A composition for preventing or treating cognitive dysfunction and intestinal dysfunction, comprising Lactobacillus paracasei NK112, a Japanese quince extract, and a dodder extract

Abstract

The present invention relates to a novel Lactobacillus paracasei NK112 strain having preventive or therapeutic effects on cognitive dysfunction and intestinal dysfunction. The present invention also relates to a composition for preventing, treating or improving cognitive dysfunction and intestinal dysfunction, comprising the Lactobacillus paracasei NK112 strain, a Japanese bean extract and a dodder extract. The strain and composition of the present invention are effective in enhancing the expression of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), improving enteritis, etc., and can therefore be effectively used for preventing or treating cognitive dysfunction and intestinal dysfunction.

Description

[Technical field]

[0001] The present invention relates to a novel Lactobacillus paracasei NK112 strain.

[0002] The present invention also relates to a composition for preventing, treating, or ameliorating cognitive dysfunction and intestinal dysfunction, comprising Lactobacillus paracasei NK112 strain, an extract of Mamedaoshi, and an extract of Cuscuta japonica. [Background technology]

[0003] As humanity develops into a more affluent society, lifestyles are rapidly becoming Westernized, and diseases such as intestinal diseases such as colitis and intestinal bacterial community disorders are increasing due to the Westernization of dietary habits centered on meat and fat, irregular meals, excessive drinking, lack of exercise, excessive stress, exposure to harmful environments, etc. These intestinal diseases and the increase in imbalance of intestinal bacterial communities lead to a rapid increase in cognitive dysfunction such as dementia, Parkinson's disease, depression, and anxiety.

[0004] Dementia and other brain degenerative diseases refer to diseases in which memory and thinking ability gradually decline due to neurodegeneration, reaching a level that interferes with daily life. With the advent of an aging society, these diseases have emerged as one of the main causes of medical, economic, and social burdens. As of 2015, there were approximately 47 million patients worldwide, and the prevalence of these diseases in the population over 60 years of age is estimated to be approximately 7% (The global prevalence of dementia: a systematic review and metaanalyisis, Alzheimers Dement, 2013, 63-75).

[0005] In addition to dementia, cognitive impairment can also be caused by diseases and symptoms common in modern life, such as high blood pressure and lack of exercise. In particular, depression, the number of patients of which has been rapidly increasing in all age groups in recent years, is known to be most correlated with cognitive impairment. Therefore, it is necessary to develop methods to improve cognitive impairment and protect neurons as a means of living a healthy daily life.

[0006] For diseases such as cognitive impairment that require continuous management while taking medication for a long time, reducing side effects is one of the important issues. For example, donepezil, which is commonly used to treat dementia in Korea, can cause side effects such as muscle pain and loss of appetite, as well as hallucinations and abdominal disorders in severe cases, so the development of new dementia treatments is urgently needed. Therefore, the development of natural medicines with fewer side effects than synthetic medicines for diseases related to cognitive impairment would be a great help to patients.

[0007] Cuscuta australis R.Br. and Cuscuta japonica Choisy are annual vines belonging to the Convolvulaceae family, also known as rabbit silk or golden silk. Dried ripe seeds of Cuscuta australis are used to replenish yang and yin energy (boosting yang and improving yin), prevent semen leakage, regulate urine (solidifying semen and reducing urine), and brighten the eyes (brightening the eyes). The reported components of Cuscuta japonica include quercetin, kaempferol, isorhamnetin, caffeic acid, thymidine, sesamin, and taraxanthin (A Review on Phytoconstituents and Biological activities of Cuscuta species. Biomed Pharmacother. 2017 Aug; 92:772-795). The components of Cuscuta japonica include resin glycosides, sugars, sterols, triterpenoids, gibberellin, alkaloids, cardiac glycosides, and steroid saponins.

[0008] On the other hand, many bacteria live in the body, and their number is greater than that of normal cells. These bacteria can be divided into beneficial bacteria that support intestinal health and harmful bacteria that harm health. Our bodies can maintain their health when beneficial bacteria such as Lactobacillus and Bifidobacterium live as the predominant bacteria in the digestive tract (Non-Patent Document 3). On the other hand, when brain diseases occur, harmful microorganisms such as Escherichia coli and Proteus mirabilis increase in the intestinal flora, which leads to overexpression of α-synuclein in intestinal cells and activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), which is known to promote the progression of Parkinson's disease and Alzheimer's disease (Oral administration of Proteus mirabilis damages dopaminergic neurons and motor functions in mice. Scientific reports, 2018, 8(1), 1275).

[0009] Live microorganisms that improve the intestinal microbial environment and have a beneficial effect on the health of the host are collectively called probiotics. Lactic acid bacteria are used as probiotics because, when ingested, they coexist in the digestive system and become important nutrients by breaking down fiber and complex proteins. It has been reported that lactic acid bacteria maintain and improve the intestinal flora, have antidiabetic effects, suppress colitis, and improve the immune system (Non-Patent Document 5). Thus, there has been active research into developing lactic acid bacteria with various physiological activities and applying them as materials for pharmaceuticals and health functional foods (Patent Document 1, etc.), but there have been no examples of them being developed as therapeutic agents for chronic diseases such as cognitive dysfunction.

[0010] Therefore, the inventors continued their research to develop a method for preventing and treating cognitive dysfunction and intestinal dysfunction, and as a result, they confirmed that the use of a novel lactic acid bacterium, Lactobacillus paracasei NK112, an extract of Japanese bean sprouts, and an extract of dodder had excellent effects on cognitive function and intestinal regulation, leading to the completion of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention aims to provide a novel Lactobacillus paracasei NK112 strain having preventive or therapeutic effects on cognitive dysfunction and intestinal dysfunction.

