Preparation method and application of ginger extracellular vesicles for treating alcoholic liver

Abstract

The invention relates to a ginger extracellular vesicle for treating alcoholic liver as well as a preparation method and application of the ginger extracellular vesicle. The ginger extracellular vesicles comprise shogaol in the ginger extracellular vesicles; the content of shogaol in the ginger extracellular vesicles is 8.0 to 14.0 [mu] g / mg. Experimental results show that the ginger extracellular vesicles provided by the invention can obviously improve the morbidity of alcoholic liver, animal liver blood indexes are obviously reduced, the liver necrosis area is obviously reduced, the animal weight is obviously increased, and alcoholic liver diseases can be effectively treated.

Description

Technical Field

[0001] The invention relates to ginger extracellular vesicles for treating acute alcoholic liver disease and a preparation method thereof, belonging to the technical field of biomedical materials. Background Art

[0002] Alcoholic liver disease (ALD), also known as alcoholic liver disease, is a liver disease caused by long-term heavy drinking. In the early stage, it usually manifests as fatty liver. If it is not diagnosed and treated in time, the liver is gradually infiltrated by alcohol and gradually develops into alcoholic hepatitis. The liver cells are damaged, immune cells gather, and the liver is swollen and tender. Long-term inflammation causes large-scale necrosis of liver cells, fibrosis and even sclerosis of the liver. Clinical manifestations include nausea, vomiting, jaundice, sclerosing of the liver and even liver failure with complications of upper gastrointestinal bleeding. This process causes irreversible necrosis of liver cells, which is difficult to recover to a large extent and can lead to life-threatening conditions.

[0003] At present, Western medicine mainly adopts methods such as nutritional support, drug therapy and liver transplantation. ALD patients have macro- and micronutrient imbalances and need good nutritional supplements, including protein, vitamin B, vitamin C, vitamin K, etc. Commonly used Western medicines mainly include glucocorticoids (CG), metadoxine, S-adenosylmethionine, polyene phosphatidylcholine, glycyrrhizic acid preparations, reduced glutathione, etc. However, this series of Western medicines are expensive and have large side effects, which seriously limits their application. Liver transplantation is also limited by a series of problems such as high cost, limited donors, and immunosuppression.

[0004] Ginger belongs to the ginger family. In daily life, ginger is often eaten as a spice. It has a unique aroma and spicy taste. It can also be used to repel insects and control pests. Studies have also shown that ginger and its active ingredients have antioxidant, anti-inflammatory, anti-cancer and antibacterial effects. Studies have shown that gingerols in ginger can regulate peroxisome proliferator-activated receptor γ (PPARγ), inhibit signaling pathways such as nuclear transcription factor κB (NF-κB) and transforming growth factor β (TGF-β), inhibit the activation of hepatic stellate cells, thereby reducing the deposition of extracellular matrix in hepatocytes, thereby exerting an anti-fibrotic effect to relieve ALD. At the same time, consuming ginger preparations can affect the expression of certain inflammatory and oxidative intermediate genes related to ALD to relieve ALD.

[0005] Plant extracellular vesicles (PEVs) are nano-sized phospholipid bilayer vesicles secreted by plant cells. They were first discovered in carrot cells in 1967. PEVs have a particle size of 50-300nm and are rich in DNA, RNA, proteins, lipids and plant-derived bioactive substances. PEVs are a cell-free system with good biocompatibility, extremely high stability, and extremely low toxicity and immunogenicity. PEVs can transfer their own active substances to the host after being internalized and engulfed by host cells, thereby exerting corresponding physiological functions. With the advantages of cell-free system, low immunogenicity and high stability, it can be used as a drug for disease treatment, or as a delivery carrier to transport related drugs to meet the needs of treatment. When used as a drug delivery carrier, PEVs can play its own advantages, improve transfection efficiency while reducing the harm caused by side effects, avoid the body's immune rejection reaction and fully reside to avoid being cleared by the body. Therefore, ginger plant extracellular vesicles rich in shogaols can not only be used as a therapeutic agent for alcoholic liver disease, but can also be used as a drug in the treatment of alcoholic liver disease. Summary of the invention

[0006] In view of the above problems, the present invention aims to provide a preparation method and application of ginger extracellular vesicles for the treatment of alcoholic liver disease, providing a new approach for the prevention and treatment of alcoholic liver disease.

