KR20230106932A - Composition for protection of brain neuronal cells comprising fermented garlic - Google Patents

Abstract

The present invention relates to a composition for improving diseases accompanying damage to brain nerve cells containing fermented garlic as an active ingredient, which can improve autoimmune diseases of the nervous system.

Description

Composition for protection of brain neuronal cells comprising fermented garlic containing fermented garlic as an active ingredient

The present invention relates to a composition containing fermented garlic as an active ingredient to prevent or improve diseases accompanying damage to brain nerve cells.

Immunity is one of the body's self-protection systems against all external macromolecules that invade or inject into living tissues. As a major component of the immune system, there are lymphocytes, which are made in the bone marrow and circulate through the blood to lymphoid tissue or organs, mainly lymph nodes, spleen, and tonsils. white blood cells that When B cells are stimulated by an appropriate antigen, they proliferate rapidly and form clones that produce special antibodies (immunoglobulin) to neutralize the antigen. Antibodies produced by B cells circulate in body fluids and carry out humoral immunity. T cells are produced in the thymus and migrate to lymphoid tissues, where they are responsible for cell-mediated immunity by directly attacking antigens. One of the most important characteristics of any normal organism is the ability to recognize, respond to, and eliminate many non-self antigens, while not reacting detrimentally to self-constituting antigenic substances. that it has Such non-response of the body to self-antigens is referred to as immunologic unresponsiveness or tolerance. If there is a problem in inducing or maintaining such self-tolerance, an immune response against self-antigens occurs, and as a result, a phenomenon of attacking one's own tissues occurs, resulting in an autoimmune disease.

The autoimmune disease is caused when the host's immune response fails to distinguish foreign antigens from its own molecules (self antigens) and induces an abnormal immune response. In autoimmune disease, the immune response to self-molecules deviate from the normal state of self-tolerance, which involves the destruction of T cells and B cells capable of responding to self-antigens and occurs early in the development of the immune system during life. has been prevented by the incident. Cell surface proteins that play a pivotal role in the regulation of the immune response through their ability to bind and present processed peptides to T cells are major histocompatibility complex (MHC) molecules. MHC exists in two forms, class I or class II, both encoded within a single gene complex. Autoimmune diseases have a marked genetic association with specific alleles of MHC class I or class II genes.

Treatment of autoimmune diseases is a disease that occurs as a result of an abnormal reaction to own cells, so drugs that suppress autoimmune functions are mainly used. However, there are many side effects, so it is difficult to use continuously, and recurrence is not sufficiently prevented, so there is a limit to treatment. There are various immunotherapies, but there is no case that has been recognized as a method that shows clear therapeutic efficacy. Various animal models of human autoimmune diseases exist, which are being used to test possible treatments.

Nerve cells transmit bio-electrical signals, and at this time, myelin, which serves as an insulator (functions like a covering of an electric wire), surrounds and protects the nerve cells to prevent the leakage of electrical signals. Recently, due to factors such as incorrect eating habits and environmental pollution, myelin sheaths in nerve cells are damaged, electrical signals are leaked, and diseases in which signal transmission problems occur are increasing. It is a representative disease, and it is a kind of autoimmune disease.

The multiple sclerosis (MS) is a demyelinating disease caused by repeated inflammation in myelin and axon of nerve cells. Multiple sclerosis occurs mainly in the younger age group between the ages of 20 and 40 and repeats relapsing and remitting, and as relapses repeat, symptoms do not improve completely and disability remains [(F.D. Lublin, S.C. Reingold, Neurology 46(4) (1996) 907-11), (J. Goverman, Nat Rev Immunol 9(6)(2009) 393-407). Currently, about 2.3 million patients around the world are suffering from this disease, but since it is an autoimmune disease with an unclear pathogenesis, they are suffering from prescription medications that relieve symptoms when it develops. Multiple sclerosis is caused by an abnormality of the autoimmune system, in which lymphocytes such as T cells and macrophages infiltrate the white matter around the ventricles and spinal cord, and these immune cells form myelinated oligodendrocytes. Demyelination of the brain and spinal cord proceeds by destroying myelin basic protein (MBP) and myelinoligodendrocyte glycoprotein (MOG) associated with cells (oligodendrocyte) [J.Patel, R. Balabanov, Int J Mol Sci 13(8), (2012), 10647-59].

Multiple sclerosis is characterized by a variety of signs and symptoms, such as worsening after remission and recovery of symptoms, the most common type being relapsing-remitting MS. As the disease progresses, damage to the nervous system accumulates and recovery becomes delayed, and it continues to deteriorate in the same way as a chronic degenerative disease. This is called secondary progressive MS. If it progresses gradually from the beginning without obvious recurrence, it is called primary progressive MS. As a representative treatment for multiple sclerosis, an oral drug approved by the FDA (dimethyl fumarte, Tecfidera) is used.

Therefore, there is a need for natural substances capable of improving or preventing diseases accompanying damage to brain nerve cells, such as multiple sclerosis.

Republic of Korea Patent No. 2328668 Republic of Korea Patent No. 2216651

An object of the present invention is to provide a pharmaceutical composition capable of preventing diseases accompanying damage to brain nerve cells, including fermented garlic.

