WO2023204358A1 - Food and pharmaceutical composition containing probiotics for preventing or treating dementia - Google Patents

Abstract

The present invention relates to a food composition or a pharmaceutical composition for preventing or treating dementia, the composition comprising Bifidobacterium bifidum BGN4 (KCCM12754P) and Bifidobacterium longum BORI (KCCM-10492).

Description

Food and pharmaceutical compositions for preventing or treating dementia containing probiotics

The present invention relates to a composition for preventing or treating dementia containing probiotics, and more specifically, to a composition containing probiotics , Bifidobacterium bifidum BGN4 (KCCM12754P) strain and Bifidobacterium longum BORI (KCCM- 10492) relates to a food composition and pharmaceutical composition for preventing or treating dementia containing the strain.

With recent advances in medical technology, human life expectancy is increasing. Dementia, which is more likely to occur as people age, devastates life in old age and brings misery to families.

Dementia is a disease characterized by overall impairment of mental function, including memory impairment and loss of judgment. In Korea, the prevalence of dementia among seniors aged 65 or older is approximately 8%, and the number of dementia patients is expected to increase rapidly as the population ages.

As the population ages, there is an urgent need to prepare measures against dementia, and in 2010, the cost of elderly dementia care increased by 32% compared to the previous year. According to the Health Insurance Review and Assessment Service, which announced the 2010 medical cost statistical index, medical expenses for seniors aged 65 years or older increased by 14.5% from the previous year to KRW 13.7847 trillion in 2010, and among them, medical expenses for dementia, which is Alzheimer's disease, increased by 32.2% to KRW 290.3 billion, the largest increase. It was reported that a high rate of increase was recorded. (Health Insurance Review and Assessment Service).

Alzheimer's disease (AD) accounts for the largest proportion of dementia types, approximately 70%. Although the cause of senile dementia disease is not clear, close observation of brain tissue in Alzheimer's disease shows that cholinergic nerves are severely damaged. Based on this, many attempts are being made to inhibit the activity of acetylcholinesterase, which induces a decrease in the function of acetylcholine.

Currently, acetylcholinesterase inhibitors such as tacrine, donepezil, rivastimine, and galantamine are used as treatments for Alzheimer's disease. However, because such compound-based drugs are accompanied by side effects, there is a need to develop safer and more effective natural product-based materials.

Meanwhile, trillions of microorganisms coexist in the human intestine, and according to several recent discoveries or papers, intestinal microorganisms are reported to have a close influence on cognitive function and emotional state while constantly communicating with the human brain. However, no specific research has yet been conducted on the impact of intestinal microorganisms and probiotics on the process of dementia.

The present invention seeks to develop and provide a composition containing probiotics with excellent dementia prevention or treatment efficacy.

The present invention provides a food composition for improving dementia containing the Bifidobacterium bifidum BGN4 (KCCM12754P) strain and the Bifidobacterium longum BORI (KCCM-10492) strain.

In addition, the present invention provides a pharmaceutical composition for preventing or treating dementia containing Bifidobacterium bifidum BGN4 (KCCM12754P) strain and Bifidobacterium longum BORI (KCCM-10492) strain. .

In the present invention, it was confirmed that the probiotic (BGN and BORI mixture) composition of the present invention has an excellent effect in improving, preventing or treating dementia. Therefore, the present invention can be usefully used as a health functional food or pharmaceutical composition for preventing or treating dementia in the elderly.

Figure 1 shows immunofluorescence results for ChAT- and NeuN-positive cells (A) and NeuN+ in the CA3 portion of the hippocampus of control or dementia mouse models (5xFAD mice) treated with B. bifidum BGN4 and B. longum BORI (BGN4/BORI). (B) and ChAT+/NeuN+(C) quantification data of neurons.

