KR20200005907A - Composition for neuroprotection containing extract or fraction of Prunus cerasoides as an effective component - Google Patents

Abstract

Provided is a composition for protecting brain nerve, containing a prunuscerasoides extract or fraction as an active ingredient on the basis of identification that the prunuscerasoides extract and fraction has an effect for neuroglobin (Ngb) activation and brain nerve protection. The composition for protecting brain nerve of the present invention can be used for a pharmaceutical composition or a food composition for treating, alleviating, and preventing stroke, brain infarction, thrombosis, brain degenerative disease, vascular dementia, hypomnesis, short term memory disorder, concentration disorder, anxiety, hypersensitivity, etc., and for brain damage protection, brain function improvement, and brain nerve protection.

Description

Composition for neuroprotection containing extract or fraction of Prunus cerasoides as an effective component}

The present invention relates to a composition for protecting the nerve of the brain, containing the extract or fraction of Prunus cerasoides as an active ingredient.

Stroke is a partial or total rapid impairment in the functioning of the brain, which lasts for a considerable period of time, and refers to cases in which no cause can be found other than cerebrovascular disease. In Chinese medicine, stroke is also referred to as 'middle wind' or 'wind', which is not a consensus because it includes diseases not included in stroke. Stroke is a term that includes both cerebral infarction (ischemic stroke) and cerebral hemorrhage (hemorrhagic stroke) caused by blood leakage into brain tissue due to rupture of the cerebrovascular vessel.

Brain tissue is usually supplied with a large amount of blood flow, but due to various causes of clogging of the blood vessels of the brain caused by a decrease in the amount of blood supplied to the brain will not function properly. When blood flow decreases for a certain period of time, brain tissue necrosis. Brain tissue necrosis leads to an unrecoverable state called cerebral infarction. Cerebral infarction includes cerebral thrombosis and embolism. Thrombosis (血栓症) refers to clogging of blood vessels by localizing the blood vessels, and when blood clots occur, the blood vessels are cut off in the blood flow, and if there is not enough compensation from other blood vessels from elsewhere, Brain tissue is killed. Embolism means that blood clots in the heart are transported to the periphery by the bloodstream and block the peripheral blood vessels. When the blood vessels are blocked by the embolism, the brain tissue of the part dies. In general, it is known that embolism tends to melt compared to blood clots, and blood flow flows again.

Degenerative diseases of the brain include Alzheimer's disease, Wilson's disease, and Parkinson's disease, which commonly indicate diffuse brain atrophy. Degenerative diseases that involve the cerebral cortex include Alzheimer's disease and Peek's disease. The main symptom is dementia.

Dementia is not a disease in itself. It is a comprehensive term that means that a state of cognitive impairment, including memory, occurs due to brain damage caused by various causes, and it is impossible to maintain a previous level of daily life. Dementia is a complex condition in which a mature brain is normally damaged or destroyed by acquired trauma or disease, and the overall cognitive and higher mental functions of intelligence, learning, and language are inferior. The most common cause of dementia is Alzheimer's disease (AD), which accounts for about 50-60%, followed by Vascular dementia (VaD), which accounts for 20-30%. Is dementia due to other causes. Vascular dementia is caused by clogged or narrowed cerebrovascular diseases such as cerebral infarction or cerebral hemorrhage.

Drugs applied to degenerative diseases of the brain include acetylcholinesterase-inhibiting drugs such as tacrine, vitamin E, which inhibits the destruction of brain cells by free radicals, and selegiline. Antioxidants and the like. However, the drugs developed so far are not known to have sufficient efficacy and are known to have a number of side effects.

In addition, the general public who complains of memory loss and concentration decreases even if the disease is not degenerative. Therefore, there is a need for drugs that can improve brain function and memory while minimizing or minimizing side effects.

Neuroglobin (Ngb) is a subtype of globin found in neurons that functions to detect, bind and transport O ₂ in neurons, and is involved in scavenge of reactive oxygen species (ROS). Ngb has been reported to promote neuronal regeneration and has been shown to be expressed at a very early stage of neuronal differentiation and to promote neuronal regeneration by preserving mitochondrial function and ATP production. In addition, Ngb plays an important role in cell hops through oxygen transport to brain tissue under normal physiological conditions. However, Ngb exhibits endogenous neuroprotective effects during brain injury. It is involved in signal transduction and is known to regulate apoptosis and cell survival by electron transfer of cytochrome C in mitochondria. Ngb has been reported to play a protective role against Alzheimer's or stroke, in particular because it is selectively expressed in cerebral neurons and is not only an endogenous neuroprotective factor but also has a function of promoting neuronal regeneration (Greenberg et al., 2008) (Yu et. al., 2013 B). Since Ngb is a pharmacologically controllable target, Ngb signal enhancement through drugs is expected to be very effective and useful for brain protection treatment (Jin et al., 2011).

On the other hand, Prunus cerasoides ( Prunus cerasoides ) is a plant of the genus Rosaceae ( Prunus ) Prunus cerasoides used in the experiments in the present invention was sold from the overseas biological material hub center. Prunus cerasoides is also called wild Himalayan cherry or sour cherry. It is known to inhabit the Himalayan Mountains, Southwestern China, Burma Province, Thailand from the Himachal Pradesh province of northern India. Prunus cerasoides Aqueous extracts of plant branches have been reported to be used in India for the purpose of preventing abortion (Kirtkar, KR et al., 1975). Prunus cerasoides contain flavonoids, steroids, terpene, and others. It contains glucogenkwanin as a flavone family, naringenin as a flavanone, and genistein and prunetin as an isoflavonoid. Steroids and terpenes include β-sitosterol and ursoric asid. Studies on components identified from Prunus cerasoides include studies on naringenin, prunetin, β-sitosterol, and ursolic acid (Kim S et al., 2013) (Tsutsui T et al., 2003) (Touillaud MS et al ., 2005) (Gu G et al., 2012).

