KR20130138037A - Lactic acid bacteria ferment of bojungikkitang and use thereof - Google Patents

Abstract

The prevent invention relates to a pharmaceutical composition containing lactic acid bacteria-fermented Bojungikkitang as an active ingredient for preventing or treating brain diseases, health functional food containing the fermented Bojungikkitang for preventing or alleviating brain diseases, and a method for prevention or alleviating brain diseases in animals except human using the fermented Bojungikkitang. The lactic acid bacteria fermented Bojungikkitang according to the present invention has antioxidative activity and neuroprotective activity, thereby being widely used for preventing or treating brain diseases as a form of pharmaceutical composition or health functional food. [Reference numerals] (AA) Survival rate (%);(BB) Bojungikkitang (Chinese traditional herbal medicine) stock solution (E0);(CC) Fermented Bojungikkitang (KERI 129);(DD) Fermented Bojungikkitang (KERI 125);(EE) Fermented Bojungikkitang (KERI 693)

Description

 [0001] The present invention relates to a fermentation product of Lactic acid bacteria and a fermentation product of Bojungikkitang and use thereof,

The present invention relates to a fermented product of a lactic acid bacterium having a brain nerve cell protecting activity and a use thereof. More specifically, the present invention relates to a fermentation product for preventing or treating a brain disease, The present invention also relates to a method for preventing or treating cerebral diseases by administering to a subject a fermented product of a lactic acid bacterium in a health functional food for preventing or ameliorating brain diseases,

Neuronal damage in the brain is directly responsible for neurodegenerative diseases such as Alzheimer's disease, Ischemia, Parkinson's disease, and Sclerosis. In particular, degenerative diseases related to the brain are diseases that cause a fatal problem in living as a human being, and are being actively studied and developed in terms of increasing social interest in the welfare of the elderly.

Although many studies have been carried out in the industry to find out the mechanism of brain damage, the causes of neuronal cell damage have not yet been clarified due to the complexity of neuronal cell damage mechanism. To date, it has been reported that excessive excitatory neurotransmitter release, generation of free radicals, inhibition of protein synthesis, gene expression abnormality and activation of immune response.

Recent reports have shown that unlike the existing theory that the nervous system is an immune privileged organ, it is associated with multiple sclerosis, trauma, Alzheimer's disease, stroke, particularly cerebral ischemic stroke and various brain injuries In addition, it has been reported that inflammation may occur in the central nervous system in the case of neurological diseases such as neurodegeneration, With this report, it is known that immunosuppression in the nervous system can inhibit brain cell damage after stroke, and a lot of research has been conducted in this regard.

For example, inhibition of cyclooxygenase-2 (COX-2) in cerebral ischemia has been shown to inhibit the formation of PGE ₂ and thereby inhibit glutamate, thereby protecting neuronal cells. In addition, many people suffering from arthritis or pain Has already been taking COX-2 inhibitors, and if epidemiological studies on the incidence of stroke in such patients are conducted, it may be a new target in future development of preventive and therapeutic agents for stroke (Iadecola C. et al., PNAS , ≪ / RTI > 30, pp1294-1299, 2001).

In addition, inflammation is also observed in the central nervous system, which is reported to be one of the main causes of neurodegeneration. The inflammatory response after cerebral ischemia is likely to be a target of a new stroke, particularly a cerebral ischemic agent, (Dirnagl U., et al., TINS., 22, pp391-397, 1999). Inhibition of neutrophil deposition may be mediated by a variety of mechanisms.

In addition, in neutrophils infiltrated into brain tissue, iNOS inducible nitric oxide synthase (iNOS) is induced, and NO is liberated from induced iNOS, resulting in brain cell damage. In addition, activation of NMDA receptor by glutamate (Vaughan CJ, et al., Stroke, 30: 1969-1973, 1999) have been reported to be associated with inhibition of iNOS expression.

However, to date, drugs with clear neuroprotective effects have not been known. Therefore, there is a desperate need to develop a drug having a new neuroprotective effect with low side effects and good efficacy.

On the other hand, Oriental medicine has been used for many years to prevent and treat various diseases. Herbal preparations have relatively few side effects in the human body and have various advantages due to the complex interactions of constituent herbal medicines. Thus, interest and demand for herbal preparations have been greatly increased. However, studies on herbal preparations that are safe and yet effective in the treatment of brain diseases are still lacking.

Therefore, the present inventors have conducted a lot of studies to find a substance capable of protecting brain cells in the course of brain diseases while suppressing the development of brain diseases. As a result, the fermented product obtained by inoculating lactic acid bacterium into a boiled- And thus the fermented product can be used to prevent or treat brain diseases. Thus, the present invention has been completed.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating brain diseases, which comprises a fermented product of lactic acid bacteria as an active ingredient in a boiled rice wort.

