Enteromorpha bioactive peptide as well as preparation and application thereof

Abstract

The invention belongs to the technical field of active peptide protein, and particularly relates to enteromorpha active peptide as well as preparation and application thereof. The enteromorpha bioactive peptide disclosed by the invention is obtained by performing enzymolysis on enteromorpha alkali-extracted protein, performing ultrafiltration and performing chromatographic separation. The preparation method of the enteromorpha active peptide is simple, convenient, efficient, green and safe, the prepared enteromorpha active peptide can efficiently remove ROS, DPPH free radicals and ABTS free radicals, lipid peroxidation is effectively inhibited, SOD activity is enhanced, excellent anti-oxidation and cell protection effects are shown, the enteromorpha active peptide is used in the fields of food, medicine, health care products or cosmetics and the like, and the enteromorpha active peptide has wide application prospects. Products with the functions of resisting oxidation and protecting cells are prepared, and a new way is opened up for resource utilization of enteromorpha.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to bioactive peptides of Ulva prolifera and their preparation and application. Background Technology

[0002] Bioactive peptides are a class of natural molecules with diverse physiological functions, typically released from parent proteins through enzymatic hydrolysis or biotechnology to exert their active effects. These molecules are not only found in plants and animals but can also be obtained through microbial synthesis or chemical recombination, encompassing a wide range of functions including antibacterial, antioxidant, antihypertensive, lipid-lowering, and anti-aging effects. Seaweed, with a protein content between 10% and 30%, is an excellent protein source for producing bioactive peptides. Algal bioactive peptides exhibit significant antioxidant potential through mechanisms such as free radical scavenging and inhibition of oxidative stress, making them natural candidates to replace synthetic antioxidants.

[0003] Ulva prolifera, belonging to the phylum Chlorophyta, order Ulvales, and family Ulvaceae, is characterized by its high protein content (16.0-33.0%) and low fat content (0.2-0.8%), giving it high nutritional value. During green tides, Ulva prolifera grows rapidly, producing a massive biomass, with annual peaks exceeding 10 million tons in the Yellow Sea. Numerous studies have shown that Ulva prolifera is rich in bioactive compounds, including polysaccharides, polyphenols, polypeptides, and other bioactive substances.

[0004] The preparation methods for algal bioactive peptides mainly include ultrasound-assisted extraction, enzyme-assisted extraction, and microwave-assisted extraction. Among these, ultrasound-assisted extraction, through the cavitation effect of ultrasound, can significantly improve the cell wall rupture efficiency, enhance the release of bioactive components, and reduce the degradation of heat-sensitive components, thus exhibiting significant advantages in peptide extraction. This method is simple to operate, quick, consumes little solvent, and has high extraction efficiency, gradually becoming an effective extraction technique. For the extracted peptides, common separation and purification methods include ultrafiltration, gel chromatography, and liquid chromatography. Ultrafiltration technology effectively removes unwanted impurities and concentrates the peptide solution through membrane separation; gel chromatography utilizes the distribution of different molecular sizes to separate and purify peptides; and liquid chromatography uses high-resolution analytical methods to accurately identify the molecular structure and function of peptides.

[0005] As a representative of large algae, the high protein content and potential for developing active ingredients in *Ulva prolifera* have not yet been fully explored. To date, research on *Ulva prolifera* has largely focused on the activity of polysaccharides and polyphenols. Existing technologies rarely disclose the extraction and application of active peptides from *Ulva prolifera*. However, it has been reported that angiotensin-converting enzyme (ACE) inhibitory peptides have been screened from *Ulva prolifera* protein degradation products.

[0006] Bioactive peptides extracted from marine algae have received considerable attention in the development of cosmetic products. However, very few seaweed bioactive peptides reach consumers. One reason is that traditional extraction methods for seaweed bioactive peptides suffer from problems such as low extraction efficiency, complex extraction processes, environmental pollution caused by the use of toxic organic solvents, and potential cytotoxicity, limiting the large-scale production and application of seaweed bioactive peptides. Another reason is that most active ingredients added to existing cosmetics rely on chemical synthesis, and these synthetic ingredients may have certain side effects and low absorption rates, posing potential risks to skin health. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention focuses on *Ulva prolifera* as the research object, isolates active peptides from *Ulva prolifera*, and explores their antioxidant activity and mechanism in vivo and in vitro. This not only expands the raw material sources for new functional foods, pharmaceuticals, health products, cosmetics, and other industries, but also provides a green solution to alleviate the nutritional and health needs under population growth, and promotes the industrial application of algal resources in the field of antioxidants.

