CN107281535B

CN107281535B - Giant salamander secretion viscous biomembrane and preparation method and application thereof

Info

Publication number: CN107281535B
Application number: CN201710681790.8A
Authority: CN
Inventors: 邢孟秋; 商海涛; 蒋坤; 魏泓
Original assignee: Individual
Current assignee: Wuhan Hualianke Biotechnology Co ltd
Priority date: 2017-08-10
Filing date: 2017-08-10
Publication date: 2020-09-15
Anticipated expiration: 2037-08-10
Also published as: CN107281535A

Abstract

The invention discloses a giant salamander secretion viscous biomembrane and a preparation method and application thereof. Through a great deal of research and study, the inventor unexpectedly finds that the giant salamander secretion has higher solubility in vitamin C solution, hydrochloric acid solution, acetic acid solution, sulfuric acid solution, phosphoric acid solution and nitric acid solution. The preparation method does not damage the original structure of the giant salamander secretion, the formed giant salamander secretion viscous biomembrane keeps the advantages of better adhesiveness, biocompatibility and the like of the giant salamander secretion, and the giant salamander secretion viscous biomembrane is uniform in quality, better in toughness and strength, capable of being used as a hemostatic membrane, a tissue adhesive membrane or a bandage and the like to cover or wrap a wound surface, and wide in application prospect.

Description

Giant salamander secretion viscous biomembrane and preparation method and application thereof

Technical Field

The invention relates to the field of biological materials, in particular to a giant salamander secretion viscous biological membrane and a preparation method and application thereof.

Background

Giant salamanders, also known as "salamanders of china", belong to the family of giant salamanders, and are one of the largest existing amphibian species. It shows tremendous research progress and valuable potential in the fields of biodiversity, genetic evolution and biochemistry. The giant salamander mucus is a giant salamander secretion after the surface of the giant salamander is stimulated, and the early-stage research finds that the giant salamander secretion has the advantages of good adhesive property, good biocompatibility, degradability and the like, and can be developed into hemostatic materials, adhesives, soft tissue filling materials and the like.

However, the traditional giant salamander secretion application form is mostly powder or gel. In clinic, a membranous material is needed to cover or wrap the wound surface, and both powder and gel cannot meet the clinical requirements. Therefore, the development of giant salamander secretion viscous biomembranes is urgently needed. However, due to the insolubility of the giant salamander secretion, researchers continuously try to prepare the giant salamander secretion into a film, but the giant salamander secretion viscous biomembrane is difficult to form on the premise of not damaging the original performances of the giant salamander secretion such as the adhesiveness, the biocompatibility and the like.

Disclosure of Invention

Based on the above, a preparation method capable of forming a giant salamander secretion viscous biomembrane without destroying the original performance of the giant salamander secretion is needed.

In addition, a giant salamander secretion viscous biomembrane and application thereof are also provided.

A preparation method of a giant salamander secretion viscous biomembrane comprises the following steps:

dissolving giant salamander secretion in an acidic solution to obtain a giant salamander secretion solution, wherein the acidic solution is at least one selected from a vitamin C solution, a hydrochloric acid solution, an acetic acid solution, a sulfuric acid solution, a phosphoric acid solution and a nitric acid solution; and

and (3) freeze-drying the giant salamander secretion solution to form a film or vacuum-drying the film to form the giant salamander secretion viscous biological film.

In one embodiment, the acidic solution is a vitamin C solution, and the concentration of vitamin C in the vitamin C solution is 0.1mol/L to 1 mol/L; and/or the presence of a catalyst in the reaction mixture,

the acid solution is hydrochloric acid solution, and the concentration of hydrogen chloride in the hydrochloric acid solution is 0.1-1 mol/L; and/or the presence of a catalyst in the reaction mixture,

the acid solution is an acetic acid solution, and the concentration of acetic acid in the acetic acid solution is 0.1-1 mol/L; and/or the presence of a catalyst in the reaction mixture,

the acid solution is a sulfuric acid solution, and the concentration of sulfuric acid in the sulfuric acid solution is 0.1-1 mol/L; and/or the presence of a catalyst in the reaction mixture,

the acid solution is phosphoric acid solution, and the concentration of phosphoric acid in the phosphoric acid solution is 0.1-1 mol/L; and/or the presence of a catalyst in the reaction mixture,

the acid solution is a nitric acid solution, and the concentration of nitric acid in the nitric acid solution is 0.1-1 mol/L.

In one embodiment, the giant salamander secretion solution is lyophilized into a membrane, and the step of lyophilizing into a membrane specifically comprises:

the giant salamander secretion-solution is cooled to minus 50 ℃ to minus 30 ℃ at a constant speed and is frozen for 0.5h to 4 h; and

and placing the frozen giant salamander secretion solution for 12-48 h under the condition that the absolute pressure is 1-10 Pa.

In one embodiment, the giant salamander secretions are dried powder of giant salamander mucus.

In one embodiment, the concentration of the giant salamander secretions in the giant salamander secretion solution is from 0.001g/mL to 1 g/mL.

