KR20110025266A - A method of producing placenta extract and a placenta extract - Google Patents

Abstract

PURPOSE: A method for preparing placenta extract containing rich physiologically active ingredients is provided to efficiently reduce contamination and to prevent liver injury. CONSTITUTION: A method for preparing placenta extract comprises: a step of pulverizing placenta in a first aqueous medium to obtain pulverized placenta solution; a step of separating the placenta solution into a first liquid part and first solid part; a step of adding the first solid part to hydrophilic solvent and separating into a second liquid part and second part; a step of mixing the first and second liquid parts; and step of adding surfactant and irradiating by UV ray.

Description

A method of producing placenta extract and a placenta extract}

The present invention relates to a method for preparing placental extract and to novel placenta extract.

The placenta is the organ that develops in the womb of a pregnant mammalian animal. The placenta is discharged from the uterus at birth and the placenta is called postpartum. The placenta contains nucleic acid components such as essential amino acids, melatonin, RNA and DNA, superoxide dismutase (SOD), hyaluronic acid (hyaluronic acid), antioxidants, cytokines, and insulin-like-growth factor Epidermal growth factor (EGFS) and senescent cell activating factors (SCAFS) are known to exist.

As a method of producing placenta extract, a method of hydrolyzing the placenta using strong acids such as 3N hydrochloric acid and strong alkali to obtain the product is known. However, this method has a disadvantage in that denatured or destroyed various physiologically active substances contained in the placenta during a long time high temperature treatment. In addition, as a method of producing a pig placenta extract for animal negative additives, the acidic hydrolysis of the pig placenta for 5 hours at 110 ℃, the hydrolyzate is filtered to remove the solid material, the placenta extract is removed After concentration and neutralization by treatment with anion exchange resin, the neutralized pig placenta extract is filtered, and the method is characterized in that it is prepared by sterilization for 15 minutes at 121 ℃.

However, even by the above method, there is still a need for a method of extracting the bioactive substance in the placental tissue without denaturing a bioactive substance such as a protein.

One embodiment of the present invention provides a method for preparing placental extract.

Another embodiment of the invention provides a placental extract.

Another embodiment of the present invention provides a pharmaceutical composition comprising the placental extract.

One embodiment of the present invention comprises the steps of grinding the separated placental tissue in a first aqueous medium to obtain a ground placental solution;

Separating the placental solution into a first liquid phase portion and a first solid phase portion;

Adding the first solid phase portion to a hydrophilic solvent in a second aqueous medium and separating the second liquid portion and the second solid phase portion;

Combining the first and second liquid portions and adding a surfactant for cell membrane degradation to obtain an extract solution; And

It provides a method for producing a placenta extract comprising; irradiating the ultraviolet light to the extract solution.

The method includes grinding the separated placental tissue in a first aqueous medium to obtain a ground placental solution. The placental tissue may be derived from an animal selected from the group consisting of human, pig and sheep. The first aqueous medium means water or a medium having water as a solvent. The first aqueous medium may be one that is not harmful to the human body. The first aqueous medium may be selected from the group consisting of water and phosphate buffered saline (PBS). The grinding may be performed using a blender or homogenizer. The grinding can make the placental tissue into small particles while physically lysing the cells in the placental tissue. The grinding may be to make the placental tissue to a size of, for example, 3mm or less. Such grinding may increase the surface area of the placental tissue into small particles, which dissolve useful components in the placental tissue, such as proteins, in the medium. The milled solution may be a homogenous solution. The grinding may be performed at 2 ℃ to 8 ℃.

The method may further comprise severing the separated placental tissue prior to obtaining the placental solution. The cutting may be performed using a miner or a chopper. Such cutting may be performed at 2 ° C to 8 ° C.

