Stem cell cryopreservation liquid based on medicinal plant extract and preparation method of stem cell cryopreservation liquid

Abstract

The invention belongs to the technical field of stem cell culture, and particularly relates to a stem cell cryopreservation solution based on a medicinal plant extract and a preparation method of the stem cell cryopreservation solution. The stem cell freezing medium based on the medicinal plant extract is prepared from the following preparation raw materials in parts by weight: 1 to 8 parts of medicinal plant extract, 50 to 70 parts of basic culture medium, 10 to 20 parts of serum, 0.1 to 0.5 part of amino acid, 1 to 5 parts of trehalose, 3 to 8 parts of glycerol and 0.1 to 1 part of buffer solution. According to the stem cell cryopreservation solution based on the medicinal plant extract, effective components in medicinal plants are scientifically extracted, and an advanced preparation process is combined, so that the problem of toxicity of a traditional stem cell cryopreservation solution is solved, and the survival rate and activity of recovered cells are improved; and a new solution is provided for long-term preservation and wide application of the stem cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cell culture, and particularly relates to a stem cell cryopreservation solution based on medicinal plant extracts and a preparation method thereof. Background Art

[0002] As an important resource in the field of biomedicine, stem cells exhibit great application potential in disease treatment, tissue repair, and regenerative medicine. However, the long-term preservation and transportation of stem cells have always been technical bottlenecks restricting their wide application. Traditional stem cell cryopreservation methods usually use chemical reagents such as dimethyl sulfoxide (DMSO) as cryoprotectants, but these chemical reagents may have potential toxic effects on cells, reducing the survival rate and activity of cells after thawing. Therefore, it is of great significance to develop a safe, efficient, and non-toxic stem cell cryopreservation solution.

[0003] In recent years, due to their rich bioactive components and natural properties, medicinal plant extracts have received increasing attention in the field of biomedicine. Many medicinal plant extracts have been proven to have various biological activities such as antioxidant, anti-inflammatory, and immunomodulatory effects. These characteristics make medicinal plant extracts potential raw materials for stem cell cryopreservation solutions. Through scientific screening and optimization combination, medicinal plant extracts can provide the necessary nutritional support for cells, reduce cell damage during freezing, and thus improve the survival rate and function of cells after thawing.

[0004] However, the current research on stem cell cryopreservation solutions based on medicinal plant extracts is still in its infancy, with problems such as complex extraction processes, unclear components, and unstable effects. Therefore, there is an urgent need to develop a stem cell cryopreservation solution based on medicinal plant extracts with a simple preparation process, clear components, and stable effects to meet the actual needs of stem cell preservation and transportation.

[0005] The present invention aims to provide a stem cell cryopreservation solution based on medicinal plant extracts and a preparation method thereof. By scientifically extracting the effective components from medicinal plants and combining advanced preparation processes, it aims to solve the toxicity problem of traditional stem cell cryopreservation solutions, improve the survival rate and activity of cells after thawing, and provide a new solution for the long-term preservation and wide application of stem cells. Summary of the Invention

[0006] The object of the present invention is to provide a stem cell cryopreservation solution based on medicinal plant extracts and a preparation method thereof, which not only solves the toxicity problem of traditional stem cell cryopreservation solutions, but also improves the survival rate and activity of cells after thawing, and provides a new solution for the long-term preservation and wide application of stem cells.

[0007] A stem cell cryopreservation solution based on medicinal plant extracts, the raw materials for its preparation, by weight, include 1-8 parts of medicinal plant extracts, 50-70 parts of basal medium, 10-20 parts of serum, 0.1-0.5 parts of amino acids, 1-5 parts of trehalose, 3-8 parts of glycerol, and 0.1-1 part of buffer solution.

[0008] Preferably, the medicinal plant extracts include one or more of astragalus extract, salvia miltiorrhiza extract, ginseng extract, angelica sinensis extract, ganoderma lucidum extract, wolfberry extract, and notoginseng extract; more preferably, they are astragalus extract, salvia miltiorrhiza extract, and ginseng extract.

[0009] Preferably, the mass ratio of the astragalus extract, salvia miltiorrhiza extract, and ginseng extract is (2-4):(1-3):1; more preferably, it is 3:2:1.

[0010] Using astragalus extract, salvia miltiorrhiza extract, and ginseng extract as medicinal plant extracts to prepare the stem cell cryopreservation solution can improve cell survival rate and recovery rate. Astragalus polysaccharide in astragalus, as a natural cryoprotectant, can improve the survival rate of cells under low temperature conditions by stabilizing the cell membrane structure, and the astragaloside contained can also reduce the oxidative stress response in a low temperature environment and protect cells from free radical damage. Tanshinone IIA in salvia miltiorrhiza can enhance the expression of angiogenic factors and contribute to the survival and functional recovery of cells after resuscitation. Components such as ginsenoside Rg1 and Rb1 in ginseng can promote cell survival by activating the PI3K / Akt signaling pathway, regulate the cell cycle to reduce apoptosis, and improve mitochondrial function to ensure that cells maintain appropriate energy supply during cryopreservation. The active ingredients in these three medicinal plant extracts work synergistically to build a powerful antioxidant network, preventing or reducing oxidative damage caused by low temperature from different angles. Ginsenosides and astragalus polysaccharide cooperate with each other to help maintain the osmotic pressure balance inside and outside the cell, prevent mechanical damage caused by ice crystal formation, and thus maintain cell homeostasis. These components also promote the DNA repair process, enhance the self-repair ability of cells after thawing, and accelerate the speed of cells returning to the normal physiological state. In addition, the compounds in astragalus and salvia miltiorrhiza have certain immunomodulatory effects, which can inhibit unnecessary inflammatory reactions and provide a microenvironment more conducive to the growth and differentiation of stem cells. However, the active ingredients in the commercially available medicinal plant extracts are less, contain a lot of impurities, and have poor efficacy. Therefore, in this application, by the way of self-preparing medicinal plant extracts, the content of the active ingredients beneficial to stem cell survival in the medicinal plant extracts is increased as much as possible, so as to improve the related efficacy of the stem cell cryopreservation solution.

