CN116098826A - Substance for protecting exosome activity, exosome and glabridin loaded by exosome - Google Patents

Substance for protecting exosome activity, exosome and glabridin loaded by exosome Download PDF

Info

Publication number
CN116098826A
CN116098826A CN202211434892.7A CN202211434892A CN116098826A CN 116098826 A CN116098826 A CN 116098826A CN 202211434892 A CN202211434892 A CN 202211434892A CN 116098826 A CN116098826 A CN 116098826A
Authority
CN
China
Prior art keywords
exosome
glabridin
activity
substance
exosomes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202211434892.7A
Other languages
Chinese (zh)
Inventor
徐佳洁
罗海浪
宋月星
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Xi'an Zhenrong Biotechnology Co ltd
Original Assignee
Xi'an Zhenrong Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Xi'an Zhenrong Biotechnology Co ltd filed Critical Xi'an Zhenrong Biotechnology Co ltd
Priority to CN202211434892.7A priority Critical patent/CN116098826A/en
Publication of CN116098826A publication Critical patent/CN116098826A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A61K8/20—Halogens; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A61K8/23—Sulfur; Selenium; Tellurium; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A61K8/24—Phosphorous; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/42—Amides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
    • A61K8/447—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof containing sulfur
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/46—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing sulfur
    • A61K8/466—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing sulfur containing sulfonic acid derivatives; Salts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
    • A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
    • A61K2800/782—Enzyme inhibitors; Enzyme antagonists
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/82—Preparation or application process involves sonication or ultrasonication

Abstract

The invention relates to a substance for protecting exosome activity, exosome and glabridin loaded by the exosome, and belongs to the technical field of biology. Based on the fact that exosomes in human saliva can maintain structural stability and functional activity for a long time at 4 ℃, the invention simulates the properties and composition characteristics of human saliva to provide a substance for protecting exosome activity, which can stably maintain exosome activity in vitro and has the characteristic of high safety. In addition, the invention also constructs a method for loading the glabridin by the exosome, and the method can improve the loading rate of the glabridin by the exosome.