The method may comprise washing the placental tissue before and / or after the trimming step. For example, the placental tissue may be washed before and / or after the cutting step by immersing the placental tissue in a medium such as sterile water and removing the liquid portion. Washing can remove blood, which takes up a large amount of placental tissue. The washing performed after the cutting step may be performed using a hemocytosis buffer, for example, an RBC lysis buffer. Thus, blood cells can be dissolved to remove blood cells, and at the same time, blood cell components can be discharged into the medium. Hemolysis lysis buffer, eg, RBC lysis buffer, may be an aqueous solution containing NH ₄ Cl, KHCO ₃ and EDTA. The RBC lysis buffer can be, for example, an aqueous solution containing 8.3 g NH ₄ Cl, 1.0 g KHCO ₃ and 1.8 ml 5% EDTA in 1000 ml of water. Such washing may be performed at 2 ° C to 8 ° C.

The method also includes separating the placental solution into a first liquid phase portion and a first solid phase portion. Separating the placental solution into a first liquid phase portion and a first solid phase portion may be separating by centrifugation. The first liquid portion may be a portion in which an soluble component is dissolved in an aqueous medium, and the first solid portion may be a portion except for the first liquid portion. The first solid portion may include cell debris, cell organelles, and the like. The separation conditions can be easily selected by those skilled in the art of separating the cell lysate into soluble and insoluble portions of the medium. For example, the centrifugation may be performed at 10,000 to 15,000 rpm. The first liquid portion may be separated from the pellet of the centrifuged sample. The first liquid phase portion obtained after separation is taken, and the first solid phase portion is further used for extraction.

The method also includes adding a first solid portion to a hydrophilic solvent in a second aqueous medium and separating the second liquid portion and the second solid phase portion. The second aqueous medium may be the same as or different from the first aqueous medium described above. The second aqueous medium can be selected from the group consisting of water and PBS. The solvent may be an alcohol having 1 to 6 carbon atoms. The alcohol may be selected from the group consisting of methanol, ethanol, propanol, isopropanol, butanol, pentanol and hexanol. The solvent may be a final concentration of 5 to 30% by volume on a volume basis. The solvent may be one containing 5% by volume to 30% by volume of ethanol. The addition of the solvent may be to add while stirring. The stirring may be made in the range of 50 rpm to 100 rpm. The separation may be to separate by centrifugation. The second liquid portion may be a portion in which a component soluble in a hydrophilic solvent in the second aqueous medium is dissolved, and the second solid phase portion may be a portion other than the second liquid portion. The second solid phase portion may include cell debris, cell organelles, and the like. The separation conditions can be easily selected by those skilled in the art of separating the cell lysate into soluble and insoluble portions of the medium. For example, the centrifugation may be performed at 10,000 rpm to 15,000 rpm. The second liquid portion may be separated from the pellet of the centrifuged sample. The second liquid portion obtained after separation can be taken and combined with the first liquid portion.

The method includes combining the first and second liquid portions and adding a surfactant to the resulting combination. The surfactant may be one capable of dissolving the cell membrane. The surfactant may dissolve the cell membrane and at the same time do not inactivate the natural activity of the components, for example, proteins associated with the cell membrane. The surfactant may be a nondenaturing detergent. The surfactant may be selected from the group consisting of nonionic surfactants, amphoteric surfactants, and combinations thereof. For example, the surfactant may be Triton-X 100 (polyethylene glycol p- (1,1,3,3-tetramethylbutyl) -phenyl ether), Triton-X 114 (polyethylene glycol p- (1,1,3) , 3-tetramethylbutyl) -phenyl ether), Tween 20 (PEG (20) sorbitan monolaurate), Tween 80 (PEG (20) sorbitan monooleate), Nonidet P-40 (octylphenoxypolye Toxylethanol), Brij-35 (polyoxyethylene (23) dodecanol), Brij-58 (polyoxyethylene (20) cetyl ether), CHAPS (3-[(3-colamidopropyl) dimethylammonio]- 1-propanesulfonate), CHAPSO (3-[(3-colamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate) and combinations thereof. The surfactant may be included in 0.1% by volume to 2% by volume based on the volume of the combination. The surfactant may be one containing Triton-X 100 and Tween 20 at a final concentration of 0.3% (v / v) and 1% (v / v), respectively. The addition may be made under stirring. The stirring may be made of 50rpm to 100rpm. The addition may be performed at 2 ℃ to 8 ℃. After the addition may include the step of leaving for a time required for cell membrane lysis. The addition of such surfactants can dissolve the membranes of cells and / or viruses. Thus, undesirable biological contamination can be reduced.

