A water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil and a method for preparing the same

Abstract

The application discloses a kind of enzymatic camellia seed oil water-in-oil transparent emulsion and preparation method thereof, and the water-in-oil transparent emulsion of enzymatic camellia seed oil is mainly obtained by mixing oil phase and water phase, the oil phase includes the following components by weight:59-96.4 parts of enzymatic camellia seed oil, 0.01-20 parts of emulsifier, the water phase includes the following components by weight:0.01-10 parts of red camellia flower polypeptide solution, 0.01-1 parts of honey extract, 0.01-10 parts of water.The application is through preferred composite emulsifier system and the multi-level cooperation between enzymatic camellia seed oil, red camellia flower polypeptide, honey extract, multi-directional mutual promotion, this kind of effect makes three form an organic whole, realizes "high oil phase ratio, optical transparent, long-term stable, skin feel refreshing, outstanding effect" comprehensive technical effect.

Description

Technical Field

[0001] This invention belongs to the field of cosmetics, and particularly relates to a water-in-oil transparent emulsion and its preparation method. Background Technology

[0002] Water-in-oil emulsions are a type of emulsification technology in cosmetics where oil is the continuous phase and water is the dispersed phase. Because the external phase is oil, it forms a hydrophobic film on the skin surface after application, providing excellent occlusive moisturizing and hydrating properties. It is widely used in highly moisturizing creams, sunscreens, and base makeup. Developing such systems requires addressing stability issues. Strongly lipophilic emulsifiers such as polyglycerol ricinoleate or alkyl polymethylsiloxane copolymers must be selected to prevent the aqueous phase droplets from coalescing. Simultaneously, rheological properties must be precisely controlled through organically modified clays or oil-phase gelling agents to ensure a balance between storage stability and a light, easily spreadable texture.

[0003] Currently, technological trends in this field are moving towards lighter, more functional, and more aesthetically pleasing formulations. On one hand, by employing nanoemulsification technology or selecting highly polar, low-viscosity synthetic oils, water-in-oil microemulsions or nanoemulsions with a lightweight feel and even a transparent appearance are being developed to enhance the user experience. On the other hand, these systems are also being designed as multi-functional carriers to encapsulate, protect, and deliver hydrophilic active ingredients, achieving targeted and sustained release to the skin. As consumers increasingly demand higher product quality and a better skin feel, stable and effective water-in-oil systems will continue to be an important direction for formulation innovation.

[0004] Chinese patent CN109481319A discloses a camellia seed oil micro / nano emulsion and its preparation method. The method uses 35-50% camellia seed oil as the oil phase, 2-8% lecithin as the emulsifier, 0.1-0.3% antioxidant, 2-10% water, and 31.7-60.9% glycerol as the aqueous phase to prepare a micro / nano emulsion. However, the proportion of camellia seed oil in this method is only 35%-50%, resulting in relatively limited moisturizing effect and room for improvement in skin feel. Furthermore, this method cannot further increase the proportion of camellia seed oil. This limitation stems from the balance between the required amount of emulsifier and the limit for maintaining the thermodynamic stability of the system. Any attempt to significantly increase the proportion of the oil phase will face the triple challenges of emulsifier dosage limits, a sharp decrease in system stability, and uncontrolled droplet size, making a transparent appearance impossible to achieve. In addition, the method uses lecithin as an emulsifier, which has weak emulsifying ability, and the transparency and stability of the material need to be improved, which is detrimental to the application of this raw material. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil with simple preparation process, refreshing skin feel, high stability and high transparency, and its preparation method.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0007] A water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil is mainly obtained by mixing an oil phase and an aqueous phase. The oil phase comprises the following components in parts by weight: 59-96.4 parts of enzymatically hydrolyzed camellia seed oil and 0.01-20 parts of emulsifier. The aqueous phase comprises the following components in parts by weight: 0.01-10 parts of red camellia flower polypeptide solution, 0.01-1 part of honey extract, and 0.01-10 parts of water, wherein the polypeptide content in the red camellia flower polypeptide solution is 0.01%-1% by mass.

[0008] In the above-mentioned water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil, preferably, the oil phase comprises the following components in parts by weight: 75-85 parts of enzymatically hydrolyzed camellia seed oil, 5-15 parts of emulsifier, and the aqueous phase comprises the following components in parts by weight: 0.1-5 parts of red camellia flower polypeptide solution, 0.05-0.5 parts of honey extract, and 1-5 parts of water.

[0009] In the above-mentioned water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil, preferably, the emulsifier includes a first emulsifier and a second emulsifier, wherein the first emulsifier is one or more of PEG-5 glyceryl triisostearate, sorbitan monooleate and polyglycerol-10 dioleate, and the second emulsifier is PPG-3 octyl ether.

[0010] In the above-mentioned water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil, preferably, the mass ratio of the first emulsifier and the second emulsifier is 1:(0.1-2).

[0011] In the above-mentioned water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil, preferably, the aqueous phase further includes 0.01-10 parts of an alcohol additive, wherein the alcohol additive is 1,2-pentanediol and 1,2-hexanediol, and the mass ratio of the two is 1:(0.5-2). The above-mentioned alcohol additive has both preservative and refractive index regulating functions to match the refractive indices of the aqueous and oil phases. The amount of alcohol additive used is preferably chosen to match the refractive indices of the aqueous and oil phases.

[0012] In the above-mentioned water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil, preferably, the oil phase further includes 0.01-5 parts of antioxidant, wherein the antioxidant is one or more of vitamin E and its derivatives, camellia flower oil extract, and camellia stem and leaf oil extract.

[0013] In the above-mentioned water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil, preferably, the extractant oil used in the extraction of the camellia flower oil extract and the camellia stem and leaf oil extract is enzymatically hydrolyzed camellia seed oil. The camellia flower oil extract and the camellia stem and leaf oil extract are obtained by drying, grinding, and sieving the above raw materials, then soaking them in enzymatically hydrolyzed camellia seed oil, followed by ultrasonic crushing, and extracting for 2-48 hours. This extraction method yields extracts with better compatibility with the oil phase.

[0014] As a general technical concept, the present invention also provides a method for preparing the above-mentioned enzymatically hydrolyzed camellia seed oil water-in-oil transparent emulsion, comprising the following steps: (1) Dissolve the lipase in water, disperse it evenly, add camellia seed oil, stir and mix, and then perform enzymatic hydrolysis; (2) After enzymatic hydrolysis, the enzymatic hydrolysis system is allowed to stand and separate into layers. The oil layer is then filtered to obtain enzymatically hydrolyzed camellia seed oil. (3) Add emulsifier to the enzymatically hydrolyzed camellia seed oil, mix well to obtain the oil phase; mix red camellia flower polypeptide solution, honey extract and water well to obtain the aqueous phase; (4) The aqueous phase is slowly added to the oil phase and stirred continuously. After the material changes from turbid to clear and transparent, it is homogenized and then stirred and allowed to stand to obtain the water-in-oil transparent emulsion.

[0015] In the above preparation method, preferably, the amount of lipase used is 0.1-10% of the mass of the camellia seed oil, and the enzymatic hydrolysis is carried out at room temperature.

[0016] In the above preparation method, preferably, the homogenization process includes high-speed homogenization, high-pressure homogenization, and microfluidic homogenization, wherein the rotation speed of high-speed homogenization is 3000-10000 rpm and the processing time is 1-20 min; the pressure of high-pressure homogenization and microfluidic homogenization is 1-500 Bar and the number of cycles is 1-5 times.

