Animal umbilical cord extract with functions of resisting inflammation, relieving and repairing skin as well as preparation method and application of animal umbilical cord extract

Abstract

The invention provides an animal umbilical cord extract with functions of resisting inflammation, relieving and repairing skin as well as a preparation method and application of the animal umbilical cord extract, and belongs to the technical field of animal umbilical cord extracts. According to the method, tissue blocks obtained from animal umbilical cords are subjected to primary culture and subculture, and then umbilical cord tissue cells subjected to subculture are subjected to high-glucose and low-oxygen pretreatment, so that the umbilical cord cells show unique stress reaction and regulation ability in a high-glucose and low-oxygen culture environment, and a large amount of various growth metabolism cell active factors are secreted; the animal umbilical cord extract containing a large number of bioactive factors is extracted through further post-treatment, and the bioactive factors have remarkable effects in the aspects of promoting damage repair, angiogenesis and inhibiting inflammatory response and can regulate expression of inflammatory factors such as IL-6 and TNF-alpha, so that the inflammatory response is regulated.

Description

Technical Field

[0001] The present invention relates to the technical field of animal umbilical cord extracts, and in particular to an animal umbilical cord extract having the functions of anti-inflammatory, soothing and skin repairing, and a preparation method and application thereof. Background Art

[0002] After the age of 25, human skin begins to show different degrees of aging characteristics, such as dry skin, fine lines, enlarged pores, and bad living habits, life pressure, external environment stimulation to the skin, dull skin, spots, poor skin metabolism and blood circulation, weakened skin self-defense ability, etc. A series of skin problems appear to varying degrees, seriously affecting modern people's physical and mental health and pursuit of quality of life. Therefore, skin care is particularly important in our lives, and the demand for anti-aging products is also growing.

[0003] At present, many anti-aging and repair facial care products on the market often add various peptide ingredients. Although they have significant anti-aging and repair effects, they also have some disadvantages. The active ingredients are single, and the peptide ingredients may cause skin allergic reactions, such as redness, swelling, itching, etc. Long-term use of peptide skin care products may cause the skin to become dependent on them. Once discontinued, the skin condition may deteriorate rapidly. Not only is it difficult to effectively care for problematic skin, but it will also cause skin damage of varying degrees.

[0004] In recent years, skin care products containing bioactive ingredients have been favored. With the increasing research on animal tissues such as placenta and umbilical cord, the physiological activities of placenta and umbilical cord of many animals (such as cattle, sheep, deer, donkeys, etc.) have been continuously discovered. Animal umbilical cord extract is a type of composite active ingredient derived from animal umbilical cord. Related studies have shown that umbilical cord extract has a variety of biological effects such as promoting peripheral blood circulation, promoting cell and tissue respiration, removing free radicals, moisturizing, immune activation and beauty. At present, the reported preparation methods of umbilical cord extraction mainly include: direct extraction method, supercritical CO2 extraction method and enzyme-assisted extraction method. However, the above-mentioned extraction method is simple and extensive, with low extraction efficiency and low raw material utilization rate. In addition, the method of extracting animal umbilical cord extract by directly enzymatic hydrolysis of placenta and other tissues will reduce the activity of cell bioactive factors or substances. Therefore, providing a preparation method of animal umbilical cord extract with high content of bioactive factors and high biological activity is a technical problem that needs to be solved in the prior art. Summary of the invention

[0005] The object of the present invention is to provide a method for preparing an animal umbilical cord extract having anti-inflammatory, soothing and skin repairing effects. The animal umbilical cord extract prepared by the preparation method provided by the present invention has a high content of bioactive factors and high biological activity.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing an animal umbilical cord extract having the functions of anti-inflammatory, soothing and skin repairing, comprising the following steps:

[0008] (1) pre-treating the animal umbilical cord, then removing the Wharton's jelly from the umbilical cord, and cutting it into pieces to obtain a tissue block;

[0009] (2) The tissue block obtained in step (1) is cultured in a complete culture medium, and when the cell confluence reaches 80-90%, the cell is digested with trypsin solution, and then an equal volume of culture medium is added to terminate the digestion, and the cell is centrifuged and washed with physiological saline to obtain a cell precipitate;

[0010] (3) resuspending the cell precipitate obtained in step (2) with complete culture medium, and then subculturing, and when the cell confluence reaches 50-60%, adding glucose solution for pretreatment, and then adjusting the culture state to hypoxic state, collecting the supernatant, and obtaining the supernatant solution of P3-P7 cells;

[0011] (4) The P3-P7 cell supernatant solution obtained in step (3) is centrifuged, tangentially filtered and concentrated to purify the cell supernatant to obtain an animal umbilical cord extract.

