Preparation method, product and application of tea tree flower extract against photoaging

Abstract

The present application relates to the technical field of cosmetic raw materials, and particularly relates to a preparation method, product and application of a tea flower extract with anti-photoaging effect. The preparation steps of the tea flower extract include: S1. pretreatment of tea flowers; S2. activation of biological enzymes; S3. first enzymolysis; S4. extraction; S5. filtration; S6. second enzymolysis; S7. purification, and the tea flower extract is obtained. The present application aims to make full use of tea flower resources, avoid the waste of tea flowers as waste, and develop a tea flower extract which can resist ultraviolet damage and maintain skin homeostasis by excavating the synergistic effect between polysaccharides and proteins in tea flowers, so as to realize more comprehensive and in-depth development and utilization of tea flower resources.

Description

Technical Field

[0001] This invention relates to the field of cosmetic raw material technology, and in particular to a method for preparing an anti-photoaging tea tree flower extract, as well as the product and its application. Background Technology

[0002] Skin is the largest organ in the human body, and its aging directly reflects the body's overall aging process. Skin aging is mainly divided into two categories: endogenous aging and exogenous aging. Endogenous aging, also known as natural aging, is primarily influenced by genetic factors and constitution. Exogenous aging, also known as photoaging, mainly refers to aging caused by environmental factors such as ultraviolet radiation, smoking, wind, sun exposure, and contact with harmful chemicals. Studies have shown that photoaging accounts for more than 80% of facial aging, with ultraviolet radiation being the most critical exogenous factor leading to photoaging. Long-term exposure to ultraviolet radiation can cause a series of problems, including abnormal pigmentation, deepening and thickening of wrinkles, dry skin, sagging, atrophy, and telangiectasia. The mechanisms of skin photoaging mainly involve immune system interference, changes in cytokines, increased production of matrix metalloproteinases, decreased mature collagen fibers, increased reactive oxygen species, damage to the extracellular matrix structure, DNA damage, and telomere destruction. Therefore, how to prevent skin photo-oxidation or slow down the damage caused by ultraviolet radiation to the skin, and how to delay skin aging, has always been a major concern. Traditional physical or chemical sunscreens often have problems such as a heavy feel on the skin, poor safety, and limited protective efficiency, and therefore cannot fully meet the needs of cosmetic applications.

[0003] Existing research has found that many plant-derived natural extracts or products have certain sun protection and UV radiation damage mitigation effects. They typically achieve these effects through the following mechanisms: possessing highly unsaturated structures with benzene rings or conjugated systems that absorb UV rays; resisting the skin's photo-oxidation process; regulating extracellular matrix synthesis and decomposition; inhibiting pigment deposition; soothing skin inflammation; and regulating autophagy. For example, curcumin, the main active ingredient in turmeric, can antagonize UVR-induced DNA damage; silymarin can induce apoptosis in photodamaged cells, affecting the cell cycle to facilitate DNA repair, thus providing UVB protection. Polyphenolic compounds such as green tea polyphenols, resveratrol, and tannins can significantly reduce intracellular ROS levels and slow down skin aging. Plant-derived polysaccharides, due to their antioxidant properties, exhibit photoaging treatment effects. For instance, Dendrobium officinale polysaccharides can upregulate the TGF-β / Smad signaling pathway in fibroblasts, thereby reducing oxidative damage caused by UV radiation. Green tea seed extract can reduce the expression of UVB-induced MMP-1, MMP-3, and MMP-9, and increase the production of type I collagen precursors; caffeine can protect the skin from oxidative stress-induced aging by regulating the A2AR / SIRT3 / AMPK pathway and activating autophagy. These naturally derived ingredients, due to their higher safety and easier skin absorption, are gradually becoming a focus of attention in the cosmetics industry's research on anti-photoaging and photodamage.

[0004] Tea tree flowers ( Camellia sinensis (L.) O. Kuntze is the flower of the tea tree, a plant belonging to the genus *Camellia* of the family Theaceae, section *Camellia*. It is one of the reproductive organs of the tea tree and is known as the "placenta of a safe plant" and "the essence of the tea tree." Tea tree flowers are rich in beneficial components and active substances such as tea polyphenols, tea polysaccharides, proteins, flavonoids, tea saponins, caffeine, amino acids, vitamins, trace elements, superoxide dismutase (SOD), and catalase (CAT). They possess certain effects such as lowering lipids and aiding weight loss, protecting against radiation, moisturizing, anti-inflammatory and antibacterial properties, and acting as surfactants. The amount of catechins in tea tree flowers is similar to that in tea leaves, while the content of polysaccharides and proteins is significantly higher than the average level in tea leaves. These components indicate that tea tree flowers are a highly bioactive plant raw material with great application potential.

[0005] Tea tree flowers typically last only two days. If pollination is not completed within two days after opening, they will naturally fall off, exhibiting characteristics of "long flowering period, abundant flowers, and few fruits." For a long time, people have primarily harvested the tender buds and leaves of tea trees for tea production, leaving the flowers to wither naturally. Some tea farmers use pruning and the application of plant growth regulators to remove fallen flowers and fruits (flowers and fruits require more than 50% of the products of tea tree photosynthesis) to achieve high yields in tea gardens. Statistics show that tea-producing areas nationwide produce 4-8 million tons of fresh tea flowers annually, indicating abundant tea tree flower resources. Utilizing only fresh tea leaves while discarding tea tree flowers is undoubtedly a huge waste of tea resources. Manually harvesting tea tree flowers ensures that more nutrients from the tea tree are supplied to the growth of tea leaves and buds, resulting in a 25%-30% increase in tea yield and quality the following year, and bringing additional economic benefits to tea farmers.

[0006] In 2013, tea tree flower (Camellia sinensis) was approved by the National Health Commission of China as a new food ingredient. However, the current application of tea tree flower is relatively narrow, mainly including tea tree flower tea, tea tree flower beverages, tea tree flower wine, tea tree flower essential oil, and tea tree flower pollen, and these products are primarily used in the food and healthcare fields. In the cosmetics industry, the application of tea tree flower extract is still in its initial stages, for example, as an additive in soaps and hand sanitizers, but its unique skincare effects, such as antioxidant, moisturizing, and antibacterial properties, have not yet been fully developed and utilized. Therefore, the potential application value of tea tree flower extract in the cosmetics field still needs further in-depth exploration and research.