[0017] The method for preparing a water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil according to the present invention, more specifically, includes the following steps: S1: Accurately weigh a certain proportion of lipase and dissolve it in deionized water. After complete dissolution, add the enzyme solution to camellia seed oil, stir and mix, and enzymatically hydrolyze at room temperature for 1-24 hours. S2: After enzymatic hydrolysis, allow the mixture to stand and separate into layers, filter and collect the oil layer. Residual water can be absorbed with absorbent material and filtered again to obtain enzymatically hydrolyzed camellia seed oil. S3: Accurately weigh a certain weight of enzymatically hydrolyzed camellia seed oil, add a certain amount of emulsifier and antioxidant, and mix evenly in a water bath at 50±2℃. Set aside. S4: Weigh a certain amount of alcohol additive, red camellia peptide solution, honey extract, and deionized water, mix evenly, and heat to about 45℃ to obtain the aqueous phase. S5: Slowly add the aqueous phase dropwise to the oil phase while continuously stirring at 100-3000 rpm for 1-60 minutes until the mixture changes from turbid to clear and transparent. S6: Homogenize the transparent water-in-oil emulsion. After homogenization, it needs to be stirred in a water bath for 1-60 minutes, and then allowed to stand to defoam, resulting in a water-in-oil transparent emulsion.

[0018] Camellia seed oil has a structure similar to sebum, making it highly skin-friendly. Its core components are oleic acid, tea polyphenols, and squalene. It strengthens the skin barrier by replenishing lipids, achieving deep hydration. Simultaneously, its rich unsaturated fatty acids repair the sebum film and improve dryness and flaking. Enzymatic hydrolysis of camellia seed oil involves a lipase-catalyzed glycerolization reaction, converting the main component, triglycerides, into diglycerides and monoglycerides. This molecular-level structural change brings multiple advantages. Studies have shown that the generated monoglycerides and diglycerides themselves possess emulsifying properties, reducing the need for exogenous emulsifiers in formulations. Enzymatically hydrolyzed camellia seed oil has higher polarity and a higher affinity for water, contributing to the stability of water-in-oil systems. Water-in-oil emulsions prepared using enzymatically hydrolyzed structured oils exhibit significantly enhanced physical stability and effectively delay phase separation. Furthermore, the increased polarity of enzymatically hydrolyzed oils enhances their hydrophilicity and simultaneously reduces their refractive index. This facilitates refractive index matching between the oil and aqueous phases when constructing transparent water-in-oil systems, promoting the formation of transparent emulsions. Consequently, a clear and transparent appearance is more easily achieved while maintaining a refreshing skin feel. Lipase enzymatically hydrolyzes camellia seed oil, breaking down triglycerides into diglycerides, monoglycerides, and fatty acids, thus reducing the molecular weight of the oil. This results in lower surface tension, significantly improved spreadability and absorption rate, leading to a refreshingr skin feel. Water-in-oil emulsions prepared using enzymatically hydrolyzed camellia seed oil as the oil phase also exhibit a refreshing skin feel.

[0019] Camellia flower peptides can promote cell repair, increase AQP3 protein expression in fibroblasts, and increase LOR protein expression in keratinocytes. They significantly promote scratch repair in keratinocytes, demonstrating significant repair, moisturizing, antioxidant, and nourishing functions. Camellia flower peptides are obtained from camellia flowers through high-pressure homogenization and pepsin hydrolysis, followed by purification. The main preparation process is as follows: Camellia flower raw materials are pulverized and passed through a 50-100 mesh sieve to obtain camellia flower powder. The camellia flower powder is added to ultrapure water at a solid-liquid ratio of 1:20. The prepared camellia flower suspension is homogenized using a high-pressure homogenizer at a pressure of 800-1000 Bar, once. After homogenization, the mixture is centrifuged at 8500 rpm for 15 minutes, and the supernatant is collected. Ammonium sulfate is added to the supernatant to 80% for salting out. The protein precipitate is collected by centrifugation at 8500 rpm for 15 minutes and resuspended in ultrapure water. The precipitate was dissolved using ultrasound, desalted by dialysis, and then freeze-dried to obtain camellia protein powder. The camellia protein powder was mixed with distilled water at a ratio of 1:20, and the pH was adjusted to the optimal pH for the protease reaction. The protease was added, and the enzymatic reaction was carried out at 50-60℃ for 4 hours. After the enzymatic reaction, the hydrolysate was placed in a boiling water bath for 10-15 minutes to inactivate the enzyme. The inactivated hydrolysate was then centrifuged at 8500 rpm for 15 minutes, and the supernatant was collected to obtain the camellia polypeptide solution. For specific details of the above method, please refer to Chinese patent application CN121181647A.

[0020] The oil-in-water transparent emulsion of this invention uses enzymatically hydrolyzed camellia seed oil as the core component in its oil phase and red camellia flower polypeptide and honey extract as the core components in its aqueous phase. The synergistic effect of these three components, combined with the optimized addition of emulsifiers, results in a transparent oil-in-water system with a refreshing feel, good moisturizing effect, and strong stability. Specific analysis is as follows: 1. The enzymatic hydrolyzed camellia seed oil, honey extract, and red camellia peptides form a synergistic mechanism that enhances the stability, transparency, and skin feel of the emulsion: Diglycerides and monoglycerides in the enzymatic hydrolyzed camellia seed oil spontaneously adsorb at the oil-water interface, forming a dense interfacial film that significantly reduces interfacial tension and prevents droplet aggregation. Simultaneously, the honey extract and red camellia peptides in the aqueous phase interact through hydrogen bonds to form a three-dimensional thickening network, enhancing the cohesive strength of the aqueous droplets. This network inhibits excessive droplet deformation and breakage during homogenization and provides steric hindrance during storage, working synergistically with the interfacial film to resist droplet aggregation. This three-part combination forms a dual stabilizing mechanism of "reinforced outer shell and stable core," enabling the emulsion to maintain transparency and stability even at an oil phase ratio as high as 96.4 parts. The ultra-low interfacial tension provided by the enzymatic hydrolyzed camellia seed oil creates the thermodynamic conditions for the formation of nano-sized droplets. The aqueous viscosity-enhancing network formed by honey extract and red camellia peptides effectively controls droplet size during homogenization, maintaining it within a narrow distribution range of 100-150nm, much smaller than the wavelength of visible light. This eliminates light scattering and imparts excellent optical transparency to the emulsion. Simultaneously, this network inhibits droplet coarsening during storage, ensuring long-term maintenance of transparency. Upon application, external force causes the nanodroplets to break down, releasing the aqueous phase and providing a refreshing, watery feel not found in traditional pure oil or high-oil-phase products. The released honey extract provides an immediate silky smooth application and long-lasting skin hydration, directly optimizing the user experience. The long-lasting hydration provided by the honey extract complements the occlusive moisturizing effect of the oil phase, keeping skin soft and supple for hours after application, significantly improving post-use comfort. Meanwhile, the monoglycerides and fatty acids in enzymatically hydrolyzed camellia seed oil have a transdermal penetration-promoting effect, which helps red camellia peptides penetrate the stratum corneum more effectively and exert their barrier repair function (promoting the expression of ABCA12 and LOR proteins), thereby reducing unpleasant skin sensations such as tightness and stinging that may occur due to dry skin or damaged barrier. This results in a multi-layered skin feel experience that is instantly hydrating, smooth to apply, and comfortable after use.

