Liquid dressing and preparation method thereof

Abstract

The invention relates to the technical field of biological materials, and discloses a liquid dressing and a preparation method thereof, and the liquid dressing comprises a lipid phase and a water phase; the lipid phase comprises lecithin, glyceryl dioleate, polysorbate and an organic solvent; the water phase comprises hyaluronic acid or a salt thereof and a buffer solution, the molecular weight of the hyaluronic acid or the salt thereof is 500-5000Da, and the mass ratio of the hyaluronic acid or the salt thereof to lecithin is (0.05-0.2): 10. The mode of combining the lipid phase and the water phase is adopted, all components in the lipid phase and the water phase have a synergistic effect, free hyaluronic acid or salt thereof is released while the carrier transports hyaluronic acid or salt thereof, the free hyaluronic acid or salt thereof preferentially reaches an action target spot, and then the hyaluronic acid or salt thereof is slowly released under the carrier effect of the lipid phase, so that the hyaluronic acid or the salt thereof is slowly released. According to the present invention, the long-term and lasting protection on the wound part is achieved, the rapid release and slow release combined purpose is achieved, the in vivo immune system is enhanced, the overall cell inflammatory factor level in the body is significantly reduced, and the rapid and continuous pain relieving effect is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, in particular to a liquid dressing and a preparation method thereof. Background Art

[0002] With the continuous advancement of technology and lifestyle, oral health issues are receiving increasing attention. Oral diseases not only cause numerous problems with patients' diet and lifestyle, but can also lead to complications such as sepsis, hypertension, and diabetes. Oral mucositis is a common complication during the treatment of malignant tumors. The incidence of oral mucositis is close to 100% in patients undergoing radiotherapy for head and neck cancers, and 20-40% in patients receiving traditional chemotherapy. Its occurrence is generally believed to be closely related to chemotherapy, molecularly targeted therapy, radiotherapy, and the patient's own risk factors. Chemotherapy-related oral mucositis is the most common complication, typically occurring within 4-7 days after the start of chemotherapy and peaking at 10-14 days. Symptoms include oral mucosal congestion, erythema, edema, erosions, and ulcers of varying degrees. Patients often experience localized pain, difficulty eating, dry mouth, and taste disturbances. Conventional medications for oral mucositis lack rapid pain relief, have slow release rates of active ingredients, and exhibit poor adhesion. Therefore, there is an urgent need for a product that can rapidly release active ingredients with strong adsorption. Summary of the Invention

[0003] In view of this, the present invention provides a liquid dressing and a preparation method thereof to solve the problems that existing drugs for treating oral mucositis cannot quickly relieve pain, have a slow release rate of active ingredients and poor adhesion.

[0004] In order to solve the above problems, the present invention adopts the following technical solutions:

[0005] On the one hand, the present invention provides a liquid dressing comprising a lipid phase and an aqueous phase; wherein the lipid phase comprises lecithin, diolein, polysorbate and an organic solvent; the aqueous phase comprises hyaluronic acid or a salt thereof and a buffer solution, the molecular weight of the hyaluronic acid or its salt is 500-5000 Da, and the mass ratio of the hyaluronic acid or its salt to the lecithin is 0.05-0.2:10.

[0006] Preferably, the mass ratio of the lecithin to the diolein is 10:0.5-2.

[0007] Preferably, the mass ratio of the lecithin to the polysorbate is 10:2-4.

[0008] Preferably, the mass ratio of the total mass of the lecithin, the diolein, and the polysorbate to the organic solvent is 1:5-10.

[0009] Preferably, the lecithin comprises one or more of soybean lecithin, egg yolk lecithin and marine lecithin.

[0010] Preferably, the polysorbate includes one or more of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0011] Preferably, the aqueous phase further comprises a plasticizer, and the mass ratio of the hyaluronic acid or its salt to the plasticizer is 0.01:0.6-5.

[0012] Preferably, the plasticizer includes one or more of glycerol, propylene glycol, sorbitol, castor oil and tributyl citrate.

[0013] Preferably, the aqueous phase further comprises trehalose, and the mass ratio of the hyaluronic acid or its salt to the trehalose is 0.01:1-2.

[0014] Preferably, the amount of the buffer solution added is 80-99% of the total mass of the aqueous phase.

[0015] Preferably, the buffer solution is phosphate buffer.

[0016] Preferably, the hyaluronic acid or its salt includes one or more of hyaluronic acid, sodium hyaluronate, and potassium hyaluronate.