[0007] In a first aspect, the present invention provides a ginger extracellular vesicle, wherein the ginger extracellular vesicle comprises shogaol in the ginger extracellular vesicle; the content of shogaol in the ginger extracellular vesicle is 8.0-14.0 μg / mg.

[0008] The particle size of the ginger extracellular vesicle is 50-200nm, and it has a double-layer phospholipid layer vesicle structure; the Zeta potential of the drug-loaded ginger extracellular vesicle is -20 to -40mV.

[0009] In a second aspect, the present invention provides a method for preparing the ginger extracellular vesicles, comprising: The ginger is peeled and juiced, then filtered, and the filtrate is subjected to gradient low-speed centrifugation and ultracentrifugation in sequence to obtain ginger extracellular vesicles.

[0010] Furthermore, the step of gradient low-speed centrifugation is: The ginger juice filtrate obtained after filtering through gauze was centrifuged at 1000-2000 g for 10-30 minutes at 4°C, and the supernatant was collected; The supernatant was centrifuged at 3000-6000 g for 20-40 minutes at 4°C to collect the supernatant; The supernatant was centrifuged at 8000-10000 g for 40-60 minutes at 4° C. to collect the supernatant, completing the gradient low-speed centrifugation operation; Furthermore, the ultracentrifugation step is: finally centrifuging the supernatant obtained by gradient low-speed centrifugation at 120,000-150,000 g for 90-120 minutes at 4°C, collecting the bottom precipitate, completing the ultracentrifugation operation, and obtaining ginger extracellular vesicles.

[0011] In a third aspect, the present invention provides a pharmaceutical composition comprising the ginger extracellular vesicles described above.

[0012] In a fourth aspect, the present invention provides an application of any one of the above-mentioned ginger extracellular vesicles, the preparation method of ginger extracellular vesicles, and the pharmaceutical composition, or a combination of at least two of them in the preparation of a product for treating alcoholic liver disease.

[0013] Beneficial Effects The ginger extracellular vesicles provided by the present invention not only have the advantages of a cell-free system, good biocompatibility, good stability, excellent drug loading capacity, etc., but also have a wide source, high yield, are economical and environmentally friendly, have natural therapeutic capabilities, and are conducive to clinical applications; The preparation method of ginger extracellular vesicles of the present invention sequentially subjects ginger juice to gradient low-speed centrifugation and ultracentrifugation, and the obtained ginger extracellular vesicles have a similar number of nanoparticles, a more concentrated particle size distribution, and a higher content of shogaol.

[0014] The present invention proves through animal experiments that the ginger extracellular vesicles provided by the present invention can significantly improve the incidence of alcoholic liver disease, the blood indicators of animal livers are significantly reduced, the area of ​​liver necrosis is significantly reduced, and the weight of animals is significantly increased, and alcoholic liver disease can be effectively treated. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 and Figure 2 The transmission electron microscopy and dynamic light scattering test results of ginger extracellular vesicles prepared in Example 1; Figure 3 The Zeta potential detection result of ginger extracellular vesicles provided by the present invention; Figure 4 is the HPLC gingerol detection result of the ginger extracellular vesicles provided by the present invention; Figure 5 This is the evaluation of the weight changes of ALD mice after oral administration of ginger extracellular vesicles to ALD mice for 11 days in Example 1 of the present invention; Figure 6The effect of oral administration of ginger extracellular vesicles on the changes in ALT content in the blood of ALD mice for 11 days in Example 1 of the present invention; Figure 7 The effect of oral administration of ginger extracellular vesicles to ALD mice on the changes in AST content in the blood of ALD mice for 11 days in Example 1 of the present invention; Figure 8 The effect of oral administration of ginger extracellular vesicles on the changes in TBIL content in the blood of ALD mice for 11 days in Example 1 of the present invention; Fig. 9 The effect of oral administration of ginger extracellular vesicles to ALD mice on the changes in γ-GT content in the blood of ALD mice for 11 days in Example 1 of the present invention; Fig.10 The effect of oral administration of ginger extracellular vesicles on the changes in ALP content in the blood of ALD mice for 11 days in Example 1 of the present invention; Fig.11 This is the effect of oral administration of ginger extracellular vesicles to ALD mice in Example 1 of the present invention on the change of TBA content in the blood of ALD mice after 11 days. Fig.12 This is an H&E staining image of the liver of ALD mice 11 days after the ALD mice orally administered ginger extracellular vesicles in Example 1 of the present invention. DETAILED DESCRIPTION