[0012] Preferably, the animal umbilical cord in step (1) is derived from at least one of pig, cattle and sheep.

[0013] Preferably, the complete culture medium in step (2) is MEM-α culture medium containing 10% fetal bovine serum.

[0014] Preferably, the primary culture time in step (2) is 7 to 10 days.

[0015] Preferably, the animal umbilical cord cells are subcultured to 3 to 6 times the age in step (3).

[0016] Preferably, in step (3), the hypoxic state is to adjust the oxygen concentration in the incubator to 5-10%; and the culture time in the hypoxic state is 12-24 hours.

[0017] Preferably, the glucose concentration in step (3) is 30-60 mM.

[0018] Preferably, the molecular weight of the membrane used for tangential flow filtration in step (4) is 2000-5000 Da; and the multiple of concentration and purification is 3-6 times.

[0019] The present invention also provides an animal umbilical cord extract prepared by the preparation method described in the above technical scheme, which has the functions of anti-inflammatory, soothing and skin repairing.

[0020] The present invention also provides the use of the animal umbilical cord extract having the anti-inflammatory, soothing and skin repairing properties described in the above technical solution in skin care products.

[0021] The present invention provides a method for preparing an animal umbilical cord extract having both anti-inflammatory, soothing and skin repairing properties, wherein the tissue block obtained from the animal umbilical cord is subjected to primary culture and subculture, and then the umbilical cord tissue cells after subculture are subjected to high sugar and hypoxia pretreatment, so that the umbilical cord cells exhibit a unique stress response and regulatory ability under a high sugar culture environment, specifically, a large amount of secretion of a variety of growth and metabolism cell active factors, namely, bioactive factors, and then further post-processing to extract an animal umbilical cord extract containing a large amount of bioactive factors, which have significant effects in promoting damage repair, angiogenesis and inhibiting inflammatory responses, and can regulate the expression of inflammatory factors such as IL-6 and TNF-α, thereby regulating inflammatory responses. Moreover, compared with normoxic culture conditions, the hypoxic conditions of cultured MSCs may lead to increased production and secretion of trophic factors, enhanced angiogenesis, and enhanced immunoregulatory activity of conditioned cells. The animal umbilical cord extract prepared by the method provided by the present invention has a high yield of bioactive substances, high biological activity, and excellent anti-inflammatory, soothing and repairing effects. The preparation method of the present invention is simple to operate, and the prepared animal umbilical cord extract can also be made into freeze-dried powder, which can better maintain the biological activity of the umbilical cord extract, is not easy to degrade during long-term storage, and is easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a bar graph of cell viability of animal umbilical cord extracts of different concentrations in the present invention;

[0023] Figure 2 It is a bar graph showing the expression of inflammatory factor IL-6 after different concentrations of animal umbilical cord extracts acted on macrophage cell line RAW264.7 in the present invention;

[0024] Figure 3 It is a bar graph showing the inhibition rate of inflammatory factor IL-6 after different concentrations of animal umbilical cord extracts act on macrophage cell line RAW264.7 in the present invention;

[0025] Figure 4 It is a bar graph showing the expression of inflammatory factor TNF-α after different concentrations of animal umbilical cord extracts acted on macrophage cell line RAW264.7 in the present invention;

[0026] Figure 5 It is a bar graph showing the inhibition rate of inflammatory factor TNF-α after different concentrations of animal umbilical cord extracts act on macrophage cell line RAW264.7;

[0027] Figure 6This is a graph showing the difference in skin moisture content of samples containing 0.05% of the animal umbilical cord extract prepared in Example 1 of the present invention;

[0028] Figure 7 This is a graph showing the difference in the red pigment content of the skin of samples containing 0.05% of the animal umbilical cord extract prepared in Example 1 of the present invention;

[0029] Figure 8 This is a graph showing the difference in transepidermal water loss rates of samples containing 0.05% of the animal umbilical cord extract prepared in Example 1 of the present invention;

[0030] Fig. 9 The figure is a statistical chart of the self-assessment satisfaction evaluation results after the sample containing 0.05% of the animal umbilical cord extract prepared in Example 1 of the present invention was applied to the face of the test subjects. DETAILED DESCRIPTION

[0031] The present invention provides a method for preparing an animal umbilical cord extract having the functions of anti-inflammatory, soothing and skin repairing, comprising the following steps:

[0032] (1) pre-treating the animal umbilical cord, then removing the Wharton's jelly from the umbilical cord, and cutting it into pieces to obtain a tissue block;

