An ovarian anti-aging cell biological preparation, a preparation method and application thereof

Abstract

The application discloses an ovarian anti-aging cell biological preparation and a preparation method and application thereof, and belongs to the technical field of biotechnology. The biological preparation is obtained by compounding ovarian stem cells and mulberry source specific polypeptides SEQ ID NO. 1 and SEQ ID NO. 2, a double mechanism of cell repair and molecular regulation is formed, animal experiments show that the efficiency of promoting E2 secretion and inhibiting FSH secretion is improved, and the follicle survival rate under a microscope is significantly improved, and the ovarian anti-aging aspect embodies a good treatment prospect. Meanwhile, the application adopts the medicinal and edible mulberry to replace the active ingredients extracted from radix falcaliae polygoni multiflori, and avoids the hepatotoxicity risk.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an ovarian anti-aging cell biological agent, its preparation method, and its application. Background Technology

[0002] The ovaries are paired solid organs located in the female pelvic cavity. They are female gonads, and their size and shape change with age: in early childhood, their surface is smooth; after puberty, due to multiple ovulations, the surface becomes scarred and uneven; the ovaries are largest during sexual maturity, and their volume decreases significantly after menopause. Their main functions are to produce and release egg cells and secrete sex hormones to promote and maintain the development of female secondary sexual characteristics.

[0003] Premature ovarian failure (POF) refers to the cessation of menstruation before the age of 40 due to ovarian dysfunction. It is characterized by primary or secondary amenorrhea accompanied by elevated levels of gonadotropins and decreased levels of estrogen, along with varying degrees of hypoestrogenic symptoms. POF has become a major factor affecting women's physical and mental health.

[0004] In the prior art, some progress has been made in the treatment of premature ovarian failure using stem cells. For example, the use of ovarian stem cells in the preparation of ovarian anti-aging drugs is described in patent publication number CN113633754A. It describes the preparation of ovarian stem cells by separation and preparation, and the isolation of active peptides with anti-aging effects from Polygonum multiflorum. After using stem cells and active peptides together to treat ovarian aging models, it was found that they can effectively promote follicle secretion and E2 secretion, and have a good anti-ovarian aging effect.

[0005] Those skilled in the art believe that Polygonum multiflorum is not a food and medicine of the same origin, and should be used with caution by those with impaired liver function or a family history of liver disease. Mulberry is a nutritious fruit, rich in various vitamins, minerals, and active ingredients, and is considered to have multiple health benefits in both traditional medicine and modern nutrition. Therefore, the applicant is attempting to find active proteins from mulberries to combine with ovarian stem cells to enhance anti-aging effects. Summary of the Invention

[0006] Therefore, this invention provides an ovarian anti-aging cell biological agent, its preparation method, and its application, in order to solve the related technical problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] According to a first aspect of the present invention, an ovarian anti-aging cell biological agent is provided, comprising:

[0009] Ovarian stem cells; and

[0010] The polypeptides shown in SEQ ID NO.1 and SEQ ID NO.2.

[0011] Furthermore, the ovarian stem cells are derived from isolated ovarian tissue.

[0012] Furthermore, the polypeptides shown in SEQ ID NO.1 and SEQ ID NO.2 are derived from mulberry extract.

[0013] Furthermore, the concentration of the added ovarian stem cells is 1-3 x 10⁻⁶. 6 per mL.

[0014] According to a second aspect of the present invention, a method for preparing an ovarian anti-aging cell biological agent is provided, comprising the following steps:

[0015] (1) Ovarian stem cells were obtained from isolated ovarian tissue through digestion and culture for later use;

[0016] (2) A polypeptide was isolated from mulberry extract, the polypeptide being shown in SEQ ID NO.1 and SEQ ID NO.2;

[0017] (3) The ovarian stem cells and polypeptides are mixed evenly in a certain proportion to obtain the preparation.

