Composition containing mesenchymal stem cells and application of composition in preparation of product for resisting premature ovarian failure

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a composition containing mesenchymal stem cells and application of the composition in preparation of products for resisting premature ovarian failure. The invention relates to a composition containing mesenchymal stem cells. The umbilical cord mesenchymal stem cell gel is prepared from the following components in percentage by weight: 10 percent of umbilical cord mesenchymal stem cells, 4 to 6 percent of human serum albumin stock solution, 1 to 2 percent of ginkgo biloba extract, 1 to 3 percent of coix seed oil, 2 to 2.5 percent of flavonol glycoside, 1.1 to 1.3 percent of vitamin E, 0.4 to 0.8 percent of glutathione, 0.5 to 1.5 percent of sodium hyaluronate, 0.5 to 1.0 percent of phycocyanin, 1.2 to 1.8 percent of zinc gluconate, 0.5 to 0.8 percent of heparin calcium, 0.1 to 0.3 percent of pH regulator and the balance of normal saline. The composition disclosed by the invention can be used for preparing products for resisting premature ovarian failure and has a very good effect.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a composition containing mesenchymal stem cells and its application in the preparation of products with anti-ovarian premature failure effect. Background Art

[0002] Ovarian premature failure refers to a disease in which a woman's ovarian function significantly declines before the age of 40, resulting in oligomenorrhea or amenorrhea, accompanied by an increase in serum gonadotropin (FSH) level and a decrease in estrogen level. This is a complex endocrine disorder disease that may affect fertility and overall health. At present, the treatment of POF mainly focuses on hormone replacement therapy, but it may increase the risk of diseases such as endometrial cancer, breast cancer, and cardiovascular and cerebrovascular diseases.

[0003] Umbilical cord mesenchymal stem cells are a type of pluripotent stem cells extracted from neonatal umbilical cord tissue. In recent years, they have received increasing attention due to their unique biological characteristics and broad application prospects.

[0004] Therefore, there is an urgent need for a composition containing mesenchymal stem cells and its application in the preparation of products with anti-ovarian premature failure effect. Summary of the Invention

[0005] The object of the present invention is to provide a composition containing mesenchymal stem cells and its application in the preparation of products with anti-ovarian premature failure effect.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: A composition containing mesenchymal stem cells, by weight percentage, includes: 10% umbilical cord mesenchymal stem cells, 4 - 6% human blood albumin stock solution, 1 - 2% ginkgo extract, 1 - 3% coix seed oil, 2 - 2.5% flavonol glycosides, 1.1 - 1.3% vitamin E, 0.4 - 0.8% glutathione, 0.5 - 1.5% sodium hyaluronate, 0.5 - 1.0% phycocyanin, 1.2 - 1.8% zinc gluconate, 0.5 - 0.8% calcium heparin, 0.1 - 0.3% pH regulator, and the balance is physiological saline.

[0007] Further, the preparation method of the umbilical cord mesenchymal stem cells is characterized by including the following steps: (1) Wash the mucus and blood stains on the surface of the umbilical cord, cut the umbilical cord into small sections of 2 - 3 cm, remove 2 arteries and venous membranes of the umbilical cord, cut it into small pieces, wash and filter, then inoculate it into a culture dish, and place the culture dish in an oven at 37°C to dry; (2) After drying, add 5 - 6 mL of culture medium to the culture dish, and statically culture it at 37°C; add 5 - 6 mL of culture medium on the third day of culture, and change the medium on the 7th - 8th day; (3)On the 12th - 14th day, gently remove the tissue blocks, add digestive fluid for digestion, and obtain umbilical cord mesenchymal stem cells.

[0008] In the composition of the present invention, umbilical cord mesenchymal stem cells have multi - directional differentiation potential and immunomodulatory functions. By secreting various growth factors, cytokines, exosomes and other substances, they promote tissue repair and regeneration. For premature ovarian failure, umbilical cord mesenchymal stem cells stimulate follicle development: by secreting growth factors (such as VEGF, IGF - 1, etc.), they promote the activation and development of residual follicles in the ovary. Regulate local inflammatory responses, reduce oxidative stress, and create favorable conditions for the restoration of ovarian function. Promote angiogenesis and improve ovarian blood supply.

[0009] Furthermore, the cleaning solution for cleaning the mucus and blood stains on the umbilical cord surface is physiological saline.

