Preparation method of coptis chinensis exosomes and use thereof in preparing anti-ulcerative colitis drugs

Abstract

The present invention discloses a method for preparing coptis chinensis exosomes and a use thereof in preparing an anti-ulcerative colitis drug, and belongs to the field of medical technology. The present invention enzymatically hydrolyzes coptis chinensis, performs differential centrifugation, ultrahigh-speed centrifugation, sucrose gradient centrifugation, and membrane filtration to obtain the coptis chinensis exosomes. Experiments have confirmed that the coptis chinensis exosomes obtained by the preparation method of the present invention can significantly increase the body weight of ulcerative colitis model mice, significantly reduce the bloody stool index and loose stool index, increase the colon length, and achieve the purpose of treating ulcerative colitis. The present invention provides a new treatment approach for the prevention and treatment of ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for preparing coptis chinensis exosomes and an application thereof in preparing an anti-ulcerative colitis drug. Background Art

[0002] Ulcerative colitis (UC) is a disease characterized by superficial, nonspecific inflammation of the rectum and colon, accompanied by damage to multiple organs outside the intestine. It is currently believed that the disease is related to genetic factors, with autoimmune mechanisms as the basis, infection and neural factors as the inducements, and the lesions are mainly in the rectum and sigmoid colon. The first is diffuse inflammatory changes in the superficial mucosa. Then congestion, edema, hypertrophy and increased fragility will occur, resulting in small ulcers, which will then develop into large ulcers. In the late stage, due to the hyperplasia of colon tissue, the intestinal wall becomes thicker, narrower, and the intestinal tube becomes shorter. The course of the disease is long, which seriously affects the patient's physical health and quality of life, and the risk of colorectal cancer increases with the extension of the course of the disease.

[0003] The treatment plans for ulcerative colitis at home and abroad are basically the same. Clinical treatment mainly uses sulfasalazine salicylic acid preparations, hormone preparations such as prednisone or dexamethasone, and immunosuppressant azathioprine, but the efficacy is poor and there are significant adverse reactions. Therefore, people have long attached great importance to the research and development of drugs for the treatment of ulcerative colitis, which is of great significance for alleviating the pain of patients and improving their quality of life.

[0004] Extracellular vesicles (EVs) are small lipid vesicles released by cells, with a diameter of 30-200nm. Extracellular vesicles are usually divided into exosomes, microvesicles or microparticles and apoptotic bodies according to their size. Exosomes are part of extracellular vesicles, with a size of 40-160nm. The plasma membrane first invaginates into early endosomes, and the endosomes fuse with each other to form late endosomes, that is, multivesicular bodies. Multivesicular bodies bud out to form intraluminal vesicles, which gradually fuse with the cell membrane and are released to the outside of the cell membrane through the Rab family guanosine triphosphatase pathway, that is, the intraluminal vesicles released to the outside of the cell through exocytosis are called exosomes. Therefore, the membrane structure of exosomes is closer to the cell membrane and has a lipid bilayer structure. Studies have found that when the Rab family guanosine triphosphatase is inhibited, a large number of intraluminal vesicles will accumulate in the cell, making it difficult to release to form exosomes.

[0005] The substances carried by exosomes include lipids, metabolites, proteins, microRNAs, mRNAs, long non-coding RNAs, and DNA. Exosomes can interact directly with neighboring cells or indirectly with distant receptor cells through systemic circulation. The interaction between exosomes and target cells can not only be through binding to cell surface receptors, but also through endocytosis to release the substances they carry into receptor cells. Traditional Chinese medicine products have the defects of low solubility and low bioavailability of active ingredients, and exosomes, as an emerging tool for intercellular communication, may play a role as a carrier of traditional Chinese medicine.

[0006] Coptis chinensis is the dried rhizome of the perennial herbaceous plant Coptis chinensis, Coptis chinensis triangularis or Coptis chinensis of the Ranunculaceae family. Coptis chinensis was first recorded in Shennong's Herbal Classic and has the effects of regulating the spleen and stomach, clearing away heat and dampness, purging fire and detoxifying. Taiping Huimin Hejijufang records that it is mainly used to treat heat in the heart meridian, internally treat blood heat vomiting and bleeding, carbuncle, furuncle, red eyes and toothache, thirst, and externally treat eczema, wet sores and other skin diseases. Coptis chinensis tastes bitter and is cold in nature. It enters the heart, liver, gallbladder, spleen, stomach and large intestine meridians. In clinical practice, processed products such as Coptis chinensis tablets, ginger coptis chinensis, wine coptis chinensis and cornel coptis chinensis are used to treat diseases. At present, 175 chemical components have been isolated and identified from the plant Coptis chinensis, including alkaloids, flavonoids, triterpenes, volatile oils, polysaccharides and lignans. Modern pharmacological research mainly focuses on alkaloid active ingredients. At present, there are no reports of exosomes derived from Coptis chinensis. Summary of the invention

[0007] The object of the present invention is to provide a method for preparing exosomes of Coptis chinensis.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] A method for preparing coptis chinensis exosomes comprises the following steps:

[0010] The coptis chinensis is subjected to enzymatic hydrolysis, the supernatant is taken after differential centrifugation, the precipitate is taken by ultra-high speed centrifugation, and then the coptis chinensis exosomes are obtained by sucrose gradient centrifugation and membrane filtration.

