阅读说明：本技术 Egcg在制备调节肠道菌群药物中的应用 (Application of EGCG in preparation of medicine for regulating intestinal flora ) 是由 刘庆军 沈鹤霄 张帆 李国龙 熊云 于 2021-08-10 设计创作，主要内容包括：本发明公开了EGCG在制备调节肠道菌群药物中的应用。本发明通过体外模拟体内环境,并收集人体粪便中的肠道微生物,进行EGCG体外干预实验,首次验证并发现了EGCG对于肠道菌群具有调节作用,尤其是可以显著或极显著上调多种肠道益生菌的丰度,包括：栖粪杆菌属Faecalibacterium,布劳特氏菌属Blautia,丁酸弧菌属Anaerostipes,纺锤链杆菌属Fusicatenibacter和毛螺旋菌属Parasutterella,因此EGCG不仅可作为肠道菌群调节药物、功能食品或保健食品,有效预防和/或治疗肠道菌群紊乱；而且还可用于辅助治疗与上述益生菌相关的如痛风、肝硬化、克罗恩、便秘、抑郁症、2型糖尿病等疾病,对于维持肠道菌群稳定以及相关疾病的辅助治疗具有重要意义,具有较大的应用价值。(The invention discloses an application of EGCG in preparing a medicine for regulating intestinal flora. According to the invention, an in-vitro simulation in-vivo environment is adopted, intestinal microorganisms in human excrement are collected, an EGCG in-vitro intervention experiment is carried out, EGCG is verified and found to have a regulating effect on intestinal flora for the first time, and particularly the abundance of various intestinal probiotics can be remarkably or extremely remarkably up-regulated, wherein the method comprises the following steps: the EGCG can be used as an intestinal flora regulating medicament, functional food or health food, and can effectively prevent and/or treat intestinal flora disorder; and can also be used for adjuvant treatment of diseases related to the probiotics, such as gout, liver cirrhosis, Crohn, constipation, depression, type 2 diabetes and the like, has important significance for maintaining the stability of intestinal flora and adjuvant treatment of related diseases, and has great application value.)

1. Application of epigallocatechin gallate in preparing medicine for regulating intestinal flora is provided.

2. Application of a pharmaceutical composition containing epigallocatechin gallate in preparing a medicine for regulating intestinal flora.

3. Application of epigallocatechin gallate in preparing functional food or health food for regulating intestinal flora is provided.

4. Use according to any one of claims 1 to 3, wherein epigallocatechin gallate promotes the proliferation of gut probiotics selected from the group consisting of: one or more of Faecalibacterium, Blautia, Anaerostipes, Fusobacterium Fusicatenibacter, Parstutterella.

5. The use according to claim 4, wherein the epigallocatechin gallate improves gout, cirrhosis, Crohn, constipation, depression and/or type 2 diabetes by increasing the abundance of Faecalibacterium.

6. The use as claimed in claim 4, wherein the epigallocatechin gallate improves gout, constipation and/or depression by increasing the abundance of Blautia Blautia.

7. The use according to claim 4, wherein the epigallocatechin gallate improves constipation and/or depression by increasing the abundance of Anaerostipes butyrate.

8. The use of claim 2, wherein the medicament further comprises a pharmaceutically acceptable excipient.

9. Use according to claim 8, wherein the pharmaceutical composition is a tablet, capsule, powder, pill, granule or solution.

10. The use according to claim 3, wherein the functional or health food is a biscuit, a cake, a beverage, an oral liquid, a granule, a beverage, a liquid milk, a milk powder.

Technical Field

The invention belongs to the technical field of medicine and health care, and particularly relates to an application of EGCG in preparation of a medicine for regulating intestinal flora.

Background

EGCG, namely epigallocatechin gallate, is a main component of green tea polyphenol, is a catechin monomer separated from tea, and is researched and found to have various beneficial effects of resisting bacteria, viruses, oxidation, arteriosclerosis, thrombosis, angiogenesis, inflammation, tumor and the like, but the EGCG has no related report on the regulation effect of intestinal microorganisms at present.

