JP7336656B2 - Spirulina genus extract and use thereof - Google Patents

Description

The present invention relates to a novel use of the extract of the genus Spirulina, in particular in the manufacture of a medicament for neuroprotection, promotion of bone formation and treatment of allergic dermatitis.

The genus Sarcodia is mainly distributed in the Indian Ocean and the western Pacific region. The genus Spirulina is a heterotrophic marine macroalgae with a high content of micronutrients. Therefore, in Japan, it is also called a longevity food, and in Europe and the United States, the genus Atsubanori is regarded as a representative of sea vegetables. At present, most of the uses of the genus Melanophyllus are edible as it is or as a food additive. The genus Aspen has a long history as food, but there are very few studies on its effects.

The skeleton is a rigid organ that constitutes the endoskeleton of vertebrates, and contains biomineralized skeletal tissue, bone marrow, periosteum, nerves, blood vessels, soft tissues such as cartilage, and a small number of skeletal cells. Skeletal defects or defects may result from congenital defects, acquired diseases, old age or trauma. A small bone defect can be spontaneously fused, but a large bone defect or a small skeletal bone defect is difficult to fuse completely. Therefore, it needs to be treated by surgery such as bone graft (including autogenous bone graft, allogenic bone graft and xenogenic bone graft), artificial bone (including bone cement and bioceramics), tissue-engineered bone, or bone transfer surgery. In other words, it is necessary to administer different drugs or perform different surgeries for various bone diseases with different factors (eg, osteoporosis, bone defect, fracture disease, etc.).

In addition, as the global population ages, the prevalence of neurodegenerative diseases continues to increase, increasing the impairment of cognitive, memory and motor skills in the elderly. According to reports, the World Health Organization predicts that neurodegenerative diseases that affect motor function will become the second leading cause of death within the next 20 years. Therefore, the development of therapeutic methods for this kind of disease has become one of the important research projects in biomedical nowadays. The expression mechanisms of many neurodegenerative diseases have not yet been elucidated, and effective therapeutic methods are currently lacking for these diseases. Also, available treatments are limited to symptom control and inhibition of disease progression.

On the other hand, atopic dermatitis is a chronic disease in which immunity and inflammation are deeply involved. Atopic dermatitis is an itchy, inflammatory skin disease as well as a chronic disease that usually begins in infancy. Atopic dermatitis has persistent itching as its main symptom, and has the property of repeating recovery and exacerbation without a specific cause. Although many studies have recently been conducted on atopic dermatitis, the cause of atopic dermatitis is not clear at present.

By the way, natural compounds purified from seaweed have been demonstrated to have numerous biological activities such as anticoagulant, antiviral, antioxidant, antiallergic, anticancer, antiinflammatory and antiobesity. Therefore, the genus Aspen, which is a type of seaweed, also has a very high potential for research and development, and there is a lot of room for research and development in medical applications.

In the present invention, the alcoholic extract is obtained by drying the genus Medicinalum and then extracting it with alcohol. Next, the alcoholic extract is immersed in water, allowed to float, and then partition-extracted with ethyl acetate. Finally, the Ethyl Acetate Extract EE from the Ethyl Acetate layer is obtained.

In a neuroprotection experiment, the Spirulina ethyl acetate extract EE was able to improve the viability of SH-SY5Y neurons injured by 6-hydroxydopamine (6-OHDA). And the Typophylla ethyl acetate extract EE was able to improve swimming ability for zebrafish with Parkinson's disease-like behavior. These results indicate that the Ethyl Acetate Extract EE of the genus Typoracea has a neuroprotective effect and can improve the symptoms of neurodegenerative diseases.

In osteogenesis experiments, the Apocynaceae ethyl acetate extract EE promoted an increase in the number of phalanges in zebrafish. In addition, the Ethyl acetate extract of the genus Typoracea was able to improve the recovery rate of cranial defects in rats. This result indicated that the Ethyl Acetate Extract EE of the genus Typophylla can be applied to the treatment of bone defects by promoting osteogenesis.

In an atopic dermatitis experiment, the Typophyllus ethyl acetate extract EE was able to improve symptoms of atopic dermatitis in mice (for example, symptoms such as erythema, edema, desquamation and dryness). In addition, the Spirulina ethyl acetate extract EE was able to reduce the increased expression of IgE due to atopic dermatitis. In addition, atopic dermatitis causes enlargement of the spleen and lymph nodes, and the administration of the Spirulina ethyl acetate extract EE was able to ameliorate swelling of the spleen and lymph nodes. This result indicated that the Ethyl Acetate Extract EE of the genus Typophyll can treat atopic dermatitis.

The terms "a" or "a" are used herein to describe members and components of the present invention. This term is only for convenience of description and to indicate the basic idea of the invention. Such descriptions should be read to include one or at least one and the singular also includes the plural unless expressly specified otherwise. Also, when used in conjunction with the term "comprising" in the claims, the term "a" can mean one or more than one.

As used herein in the claims, the term "or" means "and/or." However, this excludes cases where it is clearly indicated separately that it is limited to another choice, or where it is mutually exclusive with other choices.

The present invention provides a method for producing an extract of the genus Aspen. The method includes the following steps. That is, (a) extracting the genus Meliflora with an alcohol-based solvent to obtain an alcohol-based extract of the genus Meliflora. The alcoholic solvent is methanol or ethanol. (b) partitioning and extracting the alcoholic extract of the genus Medicinalum 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 Medicinalum. 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.

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

In a specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the alcoholic extract of the genus Meliflora is an ethanolic extract of the genus Medicinalum.

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

In a specific embodiment, the method includes step (a1) before step (a). In the step (a1), the Spirulina genus is dried. In a preferred specific embodiment, the Spirulina is dried under a temperature of 20-70°C. In a more preferred specific embodiment, the Spirulina is dried under a temperature of 40-60°C. In another specific embodiment, the Spirulina is dried under a temperature of 50°C.

In a specific embodiment, the drying time for the Spirulina is 12-48 hours. In a preferred embodiment, the drying time for the Medicinal plant is 16-32 hours. In a preferred embodiment, the drying time for the Medicinal plant is 20-24 hours.

In the present invention, after drying the genus Dispensary, it is filtered through a filtration net having a predetermined mesh pore size, and extracted with ethanol after filtration. In a specific embodiment, the method includes step (a2) prior to step (a). In the step (a2), the Spirulina is filtered through a filter net with a pore size of 30 to 70 mesh. In a preferred embodiment, the method comprises step (a2) prior to step (a). In the step (a2), the Spirulina is filtered through a filter net with a pore size of 40 to 60 mesh. In a more preferred embodiment, the method comprises step (a2) before said step (a). In the step (a2), the Spirulina is filtered through a filter net with a pore size of 50 mesh.

