Mulberry-derived extracellular vesicle particles, methods of making and using the same

Abstract

The application belongs to the technical field of traditional Chinese medicine exosomes, and particularly relates to mulberry-derived extracellular vesicle particles, a preparation method thereof and application thereof in the preparation of medicines for preventing and treating hyperlipidemia and related symptoms. The mulberry-derived extracellular vesicle particles provided by the application are prepared by using mulberries as raw materials, centrifuging the mulberries at different centrifugal speeds after juice extraction, and then performing density gradient centrifugation with a sucrose solution with a specific density. Experimental research shows that the mulberry-derived extracellular vesicle particles can significantly reduce total cholesterol, triglyceride and low-density lipoprotein in blood, inhibit the formation of aortic plaques caused by hyperlipidemia and lipid deposition in the liver, and are safe and non-toxic, and therefore can be used for the preparation of medicines for preventing and treating hyperlipidemia and related symptoms.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine exosome technology, specifically involving extracellular vesicle particles derived from mulberry, their preparation method, and their application in the preparation of drugs for the prevention and treatment of hyperlipidemia and related symptoms. Background Technology

[0002] High blood lipids pose a significant threat to the cardiovascular, cerebrovascular, and renal blood vessels, leading to atherosclerosis, coronary heart disease, stroke, and other cardiovascular and cerebrovascular diseases. They form the basis for the development of these diseases and can also cause fatty liver. However, the occurrence and development of high blood lipids is insidious and gradual, without immediately presenting obvious clinical symptoms, making it difficult to detect. This makes high blood lipids highly dangerous. Therefore, the prevention and treatment of high blood lipids are particularly important.

[0003] Currently, the main medications for treating hyperlipidemia include fibrates and statins. However, these drugs often cause various side effects. For example, common adverse reactions of fibrates include gastrointestinal reactions, nausea, and diarrhea; in severe cases, they can lead to liver damage. The most common side effects of statins are gastrointestinal reactions and liver dysfunction. Elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) may occur after taking these medications, and muscle toxicity may also occur when used in combination with other lipid-lowering drugs. Therefore, it is necessary to find more safe and effective drugs for the prevention and treatment of hyperlipidemia and related symptoms. Summary of the Invention

[0004] To address the above problems, this invention provides mulberry-derived extracellular vesicle particles (MFEVLPs), their preparation method, and their application in the preparation of drugs for the prevention and treatment of hyperlipidemia and related symptoms. The mulberry-derived extracellular vesicle particles provided by this invention can significantly reduce total cholesterol, triglycerides, and low-density lipoprotein in the blood, and can improve other symptoms caused by hyperlipidemia. Furthermore, they are safe and non-toxic, and can be used to prepare drugs for the prevention and treatment of hyperlipidemia and related symptoms.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides the application of mulberry-derived extracellular vesicle-like particles in the preparation of a drug for the prevention and treatment of hyperlipidemia and related symptoms, wherein the active ingredient of the drug includes the mulberry-derived extracellular vesicle-like particles.

[0007] This invention obtains an extracellular vesicle-like particle from mulberry, and through experiments, it was found that the extracellular vesicle-like particle from mulberry can significantly reduce total cholesterol, triglycerides and low-density lipoprotein in the blood, and is safe and non-toxic, and can be used to make drugs for the prevention and treatment of hyperlipidemia.

[0008] Meanwhile, experiments conducted in this invention have revealed that mulberry-derived extracellular vesicle-like particles have a significant inhibitory effect on the formation of aortic plaques caused by hyperlipidemia, thus delaying the development of atherosclerotic lesions; they can also significantly reduce lipid deposition in the liver caused by hyperlipidemia, thereby delaying hepatic steatosis. Therefore, mulberry-derived extracellular vesicle-like particles can also be formulated into drugs for the prevention and treatment of other symptoms related to hyperlipidemia.

[0009] In conjunction with the first aspect, the aforementioned symptoms are atherosclerosis caused by hyperlipidemia.