[0021] 2. In a more preferred embodiment, the preferred emulsifier combination of the present invention (a first emulsifier and a second emulsifier compounded in a specific ratio) forms a multi-layered synergistic effect with enzymatically hydrolyzed camellia seed oil, honey extract, and red camellia peptides: the diglycerides / monoglycerides in the enzymatically hydrolyzed camellia seed oil have self-emulsifying ability and can spontaneously adsorb onto the oil-water interface, initially reducing interfacial tension. More importantly, they can insert into the interfacial film composed of the first and second emulsifiers, reducing the required degree of order in the molecular arrangement within the film, allowing the composite emulsifier to form a complete interfacial coverage even at a lower concentration. The first emulsifier has multiple hydrophobic tail chains that can be tightly arranged at the interface to form a high-strength interfacial film. The second emulsifier, PPG-3 octyl ether, is a low-molecular-weight oil-soluble nonionic surfactant that can fill the gaps between the molecules of the first emulsifier, further reducing interfacial tension and increasing the flexibility of the interfacial film. Flavonoids, phenols, and glycoproteins in honey extract also possess surface activity, adsorbing onto interfacial defects and repairing them, thus further enhancing the long-term storage stability of the nanoemulsion. Through these mechanisms, the self-emulsifying components of enzymatically hydrolyzed camellia seed oil, together with the composite emulsifier, construct a low-interfacial-tension, high-strength mixed interfacial film. The honey extract fills interfacial defects, giving the emulsion excellent anti-agglomeration stability. The composite emulsifier and enzymatically hydrolyzed camellia seed oil achieve ultra-low interfacial tension, providing thermodynamic conditions for nanodroplet formation. PPG-3 octyl ether and enzymatically hydrolyzed camellia seed oil provide a refreshing oil-phase skin feel; honey extract provides a soft touch. Together, they create a skin feel experience of "external moisturization and internal refreshment, smooth application, and comfortable use." The combination of enzymatically hydrolyzed camellia seed oil and emulsifier synergistically enhances the transdermal penetration of red camellia peptides, improving their barrier repair efficacy.

[0022] 3. Enzymatic hydrolysis of camellia seed oil generates its own diglycerides and monoglycerides, forming a low interfacial tension (<5 mN / m) adsorption film at the oil-water interface. This allows the aqueous phase to be uniformly dispersed in the oil phase as nano-sized droplets (average particle size 100-150 nm). This microstructure provides a highly uniform, large specific surface area, and relatively isolated dispersion environment for honey extract and red camellia peptides. In other words, the nano-sized dispersion environment created by enzymatic hydrolysis of camellia seed oil is a prerequisite for the formation of a dense, thickening network between honey extract and red camellia peptides. Simultaneously, the three-dimensional thickening network formed by honey extract and red camellia peptides within the aqueous phase significantly improves the cohesive strength and resistance to deformation of the aqueous droplets. When the emulsion is subjected to shear (such as storage vibration or thermal convection caused by temperature changes), the cohesive-enhanced aqueous droplets are less prone to violent shape oscillations or breakage, thus reducing repeated impacts on the oil-water interface film. The interfacial membrane is protected from frequent mechanical disturbances, allowing its molecular arrangement to maintain its integrity and density for a longer period. Consequently, the interfacial desorption rate of diglycerides / monoglycerides in enzymatically hydrolyzed camellia seed oil is significantly reduced. This dual protection cannot be achieved by using enzymatically hydrolyzed camellia seed oil alone, or by using honey extract and red camellia peptides alone.

[0023] 4. During the emulsion application stage, the monoglycerides and free fatty acids in enzymatically hydrolyzed camellia seed oil can reversibly disrupt the orderly lipid arrangement of the stratum corneum, opening microscopic channels for the transdermal absorption of red camellia peptides. Because the red camellia peptides and honey extract are encapsulated in a network and released slowly, their contact window with the stratum corneum is extended, allowing for more efficient utilization of the channels opened by the enzymatically hydrolyzed camellia seed oil. The increased retention and absorption of peptides on the skin surface may also reduce the volatilization of free fatty acids, prolonging the duration of the penetration-promoting effect of the enzymatically hydrolyzed camellia seed oil.

[0024] In summary, this invention achieves a comprehensive technical effect of "high oil-to-phase ratio, optical transparency, long-term stability, refreshing skin feel, and outstanding efficacy" through the multi-level synergistic and multi-directional mutual promotion between the optimized composite emulsifier system and enzymatically hydrolyzed camellia seed oil, honey extract, and red camellia peptides.

[0025] The enzymatically hydrolyzed camellia seed oil emulsion of this invention is a transparent water-in-oil system with a refreshing feel, simple process, and high stability. This invention focuses on improving the stability and skin feel of the water-in-oil system. Camellia seed oil is first enzymatically hydrolyzed, and the resulting enzymatically hydrolyzed camellia seed oil, after separation and purification, is the oil phase. Then, by screening and optimizing the types and amounts of emulsifiers, a transparent water-in-oil vegetable oil system with high stability and a refreshing feel is prepared.

[0026] Compared with the prior art, the advantages of the present invention are as follows: This invention relates to a transparent water-in-oil emulsion of enzymatically hydrolyzed camellia seed oil and its preparation method. Combining the advantages of enzymatically hydrolyzed camellia seed oil and water-in-oil systems, and based on the synergistic effect of enzymatically hydrolyzed camellia seed oil, honey extract, and red camellia flower polypeptides, a transparent water-in-oil system with a simple preparation process, refreshing skin feel, high oil phase ratio, and high stability has been developed. It can not only reduce the viscosity of the system and improve the skin feel, but also improve the stability, helping it to achieve significant breakthroughs in terms of skin feel, efficacy, and stability. Moreover, the preparation process is simpler and easier to implement than existing technologies, which is conducive to its further promotion and application.

[0027] This invention relates to a water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil and its preparation method. The oil phase is enzymatically hydrolyzed camellia seed oil, and the aqueous phase consists of a red camellia peptide solution, honey extract, and water. Apart from necessary emulsifiers, the core components are enzymatically hydrolyzed camellia seed oil and red camellia peptides. The excellent antioxidant, soothing, and barrier-repairing effects of enzymatically hydrolyzed camellia seed oil and red camellia peptides will undoubtedly further enhance the cosmetic and skincare efficacy and application potential of this emulsion. Furthermore, plant oils can generate more active ingredients after enzymatic hydrolysis, which helps to enhance the efficacy of the oils and further strengthen the skincare effect of the emulsion. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a comparison chart showing the increase in ABCA12 protein levels between Examples 1-7 and Comparative Examples 1-4.

[0030] Figure 2 This is a comparison chart of the LOR protein enhancement rates of Examples 1-7 and Comparative Examples 1-4.

[0031] Figure 3 This is a comparison chart of the overall skin feel scores of Examples 1-7 and Comparative Examples 1-4.

[0032] Figure 4 This is a comparison chart of the moisture content improvement rates of Examples 1-7 and Comparative Examples 1-4. Detailed Implementation

[0033] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0034] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0035] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0036] The camellia polypeptide solution used in the following examples and comparative examples is the camellia polypeptide solution hydrolyzed by pepsin as described in Example 2 of Chinese Patent Application Publication No. CN121181647A, with a polypeptide mass concentration of 0.1%.

[0037] Example 1 A water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil and its preparation method, comprising the following preparation steps: (1) Weigh 0.5g of lipase and prepare a 1% aqueous solution. After it is evenly dispersed, add it to 100g of camellia seed oil and stir to disperse it to obtain a turbid oil-water mixture. Enzymatically hydrolyze it at 30℃ for 2h.

