Use of lactoferrin as humectant

Abstract

The present invention discloses the use of lactoferrin as a humectant in cosmetic or personal care products.

Description

Technical Field

[0001] The invention relates to the field of cosmetics or personal care products, and in particular to use of lactoferrin as a moisturizer in cosmetics or personal care products. Background Art

[0002] Lactoferrin (LF) is an 80kDa iron-binding glycoprotein produced by epithelial cells and neutrophil precursors. It is present in the milk of most mammals and in many other tissues and their secretions. The structures of lactoferrin from different sources are very similar. Bovine lactoferrin (bLF) and human lactoferrin (hLF) are composed of 689 and 691 amino acids, respectively, with a homology of 69%. Among them, bovine lactoferrin is folded by two basically symmetrical and highly homologous leaf-shaped structural polypeptide chains. Human lactoferrin is present in high levels in human milk, tear film, upper respiratory tract fluid, seminal plasma and cervical mucus plugs. It is present in low levels in earwax, saliva, small intestine, vaginal fluid and amniotic fluid. It is not present in the alveoli and skin, but is present in the sweat secreted by normal human skin. Lactoferrin has a high affinity for iron ions, and natural lactoferrin is usually only saturated with 15-20% of the iron part. Lactoferrin is known for its biological activities such as anti-inflammatory, antibacterial, anti-tumor, antiviral, whitening, repair of skin damage caused by inflammation, immunomodulation and osteogenic activity.

[0003] Lactoferrin is considered to be a protein with great development potential, and therefore has broad application prospects in the fields of food, medicine, skin care products and cosmetics. The skin is the largest organ in the human body and is a physical, chemical and immune barrier that isolates harmful substances. The efficacy of lactoferrin on the skin is particularly prominent, and it has the potential to be used as a new raw material for skin care products. There have been many studies on the effect of lactoferrin on the skin, and the prior art literature has disclosed that lactoferrin has the effects of whitening the skin and repairing skin damage caused by inflammation. However, to date, the prior art literature has not disclosed the moisturizing effect of lactoferrin on the skin. Summary of the invention

[0004] The inventors unexpectedly discovered that lactoferrin has excellent moisturizing effect when studying the various effects of lactoferrin on the skin. Specifically, the inventors unexpectedly discovered that lactoferrin leads to an increase in the key moisturizing protein AQP3 in an in vitro skin model, thereby proving that lactoferrin has moisturizing effect.

[0005] Skin moisturizing is the most basic requirement for skin care. The stratum corneum of human skin is mainly composed of anucleated keratinocytes, keratin filaments, amino acid compounds and natural moisturizing factors, which play the role of retaining moisture and providing a barrier. The source of moisture in the stratum corneum of the skin is mainly sweat secreted by sweat glands, and 20% of the water is tightly bound to the natural moisturizing factor and skin lipids. The sebum film formed by the emulsification of sweat and water and the natural moisturizing factor become the two most important factors for skin moisturizing.

[0006] Skin models are increasingly used in skin care efficacy. They provide important tools and platforms for evaluating the safety, effectiveness and mechanism of action of cosmetics, skin care products and their raw materials. Through skin models, we can deeply study the specific effects of different ingredients in skin care and understand how they act on different layers and targets of the skin. Based on the research results of skin models, customized skin care solutions can be developed for different skin types and needs to meet the personalized needs of consumers.

[0007] TEER (transcutaneous electrical resistance) is an important indicator for measuring the skin barrier function, especially its repair and integrity. TEER represents the skin's ability to repair its barrier. A higher TEER value usually means that the skin barrier function is stronger and can better prevent water loss, thereby keeping the skin moisturized. When the TEER value is high, it means that the skin barrier is intact and functions well, and can effectively block external harmful substances from entering the skin, while reducing the loss of water inside the skin, thereby enhancing the skin's moisturizing effect. In the case of damaged or dry skin, increasing the TEER value can promote the repair of the skin barrier and restore its moisturizing function. Maintaining a high TEER value for a long time can keep the skin hydrated and smooth, reduce problems such as dryness and desquamation, and thus improve skin quality.

