Highly stable multi-encapsulated hinokitiol liposomes and their preparation method and application

Abstract

This scheme provides a highly stable multi-encapsulated cypress phenol liposome and its preparation method and application. Cypress phenol is emulsified with a special glycolipid emulsifier to obtain a nano-cypress phenol emulsion, and then the nano-cypress phenol is further encapsulated with a phospholipid bilayer of phospholipids and emulsified esters to obtain a multi-encapsulated cypress phenol liposome with a special double-shell three-layer structure. The cypress phenol active substance is encapsulated by glycolipids and sandwiched between the phospholipid bilayers, which improves the water solubility and stability of the multi-encapsulated cypress phenol liposome, and has a good antibacterial effect, and can be widely used in the cosmetics field.

Description

Technical Field

[0001] The present invention relates to the technical field of hinokitiol, and in particular to a highly stable multi-encapsulated hinokitiol liposome and a preparation method and application thereof. Background Art

[0002] Hinokitiol, also known as hinokitiol, is a natural monoterpenoid compound with a tropolone skeleton extracted from natural tree trunks. It belongs to the tropolone family of compounds and exhibits excellent antibacterial, moisturizing, and pest repellent properties. It is a highly safe botanical ingredient and can be used as an antibacterial and insect repellent. Hinokitiol has been used in shower gels, cosmetics, pharmaceuticals, and agricultural products, and has been shown to be surprisingly effective against Staphylococcus aureus (MRSA). This bacterium commonly inhabits the skin and respiratory tract, causing skin infections such as sepsis, peritonitis, food poisoning, and boils and abscesses. Therefore, hinokitiol's application in skincare offers numerous potential benefits, including antibacterial, whitening, and insect repellent properties. However, its easy sublimation, accelerated decomposition by light, and poor water solubility limit its application in daily chemicals and pharmaceuticals.

[0003] Regarding the stabilization and water solubility of cypress phenol, prior art, such as Japanese Patent Publication No. 9-188620, discloses a method for adjusting the pH to 5.0-6.0 by combining with an organic acid. Prior art, Japanese Patent Publication No. 10-291906 discloses an emulsification and dispersion method using a surfactant, etc. Prior art WO1997002025 prepares cypress phenol into an aluminum salt and / or a coordination compound with an aluminum compound. However, the above methods are complex to operate, have high production costs, cause serious environmental pollution, and cannot completely solve its water solubility and stability. Another example is prior art CN 107661505 A, which prepares cypress phenol into an inclusion compound with cyclodextrin. Although this solution is relatively environmentally friendly, it is complex, time-consuming, and difficult to control quality, making it unsatisfactory for large-scale, low-cost production.

[0004] In other words, although hinokitiol has been proven to have good antibacterial effects, its poor stability and water solubility have resulted in poor results when used in skincare products. Firstly, if hinokitiol is unstable, it will easily decompose during storage, weakening its antibacterial and whitening properties and failing to achieve the desired results. Secondly, long-term storage can also cause hinokitiol to change color, taste, or texture during use, affecting the user experience. Thirdly, the addition of additional additives or storage processes to improve stability also leads to a sharp increase in the production cost of cosmetics. Therefore, existing technologies continue to improve the water solubility and stability of hinokitiol and enhance its antibacterial effects to expand its application in the cosmetics field. Summary of the Invention

[0005] The embodiments of the present application provide a highly stable multi-encapsulated cypress phenol liposome, a preparation method and application thereof, wherein cypress phenol is emulsified with a special sugar ester emulsifier to form a nano-cypress phenol emulsion, and then the nano-cypress phenol in the nano-cypress phenol emulsion is encapsulated with a special phospholipid to form a highly stable multi-encapsulated cypress phenol liposome, which has good stability and water solubility, and further improves the antibacterial effect, and can be widely used in the field of cosmetics.

[0006] In a first aspect, the present invention provides a method for preparing highly stable multi-encapsulated hinokitiol liposomes, the method comprising:

[0007] mixing a first aqueous phase solution and a second aqueous phase solution under high shear homogenization conditions, and heating to a first temperature and maintaining for a period of time to obtain a mixed aqueous phase solution, wherein the first aqueous phase solution contains at least one polyol, and the second aqueous phase solution contains at least one polyol and a sugar ester emulsifier;

[0008] Mixing the aqueous phase solution and the oil phase solution under high shear homogenization conditions, and heating to a second temperature and maintaining for a period of time to obtain a nano-hinokitiol emulsion, wherein the oil phase solution contains oil and hinokitiol;

[0009] adding phospholipids and emulsified esters to an ethanol solution to obtain a lipid mixture, rotary evaporating the lipid mixture at low pressure and removing the ethanol to form a lipid film;

[0010] The nano-hinokitiol emulsion is dissolved in a phosphate buffer solution to obtain a nano-hinokitiol dispersion, the nano-hinokitiol dispersion is added to a container containing a lipid film and stirred, and then placed in an ultrasonic oscillation device with an ice water bath for ultrasonic oscillation to obtain multi-encapsulated hinokitiol liposomes.

[0011] In a second aspect, the embodiments of the present application provide a highly stable multi-encapsulated hinokitiol liposome, which is prepared according to the preparation method of the highly stable multi-encapsulated hinokitiol liposome mentioned in the first aspect.

[0012] In a third aspect, the embodiments of the present application provide a method for applying highly stable multi-encapsulated hinokitiol liposomes, wherein the multi-encapsulated hinokitiol liposomes are used as an ingredient in antibacterial cosmetics.

[0013] In some embodiments, the type of antibacterial cosmetics of this solution is not particularly limited, and can be selected from one of cream, gel, spray, facial cleanser, lotion, lip balm, and facial mask.

