A pongamia seed extract, a preparation method and application thereof

Abstract

The application relates to a phyllium seed extract and a preparation method and application thereof, and belongs to the technical field of plant raw material extraction. The application relates to an application of a phyllium seed extract in preparation of anti-aging and soothing and repairing cosmetics. The application provides a green, efficient and efficacious soothing and repairing raw material for the cosmetic industry.

Description

Technical Field

[0001] This application relates to the field of plant raw material extraction technology, and in particular to a water croton seed extract, its preparation method and application. Background Technology

[0002] Pongamia pinnata (L.) Pierre, also known as hairless Pongamia glabra Vent., is a plant belonging to the genus Pongamia in the legume family (Fabaceae). It is widely distributed in tropical and subtropical regions of Asia, Australia, and the Pacific coast. Its seeds are rich in oils and flavonoids and have been used in traditional medicine for hundreds of years, primarily to treat skin infections, inflammation, and ulcers. Modern pharmacological studies have shown that the flavonoids in Pongamia seeds (such as quercetin and quercetin seed extract) possess various biological activities, including antioxidant, anti-inflammatory, antibacterial, and antiviral properties.

[0003] In the cosmetics field, PONGAMIA GLABRA seed oil has been included in the "Catalogue of Used Cosmetic Ingredients (2021 Edition)" under the INCI name "PONGAMIA GLABRA SEED OIL," primarily used as a moisturizer and skin conditioning agent. Existing patents (such as CN108024950A) disclose methods for enriching specific active ingredients from PONGAMIA GLABRA oil for use in cosmetic compositions; Unilever's patent application CN110022847A discloses a cosmetic composition containing chlorophyll dione and chlorophyllin for providing anti-abrasion photoprotection. However, the application of PONGAMIA GLABRA seed extract in the prior art is still mainly focused on moisturizing, sun protection, and single antioxidant effects in the form of seed oil. There are no reports of its systematic application in skin-repairing functional cosmetics, and multi-level efficacy verification from cells to animal models is also lacking. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the objectives of this application include providing a water-wampee seed extract, its preparation method, and its applications. This provides the cosmetics industry with a green, highly effective, and demonstrably potent soothing and repairing ingredient.

[0005] In one aspect, embodiments of this application provide the application of a water croton seed extract in the preparation of soothing and repairing cosmetics.

[0006] The *Wampee chinensis* seed extract provided in this application possesses excellent soothing and repairing effects, and can be effectively applied in the preparation of soothing and repairing cosmetics. This provides a novel technological pathway for the high-value utilization of *Wampee chinensis* seed extract and also offers a new option for the development of green functional raw materials in the cosmetics industry.

[0007] Secondly, embodiments of this application provide a multi-level evaluation system for the soothing and repairing effects of the water croton seed extract provided in the first aspect, including: testing the hyaluronidase inhibition rate at the biochemical level; testing the cell scratch healing rate, anti-inflammatory activity, and cell proliferation rate at the cellular level; and testing the anti-aging properties at the cellular and nematode levels.

[0008] The multi-level evaluation system for the soothing and repairing effects of the water croton seed extract provided in this application, with its multi-pathway synergistic effect, forms a complete chain of evidence for the repairing effects from "barrier protection - inflammation inhibition - proliferation repair - anti-aging".

[0009] In some embodiments of this application, the evaluation of anti-aging properties includes: testing β-galactosidase activity at the cellular level; and testing the accumulation of lipofuscin in nematodes, heat aging resistance, mitochondrial anti-aging properties, and longevity protein expression levels at the nematode level.

[0010] The anti-aging evaluation provided in this application verifies the efficacy of anti-aging from multiple dimensions and systematically studies the in vivo anti-aging properties of the extract of water croton seed.

[0011] Thirdly, embodiments of this application provide a method for preparing the water-wheat scutellaria seed extract provided in the first aspect, comprising: S1, Pre-process the raw material of water yellow peel seed to obtain water yellow peel seed powder; S2, after mixing the water croton seed powder with the extraction solvent, it is subjected to high-voltage pulse electric field treatment and then extracted; after extraction, it is separated and purified to obtain water croton seed extract.

