Nano-emulsion loaded with terpinen-4-ol and preparation method of nano-emulsion

Abstract

The invention discloses a nano emulsion loaded with terpinen-4-ol. The nano emulsion is prepared from the following components in percentage by mass: 3 to 4 percent of terpinen-4-ol, 6 to 8 percent of an amino acid surfactant, 5 to 6 percent of a cosurfactant, 0.1 to 1 percent of sorbitan fatty acid ester, 0 to 0.5 percent of N-isopropylacrylamide, 0 to 1 percent of vitamin E and the balance of water, the amino acid surfactant is prepared from one or more of sodium lauroyl sarcosinate, sodium lauroyl glutamate, N-oleoyl sodium methyl taurate and sodium cocoyl glycinate. The particle size of the nano-emulsion is 50-100 nm, the nano-emulsion is uniform in distribution and high in stability at room temperature, N-isopropylacrylamide is introduced under the action of hydrogen bonds in a two-time freeze-drying and redissolving mode, vitamin E can form a protective film on the surface of liposome, the stability of the emulsion is enhanced, and the stability of the nano-emulsion is improved. The terpinen-4-ol is loaded into the side chain of the amino acid surfactant by adopting the amino acid surfactant, so that the toxicity and irritation are reduced, and the adverse reaction of the system is reduced.

Description

Technical Field

[0001] The present patent application relates to the field of biotechnology, and in particular to a nanoemulsion loaded with terpinene-4-ol and a preparation method thereof. Background Art

[0002] Terpinen-4-ol is an essential oil extracted from the fresh branches and leaves of Melaleuca alternifolia, a shrub species of the Myrtaceae family. It has a broad-spectrum bactericidal effect. In recent decades, terpinen-4-ol has been widely used to treat various skin infections, such as furuncle, acne, and candidal infections.

[0003] Nanoemulsion, also known as microemulsion, is one of the most widely used colloidal dispersion systems for the co-delivery of fat-soluble components. It is spontaneously formed by water, oil, surfactants, co-surfactants, etc. Generally speaking, nanoemulsions are divided into three types, namely oil-in-water nanoemulsions (O / W), water-in-oil nanoemulsions (W / O), and bicontinuous nanoemulsions (BC). The use of nanoemulsions can help improve the water solubility and bioavailability of poorly soluble drugs. The emulsion preparation method uses ultrasonic emulsification. Ultrasound is based on the cavitation phenomenon, which will produce high temperature, high pressure, strong shear force and mechanical force, and obtain an emulsion with better properties under less surfactant.

[0004] However, most common surfactants are mildly toxic. Although synthetic surfactants can improve the stability of emulsions, they may be harmful to human health. The search and application of natural surfactants is a challenge. In addition, the stability of nanoemulsions may be affected by environmental factors such as pH, temperature, and ionic strength, and the formula needs to be optimized to adapt to different application environments.

[0005] In view of the unfavorable factors in the storage and volatilization of natural plant essential oils during use, we proposed a method for preparing an amino acid surfactant self-emulsifying nanoemulsion that can load terpinene-4-ol. The release rate and targeted drug delivery are controlled by encapsulating the natural mild amino acid surfactant, reducing irritation while achieving a more stable and prolonged release, thus obtaining a nanoemulsion with good biocompatibility, safety and mildness. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and to provide a nanoemulsion loaded with terpinene-4-ol and a preparation method thereof. The nanoemulsion prepared by the method of twice freeze-drying and re-dissolving has a small and uniform particle size, can ensure the effective concentration of terpinene-4-ol, realizes responsive release at 37°C, and is stably stored at a temperature lower than this, thereby enhancing the storage stability of terpinene-4-ol and greatly reducing the allergenic side effects of natural essential oils.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In one aspect, the present invention discloses a nanoemulsion loaded with terpinene-4-ol, wherein the nanoemulsion comprises the following components in percentage by mass:

[0009] Terpinen-4-ol 3-4%, amino acid surfactant 6-8%, co-surfactant 5-6%, sorbitan fatty acid ester 0.1-1%, N-isopropylacrylamide 0-0.5%, vitamin E 0-1%, and the balance is water;

[0010] The amino acid surfactant includes one or more of sodium lauroyl sarcosinate, sodium lauroyl glutamate, sodium N-oleoyl methyl taurate, and sodium cocoyl glycinate.

[0011] Preferably, the co-surfactant includes one of glycerol and isopropyl alcohol.

[0012] Preferably, the sorbitan fatty acid ester includes one of Span-40, Span-60 and Span-80.

[0013] Preferably, the nanoemulsion comprises the following components in percentage by mass:

[0014] Terpinen-4-ol 3.76%, amino acid surfactant sodium lauroyl sarcosinate 6.8%, co-surfactant glycerol 5.5%, sorbitan fatty acid ester Span-80 0.5%, N-isopropylacrylamide 0.5%, vitamin E 1%, and the balance is water.

[0015] On the other hand, the present invention discloses a method for preparing a nanoemulsion loaded with terpinene-4-ol, comprising the following steps:

[0016] S1. Preparation of primary emulsion: at room temperature, add amino acid surfactant, co-surfactant and sorbitan fatty acid ester to water in sequence according to a set ratio, stir evenly, add terpinene-4-ol, and continue stirring until the solution is clear to obtain a primary emulsion;

[0017] S2, first freeze drying and reconstitution: the primary emulsion is freeze dried for the first time, N-isopropylacrylamide and water are added to the powder after the first freeze drying, and the powder is reconstituted for the first time and ultrasonicated to obtain a reconstituted emulsion;

[0018] S3, second freeze drying and re-dissolution: the re-dissolved emulsion is freeze dried for the second time, vitamin E and water are added to the powder after the second freeze drying, and the powder is re-dissolved for the second time and ultrasonicated to obtain a nanoemulsion loaded with terpinene-4-ol.

[0019] Preferably, adding N-isopropylacrylamide and water to S2, performing the first redissolution and ultrasonication comprises: adding N-isopropylacrylamide in a set ratio and an equal amount of water to that contained in the primary emulsion of S1, performing the first redissolution in a 60° C. water bath and ultrasonication at 20° C. for 30 min.

[0020] Preferably, adding vitamin E and water to S3, performing a second redissolution and ultrasonication comprises: adding a set ratio of vitamin E and an equal amount of water to that contained in the primary emulsion of S1, performing a second redissolution in a 60° C. water bath, and ultrasonicating at 20° C. for 30 min.

