KR20200056771A - Composition for Preventing or Treating Ischemic Brain Disease Containing Extract of Mixed Extract of Angelica gigas Nakai and Oenanthe javanica - Google Patents

Abstract

Provided are uses of a mixed extract of Angelica gigas and Oenanthe javanica for preventing and/or treating ischemic brain disease, treating and/or alleviating ischemic brain disease, protecting nerve cells, and/or generating nerve cells. The present invention proposes a protective effect against nerve cell damage caused by cerebral ischemia and a damaged nerve cell regeneration (or production) effect, thereby providing a novel treatment method having an excellent effect on preventing and treating ischemic brain disease.

Description

Composition for Preventing or Treating Ischemic Brain Disease Containing Extract of Mixed Extract of Angelica gigas Nakai and Oenanthe javanica}

Provided is a pharmaceutical composition for the prevention and / or treatment of ischemic brain disease, including a mixed extract of Angelica gigas Nakai and buttercup, and a health functional food.

Stroke, one of the typical ischemic brain diseases, is a disease that causes severe dysfunction by causing nerve cell damage in a wide area of the brain due to blockage of blood flow. Stroke is largely classified into cerebral ischemia caused by clogging of blood vessels and cerebral hemorrhage caused by blood vessel rupture.

Stroke can be largely divided into ischemic stroke (cerebral infarction), where the blood vessels are blocked, and hemorrhagic stroke (brain hemorrhage), in which cerebral vessels burst, and ischemic stroke is known to be caused by arteriosclerosis or thrombus, and in case of cerebral hemorrhage, hypertension Primary brain hemorrhage caused by and arteriovenous malformation or subarachnoid hemorrhage caused by an aneurysm can be said to be important causes.

Currently, various drugs having a therapeutic effect on ischemic brain diseases such as stroke have been developed, but many side effects have been reported, and thus it is required to develop an effective and safe therapeutic agent.

Registered Patent No. 10-0757207 (September 04, 2007) Patent Publication No. 10-2012-0053604 (May 29, 2012)

An example is a pharmaceutical composition for the prevention and / or treatment of ischemic brain disease comprising a mixed extract of Angelica gigas Nakai and buttercup as an active ingredient, a pharmaceutical composition for protecting neurons, and / or regeneration of neurons (or production). It provides a pharmaceutical composition for.

Another example is ischemic brain disease comprising the step of administering a mixed extract of Angelica keiskei and buttercup to a subject in need of prevention and / or treatment of ischemic brain disease, neuronal cell protection, and / or neuronal regeneration (or production). Provided is a method of preventing and / or treating, a method of protecting neurons, and / or a method of regulating (or generating) neurons.

The mixed extract of Angelica keiskei and buttercup may be a mixture of Angelica keiskei extract and buttercup extract, extract of a mixture of Angelica keiskei and buttercup, or a combination thereof.

The ischemic brain disease may be selected from ischemic cerebrovascular disease and ischemic cranial nerve disease (degenerative neurological disease), for example, stroke (eg, cerebral infarction or cerebral hemorrhage), hypertension, dementia (eg, vascular dementia, etc.), Parkinson's disease, It can be selected from the group consisting of Huntington's disease, Lou Gehrig's disease, and the like.

The neurons may be neurons of the brain (eg, hippocampus (eg, CA1 region, etc.)), such as neurons, glial cells (eg, astrocytes, microglia, oligodendrocytes) (oligodendrocyte, etc.).

Another example provides a health functional food for improving ischemic brain disease, protecting neurons, and / or regenerating (or generating) neurons, comprising a mixed extract of Angelica Angelica and buttercup as an active ingredient. The mixed extract of Angelica keiskei and buttercup may be a mixture of Angelica keiskei extract and buttercup extract, extract of a mixture of Angelica keiskei and buttercup, or a combination thereof.

Another example provides a method for preparing a composition having an effect of preventing, treating and / or improving ischemic brain disease, comprising the step of preparing a mixed extract of Angelica Angelica and Buttercup.

Another example provides a pharmaceutical composition for concomitant administration or a kit for co-administration for the prevention and / or treatment of ischemic brain disease, neuronal cell protection, and / or neuronal cell regeneration (or production), including Angelica keiskei koidz extract and parsley extract. do. The pharmaceutical composition for co-administration may include a single Angelica extract and a parsley extract in one dosage form, or may include a dosage unit included in each different dosage form. Another example is a method for preventing and / or treating neurological diseases, comprising administering an Angelica keiskei koidus extract to a subject in need and preventing or / or treating ischemic brain disease, and administering a buttercup extract to the subject. Methods of protection and / or methods of regenerating (or generating) neurons are provided. The administration step of the Angelica keiskei kombu and the administration step of the buttercup extract may be carried out simultaneously or irrespective of the sequence.

Provided herein is the use of a mixed extract of Angelica gigas Nakai and buttercup to prevent and / or treat ischemic brain disease, and to protect and / or regenerate neurons.

Angelica is a perennial herbaceous plant belonging to the mountain family, and is mainly cultivated for medicinal purposes in Korea, Japan, and China. Angelica is divided into true Angelica (Angelica gigas Nakai), ildanggwi produced in Japan (Angelica acutiloba Kitagawa), and Chinese angelica (Angelica sinensis Diels) produced in China are produced in Korea according to its origin, its composition and pharmacological effect Is known to be different. The Angelica extract used herein may be obtained using the root of Angelica (eg, dried and shredded Angelica root).

The buttercup used in this specification may be a water dropwort ( Oenanthe javanica ). Dolphin is a perennial herb belonging to the family Apiaceae ( Umbelliferae ) and is a dicotyledonous plant. The stone parsley is wild in wetlands or watersides, and has more leaves than the water parsley, and the stem is short and the red under the stem. In addition, it has a unique flavor and is used for food, has antipyretic, soothing, and constipation prevention effects, and is known to have antibacterial effects. The buttercup extract used in the present specification may be obtained by using a whole plant (outpost) or a part of the stone buttercup, and a part of the stone buttercup may be one or more selected from the group consisting of the upper part of the stone buttercup, specifically, a flower, a stem, and a leaf. It can be a site.

As used herein, 'treatment' refers to alleviating or ameliorating symptoms, reducing the extent of a disease, delaying or alleviating disease progression, improving a condition or symptom, alleviating or stabilizing, partially or fully recovering, prolonging survival, or other beneficial benefits. It is used in a sense including all treatment results. 'Prevention' is used in a sense to include all mechanisms and / or effects that act on a subject who does not have a specific disease to prevent the specific disease from developing, or to slow the onset of the disease.

In the present specification, it is confirmed that the mixed extract of Angelica keiskei and buttercup shows a protective / neuronal regeneration effect against brain cell damage due to cerebral ischemia, and the neuronal protective effect and / or nerve cell regeneration of the mixed extract of Angelica keiskei and the buttercup (Or produce) effect, and treatment and / or prevention and / or improvement effect on ischemic brain disease.

Thus, an example is a pharmaceutical composition for the prevention and / or treatment of ischemic brain disease comprising a mixed extract of Angelica gigas Nakai and buttercup as an active ingredient, a pharmaceutical composition for protecting neurons, and / or regeneration of neurons (or Pharmaceutical composition for production). Another example is ischemic brain disease comprising the step of administering a mixed extract of Angelica Angelica and buttercup to a subject in need of prevention and / or treatment of ischemic brain disease, neuronal cell protection, and / or neuronal regeneration (or production). Provided is a method of preventing and / or treating, a method of protecting neurons, and / or a method of regenerating (or generating) neurons. The mixed extract of Angelica keiskei and buttercup may be a mixture of Angelica keiskei extract and buttercup extract, an extract of a mixture of Angelica keiskei and buttercup, or a combination thereof. The neurons may be neurons of the brain (eg, hippocampus (eg, CA1 region, etc.)), such as neurons, glial cells (eg, astrocytes, microglia, oligodendrocytes) (oligodendrocyte, etc.) may be one or more selected from the group consisting of, but is not limited thereto.

