Application of nanovesicles sourced from lysimachia christinae hance in preparation of drugs for preventing and treating atherosclerosis and antitumor drugs

Abstract

The invention provides application of nanovesicles sourced from lithospermum erythrorhizon to preparation of medicines for preventing and treating atherosclerosis and resisting tumors, which comprises the following steps: extracting and separating lithospermum erythrorhizon by crushing or wall breaking and adding an extracting agent to obtain an extracting solution, pre-centrifuging the extracting solution to obtain a supernatant, and performing ultracentrifugation on the supernatant to obtain the nanovesicles sourced from lithospermum erythrorhizon. The obtained precipitate is the nano vesicles from the lysimachia christii.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, relates to a new medicinal use of nanovesicles derived from Allium truncatum, and specifically relates to the use of nanovesicles derived from Allium truncatum in preparing drugs for preventing and treating atherosclerotic diseases and anti-tumor drugs. Background Art

[0002] The scientific name of stone leek is broad-leaved leek, which belongs to the genus Allium of the Liliaceae family. At present, there are many methods for extracting and applying plant exosomes or plant extracellular vesicles, such as ginseng, bitter melon, garlic, etc., but there is no literature introducing the extraction method and application of extracellular vesicles from stone leek.

[0003] In existing reports, some researchers extracted the substances in Allium sibiricum by physical or chemical means such as extraction and ethanol extraction, and then conducted relevant experiments on the tumor cells studied, such as detecting the survival rate of cells through CCK8 or MTT experiments; verifying the migration rate of cells through cell scratch experiments; detecting the cell cycle by flow cytometry; verifying key proteins by Western Blot, etc. However, at present, only CCK8 and MTT experiments are used to verify the survival rate of cells, and the experimental design is not rigorous. Moreover, most people only achieve the phenomenon of anti-tumor growth, without further mechanism and mechanism research.

[0004] In addition, most people use chemical means to extract the various chemical components of Allium sibiricum, and its composition is very complex, without reaching specific components or monomers. Due to the complexity of the components of the extract, the study of the mechanism of anti-tumor has become very difficult. For example, many people have not reached specific miRNA or protein, and use bioinformatics technology to explore its mechanism. It is not convincing to only use phenomena to explain that a certain chemical component has the effect of anti-tumor growth. Then, the extraction method of Allium sibiricum is complicated, time-consuming, labor-intensive and expensive. There is no simple method to extract Allium sibiricum. Finally, no one controls the quality of each extract of Allium sibiricum.

[0005] Therefore, it is meaningful to provide a method for extracting high-purity, high-quality extracellular vesicles from Allium truncatum and to conduct in-depth research on the functions of the extracted extracellular vesicles to explore their application potential in treating diseases. Summary of the invention

[0006] To achieve the above-mentioned purpose, the present invention provides the use of Allium oleiferum-derived nanovesicles (abbreviated as AH-EVLP) in the preparation of drugs for preventing and treating atherosclerotic diseases and anti-tumor drugs.

[0007] The purpose of the present invention is achieved through the following solutions:

[0008] Application of Allium truncatum-derived nanovesicles in the preparation of drugs for preventing and treating atherosclerotic diseases.

[0009] Application of Allium truncatum-derived nanovesicles in the preparation of anti-tumor drugs.

[0010] Furthermore, the method for preparing the Shilecone-derived nanovesicles includes extracting extracellular vesicles from the Shilecone using a continuous gradient high-speed centrifugation method.

[0011] Furthermore, the preparation method of the stone leek derived nanovesicles includes: extracting and separating the stone leek by crushing or breaking the wall and adding an extractant to obtain an extract, pre-centrifuging the extract to obtain a supernatant, and then ultracentrifuging the supernatant to obtain a precipitate, which is the stone leek derived nanovesicles.

