Placenta active substance for promoting secretion of testosterone as well as preparation method and application of placenta active substance

Abstract

The invention relates to a placenta active substance for promoting testosterone secretion as well as a preparation method and application of the placenta active substance. The preparation method comprises the following steps: carrying out crushing treatment on placenta tissues, mixing the crushed placenta tissues with protease and polysaccharide enzyme, carrying out enzymolysis, carrying out solid-liquid separation on an enzymolysis product, collecting clear liquid, and carrying out purification treatment on the clear liquid to obtain the placenta active factor. According to the novel placenta active substance preparation method, the mild, efficient and low-energy-consumption placenta active substance extraction process is achieved for the first time, the problems that in a traditional method, enzymolysis conditions are harsh, and active ingredients are easy to degrade are solved, and the obtained placenta active substances have high biological activity and high stability.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a placenta active substance for promoting testosterone secretion and a preparation method and application thereof. Background Art

[0002] Placental active substances (placental active factors) are a class of various immunomodulatory factors present in placental tissue. They have important research value and clinical application potential in the field of reproductive medicine.

[0003] In the field of extraction and application of placental active factors, existing technical solutions usually use physical crushing, chemical dissolution or enzymatic hydrolysis to extract active ingredients from placental tissue. Commonly used methods include acid-base dissolution, single enzyme enzymatic hydrolysis and ultrasound-assisted extraction. Acid-base dissolution method: extract active factors by acid-base dissolution of placental tissue. This method is low-cost and relatively simple to operate, but acid-base treatment can easily lead to protein and polypeptide degradation, affecting the biological effect and stability of active ingredients; single enzyme enzymatic hydrolysis method: use a single enzyme, such as trypsin or collagenase, to enzymatically hydrolyze placental tissue to release active factors such as proteins and polypeptides. This method improves the extraction rate to a certain extent, but has strict requirements on enzymatic hydrolysis conditions (temperature, pH value, etc.), the extraction efficiency and quality of active ingredients are unstable, and the types of active substances extracted are limited; ultrasound-assisted extraction: combine ultrasonic crushing technology with enzymatic hydrolysis to improve cell wall breaking efficiency and the release of active factors. This method can indeed improve the extraction efficiency, but the equipment requirements are high, and the intensity and duration of ultrasound need to be precisely controlled, otherwise it may lead to the degradation of active factors.

[0004] In summary, the existing methods for extracting placental active factors still have the following problems: (1) The active ingredients are unstable: acid-base dissolution and ultrasound-assisted extraction have a certain destructive effect on the structure of the active factors, resulting in insufficient stability and biological activity of the extracts; (2) The extraction rate and purity are low: single enzyme hydrolysis and physical crushing methods are difficult to fully release all the active ingredients in the placenta, resulting in a low extraction rate; at the same time, the impurity removal effect is not ideal, affecting the purity of the extract; (3) Poor process consistency: enzymatic hydrolysis, ultrasound-assisted and other methods have high requirements for the environment and equipment, and are easily interfered by external factors, resulting in difficulty in maintaining consistent product quality and biological activity, affecting the feasibility of large-scale production.

[0005] Therefore, there is an urgent need for an innovative extraction process that can improve the extraction efficiency of placental active factors under mild conditions and ensure the stability and purity of the active ingredients. Summary of the invention

[0006] In view of the deficiencies of the prior art and actual needs, the present invention provides a placental active substance that promotes testosterone secretion, and a preparation method and application thereof, so as to achieve efficient extraction of high-purity placental active factors under mild conditions.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for preparing a placenta active factor, the preparation method comprising:

[0009] The placental tissue is crushed, the crushed placental tissue is mixed with protease and polysaccharidase for enzymolysis, the enzymolysis product is separated into solid and liquid, the clear liquid is collected, and the clear liquid is purified to obtain the placental active factor.

[0010] In the present invention, a completely new method for extracting placental active factors is designed, including designing a dual-enzyme coordinated low-temperature enzymatic hydrolysis treatment method, which can extract placental active factors under mild conditions, improve the extraction efficiency, and obtain high-purity and high-activity placental active factors.

