Ackermann muciniphile as well as regulator and application thereof

Abstract

The invention relates to Ackermann muciniphile as well as a regulator and application thereof, and relates to the technical field of biochemistry. The invention provides application of ackermann muciniphile in preparation of drugs for treating anxiety, and the ackermann muciniphile can significantly improve exploration interest, significantly reduce anxiety level and reverse anxiety manifestation caused by chronic stress, which indicates that the ackermann muciniphile has an effect of treating anxiety. The invention also provides an application of the regulator in preparation of medicines or health-care foods for regulating intestinal flora. The regulator comprises cape jasmine and fermented soybean soup. The regulator can obviously increase the quantity of Ackerman muciniphile in the intestinal tract of a mouse, so that the intestinal health is improved, and the micro-ecological balance of the intestinal tract of a human body is regulated.

Description

Technical Field

[0001] The invention relates to the field of biochemical technology, in particular to Akkermansia muciniphila and a regulator and application thereof. Background Art

[0002] Anxiety disorders are a common mental illness that occurs when the body faces fear or stress, affecting 1% of the world's population and causing disability. In the UK, about 16.8% of people suffer from anxiety disorders, while in the US, about 400,000 adults are affected. The symptoms include body tremors, palpitations, muscle tension, mental tension, irritability, insomnia and constant worry. In addition, anxiety disorders include various forms such as generalized anxiety and paroxysmal panic states, often accompanied by dizziness, chest tightness, dyspnea, dry mouth, frequent urination, urgency, sweating, tremors, motor restlessness and sleep disorders, as well as neurological disorders. Anxiety disorders seriously affect the quality of life and physical and mental health of patients, and may cause other mental illnesses such as phobias, hypochondriasis and neurasthenia. With the acceleration of social pace and the increase of pressure, the number of patients with anxiety disorders is on the rise. Nowadays, the treatment of anxiety disorders in clinical practice mainly relies on anti-anxiety drugs, including benzodiazepines, buspirone and its analogs. However, these drugs are often accompanied by side effects, such as dependence, tolerance, and withdrawal symptoms, which limit their long-term and widespread application.

[0003] In recent years, with the deepening of microbiome research, the relationship between intestinal microorganisms and host health has received increasing attention. The intestinal microbiota interacts with the central nervous system through the gut-brain axis, affecting the host's emotions, cognition and behavior. Akkermansia muciniphila (AKK), as an important intestinal commensal bacteria, has been found to be closely related to host metabolic health, immune regulation and nervous system function. Akkermansia muciniphila is a Gram-negative strict anaerobic bacterium that is good at degrading intestinal mucins and is one of the key species in the intestinal microbiota. Studies have shown that the abundance of AKK is negatively correlated with a variety of metabolic diseases such as obesity, diabetes, and cardiovascular disease, suggesting that it may play an important role in maintaining host health. The intestinal flora is a key regulator of immune cells in the gut-brain axis. Therefore, regulating the intestinal microbiota, especially increasing the number of Akkermansia muciniphila, may provide a potential new strategy for the treatment of anxiety. Summary of the invention

[0004] In view of the above problems, the present invention provides an application of Akkermansia muciniphila in the preparation of a drug for treating anxiety disorder. Akkermansia muciniphila can significantly improve exploration interest, significantly reduce anxiety levels, and reverse anxiety-like symptoms, indicating that it has the effect of treating anxiety.

[0005] In order to achieve the above object, the present invention provides a use of Akkermansia muciniphila in preparing a drug for treating anxiety.

[0006] The inventors found through animal experiments that Akkermansia muciniphila can significantly increase exploration interest, significantly reduce the anxiety level of mice, and reverse the anxiety-like behavior caused by chronic stress, indicating that it has the effect of treating anxiety and can be used as a potential drug for the treatment of generalized anxiety disorder, providing new strategies and methods for the treatment of anxiety disorders.

[0007] In one embodiment, the anxiety disorder is generalized anxiety disorder.

[0008] The generalized anxiety disorder described above can be caused by chronic restraint stress.

[0009] In one embodiment, the dosage of the drug for treating anxiety disorder is ≥ 8×10^8 CFU / kg body weight.

[0010] The present invention also provides a medicine for treating anxiety disorder, wherein the medicine comprises a medicine active ingredient and an auxiliary material, wherein the medicine active ingredient is Akkermansia muciniphila.

[0011] In one embodiment, the auxiliary material includes at least one of an excipient, a lubricant, an antioxidant, a preservative, a binder, a filler, or a thickener.

[0012] The present invention also provides an application of a regulator in preparing a medicine or health food for regulating intestinal flora, wherein the regulator comprises gardenia and fermented black beans soup.