[0012] The method for preparing water wampee seed extract provided in this application uses a pulsed electric field to disrupt the cell membrane structure of water wampee seed powder through instantaneous high-voltage electric pulses, forming an electroporation effect, which promotes the rapid release of intracellular flavonoids and other active ingredients. This method can be carried out at room temperature, avoiding the damage of heat-sensitive components to high temperatures, and significantly increasing the total flavonoid content of the extract. It has the advantages of short preparation time, low energy consumption, and high retention rate of active ingredients, thus enabling the efficient preparation of water wampee seed extract with excellent soothing and repairing effects.

[0013] In some embodiments of this application, the electric field strength of the high-voltage pulse electric field treatment is 1~10 kV / cm and the number of pulses is 50~130.

[0014] This application employs suitable conditions to treat water wampee seed powder with a high-voltage pulsed electric field, which can efficiently produce water wampee seed extract with excellent soothing and repairing effects.

[0015] In some embodiments of this application, water croton seed powder and extraction solvent are mixed at a material-to-liquid ratio of 1:5 to 30.

[0016] This application mixes water croton seed powder and extraction solvent at a suitable material-liquid ratio to facilitate the full dissolution and extraction of water croton seed powder, thereby efficiently obtaining water croton seed extract with excellent soothing and repairing effects.

[0017] In some embodiments of this application, the extraction temperature is 30~60 ℃ and the extraction time is 60~120 min.

[0018] This application employs suitable conditions for extraction, which facilitates the full extraction of active ingredients from the water croton seed powder, thereby efficiently obtaining a water croton seed extract with excellent soothing and repairing effects.

[0019] In some embodiments of this application, the extraction process further includes stirring at a speed of 500-1000 rpm.

[0020] This application employs a suitable stirring speed during extraction to improve extraction efficiency and increase the retention rate of active ingredients, thereby efficiently obtaining a water croton seed extract with excellent soothing and repairing effects.

[0021] In some embodiments of this application, the separation and purification after extraction includes: solid-liquid separation of the extract obtained from extraction, collection of filtrate, standing of filtrate at 4°C for 20-24 hours, centrifugation at 3000-8000 rpm for 10-30 minutes at 4°C, and filtration of the middle clear layer through a ceramic membrane after separation.

[0022] After extraction, this application involves separation and purification under suitable conditions, which not only facilitates the obtaining of pure water wampee seed extract but also reduces the loss of active ingredients, thereby efficiently producing water wampee seed extract with excellent soothing and repairing effects.

[0023] Fourthly, embodiments of this application provide a water-wheat seed extract prepared by the preparation method provided in the first aspect.

[0024] The preparation method provided in this application yields a water croton seed extract with excellent soothing and repairing effects, which can be effectively applied to the preparation of soothing and repairing cosmetics.

[0025] Fifthly, embodiments of this application provide a cosmetic composition comprising the water wampee seed extract provided in the fourth aspect; the water wampee seed extract has a mass percentage of 0.1% to 10%.

[0026] The water croton seed extract provided in this application has excellent soothing and repairing effects. When applied in an appropriate amount to a cosmetic composition, a cosmetic composition with soothing and repairing effects can be obtained. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a comparison of the total flavonoid content of the extracts from Example 1 of this application and Comparative Example 1.

[0029] Figure 2 The in vitro antioxidant activity of the extracts from Example 1 and Comparative Example 1 of this application is shown.

[0030] Figure 3 This study examines the cytotoxicity of the extract at different concentrations in Example 1 of this application.

[0031] Figure 4 The hyaluronidase inhibition rate of the extract in Example 1 of this application.

[0032] Figure 5 The cell scratch healing rate at different concentrations of the extract in Example 1 of this application.

[0033] Figure 6 These are representative images from the cell scratch experiment in Example 5 of this application.

[0034] Figure 7 This study examines the effects of different concentrations of the extract from Example 1 of this application on cellular inflammatory factors.

[0035] Figure 8 This study examines the cell proliferation activity of the extract at different concentrations in Example 1 of this application.

[0036] Figure 9 This is a representative image of β-galactosidase staining in cells from Experiment Example 8 of this application.

[0037] Figure 10 The effect of the extract in Example 1 of this application on UVB-induced cell aging.

[0038] Figure 11 The effect of different concentrations of the extract in Example 1 of this application on lipofuscin from nematodes.

[0039] Figure 12 This study examines the anti-heat aging activity of different concentrations of the extract from Example 1 of this application.

[0040] Figure 13 This study examines the anti-aging activity of nematode mitochondria extracted from Example 1 of this application.

