a2), before the step (a), wherein the step (a2) includes filtering the sea fungus with a 40-60 mesh sieve. In a more preferred embodiment, the method includes a step (a2), before the step (a), wherein the step (a2) includes filtering the sea fungus with a 50-mesh sieve.

In another specific embodiment, the sea fungus is extracted with an alcohol solvent at room temperature. In a preferred embodiment, in step (a), the sea fungus is soaked and extracted three times with an alcohol solvent, and the soaking solutions combined three times are filtered and concentrated under reduced pressure to obtain the sea fungus alcohol extract.

In a specific embodiment, in step (b), the organic solution is used to partition and extract the sea fungus alcohol extract, so as to form an organic solvent layer and an aqueous layer, and then collect the organic solvent layer, To obtain the sea fungus extract. In a preferred embodiment, in step (b), the organic solution is used to carry out distribution extraction of the sea fungus alcohol extract, and the operation of distribution extraction is repeated three times, so as to form an organic solvent layer and a aqueous layer, and then collect the organic solvent layer to obtain the sea fungus extract.

In another specific embodiment, the step (b) further includes a step (b1), before the organic solution extraction, wherein the step (b1) includes soaking the sea fungus alcohol extract in water to obtain a sea fungus aqueous solution. In a preferred embodiment, the step (b1) comprises soaking the maria eucalyptus extract in water, so as to suspend the marinum fungus alcohol extract in the water. After performing step (b1), in the original step (b), the organic solvent will be used to distribute and extract the sea fungus aqueous solution obtained in step (b1), without using a mixture of the water and the organic solvent combined solution to form an organic solvent layer and an aqueous layer, and then collect the organic solvent layer to obtain the sea fungus extract. In another specific embodiment, in step (b), the organic solvent is used to distribute and extract the sea fungus aqueous solution obtained in step (b1), so as to form an organic solvent layer and an aqueous layer, and then collect The organic solvent layer is used to obtain the sea fungus extract. In a preferred embodiment, in step (b), the organic solvent is used to distribute and extract the sea fungus aqueous solution obtained in step (b1), and the operation of distribution and extraction is repeated three times to form an organic a solvent layer and an aqueous layer, and then collect the organic solvent layer to obtain the sea fungus extract.

After step (b1) is performed, in the original step (b), the supercritical extraction will be used to distribute and extract the sea fungus aqueous solution obtained in step (b1). In a specific embodiment, in step (b), the supercritical extraction is used to distribute and extract the sea fungus aqueous solution obtained in step (b1), so as to form an organic solvent layer and an aqueous layer, and then collect The organic solvent layer is used to obtain the sea fungus extract. In another specific embodiment, the supercritical extraction is CO ₂ supercritical extraction. According to the present invention, the process of _CO2 supercritical extraction and separation of nutritional components of natural products mainly uses supercritical carbon dioxide fluid to extract nutritional components from natural products under high pressure and low temperature conditions. In a specific embodiment, the CO ₂ supercritical extraction conditions are CO ₂ : flow rate ratio of 99% ethanol solution=10mL/min: 1mL/min; critical pressure range is 150-400 bar (bar); and critical temperature range 20-60°C. In a preferred embodiment, the CO ₂ supercritical extraction conditions are CO ₂ : the flow rate ratio of 95% ethanol solution=10mL/min: 1mL/min; the critical pressure is 250 bars (bar); and the critical temperature is 40 ℃. After CO ₂ supercritical extraction, it will also be divided into the raw material (aqueous layer) and the extracted material (organic solvent layer) left in the CO ₂ supercritical extraction reaction tank, and the organic solvent layer is collected to obtain the sea fungus Extracts. In another specific embodiment, in step (b), the sea fungus alcohol extract is extracted with CO ₂ supercritical to obtain the sea fungus extract.

In another specific embodiment, the ingredients of the sea fungus extract include cholesterol (cholesterol), stearic acid (stearic acid), methyl stearate (methyl stearate), glyceryl stearate (glyceryl stearate), ( 2S)-1-O-palmitoyl-3-O-β-D-galactopyranosylglycerol ((2S)-1-O-palmitoyl-3-O-β-D-galactopyranosylglycerol), (2S)1 , 2-di-O-oleoyl-3-O-β-D-galactopyranosylglycerol ((2S)1,2-di-O-oleoyl-3-O-β-D-galactopyranosylglycerol), 13 ² -Hydroxy-(13 ² S)-pheophytin a (13 ² -Hydroxy-(13 ² S)-phaeophytin a) and 13 ² -hydroxy-(13 ² R)-pheophytin a (13 ² -Hydroxy -(13 ² R)-phaeophytin a). In one embodiment, the ingredients of the sea fungus extract do not contain polysaccharides.

The present invention also provides a composition for the preparation of a drug for neuroprotection, wherein the composition comprises a sea fungus extract, wherein the preparation method of the sea fungus extract comprises the following steps: (a) using an alcoholic solvent A sea fungus is extracted to obtain a sea fungus alcohol extract, wherein the alcohol solvent is methanol or ethanol.

In a specific embodiment, the sea fungus in step (a) is washed, dried and/or pulverized.

In another specific embodiment, the preparation method of the sea fungus extract further comprises a step (b), which, after step (a), includes using an organic solution or supercritical extraction to extract the sea fungus alcohols distributing the extraction to form an organic solvent layer and an aqueous layer, and then collecting the organic solvent layer to obtain the sea fungus extract, wherein the organic solution is an organic solvent or a mixture of water and an organic solvent, the The organic solvents are ethyl acetate, propyl acetate, ethyl butyl ester, hexane, heptane, octane, nonane, methyl ether, diethyl ether, dichloromethane, chloroform or carbon tetrachloride.

In a specific embodiment, the preparation method of the sea fungus extract further includes a step (b1), before the extraction of the organic solution, wherein the step (b1) includes soaking the sea fungus alcohol extract in water to obtain An aqueous solution of sea fungus. In a preferred embodiment, in step (b), the organic solvent is used to distribute and extract the sea fungus aqueous solution obtained in step (b1), so as to form an organic solvent layer and an aqueous layer, and then Collect the organic solvent layer to obtain the sea fungus extract.

In another specific embodiment, the sea fungus is Sarcodia ceylanica .

In a specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the sea fungus alcohol extract is a sea fungus ethanol extract.

In another specific embodiment, the organic solvent is ethyl acetate. In a preferred embodiment, the sea fungus extract is a sea fungus ethyl acetate extract.

