Exosome for preventing and / or treating rheumatic arthritis as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicine preparation, in particular to an exosome for preventing and / or treating rheumatic arthritis and a preparation method and application thereof. The exosome disclosed by the invention is prepared from the following components in parts by mass: 10-20 parts of onions, 20-30 parts of fresh ginger and 10-20 parts of fistular onion stalks. The exosome prepared from the components can reduce the inflammatory factor level in a mouse with rheumatoid arthritis, and does not affect the growth of the mouse. And after the exosome is prepared into a certain dosage form, the rheumatoid arthritis can be effectively treated. A basis is provided for the preparation of the plant exosome, and a basis is provided for the safe treatment of rheumatoid arthritis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation technology, and in particular to an exosome for the prevention and / or treatment of rheumatoid arthritis, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis is a common acute or chronic inflammatory disease of connective tissue, clinically characterized by migratory aches, heaviness, and pain in the joints and muscles, causing significant disruption to patients' lives. Currently, medications for treating rheumatoid arthritis mainly include nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and immunosuppressants; however, these drugs often have significant side effects and inconsistent efficacy.

[0003] Plant exosomes are nanoscale vesicles secreted by plant cells, containing various bioactive substances such as proteins, nucleic acids, and lipids, and possessing multiple biological functions including anti-inflammatory and immunomodulatory effects. In recent years, the application of plant exosomes in disease treatment has received widespread attention, but there are no reports of their preparation as drugs for treating rheumatoid arthritis. Summary of the Invention

[0004] The purpose of this invention is to provide a drug for treating rheumatoid arthritis with plant exosomes as the active ingredient, in order to solve the problems of large side effects and unstable efficacy of existing drugs.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an exosome for the prevention and / or treatment of rheumatoid arthritis, prepared from components comprising the following parts by weight: 10-20 parts onion, 20-30 parts ginger, and 10-20 parts scallion whites.

[0006] The present invention also provides a method for preparing exosomes for the prevention and / or treatment of rheumatoid arthritis, comprising the following steps: (1) Weigh each component according to the stated mass fractions, mix them, and obtain a mixture; (2) Mix the mixture with water, sonicate to break it up, filter it, and take the filtrate to obtain extract 1; (3) Centrifuge the extract 1 at 2500~3000g for 25~30min, collect the supernatant to obtain the first centrifuged liquid; centrifuge the first centrifuged liquid at 4500~5500g for 30~40min, collect the supernatant to obtain the second centrifuged liquid; centrifuge the second centrifuged liquid at 6500~7500g for 35~45min, collect the supernatant to obtain the third centrifuged liquid; centrifuge the third centrifuged liquid at 9000~10000g for 45~60min, collect the supernatant to obtain the fourth centrifuged liquid; (4) Pass the fourth centrifuged liquid through microporous filter membranes with pore sizes of 0.4~0.6µm and 0.1~0.3µm in sequence, collect the filtrate, and obtain extract 2; (5) Mix extract 2 with polyethylene glycol-800, let stand for 12-16 hours, centrifuge, collect the precipitate, and obtain exosomes.

[0007] Preferably, the mass ratio of the mixture to water in step (2) is 1~3g:1~3g; The ultrasonic crushing power in step (2) is 400~500W, and the ultrasonic crushing time is 10~20min.

[0008] Preferably, the filtration in step (2) is a filtration of 14 to 18 layers of gauze.

[0009] Preferably, the volume ratio of the extract 2 and polyethylene glycol-800 in step (5) is 100:1~3.

[0010] Preferably, the centrifugation speed in step (5) is 15000~17000g and the centrifugation time is 45~60min.

[0011] The present invention also provides the use of the aforementioned exosomes in the preparation of health products for the treatment and / or prevention of rheumatoid arthritis.

[0012] The present invention also provides the application of the exosomes prepared by the above preparation method in the preparation of health care products for the prevention and / or treatment of rheumatoid arthritis.

[0013] The present invention also provides a medicament for the prevention and / or treatment of rheumatoid arthritis, the medicament comprising the aforementioned exosomes and excipients.