[0011] A method for preparing coptis chinensis exosomes comprises the following steps:

[0012] S1. Rhizoma Coptidis is washed, low-temperature dried, and crushed. The crushed powder is mixed with PBS buffer, and a complex enzyme is added to perform enzymolysis reaction to obtain an enzymolysis solution;

[0013] S2, performing differential centrifugation on the enzymatic hydrolysate obtained in step S1, taking the supernatant after differential centrifugation, discarding the precipitate, performing ultra-high speed centrifugation on the supernatant, taking the precipitate, and suspending it in pre-cooled PBS buffer to obtain a precipitate suspension;

[0014] S3, adding sucrose solution in a centrifuge tube according to the gradient, adding the precipitate suspension obtained in step S2 to the top layer, and ultra-high-speed centrifugation; after the centrifugation, collect each layer into a centrifuge tube, add pre-cooled PBS buffer and mix well, and ultra-high-speed centrifuge to remove sucrose to obtain a precipitate;

[0015] S4. Resuspend the precipitate obtained in step S3 with pre-cooled PBS buffer, use a disposable syringe to absorb the resuspended liquid, slowly drip it onto the cell filter membrane for filtration, and use a low-temperature centrifuge to perform low-temperature ultra-high-speed centrifugation on the filtered solution. After centrifugation, aspirate the supernatant and retain the precipitate, and resuspend the obtained precipitate with PBS buffer to obtain the extracted and separated exosome product.

[0016] A method for preparing coptis chinensis exosomes comprises the following steps:

[0017] S1. Rhizoma Coptidis is washed, low-temperature dried, and crushed. The crushed powder is mixed with PBS buffer, and a complex enzyme is added to perform enzymolysis reaction to obtain an enzymolysis solution;

[0018] S2, subjecting the enzymatic hydrolysate obtained in step S1 to differential centrifugation, centrifuging for 10 min at a centrifugal force of 1000 g, centrifuging for 30 min at a centrifugal force of 3000 g, and centrifuging for 50 min at a centrifugal force of 10000 g; taking the supernatant after differential centrifugation, discarding the precipitate, subjecting the supernatant to ultrahigh-speed centrifugation, centrifuging for 120 min at a centrifugal force of 13000 g, taking the precipitate, and suspending it in a pre-cooled PBS buffer to obtain a precipitate suspension;

[0019] S3, adding sucrose solution in a centrifuge tube according to the gradient, the sucrose concentration gradient is 8%, 30%, 45%, 60%, adding the precipitation suspension obtained in step S2 to the top layer, and centrifuging for 120 min at a centrifugal force of 13000g; after the centrifugation, collect each layer into a centrifuge tube, add pre-cooled PBS buffer and mix well, centrifuge at 13000g for 120 min, remove sucrose, and obtain a precipitate;

[0020] S4. Resuspend the precipitate obtained in step S3 with pre-cooled PBS buffer, use a disposable syringe to absorb the resuspended liquid, and slowly drip it onto a cell filter membrane with a specification of 0.22 μm for filtration. The filtered solution is centrifuged in a low-temperature centrifuge at 4°C and a centrifugal force of 130,000 g for 120 minutes. After centrifugation, the supernatant is aspirated and the precipitate is retained. The precipitate is resuspended with PBS buffer to obtain the extracted and separated exosome product.

[0021] Furthermore, in step S1, the temperature of low temperature drying is 40-60°C; and the powder is crushed to 80-120 mesh.

[0022] Furthermore, in step S1, the composite enzyme comprises 45wt% of cellulase, 20wt% of pectinase, 15wt% of hemicellulase, 10wt% of ligninase and 10wt% of wall protease; the added mass of the composite enzyme accounts for 0.1-0.4% of the mass of Coptis chinensis; and the enzymolysis time is 6-10h.

[0023] Furthermore, in step S4, the PBS buffer is 1-3 mL.

[0024] Another aspect of the present invention is to provide a Coptis chinensis exosomes prepared according to the aforementioned method.

[0025] Another aspect of the present invention is to provide the use of the Coptis chinensis exosomes in the preparation of anti-ulcerative colitis drugs.

[0026] The present invention also provides a medicine for preventing and treating ulcerative colitis, comprising coptis chinensis exosomes.