Another object of the present invention is to provide a health functional food for preventing or ameliorating a brain disease comprising a fermented product of a lactic acid bacteria in a boiled rice wort.

It is still another object of the present invention to provide a method for preventing or treating brain diseases using fermented lactic acid bacteria of boiled herbal tea.

As one embodiment for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating brain diseases, which comprises a fermented product of lactic acid bacteria in a boiled herbal extract as an active ingredient.

The term " Bocheongikgi-tang "of the present invention means that it helps the middle and helps the 气, It is a prescription to use as a physical strength enhancer. It is said that it can be used for tuberculosis, summer burning disease, fatigue after illness, improvement of weak condition, loss of appetite, cold of the weak, hemorrhoids, haemorrhage, uterine drainage, gastrointestinal tract, hyperhidrosis, etc. It is known that it is used as a physical strength enhancer when it is tired and has no appetite. However, it has not been disclosed that it can be used for prevention or treatment of brain diseases. The above-mentioned boiled-chowder can be produced according to a known method of manufacturing a boiled chowder which has been traditionally used in one room, and its constituent herb is Astragalus membranaceus Bunge), ginseng ( Panax ginseng CA Meyer), Atractylodes japonica Koidz.), licorice ( Glycyrrhiza glabra L.), Angelica gigas Nakai), dermis ( Citrus unshiu Markovich, Cimicifuga heracleifolia Kom., and Bupleurum falcatum Linne). For the purpose of the present invention, the above-mentioned boiled water hopper is preferably a fermented boiled water boiler fermented using lactic acid bacteria. The term " fermented liquid fermented lactic acid bacteria "of the present invention can be used in combination with" fermented liquid fermented hot water tanks ".

On the other hand, as the lactic acid bacteria that can be used for fermenting the above-mentioned boiled rice tanks, there may be used isolated strains or various commercially available lactic acid bacteria without limitation. But are not limited to, those selected from the group consisting of Lactobacillus sp., Streptococcus sp., Bifidobacterium sp., Lactococcus sp., Pediococcus sp. microorganisms of the genus Lactobacillus sp. or Leuconostoc sp. may be used. Preferably, microorganisms of the genus Lactobacillus can be used. More preferably, lactobacillus fermentum or lactobacillus casei strain can be used and, most preferably Lactobacillus Kasei KFRI 129 (Lactobacillus casei KFRI 129) , Lactobacillus buffer momentum KFRI 145 (Lactobacillus fermentum KFRI 145) , or Lactobacillus Kasei KFRI 693 (Lactobacillus casei KFRI 693) can be used.

The term "lactobacillus fermented product " of the present invention means a culture result obtained by inoculating a lactic acid bacterium with a culture medium in which lactic acid bacteria can grow and culturing. In the present invention, it means a dry powder of the culture supernatant obtained from a culture obtained by inoculating lactic acid bacteria into a boiled waxy tang.

The term "brain disease" in the present invention means all diseases occurring in the brain, which are caused by destruction of brain cells. Specifically, the brain diseases include neurological diseases such as neurodegenerative diseases (Alzheimer's disease, Huntington's chorea, Parkinson's disease), senile dementia, frontotemporal dementia, vascular dementia, migraine, neuropathic pain of central origin; Depression (endogenous, resistant, reactive or unexplained depression), nervous breakdown, schizophrenia, bipolar disorder, generalized anxiety disorder, stress-related disorder, panic disorder, obsessive compulsive disorder, posttraumatic stress disorder, attention deficit hyperactivity disorder, Mental disorders such as disorders (particularly bulimia, anorexia), phobias (especially agoraphobia), autism; Memory associated with neurological or psychiatric disorders, attention and attention deficit disorders.

The term "prevention" in the present invention means all the actions of inhibiting or delaying the onset of brain diseases by administration of a pharmaceutical composition for preventing or treating brain diseases comprising the fermented product of lactic acid bacteria as an active ingredient of the present invention do.

The term "treatment" in the present invention refers to all treatments for improving or alleviating symptoms of brain diseases by administering a pharmaceutical composition for preventing or treating brain diseases, which comprises the fermented product of lactic acid bacteria as an active ingredient, it means.

