An animal umbilical cord extract with anti-inflammatory soothing and skin repairing functions, and a preparation method and application thereof

Abstract

The application provides an animal umbilical cord extract with anti-inflammatory soothing and skin repairing functions, and a preparation method and application thereof, and belongs to the technical field of animal umbilical cord extracts. Tissue blocks obtained from animal umbilical cords are subjected to primary culture and subculture, and then the umbilical cord tissue cells after subculture are subjected to high-sugar and low-oxygen pretreatment, so that the umbilical cord cells exhibit unique stress response and regulation capacity in a high-sugar and low-oxygen culture environment, secrete a large amount of various growth and metabolism type cell active factors, and then are subjected to further post-treatment to obtain an animal umbilical cord extract containing a large amount of bioactive factors. The bioactive factors have remarkable effects in promoting injury repair, angiogenesis and inhibiting inflammatory response, and can regulate the expression of inflammatory factors such as IL-6 and TNF-α, so as to regulate the inflammatory response.

Description

Technical Field

[0001] This invention relates to the field of animal umbilical cord extract technology, and in particular to an animal umbilical cord extract with anti-inflammatory, soothing, and skin-repairing properties, its preparation method, and its applications. Background Technology

[0002] After the age of 25, human skin begins to show varying degrees of aging characteristics, such as dryness, fine lines and wrinkles, enlarged pores, and, coupled with poor lifestyle habits, stress, and environmental irritants, dullness, age spots, poor skin metabolism and blood circulation, and weakened skin's self-defense capabilities. A series of skin problems emerge to varying degrees, seriously affecting the physical and mental health of modern people and their pursuit of quality of life. Therefore, skin care is extremely important in our lives, and the demand for anti-aging products is increasing daily.

[0003] Currently, many anti-aging and repair facial care products on the market often contain various peptide ingredients. While these have significant anti-aging and repair effects, they also have some drawbacks. The active ingredients are limited, and peptides may trigger allergic reactions such as redness, swelling, and itching. Long-term use of peptide skincare products may lead to skin dependence, and once discontinued, skin condition may rapidly decline. Not only do they fail to effectively care for problem skin, but they can also cause varying degrees of skin damage.

[0004] In recent years, skincare products containing bioactive ingredients have become increasingly popular. With increasing research on animal tissues such as the placenta and umbilical cord, the physiological activities of the placenta and umbilical cord of many animals (such as cattle, sheep, deer, and donkeys) have been continuously discovered. Animal umbilical cord extracts are a class of complex active ingredients derived from animal umbilical cords. Related studies have shown that umbilical cord extracts have various biological functions, including promoting peripheral blood circulation, promoting cell and tissue respiration, removing free radicals, moisturizing, immune activation, and beautifying the skin. Currently, the reported methods for preparing umbilical cord extracts mainly include: direct extraction, supercritical CO2 extraction, and enzyme-assisted extraction. However, these extraction methods are simple and crude, with low extraction efficiency and low raw material utilization. Furthermore, methods that directly extract animal umbilical cord extracts using tissues such as placenta through enzymatic hydrolysis can reduce the activity of cellular bioactive factors or substances. Therefore, providing a method for preparing animal umbilical cord extracts with high bioactive factor content and high bioactivity is a technical problem that urgently needs to be solved by existing technologies. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing an animal umbilical cord extract that has anti-inflammatory, soothing, and skin-repairing properties. The animal umbilical cord extract prepared by the method provided by this invention has a high content of bioactive factors and high bioactivity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing an animal umbilical cord extract that has anti-inflammatory, soothing, and skin-repairing properties, comprising the following steps:

[0008] (1) The animal umbilical cord was pretreated, and then the Wharton's jelly was removed from the umbilical cord. After being cut into pieces, tissue blocks were obtained.

[0009] (2) The tissue blocks obtained in step (1) are cultured in a complete culture medium for primary culture. When the cell confluence reaches 80-90%, trypsin digestion is performed, and then an equal volume of culture medium is added to terminate the digestion. After centrifugation and washing with physiological saline, cell pellet is obtained.

[0010] (3) After resuspending the cell pellet obtained in step (2) with complete culture medium, it is passaged and cultured. When the cell confluence reaches 50-60%, glucose solution is added for pretreatment, and then cultured under low oxygen conditions. The supernatant is collected to obtain P3-P7 generation cell supernatant solution.

