Application of scylla serrata dodecapeptide in preparation of skin soothing composition

Abstract

The invention discloses application of scylla serrata dodecapeptide in preparation of a skin relieving composition. The amino acid sequence of the scylla serrata dodecapeptide is as shown in SEQ ID NO.01. The invention further discloses a preparation method of the scylla serrata dodecapeptide. The blue crab dodecapeptide can effectively inhibit polarization of LPS-induced mouse macrophages RAW 264.7 to type 1 macrophages, secretion of proinflammatory factors and increase secretion of anti-inflammatory factors, can effectively relieve irritation of SDS to zebra fish embryos, does not show cytotoxicity to the mouse macrophages RAW 264.7, does not have an obvious stimulation effect on the zebra fish embryos, and can be used for preparing the anti-inflammatory and anti-inflammatory drugs. The traditional Chinese medicine composition has the effect of relieving inflammation and is relatively good in safety.

Description

Technical Field

[0001] The invention belongs to the technical field of marine molecular biology, and particularly relates to the use of blue crab dodecapeptide in preparing a skin soothing composition. Background Art

[0002] Epidemiological studies have shown that the global incidence of sensitive skin is very high, accounting for about 50% of the total population, and has become a common skin problem that troubles people. According to the "Cosmetic Classification Rules and Catalog", soothing effect is defined as helping to improve skin irritation, mainly targeting sensitive skin syndrome caused by damaged skin barrier function, abnormal nerve function or inflammatory response. For people with sensitive skin, soothing products can quickly relieve uncomfortable symptoms such as skin tightness, redness, burning, stinging, itching, desquamation, etc., and improve skin comfort, so they are becoming more and more popular among consumers.

[0003] There are many types of soothing cosmetics on the market, but some cosmetics that illegally add hormones and other anti-allergic chemicals may have quick initial effects, but they are easy to become addicted to and have obvious side effects, which may lead to skin problems such as skin atrophy, sensitivity, capillary dilation and pigmentation. Many soothing raw materials of plant extracts on the market are widely recognized by consumers, but the research on the effective ingredients and mechanism of action of plant extract raw materials is still incomplete, and their addition amount, optimal compatibility combination and safety performance need further research. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects of the prior art and provide the use of blue crab dodecapeptide in preparing a skin soothing composition.

[0005] Another object of the present invention is to provide a skin care composition.

[0006] The technical solution of the present invention is as follows:

[0007] Use of blue crab dodecapeptide in preparing a skin soothing composition, the amino acid sequence of the blue crab dodecapeptide is shown as SEQ ID NO.01.

[0008] A skin soothing composition, the effective ingredient of which includes the blue crab dodecapeptide with an amino acid sequence as shown in SEQ ID NO.01.

[0009] In a preferred embodiment of the present invention, the active ingredient is the blue crab dodecapeptide.

[0010] The invention discloses a use of blue crab dodecapeptide in preparing a cosmetic composition. The amino acid sequence of the blue crab dodecapeptide is shown as SEQ ID NO.01.

[0011] A cosmetic composition, the active ingredient of which includes the blue crab dodecapeptide with an amino acid sequence as shown in SEQ ID NO.01.

[0012] In a preferred embodiment of the present invention, the active ingredient is the blue crab dodecapeptide.

[0013] The beneficial effects of the present invention are:

[0014] 1. The blue crab dodecapeptide of the present invention can effectively inhibit the polarization of mouse macrophages RAW 264.7 to type 1 macrophages induced by LPS, and has the effect of relieving inflammation.

[0015] 2. The blue crab dodecapeptide of the present invention can effectively inhibit the secretion of pro-inflammatory factors of mouse macrophage RAW264.7 and increase the secretion of anti-inflammatory factors, thus having the effect of relieving inflammation.

[0016] 3. The concentration of blue crab dodecapeptide in the present invention can effectively alleviate the irritation of SDS to zebrafish embryos and has a soothing effect.

[0017] 4. The blue crab dodecapeptide of the present invention showed no cytotoxicity to mouse macrophages RAW 264.7 and no obvious stimulation to zebrafish embryos, and had good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the toxicity evaluation results of the blue crab dodecapeptide in Example 1 of the present invention on mouse macrophages RAW 264.7.

[0019] The horizontal axis represents the concentration of blue crab dodecapeptide (μg / mL), and the vertical axis represents the cell viability (%).

[0020] Figure 2 The results of the effect of the blue crab dodecapeptide on the morphology of mouse macrophage RAW264.7 cells stimulated by LPS in Example 2 of the present invention are shown in Figure 2. There are 8 groups in total, including a blank control group (BC), a negative control group (NC), a positive control group (PC) and groups with different concentrations of blue crab dodecapeptide, in which the red arrows mark cells polarized into type 1 macrophages.

[0021] Figure 3 This is a result diagram of the effect of the blue crab twelve peptide on the secretion levels of pro-inflammatory factors TNF-α, IL-6, IL-8, PGE2 and anti-inflammatory factor IL-10 in mouse macrophage RAW264.7 under LPS stimulation in Example 3 of the present invention. The horizontal axis represents different groups, including blank control group (BC), negative control group (NC), positive control group (PC) and groups with different concentrations of blue crab twelve peptide; the vertical axis is the content of inflammatory factors (μg / mL).

