TWI881585B - Sheep fetal tissue hydrolyzed peptide, its preparation method, composition containing the same and its use - Google Patents

Abstract

The present invention provides a method for preparing a sheep fetus tissue hydrolyzed peptide, comprising: mixing sheep fetus tissue powder and a solvent to obtain a sheep fetus tissue mixed solution; centrifuging and separating the sheep fetus tissue mixed solution to obtain a first supernatant and a first precipitate respectively; adding a first hydrolase solution to the first precipitate to mix to obtain a first hydrolyzed solution; adding a second hydrolase to the first hydrolyzed solution to obtain a second hydrolyzed solution; centrifuging and separating the second hydrolyzed solution to obtain a second supernatant and a second precipitate respectively, and the second supernatant contains the sheep fetus tissue hydrolyzed peptide. The sheep fetus tissue hydrolyzed peptide has the effects of anti-oxidation, skin whitening, anti-skin aging, anti-inflammatory and anti-allergy.

Description

Sheep fetal tissue hydrolyzed peptide, its preparation method, composition containing the same and its use

The present invention relates to a preparation method, in particular to a preparation method of hydrolyzed peptides from sheep fetal tissue. The present invention also relates to a sheep fetal tissue hydrolyzed peptide prepared by the above-mentioned preparation method, a pharmaceutical composition or a cosmetic composition containing the sheep fetal tissue hydrolyzed peptide, and the use of the sheep fetal tissue hydrolyzed peptide.

The placenta is a transitional organ formed during the gestation period of mammals. One part is differentiated from the embryonic mold and connected to the embryo, and the other part comes from the mother's endometrium. The inner layer of the placenta is the amniotic sac, which contains amniotic fluid. The placenta is attached to the uterine wall and obtains nutrients and oxygen from the mother's blood and excretes waste. After the fetus is born, the uterus will contract and expel the placenta.

Placenta has a long history of being used as medicine. In traditional Chinese medicine, placenta is called Ziheche. The pharmacopoeia records that it is sweet and salty, warm in nature, and is believed to have the effects of nourishing qi and blood, nourishing the kidneys and improving essence. In Taiwan, human placentas are treated as medical waste after childbirth, but pig placentas can still be obtained through slaughterhouses. Currently, the drugs and health products containing placenta ingredients (such as placenta extract) on the market mainly come from pig placentas.

The Paul Niehend Clinic in Switzerland has been famous in the European and American upper class and aristocratic circles for nearly a hundred years for using sheep embryos as injections and beauty cosmetics. Even the Pope and the Queen of England have received rejuvenation treatments using sheep embryo injections. Due to the scarcity of sheep embryos, it is impossible to widely promote them to a larger market. They are just rare luxury products. If there is a sufficient supply of sheep embryos, it will definitely benefit more people and consumers.

Therefore, developing a cosmetic raw material composed of active ingredients from placenta and embryonic tissues must have great market potential.

In view of this, the present invention provides a preparation method, which can extract and hydrolyze substances with multiple functions in sheep fetal tissue (including placenta and embryo).

To achieve the aforementioned purpose, the present invention provides a method for preparing hydrolyzed peptides from sheep fetus tissue, comprising: Step (A): mixing a sheep fetus tissue powder and a solvent, and extracting at a temperature greater than 0 ^℃ and less than or equal to 10 ^℃ to obtain a sheep fetus tissue mixed solution, wherein the weight of the solvent is more than 8 times the weight of the sheep fetus tissue powder, and the solvent contains water; Step (B): centrifuging and separating the sheep fetus tissue mixed solution to obtain a first supernatant and a first precipitate respectively; Step (C): adding a first hydrolase solution to the first precipitate and mixing to obtain a first hydrolyzate, wherein the first hydrolase solution contains a first hydrolase, and the content of the first hydrolase is 0.4 weight percent to 0.6 weight percent based on the total weight of the first hydrolyzate; Step (D): adding a second hydrolase to the first hydrolyzate to obtain a second hydrolyzate, wherein the content of the second hydrolase is 0.1 weight percent to 10 weight percent based on the total weight of the first hydrolyzate; and Step (E): centrifuging and separating the second hydrolyzate to obtain a second supernatant and a second precipitate, respectively, and the second supernatant contains the fetal sheep tissue hydrolyzed peptide.

Preferably, in the aforementioned step (A), the weight of the solvent is 8 to 15 times the weight of the sheep fetal tissue powder. Preferably, the weight of the solvent is 9 to 12 times the weight of the sheep fetal tissue powder.

Preferably, in the step (A), the extraction is performed for 5 to 10 hours. Preferably, the extraction is performed for 6 to 8 hours.

Preferably, in the aforementioned step (A), the extraction temperature is greater than 0 ^° C and less than or equal to 10 ^° C. For example, it may be 2 ^° C, 4 ^° C, 6 ^° C, or 8 ^° C.

Preferably, the sheep fetal tissue powder comprises sheep embryo powder, sheep placenta powder or a combination thereof.

Preferably, the sheep fetus tissue powder comprises 60 to 80 weight percent of sheep embryo powder and 20 to 40 weight percent of sheep placenta powder. In one embodiment, the sheep fetus tissue powder comprises 65 to 75 weight percent of sheep embryo powder and 25 to 35 weight percent of sheep placenta powder.

Preferably, the centrifugation in the aforementioned step (B) is performed at a temperature greater than 0 ^° C and less than or equal to 10 ^° C. For example, it may be 2 ^° C, 4 ^° C, 6 ^° C, or 8 ^° C.

According to the present invention, the first supernatant contains albumin.