[0027] The present invention discloses the following technical effects:

[0028] The coptis chinensis exosomes extracted by the present invention present a teacup-like membrane vesicle structure, and the particle size is concentrated around 150 nm, which proves that the coptis chinensis exosomes have been successfully extracted.

[0029] The invention hydrolyzes coptis chinensis, performs differential centrifugation, ultra-high speed centrifugation, sucrose gradient centrifugation, and membrane filtration to obtain the coptis chinensis exosomes. Experiments have confirmed that the coptis chinensis exosomes obtained by the preparation process of the invention significantly increase the body weight of ulcerative colitis model mice, reduce the bloody stool index and loose stool index, and increase the colon length, thereby achieving the purpose of treating ulcerative colitis. The invention provides a new treatment approach for the prevention and treatment of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Electron micrograph of the exosomes of Coptis chinensis prepared in Example 1 of the present invention.

[0031] Figure 2 Nanoparticle size diagram of Coptis chinensis exosomes prepared in Example 1 of the present invention.

[0032] Figure 3 Comparison of body weight on the last day / body weight on the first day in each group of the efficacy examples of the present invention

[0033] Compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01; compared with the Example 1 group, & P<0.05, && P<0.01.

[0034] Figure 4Comparison of blood in stool index in each group of the efficacy examples of the present invention

[0035] Compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01; compared with Example 1 group, & P<0.05, && P<0.01.

[0036] Figure 5 Comparison of loose stool index in each group of the efficacy examples of the present invention

[0037] Compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01; compared with Example 1 group, & P<0.05.

[0038] Figure 6 Comparison of colon length in each group of the efficacy examples of the present invention

[0039] Compared with the normal control group, ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01; compared with Example 1 group, & P<0.05, && P<0.01. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0043] Example 1

[0044] S1. Wash the coptis root, dry it at 50°C, and grind it into 80 mesh. The powder after grinding is mixed with PBS buffer solution, and a composite enzyme is added, which includes 45wt% of cellulase, 20wt% of pectinase, 15wt% of hemicellulase, 10wt% of ligninase and 10wt% of wall protease; the added mass of the composite enzyme accounts for 0.2% of the mass of the coptis root, to carry out enzymolysis reaction, and the enzymolysis time is 8h to obtain an enzymolysis solution;

[0045] S2, subjecting the enzymatic hydrolysate obtained in step S1 to differential centrifugation, centrifuging for 10 min at a centrifugal force of 1000 g, centrifuging for 30 min at a centrifugal force of 3000 g, and centrifuging for 50 min at a centrifugal force of 10000 g; taking the supernatant after differential centrifugation, discarding the precipitate, subjecting the supernatant to ultrahigh-speed centrifugation, centrifuging for 120 min at a centrifugal force of 13000 g, taking the precipitate, and suspending it in a pre-cooled PBS buffer to obtain a precipitate suspension;

[0046] S3, adding sucrose solution in a centrifuge tube according to the gradient, the sucrose concentration gradient is 8%, 30%, 45%, 60%, adding the precipitation suspension obtained in step S2 to the top layer, and centrifuging for 120 min at a centrifugal force of 13000g; after the centrifugation, collect each layer into a centrifuge tube, add pre-cooled PBS buffer and mix well, centrifuge at 13000g for 120 min, remove sucrose, and obtain a precipitate;

[0047] S4. Resuspend the precipitate obtained in step S3 with pre-cooled PBS buffer, use a disposable syringe to absorb the resuspended liquid, and slowly drip it onto a cell filter membrane with a specification of 0.22 μm for filtration. The filtered solution is centrifuged in a low-temperature centrifuge at 4°C and a centrifugal force of 130,000 g for 120 minutes. After centrifugation, the supernatant is aspirated and the precipitate is retained. The precipitate is resuspended with 3 mL of PBS buffer to obtain the extracted and separated exosome product.

[0048] The morphology and structure of exosomes were observed using transmission electron microscopy (TEM), and the particle size of exosomes was determined by nanoparticle tracking analysis (NTA).

[0049] Electron microscopy revealed that the exosomes of Coptis chinensis contain a typical lipid bilayer membrane structure and are round in shape, which is consistent with the characteristics of plant exosomes ( Figure 1). The particle size was measured by NTA and it was found that the particle size of exosomes was concentrated around 150nm ( Figure 2 ).

[0050] The above method can be used to separate plant exosomes with vesicle structure from Coptis chinensis.

