A cepharanthin-chitosan PLGA nanoemulsion and its preparation method and application

Abstract

The present invention proposes a cephalanthin-chitosan PLGA nanoemulsion and its preparation method and application, which belong to the technical field of preparation of antibacterial and anti-inflammatory drugs. The preparation method includes: PLGA, cephalanthin and an organic solvent are stirred evenly to obtain an oil phase; the inner aqueous phase is added to the oil phase and ultrasonically obtained by water-in-oil type colostrum; chitosan is dissolved in an acid solution to obtain a chitosan solution, and a surfactant is added and stirred evenly to obtain an outer aqueous phase; the water-in-oil type colostrum is added to the outer aqueous phase and ultrasonically stirred to obtain the cephalanthin-chitosan PLGA nanoemulsion. The present invention encapsulates cephalanthin in the chitosan PLGA nanoemulsion, which can not only improve the problems of cephalanthin being difficult to dissolve in water and having a fast metabolic rate, but also chitosan and PLGA can play a synergistic role to enhance the antibacterial and anti-inflammatory effects of cephalanthin.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of antibacterial and anti-inflammatory drugs, and in particular relates to a cepharanthin-chitosan PLGA nanoemulsion and a preparation method and application thereof. Background Art

[0002] Cepharanthine (CEP) is a bis-benzylisoquinoline alkaloid extracted from the genus Cepharanthine. Cepharanthine is a positively charged, amphiphilic molecule with multiple biological activities. As an old drug that has been on the market for more than 70 years, Cepharanthine has a mature production process and is inexpensive. In addition to antiviral effects, there are reports that Cepharanthine has anti-parasitic, anti-tumor, and anti-inflammatory activities. Cepharanthine can also be used to treat a variety of acute and chronic diseases, such as snake bites, alopecia areata, and leukemia, and no serious side effects have been reported. However, the clinical use of Cepharanthine will have problems such as low bioavailability and poor water solubility. Therefore, there is an urgent need to find an effective method to solve this problem.

[0003] Poly(lactic-co-glycolide) (PLGA), also known as poly(lactide-co-glycolide), can be used as a drug delivery system. PLGA nanoparticles can increase drug solubility in solvents, promote drug absorption, enhance drug bioavailability, modify pharmacokinetic properties, effectively reduce drug dosage, and mitigate drug toxicity and side effects. Furthermore, nanoparticles easily penetrate interstitial spaces, retaining in tissues and cells for long periods and slowly releasing drugs, thus holding great potential for drug delivery. Chitosan, a deacetylated form of chitin, exhibits varying antimicrobial activity against fungi, Gram-positive bacteria, and Gram-negative bacteria. Due to its nontoxicity, strong adsorption, biodegradability, and high bioactivity, chitosan has been widely used in the food, pharmaceutical, environmental, and agricultural sectors.

[0004] However, uniform particle size, high encapsulation efficiency, and good stability are the three most important aspects of PLGA nanoparticle preparation, and they also represent the technical challenges that must be overcome in the current research and preparation of PLGA nanoparticles. Beyond these preparation challenges, their application also has certain limitations. First, their preparation techniques require stringent equipment and conditions. Second, when the encapsulated drug is water-soluble, it is prone to leakage, resulting in a low encapsulation efficiency. Finally, the difficulty in controlling the sterilization conditions of the nanoparticles is also a major obstacle to the application of PLGA nanoparticles in other therapeutic approaches.

[0005] Due to the above problems, PLGA is currently rarely used in the preparation of antibacterial drug carriers. However, by combining the advantages of chitosan, the problems of PLGA itself can be greatly improved. However, the presence of chitosan can easily affect the morphology and stability of nanoparticles. Summary of the Invention

[0006] In order to solve the problems of low utilization and poor water solubility of celastrol, the present invention proposes a celastrol-chitosan PLGA nanoemulsion and its preparation method and application. The chitosan PLGA nanoemulsion is prepared by utilizing the good biocompatibility of PLGA and the antibacterial and adhesive properties of chitosan. Then, the celastrol-chitosan PLGA nanoemulsion is obtained by encapsulating celastrol in the chitosan PLGA nanoemulsion. This not only improves the problems of celastrol itself, but also chitosan and PLGA can make up for their respective defects and simultaneously enhance the antibacterial and anti-inflammatory effects of celastrol.

[0007] One of the technical solutions of the present invention:

[0008] A method for preparing a cepharanthin-chitosan PLGA nanoemulsion comprises the following steps:

[0009] (1) PLGA, cepharanthin and an organic solvent are stirred to obtain an oil phase;

[0010] (2) adding the inner water phase to the oil phase and ultrasonically obtaining water-in-oil colostrum;

[0011] (3) dissolving chitosan in an acid solution to obtain a chitosan solution, adding a surfactant and stirring uniformly to obtain an external aqueous phase;

[0012] (4) adding the water-in-oil colostrum into the external aqueous phase and stirring with ultrasonic waves to obtain the cepharanthin-chitosan PLGA nanoemulsion.

[0013] Furthermore, the internal aqueous phase is water.

[0014] Furthermore, the PLGA is carboxyl terminated and has a relative molecular mass of 0.7-2Mw.