According to the present invention, the sea fungus extract can be used to provide neuroprotection, which means the ability to prevent or reduce the death or damage of nerve cells (including neurons and glial cells), or to rescue nerve cells or to revive nerve cells or The ability to recover, for example, from pathological or deleterious conditions of the brain, central nervous system, or peripheral nervous system. Neuroprotection involves regeneration of nerve cells, ie, the regrowth of a population of nerve cells after disease or trauma. Neuroprotection is used in the nervous system to protect against acute disease (such as stroke, brain or nervous system injury/trauma, cerebral hypoxia, spinal cord injury or peripheral nerve injury) or chronic neurodegenerative disease (such as Parkinson's Mechanisms of brain/neuron damage or degeneration caused by Alzheimer's disease, Alzheimer's disease or multiple sclerosis and strategy. The goal of neuroprotection is to limit neurological dysfunction/death after nervous system injury and to try to preserve as high an integrity of cellular interactions as possible in the brain so that neurological function is not disturbed. By providing neuroprotection, the sea fungus extract can be used to treat neuronal damage (such as cerebral apoplexy, especially ischemic stroke, brain injury, cerebral hypoxia, spinal cord injury or peripheral nerve injury) and to treat or prevent neurodegeneration disease. Therefore, in a specific embodiment, the present invention relates to the use of the sea fungus extract as an active ingredient in the preparation of medicines for nerve cell protection and/or regeneration.

In one embodiment, the neuroprotection comprises inhibiting or preventing neuronal apoptosis. In a preferred embodiment, the sea fungus extract can inhibit or prevent nerve cell apoptosis.

Therefore, the present invention finds that the sea fungus extract has some beneficial effects and functions in providing neuroprotection, so that the sea fungus extract can be used to treat neuron or brain damage and treat or prevent neurodegenerative diseases, wherein the Neurodegenerative diseases include acute or chronic neurodegenerative diseases.

The term "neurodegenerative disease" as used herein is also used to describe an acute, progressive or chronic disease caused by damage to the central nervous system, and the damage can be cured by the treatment of the sea fungus extract according to the present invention. reduce and/or alleviate. The term "acute neurodegenerative disease" means a disease or disorder of sudden onset that results in associated neuronal death or damage. Exemplary acute neurodegenerative diseases include cerebrovascular insufficiency, focal or diffuse brain trauma, spinal cord injury, cerebral ischemia or infarction (including emolic occlusion and thrombotic occlusion). occlusion), perinatal hypoxic-ischemia (perinatal hypoxic-ischemia), neonatal hypoxic-ischemic encephalopathy (neonatal hypoxia-ischaemic encephalopathy), neonatal perinatal asphyxia (perinatal asphyxia, cardiac arrest, intracranial hemorrhage, subarachnoid hemorrhage, stroke and traumatic brain injury.

In addition, examples of neurodegenerative diseases that can be treated with the sea fungus extract of the present invention include but are not limited to amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease , frontotemporal dementia, spinocerebellar atrophy, type III cerebellar spinal stem ataxia syndrome (Machado-Joseph disease, MJD), dentatorubral pallidoluysian atrophy (DRPLA), spinal cord Spinal and bulbar muscular atrophy (SBMA) or Fragile X-associated tremor and ataxia syndrome (FXTAS).

In one embodiment, the sea fungus extract provides neuroprotection to prevent or treat neurodegenerative diseases. In a preferred embodiment, the neurodegenerative disease includes amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, frontotemporal dementia, spinal cord Cerebellar atrophy, cerebellar spinospinal ataxia type III, dentate rubrum pallidomuscular atrophy, spinobulbar muscular atrophy, or Fragile X ataxia syndrome. In a more preferred embodiment, the neurodegenerative disease comprises Parkinson's disease.

As used herein, the term "treating" refers to alleviating symptoms or complications; delaying the progression of a disease, disorder or condition; alleviating or alleviating symptoms and complications; and/or curing or eliminating the disease, disorder or condition.

As used herein, the term "prevention" refers to preventing the onset, recurrence or spread of a disease or disorder or one or more symptoms thereof. In certain embodiments, these terms refer to the use of the compounds described herein, with or without one or more other additional active agents, prior to the onset of symptoms. The provided medicaments treat, or are administered to, a subject particularly at risk of a disease or condition provided herein.

The present invention further provides a composition for the preparation of drugs for the prevention or treatment of neurodegenerative diseases, wherein the composition comprises a sea fungus extract, wherein the preparation method of the sea fungus extract comprises the following steps: (a) using Extract a sea fungus with an alcohol solvent to obtain a sea fungus alcohol extract, wherein the alcohol solvent is methanol or ethanol.

In a specific embodiment, the sea fungus in step (a) is washed, dried and/or pulverized.

In another specific embodiment, the preparation method of the sea fungus extract further comprises a step (b), which, after step (a), includes using an organic solution or supercritical extraction to extract the sea fungus alcohols distributing the extraction to form an organic solvent layer and an aqueous layer, and then collecting the organic solvent layer to obtain the sea fungus extract, wherein the organic solution is an organic solvent or a mixture of water and an organic solvent, the The organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, diethyl ether, methylene chloride, chloroform or carbon tetrachloride.

In a specific embodiment, the preparation method of the sea fungus extract further includes a step (b1), before the extraction of the organic solution, wherein the step (b1) includes soaking the sea fungus alcohol extract in water to obtain An aqueous solution of sea fungus. In a preferred embodiment, in step (b), the organic solvent is used to distribute and extract the sea fungus aqueous solution obtained in step (b1), so as to form an organic solvent layer and an aqueous layer, and then Collect the organic solvent layer to obtain the sea fungus extract. .

In another specific embodiment, the sea fungus is Sarcodia ceylanica .

In a specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the sea fungus alcohol extract is a sea fungus ethanol extract.

In another specific embodiment, the organic solvent is ethyl acetate. In a preferred embodiment, the sea fungus extract is a sea fungus ethyl acetate extract.

In a specific embodiment, the neurodegenerative disease includes amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, frontotemporal dementia, spinocerebellar atrophy syndrome, type III cerebellar-spinal stem ataxia syndrome, dentate rubrum pallidomyelodystrophy, spinobulbar muscular atrophy, or X-chromosome fragile ataxia syndrome. In a preferred embodiment, the neurodegenerative disease comprises Parkinson's disease.

The present invention also provides a composition for the preparation of a drug for promoting bone formation, wherein the composition contains a sea fungus extract, wherein the preparation method of the sea fungus extract comprises the following steps: (a) using an alcoholic solvent A sea fungus is extracted to obtain a sea fungus alcohol extract, wherein the alcohol solvent is methanol or ethanol.

In a specific embodiment, the sea fungus in step (a) is washed, dried and/or pulverized.

