Application of cornu cervi degelatinatum extract in strengthening skin barrier

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of cornu cervi degelatinatum extract in strengthening a skin barrier. The cornu cervi degelatinatum extract disclosed by the invention can promote proliferation of HacaT cells and expression of tight junction protein Claudin-1, also has better capabilities of removing hydroxyl free radicals, DPPH free radicals and superoxide anions and an effect of promoting wound healing, and can protect a skin mechanical barrier and promote barrier injury repair. The cornu cervi degelatinatum extract has a good application prospect in preparation of drugs for promoting wound healing and products related to strengthening and toughening skin barriers, and resource development and utilization of cornu cervi degelatinatum are enriched.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to the application of antler frost extract in strengthening the skin barrier. Background Art

[0002] Deer antler cream (Cervi Cornu Degelatinatum) is the bone residue left after antler glue is made from red deer or sika deer antlers. It is salty, astringent, and warm in nature, and enters the liver and kidney meridians. It has the effects of warming the kidneys and supporting yang, promoting essence and blood, nourishing the marrow and strengthening bones, and astringing and stopping bleeding. Deer antler cream generally contains 80-90% calcium hydroxyphosphate and very small amounts of amino acids, polysaccharides, lipids, and other ingredients. Because its main components are minerals such as hydroxyapatite and calcium carbonate, its development in the field of comprehensive health products is limited, and research on its active ingredient preparation and pharmacological activity is relatively limited.

[0003] Deer antler cream, derived from deer antlers, is an animal medicinal material that is relatively scarce and complex to harvest and process. Currently, it's mostly prepared as a traditional Chinese medicine slice, selling for around 100 yuan per kilogram, a very low price. Furthermore, much of the deer antler cream produced by domestic rubber manufacturers remains unsatisfied by the market, leading to underutilization of the deer antler cream resource. Therefore, deep processing of deer antler cream, extracting its active ingredients, researching its pharmacological activity, and preparing it into high-value-added products are crucial for increasing its market value and developing and utilizing the resource. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides the application of deer antler frost extract in strengthening the skin barrier. The deer antler frost extract of the present invention can promote the proliferation of HacaT cells and the expression of tight junction protein Claudin-1. It also has excellent ability to scavenge hydroxyl free radicals, DPPH free radicals and superoxide anions, and promotes wound healing, thereby playing an active role in protecting the skin's mechanical barrier and promoting wound healing. Deer antler frost extract has good application prospects in the preparation of drugs that promote wound healing and products related to strengthening the skin barrier. The present invention enriches the resource development and utilization of deer antler frost.

[0005] To achieve the technical purpose of the present invention, on the one hand, the present invention provides the use of deer antler cream extract in strengthening the skin barrier, which includes promoting HacaT cell proliferation, promoting the expression of tight junction proteins and scavenging free radicals.

[0006] Furthermore, the tight junction protein is Claudin-1, and the free radicals include hydroxyl radicals, DPPH radicals and superoxide anions.

[0007] Furthermore, the antler frost is subjected to enzymatic hydrolysis to obtain the antler frost extract. The enzymatic hydrolysis adopts single enzyme hydrolysis or double enzyme hydrolysis, wherein the double enzyme hydrolysis uses trypsin and papain, and the single enzyme hydrolysis uses neutral protease or alkaline protease.

[0008] Specifically, the double-enzyme hydrolysis scheme is as follows: add 10 times the amount of water to the antler frost powder and keep it at 97°C for 20 minutes, then cool it to 40°C with cold water, add 2wt% trypsin and papain (enzyme / substrate), the mass ratio of trypsin and papain is 1:2.8, place it on a magnetic stirrer, and hydrolyze it at 44°C for 200 minutes. After the enzymatic hydrolysis reaction is completed, heat it to boiling and keep it in a 97°C water bath for 15 minutes, and rotate it at 4000r·min -1 After centrifugation for 15 minutes, the supernatant was concentrated in vacuo at 80°C, dried at 60°C, and then crushed to obtain the antler frost double enzymatic hydrolysate (MST).