[0033] (2) The tissue block obtained in step (1) is cultured in a complete culture medium, and when the cell confluence reaches 80-90%, the cell is digested with trypsin solution, and then an equal volume of culture medium is added to terminate the digestion, and the cell is centrifuged and washed with physiological saline to obtain a cell precipitate;

[0034] (3) resuspending the cell precipitate obtained in step (2) with complete culture medium, and then subculturing, and when the cell confluence reaches 50-60%, adding glucose solution for pretreatment, and then adjusting the culture state to hypoxic state, collecting the supernatant, and obtaining the supernatant solution of P3-P7 cells;

[0035] (4) The P3-P7 cell supernatant solution obtained in step (3) is centrifuged, tangentially filtered and concentrated to purify the cell supernatant to obtain an animal umbilical cord extract.

[0036] In the present invention, unless otherwise specified, the raw materials used are conventional commercial products in the art.

[0037] The invention pre-treats the animal umbilical cord, then peels off the Wharton's jelly of the umbilical cord, and cuts it into pieces to obtain the tissue block.

[0038] In the present invention, the animal umbilical cord is preferably derived from at least one of pigs, cattle, and sheep, more preferably cattle.

[0039] In the present invention, the complete culture medium is preferably MEM-α culture medium containing 10% fetal bovine serum.

[0040] In the present invention, the pretreatment preferably includes: disinfecting the animal umbilical cord with alcohol, washing it with physiological saline, and removing the arteries and veins inside the animal umbilical cord. In the present invention, the size of the tissue block is preferably 1 mm×1 mm.

[0041] After obtaining the tissue block, the present invention uses complete culture medium to carry out primary culture on the tissue block, waits for the cell confluence to reach 80-90%, performs trypsin digestion, then adds an equal volume of culture medium to terminate digestion, and obtains cell precipitation by centrifugation and washing with physiological saline.

[0042] In the present invention, the primary culture time is preferably 7 to 10 days. In the present invention, primary cells are obtained through primary culture.

[0043] In the present invention, the pancreatic enzyme used for the pancreatic digestion is preferably recombinant pancreatic enzyme; the volume ratio of the pancreatic enzyme aqueous solution to the primary cultured umbilical cord cells is preferably 1: (2-6). In the present invention, the temperature of the pancreatic digestion is preferably 25-37°C, and the time of the pancreatic digestion is preferably 5-10 minutes.

[0044] In the present invention, the cell sedimentation is centrifuged at a speed of preferably 1300-1500 rpm, and the centrifugation time is preferably 5-10 min. In the present invention, the number of times of washing with physiological saline is preferably 2-4 times.

[0045] After obtaining the cell precipitate, the present invention resuspends the cell precipitate with complete culture medium and performs subculture. When the cell fusion degree reaches 50-60%, glucose solution is added for pretreatment, and then the culture is adjusted to a hypoxic state, and the supernatant is collected to obtain a P3-P7 generation cell supernatant solution.

[0046] In the present invention, the subculture is preferably cultured to the 3rd to 7th generation of animal umbilical cord cells. The present invention obtains umbilical cord extracts containing active proteins through subculture.

[0047] In the present invention, the hypoxic state is preferably to adjust the oxygen concentration in the incubator to 5-10%; the culture time in the hypoxic state is preferably 12-24 hours.

[0048] In the present invention, the concentration of glucose in the glucose solution is preferably 5wt%, and the volume ratio of the glucose solution to the umbilical cord cell culture medium for subculture is preferably 0.5-3mL:10ml, more preferably 1.08mL:10ml. The present invention pre-treats umbilical cord tissue cells with high sugar and low oxygen, so that umbilical cord cells exhibit unique stress response and regulatory capabilities in a high sugar and low oxygen culture environment, which is specifically manifested in the secretion of a large number of growth and metabolism cell activity factors, namely bioactive factors, thereby increasing the content of bioactive factors in the prepared animal umbilical cord extract and improving its biological activity.

[0049] After obtaining the P3-P7 generation cell supernatant solution, the present invention sequentially centrifuges, tangential flow filters and concentrates and purifies the P3-P7 generation cell supernatant solution to obtain an animal umbilical cord extract.

[0050] In the present invention, the centrifugal speed is preferably 5000-8000 r / min, more preferably 6000-7500 r / min; the centrifugal time is preferably 15-30 min, more preferably 20 min.

[0051] In the present invention, the molecular weight of the membrane used for the tangential flow filtration is preferably 2000-5000Da, more preferably 3000Da; the multiple of the concentration and purification is preferably 3-6 times.

[0052] The present invention also provides an animal umbilical cord extract prepared by the preparation method described in the above technical scheme, which has the functions of anti-inflammatory, soothing and skin repairing.