[0011] The preparation method of the astragalus extract includes the following steps:

[0012] A1. Fermentation pretreatment: Wash the astragalus membranaceus, dry and crush it, add water to adjust the water content to 30% - 40% to obtain the astragalus membranaceus fermentation substrate, add the astragalus membranaceus fermentation agent, and carry out aerobic fermentation at 30 - 37 °C for 2 - 3 days, then take it out to obtain the astragalus membranaceus pretreatment product;

[0013] A2. Enzymolysis: Add the astragalus membranaceus pretreatment product to water, adjust the temperature to 45 - 55 °C, pH to 4.5 - 5.5, add cellulase, hemicellulase and xylanase, carry out enzymolysis for 2 - 4 h and then inactivate by boiling. Continue to adjust the system temperature to 40 - 50 °C, pH to 3 - 4, add pectinase, carry out enzymolysis for 1 - 3 h and then inactivate by boiling. Continue to adjust the system temperature to 35 - 40 °C, pH to 5.5 - 6.5, add glucose oxidase, carry out enzymolysis for 1 - 2 h and then inactivate by boiling. After cooling to room temperature, filter through a 0.22 μm microporous filter membrane, concentrate to 2 - 4 times the mass of the astragalus membranaceus pretreatment product, add an ethanol aqueous solution with a mass concentration of 95% and 8 - 10 times the mass of the astragalus membranaceus pretreatment product, stir evenly and then let it stand for 1 - 2 days, centrifuge and freeze-dry to obtain the crude astragalus membranaceus extract;

[0014] A3. Dissolve the crude astragalus membranaceus extract in water, add macroporous resin, oscillate at 100 - 150 r / min and 30 - 35 °C for 1 - 3 h, then filter, concentrate and dry to obtain the astragalus membranaceus extract.

[0015] Preferably, the astragalus membranaceus fermentation agent includes one or more of bacillus licheniformis, lactobacillus bulgaricus, lactobacillus plantarum, saccharomyces cerevisiae, rhodopseudomonas palustris, aspergillus niger, candida utilis; Further preferably, it is lactobacillus plantarum, saccharomyces cerevisiae, rhodopseudomonas palustris, aspergillus niger.

[0016] In some preferred embodiments, the lactobacillus plantarum has a strain number of CICC 24936; the saccharomyces cerevisiae is active dry wine yeast with a strain number of CICC 1892; the rhodopseudomonas palustris has a strain number of CICC 23812; the aspergillus niger has a strain number of CICC 41254.

[0017] Preferably, the dosage of the lactobacillus plantarum is 10 7 -10 8 CFU / g of the astragalus membranaceus fermentation substrate; the dosage of the saccharomyces cerevisiae is 10 8 -10 9 CFU / g of the astragalus membranaceus fermentation substrate; the dosage of the rhodopseudomonas palustris is 10 7 -10 8 CFU / g of the astragalus membranaceus fermentation substrate; the dosage of the aspergillus niger is 10 7 -10 8 CFU / g of the astragalus membranaceus fermentation substrate.

[0018] Using Lactobacillus plantarum, Saccharomyces cerevisiae, Rhodopseudomonas palustris, and Aspergillus niger to ferment Astragalus membranaceus first can increase the content of Astragalus polysaccharide in the finally obtained Astragalus extract. Lactobacillus plantarum can degrade the cellulose in the Astragalus cell wall through the production of metabolites, promoting the release of free polysaccharides and extracellular polysaccharides, thereby increasing the dissolution rate of Astragalus polysaccharide. During the fermentation process, Saccharomyces cerevisiae not only converts sugar into alcohol but also may produce enzymes and other metabolites, which contribute to the destruction of the Astragalus cell wall and the transformation of bound polysaccharides, making the polysaccharides easier to extract. Rhodopseudomonas palustris can produce bioactive substances that degrade the Astragalus cell wall and at the same time affect its polysaccharide structure, making it easier to extract and utilize. Aspergillus niger uses enzymes such as cellulase and hemicellulase it produces to gently decompose the Astragalus cell wall, accelerate the release of active ingredients such as polysaccharides, increase the yield of Astragalus polysaccharide, and promote the conversion of insoluble polysaccharides into soluble polysaccharides. The synergistic action of these four microorganisms can more thoroughly degrade the cell wall of Astragalus membranaceus and increase the extraction rate of polysaccharides. The metabolites (such as lactic acid and ethanol) produced by Lactobacillus plantarum and Saccharomyces cerevisiae can change the pH value and redox state of the local microenvironment, which is beneficial to the growth of other microorganisms and the manifestation of enzyme activity, further promoting the release and transformation of polysaccharides. The lactic acid produced by Lactobacillus plantarum can provide a carbon source for Rhodopseudomonas palustris; while the small molecules after Aspergillus niger decomposes complex carbohydrates are easily absorbed by other microorganisms. Through the metabolic activities of microorganisms, the structure of Astragalus polysaccharide may change, such as a decrease in molecular weight and an increase in the number of branches, which not only increases the solubility of polysaccharides but also may enhance its immunomodulatory or other health benefit characteristics.

[0019] Preferably, the mass ratio of the Astragalus pretreatment product to water is 1:(20 - 30).

[0020] Preferably, the addition amount of cellulase is 80 - 120 U / g of the Astragalus pretreatment product; the addition amount of hemicellulase is 50 - 100 U / g of the Astragalus pretreatment product; the addition amount of xylanase is 50 - 100 U / g of the Astragalus pretreatment product; the addition amount of pectinase is 150 - 250 U / g of the Astragalus pretreatment product; the addition amount of glucose oxidase is 50 - 100 U / g of the Astragalus pretreatment product.

[0021] Preferably, the mass ratio of the crude Astragalus extract to water is 1:(8 - 12).

[0022] Preferably, the addition amount of the macroporous resin is the same as the mass of the crude Astragalus extract.

[0023] Preferably, the macroporous resin is a non-polar macroporous resin with an average pore diameter of 9 - 11 nm and a pore volume of 1.5 - 1.7 mL / g.

[0024] In some preferred embodiments, the macroporous resin is purchased from Tianjin Haoju Resin Technology Co., Ltd., D101.