CN 106562293 A CN 106138740 A CN 105918495 A CN 103432247 B CN 102716212 B CN 103432247 A US 8852578 B2 TW, I353841, B

 Khan AA, Mao XO, Banwait S, Der Mardirossian CM, Bokoch GM, Jin K, Greenberg DA .; Regulation of hypoxic neuronal death signaling by neuroglobin; FASEBJ. 2008 Jun; 22 (6): 1737-47.  Yu Z, Poppe JL, Wang X .; Mitochondrial mechanisms of neuroglobin's neuroprotection; Oxid Med Cell Longev. 2013; 2013: 756989.  Jin K, Mao XO, Xie L, John V, Greenberg DA .; Pharmacological induction of neuroglobin expression; Pharmacology. 2011; 87 (1-2): 81-4.  Kirtkar KR, Basu BD .; Indian Medicinal Plants, M / s Periodicals, New Delhi, 1975; 2: 959.  Kim S, Park TI .; Naringenin: a partial agonist on estrogen receptor in T47D-KBluc breast cancer cells; Int J Clin Exp Med. 2013 Oct 25; 6 (10): 890-9.  Tsutsui T, Tamura Y, Yagi E, Someya H, Hori I, Metzler M, Barrett JC .; Cell-transforming activity and mutagenicity of 5 phytoestrogens in cultured mammalian cells; Int J Cancer. 2003 Jun 20; 105 (3): 312-20.  Touillaud MS, Pillow PC, Jakovljevic J, Bondy ML, Singletary SE, Li D, Chang S .; Effect of dietary intake of phytoestrogens on estrogen receptor status in premenopausal women with breast cancer; Nutr Cancer. 2005; 51 (2): 162-9.  Gu G, Barone I, Gelsomino L, Giordano C, Bonofiglio D, Statti G, Menichini F, Catalano S, Ando S .; Oldenlandia diffusa extracts exert antiproliferative and apoptotic effects on human breast cancer cells through ERα / Sp1-mediated p53 activation; J Cell Physiol. 2012 Oct; 227 (10): 3363-72.

The present invention provides the treatment, improvement and prevention of stroke, cerebral infarction, thrombosis, cerebral degenerative disease, vascular dementia, memory loss, short-term memory disorder, concentration disorder, anxiety, neurosensitivity, etc. through neuroglobin (Ngb) activation, It is an object of the present invention to provide a brain neuroprotective composition having an effect on improving brain function and protecting neurons.

The present invention reveals that the extracts and fractions of Prunus cerasoides have a neuroprotective effect through neuroglobin (Ngb) activation, and based on this, extracts or fractions of Prunus cerasoides are effective. An object of the present invention is to provide a composition for protecting a nerve of nerve.

It was first discovered in the present invention that Prunus cerosaides extracts and fractions exhibit neuroprotective efficacy, and in the present invention, Prunus cerosaides extracts and fractions exhibit neuroprotective efficacy through neuroglobin (Ngb) activation. Prove that.

In order to achieve the above object, in the present invention,

Prunus cerasoides extract or fractions thereof as an active ingredient,

Through neuroglobin (Ngb) activation, treatment and prevention of stroke, cerebral infarction, thrombosis, cerebral degenerative diseases, vascular dementia, memory loss, short-term memory disorders, concentration disorders, anxiety, neurosensitivity; Brain injury protection; Improving brain function; Provided is a pharmaceutical composition for protecting nerves for at least one of brain nerve cell protection.

In the present invention,

Prunus cerasoides extract or fractions thereof as an active ingredient,

Neuroglobin (Ngb) activation improves and prevents stroke, cerebral infarction, thrombosis, cerebral degenerative diseases, vascular dementia, memory loss, short-term memory disorders, concentration disorders, anxiety, neurosensitivity; Brain injury protection; Improving brain function; Provided is a food composition for protecting the nerves for at least one of the neuronal cell protection.

Prunus cerasoides extract in the composition is preferably obtained by ultrasonic extraction for 2 to 4 days at 30 ℃ to 60 ℃ using methanol or ethanol as the extraction solvent.

In the food composition, the food may have any one of tablets, capsules, powders, granules, liquids, and pills. In addition, the food may have any one form of beverages, powdered drinks, solids, chewing gum, tea, vitamin complexes, food additives.

In the present invention, the prunus cerasoides extract and fractions have been found to have neuroglobin (Ngb) activation, thereby protecting the brain nerve. The present invention, based on these results, provides an extract or fraction of Prunus cerasoides as an active ingredient, a composition for protecting the nerve of the brain.

