Mitochondrial in-vitro activity retention agent as well as preparation method and application thereof

Abstract

The invention provides a mitochondrial in-vitro activity retention agent, a preparation method and application, and belongs to the technical field of mitochondrial activity preservation. According to the mitochondrial in-vitro activity retention agent, a mitochondrial membrane structure is stabilized through the SS-31 peptide, active oxygen is removed from the source by the rosmarinic acid derivative, a reducing environment is maintained by the reduced glutathione, and an energy metabolism substrate is supplemented by the nicotinamide ribose, so that the mitochondrial in-vitro activity retention agent has a multi-mechanism synergistic effect; the biological activity of the separated mitochondria can be effectively maintained in the in-vitro extraction, purification and preservation process, and the preservation rate of key indexes such as mitochondrial membrane potential and ATP generation rate is remarkably increased to 85% or above. The mitochondrial in-vitro activity retention agent can be used for in-vitro purification, purification and preservation of mitochondria or preparation of stem cell-derived mitochondrial microvesicles, and provides core raw material guarantee for development of products such as efficient anti-aging cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of mitochondrial activity preservation technology, and relates to an in vitro mitochondrial activity preservation agent, its preparation method, and its application. Background Technology

[0002] Mitochondria, as the "energy factories" of eukaryotic cells, play a central role in vital activities such as cell metabolism, signal transduction, and programmed cell death. Exogenous supplementation of functional mitochondria or their active components can repair cellular energy metabolism disorders, showing great potential in regenerative medicine, anti-aging cosmetics, and other fields.

[0003] However, during the in vitro extraction, purification, preservation, and subsequent formulation processing of mitochondria, once they are removed from their natural intracellular environment, they are highly susceptible to damage and rapid inactivation. The main causes of inactivation include: 1) mechanical damage such as centrifugation and ultrasonic disruption can destroy the integrity of the mitochondrial membrane structure; 2) oxidative stress causes the collapse of the mitochondrial inner membrane potential, leading to the large-scale production of reactive oxygen species (ROS); and 3) depletion of key cofactors such as NAD+ and CoQ10, as well as antioxidants such as glutathione.

[0004] To maintain mitochondrial activity, osmotic protectants such as sucrose and mannitol, as well as bovine serum albumin (BSA) and ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) are usually added to the mitochondrial separation buffer. Although these measures can reduce swelling and calcium ion damage to some extent, their effect on preserving the activity of core functions such as mitochondrial oxidative phosphorylation is limited. The mitochondrial activity preservation rate after extraction is generally less than 70%, and the ATP (adenosine triphosphate) production capacity is significantly reduced. Summary of the Invention

[0005] The purpose of this invention is to provide an in vitro mitochondrial activity preservation agent, its preparation method, and its application, in order to solve the problem of poor in vitro mitochondrial activity preservation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, this application provides an in vitro mitochondrial activity retention agent, comprising a basal buffer and SS-31 peptide, rosmarinic acid derivative, reduced glutathione, nicotinamide ribose, and polyvinylpyrrolidone K30 at final concentrations of 50-200 μM, 100-500 μM, 5-20 mM, 1-5 mM, and 0.5-1.5% (w / v) in the basal buffer; wherein the basal buffer comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and mannitol, and the rosmarinic acid derivative has the structural formula: rosmarinic acid-CO-NH-(CH2)4-N+ (CH3)3.

[0007] Secondly, this application provides a method for preparing an in vitro mitochondrial activity retention agent, the method comprising: SS-31 peptide, rosmarinic acid derivative, reduced glutathione and nicotinamide ribose were dissolved in part of the basic buffer solution to form a concentrated solution of active ingredients; After mixing the concentrated active ingredient solution with another portion of the basic buffer solution, polyvinylpyrrolidone K30 was added, dissolved, mixed, and filtered to obtain the mitochondrial in vitro activity maintainer.

[0008] Thirdly, this application provides a mitochondrial kit that includes the mitochondrial in vitro activity maintainer described in the first aspect.

[0009] Fourthly, this application provides an application of a mitochondrial in vitro activity maintainer or mitochondrial kit, namely, for the in vitro purification, preservation, or preparation of stem cell-derived mitochondrial microvesicles of mitochondria.