[0053] The present invention also provides the use of the animal umbilical cord extract having the anti-inflammatory, soothing and skin repairing properties described in the above technical solution in skin care products.

[0054] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] Example 1

[0056] A method for preparing an animal umbilical cord extract having anti-inflammatory, soothing and skin repairing effects, comprising the following steps:

[0057] (1) The 10 cm umbilical cord of a 6-month-old fetal bovine was disinfected with alcohol and rinsed with saline to remove the arteries and veins inside the animal's umbilical cord. The Wharton's jelly of the umbilical cord was then peeled off and shredded to obtain a tissue block with a size of 1 mm × 1 mm;

[0058] (2) The tissue block obtained in step (1) is cultured in MEM-α complete medium for 7 to 10 days, and when the cell confluence reaches 80 to 90%, trypsin digestion is performed at 25° C. for 5 minutes, and then an equal volume of culture medium is added to terminate the digestion, and the mixture is centrifuged at a speed of 1500 rpm for 5 minutes, and then an appropriate amount of physiological saline is added to wash the cell precipitate 3 times to obtain a cell precipitate;

[0059] The pancreatic enzyme used in the pancreatic digestion is recombinant pancreatic enzyme, specifically 1× pancreatic enzyme aqueous solution; the volume ratio of the pancreatic enzyme aqueous solution to the primary cultured umbilical cord cells is 1:4;

[0060] (3) resuspending the cell precipitate obtained in step (2) with complete culture medium, subculturing the animal umbilical cord cells after 3 to 6 generations, and adding glucose solution for pretreatment when the cell confluence reaches 50 to 60%, and then adjusting the culture state to hypoxia, collecting the supernatant, and obtaining the supernatant solution of P3-P7 cells;

[0061] The concentration of glucose in the glucose solution is 5wt%, and the volume ratio of the glucose solution to the umbilical cord cell culture medium for subculture is 1.08mL:10ml; the hypoxic state is to adjust the oxygen concentration in the incubator to 5%; the hypoxic state culture time is 12h;

[0062] (4) The P3-P7 cell supernatant solution obtained in step (3) is centrifuged at a speed of 7500 rpm for 20 minutes, and then tangential flow filtered using a membrane with a molecular weight of 3000 Da, and then concentrated and purified to 3 times the P3-P7 cell supernatant solution to obtain an animal umbilical cord extract.

[0063] Comparative Example 1

[0064] The preparation method of animal umbilical cord extract comprises the following steps:

[0065] (1) The 10 cm umbilical cord of a 6-month-old fetal bovine was disinfected with alcohol and rinsed with saline to remove the arteries and veins inside the animal's umbilical cord. The Wharton's jelly of the umbilical cord was then peeled off and shredded to obtain a tissue block with a size of 1 mm × 1 mm;

[0066] (2) The tissue block obtained in step (1) is cultured in complete culture medium for 7 to 10 days, and when the cell confluence reaches 80 to 90%, trypsin digestion is performed at 25° C. for 5 minutes, and then an equal volume of culture medium is added to terminate the digestion, and the cell pellet is centrifuged at a speed of 1500 rpm for 5 minutes to obtain a cell pellet, and then an appropriate amount of physiological saline is added to wash the cell pellet three times;

[0067] The pancreatic enzyme used in the pancreatic digestion is recombinant pancreatic enzyme, specifically 1× pancreatic enzyme aqueous solution; the volume ratio of the pancreatic enzyme aqueous solution to the primary cultured umbilical cord cells is 1:4;

[0068] (3) resuspending the cell precipitate obtained in step (2) in complete culture medium, subculturing the animal umbilical cord cells after 3 to 6 generations, and when the cell confluence reaches 50 to 60%, without adding glucose solution for pretreatment, adjusting the culture state to hypoxia, collecting the supernatant, and obtaining the supernatant solution of P3-P7 cells;

[0069] The hypoxic state is to adjust the oxygen concentration in the incubator to 5%; the hypoxic state culture time is 12 hours;

[0070] (4) The P3-P7 cell supernatant solution obtained in step (3) is centrifuged at a speed of 7500 rpm for 20 minutes, and then tangential flow filtered using a membrane with a molecular weight of 3000 Da, and then concentrated and purified to 3 times the P3-P7 cell supernatant solution to obtain an animal umbilical cord extract.