[0018] Furthermore, the preparation method of mulberry extract is as follows:

[0019] (1) Raw material pretreatment: Fresh mulberries are gently rinsed with 0.5% dilute salt water to remove impurities and drained for later use;

[0020] (2) Grinding: The drained mulberries are quick-frozen with liquid nitrogen and then put into a pre-cooled cryogenic grinder to be quickly ground into frozen mulberry powder;

[0021] (3) Extraction: Add frozen mulberry powder to pre-cooled extraction buffer at a mass-volume ratio of 1:5-10. Under ice bath conditions, homogenize thoroughly for 1-3 minutes using a homogenizer. Centrifuge the homogenate at 8000 rpm for 20-40 minutes at 4°C and collect the supernatant to obtain mulberry extract.

[0022] Further, the extraction buffer is a Tris-HCl buffer with pH 7.0-8.0, and contains 10 mM DTT, 5 mM EDTA, 1 wt% PVPP and 2 mM PMSF.

[0023] According to a third aspect of the present invention, the use of the ovarian anti-aging cell biological agent as described above in the preparation of ovarian anti-aging drugs is provided.

[0024] Furthermore, it also includes pharmaceutically acceptable excipients.

[0025] The present invention has the following advantages:

[0026] By combining ovarian stem cells with mulberry-derived specific peptides (SEQ ID NO.1 / NO.2), a dual mechanism of cell repair and molecular regulation is formed. Animal experiments show that this method improves E2 secretion efficiency compared to traditional Polygonum multiflorum peptide regimens and significantly increases follicle survival rate, demonstrating promising therapeutic prospects for ovarian anti-aging. Using mulberry, a food and medicinal herb, instead of Polygonum multiflorum for extracting active ingredients avoids the risk of hepatotoxicity. Detailed Implementation

[0027] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] According to a first aspect of the present invention, an ovarian anti-aging cell biological agent is provided, comprising:

[0029] Ovarian stem cells; and

[0030] The polypeptides shown in SEQ ID NO.1 and SEQ ID NO.2.

[0031] Furthermore, ovarian stem cells are derived from isolated ovarian tissue.

[0032] Furthermore, the polypeptides shown in SEQ ID NO.1 and SEQ ID NO.2 are derived from mulberry extract.

[0033] Furthermore, the concentration of added ovarian stem cells is 1-3 x 10⁻⁶. 6 per mL.

[0034] According to a second aspect of the present invention, a method for preparing an ovarian anti-aging cell biological agent is provided, comprising the following steps:

[0035] (1) Ovarian stem cells were obtained from isolated ovarian tissue through digestion and culture for later use;

[0036] (2) Polypeptides were isolated from mulberry extract, as shown in SEQ ID NO.1 and SEQ ID NO.2;

[0037] (3) Mix ovarian stem cells and polypeptides in a certain proportion to obtain a preparation.

[0038] Furthermore, the preparation method of mulberry extract is as follows:

[0039] (1) Raw material pretreatment: Fresh mulberries are gently rinsed with 0.5% dilute salt water to remove impurities and drained for later use;

[0040] (2) Grinding: The drained mulberries are quick-frozen with liquid nitrogen and then put into a pre-cooled cryogenic grinder to be quickly ground into frozen mulberry powder;

[0041] (3) Extraction: Add frozen mulberry powder to pre-cooled extraction buffer at a mass-volume ratio of 1:5-10. Under ice bath conditions, homogenize thoroughly for 1-3 minutes using a homogenizer. Centrifuge the homogenate at 8000 rpm for 20-40 minutes at 4°C and collect the supernatant to obtain mulberry extract.

[0042] Furthermore, the extraction buffer is a Tris-HCl buffer with a pH of 7.0-8.0, containing 10 mM DTT, 5 mM EDTA, 1 wt% PVPP and 2 mM PMSF.

[0043] According to a third aspect of the present invention, the use of the above-mentioned ovarian anti-aging cell biological agent in the preparation of ovarian anti-aging drugs is provided.

[0044] Furthermore, it also includes pharmaceutically acceptable excipients.

[0045] To better illustrate the technical effects of this application, the present invention also provides the following preparation examples and embodiments.

[0046] Preparation Example 1

[0047] Preparation of stem cells

[0048] 1. Ovarian tissue is dissociated into a single-cell suspension.

