Preparation and evaluation method of ozonated oil liposomes

Abstract

The present invention provides a preparation method and evaluation method of ozonated oil liposomes. The preparation method comprises the following steps: a. preparing ozonated oil, preferably ozonated sunflower oil, as a loading object; b. mixing the ozonated oil, a surfactant, and a phospholipid to form a uniform, clear oil phase; c. adding deionized water as the aqueous phase to the oil phase to form oil-in-water ozonated oil liposomes; d. sonicating the formed oil-in-water ozonated oil liposomes using a cell ultrasonic disruptor to form uniform and stable ozonated oil liposomes; and e. storing the prepared ozonated oil liposomes at 2-8°C. The evaluation method comprises stability analysis, ozone quality analysis, and antibacterial performance analysis. After the ozonated oil is prepared into liposomes, the problems of poor stability and single dosage form of the ozonated oil can be solved.

Description

Technical Field

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

[0002] The antimicrobial properties of ozone are widely recognized and used, but its gaseous instability severely limits its practical application. To overcome this problem, researchers discovered that ozone in sunflower oil can react with unsaturated bonds to form stable and therapeutically active ozone derivatives. Therefore, ozonated sunflower oil could be used to exploit the antimicrobial potential of both components. Ozone kills bacteria primarily through its oxidizing properties, which damage bacterial cell walls and plasma membranes, increasing their permeability 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 targeted 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. Due to the advantages of their biological membrane-like structure, liposomes are highly valued in the medical field. Therefore, how to combine the antibacterial effects of plant essential oils with the advantages of nanomaterials as delivery vehicles 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, simple to prepare and made from readily available materials. Ozonated oil is inexpensive and possesses significant antibacterial properties. Therefore, the present invention utilizes liposomes as a delivery vehicle and ozonated oil as an antibacterial agent to improve the bioavailability of the ozonated oil and enhance its ability to fight pathogenic infections.

[0005] This invention uses liposomes as carriers to encapsulate and deliver ozonated oil. The research focused on investigating the effects of parameters such as liposome wall material content, ozonated oil loading, and surfactant on the stability and antibacterial properties of the ozonated oil liposomes. Regarding the preparation method, factors such as stirring speed, addition rate, ultrasonic power, and ultrasonic time were studied. The stability, ozone quality, and antibacterial properties of the ozonated oil liposomes were also evaluated. Ultimately, uniform and stable ozonated oil liposomes were obtained.

[0006] Specifically, the present invention provides a novel method for preparing ozonated oil liposomes and an evaluation method thereof. The method for optimizing the preparation of ozonated oil liposomes comprises the following steps: selecting ozonated sunflower oil with a peroxide value of 150 mmoL / kg as the ozonated oil; controlling the total volume of the reaction system to be 50 mL; adding 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 a lipid material to a reaction vessel; controlling the rotating speed of the reaction vessel to be 500-1500 rpm to uniformly mix the mixture to form a uniform oil phase; then using a syringe pump to dropwise add an appropriate amount of deionized water to the oil phase at a speed of 10-100 mL / h; and stirring the mixture to form an O / W crude emulsion. Finally, using a cell ultrasonic disruptor to ultrasonicate the crude emulsion for 5-20 minutes at an ultrasonic power of 150-450 W to form a uniform and stable ozonated oil emulsion. The present invention further describes its evaluation methods. Kinetic stability and accelerated stability were used to analyze its stability, and its peroxide value was tested using iodine titration. Furthermore, a bactericidal test was conducted using Staphylococcus aureus and Candida albicans as indicator bacteria through suspension quantitative experiments. The results showed that the product prepared under the optimized process conditions of the ozonated oil liposomes was stable, with a peroxide value of 34 mmol / kg. Antibacterial results demonstrated its strong bactericidal effect.

[0007] The present invention has two purposes: first, to provide a novel ozonated oil liposome and a preparation method thereof; second, to provide a method for evaluating 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, comprising the steps of:

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

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

[0012] c. Deionized water is added as the aqueous phase to the oil phase to form oil-in-water ozonated oil liposomes;

[0013] d. The formed oil-in-water ozonated oil liposomes were sonicated using a cell ultrasonic disruptor to form uniform and stable ozonated oil liposomes;

[0014] e. Store the prepared ozonated oil liposomes 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 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 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 to 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 described above.

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

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

[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 and the sediment layer at the bottom of the liposomes 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 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 cream layer on the top of the liposomes and the sediment layer at the bottom to determine their stability.