[0021] The invention also discloses the application of a nanoemulsion loaded with terpinene-4-ol in biomedicine, cosmetics and food.

[0022] The present invention has the following beneficial effects: the present invention obtains a nanoemulsion with a particle size of 50-100 nm through two freeze-drying and re-dissolving steps, and exhibits excellent stability at room temperature. By introducing N-isopropylacrylamide through the action of hydrogen bonds in the first freeze-drying and re-dissolving method, it can be spontaneously assembled to form micelles with a core-shell structure, enhancing the resolubility of the micelles, and can become a potential drug carrier to prolong the release of drugs. The vitamin E added in the second freeze-drying and re-dissolving method can form a protective film on the surface of the liposome, which helps to be more evenly distributed in the liposome structure and enhance the stability of the emulsion. Compared with the nanoemulsion without freeze-drying and re-dissolving steps, it has a more uniform particle size distribution and higher stability and antioxidant properties.

[0023] The electron microscope result of the nanoemulsion loaded with terpinene-4-ol of the present invention is microsphere-shaped, and the particle size distribution is uniform, which is consistent with the expected result of the water-in-oil emulsion; the amino acid surfactant used in the present invention has a hydrophobic long chain, is naturally mild, can better load turpentine, and loads terpinene-4-ol into the side chain of the amino acid surfactant, which can reduce toxicity and irritation, greatly reduce the adverse reaction of the system, and improve the use limitation of terpinene-4-ol; no irritation and inflammatory reaction are found in chicken embryo chorioallantoic membrane and rabbit eye experiments; it is slowly released and performs excellently in biocompatibility and stability experimental results. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1This is a photo of the nanoemulsion loaded with terpinene-4-ol prepared in Example 1 of Test Example 1.

[0026] Figure 2 This is a photograph of the nanoemulsion loaded with terpinene-4-ol prepared in Example 1 using the staining method in Test Example 1. Bottle A is added with methylene blue, and bottle B is added with Sudan III.

[0027] Figure 3 The particle size distribution diagrams of the nanoemulsion loaded with terpinene-4-ol prepared in Example 1 and Example 2 in Test Example 2: (a) is Example 1, and (b) is Example 2;

[0028] Figure 4 The DPPH antioxidant performance evaluation diagram of the nanoemulsion loaded with terpinene-4-ol prepared in Example 1 and Example 2 in Test Example 3: (a) is Example 1, and (b) is Example 2;

[0029] Figure 5 This is a graph for evaluating cell survival rate in the nanoemulsion loaded with terpinene-4-ol prepared in Example 1 and Example 2 in Test Example 4;

[0030] Figure 6 This is a biocompatibility evaluation diagram of the nanoemulsion loaded with terpinene-4-ol prepared in Example 1 and Example 3 in Test Example 5;

[0031] Figure 7 Comparison photos of 0.1 mol / L NaOH solution acting on CAM membrane before and after 5 minutes in Test Example 6 (positive control): (a) before the action; (b) after the action;

[0032] Figure 8 Comparison photos of 0.9% w / v saline solution acting on the CAM membrane before and after 5 minutes in Test Example 6 (negative control): (a) before the action; (b) after the action;

[0033] Fig. 9 Comparison photos of the CAM membrane before and after 5 minutes of 1% terpinene-4-ol solution acting on it in Test Example 6 (negative control): (a) before the action; (b) after the action;

[0034] Fig.10 Comparison photos of the nanoemulsion loaded with terpinene-4-ol prepared in Example 1 of Test Example 6 acting on the CAM membrane before and after 5 minutes: (a) before the action; (b) after the action;

[0035] Fig.11 Comparison photos of the nanoemulsion loaded with terpinene-4-ol prepared in Example 2 acting on the CAM membrane before and after 5 minutes: (a) before the action; (b) after the action;

[0036] Fig.12 The particle size distribution diagram of the nanoemulsion prepared in Example 1, Comparative Example 1 and Comparative Example 2;

[0037] Fig.13 This is an electron microscope photo of the nanoemulsion A prepared in Comparative Example 1;

[0038] Fig.14 This is an electron microscope photo of the nanoemulsion B prepared in Comparative Example 2;

[0039] Fig.15 This is an electron microscope photograph of nanoemulsion C prepared in comparative example 3.

[0040] The above drawings have shown clear embodiments of the present invention, which will be described in more detail below. These drawings and text descriptions are not intended to limit the scope of the present invention in any way, but to illustrate the concept of the present invention for those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the examples. The following examples are only used to more clearly illustrate the performance of the present invention, and are not limited to the following examples.

[0042] A nanoemulsion loaded with terpinene-4-ol, the nanoemulsion comprising the following components in percentage by mass:

[0043] Terpinen-4-ol 3-4%, amino acid surfactant 6-8%, co-surfactant 5-6%, sorbitan fatty acid ester 0.1-1%, N-isopropylacrylamide 0-0.5%, vitamin E 0-1%, and the balance is water;

[0044] The amino acid surfactant includes one or more of sodium lauroyl sarcosinate, sodium lauroyl glutamate, sodium N-oleoyl methyl taurate, and sodium cocoyl glycinate.

[0045] The prepared nanoemulsion loaded with terpinene-4-ol has an amino acid surfactant with a long hydrophobic chain, which can better load terpinene-4-ol. The nanoemulsion is in a microsphere shape with a particle size of 50-100 nm, which greatly reduces the allergic reaction generated when directly used. The nanoemulsion is natural and mild, easy to transport and store, and has good stability. The use effect will not be reduced, and the use limitation of terpinene-4-ol is improved.

[0046] In one possible design, the co-surfactant includes one of glycerol and isopropyl alcohol.

[0047] Preferably, the co-surfactant is glycerol.

[0048] In a possible design, the sorbitan fatty acid ester includes one of Span-40, Span-60 and Span-80.

[0049] Preferably, the sorbitan fatty acid ester is Span-80.

[0050] It is understandable that glycerol is easily soluble in water and lipophilic in the system, which can improve the flexibility and firmness of the interfacial film, thereby reducing the viscosity of the system, and as a cosurfactant, it is helpful for the absorption and utilization of drugs. Compared with isopropanol, when glycerol is used as a cosurfactant, the microemulsion area of ​​the formed nanoemulsion is larger, which is helpful for the stability and formation of the nanoemulsion.

