Preparation method and application of pinellia ternate exosome

Abstract

The invention provides a preparation method and application of pinellia ternate exosomes, and belongs to the technical field of plant biologication.The preparation method comprises the following steps that 1, pinellia ternate sterile cluster buds are inoculated into a liquid culture medium to be subjected to intermittent immersion culture, and pinellia ternate cluster seedlings are obtained; or inoculating the callus of the pinellia ternate into a liquid culture medium, and performing intermittent immersion culture to obtain loose cells of the pinellia ternate; the ventilation time of the intermittent immersion culture is 3-20 min, the retention time is 5-30 min, the deflation time is 5-15 min, the immersion interval time is 2-8 h, and the cycle number is infinite. (2) after homogenating the pinellia ternate clustered seedlings or the pinellia ternate loose cells obtained in the step (1), separating pinellia ternate exosomes; pinellia ternate clustered seedlings or loose cells obtained by a gap immersion culture method are used as raw materials, and the extracted pinellia ternate exosome is stable in component, high in quality control degree, wide in application range, low in preparation cost and short in period.

Description

Technical Field

[0001] The present invention belongs to the field of plant biotechnology, and in particular relates to a preparation method of exosomes of Pinellia ternata and an application thereof. Background Art

[0002] Exosomes are a type of nano-scale vesicles derived from the cell membrane, which are widely found in animals, plants and microorganisms. They have a phospholipid bilayer structure, contain molecules such as nucleic acids and proteins, are protected from degradation and transported to receptor cells. They also have certain biological activities, such as affecting other cell functions and regulating gene transcription, and serve as biological markers for early diagnosis of cancer, immune diseases, etc. In addition, exosomes have broad application prospects in cross-border regulation of the biological world. With the deepening of exosome research, the role of plant exosomes or plant-derived exosomes-like nanovesicles (PELN) has received widespread attention in recent years. The diameter of plant exosomes is 30 to 150 nanometers, and they have a typical saucer-like appearance under a projection electron microscope. Since the biogenesis of plant exosomes and microvesicles is not strictly separated, they can be formed at the same time, and the two have little difference in physical properties and composition, they are often called "nanovesicles", "microvesicles" and "exosome-like vesicles". At present, plant exosomes have been studied and discovered in more than 10 edible plants, including ginseng, sedum, loquat, garlic, epimedium, ginger, green pepper, garlic, mushroom, lemon, yam, grape, tomato, broccoli and onion. In contrast, the research on exosomes of Pinellia and other Chinese medicinal materials is still in its infancy. However, with the advancement of science and technology and the advancement of the modernization of traditional Chinese medicine, research in this field is bound to accelerate. It can be foreseen that in the future, the study of plant cell exosomes will gradually become one of the hot spots in biological science research.

[0003] Pinellia ternata (Thunb.) Breit, a plant of the genus Pinellia in the family Araceae, has antitussive, anti-tumor, and anti-thrombotic effects and has a long history of use. As a traditional Chinese medicine, the effectiveness and safety of Pinellia ternata have been verified historically. By studying Pinellia ternata exosomes through modern scientific and technological means, we can understand its pharmacological effects at the molecular level and provide a scientific basis for the modernization and internationalization of traditional Chinese medicine. In addition, the components of Pinellia ternata exosomes have the potential to become candidates for new drug development, especially in anti-inflammatory, immunomodulatory, and wound healing.