[0017] On the other hand, the present invention also provides a method for preparing any of the above-mentioned liquid dressings, comprising the following steps:

[0018] (1) dissolving lecithin, diolein, and polysorbate in an organic solvent to obtain a lipid phase;

[0019] (2) dissolving hyaluronic acid or a salt thereof in a buffer solution to obtain an aqueous phase;

[0020] (3) mixing the lipid phase and the aqueous phase to obtain mixed micelles;

[0021] (4) Post-processing the mixed micelles to obtain a liquid dressing.

[0022] Preferably, the particle size of the mixed micelles is 80-150 nm, and the polydispersity index PDI is less than 0.25.

[0023] Preferably, the post-treatment includes sterile filtration and freeze-drying; wherein the sterile filtration is performed using a 0.2-0.25 μm filter membrane; and the freeze-drying is performed by pre-freezing at -45 to -35°C for 3 to 5 hours, and then freeze-drying at -55 to -45°C for 20 to 28 hours.

[0024] Preferably, the post-treatment includes sterile filtration and sterilization; the sterile filtration is performed by filtering with a 0.2-0.25 μm filter membrane; the sterilization is performed by γ-ray irradiation under conditions of 20-30 kGy or wet heat sterilization at 120-125° C. for 10-20 min.

[0025] The present invention provides a liquid dressing, which has the following advantages compared with the prior art:

[0026] The present invention adopts a combination of a lipid phase and an aqueous phase. The components in the lipid phase and the aqueous phase act synergistically. While the lipid phase acts as a carrier to transport hyaluronic acid or its salt, free hyaluronic acid or its salt is released, so that it reaches the target site first. The carrier effect of the lipid phase is then used to slowly release the hyaluronic acid or its salt, thereby providing long-term and lasting protection for the wound site, achieving the purpose of combining rapid release with sustained release, enhancing the body's immune system, and significantly reducing the overall level of cellular inflammatory factors in the body, thereby achieving a rapid and sustained analgesic effect.

[0027] Specifically, the present invention uses diolein to optimize adhesion, and the formed film is not easily swallowed by mistake, forming long-term protection, while hyaluronic acid or its salt, lecithin and diolein combine to form a two-phase repair power, effectively enhancing the repair effect; the lecithin of the present invention and diolein synergistically act, greatly enhancing the adsorption of the product, while by loading small molecule (500-5000Da) hyaluronic acid or its salt, utilizing the property that small molecule hyaluronic acid or its salt is quickly penetrated in the product, greatly accelerating the release rate of the drug. In addition, the liquid dressing treatment area of ​​the present invention is large, and is non-drug analgesic, without taboos and side effects. DETAILED DESCRIPTION

[0028] The present invention will be described below by specific embodiment, and it will be appreciated by those skilled in the art that the specific embodiment below is only for illustrative purposes, and does not limit the scope of the present invention in any way. In addition, in the following examples, unless otherwise stated, the reagent and equipment used are all commercially available. If in the following examples, concrete treatment conditions and treatment process are not clearly described, then conditions and methods well known in the art can be adopted to process.

[0029] In one aspect of the present invention, a liquid dressing is provided, comprising a lipid phase and an aqueous phase.

[0030] The present invention adopts a combination of a lipid phase and an aqueous phase. The components in the lipid phase and the aqueous phase act synergistically. While the carrier transports hyaluronic acid or its salt, the free hyaluronic acid or its salt is released, so that it reaches the target site first. The carrier effect of the lipid phase is then used to slowly release the hyaluronic acid or its salt, thereby providing long-term and lasting protection for the wound site, achieving the purpose of combining rapid release with sustained release, enhancing the body's immune system, and significantly reducing the overall level of cellular inflammatory factors in the body, achieving a rapid and sustained analgesic effect. The invention can be used to alleviate and treat oral mucositis, migratory glossitis, etc.

[0031] In some embodiments of the present invention, the lipid phase includes lecithin, diolein, polysorbate, and an organic solvent. Lecithin primarily serves as a film-forming and sustained-release carrier, diolein is used to enhance adsorption and water resistance, and the synergistic effect of diolein and lecithin can further enhance adsorption, and polysorbate acts as a dispersant to prevent liposome aggregation. The lipid phase of the present invention can enhance the product's water-based film-forming properties, provide mechanical protection, and exhibit good adhesion and flexibility.

