CN-115634279-B - Deer blood peptide hydrogel and application thereof in preparation of diabetes skin injury drugs

CN115634279BCN 115634279 BCN115634279 BCN 115634279BCN-115634279-B

Abstract

The invention relates to a new application of deer blood peptide and a preparation method of deer blood peptide hydrogel, and the new application is the application of deer blood peptide in medicines or cosmetics for promoting diabetic skin repair. The preparation method comprises (1) performing enzymolysis, preparing deer blood solution, regulating pH, preheating, adding pepsin, digesting, regulating pH with NaOH, adding pancreatin, further reacting, and heating to deactivate enzyme after digestion. And (5) centrifuging to obtain an enzymolysis liquid supernatant. The preparation method of the deer blood peptide hydrogel comprises the steps of (1) preparing deer blood peptide solution, carrying out molecular interception in sequence, carrying out fractionation treatment to obtain corresponding components, carrying out desalination treatment, and carrying out freeze-drying to obtain deer blood peptide of different components, (3) adding chitosan into glacial acetic acid solution for full dissolution, adding beta-GP solution to obtain chitosan/beta-GP solution, adding sodium alginate powder to dissolve, then dripping CaCl 2 solution to obtain blank hydrogel, and then adding the deer blood peptide to obtain the deer blood peptide hydrogel. The discovery of the new application provides a new method and a beneficial basis for the treatment of the chronic skin infection diseases of diabetes.

Inventors

DING CHUANBO
HAO MINGQIAN
LIU XINGLONG
ZHAO TING
LIU WENCONG
ZHENG YINAN
ZHANG YING
MA LINA

Assignees

吉林农业科技学院

Dates

Publication Date: 20260512
Application Date: 20220414

Claims (4)