[0011] Preferably, the fragmentation process includes cutting and grinding the placental tissue.

[0012] Preferably, the cutting process comprises cutting into small pieces of 1 cm×1 cm to 2 cm×2 cm.

[0013] Preferably, the grinding process includes cryo-grinding, specifically including placing the cut placental tissue in a liquid nitrogen environment for cryo-grinding to obtain tissue particles.

[0014] Preferably, the cryo-grinding is carried out in a cryo-grinder at a frequency of 15 Hz to 25 Hz, for example, 16, 17, 18, 19, 20, 21, 22, 23 or 24 Hz, and a time of 60 s to 120 s, including but not limited to 62, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 or 118 s, etc.

[0015] Preferably, the diameter of the tissue particles is less than 500 μm.

[0016] Preferably, the protease comprises at least one of neutral protease, lysozyme or chymotrypsin.

[0017] Preferably, the polysaccharidase comprises at least one of cellulase, hyaluronidase or glucanase.

[0018] Preferably, the protease is neutral protease, lysozyme and chymosin, and the mass ratio of protease, lysozyme and chymosin is (0.5-5):(0.5-5):1, for example, it can be 2.5:1.5:1, 1.5:1:1, 2.5:1.5:1, 3:3:1, 4:4.5:1, 1:1.5:1 or 0.8:1.5:1, etc., preferably (2-3):(1-2):1.

[0019] Preferably, the mass ratio of the polysaccharidase being cellulase, hyaluronidase and glucanase is (3-10):1:(1-5), for example, it can be 6:1:3, 4:1:4, 8:1:1, 5:1:2, 4:1:5 or 9:1:3, etc., preferably (5-7):1:(2-4).

[0020] Preferably, the amount of the protease used is 1.5% to 2.5% of the wet weight of the crushed placenta tissue, for example, it can be 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.2%, 2.3% or 2.4%.

[0021] Preferably, the amount of the polysaccharidase used is 0.5% to 1.5% of the wet weight of the crushed placenta tissue, for example, it can be 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.3% or 1.4%, etc.

[0022] Preferably, the enzymatic hydrolysis time is 4 to 8 hours, for example, 4.5, 5, 5.5, 6, 6.5, 7 or 7.5 hours, etc., and the temperature is 15 to 20°C, for example, 16, 17, 18 or 19°C, etc.

[0023] Preferably, the pH condition of the enzymatic hydrolysis is 6.5-7.5, for example, it can be 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3 or 7.4.

[0024] Preferably, the purification process comprises ultrafiltration and nanofiltration.

[0025] In the present invention, a purification process is further designed, and ultrafiltration and nanofiltration are combined to efficiently separate active factors from impurities, which can better retain the biological activity and stability of placental active factors and improve product purity.

[0026] Preferably, the molecular weight cut-off of the ultrafiltration membrane used in the ultrafiltration treatment is 10 kDa to 50 kDa, for example, 20, 30 or 40 kDa.

[0027] Preferably, the molecular weight cutoff of the nanofiltration membrane of the nanofiltration treatment is 200 kDa to 1000 kDa, for example, 300, 400, 500, 600, 700, 800 or 900 kDa.

[0028] Preferably, the conditions of the ultrafiltration treatment and the nanofiltration treatment are independently: temperature 20-25°C, for example, 21, 22, 23 or 24°C, and pressure 0.1MPa-0.4MPa.

[0029] Preferably, the crushing process also includes a cleaning step before the crushing process.

[0030] Preferably, the purification process further includes the steps of concentration and drying.

[0031] Preferably, the drying process comprises freeze drying.