[0049] (1) Material selection and washing:

[0050] Mice were euthanized by cervical dislocation after CO2 asphyxiation. The abdominal cavity was opened on a sterile operating table, and both ovaries were removed and washed three times in pre-cooled sterile PBS on ice. Under a stereomicroscope, the fat and connective tissue around the ovaries were carefully removed, leaving only the ovaries themselves. The ovaries were then cut into 1mm pieces and washed repeatedly with pre-cooled PBS to remove blood.

[0051] (2) Enzyme digestion:

[0052] The cleaned ovarian granules were transferred to centrifuge tubes containing 2 mg / mL collagenase IV and 20 µg / mL Dnase I solution. The mixture was then incubated in a water bath at 37°C with shaking for about 60 minutes. Every 15 minutes, the tissue was ground with a pipette tip and gently blown to promote dissociation.

[0053] (3) Termination of digestion and preliminary filtration:

[0054] Add a large amount of pre-cooled PBS, pipette repeatedly several times to stop digestion, filter the cell suspension through a 70µm cell sieve to remove incompletely digested tissue blocks and large fragments, collect the filtrate into a new centrifuge tube, centrifuge to remove the supernatant and obtain the precipitated cells.

[0055] (4) Trypsin treatment:

[0056] Resuspend the cell pellet in an appropriate amount of 0.25% trypsin-EDTA solution, incubate at 37°C for 8 min, add serum-containing culture medium to stop digestion, and gently pipette to form a single-cell suspension.

[0057] (5) Cleaning and counting:

[0058] Filter the cell suspension again through a 40µm cell sieve to ensure a single-cell suspension. Centrifuge at 300-400g for 5 minutes and discard the supernatant. Resuspend the cells in pre-chilled PBS buffer with 2% FBS. Mix a small amount of the cell suspension with trypan blue and perform cell counting and viability assessment using a hemocytometer; viability >80%.

[0059] 2. Cell passage and culture

[0060] (1) Seed cells in a Matrigel medium diluted in cold serum-free medium, coated for 60 min and then dried. Place the culture dish in a 37°C, 5% CO2, high humidity incubator and culture. Minimize disturbance for the first 24-48 h, and then replace with fresh complete medium every 2 days thereafter.

[0061] (2) Closely observe cell morphology under an inverted microscope to obtain cells that are small in size, round or oval, highly refractive, with little cytoplasm and a high nucleocytoplasmic ratio. When the cells have been cultured for about 10 days and have grown to 70-80% confluence, they can be passaged. Aspirate the old culture medium, wash 1-2 times with PBS solution without calcium and magnesium ions, add an appropriate amount of 0.25% trypsin-EDTA solution preheated to 37℃, incubate at 37℃ for 3 min, observe closely, and when the cells become round and detach, add an equal volume of serum-containing culture medium to stop digestion, and gently pipette to form a single-cell suspension. Centrifuge at 300-400g for 5 min, discard the supernatant, resuspend the cells in fresh complete culture medium, and seed them into new coated culture dishes at a ratio of 1:2. Passage to P4 to obtain ovarian stem cells.

[0062] (3) Identification of ovarian stem cells: Ovarian stem cells were added to alkaline phosphatase and stained for 30 minutes. The colonies began to stain. After 35 minutes, the culture medium was discarded to stop the staining. PBS solution was added and the cell clusters were observed to be purple-blue under a microscope, confirming that the cells were ovarian stem cells.

[0063] Preparation Example 2

[0064] 1. Preparation of mulberry extract

[0065] (1) Raw material pretreatment: Fresh mulberries are gently rinsed with 0.5% dilute salt water to remove impurities and drained for later use;

[0066] (2) Grinding: The drained mulberries are quick-frozen with liquid nitrogen and then put into a pre-cooled cryogenic grinder to be quickly ground into frozen mulberry powder;

[0067] (3) Extraction: Frozen mulberry powder was added to pre-cooled extraction buffer at a mass-to-volume ratio of 1:8. The extraction buffer was a Tris-HCl buffer with a pH of 7.0-8.0, containing 10 mM DTT, 5 mM EDTA, 1 wt% PVPP, and 2 mM PMSF. The mixture was homogenized thoroughly for 3 min under ice bath conditions. The homogenate was then centrifuged at 8000 rpm for 40 min at 4°C, and the supernatant was collected to obtain the mulberry extract.