[0004] The premise of exosome research is to prepare exosomes with high quality control, and the most important thing for high-quality exosomes is to select high-quality control materials. At present, plant exosomes are usually prepared by squeezing tissues or tissue enzyme digestion juice after multiple high-speed centrifugation and then ultra-high-speed centrifugation; for example: when extracting ginger exosomes, the coarsely filtered ginger juice is centrifuged at 10000g for 1h at 4°C, the supernatant is taken and repeated 2-3 times to remove cell debris, and then centrifuged at 100000g for 80min to precipitate ginger exosomes. For plants that are squeezed or digested by enzymes, most of the plant raw materials currently studied are artificially planted in open-air environments. These materials are affected by the constant changes in light, temperature, pathogenic microorganisms, etc. in the open-air environment, resulting in different exosomes prepared from each batch of exosomes from the same plant, which makes the quality control of exosomes very difficult. Ultimately, it brings uncertain challenges to the research and industrialization of plant exosomes. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method for preparing Pinellia exosomes and its application, using Pinellia ternata clumping seedlings or loose cells obtained by intermittent immersion culture as raw materials, and the extracted Pinellia ternata exosomes have stable components, high quality control, wide application range, low preparation cost and short cycle.

[0006] The present invention provides a method for preparing exosomes of Pinellia ternata, comprising the following steps:

[0007] 1) inoculating the sterile clustered buds of Pinellia ternata into a liquid culture medium for intermittent immersion culture to obtain clustered Pinellia ternata seedlings;

[0008] Alternatively, the Pinellia callus is inoculated into a liquid culture medium for intermittent immersion culture to obtain the Pinellia loose cells; the intermittent immersion culture has a ventilation time of 3 to 20 minutes, a holding time of 5 to 30 minutes, a degassing time of 5 to 15 minutes, an immersion interval of 2 to 8 hours, and an unlimited number of cycles;

[0009] 2) homogenizing the Pinellia ternata clumping seedlings or Pinellia ternata loose cells obtained in step 1), and isolating Pinellia ternata exosomes;

[0010] Preferably, when culturing sterile clustered shoots of Pinellia ternata, the liquid culture medium formula is: MS+6-BA 0.5-2 mg / L+NAA 0.01-0.2 mg / L+sucrose 10-40 g / L.

[0011] Preferably, when culturing Pinellia callus, the liquid culture medium formula is: MS+6-BA 0.10-0.30 mg / L+2,4-D 1.00-2.00 mg / L+KT 0.4-0.6 mg / L+sucrose 25-35 g / L.

[0012] Preferably, the light intensity of the intermittent immersion culture is 1800-2000 lx, the light exposure time is 12-16 h / d, and the temperature is 24-26°C.

[0013] Preferably, the separation of Pinellia exosomes described in step 2) is carried out by centrifugation, and the centrifugation includes centrifugation at 1800-2200g for 8-12min; collecting the first supernatant, centrifuging the first supernatant at 4500-5500g for 8-12min, and collecting the second supernatant; centrifuging the second supernatant at 9000-11000g for 40-50min, collecting the third supernatant, centrifuging the third supernatant at 90000-110000g for 1.5-2.5h, discarding the fourth supernatant, collecting the precipitate, and resuspending to obtain Pinellia exosomes.

[0014] Preferably, during the intermittent immersion culture in step 1), terahertz waves are applied to the liquid culture medium.

[0015] Preferably, the size of the aseptic clustered buds of Pinellia ternata is (4-6)×(4-6)×(4-6) mm. 3 The inoculation quantity of the sterile clustered buds of Pinellia ternata is 30 to 120 buds / L of culture medium, preferably 60 to 90 buds / L of culture medium.

[0016] Preferably, the period of the intermittent submerged culture in step 1) is 50 to 120 days, preferably 60 to 90 days.

[0017] Preferably, after the intermittent immersion culture in step 1) is completed, the method further comprises the steps of collecting the culture fluid and isolating the Pinellia exosomes from the culture fluid.

[0018] The present invention also provides the use of the pinellia exosomes prepared by the preparation method in the preparation of medicines or cosmetics.

