Porphyra extract and uses thereof

Abstract

To provide use of Sarcodia ceylanica extract in the manufacture of drugs for treating allergic dermatitis such as atopic dermatitis.SOLUTION: Provided is use of a composition in the manufacture of a drag for treating allergic dermatitis, the composition containing Sarcodia ceylanica ethyl acetate extract, a method for manufacturing the Sarcodia ceylanica ethyl acetate extract comprising: (a) extracting Sarcodia ceylanica with ethanol to obtain Sarcodia ceylanica ethanol extract; and (b) forming an ethyl acetate layer and an aqueous layer by partitioning and extracting the Sarcodia ceylanica ethanol extract with ethyl acetate and water, and subsequently collecting the ethyl acetate layer to obtain the Sarcodia ceylanica ethyl acetate extract.SELECTED DRAWING: Figure 8

Description

[Technical field]

[0001] The present invention relates to a novel use of a Porphyra extract, in particular to the use of said Porphyra extract in the manufacture of a medicament for neuroprotection, promotion of bone formation and treatment of allergic dermatitis. [Background technology]

[0002] The genus Sarcodia is distributed mainly in the Indian Ocean and the Western Pacific Ocean. It is a heterotrophic marine macroalgae with a high content of micronutrients. For this reason, it is also known as a longevity food in Japan, and in Europe and the United States, it is considered a representative sea vegetable. Currently, Sarcodia is mostly used as it is or as a food additive. Although Sarcodia has a long history of being used as food, there is very little research on its effects.

[0003] The skeleton is a rigid organ that constitutes the endoskeletons of vertebrates, and contains biomineralized skeletal tissue, bone marrow, periosteum, nerves, blood vessels, cartilage, and other soft tissues, as well as a small number of skeletal cells. Skeletal defects or defects are thought to be due to congenital defects, acquired diseases, aging, or trauma. Small bone defects can heal naturally, but large defects or small skeletal defects are difficult to heal completely. Therefore, treatment is required by bone grafts (including autologous bone grafts, allogeneic bone grafts, and xenogeneic bone grafts), artificial bones (including bone cements and bioceramics), tissue engineered bones, or surgery such as bone transfer surgery. In other words, different drugs and surgeries are required for various bone diseases with different causes (e.g., osteoporosis, bone defects, fracture diseases, etc.).

[0004] In addition, with the aging of the global population, the number of patients with neurodegenerative diseases continues to increase, and the elderly suffer from increased damage to cognition, memory and motor skills. 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, developing treatment methods for this type of disease has become one of the important research projects in biomedical sciences these days. The manifestation mechanisms of many neurodegenerative diseases are still unclear, and at present, there is a lack of effective treatment methods for these diseases. In addition, the available treatments are limited to symptom control and inhibition of disease progression.

[0005] On the other hand, atopic dermatitis is a chronic disease in which immunity and inflammation are deeply involved. Atopic dermatitis is an inflammatory skin disease accompanied by itching, and is also a chronic disease that usually develops during infancy. The main symptom of atopic dermatitis is persistent itching, and the condition tends to improve and worsen repeatedly without a specific cause. Although much research has been done on atopic dermatitis recently, the cause of atopic dermatitis is still unclear.

[0006] Meanwhile, natural compounds purified from seaweed have been demonstrated to have numerous biological activities, such as anticoagulant, antiviral, antioxidant, antiallergic, anticancer, anti-inflammatory, and anti-obesity, etc. Therefore, the genus Porphyra, a type of seaweed, also has extremely high potential for research and development, and there is a great deal of room for research and development in medical applications. Summary of the Invention [Problem to be solved by the invention]

[0007] In the present invention, the Porphyra genus is dried and then extracted with alcohol to obtain an alcoholic extract. The alcoholic extract is then immersed in water, suspended in water, and then partitioned and extracted with ethyl acetate. Finally, the Porphyra genus ethyl acetate extract EE is obtained from the ethyl acetate layer.

[0008] In a neuroprotection experiment, the Porphyra spp. ethyl acetate extract EE could improve the survival rate of SH-SY5Y nerve cells damaged by 6-hydroxydopamine (6-OHDA). In addition, the Porphyra spp. ethyl acetate extract EE could improve the swimming ability of zebrafish with Parkinson's disease-like behavior. These results indicate that the Porphyra spp. ethyl acetate extract EE has a neuroprotective effect and can improve the symptoms of neurodegenerative diseases.

[0009] In bone formation experiments, the Porphyra genus ethyl acetate extract EE promoted an increase in the number of bone segments in zebrafish. In addition, the Porphyra genus ethyl acetate extract was able to improve the recovery rate of rat skull defects. These results indicate that the Porphyra genus ethyl acetate extract EE can be applied to the treatment of bone defects by promoting bone formation.

[0010] In atopic dermatitis experiments, the Porphyra genus ethyl acetate extract EE was able to improve the symptoms of atopic dermatitis in mice (e.g., symptoms such as erythema, edema, epidermal peeling and dryness). In addition, the Porphyra genus ethyl acetate extract EE was able to reduce the increased IgE expression level caused by atopic dermatitis. In addition, atopic dermatitis causes the spleen and lymph nodes to swell, but administration of the Porphyra genus ethyl acetate extract EE was able to improve the swelling of the spleen and lymph nodes. These results demonstrated that the Porphyra genus ethyl acetate extract EE can treat atopic dermatitis.

[0011] The term "a" or "a kind" is used in the present specification to describe the members and components of the present invention. This term is merely for convenience of description and to illustrate the basic idea of ​​the present invention. Such description should be interpreted as including one or at least one, and the singular includes the plural unless expressly stated otherwise. In addition, when used in conjunction with the term "comprising" in the claims, the term "a" may mean one or more than one.

[0012] As used herein in the claims, the term "or" means "and / or" unless otherwise expressly stated to be limited to alternative options or mutually exclusive of other options. [Means for solving the problem]

[0013] The present invention provides a method for producing a Porphyra extract, which includes the following steps: (a) extracting Porphyra with an alcoholic solvent to obtain an alcoholic Porphyra extract. The alcoholic solvent is methanol or ethanol. (b) distributively extracting the alcoholic Porphyra extract with an organic solution or supercritical extraction to form an organic solvent layer and an aqueous layer. Then, the organic solvent layer is collected to obtain the Porphyra extract. 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 Sarcodia genus is Sarcodia ceylanica.

