Anti-aging mitochondrial preparation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an anti-aging mitochondrial preparation as well as a preparation method and application thereof. The mitochondrial preparation is formed by compounding mammalian-derived mitochondria and a pharmaceutically acceptable injection solvent, the membrane potential (delta psi m) of the mammalian-derived mitochondria is kept unchanged or reduced by 50%-90% compared with that before separation, and the outer membrane integrity rate is larger than or equal to 90%. Under the state that the membrane potential is reduced, the outer membrane structure is still kept complete, and the capability of inducing host cell PINK1 / Parkin dependent mitochondrial autophagy and network remodeling is achieved, so that inferior mitochondria is selectively removed, and mitochondrial neogenesis is promoted. The preparation can induce systemic mitochondrial quality remodeling in multiple tissues, significantly improve ATP level, reduce ROS generation, increase mitochondrial DNA copy number, down-regulate aging markers p16 and p21, and improve cognition, movement and organ functions in an old animal model.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-aging mitochondrial preparation, its preparation method, and its application. Background Technology

[0002] Aging is a natural process of gradual structural and functional decline in the body under the combined influence of genetic and environmental factors. Its characteristics include decreased tissue repair capacity, disordered energy metabolism, and weakened antioxidant capacity. Aging not only affects lifespan but also significantly increases the risk of various age-related diseases such as cardiovascular disease, neurodegenerative diseases, and metabolic syndrome. Therefore, delaying aging and improving age-related symptoms have become important research directions in the fields of medicine and life sciences.

[0003] In recent years, various anti-aging agents have been developed to address aging and related diseases, including antioxidants (such as vitamin C and glutathione), hormone replacement therapies (such as melatonin), immunomodulators, bioactive peptides, and cell or organelle preparations. These agents can, to some extent, delay the decline of bodily functions and improve quality of life by scavenging free radicals, improving hormone levels, regulating immune function, and promoting tissue repair.

[0004] After the decline in mitochondrial function was confirmed as a significant driver of aging, mitochondrial-based anti-aging strategies have gained increasing attention. In recent years, mitochondrial transplantation (MT) has been considered a potential anti-aging strategy that can directly restore bioenergy. Anti-aging mitochondrial preparations refer to formulations made from exogenous healthy or functionally optimized mitochondria, prepared in a specific manner for injection or oral administration. By supplementing, replacing, or regulating host cell mitochondria, these preparations improve cellular energy metabolism and redox status, thereby delaying aging and alleviating age-related symptoms.

[0005] However, existing anti-aging mitochondrial formulations generally use mitochondria with intact membrane potential as raw materials, which require strict conditions for preparation, transportation, and storage, typically necessitating a complete cold chain to maintain mitochondrial functional activity. Therefore, there is a need to develop new mitochondrial formulations to address these issues. Summary of the Invention

[0006] To address the issue that existing anti-aging mitochondrial formulations generally use mitochondria with intact membrane potential as raw materials, requiring strict conditions for preparation, transportation, and storage, often necessitating a complete cold chain to maintain mitochondrial functional activity, this invention utilizes xeno-MTs with impaired membrane potential as signaling molecules to activate host PINK1 / Parkin-mediated mitophagy and mitochondrial fission. Through mitochondrial quality control, it aims to improve the body's aging phenotype and mitochondrial functional imbalance, providing an anti-aging mitochondrial formulation, its preparation method, and its applications. To achieve the above objectives, this invention employs the following technical solution.

[0007] Although the mechanisms of intercellular mitochondrial transfer (MT) are well understood, the effects of xeno-MT on delaying aging in middle-aged and aged mice have not been fully elucidated. This invention demonstrates that MT has therapeutic potential, improving performance and biochemical indicators in aged mice. Meanwhile, the preservation of isolated mitochondria remains a key concern in clinical MT research. This invention confirms that even mitochondria with impaired membrane potential can still activate host mitophagy and promote mitochondrial remodeling, just like functional donor mitochondria. Our findings provide clinical significance for using widely available and ethically sound xeno-MT for anti-aging. Based on this, this invention provides an anti-aging mitochondrial formulation, which is obtained by compounding mammalian-derived mitochondria with a solvent; the mass ratio of the mammalian-derived mitochondria to the volume ratio of the solvent is 2 mg to 20 mg: 100 mL.

[0008] The mitochondria of mammalian origin are isolated from mammals; the membrane potential of the mitochondria of mammalian origin remains unchanged or decreases by 50% to 90% compared to before isolation from mammals, and the outer membrane integrity is ≥90%.

[0009] The mammalian-derived mitochondria are selected from at least one of yak ear fibroblast mitochondria, human HEK293T cell mitochondria, human HepG2 cell mitochondria, and human HeLa cell mitochondria.

[0010] The solvent includes at least one of physiological saline, lactated Ringer's solution, compound electrolyte injection, glucose solution for injection, and compound electrolyte glucose injection.

[0011] The anti-aging mitochondrial formulation provided by this invention is an anti-aging formulation that does not rely on high membrane potential mitochondria to trigger systemic mitochondrial remodeling. This anti-aging mitochondrial formulation uses xenogeneic mammalian mitochondria whose membrane potential remains unchanged or decreases by 50%–90% compared to before isolation from mammals during preparation, storage, or transportation, and whose outer membrane integrity is ≥90%. It is compounded with a pharmaceutically acceptable isotonic solvent for injection. Even if the membrane potential decreases to within 10%–50% of that of fresh mitochondria, the mitochondria can still exert their anti-aging effect under structurally intact conditions. Therefore, the anti-aging mitochondrial formulation provided by this invention does not suffer from the defect of decreased membrane potential due to fluctuations in storage and transportation conditions, which would significantly weaken the efficacy of the mitochondrial formulation. Therefore, the anti-aging mitochondrial formulation provided by this invention not only does not increase production and logistics costs, but also does not limit its clinical application scope.

[0012] Furthermore, the mammalian-derived mitochondria are obtained from mammalian cells or tissues by differential centrifugation.

[0013] Furthermore, the steps for isolating the mammalian-derived mitochondria from mammalian cells or tissues are as follows: The mammalian cells or tissues are pretreated to purify and activate the cells, resulting in a cell precipitate.

[0014] The cell precipitate was disrupted in an isotonic solution, and the nuclei and large fragments were removed by differential centrifugation. The mitochondrial precipitate was collected to obtain the mammalian-derived mitochondria.

[0015] Furthermore, the preprocessing steps are as follows: Under low temperature conditions, the mammalian cells or tissues are first washed, and then enzymatically digested.

[0016] Low temperature conditions refer to 3.8℃~4.2℃.

[0017] Furthermore, the conditions for the differential centrifugation are as follows: Preliminary enucleation: 600×g~1,000×g, 5 minutes~10 minutes; Mitochondrial collection: 7,000×g~10,000×g, 10 minutes.

