Sarcodia extract and uses thereof

Abstract

To provide use of a composition comprising a Sarcodia extract in preparation of drugs for promoting osteogenesis.SOLUTION: Disclosed is a use of a composition comprising a Sarcodia extract in preparation of drugs for promoting osteogenesis, where the composition comprises an ethyl acetate extract of Sarcodia by a method comprising the steps of: (a) extracting Sarcodia with ethanol to obtain an ethanol extract of Sarcodia; and (b) conducting distribution extraction of the ethanol extract of Sarcodia with ethyl acetate and water to form an ethyl acetate layer and an aqueous layer to collect the ethyl acetate layer thereby obtaining the ethyl acetate extract of Sarcodia.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present invention relates to a novel use of an extract of the genus Sarcodia, and particularly to the use of the extract of the genus Sarcodia in the manufacture of drugs for neuroprotection, promotion of bone formation, and treatment of allergic dermatitis.

Background Art

[0002] The genus Sarcodia is mainly distributed in the Indian Ocean and the Western Pacific region. The genus Sarcodia is a heterotrophic marine macroalgae with a high content of micronutrients. Therefore, it is also called a longevity food ingredient in Japan, and in Europe and the United States, the genus Sarcodia is regarded as a representative of sea vegetables. Currently, many uses of the genus Sarcodia are for direct consumption or as food additives. Although the history of the genus Sarcodia as food is long, research on its effects is extremely scarce.

[0003] The skeleton is a solid organ that constitutes the endoskeleton of vertebrates and includes biomineralized skeletal tissues, soft tissues such as bone marrow, periosteum, nerves, blood vessels, and cartilage, and a small number of skeletal cells. Defects or deficiencies in the skeleton are thought to be caused by congenital defects, acquired diseases, aging, or trauma. When the bone defect is small, it can heal naturally, but when the bone defect is large or the bone defect of a small skeleton is difficult to completely heal. Therefore, it is necessary to treat it by surgery such as bone transplantation (including autologous bone transplantation, allogeneic bone transplantation, and xenogeneic bone transplantation), artificial bone (including bone cement and bioceramics), tissue engineering bone, or bone transfer surgery. That is, for various bone diseases with different causes (such as osteoporosis, bone defect, or fracture diseases), administration of different drugs or implementation of surgery is required.

[0004] In addition, with the global aging of the population, the number of patients with neurodegenerative diseases continues to increase, and the impairment of cognitive, memory, and motor abilities in the elderly is on the rise. According to reports, the World Health Organization predicts that neurodegenerative diseases affecting motor function will become the second leading cause of death within the next 20 years. Therefore, the development of treatment methods for this type of disease has become one of the important research projects in current biomedical research. The expression mechanisms of many neurodegenerative diseases are still not clear, and currently, there is a lack of effective treatment methods for these diseases. Moreover, the applicable treatments are limited to symptom control and suppression of disease progression.

[0005] On the other hand, atopic dermatitis is a chronic disease deeply related to immunity and inflammation. Atopic dermatitis is an inflammatory skin disease accompanied by itching and is also a chronic disease, usually occurring in infancy. Atopic dermatitis has persistent itching as its main symptom and has the property of repeating recovery and exacerbation without a specific cause. Recently, many studies have been conducted on atopic dermatitis, but the cause of atopic dermatitis is not yet clear.

[0006] By the way, natural compounds purified from seaweeds have been demonstrated to have numerous biological activities such as anticoagulation, antiviral, antioxidant, anti-allergic, anti-cancer, anti-inflammatory, and anti-obesity. Therefore, the genus Atsuanori, which is a type of seaweed, also has extremely high research and development potential and has a great deal of room for research and development in medical applications.

Summary of the Invention

Problems to be Solved by the Invention

[0007] In the present invention, after drying the genus Atsuanori, an alcohol extract is obtained by extracting with alcohol. Next, the alcohol extract is immersed in water and suspended, and then partition extraction is carried out using ethyl acetate. Finally, the ethyl acetate extract EE of the genus Atsuanori is obtained from the ethyl acetate layer.

[0008] In the neuroprotection experiment, the ethyl acetate extract EE of the genus Atsubanori was able to improve the survival rate of SH-SY5Y neurons damaged by 6-hydroxydopamine (6-OHDA). Moreover, the ethyl acetate extract EE of the genus Atsubanori was able to improve the swimming ability of zebrafish with Parkinson's disease-like behavior. From these results, it was shown that the ethyl acetate extract EE of the genus Atsubanori has a neuroprotective effect and can improve the symptoms of neurodegenerative diseases.

[0009] In the bone formation experiment, the ethyl acetate extract EE of the genus Atsubanori promoted an increase in the number of vertebral segments of zebrafish. Moreover, the ethyl acetate extract of the genus Atsubanori was able to improve the recovery rate of the skull defect site in rats. From these results, it was shown that the ethyl acetate extract EE of the genus Atsubanori can be applied to the treatment of bone defects by promoting bone formation.

[0010] In the atopic dermatitis experiment, the ethyl acetate extract EE of the genus Atsubanori was able to improve the symptoms of atopic dermatitis in mice (such as symptoms like erythema, edema, epidermal detachment, and dryness). Moreover, the ethyl acetate extract EE of the genus Atsubanori was able to decrease the increased IgE expression level caused by atopic dermatitis. In addition, atopic dermatitis enlarges the spleen and lymph nodes, but administration of the ethyl acetate extract EE of the genus Atsubanori was able to improve the enlargement of the spleen and lymph nodes. From these results, it was shown that the ethyl acetate extract EE of the genus Atsubanori can treat atopic dermatitis.

[0011] The terms "one" or "a kind of" in this text are used to describe the members and components of the present invention. This term is only for the convenience of description and to indicate the basic concept of the present invention. Such description should be interpreted as including one kind or at least one kind, and when indicated in the singular, it also includes the plural cases unless otherwise specifically specified. Also, when used in combination with the term "comprising" in the claims, this term "one" can mean 1 or greater than 1.

[0012] In this text, the term "or" used in the claims means "and / or", unless otherwise clearly indicated to be limited to another alternative or mutually exclusive with other alternatives.

Means for Solving the Problems

[0013] The present invention provides a method for producing an extract of the genus Sarcodia. The method includes the following steps. That is, (a) extracting the genus Sarcodia with an alcoholic solvent to obtain an alcoholic extract of the genus Sarcodia. The alcoholic solvent is methanol or ethanol. (b) Performing partition extraction on the alcoholic extract of the genus Sarcodia by organic solution or supercritical extraction to form an organic solvent layer and an aqueous layer. Subsequently, collecting the organic solvent layer to obtain the extract of the genus Sarcodia. The organic solution is an organic solvent or a mixture of water and an organic solvent. The organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, ethyl ether, dichloromethane, chloroform or carbon tetrachloride.

[0014] In a specific embodiment, the genus Sarcodia is Sarcodia ceylanica.

[0015] In a specific embodiment, the alcoholic solvent is ethanol. In a preferred specific embodiment, the alcoholic extract of the genus Sarcodia is an ethanol extract of the genus Sarcodia.

[0016] In another specific embodiment, the organic solvent is ethyl acetate. In a preferred specific embodiment, the extract of the genus Sarcodia is an ethyl acetate extract of the genus Sarcodia.

