A recombinant collagen composition, its uses and cosmetics

Abstract

This application belongs to the field of cosmetics and discloses a recombinant collagen composition, its uses, and cosmetics. The composition includes spermidine and recombinant humanized collagen. The recombinant humanized collagen includes a leader peptide and at least one basic repeating unit. The basic repeating unit includes the amino acid sequence shown in SEQ ID No. 1. This composition can exhibit superior skin application efficacy, such as autophagy, anti-inflammation, anti-aging, and repair.

Description

Technical Field

[0001] This invention relates to the field of new materials, and more particularly to a recombinant collagen composition, its uses, and cosmetics. Background Technology

[0002] Collagen, as a biological macromolecule, possesses abundant ligand-binding pockets, enabling it to bind with various small molecules to form composites. Through intermolecular interactions, collagen binds to physiologically active small molecules, potentially leading to superior therapeutic effects in these collagen compositions. In the biopharmaceutical field, for example, conjugating small molecule drugs to antibody proteins can endow them with targeted delivery capabilities, thereby enhancing efficacy and reducing toxicity; conjugating small molecule drugs to protein carriers can also increase drug solubility and provide a protective environment.

[0003] However, current applications of collagen in dermatology, cosmetics, and other fields are still limited to the functional properties of collagen itself. They mainly rely on the three-dimensional structure and physical properties of collagen itself to improve skin texture by injecting or delivering it into the dermis through other skin delivery methods, or to improve the skin barrier and radiance by improving the hydration of the epidermis through topical application. There is a lack of more innovative research and applications.

[0004] Current technologies mainly focus on the synthesis and preparation of collagen, its functional roles, and transdermal delivery to enhance its utilization and absorption in the skin. Research and application of combining active small molecules based on collagen's structural characteristics and anti-aging principles to achieve synergistic effects are virtually nonexistent.

[0005] A subsidiary of the same group as this applicant previously filed a patent application, publication number CN115960209A, entitled "A Recombinant Humanized Collagen and Its Applications." This application proposed a recombinant humanized collagen with the following characteristics: good solubility, low immunogenicity, easy availability, and high bioactivity. Particularly in terms of bioactivity, it exhibits excellent cell healing and proliferation-promoting abilities. Furthermore, the humanized lead peptide introduced into this recombinant humanized collagen endows the collagen with stronger cell penetration and skin permeability, making it a relatively ideal anti-aging skin ingredient.

[0006] This application was made after discovering some more peculiar phenomena during ongoing research on this collagen. Summary of the Invention

[0007] The purpose of this invention is to provide a recombinant collagen composition consisting of spermidine and recombinant humanized collagen, which can exhibit superior skin application effects, such as promoting autophagy, anti-inflammation, anti-aging, and repair.

[0008] In addition, the present invention also provides applications of the composition and cosmetics.

[0009] To achieve the above objectives, this application discloses a recombinant collagen composition comprising spermidine and recombinant humanized collagen; the recombinant humanized collagen comprising a leader peptide and at least one basic repeating unit; the basic repeating unit comprising the amino acid sequence shown in SEQ ID No. 1.

[0010] SEQ ID No.1: GPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPC;

[0011] Spermine is a low-molecular-weight polyamine that is naturally present in organisms. It mainly regulates mitochondrial function by inducing autophagy, improves the energy state of cells, effectively resists aging stimuli, and achieves anti-aging and longevity effects.

[0012] This invention utilizes the active site of the leader peptide fragment in the recombinant humanized collagen from the aforementioned patent to innovatively combine recombinant humanized collagen with spermidine molecules. This composition has a synergistic effect on the anti-aging efficacy of the skin. The collagen of this invention has the ability to promote cell proliferation and migration, improve skin repair, and also regulate the ECM structure of the dermis, thus improving wrinkles. Simultaneously, the humanized leader peptide endows the collagen with cell permeability and skin permeability, solving the problem of poor collagen absorption in the skin.

[0013] When spermidine molecules are combined with the above-mentioned collagen to form a composition, spermidine activates autophagy while collagen interacts with cells, improving the energy state of cells. Through the dual effects of collagen replenishment and spermidine energy replenishment, the overall anti-aging effect of collagen is enhanced and amplified, ultimately achieving a synergistic anti-aging effect, rather than a simple addition.

[0014] Furthermore, we discovered unexpected synergistic enhancements in atypical anti-aging effects, such as anti-inflammation and repair. Based on the collagen and spermidine combination, collagen can be further compounded with other active small molecules to achieve even greater efficacy.

