Freeze-dried flash-release tablet containing stem cell extract as well as preparation method and application of freeze-dried flash-release tablet

Abstract

The invention relates to the technical field of cell engineering, and particularly discloses a freeze-dried flash release tablet containing a stem cell extract as well as a preparation method and application of the freeze-dried flash release tablet. The freeze-dried flash release tablet containing the stem cell extract provided by the invention is prepared from the following components in parts by weight: 0.5 to 2 parts of the stem cell extract, 0.5 to 1 part of guar gum, 4 to 8 parts of trehalose, 0.5 to 1 part of nicotinamide, 1.5 to 3 parts of glycine and 5 to 10 parts of corn starch, the preparation method of the stem cell extract comprises the following steps: extracting stem cells from an umbilical cord, establishing an immortalized stem cell line, and preparing immortalized overexpressed SIRT6 stem cell outer vesicles. The freeze-dried flash-release tablet containing the stem cell extract provided by the invention has the effects of resisting aging, enhancing immunity, improving sleep quality and the like, and has a good health-care effect on human health.

Description

Technical Field

[0001] The present application relates to the field of cell engineering technology, and in particular to a freeze-dried flash-release tablet containing stem cell extracts, and a preparation method and application thereof. Background Art

[0002] With the healthy development of my country's social economy, the life expectancy of Chinese people has been continuously extended, and we have gradually entered an aging society. Products with functional functions such as anti-aging, anti-fatigue, immunity enhancement, and sleep improvement are attracting more and more attention.

[0003] As a natural resource rich in bioactive ingredients, umbilical cord holds broad application prospects in medicine and skincare. The active ingredients in umbilical cord include stem cells and exosomes. Exosomes are tiny vesicles with a double-layer lipid membrane produced by stem cells. As crucial mediators of cellular communication, exosomes can carry proteins, RNA, and other biomacromolecules, and participate in regulating various physiological and pathological processes within the body. They promote cell regeneration and regulate immune function, thus showing great potential for anti-aging, immunity enhancement, and improved sleep quality.

[0004] Therefore, how to efficiently extract the active ingredients in the umbilical cord and make them into easy-to-use pharmaceutical dosage forms is an urgent problem that needs to be solved. Summary of the Invention

[0005] In order to efficiently utilize the active ingredients in the umbilical cord and prepare them into a pharmaceutical dosage form that is easy to use, the present application provides a freeze-dried flash-release tablet containing stem cell extracts, and a preparation method and application thereof.

[0006] In a first aspect, the present application provides a freeze-dried flash-release tablet containing stem cell extract, which adopts the following technical solution: a freeze-dried flash-release tablet containing stem cell extract, comprising the following components in parts by weight: 0.5-2 parts of stem cell extract, 0.5-1 parts of guar gum, 4-8 parts of trehalose, 0.5-1 parts of niacinamide, 1.5-3 parts of glycine and 5-10 parts of corn starch.

[0007] The present application provides a freeze-dried flash-release tablet containing stem cell extract. The freeze-dried flash-release tablet uses stem cell extract as the core active ingredient, which has the effects of anti-aging, enhancing immunity and improving sleep quality; the addition of guar gum and trehalose can not only enhance the structural stability of the freeze-dried flash-release tablet, but also improve the hygroscopicity and dissolution rate of the product; the combination of niacinamide and glycine can enhance the antioxidant properties and cell repair ability of the freeze-dried flash-release tablet; the addition of corn starch optimizes the molding properties of the freeze-dried flash-release tablet, ensuring the appearance quality and ease of use of the product. By controlling the weight of each component within the above range, the freeze-dried flash-release tablet prepared in the present application can exert the synergistic effect of each component, significantly improve the stability and bioavailability of the product, and when used on the human body, it can exert its effects of anti-aging, enhancing immunity and improving sleep quality, and has a good health care effect on human health.

[0008] Optionally, the weight portion of the stem cell extract is 1-1.8 parts.

