Preparation method of placenta active factor as well as product and application of placenta active factor

Abstract

The invention relates to a preparation method of a placenta active factor as well as a product and application of the placenta active factor, and the preparation method comprises the following steps: soaking and rinsing a full-month normal delivery placenta, and homogenizing to obtain homogenate; repeatedly freezing and thawing the homogenate to collect a thaw solution, centrifuging the thaw solution, taking a supernatant, mixing the supernatant with a compound enzyme, and carrying out ultrasonic enzymolysis extraction to obtain an enzymolysis product; and centrifuging the enzymolysis product, taking the supernatant, and carrying out membrane filtration to obtain the placenta active factor. The compound enzyme comprises a combination of animal protein hydrolysis compound enzyme, alkaline protease, neutral protease and papain. The placenta tissue is extracted through cooperation of the specific compound enzyme and ultrasonic waves, the placenta tissue and the specific compound enzyme have a synergistic effect, the extraction efficiency can be further improved, the content of active ingredients in the extract is high, the biological activity is high, and the obvious anti-oxidation and anti-aging effects are achieved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a preparation method of a placenta active factor and a product and application thereof. Background Art

[0002] The placenta is a transitional organ for the exchange of substances between mother and child, formed by the embryonic membrane and the maternal endometrium during pregnancy in metatherians and eutherians. The fetus develops in the uterus and relies on the placenta to obtain nutrition from the mother, while both sides maintain considerable independence. The placenta also produces a variety of hormones that maintain pregnancy and is an important endocrine organ. In China, the placenta after childbirth is also a traditional Chinese medicine, called human afterbirth and placenta.

[0003] Human placental active factor is the most widely used drug among human placenta products. It contains a variety of active substances and enzymes necessary for human physiology, and plays an important regulatory role in maintaining the normal endocrine function of the human body. It can ensure that the human body is in a healthy state, improve the low function of various organs, delay the decline of physiological functions, and delay aging; the placental active factor is rich in a variety of nutrients, among which amino acids and minerals can supplement the various nutrients needed by the human body and activate human cells; anti-melanin factors and hyaluronic acid moisturizing factors have powerful beauty, whitening and moisturizing effects, and fundamentally delay aging; immune regulatory factors, interferon a, interferon beta, erythropoietin, nerve growth factor, interleukin, insulin-like growth factor, platelet-derived factor, transforming growth factor and colony stimulating factor have the effect of promoting cell differentiation and proliferation, and can effectively enhance immune repair; albumin, globulin, macroglobulin and a variety of placental globulin antibodies can significantly improve human immunity, effectively prevent the occurrence of diseases, and strangle the disease in its infancy; various vitamins and superoxide dismutase can effectively maintain the normal functions of various human functions, ensure normal metabolism, promptly remove aging cells and mutant cells generated at any time, eliminate the damage of free radicals to the human body, prevent and treat carcinogenesis, and effectively prevent the occurrence of cancer.

[0004] At present, the method of ethanol extraction is often used to obtain placental active factors, which easily denatures and inactivates some proteins (such as collagen) in the placental active factors, reducing the utilization effect of the placental active factors. In addition, the placental active factors prepared by this method contain ethanol, which is not suitable for use in skin care products. And the method of repeated low-temperature freezing and high-temperature fusion alternating treatment and long-term heat preservation treatment under acidic conditions not only has complicated preparation processes, but also leads to reduced biological activity of active substances in the placenta. Therefore, how to provide a preparation method of placental active factors with high biological activity has become a technical problem that needs to be solved urgently. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for preparing a placenta active factor and its product and application.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing a placenta active factor, the preparation method comprising the following steps:

[0008] (1) homogenizing a full-term normal placenta to obtain a homogenate;

[0009] (2) repeatedly freezing and thawing the homogenate to collect the thawed liquid, centrifuging the thawed liquid to obtain the supernatant, mixing it with the complex enzyme, performing ultrasonic enzymatic extraction, and obtaining the enzymatic hydrolysis product;

[0010] (3) centrifuging the enzymatic hydrolysis product, taking the supernatant and filtering it with a membrane to obtain the placental active factor;

[0011] The complex enzyme comprises a combination of animal protein hydrolyzing complex enzyme, alkaline protease, neutral protease and papain.