[0041] Figure 14The expression level of SIR-2.1 in nematodes extracted in Example 1 of this application. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0043] Currently, the application of water croton seed extract is mainly focused on moisturizing, sun protection and single antioxidant effects in the form of seed oil. There are no reports of its systematic application in skin repair functional cosmetics, and there is also a lack of multi-level efficacy verification from cells to model animals.

[0044] To provide the cosmetics industry with a green, efficient, and proven soothing and repairing ingredient, this application provides an example of the application of *Coptis chinensis* seed extract in the preparation of soothing and repairing cosmetics.

[0045] The *Wampee chinensis* seed extract provided in this application possesses excellent soothing and repairing effects, and can be effectively applied in the preparation of soothing and repairing cosmetics. This provides a novel technological pathway for the high-value utilization of *Wampee chinensis* seed extract and also offers a new option for the development of green functional raw materials in the cosmetics industry.

[0046] This application provides a multi-level evaluation system for the soothing and repairing effects of the above-mentioned *Coptis chinensis* seed extract, including: testing hyaluronidase inhibition rate at the biochemical level; testing cell scratch healing rate, anti-inflammatory activity, and cell proliferation rate at the cellular level; and testing anti-aging activity at both the cellular and nematode levels. The multi-level evaluation system for the soothing and repairing effects of the *Coptis chinensis* seed extract provided by this application, through multi-pathway synergistic effects, forms a complete chain of evidence for repairing effects from "barrier protection - inflammation inhibition - proliferation repair - anti-aging".

[0047] In some embodiments of this application, the evaluation of anti-aging efficacy includes: testing β-galactosidase activity at the cellular level; and testing the accumulation of lipofuscin in nematodes' intestines, nematode resistance to heat aging, nematode mitochondrial anti-aging, and the expression level of nematode longevity proteins at the nematode level. The evaluation of anti-aging efficacy provided in this application verifies the efficacy of anti-aging from multiple dimensions and systematically studies the in vivo anti-aging properties of *Coptis chinensis* seed extract.

[0048] Caenorhabditis elegans, as an internationally recognized model organism for aging research, has been widely used in evaluating the anti-aging efficacy of cosmetics. Existing studies have constructed anti-aging evaluation methods based on Caenorhabditis elegans, using indicators such as heat stress resistance, UV radiation resistance, and lipofuscin accumulation for comprehensive evaluation. These methods have advantages such as short growth cycle, simple experimental operation, and high conservation with mammalian aging signaling pathways. Therefore, this application utilizes the nematode model to evaluate the anti-aging properties of *Cephalotaxus fortunei* seed extract in multiple dimensions.

[0049] This application constructs a multi-level, multi-indicator efficacy evaluation system from enzymology, cells to model animals (C. elegans); through hyaluronidase inhibition, cell scratch assay, cell proliferation, cytotoxicity, anti-inflammation, β-galactosidase staining, and indicators such as nematode anti-carbonylation, anti-photoaging, longevity protein expression, and mitochondrial function, it comprehensively verifies the soothing and repairing efficacy of *C. elegans* seed extract; realizes the high-value utilization of *C. elegans* seed resources, provides the cosmetics industry with a green, efficient, and effective anti-aging and repairing raw material, and provides a methodological reference for the research and evaluation of plant-derived cosmetic raw materials.

[0050] This application provides a method for preparing the above-mentioned water croton seed extract, comprising: S1, Pre-process the raw material of water yellow peel seed to obtain water yellow peel seed powder; S2, after mixing the water croton seed powder with the extraction solvent, it is subjected to high-voltage pulse electric field treatment and then extracted; after extraction, it is separated and purified to obtain water croton seed extract.

[0051] The method for preparing water wampee seed extract provided in this application uses a pulsed electric field to disrupt the cell membrane structure of water wampee seed powder through instantaneous high-voltage electric pulses, forming an electroporation effect, which promotes the rapid release of intracellular flavonoids and other active ingredients. This method can be carried out at room temperature, avoiding the damage of heat-sensitive components to high temperatures, and significantly increasing the total flavonoid content of the extract. It has the advantages of short preparation time, low energy consumption, and high retention rate of active ingredients, thus enabling the efficient preparation of water wampee seed extract with excellent soothing and repairing effects.

