Cornu cervi degelatinatum extract as well as preparation method, composition and application thereof

Abstract

The invention relates to a cornu cervi degelatinatum extract, in particular to a cornu cervi degelatinatum extract as well as a preparation method, a composition and application thereof. The preparation method of the cornu cervi degelatinatum extract comprises the following steps: S1, mixing cornu cervi degelatinatum with water, heating to boil, preserving heat, and cooling to obtain a mixture a; s2, adding alkaline protease into the mixture a, and stirring to obtain a zymolyte; s3, heating the zymolyte until the zymolyte is boiled, preserving heat, cooling, filtering or centrifuging, taking supernate, and carrying out vacuum concentration to obtain a concentrate; and S4, adding ethanol into the concentrate, carrying out alcohol precipitation treatment, filtering or centrifuging, taking the precipitate, and drying to obtain the cornu cervi degelatinatum extract. The cornu cervi degelatinatum extract is obtained by combining alkaline proteolysis of cornu cervi degelatinatum and alcohol precipitation treatment of ethanol, is a hydrolytic peptide, contains multiple amino acids, has a certain proliferation promoting effect on HaCaT cells, and has the effects of improving the activity of fatty acid synthase FAS in the cells and increasing the HA content.

Description

Technical Field

[0001] The present invention relates to extracts of Cornu Cervi Degelatinatum, and particularly to an extract of Cornu Cervi Degelatinatum, a preparation method thereof, a composition and an application thereof. Background Art

[0002] Cornu Cervi Degelatinatum is the horn block after removing the gelatin from deer antlers, which is made by boiling to remove the glue and drying. The Chinese Pharmacopoeia records that it has the effects of warming the kidney and assisting yang, astringing and stopping bleeding, etc., and can be used for diseases such as spleen-kidney yang deficiency and excessive leucorrhea. At present, it has been reported that it contains 33 chemical components, including minerals, amino acids, polysaccharides and lipids, etc., but hydroxyapatite is the main component, accounting for more than 80-90%. Due to the main component being minerals, the development of its products is limited, and there is less research on the preparation of active substances and pharmacological activities. Related research mainly focuses on identification, processing, treatment of breast diseases and clinical applications, etc. In addition, Cornu Cervi Degelatinatum, as a traditional Chinese medicine decoction piece, has a low selling price (about 100 yuan per kilogram), and a large amount of Cornu Cervi Degelatinatum produced by the domestic glue-making industry is overstocked, and its value has not been fully utilized.

[0003] Chinese Patent CN106236692B discloses a preparation method of Cornu Cervi Degelatinatum with antioxidant activity, including grinding deer antler discs into powder, adding deionized water and performing water bath heat extraction at 100°C three times, and collecting the precipitate through a centrifugation process at 8500-12000 rpm. After drying and grinding, Cornu Cervi Degelatinatum with antioxidant activity is obtained. Regarding obtaining various active ingredients from the extract of Cornu Cervi Degelatinatum, this patent does not cover it.

[0004] Therefore, providing a preparation method of an extract of Cornu Cervi Degelatinatum that can obtain various active ingredients is the main technical problem to be solved at present. Summary of the Invention

[0005] The first aspect of the present invention provides a preparation method of an extract of Cornu Cervi Degelatinatum, including the following steps:

[0006] S1. Mix Cornu Cervi Degelatinatum with water, heat to boiling, keep warm, and cool to obtain mixture a;

[0007] S2. Add alkaline protease to mixture a and stir to obtain an enzymolysate;

[0008] S3. Heat the enzymolysate to boiling, keep warm, cool, filter or centrifuge, and take the supernatant for vacuum concentration to obtain a concentrate;

[0009] S4. Add ethanol to the concentrate for alcohol precipitation treatment, filter or centrifuge, take the precipitate and dry it to obtain the extract of Cornu Cervi Degelatinatum.

[0010] During the experiment, the inventors found that by combining alkaline protease hydrolysis of deer antler cream with ethanol precipitation treatment, the protein content of the deer antler cream extract obtained was > 50%, and it contained various amino acids. It had a certain promoting effect on the proliferation of HaCaT cells, could enhance the activity effect of fatty acid synthase FAS in cells, and was comparable to recombinant human epidermal growth factor (rhEGF). It could promote the expression of HAS2 gene, HAS3 gene, and CD44 gene, and increase the hyaluronic acid content. This enabled the deer antler cream extract to be better applied in the technical field of skin care and skin treatment products. The inventors found during the experiment that there were significant differences in the effect of the deer antler cream extract obtained with and without ethanol precipitation on the activity of fatty acid synthase FAS, and there were also differences in the increase of hyaluronic acid content. The possible reason was that the enzymolysate was exposed to ethanol for a long time, and molecular hydrophobic aggregation or local conformational changes occurred.

[0011] In one embodiment, the deer antler cream in S1 is deer antler cream powder, and the particle size of the deer antler cream powder is 60 - 100 mesh.

[0012] In one embodiment, the mass ratio of the deer antler cream to water in S1 is 1:(10 - 50).

[0013] In one embodiment, the addition amount of the alkaline protease in S2 is 2 - 10 wt% of the deer antler cream.

[0014] In one embodiment, after adding the alkaline protease to the mixture a in S2, the pH of the mixture a is adjusted to 8.5 - 11.5, and stirring enzymolysis is carried out at 30 - 50 °C for 50 - 200 min to obtain an enzymolysate.

[0015] The inventors found during the experiment that adding alkaline protease to the mixture a and then performing ethanol precipitation treatment could obtain a deer antler cream enzymolysate with the effect of improving the skin chemical barrier. However, the deer antler cream extract obtained only by alkaline protease hydrolysis could not promote the activity of FAS. The inventors found that the deer antler cream extract obtained after further ethanol precipitation treatment was required to promote the activity of FAS, and the deer antler cream extract obtained after ethanol precipitation treatment had a better effect on increasing the HA content in cells than the deer antler cream extract obtained without ethanol precipitation treatment.

[0016] In one embodiment, the centrifugation speed in S3 is 3000 - 4000 r·min -1 , and the centrifugation time is 5 - 15 min.

[0017] In one embodiment, ethanol is added to the concentrate in S4 to obtain a mixture b. The mass concentration of ethanol in the mixture b is 50 - 90%, and the ethanol includes an ethanol aqueous solution with a mass concentration of 95% or absolute ethanol.

[0018] In one embodiment, the ethanol is an aqueous ethanol solution with a mass concentration of 95%.

[0019] In one embodiment, the ethanol is anhydrous ethanol.

[0020] In one embodiment, the temperature for alcohol precipitation in S4 is 1 - 10 °C, and the time for alcohol precipitation is 12 - 60 h.

[0021] In one embodiment, the method for preparing the deer horn cream extract includes the following steps: adding deer horn cream to water to obtain a mixture, heating the mixture to boiling and maintaining for 10 - 20 min, then cooling to 40 °C, adding alkaline protease to the mixture, and adjusting the pH of the mixture to 8.5 - 11.5 with NaOH solution, stirring and enzymolyzing at 30 - 50 °C for 50 - 200 min. After the enzymolysis reaction, an enzymolyzate is obtained. Heating the enzymolyzate to boiling and maintaining for 5 - 20 min, cooling, filtering or centrifuging at a centrifugal speed of 3000 - 4000 r·min -1 for 5 - 15 min under the condition of, taking the supernatant and vacuum - concentrating it to 12 - 35% of the original volume at 55 - 90 °C to obtain a concentrate, adding anhydrous ethanol to the concentrate until the alcohol concentration is 50 - 90%, performing alcohol precipitation at 4 °C for 12 - 60 h, then filtering or centrifuging at a centrifugal speed of 3000 - 4000 r·min -1 for 5 - 15 min under the condition of, taking the precipitate, drying it to obtain the deer horn cream extract.