[0015] Meanwhile, the structures of spermidine and recombinant humanized collagen were simulated using computer simulations, as detailed in the references below. Figure 1 and Figure 2 ;

[0016] Figure 1 Computer-simulated structural diagram of recombinant humanized collagen;

[0017] Figure 2Computer-simulated structural diagrams of recombinant humanized collagen and spermidine;

[0018] As can be seen from the simulation diagram, the two form an effective composite; we believe that this possible composite structure is the main reason for the optimization of the relevant effects.

[0019] In the above composition, the leader peptide comprises the amino acid sequence shown in SEQ ID No. 2.

[0020] SEQ ID No.2: MESCPTGPQNYSPQYDSYDVKSGVAVG;

[0021] In the above composition, the number of basic repeating units is 2 to 6, preferably 3 to 6, and the recombinant humanized collagen includes the amino acid sequence shown in SEQ ID No. 3.

[0022] SEQ ID No. 3:

[0023] MESCPTGPQNYSPQYDSYDVKSGVAVGGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCHHHHHH.

[0024] In the above composition, the weight ratio of spermidine to recombinant humanized collagen is 1-10:10-100.

[0025] In the above-mentioned composition, it can also serve as a carrier for cosmetic functional ingredients to further enhance the efficacy of cosmetic functional ingredients. Specifically, the composition of the present invention also includes cosmetic functional ingredients, which are one or more combinations of ergothioneine, ectoine, solanolone A, and solanine A.

[0026] We also conducted computer simulations regarding the binding of ergothioneine to the above-mentioned composition, as detailed in the following figures. Figure 3 ;

[0027] Figure 3 Computer simulation diagram of the structure of the carrier and ergothionein binding;

[0028] The amount of the cosmetic functional ingredient used is equivalent to 1 to 100% of the weight of recombinant humanized collagen.

[0029] Furthermore, the present invention also discloses the use of the composition described above in the preparation of cosmetics.

[0030] In the above-mentioned uses, the cosmetic is a cosmetic that has at least one of the following functions after acting on the skin: promoting autophagy, anti-inflammatory, anti-aging, and repair.

[0031] Finally, the present invention also discloses a cosmetic product containing any of the compositions described above.

[0032] In the above-mentioned cosmetics, the cosmetics contain 0.0001 to 1 wt% of any of the compositions described above.

[0033] In the above-mentioned cosmetics, the cosmetics are those that act on the skin and are in the form of creams or lotions.

[0034] The present invention has the following advantages and effects compared with the prior art:

[0035] This invention innovatively combines recombinant humanized collagen with spermidine molecules to form a composition that synergistically enhances the anti-aging effects on the skin by utilizing the active site of the leader peptide fragment in recombinant humanized collagen. This collagen promotes cell proliferation and migration, improves skin repair, and regulates the ECM structure of the dermis, thus reducing wrinkles. Simultaneously, the human leader peptide endows the collagen with cell permeability and skin penetration, addressing the issue of poor collagen absorption in the skin. After forming the composition with the collagen, spermidine activates autophagy while the collagen interacts with cells, increasing cellular energy levels. Through the dual effects of collagen replenishment and spermidine energy replenishment, the overall anti-aging effects of collagen are enhanced and amplified, ultimately achieving a synergistic anti-aging effect, rather than a simple additive one. Furthermore, this composition also exhibits unexpected synergistic enhancements in atypical anti-aging effects, such as anti-inflammation and repair. Based on the patented collagen and spermidine composition, collagen can be further combined with other active small molecules to achieve even greater efficacy enhancement. Attached Figure Description

[0036] Figure 1 Computer-simulated structural diagram of recombinant humanized collagen;

[0037] Figure 2 Computer-simulated structural diagrams of recombinant humanized collagen and spermidine

[0038] Figure 3 Computer simulation diagram of the structure of the carrier and ergothionein binding;

[0039] Figure 4 This is a fluorescence imaging image from a cell autophagy experiment;

[0040] Figure 5 This is a fluorescence imaging image of type I collagen expression in cells. Detailed Implementation

[0041] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0042] Part 1: Raw Material Sources or Preparation

[0043] The recombinant humanized collagen was prepared according to Example 1 of the patent application CN115960209A, which discloses a recombinant humanized collagen and its applications. The recombinant humanized collagen comprises a leader peptide and four basic repeating units, and its sequence is shown in SEQ ID No. 3.

