A composition containing stem cell exosomes and its use in the preparation of a medicament for treating liver damage

Abstract

The application discloses a stem cell exosome-containing composition and application of the stem cell exosome-containing composition in preparation of a medicine with a liver damage treatment effect, and the stem cell exosome-containing composition comprises, by weight, 0.1-0.5 parts of umbilical cord mesenchymal stem cell exosome, 3-6 parts of pueraria extract, 0.2-1 part of honey, 0.06-0.32 parts of compound vitamin, 0.3-0.7 parts of olive oil, 0.08-0.2 parts of starch, 0.03-0.15 parts of glutamic acid, 0.03-0.15 parts of cysteine, 0.03-0.15 parts of glycine, 0.05-0.35 parts of glucuronolactone, 0.05-0.18 parts of sodium carboxymethyl cellulose and 22-37 parts of deionized water. The stem cell exosome can adjust cell apoptosis, growth, multiplication and differentiation pathways through the transcriptome and proteome of a receptor cell, thereby delaying the occurrence and development of liver diseases; the pueraria extract is rich in various flavones and isoflavones, can promote liver cell regeneration, and provides required nutrients for the liver in cooperation with other components, and effectively treats liver damage.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a composition containing stem cell exosomes and its application in the preparation of a medicament for treating liver injury. Background Technology

[0002] The liver is one of the five major organs in the human body, playing a crucial role in metabolism. Various hepatotoxic factors, such as viral infections, drug poisoning, lipid deposition, and autoimmune reactions, can cause liver damage. Continued liver damage leads to excessive accumulation of extracellular matrix, resulting in loss of normal structure and progression to liver fibrosis.

[0003] Stem cell exosomes are membrane-derived nanoscale vesicles with important biological functions such as reducing apoptosis, alleviating inflammation, and improving tissue repair. They can regulate apoptosis, growth, proliferation, and differentiation pathways by altering the extracellular matrix and the transcriptome and proteome of recipient cells, thereby delaying the occurrence and development of liver diseases.

[0004] Studies have shown that exosomes are safer and more effective than cell transplantation in treating liver injury. Based on this, the development of a drug containing stem cell exosomes has become a current research direction. Summary of the Invention

[0005] This invention first provides a composition containing stem cell exosomes and its application in the preparation of a drug with therapeutic effects on liver injury. The composition can synergistically provide the liver with the necessary nutrients, promote hepatocyte regeneration, and effectively treat liver injury.

[0006] The present invention solves the technical problem by adopting the following technical solution:

[0007] This invention provides a composition containing stem cell exosomes, characterized in that the composition containing stem cell exosomes comprises, by weight: 0.1-0.5 parts umbilical cord mesenchymal stem cell exosomes, 3-6 parts kudzu root extract, 0.2-1 part honey, 0.06-0.32 parts compound vitamins, 0.3-0.7 parts olive oil, 0.08-0.2 parts starch, 0.03-0.15 parts glutamic acid, 0.03-0.15 parts cysteine, 0.03-0.15 parts glycine, 0.05-0.35 parts glucuronide, 0.05-0.18 parts sodium carboxymethyl cellulose, and 22-37 parts deionized water.

[0008] Preferably, the composition containing stem cell exosomes comprises, by weight: 0.2-0.4 parts umbilical cord mesenchymal stem cell exosomes, 4-5 parts kudzu root extract, 0.4-0.8 parts honey, 0.18-21 parts compound vitamins, 0.4-0.6 parts olive oil, 0.12-0.16 parts starch, 0.07-0.11 parts glutamic acid, 0.07-0.11 parts cysteine, 0.07-0.11 parts glycine, 0.17-0.23 parts glucuronide, 0.09-0.14 parts sodium carboxymethyl cellulose, and 27-32 parts deionized water.

[0009] Preferably, the composition containing stem cell exosomes comprises, by weight: 0.3 parts umbilical cord mesenchymal stem cell exosomes, 4 parts kudzu root extract, 0.6 parts honey, 0.2 parts compound vitamins, 0.5 parts olive oil, 0.14 parts starch, 0.09 parts glutamic acid, 0.09 parts cysteine, 0.09 parts glycine, 0.21 parts glucuronide, 0.12 parts sodium carboxymethyl cellulose, and 29 parts deionized water.

[0010] Preferably, the method for preparing the umbilical cord mesenchymal stem cell exosomes is as follows:

[0011] S1. Take umbilical cord tissue, wash it three times in PBS buffer for 8-10 minutes each time, remove arteries and veins to obtain Walton gel, cut the Walton gel into 4-6 mm tissue blocks, rinse with deionized water for 15-30 minutes, and then wash with PBS buffer five times for 5-8 minutes each time to obtain tissue blocks.

[0012] S2. Mix the tissue block and collagenase I solution at a weight ratio of 1:(0.2-0.28) and react for 15-30 minutes to obtain the mixture.

[0013] S3. Centrifuge the mixture at 800-1200 r / min for 20-30 min, filter it through a 0.4 μm sterile filter membrane to obtain cell pellet, and wash it 3 times with PBS buffer.