[0010] Furthermore, the filter screen for filtration is an 8 - mesh filter screen, and the size of the obtained tissue blocks is between 1 - 2 mm.

[0011] Furthermore, the drying time is 2 - 3 hours.

[0012] Furthermore, the culture medium is α - MEM culture medium containing 20% fetal bovine serum.

[0013] Furthermore, the preparation method of the ginkgo extract includes the following steps: (1)Soak ginkgo leaves in water, wash, dry, and crush to obtain raw materials; (2)Add the raw materials to water to obtain a fermentation material, add Bacillus pumilus to the fermentation material, carry out fermentation, and after sterilization, obtain a fermentation product; (3)Add an ethanol aqueous solution to the fermentation product, stir, cool, stand, and filter with a filter membrane to obtain an alcohol precipitation solution; (4)Filter the alcohol precipitation solution with an ultrafiltration membrane, and freeze - dry to obtain ginkgo extract.

[0014] 2. The ginkgo extract of the present invention is rich in flavonoids and terpenoid lactones, and has the effects of antioxidant, anti - inflammatory and improving blood circulation. It may indirectly support ovarian health in the following ways: scavenging free radicals and protecting ovarian cells from oxidative stress damage. Dilating blood vessels and increasing the blood supply to ovarian tissue, thereby optimizing its physiological functions.

[0015] Furthermore, in step (2), the fermentation conditions are fermentation at 25 - 30 °C for 20 - 25 h, and the ventilation rate is 7.0 - 7.5 L / min.

[0016] Further, in step (3), an ethanol aqueous solution with a volume fraction of 60-70% is added to the fermentation product, stirred at a temperature of 60-70°C for 1-2 h, cooled to 15-20°C, allowed to stand for 2-4 h, and filtered through a 0.45 μm filter membrane to obtain an alcohol precipitation solution.

[0017] Further, in step (4), the cut-off molecular weight of the ultrafiltration membrane is 12,000-25,000 Daltons.

[0018] The present invention also provides an application of a composition containing mesenchymal stem cells in the preparation of a product with anti-ovarian premature failure.

[0019] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. The umbilical cord mesenchymal stem cells in the composition of the present invention have multi-directional differentiation potential and immunomodulatory functions, and promote tissue repair and regeneration by secreting various growth factors, cytokines, exosomes and other substances. For ovarian premature failure, umbilical cord mesenchymal stem cells stimulate follicle development: by secreting growth factors (such as VEGF, IGF-1, etc.), promoting the activation and development of residual follicles in the ovary. Regulating local inflammatory responses, reducing oxidative stress, and creating favorable conditions for ovarian function recovery. Promoting angiogenesis and improving ovarian blood supply.

[0020] 2. The ginkgo extract of the present invention is rich in flavonoids and terpene lactones, and has antioxidant, anti-inflammatory and blood circulation improving effects. It may indirectly support ovarian health in the following ways: scavenging free radicals and protecting ovarian cells from oxidative stress damage. Dilating blood vessels and increasing the blood supply to ovarian tissues, thereby optimizing its physiological functions.

[0021] 3. Umbilical cord mesenchymal stem cells and ginkgo extract play a synergistic role in the following ways to jointly improve ovarian function: Umbilical cord mesenchymal stem cells reduce inflammatory responses and promote tissue repair through immunomodulation and secretion of growth factors. Ginkgo extract further reduces oxidative stress and inflammatory damage through antioxidant and anti-inflammatory effects. The two jointly optimize the local microenvironment of the ovary and create favorable conditions for follicle development. Factors such as VEGF secreted by umbilical cord mesenchymal stem cells can directly promote ovarian angiogenesis. The two work synergistically to ensure that ovarian tissues obtain sufficient oxygen and nutrients. Ginkgo extract indirectly supports follicle development and hormone balance by improving blood circulation and reducing inflammation. The two work synergistically to improve the effect of ovarian function recovery. Detailed implementation methods

[0022] 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 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.

[0023] Example 1 This embodiment provides a composition containing mesenchymal stem cells, which includes, by weight percentage: 10% umbilical cord mesenchymal stem cells, 4% human blood albumin stock solution, 2% ginkgo extract, 1% coix seed oil, 2.5% flavonol glycosides, 1.1% vitamin E, 0.8% glutathione, 0.5% sodium hyaluronate, 1.0% phycocyanin, 1.2% zinc gluconate, 0.8% calcium heparin, 0.1% pH regulator, and the balance is physiological saline.