[0044] Spermine: Purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., purity ≥98%.

[0045] Part Two: Preparation and Performance Testing of the Composition

[0046] The method for preparing the composition of the present invention is as follows:

[0047] The purified recombinant humanized collagen solution (dissolved in 10 mM Tris-HCl aqueous solution at pH 7.8) was mixed with spermidine aqueous solution at different mass ratios. The mixture was stirred at room temperature, and the final concentration was shown in Table 1. After thorough stirring, the mixture was stored at 4°C.

[0048] The basic formulations of the embodiments and comparative examples of the present invention are shown in Table 1 below;

[0049] Table 1 Formulation Table Unit: ppm

[0050]

[0051]

[0052] Performance testing

[0053] 1. Autophagy test

[0054] Autophagy assays were conducted using NHDF cells. The experimental steps are as follows:

[0055] (1) Seeding plate: NHDF cells, 96-well black-walled transparent plate, 0.75*10 4One hole / hole.

[0056] (2) Incubation: Take out the 96-well plate, wash once with complete culture medium, add 50 μL of DAPGreen working solution (0.2 μM) to each well, and incubate in an incubator for 30 min. Wash twice with complete culture medium, add 100 μL of complete culture medium to each well, and incubate in an incubator for 16 h.

[0057] (3) Sample addition: Add 100 μL of complete culture medium to the blank group; wash the positive group 3 times with serum-free culture medium and add 100 μL of serum-free culture medium; add 100 μL of complete culture medium containing the corresponding concentration of sample to the sample group and incubate in an incubator for 24 h.

[0058] (4) Nuclear staining: Add complete culture medium containing Hoechest 33342 staining working solution (1 drop / mL) to each well and incubate for 10 min.

[0059] (5) Imaging plate reading: Take out the 96-well plate, discard the staining solution in the wells and replace it with HBSS. Detect the staining solution on the imaging instrument and calculate the relative average fluorescence intensity and enhancement rate of each cell in each group.

[0060] 2. Collagen expression test

[0061] The expression level of native type I collagen was measured using NHDF cells. The experimental steps are as follows:

[0062] (1) Seeding plate: NHDF cells, 96-well black-walled transparent plate, 0.35*10 4 One hole / hole.

[0063] (2) Sample addition: 24 h after cell plating, 100 μL of complete culture medium was added to the blank group; 100 μL of complete culture medium containing 1 ng / mTGFβ1 was added to the positive control group; and 100 μL of complete culture medium containing the corresponding concentration of sample was added to the sample group. The cells were incubated in an incubator for 72 h.

[0064] (3) Staining: After washing once with PBS, fix with paraformaldehyde for 10 min, and wash with PBS 3 times after fixation; prepare 0.1% Triton X solution in PBS, incubate for 15 min for permeation, and wash with PBS 3 times after permeation; prepare 5% BSA in PBS and incubate at room temperature for 1-2 h for blocking; dilute COL1 primary antibody with 5% BSA and incubate at room temperature for 1 h, and wash with PBS 3 times after incubation; dilute 488 secondary antibody in PBS and incubate at room temperature for 1 h, and wash with PBS 3 times after incubation; incubate DAPI at room temperature for 7 min, and wash with PBS 3 times after incubation.

[0065] (4) Imager reading: The imager detects the image and calculates the relative fluorescence intensity based on the captured image to determine the collagen expression enhancement rate.

[0066] 3. Anti-inflammatory test

[0067] The expression of TNF-α inflammatory cytokine was measured using THP-1 cells. The experimental steps are as follows:

[0068] (1) Seeding plate: Inoculate each well of a 24-well plate with 5*10 5 One THP-1 cell suspension was added to each well with complete culture medium until the total volume in the well was 0.5 mL.

[0069] (2) Sample addition and pre-incubation: Set up blank control group, model group and sample group. Add 0.5 mL of complete culture medium to the blank control group and the corresponding well. Add 0.5 mL of sample working solution with a concentration of 2 times the final test concentration to the corresponding well of the sample group. Shake to mix well and incubate in an incubator for 18-24 h.

[0070] (3) LPS stimulation: Add 10 μL of LPS working solution (100 μg / mL) to each well of the model group and sample group to make the final LPS concentration in the well about 1 μg / mL, shake to mix, and incubate in an incubator for 24 h.