[0018] Furthermore, the mammalian-derived mitochondria are yak ear fibroblast mitochondria; the yak ear fibroblast mitochondria are isolated from yak ear fibroblasts.

[0019] The method for isolating mitochondria from yak ear fibroblasts includes the following steps: Obtain raw materials for yak ear fibroblasts.

[0020] Cell pretreatment: The yak ear fibroblast raw material was pretreated at low temperature (3.8℃~4.2℃) to obtain cell precipitate.

[0021] The cell precipitate was broken up in an isotonic solution, and the mitochondrial precipitate was collected to obtain the mitochondria of the yak ear fibroblasts.

[0022] The crushing process includes the following steps: The cells were mechanically disrupted in an isotonic solution to rupture the cell membrane and release organelles. The nuclei and large fragments were then removed by differential centrifugation.

[0023] Furthermore, the differential centrifugation conditions are as follows: preliminary denucleation: 600×g~1,000×g, 5 minutes~10 minutes; mitochondrial collection: 7,000×g~10,000×g, 10 minutes.

[0024] The present invention also provides a method for preparing the aforementioned anti-aging mitochondrial preparation, comprising the following steps: The anti-aging mitochondrial preparation is obtained by resuspending the mammalian-derived mitochondria in the solvent.

[0025] The final concentration of the mammalian-derived mitochondria is 0.02 mg / mL to 0.2 mg / mL.

[0026] The present invention also provides the application of the aforementioned anti-aging mitochondrial preparation in the preparation of anti-aging drugs.

[0027] Furthermore, the anti-aging drug uses the anti-aging mitochondrial preparation as its active ingredient.

[0028] Furthermore, the anti-aging drug also includes pharmaceutically acceptable excipients.

[0029] Furthermore, the excipients include any one or more combinations of trehalose, sucrose, mannitol, glucose, glycine, histidine, sodium citrate, phosphate buffer, acetate buffer, taurine, or L-carnitine.

[0030] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides an anti-aging mitochondrial preparation, which is an anti-aging preparation that does not rely on high membrane potential mitochondria to trigger systemic mitochondrial remodeling. This anti-aging mitochondrial preparation is obtained by compounding mammalian-derived mitochondria with a solvent; the mass ratio of mammalian-derived mitochondria to solvent is 2 mg~20 mg:100 mL; wherein, the mammalian-derived mitochondria are isolated from mammals; the membrane potential of the mammalian-derived mitochondria remains unchanged or decreases by 50%~90% compared to before isolation, and the outer membrane integrity rate is ≥90%; the mammalian-derived mitochondria include at least one of yak ear fibroblast mitochondria, human HEK293T cell mitochondria, human HepG2 cell mitochondria, and human HeLa cell mitochondria; the solvent includes at least one of physiological saline, lactated Ringer's solution, compound electrolyte injection, glucose solution for injection, and compound electrolyte glucose injection. The anti-aging mitochondrial formulation provided by this invention uses xenogeneic mammalian mitochondria with a membrane potential within 10% to 50% of that of fresh mitochondria and an outer membrane integrity of ≥90%, which are combined with pharmaceutically acceptable isotonic solvents for injection. Unlike existing anti-aging mitochondrial formulations that generally require donor mitochondria to maintain a high membrane potential to sustain respiratory chain activity, the anti-aging mitochondrial formulation provided by this invention does not have the same stringent requirements for preparation, transportation, and storage conditions as existing anti-aging mitochondrial formulations, and does not require a complete cold chain to maintain mitochondrial functional activity. Fluctuations in storage and transportation conditions can lead to a decrease in membrane potential, which in turn significantly weakens the efficacy of existing anti-aging mitochondrial formulations. This does not increase production and logistics costs, nor does it limit its clinical application. However, the anti-aging mitochondrial formulation provided by this invention uses mitochondria from xenogeneic mammals, whose membrane potential is within 10% to 50% of that of fresh mitochondria and whose outer membrane integrity is ≥90%, during preparation, storage, or transportation. These mitochondria are compounded with a pharmaceutically acceptable isotonic solvent for injection. Even if the membrane potential drops to within 10% to 50% of that of fresh mitochondria, the mitochondria can still exert their anti-aging effect while maintaining structural integrity. Therefore, the anti-aging mitochondrial formulation provided by this invention does not suffer from the defect of a decrease in membrane potential due to fluctuations in storage and transportation conditions, which would significantly weaken the efficacy of the mitochondrial formulation. Therefore, the anti-aging mitochondrial formulation provided by this invention not only does not increase production and logistics costs but also does not limit its clinical application scope.

[0031] 2. This invention has revealed that even when the membrane potential drops to the aforementioned range, mitochondria can still induce host PINK1 / Parkin-dependent mitophagy and network remodeling under structurally intact conditions, achieving a dual effect of clearing damaged endogenous mitochondria and promoting the generation of new mitochondria. This breakthrough significantly reduces dependence on donor freshness and cold chain transportation, while preserving the safety, biocompatibility, and anti-aging efficacy of the formulation, thus solving the problems of harsh storage and transportation conditions, limited sources, and ethical barriers associated with existing high-membrane-potential mitochondrial formulations.

[0032] 3. The formulation of the present invention utilizes the above-mentioned mechanism to trigger the closed-loop quality control of mitochondria in the host cell without relying on direct energy supply from donor mitochondria, thus possessing the dual effects of "removing defects and promoting regeneration". The requirements for temperature and time in the preparation, storage and transportation stages are significantly reduced, and it can withstand long-term refrigeration and short-term room temperature exposure, greatly reducing logistics and storage costs. The use of xenogeneic sources further expands the range of available donors, giving it broader application potential in the fields of anti-aging and related metabolic and degenerative diseases. Attached Figure Description

[0033] Figure 1 The figures shown are phenotypic characteristic diagrams of xenogeneic mitochondrial transplanted mice and control groups in embodiments of the present invention; wherein: Figure A shows the in vivo and exogenous mitochondrial transplantation and phenotypic evaluation; where AC: aged control mice; A: aged mice treated with xenogeneic mitochondrial transplantation. Figure B is a schematic diagram of the treatment plans for each group of animals; Figure C shows the scoring of 18-month-old mice based on the degree of aging indicators shown (0 points indicates none, 3 points indicates high); n=10 biologically independent samples; Figure D shows the duration of movement in the two groups of mice during the rotarod experiment; n=10 biologically independent samples. Figure E shows the white blood cell count; n=10 biologically independent samples; F plot shows the percentage of neutrophils; n=10 biologically independent samples; G plot represents the percentage of lymphocytes; n=10 biologically independent samples. Detailed Implementation

[0034] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0035] Example 1 A method for preparing an anti-aging mitochondrial preparation includes the following steps: 1. Donor cell preparation Healthy yak ear margin fibroblasts were obtained and harvested at 75% ± 5% confluence, yielding approximately 1.5 × 10⁻⁶ cells. 7 One donor cell.