[0017] In a specific embodiment, the method includes step (a1) before step (a). In step (a1), the Atubanol genus is dried. In a preferred specific embodiment, the Atubanol genus is dried at a temperature of 20 to 70 °C. In a more preferred specific embodiment, the Atubanol genus is dried at a temperature of 40 to 60 °C. In another specific embodiment, the Atubanol genus is dried at a temperature of 50 °C.

[0018] In a specific embodiment, the drying time of the Atubanol genus is 12 to 48 hours. In a preferred specific embodiment, the drying time of the Atubanol genus is 16 to 32 hours. In a preferred specific embodiment, the drying time of the Atubanol genus is 20 to 24 hours.

[0019] In the present invention, after drying the Atubanol genus, it is filtered through a filter screen with a predetermined mesh pore size, and then extracted with ethanol after filtration. In a specific embodiment, the method includes step (a2) before step (a). In step (a2), the Atubanol genus is filtered through a filter screen with a pore size of 30 to 70 mesh. In a preferred specific embodiment, the method includes step (a2) before step (a). In step (a2), the Atubanol genus is filtered through a filter screen with a pore size of 40 to 60 mesh. In a more preferred specific embodiment, the method includes step (a2) before step (a). In step (a2), the Atubanol genus is filtered through a filter screen with a pore size of 50 mesh.

[0020] In another specific embodiment, the Atubanol genus is extracted with an alcohol-based solvent at room temperature. In a preferred specific embodiment, in step (a), the Atubanol genus is immersed in an alcohol-based solvent and extracted three times. Then, the three immersion liquids are combined, filtered, and concentrated under reduced pressure to obtain the Atubanol genus alcohol-based extract.

[0021] In a specific embodiment, in step (b), the organic solvent layer and the aqueous layer are formed by subjecting the Saururus chinensis alcohol extract to partition extraction with the organic solution. Subsequently, the organic solvent layer is collected to obtain the Saururus chinensis extract. In a preferred specific embodiment, in step (b), the Saururus chinensis alcohol extract is subjected to partition extraction with the organic solution, and the partition extraction operation is repeated three times to form the organic solvent layer and the aqueous layer. Subsequently, the organic solvent layer is collected to obtain the Saururus chinensis extract.

[0022] In another specific embodiment, step (b) further includes step (b1) before extraction with the organic solution. In step (b1), the Saururus chinensis alcohol extract is immersed in water to obtain an aqueous Saururus chinensis solution. In a preferred specific embodiment, in step (b1), the Saururus chinensis alcohol extract is immersed in water to suspend the Saururus chinensis alcohol extract in water. After the execution of step (b1), in the original step (b), the aqueous Saururus chinensis solution obtained in step (b1) is subjected to partition extraction with the organic solvent without using the mixture of water and the organic solvent, thereby forming the organic solvent layer and the aqueous layer. Subsequently, the organic solvent layer is collected to obtain the Saururus chinensis extract. In another specific embodiment, in step (b), the aqueous Saururus chinensis solution obtained in step (b1) is subjected to partition extraction with the organic solvent to form the organic solvent layer and the aqueous layer. Subsequently, the organic solvent layer is collected to obtain the Saururus chinensis extract. In a preferred specific embodiment, in step (b), the aqueous Saururus chinensis solution obtained in step (b1) is subjected to partition extraction with the organic solvent, and the partition extraction operation is repeated three times to form the organic solvent layer and the aqueous layer. Subsequently, the organic solvent layer is collected to obtain the Saururus chinensis extract.

[0023] After the execution of step (b1), in the original step (b), the aqueous solution of the genus Atsubanori obtained in step (b1) is subjected to partition extraction by the supercritical extraction. In a specific embodiment, in step (b), the aqueous solution of the genus Atsubanori obtained in step (b1) is subjected to partition extraction by the supercritical extraction to form an organic solvent layer and an aqueous layer. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Atsubanori. In another specific embodiment, the supercritical extraction is supercritical CO2 extraction. In the present invention, in the process of separating the nutritional components of natural products by supercritical CO2 extraction, mainly the supercritical carbon dioxide fluid is used to extract the nutritional components from natural products under high-pressure and low-temperature conditions. In a specific embodiment, the supercritical CO2 extraction conditions are that the flow rate ratio of CO2:99% ethanol solution = 10 mL / min:1 mL / min, the range of critical pressure is 150 to 400 bar, and the range of critical temperature is 20 to 60 °C. In a preferred specific embodiment, the supercritical CO2 extraction conditions are that the flow rate ratio of CO2:95% ethanol solution = 10 mL / min:1 mL / min, the critical pressure is 250 bar, and the critical temperature is 40 °C. Similarly, by supercritical CO2 extraction, it is separated into the raw material (aqueous layer) remaining in the supercritical CO2 extraction reaction tank and the substance to be extracted (organic solvent layer). Therefore, the extract of the genus Atsubanori is obtained by collecting the organic solvent layer. In another specific embodiment, in step (b), the extract of the genus Atsubanori is obtained by extracting the alcohol-based extract of the genus Atsubanori by supercritical CO2 extraction.

[0024] In another specific embodiment, the components of the extract of the *Atsubanori* genus include cholesterol, stearic acid, methyl stearate, glyceryl stearate, (2S)-1-O-palmitoyl-3-O-β-D-galactopyranosylglycerol, (2S)1,2-di-O-oleoyl-3-O-β-D-galactopyranosylglycerol, 132-Hydroxy-(132S)-phaeophytin a, and 132-Hydroxy-(132R)-phaeophytin a. In a specific embodiment, the components of the extract of the *Atsubanori* genus do not include polysaccharides.

[0025] The present invention further provides the use of a composition in the manufacture of a drug for neuroprotection. The composition includes an extract of the *Atsubanori* genus, and the method for manufacturing the extract of the *Atsubanori* genus includes the following steps. That is, (a) extracting the *Atsubanori* genus with an alcoholic solvent to obtain an alcoholic extract of the *Atsubanori* genus. The alcoholic solvent is methanol or ethanol.

[0026] In a specific embodiment, the *Atsubanori* genus in step (a) is subjected to washing, drying, and / or pulverization treatment.

[0027] In another specific embodiment, the method for producing the extract of the genus Atsubanori further includes step (b) after step (a). In step (b), the alcoholic extract of the genus Atsubanori is subjected to partition extraction by an organic solution or supercritical extraction to form an organic solvent layer and an aqueous layer. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Atsubanori. The organic solution is an organic solvent or a mixture of water and an organic solvent. The organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, ethyl ether, dichloromethane, chloroform or carbon tetrachloride.

[0028] In a specific embodiment, the method for producing the extract of the genus Atsubanori further includes step (b1) before the extraction with the organic solution. In step (b1), the alcoholic extract of the genus Atsubanori is immersed in water to obtain an aqueous solution of the genus Atsubanori. In a preferred specific embodiment, in step (b), the aqueous solution of the genus Atsubanori obtained in step (b1) is subjected to partition extraction with the organic solvent to form an organic solvent layer and an aqueous layer. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Atsubanori.

[0029] In another specific embodiment, the genus Atsubanori is Atsubanori (Sarcodia ceylanica).

[0030] In a specific embodiment, the alcoholic solvent is ethanol. In a preferred specific embodiment, the alcoholic extract of the genus Atsubanori is an ethanol extract of the genus Atsubanori.

[0031] In another specific embodiment, the organic solvent is ethyl acetate. In a preferred specific embodiment, the extract of the genus Atsubanori is an ethyl acetate extract of the genus Atsubanori.

