A brightleaf gmelina extract and retinol composition and uses thereof

Abstract

The present application provides a combination of extract of mallotus philippinensis and retinol and application thereof, the combination comprises retinol and extract of mallotus philippinensis, and the mass ratio of retinol and extract of mallotus philippinensis is 1: (20-200). The combination can synergistically promote the expression of collagen type I (COL I), the gene expression of collagen type III (COL III) and elastin, and further enhance the retinol firming and anti-wrinkle effects.

Description

Technical Field

[0001] This invention relates to a composition and its application, particularly a composition of *Populus gloriosa* extract and retinol with firming and anti-wrinkle effects and its application. Background Technology

[0002] *Adinandranitida* Merr. ex H. L. Li is a plant in the genus *Adinandranitida*, also known as cliff tea or stone wall tea. It mainly contains flavonoids, polyphenols, and triterpenoid saponins. Pharmacological studies have reported that *Adinandranitida* and its isolated chemical components possess antibacterial, liver and stomach damage-reducing, and lipid synthesis-inhibiting biological activities.

[0003] Retinol and its derivatives are ingredients in firming and anti-wrinkle cosmetics. These ingredients have a well-defined mechanism of action, effectively improving skin texture, reducing wrinkles, enhancing skin elasticity, and promoting collagen production.

[0004] Patent CN106728898 A discloses a pharmaceutical composition for repairing the skin barrier and its preparation method, which provides an extraction method for a composition of *Tea japonica*, *Lysimachia christinae*, *Cynanchum paniculatum*, *Ginkgo biloba* leaves, *Euphorbia pekinensis*, and *Anoectochilus roxburghii*, demonstrating that the composition can improve erythema, papules, and skin lesions caused by hormone-dependent dermatitis. Patent CN117064960A discloses a flavonoid extract based on *Tea japonica*, its preparation method, and its application, disclosing a process for preparing *Tea japonica* flavonoid extract using freezing, enzymatic hydrolysis, and pure water reflux extraction. It also found that the extracted *Tea japonica* flavonoid extract has superior in vitro antibacterial effects and a stronger immune-enhancing effect, and can be applied to the prevention and treatment of H9 subtype avian influenza in poultry. However, there are currently no research reports on the synergistic effect of *Tea japonica* extract combined with retinol components in promoting collagen production. Summary of the Invention

[0005] The purpose of this invention is to provide a composition of *Populus glomeratus* extract and retinol, and its application. It can synergistically promote the expression of type I collagen (COL I), type III collagen (COL III), and elastin genes, further enhancing the firming and anti-wrinkle effects of retinol.

[0006] The technical solution of the present invention is a composition of *Populus gloriosa* extract and retinol, wherein the composition comprises retinol and *Populus gloriosa* extract, the mass ratio of retinol to *Populus gloriosa* extract is 1:(20-200), preferably 1:20 and more preferably 1:200.

[0007] In the aforementioned composition of *Populus gloriosa* extract and retinol, the preparation method of the *Populus gloriosa* extract includes the following steps:

[0008] S1. Crude extraction: Take leaves of *Populus gloriosa*, add water at a material-to-liquid ratio of 1:20, heat and extract at 60℃~70℃, filter while hot after extraction, extract 1~3 times, combine the filtrates to obtain crude extract A.

[0009] S2. Temperature gradient treatment: The crude extract A is kept at 30℃ for 0.75-1.25 h, 65℃ for 0.25-0.75 h, and 85℃ for 1.75-2.25 h in sequence to precipitate impurities. The precipitate is removed by hot filtration with 300-mesh filter paper to obtain filtrate B.

[0010] S3. Membrane filtration purification: Filtrate B is filtered sequentially through an 800nm ​​ceramic membrane and a 50nm ceramic membrane to obtain purified filtrate C.

[0011] S4. Drying treatment: The purified filtrate C is concentrated, freeze-dried or spray-dried to obtain the extract of *Populus glossifolia*.

[0012] In the aforementioned composition of *Populus gloriosa* extract and retinol, the heating extraction temperature in S1 is 65°C, and the extraction is performed twice.

[0013] In the aforementioned composition of *Populus gloriosa* extract and retinol, the crude extract A in S2 is sequentially kept at 30°C for 1 h, 65°C for 0.5 h, and 85°C for 2 h.

[0014] In the aforementioned composition of *Populus gloriosa* extract and retinol, the *Populus gloriosa* extract contains chamomile A, the structural formula of which is: .

[0015] The use of a composition of *Polygonum bistorta* extract and retinol in the preparation of cosmetics with firming and / or anti-wrinkle effects.

[0016] In the aforementioned application of the combination of *Populus gloriosa* extract and retinol in the preparation of cosmetics with firming and / or anti-wrinkle effects, the concentration of retinol used is 0.00025% (m / m), and the concentration of *Populus gloriosa* extract used is 0.005% to 0.05% (m / m).

