KR20210065671A - Composition for preventing the ischemic stroke disease containing natural complex extract - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing ischemic cerebrovascular disease or a food composition for preventing or improving ischemic cerebrovascular disease, comprising a natural complex extract having at least one selected from the group consisting of Populus tomentiglandulosa extract, Oenanthe javanica extract, Prunus pendula extract and Angelica gigas extract as an active ingredient. It has been confirmed that the natural complex extract has excellent neuroprotective ability and neuronal apoptosis inhibitory ability, and can effectively prevent nerve damage in the CA1 region of the hippocampal tissue of the brain known to be sensitive to cerebral ischemia, and thus is useful for effectively preventing ischemic brain disease.

Description

A composition for preventing ischemic cerebrovascular disease containing a natural product complex extract as an active ingredient {COMPOSITION FOR PREVENTING THE ISCHEMIC STROKE DISEASE CONTAINING NATURAL COMPLEX EXTRACT}

The present invention relates to a composition for preventing ischemic cerebrovascular disease containing a natural complex extract as an active ingredient.

Recently, the elderly population is rapidly increasing worldwide due to the development of medicine and life sciences along with the improvement of the economic level. According to the '2017 Social Indicators of Korea' released by the National Statistical Office in 2018, by 2040, more than half of the total population will enter a 'super-aging society', with more than half of the population over the age of 65 determining the aging society. The proportion of the population aged 65 years or older accounted for 13.8% (7,076 thousand) of the total Korean population (51,466 thousand) in 2017, and is expected to continue to increase to 24.5% in 2030, 32.8% in 2040, and 40.1% in 2060. . In addition, it was confirmed that the life expectancy of Koreans was 82.4 years, a significant increase from 1970 when it was 61.9 years old. However, in terms of quality of health, the healthy life expectancy of Koreans is 64.9 years in 2016, and it is estimated that they will suffer from various diseases for 17.5 years, which is 21.2% of life expectancy.

In addition, according to the latest statistics on the National Statistical Portal, as of 2019, the proportion of the elderly aged 65 and over in Korea's total population was 14.9%, which means that Korea is already in an aging society. The proportion of the elderly population is expected to continuously increase to 15.7% in 2020, 25% in 2030, 33.9% in 2040, and 43.9% in 2060. The social cost is expected to be enormous.

As such, with the increase in life expectancy of Koreans and the aging trend of Korean society, the number of patients with geriatric diseases is increasing every year, and the suffering and social costs are also expected to increase.

As described above, with the aging of the population, the incidence and prevalence of various geriatric diseases due to aging are increasing, and in particular, central nervous system (CNS) diseases including damage to the brain or spine continue to increase. is a trend that Among them, the CNS disease with the highest proportion is cerebrovascular disease, which ranks first in the single disease mortality rate, with 60,000 cases occurring annually in Korea.

The cerebrovascular disease is a typical geriatric disease, often referred to as stroke, and is a disease caused by blockage or bursting of blood vessels in the brain. The cerebrovascular disease is characterized in that the blood vessels in the brain are blocked or burst and the circulation of a certain part of the brain is suddenly stopped due to abnormal cerebral blood flow, resulting in loss of the function of the nerve corresponding to the part. It causes damage to nerve cells and causes serious dysfunction.

The cerebrovascular disease is considered to be the first cause of permanent disability such as muscular paralysis, impaired consciousness, visual impairment and cognitive decline, unlike other diseases in which deficits are recovered during survival, and understanding of the underlying etiology and treatment technology Although the demand for development is increasing, it is known that many of them recur even after treatment, causing enormous social and economic losses.

The stroke is largely caused by a hemorrhagic stroke, which is caused by the rupture of a blood vessel supplying blood to the brain due to an impact such as a traffic accident, and temporary blockage of blood flow due to a thrombus, decreased perfusion, etc., thereby reducing the supply of oxygen and nutrients to the brain. It can be divided into two types: ischemic stroke, which occurs when it is not done. The major causes of the hemorrhagic stroke are primary cerebral hemorrhage due to hypertension and subarachnoid hemorrhage due to arteriovenous malformation or aneurysm.

The stroke may cause hemiparesis, speech impairment, loss of memory and reasoning ability, coma, and, in severe cases, even death, depending on the damaged brain region.

Recently, in Korea, the incidence of ischemic cerebrovascular disease has been continuously increasing compared to hemorrhagic cerebrovascular disease due to the influence of westernization of diet and lifestyle, etc. According to one study, ischemic cerebrovascular disease is known to account for about 80% of all cerebrovascular disease types.

As described above, in ischemic stroke, which has a high incidence among cerebrovascular diseases, thrombus and other wastes generated in the cardiovascular system cause blockage of blood vessels in the brain, or blood is not supplied to the brain in pathological conditions such as cardiac arrest. Refers to a disease caused by the induction of neuronal cell death in a specific region of the brain. In an experiment using a transient forebrain ischemic model using Mongolian gerbil, the hippocampus (4 days after induction of cerebral ischemia for 5 minutes) It has been reported that neuronal apoptosis is observed in the CA1 (conus ammonis 1) region of the hippocampus, which is called delayed neuronal death (Kirino, Brain Res., 239, pp 57 - 69, 1982).

Many mechanisms have been proposed for neuronal cell death following ischemic stroke. Specific mechanisms include excitotoxcity due to excessive increase in glutamate, oxidative stress by increased reactive oxygen species, activation of glial cells, and infiltration of peripheral inflammatory cells. An inflammatory response is known (Mitani et al ., Neuroscience., 42, pp661-670, 1991; Chu et al ., J Neuroinflammation., 9, pp 69, 2012; Wang et al ., Free Radic Biol Med., 43, 43 - 47 2016-05-04 pp 1048 - 1060, 2007), the exact mechanism of neuronal apoptosis caused by cerebral ischemia has not been elucidated, and hypotheses for new mechanisms are continuously being proposed.

Accordingly, current research for the treatment of ischemic cerebrovascular disease is progressing in the direction of effectively inhibiting neuronal cell death induced during cerebral ischemia based on the mechanism presented above, and in this regard, it is related to whether ischemia or occlusion of cerebral blood vessels. It is distinct from the treatment of degenerative cerebrovascular disease, which treats the loss or annihilation of nerve cells in a specific area or inhibition of nerve differentiation.

Currently, nimodipine, a calcium channel antagonist, flunarizine, and MK-801, a glutamate antagonist, dextraphan, and tirilazad, a free radical scavenger, are used in nerve cells. It has been developed as a protective agent and has been proven to have neuroprotective efficacy through preclinical studies, but it is difficult to see it as a drug to be used clinically as a treatment for cerebral ischemia due to the lack of efficacy and side effects in large-scale clinical trials.

Due to these side effects, etc., in the case of drug administration for the prevention of ischemic stroke, a compound extracted from natural resources is preferred rather than a single compound that may cause side effects like drugs for general diseases. According to this trend, with the recent increase in interest and acceptance of health functional foods, a number of natural substances are being marketed as health functional foods effective in preventing cerebrovascular diseases in Korea.

However, among them, many of them have not undergone scientific verification, and rather, they become a social problem because they cause misuse and abuse of health functional foods.

Therefore, by objectively verifying natural resources that have been used as food for a long time and whose safety has been proven, it is a natural substance that has therapeutic and preventive effects on cerebrovascular diseases, that is, natural substances that can inhibit or delay the death of brain nerve cells due to ischemia. The need for research on this topic is increasing.

KR 10-1601024B KR 10-1598970B KR 10-0306427B KR 10-2018-0063091A KR 10-2016-0048844A KR 10-2014-0033905A KR 10-2012-0021868A

In order to solve the above problems, the present invention effectively prevents ischemic stroke, inhibits neuronal cell death due to cerebral ischemia, as well as a composition for preventing ischemic stroke comprising an extract of a natural plant that is harmless to the human body as an active ingredient or, An object of the present invention is to provide a functional food for preventing ischemic stroke containing the composition as an active ingredient.

In order to achieve the above object, the present invention may relate to a pharmaceutical composition for preventing ischemic cerebrovascular disease comprising a natural product complex extract extract as an active ingredient.

