CN118406128A

CN118406128A - Eggshell membrane polypeptide composition, preparation method and application in wrinkle and hair loss prevention

Info

Publication number: CN118406128A
Application number: CN202211283655.5A
Authority: CN
Inventors: 王晓娟
Original assignee: Ningbo Gelukang Biotechnology Co ltd
Current assignee: Tu Zaiwen
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2024-07-30
Also published as: CN114874306B; CN114874306A

Abstract

The present application discloses an eggshell membrane polypeptide composition, a preparation method and an application in wrinkle prevention and hair loss prevention. The composition comprises at least one of the polypeptides shown in SEQ ID NO.1 to 11. The polypeptide composition has a significant effect of promoting proliferation and migration activity, inhibiting the contraction function of human skeletal muscle cells, promoting hair follicle regeneration, and preventing and treating hair loss, and has a promising application in anti-wrinkle, anti-aging and anti-hair loss cosmetics or cosmetics.

Description

Eggshell membrane polypeptide composition, preparation method and application thereof in preventing wrinkles and alopecia

Technical Field

The application relates to the technical field of eggshell membrane protein components, in particular to an eggshell membrane polypeptide composition, a preparation method and application thereof in preventing wrinkles and alopecia.

Background

Eggshell membranes are a class of bioactive substances consisting essentially of proteins, which are predominantly in the form of glycoproteins, similar to the cell membrane structure in cellular tissue. Besides glycoprotein, eggshell membrane contains a certain amount of other components, mainly including bioactive components such as collagen, keratin, ovalbumin, osteopontin, lysozyme, etc. The eggshell membrane protein has high content of polypeptide and amino acid, and is rich in sarcosine, proline, histidine, lysine, etc. These amino acids have good antioxidant activity and are essential nutrients for good quality animal hair. In addition, due to the composition components and structural functional characteristics of eggshell membranes, the eggshell membranes are used as functional components in a plurality of fields such as daily chemical industry, medicine, food health care and the like.

Disclosure of Invention

However, the further fully excavated active ingredients of eggshell membranes still have great significance in fully developing the application fields thereof.

In a first aspect, embodiments of the present application disclose an eggshell membrane polypeptide composition comprising at least one of the polypeptides shown in SEQ ID NO. 1-11.

In a second aspect, embodiments of the present application disclose an anti-wrinkling and anti-hair loss composition comprising at least one of the polypeptides shown in SEQ ID nos. 1-11, and a cosmetically or pharmaceutically acceptable salt of the polypeptides shown in SEQ ID nos. 1-11, a conjugate molecule, and at least one cosmetically or pharmaceutically acceptable excipient or adjuvant.

In a third aspect, embodiments of the present application disclose an anti-wrinkling and anti-hair loss composition comprising a cosmetically or pharmaceutically effective amount of at least one polypeptide as shown in SEQ ID No. 1-11, a cosmetically or pharmaceutically effective amount of an active agent selected from exfoliating agents, wetting agents, depigmenting or whitening agents, primary color agents, anti-glycation agents, nitric oxide synthase inhibitors, agents that stimulate the synthesis of and/or prevent the degradation of skin or epidermal molecules, agents that stimulate proliferation of fibroblasts and/or keratinocytes and stimulate differentiation of keratinocytes, agents that are intended to improve the dermis-epidermis junction, skin soothing agents, solidifying agents, anti-atmospheric and/or anti-radical agents, agents that act on capillary circulation and/or microcirculation, tranquilizers, anti-inflammatory agents, antimicrobial agents, agents that act on cellular metabolism, agents that act on capillary circulation and/or microcirculation, vitamins, chelating agents, organic or mineral photoprotectants that are resistant to uv light a and/or B, and mixtures thereof.

In a fourth aspect, the embodiment of the application discloses a wrinkle-preventing and alopecia-preventing composition nanoemulsion, which comprises, by weight, 2-8 parts of isopropyl myristate, 10-20 parts of polyoxyethylated castor oil, 12-18 parts of polyoxyethylene ether (EL-40), 1-10 parts of 1, 2-propanediol, 1-8 parts of isopropyl alcohol and 10-15 parts of at least one of polypeptides shown as SEQ ID NO.2, 4-8 and 10-11.

In a fifth aspect, the embodiment of the application discloses a wrinkle-preventing and alopecia-preventing composition nanoemulsion, which comprises, by weight, 2-8 parts of isopropyl myristate, 10-20 parts of polyoxyethylated castor oil, 12-18 parts of polyoxyethylene ether (EL-40), 1-10 parts of 1, 2-propanediol, 1-8 parts of isopropyl alcohol and 10-15 parts of a polypeptide mixture shown in SEQ ID NO. 4-8.

In a sixth aspect, the embodiment of the application discloses a wrinkle-preventing and alopecia-preventing composition nanoemulsion, which comprises, by weight, 2-8 parts of isopropyl myristate, 10-20 parts of polyoxyethylated castor oil, 12-18 parts of polyoxyethylene ether (EL-40), 1-10 parts of 1, 2-propanediol, 1-8 parts of isopropyl alcohol and 10-15 parts of polypeptide mixtures shown in SEQ ID NO. 4-8 and 10-11.

