KR101951402B1 - Fermentative product of Sagunjatang having brain neuron cell-protective activity and uses thereof - Google Patents

Abstract

The present invention relates to a fermented product of Lactobacillus thunbergii having cranial nerve cell protective activity and its use, and more particularly to a pharmacological agent for preventing or treating cranial nerve diseases including fermented products of Lactobacillus of extracts containing ginseng, A health functional food composition, and a preventive or therapeutic method.
The fermented product of Lactobacillus lactic acid bacteria obtained by fermenting Lactobacillus plantarum with an extract containing ginseng, liquorice, liquorice, and licorice, i.e., Sangunjatang, has excellent protective effect on brain nerve cells. Therefore, The composition of the present invention may be useful for preventing or treating cranial neuropathy.

Description

[0001] The present invention relates to fermentation products of lactic acid bacteria of the genus sanjitang having a brain nerve cell protective activity and uses thereof.

The present invention relates to a fermented product of Lactobacillus thunbergii having cranial nerve cell protective activity and its use, and more particularly to a pharmacological agent for preventing or treating cranial nerve diseases including fermented products of Lactobacillus of extracts containing ginseng, A health functional food composition, and a preventive or therapeutic method.

Neuronal damage in the brain is a direct cause of a variety of neurological disorders such as neurodegenerative diseases, neurological diseases, mental illnesses, and memory disorders (Yan Wang, Neurotox Res. 2015 May, 27 (4): 368-383. Merry W. Ma, Mol Neurodegener 2017, 12: 7 .; Marin O, Nat Rev Neurosci 2012 Jan 18, 13 (2): 107-20 .; Yun Chen, Front Neurol. . Therefore, although many studies have been conducted in the art to find the mechanism of brain damage, the causes of brain nerve cell damage have not yet been clarified due to the complexity of brain neuronal damage mechanism.

According to recent reports, the generation of free radicals and the release of excessive excitatory neurotransmitters have been pointed out as causes of damage to the brain nerve cells. Specifically, excessive oxidative stress accumulates Reactive Oxygen Species (ROS) such as superoxide ions and hydroxyl free radicals that exceed the antioxidant capacity of cells, Mitochondria, nuclei and endoplasmic reticulum. Such ROS are nopyige the permeability of the cells for Ca ^{2 +,} Ca ^{2 +} concentration in the cell is increased to give damage to the mitochondria, amino acids are oxidized. Eventually, the nucleus and mitochondrial DNA are oxidized, resulting in DNA damage, such as DNA strand breakage (Finkel T, Nature. 2000 Nov 9, 408 (6809): 239-47.).

In particular, the brain has a high oxygen utilization rate for metabolism and high lipid peroxide content, while the antioxidant system for active oxygen and the antioxidant agent for low molecular weight are relatively small compared to other tissues, It is very vulnerable to enemy damage. For this reason, it is known that ROS is associated with the induction of degenerative neurological diseases such as dementia caused by induction of brain neuronal cell death (PF Good, Am J Pathol 1996 Jul, 149 (1): 21-28 .; Smith MA, J. Neurochem 1998 May, 70 (5): 2212-5.)

On the other hand, herbal medicine has been used for many years for the prevention and treatment of various diseases. Recently, it has been recognized that the side effects are relatively low in the human body and that various effects are exhibited by the complex interaction of the constituent herbal medicine ingredients. Demand is increasing significantly. In this regard, there is a need for an improved herbal medicine for treating pain of the human nervous system (Korean Patent Laid-Open No. 10-1992-001150), an Alzheimer's product containing extracts of herbal medicines, And a health functional food for preventing or improving sexual dementia (Korean Patent No. 10-1029699) have been developed. However, studies on herbal preparations which are still safe and have excellent therapeutic effect on cranial nerve diseases are insufficient.

Under these circumstances, the present inventors have made intensive efforts to develop a substance capable of protecting brain cells in the course of disease development while inhibiting the development of brain neuropathy. As a result, it has been found that lactic acid bacteria fermentation of Sangunjatang including ginseng, The present inventors have accomplished the present invention by confirming that water can provide a very excellent brain cell protection effect and that the above fermented product can prevent or treat brain nerve diseases.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating cerebral neuropathy which comprises fermented product of lactic acid bacteria of an extract containing ginseng, liquorice, liquorice, licorice and licorice.