[0051] In one possible design, the nanoemulsion contains the following components in mass percentages:

[0052] Terpinen-4-ol 3.76%, amino acid surfactant sodium lauroyl sarcosinate 6.8%, co-surfactant glycerol 5.5%, sorbitan fatty acid ester Span-80 0.5%, N-isopropylacrylamide 0.5%, vitamin E 1%, and the balance is water.

[0053] A method for preparing a nanoemulsion loaded with terpinene-4-ol comprises the following steps:

[0054] S1. Preparation of primary emulsion: at room temperature, add amino acid surfactant, co-surfactant and sorbitan fatty acid ester to water in sequence according to a set ratio, stir evenly, add terpinene-4-ol, and continue stirring until the solution is clear to obtain a primary emulsion;

[0055] S2, first freeze drying and reconstitution: the primary emulsion is freeze dried for the first time, N-isopropylacrylamide and water are added to the powder after the first freeze drying, and the powder is reconstituted for the first time and ultrasonicated to obtain a reconstituted emulsion;

[0056] It should be noted that glycerol, as a component of the primary emulsion, is used as a co-surfactant to stabilize the nanoemulsion, which can enhance the emulsification of the solution, protect the structure of the liposome, reduce the damage to the liposome structure, maintain the particle size and encapsulation rate of the liposome, and is beneficial to the release and bioavailability of the drug, thereby ensuring the stability and effectiveness of the liposome during storage and use, and increasing the retention time of the drug in the body, thereby improving the efficacy of the drug; on the other hand, glycerol can also be used as a protective agent for the first freeze-drying and re-dissolution. Since the removal and re-dissolution of water during the freeze-drying and re-dissolution process will lead to the instability of the nanoemulsion, the presence of glycerol can improve the stability and rehydration of the freeze-dried nanoemulsion, so that the freeze-dried nanoemulsion can be quickly and completely re-dispersed in water.

[0057] It is understandable that glycerol, as one of the components in the nanoemulsion, does not need to be processed after the freeze-drying process, which not only reduces the loss of drug but also plays a role in stabilizing the emulsion.

[0058] S3. Second freeze drying and re-dissolution: The re-dissolved emulsion is freeze dried for the second time, vitamin E and water are added to the powder after the second freeze drying, and the powder is re-dissolved for the second time and ultrasonicated to obtain a nanoemulsion loaded with terpinene-4-ol.

[0059] It should be noted that N-isopropylacrylamide itself is temperature-responsive above 37°C. The purpose of adding N-isopropylacrylamide is to utilize the temperature-sensitive properties of NIPAM to prepare an intelligent drug delivery system and achieve precise control of drug release. At temperatures close to human body temperature, the expansion or contraction of PNIPAM can trigger the release of terpinen-4-ol. However, if N-isopropylacrylamide is added to the solution and added to the nanoemulsion by stirring, its drug loading is limited, the temperature responsiveness cannot be fully utilized, and the long-term controlled release of the drug cannot be obtained; if it is synthesized, on the one hand, the process is complicated and the yield is low, and on the other hand, it is easy to introduce unnecessary impurities, increasing the toxicity of the nanoemulsion.

[0060] After adding N-isopropylacrylamide, the present invention can form a relatively strong hydrogen bond between N-isopropylacrylamide and the hydrophobic monomer during the second freeze-drying process, thereby copolymerizing to form a thermosensitive block copolymer, which can spontaneously assemble into a core-shell structured micelle in a specific solvent, with a hydrophobic core at the center and a hydrophilic shell at the periphery. This structure can enhance the solubility of the micelle on the one hand, and make it a potential drug carrier on the other hand, which is particularly suitable for the long-term controlled release of hydrophobic drugs. At the same time, no new substances are introduced, and the product is safer.

[0061] It is understandable that vitamin E is an effective antioxidant that can protect the nanoemulsion from damage by oxidative stress. Its addition in the last step of the preparation, the re-dissolution process, can form a protective layer on the surface of the liposomes, which helps to evenly distribute vitamin E in the liposome structure and improve the stability, avoiding affecting the structure of N-isopropylacrylamide and the permeability of glycerol to liposomes.

[0062] In a possible design, N-isopropylacrylamide and water are added to S2 for the first reconstitution and ultrasonication, which includes: adding a set ratio of N-isopropylacrylamide and an equal amount of water to that contained in the primary emulsion of S1, reconstitution for the first time in a 60°C water bath, and ultrasonication at 20°C for 30 minutes.

[0063] In a possible design, vitamin E and water are added to S3, and the second reconstitution and ultrasound include: adding a set ratio of vitamin E and an equal amount of water to that contained in the primary emulsion of S1, reconstitution for the second time in a 60°C water bath and ultrasound at 20°C for 30 minutes.

[0064] The invention also discloses an application of loaded terpinene-4-ol in biomedicine, cosmetics and food.

[0065] The present invention is further described below with reference to a specific embodiment.

[0066] Unless otherwise specified, the experimental methods used in the following specific examples are conventional methods.

[0067] In the following specific examples, the operations involved in the operation without specifying the conditions were all carried out under conventional conditions or the conditions recommended by the manufacturer. The raw materials used without specifying the manufacturer and specifications were all conventional products that can be obtained through commercial purchase.

[0068] Example 1

[0069] S1. Sodium lauroyl sarcosinate, glycerol and Span-80 were added to water in sequence. After being evenly mixed with a magnetic stirrer, terpinene-4-ol was added dropwise while stirring. The speed was first fast and then slow. When the last drop was added, terpinene-4-ol floated on the surface of the solution and did not gather into oil droplets. At this time, stirring was continued at a speed of 3500r / min for 60s, and water was added dropwise with a syringe. When the solution changed from turbid to clear, the addition was stopped, and then stirred at 2500r / min for 50s to obtain a primary emulsion; the mass ratio of terpinene-4-ol, sodium lauroyl sarcosinate, glycerol and Span-80 was 3.76:6.8:5.5:0.5;

[0070] S2, first freeze drying and reconstitution: freeze the primary emulsion at -80°C overnight, then put it into a freeze dryer for first freeze drying, add N-isopropylacrylamide and water to the powder after the first freeze drying, reconstitute it in a 60°C water bath, and ultrasonicate it at 20°C for 30 minutes to obtain a reconstituted emulsion;

[0071] The mass ratio of N-isopropylacrylamide to terpinen-4-ol in S1 is 0.5:3.76; the amount of water added is the same as the amount of water contained in the primary emulsion in S1;

[0072] S3, second freeze drying and reconstitution: the reconstituted emulsion is placed in -80 ° C. and frozen overnight, and then placed in a freeze dryer for a second freeze drying. Vitamin E and water are added to the powder after the second freeze drying, and the powder is reconstituted for a second time in a 60 ° C. water bath and ultrasonicated at 20 ° C. for 30 minutes to obtain a nanoemulsion loaded with terpinene-4-ol;

[0073] Among them, the mass ratio of vitamin E to terpinene-4-ol in S1 is 1:3.76; the amount of added water is the same as the amount of water contained in the primary emulsion in S1.