In another specific embodiment, the Spirulina is extracted with an alcoholic solvent at room temperature. In a preferred embodiment, in step (a), the Spirulina is immersed in an alcoholic solvent and extracted three times. Then, the immersion liquid for three times is combined, filtered, and concentrated under reduced pressure to obtain the alcoholic extract of the genus Medicinalum.

In a specific embodiment, step (b) comprises partitioning and extracting said Medicinal alcohol-based extract with said organic solution to form an organic solvent layer and an aqueous layer. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum. In a preferred embodiment, in step (b), the alcoholic extract of the genus Medicinalum is subjected to partition extraction with the organic solution, and the partition extraction operation is repeated three times to form an organic solvent layer and an aqueous layer. . Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum.

In another specific embodiment, said step (b) further comprises step (b1) prior to the extraction with the organic solution. In the step (b1), the Spirulina genus alcoholic extract is immersed in water to obtain a Spirulina genus aqueous solution. In a preferred specific embodiment, in said step (b1), said Meliflora alcoholic extract is immersed in water to float said Meliflora alcoholic extract in water. After step (b1) is performed, in the original step (b), the Medicinal genus aqueous solution obtained in step (b1) is partition-extracted with the organic solvent without using the mixture of water and the organic solvent. . As a result, an organic solvent layer and an aqueous layer are formed, and then the extract of the genus Meliformis is obtained by collecting the organic solvent layer. In another specific embodiment, step (b) forms an organic solvent layer and an aqueous layer by subjecting the Apocynaceae genus aqueous solution obtained in step (b1) to partition extraction with the organic solvent. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum. In a preferred specific embodiment, in step (b), the aqueous solution of the genus Medicinalum obtained in step (b1) is partitioned and extracted with the organic solvent, and the partitioned extraction operation is repeated three times to obtain an organic solvent layer and an aqueous form a layer. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum.

After execution of step (b1), in the original step (b), the Apocyrus genus aqueous solution obtained in step (b1) is partition-extracted by the supercritical extraction. In a specific embodiment, in step (b), an organic solvent layer and an aqueous layer are formed by subjecting the Apocynaceae genus aqueous solution obtained in step (b1) to partition extraction by the supercritical extraction. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum. In another specific embodiment, said supercritical extraction is supercritical _CO2 extraction. In the present invention, the process of separating nutrient components from natural products by supercritical _CO2 extraction mainly utilizes carbon dioxide fluid in supercritical state to extract nutritive components from natural products under high pressure and low temperature conditions. In a specific embodiment, the supercritical CO ₂ extraction conditions are CO : 99% ethanol solution flow rate ratio = 10 mL/min: ₁ mL/min, critical pressure range 150-400 bar, and critical temperature is set to 20 to 60°C. In a preferred specific embodiment, the supercritical _CO2 extraction conditions are _CO2 : 95% ethanol solution flow rate ratio = 10 mL/min: 1 mL/min, critical pressure of 250 bar, and critical temperature of 40°C. and Similarly, the supercritical _CO2 extraction also separates the raw material (aqueous layer) remaining in the supercritical _CO2 extraction reactor and the substance to be extracted (organic solvent layer), so the organic solvent layer is collected. to obtain the extract of the genus Aspen. In another specific embodiment, in step (b), said Spirulina extract is obtained by extracting said Spirulina alcoholic extract with supercritical _CO2 extraction.

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

The invention further provides use of the composition in the manufacture of a medicament for neuroprotection. The composition comprises an extract of the genus Medicinalum, and a method for producing the extract of the genus Medicinalum includes the following steps. That is, (a) extracting the genus Meliflora with an alcohol-based solvent to obtain an alcohol-based extract of the genus Meliflora. The alcoholic solvent is methanol or ethanol.

In a specific embodiment, the Spirulina of step (a) is washed, dried and/or ground.

In another specific embodiment, the method for producing the Spirulina extract further comprises step (b) after step (a). In the step (b), an organic solvent layer and an aqueous layer are formed by partitioning and extracting the Medicinal alcohol-based extract by organic solution or supercritical extraction. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum. 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.

In a specific embodiment, the method for producing the Spirulina extract further comprises step (b1) prior to the extraction with the organic solution. In the step (b1), the Spirulina genus alcoholic extract is immersed in water to obtain a Spirulina genus aqueous solution. In a preferred specific embodiment, in step (b), an organic solvent layer and an aqueous layer are formed by partitioning and extracting the Spirulina genus aqueous solution obtained in step (b1) with the organic solvent. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum.

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

In a specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the alcoholic extract of the genus Meliflora is an ethanolic extract of the genus Medicinalum.

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

In the present invention, the Spirulina genus extract can be applied to impart a neuroprotective effect. Neuroprotection is, for example, the ability to prevent or reduce neuronal cell (including neuronal and glial cell) death or damage in conditions that may be pathologically detrimental to the cerebral, central or peripheral nervous system, or , means the ability to allow neuronal salvage or neuronal resuscitation or restoration. Neuroprotective effects include nerve cell regeneration, ie, re-growth of nerve cells after disease or trauma. Neuroprotection refers to the nervous system after acute disease (e.g. stroke, brain or nervous system injury/trauma, cerebral hypoxia, spinal cord injury or peripheral nerve injury) or after chronic neurodegenerative disease (e.g. Mechanisms and strategies to prevent cerebral/neuronal damage or degeneration caused by Parkinson's disease, Alzheimer's disease or multiple sclerosis. The purpose of neuroprotection is to prevent neuronal dysfunction/death after nervous system injury and to maintain as highly ordered cellular interactions in the cerebrum as possible so that neuronal function is not disturbed. be. The Spirulina extract provides neuroprotective effects to treat neuronal injury (e.g., stroke, particularly ischemic stroke, brain injury, cerebral hypoxia, spinal cord injury or peripheral nerve injury) and neurodegeneration. Allows treatment or prevention of disease. Therefore, in a specific embodiment, the present invention relates to the use of said Medicinal extract as an active ingredient in the manufacture of a medicament for protecting and/or regenerating nerve cells.

In specific embodiments, the neuroprotection includes inhibition or prevention of neuronal cell death. In a preferred specific embodiment, the Spirulina extract is capable of inhibiting or preventing neuronal cell death.

Thus, in the present invention, it was found that the extract of the genus Medicinalum has several beneficial actions and functions in imparting neuroprotective action. Therefore, the extract of the genus Medicinal can be applied to the treatment of neuron or brain damage, and the treatment or prevention of neurodegenerative diseases. Such neurodegenerative diseases include acute or chronic neurodegenerative diseases.