The active ingredient of the pharmaceutical composition for the prevention or treatment of brain diseases of the present invention may include a fermented product of lactic acid bacteria obtained as a result of inoculating lactic acid bacteria in a boiled herbal wax and inoculating the lactic acid bacteria, , Microorganisms such as Streptococcus sp., Bifidobacterium sp., Lactococcus sp., Pediococcus sp., Leuconostoc sp., And preferably microorganisms belonging to the genus Lactobacillus. More preferred are lactobacillus fermentum or lactose the Kasei Bacillus strain and most preferably Lactobacillus Kasei KFRI 129 (Lactobacillus casei KFRI 129), Lactobacillus fermentum KFRI 145 ( Lactobacillus fermentum KFRI 145), or Lactobacillus casei KFRI 693.

In a preferred embodiment of the present invention, it was confirmed that the fermented product fermented by inoculating Lactobacillus casei KFRI 693 into a fresh-water fermentation tank had an excellent antioxidative effect as compared with the crude fermented raw broth without fermentation (FIGS. 1 to 3). In addition, the fermented product fermented by inoculating Lactobacillus casei KFRI 129, Lactobacillus perfume KFRI 145, or Lactobacillus casei KFRI 693 into a boiled wingtip, protects HT22 cells, which are neuronal cells, from oxidative stress induced by glutamate (Fig. 4). As shown in Fig.

As a result of the behavior test of improving the cognitive ability of the animal using the fermented product fermented by inoculation with Lactobacillus casei KFRI 693 in the boiled rice goblet, It was confirmed that the cognitive ability was increased (Figs. 5 and 6).

Therefore, based on the above results, it was expected that the component changes in the biotransformation of the components of the Bojongwangtang by Lactobacillus casei KFRI 129, Lactobacillus fermentum KFRI 145, or Lactobacillus casei KFRI 693 , A pharmaceutical composition for preventing or treating a brain disease comprising the fermented product of lactic acid bacteria as an active ingredient of the present invention can be more effectively used for prevention or treatment of cerebral diseases than the undiluted crude broth, Thereby preventing or treating the symptom or complication induced thereby.

The pharmaceutical composition for the prevention or treatment of brain diseases of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent.

Examples of pharmaceutically acceptable carriers, excipients and diluents that may be used in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate , Gelatin, calcium phosphate, calcium silicate, calcium carbonate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil and the like.

The pharmaceutical composition of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral dosage forms, external preparations, suppositories, and sterile injectable solutions according to conventional methods. . When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. 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 or lactose, gelatin And the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups, and include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

In another aspect, the present invention provides a method for producing the fermented lactic acid bacteria.

The method for producing the lactic acid bacterium fermented product of the present invention includes a step of inoculating lactic acid bacteria into a boiled rice dung soup and fermenting the same. The lactic acid bacteria may be, but not limited to, microorganisms belonging to the genus Lactobacillus, Streptococcus, Bifidobacterium, Lactococcus, Pediococcus, and Leuconoste, preferably lactobacillus microorganisms , more preferably Lactobacillus buffer momentum or Kasei Lactobacillus strains, most preferably Lactobacillus Kasei KFRI 129 (Lactobacillus casei KFRI 129), Lactobacillus fermentum KFRI 145 ( Lactobacillus fermentum KFRI 145), or Lactobacillus casei KFRI 693 ( Lactobacillus casei KFRI 693) can be used. The fermentation conditions of the lactic acid bacteria are not particularly limited, but it is preferably carried out at a temperature of 35 to 45 DEG C and for a time of 1 to 3 days. More preferably, the lactic acid bacteria are inoculated into the boiled rice warts and cultured at 37 占 폚 for 48 days.

In a preferred embodiment of the present invention, in order to produce a fermented lactic acid bacterium of the present invention, the extract of the insecticidal extract is adjusted to pH 7.0 with 1 M sodium hydroxide, sterilized at 121 ° C for 15 minutes , 1% (v / v) Lactobacillus casei KFRI 129, Lactobacillus fermentum KFRI 145, or Lactobacillus casei KFRI 693 at a concentration of 1 to 5 x 10 ⁷ CFU / ml and incubated at 37 ° C for 48 hours for fermentation A culture was obtained, from which the culture supernatant was separated, and then lyophilized to prepare a lactic acid bacterium fermented product.

In another aspect, the present invention provides a health functional food for preventing or ameliorating a brain disease comprising a fermented product of lactic acid bacteria in a boiled rice hull.

