Method for preparing ganoderma lucidum spore powder exosome
The purification process was simplified by using differential centrifugation and multiple filtration, which solved the problem of low purity and concentration of Ganoderma lucidum exosomes, and enabled the preparation of high-concentration and high-purity exosomes, supporting the industrial application of exosomes.
Patent Information
- Application Number
- CN202511497457.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-20
- Publication Date
- 2025-12-16
AI Technical Summary
Existing technologies for extracting Ganoderma lucidum exosomes suffer from low purity and concentration, leading to structural damage and heterogeneous heavy particle aggregation in the prepared exosomes, making it difficult to achieve industrial application.
Differential centrifugation and multiple filtration methods, including low-speed, medium-speed and ultra-speed centrifugation combined with 0.45 µm and 0.22 µm PVDF filter membrane filtration, were used to simplify the purification steps and improve the concentration and purity of exosomes.
This study achieved the preparation of high-concentration and high-purity exosomes from Ganoderma lucidum spore powder, providing an excellent source of exosomes and laying the foundation for subsequent functional and mechanistic research.
Smart Images
Figure CN121136831A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing exosomes from Ganoderma lucidum spore powder. Background Technology
[0002] As a precious traditional Chinese medicine with a long history, Ganoderma lucidum's fruiting body, broken-cell wall spore powder, and spore oil are rich in polysaccharides, triterpenoids, proteins, and other active ingredients. Modern pharmacological studies have confirmed that it has multiple pharmacological activities, including anti-tumor, immunomodulatory, antioxidant, anti-inflammatory, and neuroprotective effects.
[0003] Besides traditional extracts, Ganoderma lucidum exosomes, as a naturally derived extracellular vesicle, have attracted much attention. Existing studies have shown that Ganoderma lucidum exosomes are not only rich in various phytochemicals, but also specifically carry miRNAs with synergistic biological effects, exhibiting multiple benefits such as anti-tumor, anti-inflammatory, and anti-aging properties.
[0004] Currently, the extraction and purification of plant exosomes requires further separation and purification after obtaining a mixture of plant exosomes through methods such as juice extraction or tissue permeation-centrifugation. The main techniques used for the separation and purification of plant exosomes include ultracentrifugation, density gradient centrifugation, ultrafiltration, and size exclusion chromatography. Existing methods for extracting Ganoderma lucidum exosomes borrow from established plant exosome extraction procedures, typically involving ultracentrifugation, which effectively removes fibrous impurities from plant tissues and promotes exosome deposition. To improve exosome purity, the obtained precipitate is usually resuspended in PBS solution and subjected to a second round of high-speed centrifugation. Although this method is low-cost, allows for large sample volumes, and has high throughput, the workload is significant and time-consuming after several centrifugation steps. More importantly, high molecular weight components in the plant juice, such as cellulose, starch, and other protein impurities, can easily lead to a decrease in exosome purity after centrifugation. Furthermore, the repeated high-intensity centrifugation during purification can cause significant structural damage and aggregation of heterogeneous heavy particles, resulting in a lower concentration of the prepared exosomes. To achieve a certain level of purity, a significant reduction in concentration is necessary. However, the lower initial concentration limits the application of further refined purification techniques, making it difficult to achieve a breakthrough in purity. This negative correlation between concentration and purity is a key obstacle preventing Ganoderma lucidum exosomes from moving from the laboratory to industrial applications.
[0005] Therefore, developing exosome preparation technologies that can simultaneously achieve high purity, high concentration, and high bioactivity retention has become an urgent need in this field. Summary of the Invention
[0006] The purpose of this invention is to effectively overcome the aforementioned problems by providing a method for preparing exosomes from Ganoderma lucidum spore powder based on differential centrifugation and multiple filtration. This method simplifies the purification steps while significantly improving the concentration and purity of the prepared exosomes.
[0007] The objective of this invention is achieved through the following means: (1) Pretreatment: Take 200-250g of Ganoderma lucidum spore powder and immerse it in 500-1000 mL of 4 °C PBS solution for 10-12 hours, then filter to obtain the original solution of Ganoderma lucidum spore powder; preferably, take 200g of Ganoderma lucidum spore powder and immerse it in about 500-800 mL of 4 °C PBS solution for 12 hours, then filter to obtain the original solution of Ganoderma lucidum spore powder; (2) Differential centrifugation The pretreated stock solution was centrifuged at 3,000×g for 40 min at low speed. The supernatant was then centrifuged at 10,000×g for 70 min at medium speed. The supernatant was then centrifuged at 130,000×g for 90 min at ultraspeed. The precipitate was completely resuspended in more than 20 mL of PBS solution and then filtered through 0.45 µm and 0.22 µm PVDF membranes, respectively. The precipitate was then centrifuged again at 130,000×g for 90 min at ultraspeed. The final precipitate was the Ganoderma lucidum spore powder exosomes.