[0010] In conjunction with the first aspect, the aforementioned symptoms are due to lipid deposition in the liver caused by hyperlipidemia.

[0011] In conjunction with the first aspect, the dosage form of the drug is an injection or an oral preparation.

[0012] Preferably, the drug for preventing and treating hyperlipidemia and related symptoms further includes pharmaceutically acceptable excipients for injection or oral preparations. Specific excipients and corresponding preparation methods can be conventionally selected according to the specific dosage form of the injection or oral preparation, as long as they do not adversely affect the pharmacological activity and physicochemical stability of the extracellular vesicle-like particles derived from mulberry and meet clinical medication requirements; this invention does not impose any limitations in this regard.

[0013] Secondly, the present invention provides a method for preparing the above-mentioned extracellular vesicle-like granules derived from mulberries, specifically including the following operations:

[0014] After washing the mulberries, juice them and centrifuge them sequentially at 1000xg for 8-12 min, 3000xg for 16-24 min, and 10000xg for 25-35 min. Centrifuge the supernatant at 100,000xg for 80-100 min, resuspend the precipitate in PBS, and centrifuge the sample in a density gradient solution of 8%, 30%, 45%, and 60% wt sucrose at 150,000xg for 2-2.5 h. Collect the middle layer of the 30%-45% sucrose solution, wash away the sucrose with PBS at 150,000xg for 1-1.2 h, discard the supernatant, and the resulting precipitate is the extracellular vesicle-like granules derived from mulberries.

[0015] In conjunction with the second aspect, the centrifugation time at 1000xg is preferably 10min.

[0016] In conjunction with the second aspect, the centrifugation time at 3000xg is preferably 20min.

[0017] In conjunction with the second aspect, the centrifugation time at 10000xg is preferably 30min.

[0018] In conjunction with the second aspect, the centrifugation time at 100,000 x g is preferably 90 min.

[0019] In conjunction with the second aspect, the optimal centrifugation time for the first centrifugation at 150,000 x g is 2 hours.

[0020] In conjunction with the second aspect, the preferred centrifugation time for the second centrifugation at 150,000 x g is 1 hour.

[0021] Thirdly, the present invention provides extracellular vesicle-like particles derived from mulberries, which are prepared according to the above-described preparation method.

[0022] Fourthly, the present invention provides a drug for preventing and treating hyperlipidemia and related symptoms, wherein the active ingredient of the drug includes the above-mentioned extracellular vesicle-like particles derived from mulberry.

[0023] In conjunction with the fourth aspect, the aforementioned related symptoms are atherosclerosis caused by hyperlipidemia.

[0024] In conjunction with the fourth aspect, the related symptoms are caused by lipid deposition in the liver due to hyperlipidemia.

[0025] In conjunction with the fourth aspect, the dosage form of the drug for preventing and treating hyperlipidemia and related symptoms is an injection or oral preparation.

[0026] Preferably, the drug for preventing and treating hyperlipidemia and related symptoms further includes pharmaceutically acceptable excipients for injection or oral preparations. Specific excipients and corresponding preparation methods can be conventionally selected according to the specific dosage form of the injection or oral preparation, as long as they do not adversely affect the pharmacological activity and physicochemical stability of the extracellular vesicle-like particles derived from mulberry and meet clinical medication requirements; this invention does not impose any limitations in this regard.

[0027] The beneficial effects of this invention are as follows: Using mulberry as raw material, the juice is extracted, centrifuged at different speeds, and then subjected to density gradient centrifugation with a sucrose solution of a specific density to obtain extracellular vesicle-like particles capable of preventing and treating hyperlipidemia and related symptoms. Animal experiments have shown that these extracellular vesicle-like particles can significantly reduce total cholesterol, triglycerides, and low-density lipoprotein in the blood, inhibit the formation of aortic plaques caused by hyperlipidemia, and reduce lipid deposition in the liver caused by hyperlipidemia. Furthermore, they are safe and non-toxic, and therefore can be used as an active ingredient in the preparation of drugs for preventing and treating hyperlipidemia and related symptoms. Attached Figure Description