[0007] Chinese patent CN102030834B discloses a method for extracting and preparing camellia polysaccharides from camellia flowers and the uses of the obtained camellia polysaccharides. The method involves enzymatic extraction, followed by alcohol precipitation and separation. The separated precipitate is dried to obtain crude camellia polysaccharides. These crude polysaccharides are then decolorized and deproteinized using resin or chemical methods, followed by gel purification or ultrafiltration to obtain the final camellia polysaccharide. Animal experiments have demonstrated that camellia polysaccharides have anti-diabetic and therapeutic effects. Chinese patent application CN105707230A discloses a camellia flower extract with stamens, its extraction method, and its uses. This technique first uses an ethanol-water solution for extraction, recovers the ethanol from the extract, and then uses petroleum ether and ethyl acetate for extraction. After solvent recovery, the camellia flower extract with stamens is obtained. The provided "camellia flower with stamens" extract exhibits significant antibacterial activity and high safety, and can be used to extend the shelf life of milk by 5-7 days. However, these existing technologies have not solved the aforementioned technical problems, hindering research into the application of camellia flower extracts in the field of skincare products.

[0008] Against this backdrop, there is an urgent need to provide a technology that can effectively develop tea tree flower resources, fully explore their skincare benefits, and develop their potential value in the cosmetics field. Summary of the Invention

[0009] This invention aims to make full use of tea tree flower resources that might otherwise be considered waste. By exploring the synergistic effect between polysaccharides and proteins in tea tree flowers, it develops a tea tree flower extract that can both resist UV damage and maintain skin homeostasis, in order to achieve a more comprehensive and in-depth development and utilization of tea tree flower resources.

[0010] The first aspect of this invention provides a method for preparing an anti-photoaging tea tree flower extract, the preparation steps of which include:

[0011] S1. Pre-treat the tea tree flowers to obtain fine powder of tea tree flowers;

[0012] S2. Activate the biological enzyme to obtain an activated enzyme solution;

[0013] S3. Add tea flower powder to the enzyme activating solution and perform one enzymatic hydrolysis to obtain hydrolysate 1;

[0014] S4. Extract the enzyme hydrolysate 1 to obtain the extract;

[0015] S5. Filter the extract to obtain liquid 1;

[0016] S6. Mix the feed solution 1 and biological enzyme 2, and perform a second enzymatic hydrolysis to obtain enzymatic hydrolysate 2;

[0017] S7. Purify the enzymatic hydrolysate 2 to obtain liquid 2, which is the tea tree flower extract.

[0018] Plant cell walls are mainly composed of cellulose and hemicellulose, and contain pectin and starch between cells. Generally, most solvents have limited swelling capacity for plant cell walls, especially in the extraction process of tea flowers, where cell wall disruption is difficult during pollen extraction, which to some extent limits the release of active ingredients from cell tissues. This invention utilizes the action of biological enzymes to maximize the extraction of biological components from tea flowers, thereby fully leveraging the skin-care benefits of tea flowers and achieving the full utilization and in-depth development of tea flower resources.

[0019] In some preferred embodiments, step S1 specifically includes: picking fresh tea tree flowers, sorting and removing calyxes, receptacles and impurities, drying at low temperature, crushing and sieving to obtain fine tea tree flower powder.

[0020] Preferably, the temperature of the low-temperature drying is ≤45°C. The drying method includes at least one of vacuum drying, freeze drying, and oven drying; more preferably, the drying method includes freeze drying or oven drying.

[0021] Preferably, the mesh size of the sieve is 20-200 mesh, more preferably 40-80 mesh.

[0022] In some preferred embodiments, step S2 specifically includes: dissolving the biological enzyme in deionized water and activating the enzyme at 30-40°C for 0.5-2 hours to obtain an activated enzyme solution.

[0023] Preferably, the bio-enzyme is a bio-complex enzyme, including at least three of the following: cellulase, pectinase, α-amylase, xylanase, tanninase, glucanase, mannanase, and glucoamylase.

[0024] More preferably, the bio-complex enzyme comprises cellulase, pectinase, α-amylase, xylanase and tanninase in a mass ratio of (0.1-3):(0.1-2):(0.1-4):(0.15-2):(0.05-1.5).

[0025] More preferably, the α-amylase is a mesophilic α-amylase.

[0026] Preferably, based on the total mass of tea flower powder, the bio-complex enzyme is added at the following concentrations: cellulase 0.2%-1.0%, pectinase 0.2%-1.0%, α-amylase 0.2%-1.0%, xylanase 0.2%-1.0%, and tanninase 0.1%-0.5%.

[0027] Preferably, the cellulase activity is (2000-5000) U / g, the pectinase activity is (40000-80000) U / g, the α-amylase activity is (1000-3000) U / g, the xylanase activity is (60000-100000) U / g, and the tanninase activity is (100-500) U / g.

[0028] More preferably, the cellulase activity is 3500 U / g, the pectinase activity is 60000 U / g, the mesophilic α-amylase activity is 1000 U / g, the xylanase activity is 80000 U / g, and the tanninase activity is 300 U / g.

[0029] Preferably, the amount of deionized water added in step S2 is 5-15 times the mass of the tea flower powder.

[0030] Tea tree flowers contain active ingredients such as xylan, pectin, tea polyphenols, amino acids, flavonoids, and alkaloids. Xylan, as a heteropolysaccharide, is a key component of plant hemicellulose; while pectin is an important substance constituting plant cell walls and also plays a role in connecting adjacent cell walls. This invention selects cellulase, pectinase, mesophilic α-amylase, xylanase, and tanninase as a bio-complex enzyme, which can efficiently degrade the cell walls of tea tree flowers, disrupting the cell structure. After cell rupture, the active ingredients are exposed and dissolved from the cells. This invention utilizes bio-complex enzyme reaction technology to prepare tea tree flower extract, which can improve the extraction efficiency of active ingredients and maintain their stability as much as possible.

[0031] Furthermore, the components in tea flowers, such as tea polyphenols, soluble proteins, polysaccharides, caffeine, and metal ions, readily interact to form large molecular complexes. Among these, ester-type catechins, containing more hydroxyl groups, are more likely to bind with other components, forming complexes that cause turbidity and precipitation in the extract, negatively impacting product stability and limiting their addition to skincare products. This invention selects cellulase, pectinase, α-amylase, xylanase, and tanninase in a mass ratio of (0.1-3):(0.1-2):(0.1-4):(0.15-2):(0.05-1.5) to effectively hydrolyze the corresponding complexes, thereby inhibiting precipitation formation. It is speculated that the specific amount of bio-complex enzymes added in this invention maximally catalyzes the hydrolysis of gallic acid catechins, generating non-ester-type catechins and gallic acid; gallic acid then combines with caffeine to form small-molecule water-soluble substances, effectively reducing precipitate formation. In addition, the formation of gallic acid also enhances the antioxidant properties of tea tree flower extract to some extent, thereby improving its overall performance.

[0032] In some preferred embodiments, step S3 specifically includes: adding fine tea flower powder to an activating enzyme solution, adjusting the pH to 4-6, and enzymatically hydrolyzing at 45-55℃ and 100-200rpm for 1-3 hours to obtain enzymatic hydrolysate 1.