[0052] In some embodiments of this application, the electric field strength of the high-voltage pulsed electric field treatment is 1~10 kV / cm, and the number of pulses is 50~130. As an example, the electric field strength of the high-voltage pulsed electric field treatment can be, but is not limited to, any value of 1 kV / cm, 2 kV / cm, 3 kV / cm, 4 kV / cm, 5 kV / cm, 6 kV / cm, 7 kV / cm, 8 kV / cm, 9 kV / cm, 10 kV / cm, or a range between these values. The number of pulses can be, but is not limited to, any value of 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, or a range between these values. By subjecting water wampee seed powder to high-voltage pulsed electric field treatment under suitable conditions, water wampee seed extract with excellent soothing and repairing effects can be efficiently obtained.

[0053] In some embodiments of this application, the water lily seed powder and extraction solvent are mixed at a material-to-liquid ratio of 1:5 to 30. As an example, the water lily seed powder and extraction solvent can be mixed at any point value or a range between the two, including but not limited to material-to-liquid ratios of 1:5, 1:6, 1:8, 1:10, 1:12, 1:15, 1:16, 1:18, 1:20, 1:22, 1:25, 1:26, 1:28, and 1:30. Mixing the water lily seed powder and extraction solvent at a suitable material-to-liquid ratio facilitates the full dissolution and extraction of the water lily seed powder, thereby efficiently obtaining a water lily seed extract with excellent soothing and repairing effects.

[0054] In some embodiments of this application, the extraction solvent is an aqueous solution. Aqueous solutions, as extraction solvents, are environmentally friendly and meet the sustainable development requirements of the cosmetics industry.

[0055] In some embodiments of this application, the extraction temperature is 30-60 °C, and the extraction time is 60-120 min. As an example, the extraction temperature can be, but is not limited to, any value of 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, or 60 °C, or a range between these values. The extraction time can be, but is not limited to, any value of 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, 95 min, 100 min, 105 min, 110 min, 115 min, or 120 min, or a range between these values. Using suitable conditions for extraction facilitates the full extraction of active ingredients from the *Hydrocotyle sibthorpioides* seed powder, thereby efficiently obtaining a *Hydrocotyle sibthorpioides* seed extract with excellent soothing and repairing effects.

[0056] In some embodiments of this application, the extraction process further includes stirring at a speed of 500-1000 rpm. As an example, the stirring speed can be, but is not limited to, any value from 500 rpm, 550 rpm, 600 rpm, 650 rpm, 700 rpm, 750 rpm, 800 rpm, 850 rpm, 900 rpm, 950 rpm, and 1000 rpm, or a range between both. Simultaneous stirring at an appropriate speed during extraction improves extraction efficiency and increases the retention rate of active ingredients, thereby efficiently obtaining a water-wheat peel seed extract with excellent soothing and repairing effects.

[0057] In some embodiments of this application, the separation and purification process after extraction includes: solid-liquid separation of the extract, collection of the filtrate, standing of the filtrate at 4°C for 20-24 hours, centrifugation at 3000-8000 rpm for 10-30 minutes at 4°C, separation of the layers, and filtration of the clear middle layer through a ceramic membrane. As an example, the centrifugation speed can be, but is not limited to, any value of 3000 rpm, 3500 rpm, 4000 rpm, 4500 rpm, 5000 rpm, 5500 rpm, 6000 rpm, 6500 rpm, 7000 rpm, 7500 rpm, or 8000 rpm, or a range between these values. The centrifugation time can be, but is not limited to, any value of 10 minutes, 12 minutes, 15 minutes, 16 minutes, 18 minutes, 20 minutes, 22 minutes, 25 minutes, 26 minutes, 28 minutes, or 30 minutes, or a range between these values. After extraction, separation and purification under suitable conditions not only facilitate the obtaining of pure water wampee seed extract but also reduce the loss of active ingredients, thus efficiently producing water wampee seed extract with excellent soothing and repairing effects.

[0058] This application provides a water-wampee seed extract prepared by the above-described preparation method. The water-wampee seed extract prepared by the method provided in this application has excellent soothing and repairing effects and can be effectively applied to the preparation of soothing and repairing cosmetics.

[0059] This application provides a cosmetic composition comprising the above-mentioned *Wampee citronella* seed extract; the mass percentage of the *Wampee citronella* seed extract is 0.1% to 10%. As an example, the mass percentage of the *Wampee citronella* seed extract can be, but is not limited to, any point value or a range between 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, and 10%. The *Wampee citronella* seed extract provided in this application has excellent soothing and repairing effects; when applied in an appropriate amount to a cosmetic composition, a cosmetic composition with soothing and repairing effects can be obtained.

[0060] The features and performance of this application will be further described in detail below with reference to the embodiments.