In addition, another object of the present invention is to provide a food composition that can improve or prevent diseases accompanying damage to brain nerve cells, including fermented garlic.

In order to achieve the above object, the pharmaceutical composition for preventing diseases accompanying damage to brain nerve cells of the present invention may contain fermented garlic as an active ingredient.

The garlic fermented product may be fermented garlic pulverized material with a strain culture medium.

The strain culture medium is Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus casei ( Lactobacillus casei ) , At least one strain selected from the group consisting of Lactobacillus pentosus , Lactobacillus sakei , Leuconostoc mesenteroides and Lactobacillus buchneri is contained. can

The fermentation may be performed for 40 to 60 hours at 35 to 45 °C.

The fermentation may be performed by mixing 0.02 to 0.09 parts by weight of the strain culture medium with respect to 100 parts by weight of the crushed garlic.

The disease accompanying damage to the brain nerve cells is an autoimmune disease, and the autoimmune disease is in the group consisting of multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, ascending myelitis, central myelitis, and chronic inflammatory demyelinating polyneuropathy. It may be one or more selected species.

In addition, the food composition for improving or preventing diseases accompanying damage to brain nerve cells of the present invention for achieving the above other object may contain fermented garlic as an active ingredient.

The composition for improving or preventing diseases accompanying damage to brain nerve cells, including the fermented garlic product of the present invention, can improve short-term memory ability and behavioral ability, and can reduce oxidative stress and inflammatory response.

In addition, the composition of the present invention reduces demyelination and increases the expression level of PLP protein constituting myelin, thereby improving or preventing diseases accompanying damage to brain nerve cells, particularly multiple sclerosis.

Therefore, the composition of the present invention contains fermented garlic as an active ingredient to improve or prevent diseases accompanying damage to brain nerve cells, and is therefore effective in manufacturing medicines and foods.

1 is a graph measuring the SAC content of fermented garlic prepared according to Example 1 of the present invention and pulverized garlic prepared according to Comparative Example 1.
2a and 2b are Y-shaped mazes for measuring the Y maze test using a control group, CPZ induction group, DMF group, Example 1 group and Comparative Example 1 group.
Figure 3 is a graph measuring Y maize test using a control group, CPZ induction group, DMF group, Example 1 group and Comparative Example 1 group.
Figure 4 is a graph measuring the rotarod test using a control group, CPZ induction group, DMF group, Example 1 group and Comparative Example 1 group.
Figure 5 is a graph measuring lipid oxide in brain tissue using a control group, CPZ induction group, DMF group, Example 1 group and Comparative Example 1 group.
6 is a graph measuring protein oxide in brain tissue using a control group, CPZ induction group, DMF group, Example 1 group and Comparative Example 1 group.
Figure 7 is a graph measuring the amount of nitric oxide (NO) produced using a control group, CPZ induction group, DMF group, Example 1 group and Comparative Example 1 group.
Figure 8a is a Western blot showing the expression of TNF-α protein in brain neurons of a control group, CPZ-induced group, DMF group, Example 1 group, and Comparative Example 1 group; Figure 8b is a graph quantitatively showing the expression level measured in Figure 8a.
Figure 9a is a Western blot showing the expression of IL-1β protein in brain neurons of the control group, CPZ-induced group, DMF group, Example 1 group, and Comparative Example 1 group; 9b is a graph quantitatively showing the expression level measured in FIG. 9a.
Figure 10a is a Western blot showing the expression of NF-κB protein in brain neurons of a control group, a CPZ-induced group, a DMF group, Example 1 group, and Comparative Example 1 group; 10b is a graph quantitatively showing the expression level measured in FIG. 10a.
Figure 11a is a Western blot showing the expression of Nrf2 protein in brain neurons of a control group, a CPZ-induced group, a DMF group, Example 1 group, and Comparative Example 1 group; 11b is a graph quantitatively showing the expression level measured in FIG. 11a.
12a is a diagram showing the expression of myelin in brain tissues of a control group, a CPZ-induced group, a DMF group, Example 1 group, and Comparative Example 1 group; 12b is a graph quantitatively showing the expression level measured in FIG. 12a.
13A is a diagram showing the expression of GFAP in brain tissues of a control group, a CPZ-induced group, a DMF group, Example 1 group, and Comparative Example 1 group; 13b is a graph quantitatively showing the expression level measured in FIG. 13a.
14A is a diagram showing the expression of PLP in brain tissues of a control group, a CPZ-induced group, a DMF group, Example 1 group, and Comparative Example 1 group; 14b is a graph quantitatively showing the expression level measured in FIG. 14a.

The present invention relates to a composition containing fermented garlic as an active ingredient to prevent or improve diseases accompanying damage to brain nerve cells.

The disease accompanying damage to the brain nerve cells is an autoimmune disease, and the autoimmune disease is in the group consisting of multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, ascending myelitis, central myelitis, and chronic inflammatory demyelinating polyneuropathy. Selected 1 or more types are mentioned.

Hereinafter, the present invention will be described in detail.

The composition capable of preventing or improving diseases accompanying damage to brain nerve cells of the present invention contains fermented garlic as an active ingredient.