[0009] In some embodiments, the weight portion of the stem cell extract can be 0.5-1 part, 0.5-1.5 parts, 0.5-1.8 parts, 1-1.5 parts, 1-1.8 parts, 1-2 parts, 1.5-1.8 parts, 1.5-2 parts, or 1.8-2 parts.

[0010] In a specific embodiment, the weight portion of the stem cell extract can also be 0.5 parts, 1 parts, 1.5 parts, 1.8 parts or 2 parts.

[0011] Optionally, the method for preparing the stem cell extract comprises the following steps: extracting stem cells from sheep umbilical cord tissue, establishing an immortalized stem cell line, and preparing immortalized SIRT6-overexpressing stem cell extracellular vesicles; Establishing an immortalized stem cell line: hUCMSCs were seeded onto culture plates, followed by the addition of TERT lentivirus and αMEM complete medium. The cells were mixed thoroughly and placed in a CO2 incubator. After 6 hours, Polybrene and αMEM complete medium were added, and the cells were cultured in a CO2 incubator for 48 ± 2 hours. The αMEM complete medium containing puromycin was then replaced for selection and expansion to obtain immortalized stem cells. Preparation of immortalized stem cell extracellular vesicles overexpressing SIRT6: Immortalized stem cells were seeded into culture plates, and SIRT6 lentivirus, complete culture medium, and cationic polymer were added in sequence for 48±2h; then, complete culture medium containing blasticidin S was replaced for screening and amplification. No antibiotics were added after 5 passages; when the degree of confluence reached more than 70%, the supernatant was removed; the precipitate was washed with PBS, and αMEM complete culture medium was added for 48±2h. The supernatant was collected in a centrifuge tube and centrifuged to obtain immortalized stem cell extracellular vesicles overexpressing SIRT6, which is the stem cell extract. Optionally, in the step of establishing the immortalized stem cell line, the TERT lentivirus titer is 8×10 8 TU / ml, the volume ratio of TERT lentivirus and DMEM / F12 culture medium was 1:(33-38).

[0012] Optionally, the concentration of puromycin in the DMEM / F12 culture medium containing puromycin is 3-5 μg / mL.

[0013] Optionally, in the step of establishing the immortalized stem cell line, when adding Polybrene and DMEM / F12 culture medium, the final concentration of Polybrene is maintained at 5-7 μg / mL.

[0014] Optionally, in the step of preparing immortalized SIRT6-overexpressing stem cell extracellular vesicles, the specific steps of centrifugation are: centrifuging the culture supernatant containing immortalized SIRT6-overexpressing stem cell extracellular vesicles at 4°C and 2000×g for 102 minutes, and taking supernatant ①; centrifuging supernatant ① at 4°C and 1000×g for 30 minutes, and taking supernatant ②; ultracentrifuging supernatant ② at 4°C and 110,000×g for 70 minutes, discarding the supernatant and taking the precipitate, adding PBS to the precipitate to resuspend it, ultracentrifuging it at 4°C and 110,000×g for 70 minutes, taking the precipitate, and obtaining immortalized SIRT6-overexpressing stem cell extracellular vesicles (stem cell extract).

[0015] In a second aspect, the present application provides a method for preparing a freeze-dried flash-release tablet, comprising the following steps: first, dispersing the stem cell extract in water, then adding guar gum, trehalose, glycine, nicotinamide and corn starch to obtain a freeze-dried liquid containing stem cell extracellular vesicles; the above-mentioned freeze-dried liquid is divided into molds, vacuum freeze-drying, and direct compression molding to obtain freeze-dried flash-release tablets.

[0016] Optionally, the vacuum freeze-drying process is: rapid freezing at -80°C for 2 hours, followed by vacuum drying at -40°C and 10 Pa for 24 hours, and then vacuum drying at 25°C and 5 Pa for 6 hours.

[0017] In a third aspect, the present application provides the use of freeze-dried flash-release tablets containing stem cell extracts in the preparation of anti-aging, immunity-enhancing and sleep quality-improving drugs.