[0032] The present invention uses diolein to optimize adhesion, and the formed film is not easily swallowed by mistake, forming long-lasting protection. At the same time, hyaluronic acid or its salt, lecithin and diolein combine to form a two-phase repair force, effectively enhancing the repair effect. In addition, the lecithin of the present invention and diolein work synergistically, greatly enhancing the adsorption of the product. At the same time, by loading small molecule hyaluronic acid or its salt, the property of small molecule hyaluronic acid or its salt that quickly penetrates in the product is utilized, greatly accelerating the release rate of the drug.

[0033] In some embodiments of the present invention, the mass ratio of the lecithin to the diolein is 10:0.5-2, specifically 10:0.5, 10:1, 10:1.5, and 10:2; the mass ratio of the lecithin to the polysorbate is 10:2-4, specifically 10:2, 10:3, and 10:4; and the mass ratio of the total mass of the lecithin, diolein, and polysorbate to the organic solvent is 1:5-10, specifically 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10. Under the above ratios, the aqueous phase and the lipid phase can combine better and faster, resulting in a stronger adsorption capacity of the final product and a longer-lasting effect of the resulting liquid dressing.

[0034] In some embodiments of the present invention, the lecithin includes one or more of soybean lecithin, egg yolk lecithin and marine lecithin, preferably soybean lecithin; the polysorbate includes one or more of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80, preferably polysorbate 80; the organic solvent can be any conventional organic solution that can be used as a solvent, for example, ethanol, propanol, dichloromethane, etc., without special limitation.

[0035] In some embodiments of the present invention, the aqueous phase includes hyaluronic acid or its salt and a buffer solution, wherein the hyaluronic acid or its salt is used to accelerate osmotic repair, and the buffer solution is used to maintain the stability of the system.

[0036] In some embodiments of the present invention, the hyaluronic acid or its salt comprises one or more of hyaluronic acid, sodium hyaluronate, and potassium hyaluronate, and the molecular weight of the hyaluronic acid or its salt is 500-5000Da, specifically 500Da, 800Da, 1000Da, 2000Da, and 5000Da, etc. While the lipid phase acts as a carrier to transport the hyaluronic acid or its salt, the free small molecule hyaluronic acid or its salt is released, so that it preferentially reaches the target site, and then the carrier effect of the lipid phase is used to slowly release the hyaluronic acid or its salt, providing long-term and lasting protection to the wound site, achieving the purpose of combining rapid release with sustained release, enhancing the body's immune system, and significantly reducing the overall level of cellular inflammatory factors in the body, achieving a rapid and sustained analgesic effect.

[0037] In some embodiments of the present invention, the mass ratio of the hyaluronic acid or its salt to the lecithin is 0.05-0.2:10, specifically 0.05:10, 0.1:10, 0.15:10, and 0.2:10, etc. By limiting the hyaluronic acid or its salt to this range, the small molecule sodium hyaluronate can quickly reach the target site and continuously produce its effect, ultimately achieving the purpose of combining rapid release with sustained release.

[0038] In some embodiments of the present invention, to improve fluidity, the aqueous phase further includes a plasticizer, which includes one or more of glycerol, propylene glycol, sorbitol, castor oil, and tributyl citrate, preferably glycerol. The mass ratio of the hyaluronic acid or its salt to the plasticizer is 0.01:0.6-5, specifically 0.01:0.6, 0.01:1, 0.01:2, 0.01:3, 0.01:4, and 0.01:5, etc.

[0039] In some embodiments of the present invention, when a lyophilized dosage form is prepared by lyophilization, the aqueous phase further comprises trehalose, and the mass ratio of the hyaluronic acid or its salt to the trehalose is 0.01:1-2, specifically 0.01:1, 0.01:1.5, and 0.01:2. Trehalose, as a lyoprotectant, can ensure the stability and safety of the lyophilized preparation.

[0040] In some embodiments of the present invention, the buffer solution is a phosphate buffer solution having a pH of 6.5-8, preferably a pH of 6.8. The amount of the buffer solution added is 80-99% of the total mass of the aqueous phase, specifically 80%, 85%, 90%, 95%, 99%, etc. A solution using a phosphate buffer solution as the aqueous phase can maintain the stability of the system.

[0041] In another aspect of the present invention, the present invention further provides a method for preparing any of the above-mentioned liquid dressings, comprising the following steps:

[0042] (1) dissolving lecithin, diolein, and polysorbate in an organic solvent to obtain a lipid phase;

[0043] (2) dissolving hyaluronic acid or a salt thereof in a buffer solution to obtain an aqueous phase;

[0044] (3) mixing the lipid phase and the aqueous phase to obtain mixed micelles;

[0045] (4) Post-processing the mixed micelles to obtain a liquid dressing.