In one embodiment, the giant salamander secretion is prepared by the following method:

scraping giant salamander mucus from the outer surface of the giant salamander;

freezing the giant salamander mucus for 0.5 to 2 hours at the temperature of minus 30 to minus 10 ℃; and

and (3) performing vacuum freeze drying on the frozen giant salamander mucus at the temperature of-50 to-30 ℃ and under the absolute pressure of 5 to 20Pa to obtain the giant salamander secretion.

In one embodiment, the giant salamander secretion has a particle size of 1 μm to 100 μm.

In one embodiment, in the step of dissolving the giant salamander secretion in the acidic solution, the dissolving operation is performed for 4 to 8 hours under stirring at a temperature of 10 to 35 ℃.

A giant salamander secretion viscous biomembrane is prepared by any one of the preparation methods of the giant salamander secretion viscous biomembrane.

The giant salamander secretion viscous biomembrane is applied to preparation of a hemostatic membrane, a tissue adhesive membrane or a bandage.

The preparation method of the giant salamander secretion viscous biomembrane comprises the steps of dissolving the giant salamander secretion in an acid solution to obtain a giant salamander secretion solution, and then freeze-drying or vacuum-drying the giant salamander secretion solution to form a membrane to obtain the giant salamander secretion viscous biomembrane. Through a great deal of research and study, the inventor unexpectedly finds that the giant salamander secretion has high solubility in a vitamin C solution, a hydrochloric acid solution, an acetic acid solution, a sulfuric acid solution, a phosphoric acid solution and a nitric acid solution, so that the giant salamander secretion can be dissolved in the acidic solution to form a giant salamander secretion solution, and then the giant salamander secretion solution is freeze-dried to form a membrane to obtain the giant salamander secretion viscous biomembrane. The preparation method does not damage the original structure of the giant salamander secretion, the formed giant salamander secretion viscous biomembrane keeps the advantages of better adhesiveness, biocompatibility and the like of the giant salamander secretion, and the giant salamander secretion viscous biomembrane is uniform in quality, better in toughness and strength, capable of being used as a hemostatic membrane, a tissue adhesive membrane or a bandage and the like to cover or wrap a wound surface, and wide in application prospect.

Drawings

FIG. 1 is a flow chart of a method for preparing a viscous biofilm of giant salamander secretions in accordance with an embodiment;

FIG. 2 is a schematic representation of the viscous biofilm of giant salamander secretions prepared in example 1;

FIG. 3 is a comparison graph of the results of the control group in the test four and the wound treated with the viscous biofilm of giant salamander secretions prepared in example 2;

FIG. 4 is a graph comparing the results of five tests in which the viscous biofilm of giant salamander secretions prepared in example 3 was implanted into the subcutaneous bursa of mice and then varied with time.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Referring to fig. 1, a method for preparing a giant salamander secretion viscous biofilm according to an embodiment includes the following steps S110 to S150.

S110, scraping giant salamander mucus from the outer surface of the giant salamander.

Specifically, fresh giant salamanders are cleaned by clear water, impurities such as molting and the like are removed, then flat objects such as a spoon are used for scraping the giant salamander body surfaces back and forth, and when a large amount of milky mucus is secreted by the giant salamander body surfaces, the giant salamander mucus is scraped and collected. About 5g of giant salamander mucus can be collected from about 5kg of adult giant salamander generally each time.

In this embodiment, 30g of giant salamander mucus was obtained by scraping in total.

S120, freezing the giant salamander mucus collected in the S110 for 0.5-2 h at the temperature of minus 30-minus 10 ℃.

Specifically, the collected giant salamander mucus is frozen in a refrigerator at-20 ℃ for 1 h.

S130, performing vacuum freeze drying on the giant salamander mucus frozen in the S120 at the temperature of minus 50 ℃ to minus 30 ℃ and under the absolute pressure of 5Pa to 20Pa to obtain the giant salamander secretion.

Specifically, the frozen giant salamander mucus is subjected to vacuum freeze drying operation under the conditions that the temperature is-50 ℃ to-30 ℃ and the absolute pressure is 5Pa to 20Pa, and the vacuum freeze drying time is 12h to 48 h.

Specifically, the absolute pressure of 5Pa to 20Pa represents that the moisture in the giant salamander mucus is removed under the vacuum condition that the actual pressure is lower than the atmospheric pressure, and the dry powder of the giant salamander mucus is obtained.

In the embodiment, the frozen giant salamander mucus is placed in a freeze dryer and dried for 48 hours at the temperature of minus 40 ℃ and under the absolute pressure of 10Pa, so that the giant salamander mucus is dried into powder.

Specifically, the freezing temperature in S120 is higher than the vacuum freeze-drying temperature in S130, so that the giant salamander mucus is gradually cooled, and the internal structure of the giant salamander mucus is prevented from being damaged by quenching.

In one embodiment, after the freeze-drying under vacuum to obtain the dry powder of the giant salamander mucus, the method further comprises crushing the dry powder of the giant salamander mucus to form the micro-particles.