[0071] Comparative Example 2

[0072] A method for preparing an animal umbilical cord extract having anti-inflammatory, soothing and skin repairing effects, comprising the following steps:

[0073] (1) The 10 cm umbilical cord of a 6-month-old fetal bovine was disinfected with alcohol and rinsed with saline to remove the arteries and veins inside the animal's umbilical cord. The Wharton's jelly of the umbilical cord was then peeled off and shredded to obtain a tissue block with a size of 1 mm × 1 mm;

[0074] (2) The tissue block obtained in step (1) is cultured in complete culture medium for 7 to 10 days, and when the cell confluence reaches 80 to 90%, trypsin digestion is performed at 25° C. for 5 minutes, and then an equal volume of culture medium is added to terminate the digestion, and the cell pellet is centrifuged at a speed of 1500 rpm for 5 minutes to obtain a cell pellet, and then an appropriate amount of physiological saline is added to wash the cell pellet three times;

[0075] The pancreatic enzyme used in the pancreatic digestion is recombinant pancreatic enzyme, specifically 1× pancreatic enzyme aqueous solution; the volume ratio of the pancreatic enzyme aqueous solution to the primary cultured umbilical cord cells is 1:4;

[0076] (3) resuspending the cell precipitate obtained in step (2) with complete culture medium, subculturing the animal umbilical cord cells after 3 to 6 generations, and adding glucose solution for pretreatment when the cell confluence reaches 50 to 60%, and then adjusting the culture state to normoxic state, collecting the supernatant, and obtaining the supernatant solution of P3-P7 cells;

[0077] The concentration of glucose in the glucose solution is 5wt%, and the volume ratio of the glucose solution to the umbilical cord cell culture medium for subculture is 1.08mL:10ml; the hypoxic state is to adjust the oxygen concentration in the incubator to about 20%; the culture time in the normoxic state is 12h;

[0078] (4) The P3-P7 cell supernatant solution obtained in step (3) is centrifuged at a speed of 7500 rpm for 20 minutes, and then tangential flow filtered using a membrane with a molecular weight of 3000 Da, and then concentrated and purified to 3 times the P3-P7 cell supernatant solution to obtain an animal umbilical cord extract.

[0079] Comparative Example 3

[0080] A method for preparing an animal umbilical cord extract, comprising the following steps:

[0081] (1) The 10 cm umbilical cord of a 6-month-old fetal bovine was disinfected with alcohol and rinsed with saline to remove the arteries and veins inside the animal's umbilical cord. The Wharton's jelly of the umbilical cord was then peeled off and shredded to obtain a tissue block with a size of 1 mm × 1 mm;

[0082] (2) homogenizing the tissue block obtained in step (1), adding 0.25% trypsin aqueous solution to the homogenized tissue to prepare a mixed solution, and incubating the mixed solution in a 37° C. incubator for 24 hours; stirring the mixed solution after incubation and centrifuging it at 7500 r for 20 minutes, collecting the supernatant, and retaining the deposited tissue for a second enzymatic hydrolysis; the volume ratio of the tissue block slurry to the 0.25% trypsin aqueous solution and water is 1:5:20;

[0083] (3) adding the trypsin aqueous solution to the deposited tissue obtained in step (2) again to repeat the enzymatic hydrolysis process in step (2), then centrifuging at 7500r / min for 20min and collecting the supernatant;

[0084] (4) The supernatants collected in step (2) and step (3) were mixed; and then the enzyme was inactivated at 60° C. for 40 min.

[0085] (5) The supernatant obtained in step 4 is passed through 100 μm, 70 μm, and 40 μm filters, and then tangentially filtered using a membrane with a molecular weight of 3000 Da, and then concentrated and purified to 3 times the supernatant solution to obtain an animal umbilical cord extract.

[0086] Comparative Example 4

[0087] A method for preparing an animal umbilical cord extract, comprising the following steps:

[0088] (1) The 10 cm umbilical cord of a 6-month-old fetal bovine was disinfected with alcohol and rinsed with saline to remove the arteries and veins inside the animal's umbilical cord. The Wharton's jelly of the umbilical cord was then peeled off and shredded to obtain a tissue block with a size of 1 mm × 1 mm;

[0089] (2) homogenizing the tissue blocks obtained in step (1), adding 0.25% trypsin aqueous solution to the homogenized tissue to prepare a mixed solution, and incubating the mixed solution in a 37°C incubator for 12 hours; stirring the mixed solution after incubation and centrifuging it at 7500r for 20 minutes, collecting the supernatant, and retaining the deposited tissue for a second enzymatic hydrolysis; the volume ratio of the tissue block slurry to the 0.25% trypsin aqueous solution and water is 1:4:20.