[0015] Furthermore, the mass ratio of the PLGA, cepharanthin and chitosan is (1-2): (1-2): (2-3).

[0016] Furthermore, the concentration of the PLGA is 40-50 mg / mL, the concentration of the cepharanthin is 10 mg / mL, and the concentration of the chitosan solution is 2-5 mg / mL. By controlling the concentration of the raw materials, the particle size of the nanoparticles in the cepharanthin-chitosan PLGA nanoemulsion can be effectively maintained, and its antibacterial effect can be maintained at a high level.

[0017] Furthermore, the volume ratio of the inner aqueous phase to the oil phase is 1:(8-12), and the volume ratio of the water-in-oil colostrum to the outer aqueous phase is 1:(10-14).

[0018] Furthermore, the organic solvent is acetone or chloroform, the surfactant is poloxamer 188 or polyvinyl alcohol (PVA), and the acid solution is acetic acid or hydrochloric acid solution.

[0019] Furthermore, in step (1), the stirring rate is 200-300 rpm and the time is 2-3 h.

[0020] Furthermore, in step (2), the ultrasonic power is 80-100W, the ultrasonic time is 3-5min, and the ultrasonication is performed for 2s on and 2s off.

[0021] Furthermore, in step (3), the stirring rate is 200-300 rpm, and the stirring time is 1-2 h.

[0022] Furthermore, in step (4), the ultrasonic power is 120-140W, the ultrasonic time is 3-5min, and the ultrasonication is performed for 2s and then stopped for 2s.

[0023] Furthermore, the ultrasonic process must be performed on ice.

[0024] The second technical solution of the present invention:

[0025] A cephalaenopsisin-chitosan PLGA nanoemulsion prepared by the above preparation method.

[0026] The third technical solution of the present invention:

[0027] The application of the cepharanthin-chitosan PLGA nanoemulsion in the preparation of antibacterial and anti-inflammatory drugs.

[0028] Compared with the prior art, the present invention has the following advantages and technical effects:

[0029] The cephalanthrin-chitosan PLGA nanoemulsion provided by the present invention uses a double emulsification-solvent evaporation method to successfully encapsulate cephalanthrin in the PLGA chitosan nanoemulsion. For the first time, a new antibacterial preparation is constructed by combining the advantages of cephalanthrin and chitosan PLGA nanoemulsion. The cephalanthrin-chitosan PLGA nanoemulsion obtained by the present invention has good stability, high encapsulation efficiency, uniform particle size, a sustained release effect, and improves the bioavailability of cephalanthrin. Compared with the use of cephalanthrin alone, it can effectively improve the solubility of the drug, enhance the antibacterial effect of cephalanthrin, affect the morphological structure of bacteria, and also improve the anti-inflammatory effect of cephalanthrin.

[0030] The present invention can not only provide new ideas for the improvement of traditional Chinese medicine dosage forms, but also provide a material basis for the development of other antibacterial drugs, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of this application. The exemplary embodiments and descriptions of this application are intended to explain this application and do not constitute an improper limitation on this application. In the accompanying drawings:

[0032] Figure 1 These are photos of the appearance of DMSO aqueous solutions of BCPN prepared in Comparative Example 1, CCPN prepared in Example 1, and CEP prepared in Comparative Example 4 at 0, 7, and 28 days, where a represents 0 day, b represents 7 days, and c represents 28 days. A represents BCPN, B represents the DMSO aqueous solution of CEP, and C represents CCPN.

[0033] Figure 2 The test results of Size, Zeta potential and PDI of BCPN prepared in Comparative Example 1 and CCPN prepared in Example 1 at 25° C., wherein A is the test result of particle size and PDI, and B is the test result of Zeta potential;

[0034] Figure 3 The test results of Size, Zeta potential, and PDI of the BCPN prepared in Comparative Example 1 and the CCPN prepared in Example 1 at 4° C. are shown in Figures A and B, respectively, for Size and PDI, and B for Zeta potential.

[0035] Figure 4 The particle size distribution and potential diagrams of BCPN and CCPN, where A and B are the particle size and potential distribution diagrams of BCPN, and C and D are the particle size and potential distribution diagrams of CCPN, respectively;

[0036] Figure 5 This is the antibacterial stability test result of CCPN;

[0037] Figure 6 Transmission electron microscopy images of the CCPN prepared in Example 1 and the BCPN prepared in Comparative Example 1, wherein A and B are BCPN at 5.00k× and 10.00k× magnifications, respectively, and C and D are CCPN at 5.00k× and 10.00k× magnifications, respectively;

[0038] Figure 7 FTIR spectra of CCPN prepared in Example 1, BCPN prepared in Comparative Example 1, and the raw materials CEP, CS, and PLGA used;

[0039] Figure 8 The effect of different concentrations of CCPN on the growth of Staphylococcus aureus;

[0040] Figure 9 The effect of different concentrations of CCPN on the growth of Escherichia coli;

[0041] Figure 10The effect of different concentrations of CCPN on the growth of Salmonella;

[0042] Figure 11 The results of the effect of CCPN prepared in Example 1 on the growth curve of S. aureus ATCC29213 at 2MIC and 4MIC;

