CN115991758A

CN115991758A - Pearl powder polypeptide for promoting diabetic wound healing and application thereof

Info

Publication number: CN115991758A
Application number: CN202211035850.6A
Authority: CN
Inventors: 兰太进; 刘鹏; 林江; 皮振宇; 李静
Original assignee: Guangxi University of Chinese Medicine
Current assignee: Guangxi University of Chinese Medicine
Priority date: 2022-08-27
Filing date: 2022-08-27
Publication date: 2023-04-21
Anticipated expiration: 2042-08-27
Also published as: CN115991758B

Abstract

The invention discloses a small molecular polypeptide derived from pearl powder and application thereof in preparing a medicament or an external dressing for promoting diabetic wound healing, and belongs to the technical field of medicines. The amino acid sequence of the polypeptide is shown as SEQ ID NO. 1; or the polypeptide is a conservative variant obtained by deleting, replacing, inserting or/and adding one to several amino acids on the basis of the amino acid sequence shown in SEQ ID NO. 1. The polypeptide provided by the invention has biological activity for promoting healing of acute and chronic skin wounds and mucosal ulcers, has good application prospect, and can be used for preparing external medicines or external dressings for promoting healing of diabetic wounds.

Description

Pearl powder polypeptide for promoting diabetic wound healing and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a small molecular polypeptide derived from pearl powder and application thereof in preparing a medicament or an external dressing for promoting diabetic wound healing.

Background

Among the complications of diabetes, impaired wound healing in diabetics is a typical complication of diabetes and is also a major cause of non-invasive lower limb amputation. This complication results in a lifetime risk of up to 32% for diabetic patients suffering from chronic foot ulcers. In the past decades, with the continuous development of various new drugs, new technologies and new treatment strategies, particularly the development of multidisciplinary comprehensive treatment modes in recent years, the treatment effect of the diabetic wound surface is improved to a certain extent, but the problems of long treatment period, low short-term wound surface healing rate and the like can not be fundamentally solved at all. Therefore, new therapeutic drugs and therapeutic methods are actively sought to shorten the wound healing time, which has important significance for improving the life quality of patients and reducing the medical cost.

Pearl powder is used as a traditional Chinese medicine and is applied in China for thousands of years. In the traditional application, the pearl powder can be used for treating various acute and chronic wound surface disunion by local external application, and is currently received in pharmacopoeia of the people's republic of China. Modern clinical observation shows that the pearl powder can obviously promote healing of various acute and chronic skin wounds and mucous ulcer, including peptic ulcer, oral ulcer, skin injury, wound ulcer, bedsore pressure sore and scald by singly or jointly using the pearl powder. The in-vivo and in-vitro animal cell experimental results show that the water-soluble component of the pearl powder has remarkable healing promoting effect on an animal wound injury model, and can play an antioxidant role by adjusting the ROS level and promote the migration of skin fibroblasts, so that the wound healing effect is accelerated. In addition, pearl powder is involved in regulating signals related to cell growth and repair closely related to wound healing, including stimulating fibroblast mitosis, enhancing cell adhesion, promoting collagen deposition, inhibiting metalloproteinase (TIMP) -1 activity, and the like.

The prior researches show that the pearl powder contains a large amount of inorganic components such as calcium carbonate, trace elements and the like and also contains organic components such as rich active peptides and the like. CN113559240a discloses a pearl polypeptide liquid and application thereof in promoting wound healing, the separation and extraction process of the pearl polypeptide liquid comprises the following steps: mixing the pearl superfine powder with PBS solution with the weight percentage of 1+/-0.5%, extracting small molecular peptide in the pearl powder by using a supercritical carbon dioxide extraction instrument with the dosage ratio of 1:4+/-0.5, collecting the extracted mixed solution, centrifuging for 10-15min by using a high-speed centrifuge with the speed of 5000+/-1000 rpm, and collecting the upper layer liquid, namely the pearl polypeptide liquid. However, although the technology of this patent obtains the pearl powder polypeptide liquid by the extraction and separation technology, the polypeptide liquid is a mixture of various small molecular peptides, and the small molecular peptides with no activity or low activity are contained in the polypeptide liquid. Based on the above, we develop the screening research of pearl powder polypeptide for promoting the chronic wound activity of diabetes, and found that the small molecular polypeptide Na-1 from pearl powder has good effect of promoting the healing of the diabetes wound.

Disclosure of Invention

In view of the shortcomings of the prior art, a first object of the present invention is to provide a polypeptide derived from pearl powder and having high activity for promoting healing of diabetic wounds.

The above object of the present invention is achieved by the following technical solutions: a polypeptide, the amino acid sequence of which is shown as SEQ ID NO. 1; or the polypeptide is a conservative variant obtained by deleting, substituting, inserting or/and adding one to several amino acids on the basis of the amino acid sequence shown in SEQ ID NO. 1.