The natural compound extract hyeonsasi trees (Populus tomentiglandulosa ) extract, water parsley ( Oenanthe javanica ) extract, Prunus pendula ) extract and Angelica gigas ) at least one selected from the group consisting of extract, preferably sagebrush extract, water parsley extract, oak extract and ginseng extract It may be composed of an Angelica extract.

The natural product complex extract may be extracted with any one extraction solvent selected from the group consisting of water, an organic solvent, and a mixture thereof, preferably ethanol or an aqueous ethanol solution.

The natural product complex extract is a mixture of several natural extracts, that is, in order to prevent damage to nerve cells in the CA1 region of the brain hippocampus tissue, that is, to improve nerve cell protection, the mixing ratio of each extract is 100 parts by weight of the As a basis, 60 parts by weight of parsley extract, 60 parts by weight of extract of Quercus oleifera, and 60 parts by weight of extract of Angelica serrata, that is, based on 100 parts by weight of extract of sagebrush, 100 parts by weight of sagebrush extract, 60 parts by weight of parsley extract, It may be prepared by mixing 60 parts by weight of the extract and 60 parts by weight of the Angelica oleracea extract.

Since the natural product complex extract has excellent protective ability of nerve cells due to cerebral ischemia, specifically, nerve cells in the CA1 region of the brain hippocampus, in this aspect, the pharmaceutical composition for preventing ischemic cerebrovascular disease is nerve cells caused by cerebral ischemia, specifically It can be used for the purpose of preventing damage to nerve cells in the CA1 region of the brain hippocampus.

The pharmaceutical composition for preventing ischemic cerebrovascular disease may be any one selected from the group consisting of ischemic stroke, cerebral infarction, cerebral hemorrhage, thrombosis, embolism, transient ischemic attack, subarachnoid hemorrhage, leukoplakia and small infarction.

The pharmaceutical composition for preventing ischemic cerebrovascular disease may include 0.001 to 99.999% by weight (w/w) of the natural product complex extract based on the total weight of the composition. Specifically, the pharmaceutical composition for preventing ischemic cerebrovascular disease is 50 mg/kg or more per day, preferably 50 mg/kg to 200 mg/kg, based on the dosage for the individual or patient in the natural product complex extract in the composition. It may be included in an amount that can be administered as In this aspect, the effective amount of the natural product complex extract may be 50 mg/kg or more per day, preferably 50 mg/kg to 200 mg/kg based on the dosage for the individual or patient.

In another aspect, the present invention may relate to a food composition for preventing or improving ischemic cerebrovascular disease comprising a natural product complex extract extract as an active ingredient.

The natural compound extract hyeonsasi trees (Populus tomentiglandulosa ) extract, water parsley ( Oenanthe javanica ) extract, Prunus pendula ) extract and Angelica gigas ) at least one selected from the group consisting of extract, preferably sagebrush extract, water parsley extract, oak extract and ginseng extract It may be composed of an Angelica extract.

The natural product complex extract may be extracted with any one extraction solvent selected from the group consisting of water, an organic solvent, and a mixture thereof, preferably ethanol or an aqueous ethanol solution.

The natural product complex extract is a mixture of several natural extracts, that is, in order to prevent damage to nerve cells in the CA1 region of the brain hippocampus tissue, that is, to improve nerve cell protection, the mixing ratio of each extract is 100 parts by weight of the As a basis, 60 parts by weight of parsley extract, 60 parts by weight of extract of Quercus oleifera, and 60 parts by weight of extract of Angelica serrata, that is, based on 100 parts by weight of extract of sagebrush, 100 parts by weight of sagebrush extract, 60 parts by weight of parsley extract, It may be prepared by mixing 60 parts by weight of the extract and 60 parts by weight of the Angelica oleracea extract.

Since the natural product complex extract has excellent protective ability of nerve cells caused by brain ischemia, specifically, nerve cells in the CA1 region of the brain hippocampus, in this aspect, the food composition for preventing or improving ischemic cerebrovascular disease is nerve cells caused by cerebral ischemia, Specifically, it can be used for the purpose of preventing damage to nerve cells in the CA1 region of the brain hippocampal tissue.

The food composition for preventing or improving ischemic cerebrovascular disease may be a health functional food.

The food composition for preventing or improving ischemic cerebrovascular disease may include 0.001 to 49.999% by weight of the natural product complex extract based on the total weight of the composition.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention are using natural resources that have secured human safety unlike the active ingredients of drugs, which have problems with existing safety and side effects, while researching substances that can prevent neuronal cell death due to ischemic stroke, natural product complex extract, Specifically, the present inventors confirmed whether the neuroprotective ability can be improved by mixing and using other natural extracts with the extract of sagebrush, which was confirmed to have neuroprotective ability in the CA1 region of the brain hippocampus, When the extract, water parsley extract, oak extract, and Angelica basil extract were used together, it was confirmed that the improvement effect was the best, and it was confirmed as a component of natural product complex extraction. As a result of additional research, the mixing ratio of the extracts was Based on 100 parts by weight, when each 60 parts by weight of water parsley extract, oak extract and Angelica basil extract are added and mixed, the improvement of the effect of preventing damage to nerve cells in the CA1 region of the brain hippocampal tissue, which is known to be vulnerable to cerebral ischemia, is the best. And it was experimentally confirmed that the most effective inhibition of neuronal cell death by inhibiting the abnormal activity of astrocytes and microglia, which are glial cells, was experimentally confirmed. Based on this, it was confirmed experimentally that the natural product complex extract was effective against cerebral ischemia through animal experiments. It was confirmed that there is a preventive, ameliorating or therapeutic effect on ischemic cerebrovascular disease that may be caused by, and the effect can be remarkably improved when administered at 50 mg/kg or more relative to the weight of the experimental animal (individual) in relation to the dosage. By confirming that there is, the present invention was completed based on this.

Unless otherwise specified in the present specification, the extract has the meaning commonly used as a crude extract in the art, but in a broad sense also includes a fraction obtained by additionally fractionating the extract. In addition, the extract includes not only those obtained using an extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various purification methods are also included in the extract of the present invention.

Unless otherwise stated herein, composition is meant to include products comprising the specified ingredients in the specified amounts, as well as products resulting directly or indirectly from combinations of the specified ingredients in the specified amounts.

Unless otherwise specified herein, an active ingredient is a composition, specifically a pharmaceutical composition or a food composition, which can effectively inhibit the treatment, improvement or prevention of ischemic cerebrovascular disease, specifically ischemic or delayed neuronal cell death. means ingredients.

Unless otherwise specified herein, the term "patient" includes both humans and animals.

Unless otherwise specified herein, an effective amount or therapeutically effective amount means an amount of the extract or composition of the present invention effective to produce an effect of treating, alleviating, alleviating, inhibiting or preventing a target disease.

Unless otherwise specified herein, nerve cell protection means to inhibit neuronal cell death in the central nervous system, for example, brain or spinal cord, or to protect from damage to nerve cells. More specifically, the protection of neurons may be to inhibit and protect neuronal cell death in a brain region induced by cerebral ischemia, for example, a hippocampal region.

Unless otherwise specified herein, glial cells are non-neuronal cells that provide nutrients to neurons and support neurons, structurally and functionally support neurons, and refer to non-neuronal cells that provide nutrients to neurons. . The glial cells are 10 times more than the number of nerve cells, occupy more than 50% of the volume of the brain and spinal cord, and play a leading and auxiliary role in neurotransmission and excitation of the nervous system. When the glial cell is abnormally active, it may rather cause damage to the nerve cell, and when the abnormal activation of the glial cell is effectively inhibited, the damage to the nerve cell may be suppressed.