[0043] Figure 12 These are scanning electron micrographs of the effect of CCPN prepared in Example 1 on the morphology of S. aureus ATCC29213, wherein A and B are untreated S. aureus at 20.00k× and 30.00k× magnifications, respectively, and C and D are S. aureus treated with CCPN at 20.00k× and 30.00k× magnifications, respectively;

[0044] Figure 13 Transmission electron micrographs showing the effect of CCPN prepared in Example 1 on the morphology of S. aureus ATCC29213, wherein A and B are untreated S. aureus at 10.00k× and 20.00k× magnifications, respectively, and C and D are S. aureus treated with CCPN at 10.00k× and 20.00k× magnifications, respectively;

[0045] Figure 14 The results show the effect of CEP on the activity of RAW264.7 cells;

[0046] Figure 15 The results show the effect of BCPN on the activity of RAW264.7 cells;

[0047] Figure 16 The results show the effect of CCPN on the activity of RAW264.7 cells;

[0048] Figure 17 These are the results of the effect of CCPN prepared in Example 1 of the present invention on the expression levels of IL-1β, IL-6, TNF-α, and IL-10 in RAW264.7 cells after LPS modeling, where a represents IL-1β, b represents IL-6, c represents TNF-α, and d represents IL-10. DETAILED DESCRIPTION

[0049] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0050] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0051] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0052] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0053] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0054] The present invention provides a method for preparing a cephalaenopsis-chitosan PLGA nanoemulsion, comprising the following steps:

[0055] (1) PLGA, cepharanthin and an organic solvent are stirred to obtain an oil phase;

[0056] (2) adding the inner water phase to the oil phase and ultrasonically obtaining water-in-oil colostrum;

[0057] (3) dissolving chitosan in an acid solution to obtain a chitosan solution, adding a surfactant and stirring uniformly to obtain an external aqueous phase;

[0058] (4) adding the water-in-oil colostrum into the external aqueous phase and stirring with ultrasonic waves to obtain the cepharanthin-chitosan PLGA nanoemulsion.

[0059] The preparation conditions of the cepharanthin-chitosan PLGA nanoemulsion directly affect its stability and encapsulation efficiency. Under some conditions, the nanoemulsion becomes flocculent, milky, or stratifies in a very short time, and has a low encapsulation efficiency. Therefore, the present invention optimizes the volume ratio of the inner aqueous phase to the oil phase, the volume ratio of the water-in-oil colostrum to the outer aqueous phase, the raw material concentration and dosage, and the ultrasonic time during the preparation process:

[0060] In some embodiments of the present invention, the internal aqueous phase is distilled water.

[0061] In some embodiments of the present invention, the PLGA is carboxyl-terminated and has a relative molecular mass of 0.7-2 Mw.

[0062] In some embodiments of the present invention, the mass ratio of PLGA to cepharanthin to chitosan is (1-2):(1-2):(2-3).

[0063] The raw material concentration will affect the nanoparticle size of the cepharanthin-chitosan PLGA nanoemulsion. In order to ensure the uniformity of the particle size and at the same time ensure that the nanoemulsion has a high antibacterial effect, in some embodiments of the present invention, the concentration of the PLGA is 40-50 mg / mL, the concentration of the cepharanthin is 10 mg / mL, and the concentration of the chitosan solution is 0.2-0.5 mg / mL.

[0064] In some embodiments of the present invention, the volume ratio of the inner aqueous phase to the oil phase is 1:(8-12), and the volume ratio of the water-in-oil colostrum to the external aqueous phase is 1:(10-14).

[0065] In some embodiments of the present invention, the organic solvent is acetone, the surfactant is poloxamer 188, and the acid solution is acetic acid solution.

[0066] In step (1) of some embodiments of the present invention, the stirring rate is 200-300 rpm and the time is 2-3 h.

[0067] In step (2) of some embodiments of the present invention, the ultrasonic power is 80-100W, the ultrasonic time is 3-5min, and the ultrasonic treatment is performed for 2s on and 2s off.

[0068] In step (3) of some embodiments of the present invention, the stirring rate is 200-300 rpm, and the stirring time is 1-2 h.

[0069] In step (4) of some embodiments of the present invention, the ultrasonic power is 120-140W, the ultrasonic time is 3-5min, and the ultrasonication is performed for 2s and then stopped for 2s.

[0070] In some embodiments of the present invention, due to the continuous heat release of the ultrasonic instrument, the ultrasonic process must be carried out on ice to ensure that the chitosan Not following There is a lot of degradation caused by the decrease in viscosity as the temperature rises.

[0071] The source of the cephalanthrin used in the examples of the present invention is commercial cephalanthrin (CEP, McLean, purity 99.9%), which was prepared according to the concentration requirements in the examples. Polylactic acid-glycolic acid copolymer (PLGA, 50:50, 0.7-2 MW) was purchased from Shandong Academy of Pharmaceutical Sciences, and chitosan (CS, 20-100 mPa / s) was purchased from Shanghai TCI Development Co., Ltd.

[0072] The technical solution of the present invention is further illustrated by the following examples.

[0073] Example 1

[0074] (1) 100 mg of PLGA (50 mg / mL) was added to 2 mL of acetone solution and vortexed. 200 mg of cepharanthin (10 mg / mL) was then added and stirred at 200 rpm for 3 h to obtain an oil phase.

