Application of bilberry anthocyanin in preparation of medicine for regulating intestinal flora and improving Alzheimer's disease

Abstract

The invention provides application of bilberry anthocyanin in preparation of a medicine for regulating intestinal flora and improving Alzheimer's disease, and belongs to the field of biological medicine. According to the present invention, with the application of the bilberry anthocyanin, the relative abundance of the flora such as Hungata heath wayi, Pseudoflavanonifr sp. An85, Dorea sp. 5-2, Anerotrunk sp. G3 (2012) and Maillard massiliensis in the intestinal tract can be improved, the improvement of the Alzheimer's disease can be achieved by adjusting the intestinal tract flora, and the verification is performed by detecting the cognitive function, the A [beta] 1-42 level and the neuroinflammation. The invention solves the technical blank that the improvement of the Alzheimer's disease by optimizing the specific flora of the intestinal tract through a natural compound is not clearly proposed in the prior art.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of bilberry anthocyanidins in the preparation of drugs for regulating intestinal flora and improving Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a highly prevalent neurodegenerative disease with a complex and incompletely understood etiology. Currently, there is a lack of effective cures. Existing treatments primarily focus on symptomatic treatments, such as alleviating cognitive decline and improving psychiatric and behavioral symptoms. Due to the lack of a multidimensional, comprehensive treatment strategy, existing therapies are unable to meet the growing clinical demand. In recent years, with the deepening of research on the regulatory mechanisms of the gut-brain axis, the gut microbiota has been shown to play a key role in the development and progression of Alzheimer's disease, providing a new research direction for Alzheimer's disease intervention. Existing studies and patents have explored modulating the gut microbiota to intervene in Alzheimer's disease. For example, Bacillus coagulans JA845 has been proposed to improve learning and memory, reduce neuronal cell damage, reduce Aβ deposition, and regulate the gut microbiota. Fecal microbiota transplantation (FMT), as an important means of regulating the gut microbiota, is also considered to have potential application value in the treatment of Alzheimer's disease. However, existing technologies have not yet clearly proposed the use of natural compounds to optimize specific intestinal flora to achieve the treatment of Alzheimer's disease. Therefore, how to achieve efficient and specific regulation of intestinal flora to obtain more significant Alzheimer's disease treatment effects remains a key issue that needs to be solved urgently. Summary of the Invention

[0003] The present invention solves the technical gap in the prior art that has not yet clearly proposed to improve Alzheimer's disease by optimizing its specific intestinal flora through natural compounds.

[0004] The invention relates to the use of bilberry anthocyanins in the preparation of a drug for regulating intestinal flora, wherein the regulation is to increase the relative abundance of the flora; the flora includes Hungatella hathewayi, Pseudoflavonifractor sp. An85, Dorea sp.5-2, Anaerotruncus sp. G3(2012) and Mailhella massiliensis.

[0005] Preferably, the concentration of bilberry anthocyanidins is 1×10 5 mg / L.

[0006] Preferably, the dosage of the bilberry anthocyanidin is 20 mg / kg.

[0007] Preferably, it is characterized in that the administration time of the bilberry anthocyanidin is 3 months.

[0008] Preferably, the bilberry anthocyanidins are administered by gavage.

[0009] A drug for regulating intestinal flora, characterized in that the drug contains bilberry anthocyanidins; the regulation is to increase the relative abundance of the flora; the flora includes Hungatella hathewayi, Pseudoflavonifractor sp.An85, Dorea sp. 5-2, Anaerotruncus sp. G3(2012) and Mailhella massiliensis.

[0010] Application of bilberry anthocyanins in the preparation of drugs for improving Alzheimer's disease.

[0011] A drug for improving Alzheimer's disease, comprising bilberry anthocyanidin.

[0012] Beneficial effects 1. The present invention increases the relative abundance of Hungatella hathewayi, Pseudoflavonifractor sp. An85, Dorea sp. 5-2, Anaerotruncus sp. G3(2012) and Mailhella massiliensis flora in the intestine through bilberry anthocyanins.

