Preparation method and application of skin tightening and wrinkle-fighting leucadendron extract

Abstract

The present application relates to the field of cosmetic technology, and discloses a preparation method and application of a leucothoe fontanesiana extract for skin tightening and wrinkle resistance. The steps of the preparation method comprise: S1: mixing leucothoe fontanesiana powder and water, placing them in a sealed container, filling in carbon dioxide, and then performing instantaneous pressure relief and synchronous temperature rise; S2: adding a tannin capturing agent to the product of S1 to capture tannins, and then adding soybean phospholipids to perform homogenization treatment, wherein the tannin capturing agent comprises gelatin and / or weak cationic polysaccharide; S3: performing alternating double-frequency ultrasonic extraction on the product of S2 at 65-70 DEG C, and the ultrasonic frequency is 20-24 kHz and 45-50 kHz. By using the preparation method, the leucothoe fontanesiana extract obtained not only has good tightening and wrinkle resistance function itself, but also can produce good synergistic effect with collagen when used in combination with collagen, thereby effectively improving the tightening and wrinkle resistance effect of collagen.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to a method for preparing and applying a skin-firming and anti-wrinkle extract of *Chimonanthus praecox*. Background Technology

[0002] The core characteristics of skin aging (especially photoaging) are abnormal collagen degradation caused by the overexpression of matrix metalloproteinases (MMPs) and a weakening of fibroblast synthesis capacity. Ultraviolet radiation induces excessive generation of reactive oxygen species (ROS), which not only directly causes oxidative damage to biomolecules but also leads to the specific degradation of dermal collagen and elastin by activating MMPs (such as MMP-1 and MMP-3). Simultaneously, the decreased proliferation and synthesis capacity of fibroblasts results in a negative metabolic balance where the degradation rate exceeds the synthesis rate, ultimately manifesting as skin laxity and deepening wrinkles.

[0003] Currently, collagen is widely used for firming and anti-wrinkle purposes, but its practical application faces significant bottlenecks: Firstly, its stability is poor; exogenously supplemented collagen is easily hydrolyzed in the high-level MMPs environment of the skin, resulting in a short residence time and difficulty in forming lasting structural support. Secondly, the microenvironment is limiting; aging skin is in a state of oxidative stress, and simply supplementing raw materials cannot reverse the metabolic passivation of fibroblasts, resulting in collagen being supplemented but not produced, leading to low overall repair efficiency. Therefore, how to effectively supplement collagen while inhibiting its degradation and optimizing the skin's regenerative microenvironment is a technical problem that urgently needs to be solved to improve anti-wrinkle efficacy.

[0004] Patent TWI734058B discloses the use of *Chimonanthus praecox* extract for delaying skin cell aging, mentioning that the extract obtained by water extraction can delay skin aging by increasing mitochondrial activity. However, while *Chimonanthus praecox* extract obtained by such conventional extraction methods has a certain effect on delaying skin aging, its effect on improving collagen firmness and anti-wrinkle properties is not ideal. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for preparing and applying a skin-firming and anti-wrinkle extract of *Chimonanthus praecox*. Using the preparation method of this invention, the obtained *Chimonanthus praecox* extract not only possesses good firming and anti-wrinkle functions on its own, but also exhibits a good synergistic effect with collagen when used in combination, effectively enhancing the firming and anti-wrinkle efficacy of collagen.

[0006] The specific technical solution of this invention is as follows: In a first aspect, the present invention provides a method for preparing a skin-firming and anti-wrinkle extract of *Chimonanthus praecox*, comprising the following steps: S1: Mix the willow-leaf wintersweet powder with water, place it in a sealed container, fill it with carbon dioxide, and then perform instantaneous depressurization and simultaneous heating. S2: A tannin-capturing agent is added to the product of S1 to capture tannins, followed by homogenization with soybean lecithin; the tannin-capturing agent includes gelatin and / or cationic polysaccharides; S3: Extract the product from S2 by alternating dual-frequency ultrasound at 65~70℃, with ultrasound frequencies of 20~24kHz and 45~50kHz.

[0007] Using the preparation method of the present invention, the prepared willow-leaf wintersweet extract can play a better role in the following two dimensions, thereby giving it a better firming and anti-wrinkle effect, and at the same time, it can also have a greater effect on the firming and anti-wrinkle effect of collagen: (1) The willow-leaf wintersweet extract obtained by the present invention can better inhibit the expression of matrix metalloproteinase 1 (MMP-1) and matrix metalloproteinase 3 (MMP-3), thereby constructing a low enzyme activity environment in the dermis to reduce the enzymatic hydrolysis rate of collagen, prolonging its residence time and physical support in the skin tissue; (2) The willow-leaf wintersweet extract obtained by the present invention can better remove reactive oxygen species (ROS), thereby relieving the oxidative inhibition state of fibroblasts, increasing the sensitivity of cells to collagen-mediated integrin receptor and transforming growth factor-β (TGF-β) signals, thereby accelerating endogenous collagen regeneration, and finally establishing a positive metabolic balance of "exogenous filling and endogenous promotion".

[0008] Specifically, in the preparation method of the present invention, the following design helps to improve the effects of the willow-leaf wintersweet extract in the above two dimensions: (1) In step S1, the instantaneous depressurization and simultaneous heating after CO2 is introduced helps to completely inactivate the endogenous protease in the willow-leaf wax plum, prevent it from degrading the exogenous collagen in the compound system, and ensure the structural integrity and stability of the collagen molecules.

[0009] (2) In step S2, by adding gelatin and / or cationic polysaccharides, the macromolecular high tannins present in the willow-leaf plum can be selectively captured, so as to avoid the macromolecular high tannins from binding with collagen and affecting the triple helix spatial structure of collagen, thus weakening the firming and anti-wrinkle effect of collagen.

[0010] (3) In steps S1 and S2, the operation of instantaneous depressurization and synchronous heating → tannin capture → addition of soybean lecithin was adopted. This specific sequential design can produce the following effects: by instantaneously depressurizing and synchronously heating after CO2 is introduced, the cell walls of *Chimonanthus praecox* can be ruptured, releasing intracellular substances. After that, the tannin capture agent is added, which helps to make the tannin capture agent fully exert its function, achieve efficient capture of macromolecular high-polymer tannins, thereby reducing the impact of macromolecular high-polymer tannins on the efficacy of exogenous collagen, while protecting the potential of *Chimonanthus praecox* extract to promote collagen synthesis. In addition, the addition of soybean lecithin after the tannin capture is completed can, on the one hand, avoid the tannins in the system from interfering with the targeted encapsulation of fat-soluble terpenes and flavonoid aglycones by soybean lecithin, thereby using the encapsulation of soybean lecithin to promote the delivery and absorption of the above-mentioned firming and anti-wrinkle functional components in the body; on the other hand, it can also avoid soybean lecithin from hindering the capture efficiency of macromolecular high-polymer tannins, thereby better avoiding the impact of macromolecular high-polymer tannins on the firming and anti-wrinkle effect of collagen.

[0011] (4) In step S3, alternating dual-frequency ultrasonic extraction is performed using specific temperature and ultrasonic frequency, which can directionally enrich high-purity oligoflavones and monomeric phenolic acids. These two types of components can play a good role in clearing ROS and inhibiting MMPs. However, if the temperature and ultrasonic frequency are not designed properly during the alternating dual-frequency ultrasonic extraction process, the extraction and enrichment effect of oligoflavones and monomeric phenolic acids may be weakened.

[0012] Optionally, in step S1, the specific process of filling with carbon dioxide includes: introducing carbon dioxide until the pressure inside the sealed container reaches 1.5~2.0MPa, heating to 55~60℃, and stirring for 30~40min.

[0013] Optionally, in step S1, the specific process of instantaneous depressurization and simultaneous heating includes: depressurizing to atmospheric pressure at a rate of 0.5~0.6MPa / s, starting heating at the same time as depressurization, raising the temperature to 95~100℃ within 25~30s, and then maintaining it for 2~5min.