FIG. 1 shows that Prunus cerosaides extract alters Ngb gene transcriptional activity in SKNSH cells transfected with Ngb-luc plasmid, and shows the increase of Ngb gene transcriptional activity by Prunus cerosaides extract. .
FIG. 2 shows that Prunus cerosaides extract alters Ngb gene transcriptional activity in N2a cells transfected with Ngb-luc plasmid. As shown in FIG. .
Figure 3 is a vehicle (0.1% DMSO), prunus cerasoides extract treated Ngb gene expression in N2a cells treated with the results.
4 is a result of confirming the neuroprotective effect of the Prunus cerasoides extract in vitro hypoxia model, cells treated at 10 ^-7 ~ 10 ^-6 g / ml of hypoxic conditions when treated with Prunus cerasoides extract The results show a significant increase in survival.
5 is a result of confirming the neuroprotective effect of the Prunus cerasoides fraction in the in vitro hypoxia model, the cell viability and cells at the concentration of 10 ^-6 g / ml of hypoxic conditions when treated with the Prunus cerasoides fraction It has been shown to effectively improve toxicity.
6 is a test result of the prunus cerasoides extract in the mouse stroke model to reduce the stroke caused by stroke, the result of measuring the size of cerebral infarction after 45 minutes obstruction of the middle cerebral artery, 24 hours reperfusion.
Figure 7 shows the results of changes in the expression of E2-reactive Ngb protein in brain tissue after oral administration of Prunus cerosaides extract for 5 weeks to the female rats from which the ovaries were extracted.
Figure 8 is the result of oral administration of Prunus cerosaides extract to the ovarian isolated rats to measure the changes in blood lipids and glucose metabolism.
Figure 9 is the result of oral administration of prunus cerasoides extract to the ovarian isolated rats to measure the changes in blood liver level.
10 is a result of evaluating the effect of Prunus cerasoides extract on neurobehavioral changes including depression and anxiety, motility, cognitive function, (A) after administration of Prunus cerasoides extract for 2 weeks Results of cruciform high labyrinth experiment, (B) is the result of open field test after administration of Prunus cerasoides extract for 2 weeks, and (C) is the result of rotarod experiment after administration of Prunus cerasoides extract for 2 weeks to be.
11 is a result of evaluating the effect of Prunus cerasoides extract on neurobehavioral changes including depression and anxiety, motility, cognitive function, (A) after administration of Prunus cerasoides extract for 4 weeks The result of open field test, (B) is the result of open field test after administration of Prunus cerasoides extract for 4 weeks, (C) the result of forced swimming test after administration of Prunus cerasoides extract for 4 weeks (D) is the tail suspension test result after administration of Prunus cerosaides extract for 4 weeks.

In the present invention, the extract of Prunus cerasoides and fractions thereof reveals a neuroprotective effect through neuroglobin (Ngb) activation. In addition, the present invention provides a composition for protecting the nerve of the brain, the extract of Prunus cerasoides or fractions thereof based on these results.

Prunus cerasoides extract and fractions

Prunus cerasoides extract in the present invention is preferably obtained by extraction at 10 ~ 80 ℃ using water, alcohol or an organic solvent having 1 to 4 carbon atoms. More preferably, extraction by adding an extraction solvent to Prunus cerasoides; Filtering the extract; Concentrating the filtrate under reduced pressure; And, if necessary, drying the concentrate. Fractions can also be obtained from the extracts thus obtained. Each step will be described in more detail as follows.

1) Extraction Step

Extracted solvent is added to the mixture of bark, throat and leaves of Prunus cerasoides. At this time, the extraction solvent may be water, alcohol or an organic solvent having 1 to 4 carbon atoms, preferably water or a lower alcohol having 1 to 3 carbon atoms. More preferably methanol or ethanol is used. The extraction solvent is preferably extracted by adding 1 to 20 times the amount of dried Prunus cerosaides, and more preferably by adding 5 to 15 times. As the extraction method, methods such as shaking extraction, soxhlet extraction, reflux extraction, ultrasonic extraction using an ultrasonic generator, and an automatic extractor can be used. Extraction temperature becomes like this. Preferably it is 10-80 degreeC, More preferably, it is 20 degreeC-70 degreeC, Most preferably, it is 30 degreeC-60 degreeC. Extraction time is not limited, but in the case of ultrasonic extraction is preferably extracted 2 to 4 days at 50 ℃, 20 ± 5 minutes if using an automatic extractor. As an automatic extractor, ASE300 accelerated extractor (DIONEX Corporation) etc. can be used, for example.

In a preferred embodiment of the present invention using 95% or more methanol and ultrasonic extraction at 50 ℃ 2 to 4 days. In another preferred embodiment, when using an ASE300 accelerated extractor (DIONEX Corporation), extraction is performed for 20 minutes at 50 ℃, 1500 psi using N2 gas.

2) filtration step

The extract obtained in the above step is filtered using a porous filter medium. Filtration is to filter out solids, foreign matters, and the like and can be used without limitation as long as it is a filter medium suitable for this purpose. For example, filter paper, glass filter, glass fiber, cotton, cotton, nylon, porous metal, metal fiber and the like can be used.

3) concentration under reduced pressure

If left for a long time after filtration may precipitate, it is preferable to concentrate immediately. Concentration under reduced pressure is preferably a vacuum vacuum concentrator or rotary evaporator, but is not limited thereto. After completely concentrated under reduced pressure, it may be put in a vial or the like, sealed under reduced pressure, or further sealed and stored after further drying.

4) drying step

After concentration, further drying may be added as necessary. The drying method is not particularly limited, but preferably a reduced pressure drying, vacuum drying, boiling drying, spray drying, freeze drying, or the like can be used.

5) obtaining fractions

Fractions were obtained by applying the extracts obtained on the stems and leaves in the same manner as described above to the open column eluted with methanol solvent. 16 fractions and 15 fractions were obtained for the stem extract and the leaf extract, respectively.