Another example provides a pharmaceutical composition for co-administration for the prevention and / or treatment of ischemic brain disease, the protection of neurons, and / or the regeneration (or production) of neurons, including the Angelica keiskei kombu extract and the buttercup extract. Another example provides a kit for concomitant administration for the prevention and / or treatment of ischemic brain disease, the protection of neurons, and / or the regeneration (or generation) of neurons, including the Angelica keiskei koidus extract and the buttercup extract. The Angelica kerosene extract and the buttercup extract contained in the pharmaceutical composition or kit for co-administration may be in the form of being formulated together (for example, in the form of a mixture), or may be respectively formulated and included in one dosage unit. Another example is the step of administering the Angelica extract to a subject in need of prevention and / or treatment of ischemic brain disease, neuronal cell protection, and / or neuronal regeneration (or production), and administering the buttercup extract to the subject. Provided is a method for preventing and / or treating ischemic brain disease, a method for protecting neurons, and / or a method for regenerating (or generating) neurons. The administration step of the Angelica keiskei kombu and the administration step of the buttercup extract may be carried out simultaneously or irrespective of the sequence.

The Angelica Angelica extract may be obtained by extracting Angelica Angelica (eg, root) with one or more extraction solvents selected from the group consisting of water and straight or branched alcohol having 1 to 4 carbon atoms. In one example, the Angelica Angelica extract is 40-100% (v / v), 50-100% (v / v), 60-100% (v / v), 70-100 of the Angelica (eg, root) % (v / v), 80-100% (v / v), 90-100% (v / v), 92-100% (v / v), 95-100% (v / v), 96-100 % (v / v), 97 to 100% (v / v), or 98 to 100% (v / v) ethanol aqueous solution (eg, 98% (v / v) ethanol aqueous solution (sold)) extract. In addition, the Angelica keiskei kombu is 10 to 80 ℃, 10 to 70 ℃, 10 to 60 ℃, 10 to 50 ℃, 20 to 80 ℃, 20 to 70 ℃, 20 to 60 ℃, 20 to 50 ℃, 30 to 80 It may be extracted at ℃, 30 to 70 ℃, 30 to 60 ℃, 30 to 50 ℃, 40 to 80 ℃, 40 to 70 ℃, 40 to 60 ℃, or 40 to 50 ℃.

The buttercup extract is extracted with one or more extraction solvents selected from the group consisting of water and straight or branched alcohols having 1 to 4 carbon atoms, for example, one or more sites selected from the group consisting of flowers, stems, leaves, and outposts. It may be obtained. In one example, the buttercup extract is 10 to 80% (v / v), 10 to 70% (v / v), 10 to 65% (of buttercup (eg, flower, stem, leaf, and / or outpost, etc.)) v / v), 10-60% (v / v), 10-55% (v / v), 10-52% (v / v), 10-50% (v / v), 10-49% ( v / v), 10-48% (v / v), 10-47% (v / v), 10-46% (v / v), 10-45% (v / v), 20-80% ( v / v), 20-70% (v / v), 20-65% (v / v), 20-60% (v / v), 20-55% (v / v), 20-52% ( v / v), 20-50% (v / v), 20-49% (v / v), 20-48% (v / v), 20-47% (v / v), 20-46% ( v / v), 20-45% (v / v), 30-80% (v / v), 30-70% (v / v), 30-65% (v / v), 30-60% ( v / v), 30-55% (v / v), 30-52% (v / v), 30-50% (v / v), 30-49% (v / v), 30-48% ( v / v), 30-47% (v / v), 30-46% (v / v), or 30-45% (v / v) ethanol extract. In addition, the buttercup extract, the pH at the time of extraction, such as 8 to 10, 8.5 to 10, 8.8 to 10, 8 to 9.5, 8.5 to 9.5, 8.8 to 9.5, 8 to 9.2, It may be obtained by adjusting to 8.5 to 9.2, or 8.8 to 9.2. By extracting the buttercup in the above pH range, an extract having a high active ingredient content in the extract can be obtained. The extraction pH can be adjusted by adding a pH adjusting agent with an extraction solvent to the buttercup. In one example, the pH adjusting agent may be at least one selected from the group consisting of sodium hydroxide, calcium hydroxide, potassium hydroxide, etc., but is not limited thereto, and may be selected from all substances capable of adjusting the pH to the above range. . In addition, the buttercup extract is 30 to 100 ℃, 30 to 98 ℃, 30 to 95 ℃, 30 to 94 ℃, 30 to 93 ℃, 30 to 92 ℃, 30 to 91 ℃, 30 to 90 ℃, 50 to 100 ℃ , 50 to 98 ℃, 50 to 95 ℃, 50 to 94 ℃, 50 to 93 ℃, 50 to 92 ℃, 50 to 91 ℃, 50 to 90 ℃, 70 to 100 ℃, 70 to 98 ℃, 70 to 95 ℃ , 70 to 94 ℃, 70 to 93 ℃, 70 to 92 ℃, 70 to 91 ℃, 70 to 90 ℃, 75 to 100 ℃, 75 to 98 ℃, 75 to 95 ℃, 75 to 94 ℃, 75 to 93 ℃ , 75 to 92 ℃, 75 to 91 ℃, 75 to 90 ℃, 80 to 100 ℃, 80 to 98 ℃, 80 to 95 ℃, 80 to 94 ℃, 80 to 93 ℃, 80 to 92 ℃, 80 to 91 ℃ , 80 to 90 ℃, 85 to 100 ℃, 85 to 98 ℃, 85 to 95 ℃, 85 to 94 ℃, 85 to 93 ℃, 85 to 92 ℃, 85 to 91 ℃, 85 to 90 ℃, 90 to 100 ℃ , It may be extracted from 90 to 98 ℃ or 90 to 95 ℃.

The extract of the mixture of Angelica Angelica and Buttercup extracts a mixture of Angelica Angelica (root) and buttercup (flower, stem, leaf, outpost, etc.) with one or more selected from the group consisting of water and straight or branched alcohol having 1 to 4 carbon atoms. It may be obtained by extraction with a solvent.

In one example, the mixed extract of the Angelica and buttercup,

(1) (i) 40 to 100% (v / v), 50 to 100% (v / v), 60 to 100% (v / v), 70 to 100% (v / v) of the Angelica (root) ), 80 to 100% (v / v), 90 to 100% (v / v), 92 to 100% (v / v), 95 to 100% (v / v), 96 to 100% (v / v) ), 97 to 100% (v / v), or 98 to 100% (v / v) ethanol aqueous solution (e.g. 98% (v / v) ethanol aqueous solution (drink)) extract and (ii) buttercup (e.g. flower , Stem, leaf, outpost, etc.) 10 to 80% (v / v), 10 to 70% (v / v), 10 to 65% (v / v), 10 to 60% (v / v), 10 To 55% (v / v), 10 to 52% (v / v), 10 to 50% (v / v), 10 to 49% (v / v), 10 to 48% (v / v), 10 To 47% (v / v), 10 to 46% (v / v), 10 to 45% (v / v), 20 to 80% (v / v), 20 to 70% (v / v), 20 To 65% (v / v), 20 to 60% (v / v), 20 to 55% (v / v), 20 to 52% (v / v), 20 to 50% (v / v), 20 To 49% (v / v), 20 to 48% (v / v), 20 to 47% (v / v), 20 to 46% (v / v), 20 to 45% (v / v), 30 To 80% (v / v), 30 to 70% (v / v), 30 to 65% (v / v), 30 to 60% (v / v), 30 to 55% (v / v), 30 To 52% (v / v), 30 to 50% (v / v), 30 to 49% (v / v), 30 to 48% (v / v), 30 to 47% (v / v), 30 To 46% (v / v), or 30 to 45% (v / v) aqueous ethanol solution (eg, 50% (v / v) ethanol aqueous solution) extract (optionally, pH 8 to 10, 8.5 to 10, 8.8 to 10, 8 to 9.5, 8.5 to 9.5, 8.8 to 9.5, 8 to 9.2, 8.5 to 9.2, or 8.8 to 9.2 extracts extracted under conditions), or

(2) 40 to 100% (v / v), 45 to 100% (v / v), 48 to 100% of the mixture of Angelica (root) and buttercup (e.g., seed, bud, flower, outpost, etc.) v / v), 50 to 100% (v / v), 60 to 100% (v / v), 70 to 100% (v / v), 80 to 100% (v / v), 90 to 100% ( v / v), 92 to 100% (v / v), 96 to 100% (v / v), or 98 to 100% (v / v) ethanol aqueous solution extract