[0010] The present invention has the following beneficial effects: (1) In this application, both SS-31 peptide and rosmarinic acid derivative target mitochondria. The combined use of the two mitochondrial-targeting reagents has a clear and complementary mechanism of action, achieving synergistic protection from "structural reinforcement" to "source oxygen removal". It can provide efficient and stable activity protection for isolated mitochondria throughout the entire in vitro operation process, significantly improving the activity preservation rate and functional integrity of mitochondria and mitochondrial-derived products.

[0011] (2) The mitochondrial in vitro activity preservation agent in this application is stable at 4°C for a long time and has good stability. It is compatible with commonly used mitochondrial extraction reagents and activity preservation agents such as PQQ, and can be directly integrated into existing production processes as an additive.

[0012] (3) When the mitochondrial in vitro activity preservation agent in this application is used for mitochondrial extraction and preservation, it can increase the preservation rate of key activity indicators such as mitochondrial membrane potential and ATP production rate to more than 85%, which is significantly better than traditional buffer system or single protective agent system.

[0013] (4) The mitochondrial in vitro activity preservation agent in this application can not only be used to prepare highly active mitochondrial microvesicles for anti-aging cosmetics, but also provide a reliable in vitro mitochondrial preservation solution for biomedical fields such as mitochondrial transplantation and mitochondrial function research. Attached Figure Description

[0014] Figure 1 Comparison of membrane potential detection for the mitochondrial in vitro activity preservation agents prepared in Examples 1 and 2 and the mitochondrial preservation buffer prepared in Comparative Example 1; Figure 2 Comparison of ATP detection results for the mitochondrial in vitro activity preservation agents prepared in Examples 1 and 2 and the mitochondrial preservation buffer prepared in Comparative Example 1; Figure 3 Comparison of ROS detection results for the mitochondrial in vitro activity preservation agents prepared in Examples 1 and 2 and the mitochondrial preservation buffer prepared in Comparative Example 1. Detailed Implementation

[0015] This application provides an in vitro mitochondrial activity retention agent, comprising a basal buffer and SS-31 peptide, rosmarinic acid derivative, reduced glutathione, nicotinamide ribose, and polyvinylpyrrolidone K30 at final concentrations of 50-200 μM, 100-500 μM, 5-20 mM, 1-5 mM, and 0.5-1.5% (w / v) in the basal buffer; wherein the basal buffer comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and mannitol, and the rosmarinic acid derivative has the structural formula: rosmarinic acid-CO-NH-(CH2)4-N + (CH3)3.

[0016] In the basal buffer, the final concentrations of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and mannitol are 10-20 mM, 2-5 mM, 120-150 mM, and 1-3% (w / v), respectively; the osmotic pressure of the basal buffer is 280-320 mOsm / kg.

[0017] In this embodiment, SS-31 peptide is a synthetically produced mitochondrial-targeting antioxidant peptide with the sequence D-Arg-Dmt-Lys-Phe-NH2, where Dmt is 2,6-dimethyltyrosine. This SS-31 peptide can specifically bind to cardiolipin in the inner mitochondrial membrane, stabilizing the structure of the inner mitochondrial membrane and inhibiting the opening of the mitochondrial permeability transition pore (mPTP).

[0018] Rosmarinic acid derivatives are mitochondrial-targeting compounds formed by chemically synthesizing rosmarinic acid by linking it to quaternary ammonium salt mitochondrial-targeting groups. By introducing these quaternary ammonium salt mitochondrial-targeting groups, rosmarinic acid derivatives can accumulate within mitochondria due to the negative potential of the mitochondrial membrane. This allows the antioxidant activity units of rosmarinic acid to function at the main sites of ROS production, improving the efficiency of scavenging endogenous ROS in mitochondria and thus eliminating ROS at its source.

[0019] Rosmarinic acid is a natural polyphenol compound that can be obtained through plant extraction or chemical synthesis. The rosmarinic acid molecule contains an aromatic ring structure and multiple phenolic hydroxyl functional groups, making it suitable for chemical modification reactions with quaternary ammonium salt mitochondrial targeting groups. These quaternary ammonium salt mitochondrial targeting groups include, but are not limited to, tetramethylammonium structures, which can interact with the mitochondrial membrane through electrostatic interactions and lipid affinity, thereby endowing the rosmarinic acid derivatives with mitochondrial targeting capabilities.