[0025] Sequential enzymatic hydrolysis with cellulase, hemicellulase, xylanase, pectinase, and glucose oxidase can further increase the content of active ingredients such as astragalus polysaccharide in the astragalus extract. First, cellulase, hemicellulase, and xylanase act together to degrade cellulose, hemicellulose, and xylan in the astragalus cell wall, weakening the structural strength of the cell wall. Pectinase degrades pectin, making the cell wall more porous. These two steps of enzymatic hydrolysis not only break the physical barrier of the cell wall but also promote the release of active ingredients such as polysaccharides inside the cell. Finally, the addition of glucose oxidase not only oxidizes reducing sugars such as glucose to produce products that help further break down the cell wall structure but also fine-tunes the structure of astragalus polysaccharide, making it more easily extractable and utilizable. These enzymes work synergistically to not only gradually degrade the components of the cell wall but also promote the release and structural improvement of astragalus polysaccharide, jointly achieving the efficient extraction of active ingredients such as astragalus polysaccharide. In addition, sequential enzymatic hydrolysis according to the different properties and optimal conditions of each enzyme not only improves the extraction efficiency but also significantly increases the content of astragalus polysaccharide, enhances the biological activity of astragalus polysaccharide, and thus improves the efficacy of the stem cell cryopreservation solution. However, the crude astragalus extract obtained only by fermentation and enzymatic hydrolysis still contains macromolecules such as inorganic salts and pigments. The crude astragalus extraction is purified by a specific non-polar macroporous resin, which adsorbs the non-polar macromolecules in the crude astragalus extract, thereby further increasing the purity of the polar molecule astragalus polysaccharide.

[0026] The preparation method of the salvia miltiorrhiza extract comprises the following steps:

[0027] B1. Wash and dry and crush salvia miltiorrhiza to obtain salvia miltiorrhiza powder, put it into an extraction kettle for supercritical carbon dioxide extraction to obtain a salvia miltiorrhiza extract, and obtain a crude salvia miltiorrhiza extract after rotary evaporation;

[0028] B2. Load the crude salvia miltiorrhiza extract onto a silica gel column and elute it with an acetonitrile aqueous solution with a mass concentration of 60%-70%. Start collecting the eluate after 20-25 minutes of elution, add ethyl acetate and let it stand for extraction. After the liquid surface is stratified, remove the aqueous phase, and after rotary evaporation, the salvia miltiorrhiza extract is obtained.

[0029] Preferably, the specific conditions for the supercritical carbon dioxide extraction are: pressure 23-28 MPa, entrainer flow rate 0.5-1.5 mL / min, extraction time 1.5-2.5 h, and extraction temperature 38-42 °C.

[0030] Preferably, the entrainer is an ethanol aqueous solution with a mass fraction of 95%.

[0031] Preferably, the surface of the silica gel column has C18 alkyl groups, with a specific surface area of 350 - 400 m 2 / g and a particle size of 15 - 25 μm.

[0032] In some preferred embodiments, the silica gel column is purchased from Sigma - Aldrich (Shanghai) Trading Co., Ltd.

[0033] Preferably, the flow rate of the sample loading is 1 - 2 mL / min.

[0034] Preferably, the flow rate of the elution is 2 - 4 mL / min.

[0035] Preferably, in step B2, the addition amounts of the acetonitrile aqueous solutions with mass concentrations of 60% - 70% are both 70 - 80 times the mass of the crude Danshen extract.

[0036] Preferably, the addition amount of ethyl acetate is consistent with the mass of the collected eluate.

[0037] First, extract Danshen by supercritical carbon dioxide and then perform specific separation on it, which can increase the content of active ingredients such as tanshinone IIA in the Danshen extract. By selecting an ethanol aqueous solution with a mass fraction of 95% as the entrainer, the extraction ability of supercritical carbon dioxide for tanshinone can be improved. This may be because tanshinone has a certain polarity, carbon dioxide is a non - polar solvent, and has a stronger solubility for non - polar or low - polarity solutes. Adding an entrainer can improve the extraction rate of tanshinone through the action of polar solvent molecules on solute molecules. The crude Danshen extract obtained after extraction is purified by silica gel column chromatography, which can further remove impurities and improve the purity of tanshinone IIA. At the same time, according to the different retention times of various tanshinones, controlling the time for collecting the eluate helps to ensure the content of active ingredients such as tanshinone IIA in the collected eluate, thereby improving the efficacy of the Danshen extract.

[0038] The preparation method of the ginseng extract includes the following steps:

[0039] C1. Enzymatic hydrolysis: Wash the ginseng, dry and crush it to obtain ginseng powder, add water to adjust the temperature to 45 - 55 °C and the pH to 4.5 - 5.5, add cellulase, after enzymatic hydrolysis for 2 - 4 h, boil to inactivate the enzyme, continue to adjust the system temperature to 50 - 65 °C and the pH to 6 - 7, add papain, after enzymatic hydrolysis for 1 - 3 h, boil to inactivate the enzyme, cool to room temperature and filter through a 0.22 μm microporous membrane filter, add an ethanol aqueous solution with a mass concentration of 90% - 95% for reflux extraction, and concentrate and dry the extract to obtain the ginseng enzymatic hydrolysate;

[0040] C2. Fermentation: Adjust the water content of the ginseng enzymatic hydrolysate to 30%-40% by adding water to obtain the ginseng fermentation substrate. Add the ginseng fermenting agent and perform aerobic fermentation at 25-35°C for 2-3 days, then take it out to obtain the ginseng fermented product. Add anhydrous ethanol in an amount 3-5 times the mass of the ginseng fermented product, stir evenly, and let it stand for 1-2 days. Remove the ethanol by vacuum distillation to obtain the crude ginseng extract;

[0041] C3. Load the crude ginseng extract onto a macroporous resin and elute it with an ethanol aqueous solution with a mass concentration of 80%-85%. Collect the eluate, concentrate it under reduced pressure, and dry it to obtain the ginseng extract.

[0042] Preferably, the mass ratio of the ginseng powder to water is 1:(20-30).

[0043] Preferably, the addition amount of the cellulase is 80-120 U / g of ginseng powder; the addition amount of the papain is 50-100 U / g of ginseng powder.