[0014] The main contributions and innovations of the present invention are as follows:

[0015] The embodiments of the present application provide a unique double-encapsulation technology, which achieves the high stability of cypress phenol in aqueous solution through double encapsulation by sugar ester emulsifier and phospholipid bilayer: the sugar ester emulsifier first forms a layer of nanoparticles that encapsulate cypress phenol, and then is encapsulated again by the phospholipid bilayer to form a special "double shell three membrane" structure, which greatly improves the stability of cypress phenol while maintaining its antibacterial activity.

[0016] The multi-encapsulated hinokitiol liposomes prepared in this protocol not only improve the stability of hinokitiol through a unique structural design but also demonstrate significant advantages in antibacterial efficacy. The liposomes' biomembrane-like structure enables them to fuse with the cell membranes of single-cell organisms, efficiently delivering the hinokitiol active ingredient into target cells, thereby enhancing antibacterial efficacy. These liposomes can be widely used in various cosmetics, such as creams, gels, sprays, cleansers, lotions, lip balms, and facial masks, providing users with highly effective, gentle, and long-lasting antibacterial effects. This potential for widespread application provides a solid foundation for the development of new antibacterial cosmetics.

[0017] The details of one or more embodiments of the present application are set forth in the following drawings and description to make other features, objects, and advantages of the present application more readily apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0019] Figure 1 1 is a flowchart of the steps of preparing highly stable multi-encapsulated hinokitiol liposomes according to an embodiment of the present application.

[0020] Figure 2 Schematic diagram of the structure of highly stable multi-encapsulated hinokitiol liposomes according to an embodiment of the present application.

[0021] Figure 3 Schematic diagram of the antibacterial process of highly stable multi-encapsulated cypress phenol liposomes according to an embodiment of the present application.

[0022] Figure 4 This is a diagram showing the inhibitory effect on Staphylococcus aureus.

[0023] Figure 5 This is a diagram showing the inhibitory effect of Malassezia furfur.

[0024] Figure 6 This is a diagram showing the inhibitory effect of Propionibacterium acnes.

[0025] Figure 7 This is a HPLC liquid phase diagram of the hinokitiol nanoemulsion of hinokitiol hexanediol solution in the initial state.

[0026] Figure 8 This is the HPLC liquid phase diagram of the hinokitiol nanoemulsion of hinokitiol hexanediol solution under 14 days of sunlight irradiation.

[0027] Figure 9 This is the HPLC liquid phase diagram of the hinokitiol nanoemulsion of hinokitiol hexanediol solution under 28 days of sunlight irradiation. DETAILED DESCRIPTION

[0028] Exemplary embodiments will be described in detail herein, with examples illustrated in the accompanying drawings. In the following description, when referring to the drawings, identical numerals in different figures represent identical or similar elements, unless otherwise indicated. The implementations described in the following exemplary embodiments are not intended to represent all implementations consistent with one or more embodiments of this specification. Rather, they are merely examples of apparatuses and methods consistent with certain aspects of one or more embodiments of this specification, as detailed in the appended claims.

[0029] It should be noted that in other embodiments, the steps of the corresponding method are not necessarily performed in the order shown and described in this specification. In some other embodiments, the method may include more or fewer steps than those described in this specification. In addition, a single step described in this specification may be broken down into multiple steps for description in other embodiments, and multiple steps described in this specification may be combined into a single step for description in other embodiments.

[0030] The present invention provides a method for preparing highly stable multi-encapsulated cypress phenol liposomes, which adopts a double-encapsulation method to obtain multi-encapsulated cypress phenol liposomes that can be stably present in solution. First, a special sugar ester emulsifier is used to emulsify cypress phenol to obtain nano-cypress phenol microparticles with cypress phenol encapsulated by a sugar ester inner layer. Then, a lipid film formed by a special phospholipid and emulsified ester is used to encapsulate the nano-cypress phenol microparticles and dispersed cypress phenol particles to obtain multi-encapsulated cypress phenol liposomes with nano-cypress phenol microparticles encapsulated by a phospholipid bilayer. The multi-encapsulated cypress phenol liposomes form a special double-shell three-membrane structure of a phospholipid bilayer composite sugar ester inner layer, which greatly improves the stability of cypress phenol in various solutions without destroying the structural characteristics of cypress phenol.

[0031] The schematic diagram of the preparation method of the highly stable multi-encapsulated cypress phenol liposomes proposed in this scheme is as follows: Figure 1 As shown, it can be seen that this scheme first uses a mixed aqueous solution to encapsulate hinokitiol to form a nano-hinokitiol emulsion, and then uses a special lipid film to encapsulate the nano-hinokitiol to form a multi-encapsulated multi-encapsulated hinokitiol liposome.

[0032] Regarding the ingredient ratio of the nano-hinokitiol emulsion of this solution:

[0033] In this solution, the weight of the first aqueous phase solution accounts for 20-70% of the nano-hinokitiol emulsion, the weight of the second aqueous phase solution accounts for 20-70% of the nano-hinokitiol emulsion, the weight of the oil phase solution accounts for 6-10% of the nano-hinokitiol emulsion, and the sum of the first aqueous phase solution, the second aqueous phase solution and the oil phase solution accounts for 100% of the nano-hinokitiol emulsion.

[0034] Specifically, the oil phase solution contains 5-9% by weight of the oil and 1-5% by weight of the hinokitiol emulsion. In some preferred embodiments, the oil phase solution of this embodiment accounts for 10% by weight of the nano-hinokitiol emulsion, wherein the oil phase solution contains 6% by weight of the oil and 4% by weight of the hinokitiol emulsion.