[0038] (2) After the enzymatic hydrolysis is completed, place the enzymatic hydrolysis system in an 80℃ water bath and stir for 10 min to complete the enzyme inactivation. Then pour the system into a separatory funnel and let it stand for more than 30 min, gradually draining the lower aqueous phase. Centrifuge the upper oil phase at 8000 rpm for 10 min. Carefully remove the centrifuge bottle and pour the upper oil phase into a clean anhydrous beaker.

[0039] (3) Dehydration: Weigh out powder-free molecular sieve packing material (1% of the total mass) at a ratio of 1:10 and introduce it into the centrifuged oil phase. After gentle stirring, let it stand to allow the water to settle and be adsorbed by the packing material. After adsorption, pour the oil phase into a vacuum filtration flask and use a 0.2 μm filter membrane to remove suspended particles from the oil phase, obtaining clean enzymatically hydrolyzed camellia seed oil.

[0040] (4) Accurately weigh 75g of enzymatically hydrolyzed camellia seed oil, and weigh 4g of PEG-5 glyceryl triisostearate, 4g of PPG-3 octyl ether, 4g of sorbitan monooleate and 5g of camellia flower oil extract. Add them to the enzymatically hydrolyzed camellia seed oil and stir and mix evenly in a water bath at 50±2℃ to obtain a uniform and transparent material A for later use.

[0041] (5) Accurately weigh 2.5g of 1,2-hexanediol and 2.5g of 1,2-pentanediol, weigh 1.5g of red camellia polypeptide solution and 0.05g of honey extract, add 1.45g of deionized water, stir and mix evenly and heat to 45℃ to obtain material B.

[0042] (6) Slowly add material B to material A, start stirring at 500 rpm, and continue stirring for 30 minutes to obtain a clear and transparent water-in-oil emulsion.

[0043] (7) The above emulsion was subjected to micro-jet treatment at a pressure of 500 Bar and a cycle of 3 times. After homogenization, it was continuously stirred for 30 minutes and allowed to stand to defoam before obtaining the material of Example 1, which is the water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil.

[0044] Example 2 A water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil and its preparation method, comprising the following preparation steps: (1) Accurately weigh 79g of enzymatically hydrolyzed camellia seed oil prepared in Example 1, and weigh 4g of PEG-5 glyceryl triisostearate, 4g of PPG-3 octyl ether, 4g of sorbitan monooleate and 1g of vitamin E. Add them to the enzymatically hydrolyzed camellia seed oil and stir and mix evenly in a water bath at 50±2℃ to obtain a uniform and transparent material A for later use.

[0045] (2) Accurately weigh 2.5g of 1,2-hexanediol and 2.5g of 1,2-pentanediol, weigh 1.0g of red camellia polypeptide solution and 0.1g of honey extract, add 1.9g of deionized water, stir and mix evenly and heat to 45℃ to obtain material B.

[0046] (3) Slowly add material B to material A, start stirring at 500 rpm, and continue stirring for 30 minutes to obtain a clear and transparent water-in-oil emulsion.

[0047] (4) The above emulsion was subjected to micro-jet treatment at a pressure of 500 Bar and a cycle of 3 times. After homogenization, it was continuously stirred for 30 minutes and allowed to stand to defoam before obtaining the material of Example 2, which is the water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil.

[0048] Example 3 A water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil and its preparation method, comprising the following preparation steps: (1) Accurately weigh 59g of enzymatically hydrolyzed camellia seed oil prepared in Example 1, and weigh 13g of PEG-5 glyceryl triisostearate, 7g of PPG-3 octyl ether and 1g of vitamin E acetate. Add them to the enzymatically hydrolyzed camellia seed oil and stir and mix evenly in a water bath at 50±2℃ to obtain a uniform and transparent material A for later use.

[0049] (2) Accurately weigh 5g of 1,2-hexanediol and 5g of 1,2-pentanediol, weigh 5g of red camellia polypeptide solution and 0.1g of honey extract, add 4.9g of deionized water, mix evenly and heat to 45℃ to obtain material B.

[0050] (3) Add material B slowly to material A and start stirring at 100 rpm. Continue stirring for 60 minutes to obtain a clear and transparent water-in-oil emulsion.

[0051] (4) The above emulsion was subjected to high-speed homogenization at a speed of 8000 rpm. After high-speed homogenization, the mixture was allowed to stand and defoamed to obtain the material of Example 3, which is the water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil.

[0052] Example 4 A water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil and its preparation method, comprising the following preparation steps: (1) Accurately weigh 96.4g of enzymatically hydrolyzed camellia seed oil prepared in Example 1, and weigh 0.5g of sorbitan monooleate, 0.5g of polyglycerol-10 dioleate, 0.5g of PPG-3 octyl ether and 0.1g of vitamin E. Add them to the enzymatically hydrolyzed camellia seed oil and stir and mix evenly in a water bath at 50±2℃ to obtain a uniform and transparent material A for later use.

[0053] (2) Accurately weigh 0.5g of 1,2-hexanediol and 0.5g of 1,2-pentanediol, weigh 0.5g of red camellia polypeptide solution and 0.01g of honey extract, add 0.49g of deionized water, mix evenly and heat to 45℃ to obtain material B.

[0054] (3) Slowly add material B to material A and start stirring at 1000 rpm for 5 minutes to obtain a clear and transparent water-in-oil emulsion.

[0055] (4) The above emulsion was subjected to high pressure homogenization at a pressure of 500 Bar, with 3 cycles and continuous stirring for 30 minutes. After standing and defoaming, the material of Example 4 was obtained, which is the water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil.

[0056] Example 5 A water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil and its preparation method, comprising the following preparation steps. (1) Accurately weigh 79g of enzymatically hydrolyzed camellia seed oil prepared in Example 1, and weigh 4g of PEG-5 glyceryl triisostearate, 4g of PPG-3 octyl ether, 4g of sorbitan monooleate and 1g of vitamin E. Add them to the enzymatically hydrolyzed camellia seed oil and stir and mix evenly in a water bath at 50±2℃ to obtain a uniform and transparent material A for later use.

[0057] (2) Accurately weigh 2.5g of 1,2-hexanediol and 2.5g of 1,2-pentanediol, weigh 2.0g of red camellia polypeptide solution and 0.1g of honey extract, add 0.9g of deionized water, stir and mix evenly and heat to 45℃ to obtain material B.

[0058] (3) Slowly add material B to material A, start stirring at 500 rpm, and continue stirring for 30 minutes to obtain a clear and transparent water-in-oil emulsion.

[0059] (4) The above emulsion was subjected to micro-jet treatment at a pressure of 500 Bar and a cycle of 3 times. After homogenization, it was continuously stirred for 30 minutes and allowed to stand to defoam before obtaining the material of Example 5, which is the water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil.

[0060] Example 6 A water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil and its preparation method, comprising the following preparation steps. (1) Accurately weigh 79g of enzymatically hydrolyzed camellia seed oil prepared in Example 1, and weigh 4g of PEG-5 glyceryl triisostearate, 4g of lecithin, 4g of sorbitan monooleate and 1g of vitamin E. Add them to the enzymatically hydrolyzed camellia seed oil and stir and mix evenly in a water bath at 50±2℃ to obtain a uniform and transparent material A for later use.

[0061] (2) Accurately weigh 2.5g of 1,2-hexanediol and 2.5g of 1,2-pentanediol, weigh 1.0g of red camellia polypeptide solution and 0.1g of honey extract, add 1.9g of deionized water, stir and mix evenly and heat to 45℃ to obtain material B.