It is another object of the present invention to provide a method for preventing or treating a brain neuropathy, which comprises administering the pharmaceutical composition to a subject other than a human.

It is another object of the present invention to provide a health functional food composition for improving cerebral neurological diseases, which comprises a fermented product of lactic acid bacteria of an extract comprising ginseng, liquorice, liquorice, licorice and licorice.

In order to achieve the above object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating cranial nerve diseases, comprising fermented product of lactic acid bacteria of an extract containing ginseng, liquorice, liquorice, licorice and licorice.

In the present invention, the fermented product of Lactobacillus japonica extract of Sangunjatang, which is an extract of ginseng, liquorice, liquorice and licorice, increases survival rate of damaged brain nerve cells by oxidative stress, stabilizes mitochondrial membrane potential and inhibits ROS production, Lt; RTI ID = 0.0 > inhibitory < / RTI > activity.

The term "ginseng" (ginseng) of the present invention is a root of ginseng ( Panax ginseng CA Meyer) of Araliaceae. It has a characteristic odor, flavor is slightly worn, and the property is slightly warm. Ginseng is known for its refreshing properties and is used for the weakness of the body, boredom, fatigue, loss of appetite, vomiting, helping the lung function and enhancing the new function.

The term " Atractylodes japonica Koidz. &Quot; of the present invention refers to atractylodes japonica Koidzumi or Atractylodes macrocephala Koidzumi) by removing the root stem or juniper. It has a peculiar smell, taste is a little bit weary, it is viscous when it is chewed, and it is characterized by its warm nature. Chronic dyspepsia, enteritis, diarrhea and is also known to excel in promoting appetite.

The term " Poria cocos Wolf " of the present invention means a medicament prepared by scarring of Poria cocos Wolf with the parasitic rootstock on the root of pine, and removing the outer layer almost. It has little smell, is a little mucous, has a taste of sweet and flat, and the quality is characterized by flatness without shifting to one side. Nephritis, cystitis, and urethritis, and also has a genetic effect, which is used to treat chronic bronchitis and bronchiectasis.

The term "licorice"(licorice; Glycyrrhiza glabra L.) is a perennial herb with roots of dicotyledonous roots and roots, characterized by a characteristic odor and sweet taste. Licorice is known to harmonize the toxicity of all medicines, to make medicinal effects appear better, to control the heat and scent of the books, to communicate all blood vessels, and to strengthen muscles and bones.

The term "extract" of the present invention refers to a preparation which is prepared by squeezing a herbal medicine with an appropriate leaching solution and concentrating by evaporating the leaching solution. The diluent, concentrate or extract of the extract obtained by the extraction treatment is dried A dried product to be obtained, a controlled preparation thereof or a purified product thereof. The extracts including ginseng, liquorice, liquorice, and licorice can be prepared by using common extraction, separation and purification methods known in the art. Examples of the extraction method include, but are not limited to, hot water extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction.

In the present invention, the extract may be one obtained by extracting a mixture of ginseng, liquorice, liquorice, licorice, and more specifically, from 0.5 to 1.5: 0.5 to 1.5: 0.5 to 1.5: 0.5 to 1.5 Weight ratio, most specifically in a weight ratio of 1: 1: 1: 1.

The extract may be selected from the group consisting of a ginseng extract, a crude extract, a ginseng extract, and a mixture of ginseng extract. More specifically, the ginseng extract, the extract extract, the ginseng extract and the licorice extract are mixed at a ratio of 0.5 to 1.5: 0.5 to 1.5: 0.5 to 1.5: To 1.5, most specifically in a weight ratio of 1: 1: 1: 1.

In the present invention, the extract may be prepared by extracting with an extraction solvent or extracting with an extraction solvent, followed by fractionation with a fraction solvent. Specifically, the extraction solvent may be water, an organic solvent, or a mixed solvent thereof. The organic solvent may be an alcohol having 1 to 4 carbon atoms, a polar solvent such as ethyl acetate or acetone, a hexane or a dichloromethane Non-polar solvents, or mixed solvents thereof. More specifically, the extract may be extracted with water, but is not limited thereto.

