Undenatured collagen from the giant salamander, its extraction method, and its medicinal applications in promoting wound healing.

Abstract

This invention belongs to the field of biomedical materials and discloses a non-denatured collagen from the giant salamander, its extraction method, and its pharmaceutical application in promoting wound healing. The non-denatured collagen is obtained from giant salamander skin powder using an acetic acid extraction system or a pepsin-assisted acetic acid extraction system. It maintains a triple helix structure, exhibits a positive peak near 221 nm and a negative peak near 197 nm in circular dichroism spectroscopy, and a denaturation peak at 54–59°C in DSC. This collagen significantly promotes repair in a zebrafish tail fin injury model and can be used to prepare drugs or topical repair materials that promote wound healing.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, specifically relating to a non-denatured collagen from the giant salamander, its extraction method, and its pharmaceutical application in promoting wound healing. Background Technology

[0002] Collagen is one of the main structural proteins in animal connective tissue. Due to its excellent biocompatibility, film-forming properties, water retention, and tissue repair capabilities, it has been widely used in biomedical materials, wound dressings, tissue engineering, and drug delivery systems. Among these, non-denatured collagen, which maintains its natural triple helix conformation, generally exhibits better biological activity and application value than denatured collagen. Giant salamander skin is rich in collagen resources, is naturally derived, and has the potential to be developed into a medical bioactive material. Existing extraction methods for giant salamander skin collagen mainly include acid extraction, alkali extraction, salt extraction, and enzyme-assisted extraction. However, different extraction conditions significantly affect the integrity of the collagen molecular chain, the degree of retention of the triple helix structure, thermal stability, and subsequent biological activity. In existing technologies, on the one hand, there is a lack of systematic comparison of the structural preservation of giant salamander collagen obtained from different extraction systems, making it difficult to screen for a more suitable optimal process for pharmaceutical applications. On the other hand, existing extraction techniques often focus more on yield or general physicochemical properties, lacking a technical solution that fully combines structural characterization and biological evaluation to determine whether the obtained collagen can maintain its non-denatured structure while also possessing wound-healing activity. Therefore, developing a non-denatured giant salamander collagen that can better maintain its triple helix structure, has high stability, and is suitable for pharmaceutical applications promoting wound healing, along with its extraction method, has significant research significance and application value. Summary of the Invention

[0003] To address the shortcomings mentioned in the background art, the present invention aims to provide a non-denatured collagen from the giant salamander, its extraction method, and its pharmaceutical application in promoting wound healing. Using giant salamander skin as raw material, a non-denatured collagen maintaining its triple-helix structure is obtained through an acetic acid extraction system or a pepsin-assisted acetic acid extraction system. This collagen exhibits good structural stability and wound-healing activity, and can be used to prepare drugs or topical repair materials that promote wound healing.

[0004] The objective of this invention can be achieved through the following technical solutions: A non-denatured collagen from giant salamanders, wherein the collagen is obtained by extracting the skin of giant salamanders using an acetic acid extraction system or a pepsin-assisted acetic acid extraction system, and retains a triple helix structure. Its circular dichroism chromatogram shows a positive absorption peak at 221±5 nm and a negative absorption peak at 197±5 nm. The denaturation endothermic peak temperature in differential scanning calorimetry is 54-59℃.

[0005] More preferably, the acid extraction system is an acetic acid extraction system, and the enzyme-assisted acid extraction system is a pepsin-assisted acetic acid extraction system.

[0006] More preferably, the ultraviolet absorption spectrum of the non-denatured collagen has a maximum absorption peak at 230–240 nm, and the infrared absorption spectrum has characteristic peaks of amide A band, amide B band, amide I band, amide II band and amide III band.

[0007] More preferably, in the infrared absorption spectrum, the amide A band is located at 3290–3350 cm⁻¹, the amide B band is located at 2900–2935 cm⁻¹, the amide I band is located at approximately 1650 cm⁻¹, the amide II band is located at 1530–1545 cm⁻¹, and the amide III band is located at approximately 1235 cm⁻¹.

