Extraction method of bovine milk exosome and application of product thereof in preparation of product for relieving or treating gingivitis or repairing skin wound

Abstract

The application provides an extraction method of bovine milk exosomes and application of a product thereof in preparation of a product for relieving or treating gingivitis or repairing skin wounds, and belongs to the field of skin care products or medicines, and particularly provides a bovine milk exosome product with functions of relieving or treating gingivitis, promoting periodontal tissue stability, wound anti-inflammation, and promoting skin wound healing and repairing. The application provides a method for quickly and massively obtaining bovine milk exosome raw materials and products. Through animal experiments, it is verified that the bovine milk exosomes prepared by the method have the effects of anti-inflammation and wound healing promotion.

Description

Technical Field

[0001] This invention relates to the field of skincare products or pharmaceuticals, specifically to a method for extracting bovine milk exosomes and the application of their products in the preparation of products for relieving or treating gingivitis or repairing skin wounds. In particular, it relates to the application of bovine milk exosomes or bovine milk exosome products in the preparation of products for relieving or treating gingivitis or promoting periodontal tissue stability, wound anti-inflammatory effects, promoting skin wound healing and / or skin wound repair, and a method for preparing bovine milk exosomes or bovine milk exosome products. Background Technology

[0002] Exosomes are multivesicular bodies produced in cells. They are membranous vesicles with a diameter of approximately 30–150 nm, secreted by living cells, and have a density of 1.13–1.19 g / ml. They typically exhibit a "cup-and-disc" morphology. Exosomes are heterogeneous; even exosomes secreted by the same cell type can have significant functional differences. Exosomes are present in almost all biological fluids and are important mediators of intercellular communication. In various diseases, exosomes provide a window into changes in cellular or tissue states, playing an irreplaceable role in disease diagnosis, prognosis, and even treatment, and have become one of the most promising biomarkers in liquid biopsy.

[0003] Studies have found that exosomes in breast milk possess a variety of biological functions, including antioxidant capacity, immunomodulatory capacity, and antibacterial activity. Due to these functions, breast exosomes play a crucial role in human health, such as protecting the intestinal barrier by regulating the microbiota and modulating bone / muscle metabolism. Currently, the functions of breast exosomes are receiving increasing attention, and research is becoming more in-depth. However, existing methods for preparing breast exosomes typically involve high-speed centrifugation, acid pretreatment, and single-phase tangential flow filtration to produce cells or breast exosomes in small batches, resulting in low exosome yields and weak efficacy. Summary of the Invention

[0004] Purpose of the invention The purpose of this invention is to provide a method for extracting bovine milk exosomes and the application of their products in the preparation of products for relieving or treating gingivitis or repairing skin wounds. Specifically, it relates to the application of bovine milk exosomes or bovine milk exosome products in the preparation of products for relieving or treating gingivitis, promoting periodontal tissue stability, reducing wound inflammation, promoting skin wound healing, and / or repairing skin wounds, as well as a method for preparing bovine milk exosomes or bovine milk exosome products. This invention provides a method for rapidly and extensively obtaining pure natural bovine milk exosome raw materials and products. Animal experiments have verified that the bovine milk exosomes prepared by this method have anti-inflammatory and wound-healing effects.

[0005] This invention employs a multi-membrane filtration and chromatography method to prepare large quantities of high-purity natural bovine milk exosomes. These exosome products have the function of relieving or treating gingivitis and repairing skin wounds. This method has advantages such as low cost, high efficiency, and being purely natural and pollution-free, and it has great application potential in the fields of skincare products and pharmaceuticals.

[0006] Solution To achieve the objectives of this invention, the technical solution adopted is as follows: In a first aspect, the present invention provides the use of milk exosomes or bovine milk exosome products in the preparation of products for relieving or treating gingival inflammation or promoting periodontal tissue stability, wound anti-inflammatory effects, promoting skin wound healing and / or skin trauma repair. Alternatively, it provides a method for relieving or treating gingival inflammation or promoting periodontal tissue stability, wound anti-inflammatory effects, promoting skin wound healing and / or skin trauma repair by administering an effective dose of bovine milk exosomes or bovine milk exosome products to a subject in need.

[0007] Furthermore, the bovine milk exosomes are extracted through the following steps: S1. Skim milk is filtered through a membrane with a pore size of 0.1μm~0.5μm; S2. Concentrate the permeate from step S1 using a 100 Da to 300 kDa organic membrane to obtain crude exosomes.