In the present invention, the extracts containing ginseng, Baekjong, Baekryeong, and licorice may be Sangunjatang. It is said that Sangunjatang is effective for stomach anorexia, vomiting, weakness of limbs, hemorrhoids, dehydration, and nocturnal enuresis. However, the protective effect of neurons in the brain of Sangunjatang is not known, and the protective effect of the fermented product of Lactobacillus fermented by Sangunjatang on the brain nerve cells is also unknown.

The term "fermented product" of the present invention means the result of fermentation of the above extract or its fraction. The term "fermentation" means a process of obtaining a substance useful for humans by using microorganisms or fungi in a narrow sense, and in a broad sense, a sugar decomposition process for obtaining energy without using oxygen in a narrow sense. For example, in the present invention, the fermentation product may be prepared by cultivating a bacterium in a substance to be fermented, and may include a substance advantageous to humans produced through various enzymatic actions and the like.

The fermented product may be a lactobacillus fermented product of an extract of a mixture containing ginseng, liquorice, liquorice, licorice, etc. In the present specification, the fermented product may be named as "fermented product of Lactobacillus lactic acid bacteria" or "fermented product of Lactobacillus" .

The term "lactic acid bacteria" of the present invention collectively refers to bacteria that decompose saccharides such as glucose to produce lactic acid, and are referred to as lactic acid bacteria, lactic acid bacteria and the like. A type of lactic acid bacteria commonly known is a microorganism belonging to the genus Lactobacillus bacteria (Lactobacillus), A streptococcus (Streptococcus), A Phedi OKO kusu (Pediococcus), A flow Pocono stock (Leuconostoc).

For the purpose of the present invention, as the lactic acid bacteria, isolated strains or various commercially available lactic acid bacteria may be used without limitation. Specifically, microorganisms of the genus Lactobacillus can be used without limitation, and more specifically, Lactobacillus zeae , Lactobacillus plantarum or Lactobacillus amylophilus can be used, Most specifically, lactobacillus plantarum can be used, but is not limited thereto.

The lactic acid bacteria may be inoculated in an amount of 0.1 to 1.9% (v / v), specifically 0.5 to 1.5% (v / v), most specifically 1% (v / v) It is not.

In addition, the lactic acid bacteria may be cultured in the extract for 40 to 55 hours, specifically 43 to 53 hours, most specifically 48 hours, but the present invention is not limited thereto.

The term "cranial nerve cell protective activity" of the present invention means blocking or reducing the degree of damage or destruction of the cranial nerve cell.

The term "cranial nerve disease" of the present invention means a disease caused by damage or destruction of the brain nerve cell. Specifically, neurodegenerative diseases such as Alzheimer's disease, Huntington's chorea, Parkinson's disease; Neurological diseases such as senile dementia, frontotemporal dementia, vascular dementia, migraine, neuropathic pain of central origin; (Especially bulimia, anorexia), phobias (particularly in the form of plaques), depression, nervous breakdown, schizophrenia, bipolar disorder, generalized anxiety disorder, stress related disorder, panic disorder, obsessive- compulsive disorder, posttraumatic stress disorder, attention deficit hyperactivity disorder Phobias), mental disorders such as autism; Memory disorders associated with neurological or mental illnesses, attention deficit disorders or arousal disorders.

In a specific example of the present invention, a mixture containing the same amount of ginseng, liquorice, liquorice, licorice and licorice was extracted with water to prepare a saunjatang. The lactic acid bacterium was inoculated at 1% (v / v) For 48 hours to prepare a fermented product of Lactobacillus acidophilus (Preparation Example 1). Among the various lactic acid bacteria, it was confirmed that the fermented product fermented with Lactobacillus amylophilus showed a marked change in the content of indices and other undefined compounds compared to that of Sangunjatang (Table 1), and Lactobacillus amylophilus or Lactobacillus amylophilus (Fig. 1 and Fig. 2). In addition, the fermentation efficiency of the fermented product was higher than that of the fermented product. In addition, the fermented product stabilized the mitochondrial cell membrane potential of the damaged brain cells and confirmed that it inhibited the production of reactive oxygen species, thereby confirming the brain cell protective activity (FIGS. 3 and 4).

This suggests that fermented lactic acid bacteria fermented with lactobacillus plantarum extract of ginseng, liquorice, liquorice, licorice and licorice may exhibit superior brain cell protective activity and thus may be useful for prevention or treatment of cerebral neuropathy will be.