[0017] In the aforementioned application of the *Populus gloriosa* extract and retinol composition in the preparation of cosmetics with firming and / or anti-wrinkle effects, the firming and / or anti-wrinkle effects are achieved by the synergistic promotion of the gene expression of type I collagen, type III collagen, and / or elastin by the *Populus gloriosa* extract and retinol.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] This application demonstrates a significant synergistic effect between *Populus glehnia littoralis* extract and retinol in a specific ratio, promoting the expression of type I collagen, type III collagen, and elastin genes. Experimental data show that the combination of 0.00025% retinol and 0.005% *Populus glehnia littoralis* extract significantly increased COL I protein expression by 116.61%, COL III gene expression by 305.26%, and elastin gene expression by 365.52%. The combination of 0.00025% retinol and 0.05% *Populus glehnia littoralis* extract alone increased COL I protein expression by 137.13%, COL III gene expression by 497.37%, and elastin gene expression by 393.10%, all significantly superior to the effects of either ingredient alone. King's Q method and the highest single-drug model validation results show that both ingredients have a synergistic effect in promoting COL I expression and the expression of COL III and elastin genes. Therefore, the synergistic effect of *Polygonum bistorta* extract and retinol can enable lower concentrations of retinol to exert a more superior anti-wrinkle effect, while also expanding the application range of *Polygonum bistorta* extract. Detailed Implementation

[0020] The present invention will be further illustrated by the following embodiments, but these embodiments are not intended to limit the scope of the invention.

[0021] Example 1. A composition of *Populus gleamingii* extract and retinol, wherein the composition comprises retinol and *Populus gleamingii* extract, the mass ratio of retinol to *Populus gleamingii* extract is 1:200, the concentration of retinol is 0.00025% (m / m), the concentration of *Populus gleamingii* extract is 0.05% (m / m), and the *Populus gleamingii* extract contains camsanthin A.

[0022] The preparation method of the aforementioned *Populus gloriosa* extract includes the following steps:

[0023] S1. Crude extraction: Take 200g of bright-leaved poplar leaves, add water at a material-to-liquid ratio of 1:20, heat and extract at 65℃, filter while hot after extraction, extract twice, combine the filtrates to obtain crude extract A.

[0024] S2. Temperature gradient treatment: The crude extract A is kept at 30℃ for 1 h, 65℃ for 0.5 h, and 85℃ for 2 h in sequence to precipitate impurities. The precipitate is removed by hot filtration with 300 mesh filter paper to obtain filtrate B.

[0025] S3. Membrane filtration purification: Filtrate B is filtered sequentially through an 800nm ​​ceramic membrane and a 50nm ceramic membrane to obtain purified filtrate C.

[0026] S4. Drying treatment: The purified filtrate C is concentrated, freeze-dried or spray-dried to obtain the extract of *Populus glossifolia*.

[0027] Example 2. A composition of *Populus gleamingii* extract and retinol, wherein the composition comprises retinol and *Populus gleamingii* extract, the mass ratio of retinol to *Populus gleamingii* extract is 1:20, the concentration of retinol is 0.00025% (m / m), the concentration of *Populus gleamingii* extract is 0.005% (m / m), and the *Populus gleamingii* extract contains camsanthin A.

[0028] Example 3. Application of a composition of *Populus glomeratus* extract and retinol in the preparation of cosmetics with firming and / or anti-wrinkle effects.

[0029] The concentration of retinol in the composition of *Polygonum bistorta* extract and retinol in the cosmetic is 0.00025% (m / m), and the concentration of *Polygonum bistorta* extract is 0.005% (m / m). The remaining ingredients in the cosmetic are excipients. The firming and / or anti-wrinkle effects are achieved by the synergistic promotion of the expression of type I collagen, type III collagen, and / or elastin by *Polygonum bistorta* extract and retinol.

[0030] Comparative Example 1. A retinol solution comprising 0.00025% (m / m) retinol.

[0031] Comparative Example 2. A solution of *Populus glossifolius*, based on the method for preparing *Populus glossifolius* extract disclosed in Example 1, comprising a *Populus glossifolius* solution with a concentration of 0.05% (m / m).

[0032] Comparative Example 3. A solution of *Populus gloriosa*, based on the method for preparing *Populus gloriosa* extract disclosed in Example 1, comprising a *Populus gloriosa* solution with a concentration of 0.005% (m / m).

[0033] I. Cytotoxicity test.

