Preparation and evaluation method of ozonized oil liposome

Abstract

The invention provides a preparation method and an evaluation method of ozonized oil liposome. The preparation method comprises the following steps: a, preparing ozonized oil, preferably ozonized sunflower seed oil, as a loading object; b, uniformly mixing and stirring ozonized oil, a surfactant and phospholipid to form a uniform and clear oil phase; c, adding deionized water serving as a water phase into the oil phase to form oil-in-water ozonized oil liposome; d, performing ultrasonic treatment on the formed oil-in-water ozonized oil lipidosome by using a cell ultrasonic crusher, so as to form uniform and stable ozonized oil lipidosome; and e, storing the prepared ozonized oil liposome in an environment of 2-8 DEG C. The evaluation method comprises the steps of stability analysis, ozone quality analysis and antibacterial performance analysis. After the ozonized oil is prepared into the liposome, the problems of poor stability and single dosage form of the ozonized oil can be solved.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and in particular relates to a preparation method of ozonated oil liposomes and an evaluation method thereof. The ozonated oil is ozonated sunflower seed oil. Background Art

[0002] The antibacterial properties of ozone are widely known and used, but the instability of the gas severely limits its practical application. To overcome this problem, researchers found that ozone in sunflower oil can react with unsaturated bonds to form stable and therapeutically active ozone derivatives. Therefore, ozonated sunflower oil can be used to exert the antibacterial potential of these two components. Ozone kills bacteria mainly through its oxidizing properties. Oxidation reactions can cause damage to the bacterial cell wall and plasma membrane, thereby increasing the permeability of bacterial cells to ozone.

[0003] In recent years, the application of nanomaterials in medicine and tissue engineering has attracted much attention. Nanomaterials have unique physicochemical properties that enable them to provide long-lasting effects and enhance their stability. The size of nanomaterials allows ingredients to pass through the skin more effectively, achieving directional effects and pore penetration. Liposomes are spherical vesicles composed of lipid amphiphilic substances (such as phospholipids) with a structure similar to that of cell membranes. Liposomes are highly valued in the medical field due to their advantages in biological membrane-like structures. Therefore, how to combine the antibacterial effects of plant essential oils with the advantages of nanomaterials as delivery carriers has become a hot research direction in the current field of new drug development. Summary of the invention

[0004] Liposomes are a safe and effective drug delivery system with simple preparation and readily available materials; ozonated oil is inexpensive and has significant antibacterial ability. Therefore, the present invention selects liposomes as delivery carriers and ozonated oil as antibacterial drugs to improve the bioavailability of ozonated oil and enhance its ability to resist pathogenic bacteria infection.

[0005] The present invention uses liposomes as carriers to embed and transport ozonated oil. The research focuses on exploring the effects of parameters such as the content of liposome wall materials, the loading of ozonated oil, and surfactants on the stability and antibacterial properties of ozonated oil liposomes. In terms of the preparation method, factors such as stirring speed, droplet addition speed, ultrasonic power and ultrasonic time are studied, and the stability, ozone quality and antibacterial properties of ozonated oil liposomes are evaluated. Finally, uniform and stable ozonated oil liposomes are obtained.

[0006] Specifically, the invention provides a novel method for preparing ozonated oil liposome and an evaluation method thereof. The specific steps of optimizing the preparation method of ozonated oil liposome are as follows: the ozonated oil selects ozonated sunflower oil with a peroxide value of 150 mmoL / kg, the total volume of the reaction system is controlled to be 50 mL, 5-20% of the ozonated oil, 2-10% of different types of surfactants (Tween 20, Tween 40, Tween 60, Tween 80) and 2.5-20% of the lipid material are added to a reaction container, the rotating speed of the reaction container is controlled to be 500-1500 rpm, so that it is mixed evenly to form a uniform oil phase, and then a syringe pump is used to drop an appropriate amount of deionized water into the oil phase at a speed of 10-100 mL / h, and then stirred to form an O / W crude emulsion. Finally, a cell ultrasonic crusher is used to control the time to be 5-20 min, and the ultrasonic power is 150-450 W to ultrasonicate the crude emulsion to form a uniform and stable ozonated oil emulsion. At the same time, the present invention further describes its evaluation means. For this, its stability is analyzed by kinetic stability and accelerated stability, its peroxide value is detected by iodine titration, and Staphylococcus aureus and Candida albicans are used as indicator bacteria, and a bactericidal experiment is carried out by suspension quantitative experiment. The results show that the product prepared under the optimized process conditions of ozonated oil liposomes is stable, the peroxide value is 34mmol / kg, and the antibacterial results show that its bactericidal effect is strong.

