A weight-loss composition containing mesenchymal stem cells, and preparation method and application thereof

Abstract

The present invention proposes a weight loss composition containing mesenchymal stem cells and its preparation method and application, belonging to the field of medical technology. Eurotium cristatum and Lactobacillus plantarum are used to ferment white tea and black tea, probiotics are collected for embedding to obtain embedded probiotics, tea residues are subjected to supercritical fluid extraction, oxidation, extraction to obtain a tea brown composition, fetal bovine serum, dexamethasone, penicillin, streptomycin, magnetic particles, interleukin-2 are added to DMEM culture medium, a culture medium is prepared, a low-frequency magnetic field culture for adipose mesenchymal stem cells is used, separation, a stem cell solution is obtained, and a stem cell microsphere is obtained by embedding, and the supernatant is freeze-dried to obtain a paracrine culture material, and the fermentation product, the tea brown composition, and the embedded probiotics are mixed uniformly to obtain a weight loss composition containing mesenchymal stem cells. Not only has good anti-oxidation, anti-inflammatory, antibacterial and blood sugar and blood lipid regulating effects, but also has a good weight loss effect and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a weight-loss composition containing mesenchymal stem cells, and a preparation method and application thereof. Background Art

[0002] The characteristics of obesity's harm to human health are gradual and persistent damage. There are no advanced medical treatments for obesity, nor are there long-term effective methods. Studies have found that obesity is always accompanied by a chronic low-grade inflammation. The visceral fat tissue of obese people can release a large number of inflammatory factors, including certain chemokines such as CCL2, CCL5, and IP-10. There are also many reports that obese patients have more IGF-1, estradiol, IL-1, IL-6, IL-8, tumor necrosis factor TNF, leptin, and inflammatory response factors in their serum. Therefore, if the overactivation of signal regulatory networks such as certain cytokines or chemokines caused by obesity can be effectively reduced or blocked, the effect of obesity in promoting the occurrence and development of tumors can be greatly delayed.

[0003] The accumulation of white adipose tissue in adipose tissue is associated with insulin resistance, dyslipidemia, diabetes, and cardiovascular disease; while brown adipose tissue is the opposite. Its most prominent physiological function is to consume energy and generate heat, and it plays an important role in regulating body temperature. Brown adipose tissue is similar to it. White adipocytes and brown adipocytes have common precursor cells. Improving the differentiation of brown adipocytes and reducing the differentiation of white adipocytes will increase energy consumption, help lose weight, and provide new treatments for metabolic disorders such as diabetes and hypertension. Therefore, adipose tissue is a complex endocrine organ. Obesity leads to changes in the level of secreted adipose factors and impaired regulation of adipose factors, which are closely related to the occurrence and development of metabolic syndrome. Adipose tissue affects the entire process of the occurrence and development of metabolic syndrome through adipose factors and fat metabolism. The cytokines secreted by adipocytes mainly include leptin, adiponectin, visfatin, and chemokines; in addition, a variety of receptors are expressed on the surface of adipocytes to mediate their differentiation, maturation, metabolism, and apoptosis. The main known receptors include insulin receptors, leptin receptors, tumor necrosis factor α receptors, peroxisome proliferator-activated receptors (PPARs), adrenaline receptors, and adrenocortical hormone receptors.

[0004] At present, the drugs for treating obesity mainly include appetite suppressants and digestion and absorption inhibitors according to their mechanism of action. Others include hypoglycemic drugs, metabolic stimulants and local lipolysis agents. Appetite suppressants act on the central nervous system and are an important type of weight loss drugs. Appetite is regulated by the satiety center in the ventromedial hypothalamus and the feeding center in the lateral hypothalamus. The central enhancement or inhibition of feeding function is mainly through changes in catecholamine neurotransmitters such as norepinephrine, dopamine, 5-hydroxytryptamine (HT), etc. One of the representative drugs is amphetamine and its analogs, including methamphetamine, benzyl methamphetamine, diethylpropion, dexamphetamine, etc. Its mechanism of action is to block the reuptake of norepinephrine by nerve endings while promoting the release of norepinephrine from the center and increasing the norepinephrine content in the synaptic cleft, thereby producing a mimetic norepinephrine effect. The main representative drug of digestion and absorption inhibitors is Orlistat, also known as Xenieal. It is currently the only non-centrally acting weight loss drug approved by the US FDA. It obtained FDA marketing authorization in 1999. Two years later, CFDA also approved its marketing in China. Orlistat is a potent and selective gastrointestinal lipase inhibitor. Its mechanism of action is to inhibit the action of pancreatic enzymes and lipase in the gastrointestinal tract, thereby reducing the decomposition and absorption of fat in food. At therapeutic doses, combined with a slightly low-calorie diet, it can reduce fat absorption by nearly 30%. This inhibitory effect can cause a negative energy balance of 200kcal, thereby reducing weight. Taking acarbose as an example of α-glucosidase inhibitor, acarbose reduces the decomposition of polysaccharides by competitively inhibiting the activity of α-glucosidase on the brush border of small intestinal epithelial cells, delaying the absorption of glucose by the intestine after a meal, and has the effect of significantly reducing postprandial blood sugar, reducing the stimulation of pancreatic β cells by excessive postprandial blood sugar, increasing insulin sensitivity, and improving IR. Metabolic stimulants include central nervous system stimulants and hormones. These drugs have many adverse reactions and are limited in clinical application. Their mechanism of action is mainly to increase metabolic rate, increase heat production and promote fat decomposition. Studies have found that drugs that stimulate β-adrenergic receptors can promote lipolysis in human adipocytes, while drugs that stimulate α2-adrenergic receptors inhibit lipolysis: suggesting that β-adrenergic agonists and α2-adrenergic antagonists can mobilize local adipose tissue to achieve weight loss. However, the effects of these therapeutic drugs need to be further improved.

[0005] Literature reports that mesenchymal cells from various tissues have good therapeutic effects on various diseases. Stem cells are cells with secretory functions, and the bioactive molecules they secrete have a repairing effect on different organs. Studies have found that stem cells derived from adipose tissue can improve the insulin resistance produced by induced differentiated 3T3L1 cells, and they can promote angiogenesis through the active substances they secrete, protect the kidneys, and resist heart damage. These results show that stem cell-derived conditioned medium has a significant improvement effect on metabolic diseases. Studies have found that stem cells derived from adipose tissue can inhibit dietary obesity in mice, and inhibit obesity in mice by improving insulin resistance and excessive hypertrophy of adipose tissue. However, at present, there are not enough studies on the use of other stem cells for the treatment of obesity. Summary of the invention

[0006] The purpose of the present invention is to provide a weight loss composition containing mesenchymal stem cells and a preparation method and application thereof, which not only has excellent antioxidant, anti-inflammatory, antibacterial and blood sugar and blood lipid regulating effects, but also has a good weight loss effect, is safe and effective, has good stability, is easy to store, has a good taste, has a good tea aroma, is highly patient-compliant, and has broad application prospects.

