Cell activity regeneration compound and preparation method thereof

Abstract

The invention discloses a cell activity regeneration compound and a preparation method thereof, and belongs to the technical field of cell regeneration. The cell activity regeneration compound comprises a component A and a component B, and the mass ratio of the component A to the component B is (5: 1)-(1: 5); the component A is prepared from the following raw materials in parts by mass: 15 to 25 parts of asplenium bauxing, 2 to 5 parts of tea oil, 15 to 35 parts of herba taraxaci, 5 to 15 parts of endive root and 1 to 5 parts of folium perillae; the component B is prepared from the following components in parts by mass: 15 to 20 parts of polycaprolactone-protein conjugate, 5 to 10 parts of cardiomyopeptide and 5 to 15 parts of amifostine mercaptan; the compound prepared from the component A and the component B according to a certain mass ratio can effectively enhance the cell activity, accelerate the regeneration capacity of cells, reduce the damage of environmental pressure to the cells, promote the cell proliferation and mobilize the reconstruction of the number of the somatic cells, so that the aging of the cells is retarded.

Description

Technical Field

[0001] The present invention relates to the technical field of cell regeneration, and more specifically to a cell vitality regeneration complex and a preparation method thereof. Background Art

[0002] The human body is made up of cells. The chemical substances that make up the cells are constantly affected by the internal and external environment during movement and are damaged, resulting in functional degeneration and aging. The aging and death of cells will cause depression and relaxation. Studies have shown that the nucleus, cytoplasm and cell membrane of aging cells have obvious changes: ① The water content in the cell decreases, the volume decreases, and the metabolic rate slows down; ② The activity of intracellular enzymes decreases; ③ The pigments in the cell accumulate; ④ The intracellular respiration rate slows down, the volume of the cell nucleus increases, the nuclear membrane folds inward, the chromatin shrinks, and the color deepens. The number of mitochondria decreases and the volume increases; ⑤ The permeability function of the cell membrane changes, which reduces the material transport function.

[0003] The proliferation ability of tissue cells is called regeneration, which can be divided into physiological regeneration and pathological regeneration. Physiological regeneration occurs under normal circumstances, which is a process in which consumed and dying cells are constantly replaced by new cells, such as the shedding of epidermal keratin cells, hair renewal, and sebaceous gland cell excretion. The nuclear division rate of regenerated cells is different during the day and at night. Pathological regeneration is when cells and tissues are necrotic or defective. If the degree of damage is mild and the damaged cells have strong regeneration ability, the same cells around the damage can proliferate and differentiate to restore the structure and function of the original epidermal material. For cells with weak cell regeneration ability, the regeneration rate is very slow if they rely solely on themselves. Therefore, in order to accelerate cell regeneration, we propose a cell vitality regeneration complex and its preparation method. Summary of the invention

[0004] The purpose of the present invention is to provide a cell vitality regeneration complex and a preparation method thereof to solve the problems raised in the above background technology:

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A cell vitality regeneration complex comprises component A and component B, wherein the mass ratio of component A to component B is 5:1-1:5;

[0007] Component A comprises, by weight: 15-25 parts of Aspleniaceae, 2-5 parts of tea oil, 15-35 parts of dandelion, 5-15 parts of chicory root, and 1-5 parts of perilla leaf;

[0008] Component B comprises, by weight: 15-20 parts of polycaprolactone-protein conjugate, 5-10 parts of myocardial peptide, 5-15 parts of amifostine thiol, and 1-3 parts of amifostine disulfide.

[0009] Preferably, dandelion is preferably 25 parts, chicory root is preferably 10 parts, and perilla leaf is preferably 3 parts.

[0010] Preferably, the concentration of myocardial peptide is 25 μg / mL-500 μg / mL, the concentration of amifostine thiol is 0.01 μM-250 μM, and the concentration of amifostine disulfide is 0.01 μM-25 μM.

[0011] Preferably, the concentration of myocardial peptide is preferably 50 μg / mL, the concentration of amifostine thiol is preferably 50 μM, and the concentration of amifostine disulfide is preferably 1 μM.

[0012] Preferably, the polycaprolactone-protein conjugate is a product obtained by combining polycaprolactone nanoparticles with active proteins derived from skin fibroblasts.