[0007] The present invention has two purposes. The first purpose is to provide a novel ozonated oil liposome and a preparation method thereof. The second purpose is to provide an evaluation method for the novel ozonated oil liposome.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] In one aspect, the present invention provides a method for preparing ozonated oil liposomes, the method comprising the following steps:

[0010] a. Prepare ozonated oil as the loading object;

[0011] b. Mix the ozonated oil, surfactant and phospholipid to form a uniform and clear oil phase;

[0012] c. adding deionized water as the aqueous phase to the oil phase to form water-in-oil ozonated oil liposomes;

[0013] d. using a cell ultrasonic disruptor to ultrasonically disrupt the formed water-in-oil ozonated oil liposomes to form uniform and stable ozonated oil liposomes;

[0014] e. The prepared ozonated oil liposomes were stored at 2-8°C.

[0015] In some embodiments, the ozonated oil is ozonated sunflower oil.

[0016] In some embodiments, the volume percentage of the ozonated oil is 5-20%.

[0017] In some embodiments, the volume percentage content of the ozonated oil is 20%.

[0018] In some embodiments, the surfactant is selected from Tween 20, Tween 40, Tween 60, and Tween 80.

[0019] In some embodiments, the surfactant is Tween 80.

[0020] In some embodiments, the volume percentage of the surfactant is 2-10%.

[0021] In some embodiments, the volume percentage content of the surfactant is 10%.

[0022] In some embodiments, the volume percentage of the phospholipids is 2.5-20%.

[0023] In some embodiments, the volume percentage of the phospholipid is 5%.

[0024] In some embodiments, the volume ratio of the water phase to the oil phase is 7:1-3:1.

[0025] In the present invention, ozonated oil, surfactant and phospholipid belong to the oil phase.

[0026] In some embodiments, the stirring speed is 500-1500 rpm.

[0027] In some embodiments, the stirring speed is 500 rpm.

[0028] In some embodiments, the dropping rate of deionized water is 10-100 mL / h.

[0029] In some embodiments, the dropping rate of deionized water is 60 mL / h.

[0030] In some embodiments, in step d, the ultrasonic power is 150-450 W, and the ultrasonic time is 5-20 min.

[0031] In some embodiments, in step d, the ultrasonic power is 350 W and the ultrasonic time is 10 min.

[0032] In another aspect, the present invention provides ozonated oil liposomes, which are prepared by the method as described above.

[0033] In another aspect, the present invention provides an evaluation method for ozonated oil liposomes, the evaluation method comprising stability analysis, ozone quality analysis and antibacterial performance analysis.

[0034] In some embodiments, the performance of the prepared ozonated oil liposomes is evaluated by particle size and polydispersity.

[0035] In some embodiments, the particle size and polydispersity coefficient analysis is performed using a particle size analyzer.

[0036] In some embodiments, the stability analysis is selected from a kinetic stability analysis and an accelerated stability analysis.

[0037] In some embodiments, the kinetic stability analysis is to transfer the liposomes into a test tube and store them at 4°C, 25°C, and 40°C for 30 days, respectively. After storage, the cream layer on the top of the liposomes and the sediment layer on the bottom are observed within 7, 14, 21, and 28 days to determine their stability.

[0038] In some embodiments, the accelerated stability analysis is to place the liposomes in a 4°C refrigerator for 48 hours, then transfer them to a constant temperature box at 45°C and place them for 48 hours to obtain one cycle. After repeating this step 6 times, centrifuge at 1500 rpm for 30 minutes, observe the cream layer on the top of the liposomes and the sediment layer on the bottom to judge their stability.

[0039] In some embodiments, ozone quality analysis is performed using iodometric analysis.

