KR101111522B1 - Composition for the treatment and/or prevention of neurological ischemic brain disease by using Laurus nobilis leaves extracts - Google Patents

Abstract

In the present invention, the laurel alcohol extract and the nucleic acid fraction have a protective effect against cell death in an artificial cerebral ischemia model in which OGD treatment of human neuroblastoma SH-SY5Y cells is performed. The laurel chloroform fraction is cerebral ischemia of human neuroblastoma SH-SY5Y cells. In the model, the cells were protected from apoptosis by inhibiting the dephosphorylation process that activates DAPK. In the cerebral cortex of tissue culture, OGD-induced hippocampus cell death was suppressed in a dose-dependent manner. It relates to a composition for the prevention and treatment of ischemic neurological brain diseases, including alcohol extract and chloroform fraction of laurel leaves, which can confirm the reduction of the infarction range.

Description

Composition for the treatment and / or prevention of neurological ischemic brain disease by using Laurus nobilis leaves extracts}

The present invention relates to a composition for the prevention and treatment of ischemic neurological brain disease containing alcohol extract and chloroform fraction of laurel leaf, more specifically, laurel alcohol extract, chloroform fraction and nucleic acid fraction is derived from oxygen glucose deficient environment By extracting the effect on brain cell death and the effect of inhibiting cerebral infarction due to the middle cerebral artery occlusion, the alcohol extract and the chloroform fraction of the bay leaf provide a composition for preventing and treating ischemic neurological brain disease.

Of the various organs of the human brain, the brain is the most energy consuming organ, but it is an organ that does not produce or store energy itself and depends solely on the blood supply. Thus, blocking the blood supply leads to immediate brain tissue damage and death of brain neurons. Such damage is associated with various ischemic brain-related diseases because it leads to impairment of brain function, and may also be accompanied by behavioral disorders, movement disorders, and perceptual disorders caused by damage to specific brain tissues. That is, by effectively preventing and treating damage to the brain tissue due to the blockage of the blood supply, it is possible to alleviate various disorders that may occur secondary.

 In particular, stroke can be divided into cerebral ischemia, which occurs when the blood supply is blocked due to constriction of blood vessels or blocked by blood clots, and cerebral hemorrhage due to intracranial hemorrhage. Among them, cerebral ischemia causes a lack of oxygen and nutrients due to blocking blood supply, resulting in a decrease in ATP in neurons and edema resulting in direct cell damage and necrosis. This edema interferes with the smooth functioning of the brain and induces necrosis of tissues, leading to extensive brain tissue damage, leading to secondary behavioral and cognitive impairments, depending on the site of injury. Therefore, most stroke patients have behavioral disorders due to brain tissue damage even after removing the thrombi, which is also an important part of stroke treatment. In addition, it is a disease frequently occurring as recently as the number 1 cause of death. The current treatment for cerebral ischemia is the FDA-approved tissue plasminogen activator (t-PA), which is a thrombolytic agent that removes clogged clots and rapidly replenishes blood. If there is a possibility of hemorrhagic cerebral ischemia at the same time, there is a limit such as that it cannot be used. That is, there are currently no medicines that treat or prevent tissue damage caused by ischemia or prevent or prevent behavioral or perceptual impairment caused by brain damage.

Laurel is an evergreen tree of the dicotyledon plant Buttercup, Camphoraceae, and has been used as a spice or ornamental water in Europe. These laurels are antioxidants [Simic, M., T. Kundakovic, and N. Kovacevic, Preliminary assay on the antioxidative activity of Laurus nobilis extracts. Fitoterapia, 2003. 74 (6): p. 613-6.], Analgesic, anti-inflammatory [Sayyah, M., et al., Analgesic and anti-inflammatory activity of the leaf essential oil of Laurus nobilis Linn. Phytother Res, 2003. 17 (7): p. 733-6.], Anticonvulsants [Sayyah, M., J. Valizadeh, and M. Kamalinejad, Anticonvulsant activity of the leaf essential oil of Laurus nobilis against pentylenetetrazole- and maximal electroshock-induced seizures. Phytomedicine, 2002. 9 (3): p. 212-6.] Is known to be effective.

The method of inducing apoptosis by oxygen-glucose deficiency (OGD) is the most similar model to apoptosis due to oxygen and nutrient deficiency by blocking blood supply in real cerebral ischemia [Goldberg, MP and DW Choi, Combined oxygen and glucose deprivation] in cortical cell culture: calcium-dependent and calcium-independent mechanisms of neuronal injury. J Neurosci, 1993. 13 (8): p. 3510-24 .; Strasser, U. and G. Fischer, Quantitative measurement of neuronal degeneration in organotypic hippocampal cultures after combined oxygen / glucose deprivation. J Neurosci Methods, 1995. 57 (2): p. 177-86.]. The OGD method treats the cultured cells with artificially glucose-free medium and seals them with 95% N ₂ and 5% CO ₂ mixed gas to induce ischemic state and supply glucose and oxygen again.

At this time, the cells are induced necrosis (necrosis) and apoptosis (apoptosis) in a state of ischemia for a certain period of time, and due to the reoxygantion of glucose and oxygen, the ATP is rapidly produced to induce apoptosis more rapidly. The direct application of this OGD model to human neuroblastoma and cerebral cortex in rats is a method of verifying the effectiveness of anticipated medicinal herbs in the environment most similar to clinical cerebral ischemia at in vitro levels [Siqueira, IR, et al. ., Neuroprotective effects of Ptychopetalum olacoides Bentham (Olacaceae) on oxygen and glucose deprivation induced damage in rat hippocampal slices. Life Sci, 2004. 75 (15): p. 1897-906; Croslan, D.R., et al., Neuroprotective effects of neuregulin-1 on B35 neuronal cells following ischemia. Brain Res, 2008. 1210: p. 39-47.]. In particular, the method using the cerebral cortex of rats has the advantage of effectively reproducing intracellular secretions and functions related to the actual cell damage because it uses the actual brain tissue. Taylor, C.P., S.P. Burke, and M.L. Weber, Hippocampal slices: glutamate overflow and cellular damage from ischemia are reduced by sodium-channel blockade. J Neurosci Methods, 1995. 59 (1): p. 121-8.].

 DAPK (death associated protein kinase) is a protein involved in apoptotic cell death in which cells die. They exist in phosphorylated state in normal brain cells and are activated by dephosphorylation in the early stages of apoptosis [Velentza, AV, et al. ., An aminopyridazine-based inhibitor of a pro-apoptotic protein kinase attenuates hypoxia-ischemia induced acute brain injury. Bioorg Med Chem Lett, 2003. 13 (20): p. 3465-70.]. In the past, research on DAPK has focused on inducing apoptosis of cancer cells by activating DAPK in cancer-related studies. However, in recent studies, DAPK is rapidly activated in the hypoxic, low-nutrient state (OGD), the brain ischemic environment. There has been research and has been reported to induce apoptosis by inhibiting or promoting the function of proteins, in particular by acting on the phosphorylation of other proteins involved in suicide. Shamloo, M., et al., Death-associated protein kinase is activated by dephosphorylation in response to cerebral ischemia. J Biol Chem, 2005. 280 (51): p. 42290-9 .; Chen, C.H., et al., Bidirectional signals transduced by DAPK-ERK interaction promote the apoptotic effect of DAPK. EMBO J, 2005. 24 (2): p. 294-304 .; Eisenberg-Lerner, A. and A. Kimchi, DAP kinase regulates JNK signaling by binding and activating protein kinase D under oxidative stress. Cell Death Differ, 2007. 14 (11): p. 1908-15.]. Therefore, inhibition of DAPK activity can inhibit cell death induced by cerebral ischemia.