[0033] This invention uses water as a solvent, avoiding the introduction of organic reagents and reducing environmental pollution and potential health risks. Pre-treatment of the raw materials by low-temperature drying before pulverization effectively preserves the active ingredients, ensuring the quality and efficacy of the final product. Using oven drying or freeze-drying methods helps protect heat-sensitive active ingredients, reduces energy consumption, and improves production efficiency. Through the implementation of this invention, the application of tea tree flowers in the cosmetics industry can be further explored, potentially expanding the application scope of tea tree flower resources.

[0034] In some preferred embodiments, step S4 specifically includes: heating the enzymatic hydrolysate 1 to 85-95°C and stirring at 100-200 rpm for 1-3 hours to obtain the extract.

[0035] In some preferred embodiments, step S5 specifically includes: filtering the extract obtained in step S4 multiple times to obtain liquid 1.

[0036] In some preferred embodiments, step S6 specifically includes: adjusting the pH of the feed solution to 3-5, adding biological enzyme II, performing secondary enzymatic hydrolysis for 0.5-2 hours at 45-55℃ and 100-200 rpm, and inactivating the enzyme at 85-92℃ to obtain enzymatic hydrolysate 2.

[0037] Preferably, the bioenzyme II includes at least one of protease, amylase, lipase, and cellulase; more preferably, it is a protease; and even more preferably, it is an acidic protease.

[0038] Preferably, the amount of the added bio-enzyme II is 0.1-3% of the total mass of the tea flower powder, more preferably 0.2-1%.

[0039] In some preferred embodiments, step S7 specifically includes: adding a clarifying agent to the enzymatic hydrolysate 2, letting it stand in a water bath at 50-60°C for 20-60 minutes, and then filtering to obtain liquid 2, which is the finished tea tree flower extract.

[0040] Preferably, the clarifying agent includes one or more of chitosan, ZTC (natural clarifying agent) type II, ZTC type III, and ZTC 101.

[0041] Preferably, the amount of clarifying agent added is 1-5% of the total mass of liquid 2.

[0042] The filtration methods of this invention can be categorized as gauze filtration, porous material filtration, membrane filtration, etc.

[0043] Preferably, step S5 employs gauze filtration, porous material filtration, and membrane filtration.

[0044] Preferably, porous material filtration and membrane filtration are used in step S7.

[0045] Preferably, the gauze filter is a 100-400 mesh gauze filter.

[0046] Preferably, the porous material includes one or more combinations of diatomaceous earth, activated carbon, zeolite, porous ceramics, and molecular sieves; more preferably, it is diatomaceous earth.

[0047] Preferably, the porous material has a particle size of 200-400 mesh.

[0048] The membrane filtration may include at least one selected from polypropylene (PP) membranes, polyethersulfone (PES) membranes, polyvinylidene fluoride (PVDF) membranes, polytetrafluoroethylene (PTFE) membranes, nylon membranes, cellulose acetate (CA) membranes, and MCE filter membranes. Preferably, the membrane filtration is a polypropylene membrane filtration.

[0049] A second aspect of the present invention provides a tea tree flower extract prepared by the method described above, wherein the tea tree flower extract comprises tea tree flower sugars and tea tree flower plant peptides.

[0050] Preferably, the total sugar content in the tea tree flower extract is ≥8.0 mg / mL; and the total peptide content in the tea tree flower extract is ≥7.0 mg / mL.

[0051] Preferably, the tea flower sugars include monosaccharides; the monosaccharides include at least two of glucose, galactose, arabinose, mannose, galacturonic acid, xylose, or fructose.

[0052] More preferably, the monosaccharide comprises glucose, galactose, arabinose, mannose, galacturonic acid and xylose in a mass ratio of (50-80):(5-35):(1-15):(1-10):(0.1-5):(0.1-3).

[0053] Preferably, the weight-average molecular weight (Mw) of the tea flower sugars is 5 × 10⁻⁶. 3 -5×10 4 .

[0054] Preferably, the tea tree flower extract contains ≥100 types of plant peptides as determined by peptide sequence analysis.

[0055] Preferably, the tea tree flower plant peptide is composed of 2 to 30 amino acids.

[0056] A third aspect of the present invention provides an application of the tea tree flower extract as described above, wherein the tea tree flower extract is applied to a skin care composition; the amount of the tea tree flower extract added to the skin care composition is 10-30 wt%.

[0057] In addition to tea tree flower extract, the skincare composition may also contain functional components such as moisturizers, skin conditioning agents, thickeners, and preservatives, depending on the user's needs.

[0058] In some preferred embodiments, the skincare composition comprises, by weight percentage, 20-30% tea tree flower extract, 10-40% glycerin, 0.1-2.0% 1,2-hexanediol, 0.1-2.0% p-hydroxyacetophenone, and the balance being water.

[0059] The preparation method of the skin care composition is not particularly limited; for example, the raw materials can be mixed and filtered to obtain the composition.

[0060] The tea tree flower extract prepared by this invention is rich in sugars, proteins, polypeptides, and tea polyphenols. Through synergistic effects of multiple pathways, it can effectively resist damage caused by ultraviolet radiation and maintain the stability of the skin. Experimental verification shows that the tea tree flower extract of this invention has the following effects: ① It possesses excellent DPPH free radical scavenging ability; ② It can significantly reduce the increase in ROS levels in HaCaT cells induced by ultraviolet radiation; ③ It can upregulate the content of type I procollagen C-terminal peptide and promote type I collagen synthesis; ④ It can reduce the expression of MMP-1 in HSF cells after UVB irradiation and inhibit abnormal extracellular matrix degradation; ⑤ In clinical applications, after continuous use for 4 weeks, it has a significant effect on improving facial skin elasticity. Therefore, the implementation of this invention realizes an in-depth exploration of the potential value of tea tree flowers and has positive research significance.

[0061] Beneficial effects:

[0062] This invention provides a tea tree flower extract with anti-photoaging properties and its preparation method, which has the following advantages:

[0063] (1) The present invention selects cellulase, pectinase, mesophilic α-amylase, xylanase and tanninase as biological complex enzymes, which can efficiently degrade the cell wall of tea flower, so that the active ingredients can be exposed and dissolved from the cells, significantly improving the extraction efficiency of active ingredients and the stability of tea flower extract.

[0064] (2) This invention optimizes the mass ratio and dosage of the bio-complex enzyme, which can maximize the hydrolysis of macromolecular complexes generated during the tea flower extraction process and inhibit the formation of precipitates during extraction. At the same time, it also enhances the antioxidant properties of the tea flower extract to a certain extent and improves the overall performance of the tea flower extract.