[0007] The technical solution of the present invention is achieved in this way:

[0008] The invention provides a method for preparing a weight-loss composition containing mesenchymal stem cells. The method comprises the following steps: using Eurotium cristatum and Lactobacillus plantarum to ferment white tea and black tea, collecting probiotics for embedding to obtain embedded probiotics, subjecting tea residues to supercritical fluid extraction and oxidation, extracting to obtain a tea-brown composition, adding fetal bovine serum, dexamethasone, penicillin, streptomycin, magnetic particles and interleukin-2 into a DMEM culture medium to prepare a culture medium, which is used for low-frequency magnetic field culture of adipose mesenchymal stem cells, separating to obtain a stem cell solution, embedding to obtain stem cell microspheres, freeze-drying the supernatant to obtain a paracrine culture substance, and uniformly mixing the paracrine culture substance with a fermentation product, the tea-brown composition and the embedded probiotics to prepare a weight-loss combination containing mesenchymal stem cells.

[0009] As a further improvement of the present invention, the following steps are included:

[0010] S1. Activation of bacterial strains: Inoculate Eurotium cristatum and Lactobacillus plantarum into slant culture medium, activate and culture, and obtain bacterial seed liquid;

[0011] S2 tea fermentation: white tea and black tea were mixed, added to water, sterilized, inoculated with Eurotium cristatum and Lactobacillus plantarum seed solution, fermented, filtered, washed with solids, concentrated and collected, the washed solids were retained, and the filtrate was dried to obtain a fermentation product;

[0012] S3. Isolation and embedding of fermentation strains: sodium alginate and carboxymethyl chitosan were added to the bacterial solution, stirred and mixed, lecithin was added dropwise to emulsify the fish oil, a mixed solution of calcium chloride and EDC was added dropwise, stirred and solidified, centrifuged, washed, and dried to obtain embedded probiotics;

[0013] S4 tea extraction: The solid in step S2 is subjected to supercritical fluid extraction to obtain an extract;

[0014] S5. Oxidation of the extract: adding the extract to water, adding polyphenol oxidase, stirring the reaction, inactivating the enzyme, and obtaining a brown oxidation product;

[0015] S6. Extraction of a brown composition: extracting by adding a mixed solvent to the brown oxidation product, adding ethanol to precipitate, filtering, washing, and drying to obtain a brown composition;

[0016] S7. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, magnetic particles, and interleukin-2 were added to DMEM medium to prepare a culture medium;

[0017] S8. Culture of adipose-derived mesenchymal stem cells: After the adipose-derived mesenchymal stem cells are revived, they are added to the culture medium and cultured in a low-frequency magnetic field. After the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated by a magnet, the residue is centrifuged, the supernatant is collected, and freeze-dried to obtain paracrine products. The solid is washed with D-Hank's solution, digested, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution;

[0018] S9. Embedding of stem cells: After the stem cell solution is revived, sodium alginate and lecithin are added, stirred and mixed evenly, added dropwise to liquid paraffin, emulsified, calcium chloride solution is added dropwise, solidified at room temperature, centrifuged, washed, and dried to obtain stem cell microspheres;

[0019] S10. Preparation of a weight loss composition containing mesenchymal stem cells: Evenly mix the fermentation product, the brown composition, the embedded probiotics, the stem cell microspheres and the paracrine product to prepare a weight loss composition containing mesenchymal stem cells.

[0020] As a further improvement of the present invention, the activation conditions of the crown-shaped spores in step S1 are 28-32°C, 100-200r / min, and the activation culture is 12-18h, the activation conditions of the plant lactobacillus are 30-35°C, 150-250r / min, and the activation culture is 18-24h, and the bacterial content of the bacterial seed liquid is 10 8 -10 9cfu / mL, the mass ratio of white tea, black tea and water in step S2 is 5-7:3-5:100, the inoculation amounts of the seed solutions of Eurotium cristatum and Lactobacillus plantarum are 3-4v / v% and 1-2v / v%, respectively, and the fermentation time is 24-30h.

[0021] As a further improvement of the present invention, the mass ratio of the bacterial liquid, sodium alginate, carboxymethyl chitosan and lecithin in step S3 is 100:4-7:5-8:0.5-1, the conditions of the supercritical fluid extraction in step S4 are pressure 27-32MPa, temperature 35-40°C, time 150-200min, the entrainer is ethyl acetate solution, the content of ethyl acetate is 5-10wt%, and the amount of entrainer added is 1-2wt%.

[0022] As a further improvement of the present invention, the mass ratio of the extract and the polyphenol oxidase in step S5 is 100:2-3, the temperature of the stirring reaction is 35-45°C, the time is 1-3h, the polyphenol oxidase is catechol oxidase and laccase, the mass ratio is 10:2-3, the mixed solvent in step S6 is a mixture of chloroform, ethyl acetate and n-butanol, the volume ratio is 3-5:5-7:2-3, ethanol is added until the ethanol content of the system is 65-75wt%, and the precipitation time is 1-2h.

[0023] As a further improvement of the present invention, the content of fetal bovine serum in the fetal culture medium in step S7 is 9-11wt%, the content of dexamethasone is 5-15nmol / L, the content of penicillin is 100-120U / mL, the content of streptomycin is 100-120μg / mL, the content of magnetic particles is 100-200μg / mL, the content of interleukin-2 is 10-20μg / mL, the magnetic particles are ferroferric oxide particles, the low-frequency magnetic field culturing time in step S8 is 1-2 weeks, the culturing conditions are 36-38°C, the CO2 concentration is 3-6v / v%, the frequency of the low-frequency magnetic field is 40-50Hz, and the stimulation time is 30-40min / d; the digestive fluid is a PBS buffer with a pH of 7.5-7.7 containing 1-2wt% trypsin and 0.5-1wt% alkaline protease.

[0024] As a further improvement of the present invention, the preparation method of the ferroferric oxide particles is as follows:

[0025] Ferric chloride and ferrous chloride are mixed and dissolved in water, and ammonia water is added dropwise under the protection of inert gas, and the mixture is heated and stirred for reaction, centrifuged, washed, dried, and calcined to obtain ferrosoferric oxide particles.

[0026] As a further improvement of the present invention, the mass ratio of the stem cell solution, sodium alginate and lecithin in step S9 is 100:13-16:1-2, and the mass ratio of the fermentation product, brown composition, embedded probiotics, stem cell microspheres and paracrine products in step S10 is 3-5:2-4:3-4:1-2:1.5-2.5.

[0027] The present invention further protects a weight loss composition containing mesenchymal stem cells prepared by the above preparation method.

[0028] The present invention further protects the use of the weight loss composition containing mesenchymal stem cells in the preparation of weight loss drugs.