[0012] The placenta includes any one of human placenta, pig placenta, cow placenta, sheep placenta or deer placenta, or a combination of at least two of them.

[0013] In the present invention, animal protein hydrolysis complex enzyme, alkaline protease, neutral protease and papain are used together to obtain a complex enzyme, and the four enzymes cooperate with each other to synergistically enhance the efficiency of placental tissue extraction. In addition, the enzymatic hydrolysis process is combined with ultrasound to further improve the extraction effect.

[0014] The preparation method of the placental active factor involved in the present invention creatively adopts a specific enzyme combination combined with ultrasound to extract the placental tissue, thereby improving the extraction efficiency. At the same time, the prepared placental active factor has a high content of active ingredients and high biological activity, especially a very rich content of hepatocyte growth factor, which has a significant improvement effect on liver damage repair.

[0015] Preferably, the mass ratio of the animal protein hydrolyzing complex enzyme, alkaline protease, neutral protease and papain is (1-5):(1-4):(2-8):(1-5);

[0016] The specific values ​​in the above “1-5” may be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.;

[0017] The specific values ​​in the above “1-4” may be, for example, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, etc.;

[0018] The specific values ​​in the above “2-8” can be, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, etc. Other specific point values ​​within the numerical range can be selected, and they will not be described one by one here;

[0019] In the present invention, when the ratio of the complex enzyme is within the above specific range, it has a better extraction effect on placental tissue.

[0020] Preferably, the dosage of the complex enzyme is 1-5‰ of the mass of the placenta after rinsing, for example, it can be 1‰, 1.5‰, 2‰, 2.5‰, 3‰, 3.5‰, 4‰, 4.5‰, 5‰, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0021] Preferably, the placenta is soaked and rinsed before being homogenized in step (1).

[0022] Preferably, the solvents for soaking and rinsing are both physiological saline.

[0023] Preferably, the ratio of the placenta to normal saline is 1g:(0.5-2)mL, for example, it can be 1g:0.5mL, 1g:0.75mL, 1g:1mL, 1g:1.25mL, 1g:1.5mL, 1g:1.75mL, 1g:2mL, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0024] Preferably, the soaking time is 3-10 min, for example, it can be 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described here one by one.

[0025] Preferably, the soaking temperature is 16-25°C, for example, it can be 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described here one by one.

[0026] Preferably, the number of rinsing is 2-5 times, for example, 2 times, 3 times, 4 times, 5 times, etc.

[0027] Preferably, the rinsing time is 4-6 min / time, for example, it can be 4 min / time, 4.5 min / time, 5 min / time, 5.5 min / time, 6 min / time, etc.

[0028] Preferably, the rinsing temperature is 16-25°C, for example, it can be 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described here one by one.

[0029] Preferably, the placenta is shredded before homogenization in step (1).

[0030] Preferably, the placenta is mixed with physiological saline before homogenization.

[0031] Preferably, the ratio of the placenta to normal saline is 1g:(0.5-2)mL, for example, it can be 1g:0.5mL, 1g:0.75mL, 1g:1mL, 1g:1.25mL, 1g:1.5mL, 1g:1.75mL, 1g:2mL, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0032] Preferably, the rotation speed of the homogenate is 20000-40000rpm, for example, it can be 20000rpm, 22500rpm, 25000rpm, 27500rpm, 30000rpm, 32500rpm, 35000rpm, 37500rpm, 40000rpm, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0033] Preferably, the homogenization time is 20-40 min, for example, it can be 20 min, 22 min, 25 min, 28 min, 30 min, 32 min, 35 min, 38 min, 40 min, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described here one by one.

[0034] Preferably, the number of repeated freeze-thaw cycles in step (2) is 2-5 times, for example, 2 times, 3 times, 4 times, 5 times, etc.

[0035] Preferably, the centrifugal speed is 2500-5000rpm, for example, it can be 2500rpm, 2750rpm, 3000rpm, 3250rpm, 3500rpm, 3750rpm, 4000rpm, 4250rpm, 4500rpm, 4750rpm, 5000rpm, etc. Other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0036] Preferably, the centrifugation time is 8-15 min, for example, it can be 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc. Other specific point values ​​within this numerical range can be selected, which will not be described here one by one.