Description

Substance for protecting exosome activity, exosome and glabridin loaded by exosome
Technical Field
The invention relates to a substance for protecting exosome activity, exosome and glabridin loaded by the exosome, belongs to the technical field of biology, and particularly relates to artificial saliva for protecting exosome activity, exosome and glabridin loaded by the exosome.
Background
Glabridin is a phytoestrogen, and its biological activities include antioxidant, antiinflammatory, neuroprotective, antiatherosclerotic, energy metabolism regulating, antitumor, nephritis resisting, etc. Since glabridin also has a very strong tyrosinase activity inhibition effect, and tyrosinase is the rate-limiting enzyme of melanin formation pathways, glabridin has a very high application value in the aspects of skin whitening, pigmentation abnormality treatment and other diseases. However, the practical application of glabridin in the aspects of treating diseases such as pigmentation abnormality, whitening skin and the like is limited due to the reasons of poor transdermal property, high in vivo degradation speed, poor cell membrane permeation capability and the like of glabridin.
In order to promote the practical application of the glabridin, researchers mostly choose to modify the glabridin by adopting a carrier technology of externally loading the glabridin, so that the glabridin can permeate cell membranes to exert the efficacy of the glabridin.
Exosomes are biologically active substances whose active function is determined by the composition of the exosomes' substances and the complex structure. General storage conditions make it difficult to maintain the complex structure of exosomes, resulting in loss of exosome activity. The exosomes such as blood, milk, urine, male semen, etc. still rapidly decline in activity under the condition of preservation at 4 ℃, and even the exosomes carry glabridin due to the instability of exosome activity, the efficacy of glabridin cannot be fully exerted.
Therefore, it is highly desired to provide a substance capable of maintaining the activity of exosomes, and further, by loading glabridin into exosomes, the advantages of glabridin can be fully exerted.
Disclosure of Invention
Since exosomes in human saliva can maintain structural stability and functional activity for nearly 20 months at 4 ℃. According to the characteristic of human saliva, in order to solve the technical problems that in the prior art, the exosome is loaded with glabridin, and the exosome activity is unstable and the efficacy of glabridin cannot be ensured, the invention simulates the characteristics and composition characteristics of human saliva, and discloses a substance for protecting the exosome activity, which can stably maintain the exosome activity in vitro and has the characteristic of high safety. The invention also constructs a method for loading the glabridin by the exosome, which can improve the loading rate of the glabridin by the exosome.
Based on the technical problems that the exosome activity is unstable and the efficacy of glabridin cannot be ensured, the novel technical scheme is as follows:
a substance that protects exosome activity, the substance being artificial saliva capable of providing a saliva environment. The artificial saliva comprises the following components in percentage by mass: phosphate buffer solution with pH of 7.0, 0.2%, anhydrous calcium chloride 0.025%, potassium chloride 0.05%, sodium chloride 0.6%, anhydrous magnesium sulfate 0.01%, mussel mucin 0.05-0.5%, albumin 0.05-0.5%, N-acetylglucosamine 0.01-0.1%, trehalose 0.01-0.1%, glycine 0.01-0.1%, L-methionine 0.01-0.1%, L-serine 0.01-0.1%, taurine 0.01-0.1%, and purified water for the rest to be added to supplement 100%.
An exosome comprises an exosome body and a substance for protecting exosome activity, wherein the mass percentage of the exosome body and the substance for protecting exosome activity is 0.01% -1%. Further, the mass percentage of the exosome body and the substance for protecting exosome activity is 0.1%. Further, the exosome body is secreted by umbilical cord mesenchymal stem cells.
The exosomes as described above are used to load glabridin.
In the process of loading glabridin on exosomes, the invention constructs a method capable of improving the loading rate of glabridin on exosomes.
The method is an emulsion ultrasonic method and specifically comprises the following steps of:
1) Mixing alcoholic solution dissolved with glabridin with exosome supernatant according to the mass ratio of (1-5): (10-20), adding tween-80 accounting for 0.1-1% of the mass of the mixed system into the mixed system, and uniformly stirring to obtain mixed solution; the exosomes include exosome bodies and substances that protect exosome activity.