[0018] In summary, this application has the following beneficial effects: 1. This application uses stem cell extract to prepare freeze-dried flash-release tablets, and the weight of the stem cell extract is controlled within the range of 0.5-2 parts. The freeze-dried flash-release tablets prepared have the effects of anti-aging, enhancing immunity and improving sleep quality, and have a good health care effect on human health.

[0019] 2. In this application, by further controlling the weight of the stem cell extract within the range of 1-1.8 parts, the freeze-dried flash-release tablets obtained have a better effect on improving the immune function and sleep quality of mice. DETAILED DESCRIPTION

[0020] The present application provides a freeze-dried flash-release tablet containing a stem cell extract, comprising the following components in parts by weight: 0.5-2 parts of stem cell extract, 0.5-1 parts of guar gum, 4-8 parts of trehalose, 0.5-1 parts of niacinamide, 1.5-3 parts of glycine, 5-10 parts of corn starch, and 80-100 parts of water; further, the weight portion of the stem cell extract is 1-1.8 parts.

[0021] The present application provides a method for preparing a freeze-dried flash-release tablet of a stem cell extract, comprising the following steps: (1) Extraction of stem cells from sheep umbilical cord: Fresh sheep umbilical cord tissue was pretreated and then minced into small pieces of tissue with a volume of approximately 1 mm × 1 mm × 1 mm. Fresh αMEM medium containing 10% FBS was used for culture at 37°C and 5% CO2, and the medium was changed every 2 to 3 days. When the cell confluence reached more than 80%, digestion was performed using digestive enzyme (Thermo Scientific). When the cells became round, αMEM complete medium was added with a pipette to terminate the digestion. Then, the cells were passaged every 3 to 4 days in αMEM complete medium to obtain mesenchymal stem cells.

[0022] (2) Establishment of immortalized stem cell lines: The extracted stem cells were inoculated into culture plates, and then TERT lentivirus and αMEM complete medium were added, mixed and placed in a CO2 constant temperature incubator; after 6 hours, Polybrene and αMEM complete medium were added, and cultured in a CO2 constant temperature incubator for 48±2h; then, αMEM complete medium containing puromycin was replaced for screening and amplification to obtain immortalized stem cells.

[0023] (3) Preparation of extracellular vesicles of immortalized stem cells overexpressing SIRT6: Preparation of extracellular vesicles of immortalized stem cells overexpressing SIRT6: Immortalized stem cells were seeded into culture plates, and SIRT6 lentivirus, complete culture medium, and cationic polymer were added in sequence, and cultured for 48±2h; then, complete culture medium containing blasticidin S was replaced for screening and amplification. After 5 generations, antibiotics were no longer added; when the degree of fusion reached more than 70%, the supernatant was removed; the precipitate was washed with PBS, and αMEM complete culture medium was added and cultured for 48±2h. The supernatant was placed in a centrifuge tube and centrifuged to obtain immortalized extracellular vesicles of stem cells overexpressing SIRT6, which is the stem cell extract.

[0024] (4) Preparation of freeze-dried flash-release tablets: First, the stem cell extract is dispersed in water, and then guar gum, trehalose, glycine, nicotinamide and corn starch are added to obtain a freeze-dried solution containing stem cell extracellular vesicles; the freeze-dried solution is divided into molds, rapidly frozen at -80°C for 2 hours, and then vacuum-dried at -40°C and 10 Pa for 24 hours, followed by vacuum drying at 25°C and 5 Pa for 6 hours, and then pressed into shape by direct compression to obtain freeze-dried flash-release tablets; The fresh sheep umbilical cord tissue used in the examples of this application was purchased from Tianjin Bainong Experimental Animal Breeding Technology Co., Ltd. The clinical examination report of the specific viruses (peste des petits ruminants, foot-and-mouth disease and brucellosis) of the sheep umbilical cord was negative; the raw materials, reagents, solvents, etc. used in this application can all be obtained commercially.