[0046] In the present invention, lecithin, diolein and polysorbate are first dissolved in an organic solvent to obtain a lipid phase.

[0047] In some embodiments of the present invention, lecithin, diolein and polysorbate are dissolved in an organic solvent at room temperature or under heating conditions of 50-80°C. It can be understood that heating can accelerate the complete dissolution. Therefore, there is no special limitation on the dissolution temperature, and the dissolution conditions can be selected according to actual conditions.

[0048] In the present invention, hyaluronic acid or a salt thereof is dissolved in a buffer solution to obtain an aqueous phase.

[0049] In some embodiments of the present invention, hyaluronic acid or a salt thereof is dissolved in a buffer solution and stirred until transparent to obtain an aqueous phase.

[0050] In some embodiments of the present invention, when it is necessary to add a plasticizer and trehalose, hyaluronic acid or a salt thereof, the plasticizer and trehalose are mixed and dissolved in a buffer solution, and stirred until transparent to obtain an aqueous phase.

[0051] In the present invention, after the lipid phase and the aqueous phase are obtained, the lipid phase and the aqueous phase are mixed to obtain mixed micelles.

[0052] In some embodiments of the present invention, the lipid phase is slowly injected into the aqueous phase and then shaken to obtain mixed micelles. The injection flow rate is 0.5-2 mL / min, specifically 0.5 mL / min, 1 mL / min, 1.5 mL / min, 2 mL / min, etc.; the aqueous phase is stirred during the injection of the lipid phase into the aqueous phase, and the stirring speed is 300-800 rpm, specifically 300 rpm, 500 rpm, 800 rpm, etc.; in addition, after the lipid phase is completely injected into the aqueous phase, the mixture is shaken for 5-10 minutes, specifically 5 minutes, 8 minutes, 10 minutes, etc., to form mixed micelles with uniform particle size distribution.

[0053] In some embodiments of the present invention, the mixed micelles have a particle size of 80-150 nm, and the polydispersity index (PDI) of the mixed micelles is less than 0.25 as verified by dynamic light scattering. This indicates that the mixed micelles prepared by the present invention have a uniform particle size distribution, which can ensure good film formation uniformity and sustained release effect.

[0054] In the present invention, after the mixed micelles are obtained, the mixed micelles are post-processed to obtain a liquid dressing.

[0055] In some embodiments of the present invention, the mixed micelles are post-processed to obtain a lyophilized liquid dressing or a liquid dressing. The liquid dressing is ready for use and suitable for portable use; the lyophilized liquid dressing is suitable for long-term storage and transportation and can be reconstituted upon use.

[0056] In some embodiments of the present invention, when a liquid dressing in liquid dosage form is obtained, the post-treatment includes sterile filtration and sterilization.

[0057] In order to remove microorganisms and large particles, the sterile filtration is performed using a 0.2-0.25 μm filter membrane, wherein the pore size of the filter membrane can be 0.2 μm, 0.22 μm, and 0.25 μm, etc., and the filter membrane is generally a polyethersulfone (PES) filter membrane. The total aerobic bacteria count in the filtrate after sterile filtration is ≤10 CFU / mL, and the mold and yeast counts are ≤10 CFU / mL.

[0058] The sterilization treatment is γ-ray irradiation or moist heat sterilization, wherein the irradiation dose of the γ-ray irradiation is 20-30 kGy, specifically 20 kGy, 25 kGy and 30 kGy, etc.; the temperature of the moist heat sterilization is 120-125 ° C, specifically 120 ° C, 121 ° C, 123 ° C and 125 ° C, etc., and the moist heat sterilization time is 10-20 minutes, specifically 10 minutes, 15 minutes and 20 minutes, etc. After sterilization, the sterility assurance level (SAL) of the liquid dressing in liquid dosage form is ≤10 -6 .

[0059] In some embodiments of the present invention, when a liquid dressing is obtained, a filling process is further included between the sterile filtration and the sterilization process. The filling process is to fill the sterile filtered filtrate into a pre-sterilized pump spray bottle, and the atomized particle size of the pump head is 50-100 μm.

[0060] It should be noted that liquid dressings in liquid dosage form should be stored below 25°C away from light after moist heat sterilization and should be used up within 30 days after opening.

[0061] In some embodiments of the present invention, when a lyophilized liquid dressing is obtained, the post-treatment comprises sterile filtration and lyophilization performed sequentially.