[0024] The preparation method of the umbilical cord mesenchymal stem cells is characterized by including the following steps: (1) Wash the mucus and blood stains on the surface of the umbilical cord, cut the umbilical cord into small sections of 2-3 cm, remove the two arteries and venous membranes of the umbilical cord, cut it into small pieces, wash and filter, then inoculate it into a culture dish, and place the culture dish in an oven at 37°C to dry. (2) After drying, add 6 mL of culture medium to the culture dish and statically culture it at 37°C; supplement 5 mL of culture medium on the third day of culture and change the medium on the eighth day. (3) On the 12th day, gently remove the tissue blocks, add digestive juice for digestion to obtain umbilical cord mesenchymal stem cells.

[0025] The cleaning solution for washing the mucus and blood stains on the surface of the umbilical cord is physiological saline.

[0026] The filter screen for filtration is an 8-mesh filter screen, and the size of the obtained tissue blocks is between 1-2 mm.

[0027] The drying time is 3 hours.

[0028] The culture medium is α-MEM culture medium containing 20% fetal bovine serum.

[0029] The preparation method of the ginkgo extract includes the following steps: (1) Soak ginkgo leaves in water, wash, dry, and pulverize to obtain raw materials. (2) Add the raw materials to water to obtain a fermentation material, add Brevibacterium to the fermentation material for fermentation, and after sterilization, obtain a fermentation product. (3) Add an ethanol aqueous solution to the fermentation product, stir, cool, stand still, and filter with a filter membrane to obtain an alcohol precipitation solution. (4) Filter the alcohol precipitation solution with an ultrafiltration membrane and freeze-dry to obtain the ginkgo extract.

[0030] In step (2), the fermentation conditions are fermentation at 25°C for 25 h with an aeration rate of 7.0 L / min.

[0031] In step (3), 70% ethanol aqueous solution by volume is added to the fermentation product, stirred at 60°C for 2 h, cooled to 15°C, allowed to stand for 4 h, and filtered through a 0.45 μm filter membrane to obtain an alcohol precipitation solution.

[0032] In step (4), the cut-off molecular weight of the ultrafiltration membrane is 12,000 - 25,000 Daltons.

[0033] Example 2 This example provides a composition containing mesenchymal stem cells, which includes, by weight percentage: 10% umbilical cord mesenchymal stem cells, 6% human blood albumin stock solution, 1% ginkgo extract, 3% coix seed oil, 2% flavonol glycosides, 1.3% vitamin E, 0.4% glutathione, 1.5% sodium hyaluronate, 0.5% astaxanthin, 1.8% zinc gluconate, 0.5% calcium heparin, 0.3% pH regulator, and the balance is physiological saline.

[0034] The preparation method of the umbilical cord mesenchymal stem cells is characterized by including the following steps: (1) Wash the mucus and blood stains on the surface of the umbilical cord, cut the umbilical cord into small segments of 2 - 3 cm, remove 2 arteries and venous membranes of the umbilical cord, cut into small pieces, wash and filter, then inoculate into a culture dish, and place the culture dish in an oven at 37°C to dry. (2) After drying, add 5 mL of culture medium to the culture dish and statically culture at 37°C; supplement 6 mL of culture medium on the third day of culture, and change the medium on the 7th - 8th day. (3) On the 12th day, gently remove the tissue blocks, add digestive juice for digestion to obtain umbilical cord mesenchymal stem cells.

[0035] The cleaning solution for washing the mucus and blood stains on the surface of the umbilical cord is physiological saline.

[0036] The filter screen for filtration is an 8 - mesh filter screen, and the size of the obtained tissue blocks is between 1 - 2 mm.

[0037] The drying time is 2 - 3 hours.

[0038] The culture medium is α - MEM culture medium containing 20% fetal bovine serum.

[0039] The preparation method of the ginkgo extract includes the following steps: (1) Soak, wash, dry, and crush ginkgo leaves to obtain raw materials. (2) Add the raw materials to water to obtain a fermentation material, add Brevibacterium to the fermentation material for fermentation, and after sterilization, obtain a fermentation product. (3) Add an ethanol aqueous solution to the fermentation product, stir, cool, let stand, and filter with a membrane filter to obtain an alcohol precipitation solution; (4) Filter the alcohol precipitation solution with an ultrafiltration membrane and freeze-dry to obtain a ginkgo extract.