[0074] Example 2

[0075] S1. Sodium cocoyl glutamate, glycerol and Span-80 were added to water in sequence. After being evenly mixed with a magnetic stirrer, terpinene-4-ol was added dropwise while stirring. The speed was first fast and then slow. When the last drop was added, terpinene-4-ol floated on the surface of the solution and did not converge into oil droplets. At this time, stirring was continued at a speed of 3500r / min for 60s, and then water was added dropwise with a syringe. When the solution changed from turbid to clear, the addition was stopped, and then stirred at 2500r / min for 50s to obtain a primary emulsion; the mass ratio of terpinene-4-ol, sodium cocoyl glutamate, glycerol and Span-80 was 3.76:6.8:5.5:0.5;

[0076] S2, first freeze drying and reconstitution: freeze the primary emulsion at -80°C overnight, then put it into a freeze dryer for first freeze drying, add N-isopropylacrylamide and water to the powder after the first freeze drying, reconstitute it in a 60°C water bath, and ultrasonicate it at 20°C for 30 minutes to obtain a reconstituted emulsion;

[0077] The mass ratio of N-isopropylacrylamide to terpinen-4-ol in S1 is 0.5:3.76; the amount of water added is the same as the amount of water contained in the primary emulsion in S1;

[0078] S3, second freeze drying and reconstitution: the reconstituted emulsion is placed in -80 ° C. and frozen overnight, and then placed in a freeze dryer for a second freeze drying. Vitamin E and water are added to the powder after the second freeze drying, and the powder is reconstituted for a second time in a 60 ° C. water bath and ultrasonicated at 20 ° C. for 30 minutes to obtain a nanoemulsion loaded with terpinene-4-ol;

[0079] Among them, the mass ratio of vitamin E to terpinene-4-ol in S1 is 1:3.76; the amount of added water is the same as the amount of water contained in the primary emulsion in S1.

[0080] Example 3

[0081] S1. Sodium lauroyl glycine, glycerol and Span-80 were added to water in sequence. After being evenly mixed with a magnetic stirrer, terpinene-4-ol was added dropwise while stirring. The speed was first fast and then slow. When the last drop was added, terpinene-4-ol floated on the surface of the solution and did not gather into oil droplets. At this time, stirring was continued at a speed of 3500 r / min for 60 s, and water was added dropwise with a syringe. When the solution changed from turbid to clear, the addition was stopped, and then stirred at 2500 r / min for 50 s to obtain a primary emulsion; the mass ratio of terpinene-4-ol, sodium lauroyl glycine, glycerol and Span-80 was 3.76:6.8:5.5:0.5;

[0082] S2, first freeze drying and reconstitution: freeze the primary emulsion at -80°C overnight, then put it into a freeze dryer for first freeze drying, add N-isopropylacrylamide and water to the powder after the first freeze drying, reconstitute it in a 60°C water bath, and ultrasonicate it at 20°C for 30 minutes to obtain a reconstituted emulsion;

[0083] The mass ratio of N-isopropylacrylamide to terpinen-4-ol in S1 is 0.5:3.76; the amount of water added is the same as the amount of water contained in the primary emulsion in S1;

[0084] S3, second freeze drying and reconstitution: the reconstituted emulsion is placed in -80 ° C. and frozen overnight, and then placed in a freeze dryer for a second freeze drying. Vitamin E and water are added to the powder after the second freeze drying, and the powder is reconstituted for a second time in a 60 ° C. water bath and ultrasonicated at 20 ° C. for 30 minutes to obtain a nanoemulsion loaded with terpinene-4-ol;

[0085] Among them, the mass ratio of vitamin E to terpinene-4-ol in S1 is 1:3.76; the amount of added water is the same as the amount of water contained in the primary emulsion in S1.

[0086] Example 4

[0087] S1. Sodium N-oleoyl methyl taurate, glycerol and Span-80 were added to water in sequence. After being evenly mixed with a magnetic stirrer, terpinene-4-ol was added dropwise while stirring. The speed was first fast and then slow. When the last drop was added, terpinene-4-ol floated on the surface of the solution and did not gather into oil droplets. At this time, stirring was continued at a speed of 3500 r / min for 60 s, and water was added dropwise with a syringe. When the solution changed from turbid to clear, the addition was stopped, and then stirring was continued at 2500 r / min for 50 s to obtain a primary emulsion; the mass ratio of terpinene-4-ol, sodium N-oleoyl methyl taurate, glycerol and Span-80 was 3.76:6.8:5.5:0.5;

[0088] S2, first freeze drying and reconstitution: freeze the primary emulsion at -80°C overnight, then put it into a freeze dryer for first freeze drying, add N-isopropylacrylamide and water to the powder after the first freeze drying, reconstitute it in a 60°C water bath, and ultrasonicate it at 20°C for 30 minutes to obtain a reconstituted emulsion;

[0089] The mass ratio of N-isopropylacrylamide to terpinen-4-ol in S1 is 0.5:3.76; the amount of water added is the same as the amount of water contained in the primary emulsion in S1;

[0090] S3, second freeze drying and reconstitution: the reconstituted emulsion is placed in -80 ° C. and frozen overnight, and then placed in a freeze dryer for a second freeze drying. Vitamin E and water are added to the powder after the second freeze drying, and the powder is reconstituted for a second time in a 60 ° C. water bath and ultrasonicated at 20 ° C. for 30 minutes to obtain a nanoemulsion loaded with terpinene-4-ol;

[0091] Among them, the mass ratio of vitamin E to terpinene-4-ol in S1 is 1:3.76; the amount of added water is the same as the amount of water contained in the primary emulsion in S1.