Specifically, crushing dry giant salamander mucus powder by using a low-temperature ultrafine crusher, adding liquid nitrogen while crushing, keeping the temperature at a low temperature, crushing to obtain tiny granular giant salamander secretions, and promoting the giant salamander secretions to be dissolved in an acid solution, thereby preparing the giant salamander secretion viscous biomembrane.

In one embodiment, the giant salamander secretions have a particle size of 1 μm to 100 μm.

In one embodiment, the giant salamander secretions have a particle size of 20 μm to 60 μm.

In this embodiment, the grain size of the giant salamander secretion is 50 μm.

S140, dissolving the giant salamander secretion obtained in the step S130 in an acid solution to obtain a giant salamander secretion solution, wherein the acid solution is at least one selected from a vitamin C solution, a hydrochloric acid solution, an acetic acid solution, a sulfuric acid solution, a phosphoric acid solution and a nitric acid solution.

Specifically, the giant salamander secretion is dry powder of giant salamander mucus.

Wherein the acidic solution is at least one selected from the group consisting of a vitamin C solution, a hydrochloric acid solution, an acetic acid solution, a sulfuric acid solution, a phosphoric acid solution and a nitric acid solution.

In one embodiment, the acidic solution is a vitamin C solution, and the concentration of vitamin C in the vitamin C solution is 0.1mol/L to 1 mol/L.

Specifically, the concentration of the vitamin C in the vitamin C solution is 0.5 mol/L-1 mol/L.

Specifically, the solvent of the vitamin C solution is water.

In one embodiment, the acidic solution is a hydrochloric acid solution, and the concentration of hydrogen chloride in the hydrochloric acid solution is 0.1mol/L to 1 mol/L.

Specifically, the concentration of the hydrogen chloride in the hydrochloric acid solution is 0.5 mol/L-1 mol/L.

Specifically, the solvent of the hydrochloric acid solution is water.

In one embodiment, the acidic solution is an acetic acid solution, and the concentration of acetic acid in the acetic acid solution is 0.1mol/L to 1 mol/L.

Specifically, the concentration of acetic acid in the acetic acid solution is 0.5 mol/L-1 mol/L.

Specifically, the solvent of the acetic acid solution is water.

In one embodiment, the acidic solution is a sulfuric acid solution, and the concentration of sulfuric acid in the sulfuric acid solution is 0.1mol/L to 1 mol/L.

Specifically, the concentration of the sulfuric acid in the sulfuric acid solution is 0.1 mol/L-0.5 mol/L.

Specifically, the solvent of the sulfuric acid solution is water.

In one embodiment, the acidic solution is a phosphoric acid solution, and the concentration of phosphoric acid in the phosphoric acid solution is 0.1mol/L to 1 mol/L.

Specifically, the concentration of phosphoric acid in the phosphoric acid solution is 0.5mol/L to 1 mol/L.

Specifically, the solvent of the phosphoric acid solution is water.

In one embodiment, the acidic solution is a nitric acid solution, and the concentration of nitric acid in the nitric acid solution is 0.1mol/L to 1 mol/L.

Specifically, the concentration of nitric acid in the nitric acid solution is 0.5 mol/L-1 mol/L.

Specifically, the solvent of the nitric acid solution is water.

Through a great deal of research and study, the inventor unexpectedly finds that the giant salamander secretion has high solubility in a vitamin C solution, a hydrochloric acid solution, an acetic acid solution, a sulfuric acid solution, a phosphoric acid solution and a nitric acid solution, so that the giant salamander secretion can be dissolved in the acidic solution to form a giant salamander secretion solution, and then the giant salamander secretion solution is freeze-dried to form a membrane to obtain the giant salamander secretion viscous biomembrane. Although the mechanism of the acidic solution for dissolving giant salamander secretions is not clear, it may be related to the ability of the H ions in the acidic solution to form ionic bonds with the giant salamander secretions. Experimental research proves that the giant salamander secretion has high solubility in the acidic solution and can reach 1g/mL at normal temperature. Therefore, the giant salamander secretion with higher dosage can be dissolved in the acidic solution, and then freeze-dried to form a membrane, so as to prepare the giant salamander secretion viscous biomembrane.

Specifically, the concentration of the giant salamander secretion in the giant salamander secretion solution is 0.1 g/mL-1 g/mL.

Preferably, the concentration of the giant salamander secretion in the giant salamander secretion solution is 0.005 g/mL-0.5 g/mL.

For example, the concentration of the giant salamander secretion is too high, the giant salamander secretion is not easy to dissolve, and the giant salamander secretion solution which is uniformly mixed is difficult to form. For example, the concentration of giant salamander secretion is too low, and the giant salamander secretion is difficult to freeze-dry to form a membranous biomaterial. The concentration of the giant salamander secretion is 0.001 g/mL-1 g/mL, the concentration is proper, and the giant salamander secretion viscous biomembrane with good toughness and strength can be prepared.

Specifically, in the step of dissolving the giant salamander secretion in the acidic solution, the dissolving operation is to stir at a temperature of 15-25 ℃ for 5-7 h.