The amino acid sequence of SEQ ID NO.1 is ISKCFIACGIGQRQSPINIV, and this polypeptide is designated Na-1. The polypeptide Na-1 provided by the invention can be directly customized to commercial peptide synthesis companies, or can be synthesized according to the operation rules of manufacturers by using a commercially available automatic synthesizer.

On the other hand, the invention also provides application of the polypeptide in preparing a medicament or an external dressing for promoting the healing of the diabetic wound.

In another aspect, the invention also provides a composition for promoting the healing of diabetic wounds, which comprises the polypeptide.

Further preferred is a composition for promoting healing of diabetic wounds as described above, which is a gel. Still further preferably, the gelling agent comprises the polypeptide, a gelling matrix, a humectant, a penetration enhancing agent, a pH adjusting agent, and water.

The gel matrix is one or more than two of carbomer, hydroxypropyl methylcellulose and sodium hyaluronate; the humectant is glycerin; the penetration enhancer is azone; the pH regulator is sodium hydroxide. In addition, the gel can also contain a preservative, wherein the preservative is one or more than two of potassium sorbate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate and sodium benzoate.

Compared with the prior art, the polypeptide provided by the invention has biological activity of promoting healing of acute and chronic skin wounds and mucosal ulcers, has good application prospect, and can be used for preparing external medicines or external dressings for promoting healing of diabetic wounds.

Drawings

Fig. 1 is a graph of the healing process of the full-cortical excision wound of diabetic rats, and the wound healing was recorded at 0, 2, 4, 6, 8, 10, 12 and 14 d.

Fig. 2 is a graph of the time course of the rate of healing of the full-cortical resection wound in diabetic rats.

FIG. 3 is a chart of the staining of each set of wound surfaces H & E and Masson 14 days after molding.

FIG. 4 is a picture of Na-1 external gel.

Detailed Description

In order to better illustrate the characteristics and essence of the invention, the following examples are used to provide the results of pharmacological tests of Na-1 in promoting the healing of the resected wound of the full cortex of diabetic rats, illustrating its use in the technical field of medicine. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. In addition, the specific technical operation steps or conditions are not noted in the examples, and are carried out according to the techniques or conditions described in the literature in the field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: na-1 source and artificial synthesis

Na-1 amino acid sequence: ISKCFIACGIGQRQSPINIV

Na-1 is a polypeptide sequence shared by a plurality of pearl proteins, and detailed information is shown in table 1. The Shanghai worker is entrusted to carry out synthetic synthesis on Na-1, and is used for observing the healing effect of the diabetic wound surface.

Table 1: positional information of Na-1 in Pearl protein sequence

Example 2: preparation of Na-1 external gel

Carbomer (0.4051 g) and hydroxypropyl methylcellulose (0.1031 g) were weighed into 60mL distilled water slowly and stirred with complete dissolution. And weighing a proper amount of sodium hydroxide, adding the sodium hydroxide into the solution, stirring while adding, and adjusting the pH to 10 to obtain colorless and transparent carbomer hydroxypropyl methylcellulose gel. Then glycerin (5.2 g) is added and stirred uniformly to obtain gel matrix. Na-1 (0.12496 g) was weighed and 20mL of water was added to disperse the mixture, thereby obtaining a Na-1 solution. Adding Na-1 solution into gel matrix, stirring, adding azone (1.1 g) into the gel matrix, adding distilled water to make up 100g, and stirring to obtain Na-1 topical gel. The product is milky gel, has stable properties, no layering, no oil bleeding, fine texture, no color, transparency, proper viscosity, easy application, easy cleaning, and no greasy feeling (figure 4).

Example 3: experiment of influence of Na-1 on healing of full cortex excision wound of diabetic rat

1. Preparation of diabetic rats

Male SD rats were intraperitoneally injected with 1% streptozotocin (streptozotocin dissolved in citrate buffer solution at pH 4.5) at 65mg/kg,6mL/kg, and after 72h following streptozotocin injection, the first tail tip blood sampling was performed to detect random blood glucose levels of the rats, blood glucose levels exceeding 16.7mmol/L were used as a standard for molding diabetic rats, rats with blood glucose levels greater than 16.7mmol/L were selected as subjects, and wound surfaces were prepared after stabilization for 5 d.

2. Preparation of diabetic rat wound surface

The method comprises the steps of anesthetizing rat diethyl ether, fixing a prone position on an operation table, removing long hairs on the back of the rat by electric pushing, then coating depilatory cream, scraping the depilatory cream after 6min, wiping residual depilatory cream on a depilatory area with warm water, wiping the residual depilatory cream with gauze, sterilizing with iodophor, spreading an operation towel, covering a 1-corner coin (diameter is 1.8 cm) after autoclaving and sterilizing on the operation area, pressurizing to form circular marks, and cutting circular skin to deep fascia layers by using surgical scissors according to the marks. Because the thickness and the tension of the skin at the back of each rat are different, the number and the positions of the prepared wound surfaces of each rat are designed to avoid misjudgment of experimental results caused by the difference between the wound surfaces: each rat is provided with 2 wound surfaces, the wound surfaces are positioned at 1.5cm positions on the left side and the right side of the spine, and the distance between the centers of the wound surfaces on the same side and the neck is half of the total length of the back.

