Active factor-rich sheep placenta extract, and preparation method and application thereof

Abstract

The present application relates to a kind of active factor-rich sheep placenta extract and its preparation method and application.The preparation method includes the following steps: S1, sheep placenta is pretreated into placenta homogenate liquid;S2, the separation and enrichment of placenta-derived active stem cell group;S3, stem cell directional activation fermentation;S4, double source merging and low-temperature controllable enzymolysis, the sheep placenta extract is obtained.The preparation method separates endogenous stem cells from fresh placenta, realizes the efficient enrichment of active cytokines by directional suspension fermentation, and maximizes the natural biological activity of cytokines;On the other hand, the waste in the conventional process such as filtration residue, centrifugal supernatant is all recycled and enzymolysis, the protein and polypeptide nutrient ingredients in placenta tissue are fully extracted, the full-component high-value utilization of placenta raw material is realized, and the problems of activity loss, material waste and production cost increase caused by step-by-step independent process are avoided.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and cosmetic raw material preparation technology, and specifically relates to a sheep placenta extract rich in active factors, its preparation method and application. Background Technology

[0002] The placenta is a temporary organ that exists within the mother's body, its main function being to regulate the transport of gases, nutrients, and waste products between the mother and fetus during pregnancy. In recent years, scientists have developed placental extracts based on its biological characteristics. Sheep placental extract is rich in amino acids, bioactive peptides, nucleic acids, growth factors, and other active substances, possessing effects such as repairing tissue damage, beautifying the skin, anti-oxidation and anti-aging, enhancing immunity, and protecting the liver. It has long been used as a functional ingredient in health foods and cosmetics.

[0003] In existing technologies, the preparation of sheep placenta extract mainly involves the following process routes, but all have significant shortcomings. The first is the high-temperature cooking or acid-base hydrolysis route. This process typically involves prolonged treatment at temperatures above 100 °C or hydrolysis under strong acid or alkali conditions. While it can yield a certain amount of amino acids and short peptides, heat-sensitive cytokines (such as EGF and NGF, which begin to denature above 60 °C) are completely inactivated during this process, resulting in the product losing its biological signal regulation function. The second is the traditional enzymatic hydrolysis combined with ultrafiltration route, such as the low-temperature protease hydrolysis method disclosed in existing patents. This involves enzymatic hydrolysis at around 43 °C for 1 to 3 hours followed by ultrafiltration. Although this process retains some peptide activity, it only achieves the hydrolysis and utilization of tissue proteins and fails to target and enrich cytokines derived from stem cells, resulting in a highly incomplete cytokine profile in the product. The third approach is a combination of low-temperature freeze-thaw and centrifugation, which involves alternating deep freezing at -20°C and thawing at room temperature (25-28°C) with high-speed centrifugation to remove impurities. While this can release some intracellular proteins, it cannot activate the secretory function of stem cells. The content of endogenous cytokines is limited by the inherent static reserves in placental tissue, resulting in very limited enrichment. The fourth approach is enzymatic fermentation, which uses Aspergillus niger, yeast, and complex proteases for co-fermentation. While microbial metabolism produces some hydrolysis products, it also consumes nutrients. Furthermore, the fermentation products are mainly small molecules produced from hydrolyzed tissue blocks, with extremely low levels of paracrine factors derived from stem cells (such as VEGF and IGF carried by exosomes). The fifth approach is stem cell culture alone, which involves isolating and culturing placental mesenchymal stem cells, collecting conditioned medium, and then concentrating the medium through dextran gel chromatography and tangential flow ultrafiltration. Although this approach can obtain cytokines such as interleukin, CSF, TNF, IFN, EGF, and TGF, it completely abandons the protein resources of sheep placental tissue itself, resulting in low raw material utilization, high costs, and long cycles, making it difficult to achieve industrial application.

[0004] While existing technologies have proposed methods to simultaneously utilize tissue hydrolysates and stem cell factors by using a triple-enzyme fractionation method to dissociate placental tissue and inducing high expression of active factors in mesenchymal stem cells through in vitro proliferation culture, these processes involve complex cell culture steps, making industrialization extremely difficult and unsuitable for practical production. Furthermore, existing low-temperature extraction processes, such as repeated freeze-thaw cycles combined with two-stage ultrafiltration, while preserving activity to some extent, lack systematic enrichment methods for stem cell factors. In contrast, processes employing pH adjustment, isoelectric point precipitation, and 55–65 °C heat treatment combined with ultrafiltration result in the inactivation of some active factors due to the heat treatment step.

[0005] In summary, current technologies have not yet established a method to simultaneously achieve efficient enzymatic hydrolysis and utilization of sheep placental tissue proteins and maximize the preservation and targeted enrichment of endogenous stem cell bioactive factors in a single process chain. With the increasing demand for a full spectrum of bioactive factors in the market for functional cosmetics and functional foods, how to fully utilize sheep placental tissue protein resources while efficiently enriching multiple stem cell-derived cytokines has become a critical technological gap that urgently needs to be addressed in this field. Summary of the Invention

[0006] The purpose of this invention is to disclose a sheep placenta extract rich in active factors, its preparation method and application, in order to solve one or more technical problems existing in the existing methods and provide at least one beneficial option or create conditions.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention is to provide a method for preparing a sheep placenta extract rich in active factors. The preparation method includes the following steps: S1. Sheep placenta pre-processed into placental homogenate; S2. The placental homogenate is filtered to remove tissue residue, and red blood cell lysis buffer is added. After lysis, at least 2 times the volume of physiological saline is added to terminate the reaction. The precipitate is collected by centrifugation at 1000-1200 rpm, resuspended in physiological saline, and then collected by centrifugation at 2000-2500 rpm to obtain placental-derived active stem cell population. S3. Add serum-free targeted activation medium to the placental-derived active stem cell population for suspension fermentation. Detect the concentration of the target active cytokines in the suspension fermentation broth at regular intervals. Terminate fermentation when the concentration reaches its peak or after 48 hours. Collect the cell pellet by centrifugation of the obtained fermentation product at 2000-3000 rpm. After lysing the cell pellet, centrifuge at 10000-12000 rpm to collect the supernatant. Combine the supernatant from the centrifuged fermentation product to obtain a stem cell-derived cytokine enrichment solution. S4. Collect the placental tissue residue filtered and retained in step S2 and the supernatant produced by each centrifugation in step S2. Combine them with the stem cell-derived cytokine enrichment solution and add protease at pH 7.5-8.0 for low-temperature enzymatic hydrolysis. The enzymatic hydrolysis supernatant is the sheep placental extract.