The temperature condition when dissolving giant salamander secretion is suitable for giant salamander secretion can dissolve in acid solution, and avoids the too high internal structure who destroys giant salamander mucus simultaneously. Through stirring treatment, the dissolution of giant salamander secretions is accelerated, so that a uniformly mixed solution is formed.

S150, freeze-drying the giant salamander secretion solution obtained in the step S140 to form a film or vacuum-drying the film to form the giant salamander secretion viscous biological film.

The giant salamander secretion is dissolved in the giant salamander secretion solution, and during freeze-drying, the acidic solution solvent is volatilized to form a membranous giant salamander secretion viscous biomembrane.

In one embodiment, the step of lyophilizing the giant salamander secretion solution into a membrane specifically includes the following steps S151 to S152.

S151, cooling the giant salamander secretion-solution to-50 ℃ to-30 ℃ at a constant rate, and freezing for 0.5h to 4 h.

Generally, the giant salamander secretion is dissolved at room temperature, for example, 10 ℃ to 35 ℃, the giant salamander secretion solution is cooled to minus 50 ℃ to minus 30 ℃ at a constant speed, and is gradually cooled, so that the internal structure of the giant salamander secretion is prevented from being damaged by quenching.

In one embodiment, the giant salamander secretion-solution is added into a forming mold, wherein the liquid thickness of the giant salamander secretion-solution in the forming mold is 0.5cm to 10 cm. Then the forming die loaded with the giant salamander secretion-solution is put into a freeze dryer, and is cooled to-50 ℃ to-30 ℃ at the speed of 0.5 ℃/min to 2 ℃/min, and is frozen for 0.5h to 4 h.

Specifically, the size and shape of the forming die can be adjusted according to actual needs.

The giant salamander secretion is kept for a period of time at the low temperature of-50 ℃ to-30 ℃ so as to improve the toughness and strength of the giant salamander secretion.

S152, placing the frozen giant salamander secretion solution in the S151 under the condition that the absolute pressure is 1-10 Pa for 12-48 h.

Under the condition that the absolute pressure is 1 Pa-10 Pa, the solvent in the giant salamander secretion solution is volatilized to obtain the membranous giant salamander secretion viscous biomembrane.

Specifically, after the giant salamander secretion viscous biological membrane is placed for 12-48 hours under the condition that the absolute pressure is 1-10 Pa, the obtained membranous giant salamander secretion viscous biological membrane has tiny pores, so that a channel can be provided for nutrient substance transportation, and the biocompatibility of the membrane is improved.

In another embodiment, the giant salamander secretion solution can be dried in vacuum to form a film. Namely, the giant salamander secretion solution is placed under a vacuum condition (lower than atmospheric pressure), and a solvent in the giant salamander secretion solution is volatilized to form a giant salamander secretion viscous biomembrane. Specifically, the temperature for vacuum drying to form a film generally does not exceed 37 ℃.

The preparation method of the giant salamander secretion viscous biomembrane comprises the steps of dissolving the giant salamander secretion in an acid solution to obtain a giant salamander secretion solution, and then freeze-drying or vacuum-drying the giant salamander secretion solution to form a membrane to obtain the giant salamander secretion viscous biomembrane. Through a great deal of research and study, the inventor unexpectedly finds that the giant salamander secretion has high solubility in an acidic solution selected from a vitamin C solution, a hydrochloric acid solution, an acetic acid solution, a sulfuric acid solution, a phosphoric acid solution and a nitric acid solution, so that the giant salamander secretion can be dissolved in the acidic solution to form a giant salamander secretion solution, and then the giant salamander secretion-solution is freeze-dried to form a membrane to obtain the giant salamander secretion viscous biomembrane. The preparation method does not damage the original structure of the giant salamander secretion, the formed giant salamander secretion viscous biomembrane keeps the advantages of better adhesiveness, biocompatibility and the like of the giant salamander secretion, and the giant salamander secretion viscous biomembrane is uniform in quality, better in toughness and strength, capable of being used as a hemostatic membrane, a tissue adhesive membrane or a bandage and the like to cover or wrap a wound surface, and wide in application prospect.

In practical applications, the preparation method of the giant salamander secretion viscous biofilm is not limited to the sequence of steps S110 to S150. Those skilled in the art can make adjustments as needed. Steps S110 to S130 can be omitted, for example, when giant salamander mucus has been extracted in advance and dried to form giant salamander secretions or obtained directly from the market.

The giant salamander secretion viscous biomembrane of an embodiment is prepared by the preparation method of the giant salamander secretion viscous biomembrane.

The giant salamander secretion viscous biomembrane is applied as a hemostatic membrane, a tissue adhesive membrane or a bandage.

The giant salamander secretion viscous biomembrane keeps the advantages of good adhesiveness, biocompatibility and the like of the giant salamander secretion, has uniform texture and good toughness and strength, can be used as a hemostatic membrane, a tissue adhesive membrane or a bandage and the like to cover or wrap a wound surface, and has wide application prospect.

The following are specific examples (the following examples, unless otherwise specified, do not contain other components not specifically indicated except for inevitable impurities)

Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, are usually carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer of the kits.