[0035] Regarding the selection of the first aqueous phase solution of this scheme:

[0036] The first aqueous phase solution of this embodiment contains a polyol selected from the group consisting of ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, butylene glycol, 1,2-butylene glycol, 1,4-butylene glycol, 2,3-butylene glycol, 1,5-pentanediol, 1,2-pentanediol, isopentyl glycol, neopentyl glycol, hexylene glycol, 1,2-hexanediol, 1,6-hexanediol, octyl glycol, octyl glycol, decylene glycol, 1,10-decylene glycol, sorbitol, xylitol, lactitol, maltitol, erythritol, mannitol, threitol, glycerol, 1,2,6-hexanetriol, dipropylene glycol, menthylene glycol, methylpropylene glycol, panthenol, and pentaerythritol, or a combination of two or more thereof. Preferably, the first aqueous phase solution contains sorbitol and glycerol. The purpose of the first aqueous phase solution provided in this scheme is to provide the necessary solvent environment, and the polyol can form a certain viscosity in the aqueous phase, thereby enhancing the stability of the nano-hinokitiol emulsion.

[0037] Regarding the selection of the second aqueous phase solution of this scheme:

[0038] The second aqueous phase solution of this embodiment contains a polyol selected from the group consisting of ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, butylene glycol, 1,2-butylene glycol, 1,4-butylene glycol, 2,3-butylene glycol, 1,5-pentanediol, 1,2-pentanediol, isopentyl glycol, neopentyl glycol, hexylene glycol, 1,2-hexanediol, 1,6-hexanediol, octyl glycol, octylene glycol, decylene glycol, 1,10-decylene glycol, sorbitol, xylitol, lactitol, maltitol, erythritol, mannitol, threitol, glycerol, 1,2,6-hexanetriol, dipropylene glycol, menthylene glycol, methylpropylene glycol, panthenol, and pentaerythritol, or a combination of two or more thereof. Preferably, the polyol in the second aqueous phase solution is butylene glycol or sorbitol.

[0039] Additionally, the glycolipid emulsifiers in the second aqueous phase solution are selected from one or a combination of two or more monorhamnolipids, disrhamnolipids, and sucrose fatty acid esters. In this protocol, sucrose fatty acid esters are used as emulsifiers to encapsulate hinokitiol to produce nano-hinokitiol. The nano-hinokitiol is then dispersed in the solution to form a nano-hinokitiol emulsion with a particle size of less than 80 nm. These glycolipid emulsifiers were specifically chosen for their high HLB values, typically ranging from 14 to 18, significantly higher than those of conventional emulsifiers (8 to 12). High HLB values ​​enhance the hydrophilicity of these emulsifiers, enabling the formation of smaller emulsified particles, such as nano-scale microemulsions.

[0040] It should be noted that when the glycolipid emulsifier is monorhamnolipid or disrhamnolipid, the monorhamnolipid or disrhamnolipid can be a monoester, diester, triester, polyester, or a mixture thereof; when the glycolipid emulsifier is sucrose fatty acid ester, the carbon chain length of the sucrose fatty acid is C4-C28. Preferably, the glycolipid emulsifier of this embodiment is sucrose fatty acid ester, and more preferably, the carbon chain length of the sucrose fatty acid ester is C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, or C22.

[0041] Regarding the selection of oil phase solution for this scheme:

[0042] The oil in the oil phase solution of this embodiment is selected from one or a combination of two or more of orange oil, bergamot oil, olive oil, cardamom nut oil, saw palmetto oil, sweet orange flower oil, clove flower oil, rose flower oil, hydrogenated sunflower oil, lecithin, hydrogenated lecithin, and cephalin. The hinokitiol in the oil phase solution of this embodiment can be derived from natural, synthetic, or semi-synthetic sources.

[0043] About the preparation process of nano-hinokitiol emulsion:

[0044] In some embodiments, the second aqueous phase solution is slowly added to the first aqueous phase solution under high-shear homogenization conditions with high-speed stirring to thoroughly mix the first and second aqueous phase solutions, and the mixture is simultaneously heated to 58-62°C and maintained for 10-20 minutes to obtain a mixed aqueous phase solution. The high-speed stirring under high-shear homogenization conditions in this embodiment ensures thorough mixing between the first and second aqueous phase solutions, uniformly distributing the polyol and glycolipid emulsifier, and avoiding localized concentrations of excessively high or low concentrations. Furthermore, heating to 58-62°C allows the glycolipid emulsifier to more effectively exert its emulsifying effect.

[0045] In some preferred embodiments, the second aqueous phase solution is slowly added to the first aqueous phase solution under high shear homogenization conditions of 8000 rpm / min to fully mix the first aqueous phase solution and the second aqueous phase solution, while heating to 60° C. and maintaining for 15 minutes.

[0046] In some embodiments, the aqueous and oily phases are mixed under high shear homogenization conditions and heated to 62-68°C for 10-20 minutes to produce a nano-hinokitiol emulsion. The second temperature in this embodiment is higher than the first temperature. This allows the glycolipid emulsifier to fully function, encapsulating the hinokitiol to form nano-hinokitiol, and dispersing the nano-hinokitiol in the solvent system to produce a stable, clear, and transparent nano-hinokitiol emulsion.

[0047] This scheme uses glycolipid emulsifiers to encapsulate cypress phenol to form nano-cypress phenol, and nano-cypress phenol is dispersed in a solvent system to form a nano-cypress phenol emulsion. In order to further increase the stability of the nano-cypress phenol emulsion and the permeability of the cypress phenol active ingredient, a small amount of free cypress phenol active molecules are encapsulated, and special phospholipids are selected to further encapsulate the nano-cypress phenol emulsion.

