KR20150133678A - Neuroprotective composition comprising extract or fractions of Schinus terebinthifolius Raddi as a active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for neuroprotection, containing an extract of Schinus terebinthifolius Raddi or a fraction thereof as an active ingredient. More specifically, the extract of Schinus terebinthifolius Raddi or the fraction thereof of the present invention has protective effects on brain cells and low cytotoxicity due to having inhibitory activities on glutamate toxicity in an N18-RE-105 cell line which possesses characteristics of the brain cells, thereby being useful for a pharmaceutical composition for neuroprotection, a pharmaceutical composition for preventing and treating cerebropathy, and health food for the same purpose.

Description

[0001] The present invention relates to a pharmaceutical composition for protecting nerve cells containing an extract of Brassica pepperii or a fraction thereof as an active ingredient. [0002] The present invention relates to a neuroprotective composition comprising extracts of fragments of Schinus terebinthifolius,

The present invention relates to a method for producing the < RTI ID = 0.0 > terebinthifolius Raddi) extract or fractions thereof as an active ingredient, and a composition for preventing and treating brain diseases.

Glutamate acts as a major excitatory neurotransmitter in the central nervous system (CNS) and acts as a metabotropic receptor and an ionotropic receptor (NMDA-, quisqualate-, kainate-type ) And exhibits various neurophysiological effects. The outside neurons release glutamate or absorbed directly re presynaptic terminals (presynaptic terminal) Na ⁺ - dependent high affinity transporter (Na ⁺ -dependent high affinity transporter), stellate cells (astrocyte), (colloid glial cell (cell by) ), And then converted to glutamine by glutamine synthase, which requires ATP, to move to the front end of the synapse.

The glutamate concentration outside the cells is maintained at a low concentration of 0.3 μM by the action of these pre-synaptic terminals and astrocytes. However, when the brain is subjected to ischemia, hypoxia, seizure, hypoglycemia, trauma, etc., the energy supply required for glutamate transportation becomes insufficient, and the extracellular glutamate concentration is reduced to a normal state 3,000, which is about 10,000 times, which causes the central nerve cell to die. These glutamate-induced neuronal cell deaths are called glutamate-induced neurotoxicity and are closely related to the oxidative stress caused by the generation of free radicals in the cells. Therefore, it has been clarified that the death of neurons by glutamate toxicity is caused by the destruction of cell components such as lipids, sugars, nucleic acids and proteins due to free radicals generated in cells excessively, and thus a free radical scavenger free radical scavenger) are very important as a new therapeutic agent for protecting brain cells.

The increase in extracellular concentration of glutamate is caused by acute diseases such as cerebral ischemia, cerebral hemorrhage, cerebral infarction, cerebral function coma, stroke, stroke, brain trauma, hypoxia, hypoglycemia, seizure, lacune, head trauma, thrombosis, Alzheimer ' s disease, AD, Huntington ' s disease (HD), Parkinson ' s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer & It is known to be involved in chronic diseases such as Creutzfeld-Jakob disease and brain circulatory metabolic disorders (S. Krobitsch, AG Kazantsev / The International Journal of Aging, Vol. Carlos Cepeda et al., Progress in (1986), Vol. 10 (1): 10-17; Carol Rocerz, et al., Folia Neuropathol 2013; Brain Research, Volume 211, ISSN 0079-6123; Nelsson, et al., Journal of Cerebral Blood Flow and Metabolism 10: 631-637; Niels C. Danbolt, Progress in Neurobiology 65 (2001) 1-105; Roukoz Chamoun, MD et al, J Neurosurg 113: 564.570, 2010; A. Zaunner et al., Acta Neurochir (1996) [Suppl] 67: 40-44; A. Zaunner et al., Acta Neurochir (1996) [Suppl] 67: 40-44; Yu Seung Woon et al., Neurobiol Dis. 2003 Dec; 14 (3): 303-17. Poly (ADP-ribose) polymerase-1 and apoptosis inducing factor in glutamate excitotoxicity; Nishizawa, Life Sci. 2001 Jun 15; 69 (4): 369-81. Glutamate release and neuronal damage in ischemia; Feng Han et al., Neuroscience Letters 430 (2008) 275.280; Sanju Lama et al., JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289, NUMBER 29, JULY 18, 2014; R.P. Clauss et al., Medical Hypotheses 75 (2010) 287-290; Sangmin et al., Inhibitory effect of bee venom on glutamate-mediated excitatory neurotoxicity, NSC-34 neuronal apoptosis inhibition, Journal of the Korean Society of Pharmaceutical Sciences, 55 (5) 385 ~ 390 (2011))

NIH research shows that stroke, one of the acute illnesses, is the third leading cause of death in the United States and is the leading cause of adult disability. About half a million patients die each year, of which 30% die, 20-30% become permanent cramps, and the rest suffer physical disabilities, body paralysis, vision loss, and memory loss. Also, in Korea, cancer is the second leading cause of death following cancer, and a therapeutic agent for such diseases is strongly required.

Glutamate binds to two receptors, an ionotropic receptor and a metabotropic receptor. Ionic receptors are associated with ion channels and act independently on nerve cells, resulting in excitotoxic damage. On the other hand, in metabolic receptors, glutamate does not cause direct neuronal death, unlike ionic receptors.