[0051] Example 2

[0052] S1. Wash the coptis root, dry it at 40°C, and grind it into 120 mesh. The powder after grinding is mixed with PBS buffer solution, and a composite enzyme is added, which includes 45wt% of cellulase, 20wt% of pectinase, 15wt% of hemicellulase, 10wt% of ligninase and 10wt% of wall protease; the added mass of the composite enzyme accounts for 0.1% of the mass of the coptis root, to carry out enzymolysis reaction, and the enzymolysis time is 10h to obtain an enzymolysis solution;

[0053] S2, subjecting the enzymatic hydrolysate obtained in step S1 to differential centrifugation, centrifuging for 10 min at a centrifugal force of 1000 g, centrifuging for 30 min at a centrifugal force of 3000 g, and centrifuging for 50 min at a centrifugal force of 10000 g; taking the supernatant after differential centrifugation, discarding the precipitate, subjecting the supernatant to ultrahigh-speed centrifugation, centrifuging for 120 min at a centrifugal force of 13000 g, taking the precipitate, and suspending it in a pre-cooled PBS buffer to obtain a precipitate suspension;

[0054] S3, adding sucrose solution in a centrifuge tube according to the gradient, the sucrose concentration gradient is 8%, 30%, 45%, 60%, adding the precipitation suspension obtained in step S2 to the top layer, and centrifuging for 120 min at a centrifugal force of 13000g; after the centrifugation, collect each layer into a centrifuge tube, add pre-cooled PBS buffer and mix well, centrifuge at 13000g for 120 min, remove sucrose, and obtain a precipitate;

[0055] S4. Resuspend the precipitate obtained in step S3 with pre-cooled PBS buffer, use a disposable syringe to absorb the resuspended liquid, and slowly drip it onto a cell filter membrane with a specification of 0.22 μm for filtration. The filtered solution is centrifuged in a low-temperature centrifuge at 4°C and a centrifugal force of 130,000 g for 120 minutes. After centrifugation, the supernatant is aspirated and the precipitate is retained. The precipitate is resuspended with 3 mL of PBS buffer to obtain the extracted and separated exosome product.

[0056] Example 3

[0057] S1. Wash the coptis root, dry it at 60°C, and grind it into 80 mesh. The powder after grinding is mixed with PBS buffer solution, and a composite enzyme is added, which includes 45wt% of cellulase, 20wt% of pectinase, 15wt% of hemicellulase, 10wt% of ligninase and 10wt% of wall protease; the added mass of the composite enzyme accounts for 0.4% of the mass of the coptis root, to carry out enzymolysis reaction, and the enzymolysis time is 6h to obtain an enzymolysis solution;

[0058] S2, subjecting the enzymatic hydrolysate obtained in step S1 to differential centrifugation, centrifuging for 10 min at a centrifugal force of 1000 g, centrifuging for 30 min at a centrifugal force of 3000 g, and centrifuging for 50 min at a centrifugal force of 10000 g; taking the supernatant after differential centrifugation, discarding the precipitate, subjecting the supernatant to ultrahigh-speed centrifugation, centrifuging for 120 min at a centrifugal force of 13000 g, taking the precipitate, and suspending it in a pre-cooled PBS buffer to obtain a precipitate suspension;

[0059] S3, adding sucrose solution in a centrifuge tube according to the gradient, the sucrose concentration gradient is 8%, 30%, 45%, 60%, adding the precipitation suspension obtained in step S2 to the top layer, and centrifuging for 120 min at a centrifugal force of 13000g; after the centrifugation, collect each layer into a centrifuge tube, add pre-cooled PBS buffer and mix well, centrifuge at 13000g for 120 min, remove sucrose, and obtain a precipitate;

[0060] S4. Resuspend the precipitate obtained in step S3 with pre-cooled PBS buffer, use a disposable syringe to absorb the resuspended liquid, and slowly drip it onto a cell filter membrane with a specification of 0.22 μm for filtration. The filtered solution is centrifuged in a low-temperature centrifuge at 4°C and a centrifugal force of 130,000 g for 120 minutes. After centrifugation, the supernatant is aspirated and the precipitate is retained. The precipitate is resuspended with 3 mL of PBS buffer to obtain the extracted and separated exosome product.

[0061] Example 4

[0062] S1. Wash the coptis root, dry it at 45°C, and grind it into 120 mesh. The powder after grinding is mixed with PBS buffer solution, and a composite enzyme is added, which includes 45wt% of cellulase, 20wt% of pectinase, 15wt% of hemicellulase, 10wt% of ligninase and 10wt% of wall protease; the added mass of the composite enzyme accounts for 0.4% of the mass of the coptis root, to carry out enzymolysis reaction, and the enzymolysis time is 8h to obtain an enzymolysis solution;

