阅读说明：本技术 一种含有多种生长因子的胎盘提取液的高效制备方法 (Efficient preparation method of placenta extract containing multiple growth factors ) 是由 姚玲玲 杨苗 罗保君 孟得龙 于 2020-04-17 设计创作，主要内容包括：本发明公开了一种含有多种生长因子的胎盘提取液的高效制备方法,采用两步提取法,其中,第一步的机械法对组织进行匀浆,获得一部分组织细胞,第二步将组织匀浆不易破碎的组织进行多种酶混合酶解,获得剩余的组织细胞,从而获得最大量的组织细胞,进而获得更多种类的活性因子,提高活性因子的获得量,所以本发明提供的制备方法既提高了活性因子的种类,又提高了活性因子的获得量。(The invention discloses a high-efficiency preparation method of placenta extract containing multiple growth factors, which adopts a two-step extraction method, wherein a mechanical method is adopted for homogenizing tissues to obtain a part of histiocytes, and a second step is adopted for carrying out mixed enzymolysis on the tissues which are not easy to break after tissue homogenization to obtain the rest histiocytes, thereby obtaining the maximum histiocytes, further obtaining more types of active factors and improving the obtaining amount of the active factors.)

1. A high-efficiency preparation method of placenta extract containing multiple growth factors is characterized by adopting a two-step extraction method, and specifically comprising the following steps:

step 1: washing placenta with normal saline in a sterile operating table and removing placental membrane tissue;

step 2: cutting placenta, adding normal saline, repeatedly centrifuging until the supernatant is colorless or yellowish, and removing supernatant to obtain placenta tissue;

and step 3: homogenizing the placenta tissue obtained by centrifuging, and filtering to obtain filtrate A;

and 4, step 4: freeze-thawing the filtrate A at-80 ℃ and room temperature, repeatedly freeze-thawing for multiple times, centrifuging the filtrate A, and filtering to obtain filtrate B;

and 5: adding pancreatin into the tissue which can not pass through the filter screen after homogenizing, performing enzymolysis for 30-150min at a constant temperature of 37 ℃ by using a shaking table, and stopping the enzymolysis to obtain an enzymolysis liquid A;

step 6: centrifuging enzymolysis solution A, adding physiological saline into the precipitate, centrifuging to obtain precipitate A, adding mixed enzyme solution composed of collagenase II, DNase and hyaluronidase into the precipitate A, performing enzymolysis at 37 deg.C for 40-200min, and stopping enzymolysis to obtain enzymolysis solution B;

and 7: filtering the enzymolysis liquid B, and centrifuging the filtrate to obtain a precipitate B;

and 8: adding erythrocyte lysate into the precipitate B, cracking for 3-10min, and centrifuging to obtain precipitate C;

and step 9: adding water for injection into the precipitate C, repeatedly freezing and thawing at-80 deg.C and normal temperature for 3-4 times, and centrifuging to obtain supernatant A;

step 10: mixing the filtrate B and the supernatant A, filtering, and concentrating the filtrate to obtain human placenta extract.

2. The method for efficiently preparing placental extract according to claim 1, wherein in step 3, physiological saline is added to the placental tissue obtained by centrifugation at a weight ratio of 1:5, and the placental tissue is homogenized by using a tissue homogenizer and then passed through a 400 mesh sieve.

3. The method for efficiently preparing placental extract containing multiple growth factors according to claim 1, wherein in step 5, the pancreatin solution is used in a mass concentration of 0.1% to 0.5%, and 0.2g edta is contained per 1L of pancreatin solution, and the weight ratio of tissue to pancreatin solution is 0.5 to 10: 1.

4. The method for preparing placental extract containing multiple growth factors according to claim 1, wherein in step 6, collagenase II is present in a mass concentration of 0.05% to 0.4% and DNase and hyaluronidase are present in a concentration of 20 IU/ml to 100IU/ml and 400 IU/ml, respectively, in the mixed enzyme solution.

5. The method for efficiently preparing placental extract containing multiple growth factors according to claim 4, wherein the weight ratio of precipitate A to mixed enzyme solution is 0.5-10: 1.