Unless otherwise specified herein, astrocytes are a type of glial cells that play a role as a supporting tissue in the central nervous system, have a small cell body and a protrusion that splits in several directions, are star-shaped, and are fibrous Or it is characterized by having a protoplasmic projection, also called astrocytes. The astrocytes are the largest and most numerous cells among the glial cells of the central nervous system, and long, thin cell projections extend from the star-shaped cell body to the brain and spinal cord, and the terminal ampulla of the cell projection is the perivascular terminal button ( end-foot) and surrounds the capillaries, and the terminal button at these sites becomes part of the blood-brain barrier (BBB) to prevent toxic substances from entering the nervous system arbitrarily. In the cytoplasm of the astrocytes, intermediate filaments composed of glial fibrillary acidic protein (GFAP) composed of a unique amino acid sequence are concentrated, and the intrinsic protein, which is the glial fibrillary acidic protein, is concentrated in the central nervous system. Because it exists only in astrocytes, it is used as an immunohistochemical marker. Because the byeolglia have a traffic block, they form a structural syncytial body, physically support the nerve cells, and help the metabolic process. In addition, the astrocytes absorb an excessive amount of potassium to provide an appropriate ionic environment, control the activity of gamma-aminobutyric acid (GABA), and inactivate neurotransmitters such as glutamate.

Unless otherwise specified herein, microglia are glial cells of the central nervous system derived from the mesoderm, also referred to as microglia. The microglia are the smallest cells among glial cells, and the shape of the nucleus is elongated and there are twigs on the protrusion, and the tissue of the central nervous system, that is, the brain and spinal cord, transports and supplies necessary substances or destroys and removes debris. It functions to create a chemical environment suitable for the activity of nerve cells, and acts as an amoebic phagocytic to form the immune system of the brain that defends the nervous system from bacteria.

Unless otherwise specified herein, ischemia means that blood flow to a tissue is reduced or blood flow is blocked. When oxygen and nutrients are depleted in the tissue due to ischemia, apoptosis (necrosis) may occur in the tissue in which ischemia occurs.

In the present invention, ischemic cerebrovascular disease refers to a general term that refers to a disease that occurs because blood vessels in the brain are blocked for some reason and the necessary blood is not supplied to the brain, and is also referred to as cerebral ischemic disease. The ischemic cerebrovascular disease is caused by the induction of delayed neuronal cell death in the nerve cells of the CA1 region of the hippocampal tissue, which is known to be sensitive to cerebral ischemia among brain cells, because the blood flow of the brain is reduced and the supply of oxygen and glucose is not made normally. Refers to a disease, and examples of the ischemic cerebrovascular disease may include ischemic cerebrovascular disease, ischemic cerebral infarction, and the like.

The present invention relates to a pharmaceutical composition for preventing ischemic cerebrovascular disease comprising a natural product complex extract extract as an active ingredient.

The natural compound extract hyeonsasi trees (Populus tomentiglandulosa ) extract, water parsley ( Oenanthe javanica ) extract, Prunus pendula ) extract and Angelica gigas ) at least one selected from the group consisting of extract, preferably sagebrush extract, water parsley extract, oak extract and ginseng extract It may be composed of an Angelica extract.

The sycamore ( Populus) tomentiglandulosa ) is a member of the Salicaceae family and is a hybrid of Populus alba L. and Populus glandulosa Uyeki . age is evident. The sagebrush grows well in valleys or under the foot of a mountain, and propagation is done by cuttings. The aspen trees are widely planted in mountainous areas across the country, and clusters of the aspen trees can be easily identified in the mountainous areas close to the sanctuary. The aspen tree reaches a height of 20 m and a circumference of 50 cm. The bark of the aspen tree is gray-white, smooth, and rhombic bark is developed, and the leaves of the aspen tree are 3 cm to 8 cm in length and have an egg-shaped phyllotaxis, and the flowers of the aspen tree are either male or female. It blooms in April, and the fruit of the aspen tree is egg-shaped and ripens in May.

The sagebrush includes all forms commonly used to obtain extracts in the field of extracts without any particular limitation on the form thereof. Specifically, the sagebrush may be various organs or parts of the sagebrush, specifically leaves, flowers, roots, stems, branches, bark, and seeds, for example, leaves, stems, and mixtures thereof that are above-ground parts or edible parts. Any one selected from the group consisting of, for example, may be a leaf, a stem, or a mixture thereof.

The water dropwort (water dropwort, Oenanthe javanica ) is a perennial herb of the umbel family that grows wild throughout Korea and is widely distributed and cultivated for food in the boreal to tropical regions such as China and Japan. The water parsley is a food ingredient characterized by its unique fragrant and soft texture, and is used as a material for various foods. It is also used to prevent constipation or to get rid of a hangover after drinking. The stem of the water parsley grows to about 20 to 50 cm, is hollow, and the branches are divided a lot from the bottom and spread to the side. The leaves of the water parsley run alternately, and have long petioles with compound leaves that are split once or twice to the right, but become shorter upwards, and small leaves are ovate and have serrated edges. The flowers of the water parsley are white, and run finely in double inflorescences from the tip of the main stem in July to September, and have 5 petals and 5 stamens. The fruit of the water parsley is an oval-shaped segment.

The water parsley includes all forms commonly used to obtain extracts in the field of extracts without any particular limitation on the form thereof. Specifically, the water parsley may be various organs or parts of the water parsley, specifically leaves, flowers, roots, stems, branches, shells, and seeds, for example, consisting of leaves, stems, and mixtures thereof that are above-ground or edible parts. Any one selected from the group, for example, may be a leaf, a stem, or a mixture thereof.

The Angelica gigas Nakai is a perennial herbaceous plant belonging to the umbel family that is produced in Korea, and is mainly cultivated for medicinal purposes in Korea, Japan, China, etc., and is mainly cultivated in Japan depending on the production area. It is distinguished from Angelica acutiloba Kitagaw or Chinese Angelica sinensis Diels grown in China, and its components and pharmacological effects are also known to be different. The said Angelica grows near streams in mountain valleys. The height is 1 to 2 m, the whole is purple, the roots are large and the scent is strong, and the stems are straight. The flower of the Angelica basil blooms from August to September, is purple, and hangs in a double inflorescence at the end of the stem, and the petals are 5 long oval, pointed at the end, and have 5 stamens. The fruit of the Angelica fruit is produced in October, has an oval shape, has wings on the edge, and has one tube between the ridges.

The chamanggui is edible young shoots from ancient times, and the roots are called angelic guinea pigs and are used as medicines. Angelica root, which is used as a medicinal product, is used as a medicine for various diseases such as pain relief, anti-cancer, alleviation of kidney toxicity, improvement of liver function, treatment of diabetic hypertension, and improvement of blood circulation.

The common Angelica basil is not particularly limited in its form, and includes any form commonly used to obtain an extract in the field of extracts. Specifically, the Angelica larvae may be various organs or parts of Angelica keis, specifically leaves, flowers, roots, stems, branches, shells, and seeds, for example, the group consisting of roots, leaves, stems, and mixtures thereof. Any one selected from among, for example, may be a root.

The cherry tree ( Prunus pendula for. ascendens) is a deciduous broad-leaved tree belonging to the Rosaceae. It is distributed in Korea and Japan, and in Korea, it is distributed in Jangsangot, Wibongsan, Jirisan, Bogildo, and Jeju. The All Cherry tree reaches 10 m in height, has thin branches, grayish-brown bark, and has hairs on small branches, leaves alternate phyllotaxis, broad lancet-shaped or oval, serrated edges, and hairs along with petioles at first. The veins on the back also have hairs and saw teeth. The flowers of the All Cherry tree bloom earlier than the leaves in April, are light red, and 2 to 5 blooms in a hieroglyphic inflorescence. Petals are oval, concave at the tip, 10 to 12 mm long, with dense hairs on the lower part of the pistil. The style has no hairs, and the lower part of the calyx tube is particularly large. The fruits are round and ripen black in June to July. Among the above-mentioned all-cherries, those whose branches droop downward like weeping willows are called drooping all-cherries and are planted in gardens or parks as ornamental trees. In addition, when the bow was an important weapon in the past, the cherry tree was used as one of the important military materials. The bark of the sagebrush has the efficacy of relieving inflammation, antitussives, and detoxification, so it has been used as a treatment for relieving, dermatitis, heartburn, and pruritus.

The cherry tree includes all forms commonly used to obtain extracts in the field of extracts without particular limitations on the form. Specifically, the All cherry tree may be various organs or parts of the All cherry tree, specifically leaves, flowers, roots, stems, branches, bark, and seeds, for example, selected from the group consisting of bark or stems and mixtures thereof. Any one, for example, may be bark.