[0008] More preferably, the non-denatured collagen is in the form of powder, solution, membrane, sponge, gel, or freeze-dried product.

[0009] A method for extracting non-denatured collagen from the giant salamander includes the following steps: S1. Pre-treat the giant salamander skin raw material and make it into giant salamander skin powder; S2. The giant salamander skin powder is mixed with an acid extraction system or an enzyme-assisted acid extraction system for extraction, and the extracted mixture is subjected to solid-liquid separation to collect the liquid phase; S3. The liquid phase is purified and subjected to one or more of the following treatments: neutralization, enzyme inactivation, concentration, dialysis, and drying, to obtain the non-denatured collagen of the giant salamander.

[0010] More preferably, in step S2, the extraction medium is an acetic acid solution with an acetic acid concentration of 0.4–0.6 mol / L, a material-to-liquid ratio of 21–23 mL / g, an extraction temperature of 18–22℃, and an extraction time of 28–32 h.

[0011] More preferably, the extraction medium in step S2 is a system composed of acetic acid solution and pepsin, wherein the concentration of acetic acid is 0.4-0.6 mol / L, the material-to-liquid ratio is 20-22 mL / g, the amount of pepsin added is 2.0%-3.0% of the mass of the mixed system, the enzymatic hydrolysis temperature is 18-22℃, and the enzymatic hydrolysis time is 18-22 h; in step S3, the pH is adjusted to 6.8-7.2 to inactivate the enzyme.

[0012] More preferably, the solid-liquid separation is performed by centrifugation under the following conditions: 4°C, 10000 r / min, and 15 min. The purification process includes one or more of the following: adjusting the pH to neutral to inactivate enzymes, dialysis, concentration, and freeze drying.

[0013] Application of undenatured collagen from the giant salamander in the preparation of drugs to promote wound healing.

[0014] The beneficial effects of this invention are: This invention uses giant salamander skin as raw material and compares and screens various extraction systems, including acid extraction, alkali extraction, salt extraction, and enzyme-assisted extraction, to obtain non-denatured collagen from giant salamanders with good structural preservation and high stability. The obtained collagen exhibits characteristic absorption of collagen in both UV and IR detection. Acid-extracted and enzyme-extracted samples show a positive absorption peak at approximately 221 nm and a negative absorption peak at approximately 197 nm in circular dichroism spectroscopy. The acetic acid-extracted sample shows a denaturation endothermic peak reaching 58.38℃ in differential scanning calorimetry, demonstrating superior thermal stability compared to other processed samples. In contrast, the alkali-extracted sample shows some degree of structural breakage. Therefore, the optimized extraction path of this invention has a significant advantage in maintaining the natural triple helix conformation of collagen. Furthermore, the collagen showed no significant toxicity at a concentration of 200 µg / ml in a zebrafish tail fin injury model and promoted regeneration and repair after tail fin injury. This gives the product both good biocompatibility and wound healing potential, making it suitable for further development into wound repair drugs or topical medical materials. Attached Figure Description

[0015] The invention will now be further described with reference to the accompanying drawings.

