Honeysuckle total extract liposome gel as well as preparation method and application thereof

Abstract

The invention provides a honeysuckle total extract liposome gel as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The honeysuckle total extract liposome is prepared by adopting a micro-fluidic method, and the obtained liposome is relatively small and uniform in particle size and has high drug loading capacity. The honeysuckle total extract is prepared into the liposome gel, so that the residence time of the honeysuckle total extract on the skin surface can be effectively prolonged, the transdermal absorbability of the honeysuckle total extract is improved, the long-acting slow release effect is achieved, the adverse drug reaction is reduced, and the drug use frequency is reduced. Compared with sulfadiazine silver cream sold in the market, the honeysuckle total extract liposome gel provided by the invention has more effective effects of treating scalded wounds and promoting wound healing, has the characteristics of high safety, low price, remarkable curative effect and the like, and shows unique advantages in treatment of burns and scalds.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a total extract liposome gel of honeysuckle and a preparation method and application thereof. BACKGROUND

[0002] Honeysuckle has been praised as a good medicine for clearing heat and detoxifying since ancient times. It is sweet and cold in nature, can disperse wind-heat and clear heat and detoxify, and can be used for the treatment of various heat diseases. Honeysuckle has rich pharmacological activities, and a large number of studies have shown that honeysuckle has significant effects on anti-inflammatory, antibacterial, antiviral, antioxidant, and immune regulation. However, the total extract of honeysuckle has poor water solubility, and its saturated solubility in water is about 3 mg / mL. Due to the low solubility of the total extract of honeysuckle, its drug property is poor, the transdermal absorption is poor, and the bioavailability is also low. Therefore, it is urgent to seek a suitable drug carrier to improve the transdermal absorption of the total extract of honeysuckle and thereby improve its bioavailability. SUMMARY

[0003] Therefore, the purpose of the present application is to provide a total extract liposome gel of honeysuckle and a preparation method and application thereof. The total extract liposome gel of honeysuckle provided by the present application has good transdermal absorption and bioavailability, and can effectively promote the healing of burn wounds.

[0004] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0005] The present application provides a total extract liposome gel of honeysuckle, which comprises a gel and total extract liposomes of honeysuckle distributed in the gel; the total extract liposomes of honeysuckle are prepared by a microfluidic method;

[0006] The total extract liposome gel of honeysuckle comprises the following preparation raw and auxiliary materials in mass percentage:

[0007]

[0008] Preferably, the phospholipid is one or more of soybean phospholipid, lecithin, hydrogenated soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidyl ethanolamine, and distearoyl phosphatidylcholine;

[0009] The stabilizer is one or more of polyoxyethylene 40 hydrogenated castor oil, vitamin E polyethylene glycol succinate, and Tween 80;

[0010] The humectant is one or more of propylene glycol, glycerol, and hyaluronic acid;

[0011] The bacteriostatic agent is one or more of sorbic acid, benzalkonium chloride, phenoxyethanol, and hydroxybenzoate;

[0012] The pH regulator is one or more of triethanolamine, sodium hydroxide and potassium hydroxide.

[0013] Preferably, the gel matrix comprises one or more of methylcellulose, hydroxypropyl methylcellulose, carbomer, alginate, tragacanth gum and gelatin.

[0014] Preferably, the pH value of the honeysuckle total extract liposome gel is 5.0-6.0.

[0015] Preferably, the particle size of the honeysuckle total extract liposome is 100 to 160 nm.

[0016] The present invention provides a method for preparing the honeysuckle total extract liposome gel, comprising the following steps:

[0017] dissolving the total honeysuckle extract in an organic solvent to obtain an organic solution of the total honeysuckle extract;

[0018] mixing cholesterol, phospholipid, stabilizer and alcohol solvent to obtain an alcohol solution;

[0019] mixing the organic solution of the total honeysuckle extract with the alcohol solution to obtain an organic phase;

[0020] Using pure water as the aqueous phase, the organic phase and the aqueous phase are injected into different inlets of a microfluidic device, and the effluent is dialyzed to obtain the honeysuckle total extract liposomes;

[0021] The honeysuckle total extract liposome, gel matrix, water, moisturizing agent, antibacterial agent and pH value regulator are mixed to obtain the honeysuckle total extract liposome gel.

[0022] Preferably, the concentration of the total honeysuckle extract in the organic phase is 1 to 25 mg / mL.

[0023] Preferably, the total flow rate of the organic phase and the aqueous phase is 2 to 18 mL / min, and the flow rate ratio of the organic phase to the aqueous phase is 1:1 to 7.

[0024] Preferably, the dialysis time is 4 to 48 hours.

[0025] The invention discloses an application of the honeysuckle total extract liposome gel in preparing a burn and scald wound dressing.