[0032] In the present invention, the extract of the genus *Atsubanoi* can be applied to impart a neuroprotective effect. The neuroprotective effect means, for example, the ability to prevent or reduce the death or damage of nerve cells (including neurons and glial cells) under conditions that can be pathologically harmful to the brain, central nervous system, or peripheral nervous system, or the ability to enable the rescue, resuscitation, or recovery of nerve cells. The neuroprotective effect includes the regeneration of nerve cells, that is, the regrowth of nerve cells after disease or trauma. The neuroprotective effect is a mechanism and strategy for preventing brain / neuron damage or degeneration caused by acute diseases (such as stroke, brain or nervous system injury / trauma, cerebral hypoxia, spinal cord injury, or peripheral nerve injury) or chronic neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, or multiple sclerosis) in the nervous system. The purpose of the neuroprotective effect is to suppress nerve dysfunction / death after nervous system injury and maintain the most highly organized cell interactions in the brain so that nerve function is not impaired. By imparting a neuroprotective effect, the extract of the genus *Atsubanoi* enables the treatment of neuron damage (such as stroke, particularly ischemic stroke, brain injury, cerebral hypoxia, spinal cord injury, or peripheral nerve injury), and the treatment or prevention of neurodegenerative diseases. Therefore, in a specific embodiment, the present invention relates to the use of the extract of the genus *Atsubanoi* as an active ingredient in the manufacture of a drug for protecting and / or regenerating nerve cells.

[0033] In a specific embodiment, the neuroprotection includes the suppression or prevention of nerve cell death. In a preferred specific embodiment, the extract of the genus *Atsubanoi* can suppress or prevent nerve cell death.

[0034] Thus, in the present invention, it has been found that the extract of the genus *Atsubanoi* has several beneficial effects and functions in imparting a neuroprotective effect. Therefore, the extract of the genus *Atsubanoi* is applicable to the treatment of neuron or brain damage, and the treatment or prevention of neurodegenerative diseases. The neurodegenerative diseases include acute or chronic neurodegenerative diseases.

[0035] As used herein, the term "neurodegenerative disease" is also used to describe acute, progressive or chronic diseases caused by damage to the central nervous system. And in the present invention, the damage can be reduced and / or alleviated by treatment with the extract of the genus Atsubanol. The term "acute neurodegenerative disease" means a sudden disease or disorder that causes associated neuronal death or damage. Examples of acute neurodegenerative diseases include cerebrovascular insufficiency, focal or diffuse brain injury, spinal cord injury, cerebral ischemia or infarction (embolic occlusion and thrombotic occlusion), perinatal hypoxic-ischemia, neonatal hypoxia-ischemic encephalopathy, perinatal asphyxia, cardiac arrest, intracranial hemorrhage, subarachnoid hemorrhage, stroke and traumatic brain injury.

[0036] In addition, representative examples of neurodegenerative diseases treatable using the extract of the genus Atsubanol 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 ataxia, Machado-Joseph disease (MJD), dentatorubral pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA), or fragile X-associated tremor and ataxia syndrome (FXTAS).

[0037] In a specific embodiment, the extract of the *Atsubanol* genus prevents or treats neurodegenerative diseases by conferring a neuroprotective effect. In a preferred specific embodiment, the neurodegenerative diseases include amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, frontotemporal dementia, spinocerebellar ataxia, spinocerebellar degeneration type 3, dentatorubral-pallidoluysian atrophy, spinal muscular atrophy, or fragile X-associated tremor / ataxia syndrome. In a more preferred specific embodiment, the neurodegenerative disease includes Parkinson's disease.

[0038] As used herein, the term "treatment" means alleviation of symptoms or complications, delay in the progression of a disease, disorder or medical condition, reduction or alleviation of symptoms and complications, and / or cure or elimination of a disease, disorder or medical condition.

[0039] As used herein, the term "prevention" means prevention of the onset, recurrence or spread of a disease or medical condition, or one or more of its symptoms. In any embodiment, these terms mean using the drugs provided herein, particularly in individuals at risk of the diseases or medical conditions presented herein, to treat, or administer the drugs provided herein, in the presence or absence of one or more other activators before the onset of symptoms.

[0040] The present invention further provides the use of a composition in the manufacture of a drug for preventing or treating neurodegenerative diseases. The composition includes an extract of the *Atsubanol* genus, and the method for manufacturing the extract of the *Atsubanol* genus includes the following steps. That is, (a) extracting the *Atsubanol* genus with an alcohol solvent to obtain an alcohol extract of the *Atsubanol* genus. The alcohol solvent is methanol or ethanol.

[0041] In a specific embodiment, the *Atsubanol* genus in step (a) is subjected to washing, drying and / or pulverization treatment.

[0042] In another specific embodiment, the method for producing the extract of the genus Sarcodia further includes step (b) after step (a). In step (b), the alcoholic extract of the genus Sarcodia is subjected to partition extraction by an organic solution or supercritical extraction to form an organic solvent layer and an aqueous layer. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Sarcodia. The organic solution is an organic solvent or a mixture of water and an organic solvent. The organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, ethyl ether, dichloromethane, chloroform or carbon tetrachloride.

[0043] In a specific embodiment, the method for producing the extract of the genus Sarcodia further includes step (b1) before the extraction with the organic solution. In step (b1), the alcoholic extract of the genus Sarcodia is immersed in water to obtain an aqueous solution of the genus Sarcodia. In a preferred specific embodiment, in step (b), the aqueous solution of the genus Sarcodia obtained in step (b1) is subjected to partition extraction with the organic solvent to form an organic solvent layer and an aqueous layer. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Sarcodia.

[0044] In another specific embodiment, the genus Sarcodia is Sarcodia ceylanica.

[0045] In a specific embodiment, the alcoholic solvent is ethanol. In a preferred specific embodiment, the alcoholic extract of the genus Sarcodia is an ethanol extract of the genus Sarcodia.

[0046] In another specific embodiment, the organic solvent is ethyl acetate. In a preferred specific embodiment, the extract of the genus Sarcodia is an ethyl acetate extract of the genus Sarcodia.

[0047] In a specific embodiment, the neurodegenerative diseases include amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, frontotemporal dementia, spinocerebellar ataxia, spinocerebellar degeneration type 3, dentatorubral-pallidoluysian atrophy, spinal muscular atrophy, or fragile X-associated tremor / ataxia syndrome. In a preferred specific embodiment, the neurodegenerative disease includes Parkinson's disease.

[0048] The present invention further provides the use of a composition in the manufacture of a drug for promoting bone formation. The composition includes an extract of the genus Atsubanol, and the method for manufacturing the extract of the genus Atsubanol includes the following steps. That is, (a) extracting the genus Atsubanol with an alcohol-based solvent to obtain an alcohol-based extract of the genus Atsubanol. The alcohol-based solvent is methanol or ethanol.

[0049] In a specific embodiment, the genus Atsubanol in step (a) is subjected to washing, drying, and / or pulverization treatment.

[0050] In another specific embodiment, the method for manufacturing the extract of the genus Atsubanol further includes step (b) after step (a). In step (b), the alcohol-based extract of the genus Atsubanol is subjected to partition extraction by organic solution or supercritical extraction to form an organic solvent layer and an aqueous layer. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Atsubanol. The organic solution is an organic solvent or a mixture of water and an organic solvent. The organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, ethyl ether, dichloromethane, chloroform, or carbon tetrachloride.

