Camellia composite oil and preparation method and application thereof, and cosmetic

Abstract

The present application relates to the technical field of cosmetic raw materials, and discloses a camellia flower composite oil, a preparation method and application thereof, and a cosmetic. The preparation method of the camellia flower composite oil comprises the following steps: (1) performing enzymatic reaction on camellia oil in the presence of lipase and a solvent, and separating to obtain enzymatic camellia oil; (2) performing extraction on camellia flower powder and the enzymatic camellia oil to obtain an extraction mixture; and (3) performing solid-liquid separation on the extraction mixture to obtain the camellia flower composite oil. The camellia flower composite oil provided by the present application has high polarity, high antioxidant property, high safety and good formulation application performance, and can significantly improve the water phase carrying capacity and long-term stability of a water-in-oil emulsion.

Description

Technical Field

[0001] This invention relates to the field of cosmetic raw material technology, specifically to a camellia compound oil, its preparation method and application, and cosmetics. Background Technology

[0002] Camellia oil, a natural plant oil, is rich in unsaturated fatty acids such as oleic acid, possessing excellent skincare benefits and widely used in the cosmetics industry. Camellia flowers are rich in polyphenols, flavonoids, and other active ingredients, exhibiting excellent antioxidant and free radical scavenging effects, making them a commonly used natural active ingredient in cosmetics. By extracting camellia flowers using camellia oil as a solvent, the synergistic skincare effects of both can be achieved, yielding a functional oil that combines the functions of an oil matrix with antioxidant activity.

[0003] However, directly extracting camellia flowers with conventional camellia oil has limited effect on improving the overall antioxidant efficacy of the oil phase. Furthermore, camellia oil has low polarity and limited compatibility with polar components such as polyols, which restricts its application in high-end water-in-oil emulsion formulations. Moreover, the high temperatures and microwaves used in the extraction process can easily damage the structure of unsaturated fatty acids in camellia oil, further affecting the product's efficacy and stability.

[0004] To enhance the functionality of camellia oil, current technologies typically employ enzymatic modification, utilizing lipases to hydrolyze the ester bonds in camellia oil triglycerides to increase their acid and hydroxyl values, thereby altering their polarity. However, this enzymatic process introduces a large amount of free fatty acids, potentially negatively impacting the oxidative stability of the oil. Furthermore, this contradicts the low-irritant and high-safety requirements for raw materials in cosmetics, thus limiting the direct application of such modified oils in cosmetics.

[0005] Existing technologies also include solutions that combine enzymatic hydrolysis and extraction. For example, CN119547828A discloses a method for extracting turmeric after enzymatic hydrolysis and purification of various vegetable oils to solve the problem of color reversion in edible oils. However, this technology is complex to operate, and the enzymatic hydrolysis and subsequent diglyceride distillation processes involve high temperatures, long times, and high energy consumption. Furthermore, this technology focuses on the food industry, maintaining the appearance and color of oils through precise component control, but it does not address or solve the comprehensive needs of the cosmetics industry for high polarity of the oil phase matrix, high antioxidant activity, and emulsion application performance.

[0006] Therefore, developing a camellia-based composite functional oil that combines high polarity, high antioxidant properties, and high safety to improve the water phase carrying capacity and long-term stability of water-in-oil emulsions is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] The purpose of this invention is to overcome the problems of existing technologies, such as the low polarity of camellia oil as a raw material for cosmetic oil phase, insufficient extraction efficacy of active ingredients from camellia flowers, and insufficient antioxidant capacity, which make it difficult to meet the comprehensive performance requirements of high-end water-in-oil emulsion formulations for the oil phase matrix.

[0008] To achieve the above objectives, the first aspect of the present invention provides a method for preparing camellia flower compound oil, the method comprising the following steps: (1) In the presence of lipase and solvent, the raw camellia oil was subjected to enzymatic hydrolysis, and the enzymatically hydrolyzed camellia oil was obtained after separation; (2) Camellia pollen is extracted with the enzymatically hydrolyzed camellia oil to obtain an extraction mixture; (3) The extraction mixture is subjected to solid-liquid separation to obtain camellia compound oil; The mass ratio of the raw material camellia oil to lipase is 100:0.25-0.75; The amount of enzymatically hydrolyzed camellia oil used is 10-20 mL relative to 1 g of camellia pollen.

[0009] A second aspect of the present invention provides a camellia compound oil prepared by the method described in the first aspect.

[0010] The third aspect of this invention provides the application of the camellia compound oil described in the second aspect in cosmetics.

[0011] The fourth aspect of the present invention provides a cosmetic product containing the camellia compound oil described in the second aspect of the present invention; The cosmetic product is selected from at least one of the following: serum, cleansing cream, moisturizing cream, and lotion.

[0012] The camellia compound oil provided by this invention has high polarity, high antioxidant properties, high safety and good formulation application performance, and can significantly improve the water phase carrying capacity and long-term stability of water-in-oil emulsions.

[0013] This invention uses enzymatically hydrolyzed camellia oil, which differs from traditional solvents, as the extraction medium to extract camellia flowers, resulting in a fat-soluble functional oil that is an in-situ composite of active ingredients from camellia flowers and an enzymatically hydrolyzed camellia oil matrix. Compared to the raw camellia oil, the camellia composite oil provided by this invention exhibits significantly increased acidity, accompanied by changes in hydroxyl value, thus enhancing the system's polarity.

[0014] More specifically, the camellia compound oil obtained by this invention has an acid value of 62-70 mg KOH / g and a hydroxyl value of 8-10 mg KOH / g. Both the acid value and hydroxyl value are within the physicochemical range that is more suitable for cosmetic applications, and it has better formulation compatibility.

[0015] The camellia compound oil provided by this invention achieves a synergistic effect of "1+1>2": the increased polarity of enzymatically hydrolyzed camellia oil promotes the dissolution and stabilization of antioxidant components such as camellia polyphenols; while the camellia components provide excellent antioxidant protection for the high acid value system, helping to improve the antioxidant performance and application stability of the high polarity oil system. Therefore, although the camellia compound oil has a high acid value, it still exhibits good cell compatibility in the constructed formulation system, with no obvious cytotoxicity observed. This breaks the traditional perception that "high acid value oils are not suitable for cosmetics," achieving a significant performance improvement under the premise of safety.

[0016] Meanwhile, the present invention uses composite oils as oil phase raw materials, which can significantly improve the water phase carrying capacity and long-term stability of water-in-oil emulsions, and maintain good compatibility with polyols.

[0017] In summary, the camellia compound oil provided by this invention combines the texture of oil with a light and thin feel on the skin, has good antioxidant properties, better compatibility with polyols, and can significantly improve the water phase carrying capacity and long-term stability of water-in-oil emulsions, providing an innovative core ingredient for high-end cosmetic formulations. Attached Figure Description

[0018] Figure 1 The state diagrams of the oil-ethanol-water system numbered 1#, 5#, and 9# in Table 4 of Test Example 2 are shown. Figure 2 This is a state diagram of the water-in-oil system for TX-1, TX-2, TX-D1, and TX-D2; Figure 3 These are cytotoxicity test results for TX-1, TX-D1, and TX-D3. Figure 4 These are scratch healing test images for TX-1, TX-D1, and TX-D3; Figure 5 This is a test chart showing the ABCA12 efficacy of TX-1, TX-D1, and TX-D3. Figure 6 This is a LOR (Life Effect) efficacy test chart for TX-1, TX-D1, and TX-D3; Figure 7 This is a test chart of the COL I-promoting efficacy of TX-1, TX-D1, and TX-D3; Figure 8 These are test charts showing the COL III-promoting efficacy of TX-1, TX-D1, and TX-D3. Figure 9 This is a test chart showing the efficacy of TX-1, TX-D1, and TX-D3 in promoting elastin. Detailed Implementation

[0019] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0020] As mentioned above, the first aspect of the present invention provides a method for preparing camellia flower compound oil, the method comprising the following steps: (1) In the presence of lipase and solvent, the raw camellia oil was subjected to enzymatic hydrolysis, and the enzymatically hydrolyzed camellia oil was obtained after separation; (2) Camellia pollen is extracted with the enzymatically hydrolyzed camellia oil to obtain an extraction mixture; (3) The extraction mixture is subjected to solid-liquid separation to obtain camellia compound oil; The mass ratio of the raw material camellia oil to the lipase is 100:0.25-0.75; The amount of enzymatically hydrolyzed camellia oil used is 10-20 mL relative to 1 g of camellia pollen.