[0026] The present invention provides a total honeysuckle extract liposome gel, comprising a gel and total honeysuckle extract liposomes distributed in the gel; the total honeysuckle extract liposomes are prepared using a microfluidics method; the total honeysuckle extract liposome gel comprises the following raw materials and auxiliary materials in percentage by weight: 0.04-0.5% total honeysuckle extract; 0.01-0.5% phospholipids; 0.01-0.5% cholesterol; 0.01-0.04% stabilizer; 0.5-3% gel matrix; 2-6% moisturizer; 0.1-0.5% antibacterial agent; 1-6% pH adjuster; and the balance is water. The lipid system refers to microvesicles formed by encapsulating a drug within a lipid bilayer. The phospholipid bilayer structure of the liposome easily fuses with the stratum corneum of the skin, enhancing drug skin permeability and improving drug bioavailability, making it an ideal carrier for topical drug administration. Gel is a transparent or translucent semisolid preparation with a three-dimensional network structure. Liposomes are dispersed in a polymer gel matrix to form a new type of gel. The present invention prepares the total extract of honeysuckle into a liposome gel, which not only retains the advantages of the lipid system and the gel, but also forms a multi-reservoir system. The adhesion performance of the gel effectively increases the retention time of the total extract of honeysuckle on the skin surface, improves its transdermal absorbability, exerts a long-lasting sustained-release effect, reduces adverse drug reactions, and reduces the number of medications. Compared with the commercially available sulfadiazine silver cream, the total extract liposome gel of honeysuckle provided by the present invention is more effective in treating burns and scalds and promoting wound healing. It has the characteristics of high safety, economical price, significant efficacy, etc., and shows unique advantages in the treatment of burns and scalds.

[0027] The present invention provides a method for preparing the above-mentioned honeysuckle total extract liposome gel. Due to the complex components in the honeysuckle total extract, it has poor solubility in many solvents, making it difficult to prepare liposomes. In addition, the preparation process of liposomes using traditional methods (such as ethanol injection method, thin film dispersion method, ultrasonic dispersion method, reverse evaporation method) is relatively cumbersome and the liposomes produced are large in particle size and uneven in particle size distribution. Large particle size also reduces the transdermal absorption rate of drugs. The present invention uses microfluidic technology to precisely control the fluid flow rate and flow rate, so that the liquid components introduced at different times can be fully mixed and highly uniform and orderly. The prepared liposomes have a small and uniform particle size, high batch reproducibility, and high drug loading. The preparation method of the honeysuckle total extract liposome gel of the present invention is simple, the quality is controllable, and it has good application and development prospects. The honeysuckle total extract liposome gel provided by the present invention can increase the retention time of the honeysuckle total extract on the skin surface and improve the transdermal permeability of the honeysuckle total extract, providing an experimental basis for the treatment of local skin infections with the honeysuckle total extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1This is the screening result of the phospholipid-cholesterol mass ratio of honeysuckle total extract liposomes;

[0029] Figure 2 This is the screening result of drug-lipid ratio of honeysuckle total extract liposome;

[0030] Figure 3 This is the particle size distribution diagram of the honeysuckle total extract liposomes prepared in Example 7;

[0031] Figure 4 This is the Zeta potential distribution diagram of the honeysuckle total extract liposomes prepared in Example 7;

[0032] Figure 5 These are typical images of burn wound healing at different stages in mice;

[0033] Figure 6 The healing rate of burn wounds in mice on the 4th day;

[0034] Figure 7 The healing rate of burn wounds in mice on the 7th day;

[0035] Figure 8 The healing rate of burn wounds in mice on the 10th day;

[0036] Figure 9 The scald wound healing rate of mice on day 14;

[0037] Figure 10 The scald wound healing rate of mice on day 17;

[0038] Figure 11 The healing rate of burn wounds in mice on the 20th day;

[0039] Figure 12 Comparison of the healing rates of back burn wounds in each group of mice at different times;

[0040] Figure 13 HE staining results of burn wounds of mice in each group;

[0041] Figure 14 These are the results of immunohistochemical staining of the pro-inflammatory factor TNF-α on the burn wounds of mice in each group. DETAILED DESCRIPTION

[0042] The present invention provides a honeysuckle total extract liposome gel, comprising a gel and honeysuckle total extract liposomes distributed in the gel; the honeysuckle total extract liposomes are prepared by a microfluidic method;

[0043] The honeysuckle total extract liposome gel comprises the following raw and auxiliary materials in percentage by weight:

[0044]

[0045] In terms of percentage by mass, the raw materials for preparing the honeysuckle total extract liposome gel include 0.04-0.5% of honeysuckle total extract, preferably 0.1-0.4%, specifically 0.04%, 0.08%, 0.1%, 0.2%, 0.3% or 0.4%, more preferably 0.3%. In the present invention, the honeysuckle total extract is a honeysuckle total extract extract or a honeysuckle total extract powder. In the present invention, the particle size of the honeysuckle total extract powder is preferably 3-5 μm, more preferably 4 μm. In the present invention, the mass content of the honeysuckle total extract in the honeysuckle total extract extract and the honeysuckle total extract powder is preferably 12-20%, more preferably 15-18%. In the present invention, the solubility of the honeysuckle total extract in the solvent (40 mg / mL) is greater than the solubility of the honeysuckle total extract powder (10 mg / mL), and the drug loading capacity of the honeysuckle total extract liposome is greater than the drug loading capacity of the honeysuckle total extract powder liposome. Therefore, the honeysuckle total extract is preferably the honeysuckle total extract extract. In the present invention, the source of the honeysuckle total extract is preferably commercially available or self-prepared. When the source is commercially available, the manufacturer purchased is preferably Shandong Yatu Biotechnology Co., Ltd.; when self-prepared, the preparation method of the honeysuckle total extract preferably includes the following steps:

[0046] Add 5 times the volume of 70% ethanol to the dried honeysuckle medicinal material, heat and reflux to extract for 1 hour, and filter to obtain a filtrate; repeat three times, and spin-dry the filtrate in a rotary evaporator to an extract to obtain a total honeysuckle extract.

[0047] The preparation method of the honeysuckle total extract powder preferably comprises the following steps:

[0048] Add 5 times the volume amount of 70% ethanol to the dried honeysuckle medicinal material, heat and reflux to extract for 1 hour, and filter to obtain a filtrate; repeat three times, spin-dry the filtrate in a rotary evaporator to an extract, vacuum-dry the extract, and crush to obtain a honeysuckle total extract powder.

