Large-scale preparation method of ginseng exosome

Abstract

The invention discloses a large-scale preparation method of ginseng exosomes, which comprises the following steps: culturing calluses: inoculating explants into an induction culture medium until ginseng calluses are generated; proliferation of the calluses: inoculating the newborn calluses into a proliferation culture medium for proliferation culture; inducing germination and rooting: after the ginseng callus is amplified, transplanting the ginseng callus into a germination induction culture medium until the ginseng callus is germinated, and then transferring the ginseng callus into a rooting culture medium for culturing and rooting; and preparing the exosome, namely grinding root hairs grown from the ginseng callus regeneration plant, uniformly mixing the ground root hairs with PBS (Phosphate Buffer Solution) or performing liquid culture on ginseng suspension cells, and finally performing centrifugal treatment to obtain the ginseng exosome. According to the large-scale preparation method of the ginseng exosome, the endotoxin pollution in the growth and development process is reduced through ginseng callus culture rooting, and then the ginseng exosome is prepared, so that the large-scale preparation requirement in modern production and processing is met.

Description

Technical Field

[0001] The present invention relates to the technical field of ginseng exosome preparation, and in particular to a large-scale preparation method of ginseng exosomes. Background Art

[0002] Ginseng exosomes, nanoscale bioactive substances extracted from ginseng, have demonstrated significant application value in anti-aging, wound healing, anti-inflammatory, antioxidant, immunomodulatory, and anti-tumor applications. For example, 1) ginseng exosomes can promote collagen production by regulating the TGF-β pathway, accelerating wound healing, reducing UV-induced skin damage, lowering melanin content, and improving skin pigmentation. 2) ginseng exosomes can significantly promote skin cell proliferation and migration and enhance fibroblast activity, thereby accelerating wound healing. 3) ginseng exosomes exhibit significant anti-inflammatory effects, reducing inflammatory cytokine levels and alleviating skin inflammation by inhibiting the NF-κB signaling pathway. 4) In cancer treatment, ginseng exosomes modulate macrophage polarization (M1 polarization), enhancing anti-tumor immune responses and inhibiting the growth of non-small cell lung cancer cells. 5) ginseng exosomes are rich in antioxidants, such as ginsenosides and antioxidant enzymes, which can neutralize free radicals and reduce oxidative stress-induced damage to the skin. ⑥ In the treatment of inflammatory bowel disease (IBD), ginseng exosomes alleviate disease symptoms by regulating immune responses; in terms of neuroprotection, it can reduce neuronal damage in Alzheimer's disease models.

[0003] At present, there are two main preparation methods for ginseng exosomes in China: for rhizome plants such as ginseng or Panax notoginseng, the extraction method is mainly to clean the plant surface, cut it into small pieces, add PBS and then break the wall, and continue to separate and extract exosomes in the tissue fluid.

[0004] However, the rhizomes of plants like ginseng and Panax notoginseng are buried deep in humus-rich soil, where a large network of bacteria and fungi adheres to the root surface. These fungi act as signal transducers, supporting the normal growth and development of ginseng. When these rhizomes are treated with traditional exosome extraction methods, endotoxin contamination is unavoidable. Endotoxins are primarily bacterially produced and consist of lipopolysaccharides (LPS), a component of the cell wall structure of Gram-negative bacteria. Removing exosomes from plant exosomes, which are biologically active, is difficult. Consequently, the accumulation of endotoxins in exosomes significantly limits their further application as biomaterials. Summary of the Invention

[0005] The purpose of the present invention is to provide a large-scale preparation method of ginseng exosomes, which reduces endotoxin contamination during the growth and development process by culturing ginseng callus tissue and then preparing ginseng exosomes, thereby meeting the large-scale preparation needs in modern production and processing.

[0006] The technical solution adopted by the large-scale preparation method of ginseng exosomes disclosed in the present invention is: A large-scale preparation method of ginseng exosomes comprises the following steps: Callus culture: The explants are inoculated into induction medium until ginseng callus is generated; Callus proliferation: inoculating the newly grown callus into a proliferation medium for proliferation culture; Inducing germination and rooting: After the ginseng callus tissue is expanded, it is transplanted into a germination induction medium until buds appear, and then transferred to a rooting medium for rooting culture; For exosome preparation, the roots grown from ginseng callus regenerated plants are ground and mixed with PBS or ginseng suspension cells are cultured in liquid, and finally centrifuged to obtain ginseng exosomes.