[0040] In the step (2), the fermentation conditions are fermentation at 30 °C for 20 h and an aeration rate of 7.5 L / min.

[0041] In the step (3), add an ethanol aqueous solution with a volume fraction of 70% to the fermentation product, stir at 70 °C for 1 h, cool to 20 °C, let stand for 2 h, and filter with a 0.45 μm membrane filter to obtain an alcohol precipitation solution.

[0042] In the step (4), the cut-off molecular weight of the ultrafiltration membrane is 12,000 - 25,000 Daltons.

[0043] Example 3 This example provides a composition containing mesenchymal stem cells, which includes, by weight percentage: 10% umbilical cord mesenchymal stem cells, 5% human blood albumin stock solution, 1.5% ginkgo extract, 2% coix seed oil, 2.2% flavonol glycosides, 1.2% vitamin E, 0.6% glutathione, 1% sodium hyaluronate, 0.7% phycocyanin, 1.5% zinc gluconate, 0.6% calcium heparin, 0.2% pH regulator, and the balance is physiological saline.

[0044] The preparation method of the umbilical cord mesenchymal stem cells is characterized by including the following steps: (1) Wash the mucus and blood stains on the surface of the umbilical cord, cut the umbilical cord into small sections of 2 - 3 cm, remove 2 arteries and venous membranes of the umbilical cord, cut into small pieces, wash and filter, then inoculate into a culture dish, and place the culture dish in an oven at 37 °C to dry; (2) After drying, add 6 mL of culture medium to the culture dish and statically culture at 37 °C; supplement 5 mL of culture medium on the third day of culture, and change the medium on the 7th - 8th day; (3) On the 14th day, gently remove the tissue blocks, add digestive juice for digestion to obtain umbilical cord mesenchymal stem cells.

[0045] The cleaning solution for washing the mucus and blood stains on the surface of the umbilical cord is physiological saline.

[0046] The filter screen for filtration is an 8 - mesh filter screen, and the size of the obtained tissue blocks is between 1 - 2 mm.

[0047] The drying time is 3 hours.

[0048] The culture medium is α - MEM culture medium containing 20% fetal bovine serum.

[0049] The preparation method of the ginkgo extract includes the following steps: (1)Soak ginkgo leaves in water, wash them, dry them, and pulverize them to obtain raw materials; (2)Add the raw materials to water to obtain a fermentation material, add Bacillus pumilus to the fermentation material, carry out fermentation, and after sterilization, obtain a fermentation product; (3)Add an ethanol aqueous solution to the fermentation product, stir, cool, stand still, and filter with a filter membrane to obtain an alcohol precipitation solution; (4)Filter the alcohol precipitation solution with an ultrafiltration membrane and freeze-dry it to obtain a ginkgo extract.

[0050] The fermentation conditions in step (2) are fermentation at 30 °C for 25 h and an aeration rate of 7.5 L / min.

[0051] In step (3), add an ethanol aqueous solution with a volume fraction of 70% to the fermentation product, stir at 70 °C for 2 h, cool to 20 °C, stand still for 3 h, and filter with a 0.45 μm filter membrane to obtain an alcohol precipitation solution.

[0052] The cut-off molecular weight of the ultrafiltration membrane in step (4) is 12,000 - 25,000 Daltons.

[0053] Comparative Example 1 The difference between this comparative example and Example 1 is: A composition containing mesenchymal stem cells, by weight percentage, includes: 11% umbilical cord mesenchymal stem cells, 5% human blood albumin stock solution, 0.5% ginkgo extract, 2% coix seed oil, 2.2% flavonol glycosides, 1.2% vitamin E, 0.6% glutathione, 1% sodium hyaluronate, 0.7% phycocyanin, 1.5% zinc gluconate, 0.6% calcium heparin, 0.2% pH regulator, and the balance is physiological saline.

[0054] Comparative Example 2 The difference between this comparative example and Example 1 is: A composition containing mesenchymal stem cells, by weight percentage, includes: 10% umbilical cord mesenchymal stem cells, 5% human blood albumin stock solution, 2% coix seed oil, 2.2% flavonol glycosides, 1.2% vitamin E, 0.6% glutathione, 1% sodium hyaluronate, 0.7% phycocyanin, 1.5% zinc gluconate, 0.6% calcium heparin, 0.2% pH regulator, and the balance is physiological saline.