A representative component of the garlic is a sulfur compound called allin. Allin has no scent, but as soon as the garlic tissue is damaged, it interacts with an enzyme called allinase in the garlic tissue to become allicin, a self-defense substance. The allicin has a strong bactericidal and antibacterial action to kill food poisoning bacteria and to kill Helicobacter pylori bacteria that cause gastric ulcers, as well as help digestion, increase immunity, and lower cholesterol levels. In addition, garlic contains various sulfur compounds other than allicin, which also acts as an antioxidant to remove active oxygen.

Garlic fermented product of the present invention is inoculated with a strain culture solution to garlic pulverized by mixing garlic and solvent, and then 35 to 45 ℃, preferably 37 to 40 ℃ for 40 to 60 hours, preferably 45 to 50 hours fermented during

Garlic and the solvent used in preparing the garlic powder are mixed in a weight ratio of 1: 1-20, preferably 1: 5-10. Examples of the solvent include water, lower alcohol having 1 to 4 carbon atoms, ethylene glycol, ethyl ether, or a mixture thereof; Examples of the lower alcohol include aqueous solutions of 20 to 99% by volume of methanol, ethanol, butanol or propanol. Preferably, the solvent includes water in that it can excellently prevent or improve diseases accompanying damage to brain nerve cells.

If the weight ratio of the garlic and the solvent is out of the above range, the culture may not be performed smoothly, so that the effect on diseases accompanying damage to brain nerve cells may be ineffective or significantly low.

Strains in the culture medium include Lactobacillus plantarum, Lactobacillus casei , At least one selected from the group consisting of Lactobacillus pentosus , Lactobacillus sakei , Leuconostoc mesenteroides and Lactobacillus buchneri , In the case of using strains other than the above strains, there may be no or low effect on diseases accompanying damage to brain nerve cells.

If the temperature and time during fermentation are less than the lower limit, the content of the physiologically active substance of garlic may be lowered, and if it exceeds the upper limit, the desired effect may not be exerted at all due to the decomposition of the physiologically active substance.

The concentration of the strain culture medium is 1X10 ⁷ to 1X10 ¹⁰ CFU, preferably 1X10 ⁸ to 1X10 ⁹ CFU. If the concentration of the culture medium is out of the above range, the desired effect cannot be expected.

The strain culture solution is used in an amount of 0.02 to 0.09 parts by weight, preferably 0.03 to 0.05 parts by weight, based on 100 parts by weight of the crushed garlic. If the content of the strain culture solution is less than the lower limit, fermentation may not be performed, and if it exceeds the upper limit, the effect on diseases accompanying damage to brain nerve cells may be significantly lowered.

The fermented garlic product of the present invention can be used in food compositions as well as pharmaceutical compositions.

In a broad sense, the fermented garlic product of the present invention is meant to include processed products of fermented garlic formulated to be administered to animals, such as fermented garlic powder. Although the present invention proceeds with fermented garlic, it will be expected by those skilled in the art that the desired effect can be achieved even in the form of a processed product of fermented garlic.

On the other hand, in the present specification, the term 'contained as an active ingredient' means containing an amount sufficient to achieve the efficacy or activity of fermented garlic. For example, the garlic fermented product is used at a concentration of 10 to 1500 μg/ml, preferably 100 to 1000 μg/ml. Since fermented garlic is a natural product and does not have side effects on the human body even when administered in excess, the upper limit of the amount of fermented garlic included in the composition of the present invention can be selected and implemented by those skilled in the art within an appropriate range.

The pharmaceutical composition of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to the above active ingredients, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, A lubricant or flavoring agent may be used.

The pharmaceutical composition may be preferably formulated as a pharmaceutical composition by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration.

Formulations of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

In compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare formulations for injections such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

Furthermore, using a method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field, it can be preferably formulated according to each disease or component.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., preferably oral administration. .

The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, route of administration, excretion rate and reaction sensitivity, A ordinarily skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.001-10 g/kg.

The pharmaceutical composition of the present invention may be prepared in unit dose form by formulation using a pharmaceutically acceptable carrier and/or excipient, or prepared by being placed in a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

In addition, the present invention provides a food composition for improving or preventing diseases accompanying damage to nerve cells containing fermented garlic as an active ingredient.

The food composition according to the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, chewing gum, ice cream, vitamin complexes, and health supplements. etc.

The food composition of the present invention may include not only fermented garlic as an active ingredient, but also ingredients commonly added during food preparation, such as proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. . Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides such as conventional sugars such as dextrins and cyclodextrins and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is made into drinks and beverages, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be further included in addition to the fermented garlic of the present invention. .

The present invention provides a health functional food comprising a food composition for improving or preventing diseases accompanying damage to nerve cells, which contains the fermented garlic as an active ingredient. Health functional food is a food made by adding fermented garlic to food materials such as beverages, teas, spices, chewing gum, confectionery, etc., or made into capsules, powders, suspensions, etc. However, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time using food as a raw material. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The amount of heat-processed processed salt added to such health functional foods varies depending on the type of target health functional food and cannot be uniformly defined, but it can be added within a range that does not impair the original taste of the food, and is usually 0.01 for the target food. to 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of pills, tablets, capsules or beverages.

Hereinafter, preferred embodiments are presented to aid understanding of the present invention, but the following examples are merely illustrative of the present invention, and various changes and modifications are possible within the scope and spirit of the present invention. It is obvious to those skilled in the art, It goes without saying that these variations and modifications fall within the scope of the appended claims.