[0008] This invention has found that the active peptides of *Ulva prolifera* can efficiently scavenge ROS, DPPH free radicals and ABTS free radicals, effectively inhibit lipid peroxidation while enhancing SOD activity, exhibiting excellent antioxidant effects and protecting cells.

[0009] This invention provides antioxidant and / or cell protection applications for *Ulva prolifera* bioactive peptides, wherein the bioactive peptides are selected from at least one of the following: (1) The enzymatic hydrolysis product of the alkaline extract of Ulva prolifera is selected from components of different molecular weights obtained by ultrafiltration centrifugation; further, it is selected from at least one of three components with molecular weights greater than 5kDa, 3-5kDa, and less than 3kDa obtained by ultrafiltration centrifugation with molecular weight cutoffs of 5kDa and 3kDa, preferably the component with a molecular weight less than 3kDa; the enzymatic hydrolysis product is selected from any one or any combination of papain hydrolysis product, alkaline protease hydrolysis product, trypsin hydrolysis product, bromelain hydrolysis product, and complex protease hydrolysis product, preferably papain hydrolysis product; the alkaline extract of Ulva prolifera is an alkaline aqueous extract of Ulva prolifera, wherein the ratio of Ulva prolifera to alkaline aqueous solution is 1g:10-60 mL, further 1g:40-60 mL, further 1g:50-55 mL. mL; the pH value of the alkaline aqueous solution is 7-10, further 9-10, further 10, and the alkali dissolved in the alkaline aqueous solution is a strong or weak alkali, including but not limited to basic sodium salts and basic potassium salts, such as sodium hydroxide, sodium bicarbonate, sodium carbonate, potassium hydroxide, potassium bicarbonate, potassium carbonate, etc.; the extraction temperature is 30-70℃, further 60±5℃, further 60-65℃, and even further 60-60.5℃; the extraction time is 0.5-4h, further 2-4h, and even further 3h.

[0010] (2) The glucan gel eluent of the proteolytic product of the alkali extract of Ulva lactuca, further the pure water eluent of the glucan gel, further the eluent contained in the earliest elution peak of the pure water elution of the glucan gel.

[0011] Furthermore, the active peptides of *Ulva prolifera* are used to prepare antioxidant products and / or cell protection products, including but not limited to food, pharmaceuticals, health products or cosmetics. The products also contain excipients that meet the requirements of food, pharmaceuticals, health products or cosmetics, and other active or auxiliary active ingredients for antioxidant and / or cell protection.

[0012] This invention provides an antioxidant and / or cell-protective product containing at least one of the following active peptides selected from: (1) The enzymatic hydrolysis product of the alkaline extract of Ulva prolifera is selected from components of different molecular weights obtained by ultrafiltration centrifugation of the alkaline extract of Ulva prolifera, and further selected from at least one of three components with molecular weights greater than 5kDa, 3-5kDa, and less than 3kDa obtained by ultrafiltration centrifugation with molecular weight cutoffs of 5kDa and 3kDa, preferably the component with a molecular weight less than 3kDa; the enzymatic hydrolysis product is selected from any one or any combination of papain hydrolysis product, alkaline protease hydrolysis product, trypsin hydrolysis product, bromelain hydrolysis product, and complex protease hydrolysis product, preferably papain hydrolysis product; the alkaline extract of Ulva prolifera is an alkaline aqueous extract of Ulva prolifera, wherein the ratio of Ulva prolifera to alkaline aqueous solution is 1g:10-60 mL, further 1g:40-60 mL, further 1g:50-55 mL. mL; the pH value of the alkaline aqueous solution is 7-10, further 9-10, further 10, and the alkali dissolved in the alkaline aqueous solution is a strong or weak alkali, including but not limited to basic sodium salts and basic potassium salts, such as sodium hydroxide, sodium bicarbonate, sodium carbonate, potassium hydroxide, potassium bicarbonate, potassium carbonate, etc.; the extraction temperature is 30-70℃, further 60±5℃, further 60-65℃, and even further 60-60.5℃; the extraction time is 0.5-4h, further 2-4h, and even further 3h.

[0013] (2) The glucan gel eluent of the proteolytic product of the alkali extract of Ulva proteoglycan is further a purified water eluent of the glucan gel, and further a eluent contained in the earliest purified water elution peak of the glucan gel at 488 nm. The purified water includes, but is not limited to, deionized water, ultrapure water, distilled water, ultrafiltered water, etc.