Validation

In the present invention, the gene reporter evaluation test (Neuroglobin reactive gene transcription experiment), the evaluation of the effect on the transcriptional activity of the endogenous hormone-responsive target genes in the cell, the neurotoxic through the cytotoxicity and viability in hypoxia (Oxygen glucose deprivation, OGD) Evaluation of protective actions, assessment of neuroprotective effects in a live ischemic stroke model through middle cerebral artery occlusion (MCAO), and neurons through changes in Ngb protein expression in animal models undergoing Ovariectomy (OVX) To evaluate the protective effect, to evaluate the effect on blood metabolism markers and hepatic values in animal models subjected to Ovariectomy (OVX), and to evaluate the effect on behavioral neuroprotection in animal models treated with extract.

In the present invention through these experiments, Prunus cerasoides extracts and fractions are neuroglobin (Ngb) activation, neuroprotective effect, brain protection effect by reducing cerebral infarct size, brain protection effect by increasing the amount of expression of Ngb, triglycerides It has been shown to improve neurobehavioral symptoms on depression, anxiety and motility in improving levels and liver counts. It is first discovered and demonstrated in the present invention that Prunus cerosaides extracts and fractions exhibit neuroprotective efficacy or neuroglobin (Ngb) activation.

In the present invention, the safety was confirmed through acute toxicity test of Prunus cerasoides extract.

Based on these experimental results, the neuroprotective composition of the present invention, through neuroglobin (Ngb) activation, stroke, cerebral infarction, thrombosis, cerebral degenerative disease, vascular dementia, memory loss, short-term memory impairment, concentration disorder, anxiety, nervousness It can be used as a pharmaceutical composition or a food composition for the treatment, improvement and prevention, etc., brain damage protection, brain function improvement, brain nerve cell protection.

In recent years, the signal correlation between Ngb and female hormone receptor (ER) has been reported in neurons. Since the action of estrogen-induced neuroglobin was first reported in neurons by the De Marinis group (De Marinis, Ascenzi et al. 2010), estrogen-estrogen receptor (ER) α binding in neurons (SKNBE, NT2) Increased Ngb expression has been reported (Guglielmotto, Reineri et al. 2016) and increased Ngb induction by p38 MAPK signaling mediated via ERβ (De Marinis, Marino et al. 2011). In addition, mitochondrial hop-promoting action as a mechanism of action of ERβ has recently been reported (Ponnusamy, Tran et al. 2016), and it is expected that the possibility of interaction between Ngb and ERβ in mitochondria is very high. Bioinformatics information suggests that Ngb is highly expressed in certain areas of the brain (locus coeruleus, occipital cortex, lobe amygdala), carotid arteries, and cardiomyocytes (Guglielmotto, Reineri et al. 2016). It can be identified as a very direct therapeutic target for cardiovascular disease studies, and is expected to be a very effective and sensitive target, particularly in brain and cardiovascular disease studies of postmenopausal women. Therefore, the composition for protecting the nerve of the present invention may be particularly suitably used for the protection of the brain of women, especially menopausal women, and may be used to alleviate menopausal symptoms related to the brain nerve.

Pharmaceutical composition

The pharmaceutical composition for protecting the nerve of the present invention includes Prunus cerasoides extract or fraction as an active ingredient.

The pharmaceutical composition of the present invention may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions in addition to the active ingredient. The pharmaceutical composition of the present invention may further include other pharmaceutically active ingredients or active mixtures.

The pharmaceutical compositions of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and the like, oral formulations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid form preparations include at least one excipient such as starch, calcium carbonate, sucrose ( Sucrose) or lactose (Lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, Whitepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, it is good to administer the Prunus cerosaides extract at 0.0001 to 1000 mg / kg per day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular (Intracerebroventricular) injections.

The term definitions of the excipients, binders, disintegrants, lubricants, copulation agents, flavoring agents, etc. of the present invention are those described in the literature known in the art and include those having the same or similar functions.

Food composition

The food composition for protecting the nerve of the brain comprises the Prunus cerasoides extract or fraction as an active ingredient.

The food composition comprises 0.0001 to 20% by weight of the active ingredient in the total weight.

The food especially includes a dietary supplement. "Health functional food" as defined in the present invention means a food prepared and processed using raw materials or ingredients having a useful functionality to the human body, "functional" is to control nutrients or physiology for the structure and function of the human body Ingestion is intended for the purpose of obtaining a beneficial effect on health uses such as a pharmaceutical action. The health functional food may have any one form of tablets, capsules, powders, granules, liquids, and pills.

In addition, the food composition of the present invention may be a food composition in the form of a functional ingredient added to various foods or beverages. The food, for example, may be in the form of any one of beverages, powdered drinks, solids, chewing gum, tea, vitamin complexes, food additives.

The food composition of the present invention is not essential in addition to other ingredients other than the above containing the active ingredient as an essential ingredient and may further contain various ingredients such as various flavors or natural carbohydrates, such as ordinary food or drink. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may include a flesh for preparing natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but is preferably selected in the range of 0 to about 20% by weight of the total weight of the food composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to specific experimental examples and examples. However, these experimental examples and examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited by these examples.

Experimental Example

Preparation of Prunus cerosaides Extracts and Fractions

99.9% methanol was used as an extraction solvent, and an ultrasonic extractor (BRANSON) was used.