[0012] Another object of the present invention is to provide a composition for preventing, treating, or ameliorating cognitive dysfunction and intestinal dysfunction, comprising Lactobacillus paracasei NK112 strain, a Chinese quince extract, and a dodder extract. [Means for solving the problem]

[0013] To achieve the above object, the present invention provides a novel Lactobacillus paracasei NK112 strain, deposited under accession number KCCM12566P.

[0014] In one embodiment, the Lactobacillus paracasei NK112 strain has a preventive or therapeutic effect on cognitive dysfunction or intestinal dysfunction.

[0015] The present invention also provides a composition for preventing, treating, or ameliorating cognitive dysfunction or intestinal dysfunction, comprising at least one selected from the group consisting of Lactobacillus paracasei NK112 strain, a Cuscuta australis R.Br. extract, and a Cuscuta japonica Choisy extract.

[0016] The composition may be a pharmaceutical composition, a food composition or an animal feed composition. In one embodiment, the composition may include Lactobacillus paracasei strain NK112, an extract of Japanese bean sprouts, and an extract of dodder.

[0017] In one embodiment, the composition may contain the extract of Japanese bean sprouts, the extract of dodder, and the Lactobacillus paracasei NK112 strain in a weight ratio of 0.3-30:1:0.05-5, a weight ratio of 1-6:1:0.1-1, or a weight ratio of 2-4:1:0.3-7, preferably a weight ratio of 3:1:0.5.

[0018] In one embodiment, the composition may promote expression of brain-derived neurotrophic factor (BDNF).

[0019] In one embodiment, the composition may promote expression of nerve growth factor (NGF).

[0020] In one embodiment, the composition may be an extract of a mixture of bean sprouts and dodder.

[0021] In one embodiment, the extract may be extracted with a solvent selected from the group consisting of water (distilled water), C1 to C6 alcohols, ethyl acetate, acetone, and mixtures thereof, and preferably with distilled water.

[0022] In one embodiment, the extract may be one obtained by extracting with a solvent in an amount 10 to 50 times, and preferably 20 to 30 times, the weight of the bean sprouts and dodder.

[0023] In one embodiment, the extract may be reflux extracted or ultrasonically extracted, preferably reflux extracted, of Mamedao and Cuscuta.

[0024] In one embodiment, the extract may be an extract of the original material (whole herbal medicine) or powder (powdered herbal medicine) of Mamedaoshi and Cuscuta, and preferably an extract of powder of Mamedaoshi and Cuscuta.

[0025] In one embodiment, the Lactobacillus paracasei NK112 strain may be killed cells, a cell wall fraction, or a cytoplasmic fraction of Lactobacillus paracasei NK112.

[0026] In one embodiment, the cognitive dysfunction of the present invention may be at least one selected from the group consisting of Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, depression, anxiety, stroke, cerebral ischemia, encephalitis, amnesia, traumatic brain injury, Wernicke-Korsakoff syndrome, electroencephalopathy, epilepsy, hippocampal sclerosis, cerebral senility, dementia, frontotemporal lobar degeneration, normal pressure hydrocephalus, memory loss, and learning disability.

[0027] In one embodiment, the intestinal dysfunction of the present invention may be at least one selected from the group consisting of inflammatory bowel disease, intestinal peristalsis disorder, intestinal obstruction, colonic inertia, irritable bowel disease, dyspepsia, abdominal bloating, diarrhea, and constipation. Effect of the Invention

[0028] The Lactobacillus paracasei NK112 strain of the present invention and a composition containing said strain, an extract of Japanese bean sprouts, and an extract of dodder are effective in enhancing the expression of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), improving enteritis, etc., and therefore can be effectively used in the prevention or treatment of cognitive dysfunction and intestinal dysfunction. [Brief description of the drawings]

[0029] [Figure 1A] FIG. 1 shows the spontaneous change behavior ratio of mice in a Y-maze test when the NK112 strain was administered to the mice. [Figure 1B] FIG. 1 shows the time spent in a bright room due to fear memory in a passive avoidance test when the NK112 strain was administered to mice. [Figure 2A] FIG. 1 shows the amount of nerve growth factor (NGF) produced when treated with a mixed composition of a Mamedaoshi extract, a Cuscuta dodder extract, and the NK112 strain. [Figure 2B] FIG. 1 shows the amount of nerve growth factor (NGF) produced when treated with a mixed composition of a Mamedaoshi extract, a Cuscuta dodder extract, and the NK112 strain. [Diagram 3] FIG. 1 shows the survival rate of nerve cells when treated with a mixed composition of an extract of Mamedaoshi, an extract of Cuscuta japonica, and the NK112 strain. [Figure 4A] FIG. 1 shows the spontaneous change behavior ratio of mice in a Y-maze test when the mice were treated with a mixed composition of a Mamedao extract, a Cuscuta dodder extract, and the NK112 strain. [Figure 4B] FIG. 1 shows cognitive indices in a new substance discovery test when mice were treated with a mixed composition of a Mamedao extract, a Cuscuta dodder extract, and the NK112 strain. [Figure 4C] FIG. 13 is a graph showing the time spent in a bright room due to fear memory in a passive avoidance behavior test when mice were treated with a mixed composition of a Mamedao extract, a Cuscuta dodder extract, and the NK112 strain. [Figure 5A] 13 is a photograph showing the hippocampal rotation area when mice were treated with a mixture of an extract of Japanese bean sprouts, an extract of dodder, and the NK112 strain. [Figure 5B] FIG. 1 shows the amount of synaptic proteins when mice were treated with a mixed composition of an extract of Mamedaoshi, an extract of Cuscuta japonica, and the NK112 strain. [Figure 6A] 13 is a photograph showing dentate gyrus cells in the hippocampus when mice were treated with a mixture of an extract of Japanese bean sprouts, an extract of dodder, and the NK112 strain. [Figure 6B] FIG. 1 shows the number of neuroblasts when mice were treated with a mixed composition of a Mamedao extract, a Cuscuta dodder extract, and the NK112 strain. [Figure 6C] FIG. 1 shows the neurite length when mice were treated with a mixed composition of Mamedaoshi extract, Cuscuta dodder extract, and NK112 strain. [Figure 7A] FIG. 1 shows the changes in intestinal length and inflammation index when mice were treated with a mixed composition of Mamedaoshi extract, Cuscuta dodder extract, and NK112 strain. [Figure 7B] FIG. 1 shows the changes in intestinal length and inflammation index when mice were treated with a mixed composition of Mamedaoshi extract, Cuscuta dodder extract, and NK112 strain. [Figure 7C] FIG. 1 shows the changes in intestinal length and inflammation index when mice were treated with a mixed composition of Mamedaoshi extract, Cuscuta dodder extract, and NK112 strain. [Figure 7D] FIG. 1 shows the changes in intestinal length and inflammation index when mice were treated with a mixed composition of Mamedaoshi extract, Cuscuta dodder extract, and NK112 strain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Hereinafter, the present application will be described in detail with reference to the accompanying drawings so that a person having ordinary skill in the art to which the present invention pertains can easily carry out the present application. However, since the present application can be carried out in various forms, it is not limited to the following embodiments and examples.