[0075] (2) Add the inner aqueous phase distilled water directly to the above oil phase, with the volume ratio of the inner aqueous phase to the oil phase being 1:9, and immediately sonicate on ice at a power of 90W for 4 minutes, with a 2-second interval and a 2-second pause, to obtain water-in-oil colostrum (PE);

[0076] (3) 1 mL of acetic acid was added to 100 mL of deionized water to prepare a 1% acetic acid solution, 0.3 g of chitosan and 0.9 g of poloxamer 188 (F68) were added, mixed, and fully dissolved on a rotary evaporator to obtain an external aqueous phase;

[0077] (4) Prepare the required amount of water-in-oil colostrum and external aqueous phase according to steps (1) to (3), then add the water-in-oil colostrum to the external aqueous phase, the volume ratio of the water-in-oil colostrum to the external aqueous phase being 1:13, and immediately sonicate on ice at a power of 120W, a sonication time of 4 minutes, and a sonication interval of 2 seconds on and 2 seconds off to obtain a stable water-in-oil-in-water emulsion, place the emulsion on a magnetic stirrer and stir for 6 hours to remove the organic solvent, and obtain a cepharanthin-chitosan PLGA nanoemulsion (CCPN), which is stored in a refrigerator at 4°C.

[0078] Example 2

[0079] (1) 100 mg of PLGA (50 mg / mL) was added to 2 mL of acetone solution and vortexed. 200 mg of cepharanthin (10 mg / mL) was then added and stirred at 200 rpm for 3 h to obtain an oil phase.

[0080] (2) Add the inner aqueous phase distilled water directly to the above oil phase, with the volume ratio of the inner aqueous phase to the oil phase being 1:12, and immediately sonicate on ice at an ultrasonic power of 80w for 3 minutes, with a sonication interval of 2 seconds and a rest period of 2 seconds, to obtain water-in-oil colostrum (PE);

[0081] (3) 1 mL of acetic acid was added to 100 mL of deionized water to prepare a 1% acetic acid solution, 0.2 g of chitosan and 0.9 g of poloxamer 188 (F68) were added, mixed, and fully dissolved on a rotary evaporator to obtain an external aqueous phase;

[0082] (4) Prepare the required amount of water-in-oil colostrum and external aqueous phase according to steps (1) to (3), then add the water-in-oil colostrum to the external aqueous phase, the volume ratio of the water-in-oil colostrum to the external aqueous phase being 1:10, and immediately sonicate on ice at a power of 120W, a sonication time of 3 minutes, and a sonication interval of 2 seconds on and off to obtain a stable water-in-oil-in-water emulsion, place the emulsion on a magnetic stirrer and stir for 6 hours to remove the organic solvent, and obtain a cepharanthin-chitosan PLGA nanoemulsion (CCPN), which is stored in a refrigerator at 4°C.

[0083] Example 3

[0084] (1) 100 mg of PLGA (50 mg / mL) was added to 2 mL of acetone solution and vortexed. 200 mg of cepharanthin (10 mg / mL) was then added and stirred at 300 rpm for 2 h to obtain an oil phase.

[0085] (2) Add the inner aqueous phase distilled water directly to the above oil phase, with the volume ratio of the inner aqueous phase to the oil phase being 1:8, and immediately sonicate on ice at a power of 100 W for 5 min, with an on / off interval of 2 s, to obtain water-in-oil colostrum (PE);

[0086] (3) 1 mL of acetic acid was added to 100 mL of deionized water to prepare a 1% acetic acid solution, 0.4 g of chitosan and 0.9 g of poloxamer 188 (F68) were added, mixed, and fully dissolved on a rotary evaporator to obtain an external aqueous phase;

[0087] (4) Prepare the required amount of water-in-oil colostrum and external aqueous phase according to steps (1) to (3), then add the water-in-oil colostrum to the external aqueous phase, the volume ratio of the water-in-oil colostrum to the external aqueous phase being 1:14, and immediately sonicate on ice at a power of 140W, a sonication time of 5 minutes, and a sonication interval of 2 seconds on and off to obtain a stable water-in-oil-in-water emulsion, place the emulsion on a magnetic stirrer and stir for 6 hours to remove the organic solvent, and obtain a cepharanthin-chitosan PLGA nanoemulsion (CCPN), which is stored in a refrigerator at 4°C.