200 g of the mixture of bark, throat and leaves of the Prunus cerasoides sample dried in the shade was extracted at 50 ° C. for 3 days using 1.5 L of methanol. {(15 minutes of ultrasonic crushing, 2 hours place) × 10 times / day} × 3 days.

After extraction, the solid material was filtered through a degreased swab funnel. After long filtration, precipitates formed, and the filtrate was concentrated under reduced pressure completely at 45 ° C. The concentrated sample under reduced pressure was sealed in a vial and used in the experiment while being stored in a low temperature room (-4 ° C).

In addition, the stem and leaf extracts obtained after extracting the stem and the leaves in the same manner as described above were applied to an open column eluted with methanol solvent to obtain 16 fractions and 15 fractions, respectively. These fractions were used for the experiment.

Experiment method

1. Gene reporter evaluation test: Neuroglobin (Ngb) reactive gene transcription test

For Ngb-luciferase assay, we used human neuroblastoma cell line (SKNSH) and mouse neuroblastoma cell (N2a) overexpressed Ngb gene and transfected with promoter plasmid. At seeding, cells were cultured in 10% FBS DMEM medium and seeded in 96-well plates at a concentration of 3 × 10 ⁴ cells / well. When cells reached about 45-50% confluency, they were changed to 2% FBS DMEM medium. In the case of Ngb-luciferase assay, when the cells reach about 80% confluency, drug treatment is performed. As a negative control group, 0.1% Dimethyl Sulfoxide (DMSO) and Prunus cerosaides (stock solution dissolved in DMSO) of various concentrations are used. Diluted in the medium and treated with cultured cells. After incubation for 24 hours, cell culture was terminated, and water-soluble cell extracts were obtained using Passive Lysis Buffer (Promega, USA). Luciferase activity present here was measured quantitatively using Luciferase Assay System (Promega; including substrate and reaction buffer of luciferase) and Luminometer (ELISA reader, Promega, Madison, WI, USA). The gene activity expressed by the test substance was set to 100% of the activity indicated by the negative control (0.1% DMSO), and the relative activity was shown.

2. Evaluation of the effect on the transcriptional activity of intracellular endogenous hormone responsive target genes

After treatment with vehicle (0.1% DMSO) and Prunus cerosaides extract in mouse neuroblastoma cell (N2a), mRNA was extracted using Trizol. cDNA was obtained by reverse transcription of a certain amount of mRNA (0.5-1 μg) using iScript cDNA synthesis kit (Bio-Rad) or superscript II reverse transcriptase kit (Invitrogen) or ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO). . The expression levels of the genes were quantitatively measured by real-time PCR using cDNA, a pair of primers capable of recognizing the cDNA of the target gene, and PCR SYBR green kit (Qiagen) reagent. In order to make up for the technical mistakes in the various stages of the reaction, the expression level of GAPDH, a housekeeping gene, was simultaneously measured, and the ratio between the expression level of the gene and the expression level of the target gene was used for the final quantitative analysis.

Primers that recognize GAPDH are forward 5'-CTCTCTGCTCCTCCTGTTCGAC and reverse 5'-TGAGCGATGTGGCTCGGCT. The expression level of human Ngb (hNgb) as a neuroprotective target gene was measured. The primers that recognize hNgb for real-time RT PCR reactions are forward 5'-TGGAAGACCTGTCCTTCACTG and reverse 5'-GAGCAGAGACTCACCCACTG. Real-time PCR was performed at about 40 cycles (95 ° C; 15 seconds, 60 ° C; 60 seconds). Each gene expression curve was expressed in logarithm and then the threshold cycle (C _T ) was mathematically obtained. The value obtained by substituting this value into 2- ^ΔΔC _T indicates the relative expression level of the specific gene. The expression level of the specific gene divided by the expression level of the house keeping gene was selected as the final value for the gene expression level, and the treatment value of the vehicle and the test material were compared.

3. Oxygen glucose deprivation, OGD Cytotoxicity in) and Survival  Assessment of neuroprotective activity

Mouse neuroblastoma cells (N2a) and primary cortical neuron cells were placed in oxygen-depleted anerobic chambers (PlasLabs) for 8 and 3 hours of OGD, respectively. In the case of primary cortical neuron cells, a pure layer of neuron was obtained from the cortex separated from the fetus of the pregnant rats on 14-16 days, treated with trypsin for 15 minutes, added with horse serum, and centrifuged. Separated. The 24 well plate was coated with poly-D-lysine to seed primary neurons and replaced with neurobasal medium containing 2% B-27 every 2-3 days. Cells were incubated for 6-7 days in an incubator containing 37 ° C in 95% air / 5% CO2 and treated with drugs. OGD-induced solutions included NaCl (110 mM), KCl (5 mM), CaCl ₂ (1.8 mM), NaHCO ₃ (44 mM), MgSO ₄ (0.8 mM) and did not contain glucose and plasma and maintained pH 7.4. Primary cortical neuron cells were treated with vehicle (0.1% DMSO), Prunus cerasoides extract and fractions in the neurobasal medium containing 1% B-27 the day before OGD induction. The wells of the cell culture plate were washed once and the neurobasal medium treated with the drug was treated with the cells. After 24 hours, the wells of the plate were washed once and treated with OGD solution to prepare 90% nitrogen, 5% hydrogen and 5% carbon dioxide. The mixed gas was passed through at least 30 minutes to completely remove oxygen in the bubbling solution. In the case of N2a cells, the wells of the cell culture plate were washed once, and the mixed gas of 90% nitrogen, 5% hydrogen, and 5% carbon dioxide was passed through the mixed gas of 90% nitrogen, 5% hydrogen, and 5% carbon dioxide for 30 minutes immediately after treatment with the drug-treated OGD solution. Oxygen was completely removed. After a certain period of time, primary cortical neuron cells were treated with neurobasal medium containing 2% B-27 and DMEM containing glucose and plasma for N2a cells and transferred to an incubator for 24 hours to reperfusion for WST assay kit ( Dojindo) and LDH assay kit (Biovision). After 24 hours of reperfusion, media were taken from the wells of the plate and WST assay was performed to evaluate cell viability through absorbance measurements at 450 nm or LDH assay to measure cytotoxicity through absorbance measurements at 490 nm and 600 nm. The cell viability and toxicity of the test substance were determined by comparing the viability and toxicity of the vehicle (0.1% DMSO) to 100%, indicating their relative activity, and finally evaluating the results.

