KR102177309B1 - Composition for Preventing or Treating Ischemic Brain Disease Containing Extract of Mixed Extract of Angelica gigas Nakai and Oenanthe javanica - Google Patents

Abstract

There is provided a use of the mixed extract of Angelica kelp and parsley in the prevention and/or treatment and/or amelioration of ischemic brain diseases, for the protection of neurons, and/or for the production of neurons.

Description

Pharmaceutical composition for preventing or treating ischemic brain disease containing a mixed extract of Angelica gigas Nakai and Oenanthe javanica}

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

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

Stroke can be divided into ischemic stroke (cerebral infarction) in which cerebrovascular is blocked (cerebral infarction) and hemorrhagic stroke (cerebral hemorrhage) in which cerebellar blood vessels are ruptured. Primary cerebral hemorrhage and subarachnoid hemorrhage due to arteriovenous malformations or aneurysms are important causes.

Currently, various drugs having a therapeutic effect on ischemic brain diseases such as stroke are being developed, but many side effects have been reported, and thus, the development of effective and safe therapeutic agents is required.

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

One example is a pharmaceutical composition for the prevention and/or treatment of ischemic brain disease, comprising a mixed extract of Angelica gigas Nakai and parsley as an active ingredient, a pharmaceutical composition for protecting nerve cells, and/or regeneration (or generation) of nerve cells It provides a pharmaceutical composition for use.

Another example is an ischemic brain disease comprising administering a mixed extract of Angelica keiskei koidz and water parsley to a subject in need of preventing and/or treating ischemic brain disease, protecting neurons, and/or regenerating (or producing) neurons. It provides a method of preventing and/or treating, a method of protecting nerve cells, and/or a method of regenerating (or producing) nerve cells.

The mixed extract of Angelica keiskei koidz and water parsley may be a mixture of Angelica kelp and water parsley extract, an extract of a mixture of Angelica kelp and water parsley, or a combination thereof.

The ischemic brain disease may be selected from ischemic cerebrovascular disease and ischemic cranial nerve disease (degenerative neurological disease), such as stroke (eg, cerebral infarction or cerebral hemorrhage), high blood pressure, dementia (eg, vascular dementia), Parkinson's disease, It can be selected from the group consisting of Huntington's disease and Lou Gehrig's disease.

The nerve cells 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.

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

Another example provides a method of preparing a composition having an effect of preventing, treating, and/or improving ischemic brain diseases, comprising preparing a mixed extract of Angelica keiskei koidz and water parsley.

Another example provides a pharmaceutical composition for combination administration or a combination administration kit for the prevention and/or treatment of ischemic brain diseases, nerve cell protection, and/or nerve cell regeneration (or production), including the extract of Korean Angelica Angelica extract and parsley extract. do. The pharmaceutical composition for co-administration may include a dosage unit including the Angelicae extract and the water parsley extract in one formulation, or in different formulations. Another example is a method for preventing and/or treating neurological diseases, including administering an extract of Angelica chinensis to a subject in need of prophylaxis and/or treatment of ischemic brain disease, and administering a parsley extract to the subject. Methods of protection, and/or methods of regenerating (or producing) nerve cells are provided. The step of administering the Angelicae extract and the step of administering the water parsley extract may be performed simultaneously or visually regardless of sequence.

In the present specification, the use of the mixed extract of Angelica gigas Nakai and water parsley is provided for the prevention and/or treatment of ischemic brain diseases, and for the protection and/or regeneration of nerve cells.

Angelica is a perennial herbaceous plant belonging to the umbel family, and is cultivated for medicinal purposes mainly 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 Are known to be different. The Angelica keiskei extract used herein may be obtained using the roots of Angelica Angelica (eg, dried and crushed substance of the Angelica root).

Water parsley used in the present specification may be water dropwort ( Oenanthe javanica ). Parsley is a perennial herb belonging to Umbelliferae and is a dicotyledon. Stone parsley is wild in wetland or waterside, and has more leaves than parsley, short stem and slightly reddish under the stem. In addition, because it has a unique flavor, it is also 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 using the whole (outpost) or part of the plant body of the stone buttercup, and a part of the stone buttercup is one or more selected from the group consisting of flowers, stems, and leaves. It can be a part.

In the present specification,'treatment' refers to relief or amelioration of symptoms, reduction of the extent of the disease, delay or alleviation of disease progression, improvement, alleviation or stabilization of disease states or symptoms, partial or complete recovery, prolongation of survival, and other beneficial It is used to include all treatment results. 'Prevention' is used as a meaning to include all mechanisms and/or effects that act on a subject who does not have a specific disease to prevent the onset of the specific disease or to delay the onset of the disease.