[0009] Specifically, the neutral protease hydrolysis scheme is as follows: add 10 times the water to the antler frost powder and heat to boiling, keep it for 20 minutes, cool it to 40°C, add 15wt% neutral protease (enzyme / substrate), place it on a magnetic stirrer, and hydrolyze it at 40°C for 200 minutes. After the enzymatic hydrolysis reaction is completed, heat it to a hydrolyzate temperature of 88°C, add 8wt% lactic acid (lactic acid / substrate), heat it to boiling, and then keep it at 97°C for 2h, pass it through 300 mesh filter cloth and filter paper in turn, collect the filtrate and concentrate it in vacuum at 80°C, dry it at 60°C, and then crush it to obtain the antler frost neutral protease hydrolyzate (SMST).

[0010] Specifically, the alkaline protease hydrolysis scheme is as follows: add 10 to 50 times water to the antler frost powder and heat to boiling, keep it for 10 to 20 minutes, then cool it to 40°C, add 2 to 10 wt% alkaline protease, and adjust the pH to 8.5 to 11.5 with NaOH solution, stir and hydrolyze at 30 to 50°C for 50 to 200 minutes, after the enzymatic hydrolysis reaction is completed, heat to boiling and keep it for 5 to 20 minutes, cool, filter or simmer at 3000 to 4000 r / min -1 The supernatant was collected and concentrated under vacuum at 55-90°C, dried at 60°C and then crushed to obtain the antler frost alkaline protease hydrolysate (JMST).

[0011] On the other hand, the present invention seeks to protect the use of antler frost extract in the preparation of a medicament for promoting wound healing.

[0012] Furthermore, the antler frost extract promotes the proliferation of HacaT cells, and the antler frost extract promotes the expression of tight junction protein, and the tight junction protein is Claudin-1.

[0013] Specifically, the present invention tested the performance of antler frost extract in protecting the activity of the skin's mechanical barrier and found that both the antler frost water extract and the antler frost hydrolysate were non-toxic to HaCaT cells, and high-dose ST (antler frost water extract) and low-dose JMST both had good proliferative effects on HaCaT cells. Both the antler frost water extract and the antler frost hydrolysate can promote the expression of Claudin-1, with low-dose JMST having the best promoting effect, followed by low-dose SMST and low-dose MST. After intervention with low-dose JMST, SMST, and MST, the Claudin-1 content was 2.33 times, 1.35 times, and 1.28 times that after intervention with low-dose ST, respectively, and the promoting effect was significantly improved. The effect of low-dose JMST in promoting the proliferation of HacaT cells and the expression of Claudin-1 were significantly better than those of Tremella heteropolysaccharide and hyaluronic acid.

[0014] Specifically, the present invention tested the performance of antler frost extract in promoting wound healing and found that both antler frost water extract and antler frost enzymatic hydrolysate can promote wound healing, with JMST having the best effect, showing a level equivalent to that of the positive drug at 24 hours and 48 hours. JMST's effect in promoting wound healing was significantly stronger than that of Tremella heteropolysaccharide and hyaluronic acid, and the 48-hour wound healing rates of MST, SMST, and ST were significantly different from those of the blank group.

[0015] On the other hand, the present invention claims protection for the use of antler frost extract in the preparation of antioxidants, wherein the antler frost extract scavenges free radicals, including hydroxyl radicals, DPPH radicals and superoxide anions.