Neuronal cell death caused by binding of glutamate to ionic receptors can be divided into Na ⁺ , Cl ^- dependent and Ca ^{2 +} dependent. Na ⁺ and Cl ^- dependent neuronal cells develop within a few minutes after exposure to glutamate and show neuronal cell death similar to the depolarizing agent treatment. Glutamate query Squelch acid receptor (quisqualate receptor) to stimulate the Na ⁺ ions and the excess flows into the cell, with the influx of Na ⁺ ions Cl ^-, finally dissolved in H ₂ O is entered into the cells, cell expansion (swelling) lt; / RTI >

Ca ^{+ 2-dependent} neuronal cell death delay DE nerve injury (delayed neuronal damage) to occur after a few hours after glutamate treatment, similar to when the processing of A23187 Ca ²⁺ ion permeable carrier (ionophore). Influx of Na ⁺ ions opens the NMDA receptor Ca ^{2 +} channel which is closed by causing depolarization (depolarization) of the cell membrane, which causes the voltage-sensitive Ca ^{2 +} channels (voltage sensitive Ca ^{2 +} channel, VSCC) and Mg ^{2 +} with do. This causes a large amount of Ca ^{+ 2} are to be introduced into the cell. Thus flowing the Ca ²⁺ ions Phospholipase _{(phospholipase A 2, PLA 2)} , nitric oxide synthase (nitric oxide synthetase, NOS), Protease (protease), endonuclease (endonuclease) Ca ^{+ 2-dependent,} such as Energize enzymes. In particular, PLA ₂ degrades phospholipids to produce arachidonic acid, and free radicals such as superoxide are produced during the metabolism of arachidonic acid.

In addition, arachidonic acid and peroxides promote glutamate release in presynaptic neurons, further increasing the extracellular glutamate concentration. Peroxides are formed by the action of xanthine oxidase. Nitric oxide produced by the action of NOS reacts with peroxides to pass through peroxynitrite and reacts with hydroxyl radicals (hydroxyl radical is generated. In other words, enzymatic action of PLA ₂ , NOS, xanthine oxidase, etc., results in excess radicals in the cells, resulting in oxidative stress resulting in DNA, protein, lipid, And nonselective destruction of the nerve cells, and also endonuclease (endonuclease) by the action of apoptosis may occur.

     To investigate the mechanism of toxicity of glutamate, N18-RE-105 cell line sensitive to glutamate toxicity has been used. The N18-RE-105 cell line is prepared by cell fusion of mouse neuroblastoma clone N18TG-2 and embryonic neural retina of embryonic day 18 fisher rat (Fisher rat).

The N18-RE-105 cell line has a receptor similar to the glutamate receptor present in the hippocampus and has the ability to inhibit the production of delocalized calcium dependent cell death resulting in oxidative damage through accumulation of intracellular free radicals delayed calcium dependent cell death, which is very similar to actual brain cell death due to glutamate toxicity. Therefore, due to these properties, the N18-RE-105 cell line is used for an in vitro ischemic model and a glutamate toxicity mechanism.

Glutamate toxicity in the N18-RE-105 cell line is known to result from the inhibition of cysteine uptake by glutamate. Cysteine is an important amino acid that constitutes glutathione in cells. In N18-RE-105 cells, the cysteine and glutamate are transported in opposite directions relative to each other through the cell membrane. If the extracellular glutamate concentration increases and the cysteine does not enter the cell, the concentration of glutathione decreases and free radicals accumulate inside the cell, eventually causing the neuron to die. This fact is also confirmed in the primary culture of the embryonic cortical neurons of the embryo, and the quisqualate-type glutamate toxicity is mainly due to oxidative stress. .

As described above, glutamate causes acute cranial nerve diseases such as stroke and trauma, and oxidative stress due to glutamate toxicity is one of the causes in chronic brain diseases such as PD, AD and HD It is known. Accordingly, antioxidant active substances such as lipid peroxidation inhibiting substances that can suppress or alleviate oxidative stress due to glutamate toxicity are expected to be therapeutic agents for acute and chronic neurological diseases. Therefore, searching for glutamate toxicity inhibitors using tissues or cultured cells in vivo is expected to create not only substances acting on known targets but also new substances having new neuronal cell protection mechanisms.

Brazilian peppercorn ( Schinus terebinthifolius Raddi.), mainly from Brazil, Argentina and Paraguay, such as anacardiaceae (Anacardiaceae) plants that live in South America, rose pepper (Rose pepper), Brazilian pepper tree (Brazilian pepper), pink pepeokon (pink peppercorn) or berry (Christmasberry ), And it is not pepper, but it has a taste similar to pepper, so it is often used as a seasoning for food.

The essential oil of plant-growth-related parts of Brazilian pepper trees exhibits non-steroidal anti-inflammatory activity by inhibiting phospholipase A2 (Pires OC et (Jain MK et al., Phytochemistry 1995 (39)), it is known that triterpenoids present in the fruit show wound healing activity (Jain MK et al. : 537-547). In addition, Brazilian peppermint essential oil contains substances of terebinthone, hydroxymasticadienoic, terebinthifolic and ursolic acid, and by this substance antifungal and antibacterial activity (Siddiqui R et al., Biochem Syst Ecol. 2003 (31): 563-572). However, the protective effect of neuroprotection and brain diseases is not known at present.