In the present specification, it was confirmed that the mixed extract of Angelica keiskei koidz and water parsley exhibited a protective/neuronal regeneration effect against brain cell damage caused by cerebral ischemia, and thus the neuronal protective effect and/or nerve cell regeneration of the mixed extract of Angelica keiskei koidz and water parsley It proposes a (or produced) effect, and a treatment and/or prevention and/or improvement effect for 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 parsley as an active ingredient, a pharmaceutical composition for protecting nerve cells, and/or a nerve cell regeneration (or It provides a pharmaceutical composition for production). Another example is an ischemic brain disease comprising administering a mixed extract of Angelica keiskei koidz and water parsley to a subject in need of preventing and/or treating ischemic brain disease, protecting neurons, and/or regenerating (or producing) neurons. It provides a method of preventing and/or treating, a method of protecting nerve cells, and/or a method of regenerating (or producing) nerve cells. The mixed extract of Angelica keiskei koidz and water parsley may be a mixture of Angelica kelp and water parsley extract, an extract of a mixture of Angelica kelp and water parsley, or a combination thereof. The nerve cells may be neurons of the brain (eg, hippocampus (eg, CA1 region, etc.)), such as neurons, glial cells (eg, astrocytes, microglia), oligodendrocytes (oligodendrocyte), etc.), but 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 diseases, nerve cell protection, and/or regeneration (or production) of nerve cells, comprising the extract of Angelica Angelica and parsley extract. Another example provides a kit for combination administration for the prevention and/or treatment of ischemic brain diseases, nerve cell protection, and/or regeneration (or production) of nerve cells, comprising the extract of Angelica Angelica and the water parsley extract. The Angelicae extract and water parsley extract included in the pharmaceutical composition or kit for co-administration may be formulated together (for example, in a mixture form), or may be formulated and included in one dosage unit. Another example is the step of administering the extract of Angelica delicacy to a subject in need of prophylaxis and/or treatment of ischemic brain disease, protection of nerve cells, and/or regeneration (or generation) of nerve cells, and administering a parsley extract to the subject. It provides a method for preventing and/or treating ischemic brain diseases, including a method, a method for protecting nerve cells, and/or a method for regenerating (or generating) nerve cells. The step of administering the Angelicae extract and the step of administering the water parsley extract may be performed simultaneously or visually regardless of sequence.

The Angelicae extract may be obtained by extracting the Angelicae (eg, root) with water and one or more extraction solvents selected from the group consisting of straight chain or branched alcohols having 1 to 4 carbon atoms. In one example, the Angelicae extract is 40 to 100% (v / v), 50 to 100% (v / v), 60 to 100% (v / v), 70 to 100 %(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) aqueous ethanol solution (eg, 98% (v/v) aqueous ethanol solution (alcohol)) extract. In addition, the Angelicae extract 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 a straight-chain or branched alcohol having 1 to 4 carbon atoms, such as water, and one or more parts selected from the group consisting of flowers, stems, leaves, outposts, etc. It may be obtained. In one example, the buttercup extract is 10 to 80% (v/v), 10 to 70% (v/v), 10 to 65% (eg, flowers, stems, leaves, and/or outposts) of the buttercups ( 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) of ethanol extract. In addition, the water parsley extract, for example, 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 water parsley in the above pH range, an extract having a high content of active ingredients in the extract can be obtained. The extraction pH can be adjusted by adding a pH adjuster together with an extraction solvent to the parsley. In one example, the pH adjusting agent may be one or more 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 within the above range. . In addition, the water parsley 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°C, 50 to 95°C, 50 to 94°C, 50 to 93°C, 50 to 92°C, 50 to 91°C, 50 to 90°C, 70 to 100°C, 70 to 98°C, 70 to 95°C , 70 to 94°C, 70 to 93°C, 70 to 92°C, 70 to 91°C, 70 to 90°C, 75 to 100°C, 75 to 98°C, 75 to 95°C, 75 to 94°C, 75 to 93°C , 75 to 92°C, 75 to 91°C, 75 to 90°C, 80 to 100°C, 80 to 98°C, 80 to 95°C, 80 to 94°C, 80 to 93°C, 80 to 92°C, 80 to 91°C , 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 at 90 to 98 ℃ or 90 to 95 ℃.

The extract of the mixture of Korean angelica and parsley is extracted from the group consisting of water and a straight chain or branched alcohol having 1 to 4 carbon atoms in a mixture of Korean angelica (root) and water parsley (flower, stem, leaf, outpost, etc.) It may be obtained by extraction with a solvent.

In one example, the mixed extract of the angelica and parsley,

(1) (i) 40 to 100% (v/v), 50 to 100% (v/v), 60 to 100% (v/v), 70 to 100% (v/v) of the Angelicae (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) aqueous ethanol solution (eg, 98% (v/v) aqueous ethanol solution (alcohol)) extract and (ii) buttercup (eg, flower , Stems, leaves, outposts, etc.) of 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) of an aqueous ethanol solution (e.g., 50% (v/v) aqueous ethanol) 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) 40-100% (v/v), 45-100% (v/v), 48-100% (e.g., seeds, sprouts, flowers, outposts, etc.) of a mixture of angelica (root) and parsley ( 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), 92-100% (v/v), 96-100% (v/v), or 98-100% (v/v) ethanol aqueous solution extract

(3) or a combination thereof

Can be

The Angelicae extract in the mixture of the Angelicae extract and the parsley extract 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°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 ; And/or the parsley 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°C, 50 to 95°C, 50 to 94°C, 50 to 93°C, 50 to 92°C, 50 to 91°C, 50 to 90°C, 70 to 100°C, 70 to 98°C, 70 to 95°C , 70 to 94°C, 70 to 93°C, 70 to 92°C, 70 to 91°C, 70 to 90°C, 75 to 100°C, 75 to 98°C, 75 to 95°C, 75 to 94°C, 75 to 93°C , 75 to 92°C, 75 to 91°C, 75 to 90°C, 80 to 100°C, 80 to 98°C, 80 to 95°C, 80 to 94°C, 80 to 93°C, 80 to 92°C, 80 to 91°C , 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 at 90 to 98 ℃ or 90 to 95 ℃. In addition, the extract of the mixture of angelica and parsley 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 It may be extracted at ℃, 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 Angelicae extract and the water parsley extract in the mixture of the Angelicae extract and the water parsley mixture, or the mixing ratio of the Angelicae and water parsley in the mixture of Korean Angelica and parsley 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 (or more, the weight of the Angelicae or the solid content of the Angelicae extract: the weight of the water parsley or the weight of the solid content of the water parsley extract). The weight of the solid content refers to the weight of the solid remaining after removing the solvent component of the extract. This is a term used to indicate that when the mixture is a mixture of Angelica kelp extract and parsley extract, the mixing ratio means the ratio between the weight of the active ingredient from which the extraction solvent has been removed so that the mixing ratio is not affected by the properties and/or concentration of the extract. .