The large number of microorganisms exist in the intestinal tract of a human body, and the microorganisms depend on the intestinal life of the human body and help the human body to complete various physiological and biochemical functions. The intestinal microorganisms play an important bridge role between diet and hosts, and can regulate human health by themselves and metabolites, so the intestinal microorganisms are closely related to human health. The intestinal microorganisms achieve microecological balance through dynamic physiological action with a host, and bacteria and endotoxin in the intestinal tract are effectively prevented from being translocated. When the normal microbial community is affected by the host and the external environment, the original balance is destroyed, the intestinal flora is disordered, the species, the quantity and the proportion of the normal microbial community in the intestinal tract are abnormally changed, and the normal microbial community is converted into a pathological combination state, so that the host is pathogenic. The current research shows that various diseases of human body are closely related to intestinal microorganisms, for example, there are many documents of coprobacteria (Faecalibacterium) which are related to Gout, liver cirrhosis, intestinal diseases or constipation, depression and diabetes, such as Guo, Zhuang et al, endogenous microbial differentiation Gout Patents from health human scientific Reports,2016, that in Gout Patients, Faecalibacterium praussnitizii is almost exhausted and intestinal flora changes are similar to those in type 2 diabetes Patients and liver cirrhosis, so Faecalibacterium can be used as one of the indicators for judging human health; meanwhile, the correlation of Faecalibacterium with Crohn's disease is reported in Fujimoto et al, Descripted absentence of Faecalibacterium praerusnitzii in the gut microbiota of Crohn's disease, journal of Gastroenterology and Hepatology,2013,28(4), 613-; the publication Zhuang, Min et al, Absolution of pharmaceuticals and butyl-Production microorganisms management characterization of Short-Chain Fatty Acids Production and Hormons selection molecular Nutrition & Food Research,2019, reports that Faecalibacterium is associated with gut function and Constipation leads to decreased abundance of the bacteria; faecalibacterium is reported to be associated with depression in the literature Simpson, Carra A et al, the gut microbiota in anxiety and depression-analytical Review. clinical psychological Review,2020. For example, Blautia in Blautia is related to diseases such as gout, constipation and depression, and antiaerostipes in vibrio is mainly related to diseases such as constipation and depression. Also, for example, fusiforme fusicuenbacter, has been reported to be associated with various intestinal diseases, and for example, helichrysum parasutenella, has been reported to be associated with liver cirrhosis diseases. Based on this, enrichment of some probiotics in vivo to increase their abundance and further reduce harmful intestinal bacteria may help to treat some diseases by changing the composition of intestinal microbial flora, and intestinal microbes are considered as one of the treatment targets of various diseases at present.

Disclosure of Invention

The invention provides the application of EGCG in preparing medicines for regulating intestinal flora aiming at the blank of the prior art, EGCG intervention experiments are carried out through in vitro simulation, the EGCG is verified to have the function of regulating the intestinal flora for the first time, particularly, the proliferation of Faecalibacterium, Blautia, butyric acid vibrio and Anterostipes, fusiforme Fusicatenibacter and Parasuturella is remarkably up regulated, and the abundance of the intestinal probiotics is increased, so that the EGCG can be used for preparing medicines, functional foods or health-care foods for regulating the intestinal flora, not only can effectively prevent and/or treat intestinal flora disorder, but also can be used for assisting in treating diseases such as gout, liver cirrhosis, Crohn, constipation, depression, type 2 diabetes and the like.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides application of epigallocatechin gallate in preparing a medicine for regulating intestinal flora.

The invention provides application of a pharmaceutical composition containing epigallocatechin gallate in preparing a medicine for regulating intestinal flora.

The invention provides application of epigallocatechin gallate in preparing functional food or health-care food for regulating intestinal flora.

Further, the epigallocatechin gallate promotes the proliferation of intestinal probiotics selected from the group consisting of: one or more of Faecalibacterium, Blautia, Anaerostipes, Fusobacterium Fusicatenibacter, Parstutterella.

Further, the epigallocatechin gallate improves gout, liver cirrhosis, crohn's disease, constipation, depression and/or type 2 diabetes by increasing the abundance of Faecalibacterium. Therefore, the compound can be prepared into a medicament for adjuvant therapy of gout, liver cirrhosis, Crohn, constipation, depression and/or type 2 diabetes.