[0014] Optionally, in step S2, before adding the tannin scavenging agent, the product of S1 is cooled to 30~35℃; the amount of the tannin scavenging agent is 0.15~0.3% of the willow-leaf wax plum powder; the positively charged group in the cationic polysaccharide is a quaternary ammonium group, and the degree of substitution of the quaternary ammonium group is 0.05~0.5, or the positively charged group in the cationic polysaccharide is a primary amino group; the specific process of tannin scavenging is stirring at 100~160 rpm for 15~20 min.

[0015] Sodium hyaluronate is a commonly used raw material in cosmetics. When the selected cationic polysaccharide uses a primary amino group as the positively charged group, the charge of the primary amino group is pH-dependent, and it is only partially protonated in the weakly acidic environment of the skin. Therefore, it can avoid strong electrostatic binding between the cationic polysaccharide and sodium hyaluronate, forming a water-insoluble polyelectrolyte complex precipitate. When the selected cationic polysaccharide uses a quaternary ammonium group as the positively charged group, controlling the degree of substitution of the quaternary ammonium group in the cationic polysaccharide to 0.05~0.5 also helps to avoid the formation of macroscopic precipitation by binding between the cationic polysaccharide and sodium hyaluronate. In summary, the present invention preferably uses cationic polysaccharides with "a quaternary ammonium group as the positively charged group, and a degree of substitution of the quaternary ammonium group of 0.05~0.5" or "a primary amino group as the positively charged group". This can prevent strong electrostatic binding between the cationic polysaccharide and sodium hyaluronate, thus avoiding macroscopic precipitation when the willow-leaf wintersweet extract is used in cosmetics containing sodium hyaluronate, thereby avoiding negative impacts on the stability of the cosmetic and the efficacy of sodium hyaluronate.

[0016] Optionally, in step S2, the cationic polysaccharide includes one or more of chitosan, cationic hydroxyethyl cellulose, and guar hydroxypropyltrimethylammonium chloride.

[0017] Optionally, in step S2, the amount of soybean lecithin used is 0.2~0.4wt% of the willow-leaf wax plum powder; the rotation speed of the homogenization treatment is 18000~20000rpm, the time is 15~20min, and the temperature of the liquid is ≤30℃ during the process.

[0018] Optionally, in step S1, the willow-leaf wintersweet powder is powder of the stems and / or leaves of the willow-leaf wintersweet, with a particle size not greater than 200 μm, and the mass-volume ratio of the willow-leaf wintersweet powder to water is 1 kg: 10~14 L.

[0019] Optionally, in step S3, the duration of the alternating dual-frequency ultrasound extraction is 30-40 minutes, during which the ultrasound frequency is switched every 3-5 seconds, and the ultrasound power is 750-850W.

[0020] Optionally, after step S3, the product of S3 is subjected to solid-liquid separation, and the separated liquid is vacuum concentrated and freeze-dried.

[0021] Optionally, in step S1, the preparation steps of the willow-leaf wintersweet powder include: soaking the dried stems and / or leaves of the willow-leaf wintersweet in liquid nitrogen, then crushing and sieving them.

[0022] Secondly, the present invention provides the application of willow-leaf wintersweet extract in the preparation of skin-firming and anti-wrinkle preparations, wherein the willow-leaf wintersweet extract is prepared by the preparation method described above.

[0023] Optionally, the skin-firming and anti-wrinkle preparation includes willow-leaf wintersweet extract and collagen.

[0024] Optionally, the mass ratio between the willow-leaf wintersweet extract and collagen is 1:10~30; the collagen includes recombinant humanized type I collagen and / or recombinant humanized type III collagen.

[0025] Optionally, the skin-firming and anti-wrinkle preparation is a cosmetic, wherein the total content of willow-leaf wax plum extract and collagen is 0.1~8wt%.

[0026] Thirdly, the present invention provides a method for improving the skin firming and anti-wrinkle effects of collagen by combining willow-leaf wintersweet extract with collagen and preparing the willow-leaf wintersweet extract using the preparation method described above.

[0027] Traditional methods for preparing *Chimonanthus praecox* extract make it difficult to form an effective synergy with collagen. However, the preparation method of this invention can enhance the effect of *Chimonanthus praecox* extract on collagen efficacy, thereby achieving an effective synergy. This results in a firming and anti-wrinkle effect when the two are combined, which is superior to that of a single component.

[0028] Compared with the prior art, the present invention has the following advantages: The preparation method of this invention enables the obtained willow-leaf wintersweet extract to have good effects in inhibiting MMPs and scavenging ROS. By downregulating MMPs, it can provide degradation protection for collagen, and by scavenging ROS, it can enhance the collagen synthesis pathway, thereby improving the firming and anti-wrinkle effects of the willow-leaf wintersweet extract itself, and enhancing its synergistic effect on the firming and anti-wrinkle effects of collagen. Attached Figure Description

[0029] Figure 1 The skin condition of two subjects in experimental group 1 in test case 3 on day 0 and day 28. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments.

[0031] In a first aspect, the present invention relates to a method for preparing a skin-firming and anti-wrinkle extract of *Chimonanthus praecox*, comprising the steps of: S1: Mix the willow-leaf wintersweet powder with water, place it in a sealed container, fill it with carbon dioxide, and then perform instantaneous depressurization and simultaneous heating. S2: A tannin-capturing agent is added to the product of S1 to capture tannins, followed by homogenization with soybean lecithin; the tannin-capturing agent includes gelatin and / or cationic polysaccharides; S3: Extract the product from S2 by alternating dual-frequency ultrasound at 65~70℃, with ultrasound frequencies of 20~24kHz and 45~50kHz.

[0032] In some specific embodiments, in step S1, the willow-leaf wintersweet powder is powder of the stems and / or leaves of the willow-leaf wintersweet, with a particle size not greater than 200 μm.

[0033] In some specific embodiments, in step S1, the mass-to-volume ratio of the willow-leaf wintersweet powder to water is 1 kg: 10~14 L.

[0034] In some specific embodiments, step S1, the preparation step of the willow-leaf wintersweet powder includes: soaking the dried stems and / or leaves of the willow-leaf wintersweet in liquid nitrogen, then pulverizing and sieving. Optionally or preferably, the mass-to-volume ratio of the dried stems and / or leaves of the willow-leaf wintersweet to the liquid nitrogen is 1 kg: 5~8 L; the soaking time is 15~20 min; the pulverization process specifically includes: pulverizing at 23000~27000 rpm in a high-speed pulverizer for 1~3 min, repeated 2~3 times.

[0035] In some specific embodiments, the specific process of filling carbon dioxide in step S1 includes: introducing carbon dioxide until the pressure inside the sealed container reaches 1.5~2.0MPa, heating to 55~60℃, and stirring for 30~40min.

[0036] In some specific embodiments, the instantaneous depressurization and simultaneous heating process in step S1 includes: depressurizing to atmospheric pressure at a rate of 0.5~0.6MPa / s, starting heating at the same time as depressurization, raising the temperature to 95~100℃ within 25~30s, and then maintaining it for 2~5min.

[0037] In some specific embodiments, in step S2, the product of S1 is cooled to 30~35°C before adding the tannin scavenging agent.

[0038] In some specific embodiments, in step S2, the amount of the tannin scavenging agent is 0.15~0.3% of the willow-leaf wax plum powder.

[0039] In some specific embodiments, in step S2, the positively charged group in the cationic polysaccharide is a quaternary ammonium group, and the degree of substitution of the quaternary ammonium group in the cationic polysaccharide is 0.05~0.5. In other specific embodiments, in step S2, the positively charged group in the cationic polysaccharide is a primary amino group.

[0040] In some specific embodiments, in step S2, the cationic polysaccharide includes one or more of chitosan, cationic hydroxyethyl cellulose, and guar hydroxypropyltrimethylammonium chloride.

[0041] In some specific embodiments, in step S2, the specific process of capturing tannins involves stirring at 100-160 rpm for 15-20 minutes.