Preferably, the centrifugation in the aforementioned step (B) is performed at 4000 g to 6000 g for 20 to 40 minutes.

Preferably, the volume ratio of the first hydrolase solution to the first precipitate is 5:1 to 10:1. More preferably, the volume ratio of the first hydrolase solution to the first precipitate is 8:1 to 10:1.

In one embodiment, the first hydrolysis solution comprises water and a first hydrolase.

Preferably, the first hydrolase and the second hydrolase are selected from any one of papain, pineapple protease, pepsin, trypsin, alkaline protease and combinations thereof. The first hydrolase may be the same as or different from the second hydrolase.

Preferably, the first hydrolase is papain, which can reduce the cost of the present invention.

Preferably, the first hydrolase is mixed with the first precipitate to perform a first hydrolysis reaction to obtain a first hydrolyzate, and the reaction temperature of the first hydrolysis reaction is 40 ^° C to 70 ^° C. More preferably, the reaction temperature of the first hydrolysis reaction is 55 ^° C to 65 ^° C.

Preferably, the reaction time of the first hydrolysis reaction is 2 to 6 days. For example, it can be 2, 3, 4, 5 or 6 days.

Preferably, the second hydrolase is pineapple protease, which can reduce the cost of the present invention.

Preferably, based on the total weight of the first hydrolyzate, the amount of the second hydrolase is 0.5 weight percent to 5 weight percent. Preferably, based on the total weight of the first hydrolyzate, the amount of the second hydrolase is 0.8 weight percent to 2 weight percent. For example, based on the total weight of the first hydrolyzate, the amount of the second hydrolase can be 0.9 weight percent, 1 weight percent, 1.1 weight percent, 1.3 weight percent, 1.5 weight percent.

Preferably, a second hydrolase is added to the first hydrolyzate to perform a second hydrolysis reaction to obtain a second hydrolyzate, wherein the reaction time of the second hydrolysis reaction is 2 to 6 days, for example, 2, 3, 4, 5 or 6 days.

Preferably, the centrifugation in step (E) is performed at a temperature of greater than 0 ^° C and less than or equal to 12 ^° C.

Preferably, the centrifugation in step (E) is performed at 4000 g to 6000 g for 20 min to 40 min.

To achieve the aforementioned purpose, the present invention further provides a sheep fetal tissue hydrolyzed peptide, which is prepared by the aforementioned preparation method.

To achieve the aforementioned object, the present invention further provides a cosmetic composition comprising the aforementioned sheep fetal tissue hydrolyzed peptide.

Preferably, the cosmetic is in the form of a solution, a suspension, a spray, a liquid lotion, a mousse, a serum, a gel, an emulsion, a microemulsion, a cream, an ointment, a stick, a powder or a patch.

To achieve the aforementioned object, the present invention further provides a pharmaceutical composition comprising the aforementioned hydrolyzed peptide from sheep fetus tissue, wherein the pharmaceutical composition contains an effective dose of the hydrolyzed peptide from sheep fetus tissue and a pharmaceutically acceptable carrier.

The "effective dose" mentioned in the present invention refers to an effective amount that can achieve the desired efficacy in terms of dosage and time period; according to the present invention, it means that by administering a specific range of sheep fetal tissue hydrolyzed peptides, it can reduce the IL-6 induced by lipopolysaccharide (LPS), promote the proliferation of collagen type I-C peptide and have anti-aging effects, scavenge 1,1-diphenyl-2-trinitrophenylhydrazine (1,1-diphenyl-2-picrylhydrazyl, DPPH) free radicals, reduce the release of β-hexosaminidase, reduce the melanin content of melanoma tumor cells, or inhibit tyrosinase activity.

In one embodiment, the concentration of the hydrolyzed peptides from sheep fetus tissue is 0.1 wt% to 10 wt%. In another embodiment, the concentration of the hydrolyzed peptides from sheep fetus tissue is 3 wt% to 10 wt%. In another embodiment, the concentration of the hydrolyzed peptides from sheep fetus tissue is 1.5 wt% to 8 wt%. In another embodiment, the concentration of the hydrolyzed peptides from sheep fetus tissue is 1.5 wt% to 7 wt%. In another embodiment, the concentration of the hydrolyzed peptides from sheep fetus tissue is 0.1 wt% to 7 wt%. In another embodiment, the concentration of the hydrolyzed peptides from sheep fetus tissue is 7.5 wt% to 15 wt%. In another embodiment, the concentration of the hydrolyzed peptides from sheep fetus tissue is 0.2 wt% to 2.5 wt%. In another embodiment, the concentration of the hydrolyzed peptides from sheep fetus tissue is 0.2 wt% to 1 wt%. In another embodiment, the concentration of the hydrolyzed peptides from sheep fetus tissue is 0.2 wt% to 7.5 wt%.

The carrier includes, but is not limited to, a solvent, an emulsifier, a suspending agent, a decomposer, a binding agent, an excipient, a stabilizing agent, a diluent, a gelling agent, a lubricant, a surfactant, and other similar or applicable carriers of the present invention.

To achieve the aforementioned purpose, the present invention further provides a use of the aforementioned sheep fetal tissue hydrolyzed peptides for anti-oxidation.

To achieve the aforementioned purpose, the present invention further provides a use of the aforementioned sheep fetal tissue hydrolyzed peptide, which is used for whitening skin.

To achieve the aforementioned purpose, the present invention further provides a use of the aforementioned sheep fetal tissue hydrolyzed peptides for preparing a drug for treating or inhibiting inflammatory diseases.

To achieve the aforementioned purpose, the present invention further provides a use of the aforementioned sheep fetal tissue hydrolyzed peptides, which is used to prepare anti-allergic or auxiliary drugs for regulating allergic constitutions.