[0088] Comparative Example 1 (blank chitosan PLGA nanoemulsion)

[0089] The preparation process is the same as that of Example 1, except that cepharanthin is not added. The preparation method is as follows:

[0090] (1) 100 mg of PLGA (50 mg / mL) was added to 2 mL of acetone solution, vortexed, and stirred at 200 rpm for 3 h to obtain the oil phase;

[0091] (2) Add the inner aqueous phase distilled water directly to the above oil phase, with the volume ratio of the inner aqueous phase to the oil phase being 1:9, and immediately sonicate on ice at a power of 90W for 4 minutes, with a 2-second interval and a 2-second pause, to obtain water-in-oil colostrum (PE);

[0092] (3) 1 mL of acetic acid was added to 100 mL of deionized water to prepare a 1% acetic acid solution, 0.3 g of chitosan and 0.9 g of poloxamer 188 (F68) were added, mixed, and fully dissolved on a rotary evaporator to obtain an external aqueous phase;

[0093] (4) Prepare the required amount of water-in-oil colostrum and external aqueous phase according to steps (1) to (3), then add the water-in-oil colostrum to the external aqueous phase, the volume ratio of the water-in-oil colostrum to the external aqueous phase being 1:9, and immediately sonicate on ice at a power of 120W for 4 minutes, with an ultrasonic cycle of 2 seconds and a rest time of 2 seconds to obtain a stable water-in-oil-in-water emulsion, place the emulsion on a magnetic stirrer and stir for 6 hours to remove the organic solvent, and obtain a chitosan PLGA nanoemulsion (BCPN), which is then stored in a refrigerator at 4°C.

[0094] Comparative Example 2

[0095] The same as Example 1, except that 100 mg of PLGA (concentration of 50 mg / mL) was added to 2 mL of acetone solution and vortexed, and then 400 mg of cepharanthin (concentration of 200 mg / mL) was added, and stirred at 200 rpm for 3 h to obtain an oil phase, that is, the mass ratio of PLGA to cepharanthin was 1:4.

[0096] Comparative Example 3

[0097] The same as Example 1, except that in step (4), the volume ratio of the water-in-oil colostrum to the external aqueous phase is 1:13.

[0098] Comparative Example 4

[0099] Preparation of DMSO aqueous solution of CEP (cepharanthin):

[0100] 200 mg / mL of CEP was dissolved in 2 mL of DMSO solution. After the solution was fully dissolved, 2 mL of CEP DMSO solution was added to 200 mL of distilled water and stirred on a magnetic stirrer for 2 h to obtain a DMSO aqueous solution of CEP.

[0101] Performance Testing

[0102] 1. Encapsulation efficiency

[0103] The encapsulation efficiency of the CCPN prepared in Examples 1-3 was detected by high performance liquid chromatography.

[0104] 1. Chromatographic conditions

[0105] The mobile phase was methanol: water: triethylamine (80:20:0.05); the detection wavelength was 283 nm; the column temperature was 30° C.; the volume flow rate was 1.0 mL / min; and the injection volume was 10 μL.

[0106] 2. Determination method:

[0107] The encapsulation efficiency and drug loading of CCPN were determined under the above chromatographic conditions. The prepared CCPN was centrifuged at high speed in a high-speed centrifuge, and the supernatant was carefully collected and diluted to an appropriate concentration. The diluted solution was filtered through a 0.45 μm filter membrane and then tested on the machine.

[0108] Encapsulation efficiency (EE) % = Ce / Cn × 100%

[0109] In the formula, Ce represents the concentration of CEP encapsulated in the nanoparticles; Cn represents the total concentration of CEP, that is, the sum of encapsulated CEP and free CEP.

[0110] Drug loading % = We / Wp × 100%

[0111] In the formula, We represents the mass of CEP encapsulated in the nanoparticles; Wp is the total mass of the drug, that is, the sum of the mass of the carrier and the drug.

[0112] The encapsulation efficiency of CCPN was determined by testing and the results are shown in Table 1.

[0113] Table 1 Encapsulation efficiency determination results

[0114] Encapsulation efficiency / % Drug loading rate / % Example 1 91.48 3.46 Example 2 71.34 2.95 Example 3 78.14 3.01

[0115] 2. Stability

[0116] The appearance of the nanoemulsions prepared in Example 1 and Comparative Example 1 was photographed at 0, 7, and 28 days respectively, and the DMSO aqueous solution of CEP prepared in Comparative Example 4 was used as a control. Figure 1 , Figure 1In the figure, a represents 0 day, b represents 7 days, and c represents 28 days. A represents BCPN, B represents the DMSO aqueous solution of CEP, and C represents CCPN.

[0117] Depend on Figure 1 It can be seen that the CCPN prepared in Example 1 can still maintain a clear emulsion for a long time, while the DMSO aqueous solution of CEP begins to separate after 7 days and becomes a precipitate after 28 days, indicating that CCPN is more stable than CEP.

[0118] 3. Particle size, zeta potential and stability analysis

[0119] The particle size, Zeta potential, PDI (polydispersity index), and anti-Staphylococcus aureus activity (MIC) of the BCPN of Comparative Example 1 and the CCPN prepared in Example 1 were tested on days 0, 7, 14, 21, and 28, respectively.

[0120] Figure 2 The test results of Size, Zeta potential and PDI of BCPN and CCPN at 25°C, where A is the test result of Size and PDI, and B is the test result of Zeta potential;

[0121] Figure 3 These are the Size, Zeta potential, and PDI test results of BCPN and CCPN at 4°C, where A is the Size and PDI test results, and B is the Zeta potential test result.

[0122] Figure 4 Figure 3 is the particle size and potential distribution diagram of BCPN and CCPN, where A and B are the particle size and potential distribution diagrams of BCPN, with a particle size of 494.00±18.61 and a potential of 62.50±0.66; C and D are the particle size and potential distribution diagrams of CCPN, with a particle size of 588.13±31.87 and a potential of 48.60±1.00.