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

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

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

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

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

[0020] In another embodiment, the Porphyra is extracted with an alcoholic solvent at room temperature. In a preferred embodiment, in step (a), the Porphyra is immersed in an alcoholic solvent and extracted three times. The three immersions are then combined, filtered, and concentrated under reduced pressure to obtain the Porphyra alcoholic extract.

[0021] In a specific embodiment, in step (b), the alcohol extract of the genus Porphyra is partition-extracted with the organic solution to form an organic solvent layer and an aqueous layer. Then, the organic solvent layer is collected to obtain the extract of the genus Porphyra. In a preferred specific embodiment, in step (b), the alcohol extract of the genus Porphyra is partition-extracted with the organic solution, and the partition-extraction operation is repeated three times to form an organic solvent layer and an aqueous layer. Then, the organic solvent layer is collected to obtain the extract of the genus Porphyra.

[0022] In another specific embodiment, the step (b) further includes a step (b1) before extraction with an organic solution. In the step (b1), the alcoholic extract of the genus Porphyra is immersed in water to obtain an aqueous solution of the genus Porphyra. In a preferred specific embodiment, in the step (b1), the alcoholic extract of the genus Porphyra is immersed in water to float the alcoholic extract of the genus Porphyra in water. After performing the step (b1), in the original step (b), the aqueous solution of the genus Porphyra obtained in the step (b1) is partition-extracted with the organic solvent without using the mixture of water and organic solvent. This forms an organic solvent layer and an aqueous layer, and the organic solvent layer is then collected to obtain the extract of the genus Porphyra. In another specific embodiment, in the step (b), the aqueous solution of the genus Porphyra obtained in the step (b1) is partition-extracted with the organic solvent to form an organic solvent layer and an aqueous layer. The organic solvent layer is then collected to obtain the extract of the genus Porphyra. In a preferred embodiment, in step (b), the aqueous solution of Porphyra obtained in step (b1) is partition-extracted with the organic solvent, and the partition-extraction operation is repeated three times to form an organic solvent layer and an aqueous layer, and then the organic solvent layer is collected to obtain the Porphyra extract.

[0023] After step (b1) is performed, in the original step (b), the aqueous solution of Porphyra obtained in step (b1) is partition-extracted by the supercritical extraction. In a specific embodiment, in step (b), the aqueous solution of Porphyra obtained in step (b1) is partition-extracted by the supercritical extraction to form an organic solvent layer and an aqueous layer. Then, the organic solvent layer is collected to obtain the Porphyra extract. In another specific embodiment, the supercritical extraction is supercritical CO2 extraction. In the process of separating nutritional components from natural products by supercritical CO2 extraction in the present invention, the nutritional components are extracted from natural products under high pressure and low temperature conditions mainly using carbon dioxide fluid in a supercritical state. In a specific embodiment, the supercritical CO2 extraction conditions are a flow rate ratio of CO2:99% ethanol solution=10mL / min:1mL / min, a critical pressure range of 150-400 bar, and a critical temperature range of 20-60°C. In a preferred embodiment, the supercritical CO2 extraction conditions are a flow rate ratio of CO2:95% ethanol solution of 10 mL / min:1 mL / min, a critical pressure of 250 bar, and a critical temperature of 40°C. Similarly, the supercritical CO2 extraction separates the raw material (aqueous layer) remaining in the supercritical CO2 extraction reaction vessel from the extracted material (organic solvent layer), and the organic solvent layer is collected to obtain the Porphyra extract. In another embodiment, in step (b), the Porphyra alcohol extract is extracted with supercritical CO2 extraction to obtain the Porphyra extract.

[0024] In another embodiment, the components of the Laminaria extract 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-(132S)-phaeophytin b. a) and 132-Hydroxy-(132R)-phaeophytin a. In a specific embodiment, the components of the Laminaria extract do not include polysaccharides.

[0025] The present invention further provides a use of the composition in the manufacture of a medicament for neuroprotection, the composition comprising a Porphyra extract, and a method for the manufacture of the Porphyra extract, comprising the steps of: (a) extracting Porphyra with an alcoholic solvent to obtain an alcoholic Porphyra extract, the alcoholic solvent being methanol or ethanol;

[0026] In a specific embodiment, the Porphyra in step (a) is subjected to a washing, drying and / or grinding process.

[0027] In another specific embodiment, the method for preparing the Porphyra extract further comprises step (b) after step (a). In step (b), the Porphyra alcohol extract is partition-extracted by organic solution or supercritical extraction to form an organic solvent layer and an aqueous layer. Then, the organic solvent layer is collected to obtain the Porphyra extract. 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 Porphyra extract further includes step (b1) before the extraction with the organic solution. In step (b1), the Porphyra alcoholic extract is immersed in water to obtain an aqueous Porphyra solution. In a preferred specific embodiment, in step (b), the aqueous Porphyra solution obtained in step (b1) is partition-extracted with the organic solvent to form an organic solvent layer and an aqueous layer. Then, the organic solvent layer is collected to obtain the Porphyra extract.

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

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

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

[0032] In the present invention, the Porphyra extract can be applied to provide neuroprotection. Neuroprotection means, for example, the ability to prevent or reduce death or damage of nerve cells (including neurons and glial cells) in conditions that may be pathologically harmful to the cerebrum, central nervous system, or peripheral nervous system, or the ability to rescue, resuscitate, or restore nerve cells. Neuroprotection includes regeneration of nerve cells, i.e., regrowth of nerve cells after disease or trauma. Neuroprotection is a mechanism and strategy in the nervous system to prevent cerebral / neuronal damage or degeneration caused by acute diseases (e.g., stroke, brain or nervous system injury / trauma, cerebral hypoxia, spinal cord injury, or peripheral nerve injury) or chronic neurodegenerative diseases (e.g., Parkinson's disease, Alzheimer's disease, or multiple sclerosis). The purpose of the neuroprotective effect is to prevent neuronal dysfunction / death after nervous system injury and to maintain as highly organized cellular interactions as possible in the cerebrum, so that neuronal function is not disturbed. The extract of the genus Caprifolius provides a neuroprotective effect, which allows the treatment of neuronal damage (e.g., stroke, in particular ischemic stroke, brain damage, 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 Caprifolius as an active ingredient in the manufacture of a medicament for protecting and / or regenerating nerve cells.