Example 1. Garlic fermented product

Garlic and water were mixed in a weight ratio of 1: 2 to sterilize the crushed garlic powder, and then mix 0.03 parts by weight of Lactobacillus plantarum BR4 culture medium (1X10 ⁹ CFU) with 100 parts by weight of the sterilized garlic powder. Fermented garlic was obtained by stationary fermentation at 37 ° C. for 48 hours.

The cultured fermented garlic is used after being heat-treated and filtered.

Comparative Example 1. Garlic Powder

Garlic and water were mixed in a weight ratio of 1:2 to obtain sterilized garlic pulverized material by sterilizing the pulverized garlic pulverized material.

<Test Example>

Test Example 1. Measurement of S-allyl-L-cysteine (SAC) content

Samples prepared for SAC quantitative analysis were filtered with a 0.45 μm membrane filter, then HPLC (Agilent 1260 Infinity, Agilent Technologies, CA, USA) and C18 column (AccucoreTM XL C18 column, 4.6 X 150 mm, 4 μm, Thermo Fisher Scientific , MA, USA). HPLC analysis conditions used mobile phase solvent A 20 mmol/L sodium dihydrogenphosphate and 10 mmol/L heptanesulfonic acid, pH 2.3) and B (acetonitrile mixing with eluent A, pH 2.3 ratio 1:1, v/v)), A :B starting with an initial ratio of 100:0, 68:32 at 5 minutes, 60:40 at 15 minutes, 0:100 at 18 minutes, 100:0 at 20 minutes, and 100:0 at 30 minutes. A gradient system with a ratio maintained for 30 minutes was used. A UV detector (208 nm) was used as a detector, and 10 μL of the reaction solution was injected and analyzed while maintaining a flow rate of 0.8 mL/min and a column temperature of 38 °C. The SAC content was measured by preparing a calibration curve using S-allyl-L-cysteine as a standard material.

The SAC does not exist in raw garlic, but is a sulfur compound produced from γ-glutamyl-S-allyl-cysteine (GSAC) by γ-glutamyl trans-peptidase (γ-GTP) during garlic processing.

1 is a graph measuring the SAC content of fermented garlic prepared according to Example 1 of the present invention and pulverized garlic prepared according to Comparative Example 1.

As shown in FIG. 1, S-allyl-L-cysteine (SAC), which is known to help prevent multiple sclerosis, was added to the garlic powder of Comparative Example 1 in which the fermented garlic product of Example 1 was not fermented. It was confirmed that it contained about 2.5 times more than that.

animal testing

6-week-old normal rats (male, wistar rats, weight 250±20 g) were subjected to an adaptation period of 1 week under a temperature of 22±1 ℃, humidity of 55±3%, and a light/dark cycle of 12 hours, and then control group and CPZ induction group , DMF group, Example 1 group and Comparative Example 1 group, except for the control group, 0.4 wt% Cuprizone was added to the feed for 5 weeks. The Cuprizone, as a strong copper chelator, induces cell damage of oligodendrocytes, resulting in demyelination and multiple sclerosis.

Animal experiments were conducted after receiving approval from the Animal Experimentation Ethics Committee at Mokpo National University. For the experiments, mice were sacrificed by euthanasia by carbon dioxide coma with a respiratory anesthetic, and then brain tissue and serum were collected. For histopathological evaluation of brain tissue, lesions were taken and fixed in 10% neutral formalin, and tissue and serum samples were stored at -70 °C until use in experiments.

-mouse-

Control: Intake of a diet containing 1.3% by weight of common salt (based on NaCl)

CPZ induction group: dietary intake containing 0.4 wt% Cuprizone

DMF group: dietary intake containing 0.4 wt% Cuprizone and oral administration of 15 mg/kg dimethyl fumarate

Example 1 Group: Dietary intake containing 0.4 wt% Cuprizone and oral administration of 5.2 g/kg of fermented garlic of Example 1

Comparative Example 1 Group: Dietary intake containing 0.4 wt% Cuprizone and oral administration of 5.2 g/kg of garlic powder of Comparative Example 1

The CPZ induction group, DMF group, Example 1 group and Comparative Example 1 group are Cuprizone-induced multiple sclerosis animal models.

Test Example 2. Y maze test (cognitive impairment)

The Y maze test is an experimental method for evaluating instantaneous spatial cognition in the form of short-term memory. Mice of each group were placed in a Y-shaped maze (FIG. 2) consisting of three branches (each branch set to A, B, and C). For the next 8 minutes, record the movement path of the passage (arm) where the mouse entered (even if it re-entered the branch it entered). If you enter three different aisles (arms) in turn, you get 1 point (actual alternation), and no points if you do not enter in succession.

Spontaneous alternation was recorded by dividing the above measured score by the total number of crossings minus 2 and entering the conversion paper again with probability.

Multiple sclerosis is a demyelinating disease caused by repetitive inflammation in the central nervous system, and symptoms include movement disorders, visual impairment, memory impairment, and cognitive dysfunction. The Y-maze test is a representative method for evaluating the short-term memory improvement effect, which is measured using a Y-shaped arm and expressed as a spontaneous alternation ratio.