[0031] Throughout this specification, when a part is said to "comprise" a certain element, this does not mean that it excludes other elements, but that it may further include other elements, unless otherwise specified.

[0032] The present invention relates to a novel lactic acid bacterium, Lactobacillus paracasei NK112 strain.

[0033] The present invention also relates to a composition for preventing, treating, or improving cognitive dysfunction or intestinal dysfunction, comprising at least one selected from the group consisting of Lactobacillus paracasei NK112 strain, a Cuscuta australis R.Br. extract, and a Cuscuta japonica Choisy extract.

[0034] The composition has the effect of promoting the expression of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). Nerve growth factor (NGF) binds to TrkA (Tropomyosin receptor kinase A) receptor to activate TrkA as pTrkA, and the nerve growth factor (NGF) and pTrkA complex migrate to the cell body to affect the nerve regeneration effect. In addition, it activates cAMP-response element binding protein (CREB), which plays an important role in regulating cell cycle, neurite growth, and synaptic plasticity. Brain-derived neurotrophic factor (BDNF) is one of the nerve growth promoting factors, and is known to play an important role in regulating neurotransmitters and neuroplasticity. Therefore, the increase in the expression of nerve growth factor and brain-derived neurotrophic factor means that it has a nerve regeneration effect.

[0035] The extract of the present invention may be produced by a production method including, but not limited to, the following steps. 1) adding an extraction solvent to the bean vine and the dodder to extract; 2) filtering the extract of step 1); 3) Concentrating the filtered extract of step 2) under reduced pressure and then drying.

[0036] In the above-mentioned method, the extraction method may be any method commonly used in the art, such as reflux extraction, ultrasonic extraction, stirring extraction, filtration, hot water extraction, and immersion extraction.

[0037] In the above method, the vacuum concentration in step 3) may be performed using, but is not limited to, a vacuum vacuum concentrator or a vacuum rotary evaporator, and the drying may be performed using, but is not limited to, vacuum drying, vacuum drying, boiling drying, spray drying, or freeze drying.

[0038] The food composition of the present invention may be a health functional food, and the term "health functional food" means a food manufactured and processed using raw materials or ingredients that have functionality useful to the human body, and "functional" means that the food is ingested for the purpose of regulating nutrients for the structure and function of the human body or obtaining useful effects for health purposes such as physiological actions.

[0039] The present invention will be described in more detail below with reference to examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. EXAMPLES

[0040] Isolation and identification of lactic acid bacteria 1-1. Isolation of lactic acid bacteria from human feces Human feces were suspended in GAM Broth (Nissui Pharmaceutical, Japan), the supernatant was then collected and transferred to BL agar medium (Nissui Pharmaceutical, Japan) and cultured anaerobically at 37°C for approximately 48 hours, after which the strains that formed colonies were isolated.

[0041] 1-2. Identification of isolated lactic acid bacteria The isolated strain was analyzed for its base sequence to confirm its physiological characteristics and phylogenetic position. It was found to be a Lactobacillus strain, with a base sequence similarity of 90-99%.

[0042] Among the above strains, the nucleotide sequence of the strain having the 16S rDNA nucleotide sequence of SEQ ID NO: 1 was analyzed using the BLAST program of the Gene Bank, and it was confirmed that the strain is a novel strain having 99% homology with the 16S rDNA sequence of Lactobacillus paracasei strain, and was named Lactobacillus paracasei NK112. The strain was deposited at the Korea Microorganism Collection on July 18, 2019, and was assigned the accession number KCCM12566P.

[0043]

number

[0044] 1-3.Biochemical identification using API kits The physiological characteristics of Lactobacillus paracasei NK112 strain were analyzed by sugar fermentation test using API 20 strep Kit (BioMerieux's, USA). The results are shown in Table 1 below. In Table 1, "+" indicates positive carbon source utilization and "-" indicates negative carbon source utilization. The analysis confirmed that NK112 showed the most similar biochemical characteristics to Lactobacillus paracasei at 56.5%.

[0045] [Table 1-1]

[0046] [Table 1-2]

[0047] [Table 1-3] EXAMPLES

[0048] Confirmation of the effect of treating NK112 strain on the expression of brain-derived neurotrophic factor (BDNF) in nerve cells Human SH-SY5Y neuronal cells were cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics, and 2 × 10 6 Then, NK112 strain (1 × 10 5 CFU / mL) and lipopolysaccharide at a concentration of 2μg / mL were added and cultured, and the expression level of brain-derived neurotrophic factor was measured by immunoblotting. As a result, it was confirmed that Lactobacillus paracasei NK112 strain has the effect of increasing the expression of brain-derived neurotrophic factor, which decreases due to aging and dementia, to a level of 10-30%. EXAMPLES

[0049] Confirmation of the anti-anxiety and anti-depressant effects of the NK112 strain To confirm the effect of Lactobacillus paracasei NK112 strain in improving cognitive function, the following experiment was conducted.