The sheep fetal tissue hydrolyzed peptide prepared by the preparation method of the present invention has anti-inflammatory effect because it can reduce the IL-6 produced by LPS induction; it has anti-aging effect because it can promote the proliferation of collagen type I-C peptide; it has antioxidant effect because it can scavenge DPPH free radicals; it has the effect of inhibiting allergies because it can reduce the release of β-hexosaminidase; and it has the effect of skin whitening because it can reduce the melanin content of melanoma tumor cells and inhibit the activity of tyrosinase.

The following figures are provided to illustrate the implementation of the present invention. Those skilled in the art can easily understand the advantages and effects that can be achieved by the present invention through the contents of this manual, and can make various modifications and changes without departing from the spirit of the present invention to implement or apply the contents of the present invention.

Preparation Example 1 Preparation of Sheep Fetal Tissue Hydrolyzed Peptides

The preparation method of the hydrolyzed peptides of sheep fetus tissue of the present invention is shown in FIG1 . First, step S1 as shown in FIG1 is performed: a sheep fetus tissue powder and a solvent are mixed and extracted to obtain a sheep fetus tissue mixed solution. Specifically, in this embodiment, 100 grams (g) of sheep fetus tissue powder is composed of 30 g of sheep placenta powder (Zhenyuebo International Co., Ltd.) and 70 g of sheep embryo powder. The sheep fetus tissue powder is suspended in 1200 g of water and slowly stirred and mixed at 4 ^○ C for 8 hours to extract to obtain a sheep fetus tissue mixed solution.

Then, the step S2 shown in FIG. 1 is performed to centrifuge and separate the sheep fetal tissue mixture to obtain a first supernatant and a first precipitate. Specifically, the sheep fetal tissue mixture is centrifuged continuously at 5500 g for 30 minutes at a temperature of 10 ^○ C to separate the first supernatant and the remaining precipitate, 93.77 g of the first precipitate. The volume of the first supernatant is about 890 milliliters (mL), and the protein content measured by Bradford protein quantification method and Western blot method is about 7.0 milligrams (mg)/mL, which contains a large amount of albumin.

Then, step S3 as shown in FIG1 is performed, a first hydrolase solution is added to the first precipitate and mixed to obtain a first hydrolyzed liquid. Specifically, a first hydrolase solution containing papain (DSM, Collupulin 200MG) with a volume 9 times the volume of the first precipitate obtained in step S2 is added to the first precipitate, and a first hydrolysis reaction is performed at 60 ^° C for 3 days to obtain a first hydrolyzed liquid with a weight of 844 g, and based on the total weight of the first hydrolyzed liquid, the content of the first hydrolase is 0.5 wt%.

Next, step S4 as shown in FIG1 is performed, and a second hydrolase is added to the first hydrolyzate to obtain a second hydrolyzate. Specifically, 8.44 g of the second hydrolase, pineapple protease (Bromelain) is added to the first hydrolyzate, and the content of the second hydrolase is 1 wt% based on the total weight of the first hydrolyzate. The second hydrolysis reaction is performed at 60 ^° C for 3 days, and a second hydrolyzate is obtained, which weighs about 852 g.

Finally, the second hydrolyzate is centrifuged and separated as shown in FIG1 , to obtain a second supernatant and a second precipitate, respectively, and the second supernatant contains the fetal sheep tissue hydrolyzed peptides. Specifically, the second hydrolyzate is separated after continuous centrifugation at 5500 g for 30 minutes at a temperature of 10 ^○ C, to obtain about 950 mL of the second supernatant and the second precipitate, respectively. The second supernatant is the fetal sheep tissue hydrolyzed peptide of the present invention, and the protein content thereof measured by the Bradford protein quantification method is about 6.9 mg/mL. The second precipitate is about 23 g after drying. For subsequent testing, phenoxyethanol and pentylene glycol were added to the second supernatant for preservation for use in subsequent tests. The concentration of phenoxyethanol in the sheep fetal tissue hydrolyzed peptide sample to which phenoxyethanol and pentylene glycol were added was 1 wt%, and the concentration of pentylene glycol was 5 wt%.

Test example 1 ：Cytotoxicity test

In order to test whether the hydrolyzed peptides from sheep fetus tissue in Preparation Example 1 would affect the growth of cells, the hydrolyzed peptides from sheep fetus tissue in Preparation Example 1 were serially diluted with a phosphate buffer solution and subjected to a cytotoxicity test in different dilution groups: 50 weight percent (wt%), 25 wt%, 12.5 wt%, 6.25 wt%, 3.125 wt%, 1.5625 wt%, 0.78125 wt%, and 0.390625 wt%. Dulbecco's Modified Eagle Medium (DMEM, GIBCO) was used as a blank control group; a cell culture medium containing 10% dimethyl sulfoxide (DMSO) was used as a positive control group.

The test was conducted according to ISO10993-5. After 6000 mouse fibroblasts (NIH-3T3) were seeded in each well of a cell culture plate, each well contained 180 μL of cell culture medium. Each group of cells was treated with 20 μL of different serial dilution groups of fetal sheep tissue hydrolyzed peptides, blank control group or positive control group and cultured in an environment of 5% carbon dioxide and 37 ^℃ for 24 hours. The solution was replaced with MTT solution and cultured in an environment of 5% carbon dioxide and 37 ^℃ for 2 hours. The plate-type enzyme immunoassay was used to detect 570 The absorbance value of each group was divided by the absorbance value of the blank control group (i.e. the absorbance value of the blank control group was 100%) to calculate the cell survival rate.