The method also includes irradiating the combination with ultraviolet light. Irradiation of the ultraviolet light may be irradiated with conditions necessary to remove the viability of the biological material which may be included in the combination. The ultraviolet light may be one having energy in the range of 1000 J / m ² to 3000 J / m ² . Ultraviolet radiation can further reduce biological material contamination.

Other embodiments of the invention are FGF-2, Flt-3 ligand, Fractalkine, GRO, IL-1ra, IL-6, IL-7, IL-8, IL-12 (p70), IL-15, IP-10 Placenta extract comprising an active ingredient selected from the group consisting of, MCP-1, MDC, MIP-1b, PDGF-AA, PDGF-AB / BB, RANTES, sCD40L, sIL-2Ra, TNFa, VEGF and combinations thereof to provide. The active ingredient may be included at a concentration of at least 2 pg / ml, at least 3 pg / ml, at least 5 pg / ml, at least 7 pg / ml, or at least 9 pg / ml.

 The placental extract is FGF-2, Flt-3 ligand, Fractalkine, GRO, IL-1ra, IL-6, IL-7, IL-8, IL-12 (p70), IL-15, IP-10, MCP- 1, MDC, MIP-1b, PDGF-AA, PDGF-AB / BB, RANTES, sCD40L, sIL-2Ra, TNFa and VEGF; FGF-2, Flt-3 ligand, GRO, IL-1ra, IL-6, IL-8, IL-15, IP-10, MCP-1, MDC, MIP-1b, PDGF-AA, RANTES, sIL-2Ra And TNFa; FGF-2, Flt-3 ligand, GRO, IL-1ra, IL-6, IL-8, IL-15, IP-10, MCP-1, MDC, MIP-1b, PDGF-AA, RANTES, sIL-2Ra And TNFa; Containing FGF-2, Flt-3 ligand, GRO, IL-1ra, IL-6, IL-8, IL-15, IP-10, MCP-1, MDC, PDGF-AA, RANTES, and sIL-2Ra It may be.

Another embodiment of the present invention provides a pharmaceutical composition for preventing or treating liver damage in a mammal, comprising a placental extract as described above and a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" may be selected from the group consisting of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives. The pharmaceutical composition may further include an additive. The additives may include flavors, vitamins, and antioxidants. The carrier may be any pharmaceutically acceptable, for example, lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol as a diluent, magnesium stearate or talc as a lubricant, and polyvinylpyrrolidone as a binder. Or hydroxypropylcellulose. In addition, as a disintegrant, calcium carboxymethyl cellulose, sodium starch glycolate, potassium polyacrylic acid, or crospovidone, sweetener as white sugar, fructose, sorbitol, or aspartame, stabilizer as carboxymethyl cellulose sodium, beta-cyclodextrin, White lead, or xanthan gum, and preservatives may be methyl paraoxybenzoate, propyl paraoxybenzoate, or potassium sorbate.

The pharmaceutical composition may be formulated in conventional pharmaceutical formulations known in the art. The pharmaceutical compositions can be formulated and administered in the form of oral dosages, injections, suppositories, transdermal dosage forms, and nasal dosages. For example, the formulation may be a formulation for oral administration such as liquids, suspensions, powders, granules, tablets, capsules, pills, or extracts.

The pharmaceutical composition has a total daily dose of 0.1 g / kg to 100 g / kg, 0.1 g / kg to 50 g / kg as the extract, in order to obtain a prophylactic or therapeutic effect of liver damage, based on an adult as an active ingredient. Or 0.1 g / kg to 30 g / kg. The dosage may be appropriately increased or decreased depending on the type of liver injury to be treated or prevented, the degree of disease progression, the route of administration, sex, age, weight, and the like. The liver injury may be acute liver injury. The mammal may be a human.