The term "prevention" of the present invention encompasses all actions that inhibit or delay the neuropathy of the brain by administration of the pharmaceutical composition of the present invention comprising the fermentation product of lactic acid bacteria of an extract containing ginseng, liquorice, it means.

The term "treatment" of the present invention means any action that improves or alters the cranial nerve disease by the administration of the pharmaceutical composition.

The term "pharmaceutical composition" of the present invention means a preparation for the purpose of prevention or treatment of disease, and each of them may be formulated into various forms according to ordinary methods. For example, it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions and syrups, and diluents or excipients such as lubricants, wetting agents, flavors, emulsifiers, suspending agents, And may be formulated into parenteral formulations and used. It can also be formulated in the form of external preparations, suppositories, and sterile injection solutions.

In addition, depending on the respective formulations, pharmaceutically acceptable carriers may be prepared, including carriers known in the art, such as buffers, anhydrous agents, solubilizers, isotonic agents, stabilizers, and the like. As used herein, the term "pharmaceutically acceptable carrier" may mean a carrier or diluent that does not disturb the biological activity and properties of the compound being injected, without irritating the organism. The kind of the carrier that can be used in the present invention is not particularly limited, but any carrier conventionally used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more. The carrier may be a non-naturally occuring carrier.

The composition of the present invention may be administered in a pharmaceutically effective amount. The pharmaceutically effective amount means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. The effective dose level may be appropriately determined depending on the health condition of the patient, the type of disease, The activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including the combined or concurrent use of the drug, and other factors well known in the medical arts. In general, 0.01 mg to 5000 mg per kg body weight per day of the individual to be administered may be administered, but the dosage may be administered once or several times a day at a predetermined time interval according to the judgment of a doctor or pharmacist.

The content of the lactic acid bacterial fermentation product of ginseng, liquorice, liquorice, licorice and licorice contained in the composition is not particularly limited but may be 0.01 to 100% by weight, specifically 1 to 80% by weight based on the total weight of the composition have.

In addition, the pharmaceutical composition of the present invention may be used alone or in combination with other pharmacologically active compounds exhibiting the effect of preventing or ameliorating cranial neuropathy.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. In addition, they can be administered singly or multiply. It is important to take into account all of the above factors and to administer an amount that can achieve the maximum effect in a minimal amount without causing side effects, which can be readily determined by one skilled in the art.

The term "administration" of the present invention means introduction of the pharmaceutical composition of the present invention to a subject by any appropriate method, and the route of administration of the composition of the present invention is not limited to a variety of oral or parenteral routes ≪ / RTI >

The mode of administration of the pharmaceutical composition is not particularly limited and may be conventionally used in the art. As a non-limiting example of the mode of administration, the composition can be administered orally or parenterally, and the frequency of administration is not particularly limited, but may be administered once a day or several times in divided doses.

Another embodiment provides a method of preventing or treating a neurological disorder, comprising administering the pharmaceutical composition to a subject.

Herein, the definitions of the cranial nerve diseases, prevention and treatment are as described above.

The term "individual" of the present invention may refer to all animals, including humans, who have developed or are likely to develop cranial neuropathy. The animal may be, but is not limited to, a mammal such as a cow, a horse, a sheep, a pig, a goat, a camel, a nutrient, a dog, a cat,

The preventive or therapeutic method of the present invention may specifically include administering the composition in a pharmaceutically effective amount to a subject suffering from or at risk of developing a cerebral neuropathy. The method of administration is as described above.

The composition of the present invention may contain, for administration, a pharmaceutically acceptable carrier, excipient or diluent in addition to the above-described effective ingredients. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, Cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Suitable dosages of the compositions of the present invention may be adjusted depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the time and the like, and may be suitably selected by those skilled in the art. Specifically, the dosage of the fermented product of lactic acid bacteria of the extract containing ginseng, liquorice, liquorice, licorice, and licorice may be 50 to 500 mg / kg, but is not limited thereto.

Another aspect of the present invention provides a health functional food composition for improving cerebral neurological diseases, comprising fermented product of lactic acid bacteria of an extract containing ginseng, liquorice, liquorice, licorice and licorice.