[0007] As a preferred embodiment, the specific steps of callus culture are: removing moisture from the surface of the ginseng explant, and using sterile instruments to cut the ginseng root explant into small pieces of 0.8 to 1 cm in length, inoculating the 0.8 to 1 cm long explant into an induction culture medium, inserting the explant into 1 / 3 of the length of the induction culture medium, and culturing in the dark, the temperature range is 23 to 27°C, the humidity is 55 to 60%, and the induction culture medium is not changed within 15 days until ginseng callus tissue is generated.

[0008] As a preferred embodiment, the induction culture medium includes Ms basal culture medium powder content of 4-4.4 g / L, hydrolyzed casein content of 0.1-0.5 mg / L, indolebutyric acid content of 0.5-1 mg / L, 2,4-dichlorophenoxyacetic acid content of 1.0-2.0 mg / L, sucrose content of 30-35 g / L, agar content of 7-9 g / L, and the pH value of the induction culture medium is 5.8.

[0009] As a preferred solution, when performing callus culture, the explant needs to be pre-treated. The specific processing steps are as follows: Explants should be collected on sunny afternoons during the growing season or after the dew on the plant surface has dried to reduce microbial contamination. Healthy roots without rot or scars should be selected as explants. After the explants are collected, they are disinfected by rinsing to remove attachments and dust on the explant surface; soaking the cleaned explants in 75% ethanol for 30 to 60 seconds, and then rinsing them with sterile water; and disinfecting them by soaking the explants in 1% sodium hypochlorite solution for 30 minutes, and then rinsing them with sterile water to remove the sodium hypochlorite solution.

[0010] As a preferred solution, the culture conditions for callus proliferation are a temperature of 23-25° C., a light duration of 11-12 hours per day, a light intensity of 1000-2000 lux, or dark culture.

[0011] As a preferred embodiment, the proliferation culture medium includes Ms basic culture medium powder content of 4-4.4 g / L, vitamin B1 content of 1-2 mg / L, indolebutyric acid content of 0.2-0.5 mg / L, 2,4-dichlorophenoxyacetic acid content of 0.5-1 mg / L, sucrose content of 30-35 g / L, agar content of 7-9 g / L, and activated carbon content of 0.5-0.7 g / L.

[0012] As a preferred embodiment, the germination induction medium includes Ms basic medium powder content of 4-4.4 g / L, indolebutyric acid content of 1-2 mg / L, NAA content of 0.1-0.5 mg / L, sucrose content of 30-35 g / L, agar content of 7-9 g / L, and activated carbon content of 0.5-0.7 g / L; The rooting culture medium comprises Ms basic culture medium powder content of 2-2.2 g / L, indolebutyric acid content of 0.1-0.2 mg / L, NAA content of 0.1-0.5 mg / L, IBA content of 0.1-0.15 mg / L, sucrose content of 20-25 g / L, agar content of 7-9 g / L, and activated carbon content of 0.5-0.7 g / L.

[0013] As a preferred embodiment, the exosome preparation step is specifically as follows: collecting ginseng roots, chopping and grinding the root tissue, mixing it with PBS, and then extracting exosomes by gradient centrifugation.

[0014] As a preferred embodiment, the exosome preparation step is specifically as follows: ginseng roots are cut into 1 mm tissue pieces, inoculated into liquid culture medium at a rate of 40 g / L, and ginseng suspension cells are cultured in liquid. After the ginseng suspension cells secrete exosomes, the cell fluid in the culture medium is collected and separated by centrifugation. Ginseng exosomes are secreted in the supernatant of the culture medium, a filter membrane with a specific pore size is selected, and the supernatant is used to obtain ginseng exosomes by a tangential flow method.

[0015] As a preferred embodiment, the liquid culture medium includes Ms basic culture medium powder content of 4-4.4 g / L, indolebutyric acid content of 3-3.5 mg / L, NAA content of 3-3.5 mg / L, and sucrose content of 30-35 g / L.