[0036] The healthy yak ear marginal fibroblasts were obtained through primary cell isolation and culture. The primary cells were extracted in the laboratory from yak ear tissue. The steps are as follows:

[0037] 1) Preparation of digestive juice Prepare DMEM (Gibco), a 22 micropore filter tip, a large syringe, and collagenase II (Gibco).

[0038] 1.1) Pour the enzyme (dry powder) into a 50mL tube, and wash the enzyme dry powder tube 3 times with DMEM, using 1mL of DMEM each time.

[0039] 1.2) Pour DMEM into a 50mL tube and add it to a final volume of 50mL.

[0040] 1.3) After shaking well, filter into a new 50mL tube and add 1mL of double antibody.

[0041] 2) Cleaning the tissue 2.1) Prepare five 50mL tubes. Fill one tube about halfway with alcohol, and fill the other two tubes about halfway with DPBS (Gibco) containing 2% double antibiotic (Gibco) at a v / v ratio. Immerse the tissue in the alcohol for 30 seconds, pour the alcohol into an empty tube, pour the tissue into the DPBS tube, pour the DPBS from that tube into an empty tube, and pour the tissue into another DPBS tube (that is, wash twice with DPBS). Then place the tissue into a culture dish and cap it.

[0042] 2.2) Keep the tissue moist at all times; a small amount of digestive fluid can be added to prevent it from drying out too much. Tilt the petri dish and mince the tissue until it becomes a paste.

[0043] 2.3) Trim the tip of the blue pipette and lightly heat it in an alcohol lamp flame. Transfer 5 mL of the digestion solution into a 15 mL tube. Transfer a small amount of the digestion solution from the tube into a culture dish, mix slightly, and then transfer the digestion solution along with the tissue into the tube. This process can be repeated several times to minimize tissue loss.

[0044] 2.4) After marking, seal the container and place it in a shaker at 37°C and 220 rpm for 2 hours to digest.

[0045] 3) Cell Culture 3.1) After digestion, there should only be a small number of large particles in the tube; otherwise, the particles were not cut finely enough. Centrifuge at 3000g for 5 minutes at 20℃.

[0046] 3.2) Carefully pour out the digestion solution, add 5 mL of DPBS DMEM (2% penicillin antibody, v / v) to wash, then centrifuge, and repeat twice.

[0047] 3.3) Add 5 mL of culture medium (2% double antibiotic, v / v), resuspend, add to a new culture flask, label, and place in an incubator.

[0048] 4) Change the medium daily. When a large number of dead cells are observed, rinse the culture flask with DPBS to remove most of the white tissue from the sidewalls. Then change the medium and continue culturing. At this point, a small number of sparse cells should be visible in the culture flask. After the cells have basically reached confluence, passage them.

[0049] 2. Mitochondrial isolation The harvested donor cells were homogenized at 4°C and then subjected to differential centrifugation at 600g×10min and 11000g×10min respectively. The precipitate was collected to obtain mitochondria (precipitate).

[0050] 3. Membrane potential detection The resulting mitochondria (precipitate) showed a partial decrease in membrane potential during routine preparation, storage or transportation (such as short-term storage or cryopreservation at 4°C).

[0051] The mitochondrial precipitate exhibiting a partial decrease in membrane potential was resuspended in physiological saline to obtain a mitochondrial suspension. Samples were taken from the mitochondrial suspension, and the membrane potential (ΔΨm) was detected using a JC-1 probe.

[0052] The test results showed that the ΔΨm of the mitochondria was reduced by about 40% compared to the freshly prepared mitochondria, and the outer membrane integrity rate was 95% (≥90%).

[0053] 4. Formulation preparation (1) Protein concentration determination The mitochondrial suspension was analyzed using the BCA method according to the kit (Beyotime, P0012): Preparation of standard solutions: Add 1.2 mL of protein standard preparation solution to one tube of protein standard (30 mg BSA), and dissolve thoroughly to prepare a 25 mg / mL protein standard solution. To prepare a 0.5 mg / mL protein standard, add 20 µL of the 25 mg / mL protein standard to 980 µL of diluent.

[0054] Preparation of working solution: Add 100µL of BCA reagent B to 5mL of BCA reagent A, mix well, and prepare 5.1mL of BCA working solution.

[0055] Protein concentration determination: Add 0 µL, 1 µL, 2 µL, 4 µL, 8 µL, 12 µL, 16 µL, and 20 µL of standard solutions to the wells of a 96-well plate. Then, add standard diluent to bring the total volume to 20 µL. This corresponds to standard concentrations of 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, respectively. Add 20 µL of sample solution to the wells of the 96-well plate. Add 200 µL of BCA working solution to each well and incubate at 37°C for 25 minutes.

[0056] Color development and reading: Measure the absorbance at A562 or other wavelengths between 540nm and 595nm using an ELISA reader, and calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0057] The results are as follows: Standard curve: y = 1.3381x - 0.2308, R² = 0.9918, and the calculated result is 2.41 mg / mL.

[0058] (2) Dilution and preparation Dilute the protein concentration to the target dose (1 mg / kg body weight), i.e., dilute with physiological saline to 0.02 mg / mL, and prepare an anti-aging mitochondrial preparation for later use. Store at 4°C for later use.

[0059] The concentration of the prepared anti-aging mitochondrial preparation was 0.02 mg / mL.

[0060] 5. Dosing regimen Administer the injection via the tail vein to aged mice, once every 48 hours, for a total of 10 times.

[0061] Among them, aged mice generally refer to naturally aged mice that are ≥18 months old, specifically 18-month-old C57BL / 6 strain mice. The dosage is 1 mg / kg body weight.

[0062] Example 2 A method for preparing an anti-aging mitochondrial preparation includes the following steps: 1. Donor cell preparation Healthy yak ear margin fibroblasts were obtained and harvested at 80% confluence, yielding approximately 1.5 × 10⁻⁶ cells. 7 One donor cell.

[0063] The healthy yak ear marginal fibroblasts were obtained through primary cell isolation and culture. The primary cells were extracted in the laboratory from yak ear tissue. The steps are as follows:

[0064] 1) Preparation of digestive juices: Prepare DMEM (Gibco), a 22 micropore filter tip, a large syringe, and collagenase II (Gibco).

[0065] 1.1) Pour the enzyme (dry powder) into a 50mL tube, and wash the enzyme dry powder tube 3 times with DMEM, using 1mL of DMEM each time.

[0066] 1.2) Pour DMEM into a 50mL tube and add it to a final volume of 50mL.

[0067] 1.3) After shaking well, filter into a new 50mL tube and add 1mL of double antibody.