[0020] In this embodiment, rosmarinic acid and quaternary ammonium salt mitochondrial targeting groups undergo a condensation reaction via the carboxyl group in rosmarinic acid and the methylene linker containing a reactive amino group in the quaternary ammonium salt mitochondrial targeting group, forming a stable amide bond structure to balance molecular flexibility and mitochondrial targeting efficiency. Therefore, the structural formula of the rosmarinic acid derivative in this embodiment is represented as: rosmarinic acid-CO-NH-(CH2)4-N + (CH3)3.

[0021] In this application, the tetramethylammonium derivative is preferably tetramethylammonium chloride or a quaternary ammonium salt. The following describes a method for preparing rosmarinic acid derivatives using tetramethylammonium chloride as an example. This method includes: S01: Under the protection of an inert gas such as nitrogen or argon, rosmarinic acid and tetramethylammonium chloride are dissolved in dichloromethane. After adding p-toluenesulfonic acid, the mixture is stirred at room temperature for 6 hours to obtain a reaction solution. During this reaction, rosmarinic acid undergoes a chemical modification reaction with tetramethylammonium mitochondrial targeting groups under the stated conditions to form rosmarinic acid derivatives. The mass ratio of rosmarinic acid, tetramethylammonium chloride, and p-toluenesulfonic acid is (30-70):(10-40):(0.5-3), preferably 50:25:1.

[0022] S02: Add 0.5-2 times the volume of deionized water to the reaction solution, shake and mix at room temperature, and separate the layers 1-3 times. Extract the residual solvent and impurities in the reaction solution with water to obtain the extract. Add 5-20 wt% anhydrous sodium sulfate to the extractant for dehydration for 10-30 min. After filtration, add the obtained organic phase to a rotary evaporator and evaporate at 30-50℃ to remove the solvent, obtaining the crude product. Add the crude product to a silica gel column, using chloroform and ethyl acetate at a volume ratio of 1:10 as eluents, and perform column chromatography at room temperature with silica gel particles of 200-400 mesh to obtain the purified rosmarinic acid derivative.

[0023] Reduced glutathione (GSH), chemically known as N-(NL-γ-glutamyl-L-cysteyl)glycine, is a core component of the endogenous antioxidant system, directly scavenging free radicals and regenerating other antioxidants. Nicotinamide riboside (NR) is a precursor to nicotinamide adenine dinucleotide (NAD+), and replenishing the NAD+ pool is crucial for maintaining mitochondrial electron transport chain function and sirtuin activity.

[0024] In the embodiments of this application, SS-31 peptide provides physical structural stability, rosmarinic acid derivative provides targeted antioxidant protection, reduced glutathione provides global reducing environment support, and nicotinamide ribose provides energy metabolism substrate supplementation. Thus, SS-31 peptide, rosmarinic acid derivative, reduced glutathione, and nicotinamide ribose constitute a multi-layered synergistic protection network to achieve the purpose of protecting mitochondria in vitro.

[0025] The mitochondrial in vitro activity retention agent in this application embodiment is a sterile, clear liquid. After being stored at 4°C in the dark for 12 months, the content retention rates of SS-31 peptide and rosmarinic acid derivative are both greater than 95%, and the content retention rate of reduced glutathione is both greater than 90%.

[0026] This application also provides a method for preparing an in vitro mitochondrial activity retention agent, the method comprising: S01: Dissolve SS-31 peptide, rosmarinic acid derivative, reduced glutathione and nicotinamide ribose into a portion of the base buffer to form a concentrated solution of active ingredients.

[0027] Disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and mannitol were added to water for injection and dissolved. The pH was adjusted to 7.2-7.4 to obtain the basal buffer. The basal buffer was divided into two portions at a volume ratio of 2-3:7-8. SS-31 peptide, rosmarinic acid derivative, reduced glutathione, and nicotinamide ribose were added to the smaller portion of the basal buffer. The mixture was stirred for 30-60 minutes under inert gas protection, at a temperature of 2-8℃, and a stirring speed of 100-200 rpm until completely dissolved, yielding a concentrated solution of the active ingredients.

[0028] S02: After mixing the concentrated active ingredient solution with another part of the basic buffer solution, add polyvinylpyrrolidone K30, dissolve, mix, and filter to obtain the mitochondrial in vitro activity retention agent.