[0016] The beneficial effects of the large-scale preparation method of ginseng exosomes disclosed in the present invention are as follows: by selecting suitable explants, callus culture, callus proliferation and germination induction are carried out in sequence, so that ginseng callus regeneration plants can be cultured on a large scale; by collecting roots for subsequent exosome preparation, the quality of the cultured roots is guaranteed and endotoxin contamination during the growth and development of ginseng is avoided; after sterile induction culture, the roots retain ginseng nutrients and exosome activity while avoiding endotoxin contamination in the traditional direct plant-based exosome extraction method. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The present invention is a flow chart of a large-scale preparation method of ginseng exosomes. DETAILED DESCRIPTION

[0018] The present invention will be further described and explained below in conjunction with specific embodiments and accompanying drawings: Please refer to Figure 1 A large-scale preparation method of ginseng exosomes comprises the following steps: The explants are pre-treated, and the specific steps are as follows: Explants should be collected on sunny afternoons during the growing season or after the dew on the plant surface has dried to reduce microbial contamination. Healthy roots without rot or scars should be selected as explants. After the explants are collected, they are disinfected by rinsing to remove attachments and dust on the explant surface; soaking the cleaned explants in 75% ethanol for 30 to 60 seconds, and then rinsing them with sterile water; and disinfecting them by soaking the explants in 1% sodium hypochlorite solution for 30 minutes, and then rinsing them with sterile water to remove the sodium hypochlorite solution.

[0019] This stage is mainly to remove most of the microorganisms in the explant, and subsequent operations are performed in a sterile clean bench to avoid infection by other bacteria or fungi.

[0020] Callus culture: Remove moisture from the surface of the ginseng explants and cut the ginseng root explants into small pieces of 0.8 to 1 cm in length using sterile instruments. Inoculate the 0.8 to 1 cm long explants into the induction medium. Insert the explants into the induction medium to 1 / 3 of its length and culture in the dark. The temperature range is 23 to 27°C and the humidity is 55 to 60%. Do not change the induction medium within 15 days until ginseng callus is generated.

[0021] The induction culture medium includes Ms basic culture medium powder with a content of 4-4.4 g / L, hydrolyzed casein with a content of 0.1-0.5 mg / L, indolebutyric acid with a content of 0.5-1 mg / L, 2,4-dichlorophenoxyacetic acid with a content of 1.0-2.0 mg / L, sucrose with a content of 30-35 g / L, and agar with a content of 7-9 g / L, and the pH value of the induction culture medium is 5.8.

[0022] Callus proliferation: inoculate the newly grown callus into a proliferation medium for proliferation culture. The culture conditions are a temperature of 23-25°C, a light intensity of 11-12 hours per day, and a light intensity of 1000-2000 lux or dark culture.

[0023] The proliferation culture medium includes Ms basic culture medium powder with a content of 4-4.4 g / L, vitamin B1 content of 1-2 mg / L, indolebutyric acid content of 0.2-0.5 mg / L, 2,4-dichlorophenoxyacetic acid content of 0.5-1 mg / L, sucrose content of 30-35 g / L, agar content of 7-9 g / L, and activated carbon content of 0.5-0.7 g / L, wherein the concentration of 2,4-dichlorophenoxyacetic acid is reduced to reduce browning.

[0024] Inducing germination and rooting, after the ginseng callus tissue is expanded, it is transplanted into the germination induction medium. During this process, the germination induction medium is changed every 15 days until buds appear, and then transferred to the rooting medium for rooting culture.

[0025] The germination induction culture medium includes Ms basic culture medium powder content of 4-4.4 g / L, indolebutyric acid content of 1-2 mg / L, NAA content of 0.1-0.5 mg / L, sucrose content of 30-35 g / L, agar content of 7-9 g / L, and activated carbon content of 0.5-0.7 g / L.

[0026] Increase indolebutyric acid content to promote bud differentiation and balance with low auxin through NAA.

[0027] The rooting culture medium includes Ms basic culture medium powder content of 2-2.2 g / L, indolebutyric acid content of 0.1-0.2 mg / L, NAA content of 0.1-0.5 mg / L, IBA content of 0.1-0.15 mg / L, sucrose content of 20-25 g / L, agar content of 7-9 g / L, and activated carbon content of 0.5-0.7 g / L.