[0014] The products include, but are not limited to, food, pharmaceuticals, health products, or cosmetics. The products also contain excipients that meet the requirements of food, pharmaceuticals, health products, or cosmetics, and other active or auxiliary active ingredients that provide antioxidant and / or cell protection benefits.

[0015] Specifically, when the cosmetic is a lotion or cream, it contains the following components by mass fraction: Span-60 or shea butter 1%-6%, cetearyl alcohol 3%-6%, caprylic / capric triglyceride 1%-2%, ethylhexyl palmitate 1%-5%, glyceryl fatty acid ester 1%-3%, glyceryl stearate and PEG-100 stearate 1%-2%, dimethyl silicone oil 0-2%, glycerin 9%-11%, 1,3-butanediol 1%-7%, sodium hyaluronate 0.1%-0.4%, Centella asiatica extract 0.1%-0.4%, EDTA-2Na 0.01%-0.1%, xanthan gum 0.2%-0.5%, Ulva prolifera peptide 0.1%-0.5%, niacinamide 0.1%-0.6%, p-hydroxyacetophenone 0.1%-0.6%, 1,2-hexanediol 0.5%-2%, peony bark extract 0.1%-0.6%, and the remainder is water. Further, the product contains the following components by weight: Span-60 or shea butter 1%-5%, cetearyl alcohol 4%-5%, caprylic / capric triglycerides 2%, ethylhexyl palmitate 2%-4%, glyceryl fatty acid esters 1%-2%, glyceryl stearate and PEG-100 stearate 2%, dimethyl silicone oil 0-2%, glycerin 10%, 1,3-butanediol 2%-6%, sodium hyaluronate 0.15%-0.25%, Centella asiatica extract 0.3%, EDTA-2Na 0.05%, xanthan gum 0.3%-0.4%, Ulva prolifera peptides 0.3%, niacinamide 0.5%, p-hydroxyacetophenone 0.5%, 1,2-hexanediol 1%, peony bark extract 0.5%, and the remainder is water. Further, a certain weight of fragrance is also added to the lotion or cream.

[0016] This invention provides a method for preparing active peptides from *Ulva prolifera*, which possess antioxidant and / or cell-protective effects. The method includes: enzymatically hydrolyzing *Ulva prolifera* alkaline extract protein under specific temperature and pH conditions to obtain the enzymatic hydrolysis product. The enzyme used for hydrolysis is selected from any one or any combination of papain, alkaline protease, trypsin, bromelain, and complex protease, preferably papain. The hydrolysis temperature for papain is 55℃ and pH is 6.5; for alkaline protease, it is 40℃ and pH is 10; for trypsin, it is 50℃ and pH is 8; for bromelain, it is 55℃ and pH is 6.5; and for complex protease, it is 50℃ and pH is 7.5. Further, the pH is first adjusted to the hydrolysis conditions and the mixture is placed at room temperature before adding the enzyme to the *Ulva prolifera* alkaline extract protein for enzymatic hydrolysis.

[0017] Further, after enzymatic hydrolysis of the alkaloid-extracted protein from *Ulva prolifera*, the enzyme was inactivated, the pH was adjusted to 7, and the supernatant was collected to obtain the enzymatic hydrolysis product of the alkaloid-extracted protein from *Ulva prolifera*. Enzyme inactivation was performed by heating in a 90℃ water bath for 15 min, followed by cooling and adjusting the pH to 7. The supernatant was collected by filtration or centrifugation, preferably centrifuging at 4000 g for 20 min.

[0018] Furthermore, the enzymatic hydrolysis product of the alkali-extracted protein from *Ulva prostrata* is separated by ultrafiltration centrifugation to obtain components with different molecular weights. Further, ultrafiltration centrifugation with a molecular weight cutoff of at least 3 kDa is used to obtain components with molecular weights less than 3 kDa; ultrafiltration centrifugation with a molecular weight cutoff of 5 kDa can also be used to obtain components with molecular weights greater than 5 kDa and those between 3 and 5 kDa. The ultrafiltration centrifugation speed is 3000-4500 × g, and the ultrafiltration centrifugation time is 20-40 min.

[0019] Further, the enzymatic hydrolysis product of the *Ulva prolifera* alkali extract protein is purified by dextran gel electrophoresis and eluted with purified water to obtain the purified water eluent of the *Ulva prolifera* alkali extract protein hydrolysis product. The eluent containing the earliest purified water elution peak at 488 nm of the *Ulva prolifera* alkali extract protein hydrolysis product is further obtained. The enzymatic hydrolysis product of the *Ulva prolifera* alkali extract protein is selected from components of different molecular weights obtained by ultrafiltration centrifugation of the *Ulva prolifera* alkali extract protein hydrolysis product. It is further selected from at least one of three components with molecular weights greater than 5 kDa, 3-5 kDa, and less than 3 kDa obtained by ultrafiltration centrifugation with molecular weight cutoffs of 5 kDa and 3 kDa, preferably the component with a molecular weight less than 3 kDa. The purified water includes, but is not limited to, deionized water, ultrapure water, distilled water, and ultrafiltered water.