[0029] The present invention has the following beneficial effects:

[0030] Eurotium cristatum belongs to probiotics, which is widely distributed in nature. It has the effects of improving bitter taste, increasing antioxidant activity, inhibiting bacteria, reducing fat and losing weight, and regulating intestinal flora. Lactobacillus plantarum is a probiotic that can secrete substances such as short-chain fatty acids and antimicrobial peptides, thereby playing the role of antioxidant, antibacterial, reducing fat and losing weight, and regulating intestinal flora. The present invention adopts Eurotium cristatum and Lactobacillus plantarum to synergistically ferment white tea and black tea. On the one hand, it can increase the yield of catechins in tea, and at the same time, it can also promote the proliferation of probiotics, improve the aroma of the composition, and reduce the content of grassy smell compounds and bitter catechins. The obtained product has better lipid-lowering and weight-loss effects.

[0031] The present invention embeds probiotics by carboxymethyl chitosan and sodium alginate, and the crosslinking agents are calcium ions and EDC. Compared with the common crosslinking agent glutaraldehyde, the present invention uses EDC to activate the carboxymethyl chitosan and generate an amide group-forming reaction, and the carboxymethyl chitosan itself has no crosslinking effect, so the toxicity of the product is greatly reduced, thereby making the product safer and more environmentally friendly, and the carboxylated chitosan can better withstand gastric acid erosion, so that the probiotics can better enter the intestines to exert their effects.

[0032] White tea has health functions such as anti-oxidation, lowering blood sugar and blood lipids, alleviating metabolic disorders, protecting the liver, and reducing cholesterol absorption. It is rich in catechins. Dark tea itself is a preliminary fermented tea, which is rich in theabrownin and catechins. Theabrownin has a complex structure. It is the product of oxidative polymerization of polyphenols and their combination with gallic acid, alkaloids, flavanols, flavonoids, purines, carboxylic acids, sugars, proteins, amino acids, lipids, etc. It has a multi-benzene ring structure. Theabrownin contains a large number of hydroxyl, carboxyl, alkyl, methyl and amino groups.

[0033] The present invention extracts tea solids through supercritical fluid, which contains rich polyphenols such as catechins, theaflavins, thearubigins and theabrownins. After being enzymatically hydrolyzed by catechol oxidase and laccase, a large number of different oxidation products can be generated. Because of its dark color, a tea-brown composition is prepared after purification, which has the effects of lowering fat and losing weight, anti-oxidation, anti-tumor and improving immunity, and has obvious effects of lowering fat and losing weight.

[0034] Ferroferric oxide is a type of magnetic substance. During the low-frequency magnetic field culture process, it responds to the magnetic field and adheres to the surface of stem cells to form a micromagnetic field, affecting the structure of the cell membrane and cell membrane-related enzymes to increase the permeability of small molecules, thereby changing cell behavior with the help of the second messenger. At the same time, it controls the opening of voltage-gated calcium channels, causing calcium ion influx, causing an increase in intracellular calcium ion concentration, and promoting the growth and development of stem cells as well as their paracrine function, so that products rich in active substances and exosomes can be obtained.

[0035] Adipose-derived mesenchymal stem cells have multidirectional differentiation potential and can differentiate into brown adipocytes, etc. They participate in the remodeling process of adipose tissue by affecting the differentiation and maturation of adipocytes. They can also affect the inflammatory state and metabolic function of adipose tissue by secreting cytokines and hormones, and then participate in the regulation of body weight and fat distribution. They can secrete a variety of cytokines and hormones, such as interleukin (IL), tumor necrosis factor α (TNF-α), etc. These factors can regulate immune response and inflammatory process, reduce obesity-related inflammation, lower blood sugar by improving pancreatic β cell function and peripheral tissue insulin resistance, and improve insulin sensitivity, so as to have better blood sugar regulation function, and can promote the transformation of white adipose tissue to beige fat. Beige fat has higher energy consumption and heat production capacity, which helps to reduce obesity. The released exosomes can reduce fat inflammation and obesity by polarizing M2 macrophages and promoting beigeization of white adipose tissue, and reduce fat storage and improve metabolic health by affecting the metabolic pathways of adipocytes, such as regulating mitochondrial function and energy metabolism.

[0036] The present invention greatly improves the stability and survival rate of stem cells by embedding stem cells, thereby improving the activity of stem cells.

[0037] The weight loss composition containing mesenchymal stem cells prepared by the present invention not only has excellent anti-oxidation, anti-inflammatory, antibacterial and blood sugar and blood lipid regulating effects, but also has excellent weight loss effect, is safe and effective, has good stability, is easy to store, has a good taste, has a good tea aroma, is highly patient-compliant, and has broad application prospects. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention are 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.

[0039] Eurotium cristatum, CICC2099, 10 billion cfu / g, was purchased from Wuhan Zhuoyan Huabiao Technology Co., Ltd., and Lactobacillus plantarum, 20 billion cfu / g, was purchased from Shanghai Zhongfeng Biotechnology Co., Ltd.

[0040] EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, is commercially available.

[0041] Catechol oxidase, 1000 U / mg, was purchased from Merck Life Sciences; laccase, 10,000 U / g, trypsin, 200,000 U / g, alkaline protease, 200,000 U / g, were purchased from Xiasheng (Beijing) Enzyme Biotechnology Development Co., Ltd.

[0042] Preparation Example 1 Preparation of FeO particles

[0043] Here’s how:

[0044] 3.24 g of ferric chloride and 1.26 g of ferrous chloride were mixed and dissolved in 500 mL of water. Under nitrogen protection, 20 mL of ammonia water was added dropwise. The mixture was heated to 90°C, stirred for reaction for 4 h, centrifuged, washed, dried, and calcined at 500°C for 2 h to obtain ferrosoferric oxide particles. Example 1

[0045] This embodiment provides a method for preparing a weight loss composition containing mesenchymal stem cells, comprising the following steps:

[0046] S1. Activation of strains: Inoculate Eurotium cristatum and Lactobacillus plantarum in slant culture medium, activate and culture, and obtain a strain with a bacterial count of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0047] The activation conditions of the cristatum cristatum are 28° C., 100 r / min, and activation culture for 18 hours;

[0048] The activation conditions of the plant lactobacillus are 30° C., 150 r / min, and activation culture for 24 hours;

[0049] S2. Fermentation of tea leaves: 5 g of white tea and 3 g of black tea were mixed and added to 100 mL of water, sterilized, inoculated with Eurotium cristatum and Lactobacillus plantarum seed solution, the inoculation amounts were 3 v / v% and 1 v / v%, respectively, and fermented for 24 h, filtered, the solids were washed, the bacterial solution was concentrated and collected, the washed solids were retained, and the filtrate was dried to obtain a fermentation product;