[0008] qPCR (real-time fluorescence quantitative polymerase chain reaction) testing is an important technical means to verify the moisturizing effect. qPCR is a nucleic acid quantitative technology developed on the basis of PCR (polymerase chain reaction). It uses fluorescent dyes or probes to monitor the generation of PCR products in real time during DNA amplification, thereby achieving accurate quantification of the expression of specific genes. qPCR has the advantages of high sensitivity, strong specificity, and good repeatability, and is widely used in molecular biology research and clinical diagnosis. When verifying the moisturizing effect, qPCR testing mainly evaluates the effect of moisturizing products by detecting the expression of genes closely related to the moisturizing function of the skin. These genes usually include aquaporin genes (such as AQP3). Aquaporin 3 (AQP3) is a protein located on the cell membrane that not only has high permeability, but is also an efficient transporter of glycerol. It is very important for barrier repair and moisturizing of the epidermis. AQP3 brings circulating water into the epidermis, forming a short water loop between the basal layer and the stratum corneum of the epidermal cells, thereby ensuring a constant water content in the epidermis. Through qPCR testing, the expression of AQP3 gene in skin cells after treatment with moisturizing products can be detected. If the moisturizing product can significantly increase the expression of AQP3 gene, then it can be considered that the product has a positive effect in promoting skin hydration and enhancing moisturizing effect.

[0009] In verifying the mechanism of moisturizing efficacy, the immunofluorescence staining test mainly plays a role by detecting and locating specific proteins or other molecules related to the moisturizing function of the skin. Immunofluorescence staining is a technique that uses specific antibodies combined with labeled fluorescent dyes to detect and locate specific proteins or other molecules in cells or tissues. Proteins closely related to the moisturizing function of the skin are selected as target proteins, such as aquaporin 3 (AQP3). These proteins play a key role in the process of skin moisturizing, such as regulating the transmembrane transport of water molecules and moisturizing factors, promoting the synthesis of moisturizing ingredients, etc. Through the immunofluorescence staining test, the expression level or distribution changes of the target protein in the skin cells before and after the use of moisturizing products can be detected. If the moisturizing product can significantly increase the expression level of the target protein or improve its distribution state, it can be considered that the product has a certain effect in promoting skin moisturizing. The immunofluorescence staining test is combined with other molecular biological techniques (such as qPCR) to further reveal the specific mechanism of action of moisturizing products. By detecting the effect of moisturizing products on the expression of the AQP3 gene, it can be understood whether the product enhances the moisturizing function of the skin by regulating the expression of these genes.

[0010] ELISA (enzyme-linked immunosorbent assay) test plays a role in verifying the mechanism of moisturizing efficacy by mainly detecting the changes in the content of biological molecules (such as hyaluronan synthase 1, HAS1) that are closely related to the moisturizing function of the skin. ELISA is a comprehensive technology that combines immune response and efficient catalytic reaction of enzymes. In the test, specific antibodies are fixed on a solid phase carrier and maintain their immunoreactivity. After the target molecules (such as hyaluronan synthase 1, HAS1) in the sample to be tested are bound to these antibodies, enzyme-labeled antibodies or antigens are added for reaction. Finally, by detecting the intensity of the color reaction (or fluorescent reaction) produced by enzyme catalysis, the content of the target molecule can be quantitatively determined. Hyaluronic acid is an important moisturizing component in the skin and has a strong ability to absorb and retain water. In the efficacy verification of moisturizing products, the ELISA test can detect the changes in the content of hyaluronic acid in the skin before and after using the product. If a moisturizing product can significantly increase the content of hyaluronan synthase 1 in the skin, it can be considered that the product has a positive effect in enhancing the moisturizing function of the skin. By comparing the changes in HA synthase 1 levels before and after using the product, the moisturizing effect of the moisturizing product can be evaluated and its mechanism of action can be understood.

[0011] Thus, according to a first aspect, the present invention provides the use of lactoferrin as a humectant in a cosmetic or personal care product.

[0012] According to a second aspect, the present invention provides use of lactoferrin in preparing a cosmetic or personal care product with moisturizing function.