The middle cerebral artery is the most frequent ischemic vessel in cerebral ischemia patients. When the blood supply is blocked, glutamate receptors are activated and the intracellular calcium concentration is rapidly increased, leading to cell death [Irving, E.A. and M. Bamford, Role of mitogen- and stress-activated kinases in ischemic injury. J Cereb Blood Flow Metab, 2002. 22 (6): p. 631-47 .; Karadottir, R., et al., NMDA receptors are expressed in oligodendrocytes and activated in ischaemia. Nature, 2005. 438 (7071): p. 1162-6.]. Therefore, local cerebral ischemia induction using middle cerebral artery occlusion (MCAO) has been used in many studies [Longa, E.Z., et al., Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989. 20 (1): p. 84-91]. The middle cerebral artery occlusion method uses a surgical nylon thread to block the inlet of the middle cerebral artery, thereby blocking the supply of blood, and then removing the thread, thereby distributing the distribution of nerve cells in the cerebral infarction center and border of the cerebral cortex. Differences clearly induce cerebral infarction and even neurological ataxia [Dirnagl, U., C. Iadecola, and MA Moskowitz, Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci, 1999. 22 (9): p. 391-7 .; Mergenthaler, P., U. Dirnagl, and A. Meisel, Pathophysiology of stroke: lessons from animal models. Metab Brain Dis, 2004. 19 (3-4): p. 151-67 .; Rodrigues, C.M., et al., Neuroprotection by a bile acid in an acute stroke model in the rat. J Cereb Blood Flow Metab, 2002. 22 (4): p. 463-71.].

In order to solve the above problems, the present invention proposes a composition for the prevention and treatment of ischemic neurological brain disease of the alcohol extract and chloroform fraction of the bay leaf.

The present invention has been made to solve the above problems, the purpose of which is the effect of laurel alcohol extract, chloroform fraction and nucleic acid fraction on brain cell death induced in oxygen glucose deficient environment, and brain due to middle cerebral artery occlusion The present invention provides a composition for the prevention and treatment of ischemic neurological brain diseases containing alcohol extract and chloroform fraction of laurel leaves by deriving an infarction inhibitory effect.

According to a feature of the present invention for achieving the above object, the first step of cutting the dried laurel medicinal herbs and then immersed in methanol 40 ~ 50 ℃ three times repeatedly filtered with filter paper; Concentrated under reduced pressure in a portion of the methanol extract to obtain a methanol extract, a portion of the extract was shaken repeatedly extracted by addition of 10% nucleic acid and fractionated in a separatory funnel to separate the nucleic acid layer and methanol layer, the nucleic acid layer was concentrated under reduced pressure nucleic acid (Hexane) fraction Fractionating the second step; The methanol layer is added again chloroform and distilled water (1: 1, v / v) and shaken repeatedly extracted twice at 30 ~ 40 ℃ and fractionated with a separatory funnel to fractionate into a chloroform layer and an aqueous layer; After the third step, the aqueous layer is concentrated under reduced pressure to fractionate the aqueous fraction, and the chloroform layer is concentrated under reduced pressure to fractionate the final chloroform fraction. It provides a composition for the prevention and treatment of ischemic nervous system brain disease containing alcohol extract and chloroform fraction.

At this time, according to an additional feature of the present invention, it is preferable that the laurel ethanol extract, the laurel hexane layer fraction or the laurel chloroform layer fraction is included as an active ingredient and mixed with a conventional pharmaceutically acceptable carrier.

In addition, according to an additional feature of the present invention, it is preferred that the pharmaceutical form used is any one selected from powders, granules, tablets, capsules, solutions, and injections.

The composition for the prevention and treatment of ischemic nervous system brain disease containing alcohol extract and chloroform fraction of laurel leaves according to the present invention has an inhibitory effect on OGD-induced brain neuron cell death in both nucleic acid and chloroform fractions including laurel alcohol extract. In addition, each fraction fractionated from the alcohol extract of laurel has a difference in degree, but because all of the effects occur, it can be used as a prevention and treatment of brain diseases related to laurel.

In addition, among the laurel fractions, especially the chloroform fraction, it effectively reduced OGD-induced apoptosis in human neuroblastoma SH-SY5Y cells, and further effectively reduced cell death induced by hypoxia and hypotrophy in rat cerebral cortical cells. And neuroprotective cells, and SH-SY5Y cells protect against brain cell death even with laurel chloroform and nucleic acid fractions of 0.16 μg / ml, even in an environment that causes about 50% cell death. Tissue experiments showed that the laurel chloroform fraction effectively inhibited the death of the most injured cerebral cortex in actual cerebral ischemia, supporting the results of in vitro experiments with cells and suggesting the possibility of in vivo experiments with animals. Neuronal sensitization due to cerebral ischemia of laurel chloroform fraction There is a possibility of death as the protective substance.

In addition, laurel chloroform fraction in cell experiments can effectively inhibit early cell apoptosis by inhibiting dephosphorylation of death associated protein kinase (DAPK) to reduce brain cell death, thereby inhibiting DAPK dephosphorylation in cerebral ischemia. As a result, DAPK may be an important regulator of cerebral ischemia-related apoptosis, and there is a possibility that DAPK's active modulator is a new therapeutic agent in the field of cerebral ischemia, which has no special treatment to date.

In addition, animal experiments induced by the cerebral ischemia of laurel chloroform fractions have excellent therapeutic effects on the behavioral disorders, which are all phenomena. Given that it can be seen as a laurel chloroform fraction can lead to prophylactic and therapeutic potential not only in direct brain disease but also in its phenomena.

In addition, the laurel chloroform fraction has a better effect at lower concentrations in all experiments compared to the previously proven neuroprotective substance carnosine, making the laurel chloroform fraction a more potent neuroprotective drug than the conventional drug. There is this.

In order to achieve the above object, the present invention is a substance that can be used in the composition for the prevention and treatment of ischemic neurological brain disease, dried by laurel leaves, containing laurel extracts, hexane fractions, chloroform fractions of laurel and finely divided into effective ingredients Characterized in that.

Hereinafter, the present invention will be described more specifically.

The composition for the prevention and treatment of ischemic neurological brain disease containing the alcohol extract and the chloroform fraction of the laurel leaf of the present invention comprises 0.5 to 50% by weight of the laurel fraction to the total weight.

The present invention provides a composition for the prevention and treatment of ischemic neurological brain diseases in which the laurel fraction may selectively use any one of a laurel methanol fraction, a laurel hexane (Hexane) fraction or a laurel chloroform fraction.