[0016] Specifically, the present invention discovered through antioxidant activity tests on antler frost extracts that antler frost enzymatic hydrolysates have superior free radical scavenging capabilities. The scavenging rates of antler frost enzymatic hydrolysates for hydroxyl radicals were all higher than those of antler frost aqueous extracts, with MST, SMST, and JMST being 1.9, 2.9, and 1.6 times those of ST, respectively. The scavenging rates of antler frost enzymatic hydrolysates for DPPH radicals were all higher than those of antler frost aqueous extracts, with MST, SMST, and JMST being 7.3, 13.2, and 13.7 times those of ST, respectively. The scavenging rates of antler frost enzymatic hydrolysates for superoxide anions were all higher than those of antler frost aqueous extracts, with MST, SMST, and JMST being 9.1, 6.2, and 7.4 times those of ST, respectively.

[0017] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0018] (1) The antler frost extract of the present invention has a better effect of strengthening the skin barrier. The antler frost extract strengthens the skin barrier by promoting the proliferation of HaCaT cells, promoting the expression of tight junction protein Claudin-1 and scavenging free radicals. In addition, it can promote wound healing. The antler frost extract has good application prospects in the preparation of drugs for promoting wound healing and products related to strengthening the skin barrier. The present invention enriches the resource development and utilization of antler frost. The present invention tested the performance of antler frost extract in protecting the activity of the skin mechanical barrier and found that high-dose ST and low-dose JMST both have a good proliferation-promoting effect on HaCaT cells. Both the antler frost water extract and the antler frost enzymatic hydrolysate can promote the expression of Claudin-1. Low-dose JMST has the best promoting effect, followed by low-dose SMST and low-dose MST. After intervention with low-dose JMST, SMST, and MST, the Claudin-1 content was 2.33 times, 1.35 times, and 1.28 times that after intervention with low-dose ST, respectively, and the promoting effect was significantly improved. The effects of low-dose JMST in promoting the proliferation of HacaT cells and the expression of Claudin-1 were significantly better than those of Tremella heteropolysaccharide and hyaluronic acid.

[0019] (2) The present invention can significantly improve the yield of antler frost extract by enzymatic hydrolysis. The yield of antler frost extract prepared by enzymatic hydrolysis is 3.1 to 10.5 times that of water extract. The yield of antler frost extract prepared by alkaline protease hydrolysis is the highest, which is 11.23 to 17.87%.

[0020] (3) The deer antler frost extract obtained by enzymatic hydrolysis of the present invention has a relatively good antioxidant effect. The scavenging ability of MST, SMST, and JMST for hydroxyl radicals are 1.9, 2.9, and 1.6 times that of ST, respectively. The scavenging rates for DPPH free radicals are 7.3, 13.2, and 13.7 times that of ST, respectively. The scavenging rates for superoxide anions are 9.1, 6.2, and 7.4 times that of ST, respectively.

[0021] (4) The antler frost extract of the present invention can promote wound healing, and JMST has the best effect, showing a level equivalent to that of positive drugs within 24 hours, and its effect is significantly stronger than that of Tremella heteropolysaccharide and hyaluronic acid. The 48-hour wound healing rates of MST, SMST, and ST are significantly different from those of the blank group. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention.

[0023] Figure 1The results are for the hydroxyl radical scavenging rate of antler frost extracts. ST is the aqueous extract of antler frost, MST is the double enzymatic hydrolysate of antler frost, SMST is the neutral protease hydrolysate of antler frost, and JMST is the alkaline protease hydrolysate of antler frost.

[0024] Figure 2 This is the DPPH free radical scavenging rate result of deer antler cream extract.

[0025] Figure 3 The results are for the superoxide anion clearance rate of deer antler cream extract.

[0026] Figure 4 The effect of antler frost extract on HacaT cells. LD indicates the low-dose group (50 μg / mL); HD indicates the high-dose group (100 μg / mL); Blank Control indicates the blank group; # indicates P < 0.05 compared with the blank group; * indicates P < 0.05 compared with the low-dose group.

[0027] Figure 5 Effect of antler frost extract on the content of tight junction protein Claudin-1.

[0028] Figure 6 These are pictures of the cell scratch areas in each treatment group after scratch treatment for different times.