6. The method for efficiently preparing the placental extract liquid containing various growth factors according to claim 1, wherein in step 7, the physiological saline is added to the enzymatic hydrolysate B at a volume ratio of 1:3-10 before filtration, and then the mixture is filtered by a 100um filter screen.

7. The method for efficiently preparing placental extract according to claim 1, wherein the volume ratio of pellet B to erythrocyte lysate in step 8 is 1: 3-10.

8. The method for efficiently preparing placental extract according to claim 1, wherein in step 10, 0.45um filter is used for filtration.

Technical Field

The invention relates to a preparation method of placenta extract, in particular to an efficient preparation method of placenta extract containing multiple growth factors, belonging to the technical field of biology.

Background

The placenta is a tissue-bound organ between mother and child, which is formed by the combined growth of embryonic germ membrane and mother endometrium during human pregnancy, and is also an important organ for exchanging substances between fetus and mother. The fetus develops in the uterus and relies on the placenta to obtain nutrition from the mother, while the two parties remain fairly independent. The placenta also synthesizes various hormones, enzymes, cytokines, etc. to maintain normal pregnancy. Placenta is also a Chinese medicine called placenta hominis, also called placenta hominis, placenta hominis.

Placental extract generally refers to an effective bioactive component extracted from the placenta. The placenta extract contains a large amount of proteins, polypeptides and cytokines with various biological activities, participates in the maintenance of cell structures, the communication of motion information and the repair and regeneration of tissues, and has good effects of resisting photoaging, resisting oxidation, resisting wrinkles, whitening skin, healing wounds, repairing cells and the like.

The placenta extract has high application value in beauty treatment, anti-aging, emergency treatment and biological treatment, and the research on the placenta extract is a topic all over the world.

At present, the preparation method of the placenta extract mainly comprises the following steps: solvent extraction, direct extraction, and enzymolysis.

Solvent extraction method: the effective components are extracted from placenta tissue by compatibility with a solvent such as ethanol.

The direct extraction method comprises the following steps: freeze-drying placenta at low temperature, grinding, and making into lyophilized powder.

An enzymolysis method comprises the following steps: the placenta tissue is subjected to enzymolysis through protease, and then the enzymolysis liquid is centrifuged to obtain the extract.

The direct extraction method uses less protein than the other two methods because the protein obtained has a large molecular weight and is not easily absorbed.

Since the solvent extraction method has a limited tissue in which the solvent can be dissolved, not only the types of the active factors that can be extracted are small, but also the amount of the active factors that can be obtained is low.

In the enzymolysis method, because the specificity of the specimen is not easy to unify in the enzymolysis process, the digestion time is too long to hurt cells, the digestion time is too short to digest tissues sufficiently, each enzyme has stronger specificity, and complete enzymolysis on the tissues cannot be realized, the types of the obtained active factors are less in the extraction process, and the obtaining amount of the active factors is relatively lower.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a high-efficiency preparation method of a human placenta extract, which can improve the variety of active factors and the obtaining amount of the active factors.

In order to achieve the above object, the present invention adopts the following technical solutions:

a high-efficiency preparation method of placenta extract containing multiple growth factors is characterized by adopting a two-step extraction method, and specifically comprising the following steps:

step 1: washing placenta with normal saline in a sterile operating table and removing placental membrane tissue;

step 2: cutting placenta, adding normal saline, repeatedly centrifuging until the supernatant is colorless or yellowish, and removing supernatant to obtain placenta tissue;

and step 3: homogenizing the placenta tissue obtained by centrifuging, and filtering to obtain filtrate A;

and 4, step 4: freeze-thawing the filtrate A at-80 ℃ and room temperature, repeatedly freeze-thawing for multiple times, centrifuging the filtrate A, and filtering to obtain filtrate B;

and 5: adding pancreatin into the tissue which can not pass through the filter screen after homogenizing, performing enzymolysis for 30-150min at a constant temperature of 37 ℃ by using a shaking table, and stopping the enzymolysis to obtain an enzymolysis liquid A;