[0008] Preferably, the process may further include step S3: obtaining exosomes by chromatographic purification of the concentrate.

[0009] Secondly, a method for extracting bovine milk exosomes is provided, comprising the following steps: S1. Skim milk is filtered through a membrane with a pore size of 0.1μm~0.5μm; S2. Concentrate the permeate from step S1 using a 100 Da~300 kDa organic membrane; S3. The concentrated solution is purified by chromatography to obtain exosomes.

[0010] In the first or second aspect mentioned above, the membrane used in step S1 is a ceramic membrane or an organic membrane; And / or, the membrane pore size is 0.1 μm to 0.45 μm. Optionally, in step S1, the concentration is increased by 2 to 5 times or 3 to 5 times.

[0011] In the first or second aspect mentioned above, the organic membrane used in step S2 is a 100 Da to 200 kDa organic membrane; And / or, in step S2, concentrate by 2 to 10 times or 5 to 10 times.

[0012] In either the first or second aspect mentioned above, in step S3, the chromatography column is a Capto Core 700.

[0013] In the first or second aspect mentioned above, the concentration of exosomes in the crude exosome product is 10. 6 ~10 8 particles / mL; And / or, exosome products 10 8 ~10 10 particles / mL.

[0014] In the second aspect above, the dosage form of the product is a liquid topical formulation or an injection; And / or, promoting periodontal tissue stability includes reducing tooth loosening; And / or, functions to relieve or treat gingival inflammation include reducing periodontal pockets or reducing the gingival bleeding index; And / or, promoting skin wound healing or repair functions, including promoting fibrous tissue generation and wound healing.

[0015] Beneficial effects The purpose of this invention is to provide a method for preparing bovine milk exosome raw materials and products. By using a multi-membrane filtration + chromatography method, whey raw materials rich in exosomes and exosome products with high purity can be quickly obtained from bovine milk.

[0016] This invention provides a method for rapidly and extensively obtaining bovine milk exosomes and their products. Animal experiments have verified that the bovine milk exosomes prepared by this method have anti-inflammatory and wound-healing effects. Attached Figure Description