[0042] In some specific embodiments, in step S2, the amount of soybean lecithin used is 0.2~0.4wt% of the willow-leaf wax plum powder; the homogenization treatment speed is 18000~20000rpm, the time is 15~20min, and the temperature of the liquid is ≤30℃ during the process.

[0043] In some specific embodiments, in step S3, the duration of the alternating dual-frequency ultrasound extraction is 30-40 minutes, during which the ultrasound frequency is switched every 3-5 seconds, and the ultrasound power is 750-850W.

[0044] In some specific embodiments, after step S3, the product of S3 is subjected to solid-liquid separation, and the separated liquid is vacuum concentrated and freeze-dried. Optionally or preferably, the solid-liquid separation is performed by ion exchange or gauze filtration; the vacuum concentration is performed at a vacuum degree < -0.08 MPa and a temperature of 50~60℃, concentrating to 1 / 5~1 / 10 of the original volume; the freeze-drying process includes: pre-freezing to -50~-40℃, followed by sublimation drying at a vacuum degree <10 Pa for 36~48 hours.

[0045] Secondly, the present invention relates to the application of willow-leaf wintersweet extract in the preparation of skin-firming and anti-wrinkle preparations, wherein the willow-leaf wintersweet extract is prepared by the preparation method described above.

[0046] In some specific embodiments, the skin-firming and anti-wrinkle preparation includes *Chimonanthus praecox* extract and collagen. Optionally or preferably, the mass ratio of the *Chimonanthus praecox* extract to collagen is 1:10-30; the collagen includes recombinant humanized type I collagen and / or recombinant humanized type III collagen.

[0047] In some specific embodiments, the total content of willow-leaf wax plum extract and collagen in the skin-firming and anti-wrinkle preparation is 0.1~8wt%.

[0048] In some specific embodiments, the skin-firming and anti-wrinkle preparation is a cosmetic, such as a toner, lotion, serum, cream, face cream, eye cream, mask liquid, or ampoule essence.

[0049] Thirdly, the present invention relates to a method for improving the skin firming and anti-wrinkle effects of collagen, which involves combining willow-leaf wintersweet extract with collagen and preparing the willow-leaf wintersweet extract using the preparation method described above.

[0050] The present invention will now be described through specific embodiments and comparative examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0051] Example 1: Preparation of Willow Leaf Chimonanthus Extract This embodiment prepares the willow-leaf wintersweet extract according to the following steps: S1: Preparation of Willow Leaf Winter Jasmine Powder S1.1: Place 1 kg of dried willow-leaf wintersweet leaves in a stainless steel freezing container, slowly pour in 7 L of liquid nitrogen to ensure complete immersion, and let stand for 18 minutes to obtain brittle leaves.

[0052] S1.2: The brittle leaves are transferred in batches into a high-speed pulverizer and pulverized at 25,000 rpm for 1 minute. This process is repeated 3 times to obtain uniform ultrafine powder. The powder is then passed through an 80-mesh sieve to obtain willow-leaf wintersweet powder.

[0053] S2: CO2-assisted enzyme inactivation S2.1: Add the willow-leaf wintersweet powder to deionized water at a ratio of 1kg:13L, transfer the resulting mixture into a high-pressure reactor, introduce CO2 until the pressure inside the reactor reaches 1.5MPa, adjust the temperature to 60℃, and stir for 30min.

[0054] S2.2: Depressurize to atmospheric pressure at a rate of 0.5 MPa / s, and start heating at the same time as depressurization. Instantly heat the liquid to 100°C within 30 seconds and maintain the temperature for 2 minutes.

[0055] S3: Tannin capture and selective membrane phase separation S3.1: Quickly cool the liquid after step S2 to 30°C, add gelatin at 0.2 wt% of the willow-leaf wax plum powder, and stir slowly at 150 rpm for 15 min.

[0056] S3.2: Add enzymatically hydrolyzed soybean lecithin (purchased from Sichuan Century Star Biotechnology) at a rate of 0.3 wt% of the amount of willow-leaf wax plum powder. Transfer the mixture to a homogenizer and homogenize continuously at 19,000 rpm for 15 minutes, while controlling the temperature of the liquid to not exceed 30°C.

[0057] S4: Alternating dual-frequency ultrasound extraction After completing step S3, the liquid was transferred into an ultrasonic extraction tank. The temperature inside the tank was kept constant at 70℃. The extraction was performed using alternating dual-frequency ultrasonic extraction at 24kHz and 45kHz for 30 minutes. The ultrasonic power was 800W. The ultrasonic frequency was switched every 3 seconds during the extraction.

[0058] S5: Solid-liquid separation and drying S5.1: Centrifuge the liquid after step S4 at 4000 rpm for 15 min and collect the supernatant.

[0059] S5.2: Transfer the supernatant into a rotary evaporator, maintain the vacuum degree below -0.08MPa, and the water bath temperature at 55℃, concentrate to 1 / 7 of the original volume to obtain an extract.

[0060] S5.3: Spread the extract in a freeze-drying pan, pre-freeze to -40°C, then maintain the vacuum degree below 10Pa and sublimate dry for 48 hours to obtain the willow-leaf wax plum extract.

[0061] Example 2: Preparation of Willow Leaf Chimonanthus Extract This embodiment prepares the willow-leaf wintersweet extract according to the following steps: S1: Preparation of Willow Leaf Winter Jasmine Powder S1.1: Place 1 kg of dried willow-leaf wintersweet leaves in a stainless steel freezing container, slowly pour in 7 L of liquid nitrogen to ensure complete immersion, and let stand for 18 minutes to obtain brittle leaves.

[0062] S1.2: The brittle leaves are transferred in batches into a high-speed pulverizer and pulverized at 25,000 rpm for 1 minute. This process is repeated 3 times to obtain uniform ultrafine powder. The powder is then passed through an 80-mesh sieve to obtain willow-leaf wintersweet powder.

[0063] S2: CO2-assisted enzyme inactivation S2.1: Add the willow-leaf wintersweet powder to deionized water at a ratio of 1kg:13L, transfer the resulting mixture into a high-pressure reactor, introduce CO2 until the pressure inside the reactor reaches 2.0MPa, adjust the temperature to 60℃, and stir for 30min.

[0064] S2.2: Depressurize to atmospheric pressure at a rate of 0.5 MPa / s, and start heating at the same time as depressurization. Instantly heat the liquid to 100°C within 30 seconds and maintain the temperature for 2 minutes.

[0065] S3: Tannin capture and selective membrane phase separation S3.1: Quickly cool the liquid after step S2 to 30°C, add chitosan at a rate of 0.3 wt% of the willow-leaf wax plum powder, and stir slowly at 100 rpm for 20 min.

[0066] S3.2: Add enzymatically hydrolyzed soybean lecithin (purchased from Sichuan Century Star Biotechnology) at a rate of 0.2 wt% of the amount of willow-leaf wax plum powder. Transfer the mixture to a homogenizer and homogenize continuously at 18,000 rpm for 20 minutes, while controlling the temperature of the liquid to not exceed 30°C.

[0067] S4: Alternating dual-frequency ultrasound extraction After completing step S3, the liquid was transferred into an ultrasonic extraction tank. The temperature inside the tank was kept constant at 70℃. The extraction was performed using alternating dual-frequency ultrasonic extraction at 22kHz and 48kHz for 30 minutes. The ultrasonic power was 750W. The ultrasonic frequency was switched every 4 seconds during the extraction.

[0068] S5: Solid-liquid separation and drying S5.1: Centrifuge the liquid after step S4 at 4000 rpm for 15 min and collect the supernatant.

[0069] S5.2: Transfer the supernatant into a rotary evaporator, maintain the vacuum degree below -0.08MPa, and the water bath temperature at 55℃, concentrate to 1 / 7 of the original volume to obtain an extract.

[0070] S5.3: Spread the extract in a freeze-drying pan, pre-freeze to -40°C, then maintain the vacuum degree below 10Pa and sublimate dry for 48 hours to obtain the willow-leaf wax plum extract.