1. The SIRT1/NF- κB signal path activator is characterized by comprising a hydrogel loaded with pilose antler blood peptide, and the preparation method of the hydrogel loaded with pilose antler blood peptide comprises the following steps: (1) Preparing deer antler blood peptide by adopting an in-vitro gastrointestinal digestion of deer antler blood powder in a two-step enzymolysis method, preparing 20mg/ml deer antler blood solution, regulating the pH value to 1.5-2.5,37 ℃ by using 1-2M HCl, preheating for 10-30min, adding pepsin according to an enzyme bottom ratio of 1:10-50, digesting 2-4 h of the solution in a constant-temperature oscillator at a speed of 37 ℃ of 120 rpm, regulating the pH value to 7.5-8.0 by using 1-2M NaOH, adding pancreatin, carrying out enzyme/substrate=1:15-25, carrying out further reaction for 4h, simulating intestinal digestion, maintaining the pH stability by using 1-2M HCl or 1-2M NaOH in the enzymolysis process, inactivating enzyme by heating for 15-30min in a boiling water bath, cooling the reactant to room temperature, centrifuging for 30min at a speed of 8000 rpm to obtain deer antler blood peptide, and then carrying out double-20 ℃ slow filtration on the supernatant for 2 times, and freeze-drying and preserving for standby; (2) Separating, freeze-drying, namely preparing 50 mg/mL of pilose antler blood peptide solution, sequentially carrying out grading treatment by ultrafiltration membranes with molecular cutoff of 10 kDa and 3 kDa to obtain components F1, F2 and F3, respectively desalting 3 components by nanofiltration membranes of 150 Da, freeze-drying and storing at-20 ℃ for later use; (3) The preparation of the hairy antler blood peptide hydrogel comprises the steps of adding 200mg of chitosan into 0.1M of 10mL of glacial acetic acid solution for full dissolution, dropwise adding 2.5mL of 55% beta-GP solution under the condition of ice water bath stirring to obtain chitosan/beta-GP solution, adding 100 mg sodium alginate powder into the prepared solution for continuous stirring to dissolve, then dripping 0.2 mL of 0.5% CaCl 2 solution, continuously stirring to obtain blank hydrogel sol, and then adding component F3 powder in the obtained hairy antler blood peptide into a blank hydrogel system for uniform mixing to obtain the hairy antler blood peptide hydrogel, wherein the concentration of the component F3 powder is 0.1%, and the method is CAVBPH for short.
2. A medicament for promoting the expression of growth factors in diabetic damaged tissues, which is characterized by comprising a hydrogel loaded with pilose antler blood peptide, wherein the preparation method of the hydrogel loaded with pilose antler blood peptide comprises the following steps: (1) Preparing deer antler blood peptide by adopting an in-vitro gastrointestinal digestion of deer antler blood powder in a two-step enzymolysis method, preparing 20mg/ml deer antler blood solution, regulating the pH value to 1.5-2.5,37 ℃ by using 1-2M HCl, preheating for 10-30min, adding pepsin according to an enzyme bottom ratio of 1:10-50, digesting 2-4 h of the solution in a constant-temperature oscillator at a speed of 37 ℃ of 120 rpm, regulating the pH value to 7.5-8.0 by using 1-2M NaOH, adding pancreatin, carrying out enzyme/substrate=1:15-25, carrying out further reaction for 4h, simulating intestinal digestion, maintaining the pH stability by using 1-2M HCl or 1-2M NaOH in the enzymolysis process, inactivating enzyme by heating for 15-30min in a boiling water bath, cooling the reactant to room temperature, centrifuging for 30min at a speed of 8000 rpm to obtain deer antler blood peptide, and then carrying out double-20 ℃ slow filtration on the supernatant for 2 times, and freeze-drying and preserving for standby; (2) Separating, freeze-drying, namely preparing 50 mg/mL of pilose antler blood peptide solution, sequentially carrying out grading treatment by ultrafiltration membranes with molecular cutoff of 10 kDa and 3 kDa to obtain components F1, F2 and F3, respectively desalting 3 components by nanofiltration membranes of 150 Da, freeze-drying and storing at-20 ℃ for later use; (3) The preparation of the hairy antler blood peptide hydrogel comprises the steps of adding 200mg of chitosan into 0.1M of 10mL of glacial acetic acid solution for full dissolution, dropwise adding 2.5mL of 55% beta-GP solution under the condition of ice water bath stirring to obtain chitosan/beta-GP solution, adding 100 mg sodium alginate powder into the prepared solution for continuous stirring to dissolve, then dripping 0.2 mL of 0.5% CaCl 2 solution, continuously stirring to obtain blank hydrogel sol, and then adding component F3 powder in the obtained hairy antler blood peptide into a blank hydrogel system for uniform mixing to obtain the hairy antler blood peptide hydrogel, wherein the concentration of the component F3 powder is 0.1%, and the method is CAVBPH for short.
3.A preparation method of a pilose antler blood peptide-loaded hydrogel is characterized by comprising the following steps: (1) Preparing deer antler blood peptide by adopting an in-vitro gastrointestinal digestion of deer antler blood powder in a two-step enzymolysis method, preparing 20mg/ml deer antler blood solution, regulating the pH value to 1.5-2.5,37 ℃ by using 1-2M HCl, preheating for 10-30min, adding pepsin according to an enzyme bottom ratio of 1:10-50, digesting 2-4 h of the solution in a constant-temperature oscillator at a speed of 37 ℃ of 120 rpm, regulating the pH value to 7.5-8.0 by using 1-2M NaOH, adding pancreatin, carrying out enzyme/substrate=1:15-25, carrying out further reaction for 4h, simulating intestinal digestion, maintaining the pH stability by using 1-2M HCl or 1-2M NaOH in the enzymolysis process, inactivating enzyme by heating for 15-30min in a boiling water bath, cooling the reactant to room temperature, centrifuging for 30min at a speed of 8000 rpm to obtain deer antler blood peptide, and then carrying out double-20 ℃ slow filtration on the supernatant for 2 times, and freeze-drying and preserving for standby; (2) Separating, freeze-drying, namely preparing 50 mg/mL of pilose antler blood peptide solution, sequentially carrying out grading treatment by ultrafiltration membranes with molecular cutoff of 10 kDa and 3 kDa to obtain components F1, F2 and F3, respectively desalting 3 components by nanofiltration membranes of 150 Da, freeze-drying and storing at-20 ℃ for later use; (3) The preparation of the hairy antler blood peptide hydrogel comprises the steps of adding 200mg of chitosan into 0.1M of 10mL of glacial acetic acid solution for full dissolution, dropwise adding 2.5mL of 55% beta-GP solution under the condition of ice water bath stirring to obtain chitosan/beta-GP solution, adding 100 mg sodium alginate powder into the prepared solution for continuous stirring to dissolve, then dripping 0.2 mL of 0.5% CaCl 2 solution, continuously stirring to obtain blank hydrogel sol, and then adding component F3 powder in the obtained hairy antler blood peptide into a blank hydrogel system for uniform mixing to obtain the hairy antler blood peptide hydrogel, wherein the concentration of the component F3 powder is 0.1%, and the method is CAVBPH for short.
4. A method of preparing a loaded pilose antler blood peptide hydrogel according to claim 3, wherein the blank hydrogel gels at 37 ℃ for 5min and the pilose antler blood peptide hydrogel gels at 2 min.