[0090] (3) adding the trypsin aqueous solution to the deposited tissue obtained in step (2) again to repeat the enzymatic hydrolysis process in step (2), then centrifuging at 7500r / min for 20min and collecting the supernatant;

[0091] (4) The supernatants collected in step (2) and step (3) were mixed; and then the enzyme was inactivated at 60° C. for 40 min.

[0092] (5) The supernatant obtained in step 4 is passed through 100 μm, 70 μm, and 40 μm filters, and then tangentially filtered using a membrane with a molecular weight of 3000 Da, and then concentrated and purified to 3 times the supernatant solution to obtain an animal umbilical cord extract.

[0093] Detection 1: Protein content determination

[0094] The protein content of the animal umbilical cord extracts prepared in Example 1 and Comparative Examples 1 to 4 but not concentrated and purified was determined using a BCA protein quantification kit. The test results are shown in Table 1.

[0095] Table 1 Protein content of animal umbilical cord extracts prepared in Examples 1 to 3

[0096] sample Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Protein content (mg / mL) 2.84 1.82 1.67 0.58 0.46

[0097] As shown in Table 1, the animal umbilical cord extract prepared in Example 1 has the best content of active protein.

[0098] The protein content of the animal umbilical cord extract concentrate prepared in Example 1 was calibrated to 3.0±0.2 mg / mL, and then packaged. Finally, the obtained animal umbilical cord extract solution was frozen at -80°C for detection.

[0099] Test 2: Safety Evaluation

[0100] Experimental animals: 70 SPF SD rats, half male and half female, weight / age: 180g-200g / 6-8 weeks old;

[0101] Poisoning method: The animal umbilical cord extract prepared in Example 1 was used as the test substance, and was administered orally after preparation, with a gavage volume of 2 mL / 100 g. The animals were poisoned 7 days a week, and the daily poisoning time period should be the same. The poisoning dose was adjusted regularly (weekly) according to body weight to maintain the poisoning level per unit body weight unchanged, and the poisoning was continued for 90 days. During the test, all animals were poisoned in exactly the same way. The control group was subjected to the same operation using pure water.

[0102] Experimental results: The animal umbilical cord extract prepared in Example 1 was used as the test substance, and was exposed to SD rats by oral gavage for 90 consecutive days. During the poisoning period, no abnormal state or animal death was observed in the test group, and the detection values ​​of various indicators in the test group did not show toxicological changes; after the poisoning, a histopathological examination was performed, and no histopathological changes related to the test substance were found. Therefore, it is believed that: under the experimental conditions, the subchronic oral toxicity of the test substance, the animal umbilical cord extract prepared in Example 1, to rats, the dose level (NOAEL) at which no adverse effects were observed was 1000 mg / kg BW / d. According to the relevant requirements of the "Technical Guidelines for Cosmetic Safety Assessment" and "Several Measures for Optimizing Cosmetic Safety Assessment Management", the relevant toxicological endpoints are: (animal) umbilical cord extract has an acute oral toxicity LD50>5000mg / kgbw for rats, which is practically non-toxic; the raw material has no multiple skin irritation and acute eye irritation, no skin sensitization, no phototoxicity and photoallergic reaction at a concentration of 100%; the raw material has no potential gene mutation and chromosome aberration, and the subchronic oral toxicity NOAEL for rats is 1000mg / kgbw / d. There is currently no other toxicological endpoint information for this raw material. According to the repeated dose toxicity tests currently retrieved, 1000mg / kgbw / d is selected to calculate the safety margin value (MoS). Under normal circumstances, when MoS≥100, it can be determined to be safe.

[0103] Test 3: Eye irritation / corrosion chicken embryo chorioallantoic membrane test

[0104] Preparation of CAM: 9-day-old chick embryos were candled and the eggshell of the air chamber was peeled off with dental serrated curved forceps to expose the white egg membrane, being careful not to damage the integrity of the egg membrane. A drop of 0.9% sodium chloride (NaCl) solution was dripped with a pipette to moisten the egg membrane, and the inner membrane was carefully removed with forceps to ensure that the vascular membrane was not damaged. At this time, the structure of the vascular system was observed again, and its integrity and suitability for the experiment were judged.

[0105] Reaction time method: Take 0.3 mL of the test substance prepared in Example 1 and apply it to the chicken embryo chorioallantoic membrane, ensuring that at least 50% of the chicken embryo chorioallantoic membrane surface is covered by the test substance. Observe the reaction of 6 chicken embryo chorioallantoic membranes and record the time of occurrence of each toxic effect within 5 minutes of exposure.

