Ruthenium-dihydromyricetin, ruthenium-dihydromyricetin-loaded apoptosis vesicle as well as preparation method and application of ruthenium-dihydromyricetin-loaded apoptosis vesicle

Abstract

The invention provides ruthenium-dihydromyricetin, ruthenium-dihydromyricetin-loaded apoptosis vesicles as well as a preparation method and application of the ruthenium-dihydromyricetin-loaded apoptosis vesicles, and belongs to the technical field of preparation of antibacterial and anti-inflammatory products. The invention provides ruthenium-dihydromyricetin. The ruthenium-dihydromyricetin is prepared from the following raw materials: 50 to 80 parts of polyvinylpyrrolidone, 20 to 30 parts of ruthenium trichloride and 5 to 15 parts of dihydromyricetin. Ruthenium-dihydromyricetin with good dispersibility is obtained, the method is simple, and the product is uniform in morphology. Besides, the ruthenium-dihydromyricetin disclosed by the invention has antibacterial and anti-inflammatory effects, and has relatively low toxicity and relatively high biological safety in the antibacterial and anti-inflammatory process. According to the prepared ruthenium-dihydromyricetin-loaded apoptosis vesicles, the apoptosis vesicles can cooperate with ruthenium-dihydromyricetin to further improve the treatment effect on bacteria and inflammation, and the use dosage of nano-drugs can be reduced. The ruthenium-dihydromyricetin-loaded apoptosis vesicle disclosed by the invention has a wide application prospect in preparation of antibacterial and anti-inflammatory products.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of antibacterial and anti-inflammatory products, and in particular relates to ruthenium-dihydromyricetin, apoptotic vesicles loaded with ruthenium-dihydromyricetin, and a preparation method and application thereof. Background Art

[0002] Bacterial infection of wounds has a high incidence in clinical practice. Whether it is a surgical incision, skin abrasion or burn, different types of trauma are susceptible to bacterial invasion. When the integrity of the skin or tissue is damaged, bacteria have the opportunity to invade the body and cause infection. These infections not only affect the healing speed of the wound, but also may trigger systemic inflammatory responses, such as sepsis, septicemia, etc., and even threaten the patient's life in severe cases. There are many types of pathogens in bacterial infection wounds, and they have different drug resistance and pathogenicity. Common pathogens include Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, etc., which can enter the body through different pathways, such as wound contamination, blood circulation, etc. These pathogens produce toxins and enzymes during the infection process, destroying the normal structure and function of the tissue, leading to symptoms such as redness, swelling, pain, and exudation of the wound. Antibiotics are the main treatment option for bacterial infections. However, antibiotics are cleared by the blood in the systemic circulation, and the physical barriers of bacterial cell walls and cell membranes hinder the penetration of antibiotics, resulting in low accumulation of antibiotics in bacterial cells. Unfortunately, because there is no effective vaccine against prevalent bacterial pathogens such as Escherichia coli and Staphylococcus aureus, antibiotic prophylaxis is the only clinically available option to prevent most bacterial infections after surgery. Although antibiotics are crucial in combating bacterial infections and eradicating resistant strains, their development is hindered by long cycles, unforeseen side effects, and the emergence of resistance. Therefore, there is an urgent need in the art to provide a strategy to address the clinical challenges of antibiotic use (unsatisfactory therapeutic effects, long-term toxicity, and induction of antibiotic resistance). Summary of the invention

[0003] In view of this, one of the objects of the present invention is to provide a ruthenium-dihydromyricetin having antibacterial and anti-inflammatory effects and a preparation method thereof. The ruthenium-dihydromyricetin prepared by the present invention has lower toxicity and higher biosafety in the antibacterial and anti-inflammatory process.

[0004] The second purpose of the present invention is to provide an apoptotic vesicle loaded with ruthenium-dihydromyricetin and a preparation method thereof. The apoptotic vesicle cooperates with ruthenium-dihydromyricetin to further enhance the therapeutic effect on bacteria and inflammation, and has a high degree of biosafety.