Example 1

The method comprises the steps of cleaning the body surface of a fresh giant salamander with clear water, removing impurities such as ecdysis and the like, scraping the body surface of the giant salamander back and forth with a spoon, and scraping and collecting giant salamander mucus when the body surface of the giant salamander secretes a large amount of milky mucus. About 5g of giant salamander mucus can be collected from about 5kg of adult giant salamander each time, and 30g of giant salamander mucus is obtained by scraping in total. And (3) freezing the collected giant salamander mucus in a refrigerator at the temperature of-20 ℃ for 2 h. And (3) putting the frozen giant salamander mucus into a freeze dryer, and carrying out vacuum freeze drying for 48h under the conditions that the temperature is-40 ℃ and the absolute pressure is 10Pa to obtain the dry powder of the giant salamander mucus. Crushing the dry giant salamander mucus powder by using a low-temperature ultrafine crusher, adding liquid nitrogen while crushing, keeping the temperature at low temperature, and crushing to obtain giant salamander secretion with the average particle size of 50 microns.

1g of giant salamander secretion prepared by the method is added into 20mL of vitamin C solution, wherein the concentration of the vitamin C in the vitamin C solution is 0.5 mol/L. Stirring at room temperature (20 deg.C) for 6h to obtain uniform giant salamander secretion solution.

Injecting 20mL of giant salamander secretion solution into a forming mold with the length multiplied by the width of 4cm multiplied by 4cm, then putting the forming mold into a freeze dryer, cooling to-40 ℃ at the speed of 1 ℃/min, and freezing and standing for 2h at the temperature of-40 ℃. And then placing the forming die under the condition of absolute pressure of 5Pa for 24h to obtain the membranous giant salamander secretion viscous biomembrane with uniform texture and microporous shape. The viscous biofilm of giant salamander secretions prepared is shown in fig. 2 (white blocks in glass dish).

Example 2

Giant salamander secretions were prepared as in example 1.

0.5g of giant salamander secretion is added into 20mL of hydrochloric acid solution, wherein the concentration of hydrogen chloride in the hydrochloric acid solution is 1 mol/L. Stirring at room temperature (20 deg.C) for 4 hr to obtain a uniform solution of giant salamander secretion.

5mL of giant salamander secretion solution is injected into a forming die with the length multiplied by the width of 1cm multiplied by 6cm, then the forming die is put into a freeze dryer, the temperature is reduced to minus 30 ℃ at the speed of 0.5 ℃/min, and the giant salamander secretion solution is frozen and placed for 0.5h at the temperature of minus 30 ℃. And then placing the forming die under the condition of absolute pressure of 10Pa for 12h to obtain the membranous giant salamander secretion viscous biomembrane with uniform texture and microporous shape.

Example 3

Giant salamander secretions were prepared as in example 1.

0.1g of giant salamander secretion is added into 20mL of acetic acid solution, wherein the concentration of acetic acid in the acetic acid solution is 0.5 mol/L. Stirring at 25 deg.C for 4h to obtain a uniform solution of giant salamander secretion.

Injecting 20mL of giant salamander secretion solution into a forming mold with the diameter of 0.6cm, then putting the forming mold into a freeze dryer, cooling to-40 ℃ at the speed of 1 ℃/min, and freezing and standing for 0.5h at the temperature of-40 ℃. And then placing the forming die under the condition that the absolute pressure is 8Pa for 12h to obtain the membranous giant salamander secretion viscous biomembrane with uniform texture and micropore shape.

Example 4

1g of the giant salamander secretion preserved by the method of example 1 was added to 20mL of a sulfuric acid solution, wherein the concentration of sulfuric acid in the sulfuric acid solution was 0.1 mol/L. Stirring at room temperature (20 deg.C) for 8 hr to obtain uniform giant salamander secretion solution.

Injecting 20mL of giant salamander secretion solution into a forming mold with the length multiplied by the width of 10cm multiplied by 2cm, then putting the forming mold into a freeze dryer, cooling to-40 ℃ at the speed of 2 ℃/min, and freezing and standing for 2h at the temperature of-40 ℃. And then placing the forming die under the condition of absolute pressure of 10Pa for 15h to obtain the membranous giant salamander secretion viscous biomembrane with uniform texture and microporous shape.

Example 5

0.02g of the giant salamander secretion preserved as prepared in example 1 was added to 20mL of a phosphoric acid solution containing 0.1mol/L of phosphoric acid. Stirring at room temperature (20 deg.C) for 4 hr to obtain a uniform solution of giant salamander secretion.

Injecting 20mL of giant salamander secretion solution into a forming mold with the length multiplied by the width multiplied by 2cm, then putting the forming mold into a freeze dryer, cooling to-40 ℃ at the speed of 2 ℃/min, and freezing and standing for 4h at the temperature of-40 ℃. And then placing the forming die under the condition of absolute pressure of 10Pa for 48h to obtain the membranous giant salamander secretion viscous biomembrane with uniform texture and microporous shape.