[0016] Figure 1 Infrared spectra of collagen extracted in Examples 1-2 and Comparative Examples 1-4; Figure 2 The images show the UV absorption spectra of collagen extracted in Examples 1-2 and Comparative Examples 1-4. Figure 3 The following are circular dichroism chromatograms of collagen extracted in Examples 1-2 and Comparative Examples 1-4; Figure 4 Differential scanning calorimetry (DSC) graphs of collagen extracted in Examples 1-2 and Comparative Examples 1-4; Figure 5 Scanning electron micrographs of collagen extracted in Examples 1-2 and Comparative Examples 1-4 are shown. A shows collagen extracted with pepsin; B shows collagen extracted with sodium chloride; C shows collagen extracted with hydrochloric acid; D shows collagen extracted with acetic acid; E shows collagen extracted with potassium hydroxide; and F shows collagen extracted with sodium hydroxide. Figure 6 The images show the effects of collagen extracted by six different methods on the repair of zebrafish tail fin damage. A is the blank control group; B is the collagen extraction group using potassium hydroxide; C is the collagen extraction group using sodium hydroxide; D is the collagen extraction group using acetic acid; E is the collagen extraction group using hydrochloric acid; F is the collagen extraction group using pepsin; and G is the collagen extraction group using sodium chloride. Figure 7 The repair rate of zebrafish tail fins after 24 hours of treatment with non-denatured collagen from different extraction processes; Figure 8 To determine the repair rate of zebrafish tail fins after 48 hours of treatment with non-denatured collagen from different extraction processes; Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] In this invention, the undenatured collagen from the giant salamander can be prepared using an acid extraction system or an enzyme-assisted acid extraction system. Preferably, in step S2, when the extraction medium is an acetic acid solution, the acetic acid concentration is 0.4–0.6 mol / L, the solid-liquid ratio is 21–23 mL / g, the extraction temperature is 18–22℃, and the extraction time is 28–32 h. Further, in step S2, when the extraction medium is a system composed of an acetic acid solution and pepsin, the acetic acid concentration is 0.4–0.6 mol / L, the solid-liquid ratio is 20–22 mL / g, the amount of pepsin added is 2.0%–3.0% of the mass of the mixed system, the enzymatic hydrolysis temperature is 18–22℃, and the enzymatic hydrolysis time is 18–22 h; in step S3, the pH is adjusted to 6.8–7.2 to inactivate the enzyme.

[0019] The present invention will be further described below with reference to specific embodiments.

[0020] Example 1: Preparation of non-denatured collagen from giant salamanders by acetic acid extraction Take 1.0 g of giant salamander skin powder, place it in a container, add 0.5 mol / L acetic acid solution as the extraction medium, control the material-to-liquid ratio at 22 mL / g, and extract by shaking at 20℃ for 30 h. After extraction, centrifuge the resulting mixture at 10000 r / min for 15 min at 4℃, and collect the supernatant. The supernatant obtained is the crude extract of giant salamander skin collagen.

[0021] The crude extract was salted out by adding sodium chloride at 4°C, causing collagen to precipitate from the liquid phase. After standing, the precipitate was collected by centrifugation. The precipitate was redissolved in 0.01 mol / L acetic acid solution and purified by salting out again. After centrifugation, the precipitate was redissolved in 0.01 mol / L acetic acid solution to obtain a purified collagen solution. The purified collagen solution was placed in a dialysis bag and dialyzed with acetic acid solution and deionized water sequentially at 4°C. After dialysis, the collagen solution was purified by low-temperature membrane separation to remove small molecule impurities and residual salts. The obtained collagen solution was then concentrated at low temperature, pre-frozen, and freeze-dried to obtain the undenatured collagen product from the giant salamander.

[0022] Example 2: Preparation of non-denatured collagen from giant salamander by pepsin-assisted acetic acid extraction Take 1.0 g of giant salamander skin powder and place it in an extraction container. Add 0.5 mol / L acetic acid solution as the extraction medium, controlling the material-to-liquid ratio at 21 mL / g. Add 2.5% (w / w) of pepsin to the mixture and hydrolyze at 20℃ for 20 h with shaking. After hydrolysis, adjust the pH of the system to neutral to terminate the enzyme reaction. Then, centrifuge at 10000 r / min for 15 min at 4℃ and collect the supernatant to obtain crude collagen extract.

[0023] The crude extract was salted out by adding sodium chloride at 4°C, causing collagen to precipitate from the liquid phase. After standing, the precipitate was collected by centrifugation. The precipitate was redissolved in 0.01 mol / L acetic acid solution and purified by salting out again. After centrifugation, the precipitate was redissolved in 0.01 mol / L acetic acid solution to obtain a purified collagen solution. The purified collagen solution was placed in a dialysis bag and dialyzed with acetic acid solution and deionized water sequentially at 4°C. After dialysis, the collagen solution was purified by low-temperature membrane separation to remove small molecule impurities and residual salts. The obtained collagen solution was then concentrated at low temperature, pre-frozen, and freeze-dried to obtain the undenatured collagen product from the giant salamander.