In a seventh aspect, the embodiment of the application discloses a wrinkle-preventing and alopecia-preventing composition nanoemulsion, which comprises, by weight, 2-8 parts of isopropyl myristate, 10-20 parts of polyoxyethylated castor oil, 12-18 parts of polyoxyethylene ether (EL-40), 1-10 parts of 1, 2-propanediol, 1-8 parts of isopropyl alcohol and 10-15 parts of polypeptide mixtures shown in SEQ ID NO.2, 4-8 and 10-11.

In an eighth aspect, the embodiment of the application discloses a preparation method of an eggshell membrane polypeptide composition, which comprises the following steps:

Obtaining eggshell membrane powder;

acidolysis, which comprises preparing suspension of eggshell membrane powder with 10m/v% acetic acid solution, adding 3-mercaptopropionic acid with final concentration of 0.5m/v% into the suspension, fully reacting, and centrifuging to obtain acidolysis solution;

the enzymolysis comprises the step of taking acidolysis solution and adopting an enzyme preparation to carry out enzymolysis to obtain a crude eggshell membrane polypeptide product, wherein the enzyme preparation comprises alpha-chymotrypsin and collagenase;

purifying, namely, carrying out Sephadex G-25 purification and RP-HPLC separation and purification on the crude eggshell membrane polypeptide.

In some embodiments, the enzymatic hydrolysis step comprises: first enzymolysis and second enzymolysis, first enzymolysis includes:

Regulating the pH value of the acidolysis solution to 5.0-5.5, separating out, centrifuging, taking the precipitate, dissolving the precipitate again with 10% acetic acid, dialyzing, taking out, and freeze-drying to obtain freeze-dried powder;

Preparing the freeze-dried powder into 50mg/mL solution by using 0.7m/v% physiological saline, adding a first enzyme preparation into the solution, treating the solution for 1 to 2.5 hours by adding 10000 to 15000U/mL enzyme, taking supernatant after enzyme deactivation treatment and centrifugation, concentrating and freeze-drying; wherein the first enzyme preparation used comprises alkaline protease 200000U/g, pepsin 30000U/g and papain 100000U/g;

The second enzymatic hydrolysis comprises: preparing 50mg/mL solution of the freeze-dried powder obtained by the first enzymolysis with 0.7m/v% physiological saline, adding a second enzyme preparation into the solution, treating the solution for 1 to 2.5 hours with the enzyme dosage of 10000 to 15000U/mL, performing enzyme deactivation treatment and centrifugation, taking supernatant, concentrating and freeze-drying to obtain a crude eggshell membrane polypeptide product; wherein the second enzyme preparation used comprises 100000U/g of alpha-chymotrypsin and 150000U/g of collagenase.

In a ninth aspect, the embodiment of the application discloses an application of the eggshell membrane polypeptide composition in the first aspect or the eggshell membrane polypeptide composition prepared by the preparation method in the eighth aspect in preparation of cosmetics for preventing wrinkles and alopecia.

According to the embodiment of the application, 11 polypeptides are prepared by carrying out enzymolysis on eggshell membrane powder, antibacterial experiments prove that the 11 polypeptides have antibacterial effects on 15 common pathogenic bacteria to different degrees, and cell experiments prove that 9 polypeptides have obvious proliferation and migration activity promotion effects on skin fibroblasts, and in addition, the eggshell membrane polypeptides have inhibition effects on the contraction function of human skeletal muscle cells, so that the eggshell membrane polypeptides can achieve the purpose of reducing muscle contraction by inhibiting the contraction of human skeletal muscle cells, and further the aim of reducing the generation of wrinkles is achieved.

According to the embodiment of the application, animal experiments are carried out, part of polypeptides in the prepared eggshell membrane polypeptides in 11 are combined to prepare the crease-resist and alopecia-preventing nanoemulsion, and skin repair experiments and alopecia repair experiments prove that the nanometer prepared by the eggshell membrane polypeptides has obvious wound healing induction, fibroblast to myofibroblast transformation promotion and collagen deposition in the fibroblast promotion, and has obvious skin tissue repair function; the eggshell membrane polypeptide prepared by the embodiment of the application has application prospects in anti-wrinkle and anti-aging cosmetics and also has application prospects in anti-hair loss cosmetics or health care products.

Drawings

FIG. 1 is a Sephadex G-25 purification elution diagram of a crude eggshell membrane polypeptide provided by an embodiment of the application.

FIG. 2 is a RP-HPLC chromatogram of the components corresponding to the elution peaks of 0.8-1.4 BV in FIG. 1.

FIG. 3 is an RP-HPLC chromatogram of the components corresponding to the elution peaks of 1.8-2.1 BV in FIG. 1.