step 6: centrifuging enzymolysis solution A, adding physiological saline into the precipitate, centrifuging to obtain precipitate A, adding mixed enzyme solution composed of collagenase II, DNase and hyaluronidase into the precipitate A, performing enzymolysis at 37 deg.C for 40-200min, and stopping enzymolysis to obtain enzymolysis solution B;

and 7: filtering the enzymolysis liquid B, and centrifuging the filtrate to obtain a precipitate B;

and 8: adding erythrocyte lysate into the precipitate B, cracking for 3-10min, and centrifuging to obtain precipitate C;

and step 9: adding water for injection into the precipitate C, repeatedly freezing and thawing at-80 deg.C and normal temperature for 3-4 times, and centrifuging to obtain supernatant A;

step 10: mixing the filtrate B and the supernatant A, filtering, and concentrating the filtrate to obtain human placenta extract.

The efficient preparation method of the placenta extract containing various growth factors is characterized in that in the step 3, normal saline is added into the placenta tissue obtained by centrifugation according to the weight ratio of 1:5, a tissue homogenizer is used for tissue homogenization, and then a 400-mesh filter screen is used for filtering.

The method for efficiently preparing the placenta extract containing various growth factors is characterized in that in the step 5, the mass concentration of the pancreatin solution is 0.1-0.5%, 0.2g of EDTA is contained in 1L of the pancreatin solution, and the weight ratio of the tissue to the pancreatin solution is 0.5-10: 1.

The method for efficiently preparing the placenta extract containing various growth factors is characterized in that in step 6, in the mixed enzyme solution, the mass concentration of collagenase II is 0.05-0.4%, the concentrations of DNase and hyaluronidase are 20-100IU/ml and 400-1000IU/ml respectively, and the weight ratio of the precipitate A to the mixed enzyme solution is 0.5-10: 1.

The efficient preparation method of the placenta extract containing multiple growth factors is characterized in that in step 7, the normal saline is added into the enzymolysis liquid B according to the volume ratio of 1:3-10 before filtration, and then the mixture is filtered by a 100um filter screen.

The efficient preparation method of the placenta extract containing various growth factors is characterized in that in the step 8, the volume ratio of the sediment B to the erythrocyte lysate is 1: 3-10.

The method for efficiently preparing the placenta extract containing various growth factors is characterized in that in the step 10, the placenta extract is filtered by a 0.45um filter screen.

The invention has the advantages that:

(1) the preparation method comprises the following steps of (1) homogenizing tissues by a mechanical method to obtain a part of histiocytes, and performing mixed enzymolysis on the tissues which are not easy to break and homogenized tissues by a mechanical method to obtain the rest histiocytes, so that the maximum amount of histiocytes is obtained, more types of active factors are obtained, and the obtaining amount of the active factors is increased, so that the preparation method provided by the invention not only improves the types of the active factors, but also improves the obtaining amount of the active factors;

(2) the preparation method provided by the invention effectively reduces the influence of hemoglobin on the placenta extract by adding the step of removing red blood cells (specifically adopting cell lysate), so that the obtained placenta extract tends to be colorless.

Detailed Description

The present invention will be described in detail with reference to the following embodiments.

Preparation of human placenta extract

The preparation method of the human placenta extract provided by the invention adopts a two-step extraction method, and mainly comprises the following steps:

step 1: processing placenta

The placenta was taken from a healthy human under aseptic conditions, transported to the laboratory within 4h, and then washed 3 times with physiological saline in an aseptic station, and the placental membrane tissue was removed.

Step 2: centrifugation

Taking proper amount of placenta, and recording the weight as W₀Shearing placenta, adding normal saline, centrifuging at 2000rpm for 5min, discarding supernatant, centrifuging repeatedly until the color of the supernatant is colorless (or yellowish), and discarding supernatant to obtain placenta tissue.

And step 3: homogenate

Adding normal saline into placenta tissue obtained by centrifugation according to the weight ratio of 1:5, homogenizing the placenta tissue by using a tissue homogenizer, then sieving the placenta tissue by using a 400-mesh sieve to obtain filtrate A, collecting tissues which cannot pass through the sieve, weighing the tissues, and recording the weight as W₁。

And 4, step 4: repeated freeze thawing

Freezing and thawing the filtrate A at-80 deg.C and room temperature, repeatedly freezing and thawing for 4 times, centrifuging the filtrate A at 5000rpm for 5min, removing precipitate to obtain supernatant, filtering the supernatant with 40um filter screen to obtain filtrate B, and standing at 4 deg.C.