[0062] (3) Slowly add material B to material A, start stirring at 500 rpm, and continue stirring for 30 minutes to obtain a clear and transparent water-in-oil emulsion.

[0063] (4) The above emulsion was subjected to micro-jet treatment at a pressure of 500 Bar and a cycle of 3 times. After homogenization, it was continuously stirred for 30 minutes and allowed to stand to defoam before obtaining the material of Example 6, which is the water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil.

[0064] Example 7 A water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil and its preparation method, comprising the following preparation steps: (1) Accurately weigh 79g of enzymatically hydrolyzed camellia seed oil prepared in Example 1, and weigh 4g of PEG-5 glyceryl triisostearate, 4g of PPG-3 octyl ether, 4g of sorbitan monooleate and 1g of vitamin E. Add them to the enzymatically hydrolyzed camellia seed oil and stir and mix evenly in a water bath at 50±2℃ to obtain a uniform and transparent material A for later use.

[0065] (2) Accurately weigh 2.5g of 1,2-hexanediol and 2.5g of 1,2-pentanediol, weigh 0.01g of red camellia polypeptide solution and 0.01g of honey extract, add 2.98g of deionized water, stir and mix evenly and heat to 45℃ to obtain material B.

[0066] (3) Slowly add material B to material A, start stirring at 500 rpm, and continue stirring for 30 minutes to obtain a clear and transparent water-in-oil emulsion.

[0067] (4) The above emulsion was subjected to micro-jet treatment at a pressure of 500 Bar and a cycle of 3 times. After homogenization, it was continuously stirred for 30 minutes and allowed to stand to defoam before obtaining the material of Example 7, which is the water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil.

[0068] Comparative Example 1 A water-in-oil emulsion and its preparation method, comprising the following preparation steps: (1) Accurately weigh 79g of enzymatically hydrolyzed camellia seed oil prepared in Example 1, and weigh 4g of PEG-5 glyceryl triisostearate, 4g of PPG-3 octyl ether, 4g of sorbitan monooleate and 1g of vitamin E. Add them to the enzymatically hydrolyzed camellia seed oil and stir and mix evenly in a water bath at 50±2℃ to obtain a uniform and transparent material A for later use.

[0069] (2) Accurately weigh 2.5g of 1,2-hexanediol and 2.5g of 1,2-pentanediol, weigh 1.0g of red camellia polypeptide solution, add 2.0g of deionized water, stir and mix evenly and heat to 45℃ to obtain material B.

[0070] (3) Slowly add material B to material A, start stirring at 500 rpm, and continue stirring for 30 minutes to obtain a clear and transparent water-in-oil emulsion.

[0071] (4) The above emulsion was subjected to micro-jet treatment at a pressure of 500 Bar and a cycle of 3 times. After homogenization, it was continuously stirred for 30 minutes and allowed to stand to defoam before obtaining the material of Comparative Example 1, which is the water-in-oil emulsion.

[0072] Comparative Example 2 A water-in-oil emulsion and its preparation method, comprising the following preparation steps: (1) Accurately weigh 79g of enzymatically hydrolyzed camellia seed oil prepared in Example 1, and weigh 4g of PEG-5 glyceryl triisostearate, 4g of PPG-3 octyl ether, 4g of sorbitan monooleate and 1g of vitamin E. Add them to the enzymatically hydrolyzed camellia seed oil and stir and mix evenly in a water bath at 50±2℃ to obtain a uniform and transparent material A for later use.

[0073] (2) Accurately weigh 2.5g of 1,2-hexanediol and 2.5g of 1,2-pentanediol, weigh 0.1g of honey extract, add 2.9g of deionized water, stir and mix evenly and heat to 45℃ to obtain material B.

[0074] (3) Slowly add material B to material A, start stirring at 500 rpm, and continue stirring for 30 minutes to obtain a clear and transparent water-in-oil emulsion.

[0075] (4) The above emulsion was subjected to micro-jet treatment at a pressure of 500 Bar and a cycle of 3 times. After homogenization, it was continuously stirred for 30 minutes and allowed to stand to defoam before obtaining the material of Comparative Example 2, which is the water-in-oil emulsion.

[0076] Comparative Example 3 A water-in-oil emulsion and its preparation method, comprising the following preparation steps: (1) Accurately weigh 79g of camellia seed oil (unhydrolyzed), and weigh 4g of PEG-5 glyceryl triisostearate, 4g of PPG-3 octyl ether, 4g of sorbitan monooleate and 1g of vitamin E. Add them to the camellia seed oil (unhydrolyzed) and stir and mix evenly in a water bath at 50±2℃ to obtain a uniform and transparent material A for later use.

[0077] (2) Accurately weigh 2.5g of 1,2-hexanediol and 2.5g of 1,2-pentanediol, weigh 1.0g of red camellia polypeptide solution and 0.1g of honey extract, add 1.9g of deionized water, stir and mix evenly and heat to 45℃ to obtain material B.

[0078] (3) Slowly add material B to material A, start stirring at 500 rpm, and continue stirring for 30 minutes to obtain water-in-oil emulsion.

[0079] (4) The above emulsion was subjected to micro-jet treatment at a pressure of 500 Bar and a cycle of 3 times. After homogenization, it was continuously stirred for 30 minutes and allowed to stand to defoam before obtaining the material of Comparative Example 3, which is the water-in-oil emulsion.

[0080] Comparative Example 4 A water-in-oil emulsion and its preparation method, comprising the following preparation steps: (1) Accurately weigh 50g of enzymatically hydrolyzed camellia seed oil prepared in Example 1, and weigh 4g of PEG-5 glyceryl triisostearate, 4g of PPG-3 octyl ether, 4g of sorbitan monooleate and 1g of vitamin E. Add them to the enzymatically hydrolyzed camellia seed oil and stir and mix evenly in a water bath at 50±2℃ to obtain a uniform and transparent material A for later use.

[0081] (2) Accurately weigh 2.5g of 1,2-hexanediol and 2.5g of 1,2-pentanediol, weigh 1.0g of red camellia polypeptide solution and 0.1g of honey extract, add 1.9g of deionized water, stir and mix evenly and heat to 45℃ to obtain material B.

[0082] (3) Slowly add material B to material A, turn on the stirrer, and stir continuously for 30 minutes at a speed of 500 rpm.

[0083] (4) The above emulsion was subjected to micro-jet treatment at a pressure of 500 Bar and a cycle of 3 times. After homogenization, it was continuously stirred for 30 minutes and allowed to stand to defoam before obtaining the material of Comparative Example 4, which is the water-in-oil emulsion.

[0084] The performance of the materials from the above embodiments and comparative examples was tested, and the test plan and results are as follows: 1. Transparency A turbidimeter is an optical instrument specifically designed for the rapid measurement of the concentration of suspended particles in a liquid, and its measurement results directly reflect the degree of turbidity of the liquid. This invention uses a turbidimeter to measure the turbidity of different samples to characterize the transparency of the material.

[0085] Turn on the instrument and warm it up for 3 minutes. During this time, clean the sample vials and ensure they are dry, and allow the samples to return to room temperature for later use. First, perform zero-point calibration. Fill the sample vials with ultrapure water and place them in the sample chamber for measurement. Once the data stabilizes, adjust the values ​​to zero. Next, dilute the standard turbidity solution to 100 NTU, fill the sample vials, and perform measurement. Once the measurement data stabilizes, adjust the value to 100 NTU to complete the instrument calibration. After cleaning the sample vials, measure the turbidity of the example and comparative samples. If the sample turbidity is too high and outside the measurement range, dilute the sample and measure again. Measure the same sample three times. The specific results are shown in Table 1 below.