[0013] A method for preparing a cell vitality regeneration complex comprises the following steps:

[0014] Wash Baoxing Asplenium, dandelion, chicory root, and perilla leaf respectively, add water, grind and filter the residue to obtain Baoxing Asplenium filtrate, dandelion filtrate, chicory root filtrate, and perilla leaf filtrate, extract the filtrate to obtain four extracts, and mix the four extracts to obtain a mixed solution;

[0015] Heat the mixture to boiling and keep it warm for 2-5 minutes, then add tea oil and stir. After stirring, cool to obtain component A solution;

[0016] Fully dissolving myocardial peptide, amifostine thiol and amifostine disulfide, and uniformly mixing them to obtain a mixed solution, and then uniformly suspending the polycaprolactone-protein conjugate in the mixed solution to obtain a component B solution;

[0017] The component A solution and the component B solution are mixed in a mass ratio of 5:1-1:5 to obtain a cell vitality regeneration complex.

[0018] Preferably, the Baoxing Asplenium, dandelion, chicory root and perilla leaf are ground for 20 minutes, and the filter mesh used for filtering has a pore size of 5 μm.

[0019] Preferably, the Baoxing Asplenium filtrate, the dandelion filtrate, the chicory root filtrate, and the perilla leaf filtrate are respectively treated with supercritical CO 2 Extraction, extraction pressure is 26-28MPa, extraction temperature is 45-48℃, CO 2 Extraction flow rate is 50-55L·h -1 The temperature of separation kettle I is 40-42°C and the pressure is 8-10MPa, the temperature of separation kettle II is 35-38°C and the pressure is 5-6MPa, and the obtained Baoxing Aspleniaceae, dandelion, chicory root and perilla leaf extracts are mixed to obtain a mixed solution.

[0020] Preferably, it also includes a gel solution, which is obtained by fully dissolving carboxymethyl cellulose, PBS buffer and glycerol;

[0021] The gel solution is added to the cell vitality regeneration complex to obtain a complex gel.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The composite prepared by component A and component B in a certain mass ratio can effectively enhance cell vitality, accelerate cell regeneration ability, reduce environmental pressure damage to cells, promote cell proliferation, mobilize the reconstruction of somatic cell number, and thus slow down cell aging. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0025] Example:

[0026] A cell vitality regeneration complex comprises component A and component B, wherein the mass ratio of component A to component B is 5:1-1:5;

[0027] Component A comprises, by mass, 15-25 parts of Aspleniaceae, 15-35 parts of dandelions, 5-15 parts of chicory roots, 1-5 parts of perilla leaves, and 2-5 parts of tea oil; wherein, dandelions are preferably 25 parts, chicory roots are preferably 10 parts, and perilla leaves are preferably 3 parts.

[0028] Specifically, Baoxing Stylem fern is rich in various antioxidants, which can eliminate free radicals in the body and protect cell health; dandelion, chicory root and perilla leaf all contain effective ingredients such as flavonoids, all of which have significant antioxidant and anti-aging effects. Dandelion is also rich in vitamins and various trace elements, which can promote cell antioxidant capacity and eliminate free radicals; chicory root is rich in antioxidants such as flavonoids and polyphenols, and has high content of vitamin C and vitamin K; perilla leaves contain minerals such as potassium, sodium, calcium and anti-aging factor SOD, which can increase the activity of superoxide dismutase in red blood cells; tea oil is rich in vitamin E, which can promote skin cell regeneration.

[0029] Component B comprises, by weight: 5-10 parts of myocardial peptide, 5-15 parts of amifostine thiol, 1-3 parts of amifostine disulfide, and 15-20 parts of polycaprolactone-protein conjugate.

[0030] In the present application, the concentration of myocardial peptide is 25 μg / mL-500 μg / mL, the concentration of amifostine thiol is 0.01 μM-250 μM, and the concentration of amifostine disulfide is 0.01 μM-25 μM. Among them, the concentration of myocardial peptide is preferably 50 μg / mL, the concentration of amifostine thiol is preferably 50 μM, and the concentration of amifostine disulfide is preferably 1 μM.