[0011] (4) The P3-P7 generation cell supernatant obtained in step (3) is subjected to centrifugation, tangential flow filtration and concentration purification to obtain animal umbilical cord extract.

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

[0013] Preferably, the complete culture medium in step (2) is MEM-α 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 in step (3) are passaged to 3-6 times their original number.

[0016] Preferably, in step (3), the low-oxygen state is achieved by adjusting the oxygen concentration in the incubator to 5-10%; the incubation time under the low-oxygen 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 concentration and purification factor 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 solution, which has anti-inflammatory, soothing and skin-repairing properties.

[0020] This invention also provides the application of the animal umbilical cord extract, which has anti-inflammatory, soothing, and skin-repairing properties as described in the above technical solution, in skin care products.

[0021] This invention provides a method for preparing an animal umbilical cord extract with anti-inflammatory, soothing, and skin-repairing properties. The method involves primary and passage culture of tissue blocks obtained from animal umbilical cords. The passaged umbilical cord cells are then pretreated with high glucose and hypoxia, causing them to exhibit unique stress responses and regulatory capabilities under high glucose conditions. Specifically, this manifests as the secretion of a large number of growth and metabolic cell-active factors, i.e., bioactive factors. Further post-processing yields an animal umbilical cord extract containing abundant bioactive factors. These bioactive factors have significant effects in promoting damage repair, angiogenesis, and inhibiting inflammatory responses. Furthermore, they can regulate the expression of inflammatory factors such as IL-6 and TNF-α, thereby modulating the inflammatory response. Moreover, compared to normoxic culture conditions, hypoxic conditions in cultured MSCs may lead to increased production and secretion of trophic factors, enhanced angiogenesis, and increased immunomodulatory activity of conditional cells. The method provided by this invention produces animal umbilical cord extracts with high bioactive content and high bioactivity, exhibiting excellent anti-inflammatory, soothing, and repairing effects. Furthermore, the preparation method of this invention is simple to operate, and the prepared animal umbilical cord extracts can also be made into freeze-dried powder, which can better maintain the bioactivity of the umbilical cord extracts, making them less prone to degradation during long-term storage, and convenient to use. Attached Figure Description

[0022] Figure 1 This is a bar graph showing the cell viability of animal umbilical cord extracts at different concentrations in this invention.

[0023] Figure 2 This is a bar graph showing the expression levels of the inflammatory factor IL-6 after different concentrations of animal umbilical cord extracts were applied to the RAW264.7 macrophage cell line in this invention.

[0024] Figure 3 The bar graph shows the inhibition rate of the inflammatory factor IL-6 after different concentrations of animal umbilical cord extracts were applied to the RAW264.7 macrophage cell line in this invention.

[0025] Figure 4 The bar graph shows the expression levels of the inflammatory factor TNF-α after different concentrations of animal umbilical cord extracts were applied to the RAW264.7 macrophage cell line in this invention.

[0026] Figure 5 The bar graph shows the inhibition rate of the inflammatory factor TNF-α after different concentrations of animal umbilical cord extracts were applied to the RAW264.7 macrophage cell line in this invention.

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

[0028] Figure 7 The graph shows the results of the differential analysis of skin red pigment content in samples containing 0.05% of the animal umbilical cord extract prepared in Example 1 in this invention.

[0029] Figure 8 The graph shows the results of the differential analysis of transepidermal water loss rate of the skin of the sample containing 0.05% of the animal umbilical cord extract prepared in Example 1 in this invention.

[0030] Figure 9 This is a statistical chart showing the self-rated satisfaction evaluation results of a sample containing 0.05% of the animal umbilical cord extract prepared in Example 1 of this invention after being applied to the face of a subject. Detailed Implementation

[0031] This invention provides a method for preparing an animal umbilical cord extract that has anti-inflammatory, soothing, and skin-repairing properties, comprising the following steps:

[0032] (1) The animal umbilical cord was pretreated, and then the Wharton's jelly was removed from the umbilical cord. After being cut into pieces, tissue blocks were obtained.

[0033] (2) The tissue blocks obtained in step (1) are cultured in a complete culture medium for primary culture. When the cell confluence reaches 80-90%, trypsin digestion is performed, and then an equal volume of culture medium is added to terminate the digestion. After centrifugation and washing with physiological saline, cell pellet is obtained.