[0012] Furthermore, the preparation method of the stone leek-derived nanovesicles includes: washing the stone leek, draining it, cutting it into sections and squeezing out the juice, centrifuging it in a low-speed centrifuge using differential centrifugation, and collecting the supernatant; centrifuging the supernatant using an ultrahigh-speed centrifuge and collecting the precipitate, resuspending the precipitate with PBS, filtering it through a membrane, removing residual non-vesicle large-size particles, and obtaining the stone leek-derived nanovesicles.

[0013] Furthermore, the cleaning method includes cleaning three times with water and then cleaning three times with deionized water.

[0014] Further, the speed and temperature selected by the differential centrifugation method are 4-10°C, 1000-2000g / min (e.g., 1100g, 1200g, 1300g, 1400g, 1500g, 1600g, 1700g, 1800g, 1900g, etc.), 10-20min; 3000-4000g / min (e.g., 3100g, 3200g, 3300g, 3400g, 3500g, etc.), 10-20min; The speed of the load should be 2000-3000 g / min (for example, 15000 g, 16000 g, 17000 g, 18000 g, 19000 g, etc.), 40-50 min; 150000 g / min, 90 min.

[0015] Further, 500 μl of PBS was added to the precipitate for re-suspending, and after re-suspending, the precipitate was filtered using a 0.22 μm filter, and then stored or freeze-dried.

[0016] Furthermore, the obtained Allium truncatum-derived nanovesicles were identified by transmission electron microscopy, Zeta potential and DLS particle size analysis.

[0017] Furthermore, the medicine comprises a therapeutically effective amount of Allium truncatum-derived nanovesicles.

[0018] Furthermore, the drugs are prepared into various pharmaceutical dosage forms by conventional methods, including tablets, capsules, oral liquids, lozenges, granules, pills, pills, suspensions, wine preparations, tinctures, drops and injections.

[0019] Furthermore, the medicine also contains one or more pharmaceutically acceptable carriers or excipients.

[0020] Furthermore, the excipients may include diluents, wetting agents, lubricants, fillers, preservatives, and the like.

[0021] Furthermore, the atherosclerotic disease may include atherosclerosis or coronary heart disease, cerebral infarction, cerebral stroke, myocardial infarction, arrhythmia or peripheral vascular disease caused by atherosclerosis.

[0022] Furthermore, the tumor diseases include liver cancer, brain glioma, cervical cancer, endometrial cancer, etc.

[0023] Compared with the prior art, the technical solution provided by the present invention has the following technical advantages:

[0024] 1. The method provided by the present invention comprises three parts: grinding, centrifugation and purification. The various parameters between the three steps, including the material-liquid ratio, centrifugal force, centrifugation time, experimental temperature, etc., are interrelated and interact with each other, and play a decisive role in the yield and biological activity of the nanovesicles derived from stone leek.

[0025] 2. The technical solution provided by the present invention first adds PBS buffer to the precipitate extracted from the stone leek. Compared with other solutions, the addition of PBS buffer can enable the stone leek-derived nanovesicles to maintain good biological activity.

[0026] 3. The extracellular vesicles prepared by the technical solution provided by the present invention have regular and intact morphology, are quasi-circular under transmission electron microscopy, have high yield, and have a particle size distribution of 100-200 nm, which is relatively concentrated. The results of the nanoparticle tracking analyzer show that the average diameter of the obtained Shileix officinalis-derived nanovesicles is 148.3 nm, and the concentration is 5.4x10 6 Pieces / mL.

[0027] 4. The technical solution provided by the present invention can verify that the nanovesicles derived from Allium truncatum can inhibit the proliferation and migration of glioma cells and cervical cancer cells, and have a certain killing effect on tumor cells.

[0028] 5. The present invention uses Allium sphaerocephalum-derived nanovesicles to conduct in vitro and in vivo experiments and finds that it has a powerful effect of protecting endothelial cells and resisting atherosclerosis, which is specifically manifested in reducing and preventing the blood lipid level and the formation of aortic plaques in atherosclerosis, providing an important basis for the application of Allium sphaerocephalum-derived nanovesicles in the prevention and treatment of atherosclerosis.