[0037] Preferably, the power of the ultrasound in step (2) is 25-50 W, for example, it can be 25 W, 28 W, 30 W, 32 W, 35 W, 38 W, 40 W, 42 W, 45 W, 48 W, 50 W, etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0038] Preferably, the time for ultrasonic enzymatic extraction is 15-30 min, for example, it can be 15 min, 18 min, 20 min, 22 min, 25 min, 28 min, 30 min, etc. Other specific point values ​​within this numerical range can be selected, and they will not be described one by one here.

[0039] Preferably, the centrifugation in step (3) is gradient centrifugation.

[0040] Preferably, the gradient centrifugation comprises: first centrifuging at a speed of 1800-3000g for 20-45min, taking the supernatant; then centrifuging at a speed of 6000-10000g for 20-45min, taking the supernatant; and finally centrifuging at a speed of 25000-30000g for 1-3h;

[0041] The above-mentioned “1800-3000g” may be, for example, 1800g, 2000g, 2200g, 2500g, 2800g, 3000g, etc.;

[0042] “20-45min” can be, for example, 20min, 25min, 30min, 35min, 40min, 45min, etc.;

[0043] “6000-10000g” may be, for example, 6000g, 7000g, 8000g, 9000g, 10000g, etc.;

[0044] “25000-30000g” may be, for example, 25000g, 26000g, 27000g, 28000g, 29000g, 30000g, etc.;

[0045] “1-3h” can be, for example, 1h, 1.2h, 1.5h, 1.8h, 2h, 2.2h, 2.5h, 2.8h, 3h, etc.

[0046] The present invention can further purify placental active factors with higher active components through the above-mentioned specific gradient centrifugation process.

[0047] Preferably, the membrane filtration is performed by filtering through 0.22 μm and 0.45 μm membranes in sequence.

[0048] In a second aspect, the present invention provides a placenta active factor, which is prepared by the preparation method described in the first aspect.

[0049] In a third aspect, the present invention provides a use of the placental active factor as described in the second aspect in the preparation of anti-aging and / or antioxidant products.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] The preparation method of the placental active factor involved in the present invention creatively adopts a specific enzyme combination combined with ultrasound to extract the placental tissue. While improving the extraction efficiency, the prepared placental active factor has a high content of active ingredients and high biological activity, especially a very rich content of hepatocyte growth factor, which has significant antioxidant, anti-aging and hemoglobin level-enhancing effects. DETAILED DESCRIPTION

[0052] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0053] The placentas involved in the following embodiments are collected from healthy pregnant women at full term, and after being tested for hepatitis B, hepatitis C, AIDS, syphilis and other viruses, the placentas with negative virus test results are screened;

[0054] The animal protein hydrolyzing complex enzyme is a product purchased from Shanghai Yuanye Biotechnology Co., Ltd. with model number S22221, the alkaline protease is a product purchased from Shanghai Yuanye Biotechnology Co., Ltd. with model number S10154, the neutral protease is a product purchased from Shanghai Yuanye Biotechnology Co., Ltd. with model number S10013, and the papain is a product purchased from Shanghai Yuanye Biotechnology Co., Ltd. with model number S10011.

[0055] The Caenorhabditis elegans used were purchased from the Caenorhabditis Genetics Center of the University of Minnesota (http: / / www.cbs.umn.edu / CGC / ).

[0056] Example 1

[0057] This embodiment provides a placenta active factor, and its preparation method is as follows:

[0058] (1) Immerse the qualified placental tissue in physiological saline at a ratio of 1 g:1 mL for 5 min at 18°C ​​to remove the blood, blood vessels, mucosa and umbilical cord from the placenta to obtain the soaked placental tissue. Rinse the soaked placental tissue in physiological saline at 18°C ​​for 2 times, 5 min each time.

[0059] (2) The rinsed placenta was cut into pieces, mixed with physiological saline at a ratio of 1 g:1 mL, and homogenized at 30,000 rpm for 30 min using a tissue homogenizer to prepare a homogenate.