In another specific embodiment, the preparation method of the sea fungus extract further comprises a step (b), which, after step (a), includes using an organic solution or supercritical extraction to extract the sea fungus alcohols distributing the extraction to form an organic solvent layer and an aqueous layer, and then collecting the organic solvent layer to obtain the sea fungus extract, wherein the organic solution is an organic solvent or a mixture of water and an organic solvent, the The organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, diethyl ether, dichloromethane, chloroform or tetrachloro carbonized.

In a specific embodiment, the preparation method of the sea fungus extract further includes a step (b1), before the extraction of the organic solution, wherein the step (b1) includes soaking the sea fungus alcohol extract in water to obtain An aqueous solution of sea fungus. In a preferred embodiment, in step (b), the organic solvent is used to distribute and extract the sea fungus aqueous solution obtained in step (b1), so as to form an organic solvent layer and an aqueous layer, and then Collect the organic solvent layer to obtain the sea fungus extract.

In another specific embodiment, the sea fungus is Sarcodia ceylanica .

In a specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the sea fungus alcohol extract is a sea fungus ethanol extract.

In another specific embodiment, the organic solvent is ethyl acetate. In a preferred embodiment, the sea fungus extract is a sea fungus ethyl acetate extract.

As used herein, the term "osteogenesis" refers to proliferation from undifferentiated stem cells and osteoblastic cell lines into osteoblasts and bone tissue (eg, synthesis and accumulation of new bone matrix). Osteogenesis also refers to the differentiation or transdifferentiation of progenitor or precursor cells into bone cells (ie, osteoblasts). Progenitor or precursor cells can be pluripotent stem cells, including, for example, mesenchymal stem cells. Progenitor cells or precursor cells can be cells that are pre-destined to form an osteoblast lineage (e.g., pre-osteoblast cells) or cells that are not pre-destined to form an osteoblast lineage (e.g., preadipocytes or myoblasts ( myoblast)).

Therefore, the sea fungus extract of the present invention can be used to treat bone diseases. The bone disease includes, but is not limited to, osteoporosis, bone defect or fracture disease. Osteoporosis is a disease that The sign is the net loss of bone mass per unit volume. The consequence of this loss of bone mass and resulting fractures is the breakdown of the skeleton to provide adequate structural support for the body, low bone mass and structural degradation of bone tissue leading to bone fragility and increased hip, spine and wrist fractures. Osteoporosis is divided into primary osteoporosis and secondary osteoporosis according to the cause of occurrence. In the molecular mechanism of primary osteoporosis, receptor activator of nuclear factor kappa B (kappa B) ligand (RANKL) on osteoblasts and receptor activator of nuclear factor kappa B on the surface of osteoclasts ( RANK) combine to activate osteoclasts, stimulate the differentiation and activity of osteoclasts, and promote the occurrence of osteoporosis. In addition, the sharp decrease in estrogen in postmenopausal women also promotes the activity of osteoclasts, which can easily lead to osteoporosis and fractures. Secondary osteoporosis is caused by long-term medication, poor living habits, endocrine disorders and other diseases (such as rheumatoid arthritis, diabetes, stroke, Parkinson's disease and cancer bone metastasis). In addition, a bone defect refers to an imbalance in the ratio of bone formation to bone resorption such that the individual exhibits a lower-than-expected skeleton, or the individual's skeleton is less complete than expected. Bone defects can also result from fractures, from surgical intervention, or from dental or periodontal disease. Bone healing includes, but is not limited to, repairing bone defects as occurs in: eg, closed, open and non-union fractures.

In one embodiment, the sea fungus extract promotes bone formation to prevent or treat bone diseases. In a preferred embodiment, the bone disease includes osteoporosis, bone defect or fracture disease. In a more preferred embodiment, the bone disease comprises a bone defect.

The present invention further provides a composition for the preparation of medicines for the prevention or treatment of bone diseases, wherein the composition comprises a sea fungus extract, wherein the preparation method of the sea fungus extract comprises the following steps: (a) using an alcohol A sea fungus is extracted with a solvent to obtain a sea fungus alcohol extract, wherein the alcohol solvent is methanol or ethanol.

In a specific embodiment, the sea fungus in step (a) is washed, dried and/or pulverized.

In another specific embodiment, the preparation method of the sea fungus extract further comprises a step (b), which, after step (a), includes using an organic solution or supercritical extraction to extract the sea fungus alcohols distributing the extraction to form an organic solvent layer and an aqueous layer, and then collecting the organic solvent layer to obtain the sea fungus extract, wherein the organic solution is an organic solvent or a mixture of water and an organic solvent, the The organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, diethyl ether, methylene chloride, chloroform or carbon tetrachloride.

In a specific embodiment, the preparation method of the sea fungus extract further includes a step (b1), before the extraction of the organic solution, wherein the step (b1) includes soaking the sea fungus alcohol extract in water to obtain An aqueous solution of sea fungus. In a preferred embodiment, in step (b), the organic solvent is used to distribute and extract the sea fungus aqueous solution obtained in step (b1), so as to form an organic solvent layer and an aqueous layer, and then Collect the organic solvent layer to obtain the sea fungus extract.

In another specific embodiment, the sea fungus is Sarcodia ceylanica .

In a specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the sea fungus alcohol extract is a sea fungus ethanol extract.

In another specific embodiment, the organic solvent is ethyl acetate. In a preferred embodiment, the sea fungus extract is a sea fungus ethyl acetate extract.

According to the present invention, the sea fungus extract can increase bone mass or promote bone growth and Restoration to treat bone disease. In a specific embodiment, the bone disease includes osteoporosis, bone defect or fracture disease. In a preferred embodiment, the bone disease comprises a bone defect.

The present invention also provides a composition for the preparation of a drug for the treatment of allergic dermatitis, wherein the composition contains a sea fungus extract, wherein the preparation method of the sea fungus extract comprises the following steps: (a) using an alcohol A sea fungus is extracted with a solvent to obtain a sea fungus alcohol extract, wherein the alcohol solvent is methanol or ethanol.

In a specific embodiment, the sea fungus in step (a) is washed, dried and/or pulverized.

In another specific embodiment, the preparation method of the sea fungus extract further comprises a step (b), which, after step (a), includes using an organic solution or supercritical extraction to extract the sea fungus alcohols distributing the extraction to form an organic solvent layer and an aqueous layer, and then collecting the organic solvent layer to obtain the sea fungus extract, wherein the organic solution is an organic solvent or a mixture of water and an organic solvent, the The organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, diethyl ether, methylene chloride, chloroform or carbon tetrachloride.

In a specific embodiment, the preparation method of the sea fungus extract further includes a step (b1), before the extraction of the organic solution, wherein the step (b1) includes soaking the sea fungus alcohol extract in water to obtain An aqueous solution of sea fungus. In a preferred embodiment, in step (b), the organic solvent is used to distribute and extract the sea fungus aqueous solution obtained in step (b1), so as to form an organic solvent layer and an aqueous layer, and then Collect the organic solvent layer to obtain the sea fungus extract.