[0029] In summary, the method for extracting nanovesicles from stone allium provided by the present invention has a simple operation process, high purity, good overall shape, high purity, concentrated particle size distribution, uniform morphology, and has good research prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to make the technical problems solved by the present invention, the technical means adopted and the technical effects achieved more clearly, the specific embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, it should be noted that the drawings described below are only drawings of exemplary embodiments of the present invention, and those skilled in the art can obtain drawings of other embodiments based on these drawings without creative work.

[0031] Figure 1 This is the experimental flow chart of Example 1.

[0032] Figure 2 This is a morphological diagram of the nanovesicles under a transmission electron microscope in Example 1.

[0033] Figure 3 This is the particle size distribution diagram of the nanovesicles analyzed by the nanoparticle tracer analyzer in Example 1.

[0034] Figure 4 This is the protein blotting of the marker proteins (TSG101, CD81, CD9) of the nanovesicles derived from Allium truncatum in Example 1.

[0035] Figure 5 This is the experimental flow chart of Example 2.

[0036] Fig. 6A Statistical comparison of total cholesterol content measured in liver homogenates of mice in each group in Example 2, *p<0.05.

[0037] Figure 6B Statistical comparison of triglyceride content measured in liver homogenate of each group of mice in Example 2, *p<0.05, **p<0.01.

[0038] Figure 6C Statistical comparison of low-density lipoprotein content measured in liver homogenates of mice in each group in Example 2. *p<0.05, ***p<0.001.

[0039] Fig.6DStatistical comparison of high-density lipoprotein content measured in liver homogenates of mice in each group in Example 2. **p<0.01, ***p<0.001, ****p<0.0001.

[0040] Figure 7 This is an Oil Red O staining image of the mouse aorta blood vessels dissected in Example 2.

[0041] Figure 8 This is an electron micrograph of cells labeled with the lipophilic dye Dil in Example 3. DAPI is a cell nucleus marker, Dil is a marker for nanovesicles derived from Allium sphaerocephalum, and Merge is a combination of the two.

[0042] Fig. 9A This is a CCK8 cell viability test graph of C33A cervical cancer cells in Example 3. The horizontal axis is the addition of different concentrations of leek-derived nanovesicles (0 μg / ml, 50 μg / ml, 75 μg / ml, 100 μg / ml, 150 μg / ml), and the vertical axis is the relative cell viability. ****p<0.0001. .

[0043] Fig. 9B This is a graph of the CCK8 cell viability test of Hela cervical cancer cells in Example 3. The horizontal axis is the addition of different concentrations of leek-derived nanovesicles (0 μg / ml, 50 μg / ml, 75 μg / ml, 100 μg / ml, 150 μg / ml), and the vertical axis is the relative cell viability. ****p<0.0001.

[0044] Fig. 9C This is a graph of the CCK8 cell viability test of normal cervical cells in Example 3. The horizontal axis represents the addition of different concentrations of leek-derived nanovesicles (0 μg / ml, 50 μg / ml, 75 μg / ml, 100 μg / ml, 150 μg / ml), and the vertical axis represents the relative cell viability. Ns p>0.05.

[0045] Fig.10 This is a diagram of the clone formation of C33A and Hela cervical cancer cells in Example 3. NC is the control group, and AH-EVLP is the group with the addition of Allium truncatum-derived nanovesicles.

[0046] Fig.11A The scratch image of C33A cervical cancer cells in Example 3. NC is the control group, and AH-EVLPs is the group with allium-derived nanovesicles. The images were taken under a microscope at 0h, 24h, and 48h. Magnification: 10x.

[0047] Fig. 11BThe scratch image of Hela cervical cancer cells in Example 3. NC is the control group, and AH-EVLPs is the group with allium-derived nanovesicles. The images were taken under a microscope at 0h, 24h, and 48h. Magnification: 10x.

[0048] Fig.12 The figure shows the clone formation of U251, T98G and U87 glioma cells in Example 3. Control is the control group, and AH-EVLPs is the group with Allium truncatum-derived nanovesicles.