4. Middle Cerebral Artery Occlusion  Surgery (middle cerebral artery occlusion, MCAO Evaluation of Neuroprotective Effect in Human Ischemic Stroke Model

To evaluate the neuroprotective effects of oxidative damage of brain tissues due to cerebrovascular occlusion and reperfusion, mouse MCAO surgery was performed to establish a stroke model. The medial neck of the 10-week-old female CD1 mouse was incised to expose the external carotid artery (ECA), and then a nylon suture was inserted through the external carotid artery into the internal carotid artery (ICA) for the middle cerebral artery. , MCA) was blocked. After 45 minutes, nylon sutures were removed to restore blood flow and induced ischemia / reperfusion damage. A transient ischemic model was produced by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

The animals were divided into vehicle (0.9% Nacl solution, saline) group and Prunus cerasoides (250 mg / kg) extract group. Before the MCAO operation, the two groups of medications were administered intraperitoneally for 4 weeks at intervals of 24 hours. After the administration, MCAO surgery was performed. The change in cerebral infarct size indicated by the test substance was evaluated based on the cerebral infarct size indicated by the vehicle (0.9% Nacl solution, saline) group.

5. ovariectomy, OVX In animal models Ngb  Evaluation of neuroprotective effect by changing protein expression level

The effects of oral administration of drugs on the ovarian isolated rats on the changes of Ngb protein expression in brain tissues were studied. To confirm the brain protective effect of postmenopausal women, ovaries of 9-week-old female SD rats were ectomy to create a hormonal environment similar to postmenopausal women. Eight-week-old female SD rats were purchased and allowed to acclimate for one week. Rats were anesthetized with isoflurane, and under the anesthesia, the abdomen was dissected to remove the ovaries. After ovarian extraction, 0.2 mg / kg of ketoprofen, an analgesic, was administered. After the operation, three animals were placed in cages for two weeks of recovery and blood estrogen wash-out. Phytoestrogen-limited diets (Harlan 2020X Teklad Global Soy Protein-Free Extruded Rodent Diet) were administered to avoid affecting the experimental results. Rats undergoing recovery were divided into Sham (no OVX), OVX control, low concentrations of Prunus cerasoides extract group, and high concentrations of Prunus cerasoides extract group. For 5 weeks, Sham (no OVX) and OVX control group were fed a formulated feed, a low concentration extract group was given a 250 mg / kg plant extract, and a high concentration extract group 500 mg / kg. At this time, the distribution of 20-25g of food per rat so that the quantitative components can be provided, and the weight change of the rat was observed by measuring the weight every week. After 5 weeks of oral administration, fasting for 24 hours, anesthetized with isoflurane, sacrificed, and extracted brain tissue to separate proteins. Proteins were isolated by adding a lysis buffer containing protease inhibitors and phosphatase inhibitors to the brain tissues of mice, and the extracted proteins were quantified by BCA method at 50 μg of 7.5% SDS-PAGE gel at 80V for 2 hours. It moved. After electrophoresis, transfer to PVDF membrane for 3 hours at 25V, and then block with phosphate buffered saline (PBS) dissolved in skim milk for 1 hour at RT and react with primary and secondary antibodies to confirm with ECL solution. It was. At this time, Ngb (RD181043050, BIOVENDER) was used as a primary antibody in a 1: 1000 ratio. Β-actin was used to correct the difference in protein amount due to experimental error.

6. Ovariactomy OVX Metabolism in Animal Models Received With markers On guard  Impact study

The effects of oral administration of extracts on ovarian-extracted rats on the changes in blood metabolic markers and liver values were studied. After 5 weeks of oral administration, fasted for 24 hours, anesthetized with isoflurane, and collected blood samples of about 4-5 ml from the heart. The collected blood samples were centrifuged at 3500 rpm (15 minutes, 4 ° C.) to obtain plasma samples, and then stored at 4 ° C. and requested to an analysis institution within 1-2 days to analyze lipid profiles in blood.

7. Receiving medication  Study on the effect on behavioral neuroprotection in animal models

Drugs were injected intraperitoneally to evaluate neurobehavioral changes, including depression and anxiety, motility and cognitive function. 10-week-old female C57bL / 6 mice and 10-month-old female C57bL / 6 mice were divided into vehicle (0.9% Nacl solution, saline) and Prunus cerasoides (250 mg / kg) extract administration groups. A total of four groups of drugs were administered intraperitoneally for 4 weeks at intervals of 24 hours. After 2 and 4 weeks of administration, behavioral tests such as open field test, cruciform high labyrinth, tail hanging test, forced swimming test, and rotarod were performed. Was carried out. During the behavioral experiment, the video was recorded and analyzed. After the experiment, the video was analyzed and evaluated, and the behavioral change represented by the test substance was measured based on the behavioral value indicated by the vehicle (0.9% Nacl solution, saline) administration group. Analysis, final comparative evaluation.