Specifically, the fermented lactic acid bacteria according to the present invention may be added to foods or beverages for the purpose of preventing brain diseases. The type of food is not particularly limited, and examples thereof include various foods such as confectionery, bakery products, Drinks such as water, soft drinks and fruit drinks, gum, tea, vitamin complex, seasoning, and health functional foods. At this time, the amount of the fermented lactic acid bacteria in the food or beverage may generally be 0.01 to 15% by weight, preferably 0.1 to 5% by weight, of the total food weight in the case of the health functional food composition of the present invention, May be added in a proportion of 0.01 to 5.0 g, preferably 0.01 to 1.0 g based on 100 g. The health functional food of the present invention thus obtained contains the fermented product of the lactic acid bacteria of the present invention and is thus a food which can sufficiently utilize the preventive or therapeutic effect of the cerebrospinal fluid of the fermented product of lactic acid bacteria.

The health functional food of the present invention can be easily produced by adding the lactic acid bacteria fermented product of the present invention described above to the base material or by adding the lactic acid bacteria fermented product of the present invention described above after the production of the food . At this time, a taste and odor correction agent may be added as needed.

In addition, the health functional food is a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the health functional food of the present invention may contain natural fruit juice and fruit flesh for the production of fruit juice drinks and vegetable drinks. Such components may be used independently or in combination. The proportion of such additives is generally selected within the range of about 20 parts by weight or less per 100 parts by weight of the health functional food of the present invention.

In another aspect, the present invention provides a method for preventing or treating brain diseases. The method comprises the step of administering to a subject in need of treatment a lactic acid bacteria fermentation product of a pharmaceutically effective amount of a boiled fish wort.

The method of treatment according to the present invention does not mean that such treatment does not work in humans. In addition, in consideration of having a brain disease in which the symptom may be improved by administering the fermented product of the lactic acid bacteria of the present invention having a brain cell protecting effect in the case of a human, it can be sufficiently used in human therapy.

The term "individual" in the present invention means a mammal, including a human, a sheep, a sheep, a pig, a goat, a camel, Dogs, and humans. By administering the fermented product of lactic acid bacteria of the present invention having a brain cell protection effect to an individual, brain diseases can be effectively prevented and treated.

The term "administering" of the present invention means introducing a predetermined substance into a patient by any appropriate method, and the administration route of the conjugate can be administered through any conventional route so long as the drug can reach the target tissue . But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, rectal, and the like. In addition, the lactic acid fermentation product may be administered by any device capable of transferring the active substance to the target cell. Specifically, the fermented lactic acid bacteria of the present invention is preferably orally administered orally.

The term "pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the patient's sex, age, The activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The fermented lactic acid bacteria of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. The fermented lactic acid bacteria of the present invention may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The preferred dosage of the fermented lactic acid bacteria according to the present invention may be appropriately selected by those skilled in the art depending on the condition and body weight of the patient, degree of disease, drug form, route of administration and period of time. However, for the desired effect, the fermented lactic acid bacteria of the present invention can be administered at 50 to 1000 mg / kg per day, preferably 100 to 500 mg / kg per day. The administration may be carried out once a day or divided into several times.

According to one embodiment of the present invention, it was confirmed that the fermented product of lactic acid bacteria in the boiled rice tanks can protect brain cells from glutamate which damages brain cells, and that such protective effect is far superior to the effect of the boiled rice hull itself 3 and Fig. 4). In addition, according to another embodiment, as a result of the cognitive-behavioral experiment based on the mouse model, the fermented product of Lactobacillus acidophilus of Bohungwanggi was able to restore the cognitive ability damaged by the cholinergic blocker Scopolamine (Example 4, Fig. 5 And FIG. 6). Based on the results of this animal behavior test, it was found that fermented lactic acid bacteria can be used to treat brain diseases.

The fermented lactic acid bacteria of the present invention can be used in the form of a pharmaceutical composition or a health functional food, and thus can be widely used for the prevention or treatment of brain diseases.