[0021] Preferably, in step (1), the conditions for the enzymatic hydrolysis reaction include: a temperature of 25-37 °C and a time of 1-3 h.

[0022] In a preferred embodiment, the solvent in step (1) is water.

[0023] Preferably, in step (1), the amount of solvent used is 25-100 mL relative to 100 g of raw camellia oil.

[0024] In a preferred embodiment, the raw camellia oil contains, based on the mass of total fatty acids, oleic acid with a mass content of 70%-90%, linoleic acid with a mass content of 5%-15%, and saturated fatty acids with a mass content of ≤15%.

[0025] In this invention, the total fatty acids refer to the sum of all fatty acids in the raw material camellia oil.

[0026] In this invention, the saturated fatty acid refers to fatty acids whose alkane chains do not contain carbon-carbon double bonds, including palmitic acid, stearic acid, arachidic acid, etc.

[0027] The present invention does not have any special requirements for the method of determining the mass content of oleic acid, linoleic acid and saturated fatty acids; for example, gas chromatography can be used.

[0028] In a preferred embodiment, in step (1), the separation method is selected from at least one of static separation and centrifugal separation.

[0029] Preferably, in step (1), the acid value of the obtained enzymatically hydrolyzed camellia oil is 72-76 mg KOH / g, and the hydroxyl value is 11-13 mg KOH / g. Using enzymatically hydrolyzed camellia oil with higher acid and hydroxyl values ​​to extract camellia pollen can ensure that the acid and hydroxyl values ​​of the final camellia compound oil fall within a range more suitable for cosmetic applications, thereby balancing extraction efficiency, antioxidant properties, and formulation compatibility.

[0030] More preferably, in step (3), the resulting camellia compound oil has an acid value of 62-70 mg KOH / g and a hydroxyl value of 8-10 mg KOH / g. When the acid value and hydroxyl value of the camellia compound oil fall within the above range, it is more suitable for cosmetic applications and exhibits better formulation compatibility.

[0031] In a preferred embodiment, in step (2), the average particle size of the camellia pollen is ≤0.6mm and the moisture content is ≤10wt%.

[0032] According to one specific embodiment, in step (2), the camellia pollen is prepared by drying camellia flowers and then pulverizing and sieving them sequentially.

[0033] The present invention does not have any special requirements for the drying method, as long as the moisture content of the dried camellia flowers is ≤10wt%. Those skilled in the art can carry out the drying according to the technical means known in the art, and this should not be construed as a limitation of the present invention.

[0034] Preferably, the average particle size of the camellia pollen is 0.18 mm to 0.6 mm.

[0035] The present invention does not have any special requirements for the specific operation of sieving. Those skilled in the art can use methods known in the art to perform the sieving. For example, a Chinese standard sieve can be used for sieving, such as passing the material through a 30-mesh sieve and an 80-mesh sieve in succession, and taking the material with a relatively more uniform particle size in the middle part.

[0036] In a preferred embodiment, in step (2), the extraction includes the following steps: mixing camellia pollen with the enzymatically hydrolyzed camellia oil and then performing ultrasonic-assisted extraction, followed by stirring extraction.

[0037] Preferably, the conditions for ultrasonic-assisted extraction include: an ultrasonic frequency of 35-45 kHz, an ultrasonic power density of 0.1-0.5 W / mL, a temperature of 20-40℃, and a time of 15-45 min.

[0038] In a preferred embodiment, the stirring and extraction process is carried out at a speed of 200-400 rpm, a temperature of 20-30°C, and a time of 1-3 h.

[0039] In a preferred embodiment, in step (3), the solid-liquid separation method is selected from at least one of centrifugation and vacuum filtration.

[0040] As previously stated, the second aspect of the present invention provides a camellia compound oil prepared by the preparation method described in the first aspect.

[0041] As previously stated, the third aspect of this invention provides the application of the camellia compound oil described in the second aspect in cosmetics.

[0042] As previously described, the fourth aspect of the present invention provides a cosmetic product containing the camellia compound oil described in the second aspect of the present invention.

[0043] Preferably, the cosmetic is selected from at least one of serum, cleansing cream, moisturizing cream, and moisturizing lotion.

[0044] In this invention, the raw materials, camellia oil and camellia flowers, are both derived from the Camellia oleifera plant, which belongs to the Camellia genus. The Camellia oleifera plant can be selected from at least one of Yunnan, Jiangxi, Hunan, and Zhejiang provinces.

[0045] The present invention will be described in detail below through examples. In the following examples, unless otherwise specified, all raw materials used are commercially available analytical grade products. Room temperature in the examples refers to 22±3℃.

[0046] Raw material camellia oil: purchased from Guangzhou Pengyuan Chemical Co., Ltd. In the raw material camellia oil, based on the mass of total fatty acids, the mass content of oleic acid is 80%, the mass content of linoleic acid is 10%, and the mass content of saturated fatty acids is 3%.

[0047] Vegetable oil: specifically meadowfoam seed oil, purchased from Shanghai Fankai Industrial Co., Ltd.; Lipase 1: Enzyme activity of 20,000 U / g, purchased from ACMEC, catalog number L17360, derived from Aspergillus niger; Lipase 2: Enzyme activity of 50,000 U / g, purchased from Adamas Life, catalog number 88911K, derived from Aspergillus niger; Lipase 3: Enzyme activity of 120,000 U / g, purchased from Yuanye Company, product number S59748, derived from Aspergillus niger; Human immortalized keratinocytes (HaCaT cells): purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number CL-0090; Thiazole blue tetrazolium bromide (MTT): purchased from Solarbio, CAS No. 298-93-1, purity ≥98%; Dimethyl sulfoxide (DMSO): purchased from SIGMA; Recombinant human transforming growth factor-β1 (TGF-β1): a recombinant protein expressed by HEK293 cells, purchased from MCE, catalog number HY-P70543; Epigallocatechin gallate (EGCG): purchased from MCE, CAS No. 989-51-5, purity 99.58%; The microplate reader used was a Thermo Fisher Scientific Variokan Lux model. Immunofluorescence images were acquired using an Olympus IX73 inverted fluorescence microscope.

[0048] Example 1 A method for preparing camellia flower compound oil, the method comprising the following steps: (1) Dissolve 0.75g of lipase in 50 mL of water, then add 100g of raw camellia oil and mix. Stir and mix under the temperature provided by the water bath. After complete enzymatic hydrolysis, separate the oil and water of the product by centrifugation to obtain enzymatically hydrolyzed camellia oil. The lipase is lipase 1; The mixing conditions were: temperature 30°C, time 1 hour, and stirring speed 500 rpm. (2) The camellia flowers are dried by hot air drying, then powdered. The powdered product is passed through a 50-mesh Chinese standard sieve. The sieve material is collected to obtain camellia flower powder. The camellia flower powder is mixed with the enzymatically hydrolyzed camellia oil and then subjected to ultrasonic-assisted extraction, followed by stirring extraction to obtain an extraction mixture. The hot air drying conditions include: a temperature of 45°C and a time of 12 hours; The camellia pollen had an average particle size of 0.3 mm and a moisture content of 6 wt%. Based on a mass of 1g of camellia pollen, the amount of enzymatically hydrolyzed camellia oil used is 10 mL; The conditions for ultrasound-assisted extraction include: an ultrasound frequency of 45 kHz, an ultrasound power density of 0.1 W / mL, a temperature of 20℃, and a time of 30 min. The stirring extraction conditions include: a rotation speed of 200 rpm, a temperature of 25°C, and a time of 2 h; (3) The extraction mixture is filtered to obtain camellia compound oil, named L1.