[0063] S2, subjecting the enzymatic hydrolysate obtained in step S1 to differential centrifugation, centrifuging for 10 min at a centrifugal force of 1000 g, centrifuging for 30 min at a centrifugal force of 3000 g, and centrifuging for 50 min at a centrifugal force of 10000 g; taking the supernatant after differential centrifugation, discarding the precipitate, subjecting the supernatant to ultrahigh-speed centrifugation, centrifuging for 120 min at a centrifugal force of 13000 g, taking the precipitate, and suspending it in a pre-cooled PBS buffer to obtain a precipitate suspension;

[0064] S3, adding sucrose solution in a centrifuge tube according to the gradient, the sucrose concentration gradient is 8%, 30%, 45%, 60%, adding the precipitation suspension obtained in step S2 to the top layer, and centrifuging for 120 min at a centrifugal force of 13000g; after the centrifugation, collect each layer into a centrifuge tube, add pre-cooled PBS buffer and mix well, centrifuge at 13000g for 120 min, remove sucrose, and obtain a precipitate;

[0065] S4. Resuspend the precipitate obtained in step S3 with pre-cooled PBS buffer, use a disposable syringe to absorb the resuspended liquid, and slowly drip it onto a cell filter membrane with a specification of 0.22 μm for filtration. The filtered solution is centrifuged in a low-temperature centrifuge at 4°C and a centrifugal force of 130,000 g for 120 minutes. After centrifugation, the supernatant is aspirated and the precipitate is retained. The precipitate is resuspended with 3 mL of PBS buffer to obtain the extracted and separated exosome product.

[0066] Comparative Example 1

[0067] Compared with Example 1, the composite enzyme in step S1 is 45 wt % of cellulase and 55 wt % of pectinase.

[0068] The remaining steps are the same as in Example 1.

[0069] S1. Rhizoma Coptidis is washed, dried at low temperature at 50°C, and crushed into 80 meshes. The crushed powder is mixed with PBS buffer, and a composite enzyme (the added mass of the composite enzyme accounts for 0.2% of the mass of Rhizoma Coptidis) is added to perform enzymolysis reaction for 8 hours to obtain an enzymolysis solution;

[0070] S2, subjecting the enzymatic hydrolysate obtained in step S1 to differential centrifugation, centrifuging for 10 min at a centrifugal force of 1000 g, centrifuging for 30 min at a centrifugal force of 3000 g, and centrifuging for 50 min at a centrifugal force of 10000 g; taking the supernatant after differential centrifugation, discarding the precipitate, subjecting the supernatant to ultrahigh-speed centrifugation, centrifuging for 120 min at a centrifugal force of 13000 g, taking the precipitate, and suspending it in a pre-cooled PBS buffer to obtain a precipitate suspension;

[0071] S3, adding sucrose solution in a centrifuge tube according to the gradient, the sucrose concentration gradient is 8%, 30%, 45%, 60%, adding the precipitation suspension obtained in step S2 to the top layer, and centrifuging for 120 min at a centrifugal force of 13000g; after the centrifugation, collect each layer into a centrifuge tube, add pre-cooled PBS buffer and mix well, centrifuge at 13000g for 120 min, remove sucrose, and obtain a precipitate;

[0072] S4. Resuspend the precipitate obtained in step S3 with pre-cooled PBS buffer, use a disposable syringe to absorb the resuspended liquid, and slowly drip it onto a cell filter membrane with a specification of 0.22 μm for filtration. The filtered solution is centrifuged in a low-temperature centrifuge at 4°C and a centrifugal force of 130,000 g for 120 minutes. After centrifugation, the supernatant is aspirated and the precipitate is retained. The precipitate is resuspended with 3 mL of PBS buffer to obtain the extracted and separated exosome product.

[0073] Comparative Example 2

[0074] Compared with Example 1, the centrifugal force in step S2 is changed.

[0075] The remaining steps are the same as in Example 1.

[0076] S1. Rhizoma Coptidis is washed, low-temperature dried at 50°C, and crushed into 80 meshes. The crushed powder is mixed with PBS buffer, and a composite enzyme (the composite enzyme is 45wt% of cellulase, 20wt% of pectinase, 15wt% of hemicellulase, 10wt% of ligninase and 10wt% of wall protease; the added mass of the composite enzyme accounts for 0.2% of the mass of Rhizoma Coptidis) is added to perform enzymolysis reaction for 8h to obtain an enzymolysis solution;

[0077] S2, subjecting the enzymatic hydrolysate obtained in step S1 to differential centrifugation, centrifuging at a centrifugal force of 800 g for 10 min, centrifuging at a centrifugal force of 2000 g for 30 min, and centrifuging at a centrifugal force of 10000 g for 50 min; taking the supernatant after differential centrifugation, discarding the precipitate, subjecting the supernatant to ultrahigh-speed centrifugation, centrifuging at a centrifugal force of 15000 g for 120 min, taking the precipitate, and suspending it in a pre-cooled PBS buffer to obtain a precipitate suspension;