(3) or combinations thereof

Can be

The Angelica extract in the mixture of the Angelica keiskei koki extract and the buttercup extract is 10 to 80 ° C, 10 to 70 ° C, 10 to 60 ° C, 10 to 50 ° C, 20 to 80 ° C, 20 to 70 ° C, 20 to 60 ° C, 20 It may be extracted from 50 to 50 ° C, 30 to 80 ° C, 30 to 70 ° C, 30 to 60 ° C, 30 to 50 ° C, 40 to 80 ° C, 40 to 70 ° C, 40 to 60 ° C, or 40 to 50 ° C ; And / or the buttercup extract is 30 to 100 ° C, 30 to 98 ° C, 30 to 95 ° C, 30 to 94 ° C, 30 to 93 ° C, 30 to 92 ° C, 30 to 91 ° C, 30 to 90 ° C, 50 to 100 ° C , 50 to 98 ℃, 50 to 95 ℃, 50 to 94 ℃, 50 to 93 ℃, 50 to 92 ℃, 50 to 91 ℃, 50 to 90 ℃, 70 to 100 ℃, 70 to 98 ℃, 70 to 95 ℃ , 70 to 94 ℃, 70 to 93 ℃, 70 to 92 ℃, 70 to 91 ℃, 70 to 90 ℃, 75 to 100 ℃, 75 to 98 ℃, 75 to 95 ℃, 75 to 94 ℃, 75 to 93 ℃ , 75 to 92 ℃, 75 to 91 ℃, 75 to 90 ℃, 80 to 100 ℃, 80 to 98 ℃, 80 to 95 ℃, 80 to 94 ℃, 80 to 93 ℃, 80 to 92 ℃, 80 to 91 ℃ , 80 to 90 ℃, 85 to 100 ℃, 85 to 98 ℃, 85 to 95 ℃, 85 to 94 ℃, 85 to 93 ℃, 85 to 92 ℃, 85 to 91 ℃, 85 to 90 ℃, 90 to 100 ℃ , It may be extracted from 90 to 98 ℃ or 90 to 95 ℃. In addition, the extract of the mixture of Angelica keiskei and buttercup is 10 to 80 ° C, 10 to 70 ° C, 10 to 60 ° C, 10 to 50 ° C, 20 to 80 ° C, 20 to 70 ° C, 20 to 60 ° C, 20 to 50 It may be extracted from ℃, 30 to 80 ℃, 30 to 70 ℃, 30 to 60 ℃, 30 to 50 ℃, 40 to 80 ℃, 40 to 70 ℃, 40 to 60 ℃, or 40 to 50 ℃.

The mixing ratio of the Angelica keiskei extract and the parsley extract in the Angelica keiskei extract and the buttercup mixture extract, or the mixing ratio of the Angelica keiskei and the buttercup in the Angelica kerosene mixture is 1: 0.1 to 10, 1: 0.1 to 5, 1: 0.1 to 4, 1 : 0.1 to 3, 1: 0.1 to 2, 1: 0.1 to 1.5, 1: 0.1 to 1.2, 1: 0.5 to 10, 1: 0.5 to 5, 1: 0.5 to 4, 1: 0.5 to 3, 1: 0.5 To 2, 1: 0.5 to 1.5, 1: 0.5 to 1.2, 1: 0.8 to 10, 1: 0.8 to 5, 1: 0.8 to 4, 1: 0.8 to 3, 1: 0.8 to 2, 1: 0.8 to 1.5 , 1: 0.8 to 1.2, or 1: 0.9 to 1.1 (above, Angelica kerosene weight or solid content weight of Angelica kerosene extract: parsley weight or parsley extract solid content weight). The solids weight means the weight of the solids remaining after removing the solvent component of the extract. This is a term used to indicate that when the mixture is a mixture of a true Angelica extract and a buttercup extract, the mixing ratio means the ratio between the weights of the active ingredients with the extraction solvent removed so as not to be affected by the properties and / or concentration of the extract. .

The mixed extract of Angelica Angelica and Buttercup included as an active ingredient in the pharmaceutical composition may be in the form of a dried product, a concentrated product, or a concentrated dried product.

The content of the mixed extract of Angelica keiskei and buttercup contained as an active ingredient in the pharmaceutical composition can be appropriately adjusted according to the type and purpose of use, the patient's condition, the type and severity of the symptoms, and 0.001 to 99.9% by weight based on the solid content weight. 0.01 to 99.9 wt%, 0.1 to 99.9 wt%, 1 to 99.9 wt%, 10 to 99.9 wt%, 30 to 99.9 wt%, 40 to 99.9 wt%, 50 to 99.9 wt%, 0.001 to 90 wt%, 0.01 to 90 wt%, 0.1 to 90 wt%, 1 to 90 wt%, 10 to 90 wt%, 30 to 90 wt%, 40 to 90 wt%, 50 to 90 wt%, 0.001 to 70 wt%, 0.01 to 70 wt% %, 0.1 to 70% by weight, 1 to 70% by weight, 10 to 70% by weight, 30 to 70% by weight, 40 to 70% by weight, 50 to 70% by weight, 0.001 to 50% by weight, 0.01 to 50% by weight, It may be 0.1 to 50% by weight, 1 to 50% by weight, 10 to 50% by weight, 30 to 50% by weight, or 40 to 50% by weight, but is not limited thereto. The solid content weight means the weight of the solid material from which the solvent component is removed from the extract as described above.

As used herein, ischemic brain disease is caused by blockage of cerebral blood flow caused by clogging of the cerebral blood vessels, resulting in brain damage (e.g., loss of nerve cells, loss of function, or damage due to loss of function, etc.). As a disease, depending on the location of a blocked cerebrovascular or damaged brain region, it may be selected from all diseases that exhibit symptoms such as loss of motor control (systemic or local paralysis), speech disorder, visual field disorder, dizziness, loss of room, and the like. The ischemic brain disease may be selected from ischemic cerebrovascular disease and ischemic cranial nerve disease (degenerative neurological disease), for example, stroke (eg, cerebral infarction or cerebral hemorrhage), hypertension, dementia (eg, vascular dementia, etc.), Parkinson's disease, It can be selected from the group consisting of Huntington's disease, Lou Gehrig's disease, and the like.

In addition, as used herein, neuronal protection, and / or neuronal regeneration (or production) may refer to neuronal protection from neuronal damage by brain ischemia and / or regeneration of damaged neurons. The neurons may be neurons of the brain (eg, hippocampus (eg, CA1 region, etc.)), such as neurons, glial cells (eg, astrocytes, microglia, oligodendrocytes) (oligodendrocyte, etc.) may be one or more selected from the group consisting of, but is not limited thereto.

The pharmaceutical composition may be administered by various routes to mammals, including humans, dogs, cats, horses, cows, pigs, goats, rabbits, mice, rats, etc., or cells, tissues, or cultures isolated therefrom. . The mode of administration may be any of the commonly used modes, for example, parenteral administration, such as oral administration, intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, topical administration of lesion sites (eg, brain, spinal cord, etc.). Can be. The pharmaceutical composition is formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. according to a conventional method, transdermal preparations, suppositories, parenteral formulations in the form of sterile injectable solutions, etc. Can be used.

The pharmaceutical composition may further contain an auxiliary agent such as a pharmaceutically acceptable carrier, excipient, and / or diluent in addition to the extract. Examples of such carriers, excipients, or diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, And one or more selected from the group consisting of microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, one or more diluents or excipients selected from the group consisting of fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are usually used may be used. Solid preparations for oral administration include one or more selected from the group consisting of tablets, pills, powders, granules, capsules, syrups, powders, suspensions, etc., and these solid preparations include at least one excipient in the extract, For example, it may be prepared by mixing one or more selected from the group consisting of starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition, lubricants such as magnesium styrene talc are used in addition to simple excipients. Liquid preparations for oral administration include one or more selected from the group consisting of suspending agents, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used, various excipients, such as wetting agents and sweeteners , One or more selected from the group consisting of fragrances, preservatives, and the like may be included. Formulations for parenteral administration include one or more selected from the group consisting of sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and transdermal preparations. As the non-aqueous solvent and suspension, one or more selected from the group consisting of propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.