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 alleviated by treatment with the Spirulina extract. The term "acute neurodegenerative disease" refers to a sudden disease or condition resulting in associated neuronal death or damage. Examples of acute neurodegenerative diseases include 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, neonatal perinatal asphyxia, cardiac arrest, intracranial hemorrhage ( intracranial hemorrhage, subarachnoid hemorrhage, stroke and traumatic brain injury.

In addition, representative examples of neurodegenerative diseases that can be treated using the extract of the genus Aspen of the present invention include 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 muscular atrophy (SBMA), or fragile X-associated tremor and ataxia syndrome (FXTAS).

In a specific embodiment, the Astragalus extract prevents or treats neurodegenerative diseases by providing neuroprotection. In a preferred 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, Includes dentate-rubropallid-luis body atrophy, spinobulbar muscular atrophy or fragile X-related tremor/ataxia syndrome. In a more preferred embodiment, said neurodegenerative disease comprises Parkinson's disease.

As used herein, the term "treatment" means alleviating symptoms or complications, slowing progression of a disease, condition or condition, alleviating or alleviating symptoms and complications, and/or curing a disease, condition or condition. Or means removal.

As used herein, the term "prevention" means prevention of the onset, recurrence or spread of a disease or condition, or one or more symptoms thereof. In any of the examples, these terms specifically refer to drugs provided herein in the presence or absence of one or more other activating agents prior to the onset of symptoms. It means treating an individual at risk for a disease or condition provided herein or administering a drug provided herein.

The present invention further provides use of the composition in the manufacture of a medicament for preventing or treating neurodegenerative diseases. The composition comprises an extract of the genus Medicinalum, and a method for producing the extract of the genus Medicinalum includes the following steps. That is, (a) extracting the genus Meliflora with an alcohol-based solvent to obtain an alcohol-based extract of the genus Meliflora. The alcoholic solvent is methanol or ethanol.

In a specific embodiment, the Spirulina of step (a) is washed, dried and/or ground.

In another specific embodiment, the method for producing the Spirulina extract further comprises step (b) after step (a). In the step (b), an organic solvent layer and an aqueous layer are formed by partitioning and extracting the Medicinal alcohol-based extract by organic solution or supercritical extraction. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum. 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.

In a specific embodiment, the method for producing the Spirulina extract further comprises step (b1) prior to the extraction with the organic solution. In the step (b1), the Spirulina genus alcoholic extract is immersed in water to obtain a Spirulina genus aqueous solution. In a preferred specific embodiment, in step (b), an organic solvent layer and an aqueous layer are formed by partitioning and extracting the Apocynaceae genus aqueous solution obtained in step (b1) with the organic solvent. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum.

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

In a specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the alcoholic extract of the genus Meliflora is an ethanolic extract of the genus Medicinalum.

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

In a specific embodiment, the neurodegenerative diseases include amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, frontotemporal dementia, spinocerebellar atrophy, spinocerebellar degeneration type 3, dental Included are nucleus ruculopallidus louis body atrophy, spinobulbar muscular atrophy or Fragile X-associated tremor/ataxia syndrome. In a preferred embodiment, said neurodegenerative disease comprises Parkinson's disease.

The invention further provides use of the composition in the manufacture of a medicament for promoting bone formation. The composition comprises an extract of the genus Medicinalum, and a method for producing the extract of the genus Medicinalum includes the following steps. That is, (a) extracting the genus Meliflora with an alcohol-based solvent to obtain an alcohol-based extract of the genus Meliflora. The alcoholic solvent is methanol or ethanol.

In a specific embodiment, the Spirulina of step (a) is washed, dried and/or ground.

In another specific embodiment, the method for producing the Spirulina extract further comprises step (b) after step (a). In the step (b), an organic solvent layer and an aqueous layer are formed by partitioning and extracting the Medicinal alcohol-based extract by organic solution or supercritical extraction. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum. 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.

In a specific embodiment, the method for producing the Spirulina extract further comprises step (b1) prior to the extraction with the organic solution. In the step (b1), the Spirulina genus alcoholic extract is immersed in water to obtain a Spirulina genus aqueous solution. In a preferred specific embodiment, in step (b), an organic solvent layer and an aqueous layer are formed by partitioning and extracting the Apocynaceae genus aqueous solution obtained in step (b1) with the organic solvent. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum.

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

In a specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the alcoholic extract of the genus Meliflora is an ethanolic extract of the genus Medicinalum.

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

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

Thus, the extract of the genus Medicinalum of the present invention is applicable to the treatment of bone diseases. Such bone diseases include (but are not limited to) osteoporosis, bone loss or fracture disease. Osteoporosis is a disease characterized by a net loss of bone mass per volume. This loss of bone mass, and the accompanying fractures, destroys the skeleton that provides structural support for the body. Low bone mass and structural deterioration of bone tissue lead to bone fragility and increased fractures of the hip, spine and wrist. Osteoporosis is classified into primary osteoporosis and secondary osteoporosis according to the cause of occurrence. In the molecular mechanism of primary osteoporosis, the receptor activator of nuclear factor κB (kappa B) ligand on osteoblasts (RANKL) and the receptor activator of nuclear factor κB on the surface of osteoclasts (RANK). binding activates osteoclasts and stimulates osteoclast differentiation and activity, thereby promoting the development 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, neglect, endocrine disorders and other diseases such as rheumatoid arthritis, diabetes, stroke, Parkinson's disease and bone metastasis of cancer. In addition, a bone defect is an imbalance in the ratio of bone formation to bone resorption. This results in the individual forming a skeleton that is less than expected, or the skeleton of the individual is incomplete compared to expectations. Bone defects can also be caused by fractures, surgical interventions, or dental or periodontal disease. Bone fusion includes (but is not limited to) repair of bone defects. For example, bone defects occur in closed, open and nonunion fractures.

In a specific embodiment, the Astragalus extract prevents or treats bone disease by promoting bone formation. In a preferred embodiment, said bone disease comprises osteoporosis, bone defect or fracture disease. In a more preferred specific embodiment, said bone disease comprises bone defect.

The present invention further provides use of the composition in the manufacture of a medicament for preventing or treating bone disease. The composition comprises an extract of the genus Medicinalum, and a method for producing the extract of the genus Medicinalum includes the following steps. That is, (a) extracting the genus Meliflora with an alcohol-based solvent to obtain an alcohol-based extract of the genus Meliflora. The alcoholic solvent is methanol or ethanol.

In a specific embodiment, the Spirulina of step (a) is washed, dried and/or ground.