[0028] Reduce indolebutyric acid concentration and promote root primordium formation.

[0029] For exosome preparation, the roots grown from ginseng callus regenerated plants are ground and mixed with PBS or ginseng suspension cells are cultured in liquid, and finally centrifuged to obtain ginseng exosomes.

[0030] The specific steps for preparing exosomes are as follows: collect ginseng roots, cut and grind the root tissue, mix it with PBS, and then extract exosomes by gradient centrifugation.

[0031] Another exosome preparation step is as follows: ginseng roots are cut into 1 mm tissue blocks, inoculated into liquid culture medium at a rate of 40 g / L, and cultured with shaking to establish a suspension cell line, and liquid culture is performed to induce ginseng callus cells. After the ginseng suspension cells secrete exosomes, the culture supernatant in the culture medium is collected and separated by centrifugation. Ginseng exosomes are secreted in the culture supernatant, and a filter membrane with a pore size of 500 nm is selected. The supernatant is used to obtain ginseng exosomes by the tangential flow method.

[0032] The liquid culture medium includes Ms basic culture medium powder content of 4-4.4 g / L, indolebutyric acid content of 3-3.5 mg / L, NAA content of 3-3.5 mg / L, and sucrose content of 30-35 g / L.

[0033] The corresponding components and functions of the above-mentioned culture media are as follows: Basic MS medium: It is the basic medium for plant tissue culture, providing the macro and trace elements required for plant growth and supporting cell division and differentiation.

[0034] Hydrolyzed casein: As an organic nitrogen source, it has a good promoting effect on the differentiation of embryoids and adventitious buds. The usual dosage is 500 mg / L.

[0035] Indolebutyric acid (IBA): A synthetic auxin that primarily promotes root formation, particularly in rooting cultures. In Pinellia ternata tissue culture, low concentrations of IBA (0.05 mg·L⁻¹) can induce high-quality roots.

[0036] 2,4-Dichlorophenoxyacetic acid (2,4-D): A potent auxin primarily used to induce callus formation, but it inhibits bud formation. It is commonly used in callus induction and differentiation culture.

[0037] Sucrose: acts as a carbon source and energy source, while regulating osmotic pressure and maintaining cell turgor and metabolic activity.

[0038] Agar: As a solidifying agent for solid culture medium, it keeps the culture medium in a stable shape, making it easier to operate and observe.

[0039] Vitamin B1: As a coenzyme, it participates in various metabolic reactions and plays an important role in the formation and vitality of callus tissue.

[0040] Activated carbon: used to adsorb harmful substances in the culture medium, such as phenolic compounds, to prevent the culture from browning, but excessive use may inhibit root growth.

[0041] NAA (Naphthaleneacetic Acid): A synthetic auxin that promotes cell division and elongation. It interacts with cytokinins to promote bud proliferation and growth. In rooting cultures, NAA is often used in combination with IBA to enhance its effectiveness.

[0042] The present invention provides a large-scale preparation method of ginseng exosomes. By selecting suitable explants, callus culture, callus proliferation and germination induction are carried out in sequence, so that ginseng callus regeneration plants can be cultured on a large scale. Roots are collected for subsequent exosome preparation, thereby ensuring the quality of the cultured roots and avoiding endotoxin contamination during the growth and development of ginseng. After sterile induction culture, the roots retain ginseng nutrients and exosome activity while avoiding endotoxin contamination in traditional direct plant-based exosome extraction methods.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A large-scale preparation method of ginseng exosomes, characterized in that: The following steps are involved: Callus culture: The explants are inoculated into induction medium until ginseng callus is generated; Callus proliferation: inoculating the newly grown callus into a proliferation medium for proliferation culture; Inducing germination and rooting: After the ginseng callus tissue is expanded, it is transplanted into a germination induction medium until buds appear, and then transferred to a rooting medium for rooting culture; For exosome preparation, the roots grown from ginseng callus regenerated plants are ground and mixed with PBS or ginseng suspension cells are cultured in liquid, and finally centrifuged to obtain ginseng exosomes.