[0022] Figure 4This is a graph showing the effects of different concentrations of blue crab dodecapeptide on 24 hpf zebrafish embryos at different times in Example 4 of the present invention. The horizontal axis is the effect of different concentrations of blue crab dodecapeptide (μg / mL) on zebrafish embryos at different times (min), and the vertical axis is the stimulation intensity (%) of zebrafish embryos.

[0023] Figure 5 This is a graph showing the soothing effect evaluation results of different concentrations of blue crab dodecapeptide on 24 hpf zebrafish embryos stimulated in Example 5 of the present invention. The horizontal axis represents different groups, including a blank control group (BC), a negative control group (NC) and groups with different concentrations of blue crab dodecapeptide; the vertical axis represents the stimulation intensity (%) of zebrafish embryos. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0025] The blue crab dodecapeptide in the following examples is composed of 12 amino acids, and the amino acid sequence is: Arg-Arg-His-Gly-His-Val-Arg-Cys-Ser-Val-Val-Val (RRHGHVRCSVVV, SEQ ID NO.01). The blue crab dodecapeptide with a purity of more than 95% can be obtained by the existing solid phase chemical synthesis method. The blue crab dodecapeptide in the examples was synthesized by a solid phase synthesis method commissioned by Gill Biochemical (Shanghai) Co., Ltd., and high performance liquid chromatography and mass spectrometry detection information is provided.

[0026] Example 1 Evaluation of the cytotoxicity of blue crab dodecapeptide on mouse macrophage RAW264.7

[0027] (1) Mouse macrophages RAW264.7 were cultured in high-glucose DMEM cell culture medium containing 10% FBS, and fresh culture medium was replaced after the cells adhered.

[0028] (2) When the cells cover 80% to 90% of the bottom of the culture flask, cell subculture is performed. Cells in good growth condition are subcultured at 1×10 5 The cells were seeded into a 96-well plate at a density of 100 cells / well and incubated overnight in a cell culture incubator.

[0029] (3) When the cell plating rate in the 96-well plate reached 40% to 60%, cell culture medium containing different concentrations of blue crab dodecapeptide was added. The solvent control group was added with complete cell culture medium, and the zero control group was added with cell culture medium only without cell inoculation. Three replicate wells were set for each group.

[0030] (4) After incubation for 24 hours, discard the supernatant, add 0.5 mg / mL MTT working solution, and incubate at 37° in the dark for 4 hours. After the incubation, discard the supernatant, add 100 μL DMSO to each well, and measure the absorbance OD with a microplate reader. 490 nm , the cell survival rate was calculated according to the following formula:

[0031]

[0032] The results are as follows Figure 1 As shown, the blue crab dodecapeptide showed no toxicity to macrophage RAW264.7 within the concentration range of 600 μg / mL.

[0033] Example 2 Effect of Scylla 12-peptide on the morphology of RAW264.7 cells stimulated by LPS

[0034] (1) Cells in good growth condition were divided into 1×10 6 The cells were inoculated into a 24-well plate at a density of 100 cells / well and incubated in a cell culture incubator for 6-8 hours.

[0035] (2) When the cell plating rate reaches 40% to 60%, the supernatant is discarded and complete cell culture medium (blank control BC), cell culture medium containing 1 μg / mL LPS (negative control NC), cell culture medium containing 1 μg / mL LPS and 100 μg / mL dexamethasone (positive control PC), and cell culture medium containing 1 μg / mL LPS and different concentrations of blue crab dodecapeptide (sample group) are added to the well plate, 1 mL is added to each well, and 3 parallels are set for each group.

[0036] (3) After adding the sample, place the cells in a cell culture incubator and continue culturing for 24 hours. Observe the cell morphology of each well under a microscope and take photos to record the results.

[0037] The results are as follows Figure 2 As shown, LPS can induce the polarization of mouse macrophages RAW264.7 into type 1 macrophages (NC group), and blue crab dodecapeptide can reduce LPS-induced macrophage type 1 polarization, indicating that blue crab dodecapeptide can effectively inhibit LPS-induced mouse macrophages RAW 264.7 polarization into type 1 macrophages and has the effect of alleviating inflammation.

[0038] Example 3 Effect of Scylla dodecapeptide on the level of inflammatory factors in RAW264.7 cells stimulated by LPS

[0039] The cell supernatant in each well in Example 2 was collected, and the contents of pro-inflammatory factors TNF-α, IL-6, IL-8 and PGE2 and anti-inflammatory factor IL-10 were determined according to the instructions of the ELISA kit.