[0019] Compared with the prior art, the present invention has the following beneficial effects: the method for preparing Pinellia exosomes provided by the present invention obtains Pinellia ternata clustered seedlings or Pinellia ternata loose cells by intermittent immersion culture, and the Pinellia ternata clustered seedlings or Pinellia ternata loose cells themselves present standardized operations during the intermittent immersion culture process, and the materials are easier to obtain and have high repeatability; the exosome components in the plant materials prepared under controllable culture conditions are stable, the quality control degree is high, and the application range is wide; the intermittent immersion culture method is adopted, and high-quality controlled plant materials can be obtained in a short time and in large quantities, with low cost, short plant growth cycle, and short preparation cycle of Pinellia ternata exosomes.

[0020] Furthermore, in the process of the intermittent immersion culture, the present invention applies terahertz waves to the liquid culture medium, which can further increase the amount of exosomes produced by the plant material and improve the yield of exosomes in Pinellia ternata. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a transmission electron microscopy image of the Pinellia exosomes prepared in Example 1;

[0022] Figure 2 This is a particle size analysis diagram of the Pinellia exosomes prepared in Example 1;

[0023] Figure 3 This is the cluster analysis of non-targeted metabolome of Pinellia ternata exosomes cultured in high-throughput bioreactor in Example 2;

[0024] Figure 4 This is an electron micrograph of the exosomes of Pinellia ternata treated with terahertz radiation in Example 2. DETAILED DESCRIPTION

[0025] The present invention provides a method for preparing exosomes of Pinellia ternata, comprising the following steps: 1) inoculating sterile clustered buds of Pinellia ternata into a liquid culture medium for intermittent immersion culture to obtain clustered seedlings of Pinellia ternata; or inoculating Pinellia ternata callus into a liquid culture medium for intermittent immersion culture to obtain loose cells of Pinellia ternata; 2) homogenizing the clustered seedlings of Pinellia ternata or the loose cells of Pinellia ternata obtained in step 1), and isolating the exosomes of Pinellia ternata.

[0026] In the present invention, the aseptic clustered buds of Pinellia ternata are inoculated into a liquid culture medium for intermittent immersion culture to obtain the aseptic clustered buds of Pinellia ternata. In the present invention, the size of the aseptic clustered buds of Pinellia ternata is preferably (4-6)×(4-6)×(4-6) mm 3 A single bud, preferably 5×5×5mm 3 The number of the sterile clustered buds of Pinellia ternata is 30 to 120 per liter of culture medium, preferably 60 to 90 per liter of culture medium. The present invention preferably inoculates the sterile clustered buds of Pinellia ternata into a liquid culture medium and observes for 1 to 5 days, more preferably 2 to 3 days, and after confirming that there is no microbial contamination, aseptically transfers the buds to an intermittent immersion culture system.

[0027] In the present invention, when culturing sterile clustered shoots of Pinellia ternata, the liquid culture medium preferably includes the following components: MS+6-BA 0.5-2mg / L+NAA 0.01-0.2mg / L+sucrose 10-60g / L; the pH value of the liquid culture medium is preferably 5.8-6.3; further preferably includes the following components: MS+6-BA 0.80-1.20mg / L+NAA 0.01-0.03mg / L+sucrose 28-31g / L, and the pH value is preferably 6.0-6.2; further preferably, it is MS+6-BA 1.00mg / L+NAA 0.02mg / L+sucrose 30g / L, and the pH value is preferably 6.1.

[0028] When culturing the callus of Pinellia ternata, the liquid culture medium preferably comprises the following components: MS+6-BA 0.10-0.30mg / L+2,4-D 1.00-2.00mg / L+KT 0.4-0.6mg / L+sucrose 25-35g / L; further preferably comprises the following components: MS+6-BA 0.15-0.25mg / L+2,4-D 1.30-1.70mg / L+KT 0.45-0.55mg / L+sucrose 28-32g / L, and further preferably comprises the following components: MS+6-BA 0.20mg / L+2,4-D 1.50mg / L+KT 0.50mg / L+sucrose 30g / L. The pH of the liquid culture medium is preferably 5.5-6.0, more preferably 5.7-5.9, and more preferably 5.8.