[0032] As a preferred technical solution, the method for preparing the placenta active factor comprises:

[0033] The placenta tissue is cut and ground, and the ground placenta tissue is mixed with a protease and a polysaccharidase, wherein the amount of the protease is 1.5% to 2.5% of the wet weight of the crushed placenta tissue, and the amount of the polysaccharidase is 0.5% to 1.5% of the wet weight of the crushed placenta tissue, and enzymolysis is performed at 15 to 20° C. for 4 to 8 hours; the enzymolysis product is centrifuged, the clear liquid is collected, the clear liquid is ultrafiltered and nanofiltered, and then concentrated and dried to obtain placental active factors.

[0034] In a second aspect, the present invention provides a placenta active factor, which is prepared by the preparation method of the placenta active factor described in the first aspect.

[0035] In a third aspect, the present invention provides use of the placenta active factor described in the second aspect in the preparation of an agent for promoting the proliferation of TM3 cells and the secretion of testosterone.

[0036] The placenta active factor prepared by the present invention has high biological activity and high stability, can promote the proliferation of TM3 cells and the secretion of testosterone, provides a new treatment method for treating male dysfunction, and provides theoretical support for the application of placenta extract products in the market.

[0037] Compared with the prior art, the present invention has at least the following beneficial effects:

[0038] The present invention provides a novel method for preparing placental active factors, which realizes for the first time a mild, efficient and low-energy placental active factor extraction process, overcomes the problems of harsh enzymatic hydrolysis conditions and easy degradation of active ingredients in traditional methods, and the obtained placental active factors have high biological activity and high stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the result diagram of TM3 cell proliferation;

[0040] Figure 2 This is the result of testosterone secretion in TM3 cells. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0042] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0043] The present invention designs a completely new placental active factor extraction method, designs dual-enzyme coordinated low-temperature enzymatic hydrolysis and membrane separation technology, and extracts high-purity placental active factors from healthy placental tissue.

[0044] 1. Double enzyme synergistic low temperature extraction:

[0045] The present invention adopts the synergistic effect of two enzymes, and applies the combination of protease and polysaccharidase to placental tissue, so that the active ingredients of proteins, polypeptides and polysaccharides in the placenta can be efficiently released under milder conditions. By precisely controlling the temperature and time of the enzymatic reaction, the damage to the active substances by high temperature is avoided, thereby protecting the biological activity of the active factors. Unlike traditional single enzyme treatment, the synergistic effect of two enzymes can decompose the placental tissue more comprehensively, improve the extraction rate, and can be carried out under low temperature conditions, which not only reduces the degradation risk of active ingredients, but also greatly reduces the loss of heat-sensitive substances.

[0046] 2. Membrane separation and purification technology:

[0047] After extraction, ultrafiltration and nanofiltration combined membrane separation technology is applied to efficiently separate active factors from impurities. Ultrafiltration membrane is used to initially remove macromolecular impurities, while nanofiltration membrane is used to further purify the target active factors. Operating at room temperature and low pressure avoids the destruction of active factors by traditional high-temperature and high-pressure concentration. Compared with traditional chemical or high-temperature purification methods, membrane separation technology can better retain the biological activity and stability of active factors, while improving product purity and real-time monitoring of key parameters such as temperature, pH, enzymatic hydrolysis time, and pressure. Ensure that the extraction results of each batch are highly consistent and meet the standards of industrial production.

[0048] In summary, the present invention realizes a mild, efficient and low-energy placental active factor extraction process by combining the synergistic action of two enzymes with low-temperature membrane separation, overcoming the problems of harsh enzymatic hydrolysis conditions and easy degradation of active ingredients in traditional methods. The placental active factor obtained by the present invention has high biological activity and high stability, and can promote the proliferation of TM3 cells and the secretion of testosterone, providing a new treatment for male dysfunction and providing theoretical support for the application of placental extract products in the market.

[0049] Example 1

[0050] This example involves extraction of placental active factors.

[0051] 1. Placental Tissue Pretreatment

[0052] (1) Raw material selection: Select healthy placental tissue and immediately store it in a frozen environment at -50°C after collection to avoid enzyme activity and degradation of biological components.