[0049] In terms of percentage by mass, the preparation excipients of the honeysuckle total extract liposome gel include 0.01-0.5% of phospholipids, preferably 0.1-0.4%, specifically 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%, more preferably 0.05%. In the present invention, the phospholipids are preferably one or more of soybean lecithin, lecithin, hydrogenated soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and distearoylphosphatidylcholine.

[0050] In terms of mass percentage, the preparation auxiliary materials of the honeysuckle total extract liposome gel include 0.01-0.5% cholesterol, preferably 0.1-0.4%, specifically 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%, more preferably 0.05%.

[0051] In the present invention, the mass ratio of phospholipid to cholesterol is preferably 1 to 3:1, more preferably 1: 1. In the present invention, when the mass ratio of phospholipid to cholesterol is 1:1, the liposome has a lower PDI (particle size polydispersity index).

[0052] In the present invention, the mass ratio of the total honeysuckle extract to phospholipids + cholesterol (drug-to-lipid ratio) is preferably 3: 1 to 1: 3, more preferably 3: 1. In the present invention, when the drug-to-lipid ratio is 3: 1, the liposome has a suitable particle size and PDI.

[0053] The preparation excipients for the honeysuckle total extract liposome gel include, by weight percentage, 0.01 to 0.04% of a stabilizer, preferably 0.01 to 0.02%. In the present invention, the stabilizer is preferably one or more of polyoxyethylene 40 hydrogenated castor oil, vitamin E polyethylene glycol succinate, and Tween 80, more preferably polyoxyethylene 40 hydrogenated castor oil. In the present invention, after using the stabilizer, the particle size of the honeysuckle total extract liposomes is smaller and more uniform. In particular, when polyoxyethylene 40 hydrogenated castor oil is used as a stabilizer, the particle size and PDI of the honeysuckle total extract liposomes are minimized. A smaller particle size is beneficial for the transdermal absorption of the honeysuckle total extract liposomes, and a smaller PDI indicates that the particle size of the honeysuckle total extract liposomes is uniform and has good dispersibility.

[0054] The preparation excipients of the honeysuckle total extract liposome gel include, by weight percentage, 0.5-3% of a gel matrix, preferably 1-2%, specifically 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% or 4%, more preferably 1%. In the present invention, the gel matrix preferably includes one or more of methylcellulose, hydroxypropyl methylcellulose, carbomer, alginate, tragacanth gum and gelatin, more preferably carbomer.

[0055] The preparation excipients of the honeysuckle total extract liposome gel include 2-6% of a moisturizing agent, preferably 3-5%, specifically 2%, 3%, 4%, 5% or 6%, more preferably 4%, by weight percentage. In the present invention, the moisturizing agent is preferably one or more of propylene glycol, glycerin and hyaluronic acid, more preferably propylene glycol.

[0056] The preparation excipients of the honeysuckle total extract liposome gel include, by weight percentage, 0.1-0.5% of an antibacterial agent, preferably 0.2-0.4%, specifically 0.1, 0.2, 0.3, 0.4 or 0.5%, more preferably 0.25%. In the present invention, the antibacterial agent is one or more of sorbic acid, benzalkonium chloride, phenoxyethanol and methylparaben, more preferably phenoxyethanol.

[0057] The preparation excipients for the honeysuckle total extract liposome gel include, by weight percentage, 1-6% of a pH adjuster, specifically 1%, 2%, 3%, 4%, 5%, or 6%, more preferably 2%. In the present invention, the pH adjuster is one or more of triethanolamine, sodium hydroxide, and potassium hydroxide, more preferably triethanolamine. In the present invention, the pH value of the honeysuckle total extract liposome gel is preferably 5.0-6.0.

[0058] In the present invention, when the mass percentage of phospholipids in the honeysuckle total extract liposome gel is 0.05% and the mass percentage of cholesterol is 0.05%, the particle size is appropriate, the PDI is minimum, the liposome particle size is more uniform, and the dispersibility is better. In the present invention, when the mass percentage of the honeysuckle total extract is 0.3%, the prepared liposome has a high drug loading capacity. In the present invention, the optimal prescription ratio of the honeysuckle total extract liposome gel is: 0.3% honeysuckle total extract extract, 0.05% phospholipids, 0.05% cholesterol, 0.01% polyoxyethylene 40 hydrogenated castor oil, 1% carbomer, 4% propylene glycol, 0.25% phenoxyethanol, 2% triethanolamine to adjust the pH to 5.0-6.0, and the balance is purified water.

[0059] In the present invention, the honeysuckle total extract liposomes are prepared by a microfluidic method, and the particle size of the honeysuckle total extract liposomes is preferably 100-160 nm, more preferably 120-140 nm.

[0060] The present invention provides a method for preparing the honeysuckle total extract liposome gel, comprising the following steps:

[0061] dissolving the total honeysuckle extract in an organic solvent to obtain an organic solution of the total honeysuckle extract;

[0062] mixing cholesterol, phospholipid, stabilizer and alcohol solvent to obtain an alcohol solution;

[0063] mixing the organic solution of the total honeysuckle extract with the alcohol solution to obtain an organic phase;

[0064] Using pure water as the aqueous phase, the organic phase and the aqueous phase are injected into different inlets of a microfluidic device, and the effluent is dialyzed to obtain the honeysuckle total extract liposomes;

[0065] The honeysuckle total extract liposome, gel matrix, water, moisturizing agent, antibacterial agent and pH value regulator are mixed to obtain the honeysuckle total extract liposome gel.