BACKGROUND ART A stroke caused by glutamate is becoming a worldwide serious disease, and development of a therapeutic agent for it is actively under way. Particularly, since free radicals are the main ultimate source of neuronal cell death due to glutamate toxicity, studies on antioxidants such as novel free radical scavengers or lipid peroxidation inhibitors have been actively conducted. Upjohn's U78517F compound, a lipid peroxidation inhibitor composed of an amine unit of 21-amino steroid and a tetramethyl-chroman ring of vitamin E, It has been reported that IRFI-016, which is a basic skeleton of benzofuran, significantly inhibits brain cell damage after recirculation after cerebral ischemia. In addition, the quinone-based idebenone known as free radical scavenging agent strongly inhibits glutamate cytotoxicity in N18-RE-105 cells, and in vivo experiments have shown that it inhibits brain sclerosis and cerebrovascular dementia It is known as a substance that is currently under clinical trials in Japan as a brain metabolism activator after cardiac surgery and after organ transplantation. The TAK-218 compound, a peroxidase inhibitor developed by Takeda in Japan in June 1996, has been reported to exhibit neuroprotective and anti-ischemic effects by eliminating free radicals and abnormal free dopamine .

In addition, in relation to glutamate toxicity, a study evaluating brain cell protective activity by observing an increase in cell survival rate of a plant extract of Pseudomonas aeruginosa from HT22 cell line, a hippocampus-derived cell line of mice induced by glutamate toxicity, was reported (Journal of the Pharmaceutical Sciences, Kor. J. Pharmacogn. 39 (3): 213-217 (2008)). In addition, 435 medicinal plant extracts naturally grown in Korea and abroad were searched for natural products having nerve cell protection effect by using neural cell line hybridoma N18-RE-105 cell line. As a result, Viola mandshurica ), And its contents have been registered (Korean Patent No. 10-0982022). In addition, the round maturation extract and the non-polar solvent soluble extract blocked the apoptosis caused by glutamate and hydrogen peroxide neurotoxicity, thus confirming the significant effect on neuronal cell protection activity (Korean Patent No. 10-0776347). However, there has been no report on the neuronal cell protection activity of the Brazilian pepper extract or its fractions.

Accordingly, the present inventors have made efforts to develop a natural substance having a nerve cell protective effect, and have found that the extract of Brassica pepper or its fraction inhibits glutamate toxicity to nerve cells and has little cytotoxicity, The present invention has been accomplished on the basis of such finding that it can be effectively used as an active ingredient of a composition for preventing and treating cancer.

It is an object of the present invention to provide a method for the preparation of Brazil pepper terebinthifolius Raddi) extract or a fraction thereof as an active ingredient, and a composition for preventing and treating brain diseases.

In order to achieve the above object, the present invention Brazil Pepper Tree (Schinus terebinthifolius Raddi) extract or a fraction thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing and treating brain diseases, which comprises a Brazilian pepper extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a health food for nerve protection containing an extract of Brazilian pepper or its fraction as an active ingredient.

In addition, the present invention provides a health food for prevention and improvement of brain diseases, which comprises a Brazilian pepper extract or a fraction thereof as an active ingredient.

The inventive Brazilian peppercorn ( Schinus terebinthifolius Raddi extract or its fractions are excellent in the effect of significantly inhibiting the toxicity of nerve cells and are not cytotoxic and thus safe for the human body. Therefore, the above-mentioned Brazil pepper extract or its fractions can be used as a neuroprotective composition or for the prevention and treatment of brain diseases It can be effectively used as an active ingredient of the composition.

Figure 1 is a diagram showing glutamate-mediated neuronal cell death pathways.
VSCC: voltage-sensitive calcium channel;
NOS: nitric oxide synthase;
PLA _2: Phospholipase A ₂ (phospholipase A _2);
AA: arachidonic acid;
DG: diacyl glycerol;
Glut: glutamate; And
IP ₃ : inositol triphosphate.
FIG. 2 is a graph showing N18-RE-105 neuronal cell protection activity of Brazilian pepper extract. FIG.
A: N18-RE-105 cells not treated with glutamate;
B: N18-RE-105 cells treated with 20 mM glutamate; And
C: N18-RE-105 cells treated with 20 mM of glutamate and 45 ug / ml of Brazilian pepper extract.
FIG. 3 is a graph showing the inhibitory effect of Brassica pepper extract and its fractions on glutamate toxicity on N18-RE-105 neuronal cell line.
(-) Glut: N18-RE-105 cell line not treated with 20 mM glutamate;
(+) Glut: N18-RE-105 cell line treated with 20 mM glutamate;
FIG. 4 is a diagram showing a process for producing Brazilian pepper wood fractions. FIG.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for producing the < RTI ID = 0.0 > terebinthifolius Raddi) extract or a fraction thereof as an active ingredient.

The Brazilian pepper extract is preferably but not limited to be produced by a process comprising the following steps:

1) extracting Brazilian black pepper with an extraction solvent;

2) filtering the extract of step 1);

3) concentrating the filtered extract of step 2) under reduced pressure and then drying to produce an extract of Brazilian black pepper; And

4) Extracting the Brazilian pepper extract of step 3) with an organic solvent to produce the Brazil pepper fraction.