The natural product complex extract of the present invention may be prepared according to a conventional method for preparing a plant extract. More specifically, in the preparation of the natural product complex extract, an extraction solvent is added to a pulverized product obtained by pulverizing a natural product or a dried product of a natural product from which impurities have been removed, or a mixture of the natural product or a mixture obtained by pulverizing a dried product of the natural product. method can be done. The extraction method using the solvent may be a hot water extraction method, a cold extraction method, a hot extraction method, a pressure extraction method, a reflux extraction method, or an ultrasonic grinding extraction method, and preferably a solvent extraction method.

In this aspect, the natural product complex extract is a solvent extract extracted with an extraction solvent, a supercritical extract, that is, an extract or an ultrasonic extract through supercritical extraction with a supercritical fluid, or an extract extracted with the extraction solvent, supercritical fluid or ultrasound. It may be a fraction obtained by adding a fractionation solvent or a purified product obtained by performing chromatography on the fraction. More specifically, it may be extracted by performing a solvent extraction method, a supercritical extraction method, or an ultrasonic extraction method on the extraction target plant from which impurities have been removed, the pulverized product of the plant, the dried product of the pulverized plant, or a mixture of plants.

As an example, the H. ash tree extract is any one selected from the group consisting of leaves, stems and mixtures, a solvent extraction method, a supercritical extraction method or an ultrasonic extraction method, preferably a solvent extraction method, for the pulverized product or the dried product of the pulverized product is extracted by performing a solvent extraction method it may have been

As another example, the water parsley extract is extracted by performing a solvent extraction method, supercritical extraction method or ultrasonic extraction method, preferably a solvent extraction method, on any one selected from the group consisting of leaves, stems and mixtures, the pulverized product or the dried product of the pulverized product. it may have been

As another example, the Angelica basil extract is a solvent extraction method, supercritical extraction method or ultrasonic extraction method for any one selected from the group consisting of leaves, young leaves, roots and mixtures, preferably roots, a pulverized product or a dried product of the pulverized product. , preferably, it may be extracted by performing a solvent extraction method.

As another example, the Alcherichia tree extract is any one selected from the group consisting of bark or stems and mixtures thereof, preferably a solvent extraction method, supercritical extraction method, ultrasonic extraction method, for a dried product of the bark, a pulverized product thereof or a pulverized product thereof, It may be extracted by performing a reflux extraction method, a leaching method, a fermentation method or a foaming method, preferably a solvent extraction method.

The solvent extract may be prepared by using a conventional solvent, specifically, an extraction solvent under conditions of conventional temperature and pressure, according to a conventional method known in the art.

The extraction solvent may be any one selected from the group consisting of water, an organic solvent, or a mixture thereof, preferably water, an alcohol having 1 to 5 carbon atoms, and mixtures thereof that can be used for extracting natural products, more preferably It could be water. The organic solvent is a linear or branched alcohol having 1 to 5 carbon atoms such as methanol and ethanol, a polar solvent such as ethyl acetate or acetone, a non-polar solvent such as nucleic acid or dichloromethane, a neutral solvent such as a ketone having 3 to 5 carbon atoms, or their It may be a mixed solvent, preferably 10% (v/v, ethanol volume/total volume of aqueous solution) to 90% (v/v) aqueous solution of linear or branched alcohol having 1 to 5 carbon atoms, such as ethanol, more preferably is 20% (v/v) to 70% (v/v) aqueous ethanol solution, more preferably 25% (v/v) to 50% (v/v) aqueous ethanol solution, most preferably 30% (v/v) aqueous solution v) to 49.9% (v/v) aqueous ethanol solution.

In this aspect, the natural product complex extract may be extracted with any one extraction solvent selected from the group consisting of water, an organic solvent, and a mixture thereof, preferably ethanol or an aqueous ethanol solution. The aqueous ethanol solution is 10% (v / v, ethanol volume / total volume of aqueous solution) to 90% (v / v) ethanol aqueous solution or 20% (v / v) to 70% (v / v) ethanol aqueous solution or 25% ( v/v) to 50% (v/v) aqueous ethanol solution or 30% (v/v) to 49.9% (v/v) ethanol aqueous solution.

The extraction solvent may be 1 to 50 times, or 2 to 30 times, or 2 to 20 times, based on the weight of the extraction target. The extraction target is for extraction or leaching in the extraction solvent for 1 hour to 2,400 hours, or 2 hours to 1,680 hours, or 3 hours to 720 hours, or 4 hours to 480 hours, or 5 hours to 240 hours, or 6 hours to 120 hours, alternatively from 12 hours to 60 hours, or from 24 hours to 48 hours.

For example, the leaching method may be performed at 4° C. to 60° C., or 6° C. to 50° C., or 10° C. to 40° C., or 15° C. to 30° C. for 6 hours to 120 hours, or 8 hours to 96 hours, or 24 hours. to 72 hours, and the extraction solvent used for the leaching method is any one selected from the group consisting of water, alcohols having 1 to 6 carbon atoms, or mixtures thereof, or water, ethanol, or mixtures thereof. Any one selected, or ethanol or an aqueous ethanol solution may be used, and the ethanol aqueous solution may be 10% to 90% ethanol aqueous solution or 20 to 80% ethanol aqueous solution or 30 to 70% ethanol aqueous solution.

For example, the ultrasonic extraction method may be performed at 20°C to 70°C, or 25°C to 60°C, or 30°C to 50°C for 10 minutes to 6 hours, or 20 minutes to 5 hours, or 30 minutes to 3 hours. And, the extraction solvent used in the ultrasonic extraction method is any one selected from the group consisting of water, alcohols having 1 to 6 carbon atoms or mixtures thereof, or any one selected from the group consisting of water, ethanol, or mixtures thereof, or ethanol or It may be an aqueous ethanol solution.

In addition, the preparation of the fraction of the natural product complex extract can be carried out by a method of obtaining a fraction according to the polarity of the fractionation solvent after adding a fractionation solvent to the extract prepared by the above extraction method, that is, the crude extract or crude extract. The method for obtaining the fraction may be performed by a separation method by layer separation or a fractionation method. More specifically, after adding a fractionation solvent having a different polarity from the extraction solvent to the extract, for example, hexane, chloroform, methylene chloride, ethyl acetate, butanol and water fractionation solvents are added in the same order as described above, followed by layer-separated hexane fraction , a chloroform fraction, a methylene chloride fraction, an ethyl acetate fraction, a butanol fraction and a water fraction.

In this aspect, the fractionation solvent may be water, an alkane having 5 to 7 carbon atoms, a linear or branched alcohol having 1 to 5 carbon atoms including butanol, ethyl acetate, methylene chloride, chloroform, hexane, or a mixture thereof.

The fractionation method by layer separation may be a method of adding to the extract in that order according to the degree of nonpolarity of the solvent, and obtaining a fraction obtained by layer separation for each application, for example, by adding hexane to the ethanol aqueous solution extract hexane fraction obtained by fractionating the layer-separated hexane layer; a methylene chloride fraction obtained by fractionating the methylene chloride layer separated by adding methylene chloride to the layer remaining after separating the hexane fraction; The ethyl acetate fraction obtained by fractionating the layer-separated ethyl acetate layer by adding ethylene acetate to the layer remaining after separating the methylene chloride fraction and the water fraction remaining after separating the ethyl acetate fraction may be sequentially obtained.

The extraction solvent or the fractionation solvent may be replaced with other conventional solvents known to those skilled in the art that can exhibit substantially the same effect.

Chromatography for purification of the fraction is silica gel column chromatography (silica gel column chromatography), thin layer chromatography (TLC), high performance liquid chromatography (high performance liquid chromatography, HPLC), such as various chromatography uses. can be done by

In addition, the extract may be concentrated or the solvent may be removed by performing extraction, fractionation or purification, followed by filtration under reduced pressure or additional concentration and/or freeze-drying. Therefore, the natural product complex extract in the present invention is used in the sense of including a dry product of an extract dried by a conventional method and a concentrate of an extract concentrated by a conventional method, a dried product of the extract or concentrate, or a diluted solution of the concentrate or dried product. . The obtained natural product complex extract may be stored in a deep freezer until use. The dried product of the extract or concentrate may be prepared in a powder state by performing an additional process such as distillation under reduced pressure, freeze drying, or spray drying on the extract or concentrate.