[0029] Figure 7 The effect of antler frost extract on the healing ability of HaCaT cells. Positive Control indicates positive control.

[0030] Figure 8 The effects of different interventions on HacaT cells.

[0031] Figure 9 The effect of different interventions on the content of tight junction protein Claudin-1.

[0032] Figure 10 These are pictures of the cell scratch areas in different intervention treatment groups after different scratch treatment times.

[0033] Figure 11 The effects of different interventions on the healing ability of HaCaT cells.

[0034] Figure 12 This is the UV-VIS scanning spectrum of JMST. DETAILED DESCRIPTION

[0035] The technical solutions of the present invention are now described with reference to the following examples. However, the present invention is not limited to the following examples. The experimental methods and detection methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0036] HaCaT cells (immortalized human epidermal cells) were purchased from Shanghai Yaji Biotechnology Co., Ltd.

[0037] Trypsin, papain, neutral protease, alkaline protease, lactic acid, hydroxyl free radical scavenging ability test kit, DPPH free radical scavenging ability test kit, and superoxide anion scavenging ability test kit were all purchased from Beijing Solebow Technology Co., Ltd.

[0038] PBS phosphate buffered saline powder was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.

[0039] CCK-8 cell proliferation / toxicity detection kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0040] Human Claudin-1 (CLDN1) ELISA kit was purchased from Wuhan Huamei Bioengineering Co., Ltd.

[0041] Tremella fuciformis polysaccharide was purchased from Shanghai Huiwen Biotechnology Co., Ltd.

[0042] Hyaluronic acid was purchased from Huaxi Freda Biopharmaceutical Co., Ltd.

[0043] Example 1

[0044] This embodiment provides a method for preparing a cervical antler frost extract.

[0045] 1. Preparation of Deer Antler Frost Water Extract

[0046] Add 8 times the amount of water to an appropriate amount of antler frost powder, extract at 90℃ in a water bath for 3h, filter, collect the filtrate, concentrate in a vacuum at 80℃, dry at 60℃ and then grind, record as ST, the yield is 1.80%.

[0047] 2. Preparation of Deer Antler Frost Enzyme Hydrolysate

[0048] (1) Preparation of antler frost double enzymatic hydrolysate: Take an appropriate amount of antler frost powder, add 10 times the amount of water, and keep it in a 97°C water bath for 20 minutes, then cool it to 40°C with cold water, add 2wt% trypsin and papain (enzyme / substrate), the mass ratio of trypsin and papain is 1:2.8, put it on a magnetic stirrer, and hydrolyze it at 44°C for 200 minutes. After the enzymatic hydrolysis reaction is completed, heat it to boiling on an electric stove and keep it in a 97°C water bath for 15 minutes, and rotate at 4000 r·min -1After centrifugation for 15 min, the supernatant was concentrated in vacuo at 80°C, dried at 60°C and then pulverized, which was recorded as MST with a yield of 5.54%.

[0049] (2) Preparation of antler frost neutral protease hydrolysate: Take an appropriate amount of antler frost powder, add 10 times water and heat to boiling on an electric stove, keep it for 20 minutes, cool it naturally to 40°C, add 15wt% neutral protease (enzyme / substrate), place it on a magnetic stirrer, and hydrolyze it at 40°C for 200 minutes. After the hydrolysis reaction is completed, heat it in a 95°C water bath until the hydrolyzate temperature is 88°C, add 8wt% lactic acid (lactic acid / substrate), heat it to boiling, and then keep it in a 97°C water bath for 2 hours, pass it through a 300-mesh filter cloth and filter paper in turn, collect the filtrate, concentrate it in a vacuum at 80°C, dry it at 60°C, and then crush it, record it as SMST, with a yield of 18.83%.