In the above method, the Brazilian pepper of step 1) may be used without limitation such as cultivated or commercially available. The Brazilian pepper can be any of leaves, stalks or roots, but is not limited thereto.

In the above method, it is preferable to use water, an alcohol or a mixture thereof in the extraction solvent of the step 1). As the alcohol, C ₁ to C ₂ lower alcohol is preferably used, and as the lower alcohol, ethanol or methanol is preferably used. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. It is preferable that the extraction solvent is added by 1 to 10 times the amount of the dried Brazil pepper tree, and it is more preferable that the extraction solvent is added by 2 to 3 times. The extraction temperature is preferably 20 占 폚 to 100 占 폚, more preferably 20 占 폚 to 40 占 폚, and most preferably room temperature, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, most preferably 24 hours, but is not limited thereto. In addition, the extraction number is preferably 1 to 5 times, more preferably 3 to 4 times, and most preferably, 3 times, but not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

In the above method, the organic solvent in step 4) is preferably n-hexane, chloroform, ethyl acetate or butanol, but is not limited thereto. The fractions are n-hexane fraction, chloroform fraction, ethyl acetate fraction, butanol fraction or water fraction obtained by suspending the Brazil pepper extract in water and sequentially fractionating the fraction with n-hexane, chloroform, ethyl acetate, butanol and water But is not limited thereto. The fraction may be obtained by repeating the fractionation process from the Brazil pepper extracts one to five times, preferably three times, followed by fractionation and concentration under reduced pressure. However, the fraction is not limited thereto.

In a specific embodiment of the present invention, the Brazilian peppercorn extract and fractions thereof were prepared using Brazil pepper (see FIG. 4).

Also, in order to confirm the protective effect of nerve cell of Brassica pepperii extract or its fractions, the present inventors treated the Brassica pepper extract or its fractions of the present invention with N18-RE-105 cell line treated with high glutamate, It was confirmed that cell death was significantly inhibited and the neuronal cell protection activity was excellent (see FIG. 2).

In addition, the concentration of Brazilian pepper extract or fraction thereof (IC ₅₀ ), which inhibits glutamate toxicity by 50%, was found to be 22.5 ug / ml of Brazilian pepper extract, which was similar to 21.5 ug / ml of vitamin E And 22.0 ug / ml and 28.4 ug / ml, respectively, of the pepper kernel fractions and the butanol fractions of Brazil pepper (Fig. 1).

In order to confirm the inhibitory effect on the glutamate toxicity according to the concentration of the Brazil pepper extract or the fraction thereof, the inventors of the present invention administered N18-RE-105 neuronal cell line with concentrations of Brassica pepper extract and its fractions (13.6, 22.7, and 45.5 占 퐂 / ml), it was confirmed that N18-RE-105 cell death was inhibited by glutamate in a concentration-dependent manner (see FIG. 3).

The present inventors also confirmed that the extract of Brassica pepperii or its fractions showed no cytotoxicity even at a high concentration of 80 / / ml, which is about four times higher than the effective concentration of Brassica pepper extract or its fraction .

Therefore, the extract of Brassica pepperii or its fractions of the present invention is excellent in the effect of significantly inhibiting the toxicity of neurons and is safe for the human body because of its cytotoxicity, so that it can be effectively used as an active ingredient of a pharmaceutical composition for neuroprotection.

The composition containing the Brazilian pepper extract of the present invention or a fraction thereof may further contain one or more active ingredients showing the same or similar functions in addition to the above components.

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, calcium stearate, , White sugar, dextrose, sorbitol and talc. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

That is, the composition of the present invention can be administered in various formulations of oral and parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, . ≪ / RTI > Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, , Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions and syrups, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are commonly used simple diluents . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The composition of the present invention may be administered orally or parenterally in accordance with the intended method, and may be administered orally, parenterally or intraperitoneally, rectally, subcutaneously, intravenously, intramuscularly, . The dosage varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity.

The dosage unit may contain, for example, 1, 2, 3 or 4 times the individual dose or may contain 1/2, 1/3 or 1/4 times the dose. The individual dosages specifically include amounts in which the active drug is administered in a single dose, which usually corresponds to the full, half, one-third, or one-fourth of the daily dose. The effective dose of the composition of the present invention may be 0.0001 to 10 g / kg, specifically 0.001 to 1 g / kg, and may be administered 1 to 6 times a day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for neuroprotection.

The present invention also provides a pharmaceutical composition for preventing and treating brain diseases, which comprises a Brazilian pepper extract or a fraction thereof as an active ingredient.

The Brazilian pepper extract is preferably but not limited to be produced by a process comprising the following steps:

1) extracting Brazilian black pepper with an extraction solvent;

2) filtering the extract of step 1);

3) concentrating the filtered extract of step 2) under reduced pressure and then drying to produce an extract of Brazilian black pepper; And

4) Extracting the Brazilian pepper extract of step 3) with an organic solvent to produce the Brazil pepper fraction.

In the above method, the Brazilian pepper of step 1) may be used without limitation such as cultivated or commercially available. The Brazilian pepper may be any of leaves, stalks or roots, but is not limited thereto.