[0024] Comparative Example 1: Preparation of non-denatured collagen from giant salamanders using sodium hydroxide extraction Take 1.0 g of giant salamander skin powder, place it in an extraction container, add 0.1 mol / L sodium hydroxide solution as the extraction solvent, mix well at a material-to-liquid ratio of 20 mL / g, and extract by shaking at 20℃ for 30 h. After extraction, centrifuge the resulting mixture at 10000 r / min for 15 min at 4℃, collect the supernatant after centrifugation, and obtain crude collagen extract.

[0025] The crude extract was salted out by adding sodium chloride at 4°C, causing collagen to precipitate from the liquid phase. After standing, the precipitate was collected by centrifugation. The precipitate was redissolved in 0.01 mol / L acetic acid solution and purified by salting out again. After centrifugation, the precipitate was redissolved in 0.01 mol / L acetic acid solution to obtain a purified collagen solution. The purified collagen solution was placed in a dialysis bag and dialyzed with acetic acid solution and deionized water sequentially at 4°C. After dialysis, the collagen solution was purified by low-temperature membrane separation to remove small molecule impurities and residual salts. The obtained collagen solution was then concentrated at low temperature, pre-frozen, and freeze-dried to obtain the alkaline-extracted collagen product.

[0026] Comparative Example 2: Preparation of non-denatured collagen from giant salamanders using potassium hydroxide extraction Take 1.0 g of giant salamander skin powder, place it in an extraction container, add 0.1 mol / L potassium hydroxide solution as the extraction solvent, mix well at a material-to-liquid ratio of 23 mL / g, and extract by shaking at 20℃ for 30 h. After extraction, centrifuge the resulting mixture at 10000 r / min for 15 min at 4℃, collect the supernatant after centrifugation, and obtain crude collagen extract.

[0027] The crude extract was subjected to precipitation treatment with sodium chloride at 4°C, causing collagen to precipitate. The precipitate was collected after centrifugation. The precipitate was then redissolved in acetic acid solution to obtain a reconstituted solution. Subsequently, the reconstituted solution was desalted and purified by dialysis to remove inorganic salts, small molecule impurities, and residual extraction medium. After dialysis, the resulting collagen solution was concentrated, pre-frozen at low temperature, and then freeze-dried to finally obtain undenatured collagen from the giant salamander.

[0028] Comparative Example 3: Preparation of non-denatured collagen from giant salamanders using hydrochloric acid extraction Take 1.0 g of giant salamander skin powder, place it in an extraction container, add 0.4 mol / L hydrochloric acid solution as the extraction solvent, mix well at a material-to-liquid ratio of 25 mL / g, and extract by shaking at 20℃ for 30 h. After extraction, centrifuge the resulting mixture at 10000 r / min for 15 min at 4℃, and collect the supernatant to obtain crude collagen extract.

[0029] The crude extract was subjected to precipitation treatment with sodium chloride at 4°C, causing collagen to precipitate. After centrifugation, the precipitate was collected and reconstituted with acetic acid solution to obtain a reconstituted solution. The reconstituted solution was then dialyzed with acetic acid solution and deionized water to remove hydrochloric acid, sodium chloride, and small molecule impurities, yielding a purified collagen solution. The purified collagen solution was then concentrated, pre-frozen, and freeze-dried to obtain non-denatured collagen from the giant salamander.

[0030] Comparative Example 4: Preparation of non-denatured collagen from giant salamanders using sodium chloride extraction Take 1.0 g of giant salamander skin powder and place it in an extraction container. Add 0.5 mol / L sodium chloride solution as the extraction medium and mix thoroughly at a material-to-liquid ratio of 23 mL / g. Extract by shaking at 20℃ for 30 h. After extraction, centrifuge the resulting mixture at 10000 r / min for 15 min at 4℃ and collect the supernatant to obtain crude collagen extract.