[0013] According to a third aspect, the present invention provides the use of lactoferrin as the sole humectant in a cosmetic or personal care product as a humectant.

[0014] According to a fourth aspect, the present invention provides use of lactoferrin as the sole humectant in preparing a cosmetic or personal care product with a moisturizing function.

[0015] In some embodiments, the cosmetic or personal care product may be selected from hand care, face care, body care, foot care, head care, nail care, or mouth care products.

[0016] In some embodiments, the amount of lactoferrin is 0.01 wt%-1 wt%, preferably 0.05 wt%-0.5 wt% of the cosmetic or personal care product, depending on the desired product quality.

[0017] According to a fifth aspect, the present invention provides a cosmetic or personal care product having moisturizing function, wherein the cosmetic or personal care product comprises lactoferrin as the only moisturizer.

[0018] In some embodiments, the cosmetic or personal care product may be selected from hand care, face care, body care, foot care, head care, nail care, or mouth care products.

[0019] In some embodiments, the amount of lactoferrin in the cosmetic or personal care product is 0.01 wt%-1 wt%, preferably 0.05 wt%-0.5 wt% of the cosmetic or personal care product, depending on the desired product quality.

[0020] The inventors have confirmed through transepidermal electrical resistance (TEER), qPCR, immunofluorescence staining and ELISA experiments that after using lactoferrin or cosmetics or personal care products containing lactoferrin in a skin model, the expression of moisturizing-related genes, proteins and moisturizing factors has been improved to a certain extent, proving the role of lactoferrin in the moisturizing process. Specifically, compared with the blank control, lactoferrin or cosmetics or personal care products containing lactoferrin can improve the expression of moisturizing-related genes, proteins and related moisturizing factors, thereby achieving the moisturizing effect of the skin.

[0021] In some embodiments, the cosmetic or personal care product of the present invention may comprise the lactoferrin according to the present invention and the excipients acceptable to skin care products shown in Table 1 below (the excipients acceptable to skin care products formula table is hereinafter referred to as "blank formula table"), that is, the lactoferrin replaces a portion of the water in the blank formula table shown in Table 1 below, thereby formulating a cosmetic or personal care product.

[0022] Table 1: Blank formula table

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : TEER test results of lactoferrin aqueous solutions with concentrations of 0.05wt%, 0.2wt%, and 0.5wt% and a cosmetic preparation containing 0.2wt% lactoferrin.

[0025] Figure 2 : The test results of RNA extracted from cells using the qPCR method for aqueous lactoferrin solutions with concentrations of 0.05wt%, 0.2wt%, and 0.5wt% and a cosmetic preparation containing 0.2wt% lactoferrin.

[0026] Figure 3 : Results of immunofluorescence staining of AQP3 in aqueous lactoferrin solutions with concentrations of 0.05wt%, 0.2wt%, and 0.5wt% and in a cosmetic preparation containing 0.2wt% lactoferrin.

[0027] Figure 4: Results of ELISA of aqueous lactoferrin solutions with concentrations of 0.05 wt%, 0.2 wt%, and 0.5 wt% and a cosmetic preparation containing 0.2 wt% lactoferrin. DETAILED DESCRIPTION

[0028] Embodiment 1:

[0029] Lactoferrin powder was purchased from Milchwerke Schwaben eG, with the trade name Mercurius-Production Lactoferrin, and the specification was 5 kg / bag. According to the formula table in Table 2 below, the above lactoferrin powder was dissolved in water to prepare three aqueous solutions A1, A2 and A3 with 0.05 wt%, 0.2 wt% and 0.5 wt% respectively, and then the subsequent experiments were carried out.

[0030] Table 2: Formulas of A1, A2 and A3

[0031]

[0032] Embodiment 2:

[0033] Cosmetic formulation A4 was prepared according to the formula in Table 3 below and the subsequent experiments were carried out.

[0034] Table 3: Formulation of cosmetic preparations

[0035]

[0036] The preparation method is as follows:

[0037] Dissolve lactoferrin in distilled water.