[0031] In the present application, the polycaprolactone-protein conjugate is the product of combining polycaprolactone nanoparticles with active proteins from skin fibroblasts. Polycaprolactone is an aliphatic polyester with poly-α-hydroxy acid groups, and is a synthetic polymer with biocompatibility and bioabsorbability; on the other hand, it is biocompatible with a variety of active ingredients from skin fibroblasts, which is beneficial for combining and retaining a variety of active ingredients and promoting a sustained-release effect. Biologically, polycaprolactone also has the function of promoting and inducing stem cells. Polycaprolactone (PCL) is a natural polymer that acts as a biomolecule carrier and scaffold. Some of its functions are used in the delivery of biomolecules and cells in tissue engineering, which can stimulate the regeneration of collagen stem cells, restore cellular immune expression, and enhance the skin's self-repair and regeneration capabilities.

[0032] Myocardial peptides are widely used in perioperative myocardial protection during cardiac surgery, and have significant protective effects in various injury models, increasing the activity of mitochondrial dehydrogenase in myocardial cells, protecting cell membrane integrity, and reducing the generation of free radicals. Amifostine has two sides, protecting normal cells from DNA damage and preventing DNA repair in cancer cells, especially the essential active metabolites of amifostine for cell protection - amifostine thiol and amifostine disulfide.

[0033] 3T3 cells in the logarithmic growth phase were cultured at 37°C with 5% CO 2 After culturing for 24 h under the culture conditions, the cells were divided into the following groups: a normal control group, and seven concentration gradient groups of myocardial peptide 500, 250, 100, 50, 25, 10, and 1 μg / mL, with 5 parallel wells in each group.

[0034] Mouse renal tubular epithelial cells in logarithmic growth phase were cultured at 37°C with 5% CO 2 After culturing for 24 h under culture conditions, the cells were grouped as follows: normal control group, seven concentration gradient groups of amifostine thiol at 250, 100, 50, 25, 10, 1, 0.1, and 0.01 μM, and eight concentration gradient groups of amifostine disulfide at 25, 10, 5, 2.5, 1, 0.5, 0.1, and 0.01 μM, with 5 parallel wells in each group.

[0035] The MTT method was used to detect the effect of myocardial peptide on the survival rate of 3T3 cells. The survival rates (%) corresponding to seven concentrations of myocardial peptide 500-1μg / mL were 118, 114, 118, 134, 115, 100, and 96, respectively. It can be seen that when the concentration of myocardial peptide was 25μg / mL-500μg / mL, it showed a significant effect in promoting the proliferation of 3T3 cells, among which the survival rate was the highest at 50μg / mL.

[0036] The MTT method was used to detect the effect of amifostine thiol on the survival rate of renal tubular epithelial cells. When the concentration of amifostine thiol was 250-0.01 μM, the survival rate (%) results were 63, 67, 112, 101, 100, 100, 101, and 97, respectively, indicating that when the concentration of amifostine thiol was 50 μM, it showed a significant effect of promoting the proliferation of renal tubular epithelial cells.

[0037] The MTT method was used to detect the effect of amifostine disulfide on the survival rate of renal tubular epithelial cells. When the concentration of amifostine disulfide was 25-0.01μM, the survival rate (%) results were 1, 1, 3, 103, 109, 99, 99, and 99, respectively, indicating that when the concentration of amifostine disulfide was 1μM, it showed a significant effect of promoting the proliferation of renal tubular epithelial cells.

[0038] PI staining and flow cytometry were used to detect the cell cycle distribution. The results showed that the administration of 50 μM amifostine thiol and 1 μM amifostine disulfide could significantly increase the cell distribution ratio of renal tubular epithelial cells in the S phase, which was manifested by an increase in the PI index and enhanced cell proliferation activity.

[0039] A method for preparing a cell vitality regeneration complex comprises the following steps:

[0040] Wash Baoxing Asplenium, dandelion, chicory root, and perilla leaf respectively, add water, grind and filter the residue to obtain Baoxing Asplenium filtrate, dandelion filtrate, chicory root filtrate, and perilla leaf filtrate, extract the filtrate to obtain four extracts, and mix the four extracts to obtain a mixed solution; wherein, Baoxing Asplenium, dandelion, chicory root, and perilla leaf are ground for 20 minutes, and the filter mesh used for filtering has an aperture of 5 μm;

[0041] Specifically, Baoxing Asplenium filtrate, dandelion filtrate, chicory root filtrate, and perilla leaf filtrate were respectively treated with ultra-critical CO 2 Extraction, extraction pressure is 26-28MPa, extraction temperature is 45-48℃, CO 2 Extraction flow rate is 50-55L·h -1The temperature of separation kettle I is 40-42°C and the pressure is 8-10MPa, the temperature of separation kettle II is 35-38°C and the pressure is 5-6MPa, and the obtained Baoxing Aspleniaceae, dandelion, chicory root and perilla leaf extracts are mixed to obtain a mixed solution.

