Preparation method and application of honeysuckle flower exosome powder

Abstract

The invention relates to extraction of exosome-like vesicles secreted from honeysuckle flower cells and application of the exosome-like vesicles. The diameter of the extracellular vesicle ranges from 50 nanometers to 200 nanometers, and the extracellular vesicle contains specific bioactive components and has the effect of relieving periodontitis. The action mechanism of relieving periodontitis is that periodontitis clinical symptoms are relieved by inhibiting inflammation, promoting gingival cell proliferation and inhibiting tooth bone loss. The preparation method comprises the steps of honeysuckle flower superfine grinding, multi-stage differential centrifugation and carbon dioxide supercritical extraction. The application method is gingival smearing, and the clinical symptom effect of periodontitis can be relieved. The invention provides a safe and effective new way for treating periodontitis by using the honeysuckle flower exosome vesicles.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology extraction, and specifically relates to a method for preparing honeysuckle exosome powder and application of the powder in anti-periodontitis products. Background Art

[0002] Periodontitis is one of the most common chronic non-communicable diseases in the world. Among adults in my country, the incidence of periodontitis is over 70%. Periodontitis can lead to the destruction of periodontal supporting tissues and even tooth loss, seriously affecting the patient's quality of life. Periodontitis is not caused by individual pathogens, but by the synergistic effects of multiple microorganisms and the imbalance of periodontal microbial ecology. At present, the world's recognized oral pathogen is Porphyromonas gingivalis. The lipopolysaccharide (LPS-PG) secreted by Porphyromonas gingivalis first stimulates gingival epithelial cells to cause the secretion of inflammatory factors, which in turn leads to redness, swelling and inflammation of the gums. Gingival epithelial cells (HGE), as the first barrier of periodontal tissue, play an important role in resisting the invasion of periodontitis bacteria. Therefore, the protection of HGE cells is a research direction for the treatment of periodontitis.

[0003] Honeysuckle, also known as "snow lotus", is rich in a variety of bioactive substances, including polysaccharides, flavonoids and alkaloids. These ingredients give honeysuckle a strong immunomodulatory ability. It can enhance the function of the human immune system and improve the body's resistance to external pathogens. Studies have shown that honeysuckle can promote the activity of immune cells, increase the production of antibodies, and thus enhance the body's immunity. In addition, honeysuckle also has a variety of health benefits such as anti-inflammatory, antioxidant and anti-tumor. It is widely used in traditional Chinese medicine to treat hepatitis, immune diseases and tumors.

[0004] Exosomes are nanoscale vesicles composed of phospholipid bimolecules, lipids, including microRNA (miRNA) and proteins and a large number of active substances. Their morphology can be observed using a transmission electron microscope, and their size is about 30-150nm. According to a large number of studies, this cell structure also exists in plants, which is called plant exosomes. Exosomes from honeysuckle have good biological stability and permeability due to their double-layer membrane structure, making them easier to be absorbed by cells. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for preparing honeysuckle exosome powder and application of the powder in preparing anti-periodontitis products.

[0006] The present invention provides a method for preparing honeysuckle exosome powder, comprising the following steps:

[0007] (1) After the honeysuckle flowers are crushed, water is added and mixed evenly, and then the mixture is boiled and centrifuged to obtain the honeysuckle exosome pretreatment solution.

[0008] (2) Drying the honeysuckle exosome pretreatment solution under low temperature and reduced pressure to obtain the honeysuckle exosome crude dry powder.

[0009] (3) The obtained crude dry powder of honeysuckle exosomes is treated by supercritical carbon dioxide extraction, with an extraction pressure of 5-20 MPa, an extraction temperature of 25-65° C., and an extraction time of 0.5-4 hours, and then dried under reduced pressure to obtain supercritically extracted honeysuckle exosome powder.

[0010] In the above method, preferably, in step (1), the mass volume ratio of honeysuckle to water is 1 (g):5 (mL), the mixing time is 6 to 12 hours, and the mixing temperature is 15°C.

[0011] The cell wall is the outer structure of plant cells, mainly composed of cellulose, which plays a role in protecting cells. At high temperatures, the cellulose in the cell wall loses stability due to heat, causing the cell wall to rupture. Then, with the further effect of temperature, the proteins and lipids in the cell membrane may change, causing the membrane structure to lose stability, eventually leading to cell membrane rupture and releasing exosomes. Preferably, in step (1), the boiling time is 1 hour.

[0012] Preferably, in step (1), the centrifugation conditions are as follows: in step (1), after the honeysuckle is crushed, water is added and mixed evenly, and then boiled, centrifuged at a speed of 10000×g for 1 hour, the centrifugation temperature is 10-25°C, and the precipitate is discarded to obtain the honeysuckle exosome pretreatment solution.