[0040] In some embodiments, the iodine titration method is to weigh 2.5000g of ozonated oil liposome sample into a 250mL iodine volumetric bottle, add 30mL of chloroform-glacial acetic acid (2:3V / V) mixture, gently shake to mix evenly, add 1mL of saturated potassium iodide (KI) solution, cover the bottle cap, gently shake for 0.5min, place in the dark for 3min, then add 100mL of distilled water, shake well and titrate with 0.1mol / L sodium thiosulfate solution, titrate to light yellow, stir vigorously during titration, add 1mL of starch indicator, the solution changes from light yellow to blue, continue to titrate until the blue disappears, which is the end point, record the titrated sodium thiosulfate volume, and repeat the test three times for each sample. Among them, sunflower oil is the blank group, ozonated oil liposome is the experimental group, the peroxide value of ozonated oil liposome is determined, and then the peroxide value-ozone mass standard curve is used to bring the peroxide value into the standard curve to calculate the ozone mass of ozonated oil liposome.

[0041] The peroxide value calculation formula is as follows:

[0042]

[0043] Where PI is the peroxide value, M is the concentration of sodium thiosulfate (0.1 mol / L), V is the volume of sodium thiosulfate consumed (mL), and m is the sample mass (g).

[0044] In some embodiments, the antibacterial performance analysis uses Staphylococcus aureus and Candida albicans as indicator bacteria.

[0045] In some embodiments, the antibacterial performance analysis includes taking 0.01 mL of a diluted bacterial suspension (concentration 10-300 CFU / mL), applying it evenly on the surface of a solid culture medium with a sterilized applicator under a sterile environment, incubating for a period of time, and then taking 0.1 mL of ozonated oil liposomes and applying it evenly on the surface of the culture medium. After treatment, place it in a 37°C incubator for 24 hours, observe the surface of the culture medium, and count the total number of colonies in CFU / mL, wherein physiological saline is set as a blank group, and ozonated oil with the same peroxide value (diluted with sunflower oil) is used as a positive control. The bactericidal rate is calculated by the formula:

[0046]

[0047] Among them: n0 is the total number of colonies in the control group, and n1 is the total number of colonies in the sample treatment.

[0048] definition

[0049] O / W: It is an oil-in-water system, an emulsified system in which oil is dispersed in water, with oil as the inner phase and water as the continuous outer phase.

[0050] PDI: Polydispersity index, represents the degree of uniform dispersion of particles, that is, the particle size distribution coefficient. The smaller the value, the more uniform the particle distribution in the solution. On the contrary, the larger the value, the wider the particle distribution in the solution.

[0051] Tween 20: also known as polyoxyethylene sorbitan monolaurate, it has strong hydrophilicity.

[0052] Tween 40: also known as polyoxyethylene sorbitan monopalmitate, is an oil / water emulsifier used as a stabilizer, solubilizer, dispersant, antistatic agent, fiber lubricant, etc.

[0053] Tween 60: also known as polyoxyethylene sorbitan monostearate, is a commonly used non-ionic surfactant with emulsifying, solubilizing and cleaning functions.

[0054] Tween 80: Also known as sorbitan monooleate polyoxyethylene ether, it is made by the polymerization of sorbitan monooleate and ethylene oxide. It is a chemical raw material.

[0055] Beneficial Effects

[0056] Pure ozonated oil is oily and can only be applied on the skin surface, and the peroxide value of ozonated oil will decrease with the extension of storage time, thereby reducing its performance. Compared with the existing technology, the present invention has the following effects: first, the present invention solves the problem that traditional ozonated oil is limited to skin application, expands the application market of ozonated oil, and secondly, the storage stability of ozonated oil is greatly increased by liposome encapsulation, and the storage time of ozonated oil is extended, and a stable and diverse dosage form (for example, ozonated oil can be prepared into liposome suspension for injection, etc.) ozonated oil nanoliposome is developed; secondly, the present invention develops a new type of ozonated oil liposome through laboratory process development, evaluation of physical and chemical properties, analysis of stability and antibacterial properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 A schematic diagram of a process according to an embodiment of the present invention is shown.

[0058] Figure 2 The effect of phospholipid materials on the physicochemical properties of ozonated oil liposomes is shown (Spass analysis, N=3).

[0059] Figure 3 The effect of oil loading on the physicochemical properties of ozonated oil liposomes is shown (Spass analysis, N=3).