[0005] The third object of the present invention is to provide the use of ruthenium-dihydromyricetin or apoptotic vesicles loaded with ruthenium-dihydromyricetin in the preparation of antibacterial and anti-inflammatory products.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a ruthenium-dihydromyricetin, which comprises the following raw materials in parts by weight: 50-80 parts of polyvinyl pyrrolidone, 20-30 parts of ruthenium trichloride and 5-15 parts of dihydromyricetin.

[0008] The present invention also provides a method for preparing the above-mentioned ruthenium-dihydromyricetin, comprising the following steps: dispersing polyvinyl pyrrolidone, ruthenium trichloride and dihydromyricetin in a liquid solvent respectively to obtain a polyvinyl pyrrolidone solution, a ruthenium trichloride solution and a dihydromyricetin solution; adding the ruthenium trichloride solution to the polyvinyl pyrrolidone solution to obtain a mixed solution; adding the dihydromyricetin solution to the mixed solution and stirring, dialyzing to obtain the dialyzed solution, purifying to obtain the precipitate, washing, and freeze-drying to obtain ruthenium-dihydromyricetin.

[0009] Preferably, the liquid solvent is at least one of water, ethanol, methanol, acetone, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, chloroform, ethylene glycol, glycerol, isopropanol and polyethylene glycol 200.

[0010] Preferably, the weight-to-volume ratio of polyvinyl pyrrolidone to the liquid solvent is 50-80 mg: 1-20 mL; the weight-to-volume ratio of ruthenium trichloride to the liquid solvent is 10-50 mg: 1-20 mL; the weight-to-volume ratio of dihydromyricetin to the liquid solvent is 5-15 mg: 1-5 mL.

[0011] Preferably, the stirring time is more than 1 hour; the dialysis method is dialysis bag dialysis, the molecular weight of the dialysis bag is 6000 to 12000 Daltons, and the dialysis time is 12 to 24 hours.

[0012] Preferably, the purification method includes centrifugation, ultrafiltration or rotary evaporation; the centrifugal speed is 8000-20000 rpm, and the centrifugal time is 5-30 min.

[0013] The present invention also provides a ruthenium-dihydromyricetin-loaded apoptotic vesicle, which is prepared by the following method: adding the above-mentioned ruthenium-dihydromyricetin or the ruthenium-dihydromyricetin prepared by the above-mentioned preparation method to culture during the process of inducing apoptosis of mesenchymal stem cells, collecting the supernatant and centrifuging it to obtain the ruthenium-dihydromyricetin-loaded apoptotic vesicle.

[0014] Preferably, the reagent for inducing apoptosis of mesenchymal stem cells includes staurosporine; when inducing apoptosis of mesenchymal stem cells for 2 to 10 hours, the ruthenium-dihydromyricetin is added; the culture time is 1 to 10 hours; the centrifugal force of the centrifugation is 10,000 to 20,000 g, and the centrifugation time is 10 to 30 minutes.

[0015] Preferably, the mesenchymal stem cells include umbilical cord mesenchymal stem cells.

[0016] The present invention also provides the use of the above-mentioned ruthenium-dihydromyricetin, the ruthenium-dihydromyricetin prepared by the above-mentioned preparation method or the above-mentioned apoptotic vesicles in the preparation of antibacterial and anti-inflammatory products.

[0017] Beneficial effects of the present invention:

[0018] The invention prepares ruthenium-dihydromyricetin with good dispersibility, the method is simple, and the product has uniform morphology. In addition, the ruthenium-dihydromyricetin prepared by the invention has antibacterial and anti-inflammatory effects, and has lower toxicity and higher biological safety in the antibacterial and anti-inflammatory process.