[0034] (3) After resuspending the cell pellet obtained in step (2) with complete culture medium, it is passaged and cultured. When the cell confluence reaches 50-60%, glucose solution is added for pretreatment, and then cultured under low oxygen conditions. The supernatant is collected to obtain P3-P7 generation cell supernatant solution.

[0035] (4) The P3-P7 generation cell supernatant obtained in step (3) is subjected to centrifugation, tangential flow filtration and concentration purification to obtain animal umbilical cord extract.

[0036] Unless otherwise specified, all raw materials used in this invention are commercially available products in the art.

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

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

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

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

[0041] After obtaining the tissue block, the present invention performs primary culture of the tissue block using complete culture medium. When the cell confluence reaches 80-90%, it is digested with trypsin solution, and then an equal volume of culture medium is added to terminate the digestion. After centrifugation and washing with physiological saline, cell pellet is obtained.

[0042] In this invention, the primary culture time is preferably 7 to 10 days. This invention obtains first-generation cells through primary culture.

[0043] In this invention, the pancreatic enzyme used for 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 this invention, the temperature of the pancreatic enzyme digestion is preferably 25-37°C, and the digestion time is preferably 5-10 min.

[0044] In this invention, the preferred centrifugation speed for the cell pellet is 1300–1500 rpm; the preferred centrifugation time is 5–10 min. In this invention, the preferred number of times the physiological saline is used for washing is 2–4 times.

[0045] After obtaining the cell pellet, the present invention resuspends the cell pellet in complete culture medium and performs passage culture. When the cell confluence reaches 50-60%, glucose solution is added for pretreatment, and then cultured under low oxygen conditions. The supernatant is collected to obtain the P3-P7 generation cell supernatant solution.

[0046] In this invention, the passage culture is preferably performed using animal umbilical cord cells cultured to the 3rd to 7th generation. This invention obtains umbilical cord extracts containing active proteins through passage culture.

[0047] In this invention, the hypoxic state is preferably adjusted to an oxygen concentration of 5-10% in the incubator; the incubation time under hypoxic conditions is preferably 12-24 hours.

[0048] In this invention, the glucose concentration in the glucose solution is preferably 5 wt%, and the volume ratio of the glucose solution to the culture medium for passaged umbilical cord cells is preferably 0.5–3 mL:10 mL, more preferably 1.08 mL:10 mL. This invention pretreats umbilical cord tissue cells with high glucose and low oxygen, enabling them to exhibit unique stress responses and regulatory capabilities under high glucose and low oxygen conditions. Specifically, this manifests as the secretion of a large number of various growth and metabolic cell-active factors, i.e., bioactive factors, thereby increasing the content of bioactive factors in the prepared animal umbilical cord extract and enhancing its biological activity.

[0049] After obtaining the P3-P7 generation cell supernatant solution, the present invention sequentially centrifuges, performs tangential flow filtration and concentration purification on the P3-P7 generation cell supernatant solution to obtain animal umbilical cord extract.

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

[0051] In this invention, the molecular weight of the membrane used for tangential flow filtration is preferably 2000-5000 Da, more preferably 3000 Da; the concentration and purification factor 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 solution, which has anti-inflammatory, soothing and skin-repairing properties.

[0053] This invention also provides the application of the animal umbilical cord extract, which has anti-inflammatory, soothing, and skin-repairing properties as described in the above technical solution, in skin care products.

[0054] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0055] Example 1

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

[0057] (1) Disinfect the umbilical cord of a 6-month-old 10cm fetal calf with alcohol, rinse with physiological saline, remove the arteries and veins inside the umbilical cord, then peel off the Wharton's jelly from the umbilical cord, cut it into pieces, and obtain a tissue block with a size of 1mm×1mm.

[0058] (2) The tissue blocks obtained in step (1) are cultured in MEM-α complete medium for 7 to 10 days. When the cell confluence reaches 80 to 90%, trypsin digestion is performed at 25°C for 5 minutes. Then, an equal volume of medium is added to terminate the digestion. The cells are centrifuged at 1500 rpm for 5 minutes. Then, an appropriate amount of physiological saline is added to wash the cell pellet 3 times to obtain the cell pellet.