The composition comprising the laurel fraction of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

It provides a composition for the prevention and treatment of cerebral nerve cell protection and Parkinson's disease and neurodegenerative brain disease, wherein the fraction of the present invention is any one selected from powders, granules, tablets, capsules, solutions, injections.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto, and modifications such as insertion, deletion, and modification, which can be easily implemented by those skilled in the art, are also included within the scope of the present invention. .

Experimental Example

Preparation of Sample

After cutting the laurel dry medicinal herbs and incubated in methanol 40 ~ 50 ℃, the process of filtration with filter paper was repeated three times. A portion of this methanol extract was concentrated under reduced pressure to obtain a methanol extract. A portion of the extract was shaken and extracted repeatedly with 10% nucleic acid. The extract was separated from a separatory funnel, and the nucleic acid layer and the methanol layer were separated, and the nucleic acid layer was concentrated under reduced pressure. Got. The methanol layer was again extracted with chloroform and distilled water (1: 1, v / v) and shaken twice at 30 to 40 ° C., fractionated with a separatory funnel, separated into a chloroform layer and an aqueous layer, and the aqueous layer was concentrated under reduced pressure. Aqueous) fractions were obtained. The chloroform layer was concentrated under reduced pressure to obtain a final chloroform fraction.

The laurel was 10 kg warmed to obtain 318.7 g of the nucleic acid fraction and 2.4 kg of methanol layer. The methanol was filtered and concentrated under reduced pressure to obtain 282.8 g of the chloroform fraction.

Cell Culture and Treatment

Human neuroblastoma SH-SY5Y cells (ATCC no. CRL-2266) were found in 10% fetal bovine serum (Gibco-BRL, cat. # 16000-044, USA) and antibiotics (Sigma-Aldrich, cat. # A5955, USA) The temperature was maintained at 37 ° C. under dulbrcco's modified eagle's medium (Sigma-Aldrich, cat. # D2902, USA) medium, and the mixture of 95% air and 5% CO ₂ was continuously supplied and incubated under appropriate conditions of cell culture. . The cells were fed fresh medium every four days. For the experiment, cells were cultured at 18 x 10 4 cells / ml in 6-well plates or 8 x 10 4 cells / ml in 96-well plates.

Induction of cell death due to oxygen-glucose deficiency

Experiments inducing apoptosis due to oxygen glucose deficiency have already been reported in previous studies. [Goldberg, M.P. and D.W. Choi, Combined oxygen and glucose deprivation in cortical cell culture: calcium-dependent and calcium-independent mechanisms of neuronal injury. J Neurosci, 1993. 13 (8): p. 3510-24.]

After culturing the cells on the plate, wash the cells with glucose-free DMEM so that the existing glucose-containing medium is sufficiently removed, and then change the medium without glucose. After changing the medium, the cells were placed in a 37 ° C. incubator with 95% air and 5% CO ₂ gas continuously for 1 hour, and after 1 hour, the laurel chloroform fraction and the nucleic acid fraction were 0.16 ㎍ / ml, respectively. After 4 μg / ml at, allow another 1 hour of acclimation. At this time, the positive control group was treated with carnosine 500 ㎍ / ㎖.

The cells are transferred to an anaerobic vessel (Modular incubator chamber (MIC-101), Billups-Rothernberg Inc., USA), filled with 95% nitrogen and 5% CO ₂ mixed gas, sealed and incubated for 20 hours in a 37 ° C incubator. . After 20 hours, each cell was reprocessed with glucose at a final concentration of 4.5 mg / L and transferred to an incubator with 95% air and 5% CO ₂ gas for reoxygenation. [Tabakman, R., et al., Neuroprotection by NGF in the PC12 in vitro OGD model: involvement of mitogen-activated protein kinases and gene expression. Ann NY Acad Sci, 2005. 1053: p. 84-96.]

Groups not treated with OGD were incubated in glucose-containing DMEM, and then exchanged with glucose-free DMEM to obtain the same conditions during reoxygenation of the OGD treated group, and the glucose was changed to a concentration of 4.5 mg / L. Treated.

Flow cytometry for measuring cell death

Flow cytometry was performed to determine the effect on the DNA of the cells. Cells treated with laurel chloroform fraction and induced death by OGD were made into single cells using trypsine, washed three times with PBS, and fixed at -20 ° C with cold 70% ethanol for 1 day. For analysis, cells were centrifuged at 1000 rpm for 5 minutes to remove ethanol, and 40 ㎍ / ml of Propidium iodide (PI, Sigma-Aldrich, cat. # P4170, USA) and PBS were stained at 4 ° C for 30 minutes and flow cytometry (FACS, Coulter EPICS XL, USA). The apoptotic cells showed decreased PI staining intensity and can be compared with normal cells, and the degree of apoptosis was expressed as a percentage compared with the normal group without OGD treatment.

MTT assay for measuring cell death

MTT reduction assay was used to treat the laurel alcohol extract and the nucleic acid fraction and to measure the survival rate of cells induced by apoptosis with OGD.

MTT (3- (4,5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) solution was treated at a concentration of 0.5 mg / ml and reacted at 37 ° C for 4 hours. . Mitochondria of living cells and MTT reacted with purple crystal cells were dissolved in 100% dimethyl sulfoxide (DMSO) solution, and UV absorbance was measured at 540 nm using a microplate reader (Thermomax, Molecular device, USA). Cell viability (%) is expressed as a percentage of the absorbance values of the normal control group not treated with OGD.

Western blot analysis

After culturing the SH-SY5Y cell, PBS was washed and protein of whole cell was extracted using PRO-PREP protein solution (Intron biotechnology, cat. # 17081, Korea), and protein assay solution (Intron biotechnology, cat. # 21011). , Korea) to quantify the total protein concentration. After electrophoresis of each 50 ㎍ total cellular protein on 10% SDS-polyacrylamide gel, the protein was transferred to PVDF membrane, and the membrane was placed in 5% skim milk dissolved in Tris buffered-0.1% tween 20 (TBST) at room temperature. Shake for 2 hours was treated to prevent other proteins stick. And the membrane was the primary antibody Monoclonal Anti-Death associated protein (DAP) Kinase clone (1: 250, Sigma-Aldrich, USA), and Monoclonal Anti-Phospho DAP-Kinase (pSer308) (1: 1000, Sigma-Aldrich, USA). It treated with and reacted at 4 degreeC for 24 hours. The next day, the membrane was washed with TBST for 30 minutes, then reacted with a secondary antibody (anti-rabbit or anti-mouse HRP-conjugated IgG; Chemicon, cat. # AP124P, USA) for 1 hour at room temperature, and washed again for 30 minutes. The analyzer was analyzed by LAS1000 (Fuji photo film, Japan). All western blot assays were repeated three times.

Induction of Oxygen Glucose Deficiency in the Cerebral Cortex of Rats

The male Sprague-Dawley white paper was sacrificed with dimethyl ether, and the brain was quickly removed and incubated for 5 minutes in a cold Krebs-Henseleit solution (pH 7.4). Krebs-Henseleit solution is (in mM); 120 NaCl, 4 NaHCO ₃ , 10 MgSO ₄ , 2 KCl, 1.18 KH ₂ PO ₄ , 0.5 CaCl ₂ , 25 HEPES, and 11 glucose, pH 7.4.