2) Under the ice bath condition, carrying out ultrasonic treatment on the mixed solution obtained in the step 1) for 30-300 s, wherein the frequency is 20KHz-100KHz;
3) Centrifuging the mixed solution after ultrasonic treatment with 5000G-10000G for 10min-30min to obtain supernatant 1, and filtering the supernatant 1 with 0.2 μm sterile filter membrane to obtain filtrate;
4) Centrifuging the filtrate with 100000G-150000G for 30min-180min, discarding supernatant, and resuspending the suspension with sterile physiological saline to obtain glabridin-loaded exosome.
Wherein, the mass ratio of the glabridin in the alcohol solution in the step 1) is 1-10%, the alcohol solution for dissolving the glabridin is one of methanol, ethanol, propanol, n-butanol or isobutanol, and the alcohol solution is anhydrous alcohol solution. The ultrasonic power in the step 2) is 0.1W/cm 3 -1W/cm 3 The pulse time is 1s-10s, and the interval time is 1s-10s.
The technical effects achieved by the technical scheme are as follows:
1. the artificial saliva has high safety, and can maintain the activity of exosomes in vitro.
2. The artificial saliva of the invention protects the activity of exosomes, so that the advantage of the liquorice loaded by exosomes can be fully exerted.
3. The invention constructs an emulsion ultrasonic method, which can rapidly extrude glabridin molecules and exosomes, and simultaneously the ultrasonic effect can increase the permeability of exosome films, and promote the extruded glabridin molecules to rapidly enter the exosomes, thereby improving the loading rate of the exosomes to glabridin.
Drawings
Fig. 1: a Westen blot exosome activity detection diagram;
fig. 2: a tyrosinase activity inhibition test effect diagram;
fig. 3: and a graph showing the effect of melanin formation inhibition test.
Detailed Description
The content of water in saliva of human body is 98.5% -99%, and the rest 1% -1.5% is solid matter, contains sodium, potassium, calcium, chlorine, sulfur and other ions, also contains various enzymes, mucin, albumin, immunoglobulin, various amino acids, glucose, urea and growth factor combination, and is developed based on these matter bases of saliva of human body.
The total content of saccharides in the solid components of the saliva of a human body can be up to 50% -65%, and the saccharides in the saliva can be effectively replaced by adding N-acetylglucosamine and trehalose, wherein the N-acetylglucosamine is the main saccharides in the saliva and reaches 30% of the main saccharides in the saliva, and the N-acetylglucosamine is the main saccharides in the saliva and reaches 30% of the saccharides in the saliva. The other type of sugar in natural human saliva is mainly glucose, but research shows that trehalose has better effect than glucose in maintaining the stability of exosomes, so that the invention replaces glucose with trehalose in the composition proportion of sugar classification. Trehalose is a widely used saccharide for freezing and preserving cells and tissues, and researches have shown that trehalose can maintain the structural stability of exosomes, so that the sugar composition adopted in the invention is a combination of N-acetylglucosamine and trehalose.
The saliva of human body contains rich composite amino acid components, the composite amino acid components in the saliva have important significance for maintaining the functions and metabolic activities of oral mucosa cells, and meanwhile, research also finds that the composite amino acid components play an important role in maintaining the membrane structural stability of the lipid bilayer. The vesicle membrane of the exosome is also of a lipid structure, so that the compound amino acid has a good stabilizing effect on the membrane structure of the exosome. Because the exosome does not have metabolic activity, the complex amino acid complex is not needed to maintain the metabolic activity of cells, but only the stability protection of the amino acid on the membrane structure is considered, so that the exosome membrane structure has a good function of maintaining the stability of the exosome membrane structure by replacing the complex amino acid in human saliva with four amino acids of glycine, methionine, serine and taurine.
The human saliva contains sodium, potassium, calcium, chlorine, sulfur and other ions which have important functions for maintaining the osmotic pressure of saliva.
The protein in human saliva accounts for 0.3% of saliva components, and salivary proteins play various physiological roles in lubrication, antibiosis. Among them, sialoadhesin and albumin play an important role in maintaining the stability of various protein structures. The main purpose of the invention is to keep the biological activity of exosomes in vitro, so that complex protein component combination is not needed, only mucin and albumin combination is adopted to replace all other protein combinations in saliva, and simultaneously, because the sialoadhesin is difficult to obtain efficiently at present, mussel mucin is adopted to replace the sialoadhesin, the invention has better effect of keeping exosome activity, and can achieve the effect of protecting exosomes by human salivary proteins.