Figure 2 shows immunofluorescence results of ChAT and NeuN positive cells (A) and quantification of NeuN+ (B) and ChAT+/NeuN+ (C) neurons in the CA1 hippocampus of control or 5xFAD mice treated with B. bifidum BGN4 and B. longum BORI. It's data.

Figure 3 shows immunofluorescence results of ChAT and NeuN positive cells (A) and quantification of NeuN+ (B) and ChAT+/NeuN+ (C) neurons in the CA2 hippocampus of control or 5xFAD mice treated with B. bifidum BGN4 and B. longum BORI. It's data.

Figure 4 shows (A) immunofluorescence results of Map2 and BDNF positive cells in the CA3 hippocampus of control or 5xFAD mice treated with B. bifidum BGN4 and B. longum BORI (BGN4/BORI), and Map2+ in the CA3 hippocampal region. This is data quantifying the number of /BDNF+ neurons (B).

Figure 5 shows (C) the results of Western blot analysis of scaffolding proteins PSD95 and Homer1 and BDNF in the hippocampus of control or 5xFAD mice treated with B. bifidum BGN4 and B. longum BORI. BDNF (B), PSD95, and Homer1 (C) expression proteins were quantified by β-actin expression levels.

Figure 6 is an immunofluorescence result showing amyloid-β42 accumulation within neurons in the CA3 hippocampus of control or 5xFAD mice treated with B. bifidum BGN4 and B. longum BORI (A), and amyloid-β42+ (B) in the CA3 hippocampal region. This is cell quantification data.

Figure 7 is an immunofluorescence result (A) showing cleaved caspase-3 positive cells within neurons in the CA3 hippocampus of control or 5xFAD mice treated with B. bifidum BGN4 and B. longum BORI, and cleaved caspase-3 in the CA3 hippocampal region. +(B) Quantification data of cells.

Figure 8 shows data showing immunodetection of the proteins APP, C99, and C83 by Western blot using protein lysates extracted from the hippocampus of control or 5xFAD mice treated with B. bifidum BGN4 and B. longum BORI. C99 (B) and C83 (C) expressed proteins were quantified by β-actin expression levels.

Figure 9 shows mRNA of neuroinflammation-related cytokines, IL-17 (A), IL-1β (B), and IL-6 (C) in the hippocampus of control or 5xFAD mice treated with B. bifidum BGN4 and B. longum BORI. This data quantifies expression through RT-PCR.

Figure 10 is a quantitative RT-PCR analysis of the anti-inflammatory cytokine, IL-10 (A) in the hippocampus of control or 5xFAD mice treated with B. bifidum BGN4 and B. longum BORI, and the gene related to the neuroinflammatory pathway, NF-kB. This is a graph showing quantitative RT-PCR analysis of COX2 (B).

Figure 11 is a graph quantifying the concentrations of TNF-α (A) and IL-12 (B) in the serum of control or 5xFAD mice treated with B. bifidum BGN4 and B. longum BORI through ELISA analysis.

Figure 12 shows immunodetection of Iba1 and Gfap proteins by Western blot analysis using protein lysates derived from the hippocampus. Iba1 and Gfap expression proteins were quantified by β-actin expression levels.

Figure 13 is (A) data showing the cognitive improvement effect through behavioral experiments in control and 5xFAD mice by treatment with B. bifidum BGN4 and B. longum BORI in the Y-maze test.

Figure 14 is (A) a representative heatmap showing mouse behavior according to each condition in the fear conditioning test.

Figure 15 is a graph quantifying the effect of (A) B. bifidum BGN4 and B. longum BORI processing on a contextual fear conditioning test.

Figure 16 is (A) a representative heatmap showing the time spent in the circular pool by control and 5xFAD mice treated with B. bifidum BGN4 and B. longum BORI in the Morris water maze test, a long-term memory test.

Figure 17 is a graph quantifying the time spent in each branch by performing a free swimming test after removing the escape zone in the (A) Morris water maze test.