[0060] Figure 4 The effect of surfactant content on the physicochemical properties of ozonated oil liposomes is shown (Spass analysis, N=3).

[0061] Figure 5 The effect of surfactant type on the physicochemical properties of ozonated oil liposomes is shown (Spass analysis, N=3).

[0062] Figure 6 The effect of stirring speed on the physicochemical properties of ozonated oil liposomes is shown (Spass analysis, N=3).

[0063] Figure 7 The effect of the volume ratio of the aqueous phase to the oil phase on the physicochemical properties of oxidized oil liposomes is shown (Spass analysis, N=3).

[0064] Figure 8 The effect of the dropping speed on the physicochemical properties of ozonated oil liposomes is shown (Spass analysis, N=3).

[0065] Fig. 9 The effect of ultrasound power on the physicochemical properties of ozonated oil liposomes is shown (Spass analysis, N=3).

[0066] Fig.10 The effect of ultrasound time on the physicochemical properties of ozonated oil liposomes is shown (Spass analysis, N=3).

[0067] Fig.11 The results of determination of lipid peroxide value of oxidized oils are shown (Spass analysis, N=3).

[0068] Fig.12 The graph shows the bactericidal effect of ozonated oil liposomes on Candida albicans (left) and Staphylococcus aureus (right) (Spass analysis, N=3).

[0069] Fig.13 The results of the study on the optimal inhibitory amount of ozonated oil liposomes against Candida albicans (left) and Staphylococcus aureus (right) are shown (Spass analysis, N=3). DETAILED DESCRIPTION

[0070] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0071] The invention adopts ozonated oil with a peroxide value of 150mmoL / kg as a loading object, mixes and dissolves an appropriate amount of phospholipid material and a surfactant with the ozonated oil, stirs evenly, and forms a uniform and transparent oil phase. An appropriate amount of deionized water is used as the water phase, and in the reaction process, the stirring speed and the dropping speed are controlled to form O / W type ozonated oil liposomes, and the properties of the ozonated oil liposomes are judged by controlling the ultrasonic power and the ultrasonic time by using a cell ultrasonic disruptor, and measuring the particle size and PDI. Finally, uniform and stable ozonated oil liposomes formed under the optimal process are obtained.

[0072] Example 1: Ozoned sunflower oil liposomes and their physicochemical properties

[0073] The ozonated oil was selected as ozonated sunflower oil, and 20% ozonated sunflower oil, 10% Tween 80, and 5% phospholipids (volume percentage) were mixed and stirred to form a uniform and transparent oil phase. The stirring speed was controlled to 500rpm, stirred at room temperature, the volume ratio of the water phase to the oil phase was 4:1, and the water phase was added to the oil phase at a drop rate of 60mL / h to form O / W type ozonated oil liposomes. Finally, a cell disruptor was used to control the ultrasonic power to 350W and the ultrasonic time to 10min.

[0074] Determination of particle size and polydispersity index (PDI): Use a particle size analyzer to measure. Take 1 mL of ozonated oil liposomes and put them into a colorimetric dish. Use a particle size analyzer to measure the particle size distribution and polydispersity index of the ozonated oil liposomes.

[0075] Example 2: Ozoned sunflower oil liposomes and their physicochemical properties

[0076] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that the 5% phospholipid material is changed to 2.5% phospholipid material.

[0077] Example 3: Ozoned sunflower oil liposomes and their physicochemical properties

[0078] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that the 5% phospholipid material is changed to 10% phospholipid material.

[0079] Example 4: Ozoned sunflower oil liposomes and their physicochemical properties

[0080] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that the 5% phospholipid material is changed to 20% phospholipid material.

[0081] Example 5: Ozoned sunflower oil liposomes and their physicochemical properties

[0082] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that 20% ozonated sunflower oil is replaced by 5% ozonated sunflower oil.

[0083] Example 6: Ozoned sunflower oil liposomes and their physicochemical properties

[0084] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that 20% ozonated sunflower oil is replaced by 10% ozonated sunflower oil.

[0085] Example 7: Ozoned sunflower oil liposomes and their physicochemical properties

[0086] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that 20% ozonated sunflower oil is replaced by 15% ozonated sunflower oil.

[0087] Example 8: Ozoned sunflower oil liposomes and their physicochemical properties

[0088] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that 10% Tween 80 is replaced by 2% Tween 80.