In the above method, it is preferable to use water, an alcohol or a mixture thereof in the extraction solvent of the step 1). As the alcohol, C ₁ to C ₂ lower alcohol is preferably used, and as the lower alcohol, ethanol or methanol is preferably used. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. It is preferable that the extraction solvent is added by 1 to 10 times the amount of the dried Brazil pepper tree, and it is more preferable that the extraction solvent is added by 2 to 3 times. The extraction temperature is preferably 20 占 폚 to 100 占 폚, more preferably 20 占 폚 to 40 占 폚, and most preferably room temperature, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, most preferably 24 hours, but is not limited thereto. In addition, the extraction number is preferably 1 to 5 times, more preferably 3 to 4 times, and most preferably, 3 times, but not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

In the above method, the organic solvent in step 4) is preferably n-hexane, chloroform, ethyl acetate or butanol, but is not limited thereto. The fractions are n-hexane fraction, chloroform fraction, ethyl acetate fraction, butanol fraction, or water fraction obtained by suspending the Brazil pepper extract in water and sequentially fractionating the fraction with n-hexane, chloroform, ethyl acetate, But is not limited thereto. The fraction may be obtained by repeating the fractionation process from the Brazil pepper extracts one to five times, preferably three times, followed by fractionation and concentration under reduced pressure. However, the fraction is not limited thereto.

The brain diseases include stroke, paralysis, dementia, Alzheimer's disease, Huntington's disease, Pick's disease, Creutzfeld-Jakob disease, thrombosis, em-bolism, transient ischemic but is not limited to, any one selected from the group consisting of stroke, attack, lacune, stroke, cerebral hemorrhage, cerebral infarction, head trauma, cerebral circulatory metabolic disturbance and brain function coma.

The Brazilian pepper extract or its fractions of the present invention are excellent in the effect of significantly inhibiting the toxicity of neurons and are safe for the human body due to the lack of cytotoxicity, and thus can be usefully used as an effective ingredient for pharmaceutical compositions for the prevention and treatment of brain diseases have.

In addition, the present invention provides a health food for nerve protection containing an extract of Brazilian pepper or its fraction as an active ingredient.

The Brazilian pepper extract is preferably but not limited to be produced by a process comprising the following steps:

1) extracting Brazilian black pepper with an extraction solvent;

2) filtering the extract of step 1);

3) concentrating the filtered extract of step 2) under reduced pressure and then drying to produce an extract of Brazilian black pepper; And

4) Extracting the Brazilian pepper extract of step 3) with an organic solvent to produce the Brazil pepper fraction.

In the above method, the Brazilian pepper of step 1) may be used without limitation such as cultivated or commercially available. The Brazilian pepper can be any of leaves, stalks or roots, but is not limited thereto.

In the above method, it is preferable to use water, an alcohol or a mixture thereof in the extraction solvent of the step 1). As the alcohol, C ₁ to C ₂ lower alcohol is preferably used, and as the lower alcohol, ethanol or methanol is preferably used. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. It is preferable that the extraction solvent is added by 1 to 10 times the amount of the dried Brazil pepper tree, and it is more preferable that the extraction solvent is added by 2 to 3 times. The extraction temperature is preferably 20 占 폚 to 100 占 폚, more preferably 20 占 폚 to 40 占 폚, and most preferably room temperature, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, most preferably 24 hours, but is not limited thereto. In addition, the extraction number is preferably 1 to 5 times, more preferably 3 to 4 times, and most preferably, 3 times, but not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

In the above method, the organic solvent in step 4) is preferably n-hexane, chloroform, ethyl acetate or butanol, but is not limited thereto. The fractions are n-hexane fraction, chloroform fraction, ethyl acetate fraction, butanol fraction or water fraction obtained by suspending the Brazil pepper extract in water and sequentially fractionating the fraction with n-hexane, chloroform, ethyl acetate, butanol and water But is not limited thereto. The fraction may be obtained by repeating the fractionation process from the Brazil pepper extracts one to five times, preferably three times, followed by fractionation and concentration under reduced pressure. However, the fraction is not limited thereto.

The Brazilian pepper extract of the present invention or its fractions are excellent in the effect of significantly inhibiting the toxicity of nerve cells and are not cytotoxic and thus safe for human body and thus can be usefully used as an effective ingredient of a health food for nerve protection.

The health food of the present invention can be used as it is, or can be used in combination with other food or food ingredients, and can be suitably used according to conventional methods.

There is no particular limitation on the kind of the health food. Examples of foods to which the Brazilian pepper extract or its fractions can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice cream, Drinks, tea, drinks, alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Such natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 of the composition of the present invention.

In addition to the above, the health food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , A carbonating agent used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the present invention provides a health food for prevention and improvement of brain diseases, which comprises a Brazilian pepper extract or a fraction thereof as an active ingredient.

The Brazilian pepper extract is preferably but not limited to be produced by a process comprising the following steps:

1) extracting Brazilian black pepper with an extraction solvent;

2) filtering the extract of step 1);

3) concentrating the filtered extract of step 2) under reduced pressure and then drying to produce an extract of Brazilian black pepper; And

4) Extracting the Brazilian pepper extract of step 3) with an organic solvent to produce the Brazil pepper fraction.