In another specific embodiment, the sea fungus is Sarcodia ceylanica .

In a specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the sea fungus alcohol extract is a sea fungus ethanol extract.

In another specific embodiment, the organic solvent is ethyl acetate. In a preferred embodiment, the sea fungus extract is a sea fungus ethyl acetate extract.

As used herein, the term "allergic dermatitis" refers to a general term for skin diseases caused by allergic reactions, characterized by chronic itching and eruptions on the face, neck, elbows and/or knees. Examples of allergic dermatitis include contact dermatitis and atopic dermatitis. "Contact dermatitis" refers to an eczematous inflammatory disease caused by the contact of foreign antigens with the skin, such as allergic contact dermatitis, photocontact dermatitis, systemic contact dermatitis and Contact hives. Furthermore, examples of antigens include metal allergens (cobalt, nickel, etc.), plant allergens (sumac, primrose, etc.), and food allergens (mango, ginkgo, etc.). "Atopic dermatitis (AD)" refers to a skin disease in which many patients have atopic tendencies. Symmetrical generalized eczema characterized by repeated exacerbations and remissions, for example, diffuse neurodermatitis, atopic eczema, atopic neurodermatitis, Besnier prurigo, acute Infantile eczema, flexural eczema, extremities childhood eczema, childhood atopic eczema, childhood eczema sicca, childhood eczema, adult atopic dermatitis, endogenous eczema, childhood dermatitis and chronic childhood eczema rash.

In a specific embodiment, the allergic dermatitis comprises a contact dermatitis or an atopic dermatitis. In a preferred embodiment, the allergic dermatitis comprises atopic dermatitis. In a more preferred embodiment, the atopic dermatitis includes diffuse neurodermatitis, atopic eczema, atopic neurodermatitis, Bernier prurigo, acute infantile eczema, flexural eczema rash, childhood eczema of extremities, atopic eczema of children, eczema sicca of children, eczema of children, atopic dermatitis of adults, endogenous eczema, dermatitis of children or chronic eczema of children.

In another embodiment, the medicament inhibits the symptoms of atopic dermatitis including erythema, edema, peeling or dry skin.

In a specific embodiment, the drug reduces the increase of IgE expression level caused by the atopic dermatitis.

In another embodiment, the drug improves the enlarged spleen or lymph nodes caused by the atopic dermatitis.

In a specific embodiment, the medicament further comprises a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is determined by the particular combination of administration and the particular method of administering the composition. The term "carrier" as used herein includes, but is not limited to, any and all solvents, dispersion media, vehicles, coatings, diluents, penetration and absorption delaying agents such as antibacterial and antifungal agents, buffers, carrier solutions, suspensions or colloid etc. Such media and agents for pharmaceutically active substances are well known in the art. Unless any conventional media or agents are incompatible with the active ingredients, their combination for treatment needs to be considered. Supplementary active ingredients can also be incorporated into the compositions. The term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce allergic or similar adverse reactions when administered to a subject. The preparation of aqueous compositions with proteins as active substances is well known in the art. Usually, the composition is prepared as a liquid solution, tablet, capsule or suspension injection; it can also be prepared as a solid form that can be dissolved or suspended for injection. In another embodiment, the pharmaceutically acceptable carrier comprises a dermatologically acceptable vehicle. "Dermatologically acceptable medium" refers to biologically appropriate substances, such as salts, esters and/or amides. That is to say, this substance, When used together with selected active ingredients, they do not cause undesired biological effects when administered to an individual. In addition, a substance that does not adversely interact with any of the components of the pharmaceutical composition in which it is contained. Likewise, "dermatologically acceptable salts" or "dermatologically acceptable esters" herein are biologically acceptable salts or esters.

According to the present invention, the formulation of the composition (comprising the sea fungus extract) and the pharmaceutically acceptable carrier may be via sterile aqueous solution or dispersion, aqueous suspension, oil emulsion, water in oil-in-emulsion, specific point emulsions, long-stay emulsions, viscous emulsions, microemulsions, nanoemulsions, liposomes, microparticles, microspheres, nanospheres, nanoparticles, micromercury, and several sustained-release natural or Synthetic polymers. The pharmaceutically acceptable carrier and the sea fungus extract can also be configured into aerosol, tablet, pill, capsule, sterile powder, suppository, lotion, cream, ointment, paste, gel, hydrogel , or other formulations that can be used for composition delivery.

In addition, compositions referred to in the present invention include compositions for topical and regional application. The term "topical" as used herein refers to the use of the composition described herein incorporated in a suitable pharmaceutical carrier and applied to a site of skin, such as an affected area of atopic dermatitis, for local action. Accordingly, such topical compositions include those pharmaceutical forms in which the compound is applied externally through direct contact with the skin surface to be treated. Conventional pharmaceutical forms for this purpose include ointments, lotions, creams, shampoos, lotions, pastes, gels, sprays, aerosols, etc., and may be presented as patches or dips depending on the part of the body to be treated. Applied in the form of an ulcer dressing. The term "ointment" includes formulations (including creams) having oily bases, water-soluble bases and emulsion-type bases such as petrolatum, lanolin, polyethylene glycols and mixtures of these combinations.

The medicament of the present invention may further comprise a pharmaceutically acceptable carrier, which can be administered to an individual through many different routes in the treatment mode known in the field related to the present invention. In a In some embodiments, the composition (comprising the sea fungus extract) and the pharmaceutically acceptable carrier will be administered externally, intravenously, intramuscularly, subcutaneously, topically, orally or by inhalation. The drug will be delivered to the target through the digestive and circulatory system.

In another embodiment, the individual is an animal, preferably a mammal, more preferably a human.

The term "effective dose" herein refers to a therapeutic dose which prevents, reduces, arrests or reverses the development of a symptom in a subject under specified conditions, or partially or completely alleviates the symptoms that were present in the subject when the treatment was started. symptoms. Those skilled in the art can also readily determine the appropriate dosage regimen for administering the sea fungus extract to an individual. For example, the sea fungus extract can be administered to the individual once or twice. When a dosing regimen comprises multiple administrations, it is understood that the effective dose of the sea fungus extract administered to the individual may comprise the total amount of product administered during all dosing regimens.

Therefore, the effective dosage of the sea fungus extract of the present invention for neuroprotection, promotion of bone formation and treatment of allergic dermatitis will be different.