The extracted brain was cut into 500 µm, and then plated with a cell culture insert (0.4 µm pore PET membrane, BD Falcon, USA), Krebs-Henseleit solution, and then at 37 ° C in 95% air and 5% CO2 incubator. Adapt for a minute. The solution for OGD induction (in mM) consisted of 137 NaCl, 0.44 MgSO ₄ , 0.34 Na ₂ HPO ₄ , 1.26 CaCl ₂ , 4 NaHCO ₃ , 5.36 KCl, 0.49 MgCl ₂ , 0.44 KH ₂ PO ₄ , 25 HEPES. In the OGD treated group, the sections were washed twice with modified Krebs-Henseleit solution (OGD-incubation solution, pH 7.4), treated with 3 ml each and treated with laurel chloroform extract at a concentration of 0.16-4 ㎍ / ml and positive control. Phosphorus carnosine was treated with 500 μg / ml. The tissue was placed in an anaerobic vessel filled with 95% nitrogen and 5% CO ₂ gas, reacted at 37 ° C. for 4 hours, removed from the vessel, replaced with glucose-containing Krebs-Henseleit solution, and then incubated with 95% air and 5% CO ₂ incubator. Reoxidize at 37 ° C for 2 hours. The normal control group was treated with Krebs-Henseleit solution containing glucose after 30 minutes of adaptation and incubated at 37 ° C. in 95% air and 5% CO ₂ incubator.

MTT assay of cortical tissue

MTT reduction assay was used to measure cell viability of cerebral cortical tissue. After the reaction was completed, the MTT (3- (4,5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) solution was treated at a concentration of 0.5 mg / ml and reacted at 37 ° C for 30 minutes. Purple crystals were dissolved in 100% DMSO (dimethyl sulfoxide) solution by MTT reaction, and UV absorbance was measured at 540 nm using a microplate reader (Thermomax, Molecular device, USA). Cell viability (%) is expressed as a percentage of the absorbance values of the normal control group not treated with OGD.

Induction of Regional Cerebral Ischemia and Drug Treatment

An animal model of focal cerebral ischemia is a method of occluding the middle cerebral artery using 290-310 g male Sprague-Dawley rats (Samtako, Korea) [Longa, EZ, et al., Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke, 1989. 20 (1): p. 84-91.]. The rats were inhaled during 2 ~ 3% isoflurane (AErane®, Baxter, USA) during anesthesia and maintained at 37.0 ± 0.5 ° C. through a heat plate.

First, the group that induces cerebral ischemia is to open the middle neck of anesthetized rats, separate the external carotid artery from the common carotid artery, and internal carotid artery located outside the cranial cavity. The pituitary artery, which is the only branch in, was isolated. Branches of the external carotid artery are cauterized using an electric pharynx to cut off blood supply, and micropores can be inserted into the external carotid artery while temporarily blocking the flow of the total carotid artery with a surgical clip. A nylon operating room (3-0 monofilament) was inserted and inserted slowly through the internal carotid artery up to about 20 mm to close the start of the middle cerebral artery.

At this time, the used nylon operating room was preheated by applying heat at one end to make it dull so as not to damage blood vessels or tissues and coated with 0.1% poly-L-lysine. The inserted nylon operating room was secured to the operating room with blood vessels, the clip was temporarily removed from the blood supply, and the rat awoke from anesthesia. Reoxidation of blood flow is actually clinically induced by the removal of blood clots and damage to the brain due to replenishment of blood flow, which is similarly induced in the local cerebral ischemia model. For reopening, the anesthesia was carefully pulled out of the nylon operating room after 1.5 hours of closure and the operating room was removed, the blood flow reopened and immediately awakened from the anesthesia.

Laurel chloroform fractions were administered through the abdominal cavity 30 minutes prior to surgery. The experimental group was divided into two groups, which treated the laurel fractions at each concentration, and the group inducing surgery only. The laurel chloroform fraction was diluted to a final concentration of 0.2 mg / kg, 1 mg / kg, 4 mg / kg in 1% ethanol / saline, and the positive control carnosine was diluted to a final concentration of 50 mg / kg. . The only group inducing surgery was intraperitoneally administered 1% ethanol / saline as a solvent.

TTC Dyeing

Mice induced cerebral ischemia were sacrificed by anesthesia with ether 24 hours after surgery, and the brain was extracted and placed on ice for about 5 minutes until the brain was slightly hardened. The extracted brain was cut into 2 mm thick coronal sections using a brain matrix (Harvard Bioscience, USA). Each section was placed in a 24-well plate containing 2% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich, cat. # T8877, USA) solution and stained at 50 ° C for about 10 minutes to make the cerebral infarction white and normal. Tissues were stained red and fixed with 4% formalin solution. Fixed tissues were photographed with a digital camera, and then the area of cerebral infarction of each section was measured using the Image J program, and the ratio of total cerebral infarct area was calculated by the following formula.

I% = (Vc -Vi) / Vc x 100%

I = cerebral ischemia rate

Vc = normal area of non-ischemic cerebral hemisphere

Vi = normal area of the ischemic cerebral hemisphere

Measurement of neurological behavioral indicator performance

The experimental group that induced focal cerebral ischemia were assessed neurological scores due to cerebral ischemia 24 hours after MCAO surgery according to the following criteria.

score Evaluation 0 No defects that can be clearly observed One Lifting the tail does not completely straighten the forelimbs opposite the cerebral ischemia 2 Circular pull out of cerebral ischemia when the tail is pulled 3 Falling opposite to cerebral ischemia 4 Immovable

Animals with zero neurological behavioral scores after MCAO surgery were excluded from the experiment.

Statistical processing

All measurements were expressed as mean ± standard error, and statistical analysis of each experimental group was performed by t-test using Sigma Plot 10.0 program. P <0.05 was considered statistically significant.

Experiment Result 1

Apoptosis of Human Neuroblastoma (SH-SY5Y) by OGD Treatment

SH-SY5Y cells induced cell death in an environment deficient in oxygen and glucose and cell survival rate due to reproductive digestion was measured over time. (FIG. 1).

Cell viability was measured by staining DNA of cells killed with PI using flow cytometry analysis. Cell viability was measured by flow cytometry and the measurement was expressed as a percentage in comparison to the normal group not treated with OGD, and the survival rate of neurons was constantly decreased as OGD induction time increased. As shown in the cell survival rate according to the OGD treatment time in SH-SY5Y. At this time, the cells induce different OGD induction time, while re-digestion was treated the same 24 hours. Reproductive digestion does not affect cell death after 20 hours of treatment. In this experiment, morphological changes in the cells can be observed. The experiment was decided based on 20 hours of OGD and 24 hours of reproductive digestion with a cell survival rate of about 55%.

Table 1. Table of cell viability with OGD treatment time in SH-SY5Y cells.