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

The content of the mixed extract of angelica and parsley contained as active ingredients in the pharmaceutical composition can be appropriately adjusted according to the type and purpose of use, the condition of the patient, the type and severity of symptoms, etc., and 0.001 to 99.9% by weight based on the weight of the solid content, 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 refers to the weight of the solid from which the solvent component has been removed from the extract, as described above.

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

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

The pharmaceutical composition may be administered to mammals, including humans, dogs, cats, horses, cows, pigs, goats, rabbits, mice, rats, etc., or cells, tissues, or cultures thereof isolated therefrom by various routes. . The mode of administration may be any mode commonly used, such as oral administration, or intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, and parenteral administration such as topical administration to the lesion site (eg, brain, spinal cord, etc.) I can. The pharmaceutical composition is formulated as a parenteral formulation in the form of a powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, or other oral formulation, transdermal formulation, suppository, and sterile injectable solution according to a conventional method. Can be used.

In addition to the extract, the pharmaceutical composition may further contain adjuvants such as a pharmaceutically suitable and physiologically acceptable carrier, excipient, and/or diluent. Examples of the carrier, excipient, or diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, At least one 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 commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants 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, and the like, and these solid preparations include at least one excipient in the extract, For example, it may be prepared by mixing at least one selected from the group consisting of starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include one or more selected from the group consisting of suspensions, liquid solutions, emulsions, syrups, etc., and various excipients, such as humectants, sweeteners, in addition to water and liquid paraffin, which are commonly used simple diluents. , At least one selected from the group consisting of fragrances, preservatives, etc. may be included. Formulations for parenteral administration include at least one selected from the group consisting of sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, and transdermal formulations. As the non-aqueous solvent and suspending agent, at least one selected from the group consisting of propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester 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 depending on factors such as formulation method, administration mode, patient's age, weight, sex, pathological condition, food, administration time, administration interval, route of administration, 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 a day or several times a day at regular time intervals according to the judgment of a doctor or pharmacist. For example, one or daily dosage 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 of the active ingredient (mixed extract of Angelica and Parsley) /kg, 0.001 to 300 mg/kg, 0.001 to 250 mg/kg, 0.01 to 1000 mg/kg, 0.01 to 500 mg/kg, 0.01 to 300 mg/kg, 0.01 to 250 mg/kg, 0.1 to 1000 mg/ 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 1000 mg/kg, 10 to 500 mg/kg, 10 to 300 mg/kg, or 10 to 250 mg/kg, but is not limited thereto. The one-time or daily dosage may be formulated as a single formulation in a unit dosage form, formulated in an appropriate amount, or prepared by incorporating into a multi-dose container. The above dosage is an example of an average case, and the dosage may be high or low depending on individual differences.

Another example provides a method of preparing a composition having an effect of preventing, treating, and/or improving ischemic brain diseases, comprising preparing a mixed extract of Angelica keiskei koidz and water parsley.

The step of preparing the mixed extract of Korean Angelica Angelica and water parsley may include 1) mixing the Korean Angelfish extract and the water parsley extract, or 2) extracting a mixture of Korean Angelica Angelica and water parsley with an extraction solvent. For the extraction solvent and extraction temperature used in the extracting step, refer to the description of the mixed extract of Korean angelfish and parsley.

For example, the step of 1) mixing the Angelica root extract and the water parsley extract

i) Angelica (e.g., root), 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°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 times by volume, 2 To 8 times by volume, or 4 to 6 times by volume of water and at least one selected from the group consisting of 1 to 4 straight chain or branched alcohols (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 an aqueous ethanol solution (e.g., 98% (v/v) aqueous ethanol (alcohol)),

ii) Buttercup (eg, flowers, stems, leaves, outposts, etc.), 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 ℃, 30 to 70 ℃, 30 to 60 ℃, 30 to 50 ℃, 30 to 45 ℃, 35 to 60 ℃, 35 to 50 ℃, 35 to 45 ℃, 35 to 60 °C, 35 to 50 °C, or 35 to 45 °C, 1 to 10 times by volume, 2 to 8 times by volume, or 4 to 6 times by volume of water and a straight chain or branched alcohol having 1 to 4 carbon atoms (e.g., ethanol) At least one 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 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) , Extracting with 30 to 46% (v/v), or 30 to 45% (v/v) of an aqueous ethanol solution (eg, 50% (v/v) of an aqueous ethanol solution), and

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

It may include.

In the step of preparing the parsley extract of step ii), optionally, the pH of the mixture of the parsley 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, 8.5 to 9.2, or 8.8 to 9.2 may be adjusted to perform, and the pH can 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, etc. It may include the above, but is not limited thereto.

The step of preparing the Angelica keiskei extract of step i) and the step of preparing the parsley extract of step ii) may be performed simultaneously or temporarily, regardless of the sequence.

The step of extracting the mixture of 2) Angelica and parsley with an extraction solvent includes i') preparing a mixture by mixing Korean Angelicae (root) and parsley (eg, seeds, sprouts, flowers, outposts, etc.), 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 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 times by volume, 2 to 8 volumes Times, or at least one selected from the group consisting of 4 to 6 times the volume of water and a straight chain or branched alcohol having 1 to 4 carbon atoms (eg, 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 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.