Figure 18 is (A) a graph comparing the microbial composition between each group (1-5 week samples) according to BGN4/BORI intake of 5xFAD or control mice. * Indicates a linear discriminant analysis (LDA) score >3.0.

Figure 19 is a table showing microorganisms with expression differences in the microbial taxa (A) between 5xFAD and BGN4/BORI-treated 5xFAD mice and the microbial taxa (B) between control and BGN4/BORI-treated control mice by LEfSe analysis. .

Figure 20 shows (A) changes in microbial composition at the genus level in each group over BGN4/BORI treatment time (1-5 weeks).

Figure 21 is (E) a heatmap showing Spearman's rank correlation showing the relationship between intestinal bacterial abundance and time according to each condition at the bacterial genus level.

The present invention provides a food composition for improving dementia containing the Bifidobacterium bifidum BGN4 (KCCM12754P) strain and the Bifidobacterium longum BORI (KCCM-10492) strain.

In addition, the present invention provides a pharmaceutical composition for preventing or treating dementia containing Bifidobacterium bifidum BGN4 (KCCM12754P) strain and Bifidobacterium longum BORI (KCCM-10492) strain. .

In the food composition or pharmaceutical composition of the present invention, the dementia may be caused by Alzheimer's disease.

The Bifidobacterium bifidum BGN4 (KCCM12754P) strain used in the present invention is a strain deposited with the Korea Microbial Conservation Center (overseas) on June 24, 2020, and the accession number is KCCM12754P.

The Bifidobacterium longum BORI (KCCM-10492) strain used in the present invention is a strain deposited with the Korea Microorganism Conservation Center (Overseas) on May 6, 2003, and the accession number is KCCM-10492.

In the present invention, the food composition is not necessarily limited to a specific formulation, and may be formulated in the form of capsules, tablets, powders, granules, liquids, pills, flakes, pastes, syrups, gels, jelly or bars. It may have been done. Additionally, it may be manufactured in the form of general food by adding it to food ingredients such as beverages, teas, spices, gum, and confectionery.

In the present invention, the pharmaceutical composition can be prepared in various forms for parenteral or oral administration according to known methods, and a representative example of the formulation for parenteral administration is an isotonic aqueous solution or suspension for injection. Injectable formulations can be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component can be dissolved in saline solution or buffer solution and formulated for injection. Additionally, dosage forms for oral administration include, but are not limited to, powders, granules, tablets, pills, and capsules.

In addition, the pharmaceutical composition formulated in the above manner can be administered in an effective amount through various routes including orally, transdermally, subcutaneously, intravenously, or intramuscularly. The 'effective amount' in the above refers to the amount that exhibits a preventive or therapeutic effect when administered to a patient. As an example, the pharmaceutical composition of the present invention can be administered at least once a day at 0.00001 to 100 mg/kg (body weight) based on the active ingredient. However, the dosage of the pharmaceutical composition according to the present invention can be appropriately selected depending on the administration route, administration target, age, gender, weight, individual differences, and disease state.

In the present invention, the Bifidobacterium bifidum BGN4 (KCCM12754P) strain and the Bifidobacterium longum BORI (KCCM-10492) strain are mixed and used at a ratio of 1 to 0.8 to 1.2. good night. According to the following examples, when mixed and used in the above ratio, the efficacy is excellent in improving, preventing or treating dementia.

Hereinafter, the contents of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, and includes modifications of the technical idea equivalent thereto.