[0025] The present application is further described in detail below with reference to preparation examples, embodiments, and performance testing.

[0026] Preparation Example 1 Preparation Example 1 provides a stem cell extract.

[0027] The above-mentioned stem cell extract is an immortalized stem cell extracellular vesicle that overexpresses SIRT6, and its preparation method comprises the following steps: (1) Extraction of stem cells from sheep umbilical cord tissue: After disinfecting the surface of the sheep umbilical cord tissue sample collection bag with 75% alcohol, move it into a biosafety cabinet; open the sample collection bag and use sterile tweezers to transfer the tissue sample from the collection bag to a centrifuge tube (if the umbilical cord is too long, it can be cut appropriately); rinse the sheep umbilical cord tissue alternately with 75% alcohol and PBS (1% penicillin-streptomycin), repeat once; use tweezers to transfer the rinsed sheep umbilical cord tissue from the centrifuge tube to a culture dish, use sterile scissors to cut off the tied ends of the umbilical cord, and cut the remaining middle section evenly, each section is 2-3 cm long; longitudinally cut the sample into small sections along the umbilical vein, remove the venous endothelium and two umbilical arteries, and peel off Wharton's jelly; use sterile tweezers to transfer the collected Wharton's jelly to a centrifuge tube, add PBS (containing 1% penicillin-streptomycin, Thermo Fisher Scientific) and add PBS (containing 1% penicillin-streptomycin, Thermo Fisher Scientific). After the supernatant was discarded with a pipette, the Wharton's gel was cut into small tissue pieces of approximately 1 mm × 1 mm × 1 mm using sterile scissors. The pieces were then placed in a 15 cm cell culture dish and fresh αMEM medium (containing 10% FBS) was slowly added. The dish was covered and placed in a CO2 constant temperature incubator (37°C, 5% CO2) for culture, with the medium changed every 2 to 3 days. When the cell confluence reached more than 80%, the culture supernatant was discarded. PBS was added using a pipette, the container was shaken, and the cells were centrifuged at 300 g for 5 min. The washing solution was discarded, and the digestion enzyme (Thermo Fisher Scientific) was pipetted with a pipette. Scientific) into the culture container and shake rapidly; observe under a microscope, and when the cells become round, add αMEM complete medium with a pipette to terminate digestion, centrifuge at 300g for 5 minutes; aspirate and discard the supernatant, add PBS with a pipette, and centrifuge at 300g for 5 minutes; remove the centrifuge tube, aspirate and discard the supernatant, evenly pipette the cell pellet, add αMEM complete medium, mix well, and then inoculate into a culture flask; place in a CO2 cell culture incubator (5% CO2, 37°C), and passage every 3-4 days to obtain mesenchymal stem cells.

[0028] (2) Establishment of immortalized stem cell lines: P2 hUCMSCs were harvested and seeded into 6-well culture plates, with 3.5 × 10 cells seeded per well. 5 The confluence of cells was about 70% when plating, and the volume of culture medium in each well was about 2 mL. The next day, TERT lentivirus (Shandong Weizhen) was taken out at -80°C and thawed in an ice bath. The titer of TERT lentivirus was 8×10 8TU / ml, the volume of lentivirus added to each well is 35 μL (MOI value is 80), 800 μL of αMEM complete medium is added to each well, mixed and placed in a carbon dioxide incubator; 6 hours later, 1200 μL of αMEM complete medium and cationic polymer are added, and the final concentration of the cationic polymer is 6 μg / mL. After mixing, the cells are placed in a CO2 constant temperature incubator at 37°C and 5% CO2 and incubated overnight; 48 hours after the addition of lentivirus, αMEM complete culture medium containing 4 μg / mL α-puromycin (Thermo Scientific) is replaced and cultured for 48 hours; 96 hours after the addition of lentivirus, the α-puromycin concentration is reduced to 2 μg / mL, and the infected cells are continued to be screened and amplified; the cells selected after α-puromycin are passaged, and 2 μg / mL α-puromycin is continued to be added to maintain resistance. After continuous screening and passage for 3 times, stem cells are obtained and frozen in liquid nitrogen.