[0033] In a specific embodiment, the neuroprotection includes inhibiting or preventing neuronal cell death. In a preferred embodiment, the Laminaria extract is capable of inhibiting or preventing neuronal cell death.

[0034] Thus, the present invention has found that the Porphyra extract has several beneficial effects and functions in providing neuroprotection, and is therefore applicable to the treatment of neuronal or brain damage, and the treatment or prevention of neurodegenerative diseases, including 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 is reduced and / or ameliorated by treatment with the Laminaria extract. The term "acute neurodegenerative disease" refers to a sudden disease or condition, which results in associated neuronal death or damage. Examples of acute neurodegenerative diseases include, but are not limited to, cerebrovascular insufficiency, focal or diffuse brain trauma, spinal cord injury, cerebral ischemia or infarction (embolic occlusion and thrombotic occlusion), perinatal hypoxic-ischemia, neonatal hypoxia-ischaemic encephalopathy, perinatal asphyxia, cardiac arrest, intracranial hemorrhage, subarachnoid hemorrhage, stroke, and traumatic brain injury.

[0036] Other representative examples of neurodegenerative diseases that can be treated using the Porphyra 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, spinocerebellar degeneration type 3 (Machado-Joseph disease, MJD), dentatorubral pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA), and fragile X-associated tremor and ataxia syndrome (FXTAS).

[0037] In a specific embodiment, the Porphyra extract provides neuroprotection to prevent or treat neurodegenerative diseases. In a preferred specific embodiment, the neurodegenerative disease includes amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, frontotemporal dementia, spinocerebellar atrophy, spinocerebellar degeneration type 3, dentatorubral-pallidoluysian atrophy, spinal-bulbar 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" refers to the alleviation of symptoms or complications, the delaying of the progression of a disease, illness or condition, the reduction or alleviation of symptoms and complications, and / or the cure or elimination of a disease, illness or condition.

[0039] The term "prevention" as used herein refers to the prevention of the onset, recurrence or spread of a disease or condition, or one or more symptoms thereof. In any embodiment, these terms refer to the treatment of an individual at risk for a disease or condition, particularly as set forth herein, using a drug provided herein or administering a drug provided herein, prior to the onset of symptoms, in the alternative presence or absence of one or more other active agents.

[0040] The present invention further provides a use of the composition in the manufacture of a medicament for preventing or treating a neurodegenerative disease, the composition comprising a Porphyra extract, and a method for the manufacture of the Porphyra extract, the method comprising the steps of: (a) extracting Porphyra with an alcoholic solvent to obtain an alcoholic Porphyra extract, the alcoholic solvent being methanol or ethanol;

[0041] In a specific embodiment, the Porphyra in step (a) is subjected to a washing, drying and / or grinding process.

[0042] In another specific embodiment, the method for preparing the Porphyra extract further comprises step (b) after step (a). In step (b), the Porphyra alcohol extract is partition-extracted by organic solution or supercritical extraction to form an organic solvent layer and an aqueous layer. Then, the organic solvent layer is collected to obtain the Porphyra extract. 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 preparing the Porphyra extract further includes step (b1) before the extraction with the organic solution. In step (b1), the Porphyra alcoholic extract is immersed in water to obtain an aqueous Porphyra solution. In a preferred specific embodiment, in step (b), the aqueous Porphyra solution obtained in step (b1) is partition-extracted with the organic solvent to form an organic solvent layer and an aqueous layer. Then, the organic solvent layer is collected to obtain the Porphyra extract.

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

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

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

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

[0048] The present invention further provides a use of the composition in the manufacture of a medicament for promoting bone formation, the composition comprising a Porphyra extract, and a method for the manufacture of the Porphyra extract, the method comprising the steps of: (a) extracting Porphyra with an alcoholic solvent to obtain an alcoholic Porphyra extract, the alcoholic solvent being methanol or ethanol;

[0049] In a specific embodiment, the Porphyra in step (a) is subjected to a washing, drying and / or grinding process.

[0050] In another specific embodiment, the method for preparing the Porphyra extract further comprises step (b) after step (a). In step (b), the Porphyra alcohol extract is partition-extracted by organic solution or supercritical extraction to form an organic solvent layer and an aqueous layer. Then, the organic solvent layer is collected to obtain the Porphyra extract. 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 preparing the Porphyra extract further includes step (b1) before the extraction with the organic solution. In step (b1), the Porphyra alcoholic extract is immersed in water to obtain an aqueous Porphyra solution. In a preferred specific embodiment, in step (b), the aqueous Porphyra solution obtained in step (b1) is partition-extracted with the organic solvent to form an organic solvent layer and an aqueous layer. Then, the organic solvent layer is collected to obtain the Porphyra extract.

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

[0053] In a specific embodiment, the alcoholic solvent is ethanol. In a preferred specific embodiment, the alcoholic extract of Porphyra is an ethanol extract of Porphyra.

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

[0055] As used herein, the term "osteogenesis" refers to the proliferation of undifferentiated stem cells and osteoblasts into osteoblasts and bone tissue (e.g., synthesis and deposition of new bone matrix). Osteogenesis also refers to the differentiation or transdifferentiation of progenitor or precursor cells 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 bone lineage (e.g., pre-osteoblast cells) or cells that cannot form osteoblasts (e.g., pre-adipocytes or myoblasts).

[0056] Thus, the Porphyra extract of the present invention is applicable to the treatment of bone diseases, including (but not limited to) osteoporosis, bone loss or fracture diseases. Osteoporosis is a disease characterized by a net loss of bone mass per volume. This loss of bone mass and the associated fractures destroy the skeleton, which provides structural support for the body. Low bone mass and structural deterioration of bone tissues lead to bone fragility and increased hip, spine and wrist fractures. Osteoporosis is divided into primary osteoporosis and secondary osteoporosis according to the cause of onset. In the molecular mechanism of primary osteoporosis, the binding of receptor activator of nuclear factor kappa B ligand (RANKL) in osteoblasts and receptor activator of nuclear factor kappa B (RANK) on the surface of osteoclasts activates osteoclasts, stimulating their differentiation and activity, and promoting the development of osteoporosis. In addition, the rapid decrease in estrogen in the body of postmenopausal women also promotes osteoclast activity, making them more susceptible to osteoporosis and fractures. Secondary osteoporosis is caused by long-term drug use, poor lifestyle, endocrine disorders, and other diseases (e.g., rheumatoid arthritis, diabetes, stroke, Parkinson's disease, and bone metastasis from cancer). In addition, bone defect refers to an imbalance in the ratio of bone formation to bone resorption. This results in an individual's skeleton being formed below expectations or the skeleton of the individual being incomplete compared to expectations. Bone defect can also be caused by fracture, surgical intervention, or dental or periodontal disease. Bone healing includes (but is not limited to) the repair of bone defects. For example, bone defect occurs in non-open, open, and non-union fractures.