Further, the epigallocatechin gallate achieves the effect of improving gout, constipation and/or depression by increasing the abundance of Blautia. Therefore, the compound can be prepared into a medicament for adjuvant therapy of gout, constipation and/or depression.

Further, the epigallocatechin gallate achieves the effect of improving constipation and/or depression by increasing the abundance of Anaerostipes of Vibrio butyricum. Therefore, the compound can be prepared into a medicament for adjuvant therapy of constipation and/or depression.

Furthermore, the medicine also comprises pharmaceutically acceptable auxiliary materials.

Further, the pharmaceutical composition is a tablet, a capsule, a powder, a pill, a granule or a solution.

Further, the functional food or the health food is biscuits, cakes, beverages, oral liquid, granules, beverages, liquid milk and milk powder.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, an in-vitro simulation in-vivo environment is adopted, intestinal microorganisms in normal human excrement are collected, an EGCG in-vitro intervention experiment is carried out, and EGCG is verified and found to have a regulating effect on intestinal flora for the first time, and particularly the abundance of various intestinal probiotics can be remarkably up-regulated, wherein the method comprises the following steps: the EGCG can be used as an intestinal flora regulating medicament, functional food or health food, and can effectively prevent and/or treat intestinal flora disorder; and can also be used for adjuvant treatment of diseases related to the probiotics, such as gout, liver cirrhosis, Crohn, constipation, depression, type 2 diabetes and the like, has important significance for maintaining the stability of intestinal flora and adjuvant treatment of related diseases, and has great application value.

Drawings

FIG. 1 is a graph showing the results of measurement and analysis of the abundance of some of intestinal microorganisms in the test group and the control group in example 1 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Considering the problem that the determination result of the flora abundance is inaccurate because the intestinal flora derived from a human body cannot be completely colonized in a mouse, an in-vivo simulation experiment is carried out in vitro, human excrement is taken as an intestinal flora sample, the effect of EGCG intervention on the intestinal flora is observed, and the specific operation process is as follows:

1. fecal sample handling

(1) Preparation of PBS buffer solution

Taking 1000ml as an example, the following drugs are weighed out separately by an electronic analysis day and poured into a 1000ml beaker, comprising: KH (Perkin Elmer)₂PO₄ 0.24g，Na₂HPO₄·12H₂O 2.90g，NaCl 8.00g，KCl 0.20g。

800ml of ultrapure water is measured by a measuring cylinder, poured into a beaker, put into a stirring rotor, put on a magnetic heating stirrer and stirred until the ultrapure water is completely dissolved, then the pH of the solution is measured by a calibrated pH meter, and the pH is adjusted to 7.4 +/-0.05 by 0.1M HCl or NaOH.

The solution completely dissolved and pH-adjusted was drained with a glass rod into a 1000ml volumetric flask, the beaker was rinsed with a small amount of ultrapure water and poured into the volumetric flask, and the procedure was repeated 3 times. The liquid in the volumetric flask is fixed to the scale mark by ultrapure water, and the flask is plugged with a cover and then turned upside down for 10 times to ensure that the solution is fully mixed.

Pouring the solution with constant volume into a clean yellow glass bottle with a cover, and putting 500ml of the solution in each 1000ml bottle to prevent the solution from being sprayed out during autoclaving. The cap of the yellow glass bottle is unscrewed and then placed into an autoclave for 15 minutes at the temperature of 121 ℃. The cap is quickly screwed down after removal. After cooling to room temperature, the solution is stored in a refrigerator at 4 ℃ for 6 months.

(2) Fecal sample handling

In the case of 10g, the final concentration is 5%.

A10 g fecal sample was weighed out on a hundredth balance, an appropriate amount of the above PBS buffer was added to the centrifuge tube using an automatic pipette and mixed well on a shaker. The completely mixed sample is then evenly distributed into new 50ml centrifuge tubes, and each tube is added with a proper amount of PBS buffer solution.

Filtering in a biological safety cabinet after uniformly mixing, and sequentially passing the diluted samples through 20-mesh, 50-mesh, 100-mesh and 200-mesh filter screens. Collecting filtrate in a centrifuge tube, placing the centrifuge tube in a centrifuge, balancing, centrifuging at 6000G and 4 deg.C for 15min, and discarding supernatant. After weighing the precipitate, the volume was determined with a PBS solution at a final concentration of 5% to obtain an intestinal microorganism sample.