[0066] The present invention dissolves the total honeysuckle extract in an organic solvent to obtain an organic solution of the total honeysuckle extract. In the present invention, the organic solvent is preferably one or more of dimethyl sulfoxide, chloroform, and N,N-dimethylformamide. In the present invention, the mass concentration of the total honeysuckle extract in the organic solution of the total honeysuckle extract is preferably 10 to 40 mg / mL, more preferably 30 to 40 mg / mL.

[0067] The present invention mixes cholesterol, a phospholipid, a stabilizer, and an alcohol solvent to obtain an alcohol solution. In the present invention, the alcohol solvent is preferably ethanol. In the present invention, the mass percentage of cholesterol in the alcohol solution is preferably 1 to 10 mg / mL, more preferably 5 to 10 mg / mL.

[0068] The present invention mixes the organic solution of the total honeysuckle extract with the alcohol solution to obtain an organic phase. In the present invention, the concentration of the total honeysuckle extract in the organic phase is preferably 1 to 25 mg / mL, more preferably 15 to 20 mg / mL, and specifically can be 3 mg / mL, 5 mg / mL, 8 mg / mL, 10 mg / mL, 15 mg / mL, or 17 mg / mL.

[0069] The present invention uses pure water as the aqueous phase, injects the organic phase and the aqueous phase into different inlets of a microfluidic device, and dialyzes the effluent to obtain the honeysuckle total extract liposomes. In the present invention, the microfluidic device is connected to a microfluidic chip, and the length of the microfluidic chip is preferably 2 to 5 mm, more preferably 3 mm, and the diameter is preferably 200 to 300 μm, more preferably 200 to 250 μm.

[0070] In the present invention, the total flow rate of the organic phase and the aqueous phase is preferably 2 to 18 mL / min, more preferably 5 to 15 mL / min, and further preferably 10 to 12 mL / min; the flow rate ratio of the organic phase to the aqueous phase is preferably 1:1 to 7, more preferably 1:2 to 5, and further preferably 1:3.

[0071] In the present invention, the dialysis is preferably performed in an aqueous solution, and the molecular weight cutoff of the dialysis is preferably 1000-8000 kDa, more preferably 3000-5000 kDa, and further preferably 3500 kDa; the dialysis time is preferably 4-48 h, more preferably 12-36 h, and further preferably 24 h.

[0072] The present invention comprises mixing the honeysuckle total extract liposomes, a gel matrix, water, a moisturizer, an antibacterial agent, and a pH adjuster to obtain a honeysuckle total extract liposome gel. The present invention has no particular requirements for the mixing method, and any mixing method known to those skilled in the art can be used, such as stirring. In the present invention, after adding the pH adjuster, the pH value of the resulting mixture is preferably 5.0 to 6.0.

[0073] The present invention provides application of the honeysuckle total extract liposome gel in preparing scald wound dressing.

[0074] The honeysuckle total extract liposome gel provided by the present invention, its preparation method and application are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0075] In the following examples, the preparation method of the total extract of honeysuckle is as follows:

[0076] Add 5 times the volume of 70% ethanol to the dried honeysuckle, heat and reflux for extraction for 1 hour, and filter to obtain a filtrate. Repeat this process three times, and then spin-dry the filtrate in a rotary evaporator until it becomes an extract, thereby obtaining a honeysuckle total extract extract, wherein the total honeysuckle extract content is 16%.

[0077] The preparation method of honeysuckle total extract powder is as follows:

[0078] Five times the volume of 70% ethanol was added to the dried honeysuckle root material, and the mixture was heated under reflux for 1 hour. The filtrate was filtered to obtain a filtrate. This process was repeated three times. The filtrate was dried in a rotary evaporator to a paste. The paste was vacuum dried and pulverized in a ball mill to obtain a total honeysuckle extract powder with a particle size of 4 μm.

[0079] Example 1

[0080] Screening of phospholipid-cholesterol mass ratio of liposomes containing total honeysuckle extract

[0081] Measure 1 mL of a 40 mg / mL dimethyl sulfoxide solution of honeysuckle total extract, 4 mL of an ethanol mixed solution of cholesterol and soybean lecithin with a total concentration of 20 mg / mL (wherein the mass ratio of phospholipids to cholesterol is 1:1, 2:1 or 3:1, respectively), and 200 μL of an ethanol solution of 20 mg / mL polyoxyethylene 40 hydrogenated castor oil are mixed as the organic phase, and purified water is used as the aqueous phase; the organic phase and the aqueous phase are placed in syringes respectively, placed in a microfluidic device, and then connected to a microfluidic chip (length 3 mm, diameter 200 μm), and the organic phase flow rate and the aqueous phase flow rate are set to 3 mL / min and 9 mL / min, and the two phases are slowly injected into the microfluidic chip through the inlet channel, and the resulting liquid is placed in a dialysis bag and dialyzed for 24 hours to remove the organic solvent, thereby preparing honeysuckle total extract liposomes with different phospholipid-cholesterol mass ratios.

[0082] Figure 1 These are the results of screening the phospholipid-cholesterol mass ratio of honeysuckle total extract liposomes. The results show that when the phospholipid-cholesterol mass ratio is 1:1, the liposome particle size is about 140nm and the PDI is less than 0.2. The liposomes prepared with other ratios have appropriate particle sizes, but the PDI is greater than 0.4. Excessively large PDI indicates that the liposome particle size distribution is too wide and the particle size uniformity is poor, so 1:1 is selected as the optimal phospholipid-cholesterol mass ratio.