FIG. 1 is a graph showing DPPH radical scavenging activity of fermented lactic acid bacteria obtained from boiled rice wort. (KFRI 693) showed DPPH radical scavenging activity in a concentration - dependent manner.
2 is a graph showing the hydrogen peroxide scavenging activity of fermented lactic acid bacteria obtained from boiled rice hulls. (KFRI 693) all showed concentration - dependent activity of hydrogen peroxide scavenging activity.
FIG. 3A is a graph comparing the amounts of ROS in the control group and the glutamate treatment, the fermentation product of the lactic acid bacteria treated with the boiling water. It was confirmed that when the glutamate treated cells were treated with the fermented product of Lactobacillus acidophilus (KFRI 693), the amount of ROS was reduced to a level close to that of the control group.
FIG. 3B is a graph comparing the amount of ROS when treating the fermented liquid lactic acid fermented with the boiled water extract solution and the boiled water boiler. It was confirmed that the cells treated with glutamate did not show a difference in the amount of ROS reduction when they were treated with the fermented liquid fermented product (KFRI 693) p < 0.05 versus glutamate treated cells; * p < 0.05 vs. E0 treated cells; ANOVA).
FIG. 4 is a graph showing the brain cell protective activity of the dried or fermented lactic acid bacteria. (* P <0.05 vs. glutamate treated cells; * p <0.05 vs. E0 treated cells; ANOVA) in the treatment of Lactobacillus fermentation broth, .
Figure 5a shows the improvement in spatial memory ability by recording the escape latency at the point of entry for the groups administered with the control (0.5% carboxymethylcellulose), Scopolaminem and Donepezil in the Morris water maze test. Scopolamine-treated rats show lower spatial memory ability than control rats.
Figure 5b shows the improvement in spatial memory ability by scopolamine administration in the Morris water maze test and the escape latency at the point of arrival at the point where the fermented product was administered at a concentration of 0.5 mg / When 200 mg / kg of KFRI 693 fermented with lactic acid bacteria (KFRI 693) was administered, the spatial memory capacity was significantly improved.
FIG. 6 is a graph showing changes in the time of movement of the mice to the avoidance box for the control group (0.5% carboxymethylcellulose), Scopolaminem, Donepezil, and Scopolamine in the passive avoidance test, The degree of improvement was evaluated. When the 200 mg / kg of KFRI 693 fermented with lactic acid bacteria (KFRI 693) was administered, the cognitive ability was significantly improved.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

Example  One: Boiled rice cake  And fermentation Boiled rice cake  Produce

To prepare the fermented product of lactic acid bacteria in the extract of Chinese cabbage according to the present invention, a lactic acid bacteria strain and a boiled herbal extract were prepared.

First, one day before inoculation, KFRI 748 ( Bifidobacterium thermophilum ) including 10 kinds of lactic acid bacteria (KFRI 129 Lactobacillus casei , KFRI 144 Lactobacillus plantarum , KFRI 145 Lactobacillus fermentum , KFRI 161 Lactobacillus amylophilus , KFRI 166 Lactobacillus ccurvatus , KFRI 229 Lactobacillus hilgardii , KFRI 238 Lactobacillus amylophilus , KFRI 658 Lactobacillus gasseri , KFRI 693 Lactobacillus casei ) were inoculated into each optimized culture medium for growth of strains. Lactobacillus strains were inoculated on MRS broth and Bifidobacterium strains were inoculated on Reinforced Clostridial Medium and incubated for 24 hours at 37 ° C in an incubator. This was used as lactic acid bacteria.

Next, Astragalus membranaceus Bunge), Panax ginseng ginseng CA Meyer), Atractylodes japonica Koidz.), licorice ( Glycyrrhiza glabra L.), Angelica gigas Nakai), dermis ( Citrus unshiu Markovich, Cimicifuga heracleifolia Kom., and Bupleurum falcatum Linne) was used in the ratio shown in Table 1 below to prepare a boiled rice tanks .

Composition of medicine Composition Hwanggi Ginseng Boiling licorice Angelica dermis riding Shiho Weight (g) 300 200 200 200 100 100 60 60

12.2L of water, which is 10 times the total weight of 1,220g of herbal medicines, was added to the herbal medicines, and after 1 hour of immersion, the hot water was extracted at 115 DEG C for 180 minutes. The extracted crude was filtered through a test sieve (106, Retsch, Haan, Germany) to prepare a raw stock solution.

 The mixture was adjusted to pH 7.0 with 1 M sodium hydroxide, sterilized at 121 占 폚 for 15 minutes under high pressure, and cooled at room temperature to prepare a medium to inoculate the lactic acid bacteria.

The culture broth was inoculated with the above-mentioned lactic acid bacteria at a concentration of 1% (v / v), and cultured at 37 ° C for 48 hours, followed by centrifugation to obtain a culture supernatant, and the resultant culture supernatant was lyophilized, Thereby producing the fermented product of lactic acid bacteria of the present invention.

Example  2: Boiled rice cake  Lactobacillus Fermented  Antioxidative effect

The DPPH radical scavenging activity, hydrogen peroxide (H ₂ O ₂ ) cleavage, and antioxidative activity of the lactic acid bacteria fermented product (KFRI 693, Lactobacillus casei 693 strain) The amount of intracellular ROS in the active and in vitro phases was measured.

Example  2-1: Boiled rice cake  Lactobacillus Fermented DPPH radical  Scavenging activity measurement

First, in order to measure the DPPH radical scavenging activity, 150 μl of the fermented broth of Lactobacillus lactic acid bacteria was treated with cells in a 96-well microplate. After this, DPPH was injected and reacted in a dark room for 30 minutes, and the absorbance was measured at 517 nm (FIG. 1). As a result, it was confirmed that the fermented product of lactic acid bacteria (KFRI 693) of DPPH radical scavenging activity was in a concentration dependent manner.