The pharmaceutical composition may be administered in a pharmaceutically effective amount. The dosage of the pharmaceutical composition is variously prescribed by factors such as formulation method, administration method, patient's age, weight, sex, morbidity, food, administration time, administration interval, administration route, excretion rate and response sensitivity. Can be. The dosage may vary depending on the patient's age, weight, sex, dosage form, health condition, and degree of disease, and may be dividedly administered once to several times a day at regular time intervals according to the judgment of a doctor or pharmacist. For example, a single or daily dose of the pharmaceutical composition is 0.0001 to 10000 mg / kg, specifically, 0.001 to 1000 mg / kg, 0.001 to 500 mg, based on the solid content weight of the active ingredient (mixed extract of Angelica kelp and buttercup) / kg, 0.001 to 300mg / kg, 0.001 to 250mg / kg, 0.01 to 1000mg / kg, 0.01 to 500mg / kg, 0.01 to 300mg / kg, 0.01 to 250mg / kg, 0.1 to 1000mg / kg, 0.1 to 500 mg / kg, 0.1 to 300 mg / kg, 0.1 to 250 mg / kg, 1 to 1000 mg / kg, 1 to 500 mg / kg, 1 to 300 mg / kg, 1 to 250 mg / kg , 10 to 1000mg / kg, 10 to 500mg / kg, 10 to 300mg / kg, or 10 to 250mg / kg, but is not limited thereto. The single or daily dosage may be formulated as a single dosage form in unit dose form, or may be prepared by appropriately dispensing, or incorporated into a multi-dose container. The above-mentioned dosage is an example of an average case, and the dosage may be high or low according to individual differences.

Another example provides a method for preparing a composition having an effect of preventing, treating and / or improving ischemic brain disease, comprising the step of preparing a mixed extract of Angelica Angelica and Buttercup.

The step of preparing a mixed extract of Angelica keiskei and buttercup may include 1) mixing an Angelica keiskei extract and a buttercup extract, or 2) extracting a mixture of Angelica keiskei and a buttercup with an extraction solvent. The extraction solvent and extraction temperature used in the extraction step may refer to the description of the mixed extract of Angelica keiskei and buttercup.

For example, the step 1) mixing the Angelica keiskei kombu extract and buttercup extract

i) Angelica (eg, root), 10 to 80 ° C, 10 to 70 ° C, 10 to 60 ° C, 10 to 50 ° C, 20 to 80 ° C, 20 to 70 ° C, 20 to 60 ° C, 20 to 50 ° C , 30 to 80 ° C, 30 to 70 ° C, 30 to 60 ° C, 30 to 50 ° C, 40 to 80 ° C, 40 to 70 ° C, 40 to 60 ° C, or 40 to 50 ° C, 1 to 10 volume times, 2 1 to 8 vol.x, or 4 to 6 vol. Of water and one or more selected from the group consisting of straight or branched alcohols having 1 to 4 carbon atoms (eg, ethanol), such as 40 to 100% (v / v), 50 to 100% (v / v), 60 to 100% (v / v), 70 to 100% (v / v), 80 to 100% (v / v), 90 to 100% (v / v), 92 to 100% (v / v), 95 to 100% (v / v), 96 to 100% (v / v), 97 to 100% (v / v), or 98 to 100% (v / v) Extracting with ethanol aqueous solution (e.g., 98% (v / v) ethanol aqueous solution (drink)),

ii) buttercups (eg, flowers, stems, leaves, outposts), 10 to 80 ° C, 10 to 70 ° C, 10 to 60 ° C, 10 to 50 ° C, 20 to 80 ° C, 20 to 70 ° C, 20 to 60 ℃, 20 to 50 ℃, 30 to 80 ℃, 30 to 70 ℃, 30 to 60 ℃, 30 to 50 ℃, 30 to 45 ℃, 35 to 60 ℃, 35 to 50 ℃, 35 to 45 ℃, 35 to 60 At ℃, 35 to 50 ° C, or 35 to 45 ° C, 1 to 10 volumes, 2 to 8 volumes, or 4 to 6 volumes of water and straight or branched alcohols having 1 to 4 carbon atoms (such as ethanol) One or more selected from the group consisting of, for example, 10 to 80% (v / v), 10 to 70% (v / v), 10 to 65% (v / v), 10 to 60% (v / v) , 10 to 55% (v / v), 10 to 52% (v / v), 10 to 50% (v / v), 10 to 49% (v / v), 10 to 48% (v / v) , 10-47% (v / v), 10-46% (v / v), 10-45% (v / v), 20-80% (v / v), 20-70% (v / v) , 20 to 65% (v / v), 20 to 60% (v / v), 20 to 55% (v / v), 20 to 52% (v / v), 20 to 50% (v / v) , 20 to 49% (v / v), 20 to 48% (v / v), 20 to 47% (v / v), 20 to 46% (v / v), 20 to 45% (v / v) , 30 to 80% (v / v), 30 to 70% (v / v), 30 to 65% (v / v), 30 to 60% (v / v), 30 to 55% (v / v) , 30 to 52% (v / v), 30 to 50% (v / v), 30 to 49% (v / v), 30 to 48% (v / v), 30 to 47% (v / v) , Extracting with 30 to 46% (v / v), or 30 to 45% (v / v) ethanol aqueous solution (e.g., 50% (v / v) ethanol aqueous solution), and

iii) mixing the Angelica kerosene extract extracted in step i) and the buttercup extract extracted in step ii)

It may include.

The step of preparing the buttercup extract of step ii), optionally, the pH of the mixture of the buttercup and the extraction solvent is 8 to 10, 8.5 to 10, 8.8 to 10, 8 to 9.5, 8.5 to 9.5, 8.8 to 9.5, 8 to 9.2, It may be performed by adjusting to 8.5 to 9.2, or 8.8 to 9.2, and the pH may be adjusted by adding a conventional pH adjusting agent, and the pH adjusting agent is one selected from the group consisting of sodium hydroxide, potassium hydroxide, calcium hydroxide, and the like. This may include, but is not limited to.

The step of preparing the Angelica keiskei koid extract in step i) and the step of preparing the buttercup extract in step ii) may be carried out simultaneously or simultaneously.

The step 2) of extracting a mixture of Angelica keiskei and a buttercup with an extraction solvent is i ') mixing Angelica kelp (root) and a parsley (eg, seed, bud, flower, outpost, etc.) to prepare a mixture, And ii ') the mixture is 10 to 80 ° C, 10 to 70 ° C, 10 to 60 ° C, 10 to 50 ° C, 20 to 80 ° C, 20 to 70 ° C, 20 to 60 ° C, 20 to 50 ° C, 30 to At 80 ° C, 30 to 70 ° C, 30 to 60 ° C, 30 to 50 ° C, 40 to 80 ° C, 40 to 70 ° C, 40 to 60 ° C, or 40 to 50 ° C, 1 to 10 volumes, 2 to 8 volumes One or more selected from the group consisting of pears or 4 to 6 volumes of water and straight or branched alcohols having 1 to 4 carbon atoms (such as ethanol), such as 40 to 100% (v / v), 45 to 100 % (v / v), 48-100% (v / v), 50-100% (v / v), 60-100% (v / v), 70-100% (v / v), 80-100 % (v / v), 90-100% (v / v), 92-100% (v / v), 95-100% (v / v), 96-100% (v / v), 97-100 % (v / v), or 98 to 100% (v / v) ethanol.

The mixing ratio between the Angelica keiskei koki extract and the buttercup extract or the mixing ratio of Angelica keiskei and the buttercup is as described above.

The extraction time of the extraction step of the Angelica kelp and / or buttercup is sufficient as long as it can be sufficiently extracted, and 1 hour or more, 2 hours or more, 3 hours or more, or 4 hours or more, such as 1 to 24 hours, 2 To 24 hours, 3 to 24 hours, 4 to 24 hours, 1 to 12 hours, 2 to 12 hours, 3 to 12 hours, 4 to 12 hours, 1 to 6 hours, 2 to 6 hours, 3 to 6 hours, or It may be set to about 4 to 6 hours, but is not limited thereto.

The extraction process used in the above method can be aquatic by all commonly used extraction methods, for example, it can be performed by one or more methods selected from the group consisting of hot water extraction, ultrasonic extraction, reflux extraction, and the like. It is not limited. The method may further include, after the extraction process, optionally drying and / or concentrating the extract in a conventional manner.

Another example provides a health functional food for preventing and / or ameliorating ischemic brain disease, protecting neurons, and / or regenerating (or producing) nerve cells containing an Angelica Angelica extract, or a mixed extract of Angelica Angelica and buttercup.