Figure 3 is a graph measuring Y maize test using a control group, CPZ induction group, DMF group, Example 1 group and Comparative Example 1 group. Values are represented as mean ± SD (n=6). **P<0.001 vs. control group

As shown in FIG. 3 , it was confirmed that the spontaneous alternation rate significantly decreased in the CPZ induction group and the DMF group (positive control group) compared to the control group. On the other hand, since it was confirmed that Example 1 group had a higher spontaneous alternation rate than the CPZ induction group, DMF group, and Comparative Example 1 group, it was determined that Example 1 group showed an effect of improving short-term memory in the multiple sclerosis induced model.

Test Example 3. Rotarod test (Rotarod, behavior disorder)

The rotarod test is an experimental method to evaluate the movement and balance of an experimental animal by having the experimental animal walk on a rotating cylinder (rotarod). After applying the mouse to the rotarod for 1 week, the speed of 6 ~ 30 rpm The mouse's behavior (balance ability, grip strength, etc.) was observed for 5 minutes, and the time the experimental animal stayed on the Rotard was measured and expressed.

Figure 4 is a graph measuring the rotarod test using a control group, CPZ induction group, DMF group, Example 1 group and Comparative Example 1 group.

As shown in FIG. 4, CPZ induction group > DMF group ≥ Comparative Example 1 group > Example 1 group ≥ control group quickly fell in order, confirming that the behavioral disorder was high.

That is, according to the results of Test Examples 2 and 3, the fermented garlic product of Example 1 was shown to improve short-term memory and behavioral ability.

Test Example 4. Measurement of oxidative stress-related factors

4-1. Measurement of lipid oxides in brain tissue: Supernatant obtained by adding 1 mL of 0.01 M Tris-HCl buffer (pH 7.4) to brain tissue (0.15 g) of each diet group, homogenizing it, and centrifuging it at 4 ° C and 5,000Xg for 10 minutes 1 mL of 0.15 M Tris-HCl buffer (pH 7.4) and 300 μL of 10 mM phosphate buffer (KH ₂ PO ₄ ) were added to 100 μL, followed by reaction at 37 ° C. for 20 minutes using a shaking water bath. . After the reaction was completed, 0.5 mL of 0.6 M HCl containing 20% trichloracetic acid and 0.5 mL of 1 M NaOH solution containing 0.67% thiobarbituric acid were added and mixed, followed by reaction at 95 °C for 15 minutes. After cooling the reaction solution, it was centrifuged at 5,000Xg for 10 minutes, and the absorbance was measured at 532 nm. Then, these absorbance values were measured using the standard curve of the prepared standard solution (malondialdehyde, MDA) to determine the lipid peroxide content in brain tissue. converted into

4-2. Protein oxide measurement in brain tissue: Protein oxide (protein carbonyl) content was measured by adding 1.5 mg of protein obtained from brain tissue to 100 μL of 2 M HCl solution containing 10 mM DNPH, followed by reaction at room temperature and dark room for 1 hour at 15-minute intervals. was vortexed. 1 mL of ice-cooled 10% trichloracetic acid (w/v) was added to the reaction solution, mixed, and centrifuged at 3,000Xg for 10 minutes to obtain protein pellets in ethanol/ethyl acetate (1:1, v /v) After washing with 2 mL of the solution three times, it was dissolved in 1.5 mL of 6 M guanidine hydrochloride (pH 2.3), reacted for 10 minutes at 37 ° C, and absorbance was measured at 370 nm. The absorbance value was converted into the protein carbonyl content of brain tissue by dividing the molar extinction coefficient (ε = 2.2X10 ⁴ /cmㅇM) of DNPH.

4-3. Measurement of nitric oxide (NO) production: NO concentration was measured in the plasma of each diet group using the Griess assay. For the measurement method, after mixing plasma and zinc sulfate (15 g/L), the supernatant was taken by centrifugation at 15,000 RPM for 5 minutes. 100 μL each of the supernatant and vanadium trichloride (8 mg/mL) were mixed in a 1:1 ratio. Next, 50 μL of each of Griess reagent 2% sulfanilamide (in 5% phosphoric acid) and 0.1% NED was mixed, stored at 37 ° C for 30 minutes, and absorbance was measured at 540 nm. These absorbance values were converted into NO content in plasma using a standard curve of a prepared standard solution (NaNO ₂ ).

Free radicals are generated by cell metabolism, imbalance of antioxidant defense system, and external factors. Excessive free radicals cause oxidative stress, which oxidizes proteins and lipids in body tissues and damages DNA, causing cancer. It is known to induce various pathological conditions such as aging, aging, etc. The iron component contained in Cuprizone causes fenton reaction to decompose hydrogen peroxide (H ₂ 0 ₂ ) in the human body and generates hydroxyl radicals, which are highly active toxic substances, causing oxidative stress. After orally administering fermented garlic to an animal model of Cuprizone-induced multiple sclerosis, lipid peroxide, protein oxide, and nitric oxide production in brain tissue were analyzed and shown in FIGS. 5 to 7 .