[0060] (3) The homogenate was repeatedly frozen and thawed twice, centrifuged at 3000 rpm for 10 min, and the supernatant was mixed with a composite enzyme (the composite enzyme was composed of animal protein hydrolysis composite enzyme, alkaline protease, neutral protease and papain in a mass ratio of 2:2:5:3). The amount of the composite enzyme was 3‰ of the mass of the rinsed placenta. After mixing, the mixture was placed in a HSOT ultrasonic extraction equipment for extraction at an ultrasonic power of 30 W. After extraction for 20 min, the enzymatic hydrolysis product was obtained.

[0061] (4) The enzymatic hydrolysis product is subjected to gradient centrifugation, specifically, centrifugation at 2500g for 30 minutes, and the supernatant is taken; centrifugation at 8500g for 30 minutes, and the supernatant is taken; centrifugation at 27000g for 2 hours, and the supernatant is filtered through a 0.22μm and 0.45μm double-layer membrane, and freeze-dried to obtain the placental active factor.

[0062] Example 2

[0063] This embodiment provides a placenta active factor, and its preparation method is as follows:

[0064] (1) Immerse the qualified placental tissue in physiological saline at a ratio of 1 g of placenta to physiological saline at 20°C for 4.5 min, remove the blood, blood vessels, mucosa and umbilical cord in the placenta to obtain the soaked placental tissue, and then rinse the soaked placental tissue in physiological saline at 20°C for 3 times, 4 min each time.

[0065] (2) The rinsed placenta was cut into pieces, mixed with physiological saline at a ratio of 1 g:0.5 mL, and homogenized in a tissue homogenizer at 20,000 rpm for 40 min to prepare a homogenate.

[0066] (3) The homogenate was repeatedly frozen and thawed 4 times, centrifuged at 2500 rpm for 15 min, and the supernatant was mixed with a composite enzyme (the composite enzyme was composed of animal protein hydrolysis composite enzyme, alkaline protease, neutral protease and papain in a mass ratio of 5:3:2:1), and the amount of the composite enzyme was 2‰ of the mass of the rinsed placenta. After mixing, the mixture was placed in a HSOT ultrasonic extraction equipment for extraction at an ultrasonic power of 45 W. After extraction for 15 min, the enzymatic hydrolysis product was obtained.

[0067] (4) The enzymatic hydrolysis product is subjected to gradient centrifugation, specifically, centrifugation at 2000g for 25 min, and the supernatant is taken; centrifugation at 9000g for 28 min, and the supernatant is taken; centrifugation at 30000g for 1.5 h, and the supernatant is filtered through a 0.22 μm and 0.45 μm double-layer membrane, and freeze-dried to obtain the placental active factor.

[0068] Example 3

[0069] This embodiment provides a placenta active factor, and its preparation method is as follows:

[0070] (1) Immerse the qualified placental tissue in physiological saline at a ratio of 1 g:2 mL for 4 min at 22°C to remove the blood, blood vessels, mucosa and umbilical cord from the placenta to obtain the soaked placental tissue. Then rinse the soaked placental tissue in physiological saline at 22°C for 4 times, 6 min each time.

[0071] (2) The rinsed placenta was cut into pieces, mixed with physiological saline at a ratio of 1 g:2 mL, and homogenized in a tissue homogenizer at 35,000 rpm for 25 min to prepare a homogenate.

[0072] (3) The homogenate was repeatedly frozen and thawed three times, centrifuged at 4000 rpm for 12 min, and the supernatant was mixed with a composite enzyme (the composite enzyme was composed of animal protein hydrolysis composite enzyme, alkaline protease, neutral protease and papain in a mass ratio of 4:1:6:4). The amount of the composite enzyme was 5‰ of the mass of the rinsed placenta. After mixing, the mixture was placed in a HSOT ultrasonic extraction equipment for extraction at an ultrasonic power of 25 W. After extraction for 25 min, the enzymatic hydrolysis product was obtained.