[0051] In a specific embodiment, the method for producing the extract of the genus Sarcodia further includes step (b1) before extraction with the organic solvent. In step (b1), the alcohol extract of the genus Sarcodia is immersed in water to obtain an aqueous solution of the genus Sarcodia. In a preferred specific embodiment, in step (b), the aqueous solution of the genus Sarcodia obtained in step (b1) is subjected to partition extraction with the organic solvent to form an organic solvent layer and an aqueous layer. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Sarcodia.

[0052] In another specific embodiment, the genus Sarcodia is Sarcodia ceylanica.

[0053] In a specific embodiment, the alcohol solvent is ethanol. In a preferred specific embodiment, the alcohol extract of the genus Sarcodia is an ethanol extract of the genus Sarcodia.

[0054] In another specific embodiment, the organic solvent is ethyl acetate. In a preferred specific embodiment, the extract of the genus Sarcodia is an ethyl acetate extract of the genus Sarcodia.

[0055] As used herein, the term "bone formation" means that undifferentiated stem cells and osteoblasts proliferate into osteoblasts and bone tissue (e.g., synthesis and accumulation of new bone matrix). Bone formation also means that progenitor or precursor cells differentiate or transform into bone cells (i.e., osteoblasts). Progenitor or precursor cells can be, for example, pluripotent stem cells including mesenchymal stem cells. Progenitor or precursor cells can be cells that can form the osteocyte lineage (e.g., pre-osteoblast cells), or cells that cannot form osteoblasts (e.g., preadipocytes or myoblasts).

[0056] Thus, the extract of the genus Atsubanol can be applied to the treatment of bone diseases. Such bone diseases include, but are not limited to, osteoporosis, bone defect or fracture diseases. Osteoporosis is a disease characterized by net loss of bone mass per unit volume. Such loss of bone mass and the accompanying fractures destroy the skeleton that structurally supports the body firmly. Due to low bone mass and structural deterioration of bone tissue, bone brittleness is brought about, and fractures of the hip joint, spine and wrist increase. Osteoporosis can be classified into primary osteoporosis and secondary osteoporosis according to the cause. In the molecular mechanism of primary osteoporosis, osteoclasts are activated by the binding of receptor activator of nuclear factor kappa B ligand (RANKL) in the nucleus of osteoblasts to receptor activator of nuclear factor kappa B (RANK) on the surface of osteoclasts, and the differentiation and activity of osteoclasts are stimulated, thus promoting the occurrence of osteoporosis. In addition, a sharp decrease in estrogen in the body of postmenopausal women also promotes the activity of osteoclasts, making osteoporosis and fractures more likely to occur. In the case of secondary osteoporosis, it is caused by long-term drug use, poor lifestyle, endocrine damage and other diseases (such as rheumatoid arthritis, diabetes, stroke, Parkinson's disease and bone metastasis of cancer). In addition, bone defect means that the ratio of bone formation to bone resorption lacks balance. As a result, a skeleton below the expectation of the individual is formed, or the skeleton of the individual becomes incomplete compared with the expectation. Bone defect can also be caused by fractures, surgical intervention, or teeth or periodontal diseases. Bone healing includes, but is not limited to, the repair of bone defects. For example, bone defects occur in closed, open and non-union fractures.

[0057] In a specific embodiment, the extract of the genus Atsubanol prevents or treats bone diseases by promoting bone formation. In a preferred specific embodiment, such bone diseases include osteoporosis, bone defect or fracture diseases. In a more preferred specific embodiment, such bone diseases include bone defect.

[0058] The present invention further provides the use of a composition in the manufacture of a drug for preventing or treating bone diseases. The composition contains an extract of the genus Sarcodia, and the method for manufacturing the extract of the genus Sarcodia includes the following steps. That is, (a) extracting the genus Sarcodia with an alcoholic solvent to obtain an alcoholic extract of the genus Sarcodia. The alcoholic solvent is methanol or ethanol.

[0059] In a specific embodiment, the genus Sarcodia in step (a) is subjected to washing, drying and / or pulverization treatment.

[0060] In another specific embodiment, the method for manufacturing the extract of the genus Sarcodia further includes step (b) after step (a). In step (b), the alcoholic extract of the genus Sarcodia is subjected to partition extraction by an organic solution or supercritical extraction to form an organic solvent layer and an aqueous layer. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Sarcodia. The organic solution is an organic solvent or a mixture of water and an organic solvent. The organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, ethyl ether, dichloromethane, chloroform or carbon tetrachloride.

[0061] In a specific embodiment, the method for manufacturing the extract of the genus Sarcodia further includes step (b1) before the extraction with the organic solution. In step (b1), the alcoholic extract of the genus Sarcodia is immersed in water to obtain an aqueous solution of the genus Sarcodia. In a preferred specific embodiment, in step (b), the aqueous solution of the genus Sarcodia obtained in step (b1) is subjected to partition extraction with the organic solvent to form an organic solvent layer and an aqueous layer. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Sarcodia.

[0062] In another specific embodiment, the genus Sarcodia is Sarcodia ceylanica.

[0063] In a specific embodiment, the alcohol solvent is ethanol. In a preferred specific embodiment, the alcohol extract of the *Atsubanol* genus is an ethanol extract of the *Atsubanol* genus.

[0064] In another specific embodiment, the organic solvent is ethyl acetate. In a preferred specific embodiment, the extract of the *Atsubanol* genus is an ethyl acetate extract of the *Atsubanol* genus.

[0065] In the present invention, the extract of the *Atsubanol* genus can treat bone diseases by increasing bone mass or promoting and repairing bone growth. In a specific embodiment, the bone diseases include osteoporosis, bone defect or fracture diseases. In a preferred specific embodiment, the bone diseases include bone defects.

[0066] The present invention further provides the use of a composition in the manufacture of a drug for treating allergic dermatitis. The composition contains an extract of the *Atsubanol* genus, and the manufacturing method of the extract of the *Atsubanol* genus includes the following steps. That is, (a) extracting the *Atsubanol* genus with an alcohol solvent to obtain an alcohol extract of the *Atsubanol* genus. The alcohol solvent is methanol or ethanol.

[0067] In a specific embodiment, the *Atsubanol* genus in step (a) is subjected to washing, drying and / or pulverization treatment.

[0068] In another specific embodiment, the manufacturing method of the extract of the *Atsubanol* genus further includes step (b) after step (a). In step (b), the alcohol extract of the *Atsubanol* genus is subjected to partition extraction by organic solution or supercritical extraction to form an organic solvent layer and an aqueous layer. Subsequently, the organic solvent layer is collected to obtain the extract of the *Atsubanol* genus. The organic solution is an organic solvent or a mixture of water and an organic solvent. The organic solvent is ethyl acetate, propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, ethyl ether, dichloromethane, chloroform or carbon tetrachloride.

[0069] In a specific embodiment, the method for producing the extract of the genus Sarcodia further includes step (b1) before the extraction with the organic solution. In step (b1), the alcohol extract of the genus Sarcodia is immersed in water to obtain an aqueous solution of the genus Sarcodia. In a preferred specific embodiment, in step (b), the aqueous solution of the genus Sarcodia obtained in step (b1) is subjected to partition extraction with the organic solvent to form an organic solvent layer and an aqueous layer. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Sarcodia.