[0061] Example 1 This embodiment provides a method for preparing a water-wampee seed extract, including: S1. Remove impurities from the water huangpi seeds, dry them, crush them, and sieve them to obtain water huangpi seed powder; S2, mix water yellow peel seed powder with water at a material-to-liquid ratio of 1:10 (g / mL), and subject it to high-voltage pulse electric field treatment under an electric field strength of 3kV / cm and a pulse number of 90; then stir and extract under conditions of 50℃ and 700rpm. S3. After filtering the extract, let it stand at 4℃ for 24 hours, then freeze and centrifuge at 4℃ and 6000rpm for 15 minutes. Take the middle layer of clear liquid and pass it through a 100nm ceramic membrane filter. After the filtrate is concentrated and freeze-dried, the water yellow peel seed extract is obtained.

[0062] Comparative Example 1 This comparative example provides a method for preparing a water croton seed extract, which differs from Example 1 in that: in step (2), water croton seed powder and aqueous solution are mixed at a ratio of 1:10 (g / mL), placed in an ultrasonic cleaner, and ultrasonically extracted for 30 min at a power of 200 W and a temperature of 50°C; then, the mixture is stirred and extracted at a stirring speed of 700 rpm and a stirring temperature of 50°C.

[0063] Experimental Example 1 This experiment determined the total flavonoid content of the extracts of *Coptis chinensis* seeds provided in Example 1 and Comparative Example 1 using the sodium nitrite-aluminum nitrate colorimetric method, with rutin as the standard. The total flavonoid content of the extracts of Example 1 (PEF) and Comparative Example 1 (UVE) was determined; the results are as follows. Figure 1 As shown.

[0064] Depend on Figure 1The results showed that the flavonoid content in the extract of *Wampee citrifolia* seeds provided in Example 1 was significantly higher (p<0.05), reaching 0.33 mg / mL, which was 25.36% higher than that in the extract of *Wampee citrifolia* seeds provided in Comparative Example 1. This indicates that the high-voltage pulsed electric field treatment used in this application can more efficiently disrupt the cell wall and cell membrane structure of *Wampee citrifolia* seeds, promoting the release and dissolution of intracellular flavonoid active substances. Flavonoids are the core material basis for the antioxidant, anti-inflammatory, and skin repair effects of *Wampee citrifolia* seeds, and their content directly determines the efficacy potential of the extract.

[0065] Experimental Example 2 This experiment tested the in vitro antioxidant activity of the extracts of *Coptis chinensis* seeds provided in Example 1 and Comparative Example 1. The in vitro antioxidant activity of the extracts from Example 1 (PEF) and Comparative Example 1 (UVE) was characterized by DPPH radical scavenging rate and ABTS radical scavenging rate. The results are as follows: Figure 2 As shown.

[0066] Depend on Figure 2 The results showed that, across multiple dilution ratios, the ABTS radical scavenging rate of the *Wampee chinensis* seed extract provided in Example 1 was significantly higher than that of the *Wampee chinensis* seed extract provided in Comparative Example 1 (p<0.05), exhibiting a clear dose-dependent effect. For instance, at a 64-fold dilution, the *Wampee chinensis* seed extract provided in Example 1 still maintained a high scavenging rate (70.49%), while the activity of the *Wampee chinensis* seed extract provided in Comparative Example 1 had significantly decreased. Simultaneously, the *Wampee chinensis* seed extract provided in Example 1 maintained high DPPH radical scavenging activity at all dilution ratios, demonstrating stronger antioxidant stability and efficacy potential. This indicates that the *Wampee chinensis* seed extract prepared using high-voltage pulsed electric field treatment in this application possesses stronger antioxidant stability and efficacy potential.

[0067] Experimental Example 3 This experiment investigated the cytotoxicity of the *Coptis chinensis* seed extract provided in Example 1. The CCK-8 assay was used to determine the effect of different concentrations of the *Coptis chinensis* seed extract provided in Example 1 (20%, 10%, 5%, 2%, 1.25%, 0.625%, 0.3125%) on the activity of HDF (human dermal fibroblasts). The results are as follows: Figure 3 As shown.

[0068] Depend on Figure 3 The results showed that within a concentration gradient range of 0.3125% to 20%, the extract of *Coptis chinensis* seed had no significant effect on cell viability. Cell viability remained above 91.14% at all tested concentrations, with no statistically significant differences between groups. This indicates that the *Coptis chinensis* seed extract provided in this application is non-cytotoxic, environmentally friendly, and safe, making it suitable as a raw material for cosmetics.

