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CN-122012366-A - Mitochondrial in-vitro activity maintaining agent, preparation method and application

CN122012366ACN 122012366 ACN122012366 ACN 122012366ACN-122012366-A

Abstract

The application provides a mitochondrial in-vitro activity maintaining agent, a preparation method and application, which belong to the technical field of mitochondrial activity preservation, the mitochondrial in-vitro activity maintaining agent stabilizes mitochondrial membrane structures through SS-31 peptide, rosmarinic acid derivatives remove active oxygen from sources, reduced glutathione maintains a reduction environment and nicotinamide riboside supplements a multi-mechanism synergistic effect of energy metabolism substrates, can effectively maintain the bioactivity of the isolated mitochondria in the processes of in vitro extraction, purification and preservation, and obviously improve the preservation rate of key indexes such as mitochondrial membrane potential, ATP generation rate and the like to more than 85 percent. The mitochondrial in-vitro activity maintaining agent can be used for in-vitro purification, preservation or preparation of stem cell-derived mitochondrial microvesicles, and provides a core raw material guarantee for developing efficient anti-aging cosmetics and other products.

Inventors

  • LIU YU
  • DAI XIAOYU
  • LI DONG
  • LI SHUJIAN

Assignees

  • 山东省泉溪生物技术有限公司

Dates

Publication Date
20260512
Application Date
20260128

Claims (10)

  1. 1. The mitochondrial in-vitro activity maintaining agent is characterized by comprising a basic buffer solution and SS-31 peptide, rosmarinic acid derivatives, reduced glutathione, nicotinamide riboside and polyvinylpyrrolidone K30, wherein the final concentration of the basic buffer solution is respectively 50-200 mu M, 100-500 mu M, 5-20mM, 1-5mM, 0.5-1.5% (w/v), and the basic buffer solution comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride and mannitol, and the rosmarinic acid derivatives have the structural formula of rosmarinic acid-CO-NH- (CH 2 ) 4 -N + (CH 3 ) 3 ).
  2. 2. The agent for maintaining mitochondrial in vitro activity according to claim 1, wherein the final concentrations of the disodium hydrogen phosphate, the sodium dihydrogen phosphate, the sodium chloride and the mannitol in the base buffer are 10 to 20mM, 2 to 5mM, 120 to 150mM, 1 to 3% (w/v), respectively, and the osmotic pressure of the base buffer is 280 to 320mOsm/kg.
  3. 3. The agent for maintaining mitochondrial in vitro activity according to claim 1, wherein the preparation method of the rosmarinic acid derivative comprises: Under the protection of inert gas, dissolving rosmarinic acid and tetramethyl ammonium chloride into methylene dichloride, adding p-toluenesulfonic acid, and stirring at room temperature for reaction to obtain a reaction solution; The rosmarinic acid derivative is obtained after the reaction liquid is subjected to water extraction, anhydrous sodium sulfate dehydration, rotary evaporation and silica gel column chromatography.
  4. 4. The agent for maintaining mitochondrial in vitro activity according to claim 3, wherein the mass ratio of rosmarinic acid, tetramethyl ammonium chloride and p-toluenesulfonic acid is (30-70): 10-40): 0.5-3.
  5. 5. The agent for maintaining the activity of mitochondria in vitro according to claim 3, wherein the deionized water content in the aqueous extraction is 0.5-2 times the volume of the reaction solution, the anhydrous sodium sulfate is 5-20wt% of the mass of the extract solution after the aqueous extraction, the spin-evaporation temperature is 30-50 ℃, the silica gel particle size in the silica gel column chromatography is 200-400 meshes, the eluent is a mixed solution of chloroform and ethyl acetate, and the temperature is room temperature.
  6. 6. The method for producing a mitochondrial in vitro activity maintenance agent according to any one of claims 1 to 5, comprising: Dissolving SS-31 peptide, rosmarinic acid derivative, reduced glutathione and nicotinamide riboside into partial basic buffer solution to form active ingredient concentrated solution; and (3) uniformly mixing the active ingredient concentrated solution with another part of basic buffer solution, adding polyvinylpyrrolidone K30, dissolving, uniformly mixing and filtering to obtain the mitochondrial in-vitro activity retention agent.
  7. 7. The method for preparing the agent for maintaining the activity of mitochondria in vitro according to claim 6, wherein the dissolution condition is inert gas protection, temperature is 2-8 ℃, stirring rotation speed is 100-200rpm, and stirring time is 30-60min.
  8. 8. A mitochondrial kit comprising the mitochondrial in vitro activity maintenance agent of any one of claims 1-5.
  9. 9. The mitochondrial kit of claim 8, further comprising a mitochondrial buffer, wherein the volume ratio of the mitochondrial in vitro activity maintainer to the mitochondrial buffer is 1 (5-50).
  10. 10. The use of a mitochondrial in vitro activity maintenance agent according to any one of claims 1 to 5 or a mitochondrial kit according to claim 8 or 9 for the in vitro purification, preservation or preparation of stem cell derived mitochondrial microvesicles.