[0049] Fig.13A The scratch image of T98G glioma cells in Example 3. Control is the control group, and AH-EVLPs is the group with allium-derived nanovesicles. The images were taken under a microscope at 0h and 48h. Magnification: 4x.

[0050] Fig. 13B The scratch image of U251 glioma cells in Example 3. Control is the control group, and AH-EVLPs is the group with allium-derived nanovesicles. The images were taken under a microscope at 0h and 48h. Magnification: 4x.

[0051] Fig. 13C The scratch image of U87 glioma cells in Example 3. Control is the control group, and AH-EVLPs is the group with allium-derived nanovesicles. The images were taken under a microscope at 0h and 48h. Magnification: 4x. DETAILED DESCRIPTION

[0052] The exemplary embodiments of the present invention will now be described more fully with reference to the accompanying drawings. Although each exemplary embodiment can be implemented in a variety of specific ways, it should not be understood that the present invention is limited to the embodiments set forth herein. On the contrary, these exemplary embodiments are provided to make the content of the present invention more complete and to facilitate the comprehensive communication of the inventive concept to those skilled in the art. Under the premise of being consistent with the technical concept of the present invention, the structure, performance, effect or other features described in a particular embodiment may be combined in any suitable manner into one or more other embodiments.

[0053] In the process of introducing specific embodiments, the detailed description of the structure, performance, effect or other characteristics is to enable those skilled in the art to fully understand the embodiments. However, it does not exclude that those skilled in the art can implement the present invention with a technical solution that does not contain the above-mentioned structure, performance, effect or other characteristics under certain circumstances.

[0054] Although the steps in the present invention are arranged with numbers, they are not used to limit the order of the steps. Unless the order of the steps is clearly stated or the execution of a certain step requires other steps as a basis, the relative order of the steps can be adjusted. It is understood that the term "and / or" used in this article involves and covers any and all possible combinations of one or more of the associated listed items.

[0055] Unless otherwise specified, the chemical reagents and materials in the present invention are purchased from the market.

[0056] One embodiment is the use of nanovesicles derived from Allium oleifera in the preparation of drugs for preventing and treating atherosclerotic diseases.

[0057] Another embodiment is the use of the Allium oleifera-derived nanovesicles in the preparation of anti-tumor drugs.

[0058] In one embodiment, the method for preparing the leek-derived nanovesicles comprises extracting extracellular vesicles from the leek using continuous gradient high-speed centrifugation.

[0059] In one embodiment, the preparation method of the stone leek derived nanovesicles includes: extracting and separating the stone leek by crushing or breaking the wall and adding an extractant to obtain an extract, pre-centrifuging the extract to obtain a supernatant, and then ultracentrifuging the supernatant to obtain the precipitate, which is the stone leek derived nanovesicles.

[0060] In one embodiment, the preparation method of the stone leek derived nanovesicles includes: washing the stone leek, draining it, cutting it into sections and squeezing out the juice, centrifuging it in a low-speed centrifuge using differential centrifugation, and collecting the supernatant; centrifuging the supernatant using an ultrahigh-speed centrifuge and collecting the precipitate, resuspending the precipitate with PBS, filtering it through a membrane, removing residual non-vesicle large-size particles, and obtaining stone leek derived nanovesicles.

[0061] In one embodiment, the cleaning method includes cleaning three times with water and then cleaning three times with deionized water.

[0062] In one embodiment, the speed and temperature selected for the differential centrifugation method are 4-10°C, 1000-2000 g / min, 10-20 min; 3000-4000 g / min, 20-30 min; 10000-20000 g / min, 40-50 min; 150000 / min, 90 min.

[0063] In one embodiment, 500 μl of PBS is added to the precipitate for re-suspending, and after re-suspending, the precipitate is filtered using a 0.22 μm filter and then stored or freeze-dried.