[0022] The deer horn cream extract is sieved through a sieve with 80 - 200 meshes.

[0023] The mass ratio of the water extract of deer horn cream to water is 1:10 - 50.

[0024] The addition amount of the alkaline protease is 2 - 10 wt% of the water extract of deer horn cream.

[0025] The second aspect of the present invention provides a deer horn cream extract, which is prepared by the method for preparing the deer horn cream extract described above.

[0026] The third aspect of the present invention provides a composition, which includes the deer horn cream extract and an active ingredient.

[0027] The preparation raw materials of the composition include the deer horn cream extract and an active ingredient. By weight, the preparation raw materials include 43 - 70 parts of the deer horn cream extract and 30 - 57 parts of the active ingredient.

[0028] The preparation method of the composition is to mix the deer horn cream extract and the active ingredient evenly to obtain the composition.

[0029] In one embodiment, the active ingredient comprises one or a combination of two of chitosan oligosamine and ectoin.

[0030] In one embodiment, the active ingredient is chitosan oligosamine, and by weight, the preparation raw materials of the composition consist of 55 - 70 parts of antler cream extract JMSTD and 30 - 45 parts of chitosan oligosamine.

[0031] In one embodiment, the active ingredient is ectoin, and by weight, the preparation raw materials of the composition consist of 43 - 58 parts of antler cream extract JMSTD and 42 - 57 parts of ectoin.

[0032] In order to promote the activity of fatty acid synthase FAS, the inventors found in the experiment that the combination of ectoin and antler cream extract JMSTD can produce a better synergistic effect and promote the activity of fatty acid synthase FAS.

[0033] In order to promote the increase in the content of hyaluronic acid HA in cells, the inventors found in the experiment that the combination of chitosan oligosamine or ectoin and antler cream extract JMSTD can produce a better synergistic effect and promote the increase in the content of hyaluronic acid HA in cells.

[0034] The fourth aspect of the present invention provides an application of an antler cream extract, and the antler cream extract is applied in the technical field of skin care products.

[0035] The inventors found in the experiment that the antler cream extract JMSTD provided by this application has a dual regulatory effect on hyaluronic acid and lipid generation. By combining with chitosan oligosamine or ectoin, the antler cream extract JMSTD can promote the activity of fatty acid synthase FAS and increase the content of hyaluronic acid HA in cells, and has high value in the application of adjuvant treatment products for skin diseases and moisturizing and barrier protection cosmetics.

[0036] Beneficial effects

[0037] 1. In this application, by combining alkaline protease hydrolysis of antler cream with ethanol precipitation treatment, the protein content in the obtained antler cream extract is > 50%, and it contains various amino acids, which has a certain promoting effect on the proliferation of HaCaT cells and can enhance the activity effect of fatty acid synthase FAS in cells.

[0038] 2. The antler cream extract obtained after ethanol precipitation treatment in this application has a better effect on increasing the content of HA in cells than the antler cream extract obtained without ethanol precipitation treatment.

[0039] 3. The composition provided by this application contains ectoine and extract of antler glue, which can cooperate to produce a better synergistic effect and further enhance the effect of activating fatty acid synthase FAS.

[0040] 4. The combination of chitosan oligosaccharide or ectoine and extract of antler glue in this application can produce a better synergistic effect, which can further increase the content of hyaluronic acid HA in cells.

[0041] 5. The extract of antler glue provided by this application has a dual regulatory effect on hyaluronic acid and lipid generation. By combining with chitosan oligosaccharide or ectoine, the extract of antler glue further enhances the effect of activating fatty acid synthase FAS and increasing the content of hyaluronic acid HA in cells, and has high value in the application of adjuvant treatment products for skin diseases and moisturizing and barrier protection cosmetics. Description of the Drawings

[0042] Figure 1 It is the result of cell proliferation test.

[0043] Figure 2 It is the result of the test on the regulation of fatty acid synthase activity by the extract of antler glue.

[0044] Figure 3 It is the result of the test on the expression of genes related to fatty acid synthase by the extract of antler glue JMSTD.

[0045] Figure 4 It is the result of the test on the expression of proteins related to fatty acid synthase by the extract of antler glue JMSTD.

[0046] Figure 5 It is the result of the test on the expression of ACCα, FASN and N-SMase proteins in fatty acid synthase.

[0047] Figure 6 It is the result of the test on the regulation of hyaluronic acid content by the extract of antler glue.

[0048] Figure 7 It is the result of the test on the influence of the extract of antler glue on genes related to hyaluronic acid synthesis.

[0049] Figure 8 It is the result of the test on the influence of the extract of antler glue JMSTD on the expression of proteins related to hyaluronic acid synthesis.

[0050] Figure 9 It is the result of the Western blot test.

[0051] Figure 10 It is the result of the test on the activity of fatty acid synthase by the composition.

[0052] Figure 11Test results of regulating the hyaluronic acid content in cells by the composition.

[0053] Figure 12 Ultraviolet absorption test results of JMSTD, an extract of Cornu Cervi Degelatinatum. Detailed implementation manners

[0054] The commercially available product of Cornu Cervi Degelatinatum used in the following experiments.

[0055] The first group of experiments

[0056] The first group of experiments provided an aqueous extract of Cornu Cervi Degelatinatum.

[0057] The first group of experiments included Example 1.

[0058] Example 1

[0059] In the first aspect of this example, a preparation method of an aqueous extract of Cornu Cervi Degelatinatum is provided, including the following steps:

[0060] Add small pieces or powder of Cornu Cervi Degelatinatum to water, extract in a water bath at 90 °C for 3 h, then filter and collect the filtrate. Concentrate the filtrate under vacuum at 80 °C until the relative density of the filtrate reaches 1.1 to end the concentration, and dry and pulverize it at 65 °C to obtain the aqueous extract of Cornu Cervi Degelatinatum.

[0061] The aqueous extract of Cornu Cervi Degelatinatum is sieved through a 100-mesh sieve.

[0062] In the second aspect of this example, an aqueous extract of Cornu Cervi Degelatinatum is provided, which is prepared by the above preparation method, and the obtained aqueous extract of Cornu Cervi Degelatinatum is labeled as ST.

[0063] The mass ratio of the Cornu Cervi Degelatinatum to water is 1:8.

[0064] The Cornu Cervi Degelatinatum is a commercially available product.

[0065] The second group of experiments

[0066] The second group of experiments provided an extract of Cornu Cervi Degelatinatum.

[0067] The second group of experiments included Example 2 and Example 3.

[0068] Example 2

[0069] In the first aspect of this example, a preparation method of an extract of Cornu Cervi Degelatinatum is provided, including the following steps:

[0070] Add the antler glue cream powder to water to obtain a mixture. Add 10 times the amount of water to the antler glue cream powder, keep it in a water bath at 97°C for 20 min, and then cool it to 40°C. Add 1.7 g of trypsin and 4.7 g of papain to the mixture, and then carry out enzymatic hydrolysis at 44°C on a stirrer for 200 min. After the enzymatic hydrolysis is completed, obtain the hydrolysate. Heat the hydrolysate to boiling, then keep it at 97°C for 15 min, and then centrifuge at a centrifugal speed of 4000 r·min -1 for 15 min under the condition of. Take the supernatant and carry out vacuum concentration at 80°C until the relative density of the supernatant is 1.1 to end the concentration. After drying, pulverize it and sieve it through a 100-mesh sieve to obtain the antler glue cream extract.