[0013] Gardenia and Douchi Decoction originated from the ancient Chinese medical classic Treatise on Febrile Diseases, written by Zhang Zhongjing, a famous medical scientist in the Han Dynasty. It is mainly used to treat symptoms such as chest and diaphragm heat, restlessness and anxiety caused by exogenous wind-heat or internal heat evil. The prescription is composed of two medicinal materials, gardenia and Douchi. Gardenia has the effects of clearing heat and cooling blood, purging fire and relieving restlessness, while Douchi can relieve the symptoms of the lungs, harmonize the middle and replenish qi. The combination of the two can not only clear the heat in the chest and diaphragm, but also harmonize the spleen and stomach. It has a significant effect on treating emotional irritability, insomnia and other symptoms caused by heat evil.

[0014] As a traditional Chinese medicine compound, the safety of Gardenia and Fermented Soya Bean Soup and its characteristics of being both a medicine and a food give it unique advantages in regulating intestinal flora. The medicinal and edible properties of Gardenia and Fermented Soya Bean Soup mean that it can provide nutrition as food and has medicinal value in regulating the health of the body. This dual role makes Gardenia and Fermented Soya Bean Soup easy to incorporate into daily diet and convenient for long-term use without worrying about drug dependence or tolerance issues. At the same time, the preparation and use of Gardenia and Fermented Soya Bean Soup follows the theory of traditional Chinese medicine, emphasizing overall harmony and yin-yang balance, which helps reduce potential side effects. Therefore, the safety of Gardenia and Fermented Soya Bean Soup and its characteristics of being both a medicine and a food make it an ideal choice for regulating intestinal flora.

[0015] In one embodiment, the preparation method of the gardenia and fermented black beans soup comprises the following steps: weighing gardenia and fermented black beans, boiling the gardenia, adding the fermented black beans, boiling, filtering, and obtaining the gardenia and fermented black beans soup.

[0016] In one embodiment, the preparation method of the gardenia and fermented black beans soup comprises the following steps: weighing gardenia and fermented black beans in a mass ratio of 1:4, boiling the gardenia in water at a temperature of 95-105°C, adding the fermented black beans, boiling, filtering, and obtaining the gardenia and fermented black beans soup.

[0017] In one embodiment, the intestinal flora includes Akkermansia muciniphila.

[0018] The present invention finds through animal experiments that oral administration of Gardenia and Licorice Decoction can significantly increase the number of Akkermansia muciniphila in the intestines of mice, thereby improving intestinal health and regulating the balance of human intestinal microecology, and the increased Akkermansia muciniphila can effectively treat anxiety, providing a new treatment method for diseases related to intestinal flora imbalance, and also providing new ideas and new methods for the clinical treatment of anxiety, which has important clinical significance for the treatment of anxiety.

[0019] In one embodiment, said modulation comprises upregulating the abundance of Akkermansia muciniphila in the intestine.

[0020] The active ingredients in Zhizi Shitang can regulate the composition of intestinal flora, especially increase the number of Akkermansia muciniphila. This regulatory effect may be achieved by inhibiting the growth of harmful flora and promoting the proliferation of beneficial flora (such as Akkermansia muciniphila). Zhizi Shitang can significantly regulate the abundance of Akkermansia muciniphila in the intestines of anxious mice, and the improvement effect increases with the increase of the dosage concentration.

[0021] In one embodiment, the use comprises taking the regulator, and the amount of the regulator is ≥ 1.8 g / kg body weight.

[0022] The present invention also provides a medicine for regulating intestinal flora, the medicine comprising a medicine active ingredient and excipients, and the medicine active ingredient is gardenia and fermented black beans soup.

[0023] The present invention also provides a health food for regulating intestinal flora. The health food comprises active ingredients and auxiliary materials. The active ingredient is gardenia and fermented black beans soup.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The Akkermansia muciniphila and its regulator and application of the present invention can significantly improve exploration interest, significantly reduce anxiety levels, and reverse anxiety-like manifestations caused by chronic stress, indicating that it has the effect of treating anxiety. Oral administration of Gardenia and Licorice Decoction can significantly increase the number of Akkermansia muciniphila in the intestine, thereby improving intestinal health, and the increased Akkermansia muciniphila can effectively treat anxiety, providing a new treatment method for treating diseases related to intestinal flora imbalance, and also providing new ideas and new methods for the clinical treatment of anxiety, which has important clinical significance for the treatment of anxiety. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a graph of the number of crossings in the central area of ​​the open field experiment in Example 1;