[0034] Experimental Methods: Fibroblasts were resuscitated and seeded into 96-well plates when the plating rate reached approximately 60%, and incubated overnight in an incubator (37℃, 5% CO2). Zeroing groups, solvent control groups, positive control groups (10% DMSO), and sample groups were set up. The sample groups, depending on the target cell, included different concentrations of retinol, different concentrations of *Populus glomeratus* extract, and different concentrations of a combination of *Populus glomeratus* extract and retinol. The zeroing group had no cell seeding. After drug administration, cells were incubated for 24 hours. Relative cell viability was measured using the MTT assay at a wavelength of 490 nm. Relative cell viability (%) = (OD200%) / (OD200%). 样品孔 -OD 调零孔 ) / (OD 溶剂对照孔-OD 调零孔 )*100%.

[0035] Table 1. Results of experiments on the toxicity of retinol to fibroblasts.

[0036]

[0037] Cell viability ≥90% indicates no cytotoxicity.

[0038] As shown in Table 1, retinol showed no significant cytotoxicity in the concentration range of 0.0005% (m / m). Therefore, 0.00025% (m / m) was selected as the concentration for subsequent experiments.

[0039] Table 2. Results of the fibroblast toxicity of *Populus glomeratus* extract.

[0040]

[0041] Cell viability ≥90% indicates no cytotoxicity.

[0042] As shown in Table 2, the extract of *Populus glomeratus* showed no significant cytotoxicity within a concentration range of 0.05% (m / m). In subsequent experiments, 0.05% (m / m) and 0.005% (m / m) were selected as the experimental concentrations.

[0043] To further demonstrate the safety of the compositions at the concentrations of Examples 1 and 2, the cytotoxicity of the retinol and *Populus glomeratus* extract compositions was tested. The results are shown in Table 3; the compositions described in Examples 1 and 2 showed no significant cytotoxicity.

[0044] Table 3. Fibroblast toxicity results of the combination of retinol and *Elaeagnus pungens* extract.

[0045]

[0046] Cell viability ≥90% indicates no cytotoxicity.

[0047] II. Firming and anti-wrinkle efficacy test.

[0048] Experimental Methods: Fibroblasts were resuscitated and randomly divided into a blank control group, a model group, a positive control group, and a drug-treated group. The cells were then seeded into 6-well plates and incubated overnight in an incubator (37℃, 5% CO2). Once the cell deposition rate reached 30%-50%, all groups except the blank control group underwent treatment with 30 J / cm² water. 2The samples were irradiated with UVA. After irradiation, they were cultured in an incubator (37℃, 5% CO2) for 24 h. Subsequently, culture medium was added to each well of the blank control group and the model group, the positive control group was given an equal volume of culture medium containing TGF-β1, and the other groups were given the corresponding concentration of culture medium containing the test sample and incubated for another 24 h. Gene detection of COL III and elastin and determination of COLI content were then performed.

[0049] Improvement rate (%) = (V 给药组 -V 模型组 ) / V 模型组 *100%

[0050] Experimental results:

[0051] Table 4. Effects of different embodiments and comparative examples on the relative expression level of COLIII gene.

[0052]

[0053] Compared with the blank control group, # P < 0.05 ## P < 0.01; compared with the model group, * P < 0.05 ** P < 0.01.

[0054] Table 5. Effects of different embodiments and comparative examples on the relative expression level of elastin gene.

[0055]

[0056] Compared with the blank control group, # P < 0.05 ## P < 0.01; compared with the model group, * P < 0.05 ** P < 0.01.

[0057] Table 6. Effects of different examples and comparative examples on COL I protein expression levels.

[0058]

[0059] Compared with the blank control group, # P < 0.05 ## P < 0.01; compared with the model group, * P < 0.05 ** P < 0.01.

[0060] COL I and COL III are two major collagen fibers in the skin. COL I forms a dense fibrous network to support the epidermis, while COL III imparts elasticity and extensibility to the tissue. Elastin is the main component of elastic fibers and, together with collagen fibers, maintains skin firmness, plumpness, and resists wrinkle formation. The results, as shown in Tables 4-6, indicate that Examples 1-2 and Comparative Examples 1-3 significantly increased the expression of COL I protein, as well as the gene expression of COL III and elastin. In particular, the combination of retinol and *Populus glehnia littoralis* extract in Examples 1 and 2 showed increases in COL I production of 137.13% and 116.61%, respectively, compared to the model group, both higher than the increases achieved by retinol or *Populus glehnia littoralis* extract alone. Compared to the model group, the combination of retinol and *Populus glehnia littoralis* extract significantly increased the relative expression levels of COL III and elastin genes. The increase rates for COL III gene expression were 497.37% and 305.26%, respectively, and the increase rates for elastin gene expression were 393.10% and 365.52%, respectively.

[0061] III. Verification of Synergistic Effects.