[0031] The crude extract was subjected to precipitation treatment with sodium chloride at 4°C, causing collagen to precipitate from the liquid phase. After standing, the precipitate was collected by centrifugation. The precipitate was then redissolved in acetic acid solution to obtain a reconstituted solution. Subsequently, the reconstituted solution was placed in a dialysis bag and dialyzed sequentially with acetic acid solution and deionized water to remove sodium chloride and small molecule impurities, yielding a purified collagen solution. The purified collagen solution was further concentrated, pre-frozen at low temperature, and then freeze-dried to finally obtain non-denatured collagen from the giant salamander.

[0032] Performance testing 1. Characterization and analysis of the structure of undenatured collagen from the Chinese giant salamander (1) Infrared spectroscopy test method: Take collagen samples obtained from each extraction process, dry them, mix them with potassium bromide and press them into tablets. Use a Fourier transform infrared spectrometer to scan and record the infrared absorption spectrum of the sample in the range of 4000 to 400 cm-1. Analyze the characteristic peaks of amide A band, amide B band, amide I band, amide II band and amide III band.

[0033] (2) Ultraviolet absorption spectroscopy test method: Collagen samples obtained from each extraction process were prepared into solutions, and the corresponding solvents were used as blank controls. Ultraviolet-visible spectrophotometers were used to scan in the wavelength range of 190 to 400 nm, and the ultraviolet absorption spectra and the positions of the maximum absorption peaks of each sample were recorded for comparison of the characteristic absorption peaks of different samples.

[0034] (3) Circular dichroism (CD) test: Collagen samples obtained from each extraction process were prepared into solutions of appropriate concentrations and scanned in the far ultraviolet region using a circular dichroism chromatograph. The characteristic absorption peaks of the samples at approximately 221 nm and 197 nm were recorded to compare the retention of the triple helix structure of the samples obtained from different extraction processes.

[0035] (4) Differential scanning calorimetry (DSC): Collagen samples obtained from each extraction process are placed in the sample cell of the differential scanning calorimeter after appropriate pretreatment. The test is performed under the set heating program, and the endothermic peak and strain temperature of the sample are recorded.

[0036] (5) Scanning electron microscopy (SEM): Collagen samples obtained from each extraction process were dried, fixed on the sample stage, and subjected to conductive treatment. The microstructure of the sample surface was observed using a scanning electron microscope, and the fiber entanglement, lamellar stacking, loose porous structure and rupture of different samples were recorded.

[0037] Results Analysis: According to the appendix Figure 1 The infrared spectral results show that all samples obtained from the six processes exhibited typical absorption bands of collagen, including amide A, amide B, amide I, amide II, and amide III. The amide A band for collagen extracted by the salt method appeared around 3350 cm⁻¹, while that extracted by the enzymatic and hydrochloric acid methods appeared around 3320 cm⁻¹. The amide A band for collagen extracted by the acetic acid method appeared around 3300 cm⁻¹, and that extracted by the alkaline method appeared around 3290 cm⁻¹. Amide B bands were all around 2930 cm⁻¹, amide I bands were all around 1650 cm⁻¹, amide II bands were between 1530 and 1545 cm⁻¹, and amide III bands were around 1235 cm⁻¹. The overall spectral patterns were quite similar, indicating that the samples obtained from different extraction systems all possessed the basic structural characteristics of collagen, and that all six extraction methods maintained the integrity of the triple helix structure of collagen.

[0038] According to the appendix Figure 2 The ultraviolet absorption spectra results show that the salt-extracted sample has the maximum absorption peak at 231.61 nm, while the other five samples have the maximum absorption peak at 237.21 nm. All of them are located near the characteristic absorption region of collagen, indicating that the samples obtained by different processes all have the typical ultraviolet absorption characteristics of collagen.