The health functional food is a food prepared using nutrients or ingredients or ingredients (hereinafter referred to as 'functional raw materials') that are useful for the human body that are easily deficient in everyday meals, and maintains health or prevents certain diseases or symptoms, and / Or means any food that helps to improve, and there are no specific restrictions on the final product form. For example, the health functional food may be selected from the group consisting of various foods, beverage compositions, and food additives, but is not limited thereto.

The content of the Angelica Angelica extract contained in the health functional food or the mixture of Angelica Angelica and buttercup is not particularly limited according to the type of food, desired use, etc., for example, 0.001 to 99% by weight of the total food weight, 0.01 To 99 wt%, 0.01 to 95 wt%, 0.01 to 90 wt%, 0.01 to 80 wt%, 0.01 to 50 wt%, 0.1 to 99 wt%, 0.1 to 95 wt%, 0.1 to 90 wt%, 0.1 to 80 Weight percent, 0.1 to 50 weight percent, 0.1 to 30 weight percent, 0.1 to 10 weight percent, 1 to 99 weight percent, 1 to 95 weight percent, 1 to 90 weight percent, 1 to 80 weight percent, 1 to 50 weight percent , 1 to 30 wt%, 1 to 10 wt%, 10 to 99 wt%, 10 to 95 wt%, 10 to 90 wt%, 10 to 80 wt%, 10 to 50 wt%, 10 to 30 wt%, 25 To 99 wt%, 25 to 95 wt%, 25 to 90 wt%, 25 to 80 wt%, 25 to 50 wt%, 25 to 30 wt%, 40 to 99 wt%, 40 to 95 wt%, 40 to 90 Wt%, 40-80 wt%, 40-50 wt%, 50-99 wt%, 50-95 wt%, 50-90 wt%, 50-80 wt%, 60-99 wt%, 60-95 wt% , 60 to 90% by weight, or 60 to 80% by weight.

The health functional foods include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic or natural flavoring agents, coloring agents, neutralizing agents (cheese, chocolate, etc.), pectic acid or salts thereof, alginic acid or salts thereof, It may further contain one or more selected from the group consisting of organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like. The ratio of these additives is generally selected from the range of 0.001 to about 20 parts by weight per 100 parts by weight of the total health functional food, but is not limited thereto.

Proposing a new treatment method with excellent effects in both prevention and treatment of ischemic brain disease by proposing a protective effect from brain cell ischemia caused by cerebral ischemia and a regenerated (or produced) effect of impaired nerve cells. do.

1 ischemic brain disease of 200 mg / kg of buttercup extract, 200 mg / kg of Angelica kerosene extract, or 200 mg / kg of mixed extract (100 mg / kg of Angelica kerosene + 100 mg / kg of buttercup extract) according to one embodiment. In order to investigate the preventive effect, it is a picture taken by staining the hippocampus tissue and the CA1 region of the hippocampus tissue with cresyl violet (CV) after 5 days of inducing brain ischemia after administration of these extracts. .
2a ischemic brain disease of 200 mg / kg buttercup extract, 200 mg / kg extract of Angelica keiskei koidz, or 200 mg / kg of mixed extract (100 mg / kg of Angelica kelp + 100 mg / kg of buttercup extract) according to one embodiment. In order to investigate the preventive effect, immune tissues using mouse anti-neuronal nuclei (NeuN) antibody, which is a marker for neurons, in the CA1 region of the hippocampus of 5 days after inducing brain ischemia after administration of these extracts This is a picture taken by staining with chemistry (immunohistochemistry), and 2b is a graph showing the counting of neurons having immunoreactivity to NeuN antibodies.
Figure 3a ischemic brain disease of 200 mg / kg of buttercup extract, 200 mg / kg of Angelica kerosene extract, or 200 mg / kg of mixed extract (100 mg / kg of Angelica kerosene + 100 mg / kg of buttercup extract) according to an embodiment In order to investigate the prophylactic effect, fluoro-jade, a marker for neurons in the process of killing or killing the CA1 region of the hippocampus of an experimental animal 5 days after inducing brain ischemia after administration of these extracts b, FJ B) is a picture taken by staining, 3b is a graph showing the counting of cells showing a positive response to FJ B.
Figure 4a ischemic brain disease of 200 mg / kg of buttercup extract, 200 mg / kg of Angelica kerosene extract, or 200 mg / kg of mixed extract (100 mg / kg of Angelica kerosene + 100 mg / kg of buttercup extract) according to an embodiment In order to investigate the preventive effect, the mouse anti-glial fibrillary acidic protein (GFAP) antibody, which is a marker for astrocytes, in the CA1 region of the hippocampus of 5 days after the induction of cerebral ischemia after administration of these extracts Is a photograph taken by staining with immunohistochemistry using, 4b is a graph showing the immune response of cells stained with GFAP antibody as a relative value for the vehicle (sham).
5A ischemic brain disease of 200 mg / kg buttercup extract, 200 mg / kg extract of Angelica keiskei koidz, or 200 mg / kg of mixed extract (100 mg / kg of Angelica kerosene + 100 mg / kg of buttercup extract) according to an embodiment. In order to investigate the prophylactic effect, rabbit anti-ionized calcium-binding adapter molecule 1, a marker for microglia, in the CA1 region of the hippocampus of an experimental animal 5 days after inducing brain ischemia after administration of these extracts (Iba-1) is a picture taken by immunohistochemistry using an antibody, and 5b is a graph showing the immune response of cells stained with Iba-1 antibody as a relative value to a normal group.

Hereinafter, the present invention will be described in more detail with reference to examples, but these are merely illustrative and are not intended to limit the scope of the present invention. It is apparent to those skilled in the art that the embodiments described below can be modified without departing from the essential gist of the invention.

Example  1: Preparation of extract

1.1. Preparation of Angelica Angelica Extract

The root of Angelica gigas Nakai was washed with clean water and dried sufficiently. After crushing the dried root of Angelica root, 5 volume fold (500 ml) of ethanol (98% (v / v) ethanol (sold)) was added to 100 g of the obtained powder, and extracted at 40-60 ° C. for 4 hours or more, followed by 1 um ( The filtrate filtered with a micrometer) filter was concentrated to warm until 10% (w / w) of the original weight. Crystalline cellulose was gradually added to the obtained concentrate while continuing to concentrate and dried completely, followed by pulverization to prepare ethanol extract of Angelica keiskei koidz (hereinafter referred to as AGE).

1.2. Preparation of buttercup extract

Buttercups from Chuncheon, Gangwon-do ( Oenanthe javanica ) After crushing 700 g, dilute NaOH was added to 3.5 L of an ethanol aqueous solution having a concentration of 50% or less, and a solvent adjusted to PH 9 was repeatedly extracted three times at 90 ° C for 24 hours in an extractor with a reflux cooler. This extract was filtered using a vacuum filter for Whatman No. 1 (Whatman Ltd, Maidstone, Kent, UK) was filtered using a filter paper, and the filtrate was concentrated under reduced pressure at 50 ° C using a rotary vacuum concentrator (EYELA, SN-1100, Tokyo, Japan). The concentrated extract was prepared using a freeze dryer (PVTFA 10AT, ILSIN, Korea) at a rapid freezing process at -70 ° C to prepare a buttercup extract in powder form (hereinafter referred to as OJE).

Example  2: Mixed extract of Angelica Angelica and Buttercup Brain ischemia  Neuroprotective effect check

In order to confirm the neuroprotective efficacy of the Angelica keiskei koki extract, parsley extract, or a mixture thereof (mixed extract of Angelica kerosene and parsley) prepared in Example 1, male Mongolian gerbil, Halan having a weight of 65 g to 75 g , USA) to expose the common carotid artery by anesthesia, and then ligation to observe the hippocampus tissue of Mongolian gerbil, which caused cerebral ischemia. Mongolian gerbil is an animal model that is mainly used in connection with neuronal cell death. Ligation of the bilateral allograft arteries of Mongolian gerbil for 5 minutes may cause brain ischemia similar to that of humans. When the brain ischemia is induced in Mongolian gerbil, cell death occurs in the hippocampus CA1 region characteristically after 4 days, and it is confirmed that delayed neuronal death occurred from the phenomenon.

