Use of a paramylon extract in infant nappy rash and eczema

Abstract

The application relates to the field of skin care products, in particular to a euglena protein extract and application thereof in skin care products. The fine euglena protein extract obtained by the method has a remarkable treatment effect on infant diaper rash and eczema.

Description

Technical Field

[0001] This invention relates to the field of skincare products, and in particular to the use of a Euglena protein extract in infantile diaper rash and eczema; Background Technology

[0002] In recent years, seaweed, represented by kelp, sea lettuce, and wakame, and microalgae, represented by golden algae, have become increasingly popular in the Japanese market. Among the microalgae, Euglena has seen significant growth, with a market size of approximately 10 billion yen.

[0003] In recent years, a product called "Euglena" has appeared in the microalgae market and has been developed and applied as a health food. This algae is Euglena slenderis (Euglena microphylla). Euglena cells are rich in a variety of bioactive substances, such as β-carotene, vitamin C, vitamin E, β-1,3-glucan, and PUFA, and can serve as a source of single-cell protein. For example, β-13-glucan has long been considered to play a role in regulating immune responses and has the effect of enhancing immunity in the human body.

[0004] Multiple studies have found that this substance has anti-cancer, anti-bacterial infection, macrophage activation, cytokine secretion induction, hematopoietic promotion, anti-radiation, burn treatment, wound healing promotion, blood lipid reduction, and antioxidant effects. Its derivatives (such as sulfated substances) even have anti-HIV effects.

[0005] The phylum Euglenophyta comprises only one class, Euglenophyceae, which includes the orders Euglenophyales and Styloenomales, and four families (Eugleaceae, Euglephantiaceae, Euglenophyceae, and Styloenomales). It contains approximately 40 genera and over 1000 species, most of which are freshwater species, typically found in large numbers in calm inland waters. Species of the genus Euglena within Euglenophyta, such as *Euglenagracils*, have a very long history of biological research and are arguably among the most thoroughly studied species, considered a type species in experimental biology. For example, *Euglenagracils* powder containing parastarch has received GRAS (Generally Recognized As Safe) certification from the US FDA, and in China, *Euglenagracils* received new resource food certification in May 2013.

[0006] However, research on the use of Euglena fusiforme in skincare products is still limited, and the product range is not very diverse. Given the strong consumer demand for non-toxic, harmless, and all-natural skincare products, the market urgently needs skincare products with Euglena fusiforme extracts, such as Euglena fusiforme protein, as the main ingredient.

[0007] Atopic dermatitis (AD) is a common inflammatory skin disease closely related to allergic reactions, caused by a variety of internal and external factors. It commonly affects infants, typically occurring between 1 and 3 months of age, and usually resolves spontaneously after 18 months. Some children develop it into childhood, with a portion progressing to atopic dermatitis. Recurrent skin lesions, itching, and secondary infections severely impair an infant's quality of life. Studies by Ivette AG et al. showed that the global incidence of eczema in China from 1990 to 2010 was approximately 2.5%. Meanwhile, a 2008 clinical epidemiological study by Liu Jie et al. in Tianjin, involving 506 infants, showed an eczema incidence of 75.7%. It has been confirmed that the etiology of AD involves both environmental and internal factors, and its pathogenesis mainly includes four aspects: genetic factors (such as FLG, SPINK5, IL-4 / IL-4R, Gene Z, etc.), Th1 / Th2 immune imbalance, skin barrier disruption (including physical, chemical, microbial, and immune barriers), and infectious factors. These four factors interact and form a complex network, jointly participating in the pathogenesis of Alzheimer's disease (AD). Histologically, infantile eczema is characterized by intercellular edema of the epidermal cells, accompanied by varying degrees of acanthosis and superficial perivascular lymphocytic infiltration. Due to intercellular edema, the intercellular spaces increase, the skin's "brick-and-mortar structure" becomes unstable, disrupting the original skin barrier and increasing transepidermal water loss. Inflammatory cell infiltration affects normal skin metabolism, reducing lipids, natural moisturizing factors, and anti-inflammatory factors, leading to dry, flaky, and more sensitive skin. Furthermore, the damaged skin barrier reduces the skin's ability to resist external stimuli, making it prone to secondary infections. Therefore, rebuilding the skin barrier while simultaneously implementing anti-inflammatory treatments is crucial in eczema treatment.

