Type I recombinant collagen, its metal chelate and whitening cosmetics

Abstract

This patent application discloses a type I recombinant collagen, a metal chelate thereof and a whitening cosmetic. The amino acid sequence of the type I recombinant collagen is shown in SEQ ID No: 1. The type I recombinant collagen and its metal chelate can inhibit both tyrosinase activity and protease activity receptor 2 activity, thereby inhibiting both new melanin generation and the transfer of generated melanin to the epidermis, thus having a dual mechanism of whitening effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and specifically relates to a type I recombinant collagen with whitening effect, a metal chelate thereof and application in whitening cosmetics. Background Art

[0002] From a scientific point of view, the color of the skin is mainly determined by the content of pigments in the skin, the most important of which is the content of melanin. The content of melanin in the skin can largely determine the depth of the skin color. Melanin is a protein that exists in everyone's skin, hair, retina, pia mater and other tissues. Melanin particles are not all pure black, but also yellow-brown, reddish-brown, tan, dark brown, etc. They work together to determine the different colors of human skin and hair.

[0003] Studies have shown that tyrosinase is a key enzyme in the production of melanin, and inhibiting the activity of tyrosinase can reduce the formation of melanin. After melanin is formed in the melanocytes in the basal layer of the skin, its life cycle also includes a series of complex processes such as migration, metabolism, degradation and excretion, and is closely related to keratinocytes and fibroblasts. The transfer of melanin to keratinocytes after production is mainly divided into two steps, namely, transfer within melanocytes and transfer between melanocytes and keratinocytes. Melanosomes are delivered to keratinocytes. The main regulator of melanosome delivery to keratinocytes is protease-activated receptor 2 (PAR-2). Both in vivo and in vitro experiments have shown that activating or inhibiting the activation of this receptor can enhance or weaken the phagocytic function of keratinocytes, thereby causing an increase or decrease in melanin delivery. At the same time, PAR-2 ​​also completes the melanization of the skin by stimulating the formation of dendrites in melanocytes. Summary of the invention

[0004] The problem to be solved by the present invention is to provide a recombinant collagen with whitening effect that can reduce melanin production and inhibit melanin transfer, a more optimal active form thereof, and the application thereof in whitening cosmetics.

[0005] The inventors conducted in-depth research to solve the above technical problems and found a new type I recombinant collagen that can inhibit both tyrosinase activity and protease activity receptor 2 activity, thereby inhibiting both new melanin production and the transfer of existing melanin to the epidermis, thus having a strong whitening effect. The inventors also found that divalent metal ion chelates are a more optimal active form of the type I recombinant collagen.

[0006] Specifically, the present invention includes:

[0007] 1. A type I recombinant collagen, the amino acid sequence of which is shown in SEQ ID No: 1.

[0008] SEQ ID No.: 1

[0009]

[0010]

[0011] 2. A nucleic acid encoding the type I recombinant collagen described in item 1.

[0012] 3. A host cell expressing the recombinant type I collagen described in item 1.

[0013] The host cell can be a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, Bacillus subtilis, Bacillus licheniformis, and eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, and plant cells. Escherichia coli and Pichia pastoris are preferred, and Escherichia coli is more preferred in consideration of yield.

[0014] 4. A chelate formed by the type I recombinant collagen described in item 1 and a divalent metal ion.

[0015] 5. The chelate according to item 4, wherein the divalent metal ion is selected from ferrous ion, zinc ion, calcium ion, copper ion and magnesium ion.

[0016] 6. The chelate according to item 4, wherein the divalent metal ion content of the chelate is 0.5% to 2% by weight, preferably 0.9% to 1.6% by weight.

[0017] 7. A method for preparing the chelate according to item 4, comprising:

[0018] Carrying out a chelation reaction between the type I recombinant collagen described in item 1 and a soluble divalent metal ion salt in a solution; and

[0019] After the chelation reaction is completed, the reaction solution is passed through a nanofiltration system to remove free divalent metal ions, and the solution is freeze-dried as needed to obtain the chelate.