[0071] Example 3: Preparation of Willow Leaf Chimonanthus Extract This embodiment prepares the willow-leaf wintersweet extract according to the following steps: S1: Preparation of Willow Leaf Winter Jasmine Powder S1.1: Place 1 kg of dried willow-leaf wintersweet leaves in a stainless steel freezing container, slowly pour in 7 L of liquid nitrogen to ensure complete immersion, and let stand for 18 minutes to obtain brittle leaves.

[0072] S1.2: The brittle leaves are transferred in batches into a high-speed pulverizer and pulverized at 25,000 rpm for 1 minute. This process is repeated 3 times to obtain uniform ultrafine powder. The powder is then passed through an 80-mesh sieve to obtain willow-leaf wintersweet powder.

[0073] S2: CO2-assisted enzyme inactivation S2.1: Add the willow-leaf wintersweet powder to deionized water at a ratio of 1kg:13L, transfer the resulting mixture into a high-pressure reactor, introduce CO2 until the pressure inside the reactor reaches 1.5MPa, adjust the temperature to 55℃, and stir for 40min.

[0074] S2.2: Depressurize to atmospheric pressure at a rate of 0.6 MPa / s, and start heating at the same time as depressurization. Instantly heat the liquid to 95°C within 25 seconds and maintain the temperature for 5 minutes.

[0075] S3: Tannin capture and selective membrane phase separation S3.1: Quickly cool the liquid after step S2 to 30°C, add guar gum hydroxypropyltrimethylammonium chloride (quaternary ammonium group substitution degree is 0.1), the amount added is 0.15wt% of the willow-leaf wax plum powder, and stir slowly at 160rpm for 15min.

[0076] S3.2: Add enzymatically hydrolyzed soybean lecithin (purchased from Sichuan Century Star Biotechnology) at a rate of 0.4 wt% of the amount of willow-leaf wax plum powder. Transfer the mixture to a homogenizer and homogenize continuously at 20,000 rpm for 15 minutes, while controlling the temperature of the liquid to not exceed 30°C.

[0077] S4: Alternating dual-frequency ultrasound extraction After completing step S3, the liquid was transferred into an ultrasonic extraction tank. The temperature inside the tank was kept constant at 65℃. The extraction was performed using alternating dual-frequency ultrasonic extraction at 20kHz and 50kHz for 40 minutes. The ultrasonic power was 850W. The ultrasonic frequency was switched every 5 seconds during the extraction.

[0078] S5: Solid-liquid separation and drying S5.1: Centrifuge the liquid after step S4 at 4000 rpm for 15 min and collect the supernatant.

[0079] S5.2: Transfer the supernatant into a rotary evaporator, maintain the vacuum degree below -0.08MPa, and the water bath temperature at 55℃, concentrate to 1 / 7 of the original volume to obtain an extract.

[0080] S5.3: Spread the extract in a freeze-drying pan, pre-freeze to -40°C, then maintain the vacuum degree below 10Pa and sublimate dry for 48 hours to obtain the willow-leaf wax plum extract.

[0081] Comparative Example 1: Preparation of Willow Leaf Chimonanthus Extract In this comparative example, after obtaining the *Chimonanthus praecox* powder according to the method in Example 1, the *Chimonanthus praecox* extract was prepared using a conventional water extraction method. The specific steps are as follows: S1: Preparation of Willow Leaf Winter Jasmine Powder S1.1: Place 1 kg of dried willow-leaf wintersweet leaves in a stainless steel freezing container, slowly pour in 7 L of liquid nitrogen to ensure complete immersion, and let stand for 18 minutes to obtain brittle leaves.

[0082] S1.2: The brittle leaves are transferred in batches into a high-speed pulverizer and pulverized at 25,000 rpm for 1 minute. This process is repeated 3 times to obtain uniform ultrafine powder. The powder is then passed through an 80-mesh sieve to obtain willow-leaf wintersweet powder.

[0083] S2: Water extraction Add the willow-leaf wintersweet powder to deionized water at a ratio of 1 kg: 20 L, heat under reflux at 100 °C for 0.5 h, and then cool to room temperature.

[0084] S3: Solid-liquid separation and drying S3.1: Centrifuge the liquid after step S2 at 4000 rpm for 15 min and collect the supernatant.

[0085] S3.2: Transfer the supernatant into a rotary evaporator, maintain the vacuum degree below -0.08MPa, and the water bath temperature at 55℃, concentrate to 1 / 7 of the original volume to obtain an extract.

[0086] S3.3: Spread the extract in a freeze-drying pan, pre-freeze to -40°C, then maintain the vacuum degree below 10Pa and sublimate dry for 48 hours to obtain the willow-leaf wax plum extract.

[0087] Comparative Example 2: Preparation of Willow Leaf Chimonanthus Extract The only difference between this comparative example and Example 3 is that guar gum hydroxypropyltrimethylammonium chloride was not added in this comparative example for tannin capture; all other raw materials and steps are the same as in Example 3. The specific steps for preparing the willow-leaf wintersweet extract in this comparative example are as follows: S1: Preparation of Willow Leaf Winter Jasmine Powder S1.1: Place 1 kg of dried willow-leaf wintersweet leaves in a stainless steel freezing container, slowly pour in 7 L of liquid nitrogen to ensure complete immersion, and let stand for 18 minutes to obtain brittle leaves.

[0088] S1.2: The brittle leaves are transferred in batches into a high-speed pulverizer and pulverized at 25,000 rpm for 1 minute. This process is repeated 3 times to obtain uniform ultrafine powder. The powder is then passed through an 80-mesh sieve to obtain willow-leaf wintersweet powder.

[0089] S2: CO2-assisted enzyme inactivation S2.1: Add the willow-leaf wintersweet powder to deionized water at a ratio of 1kg:13L, transfer the resulting mixture into a high-pressure reactor, introduce CO2 until the pressure inside the reactor reaches 1.5MPa, adjust the temperature to 55℃, and stir for 40min.

[0090] S2.2: Depressurize to atmospheric pressure at a rate of 0.6 MPa / s, and start heating at the same time as depressurization. Instantly heat the liquid to 95°C within 25 seconds and maintain the temperature for 5 minutes.

[0091] S3: Selective membrane phase separation After completing step S2, the liquid was rapidly cooled to 30°C. Enzymatically hydrolyzed soybean lecithin (purchased from Sichuan Century Star Biotechnology) was added at a rate of 0.4 wt% of the willow-leaf wax plum powder. The mixture was then transferred to a homogenizer and continuously homogenized at 20,000 rpm for 15 minutes, during which the temperature of the liquid was controlled not to exceed 30°C.

[0092] S4: Alternating dual-frequency ultrasound extraction After completing step S3, the liquid was transferred into an ultrasonic extraction tank. The temperature inside the tank was kept constant at 65℃. The extraction was performed using alternating dual-frequency ultrasonic extraction at 20kHz and 50kHz for 40 minutes. The ultrasonic power was 850W. The ultrasonic frequency was switched every 5 seconds during the extraction.

[0093] S5: Solid-liquid separation and drying S5.1: Centrifuge the liquid after step S4 at 4000 rpm for 15 min and collect the supernatant.

[0094] S5.2: Transfer the supernatant into a rotary evaporator, maintain the vacuum degree below -0.08MPa, and the water bath temperature at 55℃, concentrate to 1 / 7 of the original volume to obtain an extract.

[0095] S5.3: Spread the extract in a freeze-drying pan, pre-freeze to -40°C, then maintain the vacuum degree below 10Pa and sublimate dry for 48 hours to obtain the willow-leaf wax plum extract.

[0096] Comparative Example 3: Preparation of Willow Leaf Chimonanthus Extract The only difference between this comparative example and Example 3 is that the parameter design in the alternating dual-frequency ultrasonic extraction process has been changed; all other raw materials and steps are the same as in Example 3. The specific steps for preparing the *Chimonanthus praecox* extract in this comparative example are as follows: S1: Preparation of Willow Leaf Winter Jasmine Powder S1.1: Place 1 kg of dried willow-leaf wintersweet leaves in a stainless steel freezing container, slowly pour in 7 L of liquid nitrogen to ensure complete immersion, and let stand for 18 minutes to obtain brittle leaves.