Herein, the definitions of the fermented lactic acid bacteria, cranial nerve diseases and prevention of the extracts including ginseng, liquorice, liquorice and licorice are as described above.

The term "improvement" of the present invention means any action that at least reduces the degree of symptom associated with the condition being treated by administration of the composition of the present invention.

The term "food" of the present invention includes dairy products such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums, ice cream, various soups, drinks, tea, , A vitamin complex, a health functional food, and a health food, all of which include foods in a conventional sense.

Since the food composition of the present invention can be ingested routinely, it can be highly useful for health promotion purposes because it can expect a high cranial neuropathy improvement effect.

The term "functional food" as used herein means the same term as "food for special health use" (FoSHU). In addition to nutrition, It means food. Here, 'function (surname)' refers to the structure and function of the human body to obtain a beneficial effect for health use such as controlling nutrients or physiological action. The food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients which are conventionally added in the art. In addition, the above-mentioned food formulations can be manufactured without limitations as long as they are formulations recognized as food. The composition for food of the present invention can be manufactured in various formulations, and unlike ordinary drugs, it has advantages of being free from side effects that may occur when a natural product is used as a raw material for a long period of time, and is excellent in portability. The food of the invention can be taken as an adjuvant to enhance the improving effect of the cranial nerve diseases.

The health food refers to a food having an active health promotion or promotion effect compared with a general food, and a health supplement food refers to a food for health assistance. In some cases, the terms health functional foods, health foods, and health supplements are used.

Specifically, the health functional food is a food prepared by adding the compound of the present invention to a food material such as a beverage, a tea, a spice, a gum, confectionery, or the like, or encapsulated, powdered or suspended therein. But it has the advantage that there is no side effect that can occur when the medicine is taken for a long time by using the food as a raw material.

The food composition may further comprise a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier conventionally used in the art can be used.

In addition, the food composition may contain additional components that are commonly used in food compositions and can improve odor, taste, visual appearance, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid and the like. Minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu) and chromium (Cr); And amino acids such as lysine, tryptophan, cysteine, valine, and the like.

In addition, the food composition may contain at least one kind selected from the group consisting of preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate), disinfectants (such as bleaching powder and highly bleached white powder, sodium hypochlorite), antioxidants (butylhydroxyanisole (BHA) (Sodium nitrite), bleach (sodium sulfite), seasoning (sodium MSG glutamate, etc.), sweeteners (dicin, cyclamate, saccharin, etc.), coloring agents , Sodium, etc.), perfume (vanillin, lactones, etc.), swelling agents (alum, potassium hydrogen D-tartrate), emulsifiers, thickeners (foams), encapsulating agents, gum bases, foam inhibitors, solvents, And may include food additives. The additives may be selected and used in appropriate amounts depending on the type of food.

As an example of the food composition of the present invention, it can be used as a health beverage composition. In this case, various flavors or natural carbohydrates can be added as an additional ingredient like ordinary beverages. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose; Polysaccharides such as dextrin, cyclodextrin; Xylitol, sorbitol, erythritol, and the like. Sweeteners include natural sweeteners such as tau Martin and stevia extract; Synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 ml of the health beverage composition of the present invention.

In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acid, protective colloid thickener, pH adjuster, stabilizer, Alcohols or carbonating agents, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks, or 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 health beverage composition of the present invention.

The fermented product of Lactobacillus lactic acid bacteria obtained by fermenting Lactobacillus plantarum with an extract containing Panax ginseng, Lactobacillus japonica, Lactobacillus japonica and Lycopersicum, namely, Can be usefully used for the prevention or treatment of cranial nerve diseases.

