A firming anti-aging composition containing an extract of guettarda speciosa tree bark and its use

Abstract

The application discloses a firming and anti-aging composition containing a tree bark extract of Eucalyptus globulus and application thereof, and belongs to the technical field of cosmetics. The firming and anti-aging composition containing the tree bark extract of Eucalyptus globulus comprises the following components: a tree bark extract of Eucalyptus globulus, an extract of Jasminum mesnyi, an extract of Tremella fuciformis Berk, and a composite polypeptide, wherein the composite polypeptide is composed of carnosine, acetyl tetrapeptide-5 and tripeptide-10 citrulline with a mass ratio of 1:0.4-0.6:0.2-0.6. The firming and anti-aging composition has a good effect of inhibiting elastase and collagenase activity through the synergistic effect among the four components.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to a firming and anti-aging composition containing extract of the bark of the variegated bellflower tree and its application. Background Technology

[0002] With the increasing aging population and worsening environmental pollution, skin aging has become a global concern. Skin aging is mainly manifested as increased wrinkles, decreased elasticity, and the formation of age spots. Its core mechanisms involve free radical oxidative damage, collagen degradation, and damage to the extracellular matrix (ECM) structure. Studies have shown that external factors such as ultraviolet (UV) radiation and air pollutants induce the large-scale generation of reactive oxygen species (ROS), activating the mitogen-activated protein kinase (MAPK) signaling pathway, which in turn upregulates the expression of matrix metalloproteinases (MMPs, such as collagenase MMP-1 and elastase MMP-9), leading to irreversible degradation of collagen and elastic fibers. Furthermore, endogenous factors such as telomere shortening-induced cellular replication senescence and fibroblast functional decline further accelerate this process.

[0003] Currently, anti-aging cosmetics mainly rely on the following technological pathways: (1) antioxidant ingredients (such as vitamin C and coenzyme Q10) reduce oxidative damage by scavenging free radicals; (2) peptide ingredients (such as palmitoyl pentapeptide-4) promote collagen synthesis by activating the TGF-β signaling pathway; and (3) plant extracts (such as asiaticoside) reduce MMP activity by inhibiting the NF-κB pathway. However, single ingredients often have problems such as limited target sites, low bioavailability, or poor stability. In addition, existing technologies have limited synergistic inhibitory effects on collagenase and elastase, making it difficult to achieve multi-level anti-aging.

[0004] Based on the above problems, there is an urgent need to provide a firming and anti-aging composition that has a good inhibitory effect on both collagenase and elastase, so that consumers can choose it. Summary of the Invention

[0005] The purpose of this invention is to provide a firming and anti-aging composition containing extract of the bark of the variegated bellflower tree and its application. This firming and anti-aging composition is formulated with natural plant extracts and complex peptides, has high safety, and has significant firming and anti-aging effects.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a firming and anti-aging composition containing extract of the bark of the variegated bellflower tree, comprising the following components: extract of the bark of the variegated bellflower tree, extract of the flower of Magnolia officinalis, extract of Auricularia auricula-judae, and a complex polypeptide.

[0007] Preferably, the firming and anti-aging composition containing the bark extract of the variegated bellflower tree comprises the following components in parts by weight: Bark extract of the variegated bellflower tree: 1-3 parts; Magnolia flower extract: 0.5-0.7 parts; Golden ear fungus extract: 2-4 parts; Complex polypeptide: 0.3-0.5 parts: The complex polypeptide includes at least one of carnosine, acetyl tetrapeptide-5, tripeptide-10, and citrulline.

[0008] More preferably, the composite polypeptide is composed of carnosine, acetyl tetrapeptide-5, and tripeptide-10 citrulline in a mass ratio of 1:0.4-0.6:0.2-0.6.

[0009] Secondly, the present invention provides the use of the firming and anti-aging composition described in the first aspect in the preparation of cosmetics with firming and anti-aging effects.

[0010] Preferably, the dosage form of the cosmetic with firming and anti-aging effects is any one of face cream, lotion, or serum.

[0011] Thirdly, the present invention provides an emulsion with firming and anti-aging effects, wherein the emulsion with firming and anti-aging effects is composed of the following components by weight percentage: The firming and anti-aging composition containing extract of the bark of the variegated bellflower tree described in the first aspect: 1-3 wt%; Moisturizer: 1-5 wt% Emollient: 1-5 wt%; Emulsifier: 0.5-3 wt%; Preservative: 0.2-0.5 wt%; Add deionized water to bring the total to 100 wt%.

[0012] The humectant is one or more of methyl propylene glycol, 1,3-propanediol, and butylene glycol; The emollient is one or more of squalane, meadowfoam seed oil, shea butter, European hazelnut oil, and jojoba oil; The preservative is one or more of phenoxyethanol, sodium benzoate, p-hydroxyacetophenone, and sorbic acid; The emulsifier is one or more of C14-22 alcohol, C12-20 alkyl glucoside, and polyglycerol-3-methylglucose distearate.