[0049] The types and amounts of raw materials, reaction conditions, etc. in this embodiment are shown in Table 1.

[0050] Example 2 A method for preparing camellia flower compound oil, the method comprising the following steps: (1) Dissolve 0.5g of lipase in 25 mL of water, then add 100g of raw camellia oil and mix. Stir and mix under the temperature provided by the water bath. After complete enzymatic hydrolysis, separate the oil and water of the product by centrifugation to obtain enzymatically hydrolyzed camellia oil. The lipase is lipase 2; The mixing conditions were: temperature 25°C, time 2 hours, and stirring speed 400 rpm. (2) Camellia flowers are dried by vacuum drying, then powdered. The powdered product is passed through a 30-mesh Chinese standard sieve. The sieve material is collected to obtain camellia pollen. Camellia pollen is mixed with the enzymatically hydrolyzed camellia oil and then subjected to ultrasonic-assisted extraction, followed by stirring extraction to obtain an extraction mixture. The vacuum drying conditions include: a temperature of 45°C, a time of 12 hours, and a vacuum degree of -0.08 MPa. The camellia pollen had an average particle size of 0.6 mm and a moisture content of 8 wt%. Based on a mass of 1g of camellia pollen, the amount of enzymatically hydrolyzed camellia oil used is 20mL; The conditions for ultrasound-assisted extraction include: an ultrasound frequency of 40 kHz, an ultrasound power density of 0.3 W / mL, a temperature of 30℃, and a time of 15 min. The stirring extraction conditions include: a rotation speed of 300 rpm, a temperature of 20°C, and a time of 3 h; (3) The extraction mixture is filtered to obtain camellia compound oil, named L2.

[0051] The types and amounts of raw materials, reaction conditions, etc. in this embodiment are shown in Table 1.

[0052] Example 3 A method for preparing camellia flower compound oil, the method comprising the following steps: (1) Dissolve 0.25g of lipase in 100 mL of water, then add 100g of raw camellia oil and mix. Stir and mix under the temperature provided by the water bath. After complete enzymatic hydrolysis, separate the oil and water of the product by centrifugation to obtain enzymatically hydrolyzed camellia oil. The lipase is lipase 3; The mixing conditions were: temperature 37°C, time 3 hours, and stirring speed 300 rpm. (2) The camellia flowers are dried by freeze drying, then powdered. The powdered product is passed through an 80-mesh Chinese standard sieve. The sieve material is collected to obtain camellia flower powder. The camellia flower powder is mixed with the enzymatically hydrolyzed camellia oil and then subjected to ultrasonic-assisted extraction, followed by stirring extraction to obtain an extraction mixture. The freeze-drying conditions include: a temperature of -60°C, a time of 16 hours, and a vacuum degree of ≤20Pa; The camellia pollen had an average particle size of 0.18 mm and a moisture content of 10 wt%. Based on a mass of 1g of camellia pollen, the amount of enzymatically hydrolyzed camellia oil used is 15mL; The conditions for ultrasound-assisted extraction include: an ultrasound frequency of 35 kHz, an ultrasound power density of 0.5 W / mL, a temperature of 40 °C, and a time of 45 min. The stirring extraction conditions include: a rotation speed of 400 rpm, a temperature of 30°C, and a time of 1 h; (3) The extraction mixture is filtered to obtain camellia compound oil, named L3.

[0053] The types and amounts of raw materials, reaction conditions, etc. in this embodiment are shown in Table 1.

[0054] Example 4 This embodiment uses a method similar to that of Example 1, except that in this embodiment, the conditions are controlled so that: the acid value of the enzymatically hydrolyzed camellia oil prepared in step (1) is 61.35 mg KOH / g; the hydroxyl value is 8.92 mg KOH / g; specifically: The steps (1) of this method are as follows: (1) Dissolve 0.25g of lipase in 25 mL of water, then add 100g of raw camellia oil and mix. Stir and mix under the temperature provided by the water bath. After complete enzymatic hydrolysis, separate the oil and water of the product by centrifugation to obtain enzymatically hydrolyzed camellia oil. The lipase is lipase 1; The mixing conditions were: temperature 25°C, time 1 hour, and stirring speed 300 rpm. Steps (2) and (3) of this embodiment are the same as steps (2) and (3) of embodiment 1, respectively.

[0055] In this embodiment, a camellia compound oil was obtained and named L4.

[0056] The types and amounts of raw materials, reaction conditions, etc. in this embodiment are shown in Table 1.

[0057] Comparative Example 1 A method for preparing camellia flower compound oil, the method comprising the following steps: (1) The camellia flowers (same as in Example 1) were dried by hot air drying, and then powdered. The powdered product was passed through a 50-mesh sieve to obtain camellia flower powder. The camellia flower powder was mixed with 100g of raw camellia oil and then subjected to ultrasonic-assisted extraction, followed by stirring extraction. The hot air drying conditions include: a temperature of 45°C and a time of 12 hours; The camellia pollen had an average particle size of 0.3 mm and a moisture content of 6 wt%. Based on a mass of 1g of camellia pollen, the amount of raw camellia oil used is 10mL; The conditions for ultrasound-assisted extraction are the same as those in Example 1; The conditions for stirring and extraction are the same as those in Example 1; (2) The extraction mixture was filtered to obtain camellia compound oil, named D-L1.

[0058] The types and amounts of raw materials, reaction conditions, etc. of this comparative example are shown in Table 1.

[0059] Comparative Example 2 The enzymatically hydrolyzed camellia oil prepared in Example 1 was used as the product of this comparative example and named D-L2.

[0060] The types and amounts of raw materials, reaction conditions, etc. of this comparative example are shown in Table 1.

[0061] Comparative Example 3 This comparative example uses camellia oil, the raw material from Example 1, as the product of this comparative example, and names it D-L3.

[0062] The types and amounts of raw materials, reaction conditions, etc. of this comparative example are shown in Table 1.

[0063] Comparative Example 4 The method is similar to that in Example 1, except that in step (1), the mass ratio of the raw material camellia oil to lipase is 100:1; and the amount of raw material camellia oil is kept at 100g. The remaining steps are the same as in Example 1.

[0064] The camellia compound oil prepared in this comparative example was named D-L4.

[0065] The types and amounts of raw materials, reaction conditions, etc. of this comparative example are shown in Table 1.

[0066] Comparative Example 5 The method was similar to that in Example 1, except that in step (2), the amount of enzymatically hydrolyzed camellia oil used was 5 mL relative to 1 g of camellia pollen; and the amount of camellia pollen used was the same as that used in Example 1. The remaining steps are the same as in Example 1.

[0067] The camellia compound oil prepared in this comparative example was named D-L5.

[0068] The types and amounts of raw materials, reaction conditions, etc. of this comparative example are shown in Table 1.

[0069] Comparative Example 6 The same method as in Example 1 was used, except that in step (1), the raw material camellia oil was replaced with meadowfoam seed oil by equal mass. The remaining steps are the same as in Example 1.

[0070] The composite oil prepared in this comparative example is named D-L6.

[0071] The types and amounts of raw materials, reaction conditions, etc. of this comparative example are shown in Table 1.

[0072] Table 1

[0073] Table 1 (continued)

[0074] Test Example 1: Determination of Acid Value and Hydroxyl Value of the Product 1. Acid value determination The determination was performed using conventional titration methods in this field, referring to the standard method in General Chapter 0713 of the Chinese Pharmacopoeia. An appropriate amount of the product sample was weighed and dissolved in an ethanol-ether mixture. Using phenolphthalein as an indicator, the solution was titrated to the endpoint with 0.1 mol / L potassium hydroxide standard solution. A blank titration was performed under the same conditions using an equal volume of solvent without the sample. The acid value was calculated based on the amount of potassium hydroxide consumed in the titration, and the result is expressed as mg KOH / g.