[0008] Preferably, when taking the supernatant, a 40 µm filter membrane is used to remove impurities.
[0009] Preferably, the exosomes from the Ganoderma lucidum spore powder are resuspended in 2-5 mL of PBS solution at 4℃ to obtain a Ganoderma lucidum spore powder exosome suspension, which is then stored at -80℃.
[0010] Preferably, the PBS solution is pre-cooled at 4°C before use.
[0011] Preferably, the Ganoderma lucidum spore powder has a cell wall breakage rate of 10-80%, and most preferably it has a cell wall breakage rate of 10%.
[0012] The advantages of this invention compared with the prior art are as follows: This invention provides a method for preparing exosomes from Ganoderma lucidum spore powder, which yields high concentrations and quantities of exosomes from Ganoderma lucidum spore powder, providing an excellent source of exosomes for subsequent research on their functions and mechanisms. Attached Figure Description
[0013] Figure 1 Electron micrograph of exosomes from Ganoderma lucidum spore powder (100 nm).
[0014] In the figure, a) Ganoderma lucidum spore powder exosomes from Example 1; b) Ganoderma lucidum spore powder exosomes from Example 2; c) Ganoderma lucidum spore powder exosomes from Example 3. Detailed Implementation
[0015] The present invention will be further explained and illustrated below through specific embodiments: Example 1
[0016] (1) Take about 200g of Ganoderma lucidum spore powder with a cell wall breakage rate of 10%, immerse it in 600 mL of PBS solution (4℃) for 12h, and filter it through a 600 mesh filter to obtain the original solution of Ganoderma lucidum spore powder; (2) The pretreated stock solution was centrifuged at low speed (3,000×g, 40 min) using a refrigerated centrifuge (Thermo SORVALL RC6+). The supernatant was filtered through a 40 µm filter membrane and then centrifuged at medium speed (10,000×g, 70 min). The supernatant was then collected. (3) The solution obtained in (2) was subjected to ultracentrifugation (130,000×g, 90 min) using a low-temperature ultracentrifuge (Beckman Coulter, rotor: Type 70 T1). The supernatant was discarded, and 28 mL of PBS solution (4℃) was added to the precipitate to completely resuspend it. The precipitate was then filtered through 0.45 µm and 0.22 µm PVDF membranes, and the solution was ultracentrifuged again (130,000×g, 90 min) to obtain the final precipitate, namely Ganoderma lucidum spore powder exosomes. (4) Resuspend the precipitate obtained in (3) in 2 mL of PBS (4°C) solution and store it at -80°C for later use; the PBS solution should be pre-cooled at 4°C before use; (5) Using a 20 µL pipette, add 8-10 µL of the Ganoderma lucidum spore powder exosome suspension obtained in (4) onto the grid. After 10 minutes, gently blot the liquid dry with filter paper. Then, using a 20 µL pipette, add 10 µL of staining solution onto the grid. After approximately 2 minutes, blot off any excess staining solution with filter paper. Wait 10-20 minutes until the grid is dry. Observe the sample using a low-voltage transmission electron microscope (FEI Tecnai G2 120KV, USA). The electron microscope observation results are as follows: Figure 1 a; (6) The obtained Ganoderma lucidum spore powder exosome suspension was diluted 10,000 times with PBS solution and added to the sample cell. The size distribution and concentration of exosomes were evaluated using a nanoparticle tracking analyzer (Particle Metrix, ZetaView S / N 23-964). The test report was obtained using Software ZetaView (version 8.05.16 SP3), and the specific results are shown in Table 1. Example 2
[0017] (1) Take about 200g of Ganoderma lucidum spore powder with a cell wall breakage rate of 80%, immerse it in 800 mL of PBS solution (4℃) for 12h, and filter it through an 800 mesh filter to obtain the original solution of Ganoderma lucidum spore powder; (2) The pretreated stock solution was centrifuged at low speed (3,000×g, 40 min) using a refrigerated centrifuge (Thermo SORVALL RC6+). The supernatant was filtered through a 40 µm filter membrane and then centrifuged at medium speed (10,000×g, 70 min). The supernatant was then collected. (3) The solution obtained in (2) was subjected to ultracentrifugation (130,000×g, 90 min) using a low-temperature ultracentrifuge (Beckman Coulter, rotor: Type 70 T1). The supernatant was discarded, and 28 mL of PBS solution (4℃) was added to the precipitate to completely resuspend it. The precipitate was then filtered through 0.45 µm and 0.22 µm PVDF membranes, and the solution was ultracentrifuged again (130,000×g, 90 min) to obtain the final precipitate, namely Ganoderma lucidum spore powder exosomes. (4) Resuspend the precipitate obtained in (3) in 2 mL PBS (4℃) solution and store it at -80℃ for later use; the PBS solution should be pre-cooled at 4℃ before use.