Example 6

20g of the giant salamander secretion prepared and preserved according to the method in example 1 is added into 20mL of nitric acid solution, wherein the concentration of nitric acid in the nitric acid solution is 0.5 mol/L. Stirring at room temperature (20 deg.C) for 8 hr to obtain uniform giant salamander secretion solution.

Injecting 20mL of giant salamander secretion solution into a forming mold with the length multiplied by the width of 3cm multiplied by 3cm, then putting the forming mold into a freeze dryer, cooling to-50 ℃ at the speed of 2 ℃/min, and freezing and standing for 2h at the temperature of-50 ℃. And then placing the forming die under the condition of absolute pressure of 5Pa for 24h to obtain the membranous giant salamander secretion viscous biomembrane with uniform texture and microporous shape.

Comparative example 1

Giant salamander secretions were prepared as in example 1.

1g of giant salamander secretion prepared by the method is added into 20mL of water, and stirring treatment is carried out at normal temperature (20 ℃) for 6 hours, so that the giant salamander secretion is basically insoluble in the water, and the giant salamander secretion viscous biomembrane cannot be prepared.

Comparative example 2

Giant salamander secretions were prepared as in example 1.

1g of giant salamander secretion prepared by the method is added into 20mL of acetone, and stirring treatment is carried out for 6 hours at normal temperature (20 ℃), so that the giant salamander secretion is basically insoluble in the acetone, and the giant salamander secretion viscous biomembrane cannot be prepared.

Comparative example 3

Giant salamander secretions were prepared as in example 1.

1g of giant salamander secretion prepared by the method is added into 20mL of ethanol, and stirring treatment is carried out at normal temperature (20 ℃) for 6 hours, so that the giant salamander secretion is basically insoluble in the ethanol, and the giant salamander secretion viscous biomembrane cannot be prepared.

Comparative example 4

Giant salamander secretions were prepared as in example 1.

1g of giant salamander secretion prepared by the method is added into 20mL of acetonitrile, and stirring treatment is carried out for 6h at normal temperature (20 ℃), so that the giant salamander secretion is basically insoluble in the acetonitrile, and the giant salamander secretion viscous biomembrane cannot be prepared.

Comparative example 5

Giant salamander secretions were prepared as in example 1.

1g of giant salamander secretion prepared by the method is added into 20mL of dichloromethane and stirred at normal temperature (20 ℃) for 6 hours, so that the giant salamander secretion is basically insoluble in dichloromethane, and the giant salamander secretion viscous biomembrane cannot be prepared.

Comparative example 6

Giant salamander secretions were prepared as in example 1.

1g of giant salamander secretion prepared by the method is added into 20mL of dimethyl sulfoxide, and stirring treatment is carried out at normal temperature (20 ℃) for 6 hours, so that the giant salamander secretion is basically insoluble in the dimethyl sulfoxide, and the giant salamander secretion viscous biomembrane cannot be prepared.

Comparative example 7

Giant salamander secretions were prepared as in example 1.

1g of giant salamander secretion prepared by the method is added into 20mL of trifluoroacetic acid, stirring treatment is carried out at normal temperature (20 ℃) for 6 hours, and the giant salamander secretion is partially dissolved in the trifluoroacetic acid.

20mL of the giant salamander secretion-trifluoroacetic acid solution formed by the method is injected into a forming mold with the length multiplied by the width of 4cm multiplied by 4cm according to the method of the embodiment 1, then the forming mold is put into a freeze dryer, the temperature is reduced to minus 40 ℃ at the speed of 1 ℃/min, and the giant salamander secretion-trifluoroacetic acid solution is frozen and placed for 2h at the temperature of minus 40 ℃. The forming mould is then placed under an absolute pressure of 5Pa for 24 h. As a result, it was found that the membrane-like material formed by the giant salamander secretion-trifluoroacetic acid solution was inferior in both uniformity and viscosity to the biofilm prepared in example 1.

Comparative example 8

Giant salamander secretions were prepared as in example 1.

1g of giant salamander secretion prepared by the method is added into 20mL of trifluoroethanol, stirring treatment is carried out at normal temperature (20 ℃) for 6 hours, and the giant salamander secretion is partially dissolved in trifluoroacetic acid.

20mL of the giant salamander secretion-trifluoroethanol solution formed by the method is injected into a forming mold with the length multiplied by the width of 4cm multiplied by 4cm according to the method of the embodiment 1, then the forming mold is put into a freeze dryer, the temperature is reduced to minus 40 ℃ at the speed of 1 ℃/min, and the giant salamander secretion-trifluoroethanol solution is frozen and placed for 2h at the temperature of minus 40 ℃. The forming mould is then placed under an absolute pressure of 5Pa for 24 h. As a result, it was found that the membrane-like material formed by the giant salamander secretion-trifluoroethanol solution was inferior in both uniformity and viscosity to the biofilm prepared in example 1.