[0029] (3) Preparation of extracellular vesicles of immortalized stem cells overexpressing SIRT6: Immortalized stem cells were revived and seeded into 6-well culture plates, with 3.5×10 cells seeded into each well. 5 The confluence of cells was about 70% when plating, and the volume of culture medium in each well was 2 mL. The next day, SIRT6 lentivirus (Shandong Weizhen) was taken out at -80°C and thawed in an ice bath. The titer of SIRT6 lentivirus was 1.12×10 9TU / ml, the volume of lentivirus added to each well was 23 μL (MOI value was 80), 800 μL of αMEM complete medium was added to each well, mixed and placed in a carbon dioxide incubator; 6 hours later, 1200 μL of αMEM complete medium and cationic polymer were added, the final concentration of cationic polymer was 6 μg / mL, mixed and placed in a CO2 constant temperature incubator at 37°C and 5% CO2 for overnight incubation; 48 hours after adding lentivirus, the medium containing 2 μg / mL blasticidin S (Thermo Scientific) αMEM complete culture medium was added and cultured for 48 hours to obtain P0 generation S6-hUCMSCs; 96 hours after the addition of lentivirus, the concentration of blasticidin S was reduced to 1 μg / mL, and the infected cells were further screened and amplified. After 5 passages, antibiotics were no longer added: the immortalized stem cells were cultured until the confluence reached more than 70%, the culture supernatant was aspirated, the cells were washed twice with PBS, and αMEM complete culture medium was added. After 48 hours, the culture supernatant was collected in a centrifuge tube to obtain the culture supernatant containing extracellular vesicles of immortalized stem cells overexpressing SIRT6. The culture supernatant containing immortalized SIRT6-overexpressing stem cell extracellular vesicles was centrifuged at 4°C and 2000×g for 102 minutes, and supernatant ① was taken; supernatant ① was centrifuged at 4°C and 1000×g for 30 minutes, and supernatant ② was taken; supernatant ② was ultracentrifuged at 4°C and 110,000×g for 70 minutes, the supernatant was discarded, and the precipitate was taken. PBS was added to the precipitate for resuspending, and ultracentrifuged at 4°C and 110,000×g for 70 minutes. The precipitate was taken to obtain immortalized SIRT6-overexpressing stem cell extracellular vesicles, i.e., stem cell extract.

[0030] Example 1 Example 1 provides a freeze-dried flash-release tablet containing a stem cell extract.

[0031] The preparation method of the above-mentioned freeze-dried flash-release tablet containing stem cell extract comprises the following steps: first, dispersing 1.5 g of the stem cell extract provided in Preparation Example 1 in 90 g of water, and then adding 0.8 g of guar gum, 5 g of trehalose, 0.8 g of nicotinamide, 2 g of glycine and 8 g of corn starch to obtain a freeze-dried liquid containing stem cell extracellular vesicles; the above-mentioned freeze-dried liquid is divided into molds, rapidly frozen at -80°C for 2 hours, and then vacuum-dried at -40°C and 10 Pa for 24 hours, followed by vacuum drying at 25°C and 5 Pa for 6 hours, and then pressed into shape by direct compression to obtain freeze-dried flash-release tablets with a diameter of 1 cm and a thickness of 0.3 cm.

[0032] Examples 2-5 Examples 2-5 each provide a freeze-dried flash-release tablet containing a stem cell extract.

[0033] The difference between the above embodiments is the amount of stem cell extract added, as shown in Table 1 below.

[0034] Table 1 Addition amount of stem cell extract in Examples 2-5 Example Amount of stem cell extract added (g) 2 0.5 3 1 4 1.8 5 2 Example 6 Example 6 provides a freeze-dried flash-release tablet comprising a stem cell extract.