[0086] Table 1: Turbidity data of the examples and comparative examples

[0087] As shown in Table 1, Examples 1-7 exhibited relatively low turbidity, essentially remaining completely transparent. This is likely because the enzymatic hydrolysis of camellia seed oil enhanced the transparency of the emulsion itself. Furthermore, the enzymatic hydrolysis of monoglycerides and diglycerides in the camellia seed oil improved its emulsifying ability, contributing to greater transparency. In contrast, Comparative Example 3, containing unhydrolyzed camellia seed oil, showed decreased emulsifying ability, thus affecting the transparency of the emulsion. Comparative Example 4 suffered from a low oil phase content, causing phase inversion from the original water-in-oil system, resulting in a milky white appearance and impacting transparency. These results indicate that only by using specific emulsifiers, enzymatically hydrolyzing camellia seed oil, and within defined dosage ranges can a transparent water-in-oil emulsion be obtained.

[0088] 2. Particle size measurement - stability study Particle size can effectively characterize the stability of water-in-oil systems, primarily because the kinetic stability of small droplets is far greater than that of large droplets. According to Stokes' law, the settling rate of a droplet is proportional to the square of its radius; the smaller the particle size, the slower the phase separation caused by gravity. Simultaneously, small droplets can form a denser interfacial film, resisting aggregation. Therefore, maintaining a small and uniform particle size (typically nanometer-scale) is a prerequisite for system stability. Once aggregation or Ostwald ripening occurs, the average particle size will continue to increase, directly indicating that the emulsion is about to break down or separate. In this invention, particle size data is used to characterize the long-term stability of the material. First, the particle size D0 of the material is measured, and the sample is aliquoted and placed in a 45°C oven for observation. After 30 days, the particle size D30 is measured again, and the rates of change are compared.

[0089] First, turn on the instrument and preheat for 30 minutes, during which time prepare clean glass cuvettes. Disperse the sample in a hexane solution to prepare a 1% test solution, and measure the particle size using a NanoBrook 90PlusPALS particle size analyzer. Repeat the test three times for each sample and record the results. After one month of observation at 45℃, measure the particle size again using the same method and calculate the particle size change rate using the following formula: Particle size change rate formula: Change rate = ; According to the formula, the smaller the particle size change rate, the more stable the material is. The specific results are shown in Table 2 below.

[0090] Table 2: Particle size change rate of the examples and comparative examples

[0091] As shown in Table 2, a comparative analysis of the examples and comparative examples reveals that the particle size change rate of Examples 1-5 is small. Changing the type of emulsifier in Example 6, using unhydrolyzed camellia seed oil in Comparative Example 3, and reducing the amount of hydrolyzed camellia seed oil in Comparative Example 4 all caused significant changes in the particle size.

[0092] 3. Surface tension measurement Surface tension determines the spread and sensation of water-in-oil systems on the skin. Low surface tension (such as with the addition of silicone or fluorine-containing emulsifiers) allows droplets to spread easily, forming a thin and uniform oil film, resulting in a refreshing, silky, and quickly absorbed feel; conversely, high surface tension results in spherical accumulation and a sticky, heavy feel.

[0093] Surface tension was measured on samples from the examples and comparative examples. The specific testing procedure is as follows: First, the instrument was preheated and calibrated using pure water to ensure the accuracy of the measurement system. Then, the liquid to be tested was placed in a clean container, ensuring a smooth, bubble-free surface, and heated to the specified temperature. Next, the sensor was zeroed, the test parameters were input, and the instrument was used to extend the immersion ring to a certain depth below the liquid surface. The ring was then slowly raised until it penetrated the liquid surface, and the maximum tensile force was recorded. The same sample was measured three times, and the average value was taken. The results are shown in Table 3 below.

[0094] Table 3: Surface tension of examples and comparative examples

[0095] Table 3 shows that the surface tension of enzymatically hydrolyzed camellia seed oil is lower than that of unhydrolyzed camellia seed oil. The surface tension of Examples 1-7 and Comparative Examples 1-2 is relatively low, while the surface tension of Comparative Example 3 is closer to that of camellia seed oil. Comparative Example 4 uses the least amount of enzymatically hydrolyzed oil, and the majority of its components are water, so its surface tension is also the lowest. However, it separates into layers after standing, making the sample unreliable.

[0096] 4. Efficacy Test 4.1 ABCA12 protein (1) Experimental background This study used immortalized human keratinocytes as the research subject and measured the ATP-binding cassette transporter A12 (ABCA12) in the cells using immunofluorescence to provide experimental evidence for testing the barrier repair efficacy of the analyte. ABCA12 is a member of the ATP-binding cassette (ABCA12) transporter family and plays a crucial role in the differentiation of epidermal keratinocytes. ABCA12 is mainly expressed in the stratum corneum (SC) of the epidermis, forming an insoluble keratin capsule together with loricrin (LOR), which is essential for maintaining the integrity of the skin barrier. ABCA12 is believed to transport lipids, including ceramides, thereby forming an extracellular lipid layer in the stratum corneum, which is crucial for skin barrier function. Recombinant ABCA12 protein is an important cell membrane transporter that plays a key role in biological functions. It is mainly expressed in tissues such as the skin and functions in various biological processes. In the skin, ABCA12 is an indispensable protein in keratinization, responsible for transporting lipids and keratin to the epidermal keratinocytes, maintaining the skin barrier function.

[0097] (2) Experimental steps 1) Cell seeding: HaCaT cells were seeded at 20,000 cells / well in a 96-well plate and cultured at 37°C in a 5% CO2 incubator for 24 hours.

[0098] 2) Grouping and solution preparation: The cells were divided into blank group, negative group, positive group and sample group. The blank group was not treated. The negative group was modeled using UV. The positive group was modeled using UV and EGCG at a concentration of 20 mmol was added. The sample group was modeled using UV and different examples and comparative examples were added.

[0099] 3) Drug administration: According to the test protocol, when the cell deposition rate in the 96-well plate reaches 40%-60%, the cells are administered to the groups. After drug administration, the 96-well plate is placed in an incubator (37℃, 5% CO2) for 24 hours.

[0100] 4) Measurement of intracellular ABCA12 protein expression: 4-1) Sample collection: Discard the culture medium and wash the cells three times with PBS (300 μL / well); 4-2) Cell fixation: Discard PBS, add 4% paraformaldehyde fixative (100 μL / well) to fix cells, and incubate at room temperature for 20 min; 4-3) Washing: Wash 5 times with PBS (300μL / well) to remove residual paraformaldehyde; 4-4) Permeabilization: After removing the liquid, permeabilize the cell membrane with 0.1% Tritium-100 (100 μL / well); 4-5) Washing: Wash 5 times with PBS (300μL / well) to remove residual 0.1% Triton-100; 4-6) Blocking: Use 5% FBS blocking solution prepared with PBS, 100 μL / well, and incubate at room temperature for 1 h; 4-7) Apply primary antibody: Under light protection, use immunofluorescence dilution buffer to dilute the primary antibody of ABCA12 at a ratio of 1:50. Remove the liquid from the well plate without washing, add 50 μL / ml of primary antibody dilution buffer, and incubate overnight at 4°C in the dark. 4-8) Washing: Warm up for 30 min, remove primary antibody under dark, and wash cells 3 times with washing buffer (300 μL / well), 5 min each time; 4-9) Applying fluorescent secondary antibody: Dilute the fluorescent secondary antibody at a ratio of 1:200 using fluorescent diluent under light-protected conditions, and incubate at room temperature for 1 hour.