[0075] in,

[0076] In the formula: V1 —The volume of KOH standard solution consumed in the sample titration, in mL; V0 —The volume of KOH standard solution consumed in the blank titration, in mL; c —The concentration of the KOH standard solution, in mol / L; m—Sample mass, in grams.

[0077] 2. Hydroxyl value determination The anhydride back titration method was used, and the determination was performed according to the standard method in General Chapter 0713 of the Chinese Pharmacopoeia. An appropriate amount of product sample was weighed, and acetic anhydride-pyridine acylation reagent was added. Acylation was carried out in a water bath at 50℃±1℃ for 25 minutes. After hydrolysis with water, titration was performed to the endpoint using cresol red-thymol blue as an indicator and 1 mol / L potassium hydroxide standard solution. A blank test was performed simultaneously, and the acid value of the sample was measured for correction. The hydroxyl value is expressed as the number of milligrams of KOH consumed per gram of sample (mg KOH / g).

[0078] in, ; In the formula: B —The volume of KOH consumed in the blank titration, in mL; A —The volume of KOH consumed by the sample, in mL; N —The concentration of the KOH solution used in the hydroxyl value determination is expressed in mol / L; W —Sample mass, in grams; D —Acid value of the same sample, in mg KOH / g.

[0079] 3. Measurement Results The acid value and hydroxyl value of the enzymatically hydrolyzed camellia oil obtained in step (1) of Examples 1, 2, 3, and 4 were determined. The specific results are shown in Table 2.

[0080] Table 2

[0081] As can be seen from the results in Table 2, the solution provided by the present invention can achieve stable enzymatic hydrolysis of raw camellia oil, resulting in enzymatically hydrolyzed camellia oil with high acid value, high hydroxyl value, and high polarity. For example, in Examples 1-4, the acid value of the enzymatically hydrolyzed camellia oil obtained in step (1) is stable in the higher range of 61.35-75.29 mg KOH / g, and the hydroxyl value is stable in the higher range of 8.92-12.69 mg KOH / g.

[0082] Furthermore, by optimizing the conditions, the acid value and hydroxyl value of the enzymatically hydrolyzed camellia oil can be further improved, giving it higher polarity. For example, in Examples 1-3, the acid value of the enzymatically hydrolyzed camellia oil obtained in step (1) is stable at 72.49-75.29 mgKOH / g, and the hydroxyl value is stable at 11.53-12.69 mgKOH / g.

[0083] The acid value and hydroxyl value of the products from each example were determined. The specific results are shown in Table 3.

[0084] Table 3

[0085] As can be seen from the results in Table 3, the solution provided by this invention can obtain camellia compound oil with both high acid value and moderate hydroxyl value. For example, the acid values ​​of L1, L2, L3, and L4 are stable at 52.58-69.39 mg KOH / g; the hydroxyl values ​​are stable at 7.31-9.32 mg KOH / g.

[0086] Furthermore, when the acid value of the enzymatically hydrolyzed camellia oil obtained in step (1) is 72-76 mg KOH / g and the hydroxyl value is 11-13 mg KOH / g, the acid value and hydroxyl value of the prepared camellia compound oil can be maintained in a range more favorable for cosmetic applications, thereby better balancing polarity and formulation suitability. For example, the acid values ​​of L1, L2, and L3 are stable at 62.31-69.39 mg KOH / g and the hydroxyl values ​​are stable at 8.07-9.32 mg KOH / g.

[0087] More specifically, the acid values ​​of L1, L2, L3, and L4 were significantly higher than those of D-L1 and D-L3. This indicates that enzymatic hydrolysis is crucial for enhancing the polarity and activity of the raw camellia oil. Simultaneously, the acid values ​​of L1, L2, L3, and L4 were significantly lower than those of D-L2 and D-L4. This suggests that the immersion and solid-liquid separation processes can further regulate the acid value of enzymatically hydrolyzed camellia oil, bringing the final product's acid value into a more suitable range for application.

[0088] Meanwhile, the hydroxyl values ​​of L1, L2, L3, and L4 remained stable within a moderate range of 7.31-9.32 mg KOH / g, comparable to those of D-L1 and D-L3, but lower than those of D-L2 and D-L4. This indicates that the impregnation and subsequent solid-liquid separation processes were not simply mixing, but rather further regulated the overall physicochemical state of the enzymatically hydrolyzed oil system.

[0089] In summary, this invention can obtain camellia compound oil with a balanced acid value and hydroxyl value.

[0090] Test Example 2: Compatibility and stability test of the product oil with polyols and water 1. Compatibility test The products shown in Table 4 were used as the oil phase; dipropylene glycol, 1,2-hexanediol, 1,2-pentanediol, glycerol, and other polyols were used as the alcohol phase; and water was used as the aqueous phase. The mixtures were prepared at a mass ratio of oil phase / alcohol phase / water phase of 50:49:1 to obtain a mixed system. The mixed system was then allowed to stand for 24 hours.

[0091] The initial appearance and the appearance after standing of each mixture system were observed to determine the compatibility of the product oil with polyols and water.

[0092] The specific variables and test results are shown in Table 4.

[0093] 2. Stability Test The hybrid system was subjected to high and low temperature cycling experiments, and the specific steps included: The mixture was placed at 45°C (high temperature) for 24 hours, and then immediately transferred to -5°C (low temperature) for 24 hours. This constituted one cycle, and three cycles were performed consecutively. After the cycle was completed, the mixture was brought back to room temperature (approximately 25°C), and the presence of stratification was observed to determine the stability of the mixture.

[0094] The specific variables and test results are shown in Table 4.

[0095] Table 4

[0096] As can be seen from the results in Table 4, under the condition that the mass ratio of oil phase / alcohol phase / water phase is 50:49:1, the systems obtained by combining D-L3 (raw material camellia oil) of Comparative Example 3 with dipropylene glycol, glycerol, 1,2-hexanediol, and 1,2-pentanediol were initially turbid and separated into layers after standing. The stability was poor after high and low temperature cycling. However, the systems obtained by combining L1 of Example 1, L4 of Example 4, D-L2 of Comparative Example 2, D-L4 of Comparative Example 4, and D-L5 of Comparative Example 5 with the above polyols under the same conditions remained clear and no obvious separation was observed after standing and high and low temperature cycling.

[0097] The above results indicate that the high polarity resulting from enzymatic hydrolysis improves the compatibility of oils with polyols and water; and the camellia compound oil obtained after extracting camellia pollen also maintains good system compatibility and stability.

[0098] In contrast, D-L6 in Comparative Example 6 exhibited poor compatibility and stability in all polyol systems. These results indicate that even with a similar treatment method to Example 1, replacing camellia oil with meadowfoam seed oil fails to achieve the same system compatibility as the present invention. This further demonstrates that the use of camellia oil as the raw material and the enzymatic hydrolysis-extraction process in this invention play a crucial role in obtaining composite oils with good polyol / aqueous phase carrying capacity.

[0099] This invention provides, by way of example, Figure 1 The state of the mixed systems numbered 1#, 5#, and 9# in Table 4 is shown.

[0100] Depend on Figure 1 It can be seen that the mixed system #1 (with raw camellia oil as the oil phase) is turbid, while the mixed system #5 (with enzymatically hydrolyzed camellia oil as the oil phase) is clear and transparent. These results indicate that enzymatic hydrolysis can significantly improve the compatibility of camellia oil with polyols and the aqueous phase.