Example  2-2: Boiled rice cake  Lactobacillus Fermented  Measurement of hydrogen peroxide scavenging activity

Next, the hydrogen peroxide (H ₂ O ₂ ) scavenging activity was measured by measuring the scavenging activity of the active oxygen species in the cells. 20 μl of 10 mM H ₂ O ₂ and 100 μl of 0.1 M phosphate buffer solution (pH 5.0) were added to 80 μl of the stock solution and the lactic acid fermentation product solution, and the mixture was reacted at 37 ° C. for 5 minutes. Then, 1.25 mM ABTS (30 μl) and 30 μl of 1 U / ml peroxidase were added and reacted at 37 ° C for 10 minutes. After completion of the reaction, the reaction was applied to an enzyme-linked immunosorbent assay (ELISA) reader The OD value at 405 nm was measured and converted to the EDA (%) value (FIG. 2 and Table 2). FIG. 2 is a graph showing the hydrogen peroxide scavenging activity of the fermented product of lactic acid bacteria (KFRI 693) in the boiled-thawed wort, and Table 2 is numerical data thereof. As a result, as shown in Fig. 2, it was confirmed that the fermented product of Lactobacillus sp. In particular, it was confirmed that the IC ₅₀ of the hydrogen peroxide scavenging activity of the stock solution (E0) and the boiled lactic acid fermented product (KFRI 693) showed a higher activity than the stock solution (33%) of the fermented product. These results suggest that the fermented product of lactic acid bacteria (KFRI 693) is significantly higher in the hydrogen peroxide scavenging activity than that of the raw wastewater.

Bu hydrogen peroxide scavenging activity IC ₅₀ of ikgitang stock solution and lactic acid bacteria fermented product (KFRI 693) Experimental Sample Hydrogen peroxide scavenging activity IC ₅₀ ([mu] g / ml) The raw stock (E0) 2122.95 Bio-fermented liquid fermented lactic acid bacteria (KFRI 693) 1594.81

Example  2-3: Boiled rice cake  Lactobacillus Fermentation product  Intracellular ROS Volume measurement

In order to measure the intracellular ROS level, the HTNC cell line was inoculated into the medium and treated with the stock solution (E0) and the fermented product of lactic acid bacteria (KFRI 693) at a concentration of 100 μg / ml for 24 hours. After 1 hour, the medium was treated with glutamate and incubated at 37 DEG C for 8 hours. After incubation, 10 μM 2,7-dichlorofluorescin diacetate (DCF-DA) was added and reacted for 1 hour. After reaction with DCF-DA, the medium was removed and the cells were eluted with 1.0% Triton X-100 / PBS at 37 ° C for 10 minutes. Fluorescence was recorded at 490 nm at the exciation wavelength and the emission wavelength of 525 nm (Figure 3, Table 3 and Table 4). As a result, when the amount of ROS in the control (control) was regarded as 100%, it was confirmed that the amount of ROS increased by glutamate was closely related to that of the control group by fermenting KFRI 693 (100 μg / ml) ). FIG. 3B is a graph comparing the amounts of ROS in the treated raw broth and raw fermented broth of lactic acid bacteria (KFRI 693), and Table 4 shows the numerical data thereof. As shown in FIG. 3B, when the amount of ROS in the cells treated with glutamate alone was regarded as 100%, there was no difference in the case of the treatment with the stock solution of Ryoheyanggi. However, in the case of treating the fermented product (KFRI 693) %, Respectively. Thus, it was confirmed that the fermented product of lactic acid bacteria (KFRI 693) from the boiled zygok tuna had ROS reducing activity which was not observed in the stock solution.

Reduced activity of intracellular ROS in Lactobacillus fermented product (KFRI 693) Experimental conditions ROS amount (%) Control (control) 100 Glutamate treatment 118.23 Treatment of fermented lactic acid bacteria (KFRI 693) 108.54 Trolox treatment 66.27

Reduced activity of intracellular ROS in crude broth concentrate and fermented lactic acid bacteria (KFRI 693) Experimental conditions ROS amount (%) Glutamate treatment 100 Treated raw vessel liquid (E0) treatment 102.69 Treatment of fermented lactic acid bacteria (KFRI 693) 46.81

From these results, it was expected that the fermented product of lactic acid bacteria (KFRI 693) of the present invention could exhibit an antioxidative effect and reduce the damage of the stress caused by oxidation, and this antioxidative effect was confirmed by the biotransformation process Were expected to be exhibited by the components converted through.

