CN106977586B

CN106977586B - Periplaneta americana polypeptide for promoting tissue repair and application thereof

Info

Publication number: CN106977586B
Application number: CN201610030509.XA
Authority: CN
Inventors: 叶文才; 王磊; 刘忠; 范春林
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2016-01-15
Filing date: 2016-01-15
Publication date: 2020-06-16
Anticipated expiration: 2036-01-15
Also published as: CN106977586A

Abstract

The invention relates to the fields of traditional Chinese medicines, natural medicines and daily chemicals, in particular to a new periplaneta americana polypeptide for promoting tissue repair and application thereof. The new polypeptide for promoting tissue repair is named as Periplapetide B, and the structure of the polypeptide is shown as a formula I. The new polypeptide can promote the proliferation of mouse embryonic fibroblasts and the migration of human immortalized epidermal cells under a lower concentration, has low toxic and side effects and good application prospect, and can be used for preparing medicaments for treating wounds, scalds and ulcers or cosmetics and daily chemical products for improving the aesthetic feeling of skin. H-Ala-Ala-Pro-Pro-Ser-Asn-Leu-Lys-Glu-Val-Pro-Ile-Ile-Ala-Tyr-OH formula I.

Description

Periplaneta americana polypeptide for promoting tissue repair and application thereof

Technical Field

The invention relates to the fields of traditional Chinese medicines, natural medicines and daily chemicals, in particular to a new periplaneta americana polypeptide for promoting tissue repair and application thereof.

Background

The healing process following tissue trauma is a process of proliferation, differentiation, migration and apoptosis of various repair cells. Inflammation, ulcer, scald, trauma, surgery and congenital deformity can cause large-area skin defect, intractable ulcer and scar, and cause many local reactions with the systemic system, which is very serious in harm. The polypeptide medicine has unique advantages in promoting wound healing, preventing scars and treating chronic ulcers. The polypeptides of epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF) and the like have obvious effects of repairing wounds, protecting skin, resisting wrinkles and preventing aging, but the preparation cost is high, the stability is poor, and the application of the polypeptides is limited.

American cockroaches (Periplaneta americana L.) are insects of the genus Periplaneta (Periplaneta) of the family blattaceae (Blattidae) of the order blattaria of the phyla arthropoda, commonly known as cockroaches, and are widely distributed in China. As a traditional Chinese medicine, Periplaneta americana has a long history of entering medicine, recorded in Shen nong's herbal Jing for the earliest time, called blatto-nie, and used for treating sores, abscesses, toxic swelling, infantile malnutrition and other symptoms. Modern pharmacological studies show that the periplaneta americana has obvious effects of promoting tissue repair and regeneration, resisting tumors and hepatitis (2007, 32:2326-2331, a Chinese traditional medicine). At present, the medicines on the market which are prepared by taking the periplaneta americana as the main raw material comprise new healing liquid, Xinmailong capsules, Ganlong capsules and the like, and particularly the new healing liquid has good curative effect on the aspects of treating wounds, scalds, digestive tract ulcers and the like, but the effective components of the new healing liquid are not clear.

The periplaneta americana ethanol extract has been reported in the literature to have the effects of promoting mucosa repair (Zhejiang Chinese and western medicine combination journal, 2012,9: 689-; chinese patent ZL201210137422.4 discloses a preparation method of cockroach polypeptide substances and anti-herpes virus medical application thereof, Chinese patent ZL201210137410.1 introduces preparation of cockroach polypeptide and anti-gram-positive and anti-gram-negative bacteria medical application thereof, but the two patents do not clarify specific chemical compositions of polypeptide parts and do not relate to application of polypeptide in promoting tissue repair.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention mainly aims to provide a new periplaneta americana polypeptide for promoting tissue repair.

Another object of the present invention is to provide the use of the above novel polypeptides.

The purpose of the invention is realized by the following technical scheme:

a periplaneta americana polypeptide for promoting tissue repair is named Periplapetide B, and the structure of the polypeptide is shown as formula I:

H-Ala-Ala-Pro-Pro-Ser-Asn-Leu-Lys-Glu-Val-Pro-Ile-Ile-Ala-Tyr-OH

formula I;

the preparation method of the periplaneta americana new polypeptide for promoting tissue repair comprises the following steps:

extracting pulverized or uncrushed Periplaneta americana body with aqueous ethanol, concentrating the extract, adding 10 times volume of hot water, keeping warm, standing for layering, discarding the upper oil, concentrating the aqueous solution, performing reversed phase C-18 column chromatography, analyzing, tracking and detecting by liquid chromatography-mass spectrometry (HPLC-MS), collecting the fraction containing polypeptide compound, combining the fractions, and concentrating under reduced pressure or vacuum drying to obtain the total polypeptide extract; further separating and purifying the total polypeptide extract by reverse phase silica gel column chromatography, Sephadex LH-20 column chromatography and preparative HPLC to obtain new periplaneta americana polypeptide B for promoting tissue repair;

the temperature of the hot water is preferably 70 ℃;

the preparation of the new periplaneta americana polypeptide for promoting tissue repair can also be carried out by adopting a known method in the prior art, not only can be carried out by using an automatic polypeptide synthesizer for chemical synthesis, but also can be carried out by deducing a nucleotide sequence from a short peptide sequence and then cloning the nucleotide sequence into an expression vector for biosynthesis;

the periplaneta americana polypeptide for promoting tissue repair is applied to the preparation of products for promoting tissue repair or improving skin aesthetic feeling;

preferably, the periplaneta americana new polypeptide for promoting tissue repair is applied to preparing products for treating wounds, scalds and ulcers or improving the aesthetic feeling of skin;

the product is preferably a medicine, a cosmetic or a daily chemical product;

the medicine, the cosmetics or the daily chemical products contain Periplapeptide B with effective dose, and the balance is auxiliary materials or other compatible medicines;

the auxiliary materials refer to conventional excipients, such as solvents, disintegrants, flavoring agents, preservatives, coloring agents, adhesives and the like;

the other compatible medicines refer to other natural medicines, chemical medicines or biological medicines;

the medicine, cosmetic or daily chemical product can be in the form of tablet, capsule, injection, liposome nanoparticle, controlled release agent, gel ointment, topical liniment, patch (such as facial mask), cream (such as cream), detergent (such as shampoo, soap, foam bath or bath salt), lotion, gel, toner, etc.;

compared with the prior art, the invention has the following advantages and effects:

(1) the invention separates and identifies a new polypeptide compound Periplapetide B from periplaneta americana, and the structure of the new polypeptide compound Periplapetide B is shown as a formula I.

(2) The novel polypeptide can promote the proliferation of mouse embryonic fibroblasts and the migration of human immortalized epidermal cells under a lower concentration (0.39-0.78 mu g/mL), and has the effect of promoting tissue repair.

(3) The polypeptide Periplapeptide B has stable structure, can be used for treating peptic ulcer and oral ulcer, and has the effects of whitening, removing wrinkles, scars and color spots.

(4) The novel polypeptide Periplapeptide B has low toxicity, and no obvious toxic or side effect is observed under the condition of oral administration of 200 mg/kg.

(5) The novel polypeptide Periplapetide B is easy to synthesize and easy to prepare in large quantities.

Drawings

FIG. 1 shows Periplapetide B¹H NMR spectrum.

FIG. 2 shows Periplapetide B¹³C NMR spectrum.

FIG. 3 is a high resolution mass spectrum of Periplapetide B.

FIG. 4 is a graph showing the analysis of the results of the effect of Periplapetide B on the proliferation of Balb/c 3T3 cells.

FIG. 5 is a graph showing the analysis of the effect of Periplapetide B on the migration of Hacat cells at the cell level.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The following embodiments employ the following conditions for liquid chromatography-mass spectrometry (HPLC-MS) analysis:

the experimental instrument is an Agilent 6210LC/MSD TOF liquid chromatograph-mass spectrometer, and the mass range is as follows: 100-3000, collection mode: positive ion mode, Gas Temp: 300 ℃, drying Gas: 11L/min, Nebulizer: 35psig, Vcap: 2000V, fragment: 120V, Skimmer: and 65V. Analytical column is YMC C8 column (4.6X 250mm,5 μm), chromatographic conditions: mobile phase a (0.1% by volume formic acid), mobile phase B (acetonitrile), gradient elution from 0min (a: B ═ 95:5) to 90min (a: B ═ 10: 90).

Example 1 isolation and structural identification of the polypeptide Periplapetide B

(1) 5kg of periplaneta americana medicinal material is crushed into coarse powder, and the coarse powder is percolated and extracted for 3 times by using 80 percent (v/v) ethanol solution with the mass being 3 times that of the coarse powder, wherein each time lasts for 24 hours; mixing extractive solutions, concentrating under reduced pressure to obtain soft extract 1.5L, adding 10 times of hot water (70 deg.C), standing for 12 hr, layering, and removing upper oil; concentrating the lower aqueous solution, loading the concentrated lower aqueous solution onto a reversed phase C-18 chromatographic column, eluting 6 column volumes by using 30% methanol by volume, eluting 6 column volumes by using 50% and 75% methanol aqueous solutions by volume respectively, analyzing, tracking and detecting by using HPLC-MS (high performance liquid chromatography-mass spectrometry), collecting fractions mainly containing polypeptide compounds, merging the fractions, and concentrating under reduced pressure or drying in vacuum to obtain a total polypeptide extract (56g) of the periplaneta americana;

(2) performing reverse phase silica gel (ODS) column chromatography on 20g of the total polypeptide extract obtained in the step (1), and eluting by using methanol-water as an eluent according to an elution gradient of methanol to water volume ratio of 10:90, 20:80, 30:70, 50:50, 70:30 and 90:10 to obtain 6 main flow parts Fr.A-Fr.F;

(3) loading the fraction Fr.D (3.2g) obtained in the step (2) onto a Sephadex LH-20 chromatographic column, eluting with water as a mobile phase at the flow rate of 0.5mL/min, detecting by HPLC-MS analysis, and combining and collecting similar fractions to obtain 6 sub-fractions Fr.D 1-Fr.D 6;

(4) concentrating the sub-fraction Fr.D3 obtained in the step (3), dissolving the concentrated sub-fraction with 5% methanol by volume fraction, separating and purifying by reverse phase preparative HPLC, eluting with acetonitrile-water as an eluent at a volume ratio of 50:50 at a flow rate of 3mL/min, and collecting a chromatographic peak with a retention time of 20.3min to obtain Periplapetide B (11.6 mg).

(5) Structural characterization of Periplapetide B

A white powder; UV (MeOH) lambda_max(logε)205(3.98),276(2.81)nm；IR(KBr)ν_max3302,2966,2885,1661,1525,1449,1205,1135,835,723cm^-1；HR-ESI-MS m/z1582.8843[M+H]⁺(C₇₄H₁₂₀N₁₇O₂₁Calculated 1582.8839). Hydrogen spectrum (¹H NMR) and carbon Spectroscopy (¹³C NMR) data are shown in Table 1 (assigned by 2D-NMR). The amino acid sequencing results are: H-Ala-Ala-Pro-Pro-Ser-Asn-Leu-Lys-Glu-Val-Pro-Ile-Ile-Ala-Tyr-OH.¹H,¹³The C NMR spectrum is shown in the figure 1-2; the high resolution mass spectrum is shown in FIG. 3. According to the physicochemical and spectral data, the chemical structure of Periplapetide B is identified, and is shown as a formula I.

TABLE 1 preparation of Periplapetide B¹H (500MHz) and¹³c (125MHz) NMR data

^a)Measured at 500MHz.^b)Measured at 125MHz.^c)Overlapped signals werereported without designating

Example 2 Synthesis of a novel polypeptide Periplapetide B

The synthesis of the novel polypeptide Periplapepeptide B is completed by adopting a conventional solid phase synthesis method of a full-automatic polypeptide synthesizer and through the processes of resin swelling, deprotection, washing, amino acid dissolution, amino acid activation, condensation and the like.

EXAMPLE 3 promotion of Balb/c 3T3 cell proliferation by the novel polypeptide Periplapetide B

Cells in logarithmic growth phase (Balb/c 3T3 cell line, purchased from American Type Culture Collection, ATCC) were trypsinized, centrifuged, resuspended, counted, plated at 3000 cells/well in 96-well plates, and lightly platedBeating until the cells are evenly dispersed, and sealing with PBS. 37 ℃ and 5% CO₂The culture was carried out overnight. Removing the culture medium, and adding a starvation culture medium containing 0.5% FBS by volume fraction for treatment for 24h to synchronize cells; abandoning the starvation culture medium, adding complete culture medium to prepare polypeptide Periplapetide B with different concentrations, 37 ℃ and 5% CO₂Incubate for 48 h. Adding a CCK8 reagent according to the volume of 10 mu L/hole, incubating for 1-4 h at 37 ℃, and detecting by a microplate reader at 450nm and 630nm dual-wavelength. Calculating the cell survival rate, and repeating the experiment for at least 3 times;

the test result is shown in figure 4, the Periplapeptide B has obvious proliferation promoting effect on Balb/c 3T3 cells under lower concentration (0.39-0.78 mu g/mL), and the activity of the Periplapeptide B is obviously stronger than that of a new drug rehabilitation solution sold in the market.

Example 4 promoting Effect of the novel polypeptide Periplapetide B on Hacat cell migration

HaCAT cells (purchased from American type culture Collection, ATCC) in logarithmic growth phase were trypsinized, resuspended, and expressed at 1X 10⁶Inoculating into 12-well plate at 37 deg.C with 5% CO₂Culturing in an incubator until the culture hole is full of cells. The culture was aspirated, washed with PBS, and streaked with a sterile pipette tip from top to bottom perpendicular to the culture wells at the center of each test well. The cell clusters generated by the scratch are washed by PBS, so that the edge of the scratch is neat. Fresh medium and Periplapetide B (0.78. mu.g/mL) were added, and the photographs were taken under a microscope and placed in a cell culture incubator for further culture. The record was observed every 12 hours, and the obtained Image data was processed with Image Pro Plus 6.0. Uniformly selecting 30 points on each side edge of the scratch, taking the central line of the points to represent the edge of the scratch, measuring the scratch distance, and calculating the scratch repair rate by using the following formula: the scratch repair rate is (0h scratch width-scratch width at different time points)/0 h scratch width.

The test result shows that the migration rate of the HaCAT cells in the Periplapetide B group is as high as 91 percent (72 hours), and is obviously higher than that of the control group by 48 percent (72 hours) (figure 5), which indicates that the novel polypeptide Periplapetide B can obviously promote the horizontal migration of the HaCAT cells.

Example 5 repair of skin wound in mice with novel polypeptide Periplapetide B

40 male Kunming mice (purchased from the center of animals in Guangdong province) with the age of 8-10 weeks are selected and raised in cages, food is freely taken, and food intake is stopped one day before the experiment. An intraperitoneal injection of 0.2mL of pentobarbital sodium with the mass fraction of 1 percent is used for anesthetizing a mouse, the back is sheared, square wounds of 0.5cm multiplied by 0.5cm are symmetrically formed in the upper part and the lower part of the vertebral column epidermis skin, the whole layer of skin is sheared, the muscle layer is not injured, and the wounds stop bleeding for later use. The next day, mice were anesthetized with ether, and perilapeptide B (0.1mg/mL or 0.2mg/mL), saline and convalescent liquid, each 0.1mL, were added dropwise to the wound, and the dressing was changed once a day. Wound healing was observed at 3, 5, 10 days. The area of the wound surface is firstly drawn on a transparent film, then the transparent film is used as a template, a piece of hard paper with uniform texture is cut into the same size, and then the mass is weighed by an analytical balance. The size of the wound area is indirectly expressed by the mass of the hard paper sheet. Calculating the wound healing rate according to the formula: wound healing rate (%) (original wound area-non-healed wound area)/original wound area. The results are shown in Table 2.

TABLE 2 statistical results of wound healing rates in mice

Note: compared with the model group, P < 0.05 has significant difference

The test result shows that the novel polypeptide perilapeptide B can obviously promote the healing of the skin wound of a mouse, has significant difference compared with a control group, and is superior to a new rehabilitation liquid.

Example 6 repair of skin Scald in mice with novel polypeptide Periplapetide B

40 male Kunming mice (purchased from the center of animals in Guangdong province) with the age of 8-10 weeks are selected and raised in cages, food is freely taken, and food intake is stopped one day before the experiment. Using 13% of Na by mass fraction₂And (3) applying the solution S to remove hair, putting a 20g weight into a water bath, heating to boil, maintaining for 10min, clamping the upper end of the weight by using a forceps, quickly placing the weight on the skin of the mouse for 5S, and slightly pressurizing to form a shallow II-degree scald model with a scald area of 2cm multiplied by 2 cm. The next day, Periplapeptide B (0.1mg/mL or 0.2mg/mL), physiological saline and rehabilitation new liquid with different solubilities are added into the wound, and each amount is 0.1mLThe medicine is changed once a day. Wound healing was observed at 3, 5, 10 days. Wound healing rates were calculated as in example 5. The results are shown in Table 3.

TABLE 3 statistical results of the rate of healing of scalding in mice

Note: compared with the model group, P < 0.05 has significant difference

Test results show that the novel polypeptide perilapeptide B can obviously promote the healing of skin scalds of mice, has significant difference compared with a control group, and is superior to a new rehabilitation liquid.

Example 7 protective Effect of the novel polypeptide Periplapetide B on stress gastric ulcer in rats

The test method comprises the following steps: 50 male SD rats (purchased from Guangdong province animal center) with the weight of 180-210 g are taken and randomly divided into a blank group, a model group, a sucralfate group (1g/kg, positive drug group), a low dose group (Periplapetide B1 mg/kg) and a high dose group (Periplapetide B2 mg/kg). The administration group was administered by gavage for 7 days. The rats in each group are fixed on a rat plate, the rat heads of the groups are vertically soaked in a constant-temperature (20 ℃) water tank for 8 hours, and the water surface is flush with the xiphoid process of the rats. After the stress modeling is finished, the cervical vertebra of the rat is dislocated and killed, the abdomen is cut open, and the pylorus is ligated. 2mL of formaldehyde solution with the volume fraction of 10 percent is perfused into the stomach, the cardia is ligated, and the stomach body is taken out and placed in the formaldehyde solution for fixation for 15 min. Cut along the greater curvature of the stomach, expand after being washed by physiological saline, observe the damage of the gastric mucosa and calculate the index of gastric ulcer. Ulcer Index (UI) was calculated according to Guth criteria: the dot bleeding is counted as 1 point, the linear bleeding length is 2 points when the length is less than 1mm, 3 points when the length is 1-2 mm, 4 points when the length is 2-4 mm,5 points when the length is more than 4mm, and the width is more than 1mm, and the value is multiplied by 2. The results are shown in Table 4.

TABLE 4 statistical results of ulcer index in rats

Note: compared with the model group, P < 0.01 has significant difference

Test results show that the polypeptide Periplapetide B has obvious protection effect on stress gastric ulcer of rats, has obvious difference compared with model groups, and has better protection effect than the positive medicine sucralfate.

Example 8 protective Effect of the novel polypeptide Periplapetide B on canker sores in rats

The test method comprises the following steps: 40 healthy SD rats (purchased from the centers of animals in Guangdong province) with the weight of 180-210 g are randomly divided into a model group, a positive medicine group (new rehabilitation liquid), a low-dose group and a high-dose group, and the model is manufactured by a chemical burning method. Injecting glacial acetic acid with volume fraction of 80% into a glass tube with the inner diameter of 0.4cm and the length of 3cm, inserting cotton into one end of the glass tube, vertically fixing the cotton end on the mucous membrane surface of the lower lip of a rat for 5 seconds to ensure that the part has areola and edema, observing after 24 hours that ulcer and hyperemia and edema appear, and successfully molding when the diameter of the ulcer surface is more than 3 mm. Periplapetide B with different solubilities, physiological saline and rehabilitation new liquid are respectively dripped into ulcer surfaces of an experimental group, a model group and a positive medicine group, and the dosage is once a day. The healing of the ulcer surface was observed after 1,2 and 3 days. Ulcer integration criteria are as follows: 1 point is recorded when the diameter of the ulcer surface without congestion and edema is less than 1 mm; mild hyperemia and edema, and the diameter of the ulcer surface is 1-2 mm and is recorded as 2 points; moderate hyperemia and edema, and 3 points are recorded when the diameter of the ulcer surface is 2-3 mm; the diameter of the ulcer surface with severe hyperemia and edema is more than 3mm and 4 points are recorded. The results are shown in Table 5.

TABLE 5 rat oral ulcer healing score

Note: p < 0.05, P < 0.01, compared to model group

The test result shows that the polypeptide Periplapeptide B can obviously promote the healing of the oral ulcer of a rat, has significant difference compared with a model group, and is superior to a new rehabilitation liquid.

EXAMPLE 9 acute toxicity test of novel polypeptide Periplapetide B

The test method comprises the following steps: taking Kunming mice (purchased from Guangdong province animal center) with the weight of 18-22 gRandomly grouping 10 polypeptides per group, intragastrically administering polypeptide Periplapetide B with different doses, and calculating half lethal dose LD according to survival conditions of mice₅₀. The median lethal dose is calculated according to the following formula: half lethal dose (mg/kg) ═ dose at half death of the mice/body weight of the corresponding mice.

And (3) test results: no death of mice was observed at the administration dose of 200mg/kg for polypeptide Periplapetide B, indicating that it has low toxicity and LD₅₀Greater than 200mg/kg (Table 6).

TABLE 6 acute toxic Effect of Periplapetide B on mice

Example 10 evaluation of skin safety of novel polypeptide Periplapetide B

The test population: between 18 and 50 years of age, 18 females, 12 males, and 30 total. The skin of the subject is healthy, has no allergic history of the skin disease, and meets the voluntary selection standard of the subject.

The test method comprises the following steps: selecting a qualified patch device, dripping about 0.020-0.025 mL (2mg/mL) of polypeptide Periplapetide B into the patch device by a closed patch test method, externally applying a special adhesive tape to the back of a subject, removing the test product after 24 hours, observing skin reactions after 0.5, 6, 12, 24 and 48 hours after removal respectively, and recording the result according to the skin reaction grading standard in the cosmetic hygiene Specification.

And (3) test results: in the test, 30 subjects pass the patch test, and no skin adverse reaction is observed when skin reactions are observed for 0.5, 6, 12, 24 and 48 hours, which indicates that the novel polypeptide periplpeptide B is safe to use on the skin.

EXAMPLE 11 preparation of tablets

0.1g of polypeptide Periplapetide B, 40g of lactose, 60g of starch slurry and 0.2g of magnesium stearate are mixed, sieved, dried and tabletted. Each tablet contains 0.001g of Periplapetide B.

EXAMPLE 12 preparation of injection

0.1g of polypeptide Periplapeptide B and 50g of propylene glycol are ground, 100mL of water for injection is added for dilution, the mixture is uniformly mixed, 9g of sodium chloride is added, the water for injection is added to 1000mL after the solution is dissolved, the pH value is adjusted to 5.5-6.5, and the 1000 injection needles are obtained after filtration, encapsulation and sterilization.

Example 13 preparation of solid lipid nanoparticles

0.1g of polypeptide Periplapetide B and 500mg of soybean lecithin are dissolved in 25mL of ethanol, 200mg of stearic acid and 500mg of soybean lecithin are dissolved in 25mL of cyclohexane and are mixed and stirred uniformly; performing rotary evaporation under reduced pressure in 37 deg.C constant temperature water bath to remove organic solvent, allowing the medicine and adjuvants to form uniform lipid film on the wall of flask, standing overnight in vacuum drier, and removing organic solvent; dissolving 3750mg of polyethylene glycol monostearate in 175mL of water under stirring, adding into the film, performing ultrasonic treatment for 10min, and diluting to 250mL to obtain a light yellow transparent solution; freeze drying the solution to obtain lyophilized powder. Grinding for 24 hr with ball mill to obtain nanometer particle with homogeneous size, mixing and packing. Each bag contained Periplapetide B0.001 g.

EXAMPLE 14 preparation of controlled Release capsules

Polypeptide perilapeptide B0.1 g, lactose 40g and starch slurry 10g were loaded directly into a rotary granulator/coater to prepare granules, and plasticized ethylcellulose coating suspension diluted to 15% solids by mass was sprayed onto a rotating bed of polypeptide granules. During spraying, the particles were coated with a dispersion carrier film made of poloxamer 188 to form sustained release particles with an average particle size of about 450 μm. Mixing, and encapsulating to obtain capsule containing Periplapetide B0.001 g.

EXAMPLE 15 preparation of external gel ointment

Weighing 2g of glycerol, sequentially adding 0.75g of polyacrylic acid and 0.1g of aluminum hydroxide, fully mixing, adding 0.2g of periplapetide B, fully stirring and mixing under a vacuum condition to obtain ①, weighing 5g of purified water, dissolving 0.06g of lactic acid and 0.1g of sodium carboxymethylcellulose in water to obtain ②, adding ② into ①, fully stirring under a vacuum condition, carrying out a crosslinking reaction to obtain a medicine-containing paste body, coating the medicine-containing paste body (controlling the thickness of a medicine-containing paste body layer to be about 1.0 mm), cutting into the specification of a conventional emplastrum, wherein the medicine-containing amount of each emplastrum is 0.02g, airing and packaging.

EXAMPLE 16 preparation of the ointment

Melting stearyl alcohol 20g and white vaseline 21g in water bath, and heating to 75 deg.C. 1.46g of lauryl sodium sulfate, 0.025g of methyl hydroxybenzoate, 0.015g of propyl hydroxybenzoate, 10g of propylene glycol and 0.2g of perilapide B are sequentially dissolved in water, heated to 75 ℃, stearyl alcohol and white vaseline at 75 ℃ are added, and the mixture is stirred to be cooled to prepare an ointment containing 0.2% of perilapide B.

EXAMPLE 17 preparation of external Liniment

Dissolving 0.1g of Periplapelide B in 45mL of distilled water, filtering, adding 5mL of glycerol, 1mL of azone and distilled water into the filtrate to make up the volume to 50mL, and preparing the external liniment containing 0.2% of Periplapelide B.

EXAMPLE 18 preparation of cream

Mixing No. 26 white oil 1g, cetostearyl alcohol 1g, stearic acid 1g, monoglyceride 0.5g, 350 silicone oil 0.05g, GTCC 0.5g, and methyl paraben 0.03g, heating to 90 deg.C to obtain first semi-finished product; adding 4g of deionized water, 0.1g of Periplapetide B, 200.3g of peregal and 0.5g of glycerol into the first semi-finished product, and heating to 80 ℃ to form a second semi-finished product; and homogenizing the second semi-finished product at 80 ℃ for 2 times (rotating speed of 3000 r/min, time of 10 min), continuing stirring for 30 min, cooling to 45 ℃ to form cream, adding 0.005g of Kathon, and mixing uniformly to obtain the face cream containing 1% of Periplapetide B.

EXAMPLE 19 preparation of facial mask

Dissolving 0.1g of Periplapelide B in 10mL of deionized water, filtering, mixing the filtrate with 0.3g of glycerol-wetted carbomer, adding triethanolamine, adjusting the pH value to 6-7, and uniformly coating the mixture on facial mask paper to prepare each facial mask containing 0.01g of Periplapelide B.

EXAMPLE 20 preparation of toner

Preparing 0.1g of Periplapetide B into 0.2mg/mL aqueous solution, adding 5g of glycerol into 1g of hyaluronic acid for dispersion, adding water for dissolution, and stirring uniformly to obtain a hyaluronic acid solution, dissolving 1g of trehalose and 1g of allantoin in water, then mixing with the hyaluronic acid solution, stirring uniformly to obtain a mixed solution, sequentially adding 1g of polypeptide solution, 1g of D-panthenol, 10g of oat β -glucan, 10g of 1, 2-pentanediol and 0.05g of preservative into the mixed solution, adding water, and stirring uniformly to obtain the toner.

EXAMPLE 21 preparation of the emulsion

(1) 0.1g of Periplapetide B was prepared as a 0.2mg/mL aqueous solution;

(2) adding water into 0.05g of sodium hyaluronate for dissolving and dispersing, and uniformly stirring to obtain a hyaluronic acid solution;

(3) dissolving 5g of glycerol, 3g of butanediol, 2g of trehalose, 0.2g of allantoin and 0.1g of thickening agent in water, and heating to 75 ℃;

(4) heating 2g of jojoba seed oil, 3g of hydrogenated polydecene, 2g of polydimethylsiloxane, 3g of caprylic/capric triglyceride and 3g of emulsifier to 75 ℃, and uniformly stirring;

(5) quickly pouring the product prepared in the step (3) into the product prepared in the step (4), homogenizing at constant temperature for 3-5 min, and cooling;

(6) cooling to below 60 deg.C, adding (1) and (2), and homogenizing; cooling to below 40 deg.C, and adding antiseptic and essence to obtain emulsion.

EXAMPLE 22 preparation of cleansing gel

0.1g of Periplapetide B was prepared as a 0.2mg/mL aqueous solution; sequentially adding the polypeptide solution, 2.5g of glycerol, 3g of decyl glucoside, 3g of sodium cocoyl malate, 1.5g of sodium laureth sulfate, 0.1g of solubilizer and 0.05g of essence into water, stirring, adding 0.5g of thickening agent, and stirring until the mixture is completely swelled to obtain the cleansing gel.

Example 23 evaluation of the cosmetic Effect of polypeptide Periplapetide B emulsion

1.1 test article: polypeptide emulsion prepared in example 21 of the present invention

1.2 test population: between 18 and 55 years of age, 20 women, 10 men, and 30 people in total. The skin of the subject has wrinkles, color spots and dull color, and the skin has the unattractive factors of minimal invasion, scars after the operation and the like.

1.3 test methods: after the skin of the subject was cleaned, the emulsion prepared in example 21 was applied to the skin, and the effect of use was observed and felt.

1.4 test evaluation results are as follows:

table 7 summary of trial (8 week) feedback of polypeptide perilapeptide B emulsion

The test subjects show that the polypeptide Periplapeptide B emulsion product can improve the moisture content of the skin, enhance the moisture retention capacity and the elasticity of the skin and improve the moisture, tenderness and moist feeling of the skin. The product has effects of whitening skin, removing wrinkle, scar and mottle. The subjects did not develop allergic phenomena.

Example 24 evaluation of the cosmetic Effect of polypeptide Periplapetide B mask

1.1 test article: periplapetide B polypeptide mask prepared in embodiment 19 of the invention

1.2 test population: between 18 and 55 years of age, 22 women, 8 men, and 30 people in total. The face skin of the subject has wrinkles, color spots and dull color, and the skin has the unattractive factors of micro-wound, scars after the operation and the like.

1.3 test methods: after the face of the subject was cleaned, the perilapeptide B mask prepared in example 19 was applied to the face once a day, and the effect of use was observed and felt.

1.4 test evaluation results are as follows:

table 8 trial (8 week) feedback summary of periplaneta americana Periplapeptide B polypeptide mask

The test subjects show that the Periplapetide B polypeptide facial mask product can improve the moisture content of the skin, enhance the moisture retention capacity and the elasticity of the skin and improve the moisture, tenderness and moist feeling of the skin. The product has effects of whitening skin, removing wrinkle, scar and mottle. The subjects did not develop allergic phenomena.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A periplaneta americana polypeptide for promoting tissue repair is characterized in that: named as Periplapetide B, the structure is shown as formula I:

H-Ala-Ala-Pro-Pro-Ser-Asn-Leu-Lys-Glu-Val-Pro-Ile-Ile-Ala-Tyr-OH

formula I.

2. Use of the novel periplaneta americana polypeptide for promoting tissue repair according to claim 1 in the preparation of a product for promoting tissue repair or improving skin aesthetics.

3. The use of the novel periplaneta americana polypeptide for promoting tissue repair according to claim 2 in the preparation of products for promoting tissue repair or improving skin aesthetics, wherein:

the product is a medicine or a daily chemical product.

4. The use of the novel periplaneta americana polypeptide for promoting tissue repair according to claim 3 in the preparation of products for promoting tissue repair or improving skin aesthetics, wherein the product comprises:

the medicine or daily chemical product contains Periplapeptide B with effective dose, and the balance is auxiliary materials or other compatible medicines.

5. The use of the novel periplaneta americana polypeptide for promoting tissue repair according to claim 4 in the preparation of products for promoting tissue repair or improving skin aesthetics, wherein the product comprises:

the auxiliary materials are solvent, disintegrant, flavoring agent, preservative, colorant or adhesive.

6. The use of the novel periplaneta americana polypeptide for promoting tissue repair according to claim 3 in the preparation of products for promoting tissue repair or improving skin aesthetics, wherein the product comprises:

the medicine or daily chemical product is tablet, capsule, injection, external liniment, patch or detergent.

7. The use of the novel periplaneta americana polypeptide for promoting tissue repair according to claim 6 in the preparation of products for promoting tissue repair or improving skin aesthetics, wherein the product comprises:

the external liniment is gel ointment, cream, lotion or toner.

8. The use of the novel periplaneta americana polypeptide for promoting tissue repair according to claim 3 in the preparation of products for promoting tissue repair or improving skin aesthetics, wherein the product comprises:

the medicine or daily chemical product is a controlled release agent.

9. The use of the novel periplaneta americana polypeptide for promoting tissue repair according to claim 3 in the preparation of products for promoting tissue repair or improving skin aesthetics, wherein the product comprises:

the medicine or daily chemical product is gel.