[0020] Further, the preparation of the alkaline protein from *Ulva prolifera* includes: immersing *Ulva prolifera* in an alkaline aqueous solution for extraction, collecting the supernatant, and obtaining the alkaline protein from *Ulva prolifera*. The ratio of *Ulva prolifera* to alkaline aqueous solution is 1g:10-60 mL, more specifically 1g:40-60 mL, more specifically 1g:50-55 mL. The pH value of the alkaline aqueous solution is 7-10, more specifically 9-10, more specifically 10, and the alkali dissolved in the alkaline aqueous solution is a strong or weak alkali, including but not limited to alkaline sodium salts and alkaline potassium salts, such as sodium hydroxide, sodium bicarbonate, sodium carbonate, potassium hydroxide, potassium bicarbonate, potassium carbonate, etc. The extraction temperature is 30-70℃, more specifically 60±5℃, more specifically 60-65℃, and even more specifically 60-60.5℃. The extraction time is 0.5-4h, more specifically 2-4h, and even more specifically 3h. The seaweed is processed by cutting, crushing and other methods to be in the form of powder, granules, short strips, fibrous material, etc., with powder being preferred.

[0021] Furthermore, ultrasound was used to supplement the extraction of *Ulva prolifera* in alkaline aqueous solution. Specifically, the *Ulva prolifera* was first ultrasonically broken up in alkaline aqueous solution before extraction. For example, an ice-water bath and 600W ultrasonic disruption for 60 minutes were used.

[0022] Furthermore, the supernatant is collected by centrifugation or filtration. Specifically, centrifugation is performed at 8000-15000 r / min for 10-20 min; even further, centrifugation is performed at 8000 r / min for 15 min.

[0023] As one implementation method, a method for preparing active peptides from *Ulva prolifera* includes the following steps: first, extracting *Ulva prolifera* by immersing it in alkaline aqueous solution, collecting the supernatant to obtain alkaline protein from *Ulva prolifera*; then, adjusting the pH of the alkaline protein from *Ulva prolifera* to 6.5, adding papain for enzymatic hydrolysis, inactivating the enzyme, adjusting the pH to 7, collecting the supernatant to obtain the enzymatic hydrolysis product of the alkaline protein from *Ulva prolifera*; then, separating the enzymatic hydrolysis product of the alkaline protein from *Ulva prolifera* using ultrafiltration centrifugation with a molecular weight cutoff of 3 kDa to obtain components with a molecular weight less than 3 kDa; finally, purifying the components with a molecular weight less than 3 kDa using dextran gel electrophoresis, eluting with purified water, and obtaining the eluent containing the earliest elution peak at 488 nm, which is the active peptide from *Ulva prolifera*. Further, *Ulva prolifera* is immersed in alkaline aqueous solution, first ultrasonically disrupted, and then extracted.

[0024] The present invention also provides an active peptide of *Ulva prolifera*, which has antioxidant and / or cell protective effects, and is prepared by the above-described preparation method of an active peptide of *Ulva prolifera* provided by the present invention.

[0025] This invention provides an efficient, safe, and environmentally friendly method for extracting and purifying active peptides from Ulva prolifera. By optimizing the extraction process, the extraction efficiency of active peptides is significantly improved, while avoiding the problems existing in traditional methods, such as the use of toxic organic solvents.

[0026] This invention utilizes papain to enzymatically hydrolyze *Ulva prolifera* proteins, achieving a DPPH free radical scavenging rate of 85.34% in the hydrolysate. The extracted and purified UCP exhibits significant antioxidant activity, with its DPPH and ABTS free radical scavenging rates positively correlated with concentration. Furthermore, it demonstrates excellent antioxidant and cell-protective effects in both in vitro and in vivo experiments. UCP can increase MDA and SOD activity and reduce ROS levels both in vitro and in vivo, effectively mitigating oxidative damage and protecting cells from oxidative stress.