[0014] S4. Wash the cell pellet three times with balanced salt buffer solution, then resuspend the cells with twice the volume of balanced salt buffer solution, and purify the cell suspension using density gradient centrifugation to obtain umbilical cord mesenchymal stem cells.

[0015] S5. Seed umbilical cord mesenchymal stem cells at a density of 1×10 cells / ml into the culture medium and cultured in an incubator for 2-4 days. The number of passages should be controlled within P10. When the cell fusion rate reaches 80-90%, collect the culture supernatant, i.e., the first supernatant.

[0016] S6. Centrifuge the first supernatant at 4℃ and 1800r / min for 10-30min, collect the supernatant, concentrate it by ultrafiltration to remove small molecules, and then filter it through a 0.3μm sterile filter membrane to obtain umbilical cord mesenchymal cell precipitate.

[0017] S7. Wash the umbilical cord mesenchymal cell pellet three times with PBS buffer, seed it into serum-free culture medium at a density of 1×10 cells / ml, and incubate it in an incubator for 42-48 hours. Collect the supernatant of the culture medium, i.e., the second supernatant.

[0018] S8. Transfer the second clear liquid to a density pad with a mass concentration of 30% sucrose heavy water and centrifuge at 4°C with a density gradient of 80,000-160,000g for 120 min. Collect the bottom buffer pad.

[0019] S9. After washing the bottom buffer pad three times with PBS solution, the small molecules were removed by ultrafiltration concentration, and then filtered through a 0.25μm sterile filter membrane to obtain umbilical cord mesenchymal stem cell exosomes.

[0020] As a preferred embodiment, the PBS buffer contains 1-3 wt% penicillin and 1.2-3.6 wt% streptomycin sulfate; the culture medium is DMEM medium containing 8-12 wt% FBS; and the incubator conditions are 36-38℃ and 4-6% CO2 saturated humidity.

[0021] Preferably, the method for preparing the kudzu root extract is as follows:

[0022] S21. Wash the kudzu root with deionized water, ultrasonically treat it at 100W and 20℃ for 15 minutes, dry it, and pulverize it to 20-40 mesh to obtain kudzu root powder.

[0023] S22. Mix kudzu root powder and 65-85% ethanol solution at a weight ratio of 1:(5-8), extract at 40-50℃ for 30-90 min, centrifuge, filter, and obtain the first extract.

[0024] S23. Mix the first extract and activated carbon at a weight ratio of 1:(0.006-0.018), centrifuge, and filter to obtain the second extract;

[0025] S24. Mix the second extract and the 1.5% chitosan solution at a weight ratio of 1:(0.002-0.01), centrifuge, and filter to obtain the third extract;

[0026] S25. Pass the third extract into an adsorption column packed with macroporous resin at an elution rate of 2-3 BV / h. Wash the adsorption column with 3 times the resin column volume of deionized water at a flow rate of 1.5-2.5 V / h and 2 times the resin column volume of 50 wt% ethanol solution at a flow rate of 1-3 BV / h. Collect the ethanol solution to obtain the kudzu root extract.

[0027] As a preferred embodiment, the centrifugation conditions are 25℃, 12000r / min, and centrifugation time is 20min; the filter membrane pore size is 0.3μm; and the macroporous adsorption resin is HPD-100.

[0028] Preferably, the complex vitamin is composed of vitamin A, vitamin C, vitamin E, vitamin B6 and vitamin B12 in a weight ratio of 1.5:0.5:1:2:1.

[0029] Preferably, the method for preparing the composition containing stem cell exosomes is as follows:

[0030] S31. Dissolve honey, multivitamins, olive oil, starch, glutamic acid, cysteine, glycine, glucuronide, and sodium carboxymethyl cellulose in deionized water, and stir at 45℃ and 240-360 rpm for 30-45 minutes until completely dissolved to obtain mixture A;

[0031] S32. Add umbilical cord mesenchymal stem cell exosomes and kudzu root extract to mixture A, stir at 40℃ and 120-180 rpm for 15-30 min to obtain mixture B;

[0032] S33. Mixture B is refluxed at 37°C for 1-2 hours, filtered, concentrated to a relative density of 1.5, and freeze-dried to obtain a composition containing stem cell exosomes.

[0033] The present invention also provides the use of a composition containing stem cell exosomes in the preparation of a medicament for treating liver injury.

[0034] The beneficial effects of this invention are:

[0035] The present invention discloses a composition containing stem cell exosomes, comprising components such as complex vitamins and amino acids. The stem cell exosomes regulate cell growth and proliferation by altering the transcriptome and proteome of the recipient cells, while kudzu root extract synergistically promotes hepatocyte regeneration, providing the liver with the substances and energy required for normal physiological activities, and effectively treating liver damage. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0037] Example 1.

[0038] This embodiment provides a composition containing stem cell exosomes, which, by weight, comprises: 0.1 parts umbilical cord mesenchymal stem cell exosomes, 3 parts kudzu root extract, 0.2 parts honey, 0.06 parts multivitamins, 0.3 parts olive oil, 0.08 parts starch, 0.03 parts glutamic acid, 0.03 parts cysteine, 0.03 parts glycine, 0.05 parts glucuronide, 0.05 parts sodium carboxymethyl cellulose, and 22 parts deionized water.