[0048] About the composition of lipid film:

[0049] This scheme uses phospholipids and emulsified esters to prepare a phospholipid bilayer to increase the encapsulation rate of nano-cypress phenol and free cypress phenol active ingredients. Specifically, the phospholipids selected in this scheme are lecithin, cephalin, soybean lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylcholine, dioleoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylglycerol sodium salt, distearoylphosphatidylcholine, (2,3-dioleyloxypropyl) trimethylammonium chloride, polyquaternium-51, or a combination of two or more; the emulsified esters selected in this scheme are C8-12 acid triglyceride, C12-18 acid triglyceride, C18-36 Caprylic / Capric / Cocoa Glycerides, Caprylic / Capric Glycerides, Caprylic / Capric / Lauryl Triglyceride, Caprylic / Capric / Linoleic Triglyceride, Caprylic / Capric / Myristic / Stearic Triglyceride, Caprylic / Capric / Stearic Triglyceride, Caprylic / Capric / Succinic Triglyceride, Caprylic / Capric Triglyceride, Coco-Glycerides, Cottonseed Glycerides, C10-18 Fatty Acid Triglycerides, Glyceryl Caprate, Glyceryl Caprylate, Glyceryl Caprylate / Caprate, Glyceryl Cocoate, Glyceryl Diisopalmitate, Glyceryl Diisostearate, Glyceryl Dilaurate, Glyceryl Dilinoleate, Glyceryl Dimyristate, Glyceryl Dioleate, Glyceryl Dipalmitate, Glyceryl Distearate, Glyceryl Isostearate / Myristate, Glyceryl Laurate / Oleate, Glyceryl Palmitate / Stearate, Triethylhexanoin, Octyldodecanol Isostearate, Tocopheryl Acetate, Phytosteryl / Octyldodecanol Lauroyl Glutamate, Isononyl Isononanoate, Triethylhexanoin, Sorbitan Laurate, or a combination of two or more.

[0050] Preferably, the phospholipid of this solution is polyquaternium-51, and the emulsifying esters are caprylic / capric glyceride and tocopheryl acetate. It should be noted that caprylic / capric glyceride has a high HLB value (12.5), making it an excellent emulsifier for dispersing polyquaternium-51 and tocopheryl acetate in aqueous solution. This increases the specific surface area and probability of contact with nano-hinokitiol, while also reducing the self-aggregation of phospholipids into empty vesicles (empty liposomes), which is crucial for the efficient formation of liposomes. Tocopheryl acetate is partially encapsulated within the nano-hinokitiol, while the remaining portion is embedded in the hydrophobic layer of the phospholipid bilayer, which can enhance the hydrophobicity of the phospholipid hydrophobic segments. Its linear spatial structure interacts with the linear hydrophobic ends of the phospholipids, enhancing the strength of the phospholipid-based phospholipid bilayer structure and increasing its physical stability, playing a significant role in the stability of the liposomes. Tocopherol acetate acts as an antioxidant, absorbing external light energy in the phospholipid bilayer, inhibiting the light-absorbing denaturation of hinokitiol in the phospholipid bilayer. A very small amount of light that passes through the phospholipid bilayer and the glycolipid layer will be absorbed by the tocopherol acetate inside the glycolipid layer, thereby further protecting the active structure of hinokitiol.

[0051] In some embodiments, the weight ratio of phospholipids to emulsifying esters is 1 to 10:1. The weight ratio of phospholipids in this solution is greater than the weight ratio of emulsifying esters because too much emulsifying esters will destroy the strength of the phospholipid bilayer. Specifically, when the emulsifying esters are caprylic / capric glyceride and tocopheryl acetate, too much tocopheryl acetate will reduce the strength of the phospholipid bilayer. This is because tocopheryl acetate is a partially fat-soluble substance and does not have the strong hydrophilic group of phospholipids. It is not the main structural component of the bilayer.

[0052] In some embodiments, the concentration of phospholipids and emulsifying esters in the ethanol solution ranges from 1% to 10%.

[0053] About the preparation of lipid films:

[0054] An ethanol solution is added into a container and a magnet is placed therein, and the opening of the container is sealed. The container is placed on a magnetic stirrer for magnetic stirring, and under the magnetic stirring condition, phospholipids and emulsified esters are added to the ethanol solution and mixed evenly to obtain a lipid mixture. The ethanol solution is subjected to low-pressure rotary evaporation under a high-temperature environment, and the ethanol is removed to form a lipid film.

[0055] In some specific embodiments, at room temperature of 20°C and ventilation conditions, an ethanol solution is added to a container and a magnet is placed therein, wherein the amount of ethanol solution added is controlled to be 1 / 5 to 1 / 3 of the total volume of the container, and phospholipids and emulsified esters are added to the ethanol solution under magnetic stirring conditions of approximately 300 rpm and mixed uniformly to obtain a lipid mixture, and the ethanol solution is subjected to low-pressure rotary evaporation at 50 to 55°C to remove ethanol to form a lipid film on the inner wall of the container.

[0056] Preparation of multi-encapsulated hinokitiol liposomes:

[0057] The nano-hinokitiol emulsion is slowly poured into a phosphate buffer to dissolve the nano-hinokitiol dispersion, wherein the pH of the phosphate buffer is 6.3 and the weight ratio of the nano-hinokitiol emulsion to the phosphate buffer is 2 to 5:1. The phosphate buffer provided in this solution provides a stable pH environment to maintain the stability and activity of the nano-hinokitiol and disperses the nano-hinokitiol emulsion to obtain the nano-hinokitiol dispersion, thereby preventing agglomeration or even demulsification.