[0055] Comparative Example 3 The difference between this comparative example and Example 1 is: A composition containing mesenchymal stem cells, by weight percentage, includes: 10% umbilical cord mesenchymal stem cells, 5% human blood albumin stock solution, 3.5% ginkgo extract, 2% coix seed oil, 2.2% flavonol glycosides, 1.2% vitamin E, 0.6% glutathione, 1% sodium hyaluronate, 0.7% phycocyanin, 1.5% zinc gluconate, 0.6% calcium heparin, 0.2% pH regulator, and the balance is physiological saline.

[0056] Comparative Example 4 The difference between this comparative example and Example 1 is that the ginkgo biloba extract is fermented with Bacillus subtilis.

[0057] Performance test In vivo immune POF mouse experiment: Grouping: blank control group, positive control group, example group and comparative example group, 10 cases in each group. The blank control group was injected with normal saline.

[0058] Add 10 mg of mouse zona pellucida polypeptide 3 (ZP3) to 10 ml of triple-distilled water, and prepare an immune reagent and an immune enhancement preparation with complete Freund's adjuvant and incomplete Freund's adjuvant in a 1:1 ratio respectively. Select SPF-grade 8-week-old female BALB / c mice with normal estrous cycles, and subcutaneously inject 0.15 ml of the immune agent for the first immunization at multiple points, and then inoculate 0.15 ml of the immune enhancement reagent 2 times at 2 weeks and 4 weeks later. Vaginal exfoliative cytology was performed daily at a fixed point after the first immunization to determine whether the estrous cycle of the mice was disordered. ELISA method was used to compare the levels of serum E2, FSH and anti-zona pellucida antibody (AZPAb) in mice at each inoculation time point (successive estrous cycle disorders in 2 cycles, significant decrease in serum E2 and FSH compared with before modeling, and increase in AZPAb titer was considered successful modeling).

[0059] An equal amount (1 mL) of the composition of the present invention was injected into the tail vein once, the positive control group and the hydroxychloroquine treatment group were injected with normal saline into the tail vein once, and the hydroxychloroquine treatment group and the combination treatment group were perfused with 10 mg / kg of hydroxychloroquine every day. After 4 weeks, the mice were sacrificed to evaluate the estrous cycle and ovarian tissue pathology among the 6 groups.

[0060] The results are shown in Table 1.

[0061] Table 1 Performance test results

[0062] As can be seen from Table 1, the normal proportion of the estrous cycle of the mice in Examples 1-3 was higher than that of the positive control group, indicating that the composition prepared by the present invention can effectively prevent or treat ovarian premature failure caused by immune factors.

[0063] In Comparative Example 1, the ratio of umbilical cord mesenchymal stem cells to ginkgo biloba extract was different, and the estrous cycle decreased, indicating that umbilical cord mesenchymal stem cells and ginkgo biloba extract exerted a synergistic effect. Umbilical cord mesenchymal stem cells and ginkgo biloba extract exerted a synergistic effect and jointly improved ovarian function in the following ways: Umbilical cord mesenchymal stem cells reduced the inflammatory response and promoted tissue repair through immunomodulation and secretion of growth factors. Ginkgo biloba extract further alleviated oxidative stress and inflammatory damage through antioxidant and anti-inflammatory effects. The two jointly optimized the local microenvironment of the ovary and created favorable conditions for follicle development. Factors such as VEGF secreted by umbilical cord mesenchymal stem cells directly promoted ovarian angiogenesis. The synergistic effect of the two ensured that the ovarian tissue obtained sufficient oxygen and nutrients. Ginkgo biloba extract indirectly supported follicle development and hormonal balance by improving blood circulation and reducing inflammation. The synergistic effect of the two improved the effect of ovarian function recovery.

[0064] In Comparative Example 2, ginkgo biloba extract was not added, and the estrous cycle decreased, indicating that ginkgo biloba extract exerted an effect in the composition.

[0065] In Comparative Example 3, the addition amount of ginkgo biloba extract increased, and the estrous cycle decreased, indicating that the composition could exert a better effect under certain addition conditions.