And 5: first enzymolysis

Adding pancreatin solution with mass concentration of 0.1-0.5% (every 1L of pancreatin solution contains 0.2g of EDTA) into the tissue which can not pass through the filter screen after homogenization according to the weight ratio of 0.5-10:1, performing enzymolysis for 30-150min at a constant temperature of 37 ℃ by using a shaking table, and stopping enzymolysis to obtain enzymolysis solution A.

Step 6: secondary enzymolysis

Centrifuging the enzymolysis solution A at 2000rpm for 5min, removing the supernatant, adding physiological saline into the precipitate, centrifuging the precipitate at 2000rpm for 5min, removing the supernatant to obtain a precipitate A, weighing the precipitate A, adding a mixed enzyme solution according to the weight ratio of 0.5-10:1, wherein the mixed enzyme solution consists of collagenase II, DNase and hyaluronidase, the mass concentration of the collagenase II is 0.05-0.4%, the concentration of the DNase is 20-100IU/ml, the concentration of the hyaluronidase is 400-1000IU/ml, carrying out enzymolysis at 37 ℃ for 40-200min, and stopping the enzymolysis to obtain an enzymolysis solution B.

And 7: centrifugation

Adding normal saline into the enzymolysis solution B according to the volume ratio of 1:3-10, filtering with a 100um filter screen, and centrifuging the filtrate at 2000rpm for 5min to obtain a precipitate B.

And 8: cracking

Adding erythrocyte lysate into the precipitate B according to the volume ratio of 1:3-10, lysing for 3-10min, centrifuging at 2000rpm for 5min, and removing the supernatant to obtain precipitate C.

And step 9: freezing and thawing again

Adding 50-500ml water for injection into the precipitate C, repeatedly freezing and thawing at-80 deg.C and normal temperature for 3-4 times, and centrifuging at 5000rpm for 5min to obtain supernatant A.

Step 10: mixing, and concentrating

Mixing the filtrate B and the supernatant A, filtering with 0.45um filter screen, and concentrating the filtrate to obtain human placenta extract which is colorless.

Secondly, detecting the types and the content of active factors in the human placenta extract

The types and the contents of active factors in the prepared human placenta extract are detected, and the detection results are as follows:

FGF

VEGF

FDGF-AB

IGF-1

EGF

TGF-β1

example 1

685.2pg/ml

913.8pg/ml

139.7pg/ml

489pg/ml

64.5pg/ml

132.6pg/ml

Example 2

756.3pg/ml

1117pg/ml

159.8pg/ml

598.6pg/ml

75.9pg/ml

165.2pg/ml

Example 3

557.2pg/ml

725.8pg/ml

128.9pg/ml

312.3pg/ml

52.6pg/ml

99.8pg/ml

Solvent extraction method

100.2pg/ml

65.5pg/ml

Not detected

Not detected

Not detected

Not detected

Enzymolysis method

231.5pg/ml

131.4pg/ml

86.2pg/ml

40.5pg/ml

Not detected

Not detected

Therefore, the preparation method provided by the invention not only improves the types of active factors in the human placenta extract, but also obviously improves the content of the active factors.

The 6 active factors of FGF, VEGF, FDGF-AB, IGF-1, EGF and TGF-beta 1 can effectively act with skin cells, promote the nutrition metabolism of epithelial cells, prevent the skin from being damaged by ultraviolet rays, free radicals and the like, promote the proliferation of collagen cells in the dermis layer, accelerate the repair of the skin after operation, and have the functions of smoothing fine wrinkles, delaying skin aging and the like.

The human placenta extract prepared by the invention contains a plurality of active factors such as FGF, VEGF, FDGF-AB, IGF-1, EGF, TGF-beta 1 and the like, and the content of the active factors is higher, so the using effect of the extract prepared into a preparation is better than that of the existing preparation.

It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the protection scope of the present invention.