[0035] The difference between the above embodiment and embodiment 1 is that the vacuum freeze-drying process is: vacuum drying at -40°C and 10 Pa for 30 hours, followed by vacuum drying at 25°C and 5 Pa for 6 hours.

[0036] Comparative Example 1 Comparative Example 1 provides a freeze-dried flash-release tablet containing a stem cell extract.

[0037] The difference between the above embodiment and embodiment 1 is that the amount of stem cell extract added is 0.2 g.

[0038] Performance testing Various performance tests were performed on the freeze-dried flash-release tablets containing stem cell extracts provided in Examples 1-6 and Comparative Example 1. The results are shown in Table 2 below.

[0039] (1) Dissolution rate study: The freeze-dried flash-release tablet sample was placed in a conical flask containing 100 mL of room temperature water, and then shaken in a shaker at a speed of 150 r / min. The time required for the product to completely dissolve was calculated.

[0040] (2) Immune function: 40 NIH mice weighing 18-22 g were randomly divided into 8 groups (test groups 1-6, control group, and blank control group). The mice were administered 2 g / kg by oral gavage (test groups 1-6 were administered the freeze-dried flash-release tablets of Examples 1-6, the control group was administered the freeze-dried flash-release tablets of Comparative Example 1, and the blank control group was not administered). The administration was continued once a day for 15 consecutive days. On the 9th day after administration, each group of mice was immunized by intraperitoneal injection of 0.2 ml of 0.5% chicken RBC saline. Seven days after immunization, blood was collected from the orbital venous plexus, and the hemolysin content of each group of mice was measured by the hemolysin assay method using chicken RBC as the immunogen. The higher the hemolysin content, the better the humoral immune function of the mice.

[0041] (3) Sleeping test: 80 mice were randomly divided into 8 groups (test groups 1-6, comparison group and blank control group), with 20 mice in each group. All groups were intraperitoneally injected with 50 mg / kg of sodium pentobarbital. When the mice woke up from sleep, they were immediately gavaged with drugs at a dose of 2 g / kg (test groups 1-6 were administered with the freeze-dried flash-release tablets of Example 1-6, the comparison group was administered with the freeze-dried flash-release tablets of Comparative Example 1, and the blank control group was not administered with drugs). The mice were observed to see if they fell asleep again within 30 minutes, and the number of mice that fell asleep again was counted.

[0042] Table 2 Performance test results of each freeze-dried flash-release tablet According to the test results in Table 2, the dissolution time of the freeze-dried flash-release tablets containing stem cell extracts prepared in Examples 1-6 of the present application is less than 3 minutes. After being administered to mice, the hemolysin content of the mice is 0.561-0.902, and the number of mice that fall asleep again is 5-8; while after the freeze-dried flash-release tablets containing stem cell extracts provided in Comparative Example 1 are administered to mice, the hemolysin content of the mice is only 0.433, and the number of mice that fall asleep again is only 3. Therefore, it is shown that the present application uses stem cell extracts to prepare freeze-dried flash-release tablets, and controls the weight of the stem cell extract within the range of 0.5-2 parts, which can significantly improve the immune function and sleep quality of mice. Further comparison shows that after the stem cell extracts prepared in Examples 1 and Examples 3-4 are administered to mice, the hemolysin content of the mice is as high as 0.814-0.902, and the number of mice that fall asleep again is 7-8, indicating that the freeze-dried flash-release tablets obtained by controlling the weight of the stem cell extract within the range of 1-1.8 parts in the present application have a better effect on improving the immune function and sleep quality of mice.

[0043] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A freeze-dried flash-release tablet comprising a stem cell extract, characterized in that: The invention comprises the following components in parts by weight: 0.5-2 parts of stem cell extract, 0.5-1 parts of guar gum, 4-8 parts of trehalose, 0.5-1 parts of nicotinamide, 1.5-3 parts of glycine and 5-10 parts of corn starch.

2. The freeze-dried flash-release tablet containing stem cell extract according to claim 1, characterized in that The weight portion of the stem cell extract is 1-1.8 parts.