[0042] Heat the mixture to boiling and keep it warm for 2-5 minutes, then add tea oil and stir. After stirring, cool to obtain component A solution;

[0043] The myocardial peptide, amifostine thiol and amifostine disulfide are fully dissolved and uniformly mixed to obtain a mixed solution, and then the polycaprolactone-protein conjugate is uniformly suspended in the mixed solution to obtain a component B solution.

[0044] The component A solution and the component B solution are mixed in a mass ratio of 5:1-1:5 to obtain a cell vitality regeneration complex.

[0045] In a possible embodiment, a gel solution is also included, and the gel solution is obtained by fully dissolving carboxymethyl cellulose, PBS buffer, and glycerol; the gel solution is added to the cell vitality regeneration complex to obtain a complex gel. The complex gel can be used as a component with a filling function in the injection composition to repair tissue loss caused by physical damage to the face; on the other hand, it can be used as a guide for the polycaprolactone protein conjugate to exert its efficacy.

[0046] Embodiment 1:

[0047] Preparation of component A solution: washing 15 parts of Baoxing Asplenium wilfordii, 15 parts of dandelion, 5 parts of chicory root, and 1 part of perilla leaf, respectively, adding water, grinding for 20 minutes, filtering the residue with a filter with a pore size of 5 μm to obtain Baoxing Asplenium wilfordii filtrate, dandelion filtrate, chicory root filtrate, and perilla leaf filtrate, extracting the aforementioned filtrates to obtain four extracts, mixing the four extracts to obtain a mixed solution; heating the mixed solution to boiling, keeping it warm for 2-5 minutes, adding tea oil, stirring, and cooling after stirring to obtain component A solution;

[0048] Prepare component B solution: fully dissolve 5 parts of myocardial peptide with a concentration of 25 μg / mL, 5 parts of amifostine thiol with a concentration of 0.01 μM, and 1 part of amifostine disulfide with a concentration of 0.01 μM, and mix them evenly to obtain a mixed solution, and then evenly suspend 15 parts of polycaprolactone-protein conjugate in the mixed solution to obtain component B solution.

[0049] Solution A and solution B were mixed at a mass ratio of 5:1 to obtain a cell vitality regeneration complex, which was recorded as sample 1.

[0050] Solution A and solution B were mixed at a mass ratio of 1:5 to obtain a cell vitality regeneration complex, which was recorded as control sample 1.

[0051] Embodiment 2:

[0052] Preparation of component A solution: washing 15 parts of Baoxing Asplenium wilfordii, 15 parts of dandelion, 5 parts of chicory root, and 1 part of perilla leaf, respectively, adding water, grinding for 20 minutes, filtering the residue with a filter with a pore size of 5 μm to obtain Baoxing Asplenium wilfordii filtrate, dandelion filtrate, chicory root filtrate, and perilla leaf filtrate, extracting the aforementioned filtrates to obtain four extracts, mixing the four extracts to obtain a mixed solution; heating the mixed solution to boiling, keeping it warm for 2-5 minutes, adding tea oil, stirring, and cooling after stirring to obtain component A solution;

[0053] Prepare component B solution: fully dissolve 5 parts of myocardial peptide with a concentration of 50 μg / mL, 5 parts of amifostine thiol with a concentration of 50 μM, and 1 part of amifostine disulfide with a concentration of 1 μM, and mix them evenly to obtain a mixed solution, and then evenly suspend 15 parts of polycaprolactone-protein conjugate in the mixed solution to obtain component B solution.

[0054] Solution A and solution B were mixed at a mass ratio of 5:1 to obtain a cell vitality regeneration complex, which was recorded as sample 2.

[0055] Solution A and solution B were mixed at a mass ratio of 1:5 to obtain a cell vitality regeneration complex, which was recorded as control sample 2.