[0071] (4) RNA extraction: Collect cells from each group of wells and extract total RNA from each group of cells using an RNA extraction kit.

[0072] (5) Reverse transcription: Total RNA from each group of cells was reverse transcribed into cDNA using a reverse transcription kit.

[0073] (6) qPCR detection: Using GAPDH as an internal reference, the relative expression levels of TNF-α and IL-1β in each group were detected.

[0074] 4. Repair Test

[0075] A cell scratch assay was conducted using Hacat cells. The experimental steps are as follows:

[0076] (1) Seeding: Add 100 μL of diluted cell suspension to a 96-well plate to make its concentration 1.0 × 10⁻⁶. 4 One hole / hole.

[0077] (2) Incubation: Place in a CO2 incubator and incubate for 48 hours until the cells adhere to the wall and grow to about 80%-90% of the 96-well plate.

[0078] (3) Scratching: Scratching was performed using a fully automated scratcher, and the sample was washed three times with PBS before being added to serum-free DMEM medium containing the sample.

[0079] (4) Sample addition: 100 μL of serum-free DMEM medium was added to the blank group, 100 μL of EGF serum-free DMEM solution with a final concentration of 8 ng / ml was added to the positive group, and the corresponding sample diluent was added to the sample group.

[0080] (5) Imaging: Images of scratches at 0h were recorded in the microplate cell imaging detector, and 6 parallel replicates were taken for each group.

[0081] (6) Incubation: After imaging, place in a CO2 incubator for 48 hours.

[0082] (7) Imaging: Remove the 96-well plate from the CO2 incubator and place it in a microplate cell imaging detector to record images of the scratches for 48 hours.

[0083] (8) Data processing: The images were processed using the built-in analysis software of the microplate cell imaging detector to obtain the scratch healing rate data of each image.

[0084] The test results are shown in Table 2 below. Figure 4 and Figure 5 ;

[0085] Figure 4 This is a fluorescence imaging image from a cell autophagy experiment;

[0086] Figure 5 This is a fluorescence imaging image of type I collagen expression in cells.

[0087] Table 2 Detection Results

[0088]

[0089]

[0090] Results analysis:

[0091] For the autophagy test, higher values ​​indicate stronger autophagy. Comparative Example 1, with recombinant humanized collagen alone (100 ppm), promoted autophagy by 25%; Comparative Example 3, with spermidine alone (5 ppm), also promoted autophagy by 44%. Example 2 (100+5) showed a 96% increase in autophagy, indicating that the combination of recombinant collagen and spermidine further enhanced the overall autophagy-promoting effect, exhibiting a synergistic effect. Autophagy is an important indicator of the anti-aging process, suggesting that this combination of recombinant collagen and spermidine has a more significant anti-aging effect.

[0092] For the type I collagen expression test, higher values ​​indicate stronger type I collagen expression in cells. Comparative Example 1 showed that recombinant humanized collagen alone (100 ppm) promoted type I collagen expression by 26%, while Comparative Example 4 showed no significant effect on promoting type I collagen expression with spermidine alone (1 ppm). Example 1 (100+1) showed a 57% increase in type I collagen expression, indicating that the combination of recombinant collagen and spermidine further enhances the overall cellular collagen expression effect, exhibiting a synergistic effect. Type I collagen is an important component of the dermis and a key indicator of anti-aging, suggesting that the combination of recombinant collagen and spermidine has a more significant anti-aging effect.

[0093] For the anti-inflammatory test, lower values ​​indicate lower levels of the TNF-α inflammatory factor and better anti-inflammatory effects. Recombinant humanized collagen alone inhibited TNF-α expression, with an inhibition rate of 35% in Comparative Example 1 (100 ppm) and 29% in Comparative Example 2 (10 ppm). Spermidine alone, however, promoted TNF-α expression, with a promotion rate of 21% in Comparative Example 4 (1 ppm). The combination of recombinant collagen and spermidine showed significant anti-inflammatory effects, with an inhibition rate of 54% in Example 1 (100+1) and 43% in Example 3 (10+1), indicating that the combination of recombinant collagen and spermidine further inhibited the expression of inflammatory factors and had a more significant anti-inflammatory effect.