[0066] In Comparative Example 4, the bacteria used to prepare ginkgo biloba extract were different, indicating that the active substances in the ginkgo biloba extract prepared with Bacillus pumilus could exert a therapeutic effect. The addition amount of ginkgo biloba extract in the composition needs to be strictly controlled to ensure the maximization of its positive effect and avoid negative effects caused by too high a concentration. When the addition amount is too much, problems such as excessive antioxidant activity, enhanced cytotoxicity, excessive immunosuppression, and pharmacological imbalance may occur, thereby reducing the improvement effect on premature ovarian failure. Therefore, in the formulation design, the optimal addition amount range must be determined based on scientific experimental data to achieve the best synergistic effect and therapeutic effect.

[0067] 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 described in 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 composition containing mesenchymal stem cells, characterized in that, Comprising, by weight percentage: 10% umbilical cord mesenchymal stem cells, 4 - 6% human blood albumin stock solution, 1 - 2% ginkgo biloba extract, 1 - 3% coix seed oil, 2 - 2.5% flavonol glycosides, 1.1 - 1.3% vitamin E, 0.4 - 0.8% glutathione, 0.5 - 1.5% sodium hyaluronate, 0.5 - 1.0% phycocyanin, 1.2 - 1.8% zinc gluconate, 0.5 - 0.8% calcium heparin, 0.1 - 0.3% pH regulator, and the balance being physiological saline.

2. The preparation method of umbilical cord mesenchymal stem cells according to claim 1, wherein, Including the following steps: (1) Wash the mucus and blood stains on the surface of the umbilical cord, cut the umbilical cord into small segments of 2 - 3 cm, remove the two arteries and venous membranes of the umbilical cord, cut it into small pieces, wash and filter, then inoculate it into a culture dish, and place the culture dish in an oven at 37°C to dry. (2) After drying, add 5 - 6 mL of culture medium to the culture dish and incubate it statically at 37°C; supplement 5 - 6 mL of culture medium on the third day of culture, and change the medium on the 7th - 8th day. (3) On the 12th - 14th day, gently remove the tissue blocks, add digestive juice for digestion to obtain umbilical cord mesenchymal stem cells.

3. The preparation method of umbilical cord mesenchymal stem cells according to claim 2, characterized in that, The cleaning solution used to wash the mucus and blood stains on the surface of the umbilical cord is physiological saline.

4. The preparation method of umbilical cord mesenchymal stem cells according to claim 2, characterized in that, The filter screen for filtration is an 8 - mesh filter screen, and the size of the obtained tissue blocks is between 1 - 2 mm.

5. The preparation method of umbilical cord mesenchymal stem cells according to claim 2, characterized in that, The drying time is 2 - 3 hours; the culture medium is α - MEM culture medium containing 20% fetal bovine serum.

6. The preparation method of umbilical cord mesenchymal stem cells according to claim 1, wherein, The preparation method of the ginkgo biloba extract includes the following steps: (1) Soak ginkgo leaves in water, wash, dry, and pulverize to obtain raw materials. (2) Add the raw materials to water to obtain a fermentation material, add Brevibacterium to the fermentation material, and carry out fermentation. After sterilization, obtain a fermentation product. (3) Add an ethanol aqueous solution to the fermentation product, stir, cool, stand, and filter with a membrane filter to obtain an alcohol precipitation solution. (4) Filter the alcohol precipitation solution with an ultrafiltration membrane, and freeze - dry to obtain ginkgo biloba extract.

7. The preparation method of umbilical cord mesenchymal stem cells according to claim 6, characterized in that, The fermentation conditions in step (2) are fermentation at 25 - 30°C for 20 - 25 h, and the ventilation volume is 7.0 - 7.5 L / min.

8. The method for preparing umbilical cord mesenchymal stem cells according to claim 6, characterized in that, In step (3), add an ethanol aqueous solution with a volume fraction of 60 - 70% to the fermentation product, stir at a temperature of 60 - 70°C for 1 - 2 h, cool to 15 - 20°C, stand for 2 - 4 h, and filter with a 0.45 - μm membrane filter to obtain an alcohol precipitation solution.

9. The preparation method of umbilical cord mesenchymal stem cells according to claim 6, characterized in that, The cut - off molecular weight of the ultrafiltration membrane in step (4) is 12000 - 25000 daltons.

10. Use of a composition containing mesenchymal stem cells according to any one of claims 1 - 9 in the preparation of a product having anti - premature ovarian failure effect.

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