OGD time (hour) Cell viability (% of control) 0 100.0 ± 1.6 4 96.6 ± 1.3 8 93.0 ± 0.4 12 82.4 ± 12.8 16 71.3 ± 5.9 20 55.5 ± 16.5 24 49.8 ± 8.1

Inhibitory Effects of Laurel Alcohol Extracts on Cell Death of OGD-induced Human Neuroblastoma SH-SY5Y

In this experiment, the inhibitory effect of Laurel alcohol extract on OGD-induced cell death was analyzed by MTT assay.

As shown in FIG. 2, the laurel methanol extract was treated with OGD at refining concentration of 20 μg / ml at a final concentration of 0.8, and the inhibitory effect of the laurel alcohol extract on cell death in SH-SY5Y cells induced by OGD was It was confirmed through the MTT analysis method that the effect of inhibiting cell death in a concentration-dependent manner compared to the OGD treatment group.

That is, the OGD-induced group showed a survival rate of 60.8 ± 0.1% compared to the normal group that did not induce OGD. Laurel alcohol extract (methanol) and nucleic acid fractions were treated in SH-SY5Y cells at concentrations of 0.8 to 20 and 0.16 to 4 ㎍ / ml, respectively, during OGD and reoxidation.

When the laurel alcohol (methanol) extract was treated simultaneously with the OGD-induced environment, the survival rates of 63.6 ± 0.1, 67.3 ± 0.1, and 71.3 ± 0.2% were obtained according to the treatment of the final concentrations of 0.8, 4, and 20 ㎍ / ml ( Table 2). In other words, it showed a concentration-dependent OGD-induced apoptosis inhibitory effect according to the alcohol extract treatment.

Table 2. Inhibitory Effects of Laurel Alcohol Extracts and Nucleic Acid Fractions on Cell Death in OGD-Induced SH-SY5Y Cells.

Group Dose (µg / mL) Cell viability (%) Control - 100 OGD - 60.8 ± 0.1 Carnosine 500 85.6 ± 8.8 Alcohol extract 0.8 60.9 ± 0.6 (Methanol) 4 66.2 ± 0.7 20 73.1 ± 0.7

Group Dose (µg / mL) Cell viability (%) Control - 100 OGD - 59.2 ± 0.5 Carnosine 500 85.6 ± 8.8 Hexane extract 0.16 60.9 ± 0.6 0.8 66.2 ± 0.7 4 73.1 ± 0.7

Inhibitory Effect of Laurel Nucleic Acid Fractions on Cell Death of OGD-induced Human Neuroblastoma SH-SY5Y

The inhibitory effect on OGD-induced cell death of nucleic acid fractions was also confirmed by MTT assay.

That is, as shown in Figure 3, the laurel nucleic acid fraction was treated during reoxidation with OGD at a concentration of 0.16 to 4 ㎍ / ㎖ and confirmed by the MTT assay method that the effect of inhibiting cell death in a concentration-dependent manner compared to the OGD treatment group It was.

That is, the SH-SY5Y cells cultured in 96-well plate were induced by OGD for 18 hours and reoxidated for 24 hours to confirm the cell survival rate of 59.2 ± 0.5% in the OGD treated group compared to the normal control group.

Laurel alcohol extract (methanol) and nucleic acid fractions were treated in SH-SY5Y cells at concentrations of 0.8 to 20 and 0.16 to 4 ㎍ / ml, respectively, during OGD and reoxidation.

Treatment of laurel nucleic acid fractions during induction of OGD and reoxidation showed 60.9 ± 0.6, 66.2 ± 0.7, and 73.1 ± 07% survival rates, depending on the concentration of 0.16, 0.8, and 4 μg / ml. It showed an inhibitory effect (Table 2).

Table 2. Inhibitory Effects of Laurel Alcohol Extracts and Nucleic Acid Fractions on Cell Death in OGD-Induced SH-SY5Y Cells.

Group Dose (µg / mL) Cell viability (%) Control - 100 OGD - 60.8 ± 0.1 Carnosine 500 85.6 ± 8.8 Alcohol extract 0.8 60.9 ± 0.6 (Methanol) 4 66.2 ± 0.7 20 73.1 ± 0.7

Group Dose (µg / mL) Cell viability (%) Control - 100 OGD - 59.2 ± 0.5 Carnosine 500 85.6 ± 8.8 Hexane extract 0.16 60.9 ± 0.6 0.8 66.2 ± 0.7 4 73.1 ± 0.7

Cytotoxicity of Laurel Chloroform Fraction in Human Neuroblastoma SH-SY5Y Cells

Prior to the OGD induction experiment, an experiment was conducted to determine the toxicity of the laurel chloroform fraction (FIG. 4).

When cells were treated with laurel chloroform fraction at a concentration of 0.16 to 4 μg / ml, cytotoxicity was measured by flow cytometry, and the results were expressed in percentage compared to the normal group without treatment of the fraction.

After SH-SY5Y cells were aliquoted into the plate and incubated for 24 hours, the laurel chlorom fractions were treated with final concentrations of 0.16, 0.8, 2 and 4 ㎍ / ml, respectively. After 48 hours, all cells were collected, stained with PI, and checked for survival rate. Cytotoxicity was not observed at the concentrations applied to the experiments, and was not applied to the experiments because of the cytotoxicity at concentrations of 6 and 8 µg / ml, the concentrations of 4 µg / ml or more.

Inhibitory Effects of Laurel Chloroform Fraction and Carnosine on OGD-induced Cell Death in Human Neuroblastoma SH-SY5Y

In this experiment, the effect of Laurel chloroform fraction on OGD-induced cell death in SH-SY5Y cells was confirmed. Cells were cultured and cell death was induced by OGD induction and reproductive digestion, and laurel chloroform fractions were treated simultaneously. The cells were largely divided into normal cell group (OGD-treated group) and OGD-treated group, and OGD-treated group was further divided into laurel-treated group and laurel-treated OGD group. Laurel fractions were treated to a final concentration of 0.16 μg / ml to 4 μg / ml. The group treated with non-OGD compared to the normal control group had a cell survival rate of about 58.5 ± 4.9%, 64.9 ± 3.4% when treated with 0.16 μg / ml of the laurel fraction and 80.4 ± 0.4% at the highest concentration of 4 μg / ml. . (FIG. 5, Table 3).

That is, the laurel chloroform fraction was treated during reoxidation with OGD at a concentration of 0.16 to 4 ㎍ / ml, and it was confirmed that the cell death was inhibited in a concentration-dependent manner compared to the OGD treatment group. Carnosine, a positive control, was treated identically at a concentration of 500 μg / ml.

Although not shown in the results, the effect of inhibiting cell death was weakly at 0.032 ㎍ / ml, and at a concentration below that, there was no effect.

In addition, carnosine was tested as a positive control to compare the effect of the laurel chloroform fraction with conventional drugs. Carnosine has been reported in previous studies to have antioxidant effects as well as to inhibit brain ischemia, and in particular, has been shown to be effective on OGD-induced cell death. As shown in FIG. 3, when carnosine was treated with 500 μg / ml, the survival rate was 85.6 ± 8.8%, but the laurel chloroform fraction showed a similar effect even at a 100 times lower concentration.