8. Extract of Single  Assessment of safety effects through acute toxicity testing

Single dose acute toxicity studies were performed to increase the clinical applicability of the plant extracts. 10-week-old female CD1 mice were used and fasted 24 h before administration. Body weight (1% CMC in saline), 250mg / kg extract, 500mg / kg, 1000mg / kg doses were administered in a single oral dose to measure weight change, behavioral observation, and mortality. The experiment was carried out for a total of 2 weeks and the administration volume was within 5μl / g.

result

One. Prunus Cerasoides  Of extract Ngb  Through response genes SKNSH  Cells and N2a Intracellular  Transcriptional Activity Assay

Prunus cerosaides from SKNSH and N2a Cells Transfected with Ngb-luc plasmid Whether the extract changes the Ngb gene transcription activity and the results are shown in Figures 1 and 2. When Prunus cerasoides extract was treated with SKNSH cells at a concentration of 5 x 10 ^-6 g / ml to 5 x 10 ^-5 g / ml, the transcriptional activity of the Ngb gene was significantly decreased at all concentrations compared to vehicle. 1.4-1.6 fold increase (** p <0.01) (FIG. 1). When Prunus cerasoides extract was treated with N2a cells at a concentration of 5 x 10 ^-6 g / ml to 5 x 10 ^-5 g / ml, the Ngb gene was statistically significant at 5 x 10 ^-5 g / ml. Transcriptional activity was increased by about 1.4-fold (*** p <0.001, ** p <0.01) (FIG. 2). The experimental results showed that Prunus cerasoides extract induced Ngb transcriptional activity at the concentration of 5 x 10 ^-5 g / mL during gene transcription, and the extract was higher than Vehicle (Veh) for both cell types. It was confirmed to exhibit Ngb transcriptional activity.

2. Prunus Cerasoides  Of extract N2a  In the cell Ngb  Gene expression induction

Ngb gene expression was confirmed in N2a cells treated with Vehicle (0.1% DMSO) and Prunus cerosaides extract. The results are shown in FIG. When Prunus cerasoides extract was treated with N2a cells at concentrations of 10 ^-7 g / ml to 10 ^-6 g / ml, the expression of Ngb gene was significantly increased by about 2-fold at 10 ^-6 g / ml. (FIG. 3). These results confirm that Prunus cerosaides extract induces Ngb gene expression.

3. Prunus Cerasoides  Extracts Fraction  Neuroprotective effect on hypoxic-induced cells

The neuroprotective effect of Prunus cerasoides extract in the in vitro hypoxia model showed that N2a cells treated with Prunus cerasoides extract were viable at 10 ^-7 ~ 10 ^-6 g / ml concentration under hypoxic conditions. The degree was significantly increased, and the treatment of Prunus cerosaides extract in primary cortical neuron cells increased cell viability by approximately 2-fold at a concentration of 10 ⁻⁶ g / ml of hypoxic culture conditions (FIG. 4).

Confirming the fruit Taunus sera neuroprotective effects of soy des fractions in vitro hypoxia model result, when handling the fruit haejun Taunus sera Soy des fractions in primary cortical neuron cells, stem fraction from 10 ^-5 g / ml concentration of the low oxygen culture conditions 3, 5, 6, 10, 11, 12, 14, 16 and leaf fractions 6, 7, 8, 9, 11, 14 significantly reduced cytotoxicity by 10-30% (FIG. 5). Through the experimental results, it was confirmed that the Prunus cerasoides extract and fractions were effective in increasing cell viability and decreasing toxicity in the hypoxic culture state, and it was evaluated that the neuroprotective effect was excellent (FIG. 4, 5).

4. Prunus Cerasoides  By reducing cerebral infarct size Brain protection  effect

To determine whether Prunus cerosaides extract showed brain protective effects such as stroke reduction by stroke, female CD1 mice were administered with intraperitoneal injection for 4 weeks. The brain protection effect was confirmed by measuring the size of cerebral infarction after 45 minutes of middle cerebral artery occlusion and 24 hours of reperfusion. The results are shown in FIG. Compared to the vehicle group, Prunus cerasoides extract group reduced the size of cerebral infarction by about 80% (Fig. 6). These results show that long-term drug administration of Prunus cerasoides extract has a protective effect on brain stroke by reducing cerebral infarct size.

5. In menopause caused by ovarian extraction Prunus Cerasoides  Of extract Ngb  protein On expression  Neuroprotective effect

To determine whether Prunus cerasoides extract showed brain protection in postmenopausal women, expression of E2-reactive Ngb protein in brain tissues was obtained by oral administration of Prunus cerasoides extract for 5 weeks to female ovaries. Confirmed the change. The results are shown in FIG. Compared with the Sham operate group, the amount of Ngb protein was decreased in the OVX group, but it was confirmed that the concentration of Ngb protein was concentration-dependent in the brain tissue of the administration group treated with Prunus cerosaides extract (FIG. 7). These results show that long-term oral administration of Prunus cerasoides extract has a neuroprotective effect by increasing the expression of Ngb in brain tissues during hormonal deficiency in postmenopausal women.