3. Wound grouping and administration

The number of Normal rats is 2, 4 wounds are set as a non-diabetic wound blank group (Normal group), and the Normal rats are a natural wound healing group without any treatment. The number of the wound surfaces is 16, the wound surfaces are numbered and are grouped according to a random number table, and the number of the wound surfaces is divided into 3 groups, namely a diabetes wound surface blank group (Model group), a diabetes wound surface epidermal growth factor group (EGF group, positive control) and a diabetes wound surface Na-1 group (Na-1 group); wherein: model group, 4 wound surfaces, which are the natural healing groups of the wound surfaces of the diabetic rats, do not perform any treatment; EGF group, 4 wound surfaces, which are positive control medicine EGF intervention group, wherein the wound surfaces start to be dosed on the same day, and a certain amount of EGF gel (trade name Yifu, specification is 5 ten thousand IU (100 mu g)/10 g/branch, manufactured by Gui Linhua Norwegian gene pharmaceutical Co., ltd., national medicine standard character S20020112) is uniformly smeared on the wound surfaces of diabetic rats until the wound surfaces are just completely covered; na-1 group and 4 wound surfaces are small molecule polypeptide Na-1 intervention groups, the wound surfaces are started to be dosed on the same day, and a certain amount of Na-1 gel (prepared in example 2) is uniformly smeared on the wound surfaces of diabetic rats until the wound surfaces are just completely covered. Dosing 1 time daily in the morning of 10:00-12:00, free drinking water to eat. Photographing at 0, 2, 4, 6, 8, 10, 12 and 14 days after operation, calculating the wound area at each time point, and calculating the healing rate according to the following formula: healing rate (%) = (wound surface original area-area of each time point)/(wound surface original area×100%). After 14d, each set of wound tissues was fixed with 4% paraformaldehyde and then H & E and Masson stained. Infiltration of inflammatory cells and fibroblast number on longitudinal sections of wound tissue under a microscope can evaluate wound healing.

4. Experimental results

The results show that compared with Normal wound, the healing speed of the model group is obviously reduced (P < 0.05), which proves that the animal model used in the experiment is successfully modeled, and the accuracy of the research can be effectively ensured. Compared with the model group, the healing speed of the wound surface of the diabetes mellitus of the EGF group and the Na-1 group is obviously accelerated (P is less than 0.05), and representative images and healing rate time progress curves of the wound surface on days 0-14 are respectively shown in the figures 1 and 2. On day 14 after wound surface formation, the pathological observation results of H & E are shown in FIG. 3, compared with the normal group, the inflammatory cell infiltration of the model group is obvious, the EGF group has slightly inflammatory infiltration, and the Na-1 group has no obvious inflammatory infiltration. Collagen formation plays a key role in the wound healing process, it can promote cell migration and serve as a basis for extracellular matrix deposition during the wound healing proliferation phase, masson staining can be applied to assess collagen formation, where collagen is stained blue, while cell bodies, muscles and keratin are stained red. As shown in fig. 3, the model group has less collagen fibers, a loose reticular structure and an ulcerated epidermis; and the collagen fiber structure and the epidermis are intact in the Na-1 group, and the collagen fibers are orderly accumulated, so that the Na-1 has positive effect on wound healing, which is consistent with the drug effect results in figures 1 and 2.

Claims

1. A polypeptide, which is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1; or the polypeptide is a conservative variant obtained by deleting, substituting, inserting or/and adding one to several amino acids on the basis of the amino acid sequence shown in SEQ ID NO. 1.

2. Use of the polypeptide of claim 1 for the preparation of a medicament or a topical dressing for promoting healing of diabetic wounds.

3. A composition for promoting healing of diabetic wounds, comprising the polypeptide of claim 1.

4. A composition for promoting the healing of diabetic wounds according to claim 3, wherein the composition is a gel.

5. The composition for promoting healing of a diabetic wound according to claim 4, wherein the gel comprises the polypeptide, a gelling matrix, a humectant, a penetration enhancer, a pH adjuster, and water.

6. The composition for promoting the healing of diabetic wounds according to claim 5, wherein the gelling matrix is selected from one or more of carbomers, hydroxypropyl methylcellulose, sodium hyaluronate.

7. The composition for promoting healing of a diabetic wound according to claim 5, wherein the humectant is glycerin.

8. The composition for promoting healing of diabetic wounds according to claim 5, wherein the penetration enhancer is azone.

9. The composition for promoting healing of a diabetic wound according to claim 5, wherein the pH adjustor is sodium hydroxide.

10. The composition for promoting the healing of diabetic wounds according to claim 5, further comprising a preservative selected from one or more of potassium sorbate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate and sodium benzoate.