Table 3. Inhibitory effect of laurel chloroform fraction on cell death in OGD-induced SH-SY5Y cells and rat cortical tissue. Laurel chloroform fractions were treated in SH-SY5Y cells and rat cortical tissues at concentrations of 0.16 to 4 ㎍ / ml during OGD and redigestion, respectively.

Cell viability (%) Group Dose (µg / mL) SH-SY5Y cells Hippocampal slices Control - 100 100 OGD - 58.5 ± 4.9 79.7 ± 5.9 * Carnosine 500 85.6 ± 8.8 92.4 ± 7.8 LNCE 0.16 64.9 ± 3.4 * 83.6 ± 10.0 0.8 65.8 ± 3.7 * 96.1 ± 3.6 2 72.0 ± 0.5 * - 4 80.4 ± 0.4 ** 97.2 ± 1.9 #

Inhibitory Effects of Laurel Chloroform Fraction and Carnosine on OGD-induced Cell Death in Rat Cerebral Cortex

The cerebral cortical sections of rats were resuspended for 4 hours OGD and 2 hours with laurel chloroform fraction (0.16 ~ 4 ㎍ / mL) and carnosine (500 ㎍ / mL), and the absorbance values were measured by MTT assay. Survival is shown (Table 3).

As shown in Figure 6, the cerebral cortical tissue of rats were OGD for 4 hours and reoxidated for 2 hours to induce cell death, and the laurel chloroform fractions were treated together during OGD. Cell viability was measured using the MTT assay and the measurement was expressed as a percentage compared to normal tissues that were not treated with OGD.

When OGD-induced, tissue sections showed a cell viability of about 79.7 ± 5.9% compared to normal tissue sections, and 83.6 ± 10.0, 96.1 ± 3.6, 97.2 ± 1.9 according to the treatment of laurel chloroform by concentration (0.16 ~ 4 ㎍ / ml). The survival rate was%. These results showed that the laurel chloroform fraction showed neuronal death more than carnosine at a concentration of about 1/120 (4 µg / ml) when compared with 500 µg / ml of the positive control carnosine. It was found that more suppress. In addition, the laurel chloroform fraction showed an inhibitory effect not only at the cellular level but also at the tissue level.

DAPK and P-DAPK Activities in OGD-Induced Human Neuroblastoma SH-SY5Y Cells Treated with Laurel Chloroform Fraction.

In the present invention, the expression pattern of DAPK was confirmed when SH-SY5Y cells were treated with laurel chloroform fraction.

Proteins obtained from OGD-induced cells were quantified, and the same amount of total protein was classified by SDS-PAGE gel and identified by attaching DAPK and P-DAPK antibodies. DAPK is present in phosphorylated state in normal brain cells, and when apoptosis is induced by OGD, it is activated by dephosphorylation to induce early apoptosis. When confirming the expression pattern over time, it was found that the intensity (activity) of P-DAPK compared to DAPK was significantly decreased to 53.9 ± 0.1% compared to normal cells with OGD induction for about 16 hours (FIG. 7).

In order to confirm the relative intensity of DAPK activated by OGD and the inhibitory effect of daphophorylation of laurel chloroform fraction in SH-SY5Y cells, proteins of OGD-induced SH-SY5Y cells were electrophoresed by SDS-PAGE gel and phosphorylated with DAPK. The amount of DAPK was measured.

That is, it can be seen that dephosphorylation occurred as DAPK was inactivated and activated by induction of cell death by OGD.

 On the other hand, when Laurel chloroform fractions were treated at a concentration of 0.032 to 0.8 μg / ml, DAPK activity was 63.6 ± 0.01, 75.4 ± 0.06, 87.5 ± 0.16%. (FIG. 8).

As a result of Western blot analysis, it was found that the relative intensity of phosphorylated DAPK was decreased compared to DAPK in OGD-induced cells, and that the activity of DAPK was inhibited by increasing the intensity by treatment of Laurel chloroform by concentration. there was.

This result showed that the laurel chloroform fraction inhibited DAPK dephosphorylation in the early stages of brain cell death induced by hypoxic and hypotrophic states, thereby preventing DAPK activation and thus inhibiting cell death, thereby protecting the brain cells. It can be said that.

Effect of Laurel Chloroform Fraction on Cerebral Infarction and Neurological Behavior by MCAO Induction in Rats

Laurel chloroform fractions have shown a strong inhibitory effect on brain cell death in OGD-induced brain neuronal damage in previous studies. Therefore, we tried to induce cerebral infarction and verify the effects of laurel by directly closing the midbrain aorta of rats. Laurel chloroform fractions were administered at a concentration of 0.2, 1, 4 mg / kg through the abdominal cavity 30 minutes prior to MCAO surgery. The control group was treated with 1% ethanol dissolved the laurel chloroform fraction. Twenty four hours after MCAO surgery, neurological behavior of rats was tested. As a result, in the control group, the movement was significantly decreased on average, and when the body was raised, the body was inclined to one side, and when lifted, the body was bent severely, whereas the laurel treatment group showed that the behavioral disorder was decreased as the concentration was increased. This behavioral disorder was reduced (FIG. 9).

As an effect on the neurological behavioral indicators of the laurel chloroform fraction in the MCAO-induced rats of FIG. 9, neurological behavioral indicators were confirmed 24 hours after MCAO surgery. It was confirmed that the neurological behavior was significantly reduced by treatment of the laurel chloroform fraction. ^## P <0,01 vs control.

After testing neurological behavior, the rats were sacrificed and brains were extracted, sectioned 2 mm thick and stained with 2% TTC. As an effect on the cerebral infarction range of laurel chloroform fraction and carnosine in MCAO-derived model of rats, brain infarction of control group, carnosine and laurel chloroform fraction treatment group as shown in FIG. Results TTC staining develops red in response to the enzymes of mitochondria in living cells, so that infarcts are stained white.

As shown in FIG. 8, it can be seen that the infarct site is significantly reduced in the drug-treated group compared to the comparative group. Particularly, the concentration of 4 mg / kg of laurel chloroform fraction showed little inhibition of infarction compared to the comparison group even with the naked eye. The infarct area was quantified as previously described in the experimental method (FIG. 11).

When the area of cerebral infarction was numerically quantified using a computer image program as shown in FIG. 11, it was confirmed that the extent of infarction was reduced according to the concentration-specific treatment of the laurel chloroform fraction. ^# P <0,05, ^## P <0,01 vs control.

In the comparison group, the infarct area was 36.9 ± 5.8%, the laurel chloroform fraction 4 mg / kg was 7.8 ± 3.0%, and the concentrations of 1 and 0.2 mg / kg were 12.2 ± 5.9%, as shown in Table 4 below. And 32.0 ± 8.2%.

Table 4. Effects of Laurel Chloroform Fraction and Carnosine on Cerebral Infarct Range in MCAO Induced Model of Rats.