[0101] 5) Image acquisition: After washing (300μL / well) three times, retain 50μL of PBS in each well and observe and photograph under an inverted fluorescence microscope.

[0102] 6) Calculate the average fluorescence intensity value of the image using ImageJ software.

[0103] 7) Calculation formula: ABCA12 protein enhancement rate = ×100%.

[0104] (3) Results Analysis The test results are shown in Table 4 below. Figure 1 As shown.

[0105] Table 4: ABCA12 protein enhancement rate in the examples and comparative examples

[0106] Table 4 above and Figure 1 The results show that the samples in Examples 1-7 have a significant effect on enhancing ABCA12 protein levels, which is significantly better than the enhancement rate of the comparative samples. These results demonstrate the application potential of the transparent water-in-oil system of this invention in barrier repair.

[0107] 4.2 LOR protein (1) Test background This study used immortalized human keratinocytes as the research subject and measured loricrin (LOR) in the cells using immunofluorescence to provide experimental evidence for testing the barrier repair efficacy of the analytes. Loricrin (LOR) is a protein that plays an important role in the differentiation of epidermal keratinocytes. It is mainly expressed in the upper layer of the stratum spinosum of the epidermis and is a marker protein of keratinocyte differentiation. Loricrin acts as a scaffold in the formation of the keratinized capsule of the epidermis. It is synthesized in the stratum spinosum and then cross-linked with cells in the stratum granulosum under the action of glutaminase to form an important structural support for the skin barrier. Loricrin, together with involucrin (IVL), forms an insoluble keratinized capsule in the stratum corneum (SC) of the epidermis, constituting the unique stratum corneum barrier structure of the epidermis. This structure is crucial for maintaining the integrity of the skin barrier and helps prevent moisture loss and the invasion of external substances.

[0108] (2) Testing process 1) Cell seeding: HaCaT cells were seeded at 15,000 cells / well in a 96-well plate and cultured at 37°C in a 5% CO2 incubator for 24 hours.

[0109] 2) Grouping and solution preparation: The cells were divided into blank group, negative group, positive group and sample group. The blank group was not treated. The negative group was modeled using UV. The positive group was modeled using UV and EGCG at a concentration of 20 mmol was added. The sample group was modeled using UV and different examples and comparative examples were added.

[0110] 3) Drug administration: According to the test protocol, when the cell deposition rate in the 96-well plate reaches 40%-60%, the cells are administered to the groups. After drug administration, the 96-well plate is placed in an incubator (37℃, 5% CO2) for 24 hours.

[0111] 4) Measurement of intracellular LOR content: 4-1) Sample collection: Discard the culture medium and wash the cells 3 times with PBS (300 μL / well); 4-2) Cell fixation: Discard PBS, add 4% paraformaldehyde fixative (100 μL / well) to fix cells, and incubate at room temperature for 20 min; 4-3) Washing: Wash 5 times with PBS (300μL / well) to remove residual paraformaldehyde; 4-4) Permeabilization: After removing the liquid, permeabilize the cell membrane with 0.1% Triton-100 (100 μL / well); 4-5) Washing: Wash 5 times with PBS (300μL / well) to remove residual 0.1% Triton-100; 4-6) Blocking: Use 5% FBS blocking solution prepared with PBS, 100 μL / well, and incubate at room temperature for 1 h; 4-7) Apply primary antibody: Under light protection, dilute the primary antibody of LOR at a ratio of 1:50 using immunofluorescence diluent. Remove the liquid from the well plate without washing, add 50 μL / ml of primary antibody diluent, and incubate overnight at 4°C in the dark. 4-8) Washing: Warm up for 30 min, remove primary antibody under dark, and wash cells 3 times with washing buffer (300 μL / well), 5 min each time; 4-9) Applying fluorescent secondary antibody: Dilute the fluorescent secondary antibody at a ratio of 1:200 using fluorescent diluent under light-protected conditions, and incubate at room temperature for 1 hour.

[0112] 5) Image acquisition: After washing (300μL / well) three times, retain 50μL of PBS in each well and observe and photograph under an inverted fluorescence microscope.

[0113] 6) Calculate the average fluorescence intensity value of the image using ImageJ software.

[0114] 7) Calculation formula: LOR protein enhancement rate = ×100%.

[0115] (3) Test results The test results are shown in Table 5 below. Figure 2 As shown.

[0116] Table 5: LOR protein enhancement rate in the examples and comparative examples

[0117] Table 5 above and Figure 2 The results show that the samples in Examples 1-7 have a significant effect on increasing LOR protein levels, which is significantly better than the improvement rate of the comparative samples. These results demonstrate the application potential of the transparent water-in-oil system of this invention in the barrier repair dimension.

[0118] 4.3 Blind Skin Feel Test (1) Test background In human efficacy testing, blind testing of skin feel stems from the need for objectivity in cosmetic efficacy evaluation. Its necessity lies in the fact that consumers' subjective experiences with product texture, absorbency, stickiness, and other skin feel directly influence usage compliance and the ultimate efficacy achieved. However, non-efficacy factors such as brand identity, packaging, and scent can easily create psychological suggestions, leading to a "placebo effect" or expectation bias in conventional testing. Therefore, blinding is necessary to eliminate interference and restore the true differences in skin feel. This test follows three basic principles: the blinding principle (subjects and / or operators are unaware of the corresponding product information, identified by codes), the control principle (establishing a reference product or blank matrix with known texture to calibrate individual differences), and the randomization principle (randomizing the test sequence to avoid fatigue or sequence effects). Its principle is based on sensory physiology and behavioral statistics. It transforms the neural signals generated by human skin in response to changes in mechanical force (applying), temperature, and humidity into quantifiable subjective scores (such as a 9-point Likert scale) through standardized tactile descriptive terms (such as "film-forming sensation" and "silicone smoothness"). Then, it uses non-parametric statistical methods to compare the differences in scores between groups, thereby inferring the true impact of product formulation on skin feel while excluding cognitive biases.

[0119] (2) Testing process 1) Preparation and Coding The testing team labeled the test product and control product (including placebo or competing products) with only a random three-digit code, removing all trademarks, packaging, and other identifiable information. The packaging containers were identical in appearance and texture. Simultaneously, it was ensured that neither the person distributing the product nor the subjects were aware of the group assignments.

[0120] 2) Environment and subject pretreatment In a temperature and humidity controlled testing room (typically 22±1℃, RH 50±5%), subjects need to sit quietly for 20 minutes to acclimatize. The test area (such as the inner forearm or half of the face) is cleaned with a neutral detergent and dried with a lint-free tissue. After 10 minutes of equilibration, the skin will return to baseline comfort.

[0121] 3) Blind test execution 3-1) Random allocation: The order of application is allocated according to the randomization table (e.g., the test sample is applied to the left arm and the control sample is applied to the right arm) to prevent positional order deviation; 3-2) Standardized application: Trained volunteers (group unknown) take an equal amount of sample (e.g., 20mg) and apply it to the designated area at a constant speed and number of circles (e.g., 2 circles per second, 20 circles in total); 3-3) Immediate evaluation: Immediately after application, subjects blindly rated the smoothness, absorption speed, stickiness, and refreshing feeling using a 9-point Likert scale (1 = very weak, 9 = very strong) to avoid communication. 3-4) Delayed tracking: 30 minutes after application, re-evaluate dynamic changes in indicators such as residual feeling, tightness, and moisture.