Figure 5 is a graph measuring lipid oxide in brain tissue using a control group, CPZ induction group, DMF group, Example 1 group and Comparative Example 1 group. Values are represented as mean ± SD (n=6). **P<0.001 vs. control, *P<0.05 vs. control, ++P<0.001 vs. DMF group, $$P<0.001 vs. Comparative Example 1 group, ##P<0.001 vs. Example group 1, #P<0.05 vs. Example group 1

As shown in FIG. 5, Example 1 group had higher lipid peroxides than CPZ induction group, DMF group and Comparative Example 1 group, and it was confirmed that it was similar to the control group.

6 is a graph measuring protein oxide in brain tissue using a control group, CPZ induction group, DMF group, Example 1 group and Comparative Example 1 group. Values are represented as mean ± SD (n=6). **P<0.001 vs. control, *P<0.05 vs. control, +P<0.05 vs. DMF group

As shown in FIG. 6 , it was confirmed that the Example 1 group had higher protein oxides than the CPZ induction group, the DMF group, and the Comparative Example 1 group, similar to the control group.

That is, according to the experimental results of the lipid peroxide and the protein oxide, it was confirmed that the fermented garlic of Example 1 inhibited oxidative stress better than the garlic pulverized product.

Figure 7 is a graph measuring the amount of nitric oxide (NO) produced using a control group, CPZ induction group, DMF group, Example 1 group and Comparative Example 1 group. Values are represented as mean ± SD (n=6). **P<0.001 vs. control, *P<0.05 vs. control, ++P<0.001 vs. DMF group, $$P<0.001 vs. Comparative Example 1 group, ##P<0.001 vs. Example group 1, #P<0.05 vs. Example group 1

NO is a substance produced from L-arginine by nitric oxide synthase (NOS), and is known to exhibit various physiological actions such as signaling substances and antioxidant functions in vivo. However, NO, which is overproduced due to oxidative stress, induces DNA, protein, and lipid oxidation, causing functional damage and inflammation of cells, thereby causing disease.

As shown in FIG. 7 , it was confirmed that Example 1 group had lower NO production compared to CPZ induction group, DMF group and Comparative Example 1 group. In particular, Example 1 group confirmed that NO production was significantly lower than CPZ induction group and Comparative Example 1 group.

Accordingly, it is believed that the fermented garlic product of Example 1 can effectively inhibit NO production in brain tissue compared to the garlic pulverized product.

Test Example 5. Investigation of proteins related to the inflammatory response of brain neurons

Expression levels of TNF-α protein, IL-1β protein, NF-κB protein, and Nrf2 protein as factors related to the inflammatory response of brain neurons were measured.

The TNF-α protein, IL-1β protein, NF-κB protein and Nrf2 protein were extracted from mouse brain tissue, homogenized with lysis buffer, and then centrifuged at 15,000 rpm and 4 ° C for 1 hour to extract proteins from brain tissue. After that, the protein content was measured by the Bradford method and used in the experiment. The extracted protein was denatured by treating at 95 ° C. for 5 minutes, followed by electrophoresis with SDS-PAGE to transfer the protein to polyvinylidene fluoride membrane, blocking the membrane for 1 hour, washing twice for 10 minutes each with TBST solution, and 12 hours ( The primary antibody reaction was performed overnight). After washing with TBST solution twice for 10 minutes, secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobin G was added and reacted at room temperature for 1 hour. The membrane was reacted with an enhanced chemiluminescence (ECL) solution containing luminol, developed using a Davinch Chemisystem, and the expression level was analyzed using Image software.

Figure 8a is a Western blot showing the expression of TNF-α protein in brain neurons of a control group, CPZ-induced group, DMF group, Example 1 group, and Comparative Example 1 group; Figure 8b is a graph quantitatively showing the expression level measured in Figure 8a. Values are represented as mean ± SD (n=6). **P<0.001 vs. control, +P<0.05 vs. DMF group, $P<0.05 vs. Comparative Example 1 group, ##P<0.001 vs. Example group 1

As shown in Figure 8, Example 1 group of the present invention was significantly reduced compared to the CPZ induction group, DMF group and Comparative Example 1 group, and it was confirmed that it was similar to the control group, which indicates that the fermented garlic of Example 1 This means that it can effectively suppress the inflammatory response of brain tissue.

Figure 9a is a Western blot showing the expression of IL-1β protein in brain neurons of the control group, CPZ-induced group, DMF group, Example 1 group, and Comparative Example 1 group; 9b is a graph quantitatively showing the expression level measured in FIG. 9a. Values are represented as mean ± SD (n=6). **P<0.001 vs. control, ++P<0.001 vs. DMF group, +P<0.05 vs. DMF group, $$P<0.001 vs. Comparative Example 1 group, $P<0.05 vs. Comparative Example 1 group, ##P<0.001 vs. Example group 1, #P<0.05 vs. Example group 1

As shown in Figure 9, Example 1 group of the present invention was significantly reduced compared to the CPZ induction group, DMF group and Comparative Example 1 group, and it was confirmed that it was similar to the control group, which indicates that the fermented garlic of Example 1 This means that it can effectively suppress the inflammatory response of brain tissue.