[0057] In a specific embodiment, the Laminaria extract prevents or treats bone disease by promoting bone formation. In a preferred embodiment, the bone disease includes osteoporosis, bone loss or fracture disease. In a more preferred embodiment, the bone disease includes bone loss.

[0058] The present invention further provides a use of the composition in the manufacture of a medicament for preventing or treating a bone disease, the composition comprising a Porphyra extract, and a method for the manufacture of the Porphyra extract, the method comprising the steps of: (a) extracting Porphyra with an alcoholic solvent to obtain an alcoholic Porphyra extract, the alcoholic solvent being methanol or ethanol;

[0059] In a specific embodiment, the Porphyra in step (a) is subjected to a washing, drying and / or grinding process.

[0060] In another specific embodiment, the method for preparing the Porphyra extract further comprises step (b) after step (a). In step (b), the Porphyra alcohol extract is partition-extracted by organic solution or supercritical extraction to form an organic solvent layer and an aqueous layer. Then, the organic solvent layer is collected to obtain the Porphyra extract. 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 preparing the Porphyra extract further includes step (b1) before the extraction with the organic solution. In step (b1), the Porphyra alcoholic extract is immersed in water to obtain an aqueous Porphyra solution. In a preferred specific embodiment, in step (b), the aqueous Porphyra solution obtained in step (b1) is partition-extracted with the organic solvent to form an organic solvent layer and an aqueous layer. Then, the organic solvent layer is collected to obtain the Porphyra extract.

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

[0063] In a specific embodiment, the alcoholic solvent is ethanol. In a preferred specific embodiment, the alcoholic extract of Porphyra is an ethanol extract of Porphyra.

[0064] In another embodiment, the organic solvent is ethyl acetate. In a preferred embodiment, the Corynebacterium extract is an ethyl acetate extract of Corynebacterium.

[0065] In the present invention, the Porphyra extract can treat bone disorders by increasing bone mass or promoting and repairing bone growth. In a specific embodiment, the bone disorders include osteoporosis, bone loss or fracture disorders. In a preferred embodiment, the bone disorders include bone loss.

[0066] The present invention further provides a use of the composition in the manufacture of a medicament for treating allergic dermatitis, the composition comprising a Porphyra extract, and a method for the manufacture of the Porphyra extract, comprising the steps of: (a) extracting Porphyra with an alcoholic solvent to obtain an alcoholic Porphyra extract, the alcoholic solvent being methanol or ethanol;

[0067] In a specific embodiment, the Porphyra in step (a) is subjected to a washing, drying and / or grinding process.

[0068] In another specific embodiment, the method for preparing the Porphyra extract further comprises step (b) after step (a). In step (b), the Porphyra alcohol extract is partition-extracted by organic solution or supercritical extraction to form an organic solvent layer and an aqueous layer. Then, the organic solvent layer is collected to obtain the Porphyra extract. 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 preparing the Porphyra extract further includes step (b1) before the extraction with the organic solution. In step (b1), the Porphyra alcoholic extract is immersed in water to obtain an aqueous Porphyra solution. In a preferred specific embodiment, in step (b), the aqueous Porphyra solution obtained in step (b1) is partition-extracted with the organic solvent to form an organic solvent layer and an aqueous layer. Then, the organic solvent layer is collected to obtain the Porphyra extract.

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

[0071] In a specific embodiment, the alcoholic solvent is ethanol. In a preferred specific embodiment, the alcoholic extract of Porphyra is an ethanol extract of Porphyra.

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

[0073] The term "allergic dermatitis" as used herein is 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 and atopic dermatitis. "Contact dermatitis" refers to an eczematous inflammatory disease caused by contact of a foreign antigen with the skin, including allergic contact dermatitis, photocontact dermatitis, systemic contact dermatitis and contact urticaria. 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. It is also characterized by symmetrical generalized eczema that repeatedly worsens and improves, and examples of this include diffuse neurodermatitis, atopic eczema, atopic neurodermatitis, Besnier prurigo, acute infantile eczema, flexural eczema, childhood extremity eczema, childhood atopic eczema, pediatric dry eczema, childhood eczema, adult atopic dermatitis, endogenous eczema, childhood dermatitis, and chronic childhood eczema.

[0074] In a specific embodiment, the allergic dermatitis includes contact dermatitis or atopic dermatitis. In a preferred embodiment, the allergic dermatitis includes atopic dermatitis. In a more preferred embodiment, the atopic dermatitis includes diffuse neurodermatitis, atopic eczema, atopic neurodermatitis, prurigo venier, acute infantile eczema, flexural eczema, pediatric extremity eczema, pediatric atopic eczema, pediatric dry eczema, pediatric eczema, adult atopic dermatitis, endogenous eczema, pediatric dermatitis, or chronic pediatric eczema.

[0075] In another specific embodiment, the medicament suppresses symptoms of atopic dermatitis, including erythema, edema, epidermal peeling, or dry skin.

[0076] In a specific embodiment, the medicament reduces the increase in IgE expression resulting from the atopic dermatitis.

[0077] In another specific embodiment, the drug ameliorates spleen or lymph node enlargement resulting from the atopic dermatitis.