In another specific embodiment, the method for producing the Spirulina extract further comprises step (b) after step (a). In the step (b), an organic solvent layer and an aqueous layer are formed by partitioning and extracting the Medicinal alcohol-based extract by organic solution or supercritical extraction. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum. 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.

In a specific embodiment, the method for producing the Spirulina extract further comprises step (b1) prior to the extraction with the organic solution. In the step (b1), the Spirulina genus alcoholic extract is immersed in water to obtain a Spirulina genus aqueous solution. In a preferred specific embodiment, in step (b), an organic solvent layer and an aqueous layer are formed by partitioning and extracting the Apocynaceae genus aqueous solution obtained in step (b1) with the organic solvent. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum.

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

In a specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the alcoholic extract of the genus Meliflora is an ethanolic extract of the genus Medicinalum.

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

In the present invention, the extract of the genus Aspen can treat bone diseases by increasing bone mass or promoting and repairing bone growth. In specific embodiments, the bone disease includes osteoporosis, bone defect or fracture disease. In a preferred embodiment, the bone disease comprises bone defect.

The invention further provides use of the composition in the manufacture of a medicament for treating allergic dermatitis. The composition comprises an extract of the genus Medicinalum, and a method for producing the extract of the genus Medicinalum includes the following steps. That is, (a) extracting the genus Meliflora with an alcohol-based solvent to obtain an alcohol-based extract of the genus Meliflora. The alcoholic solvent is methanol or ethanol.

In a specific embodiment, the Spirulina of step (a) is washed, dried and/or ground.

In another specific embodiment, the method for producing the Spirulina extract further comprises step (b) after step (a). In the step (b), an organic solvent layer and an aqueous layer are formed by partitioning and extracting the Medicinal alcohol-based extract by organic solution or supercritical extraction. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum. 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.

In a specific embodiment, the method for producing the Spirulina extract further comprises step (b1) prior to the extraction with the organic solution. In the step (b1), the Spirulina genus alcoholic extract is immersed in water to obtain a Spirulina genus aqueous solution. In a preferred specific embodiment, in step (b), an organic solvent layer and an aqueous layer are formed by partitioning and extracting the Apocynaceae genus aqueous solution obtained in step (b1) with the organic solvent. Subsequently, the organic solvent layer is collected to obtain the extract of the genus Medicinalum.

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

In a specific embodiment, the alcoholic solvent is ethanol. In a preferred embodiment, the alcoholic extract of the genus Meliflora is an ethanolic extract of the genus Medicinalum.

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

As used herein, the term "allergic dermatitis" is a general term for skin conditions caused by allergic reactions characterized by chronic itching and rashes on the face, neck, elbows and/or knees. and Examples of allergic dermatitis include contact dermatitis and atopic dermatitis. "Contact dermatitis" is an eczematous inflammatory disease that develops when a foreign antigen comes into contact with the skin. Urticaria is mentioned. Antigens include, for example, metal allergens (cobalt, nickel, etc.), plant allergens (lacquer, primrose, etc.), and food allergens (mango, ginkgo, etc.). "Atopic dermatitis (AD)" refers to a skin disorder that causes atopic predisposition in many patients. In addition, it is characterized by bilaterally symmetrical generalized eczema that repeats exacerbation and relief. Extremity childhood eczema, childhood atopic eczema, childhood dry eczema, childhood eczema, adult atopic dermatitis, endogenous eczema, childhood dermatitis and chronic childhood eczema.

In specific examples, the allergic dermatitis includes contact dermatitis or atopic dermatitis. In a preferred embodiment, said allergic dermatitis comprises atopic dermatitis. In a more preferred specific embodiment, the atopic dermatitis includes diffuse neurodermatitis, atopic eczema, atopic neurodermatitis, Benier's prurigo, acute infantile eczema, eczema of the flexor, extremity pediatric eczema, and pediatric atopic dermatitis. Includes eczema, childhood dry eczema, childhood eczema, adult atopic dermatitis, endogenous eczema, childhood dermatitis or chronic childhood eczema.

In another specific embodiment, the drug reduces symptoms of atopic dermatitis including erythema, edema, peeling or dryness of the skin.

In a specific example, the drug reduces the increase in IgE expression resulting from the atopic dermatitis.

In another specific embodiment, the drug ameliorates splenic or lymph node enlargement resulting from the atopic dermatitis.

In a specific embodiment, the medicament further comprises a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein is determined by the composition applied in a particular combination and applied in a particular manner. The term "carrier" as used herein includes any and all solvents, dispersion media, vehicles, coatings, diluents, penetration and absorption retardants such as antibacterial and antifungal agents, buffers, Including, but not limited to, agents, carrier solutions, suspensions or colloids. These media and reagents used for drug active substances are known in the art. Unless any common vehicle or agent is incompatible with the active ingredient, therapeutic combinations should be considered. Supplementary active ingredients may also be incorporated into the compositions. The term "pharmaceutically acceptable" refers to the absence of allergic or similar side reactions when molecular entities and compositions are applied to a subject. The preparation of protein-active water compositions is well known in the art. Usually, the composition is prepared as a liquid/solution, tablet, capsule or suspension injection. They may also be prepared as dissolvable solids or suspension solids for use as injectables. In another specific embodiment, the pharmaceutically acceptable carrier includes a dermatologically acceptable vehicle. By "dermatologically acceptable medium" is meant a biologically suitable substance such as, for example, salts, esters and/or acid amides components. That is, the substance does not cause undesired biological effects when administered to an individual when used in conjunction with selected active ingredients. Also, the substance is a substance that does not adversely interact with any of the ingredients in the pharmaceutical composition containing this substance. Similarly, "dermatologically acceptable salts" or "dermatologically acceptable esters" as used herein are biologically suitable salts or esters.

In the present invention, formulation of the composition (including the extract of the genus Medicinalum) and a pharmaceutically acceptable carrier includes sterile aqueous solutions or dispersions, aqueous suspensions, oil emulsions, water-in-oil emulsions, Specific point emulsions, long-stay emulsions, viscous emulsions, microemulsions, nanoemulsions, liposomes, microparticles, microspheres, nanospheres, nanoparticles, micromercury, and several types of sustained-release natural or synthetic polymers are available. be. Pharmaceutically acceptable carriers and the Spirulina extract are used to deliver aerosols, tablets, pills, capsules, sterile powders, suppositories, detergents, creams, ointments, pastes, gels, hydrogels, or compositions. It may be formulated as other possible formulations.