[0053] (2) Cleaning and cutting: Use cold sterile saline solution in a clean room to rinse the placenta tissue, remove blood and impurities, and cut the placenta into small pieces of 1 cm×1 cm to 2 cm×2 cm.

[0054] (3) Cryo-grinding: The cut small pieces of placental tissue are placed in a liquid nitrogen environment for rapid freezing, and then ground into particles with a diameter of less than 500 μm using a cryo-grinder (frequency 20 Hz, time 90 s) to improve the efficiency of enzymatic hydrolysis.

[0055] 2. Dual enzyme synergistic low temperature enzymolysis

[0056] (1) Enzyme selection and ratio:

[0057] The proteases used were neutral protease, lysozyme and chymotrypsin (mass ratio was 2.5:1.5:1), and the total amount of protease added was 2% of the wet weight of the placenta;

[0058] The polysaccharidase used was cellulase, hyaluronidase and glucanase (mass ratio was 6:1:3), and the total amount of polysaccharidase added was 1% of the wet weight of the placenta.

[0059] (2) Enzymatic hydrolysis conditions:

[0060] Temperature: 17℃;

[0061] pH: 7.0;

[0062] Time: 6 hours, with slow stirring (speed 90 rpm) during the period.

[0063] (3) Enzymatic hydrolysis control and centrifugation: After the enzymatic hydrolysis is completed, the insoluble impurities are separated by centrifugation at 4000×g for 10 minutes to obtain a preliminary extracted active factor solution.

[0064] 3. Membrane separation and purification technology

[0065] (1) Ultrafiltration treatment: Use ultrafiltration membrane (molecular weight cutoff range 30kDa) to initially remove large molecular impurities and cell debris, and retain active factors such as proteins, peptides and polysaccharides.

[0066] (2) Nanofiltration treatment: Use nanofiltration membrane (molecular weight cutoff range 600Da) to further separate small molecule impurities to obtain a high-purity active factor solution.

[0067] (3) Operating conditions:

[0068] Temperature: 22℃;

[0069] Pressure: 0.25MPa.

[0070] 4. Concentration and drying

[0071] (1) Concentration: The high-purity active factor solution is concentrated under low-temperature vacuum (temperature -70°C, vacuum degree 0.09 MPa) to reduce the volume of the solution.

[0072] (2) Freeze drying: The concentrate is processed by a freeze dryer to obtain a powdered placental active factor product with a moisture content of less than 5%, which is convenient for storage and transportation.

[0073] Example 2

[0074] This example involves extraction of placental active factors.

[0075] 1. Placental Tissue Pretreatment

[0076] (1) Raw material selection: Select healthy placental tissue and immediately store it in a frozen environment at -20°C after collection to avoid enzyme activity and degradation of biological components.

[0077] (2) Cleaning and cutting: Use cold sterile saline solution in a clean room to rinse the placenta tissue, remove blood and impurities, and cut the placenta into small pieces of 1 cm×1 cm to 2 cm×2 cm.

[0078] (3) Cryo-grinding: The cut small pieces of placental tissue are placed in a liquid nitrogen environment for rapid freezing, and then ground into particles with a diameter of less than 500 μm using a cryo-grinder (frequency 25 Hz, time 60 s) to improve the efficiency of enzymatic hydrolysis.

[0079] 2. Dual enzyme synergistic low temperature enzymolysis

[0080] (1) Enzyme selection and ratio:

[0081] The proteases used were neutral protease, lysozyme and chymotrypsin (mass ratio was 3:2:1), and the total addition amount was 1.5% of the wet weight of the placenta;

[0082] The polysaccharidase selected was cellulase, hyaluronidase and glucanase (mass ratio was 5:1:2), and the total addition amount was 1.5% of the wet weight of the placenta.

[0083] (2) Enzymatic hydrolysis conditions:

[0084] Temperature: 20℃;

[0085] pH: 6.5;

[0086] Time: 8 hours, with slow stirring (speed 60 rpm) during the period.