[0014] Preferably, the dosage form of the medicine includes patches, granules, pills, ointments, powders, or tablets.

[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention extracts exosomes from a mixture of onion, scallion and ginger. The exosomes contain a variety of bioactive substances and have anti-inflammatory and immunomodulatory effects, which can effectively relieve the symptoms of rheumatoid arthritis.

[0016] (2) The exosomes of the present invention for the prevention and / or treatment of rheumatoid arthritis are derived from natural plants, have high safety and no side effects, and have fewer side effects compared with traditional chemical drugs.

[0017] (3) The exosomes of the present invention can be prepared into various dosage forms, which are convenient for patients to use and improve patient compliance. Detailed Implementation

[0018] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0019] Example 1

[0020] Weigh out 150g of onion, 300g of ginger, and 100g of scallion whites, and mix them to obtain a mixture. Mix 550g of the mixture with 1100g of water, sonicate at 500W for 15 minutes, filter through 16 layers of gauze, and collect the filtrate to obtain extract 1. Centrifuge extract 1 at 2500g for 30 minutes, collect the supernatant to obtain the first centrifuge liquid; centrifuge the first centrifuge liquid at 5000g for 35 minutes, collect the supernatant to obtain the second centrifuge liquid; centrifuge the second centrifuge liquid at 7000g for 40 minutes, collect the supernatant to obtain the third centrifuge liquid; centrifuge the third centrifuge liquid at 9000g for 50 minutes, collect the supernatant to obtain the fourth centrifuge liquid. First, pass the fourth centrifuge liquid through a 0.45µm filter membrane, collect the filtrate, and then pass it through a 0.2µm filter membrane, collect the filtrate to obtain extract 2. Extract 2 was mixed with polyethylene glycol-800 at a volume ratio of 100:1, allowed to stand at 4°C for 16 hours, centrifuged at 17000g for 50 minutes, and the precipitate was collected to obtain exosomes.

[0021] Example 2

[0022] Weigh out 100g of onion, 250g of ginger, and 200g of scallion whites, and mix them to obtain a mixture. Mix 550g of the mixture with 550g of water, sonicate at 400W for 20 minutes, filter through 16 layers of gauze, and collect the filtrate to obtain extract 1. Centrifuge extract 1 at 3000g for 25 minutes, collect the supernatant to obtain the first centrifuge liquid; centrifuge the first centrifuge liquid at 5000g for 40 minutes, collect the supernatant to obtain the second centrifuge liquid; centrifuge the second centrifuge liquid at 7500g for 40 minutes, collect the supernatant to obtain the third centrifuge liquid; centrifuge the third centrifuge liquid at 9500g for 60 minutes, collect the supernatant to obtain the fourth centrifuge liquid. First, pass the fourth centrifuge liquid through a 0.45µm filter membrane, collect the filtrate, and then pass it through a 0.2µm filter membrane, collect the filtrate to obtain extract 2. Extract 2 was mixed with polyethylene glycol-800 at a volume ratio of 100:2, allowed to stand at 4°C for 16 hours, centrifuged at 15000g for 60 minutes, and the precipitate was collected to obtain exosomes.

[0023] Example 3

[0024] Weigh out 200g of onion, 200g of ginger, and 150g of scallion whites, and mix them to obtain a mixture. Mix 550g of the mixture with 550g of water, sonicate at 400W for 20 minutes, filter through 16 layers of gauze, and collect the filtrate to obtain extract 1. Centrifuge extract 1 at 3000g for 25 minutes, collect the supernatant to obtain the first centrifuge liquid; centrifuge the first centrifuge liquid at 4500g for 40 minutes, collect the supernatant to obtain the second centrifuge liquid; centrifuge the second centrifuge liquid at 6500g for 45 minutes, collect the supernatant to obtain the third centrifuge liquid; centrifuge the third centrifuge liquid at 10000g for 45 minutes, collect the supernatant to obtain the fourth centrifuge liquid. First, pass the fourth centrifuge liquid through a 0.45µm filter membrane, collect the filtrate, then pass it through a 0.2µm filter membrane, collect the filtrate, and obtain extract 2. Extract 2 was mixed with polyethylene glycol-800 at a volume ratio of 100:1, allowed to stand at 4°C for 16 h, centrifuged at 16000 g for 60 min, and the precipitate was collected to obtain exosomes.