[0092] Example 5

[0093] S1. Sodium lauroyl glutamate, glycerol and Span-80 were added to water in sequence. After being evenly mixed with a magnetic stirrer, terpinene-4-ol was added dropwise while stirring. The speed was first fast and then slow. When the last drop was added, terpinene-4-ol floated on the surface of the solution and did not gather into oil droplets. At this time, stirring was continued at a speed of 3500r / min for 60s, and water was added dropwise with a syringe. When the solution changed from turbid to clear, the addition was stopped, and then stirred at 2500r / min for 50s to obtain a primary emulsion; the mass ratio of terpinene-4-ol, sodium lauroyl glutamate, glycerol and Span-80 was 3:6:5:0.1;

[0094] S2, first freeze drying and reconstitution: freeze the primary emulsion at -80°C overnight, then put it into a freeze dryer for first freeze drying, add N-isopropylacrylamide and water to the powder after the first freeze drying, reconstitute it in a 60°C water bath, and ultrasonicate it at 20°C for 30 minutes to obtain a reconstituted emulsion;

[0095] The mass ratio of N-isopropylacrylamide to terpinen-4-ol in S1 is 0.5:3; the amount of water added is the same as the amount of water contained in the primary emulsion in S1;

[0096] S3, second freeze drying and reconstitution: the reconstituted emulsion is placed in -80 ° C. and frozen overnight, and then placed in a freeze dryer for a second freeze drying. Vitamin E and water are added to the powder after the second freeze drying, and the powder is reconstituted for a second time in a 60 ° C. water bath and ultrasonicated at 20 ° C. for 30 minutes to obtain a nanoemulsion loaded with terpinene-4-ol;

[0097] Among them, the mass ratio of vitamin E to terpinene-4-ol in S1 is 1:3; the amount of added water is the same as the amount of water contained in the primary emulsion in S1.

[0098] Example 6

[0099] S1. Add a mixture of sodium N-oleoyl methyl taurate and sodium cocoyl glycinate, isopropyl alcohol and Span-60 in water in sequence, mix well with a magnetic stirrer, and add terpinene-4-ol dropwise while stirring at a speed first fast and then slow. When the last drop is added, terpinene-4-ol floats on the surface of the solution and does not gather into oil droplets. At this time, continue stirring at a speed of 3500r / min for 60s, then add water dropwise with a syringe, stop dropping when the solution changes from turbid to clear, and then stir at 2500r / min for 50s to obtain a primary emulsion; the mass ratio of terpinene-4-ol, sodium N-oleoyl methyl taurate, sodium cocoyl glycinate, isopropyl alcohol and Span-60 is 4:4:4:6:1;

[0100] S2, first freeze drying and reconstitution: freeze the primary emulsion at -80°C overnight, then put it into a freeze dryer for first freeze drying, add N-isopropylacrylamide and water to the powder after the first freeze drying, reconstitute it in a 60°C water bath, and ultrasonicate it at 20°C for 30 minutes to obtain a reconstituted emulsion;

[0101] The mass ratio of N-isopropylacrylamide to terpinen-4-ol in S1 is 0.3:4; the amount of water added is the same as the amount of water contained in the primary emulsion in S1;

[0102] S3, second freeze drying and reconstitution: the reconstituted emulsion is placed in -80 ° C. and frozen overnight, and then placed in a freeze dryer for a second freeze drying. Vitamin E and water are added to the powder after the second freeze drying, and the powder is reconstituted for a second time in a 60 ° C. water bath and ultrasonicated at 20 ° C. for 30 minutes to obtain a nanoemulsion loaded with terpinene-4-ol;

[0103] The mass ratio of vitamin E to terpinene-4-ol in S1 is 0.5:4; the amount of water added is the same as the amount of water contained in the primary emulsion in S1.

[0104] Example 7

[0105] S1. Sodium lauroyl sarcosinate, glycerol and Span-40 were added to water in sequence. After being evenly mixed with a magnetic stirrer, terpinene-4-ol was added dropwise while stirring. The speed was first fast and then slow. When the last drop was added, terpinene-4-ol floated on the surface of the solution and did not gather into oil droplets. At this time, stirring was continued at a speed of 3500r / min for 60s, and water was added dropwise with a syringe. When the solution changed from turbid to clear, the addition was stopped, and then stirred at 2500r / min for 50s to obtain a primary emulsion; the mass ratio of terpinene-4-ol, sodium lauroyl sarcosinate, glycerol and Span-40 was 3.76:6.8:5.5:0.5;

[0106] S2, first freeze drying and reconstitution: freeze the primary emulsion at -80°C overnight, then put it into a freeze dryer for first freeze drying, add N-isopropylacrylamide and water to the powder after the first freeze drying, reconstitute it in a 60°C water bath, and ultrasonicate it at 20°C for 30 minutes to obtain a reconstituted emulsion;

[0107] The mass ratio of N-isopropylacrylamide to terpinen-4-ol in S1 is 0.5:3.76; the amount of water added is the same as the amount of water contained in the primary emulsion in S1;

[0108] S3, second freeze drying and reconstitution: the reconstituted emulsion is placed in -80 ° C. and frozen overnight, and then placed in a freeze dryer for a second freeze drying. Vitamin E and water are added to the powder after the second freeze drying, and the powder is reconstituted for a second time in a 60 ° C. water bath and ultrasonicated at 20 ° C. for 30 minutes to obtain a nanoemulsion loaded with terpinene-4-ol;

[0109] Among them, the mass ratio of vitamin E to terpinene-4-ol in S1 is 1:3.76; the amount of added water is the same as the amount of water contained in the primary emulsion in S1.

[0110] Test Example 1 Nanoemulsion Appearance

[0111] Figure 1 This is a photo of the nanoemulsion loaded with terpinene-4-ol prepared in Example 1. It appears as a transparent and uniform liquid when observed with the naked eye. There is no precipitation or flocculation after centrifugation, and it has a Tyndall effect.

[0112] Figure 2 The dyeing method is used to identify the type of nanoemulsion. The diffusion speed of the oil-soluble dye Sudan III (red) and the water-soluble dye methylene blue (blue) in the nanoemulsion is used to determine the type of nanoemulsion. Take equal amounts of nanoemulsion in two test tubes, add appropriate and equal amounts of methylene blue (bottle A) and Sudan III (bottle B), respectively. The results show that the diffusion speed of methylene blue in the nanoemulsion (bottle A) is faster than that of Sudan III (bottle B), indicating that the nanoemulsion is an O / W type nanoemulsion.