[0008] The preparation method achieves two core objectives simultaneously through a single integrated process. On the one hand, it isolates endogenous stem cells from fresh placenta and achieves efficient enrichment of active cytokines through targeted suspension fermentation, maximizing the preservation of the natural biological activity of the cytokines. On the other hand, it recovers and enzymatically utilizes all wastes from conventional processes such as filter residue and centrifugation supernatant, fully extracting protein and polypeptide nutrients from placental tissue. This achieves high-value utilization of all components of placental raw materials, avoiding the problems of activity loss, material waste, and increased production costs caused by step-by-step independent processes.

[0009] In a further embodiment of the first aspect of the present invention, the low-temperature enzymatic hydrolysis in step S4 is a low-temperature stepwise enzymatic hydrolysis process, including the following steps: (1) Add trypsin with an enzyme activity ≥2500 U / g and hydrolyze at 40~42 ℃ for 2~3 hours; (2) Adjust the pH to 6.5~7.0, add papain with an enzyme activity ≥6000 U / g, and hydrolyze at 50~55 ℃ for 3~5 hours; (3) Adjust the pH to 7.0~7.5, add neutral protease with enzyme activity ≥50000 U / g, and enzymatically hydrolyze at 45~50 ℃ for 2~3 hours; (4) After the enzymatic hydrolysis is completed, the enzyme is inactivated by pH 4.0 acid treatment and centrifuged at 8000~10000 rpm at room temperature to obtain the enzymatic hydrolysis supernatant. The temperature during the entire enzymatic hydrolysis process should not exceed 55℃.

[0010] The low-temperature stepwise enzymatic hydrolysis process is segmented and regulated according to the optimal pH and temperature conditions of each enzyme, namely trypsin, papain, and neutral protease. This maximizes the hydrolysis efficiency of each enzyme, fully degrades placental tissue proteins and cell debris into small molecule active peptides and free amino acids, and improves the skin permeability and bioavailability of the hydrolysate. At the same time, the hydrolysis temperature is controlled to not exceed 55 °C throughout the process. Combined with the low-temperature acid precipitation method to inactivate enzymes, high temperature is avoided to prevent damage to the spatial structure and biological activity of active cytokines secreted by stem cells, thus achieving a balance between hydrolysis efficiency and activity preservation.

[0011] In a further embodiment of the first aspect of the present invention, the pretreatment in step S1 includes: collecting the placenta of a healthy sheep within 2 hours after giving birth, rinsing it at least twice with sterile physiological saline containing 0.1-0.2% heparin sodium pre-cooled at 4 ℃, and then cutting it into pieces of approximately 1 cm. 3Placental fragments were added to pre-cooled sterile saline containing 0.5-0.8% D-sorbitol. The mixture was homogenized at 2000 rpm for 1-2 minutes at 4-10 °C to break up large tissue fragments, followed by high-speed homogenization at 4000 rpm for 1-2 minutes to obtain the placental homogenate. Using sterile saline containing sodium heparin after collecting sheep placenta effectively prevents blood clotting, more thoroughly removes residual blood from placental tissue, and reduces the burden of subsequent erythrocyte lysis. Using pre-cooled sterile saline containing D-sorbitol as an osmotic pressure protectant maintains osmotic pressure balance during homogenization, reduces damage and inactivation of placental stem cells, and improves the yield and activity of subsequent viable stem cell populations. The homogenization was performed using a segmented, speed-adjusted method, which effectively dispersed the tissue fragments to release cells and avoided cell breakage and inactivation caused by prolonged high-speed homogenization.

[0012] In a further embodiment of the first aspect of the present invention, the erythrocyte lysis buffer in step S2 comprises 0.016 mol / L NH4Cl (ammonium chloride) and 0.017 mol / L Tris (tris(hydroxymethyl)aminomethane). This erythrocyte lysis buffer can efficiently lyse and remove contaminated erythrocytes under mild hypotonic conditions, while minimizing damage to placental stem cells, ensuring that the subsequently isolated viable stem cell population possesses good proliferation and secretion capabilities. The close physiological pH and osmotic pressure of Tris-NH4Cl at this concentration further reduce cell viability loss during lysis and washing, increasing the total amount of active cytokines secreted.

[0013] In a further embodiment of the first aspect of the present invention, the cell density of the suspension fermentation in step S3 is 3 × 10⁻⁶. 6 ~5×10 6 cells / mL. The suspension fermentation requires an appropriate cell density to avoid prolonged fermentation cycles and insufficient total factor secretion, as well as to prevent rapid nutrient consumption, accumulation of metabolic waste, and premature decline in cell activity caused by excessively high inoculation density, thus ensuring that cytokines can be efficiently and continuously secreted into the fermentation system.

[0014] In a further embodiment of the first aspect of the present invention, the serum-free targeted activation medium in step S3 includes a basal medium and a targeted activation composition, wherein the basal medium is selected from DMEM / F12 or α-MEM, and the targeted activation composition includes EGF, bFGF, TGF-β, dexamethasone, ascorbate phosphate, ITS supplement, glutamine, sodium pyruvate, and sodium selenite.

[0015] Furthermore, the concentrations of each component in the targeted activation composition are: 5-15 ng / mL EGF, 5-15 ng / mL bFGF, 2-8 ng / mL TGF-β1, 0.05-0.2 μmol / L dexamethasone, 50-150 μmol / L ascorbate phosphate, standard working concentration of ITS supplement, 2-4 mmol / L glutamine, 1 mmol / L sodium pyruvate, and 10-25 nmol / L sodium selenite. The various growth factors and nutritional supplements in the targeted activation composition can synergistically induce high expression and secretion of target active cytokines in sheep placental stem cells, while maintaining the undifferentiated state and high activity of the stem cells, achieving targeted enrichment of active factors.