[0122] 4) Data preparation and unblinding After all subjects have completed their tests, a third-party supervisor will provide the coding sheets to the statistical analysts. Outliers (such as data where all scores for the same individual are constant) will be removed before statistical analysis.

[0123] 5) To quickly and efficiently differentiate between test products, the skin feel test will be conducted on five dimensions: spreadability, refreshing feel, absorption speed, moisturizing feel, and appearance texture. A weighted average score will be calculated for each dimension to obtain the final total skin feel score. The calculation formula is as follows: Overall skin feel score = Spreadability × 0.25 + Refreshing feel × 0.25 + Absorption speed × 0.20 + Moisturizing feel × 0.20 + Appearance transparency × 0.10. The highest overall score is 9 points, and the lowest overall score is 1 point.

[0124] (3) Test results The test results are shown in Table 6 below. Figure 3 As shown.

[0125] Table 6: Overall Skin Feel Score for Examples and Comparative Examples

[0126] Table 6 above and Figure 3 The results show that the water-in-oil transparent systems of Examples 1-7 have a high overall skin feel score. In Comparative Examples 1-4, the absence or replacement of a certain component in the raw materials will affect the overall skin feel result.

[0127] 4.4 Moisturizing Test (1) Test background In human efficacy testing, the arm moisturizing test is primarily used to objectively assess the impact of skincare products (such as body lotions and bath oils) on the moisture content of the stratum corneum. The test's background is based on three points: First, necessity. With increasingly stringent regulations on cosmetic efficacy claims (such as China's "Cosmetics Supervision and Administration Regulations"), companies need to provide scientific evidence to support claims such as "moisturizing" to avoid false advertising. Second, in principle, the capacitance method is used to measure the moisture content of the stratum corneum—water's dielectric constant is much higher than other skin components, and professional instruments (such as Corneometer®) can non-invasively quantify changes in hydration before and after product use. Third, it adheres to core principles, including setting up blank / positive controls to eliminate interference from environmental and physiological fluctuations, employing randomization and blinding to avoid location and subjective bias, stabilizing the skin in a constant temperature and humidity environment (21±1℃, RH 50±5%), and tracking the moisturizing effect at time points (such as 30 minutes). This method provides a standardized and reproducible data foundation for formula screening, claim verification, and consumer insights.

[0128] (2) Testing process 1) Screening and preparation of subjects: 30 healthy volunteers will be recruited. Volunteers must discontinue the use of any moisturizing products 2-3 days prior to the test, and their arms must not come into contact with any skincare products on the day of the test. The testing environment must be controlled under constant temperature and humidity conditions of 20-22℃ and 40%-60% relative humidity.

[0129] 2) Cleaning and Environmental Adaptation: Subjects were required to clean the inside of their forearms with a clean, dry tissue. They were then required to sit quietly in the testing environment for at least 30 minutes, keeping their forearms exposed and relaxed to allow their skin to stabilize.

[0130] 3) Delineate test areas and baseline measurement: Mark several test areas on the inner side of both forearms. Each area is typically 3cm × 3cm, with at least 1cm between areas to avoid interference. Use a capacitive skin moisture meter (such as Corneometer®) to measure the initial value (baseline value) of the stratum corneum moisture content in each area. Measure each area three times and take the average value.

[0131] 4) Product application and random grouping: The test product and blank control (or control area without product) are randomly assigned to different test areas. Apply the sample evenly to the corresponding 3cm×3cm area using a latex finger cot at the standard dosage of (2.0±0.1) mg / cm².

[0132] 5) Real-time tracking measurement: After product application, the moisture content of the stratum corneum in each area is measured again 30 minutes later. All measurements for the same subject must be performed by the same operator using the same instrument.

[0133] 6) Calculation formula: Moisture content increase rate = ×100%.

[0134] (3) Test results The test results are shown in Table 7 below. Figure 4 As shown.

[0135] Table 7: Moisture content improvement rate of the examples and comparative examples

[0136] Table 7 above and Figure 4 The results show that the water-in-oil transparent systems of Examples 1-7 have good moisturizing effects. In Comparative Examples 1-4, the absence or substitution of any component in the raw materials will affect the overall moisturizing effect.

Claims

1. A water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil, characterized in that, The product is obtained by mixing an oil phase and an aqueous phase. The oil phase comprises the following components by weight: 59-96.4 parts enzymatically hydrolyzed camellia seed oil and 0.01-20 parts emulsifier. The aqueous phase comprises the following components by weight: 0.01-10 parts red camellia flower polypeptide solution, 0.01-1 part honey extract, and 0.01-10 parts water, wherein the polypeptide content in the red camellia flower polypeptide solution is 0.01%-1%. The enzymatically hydrolyzed camellia seed oil is obtained by enzymatic hydrolysis of camellia seed oil using lipase. The emulsifier includes a first emulsifier and a second emulsifier, wherein the first emulsifier is one or more of PEG-5 glyceryl triisostearate, sorbitan monooleate and polyglycerol-10 dioleate, and the second emulsifier is PPG-3 octyl ether.

2. The water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil according to claim 1, characterized in that, The oil phase comprises the following components in parts by weight: 75-85 parts enzymatically hydrolyzed camellia seed oil, 5-15 parts emulsifier; the aqueous phase comprises the following components in parts by weight: 0.1-5 parts red camellia polypeptide solution, 0.05-0.5 parts honey extract, and 1-5 parts water.

3. The water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil according to claim 1, characterized in that, The mass ratio of the first emulsifier to the second emulsifier is 1:(0.1-2).

4. The water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil according to claim 1, characterized in that, The aqueous phase also includes 0.01-10 parts of alcohol additives, wherein the alcohol additives are 1,2-pentanediol and 1,2-hexanediol, and the mass ratio of the two is 1:(0.5-2).

5. The water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil according to claim 1, characterized in that, The oil phase also includes 0.01-5 parts of antioxidants, which are one or more of vitamin E and its derivatives, camellia oil extract, and camellia stem and leaf oil extract.

6. The water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil according to claim 5, characterized in that, The extracts of camellia flower oil and camellia stem and leaf oil used in the extraction process are enzymatically hydrolyzed camellia seed oil.

7. A method for preparing a water-in-oil transparent emulsion of enzymatically hydrolyzed camellia seed oil as described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Dissolve the lipase in water, disperse it evenly, add camellia seed oil, stir and mix, and then perform enzymatic hydrolysis; (2) After enzymatic hydrolysis, the enzymatic hydrolysis system is allowed to stand and separate into layers. The oil layer is then filtered to obtain enzymatically hydrolyzed camellia seed oil. (3) Add emulsifier to the enzymatically hydrolyzed camellia seed oil, mix well to obtain the oil phase; mix red camellia flower polypeptide solution, honey extract and water well to obtain the aqueous phase; (4) The aqueous phase is slowly added to the oil phase and stirred continuously. After the material changes from turbid to clear and transparent, it is homogenized and then stirred and allowed to stand to obtain the water-in-oil transparent emulsion.

8. The preparation method according to claim 7, characterized in that, The amount of lipase used is 0.1-10% of the mass of the camellia seed oil, and the enzymatic hydrolysis is carried out at room temperature.

9. The preparation method according to claim 7, characterized in that, Homogenization processes include high-speed homogenization, high-pressure homogenization, and microjet homogenization. High-speed homogenization involves a rotation speed of 3000-10000 rpm and a processing time of 1-20 min. High-pressure homogenization and microjet homogenization involve pressures of 1-500 Bar and 1-5 cycles.

Images