In the above method, the Brazilian pepper of step 1) may be used without limitation such as cultivated or commercially available. The Brazilian pepper can be any of leaves, stalks or roots, but is not limited thereto.

In the above method, it is preferable to use water, an alcohol or a mixture thereof in the extraction solvent of the step 1). As the alcohol, C ₁ to C ₂ lower alcohol is preferably used, and as the lower alcohol, ethanol or methanol is preferably used. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. It is preferable that the extraction solvent is added by 1 to 10 times the amount of the dried Brazil pepper tree, and it is more preferable that the extraction solvent is added by 2 to 3 times. The extraction temperature is preferably 20 占 폚 to 100 占 폚, more preferably 20 占 폚 to 40 占 폚, and most preferably room temperature, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, most preferably 24 hours, but is not limited thereto. In addition, the extraction number is preferably 1 to 5 times, more preferably 3 to 4 times, and most preferably, 3 times, but not limited thereto.

In the above method, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in step 3), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

In the above method, the organic solvent in step 4) is preferably n-hexane, chloroform, ethyl acetate or butanol, but is not limited thereto. The fractions are n-hexane fraction, chloroform fraction, ethyl acetate fraction, butanol fraction or water fraction obtained by suspending the Brazil pepper extract in water and sequentially fractionating the fraction with n-hexane, chloroform, ethyl acetate, butanol and water But is not limited thereto. The fraction may be obtained by repeating the fractionation process from the Brazil pepper extracts one to five times, preferably three times, followed by fractionation and concentration under reduced pressure. However, the fraction is not limited thereto.

The brain diseases include stroke, paralysis, dementia, Alzheimer's disease, Huntington's disease, Pick's disease, Creutzfeld-Jakob disease, thrombosis, em-bolism, transient ischemic but is not limited to, any one selected from the group consisting of stroke, attack, lacune, stroke, cerebral hemorrhage, cerebral infarction, head trauma, cerebral circulatory metabolic disturbance and brain function coma.

* The Brazilian pepper extract or its fractions of the present invention are excellent in the effect of significantly inhibiting the toxicity of nerve cells and are safe to the human body due to the lack of cytotoxicity, so that they can be effectively used as an active ingredient of health foods for prevention and improvement of brain diseases have.

Hereinafter, the present invention will be described in detail with reference to Examples and Production Examples.

However, the following examples and preparations are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples and production examples.

Example 1 Preparation of Brassica pepper extract

Forty grams of dried bark of Brazilian black pepper obtained from Korean Plant Biotechnology Council (Daejeon) was extracted with 400 ml of aqueous methanol solution (methanol 80%, water 20%) to produce 2.5 g of Brazilian pepper extract.

Example 2: Preparation of Brazilian pepper tree fractions

The <Example 1> The Brazilian pepper tree extract the 2.5 g was suspended in 1 liter of water (liter), n- hexane, chloroform (CHCl _3), ethyl acetate (EtOAc) manufactured by, and butanol (C ₄ H ₉ OH ) Were successively extracted using 500 milliliters (ml) of each solvent to obtain 0.5 g of chloroform fraction, 0.6 g of ethyl acetate fraction and 0.6 g of butanol fraction (Fig. 4).

<Example 3> Confirm Brazilian pepper tree extract or neuroprotective activity of fractions thereof

The N18-RE-105 cell line (Tokyo University, Japan) was cultivated in Dulbesco's modified Eagle's medium (manufactured by Nippon Denshoku Kogyo Co., Ltd.) in order to examine the neuronal cell protection activity of the extracts of Brassica pepper or its fractions prepared in Examples 1 and 2 DMEM) was supplemented with HAT supplement (0.1 mM hypoxanthin, 0.04 mM aminopterin, 0.14 mM thymidine) and heat-treated 10% fetal calf serum (FCS). Then, the N18-RE-105 cell line was cultured in a T-25 flask for 48 hours, and then cultured in an incubator for 2 minutes using a trypsin solution (3 ml) to separate the cells from the flask. Thereafter, 1 ml of medium was added to neutralize trypsin, followed by centrifugation at 1000 rpm for 3 minutes. After removing the supernatant, the cells were suspended in the medium, and the number of cells was calculated using a hemocytometer, well) were dispensed into a 96-well microplate at a rate of 5 × 10 ⁴ cells / well. Cells were cultured in 5% CO ₂ at 37 ° C. for 24 hours. Then, 5 μl of glutamate (400 mM) solution dissolved in phosphate buffered saline (PBS) and the extract of Brassica pepper or its fraction dissolved in DMSO were dissolved in PBS A 10-fold diluted Brazilian pepper extract or its fraction solution was treated. Then, after incubation in an incubator for 24 hours, the protective activity of brain cytotoxicity through the glutamate toxicity inhibitory activity of the Brazil pepper seed extract or its fractions was observed using a phase contrast microscope.

As a result, as shown in Fig. 2, it was confirmed that the Brassica pepper extract of the present invention inhibits glutamate toxicity and protects nerve cells. In particular, in the glutamate-treated cells, swelling, blebbing, and lysis occurred in the N18-RE-105 cells compared with the control without glutamate treatment, However, the cells of the sample to which Brazil pepper extract (45 / / ml) was added did not change the shape of the cells, and it was confirmed that more than 80% of the cells were alive due to decrease in death (FIG. 2).