[0019] The apoptotic vesicles loaded with ruthenium-dihydromyricetin prepared by the present invention can cooperate with ruthenium-dihydromyricetin to further improve the therapeutic effect on bacteria and inflammation, and can reduce the dosage of nanomedicine. The apoptotic vesicles come from cells and also have high biosafety. Therefore, the apoptotic vesicles loaded with ruthenium-dihydromyricetin prepared by the present invention have broad application prospects in the preparation of antibacterial and anti-inflammatory products. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the transmission electron microscopy observation result of ruthenium-dihydromyricetin;

[0021] Figure 2 The results of transmission electron microscopy observation of apoptotic vesicles derived from umbilical cord mesenchymal stem cells;

[0022] Figure 3 The transmission electron microscopy observation results of apoptotic vesicles loaded with ruthenium-dihydromyricetin;

[0023] Figure 4 The results of the verification of the enzyme-like activity of ruthenium-dihydromyricetin are shown in the figure on the left for TMB results and the figure on the right for OPD results;

[0024] Figure 5 The free radical scavenging effect of ruthenium-dihydromyricetin under weak alkaline conditions, the left picture is the DPPH result, and the right picture is the ABTS result;

[0025] Figure 6 The results are for the catalase activity of ruthenium-dihydromyricetin;

[0026] Figure 7 The agar plate photographs of the in vitro antibacterial effect, where 1 is the Control group, 2 is the Apovs group, 3 is the Ru-DMY group, and 4 is the Apovs@Ru-DMY group;

[0027] Figure 8Statistical results of in vitro antibacterial effect, where *** indicates P < 0.001;

[0028] Fig. 9 The MI index of macrophages. The first to third rows from top to bottom are the staining results of DAPI, CD86 and Merge, respectively. 1 represents the Control group, 2 represents the LPS group, 3 represents the Apovs group, 4 represents the Ru-DMY group, and 5 represents the Apovs@Ru-DMY group;

[0029] Fig.10 It is the M2 indicator of macrophages. The first to third rows from top to bottom are the staining results of DAPI, CD206 and Merge, respectively. 1 represents the Control group, 2 represents the LPS group, 3 represents the Apovs group, 4 represents the Ru-DMY group, and 5 represents the Apovs@Ru-DMY group. DETAILED DESCRIPTION

[0030] The invention provides a ruthenium-dihydromyricetin, comprising the following raw materials in parts by weight: 50-80 parts of polyvinyl pyrrolidone (PVP), 20-30 parts of ruthenium trichloride (RuCl3), and 5-15 parts of dihydromyricetin.

[0031] The present invention does not specifically limit the specific sources of the above-mentioned raw materials. The ruthenium-dihydromyricetin provided by the present invention is a product of metal-organic coordination, which combines the antibacterial and anti-inflammatory capabilities of metals and natural products, and has good biosafety. In the ruthenium-dihydromyricetin provided by the present invention, the amount of PVP is preferably 55 to 75 parts, more preferably 60 to 70 parts, and further preferably 66 parts; the amount of RuCl3 is preferably 22 to 28 parts, more preferably 24 to 26 parts; the amount of dihydromyricetin is preferably 6 to 12 parts, more preferably 8 to 10 parts.

[0032] The present invention also provides a method for preparing the above-mentioned ruthenium-dihydromyricetin, comprising the following steps: dispersing PVP, RuCl3 and dihydromyricetin in liquid solvents respectively to obtain PVP solution, RuCl3 solution and dihydromyricetin solution; adding RuCl3 solution to PVP solution to obtain a mixed solution; adding dihydromyricetin solution to the mixed solution and stirring, dialyzing to obtain the dialyzed solution, purifying to obtain the precipitate, washing, and freeze-drying to obtain ruthenium-dihydromyricetin.