[0083] Example 2

[0084] Screening of drug-to-lipid ratio of liposomes containing total honeysuckle extract

[0085] 40 mg / mL dimethyl sulfoxide solution of honeysuckle total extract (volumes of 9 mL, 4.5 mL, 1.5 mL, 0.5 mL, controlling the drug-lipid ratio to 6:1, 3:1, 1:1, 1:3), 3 mL of 10 mg / mL cholesterol ethanol solution, 3 mL of 10 mg / mL phospholipid ethanol solution, and 200 μL of 20 mg / mL polyoxyethylene 40 hydrogenated castor oil ethanol solution were mixed as the organic phase, and purified water was used as the aqueous phase; the organic phase and the aqueous phase were placed in syringes respectively, placed in a microfluidic device, and then connected to a microfluidic chip (length 3 mm, diameter 200 μm), and the organic phase flow rate was set to 3 mL / min and the aqueous phase flow rate was set to 9 mL / min. The two phases were slowly injected into the microfluidic chip through the inlet channel, and the resulting liquid was placed in a dialysis bag and dialyzed for 24 hours to remove the organic solvent, thereby preparing honeysuckle total extract liposomes with different drug-lipid ratios.

[0086] Figure 2The results of drug-lipid ratio screening of honeysuckle total extract liposomes show that the particle size and PDI of liposomes with drug-lipid ratios of 3:1, 1:1, and 1:3 meet the requirements. The larger the drug-lipid ratio, the higher the drug loading capacity. However, after further increasing the drug-lipid ratio (6:1), the liposome particle size obtained is too large, PDI>0.4, the particle size is uneven, and micron-level particles exist. Therefore, the preferred drug-lipid ratio is 3:1.

[0087] Example 3

[0088] Preparation of liposomes of total honeysuckle extract using polyoxyethylene 40 hydrogenated castor oil as stabilizer

[0089] 3 mL of a 40 mg / mL dimethyl sulfoxide solution of honeysuckle total extract, 3 mL of a 10 mg / mL cholesterol ethanol solution, 3 mL of a 10 mg / mL phospholipid ethanol solution, and 200 μL of a 20 mg / mL polyoxyethylene 40 hydrogenated castor oil solution were mixed as the organic phase, and purified water was used as the aqueous phase. The organic phase and the aqueous phase were placed in syringes respectively, placed in a microfluidic device, and then connected to a microfluidic chip (length 3 mm, diameter 200 μm). The organic phase flow rate was set to 1 mL / min and the aqueous phase flow rate was set to 2 mL / min. The two phases were slowly injected into the microfluidic chip through the inlet channel. The resulting liquid was placed in a dialysis bag and dialyzed for 24 hours to remove the organic solvent, thereby preparing honeysuckle total extract liposomes stabilized with Tween 80.

[0090] The results showed that the prepared honeysuckle total extract liposomes were a clear yellow solution, the particle size was 136.32 nm and the PDI was 0.085 as measured by a particle size analyzer.

[0091] Example 4

[0092] Preparation of liposomes of total honeysuckle extract using Tween 80 as stabilizer

[0093] 3 mL of 40 mg / mL dimethyl sulfoxide solution of honeysuckle total extract, 3 mL of 10 mg / mL cholesterol ethanol solution, 3 mL of 10 mg / mL phospholipid ethanol solution, and 200 μL of 20 mg / mL Tween 80 solution were mixed as the organic phase, and purified water was used as the aqueous phase; the organic phase and the aqueous phase were placed in syringes respectively, placed in a microfluidic device, and then connected to a microfluidic chip (length 3 mm, diameter 200 μm), and the organic phase flow rate was set to 1 mL / min and the aqueous phase flow rate was 2 mL / min. The two phases were slowly injected into the microfluidic chip through the inlet channel, and the resulting liquid was placed in a dialysis bag and dialyzed for 24 hours to remove the organic solvent, thereby preparing honeysuckle total extract liposomes with Tween 80 as a stabilizer.

[0094] The results showed that the prepared honeysuckle total extract liposomes were a clear yellow solution, the particle size was 397.30 nm and the PDI was 0.146 as measured by a particle size analyzer.

[0095] Example 5

[0096] Preparation of liposomes of total honeysuckle extract using vitamin E polyethylene glycol succinate as stabilizer

[0097] 3 mL of 40 mg / mL dimethyl sulfoxide solution of honeysuckle total extract, 3 mL of 10 mg / mL cholesterol ethanol solution, 3 mL of 10 mg / mL phospholipid ethanol solution, and 200 μL of 20 mg / mL vitamin E polyethylene glycol succinate ethanol solution were mixed as the organic phase, and purified water was used as the aqueous phase; the organic phase and the aqueous phase were placed in syringes respectively, placed in a microfluidic device, and then connected to a microfluidic chip (length 3 mm, diameter 200 μm), and the organic phase flow rate was set to 2 mL / min and the aqueous phase flow rate was set to 6 mL / min. The two phases were slowly injected into the microfluidic chip through the inlet channel, and the resulting liquid was placed in a dialysis bag and dialyzed for 24 hours to remove the organic solvent, thereby preparing honeysuckle total extract liposomes with vitamin E polyethylene glycol succinate as a stabilizer.

[0098] The results showed that the prepared honeysuckle total extract liposomes were a clear yellow solution, the particle size was 364.15 nm and the PDI was 0.194 as measured by a particle size analyzer.