In terms of providing neuroprotection, in a specific embodiment, the effective dose of the sea fungus extract ranges from 0.2 mg/kg body weight to 200 mg/kg body weight. In a preferred embodiment, the effective dose of the sea fungus extract ranges from 1 mg/kg body weight to 100 mg/kg body weight. In a more preferred embodiment, the effective dose of the sea fungus extract ranges from 2 mg/kg body weight to 20 mg/kg body weight.

Regarding the effective dose for neuroprotection, in another specific embodiment, the effective dose of the sea fungus extract ranges from 0.5 ng/ml to 50 μg/ml. In a preferred embodiment, the effective dose of the sea fungus extract ranges from 1 ng/ml to 5 μg/ml. in a better implementation In one example, the effective dose of the sea fungus extract ranges from 5 ng/ml to 500 ng/ml.

In terms of promoting bone formation, in a specific embodiment, the effective dosage range of the sea fungus extract is 0.2 mg/kg body weight to 100 mg/kg body weight. In a preferred embodiment, the effective dose of the sea fungus extract ranges from 1 mg/kg body weight to 50 mg/kg body weight. In a more preferred embodiment, the effective dose of the sea fungus extract ranges from 2 mg/kg body weight to 10 mg/kg body weight.

In terms of treating atopic dermatitis, in a specific embodiment, the effective dose of the sea fungus extract ranges from 0.05 to 20 mg/ml. In a preferred embodiment, the effective dosage range of the sea fungus extract is 0.1 to 10 mg/ml. In a more preferred embodiment, the effective dosage range of the sea fungus extract is 0.5 to 2 mg/ml.

Figure 1 is a flow chart of the extraction of sea fungus.

Figure 2 shows the protective effect of sea fungus ethyl acetate extract EE on nerve cells. After SH-SY5Y nerve cells were injured by 6-hydroxydopamine (6-OHDA), different concentrations of sea fungus ethyl acetate extract EE were added to measure the cell survival rate to observe the effect of neuroprotection. Sea fungus ethyl acetate extract EE at a concentration of 0.5-500ng/ml can increase the cell survival rate of each group, which proves that sea fungus ethyl acetate extract EE has neuroprotective effect. Statistical analysis was carried out between each group and the 6-OHDA group, and those who reached a significant difference (P<0.05) were indicated by *.

Figure 3 shows the protective effect of sea fungus ethyl acetate extract EE on living nerves. Figure 3A shows the swimming paths of zebrafish in each group in unit time. Figure 3B shows the swimming speed of zebrafish. The speed of the control group was 2.47±0.26mm/s, and the speed of the 6-OHDA group dropped to 0.21±0.11mm/s. Adding concentrations of 0.5 and 5μg/ml of sea fungus ethyl acetate extract EE , the swimming speed of zebrafish rebounded To 2.03±0.65 and 1.24±0.28mm/s. Figure 3C shows the total swimming distance of the zebrafish per unit time. The swimming distance of the control group was 741.09±77.04mm, and the swimming distance of the 6-OHDA group decreased to 63.00±32.19mm, adding sea fungus acetic acid at a concentration of 0.5 and 5 μg/ml Ethyl ester extract EE, zebrafish swimming distance increased to 609.03±196.17 and 371.52±82.77mm.

Figure 4 is the effect of sea fungus ethyl acetate extract EE on bone formation. Fig. 4A is the control group, and Fig. 4B to Fig. 4E are the groups administered with different dosages of ethyl acetate extract EE of sea fungus (0.05-50 μg/ml). Figure 4F is the quantitative statistical results of the number of bony joints of zebrafish in each group. On the 7th day after fertilization, the zebrafish in each group counted the number of cartilage joints, and adding 0.5 μg/ml of sea fungus ethyl acetate extract EE significantly increased the development of cartilage joints, and the sea fungus ethyl acetate extract did not cause the development of zebrafish In an unusual situation, it was initially shown that sea fungus ethyl acetate extract was not embryotoxic. *: P<0.05, compared with the control group.

Fig. 5 is a comparison chart of the cranial appearance of different concentrations of sea fungus ethyl acetate extract EE on the curative effect on rat cranial defects.

Fig. 6 is a comparison chart of the curative effect of sea fungus ethyl acetate extract EE on rat skull defects taken by X-ray machine. Fig. 6A shows the X-ray images of the skull defects of rats in each group. Fig. 6B shows the recovery rate of rat skulls in each group.

Figure 7 shows the effect of sea fungus ethyl acetate extract EE on the skin appearance of mice with atopic dermatitis. Figure 7A shows the use of DNCB to induce atopic dermatitis. After the skin of the mice showed the symptoms of atopic dermatitis, the drug was applied daily; the DNCB-induced atopic dermatitis was photographed on the 1st, 7th, and 12th days of treatment. Skin appearance of mice, scale bar = 1 cm. The results showed that compared with the AD group, both the EEL group (low dose group) and the EEH (high dose group) group had the effect of alleviating the symptoms of atopic dermatitis, and the symptoms of epidermal edema, redness, dryness and scab were significantly improved have to relieve. Fig. 7B shows that sea fungus ethyl acetate extract EE relieves the clinical dermatitis score of AD symptoms. EE relieved AD symptoms as assessed by clinical dermatitis score. Four skin surface symptoms were used to assess the severity of dermatitis: erythema/hemorrhage, edema, desquamation/erosion, and dryness. Scores from 0 to 3 are given according to the severity of each symptom, with more scores being more severe. The total score of dermatitis is the sum of the above 4 scores, and the highest score is 12. The higher the score, the more severe the severity of the dermatitis. Data are presented as mean ± standard error of the mean (mean ± SEM). Those who achieved significant difference compared with AD group (P<0.05) are indicated by *; those who achieved significant difference compared with ADV group are indicated by #.

Figure 8 is the effect of EE of sea fungus ethyl acetate extract on serum IgE. Blood was collected from animals before sacrifice, and IgE in serum was detected. DNCB induced AD and caused IgE to rise, and after applying EE, the extract of sea fungus ethyl acetate, IgE all decreased. Data are presented as mean ± standard error of the mean (mean ± SEM). Those who achieved significant difference compared with AD group (P<0.05) are indicated by *; those who achieved significant difference compared with ADV group are indicated by #.

Figure 9 shows the effect of sea fungus ethyl acetate extract EE on the weight of enlarged spleen induced by DNCB. Spleen tissue was collected, photographed and weighed at the time of animal sacrifice. DNCB-induced atopic dermatitis caused enlargement of spleen tissue, and after applying EE, the weight of spleen decreased. Scale bar = 1 cm. Data are presented as mean ± standard error of the mean (mean ± SEM). Compared with the AD group, those with significant difference (P<0.05) are represented by *; compared with the ADV group, those with significant difference are represented by #.