Example  2-1. Breeding of experimental animals

56 light-weight male and female Mongolian gerbil (Mongolian gerbil, Meriones unguiculatus, Halan, USA) weighing 65g to 75m from 7 am to 7 pm constant light and dark cycle, 21 ℃ to 25 ℃ temperature and 45% to 65% Was raised under conditions of relative humidity. As the feed for the experimental animals, a common pellet-dried feed (Korea Experiment Animal, Korea) was used, and the feed and water were always consumed.

Example  2-2. After administration of the extract Brain tongue blood  Preparation of induced brain tissue sections

To the experimental animals reared in Example 2-1, the Angelica kerosene extract, parsley extract, or mixtures thereof (mixed extract of Angelica kelp and parsley) prepared in Example 1 were total 200 per 1 kg of each animal weight. It was dissolved in physiological saline so as to be the dose of mg (in the case of a mixture, 100 mg / kg of Angelica kerosene extract + 100 mg / kg of buttercup extract) (daily dose), and orally administered using sonde for 28 days.

After administration of the extract for 28 days, experiment animals were mixed with gas mixed with 3% (v / v) isoflurane (Baxtor, USA) in a gas mixed with nitrogen and oxygen at a ratio of 7: 3. General anesthesia was performed. The gas was mixed with 2.5% (v / v) isoflurane in the mixed gas of nitrogen and oxygen to perform an operation on the experimental animal while maintaining the anesthesia state of the experimental animal.

In order to cause cerebral ischemia, the animal's neck is shaved and disinfected, and then incised to expose both whole carotid arteries, ligated for 5 minutes using an aneurysmal clip (aneurysm clip, Staelting, USA), and then clip removed. And reperfusion. At this time, each experimental group observed the blood circulation of the central artery of the retina using an ophthalmoscope and observed complete obstruction of the entire artery.

While inducing cerebral ischemia, body temperature was measured by inserting a thermometer in the rectum, and the body temperature was kept constant at 36.8 ° C to 37.2 ° C, which is a normal body temperature, using a heating pad that is automatically adjusted according to the body temperature of the experimental animal.

After 5 days of inducing cerebral ischemia, thiopental sodium (Yuhan Corporation, Korea) was anesthetized by intraperitoneal injection of test animals at a dose of 30 mg per 1 kg of body weight, and then 4 ℃ containing 1,000 IU of heparin per 1,000 mL Physiological saline was injected into the left ventricle and perfused. Perfusion fixation was performed using the 4% paraformaldehyde (0.1 M phosphate buffer (PB), pH 7.4) at 4 ° C. for the animal, where the perfusion washing was completed.

Using a bone cutter, the brain bone was removed by opening the head bone space of the experimental animal after the perfusion was fixed. The brain of the extracted experimental animal was post-fixed for 4 hours in a 4% paraformaldehyde (0.1 M phosphate buffer (PB), pH 7.4) solution at 4 ° C using a stirrer at room temperature. After the post-fixation, the brain was placed in a 30% (w / v) sucrose solution (in 0.1 M phosphate buffer) and allowed to settle to the bottom, and then a sliding microtome (Reichert-Jung, Germany) to cut the brain tissue to a thickness of 30 ㎛ to make a tissue section, it was used as a test group in the following examples. The tissue sections were placed in a 6 well plate containing a storage solution and stored at 4 ° C. until staining was performed.

A tissue section obtained by performing the same process as above was prepared, except that only the physiological saline used to dissolve the extract as a vehicle was orally administered, and the buttercup extract and / or true Angelica extract as a normal group (sham) Tissue sections obtained without administering any substance including and without performing a process for causing cerebral ischemia were prepared.

Example  2-3. Brain ischemia  Neuroprotective efficacy measurement 1 ( Cresyl  Violet dyeing)

Among the tissue sections prepared in Example 2-2, tissues with well-produced hippocampal formation were selected, and washed three times with 10 M PBS (pH 7.4) for 10 minutes to remove the preservatives on the tissue sections. . The washed tissue sections were spread on a gelatin coated slide glass and dried sufficiently at 37 ° C. The tissue sections were immersed in distilled water for a while, followed by soaking in 2% (w / v) cresyl violet acetate (Sigma, USA) solution for 1 minute to perform staining of the tissue sections. Subsequently, the stained tissue sections are thoroughly washed with running water to remove excess dye from the slides, and after soaking in distilled water for a while, 50% (v / v), 70% (v / v), and 80% (v / v), 90% (v / v), 95% (v / v) and 100% (v / v) ethanol solutions were sequentially treated to perform dehydration and excess cresyl violet washing. After confirming that the Nissl body was visible in the tissue section, it was soaked in xylene (Junsey, Japan) and transparent, and then sealed with Canadian balsam (Kanto, Japan).

Each of the normal, control, and experimental tissues is an AxioM1 microscope (AxioM1 microscope, Carl Zeiss, Germany) with a digital camera (Axiocam, Cal Zeiss, Germany), 40 times the total hippocampus area, and 20 CA1 areas. Each tissue section is magnified by a double magnification, and the whole hippocampus area is photographed. A, B, C, D, E, F, G, and H of FIG. 1 are photographed, and the CA1 region of the hippocampus is photographed. 1, a, b, c, d, e, f, g and h, respectively.

As can be seen in FIG. 1, all normal groups (sham groups) were darkly stained with neurons purple throughout the hippocampus region (A, C, E, and G in FIG. 1), and in particular, in the CA1 region, the pyramidal layer ( stratum pyramidale, SP), it was observed that the nerve cells are dense (a, c, e and g in FIG. 1). However, in the case of the control group (vehicle-ischemia) in which only the solvent was administered and induced cerebral ischemia, neuronal cell death occurred in the CA1 region of the hippocampus due to delayed neuronal apoptosis caused by cerebral ischemia, and neuronal staining was hardly observed. (B of FIG. 1), it was confirmed that most of the neurons were lost in the pyramidal layer of the CA1 region (FIG. 1B). In addition, in the experimental group (OJE-ischemia, AGE-ischemia) in which 200 mg / kg of buttercup extract or 200 mg / kg of Angelica kerosene was administered, in the CA1 region of the hippocampus, compared to the control (vehicle-ischemia), it was added to cresyl violet. The staining property was strong, but when compared with the normal group (sham), the staining property for cresyl violet was slightly weaker (D, d, F and f in FIG. 1). On the other hand, in the case of the experimental group (OJE + AGE-ischemia) administered by mixing 100 mg / kg of buttercup extract and 100 mg / kg of Angelica kerosene extract, the control group and 200 mg / kg of buttercup extract or 200 mg / kg of true Compared to the experimental group to which the Angelica extract was administered, a large number of neurons were darkly stained in purple throughout the region of the hippocampus (H in FIG. 1), and it was observed that neurons were densely concentrated in the pyramidal layer of the CA1 region (FIG. 1 h). These results show that when the buttercup extract and the Angelica Angelica extract are administered together, compared to the case when the same amount of the Angelica Angelica extract or the Angelica Angelica extract is administered, neuronal cell death caused by cerebral ischemia is more effectively suppressed.

Example  2-4. Brain ischemia  Nerve cell protection efficacy measurement 2 (Immunohistochemical staining)

Immunohistochemical staining was performed using antibodies against NeuN, a marker of neurons, to confirm the neuroprotective effect of buttercup and Angelica keiskei kombu extract, and more specifically, the effect of inhibiting neuronal cell death due to cerebral ischemia.

More specifically, a tissue section in which hippocampal formation was well selected among the tissue sections prepared in Example 2-2 was selected and used in the experiment, and the selected tissue section was used to store the storage solution present in the tissue. After washing three times for 10 minutes with 0.01M PBS (phosphate buffered saline) for removal, it was reacted for 30 minutes in 0.3% H ₂ O ₂ (in 0.01M PBS) to remove endogenous peroxidase. . After the reaction, the tissue sections were washed three times with 0.01M PBS for 10 minutes each.

For neuronal staining, the tissue sections that were subjected to the above process were reacted with 5% normal horse serum (5% normal horse serum, in 0.01M PBS) for 30 minutes, and then the primary antibody was diluted 1: 800. Anti-NeuN antibody (mouse anti-NeuN, Chemicon, USA) was reacted at 4 ° C. for 48 hours. After the reaction, the tissue sections were reacted with anti-mouse IgG (Vector), a secondary antibody diluted at 1: 250 for 2 hours at room temperature, and then ABC as a tertiary antibody diluted at 1: 200. The solution (avidin-biotin complex, Vector) was reacted for 1 hour at room temperature, and 3,3'-DAB (diaminobenzidine) was developed as a substrate. In performing each step of the process of reacting with the antibody, the tissue sections for each step were washed three times for 10 minutes using 0.01M PBS.