[0044] Preferably, the ginseng fermenting agent includes one or more of Aspergillus niger, Aspergillus ficuum, Saccharomyces cerevisiae, Lactobacillus rhamnosus, and Bacillus subtilis; More preferably, it is Aspergillus niger, Aspergillus ficuum, and Saccharomyces cerevisiae.

[0045] In some preferred embodiments, for Aspergillus niger, the strain number is CICC 41254; for Aspergillus ficuum, the strain number is CICC 2123; for Saccharomyces cerevisiae, the strain number is CICC 1892.

[0046] Preferably, the dosage of Aspergillus niger is 10 8 -10 9 CFU / g of ginseng fermentation substrate; the dosage of Aspergillus ficuum is 10 7 -10 8 CFU / g of ginseng fermentation substrate; the dosage of Saccharomyces cerevisiae is 10 7 -10 8 CFU / g of ginseng fermentation substrate.

[0047] Preferably, the macroporous resin is a weakly polar macroporous resin with an average pore diameter of 9-10 nm and a pore volume of 0.75-0.95 mL / g.

[0048] In some preferred embodiments, the macroporous resin is purchased from Tianjin Haoju Resin Technology Co., Ltd., DM130.

[0049] Preferably, the loading flow rate is 1-3 mL / min.

[0050] Preferably, the elution flow rate is 8-10 mL / min.

[0051] Preferably, in step C3, the addition amount of the ethanol aqueous solution with a mass concentration of 80%-85% is 10-15 times the mass of the crude ginseng extract.

[0052] By first enzymolysis, then fermentation, and finally separation and purification, the content of active ingredients such as ginsenoside Rg1 in the ginseng extract can be increased. Cellulase is used to decompose the cellulose component in the cell wall, while papain can degrade proteins. The combined action of the two can destroy the ginseng cell wall structure, effectively open the cell wall barrier, make the active ingredients in the cells easier to be released, and increase the accessibility of the cell contents. Aspergillus niger, Aspergillus ficuum, and Saccharomyces cerevisiae are introduced as fermenting agents. The synergistic action of Aspergillus niger and Aspergillus ficuum can produce enzymes such as β-glucosidase that can hydrolyze the glycosyl group of ginsenoside through the fermentation process, promoting the conversion of ginsenoside. Especially in the process of converting ginsenoside Re and Rg2 to Rg1, the α-rhamnosidase of Aspergillus niger plays a key role. And Saccharomyces cerevisiae mainly promotes the generation of ginsenoside Rg1 indirectly by secreting enzymes such as β-glucosidase and acting on the precursor substances of ginsenoside. The enzyme systems of the three fermenting agents complement each other, further increasing the content of active ingredients such as ginsenoside Rg1 in the ginseng extract.

[0053] Preferably, the basal medium includes one or more of DMEM medium, MEM medium, IMEM medium, and RPMI1640 medium; more preferably, it is DMEM medium.

[0054] Preferably, the serum includes fetal bovine serum.

[0055] In some preferred embodiments, both the DMEM medium and the fetal bovine serum are purchased from Omashi (Shanghai) Biotechnology Co., Ltd., DMEM / F12 and standard fetal bovine serum.

[0056] Preferably, the amino acids include one or more of γ-polyglutamic acid, lysine, arginine, and proline; more preferably, they are γ-polyglutamic acid, lysine, and arginine.

[0057] Preferably, the mass ratio of γ-polyglutamic acid, lysine, and arginine is (1-3):(0.5-2):1; more preferably, it is 2:1:1.

[0058] Preferably, the molecular weight of γ-polyglutamic acid is 50000-100000.

[0059] In some preferred embodiments, the γ-polyglutamic acid is purchased from Shanghai Macklin Biochemical Co., Ltd.

[0060] γ - polyglutamic acid, lysine, and arginine are selected as amino acids and added to this system, which can maintain the cell recovery rate and activity while avoiding the use of toxic DMSO. This may be because γ - polyglutamic acid has excellent cryoprotective ability, can significantly improve the cell recovery rate in a glycerol cryopreservation system without DMSO, maintain the integrity of cell morphology, and enhance the activity of cells after recovery. At the same time, lysine and arginine, as essential amino acids, not only provide necessary nutritional support for cells to promote cell growth, but also further enhance the stability of cells during cryopreservation through regulating the osmotic pressure inside and outside cells and antioxidant effects. These three amino acids act synergistically in the cryopreservation solution. γ - polyglutamic acid forms a protective film to reduce ice crystal damage, while lysine and arginine, through mechanisms such as nutritional support and osmotic pressure regulation, jointly improve the cryopreservation efficiency of cells and the physiological functions after recovery.

[0061] Preferably, the buffer solution is phosphate buffer solution with a pH of 7.2, purchased from Shanghai Enzyme - linked Biotechnology Co., Ltd.

[0062] The preparation method of the stem cell cryopreservation solution based on medicinal plant extracts includes the following steps: After uniformly stirring the basal medium and serum, add glycerol and buffer solution, stir evenly, then add the medicinal plant extract, stir until dissolved, add amino acids and trehalose, stir evenly, and then perform aseptic filtration using a 0.22 - μm pore - size filter membrane to obtain the product, which is stored in a - 80°C refrigerator or liquid nitrogen tank for standby.

[0063] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0064] 1. The stem cell cryopreservation solution based on medicinal plant extracts prepared by the present invention, through scientifically extracting the effective components from medicinal plants and combining with advanced preparation processes, not only solves the toxicity problem existing in traditional stem cell cryopreservation solutions, but also improves the survival rate and activity of cells after recovery, providing a new solution for the long - term preservation and wide application of stem cells.

[0065] 2. The present invention selects astragalus extract, salvia extract, and ginseng extract as medicinal plant extracts to prepare the stem cell cryopreservation solution, which can improve the cell survival rate and recovery rate.

[0066] 3. The present invention selects Lactobacillus plantarum, Saccharomyces cerevisiae, Rhodopseudomonas palustris, and Aspergillus niger to ferment astragalus first, which can increase the content of astragalus polysaccharide in the finally obtained astragalus extract.