[0029] In the present invention, the intermittent submerged culture is preferably carried out in a BIOF high-throughput plant bioreactor. The present invention does not specifically limit the type of the high-throughput plant bioreactor, as long as intermittent submerged culture can be guaranteed. In the specific implementation of the present invention, the high-throughput plant bioreactor used is described in Chinese patent CN201510218596.7; the ventilation time of the intermittent submerged culture is preferably 3 to 20 minutes, and more preferably 4 to 15 minutes; the holding time is preferably 5 to 30 minutes, and more preferably 8 to 15 minutes, and the degassing time is preferably 5 to 15 minutes, and more preferably 8 to 12 minutes; the immersion interval is preferably 2 to 8 hours, and more preferably 3 to 6 hours / day, and the number of cycles is unlimited; the light intensity of the intermittent submerged culture is preferably 1800 to 2000 lx; ​​the illumination time is preferably 12 to 16 hours / day, and more preferably 13 to 15 hours / day, and the temperature is preferably 24 to 26°C.

[0030] In the present invention, the time of the intermittent submerged culture is preferably 50 to 120 days, more preferably 55 to 100 days, and even more preferably 60 to 90 days.

[0031] After obtaining the Pinellia ternata clumping seedlings or Pinellia ternata loose cells, the present invention homogenizes the obtained Pinellia ternata clumping seedlings or Pinellia ternata loose cells to separate Pinellia ternata exosomes. The present invention does not specifically limit the homogenization method, and a conventional homogenizer in the art can be used; in the present invention, the separation of Pinellia ternata exosomes is preferably carried out by centrifugation, and the further preferred centrifugation includes the steps of centrifugation at 1800-2200g for 8-12min; collecting the first supernatant, centrifuging the first supernatant at 4500-5500g for 8-12min, collecting the second supernatant; centrifuging the second supernatant at 9000-11000g for 40-50min, collecting the third supernatant, centrifuging the third supernatant at 90000-110000g for 1.5-2.5h, discarding the fourth supernatant, collecting the precipitate, and resuspending to obtain Pinellia ternata exosomes. In the present invention, the resuspended precipitate is preferably carried out using PBS, and it is further preferred that 500μL of PBS is used to dissolve 40-60mg of the precipitate.

[0032] In the process of the intermittent immersion culture, the present invention preferably applies terahertz waves to the liquid culture medium, and further preferably implants a terahertz ring into a high-throughput plant bioreactor. The terahertz waves are applied throughout the intermittent immersion culture process to promote the secretion of exosomes by the plant material.

[0033] After the intermittent immersion culture is completed, the present invention preferably further comprises the step of collecting the culture fluid and separating the Pinellia exosomes from the culture fluid. The present invention has no particular limitation on the method of separating exosomes from the culture fluid, and membrane filtration or centrifugation can be used.

[0034] The present invention provides application of the pinellia exosomes prepared by the preparation method in the preparation of medicines or cosmetics.

[0035] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] Example 1

[0037] Preparation of exosomes from Pinellia ternata cells

[0038] Prepare the Pinellia ternata loose cell liquid culture medium. The formula of the liquid culture medium is as follows: MS+6-BA0.20mg / L+2,4-D1.50mg / L+KT 0.5mg / L+sucrose 30g / L, pH 5.8, sterilization conditions are 121℃ sterilization for 20min, and cooling to room temperature for use.

[0039] Inoculation: 50 g of sterile Pinellia callus was aseptically inoculated into the above liquid culture medium. After 2 days of observation and no microbial contamination, it was aseptically connected to a high-throughput plant bioreactor.

[0040] Cultivation parameter setting: Connect the inoculated high-throughput plant bioreactor to the intelligent control system, and set the parameters as ventilation time 5 min, holding time 10 min, degassing time 10 min, immersion frequency 4 h / d, and number of cycles unlimited. The culture environment is light 1800 lx, light time 14 h / d, and temperature 25 ± 1 °C.