[0070] In another specific embodiment, the genus Sarcodia is Sarcodia ceylanica.

[0071] In a specific embodiment, the alcohol solvent is ethanol. In a preferred specific embodiment, the alcohol extract of the genus Sarcodia is an ethanol extract of the genus Sarcodia.

[0072] In another specific embodiment, the organic solvent is ethyl acetate. In a preferred specific embodiment, the extract of the genus Sarcodia is an ethyl acetate extract of the genus Sarcodia.

[0073] As used herein, the term "allergic dermatitis" refers to a general term for skin diseases caused by allergic reactions, characterized by chronic itching and rashes on the face, neck, elbows and / or knees. Examples of allergic dermatitis include contact dermatitis, atopic dermatitis, etc. "Contact dermatitis" is an eczematous inflammatory disease that develops when an exogenous antigen comes into contact with the skin. Examples include allergic contact dermatitis, photo-contact dermatitis, systemic contact dermatitis, and contact urticaria. Examples of antigens include metal allergens (such as cobalt, nickel, etc.), plant allergens (such as poison ivy, primrose, etc.), and food allergens (such as mango, ginkgo, etc.). "Atopic dermatitis (AD)" refers to a skin disease with an atopic tendency in many patients. It is characterized by symmetrical and generalized eczema that repeatedly worsens and remits. Examples include disseminated neurodermatitis, atopic eczema, atopic neurodermatitis, Besnier prurigo, acute infantile eczema, flexural eczema, infantile eczema of the extremities, infantile atopic eczema, infantile dry-type eczema, infantile eczema, adult atopic dermatitis, endogenous eczema, infantile dermatitis, and chronic infantile eczema.

[0074] In a specific embodiment, the allergic dermatitis includes contact dermatitis or atopic dermatitis. In a preferred specific embodiment, the allergic dermatitis includes atopic dermatitis. In a more preferred specific embodiment, the atopic dermatitis includes disseminated neurodermatitis, atopic eczema, atopic neurodermatitis, Besnier prurigo, acute infantile eczema, flexural eczema, infantile eczema of the extremities, infantile atopic eczema, infantile dry-type eczema, infantile eczema, adult atopic dermatitis, endogenous eczema, infantile dermatitis, or chronic infantile eczema.

[0075] In another specific embodiment, the drug suppresses the symptoms of atopic dermatitis including erythema, edema, epidermal detachment, or skin dryness.

[0076] In a specific embodiment, the drug reduces the increase in the IgE expression level derived from the atopic dermatitis.

[0077] In another specific embodiment, the drug improves the swelling of the spleen or lymph nodes derived from the atopic dermatitis.

[0078] In a specific embodiment, the drug further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein is determined by the composition applied in a specific combination and applied in a specific manner. The term "carrier" as used herein includes, but is not limited to, any and all solvents, dispersion media, vehicles, coatings, diluents, penetration / absorption retardants such as antibacterial and antifungal agents, buffering agents, carrier solutions, suspensions or colloids, etc. These media and reagents used for the active substances of the drug are known in the art. Combinations for treatment should be considered, except when there is a poor compatibility between any common medium or reagent and the active ingredient. Supplementary active ingredients may also be mixed into the composition. The term "pharmaceutically acceptable" means that no allergic or similar side reactions occur when the molecular entity and the composition are applied to a subject. The production of aqueous compositions with proteins as active substances is well known in the art. Usually, this composition is produced as a liquid / solution, tablet, capsule or suspension injection. It may also be produced as a soluble solid or a solid of a suspension that can be used as an injection. In another specific embodiment, the pharmaceutically acceptable carrier includes a dermatologically acceptable medium. The "dermatologically acceptable medium" means a biologically appropriate substance such as components of salts, esters and / or acid amides, etc. That is, when this substance is used in combination with the selected active ingredient, it does not cause any undesirable biological effects when administered to an individual. Also, this substance is a substance that does not cause any harmful interactions with any of the components in the pharmaceutical composition containing this substance. Similarly, the "dermatologically acceptable salt" or "dermatologically acceptable ester" in the text is a biologically appropriate salt or ester.

[0079] In the present invention, for the formulation of the composition (including the extract of the genus Atsubanol), sterile aqueous solutions or dispersions, aqueous suspensions, oil emulsions, water in oil emulsions, multiple point emulsions, long retention emulsions, viscous emulsions, microemulsions, nanoemulsions, liposomes, microparticles, microspheres, nanospheres, nanoparticles, micro-mercury, and several types of natural or synthetic polymers that are released sustainably can be used. The pharmaceutically acceptable carrier and the extract of the genus Atsubanol may be formulated as aerosol agents, tablets, pills, capsules, sterile powders, suppositories, detergents, creams, ointments, pastes, gels, hydrogels, or other formulations that can be used for the delivery of the composition.

[0080] In addition, the composition referred to in the present invention includes compositions that are administered locally and regionally to exert an effect. The term "local" as used in the present text relates to the local effect achieved by mixing the composition described in the present text with a suitable pharmaceutical carrier and applying it to a site of the skin (for example, the affected area of allergic dermatitis). Therefore, such locally used compositions of the present invention include drug forms such as compounds that are applied externally in direct contact with the surface of the skin to be treated. General drug forms for this purpose include ointments, liniments, creams, shampoos, emulsions, pastes, gels, sprays, aerosol agents, etc. Moreover, depending on the body part to be treated, it is also possible to use it in the form of a patch or a wet dressing. The term "ointment" includes preparations (including creams) having an oily matrix, a water-soluble matrix, and an emulsion matrix. The matrix is, for example, petrolatum, lanolin, polyethylene glycols, and mixtures thereof.

[0081] The drug of the present invention further includes a pharmaceutically acceptable carrier and can be administered to an individual by a number of different means by therapeutic methods well-known in the relevant field of the present invention. In some embodiments, the composition (including the extract of the genus Atsubanol) and the pharmaceutically acceptable carrier are administered topically, intravenously, intramuscularly, subcutaneously, locally, orally, or by inhalation. Also, the drug is transmitted to the target site by the digestive and circulatory systems.

[0082] In another specific embodiment, the individual is an animal, preferably a mammal, more preferably a human.

[0083] The term "effective dosage" in the present text refers to a therapeutic dosage that, under specific conditions, can prevent, reduce, arrest, or reverse the progression of the symptoms of an individual, or can partially or completely relieve the symptoms that existed under special circumstances at the time when the individual began receiving treatment. A person skilled in the art can easily determine the appropriate dosage and usage for administering the extract of the genus Actinidia to an individual. For example, the extract of the genus Actinidia can be administered to the individual once or twice. The dosage and usage include the understanding that, when administering multiple times, the effective dosage of the extract of the genus Actinidia administered to the individual (including the total amount of the product administered during the entire period of dosage and usage) can be understood.

[0084] Thus, the effective dosages of the extract of the genus Actinidia in the present invention are different when applied to neuroprotection, promotion of bone formation, and treatment of allergic dermatitis, respectively.

[0085] When provided for neuroprotection, in a specific embodiment, the range of the effective dosage of the extract of the genus Actinidia is 0.2 mg / kg body weight to 200 mg / kg body weight. In a preferred specific embodiment, the range of the effective dosage of the extract of the genus Actinidia is 1 mg / kg body weight to 100 mg / kg body weight. In a more preferred specific embodiment, the range of the effective dosage of the extract of the genus Actinidia is 2 mg / kg body weight to 20 mg / kg body weight.