[0016] The present invention is further described below by way of examples. It should be correctly understood that the examples or drawings of the present invention are only used to illustrate the present invention, rather than to limit the present invention.

[0017] First, the present invention provides a ginger extracellular vesicle. The ginger extracellular vesicle includes ginger extracellular vesicle content shogaol. The present invention uses ginger extracellular vesicles as a treatment tool, and the shogaol in the ginger extracellular vesicles can further improve the treatment effect.

[0018] In some embodiments, the drug-loaded ginger extracellular vesicles have a particle size of 50-200 nm and have a double-layer phospholipid layer vesicle structure. The Zeta potential of the drug-loaded ginger extracellular vesicles is -20 to -40 mV, preferably -20 to -30 mV. The Zeta potential within this range indicates that the system is in a relatively stable state.

[0019] The following is an exemplary description of the method for preparing ginger extracellular vesicles provided by the present invention, which mainly includes the following steps.

[0020] Ginger extracellular vesicle extraction: Ginger peeled, juiced, filtered, and the filtrate was subjected to gradient low-speed centrifugation and ultracentrifugation in sequence to obtain ginger extracellular vesicles.

[0021] In some embodiments, the step of gradient low-speed centrifugation may be: The ginger juice filtrate obtained after filtering through gauze was centrifuged at 1000-2000 g for 10-30 minutes at 4°C, and the supernatant was collected; The supernatant was centrifuged at 3000-6000 g for 20-40 minutes at 4°C to collect the supernatant; The supernatant was centrifuged at 8000-10000 g for 40-60 minutes at 4° C. to collect the supernatant, completing the gradient low-speed centrifugation operation; In some embodiments, the ultracentrifugation step can be: centrifuging the supernatant at 120,000-150,000 g for 90-120 minutes at 4°C, collecting the bottom precipitate with sterile PBS, centrifuging the PBS solution containing the bottom precipitate at 120,000-150,000 g for 90-120 minutes at 4°C, collecting the bottom precipitate with sterile PBS, purifying the ginger extracellular vesicles, and completing the ultracentrifugation operation.

[0022] The ginger juice filtrate obtained after filtration is subjected to gradient low-speed centrifugation, and the different sedimentation coefficients of various components in plant cells are utilized. Under the action of different centrifugal forces, various components are separated and the required components are obtained, and the components interfering with the extraction of ginger extracellular vesicles are removed.

[0023] The supernatant after gradient low-speed centrifugation is subjected to ultracentrifugation, and the plant extracellular vesicles are separated and obtained by utilizing the difference in centrifugal force required for separation of plant extracellular vesicles. The centrifugal force required for separation of ginger extracellular vesicles is above 100,000 g. The ginger extracellular vesicles described in the present invention have been verified by animal experiments to have the effect of repairing liver damage. The ginger extracellular vesicles effectively improve the weight loss of ALD mice, effectively alleviate the necrosis phenomenon in the liver area of ​​ALD mice, and effectively alleviate the deterioration of liver function in ALD mice. The ginger extracellular vesicles described in the present invention can be used to prepare a pharmaceutical composition. The application disclosed in the present invention provides a new safe and effective biological treatment direction for the treatment of alcoholic liver disease.

