A nanoemulsion of peppermint essential oil based on self-assembly of hydrophilic and hydrophobic deep eutectic solvents and a preparation method thereof

Abstract

The application discloses a peppermint essential oil nanoemulsion and a preparation method thereof. The preparation method comprises the following steps: S1, 0.275 mL of peppermint essential oil, 0.25 mL of polyglycerol-4-decanoate / thymol (1 / 2, mol / mol), 0.94 mL of betaine / glycerol (1 / 3, mol / mol) and 3.76 mL of ultrapure water are added into every 5.225 mL of a mixed system, the mixed system is uniformly shaken to obtain a coarse emulsion; and S2, the coarse emulsion is subjected to ultrasonic emulsification for 30 min to prepare the peppermint essential oil nanoemulsion. The peppermint essential oil nanoemulsion prepared by the method is mostly in spherical shape and is uniformly distributed, and is still in a stable state after being placed at different temperatures for 14 days, and the emulsion is stable in property. Anti-inflammatory effect test results show that, compared with the peppermint essential oil, the nanoemulsion reduces NO production in the presence of lipopolysaccharide (LPS) at a lower concentration. Bacteriostatic effect test results show that the peppermint essential oil nanoemulsion has a greater inhibitory effect on Staphylococcus aureus and Escherichia coli than the peppermint essential oil of the same concentration, and better antibacterial performance is obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant essential oil nanoemulsion, in particular to a mint essential oil nanoemulsion and a preparation method thereof. BACKGROUND

[0002] Mint is a member of the Lamiaceae family, belonging to the genus Mentha, and is widely distributed in temperate regions. It contains a large amount of essential oil components. Due to its strong antibacterial, antiviral, immunomodulatory, antitumor, neuroprotective, anti-fatigue and antioxidant activities, it is widely used in food, cosmetics, medicine and other fields. Essential oils have high volatility, are easily decomposed under the conditions of light, heat and oxygen, and have poor water solubility. These characteristics significantly reduce their functional properties and utilization efficiency, posing a serious challenge to the application of essential oils. To solve the above problems, it is urgent to develop a plant essential oil product that overcomes volatility and low water solubility.

[0003] Ultrasonic emulsification technology is one of the emerging technologies for essential oil transmission, which can improve the stability of essential oils to some extent and play a slow-release role, thereby improving the utilization rate of essential oils. The principle of ultrasonic emulsification is cavitation. In addition to the static hydraulic pressure of the ultrasonic wave acting on the medium, the ultrasonic wave also exerts a sound pressure on the liquid. The sound pressure is a sinusoidal wave that depends on time. During the negative phase of the wave, the local pressure is reduced to below the vapor pressure of the liquid, which produces bubbles. The bubbles grow during the low-pressure expansion half-cycle of the ultrasonic wave, and during the high-pressure half-cycle, when the applied pressure is high enough, the bubbles are severely squeezed and then collapse violently, producing ultrasonic microjets. Ultrasonic microjets can generate local hot spots, followed by local turbulence and flow, thereby homogenizing the multiphase liquid or emulsion.

[0004] Encapsulating essential oils can improve stability, increase solubility and improve dispersity, which is an effective method for efficient utilization of essential oils. Emulsion is a kind of multiphase dispersion in which small droplets of one liquid are dispersed in another immiscible liquid, forming a multiphase system.

[0005] Traditional nanoemulsion systems require the use of a large amount of volatile organic solvents and surfactants, which to some extent limits their application in key fields such as food, cosmetics, medicine, etc. In order to develop more environmentally friendly, biocompatible and superior performance nanoemulsion systems, researchers have tried to use deep eutectic solvents (DES) to construct nanoemulsion systems. Although the nanoemulsion system based on DES shows stronger performance, the related research is still in its infancy, and new construction strategies are still to be proposed. Current researches only focus on hydrophilic DES or hydrophobic DES, while nanoemulsion systems containing both types of DES may have both properties, which may improve performance. However, there is no report on the related emulsion design strategy and formula. SUMMARY

[0006] The present application aims to overcome the problems of essential oil volatility, low water solubility, instability, and short action time, and provides a peppermint essential oil nanoemulsion and a preparation method thereof.

[0007] The first object of the present application is to provide a preparation method of a peppermint essential oil nanoemulsion, comprising the following steps:

[0008] S1. Add 0.275 mL of peppermint essential oil, 0.25 mL of polyglycerol-4-decanoate / thymol (1 / 2, mol / mol), 0.94 mL of betaine / glycerol (1 / 2, mol / mol), and 3.76 mL of ultrapure water to every 5.225 mL of the mixed system, and shake the mixed system uniformly to obtain a coarse emulsion;

[0009] S2. Ultrasonic emulsification of the coarse emulsion for 30 min to prepare a peppermint essential oil nanoemulsion.

[0010] Preferably, the step S2 is: placing the coarse emulsion into an ultrasonic cell crusher, setting the power to 360 W, each cycle being 5 s of pulsed opening and 5 s of pulsed closing, ultrasonic emulsification for 30 min to obtain the peppermint essential oil nanoemulsion.

[0011] Preferably, the volume of the coarse emulsion for each ultrasonic emulsification is 5.225 mL.

[0012] Preferably, the shaking uniformity of the step S1 is achieved by a high-speed homogenizer.

[0013] Preferably, the peppermint is from Bozhou Traditional Chinese Medicine Market.

[0014] In the above system, polyglycerol-4-decanoate and thymol are used to form a hydrophobic DES according to a molar ratio of 1:2, and betaine and glycerol are used to form a hydrophilic DES according to a molar ratio of 1:3.

[0015] The second object of the present application is to provide a peppermint essential oil nanoemulsion prepared according to the preparation method.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] (1) The peppermint essential oil nanoemulsion provided by the present application uses hydrophobic and hydrophilic DESs, reduces the use of surfactants, and has better emulsification effect.

[0018] (2) The peppermint essential oil nanoemulsion provided by the present application reduces NO production in the presence of LPS, and has better anti-inflammatory effect. The nanoemulsion has better antibacterial performance than the same concentration of peppermint essential oil in inhibiting Staphylococcus aureus and Escherichia coli.

[0019] (3) The method for preparing the peppermint essential oil nanoemulsion is simple, time-saving, and only needs to uniformly mix the ingredients in proportion, then emulsify with a high-speed homogenizer, and the ultrasonic time only needs 30 min. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is the particle size and polydispersity index of the research scheme; wherein, "Size (d. nm)" represents the emulsion particle size, and "PDI" represents the polydispersity index of the emulsion.

[0021] Figure 2 is the influence of the peppermint essential oil content, hydrophobic DES content, hydrophilic DES content, H2O content, and ultrasonic power on the particle size and polydispersity index of the nanoemulsion (a) and the particle size distribution of the peppermint essential oil nanoemulsion (b); wherein, "Size (d. nm)" represents the emulsion particle size, and "PDI" represents the polydispersity index of the emulsion.

[0022] Figure 3 is the particle morphology of the peppermint essential oil nanoemulsion.

[0023] Figure 4 is the particle size and polydispersity index change diagram of the peppermint essential oil nanoemulsion prepared according to the formula stored for 14 days; wherein, "Size (d. nm)" represents the emulsion particle size, and "PDI" represents the polydispersity index of the emulsion.

[0024] Figure 5 is the influence of the nanoemulsion on the RAW264.7 and MPVEC cell viability (a) and the influence of the nanoemulsion on the LPS-induced NO (b). DETAILED DESCRIPTION

[0025] The following examples are further illustrations of the application and are not intended to limit the same.

[0026] The peppermint used in the following examples is from Bozhou Chinese medicine market, and the peppermint essential oil is extracted by steam distillation method. The specific preparation method of the peppermint essential oil is as follows: dry peppermint is finely ground with a grinder, and then passes through a 50 mesh sieve. Then, 250 g of the obtained powder and 2.5 L of water are added to a steam distillation device for extraction for 3 hours. Finally, the essential oil is dehydrated by anhydrous sodium sulfate and collected.

[0027] Laurylic acid (Lau), menthol (Men), thymol (Thy), betaine (Bet), L-proline (L-Pro), glycerol (Gly), ethylene glycol (Eth), and malonic acid (Mal) with a purity of more than 98% are purchased from Shanghai Aldrin Biochemical Technology Co., Ltd. (Shanghai, China).

[0028] Polyglycerol-4-caprate (P4C) with purity of 98% was purchased from Wuhan Kemike Biomedical Technology Co., Ltd. (Wuhan, China).

[0029] The high-speed homogenizer used in the following examples was a T18 digital Ultra-Turrax (IKA, Germany), and the ultrasonic cell disruptor was a SCIENTZ-IID ultrasonic cell disruptor from Ningbo Xinzhi Biotechnology Co., Ltd.

[0030] Example 1

[0031] 1. Experimental Methods

[0032] 1.1 Determination of the formulation of peppermint essential oil nanoemulsion

[0033] 1.1.1 Screening of DESs

[0034] Lauric acid (Lau), menthol (Men), thymol (Thy), betaine (Bet), L-proline (L-Pro), glycerol (Gly), ethylene glycol (Eth), and malonic acid (Mal), polyglycerol-4-caprate (P4C) were selected for screening. The hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs) were weighed and mixed in appropriate molar ratios. Then the mixture was subjected to magnetic stirring under heating at 60 °C until a homogeneous liquid was formed. The prepared DESs were collected and stored in a desiccator.

[0035] 1.1.2 Preparation of peppermint essential oil nanoemulsion

[0036] Peppermint essential oil, DES components were mixed in proportions according to the emulsion formulations F1-F12 shown in Table 1, and then ultrapure water was added to make up to 5.225 mL. The crude emulsion was obtained by using a high-speed homogenizer at 4000 rpm for 5 min.

[0037] The power of the ultrasonic cell disruptor was set to 360 W, and each cycle was 5 s of pulsed on and 5 s of pulsed off. The volume of the crude emulsion for each ultrasonic emulsification was 5.225 mL. During this process, the heat generated by the emulsion was controlled by using ice around the beaker. The emulsion formulation was mainly screened according to the particle size of the nanoemulsion.

[0038] Table 1 Formulation of DES-based nanoemulsion

[0039]

[0040]

[0041] 1.2 Optimization of the formulation of peppermint essential oil nanoemulsion

[0042] Five factors were designed to affect the nanoemulsion formulation, including the content of peppermint essential oil, the content of hydrophobic DES, the content of hydrophilic DES, the content of H2O and the ultrasonic power. The optimized nanoemulsion formulation is shown in Table 2. The coarse emulsion was obtained by using a high-speed homogenizer, and the obtained coarse emulsion was placed in an ultrasonic cell crusher for ultrasonic emulsification, and different ultrasonic powers were set: 120 W, 240 W, 360 W, 480 W and 600 W. The nanoemulsion formulation was optimized and screened mainly according to the particle size and polydispersity index of the nanoemulsion.

[0043] Table 2 Preferred formulation of peppermint essential oil nanoemulsion

[0044]

[0045] 1.3 Quality evaluation of peppermint essential oil nanoemulsion

[0046] 1.3.1 Judgment of nanoemulsion

[0047] There are three criteria for judging the formation of nanoemulsion:

[0048] (1) The liquid is transparent or translucent;

[0049] (2) There is a faint blue micro-luminescence;

[0050] (3) It has a Tyndall effect.

[0051] 1.3.2 Particle size and polydispersity index analysis of nanoemulsion

[0052] The particle size and polydispersity index (PDI) of the nanoemulsion were measured at 25°C using a Malvern Zetasizer Nano ZS-90 instrument. The particle size is described in nanometers (nm), and 3 replicates were measured at each time point. The results are calculated as mean ± standard error.

[0053] 1.3.3 Morphological observation of nanoemulsion

[0054] 20 μL of the sample to be tested was taken on a clean parafilm using a pipette, a carbon-supported copper grid was placed in the sample, and the water was absorbed from the edge of the copper grid with filter paper after 5 min. The copper grid was placed in another 20 μL of pure water, and the water was absorbed from the edge of the copper grid with filter paper. Then the copper grid was placed in phosphotungstic acid for 2-3 min for staining, and the water was absorbed from the edge of the copper grid with filter paper. After drying at room temperature, the copper grid was placed in a transmission electron microscope to observe the morphology of the nanoemulsion and take pictures.

[0055] 1.3.4 Determination of nanoemulsion embedding rate

[0056] Add 2 mL of n-hexane into 2 mL of nanoemulsion, vortex the mixture thoroughly, centrifuge at 1500 rpm for 5 minutes. Collect the supernatant, measure its absorbance value at 309 nm using UV-Vis spectrometer. Determine the amount of unencapsulated menthol oil using standard curve method, and calculate the entrapment efficiency according to the following equation.

[0057]

[0058] 1.3.5 Storage stability of nanoemulsion

[0059] The nanoemulsion of menthol oil prepared with the optimal formulation was placed in a transparent sample bottle and stored at 4±2℃, 20±2℃, 37±2℃ and 55±2℃ respectively for 0, 7, 14 days. The changes of particle size and polydispersity index of the nanoemulsion of menthol oil were determined by the method in 1.3.2 to evaluate the storage stability of the emulsion.

[0060] 1.4 Cell viability assay and nitric oxide (NO) production assay

[0061] MPVEC and RAW264.7 cells were seeded into 96-well plates at a concentration of 7.5×10 3 After 24 hours of co-culture, the cells were treated with LPS (10 μg / mL), LPS (10 μg / mL) combined with nanoemulsion of menthol oil (30 μg / mL) and pure menthol oil (30 μg / mL) for 24 h. Cell viability was evaluated using CCK-8 assay kit according to the manufacturer’s instructions, and the optical density (OD) was measured using a microplate reader. The NO content was determined using the Griess method, and the optical density (OD) was measured using a microplate reader.

[0062] 1.5 Evaluation of antibacterial effect of menthol oil and its nanoemulsion

[0063] 1.5.1 Determination of inhibition zone

[0064] According to the method of Miller et al. (J. M. Miller, C. Thornberry, C. N. Baker, Disk diffusion susceptibility test troubleshooting guide, Laboratory Medicine 15 (1984) 183-185, https: / / doi.org / 10.1093 / labmed / 15.3.183), an antibacterial test was performed using the paper disc diffusion method (Kirby-Bauer method). The target bacteria (bacterial suspension) were adjusted to 1×10 8CFU / mL. The bacterial inoculum (0.1 mL) was exchanged onto the surface of LB plates by rotating the plate 60° and repeated rubbing with a sterile cotton swab. Filter paper discs of 5.5 mm in diameter were soaked in the peppermint oil and the peppermint oil nanoemulsion for 2 hours and then were pinched with forceps onto the surface of agar plates. And these plates containing bacteria were incubated at 37 °C for 24 h. The diameter of the inhibition zone indicates the antibacterial capacity. Each experiment was performed in triplicate in the same way.

[0065] 1.5.2 Determination of minimum inhibitory concentration

[0066] According to the relevant literature (Y. Jing, J. Huang, X. Yu, Preparation, characterization, and functional evaluation of proanthocyanidin-chitosan conjugate, Carbohydr Polym 194 (2018) 139145, https: / / doi.org / 10.1016 / j.carbpol.2018.04.037). The minimum inhibitory concentration (MIC) of the sample was determined. The sample was diluted from 50 mg / mL to 0.195 mg / mL using LB broth. In a 96-well plate, 50 μL of bacterial suspension (about 1 x 10 5 CFU / mL) was mixed with 100 μL of different concentrations of samples and stored in a 37 °C incubator for 16 h. The highest dilution tube without bacterial growth was the MIC of the antibacterial agent.

[0067] 2. Experimental results

[0068] 2.1 Screening of DESs

[0069] Hydrophobic DESs were prepared with Men, Lau, Thy, P4C as components, in which Thy:P4C represented a new type of hydrophobic deep eutectic solvent. Hydrophilic DESs were prepared with Bet, L-Pro, Gly, Eth and Mal as components. DESs that were in liquid state at room temperature were selected for further formulation design. The compositions of the selected DESs are shown in Table 3.

[0070] Table 3 Compositions of DESs

[0071]

[0072] a: molar ratio of component 1 to component 2

[0073] 2.2 Screening of emulsion formulations

[0074] The particle size and polydispersity index of these 15 emulsions are shown in Table 4. Figure 1The results showed that Men and Thy incorporation effectively converted the non- emulsifying Lau system (F1) into emulsions with particle sizes of 131.4 nm (F3) and 5.123 nm (F4), respectively. In addition, the addition of Thy significantly reduced the particle size of the P4C system (F2) to 1.418 nm (F5). Therefore, hydrophobic DESs can promote the formation of emulsions and reduce particle size. The new hydrophobic Th y / P4C (2 / 1, mol / mol) found in this study has emulsion particle sizes as small as 1.418 nm. Hydrophilic DESs were introduced into the P4C system (F6, F7, F8, F9, and F10). The results showed that the five hydrophilic DESs increased the particle size of the P4C system to varying degrees (23.64 nm-3023 nm). The five hydrophilic DESs were added to the Th y / P4C system to form hydrophobic-hydrophilic DES co-assembled emulsion systems, and the particle size increased from 3.143 nm to 2441 nm (F11, F12, F13, F14, and F15). However, this increase was significantly lower than that observed in the pure P4C system. In the hydrophobic-hydrophilic DES co-assembled emulsion system, F11 had the smallest particle size and PDI, and the best formula selected in this test example was composed of peppermint essential oil, Th y / P4C (2 / 1, mol / mol), Bet / Gly (1 / 3, mol / mol), and water.

[0075] 2.3 Optimization of nanoemulsion formula

[0076] The total amount of oil phase had an effect on the nanoemulsion of peppermint essential oil. As the total amount of oil phase increased, the particle size of the emulsion decreased, and the PDI increased. When the total amount of oil phase was 0.275 mL, the particle size of the emulsion was the smallest, the PDI was relatively small, and the nanoemulsion system was stable.

[0077] The different amounts of hydrophobic DESs had an effect on the performance of the nanoemulsion of peppermint essential oil. At a dosage of 0.25 mL, it was a milky white uniform emulsion, and the particle size and PDI of the emulsion were relatively small, and the emulsion system was stable.

[0078] The different amounts of hydrophilic DESs had an effect on the performance of the nanoemulsion of peppermint essential oil. At a dosage of 0.94 mL, it was a milky white uniform emulsion, and the particle size and PDI of the emulsion were relatively small, and the emulsion system was stable.

[0079] The total amount of water phase had an effect on the nanoemulsion of peppermint essential oil. When the total amount of water phase was 3.76 mL, the particle size and PDI of the emulsion were relatively small, and the nanoemulsion system was stable.

[0080] The effect of ultrasonic power on the menthol nanoemulsion was that the particle size was more evenly dispersed as the ultrasonic power increased. Considering the emulsification effect of the emulsion and the loss of the instrument at high power, the ultrasonic power was finally selected to be 360 W.

[0081] In summary, the best formula screened by the test example was composed of 0.275 mL of menthol, 0.25 mL of polyglyceryl-4-decanoate / thymol (1 / 2, mol / mol), 0.94 mL of betaine / glycerol (1 / 2, mol / mol) and 3.76 mL of ultrapure water, and the ultrasonic power was 360 W. The average particle size of the menthol nanoemulsion prepared under this condition was 3.143 nm, and the PDI was 0.270. Figure 2 (b) was the particle size distribution diagram of the menthol nanoemulsion prepared under the best system. As can be seen from the diagram, the menthol nanoemulsion prepared under this condition showed a unimodal normal distribution in the range of 1-10 nm, indicating that the menthol nanoemulsion was uniformly distributed and had good stability.

[0082] 2.4 Observation of the morphology of the menthol nanoemulsion

[0083] As Figure 3 can be seen, the shape and particle size of the emulsion particles can be directly observed under a transmission electron microscope. Most of the emulsion droplets were monodisperse spherical and uniformly distributed.

[0084] 2.5 Determination of the entrapment efficiency of the menthol nanoemulsion

[0085] Table 4 shows that the formed nanoemulsion system can encapsulate menthol at 88.10% (hydrophobic DES), 93.79% (hydrophilic DES) and 98.28% (hydrophilic-hydrophobic DES). In the development of nanoemulsion research, the most important aspect of the effect of different surfactants and co-surfactants as drug carriers is the encapsulation efficiency. The menthol and the nanoemulsion matrix formed based on different DES combinations are complexes, achieving a high encapsulation efficiency.

[0086] Table 4 Determination of the entrapment efficiency of the nanoemulsion

[0087]

[0088] 2.6 Storage stability of the menthol nanoemulsion

[0089] As Figure 4As shown, the nanoemulsion particle size and PDI values slightly increased with the increase of storage time and temperature. However, the nanoemulsion with PDI values <0.3 was considered to be narrow distribution. This indicated that the peppermint essential oil nanoemulsion had good stability in terms of droplet size under the tested temperature and storage time. No visible emulsion layering or phase separation was observed during the storage of the peppermint essential oil nanoemulsion at any temperature (4, 20, and 37 °C). The results indicated that the peppermint essential oil nanoemulsion had good kinetic stability within the tested temperature and storage time.

[0090] 2.7 Cell viability assay and nitric oxide (NO) production assay

[0091] Figure 5 aThe results indicated that none of the tested samples showed a significant decrease in cell viability compared to the control group after 24 hours of treatment with the peppermint essential oil nanoemulsion in the cytotoxicity test (cell viability) of MPVEC cells and RAW264.7 cells.

[0092] In the NO test, the NO level in the supernatant of the cell control group was 3.260 ± 0.1547 μΜ. Induction of RAW264.7 cells with LPS increased NO secretion by 3.05-fold, reaching 9.959 ± 0.7038 μΜ Figure 5 bP < 0.0001 compared to the untreated control). At lower concentrations, the nanoemulsions reduced NO production in the presence of LPS. The inhibitory effect of the hydrophilic and hydrophobic DES peppermint essential oil nanoemulsions on NO was higher than that of the peppermint essential oil, the nanoemulsion based on hydrophobic DES, and the nanoemulsion based on hydrophilic DES at a concentration of 30 μg / mL.

[0093] 2.8 Antimicrobial effect of peppermint essential oil and its nanoemulsion

[0094] As can be seen from Table 5, the peppermint essential oil and its nanoemulsion prepared according to the formula had an inhibitory effect on both bacteria, and the degree of influence was different between different bacterial species. The minimum inhibitory concentration of peppermint essential oil on Staphylococcus aureus was 6.25 mg / mL, and the minimum inhibitory concentration on Escherichia coli was 50 mg / mL. The minimum inhibitory concentration of peppermint essential oil nanoemulsion on Staphylococcus aureus was 0.195 mg / mL, and the minimum inhibitory concentration on Escherichia coli was 0.781 mg / mL, indicating that the inhibitory effect of peppermint essential oil nanoemulsion on Staphylococcus aureus was greater. The inhibitory effect of peppermint essential oil nanoemulsion on Staphylococcus aureus and Escherichia coli was greater than that of peppermint essential oil at the same concentration. Therefore, the prepared emulsion has a wider antibacterial effect.

[0095] Table 5 Minimum inhibitory concentration of peppermint essential oil and its nanoemulsion

[0096]

[0097] The above further describes the present application in detail in connection with specific preferred embodiments, and is not intended to limit the specific embodiments of the present application to only these. For those skilled in the art of the present application, without departing from the present application, a number of simple deductions or substitutions can be made, and all should be considered to belong to the scope of patent protection determined by the claims submitted.

Claims

1. A method of preparing a nanoemulsion of a peppermint essential oil, characterized in that, The method comprises the following steps: S1. Adding 0.275 mL of mint oil, 0.25 mL of polyglyceryl-4-decanoate / thymol with a molar ratio of 1:2, 0.94 mL of betaine / glycerol with a molar ratio of 1:3 and 3.76 mL of ultrapure water into 5.225 mL of a mixed system, and oscillating the mixed system uniformly to obtain a coarse emulsion; S2. Ultrasonic emulsifying the coarse emulsion for 30 min to prepare a mint oil nanoemulsion.

2. The production method according to claim 1, characterized by, The step S2 is: placing the coarse emulsion into an ultrasonic cell crusher, setting the power to 360 W, setting each cycle to 5 s of pulse opening and 5 s of pulse closing, ultrasonic emulsifying for 30 min to prepare the mint oil nanoemulsion.

3. The production method according to claim 2, characterized by, The volume of the coarse emulsion for each ultrasonic emulsification is 5.225 mL.

4. The production method according to claim 1, characterized by, The oscillation uniformity in the step S1 is achieved by a high-speed homogenizer.

5. The production method according to claim 1, characterized by, The mint is Mentha canadensis L.

6. A mint oil nanoemulsion prepared by the preparation method according to any one of claims 1-5.

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