[0016] In a further embodiment of the first aspect of the present invention, the fermentation conditions for suspension fermentation in step S3 include 37°C, 5% CO2, saturated humidity, stirring speed of 40-80 rpm, and fermentation time of 24-48 hours. The suspension fermentation is carried out under low-speed stirring conditions to avoid cell adhesion and sedimentation, and to avoid excessive stirring shear force causing cell damage.

[0017] In a further embodiment of the first aspect of the present invention, the disruption process in step S3 is achieved by low-temperature freeze-thaw treatment or low-power ultrasonic treatment. The low-temperature freeze-thaw treatment involves freezing the cell pellet to -20°C and then restoring it to room temperature, repeating this process 1-2 times. The low-power ultrasonic treatment involves placing the cell pellet in an ice bath at a power of 200-400 W, working for 5 seconds and then resting for 5 seconds, for a total of 2-4 minutes. Both disruption methods can effectively disrupt cell membranes under low-temperature conditions, releasing intracellular active cytokines and avoiding protein denaturation and factor inactivation problems caused by high-temperature and high-intensity mechanical disruption. This maximizes the preservation of the biological activity of intracellular factors, and the process is simple to operate with controllable parameters, making it suitable for large-scale production.

[0018] A second aspect of this invention is to provide a sheep placenta extract rich in active factors prepared by the method described in the first aspect of this invention. The sheep placenta extract is enzymatically hydrolyzed by a combination of stem cell fermentation broth and tissue hydrolysate, thus enabling efficient enzymatic utilization of sheep placental tissue proteins and maximum retention and targeted enrichment of endogenous stem cell active factors in a single process chain.

[0019] The third aspect of the present invention is to provide an application direction for the sheep placenta extract described in the second aspect of the present invention, specifically for the preparation of cosmetic products, so that the products have firming and anti-wrinkle effects. Attached Figure Description

[0020] Figure 1 This is a graph showing the proliferation rate of human skin fibroblasts in Example 5. Detailed Implementation

[0021] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0022] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0023] Example 1: Preparation of hydrolyzed sheep placenta extract rich in stem cell-derived cytokines Step S1: Pretreatment and low-temperature homogenization of sheep placenta Fresh placenta was collected from healthy sheep within 2 hours of delivery, yielding approximately 500 g of fresh placenta. It was immediately placed in a sterile transport box and rinsed four times (500 mL each time) with 4°C pre-cooled sterile saline containing 0.1% heparin sodium to remove surface blood, fetal membranes, and umbilical cord. The cleaned placental tissue was then cut into pieces approximately 1 cm in size. 3 The placental homogenate was prepared by adding 0.5% D-sorbitol pre-cooled sterile saline at a material-to-liquid ratio of 1:2 (w / v) to a low-temperature homogenizer and homogenizing in two stages. Large pieces of tissue were first homogenized at 2000 rpm for 1 minute at 4 ℃ to break them up, and then homogenized at 4000 rpm for 2 minutes to obtain the placental homogenate.

[0024] Step S2: Isolation and enrichment of placental-derived viable stem cell population The homogenate was filtered sequentially through an 80-200 mesh sieve to remove large pieces of connective tissue and unbroken chorionic membrane fragments, yielding a filtrate. An equal volume of 1× erythrocyte lysis buffer (pH 7.2, containing 0.016 mol / L NH4Cl and 0.017 mol / L Tris) was added to the filtrate, and lysis was performed for 5 minutes, gently inverting to mix. Immediately after lysis, three volumes of sterile saline at 4 °C were added to terminate the reaction. The mixture was centrifuged at 1200 rpm for 10 minutes at 4 °C, and the precipitate was collected. The precipitate was washed 2-3 times with sterile saline at 4 °C, resuspended in a small amount of saline, and centrifuged at 2000 rpm for 10 minutes at 4 °C. The collected precipitate was the placental-derived active stem cell population.

[0025] Step S3: Autologous Directed Activation Fermentation The placental-derived active stem cell population was resuspended in physiological saline, transferred to a sterile fermentation vessel, and serum-free targeted activation medium was added to adjust the cell density to 3 × 10⁻⁶. 6Cells / mL. Suspension activation fermentation was carried out at 37℃, 5% CO2, saturated humidity, and low-speed stirring at 40-50 rpm for 48 hours. The serum-free targeted activation medium included a basal medium and a targeted activation composition. The basal medium was selected from DMEM / F12 or α-MEM. The targeted activation composition included a low-concentration cytokine induction complex, hormones and antioxidants, nutritional supplements, and minerals. The low-concentration cytokine induction complex included 10 ng / mL EGF, 10 ng / mL bFGF, and 5 ng / mL TGF-β1. The hormones and antioxidants included 0.1 μmol / L dexamethasone and 100 μmol / L ascorbate phosphate. The nutritional supplements included a standard working concentration (1×) of ITS supplement, 3 mmol / L glutamine, and 1 mmol / L sodium pyruvate. The minerals included 15 nmol / L sodium selenite.

[0026] During fermentation, the placental-derived active stem cell population was not subjected to adherent culture or passage expansion. Instead, it remained in suspension and released large amounts of cytokines and exosomes through autocrine and paracrine mechanisms under the stimulation of inducing factors. The concentrations of key cytokines (such as VEGF, HGF, IL-6, and TGF-β1) in the culture medium were measured every 12 hours during fermentation, and fermentation was terminated after 48 hours.

[0027] After fermentation, the fermentation broth was centrifuged at 4 ℃ and 2000 rpm for 15 minutes, and the supernatant and cell pellet were collected. The cell pellet was disrupted by low-power sonication (ice bath, power 300 W, 5 seconds on, 5 seconds off, total time 3 minutes) to release intracellular and exosome-carried cytokines. The mixture was then centrifuged at 4 ℃ and 12000 rpm for 15 minutes, and the disrupted supernatant was collected and combined with conditioned medium to obtain a stem cell-derived cytokine enrichment solution.