[0008] Currently, the internationally recommended treatment strategy for mild Alzheimer's disease (AD) focuses on basic treatments such as moisturizing to promote skin barrier function repair, anti-inflammation, and eliminating irritants. Meanwhile, commercially available skincare products for infants and young children with AD primarily focus on hypoallergenic, non-irritating, skin repair, and antibacterial / anti-inflammatory effects. Therefore, screening for active ingredients with barrier repair functions is essential for AD treatment.

[0009] Diaper dermatitis, also known as diaper rash, is a contact dermatitis that occurs in the area covered by an infant's diaper, mainly manifesting as erythema and rashes on the diaper area. Epidemiological studies show that the incidence of diaper dermatitis in infants and young children ranges from 7% to 35%. Among hospitalized infants and children, the incidence is as high as 17% to 43%. Although this skin problem is relatively common in infants, its recurring nature causes pain, crying, and poor sleep in affected children. If not properly managed, it can easily lead to the invasion of pathogenic microorganisms such as bacteria and fungi, causing infection and seriously affecting the child's normal growth and life, while also placing significant psychological pressure on parents. The environment of the diaper area is closely related to the development of diaper dermatitis. Typically, the diaper area environment is characterized by high humidity, urine and fecal residue, and friction from the diaper against the skin. Studies have shown that the level of IL-1α in the diaper area is significantly higher than in the non-diaper area. Proteases in feces can degrade proteins in the stratum corneum and trigger inflammation. Furthermore, residual urine leads to an increased pH level, disrupting the overall composition and function of stratum corneum lipids and ceramides. These factors can cause skin barrier defects, resulting in increased skin permeability and triggering irritant skin inflammation. The triggering factors for diaper dermatitis are multifaceted, and its treatment options are diverse. Treatment and prevention primarily revolve around two principles: 1) promoting the repair of damaged skin; 2) preventing recurrence. Therefore, promoting the repair of damaged skin is equally important for the treatment of diaper rash.

[0010] After years of research, the inventors made an unexpected discovery that the active protein extract from Euglena scabra can effectively relieve eczema and diaper rash in infants, and has a remarkable effect on repairing damaged skin. Moreover, this natural ingredient is non-toxic and has no side effects, making it especially suitable for use in infants and young children. Summary of the Invention

[0011] To address the aforementioned problems, this invention provides an active protein extract from Euglena spp. and its application in the preparation of skincare products.

[0012] The specific technical solutions to the above technical problems are as follows:

[0013] According to a first aspect, the present invention provides a method for extracting proteins from Euglena, specifically:

[0014] 1. A method for preparing Euglena protein, comprising the following steps:

[0015] 1) Cell disruption: Weigh 0.5g of Euglena powder, add PBS and mix well, then sonicate for 15min.

[0016] 2) Cell extract: Centrifuge at 12000 rpm at 4℃ for 20 min, and transfer the supernatant to a centrifuge tube for later use;

[0017] 3) Protein salting out: Slowly add solid ammonium sulfate to the cell extract while stirring constantly until the ammonium sulfate concentration reaches 60%. Continue stirring at room temperature for 1 hour, then place in a 4-degree refrigerator and let stand overnight.

[0018] 4) Protein purification: Centrifuge the precipitated protein solution at 4000 rpm for 20 min, discard the supernatant, and retain the precipitate for later use. Dissolve the protein precipitate with an appropriate amount of PBS solution, add it to a 100D dialysis bag, and dialyze with deionized water until no ammonium sulfate is visible in the solution;

[0019] 5) Sterilization: Filter the dialysis-purified protein through a 0.22μm filter and collect the filtrate to obtain sterile Euglena protein extract.

[0020] According to another aspect, the present invention relates to the use of the above-mentioned Euglena protein in the preparation of skin care products.

[0021] According to another aspect, the present invention relates to the use of the above-mentioned Euglena protein in the preparation of drugs / compositions / skincare products for treating infantile eczema.

[0022] According to another aspect, the present invention relates to the use of the above-mentioned Euglena protein in the preparation of diaper rash medications / compositions / skincare products.