[0024] The following examples are further listed to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are only examples in a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0025] Unless otherwise specified, all reagents and raw materials used in the present invention are commercially available products or products that can be prepared by known methods.

[0026] Example 1 Preparation of ginger extracellular vesicles: After squeezing the ginger with a juicer, the ginger juice filtrate obtained by filtering with gauze is centrifuged at 1000g for 20 minutes at 4°C to collect the first supernatant; then the supernatant is centrifuged at 40000g for 20 minutes at 4°C to collect the second supernatant; the second supernatant is centrifuged at 10000g for 60 minutes at 4°C to collect the third supernatant, completing the gradient low-speed centrifugation operation; then the third supernatant is centrifuged at 120000g for 120 minutes at 4°C, and the bottom precipitate is collected with sterile PBS buffer, the PBS solution containing the bottom precipitate is centrifuged at 150000g for 120 minutes at 4°C, and the bottom precipitate is collected with sterile PBS buffer, the ginger extracellular vesicles are purified, and the ultracentrifugation operation is completed to obtain the ginger extracellular vesicles.

[0027] Figure 1 and Figure 2 The transmission electron microscopy and dynamic light scattering test results of ginger extracellular vesicles obtained in Example 1; wherein, Figure 2 The horizontal axis is the particle size in nm, and the vertical axis is the concentration in ×108 particles / mL. Figure 1 and Figure 2 It can be seen that the ginger extracellular vesicles collected by gradient low-speed centrifugation and ultracentrifugation are spherical in shape, with a size of about 148 nm and a concentration of 6×108 particles / mL.

[0028] Figure 3 The Zeta potential detection result of ginger extracellular vesicles provided by the present invention. Figure 3 It can be seen that the Zeta potential of ginger extracellular vesicles is -24 mV, indicating that the structure of ginger extracellular vesicles collected by gradient low-speed centrifugation and ultracentrifugation is stable.

[0029] Figure 4 The HPLC test results of ginger extracellular vesicles of the present invention are as follows. Figure 4 It can be seen that the content of gingerol in ginger extracellular vesicles is 13.961 μg / mg The ginger extracellular vesicles provided by the present invention can be used to play a role in liver damage repair in ALD mice, and to improve the weight loss, liver necrosis area and liver function deterioration of ALD mice. Unless otherwise specified, the drugs described in the following mouse model experiments are administered orally.

[0030] Mouse model and grouping experiment: 8-week-old C57 male mice weighing 24-26g were selected and purchased from Slack Laboratory Animal Company. They were given free access to food and water to maintain circadian rhythm. After 1 week of adaptive feeding, they were randomly divided into ginger extracellular vesicle intervention group (PEVs group), experimental group (Alcohol group) and control group (Control group), with 12 mice in each group, for the experiment.

[0031] PEV group: The feed was changed to Lieber-DeCaril with 5% ethanol and 1012 parts / ml PEVs added, which was changed daily.

[0032] Alcohol group: The diet was changed to Lieber-DeCaril containing 5% ethanol and replaced daily.

[0033] Control group: The feed was changed to Lieber-DeCaril without ethanol and changed daily.

[0034] ALD models of mice were constructed for the PEVs group and the Alcohol group. Food and water intake were restricted and changed to Lieber-DeCaril feed supplemented with 5% ethanol, which was changed daily. After the second day of administration, 1012 particels / ml PEVs were added to the Lieber-DeCaril containing 5% ethanol in the PEVs group and feeding was continued for 11 days. On the 13th day, the mice were gavaged with 5g / kg of ethanol per mouse and were killed after 9 hours. The diet of the Control group was changed to Lieber-DeCaril without ethanol, and an equal amount of maltodextrin (5g / kg) was used for gavage on the 13th day, and the mice were killed after 9 hours; Body weight evaluation: record the weight of mice and draw a curve graph, such as Figure 5 As shown, oral administration of ginger extracellular vesicles can effectively improve the weight loss problem of ALD-induced mice.