[0028] Step S4: Combining two sources and low-temperature controlled enzymatic digestion The placental tissue residue filtered and retained in step S2, the supernatant from each centrifugation in step S2, and the washing waste liquid after erythrocyte lysis are collected and combined with the stem cell-derived cytokine enrichment solution to obtain a combined material. Sterile purified water is added to the combined material to adjust the material-to-liquid ratio to 1:3 (w / v), the pH is adjusted to 7.5, and protease is added for stepwise low-temperature enzymatic hydrolysis. The temperature throughout the enzymatic hydrolysis process does not exceed 55°C. (1) Add trypsin (enzyme activity ≥2500 U / g), the amount of enzyme added is 0.5% of the substrate mass, and enzymatically hydrolyze at 41 °C for 2.5 hours; (2) Adjust the pH to 6.5, add papain (enzyme activity ≥6000 U / g), the amount of enzyme added is 1.0% of the substrate mass, and enzymatically hydrolyze at 55 °C for 3 hours; (3) Adjust the pH to 7.0, add neutral protease (enzyme activity ≥50000 U / g, Bacillus subtilis source), the amount of enzyme added is 0.8% of the substrate mass, and enzymatically hydrolyze at 50 °C for 3 hours; (4) After the enzymatic hydrolysis is completed, the enzyme is inactivated by pH 4.0 acid treatment, and the mixture is quickly cooled to room temperature. It is then centrifuged at 8000~10000 rpm for 15~20 minutes, and the enzymatic hydrolysis supernatant is collected as the sheep placenta extract.

[0029] Example 2: Fractionation, membrane separation, enrichment, purification, and post-processing of sheep placental extract The sheep placental extract was sequentially subjected to fractionation membrane separation: 1) Microfiltration for impurity removal: Filtered through a 0.45 μm microfiltration membrane to remove residual cell debris and large particles; 2) Use an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, operating at a pressure of 0.1~0.3 MPa and a temperature of 4~10 ℃ to retain large protein molecules and incompletely hydrolyzed protein aggregates; 3) The 100 kDa permeate is processed through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa at an operating pressure of 0.15~0.35 MPa. The first retentate is collected as an enrichment of active proteins and cytokines with a molecular weight >10 kDa. 4) The 10 kDa permeate is processed through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa at an operating pressure of 0.2~0.4 MPa. The second retentate is collected as it is enriched with active peptides and small molecule cytokines with molecular weights of 3~10 kDa. At the same time, the 3 kDa permeate is collected, which is rich in amino acids, dipeptides and tripeptides.

[0030] Each stage of retentate and 3 kDa permeate can be collected separately according to product requirements.

[0031] Further post-processing including concentration and sterilization: The intermediate products obtained from fractionation were concentrated under vacuum at low temperature (temperature 40℃, vacuum degree 0.08~0.095 MPa, concentrated to a solid content of 15~30%) to obtain a concentrated extract. The extract was then sterilized by low-temperature cobalt-60 irradiation (irradiation dose 5~10 kGy).

[0032] Comparative Example 1: No pyrolysis, no activation process A preparation method similar to that used in the examples was employed, the difference being that step S2 omits red blood cell lysis and step S3, which involves autologous directed activation fermentation. Specifically, it includes: Steps D1-S1: Pretreatment and low-temperature homogenization of sheep placenta.

[0033] Steps D1-S2: Connective tissue removal The homogenate was filtered sequentially through 100-mesh and 200-mesh stainless steel sieves to remove large pieces of connective tissue and chorionic membrane fragments, yielding the filtrate. The filtrate was centrifuged at 4 ℃ and 2200 rpm for 12 minutes, and the precipitate (i.e., the crude cell population without erythrocyte lysis purification) and supernatant were collected. Tissue residue retained by the sieves was also collected. All collected materials (precipitate, supernatant, and tissue residue) were combined and used as substrate for subsequent enzymatic hydrolysis.

[0034] Steps D1-S3: Low-temperature controlled enzymatic hydrolysis Place the enzymatically hydrolyzed substrate obtained in steps D1-S2 into an enzymatic hydrolysis vessel, add sterile purified water to a total volume of approximately 3500 mL, and stir well. Adjust the pH to 7.5 and add protease for stepwise low-temperature enzymatic hydrolysis. The temperature of the entire enzymatic hydrolysis process shall not exceed 55 ℃: (1) Add trypsin (enzyme activity ≥2500 U / g), the amount of enzyme added is 0.5% of the substrate weight, and enzymatic hydrolyze at 41 ℃ for 2.5 hours; (2) Adjust the pH to 6.5, add papain (enzyme activity ≥6000 U / g), the amount of enzyme added is 1.0% of the substrate weight, and enzymatic hydrolyze at 55 ℃ for 3 hours; (3) Adjust the pH to 7.0, add neutral protease (enzyme activity ≥50000 U / g, Bacillus subtilis source), the amount of enzyme added is 0.8% of the substrate weight, and enzymatic hydrolyze at 50 ℃ for 3 hours; (4) After the enzymatic hydrolysis is completed, treat with pH 4.0 acid to inactivate the enzyme, cool rapidly to room temperature, centrifuge at 8000~10000 rpm for 15~20 minutes, and collect the enzymatic hydrolysis supernatant.

[0035] Comparative Example 2: High-Temperature Hydrolysis Process Referring to existing technologies, the one-step hydrolysis preparation method for sheep placenta extract specifically includes: S1: Pretreatment of the sheep placenta to separate the cotyledons: Take the frozen sheep placenta out from -20 ℃ and place it in a 4 ℃ freezer or thaw at room temperature. Separate the cotyledons and wash the cotyledons with PBS that has been prepared in advance and pre-cooled at 4 ℃, 150~250 mL each time, 3~4 times. Then, the placental leaf is cut into pieces with a diameter of 2-3 mm using surgical scissors, weighed and recorded as M1, and then stored at 4 ℃ until homogenized.

[0036] S2: Prepare the cotyledons into a homogenate, including: S21: Pre-rinse the homogenizer: Add 150~200 mL of pre-cooled PBS at 4 ℃ to the homogenizer's mixing cup, run the machine for 1 minute and then stop for 2 minutes, pour out the PBS, and repeat the above process twice.