In addition, mouse fibroblasts were treated with different serial dilution groups of fetal sheep tissue hydrolyzed peptides, cell culture medium (blank control group) and cell culture medium containing 10% DMSO (positive control group). After culturing for 24 hours in a 5% carbon dioxide, 37 ^℃ environment, the cells were observed under a microscope and the appearance of the cells was recorded and scored according to the standards in Table 1 below.

Table 1: Cytotoxicity test scoring criteria Level Cell responsiveness Appearance changes 0 without No obvious changes in appearance compared with the blank control group 1 Fine <20% of cells changed appearance or became rounded 2 Slight <50% of cells change appearance or become rounded 3 Moderate <70% of cells change appearance or become rounded 4 severe Severe changes, almost all cells have morphological changes

The test results are shown in Table 2 below:

Table 2: Results of cytotoxicity test of hydrolyzed peptides from sheep fetus tissue Group Test concentration 24-hour cell survival rate (%) Cell appearance scoring Cytotoxicity Assessment The hydrolyzed peptide from sheep fetus tissue of the present invention 0.390625% 100.5 ±1.25 0 No cytotoxicity 0.78125% 107.7 ±2.17 0 No cytotoxicity 1.5625% 111.7 ±1.79 ^＊ 0 No cytotoxicity 3.125% 114.8 ±1.84 ^＊ 0 No cytotoxicity 6.25% 130.5 ±0.33 ^＊ 1 No cytotoxicity 12.5% 100.3 ±2.12 3 Cytotoxic 25% 64.3 ±4.68 ^＊ 4 Cytotoxic 50% 34.1 ±5.67 ^＊ 4 Cytotoxic Positive control group DMSO 10% 2.6 ±0.16 ^＊ 4 Cytotoxic Blank control group -- 100 ±1.74 0 No cytotoxicity

Therefore, according to Table 2 above, it can be seen that the hydrolyzed peptides from sheep fetal tissue of the present invention will not reduce cell survival rate or affect cell morphology at a concentration range below 6.25%, and will not be cytotoxic to fibroblasts. Among them, the hydrolyzed peptides from sheep fetal tissue at a concentration of 6.25% only caused <20% of the cells to change in appearance or become rounded, which was only a slight change.

Test example 2 : Eye irritation test

In order to test whether the hydrolyzed peptides from sheep fetus tissue in Preparation Example 1 would cause irritation to the eyes, the hydrolyzed peptides from sheep fetus tissue in Preparation Example 1 were taken to perform the OECD TG 492 standard test for reconstructing human corneal epithelial cells. Water was used as a blank control group, and methyl acetate was used as a positive control group.

The above-mentioned fetal tissue hydrolyzed peptides, water (blank control group) and methyl acetate (positive control group) were added to the commercially available EpiOcular™ product that has passed the OECD TG 492 test standard to reconstruct the human corneal epithelial tissue model. After 30 minutes of treatment, the EpiOcular™ was washed with Dulbecco's Phosphate-Buffered Saline (DPBS) and added with DMEM culture medium (GIBCO) and placed in a 5% carbon dioxide, 37℃ cell culture incubator for 2 hours. The cell culture medium was then replaced with MTT solution and placed in a 5% carbon dioxide, 37℃ cell culture incubator for 3 hours. Finally, the absorbance at a wavelength of 70 nm was detected using a plate-type enzyme immunoassay. Divide the absorbance value of each group by the absorbance value of the blank control group, and convert the cell survival rate to the absorbance value of the blank control group as 100%. According to the judgment standard in Table 3 below (i.e., the judgment standard of OECD TG 492), judge whether the experimental substance has eye irritation.

Table 3: OECD 492 eye irritation test criteria In vitro test results Irritation Assessment Average cell survival rate ≤ 60% Irritant Average cell survival rate ＞ 60% Non-irritating

The test results are shown in Figure 2. According to the results in Figure 2, the cell survival rate treated with sheep fetal tissue hydrolyzed peptide (protein content 6.9 mg/mL) was > 60%, and there was no eye irritation.

Test example 3 : Skin irritation test

In order to test whether the hydrolyzed peptides from sheep fetus tissue in Preparation Example 1 would cause irritation to the skin, the hydrolyzed peptides from sheep fetus tissue in Preparation Example 1 were subjected to the OECD TG 439 standard test. DPBS was used as a blank control group, and 5% sodium dodecyl sulfate (SDS) was used as a positive control group.

The above-mentioned sheep fetal tissue hydrolyzed peptides, DPBS (blank control group) and 5% SDS (positive control group) were added to the commercially available product EpiDerm™ that has passed the OECD TG 439 test standard to reconstruct the human epidermal tissue model for 1 hour, then the above-mentioned EpiDerm™ was washed with Dulbecco's Phosphate-Buffered Saline (DPBS) and added to DMEM culture medium (GIBCO) and placed in a 5% carbon dioxide, 37°C cell culture incubator for 42 hours. After the cell culture medium was replaced with MTT solution, it was placed in a 5% carbon dioxide, 37°C cell culture incubator for 3 hours, and finally the absorbance at a wavelength of 570 nm was detected by a plate-type enzyme immunoassay. Divide the absorbance value of each group by the absorbance value of the blank control group (i.e., the absorbance value of the blank control group is 100%) to calculate the cell survival rate. Determine whether the experimental substance has skin irritation according to the judgment criteria in Table 4 below.

Table 4: OECD 439 Skin Irritation Test Criteria In vitro test results Irritation Assessment Average cell survival rate ≤ 50% Irritant Average cell survival rate ＞ 50% Non-irritating

The test results are shown in Figure 3. According to the results in Figure 3, the cell survival rate of the hydrolyzed peptides treated with sheep fetal tissue is greater than 50%, which is 89.98%. Therefore, it has no skin irritation and is similar to the blank control group.