[0020] 8. The preparation method according to item 7, wherein:

[0021] In the solution for the chelation reaction, the weight ratio of type I recombinant collagen to the soluble divalent metal ion salt is 1:0.01 to 1:0.05, preferably 1:0.02 to 1:0.03;

[0022] The temperature of the chelating reaction is 25°C to 70°C, preferably 30°C to 60°C;

[0023] The reaction time of the chelating reaction is 1 to 5 hours, preferably 2 to 3 hours;

[0024] The divalent metal ion content of the chelate is 0.5 wt % to 2 wt %, preferably 0.9 wt % to 1.6 wt %.

[0025] 9. Use of the recombinant collagen type I described in item 1 or the chelate described in item 4 in the preparation of cosmetics, medicines or medical devices for whitening.

[0026] 10. The use according to item 9, wherein the cosmetic, medicine or medical device is prepared in a form suitable for application to the skin.

[0027] The beneficial effect of the present invention is that it provides type I recombinant collagen with dual whitening mechanisms and a more optimal active form thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the SDS-PAGE electrophoresis of type I recombinant collagen.

[0029] Figure 2 This figure shows the expression of PAR-2 ​​in Hacat cells after protein intervention detected by Western Blot.

[0030] Figure 3 Figure 2 is a graph showing data of Western Blot protein bands. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below through specific embodiments. It should be particularly pointed out that these descriptions are merely exemplary descriptions and do not constitute limitations on the scope of the present invention.

[0032] Example 1: Preparation and purification of recombinant collagen in large intestine expression system

[0033] 1. Preparation of shuttle plasmid

[0034] According to the amino acid sequence shown in SEQ ID No: 1, the codon optimization of the yeast expression system was carried out to obtain the type I recombinant collagen target gene sequence. The obtained target gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis. The synthesized gene was connected to the pPicZαA plasmid to obtain the pPicZαA-I-α1 plasmid.

[0035] 2) Preparation of yeast expression strains

[0036] pPicZαA-I-α1 was linearized with Pme I and then transformed into Pichia pastoris X-33 competent cells. Bleomycin resistance was used as a selection marker to screen transformants to obtain a yeast expression strain.

[0037] 2. Induced expression of target protein

[0038] (1) Pick a single colony of the constructed yeast expression strain and add it to 5 ml of YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and culture it at 30°C and 200 rpm for 48 h for activation;

[0039] (2) Transfer the inoculum to a 500 ml Erlenmeyer flask (containing 200 ml of YPD culture medium) at a 1% inoculum volume and culture at 30°C, 200 rpm for 24 h to serve as the seed for the tank;

[0040] (3) Prepare 3 L of BSM medium and add it to a 5 L fermenter, sterilize at 121° C. for 20 min, and after cooling to 30° C., adjust the pH to 5.0, and add the seeds prepared in (2) to the fermenter by flame inoculation for fermentation;

[0041] (4) Cultivate until OD 600 When OD was 70, 50% glycerol was added until 600 When the temperature reaches about 120, stop adding, and wait until the dissolved oxygen rebounds to 100%, then start adding methanol for induction;

[0042] (5) During the induction process, the dissolved oxygen was controlled to be not less than 30%, the pH was 5.5, and the induction was continued for 40 hours. The fermentation was terminated, the culture medium was centrifuged at 12000 rpm for 2 minutes, and the supernatant was collected. The collected supernatant was passed through a 30KD ultrafiltration membrane, and then passed through a hollow fiber column and freeze-dried to obtain type I recombinant collagen. The SDS-PAGE electrophoresis of type I recombinant collagen is shown in Figure 1 As shown, its molecular weight is about 106kD.

[0043] Example 2 Preparation of recombinant collagen copper and recombinant collagen ferrous

[0044] The type I recombinant collagen prepared in Example 1 was mixed with copper sulfate to form a mixed solution, and the solution temperature was maintained at 60° C. After chelation reaction for 3 hours, the reaction solution was concentrated to 40% solid by an ultrafiltration system, and then spray-dried in a drying tower to obtain recombinant collagen copper, in which the chelated copper content was 1.6% by weight.