[0087] (3) Enzymatic hydrolysis control and centrifugation: After the enzymatic hydrolysis is completed, the insoluble impurities are separated by centrifugation at 4000×g for 10 minutes to obtain a preliminary extracted active factor solution.

[0088] 3. Membrane separation and purification technology

[0089] (1) Ultrafiltration treatment: Ultrafiltration membrane (molecular weight cutoff range 50 kDa) is used to initially remove large molecular impurities and cell debris, while retaining active factors such as proteins, peptides and polysaccharides.

[0090] (2) Nanofiltration treatment: Use nanofiltration membrane (molecular weight cutoff range 200Da) to further separate small molecule impurities to obtain a high-purity active factor solution.

[0091] (3) Operating conditions:

[0092] Temperature: 20℃;

[0093] Pressure: 0.1MPa.

[0094] 4. Concentration and drying

[0095] (1) Concentration: The high-purity active factor solution is concentrated under low-temperature vacuum (temperature -70°C, vacuum degree 0.08MPa) to reduce the volume of the solution.

[0096] (2) Freeze drying: The concentrate is processed by a freeze dryer to obtain a powdered placental active factor product with a moisture content of less than 5%, which is convenient for storage and transportation.

[0097] Example 3

[0098] This example involves extraction of placental active factors.

[0099] 1. Placental Tissue Pretreatment

[0100] (1) Raw material selection: Select healthy placental tissue and immediately store it in a freezing environment at -80°C after collection to avoid enzyme activity and degradation of biological components.

[0101] (2) Cleaning and cutting: Use cold sterile saline solution in a clean room to rinse the placenta tissue, remove blood and impurities, and cut the placenta into small pieces of 1 cm×1 cm to 2 cm×2 cm.

[0102] (3) Cryo-grinding: The cut small pieces of placental tissue are placed in a liquid nitrogen environment for rapid freezing, and then ground into particles with a diameter of less than 500 μm using a cryo-grinder (frequency 15 Hz, time 120 s) to improve the efficiency of enzymatic hydrolysis.

[0103] 2. Dual enzyme synergistic low temperature enzymolysis

[0104] (1) Enzyme selection and ratio:

[0105] The proteases used were neutral protease, lysozyme and chymotrypsin (mass ratio was 2:1:1), and the total addition amount was 2.5% of the wet weight of the placenta;

[0106] The polysaccharidase selected was cellulase, hyaluronidase and glucanase (mass ratio was 7:1:4), and the total addition amount was 0.5% of the wet weight of the placenta.

[0107] (2) Enzymatic hydrolysis conditions:

[0108] Temperature: 15℃;

[0109] pH: 7.5;

[0110] Time: 4 hours, with slow stirring (speed 120 rpm) during the period.

[0111] (3) Enzymatic hydrolysis control and centrifugation: After the enzymatic hydrolysis is completed, the insoluble impurities are separated by centrifugation at 4000×g for 10 minutes to obtain a preliminary extracted active factor solution.

[0112] 3. Membrane separation and purification technology

[0113] (1) Ultrafiltration treatment: Use ultrafiltration membrane (molecular weight cutoff range 10kDa) to initially remove large molecular impurities and cell debris, and retain active factors such as proteins, peptides and polysaccharides.

[0114] (2) Nanofiltration treatment: Use nanofiltration membrane (molecular weight cutoff range 1000Da) to further separate small molecule impurities to obtain a high-purity active factor solution.

[0115] (3) Operating conditions:

[0116] Temperature: 25℃;

[0117] Pressure: 0.4MPa.

[0118] 4. Concentration and drying

[0119] (1) Concentration: The high-purity active factor solution is concentrated under low-temperature vacuum (temperature -70°C, vacuum degree 0.1 MPa) to reduce the volume of the solution.

[0120] (2) Freeze drying: The concentrate is processed by a freeze dryer to obtain a powdered placental active factor product with a moisture content of less than 5%, which is convenient for storage and transportation.

[0121] Example 4

[0122] This example involves extraction of placental active factors.