2. The large-scale preparation method of ginseng exosomes according to claim 1, characterized in that: The specific steps of callus culture are: removing moisture from the surface of ginseng explants, cutting the ginseng root explants into small pieces of 0.8 to 1 cm in length using sterile instruments, inoculating the 0.8 to 1 cm long explants into an induction culture medium, inserting the explants into the induction culture medium to 1 / 3 of its length, and culturing in the dark at a temperature range of 23 to 27° C. and a humidity of 55 to 60%. The induction culture medium is not replaced within 15 days until ginseng callus is generated.

3. The large-scale preparation method of ginseng exosomes according to claim 2, characterized in that: The induction culture medium comprises Ms basic culture medium powder with a content of 4 to 4.4 g / L, hydrolyzed casein with a content of 0.1 to 0.5 mg / L, indolebutyric acid with a content of 0.5 to 1 mg / L, 2,4-dichlorophenoxyacetic acid with a content of 1.0 to 2.0 mg / L, sucrose with a content of 30 to 35 g / L, and agar with a content of 7 to 9 g / L, and the pH value of the induction culture medium is 5.

8.

4. The large-scale preparation method of ginseng exosomes according to claim 1, characterized in that: When performing callus tissue culture, the explant needs to be pre-treated. The specific processing steps are as follows: Explants should be collected on sunny afternoons during the growing season or after the dew on the plant surface has dried to reduce microbial contamination. Healthy roots without rot or scars should be selected as explants. After the explants are collected, they are disinfected by rinsing to remove attachments and dust on the explant surface; soaking the cleaned explants in 75% ethanol for 30 to 60 seconds, and then rinsing them with sterile water; and disinfecting them by soaking the explants in 1% sodium hypochlorite solution for 30 minutes, and then rinsing them with sterile water to remove the sodium hypochlorite solution.

5. The large-scale preparation method of ginseng exosomes according to claim 1, characterized in that: The culture conditions for callus proliferation are a temperature of 23-25° C., a light duration of 11-12 hours per day, and a light intensity of 1000-2000 lux or dark culture.

6. The method for large-scale preparation of ginseng exosomes according to claim 1, wherein: The proliferation culture medium comprises Ms basic culture medium powder in a content of 4-4.4 g / L, vitamin B1 content of 1-2 mg / L, indolebutyric acid content of 0.2-0.5 mg / L, 2,4-dichlorophenoxyacetic acid content of 0.5-1 mg / L, sucrose content of 30-35 g / L, agar content of 7-9 g / L, and activated carbon content of 0.5-0.7 g / L.

7. The large-scale preparation method of ginseng exosomes according to claim 1, characterized in that: The germination induction medium comprises Ms basic medium powder content of 4-4.4 g / L, indolebutyric acid content of 1-2 mg / L, NAA content of 0.1-0.5 mg / L, sucrose content of 30-35 g / L, agar content of 7-9 g / L, and activated carbon content of 0.5-0.7 g / L; The rooting culture medium comprises Ms basic culture medium powder content of 2-2.2 g / L, indolebutyric acid content of 0.1-0.2 mg / L, NAA content of 0.1-0.5 mg / L, IBA content of 0.1-0.15 mg / L, sucrose content of 20-25 g / L, agar content of 7-9 g / L, and activated carbon content of 0.5-0.7 g / L.

8. The large-scale preparation method of ginseng exosomes according to claim 1, characterized in that: The exosome preparation steps are as follows: collecting ginseng roots, chopping and grinding the root tissue, mixing it with PBS, and then extracting exosomes by gradient centrifugation.

9. The large-scale preparation method of ginseng exosomes according to claim 1, characterized in that: The exosome preparation steps are specifically as follows: ginseng roots are cut into 1 mm tissue pieces, inoculated into liquid culture medium at a rate of 40 g / L, and ginseng suspension cells are cultured in liquid. After the ginseng suspension cells secrete exosomes, the cell fluid in the culture medium is collected and separated by centrifugation. The ginseng exosomes are secreted in the supernatant of the culture medium. A filter membrane with a specific pore size is selected, and the supernatant is filtered using a tangential flow method to obtain ginseng exosomes.

10. The large-scale preparation method of ginseng exosomes according to claim 9, characterized in that: The liquid culture medium comprises Ms basic culture medium powder content of 4-4.4 g / L, indolebutyric acid content of 3-3.5 mg / L, NAA content of 3-3.5 mg / L, and sucrose content of 30-35 g / L.

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