[0062] In order to remove microorganisms and large particles, the sterile filtration is performed using a 0.2-0.25 μm filter membrane, wherein the pore size of the filter membrane can be 0.2 μm, 0.22 μm, and 0.25 μm, etc., and the filter membrane is generally a polyethersulfone (PES) filter membrane. The total aerobic bacteria count in the filtrate after sterile filtration is ≤10 CFU / mL, and the mold and yeast counts are ≤10 CFU / mL.

[0063] The freeze-drying process is to dispense the aseptically filtered filtrate into pre-sterilized vials, and then sequentially pre-freeze and freeze-dry. The pre-freezing temperature is -45 to -35°C, specifically -45°C, -40°C, and -35°C, and the pre-freezing time is 3-5 hours, specifically 3 hours, 4 hours, and 5 hours; the freeze-drying temperature is -55 to -45°C, specifically -55°C, -50°C, and -45°C, and the freeze-drying time is 20-28 hours, specifically 20 hours, 22 hours, 24 hours, 26 hours, and 28 hours; the vacuum degree is 5-10 Pa, specifically 5 Pa, 8 Pa, and 10 Pa, to obtain a white freeze-dried powder with a moisture content of less than 2% after freeze-drying. The vials are sealed with aluminum-plastic caps and stored in a dark environment below 25°C.

[0064] The biosafety and microbial limits of the liquid dressing were tested. The test results showed that the cytotoxicity of the liquid dressing (ISO 10993-5) was greater than 95% in human gingival fibroblast survival; the mucosal irritation (ISO 10993-10) was less than or equal to erythema or edema in a rabbit oral model; the total aerobic bacterial count was less than or equal to 10 CFU / mL, and the mold and yeast counts were less than or equal to 10 CFU / mL (Chinese Pharmacopoeia).

[0065] The following will be combined with specific embodiments to clearly and completely describe the technical solutions of the present invention. The embodiments of this application are only for example, and all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0066] Example 1

[0067] This embodiment provides a method for preparing a liquid dressing, and the specific steps are as follows:

[0068] (1) 1 g of lecithin, 0.1 g of diolein, 0.3 g of polysorbate 80, and 10.6 g of ethanol were mixed and heated in a 60°C water bath until completely dissolved to obtain a lipid phase;

[0069] (2) 0.01 g of hydrolyzed sodium hyaluronate (600 Da), 1.5 g of trehalose, and 4.5 g of glycerol were mixed and dissolved in 43.99 g of phosphate buffer (pH 6.8), and stirred until transparent to obtain an aqueous phase;

[0070] (3) The lipid phase was injected into the aqueous phase at a rate of 1 mL / min while stirring at 500 rpm. After all the lipid phase was injected into the aqueous phase, the mixture was shaken for 5 min to obtain mixed micelles with a particle size of 80-150 nm. The particle size distribution (PDI) of the mixed micelles was verified by dynamic light scattering to be <0.25.

[0071] (4) The mixed micelles were filtered using a 0.22 μm PES filter membrane, and the filtrate was dispensed into pre-sterilized 2 mL vials, frozen at -40 °C for 4 h, and then vacuum freeze-dried at 10 Pa and -50 °C for 24 h to obtain a white lyophilized powder, which was the liquid dressing.

[0072] Example 2

[0073] This embodiment provides a method for preparing a liquid dressing, and the specific steps are as follows:

[0074] (1) 1 g of egg yolk phospholipids, 0.2 g of diolein, 0.4 g of polysorbate 80, and 12.4 g of ethanol were mixed and heated in a 70°C water bath until completely dissolved to obtain a lipid phase;

[0075] (2) 0.05 g of hydrolyzed sodium hyaluronate (600 Da) and 3 g of castor oil were mixed and dissolved in 46.95 g of phosphate buffer (pH 6.8), and stirred until transparent to obtain an aqueous phase;

[0076] (3) The lipid phase was injected into the aqueous phase at a rate of 1.5 mL / min while stirring at 600 rpm. After the lipid phase was completely injected into the aqueous phase, the mixture was shaken for 6 min to obtain mixed micelles with a particle size of 80-150 nm. The particle size distribution (PDI) of the mixed micelles was verified by dynamic light scattering to be <0.25.

[0077] (4) The mixed micelles were filtered using a 0.22 μm PES filter membrane, and the filtrate was filled into a pre-sterilized pump spray bottle (10 mL / bottle) with a pump head atomizing particle size of 50-100 μm. The mixture was then irradiated with gamma rays (25 kGy) to obtain a liquid dressing.