[0045] The type I recombinant collagen prepared in Example 1 was mixed with ferrous chloride and VC to form a mixed solution, and the solution temperature was maintained at 30°C. After chelation reaction for 2 hours, the reaction solution was concentrated to 40% solid by ultrafiltration system, and then freeze-dried to obtain recombinant collagen ferrous iron, in which the chelated ferrous iron content was 1.2% by weight.

[0046] Example 3 Inhibition rate of tyrosinase in vitro by different concentrations of recombinant collagen copper and recombinant collagen ferrous

[0047] Test method: Refer to the group standard of Guangdong Cosmetics Association "T / GDCA 006-2021 Test method for inhibition of tyrosinase activity by cosmetic raw materials (in vitro method)". Refer to the reagent addition amount in Table 1, add L-tyrosine solution, sample solution / solvent, and PBS buffer to each well in turn, mix thoroughly, incubate at 37°C for 10 minutes, then add 20 μL of tyrosinase solution to each well in turn, mix at 37°C for 5 minutes ± 5 seconds, and then put it into the microplate reader immediately for measurement at a wavelength of 475 nm.

[0048] Table 1

[0049]

[0050]

[0051]

[0052] Experimental results:

[0053] Table 2

[0054]

[0055] The amount of CuSO4 solution was calculated according to the proportion of 1.6% copper content chelated in 0.5mg / mL recombinant collagen copper, and the amount of FeCl2 solution was calculated according to the proportion of 1.2% ferrous content chelated in 0.5mg / mL recombinant collagen ferrous iron. Each batch of experiments had three parallel samples in each group, and the average value was calculated using EXCEL. A total of three batches of experiments were performed to finally calculate the average value to obtain the tyrosinase inhibition rate after intervention of different samples. The results are shown in Table 2. It can be seen that the type I recombinant collagen, recombinant collagen copper and recombinant collagen ferrous iron can significantly reduce tyrosinase activity, and there is a concentration-dependent relationship. Recombinant collagen copper and recombinant collagen ferrous iron are more active than type I recombinant collagen, which shows that divalent metal ion chelates are the more optimal active forms of the type I recombinant collagen, which may be because chelated divalent metal ions have an effect on the conformation of type I recombinant collagen, which is more conducive to its inhibition of tyrosinase activity.

[0056] Example 4 Inhibition of tyrosinase activity of melanoma cells by different concentrations of recombinant collagen copper and recombinant collagen ferrous

[0057] Methods: Determination of tyrosinase activity The activity of intracellular tyrosinase was determined by L-DOPA oxidation method. B16F10 cells were inoculated in 96-well culture plates and pretreated with 200nmol / L α-MSH to establish the model. After 24 hours, different samples were added according to the group. After 24 hours of culture, the cells were washed once with PBS, and 100μL of PBS containing 1% (volume fraction) TritonX-100 was added to each well. The cells were placed at -80℃ for 30 minutes and incubated at 37℃ to completely rupture the cells. After 20 minutes, 50uL of 1x 10 -2 mol / L of L-DOPA, incubate at 37°C for 30 min, and measure the absorbance of each well at 475 nm.

[0058] Experimental results:

[0059] Table 3

[0060]

[0061] The amount of CuSO4 solution was calculated according to the proportion of 1.6% copper content chelated in 0.5mg / mL recombinant collagen copper, and the amount of FeCl2 solution was calculated according to the proportion of 1.2% ferrous content chelated in 0.5mg / mL recombinant collagen ferrous. Each batch of experiments had six parallel samples in each group and the average value was calculated using EXCEL. A total of three batches of experiments were performed and the average value was finally calculated to obtain the relative tyrosinase activity of B16F10 cells after intervention with different samples. The experimental results are shown in Table 3. The type I recombinant collagen, recombinant collagen copper and recombinant collagen ferrous all significantly inhibited intracellular tyrosinase activity. At the same concentration, recombinant collagen copper and recombinant collagen ferrous have a stronger inhibitory effect on intracellular tyrosinase activity than recombinant type I collagen.