[0073] (4) The enzymatic hydrolysis product is subjected to gradient centrifugation, specifically, centrifugation at 3000g for 20 minutes, and the supernatant is taken; centrifugation at 9500g for 25 minutes, and the supernatant is taken; centrifugation at 25000g for 3 hours, and the supernatant is filtered through a 0.22μm and 0.45μm double-layer membrane, and freeze-dried to obtain the placental active factor.

[0074] Comparative Example 1

[0075] This comparative example provides a placenta active factor, which is processed by the rinsed and shredded placenta in Example 1. The preparation method thereof is different from that in Example 1 only in that the compound enzyme does not contain animal protein hydrolyzing compound enzyme, and the reduced portion thereof is proportionally distributed to alkaline protease, neutral protease and papain, and then the corresponding placenta active factor is obtained after ultrasonic extraction, centrifugation and membrane filtration.

[0076] Comparative Example 2

[0077] This comparative example provides a placenta active factor, which is processed by the rinsed and shredded placenta in Example 1. The preparation method thereof is different from that in Example 1 only in that the compound enzyme does not contain alkaline protease, and the reduced amount thereof is proportionally distributed to the animal protein hydrolysis compound enzyme, neutral protease and papain, and then the corresponding placenta active factor is obtained after ultrasonic extraction, centrifugation and membrane filtration.

[0078] Comparative Example 3

[0079] This comparative example provides a placenta active factor, which is processed by the rinsed and shredded placenta in Example 1. The preparation method thereof is different from that in Example 1 only in that the composite enzyme does not contain neutral protease, and the reduced portion thereof is proportionally distributed to the animal protein hydrolysis composite enzyme, alkaline protease and papain, and then the corresponding placenta active factor is obtained after ultrasonic extraction, centrifugation and membrane filtration.

[0080] Comparative Example 4

[0081] This comparative example provides a placenta active factor, which is processed by the rinsed and shredded placenta in Example 1. The preparation method thereof is different from that in Example 1 only in that the composite enzyme does not contain papain, and the reduced amount of papain is proportionally distributed to the animal protein hydrolysis composite enzyme, alkaline protease and neutral protease, and then the corresponding placenta active factor is obtained after ultrasonic extraction, centrifugation and membrane filtration.

[0082] Comparative Example 5

[0083] This comparative example provides a placenta active factor, which is processed by the rinsed and shredded placenta in Example 1. The preparation method thereof is different from that in Example 1 only in that in step (3), the supernatant is not mixed with the complex enzyme and is directly subjected to ultrasonic extraction and centrifugal membrane filtration to obtain the corresponding placenta active factor.

[0084] Comparative Example 6

[0085] This comparative example provides a placenta active factor, which is processed by the rinsed and shredded placenta in Example 1. The preparation method thereof is different from that in Example 1 only in that the enzymatic hydrolysis process in step (3) is not carried out under ultrasonic conditions, and then the enzymatic hydrolyzate is centrifuged and membrane filtered to obtain the corresponding placenta active factor.

[0086] Test Example 1

[0087] This test example tests the DPPH-free radical scavenging and hydroxyl free radical scavenging effects of the placenta active factors prepared in Examples 1-3 and Comparative Examples 1-6.

[0088] (1) DPPH-free radical scavenging experiment

[0089] A 96-well plate was used, with three replicates in each group, and the system was 200 μL. Sample group: Take an appropriate amount of the extract sample prepared in the embodiment and comparative example, mix it with 100 μL of normal saline, so that the final concentration of the sample to be tested in the system is 1 mg / mL, and add 100 μL of 0.1 mM DPPH solution to the reaction system; control group: take 100 μL of normal saline, and then add 100 μL of 0.1 mM DPPH solution. After the reaction system is constructed, shake it in the dark for 10 minutes, and use an enzyme reader to test the absorbance at 520 nm.

[0090] The calculation method of clearance rate is as follows: Clearance rate (%) = [(A0-Ax) / A0] × 100%, A0 is the absorbance of the control group, and Ax is the absorbance of the sample group.