[0013] Fourthly, the present invention provides a method for preparing the emulsion described in the third aspect, the specific steps of which are as follows: S1: Mix the humectant, preservative and 1 / 2 deionized water, heat to 60-80℃ and stir at 100-300 rpm for 1-3 minutes; then homogenize at 3000-5000 rpm for 1-3 minutes to obtain mixture A. S2: Mix the emollient and emulsifier, heat to 60-80℃ and stir at a speed of 200-400 rpm for 1-3 minutes; then homogenize at a speed of 3000-5000 rpm for 1-3 minutes to obtain mixture B. S3: Add mixture B to mixture A, stir at a speed of 200-400 rpm for 3-5 minutes; then homogenize at a speed of 3000-5000 rpm for 3-5 minutes to obtain the mixture. S4: Mix the firming and anti-aging composition with the remaining deionized water, heat to 30-40℃ and stir, stirring speed: 100-300 rpm, stirring time: 1-3 min, to obtain mixture C; S5: After the mixture cools to 30-50℃, mix mixture C with the mixture and stir at a speed of 100-300 rpm for 1-3 minutes. Then homogenize at a speed of 1000-3000 rpm for 1-3 minutes. Continue to cool to 20-25℃ to obtain an emulsion with firming and anti-aging effects. Fill, sterilize, seal, and store at room temperature.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The firming and anti-aging composition containing extract of the bark of the variegated bellflower tree provided by the present invention achieves a multi-target and multi-level synergistic effect by compounding extracts of the bark of the variegated bellflower tree, extracts of the flower of Magnolia officinalis, extracts of Auricularia auricula-judae, and a specific complex polypeptide (i.e., carnosine, acetyl tetrapeptide-5, and tripeptide-10 citrulline in a mass ratio of 1:0.4-0.6:0.2-0.6).

[0015] Specifically, the bark extract of the variegated bellflower tree is rich in polyphenols and saponins, which have strong antioxidant properties. It can neutralize free radicals and reduce the expression of matrix metalloproteinases (MMPs, including elastase and collagenase) induced by oxidative stress. Some polyphenols can directly bind to the active site of enzymes and inhibit their catalytic function.

[0016] Magnolia flower extract contains magnolin, volatile oil and flavonoids (such as rutin), which can inhibit the release of inflammatory factors (such as TNF-α and IL-1β), and inflammatory factors are key upstream inducers of MMP transcription; at the same time, flavonoids may directly and weakly inhibit MMP activity.

[0017] The main active ingredient in the extract of Auricularia auricula-judae is polysaccharide (such as acidic heteropolysaccharide), which can reduce enzyme activity by chelating metal ions (such as Zn²⁺, Cu²⁺, essential cofactors of MMPs). In addition, the moisturizing effect of polysaccharides can maintain the hydration state of the extracellular matrix (ECM) and indirectly protect collagen / elastin.

[0018] Carnosine possesses both antioxidant (scavenging hydroxyl radicals and capturing carbonyl groups) and metal chelating (binding Cu²⁺ / Zn²⁺) capabilities, which can inhibit metal ion-dependent MMPs. At the same time, its anti-glycation effect reduces collagen cross-linking damage, indirectly maintaining the enzyme-inhibiting environment.

[0019] Acetyl tetrapeptide-5 is a metalloproteinase inhibitor peptide (such as targeting MMP-1 and MMP-9), which may inhibit MMP secretion by mimicking enzyme substrates to competitively bind to active sites or by reducing the mRNA expression of MMPs in fibroblasts.

[0020] Tripeptide-10citrulline contains citrulline, which can act as a signal peptide to promote collagen synthesis in fibroblasts; at the same time, citrulline residues can interact with key residues in the enzyme's active site, weakly inhibiting elastase or MMPs.

[0021] The above four components exert a synergistic effect through the following pathways: The bark extract of *Bretschneidera sinensis* (polyphenols), carnosine (metal chelate), and acetyl tetrapeptide-5 (competitive inhibitor) can target the active site, cofactor, or substrate binding site of elastase / MMPs, respectively, forming a "multi-site blockade." *Magnolia denudata* flower extract (inducible by anti-inflammatory reducing factors) and acetyl tetrapeptide-5 (inhibit MMP transcription) can downregulate MMP gene expression, reducing enzyme production at its source. The antioxidant effects of *Bretschneidera sinensis* bark extract (polyphenols) and carnosine can reduce ROS levels, and ROS is a significant trigger for MMP activation (e.g., through oxidative modification of enzyme precursors or activation of protease-activated receptors). The two work synergistically not only to directly inhibit enzymes but also to reduce their chance of being "activated," amplifying the inhibitory effect. Carnosine (strong metal chelation) and Auricularia auricula extract (weak chelation) can synergistically reduce the concentration of Zn²⁺ / Cu²⁺ in the local environment, significantly weakening the activity of MMPs (such as MMP-2 and MMP-9) that depend on these ions, forming a chelation effect of "1+1>2". Tripeptide-10 citrulline (promotes collagen synthesis) and other components (inhibit enzyme degradation) form a "synthesis-protection" dual pathway: on the one hand, it increases the total amount of ECM substrates (collagen / elastin), and on the other hand, it reduces their degradation, jointly delaying skin aging.