[0123] Compared with Example 1, the only difference is that the temperature of enzymolysis is 10°C.

[0124] Example 5

[0125] This example involves extraction of placental active factors.

[0126] Compared with Example 1, the only difference is that the temperature of enzymolysis is 25°C.

[0127] Comparative Example 1

[0128] This comparative example involves extraction of placental active factors.

[0129] The traditional process is used to extract the active factors of the placenta. The specific process includes:

[0130] 1. Placenta collection and preservation:

[0131] Source: Placental tissue collected after delivery of healthy fetuses.

[0132] Storage: Refrigerate (4°C) as soon as possible after collection to avoid protein degradation.

[0133] 2. Cleaning:

[0134] The placenta tissue is repeatedly rinsed with saline to remove blood, fat and other impurities. Low-concentration antibiotic solutions (such as penicillin and streptomycin) can be used for soaking to reduce microbial contamination.

[0135] 3. Crushing:

[0136] Remove excess tissue (such as amniotic membrane), then cut or crush the placental tissue (such as with a high-speed homogenizer or tissue masher), and add cold saline to prepare a placental homogenate.

[0137] 4. Centrifugation:

[0138] The insoluble matter was removed by high-speed centrifugation at 4000 × g, 4°C, for 30 min, and the supernatant was collected as the crude extract.

[0139] 5. Concentration and drying

[0140] (1) Concentration: The high-purity active factor solution is concentrated under low-temperature vacuum (temperature -70°C, vacuum degree 0.1 MPa) to reduce the volume of the solution.

[0141] (2) Freeze drying: The concentrate is processed by a freeze dryer to obtain a powdered placental active factor product with a moisture content of less than 5%, which is convenient for storage and transportation.

[0142] Test Example 1

[0143] This test example analyzes the placenta active factors prepared in the comparative examples of each embodiment.

[0144] Placenta active factor extraction rate, the test method is: ELISA detection of specific placenta active factors PLGF\HGF, VEGF, EGF. Extraction rate (%) = (the amount of active factors extracted / the total amount of active factors in the original placenta sample) × 100%

[0145] The purification efficiency is tested by high performance liquid chromatography (HPLC). High performance liquid chromatography (HPLC) can judge the purification efficiency through impurity peaks.

[0146] The results are shown in Table 1. It can be seen from Table 1 that the present invention designs a new method for extracting placental active factors, including designing a dual-enzyme synergistic low-temperature enzymatic hydrolysis treatment method, which can extract placental active factors under mild conditions and improve the extraction efficiency. In addition, controlling specific enzymatic hydrolysis conditions can further improve the extraction rate.

[0147] Table 1

[0148]

[0149]

[0150] Test Example 2

[0151] This test example verifies the efficacy of the extracted placental active factors (taking the one prepared in Example 1 as an example).

[0152] Control group experiment: Mouse testicular interstitial cell line (TM3 cells) was selected, and TM3 cells stored in liquid nitrogen were quickly thawed and inoculated into T25 culture flasks. 10mL of culture medium (DMEM / F12 culture medium containing 5% fetal bovine serum and 1% double antibody) was added, and cultured in a 37°C, 5% CO2 incubator. Fresh culture medium was replaced after the cells adhered to the wall. When the cell density reached 80%-90%, 0.25% trypsin-EDTA was used for digestion, and the ratio of 1:3 was passaged (the cell density was about 1×10 5 cells / mL). The cells were cultured in a 6-well plate, and 2 mL of culture medium was added to each well. Culture time: Cell proliferation and testosterone secretion levels were detected after 24h, 48h, and 72h.

[0153] Experimental group experiment: Based on the above control group experiment, the culture medium was replaced with DMEM / F12 culture medium containing 5% fetal bovine serum, 1% double antibody, and 1% placenta active factor lyophilized powder prepared in Example 1, and the other conditions were the same.