[0039] The method for preparing umbilical cord mesenchymal stem cell exosomes in this embodiment is as follows:

[0040] S1. Take umbilical cord tissue, wash it three times in PBS buffer for 8 minutes each time, remove arteries and veins to obtain Walton gel, cut the Walton gel into 4 mm tissue blocks, rinse with deionized water for 15 minutes, and then wash with PBS buffer five times for 5 minutes each time to obtain tissue blocks.

[0041] S2. Mix the tissue block and collagenase I solution at a weight ratio of 1:0.2 and react for 15 minutes to obtain the mixture.

[0042] S3. Centrifuge the mixture at 800 r / min for 20 min, filter it through a 0.4 μm sterile filter membrane to obtain cell pellet, and wash it 3 times with PBS buffer.

[0043] S4. Wash the cell pellet three times with balanced salt buffer solution, then resuspend the cells with twice the volume of balanced salt buffer solution, and purify the cell suspension using density gradient centrifugation to obtain umbilical cord mesenchymal stem cells.

[0044] S5. Seed umbilical cord mesenchymal stem cells at a density of 1×10 cells / ml into the culture medium and place them in an incubator for culture. Culture at 36℃ and 4% CO2 saturated humidity for 2 days. The number of passages should be controlled within P10. When the cell fusion rate reaches 80%, collect the culture supernatant, i.e., the first supernatant.

[0045] S6. Centrifuge the first supernatant at 4℃ and 1800r / min for 10min, collect the supernatant, concentrate it by ultrafiltration to remove small molecules, and then filter it through a 0.3μm sterile filter membrane to obtain umbilical cord mesenchymal cell precipitate.

[0046] S7. Wash the umbilical cord mesenchymal cell pellet three times with PBS buffer, and seed it into serum-free culture medium at a density of 1×10 cells / ml. Incubate in an incubator at 36℃ and 4% CO2 saturated humidity for 42h. Collect the supernatant of the culture medium, i.e. the second supernatant.

[0047] S8. Transfer the second clear liquid to a 30% sucrose heavy water density pad and centrifuge at 4°C with a density gradient of 80,000g for 120 min. Collect the bottom buffer pad.

[0048] S9. After washing the bottom buffer pad three times with PBS solution, the small molecules were removed by ultrafiltration concentration, and then filtered through a 0.25μm sterile filter membrane to obtain umbilical cord mesenchymal stem cell exosomes.

[0049] The PBS buffer in this embodiment contains 1 wt% penicillin and 1.2 wt% streptomycin sulfate; the culture medium in this embodiment is DMEM medium containing 8 wt% FBS.

[0050] The preparation method of kudzu root extract in this embodiment is as follows:

[0051] S21. Wash the kudzu root with deionized water, ultrasonically treat it at 100W and 20℃ for 15 minutes, dry it, and pulverize it to 20 mesh to obtain kudzu root powder.

[0052] S22. Mix kudzu root powder and 65% ethanol solution at a weight ratio of 1:5, extract at 40℃ for 30 min, centrifuge at 25℃ and 12000 r / min for 20 min, and filter through a 0.3 μm pore size membrane to obtain the first extract.

[0053] S23. Mix the first extract and activated carbon at a weight ratio of 1:0.006, centrifuge at 25℃ and 12000r / min for 20min, and filter through a 0.3μm pore size membrane to obtain the second extract.

[0054] S24. Mix the second extract and 1.5% chitosan solution at a weight ratio of 1:0.002, centrifuge at 25℃ and 12000r / min for 20min, and filter through a 0.3μm pore size membrane to obtain the third extract.

[0055] S25. Pass the third extract into an adsorption column packed with macroporous resin at an elution rate of 2 BV / h. Rinse the adsorption column with 3 times the resin column volume of deionized water at a flow rate of 1.5 BV / h and 2 times the resin column volume of 50 wt% ethanol solution at a flow rate of 1 BV / h. Collect the ethanol solution to obtain the kudzu root extract.

[0056] The macroporous adsorption resin used in this embodiment is HPD-100.

[0057] The multivitamin in this embodiment is composed of vitamin A, vitamin C, vitamin E, vitamin B6 and vitamin B12 in a weight ratio of 1.5:0.5:1:2:1.

[0058] The preparation method of the stem cell exosome-containing composition in this embodiment is as follows:

[0059] S31. Dissolve honey, multivitamins, olive oil, starch, glutamic acid, cysteine, glycine, glucuronide, and sodium carboxymethyl cellulose in deionized water, then stir at 45°C and 240 rpm for 30 minutes until completely dissolved to obtain mixture A;

[0060] S32. Add umbilical cord mesenchymal stem cell exosomes and kudzu root extract to mixture A, stir at 40℃ and 120 rpm for 15 min to obtain mixture B;

[0061] S33. Mixture B is refluxed at 37°C for 1 hour, filtered, concentrated to a relative density of 1.5, and freeze-dried to obtain a composition containing stem cell exosomes.