[0022] The aforementioned synergistic mechanism overcomes the limitations of traditional anti-aging ingredients targeting only a single point, significantly enhancing the synergistic inhibitory effect on collagenase and elastase activity. Furthermore, the scientifically formulated composition ensures the mildness and safety of the formulation, making it suitable for various dosage forms and providing technical support for the development of highly effective synergistic anti-aging cosmetics. Detailed Implementation

[0023] To better understand the present invention, the present invention will be further described below with reference to specific embodiments. The terminology used in the embodiments is for describing specific implementation schemes and does not constitute a limitation on the scope of protection of the present invention.

[0024] For the following tests, unless specific conditions are specified, the test methods are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, calculations are made by weight in percentages and parts.

[0025] Some of the raw materials and their sources are as follows: Bark of the variegated bellflower tree: purchased from Shanghai Qianfei Chemical Co., Ltd.

[0026] Magnolia flower extract: purchased from Shanghai Jiakai Biotechnology Co., Ltd.

[0027] Golden ear fungus extract: purchased from Shanghai LanHai Biotechnology Co., Ltd.

[0028] Carnosine: Purchased from Shenzhen Ruidelin Biotechnology Co., Ltd.

[0029] Acetyl tetrapeptide-5: Purchased from Shenzhen Ruidelin Biotechnology Co., Ltd.

[0030] Tripeptide-10 citrulline: purchased from Shenzhen Ruidelin Biotechnology Co., Ltd.

[0031] Tremella extract: purchased from Shanghai Huiwen Biotechnology Co., Ltd.

[0032] All other raw materials or reagents are commercially available.

[0033] The preparation method of the bark extract of the variegated bellflower tree according to the present invention comprises the following steps: S1. Air-dry the bark of the variegated bellflower tree until the moisture content is ≤5%, and grind it into coarse powder; S2. Mix coarse powder of variegated bellflower bark with rapeseed oil and polyglycerol-3 diisostearate, heat to 55°C, and stir at 300 rpm for 20 min to obtain a mixture; S3. Under N2 atmosphere, the mixture in S2 was subjected to ultrasonic-assisted extraction to obtain the extract. Extraction temperature: 55℃, ultrasonic frequency: 30 kHz, extraction time: 45 min. S4. Filter the extract to remove insoluble matter, centrifuge, and collect the supernatant to obtain the bark extract of *Bretschneidera sinensis*. In step S2, the mass ratio of coarse bark powder of *Bretschneidera sinensis*, rapeseed oil, and polyglycerol-3 diisostearate is 1:3:0.2.

[0034] The specific mass fractions of each component in the firming and anti-aging composition containing the bark extract of the variegated bellflower tree described in this invention are as follows: Firming and anti-aging composition 1: Bark extract of *Bretschneidera sinensis*: 2 parts; Flower extract of *Magnolia denudata*: 0.6 parts; Extract of *Auricularia auricula-judae*: 3 parts; Complex polypeptide: 0.4 parts; The complex polypeptide is composed of carnosine, acetyl tetrapeptide-5, and tripeptide-10 citrulline in a mass ratio of 1:0.5:0.4.

[0035] Firming and anti-aging composition 2: Bark extract of *Bretschneidera sinensis*: 3 parts; Flower extract of *Magnolia denudata*: 0.7 parts; Extract of *Auricularia auricula-judae*: 4 parts; Complex polypeptide: 0.3 parts; The complex polypeptide is composed of carnosine, acetyl tetrapeptide-5, and tripeptide-10 citrulline in a mass ratio of 1:0.6:0.2.

[0036] Firming and anti-aging composition 3: Bark extract of *Bretschneidera sinensis*: 1 part; Flower extract of *Magnolia denudata*: 0.5 part; Extract of *Auricularia auricula-judae*: 2 parts; Complex polypeptide: 0.5 parts; The complex polypeptide is composed of carnosine, acetyl tetrapeptide-5, and tripeptide-10 citrulline in a mass ratio of 1:0.4:0.6.

[0037] Firming and anti-aging composition ①: Unlike composition 1, it lacks the bark extract of the variegated bellflower tree. The missing mass portion is made up by the flower extract of Magnolia officinalis, the extract of Auricularia auricula-judae, and a complex polypeptide in a mass ratio of 0.6:3:0.4. The complex polypeptide is composed of carnosine, acetyl tetrapeptide-5, and tripeptide-10 citrulline in a mass ratio of 1:0.5:0.4.