Figure 10a is a Western blot showing the expression of NF-κB protein in brain neurons of a control group, a CPZ-induced group, a DMF group, Example 1 group, and Comparative Example 1 group; 10b is a graph quantitatively showing the expression level measured in FIG. 10a. Values are represented as mean ± SD (n=6). **P<0.001 vs. control, ++P<0.001 vs. DMF group, +P<0.05 vs. DMF group, $$P<0.001 vs. Comparative Example 1 group, $P<0.05 vs. Comparative Example 1 group, ##P<0.001 vs. Example group 1, #P<0.05 vs. Example group 1

As shown in Figure 10, Example 1 group of the present invention was significantly reduced compared to the CPZ induction group, DMF group and Comparative Example 1 group, and it was confirmed that it was similar to the control group, which indicates that the fermented garlic of Example 1 This means that it can effectively suppress the inflammatory response of brain tissue.

Figure 11a is a Western blot showing the expression of Nrf2 protein in brain neurons of a control group, a CPZ-induced group, a DMF group, Example 1 group, and Comparative Example 1 group; 11b is a graph quantitatively showing the expression level measured in FIG. 11a. Values are represented as mean ± SD (n=6). *P<0.05 vs. control, $P<0.05 vs. Comparative Example 1 group, ##P<0.001 vs. Example group 1, #P<0.05 vs. Example group 1

Nrf2 is known to regulate the expression of various antioxidant enzymes, and exhibits potentiation of antioxidant and anti-inflammatory defense systems against oxidative stress.

As shown in FIG. 11 , it was confirmed that the Nrf2 expression level was higher in the Example 1 group of the present invention compared to the control group, the CPZ induction group, the DMF group, and the Comparative Example 1 group.

That is, according to the results of Test Example 5, the garlic fermented product prepared according to Example 1 of the present invention is considered to exhibit excellent anti-inflammatory effects by lowering the expression level of inflammation-related proteins in brain tissue in CPZ-induced multiple sclerosis animal models. .

Test Example 6. Investigation of histopathological factors in brain tissue

The expression levels of myelin, GFAP, and PLP in brain tissue were investigated by immunohistochemical analysis.

The collected brain was fixed using 10% neutral formalin buffer, dehydrated with ethanol, embedded in paraffin, and sectioned using a microtome. After xylene treatment into the sectioned tissue, hydration and dehydration with ethanol, heat treatment and cooling in 10 mM citrate buffer (pH 6.0), reaction by adding 3% H ₂ 0 ₂ containing peroxidase to the slide, and then washing. . Then, it was fixed with 10% normal goat serum solution for 30 minutes, reacted with primary antibodies (myelin, PLP, GAFP) for 12 hours, reacted with secondary antibodies for 2.5 hours, and treated with avidin-biotin-peroxidase for 30 minutes Then, the color was developed with 0.1% DAB chromogen solution and observed under an optical microscope.

12a is a diagram showing the expression of myelin in brain tissues of a control group, a CPZ-induced group, a DMF group, Example 1 group, and Comparative Example 1 group; 12b is a graph quantitatively showing the expression level measured in FIG. 12a. Values are represented as mean ± SD (n=6). **P<0.001 vs. control, +P<0.05 vs. DMF group, $$P<0.001 vs. Comparative Example 1 group, $P<0.05 vs. Comparative Example 1 group, ##P<0.001 vs. Example group 1, #P<0.05 vs. Example group 1

Multiple sclerosis is a demyelinating disease caused by repeated inflammation of the myelin and axon of nerve cells. Luxol fast blue staining (LFB staining) is a representative method for measuring myelin (blue color) in brain tissue. am. Since the hippocampus (HC) region is known to be a tissue involved in learning and memory formation in the brain, the results of staining the HC region of the brain tissue are shown in FIG. 12A.

As shown in FIG. 12, it was confirmed that the myelin expression level (decreased demyelination) was significantly increased in Example 1 group of the present invention compared to the CPZ induction group, the DMF group, and the Comparative Example 1 group. This means that the fermented garlic product in No. 1 can be used as an excellent material to prevent myelin damage, which is a key problem in multiple sclerosis.

13A is a diagram showing the expression of GFAP in brain tissues of a control group, a CPZ-induced group, a DMF group, Example 1 group, and Comparative Example 1 group; 13b is a graph quantitatively showing the expression level measured in FIG. 13a. Values are represented as mean ± SD (n=6). **P<0.001 vs. control, ++P<0.001 vs. DMF group, $P<0.05 vs. Comparative Example 1 group, ##P<0.001 vs. Example group 1

Glial fibrillary acidic protein (GFAP) is a representative marker of astrocytes distributed in the central nervous system (CNS), which means that the degree of brain damage increases as the protein expression level increases. The results of staining the HC region of the brain tissue are shown in FIG. 13A.

As shown in FIG. 13, since the GFAP expression level was significantly increased in all groups compared to the control group, it was determined that the brain was seriously damaged. However, since the expression level of GFAP was decreased in Example 1 group of the present invention compared to the CPZ induction group, DMF group, and Comparative Example 1 group, the fermented garlic product of Example 1 could effectively prevent multiple sclerosis by improving brain damage caused by cuprizone. know that you can.