[0062] Example 2.

[0063] This embodiment provides a composition containing stem cell exosomes, which, by weight, comprises: 0.5 parts umbilical cord mesenchymal stem cell exosomes, 6 parts kudzu root extract, 1 part honey, 0.32 parts multivitamins, 0.7 parts olive oil, 0.2 parts starch, 0.15 parts glutamic acid, 0.15 parts cysteine, 0.15 parts glycine, 0.35 parts glucuronide, 0.18 parts sodium carboxymethyl cellulose, and 37 parts deionized water.

[0064] The method for preparing umbilical cord mesenchymal stem cell exosomes in this embodiment is as follows:

[0065] S1. Take umbilical cord tissue, wash it three times in PBS buffer for 10 minutes each time, remove arteries and veins to obtain Walton gel, cut the Walton gel into 6 mm tissue blocks, rinse with deionized water for 30 minutes, and then wash with PBS buffer five times for 8 minutes each time to obtain tissue blocks.

[0066] S2. Mix the tissue block and collagenase I solution at a weight ratio of 1:0.28 and react for 30 minutes to obtain the mixture.

[0067] S3. Centrifuge the mixture at 1200 r / min for 30 min, filter it through a 0.4 μm sterile filter membrane to obtain cell pellet, and wash it 3 times with PBS buffer.

[0068] S4. Wash the cell pellet three times with balanced salt buffer solution, then resuspend the cells with twice the volume of balanced salt buffer solution, and purify the cell suspension using density gradient centrifugation to obtain umbilical cord mesenchymal stem cells.

[0069] S5. Seed umbilical cord mesenchymal stem cells at a density of 1×10 cells / ml into the culture medium and place them in an incubator for culture at 38℃ and 6% CO2 saturated humidity for 4 days. The number of passages should be controlled within P10. When the cell confluence rate reaches 90%, the culture supernatant, i.e. the first supernatant, is collected.

[0070] S6. Centrifuge the first supernatant at 4℃ and 1800r / min for 30min, collect the supernatant, concentrate it by ultrafiltration to remove small molecules, and then filter it through a 0.3μm sterile filter membrane to obtain umbilical cord mesenchymal cell precipitate.

[0071] S7. Wash the umbilical cord mesenchymal cell pellet three times with PBS buffer, and seed it into serum-free culture medium at a density of 1×10 cells / ml. Incubate in an incubator at 38℃ and 6% CO2 saturated humidity for 48h. Collect the supernatant of the culture medium, i.e. the second supernatant.

[0072] S8. Transfer the second supernatant to a 30% sucrose heavy water density pad and centrifuge at 160,000 g density gradient for 120 min at 4°C. Collect the bottom buffer pad.

[0073] S9. After washing the bottom buffer pad three times with PBS solution, the small molecules were removed by ultrafiltration concentration, and then filtered through a 0.25μm sterile filter membrane to obtain umbilical cord mesenchymal stem cell exosomes.

[0074] The PBS buffer in this embodiment contains 3 wt% penicillin and 3.6 wt% streptomycin sulfate; the culture medium in this embodiment is DMEM medium containing 12 wt% FBS.

[0075] The preparation method of kudzu root extract in this embodiment is as follows:

[0076] S21. Wash the kudzu root with deionized water, ultrasonically treat it at 100W and 20℃ for 15 minutes, dry it, and pulverize it to 40 mesh to obtain kudzu root powder.

[0077] S22. Mix kudzu root powder and 85% ethanol solution at a weight ratio of 1:8, extract at 50℃ for 90 min, centrifuge at 25℃ and 12000 r / min for 20 min, and filter through a 0.3 μm pore size membrane to obtain the first extract.

[0078] S23. Mix the first extract and activated carbon at a weight ratio of 1:0.018, centrifuge at 25℃ and 12000r / min for 20min, and filter through a 0.3μm pore size membrane to obtain the second extract.

[0079] S24. Mix the second extract and 1.5% chitosan solution at a weight ratio of 1:0.01, centrifuge at 25℃ and 12000r / min for 20min, and filter through a 0.3μm pore size membrane to obtain the third extract.

[0080] S25. Pass the third extract into an adsorption column packed with macroporous resin at an elution rate of 3 BV / h. Wash the adsorption column with 3 times the resin column volume of deionized water at a flow rate of 2.5 BV / h and 2 times the resin column volume of 50 wt% ethanol solution at a flow rate of 3 BV / h. Collect the ethanol solution to obtain the kudzu root extract.

[0081] The macroporous adsorption resin used in this embodiment is HPD-100.

[0082] The multivitamin in this embodiment is composed of vitamin A, vitamin C, vitamin E, vitamin B6 and vitamin B12 in a weight ratio of 1.5:0.5:1:2:1.