[0099] Example 6

[0100] Preparation of liposomes containing total honeysuckle extract at a total flow rate of 4 mL / min and a flow rate ratio of 1:3

[0101] 3 mL of 40 mg / mL dimethyl sulfoxide solution of honeysuckle total extract, 3 mL of 10 mg / mL cholesterol ethanol solution, 3 mL of 10 mg / mL phospholipid ethanol solution, and 200 μL of 20 mg / mL polyoxyethylene 40 hydrogenated castor oil ethanol solution were mixed as the organic phase, and purified water was used as the aqueous phase; the organic phase and the aqueous phase were placed in syringes respectively, placed in a microfluidic device, and then connected to a microfluidic chip (length 3 mm, diameter 200 μm), and the organic phase flow rate was set to 1 mL / min and the aqueous phase flow rate was set to 3 mL / min. The two phases were slowly injected into the microfluidic chip through the inlet channel, and the resulting liquid was placed in a dialysis bag and dialyzed for 24 hours to remove the organic solvent, thereby obtaining honeysuckle total extract liposomes with a total flow rate of 4 mL / min and a flow rate ratio of 1:3.

[0102] The results showed that the prepared honeysuckle total extract liposomes were a clear yellow solution, and the particle size was 149.93 nm and the PDI was 0.166 as measured by a particle size analyzer.

[0103] Example 7

[0104] Preparation of honeysuckle total extract liposomes using honeysuckle total extract powder as raw material

[0105] 1 mL of dimethyl sulfoxide solution of 10 mg / mL honeysuckle total extract powder, 2 mL of 10 mg / mL cholesterol ethanol solution, 2 mL of 10 mg / mL phospholipid ethanol solution, and 200 μL of 20 mg / mL polyoxyethylene 40 hydrogenated castor oil ethanol solution were mixed as the organic phase, and purified water was used as the aqueous phase; the organic phase and the aqueous phase were placed in syringes respectively, placed in a microfluidic device, and then connected to a microfluidic chip (length 3 mm, diameter 200 μm), and the organic phase flow rate was set to 3 mL / min and the aqueous phase flow rate was set to 9 mL / min. The two phases were slowly injected into the microfluidic chip through the inlet channel, and the resulting liquid was placed in a dialysis bag and dialyzed for 24 hours to remove the organic solvent, thereby obtaining honeysuckle total extract liposomes.

[0106] The results showed that the prepared honeysuckle total extract liposomes were a clear yellow solution, and the particle size was 107.83 nm and the PDI was 0.102 as measured by a particle size analyzer.

[0107] Example 8

[0108] In this embodiment, the raw materials and auxiliary materials for preparing the high-dose group of honeysuckle total extract liposome gel include:

[0109] 0.3% total extract of honeysuckle, 0.05% phospholipids, 0.05% cholesterol, 0.01% polyoxyethylene 40 hydrogenated castor oil, 1% carbomer, 4% propylene glycol, 0.25% phenoxyethanol, 2% triethanolamine, and the balance is purified water.

[0110] The preparation method is as follows:

[0111] The organic phase was prepared by mixing 3 mL of a 40 mg / mL dimethyl sulfoxide solution of honeysuckle total extract, 2 mL of a 10 mg / mL cholesterol solution in ethanol, 2 mL of a 10 mg / mL phospholipid solution in ethanol, and 200 μL of a 20 mg / mL polyoxyethylene 40 hydrogenated castor oil solution in ethanol. Purified water was used as the aqueous phase. The organic and aqueous phases were placed in separate syringes and placed in a microfluidic device. The device was then connected to a microfluidic chip, with the organic phase flow rate set at 3 mL / min and the aqueous phase flow rate at 9 mL / min. Both phases were slowly injected into the microfluidic chip through the inlet channel. The resulting liquid was dialyzed in a dialysis bag for 24 hours to remove the organic solvent, thereby producing honeysuckle total extract liposomes.

[0112] 0.28 g of carbomer was added to 6 g of purified water, and then 20 g of the honeysuckle total extract liposome solution was added and stirred on a magnetic stirrer for uniform swelling to obtain a carbomer solution, and then 1.12 g of propylene glycol, 0.07 g of phenoxyethanol, and 0.56 g of triethanolamine were added and stirred evenly to obtain a high-dose group of honeysuckle total extract liposome gel.

[0113] The results showed that the prepared honeysuckle total extract liposomes were clear yellow liquid, and the particle size distribution was as shown in the figure. Figure 3 As shown in Figure 2, its average particle size is 128.28nm and PDI is 0.14. Its Zeta potential distribution is shown in Figure 2. Figure 4 As shown, the potential is -20.73 mV.

[0114] Example 9

[0115] In this embodiment, the raw materials and auxiliary materials for preparing the low-dose group of honeysuckle total extract liposome gel include:

[0116] Honeysuckle total extract 0.17%, phospholipids 0.05%, cholesterol 0.05%, polyoxyethylene 40 hydrogenated castor oil 0.01%, carbomer 1%, propylene glycol 4%, phenoxyethanol 0.25%, triethanolamine 2%, and the balance is purified water.

[0117] The preparation method is as follows:

[0118] The organic phase was prepared by mixing 1.7 mL of a 40 mg / mL dimethyl sulfoxide solution of honeysuckle total extract, 2 mL of a 10 mg / mL cholesterol solution in ethanol, 2 mL of a 10 mg / mL phospholipid solution in ethanol, and 200 μL of a 20 mg / mL polyoxyethylene 40 hydrogenated castor oil solution in ethanol. Purified water was used as the aqueous phase. The organic and aqueous phases were placed in separate syringes and placed in a microfluidic device. The device was then connected to a microfluidic chip, with the organic phase flow rate set at 3 mL / min and the aqueous phase flow rate at 9 mL / min. Both phases were slowly injected into the microfluidic chip through the inlet channel. The resulting liquid was dialyzed in a dialysis bag for 24 hours to remove the organic solvent, thereby producing honeysuckle total extract liposomes.