[0013] 2. The present invention improves the relative abundance of Hungatella hathewayi, Pseudoflavonifractor sp. An85, Dorea sp. 5-2, Anaerotruncus sp. G3 (2012) and Mailhella massiliensis in the intestine by using bilberry anthocyanins, thereby achieving the improvement of Alzheimer's disease. The present invention detects cognitive function, Aβ 1-42 Levels of inflammatory bowel disease and neuroinflammation were verified as follows: Improved cognitive function: Compared with the Pgf(APP / PSEN1) group, mice in the Pgf(APP / PSEN1-BA) group showed shorter escape latency and longer target quadrant residence time in the Morris water maze test, indicating that cognitive function was significantly improved.

[0014] Reduce Aβ 1-42 Level: Serum Aβ in Pgf(APP / PSEN1-BA) group mice 1-42The deposition level of APP was significantly lower than that of the Pgf(APP / PSEN1) group, suggesting that it can alleviate the core pathological burden of Alzheimer's disease.

[0015] Alleviate neuroinflammation: Immunohistochemistry results will show that the number of activated astrocytes in the brains of mice in the Pgf (APP / PSEN1-BA) group is reduced, and their inflammatory activation state is alleviated, thereby inhibiting neuroinflammatory responses and protecting neurons.

[0016] 3. The present invention provides a novel intervention strategy for Alzheimer's disease by pre-regulating the microbiota with non-drug (or natural product) methods for fecal microbiota transplantation, which has potential clinical translational value and provides new ideas for the prevention and improvement of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of differential intestinal flora are shown, where A is Hungatella hathewayi, B is Pseudoflavonifractor sp. An85, C is Anaerotruncus sp. G3(2012), D is Mailhella massiliensis, and E is Dorea sp. 5-2; Figure 2 The escape latency results of different experimental groups; Figure 3 Results of target quadrant movement time for different experimental groups; Figure 4 Serum Aβ in different experimental groups 1-42 Level results; Figure 5 Results are for hippocampal neuroinflammation levels; Figure 6 These are the expression results of microglia (ionized calcium binding adaptor molecule 1, IBA1) and astrocytes (glial fibrillary acidic protein, GFAP) in the prefrontal cortex. A is the immunofluorescence double-fluorescence image of the prefrontal cortex, B is the quantitative result of microglia, and C is the quantitative result of astrocytes. DETAILED DESCRIPTION

[0018] 1. Experimental methods

[0019] 1. Source and dosage of bilberry anthocyanins: Bilberry anthocyanins should be derived from standardized extracts with high purity (≥98%, pH differential method), and the optimal dosage and treatment duration for regulating the intestinal flora of APP / PSEN1 mice should be determined.

[0020] 2. Statistical analysis: The experimental data should be analyzed using appropriate statistical methods (such as ANOVA) to ensure the scientificity and reliability of the results.

[0021] Example 1. I. Preparation of experimental animals: Healthy C57BL6J mice at 6 - 8 weeks old (SCXK(Beijing)2017 - 0005) were selected as recipient mice. All mice should be housed in a specific pathogen - free (SPF) animal room to ensure environmental consistency and flora control. All recipient mice were treated with continuous intragastric administration of ceftriaxone sodium (dose: 50 mg / kg / d) for 7 days to maximize the clearance of their intestinal flora and establish a sterile or low - flora state. The mice after antibiotic treatment should be housed separately, and the fecal flora should be monitored regularly to confirm the sterile state.

[0022] II. Preparation of donor fecal suspension: Standards for fecal suspension preparation: Strict standard operating procedures (SOP) for fecal collection, treatment, preservation, and intragastric administration were formulated to ensure the consistency of flora activity and dose for each intragastric administration. Consider using liquid nitrogen to preserve the fecal suspension to ensure stability between batches.

[0023] (1) Control fecal suspension (without anthocyanin treatment): Fresh feces of APP / PSEN1 transgenic Alzheimer's disease model mice (SCXK(Beijing)2014 - 0004) were collected. Under anaerobic conditions, the fresh feces were thoroughly mixed with sterile saline at a certain ratio (10 mL of sterile saline was added to 1 g of feces) to prepare the fecal suspension. The suspension should be filtered to remove large particles and ensure the flora activity; (2) Vaccinium myrtillus anthocyanin - regulated fecal suspension: Fresh feces of APP / PSEN1 transgenic Alzheimer's disease model mice (SCXK(Beijing)2014 - 0004) that had been fed with Vaccinium myrtillus anthocyanin (concentration of Vaccinium myrtillus anthocyanin: 1×10 5 mg / L, dosage: 20 mg / kg) for 3 months were collected. Under anaerobic conditions, the fresh feces were thoroughly mixed with sterile saline at a certain ratio (10 mL of sterile saline was added to 1 g of feces) to prepare the fecal suspension. The suspension should be filtered to remove large particles and ensure the flora activity.