FIG. 4 is a RP-HPLC chromatogram of the components corresponding to the elution peaks of 2.4-2.8 BV in FIG. 1.

FIG. 5 is a further RP-HPLC chromatogram of the fractions corresponding to the elution peaks in FIG. 2 with a retention time of 21-24 min.

FIG. 6 is a graph showing the results of migration of eggshell membrane polypeptides to HSF cells provided in an embodiment of the present application.

FIG. 7 is a graph showing the result of shrinkage inhibition of HSMCs by eggshell membrane polypeptides according to an embodiment of the present application.

FIG. 8 is a graph showing the results of regeneration of hair follicles of rat skin tissues by eggshell membrane polypeptides according to an embodiment of the present application.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application. The reagents not specifically and individually described in the present application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.

Preparation of eggshell membrane polypeptides

1. Preparation of eggshell membrane powder

Fresh eggshell, peeling eggshell membrane, baking in oven at 70deg.C for 6 hr, pulverizing with pulverizer, and sieving with 80 mesh sieve to obtain eggshell membrane powder.

2. Acidolysis

Weighing the eggshell membrane powder, and acidolyzing with acetic acid. In a specific embodiment, 15G of the prepared eggshell membrane powder is accurately weighed and added into 150mL of 10% (m/v) acetic acid solution according to the solid-to-liquid ratio of 1:100 to prepare eggshell membrane suspension, 3-mercaptopropionic acid is added into the suspension to ensure that the concentration of the 3-mercaptopropionic acid is 0.5% (m/v) respectively, the suspension is placed into a constant temperature oscillator at 90 ℃ to continuously react for 8 hours, reactants are centrifuged for 10 minutes at 8000r/min, sediment is taken, the volume of the sediment is fixed to 200mL by using 0.7m/v% physiological saline, and the protein content in the solution is measured by adopting a Coomassie brilliant blue G-250 method.

3. Enzymolysis

(1) First enzymolysis

Regulating pH value of the solution of egg shell membrane protein obtained by acidolysis to about 5.0-5.5, standing at room temperature for 10min, centrifuging at 8000r/min for 10min, and discarding supernatant to obtain milky protein precipitate. Dissolving again with 10% acetic acid, dialyzing, taking out, and lyophilizing to obtain lyophilized powder.

Preparing the freeze-dried powder into 50mg/mL solution with 0.7m/v% physiological saline, taking 50mL, adding a first enzyme preparation into the solution, treating the solution for 1 to 2.5 hours with the enzyme dosage of 10000 to 15000U/mL, inactivating enzyme at 90 ℃ for 10min after enzymolysis is finished, centrifuging the solution at 5000r/min for 10min, taking supernatant, concentrating and freeze-drying; wherein the first enzyme preparation used comprises 200000U/g of alkaline protease (7.0-14.0U/mg, P8038, sigma-Aldrich), 30000U/g of pepsin (1.07185, sigma-Aldrich) and 100000U/g of papain (P4762,. Gtoreq.10U/mg, sigma-Aldrich).

(2) Second enzymolysis

The method specifically comprises the following steps: preparing 50mg/mL solution of the freeze-dried powder by using 0.7m/v% physiological saline, taking 50mL, adding a second enzyme preparation into the solution, treating for 1-2.5 h by adding 10000-15000U/mL enzyme, inactivating enzyme at 90 ℃ for 10min after enzymolysis is finished, centrifuging for 10min at 5000r/min, taking supernatant, concentrating and freeze-drying; the crude eggshell membrane polypeptide can be obtained. Wherein the second enzyme preparation used comprises 100000U/g of alpha-chymotrypsin (. Gtoreq.40 units/mg protein, C3142, sigma-Aldrich) and 150000U/g of collagenase (. Gtoreq.125 CDU/mg sol, C0130, sigma-Aldrich).

4. Purification

The application further adopts ammonium sulfate fractional precipitation and Sephadex G-25 to further purify the prepared eggshell membrane polypeptide crude product, so as to obtain an isolated polypeptide product, and the polypeptide product is detected by HPLC.

(1) Sephadex G-25 purification

A chromatographic column with the volume of 60mL and the volume of 2.0cm multiplied by 35cm is filled by a Sephadex G-25 packing (Sigma-Aldrich, G2580) in a wet method, the crude eggshell membrane polypeptide is taken and dissolved in deionized water to prepare a sample solution with the volume of 100mg/mL, 2.0mL is taken and loaded for 30min, then the sample solution is eluted at the flow rate of 0.5mL/min, the detection is carried out at 280nm, and the eluting peak is collected. The results are shown in FIG. 1. As can be seen from FIG. 1, there are 3 elution peaks at 0.8 to 1.4BV, 1.8 to 2.1BV, and 2.4 to 2.8BV in the elution profile. Multiple Sephadex G-25 purifications can be performed, and the three elution peaks are collected, combined, concentrated separately, and freeze-dried.