[0056] Embodiment 3:

[0057] Preparation of component A solution: washing 15 parts of Baoxing Asplenium wilfordii, 15 parts of dandelion, 5 parts of chicory root, and 1 part of perilla leaf, respectively, adding water, grinding for 20 minutes, filtering the residue with a filter with a pore size of 5 μm to obtain Baoxing Asplenium wilfordii filtrate, dandelion filtrate, chicory root filtrate, and perilla leaf filtrate, extracting the aforementioned filtrates to obtain four extracts, mixing the four extracts to obtain a mixed solution; heating the mixed solution to boiling, keeping it warm for 2-5 minutes, adding tea oil, stirring, and cooling after stirring to obtain component A solution;

[0058] Prepare component B solution: fully dissolve 5 parts of myocardial peptide with a concentration of 100 μg / mL, 5 parts of amifostine thiol with a concentration of 100 μM, and 1 part of amifostine disulfide with a concentration of 2.5 μM, and mix them evenly to obtain a mixed solution, and then evenly suspend 15 parts of polycaprolactone-protein conjugate in the mixed solution to obtain component B solution.

[0059] Solution A and solution B were mixed at a mass ratio of 5:1 to obtain a cell vitality regeneration complex, which was recorded as sample 3.

[0060] Solution A and solution B were mixed at a mass ratio of 1:5 to obtain a cell vitality regeneration complex, which was recorded as comparative sample 3.

[0061] Verification experiment 1: Experimental verification was conducted on samples 1-3 and comparative samples 1-3 prepared in Examples 1-3: the effects of the drug on the survival rates of normal cells (NC) and senescent cells (SC) at different concentrations of solution B at the same mass ratio were compared. The selectivity of the drug for senescent cells is specifically reflected in the ratio of the survival rate of normal cells to the survival rate of senescent cells, i.e., NC / SC. When NC / SC>1, it has a targeted clearance effect.

[0062] Primary endothelial cells were isolated from rat renal tubules and used as normal cells (NC). MTT cell viability test was performed 48 hours after drug administration. NC lost its proliferation ability after multiple passages (3 days between passages) to obtain senescent cells (SC). MTT cell viability test was performed 48 hours after drug administration, and a blank control group was set up for SABG staining experiment to obtain cell senescence.

[0063] Table 1 below shows the effects of different complexes on cell viability in Examples 1-3:

[0064]

[0065]

[0066] It can be seen from Table 1 above that in samples 1-3 and comparative samples 1-3, the composite of sample 2 and comparative sample 2 in Example 2 can reduce the survival rate of senescent cells and increase the survival rate of normal cells. By comparing the data of sample 1 and comparative sample 1, and sample 2 and comparative sample 2, it can be concluded that when solution A and solution B are mixed in a mass ratio of 1:5, the effect is better.

[0067] Embodiment 4:

[0068] Preparation of component A solution: washing 20 parts of Baoxing Asplenium wilfordii, 25 parts of dandelion, 10 parts of chicory root, and 3 parts of perilla leaf, respectively, adding water, grinding for 20 minutes, filtering the residue with a filter screen with a pore size of 5 μm to obtain Baoxing Asplenium wilfordii filtrate, dandelion filtrate, chicory root filtrate, and perilla leaf filtrate, extracting the aforementioned filtrates to obtain four extracts, mixing the four extracts to obtain a mixed solution; heating the mixed solution to boiling, keeping it warm for 2-5 minutes, adding tea oil, stirring, and cooling after stirring to obtain component A solution;

[0069] Prepare component B solution: fully dissolve 8 parts of myocardial peptide with a concentration of 25 μg / mL, 10 parts of amifostine thiol with a concentration of 50.01 μM, and 2 parts of amifostine disulfide with a concentration of 0.01 μM, and mix them evenly to obtain a mixed solution, and then evenly suspend 18 parts of polycaprolactone-protein conjugate in the mixed solution to obtain component B solution.

[0070] Solution A and solution B were mixed at a mass ratio of 5:1 to obtain a cell vitality regeneration complex, which was recorded as sample 4.

[0071] Solution A and solution B were mixed at a mass ratio of 1:5 to obtain a cell vitality regeneration complex, which was recorded as control sample 4.