[0038] Firming and anti-aging composition ②: Unlike composition 1, it lacks Magnolia officinalis flower extract, and the missing mass fraction is made up by a combination of Cypripedium variegata bark extract, Auricularia auricula-judae extract, and complex polypeptide in a mass ratio of 2:3:0.4. The complex polypeptide is composed of carnosine, acetyl tetrapeptide-5, and tripeptide-10 citrulline in a mass ratio of 1:0.5:0.4.

[0039] Firming and anti-aging composition ③: Unlike composition 1, it lacks auricularia auricula-judae extract. The missing mass fraction is made up by a combination of variegated bellflower bark extract, magnolia flower extract, and complex polypeptide in a mass ratio of 2:0.6:0.4. The complex polypeptide is composed of carnosine, acetyl tetrapeptide-5, and tripeptide-10 citrulline in a mass ratio of 1:0.5:0.4.

[0040] Firming and anti-aging composition ④: Unlike composition 1, this composition lacks a complex polypeptide, and the missing mass fraction is made up by extracts of the bark of the variegated bellflower, the flower extract of Magnolia officinalis, and the extract of Auricularia auricula-judae in a mass ratio of 2:0.6:3.

[0041] Firming and anti-aging composition ⑤: Unlike Composition 1, the complex polypeptide is replaced with an equal amount of carnosine, while the remaining raw materials and their mass proportions are the same as those in Composition 1.

[0042] Firming and anti-aging composition ⑥: Unlike Composition 1, the complex polypeptide is replaced with an equal amount of acetyl tetrapeptide-5, while the remaining raw materials and their mass proportions are the same as those in Composition 1.

[0043] Firming and anti-aging composition ⑦: Unlike Composition 1, the complex polypeptide is replaced with an equal amount of tripeptide-10 citrulline, while the remaining raw materials and their mass proportions are the same as those in Composition 1.

[0044] Firming and anti-aging composition ⑧: Unlike composition 1, the complex polypeptide is composed of carnosine, acetyl tetrapeptide-5, and tripeptide-10 citrulline in a mass ratio of 1:0.2:0.7, with the remaining raw materials and mass proportions being the same as those in composition 1.

[0045] Firming and anti-aging composition ⑨: Bark extract of *Bretschneidera sinensis*: 0.6 parts; Flower extract of *Magnolia denudata*: 3 parts; Extract of *Auricularia auricula-judae*: 0.4 parts; Complex polypeptide: 2 parts; The complex polypeptide is composed of carnosine, acetyl tetrapeptide-5, and tripeptide-10 citrulline in a mass ratio of 1:0.5:0.4.

[0046] Firming and anti-aging composition ⑩: Unlike Composition 1, it lacks *Auricularia auricula-judae* extract, and the missing mass is made up by *Tremella fuciformis* extract. The remaining ingredients and mass proportions are the same as in Composition 1. A lotion with firming and anti-aging effects has the following component mass percentages as shown in Table 1.

[0047] Table 1. Components and their mass percentage of emulsions with firming and anti-aging effects.

[0048] Note: The moisturizer is 1,3-propanediol; the emulsifier is C14-22 alcohol, C12-20 alkyl glucoside, and polyglycerol-3-methyl glucoside distearate in a mass ratio of 1:1:1; the emollient is jojoba oil, meadowfoam seed oil, and hazelnut seed oil in a mass ratio of 1:0.5:0.7; and the preservative is p-hydroxyacetophenone.

[0049] Blank lotion: The only difference between the blank emulsion and emulsion 1 is that the blank emulsion does not contain the firming and anti-aging composition, and the missing percentage is made up with deionized water.

[0050] The specific steps for preparing emulsions 1-5 and emulsions ①-⑩ are as follows: S1: Mix the humectant, preservative and 1 / 2 deionized water, heat to 70°C and stir at 300 rpm for 2 min; then homogenize at 4000 rpm for 2 min to obtain mixture A. S2: Mix the emollient and emulsifier, heat to 70°C and stir at 300 rpm for 2 minutes; then homogenize at 4000 rpm for 2 minutes to obtain mixture B. S3: Add mixture B to mixture A, stir at 300 rpm for 5 min; then homogenize at 4000 rpm for 5 min to obtain the mixture. S4: Mix the firming and anti-aging composition with the remaining deionized water, heat to 35°C and stir at 300 rpm for 2 minutes to obtain mixture C.

[0051] S5: After the mixture cools down to 40℃, mix mixture C with the mixture, stir at 300 rpm for 2 min; then homogenize at 2000 rpm for 2 min, and continue to cool down to 25℃ to obtain an emulsion with firming and anti-aging effects. Fill, sterilize, seal, and store at room temperature.