[0027] The application of UCP in cosmetics has resulted in lotions and creams with excellent physicochemical properties, skin feel, and efficacy. Various tests have shown that skincare products containing UCP can improve skin hydration, elasticity, and reduce redness. The cream, with its high content of shea butter (5%) and butylene glycol (6%), constructs a dense lipid barrier for effective moisturizing and, in conjunction with UCP, promotes collagen synthesis, significantly increasing the elasticity index to 67%. The lotion, due to the rapid penetration of ethylhexyl palmitate (4%), is more suitable for immediate hydration. Regarding the improvement of redness, the cream, relying on Centella asiatica extract and active peptides from Ulva lactuca, reduced the redness index to 3.15%. This indicates that cosmetics containing UCP have good moisturizing, anti-aging, and antioxidant effects, providing new ideas and methods for developing natural, safe, and effective cosmetics.

[0028] The above experimental results indicate that the active peptides of Ulva prolifera can be used in the field of skincare products, exhibiting excellent moisturizing, anti-aging, and antioxidant effects. This highlights the application potential of natural seaweed ingredients in the field of functional skincare, opens up new avenues for the resource utilization of Ulva prolifera, realizes its economic value, and provides some guidance and reference for the development of green and safe natural antioxidant skincare products.

[0029] The purpose of this invention is to prepare active peptides of Ulva prolifera and apply their antioxidant and cell-protective activities. The annual green tide will produce a large amount of Ulva prolifera, which can be used to produce active peptides on a large scale. The active peptides of Ulva prolifera have good safety and significant antioxidant effects, and can be safely used in food, medicine, health products or cosmetics. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the free radical scavenging ability of UCP.

[0031] Figure 2 This is a schematic diagram illustrating the protective effect of UCP on H2O2-induced HaCaT cells.

[0032] Figure 3 The effect of UCP on MDA content (A) and SOD activity (B) in H2O2-induced HaCaT cells.

[0033] Figure 4 The effect of UCP on ROS levels in H2O2-induced zebrafish juveniles.

[0034] Figure 5 Evaluation of the skin feel of the seaweed active peptide emulsion and face cream.

[0035] Figure 6 These are schematic diagrams of the actual products: the seaweed active peptide emulsion and face cream.

[0036] Figure 7 This diagram illustrates the effect of lotions and creams on skin hydration.

[0037] Figure 8 This diagram illustrates the effect of lotions and creams on the skin's elasticity index.

[0038] Figure 9 This diagram illustrates the effect of lotions and creams on the skin's redness index. Detailed Implementation

[0039] Example 1: Preparation of active peptides from *Ulva prolifera*, including the following steps: 1. Using *Ulva prolifera* powder as raw material, *Ulva prolifera* protein was extracted using ultrasound-assisted alkaline extraction. The optimal material-to-liquid ratio was 1:52 (g / mL). The mixture was placed in an ultrasonic homogenizer at 600 W and ultrasonicated for 60 min in an ice-water bath. After ultrasonic homogenization, alkaline extraction was performed. The pH of the crude *Ulva prolifera* protein extract was adjusted to the optimal pH of 10 using 1 mol / L NaOH solution. The alkaline extraction time was 3 h at 60.3 ℃. After the reaction, the mixture was centrifuged at 8000 r / min for 15 min, and the supernatant was collected as the *Ulva prolifera* protein extract. The extraction yield reached 40.594 mg / g.

[0040] 2. Adjust the pH of the above-mentioned seaweed protein extract to 6.5 with alkali, let it stand at room temperature for 1 hour, add papain, and after 2 hours of enzymatic hydrolysis, put the obtained protein hydrolysate into a 90 ℃ water bath and heat for 15 minutes to inactivate the enzyme. After cooling, adjust the pH to 7, then centrifuge at 4000 g for 20 minutes, discard the precipitate, and then vacuum filter the supernatant and freeze-dry it for later use.

[0041] 3. The enzymatic hydrolysis product of *Ulva prolifera* was prepared into a 1 mg / mL solution using ultrapure water. After thorough dissolution, the solution was separated using ultrafiltration centrifuge tubes with molecular weight cutoffs of 5 kDa and 3 kDa, centrifuged at 3000–4500 × g for 20–40 minutes each time, yielding three fractions: UP-Ⅰ (>5 kDa), UP-Ⅱ (3–5 kDa), and UP-Ⅲ (<3 kDa). The DPPH radical scavenging rates of UP-Ⅰ, UP-Ⅱ, and UP-Ⅲ were 76.3%, 82%, and 84.5%, respectively, while the ABTS radical scavenging rates were 14.7%, 20.6%, and 24.2%, respectively.