1. Exosome activity
The exosome comprises an exosome body and artificial saliva, wherein the exosome body is secreted by umbilical mesenchymal stem cells.
(1) Mass percentages of each substance for protecting exosome activity:
example 1: the artificial saliva comprises the following components in percentage by mass: phosphate buffer solution with pH of 7.0, 0.2%, anhydrous calcium chloride 0.025%, potassium chloride 0.05%, sodium chloride 0.6%, anhydrous magnesium sulfate 0.01%, mussel mucin 0.05%, albumin 0.05%, N-acetylglucosamine 0.01%, trehalose 0.01%, glycine 0.01%, L-methionine 0.01%, L-serine 0.01%, taurine 0.01%, and purified water added to the balance to supplement 100%, wherein the mass percentage of exosomes secreted by umbilical cord mesenchymal stem cells to artificial saliva is 0.01%.
Example 2: the artificial saliva comprises the following components in percentage by mass: phosphate buffer solution with pH of 7.0, 0.2%, anhydrous calcium chloride 0.025%, potassium chloride 0.05%, sodium chloride 0.6%, anhydrous magnesium sulfate 0.01%, mussel mucin 0.1%, albumin 0.1%, N-acetylglucosamine 0.02%, trehalose 0.02%, glycine 0.02%, L-methionine 0.02%, L-serine 0.02%, taurine 0.02% and the balance of purified water to supplement 100%, wherein the mass percentage of exosomes secreted by umbilical cord mesenchymal stem cells to artificial saliva is 0.1%.
Example 3: the artificial saliva comprises the following components in percentage by mass: phosphate buffer solution with pH of 7.0, 0.2%, anhydrous calcium chloride 0.025%, potassium chloride 0.05%, sodium chloride 0.6%, anhydrous magnesium sulfate 0.01%, mussel mucin 0.25%, albumin 0.25%, N-acetylglucosamine 0.05%, trehalose 0.05%, glycine 0.05%, L-methionine 0.05%, L-serine 0.05%, taurine 0.05%, and purified water added to the balance to supplement 100%, wherein the mass percentage of exosomes secreted by umbilical cord mesenchymal stem cells to artificial saliva is 0.1%.
Example 4: the artificial saliva comprises the following components in percentage by mass: phosphate buffer solution with pH of 7.0, 0.2%, anhydrous calcium chloride 0.025%, potassium chloride 0.05%, sodium chloride 0.6%, anhydrous magnesium sulfate 0.01%, mussel mucin 0.4%, albumin 0.4%, N-acetylglucosamine 0.08%, trehalose 0.08%, glycine 0.08%, L-methionine 0.08%, L-serine 0.08%, taurine 0.08%, and purified water added to the balance to supplement 100%, wherein the mass percentage of exosomes secreted by umbilical cord mesenchymal stem cells to artificial saliva is 0.1%.
Example 5: the artificial saliva comprises the following components in percentage by mass: phosphate buffer solution with pH of 7.0, 0.2%, anhydrous calcium chloride 0.025%, potassium chloride 0.05%, sodium chloride 0.6%, anhydrous magnesium sulfate 0.01%, mussel mucin 0.5%, albumin 0.5%, N-acetylglucosamine 0.1%, trehalose 0.1%, glycine 0.1%, L-methionine 0.1%, L-serine 0.1%, taurine 0.1%, and purified water added to balance to supplement 100%, wherein the mass percentage of exosomes secreted by umbilical cord mesenchymal stem cells to artificial saliva is 1%.
(2) Exosome activity assay:
the Westen blot is adopted to detect the activity of exosomes, the detected positive marker molecules are membrane-bound protein TSG1 and transmembrane molecule CD63, and the activity of umbilical mesenchymal stem cell exosomes in different examples is compared with that of umbilical mesenchymal stem cell exosomes stored for 3 months at 4 ℃. Exosomes extracted from umbilical cord mesenchymal stem cell culture solution are lysed by RIPA lysate and protein concentration is determined by BCA, then SDS-PAGE electrophoresis is performed, and after membrane transfer and antibody application, light development is performed.
As can be seen from FIG. 1, the exosome activity can be protected by 5 examples, but the protection effect of examples 2, 3 and 4 is significantly better than that of examples 1 and 5, wherein examples 3 and 4 have the best protection effect on exosome activity.
2. Exosome-loaded glabridin:
the exosome comprises an exosome body and artificial saliva, wherein the exosome body is secreted by umbilical mesenchymal stem cells.
(1) The method for exosome loading guanfacine comprises the following steps:
the exosome load guanfacine method is an innovative emulsification ultrasonic method, and comprises the following specific implementation steps:
example 6 dissolution of glabridin with an alcoholic solution to give glabridin a mass ratio of 1%; mixing alcoholic solution dissolved with glabridin with phosphate buffer solution containing exosome according to a mass ratio of 3:15, fully mixing; adding tween-80 with the mass ratio of 0.1% into the mixed system, and uniformly stirring; under ice bath condition, performing ultrasonic treatment for 30 seconds with frequency of 100KHz and power of 0.1W/cm 3 The method comprises the steps of carrying out a first treatment on the surface of the Pulse time 1 second, 1 second interval; centrifuging the supernatant with 5000G for 20min after the ultrasonic treatment is finished, collecting the supernatant after centrifugation, and discarding cell debris and other sediments; filtering the supernatant after centrifugation with a sterile filter membrane of 0.22 μm; then willCentrifuging the filtrate with 100000G for 180min, and discarding supernatant; resuspending the suspension with sterile physiological saline to obtain the glabridin-loaded exosome.
Example 7: dissolving glabridin with alcohol solution to make glabridin mass ratio 5%; mixing alcoholic solution dissolved with glabridin and phosphate buffer solution containing exosome according to a mass ratio of 1:10, fully mixing; adding tween-80 with the mass ratio of 0.2% into the mixed system, and uniformly stirring; under ice bath condition, performing ultrasonic treatment for 240 seconds with frequency of 30KHz and power of 0.2W/cm 3 The method comprises the steps of carrying out a first treatment on the surface of the Pulse time 3 seconds, 6 seconds apart; centrifuging the supernatant with 10000G for 30min after the ultrasonic treatment is finished, collecting the supernatant after centrifugation, and discarding cell debris and other sediments; filtering the supernatant after centrifugation with a sterile filter membrane of 0.22 μm; then the filtrate is centrifuged for 120min with 135000G, and the supernatant is discarded; resuspending the suspension with sterile physiological saline to obtain the glabridin-loaded exosome.
Example 8: dissolving glabridin with alcohol solution to make glabridin mass ratio 10%; mixing alcoholic solution dissolved with glabridin with phosphate buffer solution containing exosome according to a mass ratio of 5:20, fully mixing; adding tween-80 with the mass ratio of 1% into the mixed system, and uniformly stirring; under ice bath condition, performing ultrasonic treatment for 300 seconds with frequency of 20KHz and power of 1W/cm 3 The method comprises the steps of carrying out a first treatment on the surface of the Pulse time 10 seconds, 10 seconds apart; centrifuging the supernatant at 8000G for 10min after ultrasonic treatment, collecting the supernatant after centrifugation, and discarding cell debris and other sediments; filtering the supernatant after centrifugation with a sterile filter membrane of 0.22 μm; then centrifuging the filtrate with 150000G for 30min, and discarding the supernatant; resuspending the suspension with sterile physiological saline to obtain the glabridin-loaded exosome.
(2) Glabridin loaded by exosomes comprises the following substances in percentage by mass:
example 9: the glabridin loaded by exosomes comprises glabridin and exosomes, wherein the exosomes comprise exosome bodies and artificial saliva, and the mass percentage of the exosome bodies to the artificial saliva is 0.01%. The artificial saliva comprises the following components in percentage by mass: phosphate buffer solution with pH of 7.0, 0.2%, anhydrous calcium chloride 0.025%, potassium chloride 0.05%, sodium chloride 0.6%, anhydrous magnesium sulfate 0.01%, mussel mucin 0.05%, albumin 0.05%, N-acetylglucosamine 0.01%, trehalose 0.01%, glycine 0.01%, L-methionine 0.01%, L-serine 0.01%, taurine 0.01%, and the balance of purified water to 100%.
Example 10: the glabridin loaded by exosomes comprises glabridin and exosomes, wherein the exosomes comprise exosome bodies and artificial saliva, and the mass percentage of the exosome bodies to the artificial saliva is 0.1%. The artificial saliva comprises the following components in percentage by mass: phosphate buffer solution with pH of 7.0, 0.2%, anhydrous calcium chloride 0.025%, potassium chloride 0.05%, sodium chloride 0.6%, anhydrous magnesium sulfate 0.01%, mussel mucin 0.1%, albumin 0.1%, N-acetylglucosamine 0.02%, trehalose 0.02%, glycine 0.02%, L-methionine 0.02%, L-serine 0.02%, taurine 0.02%, and the balance of purified water to 100%.
Example 11: the glabridin loaded by exosomes comprises glabridin and exosomes, wherein the exosomes comprise exosome bodies and artificial saliva, and the mass percentage of the exosome bodies to the artificial saliva is 0.1%. The artificial saliva comprises the following components in percentage by mass: phosphate buffer solution with pH of 7.0, 0.2%, anhydrous calcium chloride 0.025%, potassium chloride 0.05%, sodium chloride 0.6%, anhydrous magnesium sulfate 0.01%, mussel mucin 0.25%, albumin 0.25%, N-acetylglucosamine 0.05%, trehalose 0.05%, glycine 0.05%, L-methionine 0.05%, L-serine 0.05%, taurine 0.05%, and the balance of purified water to 100%.