[0050] (3) Preparation of antler frost alkaline protease hydrolysate: Take an appropriate amount of antler frost powder, add 10 to 50 times of water and heat to boiling, keep for 10 to 20 minutes, then cool to 40 ° C, add 2 to 10 wt% alkaline protease (enzyme / substrate), and adjust the pH to 8.5 to 11.5 with NaOH solution. Stir and hydrolyze at 30 to 50 ° C for 50 to 200 minutes. After the enzymatic hydrolysis reaction is completed, heat to boiling and keep for 5 to 20 minutes, cool, filter or sieve at 3000 to 4000 r / min -1 The supernatant was collected and concentrated under vacuum at 55-90°C, dried at 60°C and then pulverized, which was recorded as JMST.

[0051] Specifically, an appropriate amount of antler frost powder was added, 10 times the amount of water was added, and the mixture was heated to boiling and maintained for 14 minutes. The mixture was then cooled to 40°C, 6 wt% alkaline protease was added, and the pH was adjusted to 9 with NaOH solution. The mixture was stirred and enzymolyzed at 30°C for 200 minutes. After the enzymolysis reaction was completed, the mixture was heated to boiling and maintained for 10 minutes. The mixture was cooled and then heated at 4000 r / min. -1 The supernatant was collected and concentrated under vacuum at 83°C, dried at 60°C and then pulverized, which was named JMST-1 with a yield of 11.23%.

[0052] Specifically, take an appropriate amount of antler frost powder, add 50 times water and heat to boiling, keep it for 10 minutes, then cool to 40°C, add 10wt% alkaline protease, adjust the pH to 11 with NaOH solution, stir and hydrolyze at 40°C for 60 minutes. After the enzymatic hydrolysis reaction is completed, heat to boiling and keep it for 10 minutes, cool, filter, vacuum concentrate at 80°C, dry at 60°C and crush, record it as JMST-2, and the yield is 17.87%.

[0053] Specifically, take an appropriate amount of antler frost powder, add 33 times water and heat to boiling, keep it for 10 minutes, then cool to 40 ° C, add 8 wt% alkaline protease, adjust the pH to 11 with NaOH solution, stir and enzymolyze at 50 ° C for 50 minutes, after the enzymolysis reaction is completed, heat to boiling and keep it for 20 minutes, cool and simmer at 3000 r·min -1 The product was centrifuged for 15 min under the same conditions, concentrated in vacuo at 55°C, dried at 60°C and then pulverized, and designated as JMST-3, with a yield of 16.64%.

[0054] Specifically, take an appropriate amount of antler frost powder, add 20 times water and heat to boiling, keep it for 10 minutes, then cool to 40 ° C, add 2 wt% alkaline protease, adjust the pH to 11 with NaOH solution, stir and enzymolyze at 35 ° C for 100 minutes, after the enzymolysis reaction is completed, heat to boiling and keep it for 5 minutes, cool and simmer at 3500 r·min -1 The product was centrifuged under the same conditions for 10 min, concentrated in vacuo at 90°C, dried at 60°C and then pulverized, and recorded as JMST-4 with a yield of 16.13%.

[0055] It can be seen that the yield of the deer antler frost extract prepared by enzymatic hydrolysis is significantly improved compared with the water extract, and the yield is 3.1 to 10.5 times that of the water extract.

[0056] Example 2

[0057] This example provides an antioxidant activity test of antler frost extract.

[0058] The ST, MST, SMST, and JMST prepared in Example 1 were used as test samples to detect their antioxidant activities, and the JMST was specifically JMST-1.

[0059] 1. Determination of hydroxyl radical scavenging ability

[0060] Weigh 8 mg of each sample and prepare it into 8 mg / mL solution with distilled water. According to the instructions of the hydroxyl free radical scavenging ability test kit, the hydroxyl free radical scavenging rate is used to represent the hydroxyl free radical scavenging ability of the sample. The test results are as follows: Figure 1 As shown. Figure 1 It can be seen that the scavenging rate of hydroxyl free radicals by antler frost enzymatic hydrolysates is higher than that by antler frost water extracts. The MST, SMST and JMST are 1.9, 2.9 and 1.6 times that of ST respectively.