[0101] The No. 9 mixed system (with camellia compound oil obtained by enzymatic hydrolysis as the oil phase) also exhibited a clear and transparent state. These results indicate that the extraction process did not significantly weaken the compatibility of enzymatically hydrolyzed camellia oil with polyols and the aqueous phase; the camellia compound oil obtained by enzymatic hydrolysis can still form a relatively stable system with polyols and a small amount of water.

[0102] 3. Compatibility expansion test of the product oil and representative polyols at different ratios The products obtained from D-L3 (raw camellia oil) of Comparative Example 3 and L1 (camellia compound oil obtained by enzymatic hydrolysis and extraction) of Example 1 were used as the oil phase; glycerol and 1,2-pentanediol were used as the alcohol phase; and water was used as the aqueous phase. The mixture was prepared according to the mass ratio of oil phase / alcohol phase / aqueous phase in Table 5 to obtain a mixed system.

[0103] Observe the appearance of each mixture system to determine the compatibility of the product oil with polyols and water in different proportions.

[0104] The specific ratios and test results are shown in Table 5.

[0105] Table 5

[0106] Note: "√" represents "clear and stable"; "×" represents "turbid / layered"; As can be seen from the results in Table 5, when glycerol and 1,2-pentanediol are used as the alcohol phase, D-L3 (raw camellia oil) of Comparative Example 3 is difficult to form a stable and homogeneous system under the various oil / alcohol / water phase ratios tested in this experiment; while L1 (camellia compound oil obtained by enzymatic hydrolysis and extraction) of Example 1 can remain clear and stable under the tested ratios.

[0107] The above results indicate that the camellia compound oil provided by the present invention has a wider range of formulation adaptability and a better polyol / aqueous phase carrying capacity than the raw camellia oil.

[0108] The above results indicate that enzymatic hydrolysis significantly improves the compatibility of camellia oil with polyols and a small amount of aqueous phase, making the system easier to form a clear, stable, and homogeneous state. Compared with raw camellia oil, the camellia compound oil provided by this invention exhibits superior compatibility, stability, and wider adaptability in formulation, making it more conducive to the introduction and carrying of polyols and water-soluble active ingredients in oily systems.

[0109] Test Example 3: DPPH Free Radical Scavenging Rate Determination The DPPH free radical scavenging rate of a subset of products was determined, specifically including the following steps: 1. Preparation of DPPH alcohol solution Each time, dissolve 0.002 g of DPPH in 50 mL of anhydrous ethanol and store in the dark. Before use, dilute with ethanol to an absorbance of approximately 0.7 to obtain a DPPH alcohol solution. The DPPH alcohol solution should be prepared and used immediately.

[0110] 2. Preparation of sample alcohol solution Take the product oil and prepare a sample solution with anhydrous ethanol at a volume concentration of 3%, thus obtaining the sample alcohol solution.

[0111] 3. Determination of free radical scavenging rate Under light-protected conditions, 100 μL of DPPH alcohol solution and 100 μL of sample alcohol solution were added to a 96-well plate and incubated at room temperature in the dark for 30 min. The absorbance of the mixed solution in each well was then measured at 517 nm. Each group in this experiment had three parallel wells: a sample group, a blank group, and a control group. The specific procedures are as follows: Sample group: Add 100 μL of sample alcohol solution and 100 μL of DPPH alcohol solution to each well; Blank group: Add 100 μL of sample alcohol solution and 100 μL of anhydrous ethanol to each well; Control group: 100 μL of DPPH alcohol solution and 100 μL of anhydrous ethanol were added to each well.

[0112] 4. Data Analysis Calculate the DPPH free radical scavenging rate for each group using the following formula: ; The DPPH removal rates of each product oil are shown in Table 6.

[0113] Table 6

[0114] As can be seen from the results in Table 6, the camellia compound oil prepared by the method provided in this invention has good antioxidant activity. For example, the DPPH scavenging rates of L1 and L4 were 63.21% and 55.39%, respectively, both higher than those of the comparative examples.

[0115] Furthermore, when the acid value of the enzymatically hydrolyzed camellia oil obtained in step (1) is 72-76 mg KOH / g and the hydroxyl value is 11-13 mg KOH / g, the antioxidant activity of the resulting camellia compound oil is more prominent. For example, the DPPH scavenging rate of L1 can reach 63.21%, which is higher than that of L4 (55.39%).

[0116] More specifically, under the same testing conditions, the DPPH scavenging rate of Comparative Example 2 (D-L2, enzymatically hydrolyzed camellia oil) was 37.55%, while that of Comparative Example 3 (D-L3, raw camellia oil) was 47.72%, with the former being 10.17 percentage points lower than the latter. These results indicate that enzymatic hydrolysis adversely affects the original antioxidant capacity of the raw camellia oil.

[0117] The DPPH scavenging rate of Comparative Example 1 (D-L1) was 45.57%, similar to, but slightly lower than, that of Comparative Example 3 (D-L3) at 47.72%. These results indicate that direct extraction of camellia flowers from unhydrolyzed raw camellia oil yields a product oil with a DPPH scavenging rate comparable to that of the raw camellia oil; extraction alone does not improve the antioxidant activity of the product oil. In contrast, the DPPH scavenging rates of Example 1 (L1) and Example 4 (L4) were 63.21% and 55.39%, respectively, significantly higher than those of Comparative Examples 3, 2, and 1. Specifically, Example 1 (L1) was 25.66 percentage points higher than Comparative Example 2 (D-L2) and 17.64 percentage points higher than Comparative Example 1 (D-L1). These results demonstrate that the combination of enzymatic hydrolysis and extraction is not a simple additive process, but rather produces a significant synergistic effect.

[0118] The DPPH scavenging rate of Comparative Example 4 (D-L4) was 49.74%, indicating that increasing the amount of lipase could not further enhance the antioxidant effect of the product oil. Combined with the high acid value and hydroxyl value of D-L4, it can be seen that excessive enzymatic hydrolysis is not conducive to obtaining a compound oil with better overall performance.

[0119] The DPPH scavenging rate of Comparative Example 5 (D-L5) was 51.04%, lower than that of Example 1. This indicates that using too low a dosage of enzymatically hydrolyzed camellia oil is not conducive to the full extraction of active ingredients from camellia flowers, thus affecting the antioxidant activity of the product oil.

[0120] The DPPH scavenging rate of Comparative Example 6 (D-L6) was 28.35%, significantly lower than that of Example 1. This indicates that replacing camellia oil with meadowfoam seed oil does not result in a product with antioxidant activity comparable to that of Example 1.

[0121] Furthermore, a 2 × 2 factorial interaction analysis was conducted on the DPPH free radical scavenging rates of the four groups of samples, using whether camellia oil was enzymatically hydrolyzed and whether camellia flower extract was performed as two factors. The DPPH scavenging rates of the camellia flower composite oil provided by this invention (Example 1, L1), enzymatically hydrolyzed camellia oil (Comparative Example 2, D-L2), camellia flower composite oil obtained by extraction (Comparative Example 1, D-L1), and raw camellia oil (Comparative Example 3, D-L3) were 63.21%, 37.55%, 45.57%, and 47.72%, respectively.

[0122] Based on the calculation method of two-factor interaction, .

[0123] Y11 The DPPH scavenging rate of the L1 camellia compound oil provided in Example 1 was 63.21%. Y10 The DPPH scavenging rate (37.55%) of D-L2 (i.e., enzymatically hydrolyzed camellia oil) provided in Comparative Example 2 was given. Y01 The DPPH removal rate (45.57%) of D-L1 (camellia compound oil obtained by extraction only) provided in Comparative Example 1. Y00 The DPPH removal rate of the raw material camellia oil was 47.72%.

[0124] Substituting into the above formula, we can obtain the result. I = 63.21% - 37.55% - 45.57% + 47.72% = 27.81%, meaning the interaction value is 27.81 percentage points. These results indicate that enzymatic hydrolysis and extraction are not simply additive, but rather exhibit a significant positive interaction.