[0050] 3-1. Creation of Escherichia coli-induced cognitive dysfunction and intestinal dysfunction models Five-week-old male C57BL / 6 mice weighing 19-21 g were divided into a normal group and an experimental group, with six mice in each group allowed to adapt to the laboratory for one week, after which the experiment was performed. 9 The mice were orally administered with 1000 mg of 10000 CFU / mouse / day once a day for 5 days, and the normal group was administered with saline.

[0051] Then 1×10 9 CFU heat-treated lactic acid bacteria (NK112 killed cells), 1 x 10 9 CFU lactic acid bacteria lysate supernatant (NK112 cytoplasmic component), 1 x 10 9 CFU Lactobacillus lysate precipitate (NK112 cell wall component) or a positive control drug (fluoxetine 1 mg / kg) was administered once daily for 5 days.

[0052] In the case of heat-treated lactic acid bacteria (killed NK112 bacteria), the precipitate obtained by centrifuging the lactic acid bacteria culture solution in MRS medium was 10 The cells were suspended at a concentration of CFU / mL and then heat-treated twice at 70°C for 10 minutes each.

[0053] For the lactic acid bacteria lysate supernatant (NK112 cytoplasmic component) and precipitate (NK112 cell wall component), the lactic acid bacteria culture solution in MRS medium was centrifuged at 5,000 g for 20 minutes, and the resulting precipitate was diluted with 1 × 10 10 The cells were suspended at a concentration of CFU / mL, sonicated 20 times for 1 min each, and then centrifuged again at 5000 g for 20 min to obtain the cells.

[0054] To evaluate the anxiolytic and antidepressant effects of Lactobacillus paracasei NK112 strain using the mouse model, the following experiment was performed.

[0055] 3-2. Elevated plus maze (EPM) test The elevated plus maze is an experimental device for measuring the degree of anxiety, depression, or stress. The elevated plus maze used in this study is a black flexi glass device with two open arms (30 x 7 cm) and two enclosed arms (30 x 7 cm) with 20 cm high walls, each 50 cm above the floor and 7 cm long extending from the central platform. The movements of the mice placed in the elevated plus maze were recorded in a room with a video camera installed above and a brightness of 20 lux.

[0056] Specifically, C57BL / 6 mice were placed in the center of the elevated plus maze facing the open aisle, and the time and number of times they spent in the open and closed aisles were measured for 5 min. Arm entry was defined as when all four paws entered the aisle.

[0057] Of the total test time, the time spent in the open aisle (OT) was calculated as "time spent in the open aisle / (time spent in the open aisle + time spent in the closed aisle)" × 100. The open arm entries (OE) were calculated as [open arm entries / (open arm entries + closed arm entries)] × 100. After the completion of each behavioral test, the remaining odor was removed with 70% ethanol.

[0058] According to known methods of analyzing test results, a decrease in time spent in the open aisle (OT) and open aisle entry (OE) was interpreted as an indication of symptoms such as anxiety or depression.

[0059] 3-3. Forced Swimming Test (FST) A tank with a height of 40 cm and a diameter of 20 cm was filled with water at a temperature of 25±1°C to a height of 30 cm. The disease model mice prepared in Example 3-1 were placed in the tank one by one and tested for a total of 6 minutes. The first 2 minutes were not measured as the adaptation time, and the immobility time of the experimental animals was then measured for 4 minutes. The floating state refers to a state in which the animals were floating upright and motionless, making only the minimum movement necessary to bring only their heads above the water surface.

[0060] 3-4. Tail Suspension Test (TST) According to the method of Steru et al. (Non-Patent Document 6), a fixation device was attached to the tip of the mouse's tail about 1 cm away, and the mouse was then suspended 50 cm from the ground in a tube 35 cm in diameter and 50 cm high. The immobility time of the experimental animals was measured for a total of 6 minutes.

[0061] The percentage of each measurement time in tests 3-2 to 3-4 is shown in Table 2 below. In addition, the blood concentration of corticosterone, which induces anxiety in each mouse, was measured and shown together.

[0062] [Table 2]

[0063] As shown in Table 2, the group administered Lactobacillus paracasei NK112 strain showed an increased time and frequency spent in the open aisle in the elevated plus maze test, and a significant decrease in the amount of corticosterone in the blood, confirming the anti-anxiety effect, compared to the group administered E. coli alone. In addition, the group administered Lactobacillus paracasei NK112 strain showed a significant decrease in the floating state time in the forced swimming test and tail suspension test, confirming the anti-depressant effect.

[0064] These results indicate that Lactobacillus paracasei NK112 strain is effective in improving depression and anxiety. EXAMPLES

[0065] Confirmation of cognitive function improvement effect of NK112 strain (E. coli induction model) Using the following test method, we confirmed whether memory was improved after treating the disease model mice prepared in Example 3-1 with Lactobacillus paracasei NK112 strain. As a positive control, we used 5 mg / kg of donepezil (DNZ), which is used as a dementia treatment drug.

[0066] 4-1.Y-maze task A Y-maze test was conducted to evaluate spatial cognitive ability. The maze was 40 cm long, 4 cm wide, and 12 cm high, and had four closed Y-shaped mazes. Three paths were designated A, B, and C, and the mouse was placed in the center. The total number of times the mouse entered was recorded after 8 minutes of observation. If the mouse entered three different branches consecutively, it was counted as one alternation number, and if the mouse did not enter consecutively, it was not counted as an alternation number. The alternation number was defined as entering all three branches consecutively, and the calculation for evaluating spatial cognitive function was performed using the following formula.