[0068] 2) Cleaning the tissue: 2.1) Prepare five 50mL tubes. Fill one tube about halfway with alcohol, and fill the other two tubes about halfway with DPBS (Gibco) containing 2% double antibiotic (Gibco) at a v / v ratio. Immerse the tissue in the alcohol for 30 seconds, pour the alcohol into an empty tube, pour the tissue into the DPBS tube, pour the DPBS from that tube into an empty tube, and pour the tissue into another DPBS tube (that is, wash twice with DPBS). Then place the tissue into a culture dish and cap it.

[0069] 2.2) Keep the tissue moist at all times; a small amount of digestive fluid can be added to prevent it from drying out too much. Tilt the petri dish and mince the tissue until it becomes a paste.

[0070] 2.3) Trim the tip of the blue pipette and lightly heat it in an alcohol lamp flame. Transfer 5 mL of the digestion solution into a 15 mL tube. Transfer a small amount of the digestion solution from the tube into a culture dish, mix slightly, and then transfer the digestion solution along with the tissue into the tube. This process can be repeated several times to minimize tissue loss.

[0071] 2.4) After marking, seal the container and place it in a shaker at 37°C and 220 rpm for 2 hours to digest.

[0072] 3) Cell culture: 3.1) After digestion, there should only be a small number of large particles in the tube; otherwise, the particles were not cut finely enough. Centrifuge at 2500g for 5 minutes at 20℃.

[0073] 3.2) Carefully pour out the digestion solution, add 5 mL of DPBS DMEM (2% double antibody, v / v) to wash, then centrifuge, and repeat once.

[0074] 3.3) Add 5 mL of culture medium (2% double antibiotic, v / v), resuspend, add to a new culture flask, label, and place in an incubator.

[0075] 4) Change the medium daily. When a large number of dead cells are observed, rinse the culture flask with DPBS to remove most of the white tissue from the sidewalls. Then change the medium and continue culturing. At this point, a small number of sparse cells should be visible in the culture flask. After the cells have basically reached confluence, passage them.

[0076] 2. Mitochondrial isolation The harvested donor cells were homogenized at 4°C and then subjected to differential centrifugation at 600g×10min and 11000g×10min respectively. The precipitate was collected to obtain mitochondria (precipitate).

[0077] 3. Membrane potential detection The resulting mitochondria (precipitate) showed a partial decrease in membrane potential during routine preparation, storage or transportation (such as short-term storage or cryopreservation at 4°C).

[0078] The mitochondrial precipitate exhibiting a partial decrease in membrane potential was resuspended in physiological saline to obtain a mitochondrial suspension. Samples were taken from the mitochondrial suspension, and the membrane potential (ΔΨm) was detected using a JC-1 probe.

[0079] The test results showed that the ΔΨm of the mitochondria was reduced by about 40% compared to the freshly prepared mitochondria, and the outer membrane integrity rate was 95% (≥90%).

[0080] 4. Formulation preparation (1) Protein concentration determination The mitochondrial suspension was analyzed using the BCA method according to the kit (Beyotime, P0012): Preparation of standard solutions: Add 1.2 mL of protein standard preparation solution to one tube of protein standard (30 mg BSA), and dissolve thoroughly to prepare a 25 mg / mL protein standard solution. To prepare a 0.5 mg / mL protein standard, add 20 µL of the 25 mg / mL protein standard to 980 µL of diluent.

[0081] Preparation of working solution: Add 100µL of BCA reagent B to 5mL of BCA reagent A, mix well, and prepare 5.1mL of BCA working solution.

[0082] Protein concentration determination: Add 0 µL, 1 µL, 2 µL, 4 µL, 8 µL, 12 µL, 16 µL, and 20 µL of standards to the wells of a 96-well plate. Add standard diluent to bring the total volume to 20 µL. This corresponds to standard concentrations of 0.5 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, respectively. Add 20 µL of sample to the wells of the 96-well plate. Add 200 µL of BCA working solution to each well and incubate at 37°C for 25 minutes.

[0083] Color development and reading: Measure the absorbance at A562 or other wavelengths between 540nm and 595nm using an ELISA reader, and calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0084] The result is: y=1.1558x-0.1441, R²=0.9977, and the calculated result is 2.24mg / mL.

[0085] (2) Dilution and preparation Dilute the protein concentration to the target dose (5 mg / kg body weight), i.e., dilute with physiological saline to 0.1 mg / mL, and prepare an anti-aging mitochondrial preparation for later use. Store at 4°C for later use.

[0086] The concentration of the prepared anti-aging mitochondrial preparation was 0.1 mg / mL.

[0087] 5. Dosing regimen Administer the injection via the tail vein to aged mice, once every 48 hours, for a total of 10 times.

[0088] Among them, aged mice generally refer to naturally aged mice that are ≥18 months old, specifically 18-month-old C57BL / 6 strain mice. The dosage is 5 mg / kg body weight.

[0089] Example 3 A method for preparing an anti-aging mitochondrial preparation includes the following steps: 1. Donor cell preparation Healthy yak ear margin fibroblasts were obtained and harvested at 80% confluence, yielding approximately 1.5 × 10⁻⁶ cells. 7 One donor cell.

[0090] The healthy yak ear marginal fibroblasts were obtained through primary cell isolation and culture. The primary cells were extracted in the laboratory from yak ear tissue. The steps are as follows:

[0091] 1) Preparation of digestive juices: Prepare DMEM (Gibco), a 22 micropore filter tip, a large syringe, and collagenase II (Gibco).

[0092] 1.1) Pour the enzyme (dry powder) into a 50mL tube, and wash the enzyme dry powder tube 3 times with DMEM, using 1mL of DMEM each time.

[0093] 1.2) Pour DMEM into a 50mL tube and add it to a final volume of 50mL.

[0094] 1.3) After shaking well, filter into a new 50mL tube and add 1mL of double antibody.

[0095] 2) Cleaning the tissue: 2.1) Prepare five 50mL tubes. Fill one tube about halfway with alcohol, and fill the other two tubes about halfway with DPBS (Gibco) containing 2% double antibiotic (Gibco) at a v / v ratio. Immerse the tissue in the alcohol for 30 seconds, pour the alcohol into an empty tube, pour the tissue into the DPBS tube, pour the DPBS from that tube into an empty tube, and pour the tissue into another DPBS tube (that is, wash twice with DPBS). Then place the tissue into a culture dish and cap it.

[0096] 2.2) Keep the tissue moist at all times; a small amount of digestive fluid can be added to prevent it from drying out too much. Tilt the petri dish and mince the tissue until it becomes a paste.

[0097] 2.3) Trim the tip of the blue pipette and lightly heat it in an alcohol lamp flame. Transfer 5 mL of the digestion solution into a 15 mL tube. Transfer a small amount of the digestion solution from the tube into a culture dish, mix slightly, and then transfer the digestion solution along with the tissue into the tube. This process can be repeated several times to minimize tissue loss.