Compositions in the context of the present invention also include compositions that are administered topically and regionally to act. The term "topical" as used herein refers to the use of the compositions described herein, mixed with a suitable medicinal carrier, and applied to a skin site (e.g., allergic dermatitis affected area). It relates to having a local effect. Such topical compositions therefore include drug forms such as compounds that are externally applied 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 and the like. Also, it can be used in the form of a patch or a wet bandage depending on the body part to be treated. The term "ointment" includes formulations (including creams) having an oleaginous base, a water-soluble base and an emulsion base. The substrates are, for example, petrolatum, lanolin, polyethylene glycols and mixtures thereof.

The drug of the invention further comprises a pharmaceutically acceptable carrier and can be administered to an individual by a number of different means, according to therapeutic modalities well known in the field relevant to the invention. In some embodiments, the composition (including the Spirulina extract) and a pharmaceutically acceptable carrier are administered topically, intravenously, intramuscularly, subcutaneously, topically, orally, or by inhalation. The drug is also delivered to the target site by the digestive and circulatory systems.

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

The term "effective dosage" is used herein to mean that, in a specified condition, the progression of symptoms in an individual can be prevented, reduced, arrested, or reversed, or when the individual begins receiving treatment, a particular dosage. A therapeutic dose capable of partially or completely relieving symptoms that exist under the circumstances. A person skilled in the art can easily determine the appropriate dosage and method of administration for administering the extract to an individual. For example, the Spirulina extract can be administered to the individual once or twice. Dosage and usage means the effective dose of the Spirulina extract administered to the individual when administering multiple doses (including the total amount of product administered over the entire duration of the dosage and administration regimen). includes getting.

As described above, the extracts of the genus Medicinalum of the present invention have different effective doses when applied to neuroprotection, promotion of bone formation, and treatment of allergic dermatitis.

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

For neuroprotection, in another specific embodiment, the effective dosage range of the Medicinal extract is 0.5 ng/ml to 50 μg/ml. In a preferred specific embodiment, the effective dose range of the Spirulina extract is 1 ng/ml to 5 μg/ml. In a more preferred specific embodiment, the effective dosage range of the Spirulina extract is from 5 ng/ml to 500 ng/ml.

For promoting osteogenesis, in a specific embodiment, the effective dosage range of the Spirulina extract is 0.2 mg/kg to 100 mg/kg body weight. In a preferred specific embodiment, the effective dosage range of the Spirulina extract is 1 mg/kg to 50 mg/kg of body weight. In a more preferred specific embodiment, the effective dosage range of the Spirulina extract is 2 mg/kg to 10 mg/kg of body weight.

When treating allergic dermatitis, in a specific embodiment, the effective dosage range of the Spirulina extract is 0.05-20 mg/ml. In a preferred specific embodiment, the effective dosage range of the Spirulina extract is 0.1-10 mg/ml. In a more preferred specific embodiment, the effective dosage range of the Spirulina extract is 0.5-2 mg/ml.

FIG. 1 is an extraction flow of the genus Aspen. FIG. 2 shows the protective effect of Spirulina ethyl acetate extract EE on nerve cells. SH-SY5Y neuronal cells were added with different concentrations of Epiphyllum ethyl acetate extract EE, then the cells were injured with 6-hydroxydopamine (6-OHDA), and finally the viability of the cells was measured to show neuroprotection. observed the effect of Spirulina ethyl acetate extract EE improved cell viability in each group at concentrations of 0.5 to 500 ng/ml. This proves that the Ethyl Acetate Extract EE of the Epiphyllum genus has a neuroprotective effect. As a result of statistical analysis of each group and the 6-OHDA group, significant differences (P<0.05) are indicated by *. FIG. 3 shows the protective effect of the Ethylacetate extract EE on nerves in vivo. (A) in the figure shows the swimming path of the zebrafish of each group within a unit time. In addition, (B) shows the swimming speed of zebrafish, and the speed of the control group was 2.47 ± 0.26 mm / s, while the speed of the 6-OHDA group was 0.21 ± 0 .11 mm/s. However, when the zebrafish ethyl acetate extract EE at concentrations of 0.5 μg/ml and 5 μg/ml was added, the swimming speed of the zebrafish was 2.03±0.65 mm/s and 1.24±0.28 mm/s. recovered to Also, (C) shows the total swimming distance of the zebrafish within a unit time. While the swimming distance of the control group was 741.09±77.04 mm, the swimming distance of the 6-OHDA group decreased to 63.00±32.19 mm. However, addition of 0.5 μg/ml and 5 μg/ml concentrations of the Ethyl Acetate Extract EE of the genus Typophyll restored the swimming distance of the zebrafish to 609.03±196.17 mm and 371.52±82.77 mm. FIG. 4 shows the effect of Spirulina ethyl acetate extract EE on bone formation. (A) in the figure shows a control group, and (B) to (E) show groups administered with different dosages of the Ethyl Acetate Extract EE (0.05 to 50 μg/ml) of the genus Meliformis. ing. In addition, (F) shows the results of quantitatively statistically quantifying the number of bony nodes in each group of zebrafish. For the zebrafish of each group, the number of bone segments was calculated on day 7 after fertilization, and when 0.5 μg/ml of the Ethylacetate extract EE of the genus Typoracea was added, the state of development of the bone segments was remarkably improved. Moreover, the Ethyl Acetate Extract of the Epiphyllum does not cause developmental abnormalities in zebrafish, which almost indicates that the Ethyl Acetate of the Epiphyllum extract has no toxicity to the development of the embryo body. *: P<0.05 compared with the control group. FIG. 5 is a comparative diagram of skull appearance showing the therapeutic effect of different concentrations of the Ethylacetate extract EE on cranial defects in rats. FIG. 6 is a comparative diagram of the therapeutic effect of the Ethylacetate extract EE on cranial defects of rats photographed with an X-ray apparatus. (A) in the figure shows an X-ray diagram of the condition of cranial defects in rats of each group. (B) shows the recovery rate of the skull of rats in each group. FIG. 7 shows the effect of Cypripedium ethyl acetate extract EE on skin appearance of mice with atopic dermatitis. In (A) in the figure, atopic dermatitis was induced with DNCB, and after the symptoms of atopic dermatitis appeared on the skin of mice, daily drug application was started, and on the 1st and 7th days of treatment. , shows the skin appearance of mice with DNCB-induced atopic dermatitis on day 12, respectively. Note that the scale bar = 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 moderating the symptoms of atopic dermatitis. Phenomena such as edema, redness, dryness and scab were all alleviated. (B) in the figure shows the clinical dermatitis score for alleviation of AD symptoms by Echinacea ethyl acetate extract EE. Alleviation of AD symptoms by EE was assessed by a clinical dermatitis score. At this time, the severity of dermatitis was evaluated using 4 skin surface symptoms: erythema/hemorrhage, edema, peeling/erosion, and dryness. A score of 0 to 3 was given according to the severity of each symptom, and the higher the score, the more severe the symptoms. The total score for dermatitis was the sum of the above four scores, with a maximum of 12 points. The higher the score, the more severe the dermatitis. Data represent the mean ± standard error of the mean (mean ± SEM). In addition, * indicates a significant difference (P<0.05) compared to the AD group, and # indicates a significant difference compared to the ADV group. FIG. 8 shows the effect of Spirulina ethyl acetate extract EE on serum IgE. Blood was collected before the animals were sacrificed and IgE was detected in the serum. IgE was elevated by DNCB-induced AD, but IgE was lowered by application of the Epiphyllum ethyl acetate extract EE. Data represent the mean ± standard error of the mean (mean ± SEM). In addition, * indicates a significant difference (P<0.05) compared to the AD group, and # indicates a significant difference compared to the ADV group. FIG. 9 shows the effect of Spirulina ethyl acetate extract EE on DNCB-induced swollen spleen weight. At the time of animal sacrifice, splenic tissue was harvested, photographed and weighed. Although the splenic tissue was swollen due to DNCB-induced atopic dermatitis, the weight of the spleen was reduced by the application of the Ethylacetate extract EE of the spp. Note that the scale bar = 1 cm. Data represent the mean ± standard error of the mean (mean ± SEM). In addition, * indicates a significant difference (P<0.05) compared to the AD group, and # indicates a significant difference compared to the ADV group. FIG. 10 shows the effect of Spirulina ethyl acetate extract EE on DNCB-induced swollen lymph node weight. Lymph node tissue was harvested, photographed and weighed at the time of animal sacrifice. The lymph node tissue was swollen due to DNCB-induced atopic dermatitis, but the weight of the lymph node was reduced by applying the Spirulina extract EE. Note that the scale bar is 0.5 cm. Data represent the mean ± standard error of the mean (mean ± SEM). In addition, * indicates a significant difference (P<0.05) compared to the AD group, and # indicates a significant difference compared to the ADV group.