[0071] The particle size of the antler glue cream powder is 60 mesh.

[0072] The mass ratio of the antler glue cream powder to water is 1:10.

[0073] The addition amount of the antler glue cream powder is 320 g.

[0074] The purchase source of the trypsin is Beijing Solarbio Science & Technology Co., Ltd.

[0075] The purchase source of the papain is Beijing Solarbio Science & Technology Co., Ltd.

[0076] In the second aspect of this example, an antler glue cream extract is provided, which is prepared by the above preparation method, and the obtained antler glue cream extract is labeled as MST.

[0077] Example 3

[0078] In the first aspect of this example, a preparation method of an antler glue cream extract is provided, including the following steps:

[0079] Add the antler glue cream powder to water to obtain a mixture. Add 10 times the amount of water to the antler glue cream powder, heat it to boiling and keep it for 20 min, and then cool it to 40°C. Add 45 g of neutral protease to the mixture, and carry out enzymatic hydrolysis at 40°C on a stirrer for 200 min. After the enzymatic hydrolysis reaction is completed, obtain the hydrolysate. When the hydrolysate is heated to 88°C, add 25 ml of lactic acid, then heat it to boiling and keep it in a water bath at 97°C for 2 h, centrifuge (4000 r·min -1 , 15 min) or filter (filter successively with a 300-mesh filter cloth and filter paper), collect the clear liquid and carry out vacuum concentration at 80°C until the relative density of the clear liquid is 1.1 to end the concentration. After drying, pulverize it and sieve it through a 100-mesh sieve to obtain the antler glue cream extract.

[0080] The particle size of the antler glue cream powder is 80 mesh.

[0081] The mass ratio of the antler glue cream powder to water is 1:10.

[0082] The addition amount of the aqueous extract of cornus cervi degelatinata is 300 g.

[0083] The purchased source of the neutral protease is Beijing Solarbio Science & Technology Co., Ltd.

[0084] In the second aspect of this example, a cornus cervi degelatinata extract is provided, which is prepared by the above preparation method, and the obtained cornus cervi degelatinata extract is labeled as SMST.

[0085] The third group of experiments

[0086] The third group of experiments provides a preparation method of a cornus cervi degelatinata extract, including the following steps: adding cornus cervi degelatinata powder to water to obtain a mixture, heating the cornus cervi degelatinata powder with an appropriate amount of water to boiling and maintaining for 10 - 20 min, then cooling to 40 °C, adding alkaline protease to the mixture, and adjusting the pH of the mixture to 8.5 - 11.5 with NaOH solution, carrying out stirring enzymatic hydrolysis at 30 - 50 °C for 50 - 200 min, obtaining an enzymatic hydrolysate after the enzymatic hydrolysis reaction ends, heating the enzymatic hydrolysate to boiling and maintaining for 5 - 20 min, cooling, filtering or centrifuging at a centrifugal speed of 3000 - 4000 r·min -1 under the condition of -1, centrifuging for 5 - 15 min, taking the supernatant and vacuum concentrating it to a relative density of 1.1 - 1.3 at 55 - 90 °C, and drying to obtain the cornus cervi degelatinata extract, labeled as JMST.

[0087] The particle size of the cornus cervi degelatinata powder is 60 - 100 mesh.

[0088] The mass ratio of the cornus cervi degelatinata powder to water is 1:10 - 50.

[0089] The addition amount of the alkaline protease is 2 - 10 wt% of the cornus cervi degelatinata powder.

[0090] The purchased source of the alkaline protease is Beijing Solarbio Science & Technology Co., Ltd.

[0091] The third group of experiments includes Example 4, Example 5, Example 6, and Example 7.

[0092] Example 4

[0093] In the first aspect of this example, a preparation method of a cornus cervi degelatinata extract is provided, including the following steps:

[0094] Heating the cornus cervi degelatinata powder with an appropriate amount of water to boiling and maintaining for 14 min, then cooling to 40 °C, adding alkaline protease to the mixture, and adjusting the pH of the mixture to 9 with NaOH solution, carrying out stirring enzymatic hydrolysis at 45 °C for 200 min, obtaining an enzymatic hydrolysate after the enzymatic hydrolysis reaction ends, heating the enzymatic hydrolysate to boiling and maintaining for 10 min, cooling to 40 °C, at a centrifugal speed of 4000 r·min -1Centrifuge for 5 minutes under the given conditions, take the supernatant, and vacuum concentrate it at 83°C until the relative density of the supernatant reaches 1.2 to end the concentration. After drying, pulverize it and sieve it through a 80-mesh sieve to obtain the Cornu Cervi Degelatinatum extract.

[0095] The particle size of the Cornu Cervi Degelatinatum powder is 60 mesh.

[0096] The mass ratio of the Cornu Cervi Degelatinatum powder to water is 1:10.

[0097] The addition amount of the Cornu Cervi Degelatinatum powder is 300 g.

[0098] The addition amount of the alkaline protease is 6 wt% of the Cornu Cervi Degelatinatum.

[0099] The purchase source of the alkaline protease is Beijing Solarbio Science & Technology Co., Ltd.

[0100] In the second aspect of this example, a Cornu Cervi Degelatinatum extract is provided, which is prepared by the above preparation method, and the obtained Cornu Cervi Degelatinatum extract is labeled as JMST.

[0101] Example 5

[0102] In the first aspect of this example, a preparation method of a Cornu Cervi Degelatinatum extract is provided, including the following steps:

[0103] Add appropriate amount of water to the Cornu Cervi Degelatinatum powder, heat it to boiling and keep it for 10 minutes, then cool it to 40°C. Then add alkaline protease to the mixture, and adjust the pH of the mixture to 11 with NaOH solution. Stir and enzymatically hydrolyze at 40°C for 60 minutes. After the enzymatic hydrolysis reaction ends, obtain the enzymatic hydrolysate. Heat the enzymatic hydrolysate to boiling and keep it for 10 minutes, cool it to 40°C, then filter to collect the filtrate. Vacuum concentrate the filtrate at 80°C until the relative density of the filtrate reaches 1.1 to end the concentration. After drying, pulverize it and sieve it through a 100-mesh sieve to obtain the Cornu Cervi Degelatinatum extract.

[0104] The particle size of the Cornu Cervi Degelatinatum powder is 80 mesh.

[0105] The addition amount of the Cornu Cervi Degelatinatum powder is 300 g.

[0106] The mass ratio of the Cornu Cervi Degelatinatum powder to water is 1:50.

[0107] The addition amount of the alkaline protease is 10 wt% of the Cornu Cervi Degelatinatum powder. ]>

[0108] The purchase source of the alkaline protease is Beijing Solarbio Science & Technology Co., Ltd.

[0109] In the second aspect of this example, a Cornu Cervi Degelatinatum extract is provided, which is prepared by the above preparation method, and the obtained Cornu Cervi Degelatinatum extract is labeled as JMST.

[0110] Example 6

[0111] In the first aspect of this example, a preparation method of pilose antler cream extract is provided, including the following steps:

[0112] The pilose antler cream powder is heated to boiling with appropriate amount of water and maintained for 10 min, then cooled to 40 °C. Then, alkaline protease is added to the mixture, and the pH of the mixture is adjusted to 11 with NaOH solution. Stirring enzymatic hydrolysis is carried out at 40 °C for 60 min. After the enzymatic hydrolysis reaction ends, an enzymatic hydrolysate is obtained. The enzymatic hydrolysate is heated to boiling and maintained for 10 min, cooled to 40 °C, then filtered to collect the filtrate. The filtrate is vacuum concentrated at 80 °C until the relative density of the filtrate reaches 1.3 to end the concentration. After drying, it is pulverized and sieved through a 120-mesh sieve to obtain the pilose antler cream extract.