[0042] 4. The fraction with the strongest free radical scavenging activity after ultrafiltration, UP-III, was selected and further purified using a G-25 dextran chromatography column. Specifically, the lyophilized UP-III fraction was dispensed into 1 mg / mL solutions. After loading, deionized water was added, and the bottom elution rate was adjusted to approximately 1.6 mL / min. One tube was collected every three minutes, and 200 μL from each tube was added to a 96-well plate. The absorbance at 488 nm was measured using a microplate reader, and elution curves were plotted. The fractions from each peak were collected, lyophilized, and stored at -20°C for later use. The DPPH and ABTS free radical scavenging rates of each fraction were determined. The DPPH free radical scavenging rates of the first and second elution peaks were 37.5% and 10.7%, respectively, and the ABTS free radical scavenging rates were 6.8% and 5.4%, respectively. The fraction containing the first elution peak exhibited the strongest free radical scavenging activity and was named UCP.

[0043] Example 2: In vitro antioxidant capacity of UCP UCP was prepared into solutions with concentrations of 2.5, 5.0, and 10.0 mg / mL using ultrapure water. The DPPH and ABTS free radical scavenging abilities of the UCP solutions (2.5, 5.0, and 10.0 mg / mL) were tested according to the instructions of the DPPH scavenging ability and total antioxidant capacity (T-AOC) kit.

[0044] The results are as follows Figure 1 As shown, at a UCP concentration of 10 mg / mL, the scavenging rates of DPPH and ABTS were 90.63% and 88.64%, respectively, demonstrating excellent antioxidant activity. Furthermore, UCP showed even better DPPH scavenging ability, with a DPPH scavenging rate exceeding 60% at a low concentration of 2.5 mg / mL, indicating that the antioxidant activity of UCP is closely related to its concentration.

[0045] Example 3: Culture of HaCaT cells HaCaT cells were incubated at 37 ℃ in a 5% CO2 incubator. A complete culture medium was prepared using 15% FBS, MEM basal medium, and 1% P / S to culture the HaCaT cells. When the cell density in the culture flask reached 80%–90%, the HaCaT cells were digested with 0.25% trypsin for 8 min. Digestion was stopped by adding complete culture medium, and the cells were centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 1 mL of MEM medium. The cells were then passaged at a density of 1:3–1:5, depending on the actual situation.

[0046] Example 4: Protective effect of UCP on H2O2-induced HaCaT cells When the cell density in the culture flask reaches 80%~90%, HaCaT cells are digested with 0.25% trypsin at a concentration of 1×10⁻⁶. 4 Cells were seeded at a density of 10 cells / well in 96-well plates, divided into a control group, an H2O2 damage group, and a bioactive peptide protection group. Cells were incubated overnight in a CO2 incubator (37℃, 5% CO2). When the cell deposition rate reached 40%–60%, 100 μL of different concentrations of UCP solution (25, 50, 100 ug / mL) were added to the bioactive peptide protection group. After 6 h of incubation, the supernatant was discarded. 100 μL of culture medium was added to the control group, while 0.7 mmol / L H2O2 solution was added to the H2O2 damage group and the bioactive peptide protection group. After 18 h of incubation, 10 μL of CCK-8 solution and 100 μL of MEM basal medium were added to each well according to the kit instructions. The cells were then incubated in the dark for 2 h. Cell viability was measured at 450 nm to determine the protective effect of UCP on H2O2-induced HaCaT cell viability.

[0047] The results are as follows Figure 2 As shown, UCP has a protective effect against H2O2-induced HaCaT cell viability, and this protective effect gradually increases with increasing UCP concentration. Figure 3 As shown, the MDA content in HaCaT cells treated with H2O2 increased significantly, indicating that H2O2 induced significant intracellular lipid peroxidation and oxidative damage. With increasing UCP concentration, the MDA (malondialdehyde) content in H2O2-induced HaCaT cells gradually decreased, especially at a UCP concentration of 100 µg / mL, where the MDA content (30.50%) was close to the control group level (13.57%), effectively inhibiting lipid peroxidation and reducing H2O2-induced HaCaT cell damage. Simultaneously, H2O2 treatment significantly reduced intracellular SOD activity, indicating a weakened antioxidant capacity. With increasing UCP concentration, SOD activity in HaCaT cells significantly increased. In conclusion, UCP mitigated H2O2-induced oxidative damage and protected HaCaT cells by reducing MDA content and increasing SOD activity.