[0023] In some embodiments, the present invention relates to a Euglena cell culture medium comprising: 0.02 g / L KH2PO4, 0.6 g / L peptone, 0.025 g / L MgSO4·7H2O, 0.4 g / L yeast extract, 0.4 g / L sodium acetate, 0.5 μg / L vitamin B12, 0.04 g / L potassium citrate, and 0.4 mg / L vitamin B1. Attached Figure Description

[0024] Figure 1 Gel electrophoresis image of Euglena protein extract

[0025] Figure 2 Results of tissue viability testing in a diaper rash skin model

[0026] Figure 3 Results of tissue morphology examination of diaper rash skin model

[0027] Figure 4 : Detection of barrier protein FLG in a diaper rash skin model

[0028] Figure 5 Diagram of LOR (Left-of-Right) barrier protein detection in a diaper rash skin model

[0029] Figure 6 Results of tissue viability testing in an eczema skin model

[0030] Figure 7Results of tissue morphology examination of eczema skin model

[0031] Figure 8 Detection of barrier-related protein FLG in an eczema skin model

[0032] Figure 9 Image of LOR (Lean On-Rate) protein detection in an eczema skin model Example

[0033] Example 1: Cultivation of Euglena slenderis

[0034] Algal species: Euglena slenderis (purchased from the Freshwater Algal Culture Collection, Chinese Academy of Sciences, code FACHB-848)

[0035] Basic culture conditions: light intensity 3000 lux, temperature 25±2℃, culture medium HUT (the following is the final concentration formula of the culture medium, pH=6.4):

[0036] 1 <![CDATA[KH2PO4]]> 1mL 20g / L 2 peptone 0.6g / L - 3 <![CDATA[MgSO4·7H2O]]> 1mL 25g / L 4 yeast extract 0.4g / L - 5 Sodium acetate 0.4g / L - 6 Vitamin B12 1mL 0.5 mg / L 7 Potassium citrate 1mL 40g / L 8 Vitamin B1 1mL 0.4g / L

[0037] Culture method: Mixed-culture. Take an appropriate amount of pure culture of Euglena filamentosa and, under aseptic conditions, inoculate it into 500 mL of sterile culture medium and incubate statically. Shake the solution three times a day to ensure even dispersion. The sterile culture medium consists of 0.02 g / L KH₂PO₄, 0.6 g / L peptone, 0.025 g / L MgSO₄·7H₂O, 0.4 g / L yeast extract, 0.4 g / L sodium acetate, 0.5 μg / L vitamin B12, 0.04 g / L potassium citrate, and 0.4 mg / L vitamin B1.

[0038] Preparation process: According to the HUT culture medium formula, weigh the solid powders of different components, dissolve them in deionized water, and prepare stock solutions of 1000 times concentration. Store at room temperature for later use. Add an appropriate amount of deionized water to a 1L volumetric flask, and simultaneously add 1mL of the stock solution of each component. Make up to 1L, shake well, and then transfer the prepared culture medium into a reagent bottle. Sterilize at 121℃ for 20 minutes to obtain sterile culture medium.

[0039] Example 2: Extraction process of Euglena protein

[0040] 1. Cell disruption: Weigh 0.5g of Euglena powder, add 30mL of PBS and mix well. Sonicate for 15min (2s on, 4s off, 60% power).

[0041] 2. Cell extraction solution: Centrifuge at 12000 rpm at 4℃ for 20 min, and transfer the supernatant to a centrifuge tube for later use;

[0042] 3. Protein salting out: Slowly add solid ammonium sulfate to the cell extract while stirring constantly until the ammonium sulfate concentration reaches 60%. Continue stirring at room temperature for 1 hour, then place in a 4-degree refrigerator and let stand overnight.

[0043] 4. Protein purification: Centrifuge the precipitated protein solution at 4000 rpm for 20 min, discard the supernatant, and retain the precipitate for later use. Dissolve the protein precipitate in an appropriate amount of PBS solution, add it to a 100D dialysis bag, and dialyze with deionized water until no ammonium sulfate is visible in the solution.

[0044] 5. Sterilization: Filter the dialysis-purified protein through a 0.22 μm filter, and collect the filtrate as the sterile Euglena protein extract. The Euglena protein extract was subjected to standard SDS-PAGE; see [link to results]. Figure 1 The results showed that the size of Euglena proteins ranged from 63 to 75 kDa.