[0094] For the repair test, higher values ​​indicate stronger cell migration. Recombinant humanized collagen alone promoted cell migration, with Comparative Example 1 (100 ppm) showing an increase of 18.2% and Comparative Example 2 (10 ppm) showing an increase of 4.7%. Comparative Example 3, with spermidine alone (5 ppm), had no promoting effect on cell migration. Example 2 (100+5) showed an increase of 28.2% in cell migration, and Example 4 (10+5) showed an increase of 16.2%, indicating that the combination of recombinant collagen and spermidine further enhances cell migration and has a more significant repair effect.

[0095] We believe the possible mechanism behind these effects is that the leader peptide structure in recombinant humanized collagen provides an active site to form a non-covalent bond with spermidine molecules, resulting in a supramolecular structure. Spermine is a signaling molecule that promotes autophagy and activates mitochondrial energy. When combined with patented recombinant humanized collagen, it amplifies the anti-aging effects of collagen on cells by regulating autophagy and energy-related physiological functions, exhibiting a synergistic effect. This enhanced anti-aging effect can further improve the anti-inflammatory and repair effects of cells.

[0096] In summary, the combination of the aforementioned recombinant humanized collagen with spermidine showed stronger enhancement effects than the monomers in promoting autophagy, collagen synthesis and expression, inhibiting inflammation, and promoting cell migration and repair.

[0097] Part Two: Preparation and Functional Verification of Compositions Containing Cosmetic Active Ingredients

[0098] The method for preparing the composition containing cosmetic active ingredients of the present invention is as follows:

[0099] The purified recombinant humanized collagen solution (dissolved in 10 mM Tris-HCl aqueous solution with pH 7.8) was mixed with spermidine and ergothioneine aqueous solution at different mass ratios. The mixture was stirred at room temperature, and the final concentration was shown in Table 3. After thorough stirring, it was stored at 4°C.

[0100] The basic formulations of embodiments of the present invention are shown in Table 3 below;

[0101] Table 3 Formulation Table Unit: ppm

[0102]

[0103] Performance testing

[0104] This section of performance testing focuses on the anti-inflammatory and repair functions tested in Part Two.

[0105] The test results are shown in Table 4 below;

[0106] Table 4 Test Results

[0107]

[0108]

[0109] Results analysis:

[0110] For anti-inflammatory tests, lower values ​​indicate lower levels of the TNF-α inflammatory factor and better anti-inflammatory effects. Specifically, Example 5 (a composition of collagen, spermidine, and ergothioneine) showed an inflammation inhibition rate of 63%, while Comparative Example 5 showed an inhibition rate of 3% for ergothioneine alone, and Example 1 showed an inhibition rate of 54%. This demonstrates that further combination of the recombinant humanized collagen and spermidine composition with ergothioneine can enhance the anti-inflammatory effect.

[0111] For the repair test, a higher value indicates a stronger cell migration effect. Among them, the cell migration improvement rate of Example 6 (a composition of collagen, spermidine, and ergothioneine) was 45%, while the improvement rate of ergothioneine alone in Comparative Example 5 was 8.9%, and the improvement rate of Example 2 was 28.2%, indicating that the combination of recombinant humanized collagen and spermidine with ergothioneine can enhance the cell repair effect.

[0112] Part Four: Application Formula

[0113] The application formula provided by this invention is for a face cream; its specific composition and processing technology are shown in Table 5.

[0114] Table 5 Application Formula

[0115]

[0116]

Claims

1. A recombinant collagen composition, characterized in that, It includes spermidine and recombinant humanized collagen, wherein the amino acid sequence of the recombinant humanized collagen is as shown in SEQ ID No. 3; the weight ratio of spermidine to recombinant humanized collagen is 1~10:10~100.

2. The composition according to claim 1, characterized in that, It also includes cosmetic functional ingredients, which are one or more combinations of ergothioneine, ectoine, solanolone A, and solanine A. The amount of the functional ingredient in the cosmetic product is 1 to 100% of the weight of the recombinant humanized collagen.

3. Use of the composition according to claim 1 or 2 in the preparation of cosmetics.

4. The use according to claim 3, characterized in that, The cosmetic product is one that, when applied to the skin, has at least one of the following effects: promoting autophagy, anti-inflammation, anti-aging, or repair.

5. A cosmetic product, characterized in that, The cosmetic contains the composition as described in claim 1 or 2.

6. The cosmetic product according to claim 5, characterized in that, The cosmetic contains 0.0001~1wt% of the composition as described in claim 1 or 2.

7. The cosmetic product according to claim 5, characterized in that, The cosmetics mentioned are cosmetics that act on the skin and are in the form of creams or lotions.

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