[0048] Example 5: Effect of UCP on ROS levels in H2O2-induced zebrafish juveniles Several well-developed zebrafish embryos (4-7 hpf) were placed in 12-well plates, 15 embryos per well. The embryos were divided into a control group (embryo culture medium only), an H2O2 damage group (3.5 mmol / mL), and a UCP protection group (25, 50, and 100 ug / mL). They were cultured in a 28±0.5℃ incubator under light, with the exposure medium changed daily and dead embryos removed. Zebrafish juveniles incubated to 96 hpf were gently washed three times with culture medium, followed by the addition of 500 μL of diluted DCFH-DA (10 μmol / L) fluorescent staining solution, and incubated in the dark for 30 minutes. After staining, the staining solution was discarded, and the juveniles were washed three times with embryo culture medium to remove any fluorescent staining that had not entered their bodies. The juveniles were anesthetized with diluted MS-222, and photographed under a fluorescence microscope. The fluorescence intensity within the juveniles was quantitatively analyzed using ImageJ software.

[0049] The results are as follows Figure 4 As shown in A and B, H₂O₂ treatment (3.5 mmol / L) significantly increased ROS levels in zebrafish juveniles, indicating that H₂O₂ induced significant oxidative stress. However, with increasing UCP concentration (25, 50, 100 µg / mL), ROS levels gradually decreased, especially at a UCP concentration of 100 µg / mL, where ROS levels were close to the control group, with no significant difference between the two. This indicates that UCP has a significant ROS scavenging ability and alleviates H₂O₂-induced oxidative stress. The antioxidant effect of UCP is dose-dependent; with increasing UCP concentration, its ROS scavenging effect gradually increases, indicating that UCP has significant effects in antioxidation and cell protection.

[0050] Example 6 The formulation of the seaweed active peptide emulsion is shown in Table 1.

[0051] Table 1. Formulation of Ulva prolifera active peptide emulsion The specific preparation method is as follows: Weigh out the oil phase and aqueous phase components according to the formula. Add the oil phase component to beaker A and heat to 80℃ until completely melted. Add the aqueous phase component to beaker B and heat to 85-90℃, stirring until all components are completely dissolved. Cool to 70℃ and set aside. Slowly pour the heated oil phase into the aqueous phase and homogenize at high speed for 5 minutes to form a homogeneous emulsion, avoiding the introduction of air bubbles during the process. After homogenization, stir at 1200 rpm in a mixer. When the emulsion cools to 40-45℃, add the active peptide extract of *Ulva prolifera*, nicotinamide, fragrance, preservatives, and other additives, continue stirring until homogeneous, and cool to room temperature before bottling. Figure 6 As shown in A and B.

[0052] Example 7 The formula for the seaweed active peptide face cream is shown in Table 2.

[0053] Table 2. Formula of Seaweed Active Peptide Face Cream The specific preparation method is as follows: Weigh out the oil phase and aqueous phase components according to the formula. Add the oil phase component to beaker A and heat to 80℃ until completely melted. Add the aqueous phase component to beaker B and heat to 85-90℃, stirring until all components are completely dissolved. Cool to 70℃ and set aside. Slowly pour the heated oil phase into the aqueous phase and homogenize at high speed for 5 minutes to form a homogeneous emulsion, avoiding the introduction of air bubbles during the process. After homogenization, stir at 1200 rpm in a mixer. When the emulsion cools to 40-45℃, add the active peptide extract of *Ulva prolifera*, nicotinamide, fragrance, preservatives, and other additives, continue stirring until homogeneous, and cool to room temperature before bottling. Figure 6 As shown in C and D.

[0054] Example 8: Evaluation of the skin feel of the *Ulva prolifera* active peptide emulsion and cream. Twenty healthy volunteers (aged 20-40) with no history of skin diseases were selected. An appropriate amount of sample was applied to the back of the hand and spread evenly in a circular motion. The skin feel was evaluated based on the following indicators: odor, thickness, feel on the skin, gloss, smoothness, spreadability, and absorption speed. The specific evaluation criteria are shown in Table 3.

[0055] Table 3. Skin feel evaluation indicators for lotions and creams The skin feel evaluation results of the seaweed active peptide emulsion and cream are as follows: Figure 5 As shown. The total score for this skin feel evaluation was 70 points, with average scores of 59.6 for the lotion and 58.35 for the cream. No redness or allergic reactions were observed in the volunteers after use, indicating that the lotion and cream developed in this study are safe and usable. Example 9: Efficacy Evaluation of Ulva prolifera active peptide emulsion and face cream Five volunteers aged 20-40 years, in good health, with no history of skin diseases, and who volunteered to participate, were selected. Before the experiment, the inner forearms of the volunteers were cleaned and dried. They sat quietly for 30 minutes in an indoor environment with stable temperature (21-26℃) and humidity (45-55% relative humidity), during which time they refrained from eating or strenuous exercise. Once the skin temperature and humidity had reached equilibrium with the environmental conditions, a 3cm x 3cm test area was drawn on the inner forearm of each volunteer. An appropriate amount of sample was applied to the test area and gently massaged in circular motions until fully absorbed. The skin moisture content, elasticity index, and redness index of the test area were measured using a high-definition intelligent skin analyzer. The measurements were repeated three times, and the average value was taken. The experiment was conducted continuously for seven days. Results Figures 7-9As shown, the seaweed active peptide emulsion and cream significantly improve skin hydration and elasticity index, as well as reduce redness.