[0065] (3) In the present invention, the extraction temperature in step S4 is preferably 85-95℃. Under the combined action of biological enzyme one and biological enzyme two, the active ingredients in tea flower are fully dissolved, and the total sugar content in the final tea flower extract is ≥8.0mg / mL; the total peptide content in the tea flower extract is ≥7.0mg / mL, and the types of tea flower plant peptides are ≥100.

[0066] (4) The extraction method of the present invention uses water as a solvent, avoiding the introduction of organic reagents and reducing environmental pollution and potential health risks. Through the implementation of the present invention, tea tree flower extract with significant antioxidant, stabilizing, and anti-aging effects can be obtained, providing a feasible solution for further in-depth exploration of the application of tea tree flowers in the cosmetics industry.

[0067] (5) The extraction method of the present invention has the advantages of being healthy and harmless, having no environmental impact, rapid extraction, and high-efficiency output. The extraction process is simple and easy to implement, and can meet the requirements of large-scale mass production.

[0068] (6) The extraction method of the present invention provides a new idea for the application of tea flower resources in the cosmetics field. It is not only conducive to enhancing the economic value of tea flowers, but also to promoting the development of related industries and creating more economic and ecological benefits for society. Attached Figure Description

[0069] Figure 1 Glucose standard curve;

[0070] Figure 2 .Results of ROS fluorescence intensity assay in HaCaT cells;

[0071] Figure 3 .Figure showing the results of the test on the content of type I procollagen C-terminal peptide in HSF cells;

[0072] Figure 4 Figure 1 shows the results of UVB-induced MMP-1 secretion in HSF cells.

[0073] Figure 5 Results of skin elasticity change test for samples from Example 1 and Comparative Example 2. Detailed Implementation

[0074] Note: The fresh tea tree flowers used in this embodiment of the invention are from Menghai County, Xishuangbanna Dai Autonomous Prefecture, Yunnan Province; the polypropylene membrane is from Haining Yibo Filter Material Factory, with a pore size of 0.22μm (φ100mm); the clarifying agent ZTC Type II is from Beijing Zhengtiancheng Clarification Technology Co., Ltd.; the concentration of concentrated sulfuric acid involved in the test method is 98%.

[0075] Unless otherwise specified, the solvent used in the solutions involved in this invention is water; and all raw materials used are commercially available.

[0076] Example

[0077] Example 1

[0078] The first aspect of this embodiment provides a method for preparing an anti-photoaging tea tree flower extract, the preparation steps of which include:

[0079] S1. Pre-treatment of tea tree flowers: Pick fresh tea tree flowers, sort and remove calyxes, receptacles and impurities, dry in an oven at 40℃, and pulverize through a 60-mesh sieve to obtain fine tea tree flower powder.

[0080] S2. Activation of biological enzyme 1: Dissolve biological enzyme 1 in deionized water at 10 times the weight of tea flower powder, and activate the enzyme at 35°C for 1 hour to obtain an activated enzyme solution.

[0081] The bio-enzyme is a bio-complex enzyme, which includes cellulase, pectinase, mesophilic α-amylase, xylanase, and tanninase. Based on the total mass of tea flower powder, the bio-complex enzyme is added at the following concentrations: 0.5% cellulase, 0.5% pectinase, 0.5% mesophilic α-amylase, 0.5% xylanase, and 0.2% tanninase.

[0082] The cellulase activity was 3500 U / g, the pectinase activity was 60000 U / g, the mesophilic α-amylase activity was 1000 U / g, the xylanase activity was 80000 U / g, and the tanninase activity was 300 U / g; all of them were from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.

[0083] S3. First enzymatic hydrolysis: Add the fine powder of tea flower to the activated enzyme solution, adjust the pH to 4.5 with 19% citric acid aqueous solution, and enzymatically hydrolyze for 2 hours at 52℃ and 150 rpm to obtain enzymatic hydrolysate 1.

[0084] S4. Extraction: Heat the enzymatic hydrolysate 1 to 92℃ and extract by stirring at 150 rpm for 2 hours to obtain the extract.

[0085] S5. Filtration: The extract obtained in step S4 is filtered sequentially through 200-mesh gauze, 300-mesh diatomaceous earth, and a 5μm polypropylene membrane to obtain liquid 1. In this step, the 300-mesh diatomaceous earth and the 5μm polypropylene membrane are used simultaneously, with the polypropylene membrane at the bottom and the diatomaceous earth placed on top of it for filtration.

[0086] S6. Secondary enzymatic hydrolysis: The pH of the feed solution 1 was adjusted to 4 using a 19% citric acid aqueous solution, biological enzyme 2 was added, and secondary enzymatic hydrolysis was carried out at 52℃ and 150rpm for 1 hour. The enzyme was then inactivated at 90℃ for 10 minutes to obtain enzymatic hydrolysate 2.

[0087] S7. Purification: Add 2% (by mass) of clarifying agent (ZTC Type II clarifying agent) to the enzymatic hydrolysate 2, let it stand in a water bath at 55°C for 30 minutes, and then filter it through a 300-mesh diatomaceous earth filter and a 5μm polypropylene membrane filter in sequence to obtain liquid 2, which is the finished tea tree flower extract.

[0088] The second aspect of this embodiment provides a tea tree flower extract prepared by the method described above, wherein the tea tree flower extract comprises tea tree flower sugars and tea tree flower plant peptides.

[0089] The tea tree flower extract was analyzed by HPLC (high performance liquid chromatography), and the main sugars in the tea tree flower extract included the following monosaccharides in the following mass ratios: glucose (64.8%), galactose (20.4%), arabinose (8.5%), mannose (3.0%), galacturonic acid (1.4%), and xylose (0.6%).

[0090] The tea tree flower extract was analyzed by GPC (gel permeation chromatography), and the weight-average molecular weight (Mw) of the sugars in the tea tree flower was 1.288 × 10⁻⁶. 4 .

[0091] Peptide sequence analysis of the tea tree flower extract revealed 111 types of plant peptides from the tea tree flower.

[0092] The third aspect of this embodiment provides the application of tea tree flower extract in skincare compositions as described above.

[0093] The skincare composition comprises, by weight percentage: 20% tea tree flower extract, 20% glycerin, 0.5% 1,2-hexanediol, 0.5% p-hydroxyacetophenone, and the balance being water.

[0094] The total sugar and total peptide content of the tea tree flower extract is shown in Table 1 below.

[0095] The preparation steps of the skin care composition include: mixing the raw materials according to the formula amount, filtering with a 0.22μm polypropylene membrane to obtain the skin care composition.

[0096] Example 2

[0097] This embodiment provides a method for preparing an anti-photoaging tea tree flower extract, as well as the product and its application. The specific implementation method is the same as in Embodiment 1; the difference is:

[0098] Step S1 involves freeze-drying and pulverizing the material through an 80-mesh sieve.

[0099] The extraction time in step S4 is 3 hours.