The mixing ratio of the angelica root extract and the water parsley extract or the mixture ratio of the angelica angelica and the water parsley are as described above.

The extraction time of the above-described extracting step of angelica and/or parsley is sufficient as long as the extraction can be sufficiently performed, and is 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 can be set to about 4 to 6 hours, but is not limited thereto.

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

Another example provides a health functional food for preventing and/or ameliorating ischemic brain disease, for protecting nerve cells, and/or for regenerating (or generating) nerve cells, containing an extract of Korean Angelica Angelica extract, or a mixed extract of red onion and water parsley.

The health functional food is a food manufactured using nutrients that are liable to be deficient in daily meals or raw materials or ingredients having useful functions for the human body (hereinafter, referred to as'functional raw materials'), and maintains health or prevents certain diseases or symptoms. And/or 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 Angelicae extract or the mixed extract of Angelicae and water parsley contained in the health functional food is not particularly limited appropriately depending on the form of the food, the desired use, etc., for example, 0.001 to 99% by weight of the total food weight, 0.01 To 99% by weight, 0.01 to 95% by weight, 0.01 to 90% by weight, 0.01 to 80% by weight, 0.01 to 50% by weight, 0.1 to 99% by weight, 0.1 to 95% by weight, 0.1 to 90% by weight, 0.1 to 80 Wt%, 0.1 to 50 wt%, 0.1 to 30 wt%, 0.1 to 10 wt%, 1 to 99 wt%, 1 to 95 wt%, 1 to 90 wt%, 1 to 80 wt%, 1 to 50 wt% , 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% by weight, 25 to 95% by weight, 25 to 90% by weight, 25 to 80% by weight, 25 to 50% by weight, 25 to 30% by weight, 40 to 99% by weight, 40 to 95% by weight, 40 to 90 Wt%, 40 to 80 wt%, 40 to 50 wt%, 50 to 99 wt%, 50 to 95 wt%, 50 to 90 wt%, 50 to 80 wt%, 60 to 99 wt%, 60 to 95 wt% , 60 to 90% by weight, or may be 60 to 80% by weight.

The health functional foods include various nutrients, vitamins, minerals (electrolytes), synthetic flavors or natural flavoring agents, flavoring agents, coloring agents, heavy agents (cheese, chocolate, etc.), pectic acid or its salt, alginic acid or its salt, It may further contain at least one selected from the group consisting of organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and carbonates used in carbonated beverages. The ratio of these additives is generally selected from 0.001 to about 20 parts by weight per 100 parts by weight of the total health functional food, but is not limited thereto.

By proposing a protective effect against nerve cell damage caused by cerebral ischemia and an effect of regenerating (or generating) damaged nerve cells of the mixed extract of Angelica Angelica and Parsley, we propose a new treatment that has excellent effects in both the prevention and treatment of ischemic brain diseases. do.

1 is an ischemic brain disease of 200 mg/kg of parsley extract, 200 mg/kg of Angelicae extract, or 200 mg/kg of mixed extract (Cham Angelica extract 100 mg/kg + water parsley extract 100 mg/kg) according to an embodiment In order to investigate the preventive effect, this is a photograph taken by staining the CA1 region of the hippocampal tissue and the hippocampal tissue of the experimental animal 5 days after inducing cerebral ischemia after administration of these extracts with cresyl violet (CV). .
FIG. 2A is an ischemic brain disease of 200 mg/kg of parsley extract, 200 mg/kg, or 200 mg/kg of mixed extract (Cham Angelica extract 100 mg/kg + water parsley extract 100 mg/kg) according to an embodiment In order to investigate the preventive effect, the CA1 region of the hippocampus of the experimental animal 5 days after inducing cerebral ischemia after administration of these extracts was treated with a mouse anti-neuronal nuclei (NeuN) antibody as a marker for nerve cells. It is a photograph taken by staining with chemistry (immunohistochemistry), and 2b is a graph showing the count of neurons with immunoreactivity to NeuN antibodies.
3A is an ischemic brain disease of 200 mg/kg of parsley extract, 200 mg/kg, or 200 mg/kg of mixed extract (Cham Angelica extract 100 mg/kg + water parsley extract 100 mg/kg) according to an embodiment In order to investigate the preventive effect, after administration of these extracts, the CA1 region of the hippocampus of the experimental animal 5 days elapsed due to cerebral ischemia was induced, and fluoro-jade B (fluoro-jade), a marker for neurons in the process of death or death. b, FJ B) is a picture taken by staining, 3b is a graph showing the counting cells showing a positive reaction to FJ B.
FIG. 4A is an ischemic brain disease of 200 mg/kg of parsley extract, 200 mg/kg of Angelicae extract, or 200 mg/kg of mixed extract (Cham Angelica extract 100 mg/kg + water parsley extract 100 mg/kg) according to an embodiment In order to investigate the preventive effect, the CA1 region of the hippocampus of the experimental animal 5 days elapsed after administration of these extracts caused cerebral ischemia, a mouse anti-glial fibrillary acidic protein (GFAP) antibody as a marker for astrocytes. It is a picture taken by staining with immunohistochemistry using, and 4b is a graph showing the immunoreactivity of cells stained with GFAP antibody as a relative value to the normal group (vehicle-sham).
FIG. 5A is an ischemic brain disease of 200 mg/kg of parsley extract, 200 mg/kg of Angelicae extract, or 200 mg/kg of mixed extract (Cham Angelica extract 100 mg/kg + water parsley extract 100 mg/kg) according to an embodiment In order to investigate the preventive effect, rabbit anti-ionized calcium-binding adapter molecule 1, a marker for microglia, was used to induce cerebral ischemia after administration of these extracts. It is a picture taken by staining with immunohistochemistry using (Iba-1) antibody, and 5b is a graph showing the immunoreactivity of cells stained with Iba-1 antibody in relative values with respect to the normal group.