[0062] Example 5 Effects of different concentrations of recombinant collagen copper and recombinant collagen ferrous on melanin content in B16F10 melanoma cells

[0063] Methods: The NaOH lysis method was used to determine the intracellular melanin content. B16F10 cells were taken and 8*10 4 / well was inoculated in a 6-well culture plate, 2 mL of culture medium was added each time, and a-MSH with a final concentration of 200 nmol / L was used for pretreatment. After 24 hours, different concentrations of protein intervention were used. After 72 hours of culture, the cells were washed once with PBS, 0.25% trypsin was added to digest the cells and collected in a 1.5 mL centrifuge tube and centrifuged at 10000 r / min for 10 minutes. The supernatant was discarded and 1 mL of 1 mol / L NaOH aqueous solution containing 10% (volume fraction) DMSO was added. The centrifuge tube was sealed with a sealing film and heated in a water bath at 80 for 30 minutes. The absorbance value of each well was measured at 405 nm. Relative melanin content (%) = (average absorbance value of each concentration ÷ average absorbance value of the control group) x 100%.

[0064] Experimental results:

[0065] Table 4

[0066]

[0067]

[0068] The amount of CuSO4 solution was calculated according to the proportion of 1.6% copper chelated in 0.5mg / mL recombinant collagen copper, and the amount of FeCl2 solution was calculated according to the proportion of 1.2% ferrous chelated in 0.5mg / mL recombinant collagen ferrous. Each batch of experiments had four parallel samples in each group, and the average value was calculated using EXCEL. A total of three batches of experiments were performed to finally calculate the average value, and the relative melanin content of B16F10 cells after intervention with different samples was obtained. After intervention with type I recombinant collagen, recombinant collagen copper, and recombinant collagen ferrous, the relative melanin content decreased, and there was a concentration-dependent relationship. At the same concentration, recombinant collagen copper and recombinant collagen ferrous had a more obvious effect on reducing melanin content than type I recombinant collagen.

[0069] Example 6: Different concentrations of recombinant collagen copper inhibit melanocyte dendrite generation and melanosome transfer by inhibiting the production of PAR-2 ​​by Hacat cells

[0070] Methods: Hacat cells with good growth status were digested with trypsin and centrifuged, and then resuspended in DMEM complete medium and the cell concentration was adjusted to 2×10 5 Each well of a six-well plate was inoculated with 2 mL of cell suspension and cultured in a 37°C, 5% CO2 incubator. The experiment was set up with a blank group, a model group, and a sample group. The model group was treated with UVB 25 mJ / cm 2 The model was induced by UVB irradiation. On the basis of modeling, the sample group was treated with 0.5 mg / mL of I recombinant collagen, different concentrations of recombinant collagen copper and copper sulfate solution, and incubated in a 37°C, 5% CO2 incubator.

[0071] After 48 hours, discard the culture medium, wash with PBS buffer solution 3 times, add lysis solution containing 1% PMSF to each well, place on ice for 30 minutes, gently blow until the cells are completely detached, and collect the sample. Centrifuge the sample at 4℃ high-speed centrifuge at 12000rpm for 25 minutes, and collect the supernatant

[0072] The total protein concentration of Hacat cells was determined by using a BCA detection kit. The absorbance was detected at 495 nm and the protein concentration was calculated. The total protein concentration of each sample was adjusted to the same concentration using lysis buffer, and then the total protein concentration was mixed with 5× loading buffer at a ratio of 4:1. The samples were boiled in 100°C boiling water for a total of 8 minutes to denature the protein. After the samples returned to room temperature, they were placed in a -20°C refrigerator for use.