[0098] 2.4) After marking, seal the container and place it in a shaker at 37°C and 220 rpm for 2 hours to digest.

[0099] 3) Cell culture: 3.1) After digestion, there should only be a small number of large particles in the tube; otherwise, the particles were not cut finely enough. Centrifuge at 3000g for 5 minutes at 20℃.

[0100] 3.2) Carefully pour out the digestion solution, add 5 mL of DPBS DMEM (2% penicillin antibody, v / v) to wash, then centrifuge, and repeat twice.

[0101] 3.3) Add 5 mL of culture medium (2% double antibiotic, v / v), resuspend, add to a new culture flask, label, and place in an incubator.

[0102] 4) Change the medium daily. When a large number of dead cells are observed, rinse the culture flask with DPBS to remove most of the white tissue from the sidewalls. Then change the medium and continue culturing. At this point, a small number of sparse cells should be visible in the culture flask. After the cells have basically reached confluence, passage them.

[0103] 2. Mitochondrial isolation The harvested donor cells were homogenized at 4°C and then subjected to differential centrifugation at 600g×10min and 11000g×10min respectively. The precipitate was collected to obtain mitochondria (precipitate).

[0104] 3. Membrane potential detection The resulting mitochondria (precipitate) showed a partial decrease in membrane potential during routine preparation, storage or transportation (such as short-term storage or cryopreservation at 4°C).

[0105] The batch of mitochondrial precipitate was resuspended in physiological saline to obtain a mitochondrial suspension. Samples were taken from the mitochondrial suspension, and the membrane potential (ΔΨm) was detected using a JC-1 probe.

[0106] The test results showed that the ΔΨm of the mitochondria was reduced by about 40% compared to freshly prepared mitochondria, and the outer membrane integrity rate was 95% ≥ 90%.

[0107] 4. Formulation preparation (1) Protein concentration determination The mitochondrial suspension was analyzed using the BCA method according to the kit (Beyotime, P0012): Preparation of standard solutions: Add 1.2 mL of protein standard preparation solution to one tube of protein standard (30 mg BSA), and dissolve thoroughly to prepare a 25 mg / mL protein standard solution. To prepare a 0.5 mg / mL protein standard, add 20 µL of the 25 mg / mL protein standard to 980 µL of diluent.

[0108] Preparation of working solution: Add 100µL of BCA reagent B to 5mL of BCA reagent A, mix well, and prepare 5.1mL of BCA working solution.

[0109] Protein concentration determination: Add 0 µL, 1 µL, 2 µL, 4 µL, 8 µL, 12 µL, 16 µL, and 20 µL of standard solutions to the wells of a 96-well plate. Then, add standard diluent to bring the total volume to 20 µL. This corresponds to standard concentrations of 0 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, respectively. Add 20 µL of sample solution to the wells of the 96-well plate. Add 200 µL of BCA working solution to each well and incubate at 37°C for 25 minutes.

[0110] Color development and reading: Measure the absorbance at A562 or other wavelengths between 540nm and 595nm using an ELISA reader, and calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0111] The result is: y=1.1148x-0.1907, R²=0.996, and the calculated result is 2.55mg / mL.

[0112] (2) Dilution and preparation Dilute the protein concentration to the target dose (10 mg / kg body weight), i.e., dilute with physiological saline to 0.2 mg / mL, and prepare an anti-aging mitochondrial preparation for later use. Store at 4°C for later use.

[0113] The concentration of the prepared anti-aging mitochondrial preparation was 0.2 mg / mL.

[0114] 5. Dosing regimen Administer the injection via the tail vein to aged mice, once every 48 hours, for a total of 10 times.

[0115] Among them, aged mice generally refer to naturally aged mice that are ≥18 months old, specifically 18-month-old C57BL / 6 strain mice. The dosage is 10 mg / kg body weight.

[0116] To illustrate the efficacy of the anti-aging mitochondrial preparation provided by this invention, the following experiment was conducted using the anti-aging mitochondrial preparation prepared in Example 1 as an example: 1) Phenotypic characteristics of mice supplemented with mitochondrial formulations compared to aged control groups In this invention, the rotating bar test is designed as a behavioral indicator to assess the fatigue resistance of mice. Throughout the experiment, the rotating bar speed is maintained at 40 rpm, and the time each animal spends on the bar is recorded as the latency period. Each mouse undergoes four consecutive trials, with a 10-minute interval between each trial to avoid stress and fatigue. The longest dwell time is used for data analysis.

[0117] The mice mentioned above are aged mice, which usually refer to naturally aged mice that are ≥18 months old, specifically 18-month-old C57BL / 6 strain mice.

[0118] The mice were divided into two groups as follows: AC: Aged control group; A: Xenogeneic mitochondrial transplantation treatment group. The aged control group is also known as aged mice. The xenogeneic mitochondrial transplantation treatment group mice are also known as mitochondrial supplementation mice, aged mice that received mitochondrial supplements.

[0119] The aged control mice were not treated in any way, while the xenogeneic mitochondrial transplantation treatment group mice were given the anti-aging mitochondrial preparation formulated in Example 1 by tail vein injection (1 mg / kg body weight).

[0120] In addition, aging parameters were scored on aged mice to assess their aging performance, with a scoring range of 0 (none) to 3 (high). Scoring indicators included hair loss, graying hair, kyphosis, and body size reduction. Whole blood was collected via retroorbital sampling for complete blood cell count analysis. Results of mitochondrial formulation supplementation of mouse phenotypic characteristics are as follows: Figure 1 As shown.

[0121] Mitochondrial supplements ( Figure 1 Figure A in the middle and Figure 1 Figure B in the diagram shows fewer signs of aging. Figure 1(Figure C in the figure), with significant differences, especially in hair removal and kyphosis. In the rotarod experiment, the residence time on the rotarod was significantly prolonged in aged mice receiving the mitochondrial preparation (316.40s±12.09s), while that in the aged control group was only 274.60±8.65s (Figure C in the figure). Figure 1 (Figure D in the diagram). Furthermore, mitochondrial preparation supplementation did not cause a white blood cell count increase (Figure D in the diagram). Figure 1 Figure E in the diagram), percentage of neutrophils ( Figure 1 (F-chart in the figure) and lymphocyte percentage ( Figure 1 The changes in the G-graph (in the graph).

[0122] As can be seen from the above, mitochondrial preparations supplement ( Figure 1 Figure A in the middle and Figure 1 Figure B in the diagram shows fewer signs of aging. Figure 1 (See Figure C in the diagram). In the rotator bar experiment, aged mice receiving the mitochondrial preparation exhibited a significantly prolonged dwell time on the rotator bar, reaching 316.40 ± 12.09 seconds, while aged control mice could only maintain this duration for 274.60 ± 8.65 seconds. Figure 1 (Figure D in the diagram). Furthermore, xenotransplantation of mitochondria did not cause a white blood cell count ( Figure 1 Figure E in the diagram), percentage of neutrophils ( Figure 1 (F-chart in the figure) and lymphocyte percentage ( Figure 1 The changes in the G-plot (in the graph) indicate the absence of an inflammatory response.