[0069] Test Example 4 This experiment investigated the hyaluronidase inhibition of the *Coptis chinensis* seed extract provided in Example 1. The extract, at a concentration of 1%, was used. An in vitro hyaluronidase inhibition model was employed to determine the inhibition rate. Common soothing and repairing drugs such as dipotassium glycyrrhizate, ectoine, and *Centella asiatica* extract were used as positive controls, all at a concentration of 1%. The results are as follows: Figure 4 As shown.

[0070] Hyaluronidase degrades hyaluronic acid in the skin, disrupting the skin barrier's integrity and releasing inflammatory mediators, thus exacerbating skin sensitivity and inflammation. Therefore, inhibiting this enzyme activity is a crucial pathway for cosmetic ingredients to exert their repairing effects. Figure 4 The results showed that the *Coptis chinensis* seed extract provided in Example 1 had an inhibition rate of 43.98% against hyaluronidase. Its inhibitory effect was not significantly different from that of the well-known soothing drug dipotassium glycyrrhizate (39.68%), but slightly weaker than that of *Centella asiatica* extract (52.34%). This indicates that the *Coptis chinensis* seed extract provided in this application has a good inhibitory effect on hyaluronidase.

[0071] Experimental Example 5 This experiment used the *Hydrocotyle vulgaris* seed extract provided in Example 1 and Comparative Example 1 to perform a cell scratch assay. HDF cells were cultured to a confluent monolayer. After scratching, culture media containing different concentrations (1%, 2%, 5%, 10%) of the *Hydrocotyle vulgaris* seed extract provided in Example 1 were added. The blank control group was given culture media without the *Hydrocotyle vulgaris* seed extract provided in Example 1. The scratch healing rate was calculated after 24 h of culture. The results are as follows: Figures 5-6 As shown.

[0072] Depend on Figures 5-6 The results showed that the scratch healing rate of the blank control group was only 12.20%, while the scratch healing rates of all treatment groups with different concentrations of *Coptis chinensis* seed extract were significantly higher than those of the blank control group (p<0.05), with the 5% concentration group showing the highest healing rate of 51.85%. This indicates that the *Coptis chinensis* seed extract provided in this application can significantly promote the migration and repair of HDF cells and has potential skin barrier repair and wound healing activity.

[0073] Experimental Example 6 This experiment investigated the anti-inflammatory activity of the *Hydrocotyle vulgaris* seed extract provided in Example 1. An HDF cell model was stimulated with LPS, and qPCR was used to detect the expression levels of inflammatory factors such as IL-6 and TNF-α in cells treated with different concentrations of *Hydrocotyle vulgaris* seed extract (0.25%, 0.50%, and 1%) provided in Example 1. The results are as follows: Figure 7 As shown.

[0074] Depend on Figure 7The results showed that the relative levels of IL-6 and TNF-α in the LPS-induced model were significantly higher than those in the blank control group (p<0.05), indicating that the inflammation model was successfully constructed. The levels of IL-6 and TNF-α in each concentration group of *Coptis chinensis* seed extract (0.25%, 0.50%, 1%) were significantly lower than those in the LPS-induced model group (p<0.05), with the 0.25% concentration group showing the lowest levels of inflammatory factors, and the levels of IL-6 and TNF-α decreasing by approximately 75% compared to the model group. This demonstrates that the *Coptis chinensis* seed extract provided in this application possesses significant anti-inflammatory activity.

[0075] Experimental Example 7 This experiment used the *Coptis chinensis* seed extract provided in Example 1 to conduct a cell proliferation experiment. The CCK-8 assay was used to determine the effect of different concentrations of the *Coptis chinensis* seed extract provided in Example 1 (10%, 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.15625%, 0.078125%) on HDF cell proliferation; the results are as follows. Figure 8 As shown.

[0076] Depend on Figure 8 The results showed that within a concentration gradient range of 0.078125% to 10%, the cell survival rate of the treatment groups treated with water croton seed extract remained at a high level, with the highest survival rate reaching 117.10%. This indicates that the water croton seed extract provided in this application has a significant proliferative effect on skin cells, and this effect tends to increase with increasing concentration.

[0077] Experimental Example 8 This experiment investigated the β-galactosidase activity of the *Clausena lansium* seed extract provided in Example 1 using a β-galactosidase staining kit. Groups included a blank control group, a UVB damage model, a positive control, and 1% *Clausena lansium* seed extract. Cell viability was also measured using the MTT assay. Results are as follows: Figure 9 As shown.