[0086] Regarding the effective dosage in the case of neuroprotection, in another specific embodiment, the range of the effective dosage of the extract of the genus Actinidia is 0.5 ng / ml to 50 μg / ml. In a preferred specific embodiment, the range of the effective dosage of the extract of the genus Actinidia is 1 ng / ml to 5 μg / ml. In a more preferred specific embodiment, the range of the effective dosage of the extract of the genus Actinidia is 5 ng / ml to 500 ng / ml.

[0087] When promoting bone formation, in a specific embodiment, the effective dosage range of the extract of the genus Atsubanori is 0.2 mg / kg body weight to 100 mg / kg body weight. In a preferred specific embodiment, the effective dosage range of the extract of the genus Atsubanori is 1 mg / kg body weight to 50 mg / kg body weight. In a more preferred specific embodiment, the effective dosage range of the extract of the genus Atsubanori is 2 mg / kg body weight to 10 mg / kg body weight.

[0088] When treating allergic dermatitis, in a specific embodiment, the effective dosage range of the extract of the genus Atsubanori is 0.05 to 20 mg / ml. In a preferred specific embodiment, the effective dosage range of the extract of the genus Atsubanori is 0.1 to 10 mg / ml. In a more preferred specific embodiment, the effective dosage range of the extract of the genus Atsubanori is 0.5 to 2 mg / ml.

Brief Description of the Drawings

[0089]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Mode for Carrying Out the Invention

[0090] The present invention includes, but is not limited to, the above and the following descriptions. Representative examples of the implementation method are shown below.

[0091] Extraction of Cultured Sarcodia In the present invention, artificially cultured Sarcodia ceylanica was used.

[0092] The Sparassis genus was dried under the condition of 50 °C for 24 hours to obtain dried Sparassis genus. 10.8 kg of the dried Sparassis genus was pulverized, filtered through a 50-mesh filter screen, and then immersed in a 95% alcohol solution at room temperature for extraction three times. Then, the immersion liquids for the three times were combined, filtered, and concentrated under reduced pressure to obtain 370.5 g of a crude alcohol extract of Sparassis genus. Next, the above-mentioned crude alcohol extract of Sparassis genus was added to 1 L of water to make it in a floating state, and then 1 L of ethyl acetate was added for partition extraction. By repeating the above partition extraction three times, an ethyl acetate layer and an aqueous layer were formed. Subsequently, the ethyl acetate layer was collected to obtain an ethyl acetate extract EE (83.2 g). The entire extraction flow of Sparassis genus is shown in Figure 1.

[0093] Also, in the above extraction step, the alcohol solution may be replaced with a methanol solution. And the step of extracting with ethyl acetate may be replaced with other organic solvents. For example, partition extraction may be performed using propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, ethyl ether, dichloromethane, chloroform or carbon tetrachloride solvent, or supercritical extraction (for example, supercritical CO2 extraction) may be performed, and an extract with the same components as EE can be obtained.

[0094] Also, water may be mixed with ethyl acetate or other organic solvents to form an organic solution, and the crude alcohol extract of Sparassis genus may be directly extracted. In this case, it is not necessary to extract water and the organic solvent separately in two steps. In addition, the step of immersing in water beforehand can be omitted, and the crude alcohol extract of Sparassis genus may be extracted directly using ethyl acetate or other organic solvents as they are. Even with the above changes in steps, an extract with the same components as EE can ultimately be obtained.

[0095] The above supercritical CO2 extraction conditions are that the flow rate ratio of CO2:95% ethanol solution = 10 mL / min:1 mL / min, the critical pressure is 250 bar, and the critical temperature is 40 °C.

[0096] Moreover, in the present invention, when the polysaccharide content of EE was measured by the phenol-sulfuric acid method, EE was found not to contain a polysaccharide component. Therefore, the polysaccharide component was not eluted by the extraction method of immersing the Atsubanori genus in alcohol in advance, and the Atsubanori genus alcohol crude extract did not contain the polysaccharide component. In addition, in the present invention, component analysis of the ethyl acetate extract EE of the Atsubanori genus was also performed. The analysis results were as shown in Table 1.

[0097]

Table 1

[0098] Experimental method (1) Evaluation of the neuroprotective effect of EE (a) Neuronal cell test SH-SY5Y cells were cultured in a 96-well cell culture plate at a cell density of 2×104 cells, and then placed in a cell incubator for culture. After the cells were completely adhered, they were pretreated with EE solutions of various different concentrations of the ethyl acetate extract of the Atsubanori genus for 1 hour. Then, 6-hydroxydopamine (6-OHDA) was added and treated for 15 hours, and 10% alamar blue was added and cultured continuously for 3 hours. Subsequently, the absorbance value was measured with an enzyme immunoassay analyzer, and the cell viability was measured. From the toxicity caused by 6-OHDA to neuronal cells, it was analyzed whether the Atsubanori genus extract had a protective effect on the neuronal cells treated with 6-OHDA.

[0099] (b) In vivo nerve test 6-OHDA was dissolved in 2% fresh L-ascorbic acid to prepare a 500 mM stock solution, which was then put into 1.5 mL brown microtubes and divided into 10 μL tubes. When adding drugs, the formulated drugs were dissolved in Hank’s buffer at the same ratio based on the concentrations of the experimental design. Then, 1 mL of the drug and 1 mL of Hank’s buffer containing larvae were added to a 24-well culture plate. The time when 6-OHDA was given to the larvae was between 48 hours and 120 hours after fertilization. In addition, no drugs were added to the control group, and a solvent group was set as a control for each experiment. By analyzing the behavioral ability of zebrafish, parameters such as the residence time, swimming speed, and swimming distance of the fish in different water layers were measured to evaluate the therapeutic effect of the extract of the genus Acanthopanax.

[0100] (2) Evaluation of the effect of EE on bone formation (a) Bone growth test of zebrafish Using a biological system, the promoting effect of the extract solution of the genus Acanthopanax on bone regeneration was tested. In addition, the effect of the extract of the genus Acanthopanax on bone and bone quality-related diseases (such as osteoporosis, etc.) was analyzed. The effect of the active substance on bone growth was analyzed by specifically staining the hard bone part of the bone with the fluorescent stain calcein and combining it with the developmental morphology of the embryo body of zebrafish. In addition, by using analysis software to convert the data, the effect of the substance on bone growth was presented. Zebrafish larvae 48 hours after fertilization were obtained, and after adding different concentrations of the extract of the genus Acanthopanax, the vertebral joints of the fish body were observed daily, and the breeding was continued after the observation. Zebrafish were stained with a 2 g / L calcein solution (pH = 7.8) for 10 minutes, then rinsed with pure water to wash off the excess stain. Then, after being anesthetized with MS-222, the fish body was placed in 1% methyl cellulose for fixation. Then, the vertebral skeleton of the fish body was observed with a fluorescence microscope, and the number of segments of the skeleton was statistically analyzed.