[0027] Figure 2 is a graph of the residence time in the central area of ​​the open field experiment in Example 1;

[0028] Figure 3 This is a graph showing the number of times the open area of ​​the elevated plus maze test in Example 1 is crossed;

[0029] Figure 4 This is a graph of the residence time in the open area of ​​the elevated plus maze test in Example 1;

[0030] Figure 5 This is the behavioral trajectory diagram of mice in Example 1;

[0031] Figure 6 is a graph showing the relative abundance of Akkermansia muciniphila in experimental mice in Example 2;

[0032] Figure 7 This is a comparative analysis of the relative abundance of the genus level of the intestinal flora of mice by the Gardenia and Fermented Soybean Soup in Example 2;

[0033] Figure 8 This is a comparative analysis of the relative abundance of species levels of intestinal flora in mice treated with Gardenia and Fermented Soya Bean Soup in Example 2. DETAILED DESCRIPTION

[0034] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0036] source:

[0037] Unless otherwise specified, the reagents, materials, and equipment used in this example are all commercially available; the experimental methods are all conventional experimental methods in the art unless otherwise specified.

[0038] Example 1

[0039] Application of Akkermansia muciniphila in the preparation of drugs for treating anxiety

[0040] 1.1 Preparation of AKK bacterial solution.

[0041] AKK freeze-dried powder was purchased from Beijing Beina Biotechnology Co., Ltd. and the operation was performed as follows according to the instructions: 0.5 mL of anaerobic deoxygenated liquid sulfate-thiol medium was injected into the AKK freeze-dried powder, dissolved, fully mixed, diluted to 10 mL, and cultured anaerobically at 37°C for 5 days to establish the first generation. Subsequently, 200 μL of bacterial solution was inoculated into 10 mL of fresh culture medium, and the culture was continued for 5 days before quantification, which was the second generation bacterial solution.

[0042] 1.2 Experimental animal grouping.

[0043] After one week of adaptive feeding of mice, 30 adult male mice were randomly divided into three groups according to their body weight, with 10 mice in each group, namely, blank group (Control), anxiety group (Anxiety), and AKK group (AKK).

[0044] 1.3 Preparation of chronic restraint mouse anxiety model.

[0045] Except for the blank group, mice in other groups were restrained with 50mL centrifuge tubes. In order to allow mice to breathe normally and be restrained effectively, four holes were drilled evenly at the head of the centrifuge tube, and an appropriate amount of cotton was stuffed into the tail. The mice were restrained for a total of 10 days, 4 hours a day (9:00-13:00) for the first six days, and 6 hours a day (9:00-15:00) for the last four days. After each restraint, all mice were returned to their original cages. Drugs were administered by gavage 1 hour before restraint every day.

[0046] 1.4 Drug administration to experimental animals.

[0047] The blank group and the anxiety group were gavaged with distilled water, and the AKK group was administered with 2×10^7 CFU per mouse (i.e., 8×10^8 CFU per kg body weight) 1 hour before stress every day for 10 consecutive days.

[0048] 1.5 Mouse behavioral index testing.

[0049] (1) Open field test: The open field test is widely used in rodent anxiety research. The open field apparatus (35cm×35cm×30cm) consists of five sides, with a white bottom and blue surroundings. A camera hanging above records the number of times each mouse enters the center within 5 minutes, the time spent exploring the center, and the distance moved in the center. After each mouse is tested, wipe the instrument with 75% alcohol and wait for the alcohol to evaporate completely before testing the next mouse. The entire experimental process is carried out in a quiet and unmanned environment. The open field test should be carried out after the stress ends on the 10th day.

[0050] (2) Cross elevated test: The cross elevated maze consists of two opposite open arms (30cm×10cm) and two opposite closed arms (30cm×10cm×20cm). The angle between the open arm and the closed arm is 90°, forming a central area in the middle, 70cm above the ground. Each mouse is placed in the central area facing the open arm one by one, and the activity of the mouse is recorded within 5 minutes, including the number of times it enters the open arm and the closed arm, the time spent in the open arm, and the distance walked in the open arm. After each mouse is tested, wipe the instrument with 75% alcohol and wait for the alcohol to evaporate completely before testing the next mouse. The entire experimental process is carried out in a quiet and unmanned environment. The cross elevated test should be carried out after the open field test.

[0051] 1.5 Results.

[0052] 1.5.1 Open field test.