[0067]

number

[0068] 4-2.Novel object recognition test (NORT) The mice were transferred to a behavioral observation room and allowed to adapt for 5 minutes. Two identical objects (one set) were placed in the box 30 cm apart, and the time the mice spent exploring the objects was measured for 3 minutes. In the retrieval test one day later, one object that had been timed during exposure and one new object that had been replaced were placed in the same box, and the time the mice spent exploring them was measured. The recognition index (%) was calculated by converting the preference for the new object to (time spent exploring the new object / total time spent exploring) x 100.

[0069] The behaviors and the proportion of time measured in the tests 4-1 and 4-2 are shown in Table 3 below. In addition, the expression level of brain-derived neurotrophic factor (BDNF) measured by the method of Example 2 is also shown.

[0070] [Table 3]

[0071] As shown in Table 3, the group administered Lactobacillus paracasei NK112 strain showed an increase in the rate of spontaneously modified behavior, the rate of novel object exploration time, and the expression level of brain-derived neurotrophic factor to levels equal to or greater than those of the donepezil-administered group.

[0072] These results indicate that Lactobacillus paracasei NK112 strain has excellent effects on improving cognitive function. EXAMPLES

[0073] Confirmation of cognitive function improvement effect of NK112 strain (scopolamine model) To confirm the cognitive function improving effect of Lactobacillus paracasei NK112, 100% killed NK112 cells and cell wall components were 9After oral administration at 1000 cfu / mouse for 5 days, the test was performed. The lactobacillus sample was orally administered 1 hour before the behavioral test, and scopolamine, a parasympathetic nerve inhibitor, was injected 30 minutes before the test to induce cognitive decline. As a positive control, donepezil was orally administered at 2 mg / kg.

[0074] The behavioral changes were evaluated using a Y-maze test and a passive avoidance behavior test. The Y-maze test was performed in the same manner as in Example 4.

[0075] Passive avoidance test (PAT) To evaluate fear learning and memory, a passive avoidance test (PAT) was performed in a passive avoidance box for 2 days. The box was divided into a lighted room and a dark room, and an electric shock could be administered on the floor of the dark room. When a mouse was placed in the box, a 50W light was turned on on one side of the box, and the mouse looked around. After 10 seconds, the door was opened to enter the dark room. As soon as the mouse entered the dark room, the door was closed and a 0.5mA electric current was applied to the stainless steel grid on the floor for 3 seconds to administer an electric shock. When the mouse was returned to the lighted room 24 hours after the electric shock, the latency time in the lighted room was measured.

[0076] As shown in Figure 1A, the results of the Y-maze test for the groups administered NK112 killed cells and cell wall components showed that the rate of spontaneous alternation was 62.23% and 67.60%, respectively, which were 8.45% and 17.81%, higher than the group administered scopolamine only, respectively, and showed similar or higher memory improvement effects compared to the donepezil-administered positive control group (PC).

[0077] Furthermore, as shown in Figure 1B, the passive avoidance behavior test showed that the time spent in a bright room due to fear memory was 101.22 seconds and 134.89 seconds in the groups administered NK112 killed bacteria and cell wall components, respectively, which were 116.37% and 188.35% higher than the group administered scopolamine only, and 19.36% and 59.07% higher than the positive control group.

[0078] From this, it was confirmed that Lactobacillus paracasei NK112 strain has an excellent effect of improving memory decline. EXAMPLES

[0079] Confirmation of the intestinal regulation effect of NK112 strain In order to evaluate the intestinal regulating effect of Lactobacillus paracasei NK112, the disease model mice prepared in Example 3-1 were anesthetized, and the intestinal length and inflammatory response indicators were measured.

[0080] 6-1. Measurement of body weight and intestinal length The body weight change of the mice before and after the test was measured, the abdomen was dissected, the large intestine was isolated, and the length of the intestine was measured.

[0081] 6-2. Measurement of myeloperoxidase (MPO) activity 100 mg of colon tissue was homogenized with 200 μL of 10 mM potassium phosphate buffer (pH 7.0) containing 0.5% hexadecyl trimethyl ammonium bromide. The mixture was centrifuged at 4°C and 10,000 g for 10 minutes to obtain a supernatant, which was used as a crude enzyme solution. 50 μL of the crude enzyme solution was added to 0.95 mL of a reaction solution containing 1.6 mM tetramethyl benzidine and 0.1 mM H2O2, and the absorbance was measured at 650 nm over time while reacting at 37°C. The activity of the myeloperoxidase was calculated as 1 unit per 1 μmol / mL of H2O2 purified as a reactant.

[0082] 6-3. Measurement of p-p65, p65, and TNF-α Inflammatory response indicators such as p-p65, p65, and TNF-α were measured. Specifically, the supernatant was obtained in the same manner as in Example 6-2, and 50 μg of the supernatant was taken and p-p65 and p65 were measured by immunoblotting. In addition, TNF-α was measured using an ELISA kit.

[0083] The results of measuring the improving effect of Lactobacillus paracasei NK112 on E. coli-induced intestinal dysfunction are shown in Table 4 below.

[0084] [Table 4]

[0085] As shown in Table 4, when treated with Lactobacillus paracasei NK112, weight loss caused by E. coli administration was improved, length was restored, and inflammation index values ​​such as MPO activity, TNF-α, and p-p65 / p65 were improved.

[0086] This indicates that the Lactobacillus paracasei NK112 strain has an excellent intestinal regulating effect. EXAMPLES

[0087] Preparation of a mixed composition containing extract of Japanese bean sprouts, extract of dodder, and Lactobacillus paracasei NK112 strain 7-1. Preparation of single herbal extracts by ultrasonic extraction In the following examples, washed and dried Cuscuta australis R.Br. and Cuscuta japonica Choisy purchased from Jeongdo Herbal Medicine Co., Ltd., a Korean herbal medicine and food distribution company, were used. 100 g of Cuscuta australis and Cuscuta japonica (whole herbal medicine) were added with 10 times distilled water or 20% ethanol, extracted at 25°C for 1 hour, concentrated under reduced pressure at 50-65°C, and then freeze-dried to obtain a total of four types of herbal extract powders. The yields are shown in Table 5 below.