[0059] The pancreatic enzyme used for digestion is a recombinant pancreatic enzyme, specifically a 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) After resuspending the cell pellet obtained in step (2) with complete culture medium, it is passaged to animal umbilical cord cells of 3 to 6 times the number of generations. When the cell confluence reaches 50 to 60%, glucose solution is added for pretreatment, and then cultured under low oxygen conditions. The supernatant is collected to obtain P3-P7 generation cell supernatant solution.

[0061] The glucose concentration in the glucose solution is 5 wt%, and the volume ratio of the glucose solution to the culture medium for passaged umbilical cord cells is 1.08 mL: 10 mL; the hypoxic state is achieved by adjusting the oxygen concentration in the incubator to 5%; the culture time under hypoxic conditions is 12 h.

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

[0063] Comparative Example 1

[0064] A method for preparing animal umbilical cord extract includes the following steps:

[0065] (1) Disinfect the umbilical cord of a 6-month-old 10cm fetal calf with alcohol, rinse with physiological saline, remove the arteries and veins inside the umbilical cord, then peel off the Wharton's jelly from the umbilical cord, cut it into pieces, and obtain a tissue block with a size of 1mm×1mm.

[0066] (2) The tissue blocks obtained in step (1) are cultured in a complete culture medium for 7 to 10 days. When the cell confluence reaches 80 to 90%, trypsin digestion is performed at 25°C for 5 minutes. Then, an equal volume of culture medium is added to terminate the digestion. The cells are centrifuged at 1500 rpm for 5 minutes to obtain cell pellets. Then, an appropriate amount of physiological saline is added to wash the cell pellets 3 times.

[0067] The pancreatic enzyme used for digestion is a recombinant pancreatic enzyme, specifically a 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) After resuspending the cell pellet obtained in step (2) with complete culture medium, it is passaged to animal umbilical cord cells of 3 to 6 times the number of generations. When the cell confluence reaches 50 to 60%, it is pretreated without adding glucose solution, then cultured under low oxygen conditions, and the supernatant is collected to obtain P3-P7 generation cell supernatant solution.

[0069] The hypoxic state was achieved by adjusting the oxygen concentration in the incubator to 5%; the incubation time under the hypoxic state was 12 hours.

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

[0071] Comparative Example 2

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

[0073] (1) Disinfect the umbilical cord of a 6-month-old 10cm fetal calf with alcohol, rinse with physiological saline, remove the arteries and veins inside the umbilical cord, then peel off the Wharton's jelly from the umbilical cord, cut it into pieces, and obtain a tissue block with a size of 1mm×1mm.

[0074] (2) The tissue blocks obtained in step (1) are cultured in a complete culture medium for 7 to 10 days. When the cell confluence reaches 80 to 90%, trypsin digestion is performed at 25°C for 5 minutes. Then, an equal volume of culture medium is added to terminate the digestion. The cells are centrifuged at 1500 rpm for 5 minutes to obtain cell pellets. Then, an appropriate amount of physiological saline is added to wash the cell pellets 3 times.

[0075] The pancreatic enzyme used for digestion is a recombinant pancreatic enzyme, specifically a 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) After resuspending the cell pellet obtained in step (2) with complete culture medium, it is passaged to animal umbilical cord cells of 3 to 6 times the number of generations. When the cell confluence reaches 50 to 60%, glucose solution is added for pretreatment, and then cultured under normoxic conditions. The supernatant is collected to obtain P3-P7 generation cell supernatant solution.

[0077] The glucose concentration in the glucose solution is 5 wt%, and the volume ratio of the glucose solution to the culture medium for passaged umbilical cord cells is 1.08 mL: 10 mL; the hypoxic state is achieved by adjusting the oxygen concentration in the incubator to approximately 20%; the culture time under normoxic conditions is 12 hours.

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

[0079] Comparative Example 3

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

[0081] (1) Disinfect the umbilical cord of a 6-month-old 10cm fetal calf with alcohol, rinse with physiological saline, remove the arteries and veins inside the umbilical cord, then peel off the Wharton's jelly from the umbilical cord, cut it into pieces, and obtain a tissue block with a size of 1mm×1mm.