[0123] Figure 5 This is the antibacterial stability test result of CCPN.

[0124] Depend on Figure 2-Figure 5 It can be seen that the CCPN prepared in Example 1 of the present invention has uniform particle size and good antibacterial stability.

[0125] 4. Transmission Electron Microscopy

[0126] The CCPN prepared in Example 1 and the BCPN prepared in Comparative Example 1 were scanned by transmission electron microscopy. Figure 6 , A and B are BCPN under 5.00k× and 10.00k× magnification, respectively, C and D are CCPN under 5.00k× and 10.00k× magnification, respectively.

[0127] Depend on Figure 6 It can be observed that BCPN has a spherical structure with uniform size and a particle size of about 200 nm, and the nanoparticles have a double-layer structure, in which the thicker chitosan layer is the outermost layer. The structure of CCPN after encapsulating the drug does not change significantly and is still a double-layer structure, but the particle size has increased. It can be observed that the CEP particles are encapsulated in the chitosan layer.

[0128] 5. Fourier transform infrared spectroscopy analysis

[0129] The FTIR spectra of CCPN prepared in Example 1, BCPN prepared in Comparative Example 1 and the raw materials CEP, CS and PLGA were measured by micro-infrared spectrometer. Figure 7 .

[0130] Depend on Figure 7 It can be seen that at 1600cm -1 The CS amino group bending vibration peak is located near 1620-1400 cm -1 The absorption peak at is attributed to the stretching vibration peak of the CEP benzene ring. The characteristic absorption peak of CEP here disappears in CCPN, indicating that CEP has been included in the inner cavity of the nanoparticles. Both BCPN and CCPN show the characteristic peak of CS, indicating that chitosan is coated on the outside of the nanoparticles. The characteristic absorption peaks of BCPN and CCPN do not change much, proving that the basic skeleton of the nanoparticles has not changed significantly.

[0131] MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) Analysis

[0132] Specific test process:

[0133] MICs against clinical isolates were determined using the 96-well plate microbroth dilution method, according to the Clinical and Laboratory Standards Institute (CLSI) standard (M100, 2019). The MICs and MBCs of the three drugs were tested against standard strains of Staphylococcus aureus (S. aureus ATCC 29213), Escherichia coli (E. coli ATCC 25922), and Salmonella (S162). BCPN prepared in Comparative Example 1, CCPN prepared in Example 1, and CEP prepared in Comparative Example 4 were prepared in DMSO aqueous solutions to a concentration of 10 mg / ml. The test was performed in a clean bench. 100 μL of MH liquid culture medium was added to all wells of a 96-well plate except the first column, and 200 μL was added to the last column (negative control). 200 μL / well of the diluted drug solution was then added to the first column, and serial two-fold dilutions were performed using a dispenser. No drug was added to the control wells. Finally, 100 μL / well of bacterial solution was added to all wells except the last column. The plates were incubated at 37°C for 16-18 hours, and the MIC results were read. Bacterial growth was observed after a further 48 hours, and the plates were plated for observation. The minimum drug concentration in the wells without sterile growth was the MBC.

[0134] The results are shown in Table 1.

[0135] Table 1 MIC and MBC results of BCPN, CEP solution and CCPN against three standard strains

[0136]

[0137]

[0138] As shown in Table 1, CCPN has a very good antibacterial effect, which is significantly improved compared with BCPN and CEP alone. Among the three standard strains, the bactericidal effect against Staphylococcus aureus is the most obvious.

[0139] VII. Effect of CCPN on Strain Growth Curve

[0140] The specific process of treating Staphylococcus aureus (S. aureus ATCC29213), Escherichia coli (E. coli ATCC25922) and Salmonella (S162) with CCPN prepared in Example 1: The experiment was divided into a drug treatment group and a blank control group. In the drug treatment group, CCPN was diluted with MH medium and then added to a certain amount of bacterial suspension so that the final drug concentrations were 1 / 4MIC, 1 / 2MIC, 1MIC, 2MIC and 4MIC, respectively. The initial bacterial content was 10 6CFU / ml; an equal amount of broth culture medium was used as a blank control (CON); the above culture medium was incubated at 37°C, 120 rpm in a shaker for 24 hours. Samples were taken every 2 hours and the absorbance was measured at 600 nm. A curve was plotted with absorbance as the y-axis and the sampling time as the x-axis. Figure 8 The effect of different concentrations of CCPN on the growth of Staphylococcus aureus is shown in Figure 2. Figure 9 The effect of different concentrations of CCPN on the growth of Escherichia coli is shown in Figure 2. Figure 10 The effect of different concentrations of CCPN on the growth of Salmonella.

[0141] Figures 8-10 As can be seen, the growth curve of Staphylococcus aureus after treatment with CCPN prepared in Example 1 of the present invention undergoes significant changes, and the effect on the growth curve also increases significantly with increasing drug concentration. Between 4 and 12 hours, the bacteria in the blank control group reproduce rapidly and remain in the logarithmic growth phase. When the drug concentration is 1 MIC, the bacterial population increases slowly, indicating that bacterial reproduction is inhibited to a certain extent. However, when the drug concentration is increased to 2 MIC and 4 MIC, the absorbance value of the bacterial solution does not change significantly and is basically consistent with the initial concentration. Among the three bacteria, CCPN has the best bactericidal effect on Staphylococcus aureus, with a strong bactericidal effect at a drug concentration of 1 / 4 MIC, significantly inhibiting its growth.