Test example 4 : Anti-inflammatory test

In order to test whether the hydrolyzed peptides from sheep fetus tissue in Preparation Example 1 have the effect of anti-inflammatory and reducing inflammatory factors, the hydrolyzed peptides from sheep fetus tissue in Preparation Example 1 were taken as samples, and serially diluted with phosphate buffer solution to form different dilution groups: 5 wt%, 2.5 wt%, 1.25 wt%, 0.625 wt% and 0.3125 wt%. DMEM culture medium (GIBCO) was used as a blank control group; cell culture medium containing 0.011% p38 MAPK inhibitor: SB203580 (Sigma) was used as a positive control group.

Mouse macrophage cell line (Raw264.7) was seeded in a cell culture plate so that each well contained 6000 cells and 180 μL of cell culture medium. Each well of mouse macrophages was treated with 20 μL of different serial dilution groups of fetal tissue hydrolyzed peptides, cell culture medium (blank control group) and positive control group (containing 0.011% SB203580 cell culture medium), and then 20 μL of lipopolysaccharide was added to induce inflammatory response in mouse macrophages. The cells were cultured in an environment of 5% carbon dioxide and 37°C for 24 hours, and the upper layer of cell culture medium was taken to measure the contents of Tumor Necrosis Factor-α (TNF-α) and Interleukin 6 (IL-6). Specifically, the mouse TNF-alpha ELISA kit (Mouse TNF-alpha ELISA kit, ab208348, abcam) and the mouse IL-6 ELISA kit (Mouse IL-6 ELISA kit, ab222503, abcam) were used with a plate-type enzyme immunoassay to measure the absorbance of each group at 450 nm. The absorbance of each group was divided by the absorbance of the blank control group to convert the cytokine content of each group, that is, the absorbance of the blank control group was taken as 100%.

After the TNF-α and IL-6 tests were performed on the upper cell culture medium, the remaining cells in the culture plate were subjected to a cytotoxicity test to confirm that the concentration of the hydrolyzed peptides from the fetal sheep tissue in this test would not affect the survival rate of mouse macrophages. Specifically, the cytotoxicity test method was similar to that described in Experimental Example 1: 20 μL of MTT solution was replaced for the mouse macrophages treated in each group, and cultured for 2 hours in an environment of 5% carbon dioxide and 37°C, and finally the absorbance at a wavelength of 570 nm was detected by a plate-type enzyme immunoassay. The absorbance value of each group was divided by the absorbance value of the blank control group (i.e., the absorbance value of the blank control group was taken as 100%) to calculate the cell survival rate.

The test results are shown in Table 5 and Figure 4:

Table 5: Results of the anti-inflammatory test of hydrolyzed peptides from sheep fetus tissue Group Test concentration TNF-α content (%) IL-6 content (%) Cell survival rate (%) The hydrolyzed peptide from sheep fetus tissue of the present invention 0.3125 wt% 99.63 ±0.24 110.76 ±0.93 100.73 ±2.83 0.625 wt% 100.31 ±0.06 108.12 ±2.25 108.32 ±2.52 1.25 wt% 100.91 ±0.87 99.58 ±0.33 107.53 ±2.3 2.5 wt% 101.15 ±0.13 85.51 ±0.4 107.86 ±0.22 5 wt% 101.37 ±0.37 63.44 ±1.18 ^＊ 110.73 ±1.39 Positive control group SB203580 0.011 wt% 50.27 ±0.58 ^＊ 8.56 ±0.15 ^＊ 81.96 ±3.49 Blank control group -- 100 ±0.09 100 ±2.93 101.27 ±3.94

According to the results in Table 5 and Figure 4, the hydrolyzed peptides from sheep fetus tissue of the present invention have an inhibitory effect on interleukin 6 produced by LPS-induced mouse macrophages at a concentration of 5%. This result means that the hydrolyzed peptides from sheep fetus tissue of the present invention can inhibit the inflammatory response caused by LPS-induced mouse macrophages at a concentration of 5%, that is, the hydrolyzed peptides from sheep fetus tissue of the present invention have an anti-inflammatory effect.

Test example 5 : Anti-aging test

In order to test whether the hydrolyzed peptides from sheep fetus tissue in Preparation Example 1 have anti-aging effects, the hydrolyzed peptides from sheep fetus tissue in Preparation Example 1 were used as samples, and after being serially diluted with a phosphate buffer solution, collagen proliferation tests were performed in different dilution groups: 4 wt%, 2 wt%, 1 wt%, 0.5 wt%, 0.25 wt%, and 0.125 wt%. Cell culture medium was used as a blank control group; DMEM medium (GIBCO) containing 0.000001 wt% of TGF-β was used as a positive control group.

Human fibroblasts (Hs68) were seeded at a density of 10,000 cells per well in a cell culture plate, and then treated with different serial dilution groups of fetal tissue hydrolyzed peptides, DMEM medium (GIBCO) (blank control group), and DMEM medium (GIBCO) containing 0.000001 wt% TGF-β (positive control group). The cells were cultured in an environment of 5% carbon dioxide and 37°C for 72 hours. The upper layer of cell culture medium was taken out and the procollagen type I C-peptide (PIP) EIA kit (TAKARA) was used to detect the expression of PIP in each group at 450 °C. The absorbance value at nm was used to convert the procollagen type I-C peptide content. The absorbance value of each group was divided by the absorbance value of the blank control group (i.e., the absorbance value of the blank control group was 100%) to convert the procollagen proliferation amount.