Hereinafter, the present invention will be described in more detail by way of examples, but these are only illustrative and are not intended to limit the scope of the present invention. It is obvious to those skilled in the art that the embodiments described below may be modified within the scope of the essential gist of the invention.

Example 1: Preparation of extract

1.1. Preparation of Angelica Angelica extract

The roots of Angelica gigas Nakai were washed with clean water and dried sufficiently. 5 volume times (500ml) of ethanol (98%(v/v) ethanol (alcohol)) was added to 100g of the powder obtained by crushing the dried Angelica roots, and after extracting at 40~60℃ for 4 hours or more, 1um( The filtrate filtered through a micrometer) filter was heated and concentrated until it became 10% (w/w) of the original weight. Crystalline cellulose was gradually added to the obtained concentrate, followed by concentration, completely dried, and powdered to prepare an ethanol extract powder (hereinafter referred to as AGE).

1.2. Preparation of Buttercup Extract

Water parsley ( Oenanthe) collected in Chuncheon-si, Gangwon-do javanica ) 700 g was minced , and the solvent adjusted to pH 9 by adding diluted NaOH to 3.5 L of 50% or less aqueous ethanol solution was repeatedly extracted three times at 90°C for 24 hours in an extractor equipped with a reflux condenser. Whatman No. 1 (Whatman Ltd, Maidstone, Kent, UK) was filtered using a filter paper, the filtrate was concentrated under reduced pressure at 50 ℃ using a rotary vacuum concentrator (EYELA, SN-1100, Tokyo, Japan). The concentrated extract was subjected to a rapid freezing process at -70°C using a freeze dryer (PVTFA 10AT, ILSIN, Korea) to prepare a powdery water parsley extract (hereinafter referred to as OJE).

Example 2: of the mixed extract of Angelica and parsley Cerebral ischemia Confirmation of nerve cell protection effect

In order to confirm the neuroprotective efficacy of the Angelicae extract, the water parsley extract, or a mixture thereof (mixed extract of Angelicae and water parsley) prepared in Example 1, a male Mongolian gerbil, Halan, weighing 65g to 75g , USA) was anesthetized to expose the common carotid artery and then ligated, and the hippocampal tissue of Mongolian Gerbil, which caused cerebral ischemia, was stained and observed. Mongolian gerbil is an animal model mainly used for neuronal cell death. If both warm neck arteries of Mongolian gerbil are ligated for 5 minutes, it can cause cerebral ischemia similar to that in humans. When cerebral ischemia is induced in Mongolian gerbil, cell death can be observed characteristically in the hippocampal CA1 region 4 days later. From this phenomenon, it is confirmed that delayed neuronal death has occurred.

Example 2-1. Breeding of experimental animals

56 male Mongolian gerbils (Meriones unguiculatus, Halan, USA) weighing 65g to 75g were subjected to a constant light and dark cycle from 7am to 7pm, a temperature of 21℃ to 25℃ and 45% to 65% It was reared under the condition of relative humidity. For the feed of the experimental animals, a general pellet-dried feed (Korea Experimental Animal, Korea) was used, and feed and water were allowed to be consumed at all times.

Example 2-2. After administration of the extract Cerebrospinal fluid Preparation of induced brain tissue sections

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

After administering the extract for the 28 days, the experimental animals were treated using a gas in which nitrogen and oxygen were mixed in a ratio of 7:3 and 3% (v/v) isoflurane (Baxtor, USA). General anesthesia was performed. The operation was performed on the experimental animal while maintaining the anesthesia state of the experimental animal using a gas obtained by mixing 2.5% (v/v) isoflurane with the above nitrogen and oxygen mixture gas.

In order to cause cerebral ischemia, hair on the neck of the experimental animal is cut and disinfected, then incision is made to expose both warm carotid arteries, ligated using an aneurysm clip (Staelting, USA) for 5 minutes, and then the clip is removed. And reperfused. At this time, each experimental group observed complete occlusion of the warm carotid artery by observing the blood circulation of the central artery of retina using an ophthalmoscope.

Body temperature was measured by inserting a rectal thermometer while inducing cerebral ischemia, 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 controlled according to the body temperature of the experimental animal.

Five days after the induction of cerebral ischemia, thiopental sodium (Yanhan Corporation, Korea) was injected intraperitoneally at a dose of 30 mg per 1 kg of body weight and anesthetized, followed by anesthesia at 4℃ containing 1,000 IU of heparin per 1,000 ml. The physiological saline was injected into the left ventricle and washed with perfusion. Perfusion fixation was performed using 4% paraformaldehyde (in 0.1 M phosphate buffer (PB), pH 7.4) at 4°C for the animal on which the perfusion washing was completed.

Using a bone cutter, the brain was removed by opening the skull space of the experimental animal after the perfusion was fixed. The brains of the extracted experimental animals were then fixed for 4 hours in 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 was completed, the brain was put in 30% (w/v) sucrose solution (in 0.1 M phosphate buffer) and settled until it settled on the floor, and then a sliding microtome (Reichert-Jung, Germany) cut the brain tissue to a thickness of 30 μm to make a tissue section, and 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.