[0125] Specifically, under non-enzymatic hydrolysis conditions, extraction did not significantly improve the DPPH scavenging rate; however, under enzymatic hydrolysis conditions, extraction increased the DPPH scavenging rate by 25.66 percentage points.

[0126] This indicates that enzymatic hydrolysis alters the oil phase system, making it more conducive to the extraction and loading of antioxidant active ingredients from camellia, thus producing a significant synergistic effect.

[0127] Test Example 4: Compatibility and Stability Test 4.1 Preparation of a water-in-oil system, specifically including: (1) At 50°C, PEG-5 (glyceryl triisostearate) and PPG-3 (octyl ether) were added to the oil in the examples or comparative examples and stirred for 30 minutes to make the materials evenly mixed; (2) Mix 1,2-hexanediol, 1,2-pentanediol and 0.1% (w / v) camellia polypeptide aqueous solution evenly to form an aqueous phase, and preheat the aqueous phase at 50°C for 10 minutes; The camellia polypeptide aqueous solution was prepared according to the method described in CN121181647A. Dried red camellia flowers were used as raw materials. After high-pressure homogenization extraction and pepsin enzymatic hydrolysis, the solid content was adjusted to 0.1% (w / v) with pure water to obtain a 0.1% camellia polypeptide aqueous solution.

[0128] (3) Slowly add the aqueous phase to the oil phase, and continue stirring for 30 minutes after the addition is complete to obtain the water-in-oil system.

[0129] The composition of each prepared water-in-oil system is listed in Table 7.

[0130] Table 7

[0131] 4.2 Compatibility Test The state of the prepared water-in-oil system was observed, and the results are as follows: Figure 2 As shown.

[0132] Depend on Figure 2 It can be seen that TX-1 and TX-2 used L1 (camellia compound oil obtained by enzymatic hydrolysis and extraction) from Example 1 as the oil phase, and the water-in-oil system remained clear and transparent under the conditions of water phase ratios of 3wt% and 10wt%, respectively; while TX-D1 and TX-D2 used D-L3 (raw camellia oil) from Comparative Example 3 as the oil phase, and the system showed obvious turbidity under the same water phase ratio.

[0133] 4.3 Stability Test The oil-in-water system was subjected to high and low temperature cycling experiments (the specific experimental steps are the same as the stability test section of Test Example 2). The test results are shown in Table 8.

[0134] Table 8

[0135] As shown in Table 8, the TX-1 and TX-2 systems maintained good appearance stability, with no obvious stratification or precipitation. The TX-D1 system was turbid but did not stratify, while the TX-D2 system showed stratification.

[0136] The above results indicate that the camellia compound oil provided by this invention has better water phase carrying capacity and system stability than the raw camellia oil, and is more conducive to the introduction and loading of water-soluble active ingredients in the oil-in-water system.

[0137] Test Example 5: Cytotoxicity Test This test example uses the MTT (thiazolyl blue tetrazolium bromide) colorimetric method to determine the cytotoxicity of a water-in-oil system. The principle is as follows: highly active succinate dehydrogenase in the mitochondria of living cells can reduce MTT to form purple formazan crystals; dead cells lack this function. The amount of formazan crystals is directly proportional to the number of living cells, and cytotoxicity can be indirectly determined by measuring the absorbance using a microplate reader.

[0138] The cytotoxicity test specifically includes the following steps: (1) Resuspend HaCaT cells in complete culture medium and adjust the cell density to 80,000 cells / well. Seed the cells in 96-well plates and incubate them in a 37°C, 5% (v / v) CO2 incubator for 24 h to allow the cells to adhere to the plate. (2) Dilute the oil-in-water system samples to be tested to 0.008%, 0.04%, 0.2% and 1% (v / v) respectively with complete culture medium, and add 100 μL / well to each well as the experimental group.

[0139] Simultaneously, a control group (containing cells, with an equal volume of complete culture medium, but no sample) and a blank group (cell-free, with only an equal volume of complete culture medium) were set up. The 96-well plates were then returned to the incubator and cultured for another 24–72 hours.

[0140] (3) Add 10 μL of MTT solution (concentration of 5 mg / mL) to each well under dark conditions, and then incubate at 37°C and 5% (v / v) CO2 for 4 hours. (4) Discard the supernatant from each well. Add 150 μL of dimethyl sulfoxide (DMSO) to each well; (5) Use an ELISA reader to measure the absorbance (OD) of each well at 490 nm.

[0141] Cell viability is calculated using the following formula:

[0142] The cytotoxicity test results of each water-in-oil system are as follows: Figure 3 As shown.

[0143] Depend on Figure 3 It was found that TX-D1, TX-D3, and TX-1 all exhibited good biocompatibility with the tested cells. Compared with the control group, the cell viability of each group remained at a high level, and no obvious toxic effects were observed.

[0144] The above results indicate that the formulations constructed using camellia oil (D-L3 in Comparative Example 3), enzymatically hydrolyzed camellia oil (D-L2 in Comparative Example 2), or camellia compound oil obtained through enzymatic extraction (L1 in Example 1) as the oil phase all exhibited good safety under the investigated conditions. Furthermore, TX-1 did not show increased cytotoxicity due to enzymatic extraction treatment.

[0145] The above results show that the solution provided by the present invention can maintain good safety while improving the application performance of compound oil formulations.

[0146] Test Example 6: Scratch Repair Test This test case uses a scratch repair experiment to evaluate the promoting effect of different water-in-oil systems on the repair capacity of HaCaT cells by observing the migration and proliferation of cells on both sides of the scratch.

[0147] The scratch repair test includes the following steps: (1) Resuspend HaCaT cells in culture medium and adjust the cell density to 60,000 cells / well. Seed the cells into the bottom of a 6-well plate with a sterile scratch insert fixed. Incubate the plate at 37°C and 5% (v / v) CO2 for 24 hours until the cells are completely attached.

[0148] (2) Remove the plug to form a scratch test area and take an initial (0h) scratch microscopic image.

[0149] (3) Dilute the test sample to a concentration of 0.04% (v / v) with serum-free medium and add it to the cell wells. No sample is added to the negative control group, but only an equal amount of medium is added. After adding the sample, the 6-well plate is placed back into a 37°C, 5% (v / v) CO2 incubator and cultured for another 24 h.

[0150] (4) Take microscopic images of the scratched area for 24 hours to record the cell migration status.

[0151] (5) The area of ​​the scratch-free region (A0) at 0 h and the area of ​​the scratch-free region at the same location after 24 h of culture were quantitatively analyzed using ImageJ software. 24 ).

[0152] Calculate the scratch healing rate percentage using the following formula: ;in, This represents the area of ​​the blank area marked by the scratch at time 0h. This indicates the area of ​​the blank area marked by the scratch after 24 hours.

[0153] The results of the scratch repair test are as follows Figure 4 As shown.

[0154] Depend on Figure 4 The results showed that the scratch healing rate in the negative group after 24 hours of culture was 27.35%, while the scratch healing rates after treatment with TX-D1, TX-D3, and TX-1 at a concentration of 0.04% (v / v) increased to 51.72%, 52.54%, and 84.44%, respectively. Among them, the scratch healing rate of TX-1 was significantly higher than that of TX-D1 and TX-D3.

[0155] The above results demonstrate that the camellia compound oil provided by this invention has superior effects in promoting HaCaT cell migration and wound repair. These results further indicate that the combination of enzymatic hydrolysis and extraction can endow the oil system with more prominent skin repair activity, thereby enhancing its application value in related products.

[0156] Test Example 7: Barrier Repair Test This test case used immunofluorescence to measure the expression levels of ABCA12 and LOR in cells to test the efficacy of the water-in-oil system in barrier repair. ABCA12 is responsible for transporting lipids such as ceramides to the extracellular space of stratum corneum cells; LOR is a key structural protein of the keratinocyte capsule. Together, they form the keratinocyte capsule, which is crucial for maintaining the integrity of the skin barrier.