[0083] The preparation method of the stem cell exosome-containing composition in this embodiment is as follows:

[0084] S31. Dissolve honey, multivitamins, olive oil, starch, glutamic acid, cysteine, glycine, glucuronide, and sodium carboxymethyl cellulose in deionized water, and stir at 45°C and 360 rpm for 45 minutes until completely dissolved to obtain mixture A;

[0085] S32. Add umbilical cord mesenchymal stem cell exosomes and kudzu root extract to mixture A, stir at 40℃ and 180 rpm for 30 min to obtain mixture B;

[0086] S33. Mixture B is refluxed at 37°C for 2 hours, filtered, concentrated to a relative density of 1.5, and freeze-dried to obtain a composition containing stem cell exosomes.

[0087] Example 3.

[0088] This embodiment provides a composition containing stem cell exosomes, which, by weight, comprises: 0.3 parts umbilical cord mesenchymal stem cell exosomes, 4 parts kudzu root extract, 0.6 parts honey, 0.2 parts multivitamins, 0.5 parts olive oil, 0.14 parts starch, 0.09 parts glutamic acid, 0.09 parts cysteine, 0.09 parts glycine, 0.21 parts glucuronide, 0.12 parts sodium carboxymethyl cellulose, and 29 parts deionized water.

[0089] The method for preparing umbilical cord mesenchymal stem cell exosomes in this embodiment is as follows:

[0090] S1. Take umbilical cord tissue, wash it three times in PBS buffer for 9 minutes each time, remove arteries and veins to obtain Walton gel, cut the Walton gel into 5 mm tissue blocks, rinse with deionized water for 22 minutes, and then wash with PBS buffer five times for 8 minutes each time to obtain tissue blocks.

[0091] S2. Mix the tissue block and collagenase I solution at a weight ratio of 1:0.28 and react for 30 minutes to obtain the mixture.

[0092] S3. Centrifuge the mixture at 1000 r / min for 25 min, filter it through a 0.4 μm sterile filter membrane to obtain cell pellet, and wash it 3 times with PBS buffer.

[0093] S4. Wash the cell pellet three times with balanced salt buffer solution, then resuspend the cells with twice the volume of balanced salt buffer solution, and purify the cell suspension using density gradient centrifugation to obtain umbilical cord mesenchymal stem cells.

[0094] S5. Seed umbilical cord mesenchymal stem cells at a density of 1×10 cells / ml into the culture medium and place them in an incubator for culture. Culture at 37℃ and 5% CO2 saturated humidity for 3 days. The number of passages should be controlled within P10. When the cell confluence rate reaches 85%, collect the culture supernatant, i.e., the first supernatant.

[0095] S6. Centrifuge the first supernatant at 4℃ and 1800r / min for 20min, collect the supernatant, concentrate it by ultrafiltration to remove small molecules, and then filter it through a 0.3μm sterile filter membrane to obtain umbilical cord mesenchymal cell precipitate.

[0096] S7. Wash the umbilical cord mesenchymal cell pellet three times with PBS buffer, and seed it into serum-free culture medium at a density of 1×10 cells / ml. Incubate in an incubator at 37℃ and 5% CO2 saturated humidity for 45h. Collect the supernatant of the culture medium, i.e., the second supernatant.

[0097] S8. Transfer the second supernatant to a 30% sucrose heavy water density pad and centrifuge at 4°C with a density gradient of 120,000g for 120 min. Collect the bottom buffer pad.

[0098] S9. After washing the bottom buffer pad three times with PBS solution, the small molecules were removed by ultrafiltration concentration, and then filtered through a 0.25μm sterile filter membrane to obtain umbilical cord mesenchymal stem cell exosomes.

[0099] The PBS buffer in this embodiment contains 2 wt% penicillin and 2.4 wt% streptomycin sulfate; the culture medium in this embodiment is DMEM medium containing 10 wt% FBS.

[0100] The preparation method of kudzu root extract in this embodiment is as follows:

[0101] S21. Wash the kudzu root with deionized water, ultrasonically treat it at 100W and 20℃ for 15 minutes, dry it, and pulverize it to 30 mesh to obtain kudzu root powder.

[0102] S22. Mix kudzu root powder and 75% ethanol solution at a weight ratio of 1:7, extract at 45℃ for 60 min, centrifuge at 25℃ and 12000 r / min for 20 min, and filter through a 0.3 μm pore size membrane to obtain the first extract.

[0103] S23. Mix the first extract and activated carbon at a weight ratio of 1:0.012, centrifuge at 25℃ and 12000r / min for 20min, and filter through a 0.3μm pore size membrane to obtain the second extract.

[0104] S24. Mix the second extract and 1.5% chitosan solution at a weight ratio of 1:0.006, centrifuge at 25℃ and 12000r / min for 20min, and filter through a 0.3μm pore size membrane to obtain the third extract.

[0105] S25. Pass the third extract into an adsorption column packed with macroporous resin at an elution rate of 2.5 BV / h. Rinse the adsorption column with 3 times the resin column volume of deionized water at a flow rate of 2 BV / h and 2 times the resin column volume of 50wt% ethanol solution at a flow rate of 2 BV / h. Collect the ethanol solution to obtain the kudzu root extract.

[0106] The macroporous adsorption resin used in this embodiment is HPD-100.