[0039] According to the appendix Figure 3The CD chromatograms show that all six samples obtained by different processes exhibit the basic characteristics of collagen CD spectra in the far-ultraviolet region: a negative peak around 198–202 nm and a weak positive peak or a shoulder peak close to zero around 220–223 nm. This indicates that all samples possess certain collagen conformational characteristics, but the degree of preservation of the triple helix structure varies significantly among different processes. The positive peaks around 220 nm are clearer in the pepsin-extracted, hydrochloric acid-extracted, and acetic acid-extracted samples. The chromatograms of acetic acid-extracted and pepsin-extracted samples are more typical, indicating better preservation of their triple helix structure. The hydrochloric acid-extracted sample also shows a relatively obvious positive peak, but its negative peak has a larger absolute value, suggesting a difference in conformational state compared to the acetic acid-extracted and pepsin-extracted samples. The sodium chloride-extracted sample only shows a weak positive peak or a shoulder peak around 220 nm, indicating that it still retains some triple helix structure, but its integrity is weaker than that of the acid-extracted and enzyme-extracted samples. The potassium hydroxide and sodium hydroxide extracted samples showed no obvious positive peaks near 220 nm, and only gradually approached the baseline as the wavelength increased, indicating that the triple helix structure of collagen was poorly preserved under alkaline extraction conditions, and there was a certain degree of conformational damage.

[0040] From the appendix Figure 4 The results show that the acetic acid-extracted sample exhibits the highest denaturation peak temperature at 58.38℃, indicating the best thermal stability. Collagen extracted with hydrochloric acid, potassium hydroxide, and enzymes shows absorption peaks at 54.93℃, 54.47℃, and 54.42℃, respectively, placing these groups in the middle range and indicating relatively good structural stability, though still lower than that of the acetic acid-extracted sample. Sodium hydroxide-extracted collagen shows an absorption peak at 49.39℃, while sodium chloride-extracted collagen shows an absorption peak at 48.96℃; both have the lowest peak temperatures, indicating relatively poor thermal stability. These findings suggest that when using sodium chloride and sodium hydroxide to extract collagen, the collagen structure is damaged and denatures, while acetic acid-extracted collagen exhibits the best thermal stability.

[0041] According to the appendix Figure 5 It can be seen that the three types of collagen extracted by enzyme, salt, and hydrochloric acid are composed of tightly intertwined filaments and thin sheets, exhibiting a loose and porous characteristic; the collagen extracted by acetic acid has a smooth surface and is composed of multiple layers of thin sheet-like structures stacked together; while the collagen extracted by alkali shows signs of rupture.

[0042] 2. Effect of undenatured collagen from giant salamander skin on zebrafish survival rate Healthy zebrafish at 4 hpf post-fertilization were placed in 12-well plates. Different collagen extraction processes were used to treat zebrafish with collagen extracted using fish tank water at a concentration of 200 µg / ml. A blank control group (fish tank water) was also included. Zebrafish were incubated in 2ml solutions at 28.5 °C in a biochemical incubator until 48 hpf. The number of surviving zebrafish was observed and the survival rate was calculated. The results are shown in Table 1 below. All six extraction processes at concentrations up to 200 µg / ml showed no toxicity to zebrafish.

[0043] Table 1. Effect of undenatured collagen from giant salamander skin on zebrafish survival rate.

[0044] 3. Evaluation of the repair effect of non-denatured collagen from giant salamander skin on zebrafish tail fin damage. Healthy zebrafish at 4 hpf post-fertilization were selected and quickly placed on agarose gelatin plates. The caudal fin was removed under a microscope, and the condition of the caudal fin was recorded by photograph. The zebrafish with removed caudal fins were randomly divided into a control group (caudal fin removed without drug treatment) and a collagen-fed group (raised in water containing 100 µg / ml collagen), with 12 fish in each group.

[0045] Zebrafish were photographed and observed 24 h and 48 h after caudal fin injury treatment, and the area of ​​the regenerated caudal fin was measured using ImageJ software. Caudal fin repair rate = (initial caudal fin area - current caudal fin area) / initial caudal fin area × 100%.