Prepare a tissue section obtained by performing the same procedure as above, except that only physiological saline used to dissolve the extract as a control (vehicle) was administered orally, and as a normal group (sham), Buttercup extract and/or Angelicae extract The obtained tissue sections were prepared without administering any substances including and without performing a process for causing cerebral ischemia.

Example 2-3. Cerebral ischemia Measurement of nerve cell protection efficacy 1 ( Cressil Violet dyeing)

Among the tissue sections prepared in Example 2-2, a tissue with hippocampal formation was selected, and washed three times for 10 minutes each with 0.01M PBS (pH 7.4) to remove the preservative solution from the tissue section. . The washed tissue section was spread on a gelatin-coated slide glass and sufficiently dried at 37°C. After immersing the tissue section in distilled water for a while, the tissue section was stained by immersing it in 2% (w/v) cresyl violet acetate (Sigma, USA) solution for 1 minute. Then, the stained tissue section is sufficiently washed with running water to remove excess dye from the slide, and after being immersed in distilled water for a while, 50% (v/v), 70% (v/v), 80% (v/ v), 90% (v/v), 95% (v/v) and 100% (v/v) ethanol solutions were sequentially treated to perform dehydration and excessive cresyl violet washing. After confirming that the Nissl body was visible in the tissue section, it was immersed in xylene (Junsey, Japan) to make it transparent, and then sealed with Canada balsam (Kanto, Japan).

Each of the tissues of the normal group, control group, and experimental group was used with an AxioM1 microscope (Carl Zeiss, Germany) equipped with a digital camera (Axiocam, Cal Zeiss, Germany) to cover the entire hippocampal area 40 times and CA1 area 20. Photographs of each tissue section were taken by magnifying each of the folds, and photographs of the entire hippocampal region are shown in A, B, C, D, E, F, G and H of Fig. 1, and a photograph of the CA1 region of the hippocampus is shown. It is shown in a, b, c, d, e, f, g and h of 1, respectively.

As can be seen in Figure 1, all the normal groups (sham groups) were stained with dark purple neurons in the entire hippocampus area (A, C, E, and G in FIG. 1), especially in the CA1 area, the pyramidal layer ( stratum pyramidale, SP), it could be observed that the neurons were concentrated (a, c, e, and g of FIG. 1). However, in the case of the control group (vehicle-ischemia) in which cerebral ischemia was induced by administration of only solvent, neuronal cell death occurred in the CA1 area of the hippocampus due to delayed neuronal death due to cerebral ischemia. (B in FIG. 1), it was confirmed that most of the neurons were lost in the pyramidal layer in the CA1 region (b in FIG. 1). In addition, in the case of the experimental group (OJE-ischemia, AGE-ischemia) administered with 200 mg/kg of parsley extract or 200 mg/kg of Angelicae extract, in the CA1 region of the hippocampus, compared to the control group (vehicle-ischemia), cresyl violet Although the dyeing property for was strong, compared to the normal group (sham), the dyeing property for cressil violet was slightly weakened (D, d, F, and f of FIG. 1). On the other hand, in the case of the experimental group (OJE+AGE-ischemia) in which 100 mg/kg of water parsley extract and 100 mg/kg of Angelicae extract were mixed and administered, the control and 200 mg/kg of parsley extract or 200 mg/kg of true When compared with the experimental group to which Angelicae extract was administered, a large number of neurons were stained dark purple in the entire hippocampus area (H in FIG. 1), and it was observed that neurons were concentrated in the pyramidal layer of the CA1 area (FIG. 1 h). These results show that when the water parsley extract and the Korean angelfish extract were administered together, compared to the case where the same amount of the water parsley extract or the Korean angelfish extract was administered, neuronal cell death caused by cerebral ischemia was more effectively suppressed.

Example 2-4. Cerebral ischemia Measurement of nerve cell protection efficacy 2 (immunohistochemical staining)

Immunohistochemical staining was performed using an antibody against NeuN, a marker of nerve cells, in order to confirm the neuronal protective effect of the extracts of Parsley and Angelica sinensis, and more specifically, inhibiting neuronal death due to cerebral ischemia.

More specifically, from the tissue sections prepared in Example 2-2, a tissue section that showed well hippocampal formation was selected and used in the experiment, and the selected tissue section was a storage solution present in the tissue. To remove it, it was washed three times with 0.01M phosphate buffered saline (PBS) for 10 minutes each, and then reacted for 30 minutes in 0.3% H ₂ O ₂ (in 0.01M PBS) to remove endogenous peroxidase. . The tissue section after the reaction was washed 3 times for 10 minutes each with 0.01M PBS.

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

After spreading the colored tissue section on gelatin-coated slide glass, it was dried at room temperature for 12 hours, and the dried tissue section was subjected to a conventional dewatering and transparent process to cover glass. ) Was used. After performing the reaction with the NeuN antibody, the encapsulated tissue section was observed using a microscope, and a photograph taken of the result is shown in FIG. 2A. In addition, the number of neurons obtained by counting neurons of tissue sections exhibiting immunoreactivity (ie, stained with anti-NeuN antibody) using an image analyzer (Optimas 6.5, USA) program for the photographed picture is shown in FIG. 2B. And a one-way ANOVA test was performed to verify the significance of each experimental group.