[0113] The particle size of the pilose antler cream powder is 100 mesh.

[0114] The mass ratio of the pilose antler cream powder to water is 1:33.

[0115] The addition amount of the pilose antler cream powder is 300 g.

[0116] The addition amount of the alkaline protease is 8 wt% of the pilose antler cream powder.

[0117] The purchase source of the alkaline protease is Beijing Solarbio Science & Technology Co., Ltd.

[0118] In the second aspect of this example, a pilose antler cream extract is provided, which is prepared by the above preparation method, and the obtained pilose antler cream extract is labeled as JMST.

[0119] Example 7

[0120] In the first aspect of this example, a preparation method of pilose antler cream extract is provided, including the following steps:

[0121] The pilose antler cream powder is heated to boiling with appropriate amount of water and maintained for 10 min, then cooled to 40 °C. Then, alkaline protease is added to the mixture, and the pH of the mixture is adjusted to 11 with NaOH solution. Stirring enzymatic hydrolysis is carried out at 35 °C for 60 min. After the enzymatic hydrolysis reaction ends, an enzymatic hydrolysate is obtained. The enzymatic hydrolysate is heated to boiling and maintained for 10 min, cooled to 40 °C, then filtered to collect the filtrate. The filtrate is vacuum concentrated at 80 °C until the relative density of the filtrate reaches 1.2 to end the concentration. After drying, it is pulverized and sieved through a 100-mesh sieve to obtain the pilose antler cream extract.

[0122] The particle size of the pilose antler cream powder is 80 mesh.

[0123] The mass ratio of the pilose antler cream powder to water is 1:20.

[0124] The addition amount of the deer antler cream powder is 300 g.

[0125] The addition amount of the alkaline protease is 4 wt% of the deer antler cream powder.

[0126] The purchase source of the alkaline protease is Beijing Solarbio Science & Technology Co., Ltd.

[0127] In the second aspect of this embodiment, a deer antler cream extract is provided, which is prepared by the above preparation method, and the obtained deer antler cream extract is labeled as JMST.

[0128] The fourth group of experiments

[0129] The fourth group of experiments provides a preparation method of a deer antler cream extract, including the following steps: adding an appropriate amount of water to the deer antler cream powder, heating to boiling and maintaining for 10 - 20 min, then cooling to 40 °C, adding alkaline protease to the mixture, and adjusting the pH of the mixture to 8.5 - 11.5 with NaOH solution, stirring and enzymatically hydrolyzing at 30 - 50 °C for 50 - 200 min. After the enzymatic hydrolysis reaction ends, an enzymatic hydrolysate is obtained. Heating the enzymatic hydrolysate to boiling and maintaining for 5 - 20 min, cooling, filtering or centrifuging at a centrifugal speed of 3000 - 4000 r·min -1 centrifuging for 5 - 15 min under the condition of, taking the supernatant and vacuum concentrating it to 12 - 35% of the original volume at 55 - 90 °C to obtain a concentrate, adding absolute ethanol to the concentrate to obtain a mixture b, the mass concentration of ethanol in the mixture b is 50 - 90%, performing alcohol precipitation at 4 °C for 12 - 60 h, then filtering or centrifuging at a centrifugal speed of 3000 - 4000 r·min -1 centrifuging for 5 - 15 min under the condition of, taking the precipitate, drying, pulverizing, and sieving through a sieve with 80 - 200 meshes to obtain a deer antler cream extract, labeled as JMSTD.

[0130] The particle size of the deer antler cream powder is 60 - 100 meshes.

[0131] The mass ratio of the deer antler cream powder to water is 1:10 - 50.

[0132] The addition amount of the alkaline protease is 2 - 10 wt% of the deer antler cream powder.

[0133] The purchase source of the alkaline protease is Beijing Solarbio Science & Technology Co., Ltd.

[0134] The fourth group of experiments includes Example 8, Example 9, Example 10, and Example 11.

[0135] Example 8

[0136] In the first aspect of this embodiment, a preparation method of a deer antler cream extract is provided, including the following steps:

[0137] The antler glue powder is heated with an appropriate amount of water to boiling and maintained for 14 min, then cooled to 40 °C. Then, alkaline protease is added to the mixture, and the pH of the mixture is adjusted to 9 with NaOH solution. Stirring enzymolysis is carried out at 45 °C for 200 min. After the enzymolysis reaction ends, an enzymolysate is obtained. The enzymolysate is heated to boiling and maintained for 10 min, then cooled to 40 °C. Centrifugation is carried out at a centrifugal speed of 4000 r·min -1 under the condition of for 5 min. The supernatant is taken and vacuum concentrated to 12% of the original volume at 83 °C to obtain a concentrate. Anhydrous ethanol is added to the concentrate to obtain mixture b. The mass concentration of ethanol in the mixture b is 50%. Alcohol precipitation is carried out at 4 °C for 12 h. Centrifugation is carried out at a centrifugal speed of 4000 r·min -1 under the condition of for 15 min. The precipitate is taken, dried, pulverized, and sieved through a 100-mesh sieve to obtain the antler glue extract.

[0138] The particle size of the antler glue powder is 60 mesh.

[0139] The mass ratio of the antler glue powder to water is 1:10.

[0140] The addition amount of the antler glue powder is 300 g.

[0141] The addition amount of the alkaline protease is 6 wt% of the antler glue powder.

[0142] The purchase source of the alkaline protease is Beijing Solarbio Science & Technology Co., Ltd.

[0143] In the second aspect of this example, an antler glue extract is provided, which is prepared by the above preparation method, and the obtained antler glue extract is labeled as JMSTD.

[0144] Example 9

[0145] In the first aspect of this example, a preparation method of an antler glue extract is provided, including the following steps:

[0146] The antler glue powder is heated with an appropriate amount of water to boiling and maintained for 10 min, then cooled to 40 °C. Then, alkaline protease is added to the mixture, and the pH of the mixture is adjusted to 11 with NaOH solution. Stirring enzymolysis is carried out at 40 °C for 60 min. After the enzymolysis reaction ends, an enzymolysate is obtained. The enzymolysate is heated to boiling and maintained for 10 min, then cooled to 40 °C, and then filtered to collect the filtrate. The filtrate is vacuum concentrated to 27% of the original volume at 80 °C to obtain a concentrate. Anhydrous ethanol is added to the concentrate to obtain mixture b. The mass concentration of ethanol in the mixture b is 60%. Alcohol precipitation is carried out at 4 °C for 20 h. Centrifugation is carried out at a centrifugal speed of 4000 r·min -1Centrifuge for 15 min under the given conditions, take the precipitate, dry it, crush it, and sieve it through a 100-mesh sieve to obtain the deer antler cream extract.

[0147] The particle size of the deer antler cream powder is 80 mesh.

[0148] The mass ratio of the deer antler cream powder to water is 1:50.

[0149] The addition amount of the deer antler cream powder is 300 g.

[0150] The addition amount of the alkaline protease is 10 wt% of the deer antler cream powder.

[0151] The purchase source of the alkaline protease is Beijing Solarbio Science & Technology Co., Ltd.

[0152] In the second aspect of this example, a deer antler cream extract is provided, which is prepared by the above preparation method, and the obtained deer antler cream extract is labeled as JMSTD.