[0017] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0018] Figure 1 The exosomes in the 0.1μm+150kDa membrane filtered milk exosome raw material (after centrifugation extraction) of Example 1 were observed and identified by transmission electron microscopy (TEM). Figure 2 The percentage of exosome particle size distribution and the average particle size value in the 0.1μm+150kDa membrane filtered milk exosome raw material (after centrifugation extraction) of Example 1; Figure 2 A represents the average particle size. Figure 2 B represents the particle size distribution; Figure 3This is a statistical chart of exosome concentration in the 0.1μm+150kDa membrane filtered milk exosome raw material (after centrifugation extraction) of Example 1; Figure 4 The exosomes in the 0.1μm+10kDa membrane filtered milk exosome raw material (after centrifugation extraction) of Example 2 were observed and identified by transmission electron microscopy (TEM). Figure 5 The percentage of exosome particle size distribution and the average particle size value in the 0.1μm+10kDa membrane filtered milk exosome raw material (after centrifugation extraction) of Example 2; Figure 5 A represents the statistical value of particle size distribution; Figure 5 B represents the particle size distribution; Figure 6 This is a statistical chart of exosome concentration in the 0.1μm+10kDa membrane filtered milk exosome raw material (after centrifugation extraction) of Example 2; Figure 7 The exosomes in the 0.1μm+200Da membrane filtered milk exosome raw material (after centrifugation extraction) of Example 3 were observed and identified by transmission electron microscopy (TEM). Figure 8 The percentage of exosome particle size distribution and average particle size value in the 0.1μm+200Da membrane filtered milk exosome raw material (after centrifugation extraction) of Example 3; Figure 8 A represents the statistical value of particle size distribution; Figure 8 B represents the particle size distribution; Figure 9 This is a statistical chart of exosome concentration in the 0.1μm+200Da membrane filtered milk exosome raw material (after ultra-high speed centrifugation extraction) of Example 3; Figure 10 The exosomes in the 0.45μm+10kDa membrane filtered milk exosome raw material (after centrifugation extraction) of Example 4 were observed and identified by transmission electron microscopy (TEM). Figure 11 The percentage of exosome particle size distribution and the average particle size value in the 0.45μm+10kDa membrane filtered milk exosome raw material (after centrifugation extraction) of Example 4; Figure 11 A represents the statistical value of particle size distribution; Figure 11 B represents the particle size distribution; Figure 12 This is a statistical chart of exosome concentration in the 0.45μm+10kDa membrane filtered milk exosome raw material (after ultra-high speed centrifugation extraction) of Example 4; Figure 13 The exosomes in the bovine milk exosome product obtained by 0.1μm+100kDa membrane filtration+chromatography in Example 5 were observed and identified by transmission electron microscopy (TEM). Figure 14The percentage of exosome particle size distribution and the average particle size value in the bovine exosome product obtained by 0.1μm+100kDa membrane filtration+chromatography in Example 5; Figure 14 A represents the statistical value of particle size distribution; Figure 14 B represents the particle size distribution; Figure 15 This is a statistical graph of exosome concentration in bovine exosome products obtained by 0.1μm+100kDa membrane filtration+chromatography in Example 5; Figure 16 Statistical analysis of wound area in each group on day 10 (A) and day 14 (B) of test case 2. Compared with the control group, P<0.05; Figure 16 A represents the statistical analysis of wound area in each group on day 10, with the vertical axis in mm. 2 ; Figure 16 B represents the statistical analysis of wound area in each group on day 14. The unit of the vertical axis is mm. 2 ; Figure 17 HE staining pathological observation of wound healing tissues from each group on day 8 and day 14 of test case 2; among which, Figure 17 Blank was the blank group, PBS was the PBS group, and MExo was the bovine milk exosome group; Figure 18 Masson staining pathological observation of wound healing tissues from each group on day 8 and day 14 of test case 2; among which, Figure 18 Blank was the blank group, PBS was the PBS group, and MExo was the bovine milk exosome group; Figure 19 Sirius red staining pathological observation of wound healing tissues from each group on day 8 and day 14 of test case 2; among which, Figure 19 Blank was the blank group, PBS was the PBS group, and MExo was the bovine milk exosome group; Figure 20 To compare and analyze the significance of IL-6 expression levels in each group on day 14 of test case 2. #: Compared with the Blank group, P<0.05; #: Compared with the PBS group, P<0.05; The unit of the vertical axis is pg / mL. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0021] In the following examples, the purity of the resuspended exosomes was determined using the following method: An appropriate amount of exosomes was taken out and diluted to a suitable multiple. 10% Triton X-100 was prepared, and 5 μL of 10% Triton X-100 was added to 45 μL of the above exosomes. The mixture was stirred well, incubated on ice for 1 h, and then directly analyzed using a Nano Analyzer N30E instrument (particle size analyzer).

[0022] Example 1: Preparation of bovine milk exosome raw material 1 (0.1μm + 150kDa membrane separation) The preparation of bovine milk exosomes in this embodiment includes the following method: (1) Take 4℃ skim milk (skim milk sterilized at 72℃ / 15s). (2) The permeate was concentrated 3 times by using a 0.1μm ceramic membrane or organic membrane for filtration to obtain whey permeate; (3) The whey permeate was concentrated using a 150 kDa organic membrane to a 10-fold concentration to obtain crude exosomes; (4) Collect the concentrated whey solution as a sample and store it at -80℃. (5) Take 50 mL of concentrated whey sample (the sample can be frozen in step (4), or thawed at 37°C, or the concentrated sample in step (3) can be used directly), extract exosomes by staged centrifugation (concentrated 250 times), and obtain 200 μL of resuspension. The staged centrifugation method is as follows: Transfer the sample to a new centrifuge tube and centrifuge at 2000×g, 4℃, for 30 min; carefully transfer the supernatant to a new centrifuge tube and centrifuge again at 10,000×g, 4℃, for 45 min to remove larger vesicles; collect the supernatant, filter it through a 0.45μm filter membrane, and collect the filtrate; transfer the filtrate to a new centrifuge tube, select an ultracentrifuge rotor, and centrifuge at 4℃, 100,000×g for 70 min; remove the supernatant, resuspend in 10 mL of pre-chilled 1×PBS, select an ultracentrifuge rotor, and centrifuge again at 4℃, 100,000×g for 70 min; remove the supernatant and resuspend in 200 μL of pre-chilled 1×PBS.

[0023] (6) Take a heavy suspension sample for testing.

[0024] Exosomes and vesicles with diameters of 30–150 nm in the resuspension samples after ultracentrifugation were imaged and observed in real time using transmission electron microscopy and nanoflow cytometry. The results are as follows: Figure 1 Exosomes were detected in the resuspension sample; Figure 2 Figures A and B show that the average particle size of exosomes is 75.8 nm. Figure 3 The exosome concentration was shown to be 1.56 × 10⁻⁶. 9 Particles / mL.