[0107] The multivitamin in this embodiment is composed of vitamin A, vitamin C, vitamin E, vitamin B6 and vitamin B12 in a weight ratio of 1.5:0.5:1:2:1.

[0108] The preparation method of the stem cell exosome-containing composition in this embodiment is as follows:

[0109] S31. Dissolve honey, multivitamins, olive oil, starch, glutamic acid, cysteine, glycine, glucuronide, and sodium carboxymethyl cellulose in deionized water, and stir at 45°C and 320 rpm for 38 minutes until completely dissolved to obtain mixture A;

[0110] S32. Add umbilical cord mesenchymal stem cell exosomes and kudzu root extract to mixture A, stir at 40℃ and 150 rpm for 22 min to obtain mixture B;

[0111] S33. Mixture B was refluxed at 37°C for 1.51 h, filtered, concentrated to a relative density of 1.5, and freeze-dried to obtain a composition containing stem cell exosomes.

[0112] Comparative Example 1.

[0113] Unlike Example 3, it does not contain umbilical cord mesenchymal stem cell exosomes.

[0114] Comparative Example 2.

[0115] Unlike Example 3, it does not contain kudzu root extract.

[0116] Comparative Example 3.

[0117] The method for preparing umbilical cord mesenchymal stem cell exosomes differs from that in Example 3, and is as follows:

[0118] S1. Take umbilical cord tissue, wash it three times in PBS buffer for 9 minutes each time, remove arteries and veins to obtain Walton gel, cut the Walton gel into 5 mm tissue blocks, rinse with deionized water for 22 minutes, and then wash with PBS buffer five times for 8 minutes each time to obtain tissue blocks.

[0119] S2. Mix the tissue block and collagenase I solution at a weight ratio of 1:0.28 and react for 30 minutes to obtain the mixture.

[0120] S3. Centrifuge the mixture at 1000 r / min for 25 min, filter it through a 0.4 μm sterile filter membrane to obtain cell pellet, and wash it 3 times with PBS buffer.

[0121] S4. Wash the cell pellet three times with balanced salt buffer solution, then resuspend the cells with twice the volume of balanced salt buffer solution, and purify the cell suspension using density gradient centrifugation to obtain umbilical cord mesenchymal stem cells.

[0122] S5. Seed umbilical cord mesenchymal stem cells at a density of 1×10 cells / ml into the culture medium and place them in an incubator for culture. Culture at 37℃ and 5% CO2 saturated humidity for 3 days. The number of passages should be controlled within P10. When the cell confluence rate reaches 85%, collect the culture supernatant, i.e., the first supernatant.

[0123] S6. Centrifuge the first supernatant at 4℃ and 1800r / min for 20min, collect the supernatant, concentrate it by ultrafiltration to remove small molecules, filter it through a 0.3μm sterile filter membrane for sterilization, transfer it to a 30% sucrose heavy water density pad and centrifuge it at 4℃ and 120000g density gradient for 120min, collect the bottom buffer pad.

[0124] S7. After washing the bottom buffer pad three times with PBS solution, the small molecules were removed by ultrafiltration concentration, and then filtered through a 0.25μm sterile filter membrane to obtain umbilical cord mesenchymal stem cell exosomes.

[0125] The PBS buffer in this embodiment contains 2 wt% penicillin and 2.4 wt% streptomycin sulfate; the culture medium in this embodiment is DMEM medium containing 10 wt% FBS.

[0126] Comparative Example 4.

[0127] The preparation method of the kudzu root extract differs from that in Example 3, and is as follows:

[0128] S21. Wash the kudzu root with deionized water, ultrasonically treat it at 100W and 20℃ for 15 minutes, dry it, and pulverize it to 30 mesh to obtain kudzu root powder.

[0129] S22. Mix kudzu root powder and 75% ethanol solution at a weight ratio of 1:7, extract at 45℃ for 60 min, centrifuge at 25℃ and 12000 r / min for 20 min, and filter through a 0.3 μm pore size membrane to obtain kudzu root extract.

[0130] S23. The kudzu root extract was concentrated by rotary evaporation at 45°C to recover ethanol. The product was extracted with ethyl acetate, centrifuged, concentrated by rotary evaporation again, and freeze-dried to obtain the kudzu root extract.

[0131] Comparative Example 5.

[0132] The preparation method of the kudzu root extract differs from that in Example 3, and is as follows:

[0133] S21. Wash the kudzu root with deionized water, ultrasonically treat it at 100W and 20℃ for 15 minutes, dry it, and pulverize it to 30 mesh to obtain kudzu root powder.

[0134] S22. Mix kudzu root powder and 75% ethanol solution at a weight ratio of 1:7, extract at 45℃ for 60 min, centrifuge at 25℃ and 12000 r / min for 20 min, and filter through a 0.3 μm pore size membrane to obtain the first extract.

[0135] S23. Mix the first extract and activated carbon at a weight ratio of 1:0.012, centrifuge at 25℃ and 12000r / min for 20min, and filter through a 0.3μm pore size membrane to obtain the second extract.