[0101] (b) Skull defect experiment in rats Skull defect surgery: Small-scale bone defects can regenerate naturally in vivo. However, when the defect size exceeds the size that can be repaired by the living body, it becomes a so-called critical sized bone defect that cannot fully recover on its own and cannot heal without external treatment. Differences in animal models and sites both affect the size of the defect to be constructed. In the rat skull defect model used in the present invention, first, the animals were anesthetized using the gas anesthetic isoflourane. Next, after shaving the top of the rat's head, the surgical site was disinfected with povidone iodine and 70% alcohol, and ampicillin was injected. Subsequently, holes were made in a sterilized paper towel, which was placed over the rat's head to expose only the surgical site. Then, the skin and periosteum directly above the rat's head were incised at a size of approximately 1.5 cm, and the skull and periosteum were separated using a periosteal elevator. One ridge existing exactly in the center of the skull is the median sagittal suture, and both sides of it are the parietal bones. Exactly in the center around this sagittal suture, two bone defects with a diameter of 4 mm were created using a perforator with a diameter of 4 mm. Subsequently, after suturing the periosteum with 5-0 cut gut, the skin was sutured with 4-0 nylon thread. If the animal woke up from the anesthetized state during the operation, the animal was made to inhale anesthetic gas with a gas anesthetic through an oxygen mask to maintain anesthesia. The rats were administered EE in a state of anesthesia three times a week. The dosage was 1.5 mg / kg / week and 7.5 mg / kg / week. Then, after 4 weeks of administration, the animals were sacrificed and the skull tissues were collected.

[0102] As an evaluation method for the healing condition of the rat skull, an X-ray device (X-OMAT V, Kodak, Rochester, New York) was used for imaging, and the negative film was developed using an automatic film processor. The images were scanned and converted into TIF files, and then loaded into ImageQuant software (ImageQuant Version 5.2 Copyright 1999, Molecular Dynamics) for subsequent analysis. Next, in the ImagQuant software, equal-area regions of each step wedge and blank areas were selected to represent the relationship between the gray-scale intensity of the image on the X-ray film and the thickness of the wedge-shaped step. Thereafter, a circular region with a diameter of 8 mm in the skull region and flat regions with an area of 1×1 mm2 at the center of the parietal bones on both sides were selected, the gray-scale intensity was measured, and substituted into the above equation. Thus, the bone density of the sample with respect to the thickness of the wedge-shaped step was calculated by interpolation. That is, the bone density of the bone defect region of the skull was defined as the density of the gray-scale image projected by the difference in the thickness of the wedge-shaped step.

[0103] (3) Evaluation of the effect of EE on atopic dermatitis The experimental animals used in the present invention were BALB / c strain mice. After the mice were anesthetized with 2.5% isoflurane, the hair on the back of the mice was removed using a shaver and depilatory cream, and marked with a silicone rubber with a diameter of 1 cm (area 78.5 mm2). Next, 26 μL of 2% 2,4-dinitrochlorobenzene (DNCB) (Sigma-Aldrich, Missouri, USA, Cat. No. #138630) was dropped into the above area once every two days. This was done a total of 8 times to induce skin lesions.

[0104] Seven days after DNCB induction, 100 μL of vehicle, the extract EE of *Artemisia princeps* Pamp., or the atopic dermatitis therapeutic agent crisaborole was applied daily. The experimental animals were divided into the following groups (n = 3 per group): (1) control group: not induced; (2) AD group: administered only 2% DNCB; (3) ADV group: administered 2% DNCB and vehicle; (4) EEL group: administered 50 μg of EE / 1% methylcellulose 100 μL after DNCB induction; (5) EEH group: administered 200 μg of EE / 1% methylcellulose 100 μL after DNCB induction; and (6) Cri group: administered 25 μg of crisaborole / 1% acetone-EtOH 100 μL after DNCB induction. Also, 5 minutes after applying DNCB, the drug was applied after waiting for DNCB to dry. Before induction or drug application, changes in skin symptoms were photographed and recorded with a digital camera.

[0105] On the 18th day, the animals were sacrificed, blood was collected, and tissues of the back skin, spleen, and lymph nodes were collected. Then, the sizes of the spleen and subiliac lymph nodes were observed, measured, and compared. Also, the severity of clinical dermatitis was evaluated based on four symptoms of the skin surface: erythema / hemorrhage, edema, excoriation / erosion, and dryness. Each symptom was assigned 0 - 3 points according to its severity, where 0 points indicated no symptoms, 1 point indicated mild symptoms, 2 points indicated moderate symptoms, and 3 points indicated severe symptoms. The total score of dermatitis was the sum of the above four scores, with a maximum of 12 points. The higher the score, the more severe the dermatitis.

[0106] Before sacrifice, mouse blood was collected into a blood collection tube (BD vacutainer-SST, New Jersey, USA) by cardiac puncture. Then, the blood was centrifuged at 3000 rpm for 10 minutes to obtain serum. The serum was stored in a -80°C refrigerator until the next use. Then, according to the manufacturer's instructions, the IgE concentration in the serum was detected (IgE-ELISA kit, Thermo Fisher Scientific, Vienna, Austria).

[0107] When an inflammation or immune-related disease occurs, the lymph nodes and spleen swell. Therefore, when sacrificing the mice, these two organs were collected, photographed, and weighed.

[0108] Statistical analysis All experimental data were expressed as mean ± standard error of the mean (mean ± SEM). To compare the data among multiple groups, statistical analysis of the data was performed by one-way analysis of variance (ANOVA). In addition, multiple comparisons of the differences between groups were performed by Duncan's Method. And when the P value was less than 0.05, there was a significant difference between groups.

[0109] Results Neuroprotective effect of EE After adding ethyl acetate extract EE of *Atsubanol* genus at different concentrations to SH-SY5Y neurons, the cells were damaged with 6-hydroxydopamine (6-OHDA), and finally the cell viability was measured to observe the neuroprotective effect. Then, while the cell viability of the control group was 100 ± 3.63%, the cell viability of the 6-OHDA group decreased to 0 ± 3.88%. However, when adding ethyl acetate extract EE of *Atsubanol* genus at concentrations of 0.5 - 500 ng / ml, the cell viability of each group became 10.72 ± 2.86%, 33.91 ± 19.67%, 7.92 ± 1.93%, and 32.69 ± 17.88% (see Figure 2) respectively, and all improved. From this result, it was proved that the ethyl acetate extract EE of *Atsubanol* genus has a neuroprotective effect.

[0110] Moreover, 6-OHDA was able to induce Parkinson's disease-like behavior in zebrafish. As a result, the zebrafish stopped swimming and stayed at the bottom. (A) of Fig. 3 shows the swimming paths of zebrafish in each group within a unit time. Also, (B) of Fig. 3 shows the swimming speed of zebrafish. The speed of the control group was 2.47 ± 0.26 mm / s, while the speed of the 6-OHDA group decreased to 0.21 ± 0.11 mm / s. However, when ethyl acetate extracts EE of the genus Atubanol were added at concentrations of 0.5 μg / ml and 5 μg / ml, the swimming speed of the zebrafish recovered to 2.03 ± 0.65 mm / s and 1.24 ± 0.28 mm / s, respectively. Moreover, (C) of Fig. 3 shows the total swimming distance of zebrafish within a unit time. The swimming distance of the control group was 741.09 ± 77.04 mm, while the swimming distance of the 6-OHDA group decreased to 63.00 ± 32.19 mm. However, when ethyl acetate extracts EE of the genus Atubanol were added at concentrations of 0.5 μg / ml and 5 μg / ml, the swimming distance of the zebrafish recovered to 609.03 ± 196.17 mm and 371.52 ± 82.77 mm, respectively. This result indicates that the ethyl acetate extracts EE of the genus Atubanol have a neuroprotective effect on nerves in vivo.