[0091] (2) Hydroxyl free radical scavenging experiment

[0092] The placenta active factor of each embodiment and comparative example in the sample group was diluted to 1 mg / mL, and the negative control group was respectively added with 0.1 part of ferrous sulfate, 0.1 part of ethanol, 0.1 part of salicylic acid, 0.1 part of sample or negative control, 1 part of plasma water, and 0.1 part of hydrogen peroxide to a 1.5mL centrifuge tube, the tube cover was tightly closed and shaken upside down, and kept warm in a 37°C water bath for 15 minutes, and the solution was moved to a 96-well plate, with three replicates per group. The absorbance at 510nm was measured by an enzyme marker. Hydroxyl radical scavenging rate (%) = {[A0-(AX-AX0)] / A0}×100%, wherein A0 is the absorbance of the negative control group, Ax is the absorbance of the sample group, and Axo is the absorbance of the sample background.

[0093] The test results are shown in Table 1.

[0094] Table 1

[0095] DPPH-free radical scavenging rate Hydroxyl radical scavenging rate Example 1 72.5％ 91.5％ Example 2 72.1％ 90.8％ Example 3 72.2％ 90.1％ Comparative Example 1 68.3％ 84.5％ Comparative Example 2 69.5％ 83.4％ Comparative Example 3 70.1％ 85.1％ Comparative Example 4 65.8％ 84.5％ Comparative Example 5 60.9％ 80.6％ Comparative Example 6 61.5％ 79.2％

[0096] According to the data in the table:

[0097] (1) It can be seen from Examples 1 to 3 that the placenta active factor prepared by the present invention has a high DPPH-free radical scavenging and hydroxyl free radical scavenging effect, showing significant antioxidant capacity.

[0098] (2) By comparing Example 1 with Comparative Examples 1-4, it can be seen that when the composite enzymes used in Comparative Examples 1-4 lack any one of the animal protein hydrolysis composite enzyme, alkaline protease, neutral protease and papain, the antioxidant effect of the placental active factor prepared therefrom is worse than that in Example 1. It can be seen that the present invention can have a better extraction effect on placental tissue by compounding and combining the four enzymes of animal protein hydrolysis composite enzyme, alkaline protease, neutral protease and papain, thereby ensuring the antioxidant effect of the extract.

[0099] (3) By comparing Example 1 with Comparative Examples 5 and 6, it can be seen that the present invention adopts the method of combining ultrasonic extraction with complex enzyme hydrolysis to further improve the extraction effect of placental tissue, and the prepared placental active factor has a higher antioxidant effect.

[0100] Test Example 2

[0101] This test example tests the effect of reducing lipofuscin concentration of the placenta active factors prepared in Examples 1-3 and Comparative Examples 1-6.

[0102] Synchronized L4 stage nematodes were cultured in a 20°C constant temperature liquid system (the culture system was a sterile aqueous solution containing 100 mM sodium chloride, 5.74 mM potassium dihydrogen phosphate, 44 mM potassium dihydrogen phosphate, 10 mM potassium citrate, 3 mM calcium chloride, 3 μM magnesium sulfate, 1.29 μM cholesterol, and an appropriate amount of inactivated OP50 Escherichia coli). The solvent control group was given an equal volume of normal saline.

[0103] Sample group: The placenta active factors prepared in Examples 1-3 and Comparative Example 6 were added to the culture system, and the concentration of the placenta active factors in the system was adjusted to 1 mg / mL.

[0104] After 5 days of culture, the nematodes were placed on a slide covered with agarose gel, anesthetized with 0.4M sodium azide, and the accumulation of lipofuscin in the nematodes was observed under a fluorescence microscope at an excitation wavelength of 405nm, and the fluorescence density was calculated. There were 20 nematodes in each group, and the experiment was repeated three times. The calculation results of the average fluorescence density change rate of lipofuscin in the nematodes are shown in Table 2.

[0105] Table 2

[0106] Mean fluorescence density reduction rate Example 1 18％ Example 2 17％ Example 3 16％ Comparative Example 1 15％ Comparative Example 2 13％ Comparative Example 3 14％ Comparative Example 4 15％ Comparative Example 5 12％ Comparative Example 6 11％

[0107] From the data in the table, we can see that:

[0108] (1) It can be seen from Examples 1 to 3 that the placental active factor provided by the present invention can effectively reduce the level of lipofuscin in nematodes, and many organisms in nature have the characteristic of lipofuscin accumulation with age, such as age spots produced after human aging. It can be seen that the product prepared by the preparation method of the placental active factor provided by the present invention has excellent anti-aging effect.