[0024] III. Detection of donor fecal suspension.

[0025] In this invention, metagenomic sequencing was performed on the Vaccinium myrtillus anthocyanin - regulated fecal suspension to detect the intestinal flora, deeply analyze how anthocyanins change the intestinal flora composition, and what metabolites are produced by these changed flora, and the results are as follows Figure 1By treating APP / PSEN1 Alzheimer's disease (AD) model mice with bilberry anthocyanins, researchers discovered for the first time that five gut microbiota species were significantly elevated in their feces: Hungatella hathewayi, Pseudoflavonifractor sp. An85, Dorea sp.5-2, Anaerotruncus sp. G3(2012), and Mailhella massiliensis. This study demonstrates that bilberry anthocyanins can effectively increase the relative abundance of these five gut microbiota species. Among them, Hungatella hathewayi, belonging to the Firmicutes phylum, may inhibit neuroinflammation (such as reducing TNF-α and IL-1β) through short-chain fatty acids (SCFAs); Pseudoflavonifractor sp. An85 may be involved in the metabolism of polyphenolic compounds and enhance the bioavailability of anthocyanins; Dorea sp. 5-2 is known to be related to intestinal immune homeostasis; Anaerotruncus sp. G3 (2012) may reduce systemic inflammation by regulating intestinal barrier function; Mailhella massiliensis can maintain intestinal flora diversity. However, none of the above five flora have been reported to be related to Alzheimer's disease (AD).

[0026] 4. Grouping and gavage: To further validate the results, the recipient mice were randomly divided into four groups after antibiotic treatment and gavage-treated for one month: a blank control group (CON): sterile saline was not sterilized and gavage-treated daily; a sterile control group (Pgf): sterile saline was gavage-treated daily after antibiotic treatment to serve as a baseline control; a fecal control group (Pgf(APP / PSEN1)): fecal suspension from APP / PSEN1 mice was gavage-treated daily after antibiotic treatment to establish an AD model and observe its pathological progression; and an anthocyanin-modulated fecal microbiota transplantation group (Pgf(APP / PSEN1-BA)): fecal suspension from APP / PSEN1 mice that had been fed bilberry anthocyanins (20 mg / kg) for three months after antibiotic treatment was gavage-treated daily. The gavage dose per mouse was 0.2 mL once daily.

[0027] 5. Behavioral evaluation: One month after oral administration, mice in each group underwent Morris water maze behavioral testing to assess their spatial learning and memory abilities. Key evaluation indicators included escape latency and target quadrant movement time.

[0028] The results of the escape latency of mice in the Morris water maze behavioral test are as follows Figure 2On the fifth day, the escape latency of the four groups of mice was ranked from longest to shortest as follows: control feces group, sterile control group, blank control group and anthocyanin-regulated fecal microbiota transplantation group. The escape latency of the mice in the anthocyanin-regulated fecal microbiota transplantation group was significantly shortened by about 50% compared with the control feces group (the fifth day of the control feces group vs Anthocyanin-regulated fecal microbiota transplantation group: 53.55±4.91s vs 29.16±4.36s, Figure 2 ).

[0029] The results of the target quadrant movement time of the four groups of mice are as follows Figure 3 The movement time of mice in the target quadrant was similar in the sterile control group, blank control group and anthocyanin-regulated fecal microbiota transplantation group, while the movement time of mice in the control feces group was significantly shortened (the control feces group vs Anthocyanin-regulated fecal microbiota transplantation group: 10.12±4.39s vs 14.77±3.25s, Figure 3 ). Therefore, compared with the Alzheimer's disease fecal microbiota transplantation group, the cognitive function of the bilberry anthocyanin-regulated fecal microbiota transplantation group was significantly improved, further proving that bilberry anthocyanin improves Alzheimer's disease by increasing the relative abundance of Hungatella hathewayi, Pseudoflavonifractor sp.An85, Dorea sp. 5-2, Anaerotruncus sp. G3(2012) and Mailhella massiliensis in the intestine.