[0029] The concentrated active ingredient solution and a larger volume of basal buffer were mixed thoroughly at a volume ratio of 1:3 to 1:1. Polyvinylpyrrolidone K30 was then added, and the mixture was stirred for 30-60 minutes under inert gas protection at 2-8°C and a stirring speed of 100-200 rpm until completely dissolved, yielding a mixture. This mixture was then filtered through sterile polyethersulfone membranes with pore sizes of 0.45 μm and 0.22 μm to obtain the mitochondrial in vitro activity retention agent. This mitochondrial in vitro activity retention agent was stored at 2-8°C under light-protected conditions for future use.

[0030] In addition, this application also provides a mitochondrial kit, which includes the above-mentioned mitochondrial in vitro activity preservation agent. The mitochondrial kit further includes a mitochondrial buffer, and the volume ratio of the mitochondrial in vitro activity preservation agent to the mitochondrial buffer is 1:(5-50), preferably 1:10.

[0031] In the embodiments of this application, the mitochondrial buffer is a mitochondrial extraction buffer and / or a mitochondrial resuspension buffer. When the mitochondrial buffer is a mitochondrial extraction buffer, an in vitro mitochondrial activity retention agent is added at a ratio of 1:10 (v / v) to a mitochondrial extraction buffer composed of 200-250 nM sucrose, 10 mM Tris-HCl (Tris(Hydroxymethyl) Aminomethane Hydrochloride), and 0.5 mM EGTA (ethylene glycol bis(α-aminoethyl ether tetraacetic acid)). This buffer is used for gentle sonication disruption and subsequent differential centrifugation of anucleate cytoplasms of human umbilical cord mesenchymal stem cells. When the mitochondrial buffer is a mitochondrial resuspension buffer, an in vitro mitochondrial activity retention agent is added at a ratio of 1:10 (v / v) to a PBS resuspension containing 10-50 nM Pyrroloquinoline quinone (PQQ). This buffer is used in the nano-sizing stage for gradient sonication treatment to encapsulate mitochondria within the cytoplasmic membrane.

[0032] The mitochondrial in vitro activity preservation agent or mitochondrial kit described in this application is used for the in vitro purification, preservation, or preparation of stem cell-derived mitochondrial microvesicles, such as mitochondrial purification and resuspension. When the mitochondrial in vitro activity preservation agent is added to the mitochondrial extraction buffer or mitochondrial resuspension buffer at a volume ratio of 1:10, it can maintain the treated mitochondrial membrane potential at >150mV, ATP generation rate at >85%, and ROS level at less than 10%.

[0033] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0034] Example 1 This application provides a mitochondrial in vitro activity retention agent E1, which includes a basal buffer with an osmotic pressure of 305 mOsm / kg and SS-31 peptide, rosmarinic acid derivative, reduced glutathione, nicotinamide ribose and polyvinylpyrrolidone K30 at final concentrations of 100 μM, 200 μM, 10 mM, 1 mM and 1.0% (w / v) in the basal buffer; wherein the basal buffer includes disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride and mannitol at final concentrations of 20 mM, 5 mM, 150 mM and 2.5% (w / v) respectively.

[0035] This application also provides a method for preparing an in vitro mitochondrial activity retention agent, the method comprising: S101: Under the protection of an inert gas such as nitrogen or argon, rosmarinic acid and tetramethylammonium chloride are dissolved in dichloromethane. After adding p-toluenesulfonic acid, the mixture is stirred at room temperature for 6 hours to obtain a reaction solution. During this reaction, tetramethylammonium chloride reacts with the hydroxyl or phenolic sites in rosmarinic acid to generate rosmarinic acid derivatives. The mass ratio of rosmarinic acid, tetramethylammonium chloride, and p-toluenesulfonic acid is 50:25:1. Twice the volume of deionized water is added to the reaction solution, and the mixture is shaken and mixed at room temperature, separating into three layers. Residual solvent and impurities in the reaction solution are extracted with water to obtain an extract. Anhydrous sodium sulfate (10 wt% by weight) is added to the extract for dehydration treatment for 20 minutes. The filtered organic phase is then added to a rotary evaporator and evaporated under reduced pressure at 45°C to remove the solvent, yielding a crude product. The crude product was added to a silica gel column and eluted with chloroform and ethyl acetate in a volume ratio of 1:10. The purified rosmarinic acid derivative was obtained by column chromatography at room temperature with silica gel particles of 300 mesh.