[0037] Add 100 mL of pre-cooled physiological saline at 4 ℃, run the machine for 1 minute and then stop for 2 minutes, then discard the physiological saline.

[0038] S22: Place the rinsed cotyledons into the homogenizer's grinding cup to prepare a homogenate. The specific steps are as follows: Place the above-mentioned lumpy tissue into a homogenizing cup, add 2 mL / g of pre-cooled physiological saline at 4 ℃, turn the speed control knob to slowly accelerate, and then homogenize for 2 minutes after reaching 2 / 3 of the maximum speed. Stop the machine, pour the homogenate into a beaker, and then add 2 mL / g of pre-cooled physiological saline at 4 ℃ to rinse the homogenizing cup. Pour the washing solution into the beaker as well.

[0039] S3: Repeatedly freeze and thaw the above homogenate until the cell disruption rate reaches 95%. Specifically, aliquot the homogenate into 15mL centrifuge tubes, 10mL per tube, and freeze at -30 to -20°C for 24 hours. Thaw the homogenate afterward. The thawing temperature should first be at room temperature below 37°C. After 1 / 4 of the homogenate has thawed, it can be rapidly thawed in a water bath at a temperature below 37°C to promote cell rupture. During the thawing process, shake the homogenate every 5 minutes, but the amplitude should not be too large, and the homogenate should not touch the mouth of the bottle.

[0040] Once the homogenate is completely or 90% thawed, it should be immediately transferred to a -30°C freezer for storage, and arranged neatly. Each completion of the above process constitutes one freeze-thaw cycle. After four freeze-thaw cycles, it is ready for centrifugation.

[0041] S4: Centrifuge the homogenate after repeated freeze-thaw cycles, and collect the supernatant after centrifugation, including: Before centrifugation, the centrifuge rotor was wiped with gauze soaked in benzalkonium chloride or alcohol, and the centrifuge was pre-cooled at 4°C. The thawed homogenate was then taken out and centrifuged at 4000~8000 rpm for 20 minutes. After that, the centrifuge was turned off and the supernatant obtained by centrifugation was collected.

[0042] S5: Take the supernatant obtained in S4 and filter it to obtain a clear filtrate. During filtration, the supernatant is clarified and filtered using a pre-prepared 500 mL culture medium filter (connected to a vacuum pump). The filter membrane has a pore size of 0.8 μm. Collect the filtrate and take 100 μL of the sample for cryopreservation and testing. Before use, soak the filter membrane in sterile water for 20 minutes and then in physiological saline for 10 minutes.

[0043] After filtration, preheat the water bath to 80 ℃, transfer the filtrate into the water bath for hydrolysis, and the treatment time is 30 minutes.

[0044] S6: Centrifuge the above hydrolysate at 4000 rpm for 15 minutes at 8 ℃. Then separate the supernatant and autoclave it at 121 ℃ for 2-3 times, 20-30 minutes each time, to obtain the extract of Comparative Example 2.

[0045] Example 3: Detection of various cytokine contents in extracts from different extraction processes Outsourced testing employed the Luminex multifactor assay and ELISA method, following the provided kit instructions, to detect the content of six key cytokines in the extraction processes of Example 1 and Comparative Examples 1 and 2. Table 1 - Detection of various cytokine contents in extracts from different extraction processes

[0046] Table 1 shows that the contents of various cytokines in the extract of Example 1 were significantly higher than those in the two comparative examples. Among the growth factors, EGF, bFGF, HGF, VEGF, and TGF-β1 were 5.6–31 times higher than those in Comparative Example 1 and 10–476 times higher than those in Comparative Example 2, respectively. In Comparative Example 2, EGF was only 12±3 pg / mL and NGF was only 8±2 pg / mL, close to the detection limit, indicating that 70 ℃ high temperature has a devastating effect on heat-sensitive factors. In Comparative Example 1, due to the omission of erythrocyte lysis purification and activation fermentation, the contents of each factor were only 3%–30% of those in Example 1. In addition, the content of hydrolyzed collagen peptides in Comparative Example 2 (4200±380 pg / mL) was slightly higher than that in Comparative Example 1 (3800±320 pg / mL), indicating that although high temperature destroys heat-sensitive factors, it is conducive to the release of small molecule peptides.

[0047] Example 4: Evaluation of in vitro antioxidant activity (1) Experimental materials: DPPH (1,1-diphenyl-2-trinitrophenylhydrazine, Sigma-Aldrich, USA, catalog number D9132). ABTS [2,2′-Azazo-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, Sigma-Aldrich, USA, catalog number A1888]; Ascorbic acid (VC, Sigma-Aldrich, USA, catalog number A5960); Potassium persulfate (Sinopharm Chemical Reagent Co., Ltd., item number 10017416); Anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd., product number 10009218); PBS buffer (pH 7.4, Beijing Solarbio Science & Technology Co., Ltd., catalog number P1020).

[0048] (2) Determination of DPPH free radical scavenging rate: DPPH working solution was prepared according to the literature method. 3.94 mg of DPPH powder was accurately weighed, dissolved in anhydrous ethanol, and diluted to a volumetric flask wrapped in aluminum foil to prepare a 0.1 mmol / L DPPH working solution.

[0049] The samples to be tested were extracts prepared in Example 1, Comparative Example 1, and Comparative Example 2. Each analyte was diluted with deionized water to a concentration gradient of 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg / mL.

[0050] Preparation of VC solution for the positive control group: Accurately weigh 0.01 g of ascorbic acid, dissolve it in 10 mL of deionized water to prepare a stock solution of 1 mg / mL. Dilute with deionized water to prepare a series of concentration gradients: 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg / mL.

[0051] Add sequentially to a 96-well plate (final volume 200 μL per well): Sample set: 100 μL of sample solutions of different concentrations and 100 μL of DPPH working solution; Negative control group: 100 μL of sample solutions of different concentrations and 100 μL of anhydrous ethanol; Blank group: 100 μL deionized water and 100 μL DPPH working solution; Positive control group: 100 μL of VC solutions at different concentrations and 100 μL of DPPH working solution.