[0097] S1.2: The brittle leaves are transferred in batches into a high-speed pulverizer and pulverized at 25,000 rpm for 1 minute. This process is repeated 3 times to obtain uniform ultrafine powder. The powder is then passed through an 80-mesh sieve to obtain willow-leaf wintersweet powder.

[0098] S2: CO2-assisted enzyme inactivation S2.1: Add the willow-leaf wintersweet powder to deionized water at a ratio of 1kg:13L, transfer the resulting mixture into a high-pressure reactor, introduce CO2 until the pressure inside the reactor reaches 1.5MPa, adjust the temperature to 55℃, and stir for 40min.

[0099] S2.2: Depressurize to atmospheric pressure at a rate of 0.6 MPa / s, and start heating at the same time as depressurization. Instantly heat the liquid to 95°C within 25 seconds and maintain the temperature for 5 minutes.

[0100] S3: Tannin capture and selective membrane phase separation S3.1: Quickly cool the liquid after step S2 to 30°C, add guar gum hydroxypropyltrimethylammonium chloride (quaternary ammonium group substitution degree is 0.1), the amount added is 0.15wt% of the willow-leaf wax plum powder, and stir slowly at 160rpm for 15min.

[0101] S3.2: Add enzymatically hydrolyzed soybean lecithin (purchased from Sichuan Century Star Biotechnology) at a rate of 0.4 wt% of the amount of willow-leaf wax plum powder. Transfer the mixture to a homogenizer and homogenize continuously at 20,000 rpm for 15 minutes, while controlling the temperature of the liquid to not exceed 30°C.

[0102] S4: Alternating dual-frequency ultrasound extraction After completing step S3, the liquid was transferred into an ultrasonic extraction tank. The temperature inside the tank was kept constant at 85℃. The extraction was performed using alternating dual-frequency ultrasonic extraction at 28kHz and 40kHz for 40 minutes. The ultrasonic power was 850W. The ultrasonic frequency was switched every 5 seconds during the extraction.

[0103] S5: Solid-liquid separation and drying S5.1: Centrifuge the liquid after step S4 at 4000 rpm for 15 min and collect the supernatant.

[0104] S5.2: Transfer the supernatant into a rotary evaporator, maintain the vacuum degree below -0.08MPa, and the water bath temperature at 55℃, concentrate to 1 / 7 of the original volume to obtain an extract.

[0105] S5.3: Spread the extract in a freeze-drying pan, pre-freeze to -40°C, then maintain the vacuum degree below 10Pa and sublimate dry for 48 hours to obtain the willow-leaf wax plum extract.

[0106] Comparative Example 4: Preparation of Willow Leaf Chimonanthus Extract The only difference between this comparative example and Example 3 is that this comparative example uses the sequence of enzymatic hydrolysis of soybean lecithin → instantaneous pressure relief and simultaneous heating → tannin capture; all other raw materials and steps are the same as in Example 3. The specific steps for preparing the willow-leaf wintersweet extract in this comparative example are as follows: S1: Preparation of Willow Leaf Winter Jasmine Powder S1.1: Place 1 kg of dried willow-leaf wintersweet leaves in a stainless steel freezing container, slowly pour in 7 L of liquid nitrogen to ensure complete immersion, and let stand for 18 minutes to obtain brittle leaves.

[0107] S1.2: The brittle leaves are transferred in batches into a high-speed pulverizer and pulverized at 25,000 rpm for 1 minute. This process is repeated 3 times to obtain uniform ultrafine powder. The powder is then passed through an 80-mesh sieve to obtain willow-leaf wintersweet powder.

[0108] S2: Add enzymatic hydrolysis of soybean lecithin Add the willow-leaf wintersweet powder to deionized water at a ratio of 1 kg: 13 L, add enzymatically hydrolyzed soybean lecithin (purchased from Sichuan Century Star Biotechnology) at a rate of 0.4 wt% of the willow-leaf wintersweet powder, transfer to a homogenizer, and continuously homogenize at 20,000 rpm for 15 min, during which the temperature of the liquid should not exceed 30℃.

[0109] S3: CO2-assisted enzyme inactivation S3.1: Transfer the liquid material after step S2 into a high-pressure reactor, introduce CO2 until the pressure inside the reactor reaches 1.5 MPa, adjust the temperature to 55℃, and stir for 40 minutes.

[0110] S3.2: Depressurize to atmospheric pressure at a rate of 0.6 MPa / s, and start heating at the same time as depressurization. Instantly heat the liquid to 95°C within 25 seconds and maintain the temperature for 5 minutes.

[0111] S4: Tannin Capture After completing step S3, the liquid was rapidly cooled to 30°C, and guar gum hydroxypropyltrimethylammonium chloride (quaternary ammonium group substitution degree of 0.1) was added at an amount of 0.15 wt% of the willow-leaf wax plum powder. The mixture was then slowly stirred at 160 rpm for 15 min.

[0112] S5: Alternating Dual-Frequency Ultrasonic Extraction After completing step S4, the liquid was transferred into an ultrasonic extraction tank. The temperature inside the tank was kept constant at 65℃. The extraction was performed using alternating dual-frequency ultrasonic extraction at 20kHz and 50kHz for 40 minutes. The ultrasonic power was 850W. The ultrasonic frequency was switched every 5 seconds during the extraction.

[0113] S6: Solid-liquid separation and drying S6.1: Centrifuge the liquid after step S5 at 4000 rpm for 15 min and collect the supernatant.

[0114] S6.2: Transfer the supernatant into a rotary evaporator, maintain the vacuum degree below -0.08MPa, and the water bath temperature at 55℃, concentrate to 1 / 7 of the original volume to obtain an extract.

[0115] S6.3: Spread the extract in a freeze-drying pan, pre-freeze to -40°C, then maintain the vacuum degree below 10Pa and sublimate dry for 48 hours to obtain the willow-leaf wax plum extract.

[0116] Comparative Example 5: Preparation of Willow Leaf Chimonanthus Extract The only difference between this comparative example and Example 3 is that this comparative example uses the sequence of tannin capture → instantaneous depressurization and simultaneous heating → enzymatic hydrolysis of soybean lecithin; all other raw materials and steps are the same as in Example 3. The specific steps for preparing the willow-leaf wintersweet extract in this comparative example are as follows: S1: Preparation of Willow Leaf Winter Jasmine Powder S1.1: Place 1 kg of dried willow-leaf wintersweet leaves in a stainless steel freezing container, slowly pour in 7 L of liquid nitrogen to ensure complete immersion, and let stand for 18 minutes to obtain brittle leaves.

[0117] S1.2: The brittle leaves are transferred in batches into a high-speed pulverizer and pulverized at 25,000 rpm for 1 minute. This process is repeated 3 times to obtain uniform ultrafine powder. The powder is then passed through an 80-mesh sieve to obtain willow-leaf wintersweet powder.

[0118] S2: Tannin scavenger Add the willow-leaf wintersweet powder to deionized water at a ratio of 1 kg: 13 L, add guar gum hydroxypropyltrimethylammonium chloride (quaternary ammonium group substitution degree of 0.1) at an amount of 0.15 wt% of the willow-leaf wintersweet powder, and stir slowly at 160 rpm for 15 min.

[0119] S3: CO2-assisted enzyme inactivation S3.1: Transfer the liquid material after step S2 into a high-pressure reactor, introduce CO2 until the pressure inside the reactor reaches 1.5 MPa, adjust the temperature to 55℃, and stir for 40 minutes.

[0120] S3.2: Depressurize to atmospheric pressure at a rate of 0.6 MPa / s, and start heating at the same time as depressurization. Instantly heat the liquid to 95°C within 25 seconds and maintain the temperature for 5 minutes.