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

[0040] In some embodiments, the iodine titration method is to weigh 2.5000g of ozonated oil liposome sample into a 250mL iodine volumetric flask, add 30mL of chloroform-glacial acetic acid (2:3V / V) mixture, shake gently to mix evenly, add 1mL of saturated potassium iodide (KI) solution, cover the bottle cap, shake gently 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. When the solution turns light yellow, stir vigorously during titration, add 1mL of starch indicator, the solution turns from light yellow to blue, and continue titrating until the blue disappears, which is the end point. Record the volume of titrated sodium thiosulfate, and repeat the test three times for each sample. Sunflower oil is the blank group and ozonated oil liposome is the experimental group. The peroxide value of the ozonated oil liposome is determined, and then the peroxide value-ozone mass standard curve is used to substitute the peroxide value into the standard curve to calculate the ozone mass of the ozonated oil liposome.

[0041] The formula for calculating peroxide value 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 mass of the sample (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) and evenly applying it on the surface of a solid culture medium with a sterile applicator under a sterile environment. The mixture is incubated for a period of time, and then 0.1 mL of ozonated oil liposomes are evenly applied on the surface of the culture medium. After treatment, the mixture is placed in a 37°C incubator for 24 hours. The surface of the culture medium is observed and the total number of colonies is counted in CFU / mL. Physiological saline is used 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 as follows:

[0046]

[0047] Where: 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 emulsification system in which oil is dispersed in water, with oil as the internal phase and water as the continuous external phase.

[0050] PDI: Polydispersity Index, which represents the degree of uniform dispersion of particles, also known as the particle size distribution coefficient. The smaller the value, the more uniform the particle distribution in the solution. Conversely, the larger the value, the more widely distributed the particles in the solution.

[0051] Tween 20: also known as polyoxyethylene sorbitan monolaurate, 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 detergency functions.

[0054] Tween 80: Also known as sorbitan monooleate polyoxyethylene ether, it is a chemical raw material made by polymerizing sorbitan monooleate with ethylene oxide.

[0055] Beneficial effects

[0056] Pure ozonated oil is oily and can only be applied to the skin surface. Moreover, the peroxide value of ozonated oil decreases with prolonged storage time, thereby reducing its performance. Compared with existing technologies, the present invention has the following advantages: first, the present invention solves the problem that traditional ozonated oil is limited to skin application, expanding the application market of ozonated oil; second, liposome encapsulation greatly increases the storage stability of ozonated oil, prolonging the storage time of ozonated oil, and develops a stable ozonated oil nanoliposome with diverse dosage forms (for example, the ozonated oil can be prepared into a liposome suspension for injection, etc.); third, 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 drop rate on the physicochemical properties of ozonated oil liposomes is shown (Spass analysis, N=3).

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

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

[0067] Figure 11 The results of the determination of lipid peroxide values ​​of oxidized oils are shown (Spass analysis, N=3).

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

[0069] Figure 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 present invention uses ozonated oil with a peroxide value of 150 mmol / kg as a loading object, mixes and dissolves an appropriate amount of phospholipid material and surfactant with the ozonated oil, and stirs uniformly to form a uniform and transparent oil phase. An appropriate amount of deionized water is used as the aqueous phase. During the reaction, the stirring speed and the dropping rate are controlled to form O / W type ozonated oil liposomes. The properties of the ozonated oil liposomes are determined by measuring the particle size and PDI using a cell ultrasonic disruptor to control the ultrasonic power and ultrasonic time. Ultimately, uniform and stable ozonated oil liposomes formed under the optimal process are obtained.

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

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

[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 it into a cuvette. 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: Ozonated 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: Ozonated 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: Ozonated 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: Ozonated 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: Ozonated sunflower oil liposomes and their physicochemical properties

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0130] The preparation process of ozonated oil liposomes was the same as that in Example 1, except that the ultrasonic time was changed from 10 min 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 types ( Figure 5 ), stirring speed ( Figure 6 ), the ratio of water phase to oil phase ( Figure 7 ), dripping speed ( Figure 8 ), ultrasonic power ( Figure 9 ) and ultrasound time ( Figure 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, 500 rpm stirring speed, 4:1 volume ratio of water phase to oil phase, 60 mL / h dropping speed, 350 W ultrasonic power and 10 min ultrasonic time. The liposomes prepared had the best effect, with a particle size of 110 nm and a PDI of 0.22.