[0106] Iritation score (IS): For experiments conducted using the reaction time method, the IS is calculated using the following formula, with the result rounded to two decimal places:

[0107] Where:

[0108] SecH (bleeding time) - the average time when bleeding begins to occur on the CAM membrane, in seconds (s);

[0109] SecL (vessellysis time) - the average time when vascular lysis begins to be observed on the CAM membrane, in seconds (s);

[0110] SecC (coagulation time) - the average time for the onset of coagulation observed on the CAM membrane, in seconds (s).

[0111] The eye irritation of the test substances is classified according to the calculated IS values ​​in Table 2. The test results are shown in Table 3.

[0112] Table 2 Evaluation of stimulation scoring method results

[0113] Stimulus score Irritation classification IS<1 Non-irritating 1≤IS<5 Mild irritation 5≤IS<10 Moderate irritation IS ≥ 10 Strong irritant / corrosive

[0114] Table 3 Chicken embryo chorioallantoic membrane test results

[0115]

[0116] It can be seen from Table 3 that the animal umbilical cord extract solution prepared in Example 1 has no irritation to the chicken embryo chorioallantoic membrane.

[0117] Test 4: In vitro anti-inflammatory and soothing - Macrophage Raw264.7 - Inflammatory factor (dual factor) evaluation

[0118] Test method: In this experiment, a model was established in which bacterial lipopolysaccharide induced the macrophage cell line RAW264.7 to secrete inflammatory factors IL-6 and TNF-α. The animal umbilical cord extract samples prepared in Example 1 with different concentrations were used as the sample group, and the sample group was Raw264.7 cells + LPS + (animal) umbilical cord extract, the negative control group was Raw264.7 cells + DMEM culture medium, the positive control group was Raw264.7 cells + LPS + Dex, and the model group was Raw264.7 cells + LPS. The anti-inflammatory and soothing effects of the samples were evaluated by testing the IL-6 and TNF-α contents in the negative control group, positive control group, sample group and model group and comparing their differences.

[0119] Cell viability test: cells in logarithmic growth phase were collected, cell density was adjusted and inoculated into 96-well plates, each well containing 100 μL of culture medium. After culturing in a CO2 cell culture incubator for 24 h, the culture medium was removed, the cells were washed twice with PBS buffer, and the animal umbilical cord extract prepared in Example 1 was diluted with physiological saline according to the final concentration shown in Table 4, and then the animals were grouped and given the drug. The test results are shown in Table 4. Figures 1 to 5 .

[0120] Table 4 Cell viability sample concentration design table

[0121]

[0122] Figure 1 The cell viability bar graph of different concentrations of animal umbilical cord extracts. Figure 1 The results showed that the cells had good viability after being treated with different concentrations of animal umbilical cord extracts.

[0123] Figure 2 This is a bar graph showing the expression of inflammatory factor IL-6 after different concentrations of animal umbilical cord extract acted on the macrophage cell line RAW264.7. Figure 3 The figure is a bar graph showing the inhibition rate of inflammatory factor IL-6 after different concentrations of animal umbilical cord extract acted on the macrophage cell line RAW264.7. Figure 2 and Figure 3 It can be seen that under the conditions of this experiment, the inhibition rates of IL-6 of the animal umbilical cord extract prepared in Example 1 at concentrations of 1.000%, 0.500%, and 0.050% were 90.5%±0.0%, 88.1%±0.1%, and 82.8%±1.4%, respectively. Compared with the model group, the P values ​​were <0.0001, <0.0001, and <0.0001, respectively, which were significantly different.

[0124] Figure 4 This is a bar graph showing the expression of inflammatory factor TNF-α after different concentrations of animal umbilical cord extract acted on the macrophage cell line RAW264.7. Figure 5 The figure is a bar graph showing the inhibition rate of inflammatory factor TNF-α after different concentrations of animal umbilical cord extract acted on the macrophage cell line RAW264.7. The inhibition rates of TNF-α of the animal umbilical cord extract prepared in Example 1 at concentrations of 1.000%, 0.500%, and 0.050% were 80.4%±0.1%, 73.3%±0.1%, and 53.7%±0.7%, respectively. Compared with the model group, the P values ​​were <0.0001, <0.0001, and <0.0001, respectively, which were significantly different, indicating that the animal umbilical cord extract prepared in Example 1 had the effect of inhibiting the expression of IL-6 and TNF-α content at concentrations of 1.000%, 0.500%, and 0.050%, and had anti-inflammatory and soothing effects.

[0125] Test 5: Evaluation of human skin repair performance

[0126] In this test, 30 people who tested positive for lactic acid stinging (18-42 years old, 15 males and 15 females) were randomly selected as subjects for lactic acid stinging screening on the face and nasolabial folds. After the subjects continued to use the sample containing 0.05% of the animal umbilical cord extract prepared in Example 1 prepared by the above-mentioned test four for 28 days, its efficacy was evaluated by the indicators in the following Table 5.