[0035] Blood index evaluation: Collect blood from mice and test relevant blood factors. The experimental results are as follows: Figure 6-11 As shown in the figure, blood tests showed that compared with the Alcohol group, the liver and kidney indicators of the PEVs group mice were significantly improved, and ALT, AST, TBIL, γ-GT, ALP, TBA, etc. were significantly decreased. This shows that ginger extracellular vesicles can inhibit liver damage in ALD mice and play a certain role in repair.

[0036] Liver injury evaluation: H&E staining of mouse livers was performed. The experimental results are shown in Fig.12As shown in the figure, the liver H&E staining results showed that the liver necrosis area of ​​mice in the PEVs group was significantly reduced compared with that in the Alcohol group, indicating that ginger extracellular vesicles can improve the degree of liver damage in ALD mice and play a certain therapeutic role.

[0037] Comparative Example 1 Preparation of ginger extracellular vesicles. After squeezing the ginger with a juicer, filter out larger particles with gauze, and centrifuge the collected ginger juice filtrate at 1000g for 10 minutes at 4°C to obtain the first supernatant; then, centrifuge the first supernatant at 3000g for 20 minutes at 4°C to obtain the second supernatant; then, centrifuge the second supernatant at 10000g for 40 minutes at 4°C to obtain the third supernatant; finally, centrifuge the third supernatant at 150000g for 120 minutes at 4°C to collect the bottom precipitate, and resuspend the bottom precipitate in sterile PBS buffer, controlling the mass ratio of the bottom precipitate to the sterile PBS buffer to be 1:1; The solution of the bottom precipitate obtained after differential centrifugation was transferred to a sucrose density gradient solution, and the concentration gradient of the sucrose solution was controlled to be 8% / 15% / 30% / 45% / 60% in sequence. The solution was centrifuged at 150000g for 120 minutes at 4°C, and the bands between the 30% / 45% solution concentration gradient layers were collected to obtain ginger extracellular vesicles. The shogaol content of the ginger extracellular vesicles was 5.88 μg / mg by HPLC.

[0038] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A ginger extracellular vesicle for treating alcoholic liver disease, characterized in that: The content of shogaol in the ginger extracellular vesicles is 8.0-14.0 μg / mg.

2. The ginger extracellular vesicle according to claim 1, characterized in that The ginger extracellular vesicle has a particle size of 50-200 nm and has a double-layer phospholipid layer vesicle structure; the Zeta potential of the ginger extracellular vesicle is -20 to -40 mV.

3. A method for preparing ginger extracellular vesicles according to claim 1 or 2, characterized in that: The method comprises the steps of peeling ginger and squeezing out juice, filtering the juice, and subjecting the filtrate to gradient low-speed centrifugation and ultracentrifugation in sequence to obtain ginger extracellular vesicles.

4. The preparation method according to claim 3, characterized in that The steps of the gradient low-speed centrifugation are: centrifuging the ginger juice filtrate obtained after filtering through gauze at 1000-2000g for 10-30 minutes at 4°C, and collecting the supernatant; centrifuging the supernatant at 3000-6000g for 20-40 minutes at 4°C, and collecting the supernatant; centrifuging the supernatant at 8000-10000g for 40-60 minutes at 4°C, and collecting the supernatant, thereby completing the operation of the gradient low-speed centrifugation.

5. The preparation method according to claim 3, characterized in that: The ultracentrifugation step comprises: centrifuging the supernatant obtained by gradient low-speed centrifugation at 120,000-150,000 g for 90-120 minutes at 4° C., collecting the bottom precipitate with sterile PBS, centrifuging the PBS solution containing the bottom precipitate at 120,000-150,000 g for 90-120 minutes at 4° C., collecting the bottom precipitate with sterile PBS, purifying the ginger extracellular vesicles, and completing the ultracentrifugation operation.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the ginger extracellular vesicles according to claim 1 or 2.

7. Use of any one or a combination of at least two of the ginger extracellular vesicles according to claim 1 or 2, the method for preparing the ginger extracellular vesicles according to any one of claims 3 to 5, and the pharmaceutical composition according to claim 6 in preparing a product for treating alcoholic liver disease.

Images