[0052] Each group has 3 replicates. After adding the sample, gently shake to mix, and incubate at room temperature in the dark for 30 minutes. Measure the absorbance of each well at 517 nm using a microplate reader. The DPPH free radical scavenging rate is calculated using the following formula: , Where A0 is the absorbance of the blank group, A1 is the absorbance of the sample group, and A2 is the absorbance of the negative control group.

[0053] The test results are shown in Tables 2 and 3: Table 2 - Half-maximal inhibitory concentration (IC50) of DPPH in each group 50 )

[0054] Table 3 - DPPH scavenging rate of extracts at different concentrations

[0055] As shown in Table 2, the extract IC of Example 150 The concentration was 0.28 ± 0.03 mg / mL, significantly lower than that of Comparative Examples 1 and 2. This indicates that the extract of Example 1 requires only a lower sample concentration to scavenge 50% of DPPH free radicals, demonstrating significantly superior antioxidant activity compared to the two comparative examples. Table 3 shows that within the concentration range of 0.0625–2.0 mg / mL, the DPPH free radical scavenging rates of Example 1, Comparative Examples 1, 2, and VC all increased with increasing sample concentration, exhibiting a clear dose-response relationship. Example 1 achieved a scavenging rate of 91.2 ± 3.8% at 2 mg / mL, which is very close to that of VC; while under the same 2 mg / mL conditions, Comparative Example 1 only achieved 48.6 ± 3.5%, and Comparative Example 2 only 31.2 ± 2.8%, showing a very significant difference in activity.

[0056] (3) Determination of ABTS free radical scavenging ability Preparation of ABTS stock solution: Accurately weigh 38.4 mg of ABTS powder and dissolve it in 10 mL of deionized water to prepare a 7 mmol / L ABTS aqueous solution. Accurately weigh 6.6 mg of potassium persulfate and dissolve it in 10 mL of deionized water to prepare a 2.45 mmol / L potassium persulfate aqueous solution. Mix the above ABTS solution and potassium persulfate solution in equal volumes and incubate at room temperature in the dark for 12–16 hours to obtain ABTS. + Free radical stock solution.

[0057] Preparation of ABTS working solution: Dilute with PBS buffer (pH 7.4) before use and adjust the absorbance at 734 nm to 0.70±0.02.

[0058] Add sequentially to a 96-well plate (final volume 200 μL per well): Sample set: 20 μL of sample solutions of different concentrations and 180 μL of ABTS working solution; Negative control group: 20 μL of sample solutions of different concentrations and 180 μL of PBS; Blank group: 20 μL deionized water and 180 μL ABTS working solution; Positive control group: 20 μL of VC solutions at different concentrations and 180 μL of ABTS working solution.

[0059] Each group has 3 replicates. After adding the sample, gently shake to mix, and incubate at room temperature in the dark for 6 minutes. Measure the absorbance of each well at 734 nm using a microplate reader. The ABTS free radical scavenging rate is calculated using the following formula: , Where A0 is the absorbance of the blank group, A1 is the absorbance of the sample group, and A2 is the absorbance of the negative control group.

[0060] The test results are shown in Table 4: Table 4 - Scavenging rate of ABTS free radicals by extracts of different concentrations

[0061] IC of Example 1 50 The value was 0.32±0.04 mg / mL. At 0.5 mg / mL, the clearance rate reached 62.5±3.5%, which was 2.5 times and 4.7 times that of Comparative Example 1 (25.2±2.0%) and Comparative Example 2 (13.2±1.4%), respectively; at 2.0 mg / mL, it reached 88.5±3.5%, which was close to that of VC (91.6±3.2%).

[0062] Example 1 showed a clear dose-dependent effect in both models, with significantly better activity than the two comparative examples, approaching the level of vitamin C. This indicates that the low-temperature process of the present invention effectively protects the heat-sensitive antioxidant components, and the stem cell-derived factors and tissue-hydrolyzed peptides synergistically endow the extract with excellent antioxidant capacity.

[0063] Example 5: Human skin fibroblast proliferation experiment (1) Experimental materials: The cells used in this experiment were human skin fibroblasts (HSF), purchased from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection Committee (Shanghai Institute of Life Sciences Cell Resource Center).

[0064] Cells from passages 4 to 8 in the logarithmic growth phase were used in the experiment. These cells exhibited a typical spindle shape, adhered to the culture vessel, and showed whorled or parallel arrangement during culture. The complete culture medium used was prepared by adding 10% (v / v) fetal bovine serum (FBS, Gibco, catalog number 10099141C, inactivated at 56 ℃ for 30 minutes before use) and 1% (v / v) penicillin-streptomycin antibiotic solution (100×, containing 10,000 U / mL penicillin and 10,000 μg / mL streptomycin, Gibco, catalog number 15140122). Cells were cultured in a Thermo Scientific 3111 incubator at 37 ℃, 5% CO2, and saturated humidity. Fresh complete culture medium was replaced every 2 to 3 days. When the cell confluence reached 80% to 90%, the cells were passaged at a 1:3 ratio. Mycoplasma testing (PCR) was performed on the cells before the experiment, and all results were negative. Cell viability was determined using the trypan blue exclusion assay to ensure that the viability was not less than 95%.

[0065] (2) Test samples: Three groups of test samples were set up in this experiment: extracts obtained from Example 1, Comparative Example 1, and Comparative Example 2. These were serially diluted to the required concentrations using serum-free DMEM medium. All three groups of test substances were uniformly set at low, medium, and high concentrations, namely 0.5%, 1.0%, and 2.0%, respectively.

[0066] (3) Reagents and preparation: The preparation method for MTT (purity ≥98.0%, Sigma-Aldrich, catalog number M5655) used in the experiment was as follows: Accurately weigh 250 mg of MTT, dissolve it in 50 mL of PBS (pH 7.4) to prepare a 5 mg / mL stock solution, filter it through a 0.22 μm filter membrane for sterilization, and then aliquot and store it at -20 ℃ protected from light. It should be used within 3 months, avoiding repeated freeze-thaw cycles more than 3 times. DMSO (Sigma-Aldrich, catalog number D2650) was cell culture grade and stored at room temperature in a sealed container for direct use. PBS (pH 7.4) (HyClone, catalog number SH30256.01) was cell culture grade and stored at 4 ℃. 0.25% trypsin-EDTA solution (1×, Gibco, catalog number 25200056) was stored at -20 ℃ and used within 2 weeks after thawing at 4 ℃.