[0067] 4. The present invention uses cellulase, hemicellulase, xylanase, pectinase, and glucose oxidase for enzymatic hydrolysis in sequence, which can further increase the content of effective components such as astragalus polysaccharide in the astragalus extract.

[0068] 5. The present invention first extracts Salvia miltiorrhiza with supercritical carbon dioxide and then performs specific separation on it, which can increase the content of active ingredients such as tanshinone IIA in the Salvia miltiorrhiza extract.

[0069] 6. The present invention can increase the content of active ingredients such as ginsenoside Rg1 in the ginseng extract by first performing enzymatic hydrolysis, then fermentation, and finally separation and purification.

[0070] 7. The present invention selects γ-polyglutamic acid, lysine, and arginine as amino acids and adds them to this system, which can maintain the resuscitation rate and activity of cells while avoiding the use of toxic DMSO. Detailed implementation manners

[0071] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0072] The raw materials used in the present invention are all commercially available. Specifically:

[0073] Lactobacillus plantarum, strain number CICC 24936; Saccharomyces cerevisiae is active dry wine yeast, strain number CICC1892; Rhodopseudomonas palustris, strain number CICC 23812; Aspergillus niger, strain number CICC 41254; Aspergillus ficuum, strain number CICC 2123.

[0074] Cellulase, enzyme activity about 11000U / g; hemicellulase, enzyme activity about 50000U / g; xylanase, enzyme activity about 80000U / g; pectinase, enzyme activity about 30000U / g; glucose oxidase, enzyme activity about 10000U / g; papain, enzyme activity about 100000U / g; all are purchased from Ningxia Xiasheng Industrial Group Co., Ltd.

[0075] The adsorption resin is a non-polar macroporous adsorption resin, with an average pore diameter of 9-11nm and a pore volume of 1.5-1.7mL / g, purchased from Tianjin Haoju Resin Technology Co., Ltd., D101.

[0076] The silica gel column has a C18 alkyl group on its surface, with a specific surface area of 350-400m 2 / g and a particle size of 15-25μm, purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0077] The macroporous resin is a weakly polar macroporous resin, with an average pore diameter of 9-10nm and a pore volume of 0.75-0.95mL / g, purchased from Tianjin Haoju Resin Technology Co., Ltd., DM130.

[0078] Both DMEM medium and fetal bovine serum were purchased from Omars (Shanghai) Biotechnology Co., Ltd., DMEM / F12 and standard fetal bovine serum.

[0079] The molecular weight of γ-polyglutamic acid is 50,000 - 100,000, and it was purchased from Shanghai Macklin Biochemical Co., Ltd.

[0080] The buffer solution is phosphate buffer with a pH of 7.2, and it was purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.

[0081] Example 1

[0082] This example provides a stem cell cryopreservation solution based on medicinal plant extracts. Its preparation raw materials, by weight, include 5 parts of medicinal plant extracts, 60 parts of basal medium, 15 parts of serum, 0.3 parts of amino acids, 3 parts of trehalose, 5 parts of glycerol, and 0.5 parts of buffer solution.

[0083] The medicinal plant extracts are astragalus extract, salvia miltiorrhiza extract, and ginseng extract, with a mass ratio of 3:2:1.

[0084] The preparation method of the astragalus extract is as follows:

[0085] A1. Fermentation pretreatment: Wash astragalus, dry and crush it, add water to adjust the water content to 35% to obtain the astragalus fermentation substrate, add the astragalus fermentation agent, and perform aerobic fermentation at 37°C for 3 days, then take it out to obtain the astragalus pretreatment product;

[0086] A2. Enzymolysis: Add the astragalus pretreatment product to water, adjust the temperature to 50°C and the pH to 5, add cellulase, hemicellulase, and xylanase, perform enzymolysis for 3 h and then boil to inactivate the enzymes. Continue to adjust the system temperature to 45°C and the pH to 3.5, add pectinase, perform enzymolysis for 2 h and then boil to inactivate the enzymes. Continue to adjust the system temperature to 37°C and the pH to 6, add glucose oxidase, perform enzymolysis for 1.5 h and then boil to inactivate the enzymes. After cooling to room temperature, filter through a 0.22 μm microporous filter membrane, concentrate to 3 times the mass of the astragalus pretreatment product, add 9 times the mass of the astragalus pretreatment product of an ethanol aqueous solution with a mass concentration of 95%, stir evenly, stand for 2 days, centrifuge and freeze-dry to obtain the crude astragalus extract;

[0087] A3. Dissolve the crude astragalus extract in water, add adsorption resin, oscillate at 130 r / min and 33°C for 2 h, then filter, concentrate and dry to obtain the astragalus extract.

[0088] The astragalus fermentation agent is Lactobacillus plantarum, Saccharomyces cerevisiae, Rhodopseudomonas palustris, and Aspergillus niger.

[0089] The dosage of Lactobacillus plantarum is 10 8CFU / g Astragalus fermentation substrate; the dosage of Saccharomyces cerevisiae is 10 9 CFU / g Astragalus fermentation substrate; the dosage of Rhodopseudomonas palustris is 10 7 CFU / g Astragalus fermentation substrate; the dosage of Aspergillus niger is 10 7 CFU / g Astragalus fermentation substrate.

[0090] The mass ratio of the Astragalus pretreatment product to water is 1:25.

[0091] The addition amount of the cellulase is 100 U / g Astragalus pretreatment product; the addition amount of the hemicellulase is 80 U / g Astragalus pretreatment product; the addition amount of the xylanase is 80 U / g Astragalus pretreatment product; the addition amount of the pectinase is 200 U / g Astragalus pretreatment product; the addition amount of the glucose oxidase is 80 U / g Astragalus pretreatment product.

[0092] The mass ratio of the crude Astragalus extract to water is 1:10.

[0093] The addition amount of the adsorption resin is consistent with the mass of the crude Astragalus extract.