[0153] Example 10

[0154] In the first aspect of this example, a preparation method of a deer antler cream extract is provided, including the following steps:

[0155] Add an appropriate amount of water to the deer antler cream powder, heat it to boiling and keep it for 10 min, then cool it to 40 °C, add alkaline protease to the mixture, and adjust the pH of the mixture to 11 with NaOH solution, stir and enzymatically hydrolyze at 40 °C for 60 min. After the enzymatic hydrolysis reaction ends, obtain the enzymolysate, heat the enzymolysate to boiling and keep it for 10 min, cool it to 40 °C, then filter to collect the filtrate, vacuum concentrate the filtrate to 35% of the original volume at 80 °C to obtain the concentrate, add absolute ethanol to the concentrate to obtain mixture b, the mass concentration of ethanol in the mixture b is 50%, perform alcohol precipitation at 4 °C for 40 h, and centrifuge at a centrifugal speed of 4000 r·min -1 Centrifuge for 15 min under the given conditions, take the precipitate, dry it, crush it, and sieve it through a 150-mesh sieve to obtain the deer antler cream extract.

[0156] The particle size of the deer antler cream powder is 100 mesh.

[0157] The mass ratio of the deer antler cream powder to water is 1:33.

[0158] The addition amount of the deer antler cream powder is 300 g.

[0159] The addition amount of the alkaline protease is 8 wt% of the deer antler cream powder.

[0160] The purchase source of the alkaline protease is Beijing Solarbio Science & Technology Co., Ltd.

[0161] In the second aspect of this embodiment, a pilose antler cream extract is provided, which is prepared by the above preparation method, and the obtained pilose antler cream extract is labeled as JMSTD.

[0162] Example 11

[0163] In the first aspect of this embodiment, a preparation method of a pilose antler cream extract is provided, including the following steps:

[0164] The pilose antler cream powder is heated to boiling with an appropriate amount of water and maintained for 10 min, then cooled to 40 °C. Then, alkaline protease is added to the mixture, and the pH of the mixture is adjusted to 11 with NaOH solution. Stirring enzymatic hydrolysis is carried out at 35 °C for 60 min. After the enzymatic hydrolysis reaction ends, the enzymatic hydrolysate is obtained. The enzymatic hydrolysate is heated to boiling and maintained for 10 min, cooled to 40 °C, and then filtered to collect the filtrate. The filtrate is vacuum concentrated to 22% of the original volume at 80 °C to obtain a concentrate. Absolute ethanol is added to the concentrate to obtain mixture b. The mass concentration of ethanol in the mixture b is 90%. Alcohol precipitation is carried out at 4 °C for 60 h, and then filtered or centrifuged at a centrifugal speed of 4000 r·min -1 for 15 min to take the precipitate, which is dried and then pulverized, and sieved through a 200-mesh sieve to obtain the pilose antler cream extract.

[0165] The particle size of the pilose antler cream powder is 100 mesh.

[0166] The mass ratio of the pilose antler cream powder to water is 1:20.

[0167] The addition amount of the pilose antler cream powder is 300 g.

[0168] The addition amount of the alkaline protease is 4 wt% of the pilose antler cream powder.

[0169] The purchase source of the alkaline protease is Beijing Solarbio Science & Technology Co., Ltd.

[0170] In the second aspect of this embodiment, a pilose antler cream extract is provided, which is prepared by the above preparation method, and the obtained pilose antler cream extract is labeled as JMSTD.

[0171] In the third aspect of this embodiment, an application of the pilose antler cream extract is provided, and the pilose antler cream extract is applied in the technical field of skin care products.

[0172] The fifth group of experiments

[0173] The fifth group of experiments provides a composition.

[0174] The preparation raw materials of the composition include the pilose antler cream extract JMSTD and active ingredients. By weight, its preparation raw materials include 43 - 70 parts of the pilose antler cream extract JMSTD and 30 - 57 parts of active ingredients.

[0175] The preparation method of the composition is to uniformly mix the antler cream extract JMSTD and the active ingredient to obtain the composition.

[0176] The active ingredient is selected from one of betaine, trehalose, N-acetylglucosamine, sodium polyglutamate, ectoin and collagen.

[0177] When the active ingredient is betaine, calculated by weight, the raw materials for its preparation consist of 55 - 70 parts of the antler cream extract JMSTD and 30 - 45 parts of betaine.

[0178] When the active ingredient is trehalose, calculated by weight, the raw materials for its preparation consist of 55 - 70 parts of the antler cream extract JMSTD and 30 - 45 parts of trehalose.

[0179] When the active ingredient is N-acetylglucosamine, calculated by weight, the raw materials for its preparation consist of 55 - 70 parts of the antler cream extract JMSTD and 30 - 45 parts of N-acetylglucosamine.

[0180] When the active ingredient is sodium polyglutamate, calculated by weight, the raw materials for its preparation consist of 43 - 58 parts of the antler cream extract JMSTD and 42 - 57 parts of sodium polyglutamate.

[0181] When the active ingredient is ectoin, calculated by weight, the raw materials for its preparation consist of 43 - 58 parts of the antler cream extract JMSTD and 42 - 57 parts of ectoin.

[0182] When the active ingredient is collagen, calculated by weight, the raw materials for its preparation consist of 43 - 58 parts of the antler cream extract JMSTD and 42 - 57 parts of collagen.

[0183] The fifth group of experiments includes Example 12, Example 13, Example 14, Example 15, Example 16 and Example 17.

[0184] Example 12

[0185] This example provides a composition, and calculated by weight, its raw materials for preparation consist of 60 parts of the antler cream extract JMSTD provided in Example 9 and 40 parts of betaine.

[0186] The preparation method of the composition provided in this example is to uniformly mix the antler cream extract JMSTD and betaine to obtain the composition.

[0187] Example 13

[0188] This embodiment provides a composition, the raw materials for its preparation are composed, by weight, of 60 parts of the antler frost extract JMSTD provided in Example 9 and 40 parts of trehalose.

[0189] This embodiment provides a method for preparing a composition, which comprises uniformly mixing the antler frost extract JMSTD and trehalose to obtain the composition.

[0190] Example 14

[0191] This embodiment provides a composition, the raw materials for its preparation are composed, by weight, of 60 parts of the antler frost extract JMSTD provided in Example 9 and 40 parts of acetyl glucosamine.

[0192] This embodiment provides a method for preparing a composition, which comprises uniformly mixing the antler frost extract JMSTD and acetyl glucosamine to obtain the composition.

[0193] Example 15

[0194] This embodiment provides a composition, the raw materials for its preparation are composed, by weight, of 58 parts of the antler frost extract JMSTD provided in Example 9 and 42 parts of sodium polyglutamate.

[0195] This embodiment provides a method for preparing a composition, which comprises uniformly mixing antler frost extract JMSTD and sodium polyglutamate to obtain the composition.

[0196] Example 16

[0197] This embodiment provides a composition, the raw materials for its preparation are composed, by weight, of 58 parts of the antler frost extract JMSTD provided in Example 9 and 42 parts of ectoine.

[0198] This embodiment provides a method for preparing a composition, which is to uniformly mix the antler frost extract JMSTD and ectoine to obtain the composition.

[0199] Example 17

[0200] This embodiment provides a composition, the raw materials for its preparation are composed, by weight, of 58 parts of the antler frost extract JMSTD provided in Example 9 and 42 parts of collagen.

[0201] This embodiment provides a method for preparing a composition, which comprises uniformly mixing antler frost extract JMSTD and collagen to obtain the composition.