[0142] 8. Effect of CCPN on the Killing Curve of S. aureus ATCC29213 (Staphylococcus aureus)

[0143] The CCPN prepared in Example 1 of the present invention with 2MIC and 4MIC was used to treat S. aureus ATCC29213 at different time points. The results are shown in FIG. Figure 11 , Figure 11 The results showed that 4MIC of CCPN could completely kill the bacteria in 10 minutes, and 2MIC of CCPN could completely kill the bacteria in 30 minutes, that is, CCPN had a good killing effect on S. aureus ATCC29213.

[0144] IX. Effects of CCPN on the Morphology of S. aureus ATCC29213 (Staphylococcus aureus)

[0145] The scanning electron microscopy images of the effects of CCPN prepared in Example 1 on the morphology of S. aureus ATCC29213 are shown in FIG. Figure 12 , where A and B are untreated Staphylococcus aureus (A: 20.00k×; B: 30.00k×); C and D are Staphylococcus aureus treated with CCPN at MIC concentration (C: 20.00k×; D: 30.00k×). Transmission electron microscopy images are shown in Figure 13 , where A and B are untreated Staphylococcus aureus (A: 10.00k×; B: 20.00k×); C and D are Staphylococcus aureus treated with MIC concentration CCPN (C: 10.00k×; D: 20.00k×).

[0146] Depend on Figure 12 It can be seen that when observing the morphological changes of S. aureus ATCC29213 under a scanning electron microscope, the bacterial morphology and structure of the blank control group were normal, presenting a spherical structure with a smooth surface. However, after being treated with 1MIC concentration of CCPN, it can be seen that most of the Staphylococcus aureus bacterial morphology structures underwent obvious wrinkling and deformation, and most of the bacteria were damaged, indicating that 1MIC concentration of CCPN had a strong destructive effect on the morphological structure of the bacteria.

[0147] Depend on Figure 13 Transmission electron microscopy observations of the morphological changes in S. aureus ATCC29213 after CCPN treatment revealed that the cell structure of the control group was intact, with evenly distributed cell walls, cell membranes, and protoplasts, plump, complete, and smooth. There was no cell damage and no leakage of dissolved substances. However, after exposure to 1 MIC CCPN, the integrity of the bacterial cell structure was disrupted, with damage to the cell walls and cell membranes. As intracellular substances were lost, the cytoplasm shrank, and obvious plasmolysis occurred. The cell walls and cell membranes of some bacterial cells were completely damaged, with cytoplasm leaking out at the ruptured sites. This indicates that the bacterial cell membrane structure was severely damaged by CCPN.

[0148] 10. Effects on RAW264.7 Cell Activity

[0149] The effects of the DMSO aqueous solutions of CCPN prepared in Example 1, BCPN prepared in Comparative Example 1, and CEP prepared in Comparative Example 4 on the activity of RAW264.7 cells were tested. The specific test process was as follows: the mouse RAW264.7 cells were adjusted to 1×10 5 Cells were seeded at 1000 μg / cm² in a 96-well plate and cultured at 37°C in a 5% CO2 atmosphere until adherence. The supernatant was discarded. CCPN was prepared at a concentration of 1000 μg / mL and serially diluted to 3.906 μg / mL. A blank control group was also treated with cell culture medium. After 24 hours of culture, 10 μl of CCK8 was added to each well using the CCK8 kit, and absorbance was measured at 450 nm. Figure 14 The results show the effect of CEP on the activity of RAW264.7 cells. Figure 15 The results show the effect of BCPN on the activity of RAW264.7 cells. Figure 16 The results show the effect of CCPN on the activity of RAW264.7 cells.

[0150] Depend on Figure 14-16 It can be seen that CEP had no significant effect on the survival rate of RAW264.7 cells at a concentration of 0.78125-3.125 μg / mL; BCPN had no significant effect on the survival rate of RAW264.7 cells at a concentration of 0.78125-12.5 μg / mL and CCPN had no significant effect on the survival rate of RAW264.7 cells at a concentration of 0.78125-6.25 μg / mL. Compared with CEP, the toxicity of CCPN was reduced, which can improve the toxicity of CEP to cells when used alone.

[0151] Effects of CCPN on the mRNA Expression Levels of IL-1β, IL-6, TNF-α, and IL-10 in RAW264.7 Cells

[0152] 1. Primer design:

[0153] The gene sequences of IL-1β, IL-6, IL-10, and TNF-α were retrieved from Gene Bank, and the upstream and downstream primers were designed using Primer5 software.