[0045] Example 3: Efficacy of Euglena protein extract for eczema and diaper rash

[0046] Experimental design: 1. To mimic the epidermal response during eczema development, i.e., in vitro skin models (e.g., skin models) were used. 1. During the hair regrowth process, stimulating factors (PolyI:C and LPS) were added to create a 3D skin model with impaired tissue vitality and barrier function, and TSLP was released to simulate the skin pathological state of eczema-like AD. The efficacy of active ingredients in improving the anti-inflammatory and barrier repair capabilities of eczema was evaluated by detecting barrier index, tissue morphology, inflammatory factors, and related protein expression. 2. Using a weakened epidermal model as the research object, a diaper rash model was established by using SLS stimulation of the epidermal model. The repair efficacy of active ingredients in diaper rash was evaluated by detecting barrier index, tissue morphology, inflammatory factors, and related protein expression.

[0047] The skin model used in this test was a 3D epidermal skin model. (Hereinafter referred to as skin model), batch numbers: ES201101, ES201102, produced and provided by Guangdong Boxi Biotechnology Co., Ltd.

[0048] Reagents and equipment

[0049] KC2500 (Guangdong Boxi), MTT (Sigma), DMSO (Sigma), Phosphate Buffered Medium (Soluble), EpiGrowth Culture Medium (Guangdong Boxi Biotechnology), Phosphate Buffered Medium (Soluble), MTT (Sigma), Isopropanol (Sinopharm), IL-1α ELISA Kit (Abcam), PolyI:C (Sigma), Lipopolysaccharide (E. coli. Sigma), TSLP ELISA Kit (Boster), 4% Paraformaldehyde (BioSharp), Xylene (Sinopharm), Anhydrous Ethanol (Sinopharm), Hematoxylin (Beyond), Eosin (Beyond), Hydrochloric Acid (Sinopharm), 50× Sodium Citrate (Xi'an Hert Biotechnology), Anti-FLG Antibody (Abcam), Anti-LOR Antibody (Abcam) ABC-Peroxidase Kits (VECTASTAIN), Anti-Mouse-488 (Goat Anti-Mouse) (Abcam), Antiquenching Agent (Beyotime), Hochest33342 (Beyotime); CO2 Incubator (Thermo, 150i), Clean Bench (Suzhou Antai, SW-CJ-1F), Fluorescence Microscope (Leica), Microoscillator (Qilinbeier), Microplate Reader (BioTek, Epoch).

[0050] 3.1 Efficacy of Euglena protein extract in treating diaper rash

[0051] Preparation of test solution

[0052] 1) MTT working fluid preparation

[0053] Prepare a 5 mg / mL MTT stock solution using PBS and store it at -20°C (storage time not exceeding 1 month). Before use, dilute the stock solution to 1 mg / mL with PBS and store at 4°C protected from light (storage time not exceeding 2 hours).

[0054] 2) Preparation of 0.4% SLS stock solution

[0055] Weigh 0.024 g of SLS and dissolve it in 6 mL of PBS solution. Filter through a 0.22 μm filter to prepare a 0.4% SLS stock solution for later use. Dilute the 0.4% SLS solution twice to prepare a 0.2% SLS working solution.

[0056] 3) Preparation of working solution for positive control group (0.01% dexamethasone)

[0057] Dissolve 100 mg of dexamethasone in 1 mL of DMSO to prepare a stock solution with a concentration of 100 mg / mL; dilute 1000 times with PBS to prepare 0.01% dexamethasone for later use.

[0058] 2) Dosage procedure

[0059] 1. Transfer the 3D model to a 6-well plate (add 0.9 mL of model culture medium beforehand), and label the 6-well plate with the test group number.

[0060] 2. Add 25 μL of the prepared drug solution to the model surface, gently shake the model to distribute the sample evenly on the model surface, and incubate in a CO2 incubator (37℃, 5% CO2, 95% RH) for 24 h.

[0061] 3. After incubation, wash the surface of the model with a wash bottle containing sterile PBS solution to remove any remaining test material, and gently wipe away any residual liquid inside and outside the model with a sterile cotton swab.