After the above-mentioned upper layer cell culture medium was tested for the content of pre-collagen, the remaining cells in the culture plate were subjected to a cytotoxicity test to ensure that the concentration of the hydrolyzed peptides from the fetal sheep tissue in this test would not affect the survival rate of human fibroblasts. The cytotoxicity test method is as described in Experimental Example 1: the human fibroblasts that have been treated in each group are replaced with MTT solution and cultured in an environment of 5% carbon dioxide and 37°C for 2 hours. Finally, the absorbance at a wavelength of 570 nm is detected by a plate-type enzyme immunoassay instrument, and the absorbance value of each group is divided by the absorbance value of the blank control group (i.e., the absorbance value of the blank control group is 100%) to calculate the cell survival rate.

The test results are shown in Table 6 and Figure 5:

Table 6: Test results of hydrolyzed procollagen peptide content in sheep fetal tissue Group Test concentration Procollagen content (%) Cell survival (proliferation) rate (%) The hydrolyzed peptide from sheep fetus tissue of the present invention 0.125 wt% 98.5 ±9.1 118.4 ±6.8 0.25 wt% 99.7 ±3.1 111.6 ±20.4 0.5 wt% 112.9 ±6.1 138.5 ±9.1* 1 wt% 123.4 ±1.4 153 ±12.4* 2 wt% 139 ±9.9* 159.5 ±16.1* 4 wt% 156.8 ±12.7* 201.7 ±21.1* Positive control group 0.000001 wt% 132.9 ±16.8* 113.8 ±7.8* Blank control group -- 100 ±9.3 100 ±6.2

According to the results in Table 6 and Figure 5, the hydrolyzed peptides from sheep fetal tissue of the present invention can induce human fibroblasts to express procollagen type I-C peptide when the concentration is higher than 2 wt%, thus having an anti-skin aging effect. And when the concentration is higher than 2 wt%, it will not reduce the cell survival rate.

Test example 6 ：Antioxidant activity test

In order to test whether the hydrolyzed peptides from sheep fetus tissue in Preparation Example 1 have antioxidant effects, the hydrolyzed peptides from sheep fetus tissue in Preparation Example 1 were used as samples, and the DPPH free radical scavenging ability test was performed in different dilution groups: 5 wt%, 2.5 wt%, 1.25 wt%, 0.625 wt%, 0.3125 wt%, and 0.15625 wt% after serial dilution with phosphate buffer solution. Water was used as a blank control group; and 0.05 wt% vitamin C was used as a positive control group.

The DPPH free radical scavenging test was performed using the DPPH Antioxidant Assay kit (ab289847, abcam) with different serial dilution groups of fetal tissue hydrolyzed peptides, water (blank control group) and 0.05 wt% vitamin C (positive control group). Specifically, 160 μL of each treatment group was reacted with 40 μL of DPPH solution for 30 minutes, and the absorbance at 570 nm was detected by a plate-type enzyme immunoassay instrument to calculate the DPPH free radical scavenging ability. The DPPH scavenging ability was calculated using the following formula: [1-(absorbance of the sample at 517 nm)/(absorbance of the blank control group at 517 nm)] × 100%. The DPPH free radical scavenging ability of the blank control group was set to 0%.

The test results are shown in Figure 6. The hydrolyzed peptides from sheep fetus tissue have the ability to scavenge DPPH free radicals when the concentration is higher than 0.15625%. The higher the concentration of the hydrolyzed peptides from sheep fetus tissue, the stronger the DPPH free radical scavenging ability, showing a dose-dependent proportional relationship.

Test example 7 : Suppression of allergy test

In order to test whether the sheep fetal tissue hydrolyzed peptides of Preparation Example 1 have the effect of inhibiting allergies, the sheep fetal tissue hydrolyzed peptides of Preparation Example 1 were taken as samples, and after serial dilution with phosphate buffer solution, different dilution groups: 10 wt%, 5 wt%, 2.5 wt%, 1.25 wt% and 0.625 wt% were used for the allergy inhibition test. Eagle's Minimum Essential Medium (EMEM, GIBCO) was used as the blank control group; the cell culture medium containing 0.003 wt% quercetin was used as the positive control group.

Rat mast cells (RBL-2H3) were seeded in a cell culture plate so that 6,000 cells were present in each well. 40 μL of 100 μM calcium carrier A23187 was added to each well as an immunostimulant to induce rat mast cells to degranulate and release β-hexosaminidase. After 60 minutes of treatment, 20 μL of different serial dilution groups of sheep fetal tissue hydrolyzed peptides, cell culture medium (blank control group) and cell culture medium containing 0.003 wt% quercetin (positive control group) were added respectively. The cells were cultured for 2 hours in an environment of 5% carbon dioxide and 37°C. The upper layer of cell culture medium was taken and the β-hexosaminidase content was measured. Specifically, the mouse beta-hexosaminidase A/ Beta Hex A ELISA kit (Assay Genie) was used with a plate-type enzyme immunoassay analyzer to measure the absorbance of each group at 450 nm. The absorbance of each group was divided by the absorbance of the blank control group (i.e., the absorbance of the blank control group was taken as 100%) to calculate the β-hexosaminidase content of each group.

After the β-hexosaminidase content test of the upper cell culture medium was performed, the remaining cells in the culture plate were subjected to a cytotoxicity test to ensure that the concentration of the hydrolyzed peptides from the fetal sheep tissue in this test would not affect the survival rate of rat mast cells. The cytotoxicity test method was as described in Experimental Example 1: After the rat mast cells treated in each group were replaced with MTT solution, they were cultured in an environment of 5% carbon dioxide and 37°C for 2 hours. Finally, the absorbance value at a wavelength of 570 nm was detected by a plate-type enzyme immunoassay (i.e., the absorbance value of the blank control group was 100%), and the absorbance value of each group was divided by the absorbance value of the blank control group to calculate the cell survival rate.