[0013] More preferably, in step (1), after the honeysuckle flowers are crushed, water is added and mixed evenly, and then the mixture is boiled, and then centrifuged at a speed of 10,000×g for 1 hour, and the centrifugation temperature is 25°C.

[0014] Preferably, in step (2), the conditions for low-temperature and reduced-pressure drying are as follows: the drying temperature is 4° C., and the drying time is 24-72 hours.

[0015] More preferably, in step (3), the obtained crude dry powder of honeysuckle exosomes is treated by supercritical carbon dioxide extraction, with an extraction pressure of 7 MPa, an extraction temperature of 31° C., and an extraction time of 0.5-2 hours, and the supercritically extracted honeysuckle exosome powder is obtained by low-temperature drying.

[0016] The beneficial effects of the present invention are:

[0017] The present invention provides application of honeysuckle exosome powder in anti-periodontitis products.

[0018] Preferably, the honeysuckle exosomes are used for HGE cells induced by LPS-PG, which has the effect of promoting the proliferation of HGE cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a transmission electron microscopic morphological observation of the honeysuckle exosomes prepared in the present invention; it can be seen from the figure that the honeysuckle exosomes have obvious membrane boundaries and present saucer structures of varying sizes.

[0020] Figure 2 This is a particle tracking analyzer particle size analysis result diagram of the honeysuckle exosomes extracted from the present invention, wherein the particle size of the honeysuckle exosomes was detected to be 170.1 nm;

[0021] Figure 3 This is a graph showing the potential analysis results of the Zeta potential analyzer of the honeysuckle exosomes extracted from the present invention, wherein the particle size of the honeysuckle exosomes was detected to be -6.37 mV;

[0022] Figure 4 This is the effect of the honeysuckle exosome preparations prepared by different methods on the survival rate of HGE cells after LPS-PG induction in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0024] Example 1

[0025] The specific steps for preparing honeysuckle exosomes are as follows:

[0026] (1) After the honeysuckle flowers were crushed by a wall breaking machine, they were mixed with water at a mass volume ratio of 1 g: 5 mL, mixed at 25°C for 6 h, boiled for 1 h after mixing evenly, and then centrifuged at 25°C and 10,000×g for 1 h, and the precipitate was discarded to obtain the honeysuckle flower exosome pretreatment solution.

[0027] (2) Drying the honeysuckle exosome pretreatment solution in step (1) at a drying temperature of 4° C. for 72 hours to obtain a crude dry powder of honeysuckle exosomes.

[0028] (3) The crude dry powder of honeysuckle exosomes obtained in step (2) was treated by supercritical carbon dioxide extraction, extracted for 2 hours at an extraction pressure of 7 MPa and an extraction temperature of 31° C., and then dried at 4° C. for 72 hours to obtain honeysuckle exosome powder. Example 2

[0029] The difference from Example 1 is that in step (1), the centrifugal temperature after the honeysuckle flowers and water are mixed is 10°C, and the other operations and conditions are the same as those in Example 1.

[0030] Example 3

[0031] The difference from Example 1 is that in step (3), when the crude dry powder of honeysuckle exosomes is treated by supercritical carbon dioxide extraction, the extraction pressure is 10 MPa, and the other operations and conditions are the same as in Example 1.

[0032] Example 4

[0033] The difference from Example 1 is that in step (2), when the honeysuckle exosome pretreatment liquid is dried at low temperature, the temperature used is 10°, and the other operations and conditions are the same as in Example 1.

[0034] Comparative Example 1

[0035] The difference from Example 1 is that in step (2), the time used for low-temperature drying of the honeysuckle exosome pretreatment liquid is 24 hours, and the other operations and conditions are the same as in Example 1.

[0036] Comparative Example 2

[0037] The difference from Example 1 is that in step (3), when the crude dry powder of honeysuckle exosomes is treated by supercritical carbon dioxide extraction, the extraction time is 0.5 h, and the other operations and conditions are the same as in Example 1.

[0038] Comparative Example 3

[0039] The biggest difference from Example 1 is that in step (3), when the crude dry powder of honeysuckle exosomes is treated by supercritical carbon dioxide extraction, the extraction time is 1 h, and the other operating steps and conditions are the same as in Example 1.

[0040] Comparative Example 4

[0041] The biggest difference from Example 1 is that in step (3), when the crude dry powder of honeysuckle exosomes is treated by supercritical carbon dioxide extraction, the extraction pressure is 20 MPa, and the other operating steps and conditions are the same as in Example 1.