1 is a graph showing the effect of increasing the brain cell survival rate of a fermented product of Lactobacillus japonica prepared according to an embodiment of the present invention. Survival rates of brain cells damaged by 50 μM of etoposide as a toxicant were measured. At this time, the white bar is a negative control not treated with toxic substance or test substance (Sagunjatang or its fermented product), the black bar is treated with a toxic substance and a positive control it means. SG showed that the experimental groups SG129, SG166 and SG238 treated with non-fermented Sogunzatang were fermented by Lactobacillus zeae , Lactobacillus plantarum and Lactobacillus amylophilus, respectively This means the experimental group treated with the fermentation product of Sagunjatang. The statistical significance of each result from one way ANOVA was as follows: * P <0.05, ** P <0.01 vs. Positive control.
FIG. 2 is a graph showing the effect of increasing the brain cell survival rate of the fermented product of Lactobacillus japonica prepared according to an embodiment of the present invention. Survival rates of brain cells damaged by 50 μM H ₂ O ₂ as a toxic substance were measured. At this time, the white bar is a negative control not treated with toxic substance or test substance (Sagunjatang or its fermented product), the black bar is treated with a toxic substance and a positive control it means. SG means an experimental group treated with fermented fermented by Lactobacillus ash, Lactobacillus plantarum and Lactobacillus amylophilus, SG129, SG166 and SG238, respectively. The statistical significance of each result from one-way ANOVA was as follows: ** P <0.01, *** P <0.001 vs. Positive control.
FIG. 3 is a graph showing the mitochondrial membrane potential protection effect of fermented Lactobacillus lactic acid fermented by Lactobacillus plantarum prepared according to an embodiment of the present invention. The degree of mitochondrial membrane potential of brain cells damaged by 50 μM of ethoxidic or H ₂ O ₂ as a toxic substance was expressed as a ratio of red / green fluorescence. At this time, the white bar is a negative control not treated with toxic substance or test substance (Sagunjatang or its fermented product), the black bar is treated with a toxic substance and a positive control it means. SG means an experimental group treated with non-fermented Sogunjatang, and SG166 means an experimental group treated with fermented fermented by Lactobacillus plantarum. The statistical significance of each result from one way ANOVA was as follows: * P <0.05, *** P <0.001 vs. Positive control.
FIG. 4 is a graph showing the effect of inhibiting ROS formation of fermented Lactobacillus lactic acid fermented by Lactobacillus plantarum prepared according to an embodiment of the present invention. The degree of ROS production of brain cells damaged by 50 μM of ethoposide or H ₂ O ₂ as a toxic substance was measured. At this time, the white bar is a negative control not treated with toxic substance or test substance (Sagunjatang or its fermented product), the black bar is treated with a toxic substance and a positive control it means. SG means an experimental group treated with non-fermented Sogunjatang, and SG166 means an experimental group treated with fermented fermented by Lactobacillus plantarum. The statistical significance of each result from one way ANOVA was expressed as: *** P <0.001 vs. Positive control.

Hereinafter, the present invention will be described in more detail with reference to examples. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Manufacturing example  1. Sangunjatang lactic acid bacteria Fermented  Produce

The lactic acid bacterial strain used in the present invention was subcultured twice in MRS medium at 37 ° C for 24 hours, and was prepared to have a concentration of 1 to 5 × 10 ⁷ CFU / ml.

Based on the Korean Pharmacopoeia and herbal medicine preparations, Sangunjatang was extracted with water containing the same amount of ginseng ( Panax ginseng CAMeyer), Atractylodes japonica Koidz., Poria cocos Wolf and licorice ( Glycyrrhiza glabra L.) . Thereafter, the mixture was adjusted to pH 8.0 with 1M sodium hydroxide, sterilized at 121 ° C for 15 minutes under high pressure, cooled at room temperature, and prepared as a medium to inoculate lactic acid bacteria. Each lactic acid bacterium was inoculated into the medium (Sagunzatang) at a concentration of 1% (v / v), and cultured at 37 ° C and aerobic condition for 48 hours. Then, the culture supernatant was obtained by centrifugation and lyophilized to prepare a lactic acid fermented product of the present invention.

Example  1. According to the type of lactic acid bacteria, Fermented  Analysis of composition change

Among the various kinds of lactic acid bacteria, we sought to select lactic acid bacteria suitable for the fermentation of Sungjangtang lactic acid bacteria.

Specifically, three kinds of lactic acid bacteria of # 129 ( Lactobacillus zeae ), # 166 ( Lactobacillus plantarum ), # 238 ( Lactobacillus amylophilus ) Each fermentation product was prepared according to Example 1.