Fig. 10 shows the effect of sea fungus ethyl acetate extract EE on the weight of lymph node enlargement induced by DNCB. Lymph node tissues were collected, photographed and weighed when animals were sacrificed. DNCB-induced atopic dermatitis caused swelling of lymph node tissue, after applying sea fungus extract EE, the weight of lymph nodes decreased. Scale bar = 0.5 cm. Data presented as mean ± standard error of the mean (mean±SEM). Those who achieved significant difference compared with AD group (P<0.05) are indicated by *; those who achieved significant difference compared with ADV group are indicated by #.

The present invention includes, but is not limited to, the above and the following descriptions. The implementation method is shown in the example below.

Extraction of cultured sea fungus

The present invention uses artificially cultured double-cracked sea fungus ( Sarcodia ceylanica ).

The sea fungus was dried at 50°C for 24 hours to obtain dried sea fungus. Crush 10.8kg of dried sea fungus, filter through a 50 mesh (mesh) sieve, soak and extract three times with 95% alcohol solution at room temperature, and combine the three times of soaking solution to filter and concentrate under reduced pressure to obtain 370.5g sea fungus. Agaric alcohol crude extract. Add the above sea fungus crude extract to 1L of water to make it into a suspension, then add 1L of ethyl acetate for distribution and extraction, repeat the above distribution and extraction three times to form an ethyl acetate layer and an aqueous layer, and then collect the acetic acid Ethyl layer to obtain ethyl acetate extract EE (83.2 g). The extraction process of the whole sea fungus is shown in Figure 1.

In addition, in the above extraction step, the alcohol solution can be replaced with methanol solution. At the same time, the extraction step with ethyl acetate can be replaced by other organic solvents, such as propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, diethyl ether, dichloromethane, Chloroform or carbon tetrachloride solvent for distribution extraction or supercritical extraction (such as CO ₂ supercritical extraction), can obtain the same composition as EE extract.

In addition, water can be mixed with ethyl acetate or other organic solvents to form an organic solution to directly extract the sea fungus alcohol crude extract, so there is no need to separate water and organic solvents into two parts. Each step is extracted separately. In addition, the sea fungus alcohol crude extract can also be directly extracted with ethyl acetate or other organic solvents, without the step of soaking in water first. The changes of the above steps can finally obtain the extract with the same composition as EE.

The above CO ₂ supercritical extraction conditions are: flow rate ratio of CO ₂ : 95% ethanol solution = 10 mL/min: 1 mL/min; critical pressure is 250 bar; and critical temperature is 40°C.

At the same time, the present invention uses the phenol-sulfuric acid method to measure the polysaccharide content of EE, and the result shows that EE does not contain polysaccharide components. Therefore, the extraction method of soaking sea fungus in alcohol first will not dissolve the polysaccharide components, so the crude sea fungus alcohol extract has no polysaccharide components. In addition, the present invention also analyzes the components of the sea fungus ethyl acetate extract EE, and the analysis results are shown in Table 1.

Table 1. Composition analysis of sea fungus ethyl acetate extract EE

experimental method

(1) Assess the neuroprotective effect of EE

(a) Nerve cell test

SH-SY5Y cells were cultured in a 96-well cell culture dish at a density of ² ×104 cells, placed in a cell culture incubator, and after the cells were completely attached, they were mixed with various concentrations of sea fungus ethyl acetate extract The EE solution was pretreated for 1 hour, then added 6-hydroxydopamine (6-Hydroxydopamine, 6-OHDA) for 15 hours, added 10% alamar blue (alamar blue), continued to cultivate for 3 hours, and then used an enzyme immunoassay analyzer Absorbance was measured to measure cell viability. The toxicity of 6-OHDA to nerve cells was used to analyze whether the extract of sea fungus had a protective effect on nerve cells treated with 6-OHDA.

(b) In vivo nerve testing

Dissolve 6-OHDA in 2% fresh L-Ascorbic acid (L-Ascorbic acid), prepare a 500mM stock solution, put it into a 1.5mL brown microcentrifuge tube, and divide it into a 10μl tube for later use. When adding medicine, dissolve the prepared medicine in the same proportion of Hank’s buffer (Hank’s buffer) according to the concentration designed in the experiment, and add the aforementioned 1mL medicine and 1mL Hank’s buffer containing juvenile fish into a 24-well culture plate. Juveniles were given 6-OHDA from 48 hours to 120 hours after fertilization. In addition to the control group without adding any drugs at all, each experiment has a solvent group as a control. Through the analysis of the mobility test of the zebrafish, the parameters such as the residence time of the fish body in different water layers, the swimming speed and the swimming distance are obtained to evaluate the therapeutic effect of the sea fungus extract.

(2) Assess the effect of EE on bone formation

(a) Zebrafish bone growth test

The in vivo system was used to test the promoting effect of sea fungus extract solution on bone regeneration. Analyze the effect of sea fungus extract on bone and bone-related diseases (such as: osteoporosis, etc.). Using Calcein fluorescent dye to specifically stain the hard bone in the bone, combined with the zebrafish embryonic development model, to analyze the effect of active substances on bone growth The effect on bone growth, and use analysis software to present the effect of substances on bone growth in a digital way. Take zebrafish juveniles 48 hours after fertilization, add different concentrations of sea fungus extract, observe the vertebral joints of the fish every day, and continue feeding after observation. After staining the zebrafish with 2 g/L calcein solution (pH=7.8) for 10 minutes, rinse with water to remove excess dye. Then anesthetized with MS-222, fixed the fish body with 1% Methyl Cellulose, examined the vertebral skeleton of the fish body under a fluorescent microscope, and analyzed the number of bone nodes.

(b) Rat skull defect experiment

Skull defect surgery:

A small-scale bone defect can be naturally regenerated in the body, but when the size of the defect exceeds the size that the body can repair, it cannot be healed by itself, so-called critical sized bone defect, which requires external treatment. heal. Different animal models and different sites affect the size of the defect that needs to be created. In the rat skull defect model used in the present invention, the animal is first anesthetized with the gas anesthetic isoflurane. After shaving the top of the rat's head, disinfect the operation area with betadine and 70% alcohol, and then inject ampicillin. Then use sterilized paper towels to cut a hole to cover the head of the rat, exposing only the surgical area. Right above the head of the rat, cut the skin and periosteum about 1.5 cm in size. Separate the skull and periosteum using a periosteal stripper. There is a bulge in the center of the skull, which is the sagittal suture of the central axis, and the parietal bones are on both sides. With this sagittal suture as the center, two bone defects with a diameter of 4 mm are made with a bone trephine saw head with a diameter of 4 mm. Then the periosteum was sutured with 5-0 catgut and the skin was sutured with 4-0 nylon suture. If the animal wakes up from the anesthesia state during the operation, the gas anesthesia machine is used to inhale the anesthesia gas through the breathing mask to maintain the anesthesia. Under the condition of anesthesia, the rats were fed with EE three times a week at a dose of 1.5 and 7.5 mg/kg/week. After 4 weeks of feeding, sacrifice was performed to obtain cranial tissue.