Example  3: Boiled rice cake  Lactobacillus Fermented  Analysis of protective effects of neuronal cells

In order to confirm the neuroprotective effect of the fermented lactic acid bacteria of the present invention, the HT22 cells, which are widely used as a study on the mechanism of glutamate-induced brain diseases, are widely used.

Specifically, HT22 cells cultured at a culture condition of 5% CO ₂ and 37 ° C were mixed in a DMEM medium containing 10% FBS at a rate of 6.7 × 10 ⁴ cells / well in a 48-well plate, Lt; / RTI &gt; The cultured HT22 cells were treated with a stock solution of raw fish root or a boiled lactic acid fermented product (100 쨉 g / ml), and after 1 hour, 30 쨉 l of glutamate was treated. The lactic acid bacteria fermented product was prepared by using Lactobacillus casei KFRI 129, Lactobacillus perfumant KFRI 145, or Lactobacillus casei KFRI 693 strain. At this time, HT22 cells treated with trolox, a treatment for dementia, were used as a positive control group. The glutamate-treated HT22 cells were cultured for 24 hours, MTT was added thereto, and the reaction was allowed to proceed for 3 hours. OD values were measured at 570 nm using an ELISA reader. The OD values were measured for the brain cell protection Relative protection (%) indicating activity (Figures 4 and 5). FIG. 4 is a graph showing the brain cell protective activity of the dried or fermented lactic acid bacteria, and Table 5 is a table showing the numerical data thereof. As a result, as shown in FIG. 4 and Table 5, the broth fermented with lactic acid bacteria showed 8.2% (KFRI) at 100 ㎍ / ㎖, 129), 6.75% (KFRI 145), and 22.05% (KFRI 693), respectively.

Effect of Glutamate on Oxidative Stress of Lactic Acid Fermentation Solution (KFRI 693) Experimental conditions Survival rate (%) Control (control) 100 Glutamate treatment 0 Trolox treatment 97.78 Treated raw vessel liquid (E0) treatment 4.18 Treatment of fermented lactic acid bacteria (KFRI 129) 8.20 Treatment of fermented lactic acid bacteria (KFRI 145) 6.75 Treatment of fermented lactic acid bacteria (KFRI 693) 22.05

From the above results, it can be seen that the fermented product of lactic acid bacteria of the present invention shows a protective effect on brain cells. Therefore, it was expected that the fermented product of lactic acid bacteria could prevent or treat brain diseases, It was expected to be indicated by the converted components.

Example  4: Mouse model Using Boiled rice cake  Lactobacillus Fermented  Measuring the improvement of cognitive ability

Example 4-1: Morris water maze test

Morris water maze test was performed to measure the cognitive improvement effect of lactic acid fermented product (KFRI 693). As shown in the following Table 6, ICR mice were administered with 0.5% carboxymethlcellulose, Scopolamine, a cholinergic blocker causing cognitive impairment, or Donepezil, a dementia treatment, as a control group. As experimental groups, ferritin (KFRI 693) (Table 6).

Morris water maze test experimental group Experimental group Administration drug Group 1 (control group) 0.5% carboxymethylcellulose (CMC) Group 2 Scopolamine (1 mg / kg) Group 3 Donepezil (1 mg / kg) Group 4 (30 mg / kg) plus scopolamine (1 mg / kg) Group 5 (100 mg / kg) + scopolamine (1 mg / kg) Group 6 (200 mg / kg) plus scopolamine (1 mg / kg)

The CMC and the boar fermented water (KFRI 693) were administered orally and scopolamine was administered subcutaneously.

After 90 minutes, the control group received subcutaneous administration of normal saline and the other group received subcutaneous administration of scopolamine. Scopolamine was administered 30 minutes later and the experiment was carried out.

ICR (male, 4 week old) was adapted to the surrounding environment for one week and then tested. The environment was maintained at 20 ~ 23 ℃ and the contrast was adjusted to 12 hours (09:00 ~ 21:00).