[0025] Example 2: Preparation of bovine milk exosome raw material 2 (0.1μm+10kDa membrane separation) The difference from Example 1 is that the membrane in step (3) is replaced with a 10kDa organic membrane, and the rest of the operation is the same.

[0026] Exosomes and vesicles with diameters of 30–150 nm in the centrifuged resuspension samples were imaged and observed in real time using transmission electron microscopy and nanoflow cytometry. The results are as follows: Figure 4 Exosomes were detected in the resuspension sample; Figure 5 Figures A and B show that the average particle size of exosomes is 80.7 nm. Figure 6 The exosome concentration was shown to be 4.86 × 10⁻⁶. 9 Particles / mL.

[0027] Example 3: Preparation of bovine milk exosome raw material 3 (0.1μm + 200Da membrane separation) The difference from Example 1 is that the membrane in step (3) is replaced with a 200 Da organic membrane, and the rest of the operation is the same.

[0028] Exosomes and vesicles with diameters of 30–150 nm in the centrifuged resuspension samples were imaged and observed in real time using transmission electron microscopy and nanoflow cytometry. The results are as follows: Figure 7 Exosomes were detected in the resuspension sample; Figure 8 Figures A and B show that the average particle size of bovine milk exosomes is 79.9 nm. Figure 9 The exosome concentration was shown to be 1.24 × 10⁻⁶. 9 Particles / mL.

[0029] Example 4: Preparation of bovine milk exosome raw material (0.45μm+10kDa membrane separation) The difference from Example 2 is that the membrane in step (1) is replaced with a 0.45 μm ceramic membrane, and the rest of the operation is the same.

[0030] Exosomes and vesicles with diameters of 30–150 nm in the resuspension samples after ultracentrifugation were imaged and observed in real time using transmission electron microscopy and nanoflow cytometry. The results are as follows: Figure 10 Exosomes were detected in the resuspension sample; Figure 11 Figures A and B show that the average particle size of bovine milk exosomes is 73.9 nm. Figure 12 The exosome concentration was shown to be 1.71 × 10⁻⁶. 9 Particles / mL.

[0031] The detection data of Examples 1 to 4 above are shown in Table 1: Table 1. Exosome detection results of bovine exosome raw materials (after ultracentrifugation extraction) using different membrane filtration methods.

[0032] As shown in Table 1, the membrane separation technology of the present invention can prepare raw materials rich in exosomes, and can quickly and in large quantities prepare crude products rich in exosomes. After centrifugation and resuspension, the exosomes are concentrated by about 250 times.

[0033] Example 5: Preparation of bovine milk exosome products (0.1μm + 100kDa membrane filtration + chromatography) The specific implementation steps are as follows: (1) Take skim milk at 4℃; (2) Using a 0.1 μm ceramic membrane, the permeate was concentrated 3 times to obtain whey permeate; (3) Take the whey permeate and concentrate it 10 times using a 100kDa organic membrane; (4) The concentrated whey was processed by a Capto Core 700 chromatography system to obtain a chromatographic solution; (5) Store the chromatography sample at -80℃; (6) Sampling and testing.

[0034] Exosomes and vesicles with diameters of 30–150 nm in exosome chromatography samples were imaged and observed in real time using transmission electron microscopy and nanofluid cytometry. The results are as follows: Figure 13 Exosomes were detected in the chromatography sample; Figure 14 Figures A and B show that the average particle size of bovine milk exosomes is 83 nm. Figure 15 The exosome concentration shown is 2.83 × 10⁻⁶. 8 The concentration per particle / mL was one to two orders of magnitude higher than that of the crude products in Examples 1-4, and even approached the concentration of Examples 1-4 after centrifugation and concentration by 250 times.

[0035] Exosome products were prepared from skim milk using membrane separation and chromatography techniques. The detection data for these exosome products are shown in Table 2. This indicates that this method can produce exosome products with high purity and concentration.

[0036] Table 2. Exosome Detection Results of Membrane-Filtered Milk Exosome Products

[0037] Test Example 1: Verifying the therapeutic effect of bovine milk exosomes (MExo) on periodontitis. Eighteen SD rats were randomly divided into three groups: blank group, model control group (PBS), and intervention group (bovine milk exosomes). The rats in the model control group and the control group underwent ligation surgery and were fixed to the surgical board under 1% tribromoethanol anesthesia. The maxillary left second molar was ligated with 0.2 mm silk thread and given a high-glucose solution to establish an acute severe periodontitis rat model. The modeling time was 11 weeks. During the feeding process, the ligature wire was checked and re-ligated if it was found to have fallen off.