[0078] β-galactosidase is a specific marker of senescent cells; positively stained cells appear blue, and the number and intensity of blue-stained cells directly reflect the degree of cellular senescence. A UVB-induced photoaging model was established, and the inhibitory effect of *Coptis chinensis* seed extract on the cellular senescence process was visually evaluated through staining for senescence-related β-galactosidase. Figure 9 The results showed that the number of blue-stained positive cells in the UVB damage model group was significantly increased, indicating that photoaging of cells was successfully induced. Compared with the model group, the number of blue-stained positive cells in the positive control group and the water wampee seed extract treatment group was significantly reduced, indicating that the water wampee seed extract provided in this application can significantly inhibit UVB-induced cell senescence.

[0079] The protective effect of *Coptis chinensis* seed extract against UVB-induced photodamage was evaluated simultaneously using the MTT assay; the results are as follows: Figure 10 As shown.

[0080] Depend on Figure 10 The results showed that, compared with the UVB damage model group, the cell viability of the water wampee seed extract treatment group was significantly improved (p<0.05), and the water wampee seed extract group recovered to 55.21%, indicating that the water wampee seed extract provided in this application can significantly reduce UVB-induced cell damage, improve the survival rate and viability of damaged cells, and has clear photoprotective and anti-photoaging activities.

[0081] Experimental Example 9 This experiment used the *Phellodendron amurense* seed extract provided in Example 1 to determine the lipofuscin content in the intestines of nematodes. Aged wild-type nematodes (day 10) cultured with different concentrations of the *Phellodendron amurense* seed extract provided in Example 1 (0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL) were analyzed for autofluorescence of intestinal lipofuscin using a fluorescence microscope, reflecting anti-carbonylation activity. The results are as follows: Figure 11 As shown.

[0082] Intestinal lipofuscin is the end product of lipid peroxidation and protein carbonylation in nematodes. Its accumulation is closely related to the aging process and the degree of oxidative damage, and it is often used as an aging biomarker. Figure 11 The results showed that the intestinal lipofuscin level was the highest in the blank control group. The levels of each concentration of water wampee seed extract were significantly lower than those in the blank control group (p<0.05), with a decrease of 15.27-27.42%, indicating that the water wampee seed extract provided in this application can effectively delay the aging process.

[0083] Experimental Example 10 This experiment investigated the heat aging resistance of nematodes to the extract of *Hymenochloa crus-galli* seeds provided in Example 1. Four-day-old TJ375 transgenic nematodes cultured with different concentrations of the extract (0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL) were subjected to heat stress at 35°C for 2 hours, followed by recovery at 20°C for 2 hours. ImageJ software was used to quantitatively analyze the fluorescence intensity of the nematodes in the images. The fluorescence intensity changes of the TJ375 transgenic nematode strain (containing a green fluorescent protein reporter gene (GFP) coupled with an hsp 16.2 promoter, exhibiting the same expression pattern as HSP-16.2) characterized the expression of the heat stress protein HSP-16.2. The change in fluorescence intensity of the nematodes treated with the extract under heat stress conditions demonstrated its heat aging resistance activity. The results are as follows: Figure 12 As shown.

[0084] HSP-16.2 is a heat shock protein expressed by nematodes under heat stress, and its expression level is positively correlated with cellular stress pressure. Figure 12The results showed that the average fluorescence intensity of the blank control group reached 864.82, indicating a severe heat stress response. In contrast, the expression level of HSP 16.2 was significantly reduced in all treatment groups treated with different concentrations of *Coptis chinensis* seed extract (p<0.05), especially in the 0.5 mg / mL extract treatment group, where the average fluorescence intensity was only 519.23, a reduction of approximately 40%. This indicates that the *Coptis chinensis* seed extract provided in this application can significantly reduce stress levels and alleviate aging damage caused by heat stimulation.

[0085] Experimental Example 11 This experiment investigated the anti-aging effects of *Cephalotaxus fortunei* seed extract provided in Example 1 on nematode mitochondria. Six-day-old CB7272 nematodes cultured with *Cephalotaxus fortunei* seed extract (1 mg / mL) provided in Example 1 were used. The fluorescence intensity of the mitochondrial respiratory chain complex was measured to assess respiratory chain function. Common mitochondrial anti-aging components such as chlorogenic acid, resveratrol, and curcumin were used as positive controls. The results are as follows: Figure 13 As shown.