[0072] Embodiment 5:

[0073] Preparation of component A solution: washing 20 parts of Baoxing Asplenium wilfordii, 25 parts of dandelion, 10 parts of chicory root, and 3 parts of perilla leaf, respectively, adding water, grinding for 20 minutes, filtering the residue with a filter screen with a pore size of 5 μm to obtain Baoxing Asplenium wilfordii filtrate, dandelion filtrate, chicory root filtrate, and perilla leaf filtrate, extracting the aforementioned filtrates to obtain four extracts, mixing the four extracts to obtain a mixed solution; heating the mixed solution to boiling, keeping it warm for 2-5 minutes, adding tea oil, stirring, and cooling after stirring to obtain component A solution;

[0074] Prepare component B solution: fully dissolve 8 parts of myocardial peptide with a concentration of 50 μg / mL, 10 parts of amifostine thiol with a concentration of 50 μM, and 2 parts of amifostine disulfide with a concentration of 1 μM, and mix them evenly to obtain a mixed solution, and then evenly suspend 18 parts of polycaprolactone-protein conjugate in the mixed solution to obtain component B solution.

[0075] Solution A and solution B were mixed at a mass ratio of 5:1 to obtain a cell vitality regeneration complex, which was recorded as sample 5.

[0076] Solution A and solution B were mixed at a mass ratio of 1:5 to obtain a cell vitality regeneration complex, which was recorded as control sample 5.

[0077] Embodiment 6:

[0078] Preparation of component A solution: washing 20 parts of Baoxing Asplenium wilfordii, 25 parts of dandelion, 10 parts of chicory root, and 3 parts of perilla leaf, respectively, adding water, grinding for 20 minutes, filtering the residue with a filter screen with a pore size of 5 μm to obtain Baoxing Asplenium wilfordii filtrate, dandelion filtrate, chicory root filtrate, and perilla leaf filtrate, extracting the aforementioned filtrates to obtain four extracts, mixing the four extracts to obtain a mixed solution; heating the mixed solution to boiling, keeping it warm for 2-5 minutes, adding tea oil, stirring, and cooling after stirring to obtain component A solution;

[0079] Prepare component B solution: fully dissolve 8 parts of myocardial peptide with a concentration of 100 μg / mL, 10 parts of amifostine thiol with a concentration of 100 μM, and 2 parts of amifostine disulfide with a concentration of 2.5 μM, and mix them evenly to obtain a mixed solution, and then evenly suspend 18 parts of polycaprolactone-protein conjugate in the mixed solution to obtain component B solution.

[0080] Solution A and solution B were mixed at a mass ratio of 5:1 to obtain a cell vitality regeneration complex, which was recorded as sample 6.

[0081] Solution A and solution B were mixed at a mass ratio of 1:5 to obtain a cell vitality regeneration complex, which was recorded as control sample 6.

[0082] Verification experiment 2: Experimental verification was performed on sample 4-6 and control sample 4-6 prepared in Example 4-6: the effects of the drug at different concentrations of solution B at the same mass ratio on the survival rates of normal cells (NC) and senescent cells (SC) were compared.

[0083] Table 2 below shows the effects of different complexes on cell viability in Examples 4-6:

[0084] Compound SC survival rate (%) NC survival rate (%) NC survival rate / SC survival rate Blank control group 92 92 1.000 Sample 4 78 93 1.192 Sample 5 50 95 1.900 Sample 6 63 94 1.492 Comparison sample 4 74 93 1.257 Comparison sample 5 45 97 2.156 Comparison sample 6 61 93 1.524

[0085] It can be seen from Table 2 above that the composite prepared in Example 5 has a better effect than the composites of Examples 4 and 6. At the same time, by comparing the data of Sample 4 and Comparative Sample 4, Sample 4 and Comparative Sample 5, and Sample 6 and Comparative Sample 6, it can be concluded that when Solution A and Solution B are mixed in a mass ratio of 1:5, the effect is relatively better.

[0086] Embodiment 7:

[0087] Preparation of component A solution: washing 25 parts of Baoxing Asplenium tiliaceum, 35 parts of dandelion, 15 parts of chicory root, and 5 parts of perilla leaf, respectively, adding water, grinding for 20 minutes, filtering the residue with a filter screen with a pore size of 5 μm to obtain Baoxing Asplenium tiliaceum filtrate, dandelion filtrate, chicory root filtrate, and perilla leaf filtrate, respectively extracting the aforementioned filtrates to obtain four extracts, and mixing the four extracts to obtain a mixed solution; heating the mixed solution to boiling, and keeping it warm for 2-5 minutes, adding tea oil, and stirring, and cooling after stirring to obtain a component A solution;

[0088] Prepare component B solution: fully dissolve 10 parts of myocardial peptide with a concentration of 25 μg / mL, 15 parts of amifostine thiol with a concentration of 0.01 μM, and 3 parts of amifostine disulfide with a concentration of 0.01 μM, and mix them evenly to obtain a mixed solution, and then evenly suspend 20 parts of polycaprolactone-protein conjugate in the mixed solution to obtain component B solution.