[0038] In this study, the control group rats received no intervention (no surgery, no drug administration), while the model control group and the intervention group rats were injected into the gingiva with 50 μL of PBS solution and the bovine milk exosome product from Example 5 (diluted to 1×10⁻⁶). 7 (particles / mL), administered every other day, twice daily, for 6 weeks. After 6 weeks of intervention, intraoral photographs of rats were taken and periodontal clinical indicators were recorded. Subsequently, the rats were euthanized by intraperitoneal injection of 2% sodium pentobarbital, and tissues were quickly collected for periodontal clinical indicator testing.

[0039] The periodontal clinical indicators include probing depth (PD), bleeding index (BI), and tooth mobility (TM). When checking probing depth, a periodontal probe of appropriate size is gently inserted into the gingival sulcus or pocket floor of the maxillary first molar. The periodontal pocket depth at six sites—mesial, central, and distal—on the buccal and palatal sides is measured and recorded, and the average value is calculated.

[0040] According to Mazza's criteria, the gingival bleeding index is divided into six levels: 0 = healthy gums, no inflammation or bleeding; 1 = gums show inflammatory changes in color, but no bleeding on probing; 2 = pinpoint bleeding after probing; 3 = bleeding on probing that spreads along the gingival margin; 4 = bleeding that fills and overflows the gingival sulcus; 5 = spontaneous bleeding.

[0041] During the mobility test, place the forceps together in the central fossa of the first molar's occlusal surface and move them towards the buccal palate, mesiodistal, and vertical directions. Observe and record the degree of tooth mobility. The mobility assessment criteria are: Grade I, mobility only in the buccal palate direction; Grade II, mobility in the buccal palate and mesiodistal directions; Grade III, mobility in the buccal palate, mesiodistal, and vertical directions.

[0042] The test results are statistically presented in Tables 3 and 4.

[0043] Table 3. Statistical analysis of clinical indicators before and after PBS intervention in the model group

[0044] Table 4. Statistical analysis of clinical indicators before and after intervention in the intervention group (bovine milk exosome group).

[0045] The results in Tables 3 and 4 show that, compared with the PBS control group, bovine exosome intervention exhibited more significant improvement in the following aspects: PBS could only clear periodontal pockets with a PD=4mm (reduction of 4.17%), and had no effect on periodontal pockets with PD=2 and PD=3mm. Bovine exosomes, on the other hand, could effectively reduce periodontal pockets with a PD=2mm (reduction of 4.73%) and clear periodontal pockets with a PD=3mm (reduction of 16.67%); effectively reduce gingival bleeding in a high-inflammatory state (BI=4); and show greater improvement in tooth mobility (especially grade II mobility) (58.33% vs. 37.5%).

[0046] Conclusion: Bovine milk exosomes have superior therapeutic potential compared to PBS in relieving gingival inflammation, promoting periodontal tissue stability, and improving clinical periodontal parameters, suggesting that they possess good anti-inflammatory and tissue repair regulation capabilities and may serve as a new strategy for adjuvant treatment of periodontitis.

[0047] Test Example 2: Verifying the therapeutic effect of bovine milk exosomes on full-thickness skin wounds. Animal experiment procedure: (1) Thirty male C57BL / 6 mice were randomly divided into three groups. Before the experiment, the mice were disinfected and shaved. The mice were anesthetized by isoflurane breathing. A wound model with a diameter of 1 cm was established by punching a hole. The non-punched group served as the blank control group. (2) The model control group and the intervention group were respectively treated with 50 μl PBS solution and the bovine milk exosome product of Example 5 (diluted to 1×10⁻⁶) at the wound site. 7 (particles / mL), administered every other day, twice daily. After establishing the wound model, record body weight, wound area changes, and drug response every 2 days. Wound area is as follows: Figure 16 As shown, Figure 16 Figures A and B in the figure show that the wound area was significantly different between the bovine milk exosome group and the control group (P<0.05). (3) At 7 and 14 days of intervention, three mice in each group were randomly euthanized, and tissue samples were collected from the edge of the wound—the annular area 2-3 mm from the visible wound boundary. The wound area was analyzed and calculated using ImageJ software.