[0078] In a specific embodiment, the drug further comprises a pharma- ceutically acceptable carrier. The term "pharma- ceutically acceptable carrier" as used herein is determined by the composition to be applied in a particular combination and in a particular manner. The term "carrier" as used herein includes, but is not limited to, any and all solvents, dispersion media, vehicles, coatings, diluents, penetration and absorption retardants such as antibacterial and antifungal agents, buffers, carrier solutions, suspensions or colloids, and the like. These media and agents used with the active substance of the drug are known in the art. Unless there is incompatibility between any of the common media or agents and the active ingredient, a combination for treatment should be considered. A supplementary active ingredient may also be mixed into the composition. The term "pharma- ceutically acceptable" refers to a molecular entity and composition that does not cause an allergic or similar side reaction when applied to a subject. The preparation of aqueous compositions with proteins as active substances is well known in the art. Usually, the compositions are prepared as liquids, solutions, tablets, capsules, or suspension injections. It may also be prepared as a soluble solid or a solid suspension that can be used as an injection. In another specific embodiment, the pharma- ceutical acceptable carrier includes a dermatologically acceptable medium. By "dermatologically acceptable medium" is meant a biologically suitable substance, such as, for example, a salt, ester, and / or acid amide component. That is, the substance, when used in conjunction with the selected active ingredient, does not cause undesired biological effects when administered to an individual. The substance is also a substance that does not adversely interact with any of the components in the pharmaceutical composition that contains the substance. Similarly, a "dermatologically acceptable salt" or a "dermatologically acceptable ester" in the text is a biologically suitable salt or ester.

[0079] In the present invention, the composition (including the Lavera extract) and the pharmaeutically acceptable carrier may be formulated as a sterile aqueous solution or dispersion, water suspension, oil emulsion, water in oil emulsion, point emulsion, long-term emulsion, viscous emulsion, microemulsion, nanoemulsion, liposome, microparticle, microsphere, nanosphere, nanoparticle, micromercury, and several natural or synthetic polymers for sustained release. The pharmaeutically acceptable carrier and the Lavera extract may be formulated as an aerosol, tablet, pill, capsule, sterile powder, suppository, detergent, cream, ointment, paste, gel, hydrogel, or other formulation that can be used to deliver the composition.

[0080] The composition as referred to in the present invention also includes compositions that are administered locally and regionally to act. The term "topical" as used herein refers to the use of the compositions described herein, mixed with a suitable pharmaceutical carrier, and applied to a skin site (e.g., an area affected by allergic dermatitis) to achieve a localized action. Thus, such topical compositions include drug forms such as compounds that are applied externally in direct contact with the surface of the skin to be treated. Common drug forms for this purpose include ointments, rubs, creams, shampoos, emulsions, pastes, gels, sprays, aerosols, etc., and may also be used in the form of patches or wet dressings depending on the body site to be treated. The term "ointment" includes preparations (including creams) with oily bases, water-soluble bases, and emulsion bases. The bases are, for example, petrolatum, lanolin, polyethylene glycols, and mixtures of these.

[0081] The medicament of the present invention further comprises a pharma- ceutically acceptable carrier and can be administered to an individual in a number of different ways, according to treatment modalities well known in the art to which the present invention pertains. In some embodiments, the composition (including the Laminaria extract) and a pharma- ceutically acceptable carrier are administered topically, intravenously, intramuscularly, subcutaneously, locally, orally, or by inhalation. The medicament can also be delivered to the target site via 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 dose" in this context refers to a therapeutic dose that can prevent, reduce, arrest or reverse the progression of symptoms in an individual in a particular condition, or partially or completely alleviate symptoms that existed under a particular circumstance when the individual began to receive treatment. A person skilled in the art can easily determine the appropriate dosage and method of administration of the Porphyra extract to an individual. For example, the Porphyra extract can be administered to the individual once or twice. The dosage and method of administration includes being able to understand the effective dosage of the Porphyra extract to be administered to the individual when administered multiple times, including the total amount of product administered during the entire dosage and method of administration.

[0084] Thus, the Porphyra extract of the present invention has different effective doses when applied to neuroprotection, promotion of bone formation, and treatment of allergic dermatitis.