[0100] The tea tree flower extract was analyzed by HPLC (high performance liquid chromatography), and the main sugars in the tea tree flower extract included the following monosaccharides in the following mass ratios: glucose (56.1%), galactose (21.6%), arabinose (10.5%), mannose (3.6%), galacturonic acid (1.7%), and xylose (0.7%).

[0101] The tea tree flower extract was analyzed by GPC (gel permeation chromatography), and the weight-average molecular weight (Mw) of the sugars in the tea tree flower was 1.054 × 10⁻⁶. 4 .

[0102] Peptide sequence analysis of the tea tree flower extract revealed 136 types of plant peptides from the tea tree flower.

[0103] The total sugar and total peptide content of the tea tree flower extract is shown in Table 1 below.

[0104] Example 3

[0105] This embodiment provides a method for preparing an anti-photoaging tea tree flower extract, as well as the product and its application. The specific implementation method is the same as in Embodiment 1; the difference is:

[0106] In step S2, the amount of deionized water added is 15 times the mass of the tea flower powder.

[0107] The skincare composition comprises: 30% tea tree flower extract, 20% glycerin, 0.5% 1,2-hexanediol, 0.5% p-hydroxyacetophenone, and the balance being water.

[0108] The tea tree flower extract was analyzed by HPLC (high performance liquid chromatography), and the main sugars in the tea tree flower extract included the following monosaccharides in the following mass ratios: glucose (69.2%), galactose (16.5%), arabinose (7.7%), mannose (3.3%), galacturonic acid (1.7%), and xylose (0.8%).

[0109] The tea tree flower extract was analyzed by GPC (gel permeation chromatography), and the weight-average molecular weight (Mw) of the sugars in the tea tree flower was 7.658 × 10⁻⁶. 3 .

[0110] Peptide sequence analysis of the tea tree flower extract revealed 151 types of plant peptides from the tea tree flower.

[0111] The total sugar and total peptide content of the tea tree flower extract is shown in Table 1 below.

[0112] Example 4

[0113] This embodiment provides a method for preparing an anti-photoaging tea tree flower extract, as well as the product and its application. The specific implementation method is the same as in Embodiment 1; the difference is:

[0114] Step S1 is freeze drying.

[0115] The temperature in step S4 is increased to 85°C.

[0116] The tea tree flower extract was analyzed by HPLC (high performance liquid chromatography), and the main sugars in the tea tree flower extract included the following monosaccharides in the following mass ratios: glucose (62.4%), galactose (20.8%), arabinose (8.9%), mannose (2.7%), galacturonic acid (1.6%), and xylose (0.5%).

[0117] The tea tree flower extract was analyzed by GPC (gel permeation chromatography), and the weight-average molecular weight (Mw) of the sugars in the tea tree flower was 3.792 × 10⁻⁶. 4 .

[0118] Peptide sequence analysis of the tea tree flower extract revealed 103 types of plant peptides from the tea tree flower.

[0119] The total sugar and total peptide content of the tea tree flower extract is shown in Table 1 below.

[0120] Example 5

[0121] This embodiment provides a method for preparing an anti-photoaging tea tree flower extract, as well as the product and its application. The specific implementation method is the same as in Embodiment 1; the difference is:

[0122] Based on the total mass of tea flower powder, the bio-complex enzymes are added at the following concentrations: cellulase 0.3%, pectinase 0.3%, mesophilic α-amylase 0.3%, xylanase 0.3%, and tanninase 0.2%.

[0123] The tea tree flower extract was analyzed by HPLC (high performance liquid chromatography), and the main sugars in the tea tree flower extract included the following monosaccharides in the following mass ratios: glucose (56.3%), galactose (24.5%), arabinose (9.3%), mannose (3.2%), galacturonic acid (1.2%), and xylose (0.4%).

[0124] The tea tree flower extract was analyzed by GPC (gel permeation chromatography), and the weight-average molecular weight (Mw) of the sugars in the tea tree flower was 2.316 × 10⁻⁶. 4 .

[0125] Peptide sequence analysis of the tea tree flower extract revealed 106 types of plant peptides from the tea tree flower.

[0126] The total sugar and total peptide content of the tea tree flower extract is shown in Table 1 below.

[0127] Comparative Example 1

[0128] This comparative example provides a method for preparing tea tree flower extract, the product, and its applications. The preparation steps include:

[0129] S1. Pre-treatment of tea tree flowers: Pick fresh tea tree flowers, sort and remove calyxes, receptacles and impurities, dry in an oven at 40℃, and pulverize through a 60-mesh sieve to obtain fine tea tree flower powder.

[0130] S2. Extraction: Mix the fine powder of tea tree flowers with 10 times the weight of deionized water, heat to 92℃, and stir at 150 rpm for 2 hours to obtain the extract.

[0131] S3. Filtration: The extract obtained in step S2 is filtered through a 200-mesh gauze, a 300-mesh diatomaceous earth filter, and a 5μm polypropylene membrane filter to obtain liquid 1. In this step, the 300-mesh diatomaceous earth and the 5μm polypropylene membrane are used simultaneously, with the polypropylene membrane at the bottom and the diatomaceous earth layered on top of it for filtration.

[0132] S4. Purification: Add 2% (by weight) of clarifying agent (ZTC Type II clarifying agent) to the liquid 1, let it stand in a water bath at 55℃ for 30 minutes, and then filter it through a 300-mesh diatomaceous earth filter and a 5μm polypropylene membrane filter in sequence to obtain liquid 2, which is the finished tea tree flower extract.

[0133] The total sugar and total peptide contents of the obtained tea flower extract are shown in Table 1 below.

[0134] The tea tree flower extract is used in a skin care composition, which, by weight percentage, comprises: 20% tea tree flower extract, 20% glycerin, 0.5% 1,2-hexanediol, 0.5% p-hydroxyacetophenone, and the balance being water.

[0135] Comparative Example 2

[0136] This comparative example provides a method for preparing tea tree flower extract, the product, and its applications. The preparation steps include:

[0137] S1. Pre-treatment of tea tree flowers: Pick fresh tea tree flowers, sort and remove calyxes, receptacles and impurities, dry in an oven at 40℃, and pulverize through a 60-mesh sieve to obtain fine tea tree flower powder.

[0138] S2. Activation of biological enzyme 1: Dissolve biological enzyme 1 in deionized water at 10 times the weight of tea flower powder, and activate the enzyme at 35°C for 1 hour to obtain an activated enzyme solution.

[0139] The bio-enzyme is a bio-complex enzyme, which includes cellulase, pectinase, mesophilic α-amylase, xylanase, and tanninase. Based on the total mass of tea flower powder, the bio-complex enzyme is added at the following concentrations: 0.5% cellulase, 0.5% pectinase, 0.5% mesophilic α-amylase, 0.5% xylanase, and 0.2% tanninase.