[0042] Comparative Example 5

[0043] The biggest difference from Example 1 is that in step (3), when the crude dry powder of honeysuckle exosomes is treated by supercritical carbon dioxide extraction, the extraction temperature is 65°C, and the other operating steps and conditions are the same as in Example 1.

[0044] The activity of the honeysuckle prepared in Examples 1-4 and Comparative Examples 1-5 was evaluated, and the concentration of miRNA was measured using a kit. The results are shown in Table 1 below.

[0045] Table 1 Concentration of miRNA in honeysuckle exosome powder prepared in each experimental group

[0046] Experimental groups micRNA concentration (ng / μL) Example 1 512.22 Example 2 454.07 Example 3 405.12 Example 4 109.41 Comparative Example 1 30.78 Comparative Example 2 25.42 Comparative Example 3 32.30 Comparative Example 4 28.74 Comparative Example 5 21.01

[0047] According to the data in the above table, the content of micRNA in the honeysuckle exosome powder obtained in Example 1 reached 512.22 ng / μL, and the highest concentration was 24.3 times that of the control group.

[0048] Cell experiments

[0049] After in vitro cell culture, HGE cells were divided into blank group, modeling group, Example 1 group, Example 2 group, Example 3 group, Example 4 group and Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group and Comparative Example 5 group. HGE cells were first treated with LPS-PG for 6 hours, and then incubated with samples of different groups for 12 hours. The cell survival rate was detected by MTT method. The results are shown in Table 2 and Attached Figure 4 shown.

[0050] Table 2 Survival rate of HGE cells in each experimental group (%)

[0051] Experimental Group Cell survival rate (%) Blank Group 100.00 Modeling Group 60.14 Example 1 134.17 Example 2 131.75 Example 3 127.50 Example 4 131.46 Comparative Example 1 61.07 Comparative Example 2 63.42 Comparative Example 3 67.01 Comparative Example 4 66.72 Comparative Example 5 61.75

[0052] Table 2 and Appendix Figure 4 The results showed that the survival rate of HGE cells in Examples 1-4 was significantly higher than that in Comparative Examples 1-5. Different concentrations of honeysuckle exosomes were obtained by different extraction methods. Our preparation method was more effective in increasing the concentration of honeysuckle exosomes, reducing impurities, and increasing the survival rate of HGE cells induced by LPS-PG, which significantly promoted the proliferation of HGE cells.

Claims

1. A method for preparing honeysuckle exosome powder, characterized in that: The following steps are involved: (1) After the honeysuckle flowers are crushed, water is added and mixed evenly, and then the mixture is boiled and centrifuged to obtain the honeysuckle exosome pretreatment solution. (2) Drying the honeysuckle exosome pretreatment solution under low temperature and reduced pressure to obtain the honeysuckle exosome crude dry powder. (3) The obtained crude dry powder of honeysuckle exosomes is treated by supercritical carbon dioxide extraction, with an extraction pressure of 5-20 MPa, an extraction temperature of 25-65° C., an extraction time of 0.5-4 hours, and low-temperature drying to obtain supercritically extracted honeysuckle exosome powder.

2. The method for preparing the honeysuckle exosome powder according to claim 1, characterized in that: In step (1), the mass volume ratio of honeysuckle flower to water is 1 (g):5 (mL), the mixing time is 6 to 24 hours, and the mixing temperature is 10 to 20°C.

3. The method for preparing honeysuckle exosomes according to claim 1, characterized in that: In step (1), the boiling time is 0.5-1h.

4. The method for preparing honeysuckle exosomes according to claim 1, characterized in that: In step (1), the centrifugation conditions are as follows: step (1) after the honeysuckle is crushed, water is added and mixed evenly, and then boiled, centrifuged at a speed of 10000×g for 1 to 2 hours, the centrifugation temperature is 10 to 25°C, and the precipitate is discarded to obtain the honeysuckle exosome pretreatment solution.

5. The method for preparing the honeysuckle exosome powder according to claim 1, characterized in that: In step (2), the conditions for low-temperature and reduced-pressure drying are as follows: the drying temperature is 4° C., and the drying time is 24-72 hours.

6. The method for preparing the honeysuckle exosome powder according to claim 1, characterized in that: The conditions for low-temperature drying described in step (3) are as follows: drying temperature is 4° C., and drying time is 72 hours.

7. A honeysuckle exosome powder prepared by the preparation method as claimed in claim 1.

8. Use of the honeysuckle exosome powder as claimed in claim 8 in the preparation of anti-periodontitis products.

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