[0078] Example 3

[0079] This embodiment provides a method for preparing a liquid dressing, and the specific steps are as follows:

[0080] (1) 1 g of lecithin, 0.05 g of diolein, 0.2 g of polysorbate 80, and 12 g of ethanol were mixed and stirred until completely dissolved to obtain a lipid phase;

[0081] (2) 0.05 g of hydrolyzed sodium hyaluronate (600 Da) and 5 g of glycerol were mixed and dissolved in 44.95 g of phosphate buffer (pH 6.8), and stirred until transparent to obtain an aqueous phase;

[0082] (3) The lipid phase was injected into the aqueous phase at a rate of 0.5 mL / min while stirring at 300 rpm. After the lipid phase was completely injected into the aqueous phase, the mixture was shaken for 5 min to obtain mixed micelles with a particle size of 80-150 nm. The particle size distribution (PDI) of the mixed micelles was verified by dynamic light scattering to be <0.25.

[0083] (4) The mixed micelles were filtered using a 0.22 μm PES filter membrane, and the filtrate was filled into a pre-sterilized pump spray bottle (10 mL / bottle) with a pump head atomizing particle size of 50-100 μm. The mixture was then sterilized by wet heat at 121 °C for 15 min to obtain a liquid dressing.

[0084] Comparative Example 1

[0085] This comparative example is basically the same as Example 1, the only difference being step (2).

[0086] 0.06 g of hydrolyzed sodium hyaluronate (600 Da), 1.5 g of trehalose, and 4.5 g of glycerol were mixed and dissolved in 43.94 g of phosphate buffer (pH 6.8), and stirred until transparent to obtain an aqueous phase.

[0087] Comparative Example 2

[0088] This comparative example is substantially the same as Example 1, with the only difference being that diolein is replaced by monoolein.

[0089] Comparative Example 3

[0090] This comparative example is basically the same as Example 1, and the only difference is that step (1) is different.

[0091] 1 g of lecithin, 0.1 g of diolein and 10.9 g of ethanol were mixed and heated in a 60° C. water bath until completely dissolved to obtain a lipid phase.

[0092] Comparative Example 4

[0093] This comparative example is basically the same as Example 1, the only difference being step (2).

[0094] 0.01 g of hydrolyzed sodium hyaluronate (10,000 Da), 1.5 g of trehalose, and 4.5 g of glycerol were mixed and dissolved in 43.99 g of phosphate buffer (pH 6.8), and stirred until transparent to obtain an aqueous phase.

[0095] Comparative Example 5

[0096] This comparative example is substantially the same as Example 1, the only difference being that hydrolyzed sodium hyaluronate is replaced with metronidazole.

[0097] Comparative Example 6

[0098] This comparative example provides a method for preparing a liquid dressing, and the specific steps are as follows:

[0099] (1) Dissolve 0.1 g of chitosan in 0.5 mL of lactic acid to form a homogeneous solution;

[0100] (2) Add 0.05 g of polyvinyl alcohol and 0.05 mL of glycerol to the homogeneous solution, stir evenly, and then add water to make up to 10 mL to obtain a liquid dressing.

[0101] The liquid dressing was tested for cell viability, DPPH free radical scavenging ability, cellular inflammatory factors and adsorption.

[0102] 1. Cell Viability Assay

[0103] The MTT assay was used to examine the effects of the liquid dressings prepared in Examples 1-3 and Comparative Examples 1-5 on macrophage viability. Macrophages from healthy rats were cultured in RPMI 1640 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) in a 37°C, 5% CO2 incubator. When the cell density reached 80-90%, trypsinization was performed. After cell counting, the cells were plated at 1×10 4 Cells were seeded at a density of 100 μl / well in a 96-well plate. After cell attachment, the medium was replaced with a serum-free medium containing 1 mL of liquid dressing, curcumin, and epigallocatechin gallate (ECCG) (no liquid dressing was added to the blank control group). The 96-well plate was incubated in an incubator for 4 hours. After washing with sterile phosphate-buffered saline (PBS), serum-free medium containing 2 μg / mL lipopolysaccharide (LPS) was added for 12 hours. The old medium was discarded, and 100 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 0.5 mg / mL) solution was added to each well. The 96-well plate was placed in an incubator and incubated for another 4 hours. The reaction solution was carefully aspirated and discarded. To fully dissolve the blue-purple formazan crystals, add 100 μL of dimethyl sulfoxide to each well. Place the 96-well plate on a shaker for 10 minutes. Measure the OD value using a multifunctional microplate reader at a wavelength of 560 nm. Calculate macrophage viability according to the following formula. Perform three tests on each group, and calculate the average value.