[0068] The extraction process described above is carried out at a low temperature throughout, which ensures that the extracted proteins or peptides do not denature or become inactive.

[0069] 2. Peptide purification

[0070] Sephacryl S-100 HR and Superdex-75 chromatography were performed sequentially, yielding multiple protein peaks. Protein solutions of different molecular weights were collected and dialyzed. Experiments showed that the peptides inhibited the downregulation of LRRC4, HCP5, and FTO expression. Five peptides with significant effects were screened, and MALDI-TOFMS was used to determine the amino acid sequences of the two peptides with the best effects, as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively (peptide sequencing was performed by Beijing Biotech Biotechnology Co., Ltd.).

[0071] The extraction process revealed that the content of SEQ ID NO.1 and SEQ ID NO.2 derived from mulberry extract was low. In order to better achieve its technical effect, the applicant synthesized peptides using chemical synthesis methods. The peptide synthesis was completed by Nanjing Genscript Biotech Co., Ltd., which provided the lyophilized powder.

[0072] Preparation Example 3

[0073] Stem cell combined with polypeptide preparation: Prepare physiological saline pre-cooled to 4°C, and add the ovarian stem cells collected in Preparation Example 1 to the physiological saline to prepare a final concentration of 1×10⁻⁶. 6The suspension was prepared by gently stirring until homogeneous during the addition process. The peptides SEQ ID NO.1 and SEQ ID NO.2 synthesized in Preparation Example 2 were added to the suspension at a final concentration of 3 mg / mL and then gently stirred until homogeneous for later use.

[0074] Stem cell preparation: Prepare physiological saline pre-cooled to 4°C, and add the ovarian stem cells collected in Preparation Example 1 to the physiological saline to prepare a final concentration of 1 x 10⁻⁶. 6 Add a suspension of cells / mL, stirring gently until well mixed.

[0075] Peptide preparation: Prepare physiological saline pre-cooled to 4°C. Add the peptides SEQ ID NO.1 and SEQ ID NO.2 chemically synthesized in Preparation Example 2 to the physiological saline at a final concentration of 3 mg / mL and stir gently until well mixed.

[0076] Experimental Example 1

[0077] Mouse premature ovarian failure modeling

[0078] Healthy female mice (C57BL / 6J) aged 10-12 weeks and in estrus, weighing 18-25g, were selected. Cyclophosphamide lyophilized powder was weighed and dissolved in 0.5% physiological saline to prepare a 50mg / mL cyclophosphamide solution. The solution was kept in the dark and stored on ice. Administration was via intraperitoneal injection. The model group received an intraperitoneal injection of 50mg / kg, while the control group received an equal volume of physiological saline. Intraperitoneal injections were administered daily at fixed times for 5 consecutive days. Serum hormone levels were measured on day 14 after the last administration. Blood was collected from mice under anesthesia via ocular sampling or cardiac puncture. After standing for 30 minutes, the blood was centrifuged at 3000 rpm for 15 minutes to collect serum. ELISA testing showed successful modeling when FSH > 20 mIU / mL and E2 < 20 pg / mL.

[0079] Biological agent injection experiment

[0080] Mice in the estrus phase who successfully developed the model were randomly divided into four groups: a model group, a stem cell group, a peptide group, a stem cell combined with peptide group, and a control group, with 20 mice in each group. Injection dosage: On day 14 of modeling, the stem cell group received 0.1 mL of the stem cell preparation from Example 3 via tail vein injection, repeated every 4 days for a total of 5 times; the peptide group received the same peptide preparation from Example 3 via tail vein injection on day 14 of modeling, repeated every 4 days for a total of 5 times; the stem cell combined with peptide group received 0.1 mL of the same stem cell combined with peptide preparation from Example 3 via tail vein injection on day 14 of modeling, repeated every 4 days for a total of 5 times; the modeling group and the control group received an equal volume of physiological saline via tail vein injection each time.

[0081] Data Analysis

[0082] Serum was obtained from mice in each group using the tail clipping method. Whole blood samples collected in serum separation tubes were incubated overnight at 4°C, then centrifuged at 1000g for 20 min, and the supernatant was collected. Follicle-stimulating hormone (FSH) and estradiol (E2) levels in mice were detected using a chemiluminescence immunoassay. Reagents were purchased from Xiamen Baotai Biotechnology Co., Ltd. The results are shown in Table 1.