Description

Mitochondrial in-vitro activity maintaining agent, preparation method and application Technical Field The invention belongs to the technical field of mitochondrial activity preservation, and relates to a mitochondrial in-vitro activity maintaining agent, a preparation method and application. Background Mitochondria serve as "energy factories" of eukaryotic cells and play a central role in vital activities such as cell metabolism, signal transduction and programmed death. Exogenous functional mitochondria or active components thereof can repair cell energy metabolism disorder, which has great potential in the fields of regenerative medicine, anti-aging cosmetics and the like. However, during the in vitro extraction, purification, preservation and even subsequent formulation processing of mitochondria, once they are removed from their natural intracellular environment, they are extremely vulnerable to damage and rapid inactivation. The main inactivation reasons include 1) that mechanical damages such as centrifugation and ultrasonic disruption can damage the structural integrity of mitochondrial membranes, 2) that oxidation stress collapses the inner mitochondrial membrane potential, that active oxygen (ROS for short) is produced in a large amount, and 3) that key cofactors such as NAD+, coQ10 and the like and antioxidant substances such as glutathione are exhausted. In order to maintain the activity of mitochondria, an osmotic pressure protective agent such as sucrose, mannitol and the like, bovine serum albumin (abbreviated as BSA), ethylene glycol-bis (beta-aminoethyl ether) -N, N, N ', N' -tetraacetic acid (abbreviated as EGTA) and the like are usually added into a mitochondrial separation buffer, and although the swelling and the damage to calcium ions can be reduced to a certain extent by these measures, the activity preservation effect on the core functions such as the oxidative phosphorylation of mitochondria is limited, the activity preservation rate of mitochondria is generally lower than 70 percent after extraction, and the ATP (AdenosineTriphosphate ) generating capacity is greatly reduced. Disclosure of Invention The invention aims to provide a mitochondrial in-vitro activity maintaining agent, a preparation method and application thereof, so as to solve the problem of poor in-vitro activity preservation effect of mitochondria. In order to achieve the above purpose, the present invention adopts the following technical scheme: In a first aspect, the application provides an in vitro mitochondrial activity maintaining agent, which comprises a basic buffer solution and SS-31 peptide, rosmarinic acid derivative, reduced glutathione, nicotinamide riboside and polyvinylpyrrolidone K30, wherein the final concentration of the basic buffer solution is respectively 50-200 mu M, 100-500 mu M, 5-20mM, 1-5mM, 0.5-1.5% (w/v), and the basic buffer solution comprises disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride and mannitol, and the rosmarinic acid derivative has the structural formula of rosmarinic acid-CO-NH- (CH 2)4-N+(CH3)3). In a second aspect, the present application provides a method for preparing a mitochondrial in vitro activity retaining agent, the method comprising: Dissolving SS-31 peptide, rosmarinic acid derivative, reduced glutathione and nicotinamide riboside into partial basic buffer solution to form active ingredient concentrated solution; and (3) uniformly mixing the active ingredient concentrated solution with another part of basic buffer solution, adding polyvinylpyrrolidone K30, dissolving, uniformly mixing and filtering to obtain the mitochondrial in-vitro activity retention agent. In a third aspect, the application provides a mitochondrial kit comprising the mitochondrial in vitro activity maintenance agent of the first aspect. In a fourth aspect, the application provides the use of a mitochondrial in vitro activity retention agent or a mitochondrial kit, i.e. for the in vitro purification, preservation or preparation of stem cell derived mitochondrial microvesicles. The invention has the following beneficial effects: (1) According to the application, both the SS-31 peptide and the rosmarinic acid derivative target mitochondria, through the combination of the two targeted mitochondrial reagents, the action mechanism is clear and complementary, the synergistic protection from 'structural reinforcement' to 'source oxygen removal' is realized, the efficient and stable activity protection can be provided for the separated mitochondria in the whole process of in vitro operation, and the activity preservation rate and the functional integrity of the mitochondria and the mitochondrial source products are obviously improved. (2) The mitochondrial in-vitro activity retention agent is stable for a long time at 4 ℃, has good stability, can be compatible with common mitochondrial extraction reagents, PQQ and other active retention agents, and can be directly used as an add