[0050] S3. Fermentation strain separation and embedding: 4 g of sodium alginate and 5 g of carboxymethyl chitosan were added to 100 g of bacterial solution, stirred and mixed for 10 min, 0.5 g of lecithin was added dropwise to 200 g of fish oil, emulsified at 8000 r / min for 15 min, 20 mL of a mixed solution containing 0.5 wt% calcium chloride and 1 wt% EDC was added dropwise, stirred and solidified for 30 min, centrifuged, washed, and dried to obtain embedded probiotics;

[0051] S4 tea extraction: The solid in step S2 is subjected to supercritical fluid extraction to obtain an extract;

[0052] The supercritical fluid extraction conditions are as follows: pressure 27 MPa, temperature 35° C., time 150 min, entrainer ethyl acetate, and the amount of entrainer added is 1 wt %;

[0053] S5. Oxidation of the extract: 100 g of the extract was added to 500 mL of water, 2 g of polyphenol oxidase was added, and the reaction was stirred at 35°C for 1 h, and the enzyme was inactivated to obtain a brown oxidation product;

[0054] The polyphenol oxidase is catechol oxidase and laccase, with a mass ratio of 10:2;

[0055] S6. Extraction of a dark brown composition: 3 times the mass of a mixed solvent was added to the brown oxidation product for extraction, ethanol was added to the system to an ethanol content of 65wt%, precipitated for 1h, filtered, washed, and dried to obtain a dark brown composition;

[0056] The mixed solvent is a mixture of chloroform, ethyl acetate and n-butanol in a volume ratio of 3:5:2;

[0057] S7. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, magnetic particles, and interleukin-2 were added to DMEM medium to prepare a culture medium;

[0058] The fetal culture medium contains 9wt% fetal bovine serum, 5nmol / L dexamethasone, 100U / mL penicillin, 100μg / mL streptomycin, 100μg / mL magnetic particles, and 100μg / mL interleukin-2. The magnetic particles are ferrosoferric oxide prepared in Preparation Example 1.

[0059] S8. Culture of adipose-derived mesenchymal stem cells: After the adipose-derived mesenchymal stem cells are revived, they are added to the culture medium and cultured in a low-frequency magnetic field. After the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated by a magnet, the residue is centrifuged, the supernatant is collected, and freeze-dried to obtain a paracrine product. The solid is washed with D-Hank's solution, digested with digestive solution, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution;

[0060] The low-frequency magnetic field culture time is 1 week, the culture conditions are 36°C, CO2 concentration is 3v / v%, the frequency of the low-frequency magnetic field is 40Hz, and the stimulation time is 30min / d;

[0061] The digestion solution is a PBS buffer with a pH of 7.5 containing 1wt% trypsin and 0.5wt% alkaline protease;

[0062] S9. Embedding of stem cells: After resuscitation of 100 g of stem cell solution, add 13 g of sodium alginate and 1 g of lecithin, stir and mix for 10 min, add dropwise to 200 g of liquid paraffin, emulsify at 8000 r / min for 15 min, add dropwise 10 g of 2 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain stem cell microspheres;

[0063] S10. Preparation of a weight loss composition containing mesenchymal stem cells: 3 g of fermentation product, 2 g of brown composition, 3 g of encapsulated probiotics, 1 g of stem cell microspheres and 1.5 g of paracrine product were stirred and mixed for 10 min to prepare a weight loss composition containing mesenchymal stem cells. Example 2

[0064] This embodiment provides a method for preparing a weight loss composition containing mesenchymal stem cells, comprising the following steps:

[0065] S1. Activation of strains: Inoculate Eurotium cristatum and Lactobacillus plantarum in slant culture medium, activate and culture, and obtain a strain with a bacterial count of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0066] The activation conditions of the cristatum cristatum are 32° C., 200 r / min, and activation culture for 12 hours;

[0067] The activation conditions of the plant lactobacillus are 35° C., 250 r / min, and activation culture for 18 h;

[0068] S2. Fermentation of tea leaves: 7g of white tea and 5g of black tea were mixed and added to 100mL of water, sterilized, inoculated with Eurotium cristatum and Lactobacillus plantarum seed solution, the inoculation amounts were 4v / v% and 2v / v%, respectively, and fermented for 30h, filtered, the solids were washed, the bacterial solution was concentrated and collected, the washed solids were retained, and the filtrate was dried to obtain a fermentation product;

[0069] S3. Fermentation strain separation and embedding: 7g sodium alginate and 8g carboxymethyl chitosan were added to 100g of bacterial solution, stirred and mixed for 10min, 1g of lecithin was added, and added dropwise to 200g of fish oil, emulsified at 8000r / min for 15min, 20mL of a mixed solution containing 1wt% calcium chloride and 2wt% EDC was added dropwise, stirred and solidified for 30min, centrifuged, washed, and dried to obtain embedded probiotics;

[0070] S4 tea extraction: The solid in step S2 is subjected to supercritical fluid extraction to obtain an extract;

[0071] The supercritical fluid extraction conditions are: pressure 32 MPa, temperature 40° C., time 200 min, entrainer is ethyl acetate, and the amount of entrainer added is 2 wt %;

[0072] S5. Oxidation of the extract: 100 g of the extract was added to 500 mL of water, 3 g of polyphenol oxidase was added, the reaction was stirred at 45 ° C for 3 h, the enzyme was inactivated, and a brown oxidation product was obtained;

[0073] The polyphenol oxidase is catechol oxidase and laccase, with a mass ratio of 10:3;

[0074] S6. Extraction of a dark brown composition: 5 times the mass of a mixed solvent was added to the brown oxidation product for extraction, ethanol was added to the system to an ethanol content of 75wt%, precipitated for 2h, filtered, washed, and dried to obtain a dark brown composition;

[0075] The mixed solvent is a mixture of chloroform, ethyl acetate and n-butanol in a volume ratio of 5:7:3;

[0076] S7. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, magnetic particles, and interleukin-2 were added to DMEM medium to prepare a culture medium;

[0077] The fetal culture medium contains 11wt% fetal bovine serum, 15nmol / L dexamethasone, 120U / mL penicillin, 120μg / mL streptomycin, 200μg / mL magnetic particles, and 20μg / mL interleukin-2. The magnetic particles are ferrosoferric oxide prepared in Preparation Example 1.