[0104] Cell viability = A 实验 / A 对照 ×100%,A 实验 is the absorbance of the experimental group; A 对照 is the absorbance of the control group.

[0105] Macrophages were treated with the liquid dressings of Examples 1-3 and Comparative Examples 1-5 for 4 hours, respectively. The results are shown in Table 1.

[0106] Table 1

[0107]

[0108]

[0109] It can be seen from Table 1 that the liquid dressing prepared by the present invention has a significant improvement in cell viability, indicating that the liquid dressing of the present invention has a good anti-inflammatory effect.

[0110] 2. Determination of DPPH free radical scavenging ability

[0111] Accurately weigh an appropriate amount of 2,2-diphenyl-1-picrohydrazine (DPPH) powder and dissolve it in a 75% volume fraction ethanol solution to prepare a 0.1 mol / L DPPH solution, and store it in the dark. Take 1 mL of each liquid dressing of Example 1-3 and Comparative Example 1-5 and add it to a centrifuge tube, then add 2 mL of DPPH solution respectively, mix well and place in a dark place, and react at room temperature for 30 minutes. Centrifuge at 3000 r / min for 10 minutes, use a spectrophotometer to measure the absorbance A of the supernatant at a wavelength of 517 nm. Add 1 mL of distilled water to 2 mL of DPPH solution as a blank sample, and measure the absorbance A 空白 ; The absorbance of the sample without adding DPPH solution was measured 样品 The calculation method of DPPH free radical scavenging rate is shown in the following formula. Each group was tested three times and the average value was taken. The test results are shown in Table 2.

[0112] DPPH free radical scavenging rate = [1-(AA 样品 ) / A 空白 ]×100%

[0113] Table 2

[0114] Group DPPH free radical scavenging rate (%) Example 1 <![CDATA[71.30±0.85 a ]]> Example 2 <![CDATA[72.00±0.24 a ]]> Example 3 <![CDATA[71.73±0.29 a ]]> Comparative Example 1 <![CDATA[44.00±0.47 c ]]> Comparative Example 2 <![CDATA[46.40±1.21 b ]]> Comparative Example 3 <![CDATA[38.47±0.95 e ]]> Comparative Example 4 <![CDATA[22.67±1.11 f ]]> Comparative Example 5 <![CDATA[41.23±0.81 d ]]>

[0115] As shown in Table 2, the liquid dressing prepared by the present invention has a scavenging rate of DPPH free radicals of more than 70%, and the liquid dressing of the present invention has good antioxidant activity.

[0116] 3. Detection of cellular inflammatory factors

[0117] (1) RNA extraction and reverse transcription

[0118] After LPS treatment of the cells for 12 hours, the macrophages were removed from the 37°C incubator, 1 mL of Trizol reagent was added, and RNA was extracted according to the instructions. Cell RNA was adjusted to a uniform concentration with double-distilled water. According to the instructions of the mRNA reverse transcription kit of Kangwei Century Company, the sample was diluted to the required concentration and mixed with the reverse transcription reagent. The extracted mRNA was reverse transcribed into cDNA using an ordinary PCR amplifier. Reverse transcription procedure: 37°C for 15 minutes (reverse transcription reaction); 85°C for 5 seconds (inactivation reaction of reverse transcriptase); cooled to room temperature, the sample was diluted 5 times with enzyme-free water, and the final sample was stored at -80°C until use.

[0119] (2) Real-time quantitative PCR assay

[0120] Real-time quantitative PCR (RT-gPCR) was performed on samples using a 20 μL reaction system using the instructions in the UltraSYBR One Step RT-gPCR Kit (ROX-free) from Kangwei Century Pharmaceuticals. The standard two-step PCR protocol was as follows: 95°C for 30 seconds, 95°C for 3 seconds, and 60°C for 30 seconds, for 40 cycles. GAPDH was used as the internal reference gene, and the 2-ΔΔCt method was used for relative quantification of target gene expression.

[0121] In order to explore the effects of the liquid dressings prepared in Examples 1-3 and Comparative Examples 1-5 on inflammatory factors, cells were seeded in 6-well plates (6*10 5 The cells were cultured for 24 h in the same manner as in Examples 1-3 and Comparative Examples 1-5, and the cells were grouped, modeled, administered, and cultured. After the culture was completed, the total RNA of each group was extracted, cDNA was synthesized, and the gene expression of GADPH and the target gene was detected by q-PCR. GADPH was used as an internal reference for gene expression to calculate the relative RNA expression of the target gene. The experimental results of each group of inflammatory factors are shown in Table 3.