[0062] Tissue vitality testing

[0063] 1) MTT incubation: Place the cleaned model into a 24-well plate containing 1 mg / mL MTT working solution. Then transfer the 24-well plate to a CO2 incubator (37℃, 5% CO2, 95% RH) and incubate for 3 hours.

[0064] 2) Isopropanol extraction: After the MTT incubation is complete, remove the model with tweezers, wipe the bottom surface of the residual MTT liquid with absorbent paper, transfer it to a new 24-well plate, add 2 mL of isopropanol, seal the 24-well plate with sealing film, and let it stand overnight at 4°C.

[0065] 3) Detection: After extraction, puncture the mold with a 200 μL pipette tip to allow the isopropanol extract to flow into a 24-well plate. Discard the punctured mold and mix the isopropanol extract in each well by pipetting three times. After mixing, take two 200 μL aliquots of the isopropanol extract from each well and add them to the corresponding wells of a 96-well plate, labeling them accordingly. Read the absorbance value at 570 nm using a microplate reader.

[0066] Inflammatory factor detection

[0067] 1) Collect the model culture medium: After incubation, collect the model culture medium in EP tubes and store it in a -80℃ refrigerator.

[0068] 2) Detection of inflammatory factor levels: The detection and analysis were performed according to the instructions of the IL-1α ELISA detection kit.

[0069] 3) Results Analysis: GraphPad Prism Program software was used for graphing, and t-tests were used for statistical analysis between groups. ＊ p < 0.05 indicates a significant difference. ＊＊ p < 0.01 indicates that the difference is highly significant.

[0070] Tissue morphology detection

[0071] The model used for tissue morphology was fixed with 4% paraformaldehyde for 24 hours. After fixation, the model was circumcised, stained with H&E, and photographed under a microscope to collect images.

[0072] Immunofluorescence detection

[0073] 1) Baking and dewaxing: Place the paraffin slices in a baking machine at 70℃ and bake for 4 hours.

[0074] 2) Dewaxing and hydration: Immerse the sections in xylene for 10 min, replace the xylene and immerse for another 10 min, then immerse in anhydrous ethanol for 5 min, 95% ethanol for 5 min, and 75% ethanol for 5 min. Wash three times with PBS buffer for 5 min each time.

[0075] 3) Antigen retrieval: Placing paraffin sections into 0.01M sodium citrate antigen retrieval solution and autoclaving were performed. After cooling, the sections were removed. The sections were washed three times with PBS buffer, 5 min each time.

[0076] 4) Blocking peroxidase: Add 1 drop of 3% H2O2 to each slice and incubate at room temperature for 30 min to block the activity of endogenous peroxidase. Wash 3 times with PBS buffer solution, 5 min each time.

[0077] 5) Serum blocking: Add serum homologous to the secondary antibody and block at 37°C for 60 minutes. No rinsing is required.

[0078] 6) Primary antibody incubation: Add primary antibody working solution and incubate overnight at 4°C. Wash 3 times with PBS buffer, 5 min each time.

[0079] 7) Secondary antibody incubation: Add the secondary antibody working solution and incubate at room temperature for 1 hour. Wash 3 times with PBS buffer, 5 minutes each time.

[0080] 8) Nuclear staining: After the secondary antibody incubation, wash three times with PBS buffer, 5 min each time. Remove the PBS solution adhering to the slides, add 100 μL of Hochest 33342 working solution to each slide, and incubate at room temperature for 5 min.

[0081] 9) Wash three times with PBS buffer, 5 min each time. Wipe off the PBS solution with absorbent paper, and mount the slide with a drop of anti-quenching agent; take a fluorescence microscope image (20×) within 24 hours.

[0082] According to the experimental results (see...) Figures 2-5 Euglena protein extract can significantly enhance tissue viability in a 3D skin-diaper rash model, demonstrating resistance to diaper rash. Figure 2 It can also significantly repair damage to the model caused by diaper rash. Figure 3It can significantly increase the expression levels of tight junction proteins FLG and LOR, and repair the loss of FLG and LOR proteins caused by the surfactant SLS. Figure 4 , Figure 5 In conclusion, the above results demonstrate that Euglena protein has a strong protective and repairing function against diaper rash and exhibits excellent therapeutic effects.