14A is a diagram showing the expression of PLP in brain tissues of a control group, a CPZ-induced group, a DMF group, Example 1 group, and Comparative Example 1 group; 14b is a graph quantitatively showing the expression level measured in FIG. 14a. Values are represented as mean ± SD (n=6). **P<0.001 vs. control, *P<0.05 vs. control, +P<0.05 vs. DMF group, $$P<0.001 vs. Comparative Example 1 group, ##P<0.001 vs. Example group 1, #P<0.05 vs. Example group 1

Multiple sclerosis is a disease caused by damage to myelin sheath, and the role of proteins such as proteolipid protein (PLP) constituting myelin is important. The results of staining the HC region of the brain tissue are shown in FIG. 14A.

As shown in FIG. 14, it was confirmed that the expression level of PLP increased significantly in Example 1 group of the present invention compared to the CPZ induction group, DMF group, and Comparative Example 1 group. This means that it can be used as an excellent material that can prevent myelin sheath damage, a key problem in multiple sclerosis.

Hereinafter, formulation examples of the composition containing the powder of the present invention will be described, but the present invention is not intended to limit them, but only to be specifically described.

Formulation Example 1. Preparation of powder

500 mg of fermented garlic obtained in Example 1

Lactose 100 mg

Talc 10 mg

A powder is prepared by mixing the above ingredients and filling them in an airtight bag.

Formulation Example 2. Preparation of tablets

300 mg of fermented garlic obtained in Example 1

Corn Starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets are prepared by tableting according to a conventional tablet manufacturing method.

Formulation Example 3. Preparation of capsule formulation

200 mg of fermented garlic obtained in Example 1

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a conventional capsule preparation method.

Formulation Example 4. Preparation of Injections

600 mg of fermented garlic obtained in Example 1

Mannitol 180 mg

Sterile Distilled Water for Injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

It is prepared with the above component content per 1 ampoule according to the conventional method for preparing injections.

Formulation Example 5. Preparation of liquid formulation

4 g of fermented garlic obtained in Example 1

Isomerized sugar 10 g

5 g mannitol

Appropriate amount of purified water

According to the conventional liquid formulation manufacturing method, each component is dissolved in purified water, lemon flavor is added in an appropriate amount, the above components are mixed, and then purified water is added to adjust the total amount to 100g, and then filled into a brown bottle to be sterilized. to prepare a liquid.

Formulation Example 6. Preparation of granules

1,000 mg of fermented garlic obtained in Example 1

Appropriate amount of vitamin mixture

Vitamin A Acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

10 μg of biotin

Nicotinamide 1.7 mg

Folic acid 50 μg

Calcium Pantothenate 0.5 mg

Appropriate amount of mineral mixture

Ferrous sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium Carbonate 25.3 mg

Potassium Phosphate Monobasic 15 mg

Dibasic Calcium Phosphate 55 mg

Potassium citrate 90 mg

Calcium Carbonate 100 mg

Magnesium Chloride 24.8 mg

Although the composition ratio of the above vitamin and mineral mixture was prepared by mixing ingredients suitable for granules in a preferred embodiment, the mixing ratio may be arbitrarily modified, and after mixing the above ingredients according to a conventional granule manufacturing method, It can be prepared and used for preparing a health functional food composition according to a conventional method.

Formulation Example 7. Manufacturing of functional beverages

1,000 mg of fermented garlic obtained in Example 1

Citric Acid 1,000 mg

100 g of oligosaccharides

2 g plum concentrate

1 g of taurine

Add purified water to total 900 mL

After mixing the above ingredients according to the normal health drink manufacturing method, stirring and heating at 85 ° C. for about 1 hour, the resulting solution is filtered and collected in a sterilized 2 L container, sealed and sterilized, and then refrigerated. It is used for preparing the functional beverage composition of the present invention.

Although the composition ratio is a mixture of ingredients suitable for a relatively favorite beverage in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the class of demand, the country of demand, and the purpose of use.

Claims (7)

A pharmaceutical composition for preventing diseases accompanying damage to brain nerve cells, containing fermented garlic as an active ingredient. According to claim 1, wherein the fermented garlic is a pharmaceutical composition for the prevention of diseases accompanied by damage to brain nerve cells, characterized in that fermented garlic pulverized with a strain culture medium. According to claim 2, wherein the strain culture medium Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus casei ( Lactobacillus casei ) , Containing at least one strain selected from the group consisting of Lactobacillus pentosus , Lactobacillus sakei , Leuconostoc mesenteroides and Lactobacillus buchneri A pharmaceutical composition for the prevention of diseases accompanied by damage to brain nerve cells. [Claim 3] The pharmaceutical composition for the prevention of diseases accompanying damage to brain nerve cells according to claim 2, wherein the fermentation is carried out at 35 to 45 °C for 40 to 60 hours. [Claim 3] The pharmaceutical composition for preventing diseases accompanying damage to brain nerve cells according to claim 2, wherein the fermentation is performed by mixing 0.02 to 0.09 parts by weight of the strain culture medium with respect to 100 parts by weight of the crushed garlic. The method of claim 1, wherein the disease accompanying damage to brain nerve cells is an autoimmune disease, and the autoimmune disease includes multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, ascending myelitis, central myelitis, and chronic inflammatory demyelinating bundle A pharmaceutical composition for the prevention of diseases accompanying damage to brain nerve cells, characterized in that at least one selected from the group consisting of neuropathy. A food composition for improving or preventing diseases accompanying damage to brain nerve cells containing fermented garlic as an active ingredient.

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