[0030] 6. Brain pathology testing: After the behavioral test, the mice were killed and brain tissues were collected for Aβ 1-42 Detection of β-amyloid protein 42 levels by ELISA (enzyme-linked immunosorbent assay) or other immunological methods to quantitatively detect Aβ in serum 1-42 levels to assess changes in core AD pathological markers.

[0031] Bilberry anthocyanins regulate serum Aβ in mice in the fecal microbiota transplantation group 1-42 The deposition level of feces was significantly lower than that of the control group ( vs Anthocyanin-regulated fecal microbiota transplantation group: 127.30±5.00 vs 102.94±4.30 μg / L, Figure 4), suggesting that it can reduce the core pathological burden of Alzheimer's disease, and further proved that the increased abundance of Hungatella hathewayi, Pseudoflavonifractor sp. An85, Dorea sp. 5-2, Anaerotruncus sp. G3(2012) and Mailhella massiliensis in the intestine helps to improve Alzheimer's disease.

[0032] 7. Neuroinflammation Observation Prefrontal cortex microglia and astrocyte staining: Immunofluorescence staining (e.g., using IBA1 and GFAP antibodies) was used to observe the activation and morphological changes of microglia and astrocytes in brain tissue and assess glial proliferation and inflammatory responses. Real-time quantitative PCR was used to measure GAPDH, TNF-α, IL-1β, and iNOS levels to assess inflammatory responses. The real-time quantitative PCR reaction system consisted of 12.5 µL of 2× quantitative PCR master mix, 0.5 µL of upstream primer (10 µM), 0.5 µL of downstream primer (10 µM), 1 µL of probe (10 µM), 1 µL of DNA, and 9.5 µL of deionized water. The reaction conditions were: 95°C for 30 s → (95°C for 5 s → 60°C for 30 s) × 40 cycles. Primer sequences for GAPDH, TNF-α, IL-1β, and iNOS are shown in Table 1.

[0033] Table 1

[0034] Immunohistochemistry results showed that anthocyanins regulate the number of activated glial cells (GFAP) in the brains of mice in the fecal microbiota transplantation group compared with mice in the control feces group. + The level of inflammatory cytokines (TNF-a, iNOS) was reduced by 77.73%. Figure 5 , Figure 6 ), suggesting that bilberry anthocyanins regulate the fecal microbiota transplantation group to inhibit neuroinflammatory response and protect neurons, indicating that by increasing the content of Hungatella hathewayi, Pseudoflavonifractor sp. An85, Dorea sp. 5-2, Anaerotruncus sp. G3(2012) and Mailhella massiliensis in the intestine, it has a good effect in improving Alzheimer's disease-related neuroinflammation.

Claims

1. Use of bilberry anthocyanins in the preparation of a drug for regulating intestinal flora, wherein the regulation is to increase the relative abundance of the flora; the flora includes Hungatella hathewayi, Pseudoflavonifractor sp. An85, Dorea sp. 5-2, Anaerotruncus sp. G3(2012) and Mailhella massiliensis.

2. The application according to claim 1, characterized in that The concentration of bilberry anthocyanidin is 1×10 5 mg / L.

3. The application according to claim 1, characterized in that The dosage of the bilberry anthocyanidin is 20 mg / kg.

4. The application according to claim 1, characterized in that The bilberry anthocyanidin is administered for 3 months.

5. The application according to claim 1, characterized in that: The bilberry anthocyanidin is administered orally.

6. A drug for regulating intestinal flora, characterized in that: The medicine contains the bilberry anthocyanin according to claim 1; the regulation is to increase the relative abundance of the bacterial flora; the bacterial flora includes Hungatella hathewayi, Pseudoflavonifractor sp. An85, Dorea sp. 5-2, Anaerotruncus sp. G3 (2012) and Mailhella massiliensis.

7. Application of bilberry anthocyanins in the preparation of drugs to improve Alzheimer's disease.

8. A drug for improving Alzheimer's disease, characterized in that: The medicine contains the bilberry anthocyanidin according to claim 1.

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