[0078] S8. Culture of adipose-derived mesenchymal stem cells: After the adipose-derived mesenchymal stem cells are revived, they are added to the culture medium and cultured in a low-frequency magnetic field. After the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated by a magnet, the residue is centrifuged, the supernatant is collected, and freeze-dried to obtain a paracrine product. The solid is washed with D-Hank's solution, digested with digestive solution, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution;

[0079] The low-frequency magnetic field culture time is 2 weeks, the culture conditions are 38°C, CO2 concentration is 6v / v%, the frequency of the low-frequency magnetic field is 50Hz, and the stimulation time is 40min / d;

[0080] The digestion solution is a PBS buffer solution with a pH of 7.7 containing 2wt% trypsin and 1wt% alkaline protease;

[0081] S9. Embedding of stem cells: After resuscitation of 100 g of stem cell solution, add 16 g of sodium alginate and 2 g of lecithin, stir and mix for 10 min, add dropwise to 200 g of liquid paraffin, emulsify at 8000 r / min for 15 min, add dropwise 10 g of 2 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain stem cell microspheres;

[0082] S10. Preparation of a weight loss composition containing mesenchymal stem cells: 5 g of fermentation product, 4 g of brown composition, 4 g of encapsulated probiotics, 2 g of stem cell microspheres and 2.5 g of paracrine product were stirred and mixed for 10 min to prepare a weight loss composition containing mesenchymal stem cells. Example 3

[0083] This embodiment provides a method for preparing a weight loss composition containing mesenchymal stem cells, comprising the following steps:

[0084] S1. Activation of strains: Inoculate Eurotium cristatum and Lactobacillus plantarum in slant culture medium, activate and culture, and obtain a strain with a bacterial count of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0085] The activation conditions of the cristatum cristatum are 30° C., 150 r / min, and activation culture for 16 hours;

[0086] The activation conditions of the plant lactobacillus are 32° C., 200 r / min, and activation culture for 21 hours;

[0087] S2. Fermentation of tea leaves: 6 g of white tea and 4 g of black tea were mixed and added to 100 mL of water, sterilized, inoculated with Eurotium cristatum and Lactobacillus plantarum seed solution, the inoculation amounts were 3.5 v / v% and 1.5 v / v%, respectively, and fermented for 27 h, filtered, the solids were washed, the bacterial solution was concentrated and collected, the washed solids were retained, and the filtrate was dried to obtain a fermentation product;

[0088] S3. Fermentation strain separation and embedding: 5.7 g of sodium alginate and 6.3 g of carboxymethyl chitosan were added to 100 g of bacterial solution, stirred and mixed for 10 min, 0.7 g of lecithin was added, and added dropwise to 200 g of fish oil, emulsified at 8000 r / min for 15 min, 20 mL of a mixed solution containing 0.7 wt % calcium chloride and 1.5 wt % EDC was added dropwise, stirred and solidified for 30 min, centrifuged, washed, and dried to obtain embedded probiotics;

[0089] S4 tea extraction: The solid in step S2 is subjected to supercritical fluid extraction to obtain an extract;

[0090] The supercritical fluid extraction conditions are as follows: pressure 30 MPa, temperature 37° C., time 180 min, entrainer ethyl acetate, and the amount of entrainer added is 1.5 wt %;

[0091] S5. Oxidation of the extract: 100 g of the extract was added to 500 mL of water, 2.5 g of polyphenol oxidase was added, the reaction was stirred at 40°C for 2 h, the enzyme was inactivated, and a brown oxidation product was obtained;

[0092] The polyphenol oxidase is catechol oxidase and laccase, with a mass ratio of 10:2.5;

[0093] S6. Extraction of a dark brown composition: 4 times the mass of a mixed solvent was added to the brown oxidation product for extraction, ethanol was added to the system ethanol content was 70wt%, precipitated for 1.5h, filtered, washed, and dried to obtain a dark brown composition;

[0094] The mixed solvent is a mixture of chloroform, ethyl acetate and n-butanol in a volume ratio of 4:6:2.5;

[0095] S7. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, magnetic particles, and interleukin-2 were added to DMEM medium to prepare a culture medium;

[0096] The fetal culture medium contains 10 wt % fetal bovine serum, 10 nmol / L dexamethasone, 110 U / mL penicillin, 110 μg / mL streptomycin, 150 μg / mL magnetic particles, and 15 μg / mL interleukin-2. The magnetic particles are ferrosoferric oxide prepared in Preparation Example 1.

[0097] S8. Culture of adipose-derived mesenchymal stem cells: After the adipose-derived mesenchymal stem cells are revived, they are added to the culture medium and cultured in a low-frequency magnetic field. After the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated by a magnet, the residue is centrifuged, the supernatant is collected, and freeze-dried to obtain a paracrine product. The solid is washed with D-Hank's solution, digested with digestive solution, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution;

[0098] The low-frequency magnetic field culture time is 2 weeks, the culture conditions are 37°C, CO2 concentration is 5v / v%, the frequency of the low-frequency magnetic field is 45Hz, and the stimulation time is 35min / d;

[0099] The digestion solution is a PBS buffer with a pH of 7.6 containing 1.5wt% trypsin and 0.7wt% alkaline protease;

[0100] S9. Embedding of stem cells: After resuscitation of 100 g of stem cell solution, add 14 g of sodium alginate and 1.2 g of lecithin, stir and mix for 10 min, add dropwise to 200 g of liquid paraffin, emulsify at 8000 r / min for 15 min, add dropwise 10 g of 2 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain stem cell microspheres;

[0101] S10. Preparation of a weight loss composition containing mesenchymal stem cells: 4 g of fermentation product, 3 g of brown composition, 3.5 g of encapsulated probiotics, 1.5 g of stem cell microspheres and 2 g of paracrine product were stirred and mixed for 10 min to prepare a weight loss composition containing mesenchymal stem cells.

[0102] Comparative Example 1

[0103] Compared with Example 3, the difference is that the seed liquid of Eurotium cristatum spawn is not inoculated in step S2.

[0104] The details are as follows:

[0105] S2. Fermentation of tea leaves: 6 g of white tea and 4 g of black tea were mixed, added into 100 mL of water, sterilized, inoculated with Lactobacillus plantarum seed solution at an inoculation rate of 5 v / v%, fermented for 27 h, filtered, washed the solids, concentrated and collected the bacterial solution, retained the washed solids, and dried the filtrate to obtain a fermentation product.

[0106] Comparative Example 2

[0107] Compared with Example 3, the difference is that the Lactobacillus plantarum seed liquid is not inoculated in step S2.

[0108] The details are as follows:

[0109] S2. Fermentation of tea leaves: Mix 6 g of white tea and 4 g of black tea, add into 100 mL of water, sterilize, inoculate with 5 v / v% of seed liquid of Eurotium cristatum, ferment for 27 h, filter, wash the solid, concentrate and collect the bacterial liquid, retain the washed solid, dry the filtrate to obtain the fermentation product.

[0110] Comparative Example 3

[0111] Compared with Example 3, the difference is that steps S1-S3 are not performed.