[0033] In the present invention, the liquid solvent is preferably at least one of water, ethanol, methanol, acetone, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, chloroform, ethylene glycol, glycerol, isopropanol and polyethylene glycol 200. In the present invention, the weight volume ratio of PVP to the liquid solvent is preferably 50-80 mg: 1-20 mL, more preferably 60-70 mg: 3-15 mL, and most preferably 66 mg: 5 mL; the weight volume ratio of RuCl3 to the liquid solvent is preferably 10-50 mg: 1-20 mL, more preferably 20-40 mg: 1-10 mL, and most preferably 30 mg: 1 mL; the weight volume ratio of dihydromyricetin to the liquid solvent is preferably 5-15 mg: 1-5 mL, more preferably 8-12 mg: 1-3 mL, and most preferably 10 mg: 1 mL. In the present invention, when the RuCl3 solution is added to the PVP solution, it is preferably added dropwise. In the present invention, the stirring time is preferably more than 1 hour, more preferably 1 to 100 hours; the dialysis method is preferably dialysis in water, and the dialysis is preferably performed using a dialysis bag, the molecular weight of the dialysis bag is preferably 6000 to 12000 Daltons, more preferably 7000 to 10000 Daltons, and most preferably 8000 Daltons, and the dialysis time is preferably 12 to 24 hours, more preferably 15 to 20 hours. After dialysis and impurity removal, the solution in the dialysis bag is purified, and the purification method preferably includes centrifugation, ultrafiltration or rotary evaporation; the centrifugation is preferably centrifuged at 8000 to 20000 rpm for 5 to 30 minutes, more preferably at 10000 to 15000 rpm for 10 to 15 minutes, and the precipitate is washed. The washing is preferably washed with anhydrous ethanol and water in sequence. In the present invention, the freeze-drying is preferably performed using a vacuum dryer, and the temperature is preferably -60°C.

[0034] The ruthenium-dihydromyricetin prepared by the present invention has good water dispersibility and can be dispersed in liquids such as water, physiological saline, PBS buffer, cell culture medium, etc. when used, and can also be dispersed in hydrogels such as sodium alginate hydrogel and F127 hydrogel.

[0035] The present invention also provides a ruthenium-dihydromyricetin-loaded apoptotic vesicle, which is prepared by the following method: adding the above-mentioned ruthenium-dihydromyricetin or the ruthenium-dihydromyricetin prepared by the above-mentioned preparation method to culture during the process of inducing apoptosis of mesenchymal stem cells, collecting the supernatant and centrifuging it to obtain the ruthenium-dihydromyricetin-loaded apoptotic vesicle.

[0036] In the present invention, the reagent for inducing apoptosis of mesenchymal stem cells preferably includes staurosporine (STS), and the concentration of STS is preferably 500nM; when inducing apoptosis of mesenchymal stem cells for 2 to 10 hours, the ruthenium-dihydromyricetin is added. In the present invention, the mesenchymal stem cells preferably include umbilical cord mesenchymal stem cells, which have the advantages of convenient material collection and wide sources. The culture medium used for the culture is preferably α-MEM culture medium. The present invention does not specifically limit the specific temperature of the culture, and the conventional culture temperature in the art can be used. In the present invention, the amount of the cell apoptosis inducer added is preferably enough to cover all the cells that grow on the bottom of the culture dish. In the present invention, when ruthenium-dihydromyricetin is added for inducing culture, the culture time is preferably 1 to 10 hours, more preferably 2 to 6 hours, and the final concentration of ruthenium-dihydromyricetin is 100 mg / mL. The supernatant is collected and centrifuged. The purpose of the centrifugation is to extract the apoptotic vesicles loaded with ruthenium-dihydromyricetin derived from umbilical cord mesenchymal stem cells. The centrifugal force of the centrifugation is preferably 10000-20000g, more preferably 12000-18000g, and the centrifugation time is preferably 10-30min, more preferably 15-20min.