Liquid chromatography (HPLC) analysis showed that the seven indicator components (Liquiritin, Ginsenoside Rg1, Liquiritigenin, Glycyrrhizin, Atactylenolide Ⅲ, Atlactolenolide Ⅱ, Atlactolenolide Ⅰ) were investigated. The weight of the fermented product of Sagunjatang and Sagunjatang was precisely weighed and dissolved at a concentration of 20 mg / ml using 100% methanol, filtered using a 0.2 ㎛ membrane filter, and injected with 20 쨉 l each. The HPLC (Dionex) used for the analysis was a pump (LPG 3X00), an autosampler (ACC-3000), a column oven (TCC-3000SD) and a diode array UV / VIS detector (DAD-3000RS) Respectively. The analysis was performed using a RS Tech C18 column (5 [mu] m, 250 x 4.60 mm) and the temperature of the column was maintained at 40 [deg.] C. As the mobile phase, water (A) and acetonitrile (B) containing 0.1% trifluoroacetic acid (TFA) were used and the flow rate of the mobile phase was 1.0 ml / min. The optimized mobile phase concentration gradient conditions are shown in Table 1 below.

Time (minutes) menstruum Mobile A (%) Mobile phase B (%) 0 90 10 25 65 35 40 55 45 55 30 70 63 30 70

At this time, the UV wavelength of the detector was set based on the maximum UV absorption wavelength value of each indicator component. The ginsenoside Rg1 is set to 200 nm, the liquiritin, liquiritianin, the art lactylenolide II and the art lactylenolide III are set to 220 nm, the glycyrrhizin is set to 254 nm, and the atactylthanolide I is set to 275 nm, Were measured.

As a result, fermented products (SG129, SG166, SG238) fermented with strains # 129, # 166, and # 238 were found to have six components Were changed. From the above results, it was confirmed that Lactobacillus ash, Lactobacillus plantarum or Lactobacillus amylopilas are lactic acid bacteria that can be usefully used for the production of the fermented product of Sajungjae.

Contents of 7 kinds of index components (mg / g) contained in the fermentation products of Sangunjatang (SG) Sample Liquiritin Ginsenoside Rg1 Liquiritigenin Glycyrrhizin Art Lactorenolide III Art Lactorenolide II Atactylenolide I SG 2.818 n.d. 0.327 1.631 0.534 0.117 0.060 SG129 0.036 nd 4.718 5.720 0.695 0.130 0.079 SG166 0.034 nd 4.904 6.186 0.694 0.124 0.082 SG238 0.031 nd 4.668 6.858 0.698 0.116 0.078

Example 2. Analysis of brain cell protection activity of fermented lactic acid bacteria

Example 2-1. Confirming the increase of cell survival rate

Through the above Example 1, we sought to select a fermented product exhibiting the best brain cell protection activity among the three kinds of fermented products of Sanggunjang (SG129, SG166, and SG238), in which distinct component changes were confirmed by lactic acid fermentation.

Specifically, the survival rate of human neuroblastoma SH-SY5Y cell line treated with the stress-inducing substance was measured after treatment with the Saccharogamushi fermentation product. First, SH-SY5Y cells were plated at a concentration of 4 × 10 ⁴ cells / ml in a 96-well plate in an amount of 100 μl per well and cultured for 12 hours. Subsequently, the cells were pretreated with either SG or SG109, SG166, or SG238, respectively, for 6 hours. Then, 50 μM of H ₂ O ₂ inducing oxidative stress was added to the cells, And treated with 50 μM of etoposide. After 24 hours, 10 μl of CCK solution was added to each well and incubated for 90 minutes. Cell viability was confirmed by measuring the absorbance at 450 nm using a microplate reader (SpectraMax i3, Molecular devices, CA, USA).

As a result, as shown in FIGS. 1 and 2, the fermented product of Lactobacillus plantarum (SG166) fermented at a relatively low concentration (250 / / ml) (SG129 or SG238) fermented with Lactobacillus or Lactobacillus amylopilas, respectively.

Example  2-2. Confirm mitochondrial membrane potential protection effect

The SG166 fermented with Lactobacillus plantarum (SG166) fermented with Lactobacillus plantarum showed the best effect on brain cell survival through Example 2-1. To verify the protective effect of SG166 on brain cells, the mitochondria The stabilization effect of mitochondrial membrane potential (MMP) was confirmed.