[0089] Solution A and solution B were mixed at a mass ratio of 5:1 to obtain a cell vitality regeneration complex, which was recorded as sample 7.

[0090] Solution A and solution B were mixed at a mass ratio of 1:5 to obtain a cell vitality regeneration complex, which was recorded as control sample 7.

[0091] Embodiment 8:

[0092] Preparation of component A solution: washing 25 parts of Baoxing Asplenium tiliaceum, 35 parts of dandelion, 15 parts of chicory root, and 5 parts of perilla leaf, respectively, adding water, grinding for 20 minutes, filtering the residue with a filter screen with a pore size of 5 μm to obtain Baoxing Asplenium tiliaceum filtrate, dandelion filtrate, chicory root filtrate, and perilla leaf filtrate, respectively extracting the aforementioned filtrates to obtain four extracts, and mixing the four extracts to obtain a mixed solution; heating the mixed solution to boiling, and keeping it warm for 2-5 minutes, adding tea oil, and stirring, and cooling after stirring to obtain a component A solution;

[0093] Prepare component B solution: fully dissolve 10 parts of myocardial peptide with a concentration of 50 μg / mL, 15 parts of amifostine thiol with a concentration of 50 μM, and 3 parts of amifostine disulfide with a concentration of 1 μM, and mix them evenly to obtain a mixed solution, and then evenly suspend 20 parts of polycaprolactone-protein conjugate in the mixed solution to obtain component B solution.

[0094] Solution A and solution B were mixed at a mass ratio of 5:1 to obtain a cell vitality regeneration complex, which was recorded as sample 8.

[0095] Solution A and solution B were mixed at a mass ratio of 1:5 to obtain a cell vitality regeneration complex, which was recorded as control sample 8.

[0096] Embodiment 9:

[0097] Preparation of component A solution: washing 25 parts of Baoxing Asplenium tiliaceum, 35 parts of dandelion, 15 parts of chicory root, and 5 parts of perilla leaf, respectively, adding water, grinding for 20 minutes, filtering the residue with a filter screen with a pore size of 5 μm to obtain Baoxing Asplenium tiliaceum filtrate, dandelion filtrate, chicory root filtrate, and perilla leaf filtrate, respectively extracting the aforementioned filtrates to obtain four extracts, and mixing the four extracts to obtain a mixed solution; heating the mixed solution to boiling, and keeping it warm for 2-5 minutes, adding tea oil, and stirring, and cooling after stirring to obtain a component A solution;

[0098] Prepare component B solution: fully dissolve 10 parts of myocardial peptide with a concentration of 100 μg / mL, 15 parts of amifostine thiol with a concentration of 100 μM, and 3 parts of amifostine disulfide with a concentration of 3 μM, and mix them evenly to obtain a mixed solution, and then evenly suspend 20 parts of polycaprolactone-protein conjugate in the mixed solution to obtain component B solution.

[0099] Solution A and solution B were mixed at a mass ratio of 5:1 to obtain a cell vitality regeneration complex, which was recorded as sample 9.

[0100] Solution A and solution B were mixed at a mass ratio of 1:5 to obtain a cell vitality regeneration complex, which was recorded as control sample 9.

[0101] Verification experiment 3: Experimental verification was performed on sample 7-9 and control sample 7-9 prepared in Example 7-9: the effects of the drug at different concentrations of solution B at the same mass ratio on the survival rates of normal cells (NC) and senescent cells (SC) were compared.

[0102] Table 3 below shows the effects of different complexes on cell viability in Examples 7-9:

[0103]

[0104]

[0105] It can be seen from Table 3 above that the composite prepared in Example 8 has better effect than the composites of Examples 7 and 9. At the same time, by comparing the data of Sample 7 and Comparative Sample 7, Sample 8 and Comparative Sample 8, and Sample 9 and Comparative Sample 9, it can be concluded that when Solution A and Solution B are mixed in a mass ratio of 1:5, the effect is relatively better.

[0106] Through three sets of experimental verification, it can be concluded from the data in Tables 1-3 that when the concentration of myocardial peptide is 50μg / mL, the concentration of amifostine thiol is 50μM, and the concentration of amifostine disulfide is 1μM, the prepared complex has a good effect on reducing the survival rate of senescent cells and increasing the survival rate of normal cells.