[0132] Example 30: Kinetic Stability Analysis

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

[0134] Example 31: Accelerated Stability Analysis

[0135] 20 mL of the ozonated oil liposomes from Example 1 were placed in a 4°C refrigerator for 48 hours, then transferred to a 45°C incubator and allowed to stand for 48 hours, thereby completing one cycle. This procedure was repeated six times, followed by centrifugation at 1500 rpm for 30 minutes. Phase separation was observed to determine 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] The peroxide value of the ozonated oil liposomes from Example 1 was determined using the iodine titration method. According to the iodine titration method, 2.5000 g of the ozonated oil liposome sample was weighed into a 250 mL iodine titration flask. 30 mL of a chloroform-glacial acetic acid (2:3, v / v) mixture was added and gently shaken to mix thoroughly. 1 mL of saturated potassium iodide (KI) solution was added, the flask was capped, and the mixture was gently shaken for 0.5 min. The mixture was placed in the dark for 3 min. 100 mL of distilled water was then added and shaken thoroughly. After titration, the mixture was titrated with 0.1 mol / L sodium thiosulfate solution until a light yellow color was obtained. Stir vigorously during the titration. 1 mL of starch indicator was added, and the solution changed from light yellow to blue. The titration was continued until the blue color disappeared, which was the endpoint. The volume of sodium thiosulfate titrated was recorded. The test was repeated 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-ozone mass standard curve was drawn with the peroxide value as the horizontal axis and the ozone mass as the vertical axis). The peroxide value was entered into the standard curve to calculate the ozone mass of the ozonated oil liposomes.

[0139] The formula for calculating peroxide value 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 mass of the sample (g).

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

[0143] Example 33: Determination of the sterilization 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 to 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 to the surface of the culture medium. After treatment, place it in a 37℃ incubator and incubate for 24 hours. Observe the surface of the culture medium and count the total number of colonies. The unit is CFU / mL. Among them, set physiological saline as the blank group, and set 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] Where: 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 Figure 12 As shown: the left figure shows the antibacterial rate of ozonated oil and ozonated oil liposomes against Candida albicans, and the right figure shows the antibacterial rate of ozonated oil and ozonated oil liposomes against Staphylococcus aureus. In this embodiment, ozonated oil and ozonated oil liposomes with a peroxide value of 34mmoL / Kg were used. Staphylococcus aureus and Candida albicans were respectively used as indicator bacteria. The results show that under the same peroxide value, the antibacterial effect of ozonated oil is significantly higher than that of ozonated oil liposome samples. It is speculated that this is because the density of oil is small and it tightly covers the surface of the culture medium, making bacterial reproduction difficult. Ozonated oil liposomes are water-in-oil suspensions, and water evaporates easily when applied to the surface of the culture medium. This results in a sterilization rate 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 changed to ozonated oil liposomes in the range of 0.1-1 mL.

[0150] The results are as follows Figure 13 As shown, the left figure shows the bactericidal rate of ozonated oil liposomes against Staphylococcus aureus, and the right figure shows the bactericidal rate of ozonated oil liposomes against Candida albicans. Using Staphylococcus aureus and Candida albicans as indicator bacteria, the bactericidal effect of varying amounts of ozonated oil liposomes against both bacteria was investigated. The results showed that the bactericidal rate reached 100% when the ozonated oil liposomes were added at a dose of 0.4 mL for both S. aureus and C. albicans, demonstrating the significant bacteriostatic effect of ozonated oil liposomes.

[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 are only specific embodiments of the present invention and are 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 as the loading object; b The ozonated oil, surfactant, lecithin mixed stirring to form a uniform, clear oil phase, the surfactant is selected from Tween 20, Tween 40, Tween 60, Tween 80; c. Deionized water was added as the aqueous phase to the oil phase to form water-in-oil ozonated oil liposomes; d. The formed water-in-oil ozonated oil liposomes were sonicated using a cell ultrasonic disruptor to form uniform and stable ozonated oil liposomes; e. The prepared ozonated oil liposomes were stored at 2-8°C; The volume percentage content of the ozonated oil is 5-20%; The volume percentage content of the surfactant is 2-10%; The volume percentage of the phospholipids is 2.5-20%; The volume ratio of water phase to oil phase is 7:1-3:1; Stirring speed is 500-1500 rpm; The ozonated oil is ozonated sunflower oil.

2. The method according to claim 1, characterized in that The surfactant is Tween 80.

3. The method according to claim 1, characterized in that The volume percentage content of the ozonated oil is 20%.

4. The method according to claim 1 or 2, characterized in that The volume percentage content of the surfactant is 10%.

5. The method according to claim 1 or 2, characterized in that The volume percentage content of the phospholipids is 5%.

6. The method according to claim 1 or 2, characterized in that The stirring speed was 500 rpm.

7. The method according to claim 1 or 2, characterized in that The drop rate of deionized water is 10-100 mL / h.

8. The method according to claim 1 or 2, characterized in that The dropping rate of deionized water was 60 mL / h.

9. The method according to claim 1 or 2, characterized in that In step d, the ultrasonic power is 150-450 W, and the ultrasonic time is 5-20 min.

10. The method according to claim 1 or 2, characterized in that In step d, the ultrasonic power is 350 W and the ultrasonic time is 10 min.

11. Ozonized oil liposomes, prepared by the method according to any one of claims 1 to 10.

Images