[0028] Figure 1 This is the intermediate layer of the 30%~45% sucrose solution in Example 1 of the present invention;

[0029] Figure 2 The morphology of MFEVLPs in Example 1 of this invention under a transmission electron microscope;

[0030] Figure 3 The changes in body weight of mice in each group in Example 2 of this invention;

[0031] Figure 4 The organ coefficients of each group of mice in Example 2 of this invention;

[0032] Figure 5 The results of serum lipid content in mice in each group in Example 2 of this invention;

[0033] Figure 6 The staining of the thoracic aorta of mice with Sudan IV thoracic tract infection in each group of mice in Example 2 of this invention and the ratio of the area of ​​thoracic aortic plaque to the area of ​​the thoracic aorta.

[0034] Figure 7 The staining status and results of the Sudan IV thoracic aorta in each group of mice in Example 2 of this invention;

[0035] Figure 8 The staining status and results of Oil Red O liver sections from mice in each group in Example 2 of this invention. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0037] High blood lipids can lead to cardiovascular and cerebrovascular diseases such as atherosclerosis, coronary heart disease, and stroke, and can also cause fatty liver. However, high blood lipids are not easily detected, making them highly dangerous and necessitating proactive prevention and treatment. Currently available medications for treating high blood lipids often cause various side effects; therefore, it is necessary to find more safe and effective drugs for the prevention and treatment of high blood lipids and related symptoms.

[0038] Mulberry is one of the traditional Chinese medicines listed in the Chinese Pharmacopoeia. It is used to treat dizziness, vertigo, lower back pain, tinnitus, premature graying of hair, insomnia, thirst due to fluid depletion, diabetes, and constipation caused by liver and kidney deficiency and blood and essence deficiency. In modern pharmacological research, the pharmacological effects of mulberry mainly focus on immune regulation, antioxidation, and anticancer effects. Currently, most research on the active components of mulberry focuses on anthocyanins, resveratrol, rutin, and polysaccharides, and there is no research on extracellular vesicle-like granules. Extracellular vesicle-like granules are membranous vesicles secreted by plant cells, with a lipid bilayer as the basic framework, encapsulating various proteins and nucleic acids and other active substances. Their composition differs significantly from the active components inside plant cells.

[0039] This invention provides an extracellular vesicle-like particle derived from mulberry that has the effect of preventing and treating hyperlipidemia and related symptoms. This extracellular vesicle-like particle can significantly reduce total cholesterol, triglycerides and low-density lipoprotein in the blood, and can be used for the prevention and treatment of hyperlipidemia; it can inhibit the formation of aortic plaques caused by hyperlipidemia, thereby delaying the development of atherosclerosis; it can also reduce lipid deposition in the liver caused by hyperlipidemia, thereby delaying hepatic steatosis.

[0040] Based on this, embodiments of the present invention provide the application of mulberry-derived extracellular vesicle-like particles in the preparation of drugs for the prevention and treatment of hyperlipidemia and related symptoms.

[0041] The present invention also provides a method for preparing mulberry-derived extracellular vesicle-like particles with the above-mentioned effects.

[0042] The present invention will be described below through specific embodiments.

[0043] Example 1

[0044] This embodiment provides a method for preparing extracellular vesicle-like particles (MFEVLPs) derived from mulberries.