According to the method for preparing the placental extract of the present invention, the placental extract including the active ingredient in the placental tissue can be efficiently produced. It can be extracted without deactivating the natural active ingredient in placental tissue, and can effectively reduce the contamination of biological material.

According to the placental extract of the present invention, it may contain a natural active ingredient in placental tissue.

According to the pharmaceutical composition of the present invention, it is possible to prevent or treat liver damage of a mammal.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1

(1) Preparation of Placental Extracts from Human Placental Tissues

Frozen human placental tissues were thawed at 2 ° C to 8 ° C. 5 kg of thawed placental tissues were washed with 50 L of water for injection. Part of the blood exposed outside the placental tissue was removed by washing. In addition, the umbilical cord and amnion were removed from placental tissue. Next, the placental tissue was cut using a mincer (Fuji Industrial Co., Ltd.). As a result, the placenta was composed of particles having a diameter of 3 mm or less. The sectioned placental tissue was washed in hemocytosis buffer containing 8.3 g NH ₄ Cl, 1.0 g KHCO ₃ and 1.8 ml 5% EDTA in 1000 ml of water to remove blood cells. The washing and cutting were carried out at 2 ℃ to 8 ℃.

Using the homogenizer (IKA), the sliced and washed placental tissue was used, and the PBS buffer (8 g NaCl, 0.2 g KCl, 1.44 g Na ₂ HPO _{4 in} 1 liter of water) corresponding to 10 times the volume of the sliced placenta tissue, and 0.24 g KH ₂ PO ₄ ) to obtain a ground placental tissue solution. The obtained solution was a substantially homogeneous solution. The grinding was carried out at 2 ℃ to 8 ℃.

Next, the ground solution was centrifuged (Beckmann, high-speed centrifuge), and centrifuged at 12,000 rpm, 4 ℃ for 20 minutes. After centrifugation, the supernatant was recovered and stored at 2 ° C to 8 ° C. The lower pellet was suspended by adding a 10% ethanol solution in PBS twice the volume of PBS used for the grinding, and left at 2 ° C. to 8 ° C. for 30 minutes. The suspension was centrifuged (Beckmann, high-speed centrifuge) and centrifuged at 12,000 rpm, 4 ° C. for 20 minutes. After centrifugation, the supernatant was recovered, combined with the already recovered supernatant, and the pellets were removed.

About 12 L of the combined supernatant was concentrated using a membrane with an average pore diameter of 10,000 Da until the protein concentration was 2 mg / ml. Each surfactant was added to the concentrated protein mixture so that the concentration was 0.3% by volume Triton X-100 and 1% by volume Tween 20. After addition the mixture was allowed to stand for 30 minutes at 2 ° C to 8 ° C.

Next, dialysis was performed using a membrane having an average pore diameter of 10,000 Da to remove the surfactant and the solvent. The mixture obtained after dialysis was irradiated with ultraviolet rays at an energy of 3000 J / m ² using UVC equipment manufactured by UVivatech. Bioactive substances in the final placenta extract thus obtained were analyzed.

(2) Component analysis of placenta extract

The cytokine content of the placenta extract obtained in this example was analyzed. Cytokine content was analyzed using Luminex instrument from Sartorius. A commercially available product (Lyneneck ^™ from Green Cross, Ltd., and Melsmon ^™ manufactured by Melsmon Corporation) was used as a control. Third party products are placental extracts extracted from placental tissue by strong acid and hydrolysis methods. Table 1 is a table showing the cytokine content in the placenta extract.