[0140] The cellulase activity was 3500 U / g, the pectinase activity was 60000 U / g, the mesophilic α-amylase activity was 1000 U / g, the xylanase activity was 80000 U / g, and the tanninase activity was 300 U / g; all of them were from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.

[0141] S3. First enzymatic hydrolysis: Add the fine powder of tea flower to the activated enzyme solution, adjust the pH to 4.5 with 19% citric acid aqueous solution, and enzymatically hydrolyze for 2 hours at 52℃ and 150 rpm to obtain enzymatic hydrolysate 1.

[0142] S4. Extraction: Heat the enzymatic hydrolysate 1 to 92℃ and extract by stirring at 150 rpm for 2 hours to obtain the extract.

[0143] S5. Filtration: The extract obtained in step S4 is filtered through a 200-mesh gauze, a 300-mesh diatomaceous earth filter, and a 5μm polypropylene membrane filter to obtain liquid 1. In this step, the 300-mesh diatomaceous earth and the 5μm polypropylene membrane are used simultaneously, with the polypropylene membrane at the bottom and the diatomaceous earth layered on top of it for filtration.

[0144] S6. Purification: Add 2% (by weight) of clarifying agent (ZTC Type II clarifying agent, Beijing Zhengtiancheng Clarification Technology Co., Ltd.) to solution 1, let it stand in a water bath at 55℃ for 30 minutes, and then filter it through a 300-mesh diatomaceous earth filter and a 5μm polypropylene membrane filter to obtain solution 2, which is the finished tea tree flower extract.

[0145] The total sugar and total peptide contents of the obtained tea flower extract are shown in Table 1 below.

[0146] The tea tree flower extract is used in a skin care composition, which, by weight percentage, comprises: 20% tea tree flower extract, 20% glycerin, 0.5% 1,2-hexanediol, 0.5% p-hydroxyacetophenone, and the balance being water.

[0147] Comparative Example 3

[0148] This comparative example provides a method for preparing tea tree flower extract, the product, and its application. The specific implementation method is the same as in Example 1; the difference is:

[0149] The bio-complex enzyme includes cellulase, pectinase, mesophilic α-amylase, and xylanase; based on the total mass of tea flower powder, the bio-complex enzyme is added at the following concentrations: 0.5% cellulase, 0.5% pectinase, 0.5% mesophilic α-amylase, and 0.5% xylanase.

[0150] The total sugar and total peptide contents of the obtained tea flower extract are shown in Table 1 below.

[0151] Comparative Example 4

[0152] This comparative example provides a method for preparing tea tree flower extract, the product, and its application. The specific implementation method is the same as in Example 1; the difference is:

[0153] The temperature in step S4 is raised to 70°C.

[0154] The total sugar and total peptide contents of the obtained tea flower extract are shown in Table 1 below.

[0155] Performance testing

[0156] I. Test of total sugar content in tea tree flowers

[0157] 1. Preparation of standard solution: Accurately weigh 10.5 mg of anhydrous glucose, add distilled water to a 100 mL volumetric flask, and prepare a standard glucose solution with a concentration of 0.105 mg / mL.

[0158] 2. Preparation of phenol solution: Weigh 5g of phenol, add distilled water to make up to 100mL in a volumetric flask, and prepare a 5% phenol solution.

[0159] 3. Construction of the standard curve: Accurately measure 0, 0.1, 0.2, 0.4, 0.8, 1.2, 1.6, and 2.0 mL of glucose standard solution into stoppered colorimetric tubes, and add water to each tube to a final volume of 2.0 mL. Add 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid to each tube, respectively. Cap the tubes, shake well, and incubate at 100°C for 20 min. Remove the tubes and quickly cool them to room temperature in a cold water bath. Using the corresponding reagent (0 mL of glucose standard solution from the previous step) as a blank, measure the absorbance at 490 nm. Plot the standard curve with glucose concentration on the x-axis and absorbance on the y-axis as shown below. Figure 1 As shown.

[0160] 4. Determination of total sugar content in tea tree flowers: The tea tree flower extracts prepared in Examples 1-5 and Comparative Examples 1-4 were diluted 20 times with distilled water. 0.20 mL of each extract was placed in a stoppered test tube, and 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid were added. The tube was placed in a 100°C water bath for 20 min, then removed and rapidly cooled to room temperature in a cold water bath. The absorbance was measured at a wavelength of 490 nm. The total sugar content of the tea tree flowers was calculated according to the standard curve. The test results of the total sugar content of the tea tree flower extracts in each example and comparative example are shown in Table 1.

[0161] II. Total Peptide Content Test of Tea Tree Flowers

[0162] 1. Preheat the spectrophotometer for 30 minutes, adjust the wavelength to 540 nm, and zero it with distilled water.

[0163] 2. Blank tube: Take a glass test tube, add 200μL of distilled water and 1000μL of biuret reagent, mix well and let stand at room temperature for 15min, then measure the color at 540nm and record it as blank tube A.

[0164] 3. Standard tube: Take a glass test tube, add 200 μL of standard solution and 1000 μL of biuret reagent, mix well and let stand at room temperature for 15 min, then measure the color at 540 nm and record it as standard tube A.

[0165] 4. Test tube: Take the tea flower extract prepared in Examples 1-5 and Comparative Examples 1-4, dilute it twice with distilled water to obtain the test solution; take a glass test tube, add 200 μL of the test solution and 1000 μL of biuret reagent, mix well and let stand at room temperature for 15 min, measure the color at 540 nm, and record it as test tube A.

[0166] Formula for calculating total peptide concentration in tea flower: Canalyte (mg / mL) = Cstandard tube × (Aanalyte tube – Ablank tube) ÷ (Astandard tube – Ablank tube) × D (Where, Cstandard: 5 mg / mL; D: dilution factor)

[0167] The test results of total peptide content in tea flower extracts of each embodiment and comparative example are shown in Table 1.

[0168] III. Stability Testing

[0169] Take 30g of tea tree flower extract from Examples 1-5 and Comparative Examples 1-4 respectively, store them in a sealed clean container, and store them in a refrigerator at -18℃ for 24 hours. After taking them out, place them at room temperature for 24 hours. Repeat this cycle 7 times and then compare them with the initial sample to evaluate whether the sample shows any abnormalities such as color change, turbidity, or precipitation.

[0170] The stability test results of each embodiment and comparative example are shown in Table 1.

[0171] Table 1

[0172]

[0173] IV. DPPH Free Radical Scavenging Activity Test

[0174] 1. Preparation of DPPH solution: Weigh DPPH powder and dissolve it in ethanol to prepare a 0.2 mM DPPH ethanol solution.