Specifically, SH-SY5Y cells were cultured in a 4-well slide chamber and then subjected to SG treatment or SG166 treatment for 6 hours, respectively. The cells were treated with 50 μM H ₂ O _2, 50 &lt; / RTI &gt; Then, to measure the mitochondrial cell membrane potential of the cells, the cells were stained with JC-1 (5,5 ', 6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolecarbocyanineiodide) The JC-1 staining solution was incubated for 15 minutes at a final concentration of 5 μg / ml, and the fluorescence intensity of JC-1 red fluorescence (excitation 490 nm), which indicates complete mitochondrial membrane potential, nm / emission 590 nm) and JC-1 green fluorescence (excitation 490 nm / emission 530 nm) indicating mitochondrial cell membrane disruption due to apoptosis were observed, respectively.

As a result, as shown in FIG. 3, it was confirmed that the SG166-treated cells increased the red / green fluorescence ratio compared to the cells treated with Sangunjatang (SG), indicating that toxicity such as H ₂ O ₂ or etoposide Regardless of the type of substance. From the above results, it can be seen that the fermented product of Sangunjatang stabilizes the mitochondrial cell membrane potential of brain cells damaged by toxic substances, thereby exhibiting brain cell protection activity.

Example  2-3. Active oxygen species  Confirming the production inhibitory effect

The SG166 fermented with Lactobacillus plantarum (SG166) showed the best effect on survival of brain cells through the above Example 2-1. In order to examine the protective effect of SG166 on brain cells, And inhibited the production of reactive oxygen species (ROS).

Specifically, SH-SY5Y cells were pretreated with either SG or SG166 for 6 hours each, and treated with 50 μM H ₂ O _{2 and} 50 μM ethocoside for 2 hours. Then, DCFDA, which is a fluorescent probe, was treated to a final concentration of 80 μM per well and cultured for 30 minutes. The intracellular ROS formed after the reaction was confirmed by measuring fluorescence (excitation 495 nm, emission 527 nm).

As a result, as shown in FIG. 4, SG166-treated cells showed a decrease in ROS production compared with SG treated cells, indicating that the type of toxic substances such as H ₂ O ₂ or etoposide Regardless of whether or not there is any effect. From the above results, it can be seen that the fermented product of Sungjun - tang showed the brain cell protection activity by reducing the oxidative stress caused by toxic substances.

Thus, the fermented product of Lactobacillus thunbergii exhibits superior brain cell protection effect as compared to that of Sangunjatang, and the fermented product of Lactobacillus plantarum can be more remarkably effective than that produced using other strains. Respectively.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Claims (11)

A pharmaceutical composition for the prevention or treatment of cerebral neurological diseases, comprising a fermented product of a lactic acid bacterium of Sagunajang extract, wherein the lactic acid bacterium is Lactobacillus plantarum .
[Claim 2] The pharmaceutical composition according to claim 1, wherein the extract is obtained by extracting a mixture comprising ginseng, liquorice, liquorice and licorice in a weight ratio of 0.5 to 1.5: 0.5 to 1.5: 0.5 to 1.5: 0.5 to 1.5.
The pharmaceutical composition according to claim 1, wherein the extract is extracted with water.
delete The pharmaceutical composition according to claim 1, wherein the lactic acid bacterium is inoculated at 0.5 to 1.5% (v / v) with respect to the extract.
The pharmaceutical composition according to claim 1, wherein the lactic acid bacterium is cultured in the extract for 43 to 53 hours.
The method of claim 1, wherein the neurological disorder is selected from the group consisting of Alzheimer's disease, Huntington's chorea, Parkinson's disease, senile dementia, frontotemporal dementia, vascular dementia, migraine, neuropathic pain, depression, neurodegeneration, schizophrenia, Wherein the disorder is a disorder, panic disorder, obsessive compulsive disorder, posttraumatic stress disorder, attention deficit hyperactivity disorder, eating disorder, fear, autism, memory disorder, attention disorder or arousal disorder.
2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition comprises the fermentation product in an amount of 0.0001 to 80% by weight based on the weight of the total composition.
A method for preventing or treating a neurological disorder, comprising administering to a subject other than a human a pharmaceutical composition according to any one of claims 1 to 3 and 5 to 8.
Wherein the lactic acid bacterium is Lactobacillus plantarum , wherein the lactic acid bacterium is Lactobacillus plantarum , wherein the lactic acid bacterium is Lactobacillus plantarum . delete

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