[0061] 2. DPPH free radical scavenging ability determination

[0062] Weigh 8 mg of each sample and prepare 8 mg / mL solution with the extract in the kit. Determine according to the instructions of the DPPH free radical scavenging ability test kit. The calculated DPPH free radical scavenging rate represents the strength of the sample's DPPH free radical scavenging ability. The test results are as follows: Figure 2 As shown. Figure 2 It can be seen that the scavenging rate of antler frost enzymatic hydrolysates on DPPH free radicals is higher than that of antler frost water extract. MST, SMST and JMST are 7.3, 13.2 and 13.7 times that of ST respectively.

[0063] 3. Determination of superoxide anion scavenging ability

[0064] Weigh 10 mg of each sample and use the extract in the kit to prepare a 50 mg / mL solution at a ratio of sample mass (mg): extract volume (μL) of 1:20. The sample is tested according to the instructions of the superoxide anion scavenging ability test kit. The calculated superoxide anion scavenging rate represents the strength of the sample's superoxide anion scavenging ability. The test results are as follows: Figure 3 As shown. Figure 3 It can be seen that the scavenging rates of superoxide anions by antler frost enzymatic hydrolysates are higher than those by antler frost water extracts. The MST, SMST and JMST are 9.1, 6.2 and 7.4 times that of ST, respectively.

[0065] Example 3

[0066] This example provides the use of antler frost extract in protecting the skin's mechanical barrier activity.

[0067] ST, MST, SMST, and JMST prepared in Example 1 were used as the samples to be tested, and JMST was specifically JMST-1. Appropriate amounts of the samples were taken and added with water to prepare sample solutions with concentrations of 50 μg / mL and 100 μg / mL, respectively, to test their performance in protecting the skin's mechanical barrier activity.

[0068] 1. Deer antler cream extract promotes HacaT cell proliferation

[0069] HaCaT cells were prepared with complete culture medium (DMEM (high glucose) medium (Gbico) + 10 vol% FBS fetal bovine serum (EXCELL) + 1 vol% P / S (penicillin / streptomycin solution)) to a volume of 5×10 4Cells / mL of single cell suspension were inoculated into 96-well plates (Corning) (100 μL / well), cultured in a CO2 cell culture incubator at 37°C and 5% CO2 for 24 hours. After adherence, the culture medium was discarded and cells were intervened with 50 μg / mL (low-dose drug group) and 100 μg / mL (high-dose drug group) sample solutions, respectively. At the same time, blank control cells (n=5) were prepared. After 48 hours of intervention, the culture medium was discarded and 100 μL of the prepared 10% CCK-8 solution was added to each well. The cells were continued to be incubated in the incubator. After 1 hour, the OD value at 450 nm was measured with a microplate reader to calculate the cell survival rate. The results are shown in the figure. Figure 4 shown.

[0070] Depend on Figure 4 It can be seen that the antler frost water extract and antler frost enzymatic hydrolysate are non-toxic to HaCaT cells, and high-dose ST and low-dose JMST have a good proliferation-promoting effect on HaCaT cells, which is beneficial to protecting the skin barrier.

[0071] 2. Deer antler cream extract promotes the expression of tight junction proteins

[0072] HaCaT cells were prepared with complete culture medium to a volume of 5 × 10 4 Cells / mL of single cell suspension were inoculated into culture flasks and cultured in a CO2 cell culture incubator at 37°C and 5% CO2 for 24 hours. After adherence, the culture medium was discarded and cells were treated with 50μg / mL (low-dose drug group) and 100μg / mL (high-dose drug group) sample solutions, respectively. At the same time, blank control cells were prepared, with 3 replicates per group. After 48 hours of intervention, cell lysates were collected and the Claudin-1 content was detected using the Human Claudin-1 (CLDN1) ELISA kit. The test results are shown in Figure 2. Figure 5 shown.