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

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

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

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

[0089] [Figure 1] Figure 1 shows the extraction flow of Porphyra genus. [Diagram 2] Figure 2 shows the protective effect of the Porphyra ethyl acetate extract EE on nerve cells. SH-SY5Y nerve cells were treated with different concentrations of the Porphyra ethyl acetate extract EE, then the cells were injured with 6-hydroxydopamine (6-OHDA), and the cell viability was measured to observe the neuroprotective effect. The Porphyra ethyl acetate extract EE improved the cell viability of each group at concentrations of 0.5 to 500 ng / ml. This demonstrated that the Porphyra ethyl acetate extract EE has a neuroprotective effect. Statistical analysis was performed on each group with the 6-OHDA group, and significant differences (P<0.05) are indicated with an *. [Diagram 3]FIG. 3 shows the protective effect of the ethyl acetate extract of Laminaria genus EE on nerves in vivo. In the figure, (A) shows the swimming path of each group of zebrafish in a unit time. (B) shows the swimming speed of the zebrafish, in which 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 the ethyl acetate extract of Laminaria genus EE was 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. (C) shows the total swimming distance of the zebrafish in 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 the ethyl acetate extract of Porphyra spp. EE was added at concentrations of 0.5 μg / ml and 5 μg / ml, the swimming distance of the zebrafish was restored to 609.03 ± 196.17 mm and 371.52 ± 82.77 mm, respectively. [Figure 4] FIG. 4 shows the effect of the Porphyra spp. ethyl acetate extract EE on bone formation. In the figure, (A) shows the control group, and (B) to (E) show groups administered with different doses of the Porphyra spp. ethyl acetate extract EE (0.05 to 50 μg / ml), respectively. (F) shows the quantitative statistical results of the number of bone segments for each group of zebrafish. For each group of zebrafish, the number of bone segments was calculated on the 7th day after fertilization, and when 0.5 μg / ml of the Porphyra spp. ethyl acetate extract EE was added, the development of bone segments was significantly improved. In addition, the Porphyra spp. ethyl acetate extract did not cause developmental abnormalities in zebrafish, which almost demonstrated that the Porphyra spp. ethyl acetate extract was not toxic to the development of the embryo proper. *: P<0.05 compared to the control group. [Diagram 5] FIG. 5 is a comparative diagram of skull appearance showing the therapeutic effect of different concentrations of Laminaria ethyl acetate extract EE on skull defects in rats. [Figure 6]Figure 6 is a comparative diagram of the therapeutic effect of the ethyl acetate extract EE of the Laminaria genus on the skull defects of rats photographed with an X-ray device. In the figure, (A) shows the X-ray image of the skull defect of rats in each group. (B) shows the skull recovery rate of rats in each group. [Figure 7] FIG. 7 shows the effect of the ethyl acetate extract EE of the Corynebacterium genus on the skin appearance of mice suffering from atopic dermatitis. (A) in the figure shows the photographs of the skin appearance of mice with DNCB-induced atopic dermatitis on the 1st, 7th, and 12th days of treatment after inducing atopic dermatitis with DNCB and starting daily drug application after the symptoms of atopic dermatitis appeared on the skin of the mice. The scale bar is 1 cm. As a result, compared with the AD group, both the EEL group (low dose group) and the EEH group (high dose group) had the effect of mitigating the symptoms of atopic dermatitis, and all phenomena such as epidermal edema, redness, dryness, and scabs were alleviated. (B) in the figure shows the clinical dermatitis score of AD symptom alleviation by the ethyl acetate extract EE of the Corynebacterium genus. The alleviation of AD symptoms by EE was evaluated by the clinical dermatitis score. The severity of dermatitis was evaluated using four skin surface symptoms: erythema / hemorrhage, edema, epidermal peeling / erosion, and dryness. Each symptom was scored from 0 to 3 according to its severity, with higher scores indicating more severe symptoms. The total dermatitis score was the sum of the four scores above, with a maximum of 12 points. The higher the score, the more severe the dermatitis. Data are shown as mean ± standard error of the mean (mean ± SEM). Significant differences compared to the AD group (P < 0.05) are indicated with *, and significant differences compared to the ADV group are indicated with #. [Figure 8] Figure 8 shows the effect of the ethyl acetate extract of Laminaria spp. EE on serum IgE. Blood was collected before the animals were sacrificed, and IgE in the serum was detected. IgE increased due to DNCB-induced AD, but it decreased when Laminaria spp. EE was applied. Data are shown as mean ± standard error of the mean (mean ± SEM). Significant differences compared to the AD group (P < 0.05) are indicated with *, and significant differences compared to the ADV group are indicated with #. [Figure 9] Figure 9 shows the effect of the ethyl acetate extract of Lavera genus EE on DNCB-induced spleen weight. Spleen tissue was collected at the time of sacrificing the animals, and was photographed and weighed. DNCB-induced atopic dermatitis caused splenic tissue swelling, but application of the ethyl acetate extract of Lavera genus EE reduced spleen weight. Scale bar = 1 cm. Data are shown as mean ± standard error of the mean (mean ± SEM). Significant differences compared to the AD group (P < 0.05) are indicated with *, and significant differences compared to the ADV group are indicated with #. [Figure 10] Figure 10 shows the effect of the Porphyra spp. ethyl acetate extract EE on the weight of DNCB-induced swollen lymph nodes. When the animals were sacrificed, lymph node tissue was collected, photographed, and weighed. DNCB-induced atopic dermatitis caused lymph node tissue swelling, but application of the Porphyra spp. extract EE reduced lymph node weight. Scale bar = 0.5 cm. Data are shown as mean ± standard error of the mean (mean ± SEM). Significant differences compared to the AD group (P < 0.05) are indicated with *, and significant differences compared to the ADV group are indicated with #. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] The present invention includes, but is not limited to, the above and following descriptions. Methods of implementation are illustrated in the following representative examples.

[0091] Extraction of cultivated Porphyra spp. In the present invention, artificially cultivated Sarcodia ceylanica was used.

[0092] The dried Porphyra was obtained by drying the Porphyra for 24 hours under the condition of 50°C. 10.8 kg of the dried Porphyra was crushed and filtered through a 50 mesh filter net, and then immersed in a 95% alcohol solution at room temperature for three extractions. The three immersion solutions were then combined and filtered, and concentrated under reduced pressure to obtain 370.5 g of Porphyra crude alcohol extract. Next, the Porphyra crude extract was added to 1 L of water to form a suspension, and then 1 L of ethyl acetate was added to perform partition extraction. The partition extraction was repeated three times to form an ethyl acetate layer and an aqueous layer. The ethyl acetate layer was then collected to obtain ethyl acetate extract EE (83.2 g). The entire extraction flow of Porphyra is shown in Figure 1.

[0093] In addition, 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, it is possible to obtain an extract containing the same components as EE by using propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, ethyl ether, dichloromethane, chloroform or carbon tetrachloride solvent for partition extraction or by supercritical extraction (e.g., supercritical CO2 extraction).

[0094] Alternatively, water may be mixed with ethyl acetate or other organic solvent to prepare an organic solution, and the crude alcohol extract of the Corynebacterium spp. may be directly extracted. In this case, it is not necessary to extract the crude alcohol extract of the Corynebacterium spp. separately in two steps using water and an organic solvent. In addition, the step of soaking in water in advance may be omitted, and the crude alcohol extract of the Corynebacterium spp. may be directly extracted using ethyl acetate or other organic solvent. By changing the above steps, it is possible to obtain an extract containing the same components as EE.

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

[0096] In addition, in the present invention, the polysaccharide content of EE was measured by the phenol-sulfuric acid method, and it was found that EE did not contain any polysaccharide components. Therefore, the polysaccharide components were not eluted by the extraction method of pre-soaking the Porphyra genus in alcohol, and no polysaccharide components were contained in the crude alcohol extract of Porphyra genus. In addition, in the present invention, the components of the ethyl acetate extract EE of Porphyra genus were also analyzed. The analysis results are shown in Table 1.

[0097] [Table 1]

[0098] Experimental Method (1) Evaluation of the neuroprotective effect of EE (a) Neuronal test SH-SY5Y cells were cultured in a 96-well cell culture plate at a cell density of 2 x 104 cells and then cultured in a cell incubator. After the cells were completely attached, they were pretreated with various concentrations of ethyl acetate extract of Lavera spp. EE solution for 1 hour. Then, 6-hydroxydopamine (6-OHDA) was added and treated for 15 hours, and 10% alamar blue was added and cultured for another 3 hours. The absorbance was measured using an enzyme immunoassay device to measure the cell viability. We analyzed whether Lavera spp. extract has a protective effect on 6-OHDA-treated neurons against the toxicity caused by 6-OHDA.