Test one

At the temperature of 20 ℃, the giant salamander secretion prepared by the method of example 1 is added into 10mL of vitamin C solution, hydrochloric acid solution, acetic acid solution, sulfuric acid solution, phosphoric acid solution and nitric acid solution respectively. The concentration of vitamin C in the vitamin C solution is 0.5mol/L, the concentration of hydrogen chloride in the hydrochloric acid solution is 0.5mol/L, the concentration of acetic acid in the acetic acid solution is 0.5mol/L, the concentration of sulfuric acid in the sulfuric acid solution is 0.5mol/L, the concentration of phosphoric acid in the phosphoric acid solution is 0.5mol/L, and the concentration of nitric acid in the nitric acid solution is 0.5 mol/L. Stirring was continued during the addition until settling occurred, and the addition of giant salamander secretions was stopped and the total amount added in each solution was recorded, with the results shown in table 1.

Table 1: solubility of giant salamander secretion in each acidic solution at 20 DEG C

Solution (10mL)	The highest amount (g) of dissolved giant salamander secretion	Solubility (g/mL)
			Vitamin C solution	15.2	1.52g/mL
Hydrochloric acid solution	12.5	1.25g/mL
			Acetic acid solution	5.6	0.56g/mL
Sulfuric acid solution	10.4	0.104g/mL
			Phosphoric acid solution	4.1	0.41g/mL
Nitric acid solution	13.1	1.31g/mL

The results show that the giant salamander secretion has very good solubility in vitamin C solution, hydrochloric acid solution, acetic acid solution, sulfuric acid solution, phosphoric acid solution and nitric acid solution. Particularly, the solubility of the vitamin C solution can reach 1.52 g/mL. Therefore, the giant salamander secretion is dissolved in the acid solution to form a uniform giant salamander secretion solution, so that the giant salamander secretion solution can be used for preparing the giant salamander secretion viscous biomembrane.

And when other solvents are adopted, such as water, acetone, ethanol, acetonitrile, dichloromethane, dimethyl sulfoxide and the like in comparative examples 1-6, the giant salamander secretion is tested to be basically insoluble by the same method.

In addition, comparative examples 7 and 8 in trifluoroacetic acid and trifluoroethanol, although the giant salamander secretions were partially soluble, the finally formed film-like material was relatively poor in uniformity.

Test two

The front and back surfaces of a giant salamander secretion viscous biological membrane prepared in example 1 are respectively adhered to two PDMS membranes, then the two PDMS membranes are respectively clamped by an upper clamp and a lower clamp of a universal tensile machine, and the shear adhesion stress of the giant salamander secretion viscous biological membrane is tested by stretching the membrane by the universal tensile machine. The shear adhesion stress of the giant salamander secretion viscous biofilm prepared in examples 2 to 6 was measured by the same method, and the results are shown in table 2 below.

Table 2: shear adhesion stress of giant salamander secretion viscous biofilm

Examples	Shear adhesion stress (KPa)
		Example 1	36.1KPa
Example 2	30.1KPa
		Example 3	24.5KPa
Example 4	25.4KPa
		Example 5	20.3KPa
Example 6	34.1KPa

The data in table 2 show that the giant salamander secretion viscous biological films prepared in examples 1 to 6 have good adhesive force, and the membranous material has good toughness and strength, can be used as a hemostatic film, a tissue adhesive film or a bandage and the like to cover or wrap a wound surface, and has wide application prospects.

Test three

Hemostasis test: 10 Wistar rats with half weight of male and female and 250g +/-20 g are taken, and 2 hind legs of each rat are divided into an experimental group and a control group. Before the experiment, rats are anesthetized by intraperitoneal injection of 2% pentobarbital sodium aqueous solution at the dose of 30mg/kg, the rats are fixed in a supine position, the inner sides of the rear legs of the rats are shaved by an electric induction generator, the hair is removed by 8% sodium sulfide, and the rats are disinfected by iodophor and ethanol with the volume fraction of 75%. Then, an incision was made with a scalpel at the medial side of the hind legs of the rat, which was 30mm long and 3mm deep and from which blood flowed out. After incision, the incision parts of the experimental groups are respectively wrapped by the giant salamander secretion adhesive biological films prepared in the examples 1 to 6, and the adhesion is firm. The control group was bandaged with gauze at the incision site and hemostasis was applied by compression. The hindlegs of the rats in the experimental group were bandaged with the viscous biofilm of giant salamander secretions prepared in examples 1 to 6, and the bleeding was stopped within 1min, and the surface of the viscous biofilm of giant salamander secretions was free from blood exudation. The bleeding stops after the control group is pressed for 3min, and the blood on the surface of the gauze obviously oozes. The viscous biomembrane of the giant salamander secretion has obvious hemostatic effect.

Test four

Wound healing test: 12 BALB/c mice were selected, half female and half male, eight months old. At the back of the mouse, two parallel and neat skin incisions are drawn, and the experimental group is as follows: the giant salamander secretion adhesive biological films prepared in the examples 1 to 6 are adhered to the wound; control group: the suture treatment was interrupted with surgical sutures. After 3 days, the adherent biofilms of the experimental group were torn and the sutures of the control group were removed. The incisions of both the experimental and control groups healed, but the control group had more pronounced redness and edema at the edges of the incision, while the wounds of the experimental group healed better. The results of one example are shown in fig. 3 (the viscous biofilm used in fig. 3 is specifically the viscous biofilm of giant salamander secretions prepared in example 2).