6. In menopause caused by ovarian extraction Prunus Cerasoides  Blood metabolism of extract Marker  And Gansu  Effect on change

The effects of oral administration of Prunus cerasoides extract on ovarian-extracted rats were investigated on the changes in blood metabolic markers and liver parameters. After five weeks of oral administration, fasting for 24 hours, blood samples were taken from the heart, and lipid profile analysis was performed. The results are shown in FIGS. 8 and 9. As a result of measuring lipid and glucose metabolism, triglyceride was compared with Ovx group and 250 mg / kg and 500 mg / kg of Prunus cerasoides extract. Triglycerides were reduced to a level similar to that of the Sham, E2 administration group (FIG. 8). As a result of analyzing ALP, AST, and ALP levels of hepatic metabolism enzymes, the group that received the Prunus cerasoides extract 500mg / kg compared with the control group and the control group who received the Prunus cerasoides extract 500mg / kg It was confirmed that the levels of ALP, AST, and ALP were reduced to levels similar to those of the Sham, E2 administration group (FIG. 9). These results confirm that Prunus cerasoides extract is effective in improving triglyceride and liver levels in the body.

7. Prunus Cerasoides  Behavioral Neuroprotective Effect by Extracts

To evaluate the effect of improving brain function after menopause, the effects of Prunus cerasoides extract on female mice were evaluated on neurobehavioral changes including depression and anxiety, motility and cognitive function. Intraperitoneal injection of Prunus cerasoides extract was divided into 10-week-old female mice and 10-month-old female mouse groups, and behavioral tests were conducted at 2 and 4 weeks, respectively. The results are shown in FIGS. 10 and 11. As a result of the two-week Prunus cerasoides extract administration, it was confirmed that the elderly group who received the Prunus cerasoides extract decreased the number of times that they entered the enclosed space in the cruciform high maze experiment compared to the vehicle administration group. In all age groups that received Prunus cerasoides extract, the number of times to go to the corner space was reduced compared to the vehicle-administered group, and the anxiety improvement effect was confirmed. In addition, the elderly group receiving the Prunus cerasoides extract was able to confirm the improvement in motility because the time until the fall in the rotarod experiment increased compared to the vehicle administration group (Fig. 10).

As a result of 4 weeks of Prunus cerasoides extract administration, in the open field test, the total distance moved by the Prunus cerasoides extract group of old age was increased compared to the vehicle administration group, and the number of times to the corner space was decreased to confirm the anxiety effect Could. In the forced swimming test, all the age groups receiving the Prunus cerasoides extract showed more time than the vehicle group, and in the tail hanging experiment, the prunus cerasoides extract group of the older group showed more movements than the vehicle group. Since the time shown increased, the effect of improving depression was confirmed. In addition, the elderly group receiving the Prunus cerasoides extract was confirmed that the time until the fall in the rotarod experiment increased compared to the vehicle administration group improved the mobility (Fig. 11). These results confirm that Prunus cerasoides extract is effective in improving neurobehavioral symptoms for depression, anxiety, and motility that are closely related to menopausal syndrome, and have an effect of improving brain function after menopause.

8. Prunus Cerasoides  For extract Single  Acute Toxicity Safety Effects

Single-dose acute toxicity experiments were performed to evaluate the in vivo toxicity of Prunus cerosaides extract. Female CD1 mice were divided into four groups, orally administered at vehicle (1% CMC saline), Prunus cerasoides extract 250 mg / kg, 500 mg / kg and 1000 mg / kg. Observed. The results are shown in Table 1 below. Animal movement was decreased immediately after administration, but normal behavior was observed after 24 hours, and feed and water intake were observed. The mortality rate was 0% in all groups, including the vehicle group, during the two-week period. The maximum dose of 1000 mg / kg oral dose did not show acute toxicity and therefore is considered to be safe (Table 1).

TABLE 1 Prunus Cerasoides  Evaluation of Acute Toxicity by Extracts

Claims (6)

Prunus cerasoides extract or fractions thereof as an active ingredient,
Through neuroglobin (Ngb) activation, treatment and prevention of stroke, cerebral infarction, thrombosis, cerebral degenerative diseases, vascular dementia, memory loss, short-term memory disorders, concentration disorders, anxiety, neurosensitivity; Brain injury protection; Improving brain function; A pharmaceutical composition for protecting the nerve of the brain for one or more of neuroprotective cells. The method of claim 1,
The prunus cerasoides extract is a pharmaceutical composition for protecting the nerves of the brain obtained by ultrasonic extraction at 30 ℃ ~ 60 ℃ for 2 to 4 days using methanol or ethanol as the extraction solvent. Prunus cerasoides extract or fractions thereof as an active ingredient,
Neuroglobin (Ngb) activation improves and prevents stroke, cerebral infarction, thrombosis, cerebral degenerative diseases, vascular dementia, memory loss, short-term memory disorders, concentration disorders, anxiety, neurosensitivity; Brain injury protection; Improving brain function; Cranial nerve protective food composition for at least one of the neuronal cell protection. The method of claim 3,
The prunus cerasoides extract is a brain nerve protective food composition obtained by ultrasonic extraction for 2 to 4 days at 30 ℃ ~ 60 ℃ using methanol or ethanol as the extraction solvent. The method according to claim 3 or 4,
The food is a dietary supplement having a form of any one of tablets, capsules, powders, granules, liquids, pills. The method according to claim 3 or 4,
The food composition is any one of the form of beverages, powdered drinks, solids, chewing gum, tea, vitamin complexes, food additives.

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