[0122] Table 3

[0123]

[0124]

[0125] TNF-α, IL-1β, IL-17, and IL-23, as proinflammatory cytokines, play an important role in inflammatory response and immune regulation. As can be seen from Table 3, compared with Comparative Examples 1-5, after using the liquid dressing of the present invention, the inflammatory response is milder, the immune system is in a relatively suppressed state, the therapeutic effect is good, and the inflammatory response can be effectively controlled.

[0126] 4. Adsorption test

[0127] The liquid dressings prepared in Examples 1-3 and Comparative Examples 1-6 were placed in a 50°C drying oven for 2 hours at a constant weight, cooled to room temperature, and weighed, which was recorded as W0. An appropriate amount of PBS solution was added to a beaker, and each group of liquid dressings was completely immersed in the PBS solution with tweezers. A timer was started at the same time, and the dressings were taken out at preset time points (1 min, 5 min, 10 min, 30 min, and 60 min). The dressings were hung vertically for 30 seconds to drain excess liquid, and the mass of the liquid dressing after adsorption was quickly weighed, which was recorded as W. t Repeat the test at least 3 times and take the average value. The calculation results are shown in Table 4.

[0128] The adsorption amount calculation formula is: ΔW=W t -W0

[0129] Table 4

[0130]

[0131]

[0132] As can be seen from Table 4, compared with Comparative Examples 1-6, the adsorption change of the liquid dressing of Example 1 is significantly smaller, indicating that the liquid dressing prepared by the present invention has good adsorption performance.

[0133] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A liquid dressing, characterized in that: including lipid phase and aqueous phase; The lipid phase comprises lecithin, diolein, polysorbate and an organic solvent; The aqueous phase comprises hyaluronic acid or a salt thereof and a buffer solution, the molecular weight of the hyaluronic acid or a salt thereof is 500-5000 Da, and the mass ratio of the hyaluronic acid or a salt thereof to the lecithin is 0.05-0.2:

10.

2. The liquid dressing according to claim 1, characterized in that The mass ratio of the lecithin to the diolein is 10:0.5-2; The mass ratio of the lecithin to the polysorbate is 10:2-4; The mass ratio of the total mass of the lecithin, the diolein, and the polysorbate to the organic solvent is 1:5-10.

3. The liquid dressing according to claim 1, characterized in that The lecithin includes one or more of soybean lecithin, egg yolk lecithin and marine lecithin; The polysorbate includes one or more of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

4. The liquid dressing according to claim 1, characterized in that The aqueous phase further comprises a plasticizer, and the mass ratio of the hyaluronic acid or its salt to the plasticizer is 0.01:0.6-5; The plasticizer includes one or more of glycerol, propylene glycol, sorbitol, castor oil and tributyl citrate.

5. The liquid dressing according to claim 4, characterized in that The aqueous phase further comprises trehalose, and the mass ratio of the hyaluronic acid or its salt to the trehalose is 0.01:1-2.

6. The liquid dressing according to any one of claims 1 to 5, characterized in that The amount of the buffer solution added is 80-99% of the total mass of the aqueous phase; The buffer solution is a phosphate buffer; The hyaluronic acid or its salt includes one or more of hyaluronic acid, sodium hyaluronate, and potassium hyaluronate.

7. A method for preparing the liquid dressing according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) dissolving lecithin, diolein, and polysorbate in an organic solvent to obtain a lipid phase; (2) dissolving hyaluronic acid or a salt thereof in a buffer solution to obtain an aqueous phase; (3) mixing the lipid phase and the aqueous phase to obtain mixed micelles; (4) Post-processing the mixed micelles to obtain a liquid dressing.

8. The method for preparing the liquid dressing according to claim 7, characterized in that: The particle size of the mixed micelles is 80-150 nm, and the polydispersity index PDI is less than 0.

25.

9. The method for preparing the liquid dressing according to claim 7 or 8, characterized in that: The post-processing includes sterile filtration and freeze-drying; The sterile filtration is performed using a 0.2-0.25 μm filter membrane; The freeze-drying process is to pre-freeze at -45 to -35°C for 3 to 5 hours, and then freeze-dry at -55 to -45°C for 20 to 28 hours.

10. The method for preparing the liquid dressing according to claim 7 or 8, characterized in that: The post-processing includes sterile filtration and sterilization; The sterile filtration is performed using a 0.2-0.25 μm filter membrane; The sterilization treatment is performed by γ-ray irradiation under the condition of 20-30 kGy or wet heat sterilization at 120-125° C. for 10-20 minutes.

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