[0073] Depend on Figure 5 Both the aqueous extract and hydrolyzed antler frost extract promoted claudin-1 expression, with low-dose JMST having the greatest effect, followed by low-dose SMST and low-dose MST. Claudin-1 levels after low-dose JMST, SMST, and MST interventions were 2.33-fold, 1.35-fold, and 1.28-fold higher, respectively, than after low-dose ST intervention, demonstrating a significant improvement in the expression of claudin-1.

[0074] Example 4

[0075] This embodiment provides the use of antler frost extract in promoting wound healing.

[0076] The ST, MST, SMST, and JMST prepared in Example 1 were used as test samples, and JMST was specifically JMST-1. A 50 μg / mL sample solution was prepared to detect its performance in promoting wound healing.

[0077] HaCaT cells were prepared with complete culture medium to a volume of 5 × 10 5 Cells / mL single cell suspension was inoculated into 24-well plates (Corning) (500 μL / well) and cultured in a CO2 cell culture incubator at 37°C and 5% CO2 for 24 hours. After adherence, cells were intervened and divided into blank group, positive control group (10 vol% FBS fetal bovine serum) and drug intervention group (50 μg / mL). After intervention for 48 hours, a straight scratch area was gently made with a pipette tip. Except for the positive control group, serum-free culture medium was replaced for the other groups. The cell scratch area ( Figure 6 ), ImageJ was used to calculate the scratch area and cell migration ability. The results are shown in Figure 2. Figure 7 shown.

[0078] Depend on Figure 7 It can be seen that both the antler frost water extract and the antler frost enzymatic hydrolysate can promote wound healing, and JMST has the best effect, showing a level equivalent to that of the positive drug within 24 hours. The 48-hour wound healing rates of MST, SMST, and ST were significantly different from those of the blank group.

[0079] Example 5

[0080] This example provides a comparison of the effects of JMST, a hydrolyzate of antler frost, and Tremella heteropolysaccharide and hyaluronic acid in protecting the skin's mechanical barrier activity and promoting wound healing.

[0081] Tremella polysaccharide has antioxidant and anti-inflammatory properties. It can promote wound healing by inhibiting the release of inflammatory factors and regulating immune responses. It can also protect intercellular tight junction proteins such as Claudin-1, thereby strengthening the skin barrier. Hyaluronic acid-based hydrogel materials are used in wound dressings, which can absorb wound exudate and create a moist environment, thereby accelerating wound healing. It also has a protective and repairing effect on skin barrier damage in skin diseases such as photoaging, sensitive skin, and atopic dermatitis.

[0082] 1. Comparison of the effects of JMST, a hydrolyzate of antler frost, Tremella heteropolysaccharide, and hyaluronic acid in protecting the skin's mechanical barrier activity

[0083] The experimental method was the same as in Example 3. The effects of JMST on promoting HacaT cell proliferation and tight junction protein expression were compared with those of Tremella heteropolysaccharide (≥1 million Dal) and hyaluronic acid (200,000-400,000 Dal). The intervention concentrations were 50 μg / mL and 100 μg / mL. The results were as follows: Figure 8 and Figure 9 shown.

[0084] Depend on Figure 8 It can be seen that low-dose JMST is more effective in promoting the proliferation of HacaT cells than Tremella heteropolysaccharide and hyaluronic acid. Figure 9 It can be seen that the effect of low-dose JMST in promoting Claudin-1 expression is significantly better than that of Tremella heteropolysaccharide and hyaluronic acid.