[0082] (2) Homogenize the tissue block obtained in step (1), add 0.25% trypsin aqueous solution to the homogenized tissue to make a mixture, and incubate in a 37°C incubator for 24 h; after incubation, stir the mixture, centrifuge at 7500 r for 20 min, collect the supernatant, and retain the deposited tissue material for two enzymatic digestions; the volume ratio of the tissue block slurry to 0.25% trypsin aqueous solution and water is 1:5:20;

[0083] (3) Add trypsin solution to the deposited tissue obtained in step (2) again to repeat the enzymatic digestion process in step (2), then centrifuge at 7500r for 20min and collect the supernatant;

[0084] (4) Mix the supernatant collected in steps (2) and (3); then inactivate the enzyme at 60°C for 40 min.

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

[0086] Comparative Example 4

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

[0088] (1) Disinfect the umbilical cord of a 6-month-old 10cm fetal calf with alcohol, rinse with physiological saline, remove the arteries and veins inside the umbilical cord, then peel off the Wharton's jelly from the umbilical cord, cut it into pieces, and obtain a tissue block with a size of 1mm×1mm.

[0089] (2) Homogenize the tissue block obtained in step (1), add 0.25% trypsin aqueous solution to the homogenized tissue to make a mixture, and incubate in a 37°C incubator for 12 h; after incubation, stir the mixture, centrifuge at 7500 r for 20 min, collect the supernatant, and retain the deposited tissue material for two enzymatic digestions; the volume ratio of the tissue block slurry to 0.25% trypsin aqueous solution and water is 1:4:20.

[0090] (3) Add trypsin solution to the deposited tissue obtained in step (2) again to repeat the enzymatic digestion process in step (2), then centrifuge at 7500r for 20min and collect the supernatant;

[0091] (4) Mix the supernatant collected in steps (2) and (3); then inactivate the enzyme at 60°C for 40 min.

[0092] (5) The supernatant obtained in step 4 was passed through 100μm, 70μm, and 40μm filters in sequence, and then subjected to tangential flow filtration using a membrane with a molecular weight of 3000Da. The supernatant was then concentrated and purified to three times its original volume to obtain the animal umbilical cord extract. 。

[0093] Test 1: Protein content determination

[0094] The protein content of the unconcentrated and unpurified animal umbilical cord extracts prepared in Example 1 and Comparative Examples 1-4 was determined using the BCA protein quantification kit. The results are shown in Table 1.

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

[0096] 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 highest content of active proteins.

[0098] The protein content of the concentrated animal umbilical cord extract prepared in Example 1 was determined to be 3.0 ± 0.2 mg / mL. The extract was then aliquoted and stored at -80°C for testing and future reference.

[0099] Test 2. Safety Evaluation

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

[0101] Method of administration: The animal umbilical cord extract prepared in Example 1 was used as the test substance. After preparation, it was administered by gavage at a volume of 2 mL / 100 g. Animals were administered the substance 7 days a week, with the same administration time each day. The dosage was adjusted regularly (weekly) according to body weight to maintain a constant level of administration per unit body weight for 90 consecutive days. All animals were administered the substance in the same manner during the experiment. The control group was administered the substance using pure water under the same procedure.

[0102] Experimental Results: The animal umbilical cord extract prepared in Example 1 was used as the test substance and administered to SD rats via oral gavage for 90 consecutive days. During the exposure period, no abnormal conditions or animal deaths were observed in the experimental group, and no toxicologically significant changes were observed in the test values ​​of any indicators. Histopathological examination after the exposure period revealed no histopathological changes related to the test substance. Therefore, it is considered that under the experimental conditions, the no-adverse-effect level (NOAEL) for the subchronic oral toxicity of the test substance, namely the animal umbilical cord extract prepared in Example 1, in rats is 1000 mg / kg BW / d. According to the relevant requirements of the "Technical Guidelines for Cosmetic Safety Assessment" and the "Several Measures for Optimizing the Management of Cosmetic Safety Assessment", the relevant toxicological endpoints are as follows: (Animal) umbilical cord extract has an acute oral LD50 of >5000 mg / kgbw in rats, indicating practically non-toxicity; at a concentration of 100%, the raw material showed no repeated skin irritation, acute eye irritation, skin sensitization, phototoxicity, or photoallergic reactions; the raw material has no potential for gene mutation or chromosomal aberration, and its subchronic oral toxicity NOAEL in rats is 1000 mg / kgbw / d. Currently, there is no information on other toxicological endpoints for this raw material. Based on the currently retrieved repeated-dose toxicity tests, a safety boundary value (MoS) of 1000 mg / kgbw / d was selected. Under normal circumstances, when MoS ≥ 100, it can be considered safe.