[0121] S4: Selective membrane phase separation After completing step S3, the liquid was rapidly cooled to 30°C. Enzymatic hydrolyzed soybean lecithin (purchased from Sichuan Century Star Biotechnology) was added at a rate of 0.4 wt% of the willow-leaf wax plum powder. The mixture was then transferred to a homogenizer and continuously homogenized at 20,000 rpm for 15 minutes, during which the temperature of the liquid was controlled not to exceed 30°C.

[0122] S5: Alternating Dual-Frequency Ultrasonic Extraction After completing step S4, the liquid was transferred into an ultrasonic extraction tank. The temperature inside the tank was kept constant at 65℃. The extraction was performed using alternating dual-frequency ultrasonic extraction at 20kHz and 50kHz for 40 minutes. The ultrasonic power was 850W. The ultrasonic frequency was switched every 5 seconds during the extraction.

[0123] S6: Solid-liquid separation and drying S6.1: Centrifuge the liquid after step S5 at 4000 rpm for 15 min and collect the supernatant.

[0124] S6.2: Transfer the supernatant into a rotary evaporator, maintain the vacuum degree below -0.08MPa, and the water bath temperature at 55℃, concentrate to 1 / 7 of the original volume to obtain an extract.

[0125] S6.3: Spread the extract in a freeze-drying pan, pre-freeze to -40°C, then maintain the vacuum degree below 10Pa and sublimate dry for 48 hours to obtain the willow-leaf wax plum extract.

[0126] Test Example 1: Yield of Willow Leaf Chimonanthus Extract In each embodiment and comparative example, the yield of the willow-leaf wintersweet extract was calculated, and the results are shown in Table 1.

[0127] Table 1. Effect of different extraction methods on yield

[0128] Test Example 2: In vitro experiment 2.1 Experimental Methods 2.1.1 General Preprocessing Procedure (1) Cell seeding: Human skin fibroblasts in the logarithmic growth phase were seeded at a concentration of 1×10⁻⁶ cells / cells. 5 Seeds were placed in 6-well plates at a density of 5 × 10⁻⁶ cells / well (for DCFH-DA fluorescent probe, ELISA, and β-galactosidase SA-β-gal staining assays) or at a density of 5 × 10⁻⁶ cells / well. 3 Cells were seeded at a density of cells / well in 96-well plates (CCK-8 experiment) and cultured in a CO2 incubator (37°C, 5% CO2) until the cells reached 80% confluence.

[0129] (2) Experimental group setup: Blank control group: Normally cultured cells, without UVB induction and without drug administration; Model group: UVB photoaging induction only, no drug administration; Group A: The test substance was extract of *Chimonanthus praecox* (prepared from Example 1); Group B: The test substance was recombinant humanized type III collagen; Group A+B: The test substance was a homogeneous mixture of willow-leaf wintersweet extract (prepared from Example 1) and recombinant humanized type III collagen in a mass ratio of 20:1. Positive control group (A+C group): The test substance was a homogeneous mixture of willow-leaf wax plum extract (prepared from Example 1) and retinol in a mass ratio of 20:1.

[0130] (3) Model making: After cells reached 80% confluence, the original culture medium was discarded. Except for the blank control group, all other groups were washed with PBS and then subjected to 30 mJ / cm² hydrochloric acid. 2 UVB irradiation was induced and terminated immediately after reaching the total dose. The irradiance (μW / cm²) of the light source at the cell plane was measured using a UVB radiometer. 2 According to the formula "Irradiation time (seconds) = Irradiation dose (mJ / cm²)",... 2 Irradiance (mW / cm) 2 ) Calculate the required time and continue irradiation until the total dose reaches 30 mJ / cm². 2 It will be terminated immediately thereafter.

[0131] (4) Drug administration: After irradiation, the PBS was discarded. The sample groups (group A, group B, group A+B, and group A+C) were treated with maintenance medium containing different concentrations of the test substance (mass concentration gradients of 0.05%, 0.1%, and 0.5%) for 2 h. The blank control group and the model group were cultured with maintenance medium without the test substance.

[0132] 2.1.2 ROS scavenging ability detection (DCFH-DA fluorescent probe method) Detection timing: Detection was performed 24 hours after UVB irradiation and drug administration, following the general pretreatment procedure.

[0133] Detection method: Add 10 μmol / L DCFH-DA probe and incubate at 37℃ in the dark for 20 min. Wash three times with PBS to remove unentered probe. Detect fluorescence intensity (F) using a multi-mode microplate reader (excitation 488nm / emission 525nm). ROS clearance rate (%) = (F...) 模型组 -F 样品组 ) / (F 模型组 -F 空白对照组 The ROS scavenging rate of each sample group was calculated by multiplying the ROS count by 100%.

[0134] 2.1.3 Detection of collagen metabolism-related indicators (ELISA method) Detection timing: Detection was performed 24 hours after UVB irradiation and drug administration, following the general pretreatment procedure.

[0135] Detection method: After collecting the culture supernatant, the cells were centrifuged at 3000 rpm for 10 min at 4℃. The supernatant was then used to detect the contents of MMP-1, MMP-3, and elastin. Cells in the wells after collecting the culture supernatant were washed with ice-cold PBS, and total protein was extracted using RIPA lysis buffer. The total protein concentration was detected by the dioctanine acid method (BCA method) to normalize the target protein content in the supernatant. The results are expressed as the target protein content per unit mass of total protein, and the corrected MMP-1 content (P0.05) is obtained. MMP-1 MMP-3 content (P) MMP-3 ) and elastin content (P EF According to the MMP-1 inhibition rate (%) = (F MMP-1模型组 -F MMP-1样品组 ) / (F MMP-1模型组 -F MMP-1空白对照组 )×100%, MMP-2 inhibition rate (%)=(F MMP-2模型组 -F MMP-2样品组 ) / (F MMP-2模型组 -F MMP-2空白对照组 )×100%, Elastin Enhancement Rate (%)=(FEF样品组 -F EF模型组 ) / (F EF空白对照组 -F EF模型组 The MMP-1 inhibition rate, MMP-2 inhibition rate, and elastin enhancement rate of each sample group were calculated by multiplying the result by 100%.

[0136] 2.1.4 Detection of proliferation and viability of human dermal fibroblasts (CCK-8 assay) This experiment was designed to evaluate the effect of enhancing the proliferation and vitality of core cells involved in collagen synthesis, providing cellular-level support for endogenous collagen synthesis.

[0137] Detection timing: Following the general pretreatment procedure, detection was performed once each at 24h, 48h, and 72h after UVB irradiation and drug administration.

[0138] Detection method: Add 10 μL of CCK-8 reagent to each well, incubate in the dark for 2 hours, and then measure the OD value at 450 nm. Cell viability (%) = (OD 样品组 -OD 空白对照组 ) / (OD 模型组 -OD 空白对照组 The cell viability rate of each sample group was calculated by multiplying the result by 100%.

[0139] 2.1.5 Cellular senescence inhibition assay (β-galactosidase SA-β-gal staining method) This experiment is the gold standard for detecting cell senescence and was used to verify the direct inhibitory effect of the composition on the senescence of dermal fibroblasts, further verifying the anti-aging effect of the composition at the cellular level.

[0140] Timing of detection: Detection was performed 72 hours after UVB irradiation and drug administration, following the general pretreatment procedure.

[0141] Detection method: Remove the culture medium and wash twice with PBS. Fix with 4% paraformaldehyde fixative for 15 min. Discard the fixative, wash again, and add freshly prepared SA-β-gal staining working solution. Incubate at 37℃ without CO2 for 12 h. Calculate the senescent cell positivity rate of each sample group according to the formula: (Model group positive rate - Sample group positive rate) / Model group positive rate × 100%.

[0142] 2.1.6 Assessment of Synergistic Effect Using the Burgi formula, when the actual test results of group A+B (E) are calculated... obs ) greater than the theoretical expected value (E) exp When the effect is determined to be synergistic between component A and component B, it is determined that component A and component B have a synergistic effect. Theoretical expected value (E) exp The formula for calculating E is: exp=(E A +E B )-(E A ×E B ) / 100, where E A and E B The results are for single-component group A and single-component group B, respectively.