[Example 1. In Alzheimer's model mice B. bifidum BGN4 and B. longum Verification of alleviation of neuronal death in the hippocampus portion of the mouse brain by BORI treatment]

To evaluate the effects of B. bifidum BGN4 and B. longum BORI (BGN4/BORI) in the 5xFAD Alzheimer's mouse model, 3-month-old 5xFAD mice were treated with BGN4/BORI for 30 days (once a day, BGN4 in 0.2ml sterile water). /BORI ( ^0.5 After BGN4/BORI treatment, a significant increase in the number of ChAT+/NeuN+ neurons was observed in the CA3 region of the hippocampus of control and 5xFAD mouse brains (Figure 1). In addition, the number of ChAT+/NeuN+ cells was significantly increased in the CA1 region of the hippocampus of the BGN4/BORI-treated 5xFAD mouse brain (Figure 2). However, no changes in NeuN+ and ChAT+/NeuN+ cell numbers were observed in the CA2 region of the hippocampus of 5xFAD mouse brains (Figure 3). These data indicate that intake of B. bifidum BGN4 and B. longum BORI inhibited neuronal cell death in the CA3 and CA1 regions of the hippocampus of the brain in a mouse model of dementia.

[Example 2. In Alzheimer's model mice B. bifidum BGN4 and B. longum Verification of alleviation of the pathogenesis of Alzheimer's disease by BORI]

Next, to evaluate the effect of B. bifidum BGN4 and B. longum BORI on functional synaptic plasticity, we evaluated the expression level of BDNF, which regulates hippocampal synaptic plasticity and plays an important role in cognition. We confirmed that the number of Map2+/BDNF+ neurons in the hippocampus of 5xFAD mice treated with BGN4/BORI (treated at the level of Example 1 above) was significantly increased compared to 5xFAD mice (FIG. 4). Quantitative Western blot analysis confirmed that protein expression of BDNF increased in the hippocampus of 5xFAD mice treated with BGN4/BORI (Figure 5). Additionally, the expression of synaptic scaffolding proteins, including PSD95 and Homer1, was significantly restored in BGN4/BORI-treated 5xFAD mice (Figure 5), suggesting that B. bifidum BGN4 and B. longum BORI suppress synaptic deterioration in an Alzheimer's mouse model. It has been proven to improve plasticity.

Next, we investigated whether treatment with B. bifidum BGN4 and B. longum BORI could improve dementia-related phenotypes in the hippocampus of 5xFAD mice. At 4 weeks after oral administration of BGN4/BORI, reduced amyloid-β42 positive cells were confirmed in the hippocampus of 5xFAD mice (Figure 6). In addition, the number of cleaved caspase-3 positive cells, a marker expressed during cell death, was confirmed to be significantly reduced in BGN4/BORI-treated 5xFAD mice (Figure 7). As a result of evaluating the expression of C99 and C83 proteins using APP c-terminal antibody, C99 protein expression was significantly decreased in the hippocampus of BGN4/BORI-treated 5xFAD mice (Figure 8). These results suggest that the pathological symptoms of Alzheimer's disease were attenuated by administration of B. bifidum BGN4 and B. longum BORI.

[Example 3. Neuroinflammatory response in 5xFAD mice B. bifidum BGN4 and B. longum Verification of the effectiveness of BORI]

Next, we investigated the effects of B. bifidum BGN4 and B. longum BORI on the brain neuroinflammatory response in Alzheimer's disease mice. First, BGN4/BORI significantly reduced the expression of IL-17 and IL-6 in 5xFAD mice (treated at the levels in Example 1 above), whereas no differences were observed in the expression of IL-1β and IL-10. (Figures 9, 10). In addition, when the expression of NF-kB and COX2, which are neuroinflammation-related pathway factors, was measured by BGN4/BORI, NF-kB expression in 5xFAD mice treated with BGN4/BORI (treated at the level of Example 1 above) was at the control level and was similarly reduced (Figure 10). In addition, it was confirmed that the expression of COX2 was significantly reduced in 5xFAD mice treated with probiotics (FIG. 10). Additionally, the concentrations of pro-inflammatory proteins TNF-α and IL-12 in serum were significantly restored in 5xFAD mice treated with BGN4/BORI (treated at the level of Example 1 above) (FIG. 11). Finally, the expression of the microglial marker, Iba1, and the astrocyte marker, Gfap, were significantly reduced in the hippocampus of 5xFAD mice treated with BGN4/BORI (treated at the level of Example 1 above) (FIG. 12). These results suggest that the neuroinflammatory response in the dementia mouse brain was improved by treatment with B. bifidum BGN4 and B. longum BORI.