[0127] Table 5 Repair test parameter description table

[0128]

[0129] Figure 6 This is a graph showing the difference in skin moisture content of samples containing 0.05% of the animal umbilical cord extract prepared in Example 1 of the present invention. Figure 7 This is a graph showing the difference in the red pigment content of the skin of samples containing 0.05% of the animal umbilical cord extract prepared in Example 1 of the present invention. Figure 8 The difference analysis result of the skin transepidermal water loss rate of the samples containing 0.05% of the animal umbilical cord extract prepared in Example 1 of the present invention is shown in FIG. Figures 6 to 8 The results of the human efficacy evaluation experiment show that 30 subjects who screened positive for lactic acid stinging continued to use a sample containing 0.05% of the animal umbilical cord extract prepared in Example 1. After 28 days, the sample was able to reduce the skin moisture loss rate and the skin red pigment content, increase the skin moisture content, and restore the skin to normal, indicating that the sample has a repairing effect on the skin.

[0130] Self-evaluation questionnaire survey of subjects: The subjects conducted a questionnaire survey 28 days after using the sample containing 0.05% of the animal umbilical cord extract prepared in Example 1. The survey results showed that there was a significant difference compared with before using the experimental sample, indicating that the sample with this dilution had a repairing effect.

[0131] Self-evaluation of subjects: The above 30 subjects evaluated their satisfaction with the experimental samples after using them on their faces for 28 days. The evaluation results of satisfaction with the use are shown in the following table: Evaluation criteria (5 minutes): 1 point - dissatisfied; 2 points - relatively satisfied; 3 - average; 4 points - satisfied; 5 points - satisfied. The statistical score ≥ 3 points is the effective number of people who choose to be satisfied, and the data is shown in the following Table 6.

[0132] Table 6 Subjects' self-assessment results satisfaction table

[0133]

[0134]

[0135] Note: Satisfaction = (number of valid people who choose satisfaction / number of valid test participants) * 100

[0136] Fig. 9 This is a statistical chart showing the results of self-assessment of satisfaction by subjects after a sample containing 0.05% of the animal umbilical cord extract prepared in Example 1 of the present invention was applied to the face of the subjects.

[0137] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an animal umbilical cord extract having both anti-inflammatory, soothing and skin repairing effects, characterized in that: The following steps are involved: (1) pre-treating the animal umbilical cord, then removing the Wharton's jelly from the umbilical cord, and cutting it into pieces to obtain a tissue block; (2) The tissue block obtained in step (1) is cultured in a complete culture medium, and when the cell confluence reaches 80-90%, the cell is digested with trypsin solution, and then an equal volume of culture medium is added to terminate the digestion, and the cell is centrifuged and washed with physiological saline to obtain a cell precipitate; (3) resuspending the cell precipitate obtained in step (2) with complete culture medium, and then subculturing, and when the cell confluence reaches 50-60%, adding glucose solution for pretreatment, and then adjusting the culture state to hypoxic state, collecting the supernatant, and obtaining the supernatant solution of P3-P7 cells; (4) The P3-P7 cell supernatant solution obtained in step (3) is centrifuged, tangentially filtered and concentrated to purify the cell supernatant to obtain an animal umbilical cord extract.

2. The preparation method according to claim 1, characterized in that: The animal umbilical cord in step (1) is derived from at least one of pig, cattle and sheep.

3. The preparation method according to claim 1, characterized in that: The complete culture medium in step (2) is MEM-α culture medium containing 10% fetal bovine serum.

4. The preparation method according to claim 1, characterized in that: The primary culture time in step (2) is 7 to 10 days.

5. The preparation method according to claim 1, characterized in that: The animal umbilical cord cells are subcultured to 3 to 6 times in the step (3).

6. The preparation method according to claim 1, characterized in that: In the step (3), the hypoxic state is to adjust the oxygen concentration in the incubator to 5-10%; the culture time in the hypoxic state is 12-24 hours.

7. The preparation method according to claim 1, characterized in that: The glucose concentration in step (3) is 30-60 mM.

8. The preparation method according to claim 1, characterized in that: The molecular weight of the membrane used for tangential flow filtration in step (4) is 2000-5000Da; the multiple of concentration and purification is 3-6 times.

9. The animal umbilical cord extract having anti-inflammatory, soothing and skin repairing properties obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the animal umbilical cord extract having anti-inflammatory, soothing and skin repairing effects as claimed in claim 9 in skin care products.

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