In addition, the concentration (IC ₅₀ ) of Brazilian pepper extracts inhibiting glutamate toxicity by 50% of the N18-RE-105 cell line, which is a neuronal hybridoma cell, was 22.5 / / ml, which was 21.5 ㎍ / ml, and the fractions of chloroform and ethylacetate of P. pepper showed 22.1 and 28.4.0 ㎍ / ml, respectively, indicating excellent neuronal cell protective activity (Table 1).

Neuroprotective activity of Brassica pepper extract or its fractions sample IC ₅₀ ([mu] g / ml) Brazilian black pepper extract 22.5 Chloroform fraction 22.1 Butanol fraction 28.4 Vitamin E 21.5

<Example 4> OK inhibiting glutamate toxicity active Brazilian pepper tree extract or a fraction of the nerve cells thereof

100 [mu] l of N18-RE-105 cells were dispensed into a 96-well microplate at 5 x 10 ⁴ cells per well. Cells were cultured in 5% CO ₂ at 37 ° C for 24 hours. Then, 1.0 mg / ml, 0.5 mg / ml and 0.3 mg / ml concentrations of the extracts of Brassica juncea L. or their fractions dissolved in DMSO were diluted 10 times with PBS Of Brassica pepper extract or its fraction solution was treated with 5 μl of a glutamate (400 mM) solution dissolved in phosphate buffered saline (PBS) for 30 minutes. Then, the cells were cultured in an incubator for 24 hours, and living cells were measured and quantified using an EZ-Cytox assay (Daeil Lab Service, Seoul, Korea). In the EZ-Cytox assay (dehydrogenase assay), 70 μl of the reaction medium per plate well was transferred to a new 96-well plate and incubated at 450 nm with a microplate reader (Molecular devices) And cell viability was measured. The neuronal cell protective activity was calculated using the following Equation (1).

As a result, as shown in Fig. 3, it was confirmed that Brazil pepper extract and its fractions inhibit the death of N18-RE-105 cells by glutamate in a concentration-dependent manner (Fig. 3). The cell viability was calculated using the following equation (1), and the results were again calculated using the following equation (2).

A is the absorbance of each well; And

Ac is the absorbance of a well not treated with glutamate.

Vc is the cell survival rate of a well not treated with glutamate (Glutamate);

Vg is the cell survival rate of a well treated with Glutamate; And

Vs is the cell survival rate of glutamate and well treated with the sample.

<Example 5> Cytotoxicity evaluation

In order to confirm the cytotoxicity of the extracts of Brassica pepper or its fractions, 5, 10, 20, 50 and 80 mg / ml were treated and treated with glutamate, Respectively.

As a result, it was confirmed that no cytotoxicity was observed even at a high concentration of 80 μg / ml, which is four times higher than the effective concentration of the extract of Brassica pepperii and its fractions.

&Lt; Preparation Example 1 > Preparation of pharmaceutical preparations

<1-1> Preparation of powder

2 g of the Brazilian pepper extract of the present invention

Lactose 1 g

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of tablets

100 mg of the Brazil pepper extract of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

100 mg of the Brazil pepper extract of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; 1-4 >

1 g of the Brazil pepper extract of the present invention

Lactose 1.5 g

* Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-5> Preparation of granules

150 mg of the Brazil pepper extract of the present invention

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

&Lt; Preparation Example 2 > Preparation of health food

<2-1> Production of flour food

0.5 to 5.0 parts by weight of the Brazilian pepper extract of the present invention or its fractions were added to wheat flour and the mixture was used to prepare bread, cake, cookies, crackers and noodles.

<2-2> Production of soups and gravies

0.1 to 5.0 parts by weight of the extract of Brazilian peppercorn extract of the present invention or its fractions were added to soups and juices to prepare meat products for health promotion, soups of noodles and juices.

<2-3> Preparation of ground beef

A ground beef for health promotion was prepared by adding 10 parts by weight of the extract of Brazil pepper of the present invention or its fraction to ground beef.

<2-4> Manufacture of dairy products

5 to 10 parts by weight of the extract of Brazil pepper of the present invention or its fractions were added to milk and various dairy products such as butter and ice cream were prepared using the milk.

&Lt; 2-5 >

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a known method, and then they were prepared into powders having a particle size of 60 mesh by a grinder.

The Brazilian pepper extract or its fractions of the present invention were concentrated under reduced pressure in a vacuum concentrator, dried by spraying and dried in a hot air drier, and pulverized to a size of 60 mesh with a pulverizer to obtain a dry powder.

The grains, seeds and the Brazilian pepper extract of the present invention or fractions thereof were prepared in the following proportions.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

The Brazilian pepper extract or its fraction (3 parts by weight) of the present invention,

(0.5 part by weight),

(0.5 parts by weight)

&Lt; Preparation Example 3 > Preparation of beverage

<3-1> Production of health drinks

(50%), water (75%) and 5 g of the extract of Brassica pepper extract of the present invention or its fractions were homogeneously mixed And then sterilized and packaged in glass bottles, plastic bottles, etc.