[0085] 2. Comparison of the effects of JMST, a hydrolyzate of antler frost, Tremella heteropolysaccharide, and hyaluronic acid in promoting wound healing

[0086] The experimental method was the same as that in Example 4. The effects of JMST on wound healing were compared with those of Tremella fuciformis polysaccharide (≥1 million Dal) and hyaluronic acid (200,000-400,000 Dal). The intervention concentration was 50 μg / mL. The cell scratch area was photographed at 0 h, 24 h, and 48 h. Figure 10 ), ImageJ was used to calculate the scratch area and cell migration ability. The results are shown in Figure 2. Figure 11 shown.

[0087] Depend on Figure 11 It can be seen that JMST has the best effect, and its ability to promote wound healing after 24h and 48h is comparable to that of positive drugs, and its effect in promoting wound healing is significantly stronger than that of Tremella heteropolysaccharide and hyaluronic acid.

[0088] Example 6

[0089] This example provides analysis of the ultraviolet absorption characteristics and amino acid composition of JMST.

[0090] 1. Analysis of UV absorption characteristics

[0091] The UV absorption characteristics of JMST were detected by UV spectrophotometry ( Figure 12 The results showed that it had a maximum absorption peak around 210nm, indicating that it contained peptide bonds. The amino acid content in JMST was determined by the kit method and the result was 339μmol / g.

[0092] 2. Amino acid composition analysis

[0093] The amino acid composition of JMST was analyzed by UHPLC-MS / MS. The main differences in the amino acid composition of JMST compared with ST, MST, and SMST are shown in Table 1.

[0094] Table 1 Main differences in amino acid composition of antler frost extracts

[0095]

[0096] Amino acid analysis revealed that JMST contains 49 amino acids, with L-Alanine being the highest at 12,726.15 nmol / g. It also contains relatively abundant amounts of Glycine, L-Alanine, L-Serine, L-Proline, L-Valine, L-Leucine, L-Aspartic acid, L-Phenylalanine, and L-Tyrosine. Compared to ST, MST, and SMST, JMST has higher levels of L-Alanine, L-Valine, and L-Tyrosine. JMST also contains L-2-Aminoadipic acid, which is absent in the other samples.

[0097] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the scope of protection determined by the present invention.

Claims

1. The application of deer antler cream extract in strengthening the skin barrier, characterized in that: The strengthening of the skin barrier includes promoting the proliferation of HacaT cells, promoting the expression of tight junction proteins and scavenging free radicals.

2. The use according to claim 1, characterized in that The tight junction protein is Claudin-1; The free radicals include hydroxyl radicals, DPPH radicals and superoxide anions.

3. The use according to claim 1, characterized in that enzymatically hydrolyzing the antler frost to obtain the antler frost extract; The enzymatic hydrolysis adopts single enzyme enzymatic hydrolysis or double enzyme enzymatic hydrolysis; The double enzyme enzymolysis uses trypsin and papain; The single enzyme enzymolysis uses neutral protease or alkaline protease.

4. The use according to claim 3, characterized in that The amount of alkaline protease added is 2-10% of the mass of the antler cream; The enzymatic hydrolysis pH of the alkaline protease is 8.5-11.

5.

5. The use according to claim 3, characterized in that The enzymatic hydrolysis temperature of the alkaline protease is 30-50° C., and the enzymatic hydrolysis time is 50-200 minutes.

6. The use according to claim 3, characterized in that The total amount of trypsin and papain added is 2% of the mass of the antler cream; The mass ratio of the trypsin to papain is 1:2.

8.

7. The use according to claim 3, characterized in that The added amount of the neutral protease is 15% of the mass of the antler cream.

8. Use of antler frost extract in the preparation of a medicament for promoting wound healing, characterized in that: The deer antler frost extract promotes HacaT cell proliferation, promotes the expression of tight junction proteins, and can also promote cell migration; The tight junction protein is Claudin-1.

9. Application of antler frost extract in the preparation of antioxidants, characterized in that: The deer antler cream extract scavenges free radicals; The free radicals include hydroxyl radicals, DPPH radicals and superoxide anions.

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