[0175] 2. Preparation of positive control solution: Weigh L-ascorbic acid powder and dissolve it in distilled water to prepare a 10 mg / mL L-ascorbic acid solution. Dilute with ethanol to a final experimental concentration of 5 μg / mL.

[0176] 3. Preparation of test sample dilution solution: Dilute the test sample (tea flower extract prepared in Examples 1-5 and Comparative Examples 1-4) with ethanol to an experimental concentration of 2% to obtain the sample solution.

[0177] 4. DPPH Free Radical Scavenging Test: Add 500 μL each of the sample solution and positive control solution to a 24-well plate (i.e., the final experimental concentration of the sample is 1%). Add 500 μL of ethanol to the DPPH tube (C) (3 replicates). Two groups are set up for each sample. Add 500 μL of 0.2 mM DPPH solution to one group, denoted as T-sample tube; as a correction for the sample's own absorbance value, add 500 μL of ethanol to the other group, denoted as T0-sample background. React for 30 min at room temperature and in the dark. Measure the absorbance at a wavelength of 517 nm and calculate the DPPH scavenging rate of the test sample and the positive control L-ascorbic acid; DPPH scavenging rate = 1 - (T - T0) / C × 100%.

[0178] Table 2 shows the DPPH free radical scavenging rate results for each embodiment and comparison; the values ​​in Table 2 are the average values ​​of the measurement results of 3 parallel samples for each sample.

[0179] Table 2

[0180]

[0181] V. HaCaT cell ROS level test

[0182] H2O2 and ultraviolet radiation can induce an increase in reactive oxygen species (ROS) levels in human keratinocytes, activate the mitogen-activated protein kinase (MAPK) signaling pathway, promote the expression of matrix metalloproteinases (MMPs), and inhibit collagen synthesis. ROS can oxidize non-fluorescent DCFH to generate fluorescent DCF, and the fluorescence intensity is directly proportional to the intracellular ROS level.

[0183] Before ROS level testing, cell viability was determined using the MTT assay to establish a safe concentration of tea tree flower extract for HaCaT cells in the examples and comparative cases. HaCaT cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin antibiotics at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase were collected, and a cell suspension was prepared at a concentration of 1×10⁻⁶ cells / cells. 5 Seeds were planted at a density of cells / well in 24-well plates and incubated in a CO2 incubator. After 24 hours of incubation, except for the control group, all wells were inoculated with a dose of 15 mJ / cm². 2The cells were irradiated with UVB. After irradiation, each well of the sample group was added with high-glucose medium containing 0.156% of the tea tree flower extract from the examples and comparative examples, while each well of the control and irradiation groups was added with 500 μL of high-glucose medium. After administration, the 24-well plates were incubated at 37°C and 5% CO2 for 24 h. The culture medium was discarded, the cell plates were washed twice with serum-free medium, and 500 μL of DCFH-DA working solution (10 μM) was added. The plates were then incubated at 37°C for 30 min. The cells were washed three times with DPBS, observed and photographed under a fluorescence microscope, and the fluorescence intensity was analyzed using ImageJ. The test results are shown in [Figure number missing]. Figure 2 .

[0184] Cellular ROS levels reflect the degree of cellular oxidative stress; the lower the fluorescence intensity of the tested sample, the stronger the effect of the surface sample in mitigating oxidative stress. For example... Figure 2 It can be seen that, compared with the control group, the ROS free radicals in HaCaT cells of the UVB irradiation group were significantly increased, indicating that the model was successfully constructed. Compared with the irradiation group, the tea tree flower extract samples of Examples 1-5 could all clear the increase of ROS free radicals caused by UVB irradiation to varying degrees, indicating that the tea tree flower extracts of Examples 1-5 can effectively reduce the oxidative stress level of UVB-induced damaged cells. The comparison shows that Examples 2 and 3 have the best effects, and overall, the tea tree flower extracts of Examples 1-5 are better than those of Comparative Examples 1-4.

[0185] VI. Test of C-terminal peptide content of type I procollagen in HSF cells

[0186] Type I procollagen synthesized intracellularly is secreted extracellularly. Under the action of endopeptidase, the propeptides attached to its N-terminus and C-terminus are cleaved, forming procollagen. These procollagen molecules then polymerize into collagen fibers, constituting the extracellular matrix. The cleaved free propeptides are soluble and can serve as a biochemical indicator reflecting the amount of collagen synthesized in the body. The free propeptide detected in this test is the C-terminal peptide of type I procollagen (PIP).

[0187] Prior to the PIP assay, cell viability was assessed using the MTT assay to determine the safe concentration of tea tree flower extract for HSF cells (human skin fibroblast-like cells) in the examples and comparative studies. HSF cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum and 1% streptomycin / penicillin at 37°C in a cell culture incubator containing 5% CO2. The medium was changed and passaged regularly, and cells in the logarithmic growth phase were used for experiments. HSF cells were cultured at 8 × 10⁸ cells / year. 3Seeds were seeded at a density of [number] samples per well in 96-well plates and cultured for 24 h. High-glucose medium containing 0.625% concentration of tea tree flower extract from the examples and comparative examples was added to each well of the sample group. The positive control group received high-glucose medium containing 100 ng / mL hEGF (human epidermal growth factor), and the control group received 100 μL of high-glucose medium per well. After drug administration, the 96-well plates were incubated at 37°C and 5% CO2 for 48 h. After incubation, the supernatant was collected, and the content of type I procollagen C-terminal peptide (PIP) was detected using the Procollagen type I C-peptide (PIP) EIA Kit (Takara) at 4°C, 12000 rpm, and 10 min. The test results are shown in [details omitted]. Figure 3 .

[0188] like Figure 3 In the results of the type I procollagen C-terminal peptide (PIP) content detection, compared with the blank control, the PIP secretion of HSF cells was significantly increased after treatment with the positive drug 100 ng / mL hEGF. The 0.625% concentration of tea tree flower extract in the examples significantly increased the PIP content of HSF cells after treatment for 48 h, indicating its efficacy in promoting type I collagen synthesis. Examples 2 and 3 showed the best effects, and overall, the tea tree flower extracts in Examples 1-5 were more effective than those in Comparative Examples 1-4.

[0189] VII. UVB-induced MMP-1 content assay in HSF cells

[0190] Human skin fibroblasts (HSF) in the logarithmic growth phase were collected and a cell suspension was prepared. The suspension was then diluted with 5 × 10⁻⁶ cells. 4 Seeds were planted at a density of cells / well in 24-well plates. After 24 hours of culture, all wells except the control group were subjected to UVB irradiation at a dose of 25 mJ / cm². 2 After irradiation, each well of the sample group was added with high-glucose medium containing 0.625% of the tea tree flower extract samples from the examples and comparative examples, while each well of the control and irradiation groups was added with 500 μL of high-glucose medium. After drug administration, the 24-well plates were incubated at 37°C and 5% CO2. After 24 hours of incubation, the culture medium was collected, and the MMP-1 content in the culture medium was detected using the Human Total MMP-1 (R&D Systems) kit instructions. The test results are shown below. Figure 4 .