[0202] Performance Testing

[0203] The test samples include: the aqueous extract ST of Cornu Cervi Degelatinatum provided in Example 1, the extract MST of Cornu Cervi Degelatinatum provided in Example 2, the extract SMST of Cornu Cervi Degelatinatum provided in Example 3, the extract JMST of Cornu Cervi Degelatinatum provided in Example 5, and the extract JMSTD of Cornu Cervi Degelatinatum provided in Example 9.

[0204] Preparation of sample solution: The test samples were added with water to prepare sample solutions with concentrations of 50 μg / mL and 100 μg / mL respectively.

[0205] The HaCaT cells used in the following performance test process were immortalized HaCaT human epidermal cells purchased from Shanghai Yaji Biotechnology Co., Ltd.

[0206] The complete medium used was DMEM (high glucose) medium, and the source model was Thermo Fisher Gbico.

[0207] The CCK8 used was the CCK-8 Cell Proliferation / Toxicity Detection Kit, and its source was Beijing TransGen Biotech Co., Ltd.

[0208] 1. Cell proliferation experiment:

[0209] Preparation of 10% (mass concentration) CCK-8 solution: It was prepared according to the ratio of 9:1 of the medium to the original CCK8 solution by mass.

[0210] Test method:

[0211] ① Cell preparation: The HaCaT cells were prepared into a single cell suspension of 5×10 4 cells / mL with the complete medium, inoculated into a 96-well plate (100 μL / well), and cultured for 24 h at 37 °C and 5% CO2. After the cells adhered, the medium was discarded.

[0212] ② Sample intervention: Prepare blank group cells (n = 5), and use the sample solutions of 50 μg / mL and 100 μg / mL as the intervention groups; after adding the sample solutions in the intervention groups to the cells and intervening for 48 h, discard the medium, add 100 μL of the prepared 10% (mass concentration) CCK-8 solution to each well, continue to incubate in the incubator, and measure the OD value at 450 nm with an enzyme-labeled instrument after 1 h, and calculate the cell survival rate. The test results are shown in Figure 1 .

[0213] Through Figure 1 the test results in, it can be seen that both the water in the blank control group and the sample solutions in the intervention groups are non-toxic to HaCaT cells, and ST, MST at 100 μg / mL and JMST at 50 μg / mL have a certain promoting effect on the proliferation of HaCaT cells.

[0214] 2. Test on the regulation of fatty acid synthase activity by Cornu Cervi Degelatinatum extract:

[0215] Test method:

[0216] ① Cell preparation: Prepare HaCaT cells into a single cell suspension of 5×10 4 cells / mL with complete medium, inoculate into a culture flask, and after culturing for 24 h to adhere at 37°C and 5% CO2, discard the medium.

[0217] ② Sample intervention: Use sample solutions of 50 μg / mL and 100 μg / mL as the intervention groups; prepare blank group cells and a positive control rhEGF intervention group (20 ng / mL), n = 3. After intervening with the cells for 48 h respectively; collect the cells for FAS activity detection, and the test results are shown in Figure 2 ; Collect the cells intervened with 50 μg / mL Cornu Cervi Degelatinatum extract JMSTD, extract total protein, and use the WB method to detect the expression of ACCα, FASN, and N-SMase proteins. The test results are shown in Figure 3 .

[0218] The preparation method of the rhEGF solution in the rhEGF intervention group is to dissolve rhEGF with PBS buffer solution to obtain a rhEGF solution with a mass concentration of 20 ng / mL.

[0219] It can be seen from Figure 2 the test results in that the aqueous extract ST of Cornu Cervi Degelatinatum at a high dose (100 μg / mL) can increase the activity of fatty acid synthase FAS. The Cornu Cervi Degelatinatum extract JMST in the test sample did not show the effect of promoting FAS activity, but the Cornu Cervi Degelatinatum extract JMSTD obtained after alcohol precipitation of the Cornu Cervi Degelatinatum extract can significantly increase the activity of fatty acid synthase FAS at low dose (50 μg / mL) or high dose (100 μg / mL) (P<0.05), and the amplitude of increasing the activity is better than that of other Cornu Cervi Degelatinatum extracts. And it can also be seen from Figure 2 that there are obvious differences in the effects of Cornu Cervi Degelatinatum extracts obtained with and without alcohol precipitation on the activity of fatty acid synthase FAS, which may be related to the hydrophobic aggregation of molecules or local conformational changes due to the long-term exposure of the enzymolysate to ethanol.

[0220] 3. Test on the expression of fatty acid synthase-related genes by Cornu Cervi Degelatinatum extract JMSTD:

[0221] Test method:

[0222] ① Cell preparation: Prepare HaCaT cells into a single cell suspension of 5×10 4A single cell suspension of

[0223] ② Sample intervention: Use the JMSTD sample solution of Cervi Cornu Degelatinatum extract at 50 μg / mL as the intervention group; prepare blank group cells and the positive control rhEGF intervention group (20 ng / mL), n = 3. After 48 h of cell intervention; discard the supernatant, add 1 mL of Trizol to each well to digest the cells, make the Trizol spread evenly on the cell layer, shake the cell culture flask repeatedly until the cells are digested, transfer them into a 1.5 mL EPP tube, extract total RNA, and use qRT-PCR to detect the expression of ACCα, FASN, and N-SMase genes in fatty acid synthase. The test results are shown in Figure 3 .

[0224] It can be seen through Figure 3 that the JMSTD extract of Cervi Cornu Degelatinatum can promote the expression of ACCα, FASN, and N-SMase genes in fatty acid synthase, and the effect of the JMSTD extract of Cervi Cornu Degelatinatum is equivalent to that of the positive drug rhEGF.

[0225] 4. Test on the expression of fatty acid synthase-related proteins by the JMSTD extract of Cervi Cornu Degelatinatum:

[0226] Test method:

[0227] ① Cell preparation: Prepare HaCaT cells into a single cell suspension of 5×10 4 cells / mL and inoculate them into a culture flask. After culturing for 24 h at 37 °C and 5% CO2 until they adhere, discard the culture medium.

[0228] ② Sample intervention: Use the JMSTD sample solution of Cervi Cornu Degelatinatum extract at 50 μg / mL as the intervention group; prepare blank group cells and the positive control rhEGF intervention group (20 ng / mL), n = 3. After 48 h of cell intervention, collect the cells. Extract the total protein in the cells to test the expression of the total protein, and use the WB method to detect the expression of ACCα, FASN, and N-SMase proteins in fatty acid synthase. The expression of the total protein is shown in Figure 4 and Figure 5 .

[0229] It can be seen through Figure 4 that both the JMSTD extract of Cervi Cornu Degelatinatum and the positive drug rhEGF can significantly promote the expression of ACCα, FASN, and N-SMase proteins in fatty acid synthase (P < 0.05).

[0230] Figure 5In this study, CON was the blank control group, POS was the positive control group, and JMSTD of Cornu Cervi Pantotrichum extract was the JMSTD treatment group. Through Figure 5 It can be seen that from the brightness of the bands, it is intuitive that the brightness of the ACCα, FASN, and N-SMase protein bands in the JMSTD group and the positive control group is higher than that in the blank control group, thus verifying that both Cornu Cervi Pantotrichum extract JMSTD and the positive drug rhEGF can significantly promote the expression of ACCα, FASN, and N-SMase proteins in fatty acid synthase.

[0231] 5. Test on the regulation of hyaluronic acid content by Cornu Cervi Pantotrichum extract:

[0232] ① Cell preparation: HaCaT cells were prepared into a single-cell suspension of 5×10 4 cells / mL with complete medium, inoculated into culture flasks, and cultured for 24 h at 37 °C and 5% CO2 until adherent, then the medium was discarded.