[0037] The apoptotic vesicles derived from umbilical cord mesenchymal stem cells provided by the present invention have certain therapeutic effects in antibacterial and anti-inflammatory aspects. The apoptotic vesicles derived from umbilical cord mesenchymal stem cells loaded with ruthenium-dihydromyricetin have good therapeutic effects on bacteria and inflammation through synergistic effects, while reducing the dosage of nanomedicines and antibacterial agents, thereby reducing the toxic and side effects of antibacterial agents.

[0038] The present invention also provides the use of the above ruthenium-dihydromyricetin, the ruthenium-dihydromyricetin prepared by the above preparation method or the above apoptotic vesicles in the preparation of antibacterial and anti-inflammatory products. In the present invention, the product preferably includes a drug.

[0039] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0042] Example 1

[0043] A ruthenium-dihydromyricetin is prepared from 66 mg PVP, 30 mg ruthenium trichloride, 10 mg dihydromyricetin and methanol; the preparation method is as follows:

[0044] 66 mg of PVP was dispersed in 5 mL of methanol to form a PVP solution; 30 mg of ruthenium trichloride was dispersed in 1 mL of methanol to form a ruthenium trichloride solution; the ruthenium trichloride solution was added dropwise to the PVP solution to obtain a mixed solution; 10 mg of dihydromyricetin was dispersed in 1 mL of methanol to form a dihydromyricetin solution; the dihydromyricetin solution was added to the mixed solution and stirred for 12 hours, and then dialyzed in water using a dialysis bag with a molecular weight of 8000 Daltons for 12 hours, the solution in the dialysis bag was taken and centrifuged at a speed of 10000 rpm for 5 minutes, the precipitate was washed with anhydrous ethanol and water in sequence for 3 times, and then freeze-dried in a vacuum dryer at -60°C to obtain ruthenium-dihydromyricetin.

[0045] The obtained ruthenium-dihydromyricetin was observed under a transmission electron microscope. Figure 1 shown.

[0046] Example 2

[0047] A ruthenium-dihydromyricetin is prepared from 80 mg of PVP, 20 mg of ruthenium trichloride, 15 mg of dihydromyricetin and methanol; the preparation method is as follows:

[0048] 80 mg of PVP was dispersed in 10 mL of acetone to form a PVP solution; 20 mg of ruthenium trichloride was dispersed in 10 mL of acetone to form a ruthenium trichloride solution; the ruthenium trichloride solution was added dropwise to the PVP solution to obtain a mixed solution; 15 mg of dihydromyricetin was dispersed in 5 mL of acetone to form a dihydromyricetin solution; the dihydromyricetin solution was added to the mixed solution and stirred for 24 hours, and then dialyzed in water using a dialysis bag with a molecular weight of 10,000 Daltons for 24 hours, the solution in the dialysis bag was taken and centrifuged at a speed of 20,000 rpm for 10 minutes, the precipitate was washed twice with anhydrous ethanol and water in sequence, and then freeze-dried in a vacuum dryer at -60°C to obtain ruthenium-dihydromyricetin.

[0049] Example 3

[0050] An apoptotic vesicle loaded with ruthenium-dihydromyricetin is prepared by the following method:

[0051] Umbilical cord mesenchymal stem cells were cultured in α-MEM medium, and then STS was added (STS was added to ensure that all cells on the bottom of the culture dish were covered) to induce apoptosis for 4 hours. Transmission electron microscopy showed that apoptotic vesicles were obtained, such as Figure 2 As shown, the ruthenium-dihydromyricetin prepared in Example 1 was then added (so that the final concentration of ruthenium-dihydromyricetin was 100 mg / mL) and cultured for 2 h. The supernatant was collected and centrifuged at 10000 g for 20 min to obtain apoptotic vesicles loaded with ruthenium-dihydromyricetin. The results of observation under a transmission electron microscope were as follows: Figure 3 shown.