[0052] Blank emulsion preparation steps: S1: Mix the humectant, preservative and 1 / 2 deionized water, heat to 70°C and stir at 300 rpm for 2 min; then homogenize at 4000 rpm for 2 min to obtain mixture A. S2: Mix the emollient and emulsifier, heat to 70°C and stir at 300 rpm for 2 minutes; then homogenize at 4000 rpm for 2 minutes to obtain mixture B. S3: Add mixture B to mixture A, stir at 300 rpm for 5 min; then homogenize at 4000 rpm for 5 min to obtain the mixture. S4: After the mixture cools down to 40°C, mix the remaining deionized water with the mixture and stir at 300 rpm for 2 min. Then homogenize at 2000 rpm for 2 min. Continue cooling down to 25°C to obtain a blank emulsion, fill, sterilize, seal, and store at room temperature.

[0053] Experiment 1: Effect of the composition on elastase activity An in vitro elastase activity inhibition assay was used to evaluate the effect of the test samples on the activity of skin elastase. A specific fluorescent substrate was used to measure changes in enzyme activity, thereby assessing the anti-wrinkle and firming effect.

[0054] The samples to be tested include firming and anti-aging compositions 1-3 and firming and anti-aging compositions ①-⑩, specifically grouped as follows: Test sample group: Add 50 μL of the test sample solution (3 wt% concentration) prepared with the buffer provided in the kit to a black 96-well plate. Then add 100 μL of 0.2 U / mL elastase working solution. After shaking and reacting for 15 min at room temperature, add 50 μL of 100 μg / mL elastase fluorescent coupler to each well. Incubate at room temperature in the dark for 30 min. Measure the fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a fluorescence microplate reader. Record this as MFI. 待测样品 ; Control group: Replace the test sample solution with an equal volume of 50 μL buffer solution, keeping everything else unchanged, and record the fluorescence intensity as MFI. 对照 ; Background group: Replace the sample solution with 50 μL buffer, replace the elastase working solution with 100 μL buffer, keep everything else unchanged, and record the fluorescence intensity as MFI. 背景 ; The inhibitory effect of different test samples on elastase activity was calculated using the following formula; Elastase activity inhibition rate / % = [1 - (MFI)] 待测样品 -MFI 背景 ) / (MFI 对照 -MFI 背景 )]×100% The experimental results are shown in Table 2.

[0055] Table 2. Effects of the composition on the inhibition of elastase activity. Firming and anti-aging composition 1 85.42 Firming and anti-aging composition 2 84.39 Firming and anti-aging composition 3 82.01 Firming and anti-aging composition ① 57.22 Firming and anti-aging composition ② 68.73 Firming and anti-aging composition ③ 62.67 Firming and anti-aging composition ④ 53.84 Firming and anti-aging composition ⑤ 60.45 Firming and anti-aging composition ⑥ 62.53 Firming and anti-aging composition ⑦ 65.26 Firming and anti-aging composition ⑧ 71.61 Firming and anti-aging composition ⑨ 70.31 Firming and anti-aging composition ⑩ 74.64 As shown in Table 2, comparing the elastase activity inhibition rates of firming and anti-aging composition 1 with those of firming and anti-aging compositions ①-④, it can be seen that the extracts of *Cyclocarya paliurus* bark, *Magnolia denudata* flower, *Auricularia auricula-judae* extract, and the complex polypeptide in the compositions have a significant synergistic effect. Comparing the results of firming and anti-aging composition 1 with those of firming and anti-aging compositions ⑤-⑦, it can be seen that the complex polypeptide with the specified mass ratio of this invention has a better inhibition rate of elastase activity compared to the use of carnosine, acetyl tetrapeptide-5, tripeptide-10, and citrulline alone. Comparing the results of firming and anti-aging composition 1 with those of firming and anti-aging composition ⑧, it can be seen that the complex polypeptide with the specified mass ratio of *Cyclocarya paliurus* bark and *Magnolia denudata* bark has a better inhibition rate than those of *Cyclocarya paliurus* bark. When the bark extract, Magnolia officinalis flower extract, and Auricularia auricula-judae extract are used together, the composition exhibits a better inhibitory effect on elastase activity. A comparison of firming and anti-aging compositions 1-3 and 9 shows that when the bark extract, Magnolia officinalis flower extract, Auricularia auricula-judae extract, and complex protease are used together according to the mass ratio of the present invention, the composition exhibits a better inhibitory effect on elastase activity. A comparison of firming and anti-aging composition 1 and 10 shows that, compared to Tremella fuciformis extract, when Auricularia auricula-judae extract is used together with complex polypeptides, Magnolia officinalis bark extract, and Magnolia officinalis flower extract, the composition exhibits a better inhibitory effect on elastase activity.

[0056] Experiment 2: Effect of the composition on collagenase activity Prepare a 3wt% sample solution of firming and anti-aging compositions 1-3 and ①-⑩ using purified water, and prepare it fresh each time you use it.

[0057] The Tricine buffer solution has a concentration of 50 mmol / L and a pH of 7.5.

[0058] A 0.8 U / mL collagenase solution and a 2 mmol / L FALGPA (N-[3-(2-furanyl)acryloyl]-leucine-glycine-proline-alanine) solution were prepared using Tricine buffer.