The test results are shown in Table 7 and Figure 7:

Table 7: Results of anti-allergy test of hydrolyzed peptides from sheep fetus tissue Group Test concentration β-hexosaminidase Cell survival rate (%) The hydrolyzed peptide from sheep fetus tissue of the present invention 0.625 wt% 102.1 ±2.14 97.21 ±3.34 1.25 wt% 105.3 ±0.83 94.58 ±1.77 2.5 wt% 99.3 ±6.3 94.46 ±2.77 5 wt% 95.9 ±4.33 97.06 ±2.1 10 wt% 48.2 ±1.75 ^＊ 97.37 ±2.4 Positive control group quercetin 0.003 wt% 3.3 ±1.2 ^＊ 106.17 ±6.39 Blank control group -- 100 ±6.13 100 ±3.04

According to the results in Table 7 and Figure 7, when the concentration of the hydrolyzed peptides from sheep fetus tissue of the present invention reaches 10 wt%, the amount of β-hexosaminidase expressed by the mouse mast cell line induced by the immunopromoter A23187 is significantly reduced to 48.2±1.75%. Therefore, when the hydrolyzed peptides from sheep fetus tissue of the present invention are at a concentration of 10%, they have the effect of inhibiting allergies.

Test example 8 : Whitening test I

In order to test whether the fetal tissue hydrolyzed peptides of Preparation Example 1 have whitening effects, the fetal tissue hydrolyzed peptide samples of Preparation Example 1 were serially diluted with a phosphate buffer solution and then tested for melanin content in mouse melanoma cells at different dilution groups: 1.25 wt%, 0.625 wt% and 0.3125 wt%. The DMEM culture medium was used as a blank control group; the cell culture medium containing 0.0125 wt% arbutin was used as a positive control group.

Mouse melanoma cells (B16-F10) were seeded in cell culture plates with 7000 cells per well. Each well of mouse melanoma cells was treated with 20 μL of different serial dilution groups of fetal tissue hydrolyzed peptides, cell culture medium (blank control group) and cell culture medium containing 0.0125 wt% arbutin (positive control group). B16-F10 cells were induced with α-Melanocyte-stimulating hormone (α-MSH) and cultured for 48 hours in a 5% carbon dioxide, 37 °C environment. The mouse melanoma cells were removed using trypsin/EDTA and dissolved in sodium hydroxide (NaOH) solution. The absorbance at 405 nm was detected using a plate-type enzyme immunoassay. The absorbance value of each group was divided by the absorbance value of the blank control group to calculate the melanin content of each group, that is, the absorbance value of the blank control group was taken as 100%.

A cytotoxicity test was performed. Specifically, according to Sissolak, Bernhard, et al. (2019). Application of the bradford assay for cell lysis Quantification: Residual protein content in cell culture supernatants. Biotechnology Journal, 14(7), 1800714, the cell solution dissolved in sodium hydroxide was added to the Bradford protein quantitative detection reagent and placed at room temperature for shaking reaction for 5 minutes. The absorbance at a wavelength of 595 nm was detected by a plate-type enzyme immunoassay. The absorbance value of each group was divided by the absorbance value of the blank control group (i.e., the absorbance value of the blank control group was 100%) to calculate the cell survival rate of each group.

The test results are shown in Table 8 and Figure 8 below:

Table 8: Melanin content test of melanoma cells using hydrolyzed peptides from sheep fetus tissue Group Test concentration Melanin content (%) Cell survival rate The hydrolyzed peptide from sheep fetus tissue of the present invention 0.3125 wt% 72.8 ±1.8* 105.5 ±2.1 0.625 wt% 59.7 ±2.6* 98.4 ±2.4 1.25 wt% 46.7 ±2.6* 89.9 ±1.6 Positive control group Arbutin 0.0125 wt% 41 ±4.3* 85.3 ±0.5 Blank control group -- 100 ±6.7 100 ±1

According to the results in Table 8 and FIG. 8 , when the hydrolyzed peptide from sheep fetal tissue of the present invention is at a concentration of 0.3125% or above, it has a good effect of inhibiting the production of melanin, that is, it has a skin whitening effect, and the inhibitory effect is dose-dependent.

Test example 9 : Whitening test II

In order to test whether the sheep fetal tissue hydrolyzed peptides in Preparation Example 1 have whitening effects, the sheep fetal tissue hydrolyzed peptide samples in Preparation Example 1 were serially diluted with a phosphate buffer solution and then subjected to a tyrosinase inhibition test in different dilution groups: 1.25 wt%, 0.625 wt% and 0.3125 wt%. The cell culture medium was used as a blank control group; the cell culture medium containing 0.136 wt% arbutin was used as a positive control group.

Different serial dilution groups of fetal tissue hydrolyzed peptides, cell culture medium (blank control group) and cell culture medium containing 0.136 wt% arbutin (positive control group) were reacted with the melanin-regulating enzyme-tyrosinase (Mushroom Tyrosinase, Sigma, 10 Units) solution for 20 minutes, and then L-tyrosine solution was added as a matrix to react for 20 minutes. Finally, the absorbance at 475 nm was detected by a plate-type enzyme immunoassay instrument and the tyrosinase activity was calculated. The absorbance value of each group was divided by the absorbance value of the blank control group (i.e., the absorbance value of the blank control group was 100%) to calculate the cell survival rate of each group.