[0086] The mitochondrial respiratory chain complex, a core component of the oxidative phosphorylation system, exhibits a significant decline in abundance with age. Respiratory chain function can be assessed by quantifying its specific fluorescence intensity. Figure 13 The results showed that the fluorescence intensity of the complex significantly increased after treatment with the *Coptis chinensis* seed extract, with an average fluorescence intensity increase of 12.92% (769.27). This effect is comparable to known mitochondrial anti-aging components such as chlorogenic acid (average fluorescence intensity 799.49), resveratrol (average fluorescence intensity 795.71), and curcumin (average fluorescence intensity 768.44). This indicates that the *Coptis chinensis* seed extract provided in this application enhances mitochondrial electron transport capacity, thereby improving mitochondrial aging and increasing energy metabolism levels.

[0087] Experimental Example 12 This experiment investigated the longevity protein of nematodes using the *Cephalotaxus fortunei* seed extract provided in Example 1. Three-day-old SIR-2.1::mNG nematodes cultured with the *Cephalotaxus fortunei* seed extract (1 mg / mL) provided in Example 1 were analyzed for average fluorescence intensity under an inverted fluorescence microscope to quantify the SIR-2.1 protein. Resveratrol was used as a positive control. The results are as follows: Figure 14 As shown.

[0088] The SIR-2.1 protein in *C. elegans* is a deacetylase that shares high structural homology and functional conservation with SIRT1, a key anti-aging protein in humans. Also known as a longevity protein, it regulates aging-related signaling pathways, repairs cell damage, and slows aging. As nematodes age, SIR-2.1 expression is continuously downregulated, leading to a decrease in the level of deacetylases and accelerating the aging process. Figure 14The results showed that the expression level of SIR-2.1 in the group treated with the seed extract of *Coptis chinensis* was significantly increased by 12.47% (p<0.05), which was comparable to the effect of resveratrol, a known sir-2.1 activator, indicating that the seed extract of *Coptis chinensis* provided in this application effectively activates endogenous anti-aging.

[0089] 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. The application of a water-wampee seed extract in the preparation of soothing and repairing cosmetics.

2. A multi-level evaluation system for the soothing and repairing effects of the water croton seed extract according to claim 1, characterized in that, include: Hyaluronidase inhibition rate was tested at the biochemical level; cell scratch healing rate, anti-inflammatory activity and cell proliferation rate were tested at the cellular level; and anti-aging activity was tested at both the cellular and nematode levels.

3. The multi-level evaluation system for the soothing and repairing effects of the water croton seed extract according to claim 2, characterized in that, The evaluation of anti-aging properties includes: testing β-galactosidase activity at the cellular level; and testing the accumulation of lipofuscin in nematodes, heat aging resistance, mitochondrial anti-aging properties, and longevity protein expression levels at the nematode level.

4. A method for preparing the extract of water croton seed as described in claim 1, characterized in that, include: S1, Pre-process the raw material of water yellow peel seed to obtain water yellow peel seed powder; S2, the water wampee seed powder is mixed with the extraction solvent and then subjected to high-voltage pulse electric field treatment, followed by extraction; after extraction, it is separated and refined to obtain water wampee seed extract.

5. The preparation method according to claim 4, characterized in that, The electric field strength of the high-voltage pulse electric field treatment is 1~10 kV / cm, and the number of pulses is 50~130.

6. The preparation method according to claim 4, characterized in that, The water yellow peel seed powder and the extraction solvent are mixed at a material-to-liquid ratio of 1:5~30.

7. The preparation method according to claim 4, characterized in that, The extraction temperature is 30~60℃, and the extraction time is 60~120 min; Optionally, the extraction process further includes stirring at a speed of 500-1000 rpm.

8. The preparation method according to claim 4, characterized in that, The separation and purification process after extraction includes: solid-liquid separation of the extract obtained from extraction, collection of filtrate, standing of the filtrate at 4°C for 20-24 hours, centrifugation at 3000-8000 rpm for 10-30 minutes at 4°C, separation of the middle clear layer and filtration through a ceramic membrane.

9. A water-rich yellow peel seed extract prepared by the preparation method according to any one of claims 4 to 8.

10. A cosmetic composition comprising the water wampee seed extract of claim 9; wherein the water wampee seed extract is present in a mass percentage of 0.1% to 10%.

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