The natural product complex extract is a mixture of several natural extracts, that is, in order to prevent damage to nerve cells in the CA1 region of the brain hippocampus tissue, that is, to improve nerve cell protection, the mixing ratio of each extract is 100 parts by weight of the Based on, 30 to 70 parts by weight of water parsley extract, 30 to 70 parts by weight of Alpine extract, and 30 to 70 parts by weight of Angelica oleifera extract, preferably, the mixing ratio of each extract is based on 100 parts by weight of sagebrush extract, parsley extract 50 to 60 parts by weight, 50 to 60 parts by weight of the extract, and 50 to 60 parts by weight of the Angelica oleifera extract, more preferably, the mixing ratio of each extract is based on 100 parts by weight of the sagebrush extract, 60 parts by weight of the water parsley extract, 60 parts by weight of the extract It may be in an amount of 60 parts by weight and 60 parts by weight of Angelica basil extract.

In this aspect, the natural product complex extract is based on 100 parts by weight of the sagebrush extract, 30 to 70 parts by weight of the water parsley extract, 30 to 70 parts by weight of the oak extract, and 30 to 70 parts by weight of the sagebrush extract, preferably the sagebrush extract. Based on 100 parts by weight, based on 100 parts by weight of the parsley extract, 50 to 60 parts by weight of the extract, 50 to 60 parts by weight of the Alpine tree extract, and 50 to 60 parts by weight of the Angelica oleifera extract, more preferably based on 100 parts by weight of the sagebrush extract, 60 parts by weight of the water parsley extract , may be prepared by mixing 60 parts by weight of an extract of Albaberry tree and 60 parts by weight of an Angelica basil extract.

Since the natural product complex extract has excellent protective ability of nerve cells due to cerebral ischemia, specifically, nerve cells in the CA1 region of the brain hippocampus, in this aspect, the pharmaceutical composition for preventing ischemic cerebrovascular disease is nerve cells caused by cerebral ischemia, specifically It can be used for the purpose of preventing damage to nerve cells in the CA1 region of the brain hippocampus.

In the present invention, ischemic stroke is a generic term for a disease that occurs because blood vessels in the brain are blocked for some reason and the blood necessary for the brain is not supplied, and is also called cerebral ischemic disease. The cerebral ischemia is clinically most commonly seen in cardiac arrest or stroke, and incurable cranial nerve cell damage is caused, which can lead to disability or coma, and in severe cases, death.

When the blood flow to the brain is reduced by the cerebral ischemia and the supply of oxygen and glucose is not made normally, the oxidative phosphorylation reaction is reduced, and as a result, anaerobic glycolysis is also stopped, and adenosine used as an energy source in the cell Adenosine triphosphate (ATP) is depleted. Due to the depletion of ATP, cell functions essential for maintaining life are lowered, and this leads to apoptosis by inducing various degenerative processes. The apoptosis occurs not only during the ischemia period but also by reactive oxygen species generated by oxygen supplied when blood flow in the ischemic region is restored and reperfusion occurs.

The apoptosis is more likely to occur in neurons in the CA1 region of the hippocampal tissue, which is known to be sensitive to cerebral ischemia among brain cells.

Specifically, in the present invention, the natural product complex extract was prepared at a dose of 200 mg/kg, administered for a certain period of time, and then anesthetized Mongolian gerbil to ligate the common carotid artery and induce cerebral ischemia. Then, in an experiment measuring the efficacy by staining the cells of the CA1 region of the brain hippocampal tissue and the hippocampal tissue, it was confirmed that the inhibitory effect of neuronal apoptosis caused by cerebral ischemia was excellent. Specifically, when the natural product complex extract was treated, it was experimentally confirmed through immunohistochemical staining using crezyl violet staining that the neuronal cell death of the hippocampal tissue was suppressed even in the occurrence of cerebral ischemia.

Since the natural product complex extract has the above effects, it may be contained as an active ingredient in a composition for the purpose of improving, preventing or treating ischemic cerebrovascular disease or ischemic stroke. In addition, the natural product complex extract of the present invention can be used as an active ingredient in a composition for protecting nerve cells, and in this aspect, the present invention is the use of the natural product complex extract, that is, the improvement, prevention or treatment of ischemic stroke of the natural product complex extract or nerve cells provides a protective purpose of

In this aspect, the present invention relates to a pharmaceutical composition for preventing ischemic cerebrovascular disease comprising a natural product complex extract as an active ingredient.

The pharmaceutical composition for preventing ischemic cerebrovascular disease may be any one selected from the group consisting of ischemic stroke, cerebral infarction, cerebral hemorrhage, thrombosis, embolism, transient ischemic attack, subarachnoid hemorrhage, leukoplakia and small infarction.

The transient ischemic attacks refer to the complete disappearance of symptoms within 24 hours after the onset of ischemic stroke symptoms caused by temporary cerebral blood flow failure, which indicates a warning or prognostic symptom that cerebral infarction may occur.

The administration of the pharmaceutical composition for preventing ischemic cerebrovascular disease may be administered once a day, or divided into several administrations. The dosage and frequency of administration are not intended to limit the scope of the present invention in any way.

The pharmaceutical composition for preventing ischemic cerebrovascular disease contains 0.001% by weight (w/w) to 99.999% by weight (w/w) or 0.1% by weight (w/w) to 99% by weight of the natural product complex extract based on the total weight of the composition. (w/w) may be included. Specifically, the pharmaceutical composition for preventing ischemic cerebrovascular disease is 50 mg/kg or more per day, preferably 100 mg/kg or more, for example 100 mg/kg or more, based on the dosage for the individual or patient in the natural product complex extract in the composition. It may be included in an amount that can be administered in mg/kg to 400 mg/kg. In this aspect, the effective amount of the natural product complex extract may be 50 mg/kg or more or 100 mg/kg to 400 mg/kg or 150 mg/kg to 300 mg/kg per day based on the dosage for the individual or patient. have.

The pharmaceutical composition for preventing ischemic cerebrovascular disease includes, in addition to the natural product complex extract, nutrients, vitamins, electrolytes, flavoring agents, coloring agents, neutralizers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stability It may further contain an agent, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like. The above components may be added independently or in combination to the pharmaceutical composition for preventing ischemic cerebrovascular disease. The content of the additional component may be preferably added in the range of 0.1 parts by weight to 20 parts by weight per 100 parts by weight of the natural product complex extract.

The pharmaceutical composition for preventing ischemic cerebrovascular disease containing the natural product complex extract as an active ingredient may further include appropriate carriers, excipients and diluents commonly used in the preparation thereof.

The pharmaceutical composition for preventing ischemic cerebrovascular disease containing the natural product complex extract as an active ingredient includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, Gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, physiological saline, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium, stearate and It may be at least one selected from the group consisting of mineral oil, dextrin, calcium carbonate, propylene glycol, and liquid paraffin, but is not limited thereto, and any conventional carrier, excipient or diluent may be used. The ingredients may be added independently or in combination to the natural product complex extract as the active ingredient.

The pharmaceutical composition for preventing ischemic cerebrovascular disease may be used in oral formulations such as powders, granules, tablets, suspensions, emulsions, and syrups, respectively, according to a conventional method.

The pharmaceutical composition for preventing ischemic cerebrovascular disease comprising the natural product complex extract as an active ingredient contains 0.001 wt% to 99.9 wt%, preferably 0.1 wt% to 99 wt%, of the natural product complex extract based on the total weight of the composition, more preferably Preferably, it can be added in the range of 0.1 parts by weight to 20 parts by weight per 100 parts by weight of the natural product complex extract.

When a composition containing the natural product complex extract as an active ingredient is medicated, a conventional filler, extender, binder, disintegrant, surfactant, anti-aggregant, lubricant, wetting agent, fragrance, emulsifier or preservative may be further included. , both oral and parenteral can be used.

Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one or more excipients in the antibacterial composition, for example, starch, calcium carbonate. , sucrose or lactose, gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

In addition, the dosage form for the pharmaceutical composition of the present invention may be in a preferred form depending on the method of use, and in particular, a method known in the art is adopted to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. can be formulated. Examples of specific formulations may be granules, powders, syrups, solutions, suspensions, ointments, needles, tablets, suppositories, injections, spirits, capsules, pills, soft or hard gelatin capsules, and the like.

The preferred dosage for the pharmaceutical composition of the present invention varies depending on the condition and body weight, the degree of disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art can Preferably, the pharmaceutical composition of the present invention is 1 mg/kg (active ingredient dose/individual body weight) to 5,000 mg/kg (active ingredient dose/individual body weight) per day based on the amount of the natural product complex extract, more In order to be effective, 10 mg/kg to 2,500 mg/kg, preferably 100 mg/kg or more, or 100 mg/kg to 400 mg/kg is preferred. Administration may be administered once a day, or may be administered in several divided doses. The number of administration is not intended to limit the scope of the present invention in any way.

A food composition for preventing or improving ischemic cerebrovascular disease or ischemic stroke according to another embodiment of the present invention includes the natural product complex extract as an active ingredient.

In the present invention, food means a natural product or processed product containing one or more nutrients, and preferably means that it is in a state that can be directly ingested through a certain amount of processing process, As a meaning, it is meant to include all health functional foods, beverages, food additives and beverage additives.

The food includes, for example, various foods, beverages, gum, tea, vitamin complex, health functional food, and the like. In addition, in the present invention, food includes special nutritional food (eg, formula milk, infant food, baby food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles), health supplements, seasoned foods ( Ex, soy sauce, soybean paste, red pepper paste, mixed soy sauce, etc.), sauces, sweets (eg snacks), dairy products (eg fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, overeating, vegetable beverages, soy milk, fermented beverages, etc.) and natural seasonings (eg, ramen soup, etc.). The food, health functional food, beverage, food additive and beverage additive may be prepared by a conventional manufacturing method.

In the present invention, health functional food refers to a food group or food composition that has added value to act and express the function of the food for a specific purpose using physical, biochemical, and bioengineering methods, etc. It refers to food that has been designed and processed to sufficiently express the bioregulatory function related to recovery, etc.

The health functional food may contain food-logically acceptable food supplement additives, and may further contain suitable carriers, excipients and diluents commonly used in the manufacture of health functional food.

The beverage refers to a generic term for drinking to quench thirst or enjoy taste, and is intended to include health functional beverages. The beverage is not particularly limited in other ingredients other than containing the natural product complex extract as an active ingredient as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. have.

Examples of the above natural carbohydrates include conventional sugars such as monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.) and polysaccharides (eg, dextrin, cyclodextrin), and xylitol, sorbitol, erythritol, etc. is a sugar alcohol of As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. . The proportion of the natural carbohydrate may be generally about 1 g to 20 g, preferably 5 g to 12 g per 100 ml of the food composition of the present invention. In addition, the composition of the present invention may further contain pulp for the production of natural fruit juice, fruit juice beverage, and vegetable beverage.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents, and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. These components may be used independently or in combination. The proportion of these additives is not so important, but may be selected from 0 to 20 parts by weight per 100 parts by weight of the natural product complex extract of the present invention.

In the present invention, a health functional beverage is a beverage that uses physical, biochemical, or bioengineering methods to act and express the function of the beverage for a specific purpose. It refers to a beverage designed and processed to sufficiently express the bioregulatory functions related to recovery and recovery.

The health functional beverage is not particularly limited in other ingredients other than containing the natural product complex extract of the present invention as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional beverages. have.

Examples of the above natural carbohydrates include conventional sugars such as monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.) and polysaccharides (eg, dextrin, cyclodextrin), and xylitol, sorbitol, erythritol, etc. is a sugar alcohol of As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. . The proportion of the natural carbohydrate may be generally about 1 g to 20 g, preferably 5 g to 12 g per 100 ml of the food composition of the present invention.

In addition, in the food composition for the purpose of preventing or improving the effect of ischemic stroke, the amount of the natural product complex extract may be suitably determined according to the purpose of use in the manufacture of food or beverage. As an example, the natural product complex extract may include 0.01% to 15% by weight of the total food weight, and in the case of a beverage composition, 0.001 g to 5 g, preferably 0.01 g, based on 100 ml of the total food weight to 1 g.

In the present invention, the composition containing the natural product complex extract as an active ingredient has an excellent neuroprotective effect in inhibiting neuronal cell death in the hippocampus region of a male Mongolian gerbil (Harlan, USA) that induced cerebral ischemia. , it was confirmed that the preventive effect of ischemic stroke was excellent.

In addition, the present invention relates to a composition for protecting nerve cells containing a natural product complex extract as an active ingredient.

In terms of inhibiting neuronal cell death, especially in the hippocampal region, the composition containing the natural product complex extract as an active ingredient has excellent neuroprotective effect. Therefore, the composition for protecting nerve cells is recognized for the preventive effect of ischemic stroke.

The composition for protecting nerve cells may be selected and applied according to the use as described in the composition for prevention comprising the natural product complex extract as an active ingredient according to its use, that is, according to its use for medicine or food.

In the natural product complex extract, the active ingredient of the present invention, each extract constituting the complex extract is a natural product-derived material, and each natural product previously used as a material for food or medicine and an ethanol aqueous solution that has secured safety, that is, alcohol is used. Since it is manufactured, toxicity or side effects are not a problem, and safety is recognized by confirming that there is no toxicity by administering the complex extract itself, and as a result of confirmation by the inventor of the present invention, the natural complex extract prevents the death of nerve cells. Since it can inhibit the damage to nerve cells caused by ischemic stroke can be effectively prevented, it can be usefully used in food or pharmaceuticals for the prevention, improvement or treatment of ischemic stroke. Therefore, the natural product complex extract can be widely used to prevent death or disability due to ischemic cerebrovascular disease in an aging society and a modern society in which circulatory diseases due to overnutrition have rapidly increased, so it is expected to be widely used in related industries.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.

Example 1. Preparation of natural product complex extract

The natural product complex extract used in the present invention was purchased through an oriental medicine store located in Gangneung-si, Gangwon-do. The natural product complex extract was prepared by preparing each natural product extract, followed by mixing in the combination of Table 1 below.

For each of the above natural products, in the case of the sagebrush extract (PE), the stem and leaves of the sagebrush tree were extracted, and in the case of the buttercup extract (DE), the leaves and stems of parsley were extracted, In this case, the root of Angelica serrata was used as the extraction target, and in the case of the cherry tree extract (AE), the bark of the cherry tree was used as the extraction target, and each natural product extraction target was dried by drying each natural product extraction target site, and then pulverized with a grinder. It was prepared as a powder and used.

2 kg of 30% (v/v) ethanol aqueous solution was added to 1 kg of the powder to be extracted of each of the natural products, and extracted at 80° C. for 2 hours. In the extraction process, the same extraction solvent was used to extract two more times at the same number of times, for a total of three extractions. The extract was filtered using a filter paper (Whatman No. 1, Whatman Ltd, UK), and then concentrated under reduced pressure using a rotary vacuum concentrator (EYELA, SN-100, Japan), and then concentrated. The extracted extract was vacuum-dried at 60° C. to prepare a powder state. The extract in powder form prepared above was used as a sample.

Each of the extracts prepared above was mixed in a combination shown in Table 1 to prepare a natural extract mixture, that is, 5 types of natural product complex extracts (MX1 to MX5). The unit in Table 1 below is g.

MX1 MX2 MX3 MX4 MX5 PE 1,000 1,000 1,000 1,000 1,000 DE 300 400 500 600 700 NE 300 400 500 600 700 AE 300 400 500 600 700

Example 2. Toxicity test of natural product complex extract through experimental animals

For the natural extract mixture prepared in Example 1, that is, the natural product complex extract, toxicity and side effects were tested using a rat repeated administration toxicity test model.