(2) RP-HPLC separation and purification

Further performing RP-HPLC separation and purification on the freeze-dried powder of the 3 elution peak components obtained by the Sephadex G-25 purification, wherein the purification conditions are as follows: the separation column is SepaxHP-C ₁₈ column (Sai Ching U.S.), the eluent A is 0.1% trifluoroacetic acid (TFA)/H ₂ O, and the eluent B is 0.1% trifluoroacetic acid (TFA)/acetonitrile; the mobile phase was 25% B, the flow rate was 1.0mL/min, the detection wavelength was 215nm, and the column oven temperature was 40 ℃. The elution peaks were collected 100. Mu.L each, and the molecular weight of the material in the elution peaks was identified by mass spectrometry. If no separate peak is obtained by the first separation, the mobile phase elution procedure is adjusted to 10-35 min, and the phase B is eluted from 10% gradient to 40%.

(3) Mass spectrometry analysis

Test article: elution peak of RP-HPLC freeze-dried samples.

Primary Mass Spectrometry (MS) and tandem Mass spectrometry (MS/MS) analyses were performed on MALDI-TOF/TOF Mass spectrometers. CCA matrix: 0.1% TFA, 50% ACN, 50% H ₂ O. Taking 1 mu L of sample and 3 mu LCCA matrix, mixing uniformly, taking 0.5 mu L of mixed solution, spotting on the plate according to a rule, air-drying, and then sending into a machine for detection. MS, positive ion detection mode, acceleration voltage of ion source 20kV, N ₂ laser wavelength 337nm, pulse width 3ns, ion delay extraction 150ns, vacuum degree 4×10 ^-7 Torr. After the MS/MS mode is converted, ions meeting MS are subjected to tandem mass spectrometry, and the m/z range of the MS/MS mode is 0-3000.

The identification of the small molecular peptide is obtained by searching tandem mass spectrum data and sequence tags of Mascot software and comparing and analyzing the amino acid sequence of eggshell membrane protein disclosed in NCBI.

As can be seen from FIG. 2, the RP-HPLC spectra of the components corresponding to the elution peaks of 0.8-1.4 BV in FIG. 1 are shown in FIG. 5, since the RP-HPLC spectra have a plurality of fused elution peaks in the retention time of 21-24 min, the elution means are adopted to separate the components in one step. As shown in figures 3 and 4, the RP-HPLC maps of the components corresponding to the elution peaks of 1.8-2.1 BV and 2.4-2.8 BV in figure 1 are respectively shown. Thus, 11 chromatographic peaks were obtained in total, and the corresponding fractions were collected, concentrated and lyophilized multiple times, mass-detected, and then compared with the amino acid sequence of known eggshell membrane proteins, and the amino acid sequences of the 11 fragments were analyzed as shown in Table 1.

TABLE 1

Cell experiment and antibacterial experiment

1. Materials and methods

1. Antibacterial Activity detection

Reference "Cao Fengyi, malaysia and Hong Kong celebration, qin Qi, mellin, zhu Genxing; antibacterial property study of basic amino acid-PAF 26 derived polypeptides [ J ]. Chemical and biological engineering, 2021.05.17 ] the antibacterial properties of F1-F19 eggshell membrane polypeptides prepared in the above examples were tested by the methods disclosed.

Experimental strains: coli (e.coli EXPEC, cat No. BMZ134803, minghuake), salmonella enterica subspecies enterica (s.e. subsp. Cat No. ZKCC-1053, beijing middle department quality control biotechnology limited), staphylococcus aureus (s.aureus, cat No. ZKCC-55, beijing middle department quality control biotechnology limited), staphylococcus hemolyticus (S.h, B94695, minghuake), staphylococcus epidermidis (S.e, cat No. bm 123772, minghuake), bacillus subtilis (b.sub, cat No. B98052, minghuake), enterococcus faecalis (e.fae, cat No. BMZ121811, minghuake), teng Huangwei cocci (m.lut, cat No. BMZ116117, minghuake), hemolytic streptococcus (strep.h, cat No. B9077, minghuake), green streptococcus (strep.v, cat No. B80796, minghuake), faecal streptococcus (strep.f, cat No. B81864, minghuake), streptococcus pyogenes (strep.p, cat No. B84159, minghuake), pseudomonas aeruginosa (chry.v, cat No. CIP 108934, minghuake), proteus (pro.v, cat No. BMZ124689, minghuake) and vibrio parahemolyticus (vib.p, cat No. B87538, minghuake).

Pre-heating LB (Luria-Bertani) liquid culture medium is added into a 96-well plate in advance, then the bacteria are inoculated into each well, polypeptides (F1-F11) are respectively added into each well after being diluted in equal proportion, the mixture is placed into a constant temperature shaking table for culturing for 12 hours at 37 ℃, an enzyme-labeled instrument is used for detecting the OD value of 600nm, a culture well without a bacterial strain is used as a control, and when the OD600nm value exceeds a blank well by more than 0.2, the antibacterial performance is not shown.