[0062] 1. Synergistic verification of COL III and elastin

[0063] Effect value E = (V 给药组 -V 模型组 ) / (V 空白对照组 -V 模型组 )*100%

[0064] Using the High Single Agent (HSA) model: If E (A+B) >max{E A E B If the combined effect is significantly different from that of the group with the highest single-drug effect, then a synergistic effect is determined.

[0065] Calculation results:

[0066] Table 7. Results of the verification of the synergistic effect between COL III and elastin

[0067]

[0068] Compared with the group with the highest single-drug effect, * P < 0.05 ** P < 0.01. As can be seen from the above, the highest single-drug effect group compared with Example 1 is Comparative Example 2, and the highest single-drug effect group compared with Example 2 is Comparative Example 3.

[0069] 2. Collaborative Validation of COL I

[0070] Effect value E = (V 给药组 -V 模型组 ) / (V 空白对照组 -V 模型组 )*100%

[0071] King's Q-value method for calculating synergistic effect: Q = E (A+B) / (E A +E B -E A *E B The effect value should be between 0 and 1. If Q ≥ 1.15, it is a synergistic effect; if 0.85 ≤ Q < 1.15, it is an additive effect; if Q < 0.85, it is an antagonistic effect.

[0072] Calculation results:

[0073] Table 8. Results of COL I Synergistic Effect Verification

[0074]

[0075] The results above indicate that the King's Q method is not applicable because some effect values ​​of COL III and elastin exceed 100%. Therefore, the HSA method was used to assist in the determination of COL III and elastin, while the King's Q method was used to determine COL I. The results are shown in Tables 7-8. The combination of 0.00025% retinol and 0.005%–0.05% of *Populus glehnia littoralis* extract showed a synergistic effect in promoting the expression of COL I, COL III, and elastin proteins. These results demonstrate that, after multi-model validation, *Populus glehnia littoralis* extract and retinol components have a synergistic effect in promoting collagen production, resulting in firming and anti-wrinkle effects. The combined use of *Populus glehnia littoralis* extract and retinol components can enhance the collagen-promoting effect of retinol components in firming and anti-wrinkle cosmetics.

Claims

1. A composition of *Populus glomeratus* extract and retinol, characterized in that, The composition comprises retinol and *Populus gleamingii* extract, wherein the mass ratio of retinol to *Populus gleamingii* extract is 1:(20-200).

2. The composition of *Populus glomeratus* extract and retinol according to claim 1, characterized in that, The preparation method of the *Populus gloriosa* extract includes the following steps: S1. Crude extraction: Take leaves of *Populus gloriosa*, add water at a material-to-liquid ratio of 1:20, heat and extract at 60℃~70℃, filter while hot after extraction, extract 1~3 times, combine the filtrates to obtain crude extract A. S2. Temperature gradient treatment: The crude extract A is kept at 30℃ for 0.75-1.25 h, 65℃ for 0.25-0.75 h, and 85℃ for 1.75-2.25 h in sequence to precipitate impurities. The precipitate is removed by hot filtration with 300-mesh filter paper to obtain filtrate B. S3. Membrane filtration purification: Filtrate B is filtered sequentially through an 800nm ​​ceramic membrane and a 50nm ceramic membrane to obtain purified filtrate C. S4. Drying treatment: The purified filtrate C is concentrated, freeze-dried or spray-dried to obtain the extract of *Populus glossifolia*.

3. The composition of *Populus glomeratus* extract and retinol according to claim 2, characterized in that: The heating extraction temperature in S1 is 65℃, and the extraction is performed twice.

4. The composition of *Populus glomeratus* extract and retinol according to claim 2, characterized in that: The crude extract A in S2 was kept at 30℃ for 1 h, 65℃ for 0.5 h, and 85℃ for 2 h in sequence.

5. The composition of *Populus glomeratus* extract and retinol according to claim 2, characterized in that: The extract of *Populus glomeratus* contains cambinin A.

6. The use of the composition of *Populus gloriosa* extract and retinol according to any one of claims 1-5 in the preparation of cosmetics with firming and / or anti-wrinkle effects.

7. The application of the composition of *Populus gloriosa* extract and retinol according to claim 6 in the preparation of cosmetics with firming and / or anti-wrinkle effects, characterized in that, The concentration of the combination of *Populus gloriosa* extract and retinol in the cosmetic product is as follows: the concentration of retinol is 0.00025% (m / m), and the concentration of *Populus gloriosa* extract is 0.005% to 0.05% (m / m).

8. The application of the composition of *Populus glomeratus* extract and retinol according to claim 6 in the preparation of cosmetics with firming and / or anti-wrinkle effects, characterized in that: The firming and / or anti-wrinkle effects are achieved by the synergistic promotion of gene expression of type I collagen, type III collagen and / or elastin by the extract of *Populus glossifolia* and retinol.

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