[0083] 3.2 Efficacy of Euglena protein extract for eczema

[0084] 1) MTT working fluid preparation

[0085] Same as 3.1 related section

[0086] 2) Preparation of induction working solution

[0087] PolyI:C and LPS induction working solution: Dissolve 480 μL of 5 mg / mL PolyI:C stock solution and 20 μL of 10 mg / mL LPS stock solution in 100 mL of skin model culture medium to make the final concentration of PolyI:C 24 μg / mL and the final concentration of LPS 20 μg / mL.

[0088] 3) Preparation of working solution for positive control group (0.01% dexamethasone)

[0089] Same as 3.1 related section

[0090] 3.2 Administration Procedure

[0091] 1) Transfer the 3D model to a 6-well plate (add 0.9 mL of model culture medium beforehand), and label the test group number on the 6-well plate.

[0092] Drug administration: Take 25 μL of the sample to be tested and add it to the surface of the model. After spreading it evenly, place it in a CO2 incubator (37℃, 5% CO2, 95% RH) and incubate for 24 h.

[0093] 3) Induction: After drug administration and incubation, remove the 6-well plates from the incubator. Gently wipe the model surface with a sterile cotton swab. Discard any remaining culture medium in the wells. Add PolyI:C and LPS induction working solution to the negative control, positive control, and sample groups, and add model culture medium to the blank control group. After completion, transfer all 6-well plates to a CO2 incubator (37℃, 5% CO2, 95% RH) for incubation for 24 hours.

[0094] 3.3 Tissue Viability Testing

[0095] 1) MTT incubation: Place the cleaned model into a 24-well plate containing 1 mg / mL MTT working solution. Then transfer the 24-well plate to a CO2 incubator (37℃, 5% CO2, 95% RH) and incubate for 3 hours.

[0096] 2) Isopropanol extraction: After the MTT incubation is complete, remove the model with tweezers, wipe the bottom surface of the residual MTT liquid with absorbent paper, transfer it to a new 24-well plate, add 2 mL of isopropanol, seal the 24-well plate with sealing film, and let it stand overnight at 4°C.

[0097] 3) Detection: After extraction, puncture the mold with a 200 μL pipette tip to allow the isopropanol extract to flow into a 24-well plate. Discard the punctured mold and mix the isopropanol extract in each well by pipetting three times. After mixing, take two 200 μL aliquots of the isopropanol extract from each well and add them to the corresponding wells of a 96-well plate, labeling them accordingly. Read the absorbance value at 570 nm using a microplate reader.

[0098] 3.4 Detection of inflammatory factors

[0099] 1) Sample collection: After 24 hours of induction and incubation, the model culture medium was collected in EP tubes and stored in a -80℃ refrigerator.

[0100] 2) Detection: Perform detection and analysis according to the instructions of the TSLP ELISA test kit.

[0101] 3) Results Analysis: GraphPad Prism Program software was used for graphing, and t-tests were used for statistical analysis between groups. ＊ p < 0.05 indicates a significant difference. ＊＊ p < 0.01 indicates that the difference is highly significant.

[0102] 3.5 Tissue Morphology Detection

[0103] The model used for tissue morphology was fixed with 4% paraformaldehyde for 24 hours. After fixation, the model was circumcised, stained with H&E, and photographed under a microscope to collect images.

[0104] 3.6 Immunofluorescence detection

[0105] 1) Baking and dewaxing: Place the paraffin slices in a baking machine at 70℃ and bake for 4 hours.

[0106] 2) Dewaxing and hydration: Immerse the sections in xylene for 10 min, replace the xylene and immerse for another 10 min, then immerse in anhydrous ethanol for 5 min, 95% ethanol for 5 min, and 75% ethanol for 5 min. Wash three times with PBS buffer for 5 min each time.

[0107] 3) Antigen retrieval: Placing the paraffin sections into 0.01M sodium citrate antigen retrieval solution, using...

[0108] After high-pressure retrieval and cooling, the sections were removed. The sections were washed three times with PBS buffer, each time for 5 minutes.

[0109] 4) Blocking peroxidase: Add 1 drop of 3% H2O2 to each slice and incubate at room temperature for 30 min to block the activity of endogenous peroxidase. Wash 3 times with PBS buffer solution, 5 min each time.