[0136] S24. Mix the second extract and 1.5% chitosan solution at a weight ratio of 1:0.006, centrifuge at 25℃ and 12000r / min for 20min, and filter through a 0.3μm pore size membrane to obtain the third extract.

[0137] S25. Pass the third extract into an adsorption column packed with macroporous resin at an elution rate of 2.5 BV / h. Rinse the adsorption column with 3 times the resin column volume of deionized water at a flow rate of 2 BV / h and 2 times the resin column volume of 60wt% ethanol solution at a flow rate of 2 BV / h. Collect the ethanol solution to obtain the kudzu root extract.

[0138] The macroporous adsorption resin used in this embodiment is HPD-100.

[0139] Since the pathogenic mechanisms of liver injury in mice and humans are similar, and the drug targets are also similar, many drugs have similar therapeutic effects. Therefore, the therapeutic effect of the drug on human liver injury was deduced based on the experimental data of the drug of this invention in mice. The drugs obtained in Examples 1-3 and Comparative Examples 1-5 were tested, and the specific experimental procedures are as follows:

[0140] 1. Eighty-eight healthy mice weighing 20±2g were selected and, after one week of acclimatization feeding, were randomly divided into a normal group, a model group, a positive group, and a drug-treated group. The drug-treated group included Examples 1-3 and Comparative Examples 1-5. Eight mice were assigned to each group. The experimental results of each group were the average of the experimental results of the mice in that group.

[0141] In the positive group, mice were given diammonium glycyrrhizate solution, with 0.1 mL of diammonium glycyrrhizate solution per 10 g body weight by gavage twice a day, once in the morning and once in the evening. The normal group and the model group were given the corresponding volume of physiological saline by gavage once a day in the morning. The drug treatment group was given the corresponding volume of the drug in the corresponding example and comparative example by gavage once a day in the morning.

[0142] Except for the normal group, the other groups were injected intraperitoneally twice a week with carbon tetrachloride olive oil solution, with 10 μl of carbon tetrachloride olive oil solution injected intraperitoneally per 10g body weight of mice to induce liver damage; the normal group was injected with olive oil, with 10 μl of olive oil injected intraperitoneally per 10g body weight of mice.

[0143] 2. Six weeks after administration, blood and liver tissue were collected from mice in each group. After centrifugation of the blood, serum was obtained, and the levels of ALT and AST were detected to evaluate the liver function of mice in each group.

[0144] 3. The test results are shown in the table below:

[0145]

[0146] As can be seen from the table above, the components of the product of this invention work synergistically to reduce inflammatory cells in the damaged liver, improve cell morphology, and effectively treat liver damage.

[0147] As can be seen from Examples 1-3, the composition containing stem cell exosomes described in this invention, wherein the umbilical cord mesenchymal stem cell exosomes and kudzu root extract are combined and work together with amino acids, vitamins and other components to promote the repair and proliferation of hepatocytes, relieve inflammation and treat damage.

[0148] Comparing Example 3 with Comparative Examples 1 and 3, it can be seen that without the addition of umbilical cord mesenchymal stem cell exosomes and with only one cell culture of umbilical cord mesenchymal stem cell exosomes, the metabolic and regulatory information of hepatocytes cannot be fully expressed, thus limiting the therapeutic effect on liver injury.

[0149] Comparing Example 3 with Comparative Examples 2, 4, and 5, it is evident that neither adding kudzu root extract nor using different extraction methods can fully extract the effective components from kudzu root. This invention, after ethanol extraction, employs a series of decolorization and impurity removal reactions, using macroporous resin to elute and separate the target product, thereby improving the purity of the kudzu root extract, regulating hepatocyte activity, and alleviating liver damage. Example 3 represents the optimal formulation; other conditions also have their own optimal values ​​under different numerical experimental conditions.