[0036] Add 3.58g disodium hydrogen phosphate, 0.78g sodium dihydrogen phosphate, 0.88g sodium chloride, and 2.5g mannitol to 80mL of water for injection. After dissolving, adjust the pH to 7.30±0.05 with 1M HCl or NaOH, and bring the volume to 100mL to obtain the basic buffer solution. Pre-cool the basic buffer solution at 4℃ and set aside. Place 20mL of the pre-cooled basic buffer solution in a 50mL glass beaker and place it on an ice bath. Continuously purge the liquid surface with high-purity nitrogen gas at a flow rate of 50mL / min. Add 1.0mg SS-31 peptide, 2.3mg rosmarinic acid derivative, 61.5mg reduced glutathione, and 6.1mg nicotinamide ribose to the glass beaker in the above proportions. Stir for 45min under continuous nitrogen purging, at 4℃, and with a stirring speed of 150rpm until completely dissolved to obtain the active ingredient concentrate.

[0037] S102: After thoroughly mixing the concentrated active ingredient solution with the remaining 80 mL of basal buffer, add 1.0 g of polyvinylpyrrolidone K30. Stir for 30 min under inert gas protection, at 4℃ and a stirring speed of 150 rpm until completely dissolved to obtain a mixture. Filter the mixture sequentially through sterile polyethersulfone membranes with pore sizes of 0.45 μm and 0.22 μm to obtain the mitochondrial in vitro activity retention agent. This mitochondrial in vitro activity retention agent is stored at 4℃ under light-protected conditions for later use.

[0038] Example 2 This application provides a mitochondrial in vitro activity retention agent E2, which includes a basal buffer with an osmotic pressure of 320 mOsm / kg and SS-31 peptide, rosmarinic acid derivative, reduced glutathione, nicotinamide ribose and polyvinylpyrrolidone K30 at final concentrations of 50 μM, 100 μM, 5 mM, 1 mM and 0.5% (w / v) in the basal buffer; wherein the basal buffer includes disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride and mannitol at final concentrations of 10 mM, 2 mM, 130 mM and 3% (w / v) respectively.

[0039] This application also provides a method for preparing an in vitro mitochondrial activity retention agent, which is the same as in Example 1.

[0040] Comparative Example 1 Mitochondrial preservation buffer was prepared using 250 mM sucrose, pH 7.4, 10 mM Tris-HCl, 0.5 mM EGTA, and 0.1% (w / v) bovine serum albumin. After sterile filtration, it was stored at 4°C.

[0041] The in vitro mitochondrial activity preservation effects of the mitochondrial activity preservation agents prepared in Examples 1 and 2 and the mitochondrial preservation buffer prepared in Comparative Example 1 were evaluated as follows: 1. Mitochondrial isolation Mitochondrial precipitates were isolated from fresh rat liver tissue using differential centrifugation. The mitochondrial precipitates were resuspended in mitochondrial preservation buffer (Comparative Example 1), and the protein concentration was adjusted to 2 mg / mL to obtain the mitochondrial resuspension.

[0042] 2. Grouping The mitochondrial in vitro activity preservation agents prepared in Examples 1 and 2 were added to the mitochondrial preservation buffer prepared in Comparative Example 1 at a ratio of 10% (v / v) to form E1 buffer and E2 buffer, respectively. The mitochondrial resuspension was divided into three groups: the blank group, experimental group E1, and experimental group E2. The mitochondrial resuspension was diluted with the mitochondrial preservation buffer, E1 buffer, and E2 buffer from Comparative Example 1, respectively, and the final concentration of mitochondrial protein in each group was 0.5 mg / mL. The samples in each group were stored at 4°C.

[0043] 3. Activity detection During the static storage at 4℃, samples were collected at 0h, 6h, 12h, 24h, 48h, and 72h. The samples were analyzed using the JC-1 fluorescent probe kit, and the results are expressed as the red-green fluorescence ratio. A higher ratio indicates a higher membrane potential. Figure 1 Table 1. Using an ATP assay kit, the mitochondrial ATP synthesis capacity in each sample was detected under succinic acid substrate conditions. Normalization was performed with the 0-hour blank group value as 100%, and the results were obtained. Figure 2 Table 1. The ROS levels of mitochondria in each sample were detected using the DCFH-DA fluorescent probe and expressed as relative fluorescence units (RFU). Figure 3 Table 1.