[0157] The barrier repair test specifically includes the following steps: (1) After digesting HaCaT cells in the logarithmic growth phase with trypsin, resuspend them in culture medium and adjust the cell density to 20,000 cells / well. Seed them in 96-well plates and incubate them in a 37°C, 5% (v / v) CO2 incubator for 24 h until the cells adhere.

[0158] (2) According to different test indicators, each sample of the water-in-oil system to be tested was diluted with culture medium to the corresponding concentration and then added to the cell wells, 100 μL per well: ABCA12 detection group: sample diluted to 0.04% (v / v); LOR detection group: Samples were diluted to 0.2% (v / v).

[0159] Simultaneously, a negative control group was set up, with no sample added but only an equal volume of complete culture medium; a positive control group was set up, with no sample added but 100 μL of epigallocatechin gallate (EGCG) at a concentration of 20 mmol / L was added. After adding the samples, the 96-well plates were returned to a 37°C, 5% (v / v) CO2 incubator for 24 h of further incubation.

[0160] (3) The expression levels of ABCA12 or LOR in cells in each sample well were tested using immunofluorescence, specifically including the following steps: 3-1 Sample collection: Discard the culture medium, add 300 μL of PBS to each well, and wash 3 times for 5 min each time; 3-2 Cell fixation: Discard PBS, add 100 μL of 4% paraformaldehyde solution (prepared with PBS, w / v) to each well, and incubate at room temperature for 20 min; 3-3 Washing: Discard the fixative, add 300 μL of PBS to each well, and wash 3 times for 5 min each time to remove residual paraformaldehyde; 3-4 Permeabilization: Discard the PBS, add 100 μL of 0.1% Triton X-100 solution (prepared with PBS, v / v) to each well, and permeabilize at room temperature for 10 min; 3-5 Washing: Discard the permeabilization buffer, add 100 μL of PBS to each well, and wash 3 times for 5 min each time to remove residual Triton X-100.

[0161] 3-6 Blocking: Discard the PBS, add 100 μL of 5% bovine serum albumin (BSA) blocking solution (prepared with PBS, w / v) to each well, and incubate at 37°C for 1 h.

[0162] 3-7 Primary antibody incubation: Discard the blocking solution, do not wash, add the corresponding primary antibody solution (50 μL per well) according to the different detection indicators, and incubate overnight at 4°C in the dark. ABCA12 detection group: Add anti-ABCA12 primary antibody (1:50, diluted with immunofluorescence diluent); LOR detection group: Add anti-LOR primary antibody (1:50, diluted with immunofluorescence diluent).

[0163] 3-8 Washing: Remove the primary antibody under light protection, add 100 μL of PBST washing buffer (PBS containing 0.05% v / v Tween-20) to each well, and wash 3 times for 5 min each time.

[0164] 3-9 Secondary antibody incubation: Discard the washing solution in the dark, add 50 μL of the corresponding fluorescently labeled secondary antibody (selected according to the species of the primary antibody, 1:200, diluted with immunofluorescence diluent) to each well, and incubate at room temperature in the dark for 1 h.

[0165] 3-10 Washing: Discard the secondary antibody, add 100 μL of PBST washing buffer to each well, and wash 3 times in the dark for 5 min each time.

[0166] 3-11 Image Acquisition: Retain 50 μL of PBS in each well and observe and acquire images under an inverted fluorescence microscope in the dark.

[0167] (4) ImageJ software was used to quantify the average fluorescence intensity of the immunofluorescence images (expressed in arbitrary units of au). All data are expressed as mean ± standard deviation (%). ± s) indicates that one-way ANOVA was used for inter-group comparisons. P A p-value of 0.05 was considered statistically significant. A difference of 0.01 is considered highly significant.

[0168] The ABCA12 efficacy test results for TX-D1, TX-D3, and TX-1 are as follows: Figure 5 As shown.

[0169] The LOR-enhancing efficacy test results for TX-D1, TX-D3, and TX-1 are as follows: Figure 6 As shown.

[0170] Depend on Figure 5The average fluorescence intensity of ABCA12 expression in the negative group was 132.62 au, while that in the positive group increased to 165.89 au. After treatment with TX-D1 (oil phase of raw camellia oil, D-L3 in Comparative Example 3) and TX-D3 (oil phase of enzymatically hydrolyzed camellia oil, D-L2 in Comparative Example 2), the average fluorescence intensity of ABCA12 expression was 150.46 au and 148.85 au, respectively, both higher than that in the negative group. These results indicate that both treatments have a certain promoting effect on the expression of skin barrier-related proteins. After treatment with TX-1 (camellia compound oil provided by this invention, L1 in Example 1), the average fluorescence intensity of ABCA12 expression further increased to 179.09 au, the highest among all groups. These results indicate that the method provided by this invention can effectively enhance the ability of camellia compound oil to promote ABCA12 expression.

[0171] Depend on Figure 6 It was found that the average fluorescence intensity of LOR expression in the negative group was 116.89 au, while that in the positive group increased to 139.84 au. After treatment with TX-D1 (oil phase of raw camellia oil, D-L3 of Comparative Example 3) and TX-D3 (oil phase of enzymatically hydrolyzed camellia oil, D-L2 of Comparative Example 2), the average fluorescence intensity of LOR expression was 131.18 au and 129.74 au, respectively, both higher than that in the negative group, indicating that they have a certain promoting effect on the expression of skin barrier-related protein LOR. After treatment with TX-1 (camellia compound oil provided by the present invention, L1 of Example 1), the average fluorescence intensity of LOR expression further increased to 141.55 au, which was the highest among all groups.

[0172] The above results indicate that the solution provided by this invention can effectively enhance the ability of camellia compound oil to promote LOR expression, giving it a more prominent skin barrier repair potential and making it superior to raw camellia oil and enzymatically hydrolyzed camellia oil in improving skin barrier function.

[0173] The above results indicate that the camellia compound oil provided by this invention is superior to raw camellia oil and enzymatically hydrolyzed camellia oil in promoting cell migration and repair, as well as upregulating the expression of skin barrier-related proteins ABCA12 and LOR. The combined effect of enzymatic hydrolysis and extraction can significantly enhance the skin barrier repair capacity of the samples. Therefore, the camellia compound oil provided by this invention has higher application value in skin barrier improvement, stratum corneum structure enhancement, and skin repair-related applications.

[0174] Test Example 8: Anti-aging Efficacy Test This test case uses immunofluorescence to measure the expression levels of type I collagen (COL I), type III collagen (COL III), and elastin in cells to test the anti-aging efficacy of the water-in-oil system.

[0175] Type I collagen (COL 1) is the most abundant collagen in the skin and is the most important component of collagen fibers, playing a vital role in maintaining the structural support and appearance of the skin. Type III collagen (COL III) works in conjunction with new cells and tissues to repair damage and remodel collagen. At the same time, its triple helix structure can lock in moisture and maintain the skin's hydration. Elastin is the main component of elastic fibers, which provides elasticity and resilience to the skin. The normal production and orderly integration of elastin are important foundations for maintaining healthy skin structure, function and youthful appearance.

[0176] The anti-aging efficacy test includes the following steps: (1) Resuspend the fibroblasts in complete culture medium and adjust the cell density to 10,000 cells / well. Seed them in equal amounts in 96-well plates and incubate them in a 37°C, 5% (v / v) CO2 incubator for 24 h until the cells adhere.

[0177] (2) According to different detection indicators, each sample of the oil-in-water system to be tested was diluted with complete culture medium to the corresponding concentration and then added to the cell wells, 100 μL per well: COL I detection group: sample diluted to 0.2% (v / v); COL III detection group: sample diluted to 0.2% (v / v); Elastin assay group: Samples were diluted to 0.04% (v / v); Simultaneously, a blank group was set up, with no sample added but only an equal volume of complete culture medium; a positive group was set up, with no sample added but an equal volume of 5 ng / mL recombinant human transforming growth factor-β1 (TGF-β1) added. After adding the samples, the 96-well plate was returned to a 37℃, 5% (v / v) CO2 incubator for 24 h of further incubation.

