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CN-121555402-B - Saffron exosome and preparation method and application thereof

CN121555402BCN 121555402 BCN121555402 BCN 121555402BCN-121555402-B

Abstract

The invention belongs to the technical field of exosomes, and particularly relates to a saffron exosome, and a preparation method and application thereof. The preparation method comprises the steps of collecting stigma croci and petals of saffron, washing and drying, crushing to 80-100 meshes to obtain saffron powder, soaking the saffron powder in a specific solvent according to a feed liquid ratio of 1:6-10, then carrying out ultrasonic extraction to obtain an extracting solution, wherein the ultrasonic power is set to be 200W-300W, the time is 30 min-60 min, the temperature is controlled to be 4-8 ℃, the specific solvent formula comprises the steps of adding 0.005-0.01 g of potassium nitrate or 0.01-0.02 g of potassium sulfate into each liter of PBS buffer solution, centrifuging the extracting solution at a rotating speed of 800-10000 g to remove impurity precipitates, filtering the supernatant through a 100 kDa-120 kDa ultrafiltration membrane, collecting filtrate, and carrying out 1000 g-110000 g ultracentrifugation for 70 min-90 min to obtain exosome precipitates, namely the saffron exosome. The invention can stimulate the release of the exosomes, stabilize and retain the crocus exosomes, and finally remarkably improve the extraction efficiency of the crocus exosomes, thereby achieving the optimal activity.

Inventors

  • FENG HAIMIN
  • WU ZHENZHONG
  • LI YIN

Assignees

  • 上海春申外泌体生物医学有限公司

Dates

Publication Date
20260508
Application Date
20251212

Claims (8)

  1. 1. The preparation method of the saffron exosome is characterized by comprising the following steps: collecting stigma croci and petals of saffron, cleaning, drying, and pulverizing to 80-100 meshes to obtain saffron powder; soaking saffron powder in a specific solvent according to a feed liquid ratio of 1:6-10, and then carrying out ultrasonic extraction to obtain an extracting solution, wherein the ultrasonic power is set to be 200W-300W, the time is 30 minutes-60 minutes, and the temperature is controlled to be 4-8 ℃, and the specific solvent has the formula that 0.005 g-0.01 g potassium nitrate or 0.01 g-0.02 g potassium sulfate is added into each liter of PBS buffer solution; Centrifuging the extracting solution at a rotating speed of 8000-10000 g to remove impurity precipitates, filtering the obtained supernatant by a 100 kDa-120 kDa ultrafiltration membrane, collecting filtrate, and ultracentrifugating the filtrate at 100000 g-110000 g for 70-90 min to obtain exosome precipitates, namely saffron exosome.
  2. 2. The method for preparing saffron exosome according to claim 1, wherein the mass of potassium nitrate added per liter of PBS buffer is 0.007g; the mass of potassium sulfate added to each liter of PBS buffer was 0.015g.
  3. 3. The preparation method of saffron exosomes according to claim 1, wherein each liter of PBS buffer comprises 8g of sodium chloride, 0.2g of potassium chloride, 2.9g of disodium hydrogen phosphate, 0.24g of potassium dihydrogen phosphate, and 1L of deionized water; ph=7.2 to 7.4 of PBS buffer.
  4. 4. The method for preparing saffron exosomes according to claim 1, wherein the exosome precipitate is freeze-dried to obtain exosome freeze-dried powder.
  5. 5. The preparation method of the saffron exosome according to claim 1, further comprising the step of biologically modifying the exosome, wherein protease accounting for 0.5% -1% of the mass of the extract is added into the extract, and enzymolysis is carried out for 1-2 hours at 40 ℃ -50 ℃ to obtain an enzymolysis product; and (3) after the enzymolysis product is obtained, performing centrifugal separation at a rotating speed of 8000 g-10000 g to remove impurity precipitation.
  6. 6. The method for preparing saffron exosomes according to claim 5, wherein the protease is trypsin or papain.
  7. 7. The method for preparing saffron exosomes according to claim 1, wherein the washing and drying conditions are washing with tap water and drying at 0 ℃.
  8. 8. The application of the saffron exosome prepared by the preparation method according to any one of claims 1 to 7 in preparing an antibacterial agent, wherein the antibacterial agent is resistant to escherichia coli (ESCHERICHIA COLI), staphylococcus aureus (staphylococcus aureus) and drug-resistant staphylococcus aureus VISA.