[0110] 5) Serum blocking: Add serum homologous to the secondary antibody and block at 37°C for 60 minutes. No rinsing is required.

[0111] 6) Primary antibody incubation: Add primary antibody working solution and incubate overnight at 4°C. Wash 3 times with PBS buffer, 5 min each time.

[0112] 7) Secondary antibody incubation: Add the secondary antibody working solution and incubate at room temperature for 1 hour. Wash 3 times with PBS buffer, 5 minutes each time.

[0113] 8) Nuclear staining: After the secondary antibody incubation, wash three times with PBS buffer, 5 min each time. Remove the PBS solution adhering to the slides, add 100 μL of Hochest 33342 working solution to each slide, and incubate at room temperature for 5 min.

[0114] 9) Wash three times with PBS buffer, 5 min each time. Wipe off the PBS solution with absorbent paper and mount the slide with one drop of anti-quenching agent. Take a fluorescence microscope image (20×) within 24 hours.

[0115] The experimental results show that Euglena protein can significantly downregulate TSLP, a key inflammatory factor in eczema, thereby inhibiting inflammation. Figure 6 Furthermore, facultative anaerobic neutral Euglena proteins can significantly repair damage caused by eczema in the model. Figure 7 It can significantly increase the expression levels of tight junction proteins FLG and LOR, and repair the loss of FLG and LOR proteins caused by the surfactant SLS. Figure 8 , Figure 9 In summary, the above results demonstrate that Euglena protein is effective in treating infantile eczema and diaper rash.

[0116] While diaper rash has a strong repair function, its effects are slightly less pronounced than those of corticosteroids like dexamethasone. However, dexamethasone has significant side effects; long-term use or improper dosage can lead to serious consequences such as developmental disorders and growth inhibition in infants. Euglena protein, derived from pure natural algae, is non-toxic, gentle on the skin, and more suitable for infants, achieving unexpectedly effective treatment and relief of eczema and diaper rash.

Claims

1. The use of a Euglena protein extract in the preparation of a medicament for treating diaper rash or eczema in infants, wherein the preparation method of the Euglena protein extract includes: 1) Cell disruption: Weigh 0.5g of Euglena powder, add PBS and mix well, then sonicate for 15min. 2) Cell extraction solution: Centrifuge at 12000 rpm at 4℃ for 20 min, and transfer the supernatant to a centrifuge tube for later use; 3) Protein salting out: Slowly add solid ammonium sulfate to the cell extract while stirring constantly until the ammonium sulfate concentration reaches 60%. Continue stirring at room temperature for 1 hour, then place in a 4-degree refrigerator and let stand overnight. 4) Protein purification: Centrifuge the precipitated protein solution at 4000 rpm for 20 min, discard the supernatant, and keep the precipitate for later use; dissolve the protein precipitate with an appropriate amount of PBS solution, add it to a 100D dialysis bag, and dialyze with deionized water until no ammonium sulfate is present in the solution; 5) Sterilization: Filter the dialysis-purified protein through a 0.22μm filter and collect the filtrate to obtain sterile Euglena protein extract.

2. The application of a Euglena protein extract in the preparation of skincare products for relieving diaper rash or eczema in infants, wherein the preparation method of the Euglena protein extract includes: 1) Cell disruption: Weigh 0.5g of Euglena powder, add PBS and mix well, then sonicate for 15min. 2) Cell extraction solution: Centrifuge at 12000 rpm at 4℃ for 20 min, and transfer the supernatant to a centrifuge tube for later use; 3) Protein salting out: Slowly add solid ammonium sulfate to the cell extract while stirring constantly until the ammonium sulfate concentration reaches 60%. Continue stirring at room temperature for 1 hour, then place in a 4-degree refrigerator and let stand overnight. 4) Protein purification: Centrifuge the precipitated protein solution at 4000 rpm for 20 min, discard the supernatant, and keep the precipitate for later use; dissolve the protein precipitate with an appropriate amount of PBS solution, add it to a 100D dialysis bag, and dialyze with deionized water until no ammonium sulfate is present in the solution; 5) Sterilization: Filter the dialysis-purified protein through a 0.22μm filter and collect the filtrate to obtain sterile Euglena protein extract.

Images