Test five

Degradation test: 6 BALB/c mice were selected, half female and half male, eight months old. Giant salamander secretion adhesive biofilms with diameters of 0.6cm prepared in example 3 were implanted into subcutaneous sacs, respectively. Some mice were prepared at 1 and 3 weeks post-implantation in tissue sections and examined by HE staining. The results are shown in fig. 4, c1 shows the viscous biomembrane subcutaneous implantation test of giant salamander secretion, c2 shows the HE staining micrograph after 1 week of implantation, c3 shows the HE staining micrograph after 3 weeks of implantation, and c4 shows the partial degradation of the giant salamander mucous membrane after 9 weeks of implantation. As can be seen in the figure, no erythema, swelling, ulceration and bleeding or spreading was visible after the implantation of the giant salamander secretion adhesive biofilm. Panel c2 shows a tissue section 1 week after implantation with no visible swollen/congested area visible to the eye near the implant membrane. No obvious inflammatory cell infiltration phenomenon appears on the surface of the implanted membrane, and no fibroblast appears. As shown in fig. c3, after 3 weeks, there was no inflammatory cell infiltration phenomenon, while many pores were found on the implanted membrane with cell growth, indicating that the viscous biofilm of giant salamander secretions was gradually degraded in vivo and its biocompatibility with tissues was good. As shown in fig. c4, after 9 weeks, the viscous biofilm of giant salamander secretions gradually degraded and the residue remained visible, while there was still no erythema and swelling visible to the naked eye. Therefore, subcutaneous implantation experiments show that the giant salamander secretion viscous biomembrane has good histocompatibility in a mouse body, does not stimulate obvious inflammation, and can be naturally degraded.

The experimental results show that the giant salamander secretion viscous biological film prepared in the embodiments 1 to 6 has the toughness and strength of a film-shaped material and good adhesion, the original performance of the giant salamander secretion after the film is prepared is not damaged, and the advantages of good adhesion and biocompatibility of the giant salamander secretion are retained.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A preparation method of a giant salamander secretion viscous biomembrane is characterized by comprising the following steps:

dissolving giant salamander secretions in an acidic solution to obtain a giant salamander secretion solution, wherein the acidic solution is at least one selected from a vitamin C solution, a hydrochloric acid solution, a sulfuric acid solution and a nitric acid solution, the concentration of vitamin C in the vitamin C solution is 0.1-1 mol/L, the concentration of hydrogen chloride in the hydrochloric acid solution is 0.1-1 mol/L, the concentration of sulfuric acid in the sulfuric acid solution is 0.1-1 mol/L, and the concentration of nitric acid in the nitric acid solution is 0.1-1 mol/L; and

2. The preparation method of the giant salamander secretion viscous biomembrane according to claim 1, wherein the acidic solution is a vitamin C solution, and the concentration of vitamin C in the vitamin C solution is 0.5-1 mol/L; and/or the presence of a catalyst in the reaction mixture,

the acid solution is hydrochloric acid solution, and the concentration of hydrogen chloride in the hydrochloric acid solution is 0.5-1 mol/L; and/or the presence of a catalyst in the reaction mixture,

the acid solution is a sulfuric acid solution, and the concentration of sulfuric acid in the sulfuric acid solution is 0.1-0.5 mol/L; and/or the presence of a catalyst in the reaction mixture,

the acid solution is a nitric acid solution, and the concentration of nitric acid in the nitric acid solution is 0.5-1 mol/L.

3. The preparation method of the giant salamander secretion viscous biomembrane according to claim 1, wherein the giant salamander secretion solution is lyophilized into a membrane, and the step of lyophilizing into a membrane specifically comprises:

4. The method for preparing the giant salamander secretion viscous biomembrane according to claim 1, wherein the giant salamander secretion is dry powder of giant salamander mucus.

5. The method for preparing the giant salamander secretion viscous biomembrane according to claim 1, wherein the concentration of the giant salamander secretion in the giant salamander secretion solution is 0.001-1 g/mL.

6. The preparation method of the giant salamander secretion viscous biomembrane according to claim 1, characterized in that the giant salamander secretion is prepared by the following method:

scraping giant salamander mucus from the outer surface of the giant salamander;

7. The preparation method of the giant salamander secretion viscous biomembrane according to claim 1, characterized in that the particle size of the giant salamander secretion is 1-100 μm.

8. The method for preparing the giant salamander secretion viscous biomembrane according to claim 1, wherein in the step of dissolving the giant salamander secretion in the acidic solution, the dissolving operation is performed for 4 to 8 hours under stirring at a temperature of 10 to 35 ℃.

9. A giant salamander secretion viscous biomembrane, which is prepared by the preparation method of the giant salamander secretion viscous biomembrane of any one of claims 1 to 8.

10. Use of the giant salamander secretion viscous biofilm according to claim 9 in the preparation of a hemostatic membrane, a tissue adhesive membrane or a bandage.