[0053] The basis of the open field experiment is to evaluate the exploratory behavior of mice under the influence of anxiety. Generally, mice with lower anxiety levels will have a decreased ability to explore in an open space after leaving the group. Experimental observations have shown that mice that have not experienced restraint stress usually have bright fur and tight skin. When they are placed in an open field test box, they show strong activity and will not stay in the corner for a long time. Instead, they tend to move and explore the central area of ​​the open field. In contrast, mice that have experienced long-term restraint stress have dull and matte fur, loose skin, and reduced activity in the open field box. They tend to curl up in corners or walk along the edge of the open field, showing a lower desire to explore. Figure 1 , 2As shown, compared with the blank group, after 10 days of repeated restraint, the number of times the anxious group mice entered the central area of ​​the open field, the exploration time in the central area and the walking distance were significantly reduced, while after oral administration of AKK bacteria, the number of times, time and distance of exploring the center of the open field were significantly increased.

[0054] 1.5.2 Elevated plus maze test.

[0055] The elevated plus maze test is a common method for assessing the anxiety level of mice. In the experiment, it was observed that mice with lower anxiety levels tended to explore the open arm areas frequently, stayed in these areas for longer periods of time, walked farther, and entered the open arms more often. In contrast, mice with higher anxiety levels tended to stay in the closed arms or move slowly in the closed arm areas. They rarely approached the open arms, and even if they occasionally entered, they would quickly return to the closed arms, showing lower exploratory behavior. Figure 3 , 4 As shown in the figure, compared with the blank group, the frequency of entering the open arm, the exploration time in the open arm and the walking distance of the anxious group were all reduced, while after the mice were gavaged with AKK bacteria, the frequency, time and distance of entering the open arm were significantly increased. Figure 5 shown.

[0056] Experimental conclusion: Akkermansia muciniphila can significantly reduce the anxiety-like behavior of mice, has the effect of treating anxiety, and can be used as a potential drug for the treatment of generalized anxiety disorder.

[0057] Example 2

[0058] Application of Zhizishi Decoction in Promoting the Proliferation of Akkermansia muciniphila

[0059] 1. Experimental method.

[0060] 1.1 Preparation of Gardenia and Fermented Soybean Soup.

[0061] According to the preparation method of Gardenia and Fermented Black Bean Soup in Zhang Zhongjing's Treatise on Febrile Diseases, gardenia and fermented black beans are mixed in a certain proportion, the gardenia is boiled first, the fermented black beans are added later, and the soup is filtered out.

[0062] The specific preparation method is as follows: weigh gardenia and light fermented black beans in a mass ratio of 1:4, first add 800 mL of water to the gardenia, boil at 100°C until the water reaches 500 mL, then add the light fermented black beans, boil for 10 minutes, filter, and obtain the gardenia and fermented black bean soup.

[0063] 1.2 Experimental animal grouping.

[0064] After one week of adaptive feeding of mice, 50 adult male mice were randomly divided into 5 groups according to their body weight, with 10 mice in each group, namely blank group (Control), model group (Anxiety), low-dose Gardenia and Soya Bean Decoction group (ZZCDlowdoes, ZZCD-L), medium-dose Gardenia and Soya Bean Decoction group (ZZCDmediumdose, ZZCD-M), and high-dose Gardenia and Soya Bean Decoction group (ZZCDhighdoes, ZZCD-H).

[0065] 1.3 Drug administration to experimental animals.

[0066] The blank group and the model group were gavaged with distilled water, and the low-dose group of Zhizishitang (1.8 g / kg), the medium-dose group of Zhizishitang (3.6 g / kg), and the high-dose group of Zhizishitang (7.2 g / kg) were gavaged. The administration time was 1 hour before stress every day for 10 consecutive days.

[0067] 1.4 Preparation of chronic restraint mouse anxiety model.

[0068] Except for the blank group, mice in other groups were restrained with 50mL centrifuge tubes. In order to allow mice to breathe normally and be restrained effectively, four holes were drilled evenly at the head of the centrifuge tube, and an appropriate amount of cotton was stuffed into the tail. The mice were restrained for a total of 10 days, 4 hours a day (9:00-13:00) for the first six days, and 6 hours a day (9:00-15:00) for the last four days. After each restraint, all mice were returned to their original cages. Drugs were administered by gavage 1 hour before restraint every day.

[0069] 1.5 Extraction of mouse cecal contents.

[0070] The mice were intraperitoneally injected with anesthetics. After the mice were fully anesthetized, the cecum was opened, and the contents of the cecum were taken out on ice, immediately frozen in liquid nitrogen, and stored at -80°C.

[0071] 1.6 Extraction and determination of total DNA from cecal extract.

[0072] 1.6.1 Pretreatment of cecal contents.