[0018] The electron microscopy observation results are as follows Figure 1 b. Example 3
[0019] (1) Take about 200g of Ganoderma lucidum spore powder with a cell wall breakage rate of 95%, immerse it in 800 mL of PBS solution (4℃) for 12h, and filter it through an 800 mesh filter to obtain the original solution of Ganoderma lucidum spore powder; (2) The pretreated stock solution was centrifuged at low speed (4,000×g, 40 min) using a refrigerated centrifuge (Thermo SORVALL RC6+). The supernatant was filtered through a 40 µm filter membrane and then centrifuged at medium speed (10,000×g, 70 min). The supernatant was then collected. (3) The solution obtained in (2) was subjected to ultracentrifugation (140,000×g, 90 min) using a low-temperature ultracentrifuge (Beckman Coulter, rotor: Type 70 T1). The supernatant was discarded, and 28 mL of PBS solution (4℃) was added to the precipitate to completely resuspend it. The precipitate was then filtered through 0.45 µm and 0.22 µm PVDF membranes, and the precipitate was ultracentrifuged again (140,000×g, 90 min) to obtain the final precipitate, namely Ganoderma lucidum spore powder exosomes. (4) Resuspend the precipitate obtained in (3) in 2 mL PBS (4℃) solution and store it at -80℃ for later use; the PBS solution should be pre-cooled at 4℃ before use.
[0020] The electron microscopy observation results are as follows Figure 1 c.
[0021] The low-pressure transmission electron microscopy (TEM) detection method for exosomes from Ganoderma lucidum spore powder described in the above embodiments is as follows: Lay the sealing film flat on the petri dish, place the screen on the sealing film, and use a pipette to add 8-10 µL of Ganoderma lucidum spore powder exosome suspension onto the screen. Time for 10 minutes, then gently blot dry with filter paper. Use a pipette to add 10 µL of staining solution onto the screen, and time for approximately 2 minutes. Blot dry with filter paper and wait 10-20 minutes until the screen is dry before microscopic observation. The observed exosomes should have intact structures, exhibit a typical cup-shaped morphology, and be relatively uniform in shape.
[0022] Table 1. Detection results of NTA in exosomes from Ganoderma lucidum spore powder source Concentration (Particles / mL) Average diameter (nm) percentage(%) Example 1 2.63E+12 166.7 80.0 Example 2 1.35E+12 169.5 64.1 Example 3 1.35E+12 169.5 51.6
Claims
1. A method of preparing Ganoderma spores powder exosomes, characterized by The method comprises the following steps: (1) Pretreatment: 200-250g of Ganoderma lucidum spore powder is immersed in about 500-1000 mL of 4°C PBS solution for 10-12h, and then filtered to obtain a Ganoderma lucidum spore powder stock solution; (2) Differential centrifugation After low-speed centrifugation of the stock solution obtained in the pretreatment at 3,000×g for 40 min, the supernatant is taken and subjected to medium-speed centrifugation at 10,000×g for 70 min, and the obtained supernatant is subjected to ultracentrifugation at 130,000×g for 90 min. After the obtained precipitate is completely resuspended in 20 mL or more of PBS solution, it is filtered in sequence using 0.45 µm and 0.22 µm PVDF filter membranes, and the solution is again subjected to ultracentrifugation at 130,000×g for 90 min. The final precipitate is the Ganoderma lucidum spore powder exosome.
2. The method of claim 1, wherein the Ganoderma lucidum spore powder exosome is prepared by the steps of: The Ganoderma lucidum spore powder has a broken wall rate of 10-80%. 3. The method for preparing Ganoderma lucidum spore powder exosomes according to claim 1, characterized in that... When the supernatant is taken, a 40 µm filter membrane is used to remove impurities.
4. The method for preparing Ganoderma lucidum spore powder exosomes according to claim 1, characterized in that... The Ganoderma lucidum exosome is resuspended in 2-5 4°C PBS solution to obtain a Ganoderma lucidum spore powder exosome suspension, which is stored at -80°C.
5. The method for preparing Ganoderma lucidum spore powder exosomes according to claim 3, characterized in that... The PBS solution is pre-cooled at 4°C before use.