[0088] [Table 5]

[0089] As shown in Table 5, it was confirmed that the yield of bean sprouts and dodder was increased when distilled water was used for extraction compared to when 20% ethanol was used during ultrasonic extraction.

[0090] 7-2. Preparation of single herbal extracts by reflux extraction 100g of the original materials (whole herbal medicines) of Japanese bean sprouts and dodder were mixed with 10 times the amount of distilled water or 20% ethanol, and extracted by refluxing at 80-100℃ for 3 hours, concentrated under reduced pressure at 50-65℃, and then freeze-dried to obtain a total of four types of herbal extract powders. The yields are shown in Table 6 below.

[0091] [Table 6]

[0092] As shown in Table 6, even during reflux extraction, the yield of bean sprouts and dodder was higher when extracted with distilled water than when 20% ethanol was used, and it was confirmed that when the reflux extraction method was used, the yield of bean sprouts and dodder was similar or higher than that of the ultrasonic extraction of Example 7-1.

[0093] 7-3. Preparation of single herbal extracts by grinding degree 100g of the original (whole herb) and powder (powdered herb) of Japanese bean sprouts and dodder were mixed with 10 times the amount of distilled water, extracted by refluxing at 80-100℃ for 3 hours, concentrated under reduced pressure at 50-65℃, and then freeze-dried to obtain a total of 4 types of herb extract powders. The yields are shown in Table 7 below.

[0094] [Table 7]

[0095] As shown in Table 7, when reflux extraction was performed using distilled water, it was confirmed that the yield was increased by approximately two-fold or more when powder was used compared to when the original material of bean sprouts or dodder was used.

[0096] 7-4. Preparation of complex herbal extracts with various solvent amounts Powders of Japanese bean sprouts and dodder (powdered herbal medicines) were mixed in a 3:1 weight ratio (w / w) to a total of 100g, then 10x, 20x, 30x, and 50x distilled water were added, and the mixture was refluxed at 80-100°C for 3 hours, concentrated under reduced pressure at 50-65°C, and then freeze-dried to obtain a total of four types of herbal extract powders. The yields are shown in Table 8 below.

[0097] [Table 8]

[0098] As shown in Table 8, when powders of bean sprouts and dodder were reflux-extracted using distilled water, it was confirmed that the highest yield was obtained when 20 to 30 times the amount of distilled water was added for extraction.

[0099] 7-5. Preparation of NK112 strain and mixed composition Lactobacillus paracasei NK112 strain was cultured in MRS medium, centrifuged at 5,000xg for 20 minutes at 4°C, and then washed with saline. Killed cells were produced by heating twice at 90°C for 30 minutes, followed by ultrasonic extraction and centrifugation at 5,000xg for 20 minutes at 4°C. The precipitate was freeze-dried to obtain cell wall components (1×10 9 CFU) were obtained. EXAMPLES

[0100] Confirmation of the nerve regeneration effect of the mixed composition (ability to induce nerve growth factor (NGF) secretion) 1 × 10 N2a cells 5 The cells were dispensed into a 24-well plate at cell / well, treated with a composition in which the extract of Mamedaoshi, the extract of Cuscuta japonica, and NK112P were mixed at a ratio of 3:1:0.1 or 3:1:0.5, and cultured for 24 hours. The culture medium was then collected, and the amount of nerve growth factor (NGF) produced in the culture supernatant of C6 glioma cells was quantified and measured using a Competitive Enzyme-Linked Immuno Assay (ELISA) kit (R&D systems, Minneapolis, MN, USA), which is an enzyme immunoassay method. The amount of nerve growth factor (NGF) produced by the mixed composition compared to the amount of nerve growth factor produced by the untreated control group (Ctl) was calculated (%). The results are shown in Figures 2A and 2B.

[0101] As shown in Figures 2A and 2B, the compositions containing the mixture of the extract of the Japanese laurel tree, the extract of the dodder, and NK112P in a ratio of 3:1:0.1 or 3:1:0.5 increased the production of nerve growth factor by 202.07% and 183.90% compared to the control group. This indicates that the mixture containing the extract of the Japanese laurel tree, the extract of the dodder, and the Lactobacillus paracasei NK112 strain has a nerve regeneration effect by inducing the secretion of nerve growth factor. EXAMPLES

[0102] Confirmation of anti-Alzheimer's effect (neuronal protection effect) of the mixed composition HT22 cells 4 × 10 4 Cells were dispensed into 96-well plates at cell / well and allowed to stabilize for 24 hours, after which they were treated for 1 hour with a mixture of extracts of Japanese bean sprouts, extracts of dodder and NK112P in ratios of 3:1:1, 3:1:0.5, 3:1:0.3 or 3:1:0.1, and then further treated with 3 μM of amyloid beta oligomer (AβO), a neurotoxin, and cultured for 23 hours. After removing the medium, the cells were treated with 1 mg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution for 4 hours, and the reduced formazan was dissolved in 100 μL of DMSO, and cell viability was measured using a microspectrophotometer at a wavelength of 570 nm.

[0103] As shown in FIG. 3, it was confirmed that the mixed composition of the Mamedaoshi extract, the Cuscuta extract, and the NK112P showed a superior neuroprotective effect on nerve cells compared to the control group at all mixing ratios. Specifically, the cell viability was increased by 31.94% in the composition with a ratio of 3:1:0.5, 20.96% in the composition with a ratio of 3:1:1, 20.82% in the composition with a ratio of 3:1:0.3, and 20.96% in the composition with a ratio of 3:1:0.1, compared to the control group. From this, it was found that the mixture containing the Mamedaoshi extract, the Cuscuta extract, and the Lactobacillus paracasei NK112 strain protects nerve cells and has a superior anti-Alzheimer's effect, and these effects are particularly superior in the composition containing the Mamedaoshi extract, the Cuscuta extract, and the NK112 in a ratio of 3:1:0.5. EXAMPLES

[0104] Confirmation of the cognitive function improving effect of the mixed composition (scopolamine model) In order to confirm the cognitive function improving effect of the composition in which the bean extract, the dodder extract, and NK112P were mixed in a ratio of 3:1:0.5, the composition was orally administered to mice for 5 days and then behavioral tests were performed. The composition was orally administered 1 hour before the behavioral test, and scopolamine was injected 30 minutes before the test to induce cognitive decline. As a positive control group, donepezil (DNZ) 2 mg / kg was orally administered.