[0038] Distilled water and olive oil dissolved with lactoferrin were heated to 65° C. to obtain a water phase and an oil phase, respectively.

[0039] Add the water phase into the blending kettle, then add the oil phase, mix well, and emulsify under vacuum at 80°C for 10 minutes.

[0040] Heat polysorbate 80 to 80°C and mix thoroughly.

[0041] Then, the mixture was added to a blending kettle and vacuum emulsified at 80° C. for 10 min to obtain cosmetic preparation A4.

[0042] Epidermal model construction

[0043] 1) Resuscitation cells: P1 generation keratinocytes (KC) were used to construct a 3D epidermal model.

[0044] 2) After the collagen type I was laid for 1 hour, 0.5M / transwell was inoculated, and 500 μL of culture medium was added to the top and bottom of the well and cultured at 37°C. The medium was changed every other day.

[0045] 3) On the third day, the upper and lower culture media were replaced with CnT-PR-3D culture media and cultured at 37°C overnight.

[0046] 4) On the fourth day, liquid phase culture was performed and the lower culture medium was replaced every other day until the culture was completed.

[0047] 5) 1 mL of lactoferrin aqueous solution samples A1, A2 and A3 prepared in Example 1 and lactoferrin-containing cosmetic preparation sample A4 prepared in Example 2, as well as 1 mL of 0.5 wt % vaseline as a positive control (PC) were added respectively and placed at 37° C. for 24 h.

[0048] TEER test

[0049] The experimental steps of TEER test are as follows:

[0050] After treating the epidermal model with the lactoferrin aqueous solution samples A1, A2 and A3 prepared in Example 1 and the cosmetic preparation sample A4 containing lactoferrin prepared in Example 2 for 24 hours, the epidermal model was rinsed with PBS 5 times, 2 mL each time, and the skin model after 24 hours of sample addition was measured by a resistance meter to obtain TEER.

[0051] The experimental results are as follows Figure 1 shown.

[0052] like Figure 1 As shown, the TEER test shows that compared with the blank control, each concentration of lactoferrin aqueous solution samples A1, A2 and A3 and the cosmetic preparation sample A4 can significantly increase TEER; compared with the positive control, the relative TEER increase rate of 0.2wt% lactoferrin aqueous solution sample A2 and the cosmetic preparation sample A4 is equivalent to or slightly higher than that of 0.5wt% vaseline, and the relative TEER increase rate of 0.5wt% lactoferrin aqueous solution sample A3 is even slightly higher than that of 5mg / mL vaseline.

[0053] qPCR testing

[0054] Real-time quantitative fluorescence PCR is to achieve quantitative and qualitative analysis of the starting template by real-time detection of the fluorescence signal corresponding to each cycle of PCR amplification products. This experiment adopts the SYBR fluorescent dye method. In the PCR reaction system, SYBR fluorescent dye is added. After SYBR fluorescent dye is specifically incorporated into the DNA double strand, it emits a fluorescent signal, while SYBR dye molecules that are not incorporated into the strand will not emit any fluorescent signal, thereby ensuring that the increase of the fluorescent signal is completely synchronized with the increase of the PCR product. The specific steps for semi-quantitative analysis of genes detected by fluorescent quantitative PCR are as follows:

[0055] The RNA extraction procedure is as follows:

[0056] 1. Collect the epidermal model after sample addition, cut it in half, add 1 ml Trizol, and place it at room temperature for 5 minutes to fully lyse it. It can be stored at -80℃ for a long time (if the sample is tissue, it needs to be ground with liquid nitrogen and then lysed with Trizol).

[0057] 2. Centrifuge at 12000g for 5 min at 4°C and transfer the supernatant to a new 1.5 ml centrifuge tube.

[0058] 3. Add chloroform at a concentration of 1 / 5 chloroform / ml Trizol, invert for 30 seconds, and leave at room temperature for 5 minutes. Disable the oscillator to prevent the genomic DNA from breaking.

[0059] Centrifuge at 4.4°C, 12,000 g for 15 min.