[0094] The preparation method of the Salvia miltiorrhiza extract comprises the following steps:

[0095] B1. Wash and dry and crush Salvia miltiorrhiza to obtain Salvia miltiorrhiza powder, put it into an extraction kettle for supercritical carbon dioxide extraction to obtain a Salvia miltiorrhiza extract solution, and obtain a crude Salvia miltiorrhiza extract after rotary evaporation;

[0096] B2. Load the crude Salvia miltiorrhiza extract, pass through a silica gel column, and elute with an acetonitrile aqueous solution with a mass concentration of 65%. Start collecting the eluate after 22 minutes of elution, add ethyl acetate and let it stand for extraction. After the liquid surface is layered, remove the aqueous phase, and after rotary evaporation, the Salvia miltiorrhiza extract is obtained.

[0097] The specific conditions of the supercritical carbon dioxide extraction are: pressure 25 MPa, entrainer flow rate 1 mL / min, extraction time 2 h, and extraction temperature 40 °C.

[0098] The entrainer is an ethanol aqueous solution with a mass fraction of 95%.

[0099] The loading flow rate is 1.5 mL / min.

[0100] The elution flow rate is 3 mL / min.

[0101] In the step B2, the addition amount of the acetonitrile aqueous solution with a mass concentration of 65% is 75 times the mass of the crude Salvia miltiorrhiza extract.

[0102] The addition amount of the ethyl acetate is consistent with the mass of the collected eluate.

[0103] The preparation method of the ginseng extract comprises the following steps:

[0104] C1. Enzymatic hydrolysis: Wash the ginseng, dry and crush it to obtain ginseng powder, add water to adjust the temperature to 50 °C and the pH to 5, add cellulase, after enzymatic hydrolysis for 3 h, boil to inactivate the enzyme, continue to adjust the system temperature to 55 °C and the pH to 7, add papain, after enzymatic hydrolysis for 2 h, boil to inactivate the enzyme, cool to room temperature, filter through a 0.22 μm microporous membrane filter, add an ethanol aqueous solution with a mass concentration of 95% for reflux extraction, concentrate and dry the extract to obtain the ginseng enzymatic hydrolysate;

[0105] C2. Fermentation: Add water to the ginseng enzymatic hydrolysate to adjust the water content to 35% to obtain the ginseng fermentation substrate, add the ginseng fermentation agent, carry out aerobic fermentation at 30 °C for 3 days and then take out to obtain the ginseng fermented product, add anhydrous ethanol 4 times the mass of the ginseng fermented product, stir evenly and then stand for 2 days, and remove the ethanol by vacuum distillation to obtain the crude ginseng extract;

[0106] C3. Load the crude ginseng extract onto a macroporous resin, elute with an ethanol aqueous solution with a mass concentration of 85%, collect the eluate, concentrate under reduced pressure and dry to obtain the ginseng extract.

[0107] The mass ratio of the ginseng powder to water is 1:25.

[0108] The addition amount of the cellulase is 100 U / g of ginseng powder; the addition amount of the papain is 80 U / g of ginseng powder.

[0109] The ginseng fermentation agent is Aspergillus niger, Aspergillus ficuum, Saccharomyces cerevisiae.

[0110] The dosage of Aspergillus niger is 10 9 CFU / g of ginseng fermentation substrate; the dosage of Aspergillus ficuum is 10 7 CFU / g of ginseng fermentation substrate; the dosage of Saccharomyces cerevisiae is 10 8 CFU / g of ginseng fermentation substrate.

[0111] The flow rate of loading is 2 mL / min.

[0112] The flow rate of elution is 9 mL / min.

[0113] In step C3, the addition amount of the ethanol aqueous solution with a mass concentration of 85% is 12 times the mass of the crude ginseng extract.

[0114] The basic medium is DMEM medium.

[0115] The serum is fetal bovine serum.

[0116] The amino acids are γ-polyglutamic acid, lysine, and arginine, and the mass ratio is 2:1:1.

[0117] The preparation method of the stem cell cryopreservation solution based on the medicinal plant extract comprises the following steps: After uniformly stirring the basal medium and the serum, add glycerol and buffer solution, stir evenly, then add the medicinal plant extract, stir until dissolved, add amino acids and trehalose, stir evenly, and then perform aseptic filtration using a filter membrane with a pore size of 0.22 μm to obtain the product, which is stored in a -80°C refrigerator or a liquid nitrogen tank for standby.

[0118] Example 2

[0119] The difference between this example and Example 1 is that the medicinal plant extracts are astragalus extract, salvia miltiorrhiza extract, and ginseng extract, and the mass ratio is 2:2:1.

[0120] Comparative Example 1

[0121] The difference between this comparative example and Example 1 is that the medicinal plant extracts are astragalus extract and salvia miltiorrhiza extract, and the mass ratio is 3:2.

[0122] Comparative Example 2

[0123] The difference between this comparative example and Example 1 is that the astragalus ferment is Lactobacillus plantarum, Saccharomyces cerevisiae, and Aspergillus niger.

[0124] The dosage of Lactobacillus plantarum is 10 8 CFU / g of astragalus fermentation substrate; the dosage of Saccharomyces cerevisiae is 10 9 CFU / g of astragalus fermentation substrate; the dosage of Aspergillus niger is 10 8 CFU / g of astragalus fermentation substrate.

[0125] Comparative Example 3

[0126] The difference between this comparative example and Example 1 is that the preparation method of the astragalus extract comprises the following steps:

[0127] A1. Fermentation pretreatment: Wash astragalus, dry and crush it, add water to adjust the water content to 35% to obtain an astragalus fermentation substrate, add an astragalus ferment, and perform aerobic fermentation at 37°C for 3 days, then take it out to obtain a pretreated astragalus product;

[0128] A2. Enzymolysis: Add the pretreated astragalus product to water, adjust the temperature to 50°C and the pH to 5, add cellulase, hemicellulase, xylanase, pectinase, and glucose oxidase, perform enzymolysis for 6 h, then boil to inactivate the enzymes, cool to room temperature, filter through a 0.22 μm microporous filter membrane, concentrate to 3 times the mass of the pretreated astragalus product, add an ethanol aqueous solution with a mass concentration of 95% and 9 times the mass of the pretreated astragalus product, stir evenly, stand for 2 days, and perform centrifugation and freeze-drying to obtain the crude astragalus extract;

[0129] A3. Dissolve the crude astragalus extract in water, add an adsorption resin, shake for 2 h at 130 r / min and 33 °C, then filter, concentrate and dry to obtain the astragalus extract.