[0154] Testing indicators and methods:

[0155] (1) Cell proliferation detection: CCK-8 kit was used to detect cell proliferation activity, and the absorbance value (OD value) was measured at a wavelength of 450 nm;

[0156] (2) Testosterone secretion detection: The culture supernatant was collected and the testosterone concentration was determined using an ELISA kit and calculated based on the standard curve.

[0157] The results are as follows Figure 1 and Figure 2 As shown, the placenta growth factor prepared by the present invention can effectively promote the proliferation of TM3 cells and the secretion of testosterone.

[0158] In summary, the present invention realizes for the first time a mild, efficient and low-energy placental active factor extraction process through the combination of dual enzyme synergy and low-temperature membrane separation, overcoming the problems of harsh enzymatic hydrolysis conditions and easy degradation of active ingredients in traditional methods. The obtained placental active factor has high biological activity and high stability, and can promote the proliferation of TM3 cells and the secretion of testosterone, providing a new therapeutic means for the treatment of male dysfunction and providing theoretical support for the application of placental extract products in the market.

[0159] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing a placenta active factor, characterized in that: The preparation method comprises: The placental tissue is crushed, the crushed placental tissue is mixed with protease and polysaccharidase for enzymolysis, the enzymolysis product is separated into solid and liquid, the clear liquid is collected, and the clear liquid is purified to obtain the placental active factor.

2. The method for preparing placental active factors according to claim 1, characterized in that: The fragmentation process includes cutting and grinding the placenta tissue; Preferably, the grinding process includes cryo-grinding, specifically including placing the cut placental tissue in a liquid nitrogen environment for cryo-grinding to obtain tissue particles.

3. The method for preparing the placenta active factor according to claim 1 or 2, characterized in that: The protease comprises at least one of neutral protease, lysozyme or chymotrypsin; Preferably, the polysaccharidase comprises at least one of cellulase, hyaluronidase or glucanase.

4. The method for preparing the placenta active factor according to any one of claims 1 to 3, characterized in that: The amount of the protease used is 1.5% to 2.5% of the wet weight of the placenta tissue after the crushing process; Preferably, the amount of the polysaccharidase used is 0.5% to 1.5% of the wet weight of the placenta tissue after the crushing process.

5. The method for preparing the placenta active factor according to any one of claims 1 to 4, characterized in that: The enzymatic hydrolysis time is 4 to 8 hours, and the temperature is 15 to 20°C; Preferably, the pH condition of the enzymatic hydrolysis is 6.5-7.

5.

6. The method for preparing the placenta active factor according to any one of claims 1 to 5, characterized in that: The purification process includes ultrafiltration and nanofiltration; Preferably, the molecular weight cutoff of the ultrafiltration membrane used in the ultrafiltration treatment is 10 kDa to 50 kDa; Preferably, the molecular weight cutoff of the nanofiltration membrane of the nanofiltration treatment is 200 kDa to 1000 kDa.

7. The method for preparing the placenta active factor according to any one of claims 1 to 6, characterized in that: The crushing process also includes a cleaning step before the crushing process; Preferably, the purification process further includes the steps of concentration and drying; Preferably, the drying process comprises freeze drying.

8. The method for preparing the placenta active factor according to any one of claims 1 to 7, characterized in that: The preparation method comprises: Cutting and grinding the placenta tissue, mixing the ground placenta tissue with a protease and a polysaccharidase, wherein the amount of the protease is 1.5% to 2.5% of the wet weight of the broken placenta tissue, and the amount of the polysaccharidase is 0.5% to 1.5% of the wet weight of the broken placenta tissue, and performing enzymolysis at 15 to 20° C. for 4 to 8 hours; The enzymatic hydrolysis product is centrifuged, the clear liquid is collected, the clear liquid is subjected to ultrafiltration and nanofiltration, and is concentrated and dried to obtain the placental active factor.

9. A placenta active factor, characterized in that: The placenta active factor is prepared by the preparation method of the placenta active factor according to any one of claims 1-8.

10. Use of the placenta active factor according to claim 9 in the preparation of an agent for promoting the proliferation of TM3 cells and the secretion of testosterone.

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