[0233] ② Sample intervention: Sample solutions of 50 μg / mL and 100 μg / mL were used as the intervention groups; blank group cells and positive control rhEGF intervention group (20 ng / mL) were prepared, n = 3. After intervening with the cells for 48 h respectively; the cell supernatants were collected for hyaluronic acid detection, and the test results are shown in Figure 6 .

[0234] Through Figure 6 The test results in the figure show that Cornu Cervi Pantotrichum extract JMSTD has a better effect on increasing the hyaluronic acid content in cells compared with aqueous extract ST of Cornu Cervi Pantotrichum, MST and SMST of Cornu Cervi Pantotrichum extract. And compared with the positive control rhEGF intervention group, the hyaluronic acid contents after cell intervention with 50 μg / mL and 100 μg / mL of Cornu Cervi Pantotrichum extract JMSTD are 1.39 times and 1.45 times that of the positive control rhEGF respectively. Moreover, Cornu Cervi Pantotrichum extract JMSTD is superior to Cornu Cervi Pantotrichum extract JMST at both 50 μg / mL and 100 μg / mL doses, indicating that alcohol precipitation treatment enhances the effect of JMSTD on increasing the HA content.

[0235] 6. Test on the expression of genes related to hyaluronic acid synthesis by Cornu Cervi Pantotrichum extract JMSTD:

[0236] ① Cell preparation: HaCaT cells were prepared into a single-cell suspension of 5×10 4 cells / mL with complete medium, inoculated into 6-well plates, and cultured for 24 h at 37 °C and 5% CO2 until adherent, then the medium was discarded.

[0237] ② Sample intervention: Use the JMSTD sample solution of Cervi Cornu Degelatinatum extract at 50 μg / mL as the intervention group; prepare blank group cells and the positive control rhEGF intervention group (20 ng / mL), n = 3. After intervening with the cells for 48 h respectively; discard the supernatant, add 1 ml of Trizol to each well to digest the cells, make the Trizol spread evenly on the cell layer, shake the cell culture flask repeatedly until the cells are digested, and transfer them into 1.5 ml EP tubes. Extract the total RNA in the cells and use qRT-PCR to detect the expression of HAS2, HAS3, and CD44 genes. The test results are shown in Figure 7 。

[0238] It can be seen through Figure 7 that the Cervi Cornu Degelatinatum extract JMSTD can promote the expression of HAS2, HAS3, and CD44 genes, and there is a significant difference in its promoting effect compared with the positive drug rhEGF (P < 0.05).

[0239] 7. Test on the expression of hyaluronic acid synthesis-related proteins by Cervi Cornu Degelatinatum extract JMSTD:

[0240] ① Cell preparation: Prepare a single-cell suspension of HaCaT cells at 5×10 4 cells / mL with complete medium, inoculate it into a culture flask, and after culturing for 24 h at 37 °C and 5% CO2 until adherent, discard the medium.

[0241] ② Sample intervention: Use the JMSTD sample solution of Cervi Cornu Degelatinatum extract at 50 μg / mL as the intervention group; prepare blank group cells and the positive control rhEGF intervention group (20 ng / mL), n = 3. After intervening with the cells for 48 h respectively; collect the cells, extract the total protein, and use Western blotting (WB method) to detect the expression of HAS2, HAS3, and CD44 proteins. The test results are shown in Figure 8 and Figure 9 。

[0242] Figure 8 are the test results of the effect of Cervi Cornu Degelatinatum extract JMSTD on the expression of hyaluronic acid synthesis-related proteins Figure 9 are the test results of Western blotting.

[0243] It can be seen through Figure 8 that the Cervi Cornu Degelatinatum extract JMSTD can significantly promote the expression of HAS2, HAS3, and CD44 proteins, and the promoting effect of the Cervi Cornu Degelatinatum extract JMSTD is similar to that of the positive drug rhEGF.

[0244] It can be seen through Figure 9It can be seen that the band brightness of HAS2, HAS3 and CD44 proteins in the JMSTD group and the positive control group is higher than that in the blank control group, verifying that the extract of Cervi Cornu Degelatinatum JMSTD and the positive drug rhEGF can both significantly promote the expression of these three proteins, namely HAS2, HAS3 and CD44.

[0245] 8. Test on the activity of fatty acid synthase of the composition:

[0246] The test samples include: the composition provided in Example 12, the composition provided in Example 13, the composition provided in Example 14, the composition provided in Example 15, and the composition provided in Example 16.

[0247] Preparation of the sample solution: The test sample is added with water to prepare a sample solution with a concentration of 50 μg / mL.

[0248] Preparation of the control group: The extract of Cervi Cornu Degelatinatum JMSTD provided in Example 9 is added with water to prepare a control group test solution with a concentration of 50 μg / mL.

[0249] Test method:

[0250] ① Cell preparation: HaCaT cells are prepared into a single cell suspension of 5×10 4 cells / mL with complete medium, inoculated into a culture flask, and cultured for 24 h at 37°C and 5% CO2 until adherent, then the medium is discarded.

[0251] ② Sample intervention: The sample solution is used as the intervention group; prepare blank group cells and the control group, and after intervening the cells for 48 h respectively; collect the cells for detecting the activity of fatty acid synthase FAS, and the test results are shown in Figure 10 .

[0252] Through Figure 10From the test results, it can be seen that there is no significant difference in the regulation of fatty acid synthase FAS activity by the compositions provided in Example 14, Example 15, and Example 17 compared to the blank group. The compositions provided in Example 12, Example 13, and Example 16 can all promote the activity of fatty acid synthase FAS (P < 0.05). For the composition provided in Example 12, which contains betaine and Cervi Cornu Degelatinatum extract JMSTD, the promoting effect of the composition on the activity of fatty acid synthase FAS is equivalent to that of Cervi Cornu Degelatinatum extract JMSTD, and the combination of betaine and Cervi Cornu Degelatinatum extract JMSTD does not improve the promoting effect on the activity of fatty acid synthase FAS. In Example 13, the combination of trehalose and Cervi Cornu Degelatinatum extract JMSTD will reduce the promoting effect of Cervi Cornu Degelatinatum extract JMSTD on the activity of fatty acid synthase FAS. The composition provided in Example 16 is composed of ectoine and Cervi Cornu Degelatinatum extract JMSTD, and its promoting effect on the activity of fatty acid synthase FAS is 2.2 times that of using Cervi Cornu Degelatinatum extract JMSTD alone. It can be seen that the combination of ectoine and Cervi Cornu Degelatinatum extract JMSTD can produce a better synergistic effect and promote the activity of fatty acid synthase FAS.

[0253] 9. Test on the regulation of cellular hyaluronic acid content by the composition:

[0254] Test method:

[0255] The test samples include: the composition provided in Example 12, the composition provided in Example 13, the composition provided in Example 14, the composition provided in Example 15, and the composition provided in Example 16.

[0256] Preparation of sample solution: The test samples are added with water to prepare a sample solution with a concentration of 50 μg / mL.

[0257] Preparation of control group: The Cervi Cornu Degelatinatum extract JMSTD provided in Example 9 is added with water to prepare a control group test solution with a concentration of 50 μg / mL.

[0258] Test method:

[0259] ① Cell preparation: The HaCaT cells are prepared into a single cell suspension of 5×10 4 cells / mL with complete medium, inoculated into a culture flask, and cultured for 24 h at 37 °C and 5% CO2 until adherent, then the medium is discarded.