Example 12: the glabridin loaded by exosomes comprises glabridin and exosomes, wherein the exosomes comprise exosome bodies and artificial saliva, and the mass percentage of the exosome bodies to the artificial saliva is 0.1%. The artificial saliva comprises the following components in percentage by mass: phosphate buffer solution with pH of 7.0, 0.2%, anhydrous calcium chloride 0.025%, potassium chloride 0.05%, sodium chloride 0.6%, anhydrous magnesium sulfate 0.01%, mussel mucin 0.4%, albumin 0.4%, N-acetylglucosamine 0.08%, trehalose 0.08%, glycine 0.08%, L-methionine 0.08%, L-serine 0.08%, taurine 0.08%, and the balance of purified water to 100%.
Example 13: the glabridin loaded by the exosome comprises glabridin and the exosome, wherein the exosome comprises an exosome body and artificial saliva, and the mass percentage of the exosome body and the artificial saliva is 1%. The artificial saliva comprises the following components in percentage by mass: phosphate buffer solution with pH of 7.0, 0.2%, anhydrous calcium chloride 0.025%, potassium chloride 0.05%, sodium chloride 0.6%, anhydrous magnesium sulfate 0.01%, mussel mucin 0.5%, albumin 0.5%, N-acetylglucosamine 0.1%, trehalose 0.1%, glycine 0.1%, L-methionine 0.1%, L-serine 0.1%, taurine 0.1%, and the balance of purified water to 100%.
(3) Tyrosinase activity inhibition assay:
to test the efficacy of glabridin after loading glabridin on exosomes in the above examples, examples 9 to 13 were prepared, and placed at 4℃for 3 months, and then sampled for tyrosinase activity inhibition detection. Firstly, 3 times of absolute alcohol is added in different examples, then centrifugation is carried out for 60 minutes at 10 ten thousand G/min, supernatant liquid is poured out, glabridin released due to unstable exosomes is also poured out, and the exosomes at the bottom are resuspended by phosphate buffer solution, so that tyrosinase activity inhibition test is carried out.
L-tyrosine is used as a substrate, mushroom tyrosinase is used as a reaction enzyme, the above examples 1-5 are used as test substances, sodium chloride is used as a negative control, and after reaction for 2 hours at 37 ℃ under the condition of avoiding light, the reaction is detected by an enzyme-labeled instrument at the wavelength of 490 nm.
As can be seen from fig. 2, the effect of exosome-loaded glabridin in example 9 on inhibiting tyrosinase activity is better than that of example 10, and the effect of exosome-loaded glabridin examples 10, 11 and 12 are not greatly different, and the effect of exosome-loaded glabridin in example 13 on inhibiting tyrosinase activity is weaker than that of other examples, and according to the most economical and most efficient principle, the invention suggests that the effect of exosome-loaded glabridin in example 10 can be fully exerted.
(4) Melanin formation inhibition assay:
to test the protective effect of each example on glabridin-loaded exosome activity, examples 1 to 5 were prepared, and then placed at 4℃for 3 months, followed by sampling for melanin formation inhibition test. Firstly, 3 times of absolute alcohol is added in different examples, then centrifugation is carried out for 60 minutes at 10 ten thousand G/min, supernatant is poured off, glabridin released due to unstable exosomes is also poured off, and the exosomes at the bottom are resuspended by phosphate buffer solution, so that a melanin formation inhibition test is carried out.
B16 melanocytes were used in melanogenesis inhibition assays at B16 cell concentrations of 1x10 4 Plating the holes, culturing for 12 hours, changing the liquid, adding 0.1 mug/ml alpha-MSH of a melanin inducer and artificial saliva of the invention, adding 0.1 mug/ml alpha-MSH of a melanin inducer and sodium chloride of a control group, continuously culturing for 48 hours, taking the supernatant of the culture liquid, detecting the melanin content at the wavelength of 405nm by using an enzyme-labeling instrument, and calculating the inhibition rate.
As can be seen from fig. 3, in accordance with the results of tyrosinase activity inhibition test, the inhibition effects of glabridin in examples 10, 11 and 12 were not greatly different from each other, and were substantially superior to those of examples 9 and 13, and the present invention suggests that the glabridin of example 10 as exosome load can be used in a mass composition ratio that can fully exert the effects thereof according to the most economical and most efficient principle.