[0064] Example 1: Extraction method of nanovesicles from Allium truncatum

[0065] 1. Experimental Methods (see Figure 1 ):

[0066] Wash the leek three times with water and three times with deionized water, drain and cut into sections to extract juice, centrifuge in a low-speed centrifuge using differential centrifugation (4°C, 1000g / min, 10min; 3000g / min, 20min; 10000g / min, 40min), and take the supernatant. Centrifuge in an ultra-high-speed centrifuge (150000g / min, 90min) and take the colloid around the precipitate, add 500μl PBS to each tube of precipitate to resuspend, filter with a 0.22μm pinhole filter, and store for later use.

[0067] 2. Experimental results:

[0068] Characterization of Nanovesicles from Allium truncatum:

[0069] The morphology, particle size, purity and chemical composition of the nanovesicles extracted in Example 1 were characterized.

[0070] (1) Observe the morphology of nanovesicles under a transmission electron microscope, such as Figure 2 As shown: the surface is concave, showing a typical cup-shaped and disc-shaped vesicle structure, the cell membrane structure is clearly visible, the membrane boundary is clear and sharp, the staining background is clean, and the contrast is obvious.

[0071] (2) Analyze the particle size distribution of nanovesicles using a nanoparticle tracer analyzer, such as Figure 3 As shown: The particle size is 148.3nm, between 100-200nm, which is consistent with the extracellular vesicle particle size range reported in the literature, and the concentration is 5.4x10 6 Pieces / mL.

[0072] (3) Figure 4 The Western blot of the marker proteins of nanovesicles from Allium truncatum (TSG101, CD81, CD9) is shown. The blot is clear and it can be clearly seen that the nanovesicles from Allium truncatum have been successfully extracted.

[0073] Example 2: Biological effects of Allium truncatum-derived nanovesicles in an animal model of atherosclerosis

[0074] 1. Experimental Methods (see Figure 5 ):

[0075] Male C57 mice were used as the control group, with 5 mice in each group, and were given a normal diet. Male 8-week-old APOE knockout mice were fed a high-fat diet for 10 weeks, and their eye blood was taken to measure the levels of their landmark indicators TG, TC, HDL, and LDL, and compared with mice fed only with ordinary feed. The aorta was perfused by heart and the plaques were observed by Oil Red O staining to determine whether the model was successful. After the model was successfully established, it was divided into four groups, and physiological saline and different concentrations of extracellular vesicles from Shilecone (200mg / ml, 400mg / ml, 800mg / ml) were injected into the tail vein once every other day for four weeks. After the end, the levels of TG, TC, HDL, and LDL in the mouse liver, the levels of ICAM1 and VCAM1 mRNA in the aorta, and the area of ​​aortic plaques were detected.

[0076] 2. Experimental Results

[0077] like Fig. 6A -D, the total cholesterol content, triglyceride content, and low-density lipoprotein content in the liver homogenate of mice in each treatment group were lower than those in the single model group, while the high-density lipoprotein content was relatively increased. Figure 7 As shown, according to the Oil Red O staining model of the aorta of mice, the number and area of ​​plaques on the blood vessels of the model group increased, and the number of plaques on the blood vessels of the treatment group decreased compared with the modeling group. This shows that the nanovesicles derived from Shiitake mushrooms have a good improvement effect on arteriosclerosis in mouse blood vessels.

[0078] This indicates that AH-EVLP reduces blood lipid levels and aortic plaque formation in atherosclerotic mice.

[0079] Example 3: Biological effects of Allium truncatum-derived nanovesicles in cervical cancer cell experiments

[0080] The lipophilic dye Dil-labeled AH-EVLP was co-cultured with cervical cancer cells by conventional methods to confirm that AH-EVLP could be internalized, such as Figure 8 As shown, the nanovesicles derived from Allium oleraceum were internalized by C33A and Hela cervical cancer cells. This figure shows that the nanovesicles derived from Allium oleraceum can enter the cervical cancer cells and exert their effects.

[0081] AH-EVLP inhibits the viability of cervical cancer cells but does not affect the viability of normal cervical cells. Fig. 9A As shown in Figure 5-C, AH-EVLP treatment significantly inhibited the CCK8 cell viability of C33A and Hela cervical cancer cells, but did not affect the viability of normal cervical cells.