Description

Deer blood peptide hydrogel and application thereof in preparation of diabetes skin injury drugs Technical Field The invention belongs to the field of medicines, and relates to a preparation method of deer blood peptide hydrogel and a novel medical application thereof. Background The deer antler blood (VB) is blood obtained by cutting red deer or spotted deer antler, has long medicinal history, is recorded in multiple herbal medicines such as Shennong's herbal medicine channels, ming's medical miscellaneous records, ben Cao gang mu and Shennong's herbal medicine channel Bai Zhong Ji, has the effects of tonifying kidney and strengthening yang, strengthening tendons and bones, generating sperm and benefiting marrow, and is known as a' Renzhen 'product'. VB contains a large amount of protein, polypeptide and amino acid components, and is similar to the pharmacological activity of pilose antler in traditional Chinese medicine, so that the compound can resist fatigue, resist oxidation, improve immunity, resist aging, reduce blood pressure, resist tumors and relieve osteoporosis. At present, polypeptide components including deer antler peptide show excellent activity in terms of antioxidation and anti-inflammation, but deer blood peptide (VBPs) is reported in the above fields less. Skin is the largest, most exposed, most sensitive and most vulnerable tissue of the human body and plays a vital role in various processes of preventing dehydration, protecting against harmful substances and pathogens, initiating vitamin D synthesis, excretion and thermoregulation. Skin wounds have become an important medical problem to be solved because of the variety of etiologies. In modern skin dressing, hydrogel becomes an ideal candidate product of wound dressing due to its good hydrophilicity, structure similar to extracellular matrix (ECM), good biodegradability, biocompatibility, adhesiveness, air permeability and other characteristics. The research shows that the addition of bioactive peptide to hydrogel can raise the skin repairing capacity of hydrogel in several aspects of resisting inflammation, inhibiting bacteria, promoting cell proliferation, inducing angiogenesis, etc. Currently, diabetic wound healing and angiogenesis remain a major challenge in clinical medicine, and improper care can be extremely prone to gangrene, amputation and even death of the patient. Traditional dressings, such as gauze, may adhere to new granulation tissue, cause pain and affect tissue integrity when removed, and do not possess bacteriostatic, antioxidant, or other active functions. In modern wound dressings including films, nanofibers, hydrogels, and sponges, hydrogels have become ideal candidates for wound dressings because of their three-dimensional network structure, good biodegradability, biocompatibility, adhesion, and breathability, and the maintenance of a moist environment required for cell migration. The invention aims to disclose CAVBPH the regulation and control effect on chronic skin wounds and skin flora related to diabetes. The invention relates to a method for preparing deer blood peptide-loaded hydrogel, which comprises the steps of cross-linking deer blood peptide with hydrogel to obtain deer blood peptide-loaded hydrogel, and determining that the deer blood peptide can regulate and control the structure of skin flora and improve the richness, diversity and uniformity of injured skin flora through the research on regulating and controlling the skin wound flora by using the deer blood peptide hydrogel. The deer blood peptide is used for preparing medicines and/or cosmetics for regulating and controlling the structure of the flora of the damaged skin and improving the richness and diversity of the flora of the damaged skin. The deer blood peptide is used for preparing medicines and/or cosmetics for regulating and controlling the structure of the flora of the damaged skin and improving the uniformity of the flora of the damaged skin. As a preferred aspect of the present invention, when deer blood peptide is used for regulating the structure of skin flora, deer blood peptide is loaded in hydrogel and then coated on skin by coating. In addition, the invention researches on the deer blood peptide hydrogel for promoting the repair process of diabetic wound skin by regulating SIRT1/NF- κB signal pathway, and determines that the deer blood peptide hydrogel is used for promoting wound skin repair by activating SIRT1/NF- κB signal pathway, thus, a second object of the invention is to provide an SIRT1/NF- κB signal pathway activator, which comprises a deer blood peptide-loaded hydrogel. The invention researches the influence of VEGF, HIF-a, PCNA, CD and CD68 in diabetic wound tissues through deer blood peptide hydrogel, and determines that deer blood peptide can promote the expression of growth factors in diabetic wound tissues to accelerate wound repair, so that the third object of the invention is to provide a medicament for promoting