[0040] The results are as follows Figure 3 As shown in the results, compared with the NC group, the secretion of proinflammatory factors TNF-α, IL-6, IL-8 and PGE2 in macrophages RAW264.7 was significantly reduced (P<0.05), and the secretion of anti-inflammatory factor IL-10 was significantly increased (P<0.01) when the concentrations of blue crab dodecapeptide were 25μg / mL, 50μg / mL, 100μg / mL, 300μg / mL and 500μg / mL, indicating that blue crab dodecapeptide can reduce the LPS-induced macrophage inflammatory response by reducing the secretion levels of proinflammatory factors TNF-α, IL-6, IL-8 and PGE2 and increasing the secretion level of anti-inflammatory factor IL-10 at the above treatment concentrations.

[0041] Example 4 Toxicity evaluation of blue crab dodecapeptide on zebrafish embryos

[0042] (1) Use Holt-Buffer culture medium to dilute the blue crab dodecapeptide to the concentrations of 100 μg / mL, 50 μg / mL, 25 μg / mL, and 12.5 μg / mL in sequence.

[0043] (2) Randomly select healthy zebrafish embryos at 24 hpf with consistent developmental status and place them in a 96-well plate, with 6 embryos per well.

[0044] (3) Add the above concentrations of blue crab dodecapeptide, 200 μL per well. At the same time, add Holt-Buffer culture medium as a blank control group, and set up 3 parallels for each group.

[0045] (4) After the sample was added, video was recorded every 15 minutes for 1 hour using a stereo microscope equipped with an imaging system to record the number of spin movements of the zebrafish embryo within 30 seconds.

[0046] (5) The stimulation intensity of the embryo is calculated by the number of spin movements of the zebrafish embryo. The stimulation intensity calculation formula is as follows:

[0047] Stimulation intensity (%) = N / M × 100%

[0048] Where N is the number of spin movements of zebrafish embryos in the experimental group; M is the average number of spin movements of zebrafish embryos in the blank control group.

[0049] (6) The toxicity of different concentrations of blue crab dodecapeptide to zebrafish embryos was preliminarily evaluated by the stimulation intensity of each group of zebrafish embryos and the survival of the embryos.

[0050] The results are as follows Figure 4As shown, compared with the blank control group, after treating the embryos for 15min, 30min, and 45min, the stimulation intensity of zebrafish embryos in the four concentration groups did not change significantly (P>0.05), and no death of zebrafish embryos occurred, indicating that blue crab dodecapeptide had no obvious toxicity to zebrafish embryos within the concentration range of 100μg / mL.

[0051] Example 5 Evaluation of the soothing effect of blue crab dodecapeptide on zebrafish embryos stimulated by SDS

[0052] A zebrafish embryo stimulation model was established based on zebrafish embryo behavior. Different concentrations of SDS were used to stimulate 24hpf zebrafish embryos, and the number of embryonic tumbling times was recorded at different times to determine the optimal modeling concentration of SDS. On this basis, the soothing effect of blue crab dodecapeptide on zebrafish embryos stimulated by SDS was evaluated. The experimental method is as follows:

[0053] (1) Randomly select healthy zebrafish embryos at 24 hpf with consistent developmental status and place them in a 96-well plate, with 6 embryos per well.

[0054] (2) Different concentrations of blue crab dodecapeptide were added, 200 μL per well, and 4 parallels were set for each concentration. A blank control group BC and a negative control group NC were also set up.

[0055] (3) After incubating the 96-well plate in a 28°C constant temperature biochemical incubator for 45 min, the solution in the 96-well plate was removed and a mixture of different concentrations of blue crab dodecapeptide and SDS was added to the experimental group at a volume ratio of 1:1, the BC group was added with Holt-Buffer culture solution, and the NC group was added with SDS working solution.

[0056] (4) The video recording, recording method and stimulation intensity calculation formula were the same as those described in Example 3. The soothing effect of blue crab dodecapeptide was evaluated by comparing the changes in stimulation intensity of zebrafish embryos in each group.

[0057] The results are as follows Figure 5 As shown in the results, compared with the negative control group, the stimulation intensity of zebrafish embryos was significantly reduced when the concentration of blue crab dodecapeptide was 50μg / mL and 100μg / mL (P<0.01), and the effect of reducing stimulation was dose-dependent with the concentration of dodecapeptide. This shows that blue crab dodecapeptide has a soothing effect on zebrafish embryos stimulated by SDS.

[0058] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. Use of blue crab dodecapeptide in preparing a skin soothing composition, characterized in that: The amino acid sequence of the blue crab dodecapeptide is shown in SEQ ID NO.

01.

2. A skin soothing composition, characterized in that: The effective component thereof comprises the blue crab dodecapeptide with an amino acid sequence as shown in SEQ ID NO.

01.

3. A skin soothing composition according to claim 2, characterized in that: The effective ingredient is the blue crab dodecapeptide.

4. Use of blue crab dodecapeptide in preparing a cosmetic composition, characterized in that: The amino acid sequence of the blue crab dodecapeptide is shown in SEQ ID NO.

01.

5. A cosmetic composition, characterized in that: The effective component thereof comprises the blue crab dodecapeptide with an amino acid sequence as shown in SEQ ID NO.

01.

6. A cosmetic composition according to claim 5, characterized in that: The effective ingredient is the blue crab dodecapeptide.

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