[0041] Sampling: Material extraction can be carried out after the loose cells have grown for 90 days and covered the partitions of the high-throughput plant bioreactor.

[0042] Homogenization: Take out 100 g of loose cells and homogenize them using a homogenizer, and collect all the homogenate in a centrifuge tube.

[0043] Centrifugation to prepare exosomes: Use PBS solution to balance, centrifuge at 2000g for 10 minutes. Transfer the supernatant to a new centrifuge tube, remove the precipitate, balance the supernatant with PBS, and centrifuge at 5000g for 10 minutes. Transfer the supernatant to a new centrifuge tube, balance it with PBS, and centrifuge at 10000g for 45 minutes. Transfer the supernatant to an ultracentrifuge tube, balance it with PBS, centrifuge at 100000g for 2 hours, remove the supernatant, and resuspend the precipitate in each ultracentrifuge tube with 200μL of PBS.

[0044] The prepared Pinellia exosomes were subjected to transmission electron microscopy observation, particle size analysis and non-targeted metabolome cluster analysis. Figures 1-2 As shown, Figure 1 This is a transmission electron micrograph. The exosome sample was negatively stained with 5 μl uranyl acetate and then placed on a copper grid, which was then photographed under a HITACHI transmission electron microscope. The results showed that the preparation had a typical exosome saucer-like structure. Figure 2 This is a flow cytometry analysis of exosomes. The results show that the average particle size of the exosomes obtained is 81.2 nm.

[0045] Example 2

[0046] Preparation of exosomes from Pinellia ternata seedlings under terahertz treatment

[0047] Terahertz processing group:

[0048] Preparation of liquid culture medium: The formula of liquid culture medium is MS+6-BA 1.00mg / L+NAA 0.02mg / L+sucrose 30g / L, pH 6.2, sterilize at 121℃ high temperature and high pressure for 20min, cool to room temperature for use.

[0049] Sterilization of the terahertz ring: implant the terahertz ring into a high-throughput plant bioreactor, sterilize it at 121°C for 20 minutes, and cool it for later use.

[0050] Inoculation of high-throughput plant bioreactor: Aseptically cut the aseptic clustered shoots of Pinellia ternata into 5×5×5 mm3 120 of the size were placed in liquid culture medium and observed for 2 days. After no microbial contamination was found, they were aseptically transferred to a high-throughput plant bioreactor sterilized with high temperature and high pressure.

[0051] Cultivation parameter setting: Connect the inoculated high-throughput plant bioreactor to the intelligent control system, and set the parameters as ventilation time 5 min, holding time 10 min, degassing time 10 min, immersion frequency 4 h / d, and number of cycles unlimited. The culture environment is 2000 lx of light, 14 h / d of light time, and 25 ± 1 °C of temperature.

[0052] Material extraction: After the cluster buds have grown for 90 days and turned into stably growing cluster seedlings, material extraction can be carried out.

[0053] Homogenization: 100 g of the clump seedlings were taken out and homogenized using a homogenizer, and all the homogenate was collected in a centrifuge tube.

[0054] Centrifuge to prepare exosomes: Use PBS solution to balance, centrifuge at 2000g for 10 minutes. Transfer the supernatant to a new centrifuge tube, remove the precipitate, balance the supernatant with PBS, and centrifuge at 5000g for 10 minutes. Transfer the supernatant to a new centrifuge tube, balance it with PBS, and centrifuge at 10000g for 45 minutes. Transfer the supernatant to an ultracentrifuge tube, balance it with PBS, centrifuge at 100000g for 2 hours, remove the supernatant, and resuspend the precipitate in each ultracentrifuge tube with 500μL of PBS. Aliquot into 100μL / tube. Store at -80℃ for use.

[0055] Control group:

[0056] Except that terahertz treatment was not performed during the culture process, other treatment steps and parameters were exactly the same as those of the terahertz treatment group.