[0105] The behavioral changes were evaluated by the Y-maze test and novel object exploration test similar to those in Example 4, and the passive avoidance behavior test similar to those in Example 5.

[0106] As shown in Figures 4A to 4C, when the rate of spontaneous altered behavior in a Y-maze was measured, the group administered the composition showed a rate of 65.67%, which was 21.01% higher than the scopolamine-treated group; when the cognitive index in a novel object exploration test was measured, the group administered the composition showed a rate of 77.30%, which was 28.90% higher than the scopolamine-treated group; and when the retention time due to fear memory in a passive avoidance behavior test was measured, the group administered the composition showed a rate increased by 108.33% compared to the scopolamine-treated group.

[0107] Furthermore, in all of the above tests, the mixed composition of Mamedaoshi extract, Cuscuta extract, and NK112 showed results equal to or better than those of the donepezil-administered positive control group, confirming that the mixed composition of Mamedaoshi extract, Cuscuta extract, and Lactobacillus paracasei NK112 has excellent effects in improving cognitive decline. EXAMPLES

[0108] Confirmation of the cognitive function improving effect of the mixed composition (E. coli induction model) In order to confirm the cognitive function improving effect of the composition in which the above-mentioned Mamedao extract, Cuscuta extract, and NK112P were mixed in a ratio of 3:1:0.5, a Y-maze test and a novel object exploration test were performed on the mouse model of Example 3-1 in the same manner as in Example 4, and the following Barnes maze task (BMT) was also performed.

[0109] Barnes maze task (BMT) The Barnes maze test is a test to measure learning and memory abilities. The maze is a white disk with a diameter of 89 cm and a height of 140 cm, which has 20 escape holes with a diameter of 5 cm. One of the escape holes has a black safety zone at the bottom, which is designed to be used as an escape area for experimental animals.

[0110] The disease model mouse prepared in Example 3-1 was placed in the center of the disk in the test maze prepared above. After that, strong light was irradiated, and the time it took for the mouse to avoid the light and find the dark safe area was measured. This was repeated once a day for 4 days.

[0111] Furthermore, the expression of brain-derived neurotrophic factor (BDNF) in the mouse hippocampus and blood was measured by the method of Example 2. The results are shown in Table 9 below.

[0112] [Table 9]

[0113] As shown in Table 9, the group administered the mixed composition of Mamedao extract, Cuscuta extract, and NK112 showed improved results compared to the control group in the Y-maze test, novel object exploration test, and Barnes maze test, and it was confirmed that the expression of brain-derived neurotrophic factor (BDNF), which decreases in aging and dementia, was also increased in a dose-dependent manner. This shows that the mixed composition of Mamedao extract, Cuscuta extract, and Lactobacillus paracasei NK112 has an excellent effect of improving cognitive function. EXAMPLES

[0114] Confirmation of synapse strengthening effect of the mixed composition A composition of 3:1:0.5 mixture of Mamedaoshi extract, Cuscuta japonica extract, and NK112P was orally administered to mice at 10, 30, or 100 mg / kg for 21 days, and 200 mg / kg of piracetam, an agent for improving cognitive impairment symptoms, was intraperitoneally administered to the positive control group. The mice were perfused, fixed with paraformaldehyde, and the brains were removed. Frozen sections were prepared, and then the hippocampal area was selected, washed three times with PBS, treated with hydrogen peroxide to remove endogenous peroxidase, and reacted with mouse anti-synaptophysin antibody, the primary antibody, overnight. The tissue was then reacted with a secondary antibody, biotinylated anti-mouse, for 90 minutes, then subjected to an ABC reaction (1:100 dilution) for 1 hour, and developed with DAB solution (3,3-diaminobenzidine tetrahydrochloride) for 3 minutes. Between each step, the tissue was washed three times with PBS. After the staining reaction, the tissue was placed on a gelatin-coated slide and dehydrated with 70-100% ethanol and xylene, after which the tissue was covered with a cover slide and stored.

[0115] The stained brain tissue was observed in the Cornu Ammonis region of the hippocampus under an optical microscope (Olympus Microscope System BX51; Olympus, Tokyo, Japan). The photograph is shown in Figure 5A. The amount of synaptic protein is shown in the graph in Figure 5B.

[0116] As shown in Figures 5A and 5B, the amount of synaptic protein in the groups administered 10, 30, and 100 mg / kg of the composition obtained by mixing the above-mentioned Mamedao extract, Cuscuta extract, and NK112P in a ratio of 3:1:0.5 increased by 134.6%, 120.21%, and 132.37%, respectively, compared to normal mice. This confirmed that the mixed composition containing Mamedao extract, Cuscuta extract, and Lactobacillus paracasei NK112 has excellent effects on improving cognitive function and memory by promoting synapse formation. EXAMPLES

[0117] Confirmation of the nerve regeneration effect of the mixed composition (increase in the number of neuroblast cells and the length of neurites) The test was performed as in Example 12, except that goat anti-doublecortin (DCX) antibody was used as the primary antibody and biotinylated anti-goat antibody was used as the secondary antibody. The stained brain tissue was observed as in Example 12, but the effects on proliferation of dentate gyrus (DG) cells, neuronal differentiation, and neurite outgrowth were observed. A photograph of doublecortin-positive cells observed at 200x magnification is shown in Figure 6A, and the number of neuroblasts and the length of neurites are shown in Figures 6B and 6C.