[0154] 2. RNA extraction:

[0155] Mouse RAW264.7 cells were adjusted to 2 × 10 6 / cm 2 Cells were seeded in six-well plates and cultured at 37°C in a 5% CO2 atmosphere. After cell attachment, the supernatant was discarded and CEP (3.125 μg / mL), BCPN (3.125 μg / mL), and CCPN (6.25 μg / mL and 3.125 μg / mL) were added, along with LPS (1 μg / mL) for a positive control group and a blank control group (control group). LPS was also added to each group except the control group at a final concentration of 1 μg / mL. After 24 hours of drug exposure, the supernatant was discarded and the cells were washed once with PBS. 500 μL of RNA-easy was added and the samples were shaken vigorously to fully lyse. 200 μL of RNase-free ddH2O was added to the lysate, the cells were mixed by inversion, and the cells were allowed to stand at room temperature for 5 minutes. The cells were centrifuged at 12,000 × g for 15 minutes at room temperature. 500 μL of the upper aqueous phase was transferred to a new centrifuge tube. 500 μL of isopropanol was added, the cells were mixed by inversion, and the cells were allowed to stand at room temperature for 10 minutes. Centrifuge at 12,000 × g for 10 minutes at room temperature and discard the supernatant. Add 500 μL of 75% ethanol, gently flick the bottom of the tube to resuspend the pellet, and invert the tube several times. Centrifuge at 8,000 × g for 3 minutes at room temperature and discard the supernatant. Repeat twice. Allow to air dry at room temperature. Finally, add 20 μL of RNase-free ddH2O to dissolve the precipitate and vortex for 1 minute to fully dissolve the RNA precipitate. The extracted RNA product can be stored long-term at -80°C, but only short-term at -20°C.

[0156] 3.cDNA preparation:

[0157] The extracted RNA was tested for concentration and purity using a meter. The usable RNA was adjusted to an appropriate concentration. Reverse transcription was then performed according to the reverse transcription kit instructions, using the extracted RNA as a template. The cDNA was stored at -20°C until ready for use.

[0158] 4. RT-PCR assay:

[0159] All cDNA samples were amplified by PCR using GADPH as the internal reference gene. The reaction system and amplification procedure are as follows:

[0160] Table 2 RT-PCR reaction system

[0161] Components Volume (μL) Premixed solution for real-time quantitative PCR amplification 10 Upstream primer (10 μM) 0.4 Downstream primer (10 μM) 0.4 Template DNA 6 Sterile ultrapure water 3.2

[0162] Table 3 RT-PCR amplification procedure

[0163]

[0164] Figure 17 The results show the effect of CCPN prepared in Example 1 of the present invention on the expression levels of IL-1β, IL-6, TNF-α and IL-10 in RAW264.7 cells after LPS modeling, where a represents IL-1β, b represents IL-6, c represents TNF-α, and d represents IL-10.

[0165] Depend on Figure 17 As can be seen, the relative expression levels of inflammation-related gene mRNA in cells after LPS modeling were determined by RT-PCR. Compared with the control group, the mRNA expression levels of IL-1β, IL-6, and TNF-α in the LPS-treated group were significantly increased (P<0.05). After drug treatment, the relative expression levels of related pro-inflammatory gene mRNAs were significantly reduced, with CCPN having the best effect. The pro-inflammatory factor IL-10 showed an opposite trend, with no significant difference compared to the LPS group. These results indicate that CCPN has a significant anti-inflammatory effect, and its anti-inflammatory effect is significantly enhanced compared to the CEP group and BCPN group alone.

[0166] The above are merely preferred embodiments of the present application, but the scope of protection of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A method for preparing a cepharanthin-chitosan PLGA nanoemulsion, characterized in that: The following steps are involved: (1) PLGA was added to 2 mL of acetone solution and vortexed, and then 200 mg of cepharanthin was added and stirred at 200 rpm for 3 h to obtain an oil phase, wherein the concentration of PLGA was 50 mg / mL and the concentration of cepharanthin was 10 mg / mL; (2) Add the inner aqueous phase distilled water directly to the above oil phase, with the volume ratio of the inner aqueous phase to the oil phase being 1:9, and immediately sonicate on ice at a power of 90W for 4 minutes, with a 2-second on / off sonication cycle, to obtain water-in-oil colostrum; (3) 1 mL of acetic acid was added to 100 mL of deionized water to prepare a 1% acetic acid solution, 0.3 g of chitosan and 0.9 g of poloxamer 188 were added, mixed, and fully dissolved on a rotary evaporator to obtain an external aqueous phase; (4) According to steps (1) to (3), the required amount of water-in-oil colostrum and external aqueous phase are prepared, and then the water-in-oil colostrum is added to the external aqueous phase, and the volume ratio of the water-in-oil colostrum to the external aqueous phase is 1:

13. The colostrum is immediately sonicated on ice, with an ultrasonic power of 120W and an ultrasonic time of 4 minutes, with an ultrasonication of 2 seconds on and 2 seconds off, to obtain a stable water-in-oil-in-water emulsion, which is placed on a magnetic stirrer and stirred for 6 hours to remove the organic solvent, thereby obtaining the cepharanthin-chitosan PLGA nanoemulsion; The PLGA is carboxyl-terminated and has a relative molecular mass of 0.7-2Mw.

2. A cepharanthin-chitosan PLGA nanoemulsion prepared by the preparation method according to claim 1.

3. Use of the cepharanthin-chitosan PLGA nanoemulsion according to claim 2 in the preparation of antibacterial and anti-inflammatory drugs.

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