[0059] Reaction solution without sample: Mix 40 μL of purified water with 100 μL of Tricine buffer, then add 20 μL of 0.8 U / mL collagenase solution, mix well, and incubate at 25 °C for 15 min. Then add 40 μL of 2 mmol / L FALGPA solution. After 15 min, measure the absorbance at 335 nm. Perform three parallel measurements and take the average value.

[0060] Enzyme-free reaction solution: Mix 40 μL of sample solution with 100 μL of Tricine buffer, then add 20 μL of Tricine buffer, mix well, and incubate at 25 °C for 15 min. Then add 40 μL of 2 mmol / L FALGPA solution. After 15 min, measure the absorbance at 335 nm. Perform three parallel measurements and take the average value.

[0061] Reaction solution without sample and enzyme: Mix 40 μL of purified water with 100 μL of Tricine buffer, then add 20 μL of Tricine buffer, mix well, and incubate at 25 °C for 15 min. Then add 40 μL of 2 mmol / L FALGPA solution. After 15 min, measure the absorbance at 335 nm. Perform three parallel measurements and take the average value.

[0062] Reaction solution containing sample and enzyme: Mix 40 μL of sample solution with 100 μL of Tricine buffer, then add 20 μL of 0.8 U / mL collagenase solution. Mix well and incubate at 25 °C for 15 min. Then add 40 μL of 2 mmol / L FALGPA solution. After 15 min, measure the absorbance at 335 nm. Perform three parallel measurements and take the average value. Calculate the inhibition rate of collagenase by the sample according to the formula.

[0063] Inhibition rate / % = [1 - (CD) / (AB)] × 100% In the formula: A is the absorbance of the reaction solution without the sample; B is the absorbance of the reaction solution without the sample and enzyme; C is the absorbance of the reaction solution containing the sample and enzyme; D is the absorbance of the reaction solution without the enzyme.

[0064] The experimental results are shown in Table 3.

[0065] Table 3 Effect of the composition on collagenase activity Firming and anti-aging composition 1 65.24 Firming and anti-aging composition 2 62.64 Firming and anti-aging composition 3 63.72 Firming and anti-aging composition ① 36.31 Firming and anti-aging composition ② 41.43 Firming and anti-aging composition ③ 45.62 Firming and anti-aging composition ④ 39.26 Firming and anti-aging composition ⑤ 49.21 Firming and anti-aging composition ⑥ 51.57 Firming and anti-aging composition ⑦ 50.91 Firming and anti-aging composition ⑧ 54.33 Firming and anti-aging composition ⑨ 52.29 Firming and anti-aging composition ⑩ 55.93 As shown in Table 3, comparing the collagenase activity inhibition rates of firming and anti-aging composition 1 with those of firming and anti-aging compositions ①-④, it can be seen that the extracts of *Cyclocarya paliurus* bark, *Magnolia denudata* flower, *Auricularia auricula-judae* extract, and the complex polypeptides in the compositions have a significant synergistic effect. Comparing the results of firming and anti-aging composition 1 with those of firming and anti-aging compositions ⑤-⑦, it can be seen that the complex polypeptides with the specified mass ratio of this invention have a higher inhibition rate of collagenase activity compared to the use of carnosine, acetyl tetrapeptide-5, tripeptide-10, and citrulline alone. Comparing the results of firming and anti-aging composition 1 with those of firming and anti-aging composition ⑧, it can be seen that the complex polypeptides with the specified mass ratio of this invention, combined with *Cyclocarya paliurus* bark extract, have a higher inhibition rate of collagenase activity. When bark extract, Magnolia officinalis flower extract, and Auricularia auricula-judae extract are used together, the composition exhibits a higher inhibition rate of collagenase activity. A comparison of firming and anti-aging compositions 1-3 and 9 shows that when the bark extract of Bellflower variegatedus, Magnolia officinalis flower extract, Auricularia auricula-judae extract, and complex protease are used together according to the mass ratio of the present invention, the composition exhibits a better inhibition rate of collagenase activity. A comparison of firming and anti-aging composition 1 and 10 shows that, compared to Tremella fuciformis extract, the combination of Auricularia auricula-judae extract with complex peptides, Bellflower variegatedus bark extract, and Magnolia officinalis flower extract has a better inhibitory effect on collagenase activity.

[0066] Experiment 3: Human Efficacy Test 3-1. Human skin patch test Test substance: Emulsions 1-5.

[0067] Subject selection: Healthy volunteers aged 21-57 years were selected who voluntarily participated and signed an informed consent form, and were able to complete the trial as required during the testing period. They were randomly assigned to groups of 30 subjects each.

[0068] Experimental method: Qualified patch equipment was selected, and the closed patch test method was used. 2 mL of the test substance was placed in the patch applicator and spread evenly. Then, a topical hypoallergenic adhesive tape was applied to the flexor side of the subject's forearm. The test substance was removed after 24 hours. Skin reaction was observed and recorded at 0.5, 24, and 48 hours after removal.