[0103] Test 3: Chicken embryo chorioallantoic membrane test for eye irritation / corrosiveness

[0104] Preparation of CAM: Candling of 9-day-old chicken embryos was performed. The air cell portion of the shell was removed using dental serrated forceps to expose the white membrane, handling it carefully to avoid damaging its integrity. A drop of 0.9% sodium chloride (NaCl) solution was added with a pipette to moisten the membrane, and the inner membrane was carefully removed with forceps, ensuring the vascular membrane remained undamaged. The structure of the vascular system was then observed again, and its integrity and suitability for the experiment were assessed.

[0105] Reaction time method: 0.3 mL of the test substance prepared in Example 1 was applied to the chorioallantoic membrane of chicken embryos, ensuring that at least 50% of the membrane surface was covered by the test substance. The reaction of the membranes of 6 chicken embryos was observed, and the time of occurrence of each toxic effect within 5 minutes of application was recorded.

[0106] The stimulus score (IS) is used in experiments conducted using the reaction time method. The stimulus score (IS) is calculated using the following formula, and the result is rounded to two decimal places:

[0107] In the formula:

[0108] SecH (hemorrhage time) -- the average time in which bleeding begins to occur as observed on the CAM membrane, measured in seconds (s);

[0109] SecL (vessellysis time) – the average time, measured in seconds (s), during which vascularization begins to occur as observed on the CAM membrane;

[0110] SecC (coagulation time) – the average time it takes for coagulation to begin as observed on a CAM membrane, measured in seconds (s).

[0111] Based on the calculated IS values, the eye irritation of the test substances was classified according to Table 2 below, and the test results are shown in Table 3.

[0112] Table 2 Evaluation of Stimulus Rating Method Results

[0113] IS<1 Non-irritating 1≤IS<5 Mild irritation 5≤IS<10 moderate irritation IS≥10 Strongly irritating / corrosive

[0114] Table 3 Results of the Chicken Embryo Vulonallomel Test

[0115]

[0116] As shown in Table 3, the animal umbilical cord extract solution prepared in Example 1 is non-irritating to the chorioallantoic membrane of chicken embryos.

[0117] Detection 4: In vitro anti-inflammatory and soothing evaluation of macrophage Raw264.7-inflammatory factors (dual factor)

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

[0119] Cell viability assay: Logarithmic growth phase cells were collected and seeded into 96-well plates at adjusted cell density, with 100 μL of culture medium per well. After culturing in a CO2 cell incubator for 24 h, the culture medium was aspirated, and the cells were washed twice with PBS buffer. The animal umbilical cord extract prepared in Example 1 was diluted with physiological saline according to the final concentration shown in Table 4. The cells were then administered to different groups, and the results are shown in the table below. Figures 1-5 .

[0120] Table 4. Cell viability sample concentration design table

[0121]

[0122] Figure 1 Bar graph showing cell viability of animal umbilical cord extracts at different concentrations. Figure 1 This indicates that different concentrations of animal umbilical cord extracts resulted in good cell viability after being applied to cells.

[0123] Figure 2 The bar graph shows the expression level of the inflammatory cytokine IL-6 after different concentrations of animal umbilical cord extract were applied to the RAW264.7 macrophage cell line. Figure 3 Bar graph showing the inhibition rate of the inflammatory cytokine IL-6 after different concentrations of animal umbilical cord extracts were applied to the RAW264.7 macrophage cell line. Figure 2 and Figure 3 It can be seen that, under the experimental conditions, 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, showing significant differences.

[0124] Figure 4 The bar graph shows the expression levels of the inflammatory factor TNF-α in RAW264.7 macrophage cell line after different concentrations of animal umbilical cord extracts were applied. Figure 5The bar graph shows the inhibition rate of the inflammatory factor TNF-α after different concentrations of animal umbilical cord extract were applied to the RAW264.7 macrophage cell line. The inhibition rates of TNF-α by 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, showing significant differences. This indicates that the animal umbilical cord extract prepared in Example 1 has the effect of inhibiting the expression of IL-6 and TNF-α at concentrations of 1.000%, 0.500%, and 0.050%, and has anti-inflammatory and soothing effects.