[0178] (3) The expression levels of type I collagen (COL I), type III collagen (COL III), and elastin in each well cell were detected by immunofluorescence. The primary antibodies used in this test example were anti-COL I primary antibody (1:50, diluted with immunofluorescence diluent), anti-COLIII primary antibody (1:50, diluted with immunofluorescence diluent), and anti-elastin primary antibody (1:50, diluted with immunofluorescence diluent); the secondary antibody was selected according to the species of the primary antibody (1:200, diluted with immunofluorescence diluent). The remaining operating steps were the same as step (3) in test example 7.

[0179] (4) ImageJ software was used to quantify the average fluorescence intensity of the immunofluorescence images (expressed in arbitrary units of au). All data are expressed as mean ± standard deviation (%). Mean ± s) indicates the mean, and one-way ANOVA was used for comparisons between groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly significant.

[0180] The results of the COL I-promoting efficacy test for TX-D1, TX-D3, and TX-1 are as follows: Figure 7 As shown.

[0181] The COL III-promoting efficacy test results of TX-D1, TX-D3 and TX-1 are as follows: Figure 8 As shown.

[0182] The elastin-promoting efficacy test results of TX-D1, TX-D3, and TX-1 are as follows: Figure 9 As shown.

[0183] Depend on Figure 7 It can be seen that the average fluorescence intensity of type I collagen expression in the blank group was 108.40 au, while that in the positive group increased to 120.90 au; After treatment with TX-D1 (oil phase was raw material camellia oil, and D-L3 of Comparative Example 3), the average fluorescence intensity of COL1 expression was 109.19 au, which was close to that of the blank group; After treatment with TX-D3 (oil phase was enzymatically hydrolyzed camellia oil, and D-L2 in Comparative Example 2), the average fluorescence intensity of COL1 expression increased to 116.79 au; After treatment with TX-1 (the oil phase was camellia compound oil obtained by enzymatic hydrolysis and extraction, L1 in Example 1), the average fluorescence intensity of COL1 expression increased to 120.38 au, which was similar to the level of the positive group (120.90 au).

[0184] The above results indicate that the solution provided by the present invention can effectively enhance the ability of camellia compound oil to promote the expression of type I collagen, making it superior to raw camellia oil and enzymatically hydrolyzed camellia oil in terms of skin anti-aging and dermal structure maintenance.

[0185] Depend on Figure 8 It can be seen that the average fluorescence intensity of type III collagen expression in the blank group was 107.39 au, while that in the positive group increased to 134.49 au; After treatment with TX-D1 (oil phase was raw material camellia oil, and D-L3 of Comparative Example 3), the average fluorescence intensity of COL III expression was 93.22 au, which was lower than that of the blank group; After treatment with TX-D3 (oil phase was enzymatically hydrolyzed camellia oil, and D-L2 in Comparative Example 2), the average fluorescence intensity of COL III expression increased to 115.57 au; After treatment with TX-1 (the oil phase was camellia compound oil obtained by enzymatic hydrolysis and extraction, L1 in Example 1), the average fluorescence intensity of COL III expression increased to 117.89 au.

[0186] The above results indicate that the solution provided by the present invention can effectively enhance the ability of camellia compound oil to promote the expression of type III collagen, making it superior to raw camellia oil and enzymatically hydrolyzed camellia oil in improving the dermal matrix and anti-aging of the skin.

[0187] Depend on Figure 9 It can be seen that the average fluorescence intensity of elastin expression in the blank group was 100.85 au, while that in the positive group increased to 124.45 au; After treatment with TX-D1 (oil phase was raw camellia oil, D-L3 of Comparative Example 3) and TX-D3 (oil phase was enzymatically hydrolyzed camellia oil, D-L2 of Comparative Example 2), the average fluorescence intensity of elastin expression was 116.30 au and 120.61 au, respectively, both higher than that of the blank group. After treatment with TX-1 (the oil phase was camellia compound oil obtained by enzymatic hydrolysis and extraction, L1 in Example 1), the average fluorescence intensity of elastin expression was further increased to 132.27 au, which was the highest among all groups.

[0188] The above results indicate that the solution provided by the present invention can effectively enhance the ability of camellia compound oil to promote elastin expression, making it superior to raw camellia oil and enzymatically hydrolyzed camellia oil in improving skin elasticity, maintaining skin structural integrity, and anti-aging.

[0189] Based on the combined detection results of type I collagen (COL 1), type III collagen (COL III), and elastin, the water-in-oil system (TX-1) with camellia compound oil (L1) of the present invention as the oil phase is superior to the water-in-oil system (TX-D1) with raw camellia oil (D-L3) as the oil phase and the water-in-oil system (TX-D3) with enzymatically hydrolyzed camellia oil (D-L2) as the oil phase in promoting the expression of COL1, COLIII, and elastin.

[0190] The above results demonstrate that the camellia compound oil prepared by the method provided in this invention has a good regulatory ability on the components related to the dermal matrix of the skin. Since collagen and elastin are closely related to skin firmness, elasticity, suppleness, and anti-wrinkle ability, this invention has higher application value in improving skin aging-related conditions, maintaining skin structural integrity, and enhancing skin anti-aging effects.

[0191] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for preparing camellia flower compound oil, characterized in that, Includes the following steps: (1) In the presence of lipase and solvent, the raw camellia oil was subjected to enzymatic hydrolysis, and the enzymatically hydrolyzed camellia oil was obtained after separation; (2) Camellia pollen is extracted with the enzymatically hydrolyzed camellia oil to obtain an extraction mixture; (3) The extraction mixture is subjected to solid-liquid separation to obtain camellia compound oil; The mass ratio of the raw material camellia oil to the lipase is 100:0.25-0.75; The enzymatically hydrolyzed camellia oil has an acid value of 72-76 mg KOH / g and a hydroxyl value of 11-13 mg KOH / g. The amount of enzymatically hydrolyzed camellia oil used is 10-20 mL relative to 1 g of camellia pollen.

2. The method according to claim 1, characterized in that, In step (1), the conditions for the enzymatic hydrolysis reaction include: a temperature of 25-37 °C and a time of 1-3 h.

3. The method according to claim 1 or 2, characterized in that, In step (1), the amount of solvent used is 25-100 mL relative to 100 g of raw camellia oil; And / or, in the raw camellia oil, based on the mass of total fatty acids, the mass content of oleic acid is 70%-90%, the mass content of linoleic acid is 5%-15%, and the mass content of saturated fatty acids is ≤15%.

4. The method according to claim 1 or 2, characterized in that, In step (2), the average particle size of the camellia pollen is ≤0.6mm and the moisture content is ≤10wt%.

5. The method according to claim 1 or 2, characterized in that, In step (2), the extraction operation includes: mixing the camellia pollen with the enzymatically hydrolyzed camellia oil and then performing ultrasonic-assisted extraction, followed by stirring extraction.

6. The method according to claim 5, characterized in that, The conditions for ultrasound-assisted extraction include: an ultrasound frequency of 35-45 kHz, an ultrasound power density of 0.1-0.5 W / mL, a temperature of 20-40℃, and a time of 15-45 min. And / or, the stirring extraction speed is 200-400 rpm, the temperature is 20-30℃, and the time is 1-3 h.

7. Camellia compound oil prepared by the method according to any one of claims 1-6.

8. The application of the camellia compound oil according to claim 7 in cosmetics.

9. A cosmetic product, characterized in that, The cosmetic contains the camellia compound oil as described in claim 7; The cosmetic product is selected from at least one of the following: serum, cleansing cream, moisturizing cream, and lotion.

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