[0073] Weigh 200g of cecal contents into a clean and sterilized centrifuge tube, add 1mL of 70% ethanol, shake and mix, centrifuge (1000rpm / min, -4℃, 3min), and discard the supernatant. Add 1mL of 1×PBS solution to the precipitate, mix, centrifuge (1000rpm / min, -4℃, 3min), and discard the supernatant. Wait for the liquid in the precipitate to evaporate completely.

[0074] 1.6.2 Extraction of total DNA from cecal contents.

[0075] Add 800μL Buffer SLX Mlus solution to the sample that has been processed, and shake to mix. Add 80μL Buffer DS solution, and shake to mix. Lyse in a 70℃ constant temperature metal bath for 10min, centrifuge (13000rpm / min, -4℃, 5min), and take the supernatant. Take 600μL supernatant, add 200μL Buffer SP2, and shake to mix. Add 100μL HTR Reagent, shake to mix, ice bath for 5min, centrifuge (13000rpm / min, -4℃, 5min). Take 400μL supernatant, add 450μL BindingBuffer solution and 40μL Magsi Particles solution, shake to mix, and let stand for 2min. Adsorb on the magnetic stand for 5min, remove the supernatant, add 500μL Binding Buffer solution, shake to mix, and let stand for 2min. Adsorb on the magnetic stand for another 5min, and remove the supernatant. Add 1mL Buffer PHB solution, shake and mix, adsorb on the magnetic stand for 5min. Add 1mL SPM WashBuffer solution, shake and mix, adsorb on the magnetic stand for 5min, and discard the supernatant. Add 1mL SPM WashBuffer solution again, shake and mix, adsorb on the magnetic stand for 5min, discard the supernatant, and dry. Add 60μL Elution Buffer solution, shake and mix, and place in a 65℃ metal bath for 10min. Adsorb on the magnetic stand for 5min and remove 1.5mL supernatant.

[0076] 1.6.3 Detection of sample DNA concentration and integrity.

[0077] The sample concentration was determined by an ELISA instrument, and the sample integrity was tested by gel electrophoresis. The detection of Akkermansia muciniphila was performed by 16SrRNA gene sequencing.

[0078] 1.7 Results.

[0079] like Figure 6 As shown in the results, it was found that anxiety caused a significant decrease in the abundance of AKK flora in the intestines of mice. After feeding a low dose of Gardenia and Soya Bean Soup, the improvement was not obvious; as the concentration of the drug increased, the improvement effect increased. This indicates that Gardenia and Soya Bean Soup has a regulatory effect on the abundance of AKK flora in the intestines of anxious mice.

[0080] like Figure 7 , 8 As shown in the figure, compared with the blank group, the content of AKK bacteria in the intestinal flora of the anxiety group mice was significantly reduced. Compared with the anxiety group, after feeding Gardenia and Chia Bean Soup, the content of AKK bacteria was significantly increased, among which the changes in the medium and high dose groups of Gardenia and Chia Bean Soup were the most obvious.

[0081] Experimental conclusion: Gardenia and Licorice Decoction can significantly regulate the abundance of Akkermansia muciniphila in the intestines of anxious mice, and the improvement effect increases with the increase of administration concentration.

[0082] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0083] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of Akkermansia muciniphila in the preparation of drugs for treating anxiety disorders.

2. The use according to claim 1, characterized in that: The anxiety disorder is generalized anxiety disorder.

3. The use according to claim 1, characterized in that: The dosage of the drug for treating anxiety disorder is ≥8×10^8 CFU / kg body weight.

4. A drug for treating anxiety, characterized in that: The medicine comprises active pharmaceutical ingredients and excipients, and the active pharmaceutical ingredient is Akkermansia muciniphila.

5. Use of a regulator in the preparation of a medicine or health food for regulating intestinal flora, characterized in that: The regulator includes Gardenia and Licorice Decoction.

6. The use according to claim 5, characterized in that: The preparation method of the gardenia and fermented black beans soup comprises the following steps: weighing gardenia and fermented black beans, boiling the gardenia in water, adding the fermented black beans, boiling in water, filtering, and obtaining the gardenia and fermented black beans soup.

7. The use according to claim 5, characterized in that: The intestinal flora includes Akkermansia muciniphila.

8. The use according to claim 7, characterized in that: The modulation includes upregulating the abundance of Akkermansia muciniphila in the intestine.

9. A drug for regulating intestinal flora, characterized in that: The medicine comprises active pharmaceutical ingredients and auxiliary materials, and the active pharmaceutical ingredient is gardenia and fermented black beans soup.

10. A health food for regulating intestinal flora, characterized in that: The health food comprises active ingredients and auxiliary materials, and the active ingredient is gardenia and fermented black beans soup.

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