[0112] The details are as follows:

[0113] S1. Extraction of tea leaves: 6 g of white tea and 4 g of black tea were mixed and subjected to supercritical fluid extraction to obtain an extract;

[0114] The supercritical fluid extraction conditions are as follows: pressure 30 MPa, temperature 37° C., time 180 min, entrainer ethyl acetate, and the amount of entrainer added is 1.5 wt %;

[0115] S2. Oxidation of the extract: 100 g of the extract was added to 500 mL of water, 2.5 g of polyphenol oxidase was added, the reaction was stirred at 40°C for 2 h, the enzyme was inactivated, and a brown oxidation product was obtained;

[0116] The polyphenol oxidase is catechol oxidase and laccase, with a mass ratio of 10:2.5;

[0117] S3. Extraction of a dark brown composition: 4 times the mass of a mixed solvent was added to the brown oxidation product for extraction, ethanol was added to the system to an ethanol content of 70wt%, precipitated for 1.5h, filtered, washed, and dried to obtain a dark brown composition;

[0118] The mixed solvent is a mixture of chloroform, ethyl acetate and n-butanol in a volume ratio of 4:6:2.5;

[0119] S4. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, magnetic particles, and interleukin-2 were added to DMEM medium to prepare a culture medium;

[0120] The fetal culture medium contains 10 wt % fetal bovine serum, 10 nmol / L dexamethasone, 110 U / mL penicillin, 110 μg / mL streptomycin, 150 μg / mL magnetic particles, and 15 μg / mL interleukin-2. The magnetic particles are ferrosoferric oxide prepared in Preparation Example 1.

[0121] S5. Culture of adipose-derived mesenchymal stem cells: After the adipose-derived mesenchymal stem cells are revived, they are added to the culture medium and cultured in a low-frequency magnetic field. After the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated by a magnet, the residue is centrifuged, the supernatant is collected, and freeze-dried to obtain the paracrine product. The solid is washed with D-Hank's solution, digested with digestive solution, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution;

[0122] The low-frequency magnetic field culture time is 2 weeks, the culture conditions are 37°C, CO2 concentration is 5v / v%, the frequency of the low-frequency magnetic field is 45Hz, and the stimulation time is 35min / d;

[0123] The digestion solution is a PBS buffer with a pH of 7.6 containing 1.5wt% trypsin and 0.7wt% alkaline protease;

[0124] S6. Embedding of stem cells: After resuscitation of 100 g of stem cell solution, add 14 g of sodium alginate and 1.2 g of lecithin, stir and mix for 10 min, add dropwise to 200 g of liquid paraffin, emulsify at 8000 r / min for 15 min, add dropwise 10 g of 2 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain stem cell microspheres;

[0125] S7. Preparation of a weight loss composition containing mesenchymal stem cells: 4 g of fermentation product, 3 g of brown composition, 3.5 g of encapsulated probiotics, 1.5 g of stem cell microspheres and 2 g of paracrine product were stirred and mixed for 10 min to prepare a weight loss composition containing mesenchymal stem cells.

[0126] Comparative Example 4

[0127] Compared with Embodiment 3, the difference is that step S5 is not performed.

[0128] The details are as follows:

[0129] S1. Activation of strains: Inoculate Eurotium cristatum and Lactobacillus plantarum in slant culture medium, activate and culture, and obtain a strain with a bacterial count of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0130] The activation conditions of the cristatum cristatum are 30° C., 150 r / min, and activation culture for 16 hours;

[0131] The activation conditions of the plant lactobacillus are 32° C., 200 r / min, and activation culture for 21 hours;

[0132] S2. Fermentation of tea leaves: 6 g of white tea and 4 g of black tea were mixed and added to 100 mL of water, sterilized, inoculated with Eurotium cristatum and Lactobacillus plantarum seed solution, the inoculation amounts were 3.5 v / v% and 1.5 v / v%, respectively, and fermented for 27 h, filtered, the solids were washed, the bacterial solution was concentrated and collected, the washed solids were retained, and the filtrate was dried to obtain a fermentation product;

[0133] S3. Fermentation strain separation and embedding: 5.7 g of sodium alginate and 6.3 g of carboxymethyl chitosan were added to 100 g of bacterial solution, stirred and mixed for 10 min, 0.7 g of lecithin was added, and added dropwise to 200 g of fish oil, emulsified at 8000 r / min for 15 min, 20 mL of a mixed solution containing 0.7 wt % calcium chloride and 1.5 wt % EDC was added dropwise, stirred and solidified for 30 min, centrifuged, washed, and dried to obtain embedded probiotics;

[0134] S4 tea extraction: The solid in step S2 is subjected to supercritical fluid extraction to obtain an extract;

[0135] The supercritical fluid extraction conditions are as follows: pressure 30 MPa, temperature 37° C., time 180 min, entrainer ethyl acetate, and the amount of entrainer added is 1.5 wt %;

[0136] S5. Extraction of brown composition: 4 times the mass of the mixed solvent was added to the extract, ethanol was added to the ethanol content of the system to be 70wt%, precipitated for 1.5h, filtered, washed and dried to obtain a brown composition;

[0137] The mixed solvent is a mixture of chloroform, ethyl acetate and n-butanol in a volume ratio of 4:6:2.5;

[0138] S6. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, magnetic particles, and interleukin-2 were added to DMEM medium to prepare a culture medium;

[0139] The fetal culture medium contains 10 wt % fetal bovine serum, 10 nmol / L dexamethasone, 110 U / mL penicillin, 110 μg / mL streptomycin, 150 μg / mL magnetic particles, and 15 μg / mL interleukin-2. The magnetic particles are ferrosoferric oxide prepared in Preparation Example 1.

[0140] S7. Culture of adipose-derived mesenchymal stem cells: After the adipose-derived mesenchymal stem cells are revived, they are added to the culture medium and cultured in a low-frequency magnetic field. After the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated by a magnet, the residue is centrifuged, the supernatant is collected, and freeze-dried to obtain the paracrine product. The solid is washed with D-Hank's solution, digested with digestive solution, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution;

[0141] The low-frequency magnetic field culture time is 2 weeks, the culture conditions are 37°C, CO2 concentration is 5v / v%, the frequency of the low-frequency magnetic field is 45Hz, and the stimulation time is 35min / d;

[0142] The digestion solution is a PBS buffer with a pH of 7.6 containing 1.5wt% trypsin and 0.7wt% alkaline protease;

[0143] S8. Embedding of stem cells: After resuscitation of 100 g of stem cell solution, add 14 g of sodium alginate and 1.2 g of lecithin, stir and mix for 10 min, add dropwise to 200 g of liquid paraffin, emulsify at 8000 r / min for 15 min, add dropwise 10 g of 2 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain stem cell microspheres;

[0144] S9. Preparation of a weight loss composition containing mesenchymal stem cells: 4 g of fermentation product, 3 g of brown composition, 3.5 g of encapsulated probiotics, 1.5 g of stem cell microspheres and 2 g of paracrine product were stirred and mixed for 10 min to prepare a weight loss composition containing mesenchymal stem cells.

[0145] Comparative Example 5

[0146] Compared with Example 3, the difference is that the polyphenol oxidase is a single catechol oxidase.