[0060] 5. Take as much of the upper aqueous phase as possible and transfer it to another centrifuge tube (here use a 1.5 ml EP tube). Note: Do not take the middle interface. If you need to extract DNA and protein at the same time, keep the lower liquid at 4°C.

[0061] 6. Add isopropanol at a concentration of 0.5-1 times isopropanol / ml Trizol, invert and incubate at room temperature for 10 minutes.

[0062] Centrifuge at 7.4℃, 12000g for 10min, discard the supernatant, and let the RNA sink to the bottom of the tube. (During the centrifugation process, place the large end of the ep tube cap outwards. After centrifugation, the RNA will stick to the bottom of this side. When aspirating the supernatant, you can aspirate the other side to prevent the RNA from being aspirated.)

[0063] 8. Wash the precipitate with an equal amount of 75% ethanol as Trizol. Centrifuge at 7500g, 4°C for 5 min. Discard the supernatant and keep the precipitate.

[0064] 9. Repeat step 8;

[0065] 10. Dry at room temperature or vacuum dry for 5-10 minutes.

[0066] 11. Use an appropriate amount of DEPC-treated water to dissolve the RNA sample.

[0067] The steps for reverse transcription of RNA extracted by Trizol into cDNA using reverse transcription reagent are as follows:

[0068] First-strand cDNA synthesis

[0069] 1. Remove gDNA (the reaction solution should be prepared on ice).

[0070]

[0071] Mix gently, place in PCR instrument at 42°C for 3 min, and place on ice.

[0072] 2. Prepare the reverse transcription system mixture:

[0073]

[0074] After gentle mixing, perform the reverse transcription reaction under the following conditions:

[0075] 42℃15min (reverse transcription reaction)

[0076] 95℃3min (inactivation reaction of reverse transcriptase)

[0077] 4℃(storage).

[0078] cDNA was semi-quantitatively detected using fluorescent quantitative PCR. The steps are as follows:

[0079] Fluorescence quantitative QPCR detection

[0080] The template used on the machine is diluted 5 times

[0081] qPCR reaction system:

[0082]

[0083]

[0084] qPCR reaction conditions: 3-step amplification

[0085]

[0086] CT detection limit: 45 cycles;

[0087] The experimental results are as follows Figure 2 shown.

[0088] like Figure 2 As shown, the detection of RNA extracted from the epidermal model by qPCR method showed that compared with the blank control, the lactoferrin aqueous solution samples A1, A2 and A3 and the cosmetic preparation sample A4 at various concentrations could significantly increase the expression of AQP3; compared with the positive control, the CT values ​​of 0.2wt% lactoferrin aqueous solution sample A2 and the cosmetic preparation sample A4 were equivalent to or slightly lower than 0.5wt% vaseline, and the CT value of 0.5wt% lactoferrin aqueous solution sample A3 was even slightly higher than 0.5wt% vaseline.

[0089] Immunofluorescence staining

[0090] The experimental steps for immunofluorescence staining of AQP3 protein are as follows:

[0091] 1) The other half of the epidermal model treated with the lactoferrin aqueous solution samples A1, A2 and A3 prepared in Example 1 and the cosmetic preparation sample A4 containing lactoferrin prepared in Example 2 was collected, embedded with an embedding agent and frozen at -80°C for 24 hours. After being completely frozen, the model was taken out and sliced ​​with a microtome and placed at room temperature for more than 2 hours to prevent tissue shedding.

[0092] 2) Fix with 4% paraformaldehyde for 20 min and wash twice with PBS.

[0093] 3) Block with BSA for 1 hour and wash twice with PBS.

[0094] 4) Incubate with primary antibody (AQP3, PA5-78811, Invitrogen) at 4°C overnight.

[0095] 5) Incubate with secondary antibody (Rb 488, A0423, Biotech) for 1 hour at room temperature in the dark. Wash twice with PBS.

[0096] 6) Seal the slides with a mounting medium containing DAPI.

[0097] 7) Take pictures using a fluorescence inverted microscope and analyze the results.

[0098] The experimental results are as follows Figure 3 shown.