[0123] 2) Detection of exogenous mitochondrial DNA Eleven organs or tissues were collected to detect the presence of exogenous mitochondria, and the results are shown in Table 1. Tissue DNA was extracted according to the instructions of the universal genomic DNA extraction kit (Aidlab, Beijing, China). Before ddPCR detection, the genomic DNA of each sample was diluted to 100 ng / µL. Based on the mitochondrial genome sequence of yak (Bos grunniens) listed in GenBank accession number KU891851, the NADH dehydrogenase subunit 1 (ND1) gene sequence was analyzed using Primer Premier 5.0 software, and one probe (ND1-probe) and a pair of specific primers (ddPCR-ND1) were designed. Their sequences are listed in Table 1.

[0124] Table 1. Detection sequences of exogenous mitochondria The ddPCR reaction system is as follows: 10 µL of ddPCR Supermix for probe (no dUTP), 1.8 µL each of forward and reverse primers (ddPCR-ND1, 10 µM), 0.5 µL of probe (10 µM), 2 µL of template DNA, and ddH2O to a final volume of 20 µL. The amount of nucleic acid added to each system must not exceed the specified detection range (1–20,000 copies of intact genomic DNA).

[0125] Add 20 µL of sample reaction mixture to the eight wells in the middle row of a DG8 reaction plate. Add 70 µL of oil to the oil bath of the DG8 reaction plate to form an oil barrier. After covering with a gasket, place the plate into a droplet generator to generate droplets. Then transfer the droplets to a 96-well plate and seal with a PX1 heat sealer for PCR amplification.

[0126] The PCR amplification program was as follows: 95°C for 10 min; 40 cycles (94°C for 30 s, 60°C for 60 s); 98°C for 10 min.

[0127] After PCR amplification, the 96-well plate was placed in a droplet detector (QX200) for data reading, and the data was analyzed using QXManager 1.2 Standard Edition software (Bio-Rad). The results were expressed as the absolute quantitative value of the target DNA in copies per microliter (copies / µL, as shown in Table 2) during the ddPCR reaction.

[0128] Table 2. Copy number of exogenous mitochondrial DNA per microliter of genomic DNA Note: n = 10 biologically independent samples, *p < 0.05.

[0129] The results showed that all mice that received mitochondrial supplementation had a large number of exogenous mitochondria in all observed tissues. 3) Autophagy effect identification Proteins were extracted from tissue samples using a protein extraction kit (GenePool, GPP1815) according to the manufacturer's instructions. The protein concentration was adjusted with water and 5×SDS-PAGE Loading Buffer (GenePool, GPP1820) as needed, and the samples were denatured at 100°C for 10 minutes. Separating gels (12%) and stacking gels (5%) were prepared according to an SDS-PAGE gel preparation kit (GenePool, GPP1816). 35 μg of sample was loaded into each well. Electrophoresis was performed at 80 V on the stacking gel and at 120 V on the separating gel until the bromophenol blue front reached the bottom of the gel.

[0130] During protein transfer, the 0.22 μm PVDF membrane was pre-soaked in methanol for 30 seconds, then placed in WB transfer buffer (GenePool, GPP1817), and assembled with the gel and filter paper in a transfer sandwich manner to ensure no air bubbles. The wet transfer conditions were constant current 300 mA for 2 hours.

[0131] After transfer, the membrane was blocked at room temperature for 1 hour in 5% (m / v) skim milk powder as blocking buffer. Primary antibodies LC3B (Abcam, ab48394, 1:1000), p62 (CST, 39749, 1:1000), and BNIP3 (Proteintech, ab189846, 1:1000) were then incubated overnight at 4°C with shake. After washing (TBST, 5 min × 3 times), HRP-labeled secondary antibody (1:5000 dilution, anti-rabbit or anti-mouse) was added and incubated at room temperature for 50 minutes, followed by another wash (TBST, 5 min × 4 times).

[0132] Finally, add ECL developer (GenePool, GPP1824) and react for 1 minute, then expose and develop with film in a darkroom.

[0133] Table 3. Quantification of Mitochondrial Autophagy-Related Proteins Note: n = 4 biologically independent samples, *p < 0.05.

[0134] 4) Detection of biochemical indicators in mouse tissues The levels of ATP and ROS in various cells and tissues (heart, liver, spleen, lung, kidney, ovary, stomach, brain, hypothalamus, and skeletal muscle) of mice were determined using commercially available kits (Beyotime; ATP assay kit, S0026; ROS assay kit, S0033S). ATP levels were determined spectrophotometrically, and ROS levels were determined using the DCFH-DA fluorescence method.

[0135] (1) ATP detection: Add lysis buffer at a ratio of approximately 100-200 μL per 20 mg of tissue, then homogenize using a glass homogenizer or other homogenizing equipment. Thorough homogenization ensures complete tissue lysis. After lysis, centrifuge at 12000g for 5 minutes at 4°C, and collect the supernatant for subsequent assays. Thaw the reagents on ice. Dilute the ATP standard solution with ATP assay lysis buffer to appropriate concentration gradients: 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, and 10 μM. Prepare an appropriate amount of ATP assay working solution, requiring 100 mL for each sample or standard. Thaw the reagents on ice. Add 100 μL of ATP assay reagent to 900 mL of ATP assay reagent diluent to prepare 1 mL of ATP assay working solution. Add 100 mL of ATP assay working solution to the test well or test tube. Incubate at room temperature for 4 minutes to allow all background ATP to be consumed, thereby reducing the background. Add 20 mL of sample or standard to the test well or test tube, mix quickly with a pipette (micropipette), and after at least 2 seconds, measure the RLU value with a chemiluminescence analyzer.

[0136] (2) ROS detection: Fresh tissue samples were washed with PBS. 50 mg of tissue was accurately weighed and added to 1 mL of homogenization buffer A. The mixture was homogenized thoroughly using a glass homogenizer. Centrifuged at 100 × g, 4 °C for 3 minutes, and the precipitate was discarded. The supernatant was collected. 200 µL of the homogenized supernatant and 2 µL of DHE probe were added to a 96-well plate and mixed thoroughly by pipetting. The plate was incubated at 37 °C in the dark for 25 minutes. The plate was then placed in a fluorescence microplate reader, and the fluorescence intensity was detected at an excitation wavelength of 488 nm–535 nm and an emission wavelength of 610 nm. Another 50 µL of the supernatant homogenate was diluted approximately 30 times with PBS. 100 µL of this solution was used for protein quantification. The tissue reactive oxygen species intensity was expressed as fluorescence intensity (RFU) / protein concentration (mg protein).