[Example 4. In 5xFAD mouse B. bifidum BGN4 and B. longum [Verification of cognitive and memory improvement effects after BORI treatment]

To investigate whether cognitive deficits can be recovered by treatment with B. bifidum BGN4 and B. longum BORI, the Y-maze test was performed to evaluate spatial recognition memory. Surprisingly, 5xFAD mice treated with BGN4/BORI (treated at the level of Example 1 above) showed improved cognitive behavior compared to untreated 5xFAD mice (FIG. 13). Additionally, in a contextual fear conditioning test, we found that the freezing rate was significantly increased in 5xFAD mice treated with BGN4/BORI (treated at the level of Example 1 above) (FIGS. 14, 15). Next, we evaluated whether dementia-related memory loss could be alleviated by BGN4/BORI using the Morris water maze test. Importantly, in platform-free probe testing, we found that BGN4/BORI treated (treated at the level of Example 1 above) 5xFAD mice increased the time spent in the target quadrant compared to untreated 5xFAD mice. (Figures 16, 17). These results demonstrated that treatment with B. bifidum BGN4 and B. longum BORI was an effective invention for improving Alzheimer's-related memory deficits.

[Example 5. In 5xFAD mouse B. bifidum BGN4 and B. longum [Verification of differences in intestinal microbial community after BORI treatment]

To evaluate the effects of B. bifidum BGN4 and B. longum BORI on the intestinal bacterial community, mouse fecal microbiota profiles were analyzed for 5 weeks. To collect mouse stool samples, stool samples were collected directly from mice treated with BGN4/BORI (treated at the level of Example 1 above) once a week for 5 weeks. After analyzing the microbiome in the samples, we found that the 5xFAD mouse group treated with BGN4/BORI (treated at the level of Example 1 above) showed a decrease in the bacterial genera Parvibacter, Incertae_Sedis and Oscillibacter, while the control group showed a decrease in Akkermansia and Faecalibacterium. , Erysipelatoclostridium and Candidatus_Stoquefichu were confirmed to increase (Figure 18). In addition, control mice treated with BGN4/BORI (treated at the level of Example 1 above) showed a lower relative abundance of NK4A214_group, Alistipes, Lachnoclostridium, Desulfovibrio and Peptococcaceae families compared to untreated control mice, The most relevant change was confirmed to be an increase in the Akkermansia genus (Figure 19). In addition, it was shown that the intestinal bacterial species varied depending on the treatment period of B. bifidum BGN4 and B. longum BORI (Figure 20). In particular, Akkermansia was reduced in BGN4/BORI-treated 5xFAD mice (treated at the level of Example 1 above) (0.25%) compared to 5xFAD mice not treated with BGN4/BORI (1.21%). It tended to increase at weeks 1-3 in 5xFAD mice treated with BGN4/BORI (1.83-5.86%) and to decrease in the untreated 5xFAD group (0.15-0.51%) (FIG. 20). The relative abundance of Akkermansia was strongly correlated with the duration of probiotic treatment (ρ = -0.81), and the significant decline was alleviated in the group of 5xFAD mice treated with BGN4/BORI (ρ = -0.26) (Figure 21). Treatment with B. bifidum BGN4 and B. longum BORI alters the gut microbiome genome in 5x FAD mice, suggesting their impact on Alzheimer's pathogenesis and cognitive improvement.

Claims (2)

A food composition for improving dementia comprising Bifidobacterium bifidum BGN4 (KCCM12754P) strain and Bifidobacterium longum BORI (KCCM-10492) strain. A pharmaceutical composition for preventing or treating dementia comprising Bifidobacterium bifidum BGN4 (KCCM12754P) strain and Bifidobacterium longum BORI (KCCM-10492) strain.

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