Table 1.

number Analysis target
Third party product
10nm filtration 10KDa Filtration Concentrate
Analyte Name Blank Lai neck ^TM Melsmon ^TM I'm after Enrichment After concentration One EGF 5 5 4 <3 2 <3 <3 2 Eotaxin 5 7 5 4 4 4 4 3 FGF-2 <3 <3 <3 2,889 866 3,544 3,470 4 Flt-3
Ligand 3 3 3 58 42 91 96 5 Fractalkine <3 <3 <3 22 <3 81 <3 6 G-CSF <3 <3 <3 <3 <3 <3 <3 7 GF-CSF 0 One 0 2 One 2 One 8 GRO <3 <3 <3 308 30 427 141 9 IFNa2 <3 <3 <3 <3 <3 <3 <3 10 IFNr <3 <3 <3 <3 <3 <3 <3 11 IL-1a One One One One 2 2 One 12 IL-1b <3 <3 <3 <3 <3 <3 <3 13 IL-1ra <3 <3 <3 87 44 140 105 14 IL-2 <3 <3 <3 <3 <3 <3 <3 15 IL-3 8 11 8 <3 <3 <3 7 16 IL-4 0 2 4 One One One 2 17 IL-5 3 3 3 3 3 3 3 18 IL-6 2 2 2 17 13 23 24 19 IL-7 <3 <3 <3 10 <3 13 9 20 IL-8 2 2 2 269 24 343 77 21 IL-9 One One One 2 One 2 One 22 IL-10 One One One 3 0 5 4 23 IL-12 (p40) <3 <3 <3 <3 <3 <3 <3 24 IL-12 (p70) 2 2 2 8 2 12 6 25 IL-13 One One 0 0 0 One One 26 IL-15 One 2 2 29 20 40 41 27 IL-17 2 2 2 2 2 2 2 28 IP-10 <3 5 One 229 16 355 133 29 MCP-1 4 5 3 567 150 766 455 30 MIP-3 6 6 8 10 6 10 8 31 MDC 8 8 9 25 10 42 33 32 MIP-1a 4 5 7 <3 <3 <3 <3 33 MIP-1b <3 <3 <3 51 6 72 32 34 PDGF-AA 2 3 2 59 14 78 62 35 PDGF-AB / BB 22 <3 <3 2 12 34 75 36 RANTES <3 <3 <3 1,299 217 2,120 1,866 37 sCD40L <3 <3 <3 131 <3 271 38 38 sIL-2Ra <3 <3 <3 18 9 28 28 39 TGFa 2 2 2 2 2 2 2 40 TNFa 3 3 3 4 4 4 4 41 TNFb <3 <3 <3 <3 <3 <3 <3 42 VEGF <3 5 <3 5 <3 18 5

In Table 1, the unit is pg / ml. In Table 1, 10 nm filtration is a result of measuring cytokines in the filtrate before and after filtering the final placental extract obtained in (1) using a membrane having a pore having an average pore diameter of 10 nm. The results of measurement of cytokines in the filtrate before and after performing filtration concentration using a membrane having a pore having an average pore size of 10 KDa during the extraction of the placenta extract of (1). Further, in Table 1, and melt seumon Lai neck ^{TM TM} represents a cytokine contents contained in the license-neck ^TM and Mel seumon ^TM (control) on the market. As shown in Table 1, the placental extract extracted by the method of Example 1 has a variety of cytokines and its content is higher than the control group. In particular, placental extract extracted by the method of Example 1 is FGF-2, Flt-3 ligand, Fractalkine, GRO, IL-1ra, IL-6, IL-7, IL-8, IL-12 (p70), IL -15, IP-10, MCP-1, MDC, MIP-1b, PDGF-AA, PDGF-AB / BB, RANTES, sCD40L, sIL-2Ra, TNFa, VEGF and combinations thereof But not included in the control group. Placental extract extracted by the method of Example 1 is FGF-2, Flt-3 ligand, Fractalkine, GRO, IL-1ra, IL-6, IL-7, IL-8, IL-12 (p70), IL- 15, at least 2 pg / ml, at least 3 pg / ml of IP-10, MCP-1, MDC, MIP-1b, PDGF-AA, PDGF-AB / BB, RANTES, sCD40L, sIL-2Ra, TNFa and VEGF, At least 5 pg / ml, at least 7 pg / ml, or at least 9 pg / ml.