[0099] (b) In vivo neuronal testing 6-OHDA was dissolved in 2% fresh L-ascorbic acid to prepare a 500 mM stock solution, which was then placed in a 1.5 mL brown microtube and divided into 10 μL tubes. When adding drugs, the mixed drugs were dissolved in Hank's buffer in the same ratio based on the concentration of the experimental design. Then, 1 mL of the drug and 1 mL of Hank's buffer containing juvenile fish were added to a 24-well culture plate. The time when 6-OHDA was given to the juvenile fish was between 48 and 120 hours after fertilization. In addition, no drugs were added to the control group, and a solvent group was set up as a control for each experiment. The behavioral ability of zebrafish was analyzed to measure parameters such as the time spent in each water layer, swimming speed, and swimming distance of the fish, and the therapeutic effect of the Laver extract was evaluated.

[0100] (2) Evaluation of the effect of EE on bone formation (a) Zebrafish bone growth test Using a living system, the promoting effect of the Porphyra extract solution on bone regeneration was tested. The effect of the Porphyra extract on bone and bone quality-related diseases (e.g., osteoporosis, etc.) was also analyzed. The effect of the active substance on bone growth was analyzed by exclusively staining the hard bone part of the bone with the fluorescent dye calcein and combining it with the developmental morphology of the zebrafish embryo. In addition, the effect of the substance on bone growth was presented by digitalizing it using analysis software. Zebrafish larvae were obtained 48 hours after fertilization, and different concentrations of Porphyra extract were added, and the vertebral joints of the fish were observed every day, and the fish were continued to be raised after the observation. The zebrafish were stained with 2g / L calcein solution (pH=7.8) for 10 minutes, and then rinsed with purified water to wash off excess dye. The fish were then anesthetized with MS-222 and placed in 1% methyl cellulose to fix them. The vertebrae of the fish were then observed under a fluorescent microscope, and the number of skeletal segments was statistically analyzed.

[0101] (b) Skull defect experiment in rats Skull Defect Surgery: Small bone defects can be naturally regenerated in vivo. However, if the defect size exceeds the size that can be repaired in the body, it becomes a so-called critical sized bone defect that cannot be completely healed by itself, and cannot heal without external treatment. Differences in animal models and sites all affect the size of the defect to be constructed. In the rat skull defect model used in the present invention, the animals were first anesthetized using isoflurane, a gas anesthetic. Next, the top of the rat's head was shaved, the surgical site was disinfected with povidone iodine and 70% alcohol, and ampicillin was injected. Next, a hole was made in a sterilized paper towel and 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 to a size of about 1.5 cm, and the skull and periosteum were separated with a periosteum scraper. The protuberance exactly in the center of the skull is the midline sagittal suture, and both sides of it are the parietal bones. Two bone defects, each 4 mm in diameter, were created using a 4 mm diameter trepan in the exact center of the sagittal suture. The periosteum was then sutured with 5-0 catgut, and the skin was sutured with 4-0 nylon thread. If the animals woke up from anesthesia during surgery, they were maintained under anesthesia by inhaling anesthetic gas through an oxygen mask using a gas anesthesia machine. The rats were administered EE three times a week under anesthesia. The doses were 1.5 mg / kg / week and 7.5 mg / kg / week. After 4 weeks of administration, the animals were sacrificed and cranial tissue was collected.

[0102] The rat skull healing status was evaluated by taking X-ray images (X-OMAT V, Kodak, Rochester, NY) and developing the negative film with an automatic processor. The images were scanned and converted to TIF files, and then loaded into ImageQuant software (ImageQuant Version 5.2 Copyright 1999, Molecular Dynamics) for further analysis. Next, in the ImagQuant software, equal-area areas of each step wedge and blank area were selected to express the relationship between the grayscale intensity of the image on the X-ray film and the thickness of the wedge-shaped step. Then, a circular area with a diameter of 8 mm in the skull area and a flat area with an area of ​​1 × 1 mm2 in the center of the parietal bone on both sides were selected, and the grayscale intensity was measured and substituted into the above equation. Thus, the bone density of the sample relative to the thickness of the wedge-shaped step was calculated by interpolation. In other words, the bone density of the bone defect area of ​​the skull was defined as the density of the grayscale 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 this invention were BALB / c mice. After anesthetizing the mice with 2.5% isoflurane, the hair on the back of the mice was removed with a shaver and hair removal cream, and a mark with silicone rubber of 1 cm in diameter (area 78.5 mm2) was made. Next, 26 μL of 2% 2,4-dinitirochlorobenzene (DNCB) (Sigma-Aldrich, Missouri, USA, Cat. No. #138630) was dripped into the above area once every two days. This was repeated a total of eight times to induce skin lesions.

[0104] Seven days after DNCB induction, 100 μL of vehicle, Laminaria extract EE, or crisaborole, a treatment for atopic dermatitis, was applied daily. The animals were divided into the following groups (3 animals per group): (1) control group: uninduced, (2) AD group: administered 2% DNCB only, (3) ADV group: administered 2% DNCB and vehicle, (4) EEL group: administered 50 μg EE / 100 μL 1% methylcellulose after DNCB induction, (5) EEH group: administered 200 μg EE / 100 μL 1% methylcellulose after DNCB induction, and (6) Cri group: administered 25 μg crisaborole / 100 μL 1% acetone-EtOH after DNCB induction. Five minutes after DNCB application, the DNCB was allowed to dry before applying the medication. Before induction or drug application, changes in skin symptoms were photographed and recorded using a digital camera.

[0105] On the 18th day, the animals were sacrificed, blood was collected, and tissues of the dorsal skin, spleen, and lymph nodes were collected. The size of the spleen and subiliac lymph nodes were observed, measured, and compared. The severity of clinical dermatitis was evaluated based on four symptoms on the skin surface: erythema / hemorrhage, edema, excoriation / erosion, and dryness. Each symptom was scored from 0 to 3 according to its severity, with 0 being no symptoms, 1 being mild, 2 being moderate, and 3 being severe. The total score of dermatitis was calculated by adding up the four scores above, with a maximum of 12 points. The higher the score, the more severe the dermatitis.

[0106] Before sacrifice, blood was collected from the mice by cardiac blood collection into blood collection tubes (BD vacutainer-SST, New Jersey, USA). Blood was then centrifuged at 3000 rpm for 10 min to obtain serum. Serum was stored in a refrigerator at -80°C until further use. IgE concentrations in serum were then detected (IgE-ELISA kit, Thermo Fisher Scientific, Vienna, Austria) according to the manufacturer's instructions.