[0046] From the appendix Figure 6 , Figure 7 , Figure 8 It can be seen that the non-denatured collagen from giant salamander skin has a clear promoting effect on the repair of zebrafish tail fin damage. The repair performance of each treatment group at 24 h and 48 h was generally better than that of the control group, indicating that the collagen obtained by this invention has the ability to promote the regeneration and repair of damaged tissue. Further comparison shows that the pepsin-assisted extraction group and the acetic acid extraction group showed better repair effects at both observation points, with a more significant advantage in the early repair stage, indicating that using a milder acid extraction system or an enzyme-assisted acid extraction system is more conducive to obtaining collagen with strong repair-promoting activity. The hydrochloric acid extraction group and the alkali extraction group also showed some promoting effect, but the overall effect was lower than that of the extraction process in the example; although the sodium chloride extraction group also had a repair-promoting effect, its overall performance was relatively weak. With the extension of the treatment time, the repair effect of each treatment group was further enhanced, indicating that the collagen obtained by this invention can not only play a promoting role in the early stage of injury, but also has the ability to continuously promote tail fin regeneration.

[0047] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0048] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A non-denatured collagen from the giant salamander, characterized in that, The collagen is obtained from giant salamander skin through an acetic acid extraction system or a pepsin-assisted acetic acid extraction system, and maintains a triple helix structure. Its circular dichroism chromatogram shows a positive absorption peak at 221±5 nm and a negative absorption peak at 197±5 nm. The denaturation endothermic peak temperature in differential scanning calorimetry is 54-59℃.

2. The undenatured collagen from the giant salamander according to claim 1, characterized in that, The acid extraction system is an acetic acid extraction system, and the enzyme-assisted acid extraction system is a pepsin-assisted acetic acid extraction system.

3. The undenatured collagen from the giant salamander according to claim 1 or 2, characterized in that, The ultraviolet absorption spectrum of the non-denatured collagen has a maximum absorption peak at 230–240 nm, and the infrared absorption spectrum has characteristic peaks of amide A, amide B, amide I, amide II, and amide III bands.

4. The undenatured collagen from the giant salamander according to claim 3, characterized in that, In the infrared absorption spectrum, the amide A band is located at 3290–3350 cm⁻¹, the amide B band is located at 2900–2935 cm⁻¹, the amide I band is located at approximately 1650 cm⁻¹, the amide II band is located at 1530–1545 cm⁻¹, and the amide III band is located at approximately 1235 cm⁻¹.

5. The undenatured collagen from the giant salamander according to any one of claims 1-4, characterized in that, The non-denatured collagen is in the form of powder, solution, membrane, sponge, gel, or freeze-dried product.

6. A method for extracting non-denatured collagen from the giant salamander according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Pre-treat the giant salamander skin raw material and make it into giant salamander skin powder; S2. The giant salamander skin powder is mixed with an acid extraction system or an enzyme-assisted acid extraction system for extraction, and the extracted mixture is subjected to solid-liquid separation to collect the liquid phase; S3. The liquid phase is purified and subjected to one or more of the following treatments: neutralization, enzyme inactivation, concentration, dialysis, and drying, to obtain the non-denatured collagen of the giant salamander.

7. The extraction method according to claim 6, characterized in that, In step S2, the extraction medium is an acetic acid solution with an acetic acid concentration of 0.4–0.6 mol / L, a material-to-liquid ratio of 21–23 mL / g, an extraction temperature of 18–22℃, and an extraction time of 28–32 h.

8. The extraction method according to claim 6, characterized in that, The extraction medium in step S2 is a system composed of acetic acid solution and pepsin, wherein the concentration of acetic acid is 0.4-0.6 mol / L, the material-to-liquid ratio is 20-22 mL / g, the amount of pepsin added is 2.0%-3.0% of the mass of the mixed system, the enzymatic hydrolysis temperature is 18-22℃, and the enzymatic hydrolysis time is 18-22 h; in step S3, the pH is adjusted to 6.8-7.2 to inactivate the enzyme.

9. The extraction method according to any one of claims 6-8, characterized in that, The solid-liquid separation is performed by centrifugation at 4°C, 10,000 r / min, and 15 min. The purification process includes one or more of the following: adjusting the pH to neutral to inactivate enzymes, dialysis, concentration, and freeze drying.

10. The use of the undenatured collagen from the giant salamander according to any one of claims 1-5 in the preparation of a medicament for promoting wound healing.

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