Group Dose (mg / kg) Infarct size (%) Vehicle - 36.9 ± 5.8 Carnosine 50 31.3 ± 6.1 # LNCE 0.2 32.0 ± 8.2 One 12.2 ± 5.9 ## 4 7.8 ± 3.0 ##

On the other hand, carnosine, a positive control with a proven neuroprotective effect, showed an infarction of 31.3 ± 6.1% with 50 mg / kg of treatment, which was higher at a concentration of about 12.5 times as compared to the laurel chloroform fractionation layer. It showed less protective effect, indicating that the laurel chloroform fractionation layer had a better effect in less amount than the conventional neuroprotective material.

Test Result 2

In the present invention, in vitro and in vivo models were selected that are most clinically similar to human cerebral ischemia, and the neuronal cell death suppression and ischemic brain disease using alcohol fraction, chloroform fraction and nucleic acid fraction of laurel were investigated. The prophylactic effect and therapeutic potential of

The model of induction of cell death through oxygen-glucose deprivation (OGD) is similar to the ischemic state caused by the interruption of blood supply, which induces brain neuron death. First, human neuroblastoma SH-SY5Y cells were stopped from supplying glucose and serum and induced cell death in an oxygen deficient environment. At the same time, Laurel alcohol extract, chloroform, and nucleic acid fractions were treated at different concentrations, and cell death was measured by MTT assay and flow cytometry analysis. The OGD-treated group showed a survival rate of 60.8 ± 0.1% compared to the non-OGD-treated group, and the alcohol extract-treated group showed 63.6 ± 0.1 at 0.8 µg / ml, 67.3 ± 0.1% at 4 µg / ml, The survival rate was 71.3 ± 0.2% at 20 μg / ml. The group treated with chloroform fraction showed 81.5 ± 0.8 at 4 µg / ml and 67.1 ± 3.7% at 0.8 µg / ml. For laurel nucleic acid fractions, cell viability was confirmed by MTT analysis. As the nucleic acid fractions were treated at concentrations of 0.16, 0.8, and 4 μg / ml, survival rates of 60.9 ± 0.6, 66.2 ± 0.7, and 73.1 ± 07% were obtained. It was confirmed that there is a protective effect against death.

In addition, OGD induced the cerebral cortex of the directly isolated white paper, and the chloroform fraction was treated, and the degree of cell death was measured by MTT analysis. When the survival rate according to OGD induction was 83.9 ± 4.8%, viability of 98.5 ± 2.7% was shown at the highest concentration of 4 µg / ml in the chloroform fraction.

This organotypic culture method supports the neuronal cell protective effect of the laurel fraction in the experiment using cell line, and furthermore, it can be said that the result shows the direct protective effect in the actual animal tissue. That is, the effect of the laurel chloroform fraction in the experiment of the animal tissue level at the cellular level is proved that the laurel chloroform fraction has a protective effect not only in brain cell damage but also in brain tissue damage by OGD induction.

Death associated protein kinase (DAPK) is a calcium / calmoduline-dependent serine / threonine kinase that is activated in anaerobic conditions such as OGD and induces apoptosis. In this study, OGD treatment of SH-SY5Y cells induced cell death. At that time, dephosphorylation of DAPK was confirmed. As a result of comparing the intensity of P-DAPK / DAPK for each experimental group, it was confirmed that the intensity decreased about 30% after OGD treatment, compared to normal cells without OGD treatment, and after treatment with laurel fractions 0.032, 0.16, 0.8 µg / ml, It was confirmed that the intensity increased depending on each concentration. That is, it was confirmed that the phosphorylation state was maintained by preventing activation of DAPK, which is an apoptosis inducing protein, so that apoptosis did not occur.

Based on the results of this in vitro model, in vivo model was induced cerebral ischemia by closing the middle cerebral artery (MCAO) of rats and blocking blood supply, and chloroform fractions were treated by concentration 30 minutes before surgery. The middle cerebral artery is a blood vessel in which the majority of patients with cerebral ischemia cause ischemia, which causes an infarction of 35.3 ± 7.1% with an artificial blood supply block of about 90 minutes. As the laurel fraction was treated with 0.2, 1, 4 mg / kg, the infarct site was reduced to 34.2 ± 9.7, 12.2 ± 5.9, 7.8 ± 3.0%, and neurobehavioral symptoms were alleviated.

Therefore, Laurel alcohol extract and nucleic acid fractions showed a protective effect against cell death in an artificial brain ischemia model treated with human neuroblastoma SH-SY5Y cells by OGD. Laurel chloroform fraction, in the brain ischemia model of human neuroblastoma SH-SY5Y cells, protected cells from apoptosis by inhibiting DAPK-activated dephosphorylation.In the tissue cultured cerebral cortex, the concentration was dependent on the concentration of OGD. Inhibitory effect of hippocampus on cell death was observed, and the MCAO model of white paper effectively reduced the infarct range.

Therefore, the laurel fraction showed an inhibitory effect on apoptosis induced most similar to the actual cerebral ischemia, and was effective in preventing and treating ischemic stroke, and furthermore, as a result of various brain diseases and symptoms caused by brain tissue damage. It has the effect of alleviating behavioral disorders, suggesting that it is a potential therapeutic agent for brain-related diseases.

Test Result 3

Cerebral ischemia induces cell death because brain cells do not receive oxygen and nutrients normally due to the blockage of the blood supply, and brain blood supply is very important because brain tissue death causes various functional brain disorders. Able to know. Thus, the smooth supply of oxygen and nutrients is key to preventing brain cell damage. The OGD model and MCAO model used in the present invention are models that induce blood supply to the brain in a clinically similar environment, and have great significance not only in the study of general brain damage and its phenomena, but also in the study of brain ischemia. Therefore, it is important to study the protective effect against neuronal damage caused by cerebral ischemia.

In the present invention, we tried to verify the preventive and therapeutic effects of neuronal cell death and various shapes related to brain disease of laurel alcohol extract, chloroform fraction and nucleic acid fraction in various brain ischemia models.

In the present invention has the following important features. First, the inhibitory effect on OGD-induced brain neuronal cell death was observed in all of the nucleic acid and chloroform fractions including the laurel alcohol extract. Each fraction fractionated from the alcohol extract of laurel showed a degree of difference, but all showed the effect, confirming the possibility of prevention and treatment of brain disease related to Wolgesu.

Second, among the laurel fractions, especially the chloroform fraction, it effectively reduced OGD-induced apoptosis in human neuroblastoma SH-SY5Y cells, and further reduced the apoptosis-induced hypoxic, hypotrophic cell death in rat cortical cells. And protected neurons.

In particular, in SH-SY5Y cells, even in an environment causing about 50% cell death, 0.16 μg / ml of laurel chloroform and nucleic acid fractions showed a protective effect of brain cell death. In experiments with rat tissues, the laurel chloroform fraction effectively inhibited the death of the most damaged cerebral cortex, which supports the results of in vitro experiments using cells and in vivo experiments with animals. The possibilities are presented. In addition, the potential of laurel chloroform fraction as neuroprotective agent for neuronal cell death due to cerebral ischemia has been suggested.