[0107] In the event of inflammation or immune-related disease, the lymph nodes and spleen will swell, so at the time of sacrifice, these two organs were harvested, photographed, and weighed.

[0108] statistical analysis All experimental data are shown as mean ± standard error of the mean (mean ± SEM). To compare data between multiple groups, one-way analysis of variance (ANOVA) was used for statistical analysis. In addition, Duncan's method was used for multiple comparison of differences between groups. A P value of less than 0.05 was considered to indicate a significant difference between groups.

[0109] result Neuroprotective effect of EE We added different concentrations of the ethyl acetate extract EE of the Corynebacterium genus to SH-SY5Y neuronal cells, then injured the cells with 6-hydroxydopamine (6-OHDA), and finally measured the cell viability to observe the neuroprotective effect. The cell viability of the control group was 100±3.63%, while that of the 6-OHDA group decreased to 0±3.88%. However, when the ethyl acetate extract EE of the Corynebacterium genus was added at concentrations of 0.5 to 500 ng / ml, the cell viability of each group was 10.72±2.86%, 33.91±19.67%, 7.92±1.93%, and 32.69±17.88%, respectively (see Figure 2), and all of them were improved. These results proved that the ethyl acetate extract EE of the Corynebacterium genus has a neuroprotective effect.

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

[0111] (2) Effect of EE on bone formation The ethyl acetate extract of Porphyra EE could promote bone formation. In a zebrafish model, the control group juveniles formed 10±0.63 bone segments, whereas the Porphyra EE groups at concentrations of 0.05-50μg / ml increased the number of bone segments to 13.8±0.80, 15.33±0.68, 15.00±0.84, and 15.4±0.68, respectively (see Figure 4).

[0112] In the rat skull defect experiment, EE was administered three times a week. The doses were 1.5 mg / kg / week and 7.5 mg / kg / week. After 4 weeks, the animals were sacrificed, and the skull tissue was collected and the area was quantified using images taken with an X-ray device. Figure 5 is a comparison diagram of the skull appearance showing the therapeutic effect of different concentrations of the ethyl acetate extract of Laminaria genus EE on the skull defect in rats. In addition, Figure 6 (A) shows an X-ray diagram of the skull defect state of rats in each group. Figure 6 (B) shows the skull recovery rate of rats in each group. As is clear from the results, the skull recovery rate of the control group (medium solution) was 51.90 ± 10.01%, while the skull recovery rates of the rats administered 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 Symptoms of DNCB-induced atopic dermatitis (AD) in mice included erythema, edema, epidermal peeling, dryness, and lichenification. After the mice showed obvious symptoms such as erythema, edema, epidermal peeling, and dryness, EE or crisaborole was applied daily to test whether EE has a therapeutic effect on atopic dermatitis. The symptoms of the mouse skin appearance were observed, photographed, and recorded (see Figure 7A), and the therapeutic effect of EE was quantified by clinical dermatitis score (see Figure 7B). The control group had a dry skin surface due to a lack of hair, and the score was 0.75 ± 0.25. The AD and ADV groups showed proliferation of keratinocytes on the skin surface, forming thick scabs. As the days went by, some of the scabs peeled off, and dryness of the skin around the induction was observed. In addition, wrinkles were observed on the skin. In addition, inflammation caused erythema and edema on the skin. However, keratinocyte proliferation continued, and scabs were also present on the skin surface. The clinical dermatitis scores of these two groups were significantly increased compared to the control group, reaching 9.17±0.40 and 9.50±0.56, respectively. In contrast, the EE and Crisaborole (Cri) groups showed dryness, erythema, and edema after the scabs peeled off, but the degree of keratinocyte proliferation was less than that of the AD group. Therefore, before the animals were sacrificed, the dermatitis scores of the EEL and EEH groups were significantly decreased compared to the AD group (4.50±0.76 and 3.67±0.49). Meanwhile, 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 an effect of alleviating AD symptoms.

[0114] IgE is one of the clinical diagnostic indicators of atopic dermatitis. Before the mice were sacrificed, serum was collected and the IgE content was measured, and the results are shown in Figure 8. The IgE content in serum of the control group was 1.71 ± 0.14 μg / mL. In addition, the IgE concentration in the AD and ADV groups was obviously increased (68.89 ± 4.12 μg / mL and 68.95 ± 2.60 μg / mL). In contrast, the IgE concentration in the EEL and EEH groups was only slightly decreased, to 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 of the Cri group was decreased to 65.61 ± 1.74 μg / mL. The serum IgE level, which increased with atopic dermatitis, could be decreased by applying EE.

[0115] The results of spleen weight are shown in Figure 9. The spleen weight of the control group was 91.08 ± 3.61 mg. The spleens of the AD and ADV groups were obviously enlarged, becoming 2.5 times larger than that of the control group (234.46 ± 6.56 mg and 221.14 ± 13.32 mg). In contrast, the spleen weights of the EEL and EEH groups were significantly decreased, becoming 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, becoming 167.46 ± 10.61 mg.

[0116] The results of the total weight of 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 and ADV groups were obviously enlarged, three times that of the control group (12.07 ± 0.33 mg and 11.61 ± 0.54 mg). In contrast, the lymph node weights in the EEL and EEH groups were significantly reduced to 9.10 ± 0.68 mg and 6.50 ± 0.59 mg, respectively. This was dose dependent. On the other hand, the lymph node weight in the Cri group was significantly reduced to 9.49 ± 0.81 mg. From the above results, it was possible to suppress the above phenomenon in the lymph nodes and spleen that were enlarged due to inflammation or immune reaction caused by atopic dermatitis by applying EE.

[0117] It is properly described that the present invention can be carried out under conditions or restrictions not specifically disclosed herein. The terms used for description are not intended to be limiting. 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 can be modified. Therefore, although the present invention has been described in the embodiments and other situations, the contents disclosed in the present invention can be supplemented and modified by those skilled in the art, and such modifications and modifications are considered to be within the scope of the present invention.

Claims

1. A use of a composition in the manufacture of a medicament for treating allergic dermatitis, 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 a 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. 2. The use according to claim 1, wherein the allergic dermatitis includes atopic dermatitis.

3. The use according to claim 2, wherein the drug reduces the increase in IgE expression resulting from the atopic dermatitis.

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