[0082] At the same time, clone formation experiment and scratch experiment verified that AH-EVLP can inhibit the proliferation and migration of cervical cancer cells. Fig.10As shown in the figure, compared with the control group, the addition of Isoleuca-derived nanovesicles can significantly inhibit the proliferation of C33A and Hela cervical cancer cells. Fig.11A As shown in Figure 2-B, compared with the control group, the addition of Isoleuca-derived nanovesicles can significantly inhibit the migration of C33A and Hela cervical cancer cells. Fig.12 As shown in the figure, compared with the control group, the addition of Allium sphaerocephalum-derived nanovesicles can significantly inhibit the proliferation of U251, T98G, and U87 glioma cells. Fig.13A As shown in Figure 5-C, compared with the control group, the addition of Allium truncatum-derived nanovesicles can significantly inhibit the migration of T98G, U251, and U87 glioma cancer cells.

[0083] The technical features of the above embodiments may be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0084] It should be noted that, for the above-mentioned method embodiments, for the sake of simplicity, they are all described as a series of action combinations, but those skilled in the art should know that the present invention is not limited by the described action sequence, because according to the present invention, certain steps can be performed in other sequences or simultaneously. Secondly, those skilled in the art should also know that the embodiments described in the specification are all preferred embodiments, and the actions and modules involved are not necessarily required by the present invention.

[0085] In the above embodiments, the endpoints and any values ​​of the disclosed ranges are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0086] In the above embodiments, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0087] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

[0088] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application.

[0089] Therefore, the scope of protection of this patent application should be based on the attached claims.

Claims

1. Application of nanovesicles derived from Allium truncatum in the preparation of drugs for the prevention and treatment of atherosclerotic diseases.

2. Application of nanovesicles derived from Allium truncatum in the preparation of anti-tumor drugs.

3. The use according to any one of claims 1 to 2, characterized in that: The preparation method of the leek-derived nanovesicles comprises extracting extracellular vesicles from the leek using a continuous gradient high-speed centrifugation method.

4. The use as claimed in claim 3, characterized in that The preparation method of the nanovesicles derived from the stone leek comprises: extracting and separating the stone leek by crushing or breaking the wall and adding an extractant to obtain an extract, pre-centrifuging the extract to obtain a supernatant, and ultracentrifuging the supernatant to obtain the precipitate, which is the nanovesicles derived from the stone leek.

5. The use as claimed in claim 4, characterized in that The preparation method of the stone leek-derived nanovesicles comprises: washing the stone leek, draining it, cutting it into sections and squeezing out juice, centrifuging it in a low-speed centrifuge using differential centrifugation, and collecting the supernatant; centrifuging the supernatant using an ultrahigh-speed centrifuge and collecting the precipitate, resuspending the precipitate with PBS, filtering it with a membrane, removing residual non-vesicle large-size particles, and obtaining the stone leek-derived nanovesicles.

6. The use as claimed in claim 5, characterized in that The speed and temperature selected for the differential centrifugation method are 4-10°C, 1000-2000g / min, 10-20min; 3000-4000g / min, 20-30min; 10000-20000g / min, 40-50min; 150000g / min, 90min.

7. The use as claimed in claim 5, characterized in that 500 μl of PBS was added to the precipitate for re-suspending, and the precipitate was filtered using a 0.22 μm filter after re-suspending.

8. The use according to any one of claims 1-2, characterized in that: The medicine comprises a therapeutically effective amount of Allium truncatum-derived nanovesicles.

9. The use according to any one of claims 1 to 3, characterized in that: The medicine is prepared into various pharmaceutical dosage forms by conventional methods, including tablets, capsules, oral liquids, lozenges, granules, pills, pills, suspensions, wine preparations, tinctures, drops and injections.

10. The use according to any one of claims 1 to 3, characterized in that: The medicine also contains one or more pharmaceutically acceptable carriers or excipients.

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