[0058] In addition, the nano-hinokitiol dispersion is quickly added to a container containing a lipid film and stirred, maintaining a weight ratio of the lipid film to the hinokitiol in the nano-hinokitiol dispersion at 1-2:1. In this protocol, the nano-hinokitiol is quickly added to the container containing the lipid film and magnetically stirred for 10-15 minutes. The container is then transferred to an ultrasonic oscillator with an ice-water bath, equilibrated for 15-25 minutes, and ultrasonically oscillated for 20-30 minutes to produce multi-encapsulated hinokitiol liposomes.

[0059] In some specific embodiments, nano-hinokitiol is added to a container containing a lipid film as quickly as possible and magnetically stirred for 10 minutes. The container is transferred to an ultrasonic oscillation device with an ice-water bath, temperature is balanced for 20 minutes, and ultrasonic oscillation is performed for 25 minutes to obtain multi-encapsulated hinokitiol liposomes.

[0060] This approach uses a combination of magnetic stirring and ultrasonic vibration to achieve smaller and more uniform particle sizes for the resulting multi-encapsulated hinokitiol liposomes. This also improves the stability and encapsulation efficiency of the hinokitiol in the multi-encapsulated hinokitiol liposomes. Specifically, magnetic stirring allows the phospholipids to disperse freely and prepare to self-aggregate into vesicular liposomes. After a short period of mechanical stirring, ultrasonic vibration is used to encapsulate as much nano-hinokitiol as possible within the phospholipid bilayer. The small amount of empty vesicles that form are shattered and then reassembled until they encapsulate the nano-hinokitiol and stabilize, forming a stable, high-encapsulation-efficiency, special double-shell, three-membrane structure.

[0061] This scheme uses water-soluble nano-cypressol formed by sugar ester encapsulating cypressol as the base material, and further encapsulates a small amount of free cypressol molecules into the phospholipid bilayer to form a special double-shell three-membrane coating structure to increase the high-temperature and light stability of the active substance, and has greater potential in improving the skin penetration of the active substance.

[0062] The highly stable multi-encapsulated hinokitiol liposomes of this embodiment are prepared according to the above-mentioned method for preparing highly stable multi-encapsulated hinokitiol liposomes. Figure 2As shown, the multi-encapsulated hinokitiol liposomes prepared in this embodiment include a phospholipid bilayer and a glycolipid inner layer arranged from the outside to the inside. The glycolipid inner layer encapsulates the hinokitiol active ingredient, and the phospholipid bilayers encapsulate the hinokitiol active ingredient. In other words, the glycolipid inner layer encapsulates a portion of the hinokitiol particles, the phospholipid bilayer encapsulates the glycolipid inner layer, and a portion of the dispersed hinokitiol is also encapsulated between the two phospholipid bilayers. This has the advantage of stably encapsulating the hinokitiol.

[0063] In addition, this prescription can also be used to inhibit bacteria by using the highly stable multi-encapsulated cypress phenol liposomes. Figure 3 As shown, the biomembrane-like structure of the phospholipid bilayer of the highly stable multi-encapsulated cypressin liposomes is highly similar to the phospholipid bilayer of unicellular organisms. When the highly stable multi-encapsulated cypressin liposomes are close to the unicellular organisms, the phospholipid bilayers easily fuse with each other, and the cypressin active substances in the vesicles are completely encapsulated into the unicellular organisms. Due to the dense encapsulation of cypressin by the sugar ester nanoparticles, the cypressin active substances are almost not lost during the fusion process, which further improves the utilization rate of the active substances and the bactericidal effect.

[0064] Example 1:

[0065] Ingredient selection: a solution of sorbitol and glycerol in a volume ratio of 3:2 was selected as the first aqueous phase solution, a solution of butylene glycol and sucrose monolaurate in a volume ratio of 8:1 was selected as the second aqueous phase solution, and a solution of clove oil and hinokitiol in a volume ratio of 6:4 was selected as the oil phase solution;

[0066] Proportion selection: the first aqueous phase solution accounts for 25% of the total proportion, the second aqueous phase solution accounts for 65% of the total proportion, and the oil phase solution accounts for 10% of the total proportion;

[0067] Under the action of a high shear homogenizer at 8000 rpm / min, the second aqueous phase solution was slowly poured into the first aqueous phase solution to obtain a mixed aqueous phase solution, and heated to 60°C and maintained for 15 minutes. After the oil phase solution was slowly added to the mixed aqueous phase solution, the temperature was raised to 65°C and maintained for 15 minutes to obtain a nano-hinokitiol emulsion.

[0068] The nano-hinokitiol particle size and polydispersity index in the nano-hinokitiol emulsion prepared in Example 1 were regularly tested using a nano-particle size analyzer. The test results are shown in Table 1 below:

[0069] Table 1 Test data of nano-hinokitiol emulsion of Example 1

[0070] .

[0071] Example 2

[0072] The other conditions are the same as those in Example 1, except for the selection of the proportions: the first aqueous phase solution accounts for 35% of the total proportion, the second aqueous phase solution accounts for 55% of the total proportion, and the oil phase solution accounts for 10% of the total proportion. The test data table of the nano-hinokitiol emulsion of Example 2 is shown in Table 2 below:

[0073] Table 2 Test data of nano-hinokitiol emulsion of Example 2

[0074] .

[0075] Example 3:

[0076] Other conditions are the same as those in Example 1, except for the selection of ingredients: a solution of sorbitol and glycerol in a volume ratio of 3:2 is selected as the first aqueous phase solution, a solution of butanediol and disrhamnolipid in a volume ratio of 8:1 is selected as the second aqueous phase solution, and a solution of lilac oil and hinokitiol in a volume ratio of 6:4 is selected as the oil phase solution. The test data table of the nano-hinokitiol emulsion corresponding to Example 3 is shown in Table 3 below:

[0077] Table 3 Test data of nano-hinokitiol emulsion of Example 3

[0078] .