The rat cranial healing was evaluated in the following way: photographs were taken by an X-ray machine (X-OMAT V; Kodak, Rochester, NY), and the negatives were imaged by an automatic film processor. The image is converted into a TIF image file through scanning, and then the image is transferred to the ImagQuant software (ImageQuant Version 5.2 Copyright ^© 1999, Molecular Dynamics) for subsequent analysis and processing. In the ImagQuant software, select each step wedge (step wedge) and an area of equal area in the blank to represent the correlation between the gray scale intensity of the image on the X-ray film and the wedge-shaped step thickness. Then select a circular area with a diameter of 8 mm in the skull area, and a flat area of 1 x 1 mm square in the center of the parietal bone on both sides, measure the gray scale intensity, substitute it into the above equation, and calculate the relative bone density of the sample by interpolation. The thickness of the wedge-shaped ladder. That is, the bone density in the bone defect area of the skull is defined as the gray-scale image density projected by the different thicknesses of the wedge-shaped steps.

(3) To evaluate the effect of EE on atopic dermatitis

The experimental animals used in the present invention are BALB/c strain mice. The mouse was anesthetized with 2.5% isoflurane (isoflurane), and the hair on the back of the mouse was shaved using a razor and hair removal cream, and a silicone ring with a diameter of 1 cm was used to mark (the area size was 78.5 mm ² ). Once a day, drop 26 μL of 2% 2,4-dinitrochlorobenzene (2,4-dinitirochlorobenzene, DNCB) (Sigma-Aldrich Co., MO, USA; Cat.No.#138630) within this range once a day, A total of 8 times to induce skin lesions.

After the 7th day of DNCB induction, 100 μL of vehicle, sea fungus extract EE or crisaborole, a drug for treating atopic dermatitis, was applied every day. Experimental animals were grouped as follows (3 animals in each group): (1) Control group: no induction; (2) AD group: only given 2% DNCB; (3) ADV group: given 2% DNCB and vehicle (Vehicle); ( 4) EEL group: after DNCB induction, give 50 μg EE/100 μL 1% methyl fiber; (5) EEH group: after DNCB induction, Administer 200 μg EE/100 μL 1% methylcellulose; and (6) Cri group: after DNCB induction, give 25 μg crisborole/100 μL 1% acetone-EtOH. 5 minutes after applying DNCB, wait for the DNCB to dry before applying the medicine. Before inducing or applying the medicine, take pictures with a digital camera to record the changes of skin symptoms.

On the 18th day, the animals were sacrificed, blood was collected, and the back skin, spleen and lymph node tissues were collected. Observe the size of the spleen and subiliac lymph nodes, and weigh them for comparison. Four skin surface symptoms were used to assess the severity of clinical dermatitis: erythema/hemorrhage, edema, excoriation/erosion, and dryness. According to the severity of each symptom, 0-3 points are given individually: 0 is asymptomatic; 1 is mild; 2 is moderate; 3 is severe. The total score of dermatitis is the sum of the above 4 scores, and the highest score is 12. Higher scores indicate more severe dermatitis.

Before sacrifice, the blood of the mice was collected into blood collection tubes (BD vacutainer-SST, NJ, USA) by means of cardiac blood collection. The blood was then centrifuged at 3000 rpm for 10 minutes to obtain serum, which was stored in a -80°C refrigerator until further use. IgE concentrations in serum were measured according to the manufacturer's instructions (IgE-ELISA kit, Thermo Fisher Scientific Inc., Vienna, Austria).

If inflammation or immune-related diseases occur, the lymph nodes and spleen will swell, so when the mice are sacrificed, these two organs are collected for photography and weighed.

Statistical Analysis

All experimental data are presented as mean ± standard error of the mean (mean ± SEM). For data comparison among multiple groups, one-way analysis of variance (ANOVA) was used for statistical analysis of data, and the difference between multiple groups was compared according to Duncan's Method. When the P value is less than 0.05, it means that there is a significant difference between groups sexual difference.

result

(1) The protective effect of EE on nerve

After SH-SY5Y nerve cells were injured by 6-hydroxydopamine (6-OHDA), different concentrations of sea fungus ethyl acetate extract EE were added to measure the cell survival rate to observe the effect of neuroprotection. The cell survival rate of the control group was 100±3.63%, and the cell survival rate of the 6-OHDA group dropped to 0±3.88%. Adding sea fungus ethyl acetate extract EE at a concentration of 0.5-500ng/ml all improved the cell survival rate of each group. The survival rates were 10.72±2.86, 33.91±19.67, 7.92±1.93 and 32.69±17.88%, respectively (as shown in Figure 2). The results proved that sea fungus ethyl acetate extract EE has neuroprotective effect.

In addition, 6-OHDA can induce Parkinson's-like behavior in zebrafish, so that zebrafish do not swim and perch on the bottom. Figure 3A shows the swimming paths of zebrafish in each group in unit time. Figure 3B shows the swimming speed of zebrafish. The speed of the control group was 2.47±0.26mm/s, and the speed of the 6-OHDA group dropped to 0.21±0.11mm/s. Adding concentrations of 0.5 and 5μg/ml of sea fungus ethyl acetate extract EE , the swimming speed of zebrafish rose to 2.03±0.65 and 1.24±0.28mm/s. Figure 3C shows the total swimming distance of the zebrafish per unit time. The swimming distance of the control group was 741.09±77.04mm, and the swimming distance of the 6-OHDA group decreased to 63.00±32.19mm, adding sea fungus acetic acid at a concentration of 0.5 and 5 μg/ml Ethyl ester extract EE, zebrafish swimming distance increased to 609.03±196.17 and 371.52±82.77mm. The results show that the extract of sea fungus ethyl acetate EE has a neuroprotective effect on living nerves.

(2) Effect of EE on bone formation

Sea fungus ethyl acetate extract EE can promote bone formation. In the zebrafish pattern Among them, juveniles in the control group produced 10±0.63 joints, while the groups of sea fungus extract EE with a concentration of 0.05-50 μg/ml all increased the number of joints, which were 13.8±0.80, 15.33±0.68, 15.00±0.84 and 15.4±0.68 Joints (as shown in Figure 4).