[0119] 0.34 g of carbomer was added to 11.5 g of purified water, and then 20 g of the honeysuckle total extract liposome solution was added and stirred on a magnetic stirrer for uniform swelling to obtain a carbomer solution. Then 1.36 g of propylene glycol, 0.085 g of phenoxyethanol, and 0.68 g of triethanolamine were added and stirred evenly to obtain a low-dose group of honeysuckle total extract liposome gel.

[0120] Example 10

[0121] Evaluation of the efficacy of honeysuckle total extract liposome gel in the treatment of deep second-degree burns using a Balb / c mouse burn model

[0122] First, the mice were divided into groups. They were blank control group, model group, silver sulfadiazine cream group, low-dose group of total honeysuckle extract liposome gel (0.17%), and high-dose group of total honeysuckle extract liposome gel (0.3%). One day before the experiment, the backs of the mice were depilated with a depilatory cream. Afterwards, a deep second-degree burn was performed on the backs of the mice using a burn instrument (the control group was not treated). Then, the corresponding medicine was applied to the burned area on the backs of the mice every day, with a dosage of 0.15g / cm 2 . The healing status of the burn wound (including color and hardness) was recorded regularly and photographed, and the wound area of ​​the animals was measured using Image J software to calculate the wound healing rate. When the wound on the back of the mouse was healed, the mouse was killed. The skin of the scalded area on the back of the mouse was taken for HE staining, and the expression level of the inflammatory factor TNF-α in the wound of each group of mice was detected by immunohistochemistry or enzyme-linked immunosorbent assay to evaluate the anti-inflammatory effect of the test drug on the burn wound.

[0123] Figure 5 These are typical images of burn wound healing at different stages in mice. Figure 5 The morphological changes in the wounds of mice in each group after modeling are shown. The wounds appeared evenly distributed, with a circular white border zone forming at the edges. The surrounding tissue was flesh-colored and exhibited significant edema. By day 4, scabs were observed to form on the wounds, most appearing light yellow. The wounds of mice in the low-dose liposome gel group were darker and appeared light red. By day 7, the scabs were largely intact, with clear edges, a light yellow hue, a rough surface, and a firm texture, tightly adhering to the burn wound. The wound area of ​​mice in the low- and high-dose liposome gel groups was significantly reduced, demonstrating that liposome gel significantly promotes burn wound healing. By day 10, the wounds of mice in the model group and the silver sulfadiazine cream group remained light yellow, with a rough surface and a firm texture. In contrast, the wounds of mice in the low- and high-dose liposome gel groups had turned light red. This result shows that the effect of honeysuckle total extract liposome gel in promoting wound healing is significantly better than that of sulfadiazine silver cream group. On the 14th and 17th days, the scabs of the wounds of all mice continued to fall off, the scabs at the edges of the wounds hardened and curled up, and the wound area was significantly reduced. Among them, the wound of the high-dose honeysuckle total extract liposome gel group was the most significantly reduced, further confirming its strong healing-promoting ability. By the 20th day, the wounds of mice in each group had basically reached a healed state. Therefore, honeysuckle total extract liposome gel showed significant therapeutic effects in promoting the healing of burn wounds, and its effect was better than traditional sulfadiazine silver cream.

[0124] Figure 6 The healing rate of burn wounds in mice on the 4th day. Figure 6On day 4, the wound healing rates of mice in the model group were approximately 10%, those in the silver sulfadiazine cream group were approximately 15%, those in the low-dose honeysuckle total extract liposome gel group were approximately 30%, and those in the high-dose honeysuckle total extract liposome gel group were approximately 40%. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0125] Figure 7 The healing rate of burn wounds in mice on the 7th day. Figure 7 On day 7, the wound healing rates of mice in the model group were approximately 28%, those in the silver sulfadiazine cream group were approximately 33%, those in the low-dose honeysuckle total extract liposome gel group were approximately 45%, and those in the high-dose honeysuckle total extract liposome gel group were approximately 55%. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0126] Figure 8 The healing rate of burn wounds in mice on the 10th day. Figure 8 The results showed that on the 10th day, the burn wound area of ​​mice in all groups was significantly reduced. The wound healing rate of mice in the model group was approximately 40%, the wound healing rate of mice in the silver sulfadiazine cream group was approximately 47%, the wound healing rate of mice in the low-dose honeysuckle total extract liposome gel group was approximately 66%, and the wound healing rate of mice in the high-dose honeysuckle total extract liposome gel group reached approximately 68%. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0127] Figure 9 The healing rate of burn wounds in mice on the 14th day. Figure 9 On day 14, the wound healing rates of mice in the model group were approximately 65%, those in the silver sulfadiazine cream group were approximately 75%, those in the low-dose honeysuckle total extract liposome gel group were approximately 84%, and those in the high-dose honeysuckle total extract liposome gel group reached approximately 87%. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0128] Figure 10 The healing rate of burn wounds in mice on the 17th day. Figure 10 On day 17, the wound healing rates of mice in the model group were 84%, those in the silver sulfadiazine cream group were 90%, those in the low-dose honeysuckle total extract liposome gel group were 97%, and those in the high-dose honeysuckle total extract liposome gel group were approximately 95%. Compared with the model group, *P<0.05, **P<0.01, ***P<0.001.