The invention includes, but is not limited to, the descriptions above and below. The method of implementation is illustrated in the representative examples below.

Extraction of Cultivated Type Sarcodia ceylanica In the present invention, artificially cultured Sarcodia ceylanica was used.

The genus Spirulina was dried under conditions of 50° C. for 24 hours to obtain a dry Spirulina genus. 10.8 kg of dry Dispensary was pulverized, filtered through a 50-mesh filtration screen, and then immersed in 95% alcohol solution at room temperature for three extractions. Then, the immersion liquids for three times were combined, filtered, and concentrated under reduced pressure to obtain 370.5 g of a Medicinal alcohol crude extract. Next, the above-mentioned Medicinal medicinal crude extract was added to 1 L of water to float, and then 1 L of ethyl acetate was added to perform partition extraction. The above partition extraction was repeated three times to form an ethyl acetate layer and an aqueous layer. Subsequently, the ethyl acetate layer was collected to obtain ethyl acetate extract EE (83.2 g). Fig. 1 shows the total extraction flow of the genus Aspen.

Also, in the above extraction step, the alcoholic solution may be replaced with a methanolic solution. And the step of extracting with ethyl acetate may be replaced with other organic solvents. Partition extraction using e.g. propyl acetate, butyl acetate, hexane, heptane, octane, nonane, methyl ether, ethyl ether, dichloromethane, chloroform or carbon tetrachloride solvents or supercritical extraction (e.g. supercritical _CO2 extraction) , it is possible to obtain an extract with the same components as EE.

Alternatively, water may be mixed with ethyl acetate or other organic solvent to form an organic solution, and the Crude alcohol extract may be directly extracted. In this case, it is not necessary to divide the water and the organic solvent into two steps and extract them separately. In addition, the step of pre-immersing in water may be dispensed with and ethyl acetate or other organic solvent may be directly used to extract the Crude alcoholic extract of the genus Medicinalum. Even by changing the above steps, it is possible to finally obtain an extract with the same components as EE.

The above supercritical _CO2 extraction conditions are _CO2 : 95% ethanol solution flow rate ratio = 10 mL/min: 1 mL/min, critical pressure of 250 bar, and critical temperature of 40°C.

In addition, according to the present invention, when the polysaccharide content of EE was measured by the phenol-sulfuric acid method, the EE did not contain polysaccharide components. Therefore, polysaccharide components were not eluted by the extraction method of preliminarily soaking the genus Dispensary in alcohol, and polysaccharide components were not contained in the crude alcoholic extract of the genus Dispensary. In addition, according to the present invention, component analysis of the Ethylacetate extract EE of the genus Medicinalum was also conducted. The analysis results are shown in Table 1.

Experimental method (1) Evaluation of 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 2×10 ⁴ cells, and then placed in a cell incubator. . Then, after the cells were completely adhered, they were pretreated for 1 hour with EE solutions of the Ethylacetate extract of the genus Epiphyllum at different concentrations. Then, 6-Hydroxydopamine (6-OHDA) was added for 15 hours, and 10% alamar blue was added for 3 hours of incubation. Subsequently, the absorbance value was measured with an enzyme immunoassay to measure the viability of the cells. Based on the toxicity induced by 6-OHDA on neurons, it was analyzed whether or not the extract of the genus Aspen has a protective effect on neurons treated with 6-OHDA.

(b) In vivo nerve test Dissolve 6-OHDA in 2% fresh L-ascorbic acid to prepare a 500 mM stock solution and place it in a 1.5 mL brown microtube and add 10 μl It was divided into tubes of . When adding drugs, the formulated drugs were dissolved in the same ratio of Hank's buffer based on the concentration of the experimental design. Then, 1 mL of Hank's buffer containing 1 mL of drug and fry was added to the 24-well culture plate. The time the fry were given 6-OHDA was between 48 and 120 hours after fertilization. No chemicals were added to the control group, and a solvent group was provided as a control for each experiment. By analyzing the behavioral ability of zebrafish, we measured parameters such as residence time for each water layer, swimming speed, and swimming distance, and evaluated the therapeutic effect of the extract of the genus Dispensary.