[0079] Example 4:

[0080] Other conditions are the same as in Example 1, except for the selection of the component ratio:

[0081] A solution containing sorbitol and glycerol in a volume ratio of 3:2 was selected as the first aqueous phase solution, a solution containing butanediol and monorhamnolipid in a volume ratio of 8:1 was selected as the second aqueous phase solution, and a solution containing lilac oil and hinokitiol in a volume ratio of 6:4 was selected as the oil phase solution. The test data table of the nano-hinokitiol emulsion of Example 4 is shown in Table 4 below:

[0082] Table 4 Test data of nano-hinokitiol emulsion of Example 4

[0083] .

[0084] The nano-hinokitiol emulsions of Examples 1 and 4 were selected as representative emulsions. In addition, a hinokitiol hexanediol solution in which hinokitiol was directly dissolved in hexanediol and a hinokitiol dipropylene glycol solution in which hinokitiol was directly dissolved in dipropylene glycol were selected as comparative examples to investigate the stability of hinokitiol activity. The content of hinokitiol active matter was detected by HPLC. The HPLC conditions were as follows: main chromatographic conditions: C18 column, flowability acetonitrile: water: trifluoroacetic acid (500:500:0.5), hinokitiol with a retention time of about 4.16 min, and decomposition products with retention times of 5.2 and 10.7 min (the same HPLC conditions were used in the following examples). The decay of the hinokitiol active matter was tested under heating conditions at 54 degrees, and the content change table of the hinokitiol active matter was shown in Table 5:

[0085] Table 5 Changes in the content of hinokitiol active ingredients under heating conditions at 54 degrees

[0086] .

[0087] Note: A 14-day attenuation rate of less than 10% at 54°C is the key to determining whether the shelf life of the active ingredient can reach 1 year.

[0088] The attenuation of hinokitiol active ingredients was tested under sunlight, and the content change of hinokitiol active ingredients was shown in Table 6:

[0089] Table 6 Changes in the content of hinokitiol active ingredients under sunlight

[0090]

[0091] The HPLC liquid phase diagrams of the cypress phenol hexanediol solution in the initial state, 14 days of light and 28 days of light are as follows: Figures 7 to 9 As shown, the absorption peaks of hinokitiol and its degradation products appeared at 240 nm.

[0092] Example 5

[0093] Ingredient selection: The nano-hinokitiol emulsion prepared in Example 1, the phospholipid selected was polyquaternium-51, and the emulsifying ester selected was a mixture of caprylic / capric glyceride and tocopheryl acetate in a ratio of 3:1;

[0094] Ratio selection: the ratio of phospholipids to emulsified esters is 1:1, the weight ratio of lipid film to total hinokitiol is 1.5:1, and the weight ratio of nano-hinokitiol emulsion to phosphate buffer PBS is 4:1.

[0095] Preparation method: 1) Control the room temperature at 20°C. In a fume hood, add ethanol to a spherical flask, controlling the amount of ethanol added to 1 / 5. Place a magnet in the flask, attach a magnetic stirrer to the flask, and cover the flask with a film. Under magnetic stirring (approximately 300 rpm), add phospholipids and emulsified esters to the ethanol solvent at a concentration range of 1-10%, and dissolve evenly. Ethanol solution is rotary evaporated at low pressure at 55°C to remove ethanol, forming a lipid film on the inner wall of the flask. Slowly pour the prepared nano-hinokitiol emulsion into phosphate buffered saline (PBS) (pH 6.3), mix thoroughly, and quickly pour into the flask with the lipid film formed on the inner wall. Magnetic stirring is performed for 10 minutes, followed by equilibration of the temperature for 20 minutes in an ultrasonic oscillator with an ice-water bath, and then oscillation for 20 minutes to obtain liposomes.

[0096] The encapsulation efficiency of hinokitiol was tested by ultrafiltration centrifugation: 1 mL of a 100-fold diluted liposome suspension was added to ultrafiltration centrifuge tubes with a molecular weight cutoff of 5 kD, 10 kD, and 30 kD. The tubes were centrifuged at 5,000 r / min for 10 minutes. The filtrates were collected regularly for HPLC analysis. The results of the comparison of the retention effect and the calculated encapsulation efficiency are shown in Table 7 below:

[0097] Table 7 Liposome test data of Example 5

[0098] .

[0099] Example 6:

[0100] Other conditions were the same as those in Example 5, except that the weight ratio of the nano-hinokitiol emulsion to the phosphate buffer solution (PBS) was set to 4:1, the ratio of the phospholipid to the emulsifying ester was set to 2:1, and ultrasonic vibration was performed for 25 minutes. The filtrate was collected regularly for HPLC analysis. The test results of the comparison of the interception effect and the calculation of the encapsulation efficiency are shown in Table 8 below:

[0101] Table 8 Liposome test data of Example 6

[0102] .

[0103] Embodiment seven:

[0104] Other conditions were the same as those in Example 5, except that the weight ratio of the nano-hinokitiol emulsion to the phosphate buffer solution (PBS) was set to 1:1, the ratio of the phospholipid to the emulsifying ester was set to 4:1, and ultrasonic vibration was performed for 25 minutes. The filtrate was collected regularly for HPLC analysis. The test results of the comparison of the interception effect and the calculation of the encapsulation efficiency are shown in Table 9 below:

[0105] Table 9 Liposome test data of Example 7

[0106] .

[0107] Embodiment 8:

[0108] Other conditions were the same as those in Example 5, except that the weight ratio of the nano-hinokitiol emulsion to the phosphate buffer solution (PBS) was set to 1:1, the ratio of the phospholipid to the emulsifying ester was set to 6:1, and ultrasonic vibration was performed for 30 minutes. The filtrate was collected regularly for HPLC analysis. The test results of the comparison of the interception effect and the calculation of the encapsulation efficiency are shown in Table 10 below:

[0109] Table 10 Liposome test data of Example 8

[0110] .