[0099] As attached Figure 3 As shown, the immunofluorescence staining results of AQP3 showed that compared with the blank control, the lactoferrin aqueous solution samples A1, A2 and A3 and the cosmetic preparation sample A4 at various concentrations could significantly increase the relative positive rate and relative fluorescence intensity of AQP3; compared with the positive control, the relative positive rate of AQP3 of 0.5wt% lactoferrin aqueous solution sample A3 was equivalent to that of 5mg / mL vaseline, and the relative positive rate of AQP3 of 0.2wt% lactoferrin aqueous solution sample A2 and the cosmetic preparation sample A4 was slightly lower than that of 5mg / mL vaseline; and the relative fluorescence intensity of 0.5wt% lactoferrin concentration was slightly lower than that of 0.5wt% vaseline.

[0100] ELISA test

[0101] The experimental steps of ELISA test are as follows:

[0102] 1) Collect the culture medium of the epidermal model for HAS1 ELISA (ml057977, ELISA) test.

[0103] 2) Take out the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes.

[0104] 3) Set up a HAS1 standard well, a blank control well, a positive control well (vaseline 5 mg / mL), lactoferrin aqueous solution samples A1, A2 and A3 prepared in Example 1, and a cosmetic preparation sample A4 containing lactoferrin prepared in Example 2, each with 50 μL.

[0105] 4) Add 100 μL of HAS1 detection antibody labeled with horseradish peroxidase (HRP), seal the reaction wells with sealing film, and incubate at 37°C for 60 min.

[0106] 5) Wash the plate three times, add 50 μL of TMB substrate A and B to each well, and shake at room temperature in the dark for 15 minutes.

[0107] 6) Add 50 μL of hydrochloric acid stop solution and measure the OD value of each well at a wavelength of 450 nm within 15 minutes.

[0108] The experimental results are as follows Figure 4 shown.

[0109] like Figure 4 As shown, the results of ELISA indicate that, compared with the blank control, each concentration of lactoferrin aqueous solution samples A1, A2 and A3 and the cosmetic preparation sample A4 can significantly increase the content of HAS1; compared with the positive control, the relative concentration of HAS1 in the 0.5wt% lactoferrin aqueous solution sample A3 is slightly higher than that in the 0.5wt% vaseline.

[0110] The above transepidermal electrical resistance (TEER), qPCR, immunofluorescence staining and ELISA experimental results are summarized in Table 4 below:

[0111]

[0112] The results in Table 4 confirm that after using lactoferrin aqueous solution or cosmetics or personal care products containing lactoferrin in the skin model, the expression levels of moisturizing-related genes, proteins and moisturizing factors are all increased to a certain extent, proving that lactoferrin has a moisturizing effect.

Claims

1. Use of lactoferrin as a moisturizer in cosmetics or personal care products.

2. Use of lactoferrin in preparing cosmetics or personal care products with moisturizing function.

3. Use of lactoferrin as a sole humectant in cosmetic or personal care products.

4. Use of lactoferrin as the sole moisturizer in the preparation of cosmetics or personal care products with moisturizing function.

5. The use according to any one of claims 1 to 4, wherein the cosmetic or personal care product is selected from hand care, face care, body care, foot care, head care, nail care or mouth care products.

6. The use according to any one of claims 1 to 4, wherein the amount of lactoferrin is 0.01 wt% to 1 wt% of the cosmetic or personal care product.

7. The use according to claim 6, wherein the amount of lactoferrin is 0.05 wt% to 0.5 wt% of the cosmetic or personal care product.

8. A cosmetic or personal care product having moisturizing function, wherein the cosmetic or personal care product comprises lactoferrin as the only moisturizer.

9. The cosmetic or personal care product according to claim 8, wherein the cosmetic or personal care product is selected from hand care, face care, body care, foot care, head care, nail care or mouth care products.

10. The cosmetic or personal care product according to claim 8 or 9, wherein the amount of lactoferrin is 0.01 wt% to 1 wt% of the cosmetic or personal care product.

11. The cosmetic or personal care product according to claim 10, wherein the amount of lactoferrin is 0.05 wt% to 0.5 wt% of the cosmetic or personal care product.

Images