(3) Analysis of efficacy of placenta extract

In order to confirm the effect of placental extract extracted in this example on acute liver disease, rats with acute liver injury obtained by administering carbon tetrachloride (CCl ₄ ) to rats were administered with placental extract and GOT (glutamate oxalacetate transaminase in serum). ), Enzyme activity and content of GPT (glutamate pyruvate transaminase), ALP (alkaline phosphatase) and T-BIL (total-bilirubin) were analyzed, and histopathological analysis was performed.

(3.1) Biochemical Analysis

Sprague-Dawley male rats (190 ± 10 g body weight) were randomly divided into 10 animals in each group as follows. 1st group: normal control group 2nd control group, 3rd group: 10 micrograms / horse extract extract group of this Example, 4th group: 50 micrograms / horse extract extract group of this example.

Rats of Groups 2 to 4 except Group 1 were administered twice a week for 6 weeks at a dose of 1 ml / kg of carbon tetrachloride on the first day of the experiment to cause acute liver injury in rats. Next, for the third group, subcutaneous administration to each rat for 2 weeks at a dose of 10 μg / 500 μl PBS / horse extract obtained in this example (0 (day 6 weeks after carbon tetrachloride administration), 1 day, 2 days) , 4 days, 1 week, 2 weeks), and the fourth group was administered subcutaneously to each rat for 2 weeks at a dose of 50 µg / horse extract obtained in this example (0 (day 6 weeks after carbon tetrachloride), 1 day, 2 days, 4 days, 1 week, 2 weeks). For group 1 (normal control group), rats administered physiological saline instead of carbon tetrachloride were administered subcutaneously with 500 μl of saline for 2 weeks (0 (the day after 6 weeks after carbon tetrachloride), 1, 2, 4, 1 week, 2 weeks). As for the second group (control), rats administered with carbon tetrachloride were subcutaneously administered with 500 μl of saline for 2 weeks (0 (day 6 weeks after carbon tetrachloride), 1, 2, 4, 1 week). , 2 weeks). Eight hours after the last dose on the last day of administration, blood was collected from the orbital vein of the experimental animals, and the activity or content of GOT, GPT, alkaline phosphatase (ALP) and T-BIL (total-bilirubin) was measured by a serum analyzer (ADVIA, BAYER). Measured. Table 2 shows the results of measuring the activity or content of GOT, GPT, ALP and T-BIL.

Table 2.

Experimental group The number of animals GOT GPT ALP T-BIL p (*) First group 10 153 ± 8.6 148 ± 9.4 98 ± 4.9 0.21 ± 0.04 2nd group 10 1615 ± 45.5 1659 ± 49.5 255 ± 13.8 0.35 ± 0.02 <0.05 3rd group 10 1135 ± 26.5 1015 ± 88.5 180 ± 9.8 0.3 ± 0.01 <0.05 4th group 10 771 ± 38.9 847 ± 21.5 328 ± 8.7 0.28 ± 0.01 0.0510

As shown in Table 2, the GOT, GPT, ALP and T-BIL levels were significantly higher in the control group (group 2) in which acute liver injury was induced by carbon tetrachloride (p <0.05). ). This indicates that acute damage to the liver was induced in rats by administration of carbon tetrachloride. In addition, the third and fourth groups to which the placental protein extract was administered showed a significant decrease in GOT, GPT, ALP and T-BIL levels (p <0.05) compared to the control group (p <0.05). In ALP). In addition, the value of the fourth group administered at 50 μg / horse was a little closer to the normal group compared to the third group administered at the dose of 10 μg / horse placenta extract obtained in this Example (p <0.05). This indicates that the placental extract obtained in this example lowers the elevated GOT, GPT, ALP and T-BIL levels of the rats induced by carbon tetrachloride.