[0111] (2) Effect of EE on bone formation The ethyl acetate extracts EE of the genus Atubanol were able to promote bone formation. In the zebrafish model, while 10 ± 0.63 bone joints were formed in the larvae of the control group, the number of bone joints in the ethyl acetate extracts EE groups of the genus Atubanol at concentrations of 0.05 - 50 μg / ml increased to 13.8 ± 0.80, 15.33 ± 0.68, 15.00 ± 0.84, and 15.4 ± 0.68, respectively (see Fig. 4).

[0112] In the rat skull defect experiment, EE was administered three times a week. The dosage was 1.5 mg / kg / week and 7.5 mg / kg / week. After 4 weeks, the animals were sacrificed, skull tissues were collected, and area quantification was performed using the images taken with an X-ray device. Figure 5 is a comparison diagram of the skull appearance showing the therapeutic effects of ethyl acetate extracts EE of the genus Atsubano with different concentrations on rat skull defects. Also, (A) in Figure 6 shows the X-ray diagram of the skull defect situation of the rats in each group. (B) in Figure 6 shows the recovery rate of the skulls of the rats in each group. As is clear from these results, while the recovery rate of the skulls in the control group (vehicle) was 51.90 ± 10.01%, the recovery rates of the skulls administered with EE at 1.5 mg / kg / week and 7.5 mg / kg / week were 97.47 ± 4.86% and 15.47 ± 15.86%, respectively. The above results indicate that EE can promote bone formation.

[0113] (3) Therapeutic effect of EE on atopic dermatitis The symptoms of DNCB-induced atopic dermatitis (AD) in mice included phenomena such as erythema, edema, epidermal detachment, dryness, and lichenification. After obvious symptoms such as erythema, edema, epidermal detachment, and dryness appeared in the mice, EE or crisaborole was applied daily to test whether EE had a therapeutic effect on atopic dermatitis. The symptoms of the skin appearance of the mice were observed, photographed, and recorded (see Figure 7A), and the therapeutic effect of EE was quantified with a clinical dermatitis score (see Figure 7B). In the control group, the skin surface was dry due to less hair, and the score was 0.75 ± 0.25. In the AD group and the ADV group, keratinocytes proliferated on the skin surface, and a thick scab was formed. As days passed, some of the scabs peeled off, and dryness of the skin around the induction was observed. Also, wrinkles were seen on the skin. Moreover, due to inflammation, erythema and edema appeared on the skin. However, because keratinocytes continued to proliferate, there were also scabs on the skin surface. The clinical dermatitis scores of these two groups were significantly higher than those of the control group, being 9.17 ± 0.40 and 9.50 ± 0.56 respectively. In contrast, in the groups treated with EE and crisaborole (Cri) daily, although dryness, erythema, and edema were observed after the scabs peeled off, the degree of keratinocyte proliferation was considered milder than that in the AD group. Therefore, before sacrificing the animals, the dermatitis scores of the EEL group and the EEH group were significantly lower than those of the AD group (4.50 ± 0.76 and 3.67 ± 0.49). On the other hand, the dermatitis score of the Cri group was 4.80 ± 0.37, which was statistically significant compared to the AD group. The above results indicate that EE has a mitigating effect on AD symptoms.

[0114] IgE is one of the clinical diagnostic indicators for atopic dermatitis. Before sacrificing the mice, serum was collected to measure the IgE content, and the results were as shown in Figure 8. The IgE content in the serum of the control group was 1.71 ± 0.14 μg / mL. Also, the IgE concentrations in the AD group and the ADV group were significantly increased (68.89 ± 4.12 μg / mL and 68.95 ± 2.60 μg / mL). In contrast, the IgE concentrations in the EEL group and the EEH group were only slightly decreased, being 59.33 ± 2.11 μg / mL and 59.35 ± 2.05 μg / mL respectively. However, it was statistically significant. On the other hand, the serum IgE content in the Cri group decreased to 65.61 ± 1.74 μg / mL. The serum IgE that increased with atopic dermatitis could be decreased by applying EE.

[0115] The results of the spleen weight were as shown in Figure 9. The spleen weight of the control group was 91.08 ± 3.61 mg. Also, the spleens of the AD group and the ADV group were significantly enlarged, becoming 2.5 times that of the control group (234.46 ± 6.56 mg and 221.14 ± 13.32 mg). In contrast, the spleen weights of the EEL group and the EEH group were significantly decreased, being 160.74 ± 9.10 mg and 167.41 ± 10.39 mg respectively. On the other hand, the spleen weight of the Cri group was significantly decreased, being 167.46 ± 10.61 mg.

[0116] The results of the total weight of the lymph nodes were as shown in Figure 10. The total weight of the two lymph nodes in the control group was 4.46 ± 0.29 mg. Also, the lymph nodes of the AD group and the ADV group were significantly enlarged, becoming 3 times that of the control group (12.07 ± 0.33 mg and 11.61 ± 0.54 mg). In contrast, the lymph node weights of the EEL group and the EEH group were significantly decreased, being 9.10 ± 0.68 mg and 6.50 ± 0.59 mg respectively. And it had dose dependence. On the other hand, the lymph node weight of the Cri group was significantly decreased, being 9.49 ± 0.81 mg. From the above results, the enlarged lymph nodes and spleen associated with the inflammation or immune reaction caused by atopic dermatitis could suppress the above phenomena by applying EE.

[0117] The present invention is appropriately described as being capable of being implemented under requirements or restrictions not specifically disclosed in the text. The terms used for the description are not intended to be restrictive. There is no difference in the expressions and descriptions using these terms and any other equivalents, but it should be recognized that the rights in the present invention may be modified. Therefore, although examples and other situations have been described in the present invention, the content disclosed in the text can be supplemented and modified by those skilled in the art, and such modifications and deformations are considered to be within the scope of the rights of the present invention.

Claims

1. A use of a composition in the manufacture of a medicament for promoting bone formation, the composition comprising an ethyl acetate extract of Laminaria sp., and a method for the manufacture of the ethyl acetate extract of Laminaria sp., comprising: (a) extracting Porphyra spp. with ethanol to obtain an Porphyra spp. ethanol extract; And, (b) partitioning the ethanol extract of Capillaris with ethyl acetate and water to form an ethyl acetate layer and an aqueous layer, and then collecting the ethyl acetate layer to obtain the ethyl acetate extract of Capillaris; Use including steps.

2. The use according to claim 1, wherein the ethyl acetate extract of Laminaria genus is used to prevent or treat bone diseases by promoting bone formation.

3. The use according to claim 2, wherein the bone disease comprises osteoporosis, bone loss or fracture disease.

4. 1. Use of a composition in the manufacture of a medicament for preventing or treating a bone disease, the composition comprising an ethyl acetate extract of Laminaria sp., and a method for the manufacture of the ethyl acetate extract of Laminaria sp., comprising: (a) extracting Porphyra spp. with ethanol to obtain an Porphyra spp. ethanol extract; And, (b) partitioning the ethanol extract of Capillaris with ethyl acetate and water to form an ethyl acetate layer and an aqueous layer, and then collecting the ethyl acetate layer to obtain the ethyl acetate extract of Capillaris; Use including steps.

5. The use according to claim 4, wherein the bone disease comprises osteoporosis, bone loss or fracture disease.

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