[0143] 2.2 Experimental Results 2.2.1 Effects on inhibiting oxidative stress and inflammatory senescent signaling The ROS scavenging rate detection results and synergistic effect judgment results for each sample group are shown in Table 2.

[0144] Table 2. Results of ROS clearance rate detection

[0145] The experimental results show that: (1) At three dosage concentrations of 0.05%, 0.1%, and 0.5%, both Group A (Willow Leaf Chimonanthus extract) and Group B (Recombinant Humanized Type III Collagen) exhibited certain free radical scavenging activity and showed a clear dose-response relationship. Among them, the scavenging rate of Willow Leaf Chimonanthus extract at each concentration was significantly higher than that of recombinant collagen, which verified its efficacy as a natural antioxidant active ingredient.

[0146] (2) Calculate the theoretical expected value (E) of the composition according to the Burgi formula. exp ), and compared with the actual test results (E obs In a comparative study, across all tested concentration ranges, the actual clearance rates of groups A and B were significantly higher than the theoretical expected values, with the most significant synergistic effect observed at a concentration of 0.1% (the measured value was 18.65% higher than the theoretical value). This demonstrates that the extract of *Chimonanthus praecox* and recombinant humanized collagen have a significant synergistic effect in blocking upstream signaling pathways of skin aging, enabling more efficient clearance of oxidative damage through both endogenous and exogenous pathways, thus providing technical support for maintaining the homeostasis of the dermal microenvironment.

[0147] 2.2.2 Effects on inhibiting MMP expression and promoting elastin synthesis The results of MMP-1 inhibition rate, MMP-2 inhibition rate and elastin enhancement rate of each sample group, as well as the results of synergistic effect judgment, are shown in Table 3.

[0148] Table 3 Results of Collagen Metabolism Related Indicators

[0149] The experimental results show that: UVB irradiation induced significant overexpression of MMP-1 and MMP-3 in fibroblasts. In group A+B, at a concentration of 0.1%, the inhibition rate of MMP-1 reached 81.6%, significantly exceeding the theoretical expected value. exp (71.9%), also superior to the positive control group (A+C group). At the elastin synthesis end, recombinant humanized type III collagen played a dominant role in promoting elastin production, while the addition of *Chimonanthus praecox* extract produced a significant non-linear enhancement: at a concentration of 0.5%, the elastin production rate in group A+B reached 86.8%, superior to the single-group combination (groups A and B) and the positive control group (A+C group). This indicates that *Chimonanthus praecox* extract substantially optimizes the fibroblast microenvironment by neutralizing oxidative stress, thereby amplifying the bioactivity of exogenous collagen in the extracellular matrix (ECM) remodeling process.

[0150] 2.2.3 Anti-aging and wrinkle-reducing effects at the cellular level The cell viability test results and synergistic effect judgment results for each sample group are shown in Table 4.

[0151] Table 4. Cell viability test results

[0152] The results of cell senescence inhibition rate detection and synergistic effect judgment for each sample group are shown in Table 5.

[0153] Table 5 Results of Cell Senescence Inhibition Rate Detection

[0154] The experimental results show that: SA-β-gal (acid β-galactosidase) staining revealed that group A+B significantly reduced the proportion of UVB-induced senescent positive cells, and this inhibitory effect was statistically superior to groups A and B (p<0.01). Compared with the positive control group (group A+C), group A+B exhibited similar anti-aging activity while demonstrating better cell compatibility, providing a safer cellular microenvironment for long-term anti-aging.

[0155] 2.2.4 Summary Multidimensional results indicate that the combination of *Chimonanthus praecox* extract and exogenous collagen exhibits a confirmed synergistic effect in both "inhibiting matrix degradation" and "promoting structural protein synthesis." Compared to retinol, a traditional anti-aging ingredient, the composition of *Chimonanthus praecox* extract and exogenous collagen has a higher upper limit of bioactivity in maintaining the structural integrity of the dermal extracellular matrix (ECM) and avoids the potential irritation risks associated with retinol, demonstrating superior potential for firming and anti-wrinkle applications.

[0156] Test Example 3: Human Efficacy Evaluation 3.1 Preparation methods of cosmetics This test case involves preparing different test substances into cosmetics and then evaluating their efficacy on human subjects. The preparation steps for the cosmetics are as follows: (1) Prepare materials according to the formula in Table 6 and mix the corresponding components to obtain phase A, phase B and phase C.

[0157] (2) Heat phase A and phase B to 80°C respectively until completely dissolved. Then add phase B to phase A and homogenize for 2 minutes. Continue stirring and cooling. When the system temperature drops to 40°C, add phase C, stir evenly, and then discharge to obtain cosmetics.

[0158] Table 6 Cosmetic Formulations

[0159] 3.2 Test methods for firming and anti-wrinkle effects Referring to the industry standards "Evaluation Methods for Firming Efficacy of Cosmetics" and "Evaluation Methods for Anti-wrinkle Efficacy of Cosmetics", a randomized, double-blind, controlled trial design was adopted to recruit healthy subjects (aged 35-55 years, with signs of aging such as facial wrinkles and sagging). They were randomly divided into groups of 5 people each. Cosmetics containing the test substance were used as the experimental group, and cosmetics without the test substance were used as the blank control group. The test period was 28 days.

[0160] Before the experiment, the subjects cleaned their faces 30 minutes in advance and sat quietly in a constant temperature and humidity environment (temperature 22±2℃, relative humidity 50±5%). Then, the baseline value of the experimental area was measured and defined (referred to as "D0"). During the experiment, the subjects applied the test sample once in the morning and once in the evening. The index was measured on the 14th day and the 28th day (referred to as "D14" and "D28" respectively).

[0161] Firming and Anti-wrinkle (Skin Elasticity): Using a skin elasticity tester (Cutometer MPA580), the total elasticity (R2 value) and net elasticity (R5 value) of the skin are tested to quantify and verify the macroscopic firming and anti-wrinkle effects brought about by collagen supplementation, degradation inhibition and 3D network remodeling.

[0162] 3.3 Test results of different test substances According to the test substances and their addition amounts in cosmetics as shown in Table 7, a blank control group and each experimental group were set up. The firming and anti-wrinkle efficacy of each group of cosmetics was tested, and the results are shown in Tables 8 and 9 (Tables 8 and 9, “…”). # "" indicates that p < 0.05 compared to D0. ## "Indicates that p < 0.01 compared to D0".

[0163] Table 7 Design of Different Groups

[0164] Table 8 Results of Skin Total Elasticity (R2 value) Test

[0165] Table 9 Results of Skin Net Elasticity (R5 value) Test

[0166] The experimental results show that: (1) Compared with the blank control group, experimental groups 1-4 and 7-8 showed a non-linear effect on improving skin elasticity. Both total skin elasticity (R2 value) and net elasticity (R5 value) were significantly improved. The significant increase in R5 value proved that the combination of willow-leaf wax plum extract and collagen not only worked on the surface but also penetrated deep into the dermis to promote the network remodeling of collagen fibers and elastin, thus clinically manifesting as a reduction in wrinkle depth and an improvement in contour (e.g. Figure 1 (As shown).

[0167] (2) Compared with experimental groups 5 and 6 which used a single component, experimental groups 1-4 which used a combination of two components showed better improvement in total skin elasticity (R2 value) and net elasticity (R5 value), indicating that the willow-leaf wax plum extract of the present invention can synergize with exogenous collagen to achieve better firming and anti-wrinkle effects.