[0137] (3) Mitochondrial DNA copy number detection: The copy number of the mitochondrial genome was determined by detecting the copy number of the ND1 fragment using qPCR (LightCycler480 II, Roche, Rotkreuz, Switzerland) with reference to the copy number of the diploid nuclear genome. Nuclear DNA content was determined by amplifying a fragment of the single-copy gene GAPDH. Specifically, a 126 bp primer for ND1 (qPCR-ND1) and an 85 bp primer for GAPDH (qPCR-GAPDH) were designed based on GenBank sequences DQ106412 and GU214026, respectively (Table 4). The amplification products were obtained according to standard PCR procedures, purified, and cloned into the pMD18-T vector (Sangon Co., China). Standards were prepared at 10... 7 Ten-fold serial dilutions were performed to a 10² copy number for absolute quantification of mitochondrial DNA copy number per cell in each sample. The mtDNA copy number per cell in mice was calculated by multiplying the ratio of ND1 to GAPDH quantification by 2.

[0138] Table 4 Mitochondrial DNA Detection Sequences (4) Telomere length detection: For aged mice, the relative telomere length of brain tissue and muscle was assessed using a relative mouse telomere length quantification qPCR assay kit (M8908, ScienceCell, USA).

[0139] First, restore the lyophilized telomere primers (Cat #8908a) and single-copy reference gene primers (Cat #M8908b): add 200µL of nuclease-free water (Cat #8908c) to each bottle, mix thoroughly, aliquot, and store at -20°C, avoiding repeated freeze-thaw cycles. Before the experiment, bring the primer reagents to room temperature and centrifuge at 1500×g for 1 minute. Two sets of qPCR reactions are required for each genomic DNA sample: one set with telomere primers and the other with single-copy reference gene primers. The total reaction volume per well is 20µL, including: 1µL genomic DNA template (0.2ng / µL~2ng / µL), 2µL primer solution, 10µL 2× GoldNStart TaqGreenqPCR Master Mix (Cat #MB6018a-1), and 7µL nuclease-free water. After sealing, centrifuge for 15 seconds. It is recommended to perform at least 3 replicates per set. The qPCR reaction program was as follows: pre-denaturation at 95°C for 10 minutes; followed by 32 cycles, each cycle consisting of 95°C for 20 seconds, 52°C for 20 seconds, and 72°C for 45 seconds, with fluorescence acquisition at 72°C; finally, melting curve analysis was performed. The relative telomere lengths between samples were calculated using the ∆∆Cq method.

[0140] (5) SOD and MDA detection: In addition, in accordance with the manufacturer's instructions, the total SOD activity and MDA content in the tissues were determined using the Total SOD Activity Assay Kit (NBT Method) (Beyotime, S0101) and the Lipid Peroxidation (MDA) Assay Kit (Beyotime, S0131), respectively.

[0141] Sample preparation includes the following steps: After perfusion of animals with heparinized saline to remove blood, tissue samples were obtained. Pre-chilled PBS was added for homogenization. The homogenate was centrifuged at 4°C, and the supernatant was used as the test sample. Protein concentration was first determined using a BCA protein assay kit. Kit preparation included: First, preparing the NBT / enzyme working solution by mixing 158µL of SOD detection buffer, 1µL of NBT, and 1µL of enzyme solution in a 160µL volume per reaction, and preparing an appropriate amount of NBT / enzyme working solution according to the number of test samples. This working solution was stored at 4°C. Then, the reaction initiation working solution was prepared by diluting the reaction initiation solution (40X) at a ratio of 1µL reaction initiation solution to 39µL SOD detection buffer, using freshly prepared solutions. Sample wells and blank control wells were set up according to the 96-well plate, and the test samples and solutions were added sequentially. After adding the reaction initiation working solution, the mixture was thoroughly mixed and incubated at 37°C for 30 minutes. Afterwards, the absorbance was measured at 560nm, and 600nm was selected as the reference wavelength. Finally, the total SOD activity in the sample was calculated using the inhibition percentage formula, and the calculated SOD enzyme activity units were converted between the inhibition percentage and (1 - inhibition percentage).

[0142] The formula for calculating the inhibition percentage is: (A) 空白对照1 -A 空白对照2 )-(A 样品 -A 空白对照3 ) / (A 空白对照1 -A 空白对照2 ) × 100%.

[0143] Tissue samples were homogenized using PBS. The tissue weight accounted for 10% of the homogenate or lysis buffer. After homogenization or lysis, the samples were centrifuged at 12,000g for 10 minutes, and the supernatant was used for subsequent assays. After preparation, the protein concentration was determined using a BCA protein assay kit to provide data for calculating the MDA content per unit protein weight. TBA stock solution was prepared at a concentration of 0.37%. To accelerate TBA dissolution, it was heated to 70°C and mixed using a Vortex mixer. The prepared stock solution should be stored at room temperature, protected from light. Next, the MDA detection working solution was prepared, with an appropriate amount prepared according to the number of samples. Finally, standards were diluted with distilled water to the required concentrations (1μM, 2μM, 5μM, 10μM, 20μM, and 50μM) for subsequent standard curve preparation. PBS was added to centrifuge tubes as a blank control, followed by the standards and the sample to be tested. Then, the MDA detection working solution was added, mixed, and heated in a 100°C water bath for 15 minutes. After heating, cool to room temperature in a water bath and centrifuge at 1000g for 10 minutes at room temperature. Add 200 μL of supernatant to a 96-well plate and measure the absorbance at 532 nm using a microplate reader. MDA content is calculated based on the standard curve results; for tissue samples, MDA content is expressed as protein per unit or tissue weight.

[0144] (6) Detection of WB-related indicators: Tissue proteins were extracted using a tissue protein extraction kit (GenePool, GPP1815) according to the manufacturer's instructions. The protein concentration was adjusted with water and 5×SDS-PAGE Loading Buffer (GenePool, GPP1820) as needed for loading, and the protein was denatured at 100°C for 10 minutes. Separating gels (15%) and stacking gels (5%) were prepared according to an SDS-PAGE gel preparation kit (GenePool, GPP1816). 35 μg of sample was loaded into each well. Electrophoresis was performed at 80 V on the stacking gel and at 120 V on the separating gel until the bromophenol blue front reached the bottom of the gel.

[0145] During protein transfer, a 0.22 μm pore size PVDF membrane was pre-soaked in methanol for 30 seconds, then placed in WB transfer buffer (GenePool, GPP1817), and assembled with gel and filter paper in a transfer sandwich manner to ensure no air bubbles. The wet transfer conditions were constant current 300 mA for 2 hours.