[0052] Example 4

[0053] An apoptotic vesicle loaded with ruthenium-dihydromyricetin is prepared by the following method:

[0054] Umbilical cord mesenchymal stem cells were cultured in α-MEM medium, and then STS was added (STS was added to ensure that all cells growing on the bottom of the culture dish were covered) to induce apoptosis for 6 hours, and then ruthenium-dihydromyricetin prepared in Example 2 was added (so that the final concentration of ruthenium-dihydromyricetin was 100 mg / mL) and cultured for 4 hours, and the supernatant was collected and centrifuged at 18000g for 10 minutes to obtain apoptotic vesicles loaded with ruthenium-dihydromyricetin.

[0055] Example 5

[0056] Example 1: Verification of the enzyme-like activity of the obtained ruthenium-dihydromyricetin

[0057] At pH = 5.5, different concentrations (0, 40, 80, 120, 160, 200 μg / mL) of ruthenium-dihydromyricetin and o-phenylenediamine (OPD) were mixed in PBS saline buffer and incubated at room temperature (25°C) for 0.5 h. The color of the solution was imaged and the UV-Vis-NIR spectrum was measured. The results are shown in Figure 4 As shown in the right figure.

[0058] At pH 5.5, different concentrations (0, 20, 40, 60, 80, 100 μg / mL) of ruthenium-dihydromyricetin and 3,3,5,5-tetramethylbenzidine (TMB) were mixed in PBS saline buffer and incubated at room temperature (25°C) for 0.5 h. The color of the solution was imaged and the UV-Vis-NIR spectra were measured. The results are shown in Figure 4 As shown in the left figure.

[0059] It can be seen that ruthenium-dihydromyricetin has enzyme-like activity, can produce hydroxyl radicals, and has antibacterial properties.

[0060] Example 6

[0061] The free radical scavenging effect of ruthenium-dihydromyricetin obtained in Example 1 under weak alkaline conditions

[0062] DPPH was dissolved in anhydrous ethanol to obtain a DPPH free radical test solution, whose absorption peak was at 519nm. The absorbance at 519nm was measured and adjusted to 0.6-1.0 and it could be used immediately. Ruthenium-dihydromyricetin solution (the solution for dissolving ruthenium-dihydromyricetin was neutral PBS) of different concentrations (0, 1, 2, 3, 4, 5 μg / mL) was added to the DPPH free radical test solution. Then the absorbance was measured at 519nm. The results are as follows: Figure 5 As shown in the left figure.

[0063] The ABTS free radical working solution was diluted with 0.01M PBS (pH 7.4), and its absorbance at 734nm was measured and adjusted to 0.6-1.0. Ruthenium-dihydromyricetin solution of different concentrations (0, 10, 20, 30, 40, 50 μg / mL) was added, and the absorbance was measured at 734nm. The results are shown in Figure 5 As shown in the right figure.

[0064] It can be seen that the ruthenium-dihydromyricetin prepared by the present invention can scavenge nitrogen free radicals and has anti-inflammatory properties.

[0065] Example 7

[0066] Example 1 Catalase (CAT enzyme) activity of ruthenium-dihydromyricetin obtained

[0067] The CAT enzyme activity of ruthenium-dihydromyricetin was determined using a CAT enzyme kit. According to the instructions in the kit, ruthenium-dihydromyricetin solutions of different concentrations (20, 40, 60, 80, 100 μg / mL) were reacted with the test solution at 37°C for 20 minutes. The absorbance was measured at 550 nm to determine the scavenging efficiency of H2O2. The results are shown in Figure 2. Figure 6 As shown. It shows that ruthenium-dihydromyricetin can scavenge hydrogen peroxide to produce oxygen.