* <3-2> Manufacture of vegetable juice

Vegetable juice was prepared by adding 5 g of the Brazil pepper extract of the present invention or its fraction to 1,000 ml of tomato or carrot juice.

<3-3> Production of fruit juice

Fruit juice was prepared by adding 1 g of the Brazil pepper extract of the present invention or a fraction thereof to 1,000 ml of apple or grape juice.

&Lt; Preparation Example 4 > Preparation of health food

<4-1> Production of flour food

0.5 to 5.0 parts by weight of the Brazilian pepper extract of the present invention or its fractions were added to wheat flour and the mixture was used to prepare bread, cake, cookies, crackers and noodles.

<4-2> Manufacture of soups and gravies

0.1 to 5.0 parts by weight of the extract of Brazilian peppercorn extract of the present invention or its fractions were added to soups and juices to prepare meat products for health promotion, soups of noodles and juices.

<4-3> Preparation of ground beef

A ground beef for health promotion was prepared by adding 10 parts by weight of the extract of Brazil pepper of the present invention or its fraction to ground beef.

<4-4> Manufacture of dairy products

5 to 10 parts by weight of the extract of Brazil pepper of the present invention or its fractions were added to milk and various dairy products such as butter and ice cream were prepared using the milk.

<4-5> Manufacturing of Sunshine

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a known method, and then they were prepared into powders having a particle size of 60 mesh by a grinder.

The Brazilian pepper extract or its fractions of the present invention were concentrated under reduced pressure in a vacuum concentrator, dried by spraying and dried in a hot air drier, and pulverized to a size of 60 mesh with a pulverizer to obtain a dry powder.

The grains, seeds and the Brazilian pepper extract of the present invention or fractions thereof were prepared in the following proportions.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

The Brazilian pepper extract or its fraction (3 parts by weight) of the present invention,

(0.5 part by weight),

(0.5 parts by weight)

&Lt; Preparation Example 5 > Preparation of beverage

<5-1> Production of health drinks

(50%), water (75%) and 5 g of the extract of Brassica pepper extract of the present invention or its fractions were homogeneously mixed And then sterilized and packaged in glass bottles, plastic bottles, etc.

<5-2> Preparation of vegetable juice

Vegetable juice was prepared by adding 5 g of the Brazil pepper extract of the present invention or its fraction to 1,000 ml of tomato or carrot juice.

<5-3> Preparation of fruit juice

Fruit juice was prepared by adding 1 g of the Brazil pepper extract of the present invention or a fraction thereof to 1,000 ml of apple or grape juice.

Claims (12)

Brazilian peppercorn ( Schinus terebinthifolius Raddi) extract or a fraction thereof as an active ingredient and inhibiting the toxicity of glutamate, which is a pharmaceutical composition for protecting nerve cells from glutamate.
The pharmaceutical composition for neuroprotection according to claim 1, wherein the extract is extracted with water, a C ₁ to C ₂ lower alcohol or a mixture thereof as a solvent.
The pharmaceutical composition for neuroprotection according to claim 2, wherein the lower alcohol is ethanol or methanol.
The pharmaceutical composition for neuroprotection according to claim 1, wherein the fraction is further extracted from a Brazil pepper extract with one selected from the group consisting of hexane, chloroform, ethyl acetate and butanol.
A pharmaceutical composition for preventing and treating glutamate-induced brain diseases, which comprises a Brazilian pepper extract or a fraction thereof as an active ingredient and inhibits the toxicity of glutamate.
The pharmaceutical composition according to claim 5, wherein the extract is extracted with water, a C ₁ to C ₂ lower alcohol or a mixture thereof as a solvent.
The pharmaceutical composition according to claim 5, wherein the lower alcohol is ethanol or methanol.
[Claim 7] The pharmaceutical composition according to claim 5, wherein the fraction is prepared by further extracting a Brazilian pepper extract with one selected from the group consisting of hexane, chloroform, ethyl acetate and butanol. Composition.
6. The method of claim 5, wherein the brain disease is selected from the group consisting of stroke, stroke, dementia, Alzheimer's disease, Huntington's disease, Pick's disease, Creutzfeld-Jakob disease, thrombosis, em- Wherein the compound is any one selected from the group consisting of transient ischemic attack, lacune, stroke, cerebral hemorrhage, cerebral infarction, head trauma, brain circulatory metabolic disorder, A pharmaceutical composition for preventing and treating brain diseases.
A health food containing a Brazilian pepper extract or a fraction thereof as an active ingredient and containing a pharmaceutical composition for inhibiting the toxicity of glutamate, which is a health food for protecting the nerve from glutamate.
A health food containing a Brazilian pepper extract or a fraction thereof as an active ingredient and inhibiting the toxicity of glutamate, which is a health food for preventing and improving glutamate-induced brain diseases.
12. The method of claim 11, wherein the brain disease is selected from the group consisting of stroke, stroke, dementia, Alzheimer's disease, Huntington's disease, Pick's disease, Creutzfeld-Jakob disease, thrombosis, em- Wherein the brain is one selected from the group consisting of transient ischemic attack, lacune, stroke, cerebrospinal fluid, cerebral infarction, head trauma, cerebral circulatory metabolic disorders, Health food for prevention and improvement of disease.

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