[0111] Example 9

[0112] Other conditions were the same as those in Example 5, except that the weight ratio of the nano-hinokitiol emulsion to the phosphate buffer solution (PBS) was set to 2:1, the ratio of the phospholipid to the emulsified ester was set to 8:1, and ultrasonic vibration was performed for 30 minutes. The filtrate was collected regularly for HPLC analysis. The test results of the comparison of the interception effect and the calculation of the encapsulation efficiency are shown in Table 11 below:

[0113] Table 11 Liposome test data of Example 9

[0114] .

[0115] Example 10

[0116] Other conditions were the same as those in Example 5, except that the weight ratio of the nano-hinokitiol emulsion to the phosphate buffer solution (PBS) was set to 2:1, the ratio of the phospholipid to the emulsified ester was set to 10:1, and ultrasonic vibration was performed for 30 minutes. The filtrate was collected regularly for HPLC analysis. The test results of the comparison of the interception effect and the calculation of the encapsulation efficiency are shown in Table 12 below:

[0117] Table 12 Liposome test data of Example 10

[0118] .

[0119] Example 11

[0120] Other conditions were the same as those in Example 5, except that no emulsifying ester was added, only phospholipids were selected, and the weight ratio of phospholipids to total hinokitiol was set to 1.5:1, the weight ratio of nano-hinokitiol emulsion to phosphate buffer solution (PBS) was set to 2:1, and ultrasonic vibration was performed for 20 minutes. The filtrate was regularly collected for HPLC analysis. The test results of comparison of the interception effect and calculation of the encapsulation efficiency are shown in Table 13 below:

[0121] Table 13 Liposome test data of Example 11

[0122] .

[0123] The liposomes prepared in Examples 5, 6, 7, 9, and 11 were heated at 54 degrees Celsius to measure the decay of the hinokitiol active ingredient. The hinokitiol active ingredient content was detected by HPLC, and the hinokitiol content was used to represent the decay. The results are shown in Table 14 below:

[0124] Table 14: Attenuation of hinokitiol activity measured under heating conditions at 54 degrees in different examples

[0125]

[0126] The liposomes prepared in Examples 5, 6, 7, 9, and 11 were tested for attenuation of the hinokitiol active ingredient under sunlight irradiation conditions. The hinokitiol active ingredient content was detected by HPLC, and the hinokitiol content was used to represent the attenuation. The results are shown in Table 15 below:

[0127] Table 15 Attenuation of hinokitiol active ingredients measured under sunlight in different examples

[0128] .

[0129] The test results of Examples 1 to 11 above demonstrate that the multi-encapsulated hinokitiol liposomes prepared by this solution have high stability. In addition to the stability test, this solution also tested the antibacterial effect of the multi-encapsulated hinokitiol liposomes. The test process is as follows:

[0130] (1) Preparation of antibacterial sheets: For the liquid antibacterial agent (liposomes of Example 9, nano-hinoki phenol emulsion of Example 1, and dipropylene glycol solution containing hinoki phenol are selected as liquid antibacterial agents), sterile and dry filter paper sheets are taken, 20 μl of antibacterial agent solution of actual use concentration is added to each sheet, and then the filter paper sheets are placed flat in a clean sterile plate, opened and placed in an incubator (37°C) to dry, or placed at room temperature to dry naturally for use.

[0131] (2) Preparation of negative control samples: Take sterile dry filter paper pieces, add 20 μl of sterile distilled water to each piece, dry and set aside.

[0132] (3) Inoculation of test bacteria: Use a sterile cotton swab to inoculate a sample with a concentration of 5×10 5 cfu / ml~5×10 6 Apply a 500 cfu / ml suspension of test bacteria (Staphylococcus aureus, Malassezia furfur, and Propionibacterium acnes) evenly to the surface of a nutrient agar plate three times. Rotate the plate 60° after each application, and finally swab the swab around the edge of the plate. Cover the plate and allow it to dry at room temperature for 5 minutes.

[0133] (4) Placement of antibacterial sample patches: Place one infected plate for each test, with four test samples and one negative control sample placed on each plate, for a total of five. Use sterile tweezers to place the sample patches on the plate surface. The centers of the sample patches should be at least 25 mm apart and at least 15 mm away from the edge of the plate. After placement, use sterile tweezers to gently press the sample patches against the plate surface. Cover the plate, place in a 37°C incubator, and incubate for 16-18 hours before observing the results. Use a vernier caliper to measure the diameter of the antibacterial ring (including the patch) and record it.

[0134] The inhibition results corresponding to Staphylococcus aureus, Malassezia furfur and Propionibacterium acnes are shown in the figure below. Figures 4 to 6 As shown, Figure 4 (a) corresponds to liposomes, (b) corresponds to nano-hinokitiol emulsion, and (c) corresponds to dipropylene glycol solution dissolved with hinokitiol; Figure 5 (a) corresponds to liposomes, (b) corresponds to nano-hinokitiol emulsion, and (c) corresponds to dipropylene glycol solution dissolved with hinokitiol; Figure 6 (a) corresponds to liposomes, (b) corresponds to nano-hinokitiol emulsion, and (c) corresponds to dipropylene glycol solution dissolved with hinokitiol. It can be seen that the double-shell three-membrane structure of liposomes can better increase the stability of active ingredients and improve the microbial inhibition effect of hinokitiol compared to nano-hinokitiol emulsion, which has positive significance for extending the shelf life of the product and the utilization rate of active ingredients.