[0067] The positive control used human epidermal growth factor (hEGF, recombinant protein, purity ≥97%, PeproTech, catalog number AF-100-15), prepared as a 10 μg / mL stock solution with PBS containing 0.1% BSA, aliquoted and stored at -80 ℃, with a working concentration of 10 ng / mL for use. Fetal bovine serum (FBS, superior grade, Gibco, catalog number 10099141C) was stored at -20 ℃ and inactivated in a 56 ℃ water bath for 30 minutes before use. Trypan blue staining solution (0.4%, Sigma-Aldrich, catalog number T8154) was stored at room temperature protected from light.

[0068] (4) Experimental grouping: 96-well plates, 200 μL system per well, as shown in Table 5.

[0069] Table 5 - Grouping Operation Table for Proliferation Experiment

[0070] The time-effect relationship of cell proliferation was observed at 24, 48, and 72 hours after drug treatment.

[0071] Cell viability / proliferation rate: .

[0072] Relative proliferation promotion rate (based on the negative control group): .

[0073] (5) Experimental results: The proliferation rates of each extract group after 24, 48, and 72 hours of treatment on human skin fibroblasts are shown in Tables 6-8. Table 6 - Effects of each extract group on the proliferation of human skin fibroblasts after 24 hours of treatment.

[0074] Table 7 - Effects of each extract group on the proliferation of human skin fibroblasts after 48 hours of treatment.

[0075] Table 8 - Effects of each extract group on the proliferation of human skin fibroblasts after 72 hours of treatment.

[0076] The results of the human skin fibroblast proliferation experiment showed that the cytokine-rich extract prepared in Example 1 of this invention significantly promoted the proliferation of human skin fibroblasts at a concentration range of 0.5-2.0%, exhibiting obvious dose- and time-dependent effects. At concentrations of 0.5-2.0% and for 48-72 hours, the relative cell proliferation rate increased by 35-68%, demonstrating a significantly better proliferative effect than the extracts prepared using the non-lysis, non-activation process (Comparative Example 1) and the high-temperature hydrolysis process (Comparative Example 2). Furthermore, its proliferative activity was on the same order of magnitude as that of the positive control hEGF (10 ng / mL). Figure 1 (As shown).

[0077] Example 6: Detection of elastin and hyaluronic acid content (1) Experimental materials: The cells used in this experiment were human skin fibroblasts (HSF). The complete culture medium, culture conditions, and passage methods used for cell culture were the same as in Example 5. Cells in the logarithmic growth phase from the 5th to the 7th generation were selected for the experiment, and cell viability was determined to be no less than 95% using the trypan blue exclusion assay.

[0078] (2) Test sample: The test samples used in this experiment were the same as those in Example 5, including the extracts from Example 1, Comparative Example 1, and Comparative Example 2. All three groups of samples were compared in parallel using a high concentration of 2.0% (w / v), with working solutions prepared in serum-free DMEM medium. The positive control was human epidermal growth factor (hEGF, PeproTech, catalog number AF-100-15) at a working concentration of 10 ng / mL.

[0079] (3) Reagents and preparation: Human elastin ELISA kit (Wuhan Boster Biological Engineering Co., Ltd., catalog number EK0518); Human hyaluronic acid (HA) ELISA kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd., catalog number Ml057); RIPA high-efficiency lysis buffer (Solarbio, catalog number R0010, containing PMSF); BCA protein concentration assay kit (Thermo Fisher, catalog number 23225); PBS (pH 7.4, HyClone, catalog number SH30256.01); serum-free DMEM medium (Gibco, catalog number 11965092). All kit components were stored at 4 ℃ or -20 ℃ according to the instructions. The chromogenic substrate TMB should be stored protected from light.

[0080] (4) Experimental grouping: The 6-well plate was laid with 2 mL per well, as shown in Table 9.

[0081] Table 9 - Grouping Operation Table for Content Detection Experiment

[0082] After cell adhesion, the cells were treated with the drug for 48 hours, consistent with the treatment time in Experiment 3. After treatment, the cell culture supernatant was collected for ELISA detection, and the cell lysate was collected for total protein quantification.

[0083] (5) Experimental methods: Human skin fibroblasts in the logarithmic growth phase (passage 6) were harvested, digested, counted, and their cell density adjusted to 2 × 10⁶ cells / year. 5 Cells / mL were seeded into 6-well cell culture plates, with 2 mL seeded into each well (i.e., 4 × 10⁶ cells / well). 5 (Number of cells). Incubate at 37 ℃ in a 5% CO2 incubator for 24 hours. When cells are fully adhered and reach 60-70% confluence, discard the old culture medium and add 2 mL / well of fresh complete culture medium containing the corresponding concentration of the test sample according to the group. The negative control group received 2 mL / well of complete culture medium, and the positive control group received 2 mL / well of complete culture medium containing 10 ng / mL hEGF. Each group had 3 replicates. Return the 6-well plate to the incubator and continue incubation for 48 hours.

[0084] Remove the cells from the 6-well plate and collect the cell culture supernatant from each well into a 1.5 mL sterile EP tube. Centrifuge at 3000 rpm for 10 minutes at 4 °C to remove any cell debris. Aliquot the supernatant and store at -80 °C for later analysis. Simultaneously, wash the cells in each well three times with pre-chilled PBS. Add 100 μL of pre-chilled RIPA lysis buffer to each well and lyse on ice for 30 minutes, gently shaking the culture plate for 1 minute every 10 minutes during lysis. Scrape the cells off with a cell scraper and collect the lysis buffer into an EP tube. Centrifuge at 12000 rpm for 15 minutes at 4 °C. The supernatant is the total cell protein extract and is stored at -80 °C for later analysis.