[0125] Test 5: Evaluation of Human Skin Repair Performance

[0126] In this test, 30 individuals who tested positive for lactic acid stinging (aged 18-42, 15 males and 15 females) were randomly selected as subjects. They underwent lactic acid stinging screening on the face and nasolabial folds. After the subjects used the sample containing 0.05% of the animal umbilical cord extract prepared in Example 1 for 28 days, the efficacy was evaluated using the indicators in Table 5 below.

[0127] Table 5. Description of Repair Test Parameters

[0128]

[0129] Figure 6 This is a graph showing the results of the differential analysis of skin moisture content in samples containing 0.05% of the animal umbilical cord extract prepared in Example 1 in this invention. Figure 7 This is a graph showing the results of the differential analysis of skin red pigment content in samples containing 0.05% of the animal umbilical cord extract prepared in Example 1 in this invention. Figure 8 This graph shows the results of a differential analysis of transepidermal water loss rate in the skin of samples containing 0.05% of the animal umbilical cord extract prepared in Example 1, as described in this invention. Figures 6-8 The results of the human efficacy evaluation experiment showed that after 30 subjects who tested positive for lactic acid stinging continued to use a sample containing 0.05% of the animal umbilical cord extract prepared in Example 1, the sample could reduce the rate of skin moisture loss and the content of skin red pigment, increase the skin moisture content, and restore the skin to a normal state after 28 days, indicating that the sample has a repairing effect on the skin.

[0130] Subject self-assessment questionnaire: Subjects completed a questionnaire 28 days after using the sample containing 0.05% of the animal umbilical cord extract prepared in Example 1. The results showed a significant difference compared to before using the experimental sample, indicating that the sample at this dilution has restorative effects.

[0131] Subject self-assessment: The 30 subjects evaluated their satisfaction with the experimental sample after using it on their faces for 28 days. The results of the satisfaction assessment are shown in the table below: Evaluation criteria (5 minutes): 1 point - Unsatisfied; 2 points - Somewhat satisfied; 3 points - Neutral; 4 points - Satisfied; 5 points - Satisfied. The number of valid participants who scored ≥3 points was counted, as shown in Table 6 below. 。

[0132] Table 6. Subject Self-Rating Satisfaction Table

[0133]

[0134]

[0135] Note: Satisfaction rate = (Number of valid participants who selected "satisfied" / Number of valid test participants) * 100

[0136] Figure 9 The statistical chart shows the self-rated satisfaction results of the subjects after applying a sample containing 0.05% of the animal umbilical cord extract prepared in Example 1 to the face of the subjects.

[0137] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an animal umbilical cord extract with anti-inflammatory, soothing, and skin-repairing properties, characterized in that, Includes the following steps: (1) The animal umbilical cord was pretreated, and then the Wharton's jelly was removed from the umbilical cord. After being cut into pieces, tissue blocks were obtained. (2) The tissue blocks obtained in step (1) are cultured in a complete culture medium for primary culture. When the cell confluence reaches 80-90%, trypsin digestion is performed, and then an equal volume of culture medium is added to terminate the digestion. After centrifugation and washing with physiological saline, cell pellet is obtained. (3) After resuspending the cell pellet obtained in step (2) with complete culture medium, it is passaged and cultured. When the cell confluence reaches 50-60%, glucose solution is added for pretreatment, and then cultured under low oxygen conditions. The supernatant is collected to obtain P3-P7 generation cell supernatant solution. (4) The P3-P7 generation cell supernatant obtained in step (3) was subjected to centrifugation, tangential flow filtration and concentration purification to obtain animal umbilical cord extract; The concentration of glucose in step (3) is 5 wt%, and the oxygen concentration in the hypoxia state in step (3) is 5%. The primary culture time in step (2) is 7-10 days; The animal umbilical cord cells in step (3) are passaged to 3-6 times the original number of generations.

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 pigs, cattle, and sheep.

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

4. The preparation method according to claim 1, characterized in that, The hypoxic culture time in step (3) is 12-24 hours.

5. 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 2000~5000 Da; the concentration and purification factor is 3~6 times.

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