2. Cell culture

Taking out skin fibroblasts (HSF, stock HTX2132, ATCC) frozen in a liquid nitrogen tank, putting the skin fibroblasts into a preheated constant-temperature water bath kettle with the temperature of 37 ℃ for rapid stirring and thawing, wiping the surface of the frozen tube with alcohol cotton, transferring the cells from the frozen tube to a centrifuge tube in an ultra-clean workbench for centrifugation at 1000rpm for 5min, discarding the supernatant, re-suspending the cells by using a culture solution, transferring the cells into a culture flask, adding 5mL of DMEM culture solution containing 10% FBS, putting the cells into a cell culture box, culturing at the temperature of 37 ℃ with the saturated humidity of 5% CO ₂ until the cell fusion degree reaches 80%, discarding the original culture medium, washing the cells twice by using PBS with the temperature of 0.25% trypsin+0.25% EDTA (Gibco) solution, adding the cells into the cell culture bottle according to the ratio of 1mL/25cm ², completely covering the cells at the bottom of the cell culture bottle, keeping the temperature of 37 ℃ for 2-3 min under a microscope, observing the condition, adding the cells into a medium for shaking and transferring the medium, and stopping the cell culture flask, and repeatedly filling the medium into a proper amount of culture flask for shaking culture flask when the cells are separated one by one, and stopping the cell culture flask, and filling the whole culture flask with the medium, and sealing the medium, and stopping the cell culture flask, and filling the medium, and the medium.

3. Cell migration Activity assay

HSF cells in the logarithmic growth phase were digested with 0.25% trypsin, and a cell suspension was prepared from a culture solution containing 10% fetal bovine serum and 200. Mu.g/mL of one of eggshell membrane polypeptides F1 to F11, inoculated into a 6-well plate at a density of 1/10 ⁶/well, cultured at 37℃under 5% CO ₂ until the cells were full, the culture solution was aspirated, and washed 1 time with PBS. A20. Mu.L sterile gun head was used to scribe an isolation line in the center of each well, carefully wash away the culture medium, and wash away the cell mass from the scratches several times with PBS to keep the edges of the scratches clean. After careful removal of the PBS, fresh medium without serum and samples of appropriate concentration are added and photographed under a microscope. Culturing in a cell culture box. During the culture, observations were made every 12h and recorded by photographing.

4. Cell proliferation activity assay

Proliferation of Human Skin Fibroblasts (HSF) was examined by MTT method. Experimental group: the eggshell membrane polypeptides F1 to F11 prepared in the above examples were dissolved in serum-free plain high sugar culture DMEM (containing 100U/mL penicillin and 100mg/mL streptomycin) respectively so that the concentrations of F1 to F11 were 200. Mu.g/mL, respectively, to be used as a culture solution for HSF cell culture. Control group: the culture broth was added to cells in 96-well plates (2×10 ⁴ cells/well) with serum-free DMEM medium (without F1 to F11) as a control. After the experimental group and the control group were cultured at 37℃in a 5% CO ₂ environment for 24 hours, 2. Mu.L of 5mg/mL MTT solution was added to each well, the culture was continued for 4 hours, and finally, the cells were dissolved in 200. Mu.L of DMSO solution, and absorbance at 570nm was measured on an ELISA reader. Cell proliferation rate = (experimental well OD 570-control well OD 570)/control well OD570 x 100% was calculated.

5. Muscle cell contraction detection

Contractility of muscle cells was evaluated using Cell contraction detection kit (Collagen-based Contraction Assay Kit, CBA-201) from Cell Biolabs. The experimental method comprises the following steps:

(1) Human skeletal muscle cells (HSMCs, american ATCC) were cultured in skeletal muscle cell medium (Punuocele, inc.) at 37℃under 5% CO ₂, and the harvested cells were resuspended to a final cell concentration of 10 ⁶ cells/mL medium.

(2) Mixing 100 μl of cell suspension with 400 μl of precooled collagen gel solution (model 5074,Advanced BioMatrix), transferring into 24-well plate, and incubating at 37deg.C for 1 hr; after collagen polymerization, 1.0mL of culture medium was added to the gel lattice and the culture was continued for 48 hours.

(3) Dividing the cells cultured in the step (2) for 48 hours into 11 groups (experimental groups), treating the cells with F1-F11 polypeptide solution with the concentration of 100 mu mol/L for 1 hour and 24 hours respectively before releasing the shrinkage matrix, and measuring the change of the size of the collagen gel by using a ruler; setting a control group, and taking the cells treated in the step (2) as a negative control. The shrinkage of human skeletal muscle cells was calculated for each group (mm = diameter of pre-release collagen gel (control group) -diameter of collagen gel after 24h release (experimental group).

6. Statistical analysis

All test data are expressed in mean and standard deviation, data were processed using SPSS13.0 software and data were subjected to multiple comparisons and significance differential labeling.

2. Results

The results of the observation that the concentrations of F1 to F11 reached 200. Mu.g/mL in this experiment are shown in Table 2.