As shown in Figure 2a, in all the normal groups (sham groups), neurons exhibiting immunoreactivity to anti-NeuN antibodies in the CA1 region of the hippocampus are well observed (A, C, E, and G in Figure 2A), and the solvent In the case of the vehicle in which cerebral ischemia was induced, neuronal cells showing immune reactivity to anti-NeuN antibodies occurred in the CA1 area of the hippocampus due to delayed neuronal death due to cerebral ischemia. It was hardly 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 in which cerebral ischemia was induced after administration of Buttercup extract (200 mg/kg; OJE) and Angelicae extract (200 mg/kg; AGE) alone, immunity against anti-NeuN antibodies in the CA1 region of the hippocampus Neurons exhibiting reactivity were significantly decreased compared to the normal group (D, F, and I in FIG. 2A). On the other hand, in the case of the experimental group (OJE+AGE) inducing cerebral ischemia after administration of a mixture (200 mg/kg) of parsley extract (100 mg/kg) and Angelicae extract (100 mg/kg), CA1 was similar to that of the normal group. In the region, it was observed that neurons exhibiting immunoreactivity to anti-NeuN antibodies were concentrated (H and I in Fig. 2A). These results can be quantitatively confirmed through the graph of FIG. 2B. The above results show that in the experimental group inducing cerebral ischemia after administration of the parsley extract and the Angelica keiskei extract together, the neuronal protective effect is remarkably superior compared to the non-administration group and the extract alone administration group.

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

Fluoro-Jade B (FJ B), a marker for dead neurons or neurons in the process of death, was used to confirm the neuronal protective effect of the Buttercup extract, the Buttercup extract, or the mixed extract of Buttercup and the Buttercup Thus, tissue fluorescence staining was performed.

More specifically, from the tissue sections prepared in Example 2-2, a tissue section showing a hippocampal complex was 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 in 0.01M PBS for 10 minutes each, spread on gelatin-coated slides, and dried at room temperature for 12 hours. The tissue sections were washed three times with distilled water for 5 minutes, and then reacted with 0.06% potassium permanganate solution for 20 minutes. Thereafter, after washing for 2 minutes with distilled water, fluorescent 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 3 times for 1 minute, dried for 20 minutes in a 50°C slide warmer, immersed in xylene (xylene, Junsei, Japan), and then sealed with DPX (Sigma, USA). The enclosed tissue was observed using a fluorescence microscope (Ex: 385nmm Em: 425nm), and a photograph of the result is shown in FIG. 3A. In addition, stained nerve cells were counted using an image analyzer (Optimas 6.5, USA) program for the photographed picture, and the counting results were shown in FIG. 3B as a relative value to the control group (Vehicle administration group).

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

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

In order to further confirm the neuroprotective effect of the water parsley extract, the Korean Angelica Angelica extract, or the mixture of the water parsley and the Korean angelica root extract, more specifically, the effect of preventing the death of neurons due to cerebral ischemia, with respect to the tissue section prepared in Example 2-2 In addition, astrocytes through immunohistochemistry staining using antibodies against GFAP (glial fibrillary acidic protein), a marker of astrocytes, and Iba-1 (ionized calcium-binding adapter molecule-1), a marker of microglia (astrocyte) and microglia activity changes were confirmed.

More specifically, from the tissue sections prepared in Example 2-2, a tissue section showing well hippocampal formation was selected and used in the experiment, and the selected tissue section removed a storage solution existing in the tissue. In order to do this, after washing three times with 0.01M phosphate buffered saline (PBS) for 10 minutes each, it was reacted for 30 minutes in 0.3% H ₂ O ₂ (in 0.01M PBS) to remove endogenous peroxidase. The tissue section after the reaction was washed 3 times for 10 minutes each with 0.01M PBS.

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

After spreading the colored tissue section on gelatin-coated slide glass, it was dried at room temperature for 12 hours, and the dried tissue section was subjected to a conventional dewatering and transparent process to cover glass. ) Was used. After performing the reaction with the GFAP or Iba-1 antibody, the encapsulated tissue section was observed using a microscope, and photographs of the observation results are shown in Figs. 4A (asterisk cells stained with antibodies to GFAP) and Fig. 5A. It is shown in (microglia stained with Iba-1 antibody).

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

As shown in FIG. 4A, in the case of all the sham groups, astrocytes exhibiting immunoreactivity to the anti-GFAP antibody were observed in a dormant state (A, C, E, and G of FIG. 4A). On the other hand, in the case of the control group (vehicle-ischemia) to which only the solvent was administered, astrocytes exhibiting immunoreactivity to the anti-GFAP antibody were increased compared to the normal group, and the cytoplasm became enlarged (B and I in FIG. 4A). In addition, astrocytes showing immune reactivity to anti-GFAP antibodies in the experimental group that caused cerebral ischemia after administration of Buttercup extract (200 mg/kg; OJE) and Angelicae extract (200 mg/kg; AGE) alone were control ( vehicle-ischemia), the degree of activation was decreased (D, F, and I in FIG. 4A). On the other hand, in the case of the experimental group inducing cerebral ischemia after administering a mixture (200 mg/kg) of parsley extract (100 mg/kg) and Korean angelfish extract (100 mg/kg), immunoreactivity to anti-GFAP antibodies was observed. The visible astrocytes were observed in the form of a dormant state like the normal group (H and I in FIG. 4A).

As shown in Figure 5a, in the case of all the normal groups (sham groups), it was observed that microglia cells exhibiting immunoreactivity to the anti-Iba-1 antibody were evenly distributed in a dormant state (Figure 5A, A, C, E and G). In the case of the control group (vehicle-ischemia) to which only the solvent was administered, microglia cells exhibiting immunoreactivity to anti-Iba-1 antibodies were observed in an activated form, particularly, in a form gathered in a pyramidal layer (B in FIG. 5A). ). In addition, microglia showing immunoreactivity to anti-Iba-1 antibodies in the experimental group that caused cerebral ischemia after administration of Buttercup extract (200 mg/kg; OJE) and Angelicae extract (200 mg/kg; AGE) alone. The degree of activation was decreased compared to the control (vehicle-ischemia) (D, F, and I of FIG. 5A). On the other hand, in the case of the experimental group in which cerebral ischemia was induced after administration of a mixture (200 mg/kg) of parsley extract (100 mg/kg) and Korean angelfish extract (100 mg/kg), a dormant state similar to that of the normal group (Sham) Was observed in the form of (H and I in FIG. 5A).