[0068] Example 8

[0069] In vitro antibacterial effect of apoptotic vesicles loaded with ruthenium-dihydromyricetin obtained in Example 3

[0070] The plate counting method was used to evaluate the in vitro antibacterial activity of umbilical cord mesenchymal stem cell-derived ruthenium-dihydromyricetin-loaded apoptotic vesicles. The methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli (E.Coli) used were typical Gram-positive and Gram-negative bacteria, respectively. The experiment was divided into the following four groups:

[0071] Control group (denoted as group 1): bacteria were treated with PBS;

[0072] Apovs group (denoted as group 2): apoptotic vesicles (prepared in the same manner as in Example 3, except that ruthenium-dihydromyricetin was not added, and the rest was the same as in Example 3) were added (dissolved in PBS at a concentration of 1.0×10 10 particles / mL);

[0073] Ru-DMY group (denoted as group 3): ruthenium-dihydromyricetin obtained in Example 1 (dissolved in PBS, concentration of 100 mg / mL) was added;

[0074] Apovs@Ru-DMY group (denoted as group 4): apoptotic vesicles loaded with ruthenium-dihydromyricetin obtained in Example 3 (dissolved in PBS, with apoptotic vesicle concentration of 1.0×10 10 particles / mL, ruthenium-dihydromyricetin concentration 100 mg / mL);

[0075] Take the MRSA and E. coli bacterial suspensions and adjust the OD value to 0.6 (1.0×10 7 CFU / mL), prepare different groups of working solutions, add 20μL MRSA bacterial suspension and 20μL E. coli bacterial suspension to 96-well plates, add 180μL of different groups of working solutions to each well, incubate at 37℃ for 4h, dilute the incubated bacterial suspension by 10 3 Times, take 20 μL of the diluted bacterial suspension and add it to the broth agar plate, spread it evenly with glass beads and place it in a 37°C incubator for incubation for 12 h, take out the agar plate and take pictures.

[0076] The results are as follows Figure 7 and Figure 8 As shown, it is shown that the ruthenium-dihydromyricetin and the apoptotic vesicles loaded with ruthenium-dihydromyricetin of the present invention both have significant antibacterial effects, among which the antibacterial effect of the apoptotic vesicles loaded with ruthenium-dihydromyricetin is significantly better than that of the ruthenium-dihydromyricetin group and the simple apoptotic vesicle group. The apoptotic vesicles loaded with ruthenium-dihydromyricetin of the present invention play a synergistic role.

[0077] Example 9

[0078] Example 3 Immunofluorescence staining of mouse mononuclear macrophage leukemia cells (RAW264.7) using ruthenium-dihydromyricetin-loaded apoptotic vesicles derived from umbilical cord mesenchymal stem cells

[0079] The experiments were divided into the following five groups:

[0080] Control group (denoted as group 1): no treatment was given to the cells;

[0081] LPS group (denoted as group 2): LPS at a concentration of 100 ng / mL was added to the cells;

[0082] Apovs group (referred as group 3): LPS group at a concentration of 100 ng / mL was added to the cells, and apoptotic vesicles (preparation method is the same as in Example 3, except that ruthenium-dihydromyricetin is not added, and the rest is the same as in Example 3) were added (dissolved with PBS);

[0083] Ru-DMY group (denoted as group 4): LPS group with a concentration of 100 ng / mL was added to the cells, and ruthenium-dihydromyricetin obtained in Example 1 (dissolved in PBS) was added;

[0084] Apovs@Ru-DMY group (denoted as group 5): LPS at a concentration of 100 ng / mL was added to the cells, and the apoptotic vesicles loaded with ruthenium-dihydromyricetin prepared in Example 3 (dissolved in PBS) were added.