[0168] (3) Comparing the test results of experimental groups 2, 5-6 and 9-10, it can be seen that, compared with the traditional method of preparing willow leaf wax plum extract, the preparation method of the present invention can not only enhance the firming and anti-wrinkle effect of willow leaf wax plum extract itself, but also effectively enhance its synergistic effect with collagen, and produce a better enhancement effect on the firming and anti-wrinkle effect of collagen. Furthermore, when the willow-leaf wintersweet extract prepared using traditional methods was combined with collagen (experimental group 9), the improvement effect on total skin elasticity (R2 value) and net elasticity (R5 value) was not as good as that of collagen alone (experimental group 6). The reason may be that the willow-leaf wintersweet extract prepared using traditional methods has a low content of functional components that can protect exogenous collagen (such as functional components that can inhibit the expression of MMP-1 and MMP-3), which dilutes the efficacy of exogenous collagen when introduced. At the same time, the combination of macromolecular high-polymer tannins with collagen will affect the triple helix spatial structure of collagen, resulting in a weakening of the firming and anti-wrinkle effect of collagen.

[0169] (3) The improvement effect of total skin elasticity (R2 value) and net elasticity (R5 value) in experimental group 8 was significantly better than that in experimental group 11. This indicates that by adding gelatin at a specific stage during the preparation of the willow-leaf plum extract, the prepared willow-leaf plum extract can achieve better firming and anti-wrinkle effects after being combined with collagen. This is because gelatin can preferentially and selectively capture the macromolecular high-polymer tannins present in the willow-leaf plum, and avoid the macromolecular high-polymer tannins in the compound system from binding with exogenous collagen, affecting the triple helix spatial structure of collagen and causing the collagen efficacy to weaken.

[0170] (4) The improvement effect of total skin elasticity (R2 value) and net elasticity (R5 value) in experimental group 8 was significantly better than that in experimental group 12. This indicates that the temperature and ultrasonic frequency design during the alternating dual-frequency ultrasonic extraction process of the willow-leaf wax plum extract will affect the firming and anti-wrinkle effect of the willow-leaf wax plum extract combined with exogenous collagen. This is because these two parameters will affect the effect of targeted enrichment of oligoflavonoids and monomeric phenolic acids, and these two types of components can play a good role in clearing ROS and inhibiting MMPs.

[0171] (5) The improvement effect of total skin elasticity (R2 value) and net elasticity (R5 value) in experimental group 8 was significantly better than that in experimental group 13. This indicates that if the timing of adding soybean lecithin is set before the instantaneous pressure relief and synchronous heating in the preparation process of the willow-leaf wintersweet extract, the firming and anti-wrinkle effect of the willow-leaf wintersweet extract combined with exogenous collagen will be weakened. This is because: although in the order of Comparative Example 4 (adding soybean lecithin → instantaneous pressure relief and synchronous heating → tannin capture), soybean lecithin can be used to pre-encapsulate the heat-sensitive components. To prevent it from failing due to heat during instantaneous pressure relief and simultaneous heating, this invention targets the firming and anti-wrinkle effects. The role of soybean lecithin is to encapsulate fat-soluble, heat-resistant terpenes and flavonoid aglycones, promoting the body's absorption of these firming and anti-wrinkle ingredients. In addition, this invention uses a tannin scavenger to capture tannins. In this case, if the order in Comparative Example 4 is used, the pre-added soybean lecithin will hinder the tannin capture efficiency and cause the soybean lecithin to indiscriminately encapsulate non-target substances, weakening the targeted loading and delivery of fat-soluble anti-wrinkle ingredients.

[0172] (6) The improvement effect of total skin elasticity (R2 value) and net elasticity (R5 value) in experimental group 8 was significantly better than that in experimental group 14. This indicates that if the timing of adding the tannin scavenger is set before the instantaneous depressurization and synchronous heating in the preparation process of the willow-leaf wax plum extract, the firming and anti-wrinkle effect of the willow-leaf wax plum extract combined with exogenous collagen will be weakened. This is because: by performing instantaneous depressurization and synchronous heating after filling with CO2, the cell wall of the willow-leaf wax plum can be ruptured and intracellular substances can be released. Adding the tannin scavenger afterward is conducive to the full play of the tannin scavenger, achieving efficient capture of macromolecular high-polymer tannins, thereby reducing the influence of macromolecular high-polymer tannins on the efficacy of exogenous collagen, while protecting the potential of the willow-leaf wax plum extract to promote collagen synthesis. Furthermore, the firming and anti-wrinkle effect of experimental group 14 was even lower than that of experimental group 11, which did not use a tannin scavenger in the preparation of the willow-leaf wax plum extract. This may be because the tannin scavenger was added too early in experimental group 14, which would hinder the rupture of plant cell walls during the subsequent process of instantaneous depressurization and synchronous heating by charging CO2.

Claims

1. A method for preparing a skin-firming and anti-wrinkle extract of *Chimonanthus praecox*, characterized in that the steps include... include: S1: Mix the willow-leaf wintersweet powder with water, place it in a sealed container, fill it with carbon dioxide, and then perform instantaneous depressurization and simultaneous heating. S2: A tannin-capturing agent is added to the product of S1 to capture tannins, followed by homogenization with soybean lecithin; the tannin-capturing agent includes gelatin and / or cationic polysaccharides; S3: Extract the product from S2 by alternating dual-frequency ultrasound at 65~70℃, with ultrasound frequencies of 20~24kHz and 45~50kHz.

2. The preparation method according to claim 1, characterized in that, In step S1: The specific process of introducing carbon dioxide includes: introducing carbon dioxide until the pressure inside the sealed container reaches 1.5~2.0MPa, heating to 55~60℃, and stirring for 30~40min; The specific process of instantaneous depressurization and simultaneous heating includes: depressurizing to atmospheric pressure at a rate of 0.5~0.6MPa / s, starting heating at the same time as depressurization, raising the temperature to 95~100℃ within 25~30s, and then maintaining it for 2~5min.

3. The preparation method according to claim 1, characterized in that, In step S2, before adding the tannin scavenging agent, the product from S1 is cooled to 30-35°C; the amount of the tannin scavenging agent is 0.15-0.3% of the willow-leaf wax plum powder; the positively charged group in the cationic polysaccharide is a quaternary ammonium group, and the degree of substitution of the quaternary ammonium group is 0.05-0.5, or the positively charged group in the cationic polysaccharide is a primary amino group; the specific process of tannin scavenging is stirring at 100-160 rpm for 15-20 min.

4. The preparation method according to claim 1, characterized in that, In step S2, the amount of soybean lecithin used is 0.2~0.4wt% of the willow-leaf wax plum powder; the homogenization treatment speed is 18000~20000rpm, the time is 15~20min, and the temperature of the liquid is ≤30℃ during the process.

5. The preparation method according to claim 1, characterized in that, In step S1, the willow-leaf wintersweet powder is powder from the stems and / or leaves of the willow-leaf wintersweet, with a particle size not exceeding 200 μm, and the mass-to-volume ratio of the willow-leaf wintersweet powder to water is 1 kg: 10~14 L; in step S3, the duration of the alternating dual-frequency ultrasonic extraction is 30~40 min, during which the ultrasonic frequency is switched every 3~5 s, and the ultrasonic power is 750~850 W; after step S3, the product of S3 is subjected to solid-liquid separation, and the separated liquid is vacuum concentrated and freeze-dried.

6. The application of willow-leaf wintersweet extract in the preparation of skin-firming and anti-wrinkle formulations, characterized in that, The willow-leaf wintersweet extract was prepared using the preparation method described in any one of claims 1 to 5.

7. The application according to claim 6, characterized in that, The skin-firming and anti-wrinkle preparation includes willow-leaf wintersweet extract and collagen.

8. The application according to claim 7, characterized in that, The mass ratio between the willow-leaf wintersweet extract and collagen is 1:10~30; the collagen includes recombinant humanized type I collagen and / or recombinant humanized type III collagen.

9. The application according to claim 7 or 8, characterized in that, The skin-firming and anti-wrinkle preparation is a cosmetic, wherein the total content of willow-leaf wax plum extract and collagen is 0.1~8wt%.

10. A method for improving the skin-firming and anti-wrinkle effects of collagen, characterized in that, The extract of *Chimonanthus praecox* is compounded with collagen and prepared by the preparation method described in any one of claims 1 to 5.

Images