[0191] like Figure 4The MMP-1 content detection results shown indicate that, compared with the blank control, UVB irradiation significantly increased the secretion of MMP-1 in HSF cells, indicating successful model construction. After 24 hours of treatment with 0.625% concentration of tea tree flower extract in the examples, the MMP-1 content of HSF cells significantly decreased, demonstrating the effect of reducing abnormal degradation of the extracellular matrix. Examples 2 and 3 showed the best results, and overall, the tea tree flower extracts in Examples 1-5 were more effective than those in Comparative Examples 1-4.

[0192] VIII. Safety Testing of Skin Patches

[0193] Following the skin occlusive patch test method in the "Cosmetic Safety Technical Specifications" (2015 edition), 31 eligible subjects aged 20-55 years were selected. The tea tree flower extracts from Examples 1 and 3 were prepared into sample solutions with mass fractions of 1%, 2%, and 5% respectively using distilled water. A 50mm² area was selected as the sample solution. 2 A patch testing device with a depth of approximately 1 mm was used. 20 μL of sample solution was added to each compartment of the patch testing device, with the blank control group consisting of an equal volume of distilled water. The patch testing device containing the test substance was applied to the flexor side of the subject's forearm using hypoallergenic adhesive tape, and gently pressed with the palm of the hand to ensure even application to the skin, for 24 hours. After removing the patch testing device, skin reactions were observed at 30 minutes (after the indentation disappeared), 24 hours, and 48 hours according to the standards in Table 3, and the results were recorded. The grading criteria for skin reactions in the closed patch test are shown in Table 3, and the results of the human closed patch test are shown in Table 4. The results of the skin patch test showed no adverse skin reactions in 31 participants, indicating that the tea tree flower extract provided by this invention has excellent safety.

[0194] Table 3

[0195]

[0196] Table 4

[0197]

[0198] IX. Clinical Anti-aging Tests

[0199] Sixteen healthy subjects were selected. After washing their hands and faces and drying their skin with tissues, the subjects entered the testing environment (constant temperature and humidity: temperature 22±2℃, humidity 50±10%RH) and rested for 20 minutes before entering the test. The sample application method involved applying 2% of the base lotion from Example 1 and Comparative Example 2 to the left and right sides of the face respectively (once in the morning and once in the evening) for 4 weeks. Using the ANTSCI Callegari Soft-PLUS elasticity testing probe, the skin elasticity of the left and right cheekbones was tested at days 0 (D0), 14 (D14), and 28 (D28) (a higher value indicates better surface skin elasticity). Three parallel measurements were taken, and the average value was recorded. The test results are shown below. Figure 5 (Statistical differences in the test data were analyzed simultaneously; *P < 0.05 compared to D0). The base emulsion formulation, by weight percentage, is as follows: 2% tea tree flower extract prepared in Example 1 or Comparative Example 2, 3% soybean oil, 1.5% glyceryl stearate (A165), 0.5% 1618 alcohol, 0.2% carbomer 940, 3% butylene glycol, 0.5% p-hydroxyacetophenone, 0.5% 1,2-hexanediol, pH adjusted to 5.5 with NaOH, and water added to 100%.

[0200] Note: The clinical anti-aging test used a half-face test, where 16 subjects used the product containing Example 1 on one side of their face and the product containing Comparative Example 2 on the other side. All data from all subjects were averaged, and statistical significance was calculated compared to day 0.

[0201] like Figure 5 As shown, when subjects applied a base lotion containing 2% of the tea tree flower extract (Example 1) to their faces, skin elasticity increased at 14 and 28 days, with increases of 6.93% and 9.42%, respectively, both significantly different from the initial level (day 0) (P<0.05). However, after using the base lotion of Comparative Example 2, the change in skin elasticity was slightly higher than the initial level, but not significantly different (P>0.05).

Claims

1. A method for preparing a tea tree flower extract with anti-photoaging properties, characterized in that, The preparation steps include: S1. Pre-treat the tea tree flowers to obtain fine powder of tea tree flowers; S2. Activate the biological enzyme to obtain an activated enzyme solution; S3. Add tea flower powder to the enzyme activating solution and perform one enzymatic hydrolysis to obtain hydrolysate 1; S4. Extract the enzyme hydrolysate 1 to obtain the extract; S5. Filter the extract to obtain liquid 1; S6. Mix the feed solution 1 and biological enzyme 2, and perform a second enzymatic hydrolysis to obtain enzymatic hydrolysate 2; S7. Purify the enzymatic hydrolysate 2 to obtain tea tree flower extract; The bio-enzyme is a bio-complex enzyme, comprising cellulase, pectinase, α-amylase, xylanase, and tanninase in a mass ratio of (0.1-3):(0.1-2):(0.1-4):(0.15-2):(0.05-1.5). The biological enzyme II includes at least one of protease, amylase, lipase, and cellulase; The S4 step specifically includes: heating the enzymatic hydrolysate 1 to 85-95℃ and stirring at 100-200 rpm for 1-3 hours to obtain the extract.

2. The method for preparing the anti-photoaging tea tree flower extract according to claim 1, characterized in that, Based on the total mass of tea flower powder, the bio-complex enzyme is added at the following concentrations: cellulase 0.2%-1.0%, pectinase 0.2%-1.0%, α-amylase 0.2%-1.0%, xylanase 0.2%-1.0%, and tanninase 0.1%-0.5%.

3. The method for preparing the anti-photoaging tea tree flower extract according to claim 1, characterized in that, The S1 step specifically includes: picking fresh tea tree flowers, sorting and removing the calyx and receptacle, drying at low temperature, crushing and sieving to obtain fine tea tree flower powder.

4. The method for preparing the anti-photoaging tea flower extract according to claim 3, characterized in that, The drying method includes at least one of vacuum drying, freeze drying, and oven drying.

5. The method for preparing the anti-photoaging tea tree flower extract according to claim 4, characterized in that, The sieve mesh size is 20-200 mesh.

6. The method for preparing the anti-photoaging tea tree flower extract according to claim 5, characterized in that, The sieve mesh size is 40-80 mesh.

7. A tea tree flower extract prepared by the method according to any one of claims 1-6, characterized in that, The tea tree flower extract contains tea tree flower sugars and tea tree flower plant peptides; The tea tree flower extract was analyzed for peptide sequences, and the number of tea tree flower plant peptides was ≥100.

8. The application of the tea tree flower extract according to claim 7 in the preparation of a skin care composition, characterized in that, The amount of tea tree flower extract added to the skin care composition is 10-30 wt%.

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