[0089] Example 9: Ozoned sunflower oil liposomes and their physicochemical properties

[0090] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that 10% Tween 80 is replaced by 4% Tween 80.

[0091] Example 10: Ozoned sunflower oil liposomes and their physicochemical properties

[0092] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that 10% Tween 80 is replaced by 6% Tween 80.

[0093] Example 11: Ozoned sunflower oil liposomes and their physicochemical properties

[0094] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that 10% Tween 80 is replaced by 10% Tween 60.

[0095] Example 12: Ozoned sunflower oil liposomes and their physicochemical properties

[0096] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that 10% Tween 80 is replaced by 10% Tween 40.

[0097] Example 13: Ozoned sunflower oil liposomes and their physicochemical properties

[0098] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that 10% Tween 80 is replaced by 10% Tween 20.

[0099] Example 14: Ozoned sunflower oil liposomes and their physicochemical properties

[0100] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that 500 rpm is changed to 800 rpm.

[0101] Example 15: Ozoned sunflower oil liposomes and their physicochemical properties

[0102] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that the speed is changed from 500 rpm to 1200 rpm.

[0103] Example 16: Ozoned sunflower oil liposomes and their physicochemical properties

[0104] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that the speed is changed from 500 rpm to 1500 rpm.

[0105] Example 17: Ozoned sunflower oil liposomes and their physicochemical properties

[0106] The preparation process of ozonated oil liposomes is the same as that in Example 1, except that the ratio of 4:1 is changed to 7:1.

[0107] Example 18: Ozoned sunflower oil liposomes and their physicochemical properties

[0108] The preparation process of ozonated oil liposomes is the same as that in Example 1, except that the ratio is changed from 4:1 to 6:1.

[0109] Example 19: Ozoned sunflower oil liposomes and their physicochemical properties

[0110] The preparation process of ozonated oil liposomes is the same as that in Example 1, except that the ratio is changed from 4:1 to 5:1.

[0111] Example 20: Ozoned sunflower oil liposomes and their physicochemical properties

[0112] The preparation process of ozonated oil liposomes is the same as that in Example 1, except that the ratio of 4:1 is changed to 3:1.

[0113] Example 21: Ozoned sunflower oil liposomes and their physicochemical properties

[0114] The preparation process of ozonated oil liposomes is the same as that in Example 1, except that the flow rate is changed from 60 mL / h to 10 mL / h.

[0115] Example 22: Ozoned sunflower oil liposomes and their physicochemical properties

[0116] The preparation process of ozonated oil liposomes is the same as that in Example 1, except that 60 mL / h is changed to 30 mL / h.

[0117] Example 23: Ozoned sunflower oil liposomes and their physicochemical properties

[0118] The preparation process of ozonated oil liposomes is the same as that in Example 1, except that the flow rate is changed from 60 mL / h to 100 mL / h.

[0119] Example 24: Ozoned sunflower oil liposomes and their physicochemical properties

[0120] The preparation process of ozonated oil liposomes is the same as that in Example 1, except that the ultrasonic power of 350 W is changed to 150 W.

[0121] Example 25: Ozoned sunflower oil liposomes and their physicochemical properties

[0122] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that the ultrasonic power of 350W is changed to 250W.

[0123] Example 26: Ozoned sunflower oil liposomes and their physicochemical properties

[0124] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that the ultrasonic power of 350 W is changed to 450 W.

[0125] Example 27: Ozoned sunflower oil liposomes and their physicochemical properties

[0126] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that the ultrasonic time of 10 min is changed to 5 min.

[0127] Example 28: Ozoned sunflower oil liposomes and their physicochemical properties

[0128] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that the ultrasonic time of 10 min is changed to 15 min.

[0129] Example 29: Ozoned sunflower oil liposomes and their physicochemical properties

[0130] The preparation process of ozonated oil liposomes is the same as that of Example 1, except that the ultrasonic time of 10 min is changed to 20 min.