2. EGCG intervention experiment

(1) Preparation of basic culture medium

Preparing a basic culture medium for culturing the intestinal microorganisms: GAM medium, 1000ml for example. 60g of the modified GAM broth was weighed on an electronic analytical balance and poured into a 1000ml beaker. 800ml of ultrapure water is weighed by a measuring cylinder, poured into a beaker, put into a stirring rotor and put on a magnetic heating stirrer to be stirred until the ultrapure water is completely dissolved. The above completely dissolved solution was drained to a 1000ml volumetric flask with a glass rod, and the beaker was rinsed with a small amount of ultrapure water and poured into the volumetric flask, and repeated 3 times. The liquid in the volumetric flask is fixed to the scale mark by ultrapure water, and the flask is plugged with a cover and then turned upside down for 10 times to ensure that the solution is fully mixed.

Pouring the solution with constant volume into a clean yellow glass bottle with a cover, and putting 500ml of culture medium in each 1000ml bottle to prevent the solution from being sprayed out during autoclaving. The glass bottle with the yellow cap containing the culture medium was unscrewed and placed in a pulse autoclave using the liquid program at 121 ℃ for 15 minutes. After sterilization, the bottle cap is immediately screwed down and cooled to room temperature.

(2) Preparation of EGCG-GAM culture medium

On a sterile operating table, the GAM medium was dispensed into glass tubes with 2ml per tube using a 1ml pipette. The black cap was placed over the bottle mouth and masked slightly. Then, a 1ml pipette was used to take out EGCG solution (EGCG purchased from Hengheng Biotech Co., Ltd., purity 90%, dissolved in PBS buffer solution to a mass percent concentration of 5%), and the EGCG solution was added to the above glass tube containing GAM medium at 2ml per tube to prepare a test group. Meanwhile, a blank control group is also arranged, each group has 3 repetitions, and the grouping is as follows:

test groups: 2mL of GAM culture medium, 2mL of EGCG solution and 1mL of PBS buffer solution;

control group: 2mL of GAM medium +3mL of PBS buffer;

after confirming that each glass tube cover is screwed, the glass tube cover is transferred to an anaerobic box transfer box and needs to be disinfected by 84 disinfectant before being placed. After the anaerobic tank is placed, the bottle cap is unscrewed to replace oxygen, and the replacement is carried out for 12 hours.

The intestinal microorganism samples obtained after the treatment are respectively inoculated into a test group (EGCG-GAM culture medium) and a blank control group (GAM culture medium-PBS buffer solution) with 1ml per tube.

After culturing for 72 hours in an anaerobic box, observing the growth condition of the flora, photographing and keeping.

And centrifuging the test group sample and the control group sample at 10000rpm for 3min, discarding the supernatant, treating the precipitate with liquid nitrogen, sending to Wuhan Aikanjian biotechnology limited, and respectively measuring the abundance of the intestinal flora in the test group and the control group, namely the percentage of different intestinal microorganisms in all the detected strains, wherein the measurement result of the abundance of part of the intestinal flora is shown in Table 1.

And the abundance changes of partial intestinal flora before and after EGCG intervention are subjected to significance analysis.

The results of the analysis are shown in fig. 1, where denotes P < 0.01, i.e. there are very significant differences in abundance changes; p < 0.05, i.e. there was a significant difference in abundance change.

TABLE 1 abundance of intestinal flora in different treatment groups

The results show that, after EGCG desiccation, a proportion of the probiotics in the gut flora, including: the abundances of Faecalibacterium, Blautia, Anaerobiospecies and Fusicatenibacterium are remarkably improved compared with those of a control group, and the abundance of Paraspiraea parasutella is also slightly increased and is remarkably different from that of the control group. The results show that the EGCG can regulate the intestinal flora, effectively prevent and/or treat intestinal flora disorder, and can be used for adjuvant treatment of diseases related to the probiotics, such as gout, liver cirrhosis, Crohn, constipation, depression, type 2 diabetes and the like.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.