[0130] Comparative Example 4

[0131] The difference between this comparative example and Example 1 is that: the adsorption resin is a weakly polar macroporous resin with an average pore diameter of 9 - 10 nm, a pore volume of 0.75 - 0.95 mL / g, and is purchased from Tianjin Haoju Resin Technology Co., Ltd., DM130.

[0132] Comparative Example 5

[0133] The difference between this comparative example and Example 1 is that: the preparation method of the salvia miltiorrhiza extract has the following steps:

[0134] B1. Wash and dry the salvia miltiorrhiza, then crush it to obtain salvia miltiorrhiza powder, put it into an extraction kettle for supercritical carbon dioxide extraction to obtain a salvia miltiorrhiza extract solution, and obtain the crude salvia miltiorrhiza extract after rotary evaporation;

[0135] B2. Load the crude salvia miltiorrhiza extract, pass it through a silica gel column, and elute with an acetonitrile aqueous solution with a mass concentration of 65%. Start collecting the eluate after 22 min of elution, add ethyl acetate and let it stand for extraction. After the liquid surface is layered, remove the aqueous phase, and then perform rotary evaporation to obtain the salvia miltiorrhiza extract.

[0136] The specific conditions for the supercritical carbon dioxide extraction are: pressure 25 MPa, extraction time 2 h, and extraction temperature 40 °C.

[0137] Comparative Example 6

[0138] The difference between this comparative example and Example 1 is that: the preparation method of the salvia miltiorrhiza extract has the following steps:

[0139] B1. Wash and dry the salvia miltiorrhiza, then crush it to obtain salvia miltiorrhiza powder, put it into an extraction kettle for supercritical carbon dioxide extraction to obtain a salvia miltiorrhiza extract solution, and obtain the crude salvia miltiorrhiza extract after rotary evaporation;

[0140] B2. Load the crude salvia miltiorrhiza extract, pass it through a silica gel column, and elute with an acetonitrile aqueous solution with a mass concentration of 65%. Collect the eluate, add ethyl acetate and let it stand for extraction. After the liquid surface is layered, remove the aqueous phase, and then perform rotary evaporation to obtain the salvia miltiorrhiza extract.

[0141] Comparative Example 7

[0142] The difference between this comparative example and Example 1 is that: the ginseng fermenting agent is Aspergillus ficuum and Saccharomyces cerevisiae.

[0143] The dosage of Aspergillus ficuum is 10 9CFU / g of ginseng fermentation substrate; the dosage of Saccharomyces cerevisiae is 10 8 CFU / g of ginseng fermentation substrate.

[0144] Comparative Example 8

[0145] The difference between this comparative example and Example 1 is that the amino acids are lysine and arginine, and the mass ratio is 1:1.

[0146] Performance test

[0147] Taking human mesenchymal stem cells as an example, referring to the method in the paper "Development of DMSO-FREE Cell Cryopreservation Solution" published by Chen Huanyun, the human mesenchymal stem cells were immersed in the stem cell cryopreservation solution prepared in this application. After being placed at -80°C for 3 months, they were taken out and revived. Calculate the stem cell revival rate. The stem cell revival rate = the number of cells after cryopreservation revival / the original number of cells × 100%, %; after culturing the revived stem cells for 24 hours, test the cell viability. The cell viability = the number of cells after culturing for 24 hours / the number of cells after cryopreservation revival × 100%, %. The results are shown in Table 1.

[0148] Table 1 Measurement results

[0149] Stem cell resuscitation rate / % Cell viability / % Example 1 99.21 99.86 Example 2 98.90 99.49 Comparative Example 1 81.03 89.52 Comparative Example 2 93.86 97.75 Comparative Example 3 91.99 96.37 Comparative Example 4 78.23 88.04 Comparative Example 5 89.84 95.31 Comparative Example 6 90.17 96.16 Comparative Example 7 87.16 93.27 Comparative Example 8 82.05 90.92

[0150] According to statistics, the stem cell cryopreservation solution based on medicinal plant extracts prepared in Examples 1-2 of the present invention has a high stem cell revival rate and high viability of the revived stem cells, indicating that the stem cell cryopreservation solution provided by the present invention can significantly improve the revival rate of stem cells after cryopreservation revival and the viability of the revived cells, while reducing the damage to stem cells caused by traditional DMSO cryopreservation solutions. In Comparative Example 1, ginseng extract was not added. In Comparative Example 2, Rhodopseudomonas palustris was not added to ferment Astragalus membranaceus. In Comparative Example 3, stepwise enzymatic hydrolysis was performed on the Astragalus membranaceus pretreatment. In Comparative Example 4, the resin for adsorbing Astragalus polysaccharide was not a non-polar macroporous resin. In Comparative Example 5, when supercritical carbon dioxide was used to extract Salvia miltiorrhiza, an aqueous ethanol solution with a mass fraction of 95% was not added as an entrainer. In Comparative Example 6, other components in the crude extract of Salvia miltiorrhiza were not eluted, reducing the content of effective components such as tanshinone IIA in the Salvia miltiorrhiza extract. In Comparative Example 7, Aspergillus niger was not added as a ginseng fermenting agent. In Comparative Example 8, γ-polyglutamic acid was not added as an amino acid. The prepared stem cell cryopreservation solution has a poor cryopreservation effect on stem cells, and both the stem cell revival rate and cell viability are relatively low. Therefore, the stem cell cryopreservation solution prepared by using the raw materials and methods described in this application not only solves the toxicity problem existing in traditional stem cell cryopreservation solutions, but also improves the survival rate and viability of cells after revival, providing a new solution for the long-term preservation and wide application of stem cells.

[0151] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A stem cell cryopreservation solution based on medicinal plant extracts, characterized in that: The raw materials for its preparation include, by weight, 1-8 parts of medicinal plant extracts, 50-70 parts of basal culture medium, 10-20 parts of serum, 0.1-0.5 parts of amino acids, 1-5 parts of trehalose, 3-8 parts of glycerol, and 0.1-1 parts of buffer; the medicinal plant extracts include one or more of astragalus extract, salvia miltiorrhiza extract, ginseng extract, angelica extract, ganoderma lucidum extract, wolfberry extract, and Panax notoginseng extract.