[0025] Experimental Example 1

[0026] Twenty-four healthy C57BL / 6 mice were randomly divided into four groups: a blank control group, a model control group, the Example 1 group, and a control group. Complete Freund's adjuvant was fully emulsified and prepared for use. Mice in the model control group, Example 1 group, and control group received a subcutaneous injection of 0.01 mL of the emulsified complete Freund's adjuvant into the paw pads of their left and right hind limbs. Mice in the blank control group received no treatment. Simultaneously, mice in the model control group, Example 1 group, and control group received a subcutaneous injection of 0.1 mg / kg epinephrine hydrochloride solution into their backs. Mice in the blank control group received no injection. Two hours after injection, mice in each group were placed in 4°C cold water for 10 minutes, once daily for 7 consecutive days, to complete the model establishment.

[0027] After modeling, mice in the blank control group and the model control group were not given any drugs by gavage. Mice in Example 1 were gavage with exosomes from Example 1 at 1 mg / kg / day, while mice in the control group were gavage with ibuprofen at 1.5 mg / kg / day. Gavage was continued for 14 days.

[0028] After the experiment, the weight of mice in each group was measured, and the results are shown in Table 1.

[0029] Table 1. Changes in mouse body weight after treatment with different drugs.

[0030] As can be seen, the body weight of mice in Example 1 group was close to that of mice in the blank control group, which indicates that the exosomes of the present invention do not affect the body weight of mice.

[0031] The thickness of the left and right hind paws of mice was measured with a vernier caliper on days 7 and 14 after gavage administration. The results are shown in Table 2.

[0032] Table 2. Treatment effects of different drugs on the hind paws of mice.

[0033] As can be seen, after 14 days of gavage administration of exosomes from Example 1, the thickness of the left and right hind paws of mice was significantly reduced, indicating that the exosomes from Example 1 of this invention can reduce the inflammatory response of rheumatoid arthritis induced by complete Freund's adjuvant and epinephrine hydrochloride.

[0034] Fourteen days after administration of the drug via gavage, tissues from both ankles were collected, ground, and centrifuged at 3000 rpm for 10 min. The supernatant was collected, and the levels of IL-1β, IL-6, and TNF-α were detected according to the instructions of the ELISA kit for mice. The results are shown in Table 3.

[0035] Table 3. Changes in the concentration of inflammatory factors in mice after treatment with different drugs.

[0036] As can be seen, the inflammatory factors in the ankle tissue of mice fed with exosomes of Example 1 were lower than those in the group fed with ibuprofen. This indicates that the exosomes of Example 1 of the present invention can reduce inflammatory factors in mice and have anti-inflammatory effects. They can be used for the treatment of rheumatoid arthritis.

[0037] Application Example 1

[0038] Take 10 parts of exosomes prepared in Example 2, 10 parts of carbomer, 2 parts of azone, and 80 parts of water, mix them, stir evenly, coat them on non-woven fabric, cover them with polyethylene film, and obtain the patch.

[0039] Twenty patients (regardless of gender) with rheumatoid factor ≥20 IU / mL, C-reactive protein ≥10 mg / L, anti-streptolysin O ≥200 IU / mL, and experiencing joint stiffness and swelling were selected for treatment. The patch prepared according to this application example was used for treatment; the patch was applied to the affected joint and changed every 8 hours. Patients were followed up weekly, and rheumatoid factor levels were measured every 2 weeks.

[0040] As a result, none of the 20 patients experienced allergic reactions during the use of the patch, and 8 patients (with a disease duration of less than 2 years) showed a significant reduction in swelling and pain after one week of use. The remaining patients (with a disease duration of more than 2 years) showed a significant reduction in swelling and pain after two weeks of use. After 2 weeks of use, tests showed that the rheumatoid factor levels in all patients decreased. This indicates that the patch of the present invention can treat rheumatoid arthritis.