[0107] Among them, Example 1, Example 4, Example 7; Example 2, Example 5, Example 8; Example 3, Example 6, Example 9; are three groups of complexes with the same concentration but different mass proportions. It can be concluded from the data in Tables 1-3 that the complexes prepared with different mass proportions have slightly different effects. When the A solution contains 25 parts of dandelion, 10 parts of chicory root, 3 parts of perilla leaf, and 20 parts of Aspleniaceae, and the B solution contains 8 parts of myocardial peptide, 10 parts of amifostine thiol, and 2 parts of amifostine disulfide, the complexes prepared by solution A and solution B have the best effect.

[0108] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A cell vitality regeneration complex, characterized in that: The invention comprises component A and component B, wherein the mass ratio of component A to component B is 5:1-1:5; The component A comprises, by weight: 15-25 parts of Aspleniaceae, 15-35 parts of dandelion, 5-15 parts of chicory root, 1-5 parts of perilla leaf, and 2-5 parts of tea oil; The component B comprises, by weight: 5-10 parts of myocardial peptide, 5-15 parts of amifostine thiol, 1-3 parts of amifostine disulfide, and 15-20 parts of polycaprolactone-protein conjugate.

2. A cell vitality regeneration complex according to claim 1, characterized in that: The dandelion is preferably 25 parts, the chicory root is preferably 10 parts, and the perilla leaf is preferably 3 parts.

3. The cell vitality regeneration complex according to claim 1, characterized in that: The concentration of myocardial peptide is 25 μg / mL-500 μg / mL, the concentration of amifostine thiol is 0.01 μM-250 μM, and the concentration of amifostine disulfide is 0.01 μM-25 μM.

4. A cell vitality regeneration complex according to claim 3, characterized in that: The concentration of myocardial peptide is preferably 50 μg / mL, the concentration of amifostine thiol is preferably 50 μM, and the concentration of amifostine disulfide is preferably 1 μM.

5. The cell vitality regeneration complex according to claim 1, characterized in that: The polycaprolactone-protein conjugate is a product obtained by combining polycaprolactone nanoparticles with active proteins from skin fibroblasts.

6. A method for preparing a cell vitality regeneration complex according to any one of claims 1 to 5, characterized in that: The steps include: Wash Baoxing Asplenium, dandelion, chicory root, and perilla leaf respectively, add water, grind and filter the residue to obtain Baoxing Asplenium filtrate, dandelion filtrate, chicory root filtrate, and perilla leaf filtrate, extract the filtrate to obtain four extracts, and mix the four extracts to obtain a mixed solution; Heat the mixture to boiling and keep it warm for 2-5 minutes, then add tea oil and stir. After stirring, cool to obtain component A solution; Fully dissolving myocardial peptide, amifostine thiol and amifostine disulfide, and uniformly mixing them to obtain a mixed solution, and then uniformly suspending the polycaprolactone-protein conjugate in the mixed solution to obtain a component B solution; The component A solution and the component B solution are mixed in a mass ratio of 5:1-1:5 to obtain a cell vitality regeneration complex.

7. The method for preparing a cell vitality regeneration complex according to claim 6, characterized in that: The Baoxing Asplenium, dandelion, chicory root and perilla leaf are ground for 20 minutes, and the filter mesh used for filtering has an aperture of 5 μm.

8. The method for preparing a cell vitality regeneration complex according to claim 6, characterized in that: The Baoxing Asplenium filtrate, dandelion filtrate, chicory root filtrate and Perilla leaf filtrate are respectively extracted by supercritical CO2, with an extraction pressure of 26-28MPa, an extraction temperature of 45-48°C, and a CO2 extraction flow rate of 50-55L·h -1 The temperature of separation kettle I is 40-42°C and the pressure is 8-10MPa, the temperature of separation kettle II is 35-38°C and the pressure is 5-6MPa, and the obtained Baoxing Aspleniaceae, dandelion, chicory root and perilla leaf extracts are mixed to obtain a mixed solution.

9. The method for preparing a cell vitality regeneration complex according to claim 6, characterized in that: Also included is a gel solution, which is obtained by fully dissolving carboxymethyl cellulose, PBS buffer and glycerol; The gel solution is added to the cell vitality regeneration complex to obtain a complex gel.

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