Fill the water maze pool (90 cm in diameter, 40 cm in height) with water (20 ± 1 ° C) and make the platform opaque by mixing milk invisibly. The pool was divided into four sections and a platform (diameter 10 cm, height 26 cm) was placed 1 cm under the water in the middle of one section. Repeated 2 trials (trial 1 and trial 2) for 4 days and 20 minutes between trials. The point of arrival was different every day, and the position of the platform was fixed during the experiment. On the first day of the experiment, I swam for 60 seconds without the platform. When the mouse reaches the platform, it is allowed to stay on the platform for 10 seconds. If the platform can not be found for 120 seconds, the experiment is stopped and the mouse is placed on the platform. The degree of improvement in spatial memory ability was evaluated by recording the escape latency at the entry point (FIGS. 5A and 5B). As a result, as shown in FIG. 5A, the scopolamine-treated rats show lower spatial memory ability than the control rats. In FIG. 5B, when the 30 mg / kg or 100 mg / kg of the lactic acid fermented product (KFRI 693) was administered, the effect of improving the spatial memory capacity was not observed. However, when administered at 200 mg / kg, I could see improvement.

Example 4-2: Passive avoidance test

Passive avoidance test was performed to measure the cognitive ability improvement effect of lactic acid fermented product (KFRI 693). As a control, ICR mice were treated with 0.5% carboxymethlcellulose, Scopolamine, a cholinergic blocker causing cognitive impairment, or Donepezil, a treatment for dementia, as shown in the following Table 7. As a control group, ferritin (KFRI 693) (Table 7).

Passive avoidance test group Experimental group Administration drug Group 1 (control group) 0.5% carboxymethylcellulose (CMC) Group 2 Scopolamine (1 mg / kg) Group 3 Donepezil (1 mg / kg) Group 4 (30 mg / kg) plus scopolamine (1 mg / kg) Group 5 (100 mg / kg) + scopolamine (1 mg / kg) Group 6 (200 mg / kg) plus scopolamine (1 mg / kg)

The CMC and the boar fermented water (KFRI 693) were administered orally and scopolamine was administered subcutaneously.

After 90 minutes, the control group received subcutaneous administration of normal saline and scopolamine was subcutaneously administered to the other group. Scopolamine was administered 30 minutes later and the experiment was carried out. After 24 hours, the same experiment was carried out, and when there was no movement for 180 seconds, the experiment was stopped.

The passive avoidance system is equipped with a door for the mouse to move between the two acrylic boxes, and a stainless steel rod is placed on the floor so that electric shock can be applied. After one minute of adapting to the dark box for the first time, the lights were turned on through the lights and the noise caused the mice to move to the opposite avoiding box. After that, the mice were placed in a dark box again, and after 20 seconds, the lights were again turned on and caused noise, and the mice entered the evasive box and gave electric shock (0.1 mA / 10 g body) 2 seconds later. The time of movement of the mouse to the dodge box was measured by escape latency time (Fig. 6). As a result, as shown in FIG. 6, the scopolamine-treated rats show lower situational awareness than the control rats. In the case of administration of 30 mg / kg or 100 mg / kg of lactic acid fermented product (KFRI 693), improvement of cognitive ability was not observed compared to the control group. However, when administered at 200 mg / kg, .

These results show that the fermented lactic acid fermented product of the present invention can significantly improve the cognitive ability in an animal model, suggesting that it is effective in preventing or treating brain diseases.

Claims (8)

A pharmaceutical composition for the prevention or treatment of brain diseases, including the lactic acid bacteria fermentation product of Bojungikgi-tang as an active ingredient.
The method of claim 1,
Wherein the lactic acid bacteria fermented product is obtained by inoculating lactic acid bacteria into a boiled waxy fermentation broth and fermenting at a temperature of 35 ° C to 45 ° C for 1 to 3 days.
3. The method according to claim 1 or 2,
The lactic acid bacteria are Lactobacillus sp., Streptococcus sp., Bifidobacterium sp., Lactococcus sp., Pediococcus sp. , And Leuconostoc sp. Microorganisms.
4. The composition of claim 3, wherein the lactic acid bacteria is one selected from the group consisting of Lactobacillus casei KFRI 129, Lactobacillus permanent KFRI 145, or Lactobacillus casei KFRI 693.
The method of claim 1,
The brain diseases include Alzheimer's disease, senile dementia, prefrontal dementia, vascular dementia, Huntington's chorea, Parkinson's disease, migraine, depression, nervous breakdown, schizophrenia, bipolar syndrome, general anxiety disorder, stress-related disorders, panic disorder, OCD, Posttraumatic stress disorder, attention deficit hyperactivity disorder, dietary disorder, phobia, autism, attention disorder, arousal disorder or cognitive impairment.
The method of claim 1,
Wherein the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent.
Health functional foods for the prevention or improvement of brain diseases, including fermented lactic acid bacteria of Bojungikgi-tang.
A method for preventing or treating brain diseases in animals other than humans, comprising administering a pharmaceutically effective amount of a lactobacillus fermentation product of Bojungikgi-tang to an individual in need thereof.

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