From the above results, compared with the control group and the case where the Buttercup extract and the Buttercup extract are administered alone, the neurons in the test animal inducing cerebral ischemia after the Buttercup extract and the Buttercup extract are administered together, in particular, the hippocampus region is crezil violet. As a result of observing the neural tissue section through staining and immunohistochemical staining using an anti-NeuN antibody, which is a marker of nerve cells, it was confirmed that neuronal cell death was suppressed in the entire hippocampus region, specifically in the CA1 region of the hippocampus. , As a result of confirming through FJ B, a marker for neurons that have died or are in the process of death, it was confirmed that neuronal death was remarkably suppressed, and anti-GFAP and anti-Iba- markers of astrocytes and microglia 1 As a result of confirming through antibody, it was confirmed that it can very excellently inhibit abnormal activation of glial cells. These results demonstrate that when a mixture of Buttercup extract and Angelica kelp extract are administered in combination, they show a remarkable synergistic effect in protecting neurons and inhibiting abnormal activation of glial cells compared to when administered alone.

Claims (17)

Contains a mixed extract of Angelica gigas Nakai and water parsley as an active ingredient,
The mixed extract of the angelica and parsley,
Angelica Angelica ethanol extract extracted from 40 to 80 ℃ with 90 to 100% (v / v) aqueous ethanol solution, and water parsley ethanol extract extracted from 30 to 100 ℃ with 10 to 80% (v / v) ethanol aqueous solution Contains, or
It is an extract of a mixture of angelica and water parsley extracted with 40 to 100% (v/v) ethanol aqueous solution at 30 to 80°C,
The mixing ratio between the Angelica keiskei or Angelica kelp extract and the water parsley or water parsley extract in the mixed extract is 1:0.8 to 1.2 by weight (weight of the Korean Angelica Angelica or Angelica root extract: the weight of water parsley or parsley extract),
Pharmaceutical composition for the prevention or treatment of ischemic brain disease. The method of claim 1, wherein the water parsley is Oenanthe javanica ) phosphorus, ischemic brain disease prevention or treatment pharmaceutical composition. The pharmaceutical composition for preventing or treating ischemic brain disease according to claim 1, wherein the water parsley ethanol extract is extracted under pH 8 to 10 conditions. The method according to claim 1, wherein the mixing ratio between the Angelica keisensis or Angelica kelp extract and the water parsley or water parsley extract is 1:0.9 to 1.1 by weight (weight of the Korean angelica or Korean angelica extract: the weight of the water parsley or parsley extract) , A pharmaceutical composition for preventing or treating ischemic brain disease. The method of claim 1, wherein the mixed extract of Korean Angelica and Parsley,
Angelica kelp ethanol extract extracted from 40 to 60 ℃ with 95 to 100% (v / v) ethanol aqueous solution, and water parsley ethanol extract extracted from 80 to 100 ℃ with 30 to 60% (v / v) ethanol aqueous solution Containing,
Pharmaceutical composition for the prevention or treatment of ischemic brain disease. 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, high blood pressure, dementia, Parkinson's disease, Huntington's disease, or Lou Gehrig's disease. delete delete delete delete delete Contains a mixed extract of Angelica gigas Nakai and water parsley as an active ingredient,
The mixed extract of the angelica and parsley,
Angelica Angelica ethanol extract extracted from 40 to 80 ℃ with 90 to 100% (v / v) aqueous ethanol solution, and water parsley ethanol extract extracted from 30 to 100 ℃ with 10 to 80% (v / v) ethanol aqueous solution Contains, or
It is an extract of a mixture of angelica and water parsley extracted with 40 to 100% (v/v) ethanol aqueous solution at 30 to 80°C,
The mixing ratio between the Angelica keiskei or Angelica kelp extract and the water parsley or water parsley extract in the mixed extract is 1:0.8 to 1.2 by weight (weight of the Korean Angelica Angelica or Angelica root extract: the weight of water parsley or parsley extract),
Health functional food for preventing or improving ischemic brain disease, protecting nerve cells, or regenerating nerve cells. The method of claim 12, wherein the parsley is Oenanthe javanica ) phosphorus, health functional food. The health functional food according to claim 12, wherein the water parsley ethanol extract is extracted under pH 8 to 10 conditions. The method according to claim 12, wherein the mixing ratio between the Angelica kelp or Angelica kelp extract and the water parsley or water parsley extract is 1:0.9 to 1.1 by weight (weight of the Korean Angelica kelp or Angelica kelp extract: the weight of the water parsley or parsley extract) , Health functional food. The method of claim 12, wherein the mixed extract of Korean Angelica and Parsley,
Angelica kelp ethanol extract extracted from 40 to 60 ℃ with 95 to 100% (v / v) ethanol aqueous solution, and water parsley ethanol extract extracted from 80 to 100 ℃ with 30 to 60% (v / v) ethanol aqueous solution Containing,
Health functional food. The health functional food according to any one of claims 12 to 16, wherein the ischemic brain disease is stroke, high blood pressure, dementia, Parkinson's disease, Huntington's disease, or Lou Gehrig's disease.

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