Description

Saffron exosome and preparation method and application thereof Technical Field The invention belongs to the technical field of exosomes, and particularly relates to a saffron exosome, and a preparation method and application thereof. Background Exosomes are nanoscale vesicles secreted by cells and play a critical role in the organism. According to prior art studies, the physiological effects of exosomes are mainly manifested in the following ways: (1) The exosomes can carry ligands, receptors or small molecule RNAs and the like, so as to achieve the effect of regulating signal paths, thereby regulating physiological functions. (2) Immunomodulation, that is, some exosomes can induce T cell immune response, inhibit secretion of inflammatory factors IL-10, TGF-beta and the like, thereby achieving the effects of inhibiting inflammation and regulating immunity. (3) Tissue repair, namely, the exosome can be enriched with factors for promoting the growth of skin, so that the effects of promoting angiogenesis, repairing skin injury, resisting aging and the like are achieved. For the above reasons, exosomes are widely used in many biological research. Such as mesenchymal stem cell exosomes, neural stem cell exosomes, blood cell exosomes, etc. There are also many methods for extracting exosomes in this connection, such as ultracentrifugation, density gradient centrifugation, ultrafiltration, etc. However, these methods mainly separate exosomes from cellular secretions. Saffron is a valuable medicinal plant, and its extract has wide application in medicine, cosmetics and other fields. However, there are relatively few studies on saffron exosomes at present, especially in the extraction process. Research shows that when the traditional extraction method is applied to saffron exosomes, the extraction efficiency is often low, and high-purity exosomes are difficult to obtain, so that the application of saffron exosomes is limited. Disclosure of Invention In order to solve the technical problems, the invention provides a saffron exosome, and a preparation method and application thereof. Can extract saffron exosomes with high purity and size of 30-150 nm, and lays a foundation for wide application. The invention provides a preparation method of saffron exosomes, comprising the following steps: collecting stigma croci and petals of saffron, cleaning, drying, and pulverizing to 80-100 meshes to obtain saffron powder; soaking saffron powder in a specific solvent according to a feed liquid ratio of 1:6-10, and then carrying out ultrasonic extraction to obtain an extracting solution, wherein the ultrasonic power is set to be 200W-300W, the time is 30 minutes-60 minutes, and the temperature is controlled to be 4-8 ℃, and the specific solvent has the formula that 0.005 g-0.01 g potassium nitrate or 0.01 g-0.02 g potassium sulfate is added into each liter of PBS buffer solution; Centrifuging the extracting solution at a rotating speed of 8000-10000 g to remove impurity precipitates, filtering the obtained supernatant by a 100 kDa-120 kDa ultrafiltration membrane, collecting filtrate, and ultracentrifugating the filtrate at 100000 g-110000 g for 70-90 min to obtain exosome precipitates, namely saffron exosome. Preferably, in the preparation method of saffron exosomes, the mass of potassium nitrate added to each liter of PBS buffer is 0.007g, or the mass of potassium sulfate added to each liter of PBS buffer is 0.015g. Preferably, the preparation method of the saffron exosome comprises the following steps of preparing 8g of sodium chloride, 0.2g of potassium chloride, 2.9g of disodium hydrogen phosphate, 0.24g of monopotassium hydrogen phosphate and supplementing 1L of deionized water, wherein the pH value of the PBS buffer is=7.2-7.4. Preferably, in the preparation method of the saffron exosome, the exosome precipitate is freeze-dried to obtain the exosome freeze-dried powder. Preferably, the preparation method of the saffron exosome further comprises the step of biologically modifying the exosome, wherein protease which is 0.5-1% of the mass of the extracting solution is added into the extracting solution, and enzymolysis is carried out for 1-2 hours at 40-50 ℃ to obtain an enzymolysis product; And (3) after the enzymolysis product is obtained, carrying out centrifugal impurity precipitation removal at the rotating speed of 8000 g-10000 g. Preferably, the preparation method of the saffron exosome comprises that the protease is trypsin or papain. Preferably, the preparation method of the saffron exosomes comprises the steps of washing with tap water and drying at 0 ℃. The invention provides a saffron exosome prepared by the preparation method. Use of saffron exosomes in the preparation of antibacterial and/or antioxidant agents. Preferably, the antibacterial means against escherichia coli, staphylococcus aureus and drug-resistant staphylococcus aureus; the antioxidation refers to free radical removal. Compared with the prior art, th