[0118] As shown in Figure 6B, the number of neuroblasts in the groups administered with the composition at 30 and 100 mg / kg was increased by 173.63% and 175.42%, respectively, compared to normal mice, which was 30.38% and 31.73% higher than the positive control group administered with piracetam. Also, as shown in Figure 6C, the length of the neurites of neuroblasts in the groups administered with the composition was increased by 132.27% and 137.82%, respectively, which was similar to the positive control group.

[0119] From this, it can be seen that the mixed composition of Japanese bean extract, dodder extract, and Lactobacillus paracasei NK112 has an excellent effect on improving cognitive function by promoting the proliferation of nerve cells. EXAMPLES

[0120] Confirmation of the intestinal regulation effect of the mixed composition The mice subjected to behavioral evaluation in Example 11 were anesthetized, and the intestinal length and inflammatory response indicator substances were measured in the same manner as in Example 6. The results are shown in Figures 7A to 7D.

[0121] As shown in Figures 7A to 7D, in the group administered E. coli (EcV), intestinal length decreased and inflammation index values ​​such as MPO activity, TNF-α, IL-6, and p65 worsened, whereas in the group administered E. coli together with a composition containing a mixture of 3:1:0.5 ratio of bean sprout extract, dodder extract, and NK112P at 10 or 50 mg / kg (TSL), intestinal length recovered to the same level as the normal group (CoV), and inflammation index values ​​improved.

[0122] Therefore, it is clear that the mixed composition of Japanese bean extract, dodder extract, and Lactobacillus paracasei NK112 has an excellent intestinal regulating effect.

[0123] [Table 10]

Claims

1. Lactobacillus paracasei strain NK112, deposited under accession number KCCM12566P.

2. The Lactobacillus paracasei NK112 strain according to claim 1, characterized in that it has a preventive or therapeutic effect on cognitive dysfunction or intestinal dysfunction.

3. The Lactobacillus paracasei NK112 strain according to claim 1, characterized in that the cognitive dysfunction is at least one selected from the group consisting of Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, depression, anxiety, stroke, cerebral ischemia, encephalitis, amnesia, traumatic brain injury, Wernicke-Korsakoff syndrome, electroencephalopathy, epilepsy, hippocampal sclerosis, cerebral senility, dementia, frontotemporal lobar degeneration, normal pressure hydrocephalus, memory loss, and learning disability.

4. The Lactobacillus paracasei NK112 strain according to claim 1, characterized in that the intestinal functional disorder is at least one selected from the group consisting of inflammatory bowel disease, intestinal peristalsis disorder, intestinal obstruction, colonic inertia, irritable bowel disease, dyspepsia, abdominal distension, diarrhea, and constipation.

5. A pharmaceutical composition for preventing or treating cognitive dysfunction or intestinal dysfunction, comprising at least one selected from the group consisting of Lactobacillus paracasei NK112 strain, Cuscuta australis R.Br. extract, and Cuscuta japonica Choisy extract.

6. The pharmaceutical composition for preventing or treating cognitive dysfunction or intestinal dysfunction according to claim 5, comprising Lactobacillus paracasei NK112 strain, an extract of Japanese bean sprouts, and an extract of dodder.

7. The pharmaceutical composition for preventing or treating cognitive dysfunction or intestinal dysfunction according to claim 6, characterized in that it contains a weight ratio of 1-6:1:0.1-1 of a mixture of a dodder extract, a dodder extract, and a Lactobacillus paracasei NK112 strain.

8. The pharmaceutical composition for preventing or treating cognitive dysfunction or intestinal dysfunction according to claim 6, characterized in that it contains a weight ratio of 2 to 4:1:0.3 to 0.7 of a mixture of a dodder extract, a dodder extract, and a Lactobacillus paracasei NK112 strain.

9. The pharmaceutical composition for preventing or treating cognitive dysfunction or intestinal dysfunction according to claim 5, characterized in that it promotes the expression of brain-derived neurotrophic factor (BDNF).

10. The pharmaceutical composition for preventing or treating cognitive dysfunction or intestinal dysfunction according to claim 5, characterized in that it promotes expression of nerve growth factor (NGF).

11. 6. The pharmaceutical composition for preventing or treating cognitive dysfunction or intestinal dysfunction according to claim 5, wherein the cognitive dysfunction is at least one selected from the group consisting of Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Pick's disease, Creutzfeldt-Jakob disease, depression, anxiety, stroke, cerebral ischemia, encephalitis, amnesia, traumatic brain injury, Wernicke-Korsakoff syndrome, electroencephalopathy, epilepsy, hippocampal sclerosis, cerebral senility, dementia, frontotemporal lobar degeneration, normal pressure hydrocephalus, memory loss, and learning disability.

12. 6. The pharmaceutical composition for preventing or treating cognitive impairment or intestinal dysfunction according to claim 5, wherein the intestinal dysfunction is at least one selected from the group consisting of inflammatory bowel disease, intestinal peristalsis disorder, intestinal obstruction, colonic inertia, irritable bowel disease, dyspepsia, abdominal distension, diarrhea, and constipation.

13. A food composition for preventing or improving cognitive dysfunction or intestinal dysfunction, comprising at least one selected from the group consisting of Lactobacillus paracasei NK112 strain, a Japanese quince extract, and a dodder extract.

14. The food composition for preventing or improving cognitive dysfunction or intestinal dysfunction according to claim 13, characterized in that it contains Lactobacillus paracasei NK112 strain, a Japanese quince extract, and a dodder extract.

15. An animal feed composition for preventing or improving cognitive dysfunction or intestinal dysfunction, comprising at least one selected from the group consisting of Lactobacillus paracasei NK112 strain, a Japanese bean extract, and a dodder extract.

16. The animal feed composition for preventing or improving cognitive dysfunction or intestinal dysfunction according to claim 15, characterized in that it contains Lactobacillus paracasei NK112 strain, a Japanese bean extract, and a dodder extract.

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