[0260] ② Sample intervention: The sample solution is used as the intervention group; prepare blank group cells and the control group, and after intervening the cells for 48 h respectively; collect the cell supernatant for hyaluronic acid detection, and the test results are shown in Figure 11 . Figure 11 It is the test result of the composition regulating the cellular hyaluronic acid content.

[0261] Through Figure 11 From the test results, it can be seen that the composition provided in Example 15 did not promote the increase in the content of hyaluronic acid (HA) in cells, while the compositions provided in Example 12, Example 13, Example 14, Example 16 and Example 17 could all significantly increase the content of hyaluronic acid (HA) in cells (P < 0.05); the promoting effects of the compositions provided in Example 12, Example 13 and Example 17 on the content of hyaluronic acid (HA) in cells were comparable to those of the extract JMSTD of Cornu Cervi Degelatinatum; the promoting effects of the compositions provided in Example 14 and Example 16 on the content of hyaluronic acid (HA) in cells were 1.6 times that of using the extract JMSTD of Cornu Cervi Degelatinatum alone. It can be seen from this that the combination of betaine, trehalose, sodium polyglutamate or collagen with the extract JMSTD of Cornu Cervi Degelatinatum did not produce a synergistic effect; the combination of chitosan oligosaccharide or ectoin with the extract JMSTD of Cornu Cervi Degelatinatum could produce a better synergistic effect and promote the increase in the content of hyaluronic acid (HA) in cells.

[0262] 10. UV test:

[0263] The UV absorption characteristics of the extract JMSTD of Cornu Cervi Degelatinatum provided in Example 9 were detected by UV spectrophotometry, and the test results are shown in Figure 12 .

[0264] Through Figure 12 From the test results in [reference], it can be seen that the extract of Cornu Cervi Degelatinatum has a maximum absorption peak at about 210 nm, indicating that the extract of Cornu Cervi Degelatinatum contains peptide bonds and belongs to hydrolyzed peptides.

[0265] 11. Determination of protein content:

[0266] The protein content in JMSTD was determined using a Lowry method protein concentration assay kit. The results showed that the protein content of the samples in Example 8, Example 9, Example 10 and Example 11 was > 50%.

[0267] 12. Amino acid composition analysis:

[0268] The amino acid compositions of the water extract ST of Cornu Cervi Degelatinatum provided in Example 1, the extract MST of Cornu Cervi Degelatinatum provided in Example 2, the extract SMST of Cornu Cervi Degelatinatum provided in Example 3, the extract JMST of Cornu Cervi Degelatinatum provided in Example 5 and the extract JMSTD of Cornu Cervi Degelatinatum provided in Example 9 were determined by UHPLC-MS / MS method. The test results of the amino acid composition of the extract JMSTD of Cornu Cervi Degelatinatum are shown in Table 1; the comparison results of the amino acid compositions of the water extract ST of Cornu Cervi Degelatinatum, the extract MST of Cornu Cervi Degelatinatum, the extract SMST of Cornu Cervi Degelatinatum, the extract JMST of Cornu Cervi Degelatinatum and the extract JMSTD of Cornu Cervi Degelatinatum are shown in Table 2.

[0269] Table 1 provides the contents of 38 amino acids contained in Cervi Cornu Degelatinatum extract JMSTD (a total of 60, and Table 1 provides the contents of 38 with a content > 5 nmol / g).

[0270] Cervi Cornu Degelatinatum extract JMSTD contains 60 amino acids. From the analysis results of the amino acid composition in Table 1, it can be seen that the content of amino acid L-Glutamic acid is the highest, reaching 3670.44 nmol / g, and it contains relatively abundant amino acids such as L-Serine, L-Proline, L-Valine, L-Threonine, trans-L-Hydroxyproline, L-Leucine, L-Aspartic acid, L-Glutamine, L-Glutamic acid, L-Arginine, and L-Lysine.

[0271] There are obvious differences in the amino acid compositions of Cervi Cornu Degelatinatum extract JMSTD, aqueous extract ST of Cervi Cornu Degelatinatum, extract MST of Cervi Cornu Degelatinatum, extract SMST of Cervi Cornu Degelatinatum, and extract JMST of Cervi Cornu Degelatinatum. It can be seen from Table 2 that compared with aqueous extract ST of Cervi Cornu Degelatinatum, extract MST of Cervi Cornu Degelatinatum, extract SMST of Cervi Cornu Degelatinatum, and extract JMST of Cervi Cornu Degelatinatum, the content of amino acid L-Glutamine in Cervi Cornu Degelatinatum extract JMSTD is 2.13 - 11.8 times that of ST, MST, SMST, and JMST, and the amino acid L-Glutamic acid in Cervi Cornu Degelatinatum extract JMSTD is 3.4 - 779.3 times that of ST, MST, SMST, and JMST. And JMSTD contains D-Aspartic acid, D-Glutamic acid, L-Kynurenine, L-Cysteine, D-Cysteine, L-Cystine, D-Cystine, and L-Homocystine which are not contained in the samples of ST, MST, SMST, and JMST.

[0272] Table 1

[0273]

[0274]

[0275] Table 2

[0276]

[0277] It can be seen from the test results in Table 1 and Table 2 that the types and contents of amino acids contained in the Cornu Cervi Degelatinatum extract JMSTD provided by this application make the application of the Cornu Cervi Degelatinatum extract JMSTD in skin disease adjuvant therapy products and moisturizing and barrier protection cosmetics have high value.

Claims

1. A preparation method of pilose antler cream extract, characterized in that It includes the following steps: S1. Mix deer antler cream with water, heat to boiling, keep warm, and cool to obtain mixture a; S2. Add alkaline protease to mixture a and stir to obtain an enzymolysate; S3. Heat the enzymolysate to boiling, keep warm, cool, filter or centrifuge, and take the supernatant for vacuum concentration to obtain a concentrate; S4. Add ethanol to the concentrate for alcohol precipitation treatment, filter or centrifuge, take the precipitate, dry it, and obtain deer antler cream extract.

2. The preparation method of the pilose antler cream extract according to claim 1, characterized in that, In S1, the mass ratio of deer antler cream to water is 1:(10 - 50).

3. The method for preparing the antler frost extract according to claim 1, wherein: In S2, the addition amount of alkaline protease is 2 - 10 wt% of deer antler cream.

4. The preparation method of the pilose antler cream extract according to claim 1, characterized in that, In S2, after adding alkaline protease to mixture a, adjust the pH to 8.5 - 11.5, and carry out stirring enzymolysis at 30 - 50 °C for 50 - 200 min to obtain an enzymolysate.

5. The preparation method of the pilose antler cream extract according to any one of claims 1-4, characterized in that, In S4, add ethanol to the concentrate to obtain mixture b. The mass concentration of ethanol in mixture b is 50 - 90%, and the ethanol includes an ethanol aqueous solution with a mass concentration of 95% or absolute ethanol.

6. The preparation method of the pilose antler cream extract according to claim 1, characterized in that, In S4, the temperature for alcohol precipitation treatment is 1 - 10 °C, and the time for alcohol precipitation treatment is 12 - 60 h.

7. A pilose antler cream extract, characterized in that, The deer antler cream extract is prepared by the preparation method of the deer antler cream extract according to any one of claims 1 - 6.

8. A composition, characterized in that, The composition includes the deer antler cream extract according to claim 7 and an active ingredient.

9. The composition according to claim 8, wherein, The active ingredient includes one or a combination of two of N-acetylglucosamine and ectoin.

10. Use of the extract of Cornu Cervi Degelatinatum as described in claim 7, characterized in that, The deer antler cream extract is applied in the technical field of skin care products.

Images