[0109] (2) By comparing Example 1 with Comparative Examples 1-4, it can be seen that when the composite enzymes used in Comparative Examples 1-4 lack any one of the animal protein hydrolysis composite enzyme, alkaline protease, neutral protease and papain, the placenta active factor prepared therefrom has a significantly inferior effect on reducing the lipofuscin level than Example 1. It can be seen that the present invention can have a better extraction effect on placental tissue by compounding and combining the four enzymes of animal protein hydrolysis composite enzyme, alkaline protease, neutral protease and papain, thereby enhancing the anti-aging effect of the extract.

[0110] (3) By comparing Example 1 with Comparative Examples 5 and 6, it can be seen that the present invention adopts the method of combining ultrasonic extraction with complex enzyme hydrolysis to further improve the extraction effect of placental tissue, and the prepared placental active factor has a higher anti-aging effect.

[0111] Test Example 3

[0112] This test example tests the effect of the placenta active factors prepared in Examples 1-3 and Comparative Examples 1-6 on increasing the hemoglobin level.

[0113] Anemia rat model establishment: 3-week-old weaned SPF female SD rats, 3 per cage. The rats were raised at a temperature of 25±2°C and a humidity of 50±10%. Six rats were randomly selected as the control group and fed with normal feed throughout the experiment. The remaining rats were fed with low-iron feed and deionized water for 3 weeks, 3-5 times in a row until the hemoglobin content was about 60g / L, and anemia model rats were obtained.

[0114] Grouping: Experimental group (anemic rats, 6 rats / group, low iron feed): Example 1-3 group (placenta active factor samples prepared in Example 1-3 group, 5 mg / kg / day), comparative example 1-6 group (placenta active factor samples prepared in comparative example 1-3 group, 5 mg / kg / day), the administration method was oral gavage;

[0115] Normal control group: normal rats, 6 rats / group, fed with normal diet and gavaged with the same volume of saline as the experimental group;

[0116] Anemia control group: anemic rats, 6 rats / group, fed low-iron diet and gavaged with the same volume of saline as the experimental group.

[0117] The intragastric administration was continued for 5 weeks, and the rats in each group were fasted for 12 hours after the last intragastric administration.

[0118] Injection method: 3% sodium pentobarbital was injected at a dose of 0.2 mL / 100 g according to the rat weight before dissection to anesthetize the rat, and the abdominal aorta blood was taken after the rat was anesthetized. The rat blood hemoglobin (HGB) and red blood cell count (RBC) were measured, and the test results are shown in Table 3.

[0119] Table 3

[0120] Hemoglobin (g / L) <![CDATA[Red blood cell count (1×10 12 / L)]]> Example 1 121.6 5.5 Example 2 120.4 5.1 Example 3 119.7 5.2 Comparative Example 1 113.6 4.9 Comparative Example 2 115.6 4.8 Comparative Example 3 113.6 4.9 Comparative Example 4 114.6 4.5 Comparative Example 5 107.6 4.3 Comparative Example 6 109.6 4.4 Normal control group 125.1 5.5 Anemia control group 52.4 3.3

[0121] According to the data in the table:

[0122] (1) It can be seen from Examples 1 to 3 that the placenta active factor prepared by the present invention has a significant effect of increasing hemoglobin levels and promoting blood cell regeneration.

[0123] (2) By comparing Example 1 with Comparative Examples 1-4, it can be seen that when the composite enzymes used in Comparative Examples 1-4 lack any one of the animal protein hydrolysis composite enzyme, alkaline protease, neutral protease and papain, the therapeutic effect of the placenta active factor prepared therefrom is worse than that in Example 1. It can be seen that the present invention can have a better extraction effect on placental tissue by compounding and combining the four enzymes of animal protein hydrolysis composite enzyme, alkaline protease, neutral protease and papain.

[0124] (3) By comparing Example 1 with Comparative Examples 5 and 6, it can be seen that the present invention uses a combination of ultrasonic extraction and compound enzyme hydrolysis to further improve the extraction effect of placental tissue, and the prepared placental active factor has a better effect.