[0041] Application Example 2

[0042] Take 10 parts of exosomes prepared in Example 3, 5 parts of sodium carboxymethyl cellulose, 50 parts of lactose, 10 parts of sucrose and 25 parts of water, mix them, stir evenly, place them in a granulator to granulate, and dry them at 60°C to obtain granules.

[0043] Ten patients with rheumatoid factor (≥20 IU / mL), C-reactive protein (≥10 mg / L), and anti-streptolysin O (≥200 IU / mL), exhibiting joint stiffness and swelling, were selected and treated with the granules prepared in this application example. The dosage was 1 g each time, twice daily, once in the morning and once in the evening. After 7 days of treatment, the patients reported significant relief from swelling and pain, and testing of rheumatoid factor levels revealed a decrease in all rheumatoid factor indicators. After 10 days of treatment, the rheumatoid factor levels in 8 patients (with a disease duration of less than 2 years) returned to normal. In the remaining 2 patients with a disease duration of more than 2 years, the rheumatoid factor indicators also returned to normal after 20 days of treatment.

[0044] As demonstrated by the above embodiments, this invention provides a food-derived exosome that can reduce the levels of inflammatory factors in mice with rheumatoid arthritis without affecting their growth. Furthermore, when the exosome is prepared into a specific drug formulation, it can effectively treat rheumatoid arthritis. This provides a basis for the preparation of plant exosomes and for the safe treatment of rheumatoid arthritis.

[0045] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An exosome for the prevention and / or treatment of rheumatoid arthritis, characterized in that, It is prepared from components comprising the following parts by mass: 10-20 parts onion, 20-30 parts ginger, and 10-20 parts scallion whites.

2. A method for preparing exosomes for the prevention and / or treatment of rheumatoid arthritis, characterized in that, Includes the following steps: (1) Weigh each component according to the mass fractions specified in claim 1, mix them, and obtain a mixture; (2) Mix the mixture with water, sonicate to break it up, filter it, and take the filtrate to obtain extract 1; (3) Centrifuge the extract 1 at 2500~3000g for 25~30min, take the supernatant, and obtain the first centrifuged liquid; Centrifuge the first centrifuge solution at 4500~5500g for 30~40min, collect the supernatant, and obtain the second centrifuge solution; Centrifuge the second centrifuge solution at 6500~7500g for 35~45min, collect the supernatant, and obtain the third centrifuge solution; Centrifuge the third centrifuge solution at 9000~10000g for 45~60min, collect the supernatant, and obtain the fourth centrifuge solution; (4) Pass the fourth centrifuged liquid through microporous filter membranes with pore sizes of 0.4~0.6µm and 0.1~0.3µm in sequence, collect the filtrate, and obtain extract 2; (5) Mix extract 2 with polyethylene glycol-800, let stand for 12-16 hours, centrifuge, collect the precipitate, and obtain exosomes.

3. The preparation method according to claim 2, characterized in that, The mass ratio of the mixture to water in step (2) is 1~3g:1~3g; The ultrasonic crushing power in step (2) is 400~500W, and the ultrasonic crushing time is 10~20min.

4. The preparation method according to claim 2, characterized in that, The filtration in step (2) is a filtration of 14 to 18 layers of gauze.

5. The preparation method according to claim 2, characterized in that, In step (5), the volume ratio of the extract 2 to polyethylene glycol-800 is 100:1~3.

6. The preparation method according to claim 2, characterized in that, In step (5), the centrifugation speed is 15000~17000g and the centrifugation time is 45~60min.

7. The use of the exosomes according to claim 1 in the preparation of health care products for the treatment and / or prevention of rheumatoid arthritis.

8. The use of exosomes prepared by the preparation method according to any one of claims 2 to 6 in the preparation of health care products for the prevention and / or treatment of rheumatoid arthritis.

9. A medicament for the prevention and / or treatment of rheumatoid arthritis, characterized in that, The pharmaceutical preparation includes the exosomes as described in claim 1 and excipients.

10. The pharmaceutical preparation according to claim 9, characterized in that, The dosage forms of the medicine include patches, granules, pills, ointments, powders, or tablets.

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