Claims

1. The antioxidant and / or cell protection applications of *Ulva prolifera* active peptides, characterized in that, The active peptides of *Ulva prolifera* are selected from at least one of the following: (1) Enzymatic hydrolysis products of alkaloid-extracted protein from Ulva prolifera; (2) Dextran gel eluent of the proteolytic product of Ulva alkaline extract.

2. The application according to claim 1, characterized in that, The active peptides from *Ulva prolifera* are used to prepare antioxidant products and / or cell protection products, wherein the products are selected from at least one of food, pharmaceutical, health products or cosmetics.

3. A product for antioxidant and / or cell protection, characterized in that, Contains at least one of the following active peptides from *Ulva prolifera*: (1) Enzymatic hydrolysis products of alkaloid-extracted protein from Ulva prolifera; (2) Dextran gel eluent of the proteolytic product of Ulva alkaline extract.

4. The product according to claim 3, characterized in that, The product is selected from at least one of food, medicine, health products or cosmetics.

5. The product according to claim 4, characterized in that, When the product is a lotion or cream in cosmetics, it contains the following components by mass fraction: Span-60 or shea butter 1%-6%, cetearyl alcohol 3%-6%, caprylic / capric triglyceride 1%-2%, ethylhexyl palmitate 1%-5%, glyceryl fatty acid ester 1%-3%, glyceryl stearate and PEG-100 stearate 1%-2%, dimethyl silicone oil 0-2%, glycerin 9%-11%, 1,3-butanediol 1%-7%, sodium hyaluronate 0.1%-0.4%, Centella asiatica extract 0.1%-0.4%, EDTA-2Na 0.01%-0.1%, xanthan gum 0.2%-0.5%, Ulva prolifera polypeptide 0.1%-0.5%, niacinamide 0.1%-0.6%, p-hydroxyacetophenone 0.1%-0.6%, 1,2-hexanediol 0.5%-2%, peony bark extract 0.1%-0.6%, and the remainder is water.

6. A method for preparing active peptides from *Ulva prolifera*, characterized in that, The active peptides of *Ulva prolifera* have antioxidant and / or cell-protective effects. The steps include: enzymatically hydrolyzing *Ulva prolifera* alkali extract protein under enzymatic hydrolysis temperature and pH conditions to obtain the enzymatic hydrolysis product of *Ulva prolifera* alkali extract protein.

7. The preparation method according to claim 6, characterized in that, The enzymatic hydrolysis products of the alkali-extracted protein from *Ulva prostrata* should be subjected to ultrafiltration centrifugation with a molecular weight cutoff of at least 3 kDa to obtain components with a molecular weight of less than 3 kDa.

8. The preparation method according to claim 6, characterized in that, The enzymatic hydrolysis products of the alkaloid-extracted protein from *Ulva prolifera* were separated and purified using dextran gel electrophoresis.

9. The preparation method according to claim 6, characterized in that, First, *Ulva prolifera* was extracted by soaking in alkaline water, and the supernatant was collected to obtain *Ulva prolifera* alkaline protein. Then, the pH of the alkaline protein was adjusted to 6.5, and papain was added for enzymatic hydrolysis. After enzyme inactivation, the pH was adjusted to 7, and the supernatant was collected to obtain the enzymatic hydrolysis product of the alkaline protein. Next, the enzymatic hydrolysis product of the alkaline protein was separated by ultrafiltration centrifugation with a molecular weight cutoff of 3 kDa to obtain components with a molecular weight less than 3 kDa. Finally, the components with a molecular weight less than 3 kDa were purified by dextran gel electrophoresis, eluted with purified water, and the eluent containing the earliest elution peak at 488 nm was obtained, which is the active peptide of *Ulva prolifera*.

10. A type of active peptide from *Ulva prolifera*, characterized in that, It has antioxidant and / or cell-protective effects and is prepared by the preparation method according to any one of claims 6-9.

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