[0073] Thaw the sample to be tested in a 4°C refrigerator. Prepare the gel according to the preparation method of the separation gel and the concentrated gel. After solidification, add the sample, electrophoresis at a voltage of 80V for 30 minutes, and then switch to a voltage of 120V to continue electrophoresis for 1.5 hours before stopping. Transfer the separated gel to a polyvinylidene fluoride (PVDF) membrane. After the transfer is completed, prepare 10% skim milk powder (TBST dissolves), and block the PVDF membrane for 2 hours on a shaker at room temperature. Wash the blocked PVDF membrane with TBST 2 to 3 times, then place the PVDF membrane in the primary antibody prepared with skim milk powder, and incubate it in a shaker at 4°C overnight. Wash the PVDF membrane 3 to 5 times with TBST, and after washing, place the membrane in the secondary antibody prepared with skim milk powder, and incubate it on a shaker at room temperature for 2 hours. Then repeat the above membrane washing method, prepare the developer for development, and use ImageJ to analyze the gray value of the protein band.

[0074] Experimental results:

[0075] Table 5

[0076] Sample intervention concentration Relative to the model group down 0.5mg / mL recombinant collagen type I 27.82％*** 0.5mg / mL recombinant collagen copper 69.75％*** 0.25mg / mL recombinant collagen copper 46.79％*** 0.1mg / mL Recombinant Collagen Copper 45.63％*** CuSO4 solution 40.11％***

[0077] *Indicates significant difference compared with the model group, *p<0.05, **p<0.01, ***p<0.001.

[0078] Protein electrophoresis diagram of Western Blot detection Figure 2 As shown, protein bands were analyzed by ImageJ software, and data were plotted using GraphPad, as shown in Figure 3As shown, EXCEL was used to analyze the data, calculate STDEV and T test, and after UVB modeling, the expression of PAR-2 ​​in the model group decreased significantly compared with the normal group. After intervention with 0.1, 0.25, and 0.5 mg / mL of recombinant collagen copper, the expression was downregulated by 45.63%, 46.79%, and 69.75% respectively compared with the model group. At the same concentration of 0.5 mg / mL, recombinant collagen copper had a stronger inhibitory effect on PAR-2 ​​than type I recombinant collagen, and could better inhibit the phagocytosis of the generated melanin by Hacat cells.

Claims

1. A type I recombinant collagen, the amino acid sequence of which is shown in SEQ ID No:

1.

2. A nucleic acid encoding the recombinant type I collagen according to claim 1.

3. A host cell expressing the recombinant type I collagen according to claim 1.

4. The chelate formed by the type I recombinant collagen and divalent metal ions according to claim 1.

5. The chelate according to claim 4, wherein The divalent metal ions are selected from ferrous ions, zinc ions, calcium ions, copper ions and magnesium ions.

6. The chelate according to claim 4, wherein The content of the divalent metal ion in the chelate is 0.5 wt % to 2 wt %.

7. The chelate according to claim 4, wherein The content of the divalent metal ion in the chelate is 0.9 wt % to 1.6 wt %.

8. The method for preparing the chelate according to claim 4, comprising: Carrying out a chelation reaction between the type I recombinant collagen described in claim 1 and a soluble divalent metal ion salt in a solution; and After the chelation reaction is completed, the reaction solution is passed through a nanofiltration system to remove free divalent metal ions, and the solution is freeze-dried as needed to obtain the chelate.

9. The preparation method according to claim 8, wherein: In the solution for the chelation reaction, the weight ratio of type I recombinant collagen to soluble divalent metal ion salt is 1:0.01 to 1:0.05; The temperature of the chelation reaction is 25°C to 70°C; The reaction time of the chelation reaction is 1 to 5 hours; The divalent metal ion content of the chelate is 0.5 wt % to 2 wt %.

10. The preparation method according to claim 8, wherein: In the solution for the chelation reaction, the weight ratio of type I recombinant collagen to soluble divalent metal ion salt is 1:0.02 to 1:0.03; The temperature of the chelating reaction is 30°C to 60°C; The reaction time of the chelation reaction is 2 to 3 hours; The divalent metal ion content of the chelate is 0.9 wt % to 1.6 wt %.

11. Use of the type I recombinant collagen according to claim 1 or the chelate according to claim 4 in the preparation of cosmetics, medicines or medical devices for whitening.

12. The use according to claim 11, wherein The cosmetic, medicine or medical device is prepared in a form suitable for application to the skin.

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