[0129] Figure 11 The healing rate of burn wounds in mice on the 20th day. Figure 11 It showed that on the 20th day, the wound healing rate of mice in each group was close to 100%, and the wounds were tending to be healed.

[0130] Figure 12 Comparison of back burn wound healing rates in each group of mice at different times. Overall, the wound healing rate in the model group was lower than that in the silver sulfadiazine cream group, lower than that in the low-dose honeysuckle total extract liposome gel group, and lower than that in the high-dose honeysuckle total extract liposome gel group. At observation time points of 4 and 7 days, the wound healing rate in the high-dose honeysuckle total extract liposome gel group was significantly higher than that in the silver sulfadiazine cream group (p < 0.05).

[0131] Figure 13 HE staining results of the burn wounds of mice in each group, where the green arrow indicates that the epidermis of the mouse skin is intact, the yellow arrow indicates the infiltration of inflammatory cells, the black arrow indicates the edema and inflammation of the dermis, and the blue arrow indicates the dermis that has recovered after injury. Figure 13 In the control group, cells were arranged regularly, the dermis was intact and thicker than the epidermis, no inflammatory cell infiltration was observed, and relatively intact muscle tissue was visible under the skin. In the model group, the dermis was damaged, with many inflammatory cells infiltrating and obvious edema and inflammation in the dermis. Inflammation of the back skin of mice in the sulfadiazine silver cream group and the honeysuckle total extract liposome gel group was reduced. In particular, the high-dose honeysuckle total extract liposome gel group had significantly reduced inflammation of the back skin of mice, with good repair of the dermis and intact muscle tissue. The effect was significantly better than that of the traditional burn treatment drug sulfadiazine silver cream group.

[0132] Figure 14 The results of immunohistochemical staining of TNF-α, a pro-inflammatory factor, on the burn wounds of mice in each group are shown. Figure 14 As can be seen, the control group produced virtually no inflammatory cytokine TNF-α. However, the model group expressed significant amounts of TNF-α. Compared to the control group, the high-dose honeysuckle extract liposome gel group significantly reduced TNF-α. Overall, TNF-α expression levels were in the order of control group < high-dose honeysuckle extract liposome gel group < low-dose honeysuckle extract liposome gel group < silver sulfadiazine cream group < model group. This result is consistent with the trend in burn wound healing rates in mice.

[0133] The above results show that the liposome gel of total extract of honeysuckle has the ability to significantly inhibit inflammation and promote the healing of burn wounds.

[0134] 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A liposome gel of total honeysuckle extract, characterized in that: The invention comprises a gel and liposomes of a total honeysuckle extract distributed in the gel; the liposomes of the total honeysuckle extract are prepared by a microfluidic method; The honeysuckle total extract liposome gel comprises the following raw and auxiliary materials in percentage by weight:

2. The honeysuckle total extract liposome gel according to claim 1, characterized in that The phospholipid is one or more of soybean lecithin, lecithin, hydrogenated soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and distearoylphosphatidylcholine; The stabilizer is one or more of polyoxyethylene 40 hydrogenated castor oil, vitamin E polyethylene glycol succinate and Tween 80; The moisturizing agent is one or more of propylene glycol, glycerin and hyaluronic acid; The antibacterial agent is one or more of sorbic acid, benzalkonium chloride, phenoxyethanol and methylparaben; The pH regulator is one or more of triethanolamine, sodium hydroxide and potassium hydroxide.

3. The honeysuckle total extract liposome gel according to claim 1 or 2, characterized in that The gel matrix includes one or more of methylcellulose, hydroxypropyl methylcellulose, carbomer, alginate, tragacanth gum and gelatin.

4. The honeysuckle total extract liposome gel according to claim 1, characterized in that The pH value of the honeysuckle total extract liposome gel is 5.0-6.

0.

5. The honeysuckle total extract liposome gel according to claim 1, characterized in that The particle size of the honeysuckle total extract liposome is 100-160 nm.

6. The method for preparing the liposome gel of the total extract of honeysuckle according to any one of claims 1 to 5, characterized in that: The following steps are involved: dissolving the total honeysuckle extract in an organic solvent to obtain an organic solution of the total honeysuckle extract; mixing cholesterol, phospholipid, stabilizer and alcohol solvent to obtain an alcohol solution; mixing the organic solution of the total honeysuckle extract with the alcohol solution to obtain an organic phase; Using purified water as the aqueous phase, the organic phase and the aqueous phase are injected into different inlets of a microfluidic device, and the effluent is dialyzed to obtain liposomes of the total honeysuckle extract; The honeysuckle total extract liposome, gel matrix, water, moisturizing agent, antibacterial agent and pH value regulator are mixed to obtain the honeysuckle total extract liposome gel.

7. The preparation method according to claim 6, characterized in that The concentration of the total honeysuckle extract in the organic phase is 1-25 mg / mL.

8. The preparation method according to claim 6, characterized in that The total flow rate of the organic phase and the aqueous phase is 2 to 18 mL / min, and the flow rate ratio of the organic phase to the aqueous phase is 1:1 to 7.

9. The preparation method according to claim 6, characterized in that The dialysis time is 4 to 48 hours.

10. Use of the honeysuckle total extract liposome gel according to any one of claims 1 to 5 or the honeysuckle total extract liposome gel prepared by the preparation method according to any one of claims 6 to 9 in preparing burn and scald wound dressings.

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