[0045] Fresh mulberries (501.5g) were weighed and washed, then juiced using a juicer. The resulting mulberry juice was centrifuged sequentially at 1000xg for 10 min, 3000xg for 20 min, and 10000xg for 30 min at 4℃ using differential centrifugation. The supernatant was collected. The supernatant was then ultracentrifuged at 100,000xg for 90 min, and the precipitate was resuspended in 1 ml PBS. The sample was then centrifuged at 150,000xg for 2 h using density gradient centrifugation in 8%, 30%, 45%, and 60% wt sucrose solutions. The intermediate layer of the 30%–45% sucrose solution (e.g., [missing information]) was collected. Figure 1 As shown), add PBS (1 ml of the above mulberry exudate resuspension plus 28 ml of PBS), centrifuge at 150000xg for 1 h to wash away sucrose, discard the supernatant, and the resulting precipitate is the extracted MFEVLPs. Resuspend the precipitate in 300 μl of PBS pre-cooled at 4℃, collect it in a 1.5 ml EP tube, and store it in a -80℃ refrigerator.

[0046] The isolated MFEVLPs, under a transmission electron microscope, appear as irregularly round, saucer-like structures with one side concave, exhibiting a typical double-membrane structure, similar to the saucer or cup shape of typical exosomes. Figure 2 As shown.

[0047] Example 2

[0048] This embodiment demonstrates the preventive and ameliorative effects of mulberry-derived extracellular vesicle-like particles prepared in Example 1 on dyslipidemia and atherosclerosis.

[0049] 1. Animal experiments

[0050] Select males aged 6-8 weeks with ApoE - / - Mice (strain C57BL / 6J) were randomly divided into a blank control group (Control), a model group (Model), a low-dose MFEVLPs group (MFEVLPs-L), a high-dose MFEVLPs group (MFEVLPs-H), and a positive control group after one week of acclimatization. Mice in the blank control group were fed a standard diet, while mice in the other groups were fed a high-fat diet (21% fat, 0.15% cholesterol, 3.5 g / mouse / day). The experiment lasted for 12 weeks. Mice in the low-dose MFEVLPs group, the high-dose MFEVLPs group, and the positive control group were given prophylactic administration, i.e., MFEVLPs and the positive control drug were administered concurrently with the high-fat diet until the end of week 12. The low-dose MFEVLPs group was administered 5 μg / d (based on the protein content in the MFEVLPs solid-liquid mixture prepared in Example 1; the MFEVLPs solid-liquid mixture was diluted before injection to make an injection volume of 150 μl), and the high-dose MFEVLPs group was administered 50 μg / d (based on the protein content in the MFEVLPs solid-liquid mixture prepared in Example 1; the MFEVLPs solid-liquid mixture was diluted before injection to make an injection volume of 150 μl), both administered intraperitoneally. The positive control group was given atorvastatin at a dose of 10 mg / kg / d by gavage. The model group was intraperitoneally injected with an equal volume (150 μl) of physiological saline.

[0051] 2. Experimental methods:

[0052] (1) Record weight weekly and test organ coefficient after 12 weeks.

[0053] (2) Blood lipid level detection

[0054] Twelve weeks later, after stabilizing the mice, gently press the skin around the eyes to be removed to cause the eyeballs to become congested and protrude. Trim the mice's whiskers with scissors to prevent blood from flowing out and causing hemolysis. Use curved forceps to grasp the eyeballs and quickly remove them, allowing blood to flow from the eye socket into a 1.5 ml EP tube. Let the blood sample stand at room temperature for 30 minutes, then centrifuge at 4°C and 4000 rpm for 15 minutes. Collect the supernatant, aliquot it into serum, and store it at -80°C.

[0055] ApoE was detected using a fully automated biochemical analyzer. - / -The blood lipid levels in mice, namely total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL).

[0056] (3) Sudan IV staining for detection of thoracic aortic plaques

[0057] 1) Aortic sampling

[0058] After blood collection, mice were placed on a board, secured, and their abdomens were dissected to expose the heart. Pre-cooled PBS buffer was infused into the heart from the apex until the liver changed from light red to light white. The brachiocephalic artery, carotid artery, and subclavian artery were isolated, and the abdominal organs were removed, preserving the kidneys and the abdominal aorta along the spine. The adipose tissue surrounding the abdominal aorta and renal arteries was carefully removed. Using forceps, the heart, along with the brachiocephalic artery, carotid artery, subclavian artery, and the entire aorta, was cut along the mouse spine, severing below the bifurcation of the common iliac artery. The pieces were then incubated overnight in 4% fixative.