[0146] After transfer, the membrane was blocked in blocking buffer (5% (m / v) skim milk powder) at room temperature for 1 hour. Primary antibodies p16 (Abcam, ab189034, 1:500), p21 (Proteintech, 28248-1-AP, 1:500), DRP1 (Abcam, ab184247, 1:1000), and MFN2 (Abcam, ab124773, 1:2000) were used, and the membranes were incubated overnight at 4°C with shake. After washing (TBST, 5 min × 3 times), HRP-labeled secondary antibody (1:5000 dilution, anti-rabbit or anti-mouse) was added, and the membranes were incubated at room temperature for 50 minutes, followed by washing (TBST, 5 min × 4 times).

[0147] Finally, add ECL developer (GenePool, GPP1824) and react for 1 minute, then expose and develop with film in a darkroom.

[0148] The energy production, ROS content, mitochondrial DNA copy number, telomere length, antioxidant capacity, mitochondrial kinetics, and aging marker content of each tissue are shown in Tables 5 to 11.

[0149] Table 5. ATP content (nmol / mg protein) in multiple organs / tissues Note: n = 10 biologically independent samples, * p <0.05.

[0150] Table 6. ROS content (au / µg protein) in multiple organs / tissues Note: n = 10 biologically independent samples, * p <0.05.

[0151] Table 7 mtDNA copy number in multiple organs / tissues Note: n = 10 biologically independent samples, * p <0.05.

[0152] Table 8. Relative length of telomeres in skeletal muscles Note: n = 10 biologically independent samples, * p <0.05.

[0153] Table 9. SOD activity and MDA content (nmol / mg protein) Note: n = 3 biologically independent samples, * p <0.05.

[0154] Table 10 Detection of mitochondrial fission / fusion-related proteins Note: n = 3 biologically independent samples, * p <0.05.

[0155] Table 11 Detection of Aging Biomarkers Note: n = 3 biologically independent samples, * p <0.05.

[0156] The experimental results above show that, regardless of whether the mitochondrial membrane potential decreases to some extent during storage and transportation after preparation, the mitochondrial preparation significantly restores energy production in the brain, heart, hypothalamus, liver, skeletal muscle, and ovaries of aged mice, and reduces ROS levels in all organs except the lungs and spleen. The mtDNA copy numbers in the brain, heart, hypothalamus, kidneys, lungs, skeletal muscle, ovaries, and spleen are all significantly increased. In skeletal muscle, the relative telomere length of mice in the preparation-treated group was also significantly longer than that of mice in the control group. Multi-tissue analysis results showed that mice in the preparation-treated group had stronger antioxidant capacity. Simultaneously, mitochondrial preparation supplementation can promote mitochondrial division, inhibit mitochondrial fusion, and induce the division of damaged mitochondria, helping to clear damaged mitochondria. Ultimately, this achieves the goal of significantly reducing the levels of aging markers.

[0157] In summary, the anti-aging mitochondrial formulation provided by this invention can significantly improve aging phenotypes and enhance antioxidant capacity in aged animal models, even if the membrane potential is reduced during the conventional preparation and storage process, and achieve lasting metabolic optimization and aging reversal.

[0158] Unlike existing anti-aging mitochondrial formulations, which generally require donor mitochondria to maintain a high membrane potential to sustain respiratory chain activity, the anti-aging mitochondrial formulation provided by this invention has less stringent requirements for preparation, transportation, and storage conditions, and does not require a continuous cold chain to maintain mitochondrial functional activity. Fluctuations in storage and transportation conditions can lead to a decrease in membrane potential, significantly weakening the efficacy of existing anti-aging mitochondrial formulations. This not only increases production and logistics costs but also limits their clinical application. However, the anti-aging mitochondrial formulation provided by this invention uses xenogeneic mammalian mitochondria that, during preparation, storage, or transportation, remain unchanged or decrease by 50%–90% compared to their pre-isolation state, with an outer membrane integrity ≥90%, and is compounded with a pharmaceutically acceptable isotonic solvent for injection. Even if the membrane potential decreases to within 10%–50% of that of fresh mitochondria, the mitochondria can still exert their anti-aging effects while maintaining structural integrity. Therefore, the anti-aging mitochondrial formulation provided by this invention does not suffer from the drawback of decreased membrane potential due to fluctuations in storage and transportation conditions, which could lead to a significant reduction in the efficacy of the mitochondrial formulation. Therefore, the anti-aging mitochondrial preparation provided by this invention will not increase production and logistics costs, nor will it limit its clinical application scope.

[0159] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0160] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. An anti-aging mitochondrial preparation, characterized in that, The anti-aging mitochondrial preparation is obtained by compounding mammalian mitochondria and a solvent; the mass ratio of the mammalian mitochondria to the volume ratio of the solvent is 2 mg to 20 mg: 100 mL. Among them, the membrane potential of the mammalian-derived mitochondria remains unchanged or decreases by 50% to 90% compared to before separation, and the outer membrane integrity is ≥90%; The mammalian-derived mitochondria are selected from at least one of yak ear fibroblast mitochondria, human HEK293T cell mitochondria, human HepG2 cell mitochondria, and human HeLa cell mitochondria. The solvent includes at least one of physiological saline, lactated Ringer's solution, compound electrolyte injection, glucose solution for injection, and compound electrolyte glucose injection.

2. The anti-aging mitochondrial preparation according to claim 1, characterized in that, The mammalian-derived mitochondria were obtained by separating them from mammalian cells or tissues using differential centrifugation.

3. The anti-aging mitochondrial preparation according to claim 2, characterized in that, The steps for isolating the mammalian-derived mitochondria from mammalian cells or tissues are as follows: The mammalian cells or tissues are pretreated to purify and activate the cells, thereby obtaining a cell precipitate. The cell precipitate was disrupted in an isotonic solution, and the nuclei and large fragments were removed by differential centrifugation. The mitochondrial precipitate was collected to obtain the mammalian-derived mitochondria.

4. The anti-aging mitochondrial preparation according to claim 3, characterized in that, The preprocessing steps are as follows: Under low temperature conditions, the mammalian cells or tissues are first washed, and then enzymatically digested. Low temperature conditions refer to 3.8℃~4.2℃.

5. The anti-aging mitochondrial preparation according to claim 3, characterized in that, The conditions for differential centrifugation are as follows: Preliminary enucleation: 600×g~1,000×g, 5 minutes~10 minutes; Mitochondrial collection: 7,000×g~10,000×g, 10 minutes.

6. A method for preparing the anti-aging mitochondrial preparation according to any one of claims 1 to 5, characterized in that, Includes the following steps: The anti-aging mitochondrial preparation is obtained by resuspending the mammalian-derived mitochondria in the solvent.

7. The use of the anti-aging mitochondrial preparation according to claim 1 in the preparation of anti-aging drugs.

8. The application according to claim 7, characterized in that, The anti-aging drug uses the anti-aging mitochondrial preparation as its active ingredient.

9. The application according to claim 8, characterized in that, The anti-aging drugs also include pharmaceutically acceptable excipients.

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