[0150] Based on the above-described preferred embodiments of the present invention, it should be noted that the technical scope of the present invention is not limited to the contents of the specification. For those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition comprising stem cell exosomes, characterized in that, The stem cell-containing exosome composition comprises, by weight parts: 0.1-0.5 parts of umbilical cord mesenchymal stem cell exosomes, 3-6 parts of pueraria extract, 0.2-1 part of honey, 0.06-0.32 parts of compound vitamins, 0.3-0.7 parts of olive oil, 0.08-0.2 parts of starch, 0.03-0.15 parts of glutamic acid, 0.03-0.15 parts of cysteine, 0.03-0.15 parts of glycine, 0.05-0.35 parts of glucuronolactone, 0.05-0.18 parts of sodium carboxymethyl cellulose, 22-37 parts of deionized water; The preparation method of the umbilical cord mesenchymal stem cell exosome is: S1, take the umbilical cord tissue, wash it in PBS buffer for 3 times, 8-10 min each time, remove the arteriovenous to get the Walton glue, cut the Walton glue into 4-6 mm tissue pieces, rinse with deionized water for 15-30 min, then wash with PBS buffer for 5 times, 5-8 min each time, to get the tissue pieces; S2, mix the tissue pieces and collagenase I solution according to the weight ratio of 1:(0.2-0.28), react for 15-30 min to get the mixed material; S3, centrifuge the mixed material at 800-1200 r / min for 20-30 min, pass through a 0.4 μm sterile filter membrane to get the cell precipitate, wash it with PBS buffer for 3 times; S4, wash the cell precipitate with balanced salt buffer solution for 3 times, resuspend the cells with 2 times the volume of balanced salt buffer solution, purify the cell suspension by density gradient centrifugation to get the umbilical cord mesenchymal stem cells; S5, inoculate the umbilical cord mesenchymal stem cells into the culture medium at a density of 1×10 individual / ml, place it in the incubator for 2-4 d, control the passage number within P10, when the cell fusion rate reaches 80-90%, collect the culture supernatant, which is the first supernatant; S6, centrifuge the first supernatant at 4℃, 1800 r / min for 10-30 min, collect the supernatant, remove small molecules by ultrafiltration and concentration, filter through a 0.3 μm sterile filter membrane to sterilize, to get the umbilical cord mesenchymal cell precipitate; S7, wash the umbilical cord mesenchymal cell precipitate with PBS buffer for 3 times, inoculate it into the serum-free culture medium at a density of 1×10 individual / ml, place it in the incubator for 42-48 h, collect the culture supernatant, which is the second supernatant; S8, move the second supernatant to a 30% sucrose heavy water density pad, centrifuge at 80000-160000 g for 120 min at 4℃, collect the bottom buffer pad; S9, wash the bottom buffer pad with PBS solution for 3 times, remove small molecules by ultrafiltration and concentration, filter through a 0.25 μm sterile filter membrane to sterilize, to get the umbilical cord mesenchymal stem cell exosome; The preparation method of the pueraria extract is: S21, wash the pueraria with deionized water, ultrasonic treat at 100 W, 20℃ for 15 min, dry, crush to 20-40 mesh to get the pueraria powder; S22, the pueraria powder, 65-85% ethanol solution is mixed according to the weight ratio 1: (5-8), 40-50 ℃ under extraction 30-90 min, centrifugal, filtration, the first extract is obtained; S23, the first extract and activated carbon are mixed according to the weight ratio 1: (0.006-0.018), centrifugal, filtration, the second extract is obtained; S24, the second extract and 1.5% chitosan solution are mixed according to the weight ratio 1: (0.002-0.01), centrifugal, filtration, the third extract is obtained; S25, the third extract is passed into the adsorption column filled with macroporous resin, the elution speed is 2-3 BV / h, 3 times the resin column volume of deionized water is used to flush the adsorption column at 1.5-2.5 V / h, 2 times the resin column volume of 50wt% ethanol solution is used to flush the adsorption column at 1-3 BV / h, the ethanol solution is collected, and the pueraria extract is obtained.

2. The stem cell exosome-containing composition of claim 1, wherein, The composition containing stem cell exosomes comprises, by weight: 0.3 parts of umbilical cord mesenchymal stem cell exosomes, 4 parts of pueraria extract, 0.6 parts of honey, 0.2 parts of compound vitamin, 0.5 parts of olive oil, 0.14 parts of starch, 0.09 parts of glutamic acid, 0.09 parts of cysteine, 0.09 parts of glycine, 0.21 parts of glucuronolactone, 0.12 parts of sodium carboxymethyl cellulose, and 29 parts of deionized water.

3. The stem cell exosome-containing composition of claim 1, wherein, The PBS buffer contains 1-3wt% of penicillin and 1.2-3.6wt% of streptomycin sulfate; the culture medium is DMEM culture medium containing 8-12wt% FBS; and the culture box culture condition is 36-38℃, 4-6% CO2 saturated humidity.

4. The composition containing stem cell exosomes according to claim 1, wherein the centrifugal condition is 25℃, 12000r / min, and the centrifugal time is 20min; the filter membrane pore size is 0.3μm; and the macroporous adsorption resin model is HPD-100.

5. The stem cell containing exosome composition of claim 1, wherein, The compound vitamin is composed of vitamin A, vitamin C, vitamin E, vitamin B6 and vitamin B12 according to the weight ratio 1.5:0.5:1:2:

1.

6. The stem cell containing exosome composition of claim 1, wherein, The preparation method of the composition containing stem cell exosomes is as follows: S31, the honey, compound vitamin, olive oil, starch, glutamic acid, cysteine, glycine, glucuronolactone and sodium carboxymethyl cellulose are dissolved in deionized water, and then stirred at 45℃ and 240-360rmp for 30-45min to make them completely dissolved, to obtain mixture A; S32, the umbilical cord mesenchymal stem cell exosomes and pueraria extract are added into mixture A, and then stirred at 40℃ and 120-180rmp for 15-30min to obtain mixture B; S33, mixture B is refluxed at 37℃ for 1-2h, filtered, concentrated to the relative density of 1.5, and then freeze-dried to obtain the composition containing stem cell exosomes.

7. The composition containing stem cell exosomes according to any one of claims 1-6 for use in the preparation of a medicament for treating liver injury.

Images