(3.2) Histopathological Analysis

Eight hours after the last dose on the last day of placenta extract administration, animals were sacrificed, liver tissue was removed, fixed with formalin, and the lesion site was observed under a microscope. As a result, a large number of fibrin was observed in the control group (group 2), and the fibrin was decreased in the placenta protein extract administration group (groups 3 and 4). In addition, the control group was found to have a dark cerise cytoplasm and a distinct nucleus, but the placental protein extract administration group had a smaller nucleus and nucleolus, and the cytoplasmic eosinophility was decreased. In addition, the high dose administration group (Group 4) improved so that inflammatory reaction, proliferation of oocytes, and nodule formation were hardly observed.

According to the embodiment of the present invention, since the treatment method selected from the group consisting of strong acid, strong alkali and high temperature treatment is not included, the bioactive component in the placental tissue can be extracted in an active state without denaturation. In addition, it is possible to effectively reduce biological material contamination such as viruses through ultraviolet irradiation while using a surfactant that destroys the cell membrane during the extraction step.

Claims (13)

Grinding the separated placental tissue in a first aqueous medium to obtain a ground placental solution; Separating the placental solution into a first liquid phase portion and a first solid phase portion; Adding the first solid phase portion to a hydrophilic solvent in a second aqueous medium and separating the second liquid portion and the second solid phase portion; Combining the first and second liquid portions and adding a surfactant for cell membrane degradation to the resulting combination; And Irradiating ultraviolet light to the combination; a method of producing a placental extract comprising a. The method of claim 1, wherein the first aqueous medium is water or PBS. The method of claim 1, wherein separating the placental solution into a first liquid portion and a first solid portion is by centrifugation. The method of claim 1, wherein in the separating into the second liquid portion and the second solid phase portion, the second aqueous medium is the same as or different from the first aqueous medium. The process of claim 1 wherein in the separating into a second liquid phase portion and a second solid phase portion, the solvent is an alcohol having 1 to 6 carbon atoms. The method of claim 5, wherein the solvent has a final concentration of 5-30% (v / v) by volume. The method of claim 1 wherein the separating into the second liquid phase portion and the second solid phase portion is by centrifugation. The method of claim 1, wherein the surfactant for cell membrane degradation is Triton-X 100 (polyethylene glycol p- (1,1,3,3-tetramethylbutyl) -phenyl ether), Triton-X 114 (polyethylene glycol p- ( 1,1,3,3-tetramethylbutyl) -phenyl ether), Tween 20 (PEG (20) sorbitan monolaurate), Tween 80 (PEG (20) sorbitan monooleate), Nonidet P-40 ( Octyl phenoxypolyethoxylethanol), Brij-35 (polyoxyethylene (23) dodecanol), Brij-58 (polyoxyethylene (20) cetyl ether), CHAPS (3-[(3-colamidopropyl) Dimethylammonio] -1-propanesulfonate), CHAPSO (3-[(3-colamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate) and combinations thereof How to be. The method of claim 1 wherein the ultraviolet light is irradiated from the method of 1000J / m ² to 3000J / m ² range energy. FGF-2, Flt-3 ligand, Fractalkine, GRO, IL-1ra, IL-6, IL-7, IL-8, IL-12 (p70), IL-15, IP-10, MCP-1, MDC, Placenta extract comprising an active ingredient selected from the group consisting of MIP-1b, PDGF-AA, PDGF-AB / BB, RANTES, sCD40L, sIL-2Ra, TNFa, VEGF and combinations thereof. The composition of claim 10 wherein the FGF-2, Flt-3 ligand, GRO, IL-1ra, IL-6, IL-8, IL-15, IP-10, MCP-1, MDC, PDGF-AA, RANTES, and Placenta extract containing sIL-2Ra. 11. The placenta extract of claim 10, wherein said active ingredient is contained at a concentration of at least 2 pg / ml. A pharmaceutical composition for preventing or treating liver damage in a mammal, comprising a placenta extract according to any one of claims 10 to 12 and a pharmaceutically acceptable carrier.