The test results are shown in Figure 9. It can be seen that after each dilution group is treated, the activity of tyrosinase is lower than that of the blank control group. Therefore, the hydrolyzed peptide of sheep fetal tissue of the present invention has a whitening effect. And the concentration is above 0.3125 wt%, which is significantly different from the blank control group. The concentration of 0.625 wt% can achieve an effect similar to that of the positive control group, and above 1.25 wt% is better than the positive control group.

In summary, the sheep fetal tissue hydrolyzed peptide prepared by the preparation method of the present invention has anti-inflammatory effect because it can reduce the IL-6 produced by LPS; it has anti-aging effect because it can promote the proliferation of type I collagen-C peptide; it has antioxidant effect because it can scavenge DPPH free radicals; it has the effect of inhibiting allergies because it can reduce the release of β-hexosaminidase; and it has the effect of skin whitening because it can reduce the melanin content of melanoma tumor cells and inhibit tyrosinase.

Therefore, the above is only an embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any technician familiar with the profession can make some changes or modifications to the technical contents disclosed above into equivalent embodiments within the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

without

FIG. 1 is a flow chart of the preparation method of the hydrolyzed peptides of sheep fetal tissue of the present invention; FIG. 2 is the eye irritation test result of the hydrolyzed peptides of sheep fetal tissue of the present invention, * P <0.05, indicating that there is a statistically significant difference from the blank control group; FIG. 3 is the skin irritation test result of the hydrolyzed peptides of sheep fetal tissue of the present invention, * P <0.05, indicating that there is a statistically significant difference from the blank control group; FIG. 4 is the anti-inflammatory test result of the hydrolyzed peptides of sheep fetal tissue of the present invention, * P <0.05, indicating that there is a statistically significant difference from the blank control group; FIG. 5 is the collagen proliferation test result of the hydrolyzed peptides of sheep fetal tissue of the present invention, * P <0.05, indicating that there is a statistically significant difference from the blank control group; FIG. 6 is the DPPH scavenging ability test result of the hydrolyzed peptides from sheep fetal tissue of the present invention, * P <0.05, indicating that there is a statistically significant difference from the blank control group; FIG. 7 is the β-hexosaminidase content inhibition test result of the hydrolyzed peptides from sheep fetal tissue of the present invention, * P <0.05, indicating that there is a statistically significant difference from the blank control group; FIG. 8 is the melanin production inhibition test result of the hydrolyzed peptides from sheep fetal tissue of the present invention on melanoma tumors, * P <0.05, indicating that there is a statistically significant difference from the blank control group; FIG. 9 is the tyrosinase inhibition test result of the hydrolyzed peptides from sheep fetal tissue of the present invention, * P <0.05, indicating that there is a statistically significant difference from the blank control group.

without

Claims (10)

A method for preparing hydrolyzed peptides from sheep fetus tissue comprises: step (A): mixing sheep fetus tissue powder and a solvent, and extracting at a temperature of greater than 0 ^° C to less than or equal to 10 ^° C to obtain a sheep fetus tissue mixed solution, wherein the sheep fetus tissue powder comprises 60 weight percent to 80 weight percent of sheep embryo powder and 20 weight percent to 40 weight percent of sheep placenta powder, the weight of the solvent is more than 8 times the weight of the sheep fetus tissue powder, and the solvent comprises water; step (B): centrifuging and separating the sheep fetus tissue mixed solution to obtain a first supernatant and a first precipitate respectively; Step (C): adding a first hydrolase solution to the first precipitate to carry out a first hydrolysis reaction for 2 to 6 days to obtain a first hydrolyzed liquid, wherein the first hydrolase solution comprises a first hydrolase in an amount of 0.4 to 0.6 weight percent based on the total weight of the first hydrolyzed liquid, and the first hydrolase is papain; Step (D): adding a second hydrolase to the first hydrolyzate to carry out a second hydrolysis reaction for 2 to 6 days to obtain a second hydrolyzate, wherein the content of the second hydrolase is 0.1 weight percent to 10 weight percent based on the total weight of the first hydrolyzate, and the second hydrolase is pineapple protease; and Step (E): centrifuging and separating the second hydrolyzate to obtain a second supernatant and a second precipitate, respectively, and the second supernatant contains the sheep fetal tissue hydrolyzed peptides. A sheep fetal tissue hydrolyzed peptide prepared by the method as described in claim 1. A cosmetic composition comprising the hydrolyzed peptide from sheep fetus tissue as described in claim 2. A cosmetic composition as described in claim 3, wherein the dosage form of the cosmetic composition comprises a solution, a suspension, a spray, a liquid lotion, a mousse, a serum, a gel, an emulsion, a microemulsion, a cream, an ointment, a stick, a powder or a patch. A pharmaceutical composition comprising the hydrolyzed peptide from sheep fetal tissue as described in claim 2, wherein the pharmaceutical composition contains an effective dose of the hydrolyzed peptide from sheep fetal tissue and a pharmaceutically acceptable carrier. A use of the hydrolyzed peptides from sheep fetal tissue as described in claim 2, which is used for preparing antioxidant drugs or cosmetics. A use of the hydrolyzed peptide from sheep fetal tissue as described in claim 2, which is used for preparing skin whitening drugs or cosmetics. A use of the hydrolyzed peptide from sheep fetal tissue as described in claim 2, which is used for preparing anti-skin aging drugs or cosmetics. A use of the hydrolyzed peptides from sheep fetal tissue as described in claim 2, which is used to prepare a drug for treating or inhibiting inflammatory diseases. A use of the hydrolyzed peptides from sheep fetal tissue as described in claim 2, which is used to prepare drugs for anti-allergic or auxiliary regulation of allergic constitution.