[0078] S3, adding sucrose solution in a centrifuge tube according to the gradient, the sucrose concentration gradient is 8%, 30%, 45%, 60%, adding the precipitation suspension obtained in step S2 to the top layer, and centrifuging for 120 min at a centrifugal force of 13000g; after the centrifugation, collect each layer into a centrifuge tube, add pre-cooled PBS buffer and mix well, centrifuge at 13000g for 120 min, remove sucrose, and obtain a precipitate;

[0079] S4. Resuspend the precipitate obtained in step S3 with pre-cooled PBS buffer, use a disposable syringe to absorb the resuspended liquid, and slowly drip it onto a cell filter membrane with a specification of 0.22 μm for filtration. The filtered solution is centrifuged in a low-temperature centrifuge at 4°C and a centrifugal force of 130,000 g for 120 minutes. After centrifugation, the supernatant is aspirated and the precipitate is retained. The precipitate is resuspended with 3 mL of PBS buffer to obtain the extracted and separated exosome product.

[0080] Comparative Example 3

[0081] Compared with Example 1, step S3 sucrose gradient centrifugation was not performed.

[0082] The remaining steps are the same as in Example 1.

[0083] S1. Rhizoma Coptidis is washed, low-temperature dried at 50°C, and crushed into 80 meshes. The crushed powder is mixed with PBS buffer, and a composite enzyme (the composite enzyme is 45wt% of cellulase, 20wt% of pectinase, 15wt% of hemicellulase, 10wt% of ligninase and 10wt% of wall protease; the added mass of the composite enzyme accounts for 0.2% of the mass of Rhizoma Coptidis) is added to perform enzymolysis reaction for 8h to obtain an enzymolysis solution;

[0084] S2, subjecting the enzymatic hydrolysate obtained in step S1 to differential centrifugation, centrifuging for 10 min at a centrifugal force of 1000 g, centrifuging for 30 min at a centrifugal force of 3000 g, and centrifuging for 50 min at a centrifugal force of 10000 g; taking the supernatant after differential centrifugation, discarding the precipitate, subjecting the supernatant to ultrahigh-speed centrifugation, centrifuging for 120 min at a centrifugal force of 13000 g, taking the precipitate, and suspending it in a pre-cooled PBS buffer to obtain a precipitate suspension;

[0085] S3. Use a disposable syringe to absorb the resuspended liquid and slowly drip it onto a cell filter membrane with a specification of 0.22 μm for filtration. Use a low-temperature centrifuge to centrifuge the filtered solution at 4°C and a centrifugal force of 130,000 g for 120 min. After centrifugation, aspirate the supernatant and retain the precipitate. Use 3 mL of PBS buffer to resuspend the obtained precipitate, which is the extracted and separated exosome product.

[0086] Comparative Example 4

[0087] Compared with Example 1, step S4 membrane filtration is not performed.

[0088] The remaining steps are the same as in Example 1.

[0089] S1. Rhizoma Coptidis is washed, low-temperature dried at 50°C, and crushed into 800 meshes. The crushed powder is mixed with PBS buffer, and a composite enzyme (the composite enzyme is 45wt% of cellulase, 20wt% of pectinase, 15wt% of hemicellulase, 10wt% of ligninase and 10wt% of wall protease; the added mass of the composite enzyme accounts for 0.2% of the mass of Rhizoma Coptidis) is added to perform enzymolysis reaction for 8h to obtain an enzymolysis solution;

[0090] S2, subjecting the enzymatic hydrolysate obtained in step S1 to differential centrifugation, centrifuging for 10 min at a centrifugal force of 1000 g, centrifuging for 30 min at a centrifugal force of 3000 g, and centrifuging for 50 min at a centrifugal force of 10000 g; taking the supernatant after differential centrifugation, discarding the precipitate, subjecting the supernatant to ultrahigh-speed centrifugation, centrifuging for 120 min at a centrifugal force of 13000 g, taking the precipitate, and suspending it in a pre-cooled PBS buffer to obtain a precipitate suspension;

[0091] S3. Add sucrose solution in a centrifuge tube according to the gradient, and the sucrose concentration gradient is 8%, 30%, 45%, and 60%. Add the precipitation suspension obtained in step S2 to the top layer, and centrifuge for 120 minutes at a centrifugal force of 13000g; after the centrifugation, collect each layer into a centrifuge tube, add pre-cooled PBS buffer and mix well, centrifuge at 13000g for 120 minutes, remove sucrose, and obtain a precipitate, which is the extracted and separated exosome product.