[0131] Conclusion: In the process of preparing ozonated oil liposomes, the lipid material ( Figure 2 )、Oil capacity( Figure 3 ), surfactant content ( Figure 4 ), surfactant type ( Figure 5 )、Stirring speed( Figure 6 ), water phase to oil phase ratio ( Figure 7 )、Drop speed( Figure 8 ), ultrasonic power ( Fig. 9 ) and ultrasound time ( Fig.10 ) Several process parameters were optimized and the optimal process parameters of ozonated oil liposomes were determined by the changes in particle size and PDI. The results showed that as shown in the figure, the optimal process formula was 20% oil loading, 10% Tween 80, 5% lipid material, 500rpm stirring speed, 4:1 volume ratio of water phase to oil phase, 60mL / h dropping speed, 350W ultrasonic power and 10min ultrasonic time. The liposomes prepared had the best effect, with a particle size of 110nm and a PDI of 0.22.

[0132] Example 30: Kinetic stability analysis

[0133] The ozonated oil liposomes of Example 1 were used to evaluate their stability by dynamic stability. The nanoliposomes were transferred into test tubes and stored at 4°C, 25°C, and 40°C for 30 days. After storage, the cream layer on the top of the nanoliposomes and the sediment layer on the bottom were observed within 7, 14, 21, and 28 days to determine their stability.

[0134] Example 31: Accelerated Stability Analysis

[0135] Take 20 mL of ozonized oil liposomes of Example 1 and place them in a 4°C refrigerator for 48 hours, transfer them to a 45°C incubator and place them for 48 hours, thereby obtaining one cycle. After repeating this step 6 times, centrifuge at 1500 rpm for 30 minutes, observe the phase separation, and judge its stability.

[0136] The test results show that after the ozonated oil liposomes in Example 1 were subjected to kinetic stability tests and accelerated stability tests, no stratification or system inhomogeneity was found in the ozonated oil liposomes. The stability of the ozonated oil is judged based on the change in peroxide value, and its peroxide value decreases with increasing storage time.

[0137] Example 32: Determination of peroxide value of ozonated oil liposomes

[0138] Take the ozonated oil liposome of Example 1 and determine its peroxide value by iodine titration. According to the iodine titration determination requirements, weigh 2.5000g of ozonated oil liposome sample in a 250mL iodine volumetric bottle, add 30mL of chloroform-glacial acetic acid (2:3, V / V) mixed solution, shake gently to mix evenly, add 1mL of saturated potassium iodide (KI) solution, cover the bottle cap, shake gently for 0.5min, place in a dark place for 3min, then add 100mL of distilled water, shake well and titrate with 0.1mol / L sodium thiosulfate solution, titrate to light yellow, stir vigorously during titration, add 1mL of starch indicator, the solution changes from light yellow to blue, continue to titrate until the blue disappears, which is the end point. Record the titrated sodium thiosulfate volume, and repeat the test three times for each sample. The peroxide value of the ozonated oil liposomes was determined, and then the peroxide value-ozone mass standard curve was used (the peroxide value was used as the horizontal axis and the ozone mass was used as the vertical axis to draw the peroxide value-ozone mass standard curve), and the peroxide value was inserted into the standard curve to calculate the ozone mass of the ozonated oil liposomes.

[0139] The peroxide value calculation formula is as follows:

[0140]

[0141] Where PI is the peroxide value, M is the concentration of sodium thiosulfate (0.1 mol / L), V is the volume of sodium thiosulfate consumed (mL), and m is the sample mass (g).

[0142] The test results are as follows Fig.11 As shown, the peroxide value of the ozonated oil liposome sample was determined by iodine titration. It can be seen from the figure that the peroxide value of the ozonated oil liposome prepared under the optimal process is 34mmol / Kg, which is significantly different from the blank control group (3.99mmol / Kg). It can be proved that the ozonated oil liposome contains ozonated oil.

[0143] Example 33: Determination of bactericidal rate of ozonated oil liposomes

[0144] Take the ozonated oil liposomes of Example 1 and use Staphylococcus aureus and Candida albicans as indicator bacteria, take 0.01 mL of bacterial suspension, the concentration of bacterial suspension is 10 6 ~10 8CFU / mL, in a sterile environment, use a sterilized applicator to evenly apply it on the surface of the solid culture medium, incubate for a period of time, then take 0.1mL of ozonated oil liposomes and evenly apply it on the surface of the culture medium, place it in a 37℃ incubator after treatment, culture it for 24h, observe the surface of the culture medium, count the total number of colonies, and the unit is CFU / mL. Among them, set physiological saline as the blank group, and set the ozonated oil with the same peroxide value (diluted with sunflower oil) as the positive control. The formula for calculating the sterilization rate is:

[0145]

[0146] Among them: n0 is the total number of colonies in the positive control group, and n1 is the total number of colonies in the sample treatment.