The colored tissue sections were smeared on a gelatin coated slide glass, dried for 12 hours at room temperature, and the dried tissue sections were subjected to a normal dehydration and clarification process to cover glass. ). After performing the reaction with the NeuN antibody, the encapsulated tissue section was observed using a microscope, and the photographed result is shown in FIG. 2A. In addition, the number of neurons obtained by counting neurons in tissue sections (i.e., stained with anti-NeuN antibody) showing immunoreactivity using an image analyzer (Optimas 6.5, USA) program for the photographed picture is shown in FIG. 2B. In order to verify the significance for each experimental group, a one-way ANOVA test was performed.

As shown in Figure 2a, in all normal groups (sham groups), neurons showing immune response to anti-NeuN antibodies in the CA1 region of the hippocampus are well observed (A, C, E and G in Figure 2a), solvent In the case of the control group (vehicle) in which only is administered and induced cerebral ischemia, neuronal cell death occurs in the CA1 region of the hippocampus due to delayed neuronal apoptosis caused by cerebral ischemia, resulting in neurons showing immunoreactivity to anti-NeuN antibodies. Little was observed, and it was confirmed that most of the neurons were lost in the pyramidal layer of the CA1 region (FIG. 2A B). In addition, in the case of the experimental group that induced cerebral ischemia after separately administering the buttercup extract (200 mg / kg; OJE) and Angelica keiskei koidz (200 mg / kg; AGE), immunity to anti-NeuN antibodies in the CA1 region of the hippocampus Reactive neurons decreased significantly compared to the normal group (D, F and I in FIG. 2A). On the other hand, in the experimental group (OJE + AGE) that induced cerebral ischemia after administration of a mixture (200 mg / kg) of a buttercup extract (100 mg / kg) and a true Angelica extract (100 mg / kg), CA1 It was observed that clusters of neurons showing immunoreactivity to the anti-NeuN antibody were concentrated in the region (H and I in FIG. 2A). This result can be confirmed quantitatively through the graph of FIG. 2B. The above results show that the neuroprotective effect in the experimental group inducing cerebral ischemia after administration of the buttercup extract and the true Angelica extract was significantly superior to that of the non-administered group and the single administered group of the extract.

Example  2-5. Brain ischemia  Neuronal cell protective efficacy measurement 3 (tissue fluorescence staining)

Fluoro-Jade B (FJ B), a marker for killed neurons or neurons in the process of death, is used to confirm the neuroprotective effect of a buttercup extract, a true Angelica extract, or a mixed extract of a buttercup and a true Angelica. Tissue fluorescence staining was performed.

More specifically, the tissue fragments in which the hippocampal complex was well selected from the tissue fragments prepared in Example 2-2 were selected and used in the experiment, and tissue fluorescence staining was performed using F-J B, a marker of degenerative neurons. The tissue sections were washed three times for 10 minutes in 0.01M PBS, and then spread on a gelatin coated slide and dried at room temperature for 12 hours. The tissue sections were washed three times for 5 minutes with distilled water, and then reacted with a 0.06% potassium permanganate solution for 20 minutes. After washing with distilled water for 2 minutes, fluorescence staining was performed for 40 minutes in a 0.001% F-J B (Histochem, Jefferson, AR, USA) solution containing 0.1% acetic acid. The dyed tissue was washed with distilled water three times per minute, dried in a 50 ° C slide warmer for 20 minutes, and then immersed in xylene (xylene, Junsei, Japan), and then sealed with DPX (Sigma, USA). The encapsulated tissue was observed using a fluorescence microscope (Ex: 385nmm Em: 425nm), and a photograph of the result is shown in FIG. 3A. In addition, the stained neurons were counted using the image analyzer (Optimas 6.5, USA) program for the photographed picture, and the counted results are shown in FIG. 3B as relative values to the control group (Vehicle administration group).

As shown in FIG. 3A, all the normal groups (sham groups) were not stained by FJ B, which is a marker for the nerve cells in which the entire CA1 region of the hippocampus is killed or nerve cells in the process of death (A in FIG. 3A). C, E and G). On the other hand, in the case of the control group (vehicle-ischemia) only administered with a solvent, it was confirmed to be darkly fluorescently stained in the CA1 region of the hippocampus, and it was confirmed that a number of neurons are in the process of killing or killing (FIG. However, in the experimental group in which the buttercup extract (200 mg / kg; OJE) and the Angelica kerosene extract (200 mg / kg; AGE) were administered alone, some neurons stained by FJ B were observed in the CA1 region of the hippocampus. (D, F and I in Figure 3a). On the other hand, in the experimental group (OJE + AGE) that caused cerebral ischemia after administering a mixture (200 mg / kg) of a buttercup extract (100 mg / kg) and a true Angelica extract (100 mg / kg), it was either killed or in the process of being killed. It was observed that the number of nerve cells was significantly reduced as compared to the control group (Vehicle) as well as the single administration group (OJE and AGE) of the buttercup extract and Angelica kerosene extract (H and I in FIG. 3A). These results can be quantitatively confirmed through the graph of FIG. 3B.

Example  2-6. Brain ischemia  Confirmation of changes in glial cell activity

To further confirm the protective effect of the nerve cells of the buttercup extract, the Angelica Angelica extract, or the mixture of the Angelica and Angelica Angelica, more specifically, the tissue fragment prepared in Example 2-2, in order to further confirm the effect of preventing the death of nerve cells by ischemia. Additionally, astrocytes through immunohistochemistry staining using antibodies to glioma fibrillary acidic protein (GFAP), a marker of astrocytes, and ionized calcium-binding adapter molecule-1 (Iba-1), a marker of microglia (astrocyte) and microglia (microglia) activity changes were confirmed.

More specifically, a tissue section in which hippocampal formation was well selected from the tissue sections prepared in Example 2-2 was selected and used in the experiment, and the selected tissue section removed the storage solution present in the tissue. In order to remove the endogenous peroxidase, the mixture was washed with 0.01M PBS (phosphate buffered saline) three times for 10 minutes, and then reacted for 30 minutes in 0.3% H ₂ O ₂ (in 0.01M PBS). After the reaction, the tissue sections were washed three times with 0.01M PBS for 10 minutes each.

Regarding immunohistochemical staining to check for changes in astrocytes, the tissue sections subjected to the above process are reacted in 5% (v / v) of normal horse serum (in 0.01M PBS) for 30 minutes. In the case of immunohistochemical staining to confirm the change in microglia, the tissue sections that were subjected to the above process were treated with 5% (v / v) normal goat serum (5% normal goat serum, in 0.01M PBSS). The reaction was carried out for a minute. After completion of the reaction, the tissue sections were each diluted with a 1: 800 ratio of primary antibodies, mouse anti-GFAP antibodies (mouse anti-GFAP, Chemicon, USA) and rabbit anti-Iba-1 antibodies (rabbit anti-Iba-1). , Wako, Japan) at 4 ° C for 48 hours. The tissue fragments reacted with the primary antibody were 2 at room temperature with anti-mouse IgG (Vector) and anti-rabbit IgG (Vector) and anti-mouse IgG (Vector), respectively, diluted 1: 250. After reacting for 1 hour, the reaction was performed for 1 hour at room temperature with ABC solution (avidin-biotin complex, Vector), a tertiary antibody diluted 1: 200, and 3,3'-DAB (diaminobenzidine) was developed as a substrate. In performing each step of the process of reacting with the antibody, the tissue sections for each step were washed three times for 10 minutes using 0.01M PBS.

The colored tissue sections were smeared on a gelatin coated slide glass, dried for 12 hours at room temperature, and the dried tissue sections were subjected to a normal dehydration and clarification process to cover glass. ). After performing the reaction with the GFAP or Iba-1 antibody, the encapsulated tissue fragments were observed using a microscope, and photographs of the observation results are shown in FIGS. 4A (astrocytes stained with antibodies to GFAP) and 5A, respectively. (Microglia stained with Iba-1 antibody).

In addition, using the image analyzer (Optimas 6.5, USA) program for the photographed picture, the immunoreactivity of astrocytes stained with antibodies to GFAP and microglia cells stained with Iba-1 antibody was measured. The measurement results are shown in Fig. 4b (glia cells) and 5b (glia cells), respectively.