[0092] Comparative Example 5 Coptis chinensis extract

[0093] Take the medicinal material of Coptis chinensis, dry it at low temperature and then crush it; soak the powder of the medicinal material of Coptis chinensis in an extraction solvent (water, 50% ethanol, 75% ethanol, or 95% ethanol) with a volume of 10 times the weight of the medicinal material for 30 minutes, and heat and reflux for extraction for 60 minutes; cool it to room temperature and filter it to collect the Coptis chinensis extract; add the extraction solvent with a volume of 10 times the weight of the medicinal material to the residue, heat and reflux for extraction for 60 minutes; cool it to room temperature and filter it to collect the Coptis chinensis extract; combine the two extracts, concentrate them under reduced pressure, and freeze-dry them to obtain the Coptis chinensis extract.

[0094] Efficacy Example 1 Study on the effect of Coptis chinensis exosomes on ulcerative colitis

[0095] 1. Establishment of ulcerative colitis model

[0096] 72 male SPF C57BL / 6J mice, weighing 18-22g, were provided with free access to water and diet. The room temperature was maintained at 25±1°C, and the light-dark cycle was 12 hours. After one week of adaptive feeding, the mice were randomly divided into a normal control group, a model control group, an embodiment 1 group, an embodiment 2 group, a comparative example 1 group, a comparative example 2 group, a comparative example 3 group, a comparative example 4 group, and a Coptis chinensis extract group according to body weight, with 8 mice in each group.

[0097] The mice in the normal control group freely drank normal water, and the mice in the other groups freely drank 1.5% DSS solution (prepared by dissolving 1.5g DSS in 100mL distilled water) for modeling. Seven days after modeling, the modeling groups freely drank normal water for 7 days, and this 14-day modeling cycle was repeated for four cycles. The drugs were administered at the same time as modeling, and the drugs were administered for four cycles. The autopsy was performed on the fourth day of the recovery period of the fourth cycle.

[0098] The Example 1 group was given 15 mg / kg of the exosomes of Example 1 by gavage every day. The Example 2 group was given 15 mg / kg of the exosomes of Example 2 by gavage every day. The Comparative Example 1 group was given 15 mg / kg of the product of Comparative Example 1 by gavage every day. The Comparative Example 2 group was given 15 mg / kg of the product of Comparative Example 2 by gavage every day. The Comparative Example 3 group was given 15 mg / kg of the product of Comparative Example 3 by gavage every day. The Comparative Example 4 group was given 15 mg / kg of the product of Comparative Example 4 by gavage every day. The Coptis chinensis extract group was given 15 mg / kg of the Coptis chinensis extract (prepared according to the method of Comparative Example 5) by gavage every day.

[0099] The mice in the normal control group and the model control group were intragastrically administered with an equal volume of normal saline.

[0100] 2. Model observation and indicator detection

[0101] 2.1 Stool index and blood in stool index

[0102] After the start of drug administration, the mice were weighed, and the feces and blood in the feces were observed. The scores were scored using the literature method (ChenL, Jiao T, Liu W, Luo Y, Wang J, Guo X, Tong X, Lin Z, Sun C, Wang K, He Y, Zhang Y, Xu H, Wang J, Zuo J, Ding Q, He S, Gonzalez FJ, Xie C. Hepaticcytochrome P450 8B1 and cholic acid potentiate intestinal epithelial injury in colitis by suppressing intestinal stem cell renewal. Cell Stem Cell. 2022Sep 1;29(9):1366-1381.e9. doi: 10.1016 / j.stem.2022.08.008). The scoring rules are as follows:

[0103] Stool looseness index: Normal formed pelleted stool consistency is scored as 0, soft but still formed stool is scored as 1, soft but still formed stool is scored as 2, very soft and wet stool is scored as 3, and watery diarrhea is scored as 4.

[0104] The blood in stool index was as follows: negative blood in stool was 0 points, weakly positive blood in stool was 1 point, positive blood in stool was 2 points, visible blood in stool was 3 points, and heavy bleeding was 4 points.

[0105] The stool index and blood in stool index of each group were recorded.

[0106] 2.2 Colon length

[0107] After 56 days of treatment, the colon of mice was isolated, and photos were taken to record the colon length.

[0108] 3. Statistical methods

[0109] SPSS22.0 software was used for data processing. The data were expressed as mean ± standard deviation (`x±s). Normal distribution test and variance homogeneity test were performed on the data of each group. One-way analysis of variance was used for inter-group comparison. P < 0.05 was considered statistically significant.

[0110] 4. Experimental Results

[0111] 4.1 Effect of Coptis chinensis exosomes on the body weight of ulcerative colitis model mice

[0112] Compared with the normal control group, the body weight of the mice in the model control group decreased significantly. Compared with the model control group, the body weight of the Example 1 group and the Example 2 group increased significantly. The effect of body weight increase of the Example 1 group was better than that of the Comparative Example 1 group, the Comparative Example 2 group, the Comparative Example 3 group, and the Comparative Example 4 group. Figure 3 .