(2) Evaluation of the effect of EE on osteogenesis (a) Test of zebrafish bone growth Utilizing a biological system, the stimulating effect of the extract solution of the genus Aspilum on bone regeneration was tested. In addition, the effect of the extract of the genus Spirulina on bone and bone quality-related diseases (eg, osteoporosis) was analyzed. The effect of the active substance on bone growth was analyzed by exclusively staining the bone bone part with a fluorescent stain Calcein and combining the developmental morphology of the zebrafish embryo body. In addition, the effects of substances on bone growth were presented by converting them into data using analysis software. Juvenile zebrafish 48 hours after fertilization were obtained, and after adding extracts of the genus Aspen of different concentrations, the vertebral joints of the fish body were observed every day, and breeding was continued after the observation. Zebrafish were stained with a 2 g/L calcein solution (pH=7.8) for 10 minutes and then rinsed with clean water to remove excess stain. Then, after anesthetizing with MS-222, the fish was placed and fixed in 1% Methyl Cellulose. Then, the vertebrae of the fish body were observed with a fluorescence microscope, and the number of skeletal joints was statistically analyzed.

(b) Rat cranial defect experiment Cranial defect surgery:
Small bone defects are naturally regenerative in vivo. However, if the defect size exceeds the size that can be repaired by the body, it becomes a so-called critical sized bone defect that cannot be fully healed by itself, and cannot be healed without external treatment. . Differences in animal models and differences in site both affect the size of the defect to be constructed. In the rat skull defect model used in the present invention, animals were first anesthetized using the gaseous anesthetic isoflurane. Then, after shaving the rat's crown, the surgical site was disinfected with povidone-iodine and 70% alcohol, and ampicillin was injected. A sterile paper towel was then perforated and placed over the rat's head, exposing only the surgical site. Then, an incision of about 1.5 cm was made in the skin and periosteum just above the head of the rat, and the skull and periosteum were separated with a periosteal remover. One ridge on the skull, located just in the middle, is the midline sagittal suture, flanked by parietal bones. Two 4 mm diameter bone defects were created using a 4 mm diameter trephine exactly centered on the sagittal suture. Subsequently, the periosteum was sutured with 5-0 catgut, and the skin was sutured with 4-0 nylon thread. When animals woke up from anesthesia during surgery, they were made to inhale anesthetic gas with a gas anesthesia machine through an oxygen mask to maintain anesthesia. Rats were dosed with EE three times weekly under anesthesia. Doses were 1.5 mg/kg/week and 7.5 mg/kg/week. After administration for 4 weeks, the animals were sacrificed, and the cranial tissue was collected.

As a method for evaluating the state of recovery of the rat skull, the image was taken with an X-ray apparatus (X-OMAT V, Kodak, Rochester, NY), and the negative film was developed with an automatic processor. Images were scanned and converted to TIF files and then loaded into ImageQuant software (ImageQuant Version 5.2 Copyright 1999, Molecular Dynamics) for later analysis. Equal area areas of each step wedge and blank were then selected in the ImagQuant software to represent the relationship between the grayscale intensity of the image on the x-ray film and the wedge-shaped step thickness. Then, a circular area with a diameter of 8 mm in the skull area and a flat area with an area of 1×1 mm ² in the middle of the parietal bones on both sides were selected, and the grayscale intensity was measured and substituted into the above equation. Thereby, the bone density of the sample with respect to the thickness of the wedge-shaped step was calculated by interpolation. That is, we defined the bone density of the bone defect region of the skull as the density of the grayscale image projected by the difference in the thickness of the wedge-shaped steps.

(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 with a shaver and depilatory cream, and marked with a silicone rubber with a diameter of 1 cm (78.5 mm ² area). Then, once every two days, drop 26 μL of 2% 2,4-dinitirochlorobenzene (DNCB) (Sigma-Aldrich, Missouri, USA, Cat. No. #138630) into the above area. did. This was done a total of 8 times to induce skin lesions.

Seven days after DNCB induction, 100 μL of Vehicle, Dispensary extract EE, or atopic dermatitis therapeutic drug crisaborole were applied daily. Experimental animals: (1) Control group: uninduced, (2) AD group: 2% DNCB only administered, (3) ADV group: 2% DNCB and vehicle (Vehicle) administered, (4) EEL group: (5) EEH group: 200 μg EE/100 μL of 1% methylcellulose after DNCB induction; and (6) Cri group: 25 μg crisabolol/1% after DNCB induction. 100 μL of acetone-EtOH was administered and divided into groups (3 animals in each group). Five minutes after the application of DNCB, the drug was applied after waiting for the DNCB to dry. Changes in skin symptoms were photographed and recorded with a digital camera before induction or drug application.

Animals were sacrificed on day 18, bled, and dorsal skin, spleen and lymph node tissues were collected. Then, the sizes of the spleen and subiliac lymph nodes were observed, weighed and compared. In addition, the severity of clinical dermatitis was evaluated from four symptoms of erythema/hemorrhage, edema, excoriation/erosion, and dryness on the skin surface. . A score of 0 to 3 was assigned according to the severity of each symptom, with 0 being asymptomatic, 1 being mild, 2 being moderate and 3 being severe. The total score for dermatitis was the sum of the above four scores, with a maximum of 12 points. The higher the score, the more severe the dermatitis.

Before sacrifice, mice were bled into blood collection tubes (BD vacutainer-SST, NJ, USA) by heart bleeding method. Blood was then centrifuged at 3000 rpm for 10 minutes to obtain serum. Serum was stored in a −80° C. refrigerator until further use. Serum IgE levels were then detected according to the manufacturer's instructions (IgE-ELISA kit, Thermo Fisher Scientific, Vienna, Austria).

The lymph nodes and spleen swell when inflammation or immune-related disease occurs. Therefore, when the mice were sacrificed, these two organs were harvested, photographed, and weighed.

Statistical Analysis All experimental data are presented as mean±standard error of the mean (mean±SEM). Statistical analysis of data was performed by one-way analysis of variance (ANOVA) to compare data between multiple groups. In addition, multiple comparisons between groups were performed using Duncan's method. Moreover, when the P value was smaller than 0.05, it was considered that there was a significant difference between the groups.

Results Neuroprotective effect of EE SH-SY5Y nerve cells were added with different concentrations of Ethylacetylacetate extract EE, then the cells were injured with 6-hydroxydopamine (6-OHDA), and finally the viability of the cells was measured. By doing so, the effect of neuroprotection was observed. 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 a concentration of 0.5-500 ng/ml of Ethyl Acetate Extract EE of the genus Typoracea was added, the cell viability in each group was 10.72±2.86%, 33.91±19.67%, respectively. They were 7.92±1.93% and 32.69±17.88% (see FIG. 2), both of which were improved. This result proved that the Ethylacetate extract EE of the genus Epiphyllum had a neuroprotective effect.