3. The freeze-dried flash-release tablet containing stem cell extract according to claim 1, characterized in that The method for preparing the stem cell extract comprises the following steps: extracting stem cells from sheep umbilical cord tissue, establishing an immortalized stem cell line, and preparing immortalized SIRT6-overexpressing stem cell extracellular vesicles; Establishing immortalized stem cell lines: Extracted stem cells were plated onto culture plates, followed by the addition of TERT lentivirus and αMEM complete medium. Mix well and place in a CO2 incubator. After 6 hours, Polybrene and αMEM complete medium were added, and the cells were cultured in a CO2 incubator for 48 ± 2 hours. The αMEM complete medium containing puromycin was then replaced for selection and expansion to obtain immortalized stem cells. Preparation of extracellular vesicles of immortalized stem cells overexpressing SIRT6: Immortalized stem cells were seeded into culture plates, and SIRT6 lentivirus, complete culture medium, and cationic polymer were added in sequence for 48±2 hours. Then, complete culture medium containing blasticidin S was replaced for screening and amplification. After 5 passages, antibiotics were no longer added. When the degree of confluence reached more than 70%, the supernatant was removed. The precipitate was washed with PBS and added with αMEM complete culture medium for 48±2 hours. The supernatant was collected and placed in a centrifuge tube. After centrifugation, the immortalized stem cell overexpressing SIRT6 extracellular vesicles, i.e., the stem cell extract, were obtained.

4. The freeze-dried flash-release tablet containing stem cell extract according to claim 3, characterized in that In the step of establishing the immortalized stem cell line, the TERT lentivirus titer is 8×10 8 TU / ml, the volume ratio of TERT lentivirus and αMEM complete culture medium is 1: (33-38).

5. The freeze-dried flash-release tablet containing stem cell extract according to claim 3, characterized in that The concentration of puromycin in the αMEM complete medium containing puromycin is 3-5 μg / mL.

6. The freeze-dried flash-release tablet containing stem cell extract according to claim 3, characterized in that In the step of establishing the immortalized stem cell line, when adding Polybrene and αMEM complete medium, the final concentration of Polybrene is maintained at 5-7 μg / mL.

7. The freeze-dried flash-release tablet containing stem cell extract according to claim 3, characterized in that In the step of preparing immortalized SIRT6-overexpressing stem cell extracellular vesicles, the specific steps of centrifugation are: centrifuging the culture supernatant containing immortalized SIRT6-overexpressing stem cell extracellular vesicles at 4°C and 2000×g for 102 minutes, and taking supernatant ①; centrifuging supernatant ① at 4°C and 1000×g for 30 minutes, and taking supernatant ②; ultracentrifuging supernatant ② at 4°C and 110,000×g for 70 minutes, discarding the supernatant and taking the precipitate, adding PBS to the precipitate for resuspending, ultracentrifuging at 4°C and 110,000×g for 70 minutes, and taking the precipitate to obtain immortalized SIRT6-overexpressing stem cell extracellular vesicles.

8. The method for preparing a freeze-dried flash-release tablet containing a stem cell extract according to any one of claims 1 to 7, wherein: The method comprises the following steps: firstly dispersing the stem cell extract in water, then adding guar gum, trehalose, glycine, nicotinamide and corn starch to obtain a freeze-dried liquid containing stem cell extracellular vesicles; dividing the freeze-dried liquid into molds, vacuum freeze-drying and direct compression molding to obtain freeze-dried flash-release tablets.

9. The method for preparing a freeze-dried flash-release tablet containing stem cell extract according to claim 8, characterized in that: The vacuum freeze-drying process is: rapid freezing at -80°C for 2 hours, followed by vacuum drying at -40°C and 10 Pa for 24 hours, and then vacuum drying at 25°C and 5 Pa for 6 hours.

10. Use of the freeze-dried flash-release tablet comprising a stem cell extract according to any one of claims 1 to 7 in the preparation of medicines for anti-aging, immunity enhancement and sleep quality improvement.

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