[0113] 4.2 Effect of Coptis chinensis exosomes on the bloody stool index of ulcerative colitis model mice

[0114] Compared with the normal control group, the blood stool index of mice in the model control group increased significantly. Compared with the model control group, the blood stool index of mice in Example 1 and Example 2 groups decreased significantly. The effect of improving the blood stool index in Example 1 group was better than that in Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4. The results are shown in Figure 4 .

[0115] 4.3 Effect of Coptis chinensis exosomes of the present invention on loose stool index of ulcerative colitis model mice

[0116] Compared with the normal control group, the loose stool index of mice in the model control group increased significantly. Compared with the model control group, the loose stool index of mice in Example 1 and Example 2 groups decreased significantly. The effect of improving the loose stool index in Example 1 group was better than that in Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4. Figure 5 .

[0117] 4.4 Effect of Coptis chinensis exosomes of the present invention on colon length in ulcerative colitis model mice

[0118] Compared with the normal control group, the colon length of the mice in the model control group was significantly shortened. Compared with the model control group, the colon length of the Example 1 group and the Example 2 group was significantly increased. The effect of increasing the colon length of the Example 1 group was better than that of the Comparative Example 1 group, the Comparative Example 2 group, the Comparative Example 3 group, and the Comparative Example 4 group. Figure 6 .

[0119] The coptis chinensis exosomes of the present invention significantly increase the body weight of ulcerative colitis model mice, reduce the bloody stool index and loose stool index, and increase the colon length, thereby achieving the purpose of treating ulcerative colitis. The coptis chinensis exosomes of the present invention have a therapeutic effect on ulcerative colitis that is better than the products of comparative example 1, comparative example 2, comparative example 3, and comparative example 4. The coptis chinensis exosomes of the present invention have a therapeutic effect on ulcerative colitis that is better than the coptis chinensis extract.

[0120] The above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description, and the obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A method for preparing exosomes of Coptis chinensis, characterized in that: The method for preparing the coptis chinensis exosomes comprises the following steps: S1. Rhizoma Coptidis is washed, dried at low temperature, and crushed. The crushed powder is mixed with PBS buffer, and a composite enzyme is added to perform enzymolysis reaction to obtain an enzymolysis solution; the composite enzyme is 45wt% of cellulase, 20wt% of pectinase, 15wt% of hemicellulase, 10wt% of ligninase, and 10wt% of wall protease; S2, subjecting the enzymatic hydrolysate obtained in step S1 to differential centrifugation, centrifuging for 10 min at a centrifugal force of 1000 g, centrifuging for 30 min at a centrifugal force of 3000 g, and centrifuging for 50 min at a centrifugal force of 10000 g; taking the supernatant after differential centrifugation, discarding the precipitate, subjecting the supernatant to ultrahigh-speed centrifugation, centrifuging for 120 min at a centrifugal force of 13000 g, taking the precipitate, and suspending it in a pre-cooled PBS buffer to obtain a precipitate suspension; S3, adding sucrose solution in a centrifuge tube according to the gradient, the sucrose concentration gradient is 8%, 30%, 45%, 60%, adding the precipitation suspension obtained in step S2 to the top layer, and centrifuging for 120 min at a centrifugal force of 13000g; after the centrifugation, collect each layer into a centrifuge tube, add pre-cooled PBS buffer and mix well, centrifuge at 13000g for 120 min, remove sucrose, and obtain a precipitate; S4. Resuspend the precipitate obtained in S3 with pre-cooled PBS buffer, use a disposable syringe to absorb the resuspended liquid, and slowly drip it onto a cell filter membrane with a specification of 0.22 μm for filtration. Use a low-temperature centrifuge to centrifuge the filtered solution at 4°C and a centrifugal force of 130,000 g for 120 min. After centrifugation, aspirate the supernatant and retain the precipitate, and resuspend the obtained precipitate with PBS buffer to obtain the extracted and separated Coptis chinensis exosomes.

2. The method for preparing exosomes of Coptis chinensis according to claim 1, characterized in that: In the step S1, the temperature of low temperature drying is 40-60° C. and the powder is crushed to 80-120 mesh.

3. The method for preparing exosomes of Coptis chinensis according to claim 1, characterized in that: The added mass of the complex enzyme in step S1 accounts for 0.1-0.4% of the mass of Coptis chinensis; and the enzymolysis time is 6-10 hours.

4. The method for preparing exosomes of Coptis chinensis according to claim 1, characterized in that: The amount of PBS buffer used in step S4 is 1-3 mL.

5. A Coptis chinensis exosome, characterized in that: The exosomes are obtained by the method for preparing coptis chinensis exosomes as described in claim 1.

6. Use of the Coptis chinensis exosomes according to claim 5 in the preparation of anti-ulcerative colitis drugs.

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