2. The stem cell cryopreservation solution based on medicinal plant extracts according to claim 1, characterized in that: The preparation method of the astragalus extract comprises the following steps: A1. Fermentation pretreatment: Wash the astragalus, dry and crush it, add water to adjust the water content to 30% to 40% to obtain an astragalus fermentation substrate, add an astragalus fermentation agent, aerobically ferment at 30-37°C for 2-3 days, and then take it out to obtain an astragalus pretreatment product; A2, enzymolysis: add water to the astragalus pretreatment, adjust the temperature to 45-55 ° C, pH to 4.5-5.5, add cellulase, hemicellulase and xylanase, enzymolysis for 2-4 hours and then boil and inactivate, continue to adjust the system temperature to 40-50 ° C, pH to 3-4, add pectinase, enzymolysis for 1-3 hours and then boil and inactivate, continue to adjust the system temperature to 35-40 ° C, pH 5.5-6.5, add glucose oxidase, enzymolysis for 1-2 hours and then boil and inactivate, cool to room temperature and filter with a 0.22 μm microporous filter membrane, concentrate to 2-4 times the mass of the astragalus pretreatment, add 8-10 times the mass of the astragalus pretreatment with a mass concentration of 95% ethanol aqueous solution, stir evenly and let stand for 1-2 days, centrifuge and freeze-dry to obtain a crude astragalus extract; A3. Dissolve the crude extract of Astragalus membranaceus in water, add macroporous resin, shake at 100-150 r / min and 30-35° C. for 1-3 hours, filter, concentrate and dry to obtain the Astragalus membranaceus extract.

3. The stem cell cryopreservation solution based on medicinal plant extracts according to claim 2, characterized in that: The astragalus fermentation agent comprises one or more of Bacillus licheniformis, Lactobacillus bulgaricus, Lactobacillus plantarum, Saccharomyces cerevisiae, Rhodopseudomonas palustris, Aspergillus niger and Candida utilis.

4. The stem cell cryopreservation solution based on medicinal plant extracts according to claim 2, characterized in that: The added amount of the cellulase is 80-120U / g of the astragalus pretreatment; the added amount of the hemicellulase is 50-100U / g of the astragalus pretreatment; the added amount of the xylanase is 50-100U / g of the astragalus pretreatment; the added amount of the pectinase is 150-250U / g of the astragalus pretreatment; and the added amount of the glucose oxidase is 50-100U / g of the astragalus pretreatment.

5. The stem cell cryopreservation solution based on medicinal plant extracts according to claim 1, characterized in that: The preparation method of the salvia miltiorrhiza extract comprises the following steps: B1, washing and drying the salvia miltiorrhiza and crushing it to obtain salvia miltiorrhiza powder, putting it into an extraction kettle for supercritical carbon dioxide extraction to obtain salvia miltiorrhiza extract, and rotary evaporating to obtain salvia miltiorrhiza crude extract; B2. Load the crude extract of Salvia miltiorrhiza, pass it through a silica gel column, and elute it with an acetonitrile aqueous solution with a mass concentration of 60%-70%. After elution for 20-25 minutes, start collecting the eluate, add ethyl acetate and let it stand for extraction. After the liquid surface is separated, the aqueous phase is removed and the Salvia miltiorrhiza extract is obtained after rotary evaporation.

6. The stem cell cryopreservation solution based on medicinal plant extracts according to claim 5, characterized in that: The specific conditions of the supercritical carbon dioxide extraction are: pressure 23-28 MPa, entrainer flow rate 0.5-1.5 mL / min, extraction time 1.5-2.5 h, extraction temperature 38-42° C.

7. The stem cell cryopreservation solution based on medicinal plant extracts according to claim 1, characterized in that: The preparation method of the ginseng extract comprises the following steps: C1. Enzymolysis: Wash the ginseng, dry it, and crush it to obtain ginseng powder. Add water to adjust the temperature to 45-55°C and the pH to 4.5-5.

5. Add cellulase, enzymolyze for 2-4 hours, and then boil it to inactivate it. Continue to adjust the system temperature to 50-65°C and the pH to 6-7. Add papain, enzymolyze for 1-3 hours, and then boil it to inactivate it. After cooling to room temperature, filter it with a 0.22μm microporous filter membrane, add ethanol aqueous solution with a mass concentration of 90%-95%, reflux and extract it, concentrate and dry the extract to obtain ginseng enzymolysis product. C2, fermentation: add water to the ginseng hydrolysate to adjust the water content to 30% to 40% to obtain a ginseng fermentation substrate, add a ginseng fermentation agent, aerobically ferment at 25-35°C for 2-3 days, then take out to obtain a ginseng fermentation product, add anhydrous ethanol 3-5 times the mass of the ginseng fermentation product, stir evenly, let stand for 1-2 days, and remove the ethanol by vacuum distillation to obtain a crude ginseng extract; C3. The crude ginseng extract is loaded, passed through a macroporous resin, and eluted with an ethanol aqueous solution having a mass concentration of 80%-85%, the eluate is collected, concentrated under reduced pressure, and dried to obtain the ginseng extract.

8. The method for preparing a stem cell cryopreservation solution based on medicinal plant extracts according to claim 7, characterized in that: The ginseng fermentation agent comprises one or more of Aspergillus niger, Aspergillus ficifolius, Saccharomyces cerevisiae, Lactobacillus rhamnosus and Bacillus subtilis.

9. The stem cell cryopreservation solution based on medicinal plant extracts according to claim 1, characterized in that: The amino acids include one or more of γ-polyglutamic acid, lysine, arginine and proline.

10. A method for preparing a stem cell cryopreservation solution based on a medicinal plant extract according to any one of claims 1 to 9, characterized in that: The method comprises the following steps: after the basal culture medium and serum are stirred evenly, glycerol and buffer are added, after the stirring is evenly done, medicinal plant extracts are added, after the stirring is dissolved, amino acids and trehalose are added, after the stirring is evenly done, aseptic filtration is performed using a filter membrane with a pore size of 0.22 μm, and the product is stored in a -80°C refrigerator or a liquid nitrogen tank for later use.