[0085] The results are as follows Fig. 9 and Fig.10 As shown, in Fig. 9 The color becomes lighter from left to right, which means that fewer macrophages are transformed into M1, thus showing a good anti-inflammatory effect. Fig.10 The color gets darker from left to right, which means that more and more macrophages are transformed into M2, thus showing a good anti-inflammatory effect. CD86 is an indicator of M1, and CD206 is an indicator of M2. More macrophages are transformed into M2 and fewer are transformed into M1, indicating that it has a good anti-inflammatory effect. Fig. 9 and Fig.10 It can be seen that the apoptotic vesicle group alone, the ruthenium-dihydromyricetin group alone and the apoptotic vesicle group loaded with ruthenium-dihydromyricetin can all promote the polarization of macrophages to M2 rather than M1, indicating that the ruthenium-dihydromyricetin group and the apoptotic vesicle loaded with ruthenium-dihydromyricetin provided by the present invention both have anti-inflammatory effects.

[0086] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A ruthenium-dihydromyricetin, characterized in that The invention comprises the following raw materials in parts by weight: 50-80 parts of polyvinyl pyrrolidone, 20-30 parts of ruthenium trichloride and 5-15 parts of dihydromyricetin.

2. The method for preparing ruthenium-dihydromyricetin according to claim 1, characterized in that: The method comprises the following steps: dispersing polyvinyl pyrrolidone, ruthenium trichloride and dihydromyricetin in liquid solvents respectively to obtain polyvinyl pyrrolidone solution, ruthenium trichloride solution and dihydromyricetin solution; adding the ruthenium trichloride solution to the polyvinyl pyrrolidone solution to obtain a mixed solution; adding the dihydromyricetin solution to the mixed solution and stirring, dialyzing to obtain the dialyzed solution, purifying to obtain the precipitate, washing, and freeze-drying to obtain ruthenium-dihydromyricetin.

3. The preparation method according to claim 2, characterized in that: The liquid solvent is at least one of water, ethanol, methanol, acetone, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylformamide, dichloromethane, chloroform, ethylene glycol, glycerol, isopropyl alcohol and polyethylene glycol 200.

4. The preparation method according to claim 2, characterized in that: The weight-to-volume ratio of polyvinyl pyrrolidone to the liquid solvent is 50-80 mg: 1-20 mL; the weight-to-volume ratio of ruthenium trichloride to the liquid solvent is 10-50 mg: 1-20 mL; and the weight-to-volume ratio of dihydromyricetin to the liquid solvent is 5-15 mg: 1-5 mL.

5. The preparation method according to claim 2, characterized in that: The stirring time is more than 1 hour; the dialysis method is dialysis bag dialysis, the molecular weight of the dialysis bag is 6000-12000 Daltons, and the dialysis time is 12-24 hours.

6. The preparation method according to claim 2, characterized in that: The purification method includes centrifugation, ultrafiltration or rotary evaporation; the centrifugal speed is 8000-20000 rpm, and the centrifugal time is 5-30 minutes.

7. An apoptotic vesicle loaded with ruthenium-dihydromyricetin, characterized in that: The method is prepared by the following method: during the process of inducing apoptosis of mesenchymal stem cells, ruthenium-dihydromyricetin according to claim 1 or ruthenium-dihydromyricetin prepared by any one of the preparation methods according to claims 2 to 6 is added for culturing, the supernatant is collected and centrifuged to obtain apoptotic vesicles loaded with ruthenium-dihydromyricetin.

8. The apoptotic vesicle according to claim 7, characterized in that The reagent for inducing apoptosis of mesenchymal stem cells includes staurosporine; when inducing apoptosis of mesenchymal stem cells for 2 to 10 hours, the ruthenium-dihydromyricetin is added; the culture time is 1 to 10 hours; the centrifugal force of the centrifugation is 10,000 to 20,000 g, and the centrifugation time is 10 to 30 minutes.

9. The apoptotic vesicle according to claim 7, characterized in that The mesenchymal stem cells include umbilical cord mesenchymal stem cells.

10. Use of the ruthenium-dihydromyricetin according to claim 1, the ruthenium-dihydromyricetin prepared by the preparation method according to any one of claims 2 to 6, or the apoptotic vesicle according to any one of claims 7 to 9 in the preparation of antibacterial and anti-inflammatory products.

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