Claims (10)

1. A substance that protects exosome activity, the substance being artificial saliva capable of providing a saliva environment.
2. A substance for protecting exosome activity according to claim 1, wherein the artificial saliva comprises the following components in mass percent: phosphate buffer solution with pH of 7.0, 0.2%, anhydrous calcium chloride 0.025%, potassium chloride 0.05%, sodium chloride 0.6%, anhydrous magnesium sulfate 0.01%, mussel mucin 0.05-0.5%, albumin 0.05-0.5%, N-acetylglucosamine 0.01-0.1%, trehalose 0.01-0.1%, glycine 0.01-0.1%, L-methionine 0.01-0.1%, L-serine 0.01-0.1%, taurine 0.01-0.1%, and purified water for the rest to be added to supplement 100%.
3. The exosome is characterized by comprising an exosome body and a substance for protecting exosome activity, wherein the mass percentage of the exosome body to the substance for protecting exosome activity is 0.01% -1%.
4. An exosome according to claim 3 wherein the mass percent of exosome body to exosome activity protecting substance is 0.1%.
5. An exosome according to claim 3 or 4 wherein the exosome body is secreted by umbilical mesenchymal stem cells.
6. Use of the exosome according to claim 3 or 4 or 5 for loading glabridin.
7. The method for loading glabridin on exosomes is characterized by comprising the following steps of:
1) Mixing alcoholic solution dissolved with glabridin with exosome supernatant according to the mass ratio of (1-5): (10-20), adding tween-80 accounting for 0.1-1% of the mass of the mixed system into the mixed system, and uniformly stirring to obtain mixed solution; the exosomes comprise exosome bodies and substances for protecting exosome activity;
2) Under the ice bath condition, carrying out ultrasonic treatment on the mixed solution obtained in the step 1) for 30-300 s, wherein the frequency is 20KHz-100KHz;
3) Centrifuging the mixed solution after ultrasonic treatment with 5000G-10000G for 10min-30min to obtain supernatant 1, and filtering the supernatant 1 with 0.2 μm sterile filter membrane to obtain filtrate;
4) Centrifuging the filtrate with 100000G-150000G for 30min-180min, discarding supernatant, and resuspending the suspension with sterile physiological saline to obtain glabridin-loaded exosome.
8. The method for loading glabridin an exosome according to claim 7, wherein the mass ratio of glabridin in the alcohol solution in step 1) is 1% -10%.
9. The method according to claim 7, wherein the alcohol solution in which glabridin is dissolved in step 1) is one of methanol, ethanol, propanol, n-butanol or isobutanol, and the alcohol solution is an anhydrous alcohol solution.
10. The method for exosome-loaded glabridin according to claim 7, wherein the ultrasonic power in step 2) is 0.1W/cm 3 -1W/cm 3 The pulse time is 1s-10s, and the interval time is 1s-10s.
CN202211434892.7A 2022-11-16 2022-11-16 Substance for protecting exosome activity, exosome and glabridin loaded by exosome Pending CN116098826A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202211434892.7A CN116098826A (en) 2022-11-16 2022-11-16 Substance for protecting exosome activity, exosome and glabridin loaded by exosome