[0135] Those skilled in the art should understand that the technical features of the above embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0136] The above embodiments merely illustrate several embodiments of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present application. It should be noted that a person of ordinary skill in the art may make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present application shall be determined by the appended claims.

Claims

1. A method for preparing highly stable multi-encapsulated hinokitiol liposomes, characterized in that: The following steps are involved: Mixing a first aqueous phase solution and a second aqueous phase solution under high shear homogenization conditions, and simultaneously heating to 58-62° C. and maintaining for 10-20 minutes to obtain a mixed aqueous phase solution, wherein the first aqueous phase solution contains at least one polyol, and the second aqueous phase solution contains at least one polyol and a glycolipid emulsifier, wherein the glycolipid emulsifier in the second aqueous phase solution is selected from one or a combination of two or more of monorhamnolipid, disrhamnolipid, and sucrose fatty acid ester, and when the glycolipid emulsifier is selected from sucrose fatty acid ester, the carbon chain length of the fatty acid is C4-C28; The aqueous phase solution and the oil phase solution are mixed under high shear homogenization conditions, and heated to 62-68° C. for 10-20 minutes to obtain a nano-hinokitiol emulsion, wherein the oil phase solution contains oil and hinokitiol, and the weight of the oil phase solution accounts for 6-10% of the nano-hinokitiol emulsion. The oil phase solution contains oil accounting for 5-9% by weight of the nano-hinokitiol emulsion and hinokitiol accounting for 1-5% by weight of the nano-hinokitiol emulsion; adding phospholipids and emulsified esters to an ethanol solution to obtain a lipid mixture, wherein the weight ratio of phospholipids to emulsified esters is 1 to 10:1, and subjecting the lipid mixture to low-pressure rotary evaporation and removing ethanol to form a lipid film, wherein the emulsified esters are a mixture of caprylic / capric glycerides and tocopheryl acetate in a ratio of 3:1; Dissolving the nano-hinokitiol emulsion in a phosphate buffer to obtain a nano-hinokitiol dispersion, adding the nano-hinokitiol dispersion to a container containing a lipid film and stirring, controlling the weight ratio of the lipid film to the hinokitiol in the nano-hinokitiol dispersion to be 1-2:1, and ultrasonically vibrating the mixture in an ultrasonic oscillator with an ice-water bath to obtain multi-encapsulated hinokitiol liposomes; The multi-encapsulated cypress phenol liposomes include a phospholipid bilayer and a glycolipid inner layer arranged from the outside to the inside, wherein a portion of the cypress phenol particles are encapsulated in the glycolipid inner layer, the phospholipid bilayer encapsulates the glycolipid inner layer, and a portion of the dispersed cypress phenol is also encapsulated between the two molecular layers of the phospholipid bilayer.

2. The method for preparing highly stable multi-encapsulated hinokitiol liposomes according to claim 1, characterized in that: The weight proportion of the first aqueous phase solution is 20-70% of the nano-hinokitiol emulsion, and the weight proportion of the second aqueous phase solution is 20-70% of the nano-hinokitiol emulsion.

3. The method for preparing highly stable multi-encapsulated hinokitiol liposomes according to claim 1, characterized in that: The first aqueous phase solution contains a polyol selected from one or more of ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, 1,2-butylene glycol, 1,4-butylene glycol, 2,3-butylene glycol, 1,5-pentanediol, 1,2-pentanediol, isopentyl glycol, neopentyl glycol, 1,2-hexanediol, 1,6-hexanediol, octanediol, decanediol, sorbitol, xylitol, lactitol, maltitol, erythritol, mannitol, threitol, glycerol, 1,2,6-hexanetriol, dipropylene glycol, menthylene glycol, methylpropylene glycol, panthenol, and pentaerythritol.

4. The method for preparing highly stable multi-encapsulated hinokitiol liposomes according to claim 1, characterized in that: The polyol contained in the second aqueous phase solution is selected from one or a combination of two or more of ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, 1,2-butylene glycol, 1,4-butylene glycol, 2,3-butylene glycol, 1,5-pentanediol, 1,2-pentanediol, isopentyl glycol, neopentyl glycol, 1,2-hexanediol, 1,6-hexanediol, octanediol, decanediol, sorbitol, xylitol, lactitol, maltitol, erythritol, mannitol, threitol, glycerol, 1,2,6-hexanetriol, dipropylene glycol, menthylene glycol, methylpropylene glycol, panthenol, and pentaerythritol.

5. The method for preparing highly stable multi-encapsulated hinokitiol liposomes according to claim 1, characterized in that: The phospholipids are selected from one or a combination of two or more of lecithin, cephalin, soybean lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylcholine, dioleoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dipalmitoylphosphatidylglycerol sodium salt, distearoylphosphatidylcholine, (2,3-dioleyloxypropyl)trimethylammonium chloride, and polyquaternium-51.

6. A highly stable multi-encapsulated hinokitiol liposome, characterized in that: The method for preparing the highly stable multi-encapsulated hinokitiol liposomes according to any one of claims 1 to 5 is disclosed.

7. The highly stable multi-encapsulated hinokitiol liposome according to claim 6, characterized in that: It includes a phospholipid bilayer and a glycolipid inner layer arranged from the outside to the inside, wherein the glycolipid inner layer wraps a part of the cypress phenol particles, the phospholipid bilayer wraps the glycolipid inner layer, and at the same time, a part of the dispersed cypress phenol is also wrapped between the two molecular layers of the phospholipid bilayer.

8. A method for using highly stable multi-encapsulated hinokitiol liposomes, characterized in that: The highly stable multi-encapsulated hinokitiol liposomes according to claim 6 are used as an ingredient of antibacterial cosmetics.

Images