Specifically, 110 6-week-old rats of the SD strain were divided into 10 mice (5 males and females) in each group, and the natural product complex extract (MX1 to MX5) of Example 1 administered group and non-administered group were classified into a total of 11 groups. , The natural product complex extract (MX1 to MX5) of Example 1 was dissolved in physiological saline so as to have a total dose of 100 mg or 200 mg per 1 kg of body weight of each experimental animal to the reared experimental animals, and once daily at the same morning time Orally administered for 13 weeks. For example, when administering a total of 200 mg of the natural product complex extract per 1 kg of the body weight of each experimental animal, MX1 is quinceanus extract (PE) 105.26 mg, water parsley extract (DE) 31.58 mg, and Angelica oleracea extract (NE) 31.58 mg and 31.58 mg of Alsakura extract (AE), MX2 is 90.91 mg of H. asiatica extract (PE), 36.36 mg of buttercup extract (DE), 36.36 mg of Angelica serrata extract (NE) and 36.36 mg of Alpine extract (AE). , MX3 is to administer 80 mg of sagebrush extract (PE), 40 mg of water parsley extract (DE), 40 mg of Angelica oleifera extract (NE), and 40 mg of oleifera extract (AE), and MX4 is sagebrush 71.43 mg of extract (PE), 42.86 mg of water parsley extract (DE), 42.86 mg of Angelica asiatica extract (NE), and 42.86 mg of oleifera extract (AE) 42.86 mg, MX5 is an extract of sagebrush (PE) 64.52 mg, water parsley extract (DE) 45.16 mg, Angelica oleracea extract (NE) 45.16 mg, and Arachnid serratus extract (AE) 45.16 mg are administered.

As a control group, only physiological saline was administered at the same amount for 13 weeks. During the 13 weeks, not only the control group administered with only physiological saline, but also the natural product complex extract of Example 1 was administered to a total dose of 100 mg or 200 mg per 1 kg of body weight of each experimental animal. did not occur, and body weight, sample and water intake were confirmed to be similar.

From the above results, it was confirmed that all of the natural product complex extracts of Example 1 did not pose a problem in toxicity.

Example 3. Confirmation of neuronal protective effect during cerebral ischemia of natural product complex extract through experimental animals

In order to confirm the neuroprotective effect of the natural product complex extract prepared in Example 1, the safety of which was confirmed in Example 2, a male Mongolian gerbil weighing 70 g to 80 g as an experimental animal (Mongolian gerbil, Harlan, USA) was used. was used. The Mongolian gerbil was orally administered with a natural product complex extract before inducing cerebral ischemia, and the effect of preventing cerebral ischemia was confirmed depending on whether the natural product complex extract was used. Cerebral ischemia induction of the Mongolian gerbil was performed by anesthetizing the Mongolian gerbil to exposing the common carotid artery and then ligating the Mongolian gerbil to induce cerebral ischemia. After inducing the cerebral ischemia, the effect of inhibiting neuronal cell death due to cerebral ischemia of the natural product complex extract was confirmed through the method of staining and observing the hippocampal tissue of the Mongolian gerbil.

Example 3-1. Breeding of laboratory animals

Seventy male Mongolian gerbils (Meriones unguiculatus Harlan, USA) weighing 70 g to 80 g at 6 months of age were subjected to a constant light-dark cycle from 7 am to 7 pm, room temperature of 20°C to 25°C, and 45% to 65% relative humidity conditions. For the feed of the experimental animals, general pellet dry feed (Korea Experimental Animal, Korea) was used, and feed and water were allowed to be consumed at all times.

Example 3-2. Measurement of nerve cell protective effect during cerebral ischemia of natural product complex extract

In order to confirm the neuroprotective effect of the natural product complex extract, more specifically, the prevention or improvement effect of neuronal cell death due to cerebral ischemia, the natural product complex extract of Example 1 was tested on the experimental animals bred in Example 3-1. It was dissolved in physiological saline so as to be 200 mg/kg of animal body weight. The administration of the natural product complex extract was performed by oral administration every day for 5 days until brain ischemia was induced using an oral feeding needle.

Drugs were administered for 5 days before inducing cerebral ischemia, and then, using a gas mixed with nitrogen and oxygen in a ratio of 7:3 and 3% isoflurane (isoflurane, Baxtor, USA), experimental animals were treated. was under general anesthesia. Brain ischemia-induced surgery was performed on the experimental animal while maintaining the anesthesia state of the experimental animal by using a gas obtained by mixing 2.5% isoflurane in the nitrogen-oxygen mixed gas.

The operation on the experimental animal was performed by removing the hair from the neck and sterilizing it, then making an incision to expose both common carotid arteries, and ligating for 5 minutes using a non-traumatic aneurysm clip (Staelting, USA) for cerebral ischemia. After induction, the clip was removed and reperfusion was performed. At this time, each experimental group observed the blood circulation of the central artery of retina using an ophthalmoscope to confirm complete ligation of the common carotid artery.

During the operation of the experimental animal, the body temperature was measured by inserting a rectal thermometer, and the body temperature was kept constant so that the normal body temperature of 36.8°C to 37.2°C was used using a heating pad that was automatically adjusted according to the body temperature of the laboratory animal. .

The experimental animal was anesthetized by intraperitoneal injection of thiopental sodium (Yuhan Corporation, Korea) at a dose of 30 mg per 1 kg of body weight 5 days after inducing cerebral ischemia, and then anesthetized with heparin 1000 IU per 1000 ml. Physiological saline at 4°C was injected into the left ventricle for perfusion washing. Perfusion fixation was performed using 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4) at 4° C. for the animals after the perfusion wash was completed.

Using a bone cutter, the brains of the experimental animals that had been perfused and fixed were removed and brains were extracted. The brains of the extracted experimental animals were post-fixed for 4 hours using 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4) at 4°C. After the post-fixation is completed, the brain is put in a 30% sucrose solution (in 0.1 M phosphate buffer, pH 7.4) for cryoprotection and sedimented until it sinks to the bottom, and then a slide microtome (Reicert-) Jung, Germany) was used to cut the brain tissue to a thickness of 30 μm to make tissue sections, and the tissue sections were placed in a 6-well plate containing a storage solution and stored at 4° C. until staining was performed. .

Among the tissue sections made above, a tissue in which the hippocampal formation was well exposed was selected, and washed 3 times for 10 minutes with 0.01M PBS (pH 7.4) to remove the preservative solution attached to the tissue section.

In order to confirm the neuroprotective effect of the extract, specifically the effect of preventing neuronal cell death due to cerebral ischemia, staining was performed using cresyl violet acetate (Sigma) capable of staining nerve cells.

Specifically, the washed tissue sections were spread on gelatin-coated slides and sufficiently dried at 43°C. After immersing the sufficiently dried tissue sections in distilled water for a while, the tissue sections were stained by immersion in 2% cresyl violet acetate (cresyl violet acetate, Sigma, USA) solution for 1 minute. Then, the stained tissue sections are washed thoroughly in running water to remove excess dye on the slides, and after immersion in distilled water for a while, sequentially use 50%, 70%, 80%, 90%, 95% and 100% ethanol solutions. dehydration and washing with excess cresyl violet. After confirming that the Nissl body was visible in the tissue section, it was immersed in xylene (Junsei, Japan) to make it transparent and then encapsulated with Canada Balsam (Kanto, Japan).

In addition, the control group (solvent administration group, vehicle-ischemia group) subjected to ischemia-reperfusion was prepared in the same manner as above, except that only physiological saline used to dissolve the natural product complex extract was administered. prepared in this way. In addition, the normal group (sham group) did not administer any substance, the operation for causing the brain ischemia was not performed, and tissue sections of the normal group were prepared in the same manner as above.

For each tissue section of each experimental group, the total hippocampal area was 40 times larger and the CA1 area of the hippocampus was measured with an AxioM1 optical microscope (AxioM1 microscope, Carl Zeiss, Germany) equipped with a digital camera (Axiocam, Cal Zeiss, Germany). Each tissue section was magnified by 200 times to photograph each tissue section, and the results were confirmed.