[0147] Comparative Example 6

[0148] Compared with Example 3, the difference is that the polyphenol oxidase is a single laccase.

[0149] Comparative Example 7

[0150] Compared with Example 3, the difference is that no magnetic particles are added in step S7.

[0151] The details are as follows:

[0152] S7. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, and interleukin-2 were added to DMEM medium to prepare a culture medium;

[0153] The fetal culture medium contains 10 wt % fetal bovine serum, 10 nmol / L dexamethasone, 110 U / mL penicillin, 110 μg / mL streptomycin, and 15 μg / mL interleukin-2.

[0154] Comparative Example 8

[0155] Compared with the embodiment 3, the difference is that step S9 is not performed.

[0156] The details are as follows:

[0157] S1. Activation of strains: Inoculate Eurotium cristatum and Lactobacillus plantarum in slant culture medium, activate and culture, and obtain a strain with a bacterial count of 10 8 -10 9 cfu / mL of bacterial seed solution;

[0158] The activation conditions of the cristatum cristatum are 30° C., 150 r / min, and activation culture for 16 hours;

[0159] The activation conditions of the plant lactobacillus are 32° C., 200 r / min, and activation culture for 21 hours;

[0160] S2. Fermentation of tea leaves: 6 g of white tea and 4 g of black tea were mixed and added to 100 mL of water, sterilized, inoculated with Eurotium cristatum and Lactobacillus plantarum seed solution, the inoculation amounts were 3.5 v / v% and 1.5 v / v%, respectively, and fermented for 27 h, filtered, the solids were washed, the bacterial solution was concentrated and collected, the washed solids were retained, and the filtrate was dried to obtain a fermentation product;

[0161] S3. Fermentation strain separation and embedding: 5.7 g of sodium alginate and 6.3 g of carboxymethyl chitosan were added to 100 g of bacterial solution, stirred and mixed for 10 min, 0.7 g of lecithin was added, and added dropwise to 200 g of fish oil, emulsified at 8000 r / min for 15 min, 20 mL of a mixed solution containing 0.7 wt % calcium chloride and 1.5 wt % EDC was added dropwise, stirred and solidified for 30 min, centrifuged, washed, and dried to obtain embedded probiotics;

[0162] S4 tea extraction: The solid in step S2 is subjected to supercritical fluid extraction to obtain an extract;

[0163] The supercritical fluid extraction conditions are as follows: pressure 30 MPa, temperature 37° C., time 180 min, entrainer ethyl acetate, and the amount of entrainer added is 1.5 wt %;

[0164] S5. Oxidation of the extract: 100 g of the extract was added to 500 mL of water, 2.5 g of polyphenol oxidase was added, the reaction was stirred at 40 ° C for 2 h, the enzyme was inactivated, and a brown oxidation product was obtained;

[0165] The polyphenol oxidase is catechol oxidase and laccase, with a mass ratio of 10:2.5;

[0166] S6. Extraction of a dark brown composition: 4 times the mass of a mixed solvent was added to the brown oxidation product for extraction, ethanol was added to the system ethanol content was 70wt%, precipitated for 1.5h, filtered, washed, and dried to obtain a dark brown composition;

[0167] The mixed solvent is a mixture of chloroform, ethyl acetate and n-butanol in a volume ratio of 4:6:2.5;

[0168] S7. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, magnetic particles, and interleukin-2 were added to DMEM medium to prepare a culture medium;

[0169] The fetal culture medium contains 10 wt % fetal bovine serum, 10 nmol / L dexamethasone, 110 U / mL penicillin, 110 μg / mL streptomycin, 150 μg / mL magnetic particles, and 15 μg / mL interleukin-2. The magnetic particles are ferrosoferric oxide prepared in Preparation Example 1.

[0170] S8. Culture of adipose-derived mesenchymal stem cells: After the adipose-derived mesenchymal stem cells are revived, they are added to the culture medium and cultured in a low-frequency magnetic field. After the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated by a magnet, the residue is centrifuged, the supernatant is collected, and freeze-dried to obtain a paracrine product. The solid is washed with D-Hank's solution, digested with digestive solution, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution;

[0171] The low-frequency magnetic field culture time is 2 weeks, the culture conditions are 37°C, CO2 concentration is 5v / v%, the frequency of the low-frequency magnetic field is 45Hz, and the stimulation time is 35min / d;

[0172] The digestion solution is a PBS buffer with a pH of 7.6 containing 1.5wt% trypsin and 0.7wt% alkaline protease;

[0173] S9. Preparation of a weight loss composition containing mesenchymal stem cells: 4 g of fermentation product, 3 g of brown composition, 3.5 g of encapsulated probiotics, 1.5 g of stem cell solution (before resuscitation) and 2 g of paracrine product were stirred and mixed for 10 min to prepare a weight loss composition containing mesenchymal stem cells.

[0174] Test Example 1

[0175] The percentage of the brown composition prepared in Examples 1-3 and Comparative Examples 4-6 in the total weight of white tea and black tea was tested. The results are shown in Table 1.

[0176] Table 1

[0177]

[0178] It can be seen from the above table that the use of catechol oxidase and laccase for synergistic oxidation in Examples 1-3 of the present invention can significantly increase the yield of the dark brown composition.

[0179] Test Example 2

[0180] The experimental animals were raised at room temperature (23±2)℃, relative humidity (65±5)%, and 12h / 12h light-dark cycle. After one week of adaptive feeding, C57BL / 6J male mice were randomly divided into 4 groups (n=10), including a normal diet group (normal feed + distilled water gavage), a high-fat diet group (high-fat feed + distilled water gavage), and Example 1-3 or Comparative Example 1-8 group (high-fat feed + product gavage prepared by Example 1-3 or Comparative Example 1-8). The experimental mice were gavaged once every 24h for 9 weeks. After the last gavage, the mice were fasted but not watered for 24h. The mice were anesthetized and blood was collected from the eyeballs. The mice were killed by dislocation of the neck, and the liver, colon, and white fat (epididymal fat, perirenal fat, mesenteric fat) tissues were dissected and the contents of the cecum were collected aseptically. Part of the liver, colon, and epididymal fat tissues were fixed with a fixative, and the remaining tissues were quickly frozen in liquid nitrogen and stored at -80℃.

[0181] Determination of basic indexes of mice: The mice were weighed once on the 0th day and at the end of the experiment to calculate the weight gain ratio. During the autopsy, the liver and white fat of the mice were taken, washed and quickly weighed to calculate the organ index.

[0182] Weight gain rate = (weight at the end of the experiment - weight on the 0th day) / weight at the end of the experiment - weight on the 0th day × 100%

[0183] Organ index = organ wet weight / mouse body weight × 100%

[0184] The results are shown in Table 2.

[0185] Table 2

[0186]

[0187] Notes: Compared with the normal diet group, P<0.05; # compared with the high-fat diet group, P<0.05.