As shown in FIG. 4A, in the case of all normal groups (sham groups), astrocytes showing immunoreactivity to anti-GFAP antibodies were observed in a dormant form (A, C, E and G in FIG. 4A). On the other hand, in the case of a control group (vehicle-ischemia) administered only with a solvent, astrocytes showing an immune response to an anti-GFAP antibody increased compared to the normal group, and the cytoplasm was enlarged (B and I in FIG. 4A). In addition, astrocytes showing immunoreactivity to anti-GFAP antibodies in the experimental group that caused cerebral ischemia after administration of the buttercup extract (200 mg / kg; OJE) and the Angelica keiskei koidz extract (200 mg / kg; AGE), respectively, were the control group ( vehicle-ischemia), the degree of activation was reduced (D, F and I in FIG. 4A). On the other hand, in the experimental group inducing cerebral ischemia after administration of a mixture mixture (200 mg / kg) of a buttercup extract (100 mg / kg) and a true Angelica extract (100 mg / kg), the immunoreactivity to anti-GFAP antibodies was observed. Visible glial cells were observed in the dormant form as in the normal group (H and I in FIG. 4A).

As shown in FIG. 5A, in the case of all normal groups (sham groups), it was observed that microglial cells showing immune response to anti-Iba-1 antibody were evenly distributed in a dormant form (A in FIG. 5A). C, E and G). In the case of the control group (vehicle-ischemia) in which only the solvent was administered, microglia showing immune response to the anti-Iba-1 antibody was observed in an activated form, particularly in a form gathered in a pyramidal layer (B in FIG. 5A). ). In addition, microglial cells showing immunoreactivity to anti-Iba-1 antibodies in the experimental group that caused cerebral ischemia after administering each of the buttercup extract (200 mg / kg; OJE) and Angelica keiskei koidz (200 mg / kg; AGE), respectively. Compared to the control group (vehicle-ischemia), the degree of activation decreased (D, F and I in FIG. 5A). On the other hand, in the experimental group inducing cerebral ischemia after administration of a mixture (200 mg / kg) of a buttercup extract (100 mg / kg) and a true Angelica extract (100 mg / kg), a dormant state similar to the normal group (Sham) It was observed in the form of (H and I in Figure 5a).

From the above results, compared to the case where the control group and the parsley extract and the Angelica keiskei koi extract were administered alone, the neurons, especially the hippocampus region, of the hippocampus violet in the test animal that caused brain ischemia after administration of the parsley extract and the Angelica keiskei koidz As a result of observing the nerve tissue sections through staining and immunohistochemical staining with the neuronal marker anti-NeuN antibody, it was confirmed that neuronal cell death in the whole region of the hippocampus, specifically, the CA1 region of the hippocampus was suppressed. , As a result of confirming through FJ B, which is a marker for the neurons that are killed or in the process of death, it was confirmed that neuronal cell death was markedly suppressed, and anti-GFAP and anti-Iba- markers of astrocytes and microglia were marked. As a result of confirming through 1 antibody, it was confirmed that abnormal activation of glial cells can be very excellently suppressed. These results demonstrate that when the buttercup extract and Angelica keiskei koidz are mixed, they show a significant synergistic effect in the protection of neuronal cells and the suppression of abnormal activation of glial cells, compared to administration alone.

Claims (17)

Contains a mixed extract of Angelica gigas Nakai and buttercup as an active ingredient,
The mixed extract of the Angelica Angelica and buttercup,
Angelica ethanol extract extracted from Angelica Angelica at 90 to 100% (v / v) ethanol solution at 40 to 80 ° C, and buttercup ethanol extract extracted from Angelica kerosene at 10 to 80% (v / v) ethanol solution at 30 to 100 ° C Or
The mixture of Angelica Angelica and buttercup, extracted from 30 to 80 ° C with 40 to 100% (v / v) ethanol aqueous solution,
Pharmaceutical composition for preventing or treating ischemic brain disease. The method of claim 1, wherein the buttercup ( Oenanthe javanica ), a pharmaceutical composition for preventing or treating ischemic brain disease. The pharmaceutical composition for preventing or treating ischemic brain disease according to claim 1, wherein the buttercup ethanol extract is extracted under pH 8 to 10 conditions. According to claim 1, The mixture ratio between the Angelica or Angelica Angelica extract and the buttercup or buttercup extract in the mixed extract is 1: 0.1 to 10 by weight (weight of Angelica or Angelica extract: weight of Parsley or Parsley extract). , Ischemic brain disease prevention or treatment pharmaceutical composition. According to claim 4, Mixing ratio between the Angelica or Angelica Angelica extract and the buttercup or buttercup extract in the horn mixed extract is 1: 0.2 to 5 by weight (weight of Angelica or Angelica keiskei extract: weight of Parsley or Parsley extract) Phosphorus, ischemic brain disease prevention or treatment pharmaceutical composition. The pharmaceutical composition for preventing or treating ischemic brain disease according to any one of claims 1 to 5, wherein the ischemic brain disease is stroke, hypertension, dementia, Parkinson's disease, Huntington's disease, or Lou Gehrig's disease. Contains a mixed extract of Angelica gigas Nakai and buttercup as an active ingredient,
The mixed extract of the Angelica Angelica and buttercup,
Angelica ethanol extract extracted from Angelica Angelica at 90 to 100% (v / v) ethanol solution at 40 to 80 ° C, and buttercup ethanol extract extracted from Angelica kerosene at 10 to 80% (v / v) ethanol solution at 30 to 100 ° C Or
The mixture of Angelica Angelica and buttercup, extracted from 30 to 80 ° C with 40 to 100% (v / v) ethanol aqueous solution,
Pharmaceutical composition for neuronal cell protection or neuronal cell regeneration. The method of claim 7, wherein the buttercup ( Oenanthe javanica ) phosphorus, a pharmaceutical composition for protecting nerve cells or regenerating nerve cells. The pharmaceutical composition for protecting neurons or regenerating neurons according to claim 7, wherein the buttercup ethanol extract is extracted under pH 8 to 10 conditions. According to claim 7, The mixing ratio between the Angelica or Angelica Angelica extract and the buttercup or buttercup extract in the mixed extract is 1: 0.1 to 10 by weight (weight of Angelica or Angelica extract: weight of Parsley or Parsley extract). , Neuron protection or pharmaceutical composition for regeneration of neurons. 11. The method of claim 10, Mixing ratio between the Angelica or Angelica Angelica extract and the buttercup or buttercup extract in the horn mixed extract is 1: 0.2 to 5 by weight (weight of Angelica or Angelica kerosene extract: weight of Parsley or Parsley extract) Phosphorus, nerve cell protection or nerve cell regeneration pharmaceutical composition. Contains a mixed extract of Angelica gigas Nakai and buttercup as an active ingredient,
The mixed extract of the Angelica Angelica and buttercup,
Angelica ethanol extract extracted from Angelica Angelica at 90 to 100% (v / v) ethanol solution at 40 to 80 ° C, and buttercup ethanol extract extracted from Angelica kerosene at 10 to 80% (v / v) ethanol solution at 30 to 100 ° C Or
The mixture of Angelica Angelica and buttercup, extracted from 30 to 80 ° C with 40 to 100% (v / v) ethanol aqueous solution,
Health functional food for preventing or improving ischemic brain disease, protecting nerve cells, or regenerating nerve cells. The method of claim 12, wherein the buttercup ( Oenanthe javanica ), a health functional food. 13. The functional food of claim 12, wherein the buttercup ethanol extract is extracted under pH 8 to 10 conditions. The mixing ratio of the Angelica Angelica or Angelica Angelica extract and the buttercup or buttercup extract in the mixed extract is 1: 0.1 to 10 (weight of Angelica or Angelica kerosene extract: weight of Parsley or Parsley extract) by weight. , Health functional food. 16. The method of claim 15, wherein the mixture ratio between the Angelica Angelica or Angelica Angelica extract and the buttercup or buttercup extract in the horn mixed extract is 1: 0.2 to 5 by weight (weight of Angelica Angelica or Angelica Angelica extract: weight of Parsley or Angelica extract) Phosphorus, health functional food. The health functional food according to any one of claims 12 to 16, wherein the ischemic brain disease is stroke, hypertension, dementia, Parkinson's disease, Huntington's disease, or Lou Gehrig's disease.

Images