In addition, in order to further confirm the neuroprotective effect of the extract, specifically the effect of preventing apoptosis of neurons due to cerebral ischemia, astrocyte markers to confirm the activity change of astrocytes and microglia Immunohistochemistry staining was performed using phosphorus glial fibrillary acidic protein (GFAP) and an ionized calcium-binding adapter molecule-1 (Iba-1) antibody, a marker of microglia.

More specifically, first, the washed tissue sections were _{reacted in 0.3% H 2} O ₂ (in 0.01M PBS) for 30 minutes to remove endogenous peroxidase, and then the reaction was finished. Tissue sections were washed 3 times for 10 minutes with 0.01M PBS. The washed tissue sections were reacted for 30 minutes in 5% normal horse serum (in 0.01M PBS) in order to check the changes in astrocytes, and in order to check the changes in microglia The washed tissue sections were reacted in 5% normal goat serum (5% normal goat serum, in 0.01M PBS) for 30 minutes. The primary antibody, mouse anti-GFAP (Chemicon, USA) or rabbit anti-Iba-1 (rabbit anti-Iba-1), is a primary antibody obtained by diluting each of the reaction-finished tissue sections at a ratio of 1:800, respectively. , Wako, Japan) at 4° C. for 48 hours. The tissue sections reacted with the primary antibody were each diluted 1:250 with the secondary antibody, anti-mouse IgG (anti-mouse IgG, Vector) or anti-rabbit IgG (anti-rabbit IgG, Vector), at room temperature for 2 After reacting for an hour, the tertiary antibody, ABC solution (avidin-biotin complex, Vector) diluted 1:200, was used to react at room temperature for 1 hour. After each reaction, the tissue sections were colored with 3,3'-DAB (diaminobenzidine) as a substrate. In performing each step of the reaction process with the antibody, the tissue sections were washed 3 times for 10 minutes each using 0.01M PBS for each step.

After the color development was finished, the tissue sections were spread on a gelatin-coated slide glass, dried at room temperature for 12 hours, and the dried tissue sections were subjected to a conventional dehydration and translucency process to cover glass (cover glass). ) was used for encapsulation.

As a result of the above confirmation, in the normal group (sham group) to which the ischemic surgery was not administered, neurons were darkly stained purple in the entire hippocampus, and in particular, in the CA1 region, neurons were concentrated in the stratum pyramidale (SP). could be observed However, in the control group (vehicle-ischemia), in which ischemia surgery was performed after administration of only a solvent, that is, physiological saline, nerve cell death occurred in CA1 area of the hippocampus due to delayed nerve cell death due to cerebral ischemia. Staining with Crezyl Violet was hardly observed, and it was confirmed that most neurons were lost in the pyramidal layer of the CA1 region.

On the other hand, in the experimental group, where 200 mg/kg of natural product complex extract was administered and then ischemia surgery was performed, a large number of neurons were dyed dark purple in the entire hippocampus, and neurons were also concentrated in the pyramidal layer of the CA1 region of the hippocampus. could be observed to have been

In the area where the nerve cells were stained, the number of stained and counted nerve cells in the pyramidal layer among CA1 areas of the normal group (Vehicle-Sham group) that was not administered with ischemia surgery after administration of only a solvent was 100%. After designating as a point, whenever the number of counted neurons in each experimental group decreased by 10%, the corresponding score was deducted by 1 point, and the protective effect of each experimental group was expressed as 1 to 10. The results indicated by the above criteria are shown in Table 2 below.

In addition, based on the results of immunohistochemical staining using a marker for glia in the tissue section of the hippocampal CA1 region, the relative optical density (ROD) was measured and calculated, and after administration of only the solvent After setting the optical density (ROD) for glial cells as 100% in the tissue section of the CA1 region of the normal group (Vehicle-Sham group) without ischemia surgery and assigning it to 10 points, the number of glial cells in each experimental group Each time the optical density (ROD) decreased by 10%, the protective effect of each experimental group was expressed as 1 to 10 in the form of deducting the corresponding score by 1 point. The results indicated by the above criteria are shown in Table 2 below.

division MX1 MX2 MX3 MX4 MX5 Neuroprotective effect during cerebral ischemia 5 6 8 8 7 Changes in glial cell activity during cerebral ischemia 5 6 7 8 6

As shown in Table 2, compared to MX1, it was confirmed that MX2 to MX5 showed a neuroprotective effect against cerebral ischemia equal to or greater than that of MX5, and in particular, the most excellent protective effect was confirmed in MX4. In particular, when the inhibitory effect of glial cells was confirmed, the most excellent inhibitory effect was confirmed in MX4.

From the above results, up to 60 parts by weight of other natural extracts compared to 100 parts by weight of the sagebrush extract, other natural extracts, specifically water parsley extract, Angelica asiatica extract, and cherry tree extract, the more the mixed content of the extract increases, the neuroprotective effect is improved. This was confirmed, but it was confirmed that the neuroprotective effect is rather reduced when it exceeds this, and the optimal combination in terms of the neuroprotective effect is 60 parts by weight of parsley extract, 60 parts by weight of parsley extract, 60 parts by weight of water parsley extract compared to 100 parts by weight of sagebrush extract It was confirmed to be mixed with parts by weight and 60 parts by weight of the extract of the cherry tree extract.

Although the preferred embodiment of the present invention has been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims are also provided. is within the scope of the

Claims (10)

Populus tomentiglandulosa ) extract, water parsley ( Oenanthe javanica ) extract, Prunus pendula ) extract, and Angelica gigas extract containing as an active ingredient at least one selected from the group consisting of ischemic containing a natural product complex extract as an active ingredient A pharmaceutical composition for preventing cerebrovascular disease. The method of claim 1,
The natural compound extract hyeonsasi trees (Populus tomentiglandulosa ) extract, water parsley ( Oenanthe javanica ) extract, Prunus pendula ) Extract and Angelica gigas ) A pharmaceutical composition for preventing ischemic cerebrovascular disease, characterized in that it consists of an extract. The method of claim 1,
The natural product complex extract is a pharmaceutical composition for preventing ischemic cerebrovascular disease, characterized in that it is extracted using any one extraction solvent selected from the group consisting of water, organic solvents, and mixtures thereof. 4. The method of claim 3,
The extraction solvent is a pharmaceutical composition for preventing ischemic cerebrovascular disease, characterized in that ethanol or an aqueous ethanol solution. The method of claim 1,
The pharmaceutical composition for preventing ischemic cerebrovascular disease is ischemic stroke, cerebral infarction, cerebral hemorrhage, thrombosis, embolism, transient ischemic attack, subarachnoid hemorrhage, ischemic cerebrovascular disease, characterized in that any one selected from the group consisting of leukodystrophy and small infarction A pharmaceutical composition for prophylaxis. The method of claim 1,
The natural product complex extract is a pharmaceutical composition for preventing ischemic cerebrovascular disease, characterized in that it protects nerve cells in the CA1 region of the brain hippocampus. Populus tomentiglandulosa ) extract, water parsley ( Oenanthe javanica ) extract, Prunus pendula ) extract, and Angelica gigas extract containing as an active ingredient at least one selected from the group consisting of ischemic containing a natural product complex extract as an active ingredient A food composition for preventing or improving cerebrovascular disease. 8. The method of claim 7,
The natural compound extract hyeonsasi trees (Populus tomentiglandulosa ) extract, water parsley ( Oenanthe javanica ) extract, Prunus pendula ) Extract and Angelica gigas ) Food composition for preventing or improving ischemic cerebrovascular disease, characterized in that it consists of an extract. 9. The method of claim 8,
The natural compound extract hyeonsasi trees (Populus tomentiglandulosa ) extract on sagebrush ( Populus ) tomentiglandulosa ) Based on 100 parts by weight of the extract, water parsley ( Oenanthe javanica ) extract 60 parts by weight, Prunus pendula extract 60 parts by weight and Angelica gigas extract 60 parts by weight ischemic brain, characterized in that it is prepared by mixing A food composition for preventing or improving vascular disease. 8. The method of claim 7,
The food composition for preventing or improving ischemic cerebrovascular disease is a food composition for preventing or improving ischemic cerebrovascular disease, characterized in that it is a health functional food.