[0048] (4) Skin tissues collected on the seventh and fourteenth days were fixed in 10% formalin neutral buffer solution and further embedded in paraffin for H&E / Masson trichrome staining. The tissue sections were then observed. Serum inflammatory factors TNF-α, IL-6, and IL-1β were measured at the same time.

[0049] (5) The deposition of collagen I / III and the proliferation of fibroblasts were determined by immunohistochemical assays and HE / Masson staining results.

[0050] The results are as follows Figures 17-19 As shown, the blue arrows indicate epidermal thickening; the red arrows indicate fibrous tissue hyperplasia. After 7 days of treatment with bovine milk exosomes, the epidermis at the damaged site was significantly thickened, dermal hair follicles disappeared, fibrous tissue hyperplasia occurred, inflammatory cell infiltration was observed, and the inflammatory response decreased. On day 14 of treatment, after bovine milk exosome treatment, the fibrous tissue thickened, the structure was clear, and it had basically returned to normal. Furthermore, serum IL-6 was significantly lower than that in the blank group and the PBS group. Figure 20 As shown.

[0051] Conclusion: Among all groups, bovine exosomes showed the best prognostic effect, indicating a good healing-promoting ability and properties. Animal experiments showed that bovine exosomes can reduce the inflammatory response and restore it to normal inflammatory levels, promoting wound healing. This suggests that bovine exosomes can be used as a topical medication to promote wound healing, with effects on promoting fibrosis and wound healing, as well as anti-inflammatory functions.

[0052] This invention discloses a rapid method for preparing bovine milk exosome raw materials and products. The method includes the following steps: separation using a 0.1μm~0.5μm ceramic membrane to obtain whey permeate; concentration of the whey permeate using a 100Da~300kDa organic membrane to prepare exosome raw materials; and finally, processing with a chromatography device to prepare exosome products with high concentration and purity. Animal experiments have verified the good anti-inflammatory and tissue repair regulation effects of the bovine milk exosomes prepared by this method. Bovine milk exosomes prepared by this method have broad application prospects in wound anti-inflammatory treatment and as an adjunct to periodontitis treatment.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of a bovine milk exosome or bovine milk exosome product in the preparation of products that relieve or treat gingival inflammation or promote periodontal tissue stability, wound anti-inflammatory effects, promote skin wound healing and / or skin trauma repair.

2. The application according to claim 1, characterized in that, The bovine milk exosomes were extracted through the following steps: S1. Skim milk is filtered through a membrane with a pore size of 0.1μm~0.5μm; S2. The permeate from step S1 is concentrated using a 100 Da to 300 kDa organic membrane to obtain crude exosomes.

3. The application according to claim 2, characterized in that, It also includes step S3: the concentrate is purified by chromatography to obtain exosomes.

4. A method for extracting bovine milk exosomes, characterized in that, The steps include the following: S1. Skim milk is filtered through a membrane with a pore size of 0.1μm~0.5μm; S2. Concentrate the filtrate from step S1 using a 100 Da~300 kDa organic membrane; S3. The concentrated solution is purified by chromatography to obtain exosomes.

5. The extraction method according to claim 4, characterized in that, In step S3, the chromatography column is a CaptoCore 700.

6. The application according to claim 2 or 3, or the extraction method according to claim 4 or 5, characterized in that, The membrane used in step S1 is a ceramic membrane or an organic membrane with a pore size of 0.1 μm to 0.45 μm; And / or, in step S1, the concentration is increased by 2 to 5 times or 3 to 5 times.

7. The application according to claim 2 or 3, or the extraction method according to claim 4 or 5, characterized in that, The organic membrane used in step S2 is a 100 Da~200 kDa organic membrane; And / or, in step S2, the concentration factor is 2 to 10 times or 5 to 10 times.

8. The application according to claim 2 or 3, or the extraction method according to claim 4 or 5, characterized in that, The concentration of crude exosomes was 10. 6 ~10 8 particles / mL; And / or, concentration of 10 in exosome products 8 ~10 10 particles / mL.

9. The application according to claim 1 or 3, or the extraction method according to claim 4 or 5, characterized in that, The dosage form of the product is a liquid topical formulation.

10. The application according to claim 1 or 3, or the extraction method according to claim 4 or 5, characterized in that, Promoting periodontal stability includes reducing tooth loosening; And / or, relief or treatment of gingivitis includes reducing periodontal pockets or reducing the gingival bleeding index; And / or, promoting skin wound healing or repair functions, including promoting fibrous tissue generation and wound healing.

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