[0069] Results evaluation: Adverse skin reactions were graded according to the classification of adverse skin reactions in human trials as specified in the "Cosmetic Safety Technical Specifications" (2015 edition). The grading criteria are shown in Table 4 and the results are shown in Table 5.

[0070] Table 4. Grading Criteria for Adverse Skin Reactions Level 0 No skin reaction Level 1 Light red spots appeared on the skin Level 2 Skin shows erythema, infiltration and papules Level 3 Skin erythema, edema, papules and vesicles Level 4 Skin erythema, edema and blisters Table 5 Results of human skin patch test

[0071] 3-2. Human skin patch test for sensitive skin Test substances: Emulsions 1-5, Emulsions ①-⑩.

[0072] Subject selection: Healthy volunteers aged 21-44 years who were positive for lactic acid stinging test, voluntarily participated and signed informed consent form, and were able to complete the test as required during the test period were selected and randomly assigned to groups of 10.

[0073] Experimental Methods: Volunteers randomly divided their faces into test and control sides. After cleansing, the product was applied evenly to the test side according to the instructions. No product was applied to the control side. After 28 days of use, volunteers reported local skin reactions. If adverse reactions were observed, a dermatologist evaluated the situation.

[0074] Results evaluation: The grading of adverse skin reactions was determined according to the grading of adverse skin reactions in human trials as specified in the "Cosmetic Safety Technical Specifications" (2015 edition). The grading criteria are shown in Table 4, and the results are shown in Table 6.

[0075] Table 6 Results of Human Trial

[0076] Based on the results of human skin patch tests, lotion 1-5 showed good safety during the testing period, and no adverse reactions were observed in the subjects.

[0077] Based on the results of the sensitivity skin suitability test, no adverse skin reactions were observed in any subjects during the test period for lotions 1-5 and ①-⑩, indicating that lotions 1-5 and ①-⑩ are safe, gentle and non-irritating, and suitable for people with sensitive skin.

[0078] 3-3 Skin elasticity test Test samples: Emulsions 1-5, Emulsions ①-⑩, and blank emulsion.

[0079] Test Method: Volunteers with wrinkles and healthy, undamaged skin, aged 30-45 years on average, were randomly divided into 16 groups of 10 participants each. Each group used the test sample described above. Participants applied 2g of lotion to their face twice daily, morning and evening, for 30 consecutive days.

[0080] During the experiment, subjects were not allowed to apply any other cosmetics to the test areas. Data were collected using a Cutometer MPA580 skin elastometer on day 0 and day 30, with the test area being the cheekbone. Measurements were taken three times on the same test area, and the average value was recorded.

[0081] The test parameter is R2 (the ratio of skin elasticity Ua without negative pressure to maximum stretch Uf with negative pressure). The closer R2 is to 1, the better the skin elasticity. The R2 change rate = (R2 value on day 30 - R2 value on day 0) / R2 value on week 0. The test results are shown in Table 7.

[0082] Table 7 Effects of the composition on skin elasticity Emulsion 1 0.553 0.688 24.41 Emulsion 2 0.517 0.634 22.63 Emulsion 3 0.586 0.731 24.74 Emulsion 4 0.561 0.693 23.53 Emulsion 5 0.532 0.655 23.12 Emulsion ① 0.575 0.652 13.39 Emulsion ② 0.547 0.631 15.36 Emulsion ③ 0.563 0.641 13.85 Emulsion ④ 0.548 0.619 12.96 Emulsion ⑤ 0.524 0.598 14.12 Emulsion ⑥ 0.536 0.602 12.31 Emulsion ⑦ 0.567 0.641 13.05 Emulsion ⑧ 0.511 0.597 16.83 Emulsion 9 0.542 0.627 15.68 Emulsion 10 0.539 0.635 17.81 Blank lotion 0.525 0.553 5.33 As shown in Table 7, comparing the results of emulsions 1-3, emulsions ①-⑩, and the blank emulsion, it can be seen that the emulsion containing the firming and anti-aging composition of the present invention has the effect of improving skin elasticity; comparing the results of emulsions 1-3, it can be seen that when the amount of the firming and anti-aging composition added to the emulsion is in the range of 1wt%-3wt%, all the emulsions have the effect of improving skin elasticity, and the cost-effectiveness is the highest when the amount of the firming and anti-aging composition added to the emulsion is 2wt%; comparing the results of emulsion 1 and emulsions 4-5, it can be seen that the emulsions obtained by compounding the raw materials according to the dosage ratio specified in the present invention all have the effect of improving skin elasticity; comparing emulsion 1 and emulsion ① Results 4 and 9 show that when the bark extract of *Bretschneidera sinensis*, flower extract of *Magnolia denudata*, extract of *Auricularia auricula-judae*, and complex polypeptides in the firming and anti-aging composition are compounded in a specific ratio and used in the emulsion, the emulsion has a better effect on improving skin elasticity. Comparison of emulsion 1 and emulsions 5-8 shows that the composition and ratio of complex polypeptides in the firming and anti-aging composition affect the effect of the emulsion on improving skin elasticity, and the emulsion using the complex polypeptides of this invention has the best effect on improving skin elasticity. Comparison of emulsion 1 and emulsion 10 shows that the types of raw materials in the firming and anti-aging composition affect the efficacy of the composition, thereby affecting the efficacy of the emulsion.