TABLE 2 200. Mu.g/mL

F1

F2

F3

F4

F5

F6

F7

F8

F9

F10

F11

E.coli EXPEC

+

－

+

S.e.sub.

－

+

S.aur.

－

+

S.h

－

+

－

+

S.e

－

+

－

B.sub

－

E.fae

－

+

－

M.lut

－

+

－

+

－

+

Strep.h

－

+

Strep.v

－

+

－

+

Strep.f

－

+

－

Strep.p

－

+

－

+

－

+

Chry.v

－

+

－

Pro.v

－

+

－

+

Vib.p

－

+

－

+

－

Table 2 shows the bacteriostatic effect of eggshell membrane polypeptides F1-F11 prepared by the examples of the application on 15 common pathogenic bacteria, wherein "+" indicates 200 mug/mL has bacteriostatic effect and "-" indicates 200 mug/mL has no bacteriostatic effect. As can be seen from Table 2, F2 to F8, F10 and F11 have a broader antibacterial spectrum.

As shown in FIG. 6, the detection of the migration activity of 200. Mu.g/mL F1-F11 culture solution on HSF cells shows that after 9 egg shell membrane polypeptides except 200. Mu.g/mL F1 and 200. Mu.g/mL F9 were co-cultured with HSF cells in the scratch area, obvious growth of HSF cells was seen in the scratch area, thus demonstrating that the 9 egg shell membrane polypeptides have obvious activity of promoting the migration of HSF cells.

TABLE 3 200. Mu.g/mL

As can be seen from Table 3, F2 and F11 alone had the highest proliferation rates for HSF cells, while F1 and F9 alone had the lowest proliferation rates for HSF cells. As shown in FIG. 7 and Table 3, F2, F8 and F10 had higher shrinkage and other eggshell membrane polypeptides had relatively lower shrinkage. However, the eggshell membrane polypeptide prepared by the embodiment of the application has a certain inhibition effect on the contraction function of human skeletal muscle cells, which indicates that the eggshell membrane polypeptide can reduce muscle contraction by inhibiting the contraction of human skeletal muscle cells, thereby achieving the purpose of reducing the occurrence of wrinkles.

Wrinkle-preventing and alopecia-preventing composition and animal experiment

In order to further verify the application of the eggshell membrane polypeptide prepared in the embodiment of the application in the fields of beauty and cosmetics such as wrinkle resistance, the embodiment of the application also provides a wrinkle resistance and alopecia resistance composition, wherein the composition comprises at least one of the polypeptides shown in SEQ ID NO. 1-11, cosmetic or pharmaceutically acceptable salts of the polypeptides shown in SEQ ID NO. 1-11, a conjugate molecule and at least one of a cosmetic or pharmaceutically acceptable excipient or adjuvant.

In some embodiments, the polypeptide is contained in a cosmetically or pharmaceutically acceptable sustained release system or carrier selected from the group consisting of liposomes, nanocapsules, microcapsules, nanocapsules, sponges, vesicles, micelles, lipid globules, microemulsions, nanoemulsions, nanoparticles, microparticles, and nanoparticles.

In some embodiments, the polypeptide is adsorbed onto a cosmetically or pharmaceutically acceptable organic polymer or solid mineral carrier selected from talc, soap clay, silica, starch, or maltodextrin.

To this end, embodiments of the present application also disclose a wrinkle-and hair-loss-preventing composition comprising a cosmetically or pharmaceutically effective amount of at least one polypeptide as shown in SEQ ID No.1 to 11, a cosmetically or pharmaceutically effective amount of an active agent selected from the group consisting of exfoliating agents, wetting agents, depigmenting or whitening agents, primary color agents, anti-glycating agents, nitric oxide synthase inhibitors, agents that stimulate the synthesis of skin or epidermal molecules and/or prevent their degradation, agents that stimulate proliferation of fibroblasts and/or keratinocytes and stimulate differentiation of keratinocytes, agents that are intended to improve the dermis-epidermis junction, skin soothing agents, solidifying agents, anti-atmospheric pollution and/or anti-free radical agents, agents that act on capillary circulation and/or microcirculation, tranquilizers, anti-inflammatory agents, antimicrobial agents, agents that act on cellular metabolism, agents that act on capillary circulation and/or microcirculation, vitamins, chelating agents, organic or mineral photoprotectors that are resistant to uv light a and/or B, and mixtures thereof.

The anti-wrinkling and anti-hair loss composition object disclosed by the embodiment of the application can be applied to a body area needing treatment or care, and can achieve the aim of penetrating more effective components by a smearing, vapor wrapping or coating mode or an iontophoresis mode. The application area is the forehead area with expression lines and wrinkles and fine lines between the eyebrows, around the mouth and/or around the eyes, as well as scalp areas.

The anti-wrinkling and anti-hair loss compositions disclosed in the embodiments of the present application may contain or may be administered in combination with analgesic compounds and/or anti-inflammatory compounds, with the aim of reducing swelling and irritation of sensitive skin. Steroid-type compounds such as hydrocortisone, non-steroid-type compounds such as acetaminophen or acetylsalicylic acid or natural extracts or essential oils with intrinsic analgesic and anti-inflammatory activity.

Accordingly, other aspects of the present disclosure relate to the use of at least one peptide of formula (I) for the preparation of a cosmetic or pharmaceutical composition for the treatment of conditions in those mammals, particularly humans, in which neuronal exocytosis modulation is desired. Other aspects of the application relate to the use of at least one of the polypeptides shown in SEQ ID No.1 to 11 for the preparation of an anti-wrinkle and/or anti-hair loss cosmetic composition for treating, cleansing or caring for the skin.

In order to further verify the application of the eggshell membrane polypeptide prepared in the embodiment of the application in wrinkle resistance and alopecia resistance, the embodiment of the application also discloses a wrinkle resistance and alopecia resistance composition nanoemulsion, which comprises, by weight, 2-8 parts of isopropyl myristate, 10-20 parts of polyoxyethylated castor oil, 12-18 parts of polyoxyethylene ether (EL-40), 1-10 parts of 1, 2-propanediol, 1-8 parts of isopropanol and 10-15 parts of at least one polypeptide shown in SEQ ID NO. 1-11. The term "part" is used to indicate the ratio of each component, and the absolute weight of each component is not limited, and may be any weight ranging from 0.0001 kg to 1000kg or other weight ranges.

The embodiment of the application also discloses a preparation method of the crease-resist and alopecia-preventing composition nanoemulsion, which specifically comprises the steps of respectively preparing an oil phase and a water phase, fully mixing the oil phase comprising isopropyl myristate, polyoxyethylated castor oil, polyoxyethylene ether, 1, 2-propanediol and isopropanol in the weight ratio to obtain an oil phase, weighing at least one polypeptide shown in SEQ ID NO. 1-11 according to the formula amount, dissolving the polypeptide with pure water (the concentration is 10-25 wt%) to obtain the water phase, mixing the oil phase solution into the water phase, and fully stirring to obtain the emulsion.

1. Materials and methods

1. Experimental animal

SD rats, kai student science and technology (Shanghai) Limited, about 250g, normal diet and feeding conditions.

2. Test article

In an embodiment of the specific wrinkle-preventing and hair-loss-preventing composition nanoemulsion, the wrinkle-preventing and hair-loss-preventing composition comprises, by weight, 4.5% of isopropyl myristate, 17.5% of polyoxyethylated castor oil, 15.8% of polyoxyethylene ether, 5.5% of 1, 2-propanediol, 6.5% of isopropanol, 14% of at least one polypeptide shown in SEQ ID NO. 1-11, and the balance of water. The specific composition of the more specific wrinkle-preventing and hair-loss-preventing composition nanoemulsion as a test sample for animal experiments is shown in table 4, wherein each example and comparative example only differ in the eggshell membrane polypeptide, and the other components and weight ratios are the same.

TABLE 4 Table 4

3. Skin repair experiment

After SD rats are anesthetized, mao Tichu of the back skin of the rats is cleaned, then 75% alcohol is used for wiping the skin for sterilization, finally, the spine of the rats is taken as a central line, two wound surfaces with the same diameter of 6mm and symmetrical positions are penetrated out of the skin at the left side and the right side of the back of the rats by using a puncher as a model area, and the whole skin is removed. After operation, the rat is placed in a mouse cage, and the indoor temperature is properly adjusted up until the anesthesia effect disappears, and the rat wakes up.

SD rats with a model area were divided into a model group, an experimental group and a positive control group, the experimental group was respectively smeared with the anti-wrinkle and anti-hair-loss composition nanoemulsions (100 mg per day) provided in examples 1 to 3 and comparative examples 1 to 4 according to Table 4, respectively, and the model group rats were not treated.

4. Skin tissue section and staining

Until the wound heals completely, the skin of the model area of each group of rats in the skin repair experiment is sampled, paraffin sections are manufactured, HE-stained and then placed into a photo-microscope to photograph, and the granulation tissue thickness and the epithelialization percentage in the skin tissue are measured by using an Image Pro Plus.

Immunohistochemical detection of paraffin sections: paraffin sections were incubated overnight at room temperature with anti-a SMA (Abcam) added dropwise after blocking endogenous peroxidase and non-specific binding within 3mm, followed by incubation with goat serum IgG (Abcam) for 1h at room temperature. The negative control was treated with a buffer without antibody. a-smooth actin (a-SMA) is a marker of smooth muscle cell differentiation, and is used for marking myofibroblasts, the myofibroblasts can be stained brown, and the brown area in an Image Pro Plus detection view can be used for indirectly detecting the expression quantity of a-SMA and the myofibroblast content in granulation tissues; simultaneously, the skin tissue is stained by a Masson Trichrome method, collagen bundles are blue, and the amount of collagen in the skin tissue of the model area is estimated according to the blue area.

5. Anti-hair loss experiment

And mixing, heating and melting paraffin and rosin according to a volume ratio of 1:1, cooling to a proper temperature, uniformly coating the mixture on the back of the rat, and then continuously removing the mixture after the colloid is solidified to remove the back hair of the rat, thereby forming a model area, wherein the area of the model area is 2cm multiplied by 2cm.

SD rats with model regions were divided into model and test groups. The model is not administered after modeling, and is normally eaten. The SD rats in the test group were applied with 200mg daily in the model area for 30 consecutive days. Taking each group of rats, killing the rats, taking the skin of the dehairing area with the same surface area and the same size on the backs of the rats, scraping the hair on the skin epidermis by using a scalpel, precisely weighing the skin, and analyzing the hair quality condition of the rats; the skin of the model area is taken, paraffin sections are made, rat skin tissues are observed, the number of hair follicles of three non-overlapping areas is counted under 200 times of visual field, an average value is taken to represent the number of animal hair follicles, and analysis is carried out.

6. Skin irritation/corrosiveness detection

The test article was tested for the irritation/corrosiveness criteria of SD rats according to the skin irritation/corrosiveness test method in the toxicology test method of the 2015 edition of the cosmetic safety technical Specification.

7. Statistical analysis

2. Results

TABLE 5

In the skin repair experiment, after the nanoemulsion of the skin anti-wrinkling and anti-alopecia composition with the model area is further administered, the wound healing time of each group of rats is about 10 days on average, and the epithelialization rate and the granulation tissue thickness of each group of rats after the model area is healed after 10 days are listed in table 5. As is clear from Table 5, the epithelialization rate and the granulation tissue thickness of the skin of the model area of the model group rat were significantly reduced compared to the normal group, and the healing condition of the model area of the wrinkle-and hair-loss-preventing composition nanoemulsion provided in examples 1 to 3 was superior to that of comparative examples 1 to 4.

In addition, table 5 also shows the a-SMA expression and collagen deposition of the healed rat model regions of each group after 10 days. As can be seen from Table 5, the expression of a-SMA and the collagen content of the skin in the model area of the rat model group were significantly reduced compared with the normal group, and the effects of the anti-wrinkling and anti-alopecia composition nanoemulsion provided in examples 1 to 3 on the model area were superior to those of comparative examples 1 to 4, and the effects of the composition nanoemulsion were capable of significantly inducing a-SMA after wound healing, promoting the conversion of fibroblasts into myofibroblasts, promoting collagen deposition in fibroblasts, and having an obvious skin tissue repair function.

TABLE 6

Fig. 8 shows HE staining patterns of the rat model regions of each group, in which the arrow-type regions are hair follicles. Table 6 shows the hair weight, number of hair follicles, and skin irritation scores for each group of rat model area healed in the anti-hair loss experiment. As a result, it was found that the anti-wrinkling and anti-hair loss composition nanoemulsions provided in examples 1 to 3 were able to promote hair follicle regeneration in rat model areas and protect hair follicles, relative to model groups, so that the hair weights were significantly higher than in model groups and comparative examples 1 to 4.

In summary, 11 polypeptides are prepared by enzymolysis of eggshell membrane powder, antibacterial experiments prove that the 11 polypeptides have antibacterial effects on 15 common pathogenic bacteria to different degrees, and cell experiments prove that 9 polypeptides have obvious proliferation and migration activity promotion effects on skin fibroblasts, and in addition, the eggshell membrane polypeptides have inhibition effects on the contraction function of human skeletal muscle cells, so that the eggshell membrane polypeptides can reduce muscle contraction by inhibiting the contraction of human skeletal muscle cells, and the aim of reducing the generation of wrinkles is fulfilled.

The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.

Claims

1. A eggshell membrane polypeptide is characterized in that the sequence is shown in SEQ ID NO. 1-11.

2. Use of the eggshell membrane polypeptide of claim 1 for the preparation of an anti-wrinkle and/or anti-hair loss cosmetic composition for treating, cleansing or caring for skin.

3. The method for preparing the eggshell membrane polypeptide composition of claim 1, comprising the steps of:

Obtaining eggshell membrane powder;

Purifying, namely, purifying the eggshell membrane polypeptide crude product by Sephadex G-25 and separating and purifying by RP-HPLC;

Wherein, the enzymolysis step includes: first enzymolysis and second enzymolysis, first enzymolysis includes:

The second enzymolysis comprises: preparing the freeze-dried powder obtained by the first enzymolysis into 50mg/mL solution by using 0.7m/v% physiological saline, adding a second enzyme preparation into the solution, treating the solution for 1 to 2.5 hours by adding 10000 to 15000U/mL enzyme, taking supernatant after enzyme deactivation treatment and centrifugation, concentrating and freeze-drying to obtain a crude eggshell membrane polypeptide product; wherein the second enzyme preparation used comprises 100000U/g of alpha-chymotrypsin and 150000U/g of collagenase.

4. The use of the eggshell membrane polypeptide of claim 1 for the preparation of anti-wrinkle, anti-hair loss, bacteriostatic cosmetics.