[0059] 2) Sudan V gross staining of the aorta

[0060] The aorta was removed from formalin, and the heart was separated from the aorta. The aorta was fixed in a black dish containing pre-cooled PBS using acupuncture needles. The surrounding adipose tissue was separated. Spring scissors were inserted into the aortic lumen from the cut in the ascending aorta, and the entire aorta was longitudinally dissected from the greater curvature towards the distal end. The dissected aorta was unfolded with the intima facing upwards and carefully fixed at the vessel edge using acupuncture needles. 500 mL of acetone and 500 mL of 70% ethanol were mixed in a 1 L conical flask, and 5 g of Sudan IV staining solution was added and stirred for 30 minutes until homogeneous. The tissue was infiltrated with Sudan IV staining solution for 15 minutes, and the black dish was placed on a horizontal shaker and slowly and regularly shaken for staining. The tissue was then placed on a horizontal shaker, infiltrated with 80% ethanol solution, and slowly and regularly shaken for 30 minutes to differentiate the stained tissue. The 80% ethanol solution was discarded, and the tissue was rinsed in tap water for 1 hour before being photographed under a stereomicroscope. ImageJ was used to measure and statistically analyze the area of ​​plaques within the aorta and the total area of ​​the aorta, and the ratio of the area of ​​plaques in the thoracic aorta to the total area of ​​the thoracic aorta was calculated.

[0061] (4) Oil Red O staining to detect plaques in aortic root and liver sections

[0062] 1) Frozen section of the aortic root

[0063] The heart was placed in a 25% sucrose solution for dehydration. Dehydration was complete when the heart sank to the bottom of the centrifuge tube. The heart was then removed with forceps and blotted dry with filter paper. Two-thirds of the apical portion was transversely removed using a blade. The remaining one-third was placed cut-side down in the center of a frozen section embedding cassette. OCT was added until the remaining tissue was completely submerged. The embedding cassette was then frozen at -20°C. After complete freezing, it was transferred to a -80°C freezer for storage. The microtome was pre-cooled to -20°C, and sections were cut to a thickness of 8 μm until the entire tricuspid valve was removed. These sections were then stored at -80°C for later use.

[0064] 2) Frozen sections of liver

[0065] Liver frozen sections were prepared from the same location on the liver. After dehydrating the liver tissue in 25% sucrose solution, the sections were embedded in OCT embedding medium and sectioned to a thickness of 5 μm. The sections were stored at -80°C for later use.

[0066] 3) Oil Red O staining of the aortic root and liver

[0067] Staining was performed using a modified Oil Red O staining kit. First, frozen sections were removed and equilibrated at room temperature for 30 minutes. The sections were then rinsed briefly in ultrapure water to remove the embedding agent, rinsed in 60% isopropanol for 25 seconds, stained with Oil Red O staining solution for 15 minutes, washed with 60% isopropanol to remove excess staining solution, rinsed briefly in ultrapure water, stained with hematoxylin for 30 seconds, rinsed with tap water for 10 minutes, blotted dry with filter paper, and then slightly dried before mounting with glycerin gelatin.

[0068] 3. Experimental Results

[0069] (1) Body weight and organ coefficient

[0070] The changes in body weight of mice in each group are as follows: Figure 3 As shown, atorvastatin can cause ApoE. - / - Mice experienced weight loss, while MFEVLPs had an effect on ApoE. - / - The mouse body weight was not affected.

[0071] Organ coefficients of mice in each group are as follows Figure 4 As shown (* indicates p < 0.05 compared to the control group), both atorvastatin and MFEVLPs were effective against ApoE. - / - No adverse effects were observed on the organ coefficient in mice.

[0072] The above results indicate that extracellular vesicle-like particles derived from mulberries are safe and non-toxic.