[0113] Test Example 2 Particle Size Distribution of Nanoemulsion

[0114] The average particle size of the terpinene-4-ol nanoemulsion was measured using a Malvern particle size analyzer. The particle size directly affects the stability and application effect of the nanoemulsion. The particle size of the nanoemulsion is usually 50-500nm, and a PDI value of less than 0.3 is generally considered to be a good particle size distribution uniformity.

[0115] Figure 3 It is the particle size distribution diagram of embodiment 1 and 2, and the particle size analysis result shows that the average particle size of the nanoemulsion is 50-100nm. Table 1 is the particle size distribution and PDI of the nanoemulsion prepared by different amino acid surfactants in embodiment 1-4. It can be seen from Table 1 that the nanoemulsion particle size is about 80nm, indicating that the nanoemulsion system is evenly distributed and no obvious aggregation occurs. Smaller particle size usually means higher stability and better biocompatibility, and can provide a larger surface area, thereby enhancing the solubility and bioavailability of terpinene-4-ol.

[0116] Table 1 Particle size distribution and PDI of nanoemulsions prepared from different amino acid surfactants in Examples 1-4

[0117]

[0118] Test Example 3 DPPH Antioxidant Activity of Nanoemulsion

[0119] DPPH·+ is a stable free radical dissolved in organic solutions. Its methanol or ethanol solution will appear purple, reaching its maximum peak at a wavelength of 517nm. When a free radical scavenger appears, the single electron of DPPH·+ will be captured by it, and its color will become lighter or even colorless, so it can be used to judge the antioxidant function of the experimental product.

[0120] This test example uses ascorbic acid as a control. Figure 4 It can be seen that within the experimental concentration range (0.1-2 mg·L -1 ), the concentration of sodium lauroyl sarcosinate nanoemulsion ranged from 0.1 mg·L -1 Increase to 2 mg·L -1 , the DPPH antioxidant activity increased by about 40%, and the concentration of sodium cocoyl glutamate nanoemulsion increased from 0.1 mg·L -1 Increase to 2 mg·L -1, DPPH antioxidant activity is improved by about 35%, and the nanoemulsion prepared by the present invention can improve its own scavenging ability as the concentration increases. The antioxidant performance has been improved to a certain extent. When the amino acid surfactant reaches a certain concentration in the aqueous solution, micelles will be formed. These micelles can provide a microenvironment to promote the reaction between the antioxidant and the DPPH free radical. In addition, compared with other surfactants, the amino acid surfactant has a stronger hydrogen bonding force, which helps to improve the antioxidant performance.

[0121] Test Example 4 Cytotoxicity Experiment of Nanoemulsion

[0122] The cytotoxicity of nanoemulsion to HSF (human epidermal fibroblasts) was determined by MTT method. Figure 5 It can be seen that within the experimental concentration range (0.1-4.0 mg·L -1 ) has a proliferation effect on HSF cells, indicating that 0.1-4.0 mg·L -1 Within the range of , the nanoemulsion is very safe for HSF cells.

[0123] Test Example 5 Biocompatibility Experiment of Nanoemulsion

[0124] The collected blood was placed in a vacuum tube containing an anticoagulant to prevent blood clotting. Then 1 mL of blood was transferred to a centrifuge tube and centrifuged at 1000 rpm / min. -1 Centrifuge at a speed of 5 minutes to separate the blood, and repeatedly rinse the separated red blood cells with physiological saline until they become colorless. Place the nanoemulsions of each concentration gradient and the separated red blood cells in a centrifuge tube in a water bath preheated to 37°C, and set the heating time to 30 minutes; then centrifuge the sample, take out the supernatant, and measure the sample's absorption peak at 540nm with an enzyme marker. The samples were measured three times in parallel, with 1% Triton X-100 and PBS as the positive and negative control groups, respectively, and the hemolysis rate was calculated. Figure 6 It can be seen that the hemolysis rate of rabbit red blood cells by nanoemulsion is less than 5%. Amino acid surfactant (AAS) is an environmentally friendly surfactant based on biomass, with an amino acid skeleton structure in the molecule. This type of surfactant has a wide source of biomass raw materials, low toxicity and side effects, mild performance, low irritation and good biodegradability.

[0125] Test Example 6 Chicken Embryo Chorioallantoic Membrane Experiment

[0126] (1) Comparison of 0.1 mol / L NaOH solution before and after 5 minutes of exposure to CAM membrane (positive control)

[0127] Check whether the fertilized chicken embryo is damaged, clean the outer surface with 75% ethanol, place it with the narrow end facing down in a constant temperature and humidity chamber, and incubate it at 37°C and 50% to 70% humidity for 7-9 days. 2 Pour 0.1 mol / L NaOH solution into the test ring through the window, observe the changes in blood vessels, take pictures and assign points to the stimulation level according to the provisions of the national standard. Figure 7 These are comparison photos taken after 0.1 mol / L NaOH solution acted on the CAM membrane for 5 minutes (positive control).

[0128] (2) Comparison of 0.9% w / v saline solution before and after 5 minutes of exposure to CAM membrane (negative control)

[0129] Check whether the fertilized chicken embryo is damaged, clean the outer surface with 75% ethanol, place it with the narrow end facing down in a constant temperature and humidity chamber, and incubate it at 37°C and 50% to 70% humidity for 7-9 days. 2 Pour 0.9% w / v saline into the test ring through the window, observe the changes in blood vessels, take pictures and assign points to the stimulation level according to the provisions of the national standard. Figure 8 These are comparison photos taken after 0.9% w / v saline was applied to the CAM membrane for 5 minutes (negative control).

[0130] (3) Comparison of 1% terpinene-4-ol solution before and after 5 minutes of exposure to CAM membrane

[0131] Check whether the fertilized chicken embryo is damaged, clean the outer surface with 75% ethanol, place it with the narrow end facing down in a constant temperature and humidity chamber, and incubate it at 37°C and 50% to 70% humidity for 7-9 days. 2 1% terpinene-4-ol solution was dripped into the test ring through the window, the changes in blood vessels were observed, photos were taken and the stimulation level was assigned points according to the provisions of the national standard. Fig. 9 These are comparison photos taken after 1% terpinene-4-ol solution was applied to the CAM membrane for 5 minutes.

[0132] (4) Comparison of the effect of the nanoemulsion loaded with terpinene-4-ol prepared in Example 1 on the CAM membrane before and after 5 minutes

[0133] Check whether the fertilized chicken embryo is damaged, clean the outer surface with 75% ethanol, place it with the narrow end facing down in a constant temperature and humidity chamber, and incubate it at 37°C and 50% to 70% humidity for 7-9 days. 2 The prepared amino acid surfactant nanoemulsion is added dropwise into the test ring through the window, the changes in blood vessels are observed, photos are taken, and the stimulation level is assigned points according to the provisions of the national standard. Fig.10Comparison photos of the nanoemulsion loaded with terpinene-4-ol prepared in Example 1 acting on the CAM membrane before and after 5 minutes

[0134] (5) Comparison of the nanoemulsion loaded with terpinene-4-ol prepared in Example 2 acting on the CAM membrane before and after 5 minutes

[0135] Check whether the fertilized chicken embryo is damaged, clean the outer surface with 75% ethanol, place it in a constant temperature and humidity chamber with the narrow end facing down, and incubate it at 37°C and 50% to 70% humidity for 7-9 days. 2 The prepared amino acid surfactant nanoemulsion is added dropwise into the test ring through the window, the changes in blood vessels are observed, photos are taken, and the stimulation level is assigned points according to the provisions of the national standard. Fig.11 Comparison photos of the nanoemulsion loaded with terpinene-4-ol prepared in Example 2 acting on the CAM membrane before and after 5 minutes.

[0136] Table 2 lists the results of the chick embryo chorioallantoic membrane experiment. Fig. 9 It can be seen that after 1% terpinene-4-ol solution acted on the CAM membrane, the blood vessels showed obvious rupture and bleeding. According to the experimental observations and the results calculated in the national standard, moderate stimulation was caused to the blood vessels.

[0137] After the nanoemulsion prepared by using 1% terpinene-4-ol in the embodiment 1 and 2 acts on the CAM membrane, no bleeding, coagulation and other phenomena occur in the blood vessels, and the calculation verification is mild stimulation, which meets the national standard safety standard. This is because the droplet size of the nanoemulsion loaded with terpinene-4-ol is small, about 80nm, with a large specific surface area and a high encapsulation rate, which can improve the transmission efficiency of terpinene-4-ol and reduce the amount of direct contact of the drug with the skin or mucous membrane, thereby reducing irritation. The nanoemulsion of the present invention is equipped with low-irritation raw materials, which can achieve the control effect of long-acting sustained release, is more conducive to the transmission and transdermal absorption of terpinene-4-ol in the internal phase, can reduce the use of penetration enhancers, thereby reducing potential irritation.

[0138] Table 2 Chicken embryo chorioallantoic membrane experimental results

[0139]

[0140] Comparative Example 1

[0141] Sodium lauroyl sarcosinate, glycerol and Span-80 were added to water in sequence, and after being mixed evenly with a magnetic stirrer, terpinene-4-ol was added dropwise while stirring, at a speed first fast and then slowly. When the last drop was added, terpinene-4-ol floated on the surface of the solution and did not gather into oil droplets. At this time, stirring was continued at a speed of 3500 r / min for 60 seconds, and water was added dropwise with a syringe. When the solution changed from turbid to clear, the addition was stopped, and then stirring was continued at 2500 r / min for 50 seconds, and then N-isopropylacrylamide and vitamin E were added, and stirring was continued at 2500 r / min for 120 seconds until the solution was clear again, to obtain nanoemulsion A; wherein the mass ratio of terpinene-4-ol, sodium lauroyl sarcosinate, glycerol, Span-80, N-isopropylacrylamide and vitamin E was 3.76:6.8:5.5:0.5:0.5:1;

[0142] Comparative Example 2

[0143] S1. Sodium lauroyl sarcosinate, glycerol and Span-80 were added to water in sequence. After being evenly mixed with a magnetic stirrer, terpinene-4-ol was added dropwise while stirring. The speed was first fast and then slow. When the last drop was added, terpinene-4-ol floated on the surface of the solution and did not gather into oil droplets. At this time, stirring was continued at 3500r / min for 60s, and water was added dropwise with a syringe. When the solution changed from turbid to clear, the addition was stopped, and then stirring was continued at 2500r / min for 50s to obtain a primary emulsion.

[0144] Among them, the mass ratio of terpinene-4-ol, sodium lauroyl sarcosinate, glycerol and Span-80 is 3.76:6.8:5.5:0.5;

[0145] S2, first freeze drying and reconstitution: the primary emulsion was placed in -80 ° C for freezing overnight, and then placed in a freeze dryer for the first freeze drying. N-isopropylacrylamide, vitamin E and water were added to the powder after the first freeze drying, and the first reconstitution was carried out in a 60 ° C water bath and ultrasonicated at 20 ° C for 30 min to obtain nanoemulsion B;

[0146] Among them, the mass ratio of N-isopropylacrylamide, vitamin E and terpinen-4-ol in S1 is 0.5:1:3.76; the amount of water added is the same as the amount of water contained in the primary emulsion in S1.

[0147] Comparative Example 3

[0148] S1. Sodium lauroyl sarcosinate, glycerol and Span-80 were added to water in sequence. After being evenly mixed with a magnetic stirrer, terpinene-4-ol was added dropwise while stirring. The speed was first fast and then slow. When the last drop was added, terpinene-4-ol floated on the surface of the solution and did not gather into oil droplets. At this time, stirring was continued at a speed of 3500r / min for 60s, and water was added dropwise with a syringe. When the solution changed from turbid to clear, the addition was stopped, and then stirred at 2500r / min for 50s to obtain a primary emulsion; the mass ratio of terpinene-4-ol, sodium lauroyl sarcosinate, glycerol and Span-80 was 3.76:6.8:5.5:0.5;

[0149] S2, first freeze drying and reconstitution: freeze the primary emulsion at -80°C overnight, then put it into a freeze dryer for first freeze drying, add water in the same amount as that of the primary emulsion in S1 to the powder after the first freeze drying, reconstitute it for the first time in a 60°C water bath and ultrasonicate it at 20°C for 30 minutes to obtain a reconstituted emulsion;

[0150] S3. Second freeze drying and reconstitution: The reconstituted emulsion was frozen at -80°C overnight, and then placed in a freeze dryer for a second freeze drying. The same amount of water as that contained in the primary emulsion in S1 was added to the powder after the second freeze drying, and the first reconstitution was carried out in a 60°C water bath and ultrasonicated at 20°C for 30 min to obtain nanoemulsion C.

[0151] The nanoemulsion A of comparative example 1 was not subjected to freeze drying and reconstituting process, the nanoemulsion B of comparative example 2 was subjected to only one freeze drying and reconstituting step, and the nanoemulsion C of comparative example 3 was subjected to two freeze drying and reconstituting steps, but N-isopropylacrylamide and vitamin E were not added.

[0152] pass Figure 13-15 From the transmission electron microscope photos, we can see that the particle size of nanoemulsion A is about 500nm, which is obviously too large. The appearance and texture do not meet expectations and the stability is poor. Nanoemulsion B has droplet aggregation or fusion. On the one hand, this aggregation may be caused by the formation and growth of ice crystals during the freeze-drying process. Insufficient freeze-drying cannot effectively control the size and distribution of ice crystals, thereby affecting the colloidal stability of nanoemulsion A. On the other hand, the simultaneous addition of vitamin E and N-isopropylacrylamide may affect the structure of N-isopropylacrylamide and the permeability of glycerol to liposomes, resulting in poor stability of the nanoemulsion. The nanoemulsion C has poor solubility after freeze-drying, which affects its solubility and dispersion during reconstitution and shortens its storage period.

[0153] pass Fig.12It can be seen from the particle size distribution diagram that the particle size distribution of nanoemulsions B and C is wider than that of Example 1, and the particle sizes are larger and uneven, which will affect the stability and release characteristics of terpinene-4-ol, accelerate the release rate, and thus reduce the efficacy; and in the absence of an antioxidant, the nanoemulsion C is easily oxidized, thereby accelerating the destruction of the stability of the nanoemulsion.

[0154] The nanoemulsion loaded with terpinene-4-ol and the preparation method thereof can improve water solubility, achieve better rehydration property through two freeze-drying and redissolution, and significantly improve its water solubility through nanoemulsion technology, thereby expanding its application range in the field of drug delivery and the like. The nanoemulsion system is a thermodynamically stable dispersion system with small and uniform droplet size, high stability, no stratification and no demulsification after long-term storage, which helps to maintain the activity of terpinene-4-ol and prolong the shelf life of the product; the nanoemulsion can be used as a sustained-release carrier, and the temperature responsiveness of N-isopropylacrylamide and the strong hydrogen bonding between the nanoemulsion and the hydrophobic monomer can be used to coordinate the nanoemulsion to control the release rate of terpinene-4-ol, so that terpinene-4-ol can be delivered to the lesion site more accurately, improve the efficacy and reduce the side effects on healthy tissues, thereby providing a more lasting effect, which is particularly important for drug delivery, can reduce the frequency of administration, and improve patient compliance; due to the small size of the nanoemulsion, the bioavailability of terpinene-4-ol can be improved, so that it can be more effectively absorbed and utilized by the human body; a natural and harmless amino acid surfactant is used in combination with terpinene-4-ol to prepare a long-term stable nanoemulsion, while ensuring the safety of the environment and personnel; the method of the invention is simple to prepare, pollution-free, avoids multi-step operation, simplifies the production process, and reduces costs.

[0155] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanoemulsion loaded with terpinene-4-ol, characterized in that: The nanoemulsion comprises the following components in percentage by weight: Terpinen-4-ol 3-4%, amino acid surfactant 6-8%, co-surfactant 5-6%, sorbitan fatty acid ester 0.1-1%, N-isopropylacrylamide 0-0.5%, vitamin E 0-1%, and the balance is water; The amino acid surfactant includes one or more of sodium lauroyl sarcosinate, sodium lauroyl glutamate, sodium N-oleoyl methyl taurate, and sodium cocoyl glycinate.

2. The nanoemulsion loaded with terpinene-4-ol as claimed in claim 1, characterized in that: The co-surfactant includes one of glycerol and isopropyl alcohol.

3. The nanoemulsion loaded with terpinene-4-ol as claimed in claim 1, characterized in that: The sorbitan fatty acid ester includes one of Span-40, Span-60 and Span-80.

4. The nanoemulsion loaded with terpinene-4-ol as claimed in claim 1, characterized in that: The nanoemulsion comprises the following components in percentage by weight: Terpinen-4-ol 3.76%, amino acid surfactant sodium lauroyl sarcosinate 6.8%, surfactant glycerol 5.5%, sorbitan fatty acid ester Span-80 0.5%, N-isopropylacrylamide 0.5%, vitamin E 1%, and the balance is water.

5. The method for preparing the nanoemulsion loaded with terpinene-4-ol as claimed in claim 1, characterized in that: The following steps are involved: S1. Preparation of primary emulsion: at room temperature, add amino acid surfactant, co-surfactant and sorbitan fatty acid ester to water in sequence according to a set ratio, stir evenly, add terpinene-4-ol, and continue stirring until the solution is clear to obtain a primary emulsion; S2, first freeze drying and re-dissolving: the primary emulsion is subjected to first freeze drying, N-isopropylacrylamide and water are added to the powder after the first freeze drying, and the powder is re-dissolved for the first time and subjected to ultrasound to obtain a re-dissolved emulsion; S3, second freeze drying and re-dissolution: the re-dissolved emulsion is freeze dried for the second time, vitamin E and water are added to the powder after the second freeze drying, and the powder is re-dissolved for the second time and ultrasonicated to obtain a nanoemulsion loaded with terpinene-4-ol.

6. The method for preparing the nanoemulsion loaded with terpinene-4-ol as claimed in claim 5, characterized in that: The step of adding N-isopropylacrylamide and water to S2 for the first redissolution and ultrasonication includes: adding N-isopropylacrylamide in a set ratio and water in an amount equal to that contained in the primary emulsion of S1, performing the first redissolution in a 60° C. water bath and ultrasonicating at 20° C. for 30 minutes.

7. The method for preparing the nanoemulsion loaded with terpinene-4-ol as claimed in claim 5, characterized in that: The step of adding vitamin E and water to S3 and performing a second redissolution and ultrasonication includes: adding a set ratio of vitamin E and an amount of water equal to that contained in the primary emulsion of S1, performing a second redissolution in a 60° C. water bath, and performing ultrasonication at 20° C. for 30 minutes.

8. Use of the nanoemulsion loaded with terpinene-4-ol according to any one of claims 1 to 4 in biomedicine, cosmetics and food.

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