[0083] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A firming and anti-aging composition containing extract of the bark of the variegated bellflower tree, characterized in that, The firming and anti-aging composition comprises the following components in parts by weight: Bark extract of the variegated bellflower tree: 1-3 parts; Magnolia flower extract: 0.5-0.7 parts; Golden ear fungus extract: 2-4 parts; Complex polypeptide: 0.3-0.5 parts The preparation method of the bark extract of the variegated bellflower tree includes the following specific steps: S1. Air-dry the bark of the variegated bellflower tree until the moisture content is ≤5%, and grind it into coarse powder; S2. Mix coarse powder of variegated bellflower bark with rapeseed oil and polyglycerol-3 diisostearate, heat to 55°C, and stir at 300 rpm for 20 min to obtain a mixture; S3. Under N2 atmosphere, the mixture in S2 was subjected to ultrasonic-assisted extraction to obtain the extract. Extraction temperature: 55℃, ultrasonic frequency: 30 kHz, extraction time: 45 min. S4. Filter the extract to remove insoluble matter, centrifuge, and collect the supernatant to obtain the bark extract of *Bretschneidera sinensis*. In step S2, the mass ratio of coarse bark powder of *Bretschneidera sinensis*, rapeseed oil, and polyglycerol-3 diisostearate is 1:3:0.

2. The complex polypeptide is composed of carnosine, acetyl tetrapeptide-5, and tripeptide-10 citrulline in a mass ratio of 1:0.4-0.6:0.2-0.

6.

2. The application of the firming and anti-aging composition according to claim 1 in the preparation of cosmetics with firming and anti-aging effects.

3. The application according to claim 2, characterized in that, The dosage form of the cosmetic with firming and anti-aging effects is any one of face cream, lotion, or serum.

4. A lotion with firming and anti-aging effects, characterized in that, The emulsion contains 1-3 wt% of the firming and anti-aging composition as described in claim 1.

5. The emulsion with firming and anti-aging effects according to claim 4, characterized in that, The emulsion also contains the following components by weight percentage: Moisturizer: 1-5 wt% Emollient: 1-5 wt%; Emulsifier: 0.5-3 wt%; Preservative: 0.2-0.5 wt%; Add deionized water to bring the total to 100 wt%.

6. The emulsion with firming and anti-aging effects according to claim 5, characterized in that, The humectant is one or more of methyl propylene glycol, 1,3-propanediol, and butylene glycol; the preservative is one or more of phenoxyethanol, sodium benzoate, p-hydroxyacetophenone, and sorbic acid.

7. The emulsion with firming and anti-aging effects according to claim 5, characterized in that, The emulsifier is one or more of C14-22 alcohol, C12-20 alkyl glucoside, and polyglycerol-3-methylglucose distearate.

8. The emulsion with firming and anti-aging effects according to claim 5, characterized in that, The emollient is one or more of squalane, meadowfoam seed oil, shea butter, European hazelnut oil, and jojoba oil.

9. A method for preparing an emulsion with firming and anti-aging effects according to any one of claims 5-8, characterized in that, Prepared by the following steps: S1: Mix the humectant, preservative and 1 / 2 deionized water, heat to 60-80℃ and stir at 100-300 rpm for 1-3 minutes; then homogenize at 3000-5000 rpm for 1-3 minutes to obtain mixture A. S2: Mix the emollient and emulsifier, heat to 60-80℃ and stir at a speed of 200-400 rpm for 1-3 minutes; then homogenize at a speed of 3000-5000 rpm for 1-3 minutes to obtain mixture B. S3: Add mixture B to mixture A, stir at a speed of 200-400 rpm for 3-5 minutes; then homogenize at a speed of 3000-5000 rpm for 3-5 minutes to obtain the mixture. S4: Mix the firming and anti-aging composition with the remaining deionized water, heat to 30-40℃ and stir, stirring speed: 100-300 rpm, stirring time: 1-3 min, to obtain mixture C; S5: After the mixture cools to 30-50℃, mix mixture C with the mixture and stir at a speed of 100-300 rpm for 1-3 minutes. Then homogenize at a speed of 1000-3000 rpm for 1-3 minutes. Continue to cool to 20-25℃ to obtain an emulsion with firming and anti-aging effects. Fill, sterilize, seal, and store at room temperature.

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