[0073] (2) Blood lipid level detection

[0074] Serum lipid content results as follows Figure 5As shown in Table 1 (** indicates p < 0.01 compared to the Control group, *** indicates p < 0.001 compared to the Control group; # indicates p < 0.05 compared to the Model group, ## indicates p < 0.01 compared to the Model group), MFEVLPs can improve ApoE induced by a high-fat diet. - / - Mice showed abnormal blood lipids, with decreased levels of TC, TG, and LDL, indicating that MFEVLPs have a lipid-lowering effect.

[0075] Table 1. Comparison of serum TC, TG, and LDL levels in each group (Mean ± SD)

[0076]

[0077] (3) Results of Sudan IV thoracic aortic staining and Oil Red O aortic root staining

[0078] The staining status of the thoracic aorta of Sudan IV mice in each group and the ratio of thoracic aortic plaque area to thoracic aortic area are shown in the figure below. Figure 6 As shown in Table 2 (* indicates p < 0.05 compared to the Control group, ** indicates p < 0.01 compared to the Control group).

[0079] Table 2 Comparison of Sudan IV thoracic aortic staining results (Mean±SD)

[0080]

[0081] Oil Red O staining of the aortic root in each group of mice and the staining results are as follows: Figure 7 As shown in Table 3 (* indicates p < 0.05 compared to the Control group).

[0082] Table 3 Comparison of Oil Red O staining results for the aortic root (Mean±SD)

[0083]

[0084] Analysis combining Sudan IV thoracic aortic staining and Oil Red O aortic root staining showed consistent results: compared with the model group, the plaque area was significantly reduced in the low-dose MFEVLPs group, the high-dose MFEVLPs group, and the Atovastatin group, indicating that MFEVLPs can inhibit the formation of atherosclerotic plaques and delay the development of atherosclerotic lesions.

[0085] (4) Results of Oil Red O staining of liver sections

[0086] The staining status and results of liver sections stained with Oil Red O are as follows: Figure 8 As shown in Table 4 (* indicates p < 0.05 compared to the Control group).

[0087] Table 4 Comparison of Oil Red O staining results for liver sections (Mean±SD)

[0088]

[0089] The results showed that the model mice developed severe macrovesicular steatosis and extensive intracellular lipid accumulation in the liver. The low- and high-dose MFEVLPs groups and the Atovastatin group significantly reduced lipid deposition in the liver and significantly alleviated hepatic steatosis.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of mulberry-derived extracellular vesicle-like granules in the preparation of drugs for the prevention and treatment of hyperlipidemia and related symptoms, characterized in that... The active ingredient of the drug includes the extracellular vesicle-like particles derived from mulberry, and the dosage form of the drug is an injection or oral preparation. The preparation method of the extracellular vesicle-like particles derived from mulberry specifically includes the following steps: Mulberries are washed and juiced, then centrifuged sequentially at 1000xg for 8-12 min, 3000xg for 16-24 min, and 10000xg for 25-35 min. The supernatant is centrifuged at 100,000xg for 80-100 min, and the precipitate is resuspended in PBS. The sample is then centrifuged at 150,000xg for 2-2.5 h using a density gradient centrifugation method in 8%, 30%, 45%, and 60% wt sucrose density gradient solutions. The intermediate layer of the 30%-45% sucrose solution is collected and centrifuged with PBS at 150... Centrifuge at 000xg for 1-1.2h to wash away sucrose, discard the supernatant, and the resulting precipitate is the extracellular vesicle-like particles derived from mulberry; the related symptoms are atherosclerosis caused by hyperlipidemia or liver lipid deposition caused by hyperlipidemia.

2. The application according to claim 1, characterized in that, Specifically, the following steps are included: Centrifugation at 1000 x g for 10 min; and / or Centrifugation at 3000 x g for 20 min; and / or Centrifugation at 10000 x g for 30 min; and / or Centrifugation at 100,000 x g for 90 min; and / or The first centrifugation at 150,000 x g took 2 hours; and / or The second centrifugation at 150,000 x g took 1 hour.

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