6-OHDA was also able to induce Parkinsonian-like behavior in zebrafish. This allowed the zebrafish to stay on the bottom without swimming. FIG. 3A shows the swimming paths of zebrafish in each group within a unit time. In addition, (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 was 0.47±0.26 mm/s. It dropped to 21±0.11 mm/s. However, upon addition of 0.5 μg/ml and 5 μg/ml concentrations of Ethyl Acetate Extract EE of the zebrafish, the swimming speed of the zebrafish was 2.03±0.65 mm/s and 1.24±0.28 mm/s. recovered to s. In addition, (C) of FIG. 3 shows the total swimming distance of the zebrafish within a unit time. While the swimming distance of the control group was 741.09±77.04 mm, the swimming distance of the 6-OHDA group decreased to 63.00±32.19 mm. However, addition of 0.5 μg/ml and 5 μg/ml concentrations of the Ethyl Acetate Extract EE of the genus Typophyll restored the swimming distance of the zebrafish to 609.03±196.17 mm and 371.52±82.77 mm. This result indicates that the Ethylacetate extract EE of the genus Typoracea has a neuroprotective effect on nerves in vivo.

(2) Effect of EE on Osteogenesis Dispensary ethyl acetate extract EE was able to promote osteogenesis. In the zebrafish model, 10±0.63 bone nodules were formed in juveniles of the control group, whereas 13.8±0.63 osteomes were formed in the EE groups with the 0.05-50 μg/ml extract of the troglodytes extract, respectively. The number of phalanges increased to 80, 15.33±0.68, 15.00±0.84 and 15.4±0.68 (see Figure 4).

In rat skull defect experiments, EE was administered three times weekly. Doses were 1.5 mg/kg/week and 7.5 mg/kg/week. After 4 weeks, the animals were sacrificed, skull tissue was collected, and area quantification was performed using images taken with an X-ray device. FIG. 5 is a comparison of cranial appearance showing the therapeutic effect of different concentrations of Ethylacetylacetate extract EE on cranial defects in rats. In addition, FIG. 6(A) shows an X-ray diagram of the condition of cranial defects in the rats of each group. (B) of FIG. 6 shows the recovery rate of the skull of rats in each group. As is clear from these results, the recovery rate of the skull in the control group (vehicle) was 51.90±10.01%, whereas EE was administered at 1.5 mg/kg/week and 7.5 mg/kg. /week dosing skull recovery rates were 97.47±4.86% and 15.47±15.86%, respectively. The above results indicate that EE can promote osteogenesis.

(3) Therapeutic effect of EE on atopic dermatitis Symptoms of DNCB-induced atopic dermatitis (AD) in mice included phenomena such as erythema, edema, exfoliation, dryness and lichenification. . After the mice showed obvious symptoms such as erythema, edema, exfoliation and dryness, EE or crisaborole was applied daily to test whether EE has a therapeutic effect on atopic dermatitis. The symptoms of skin appearance of mice were observed, photographed and recorded (see FIG. 7A), and the clinical dermatitis score was used to quantify the therapeutic effect of EE (see FIG. 7B). The control group had a dry skin surface due to less hair, and the score was 0.75±0.25. Corneocytes proliferated on the skin surface of the AD group and the ADV group, forming thick scabs. Some scabs were peeled off day by day, and dryness of the skin around the induction was observed. In addition, wrinkles were observed on the skin. In addition, erythema and edema appeared on the skin due to inflammation. However, keratinocytes continued to proliferate, and scabs were also present on the skin surface. The clinical dermatitis scores of these two groups were significantly elevated compared to the control group to 9.17±0.40 and 9.50±0.56, respectively. On the other hand, in the group to which EE and crisabolol (Cri) were applied day by day, dryness, erythema and edema were observed after peeling of the scab, but the degree of keratinocyte proliferation was considered to be milder than in the AD group. was taken. Therefore, dermatitis scores in the EEL and EEH groups were significantly lower than in the AD group (4.50±0.76 and 3.67±0.49) before animal sacrifice. On the other hand, the Cri group had a dermatitis score of 4.80±0.37, which was statistically significant to the AD group. The above results indicate that EE has a mitigating effect on AD symptoms.

IgE is one of clinical diagnostic indicators for atopic dermatitis. Serum was collected and the IgE content was measured before killing the mice, and the results are shown in FIG. The serum IgE content of the control group was 1.71±0.14 μg/mL. Also, the IgE concentration in the AD group and the ADV group was clearly elevated (68.89±4.12 μg/mL and 68.95±2.60 μg/mL). In contrast, the IgE concentrations in the EEL and EEH groups decreased only slightly, 59.33±2.11 μg/mL and 59.35±2.05 μg/mL, respectively. However, it was statistically significant. On the other hand, serum IgE content in the Cri group decreased to 65.61±1.74 μg/mL. Serum IgE, which increased with atopic dermatitis, could be reduced by applying EE.

Spleen weight results were as shown in FIG. The spleen weight of the control group was 91.08±3.61 mg. In addition, the spleens of the AD and ADV groups were clearly swollen, 2.5 times that of the control group (234.46±6.56 mg and 221.14±13.32 mg). In contrast, spleen weights in the EEL and EEH groups were significantly reduced to 160.74±9.10 mg and 167.41±10.39 mg, respectively. On the other hand, the spleen weight in the Cri group was significantly reduced to 167.46±10.61 mg.

The total lymph node weight results were as shown in FIG. The total weight of the two lymph nodes in the control group was 4.46±0.29 mg. In addition, the lymph nodes of the AD group and the ADV group were clearly swollen, three times as large as the control group (12.07±0.33 mg and 11.61±0.54 mg). In contrast, 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. And it 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 phenomena by applying EE to swollen lymph nodes and spleen associated with inflammation or immune reaction due to atopic dermatitis.

The present invention is properly described as being capable of being practiced subject to requirements or limitations not specifically disclosed herein. The terms used for description are not intended to be limiting. While there is no difference in the representation and description using these terms and any other equivalents, it should be recognized that rights in the present invention may be modified. Therefore, although the present invention describes embodiments and other situations, the contents disclosed herein can be supplemented and modified by those skilled in the art, and such modifications and variations are within the scope of the present invention. is considered.

Claims (4)

Use of a composition in the manufacture of a medicament for neuroprotection, said composition comprising a Spirulina ethyl acetate extract, a method for producing said Spirulina ethyl acetate extract comprising:
(a) extracting the spp. with ethanol to obtain an ethanol extract of the spp .
(b) partitioning and extracting the Meliforms ethanol extract 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 Meliformes ethyl acetate extract; Uses that include the steps of and. Use according to claim 1, wherein said neuroprotection includes inhibition or prevention of neuronal cell death. 2. The use according to claim 1, wherein said Spirulina ethyl acetate extract is neuroprotective to prevent or treat neurodegenerative diseases. 4. Use according to claim 3, wherein said neurodegenerative disease comprises Parkinson's disease.