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211434892.7A CN116098826A (en) 2022-11-16 2022-11-16 Substance for protecting exosome activity, exosome and glabridin loaded by exosome

Publications (1)

Publication Number Publication Date
CN116098826A true CN116098826A (en) 2023-05-12

Family

ID=86255029

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211434892.7A Pending CN116098826A (en) 2022-11-16 2022-11-16 Substance for protecting exosome activity, exosome and glabridin loaded by exosome

Country Status (1)

Country Link
CN (1) CN116098826A (en)

Similar Documents

Publication Publication Date Title
JP2809735B2 (en) Lyophilization of cells
EP1948259B1 (en) Acellular bioabsorbable tissue regeneration matrices produced by incubating acellular blood products
EP0101341B1 (en) Process and device for the encapsulation in erythrocytes of at least one biologically active substance, in particular hemoglobin allosteric effectors, and erythrocytes so obtained
EP2512432B1 (en) Injectable compositions for intra-articular use combining a viscosupplementation agent and a fibroblast growth medium
CN106821938A (en) A kind of preparation method of human mesenchymal stem cell freeze-dried powder
US4338301A (en) Lung tissue extract useful for treating hyaline-membrane disease and method for producing the extract
CA2887583C (en) Method for preserving placental blood
EP0221123A1 (en) Synthetic solution for the prolonged storage of erythrocytary concentrates
CN109350557A (en) Anti-apolexis composition, skin care item comprising NADH and ceramide and the preparation method and application thereof
EP3795674A1 (en) Composition, cell storage composition, cell culture composition, cell formulation, method for producing object containing microbubble, cell storage method, cell culture method, and cell formulation production method
TR201816125T4 (en) Composition and method for protecting, transporting and storing living biological materials.
EP2748306B1 (en) Use of annelid haemoglobin for maintaining stem cells in the undifferentiated state
CN108888634A (en) The preparation method and application of hair follicle stem cells extract freeze-drying powder
CN116458493A (en) Stem cell preservation solution without DMSO (dimethyl sulfoxide), and preparation method and application method thereof
CN116098826A (en) Substance for protecting exosome activity, exosome and glabridin loaded by exosome
TW201832775A (en) Uses of Camellia sinensis callus extract in protecting skin
Li et al. Harnessing the therapeutic potential of mesenchymal stem cell-derived exosomes in cardiac arrest: Current advances and future perspectives
Turesky et al. A histochemical study of protein-bound sulfhydryl and disulfide groups in normal and inflamed human gingiva
EP3607049A1 (en) Bacterial secretome for use in the treatment of skin lesions
Ali et al. The Correlation between oxidative stress and thyroid hormones in serum and tissue homogenized of hypothyroidism patients
CN108815528B (en) Application of nereis extracellular hemoglobin in preparation of callus drugs
Pidlisetsky et al. Administration of platelet-rich plasma or concentrated bone marrow aspirate after mechanically induced ischemia improves biochemical parameters in skeletal muscle
CN103202794B (en) Preparation method and application of skin stem cell active element
KR100363197B1 (en) the extracting method of earthworm's body fluids using water contained activated oxygen
Ciftci et al. Effects of some drugs on enzymatic activity of glucose 6-phosphate dehydrogenase from chicken erythrocytes in vitro

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination