CN118834268A - Lumbricus polypeptide group and preparation method and application thereof - Google Patents

Abstract

The present invention belongs to the field of polypeptide preparation, and particularly relates to a group of earthworm polypeptides, and a preparation method and application thereof. The amino acid sequences of the earthworm polypeptide group are VALSVIQR (SEQ ID NO.1) and VIPSLNGK (SEQ ID NO.2). The present invention performs secondary enzymolysis on earthworms after crushing, screening, heating and boiling, ultrafiltration and desalting of the supernatant, separation and purification, and analyzes the earthworm peptides by LC-TOF-MS to obtain the active polypeptide structure; the two small molecule biologically active polypeptides described in the present invention are discovered for the first time and no relevant reports have been found. The research on the earthworm polypeptides in the present invention confirms their medicinal value for treating skin wounds and scars, provides a solid foundation for the clinical application of earthworm polypeptides to treat skin wounds and scars, and provides data support for the development of active peptide substances for animal medicines.

Description

A kind of earthworm polypeptide group and its preparation method and application

Technical Field

The invention belongs to the field of polypeptide preparation, and specifically relates to an earthworm polypeptide group and a preparation method and application thereof.

Background Art

The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

Wound failure is a difficult problem in surgical treatment. According to statistics, it accounts for 1.5% to 3% of the total number of surgical hospitalizations. Scar formation is an inevitable product of the process of human tissue wound repair. Its histological characteristics are excessive proliferation of fibroblasts and excessive precipitation of extracellular matrix. Excessive healing of scar tissue in the early stage or excessive atrophy in the later stage will have adverse effects on the body, such as changes in skin appearance and functional disorders. Refractory wounds on the lower limbs have the characteristics of easy ulceration, difficult to heal, easy to recur, long course of disease, osteomyelitis in severe cases, and the possibility of cancer in a few cases. They seriously affect the quality of life and physical and mental health of patients and have become a clinical problem that needs to be solved urgently.

Western medicine focuses on anti-infection, biological dressings, negative pressure suction therapy, antibiotic bone cement, hyperbaric oxygen, skin grafting and flap repair, surgery and other treatment measures for wounds. These measures have certain effects, but lack safe and effective measures to accelerate wound healing. Therefore, it is crucial to find a mild alternative medicine for wound treatment.

Summary of the invention

In order to overcome the defects of the prior art, the present invention provides a lumbar polypeptide group and a preparation method and application thereof. The lumbar polypeptide group can be used to prepare a skin wound healing or scar elimination drug. The application eliminates the immunogenic reaction caused by taking foreign proteins and reduces the incidence of adverse reactions.

To achieve the above object, the present invention adopts the following technical solutions:

A pluvialis polypeptide group, the polypeptide group consists of a characteristic polypeptide 1 and a characteristic polypeptide 2; wherein the amino acid sequence of the characteristic polypeptide 1 is VALSVIQR (SEQ ID NO.1), and the amino acid sequence of the characteristic polypeptide 2 is VIPSLNGK (SEQ ID NO.2).

Furthermore, the preparation method of the above-mentioned earthworm polypeptide group adopts the following steps:

(1) Crush and sieve the earthworms, add water, and heat to boil;

(2) cooling the mixed solution obtained in step (1), adjusting the pH value, performing enzymatic hydrolysis, boiling, and then separating and purifying;

(3) The purified sample obtained in step (2) was processed and then analyzed by LC-TOF-MS to obtain the active polypeptide VALSVIQR (SEQ ID NO. 1) and the active polypeptide VIPSLNGK (SEQ ID NO. 2).

Furthermore, the VALSVIQR (SEQ ID NO. 1), named VR-8, namely isovaline-alanine-leucine-serine-valine-isoleucine-glutamine-arginine, has a molecular weight of 884.54 Da, an isoelectric point of 11.11, a hydrophobic index of 8.15 Kcal·moL ^-1 , and good water solubility.

Furthermore, the VIPSLNGK (SEQ ID NO. 2), named VK-8, namely valine-isoleucine-proline-serine-leucine-asparagyl-glycine-lysine, has a molecular weight of 826.49 Da, an isoelectric point of 10.14, a hydrophobic index of 10.47 Kcal·moL ^-1 , and good water solubility.

Furthermore, in step (2), the enzymatic hydrolysis method is as follows: after the mixed solution is heated and boiled, it is cooled to about 35-45°C, the pH value is adjusted to 1.0-3.0, 0.5-2% pepsin is added, and enzymatic hydrolysis is carried out at 40°C for 1 hour, the pH value is adjusted to 7.0-9.0, 0.5-2% trypsin is added, and the mixture is kept warm for 3-5 hours and boiled for 10-15 minutes; the separation and purification is centrifuged at 3500-4500 rpm for 15-20 minutes, and after desalting by electrodialysis or dialysis bag, the supernatant is ultrafiltered through a 3 KD ultrafiltration membrane, and semi-preparative separation and purification is carried out by cationic resin and RP-HPLC.

Furthermore, the nano-liquid chromatography column conditions for the LC-TOF-MS analysis in step (3) are as follows: C18 reverse phase analytical column 75 mm×20 cm×3 µm; mobile phase A: a mixture of 99.9% water and 0.1% formic acid, mobile phase B: a mixture of 80% acetonitrile and 0.1% formic acid; liquid phase gradient elution: 0-2 min, 2-6% B; 2-95 min, 6-20% B; 95-107 min, 20%-32% B; 107-108 min, 32-100% B; 108-120 min, 100% B; mobile phase flow rate of 300 nL/min; ESI+ mode, using data dependent scanning mode, at a resolution of 70000 (AGC Full scan acquisition (m/z 350-1800) was performed in an orbitrap with a 3e6) flow cytometer, the normalized collision energy (NCE) was 28.0, the capillary temperature was 275 °C, the spray voltage was 1800 V, the MS ² resolution was 17500 (AGC 1e5), the maximum fill time for full scan and MS ² scan was set to 50 ms and 45 ms, respectively, and the dynamic exclusion time was set to 30 s.

Furthermore, in step (1), the standard mesh number of the earthworm crushed and sieved is 80 mesh; the mass of the added water is 8 to 12 times the mass of the earthworm; and the heating and boiling time is 15 to 30 minutes.

An application of the above-mentioned earthworm characteristic polypeptide group is characterized in that the earthworm peptide polypeptide group can be used to prepare a drug for healing skin wounds or eliminating scars.

Furthermore, the medicine comprises the characteristic peptide group of earthworms and pharmaceutically acceptable excipients.

Furthermore, the auxiliary material is an auxiliary material required for preparing an aqueous extract, a powder, a lotion, a tincture, an oil, an emulsion, an ointment, a plaster or an aerosol.

An application of the above-mentioned earthworm characteristic polypeptide group, wherein the earthworm peptide polypeptide group is used to prepare a medicine for healing skin wounds or eliminating scars.

Furthermore, the medicine comprises the characteristic peptide group of earthworms and pharmaceutically acceptable excipients.

Preferably, the auxiliary material is an auxiliary material required for preparing an aqueous extract, a powder, a lotion, a tincture, an oil, an emulsion, an ointment, a plaster or an aerosol.

Beneficial effects: (1) The polypeptides VR-8 and VK-8 described in the present invention are of high purity, safe and effective, eliminate the immunogenic reaction caused by taking foreign proteins, and reduce the incidence of adverse reactions.

(2) There is no public report in the prior art on the combined use of VR-8 peptide and VK-8 peptide for the treatment of skin wounds and scars. The present invention provides a reference for the research and development of a new type of therapeutic drug.

(3) The two polypeptide structures of the present invention are high-content, high-activity small molecule bioactive peptide components obtained by refining and purifying small molecule peptides of earthworm biomimetic enzymatic hydrolysis and analyzing. These two small molecule bioactive peptide components are discovered for the first time and no relevant reports have been found.

(4) The research on earthworm peptides in the present invention confirms its medicinal value in treating skin wounds and scars, which provides a solid foundation for the clinical application of earthworm peptides in treating skin wounds and scars, and provides data support for the development of active peptide substances for animal medicines.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and their description are used to explain the present invention, and do not constitute an improper limitation of the present invention.

Figure 1 is a diagram of discontinuous intermittent constant volume operation;

FIG2 is a diagram showing the separation effect of Sephadex G15 at 75% ethanol elution;

Figure 3 shows the effects of the earthworm polypeptide group on the serum TGF-β1 and IL-10 levels of mice with deep second-degree scalds after modeling; A shows the effects of the earthworm polypeptide group on the serum TGF-β1 level of mice with deep second-degree scalds 8 days after modeling; B shows the effects of the earthworm polypeptide group on the serum IL-10 level of mice with deep second-degree scalds 8 days after modeling; C shows the effects of the earthworm polypeptide group on the serum TGF-β1 level of mice with deep second-degree scalds 30 days after modeling; D shows the effects of the earthworm polypeptide group on the serum IL-10 level of mice with deep second-degree scalds 30 days after modeling; GP: blank group; MP: model group; DP: polysulfonated mucopolysaccharide group; VR: VR-8 polypeptide group; VK: VK-8 polypeptide group; SP: earthworm polypeptide combination group;

Figure 4 shows the effect of the earthworm polypeptide group on the ratio of type I and type III procollagen in the tissue fluid of mice with deep second-degree scalds after modeling; A is the effect of the earthworm polypeptide group on the ratio of type I and type III procollagen in the tissue fluid of mice with deep second-degree scalds 8 days after modeling; B is the effect of the earthworm polypeptide group on the ratio of type I and type III procollagen in the tissue fluid of mice with deep second-degree scalds 30 days after modeling; GP: blank group; MP: model group; DP: polysulfate mucopolysaccharide group; VR: VR-8 polypeptide group; VK: VK-8 polypeptide group; SP: earthworm polypeptide combination group.

DETAILED DESCRIPTION

The present invention is further illustrated by specific examples below. It should be understood that the preparation methods in the examples of the present invention are only used to illustrate the present invention, rather than to limit the present invention. Under the premise of the concept of the present invention, simple improvements to the preparation methods of the present invention fall within the scope of protection required by the present invention.

It should also be noted that the various preferred technical features of the method of the present invention mentioned above and the various specific technical features in the embodiments described in detail below can be combined together, and all numerical ranges with the numerical values specifically disclosed in the present invention as upper and lower limits and the like of various combinations of all these technical features fall within the scope of the present invention.

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources or synthesized from raw materials obtained from commercial sources.

The specific embodiments of the present invention are described in detail below in conjunction with the technical solutions, but the process conditions are not limited to these embodiments.

The present invention will be further described below in conjunction with specific embodiments:

Example 1 Preparation of Pheretima polypeptide group

(1) Preparation of earthworm biomimetic enzymatic hydrolysate

① Crush 200 g of earthworm and pass it through a 80-mesh sieve, add 10 times its weight of water, heat and boil for 25 min, cool to about 40°C, adjust the pH to 2.0, add 1.5% pepsin, and hydrolyze at 40°C for 1 h, adjust the pH to 8.0, add 1.5% trypsin, keep warm for 4 h, boil for 12 min, centrifuge at 4000 rpm for 18 min, and take the supernatant to obtain the earthworm bionic enzymatic extract.

② Take the earthworm bionic enzymatic hydrolysate, dilute it 1-fold and adjust the pH to 7.0, pour it into the sample tank of the electrodialysis instrument, add tap water and deionized water to the electrode tank and desalination tank respectively. Adjust the voltage to 20 V, control the flow rate to 10 L·h ^-1 , and use the change in the conductivity value of the enzymatic hydrolysate in the sample tank as the detection index. When the conductivity value approaches stability, stop the electrodialysis desalination operation and collect the desalted enzymatic hydrolysate;

③ Take the desalted enzymatic hydrolysate prepared in step ②, and use a 3000 Da ultrafiltration membrane to separate it by discontinuous intermittent constant volume operation at 0.8 MPa (see Figure 1). The enzymatic hydrolysate that has not passed through the membrane is a polypeptide solution of >3000 Da, and the enzymatic hydrolysate that has passed through the membrane is a polypeptide solution of <3000 Da. Take the polypeptide solution of <3000 Da and continue to use a 1000 Da nanofiltration membrane to separate it by discontinuous intermittent constant volume operation at 0.8 MPa to obtain a 1000-3000 Da polypeptide solution that has not passed through the membrane and a <1000 Da polypeptide solution that has passed through the membrane. The above polypeptide solutions are respectively concentrated under reduced pressure and freeze-dried to obtain <1000 Da, 1000-3000 Da, and >3000 Da polypeptide freeze-dried powders for use.

④ Take the peptide fragment <1000Da prepared in step ③ and prepare it into 20 mg·mL ^-1 with distilled water. Load the sample continuously at a flow rate of 2 BV·h ^-1 . A _220nm ≥0.10 is the loading end point. Collect the effluent and adsorb it statically for 3 hours. Elute it with water until A _220nm ≤0.05. Combine the eluate and the effluent as the water eluate. Elute it with 25% ethanol, 50% ethanol, and 75% ethanol respectively until A _220nm ≤0.05. Collect the eluates, concentrate them under reduced pressure, freeze-dry them, and set them aside.

⑤ Take an appropriate amount of Sephadex G15 gel, activate it, and load it into a column (1.6×75 cm). Prepare the lyophilized powder of the 75% ethanol elution portion into 8 mg·mL ^-1 , apply 0.02 BV to the 0.45 μm filter membrane, elute with deionized water, control the flow rate to 2.0 mL·min ^-1 , collect one test tube for every 2.0 mL, number each test tube in the order of collection and measure the absorbance at 280 nm and 220 nm under ultraviolet light, and plot the test tube number and the absorbance change value. The results are shown in Figure 2. As shown in Figure 2, the samples showed three main peaks after Sephadex G15 separation, named F1, F2 and F3 respectively. The same peaks were combined, concentrated under reduced pressure, freeze-dried, and set aside.

⑥ The samples of each chromatographic peak in step ⑤ were prepared with deionized water to 20 mg ml ^-1 and the polypeptide content of each peak was determined. The results are shown in Table 1.

Table 1 Results of peptide content determination of each sample

(2) LC-TOF-MS separation

Take 1 mg of the purified sample F2 in step (1) and add 200 µL of 25 mM ammonium bicarbonate solution to dissolve it. Add 2 µL of 1 M DTT and place it in a 60°C water bath for >40 min. Then add 8 µL of freshly prepared 1 M iodoacetamide and place it at room temperature in the dark for 30 min. Desalt the sample using a micro desalting column. Add 10 µL of loading buffer to the dried sample and shake it to dissolve it. Determine the composition using the following method:

Nano-liquid chromatography column: C18 reverse phase column (75 mm×20 cm×3 µm); mobile phase A: 99.9% water and 0.1% formic acid mixture, mobile phase B: 80% acetonitrile and 0.1% formic acid mixture. Liquid phase gradient elution method: 0-2 min, 2-6%B; 2-95 min, 6-20%B; 95-107 min, 20%-32%B; 107-108 min, 32-100%B; 108-120 min, 100%B. The mobile phase flow rate was 300 nL/min. Full scan acquisition (m/z 350–1800) was performed in ESI+ mode with data-dependent scan mode in the orbitrap at a resolution of 70,000 (AGC 3e6), a normalized collision energy (NCE) of 28.0, a capillary temperature of 275 °C, a spray voltage of 1800 V, an MS ² resolution of 17,500 (AGC 1e5), and maximum fill times of 50 ms and 45 ms for full and MS ² scans, respectively, with a dynamic exclusion time of 30 s.

The active peptides were analyzed by LC-TOF-MS and the structures were VALSVIQR (SEQ ID NO.1) and VIPSLNGK (SEQ ID NO.2). Based on the amino acid sequence of the active peptides identified by LC-TOF-MS, the active peptide raw materials can be prepared by enzymatic separation of earthworm raw materials. The active peptides in the later stage of this experiment were synthesized by Shanghai Jier Biochemical Co., Ltd.

VALSVIQR, named VR-8, isovaline-alanine-leucine-serine-valine-isoleucine-glutamine-arginine, has a molecular weight of 884.54 Da, an isoelectric point of 11.11, a hydrophobic index of 8.15 Kcal·moL ^-1 , good water solubility, and the structural formula is as follows: .

VIPSLNGK, named VK-8, namely valine-isoleucine-proline-serine-leucine-asparagyl-glycine-lysine, has a molecular weight of 826.49 Da, an isoelectric point of 10.14, a hydrophobic index of 10.47 Kcal·moL ^-1 , good water solubility, and the structural formula is as follows:

.

Example 2 Study on the treatment of skin wounds and scars with earthworm peptides

1 Experimental content

1.1 Experimental animals

Sixty male Kunming mice, 6 to 8 weeks old, weighing 18 to 22 g, were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd. and housed in the SPF animal room of Shandong University of Traditional Chinese Medicine. The environmental conditions were controlled at a temperature of 23 to 25 °C, a relative humidity of 55% to 65%, a light/dark cycle of 12 h each, and no restriction on food and water.

1.2 Animal grouping, modeling, and drug administration

Rats were randomly divided into six groups, namely, blank group, model group, polysulfonated mucopolysaccharide group, VR-8 polypeptide group, VK-8 polypeptide group and earthworm polypeptide combination group, with 10 rats in each group. Each group was housed in cages, maintained free access to drinking water and standard diet, and cleaned and disinfected regularly.

1.3 Modeling

Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (400 mg/kg), and 8% sodium sulfide solution was used to remove 2 cm×4 cm of the upper back. 24 hours later, the mice were disinfected with 70% ethanol on the depilatory area, the water temperature of the constant temperature water bath was raised to 95°C, and the depilatory area of the mice was placed 10 cm above the steam outlet for 10 seconds before being removed. The scald area was equal to the depilatory area. A deep II degree scald model of mice was established (confirmed by pathological section observation). After modeling, 2 mL of normal saline was injected intraperitoneally to resist shock.

1.4 Treatment options

During the treatment period, mice in each group were housed in separate cages and had free access to water and standard diet. The model group was massaged with a 50 μL saline cotton swab; the polysulfonated mucopolysaccharide group was massaged with a 2 g Hirudo cotton swab; the VR-8 peptide group was massaged with a 50 μL 100% VR-8 cotton swab; the VK-8 peptide group was massaged with a 50 μL 100% VK-8 cotton swab; and the earthworm peptide combination group (peptide composition ratio VR-8 was 55% and VK-8 was 45%) was massaged with a 50 μL cotton swab. The drug was administered twice a day, with an interval of 12 hours, for 28 consecutive days.

1.5 Observations and indicators

The changes in the area of the scald, the inflammatory infection, the scab and its shedding, and the changes in color, area, and hardness of the scar after formation were observed. Blood was collected from the tail of the mouse on the 7th day and from the eyeball on the 28th day, and part of the scar tissue was cut off. The tail blood and eyeball blood were centrifuged at 3400 r/min for 15 min, and the supernatant was separated. The blood samples and tissue fluid samples were stored in a refrigerator at -80℃ for later use. The ELISA kit was used to detect the changes in the content of TGF-β1, type I/III procollagen, and IL-10. The specific detection method was measured according to the instructions of the kit.

1.6 Statistical analysis

SPSS 16.0 statistical software was used, and the calculated data were expressed as x̅±s. The t-test and one-way analysis of variance were used for comparison among the groups. P < 0.05 indicated statistically significant differences.

2 Experimental results

2.1 Observation of skin lesion changes and scars

The changes in skin lesions and scars of mice after treatment were observed: on the 3rd day after treatment, the wounds in the model group had purulent secretions. There was a little secretion in the wounds of the polysulfonated mucopolysaccharide group, and the inflammatory reaction in the essential oil group was mild; on the 8th day after modeling treatment, most of the skin lesions in the model group were necrotic, with exudation, thick and hard scabs, and obvious redness and swelling at the wound edge. Most of the wounds in the polysulfonated mucopolysaccharide group were scabs, with a small amount of granulation tissue and no exudation. A small part of the wounds in the VR-8 polypeptide group and the VK-8 polypeptide group were scabs, with a small amount of granulation tissue and a little exudation. A large amount of granulation tissue was visible in the skin lesions of the earthworm polypeptide combination group, without exudation. On the 30th day, the skin lesions in the earthworm polypeptide combination group were clean and scab-free, completely healed, and hair grew. The scabs of the skin lesions in the VR-8 polypeptide group, the VK-8 polypeptide group, and the polysulfonated mucopolysaccharide group partially fell off, and a few wounds were not healed. Most of the skin lesions in the model group were covered with scabs, which were hard, thick and tough, and some wounds were healed.

2.2 Serum TGF-β1 and IL-10 concentrations and changes are shown in Table 2 and Figure 3

On the 8th day of treatment, the levels of TGF-β1 and IL-10 in the serum of the model group rats were higher than those in the polysulphonic acid mucopolysaccharide group ( P <0.01). Compared with the model group, topical application of Xiruotuo can increase the levels of TGF-β1 and IL-10 in serum ( P <0.05). VR-8 earthworm polypeptide and VK-8 earthworm polypeptide can increase the levels of TGF-β1 and IL-10 in serum, but not significantly. The topical application of earthworm polypeptide combination can significantly increase the levels of TGF-β1 and IL-10 in serum, and there are significant differences between the polysulphonic acid mucopolysaccharide group and the blank group ( P <0.05). On the 30th day of treatment, TGF-β1 and IL-10 in the earthworm polypeptide combination group basically dropped to normal levels, and the levels in the VR-8 polypeptide group, VK-8 polypeptide group and polysulphonic acid mucopolysaccharide group were slightly higher than the normal levels, while the model group showed a continuous upward trend ( P <0.05).

Table 2 Effects of earthworm peptide on serum TGF-β1 and IL-10 levels in mice with deep second-degree scald (x̅±s, ng/mL)

* P ＜0.05 compared with the blank group, * ^# P ＜0.05 compared with the model group, * ^& P ＜0.05 compared with the polysulfate mucopolysaccharide group.

3. Changes in the ratio of type I to type III procollagen in tissues

See Table 3 and Figure 4. From the first day to the 28th day of treatment, the ratio of type I/III procollagen in the tissues of mice in the model group gradually increased ( P < 0.05). Compared with the model group, topical application of Hirudo can reduce the ratio of type I/III procollagen in tissues ( P < 0.05). VR-8 earthworm polypeptide and VK-8 earthworm polypeptide can reduce the ratio of type I/III procollagen in tissues, but not significantly. The topical application of earthworm polypeptide combination can significantly reduce the ratio of type I/III procollagen in tissues, which is significantly different from the blank group and Hirudo group ( P < 0.05). On the 30th day of treatment, the ratio of type I/III procollagen in the earthworm polypeptide combination group dropped to normal levels. The VR-8 polypeptide group, VK-8 polypeptide group and polysulfonated mucopolysaccharide group were still slightly higher than the normal level, while the model group continued to increase.

Table 3 Effect of earthworm peptide on the ratio of type I and type III procollagen in the tissue fluid of mice with deep second-degree scald (x̅±s)

Group Before modeling 8 days after modeling 30 days after modeling Blank group 1.490.07 Model group (MP) 4.450.37* 8.210.41* Polysaccharide polysaccharide group (DP) 3.320.46*# 2.540.33*# VR-8 peptide group (VR) 4.231.97 2.460.75*# VK-8 peptide group (VK) 4.172.28 2.380.59*# Pteris lobata polypeptide combination group (SP) 2.860.28*& 1.610.56*&

* P ＜0.05 compared with the blank group, * ^# P ＜0.05 compared with the model group, * ^& P ＜0.05 compared with the polysulfate mucopolysaccharide group.

4. Observe the epidermal thickness, scar thickness and hair follicles under a microscope

The results showed that external application of the earthworm polypeptide combination can significantly inhibit the proliferation of the wound epidermis and hair follicles, and can significantly inhibit the formation of scars. Its effect is better than that of VR-8 polypeptide and VK-8 polypeptide used alone, as shown in Table 4, Table 5 and Table 6.

Table 4 Comparison of epidermal thickness among groups

Group x̅s F P Blank Group 24.811.3#&+ 8.4 <0.001 Model Group 78.228.4*+ Polysaccharide group 73.721.9*+ VR-8 peptide group (VR) 72.1118.75*+ VK-8 peptide group (VK) 70.8920.11*+ Pteris edulis polypeptide combination group (SP) 58.515.3*#&

Compared with the blank group, the other three groups were significantly different, * P <0.05; compared with the model group, the difference was statistically significant, ^# P <0.05; compared with the polysulfated mucopolysaccharide group, the difference was statistically significant, ^& P <0.05; compared with the earthworm peptide group and the other three groups, the difference was statistically significant, ⁺ P <0.05.

Table 5 Comparison of scar thickness among groups

Group x̅s F P Blank Group 220.441.8#& 18.9 <0.001 Model Group 1262.2531.8*+ Polysaccharide group 990.7427.8*+ VR-8 peptide group (VR) 957.2325.1*+ VK-8 peptide group (VK) 962.77364.2*+ Pteris edulis polypeptide combination group (SP) 562.7175.3#&

*: Compared with the normal skin group, the difference was statistically significant; #: Compared with the model group, the difference was statistically significant; &: Compared with the polysulfate mucopolysaccharide group, the difference was statistically significant; +: Compared with the essential oil group, the difference was statistically significant.

Table 6 Comparison of hair follicles in each group

Group M P25 P75 F P Blank Group 1 1 1 19.216 <0.001 Model Group 3.2 2.9 3.8 Polysaccharide group 3 2.2 3 VR-8 peptide group (VR) 3 2.1 3.1 VK-8 peptide group (VK) 2.8 2 2.7 Pteris edulis polypeptide combination group (SP) 1 0 1.8

There was no statistically significant difference between the earthworm polypeptide combination group and the blank group; there was no statistically significant difference between the model group and the VR-8 polypeptide group, VK-8 polypeptide group, and polysulfonated mucopolysaccharide group; the differences between the other groups were statistically significant.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. A earthworm polypeptide group, which is characterized by consisting of a characteristic polypeptide 1 and a characteristic polypeptide 2; wherein the amino acid sequence of the characteristic polypeptide 1 is VALSVIQR, and the amino acid sequence of the characteristic polypeptide 2 is VIPSLNGK.

2. A method for preparing the earthworm polypeptide set as in claim 1, characterized by the following steps:

(1) Pulverizing Lumbricus, sieving, adding water, and boiling;

(2) Cooling the mixed solution obtained in the step (1), regulating the pH value, carrying out enzymolysis, boiling, and separating and purifying;

(3) And (3) processing the purified sample obtained in the step (2) and then carrying out LC-TOF-MS analysis to obtain the active polypeptide structures VALSVIQR and VIPSLNGK.

3. The method according to claim 2, wherein in the step (2), the method of enzymolysis comprises: the mixed solution is heated and boiled, cooled to 35-45 ℃, the pH value is regulated to 1.0-3.0, 0.5-2% pepsin is added, enzymolysis is carried out at 40 ℃ to 1h, the pH value is regulated to 7.0-9.0, 0.5-2% trypsin is added, the temperature is kept to 3-5 h, and the mixture is boiled to 10-15 min.

4. The method according to claim 2, wherein in the step (2), the separation and purification method is to perform semi-preparative separation and purification of the supernatant by cation resin and RP-HPLC by ultrafiltration with 3 KD ultrafiltration membrane after desalting by using 3500 to 4500 rpm centrifugal 15 to 20 min electrodialysis or dialysis bag.

5. The method of claim 2, wherein in step (3), the LC-TOF-MS analysis uses a nanoliter liquid chromatography column under the following conditions: c18 reverse phase analytical column 75 mm X20 cm X3 μm; mobile phase a:99.9% water and 0.1% formic acid, mobile phase B: a mixture of 80% acetonitrile and 0.1% formic acid; the liquid phase gradient elution is as follows: 0-2 min,2-6%B;2-95 min,6-20% B;95-107 min,20% -32% B;107-108 min,32-100% B;108-120 min,100% B; the mobile phase flow rate was 300 nL/min.

6. The method according to claim 2, wherein in the step (3), the purified sample obtained in the step (2) is processed by: taking 1-5 mg purified samples, adding 200 [ mu ] L of 25 mM ammonium bicarbonate solution for dissolution, adding 2 [ mu ] L of 1M of DTT, placing in a water bath at 60 ℃ for >40 min, adding 8 [ mu ] L of freshly prepared 1M iodoacetamide, and placing in a light-shielding place at room temperature for 30 min.

7. The method according to claim 2, wherein in the step (1), the standard mesh number of the ground and sieved earthworms is 80 mesh; the water adding operation is to add water with the mass of 8-12 times of that of the earthworms; the heating boiling time is 15-30 min.

8. Use of the lumbricus polypeptide group as claimed in claim 1, wherein the lumbricus polypeptide group is used for preparing a medicament for healing skin wound or eliminating scar.

9. The use according to claim 8, wherein the medicament comprises the group of earthworm polypeptides and pharmaceutically acceptable excipients.

10. The use according to claim 9, wherein the adjuvant is one of the required adjuvants for the preparation of a water-infusion, a powder, a lotion, a tincture, an oil, an emulsion, an ointment, a plaster or an aerosol.