[0147] The results are as follows Fig.12 As shown: the left figure is the antibacterial rate of ozonated oil and ozonated oil liposome to Candida albicans, and the right figure is the antibacterial rate of ozonated oil and ozonated oil liposome to Staphylococcus aureus. In the present embodiment, ozonated oil and ozonated oil liposome with peroxide value of 34mmoL / Kg are adopted. Staphylococcus aureus and Candida albicans are respectively adopted as indicator bacteria, and the results show that under the same peroxide value, the antibacterial effect of ozonated oil is significantly higher than that of ozonated oil liposome sample. It is speculated that because the density of oil is small, it is tightly covered on the surface of the culture medium, causing difficulty in bacterial reproduction, and ozonated oil liposome is an oil-in-water suspension, and it is easy to evaporate water when applied on the surface of the culture medium. Cause the bactericidal rate to be lower than that of ozonated oil.

[0148] Example 34: Determination of the optimal antibacterial amount of ozonated oil liposomes

[0149] The determination of the optimal antibacterial amount of ozonated oil liposomes was the same as in Example 29, except that 0.1 mL of ozonated oil liposomes was replaced with ozonated oil liposomes in the range of 0.1-1 mL.

[0150] The results are as follows Fig.13 As shown, the left figure is the bactericidal rate of ozonated oil liposomes against Staphylococcus aureus, and the right figure is the bactericidal rate of ozonated oil liposomes against Candida albicans. Staphylococcus aureus and Candida albicans were used as indicator bacteria to explore the killing effect of different amounts of ozonated oil liposomes on the two bacteria. The results showed that for both Staphylococcus aureus and Candida albicans, when the antibacterial amount of ozonated oil liposomes was 0.4mL, the bactericidal rate was the highest, reaching 100%, proving that ozonated oil liposomes have a significant antibacterial effect.

[0151] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing ozonated oil liposomes, characterized in that: The method comprises the following steps: a. Prepare ozonated oil preferably ozonated sunflower oil as the loading object; b. The ozonated oil, surfactant and phospholipid are mixed and stirred to form a uniform, clear oil phase. Preferably, the surfactant is selected from Tween 20, Tween 40, Tween 60, Tween 80, preferably Tween 80; c. adding deionized water as the aqueous phase to the oil phase to form water-in-oil ozonated oil liposomes; d. using a cell ultrasonic disruptor to ultrasonically disrupt the formed water-in-oil ozonated oil liposomes to form uniform and stable ozonated oil liposomes; e. The prepared ozonated oil liposomes were stored at 2-8°C.

2. The method according to claim 1, characterized in that The volume percentage of the ozonated oil is 5-20%, preferably 20%.

3. The method according to claim 1 or 2, characterized in that: The volume percentage of the surfactant is 2-10%, preferably 10%.

4. The method according to any one of claims 1 to 3, characterized in that The volume percentage of the phospholipids is 2.5-20%, preferably 5%.

5. The method according to any one of claims 1 to 4, characterized in that The volume ratio of the water phase to the oil phase is 7:1-3:

1.

6. The method according to any one of claims 1 to 5, characterized in that The stirring speed is 500-1500 rpm, preferably 500 rpm.

7. The method according to any one of claims 1 to 6, characterized in that The dropping speed of deionized water is 10-100 mL / h, preferably 60 mL / h.

8. The method according to any one of claims 1 to 7, characterized in that In step d, the ultrasonic power is 150-450 W, and the ultrasonic time is 5-20 min. Preferably, the ultrasonic power is 350 W, and the ultrasonic time is 10 min.

9. Ozoned oil liposomes prepared by the method according to any one of claims 1 to 8.

10. A method for evaluating ozonated oil liposomes, characterized in that: The evaluation method includes stability analysis, ozone quality analysis and antibacterial performance analysis. Preferably, the stability analysis is selected from kinetic stability analysis and accelerated stability analysis.

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