In the skull defect experiment of rats, EE was fed 3 times a week at doses of 1.5 and 7.5 mg/kg/week. After 4 weeks, the animals were sacrificed, and the skull tissue was obtained, and the area was quantified using pictures taken by an X-ray machine. Figure 5 is a comparison of the cranial appearance of different concentrations of sea fungus ethyl acetate extract EE on the curative effect of rat skull defects. Fig. 6A shows the X-ray images of the skull defects of rats in each group. Fig. 6B shows the recovery rate of rat skulls in each group. The results showed that the skull recovery rate of the control group (vehicle) was 51.90±10.01%, while the skull recovery rates of feeding EE 1.5 and 7.5mg/kg/week were 97.47±4.86 and 15.47±15.86%, respectively. The above results show that EE can promote bone formation.

(3) Curative effect of EE on atopic dermatitis

DNCB was used to induce atopic dermatitis (AD) symptoms in mice, including erythema, edema, exfoliation, dryness and lichenification. After the mice showed obvious symptoms such as erythema, edema, peeling and dryness, EE or crisaborole was applied daily to test whether EE has the effect of treating atopic dermatitis. The skin appearance of the mice was observed and photographed (as shown in FIG. 7A ), and the therapeutic effect of EE was quantified by the clinical dermatitis score (as shown in FIG. 7B ). The control group had a dry skin surface due to the lack of hair covering, so the score was 0.75 ± 0.25. The skin surface of AD group and ADV group formed a thick layer of scab due to keratinocyte hyperplasia. As the number of days increases, after part of the scab peels off, it can be observed that the skin around the induction is dry, and the skin has wrinkles; also because of the inflammation symptoms, the skin has erythema and edema. The keratinocytes are still proliferating, so there is still a layer of scab on the skin surface. The clinical dermatitis scores of these two groups were significantly increased compared with the control group, which were 9.17±0.40 and 9.50±0.56, respectively. with On the next day, in the EE and Criborole (Cri) group, after the scab peeled off, although dryness, erythema, and swelling could still be observed, the degree of keratinocyte hyperplasia seemed to be milder than that of the AD group, so before animal sacrifice , the dermatitis scores of the EEL and EEH groups were significantly lower than those of the AD group (4.50±0.76 and 3.67±0.49), while the dermatitis scores of the Cri group were 4.80±0.37, which was statistically significant compared with the AD group. The above results show that EE has the effect of relieving AD symptoms.

IgE is one of the indicators for clinical diagnosis of atopic dermatitis. Before the mice were sacrificed, serum was collected to detect the content of IgE, and the results are shown in Figure 8. The IgE content in the serum of the control group was 1.71±0.14μg/mL. The IgE concentration of AD group and ADV group increased significantly (68.89±4.12 and 68.95±2.60μg/mL). The IgE concentrations in the EEL and EEH groups decreased only slightly, 59.33±2.11 and 59.35±2.05 μg/mL, respectively, but still statistically significant, while the serum IgE levels in the Cri group decreased to 65.61±1.74 μg/mL. Applying EE can reduce the IgE in the serum which is increased due to atopic dermatitis.

The results of the spleen weight are shown in Fig. 9, the spleen weight of the control group was 91.08±3.61 mg. The spleens of the AD group and the ADV group were significantly enlarged, which was 2.5 times that of the control group (234.46±6.56 and 221.14±13.32mg). The spleen weights of the EEL and EEH groups decreased significantly, 160.74±9.10 and 167.41±10.39 mg, respectively, while the spleen weights of the Cri group decreased significantly to 167.46±10.61 mg.

The results of the total weight of the lymph nodes are shown in Figure 10, the total weight of the two lymph nodes in the control group was 4.46±0.29 mg. The lymph nodes in the AD group and the ADV group were significantly enlarged, which was three times that of the control group (12.07±0.33 and 11.61±0.54mg). The weight of lymph nodes in EEL and EEH groups decreased significantly, 9.10±0.68 and 6.50±0.59 mg, respectively, with a dose-dependent effect. The weight of lymph nodes in Cri group decreased significantly to 9.49±0.81 mg. The results showed that atopic dermatitis For the enlarged lymph nodes and spleen caused by inflammation or immune reaction, applying EE can inhibit the aforementioned phenomenon.

The invention suitably described can be practiced subject to provisos or limitations not specifically disclosed herein. The terms that have been used to describe are not limiting. There is no difference in expression or description using these terms and any equivalents otherwise, but it should be recognized that rights within the invention may be modified. Therefore, while the present invention has described embodiments and others, the disclosure herein may be modified and changed by those skilled in the art, and such modifications and changes are considered to be within the scope of the present invention.

Claims (12)

A composition for the preparation of a medicine for neuroprotection, wherein the composition comprises a sea fungus extract, wherein the preparation method of the sea fungus extract comprises the following steps: (a) treating a sea fungus with an alcoholic solvent Extraction is carried out to obtain a sea fungus alcoholic extract, wherein the alcoholic solvent is methanol or ethanol. The use according to claim 1, wherein the neuroprotection comprises inhibiting or preventing neuronal apoptosis. The use as claimed in item 1, wherein the sea fungus extract provides neuroprotection to prevent or treat neurodegenerative diseases. The use as described in claim 3, wherein the neurodegenerative disease comprises Parkinson's disease. A composition for the preparation of a drug for promoting bone formation, wherein the composition comprises a sea fungus extract, wherein the preparation method of the sea fungus extract comprises the following steps: (a) treating a sea fungus with an alcoholic solvent Extraction is carried out to obtain a sea fungus alcoholic extract, wherein the alcoholic solvent is methanol or ethanol. The use as described in claim 5, wherein the sea fungus extract promotes bone formation to prevent or treat bone diseases. The use as described in claim 6, wherein the bone disease comprises osteoporosis, bone defect or fracture disease. A composition is used to prepare a medicine for treating allergic dermatitis, wherein the composition comprises a sea fungus extract, wherein the preparation method of the sea fungus extract comprises the following steps: (a) using an alcoholic solvent for an The sea fungus is extracted to obtain a sea fungus alcohol extract, wherein the alcohol solvent is methanol or ethanol. The use as claimed in item 8, wherein the allergic dermatitis comprises atopic dermatitis. The use as described in any one of claim 1, 5 or 8, wherein the preparation method of the sea fungus extract further comprises a step (b), which, after step (a), comprises using an organic solution to the sea fungus The fungus alcohol extract is partitioned and extracted to form an organic solvent layer and an aqueous layer, and then the organic solvent layer is collected to obtain the sea fungus extract, wherein the organic solution is a mixture of water and an organic solvent, The organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, diethyl ether, dichloromethane, chloroform or carbon tetrachloride. The use as described in any one of claim 1, 5 or 8, wherein the alcoholic solvent is ethanol. Use as described in claim item 10, wherein the organic solvent is ethyl acetate.

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