[0083] Table 1: Serum FSH and E2 levels in mice of each group (Mean±SD, n=20)

[0084]

[0085] As shown in Table 1, the model group exhibited high FSH secretion levels and low E2 secretion levels, indicating significant ovarian failure in the mice and successful model establishment. Treatment with the stem cell group and peptide group, respectively, resulted in decreased FSH secretion and increased E2 secretion in the mice, suggesting that both stem cell and peptide preparations alone have some effect on alleviating premature ovarian failure, but cannot achieve complete recovery. The combined stem cell and peptide treatment group showed significantly better efficacy than either stem cell or peptide injection alone, and its efficacy was close to that of the control group. This indicates that the combination of stem cells and peptides in biological agents has a certain effect on improving or treating premature ovarian failure and holds promise for further development.

[0086] Mice in each group of the above experiments were sacrificed, bilateral ovarian tissue was dissected, and the fat and connective tissue around the ovaries were carefully removed. Frozen sections were examined under a fluorescence microscope, and the results are shown in Table 2.

[0087] Table 2: Results of follicle detection under a microscope

[0088]

[0089] As shown in Table 2, the number of follicles in the model group was significantly lower than that in the normal (control group) mice, indicating successful model establishment. After treatment with stem cell and peptide preparations, respectively, the number of follicles in the stem cell group and peptide group significantly increased, indicating that both stem cell and peptide preparations alone have some effect on alleviating premature ovarian failure, but cannot achieve complete recovery. The treatment effect of the stem cell combined with peptide group was significantly better than that of stem cell and peptide preparation injections alone, and was essentially close to the control group level, indicating that the combination of stem cells and peptides has a certain effect on improving or treating premature ovarian failure and has potential for further development.

[0090] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An ovarian anti-aging cell biological agent, characterized in that, include: Ovarian stem cells; and The polypeptides shown in SEQ ID NO.1 and SEQ ID NO.2 are derived from mulberry extract.

2. The ovarian anti-aging cell biological agent as described in claim 1, characterized in that, The ovarian stem cells were derived from isolated ovarian tissue.

3. The ovarian anti-aging cell biological agent as described in claim 1, characterized in that, The concentration of the added ovarian stem cells is 1-3 x 10⁻⁶. 6 per mL.

4. A method for preparing an ovarian anti-aging cell biological agent, characterized in that, Includes the following steps: (1) Ovarian stem cells were obtained from isolated ovarian tissue through digestion and culture for later use; (2) A polypeptide was isolated from mulberry extract, the polypeptide being shown in SEQ ID NO.1 and SEQ ID NO.2; (3) The ovarian stem cells and polypeptides are mixed evenly in a certain proportion to obtain the preparation.

5. The method for preparing the ovarian anti-aging cell biological agent as described in claim 4, characterized in that, The preparation method of mulberry extract is as follows: (1) Raw material pretreatment: Fresh mulberries are gently rinsed with 0.5% dilute salt water to remove impurities and drained for later use; (2) Grinding: The drained mulberries are quick-frozen with liquid nitrogen and then put into a pre-cooled cryogenic grinder to be quickly ground into frozen mulberry powder; (3) Extraction: Add frozen mulberry powder to pre-cooled extraction buffer at a mass-volume ratio of 1:5-10. Under ice bath conditions, homogenize thoroughly for 1-3 minutes using a homogenizer. Centrifuge the homogenate at 8000 rpm for 20-40 minutes at 4°C and collect the supernatant to obtain mulberry extract.

6. The method for preparing the ovarian anti-aging cell biological agent as described in claim 5, characterized in that, in, The extraction buffer is a Tris-HCl buffer with a pH of 7.0-8.0, containing 10 mM DTT, 5 mM EDTA, 1 wt% PVPP and 2 mM PMSF.

7. The use of the ovarian anti-aging cell biological agent as described in any one of claims 1-3 in the preparation of ovarian anti-aging drugs.

8. The application as described in claim 7, characterized in that, It also includes pharmaceutically acceptable excipients.

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