[0188] It can be seen from the above table that the weight loss compositions containing mesenchymal stem cells prepared in Examples 1-3 of the present invention can significantly reduce the body weight of mice, and reduce white fat and liver indexes.

[0189] Biochemical index determination: After blood was collected from the eyeball, it was placed in an EP tube, left to stand for 2 hours, centrifuged, and the supernatant was taken for testing. The liver and colon were homogenized with normal saline at a mass ratio of 1:9 to prepare a 10% mass fraction homogenate, centrifuged, and the supernatant was taken for testing. The serum TC and TG levels, liver SOD and MDA levels, and colon TNF- α , IL-6 levels.

[0190] The results are shown in Table 3.

[0191] Table 3

[0192]

[0193] Notes: Compared with the normal diet group, P<0.05; # compared with the high-fat diet group, P<0.05.

[0194] As can be seen from the above table, the weight loss composition containing mesenchymal stem cells prepared in Examples 1-3 of the present invention can significantly reduce the levels of TG, TC, MDA, TNF- α , IL-6 levels, and increased SOD levels.

[0195] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a weight loss composition containing mesenchymal stem cells, characterized in that: The following steps are involved: S1. Activation of bacterial strains: Inoculate Eurotium cristatum and Lactobacillus plantarum into slant culture medium, activate and culture, and obtain bacterial seed liquid; S2. Fermentation of tea leaves: mixing white tea and black tea, adding to water, sterilizing, inoculating seed solution of Eurotium cristatum and Lactobacillus plantarum, fermenting, filtering, washing solids, concentrating and collecting the bacterial solution, retaining the washed solids, and drying the filtrate to obtain a fermentation product; the mass ratio of white tea, black tea and water is 5-7:3-5:100, the inoculation amount of Eurotium cristatum and Lactobacillus plantarum seed solution is 3-4v / v% and 1-2v / v%, respectively, and the fermentation time is 24-30h; S3. Isolation and embedding of fermentation strains: sodium alginate and carboxymethyl chitosan were added to the bacterial solution, stirred and mixed, lecithin was added dropwise to emulsify the fish oil, a mixed solution of calcium chloride and EDC was added dropwise, stirred and solidified, centrifuged, washed, and dried to obtain embedded probiotics; S4. Extraction of tea leaves: extracting the solid in step S2 by supercritical fluid extraction to obtain an extract; the supercritical fluid extraction conditions are pressure 27-32MPa, temperature 35-40°C, time 150-200min, entrainer is ethyl acetate solution, the ethyl acetate content is 5-10wt%, and the amount of entrainer added is 1-2wt%; S5. Oxidation of the extract: adding the extract to water, adding polyphenol oxidase, stirring the reaction, inactivating the enzyme, and obtaining a brown oxidation product; S6. Extraction of a brown composition: extracting by adding a mixed solvent to the brown oxidation product, adding ethanol to precipitate, filtering, washing, and drying to obtain a brown composition; S7. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, magnetic particles, and interleukin-2 were added to DMEM medium to prepare a culture medium; S8. Cultivation of adipose-derived mesenchymal stem cells: After the adipose-derived mesenchymal stem cells are revived, they are added to the culture medium prepared in step S7, and cultured in a low-frequency magnetic field. After the cells adhere to the wall and grow to 80-90% fusion, the magnetic material is separated by a magnet, the residue is centrifuged, the supernatant is collected, and freeze-dried to obtain a paracrine product. The solid is rinsed with D-Hank's solution, digested, resuspended in DMEM culture medium, and frozen to obtain a stem cell solution. The low-frequency magnetic field culture time is 1-2 weeks, the culture conditions are 36-38°C, the CO2 concentration is 3-6v / v%, the frequency of the low-frequency magnetic field is 40-50Hz, and the stimulation time is 30-40min / d. S9. Embedding of stem cells: After the stem cell solution is revived, sodium alginate and lecithin are added, stirred and mixed evenly, added dropwise to liquid paraffin, emulsified, calcium chloride solution is added dropwise, solidified at room temperature, centrifuged, washed, and dried to obtain stem cell microspheres; S10. Preparation of a weight loss composition containing mesenchymal stem cells: Evenly mix the fermentation product, the brown composition, the embedded probiotics, the stem cell microspheres and the paracrine product to prepare a weight loss composition containing mesenchymal stem cells.

2. The preparation method according to claim 1, characterized in that: The activation conditions of the cosmidia in step S1 are 28-32°C, 100-200r / min, and activation culture for 12-18h, the activation conditions of the plant lactobacillus are 30-35°C, 150-250r / min, and activation culture for 18-24h, and the bacterial content of the bacterial seed liquid is 10 8 -10 9 cfu / mL.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the bacterial solution, sodium alginate, carboxymethyl chitosan and lecithin in step S3 is 100:4-7:5-8:0.5-1.

4. The preparation method according to claim 1, characterized in that: The mass ratio of the extract and the polyphenol oxidase in step S5 is 100:2-3, the temperature of the stirring reaction is 35-45°C, the time is 1-3h, the polyphenol oxidase is catechol oxidase and laccase, the mass ratio is 10:2-3, the mixed solvent in step S6 is a mixture of chloroform, ethyl acetate and n-butanol, the volume ratio is 3-5:5-7:2-3, ethanol is added until the ethanol content of the system is 65-75wt%, and the precipitation time is 1-2h.

5. The preparation method according to claim 1, characterized in that: In step S7, the content of fetal bovine serum in the culture medium is 9-11wt%, the content of dexamethasone is 5-15nmol / L, the content of penicillin is 100-120U / mL, the content of streptomycin is 100-120μg / mL, the content of magnetic particles is 100-200μg / mL, the content of interleukin-2 is 10-20μg / mL, the magnetic particles are ferroferric oxide particles, and the digestion solution used in the digestion in step S8 is a PBS buffer with a pH of 7.5-7.7 containing 1-2wt% trypsin and 0.5-1wt% alkaline protease.

6. The preparation method according to claim 1, characterized in that: The preparation method of the magnetic particles is as follows: Ferric chloride and ferrous chloride are mixed and dissolved in water, and under the protection of inert gas, ammonia water is added dropwise, heated and stirred for reaction, centrifuged, washed, dried, and calcined to obtain magnetic particles.

7. The preparation method according to claim 1, characterized in that: The mass ratio of the stem cell solution, sodium alginate and lecithin in step S9 is 100:13-16:1-2, and the mass ratio of the fermentation product, the brown composition, the embedded probiotics, the stem cell microspheres and the paracrine product in step S10 is 3-5:2-4:3-4:1-2:1.5-2.

5.

8. A weight loss composition containing mesenchymal stem cells obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the weight loss composition containing mesenchymal stem cells as claimed in claim 8 in the preparation of weight loss drugs.

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