[0044] Table 1: Data on key in vitro mitochondrial activity indicators From Table 1 and Appendix Figure 1-3 As can be seen, after 72 hours of storage at 4°C, the membrane potential of mitochondria in the blank group decreased by about 65%, and the ATP production rate decreased to about 40%; in experimental group E1, the membrane potential of mitochondria remained at 82% of the initial value, the ATP production rate remained at 85%, and the ROS level was effectively suppressed to a low level; in experimental group E2, the membrane potential of mitochondria remained at 75% of the initial value, the ATP production rate remained at 78%, and the ROS level was effectively suppressed to a low level. This indicates that the mitochondrial in vitro activity preservative prepared in this application can significantly delay the activity decay of mitochondria during in vitro storage.

[0045] In summary, the mitochondrial activity preservation agent provided in this application can effectively maintain the structural and functional integrity of isolated mitochondria through the synergistic effect of SS-31 peptide, rosmarinic acid derivative, reduced glutathione, and nicotinamide ribose, significantly improving its activity preservation rate during in vitro operation and storage.

[0046] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A mitochondrial in vitro activity preservative, characterized in that, The product includes a basal buffer and SS-31 peptide, rosmarinic acid derivative, reduced glutathione, nicotinamide ribose, and polyvinylpyrrolidone K30 at final concentrations of 50-200 μM, 100-500 μM, 5-20 mM, 1-5 mM, and 0.5-1.5% (w / v) in the basal buffer; wherein the basal buffer comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and mannitol, and the rosmarinic acid derivative has the structural formula: rosmarinic acid-CO-NH-(CH2)4-N + (CH3)3.

2. The mitochondrial in vitro activity maintainer according to claim 1, characterized in that, In the basal buffer solution, the final concentrations of the disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and mannitol are 10-20 mM, 2-5 mM, 120-150 mM, and 1-3% (w / v), respectively; the osmotic pressure of the basal buffer solution is 280-320 mOsm / kg.

3. The mitochondrial in vitro activity maintainer according to claim 1, characterized in that, The preparation method of the rosmarinic acid derivative includes: Rosmarinic acid and tetramethylammonium chloride were dissolved in dichloromethane under an inert gas atmosphere. After adding p-toluenesulfonic acid, the mixture was stirred at room temperature to obtain a reaction solution. The reaction solution was subjected to water extraction, dehydration with anhydrous sodium sulfate, rotary evaporation, and silica gel column chromatography to obtain rosmarinic acid derivatives.

4. The mitochondrial in vitro activity maintainer according to claim 3, characterized in that, The mass ratio of the rosmarinic acid, the tetramethylammonium chloride and the p-toluenesulfonic acid is (30-70):(10-40):(0.5-3).

5. The mitochondrial in vitro activity maintainer according to claim 3, characterized in that, The amount of deionized water in the water extraction is 0.5-2 times the volume of the reaction liquid; the amount of anhydrous sodium sulfate is 5-20 wt% of the mass of the extract after water extraction; the rotary evaporation temperature is 30-50℃; the silica gel particle size in the silica gel column chromatography is 200-400 mesh, the eluent is a mixture of chloroform and ethyl acetate, and the temperature is room temperature.

6. A method for preparing the mitochondrial in vitro activity maintainer according to any one of claims 1-5, characterized in that, include: SS-31 peptide, rosmarinic acid derivative, reduced glutathione and nicotinamide ribose were dissolved in part of the basic buffer solution to form a concentrated solution of active ingredients; After mixing the concentrated active ingredient solution with another portion of the basic buffer solution, polyvinylpyrrolidone K30 was added, dissolved, mixed, and filtered to obtain the mitochondrial in vitro activity maintainer.

7. The method for preparing the mitochondrial in vitro activity retention agent according to claim 6, characterized in that, The dissolution conditions are: inert gas protection, temperature 2-8℃, stirring speed 100-200 rpm, and stirring time 30-60 min.

8. A mitochondrial reagent kit, characterized in that, Includes the mitochondrial in vitro activity maintainer as described in any one of claims 1-5.

9. The mitochondrial reagent kit according to claim 8, characterized in that, The mitochondrial kit further includes a mitochondrial buffer, and the volume ratio of the mitochondrial in vitro activity maintainer to the mitochondrial buffer is 1:(5-50).

10. The mitochondrial in vitro activity preservation agent according to any one of claims 1-5 or the mitochondrial kit according to claim 8 or 9 is used for the in vitro purification, preservation or preparation of stem cell-derived mitochondrial microvesicles of mitochondria.

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