[0085] Follow the instructions for the BCA protein concentration assay kit. Prepare a series of bovine serum albumin (BSA) standard concentrations (0, 0.125, 0.25, 0.5, 1.0 mg / mL). Add 20 μL of cell lysate and standard to each well of a 96-well plate, and add 200 μL of BCA working solution to each well. Incubate at 37 ℃ for 30 minutes, measure the absorbance at 562 nm, plot a standard curve, and calculate the total protein concentration of each group of cells.

[0086] Follow the instructions for the Human Elastin ELISA Kit and Hyaluronic Acid Kit.

[0087] (6) Experimental results: Table 10 - Elastin Content Detection

[0088] Table 11 - Hyaluronic Acid Content Detection

[0089] Elastin secretion promotion effect: The 2.0% concentration of the extract in Example 1 of this invention significantly promoted the secretion of elastin by human skin fibroblasts, with a 70.1% increase in secretion compared to the negative control group (P<0.01), and the effect was significantly better than that of the positive control hEGF (44.8% increase). Comparative Example 1 showed a weaker promoting effect (24.9% increase, P<0.05), while Comparative Example 2 showed no significant difference from the negative control group (P>0.05).

[0090] Hyaluronic acid secretion promotion effect: The 2.0% concentration of the extract in Example 1 of this invention significantly promoted hyaluronic acid secretion, with a secretion amount increasing by 65.0% compared to the negative control group (P<0.01), and the effect was better than that of the positive control hEGF (increased by 40.0%). The high concentration of Comparative Example 1 showed a slight promoting effect (increased by 22.0%, P<0.05), while Comparative Example 2 had no significant effect.

[0091] Intergroup comparison: Under the same high concentration conditions, the secretion-promoting effect of Example 1 of the present invention on elastin and hyaluronic acid was significantly better than that of Comparative Example 1 and Comparative Example 2 (P<0.01).

[0092] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A method for preparing a sheep placenta extract rich in active factors, characterized in that, Including the following steps: S1. Sheep placenta pre-processed into placental homogenate; S2. The placental homogenate is filtered to remove tissue residue, and red blood cell lysis buffer is added. After lysis, at least 2 times the volume of physiological saline is added to terminate the reaction. The precipitate is collected by centrifugation at 1000-1200 rpm, resuspended in physiological saline, and then collected by centrifugation at 2000-2500 rpm to obtain placental-derived active stem cell population. S3. Add serum-free targeted activation medium to the placental-derived active stem cell population for suspension fermentation. Detect the concentration of the target active cytokines in the suspension fermentation broth at regular intervals. Terminate fermentation when the concentration reaches its peak or after 48 hours. Collect the cell pellet by centrifugation of the obtained fermentation product at 2000-3000 rpm. After lysing the cell pellet, centrifuge at 10000-12000 rpm to collect the supernatant. Combine the supernatant from the centrifuged fermentation product to obtain a stem cell-derived cytokine enrichment solution. S4. Collect the placental tissue residue filtered and retained in step S2 and the supernatant produced by each centrifugation in step S2. Combine them with the stem cell-derived cytokine enrichment solution and add protease at pH 7.5-8.0 for low-temperature enzymatic hydrolysis. The enzymatic hydrolysis supernatant is the sheep placental extract.

2. The preparation method according to claim 1, characterized in that, Step S4 describes a low-temperature stepwise enzymatic hydrolysis process, which includes the following steps: (1) Add trypsin with an enzyme activity ≥2500 U / g and hydrolyze at 40~42 ℃ for 2~3 hours; (2) Adjust the pH to 6.5~7.0, add papain with an enzyme activity ≥6000 U / g, and hydrolyze at 50~55 ℃ for 3~5 hours; (3) Adjust the pH to 7.0~7.5, add neutral protease with enzyme activity ≥50000 U / g, and enzymatically hydrolyze at 45~50 ℃ for 2~3 hours; (4) After the enzymatic hydrolysis is completed, the enzyme is inactivated by pH 4.0 acid treatment and centrifuged at 8000~10000 rpm at room temperature to obtain the enzymatic hydrolysis supernatant. The temperature during the entire enzymatic hydrolysis process should not exceed 55℃.

3. The preparation method according to claim 1, characterized in that, The pretreatment in step S1 includes: collecting the placenta within 2 hours after the healthy sheep give birth, rinsing it at least twice with sterile physiological saline containing heparin sodium at 4 ℃, then cutting it into small pieces, adding pre-cooled sterile physiological saline containing D-sorbitol, and homogenizing it in a homogenizer at 2000 rpm for 1-2 minutes, and then at 4000 rpm for 1-2 minutes to obtain the placental homogenate.

4. The preparation method according to claim 1, characterized in that, The red blood cell lysis buffer in step S2 comprises 0.016 mol / L NH4Cl and 0.017 mol / L Tris.

5. The preparation method according to claim 1, characterized in that, The cell density for suspension fermentation in step S3 is 3 × 10⁻⁶. 6 ~5×10 6 cells / mL.

6. The preparation method according to claim 5, characterized in that, The serum-free targeted activation medium in step S3 includes a basal medium and a targeted activation composition. The basal medium is selected from DMEM / F12 or α-MEM, and the targeted activation composition includes EGF, bFGF, TGF-β, dexamethasone, ascorbate phosphate, ITS supplement, glutamine, sodium pyruvate, and sodium selenite.

7. The preparation method according to claim 6, characterized in that, The fermentation conditions for suspension fermentation described in step S3 include 37°C, 5% CO2, saturated humidity, stirring speed of 40~80 rpm, and fermentation time of 24~48 hours.

8. The preparation method according to claim 1, characterized in that, The disruption process described in step S3 is achieved by low-temperature freeze-thaw treatment or low-power ultrasonic treatment. The low-temperature freeze-thaw treatment involves freezing the cell pellet to -20°C and then restoring it to room temperature, repeating this process 1 to 2 times. The low-power ultrasonic treatment involves placing the cell pellet in an ice bath environment at a power of 200 to 400 W, working for 5 seconds and then resting for 5 seconds, for a total of 2 to 4 minutes.

9. A sheep placenta extract rich in active factors, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the sheep placenta extract according to claim 9 in cosmetic products.

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