Third, in the cell-based experiments, the laurel chloroform fraction reduced brain cell death by inhibiting dephosphorylation of death associated protein kinase (DAPK). In the present invention confirmed the activation of DAPK due to OGD induction. In normal brain cells, when DAPK is inactivated and phosphorylated DAPK is inactivated, and apoptosis signal is transmitted into the cell due to OGD induction, which is similar to cerebral ischemia, it is rapidly activated and dephosphorylated to induce early apoptosis. . Laurel chloroform fraction effectively inhibited early cell apoptosis by inhibiting dephosphorylation of DAPK even in cerebral ischemia.

These results indicate that DAPK may be an important regulator of cerebral ischemia-related apoptosis, and that DAPK's active modulators have potential as new therapeutic agents in the field of cerebral ischemia, where there is no specific therapeutic agent to date.

Fourth, the laurel chloroform fraction substantially reduced the site of cerebral infarction in an animal brain ischemia model. The middle cerebral artery used in the MCAO experiment is the most abundant ischemia in cerebral ischemia patients, and the induction of cerebral ischemia using this vessel is of great clinical value. The reduction of cerebral infarction area after treatment of the laurel chloroform fraction was remarkable.

In addition, by showing the effect also through the abdominal cavity, it can be seen that the laurel chloroform fraction effectively passed the blood brain barrier (blood brain barrier). Although this is effective in experiments at the cellular level, the material that cannot cross the blood brain barrier is of great value in the future, considering that there is a great difficulty in the actual situation of the patient. In addition, the laurel chloroform fraction showed effective neuroprotective effect in the animal model, suggesting that there is a therapeutic potential for damage caused by ischemia.

Fifth, the laurel chloroform fraction showed an excellent therapeutic effect on behavioral disorders, which is a common phenomenon in animal experiments induced by cerebral ischemia. This can be broadly interpreted as the laurel chloroform fraction is not only a direct brain disease, as the damage to human brain tissue can take many forms, including behavioral disorders, speech disorders, cognitive disorders, memory disorders, etc. In addition, it can be said that there is a possibility of prevention and treatment.

Finally, the laurel chloroform fraction showed better effects at lower concentrations in all experiments compared to carnosine, a previously proven neuroprotective substance.

These results indicate that the laurel chloroform fraction has potential as a more potent neuroprotective therapeutic drug than existing drugs.

In conclusion, Laurel alcohol extract, chloroform and nucleic acid fractions showed effects on brain cell death in human blastoma SH-SY5Y cells. In particular, chloroform fractions inhibited brain tissue cell death by OGD induction of cerebral cortical tissue in rats. It effectively reduced the area of cerebral infarction induced by MCAO and effectively reduced behavioral disorders. Thus, alcohol extracts, chloroform fractions, and nucleic acid fractions of laurels have the potential to be used as neuroprotective drugs to treat patients suffering from brain disease as well as its phenomena.

Hereinafter, an example of the preparation of a pharmaceutical composition for neuronal cell death and cerebral ischemia protection according to the present invention will be described, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Formulation Example 1 Preparation of Powder

According to the preparation method of powder in the pharmacopeia formulation, it is prepared in the following ingredient content per one packet.

Ginseng fraction ㆍ ······ 300 mg

Lactose ··········· 100 mg

Talc 10 mg

Formulation Example 2 Preparation of Tablet

According to the preparation method of tablets in the pharmacopeia formulation, it is prepared in the following component content per tablet.

Ginseng fraction ㆍ ······· 300 mg

Corn Starch ㆍ ········· 100 mg

Lactose lactic acid 100 mg

Magnesium stearate 2 mg

Formulation Example 3 Preparation of Capsule

According to the preparation method of the capsule in the general formulation of the pharmacopeia, it is prepared in the following component content per capsule.

Ginseng fraction ·········· 300 mg

Corn starch ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ 100 mg

Lactose · ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ 100 mg

Magnesium stearate 2 mg

Formulation Example 4 Preparation of Injection

According to the method for preparing an injection in the pharmacopeia formulation, it is prepared in the following component contents per ampoules (2 ml).

Ginseng fraction ·········· 300 mg

Sterile distilled water for injection ㆍ ······

pH regulator ㆍ ···········

Formulation Example 5 Preparation of Liquid

According to the method for preparing a liquid formulation in the Pharmacopoeia General Formulation, it is prepared in the following component content per 100 ml of liquid formulation.

Red ginseng fraction ············· 1 g

Heterosaccharides ·············· 10 g

Mannitol ················ 5 g

Purified water ㆍ ··············

Although the said composition ratio mixed composition which is comparatively suitable for manufacture of a pharmaceutical agent in a preferable embodiment, the compounding ratio can be arbitrarily modified according to demand-based layer, a use use, etc.

Figure 1 is a graph showing the cell survival rate by time (0 ~ 24 hours) oxygen-glucose deficiency (OGD) and reproductive digestion in SH-SY5Y cells.

Figure 2 is a graph showing the inhibitory effect of laurel alcohol extracts on cell death in OGD-induced SH-SY5Y cells.

Figure 3 is a graph showing the inhibitory effect of laurel nucleic acid fractions on cell death in OGD-induced SH-SY5Y cells.

Figure 4 is a graph showing the cytotoxicity test results according to the treatment of laurel chloroform fraction in SH-SY5Y cells.

5 is a graph showing the inhibitory effect of the laurel chloroform fraction on cell death in OGD-induced SH-SY5Y cells

6 is a graph showing the inhibitory effect of the laurel chloroform fraction on neuronal cell death induced by OGD in rat cortical tissue.

7 is a diagram showing the effect of inhibiting daphophorylation of DAPK relative intensity and laurel chloroform fraction activated by OGD in SH-SY5Y cells.

FIG. 8 is a graph of Western blot analysis of DAPK relative intensity and laurel chloroform fractions activated by OGD in SH-SY5Y cells.

9 is a graph showing the effect on the neurological behavioral indicators of the laurel chloroform fraction in MCAO-induced rats.

Figure 10 is a diagram showing the effect on the range of cerebral infarction of laurel chloroform fraction and carnosine in the MCAO induction model of rats.

11 is a graph showing the results of FIG. 10.

Claims (3)

A first step of cutting dried laurel medicines and then incubating with methanol at 40˜50 ° C. and filtration with filter paper three times repeatedly; Concentrated under reduced pressure in a portion of the methanol extract to obtain a methanol extract, a portion of the extract was shaken repeatedly extracted by addition of 10% nucleic acid and fractionated in a separatory funnel to separate the nucleic acid layer and methanol layer, the nucleic acid layer was concentrated under reduced pressure nucleic acid (Hexane) fraction Fractionating the second step; The methanol layer is added again chloroform and distilled water (1: 1, v / v) and shaken repeatedly extracted twice at 30 ~ 40 ℃ and fractionated with a separatory funnel to fractionate into a chloroform layer and an aqueous layer; After the third step, the aqueous layer is concentrated under reduced pressure to fractionate the aqueous fraction, and the chloroform layer is concentrated under reduced pressure to fractionate the final chloroform fraction. A composition for the prevention and treatment of ischemic nervous system brain disease, comprising alcohol extract and chloroform fraction. delete delete

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