[0057] result:

[0058] The results of non-targeted metabolome cluster analysis (PCA diagram) of the terahertz treatment group and the control group are shown in Figure 3 As shown, TDZ is the control group and TZ is the terahertz treatment group. The results show that the two samples are separated, the samples within the two groups are clustered together, and the quality control within the sample group is good; this indicates that the quality control degree of the Pinellia exosomes prepared by the method described in the present invention is high.

[0059] Electron microscopic image of Pinellia exosomes treated by terahertz Figure 4 As shown, the obtained exosomes have a typical exosome saucer-like structure; compared with the control group, the number of exosomes in the Pinellia ternata cluster seedlings in the terahertz treatment group is greater; the final concentration is detected to be 1.45E+10 particles / mL; the control group is 1.22E+10 particles / mL.

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing exosomes from Pinellia ternata. It is characterized in that The following steps are involved: 1) inoculating the sterile clustered buds of Pinellia ternata into a liquid culture medium for intermittent immersion culture to obtain clustered Pinellia ternata seedlings; Alternatively, the pinellia callus is inoculated into a liquid culture medium for intermittent immersion culture to obtain the pinellia loose cells; The ventilation time of the intermittent immersion culture is 3 to 20 minutes, the holding time is 5 to 30 minutes, the degassing time is 5 to 15 minutes, the immersion interval is 2 to 8 hours, and the number of cycles is unlimited; 2) The Pinellia ternata clustered seedlings or the Pinellia ternata loose cells obtained in step 1) are homogenized to separate the Pinellia ternata exosomes.

2. The preparation method according to claim 1, It is characterized in that When culturing aseptic clustered buds of Pinellia ternata, the formula of the liquid culture medium is: MS+6-BA 0.5-2 mg / L+NAA 0.01-0.2 mg / L+sucrose 10-40 g / L.

3. The preparation method according to claim 1, It is characterized in that When culturing pinellia callus, the liquid culture medium formula is: MS+6-BA 0.10-0.30 mg / L+2,4-D 1.00-2.00 mg / L+KT 0.4-0.6 mg / L+sucrose 25-35 g / L.

4. The preparation method according to any one of claims 1 to 3, It is characterized in that The intermittent immersion culture has a light intensity of 1800-2000 lx, a light duration of 12-16 h / d, and a temperature of 24-26°C.

5. The preparation method according to claim 1 or 2, It is characterized in that The separation of Pinellia exosomes in step 2) is performed by centrifugation, wherein the centrifugation comprises centrifugation at 1800-2200 g for 8-12 min; The first supernatant was collected, centrifuged at 4500-5500 g for 8-12 min, and the second supernatant was collected; the second supernatant was centrifuged at 9000-11000 g for 40-50 min, and the third supernatant was collected, centrifuged at 90000-110000 g for 1.5-2.5 h, the fourth supernatant was discarded, the precipitate was collected, and the precipitate was resuspended to obtain Pinellia exosomes.

6. The preparation method according to claim 1, It is characterized in that During the intermittent immersion culture in step 1), terahertz waves are applied to the liquid culture medium.

7. The preparation method according to claim 1, It is characterized in that The size of the aseptic clustered buds of Pinellia ternata is (4-6)×(4-6)×(4-6) mm 3 The inoculation quantity of the sterile clustered buds of Pinellia ternata is 30 to 120 buds / L of culture medium.

8. The preparation method according to claim 1, It is characterized in that The period of intermittent immersion culture in step 1) is 50 to 120 days.

9. The preparation method according to claim 1, It is characterized in that After step 1) the intermittent immersion culture is completed, the method further comprises the steps of collecting the culture fluid and isolating the Pinellia exosomes from the culture fluid.

10. Use of the Pinellia exosomes prepared by the preparation method according to any one of claims 1 to 9 in the preparation of medicines or cosmetics.

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