[0125] Test Example 4

[0126] This test example tests the composition and content of the placenta active factor prepared in Example 1.

[0127] The composition and content of the placenta active factor prepared in Example 1 were tested by the protein chip method, and the results are shown in the following table.

[0128] Table 4

[0129]

[0130] It can be seen from the data in the above table that the placenta active factor prepared by the present invention is rich in effective ingredients, including hyaluronic acid, collagen, albumin, various amino acid components, and active factors SCF and bFGF and hepatocyte growth factor, etc., especially the hepatocyte growth factor is extremely rich, indicating that the placenta active factor prepared by the present invention can have a significant improvement effect on liver damage repair.

[0131] Test Example 5

[0132] This test example analyzes the safety of the placenta active factor prepared in Example 1, and the results are shown in the following table.

[0133] Table 5

[0134]

[0135] In summary, the method for preparing placental active factors involved in the present invention creatively adopts a specific enzyme combination combined with ultrasound to extract placental tissue. While improving the extraction efficiency, the prepared placental active factors have a high content of active ingredients and high biological activity, especially a very rich content of hepatocyte growth factor, which has significant antioxidant, anti-aging and hemoglobin level-enhancing effects.

[0136] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0137] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0138] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for preparing a placenta active factor, characterized in that: The preparation method comprises the following steps: (1) homogenizing a full-term normal placenta to obtain a homogenate; (2) repeatedly freezing and thawing the homogenate to collect the thawed liquid, centrifuging the thawed liquid to obtain the supernatant, mixing it with the complex enzyme, performing ultrasonic enzymatic extraction, and obtaining the enzymatic hydrolysis product; (3) centrifuging the enzymatic hydrolysis product, taking the supernatant and filtering it with a membrane to obtain the placental active factor; The complex enzyme comprises a combination of animal protein hydrolyzing complex enzyme, alkaline protease, neutral protease and papain.

2. The preparation method as claimed in claim 1, characterized in that The mass ratio of the animal protein hydrolyzing complex enzyme, alkaline protease, neutral protease and papain is (1-5):(1-4):(2-8):(1-5); Preferably, the dosage of the complex enzyme is 1-5‰ of the weight of the placenta after rinsing.

3. The preparation method as claimed in claim 1, characterized in that The placenta in step (1) is further soaked and rinsed before being homogenized; Preferably, the solvents for soaking and rinsing are both physiological saline; Preferably, the ratio of placenta to saline is 1 g: (0.5-2) mL; Preferably, the soaking temperature is 16-25°C and the soaking time is 3-10 minutes; Preferably, the rinsing is performed 2-5 times, the time is 4-6 min / time, and the temperature is 16-25°C.

4. The preparation method according to claim 1, characterized in that: Before the homogenization in step (1), the placenta is cut into pieces; Preferably, the placenta is mixed with physiological saline before homogenization; Preferably, the ratio of placenta to saline is 1 g: (0.5-2) mL; Preferably, the homogenization speed is 20000-40000 rpm, and the homogenization time is 20-40 min.

5. The preparation method according to claim 1, characterized in that: The number of repeated freezing and thawing in step (2) is 2-5 times; Preferably, the centrifugal speed is 2500-5000 rpm and the time is 8-15 min.

6. The preparation method according to claim 1, characterized in that: The power of the ultrasound in step (2) is 25-50W; Preferably, the ultrasonic enzymatic extraction time is 15-30 min.

7. The preparation method according to claim 1, characterized in that: The centrifugation in step (3) is gradient centrifugation; Preferably, the gradient centrifugation comprises: first centrifuging at a speed of 1800-3000g for 20-45min and taking the supernatant; then centrifuging at a speed of 6000-10000g for 20-45min and taking the supernatant; and finally centrifuging at a speed of 25000-30000g for 1-3h.

8. The preparation method according to claim 1, characterized in that: The membrane filtration is performed by filtering through 0.22 μm and 0.45 μm membranes in sequence.

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

10. Use of the placenta active factor according to claim 9 in the preparation of anti-aging and / or antioxidant products.

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